Cytochrome p450 Protocols

1 The Role of Methods in the Discovery of the Cytochromes P450 Ronald W. Estabrook 1. Introduction The progress of science is frequently the result of the mtroductlon of new types of instruments, and new instruments bring with them new techmques that must be mastered if one 1sto optimize new methods when seeking answers to specific questlons. The hlstory of cytochrome P450 (P450) research 1s mtlmately woven with seminal discoveries resulting from the development and application of new methodologies and new instruments. It is the purpose of this article to reflect back on research in the premolecular biology era by describing the impact of a few specific methodologic advances that played a crucial role m the discovery of the P45Os.In this article, I have chosen to introduce personal reflections, m the form of anecdotes, of experiences I recall. Most of these occurred nearly 40 years ago when the group at the University of Pennsylvania set out on the journey that led to the discovery of the functions of P45Os. For some readers, this article may provide some answers to the questions often posed to a grandfather who enjoys describing what life was like before television and computers. 2.50 Years Ago Today, we take for granted many of the procedures that now serve as the foundation of much of the research on P45Os. Let me describe a few general examples of condltlons in the laboratory m the 195Os,conditions that we recognize today to be essential for research but whose history is poorly appreciated. 2.1. The A vailability of High Quality Chemicals My mtroduction to biochemical research as a graduate student involved the isolation and characterization of diphosphopyridine nucleotide (DPN+) [NicoFrom Methods m Molecular Biology, Vol 107 Cytochrome P450 Protocols Edlted by I R Phtlkps and E A Shephard 0 Humana Press Inc , Totowa, NJ 1

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tinamtde adenine dinucleotide (NAD+)] and triphosphopyridine nucleottde (TPN’) [Nicotinamide adenine dmucleotide (NADP+)]. I vividly remember the smell of boiling pig liver mmce as tt fell from the old commercial hamburger grinder into a boiling buffer solution held m a large metal trash can, the temperature of which was maintained hot by the introduction of a continuous jet of superheated steam; and the subsequent need to squeeze this hot denatured mince of liver through cheesecloth in order to obtain the fluid, which was then mixed with acid-washed charcoal to adsorb the pyridine nucleotides. After isolating sufficient amounts of these coenzymes, it was necessary to synthesize sodium isocitrate from chloral hydrate and succmate anhydride m order to reduce the isolated NADP+ to form mcotinamide adenme dmucleotide phosphate (NADPH), by using a crude extract contammg isocttrate dehydrogenase In this way, one could study electron transport by the cytochromes present m Keilin-Hartree heart-muscle extracts. The luxury of going to a catalog, finding the order number for a chemical from a listing of many choices of purity and sources, followed by a telephone call to a vendor m order to request delivery by overnight mail, was not included m anyone’s dreams of the future. 2.2. Centrifuges I remember as a young post-doctoral fellow at the University of Pennsylvania (circa 1954) being introduced to the “high-speed” centrifuge that was needed for isolating mitochondria and microsomes from liver homogenates. This high-speed centrifuge was an mstrument of terror-it was free-standing on the bench in the cold room encased in a flimsy wire cage-but the cage was not to protect one from the flying missile that would result if the rapidly spmning rotor disconnected from the motor shaft, but rather to protect one from catching a necktie or shirt sleeve in the rapidly spinning instrument. The noise was deafening, and one avoided the confined area of the cold room when homogenates were being fractionated by centrtfugation. Today, we apply the methodology of centrifugation as routine, but in the past it was a challenging and daunting expertence. 2.3. The Quality of Water One might expect that something as basic as the quality of the water used in experiments would not be a problem. However, there were secret formulas and magical qualities associated with the preparation of sufficiently pure water that could be used for the preparation of buffers and other reagents. The glass still, filled with a purple solution of permanganate, was always boiling above the sink and the condensate collected as a steady drip into a large reservoir vessel. Part of the lore associated with the preparation of mitochondria and microsomes from liver homogenates was the need to use “freshly distilled” water (because

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of the presumed lower content of absorbed carbon dioxide). I remember those flashes of anxiety that overwhelmed one on the trip home from the laboratory m the late eveningdid I forget to shut off the still before leaving the lab? Some of the problems associated with contaminants in water remain today and one has to be on constant vigil that spurious results are not the consequence of an adventitious unwanted agent associated with the use of impure water. Of equal importance to the purity of water is the cleanliness of the glassware used for an experiment. Gone are the days of the “acid-bath” (that treacherous mixture of hot nitric and hydrochloric acids) that was used to sear unwanted residues from the glassware. The holes in many pans of pants and shirts serve as scars formed when a dirty flask with a trace of water splattered acid as it was dipped into this hot aqua regia. Replacement of this technique by soaking glassware in large Jars of dark greenish-brown solutions of potassnun chromate dissolved in concentrated sulfuric acid accomplished the same purpose, but thankfully was stopped because of the toxicity of the chromate that was washed into the sewer system. 2.4. Animals By today’s standards, the care and feeding of animals (rodents) used for experiments was primitive in the 19.50s.I recall having to keep rats in wire cages on the roof of the Hospital of the University of Pennsylvania exposed to all the elements. In the summer, when the temperature approached 100°F in Philadelphia, it was impossible to do experiments because the exposed animals were severely stressed and the liver homogenates prepared from these animals were tinted green from bile pigments. One can tell many additional tales of “hardships” compared to the ease of conducting experiments today. However, the key to the discovery of the P45Os was not the availability of chemical reagents, the ease of fractionating homogenates by centrifugation, the purity of water, or the cleanliness of glassware, or the well-being of the rats used as the source of microsomes-it was the development and application of unique instruments. 3. Discovery of the P45Os 3.7. Difference Spectrophofometry In the early 1950s Britton Chance (1) and his colleagues (2) designed and developed a new class of spectrophotometer. The design of these instruments was based on the principle of “difference measurements.” These instruments allowed one to study small absorbance changes in the spectral properties of cellular pigments (e.g., reduction of hemeproteins or bleaching of carotinoids). Briefly (see Fig. lA), the heart of the instrument is a mechanical means of “choppmg “ a monochromatic light beam so that it alternately passes, with

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Fig. 1. (A) Schematic drawmg of the “split-beam” monochromater used for wavelength scannmg difference spectrophotometry. Light from a tungsten light source 1s focused on a divided grating generating two nonparallel light beams that are separated by the partitioned disk mounted on a 60-rpm motor (light-beam attenuator). The two separated light beams pass alternately through each cuvet, where the photomultlplier detects the amount of light transrmtted Note that the placement of the cuvets close to the surface of the photomultipher reduced the solid angle of scattered light. (Adapted with permlsslon from ref. 8). (B) Difference spectra showmg the measurement of P450 using different preparations of rat-liver microsomes diluted to 1 mg of protein/ml of reaction volume. Each sample was diluted in 0.1 A4 potassmm phosphate buffer, pH 7.4, and a small amount of sodium chthiomte added as a reducing agent Then the diluted sample was divided equally mto two cuvets and a baseline of equal light absorbance established. The contents of one cuvet were then gassed with carbon monoxide for 30 s and the difference spectra recorded The pretreatment of animals before the preparation of liver mlcrosomes is indicated

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equal light intensity, through two cuvets (sample and reference cuvets) in the “wavelength-scanning mode.” In a second design, the “dual beam mode,” light of two wavelengths balanced mechanically to give beams of equal light intensity, passed through a single cuvet. In both cases, the two beams of light impinged on the front surface of a photomultrplier generating a voltage. The synchronous electronic demodulation of the voltages generated by the photomultiplier permitted the measurement of a “difference” m light absorbed. This method had two immediate positive advantages: light scattering associated with the turbidity of particulate material in the cuvets IS self-compensated, and the small differences in the voltages generated by the photomultiplier can be continuously amplified without the need to adjust an external compensating voltage negating the “background” absorbance of light or the wavelengthdependent change m light intensity of the monochromatic beams of light. A schematic representation of a commercial adaptation of thus origmal instrument is shown m Fig. 1A. It was this unique mstrument that permitted Martin Klingenberg (3) to first observe spectrophotometrically the presence of a ptgment of rat liver microsomes absorbing light at 450 nm. The power of Chance’s spectrophotometric technique was immediately recognized and the Johnson Research Foundation (Department of Biophysics of the School of Medicine, University of Pennsylvama, Philadelphta) became a Mecca for sctenttsts from around the world interested in monitoring absorbance changes associated with a large variety of intracellular colored pigments (e.g., chlorophyll, cytochromes, peroxidases, flavoproteins, carotinoids, and so on). It was this environment that led Klingenberg to discover (3) the carbon monoxrde (CO) binding pigment in liver microsomes, a prgment that we know today as P450. The successof Klingenberg’s studies is directly attributable to the availability of the wavelength-scanning spectrophotometer developed by Chance and his colleagues. Earlier studies by Ryan and Engel (4), and Strittmatter and Ball (5) characterized a hemeprotem of microsomes, which they recognized as distinct from the cytochromes of mitochondria. However, they failed to observe the presence m microsomes of a CO-binding pigment because they had to use detergents to clarify the turbid microsomal suspenstonsfor spectrophotometric studies by a conventional spectrophotometer, a procedure we know today requn-es great caution to avoid denaturation of the P450 by a detergent. For histortcal accuracy, rt is worth noting that G. R. Williams (a colleague at the Johnson Foundation who was studying changes in the steady-state reduction of liver mitochondrial cytochromes with B. Chance) first observed the P450 CO-binding pigment of liver microsomes. Klingenberg acknowledged this contribution by a footnote m his initial paper (3). Even so, Klingenberg deserves full credit for the characterizatron of the CO-binding pigment associated with the microsomal fraction of liver since he was the first to demonstrate

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its enzymatic reduction and the details of its spectrophotometric properties David Garfinkel (6) extended the original studies of Klingenberg by showing the consequences of exposure of mtcrosomes to acidic pH, digestion with pancreatm, and sodium cholate, leading to a denaturation of the P450. Of interest IS the report by Patgen (7), who claimed that “the red color of Isolated microsomes results from the presence of adsorbed hemoglobin.” The pitfalls associated with spectrophotometrtc measurements m the presence of bound hemoglobin, as well as cytochromes present in contammatmg fragments of mitochondrial membranes, has been described in detail by Estabrook et al. (s). The technique of difference spectrophotometry remains today the method of choice for quantifying P45Osm tissue extracts, and for momtormg changes m oxidation/reduction or the spin state of P45Os or cytochrome bS as they function m the membrane-bound form Figure 1B presents an example of the use of drfference spectrophotometry to measure changes m the content of P450 m hver microsomes exposed to different mducmg agents as well as the differences in the spectral properties of the CYPlA family of induced P45Os.The observation (9) that the absorbance maximum (448 nm) of the CO-complex of reduced P450 present in liver microsomes from animals treated with methylcholanthrene IS shifted to lower wavelengths when compared to the P450 of liver microsomes of phenobarbital-treated animals (450 run), was one of the early indicators that many different types of P45Osexist. 3.2. Oxygen Electrode A second technique championed by Chance was the application of the oxygen electrode to momtor changes in oxygen utilization during reactions of biological oxidations. This polarographic technique had a histortcal relattonship to the Johnson Research Foundation. The preparation of glass-encased platinum electrodes that could be inserted mto nerve tissue for the measurement of changes m oxygen tension followmg a nerve impulse was described by Bronk et al. m 1941 (10). (Detlev Bronk was the Director of the Johnson Research Foundation m the 1940s). It was Davies and Brmk (II), colleagues of Bronk and members of the Johnson Research Foundatron, who first described the general use of the oxygen electrode for measurmg tissue oxygen tension and changes in oxygen concentrations m stn-red and unstirred solutions. The use of an oxygen electrode is very simple, and it has replaced the tedium associated with the use of the Warburg manometer for measuring respiration. As shown in Fig. ZA, a membrane-covered electrode of the type developed by Clark et al. (12) is inserted in a glass or plastic container that is closed to prevent back diffusion of oxygen from air into the reaction solution. The oxygen electrode is connected to a simple electronic system that consists of a source of polarizing voltage and a means of measuring small changes in voltage associ-

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Fig. 2 (A) Schematic drawing of the oxygen electrode mounted in a closed reactton vessel The electrode 1sconnected to a polarizing voltage and an amphfying ctrcutt to measure the small changes in voltage associated with the uptake of oxygen. Additions to the reaction chamber are made with mlcroptpets inserted mto the small openmg m the removable plug. (Adapted with permtssion from ref. 13). (B) Measurement of oxygen utthzatton by purified CYP3A4 reconstituted with NADPH-P450 reductase Trace A shows the rate and extent of oxygen utthzed followmg the addrtron of 150 ~LV NADPH. Trace B is the result obtained when the experiment IS repeated with catalase mrtially present m the reaction mixture The addition of catalase after 10 min for expenment A shows the accumulation of hydrogen peroxide durmg the P450 reaction

ated with the changes m oxygen concentration (13) These electrodes are readily available commercially at a very modest cost. It remams a puzzle (to me) why so few people working on P450 ever use an oxygen electrode to measure oxygen uptake. Because the P45Os are monooxygenases and atmospheric oxygen is the primary substrate for a P450 reaction, it would seem important to monitor oxygen utilization. The oxygen electrode method played a key role m the discovery of P450 functions. The mitial collaboratrve studies (14) carried out with Cooper and Rosenthal

mvolved

the measurement

of the stolchlometry

of the mono-

oxygenase (mixed-function oxidase) reaction of steroid C2 1-hydroxylation as catalyzed by bovine adrenal microsomes in the presence of NADPH. The successof these experiments was dependent on the accurate measurement of oxygen uptake using an oxygen electrode. Small changes m the rate of oxygen uptake following the addition of the substrate, 17a-hydroxyprogesterone, showed that the reaction did conform to the proposed stoichiometry of a mixed function oxidation reaction (15), i.e. 1 mole of NADPH is oxidized for each mole of oxygen consumed during the formation of 1 mole of product. This was the first demonstration that a P450 catalyzed reaction was a mixed-functton oxidation; ironically, very few other examples exist m the literature confirm-

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ing this stoichiometry, although rt 1s generally accepted that P450 reactions comply with this equatton. The usefulness of measurmg oxygen consumptron during studies of P450catalyzed reactions is of greatest importance when attempting to determine the extent of “uncoupling” of the P450. The factors that influence the degree of uncoupling of the cycle of P450 function and the “short-circuiting” of this cycle for the formation of oxygen radicals and hydrogen peroxide 1s still not fully understood. As shown in Fig. 2B, the presence of catalase changes the measured rate of oxygen uptake (in this case using purified CYP3A4 reconstituted with NADPH-P450 reductase) showing the accumulation of hydrogen peroxide during the reaction. Likewise, measurements with the oxygen electrode will munediately reveal the extent of lipid peroxidation occurring durmg a P450 reaction. Individuals frequently forget that oxygen 1san essential reactant for P450 and that consequently the hmitation of oxygen can lead to an underestimate of the measured activity of a P450. One example of this pitfall occurred during recent studies (16) m our own laboratory, where we wanted to determine the properties, in vivo, of a recombmant P450 expressed at high levels in Escherichia coli as it catalyzed the I7a-hydroxylation of progesterone. Earher studies had failed to show that metabohsm could occur m viva by recombmant P45Osexpressed at high levels m bacteria, because one did not fully appreciate that a limrtatron of oxygen occurs rapidly as a result of the large background respiration of E. coli. Only by the contmuous monitormg of the oxygen concentration in the reaction medium were the experiments successfully carried out. Again, this is an example where use of the oxygen electrode to measure oxygen concentrations was essential. 3.3. The Photochemical Action Spectrum In the early 1960s we had established that the CO-binding pigment of Klingenberg was present in microsomes prepared from the adrenal cortex and we had established that the stoichiometry for the 21-hydroxylation of 17ahydroxyprogesterone in the presence of NADPH, atmospheric oxygen, and bovine adrenal mtcrosomes conformed to that of a mixed-function oxidase as proposed by Mason (15). Ryan and Engel had earlier reported (4) that the 2 1-hydroxylation of progesterone catalyzed by bovine adrenal mrcrosomes was inhibited by CO. The next step appears m retrospect, obvious, measure the photochemical action spectrum for the light reversal of carbon monoxide inhibition of the steroid hydroxylation reaction by followmg the procedure developed many years earlier by Warburg (17). But it was not that simple. First, one had to obtain various mixtures of CO and oxygen in order to establish an atmosphere of mixed gassesthat would result in different degrees of mhibition of the steroid hydroxylation reaction. Here the persuasrve powers of Cooper were

Discovery of P450 essential (remember that Pennsylvania is home to a large number of coal mines, and mixtures of CO and oxygen were well-known to be explosive). Second, the Harrison Department of Surgical Research (where Cooper and Rosenthal had their laboratory) was in a hospital and we realized the impropriety of releasing CO into the atmosphere without adequate precautions. And thud, it was deemed necessary to work in subdued light to reduce background photodissociation of the P450-CO complex. We elected to first try a simple approach. Using special Thunberg-type anaerobic cuvets designed by Cooper, the steroid substrate was added to the microsomal suspension and an atmosphere of CO/oxygen established by repetitive evacuation and gassing of the sealed cuvet. The reaction was mitiated by tipping in a solution of NADPH from the sidearm of the modified Thunberg tube. The fact that the reaction system was contained m a cuvet allowed us to insert the cuvet into a spectrophotometer set for a specific wavelength of monochromatic light. By this method, we could irradiate the sample for designated periods of time m hopes of measuring a reversal of the mhibinon by CO. Also, m this way we could easily change the wavelength of light by a simple adjustment of the monochromator and thereby obtam (we hoped) our photochemical action spectrum. However, this didn’t work Even using the new grating monochromators that Chance had purchased to give a higher intensity of light was unsuccessful, as was the use of a high-Intensity slide projector with filters taped on the front surface of the focusing lens. We then turned to using an old Warburg apparatus (that Rosenthal had brought with him to the United States when he fled Berlin in the mid-1930s), an Osram zenon lamp (the type that light up football stadiums at night), and a series of borrowed hght filters to give monochromatic light of high intensity and purity (a diagram of an early version of this apparatus is shown in Fig. 3A). The design that evolved from this original experiment has been described by Rosenthal and Cooper (18) and Cooper (19). This apparatus provided success (ZU), irradiation of a sample of adrenal microsomes incubated with NADPH and steroid under an atmosphere of CO and oxygen showed a reversal of inhibition. These experiments were not without moments of crisis. I recall the intense heat generated by the xenon lamp and the associated problems of mamtaining the temperature of the reaction mixture m the shaking Warburg manometer vessel. A filter made up of cupric chloride dissolved in concentrated sulfuric acid was devised to remove the infrared contribution of the incident light beam, a situation that often resulted in generating boilmg sulfuric acid after a full day of experiments. An additional problem that soon became apparent was the need to quantify and normalize the energy of the incident light striking the reaction vessel. The location and purchase of a bolometer from Germany was a frustrating adventure solved by Rosenthal. As one can

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Fig. 3 (A) Schematic drawing of the photochemtcal action spectra irradiation apparatus used to study the role of P450 m xenobtottc metabolism by liver mtcrosomes. Light from a htgh-mtenstty xenon lamp passes through a heat filter made of a coppersulfate solutron and then through narrow-band interference filters. The monochromatic light 1s focused on the bottom of a Warburg respu-ometer vessel by a reflectmg mm-or. Various gas atmospheres made of different CO/Oz ratios flow through the manometer tubmg (Adapted with permission from ref. 20). (B) The photochemtcal action spec-

trum for reversal of CO mhlbttton of the 21-hydroxylatton of 17o+hydroxyprogesterone catalyzed by bovine adrenal mtcrosomes Each point represents an experiment using a different wavelength of light. The partition coefficient at 452 nm was 0.64 when using a ratto of CO/O2 of 2 2 Results are normahzed by destgnating the extent of reversal at 452 nm as 1 (Adapted with perrmsston from ref. 21)

appreciate, the preparation for one day’s experiments was long and tedious, and conducting an experiment required a symphony of efforts by a number

of individuals. The discovery that Klmgenberg’s pigment was the terminal oxtdase for steroid hydroxylatron reactions was announced in 1963 m a volume of Biochemicha Zeitschrtft dedrcated to Warburg (20). The use of the photochemical action spectrum method to show the role of P450 m drug and xenobrotlc metabolism (Fig. 3B) was published m 1965 (21). At this time Orrenms, Dallner, and Ernster (22) showed that the oxrdative demethylation of amlnopyrme catalyzed by liver microsomes was mhrbrted by CO. Thus the link was firmly established that P450 served as the termmal oxrdase for a vartety of reacttons. The photochemical action spectrum method was not new, it was earher applied by Warburg for the study of CO inhrbltion of cytochrome oxtdase. What was new was the use of this methodology to provide the defimttve

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proof of the role of P450 in the oxygenation of many different types of chemtcals. Also, studies of P450 by this method presented a greater challenge because of the difficulty m photo-dissociating the CO from the reduced heme of P450. High intensities of light, a high ratio of oxygen to CO m the gas phase of the reaction chamber, and a sensitive means of monitoring product formation during the enzymatic reaction, were all essential to the success of this experimental protocol. One can understand why this method did not become a general procedure for studying P450 and its role in many oxygenation reactions. 4. Reflections

Forward

It has been over 40 years since Klingenberg first recognized the presence in liver mtcrosomes of an enzymattcally reducible pigment that absorbs hght at 450 nm in the presence of carbon monoxide. Critical to this discovery was the use of the wavelength-scannmg recording spectrophotometer capable of measurmg small changes m light absorbance of turbid particulate suspensions. Would P450 have been discovered if such a spectrophotometer was not available? I think the discovery of P450 would have been delayed by at least a decade as evidenced by the work of Omura and Sato (23) in the mid- 1960s. It was not new techniques that were critical in defining the role of P450 in many oxygenation reactions. The “oxygen electrode” was firmly established 20 years earlier as a convenient method to monitor and quantify oxygen utihzation. Likewise, the application of the photochemical-actton spectrum was demonstrated by Warburg three decades earlier. These methods were not new, what was new was the application of these methods to the study of the spectrophotometric curiostty we now call P450. The ability to establish a stoichiometry for oxygen used and to demonstrate directly the reversal of CO mhibition by irradiation of samples with monochromatic light in the blue region of the spectrum (i.e., at 450 nm) all provided the essential elements for discovery. The intervening years have seen similar discoveries that serve as landmarks in the progress made m understanding the many roles of different P45Os m metabolism. In the 197Os,the ability to resolve different P45Osby gel-electrophoresis techniques, coupled with the purification of P45Os,led to the development of specific antibodies for P45Os.It was the availability of these antibodies that served as an essential element for ushering in the overwhelmmg progress made by applying the techniques of molecular biology. Today, using wellestablished techniques of clonmg and recombinant expresston, an mvestigator can easily purify a gram of a human P450 for detailed biochemical or biophysical studies

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It is rewarding to see how far science has progressed in the last four decades from those simple experiments carried out under less than ideal conditions. We stand at the edge of understanding the molecular details of how a P450 functions and what small changes are needed to alter substrate specificity or rates of interaction with its partners essential for electron transport. The techniques of crystallography, mutagenesis, nuclear magnetic resonance (NMR) analysis, and electrochemically-driven reactions will provide the next generation of researchers studying P45Os an exciting opportunity for new dtscovertes. Acknowledgments Supported m part by a grant from the National Institutes of Health (GM16488). References 1. Chance, B. (1947) Stable spectrophotometry of small density changes Rev Scz Instrum 18,601-609 2. Yang, C. C and Legallats, V (1954) A rapid and sensitive recording spectrophotometer for the vtsible and ultraviolet regton 1. description and performance Rev Scl Instrum 25,801~807 3. Klmgenberg, M (1958) Pigments of rat liver microsomes. Arch Bzochem Biophys. 75,376-386. 4. Ryan, K. J and Engel, L I (1957) Hydroxylation of steroids at carbon 2 1. J Bzol Chem. 225, 103-l 14 5 Strittmatter, C F and Ball, E G. (1952) A hemochromogen component of liver microsomes. Proc Nat1 Acad SCI 38, 19-25. 6 Garfinkel, D. (1958) Studies on pig liver mtcrosomes. I. enzymic and pigment composttion of different microsomal fractions. Arch Blochem Blophys 77, 493-509. 7 Patgen, K. (1956) Hemoglobm as the red pigment of mtcrosomes. Bzochem Blophys Acta 19,297-299. 8. Estabrook, R. W , Peterson, J A., Baron, J., and Hildebrandt, A (1971) The spectrophotometric measurement of turbid suspensions of cytochromes associated with drug metabolism, in Methods zn Pharmacology vol 2 (Chignell, C F , ed ), Appleton-Century-Crofts, pp. 303-350. 9. Alvares, A. P , &hilling, G., Levin, W , and Kuntzman, R (1967) Studies on the induction of CO-bmdmg ptgments m liver microsomes by phenobarbital and 3-methylcholanthrene Blochem Blophys Res Commun 27,462-468 10 Bronk, D. W., Brink, F , and Davies, P W (1941) Chemical control of respiration and activity in peripheral nerve Am J Physlol. 133,224,225 11 Davies, P. W. and Brink, F (1942), Mtcroelectrodes for measuring local oxygen tension in animal &sues. Rev SCL Instruments 13,524-533 12. Clark, L. C., Wolf, R., Granger, D and Taylor, Z. (1954) Continuous recording of blood oxygen tensions by polarography. J Applied Physzol 6, 189-l 93

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13. Estabrook, R. W (1967) Mitochondnal respiratory control and the polarographic measurement of ADP/O ratios. Methods Enzymol. 10,4147. 14. Cooper, D. Y., Estabrook, R W , and Rosenthal, 0 (1963), The stoichiometry of C21 hydroxylation of steroids by adrenocorticol microsomes. J Biol Chem 238, 1320-1323. 15 Mason, H S (1957) Mechanisms of oxygen metabolism, m Advances zn Enzymology, vol. 19 (Nerd, F. F., ed.), Interscience, NY, pp. 79-233 16. Shet, M S., Fisher, C. W., and Estabrook, R. W (1996) The function of recombinant cytochrome P45Os m intact E. coli cells: the 17whydroxylation of progesterone and pregnenolone by P45Oc 17 Arch Blochem Bzophys 339,2 18-225. 17. Warburg, 0. (1949) Heavy Metal Prosthetzc Groups and Enzyme Action Oxford University Press, Clarendon, London. 18. Rosenthal, 0. and Cooper, D. Y (1967) Methods of determining the photochemical action spectrum. Methods Enzymol 703-707. 19. Cooper, D. (1973) Dtscovery of the function of the hemeprotem P-450: a systematic approach to scientific research. Life Sci. 13, 115 l-l 16 1. 20. Estabrook, R W., Cooper, D. Y., and Rosenthal, 0. (1963), The light reversible carbon monoxide mhibition of the steroid C2 1-hydroxylase system of the adrenal cortex. Blochem. Zelt 338,741-755. 21 Cooper, D. Y , Levm, S., Narasimhulu, S , Rosenthal, O., and Estabrook, R. W. (1965) Photochemical action spectrum of the terminal oxidase of mixed function oxtdase systems. Science 147,400-402 22. Orremus, S., Dallner, G., and Ernster, L. (1964) Inhibition of the TPNH-lurked lipid peroxidation of liver microsomes by drugs undergoing oxidative demethylation, Blochem Blophys Res Commun. 14,329-334 23. Omura, T and Sato, R (1964a), The carbon monoxide-binding pigment of liver microsomes. I. evidence for its hemoprotein nature. J Blol Chem. 239,2370-2378

Cytochrome P450 Nomenclature David R. Nelson 1. Introduction 1.1. Approaching 1000 With genome projects spewing forth DNA sequence at tens of Megabases per year, the problem of genetic nomenclature becomes daunting. In the field of cytochrome P450 (P450), there are more than 750 sequences and they are accumulating rapidly. At the current rate, there will be more than 1000 P450 sequencesby late 1998 or early 1999. For these data to be accessible and useful they must be sorted and categorized in some meaningful way. To that end, a cytochrome P450 nomenclature system was devised (I). This system relied on evolutionary relationships as depicted in phylogenetic trees or dendrograms derived from the P450 protein sequences. The whole collection of sequences represents the P450 superfamily, with families and subfamilies being arbitrarily defined as distinct clusters on the tree. Orrgmally, 40% identity was used as a cutoff for family membership and 55% was used for subfamily membership. Since the inception of the system, both numbers have crept downward. The actual decision to mclude a sequence m an existing group largely depends on how it clusters on a tree and not so much on the absolute percent identity, which is more or less a rule of thumb. The most recent published compilation of the nomenclature is given m Nelson et al. (1996) (2). More up-to-date mformation is available at http://drnelson.utmem.edu/ne1sonhomepage.html. 1.2. Looking to the Future This system has been adopted by the P450 research commumty and survived intact for
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of sequences.One of the worst of these is the lack of two digit numbers for new P450 families. The original scheme reserved blocks of numbers for different groups of organisms. CYP families 149 were reserved for animals, families 5 1-69 for lower eukaryotes, 7 l-99 for plants, and 101 and up for bacteria. The plant P450 field has exploded recently and family numbers for plants in the two digit range have already run out. Lower eukaryotes are soon going to have the same problem, with animals not far behind. The solutron has been to assign blocks of three digit numbers to accommodate the overflow. Lower eukaryotes will be assigned numbers m the 501-699 range. Plants ~111take 701-999. Ammals will have to jump to the 301-499 range because bacteria are already in the lower hundreds, 101-299. It is conceivable that three digit numbers will still not be enough, but it should hold us well into the next decade. 2. The Process of Assigning a Name New P450 sequences frequently appear m Genbank. It is a time-consummg process to search Genbank and sort out new sequences from those that have already been named. This is especially difficult for the expressed sequencetags (ESTs), because the protein sequence has to be derived from the nucleotide sequence and there are many frameshifts in the EST data. Highest priortty goes to sequences submitted by researchers for naming. These sequences undergo an initial scan that ahgns them against the appropriate subset of P450 sequences. The alignment algorithm is part of the MacVector package of sequence analysis software (IBI). A new sequence can be ahgned with a folder containing about 200 sequences m less than a min. The folders contam bacterial (55), plant (200), insect (104), CYPl, 2, 17, and 21 (168), and other (236) sequences,a combined total of 763 sequences.A few quick scansrdentrfies the best match to a known P450 sequence. The new sequence is then added to a master alignment containing the best match. The revised ahgnment is converted to an integer array 730 positions long by the number of sequences m the ahgnment. A 200 sequence alignment m Integer form occupies 584 kb. A program called COMPARE is used to compare the new sequence to every other sequence in the array. The percent identity 1s given for each sequence. Often, a name can be assigned based on these numbers alone. If a sequence is 85% identical to a known sequence, then it will clearly fall mto the same subfamrly and a name can be given without further analysis. If the best matches are less obvious, say in the 40-50% range, then it is usually necessary to construct a tree. A difference matrrx is computed from the array by comparing every sequence with every other sequence, a process that can take a while for a large array. The difference matrix 1sstored and used to compute a tree by the UPGMA method. There is the option to use several different scoring matrices

Cytochrome P450 Nomenclature

17

m computmg the difference matrix, but, for simphcity, it is usually best to use a unit matrix that only counts identities. The numbers are later adjusted for multiple mutations at the same site as described m Nelson and Strobe1 (3). A tree will show the relationships between sequences better than the numbers from the COMPARE program. Examination of the tree in addition to the COMPARE output is sufficient to assign a name. Some sequences naturally fall midway between existing families or subfamilies. These sequencesare difficult to assign, because they are sensitive to small changes m the alignment or to the choice of scoring matrix. If two different outcomes are found with the corrected unit matrix data or a PAM250 tree, choose the PAM250 tree over the unit-matrix tree. This has been the case with the bacterial CYPlO5 family. Sometimes this is just a judgment call and the result could go either way. The results are partly historical. For example, the CYP2D subfamily is quite different from the other CYP2 family members. When CYP2Dl was first reported, there was a fairly large gap between the CYP2 family and the CYPl family. Some, but not all, of the 2D sequences were more than 40% identical to other CYP2 family members. At the time it seemed prudent to keep the 2D cluster within the CYP2 family. As more sequences have been reported, the break between the CYPI and CYP2 families is less clear, and it is now apparent that the 2D subfamily should have been given full family status Because of the large number of factors that go mto assigning a name and the possible involvement of confidential sequences, the Committee on Standardized Cytochrome P450 Nomenclature asks that individuals do not name their own P450 genes. This will avoid the possibility of assignmg identical names for different sequences Instead, individuals are encouraged to request help in naming a new gene, by submitting the sequence by e-mail or fax. The appropriate addresses are given on the P450 web page at http://drnelson.utmem.edu/ nelsonhomepage.html. These sequences will be held in confidence until they are published or appear m Genbank. The name that is assigned will be posted as well as the species that it is from and the name of the person or lab submitting the sequence. If the sequence is already in the database, the individual who submitted the sequence will be notified. In the caseof Arabidopsis, where there are about 175 EST sequences for P45Os, the matching ESTs may be crossreferenced as time permits. If one has concerns about patents and does not want the sequence he or she is working on to be known, then that individual should not submit the sequence for naming. Private names, for sequenceswithout species given or with explicit requests to withhold EST cross-references, will not be honored. A nomenclature cannot be secretive. If that is not compatible with one’s wishes, then that individual must hold his or her sequence back until these matters have been resolved.

18

Nelson

3. Benchmarks for P450 Sequence Alignment The construction of a tree is dependent on the sequence alignment and also on what parts of a sequence are included m the analysts. This is becoming even more clear as phylogemes are compared from different sets of sequence data and even nonsequence data using cladtstics. In a compartson of mitochondrtal genes coding for 13 different protein sequences over a wide range of organisms, analysis of all the sequence data gave the wrong tree, wtth good statistical support (4). Using Just hydrophobtc ammo-actd codons gave poor results, hydrophilic and charged amino-acid codons gave better results. The best tree, which was in agreement with expected relationships among these organisms, was produced using only the parts of the genes that coded for important threedimensional features m these protems. These findings have implicattons for P450 sequence alignments and tree building for nomenclature purposes. More reliable results may be obtained by leaving out the poorly conserved regions of the sequencesthat are the most drfficult to align. The regions remammg should include the sequence benchmarks that are used to align the sequences. These benchmarks are few in the N-terminal region, and more abundant m the C-termmal from the I-helix to the end of the P450 molecule. Many of these benchmark regions are specific for families or higher taxonomrc groups, like bacterial or mttochondrial P45Osthat tend to cluster together. When aligning difficult sequenceswith low simtlarity to other sequences,pay particular attention to these benchmark regions. Ahgmnent algorithms cannot use this information, and they are often quite far off when it comes to distantly related P450 sequences.This 1sunderstandable when a bacterial sequencemay be only 89% identical to a mammalian sequence. At the N-terminal of many plant and animal P45Osis a prolme-rich sequence. It 1soften PPGP, but it may be (hydrophobic; hdr)PGP. This sequence is usually followed four residues later by P(hdr)(hdr)G(polar)(hdr) as m PIIGNL. This region follows the N-termmal membrane-anchor sequence, which 1susually not conserved and cannot be aligned with any certamty. About 20 ammoacid residues later, there is a common sequence, KYG or RYG. This tripeptide is present in CYP51, lanosterol-14a-demethylase, which is one of the more ancient eukaryotic P45Osinvolved m cholesterol biosynthests. It is also present m CYP7, CYP8, and CYP19 sequences,but not m CYP4 or CYP52 (fatty-acid and alkane hydroxylases) nor in bacterial-like sequences The two basic P450 forms are called E-like and B-like P45Os for eukaryote and bacterial-like, respectively. The ancient origin of the KYG motif 1sfurther confirmed by its presence m CYPl 10 from Anabaena, a cyanobactermm. In modified form (ELG) it 1s also seen in CYPl 1B and CYP102 (P450BM-s) from Bacillus megaterium, where it occurs Just before the p l-l strand. Both CYPl 10 and

Cytochrome P450 Nomenclature

19

CYP102 are E-like bacterial P45Os,suggestmg the fundamental split between E-like and B-like P450 structures occurred before eukaryotes developed. One E-like P450 crystal structure is known of CYP102, and several B-like P450 crystal structures are known of CYPlOl (P450,,,) and CYP108 (P450,,,). For a comparison of these structures, see Peterson and Graham-Lorence (5). In bacterial B-like P45Os,near amino acid 50 there 1sa conserved trp residue, often seen m the pattern WXXT(R,K), as in WIATK or WLVTR. This IS the first region to look for near the N-terminus of a new bacterial sequence. About 100 ammo-acid restdues from the N-terminus of bacterial sequences, there is another region, around the C-helix, that is critical when aligning bacterial sequences with eukaryotic sequences. The mottf is DPPXHXXXR. This motif corresponds to a C-helix sequence, WXXXR, conserved in most eukaryotic P45Os.The R is often followed by another basic group as m WREQRR. Some lower eukaryottc P45Os (CYP53, 57, and 58) have the H instead of the W seen m most eukaryotes After the C-helix, there is a long nonconserved stretch with few alignment cues. One worth mentioning is the (N,D,S)(V,I,T)(V,I) sequence around positions 175-l 80 m eukaryotic sequences.This E-helix region can be diagnostic for some families, as in the 3A subfamily, where the sequence reads GAYSMD(V,I)I, or the 4A subfamily SLMTLDT(I,V). After this region, the next well-conserved area is the I-helix. From here (near posmon 300) the P45Os are much more strongly conserved and alignments are much easier. The I-helix has a characteristic sequence A(A,G)X(E,D)T, where T is part of the molecular oxygen-binding site. This region is not conserved m enzymes like allene oxide synthase (CYP74) that use peroxides as substrates, because they do not have to bmd molecular oxygen. Consequently, the CYP74 family is one of the most distant branches on the P450 tree. This may not reflect its true evolutionary history, because one of the key regions used in computmg trees 1snot conserved. Exactly 16 amino-acid residues from the T previously mentioned is a conserved P. This P is present even in many bacterial sequences so tt is a good marker for the junction between the I and J helices. The sequence conservation contmues for another 15 residues in eukaryotes to a highly conserved G or P residue. At this pomt, there seems to be some sequence length variatton m the region between the J-helix and the K-helix. The K-helix is the best conserved feature m P45Os,with its invariant EXXR charge pan-. About 22 ammo-acid residues beyond the K-helix R, there 1s an (aromatic)X(I,V,L)P(K,A)G sequence that spans the connection between the p-2-2 strand and the p-l-3 strand (nomenclature of Peterson and Graham-Lorence, ref. 5). The P(K,A) pair lies in this gap and causesa sharp change m direction. The region beyond thusarea is not structurally well-defined, but 17 residues from the G there 1sa

20

Nelson

sequence, (aromatic)XX(P,D), that is very helpful in making alignments. It is followed 4-5 residues later by the PERF sequence, with some variattons as in PSRF, PDNF, PQRW, and so on. The PERF motif is not present m bacterial B-like P450 sequences,though it is m CYP102, CYPl 10, and CYPI 18 bacterial E-like P45Os. It is part of the meander before the heme thiolate ligand (nomenclature of Peterson and Graham-Lorence, ref. 5). After this point, there are length variations before reaching the signature sequence of all P45Os, FXXGXXXCXG, with some variations allowed except at Cys. Beyond the signature, 18 residues from the Cys, there is a conserved tetrapeptide, LQNF, or variants of It. The C-terminal region is quite variable with an unexplained occurrence of a PR approx 33-34 resrdues from the F m LQNF. In many sequences, these are the last two amino-acid residues, whereas in others there is an extension beyond the PR sequence. These form the mam alignment benchmarks that are used m making P450 sequence alignments. Often the distance between benchmarks IS absolutely the same and a cursory check for reasonable homologies between the segments can confirm that the alignment is probably correct without introducing gaps. Of course, this IS most difficult to do in the N-terminal region and when aligning bacterial or lower eukaryotic sequences that are very dtvergent from the other sequences. 4. Whole Genome Collections: Yeast, C. elegans, and Humans Subfamilies of P450 genes were thought to represent tightly linked clusters of genes (6). The nomenclature should then reflect physical proximity on chromosomes. At one point, we considered revismg the nomenclature when these clusters were sequenced to accurately reflect the sequence order of genes within a given cluster. This approach was not implemented, and it seems to have been based on erroneous assumptions. The genome projects are begmning to show that sequences from the same subfamily are not always linked. In C. eleguns, the CYP13A subfamily exists on two different chromosomes, and this is also true for the CYP25 family. Therefore, the notion of tightly clustered subfamilies is at least parttally mcorrect. Sequencmg of genomic DNA also shows that P45Osfrom different subfamilies can occur mixed in the same cluster. Human chromosome 19q 13.2 contains the followmg linear arrangement of P450 genes: CYP2F1, CYP2A6, CYP2A7P, CYP2B7P, CYP2B6, CYP2A12, and CYP2FlP, as shown on the Lawrence Livermore National Laboratories genome project home page at http://www-bio.llnl.gov/genome/html/ gene-ideogram.html. Clearly the subfamilies identified by sequence analysis are not discrete clusters in the genome. Chromosomal evolution seems to be

Cytochrome P450 Nomenclature

21

much more rapid than gene evolution, leading to the scrambling of gene clusters on a single chromosome and between chromosomes. The most completely sequenced animal genome is that of C. elegans. When the sequence of this genome was about 62% complete (April, 1997), an extensive search for P450 genes identified 70 different sequences, leading to an estimate of about 100 P450 genes m C elegans. For a list of these sequences, see http://drnelson.utmem.edu/CEP45Os.html. For translations of the genes, see http://drnelson.utmem.edu/CEP450spep.html. This genome proJect should be finished by the end of 1998, so a complete set of P45Osfrom an animal will then be available. There 1sa great increase m the number of P45Os from yeast to C. elegans. The whole yeast genome has only three P45Os,CYP51, CYP56, and CYP61, whereas animals have about 30 times that number. Their function is clearly not required for a eukaryotic existence, except to make cholesterol. They seem to be more important for the multicellular state. Human P450 genes are best represented m the dbEST section of Genbank. A search through about 800 ESTs in December 1995 identified 12-14 sequences that were as yet unknown P45Osm humans. Since then, two or three of these have appeared in the database as newly identified P450 genes, but there are still about 10 that have yet to be described. The author’s estimate at that time for human P45Oswas about 54. That assumed 83% coverage of all human genes m the EST database. Since then, the extent of coverage has been revised to 50-60%. Therefore, the estimate of human P450 genes was probably low and it may be closer to 75-90, similar to the number in the C eleguns genome. It should not be assumed that the same genes will be present m both organisms. So far, there do not appear to be any mitochondrial P45Os or steroidogenic P45Os in C. elegans; however, a CYP51 gene fragment is present, and the CYP29 and 31 families are most similar to CYPI. A new search of the EST database is warranted, as the number of entries has more than doubled since 1995 (301,00&709,000). The sheer numbers of hits makes this search a time consuming and difficult task. In 1995 the query (~450 AND human[LIB]) NOT reductase gave 1375 hits. One problem that soon became apparent while looking at these data was the presence of alu repeats. CYP2D6 and CYPlA2 have alu repeats, so every alu repeat m dbEST had the potential to give a spurious P450 hit in a blast search. By adding NOT alu to the query, the number of hits dropped from 1375 to 787. The same query done on May 20, 1997 (without [LIB], owing to changes in the database organization) gave 1635 hits. Clearly, this will take a major commltment to sort through, but the rewards may be identification of nearly all expressed human P450 genes.

22

Nelson

5. The Problems of Short ESTs or PCR Products The dbEST section of Genbank as of May 16, 1997 has 709,000 human ESTs, 182,000 mouse ESTs, and 3 1,000 Arabidopsis ESTs. A search of this database http://www2.ncbl.nlm.nlh.gov/dbST/dbest_query.html on July 9, 1996 for Arabidopsis P450 matches by the followmg expression (P450 AND Arabidopsis[LIB]) NOT reductase gave 163 fragments. These have all been translated and compared to the plant database that the author had at that time. Although some of the fragments could not be identified as P45Os,most were assigned to specific sequences or families. These are listed in the P450 Web page at http://dmelson.utmem.edu/ArabESTS.html. An identical search done on May 19,1997 gave 227 ESTs, 64 more than the previous year. However, 48 of these were matches to a tobacco sequence, D64052, that shows a P450 spectrum and P450 activity, but does not appear to be a P450 family member based on descent from a common ancestor (7). In addition, all of these ESTs had a high probabtlity of bemg rtbosomal RNAs, so these 48 sequences probably do not represent P450 sequences. Only 14 or 15 of the new ESTs that appeared smce July, 1996 seem to be legitimate P450 ESTs. When these sequences are translated and aligned with existmg full-length sequences and other ESTs, they can be assignedto families only If they are very similar to existing sequences. If they are only moderately similar they cannot be named because the percent identity will change when full-length sequences become known. The N-terminal region of P450 sequences is less conserved than the C-termmus, so a polymerase cham reaction (PCR) fragment or an EST fragment that is from the C-terminal region and is about 55% identical to a known sequence may have lessthan 40% identity when the whole sequence is known. The 40% value is the approximate cutoff for mclusion m a P450 family. To make the problem worse, some N-terminal fragments may be from the same gene as a C-terminal fragment m the collection, but it is not possible to know this without the overlappmg region. Therefore, naming of short fragments IS dangerous and may lead to multiple names for the same gene. Some laboratories have been usmg degenerate PCR quite successfully to amplify and sequence dozens of P450 fragments from the same species or related species, and they would like to have these named. One lab amplified the region from the I-helix to the heme signature region and sent the sequences of dozens of insect PCR fragments to be named. Another lab did the same from the heme signature region to the end of the P450 gene and submitted the sequences of more than 100 insect DNA fragments. The two groups’ fragments do not overlap, making it impossible to compare them with each other. The I-helix to heme signature fragments were longer and more informative,

Cykdmme

P450 Nomenclature

23

and they could be used to assign names, though it may be necessary to revise these later as the full-length sequences are determined. The short fragments of P450 sequencescannot be used m tree building with complete sequences, because the results are very mtsleadmg. The trees that result from such mixtures of short and long sequences~111probably cluster the short fragments correctly with their closest related sequence. However, that longer sequence may now be shifted to a completely wrong location on the tree. This happens because the difference matrix does not make adjustments for short sequences.All percent identities are treated as tf they were from same length comparisons. This would be acceptable if the percent identity were nearly constant over the length of P45Os, but it is not. One solution to thts problem is to make trees from segments of the sequence alignment. If all there 1sto work with is C-termmal fragments, such as the PCR fragments from the I-helix to the heme stgnature, then use just this region from all the sequences, including full-length sequences. Another possiblhty used by Rene Feyereisen, is to make an estimation of the percent identity for the full-length sequence based on a polynomial regression between percent identities for fulllength sequences vs percent identities for the desired region from the same sequences (8). Both methods result m difference matrices that are adjusted to same length sequences 6. Weaknesses of the Nomenclature System The nomenclature system depends on evolutionary relationships between stmilar sequences bemg correctly or at least consistently determined. The mechanisms for doing this have improved over the last 10 yr, so that there are better algorithms than UPGMA and better scoring matrices than a unit matrix adjusted for multiple hits at a given site. The neighbor-jommg method of Saitou and Ner (9) is generally considered better at reconstructmg phylogenies than UPGMA, and the BLOSUM 62 matrix (10) is the usual default m many applications today. The trees built from P450 sequences would be better if these newer methods were used. The drawback of making this change is the potential to significantly alter the existmg nomenclature, which has been developed over a IO-yr period. Perhaps this should be done anyway to achieve the benefits of more accurate tree-burldmg methods. There is a good probabthty that most of the branching pattern changes would be at deep nodes on the trees and these would not affect the nomenclature, because family assignment is based on identities of -3540% or greater. The clusters at these levels should not change much no matter what tree-buildmg methods are used. This was seen m a comparison of P450 trees made by neighbor-joining and UPGMA programs before the nomenclature system was adopted (3). Only 1 out of 34 branches

24

Nelson

changed position and this would not have altered the nomenclature. In the future, a switch should be made, If not too disruptive, to more sophlstlcated tree-making algorithms and scoring matrices. References 1. Nebert, D. W., Adesmk, M., Coon, M J , Estabrook, R. W , Gonzalez, F. J., Guengerrch, F. P., Gunsalus, I. C., Johnson, E F., Kemper, B , Levm, W., Phillips, I. R., Sato, R., and Waterman, M R. (1987) The P450 gene superfamily: recommended nomenclature. DNA 6, l-l 1. 2. Nelson, D. R., Koymans, L , Kamataki, T , Stegeman, J J , Feyereisen, R., Waxman, D. J , Waterman, M R , Gotoh, 0 , Coon, M J., Estabrook, R. W., Gunsalus, I. C , and Nebert, D. W. (1996) P450 Superfamily* update on new sequences, gene mapping, accession numbers and nomenclature Pharmacogenetics 6, 14 1. 3. Nelson, D. R. and Strobe& H. W. (1987) Evolution of cytochrome P-450 proteins Mel Blol Evol 4,572-593 4 Balter, M (1997) Morphologists learn to hve with molecular upstarts. Sczence 276,1032-1034. 5 Peterson, J A, and Graham-Lorence, S E (1995) Bacterial P45Os Structural similarities and functional differences, m Cytochrome P450 Structure, Mechanzsm and Bzochemutry, 2nd ed. (Orttz de Montellano, P R, ed ), Plenum, NY, pp. 151-180 6 Nelson, D R., Kamataki, T , Waxman, D. J., Guengerich, F P , Estabrook, R W , Feyereisen, R., Gonzalez, F J Coon, M. J., Gunsalus, I. C., Gotoh, O., Okuda, K , and Nebert, D W (1993) The P450 superfamily* update on new sequences, gene mapping, accession numbers, early trivial names of enzymes and nomenclature DNA and Cell Blol 12, l-5 1 7 Sugiura, M , Sakaki, T , Yabusaki, Y , and Ohkawa, H. (1996) Clonmg and expression in Escherzchla co11 and Saccharomyces cerevislae of a novel tobacco cytochrome P-450-like cDNA. Bzochzm Bzophys Acta 1308,23 l-240. 8. Scott, J. A., Collins, F. H., and Feyereisen, R. (1994) Diversity of cytochrome P450 genes m the mosquito, Anopheles albzmanus. Blochem Blophys. Res Commun. 205,1452-1459 9 Saitou, M. and Nei, M (1987) The neighbor-joining method: a new method for reconstructing phylogenettc trees. MOE Blol Evol. 4,406-425. 10. Henikoff, S., and Hemkoff, J G (1992) Ammo acid substitution matrices from protein blocks. Proc Nat1 Acad Scz USA 89, 10,915-10,919

3 Spectral Analyses John B. Schenkman

of Cytochromes

P450

and lngela Jansson

1, Introduction Cytochrome P450 was first shown to be a hemoprotem m 1964 (1). It has as its prosthetic group a noncovalently bound iron protoporphyrin IX, a form of heme found in b-type cytochromes, like cytochrome b, hemoglobin, and myoglobm. This type of heme prosthetic group can be removed from the protein by acidic acetone or by alkali and pyridine. There are a number of methods that can be used for spectrophotometrrc analysis of hemoproteins, each useful for a different purpose. In this chapter, we will provide methods for quantifying cytochrome P450, as well as methods for studying substrate interaction with the purified protein, the hemoprotem in membranes, and in tissue homogenates. The ability of those hemoproteins with an available 6thcoordination position to bind small molecules such as cyanide, carbon monoxide, ethyl isocyanide, etc., has provided means for their analysis and quantification. In the case of cytochrome P450, the binding of carbon monoxide permitted demonstration of its mvolvement m the monooxygenase reaction. 2. Materials 2.7. Heme Determination 0.4 A4NaOH containing 40% pyridme. 2.2. Hemoprotein Determination 1, Buffer for microsomes*50 mA4Tris-HCI, pH 7.25 2. Buffer for purified cytochrome P450* 50 mA4 sodium phosphate, pH 7 25, 20% glycerol. 3. Oxygen-free carbon monoxide tank with an appropriate two-stagevalve to permit very slow bubbling of the gas into cytochromeP450-containing solutions, From Methods m Molecular Ecology, Vat Ed&d by I R PhWps and E A Shephard

25

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Cytochrome

0 Humana

P450

Protocols

Press Inc , Totowa,

NJ

Schenkman and Jansson

26

4 Dtlauroylphosphattdylcholme, 8 mM (5 mg/mL) m 50 mA4 sodmm phosphate, pH 7 25, 20% glycerol, 0 1 M NaCI, 5 mA4 ethylenedraminetetra-acetic acrd (EDTA) This IS best prepared fresh and dispersed m a bath sonifier until the mixture clarrfies to a mild opalescence. 5 Dual-beam spectrophotometer or machine capable of yielding a difference spectrum 6 Dtthionite (sodmm hydrosulfite, Na,S,O,) 7 0 5 mMDrthiomte solution This must be prepared fresh and is best accomphshed by adding dry sodium dithromte to a small rubber-stoppered vial, gassing with argon or nitrogen through needles to remove oxygen, and then mJectrngdegassed

buffer into the vial to dissolve the dithronrte 2.3. Substrate Binding Microsomes or purified cytochrome P450 at a concentration of 1 pA4cytochrome P450/mL or higher m buffer as described in Subheading 2.2. Substrates of cytochrome P450, 100 mA4 m water: ethylbenzene, hexobarbttal, ammopyrine, ethylmorphine, or any of a very wide variety of drugs or organic chemical compounds. 3. Methods 3.1. Heme and Hemoprotein Quantification 3.1 1. Pyridine Hemochromogen (see Notes 14) Cytochrome P450, like many b-type hemoprotems, contains a smgle-heme prosthetic group that can be displaced and complexed with alkaline pyridine. This provides a means to quantify the enzyme when it is not possible to do so by other methods. Interestingly, it was the ability of this method to quantify heme m liver microsomes that provided the first clue that a hemoprotein other than cytochrome b5 was present m the microsomes. Dilute a sample of mtcrosomes or purtfied cytochrome P450 to 2 mL m a final concentration of 0 2 A4 NaOH, 20% pyrtdme (see Note 1). Divide the sample into two cuvets and record the baseline of equal hght absorption between them from 600 nm to 500 nm in a dual-beam spectrophotometer (see Note 2). Add a few mg of sodium dithtomte on the tip of a spatula only to the sample cuvet and record a spectrum from 600 nm to 500 nm. The drfference m hght absorptron changes at 557 nm relative to 575 nm (AA,,,,,,) IS read directly from the recording (see Fig. 1) Calculate the heme concentratron (c) using Beer’s Law: A = E c 1 A would be the difference m light absorption changes at 557 nm relative to 575 nm. The light path (1) of the cuvet is 1 cm, and the difference extmctlon coefficrent (s5s7-s7s)for non protoporphyrm (heme) 1s 32 4 rmW’ cm-t (1,2) (see Note 3)

Cytochrome P450 Analyses

i? 5 e P!I u

27

0 O 0 0 0 -0 01 J 475

I 500

525 550 575 Wavelength (nm)

600

Fig. 1. Pyridine hemochromogen. Sample dissolved m 20% pyrldine m 0.2 A4 NaOH. Vertrcal lines indicate 557 nm (long lme) and 575 nm. There is a AA of 0.041 indicating a heme content of 1 27 pM

5 If the hemoprotem has been purified and its protein content has been measured, it is possible to determme the extent of purity by expressing the data in terms of nmoles of heme/mg protein (see Note 4).

3.1.2. Carbon Monoxide Complex (see Notes 5-8) 3.1.2.1.

CYTOCHROME P450 IN MICROSOMES

1 Suspend an ahquot of a mrcrosomal preparation (or purified preparation, see Fig. 2) m 2 mL of buffer (see Note 5) and divide it mto two cuvets. 2 Record a baseline spectrum of equal light absorptron between the cuvets m a dual beam recording spectrophotometer between 500 nm and 400 nm. 3 Bubble carbon monoxide slowly mto the sample cuvet for about 30 s (see Note 6) 4. Add a few mg of sodium dithionite on the tip of a spatula to the sample cuvet, stir, and wan about 1 minute, then record the difference spectrum (see Note 7, and Fig. 2). 5 Calculate the concentration of cytochrome P450 in the cuvet from the absorption change at 450 nm relative to the absorbance change at 490 nm, using the above Beer’s Law equation and the extinction coefficient, s45M90 = 91 n-nW1 cm-’ (I). 3.1.2.2.

C~TOCHROME P450 SPECTRUM IN TISSUE HOMOGENATES

Because there are a number of different pigments in cells, including mttochondrtal flavoproteins and hemoproteins, plus cytoplasmic pigments, such as the endoplasmic reticulum cytochrome b5, caution must be exercised m attempting to quantify cytochrome P450 in whole homogenates. The methods described below allow one to obviate the mfluences of such pigments on the cytochrome P450 spectrum m homogenates, or mrtochondrial preparations, and even allow monitoring

of this pigment

m tissue slices (3).

Schenkman and Jansson

28 0 08 0 06 $

0 04

5

002

$

0

9

-0 02 -0 04 -0 06 400

425

450

Wavelength

475

500

(nm)

Fig 2 Carbon monoxide difference spectrum of purified cytochrome P450 2B4 m 50 mM sodmm phosphate, pH 7 25, 20% glycerol, 0 2% Emulgen 911 The absorbance difference between 450 nm and 490 nm 1s 0 062, indlcatmg a concentration of 0.68 pM

1 Prepare a 1: 10 (g/mL) homogenate of hver m sucrose buffer. 2. Transfer one ahquot mto each of two cuvets and record a baseline of equal light absorption in the dual-beam spectrophotometer between 500 nm and 400 nm. 3. Bubble nitrogen into the reference cuvet and carbon monoxide mto the sample cuvet, each for 30 s. 4 Stir an equal amount (10 pL,) of dithiomte solution into each cuvet and record the spectrum (see Note 8) 3.2. Hemoprotein

Spectra

3.2.1. Absolute Spectra of Cytochrome P450 (see Notes 9-17) 3.2.1.1. FERRIC CYTOCHROME P450 1 With purified hemoprotem, dilute to a concentration of about 1 l.GM m 50 mA4 sodium phosphate buffer, pH 7.25, contammg 20% glycerol (see Note 9) 2. Fill two cuvets with the buffer and record a baseline of hght absorption of the buffer m the dual-beam spectrophotometer from 670 nm to 250 nm 3. Replace the buffer in the sample cuvet with hemoprotem m the same buffer and record the spectrum (see Note 10). A spectrum of CYP2B4 IS shown m Fig. 3. 3.2.1.2. FERROUS CYTOCHROME P450 1 Fill two anaerobic cuvets with the buffer and record a baselme of equal hght absorption of the buffer m the dual-beam spectrophotometer from 670 nm to 300 nm 2. Replace the buffer m the sample cuvet with hemoprotein m the same buffer, ‘degas the cuvet to remove oxygen, and record the ferric hemoprotein absolute spectrum 3. Inject an amount (a few pL) of dlthlomte solution sufficient to reduce the cytochrome P450 (equimolar) to each cuvet (see Note ll), and record the spectrum at

29

Cytochrome P450 Analyses

3

350

400

Wavelength

450

500

550

600

(nm)

Fig 3 Absolute spectrum of 0.96 @4 CYP2B4 m 20% glycerol, 50 rnM sodmm phosphate, pH 7.25. In the absence of substrate and at room temperature approx 95% of the hemoprotein is m the low-spm state. The charge transfer band at 650 nm (not shown) is not detectable at this concentration of cytochrome P450 2-min intervals until no further reduction is seen Add an additional mlection of dithiomte to ensure full reductton. 3.2.1.3

CARBON

MONOXIDE

COMPLEX

OF CYTOCHROME

P450

1 Fill two anaerobic cuvets with the buffer and record a baseline of equal light absorption of the buffer m the dual-beam spectrophotometer from 670 nm to 300 nm 2. Replace the buffer m the sample cuvet with hemoprotem m the same buffer. 3 Degas the cuvet and replace the gas phase with oxygen-free carbon monoxide 4 Inject an amount (a few pL) of dithiomte solution sufficient to reduce the hemoprotein (equimolar) to each cuvet, and record spectra at 2-mm mtervals until no further reduction (see Note 11). Add an additional mjection of dithiomte to ensure full reduction.

3.2.2. Substrate-Binding

Difference Spectra (see Notes 12-16)

Ferric cytochrome P450 interacts wtth a number of its substrates with a concomitant shift m the equilibrtum between the high-spin (390 nm absorption maximum) and low-spm (417 nm absorption maximum) configurations. The spectral changes seen are small compared with the absolute spectral absorption, and are best monitored in difference spectra (see Note 12, and Fig. 4). I. Prepare a suspension of microsomes m buffer, or purified hemoprotem reconstttuted with phosphohprd in glycerol-contammg buffer, with the cytochrome P450 concentration at least 1 p.M m buffer. If purified cytochrome P450 is used, add tt to the somcated phosphohpid at a molar ratio of P450 to lipid of 1.160 and allow it to interact at room temperature for a least 30 mm (see Note 13).

Schenkman and Jansson

30 004 0 03 0 02 24 001 af 0 ij) -0 01 $ -0 02 -0 03 -0 04 -0 05

-------360

400

440 Wavelength

460

520

(nm)

Fig 4 Substrate-mduced difference spectra Titration of spectral changes induced by addltlon of increasing concentrations of hexobarbltal to rat-liver mlcrosomes Concentrations were 0 08 mM, 0 33 mA4 and 0 66 mM, respectively

2 Record a baseline of equal light absorption m the region of stu,dy (usually 450-350 nm) 3 Add 0. l-l &of the concentrated substrate solution, recording a difference spectrum after each addition The total volume of the additions should not exceed 2% of the volume m the cuvet (see Notes 14 and 15) 4. Notes 1 Smaller or larger volumes can be used as needed 2 Set the sensitivity of the machme (absorbance range) appropriately. For example, a 1-nmol sample of hemoprotem m 1 mL would have a AA of 0.0324. a One may also record absolute spectra, instead of difference spectra, thereby usmg half the amount of material. First record a baseline of solvent m each of two cuvets Place sample m one cuvet and record the absolute spectrum of the ferric pyrldme complex versus the alkaline pyndme. Add a few mg of dithionite to the sample- and solvent-containing cuvets and record the spectrum Draw vertical lines between the oxidized and dlthlomte reduced spectra at 557 nm and at 575 nm. Subtract the differences m absorbance between the two wavelengths This IS the AA557-575 b. One can also do a pyrldme hemochrome measurement without a dual-beam spectrophotometer, for example with a Gllford spectrophotometer, using one cuvet at a time: Prepare enough sample in NaOH and pyridine for two cuvets Zero the light absorption of the sample m the first cuvet at 575 nm, add a few mg of sodium dlthiomte, and record the absorbance change at 575 nm Repeat the procedure with the other cuvet at 557 nm. Subtract the absorbance change (AA) at 575 from that at 557 nm. This will give the AA557-575.

Cytochrome P450 Analyses

31

3 A is really AA, the absorbance difference of a wavelength couple. If the difference m absorbance changes at 557 nm relative to 575 nm was 0.1, then the heme concentration of the mixture would be 0.003 1 M, or 3 1 nmol/mL 4 With purified cytochrome P450 forms the specific content values obtained are between 17 and 20 nmoles/mg protein 5 If purified cytochrome P450 IS to be used a detergent (e g ,0.2% Emulgen 911 or Triton NlOl) is added to dimimsh aggregation, unless phospholrpid (e.g., dilauroylphosphatidylchohne, 160: 1 molar ratio) IS present, and 20% glycerol is included to stabilize the hemoprotein from conversion to P420, a breakdown product. For kinetic measurements of cytochrome P450 reductton, one can momtor the absorption changes at 450 nm relative to the isosbestic wavelength of 490 nm. 6. The dissolved carbon monoxide will reach 1 mA4 concentration It is best to bubble carbon monoxide mto the sample before adding the chemical reductant, because lower values are obtained if the sequence is reversed. The ferrous hemoprotein is somewhat unstable and is destroyed, perhaps by hydrogen peroxrde generated by reduction of oxygen during the bubbling 7. The reduction of cytochrome P450 IS not a fast reaction, even with the strong reductant sodium dithiomte, hence the need to wait at least 1 mm. It is often useful to wait an additional minute and repeat the scan to ensure that under your conditions reduction has gone to completion. It is possible, if desired, to measure the amount of cytochrome b, m microsomes prior to quantifymg the cytochrome P450 content. To do thus, add NADH (to a final concentration of 0.1 mM) Just to the sample cuvet before the addition of the dithiomte This will rapidly and completely reduce cytochrome bs (only mmrmally reducing cytochrome P450), yielding a reduced minus oxidized spectrum To quantify the concentration of this hemoprotem Use &42u90 = 112 ti’CI& (4). 8. By reducing both cuvets, we cancel out contribution of mrcrosomal cytochrome b, and all of the mitochondrial pigments except cytochrome oxidase The concentration of cytochrome P450 in liver tissue is about five times that of any mdividual mrtochondrial pigment, and the difference spectrum of the carbon monoxide complex of cytochrome oxidase has mmtmal impact on it However, if the concentration of cytochrome P450 is low relatrve to the other cellular carbon monoxide-bmdmg pigments, like cytochrome oxidase m the mitochondrra, it may be necessary to cancel such pigments with another hgand, such as cyanide. Cyamde binds tightly to cytochrome oxidase, but has a low affmity for cytochrome P450 (dissociation constant is 2.1 mA4), hence addition of 1 mA4 KCN to both cuvets before bubblmg m carbon monoxide ~111 diminish the contribution of cytochrome oxidase to the difference spectrum of cytochrome P450. 9. The Soret extmction coefficient of most hemoproteins is in the range of 100 mM-‘cm-‘, so to obtain a good spectrum, one must choose a suitable hemoprotem concentration and an appropriate sensitivity setting For example, if the concentration of cytochrome P450 was 1 p&f, an absorption of about 0.1 for the

32

10.

11

12

13.

14

15.

Schenkman and Jansson Soret peak would be expected. It 1sbest not to add a nomornc detergent because its absorption in the ultravtolet wtll obhterate the spectrum below 400 nm. An absorbance ratio of A4,7/A280 between 1 and 2 1sa good indicator of purity of the hemoprotein, with the 280 nm absorption owing to the aromatrc ammo acids, and the 417 nm absorptton to the heme complex. The hemoprotems will have different molar amounts of aromattc residues, and the spin equilibrium will, predictably, influence the ratio The ferric hemoprotem has a small 650 nm charge transfer absorption band attributable to the high-spm configuratton, and a- and /3-absorptron bands at 570 and 535 nm, respectively in the low-spur configuratron. Its Soret (y-band) 1s at 417 nm, and 1s typical of a low-spin state, but is m equilibrmm with a variable proportton of high-spin configuratton that has an absorptron peak at about 395 mn. The proportton of htgh-spin hemoprotem can be Increased by addttton of certain substrates or by elevation of the temperature of the solutron as the spin equrhbrium 1s also temperature-dependent Sodmm dtthtomte strongly absorbs m the ultraviolet, hence it IS advrsable to use the mnnmal amount that will reduce the cytochrome P450 and provide an equal amount to the reference cuvet For example, tf the cytochrome P450 concentration is 1 pA4, addition of 2 pL of the dtthtonite solutton would be Just sufficient to reduce the hemoprotem without causing a perturbatton of the absolute spectrum The magnitude of the absorptton change induced by substrate 1s rather small, generally less than 20% of the overall absorption of the membranous cytochrome P450 m the Soret region, hence the need for suffictent amount of hemoprotem or a spectrophotometer with suffictent sensrtivtty and ability to monitor the spectral changes m difference spectrum. Bacterial cytochrome P450,,, undergoes a full shift m its low-spm peak, from 417 nm to 390 nm (high spin) on addttton of its substrate, camphor Purified mammahan cytochromes P450 tend to aggregate, and m the absence of phosphohprd tt is generally not possible to obtain substrate induced shifts in the spin equilibrium Because the spectral changes are relatively small, addition of larger volumes of liquid to the cuvet can result in minor dilutton differences between the cuvets that can give rise to an erroneous difference spectrum. For example, with a concentration of cytochrome P450 of 1 $4, an imbalance m the dilution of 1% could cause a spectral imbalance of 0 001 absorbance, a value of the order of magnitude of changes invoked by the addition of some substrates Some substrates are poorly water soluble. If added m another solvent, such as ethanol, be aware that many organic solvents are able to perturb the spectrum of cytochrome P450 Although these may be blanked out by the additton of a similar amount of solvent to the reference cuvet, such addrtrons can alter data and calculattons such as that of a drssoctation constant. Substrate concentrations generating maximal spectral changes with different substrates range from 0. l-l n&L

Cytochrome P450 Analyses

33

References 1. Omura, T. and Sato, R. (1964) The carbon monoxtde-bmdmg pigment of liver microsomes I Evidence for its hemoprotein nature. J BIOI Chem 239,2370-2378. 2. Paul, K., Theorell, H , and Akeson, A. (1953) The molar ltght absorptton of pyrtdine ferroprotoporphyrm (pyridine haemochromogen) Acta Chem Stand 7, 1284-1287 3. Cmtr, D and Schenkman, J (1972) Hepattc organelle mteractton. 1. Spectral mvestigation during drug biotransformatton. Mol. Pharmacol 8,327-338 4 Raw, I. and Mahler, H (1959) Studies of electron transport enzymes III. Cytochrome b5 of pig liver mitochondrta J Biol Chem 234, 1867-1873

4 Purification

of Cytochromes

P450

Rat and Human Hepatic Forms F. Peter Guengerich

and Martha V. Martin

1. Introduction 1.1. General Introduction Purification of cytochrome P450 enzymes has played an important role in the development of the field. Although purification of these enzymes IS not requisite to many types of investigation, purified enzymes are still required for studies on structure and mechanism. Purified enzymes are also needed for preparation of antibodies, as standards in assays, and for other purposes that may often be overlooked. The focus of the first part of this chapter will be on details of purification of microsomal cytochromes P450 from rat and human liver. Most of the methods are also applicable to liver samples derived from other species. Chapter 7 will deal with methods for the purification of microsomal cytochromes P450 expressed in recombinant vector systems, specifically bacteria. 1.2. History and Development of Methods Efforts to purify cytochrome P450 enzymes began shortly after the discovery of the protem by Omura and Sato (1,2). At that time, the technology for purification of mtrmsic membrane proteins was not very well-developed, and most of the early efforts were plagued with problems of instability. A major advance was the observation of Ichtkawa and Yamano (3) that glycerol stabilizes cytochrome P450, a pomt now apprecrated with many enzymes. In 1968, Lu and Coon were successful m separating fractions contaming cytochrome P450, nicotinamide adamne dinucleotide phosphate (NADPH)-cytochrome P450 From Methods In Molecular Srology, Vol 107 Cytochrome P450 Protocols Edlted by I R PhWps and E A Shephard 0 Humana Press Inc , Totowa, NJ

35

Guengench and Mart/n

36

reductase, and phospholipld from rabbit liver mlcrosomes and reconstituting catalytic activity (laurate 12-hydroxylation) by mixing the three components together (4,5’. Key aspects of the procedure involved the stablhzation of enzyme activity with glycerol, the use of deoxycholate to solubllize the membranes and prevent aggregation during subsequent steps, and separation of the proteins by diethylammoethyl (DEAE)-chromatography. Subsequent advances in the field of purification involved the mtroductlon of more chromatography systems, although it should be emphasized that only a few methods have been particularly successful for use with cytochrome P450 enzymes. These generally include anion exchange chromatography (especially DEAE), catlon exchange chromatography (especially carboxymethyl [CM]), and hydroxylapatlte chromatography. Another approach used is chromatography on alkyl- or alkylamino agarose columns, utilizing the principle of hydrophobic chromatography (posstbly mixed with ion exchange m the case of alkylamino agarose). Another major technological development was the mtroductlon of new detergents, particularly the nonionic ones that Improved column resolution. Combinations of these basic procedures were used to purify most of the rat liver cytochromes P450 and some of the human cytochromes P450 to apparent homogeneity. (For lead papers and reviews from this period, see refs. 6-12). It should be emphasized that many of the techniques commonly used to purify soluble proteins (e.g., gel filtration, blo-specific affinity, and immunoaffinity chromatography) have proved to be of little value. The purified proteins were used m many physical and biological studies and also used to raise polyclonal antibodies, which were used m most of the early cloning strategies. With the cloning and expression of cytochromes P450, most of the interest m purlficatlon has shlfted to recombmant proteins. Construction of vectors and overexpresslon are described elsewhere m this volume, and Chapter 7 will emphasize purification from bacterial expression systems, because this approach is gaming in popularity and, to a large extent, reducing the need to purify enzymes from ammal and human tissue on a routine basis. 1.3. General Procedures from Liver Microsomes

Used for Purification

The authors have elaborated a procedure for a single cytochrome P450 (rat 2Bl /13,14/) from start to finish and describe typical operating procedures used with each step. These steps are generally useful in the purification of other cytochromes P450 and may vary in the order in which they are used, with advantages to using certain sequences of steps. Therefore, this general procedure may be applied to different cytochromes P450.

Purification

37

of Liver P45Os

At the outset, It should be strongly emphasized that considerable care must be used in the initial handling steps with human liver samples. Human tissue should be tested for hepatitis B and human immunodeficiency virus (HIV) and, although negative, all samples should be handled wtth care because of the possibility of a test mistake and subsequent mfectron. Researchers should consult the local authorrtres regarding details of suggested procedures. Personnel should always use protecttve gloves and clothing. All homogemzatton work

should be done in a fume hood and centrifuge tubes should be capped to reduce aerosols. All glassware used m the initial stages of handling should be decontaminated using an appropriate disinfectant. Laboratory personnel should be tmmumzed

against hepatitis

B vu-us.

Unless noted otherwise, most steps are usually done at 4°C This is not always really necessary. As reported in the literature, some of the steps have been done at room temperature (e.g., 13,15,16), although fractions are usually stored at 4°C for more extended periods of time. Almost all of the procedures are done in the presence of 20% (v/v) glycerol, It is generally advisable not to repeatedly freeze and thaw cytochrome P450 samples They are usually

kept at 4OC during preparation and divided into small aliquots for storage at -20” or -70°C. 2. Materials 2.1. Solubilization

of Microsomes

1. Lrver mrcrosomes’ Prepared from phenobarbital-treated rats, in the case of preparation of P450 2B 1 (23,14). 2. Mrcrosome storage buffer: 20 mA4 Trrs-HCl buffer, pH 7.4, containing 1.0 M ethylenediaminetetra-acetic acid (EDTA) and 20% (v/v) glycerol 3 1.0 Mpotassrum phosphate buffer, pH 7.25. 4 0.1 M sodmm EDTA, pH 7.4. 5 20 mM butylated hydroxytoluene, in acetone 6. Phenylmethylsulfonyl fluoride (PMSF): 100 m&I dissolved in dry n-propanol (store at -20°C and add Just prior to use) 7. Sodium cholate. The preparation of the sodrum cholate IS important rf n-octylammo-Sepharose chromatography is to be used m the next step The followmg procedure 1srecommended whenever the quality of the sodium cholate IS an issue

Chohc acid (Sigma, St. Louis, MO) is dtssolved m 50% C2HSOH (v/v, 100 g/ 750 mL) by heatmg to the boilmg point, with stirring. Activated charcoal (12 g), 0.5 g EDTA, and 5 g of Celite (diatomaceous earth) is added and heating is continued at nearly boiling temperature for several minutes. The hot solution is poured through a Buchner funnel containing a sheet of filter paper. Aspirator vacuum is applied to the system (Buchner flask) to rapidly pull the liquid through the funnel. The liquid 1s left overmght

at room temperature,

and

Guengerich and Martin

38

crystals of white chohc acid form. These are collected by stirring the solution (with a glass rod to free crystals adsorbed to the glass) and pouring the suspension through a Buchner funnel (with a paper filter) attached to a Buchner flask, with aspirator vacuum applied. The crystalline chohc acid is collected and the process is repeated. The chohc acid crystals are dried by leaving them m a crystalhzmg dish m a desiccator (filled with Drierlte adsorbent) attached to a medium-quality vacuum pump (protected by an acetone/dry Ice trap). The overall yield of chohc acid (white) IS -80% after the two recrystalhzations. Chollc acid (100 g/400 mL H20) IS mixed with 10 g of NaOH and the solution IS stirred with a pH electrode in the solution. The pH is adjusted to 7.2-7.5 by the careful addition of glacial CH$OOH. The volume IS adjusted to give a final concentration of 20% (w/v), based upon cholic acid. This solutlon IS stable when stored at 4°C. 2.2. Preparation of Octylamino-Sepharose 2.2.1. Materials 1 400 mL Sepharose4B (Pharmacla, Plcscataway, NJ) packed gel 2

100 g CNBr dissolved m 100 mL 1,4-dloxane

Cuutzon Handle with care

3 8NNaOH

4 5 6 7 8

115 g 1,8-diaminooctane (Aldrich, Milwaukee, WI), dissolved m 400 mL Hz0 GNHCl. 0 10 Mpotassmm phosphate, pH 7 25, containing 1 0 mMEDTA 20% (w/v) Emulgen 913 (see Note 1) 2.5 x 50 cm chromatography column

2.2.2. Method of Preparation 1, A procedure is outlined for the use of n-octylammo-Sepharose 4B In some cases n-hexylammo-Sepharose has been used These materials may be purchased from commercial sources, but the cost IS high. n-Octylammo-Sepharose 4B may be prepared by the followmg procedure, which may be scaled accordmgly. 2 Sepharose 4B 1s suspended in Hz0 (1200 mL/400 mL packed gel volume) by stirring with a glass rod and collected m a smtered glass (coarse) funnel (with aspirator vacuum apphed to a Buchner flask) The process 1s repeated several

times, in order to wash the gel free of preservatives 3. The 400 mL of washed Sepharose 4B is suspended m 400 mL of cold Hz0 and gently stirred with a magnetic device (stirring bar), and with the electrode of a pH meter m the suspension Cautlon This should be done m a fume hood 4. A solution of 100 g CNBr dissolved in 100 mL of 1,4-dloxane is poured mto the suspension m one transfer and 8 N NaOH is added to the suspension over a period of 20 min to maintam pH 11.0. Cuutzon. Keep solution m a fume hood while open.

39

Purification of Liver P45Os

5. The reactron is quenched by the addition of -400 g of ice, and the gel IS collected by pouring rt into a smtered glass funnel attached to a Buchner flask (attached to aspn-ator or house vacuum, m the fume hood to avoid exposure to HCN or CNBr) 6. The collected gel IS washed once with cold H,O. 7. The collected gel 1sadded to 115 g of 1&drammooctane, drssolved m 400 mL of H,O 8 The pH 1s adjusted to 10 0 with 6 N HCl 9. The beaker 1s covered with plastic wrapping and the suspension IS stirred as slowly as possible at 4°C overnight. 10 The gel IS recovered on a smtered glass funnel and repeatedly washed with H,O and then several times with 0.10 M potassmm phosphate buffer, pH 7.25, contaming 1 mMEDTA. 11 The gel 1sthen recovered from the funnel and suspended m the same buffer. (At this point gel can be mdefimtely stored at 4 “C). 12. A 2.5 x 50 cm chromatography column IS set up, according to the manufacturer’s recommendations, and the lines are filled with degassed H20. (see Notes 2 and 3). 13 An amount of gel (at 25°C) sufficient to fill the column is placed m a Buchner flask (~0.5 total volume), m 0 10 A4 potassmm phosphate buffer, pH 7 25, and capped 14 Aspirator vacuum IS applied for 10 min with intermittent swn-lmg of the flask (to disperse an bubbles) 15 The slurry 1spoured mto the column and allowed to stand for -10 mm, at which time the outlet valve IS opened 16. More gel IS added to the top of the column, until the column 1s filled, without allowing the top of the column to dry. 17. After the column IS filled, the top valves are inserted, and the “head” of lrqmd above the gel is reduced to -1 cm (see Note 2)

2.3. Buffers

for Octylamino

Sepharose

Chromatography

1 Buffer A: 0 10 M potassium phosphate buffer, pH 7.25, contammg 1 0 mM EDTA, 20% (v/v) glycerol, and 0 60% (w/v) sodmm cholate (recrystalhzed), 1000 mL 2. Buffer B: 0.10 A4 potassium phosphate buffer, pH 7 25, containing 1 0 m&f EDTA, 20% (v/v) glycerol, and 0.42 % (w/v) sodmm cholate (recrystallrzed), 800 mL. 3. Buffer C 0.10 M potassium phosphate buffer, pH 7.25, contaming 1.0 mM EDTA, 20% (v/v) glycerol, 0.33% (w/v) sodmm cholate (recrystallized), and 0.06% (w/v) Emulgen 9 13.

2.4. DEAE Chromatography 1. DEAE-Sephacel, Pharmacia (see Note 4). 2 Chromatography column, 2 5 x 20 cm.

Guengerich and Martin

40

3. Buffer D. 5 mkfpotassium phosphate buffer, pH 7.7, containing 0.1 mA4EDTA, 20% (v/v) glycerol, and 0.20% (w/v) sodium cholate (not recrystalhzed). 4. Sodmm cholate, 20% (w/v) (Sigma, St Louis, MO), not recrystallized, pH 7 7 5. Buffer E: 5 mA4 potassium phosphate buffer, pH 7.7, contaming 0 1 mk! EDTA, 20% (v/v) glycerol, 0.5% (w/v) sodium cholate (not recrystallized), and 0.20% (w/v) Emulgen 911 (see Note 5). 6. Buffer F 5 Wpotassmm phosphate buffer, pH 7.7, contammg 0 1 WEDTA, 20% (v/v) glycerol, 0.5% (w/v) sodium cholate (not recrystallized), 0.20% (w/v) Emulgen 9 11, and 200 mM NaCl (see Note 5).

2.5. CM Chromatography 1, Buffer G: 5 mA4potassium phosphate buffer, pH 6.5, contaimng 0.1 mM EDTA and 20% (v/v) glycerol 2 CM-Sephacel (Pharmacla, Pxscataway, NJ) (see Note 6). 3 2 5 x 10 cm chromatography column. 4 Buffer H* 5 mM potassium phosphate buffer, pH 6 5, contammg 0 1 mM EDTA, 20% (v/v) glycerol, and 0.10% (w/v) Emulgen 9 11 5 Buffer I: 5 rnU potassium phosphate buffer, pH 6.5, contaming 0.1 n&I EDTA, 20% (v/v) glycerol, 0.10% (w/v) Emulgen, and 150 mM NaCl.

2.6. Hydroxylapatite

Chromatography

1. Blogel HTP-A (BloRad, Hercules, CA) (see Note 7) 2 Buffer J* 5 mMpotassmm phosphate buffer, pH 7.4, containing 0.05 mA4 EDTA and 20% (v/v) glycerol (see Notes 8 and 9). 3 Buffer K. 10 rmI4 potassium phosphate buffer, pH 7 4, containing 0.05 mA4 EDTA, 20% (v/v) glycerol, and 0 30% (w/v) Emulgen 913 (see Notes 8 and 9) 4. Buffer L: 300 rnkfpotassium phosphate buffer, pH 7.4, contaming 0.05 WEDTA, 20% (v/v) glycerol, and 0.30% (w/v) Emulgen 913 (see Notes 8 and 9). 5. Buffer M: 20 mM potassium phosphate buffer, pH 7.4, contammg 0.05 mA4 EDTA, 20% (v/v) glycerol, and 0.30% (w/v) Emulgen 913. 6. Buffer N: 40 nxI4 potassmm phosphate buffer, pH 7.4, containing 0 05 mA4 EDTA, 20% (v/v) glycerol, and 0 30% (w/v) Emulgen 9 13. 7. Buffer 0: 90 nuI4 potassmm phosphate buffer, pH 7.4, containing 0.05 mM EDTA, 20% (v/v) glycerol, and 0 30% (w/v) Emulgen 913 8. Buffer P: 150 rr&potassnun phosphate buffer, pH 7 4, contammg 0.05 mM EDTA, 20% (v/v) glycerol, and 0.30% (w/v) Emulgen 9 13. 9. Buffer Q: 300 mA4 potassmm phosphate buffer, pH 7 4, containmg 0.05 mM EDTA, 20% (v/v) glycerol, and 0.30% (w/v) Emulgen 913 10. Buffer R: 300 mM potassium phosphate buffer, pH 7.4, containing 0.05 mM EDTA and 20% (v/v) glycerol. 11. Buffer S: 300 mMpotassmm phosphate buffer, pH 7.4, containing 0.05 mMEDTA, 20% (v/v) glycerol, and 0.15% (w/v) sodium cholate (recrystallized).

41

Purification of liver P45Os 3. Methods

3.1, Solubilization of Microsomes Chapter 5 deals with methods for preparing microsomes. Samples are usually stored at -8OOC m microsome storage buffer (Subheading 2.1.). 1. The general method of solublhzatlon IS to stir the mlcrosomes, at a protein concentration of 2 mg protein/ml (as estimated from the bicmchoninic acid, or BCA, assay, Pierce, Rockford, IL) in 0.10 A4 potassmm phosphate buffer, pH 7.25, containing 1.OmMEDTA, 20% (v/v) glycerol, 20 w butylated hydroxytoluene, and 0.10 mM PMSF (added only in the case of human liver). 2. A sufficient amount of a solution of 20% (w/v) sodium cholate is added to yield a final concentration of 0.60%, which is a 3: 1 ratio of cholate to protein (w/w) 3 The solution is allowed to stir for 30 min at 4°C and then subjected to centnfugation at 10sg (3.5 x lo4 rpm in a Beckman 45 Ti rotor) for 60 mm. The pellet, which contains very little cytochrome P450, 1s discarded.

3.2, Octylamino-Sepharose

Chromatography

1. The column IS equilibrated by pumping through about 1000 mL of Buffer A, at a flow rate of -1 mL/min. 2 The cholate-solubrlized liver microsomes are applied to the column at the same

rate (at 4T). 3. The column is washed wtth -800 mL of Buffer B. 4. The buffer is then changed to 2000 mL of Buffer C 5. As the elution buffer IS passed through the column, a dark band should form and move down the column. At this point, fractions (-15 mL) are collected with an automated device. Most of the cytochrome P450 should be eluted in a sharp peak (measured at A417). (see Notes 10-12).

3.3. DEAE Chromatography 1 The cytochrome P450 (2B 1) fraction recovered from the n-octylammo-Sepharose column IS dialyzed extensively against Buffer D (4 changes of 20 volumes, 28 h per dialysis) (see Notes 13 and 14). 2. A 2.5 x 20 cm column of DEAE-Sephacel is prepared m the same manner as described for the octylamino-Sepharose column, taking care to remove bubbles (Subheading 2.2.2., items 14-17). It is useful to adjust the pH of the gel before packing the column. A column of this size might be suitable for the application of 1000 nmol of cytochrome P450. 3. The column is equilibrated by pumping through 1000 mL of Buffer E. 4. The sample is applied at a flow rate of l-2 mL/min (same as used for equihbratlon). 5. The column 1sthen washed with the Buffer E (500-1000 mL), and some colored bands will probably move down the column. If colored fractions appear, the column 1swashed until the effluent is clear.

Guengerich and Martin

42

6. A linear gradient of increasmg NaCl IS applied to the column by using 750 mL of Buffer E m one flask and 750 mL of Buffer F in the second (with the flasks connected by a tubing bridge) 7 The elution of cytochromes P450 from the column 1s monttored by measurmg A4t7 (heme), either with a contmuous monitor or m each fraction (tubes of 510 mL) (see Note 15) 8. Ahquots of fractions are analyzed by sodmm dodecyl sulfate-polyacrylamide gel electrophoresrs (SDS-PAGE). A modified version of the original procedure of Laemmh (17) is described m detail elsewhere (18’ (see Notes 16 and 17)

3.4. CM Chromatography 1 The most pure cytochrome P450 fractions from the DEAE step are pooled, adJusted to pH 6 5, and extensively dialyzed against Buffer G (20 volumes, 3 x 8 h) (see Note 18) 2 A 2 5 x 10 cm CM-Sephacel column is prepared m the same general manner as described earlier, adjusting the pH to 6 5 prior to packing (Subheading 2.2.2., items 14-17). 3. The column IS equilibrated with 500 mL of Buffer H at a flow rate of -1 mL/ mm 4 The dialyzed cytochrome P450 sample, adjusted to pH 6 5,rs applied to the column at the same flow rate. Most of the cytochrome P450 should bind to the top of the column 5 The column IS washed with -400 mL of the equilibration buffer. 6. Elution 1s accomplished with a linear NaCl gradient made by using two flasks, one containing 350 mL of Buffer H and the second containing 350 mL of Buffer I (connected by a tubing bridge). 7 Fractions (-5 mL) are collected and analyzed for A,,, and by SDS-PAGE (see Notes 19 and 20)

3.5. Hydroxylapatite Chromatography 3.5.1. Separation of Proteins (in Presence of Detergent) 3.5.1.1.

LINEAR GRADIENT METHOD

1. The fraction from the previous step is extensively dialyzed against Buffer J. 2 Hydroxylapatrte IS degassed and used to pack a 2.5 x 10 cm column m the manner described above m Subheading 2.2.2., items 14-17. 3 The cytochrome P450 preparation (up to -400 nmol) is loaded on the column at a rate of 1 mL/mm, taking care not to have color spread over more than the top l/3 of the column. 4. The column is washed with Buffer J. If any color IS eluted, washing is continued until the point where the effluent is clear 5. Elutton of the cytochrome P450 from the column is with a linear gradient, using 500 mL of Buffer K and 500 mL of Buffer L. 6. Cytochromes P450 are monitored by A4i7 measurements and peaks are analyzed for purity using SDS-PAGE (see Note 21)

Purification of Liver P45Os

43

3.5.1 2. STEPWISE GRADIENT METHOD

This is an alternative to the linear gradient previously described m Subheading

3.5.1.1.

1 The fraction from the previous step is extensively dialyzed (against three changes of 20 volumes of Buffer J, 78 h/change). 2. The sample is applied to the hydroxylapatite column, again taking care not to cover more than the upper l/3 with color owmg to cytochrome P450 3. The column 1s sequentially eluted with Buffers K, M, N, 0, P, and Q. At every step, enough buffer is used to elute some cytochrome P450 and then to lower the A4,, in effluent to near zero, or at least to ~10% of the peak absorbance 4. Fractions are momtored for A4t7 and by SDS-PAGE, taking care to dilute samples when necessary to avoid potassium dodecyl sulfate preciprtates.

3.5.2. Use in Removal of Detergent The level of detergent can be lowered by stirring cytochrome

P450 solutions

with polystyrene beads, such as Amberlite XAD-2 or Bio-Beads SM-2. In general, a ratio of -10: 1 (beads:protein, w/w) is used, with gentle stirrmg for 1 h at 4°C and filtration through glass wool (polystyrene beads can be freed of detergent by treatment with CHsOH and reused). This approach may not be useful at low protein concentrations, as proteins can be adsorbed to the beads. In general, the detergent concentration cannot be lowered as much as by the hydroxylapatite method. 1. A small column of hydroxylapatite (-1.2 x 5 cm) is poured m the usual manner. If the cytochrome P450 binds well, such a column can easily bmd 100 nmol of cytochrome P450. 2. The sample should be dtalyzed vs Buffer J to reduce the phosphate concentration to - 10 mM before application to the column (see Note 22). 3 The sample is apphed to the column at a flow rate of - 1 mL/min It IS possible to fill most of the column with color, because resolution IS not a particular issue 4 The column is extenstvely washed with Buffer J 5 The effluent is monitored at 280 nm, at which most nonionic detergents absorb, 6. Washing is contmued until the Azsoof the effluent 1s ~0 02 7. The column is eluted with Buffer R and cytochrome P450 should be eluted 8. If any (brown) color remains on the column (owmg to cytochrome P450), elution 1s continued wrth Buffer S (see Notes 23 and 24).

3.6. Other Methods Alternative chromatography procedures for the isolatton of cytochromes P450 and for their storage are described in Notes 25-34 See Notes 35 and 36 for the purification of NADPH-cytochrome P450 reductase and cytochrome bg.

44

Guengerich and Martin

4. Notes 1. Emulgen 9 13 1s a nomonic detergent that can be obtamed by request from the Kao-Atlas (Tokyo, Japan) Several other nonionic detergents can generally be substituted, mcludmg Emulgen 9 11, Renex 690, Triton N- 10 1 (Sigma), Tergitol NP- 10, Nonidet NP-40, and Lubrol PX (14,18). The latter has the advantage that tt does not have ultraviolet (UV) absorbance at 280 nm. However, some of these detergents may yield different results with particular cytochromes P450. These detergents are prepared as stock 20% (w/v) soluttons by sttrrmg m warm water and can then be stored at 4°C. (One way to avoid excessive heat is to stir the detergent mixture m an ice bucket filled with hot tap water) Many of these detergents are capable of forming hydroperoxtdes, which may be a problem m some settings, and there are (laborious) ways of removing these (19,20). 2. This general procedure IS used to prepare other columns as well. 3 In general, a column of this size will hold -1000 mg of solubthzed protem A reasonable guide is to restrict the densely colored top region of the column to
Purification of Liver P45Os

8. 9.

10.

11

12.

13.

14. 15. 16. 17.

18.

19.

ally a problem now, but some care should be exercised with regard to untial flow rates. A precaution is to keep concentrations of EDTA low (-0.05 mM), because the material can interact with the calcium in the column and cause clogging. In general, the pH 1snot particularly tmportant wtth hydroxylapatite columns and these columns do not seem to require equilibration, because most cytochromes P450 of interest are adsorbed Some cytochromes P450 are more tightly bound to the column and are eluted by mcreasmg the Emulgen 9 13 concentration to 0.20% (w/v) However, some cytochrome b5 is usually eluted m these fractions as well. If the cytochrome bs and NADPH-cytochrome P450 reductase are desired, both can be eluted by further changing the buffer detergent concentrations (14,18). However, it is generally more efficient to prepare these protems by mdependent methods. The gel may be reused, with some loss of efficiency. If it is, the procedure is to use a sintered-glass funnel to collect the gel and wash (the gel extruded from a 2 5 x 50 cm column) sequentially wrth 2 x 1000 mL of 0.10 M potassium phosphate buffer containing 0 5% (w/v) Triton N- 10 1 and 0 5 M NaCl, 3 x 1000 mL of H20; 2 x 1000 mL of 50% (v/v) aqueous 1,4-dtoxane (to remove detergent); 3 x 1000 mL of H,O; and 1000 mL of 0 10 A4 potassium phosphate buffer contaming 1.O mM EDTA and 0 02% (w/v) NaN,, in which the gel can be stored at 4OC for future use. In practice, we heat new dialysis tubmg in 10 mM EDTA at 70-80°C for 30 min and rinse with Milli-Q H20, with storage at 4°C m 1 WEDTA. This procedure removes any trace metals It is often useful to monitor the conductivity with a meter before applymg the sample to a column, if the binding is a critical step It is not very useful to momtor A2s0 as a measure of total protein because of the high absorbance of the noniomc detergent Some detergents (e g., Lubrol PX) do not absorb and may be used (14). Staining with an AgN0JNH40H solution (21) is raped and sensitive. One reason to use NaCl as the salt m the gradient is to avoid the precipitation of potassium dodecyl sulfate If the protein of interest does not bind to the DEAE column, it may be of use to lower the detergent concentrations (for instance, to 0.10% nonionic detergent and 0 20% sodmm cholate (131) or raise the pH. Elutton with gradients of changes in pH have not been particularly useful (although some success with chromatofocusmg systems has been reported /22/) Cholate is usually omitted from CM separations because it is thought to compete for binding to the protems (with the CM gel). Therefore, protein samples containing cholate (from previous steps) are extensively dialyzed to remove most of the cholate. In general, the resolution seen with CM chromatography is not as good as with DEAE chromatography Most cytochromes P450 bmd to the matrix when applied,

Guengench and Mart/n

46

20.

21

22 23 24

but sometrmes there are problems with inhibition of bmdmg by detergents The resolution of cytochromes P450 from each other, when they do bmd to the column, 1sgenerally not as good as with DEAE. Nevertheless, CM chromatography has proven to be a valuable step m the purification of some cytochromes P450 The reader is referred to a method m which rabbit P450 1A2 can be purified from liver mtcrosomes using a combmatton of only DEAE and CM chromatography (23). This procedure has been employed m our own laboratory with minor modrficatron (24) The material eluted from the DEAE column binds tightly to the CM column Other proteins are removed by a wash with low romc strength plus detergent, and detergent is then removed by washing with buffer. Purified P450 IA2 is eluted with 300-500 mMpotassium phosphate buffer, ready for use However, cytochrome P450 peaks eluted from hydroxylapattte columns are usually as well-resolved as with ton-exchange columns The nomomc detergents cholate and CHAPS (3-[3-cholam~dopropyl)d~methylammon~o]-1-propanesulfonate) are usually not very effective m replacing the nomomc detergent Sometimes treatment of the cytochrome P450 sample with polystyrene beads to lower the detergent concentratton may be useful rf bmdmg 1s a problem. The ease of elution of cytochrome P450 from hydroxylapatrte 1s somewhat dependent upon the particular commercial preparation The concentratron of cholate or CHAPS can be 0 1 to 0 2% (w/v) Ionic detergents can then be removed by dralysrs, because of therr hrgh critical mrcelle concentrations (e.g , 0 3%, w/v, for cholate), but nomomc detergents-e g., Emulgens, Tritons, Lubrols, and so on-have very low crtttcal mrcelle concentratrons (-0 005%, w/v) and cannot be removed by dralysts, ultrafiltratton, and so on (24,25)

4.1. Other Chromatography

Methods

25 Many of the rat, rabbit, and human liver cytochromes P450 have been purrfied using various combmatrons of alkylammo-Sepharose, DEAE, CM, and hydroxylapatrte chromatography (12,12,18). The sequence, the exact number of steps, and the yields are variable, depending upon the abundance of the particular P450.

4.2. FPLC Systems 26. All of the chromatography methods previously menttoned have been described with the use of “open-bed” systems, but all of these resins are available m medium-pressure FPLC columns Funae and his group were among the first to develop such technology (16,26,27) The general approach has been to fractionate solubrhzed mtcrosomes with open-bed alkylammo-Sepharose chromatography as described and then do further mampulattons on vartous ton-exchange and hydroxylapatrte columns The results can be very good, with very hrgh resolution observed m some cases. Usually, it IS preferable to use an apparatus designed to operate at medium pressure (e.g , Pharmacia FPLC system). The effluent is readily monitored continuously, and the columns may have very good capacity. Columns are expen-

Purificatron of Liver P45Os

47

sive, and care must be used in cleaning, and so on It is also of Interest to note that these columns can show dramatically different resolution with very similar detergents (16) A number of columns are available from several commerctal sources (and these also show variation)

4.3. Miscellaneous 4.3.1. General

Methods

27. Some other methods will be mentioned, particularly some that have not been very applicable. Ftrst of all, tt should again be emphasized that cytochromes P450 are intrinsic membrane protems and almost all chromatography must be done m the presence of detergents.

4.3.2. Preclpitatlon Methods 28. Certain bulk precipitation methods provide some purification, usually at early steps Ammomum sulfate fracttonatton of cholate-solubthzed mtcrosomes was used in early work on the purtfication of P45Os (28) Some separation IS provided, although the procedure has a tendency to generate cytochrome P420 (P420), an macttve form of cytochrome P450 m whtch the cystemyl ligand to the heme is broken and the Soret band of the ferrous-CO complex shifts from 450 to 420 nm (2). A milder procedure involves stepwise precipitation of cholate-solubihzed microsomes with mcreasmg concentration of polyethylene glycol 6000 (28) In general, these methods have been abandoned in favor of direct apphcation to chromatographic steps.

4.3.3. Gel Filtration 29. Gel filtratton is not very effective m the presence or absence of detergent Without detergents the aggregated cytochrome P450 elutes m the void volume, along with other membrane proteins. In the presence of detergents, the apparent size of the protem-detergent complex is dominated by the detergent micelles.

4.3.4. Affinity Chromatography 30 In general, ligand affinity methods have not been very successful. The n-octylamino-Sepharose procedure was developed because of the ability of n-octylamme to form a ligand wtth cytochrome P450 heme, but the interaction m the chromatographic separation appears to be hydrophobic Other reported affimty separations are probably not specific and also depend on hydrophobtctty (29)

4.4. Other Procedures Associated with Purification and Handling of Cytochromes P450 4.4.1. Dialysis and Related Methods 3 1 In many cases tt is necessary to exchange the medium that cytochrome P450 (or cytochrome bS or NADPH-cytochrome P450 reductase) IS m. This 1s usually accomplished by direct dialysis, using membranes with an M, cutoff of 10,000

48

Guengerich and Martin Exchanging the components of a solution 1smore effective if multiple dlalyses are done, with the first dlalysls steps being shorter (2-6 h) and the latter ones longer (8-16 h), as opposed to a single dialysis against a large volume of buffer, owing to Flck’s first law (30). The dialysis buffer should be 10-20 volumes of the protein solution. Some cytochromes P450 (e.g., all of the P450 lA2 proteins from drfferent species) are prone to precipitate at low salt concentrations m the absence of detergent. The phosphate concentratron IS kept at 100 mA4 for P450 lA2 enzymes It should be pointed out again that cholate can be removed by extended dialysis but nomomc detergents cannot (31)

4.4.2. Concentration

of Cytochrome P450

32 The most generally used method 1sultrafiltration We use devices from Amlcon (Lexington, MA) The solvent 1s forced through a semiporous membrane under NZ pressure. Because cytochromes P450 aggregate, most M, cutoff membranes can be used (Centricon devices, also made by Amicon, can be used with smaller samples.) It is very important to wash the membranes after use (with a comblnation of high salt and detergent to remove bound proteins, followed by removal of the salt and detergent with HZ0 and storage m 10% aq. C,HSOH). In practice, we record the sample history of each membrane and restrict the use of an individual membrane to a particular cytochrome P450 m order to avoid contamination. There are other, less convenient, ways to concentrate cytochromes P450 These Include repeating the hydroxylapatlte chromatography procedure previously described for removing detergent or using batchwise absorption onto calcium phosphate gel and elutlon (28), which is the functional equivalent Vacuum dlalysls and other, older procedures can be used but are not as efficient as ultrafiltration (32) Another procedure is to dialyze against a high concentration of polyethylene glycol, or even glycerol A dialysis tube can also be packed m Aquaclde (high 1M, carboxymethylcellulose, CalBiochem, San Diego, CA) to draw out HZ0 (see manufacturer’s catalog)

4.4.3. Storage of Cytochromes P450 33. As mentioned earlier, cytochromes P450 can be handled at room temperature for short periods of time In practice, most chromatography steps are done at 4’C Repeated freezing and thawing should be avoided, so when a cytochrome P450 preparation 1s purified it should be divided into small aliquots and frozen We generally store cytochromes P450 at -20°C and do not feel that -70°C 1s necessary (although the lower temperature IS preferred for long-term storage of human liver samples and mlcrosomes) Solutions should contam 20% (v/v) glycerol for freezing. A higher concentration might be beneficial but, depending upon the extent of dilution mto assays,

Purification of Liver P45Os

49

the glycerol could interfere (33). If such a problem 1santicrpated, glycerol can be removed by a brief dialysis prior to use. Many cytochrome P450 purification and storage procedures Including many of our own, call for the use of dithiothreitol (DTT) This is a precautionary procedure and was implemented during problems in purifying catalytically active P450 3A4 (34). However, there seems to be little systematic evaluation of the need to use DTT or other thiols m cytochrome P450 puriticatton, and many of the early rat protocols were done without this. However, tt is possible that some cytochromes P450 may requtre thiol stabihzatton and the authors leave this to the reader to investigate with each partrcular cytochrome P450. It should be pointed out that DTT may not always be innocuous. For instance, m collaborattve studres with Prof. P. R. Ortrz de Montellano we found that residual DTT was present m the cytochrome P450 active site and interfered with heme modtfication by hydrazines (35) Thus, DTT could be a competittve inhibitor

4.4.4. Estimation of Protein Concentrations 34. Estimation of protein concentrations provides a means of evaluating the progress of enzyme purifications and the purity of final preparations In principle, the content of cytochrome P450 heme should be about 18 nmol per mg protein, corresponding to an M, of 55,000. Analysis by UV absorbance is rather unsatisfactory because of the presence of at least trace amounts of mterfermg noniomc detergents. Colortmetrtc assays are often used. The bmret assay 1s relatively msenstttve and not particularly well-suited. The Lowry assay (36) is widely used but SubJect to many interferences, particularly from detergents and buffers. The same holds for the Coomassie and other dye-binding systems (37). We routmely use the BCA assay in our laboratory, following the procedures of the supplier (Pierce, Rockford, IL) This 1sraptd, sensitive, and less sensittve to interference, m our own experience. It should be emphasized that all of these procedures provide only rough esttmates, because the methods rely on properties such as the content of partrcular ammo acids, hydrophobrctty, and so on, and are all somewhat subject to mterference, despite the best efforts at correction If the exact protein concentratton 1s considered to be an important issue, then the recommended procedure is to do quantitative ammo acid hydrolysis on a hydrolyzed sample. The sample (-1 pmol actually used m the analysis) ts hydrolyzed m 6 N HCl at 110°C for 24 h (under vacuum) and the resulting ammo acids are derrvattzed with phenyltsothtocyanate and their concentrations esttmated from compartson with external standards following separation by high-pressure liquid chromatography (HPLC), e g , with the Waters “Prco-Tag” system (Waters-Mrlhpore, Milford, MA). The method does not account for Cys and Trp, but these may be corrected rf the sequence is known The estimates are much more reliable than any colortmetrtc procedures (38,39)

50

Guengerich and Martin

4.5. Auxiliary Enzymes 4.5.7. Purification of NADPH-Cytochrome

P450 Reductase

35 NADPH-cytochrome P450 reductase can be recovered from n-octylammoSepharose columns followmg the elution of cytochromes P450 and other mtcrosomal proteins (10,14). However, this procedure is not very efficient if large amounts of the enzyme are needed Larger amounts of the enzyme can be prepared from rat or rabbit liver mtcrosomes using the basic procedure of Yasukochi and Masters (40) (some modificattons have been Introduced in this laboratory [ZS]) (See Chapter 5 (Subheading 4., note 13) If this procedure is to be used, it is useful to induce the enzyme by treating the animals with phenobarbital (by adding sodmm phenobarbital to 0.1% (w/v) m the drmkmg water for 5 d) Recently we have used a bacterial recombinant system to produce rat NADPHcytochrome P450 reductase for use m reconstitution studies with cytochrome P450 (see Chapter 8)

4.5.2. Purification of Cytochrome &Y~ 36. Cytochrome bS is rnvolved m reactions catalyzed by a number of cytochromes P450 Its role is thought to involve transfer of an electron to the Fe2+ O2 substrate complex (42,43), although alternative mechanisms also seem possible (44). The enzyme can be produced m recombmant systems (45) In this laboratory we routmely purify the enzyme from rabbit liver microsomes usmg a modification of the method of Strittmatter (46,47). (See Chapter 5, Subheading 4., note 15)

Acknowledgments The authors are grateful to I. H. Hanna and P. F. Hollenberg srty of Michigan for providing several useful modifications procedure (41).

of the Untverof the origmal

References 1. Omura, T and Sato, R (1962) A new cytochrome m liver microsomes. J Bzol Chem. 237,1375,1376. 2. Omura, T. and Sato, R. (1967) Isolation of cytochromes P-450 and P-420 Methods Enzymol. 10,556-561. 3. Ichikawa, Y and Yamano, T. (1967) Reconversion of the detergent- and sulthydry1 reagent-produced P-420 to P-450 by polyols and glutathtone Bzochzm Bzophys. Acta 131,49@-497 4 Lu, A Y. H. and Coon, M J. (1968) Role of hemoprotem P-450 m fatty acid o-hydroxylation m a soluble enzyme system from liver mtcrosomes J. Bzol Chem 243,1331,1332 5. Lu, A. Y. H., Junk, K W , and Coon, M. J. (1969) Resolution of the cytochrome P-450-contamlng w-hydroxylation system of liver microsomes mto three components. J Bzol Chem 244,3714-3721.

Purification of Liver P45Os

51

6. Imat, Y. and Sato, R. (1974) A gel-electrophoretically homogeneous preparation of cytochrome P-450 from liver mtcrosomes of phenobarbital-pretreated rabbtts Biochem. Biophys Res. Commun. 60,814. 7. Haugen, D A and Coon, M J. (1976) Properttes of electrophorettcally homogenous phenobarbital-mduclble and P-naphthoflavone-mducible forms of liver microsomal cytochrome P-450 J. Biol. Chem 251,7929-7939 8 Levin, W., Ryan, D., West, S., and Lu, A. Y. H. (1974) Preparation of partially purified, lipid-depleted cytochrome P-450 and reduced nicotmamtde adenme dmucleotide phosphate-cytochrome c reductase from rat liver microsomes J Bzol Chem 249,1747-1754 9. Guengerich, F P. (1977) Separation and purification of multiple forms of microsomal cytochrome P-450. activities of different forms of cytochrome P-450 towards several compounds of envtronmental interest. J Biol Chem 252,3970-3979. 10. Guengerich, F. P. (1982) Microsomal enzymes involved m toxtcology analysis and separation, in Principles and Methods of Tox~ology (Hayes, A W., ed ), Raven, NY, pp. 609-634. 11 Coon, M. J , van der Hoeven, T A , Dahl, S B , and Haugen, D A. (1978) Two forms of liver microsomal cytochrome P-450, P-450LMT2, and P-40&, (rabbit hver) Methods Enzymol 52, 109-l 17 12. Ryan, D. E. and Levm, W (1990) Purification and characterization of hepattc mtcrosomal cytochrome P-450 Pharmacol Ther 45, 153-239 13. Guengertch, F P., Dannan, G. A., Wright, S. T , Martin, M V , and Kammsky, L. S. (1982) Purtficatton and charactertzatton of liver microsomal cytochromes P450: electrophoretic, spectral, catalyttc, and tmmunochemical properties and mductbility of eight isozymes Isolated from rats treated with phenobarbital or P-naphthoflavone Biochemistry 21,6019-6030. 14. Guengerich, F P and Martm, M. V. (1980) Purification of cytochrome P-450, NADPH-cytochrome P-450 reductase, and epoxide hydratase from a single preparation of rat hver mtcrosomes Arch Biochem Bzophys 205,365-379 15. Warner, M , LaMarca, M. V , and Nelms, A H. (1978) Chromatographic and electrophoretic heterogeneity of the cytochromes P-450 solubihzed from untreated rat ltver Drug Metab. Dlspos. 6, 353-362. 16 Imaoka, S and Funae, Y (1986) Ion-exchange high-performance liquid chromatography of membrane-bound protem cytochrome P-450. J Chromatogr. 375, 83-90.

17 Laemmli, U. K. (1970) Cleavage of struatural protems during the assembly of the head of bacteriophage T4 Nature 227,68&685 18 Guengertch, F. P. (1994) Analysis and characterization of enzymes, m Prznclples and Methods of Toxzcology, 3rd ed (Hayes, A W , ed.), Raven, NY, pp 1259-1313. 19. Chang, H. W and Bock, E. (1980) Pitfalls m the use of commercial nomomc detergents for the solubilization of integral membrane proteins. sulfhydryl oxidizing contammants and their elimination Anal Bzochem. 104, 112-l 17.

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20. Asham, Y. and Catravas, G N (1980) Highly reactive impurities in Triton X- 100 and Brig 35: partial characterization and removal Anal Blochem 109,55-62 21 Wray, W , Boulikas, T , Wray, V P., and Hancock, R (1981) Silver staining of proteins in polyacrylamide gels. Anal Bzochem 118, 197-203. 22. Pasanen, M and Pelkonen, 0 (1985) Resolution of multiple P-450 forms separation of aryl hydrocarbon hydroxylase and aminopyrme N-demethylase-associated P-450 isoenzymes by chromatofocusing. Gen. Pharmacol. 16,361-366. 23. Alterman, M. A. and Dowgii, A. I (1990) A simple and rapid method for the purification of cytochrome P-450 (form LM4) Blamed Chromatog 4, 221,222. 24. Sandhu, P., Guo, Z , Baba, T., Martin, M. V., Tukey, R. H , and Guengerich, F P (1994) Expression of modified human cytochrome P450 lA2 in Escherzchza coli* stabilizatron, purification, spectral characterization, and catalytic activities of the enzyme. Arch Biochem Brophys. 309, 168-177. 25 Furth, A J., Bolton, H., Potter, J., and Prrddle, J. D. (1984) Separating detergent from proteins Methods Enzymol 104,3 18-329 26 Funae, Y., Seo, R., and Imaoka, S. (1986) Two-step purrfication of cytochrome P-450 from rat liver mrcrosomes using high-performance liquid chromatography. J Chromatogr 374,271-278. 27 Funae, Y., Seo, R., and Imaoka, S (1985) Multiple forms of cytochrome P-450 from mtcrosomes of rat liver, kidney, and lung resolved by high-performance hquid chromatography Bzochem Internat. 11, 523-53 1 28 van der Hoeven, T A. and Coon, M. J. (1974) Preparation and propertres of partially purified cytochrome P-450 and reduced nicotinamrde adenme dmucleottde phosphate-cytochrome P-450 reductase from rabbit liver microsomes. J Biol Chem 249,6302-63 10 29 Imai, Y and Sato, R. (1974) An affinity column method for partial purification of cytochrome P-450 from phenobarbital-induced rabbit liver microsomes J Blochem. 75,689-697. 30. Damels, F. and Alberty, R A. (1966) Physical Chemzstry, 3rd ed., John Wiley & Sons, NY, p. 40 1. 3 1. Furth, A. J (1980) Removing unbound detergent from hydrophobic proteins. Anal Biochem 109,207-215. 32. Blatt, W. F. (1971) Ultrafiltration for enzyme concentration Methods Enzymol. 22,39-49. 33. Yoo, J. S H., Cheung, R. J., Patten, C. J., Wade, D , and Yang, C. S. (1987)

Nature of N-nitrosodimethylamme

demethylase and its inhibitors.

Cancer Res

47,3378-3383

34 Guengerich, F P , Martin, M V , Beaune, P. H., Kremers, P., Wolff, T., and Waxman, D. J. (1986) Characterization of rat and human liver microsomal cytochrome P-450 forms involved m nifedipine oxidation, a prototype for genetic polymorphism in oxidatrve drug metabolism. J Biol Chem. 261, 5051-5060.

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53

35. Ma&man, R , Guo, Z , Guengerrch, F. P., and Ortiz de Montellano, P R (1995) Active site topology of human cytochrome P450 2E 1. Chem. Res Tox~ol 9, 223-226. 36. Lowry, 0. H., Rosebrough, N. J., Farr, A L., and Randall, R J. (195 1) Protem

measurement with the Folin phenol reagent. J Biol Chem 243, 133 1,1332 37. Bradford, M M (1976) A rapid and sensitrve method for the quantitatron of microgram quantittes of protem utihzing the principle of protein-dye bmdmg. Anal Biochem 12,248-254. 38. Chun, Y-J., Shtmada, T., and Guengerich, F. P (1996) Construction of a human cytochrome P450 1Al rat NADPH-P450 reductase fusion protein cDNA, expression in Escherichla ~011, purificatron, and catalytic properties of the enzyme in bacterial cells and after purification. Arch Btochem. Biophys 330,48-58 39. Parikh, A. and Guengertch, F P. (1997) Expression, purificatron, and characterization of a catalytically active human cytochrome P450 lA2.NADPH-cytochrome P450 reductase fitston protem. Protein Express Purlf 9, 346-354 40 Yasukochi, Y and Masters, B S S. (1976) Some properties of a detergent-solubtlized NADPH-cytochrome c (cytochrome P-450) reductase purified by brospecitic affinity chromatography J Btol Chem 251, 5337-5344. 41. Shen, A. L , Porter, T D , Wilson, T E , and Kasper, C B (1989) Structural analysts of the FMN bmdmg domain of NADPH-cytochrome P-450 oxtdoreductase by site-directed mutagenesis J Blol Chem. 264, 7584-7589. 42 Peterson, J. A. and Prough, R. A. (1986) Cytochrome P-450 reductase and cytochrome bs m cytochrome P-450 catalysis, in Cytochrome P-450 (Orttz de Montellano, P. R., ed.), Plenum Press, NY, pp. 89-l 17 43 Pompon, D. (1987) Rabbit hver cytochrome P-450 LM2 roles of substrates, inhibitors, and cytochrome b, m modulatmg the partmon between productive and abortive mechanisms Blochemlstry 26,6429-6435 44 Yamazakt, H., Johnson, W W., Ueng, Y -F , Shrmada, T , and Guengerrch, F P (1996) Electron transfer from cytochrome b5 is not obligatory for strmulation of catalyttc acttvtttes of cytochrome P450 3A4: charactertzatron of a reconstrtuted cytochrome P450 3A4/NADPH-cytochrome P450 reductase system and studres wrth apo-cytochrome b, J Blol Chem 271,27,438-27,444 45. Holmans, P. L., Shet, M S , Martm-Wtxtrom, C. A., Fisher, C. W , and Estabrook, R W. (1994) The htgh-level expression in Escherlchra co11 of the membranebound form of human and rat cytochrome b5 and studies on then mechamsm of functton. Arch Biochem. Blophys. 312,554-565 46. Strittmatter, P., Fleming, P., Connors, M., and Corcoran, D (1978) Purrficatton of cytochrome b,. Methods Enzymol 52,97-101 47. Shimada, T., Misono, K. S., and Guengertch, F. P. (1986) Human liver mrcrosoma1 cytochrome P-450 mephenytom 4-hydroxylase, a prototype of genettc polymorphtsm in oxidattve drug metabolism: purttication and charactertzatlon of two similar forms involved in the reaction. J. Biol. Chem 261,909-921

Isolation and Purification of Constitutive of Microsomal Cytochrome P450 John B. Schenkman

Forms

and lngela Jansson

1. Introduction The ability to purtfy cytochrome P450 from liver mlcrosomes was the result of the collective efforts of a number of laboratories, borrowing from each others’ efforts and observatrons. Early efforts to solubrlize the hemoprotein from mtcrosomes through the use of proteases were not successful, resulting merely in the solubilization of a number of other mtcrosomal proteins. The breakthrough event was the solubrlizatron of rabbit liver mtcrosomal cytochrome P450 using the ionic detergent, deoxycholate (I), allowing the monooxygenase system to be separated mto three components, a reductase, a lipid-contammg fraction and a cytochrome P450-containing fraction. Diethylammoethyl (DEAE)-chromatography was used m subsequent studies to purify further the phenobarbital-induced hemoprotem fraction (2) (later known as CYP2B4), and eventually polyethylene glycol precipitation was used to fractionate the mtcrosomal proteins (3). Various induced forms of rat cytochrome P450 were likewise detergent-solubrhzed, using sodium cholate, and polyethylene glycol-fractionated, and subjected to DEAE-chromatography with the added step of hydroxylapatite chromatography in the presence of nomomc detergent (4). Constitutive forms of cytochrome P450 were also isolated, using variatrons of these procedures, employing gentler nomomc detergents and a substrateliganded affinity column ($6). All of the procedures depend heavily upon the ability to selecttvely separate the proteins based upon differences in their abilities to bind to different column packing materials, Minor forms of cytochrome P450 can be separated by sequential passage of protem mrxtures through an anion exchange column, a cation exchange column, an hydroxylapatite or hydrophobic column. Separatrons on molecular excluston columns are not too From Methods m Molecular Btology, Vol 107 Cytochrome P450 Protocols Edlted by I R PhMps and E A Shephard 0 Humana Press Inc , Totowa, NJ

55

Schenkman and Jansson

56

effective owing to the tendency of these proteins to form large aggregates composed of different forms of cytochrome P450 and other proteins. 2. Materials 2.7. Preparation

of Microsomes

1. 0 9% NaCl for perfuston of livers. The solutton will last mdetimtely at 4°C. 2. 0.25 Msucrose containing 0.67 mMphenylmethylsulfony1 fluoride (PMSF). The sucrose solution will keep for about 2 mo at 4’C, but PMSF is not stable m aqueous solution and has to be added at time of use from a 0.67 A4 ethanol solutton while stirring. (The ethanol solution of PMSF is stable at 4°C for months ) 3. 0.154 M KC1 for washing of the microsomal fractton This solution will last indefinitely at 4°C

2.2. Solubilization of Microsomes Solubllization buffer. 50 m&I sodium phosphate, pH 7.25, containing 0.2% Emulgen 911 (Kao, Tokyo), 20% glycerol, and 0.67 mA4 PMSF. This buffer (m the absence of PMSF) IS stable for 2 mo at 4°C. PMSF should be added Immediately before use from a 0.67 A4solution in ethanol. 2.3. Column Chromatography 2.3.7. Laurate Sepharose (LS) Chromatography 1. Materials for preparation of the lauric acid EAH-Sepharose 4B (for one 2 6 x 36 cm column) a. 60 g Ammohexyl (EAH-) Sepharose 4B (Pharmacta, Btotech, Ptscataway, NJ) b 4L05MNaCl c 12.1 g 1-ethyl-3(3-dtmethylaminopropyl) carbodiimide (EDC) d 10 L dtmethylformamide (DMF) e. 4.4 g laurtc acid (free acid). 2. LS Equiltbratton buffer. 50 mM sodium phosphate, pH 7 25, contammg 20% glycerol 3. Cholate buffer: Equiltbratton buffer contammg 0.4% sodium cholate, 1 mM dtthiothrettol (DTT) and 1 mk! EDTA 4. Emulgen buffer: Cholate buffer containing 0.8 % Emulgen 911. 5 Emulgen-NaCl buffer: Emulgen buffer containing 1 M NaCl 6 LS Dtalysis buffer I: 25 mM sodium phosphate, pH 7.25, 20% glycerol , 1 mM DTT, 1 mMEDTA. 7 LS Dialysis buffer II* 5 mM sodium phosphate, pH 6 5, 20% glycerol, 0.1 mM DTT, 0.1 mM EDTA

2.3.2. CM-Sepharose

Chromatography

(2.6

x

17 cm column)

1 75 mL of Carboxymethyl Sepharose CL 6B suspension (Pharmacta). 2 CM Equihbratton buffer: 5 mM sodium phosphate, pH 6 5,20% glycerol, 0.1 mM DTT, 0 1 mM EDTA (same as Dialysis buffer II).

Purification of Constitutive Microsomal P450

57

3. CM1 buffer: 30 mM sodium phosphate, pH 6.8, 20% glycerol, 0.5% Emulgen 911,O 1 mA4DTT, 0 1 mMEDTA. 4. CM11 buffer: 45 mMsodmm phosphate, pH 6.8,20% glycerol, 0 5% Emulgen 9 11, 0.1 mMDTT, 0 I mM EDTA 5. CM111 buffer: 90 mA4 sodium phosphate, pH 6.8, 20% glycerol, 0.5% Emulgen 911,O 1 mMDTT, 0.1 mMEDTA. 6. CM Washing buffer: 1 MNaCl in 60 mA4 sodium phosphate, pH 6.8,20% glycerol, 0 5% Emulgen 9 11,O 1 mM DTT, 0.1 mM EDTA.

2.3.3. Hydroxylapatite

Chromatography

(1.5 x 12 cm column)

1. Hydroxylapatite (HA) Equilibration buffer and dralysis buffer: 5 mM sodium phosphate, pH 7.25, 20% glycerol. 2. HA elutron buffer I: 10 mh4 sodium phosphate, pH 7.25, 20% glycerol, 0.5% Emulgen 911, 0 2% sodmm cholate, 0.1 mM DTT, 0 1 mMEDTA. 3. HA elution buffer II* 80 mA4 sodium phosphate, pH 7 25, 20% glycerol, 0 5% Emulgen 911, 0.2% sodium cholate, 0.1 mM DTT, 0.1 mM EDTA. 4. Concentratmg Elution buffer: 300 mMpotassmm phosphate, pH 7.25,20% glycerol 5. Protem Dialysis buffer (final). 25 mMsodu.tmphosphate, pH 7.25,20% glycerol

2.3.4. DEA E-Sepharose

Chromatography

(I. 5 x 25 cm column)

1. DEAE-Sepharose suspenston (Pharmacra) 2. DEAE Dialysis buffer: 5 mM sodium phosphate, pH 7.4,20% glycerol. 3. DEAE elution buffer I* Dialysis buffer containmg 0.5% Emulgen 911, 0.1 mM DTT, 0 1 mMEDTA. 4 DEAE elution buffer II* 45 mA4 sodium phosphate, pH 7.4, 20% glycerol, 0 5% Emulgen 911,0.1 mMDTT, 0.1 mMEDTA 5 DEAE washing buffer: DEAE Elution buffer II containing 1 M NaCl

3. Methods 3.1. Preparation by Carbodiimide

of a Laurate-Sepharose 48 Column Condensation Reaction (5)

1. Swell the EAH-Sepharose 4B in 500 mL of 0.5 M NaCl for 30 mm at room temperature. Wash with 3.5 L of 0 5 A4 NaCl using a sintered glass funnel and vacuum Do not allow the EAH-Sepharose 4B to become dry. 2 Wash the EAH-Sepharose 4B with 1 L water, pH 4 5 (add a few drops of 1 A4 HCl to the water). 3. Take up the EAH-Sepharose m a mixture of 220 mL DMF and 220 mL water, pH45. 4. Dissolve the lauric acid m 55 mL of DMF and add rt to the EAH-Sepharose. 5. Dissolve the EDC m 55 mL water, pH 4.5, and slowly add it to the EAHSepharose and lauric acid over 10 mm with gentle stirring. Stir for 60 mm, then leave overnight at room temperature.

58

Schenkman and Jansson

6 Pour the laurate Sepharose mto a glass column with 5-l 0 cm diameter and wash sequentially with about 16 L of 50% DMF m water, 8 L water, 4 L 50% DMF m water, 10 L water, and finally with 2 L of LS Equihbratlon buffer The washing will take 3-4 d A scum on top of the column 1sunbound laurlc acid that slowly gets washed away. When finished, pack the laurate Sepharose mto a 2.6 x 40 cm column and wash with another L of equilibration buffer

3.2. Preparation

of Carboxymethyl

Sepharose

CL6B Column

1. The CM Sepharose (Pharmacla) 1savailable in suspension. Mix well and dilute 75 mL (equivalent to 60 mL bed volume) with an equal volume of 200 mM sodium phosphate buffer, pH 6.5. Stir gently and let the suspension settle Check the pH 2. Repeat the washing of the gel twice Final pH should be 6 5 3. Wash the gel three times with equal volumes of Dlalysls buffer II Check pH of supernatant after the gel settles 4 Pack a 2.6 x 11 cm column

3.3. Preparation of DEA E-Sepharose Column Prepare a slurry of about 60 mL DEAE-Sepharose m 200 mA4sodmm phosphate, pH 7.4. Allow the column material to sediment, check the pH, and decant the supernatant. If the pH is not 7.4, repeat. Equilibrate the slurry in 5 mM sodium phosphate, pH 7.4,20% glycerol, and pour into a 1.5 x 25 cm column.

3.4. Preparation

Wash with 100 mL of the dlalysls buffer.

of Rat Liver Microsomes

(see /Votes 14)

1. Livers are removed from 10 male rats of approx 200 g (see Note 1). 2 The 10 livers (- 100 g) are each perfused retrogradely with Ice-cold isotonic salme (0 9% sodium chloride, 4’C) to flush out blood (see Notes 2-3). 3 Prepare a 1: 10 homogenate (w/v) of the livers m 0.25 M sucrose containing 0 67 mA4 PMSF (see Note 4). 4. Differential centrlfugation 1sused to separate the subcellular fractions. An mltlal centrlfugation at 6008 m a refrigerated centrifuge for 5 min sediments the cell debris, nuclei, and unbroken cells (see Note 5) 5. Centrifuge the supernatant (and any washings) at 6500g for 10 mm to sediment the mltochondrlal fraction (see Note 6). 6 Centrifuge the supernatant (and any washings) at 12,000g for 10 mm to sediment any broken mltochondrlal fragments (see Note 7) 7 The supernatant is carefully decanted from the pellets and 1s centrifuged m a refrigerated ultracentrifuge at 100,OOOgfor 1 h (see Note 8) 8. The mlcrosomal pellets are separated from the clear transparent glycogen pellet and washed. This is accomplished by addition of about 5 mL of isotonic KC1 into the centrifuge tube and sharply swlrlmg the tube contents (sometimes It is necessary to tap the tube sharply on the edge of the laboratory bench to slide the

Purification of Constitutive Microsomal P450

59

microsomal pellet from the glycogen pellet). The red mtcrosomal pellets are then homogenized m a volume of the ice-cold KC1 equal to the ortgmal suspension volume 9. Centrifuge the microsomal suspension at 100,OOOg for 30 mm to obtain the washed microsomal fraction Decant the supematant from the pellets and discard it 10. Homogenize the pellets to yield 100 mL in 0.25 M sucrose for protem determmanon (see Note 9).

3.5. Solubilization

of the Microsomal

Proteins

Dilute the microsomes to 2.5 mg protein/ml in Solubilization buffer and stir slowly under nitrogen on ice or in a cold room for 30 min. Measure the volume of the preparation and remove a 2-mL ahquot of the suspension for measurement of the starting cytochrome P450 content. Centrifuge the preparation at 113,OOOgfor 1 h and remove the supernatant from any pelleted material. The supernatant is a reddish-brown color. This procedure solubilizes about 50% of the microsomal protein and essenttally all of the cytochrome P450. 3.6. Laura te EAH-Sepharose 4B Chromatography (see Notes 10-16) This is a hydrophobic column and owes its properties to the covalent hnkage of lauric acid to the aminohexyl bridge on the Sepharose 4B support. Laurrc acid is actually a substrate of many forms of cytochrome P450, which may, m part, explain the strong binding of cytochrome P450 to thts material. 1. Apply half of the supernatant to a 2.5 x 35 cm Laurate EAH-Sepharose 4B column (or half each to two columns) that has been extensively washed with Equihbration buffer m the cold Load the column at a steady rate of 20 mL/h (about 6 drops/min) (see Note 10) 2. When all of the supernatant has been loaded, a red coloration is concentrated at the top of the column. The column is then washed with 100 mL of Equrhbratton buffer at a rate of 12 mL/h. 3. Elution is initiated by passing 500 mL of Cholate buffer through the column at the rate of 10-12 mL/h (5). Hemoprotem absorbance in the eluate fracttons is measured at 417 nm (see Notes 11 and 12). The hemoprotein peak that elutes is P450 I. 4. The column development contmues with a linear gradient of O-l MNaCl m Cholate buffer (75 mL of Cholate buffer and 75 mL of Cholate buffer contammg 1 A4 NaCl in respective gradient flasks) (see Note 13) 5. The column is next developed with 500 mL of Emulgen buffer The P450 II fraction elutes here (see Note 14). 6. The column 1s then washed with about 2 L of Emulgen buffer contaming 1 M NaCl. Cytochrome b, elutes here (see Note 15).

SchenkmanandJansson

60

7. The column may then be washed with 500 mL of Equilibration for the next use (see Note 16).

3.7. Carboxymethyl-Sepharose (see Notes 77-27)

buffer and stored

CL-6B Column Chromatography

To purify forms of cytochrome P450 in the P45011 fraction CM-Sepharose CL6B has proven to be extremely helpful. In contrast to the hydrophobic Laurate EAH-Sepharose 4B column, this 1sa cation exchange column. It acts to bind catlomc functional groups, separatmg protems on the basis of catlomc charge. To ensure efficient binding, it is necessary to dialyze fractions before loading on the column. 1 Divide the pooled P45011 fractions from the Laurate-Sepharose column into aliquots of 300-400 nmol and dialyze each twice vs 20 volumes of Dialysis buffer II at 0-4’C (see Note 17). 2. Dilute the cytochrome P450 concentration to 1 w using the CM Equilibration buffer 3 Load the cytochrome P450 onto the CM Sepharose CL6B using a flow rate of 12 mL/h. Wash the column with 50 mL of CM Equilibration buffer Cytochrome P450 is then 4. eluted stepwise using buffers with increasmg sodium phosphate concentrations. 5 Using 250 mL of the 30 mM CM1 buffer as the first step elutes a sharp peak, followed by slow tailing from the CM column (see Note 18). Pool the fractions followmg the sharp peak (CMI) The second step of the gradient utilizes 250 mL of the 45 mM CM11 buffer. Collect and pool the hemoprotem-containing fractions (see Note 19) The third step of the gradient utihzes 250 mL of the 90 mM CM111 buffer (see Note 20)

The remammg proteins are eluted from the CM Sepharose CL6B with 200 mL of CM Washing buffer as CMIV (see Note 21). The CMIV fraction that elutes must be dialyzed immediately vs 20 volumes of LS Dralysis buffer I to prevent damage to the hemoprotem by the high salt After dialysis, the fraction may be stored at -80°C until use. 10 The column is then washed with an addmonal500 mL of LS Dialysis buffer I to ensure it is cleaned, and then washed with 200 mL of CM Equilibration buffer and stored m that buffer for reuse.

3.8. HA Chromatography

of CMII (see Notes 22-24)

1 Stir about 20 mL of HA mto a slurry with HA equilibration buffer and pour mto a 15 x 10 cmcolumn. 2. Equilibrate the column by passing 200 mL of HA Equilibration buffer through it. 3. Load about 150 nmol of CM11 cytochrome P450 on the column (see Notes 22 and 23)

Purifica t/on of Cons titu tive Microsomal P450

61

4. Elute the cytochrome P450 using a lmear gradient of 10-80 mM sodium phosphate, pH 7.25. This is accomphshed by using 175 mL each of HA elutlon buffer I and HA elution buffer II to generate the gradient (see Note 24) The first peak elutmg, CYP2Cl 1, should be pure as Judged by SDS-polyacrylamide gel electrophoresls.

3.9. DEAE-Sepharose

Chromatography

of CM/l (see Note 25)

DEAE-Sepharose chromatography must be carried out on the combmed middle and last peaks of the HA column in order to obtain these proteins in pure form (7). This is an anton exchange column, which acts by weakly bmding anionic functional groups, separating proteins on the basis of anionic charge. 1. Fractions containing a mixture of proteins from HA are pooled (these are the middle and last peaks) and dialyzed overnight vs 20 volumes of DEAE DlalySIS buffer. 2. Load the dialyzed cytochrome P450 pool onto the DEAE-Sepharose column 3. Elute with two column volumes of DEAE elutlon buffer I Collect the first weakly binding hemoprotein fraction (CYP2C6) being careful to exclude the trailmg hemoprotem, if any (see Note 25) 4 Elute CYP2CI 3 with DEAE elutlon buffer II. 5 Wash the column with 100 mL of DEAE washing buffer to clean It, 6. Equilibrate the column with Dialysis buffer and store it until next use

3.10. Removal of Detergent of the Purified Proteins

and Concentration

1 Dialyze the purified protein fractions obtained from the HA or DEAE-Sepharose columns overnight against 20 volumes of HA Equihbratlon buffer 2 Apply the dialyzed sample to a small hydroxylapatlte column (0.9 x 1,5 cm) It will bmd m a thin, tight red band. 3. Detergent is removed by washing the column wtth HA Equllibratlon buffer until the absorbance at 280 nm drops to 0.00. 4. The protein is then eluted with Concentrating Elutlon buffer. 5 Immediately dialyze the concentrated cytochrome P450 against Protein Dialysis buffer overnight and store the protein at -80°C m allquots for use

4. Notes 1, The preparation of microsomes is essentially the same for a number of mammalian species (including human) and for different ttssues. The yields, however, will differ. Using 10 male rats of approx 200 g each, the yield of liver will be approx 10 g per rat. The final yield of microsomes ~111be approx 15 mg per g of liver, or more, depending upon whether the different centrifugatton fractions of the preparation are washed and the washings combined to increase yield

62

Schenkman and Jansson

2. When red cells are broken, released hemoglobin ~111 adsorb to the microsomes, mcreasmg protein content and also interfermg with spectral analyses 3 After perfusion, the livers weigh approx 30% more, so if yield is to be calculated per g of liver, it is best to weigh them before 4. A 1: 10 homogenate (w/v) provides optimal separation of subcellular particles and the greatest yield of microsames. PMSF is a serme protease mhibitor This mhibitor dimmtshes proteolytic action agamst proteins durmg preparation. 5 To enhance the yield of microsomes, if desired, the sediment may be rehomogenized in a volume of sucrose-PMSF equal to that used m the prior step and the washmgs combined with the origmal supernatant As much as 40% Increase m yield can be obtamed by washing this fraction and the subsequent fraction 6 The pellet of mitochondria will have a dark brown appearance A pink outer edge to the pellet is owmg to the presence of the less dense, heavy microsomal membranes that have also sedimented The pellet may be discarded or washed by resuspendmg m the sucrose-PMSF solution followed by centrifugation at 65OOg for 10 min; combme the washings with the supernatant 7. These are denser and will appear as a brown pellet in the center of less tightly packed, red microsomal membranes The latter will generally constitute a small amount, not enough to bother with 8 One may use greater force for shorter periods. The suggested parameter, however, is opttmal for separation of the microsomal fraction from glycogen During column chromatography, the glycogen may impair chromatographrc separation. 9 The protein content is sufficiently high for a bmret analysis if desired Biuret protein analysis may be performed on the microsomal fraction. However, as purification proceeds a more sensmve procedure, such as the Lowry method or BCA analysis (Pierce, Rockford, IL) ~111be necessary, as these require considerably less protein. 10 It will take about 24 h to load the 500 mL of solubilized mrcrosomal proteins It is best not to overload the column, and a good separation is achieved when the amount of cytochrome P450 loaded does not exceed 800 nmol The column is then eluted m step-wise manner, as depicted in Fig. 1. Il. Because of the strong absorbance at 280 nm of the nomomc detergent used to solubilize the microsomes, it is not possible to monitor protein at this wavelength 12 The first peak that elutes (P450 I) contams about 5% of the applied cytochrome P450. 13. The NaCl gradient elutes a double peak, the first part containing about 2% P450 and the second contammg NADPH-cytochrome P450 reductase (FpT, 45-55%) The volume of this gradient is less than one column volume, but once released, the reductase will elute, when the next buffer is applied, precedmg P450 II The NADPH-cytochrome P450 reductase can be easily purified if desired (8): a. Apply the pooled, pigmented peak to a 2.6 x 35 cm DEAE-Sephacel column pre-eqmlibrated with 25 mM Tris-HCl, pH 7.7, containing 0.8% Emulgen 911, 0.1% sodium cholate, 0.05 mMDTT, 0.05 mM EDTA, and 20% glyc-

63

Purification of Constitutive Microsomal P450

01 0 0

20

40

60

80

Fraction

100

120

140

160

180

number

Fig. 1. Isolatron of cytochrome P450-contaming fractions of rat hver microsomes using Laurie acid EAH-Sepharose chromatography.

b. c. d

e.

f. g. h.

1

erol. Durmg loading, some cytochrome P450 will pass through the column m the loading volume Wash the column with 450 mL of the equilibration buffer Wash the column with 450 mL of the eqmhbration buffer containing 150 mM Tris-HCI, pH 7.7. Some additional cytochrome P450 will elute. Apply a linear gradient of KC1 using 250 mL of the 150 mM Trrs-HCI equilibration buffer and 250 mL of that medium containmg 0 5 M KCl. The NADPH-cytochrome P450 reductase should elute m the first 300 mL of the gradient. Pool the reductase-contammg fractions and apply directly to a 0 9 x 12 cm column containing a bed volume of 8 mL of 2’,5’-ADP-Sepharose 4B that has been previously equihbrated with 10 &potassium phosphate buffer, pH 7.7, contammg 20% glycerol, 0 1% Emulgen 9 11,0.02 mM EDTA, and 0.2 mJ4 DTT (9) Wash the column wrth 200 mL of the same medmm except for an increase m the phosphate buffer concentration to 200 mA4 Wash the column with 45 mL of equilibration buffer (10 mM phosphate buffer). Elute the NADPH-cytochrome P450 reductase with 25 mL of equilibration buffer contammg 0 7 mM 2’-AMP It will appear as a deep yellow band moving down the column Collect the reductase-containing fractions and load on a small (0 9 x 2 cm) HA column previously equilibrated with 5 mA4 sodium phosphate, pH 7.25, contammg 20% glycerol.

64

Schenkman and Jansson j

14.

15

16 17.

Wash the column with this buffer untrl A,,, drops to zero. Elute the reductase with 300 &potassium phosphate buffer, pH 7 25, contaming 20% glycerol The reductase is detergent free. Dialyze against 25 mA4 sodium phosphate, buffer pH 7.25, containing 20% glycerol, and store at -80°C The reductase will have a specific content of 9 3 nmol/mg and a specific activity of 3 pm01 cytochrome c reduced per min/nmol. Examme by 9% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis to ensure purity Between 55 and 65% of the cytochrome P450 applied to the Laurate Sepharose column elutes as a large peak followmg the NADPH-cytochrome P450 reductase. This peak is the P450 II fraction Most of the NADH-cytochrome bs reductase activity (70-80%) is also present m this fraction The Emulgen-NaCl buffer will elute 100% of cytochrome b, as well as wash all of the remarnmg protem from the column. If desired, the cytochrome b, can be purified directly from this fraction (8). a Dialyze the b,-contaming fractions vs 4 L of 10 mM Tris-acetate, pH 8 1, containing 0.1 mA4 EDTA (Note volume will increase during dialysis, so leave room for expansion.) b After dialysis, apply to a 2 6 x 35 cm DEAE-Sephacel (Pharmacta) column that has been previously equtlibrated with the dialysis buffer c Wash with dialysis buffer to remove Emulgen 911 until A2s,,drops below 0 1 d Elute the cytochrome b, m the same buffer contammg 0 25% sodium deoxycholate and 0.25 Mpotassium thtocyanate The cytochrome bs ~111 elute in 1 column volume. e Pool b5 fractions with an A413/A28,-,> 0 2 and concentrate on an Amicon PM30 membrane; in this buffer the cytochrome bs exists m an aggregated state f Apply the concentrated cytochrome b, to a 2 6 x 68 cm Sephadex G-100 (Pharmacia) column that has been previously equilibrated with the dialysis buffer contarnmg 0 25% sodium deoxycholate and 0 25 M potassium throcyanate g. Pool fractions with an Ad13IA 280> 1 0, concentrate on the PM 30 membrane to 5 mL, and reapply to the Sephadex G-100 column h Pool fractions with an A413/A28,,> 2.5 i Concentrate the pooled fractions to 2 mL and dialyze vs 2 L of 20 rtuV Tris acetate, pH 8 1, 0.2 mMEDTA. J. Apply the cytochrome bs to a Sephadex G-25 column previously equilibrated wtth the same buffer. Collect the cytochrome b5 peak and concentrate it to about 200 flon the Amicon membrane. The preparatron ~111 be pure if the A4JAz8,, is > 2 5 It will be 48 nmol/mg. Run a 12% SDS-polyacrylamide gel to verify purity If washed extensively after each use with high salt, this laurate Sepharose column will last for several years. For convenience, allow the first dialysis to proceed overnight, then transfer to the second dialysis to be completed the following morning The pH of the fraction should be adjusted after dialysis to 6.8 with 200 n& NaH2P04 For efficient

65

Purification of Constitutive Microsomal P450 _-“.

__-

I_-

__I_--

_

-

__”

_

_““_

-“-_-l-l--

-.

-

----

-.-

-_

-

-

--

--_--_

_

“_

-

-

035

“^_-_._-_

CM

CM

IV

II CM

III

0 05 0 0

20

40

80

80

Fraction

100

120

140

160

180

number

Fig. 2 Chromatographic separation on Carboxymethyl Sepharose CL6B of the cytochrome P450 forms of the P450 II fraction from laurate Sepharose chromatography

18

19.

20.

21. 22. 23. 24.

separation on the CM-Sepharose, it is better to be slightly below pH 6.8 than above it Figure 2 shows the elutlon from CM Sepharose. The first sharp peak contains NADH-cytochrome b5 reductase and the followmg fractions contam approx 5% of the applied cytochrome P450 (CMI). CYP2A2 (RLM2) was purified from this fraction (10) using HA. Alternatively, CYP2A2 can be purified on DEAESepharose using a 5-35 mM sodium phosphate gradient (the buffers contam 5 m&f or 35 mM sodmm phosphate, pH 7.4, 20% glycerol, 0 5% Emulgen 911, 0.1 mM DTT, and 0 1 mA4 EDTA). The 45 mM buffer elutes a sharp peak (CMXI) containing forms Identified as CYP2C13 and 2C11 (6) and CYP2C6 (11) Approximately 25% of the applied cytochrome P450 ~111 be recovered m this fraction. The sharp peak that elutes in 90 Wbuffer (CMIII) contains -20% of the applied cytochrome P450, and includes a form of cytochrome P450 that was Identified as CYP2El (12). This fractton contains about 15% of the cytochrome P450 apphed to the column. Among the forms of cytochrome P450 it contains IS CYP2C7 (12) The cytochrome P450 binds tightly to the top 3 mm of the column forming a deep red band The cytochrome P450 in fractions CMI, CMIII, and CMIV also adsorb tightly to this column and it may be used to separate these forms as well. Figure 3 shows the elution peaks of the CM11 on HA The elution IS of two peaks with a smaller peak between and overlappmg these. The first part of the first peak contams electrophoretlcally pure CYP2C 11. The last peak contains CYP2C 13 and the middle peak contains CYP2C6. These are difficult to separate from each

Schenkman and Jansson

66 0 16

70

014

60

0 12

50

r^ E

01

40

0 06 30 0 06

d 2 E: 2 CL

E 20

004

.2 B rn

10

0 02 0

0 1

25

50

65

78

86

Fraction

97

108

115

130

160

number

Fig. 3. Chromatographtc separation of cytochrome P450 forms of the CM II on HA other and from traces of CYP2CI 1 on the HA column, and IS best accomplished on DEAE-Sepharose. 25 CYP2C6 is not retained m this buffer, but elutes nnmedtately after the void volume Any CYP2Cll that was present follows shortly thereafter, and any CYP2C13 that is present is retained and can be eluted using DEAE Elutton buffer II.

References 1. Lu, A. and Coon, M. (1968) Role of hemoprotem P-450 m fatty acid w-hydroxylation m a soluble enzyme system from liver mtcrosomes J Btol Chem 243, 1331,1332 2. Lu, A., Junk, K , and Coon, M. (1969) Resolution of the cytochrome P-450-contaming w-hydroxylatton system of liver mtcrosomes mto three components. J Blol Chem 244,3114-3721

3. Haugen, D and Coon, M. (1976) Properties of electrophorettcally homogeneous phenobarbital-inducible and B-naphthoflavone-inducible forms of liver microsoma1 cytochrome P-450. J Biol Chem 251,7929-7939 4. Ryan, D., Thomas, P , and Levm, W. (1982) Purrfication and characterization of a minor form of hepatic microsomal cytochrome P-450 from rats treated with polychlorinated brphenyls Arch Biochem Blophys 216,272-288. 5. Gibson, G. and Schenkman, J. (1978) Purtficatton and properties of cytochrome P-450 obtained from liver microsomes of untreated rats by laurtc acid affinity chromatography. J B~ol. Chem 253,5957-5963

Purification of Constitutive Microsomal P450

67

6. Cheng, K.-C. and Schenkman, J. (1982) Purification and characterization of two constitutive forms of rat liver microsomal cytochrome P-450 J. Bzol Chem 257, 2378-2385. 7. Schenkman, J., Favreau, L., Mole, J , Kreutzer, D and Jansson, I (1987) Fmger-

8

9

10

11.

12.

prmtmg rat liver mtcrosomal cytochromes P-450 as a means of delineating sexually distmctive forms. Arch ToxzcoZ 60,43-5 1 Tamburmi, P., White, R and Schenkman, J. (1985) Chemical characterization of protein-protein mteractions between cytochrome P-450 and cytochrome b, J Blol Chem 260,4007-4015 Yasukochi, Y. and Masters, B (1976) Some properties of a detergent-solubihzed NADPH-cytochrome c (cytochrome P-450) reductase purified by biospecific affinity chromatography J Biol Chem 251, 5337-5344 Jansson, I , Mole, J. and Schenkman, J. (1985) Purification and characterization of a new form (RLM2) of liver microsomal cytochrome P-450 from untreated rat. J Blol Chem 260,7084-7093 Jansson, I., Tamburmi, P., Favreau, L. and Schenkman, J (1985) The mteraction of cytochrome b5 with four cytochrome P-450 enzymes from the untreated rat Drug Metab Drip 13,453-458. Favreau, L , Malchoff, D , Mole, J and Schenkman, J. (1987) Responses to msulin by two forms of rat hepatic microsomal cytochrome P-450 that undergo maJor (RLM6) and minor (RLMSb) elevations in drabetes. J Brol. Chem 262, 14,3 1914,326

6 Purification

of Extrahepatic

Cytochromes

P450

Margaret Warner and Jan-Ake Gustafsson 1.

Introduction

Identtfication of the cytochrome P450 profiles in extrahepatic tissues such as the brain, breast, and prostate is a prerequisite for evaluation of the role of these enzymes in in situ activation of procarcinogens and tissue-specific metabolism of hormones and pharmaceuticals. The mam difficulty encountered in such efforts is the low level of cytochrome P450 m these tissues. Normally, in tissues where cytochromes P450 are abundant, then presence can be detected m microsomal fractions by carbon monoxide (CO) difference spectrophotometry (1) (see Chapter 3) and by Western blotting (see Chapter 43). The limit of spectrophotometric detection is 10 pmol P450/mL. In tissues where the amount is low (l-10 pmol/mg microsomal protein), problems of turbidity and interfering chromophores make it impossible to quantify cytochrome P450 by CO difference spectrophotometry. With Western blots, the limit of sensitrvity for detection of an individual form of cytochrome P450 is 0.5 pmol/lane. In the case of tissues in which the cytochrome P450 content is as low as 1 pmol/mg, even tf all of the cytochrome P450 were a single form, it would be necessary to load 0.5 mg microsomal protein per lane m order to detect it. Such high concentrations of protein usually result m a very poor signal-to-noise ratio on Western blots. Reverse transcription-polymerase cham reaction (RT-PCR) is being used extensively to identify cytochrome P450 forms in extrahepatic tissues (2-5). Caution has to be used in mterpretation of data from this technique both because of its extreme sensitivity and, more importantly, because it gives no information about whether the mRNAs detected are translated into proteins in the tissue. From Methods m Molecular B/ology, Vol Edtted by I R Phillips and E A Shephard

69

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Cytochrome

0 Humana

P450

Protocols

Press Inc , Totowa,

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Warner and Gustafsson

The main aim of purification of extrahepatic cytochromes P450 should perhaps not be purification to homogeneity but only purification to a stage where the cytochromes P450 can be identified by Western blotting (6) or by microsequencing of the protems blotted onto polyvmylidme difluoride (PVDF) membranes (7). From such information, cDNA cloning and expression of cytochromes P450 can be achieved by methods that are more productive and less tedious than protem purification techniques. The two main prerequisites for methods for the isolation of extrahepatic cytochrome P450 are therefore, that the heme of the cytochrome P450 should be retained intact so that CO difference spectrophotometry can be used for quantification and that the yield or recovery of cytochrome P450 must be high so that the cytochromes P450 extracted are representative of the cytochromes P450 m the tissue. In this chapter, the authors describe a method that has been used extensively for extraction of cytochrome P450 from tissues such as the brain, breast, abdominal fat, and ventral prostate, where cytochrome P450 content is low (8-IO), and from the pituitary gland (II), where the amount of tissue is small. The method involves two steps: solubilizatton of cytochrome P450 from whole tissue or subcellular fractions in a combmation of ionic and nonionic detergents that preserve the integrity of the cytochrome P450, followed by hydrophobic chromatography on columns of Sepharose coupled to p-chloroamphetamine. 2. Materials 2.1. Solubilization Buffers are selected to give optimal yields of cytochrome P450 from tissue or subcellular fractions and to permit optimal binding top-chloroamphetamme Sepharose. All of these buffers can be kept for weeks at 4°C. 1 Solubilization buffer 800 mL phosphate-buffered saline (PBS), 200 mL glycerol, 744 mg EDTA (2 mA4), 5 g sodrum cholate, 2 mL Emulgen

9 11 (see

Note 1).

2. Drlutron buffer 800 mL PBS, 200 mL glycerol, 2 mA4 EDTA 3. Equrlibratron buffer 250 mL solubrlrzatron buffer, 750 mL drlutton buffer. 4. Phenylmethylsulfonyl fluoride (PMSF) 200 miM (34.8 mg/mL) m ethanol This can be stored for 1 wk m the freezer rf it 1skept morsture free.

2.2. Preparation

of p-Chloroamphetamine

Columns

1 0 1 M sodium btcarbonate, made just before use 2. p-Chloroamphetamme (Srgma, St Louis, MO) solution (412 mg in 100 mL sodmm brcarbonate), made just before use. Cautzon’ p-Chloroamphetamine is a neurotoxin. Handle with care Do not inhale. 3 1 rnA4HCl.

71

Extrahepatlc Cytochromes P450 4. pH paper. 5 Activated CH-Sepharose 4B from Pharmacta or AffiGel 10 from BioRad.

2.3. Concentration

of Cytochrome

P450

Dialysis tubing (2.5 cm width). This should be prepared sodium carbonate followed by extensive washing in distilled Aquacide 2 (Calbiochem). This is a high molecular weight which absorbs water and 1s used to concentrate cytochrome

3. Methods 3.1. Handling

by bollmg m 3% water. (500K) cellulose P450 fractions.

of Tissues (see Note 2)

1. Remove tissue of Interest and place m a preweighed beaker contammg 10-20 mL ice-cold dtlutton buffer It IS not necessary to perfuse the tissue before removal In the case of the brain, blood vessels can be removed with a good pair of forceps, m order to reduce potential problems for the subsequent analysis of cytochrome P450 that may result from hemoglobin contammatton In order to measure cytochrome P450 spectrally, it 1sadvtsable to start with tissue from 10 animals. 2. Weigh the beakers with the tissue. It 1s most convenient to express the cytochrome P450 content of these tissues on the basis of the wet weight of the tissue Soft tissue such as the bram and pmutary glands do not require additional handling prior to homogemzation Organs that contain large amounts of connecttve tissue need to be cut mto tine pieces with scissors before homogenization. 3. If cytochrome P450 is bemg prepared from the whole tissue, replace the dilutton buffer with solubihzatton buffer. Use approx 1 g tissue per 10 mL buffer and homogenize 1 g at a time. Homogenize with a polytron at mid-speed for 5 s. Add 200 mMPMSF (2 uL/mL of homogenate) and contmue homogenization for 30 s. In the case of the breast it is necessary to remove pieces of connective tissue by strammg the homogenate through two layers of gauze. The homogenate can be left at 4°C overnight or can be processed after 2 h 4. An alternative to using total tissue homogenate is preparation of cytochrome P450 from total membrane fraction. This has the advantages of: a. removal of cytosolic protems, which may cause background problems on Western blots; b. removal of much of the tubulin, which 1s a major protein m the molecularweight range of 50K and is a major contaminant if mtcrosequencmg of cytochrome P450 from sodmm dodecyl sulfate (SDS)-polyacrylamide gels is attempted; c tt avoids the losses associated with subcellular fractionation. To prepare a total membrane fraction, homogenize the tissue m dilution buffer instead of solubihzatton buffer. Centrifuge the homogenate at 100,OOOg for 1 h. Discard the supernatant and resuspend the pellet in solubihzatton buffer (1 g wet weight tissue/ml) with the use of a polytron Add 200 @4 PMSF again Allow the membrane suspension to stand at 4°C for 2 h or overmght

Warner and Gustafsson

72 3.2. Extraction of Cytochrome P450 by Hydrophobic Chromatography

1. After 2-12 h solubllizatlon, remove insoluble material by centrlfugatlon at 100,OOOg for 1 h Carefully decant the supernatant The brain pellet IS usually not very compact and some care must be taken to avold contammation of the supernatant with the pellet The pellet contains large quantltles of DNA, which can clog the column or significantly reduce its flow rate 2 Measure the volume of the supernatant and add three volumes of dllutlon buffer to it The extract is now ready for chromatography and 1s stable for at least 2 d at 4’C 3 Pour the extract onto a column ofp-chloroamphetamme-Sepharose (2 5 x 10 cm) and allow it to flow through at a rate of 1 mL/mm at 4“C The flow rate may be reduced and the column left to run overnight, but care must be taken to avoid the column running dry while the sample is on 4 Wash the column with 50 mL equihbratlon buffer. If more than 10 brains have been processed, it may be necessary to wash with a further 50 mL until the eluate is clear 5. Elute the column with solubihzatlon buffer and collect 3 mL fractions. Cytochrome P450 starts elutmg at approx fraction 7 and most has eluted by fraction 14 6 Wash the column with 50 mL KC1 (1 M), The column can be stored m KC1 or can be immediately used for another sample by washing with 50 mL equllibratlon buffer 7 Measure the CO difference spectra of cytochrome P450 rn the eluted fractions and pool those contammg cytochrome P450. If 10 rats have been used, measurement of cytochrome P450 from the brain, prostate, pregnant or lactating breast will be easy. In adult vlrgm or post-lactating breasts, it may be necessary to concentrate the cytochrome P450 before a CO-difference spectrum can be obtained If small brain regions have been used, concentration will also be necessary. As a rule, the yield of cytochrome P450 from the brain is 30-50 pmol/g. The olfactory lobes from 10 rats can therefore yield only 30-50 pmol If this has eluted m 24 mL the content 1s l-2 pmol/mL and this needs to be concentrated 5- to lofold before it can be accurately measured. 8. Concentrate cytochrome P450 by pouring the pooled fractions mto dialysis tubing with a cut-off of M, 10K. Secure the ends of the tubing with knots or dlalysls tubing chps and lay the dialysis bag in a dish containing a layer of Aquaclde It 1s best to have the cytochrome P450 exposed to the smallest surface area to avoid losses on the dlalysls membrane. Dialysis tubing 2.5 cm m width 1susually good Place the dish in the refrigerator overnight. This should be enough time to reduce 24 mL to 3 mL (see Note 3)

3.3. Preparation Because as a dilute detergents acid (TCA)

of Cytochrome

P450 for Electrophoresis

the cytochrome P450 1seluted fromp-chloroamphetamine columns solution, it is necessary to preclpltate the protem and remove the prior to electrophoresis. Standard methods such as trichloroacetic precipitation are inefficlent because of the detergents. This is par-

73

Extrahepatic Cytochromes P450

titularly true when the cytochrome P450 has been concentrated, because the Emulgen 911 concentrates along with the cytochrome P450 and can prevent efficient precipitation. In many extrahepatic tissues, the major isoforms of cytochrome P450 are different from those m the liver. If the aim of the study 1s to demonstrate the presence of a specific cytochrome P450 isoforrn, which may represent only a small fraction of the cytochrome P450 m the tissue under investigation, it may be necessary to load 30-50 pmol cytochrome P450 m each lane. Proteins are efficiently precipitated from the detergent solutions by methanol. 1. To 30-mL corex tubes, add 1-3 mL cytochrome P450 solution. 2. Add 20 mL methanol and place the tube m a bucket containing crushed frozen CO2 (dry ice). 3. After 30 min on dry ice, recover the protems by centrifugation at 10,OOOg for 20 min. 4. Discard the supematant and allow the contents of the tube to air-dry or dry under a stream of nitrogen. 5 Resuspend the protein pellet m SDS gel loading buffer with the ald of a small spatula. With some tissues, like the brain, the pellet may be large and much of It ~111 be msoluble. If there are doubts about recovery, it 1s a good Idea to add an irrelevant marker protem to the cytochrome P450 solution prior to preclpitatlon and follow its recovery on Western blots. 6 After resuspension and heating m boiling water for 1 min, centrifuge to remove msoluble material and proceed with electrophoresls.

3.4. Preparation

and Care of p-Chloroamphetamine

Columns

Coupling ofp-chloroamphetamine to activated Sepharose is done according to the manufacturer’s recommendahons. We have used both Pharmacia CH Sepharose 4B and BioRad Affigel 10 with equal success. p-Chloroamphetamine has a free ammo group and IS soluble in sodium bicarbonate, pH 8.8, which 1s Ideal for the coupling reaction. 1. Weigh out 4 12 mg (2 mmol) p-chloroamphetamine and add it to 100 mL 0 1 M sodium bicarbonate. 2. Weigh out 5 g activated Sepharose m a beaker and add 100 mL 1 mM HCl 3 Wash the activated Sepharose with 500 mL 1 mMHC1 on a sintered-glass funnel Do not allow the gel to completely dry 4. With a spatula, add the gel to the p-chloroamphetamine solution. Check the pH of the mixture. It must be mamtamed at around pH 8 0 for efficient coupling The pH can be adjusted with 0.1 M sodium hydroxide 5. Place the mixture on a shaker for 3-24 h The reaction can be done m a screw capped tube or a 500-mL beaker depending on what type of shaker 1s avallable Magnetic sttrrmg bars should not be used because they can damage the gel. Because the hgand 1sm large excess, it is not necessary to block unreacted groups.

74

Warner and Gustafsson

6. Wash the coupled gel on a smtered glass funnel with 500 mL 0 1 A4 sodmm bicarbonate, followed by 500 mL water and 100 mL dilution buffer. 7 Remove the gel with a spatula and place it m a beaker contammg equilibration buffer. 8. Pour the gel into a column 2.5 x 50 cm Although there will only be 15 mL of gel, the long column is convement because it permits the sample to be poured onto the column without the need for pumps. The column 1s now ready for use.

3.5. Care of the Column Because the hgandp-chloroamphetamme is coupled to the gel by a peptide bond, it IS necessary to protect the column from proteases. In the tissues we have used, 200 mM PMSF offers sufficient protection. Although the matrix can last indefinitely, it IS necessary to remove the gel from the column and carefully wash it at least once a wk when it is in constant use. One of the main problems is clogging owing to DNA when total tissues are used for extraction of the cytochrome P450. 1 Remove gel from the column into a beaker of water 2. Use a broad flat spatula to disperse the gel. 3. Pour the gel onto a sintered-glass funnel and wash with 1 L of distilled water, followed by 200 mL ethanol, then 500 mL water and finally 500 mL 1 M KCl. The gel can be repacked mto the column m 1 M KC1 and stored at 4°C.

4. Notes 1 There are many nomomc detergents available that can be used m place of Emulgen 911 We have used Emulgen 911 for more than 20 yr. It is avarlable from Kao Atlas, Japan. 2 When the cytochrome P450 profile of the tissue under study has not been characterized, the cytochrome P450 should be extracted from the whole tissue with no subcellular fracttonation. Methods for subcellular fractionation by differential centrifugatton that work well in the liver do not necessarily produce the same well-defined fractions in other tissues. Loss of mtcrosomes in the mitochondrial fraction can be great, particularly in the case of brain and prostate. In addition, for the brain, homogemzation procedures may not be efficient to disrupt some of the smaller cells and this will lead to a low yield and perhaps loss of certam cytochrome P450 isoforms. Optimal homogemzation and differential centnfugatton techmques must be experimentally determined for individual tissues 3. Cytochrome P450 prepared as prevrously described can be used as starting material for further purificatton. Methods described for purification of hepatic cytochromes P450 can be used (see Chapters 4 and 5) If N-terminal sequencmg is the goal, one further purification step through ion exchange or hydroxyapattte (HA) columns is usually sufficient to permit identification of cytochromes P450 after resolution on SDS-polyacrylamide gels.

Extrahepatlc Cytochromes P450

75

References 1. Omura, T. and Sato, R (1964) The carbon monoxtde-bmdmg pigment of liver mtcrosomes I Evidence for its hemoprotem nature. J Bzol Chem 239,2370-2378. 2. Zaphiropoulos, P G. and Wood, T. (1993) Identtficatton ofthe major cytochromes P450 of the 2C subfamtly that are expressed m brain of female and m olfactory lobes of ethanol treated male rats Biochem. Brophys Res. Commun 193, 1006-l 0 13. 3. Kawashima, H. and Strobel, H. W. (1995) cDNA cloning of three new forms of rat bram cytochrome P450 belongmg to the CYP4F subfamily Bzochem Bzophys Res Commun 217,1137-l 144. 4. Wang, H. M , Kawashtma, H , and Strobel, H. W (1996) cDNA cloning of a novel CYP3A from rat bram Bzochem. Bzophys Res. Commun. 221, 157-l 62 5. Stapleton, G., Steel, M , Richardson, M., Mason, J. 0 , Rose, K A., Moms, R G M., and Lathe, R. ( 1995) A novel cytochrome P450 expressed prtmartly m brain J Biol Chem. 270,29,739-29,745 6. Wyss, A , Gustafsson, J.-A , and Warner, M. (1995 ). Cytochromes P450 of the 2D subfamily m rat brain. Mel Pharmacol 47, 1148-l 155. 7 Warner, M and Gustafsson, J.-A (1994) Induction of cytochrome P450 in the rat bram by ethanol Proc Natl. Acad. Sci USA 91, 1019-1023 8 Hedlund, E., Wyss, A , Kamu, T., Backlund, M., Kohler, C , Pelto-Hmkko, M , Gustafsson, J.-A , and Warner, M.( 1996) Cytochrome P450 2D4 m the brain* specific neuronal regulation by clozapme and toluene Mel Pharmacol 50,342-350. 9. Hellmold, H., Lamb, J. G , Wyss, A., Gustafsson, J -A , and Warner, M (1995) Developmental and endocrme regulation of P450 tsoforms m rat breast Mel Pharmacol 48,630-638. 10. Sundm, M , Warner, M , Haaparanta, T , and Gustafsson J.-A (1987) Isolation and catalytrc activity of cytochrome P450 from ventral prostate of control rats J B1o1 Chem. 262, 12,293-l 2,297 11. Warner, M , Tollet, P , Stromstedt, M , Carlstrom, K., and Gustafsson, J -A (1989) Endocrine regulation of cytochrome P450 m the rat brain and pttuttary gland J Endocrlnol 122,341-349

7 Purification of Cytochromes P450: Products of Bacterial Recombinant Expression Systems F. Peter Guengerich,

Natalie A. Hosea, and Martha V. Martin

1. Introduction A procedure is outlmed that IS used for purification of heterologously expressed P450 3A4 from Escherichia colz membranes. Details of constructron of the particular plasmid expressed (NF 14) are presented elsewhere (I, 2). The general procedure can be utilized for other cytochromes P450, with some modification, as described elsewhere (1,3-7). Several general purification techniques are described rn detarl m Chapters 4 and 5 of this volume.

2. Materials 2.1. Solubilization 1. Buffer A* 0 1 A4 Trts-acetate buffer, pH 7 6, containing 0.5 M sucrose and 0.5 rnA4ethylenediamme tetra-acetic acid (EDTA) 2 Lysozyme (50 mg/mL) 3 Buffer B* 0.1 A4 potassmm phosphate buffer, pH 7 4, containing 6 mA4 magnesium acetate, 20% (v/v) glycerol, and 0.1 mM drthrothrertol (DTT) 4. 0.1 M Phenylmethylsulfonyl fluoride (PMSF), m n-propanol (stored at -2OOC) 5. 0.2 mA4 Leupeptm, m HzO. 6. 0.1 mMBestatin, in H20. 7. Aprotinin (4 U/mL). 8. Buffer C 50 m&I Trrs-acetate buffer, pH 7.6, containing 0 25 M sucrose and 0 25 mMEDTA

2.2. Chromatography 1. Sodium cholate, 20% (w/v) (prepared from recrystallized chohc acid as described in Chapter 4, Subheading 2.1.). From Methods tn Molecular Edlted by I R PhWps and

Biology, Vol E A Shephard

77

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P450 Protocols Inc , Totowa.

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2 Triton N-101,20% (w/v) (prepare with gentle warmmg, <6O”C) (see Note 1). 3 Buffer D: 20 mMTrts-acetate buffer, pH 7.5, contammg 20% (v/v) glycerol, 1 nuI4 EDTA, 0 1 mMDTT, 0 62% (w/v) sodium cholate, and 0 6% (w/v) Trtton N-101 (see Note 1). 4 0.1 M sodmm EDTA, pH 7.5 5 Dtethylaminoethyl (DEAE)-Sephacel, adJusted to pH 7 5 (Pharmacia, Prscataway, NJ) (see Note 2) 6 Hydroxylapatrte (HA), BroRad Btogel HTP (BtoRad, Hercules, CA) (see Note 2) 7. Buffer E: 10 mMpotassium phosphate buffer, pH 7 7, containing 0.1 WEDTA, 20% (v/v) glycerol, and 0.1 n&I DTT. 8 Buffer F. 300 m&f potassium phosphate buffer, pH 7.7, contammg 0 05 mM EDTA, 20% (v/v) glycerol, 0 1 mMDTT and 0.05% (w/v) sodium cholate. 9. Buffer G: 500 r@potassmmphosphate buffer, pH 7.7, contammg 0.05 mMEDTA, 20% (v/v) glycerol, 0.1 mA4 DTT and 0.05% (w/v) sodmm cholate 10. Buffer H. 500 nnI4 potassium phosphate buffer, pH 7.7, containing 0 05 nuI4 EDTA, 20% (v/v) glycerol, 0 1 rrnI4 DTT, and 0 15% (w/v) sodmm cholate

3. Methods General note: all steps are done at 04°C.

3.1. Preparation

of Bacterial Membranes

1. Bacterial cells are recovered by centrrfugatton (4 x lo3 g, 15 mm) and resuspended m Buffer A at a concentratton of -70 mg wet cells/ml 2 The suspenston IS diluted twofold with H20, and lysozyme IS added to 0.1 mg/mL 3 The suspenston is gently shaken and mcubated on ice for 30 mm m order to hydrolyze the outer membrane. 4 The resultmg spheroplasts are recovered by centrtfugatton at 4 x lo3 g for 15 min, the pellet is resuspended in Buffer B at a concentration of -0 5 g/mL At thts point the preparation can be stored frozen at -70°C until further use (2). 5. The frozen spheroplasts are thawed m a water bath at room temperature. During the thawing process, protease mhtbttors are added to the spheroplasts to the following final concentrattons. PMSF, 1 mM; leupeptm, 2 )uV, bestatm, 1 w, aprotmin, 0.04/U mL 6 Cells, m a Rosett Cell packed m me, are lysed usmg a Branson somcator (at -70% full power) for approx 15 mm or until the cell lysate contams no clumps. 7 The lysate is subJected to centrtfugation (104g, 20 mm) and the pellet is dlscarded. 8. The supernatant IS then centrtfuged at 105g for 60 mm (3.5 x lo4 rpm m a Beckman 45 TI rotor). 9. The pelleted membranes are resuspended m a mmimum volume of Buffer C, quickly frozen m liquid N2, and stored at -70°C (unless further purtticatton 1s started munediately).

Purification

of Recombinant

3.2. Membrane

Solubilization

P45Os

79

and DEAE Chromatography

1. The E. cob membranes prepared as above are diluted to a protein concentration of -2 0 mg/mL in Buffer D. 2. The mtxture IS stirred for 60 mm and centrifuged at 105g for 60 mm; the pellet IS discarded. 3 The resulting supernatant is applied to a 2.5 x 10 cm column of DEAE-Sephacel (suitable for -1000 mL of solubilized membranes) that has been equrhbrated with Buffer D, and 10 mL fractions are collected. 4 After all of the sample has been applied, the column is washed with -3 bed volumes of Buffer D and the fractions contammg red color (cytochrome P450) are pooled 5 A spectral assay IS done (8,9) (see Chapter 3) to estimate recovery, and rt may be useful also to monitor the progress of purification at thus point by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (9,ZO).

3.3. Hydroxylapatite

(HA) Chromatography

1. The pooled material 1s applied directly to a 2.5 x 7 cm column of HA (equrhbrated with Buffer E), at a flow rate of -1 mL/mm Most of the brown color should be adsorbed to the column. 2. The column is washed with 10 column volumes of Buffer E (or more tf any color is eluted) and contmued until the &so IS ~0.02. 3. The column is then washed sequenttally with 1 bed volume each of Buffers F, and G, collecting u 10 mL fractions 4 The protein (cytochrome P450) is then eluted from the column with Buffer H, collectmg lo-mL fractions. 5 The fractions are analyzed for A,,, and by SDS-PAGE, taking care to dilute samples to avoid potassium dodecyl sulfate precipitates. 6. The cytochrome P450 fractions (as judged by Ad17) that are homogeneous (>95%) as judged by SDS-PAGE are pooled and dialyzed extensively (4X, >6 h each time) against Buffer E, either before or after concentration with an Amicon ultrafiltration system and a PM-30 membrane (see Note 3). The concentration of cytochrome P450 is estimated spectrally (8,9) (see Chapter 3)

3.4. Other Methods Other procedures for purtfication of recombinant cytochromes P450 are described in Notes 4-8. 4. Notes 1 See Chapter 4 (Subheading 4, items 4, 6, and 7) regardmg substrtutron of other detergents. 2 See Chapter 4 (Subheadings 4.4.1. and 4.4.2.) regarding substrtutron of other chromatography media. 3. See Chapter 4 (Subheadings 4.4.1. and 4.4.2.) regarding dialysis and various methods of protein concentration.

80

Guengerich, Hosea, and Martin

4.1. Other Procedures for Purification of Cytochromes from Bacterial Expression Systems 4.1-l. Other Ion-Exchange Chromatography Systems

P450

4. Heterologously expressed P450 lA2 has been purified from bacterlal membranes using a combmatlon of only dlethylaminoethyl (DEAE) and carboxymethyl (CM) chromatography (4). Sometimes a two-step system 1ssufficient to purify the protein and remove detergent (e.g , P450 lA2 141) In other cases, a sequence of DEAE/CM/HA has been used (I,3,5-7)

4.1.2. Phase Separation 5. Some detergents have temperature-dependent cloudpomts. It 1spossible to fractionate protein mixtures between the “detergent” and “soluble” phases with some detergents, e.g , Trlton X-l 14 (II,I2) This approach 1s relatively easy and has been successfully applied to the fractlonatlon of cytochromes P450 expressed m bacteria (13-15).

4.1.3. Flavodoxin Affinity Chromatography 6 Jenkins and Waterman reported that E coli flavodoxm is the source of electrons used by recombmant cytochromes P450 expressed m the bacteria (13) They also found that an affinity column made of immobilized flavodoxin could selectively bind mammahan cytochromes P450 (13). This approach has been utlhzed in the puritkatlon of recombinant P450 2D6 (14) Bacterial membranes were subJected to the phase separation previously described and then applied to a flavodoxm affinity column, which was eluted with a gradient of increasing NaCl concentration m the presence of noniomc detergent (13,14).

4.1.4. Histidine Tag Methods 7. One method often used m recombinant DNA technology mvolves the attachment of a poly-HIS region usually at the N- or C-terminus to faclhtate protein punfication (16) The free His residues (usually His, or His& can chelate Ni2+; thus, an Nl*+-chelate affimty column can be used for rapid purification (27) Such approaches have been used with cytochromes P450, with His tags at either the C (13,18) or N (19) terminus. Detergent IS needed to solublhze the

membranes and keep the proteins disaggregated during chromatography, and the detergent must be removed m a subsequent step

4.1.5. Fusion Proteins 8 Two types of cytochrome P450 fusion proteins have been constructed and punfied. Cytochrome P450:nicotinamlde ademne dmucleotlde phosphate (NADPH) cytochrome P450 reductase fusion proteins and close relatives occur naturally (2U-22) and have facilitated internal electron transfer. Such artificial constructs

Purification of Recombinant P45Os

81

were first prepared in yeast (23) and later in bacteria (24) Purification is facilitated by the ability to use 2’S’-ADP agarose affinity chromatography on the reductase portion. The typical procedure involves Initial separation on DEAE followed by the 2’,5’-ADP column, plus steps to remove detergents (see Chapter 4, Subheading 4., item 13). This approach has been used to purify protems containing human cytochromes P450 lA1, lA2, and 3A4 fused with the reductase (25-27), as well as some animal cytochromes P450 (24,25,28). The other kind of fusion protein has glutathione transferase (GST) attached to the N-terminus of a cytochrome P450 (2B4) (29). The presence of a GST renders the protein soluble and enables the use of a glutathione-based affinity matrix for purification (30). The construction of a thrombin-sensitive site between the GST and P450 domains facilitates the cleavage of the GST portion to yield the P450.

References 1, Gillam, E. M. J., Baba, T., Kim, B-R., Ohmori, S., and Guengerich, F. P (1993) Expression of modified human cytochrome P450 3A4 m Escherzchza colz and purification and reconstitution of the enzyme. Arch Biochem BiophJw. 305,123-l 3 1 2 Guengerich, F P., Martin, M. V., Guo, Z., and Chun, Y-J (1996) Purification of recombinant human cytochrome P450 enzymes expressed in bacteria Methods Enzymol. 272,35-44.

3. Sandhu, P., Baba, T., and Guengerich, F. P (1993) Expression of modified cytochrome P450 2C10 (2C9) m Escherichza coEl, purification, and reconstitution of catalytic activity. Arch Bzochem. Bzophys. 306,443-450. 4 Sandhu, P., Guo, Z., Baba, T., Martin, M. V , Tukey, R. H., and Guengerich, F. P (1994) Expression of modified human cytochrome P450 lA2 m Escherzchra colz: stabilization, purification, spectral characterization, and catalytic activities of the enzyme Arch Biochem Bzophys 309, 168-177 5 Gillam, E. M J., Guo, Z , and Guengench, F. P. (1994) Expression of modified human cytochrome P450 2El in Escherzchza colz, purification, and spectral and catalytic properties Arch Bzochem Bzophys 312,59-66 6. Gillam, E. M. J., Guo, Z., Ueng, Y-F., Yamazaki, H., Cock, I., Reilly, P E B , Hooper, W. D., and Guengerich, F P. (1995) Expression of cytochrome P450 3A5 in Escherzchza colz. effects of 5’ modifications, purification, spectral characterization, reconstitution conditions, and catalytic activities Arch Bzochem Bzophys 317,374-384. 7. Guo, Z., Gillam, E. M. J., Ohmori, S., Tukey, R. H., and Guengerich, F. P (1994) Expression of modified human cytochrome P450 1A 1 m Escherzchza colz effects of 5’ substitution, stabilization, purification, spectral characterization, and catalytic properties. Arch Bzochem Bzophys 312,436-446. 8 Omura, T. and Sato, R. (1964) The carbon monoxide-bmdmg pigment of liver mmrosomes. I. Evidence for its hemoprotein nature. J Bzol Chem 239,2370-2378 9 Guengerich, F P (1994) Analysis and characterization of enzymes, in Prznczples and Methods of Toxicology (3rd ed.) (Hayes, A. W., ed.), Raven, NY, pp. 1259-1313.

82

Guengerich, Hosea, and Martin

10. Laemrnh, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680-685. 11 Bordter, C. (1981) Phase separation of integral membrane proteins m Triton X-l 14 solution. J Biol Chem 256, 1604-1607 12 Sanchez-Ferrer, A., Bru, R., and Garcia-Carmona, F (1994) Phase separation of biomolecules m polyoxyethylene glycol noniomc detergents Crzt Rev Blochem Mel Biol 29,275-3 13. 13 Jenkms, C M. and Waterman, M R. (1994) Flavodoxm and NADPH-flavodoxm reductase from Escherzchza colz support bovine cytochrome P45Oc17 hydroxylase activities J Blol Chem. 269,27,401-27,408 14. Gillam, E. M J., Guo, Z., Martin, M. V , Jenkins, C. M , and Guengerich, F P (1995) Expression of cytochrome P450 2D6 in Escherzchza colz, purification, and spectral and catalytic characterization. Arch Blochem Biophys 319,54&550 15. Halkier, B A., Nielsen, H. L , Koch, B , and Moller, B L. (1995) Purification and characterizatton of recombinant cytochrome P450TYR expressed at htgh levels in Escherrchla

co11 Arch. Blochem Btophys

16. Porath, J. (1992) Immobilized Express

Purzf 3,263-28

322,369-377

metal ton affinity

chromatography.

Protean

1

17. Porath, J., Carlsson, J , Olsson, I., and Belfrage, G. (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258, 598-599 18 Imai, T., Globerman, H., Gertner, J. M , Kagawa, N., and Waterman, M R. (1993) Expression and purification of functtonal human 17a-hydroxylasell7,20-lyase (P45Oc17) in Escherichra colr Use of thts system for study of a novel form of combined 17a-hydroxylasell7,20-lyase deficiency. J Bzol Chem 268, 19,681-19,689 19. Kempf, A., Zanger, U M., and Meyer, U. A (1995) Truncated human P450 2D6: expression m Escherzchza co11 Nl 2+-chelate affinity purification, and characterization of solubility and aggregation Arch Biochem Biophys. 321,277-288 20. Fulco, A. J and Ruettmger, R. T. (1987) Occurrence of a barbiturate-mducible catalyttcally self-sufficient 119,000 Dalton cytochrome P-450 monooxygenase m Bacilli. Life SCI. 40, 1769-1775. 2 1 White, K. A. and Marletta, M. A. (1992) Nitric oxide synthase is a cytochrome P-450 type hemoprotein. Brochemistry 31,6627-663 1. 22. McMillan, K , Bredt, D S., Hirsch, D. J , Snyder, S H., Clark, J E , and Masters, B S. S. (1992) Cloned, expressed rat cerebellar mtric oxide synthase contams stoichiometrtc amounts of heme, which binds carbon monoxide Proc Nut1 Acad Scz USA 89, 11,141-l 1,145 23. Murakami, H , Yabusaki, Y., Sakaki, T., Shibata, M., and Ohkawa, H. (1987) A genetically engineered P450 monooxygenase. construction of the functional fused enzyme between rat cytochrome P45Oc and NADPH-cytochrome P450 reductase DNA 6,189-197.

24. Fisher, C W., Shet, M. S., Caudle, D. L., Martm-Wixtrom, C. A , and Estabrook, R. W (1992) High-level expression m Escherzchza cola of enzymatically active fusion proteins containing the domains of mammalian cyto-

Purification of Recombrnant P450.5

25.

26.

27

28.

29

30.

83

chromes P450 and NADPH-P450 reductase flavoprotem Proc Nat1 Acad Set USA 89, 10,817-10,821 Shet, M. S., Fisher, C. W., Arlotto, M. P., Shackleton, C. H L,, Holmans, P. L., Martin-Wixtrom, C. A , Saekl, Y., and Estabrook, R. W (1994) Purification and enzymatic properties of a recombmant fusion protem expressed m Escherichla co/i contaming the domains of bovine P450 17A and rat NADPH-P450 reductase. Arch Biochem Biophys 311,402-4 17. Chun, Y-J., Slumada, T , and Guengerich, F. P (1996) Construction of a human cytochrome P450 lAl:rat NADPH-P450 reductase fusion protein cDNA, expression m Escherlchla toll, purification, and catalytic propertles of the enzyme m bacterial cells and after purification Arch Blochem Biophys 330,48-58. Pankh, A. and Guengench, F. P. (1996) Expression, purification, and characterization of a catalytically active human cytochrome P450 lA2*NADPH-cytochrome P450 reductase fusion protein. Protean Express. Purif 9,346-354. Alterman, M. A., Chaurasla, C. S., Lu, P , Hardwick, J. P , and Hanzhk, R P (1995) Fatty acid dlscrlmmatlon and w-hydroxylatlon by cytochrome P4540 4Al and a cytochrome P4504Al/NADPH-P450 reductase fusion protein. Arch Blochem Biophys 320,289-296 Vaz, A D N , Pernecky, S J , Raner, G M , and Coon, M J (1996) Peroxo-iron and oxenmd-iron species as alternative oxygenating agents in cytochrome P450catalyzed reactions: swltchmg by threonme-302 to alanine mutagenesis of cytochrome P450 2B4 Proc Nat1 Acad. Sci. USA 93,4644-4648. Smith, D. B. and Johnson, K. S. (1988) Single-step purification of protems expressed m Escherzchza co/z as fusions with glutathlone S-transferase Gene 67,3 I-40

Cytochrome P450 Reconstitution Systems Tsutomu

Shimada and Hiroshi Yamazaki

1. Introduction

Multiple forms of cytochrome P450 (CYP) exist m liver mrcrosomes and these P450 forms have been shown to play important roles m the oxidation of structurally diverse xenobrotrc chemicals such as drugs, toxic chemicals, and carcinogens, as well as endobiottc chemicals including steroids, fatty acids, fat-soluble vitamins, and prostaglandins (1). Major cytochrome P450 enzymes in human liver microsomes identified to date mclude CYPlA2,2A6,2B6,2C8, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5 (and 3A7 m fetal livers) (2). CYPlAl and 1B 1 have also been reported to be involved m the oxidation of drugs, toxic chemicals, and carcinogens in extrahepatic tissues (3). There are large mtermdividual variations m the amounts of each of these cytochrome P450 enzymes, and these variations are considered to be one of the major factors m contributing to differences in susceptibilities of humans towards actions and toxicities of drugs, toxic chemicals, and carcmogens (2). Recently, in order to investigate the consequences for enzymic actrvity of genetic polymorphisms of individual cytochrome P450 enzymes in humans, many investigators have carried out studies with cytochrome P450 proteins purified from heterologous organisms rn which native and modified human cytochrome P450 cDNAs have been introduced (4). The conditions required to achieve maximal catalytic activrties in systems m which liver microsomal cytochrome P450 proteins are reconstituted with nicotinamide adenine dinuclotide phosphate (NADPH)-cytochrome P450 reductase in synthetic phospholipid vesicles have been shown to depend upon the particular cytochrome P450 used (5’. In some cases, particularly when CYP3A enzymes are used for reconstitution, several factors, including a particular lipid-membrane environment, cytochrome b5, divalent metal ions, such From Methods m Molecular B/ology, Vol E&ted by I R Phllllps and E A Shephard

85

107

Cytochrome

0 Humana

P450

Protocols

Press Inc , Totowa,

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Shlmada and Yamazaki

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as Mg2+, and reduced glutathione (GSH) have been shown to be requned for maximal catalytic actrvrty (6-8). Other human cytochromes P450 that have been shown to require cytochrome b, and a particular lrpid environment for optrmal reconstrtutron include CYP2El and, possibly, CYP2C9 and CYP2C 19, but the mechanisms underlying strmulation by cytochrome b, have been reported to differ depending on the cytochrome P450 enzymesexamined (9,IO) (see Note 1).

In this chapter, we describe the detailed condmons for reconstrtutron of drug oxidation activities catalyzed by human cytochrome P450 enzymes, including: 1 7-Ethoxyresorufin 0-deethylatlon catalyzed by CYPlA2, which does not requrre cytochrome b, and a partrcular lipid environment for maximal catalytic activities, 2 The chlorzoxazone 6-hydroxylatron catalyzed by CYP2E1, which requtres cytochrome bS and a particular lipid environment, but does not require factors such as Mg2+ and GSH (IO), and 3 Testosterone 6P-hydroxylatron catalyzed by CYP3A4, whrch requrres cytochrome b,, a particular lipid environment, Mg2+ and GSH for tmprovmg catalytrc activities (9)

2. Materials 2.1. Solutions 1. 1.O A4 Potassium phosphate buffer, pH 7.4 2. 1 OMPotasslumN-(2-hydroxyethyl)plperazlne-N’-(2-ethanesulfonate) (HEPES) buffer, pH 7.4. 3 TGE buffer. 10 m&I Tris-HCl, pH 7.4, contammg 20% (v/v) glycerol and 1 n-&I ethylenedraminetetraacetrc acrd (EDTA). 4. DLPC (L-a-drlauroyl-sn-glycero-3-phosphochohne). make a solutron of 1 mg of DLPCImL of 10 nnI4 potassmm-phosphate buffer, and somcate rt with an ultrasonic drsruptor (4 cycles for 20 s each) at 4°C before use The stock solution can be kept at -20°C for more than 1 mo, but needs to be somcated again before use (see Note 2).

5 The phosphohpid mixture (L-a-drlauroyl-sn-glycero-3-phosphochoiine, ~-adioleoyl-sn-glycero-3-phosphocholme, and L-o-phosphatrdyl-t,-serme). make separate solutrons of each of the three phosphohprds (1 mg/mL 10 mMpotassmm phosphate buffer, pH 7 4), somcate as previously descrrbed (see item 4), and then mix the three solutrons and sonicate again The stock solutton can be kept at -2O’C as prevrously described (see item 4) (see Notes 2 and 3) 6. An NADPH-generatmg system consisting of 5 mA4 NADP+, 50 mM glucose 6-phosphate, and glucose 6-phosphate dehydrogenase (50 umts/mL) (see Note 4) 7. 20% (w/v) Sodium cholatee Dissolve 20 g of recrystallized chollc acrd u-r 100 mL of 0.05 NNaOH and neutralize with 0.1 NHCI to pH 7.4 (cholic acid of commercial origm needs to be recrystallized, see Chapter 4, Subheading 2.1., item 7). 8. 150 m&I GSH m 10 mM potassium phosphate buffer, pH 7 4

ReconstGut/on Systems

87

9 1.O A4 MgCl,. 10 2 mM7-ethoxyresorufin. Dissolve 0.48 mg of 7-ethoxyresorufin (Sigma Chemical, St LOUIS, MO) in 1.O mL of methanol or dimethylsulfoxide 11. 50 mM chlorzoxazone: Dissolve 8 5 mg of chlorzoxazone (Sigma) m 1 0 mL of methanol. 12. 20 titestosterone Dissolve 5 8 mg oftestosterone (Sigma) m 1.OmL of methanol. 13 0.5 mA4resorufin: dissolve 1.07 mg of resorufin (Sigma) m 10 mL of methanol. 14. 10 pA4 6-hydroxychlorzoxazone: dissolve 1.9 mg of 6-hydroxychlorzoxazone (Ultra Fme Chemical, Manchester, UK) in 1.O mL of methanol Dilute IOOO-fold with methanol. 15 10 pA4 6P-hydroxytestosterone: dissolve 3.0 mg of 6P-hydroxytestosterone (Sigma) m 1.0 mL of methanol. Drlute lOOO-fold with methanol.

1 CYPlA2. This enzyme has been purified from human liver microsomes (II) Alternatively, recombmant human CYPlA2, purified from membranes of Escherzchra ~011 (E colz) into which modified CYPlA2 cDNA has been mtroduced (221, IS commercially available (Oxford Biomedical Research, Oxford, MI) (see Note 5). 2 CYP2El: This enzyme has been purified from human liver microsomes (13) Recombinant human CYP2E1, purified from membranes of E coli mto which modified CYP2El cDNA has been introduced (14), is available from Oxford Biomedical Research. Purified recombinant human CYP2El is also available from Panvera (Madison, WI) (see Note 5) 3 CYP3A4: This enzyme has been purified from human liver microsomes (15) Recombinant human CYP3A4, purified from membranes of E co11 into which modified CYP3A4 cDNA has been introduced (16), is also avatlable from Oxford Biomedical Research Purified recombinant human CYP3A4 is also available from Panvera (see Note 5) 4. NADPH-cytochrome P450 reductase has been purified from liver microsomes of various animals including mice, rats, rabbtts, and humans (17-l 9). Because the use of this enzyme from different species results in no significant differences m drug-oxidizmg activities in cytochrome P450 reconstitution systems, many mvestigators use the enzyme isolated from liver mmrosomes of phenobarbitaltreated rabbits ($19). Recombinant human NADPH-cytochrome P450 reductase may be purchased from Oxford Biomedical Research and Panvera and recombinant rat NADPH-cytochrome P450 reductase is available from Panvera (see Note 6). 5. Cytochrome b5 has been purified from liver microsomes of various species including mice, rats, rabbits, and humans (19-21). As is the case for NADPHcytochrome P450 reductase, cytochrome b, isolated from liver microsomes of phenobarbital-treated rabbits is very useful for reconstitution experiments Recombinant purified cytochrome bs is available from Panvera (see Note 7)

Shimada and Yamazahi

88 3. Methods 3.7. Reconstitution by CYPlA2

of 7-Efhoxyresorufin

0-Deefhylation

1 Incubation mixture (final volume of 1.0 mL) consists of CYPlA2 (25 pmol), NADPH-cytochrome P450 reductase (50 pmol; usually a twofold molar excess with respect to cytochrome P450), and DLPC (20 pg) m 100 mA4potassmm phosphate buffer, pH 7 4, containing an NADPH-generating system (0 25 pm01 of NADP+, 2 5 pm01 of glucose 6-phosphate, and 0 25 unit of glucose 6-phosphate dehydrogenase) and 7-ethoxyresorufin (10 nmol) a Mix CYP 1A2, NADPH-cytochrome P450 reductase, and DLPC b. Incubate at 25’C for 5 min with shaking. c Add a solution of 105 pL of the NADPH-generating system (50 p.L of 5 mM NADP+, 50 pL of 50 &glucose 6-phosphate, and 5 & of glucose 6-phosphate dehydrogenase (50 umts/mL), 100 pL of 1 0 M potassium phosphate buffer, and water (to give a final volume of 1.OmL). d Add 5 0 pL of 2 mM 7-ethoxyresorufin. 2 Incubate at 37°C for 5-10 min with vigorous shaking (-100 cycles/mm) 3. Terminate the reaction by adding 2 mL of methanol 4 Centrifuge at 9OOg for 5 mm. 5. Measure the formation of resorulin fluorometrically m a spectrofluorimeter at an excitation wavelength of 550 nm and an emission wavelength of 585 nm

3.2. Reconstitution

of Chlorzoxazone

&Hydroxylation

by CYPZEl

1 Incubation mixture (final volume of 1.0 mL) consists of CYP2El (25 pmol), NADPH-cytochrome P450 reductase (50 pmol), cytochrome bs (50 pmol), sodium cholate (500 nmol), and the phospholipid mixture (20 pg) described above (see Subheading 2.1., item 5) in 100 &potassium phosphate buffer, pH 7.4, contammg the NADPH-generating system (see Subheading 3.1., item l), and chlorzoxazone (0.5 prnol) a. Mix CYP2E 1, NADPH-cytochrome P450 reductase, cytochrome b5, sodium cholate, and the phosphohpid mixture. b. Incubate at 25’C for 5 mm with shaking. c Add a solution of 105 pL of the NADPH-generating system (see Subheading 3.1., item l), 100 p.L of 1.0 Mpotassium phosphate buffer, and water (to give a final volume of 1.O mL) d Add 10 p.L of 50 mM chlorzoxazone (see Note 8) 2 Incubate at 37’C for 10 min with vigorous shakmg (-100 cycles/mm) 3. Terminate the reaction by adding 100 pL of 43% (w/v) H,PO, and 3 0 mL of CH&. 4. Centrifuge the mixtures at 9OOg for 5 mm and desiccate ahquots (1.5 mL) of the organic layer under an N2 stream 5. Dissolve the residues m 100 pL of 27% (v/v) CH3CN in 0.5 % (w/v) H3P04

Reconstttution Systems

89

6. Product formatton 1s determmed by high performance lrqurd chromatography with a 4.6 x 150 mm Nucleosrl octylsilyl (C8) reverse-phase column (Chemco Scientific, Osaka, Japan), Elutlon is achieved wrth a mtxture of 27% (v/v) CH,CN in 0.5 % (w/v) aqueous H,PO,, with a flow rate of 1.5 mL/mm Detection is by ultraviolet (UV) absorbance at 287 run.

3.3. Reconstitution

of Testosterone Hydroxylation

by CYP3A4

1. Incubation mixtures (final volume of 1.0 mL) consists of CYP3A4 (25 pmol), NADPH-cytochrome P450 reductase (50 pmol), cytochrome b, (50 pmol), sodium cholate (500 nmol), and the phosphohpid mixture (20 pg) described above (see Subheading 2.1., item 5) m 50 &potassium HEPES buffer, pH 7.4, containing the NADPH-generating system (see Subheading 34 item l), MgC12 (30 pool), GSH (3 pool), and testosterone (0.2 pool) (see Notes 9 and 10) a. Mix CYP3A4, NADPH-cytochrome P450 reductase, cytochrome b5, sodmm cholate, and the phospholipid mixture. b. Incubate at 25°C for 5 min with shaking. c Add a solutton of 50 pL of 1.O A4 potassium HEPES buffer, 105 pL of the NADPH-generating system (see Subheading 3.1., item l), 20 & of 150 mM GSH, water (to give a final volume of 1 0 mL), and finally 30 $ of 1 0 M MgC12 (see Notes 11-13) d Add 10 pL of 20 mM testosterone. 2. Incubate at 37’C for 10 min with vigorous shaking (-100 cycles/mm) 3. Terminate the reaction by adding 100 pL of 1.0 N HCl containmg 2 0 MNaCl and 3.0 mL of CH$l, 4. Centrifuge the mixtures at 9008 for 5 mm and desiccate ahquots (1 5 mL) of the orgamc layer under a N2 stream. 5 Dissolve the restdues m 100 pL of 64 % (v/v) aqueous methanol 6 Product formation 1s determined by HPLC with a 4.6 x 150 mm Nucleostl octyldecylsilyl (C 18) reverse-phase column (Chemco) Elutton is achieved using a mixture of 64% (v/v) aqueous methanol with a flow rate of 1 5 mL/mm and the detection is by UV absorbance at 240 nm. 4.

Notes

1. Human cytochrome P450 enzymes that require cytochrome b5 for demonstratron of maximal catalytic activities m reconstttuted monooxygenase systems include CYP3A4, CYP3A5, CYP2E1, CYP2C19 and CYP2C9 (9,10,21,23). However, the mechanisms underlying stimulation by cytochrome b5 have been shown to doffer depending upon the cytochrome P450 enzymes used (9,lO). For example, apo-cytochrome b5 as well as nattve cytochrome b5have been shown to be effective in stimulatton of testosterone 6P-hydroxylation and mfedtpme oxtdatron by CYP3A4, but such apo-cytochrome b5 stimulatton could not be detected m reconstituted monooxygenase systems containing CYP2El (9,10,22). 2. In all ofthe somcated lipid stock solutions (DLPC, the phosphohptd mixture, and the mtcrosomal lipids), sodmm cholate (final concentration of 10 mM) 1s

90

3 4 5.

6

7

8.

9.

10

I 1. 12.

Shimada and Yamazaki included. When the sodium cholate 1s present the stock solutions are clear after thawmg and may be used for the reconstitution experiments without further somcatton The phospholipid mixture can be replaced by Isolated llplds from liver mlcrosomes of rats, rabbas, and humans (unpubhshed results). The NADPH-generating system can be replaced by 1 mM NADPH (final concentration), when the mcubation 1sdone at 37’C wlthm 10 mm The purltied cytochrome P450 enzymes (suspended in TGE buffer after removmg the detergents used for the purification steps) are stable at -80°C for at least 1 yr Concentrated enzymes (>5 @4cytochrome P450) are more stable and useful for the reconstitution experiments. When required, the concentrated cytochrome P450 solution can be diluted with TGE buffer. The purified NADPH-cytochrome P450 reductase (suspended in TGE buffer after removing the detergents used for the purlflcatlon steps) is stable at -80°C for at least 1 yr Concentrated enzymes (>lO flcytochrome P450 reductase) are more stable and useful for the reconstitution experiments When required, the concentrated NADPH-cytochrome P450 reductase solution can be diluted with TGE buffer. Purified cytochrome b, (suspended in TGE buffer or 100 mM potassium phosphate buffer, pH 7 4, after removing the detergents used for the purification steps) IS stable at -8O’C for at least 1 yr Concentrated protein (>lO ~uU cytochrome 6,) 1smore stable and useful for the reconstltutlon experiments. When required, the concentrated cytochrome b5 solution can be diluted with TGE buffer In the original method by Peter et al (24), the stock solution of chlorzoxazone (50 mM) was dissolved in 60 mA4 potassium hydroxide (freshly prepared), m order to avoid incluston of organic solvents in the reaction mixture (organic solvents can interfere with CYP2El activities) With testosterone 6P-hydroxylation and mfedipme oxidation reactions catalyzed by CYP3A4 in reconstituted systems, the followmg order of mixmg was found to be optimal. CYP3A4, NADPH-cytochrome P450 reductase, cytochrome bg, sodium cholate, and a phosphohpld mixture. These components are mixed at room temperature, followed by the addition of potassium HEPES buffer, pH 7 4, the NADPH-generating system, GSH (final concentration 3 0 mM), MgC12 (final concentration 30 mM) and substrate (testosterone or mfedlpme) (22). The stlmulatory effects of cytochrome b, m reconstituted monooxygenase systems contammg CYP3A4 are not observed when N-demethylatlon of ethylmorphine or benzphetamine are measured. The mechanism by which cytochrome b, stimulates some, but not other, CYP3A4-dependent reactions 1sstill unclear (22) MgC& should be added Just before the initiation of the reaction (with substrate), m order to avoid the formation of a precipitate. The mechanisms by which MgC12 and GSH stimulate the testosterone 6@hydroxylatlon activity by CYP3A4 m reconstituted systems are not known, although Mg2+ and other dlvalent metal ions seem to stimulate reduction of cytochrome b5 (5). The effect of GSH 1s less pronounced when Mg2+ is present

Reconstitution Systems

91

Table 1 Effects of MgQ on Testosterone 6P-Hydroxylation Catalyzed by Recombinant CYP3A4 in Potassium HEPES (HEPES) and Phosphate (Kpi) Buffersa System Buffer

W4

HEPES

50 50 200 200 50 50 200 200

Kpi

M&l,

(30 mM) -

+ + + +

Testosterone 6j3-hydroxylatlon (nmol/mm/nmol P450) 21 16 34 17 4.1 12 11 12

aThe standard reaction mixtures (final volume of 1.0 mL) contamed CYP3A4 (25 pmol), NADPH-P450 reductase (50 pmol), bS(50 pmol), sodmm cholate (500 nmol), and the phosphohpld mtxture (20 pg) in 50 mM potassmm HEPES buffer, pH 7 4, contammg the NADPH-generatmg system, MgCl, (30 pool), GSH (3 pool), and testosterone (0 2 ~01)

13. When 50 mMpotassmm HEPES buffer, pH 7.4, was replaced by 50 Npotasslum phosphate buffer, pH 7 4, the CYP3A4-dependent testosterone 6P-hydroxylatlon actlvltles were also stimulated by MgCl, (Table 1) However, such stimulatory effects by MgC12 could not be determmed when the potassium phosphate concentration was increased to 200 mM

References 1. Guengench, F. P and Shlmada, T. (1991) Oxidation of toxic and carcmogemc chemicals by human cytochrome P-450 enzymes. Chem Res Toxlcol 4,391-407. 2. Shimada, T., Yamazakl, H., Mlmura, M., Inul, Y., and Guengench, F P (1994) Interrndlvldual variations m human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcmogens and toxic chemicals. Studies with liver mlcrosomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 270, 414-423 3. Shimada, T , Hayes, C. L., Yamazakl, H , Amm, S., Hecht, S. S , Guengench, F P., and Sutter, T. R. (1996) Activation of chemically diverse procarcinogens by human cytochrome P450 1B 1 Cancer Res 56,2979-2984 4 Gonzalez, F. J and Korzekwa, K. R (1995) Cytochromes P450 expression systems. Ann Rev Pharmacol Toxlcol. 35,369-390. 5 Yamazakl, H., Ueng, Y.-F., Shimada, T., and Guengench, F. P (1995) Roles of dlvalent metal ions m oxidations catalyzed by recombinant cytochrome P450 3A4

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and replacement ofNADPH-cytochrome P450 reductase with other flavoprotems, ferredoxin, and oxygen surrogates. Bzochemistry 34,8380-8389. 6 Brian, W R., Sari, M. A, Iwasaki, M , Shimada, T., Kammsky, L. S , and Guengench, F. P. (1990) Catalytic activities of human liver cytochrome P-4501IIA4 expressed m Saccharomyces cerevzszae Bzochemzstzy 29, 11,280-l 1,292. 7. Shet, M. S., Ftsher, C. W , Holmans, P. L., and Estabrook, R. W. (1993) Human cytochrome P450 3A4. enzymatic properties of a purified recombinant fusion protein contammg NADPH-P450 reductase Proc Natl. Acad Scz. USA 90, 11748-l 1752. Shet, M S., Faulkner, K. M , Holmans, P L., Fisher, C. W., andEstabrook, R W (1995) The effects of cytochrome bg, NADPH-P450 reductase, and lipid on the rate of 6P-hydroxylation of testosterone as catalyzed by a human P450 3A4 fusion protein. Arch Bzochem Bzophys. 318,314-321. Yamazaki, H , Nakano, M., Imar, Y., Ueng, Y.-F , Guengerrch, F. P., and Shrmada, T (1996) Roles of cytochrome bS m the oxidation of testosterone and mfedtpme by recombmant cytochrome P450 3A4 and by human liver mtcrosomes Arch Bzochem Bzophys. 325, 174-l 82. Yamazaki, H , Nakano, M., Gillam, E. M. J., Bell, L C., Guengertch, F. P., and 10. Shtmada, T. (1996) Requirements for cytochrome b, m the oxidatton of 7ethoxycoumarin, chlorzoxazone, aniline, and N-mtrosodimethylamme by recombmant cytochrome P450 2El and by human liver microsomes. Bzochem Pharmacol

52,301-309.

11 Distlerath, L M , Reilly, P., Martm, M V , Davts, G. G., Wilkmson, G R , and Guengerich, F P (1985) Purl&cation and characterization of the human liver cytochromes P-450 involved m debrisoquine 4-hydroxylation and phenacetm 0-deethylatton, two prototypes for genetic polymorphism m oxidatlve drug metabolism. J. Biol Chem 260,9057-9067. 12. Sandhu, P., Guo, Z., Baba, T , Martin, M V., Tukey, R H , and Guengerich, F P (1994) Expression of modified human cytochrome P450 lA2 in Escherzchza colz. Stabrlizatton, purification, spectral characterizatton, and catalytic activities of the enzymes Arch Biochem Bzophys 309, 168-177. 13 Guengerich, F, P , Kim, D H , and Iwasakt, M (199 1) Role of human cytochrome P-450 IIEl m the oxidation of many low molecular weight cancer suspects Chem Res Toxzcol 4, 168-179. 14 Gillam, E. M J , Guo, Z., and Guengerrch, F P (1994) Expression of modified human cytochrome P450 2El m Escherzchza ~011, purification, and spectral and catalytic properties. Arch Bzochem Bzophys 312,59--66. 15 Guengerich, F. P , Martin, M V , Beaune, P. H , Kremers, P , Wolff, T , and Waxman, D. J (1986) Charactertzation of rat and human liver mrcrosomal cytochrome P-450 forms involved in nifedipine oxidation, a prototype for genetic polymorphism in oxidattve drug metabolism. J. Bzol Chem 261,505 l-5060. 16 Grllam, E., Baba, T., Kim, B R., Ohmort, S., and Guengerich, F P (1993) Expression of modified human cytochrome P450 3A4 in Escherichza co11 and purification and reconstitution of the enzyme. Arch Bzochem. Biophys 305,123-13 1.

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17 Willlams, C. H. Jr., and Kamm, H. (1962) Microsomal trtphosphopyridme nucleotide-cytochrome c reductase of liver. J Biol Chem. 237,587-595 18 Guengench, F. P. (1977) Preparation and properties of htghly-purtfied cytochrome P-450 and NADPH-cytochrome P-450 reductase from pulmonary mtcrosomes of untreated rabbits. Mol Pharmacol 13,9 1 l-923. 19 Tamgucht, H , Imat, Y , Iyanagi, T , and Sato, R (1979) Interaction between NADPH-cytochrome P-450 reductase and cytochrome P-450 m the membrane of phosphatidylcholme vesicles. Bzochzm. Blophys. Acta 550,341-356. 20 Ryan, D E., and Levm, W. (1990) Purificatton and characterization of hepatic mtcrosomal cytochrome P-450. Pharmacol. Ther. 45, 153-239. 2 1. Shrmada, T., Misono, K. S., and Guengerich, F. P (1986) Human liver mtcrosoma1 cytochrome P-450 mephenytoin 4-hydroxylase, a prototype of genetic polymorphism in oxidative drug metabolism. Purificatton and characterizatton of two similar forms involved in the reaction. J. BzoZ Chem. 261,909-92 1, 22. Yamazaki, H., Johnson, W. W., Ueng, Y -F., Shimada, T , and Guengerich, F P. (1996) Lack of electron transfer from cytochrome bS m sttmulatton of catalytic activities of cytochrome P450 3A4: charactertzatton of a reconstituted cytochrome P450 3A4/NADPH-cytochrome P450 reductase system and studies with apo cytochrome bS. J Biol Chem 271,27,438-27,444. 23. Yamazaki, H , Gillam, E M. J., Dong, M. -S., Johnson, W. W., Guengerich, F P., and Shimada, T (1997) Reconstttution of tolbutamide methyl hydroxylation and S-warfarin 7-hydroxylation by recombinant human CYP2C 1O(2C9): compartson of reconstitution conditions with systems contaimng CYPlAl, lA2, 2D6, 2E1, and 3A4 Arch Blochem Blophys 342,329-337 24 Peter, R., Backer, R , Beaune, P. H., Iwasakt, M , Guengertch, F P , and Yang, C S. (1990) Hydroxylatton of chlorzoxazone as a spectfic probe for human liver cytochrome P-450 IIEl Chem Res Toxic01 3,566-573

9 Catalytic Assays for Human Cytochrome Thomas

P450

K. H. Chang and David J. Waxman

1. Introduction Cytochrome P450 (P450) enzymes catalyze the blotransformatlon of a broad range of structurally diverse foreign chemicals and endogenous substances. Many P450 enzymes have been isolated and identified m liver and other tlssues, including kidney, lung, intestines, and brain. Although these enzymes have a high degree of similarity in their ammo-acid sequences, many of them are subject to differential regulation and have distinct catalytic functions With the development of P450 form-specific (or P450 subfamlly-specific) inhlbltory antibodies, the discovery of enzyme-selective chemical inhibitors, and the general availability of catalytically active, mdivldual P450 cDNA-expressed proteins, several substrates, including drugs and endogenous substances,have been identified as useful catalytic monitors for specific P450 enzymes or subfamilies (see Table 1). These catalytic monitors can serve as useful expenmental tools m a variety of studies. These include studies designed: 1 To identify individual

P45Os that catalyze the actlvatlon or detoxification

of a

particular compound, 2 To quantify the expression and catalytic activity of an mdlvrdual P450 in tissue

samplesand In cultured cells, 3. To analyzethe enzymekinetics of a particular P450; and 4 To eluctdate the mechanism of interaction between xenoblotlcs P450 enzymes

and mdlvldual

Each of the substrate oxidation reactions listed m Table 1 IS suitable for measuring the catalytic activity of the corresponding cDNA-expressed P450 enzyme. However, errors can sometimes be made when interpreting activity data derived from cDNA-expressed enzyme studies. The fact that a cDNAexpressed human P450 can catalyze a particular substrate oxidation reaction From Methods m Mo/ecutar B/ology, Vol Edlted by I R Phllllps and E A Shephard

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96 Table 1 Catalytic Enzyme CYPl Al/2

CYPlB 1 CYP2A6 CYP2B6 CYP2C8 CYP2C9

CYP2C 19 CYP2D6

CYP2El

CYP3 A415 CYP4All CYP7Al

Chang Monitors

for Individual

Human

Cytochrome

and Waxman

P450 Enzymes

Catalyttc Monitor

Assay Method

7-ethoxyresorufin 0-deethylatlon (12) phenacetm 0-deethylatlon (13) (see Note 1) caffeine N3-demethylatlon (7) 7-ethoxyresorufin 0-deethylatlon (15) (see Note 2) 17P-estradlol I-hydroxylatlon (16) coumarin 7-hydroxylation (19) 7-ethoxy-4-tnfluoromethylcoumarm 0-deethylatlon (20) (see Note 3) pachtaxel Ga-hydroxylatlon (21) (see Note 4) diclofenac 4’-hydroxylation (22) tolbutamide methylhydroxylatton (23,24) (S)-warfarm 7-hydroxylatlon (26) (S)-mephenytoin 4’-hydroxylation (29) bufuralol l’-hydroxylatlon (30) (see Note 5) debrisoqume 4-hydroxylatlon (13) dextromethorphan 0-demethylatlon (32) p-nitrophenol hydroxylatlon (34) (see Note 6) chlorzoxazone 6-hydroxylatlon (2,35) (see Note 7) launc acid 11-hydroxylatlon (3 7) N-mtrosodimethylamme N-demethylatlon (38,39) testosterone 6P-hydroxylatlon (41) (see Note 8) mfedipme oxldatlon (42,43) lauric acid 12-hydroxylatlon (44) (see Note 9) cholesterol 7a-hydroxylatlon (45)

Chapter 10 ref. 13 ref. I4 Chapter 10 ref. 17 and 18 Chapter 11 Chapter 12 Chapter 13 Chapter 14 ref. 25 ref. 27 and 28 Chapter 15 Chapter 16 ref. 31 ref. 31 and 33 Chapter 17 ref. 36 ref. 3 7 ref. 40 Chapter 18 ref. 42 Chapter 19 Chapter 20

does not necessarily mean that m human tissues the correspondmg enzyme will be an important catalyst m the same oxldatlon reaction. For example, cDNA-expressed CYP 1A 1 is active in chlorzoxazone 6-hydroxylatlon (1). However, based on enzyme kinetic conslderatlons and the tissue-specific dlstribution of human CYPl Al, this P450 is not expected to make a significant contribution to chlorzoxazone 6-hydroxylation m human liver mlcrosomes (2). Caution should be exercised when using these catalytic monitors as probes for human microsomal P450 actlvltles for the followmg reasons: First, the validity of using a particular P450 substrate as a “diagnostic” catalytic monitor for a specific human microsomal P450 depends largely on the P450 form-speclficltles of the an&P450 antibodies and of the chemical inhibitors employed to define the P450 form-specificity of the enzyme reaction. Potential cross-reactivity with, or inhibitlon of, uncharacterized P450 enzymes or closely related

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P450 forms needs to be considered. For example, polyclonal antlbodres that are immunoreactive with CYP2C9 typically cross-react with other CYP2C forms such as CYP2C8 and CYP2C19 (e.g., ref. 3) and the macrolide antibiotic triacetyloleandomycin (TAO) is an inhibitory probe of both CYP3A4 and CYP3A.5 (4). Thus, the catalytrc contributions made by the mdrvrdual CYP2C or the individual CYP3A enzymes cannot be dtstinguished using these methodological tools. Furthermore, the definition of P450 form specificity in isolated ttssue microsomes will frequently depend on the substrate concentranon used in the in vitro assay. For example, phenacetm O-deethylase acttvrty IS a marker for human hepatrc mrcrosomal CYPlA2 only rf the assay IS performed at low micromolar (e.g., 4 @4) concentrations (5). This IS because btphasic enzyme kmetrcs are observed in phenacetm O-deethylatton catalyzed by human liver microsomes (6), with CYPlA2 correspondmg to a low-Km form (7). Conducting the assay at hrgher substrate concentrations results m a loss of specificrty of the catalytic monitor (see Notes 5 and 9 for other examples). Fmally, the selectivity of a given P450 substrate probe can be tissue-specific. For example, whereas CYPlA2 accounts for the maJorrty of microsomal phenacetin O-deethylase activity in human hver, this P450 makes little or no contribution to phenacetin O-deethylase activity in human placenta mtcrosomes (8). Although certain compounds may be considered to be reasonably specific substrates for a particular P450 enzyme (or group of P450 enzymes in the same P450 subfamily), in the case of other P450 substrates, multiple enzymes may participate in then metabohsm. Experimentally, this characteristic of a general (P450 form nonspecific) substrate provides a convenient approach to verrfymg that each P450 in a panel of recombinant P450 enzymes is indeed expressed m a catalytically active form (3,941). P450 substrates that are metabolized by many P45Osmay also be useful m screening for potential interactions between a compound of interest and microsomal P450 enzymes. The following chapters in this volume describe detailed experimental protocols using drugs and other xenobiotics as substrates for assaying individual P450 enzymes in the CYP 1 (Chapter 10) and CYP2 families (Chapters 1l-l 7), endogenous substances as substrates for assaymg CYP3A, CYP4Al1, and CYP7AI (Chapters I8-20), and a general substrate for assaying multiple P450 enzymes m the CYPl, CYP2, and CYP3 families (Chapter 21). 2. Notes 1. Phenacetm O-deethylase activrty 1s a marker for human hepatrc mrcrosomal CYPlA2 only rfthe assayis performed at a suitablesubstrateconcentratron(e.g., 4 pM) (5). Conducting the assayat higher concentrationswill result in loss of

Chang and Waxman

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2

3

4

5.

6

7.

8

9

specificity of the substrate probe because of contrlbutlon of higher Km forms to the activity. cDNA-expressed CYPlBl is active in 7-ethoxyresorufin 0deethylatlon (15). However, human hepatlc mlcrosomal ‘I-ethoxyresorufin 0-deethylase activity largely reflects CYP 1A2 m human liver (12,46). Several cDNA-expressed human P45Os, mcludmg CYP2B6, are catalytically active m 7-ethoxy-4-tnfluoromethylcoumarin 0-deethylatlon (20) A method has been developed for estimatmg the CYP2B6-component of 7-ethoxy4-trifluoromethylcoumarin 0-deethylatlon activity m human liver mlcrosomes (see ref. 20 and Chapter 12). Among the 14 mdivldual cDNA-expressed human P450 enzymes examined to date, only CYP2C8 1s active m pachtaxel 6a-hydroxylatlon (21,47,48). However, whether human hepatlc mlcrosomal pachtaxel6a-hydroxylase activity can be used as a diagnostic catalytic monitor for hepatlc CYP2C8 has yet to be determmed. When usmg bufuralol l’-hydroxylatlon activity as a marker of CYP2D6 m human liver mlcrosomes, it 1snecessary to conduct the assay at a sultable substrate concentration (e g., 25 CLM)(see Chapter 16 ). Performmg the assay at higher concentrations will result m loss of speclficlty of this substrate probe because of contribution of higher Km forms such as CYPlA2 (49) Exercise caution when usmg hepatlc mlcrosomal p-mtrophenol hydroxylase activity as a diagnostic catalytic monitor for CYP2E 1 m human hver because of the potential contrlbutlon of CYP2A6 to this activity (see ref. 50 and Chapter 17) A recent report showed that human hepatlc mlcrosomal chlorzoxazone 6-hydroxylation activity reflects not only CYP2El but also CYP3A m human hver (51), mdicating that this activity m vitro 1snot specific for hepatlc CYP2El According to a recent lmmunomhlbltlon experiment with mhibitory antlpeptlde antibodies, hepatlc CYP3A4 accounts for nearly all of the testosterone 6phydroxylase activity m human hepatlc microsomes, whereas hepatlc microsomal CYP3A5 makes httle or no contrlbutlon to this actlvlty (52). When usmg hepatic mlcrosomal lauric acid 12-hydroxylation activity as a marker of CYP4All m human liver, it 1s necessary to conduct the assay at a suitable substrate concentration (e.g., 100 pM) (see Chapter 19). Performing the assay at higher concentrations will result in loss of specificity of this substrate probe because of contrlbutlon of higher Km forms such as CYP2E 1 (53).

Acknowledgments Supported m part by the British Columbia Health Research Foundation (grant 119(95-l) to Thomas K. H. Chang) and the National Institutes of Health (grant ES0738 1 to David J. Waxman). T.K.H.C. 1s the recipient of a Research Career Award in the Health Sciences from the Pharmaceutical Manufacturers Association of Canada - Health Research Foundation and the Medical Research Council of Canada.

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References 1. Carrrere, V., Goasduff, T., Ratanasavanh, D., Morel, F., Gautter, J. C , Guillouzo, A., Beaune, P., and Bet-thou, F. (1993) Both cytochromes P450 2El and IA1 are mvolved m the metabolism of chlorzoxazone Chem. Res Toxzcol 6, 852-857. 2. Yamazaki, H., Guo, Z., and Guengerich, F. P. (1995) Selectivtty of cytochrome P450 2E1 in chlorzoxazone 6-hydroxylation. Drug Metab Dispos 23,438-440. 3 Chang, T K. H., Yu, L , Goldstein, J. A , and Waxman, D J. (1997) Identification of the polymorphically expressed CYP2C19 and the wild-type CYP2C9-Ile359 allele as low-Km catalysts of cyclophosphamtde and ifosfamide acttvatron. Pharmacogenetzcs 7,2 1 l-22 1. 4. Chang, T. K. H., Gonzalez, F. J., and Waxman, D J. (1994) Evaluatron of tnacetyloleandomycm, a-naphthoflavone and diethyldithiocarbamate as selective chemical probes for mhtbitton of human cytochromes P450 Arch Bzochem Bzophys. 311,437-442

5. Sesardic, D , Boobis, A. R., Edwards, R. J., and Davies, D S (1988) A form of cytochrome P450 in man, orthologous to form d m the rat, catalyses the Odeethylation of phenacetin and IS inducible by cigarette smoking Br J Clzn Pharmacol

26,363-372.

6. Boobis, A. R., Kahn, G. C., Whyte, C., Brodie, M J , and Davies, D. S (198 1) Biphastc 0-deethylation of phenacetin and 7-ethoxycoumarin by human and rat liver mtcrosomal fractions. Bzochem. Pharmacol. 30,245 l-2456 7 Butler, M. A., Iwasaki, M., Guengerich, F. P., and Kadlubar, F. F (1989) Human cytochrome P-450PA (P-450JA2), the phenacetin 0-deethylase, 1s prtmarily responsible for the hepatic 3-demethylation of caffeine and N-oxidation of carctnogenrc arylamines Proc Nat1 Acad Scz USA 86,7696-7700 8 Sesardic, D., Pasanen, M., Pelkonen, 0 , and Boobrs, A R. (1990) Differential expression and regulation of members of the cytochrome P450 IA gene subfamily m human tissues Carcznogeneszs 11, 1183-l 188. 9. Waxman, D. J , Lapenson, D P., Aoyama, T , Gelbom, H V., Gonzalez, F. J., and Korzekwa, K. (1991) Steroid hormone hydroxylase specificities of eleven cDNAexpressed human cytochrome P45Os Arch Biochem Bzophys 290, 160-166. 10. Chang, T. K H., Weber, G. F., Crespi, C L., and Waxman, D. J. (1993) Dtfferenteal activation of cyclophosphamtde and ifosphamide by cytochromes P-450 2B and 3A in human liver microsomes. Cancer Res. 53,5629-5637. 11. Butler, A. M and Murray, M (1997) Biotransformation of parathion m human liver: participation of CYP3A4 and its inactivation during mtcrosomal parathion oxtdation J Pharmacol Exp Ther. 280,966-973 12 Burke, M D , Thompson, S , Weaver, R. J , Wolf, C. R., and Mayer, R T. (1994) Cytochrome P450 spectticities of alkoxyresorufin 0-dealkylatron m human and rat liver. Bzochem. Pharmacol 48,923-936 13 Distlerath, L. M., Reilly, P. E. B., Martin, M. V., Davis, G. G , Wilkmson, G R., and Guengertch, F P. (1985) Purification and characterizatton of the human ltver cytochrome P-450 involved n-r debrisoquine 4-hydroxylation and phenacetin

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0-deethylatton, two prototypes for genetic polymorphtsm m oxrdatlve drug metabolism. J Brol. Chem. 260,9057-9067 Tang, B. K. and Kalow, W. (1996) Assays for CYPlA2 by testmg zn vzvo metabolism of caffeme in humans Methods Enzymol 272, 124-13 1. Shimada, T., Grllam, E. M. J., Sutter, T R., Strickland, P T., Guengench, F. P., and Yamazaki, H (1997) Oxidation of xenobiottcs by recombinant human cytochrome P450 1Bl. DrugMetab Dzspos 29,617-622. Spink, D. C., Hayes, C L , Young, N. R., Christou, M., Sutter, T. R , Jefcoate, C. R., and Grerthy, J. F. (1994) The effects of 2,3,7,8-tetrachlorodtbenzo-p-dtoxm on estrogen metabolism in MCF-7 breast cancer cells. evidence for mductton of a novel 17 beta-estradiol4-hydroxylase. J Steroid Blochem Mol. Btol 51,25 l-258 Aoyama, T., Korzekwa, K., Nagata, K., Grllette, J , Gelbom, H. V., and Gonzalez, F. J. (1990) Estradtol metabolism by complementary deoxyrtbonuclerc acidexpressing human cytochrome P45Os Endocrznology 126,3 101-3 106. Suchar, L. A., Chang, R L., Rosen, R. T , Lech, J , and Conney, A. H (1995) High-performance hqutd chromatography separation of hydroxylated estradtol metabolites: Formatton of estradtol metabohtes by liver mrcrosomes from male and female rats. J Pharmacol Exp Ther 272, 197-206 Yun, C H , Shtmada, T., and Guengertch, F. P. (1991) Purrficatron and characterization of human liver microsomal cytochrome P-450 2A6 Mol. Pharmacol 40,

679-685 20. Code, E L., Crespt, C L , Penman, B W., Gonzalez, F J., Chang, T K. H , and

2 1.

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Waxman, D. J. (1997) Human cytochrome P450 2B6: Intermdtvldual hepattc expression, substrate specificity and role m procarcmogen activation Drug Metab D~spos. 25,985-993 Rahman, A., Korzekwa, K. R., Grogan, J., Gonzalez, F. J , and Harris, J W. (1994) Selective btotransformatton of taxol to 6cL-hydroxytaxol by human cytochrome P450 2C8. Cancer Res. 54,5543-5546 Leemann, T., Transon, C , and Dayer, P (1992) Cytochrome P450TB (CYP2C) a maJor monooxygenase catalyzing dtclofenac 4’-hydroxylatron m human liver. Q% Scz 52,29-34 Brian, W R., Srivastava, P K , Umbenhauer, D. R , Lloyd, R S., and Guengertch, F P. (1989) Expression of a human liver cytochrome P-450 protein with tolbutamtde hydroxylase activity m Saccaromyces cerevwae. Bzochemlstry 28,4993-4999. Doecke, C. J , Veronese, M. E., Pond, S. M , Miners, J O., Btrkett, D J., Sansom, L. N., and McManus, M E. (1991) Relationship between phenytom and tolbutamtde hydroxylattons m human liver mtcrosomes. Br J Clin. Pharmacol. 31, 125-130 Miners, J. 0 and Bukett, D J. (1996) Use of tolbutamrde as a substrate probe for human hepattc cytochrome P450 2C9.Methods Enzymol 272, 139-145 Rettie, A. E., Korzekwa, K R , Kunze, K. L , Lawrence, R F , Eddy, A C , Aoyama, T., Gelboin, H. V., Gonzalez, F. J., and Trager, W. F. (1992) Hydroxylatton of warfarin by human cDNA-expressed cytochrome P-450: a role for P-450 2C9 in the etiology of (S)-warfarm drug tnteractlons. Chem. Res. Toxzcol 5,54-59.

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27 Kaminsky, L S., Fasco, M J , and Guengerich, F P (1981) Productton and apphcatton ofanttbodtes to rat liver cytochrome P-450 Methods Enzymol 74, 262-213 28. Lang, D and Backer, R. (1995) Highly sensitive and specific high-performance liquid chromatographtc analysts of 7-hydroxywarfarm, a marker for human cytochrome P-4502C9 activity. J. Chromatogr 672, 305-309. 29 Goldstein, J A., Faletto, M. B., Romkes-Sparks, M., Sullivan, T , Kttareewan, S , Raucy, J. L., Lasker, J M , and Ghanayem, B I (1994) Evidence that CYP2Cl9 1s the major (S)-mephenytom 4’-hydroxylase in humans. Bzochemzstry 33, 1743-1752 30 Gut, J , Catin, T , Dayer, P., Kronbach, T., Zanger, U., and Meyer, U. A (1986) Debrisoqume/sparteme-type polymorphtsm of drug oxldatton purtficatton and characterizatton of two functtonally different human liver cytochrome P-450 isozymes mvolved m impaired hydroxylatton of the prototype substrate bufuralol J Blol Chem 261, 11,734-l 1,743. 3 1. Kronbach, T. (199 1) Bufuralol, dextromethorphan and debrisoqume as prototype substrates for human P450IID6. Methods Enzymol 206, 509-5 17. 32. Dayer, P , Leemann, T., and Striberni, R. (1989) Dextromethorphan O-demethylatton m liver microsomes as a prototype reaction to monitor cytochrome P-450 db, activity Clm. Pharmacol Ther 45,34-40. 33 Rodrtgues, A. D. (1996) Measurement of human liver mtcrosomal cytochrome P450 2D6 actrvrty usmg [O-methyl-14C]dextromethorphan as substrate Methods Enzymol 272, 186-195 34 Tassaneeyakul, W , Veronese, M E., Btrkett, D J., Gonzalez, F. J., and Mmers, J 0 (1993) Validation of 4-mtrophenol as an zn vitro substrate probe for human liver CYP2El using cDNA expresston and microsomal kinetic techniques Bzochem Pharmacol 46,1975-l 98 1. 35 Peter, R , Backer, R., Beaune, P. H., Iwasaki, M , Guengertch, F P., and Yang, C S (1990) Hydroxylation of chlorzoxazone as a specific probe for human liver cytochrome P-45011El Chem Res Tox~col 3,566-573 36. Lucas, D., Menez, J F., and Bet-thou, F. (1996) Chlorzoxazone an zn vitro and zn vlvo substrate probe for liver CYP2El. Methods Enzymol 272, 115-123 37. Amet, Y., Berthou, F., Band, S., Dreano, Y., Bad, J P , and Menez, J. F (1995) Validation of the (o- I)-hydroxylation of lauric acid as an zn vitro substrate probe for human liver CYP2El Bzochem. Pharmacol 50, 1775-1782. 38. Wrtghton, S. A., Thomas, P. E., Molowa, D. T., Hanm, M., Shively, J. E., Mames, S. L., Watkins, P B., Parker, G., Mendezptcon, G., Levm, W., and Guzehan, P. S. (1986) Characterization of ethanol-inducible human liver N-nitrosodtmethylamine demethylase. Bzochemzstry 25, 673 l-6735. 39. Guengerich, F. P , Ktm, D. H., and Iwasakt, M (1991) Role of human cytochrome P-450 IIEl m the oxtdatton of many low molecular weight cancer suspects. Chem Res. Tox~ol 4, 168-179 40 Yoo, J. S. H., Guengertch, F. P., and Yang, C S. (1988) Metabolism of N-mtrosodtalkylammes by human ltver mtcrosomes Cancer Res 88, 1499-1504.

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41 Waxman, D. J , Attisano, C., Guengerich, F. P , and Lapenson, D P. (1988) Human hver microsomal steroid metabolism: Identificatton of the maJor microsoma1 steroid hormone Go-hydroxylase cytochrome P-450 enzyme Arch Blochem Bzophys. 263,424-436. 42 Guengertch, F P , Martin, M V , Beaune, P H , Kremers, P , Wolff, T , and Waxman, D. J. (1986) Characterization of rat and human liver mtcrosomal cytochrome P-450 forms involved m mfedipme oxidation, a prototype for genetic polymorphism m oxidative drug metabolism. J Bzol Chem 261, 505 l-5060 43. Gonzalez, F. J., Schmid, B., Umeno, M., McBride, 0. W., Hardwtck, J. P , Meyer, U A , Gelbom, H V , and Idle, J R (1988) Human P450PCNl sequence, chromosome localization and direct evidence through cDNA expression that P450PCNl IS mfedtpme oxidase DNA 7,79-86. 44. Powell, P. K., Wolf, I., and Lasker, J. M. (1996) Identrficatton of CYP4All as the maJor lauric acid o-hydroxylase m human liver microsomes Arch Bzochem Blophys. 335,2 19-226 45 Maeda, Y., Eggertsen, G., Nyberg, B., Setogucht, T , Okuda, K , Emarsson, K , and Bjorkhem, I. (1995) Immunochemical determination of human cholesterol 7a-hydroxylase. Eur J Biochem 228, 144-148 46 Bourdt, M , Larrey, D , Nataf, J , Bernuau, J., Pessayre, D , Iwasaki, M , Guengerich, F P , and Beaune, P H. (1990) Anti-liver endoplasmic reticulum autoantibodtes are directed against human cytochrome P-4501A2: a specific marker of dihydralazme-induced hepatms J. Clan Invest 85, 1967-1973. 47 Sonnichsen, D S , Lm, Q., Schuetz, E. G., Schuetz, J. D , Pappo, A., and Relling, M V (1995) Variabihty m human cytochrome P450 pachtaxel metabolism J Pharmacol. Exp Ther 275, 566575. 48 Richardson, T. H , Jung, F , Griffin, K J , Wester, M , Raucy, J L , Kemper, B , Bornheim, L. M., Hassett, C., Omiecmski, C J , and Johnson, E. F. (1995) A universal approach to the expresston of human and rabbit cytochrome P45Os of the 2C subfamily m Escherlchla COIL Arch. Blochem Biophys 323, 87-96. 49 Yamazakt, H., Guo, Z., Persmark, M , Mimura, M., Inoue, K., Guengerich, F. P , and Shimada, T. (1994) Bufuralol hydroxylation by cytochrome P450 2D6 and IA2 enzymes in human liver microsomes. Mol Pharmacol 46,568-577 50 Draper, A. J., Madan, A., and Parkinson, A. (1997) Inhibttton of coumarm 7hydroxylase activity m human liver microsomes Arch Blochem Bzophys 341, 47-61 51 Gorski, J C , Jones, D. R., Wrighton, S A , and Hall, S D (1997) Contributton of human CYP3A subfamtly members to the 6-hydroxylation of chlorzoxazone Xenoblotica 27,243-256 52 Wang, R W and Lu, A Y H (1997) Inhibitory anti-peptide antibody against human CYP3A4. Drug Metab Dispos 25,762-767. 53 Clarke, S E., Baldwin, S. J., Bloomer, J C., Ayrton, A. D., Sozto, R S., and Chenery, R. J (1994) Laurie acid as a model substrate for the simultaneous determmation of cytochrome P450 2El and 4A in hepattc microosomes. Chem Res. Toxlcol. 7, 836-842.

Enzymatic Analysis of cDNA-Expressed Human CYPIAI, CYPlA2, and CYPIBI with 7-Ethoxyresorufin as Substrate Thomas

K. H. Chang and David J. Waxman

1. Introduction The human CYPl family consists of at least three proteins, CYPl Al, CYPlA2, and CYPlBl (I), CYPIAI IS absent or present at very low levels in human liver (2,3), but its expression IS readily detectable m lung (4,s). By contrast, CYPlA2 is constttutively expressed m human liver (2,3) and is absent m lung (#,5). CYPlBl IS primarily an extrahepatic P450, as suggested by the findmg that CYPIB 1 mRNA is present in much greater abundance m tissues such as kidney, prostate, and breast than m liver (6). Exposure to polycyclrc aromatic hydrocarbons such as those found m cigarette smoke induces the expression of both CYPlAl and CYPlA2 (3,7). CYPIBI is also mducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), as demonstrated by cell-culture experiments (8,9). The chemical inhibitor a-naphthoflavone, which is a potent inhibitor of CYPlAl and CYPlA2 (ZO), also inhibits CYPlBl and with semilar efficacy and potency (ZZ). cDNA-expressed CYPl Al, CYPl A2 and CYPlB 1are each active in the oxidation of theophyllme (II), caffeine (ZZ,Z2), estradiol (Z3,14), benzo[a]pyrene (ZZ) and 7-ethoxyresorufin (ZZ). With 7ethoxyresorutin as substrate, the rank order of specific activity (pmol/min/nmol P450) is CYPlAl > CYPlBl > CYPIA2 (ZZ). 7-Ethoxyresorufin O-deethylation activity can be a useful and convenient catalytic monitor when conductmg a comparative study usmg a panel of these three recombinant CYP 1 enzymes (see Note 1). Immunoinhibition experiments with mhibitory CYPl A-selective antibodies have suggested that CYPlA2 is a major contributor to 7-ethoxyresorufin O-deethylase activity in human hver microsomes (Z5,16). AccordFrom Methods m Molecular Biology, Vol 107 Cytochrome P450 Protocols Edtted by I R Phlllfps and E A Shephard 0 Humana Press Inc , Totowa, NJ

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mgly, this activity IS frequently used as a marker for human hepatic CYPlA2 (see Note 2). This chapter describes a 7-ethoxyresorufin 0-deethylase assay (see Note 3) that is based on a modification (17) of the spectrofluorometric method of Burke and Mayer (18) for the determination of alkoxyresorufin Odealkylatton. Thts reaction can also be measured by high-performance hquld chromatography (HPLC) with fluorescence detectlon (16). 2. Materials 1 Substrate: 7-ethoxyresorufin (MW = 241 2) (Molecular Probe, Eugene, OR). Prepare a 1 m&I (0 24 mg/mL) stock solution dissolved m dlmethylsulfoxlde (DMSO) (see Notes 4 and 5). 2 Metabollte standard* resorufin (sodmm salt, MW = 235 2) (Molecular Probes) 3. Assay buffer 100 mMHEPES, pH 7.8, containmg 5 n&I MgC& 4 Cofactor. 50 mM (42 mg/mL) mcotmamlde adenme dmucleotlde phosphate (NADPH) stock solution Prepare fresh and store on Ice (see Notes 6-8). 5 Enzymes. e g. cDNA-expressedCYP1 Al, CYPIA2, CYPlBl (Gentest, Wobum, MA), or human hver mlcrosomes. Dilute in assay buffer to a working concentration of 2 mg protein/ml and place on ice (see Note 9). 6. Equipment includes a spectrofluorometer connected to a circulating water bath

3. Methods 1 Add the followmg to a cuvet (see Note 10) placed m a spectrofluorometer which 1s connected to a circulating water bath set at 37°C. a 1 93 mL of assay buffer. b 10 clr, of 1 mM 7-ethoxyresorufin (5 @4 final concentration) (see Note 11) c 50 & of diluted enzymes (100 pg protem) (see Notes 12 and 13) 2. Record the background fluorescence reading of the sample at an excitation wavelength of 530 nm and an emlsslon wavelength of 582 nm (see Note 14). 3 Add 10 $ of 50 mM NADPH (0 25 mA4 final concentration) to the cuvet and mix to initiate the enzymatic reaction 4 Record the progress of the enzymatic reaction for up to 5 min (see Note 15). Calculate a reaction rate (fluorescent units of resorufin product formed per unit time) from the linear portion of the progress curve. 5 Calculate the net fluorescence of each unknown sample by subtracting the background fluorescence reading from the reading for each unknown sample. 6. Prepare standards by adding 10 pL of a known amount (e g , 0,O I,0 25,O 5, 1, and 2 nmol) of authentic resort& metabohte (dissolved m DMSO) to a separate cuvet containing the complete mcubatlon mixture but with heat-inactivated enzymes. Record the fluorescence reading.

7. Calculate the net fluorescenceof each standardsampleby subtractingthe background fluorescence reading from the reading for each standard sample 8 Plot a standard curve of net fluorescence vs nmol of authentic resorutin metabohte Calculate slope and intercept by linear regression analysis.

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9. Use the standard curve to calculate 7-ethoxyresorufin 0-deethylase activity for each unknown sample Express the results as nmol product formed/mm/mg mlcrosomal protein or as nmol product formed/mm/nmol total P450 4. Notes 1 7-Ethoxycoumarm 0-deethylation can also be used to assay the catalytic activity of each of the three recombmant CYPl enzymes, CYPlAl, CYPlA2, and CYPlBl. However, CYPl Al, CYPlA2, and CYPlBl are, respectively, -3-fold, 2-fold, and 6-fold more active m 7-ethoxyresorufin 0-deethylation than m 7ethoxycoumarin 0-deethylatlon (II) 2 The apparent Km values for 7-ethoxyresorufin 0-deethylatlon catalyzed by cDNA-expressed human CYPl Al and CYPlA2 are 0 017 fl and 1.7 w, respectively (12). CYPl Al IS likely to account for the majority of 7-ethoxyresorufin 0-deethylase activity m human lung mlcrosomes because It IS CYPl Al, rather than CYPlA2, that 1s expressed in human lung (4,5) By contrast, CYPlA2 IS likely the primary 7-ethoxyresorufin 0-deethylase m human liver mlcrosomes because CYPlA2 is expressed m human liver (2,3,19), whereas CYPlAl IS either absent or present at very low levels m this tissue (2,19). Whether hepatlc CYP 1B 1 contributes significantly to mlcrosomal7-ethoxyresorufin 0-deethylase activity remams to be determined. 3 The methods described using 7-ethoxyresorufin as substrate can be extended to three other alkoxyresorufins, namely 7-methoxyresorufin, 7-benzyloxyresorufin, and 7-pentoxyresorufin, whose P450-catalyzed 0-dealkylatlon actlvltles can be assayed using essentially the same method described here All four alkoxyresorufins yield the same fluorescent metabohte, resorufin CYPlA2 appears to account for the majority of human hepatic microsomal 7-methoxyresorufin 0-dealkylase activity, as judged by lmmunomhlbltlon experiments with heterologous anti-CYPlA2 antibodies (16). However, recombinant human CYP 1A 1 and CYPlA2 both metabolize 7-methoxyresorufin and with similar apparent Km (0.2-0.4 clM> and similar apparent Vmax (approximately 0.5 nmol/min/mg microsomal protem) values (12). CYP3A, CYPlA, and CYP2A can all contnbute to human hepatic microsomal 7-benzyloxyresorufin 0-dealkylase activity (16), which is consistent with the observation that multiple recombinant human P450 enzymes are active catalysts m the oxldatlon of 7-benzyloxyresorufin (20,21) 7-Pentoxyresorufin 0-dealkylase activity IS very low but detectable m human liver mlcrosomes (-3% the rate of ‘I-ethxoyresorufin 0-dealkylase activity) (221, but it IS not known which human hepatlc P450 enzyme(s) IS responsible for this activity. 4 7-Ethoxyresorufin and resorufin are light-sensltlve. Store these chemicals m the dark 5 Resorufin and alkoxyresorufin stock solutions prepared m DMSO can be stored in the dark at room temperature for up to 1 yr without degradation (18) 6. Prepare fresh solutions of NADPH for each experiment. NADPH is light-sensitive and pH-sensitive

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7 Alkoxyresorutin 0-dealkylase assays can also be carried out with an NADPHgenerating system (e.g., NADP+, u-glucose-6-phosphate and glucose-6-phosphate dehydrogenase) in place of NADPH. 8. Artificially high 7-ethoxyresorufin 0-deethylase activity is obtained with assay protocols that use acid to prectpitate mtcrosomal protein because NADPH reacts with 7-ethoxyresorufin m the presence of acid to form a product that fluoresces at the same wavelength as resorufin (23) 9. Prepare only sufficient amounts of dilute human liver microsomes for each experiment. The remamder of the undiluted microsomes can be stored at -80°C for future use (24) 10. 7-Ethoxyresorufin 0-deethylase activity in cultured cells (e.g , hepatocytes) can be measured directly in culture plates by usmg a fluorescence multiwell-plate reader (25,26) 11. Optimal 7-ethoxyresorufm concentration 1s 5-10 w. Reduced activtty 1s observed at a substrate concentration >lO uiV owmg to mhtbition of 7-ethoxyresorufin 0-deethylase activity by the enzymatic product, resorufin (23) 12. With some P450 protem expression systems, it IS necessary to reconstitute the recombinant P450 protein with NADPH-cytochrome P450 reductase, cytochrome b, and liptd prior to mtttatmg substrate oxidatton (11,27,28). Conduct preliminary experiments to estabhsh the amount of each component needed for optimal enzyme activity 13 Conduct preliminary experiments to ensure that the assay is lmear with respect to microsomal protem concentration 14 Determine the optimal excitatton wavelength and emtssion wavelength to be used with each particular spectrofluorometer by exammmg the excitation and emission spectra (29) of 7-ethoxyresorufln and resorufin generated by that mstrument 15 7-Alkoxyresorufin 0-dealkylase assays can also be carried out using a modified protocol, where methanol is used to stop the enzymatic reaction after a fixed mcubation time, and the precipitated protein is centrifuged and the fluorescence of the supernatant then measured (2I,23).

Acknowledgments Supported in part by the British Columbia Health Research Foundatton (grant 119(95-l) to Thomas K. H. Chang) and by the National Institutes of Health (grant ES07381 to David J. Waxman). Thomas K. H. Chang 1s the recrpient of a Research Career Award in the Health Sciences from the Pharmaceutical Manufacturers Association of Canada - Health Research Foundation and the Medtcal Research Council of Canada.

References 1 Nelson, D. R , Koymans, L , Kamataki, T., Stegeman, J J., Feyereisen, R., Waxman, D J , Waterman, M R., Gotoh, O., Coon, M J , Estabrook, R. W., Gunsalus, I C., and Nebert, D W. (1996) P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature Pharmacogenetm 6, l-42

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2. Murray, B. P , Edwards, R. J , Murray, S , Singleton, A M , Davies, D. S , and Boobis, A. R. (1993) Human hepatrc CYPlAl and CYPIA2 content, determined with specific anti-pepttde antibodies, correlates with the mutagenic activation of PhIP. Carcznogenesls 14,585-592. 3 Schwetkl, H., Taylor, J. A., Kitareewan, S., Lmko, P , Nagorney, D , and Goldstein, J. A. (1993) Expression of CYPlAl and CYPlA2 genes in human liver. Pharmacogenetlcs 3,239-249 4 Wheeler, C. W., Park, S. S., and Guenthner, T. M. (1990) Immunochemrcal analysis of a cytochrome P-450IAl homologue m human lung microsomes. Mol. Pharmacol.

38,634-643.

5. Shimada, T., Yun, C H., Yamazaki, H , Gautrer, J C., Beaune, P H , and Guengerich, F. P. (1992) Characterization of human lung microsomal cytochrome P-450 IA1 and its role m the oxidation of chemrcal carcmogens A401 Pharmacol. 41,856-864. 6. Shimada, T., Hayes, C. L., Yamazaki, H., Amm, S., Hecht, S. S , Guengerich, F. P., and Sutter, T. R. (1996) Activation of chemically diverse procarcmogens by human cytochrome P-450 1B 1, Cancer Res 56,2979-2984. 7. Sesardic, D , Boobu, A. R., Edwards, R J , and Davies, D S. (1988) A form of cytochrome P450 m man, orthologous to form d m the rat, catalyses the Odeethylation of phenacetm and is inducible by cigarette smoking. Br J Clan Pharmacol.

X,363-372

8 Dohr, 0 , Vogel, C., and Abel, J. (1995) Different response of 2,3,7,8-tetrachlorodrbenzo-p-dioxin (TCDD)-sensitive genes m human breast cancer MCF-7 and MDA-MB 23 1 cells. Arch. Blochem Biophys 321,405-412. 9 Kress, S and Greenlee, W F (1997) Cell-specific regulation of human CYPI Al and CYPlBl genes. Cancer Res 57, 1264-1269. 10 Chang, T. K. H , Gonzalez, F. J , and Waxman, D. J. (1994) Evaluation of triacetyloleandomycm, a-naphthoflavone and diethyldithiocarbamate as selective chemical probes for inhibition of human cytochromes P450 Arch Biochem Biophys

311,437-442.

11 Shimada, T., Gillam, E. M. J., Sutter, T. R., Strickland, P T , Guengerich, F P , and Yamazaki, H. (1997) Oxidation of xenobiotics by recombinant human cytochrome P450 1B 1. Drug Metab. Dupes 29,6 17-622. 12. Eugster, H. P., Probst, M., Wurgler, F. E., and Sengstag, C. (1993) Caffeme, estradrol, and progesterone interact with human CYP 1A 1 and CYP 1A2: evidence from cDNA-directed expression in Saccharomyces cerevlszae. Drug Metab Dispos. 21,43-49.

13. Guo, Z , Gillam, E. M. J , Ohmori, S., Tukey, R H., and Guengerich, F. P. (1994) Expression of modrfied human cytochrome P450 1Al in Escherzchia ~011: effects of 5’ substitution, stabrlization, purrfication, spectral characterization, and catalytic properties. Arch Biochem. Biophys 312,436-446. 14. Hayes, C. L., Spink, D. C., Spink, B. C., Cao, J. Q., Walker, N. J., and Sutter, T. R. (1996) 17j3-Estradiol hydroxylation catalyzed by human cytochrome P450 1B 1 Proc. Nat1 Acad Scz. USA 93,9776-978 1.

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15, Bourdi, M., Larrey, D., Nataf, J., Bernuau, J., Pessayre, D., Iwasaki, M , Guengerich, F P , and Beaune, P. H (1990) Antt-liver endoplasmtc reticulum autoanttbodtes are directed agamst human cytochrome P-4501A2: a specific marker of dlhydralazine-induced hepatitis. J Clan. Invest. 85, 1967-1973. 16. Burke, M. D., Thompson, S., Weaver, R. J , Wolf, C. R., and Mayer, R. T (1994) Cytochrome P450 specificities of alkoxyresornfin 0-dealkylation in human and rat liver Biochem. Pharmacol 48,923-936 17. Chang, T., Levine, M., Bandtera, S. M., and Bellward, G. D. (1992) Selective inhibition of rat hepatic mtcrosomal cytochrome P-450 I. Effect of the zn vzvo administration of cimetidine. J Pharmacol Exp. Ther 260, 1441-1449. 18. Burke, M D. and Mayer, R T (1983) Dtfferential effects of phenobarbitone and 3-methylcholanthrene induction on the hepattc mtcrosomal metabolism and cytochrome P-450-binding of phenoxazone and a homologous series of its n-alkyl ethers (alkoxyresoruflns) Chem -Btol Interact 45,243-258. 19 McManus, M. E , Burgess, W. M , Veronese, M. E., Huggett, A , Quattrochi, L C., and Tukey, R. H (1990) Metaboltsm of 2-acetylammofluorene and benzo[a]pyrene and activation of food-derived heterocyclic amme mutagens by human cytochromes P-450 Cancer Res 50,3367-3376. 20. Lee, Q. P., Fantel, A G., and Juchau, M R (1991) Human embryonic cytochrome P45Os: phenoxazone ethers as probes for expression of functional isoforms durmg organogenests. Blochem Pharmacol. 42,2377-2385. 21 Waxman, D. J , Lapenson, D P , Aoyama, T , Gelbom, H. V , Gonzalez, F J., and Korzekwa, K. (199 1) Steroid hormone hydroxylase spectfictties of eleven cDNAexpressed human cytochrome P45Os Arch. Biochem Biophys 290, 160-l 66. 22. Stevens, J. C , Shipley, L. A., Cashman, J. R., VandenBranden, M , and Wrighton, S. A. (1993) Comparison of human and rhesus monkey in vztro phase I and phase II hepatic drug metabolism activities. Drug Metab Dlspos. 21, 753-760. 23. Pohl, R J. and Fouts, J R. (1980) A rapid method for assaying the metabohsm of 7-ethoxyresorufin by mlcrosomal subcellular fractions. Anal Bzochem. 107,150-l 55 24 Pearce, R E., McIntyre, C J., Madan, A., Sanzgtrt, U., Draper, A. J., Bullock, P. L , Cook, D. C., Burton, L A., Latham, J , Nevms, C , and Parkinson, A (1996) Effects of freezing, thawing, and storing human liver microsomes on cytochrome P450 activity Arch Bzochem Bzophys 331, 145-169 25 Donato, M. T., Gomez-Lechon, M J., and Castell, J V (1993) A microassay for measuring cytochrome P4501A 1 and P450IIB 1 activities in intact human and rat hepatocytes cultured on 96-well plates. Anal Btochem 213,29-33. 26. Kennedy, S. W., Lorenzen, A., James, C. A , and Collms, B. T. (1993) Ethoxyresorufin 0-deethylase and porphyrm analysts m chicken embryo hepatocyte cultures with a fluorescence multiwell plate reader. Anal Bzochem 211, 102-l 12 27. Buters, J. T., Shou, M., Hardwtck, J. P., Korzekwa, K. R., and Gonzalez, F J. (1995) cDNA-directed expression of human cytochrome P450 CYPl Al using baculovirus. Purttication, dependency on NADPH-P450 oxidoreductase, and reconstitution of catalytic properties without purificatton Drug Metab Dupes 23,696-701.

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28 Sandhu, P , GUO, Z., Baba, T., Martm, M. V., Tukey, R. H., and Guengerrch, F. P. (1994) Expresston of modified human cytochrome P450 lA2 m Escherichza coZi* Stabrlizatron, purification, spectral characterization, and catalytrc actrvttres of the enzyme Arch Biochem Bzophys, 309, 168-177. 29. Burke, M. D. and Mayer, R. T. (1974) Ethoxyresorufin direct fluortmetrrc assay of a microsomal 0-dealkylatron which IS preferentially Inducible by 3-methylcholanthrene Drug Metab Dwpos 2, 583-588.

11 Spectrofluorometric Analysis of CYP2A6-Catalyzed Coumarin

7-Hydroxylation

David J. Waxman and Thomas K. H. Chang 1. Introduction CYP2A6 has been isolated and purified to apparent homogeneity from human liver (1,2). The level of CYP2A6 protein expressed m liver is low (-4% of total hepatic P450 content) (3), although substantial intermdividual variation exists (3-5). This P450 is primarily a hepatic protein, as suggested by its absence from several extrahepatic tissues, including adult human lung, colon, breast, kidney, and placenta microsomes (2). According to experiments with primary cultures of human hepatocytes, CYP2A6 is mducible by phenobarbital, dexamethasone, and rifampm (rifampicin) (6). Experiments wrth individual cDNA-expressed human P450 forms have indicated that CYP2A6 is a major catalyst of coumarm 7-hydroxylation (7). Among the other recombmant human P45Os examined, CYPlAl, lA2,2C8,2C9,2D6,2El, 3A3,3A4, and 3A5 are catalytically inactive with respect to coumarm 7-hydroxylation, whereas CYP2B6 is active at only -5% the rate of CYP2A6 (7). Immunomhibition studies have validated the use of coumarm 7-hydroxylase activity as a diagnostic catalytic marker for human hepatic CYP2A6. Antihuman CYP2A6 IgG inhibits coumarm 7-hydroxylase activity by >90% m human liver microsomes (21, indicating that CYP2A6 is the prmcipal and perhaps the sole catalyst of human liver microsomal coumarin 7-hydroxylase activity. This activity can be inhibited by a variety of compounds, includmg Smethoxypsoralen (B), tranylcypromme (8), a-naphthoflavone (8-11) and diethyldithiocarbamate @J/9,11). This chapter describes a modification (12) of a spectrofluorometric method (13) for the determination of coumarm 7-hydroxylase activity.

From Methods m Molecular Bology, Vol 107 Cytochrome P450 Protocols Edtted by I R PhIllIps and E A Shephard 0 Humana Press Inc , Totowa, NJ

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2. Materials 1 Assay buffer 100 mM potassmm phosphate, pH 7 4, containing 0 1 mM EDTA (see Note 1) 2 Substrate, coumarin (1,2-benzopyrone, MW = 146.1) (Aldrich Chemical, MIXwaukee, WI) Prepare a 1 mM (0 15 mg/mL) stock solution dissolved m assay buffer 3. Metabolite standard: 7-hydroxycoumarin (umbelliferone, MW = 162 1) (Aldrich). 4 Cofactor. 10 mA4 (8 3 mg/mL) mcotmamlde ademne dmucleotlde phosphate (NADPH) stock solution. Prepare fresh and keep on ice (see Notes 2-4) 5. Enzymes: cDNA-expressed CYP2A6 (Gentest, Worurn, MA) or human liver mlcrosomes. Dilute m assay buffer to a workmg concentration of 1 mg protein/ mL and keep on ice (see Note 5) 6 Deprotemlzmg agent 2 A4 HCl 7. Extraction solvent. chloroform 8. Back-extraction solution: 30 mM (6 mg/mL) sodium borate (Na2B407), pH 9 2

3. Methods 1 Add the followmg to each mcubatlon tube (total incubation volume of 200 6) (see Note 6): a 150 pL of assay buffer b 10 pL of 1 m/U coumarm (50 w final concentration) (see Notes 7 and 8). c 20 pL of diluted enzymes (to give 20 pg protem) (see Notes 9 and 10) 2. Prewarm mcubatlon tubes to 37°C and add 20 pL of 10 mA4NADPH (1 mM final concentration) to mltlate enzymatic reaction (stagger each incubation with 15-s delay intervals). 3. Incubate samples at 37°C for 15-30 mm m a shaking water bath (see Note 11). 4. Add 25 p.L ice-cold 2 MHCl to stop enzymatic reactlon and place the mcubatlon tube on ice 5 Extract with 450 $ chloroform while vortexmg for 30 s (see Note 12). 6. Centrifuge extraction tubes at 3000g for 5 min 7. Transfer 300 pL of the organic phase (bottom layer) to a clean test tube and backextract with 1 mL of 30 mM sodium borate, pH 9 2 8 Repeat step 6. 9. Remove the top layer and measure the fluorescence at an excltatlon wavelength of 370 nm and an emlsslon wavelength of 450 nm (see Note 13) 10 Prepare blank incubation tubes by adding the complete incubation mixture but with heat-inactivated enzymes. Process the blank mcubatlon tubes as per steps 3-9. 11. Prepare standards by adding a known amount (e g , 0, 0.1, 0 2, 0.4, 0 8, and 1.2 nmol) of authentic 7-hydroxycoumarm metabohte to tubes containing complete incubation mixture, but with heat-inactivated enzymes. Process the mcubatlon tubes containing the standards as per steps 3-9. 12. Calculate net fluorescence of each unknown sample and standard by subtracting the fluorescence reading of the blank from that of the unknown or standard.

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13. Plot a standard curve of net fluorescence vs amount of authentic ‘I-hydroxycoumarm (e.g., 0,O 1,0.2, 0.4, 0.8, and 1.2 nmol) and determine the amount of product formatton in each unknown sample by linear regression analysis. 14. Calculate coumarm 7-hydroxylase activity and express tt as nmol product formed/ min/mg mrcrosomal protein or as nmol product formed/min/nmol total P450 (see Notes 14 and 15).

4. Notes 1. Coumarin 7-hydroxylase activrty is highly dependent on the romc strength of the buffer system (4,Z4). 2. Prepare a fresh solutton of NADPH for each experiment NADPH IS hght-sensltrve and pH-sensmve. 3. Coumarin 7-hydroxylation assays can also be carried out using an NADPH-generatmg system (e.g., NADP+, n-glucose-6-phosphate and glucose-6-phosphate dehydrogenase (4) or NADP+, isocitrrc acid, and rsocttric acid dehydrogenase /ZS/) m place of NADPH. 4. CYP2A6 IS substantrally less active (30-50% lower achvrty) when assayed with some lots of NADP+ (Sigma, St Louis, MO, cat no. NOSOS) and also with the monosodium salt of o-glucose 6-phosphate (Sigma) A similar inhibitory effect has been observed for rat CYP2A 1-catalyzed testosterone 7a-hydroxylase actrvrty (C Crespl, unpublrshed observatton) 5. Dilute only sufficient amounts of mrcrosomes for each experiment The remamder of the undiluted mrcrosomes can be stored at -80°C for future use (16) 6. Formatron of 7-hydroxycoumarm m cultured CYP2AG-expressmg cells can be directly measured by a fluorescence multtwell plate reader after mcubatmg the cells wrth coumarm m a multiwell plate at 37’C for 1 h (Z 7) 7 According to results from nnmunoinhibitton experiments, coumarm 7-hydroxylase activity IS selective for human liver microsomal CYP2A6 regardless of whether the assay 1s performed at a substrate concentration of 0 02 mM (2), 0 05 mA4(4), 0.6 mA4 (Z5), or 1 mM (IS). 8. The apparent Km value for coumarin 7-hydroxylatlon catalyzed by cDNAexpressed CYP2A6 or by human liver microsomes 1s-0.5 p&$ (8). Therefore, the suggested substrate concentration (50 pA4) to be used m this assay 1s -loo-fold greater than the apparent Km. 9. Conduct preliminary expertments to ensure that the assay IS linear with respect to microsomal protein concentration. 10. With some P450 protein expression systems, tt is necessary to reconstitute the recombinant P450 protein with NADPH-cytochrome P450 reductase, cytochrome bs and lipid prior to initiating substrate oxidation (14) Conduct preliminary experiments to establish the amount of each component requrred for optimal catalytic actrvity. 11. Conduct preliminary experiments to ensure that the assay is linear with respect to incubation time. 12. “Wet” plpet tips with organic solvent prior to use.

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13 Determine the opttmal excitation wavelength and emtssion wavelength to be used with each particular spectrofluorometer by examining the excitation and emtssion spectra of coumarin and 7-hydroxycoumarm (19) generated by that instrument 14 Typical coumarm 7-hydroxylase activity levels found in human liver mlcrosomes range from CO 0 1 to 4.75 nmol/min/mg microsomal protein (20) 15 Methanol at a final concentratton of l-5% (v/v) does not inhtbtt human hver mrcrosomal coumarm ‘I-hydroxylase activtty when the assay 1s conducted at a substrate concentratton of 50 @4 (8) However, at a lower substrate concentration (0 5 @4), methanol at a final concentration of >l% (v/v) decreases this activity By comparison, mclusion of a 1% (v/v) final concentratton of dlmethylsulfoxtde, dtmethylformamide, 2-propanol, ethanol, acetonitrtle, acetone, tetrahydrofuran, or dioxane in the mcubatton mixture results m inhibitron of coumarin 7-hydroxylase activity m human liver mtcrosomes, especially at 0 5 @4 substrate concentratton.

Acknowledgments Supported in part by the National Instttutes of Health (grant CA49248 to David J. Waxman) and the British Columbia Health Research Foundation (grant 119(95-l) to Thomas K H. Chang). Thomas K. H Chang 1s the recrptent of a Research Career Award in the Health Sciences from the Pharmaceutlcal Manufacturers Association of Canada-Health Research Foundation and the Medical Research Council of Canada.

References 1 Maurice, M , Emtliani, S., Dalet-Beluche, I., Derancourt, J., and Lange, R (1991) Isolation and characterization of a cytochrome P450 of the IIA subfamily from human liver mtcrosomes. Eur J Blochem. ZOO,5 1 l-5 17. 2 Yun, C. H., Shimada, T , and Guengerrch, F. P. (1991) Purtficatlon and characterization of human liver mrcrosomal cytochrome P-450 2A6 Mel Pharmacol 40, 679685.

3 Shimada, T , Yamazakt, H , Mlmura, M., Inm, Y., and Guengerrch, F P. (1994) Interindivrdual variations m human liver cytochrome P-450 enzymes mvolved m the oxidation of drugs, carcinogens and toxic chemtcals. studies with liver mlcrosomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 270, 414-423. 4. Pearce, R., Greenway, D., and Parkinson, A. (1992) Species differences and mterindlvrdual variation m liver mmrosomal cytochrome P450 2A enzymes effects on coumarm, dtcumarol, and testosterone oxtdatron Arch Blochem Biophys. 298,21 l-225. 5 Imaoka, S., Yamada, T., Hiroi, T., Hayasht, K., Sakaki, T , Yabusakt, Y , and Funae, Y (1996) Multiple forms of human P450 expressed m Saccharomyces cerevulae- systematic charactertzatton and comparison with those of the rat Bzochem Pharmacol 51, 1041-1050.

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6 Dalet-Beluche, I., Boulenc, X., Fabre, G., Maurel, P., and Bonfils, C (1992) Purificatton of two cytochrome P450 isozymes related to CYP2A and CYP3A gene families from monkey (baboon, Papzo papzo) liver microsomes: cross reacttvtty with human forms. Eur J. Biochem. 204,641-648. 7. Waxman, D. J., Lapenson, D P., Aoyama, T , Gelbom, H. V., Gonzalez, F. J., and Korzekwa, K. (1991) Sterotd hormone hydroxylase specificities of eleven cDNAexpressed human cytochrome P45Os. Arch Biochem Biophys 290, 160-166. 8. Draper, A. J , Madan, A., and Parkmson, A. (1997) Inhibition of coumarin 7-hydroxylase activity in human liver microsomes. Arch Blochem Biophys 341,47--d 1. 9. Pelkonen, O., Sotamemt, E A., and Ahokas, J T (1985) Coumarm 7-hydroxylase acttvtty m human liver mtcrosomes: properttes of the enzyme and interspecies comparisons. Br J Clm Pharmacol 19, 59-66. 10 Yamazaki, H., Inm, Y., Yun, C. H., Mimura, M., Guengerich, F P., and Shimada, T (1992) Cytochrome P450 2El and 2A6 enzymes as maJor catalysts for metabolic actrvatton of N-mtrosodialkylamines and tobacco-related mtrosamines in human liver microsomes. Carclnogenesls 13, 1789-I 794. 11 Chang, T. K. H., Gonzalez, F J., and Waxman, D. J (1994) Evaluatton of trtacetyloleandomycm, a-naphthoflavone and diethyldithiocarbamate as selective chemical probes for mhtbitron of human cytochromes P450. Arch Blochem Blophys 311,437-442 12 Waxman, D J and Walsh, C (1982) Phenobarbital-induced

13.

14

15.

16

17

rat liver cytochrome P450: puriticatton and charactertzatton of two closely related isozymic forms J Biol Chem 257, 10,44610,457. Greenlee, W. F. and Poland, A. (1978) An Improved assay of 7-ethoxycoumarin 0-deethylase acttvity. Induction of hepatic enzyme activity m C57BL/6J and DBA/2J mice by phenobarbttal, 3-methylcholanthrene and 2,3,7,8,-tetrachlorodibenzo-p-dioxm J. Pharmacol Exp Ther. 205,596-605 Tan, Y , Patten, C. J., Smith, T., and Yang, C S. (1997) Competitive mteracttons between cytochromes P450 2A6 and 2El for NADPH-cytochrome P450 oxidoreductase m the microsomal membranes produced by a baculovuus expression system. Arch Blochem. Blophys 342,82-g 1. Crespi, C. L., Penman, B. W., Leakey, J. A. E., Arlotto, M P., Stark, A , Parkinson, A., Turner, T , Steimel, D T., Rudo, K , Davies, R. L., and Langenbach, R. (1990) Human cytochrome P450IIA3: cDNA sequence, role of the enzyme in the metabolic activation ofpromutagens, comparison to mtrosamine activation by human cytochrome P450IIEl Carcznogenesls 11, 1293-l 300. Pearce, R E., McIntyre, C J., Madan, A , Sanzgm, U., Draper, A. J., Bullock, P L., Cook, D. C., Burton, L. A., Latham, J., Nevins, C , and Parkmson, A (1996) Effects of freezing, thawing, and storing human liver microsomes on cytochrome P450 activity. Arch Blochem Blophys 331, 145-169. Chen, L., Buters, J. T M., Hardwick, J. P., Tamura, S., Penman, B W , Gonzalez, F. J , and Crespi, C. L. (1997) Coexpression of cytochrome P450 2A6 and human NADPH-P450 oxtdoreductase m the baculovuus system Drug Metab Dzspos 25,399-405

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18. Chang, T. K. H., Weber, G. F., Crespi, C. L., and Waxman, D. J. (1993) Drfferentral activation of cyclophosphamide and ifosphamide by cytochromes P-450 2B and 3A in human liver microsomes. Cancer Res. 53,5629-5637. 19. Prough, R. A., Burke, M. D., and Mayer, R. T. (1978) Direct fluorometric methods for measurmg mixed-functton oxidase activity. Methods Enzymol 52,372-377. 20. Chang, T. K. H. and Waxman, D. J. (1996) The CYP2A subfamily, m Cytochromes P450 Metabolzc and Toxrcological Aspects (Ioanmdes, C. ed ), CRC, Boca Raton, FL, pp. 99-134.

12 Determination of the CYP2B6 Component of 7-Ethoxy-4-Trifluoromethylcoumarin O-Deethylation Activity in Human Liver Microsomes Thomas K. H. Chang, Charles L. Crespi, and David J. Waxman 1. Introduction CYP2B6 protein has been isolated and purified from human liver (1,2). In a panel of 60 individual human liver microsome samples, this P450 accounts for
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Recent studies have indicated the involvement of CYPlA, CYP2B6, CYP2C, and CYP2El in 7-ethoxy-4-trifluoromethylcoumarm O-deethylation m human hver microsomes (4,13). However, selectivity of this reaction for CYP2B6 can be achieved by use of a low substrate concentration (5 @4) and by preincubation of human liver microsomes with a mixture of mhibitory antibodies to block the activities of the CYPlA, CYP2C, and CYP2El enzymes (4). This chapter describes a spectrofluorometric method for the determmation of the CYP2B6 component of human liver microsomal 7-ethoxy-4-trifluoromethylcoumarm O-deethylation activity. Simple modifications of this assay method can be introduced to measure cDNA-expressed CYP2B6-catalyzed 7-ethoxy-4-trifluoromethylcoumarin O-deethylation (4).

2. Materials 1 Assay buffer: 100 mM potassium phosphate, pH 7.4. 2 Substrate 7-ethoxy-4-trifluoromethylcoumarm (MW = 258 2) (Enzyme Systems Products, Livermore, CA) (see Note 1) Prepare a 25 mM (6 4 mg/mL) stock solution dissolved m methanol Dilute to 50 pA4 with assay buffer (see Notes 2 and 3). 3. Metabohte standard* 7-hydroxy-4-trrfluoromethylcoumarm (MW = 230 2) (Enzyme Systems Products) (see Note 1). 4 Cofactor generating system: 26 mA4 (20 mg/mL) NADP+ (see Note 4), 66 KIM (20 mg/mL) o-glucose-6-phosphate, 66 mM (13 3 mg/mL) magnesium chloride (MgC12 * 6H20). Glucose-6-phosphate dehydrogenase (40 U/mL) m 5 mA4 (1.5 mg/mL) sodium citrate (C,H,07Nas * 2H,O) 5. Enzymes: Dilute human lrver mrcrosomes in assay buffer to a working concentration of 1 mg microsomal protein/ml and keep on ice (see Note 5) cDNAexpressed BY2B6 (GENTEST, Woburn, MA). 6 Antibodies. a. Anti-CYPlA2 (polyclonal antibody developed m rabbit, Danchi Pure Chemicals, Tokyo, Japan, and is available from GENTEST) b Anti-CYP2C (polyclonal antibody developed m goat, Danchi Pure Chemicals, and is available from GENTEST) c Anti-CYP2El (monoclonal antibody developed m mouse, GENTEST, Woburn, MA ) Premix the mhrbitory antibodies m the followmg proportions: 10 pL of monoclonal anti-CYP2E 1,20 pL of polyclonal anti-CYP 1A2 and 90 pL of polyclonal anti-CYP2C 7 Deproteimzing agent: 20% (w/v) trichloroacetic acid. 8 100 mM Trrs-HCl, pH 9.0. 9 Equipment includes a spectrofluorometer

3. Methods 1. Mix 10 pL of the diluted human liver microsomes with 4 pL of the antibody mixture (see Subheading 2., item 6). Incubate for 30 mm on ice

7-Ethoxy-4-Triflouromethylcoumarin

0-Deethylatlon

Assay

719

2. Add the followmg to each mcubation tube (total mcubation volume of 200 pL)* a. 154 pL of assay buffer b 20 pL of 50 @4 7-ethoxy-4-trifluoromethylcoumarin (5 @4 final concentration, see Note 6) c. 10 pL of a solution contaming 26 mM NADP+, 66 mA4 o-glucosed-phosphate, and 66 mM magnesmm chloride d 2 pL of glucose-6-phosphate dehydrogenase (40 U/mL) m 5 mA4sodmm citrate 3. Prewarm incubation tubes to 37°C and add 14 pL of the mrxture contammg the human liver microsomes and the inhibitory antibodies to mttiate the enzymatic reaction (stagger each incubation with 15-s delay intervals) (see Note 7) 4. Incubate samples at 37°C for 20-30 mm in a water bath (see Note 8). 5. Add 40 pI-. of 20% (w/v) trtchloroacetic acid to stop enzymatic reaction and place the incubation tube on ice (see Note 9). 6. Centrifuge reaction mtxture at 10,OOOgfor 3 mm. 7. Transfer 100 & of the supernatant to a clean test tube containmg 1 9 mL of 100 mMTris-HCI, pH 9.0, and vortex. 8. Measure the fluorescence at an excitation wavelength of410 nm and an emission wavelength of 5 10 nm (see Note 10) 9 Prepare blank incubation tubes by adding the complete mcubatton mixture but with heat-mactivated mmrosomes Process the blank mcubation tubes as per steps 4-8 10 Prepare standards by addmg a known amount (e.g , 0 01,0.02,0 05,O 1,0.2,0 5, and 1 nmol) of authentic 7-ethoxy-4-trifluoromethylcoumarm metabohte to tubes containing the complete mcubation mixture but with heat-mactivated microsomes. Process the mcubation tubes contammg the standards as per steps 4-8 11. Calculate net fluorescence of each unknown sample and standard by subtracting the fluorescence reading of the blank from that of the unknown or standard. 12 Plot a standard curve of net fluorescence against amount of authentic 7-hydroxy4-trtfluoromethylcoumarm and determine the amount of product formation m each unknown sample by linear regression analysis. 13 Calculate 7-ethoxy-4-trifluoromethylcoumarm O-deethylatton acttvity and express it as nmol product formed/mm/mg microsomal protein or as nmol product formed/mm!nmol total P450 (see Note 11) 4. Notes 1 7-Ethoxy-4-trifluoromethylcoumarm and 7-hydroxy-4-trifluoromethylcoumarm are light sensitive 2. The limit of solubiltty of 7-ethoxy-4-trifluoromethylcoumarm m potassium phosphate buffer is -50 pM, but this can be increased to -100 $4 by using dimethylsulfoxide (DMSO) (0.2%, v/v final concentration) as a vehicle. This concentration of DMSO results in an -5% decrease m enzyme acttvity (14) Ethanol IS highly inhibitory to CYP2B6. 3 Stock solutions of 7-ethoxyd-trifluoromethylcoumarin are stable for at least 1 yr when stored at -20°C (14).

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4. 7-Ethoxy-4-trifluoromethylcoumarm 0-deethylase assays can also be carried out with nicotinamide adenme dinucleotide phosphate (NADPH) (e.g , 1 mA4 final concentration) instead of an NADPH-generating system (e g , NADP+, D-glucose-6-phosphate, glucose-6-phosphate dehydrogenase). 5. Dilute only sufficient amounts of mlcrosomes for each experiment The remamder of the undiluted mlcrosomes can be stored at -80°C for future use (15). 6. The apparent Km value for cDNA-expressed CYP2B6-catalyzed 7-ethoxy-4trifluoromethylcoumarm O-deethylatlon is -3 @4 (4). The assay IS conducted at 5 @4 substrate concentration in order to faclhtate the efficacy of the antibodies against the higher Km forms such as CYPZC 19 (apparent Km = 17 @4), CYP2C9 (apparent Km = 35 @4), CYP2El (apparent Km = 46 CUM)and CYP2A6 (apparent Km >50 ClM). 7. Conduct preliminary experiments to ensure that the assay is linear with respect to microsomal protein concentration. 8 Conduct preliminary experiments to ensure that the assay 1slinear with respect to mcubatlon time. 9. The 7-ethoxy-4-trifluoromethylcoumarin U-deethylation assay can also be performed using a direct contmuous spectrofluorometnc method (13,14) 10. Determine the optimal excltatlon wavelength and emlsslon wavelength to be used with each particular spectrofluorometer by exammlng the excltatlon and emlssion spectra (14) of 7-ethoxy-4-trifluoromethylcoumarin and 7-hydroxy-4trifluoromethylcoumarin generated by that instrument. 11. The CYP2B6 component of human liver microsomal 7-ethoxy-4-tnfluoromethylcoumarin 0-deethylatlon activity at 5 pA4 substrate concentration m a panel of 17 individual human liver microsome samples ranged from 0 02-0.5 nmol/min/mg microsomal protein (4).

Acknowledgments Supported m part by the British Columbia Health Research Foundation (grant 119(95- 1) to Thomas K. H. Chang) and the National Institutes of Health

(grant CA49248 to David J. Waxman). Thomas K. H. Chang 1sthe recipient of a Research Career Award in the Health Sciences from the Pharmaceutical Manufacturers

Association

of Canada-Health

Research Foundation

and the

Medical Research Council of Canada. References 1. Mlmura, M , Baba, T., Yamazakl, H., Ohmon, S., Inui, Y., Gonzalez, F. J , Guengerich, F P., and Shimada, T (1993) Characterlzatlon of cytochrome P-450 2B6 in human liver microsomes. Drug Metab. Dispos 21, 1048-1056. 2. Shlmada, T., Yamazakl, H., Mimura, M., Inui, Y , and Guengench, F. P. (1994) Interindividual variations m human liver cytochrome P-450 enzymes involved m the oxidation of drugs, carcinogens and toxic chemicals: studies with liver mlcrosomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 270,414-423.

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121

3. Yamano, S., Nhamburo, P. T., Aoyama, T., Meyer, U. A., Inaba, T., Kalow, W , Gelboin, H. V., McBride, W. O., and Gonzalez, F. J. (1989) cDNA cloning and sequence and cDNA-directed expression of human P450IIB 1: identification of a normal and two variant cDNAs derived from the CYP2B locus on chromosome 19 and differential expression in human liver. Biochemistry 28, 7340-7348 4 Code, E. L., Crespi, C. L., Penman, B. W , Gonzalez, F J , Chang, T K H , and Waxman, D. J. (1997) Human cytochrome P450 2B6. intermdividual hepatic expression, substrate specificity and role in procarcmogen activation. Drug Metab. Dlspos

25,985-993.

5. Chang, T. K. H., Yu, L., Maurel, P., and Waxman, D. J. (1997) Enhanced cyclophosphamide and ifosfamide activation in primary human hepatocyte cultures: response to cytochrome P-450 inducers and automduction by oxazaphosphormes. Cancer Res 57,1946-l 954 6. Strom, S. C , Pisarov, L. A , Dorko, K., Thompson, M. T , Schuetz, J. D., and Schuetz, E. G. (1996) Use of human hepatocytes to study P450 gene induction. Methods Enzymol 272, 388-401. 7 Waxman, D. J., Lapenson, D. P., Aoyama, T , Gelbom, H. V , Gonzalez, F. J., and Korzekwa, K (199 1) Steroid hormone hydroxylase specificities of eleven cDNAexpressed human cytochrome P45Os Arch Blochem. Biophys 290, 160-166. 8 Chang, T K H., Weber, G. F , Crespi, C. L., and Waxman, D. J (1993) Differential activation of cyclophosphamide and ifosphamide by cytochromes P-450 2B and 3A m human liver microsomes. Cancer Res 53,5629-5637. 9. Shou, M., Korzekwa, K. R., Crespi, C. L., Gonzalez, F J., and Gelbom, H. V. (1994) The role of 12 cDNA-expressed human, rodent and rabbit cytochromes P450 in the metabolrsm of benzo[a]pyrene and benzo[a]pyrene trans-7,8-diol. Carcznogenesis 10, 159-l 68. 10 Shou, M., Korzekwa, K. R , Krausz, K. W., Crespi, C L , Gonzalez, F. J., and Gelbom, H. V. (1994) Regio- and stereo-selective metabohsm of phenanthrene by twelve cDNA-expressed human, rodent and rabbit cytochromes P-450. Cancer Lett 83,305-3 13. 11. Dehal, S. S. and Kupfer, D. (1994) Metabolism of the proestrogemc pesticide methoxychlor by hepatic P450 monooxygenases in rat and humans. Dual pathways mvolvmg novel ortho rmg-hydroxylation by CYP2B. Drug Metab Dispos. 22,937-946. 12. Imaoka, S., Yamada, T , Hiroi, T., Hayashi, K., Sakaki, T., Yabusaki, Y., and Funae, Y. (1996) Multiple forms of human P450 expressed in Saccharomyces cerevwae. Systematic characterization and comparison with those of the rat Blochem Pharmacol 51,1041-1050. 13 Buters, J. T. M., Schiller, C D , and Chou, R. C. (1993) A highly sensitive tool for the assay of cytochrome P450 enzyme activity in rat, dog and man’ direct fluorescence momtormg of the deethylation of 7-ethoxy%trifluoromethylcoumarm Biochem. Pharmacol 46, 1577-1584. 14. DeLuca, J. G., Dysart, G. R., Rasnick, D., and Bradley, M. 0. (1988) A direct, highly sensitive assay for cytochrome P-450 catalyzed O-deethylation using a novel coumarm analog. Blochem. Pharmacol. 37,173 l-l 739.

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15 Pearce, R E , McIntyre, C J , Madan, A., Sanzgiri, U , Draper, A. J , Bullock, P L , Cook, D C , Burton, L A , Latham, J., Nevins, C., and Parkinson, A (1996) Effects of freezmg, thawing, and storing human liver mlcrosomes on cytochrome P450 activity. Arch Bzochem Bzophys 331, 145-169

13 High-Performance Liquid Chromatographic Analysis of CYP2CSCatalyzed Paclitaxel Ga-Hydroxylation Charles L. Crespi, Thomas K. H. Chang, and David J. Waxman 1. Introduction CYP2C8 IS a major CYP2C protein expressed m human liver (1-l). Considerable interindividual differences (-20-fold) have been observed m hepatic CYP2C8 content (5) and a blmodal dlstrlbutlon in CYP2C8 protein amounts m a panel of human liver mlcrosomes has been reported (6). Experiments with primary cultures of human hepatocytes have indicated that CYP2C8 1ssubject to inductton by phenobarbital, dexamethasone, and rifampin (rifampicm) (4). Little is known about the function of CYP2C8, although studies with lmmunologically purified or cDNA-expressed CYP2C8 have indicated that this P450 catalyzesthe metabohsm of retmol(6), retinolc acid (6), arachldonic acid (7,8), carbamazepine (9) and paclitaxel (10-12). Oxldatlon of the anticancer drug paclitaxel to 6a-hydroxypachtaxel appears to be selectively catalyzed by CYP2C8 because cDNA-expressed human CYP2C8 1sactive m this reaction, whereas CYPlA2,2A6, 2B6,2C8, 2C9-Ile359, 2C9-Cys’44, 2C18, 2C19, 2D6, 2EI,3A3,3A4 and 3A5 are inactive (10-12). Paclitaxel Ga-hydroxylase actlvlty may therefore be a potentially useful dlagnostlc catalytic marker for human hepatic CYP2C8 (see Note 1). This chapter describes a high-performance liquid chromatographic (HPLC) assay for the determination of paclltaxel 6a-hydroxylase activity. 2. Materials 2.7. Assay

1. Substrate. paclltaxel (MW = 853.9) (see Notes 2 and 3) (Calblochem Biochemicals, LaJolla, CA). Preparea 5 mA4(4.3 mg/mL) stock solution dlssolved m ethanol. From Methods m Molecular Biology, Vol 107 Cytochrome P450 Protocols Edlted by I R PhIllIps and E A Shephard 0 Humana Press Inc , Totowa, NJ

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2. Metabolite standard: 6a-hydroxypaclitaxel (MW = 869.9) (see Note 4) (GENTEST, Woburn, MA). 3. Assay buffer: 100 mM potassium phosphate, pH 7.4. 4 Cofactor. 26 mM (20 mg/mL) NADP+ (see Note 5), 66 mM (20 mg/mL) n-glucose-6-phosphate, 66 mM (13.3 mg/mL) magnesium chloride (MgCl, * 6H,O). Glucose-6-phosphate dehydrogenase (40 U/mL) m 5 mM (1.5 mg/mL) sodium citrate (C6HS0,Na, * 2H,O) 5 Enzymes e.g., cDNA-expressed CYP2C8 (GENTEST, Woburn, MA) or human liver microsomes. Dilute m assay buffer to a workmg concentration of 5 mg protem/mL and keep on ice (see Note 6). 6 Deprotemtzing agent: 100% acetomtrile.

2.2. HPLC 1. Mobrle phase A 10% (v/v) methanol (see Note 7). 2. Mobile phase B: 100% methanol (see Note 7). 3. Column. Nucleostl Cl8 column, 4 6 x 250 mm, 5 pm particle size (Stgma, St. Louis, MO), (see Note 8). 4 Detector: ultraviolet (UV) at 230 nm

3. Methods 1 Add the following to each incubation tube (total mcubatron volume of 200 pL) a. 178 pL of assay buffer, b. 0.4 @ of 5 mM pachtaxel(l0 @4 final concentration, see Notes 9 and lo), c. 10 & of a solution contammg 26 mM NADP+, 66 mA4 o-glucose-6-phosphate, and 66 miM magnesium chloride, d. 2 ltL of glucose-6-phosphate dehydrogenase (40 U/mL) in 5 mM sodium citrate 2 Prewarm incubation tubes to 37°C and add 10 pL of dtluted enzymes (50 pg protein) to initiate enzymatic reactton (stagger each incubation with 15-s delay mtervals) (see Notes 11 and 12). Incubate samples at 37’C for 30 mm m a water bath (see Note 13). Add 50 pL Ice-cold 100% acetomtrile to stop enzymattc reactton and place mcubatton tube on ice. Centnfuge reaction mixture at 12,000g for 4 mm. Inject 50-150 JJL of supernatant onto HPLC column Run column at 45°C (see Note 14) at a flow rate of 1 mL/mm with a linear gradient of 45% mobile phase A, 55% mobile phase B to 35% phase A, 65% phase B over 20 mm, then for a further 5 mm with 35% phase A, 65% phase B Under these condrtions retention times are 22 mm for 6a-hydroxypaclitaxel and 25.5 min for paclitaxel 8. Prepare blank incubation tubes by addmg the complete mcubatron mixture but with heat-inactivated enzymes. Process the blank incubation tubes as per steps 3-7.

PacMaxel6a-Hydroxylation

Assay

125

9. Prepare standards by adding a known amount (e.g., 0.1, 0.2, 0 5, 1 nmol) of authentic 6a-hydroxypaclttaxel metabolrte to tubes containing the complete mcubatton mixture but with heat-inactivated enzymes (see Note 15) Process the incubation tubes containing the standards as per steps 3-7 10. Plot a standard curve of the peak area (or peak height) of metabolite standard vs amount of authentic standard added. Determine the amount of product formatron in each unknown sample by linear regression analysts 11. Calculate paclitaxel Go-hydroxlase activity and express it as nmol product formed/ min/mg microsomal protein or as nmol product formed/mm/nmol total P450 12. Calculate net enzyme activity by subtracting the acttvrty in the blank from each of the unknown samples (see Note 16).

4. Notes 1. Exercise cautron when utilizing hepatic microsomal paclitaxel 6a-hydroxylase activity as a diagnostic catalytic marker for CYP2C8 m human liver because tt has not yet been confirmed that paclitaxel 6a-hydroxylation IS exclustvely catalyzed by CYP2C8 in human liver. This determinatton would require the development of CYP2C8-specific mhtbltory antibodies or CYP2C8-spectfic chemical inhibrtors. 2. Store pachtaxel at 4°C Pachtaxel is light-sensmve 3. Paclitaxel solutton may be stored up to 1 yr at -20°C. A significant amount of the drug adsorbs to glass, polypropylene, and polystyrene containers (13). 4. Storage of solid and solutions of 6a-hydroxypaclitaxel metabolite standard at G-20°C is recommended. A methanol solution of 6a-hydroxypaclitaxel stored at room temperature shows -30% degradation after 14 d. No degradation of an acetonitrile solution or the dry solid is observed under the same room-temperature condttron. 5. Paclitaxel 6cL-hydroxylase assays can also be carried out with NADPH (e g , 1 nnt4 final concentration) instead of an NADPH-generatmg system (e.g , NADP+, o-glucose-6-phosphate, glucose-6-phosphate dehydrogenase) 6. Prepare only sufficient amounts of dilute mtcrosomes for each experiment. The remainder of the undiluted mrcrosomes can be stored at -80°C for future use (14). 7. Mobile phases containing acetonitrile may be used instead of methanol, but this requires admstment of mobile phase gradient condrtions. 8. Paclitaxel and 6a-hydroxypachtaxel can be separated on C 18 columns purchased from other manufacturers, but this may require admstment of mobile phase gradient conditions. 9. Paclitaxel concentrations of >20 @4 result in decreased metabolite formatton by human liver mrcrosomes (II). 10. The apparent Km for pachtaxel6a-hydroxylation by cDNA-expressed CYP2C8 and by human liver microsomes is -5 pJ4 (IO). 11. With some P450 protein expression systems, it is necessary to reconstitute the recombinant P450 protein with NADPH-cytochrome P450 reductase, cytochrome

126

12 13. 14

15.

16

Crespi, Chang, and Waxman b,, and lipid before mitiatmg substrate oxidation (3). Conduct preliminary experiments to establish the amount of each component required for optimal catalytic activity. Conduct preliminary experiments to ensure that the assay IS linear with respect to microsomal protein concentration. Conduct prehmmary experiments to ensure that the assay is lmear with respect to incubation time. Column temperature can range from room temperature to 50°C. The use of a controlled, elevated temperature provides greater reproducibility m retention times and lower column back pressures The final concentration of 6a-hydroxypachtaxel metabohte standard can be determined by absorbance at 230 nm using the molar extmction coefficient 28.2 cm-‘mA&’ After determination of absorbance, dilute standard to 50% methanol/ 50% water before use. Diluted standard can be stored at -20°C. The median pachtaxel 6a-hydroxylase activity in a panel of 49 human liver microsomes was 1.09 nmol/h/mg protein at 20 pA4 substrate concentration (II).

Acknowledgments Supported m part by the British Columbia Health Research Foundation (grant 02(97-l) to Thomas K. H. Chang) and the National Institutes of Health (grant CA49248 to David J. Waxman). Thomas K. H. Chang is the recipient of

a Research Career Award m the Health Sciences from the Pharmaceutical Manufacturers

Association

of Canada-Health

Research Foundation

and the

Medical Research Council of Canada. References 1 Romkes, M , Faletto, M B , Blaisdell, J A , Raucy, J L., and Goldstem, J A (1991) Cloning and expression of complementary DNAs for multiple members of the human cytochrome P45OIIC subfamily Blochemutry 30,3247-3255 2 Wrighton, S. A., VandenBranden, M., Stevens, J. C., Shipley, L. A , and Ring, B. J. (1993) In vitro methods for assessmg human hepatic drug metabohsm: their use m drug development. Drug Metab Rev 25,453-484. 3 Goldstein, J A., Faletto, M. B., Ron&es-Sparks, M , Sullivan, T , Kitareewan, S , Raucy, J L , Lasker, J M., and Ghanayem, B I (1994) Evidence that CYP2C19 ts the maJor (S)-mephenytom 4-hydroxylase m humans Blochemzstry 33,1743-l 752 4 Chang, T K. H , Yu, L , Maurel, P., and Waxman, D. J (1997) Enhanced cyclophosphamide and ifosfamide activation in primary human hepatocyte cultures. response to cytochrome P-450 inducers and autoinduction by oxazaphosphormes Cancer Res. 57,1946-1954. 5. Wrighton, S A, Thomas, P E., Willis, P , Mames, S L., Watkins, P. B , Levm, W., and Guzelran, P S. (1987) Purification of a human hver cytochrome P-450 immunochemically related to several cytochromes P-450 purified from untreated rats. J. Clan Invest. 80, 1017-1022.

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6. Leo, M A., Lasker, J. M., Raucy, J. L., Kim, C. I., Black, M., and Lieber, C. S (1989) Metabolism of retmol and retinoic acid by human liver cytochrome P4501IC8. Arch. Blochem Blophys 269,305-312. 7. Daikh, B E., Lasker, J M., Raucy, J. L , and Koop, D. R. (1994) Regio- and stereoselective epoxidation of arachidonic acid by human cytochromes P450 2C8 and 2C9. J. Pharmacol. Exp Ther 271,1427-1433. 8 Ritkmd, A. B., Lee, C , Chang, T. K. H , and Waxman, D J. (1995) Arachidomc acid metabolism by human cytochrome P45Os 2C8, 2C9, 2E1, and lA2. Regioselective oxygenation and evidence for a role for CYP2C enzymes m arachidonic actd epoxygenatron m human liver mtcrosomes. Arch. Bzochem Blophys. 320,38&389 9 Kerr, B. M., Thummel, K. E , Wurden, C. J , Klein, S. M., Kroetz, D L., Gonzalez,

10

11

12

13. 14

F J., and Levy, R. H. (1994) Human hver carbamazepme metabolism. Role of CYP3A4 and CYP2C8 m lO,ll-epoxide formation. Bzochem Pharmacol 47, 1969-1979 Rahman, A., Korzekwa, K R., Grogan, J , Gonzalez, F. J , and Harris, J. W (1994) Selective biotransformation of taxol to 6a-hydroxytaxol by human cytochrome P450 2C8 Cancer Res 54,5543-5546. Sonrnchsen, D. S., Lm, Q., Schuetz, E. G , Schuetz, J. D., Pappo, A., and Rellmg, M. V (1995) Variability m human cytochrome P450 paclitaxel metabolism. J Pharmacol Exp. Ther 275,566-575. Rrchardson, T. H , Jung, F , Griffin, K. J., Wester, M., Raucy, J. L., Kemper, B , Bornheim, L. M , Hassett, C., Omtecinskr, C. J., and Johnson, E. F (1995) A universal approach to the expression of human and rabbit cytochrome P45Os of the 2C subfamily m Escherlchla colz. Arch Blochem Blophys 323, 87-96 Song, D , Hsu, L F., and Au, J L. S. (1996) Bmding of Taxol to plastic and glass containers and protein under in vitro condmons J Pharm Scl 85,29-3 1 Pearce, R. E , McIntyre, C J., Madan, A , Sanzgni, U., Draper, A J , Bullock, P L , Cook, D. C., Burton, L A., Latham, J , Nevms, C., and Parkmson, A. (1996) Effects of freezing, thawing, and storing human liver microsomes on cytochrome P450 activity. Arch Blochem Bzophys 331, 145-169

Determination of CYP2CSGatalyzed Diclofenac 4’-Hydroxylation by High-Performance Liquid Chromatography Charles L. Crespi, Thomas K. H. Chang, and David J. Waxman 1. Introduction CYP2C9 is a major CYP2C enzyme expressed m human liver (I-3). Recent experiments with primary cultures of human hepatocytes have suggested that CYP2C9 protein expression can be increased m cells treated with a P450 inducer such as phenobarbital, dexamethasone, or rlfampin (rifampicin) (3). This is consistent with the clmical finding that rlfampin increases the total body clearance of tolbutamide (4), which is metabolized almost entirely by hepatic CYP2C9 (5). Other substrates for CYP2C9 include phenytom (5,6), warfarin (7) and diclofenac (8). Sulfaphenazole is a CYP2C9-selective chemical mhlbitor (9-12) and inhibition experiments with this sulfur-containing compound have suggested that human liver microsomal diclofenac 4’-hydroxylatlon IS selectively catalyzed by CYP2C9 (8). This chapter describes a high-performance liquid chromatographic (HPLC) assay for the determination of diclofenac 4’-hydroxylase activity. Methods for other CYP2C9 assayssuch as tolbutamide methylhydroxylase (13) and (S)-warfarin 7-hydroxylase (14,15) can be found in the cited references. 2. Materials 2.1. Assay 1 Assay buffer: 100 mMTris-HCl, pH 7.5. 2. Substrate: dlclofenac (sodium salt, MW = 318 1) (Sigma, St Louis, MO) Prepare a 0.4 mA4 (0.127 mg/mL) stock solution dissolved m assay buffer (see Note 1) 3 Metabolite standard: 4’-hydroxydiclofenac (GENTEST, Woburn, MA) From Methods m Molecular Bology, Vol 107. CytOChrOm8 P450 Protocols Edlted by I R Phllllps and E A Shephard 0 Humana Press Inc , Totowa, NJ

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4 Cofactor generating system* 26 mM (20 mg/mL) NADP+ (see Note 2), 66 mM (20 mg/mL) o-glucose-6-phosphate, 66 mA4 (13 3 mg/mL) magnesmm chloride (MgCl, 6HzO) Glucose-6-phosphate dehydrogenase (40 U/mL) m 5 mM (1.5 mg/mL) sodium citrate (C,H,O,Na, * 2H,O). 5 Enzymes: e.g , cDNA-expressed CYP2C9 (GENTEST, Woburn, MA) or human liver microsomes. Dilute m assay buffer to a working concentration of 2.5 mg protein/ml and keep on ice (see Note 3) 6 Deprotemizing agent 94% acetomtrtle/6% glacial acetic acid.

2.2. HPLC 1 Mobile phase A. 30% acetomtrile, 70% water, 2 tiperchloric acid (m water) (see Note 4). 2 Mobile phase B* 100% methanol 3 Column. Nucleostl Cl8 column, 4 6 x 250 mm, 5-pm particle size (Sigma, St Louis, MO) (see Note 5) 4. Detector. ultraviolet (UV) at 280 nm

3. Methods 1 Add the following to each mcubation tube (total mcubation volume of 200 uL)* a. 118 pL of assay buffer, b 50 pL of 0.4 mM diclofenac (100 @4 final concentration, see Note 6), c. 10 pL of a solution containing 26 rnA4 NADP+, 66 mM n-glucose-6-phosphate and 66 mM magnesium chloride, d. 2 pL of glucose-6-phosphate dehydrogenase (40 U/mL) m 5 mM sodium citrate 2 Prewarm mcubation tubes to 37’C and add 20 pL of diluted enzymes (50 pg protein) to initiate the enzymatic reaction (stagger each incubation with 15-s delay intervals) (see Notes 7 and 8) 3 Incubate samples at 37°C for 20-30 min (see Note 9) in a water bath 4 Add 50 pL of a mixture of 94% acetonitrile/6% glacial acetic acid to stop enzymatic reaction, and place mcubation tube on ice 5. Centrifuge reaction mixture at 12,000g for 4 mm 6. Inject 50 to 150 $ of the supernatant onto HPLC column. 7 Run column at 45-50°C (see Note 10) at a flow rate of 1 mL/mm with a lmear gradient from 70% mobile phase A, 30% mobile phase B to 100% phase B over 20 min Under these conditions retention times are 11 mm for 4’-hydroxydiclofenac and 15 mm for dtclofenac. 8. Prepare blank incubation tubes by adding the complete incubation mixture but with heat-mactivated enzymes Process the blank incubation tubes as per steps 3-7. 9. Prepare standards by adding a known amount (e.g., 0, 1,3,6, 12 nmol) of authenttc 4’-hydroxydiclofenac metabohte to tubes contammg the complete mcubation mixture but with heat-inactivated enzymes Process the standard mcubation tubes as per steps 3-7.

Diclofenac

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10. Plot a standard curve of the peak area (or peak height) of metabohte standard vs amount of authentic standard added. Determme the amount of product formation m each unknown sample by linear regressron analysts I 1. Calculate dtclofenac 4’-hydroxylase activity and express it as nmol product formed/ min/mg microsomal protein or as nmol product formed/mm/nmol total P450 12. Calculate net enzyme activity by subtracting the activity m the blank from each of the unknown samples.

4. Notes 1. Dtclofenac solutions are stable for at least 1 mo when stored at SC (26) 2. Dtclofenac 4’-hydroxylase assays can also be carried out with NADPH (e.g., 1 mM final concentration) instead of an NADPH-generating system (e g , NADP+, n-glucose-6-phosphate, glucose-6-phosphate dehydrogenase) 3. Prepare only sufficient amounts of dilute mtcrosomes for each experiment. The remainder of the undiluted microsomes can be stored at -80°C for future use (17). 4. Diclofenac and 4’-hydroxydiclofenac are chromatographed at acidic pH with the carboxylic acid group protonated. Separation can also be achieved at sltghtly alkaline pH (pH 7 4) when the carboxyhc acid group is ionized, but this may adversely affect column life 5. Dtclofenac and 4’-hydroxydiclofenac can be separated on other brands of C 18 columns, but this may require admstment of mobile phase gradient conditions. 6. A final concentration of 100 l.&f dtclofenac in the incubatton mixture IS a saturatmg substrate concentration because the apparent Km for diclofenac 4’-hydroxylatton catalyzed by human liver microsomes IS -6 @4 (8) 7. With some P450 protein expression systems (18), it is necessary to reconstitute the recombinant P450 protein with NADPH-cytochrome P450 reductase, cytochrome b5, and lipid before mttiatmg substrate oxidatton Conduct preliminary experiments to establish the amount of each component required for optimal catalytic activity 8 Conduct preliminary experiments to ensure that the assay is linear with respect to microsomal protein concentration. 9. Conduct preliminary experiments to ensure that the assay is linear with respect to incubation time. 10. Column temperature can range from room temperature to 50°C. The use of a controlled, elevated temperature provides greater reproducibility m retention times and lower column back pressures

Acknowledgments Supported in part by the Brrtrsh Columbia Health Research Foundatron (grant 119(95- 1) to Thomas K. H. Chang) and the National Instttutes of Health (grant CA49248 to David J. Waxman). Thomas K. H. Chang 1s the rectprent of a Research Career Award m the Health Sciences from the Pharmaceutical Manufacturers Assoclatton of Canada-Health Research Foundatton and the Medical Research Council of Canada.

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References 1. Romkes, M., Faletto, M B., Blaisdell, J. A., Raucy, J L., and Goldstein, J. A (1991) Clonmg and expression of complementary DNAs for multiple members of the human cytochrome P45OIIC subfamily. Bzochemrstry 30,3247-3255 2 Wrighton, S A , VandenBranden, M., Stevens, J. C., Shipley, L A., and Ring, B. J (1993) In vztro methods for assessmg human hepattc drug metabohsm Their use m drug development. Drug Metab Rev 25,453-484 3. Chang, T. K. H , Yu, L., Maurel, P , and Waxman, D J. (1997) Enhanced cyclophosphamtde and tfosfamtde acttvation m primary human hepatocyte cultures: Response to cytochrome P-450 inducers and automduction by oxazaphosphormes Cancer Res. 57, 1946-l 954. 4 Ztlly, W., Breimer, D D., and Richter, E. (1975) Induction of drug metabohsm m man after rifampicm treatment measured by increased hexobarbttal and tolbutamtde clearance. Eur. J. Clan Pharmacol 9,219-227. 5. Doecke, C. J., Veronese, M. E , Pond, S. M., Miners, J. O., Btrkett, D. J., Sansom, L. N., and McManus, M. E. (1991) Relationship between phenytom and tolbutamtde hydroxylations in human liver microsomes. Br J Clw Pharmacol 31, 125-130 6 Veronese, M. E., MacKenzie, P. I., Doecke, C J , McManus, M E., Miners, J 0 , and Birkett, D J (1991) Tolbutamide and phenytom hydroxylations by cDNAexpressed human liver cytochrome P450 2C9 Biochem Brophys Res Commun 175, 1112-1118. 7 Rettie, A E , Korzekwa, K R., Kunze, K. L , Lawrence, R. F , Eddy, A C , Aoyama, T., Gelboin, H. V., Gonzalez, F. J., and Trager, W. F. (1992) Hydroxylation of warfarm by human cDNA-expressed cytochrome P-450 A role for P-450 2C9 m the ettology of (S)-warfarm drug Interactions Chem Res Tox~ol 5,54-59. 8. Leemann, T., Transon, C., and Dayer, P. (1992) Cytochrome P450TB (CYP2C) a major monooxygenase catalyzmg diclofenac 4’-hydroxylation in human liver Life Scr 52,29-34. 9 Baldwin, S. J., Bloomer, J C., Smith, G. J., Ayrton, A. D , Clarke, S. E., and Chenery, R J (1995) Ketoconazole and sulphaphenazole as the respective selective mhibttors of P4503A and 2C9. Xenobzotzca 25,261-270. 10 Newton, D J., Wang, R. W., and Lu, A Y. H (1995) Cytochrome P450 mhtbitors: evaulation of specificities m the zn vztro metabolism of therapeutic agents by human liver microsomes Drug Metab Dlspos 23, 154-l 58 11. Bourrie, M., Meunier, V., Berger, Y , and Fabre, G. (1996) Cytochrome P450 tsoforrn mhtbitors as a tool for the mvestigation of metabolic reactions catalyzed by human liver microsomes J Pharmacol. Exp Ther 277,321-332. 12. Ono, S., Hatanaka, T., Hotta, H , Satoh, T., Gonzalez, F J , and Tsutsui, M. (1996) Spectfictty of substrate and mhibitor probes for cytochrome P45Os evaluation of in vitro metabolism using cDNA-expressed human P45Os and human liver microsomes. Xenobiotica 26,68 l-693.

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13 Miners, J 0. and Birkett, D J (1996) Use of tolbutamide as a substrate probe for human hepattc cytochrome P450 2C9. Methods Enzymol 272, 139-145. 14 Kaminsky, L S., Fasco, M J , and Guengerich, F. P. (1981) Productton and application of antibodies to rat liver cytochrome P-450. Methods Enzymol 74, 262-273 15 Lang, D. and Backer, R (1995) Highly sensttive and specific high-performance liquid chromatographic analysts of 7-hydroxywarfarm, a marker for human cytochrome P-4502C9 activity J Chromatogr 672,305-309 16 Godbtllon, J., Gauron, S., and Metayer, J. P. (1985) High-performance hqmd chromatographrc determmatton of diclofenac and tts monohydroxylated metabolites m biologtcal fluids. J. Chromatogr. 338, 15 1-159 17 Pearce, R E., McIntyre, C. J , Madan, A., Sanzgiri, U , Draper, A J , Bullock, P L., Cook, D. C , Burton, L. A , Latham, J , Nevms, C , and Parkinson, A. (1996) Effects of freezing, thawing, and storing human liver microsomes on cytochrome P450 activity. Arch. Biochem Bzophys 331, 145-169 18 Goldstein, J. A., Faletto, M. B., Romkes-Sparks, M., Sulhvan, T., Knareewan, S., Raucy, J. L , Lasker, J M., and Ghanayem, B. I. (1994) Evidence that CYP2C 19 1sthe major (S)-mephenytom 4’-hydroxylase m humans. Bzochemzstry 33,1743-1752.

15 CYP2Cl g-Mediated (S)-Mephenytoin 4’-Hydroxylation Assayed by High-Performance Liquid Chromatography with Radiometric Detection Charles L. Crespi, Thomas K. H. Chang, and David J. Waxman 1. Introduction CYP2C 19 is one of at least three CYP2C enzymes expressed in human hver (1,2), although the abundance of this P450 is generally less than that of etther CYP2C8 or CYP2C9 (3). Considerable interindividual differences occur in hepatic CYP2C19 content (1,2), primarily owmg to a genetic polymorphism m this enzyme (4). Approximately 15-20% of Orientals and 3% of Caucasians exhibit the CYP2C19 poor-metabolizer phenotype, which is associated with at least two mutant alleles, designated CYP2C 19ml and CYP2C 19m2 (4). Studies with cDNA-expressed CYP2C 19 have identified several drugs as substrates for this polymorphically expressed P450, including omeprazole (5,6), diazepam (7), cyclophosphamlde (3), and lfosfamide (3). However, the precise role of human hepatic CYP2C19 in drug biotransformation is poorly understood because mhibitory monospecific antiCYP2C19 antibodies are not available and a CYP2C19-specific chemical inhibitor has yet to be identified. Recent correlational analyses have led to the conclusion that CYP2C19 is a major P450 responsible for (S)-mephenytoin 4’-hydroxylation activity in human liver microsomes (1,2). This is supported by the finding that cDNA-expressed CYP2C19 has a 50- to lOO-fold greater turnover number than CYP2C8, CYP2C9, or CYP2C 18 for (S)-mephenytoin 4’-hydroxylatlon (2). As a result, (S)-mephenytom 4’-hydroxylase activity is frequently used as a catalytic monitor for human hepatic CYP2C19. In addmon, (S)-mephenytom 4’-hydroxylation is a useful indicator of the catalytic activity of cDNA-expressed CYP2C 19 From Methods m Molecular Bology, Vol 107 Cytochrome P450 Protocols Edlted by I R Phllllps and E A Shephard 0 Humana Press Inc , Totowa, NJ

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(2). Varrous analytical methods have been developed to measure the enzymatic formation of 4’-hydroxymephenytoin, including isocratrc, reversed-phase hrgh performance hqutd chromatography (8). This chapter describes a method for the determination of (S)-mephenytoin 4’-hydroxylation by gradient, reversedphase high performance llqurd chromatography using 14C-labeled substrate and radiometrrc detection.

2. Materials 2.1. Assay 1. Substrate: [i4C]-(S)-mephenytoin (Amersham, Arlington Heights, IL). (S)mephenytom (MW = 2 18.3) m methanol (Salford Ultrafine Chermcals, Manchester, UK) (available from GENTEST, Wobum, MA) Prepare a 10 mA4 (2 18 mg/mL) stock solution of unlabeled (S)-mephenytoin in methanol (see Notes 1 and 2). Evaporate the ethanol from the 14C-(S)mephenytoin under vacuum or under a stream of nitrogen. Redissolve the residue m methanol at a final concentration of 10 &. Add unlabeled (S)-mephenytoin to give a 10 mM working solution at 5 mCi/mmol. 2 Metabolite standards: 4’-hydroxymephenytom (MW = 234.3) Nuvanol (MW = 204.2). (Both available from Salford Ultrafine Chemicals and GENTEST ) 3. Assay buffer: 100 tipotassium phosphate, pH 7.4. 4 Cofactor generating system: 26 mM (20 mg/mL) NADP+ (see Note 3), 66 mA4 (20 mg/mL), n-glucose-6-phosphate, 66 mM (13 3 mg/mL) magnesium chloride WgCl, * 6H2O) Glucose-6-phosphate dehydrogenase (40 U/mL) m 5 mA4 (1.5 mg/mL) sodium citrate (&H,O,Na, +2H,O) 5 Enzymes. e.g., cDNA-expressed CYP2C19 (GENTEST, Woburn, MA) or human liver microsomes Dilute m assay buffer to a working concentration of 5 mg protein/ml and keep on rce (see Note 4). 6 Deproteinizing agent. 100% acetomtrile. 2.2. HPLC 1. Mobile phase A: 10% (v/v) methanol (see Note 5) 2. Mobile phase B: 100% methanol (see Note 5). 3 Column: Nucleosil Cl8 column, 4.6 x 250 mm, 5-pm particle size (Sigma, St. Louis, MO) (see Note 6) 4. Scintillation fluid: Ultima Flo M (Packard Instruments, Downers Grove, IL). 5 Detector: radiometrtc-flow scintillation detector (Packard).

3. Methods 1. Add the followmg to each incubation tube (total mcubation volume of 200 pL)* a. 166 pL of 100 mM assay buffer, b. 2 pL of 10 w(S)-mephenytoin (100 pi!4 final concentration) (prepared as described m Subheading 2.1., item 1) (see Note 7),

(S)-Mephenytoin

2.

3. 4. 5. 6 7.

8. 9

4’-Hydroxylation

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137

c. 10 $ of a solution containing 26 rnA4NADP+, 66 mMn-glucose-6-phosphate and 66 mA4 magnesium chloride, d. 2 pL of glucosed-phosphate dehydrogenase (40 U/mL) in 5 mM sodium citrate. Prewarm incubation tubes to 37°C and add 20 pL of diluted enzymes (100 pg protein) to initiate the enzymatic reaction (stagger each incubation with 15-s delay intervals) (see Notes 8 and 9). Incubate samples at 37OC for 45-60 mm m a water bath (see Note 10). Add 50 pL of 100% acetomtrile to stop enzymatic reaction and place incubation tube on ice. Centrifuge reaction mixture at 12,000g for 5 mm. Inject 50-150 pL of supernatant onto HPLC column. Run column at 45-50°C (see Note 11) at a flow rate of 1 mL/min with a lmear gradient from 80% mobile phase A, 20% mobile phase B to 100% phase B over 10 min, then for a further 10 mm with 100% phase B. Under these conditions retention times are 9 mm for 4’hydroxymephenytom (see Note 12) and 16 mm for (S)-mephenytom. Prepare blank mcubation tubes by adding the complete mcubation mixture but with heat-inactivated enzymes Process the blank mcubation tubes as per steps 3-7 Quantification is achieved using a flow-scintillation detector using scintillation fluid at a flow rate of 3 mL/mm with a 0.5-mL flow cell. Countmg efficiency should be determmed using a known quantity of 14C with scmtillation fluid and HPLC mobile phase m the same proportions as will be present during an analytical run (final volume 20-50 mL) (see Note 13). Using the scmtillation-fluid pump, fill the flow cell of the detector with this mixture. Measure the radioactivity (cpm) The counting efficiency is given by cpm observed divided by the dpm present in 0.5 mL (the volume of the flow cell) Activity is calculated according to the followmg equation. ([dpm metabohte] x [total volume of mcubation plus stop addition])/ ([countmg efficiency] x (2200 dpm/nCi] x (specific activity in nCi/nmole] x [mg protein] x time x [volume injected for analysis])

10. Calculate (S)-mephenytom 4’-hydroxylase activity and express it as nmol product formed/min/mg microsomal protein or as mnol product formed/min/nmol total P450. 11 Calculate net enzyme activity by subtractmg the activity in the blank from each of the unknown samples

4. Notes 1. Ethanol is much more inhibitory to CYP2C 19 than IS methanol. Ethanol should be avoided as a solvent 2. Stock solution of (S)-mephenytoin can be stored up to 1 yr at -20°C. 3 (S)-mephenytoin 4’-hydroxylase assays can also be carried out with mcotmamide adenme dinucleotide phosphate (NADPH) (e.g., 1 mM final concentration) instead of an NADPH-generating system (e.g., NADP+, D-glucose-6-phosphate, glucose-6-phosphate dehydrogenase).

Crespi, Chang, and Waxman

738 4 5

6.

10. 11

12

13

Prepare only sufficient amounts of dilute microsomes for each experiment The remainder of the undiluted mlcrosomes can be stored at -80°C for future use (9) Mobile phases containing acetonitrile may be used instead of methanol, but this requires adjustment of mobile-phase gradient conditions. However, the 4’hydroxymephenytom peak is often broad and poorly defined with many Cl8 HPLC columns. This problem can become more pronounced as a column ages with usage The method described here sharpens the 4’-hydroxymephenytoin peak by incorporating a rapid gradient. However, the combmatlon of the methanol/ water mobile phase and the need to use 205 mn wavelength for optimal ultraviolet (UV) detection preclude assay quantlficatlon by absorbance detectlon. If absorbance detection 1s preferred, an acetomtnle/water mobile phase system should be used. Also, use of an alternative HPLC column (Jsphere ODS-L80, cat no. JL080s04-2546WT, YMC, Wilmington, NC) can provide improved metabohte peak shapes. (S)-mephenytoin and 4’-hydroxymephenytom can be separated on Cl 8 columns from other manufacturers, but this may require adJustment of mobile-phase gradient condltlons to optimize resolution. In a panel of 14 mdlvrdual human liver m;crosome samples, the average apparent Km value for (S)-mephenytom 4’-hydroxylatlon IS - 100 fl(10) Conduct preliminary experiments to ensure that the assay IS linear with respect to microsomal protem concentration. With CYP2C 19 expressed m yeast and some other expression systems, it 1s necessary to reconstitute the recombinant P450 protein with NADPH-cytochrome P450 reductase, cytochrome b,, and lipid before mltltatlon of substrate oxldatlon (2) Conduct prehmmary experiments to establish the amount of each of these required for optlmal catalytic actlvlty Conduct preliminary experiments to ensure that the assay 1slinear with respect to incubation time. Column temperature can range from room temperature to 5O“C. The use of a controlled, elevated temperature provides greater reproduclblhty in retention times and lower column back pressures The other myor, ldentlfied metabohte from (S)-mephenytom m mrvanol The HPLC mobility of mrvanol under the specific chromatography conditions should be verified to ensure that It does not cochromatograph with 4hydroxymephenytom Counting efficiency should be determined m the presence of each of several different mobile phase solvents because quenching may vary based on solvent composition

Acknowledgments Supported in part by the British Columbia Health Research Foundation (grant 119(95-l) to Thomas K. H. Chang) and the National Institutes of Health (grant CA49248 to David J. Waxman). Thomas K. H. Chang is the recipient of

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a Research Career Award m the Health Sciences from the Pharmaceutical Manufacturers Assoclatlon of Canada-Health Research Foundation and the Medical Research Council of Canada. References 1. Wrighton, S. A, Stevens, J. C , Becker, G. W , and VandenBranden, M (1993) Isolation and characterization of human hver cytochrome P450 2C 19 correlation between 2C 19 and S-mephenytom 4’-hydroxylation. Arch Blochem Btophys 306, 240-245. 2 Goldstein, J. A., Faletto, M. B., Romkes-Sparks, M , Sullivan, T , Kltareewan, S., Raucy, J. L , Lasker, J M , and Ghanayem, B. I (1994) Evidence that CYP2C19 1s the maJor (S)-mephenytom 4’-hydroxylase m humans Bzochemzstry 33, 1743-1752. 3. Chang, T K. H., Yu, L , Goldstein, J A., and Waxman, D. J (1997) Identification of the polymorphlcally expressed CYP2C19 and the wild-type CYP2C9-Ile359 allele as low-Km catalysts of cyclophosphamide and ifosfamlde activation. Pharmacogenetzcs 7,2 1 l-22 1 4. Goldstein, J. A. and de Morals, S. M. F. (1994) Bzochemzstry and molecular blology of the human CYP2C subfamily Pharmacogenetzcs 4,285--299. 5. Karam, W G , Goldstein, J A., Lasker, J. M., and Ghanayem, B I (1996) Human CYP2C19 1s a major omeprazole 5-hydroxylase, as demonstrated with recomblnant cytochrome P450 enzymes. Drug Metab DLSPOS 24,1081-1087 6 Ibeanu, G. C , Ghanayem, B I., Lmko, P , Ll, L , Pedersen, L G , and Goldstein, J A. (1996) Identification of residues 99, 220, and 221 of human cytochrome P450 2C 19 as key determinants of omeprazole hydroxylase activity J Blol Chem 271, 12,496-12,501. 7 Jung, F., Richardson, T H , Raucy, J L., and Johnson, E. F (1997) Dlazepam metabolism by cDNA-expressed human 2C P45Os. ldentlficatlon of P450 2C 18 and P450 2C19 as low Km dlazepam N-demethylases. Drug Metab Dlspos 25, 133-139 8 Chlba, K , Manabe, K., Kobayashl, K., Takayama, Y , Tam, M , and Ishizakt, T (1993) Development and preliminary application of a simple assay of Smephenytom 4-hydroxylase activity in human liver mlcrosomes. Eur. J Clan Pharmacol. 44,559-562. 9. Pearce, R E., McIntyre, C J., Madan, A., Sanzgiri, U , Draper, A. J , Bullock, P L , Cook, D. C., Burton, L. A., Latham, J., Nevins, C , and Parkinson, A. (1996) Effects of freezing, thawing, and stormg human liver mlcrosomes on cytochrome P450 actlvlty. Arch Biochem Bzophys. 331, 145-169. 10. Chiba, K., Kobayashi, K , Manabe, K , Tani, M , Kamatakl, T , and Ishlzakl, T (1993) Oxidatlve metabolism of omeprazole in human liver microsomes: cosegregation with S-mephenytom 4’-hydroxylation. J Pharmacol Exp. Ther 266,52-59.

16 CYP2D6-Dependent Bufuralol 1‘-Hydroxylation Assayed by Reversed-Phase Ion-Pair High-Performance Liquid Chromatography with Fluorescence Detection Charles L. Crespi, Thomas K. H. Chang, and David J. Waxman 1. Introduction CYP2D6 is an enzyme that is subject to genetic polymorphtsm (I). This P450 is absent in individuals with the poor metabohzer CYP2D6 phenotype (2,3), resulting in a substantially reduced capacity to metabolize a large number of clinically useful drugs, including metoprolol, propafenone, haloperrdol, dextromethorphan and codeine (I). Quinidine competrtively inhibits CYP2D6, with a K, of 3-30 nA4(3-5). Because of its preferential inhibition of CYP2D6 (6,7), quinidine is frequently used as an inhibitory chemical probe of this polymorphically expressed P450. Diagnostic catalytic monitors of human hepatic CYP2D6 include debrisoquine 4-hydroxylase (4), dextromethorphan Odemethylase (8), and bufuralol I’-hydroxylase activities (24). Advantages of using bufuralol as a CYP2D6-selective substrate include the high sensitivity of the assay owing to the highly fluorescent l’-hydroxybufuralol metabohte and the fact that the use of radiolabeled substrateis not required. This chapter describes a modification of a reversed-phase ton-pan high-performance hqurd chromatographic (HPLC) assay (9) with fluorescence detection for the determmation of bufuralol I’-hydroxylation activity. Methods for other CYP2D6 assayssuch as debrisoquine 4-hydroxylase (9) and dextromethorphan 0-demethylase (9,ZO) can be found in the cited references.

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2. Materials 2.1. Assay 1 Substrate. (+/-) bufuralol HCl (MW = 297 8) (see Note 1) (Salford Ultrafine Chemicals, Manchester, UK) (available from GENTEST (Woburn, MA) Prepare a 1 mM (0 3 mg/mL) stock solution of bufuralol HCl dissolved m assay buffer (see item 3) (see Note 2) 2 Metabollte standard. l’-hydroxybufuralol maleate (MW = 393 4) (Salford Ultrafine Chemicals, available from GENTEST) 3 Assay buffer 100 mA4 potassium phosphate, pH 7.4 4 Cofactor generating system 26 mM (20 mg/mL) NADP+ (see Note 3), 66 mM (20 mg/mL) o-glucose-6-phosphate, 66 mM (13 3 mg/mL) magnesium chloride (MgCl* . 6H20). Glucose-6-phosphate dehydrogenase (40 U/mL) m 5 mM (1.5 mg/mL) sodium citrate (C,H,O,Na, * 2H20) 5. Enzymes: e.g., cDNA-expressed CYP2D6 (GENTEST) or human liver microsomes Dilute m assaybuffer to a workmg concentration of 5 mg protem/mL and keep on Ice (see Note 4). 6 Deprotemlzmg agent. 70% (v/v) perchlorlc acid

2.2. HPLC 1 Mobile phase. 30% (v/v) acetomtnle, 2 mM perchlorlc acid (see Note 5). 2. Column: Nucleosil Cl8 column, 4.6 x 250 mm, 5-p particles size (Sigma, St. Louis, MO, cat. no. 222618) (see Note 6). 3 Detector: fluorescence, excitation wavelength: 252 nm, emission wavelength 302 nm, slit width 5-10 nm

3. Methods 1 Add the followmg to each incubation tube (total mcubatlon volume of 200 pL)* a. 163 pL, of assay buffer, b 5 pL of 1 mM (+/-) bufuralol (25 w final concentration, see Note 7), c 10 pL of a solution containing 26 mM NADP+, 66 mM D-glucose-6-phosphate, and 66 mM magnesium chloride, d 2 pL of glucosed-phosphate dehydrogenase (40 U/mL) m 5 mM sodium citrate. 2 Prewarm incubation tubes to 37°C and add 20 pL of diluted enzymes (100 pg protein) to initiate the enzymatic reaction (stagger each mcubatlon with 15-s delay Intervals) (see Notes 8 and 9) 3 Incubate samples at 37°C for 10 mm in a water bath (see Note 10) 4. Add 20 & of 70% (v/v) perchlonc acid to stop enzymatic reaction and place incubation tube on Ice 5. Centrifuge reaction mixture at 12,000g for 4 mm. 6 Iqect 10-50 $ of supernatant onto HPLC column 7. Run column at 45-50°C (see Note 11) with 30% (v/v) acetomtnle, 2 mM perchlorlc acid at a flow rate of 1 mL/min Under these condltlons retention times

Bufuralol

8 9

10.

11.

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1 ‘-Hydroxylation

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are 6 mm for l’-hydroxybufuralol and 20 min for bufuralol Momtor fluorescence (excrtatron wavelength 252 nm, emrsston wavelength 302 nm). Prepare blank incubation tubes by adding the complete incubation mrxture but with heat-inactivated enzyme Process the incubation tubes as per steps 3-7 Prepare standards by adding a known amount (e.g., 0, 0.05, 0.1, 0 2, 0 5, and 1 nmol) of authentic l’-hydroxybufuralol metabohte to tubes contammg the complete incubatron mixture but with heat-macttvated enzymes Process the mcubanon tubes contammg the standards as per steps 3-7 Plot a standard curve of peak height (or peak area) of metabolite standard vs amount of authentic metabolite added Determine the amount of product formation m each unknown and blank sample Calculate bufuralol I’-hydroxylase activity and express it as nmol product formed/mm/mg mmrosomal protein or as nmol product formed/mm/nmol total P450. Calculate net enzyme acttvtty by subtracting the acttvtty in the blank from each of the unknown samples (see Note 12).

4. Notes 1 The metaboltsm of bufuralol by CYP2D6 is enantroselectrve. The apparent k,,, values for (+) bufuralol I’-hydroxylatron and (-) bufuralol I’-hydroxylatton catalyzed by purified, reconstituted CYP2D6 are 3 7 nmol/mm/nmol P450, I e , 3.7/mm for the (+) substrate, and 0.6 nmol/mm/nmol P450, i.e , 0 6/mm, for the (-) substrate (4). 2. Bufirralol stock solutrons may be stored at -20°C for up to 2 yr. 3 Bufuralol l’-hydroxylase assays can also be carried out with mcotmamrde adenme dmucleotide phosphate (NADPH) (e.g., 1 mA4 final concentratron) instead of an NADPH-generating system (e.g , NADP+, n-glucose-6-phosphate, glucose-6 phosphate dehydrogenase) 4. Prepare only sufficient amounts of dilute mtcrosomes for each experiment The remainder of the undiluted mrcrosomes can be stored at -80°C for future use (11). 5 Mobile phase must include perchloric acid because the metabohte chromatographs as an ion pan 6. Bufuralol and I’-hydroxybufuralol can be separated on C 18 columns from other manufacturers, but may require adjustment of the acetomtrile content of the mobile phase. 7 Apparent Km values reported for bufuralol I’-hydroxylatton catalyzed by cDNAexpressed CYP2D6 range from 5 to 40 p~I4(12-15). A substrate concentratron of 25 @4 1s recommended when bufuralol l’-hydroxylase acttvtty 1s used as a marker for hepattc CYP2D6 because thus P450 accounts for nearly all of the bumralol I’-hydroxylase activity in human liver mtcrosomes when the assay IS carried out at this substrate concentratron (13). However, as the substrate concentration increases, the contrtbutron of CYP2D6 to the mrcrosomal activity

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8

9. 10. 11

12.

decreases because of the increased role of a higher Km form such as CYPlA2. This is conststent with the reported nonlmear Eadre-Hofstee plot for bufuralol l’-hydroxylation catalyzed by microsomes isolated from livers associated with the CYP2D6 extensive metabobzer phenotype (9). The addttion of qumldme (1 @4 final concentration from a 1 mM stock solution prepared m buffer) can be used to confirm the specificity of catalysis by CYP2D6 With some P450 expression systems, tt is necessary to reconstitute the recombinant P450 protein with NADPH-cytochrome P450 reductase, cytochrome b,, and lipid before mitiatmg substrate oxidation (13,14) Conduct prebmmary experiments to establish the amount of each component required for optimal catalytic activity. Conduct prehmmary experiments to ensure that the assay is linear with respect to microsomal protein concentration. Conduct preliminary experiments to ensure that the assay is linear with respect to mcubation time Column temperature can range from room temperature to 50°C The use of a controlled, elevated temperature provides greater reproducibility m retention times and lower column back pressures. In a panel of 60 human liver microsomes, bufuralol l’-hydroxylase activity ranged from 0 to 0.04 nmol/min/mg protein (13).

Acknowledgments Supported in part by the British Columbia Health Research Foundation (grant 119(95-l) to Thomas K. H. Chang) and by the National Institutes of

Health (grant ES07381 to David J. Waxman). Thomas K. H. Chang IS the recipient of a Research Career Award in the Health Sciences from the Pharmaceutical Manufacturers Assoclatlon of Canada-Health Research Foundation and the Medical Research Council of Canada. References 1 Bertilsson, L. (1995) Geographical/mterracial differences m polymorphic drug oxidation. Current state of knowledge of cytochromes P450 (CUP) 2D6 and 2C 19 Clan Pharmacobnet 29,192-209 2. Gonzalez, F. J., Skoda, R. C., Kimura, S., Umeno, M , Zanger, U M., Nebert, D W., Gelboin, H V., Hardwick, J P , and Meyer, U. A. (1988) Characterization of the common genetic defect m humans deficient in debrisoqume metabolism. Nature 331,442-446 3. Zanger, U. M., Vilbois, F., Hardwick, J. P., and Meyer, U. A (1988) Absence of hepatic cytochrome P450bufI causes genetically deficient debrisoqume oxtdation m man. Biochemistry 27,544X5454. 4 Gut, J , Catin, T , Dayer, P , Kronbach, T., Zanger, U., and Meyer, U. A (1986) Debrisoqume/sparteine-type polymorphism of drug oxidation Purification and

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5.

6

7.

8.

9.

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charactertzation of two functionally different human hver cytochrome P-450 isozymes involved m impaired hydroxylation of the prototype substrate bufuralol. J Bzol Chem 261, 11,734-l 1,743. Dayer, P., Kronbach, T., Eichelbaum, M., and Meyer, U. A (1987) Enzymatic basis of the debrisoquine/sparteine-type genetic polymorphism of drug oxidation Characterization of bufuralol I’-hydroxylation m liver microsomes of zn VEVOphenotyped carriers of the genetic deficiency. Blochem Pharmacol 36, 4145-4152. Newton, D. J., Wang, R. W., and Lu, A. Y. H (1995) Cytochrome P450 inhibitors. Evaulatton of specificities m the m vitro metabolism of therapeutic agents by human liver microsomes Drug Metab Dispos 23, 154-l 58 Bourrie, M., Meumer, V., Berger, Y., and Fabre, G. (1996) Cytochrome P450 isoform mhibttors as a tool for the investigation of metabolic reactions catalyzed by human liver microsomes. J Pharmacol Exp. Ther 277,321-332 Dayer, P., Leemann, T , and Striberni, R (1989) Dextromethorphan Odemethylation in liver microsomes as a prototype reaction to monitor cytochrome P-450 dbl activity. Clm. Pharmacoi. Ther 45,34-40. Kronbach, T., Mathys, D., Gut, J., Catin, T , and Meyer, U A (1987) Highperformance liquid chromatographtc assays for bufuralol I’-hydroxylase, debrisoquine 4-hydroxylase, and dextromethorphan 0-demethylase m microsomes and purified cytochrome P-450 tsozymes of human liver Anal. Biochem. 162,24-32.

10. Rodrigues, A. D. (1996) Measurement of human liver microsomal cytochrome P450 2D6 activity using [0-methyl-t4C]dextromethorphan as substrate. Methods Enzymol. 272, 186-l 95. 11. Pearce, R E., McIntyre, C. J , Madan, A., Sanzgni, U , Draper, A. J , Bullock, P L., Cook, D C., Burton, L A , Latham, J., Nevins, C , and Parkmson, A (1996) Effects of freezing, thawing, and storing human liver microsomes on cytochrome P450 activity. Arch Blochem Biophys. 331, 145-169 12 Crespi, C L., Gonzalez, F. J., Steimel, D. T , Turner, T. R., Gelbom, H. V., Penman, B W., and Langenbach, R. (1991) A tobacco smoke-derived nitrosamme, 4-(methylnitrosammo)1-(3-pyridyl)- 1-butanone, is activated by multiple human cytohrome P45Os including the polymorphic cytochrome P450 2D6. Carcinogeneszs12, 1197-1201 13. Yamazaki, H., Guo, Z., Persmark, M., Mimura, M , Inoue, K., Guengerich, F P , and Shimada, T. (1994) Bufuralol hydroxylation by cytochrome P450 2D6 and lA2 enzymes in human liver microsomes. Mol. Pharmacol. 46,568-577 14 Gillam, E. M. J., Guo, Z., Martin, M. V., Jenkins, C. M., and Guengerich, F P. (1995) Expression of cytochrome P450 2D6 m Escherwhia colz, purification, and spectral and catalytic characterization. Arch Biochem Blophys. 319,540-550 15. Paine, M. J. I., Gilham, D., Roberts, G. C. K., and Wolf, C. R (1996) Functional high level expression of cytochrome P450 CYP2D6 using baculovirus expression systems. Arch. Blochem. Biophys. 328, 143-150.

17 Spectrophotometric Analysis of Human CY P2El -Catalyzed pNitrophenol Hydroxylation Thomas

K. H. Chang, Charles L. Crespi, and David J. Waxman

1. Introduction CYP2El is expressed in adult (1,2) and fetal (3) human liver m addition to extrahepatrc tissues such as lung and placenta (If). Treatment of primary cultures of human hepatocytes with ethanol induces CYP2El protein (5) and this is consistent with the finding that hepatic CYP2El protein (6) and mRNA (7) amounts are increased in alcoholics. Although only a few drugs (e.g., acetamtnophen 181) have been identified as substrates for CYP2E1, many low molecular weight procarcmogens are activated by this P450 (9). Chlorzoxazone 6-hydroxylation (19-12), N-nitrosodimethylamine N-demethylation (11,13,14), andp-nitrophenol hydroxylation (12,14) reacttons can be used to measure the catalytic activity of cDNA-expressed CYP2El. Each of these activities IS also frequently used as an enzyme-selective catalytic monitor for human hepatic CYP2El (see Notes 1 and 2). Experiments with inhrbttory antrCYP2E 1 antibodies and CYP2El -selective chemrcal inhibitors suggest that CYP2E 1 contributes extensively to these activities m human liver mrcrosomes (9,15-18). Recently, lauric acid 1I-hydroxylatron was identified as an alternative probe for human hepatic CY2El (19). However, an advantage of using p-nitrophenol hydroxylation to measure CYP2E 1 actrvrty is that the formation of p-nitrocatechol can be measured by a simple spectrophotometric assay, although high-performance liquid chromatographic (HPLC) assayshave also been developed to quantify the p-nitrocatechol metabolite (20,21). This chapter describes a modrficatron of a spectrophotometric method (22) for the determination of p-nitrophenol hydroxylase activity. Methods for other CYP2El assays such as chlorzoxazone 6-hydroxylase (23), N-nitrosodtmethylamine From Methods m Molecular Biology, Vol 107 Cytochrome P450 Protocols Edlted by I R PhIllIps and E A Shephard 0 Humana Press Inc , Totowa, NJ

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N-demethylase (24) and lauric acid 1I-hydroxylase (19) can be found m the cited references. 2. Materials 1 Assay buffer 50 rmVpotassmm phosphate, pH 7.4 2. Substrate. p-mtrophenol (MW = 139.1, see Note 3) (Aldrich, Milwaukee, WI). Prepare a 5 mA4 (0.70 mg/mL) stock solution dissolved m assay buffer (see Note 4). 3 Metabohte standard. p-mtrocatechol (MW = 155.1, see Note 3) (Aldrich) 4. Cofactor generating system* 26 mM (20 mg/mL) NADP+ (see Note 4), 66 mM (20 mg/mL) o-glucose-6-phosphate, 66 mA4 (13 3 mg/mL) magnesium chloride (MgCl* 6H20) Glucose-6-phosphate dehydrogenase (40 U/mL) m 5 mM (1.5 mg/mL) sodium citrate (C,HSO,Na, * 2H20). 5. Enzymes e.g., cDNA-expressed CYPZEI or human liver microsomes Drlute in assay buffer to a working concentration of 5 mg protem/mL and keep on ice (see Note 6). 6 Deprotemizmg agent. 20% (w/v) trichloroacetic acid 7. 2 MNaOH. 8. Equipment mcludes vortex mixer and spectrophotometer.

3. Methods 1 Add the followmg to each incubation tube (total mcubation volume of 500 pL)* a 440 pL of assay buffer b. 10 pL of 5 mMp-mtrophenol (100 luV final concentration, see Note 7) c 25 pL of a solution containing 26 mM NADP+, 66 mM n-glucose-6-phosphate and 66 mA4 magnesium chloride d. 5 & of glucose-6-phosphate dehydrogenase (40 U/n&) in 5 mA4 sodnnn citrate 2 Prewarm mcubation tubes to 37°C and add 20 uL of diluted enzymes (100 ~18 protein) to initiate the enzymatic reaction (stagger each incubation with 15-s delay Intervals) (see Notes 8 and 9). 3. Incubate samples at 37°C for 30 min m a water bath (see Note 10) 4. Add 100 pL of 20% (w/v) trichloroacetic acid to stop enzymatic reaction and place incubation tube on ice 5 Centrifuge reaction mixture at 10,OOOgfor 5 mm. 6. Transfer 0.5 mL of the supernatant to a clean test tube containing 0 25 mL of 2 A4 NaOH and vortex 7. Measure absorbance at 535 nm with a spectrophotometer (see Note 11). 8 Prepare blank incubation tubes by adding the complete incubation mixture but with heat-mactivated enzymes. Process the blank mcubatron tubes as per steps 3-7. 9. Prepare standards by adding a known amount (e.g., 0, 1, 2, 5, 10, 20, nmol) of authentic p-nitrocatechol metabohte (see Note 4) to tubes containing the complete incubation mixture but with heat-inactivated enzymes. Process the standard tubes as per steps 3-7.

p-Nitrophenol Hydroxyation Assay

749

10. Calculate the net absorbance of each unknown sample and standard by subtracting the absorbance of the blank tubes from that of the unknown sample or standard. 11 Plot a standard curve of net absorbance vs amount of authentic p-nitrocatechol standard and determme the amount of product formed m each unknown sample by linear regression analysis. 12. Calculate p-nitrophenol hydroxylase activity and express it as nmol product formed/mm/mg microsomal protein or as nmol product formed/min/nmol total P450 (see Note 12).

4. Notes 1. Exercise caution when using hepatlc mlcrosomal chlorzoxazone 6-hydroxylase activity as a diagnostic catalytic marker for human CYP2El because of the reported contribution of CYP3A to this activity in human hepatlc mlcrosomes (25). In addition to cDNA-expressed CYP2E 1, recombinant human CYP 1A 1, CYP3A4, and CYP2D6 have significant catalytic activity toward chlorzoxazone 6-hydroxylatlon (25,26). 2. The conclusion that mlcrosomalp-mtrophenol hydroxylase activity 1sa dlagnostic marker for human hepatic CYP2El 1s largely based on the observation that dlethyldithiocarbamate, regarded by many as a selective inhibitor of CYP2El (91, extensively inhibits p-nitrophenol hydroxylase activity in human liver microsomes, whereas other P450 enzyme (or subfamily) selective mhibltors such as triacetyloleandomycin, sulfaphenazole, and qumidme have no effect on this activity (17). However, recent evidence indicates that dlethyldrthlocarbamate 1s not selective for CYP2El but is an inhibitor of CYP2A6-catalyzed substrate 0x1dation as well (27-30) Moreover, we have recently observed that cDNAexpressed CYP3A4 can catalyze p-nitrophenol hydroxylation (unpublished results). Therefore, exercise caution when using p-mtrophenol hydroxylase activity m human liver microsomes as a diagnostic probe for CYP2E 1. 3. p-Nitrophenol and p-nitrocatechol are light-sensitive. 4. Stock solutions ofp-mtrophenol andp-nitrocatechol can be stored for up to 1 yr at -2O’C. 5. p-Nitrophenol hydroxylase assays can also be carried out with nlcotmamlde adenine dmucleotide phosphate (NADPH) (31) instead of an NADPH generating system (e.g., NADP+, o-glucose-6-phosphate, glucose-6-phosphate dehydrogenase). 6 Prepare only sufficient amounts of diluted human liver microsomes for each experiment. The remainder of the undiluted mlcrosomes can be stored at -80°C for future use (32). 7 A substrate concentration of 100 w is suggested because the apparent Km value for p-nitrophenol hydroxylation by cDNA-expressed CYP2El and by human liver microsomes is -20-30 ~JV (17) and substrate mhlbltion 1s observed at >200 wp-nitrophenol (22)

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8. With some P450 protein expression systems, it is necessary to reconstitute the recombmant P450 protein with NADPH-cytochrome P450 reductase, cytochrome b,, and lipid before mitiating substrate oxidation (12). Conduct prelimmary experiments to establish the amount of each component required for optimal catalytic activity. 9 Conduct preliminary experiments to ensure that the assay 1slinear with respectto microsomal protein concentration 10 Conduct prehmmary experiments to ensure that the assay is linear wrth respect to incubation time 11. Read absorbance immediately upon mixing with 2 MNaOH. Over time, the alkalme pH increases the background m the assay 12. In a panel of 10 mdtvidual human liver microsome samples, p-mtrophenol hydroxylase activity ranged from 0 25-3 3 nmol/mm/mg protein (31)

Acknowledgments Supported m part by the British Columbia Health Research Foundation (grant 119(95-l) to Thomas K. H. Chang) and the National Institutes of Health (grant ES07381 to David J. Waxman). Thomas K. H. Chang is the recipient of a Research Career Award m the Health Sciences from the Pharmaceutrcal Manufacturers Association of Canada-Health Research Foundation and the Medical Research Councrl of Canada. References 1. Shimada, T , Yamazaki, H , Mimura, M., Inui, Y , and Guengerich, F P. (1994) Intermdivtdual variations m human liver cytochrome P-450 enzymes involved m the oxidation of drugs, carcinogens and toxic chemicals studies with ltver mtcrosomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp. Ther. 270, 414-423 2 Imaoka, S , Yamada, T , Hiroi, T., Hayasht, K , Sakaki, T., Yabusaki, Y , and Funae, Y. (1996) Multiple forms of human P450 expressed m Succharomyces cerevzszae: systematic characterization and comparison with those of the rat Biochem Pharmacol. 51,1041-1050. 3 Carpenter, S P., Lasker, J M , and Raucy, J L. (1996) Expression, mduction, and catalytic activity of the ethanol-inducible cytochrome P450 (CYPZEI) m human fetal liver and hepatocytes. Mol Pharmacol. 49,26&268. 4 Botto, F , Seree, E., Khyari, S. E., Desousa, G., Massacrter, A., Placidi, M., Cau, P., Pellet, W., Rahmam, R., and Barra, Y. (1994) Tissue-specific expression and methylation of the human CYP2E 1 gene Blochem. Pharmacol 48, 1095-l 103 5. Kostrubsky, V. E., Strom, S. C , Wood, S G , Wrighton, S. A , Sinclair, P R , and Smclair, J. F. (1995) Ethanol and tsopentanol increase CYP3A and CYP2E m primary cultures of human hepatocytes. Arch. Blochem Bzophys 322,5 16-520 6. Perrot, N., Nalpas, B., Yang, C. S , and Beaune, P H. (1989) Modulation of cytochrome P450 tsozymes in human liver, by ethanol and drug intake. Eur J Clan Invest 19, 549-555.

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7. Takahashi, T., Lasker, J M., Rosman, A. S., and Lieber, C. S. (1993) Induction of cytochrome P-450 2E 1 in the human liver by ethanol is caused by a corresponding increase in encoding messenger RNA Hepatology 17,236-245 8. Raucy, J. L., Lasker, J. M , Lieber, C. S , and Black, M. (1989) Acetammophen activation by human liver cytochromes P450IIE 1 and P450 1A2 Arch. Biochem Biophys 271,270-283. 9. Guengerich, F. P., Kim, D. H., and Iwasaki, M (1991) Role of human cytochrome P-450 IIE 1 m the oxidation of many low molecular weight cancer suspects Chem. Res. Toxic01 4, 168-179 10. Gillam, E. M. J., Guo, Z., and Guengertch, F. P. (1994) Expression of modified human cytochrome P450 2El in Escherzchia coli, purification, and spectral and catalytic properties. Arch. Blochem. Biophys. 312,59-66. 11. Yamazaki, H., Nakano, M., Gillam, E. M. J., Bell, L. C., Guengerich, F P., and Shimada, T. (1996) Requirements for cytochrome b, m the oxidation of 7-ethoxycoumarin, chlorzoxazone, aniline and N-mtrosodimethylamine by recombinant cytochrome P450 2E 1 and by human liver microsomes Bzochem Pharmacol. 52,30 l-309. 12. Chen, W , Peter, R M , McArdle, S., Thummel, K E , Sigle, R. 0 , and Nelson, S D. (1996) Baculovirus expression and purification of human and rat cytochrome P450 2El Arch Blochem. Btophys 335,123-130 13. Umeno, M., McBride, W. 0 , Yang, C S., Gelbom, H. V., and Gonzalez, F J. (1988) Human ethanol-inducible P450 IIEl: complete gene sequence, promoter characterization, chromosome mapping and cDNA-directed expression. Biochemistry 27,9006-90 13. 14. Patten, C. J , Ishizaki, H , Aoyama, T., Lee, M., Nmg, S. M , Huang, W , Gonzalez, F J., and Yang, C. S (1992) Catalytic properties of the human cytochrome P450 2E 1 produced by cDNA expression in mammalian cells. Arch Bzochem. Blophys 299, 163-171. 15 Wrighton, S A., Thomas, P. E , Molowa, D. T , Haniu, M , Shively, J E , Maines, S L., Watkins, P. B., Parker, G , Mendezpicon, G , Levin, W , and Guzehan, P. S (1986) Characterization of ethanol-inducible human liver N-nitrosodimethylamme demethylase. Biochemistry 25,673 l-6735. 16. Peter, R., Backer, R., Beaune, P. H., Iwasaki, M., Guengerich, F. P., and Yang, C. S. (1990) Hydroxylation of chlorzoxazone as a specific probe for human liver cytochrome P-45011El. Chem Res Towel. 3,566-573 17. Tassaneeyakul, W., Veronese, M. E., Birkett, D. J., Gonzalez, F. J., and Miners, J 0. (1993) Validation of 4nitrophenol as an in vitro substrate probe for human liver CYP2El using cDNA expression and microsomal kinetic techniques Blochem Pharmacol. 46,1975-198 1. 18. Yamazaki, H., Guo, Z., and Guengerich, F. P. (1995) Selectivity of cytochrome P450 2El in chlorzoxazone 6-hydroxylation. Drug Metab Dispos. 23,438440. 19. Amet, Y., Berthou, F , Baird, S., Dreano, Y., Bail, J. P., and Menez, J. F (1995) Validation of the (o- l)-hydroxylation of lauric acid as an zn vztro substrate probe for human liver CYP2El. Bzochem. Pharmacol 50, 1775-1782.

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20 Tassaneeyakul, W , Veronese, M. E., Btrkett, D. J., and Miners, J 0. (1993) Highperformance liquid chromatography assay for 4-mtrophenol hydroxylation, a putative cytochrome P450 2EI activity, in human liver microsomes. J Chromatogr 616,73-78. 21 Mishm, V. M., Kotvtsto, T., and Lteber, C S. (1996) The determmatton of cytochrome P450 2E 1-dependent p-mtrophenol hydroxylatton by high-performance liquid chromatography with electrochemical detection. Anal. Btochem. 233, 212-215. 22. Reinke, L. A and Moyer, M J (1985) p-Nitrophenol hydroxylation. A microsoma1 oxtdation which 1s highly mducible by ethanol. Drug Metab Dispos 13, 548-552. 23. Lucas, D., Menez, J F , and Bet-thou, F (1996) Chlorzoxazone. An zn vztro and in

vivo substrate probe for liver CYP2E 1 Methods Enzymol 272, 115-l 23. 24. Yoo, J. S. H., Guengerich, F. P., and Yang, C S. (1988) Metabohsm of N-mtrosodialkylammes by human liver mtcrosomes. Cancer Res 88, 1499-l 504. 25. Gorski, J. C., Jones, D. R., Wrighton, S A , and Hall, S D. (1997) Contributton of human CYP3A subfamily members to the 6-hydroxylatton of chlorzoxazone. Xenobiotica 27,243-256. 26. Carriere, V., Goasduff, T , Ratanasavanh, D , Morel, F., Gautier, J. C., Guillouzo, A., Beaune, P., and Berthou, F. (1993) Both cytochromes P450 2E1 and 1Al are mvolved in the metabohsm of chlorzoxazone. Chem. Res Toxxol 6,852-857 27 Yamazaki, H., Inui, Y., Yun, C. H., Mtmura, M., Guengerich, F P., and Shimada, T (1992) Cytochrome P450 2E 1 and 2A6 enzymes as major catalysts for metabolic activation of N-nitrosodialkylammes and tobacco-related nitrosammes m human liver microsomes. Carcznogenesis 13, 1789-1794. 28. Chang, T. K. H., Gonzalez, F. J., and Waxman, D J (1994) Evaluatton of triacetyloleandomycm, a-naphthoflavone and diethyldithiocarbamate as selective chemical probes for inhibition of human cytochromes P450. Arch. Blochem Bzophys. 311,437-442 29. Ono, S., Hatanaka, T., Hotta, H., Satoh, T., Gonzalez, F. J , and Tsutsui, M. (1996) Specifictty of substrate and inhibitor probes for cytochrome P45Os. evaluatton of in vztro metabolism using cDNA-expressed human P45Os and human liver mtcrosomes. Xenoblotrca 26,68 l-693. 30. Draper, A. J., Madan, A., and Parkinson, A. (1997) Inhibition of coumarm 7hydroxylase activity m human liver mtcrosomes. Arch Biochem Bzophys 341, 47-61. 3 1 Seaton, M. J., Schlosser, P M , Bond, J. A., and Medmsky, M A (1994) Benzene metabolism by human liver microsomes in relation to cytochrome P450 2E1 activity. Carcznogeneszs 15, 1799-l 806. 32. Pearce, R. E., McIntyre, C. J., Madan, A., Sanzgiri, U., Draper, A J , Bullock, P L , Cook, D. C., Burton, L A , Latham, J., Nevins, C., and Parkinson, A. (1996) Effects of freezing, thawing, and storing human liver microsomes on cytochrome P450 activity. Arch Blochem. Biophys 331,145-169.

Thin-Layer Chromatographic Analysis of Human CYP3A-Catalyzed Testosterone 6P-Hydroxylation David J. Waxman and Thomas K. H. Chang 1. Introduction At least two cytochromes P450 belongmg to the CYP3A subfamily may be expressed m adult human liver (I), CYP3A4 and CYP3A5. A third enzyme, CYP3A7, is expressed m human fetal liver (2). The CYP3A enzymes account for an estimated -30% of total human cytochrome P450 content in adult hver (3), although large inter-individual differences exist in hepatic CYP3A content. CYP3A4 is present in all adult human livers and is inducible by drugs such as nfampm (rifampicin) and dexamethasone (&). By contrast, CYP3A5 is expressed m only -lO-30% of liver samples (7) and does not respond to typical CYP3A inducers (5,6). Triacetyloleandomycin (8,9) and gestodene (9) are CYP3A-selective chemical inhibitors. Many commonly used drugs are substrates for CYP3A, including erythromycin (IO), mfedipine (11) and midazolam (12). Immunoinhibition experiments with CYP3A subfamily-specific antibodies have established several microsomal enzyme activities, includmg nifedipme oxtdase (11,13) and testosterone 6P-hydroxylase (14,15), as useful catalytic monitors for hepatic CYP3A In a recent study, an inhibitory antipeptide antibody against CYP3A4, which did not cross-react with cDNAexpressed CYP3A5 as judged by Western-blot analysis and did not mhibit cDNA-expressed CYP3A5-catalyzed testosterone 6P-hydroxylation, was found to inhibit virtually all of the testosterone 6P-hydroxylase activity m human liver microsomes (16), suggesting that this activity has a high spectlicity for hepatic CYP3A4. This is consistent with previous findings that: 1 CYP3A4 is expressedat a much higher level than CYP3A5 in human liver (17), 2. cDNA-expressedCYP3A4 is much more active than cDNA-expressedCYP3A5 in testosterone6P-hydroxylation (Z&29); and From Methods m Molecular Bology, Vol 107 Cytochrome P450 Protocols Edlted by I R PhIllIps and E A Shephard 0 Humana Press Inc , Totowa, NJ

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3 cDNA-expressedCYP3A5 exhibits an approx 1O-fold greater apparent Km than cDNA-expressed CYP3A4 in testosterone 6P-hydroxylation

(19).

Several analytical methods are available for determmmg testosterone 6phydroxylation, including isocratic (20) and gradient (21,22) high-performance liquid chromatographrc methods. This chapter describes a radrometrtc thmlayer chromatographic (TLC) assay for the determmation of testosterone 6phydroxylase activity. As mdrcated in the Notes sectron, this assay method can be used to determine P450-catalyzed 6P-hydroxylation of related steroids, such as androstenedrone and progesterone and to assay testosterone hydroxylatton activity at positions other than C-6p. Methods for other CYP3A assayssuch as nifedipine oxidase can be found m the cited reference (11). 2. Materials 1. Substrate: [4-‘“Cltestosterone, 50-62 mCi/mmol, 50 mCi/mL (Amersham, Arlington Heights, IL) Testosterone (MW = 288 4) (Stgma, St. Louts, MO). Dilute [4-t4C]testosterone with unlabeled testosterone dissolved m toluene to gave a working solutron at 5-7 mCt/mmo1(4000-6000 cpm/pL) 2. Metabolite standards. 2a-hydroxytestosterone (Stgma). 2P-hydroxytestosterone (Sigma). 6a-hydroxytestosterone (Sigma). 6P-hydroxytestosterone (Srgma) 7a-hydroxytestosterone (Steralotds, Wilton, NH) 11 P-hydroxytestosterone (Sigma) 14a-hydroxytestosterone (Sigma) 1So-hydroxytestosterone (Sigma) 15p-hydroxytestosterone (Searle, Skokre, IL) 16a-hydroxytestosterone (Sigma) 16P-hydroxytestosterone (Steralords) 19-hydroxytestosterone (Steraloids) 3. Assay buffer 100 mA4HEPES, pH 7 4, containing 0.1 n-&f ethylenediamme tetraacetic acid (EDTA). 4. Cofactor. 10 mMNADPH (8.3 mg/mL) stock solutton. Prepare fresh and keep on

ice (seeNotes 1 and 2) 5. Enzymes. e.g , cDNA-expressed CYP3A4, CYP3A5, (GENTEST, Woburn, MA) or human liver mrcrosomes. Dtlute m assay buffer to a working concentration of 1 5 mg protem per mL and keep on me (see Note 3). 6. Extractton solvent: ethyl acetate. 7. TLC plates: Aluminum-backed sihca gel 60 F-254 plates, 20 x 20 cm (EM Separations Technology, Gibbstown, NJ).

8. GlassTLC tank approx 10 x 30 cm, 25 cm tall. 9 TLC solvent mixtures, prepare before use:

Testosterone 6p-Hydroxylation Assay

10. 11. 12 13 14.

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a. 80 mL methylene chloride, 20 mL acetone b. 80 mL chloroform, 20 mL ethyl acetate, 14 mL absolute ethanol c. 80 mL dichloromethane, 20 mL acetone. Whatman 3MM chromatography paper. X-Ray film: Kodak X-OMAT AR X-Ray film holder. Scintillation cocktail. Betafluor (National Diagnostics, Manvtlle, NJ) or equivalent. Equipment includes a liquid-scintillation counter.

3. Methods 3.7. Enzyfnafic

Reaction

1. To each incubation tube (13 x 100 mm test tube) add 10 nmol of [4-r4C]testosterone at 5-7 mCl/mmol (50 @4 steroid m a final assay volume of 0.2 mL) and evaporate the solvent under a gentle stream of nitrogen 2. Add the following to each incubation tube (total incubation volume of 200 pL): 160 pL of assay buffer and 20 pL of diluted enzymes (30 pg protein) (see Notes 4 and 5) 3 Prewarm mcubation tubes to 37°C and add 20 pL of 10 mMNADPH (1 mMfina1 concentration) to mitiate the enzymatic reaction (stagger each incubation with 15-s delay intervals). 4 Incubate samples at 37’C for 10-20 min m a shaking water bath (see Note 6) 5 Add 1 mL ice-cold ethyl acetate to stop the enzymatic reaction, vortex briefly, and place the incubation tube on ice untrl all samples have been collected 6. Vortex for 30 s (to extract with ethyl acetate). 7. Centrifuge reaction mixture at 3000g for 5 mm. 8. Transfer the organic phase (upper layer) to a clean test tube. 9. To the aqueous phase add 1 mL ethyl acetate and repeat steps 6 and 7 10. Combme the orgamc extracts and dry under a gentle stream of nitrogen, 1 I. Process blank incubation tubes containing the complete mcubatron mixture but with heat-inactivated enzyme as per steps 4-10

3.2. TLC and Autoradiography 1, Activate aluminum-backed, silica-gel TLC plates by heatmg at 100°C for 1O-1 5 min. Allow to cool for 5 min (see Note 7). 2. Add solvent mixture (a) (80 mL methylene chloride and 20 mL acetone) to a glass TLC tank Add one sheet of Whatman 3MM chromatography paper to the tank to help saturate the air with solvent vapors. Equilibrate for at least 20 mm. 3. Reconstitute the dried organic extracts containing 14C-labeled enzymatic products (see Subheading 3.1., step 10) by addition of 30 & ethyl acetate to each assay tube. 4. Apply the reconstituted enzymatic products with a 10 pL microprpet in several portions as individual spots Each spot should be 12 mm m diameter, and is

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5 6. 7 8 9. 10. 11

12 13.

Waxman and Chang applied at the origin, on a line drawn 2 cm up from the bottom of a heat-activated TLC plate (see Note 8). Repeat step 3 to wash out the tube and Increase the recovery of i4C-labeled metabohtes Repeat step 4. Place the TLC plate m solvent-pre-equilibrated TLC tank (see step 2) Remove the plate from the TLC tank when the solvent front is approx 2 cm from the top Air-dry the plate for -10 mm Place the TLC plate m a second TLC tank pre-equilibrated with solvent mixture (b) (80 mL chloroform, 20 mL ethyl acetate and 14 mL absolute ethanol) (see Note 9) Repeat step 8. Mark the corners of each TLC plate with 3-4 small spots drawn m a characteristic pattern using either a fluorescent marker or radioactive mk (“hot mk spots”) These spots will expose the X-ray tilm (see step 12) in a characteristic pattern that will then be used to align the film with the TLC plate for quantification (see Subheading 3.3.) Expose the TLC plate to X-ray film in a film holder overnight at room temperature Develop X-ray film to visualize the pattern of substrate and metabolites (see Notes 10 and 11)

3.3. Metabolite

Quantification

1. Align the “hot mk spots” on the corners of the X-ray film with those seen on the TLC plate. 2. Identify the location of each metabohte on the TLC plate by drawing a rectangular grid, m pencil, markmg the regions of the plate that encompass each mdividual i4C-labeled spot visible on the X-ray film. 3. Use scissors to cut out each of these marked rectangular areas from the TLC plate. 4 Place each rectangular piece of the alummum-backed plate m a labeled 20-mL scintillation vial containing 7 mL of Betafluor scmtillation cocktail with the silica-gel side facing up (see Note 12) 5 Determine counts per mmute (cpm) m each vial using a liquid-scmtiilation counter (see Note 13). 6 Determine background cpm for each region of the TLC plate by counting the corresponding silica gel pieces cut from a control (no mmrosomes or no NADPH blank) incubation lane. Calculate net cpm by subtracting the cpm of the blank from that of the unknown. Calculate enzyme activity (nmol/min/mg protein) for each hydroxytestosterone metabolite as follows (see Notes 14 and 15) enzyme activity = net cpm of product x nmol substrate/ (total net cpm of all spots in a lane) x (mg protein) x (mcubation time) Alternatively, tf the total P450 content of the sample has been determined, testosterone 6P-hydroxylase activity may be expressed as m-no1product formed/mm/ nmol total P450.

157

Testosterone G/3-Hydroxylation Assay 3.4. Identification

of 6pHydroxytestosterone

Metabolite

1 Dissolve authentic 6P-hydroxytestosterone m ethyl acetate at a concentration of approx 0 3-l mg/mL 2. Activate aluminum-backed, silica-gel TLC plate by heating at 100°C for 10-l 5 min. Allow to cool for 5 mm. 3 Apply the authentic monohydroxytestosterone metabolite onto a heat-activated TLC plate as per step 4, Subheading 3.2. 4 Apply a portion of the r4C-labeled testosterone metabolites generated from Subheading 3.1. directly on top of a spot containing authentic metabolite, applied m step 3 above. AlternatIvely, the 14C-labeled metabolite may be mixed with the unlabeled metabolite standard and then the mixture applied to the TLC plate. 5. Carry out TLC analysrs as per steps 7-13, Subheading 3.2. 6 After developing the X-ray film, visualize the location of the authentic 6phydroxytestosterone with an ultravrolet (UV) lamp and lightly outline its location on the TLC plate using a pencil The unlabeled hydroxytesterone IS visible on the TLC plate as a dark spot on a background of green fluorescence 7. Align the “hot mk” spots on the corners of the X-ray film with those on the TLC plate and overlay the X-ray film precrsely over the TLC plate Comcrdence of the 14C-labeled X-ray film spot with the unlabeled UV-active hydroxytestosterone metabohte provrdes supporting evidence for product rdentrficatron 8. To further confirm product rdentrfication, repeat steps 1-7 using a different TLC solvent system* two sequential developments m solvent mixture (c) (80 mL drchloromethane + 20 mL acetone) 9 Repeat steps l-7 again, but perform the TLC with a third solvent system: two sequential developments rn solvent mixture (b) (80 mL chloroform, 20 mL ethyl acetate, and 14 mL absolute ethanol). An unknown metabohte IS deemed to be “identified” as 6P-hydroxytestosterone rf cochromatography of radrolabeled metabolite with unlabeled authentic 6P-hydroxytestosterone standard is evident in each of the three independent TLC solvent systems. 10 To rule out the possibihty that the unknown metabohte IS a metabolite other than 6P-hydroxytestosterone, repeat steps 1-9 but with unlabeled authentic monohydroxytestosterone metabolites such as those listed m Subheading 2., item 2

4. Notes 1. Prepare fresh solutions of NADPH for each experiment. Note that NADPH is light-sensitive and pH-sensitive 2. Testosterone 6P-hydroxylase assays can also be carried out with an NADPHgenerating system (NADP+, n-glucose-6-phosphate, glucose-6-phosphate dehydrogenase) m place of NADPH (23). 3. Prepare only sufficient amounts of dilute mrcrosomes for each experrment. The remainder of the undrluted microsomes can be stored at -8O’C for future use (24) 4 Conduct preliminary experrments to ensure that the assay IS lmear with respect to microsomal protein concentration.

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5. Recombmant CYP3A4 enzymes, such as those produced in a yeast expression system, need to be reconstituted with exogenous NADPH-cytochrome P450 reductase, cytochrome b,, and dilauroylphosphatidylcholme (DLPC) before mltiation of substrate oxtdation (25,26). Prelimmary experiments will be required to determine the optimal amounts of NADPH-cytochrome P450 reductase, cytochrome b,, and DLPC to be used. However, a high level of catalytic activity can be achieved by coexpression of recombinant CYP3A4 with NADPH-cytochrome P450 reductase and/or cytochrome 6, (27,28). 6. Conduct preliminary experiments to ensure that the assay is linear with respect to incubation time. 7. To prevent curling of the TLC plate during heating at lOO”C, cover the edges of each plate with aluminum foil. 8 To mmimize “end lane” effects, do not apply samples withm 2-3 cm from the edge of each TLC plate 9 The assay described here can be used to determine human liver microsomal steroid 6P-hydroxylase activity for two related steroids, androstenedione and progesterone, by modifications of the TLC solvent system. Androstenedione metabohtes are resolved by two sequential developments of the TLC plate m chloroform/ethyl acetate (1:2, v/v), and progesterone metabolites are resolved by TLC using ethyl acetate/n-hexanelacetic acid (19:5.1, v/v/v) (14,29). 10 Rapid and sensitive quantification of the i4C-labeled metabohtes can also be achieved using a phosphorimager instrument [Molecular Imager, BioRad, Hercules, CA), or STORM 840 Imager, Molecular Dynamics, Sunnyvale, CA)], thus eliminating the X-ray film exposure, TLC cutting, and liquid-scmtillation counting steps 11. Although 6P-hydroxytesterone is the major hydroxytestosterone metabolite of human liver microsomes, several other hydroxytestosterone metabolites are also formed (I4) These metabolites can also be determined alongside 6j3hydroxytestosterone in the same TLC assay. 12. Significant quenching (-25% decrease m cpm) will occur if the alummum-backed side of the cut TLC piece faces up during liquid-scintillation counting. 13 The overall recovery of cpm mitially added to each mcubation tube is 65-85% 14. Duplicate activity analyses generally agree within 5-7% 1.5 In a panel of SIX human liver microsomes, this activity ranged from 0.09-l 48 nmol/mm/mg microsomal protein (14), whereas m another panel of 10 microsome samples, it ranged from 0.56-8 41 nmol/mm/mg microsomal protein (7).

Acknowledgments Supported in part by the National Institutes of Health (grant DK33765 to David J. Waxman) and the British Columbia Health Research Foundation (grant 119(95-l) to Thomas K. H. Chang). Thomas K. H. Chang IS the recrpient of a Research Career Award in the Health Sciences from the Pharmaceutrcal Manufacturers Assocratron of Canada-Health Research Foundation and the Medical Research Councrl of Canada.

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References 1. Nelson, D. R , Koymans, L., Kamataki, T., Stegeman, J. J., Feyeretsen, R., Waxman, D J., Waterman, M. R., Gotoh, 0 , Coon, M. J , Estabrook, R W., Gunsalus, I C , and Nebert, D. W. (1996) P450 superfamily: update on new sequences, gene mappmg, accesston numbers and nomenclature Pharmacogenetics 6, l-42 2 Kitada, M , Kamataki, T., Itahasht, K., Rtkihtsa, T., and Kanakubo, Y. (1987) P450 HFLa, a form of cytochrome P-450 purified from human fetal livers, 1sthe 16a-hydroxylase of dehydroepiandrosterone 3-sulfate. J Blol Chem 262, 13534-13537. 3 Shimada, T., Yamazakl, H., Mimura, M , Inui, Y., and Guengertch, F. P (1994) Interindividual variations in human liver cytochrome P-450 enzymes mvolved m the oxidation of drugs, carcinogens and toxic chemicals: studies with liver mtcrosomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp. Ther 270, 414-423. 4. Pichard, L., Fabre, I , Fabre, G , Domergue, J., Samt Aubert, B , Mourad, G , and Maurel, P. (1990) Cyclosporin A drug interactions: screening for inducers and mhibltors of cytochrome P-450 (cyclosporm A oxidase) m primary cultures of human hepatocytes and m liver microsomes Drug Metab. Dispos 18, 595-606 5 Schuetz, E. G , Schuetz, J. D., Strom, S C., Thompson, M T, Fisher, R. A, Molowa, D. T , Li, D , and Guzelian, P. S. (1993) Regulation of human liver cytochromes P-450 in family 3A in primary and continuous culture of human hepatocytes. Hepatology 18, 1254-l 262 6. Chang, T. K. H., Yu, L , Maurel, P., and Waxman, D. J (1997) Enhanced cyclophosphamide and ifosfamide activation m primary human hepatocyte cultures Response to cytochrome P-450 inducers and autoinductton by oxazaphosphorines Cancer Res 57, 1946-1954. 7 Wrighton, S. A., Ring, B. J., Watkins, P B., and VandenBranden, M. (1989) Identification of a polymorphically expressed member of the human cytochrome P450111 family. Mol. Pharmacol 36,97-105. 8. Chang, T. K H , Gonzalez, F. J., and Waxman, D. J. (1994) Evaluation of triacetyloleandomycm, a-naphthoflavone and diethyldithiocarbamate as selective chemical probes for mhibrtion of human cytochromes P450. Arch Blochem Biophys

311,437-442.

9. Newton, D. J., Wang, R. W , and Lu, A. Y H (1995) Cytochrome P450 mhibttors. Evaluation of specificities in the zn vztro metaboltsm of therapeutic agents by human liver microsomes. Drug Metab Dispos 23, 154-I 58. 10. Watkins, P B., Wrighton, S. A., Maurel, P , Schuetz, E. G , Mendez-Picon, G , Parker, G. A., and Guzeban, P S. (1985) Identtflcation of an inducible form of cytochrome P-450 m human liver. Proc Natl. Acad. SCL USA 82,63 10-63 14 11. Guengerich, F P., Martin, M. V., Beaune, P. H., Kremers, P., Wolff, T., and Waxman, D. J. (1986) Characterization of rat and human liver microsomal cytochrome P-450 forms involved m nifedipine oxtdation, a prototype for genetic polymorphism in oxidative drug metaboltsm. J Blol. Chem 261, 5051-5060.

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12 Gorski, J C., Hall, S. D., Jones, D. R , VandenBranden, M., and Wrighton, S. A. (1994) Regtoselective brotransformatton of mtdazolam by members of the human cytochrome P450 3A (CYP3A) subfamily Blochem Pharmacol 47,1643-1653. 13 Gonzalez, F. J., Schmid, B., Umeno, M., McBride, 0 W., Hardwick, J. P , Meyer, U A., Gelboin, H V., and Idle, J. R. (1988) Human P450PCNl. Sequence, chromosome localrzatron and direct evidence through cDNA expression that P450PCNl is nifedrpme oxrdase. DNA 7,79-86. 14. Waxman, D. J., Attisano, C., Guengerich, F. P., and Lapenson, D P. (1988) Human liver microsomal steroid metabolism* Identification of the maJor mrcrosoma1 steroid hormone 6/3-hydroxylase cytochrome P-450 enzyme Arch. Bzochem Bzophys 263,424-436 15. Gelbom, H V., Krausz, K W , Goldfarb, I., Buters, J. T M , Yang, S. K , Gonzalez, F J , Korzekwa, K R., and Shou, M (1995) Inhibitory and non-mhibitory monoclonal antibodies to human cytochrome P450 3A3/4. Btochem Pharmacol 50,184 l-l 850 16. Wang, R W. and Lu, A. Y. H (1997) Inhibitory anti-peptide antibody against human CYP3A4 Drug Metab Dispos 25,762-767. 17 Wrtghton, S. A , Brian, W R , Sari, M A., Iwasaki, M , Guengerrch, F. P , Raucy, J L , Molowa, D. T , and VandenBranden, M (1990) Studies on the expression and metabolic capabilities of human liver cytochrome P450IIIA3 (HLp3). Mel Pharmacol. 38,207-2 13 18. Aoyama, T , Yamano, S , Waxman, D J , Lapenson, D. P., Meyer, U. A , Fischer, V , Tyndale, R., Inaba, T., Kalow, W., Gelboin, H. V., and Gonzalez, F. J. (1989) Cytochrome P-450 hPCN3, a novel cytochrome P-450111A gene product that is differentially expressed in adult human liver cDNA and deduced ammo acid sequence and distmct specificites of cDNA-expressed hPCN 1 and hPCN3 for the metabolism of steroid hormones and cyclosporine J B1o1 Chem 264, 10,388-10,395 19 Waxman, D. I., Lapenson, D. P , Aoyama, T., Gelbom, H V., Gonzalez, F J., and Korzekwa, K. (1991) Sterotd hormone hydroxylase specrticitres of eleven cDNAexpressed human cytochrome P45Os. Arch Bzochem Blophys 290, 160-166. 20 Sanwald, P., Blankson, E. A , Dulery, B D., Schoun, J , Huebert, N. D , and Dow, J. (1995) Isocratrc high-performance liquid chromatographic method for the separation of testosterone metabolites. J Chromatogr 672,207-2 15. 21. Wood, A. W., Ryan, D. E., Thomas, P E., and Levm, W. (1983) Regio- and stereoselective metabolism of two C 19 steroids by five highly purified and reconstituted rat hepatic cytochrome P-450 isozymes J Blol Chem 258, 8839-8847. 22. Arlotto, M P., Trant, J M , and Estabrook, R. W (199 1) Measurement of steroid hydroxylation reactions by high-performance liquid chromatography as indicator of P450 identity and function Methods Enzymol 206,454462. 23 Pearce, R., Greenway, D , and Parkinson, A. (1992) Species differences and mterindrvidual variation in liver microsomal cytochrome P450 2A enzymes: effects on coumarin, dicumarol, and testosterone oxidation. Arch Bzochem Blophys, 298,2 1 l-225

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24 Pearce, R. E., McIntyre, C. J , Madan, A, Sanzgut, U., Draper, A J , Bullock,

25

26

27

28.

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P. L., Cook, D. C., Burton, L A., Latham, J , Nevins, C , and Parkinson, A (1996) Effects of freezing, thawing, and storing human liver mtcrosomes on cytochrome P450 activity. Arch Blochem. Biophys. 331, 145-169. Brian, W. R , San, M A , Iwasaki, M , Shimada, T., Kaminsky, L S., and Guengerich, F P. (1990) Catalytic activtttes of human hver cytochrome P-450 IIIA4 expressed m Saccharomyces cerevuiae. Blochemlstry 29, 11280-l 1292. Buters, J. T. M , Korzekwa, K R., Kunze, K. L., Omata, Y , Hardwrck, J P , and Gonzalez, F. J. (1994) cDNA-directed expression of human cytochrome P450 CYP3A4 using baculovnus. Drug Metab Dlspos 22,688-692 Peyronneau, M., A , Renaud, J. P., Truan, G , Urban, P., Pompon, D , and Mansuy, D (1992) Opttmizatton of yeast-expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH-cytochrome P450 reductase and cytochrome b, Eur J Blochem 207,109-l 16 Lee, C A., Kadwell, S H , Kost, T. A., and Serabjit-Smgh, C J. (1995) CYP3A4 expressed by insect cells infected with a recombinant baculovuus contammg both CYP3A4 and human NADPH-cytochrome P450 reductase 1s catalytically similar to human liver microsomal CYP3A4 Arch Biochem Bzophys 319,157-167 Waxman. D. J., (1991) P450-catalyzed steroid hydroxylation: assay and product identification by thin layer chromatography Methods Enzymol 206,462-476

19 Determination of CYP4All -Catalyzed Laurie Acid 12-Hydroxylation by High-Performance Liquid Chromatography with Radiometric Detection Charles L. Crespi, Thomas K. H. Chang, and David J. Waxman 1. Introduction The human CYP4.411 gene encodes a P450 protein that has been isolated from human kidney (1,2) and liver (3) and purified to apparent homogeneity. CYP4All mRNA is present m greater abundance in kidney than in liver, whereas it is absent m lung (3). Various fatty acids, including lauric acid (2-4), are substrates for recombinant CYP4All. Expenments with a panel of mdividual cDNA-expressed P450 enzymes indicate that CYP4All is a major lauric acid 12-hydroxylase (5). Immunoinhibition experiments with heterologous and antihuman CYP4All antibodies have further established that CYP4All contributes a major fraction of the lauric acid 12-hydroxylase actlvlty in human liver microsomes (3,5,6). Accordingly, microsomal lauric acid 12-hydroxy lase activity is used as a marker for human hepatic CY P4A 11. Although CY P4A 11 is a major lauric acid 12-hydroxylase in human liver, lauric acid 11-hydroxylation in this tissue is catalyzed by CYP2El and not CYP4All (7,s). Several analytical methods are available for the quantification of 12-hydroxylauric acid, including the use of nonradioactive lauric acid as the substrate by a highperformance hquid chromatographic (HPLC) assaywith fluorescence detection (9,10). This chapter describes a radiometric HPLC assay for the determinatron of lauric acid 12-hydroxylase activity.

2. Materials 2.1. Assay 1 Assaybuffer: 100 mMTris-HCl, pH 7 5. 2. Substrate: [l-*4C]lauric acid (50-62 mCi/mmol, 50 mCi/mL) (Amersham, From. Methods m Molecular &o/ogy, Vol 107 Cytochrome P450 Protocols Edlted by. I R PhMps and E A Shephard Q Humana Press Inc , Totowa, NJ

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3.

4.

5. 6.

Arlmgton Heights, IL). Laurie acid (sodium salt, MW = 222.3) (Sigma, St Louis, MO) Prepare unlabeled lauric acid stock (5 mM m 100 mM Tris-HCl, pH 7 5) Evaporate hexane solvent from [r4C]lauric acid. Dissolve m a small volume of 100 mA4 Tris-HCl, pH 7 5. Dilute [1-i4C]lauric acid with unlabeled laurtc actd dissolved tn 100 mA4 Tris-HCl, pH 7.5, to give a working solution at 5 mCi/mmol (1 mA4). Cofactor generating system: 26 mA4 (20 mg/mL) NADP+, 66 mM(20 mg/mL) o-glucose-6-phosphate and 66 mM (13 mg/mL) magnesium chloride (MgCl, * 6H,O) Glucose-6-phosphate dehydrogenase (40 U/mL) in 5 mM (1 5 mg/mL) sodium citrate (C6H,0,Na, 2H,O) (see Note 1). Enzymes. e.g., cDNA-expressed CYP4All (GENTEST, Wobum, MA) or human hver microsomes. Dilute m assay buffer to a working concentration of 2 5 mg protein/ml and keep on ice (see Note 2). Deprotemizmg agent: 94% acetomtrtle/6% glactal acettc acid. Scinttllatton fluid: Ulttma Flo M (Packard, Downers Grove, IL)

2.2. HPLC 1 Mobile phase A. 30% acetomtrile 2 mMperchloric acid 2. Mobile phase B. 100% methanol 3. Column: Nucleosil Cl8 column, 4 6 x 250 mm, 5+m particle size, (Sigma, St Louis, MO). 4. Detector: Radiometric

3. Methods 1. To each 1 5-mL microcentrifuge (mcubation) tube add 10 nmol of 5 mCt/mmol [ 1-i4C]launc acid (100 @4 final concentration) (prepared as descrtbed m Subheading 2.1., item 2) (see Note 3) 2. Add the following to each incubatton tube (total mcubatlon volume of 100 pL). a. 64 )JL of assay buffer b 5 pL of a solution contammg 26 mM NADP+, 66 mM n-glucose-6-phosphate, and 66 mM magnesium chloride c 1 pL of glucose-6-phosphate dehydrogenase (4 U/mL) m 5 mA4 sodium citrate 3 Prewarm incubation tubes to 37°C and add 20 pL of diluted enzymes (50 c(g protein) to Initiate the enzymatic reaction (stagger each mcubatlon with 15-s delay Intervals) (see Notes 4 and 5). 4. Incubate samples at 37°C for 15-30 min in a water bath (see Note 6). 5. Add 50 pL ice-cold 94% acetomtnle/6% glacial acetic acid to stop enzymatic reaction and place incubation tube on ice. 6. Centrifige reaction mixture at 10,OOOg for 3 mm 7. Inject 100 pL of supematant onto HPLC column 8. Run column at 45-50°C (see Note 7) at a flow rate of 1 mL/mm with a lmear gradrent from 75% mobile phase A, 25% mobile phase B to 53% phase A, 47%

Laurie Acid 12-Hydroxylation

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165

phase B over 23 mm. The mobile phase is then changed to 100% phase B over 1 min and the column IS then run for a further 9 mm with 100% phase B Under these conditions, retention times are 20 min for 11-hydroxylaurtc acid, 2 1.5 mm for 12-hydroxylauric acid, and 30 mm for lauric acid (see Note 8). 9. Prepare blank mcubation tubes by adding the complete mcubation mixture but with heat-inactivated enzymes. Process the blank mcubation tubes as per steps 4-8. 10. Quanttfication ts achieved using a flow-scmtillation detector (Packard Instruments) using scintillation fluid at a flow rate of 3 mL/min with a 0 5-mL flow cell Countmg efficiency should be determmed using a known quantity of 14C with scmtillation fluid and HPLC mobile phase in the same proportions as will be present during an analytical run (final volume 20-50 mL) (see Note 9). Using the scmtillation-fluid pump, fill the flow cell of the detector with this mixture Measure the radioactivity (cpm). The counting efficiency is given by cpm observed divided by the dpm present m 0.5 mL (the volume of the flow cell) Activity is calculated according to the following equation: [(dpm metabolite) x (total volume of mcubation plus stop addition)]/ [(counting efficiency) x (2200 dpm/nCi) x (specific activity in nCi/mnole) x (mg protein) x time x (volume injected for analysis)] 11 Calculate lauric acid 12-hydroxlase activity and express it as nmol product formed/ mm/mg microsomal protein or as nmol product formed/mm/nmol total P450. 12. Calculate net enzyme activity by subtractmg the activity m the blank from each of the unknown samples (see Note 10)

4. Notes 1 Laurie acid 12-hydroxylase assays can also be carried out with NADPH (e g , 1 rmV fInal concentration) in place of an NADPH-generatmg system (NADP+, o-glucose-6-phosphate, glucose-6-phosphate dehydrogenase) 2. Prepare only sufficient amounts of dilute microsomes for each experiment. The remainder of the undiluted microsomes can be stored at -80°C for future use (21). 3 Reported apparent Km values for launc acid 12-hydroxylation by human hver mtcrosomes range from 13-49 @4 (5,7). The assay is conducted at a substrate concentration of 100 pA4 in order to minimize the contribution of a higher Km form (apparent Km -550 @4) of lauric acid 12-hydroxylase in human liver microsomes (7) 4. With some P450 expression systems, it is necessary to reconstitute the recombinant P450 protein with NADPH-cytochrome P450 reductase, cytochrome b,, and lipid before initiating substrate oxidation (4). Conduct preliminary experiments to establish the amount of each component required for optimal catalytic acttvity 5. Conduct preliminary experiments to ensure that the assay is linear with respect to microsomal protein concentration. 6. Conduct prehmmary experiments to ensure that the assay is lmear with respect to incubation time.

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7. Column temperature can range from room temperature to 50°C The use of a controlled, elevated temperature provides greater reproducibility m retention times and lower column back pressures. 8 These gradient and column conditions provide a baseline separation of 11-hydroxylauric acid and 12-hydroxylauric acid using the followmg HPLC system. a Waters 7 17 automjector, three Waters model 5 10 pumps, Packard Instruments model 150TR flow-scintillation detector, and Waters Millenmm software package (version 2.15.01) Some adJustment m gradient conditions may be necessary for other analytical systems. The small difference in retention times between 1 l- and 12-hydroxylauric acid (-1.5 min) makes detection m HPLC column effluent by fraction collection and scintillation counting impractical 9. Counting efficiency should be determined m the presence of each of several different HPLC mobile-phase solvents, because quenching may vary based on solvent composition. In our experience the counting efficiency is constant at approx 0.84 across the mobile phase gradient described m this application 10 Laurie acid 12-hydroxylase activity m a panel of 17 human liver mmrosomes ranged from 0.3-l .9 nmol/mm/mg microsomal protein at 100 uA4 substrate concentration (C. Crespi, unpublished results).

Acknowledgments Supported m part by the British Columbia Health Research Foundatton (grant 119[95-l] to Thomas K. H. Chang) and the National Institutes of Health (grant DK33765 to David J. Waxman). Thomas K. H. Chang is the recipient of a Research Career Award m the Health Sciences from the Pharmaceutical

Manufacturers Association of Canada-Health Research Foundation and the Medical Research Council of Canada. References 1 Kawashima, H., Kusunose, E., Kubota, I , Maekawa, M , and Kusunose, M. (1992) Purification and NH*-termmal ammo acid sequences of human and rat kidney fatty acid w-hydroxylases. Biochlm Biophys. Acta 1123, 156462. 2 Imaoka, S., Ogawa, H., Kimura, S., and Gonzalez, F. J (1993) Complete cDNA sequence and cDNA-directed expression of CYP4A 11, a fatty acid o-hydroxylase expressed m human kidney DNA Cell BEOI 12, 893-899. 3. Kawashima, H., Kusunose, E., Kikuta, Y., Kinoshna, H , Tanaka, S., Yamamoto, S., Kishlmoto, T., and Kusunose, M. (1994) Purification and cDNA clonmg of human liver CYP4A fatty acid o-hydroxylase. J Biochem 116,74-80 4 Palmer, C.N.A., Richardson, T.H , Griffin, K.J., Hsu, M H., Muerhoff, A S., Clark, J E., and Johnson, E.F (1993) Characterization of a cDNA encoding a human kidney, cytochrome P450 4A fatty acid o-hydroxylase and the cognate enzyme expressed in Escherzchia colr. Biochim Blophys Acta 1172, 16 l-l 66. 5. Powell, P. K., Wolf, I., and Lasker, J.M. (1996) Identification of CYP4All as the maJor lauric acid o-hydroxylase m human liver microsomes. Arch Blochem Bzophys. 335,2 19-226.

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6. Castle, P. J., Merdmk, J. L., Okita, J. R., Wrighton, S A., and Okita, R. T. (1995) Human liver lauric acid hydroxylase activities. Drug Metub Dispos. 23,1037-1043. 7 Clarke, S. E., Baldwin, S J , Bloomer, J C., Ayrton, A. D., Sozio, R. S , and Chenery, R J (1994) Laurrc acid as a model substrate for the simultaneous determination of cytochrome P450 2El and 4A m hepatic mrcrosomes. Chem Res Toxlcol 7,836-842.

8 Amet, Y., Berthou, F., Baird, S., Dreano, Y., Bail, J. P., and Menez, J. F (1995) Validation of the (o-1)-hydroxylation of lauric acid as an zn wtro substrate probe for human liver CYP2El Biochem Pharmacol 50, 1775-l 782 9. Dirven, H. A. A. M., Peters, J. G. P., Gibson, G. G., Peters, W. H. M., and Jongeneelen, F. J (1991) Laurie acid hydroxylase activity and cytochrome P45OIV family proteins m human liver microsomes. Blochem. Pharmacol 42, 1841-1844 10. Amet, Y , Berthous, F., and Menez, J F. (1996) Simultaneous radiometric and fluorrmetric detection of laurtc acid metabolites using high-performance hqurd chromatography following esteriflcation with 4-bromomethyl-6,7-drmethoxycoumarm m human and rat liver microsomes. J, Chromatogr 681,233-239. 11 Pearce, R E , McIntyre, C J , Madan, A , Sanzgirr, U , Draper, A J , Bullock, P L., Cook, D C , Burton, L. A , Latham, J., Nevms, C , and Parkmson, A. (1996) Effects of freezing, thawing, and storing human liver microsomes on cytochrome P450 activity Arch Blochem Bzophys. 331, 145-169.

20 An Isocratic High-Performance Liquid Chromatographic Assay for CYP7Al-Catalyzed Cholesterol 7wHyciroxylation David J. Waxman and Thomas K. H. Chang 1. Introduction CYP7A1, which is the only CYP7A subfamily P450 tdentified to date (I), catalyzes cholesterol 7a-hydroxylation, the first, and rate-limiting, step m the converston of cholesterol to bile acids. CYP7Al has been Isolated from human liver and purified to apparent homogeneity (2). Rodent-model studies have established that CYP7Al is highly regulated by physiological factors that influence hepatic-bile-acid biosynthesis, including cholesterol feedmg, dmrnal factors, and bile acids, whtch feedback-inhibit the overall btosynthetlc pathway in large part at the level of CYP7AI gene expression (3). Relatrvely little is known about the regulation of human CYP7A1, although liver biopsy analyses have shown that hepatic CYP7A 1protem content is elevated m patients treated with the bile-acid sequesterant cholestyramme (4). Purified CYP7Al is catalytically active in cholesterol ‘la-hydroxylation (2) and immunoinhibition experiments with antihuman CYP7Al antibodies have shown that CYP7Al accounts for most of the cholesterol 7a-hydroxylase acttvtty in human liver microsomes (4). Thus, mlcrosomal cholesterol 7a-hydroxylase activity can be used as a marker for human liver CYP7Al. Several analytical methods have been developed to quantify hepatrc mrcrosomal cholesterol 7a-hydroxylation, including reversed-phase high-performance hquid chromatography (RPHPLC) (S), isotope dilution-mass spectrometry (6) and thin-layer chromatography (TLC) (7). This chapter describes a normal-phase, isocratic HPLC assay for the determination of cholesterol 7a-hydroxylase acttvity based on the conversion by cholesterol oxidase of the primary P450 metabohte From Methods m Molecular Brology, I/o/ 107 Cytochrome P450 Protocols Edlted by I R PhIllIps and E A Shephard 0 Humana Press Inc , Totowa, NJ

169

770 7a-hydroxycholesterol detected at 254 nm.

Waxman and Chang to 7a-hydroxy-4-cholesten-3-one,

which can be

2. Materials 2.1. Assay 1. Substrate: Cholesterol (MW = 386.7) (Sigma, St. Louis, MO) Prepare a 1 r&I (0.4 mg/mL) stock solution dissolved m toluene (see Notes 1 and 2) To prepare a 1 mA4 somcated aqueous suspension of cholesterol, dry down an aliquot of the stock solution, add distilled water, and sonlcate until a fine suspension IS achieved. 2 Specialty reagents. 7a-hydroxycholesterol (Steralolds, Wllton, NH) Cholesterol oxldase (Sigma, St. LOUIS, MO) Reconstitute to give a stock solution of 12 5 U/mL m 10 mMpotassmm phosphate buffer, pH 7.4, 1 mM dithiothreltol (DTT), 20% (v/v) glycerol. 3. Metabollte standard: 7a-hydroxy-4-cholesten-3-one (enzymatlcally produced from 7a-hydroxycholesterol by cholesterol oxldase) 4. Assay buffer: 100 mM HEPES, pH 7.4, contammg 0 025 nuI4 ethylenediamine tetra-acetic acid (EDTA) and 50 mA4NaF. 5. Cofactor: Prepare a 10 n-&I (8 3 mg/mL) stock solution of mcotmamide adenine dmucleotide phosphate (NADPH) and keep on ice (see Notes 3 and 4) 6 Enzymes: e.g., cDNA-expressed CYP7Al or human liver mlcrosomes Dilute in assay buffer to a working concentration of 10 mg protein/ml and keep on ice (see Note 5) 7. 5% (w/v) sodium cholate 8. Deprotemlzmg agent. methanol. 9. Extraction reagent: petroleum ether

2.2. HPLC Conditions 1 Mobile phase: hexane/2-propanol(80 20). 2 Column: Alltech silica column, 4.6 x 250 mm, 5-w particle size. 3. Detector: Ultraviolet (UV) at 254 nm.

3. Methods 1 Add the following to each assay tube (total incubation volume of 100 pL): a 50 & of assay buffer b. 10 pL of 1 miI4 somcated aqueous cholesterol suspension (100 pA4 final concentration) c. 20 pL of P450-contammg enzyme sample (to give 200 pg protein, see Notes 6 and 7). 2. Prewarm incubation tubes to 37OC and add 20 pL of 10 mA4NADPH (1 r&I4 final concentratton) to mltlate the enzymatic reaction (stagger each incubation with 15-s delay intervals). 3. Incubate samples at 37°C for 20-30 min in a shaking water bath (see Note 8).

Cholesterol 7a-Hyydroxylation

Assay

171

4. Add 150 pL dIstIlled water, 30 pL 5% (w/v) sodium cholate and 20 & cholesterol oxidase 5 Incubate at 37°C for 10 min (see Note 9) 6. Add 300 pL ice-cold methanol to stop the enzymatic reaction and then place incubation tube on ice. 7. Extract with 3 mL petroleum ether. 8. Centrifuge reaction mixture at 5OOOgfor 5 mm. 9. Transfer extract to a clean test tube and evaporate under a gentle stream of nitrogen 10. Reconstitute the residue with 100 p.L hexane/2-propanol(80:20). 11. Inject 20-40 p.L per 100 $ of reconstituted sample onto the HPLC column 12 Run the column with a mobile phase of hexane/%-propanol(80:20) at a flow rate of 1 mL/mm. Monitor eluant at 254 nm. Under these conditions the retention times are 4.8 mm for cholesterol and 6 4 min for 7a-hydroxy-4-cholesten-3-one. 13. Measure the background activity in blank incubation tubes containing the complete incubation mixture but heat-inactivated enzymes Process the blank incubation tubes as per steps 3-12. 14. Prepare standards by adding a known amount of authentic 7a-hydroxycholesterol metabolite to tubes containing the complete incubation mixture but with heatmactlvated enzymes. Process the mcubatlon tubes as per steps 3-12. 15. Determine the amount of product formation by comparison to a standard curve derived from 7a-hydroxy-4-cholesten-3-one. 16. Calculate cholesterol 7a-hydroxlase activity and express it as nmol product formed/mm/mg mlcrosomal protein or as nmol product formed/mm/nmol total P450 (see Note 10).

4. Notes 1 Cholesterol can also be added as a Tween-80 solution (7) or directly as lowdensity hproprotein (LDL)-bound cholesterol (0.4 mg cholesterol/mg LDL). 2. Use a freshly prepared aqueous suspension of cholesterol for each experiment 3. Prepare fresh solutions of NADPH for each experiment. Note that NADPH IS light-sensitive and pH-sensitive. 4. Cholesterol 7a-hydroxylase assays can also be carried out with an NADPH-generatmg system (e g., NADP+, D-glucose-6-phosphate, glucose-6-phosphate dehydrogenase) m place of NADPH. 5. Prepare only sufficient amounts of dilute microsomes for each experiment The remainder of the undiluted mlcrosomes can be stored at -80°C for future use (8) 6. With some P450 protein-expression systems, it ISnecessary to reconstitute the recombinant P450 protein with NADPH-cytochrome P450 reductase, cytochrome b,, and lipid before mitiatmg substrate oxidation (9). Conduct preliminary experiments to estabhsh the amount of each component required for optimal catalytic activity. 7. Conduct preliminary experiments to ensure that the assay is linear with respect to microsomal protem concentration 8. Conduct prelimmary experiments to ensure that the assay 1slmear with respect to incubation time.

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9 The enzymatically produced 7a-hydroxycholesterol metabolite is converted to the UV-active 7a-hydroxy-4-cholesten-3-one by cholesterol oxidase m order to increase the detection sensitivity of the assay (10) The latter standard is produced enzymatically by incubating known amounts of 7a-hydroxycholesterol with cholesterol oxrdase. For this method to be valid, it IS essential to verify that the cholesterol oxtdase effects a quantitative conversion of 7a-hydroxycholesterol to 7a-hydroxy-4-cholesterol-3-one 10. Typical level of cholesterol 7a-hydroxylase activity m human ltver mtcrosomes is approx 0.02 nmol/min/nmol total P450 content (2).

Acknowledgments Supported in part by the National Institutes of Health (grant DK33765 to David J. Waxman) and the British Columbia Health Research Foundation (grant 119(95-l) to Thomas K. H. Chang). Thomas K. H. Chang is the recipient of a Research Career Award m the Health Sciences from the Pharmaceutical Manufacturers Association of Canada-Health Research Foundation and the Medical Research Council of Canada.

References Nelson, D R , Koymans, L , Kamataki,

T., Stegeman, J. J., Feyereisen, R ,

Waxman, D J., Waterman, M R., Gotoh, O., Coon, M. J., Estabrook, R. W., Gunsalus, I C , and Nebert, D W (1996) P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature Pharmacogenetus 6, l-42 Nguyen, L B , Shefer, S , Salen, G , Ness, G., Tanaka, R. D , Packin, V., Thomas, P., Shore, V , and Batta, A. (1990) Purification of cholesterol 7a-hydroxylase from human and rat liver and production of mhibitmg polyclonal antibodies. J. Blol. Chem. 265,454 1-4546. Waxman, D. J. (1992) Regulation of liver-specific steroid metabohzmg cyto-

chromesP450: Cholesterol7a-hydroxylase,bile acid 6l3-hydroxylase,and growth hormone-responsive steroid hormone hydroxylases J Steroid Blochem A401 B~ol 43,1055-1072. Maeda, Y , Eggertsen, G , Nyberg, B , Setoguchr, T , Okuda, K , Emarsson, K , and Bjorkhem, I (1995) Immunochemtcal determmation of human cholesterol 7a-hydroxylase Eur J. Blochem 228, 144-148 Chiang, J. Y. L. (199 1) Reversed-phase high-performance hquid chromatography assay of cholesterol 7a-hydroxylase. Methods Enzymol 206,483-49 1. Emarsson, K , Angelm, B., Ewerth, S., Nilsell, K , and Bjorkhem, I (1986) Bile acid synthesis m man. Assay of hepatrc microsomal cholesterol 7a-hydroxylase activity by isotope dilutron-mass spectrometry. J Lipid Res 27, 82-88. Waxman, D. J. (1986) Rat hepatic cholesterol 7a-hydroxylase: Biochemical properties and comparison to constitutive and xenobiottc-mducible cytochrome P-450 enzymes Arch. Biochem. Blophys. 247,335-345.

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8. Pearce, R. E., McIntyre, C. J., Madan, A., Sanzgiri, U., Draper, A. J., Bullock, P L., Cook, D. C., Burton, L. A., Latham, J., Nevins, C., and Parkinson, A (1996) Effects of freezing, thawing, and storing human ltver mtcrosomes on cytochrome P45O activrty. Arch Biochem Biophys 331, 145-169 9. Karam, W. G. and Chlang, J. Y L. (1994) Expression and purrfication of human cholesterol 7a-hydroxylase m Escherichia co11 J. Lipid Res 35, 1222-l 23 1 10. Ogishima, T. and Okuda, K. (1986) An improved method for assay of cholesterol 7a-hydroxylase activity Anal. Biochem 158,228-232.

21 Use of 7-Ethoxycoumarin to Monitor Multiple Enzymes in the Human CYPI, CYP2, and CYP3 Families David J. Waxman and Thomas K. H. Chang 1. Introduction Chapters 10-20 describe assay methods for determmmg enzyme activities that are frequently employed as markers for specific cytochrome P450 (P450) enzymes m human tissues. However, under certain circumstances, e.g., when the objective is to verify that individual cDNA-expressed P450 enzymes are expressed in catalytically active form, it is often advantageous to assay the panel of enzymes using a “general” P450 substrate, one that 1smetabolized by multiple P450 enzymes. An example of a general P4.50 substrate IS 7-ethoxycoumarin. Enzyme kinetic analysis with human liver microsomes indicates that 7-ethoxycoumarin IS metabohzed by at least two P450 enzymes or by two groups of P450 enzymes that are kinetically distmgulshable (1,2). The apparent Km and maximal specific activity (i.e., specific activity at saturating substrate concentration) values for ‘I-ethoxycoumarin U-deethylation by human liver mlcrosomes are 1l-27 @I and 0.05-0.2 1 nmol/mm/mg mlcrosoma1protem, respectively, for the low Km component. By contrast, for the high Km component, the apparent Km is approx 150 @4 and the maximal specific activity is 0.35-0.95 nmol/min/mg microsomal protem (2). Experiments with recombmant human P45Os have shown that multiple enzymes in the human CYPl, CYPZ, and CYP3 families are active catalysts of 7-ethoxycoumarm O-deethylation (3). Consequently, the 7-ethoxycoumarin O-deethylase assay has been used to verify the catalytic activity of a panel of recombinant human P45Os in the CYPl, CYP2 and CYP3 families (46). This chapter describes a modification (7) of a widely used spectrofluorometric method (8) for the deterFrom Methods m Molecular Biology, I/o/ IO7 Cytochrome P450 Protocols E&ted by I R Phlll!ps and E A Shephard 0 Humana Press Inc , Totowa, NJ

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mmatlon of 7-ethoxycoumarrn O-deethylase activity. The formation of 7-hydroxycoumarin from 7-ethoxycoumarm can also be quantified by highperformance liquid chromatography (HPLC) with fluorescence detection (9JO). 2. Materials 1. Substrate: 7-ethoxycoumarin (MW = 190.2) (Aldrich, Mdwaukee, WI) Prepare a fresh 50 mA4 (9 5 mg/mL) stock solution m methanol (see Note 1) 2. Metabohte standard. 7-hydroxycoumarm (umbelhferone, MW = 162. 1) (Aldrich). 3. Assay buffer. 100 mMpotassium phosphate, pH 7.4, containing 20% (v/v) glycerol and 0.1 mM ethylenediamine tetra-acetic acid (EDTA) (see Note 2) 4 Cofactor: 10 mM (8.3 mg/mL) mcotmamide ademne dmucleotide phosphate (NADPH) stock solution. Prepare fresh and keep on ice (see Notes 3 and 4). 5 Enzymes: Recombmant human P450 or human lrver mlcrosomes Dilute m assay buffer to a working concentration of 1 or 2 mg protein/ml and keep on ice (see Note 5). 6 Deproteinizing agent. 2 A4 HCl. 7. Extraction solvent. chloroform 8. Back-extractron solutton: 30 nn’t4 (6 mg/mL) sodium borate (Na,B407), pH 9.2 9. Equipment includes a spectrofluorometer

3. Methods 1. Add the following to each mcubation tube at room temperature (see Note 2) (total mcubatton volume of 200 &) a. 156 I& of assay buffer b 4 p.L 50 mA4 7-ethoxycoumarin (1 r&4 final concentration) c. 20 & diluted recombinant P450 (or human liver microsomes) (20 or 40 ~18 protein) (see Notes 6 and 7) 2. Prewarm incubation tubes to 37°C and add 20 & of 10 mMNADPH (1 mA4 final concentration) to initiate the enzymattc reaction (stagger each mcubatton with 15-s delay intervals). 3. Incubate samples at 37°C for 30 min m a shaking water bath (see Note 8). 4. Add 25 pL me-cold 2 M HCl to stop enzymatic reaction and place the mcubation tube on ice. 5 Extract with 450 pL chloroform (see Note 9). 6. Centrifuge reaction mixture at 3000g for 5 mm 7. Transfer 300 I.~Lof the organic phase (bottom layer) to a clean test tube and backextract with 1 mL of 30 mA4 sodium borate, pH 9.2. 8. Centrifuge at 3000g for 5 mm. 9. Remove the aqueous (top) layer and measure the fluorescence at an excitation wavelength of 370 nm and an emrsston wavelength of 450 mn (see Note 10) 10. Prepare blank incubation tubes by adding the complete incubation mixture but with heat-inactivated recombinant P450 (or human liver mtcrosomes) Process the blank incubation tubes as per steps 3-9.

7-Ethoxycoumarin

0-Deethylation

Assay

177

11. Prepare standards by addmg a known amount (e.g , 0, 0.1, 0.2, 0,4, 0.8, and 1 2 nmol) of authentic 7-hydroxycoumarin metabohte to tubes contammg the complete mcubatron mixture but wrth heat-inactivated recombinant P450 (or human liver microsomes). Process the incubatron tubes contammg the standards as per steps 3-9 12. Calculate the net fluorescence of each unknown sample and standard by subtracting the fluorescence reading of the blank from that of the unknown sample or the standard. 13. Plot a standard curve of net fluorescence agamst amount of authenttc 7-hydroxycoumarm and determine the amount of product formatton in each unknown sample by linear regressron analysts. 14 Calculate 7-ethoxycoumarin O-deethylase activity and express It as nmol product formed/min/mg mrcrosomal protein or as nmol product formed/min/nmol total P450

4. Notes 1. Use a freshly prepared stock solutron of 7-ethoxycoumarm for each experiment 2 Solubdity considerations requrre that the assay tubes be prepared at room temperature, rather than on ice, and that glycerol be included in the incubation mrxture in order to achieve true solubrhty for 1 mA47-ethoxycoumarin. Alternatively, the final concentratron of 7-ethoxycoumarin may be decreased to 0 2 or 0 5 II&I, m which case the glycerol may be eliminated. Some P45Os exhibit higher activity when assayed m the absence of glycerol. 3 Prepare fresh solutions of NADPH for each experiment. Note that NADPH is light-sensrtrve and pH-sensitive. 4. The 7-ethoxycoumarm O-deethylase assay can also be carried out with an NADPH-generating system (e.g., NADP+, n-glucose-6-phosphate and glucose6-phosphate dehydrogenase) m place of NADPH. 5. Prepare only sufticrent amounts of dilute human liver microsomes for each experiment. The remamder of the undiluted mrcrosomes can be stored at -80°C for future use (II). 6. With yeast and some other P450 protein-expression systems, rt IS necessary to reconstitute the recombinant P450 protein with NADPH-cytochrome P450 reductase, cytochrome b5, and hpid (e.g., drlauroylphosphatrdylcholme) before initiating substrate oxidation (5,12) 7 Conduct prehmmary experiments to ensure that the assay IS linear with respect to microsomal protein concentratron 8. Conduct preliminary experiments to ensure that the assay 1slinear with respect to incubation time 9 “Wet” prpet tips with the organic solvent before use 10 Determine the optimal excttation wavelength and emtsston wavelength to be used with each particular fluorimeter by examining the excitation and emission spectra of 7-ethoxycoumarm and 7-hydroxycoumarin (13) generated by that instrument

Waxman and Chang

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Acknowledgments Supported in part by the National Institutes of Health (grant ES07381 to David

J Waxman)

and the British

Columbia

Health

Research

Foundation

(grant 119(95-l) to Thomas K. H. Chang). Thomas K. H. Chang 1sthe recipient of a Research Career Award in the Health Sciences from the Pharmaceutical Manufacturers Association of Canada-Health Research Foundation and the Medtcal

Research Counctl of Canada.

References 1. Boobis, A. R and Davies, D S. (1984) Human cytochromes P-450 Xenobiotica 14,151-185

2. Yamazaki, H., Inoue, K., Mimura, M., Oda, Y., Guengerich, F P., and Shimada, T. (1996) 7-Ethoxycoumarm 0-deethylation catalyzed by cytochromes P450 lA2 and 2El m human liver mtcrosomes Bzochem. Pharmacol 51,3 13-3 19. 3. Waxman, D. J., Lapenson, D P , Aoyama, T., Gelbom, H V., Gonzalez, F. J., and Korzekwa, K. (199 1) Steroid hormone hydroxylase specificities of eleven cDNAexpressed human cytochrome P45Os. Arch Blochem Bzophys 290, 160-l 66 4. Chang, T. K H., Weber, G. F , Crespi, C L , and Waxman, D J (1993) Differential activation of cyclophosphamide and ifosphamide by cytochromes P-450 2B and 3A in human liver microsomes Cancer Res. 53,5629-5637 5. Chang, T. K. H., Yu, L., Goldstein, J. A., and Waxman, D. J. (1997) Identtficatton of the polymorphically expressed CYP2C 19 and the weld-type CYP2C9-Ile35g allele as low-Km catalysts of cyclophosphamide and ifosfamide activation. Pharmacogenetics 7,2 1 l-22 1 6. Butler, A. M. and Murray, M. (1997) Biotransformation of parathion in human liver: Participation of CYP3A4 and its mactivation during microsomal parathion oxidation. J Pharmacol Exp Ther 280,966-973. 7 Waxman, D. J and Walsh, C (1982) Phenobarbital-induced rat liver cytochrome P450: purification and characterization of two closely related isozymtc forms. J Biol Chem. 257, 10,446-10,457 8. Greenlee, W. F. and Poland, A. (1978) An improved assay of 7-ethoxycoumarm 0-deethylase activity: Induction of hepattc enzyme acttvtty in C57BL/6J and DBA/2J mice by phenobarbital, 3-methylcholanthrene and 2,3,7,8,-tetrachlorodibenzo-p-dioxin. J. Pharmacol. Exp. Ther 205,596-605. 9. Zitting, A. (1981) A sensitive liquid chromatographic assay of ethoxycoumarin deethylase with fluorescence detection. Anal. Blochem 115, 177-180. 10. Evans, R. R and Rellmg, M. V. (1992) Automated high-performance liquid chromatographic assay for the determmation of 7-ethoxycoumarm and umbelhferone J. Chromatogr 578,141-145. 11 Pearce, R. E., McIntyre, C J , Madan, A , Sanzgtri, U., Draper, A. J., Bullock, P. L., Cook, D. C., Burton, L. A., Latham, J., Nevms, C , and Parkinson, A. (1996) Effects of freezing, thawmg, and storing human liver mrcrosomes on cytochrome P450 activity. Arch Biochem Blophys 331, 145-169.

7-Ethoxycoumarin

0-Deethylation

Assay

179

12 Yamazaki, H , Nakano, M , Gillam, E. M J., Bell, L C., Guengerrch, F P , and Shimada, T. (1996) Requirements for cytochrome b, in the oxidation of 7-ethoxycoumarin, chlorzoxazone, aniline and N-nitrosodimethylamine by recombmant cytochrome P450 2E1 and by human liver mrcrosomes. Biochem Pharmacol 52,301-309.

13 Prough, R. A., Burke, M. D., and Mayer, R. T. (1978) Direct fluorometnc methods for measuring mrxed-functton oxidase actwny. Methods Enzymol. 52,372-377

22 Expression in E. co/i Christopher

of Eukaryotic M. Jenkins,

Cytochromes

P450

lrina Pikuleva, and Michael R. Waterman

1, Introduction Although much has been learned from the study of relatively abundant cytochromes P450 (e.g., P450,,, from Pseudomonasputida cytosol, P4501A2 and P4502B4 from rabbit liver microsomes, and P45Osccfrom bovine adrenal cortex mitochondrra), the development of cytochrome P450 heterologous expression systems (l-3), especially in Escherzchza coli (4), has provided an opportunity to address questions relating to the structure/fi,mctron of less abundant forms of cytochrome P450 whose cDNAs (CYP cDNAs) have been isolated by recombinant DNA technologies. Among the advances achieved through cytochrome P450 heterologous expression systemsare: 1 the abrlity to study cytochromes P450 ldentlfied only by molecular cloning techniques, 2. the abihty to study a single cytochrome P450 in the absenceof contaminatmg forms, and

3. the ability to produce large quantitiesof both wild-type and mutant cytochromes P450 for b~ochem~callbiophysrcal characterrzation. This chapter will first focus on a general strategy for preparing a CYP cDNA of interest for high-level hemoprotein expression in E. coli by describing modrfkatlons of two mammalran cytochromes P450 as examples, P45Oc17 (a microsomal sterordogenic P450) and P45Oscc(a mitochondrial steroidogenic P450). Sections on expressron conditrons, cytochrome P450 quanttficatron m whole cells, and preparation of cytochrome P450-containing spheroplasts and E. coli membranes will follow. Finally, a relatively simple solubillzatron/purrfication procedure for histidine-tagged P45Oc17 will be described. From Methods m Molecular Bology, Vol Edlted by I Fl Phtlllps and E A Shephard

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107 Cytochrome 0 Humana

Press

P450 Protocols Inc , Totowa,

NJ

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Jenkins, Pikuleva, and Waterman

2. Materials 2.1. Expression

Vector Construction

1 General molecular cloning supplies/equipment An excellent reference hstmg these is Molecular Cloning . A Laboratory Manual (5) 2. Appropriate restrictton enzymes, T4 DNA ligase, native Pfu DNA polymerase (Stratagene, La Jolla, CA) or similar thermostable DNA polymerase, and buffers 3 Cytochrome P450 (CUP) cDNA of interest. 4 Recommended expression vectors. pCWOrt+ (6), pSP 19g 1OL (Gibco-BRL, Gaithersburg, MD), pKK233-2 (Pharmacta Biotech, Ptscataway, NJ), pTrc99A (Pharmacia Btotech, Ptscataway, NJ). 5 Appropriate mutagenic oligonucleotides (see Subheading 3.1.).

2.2. E. coli Strains and Media 1 Recommended E colz strams: JM109 (Stratagene, La Jolla, CA), TOPP3 (Stratagene), DHSa (Clontech, Palo Alto, CA), and XL-l Blue (Stratagene). 2. Luria Bertam (LB) Medium. 10 g/L NaCl, 10 g/L peptone, 5 g/L yeast extract Dissolve m water and autoclave 3 Terrific Broth (TB) Medium. 1 L TB medmm 1sprepared by dissolvmg 12 g of Bacto-Tryptone (Dtfco, Detroit, MI), 24 g of Bacto-Yeast Extract (Dtfco), 2 g of Bacto-Peptone (Difco), and 8 mL of 50% (v/v) glycerol m 860 mL of distilled water. Autoclave for 25 mm m a 2 8 L Fembach flask (remove medium munedtately after autoclaving) and allow to cool Immediately before starting the culture, add 100 mL of a sterile 1OX potassium phosphate solution (0.17 MKH*PO,, 0.72 A4 KZHP04, pH 7.4) and amptcillm to 50 ug/mL. If JM109 cells are being used, the medium should be supplemented with 1 mM thiamine. 4. Glycerol (store at room temperature). 5. Sterile filtered stocks of amptctllm (Amp), 50 mg/mL, (store at -2O’C). 6. 1 M Isopropyl-1-thio-P-o-galactopyranoside (IPTG) (store at -20°C) 7 200 m/r4 &Aminolevulmtc acid (&ALA), (store at -20°C) 8 1 M thiamine (store at 4°C). 9 Equipment includes Erlenmeyer flasks (250 mL and 1 L for 50 and 200 mL cultures, respectively), 2.8 L Fembach flasks, Innova 4300 Incubator Shaker (New Brunswick Sctenttfic, Edison, NJ) or equivalent.

2.3. Reagents,

Chromatography

Resins, and Equipment

1 For measuring reduced-CO/reduced difference spectra: Double-beam

scanning

spectrophotometer(Cary, Ammco or equivalent), carbon monoxide (Danger: poisonous gas), quartz cuvets (1.4 mL), and sodmm hydrosulftte (sodium dithionite, Sigma, St. Louts, MO) 2. Detergents. 10% stocks of Trnon X-100 (Boehrmger-Mannhelm, Indtanapohs, IN), previously precondensed (7), Triton X- 114 (Boehringer-Mannheim) sodmm cholate (Sigma, St. Louis, MO). Store all stocks at 4’C

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3. Resins: N12+ mtrilotriacetic acid agarose (NTA agarose, Qiagen, Chatsworth, CA), BioGel HTP hydroxylapatite (HA) (Bio-Rad, Hercules, CA). 4. 1 MK,HPO, (store at 4°C). 5. 1 MKH,PO, (store at 4’C) 6. 5 M NaCl (store at room temperature). 7. 2.5 M Glycme, pH 7.4 (store at room temperature). 8. 0.5 M EDTA, pH 8.0 (store at room temperature). 9. 1 M dithiothreitol (DTT) (store at -2O’C). 10. 50 mMphenylmethyIsulfony1 fluoride (PMSF): dissolve in isopropanol and store at -20°C). 11 TES buffer: 100 mA4 Tris-acetate, pH 7.6, 500 mM sucrose, 1 mM EDTA (store at 4’C). 12 50 mg/mL Lysozyme (in water) (store at -20°C) 13. KMDG buffer: 100 n&f potassium phosphate, pH 7.4,6 n&f magnesium acetate, 0.1 n-J4 DTT, 20% (v/v) glycerol 14. KP, Buffer A* 50 &potassium phosphate, pH 7.4,30% glycerol (v/v), 0 1 mM DTT, 0.1 mM EDTA, P450 substrate (40 l&f progesterone for P45Oc 17), 0 1 mA4 PMSF, DNase (1 pg/mL) (Boehrmger Mannhelm, Indianapolis, IN, prepare as a 10 mg/mL stock in 50% (v/v) glycerol, 1 mA4 CaCl, and store at -2O’C) 15 KPi Buffer B: 50 tipotassmm phosphate, pH 7.4,20% (v/v) glycerol, 0.1 mM PMSF, 0.1% (v/v) Triton X- 100,O 1% (w/v) sodium cholate, P450 substrate (40 fl progesterone for P45Oc 17) 16. KPi Buffer C: 50 mM potassium phosphate, pH 7 4, 20% (v/v) glycerol, 0 1 M NaCl, 50 Wglycine, 0.2% (v/v) Triton X- 100,0.2% (w/v) sodium cholate, P450 substrate (40 flprogesterone for P45Oc17), 0.2 M histidme, pH 7.4 (store at 4°C). 17. KPi Buffer D: 1 mA4 potassium phosphate, pH 7.4, 20% (v/v) glycerol, 0 1% (v/v) Triton X- 100 18. Equipment includes Branson 250 somcator or equivalent, low-pressure chromatography columns (e g , Econo-Columns from Bio-Rad), RediFrac fraction collector (Pharmacia Biotech) or equivalent.

3. Methods 3.1. Modification

of a CYP cONA for Subcloning

and Expression

Of the eukaryotic CYP cDNAs that have been highly expressed in E colz, almost all have been mutated in a similar way which increases mRNA translational efficiency. This section outlines a general strategy to modify the 5’

and 3’-ends of a given CYP cDNA of interest, using P45Oc17 and P45Oscc as examples. 1 5’ End Modification. The 5’-primer should be designed to a. introduce a unique restriction site at the first codon of the sequence (for P45Oc17, this is an NdeI site and for P45Oscc this is an NcoI site, both containing the methionine codon),

184

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3.

4.

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Jenkins, fikuleva,

and Waterman

b. introduce an alanme codon (GCU) m the second positton of the sequence, and c. Increase the AT content of the S-end, particularly codons 4 and 5, by stlent mutagenesis. This reduces the potential for mRNA secondary structure which may mhibtt ribosome binding/translation. The GCU codon has been incorporated m the second position m both P45Ocl7 and P45Oscc (Figs. 1 and 2) because many highly expressed genes m E. co11have been found to have this as a second codon (8). In addition, nucleotides at positions +l 1 to +14 were found to be 70% AiT m these genes. For example, two primers that were designed for modifymg/subclonmg the nattve bovine CYP17 cDNA mto pCWOri+ (4,9,20) are shown m Fig. 1 Mttochondrial cytochrome P450 sequences should be modified so as to introduce alanme as the second codon and to ehminate the nutochondnal signal pepttde thereby producmg the mature form of the enzyme in E colr (11-13) (Fig. 2) (see Note 1) 3’-End Modificatton. The 3’-primer should be destgned to introduce a unique restrtctton site at the 3’-end of the cDNA, thereby eliminating any extraneous 3’noncoding sequence, which, m at least two cases, has been found to suppress expressed cytochrome P450 protein levels (24). Four histidine residues (a His tag) can be introduced at the C-terminus of the protein by mutating the original stop codon (to histidine) and msertmg three histidme codons and a new stop codon, followed by a umque restrictton site (Fig. 1) (see Note 2) Polymerase Chain Reaction (PCR). Ideally, a high-fidelity thermostable polymerase should be used. We have had the best results with native Pfu polymerase (Stratagene). A typical PCR sample should contam 0.2-0.5 uJ4 of each oligonucleotide primer, 1O-l 00 ng of double-stranded template DNA, 0.2 mM dNTPs (0.05 mA4of each dNTP), Pfu polymerase (2.5 U), and buffer. Depending on the complementartty and AT content of the oligonucleotides (approx 40 bases m length), condttions for PCR (12-18 cycles) should be approximately as follows. 30 s at 95“C (initial denaturing step), 30 s at 95’C (cycle denaturing step), 1 mm at 30-4O”C (cycle annealing step), and 2 mm at 68°C (cycle extension step) Followmg PCR, Klenow fragment of DNA polymerase I (1 U) should be added and the mixture incubated at 37°C for 10 mm to fill in ends of PCR products The products should then be purified by agarose gel electrophorests (the authors use the Qtaex II kit from Qiagen to remove the products from the gel after electrophoresis), digested with the appropriate restriction enzymes, and ltgated mto the appropriately digested expression vector (e.g , pCWOri+ for P45Oc17) using T4 DNA ltgase (see Note 3). At this point, the ligation mixture can be directly used to transform heat-shock-competent E colt cells (see Note 4). Several clones should be selected, their plasmids purtfied and analyzed with the restriction enzymes that cut at the introduced sues. Clones that have inserts of the appropriate size should be imttally sequenced using universal and reverse primers (to sequence the 5’ and 3’ ends of the insert, because these regions are most susceptible to mutations) We have found the SequiThermTM cycle sequencing kit (Eptcentre, Madison, WI) to be very reliable. The remainder of a potentially correct clone can be directly sequenced using

Cytochrome f45U in E. coli

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5’ Modification A L L GCT CTG TTA

M

5'-GG Native Bovine P45Ocl7

CATATG

22

3'-TAC M

L TTA

A V GCA GTT

F TTT

L L T CTG CTC ACC C-3'

ACC GAC GAG GAC CGA CAG AAA GAC GAG TGG G-5' W L L L A V F L L T

3’ Modification Native Bovine P45Ocl7

3’ Antisense Primer

E 5'-GAG

0 S T P stop GGT AGC ACC CCA TGA -3’

3'-CTC E

CCA TCG TGG GGT GTA GTG GTA GTG ACITTCGAAIT-5' G S T P H Ii Ii H stop

Hind III I

Fig. 1 Alignment of 5’-and 3’-P45Oc 17 Mutagenic Primers with the Native Bovine P45Oc17 Sequence. Restrrctton sites are boxed and labeled. Underlined bases indicate modifications Ammo acid residues for each codon are given m bold m the smgleletter code

5’ Sense Primer

5’ Modification Ncol

EcoRIM A S T K 5'-GGGAATTCC ATG GCT AGT ACA w Mature Bovine P45Oscc

3'-TAG I

T P R P Y S E ACT CCA CGC CCC TAC AGT GAG AT-3'

AGG TGT TTC TGG GGA GCG GGG ATG TCA CTC TA-5' S T K T P R P Y S E

Ftg. 2. Alignment of the 5’-P45Oscc Mutagenic Primer with the Native Bovine P45Oscc Sequence. Restriction sttes are as labeled. Underlined bases indicate modifications. The first ammo acid residue shown (I) for P45Oscc corresponds to the first amino acid in the mature protein (residue 40 m the precursor sequence) internal prtmers. Alternatively, a large internal fragment (defined by unique restrictron sites) of the original CYP cDNA can be substituted into a potentially correct clone (expression vector containing the PCR-derived CYP cDNA) This can obviate sequencing the majority of the PCR-derived insert.

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3.2. Growth Conditions for Expression of Cytochromes P450 in E. coli (see Note 5) 1. Prior to starting the expression culture, small cultures (2-10 mL) of bacteria should be grown overnight in LB medium containing ampicillin (100 pg/mL). 2 Add between 1 mL (for P45Oc 17) and 10 mL (for P45Oscc) of LB culture to each L of TB medium (l*lOOO to 1.100 dilution) (see Note 6) 3 Grow the cells in a shaker Incubator for approx 2-3 h at 250 rpm, 37°C (ODhoO = 0.3-0.5) (see Note 7). At this point, IPTG and &ALA (for otherwise lowexpressing cytochromes P450) should be added to the medium to final concentrations of 1 mA4 and 0 5 mM, respectively (see Notes g-10). 4 Grow the cells for an addltional48 h at 30°C (see Notes 11 and 12) For P45Oc 17, we have found that a low shaking speed (150 rpm) IS optimal, whereas for P45Oscc, a higher shaking speed (2 10 rpm) results m substantially increased levels of expression (see Notes 13 and 14).

3.3. Quantification of Expressed Cytochromes P450 in Whole Cells (see Notes IS-18) 1 Centrifuge 4 mL of the culture at 2000g for 10 mm, Place remainder at 4°C 2 Resuspend the pellet of cells in an equal volume of 100 mM potassium phosphate, pH 7.4,20% (v/v) glycerol, 10 Wglucose at 4°C. 3. Add a small amount of sodium dlthlonite (a few milligrams) and mix to dissolve. 4. Add l-l .2 mL of resuspended cells to each quartz cuvet, place the cuvets m the spectrophotometer, blank the instrument between 400 and 500 nm, and record a baseline 5. Remove the sample cuvet and bubble in carbon monoxide at a moderate rate (l-2 bubbles/s) for 30-60 s Mix the sample by mverslon using a piece of parafilm. Record spectra until the absorbance at 450 nm no longer increases. 6 The cytochrome P450 concentration present 1s calculated from the difference between the absorbance at 450 nm (peak) and 490 nm using an extmctlon coeffcient of 91 (mW’cm-‘) according to Omura and Sato (IS).

3.4. Preparation of Spheroplasts 1 Centrifuge remaining cells (see Subheading 3.3., item 1) at 2000g for 15 min at 4°C and discard supematant. Determine wet cell mass (typically 15 g/L of culture) and thoroughly resuspend cells with an electronic pipettor in cold TES buffer, 2-3 mL per gram of wet cell mass. Dilute this suspension with TES buffer to 10 mL per gram of wet cell mass. 2. Add lysozyme to 0.5 mg/mL and slowly add an equal volume of ice-cold 0 1 mM EDTA, pH 8.0, while stirring moderately with a magnetic stirrer 3. Slowly stir for 20-30 min at 4°C. 4. Pellet spheroplasts at 5OOOgfor 15 mm at 4°C and discard supematant

Cytochrome P450 in E. coli 3S.lso/stion

187

of E. coli Membranes

1 Using a teflon homogenizer, thoroughly resuspend spheroplasts m 2 mL of icecold KMDG buffer per gram of spheroplasts and transfer to a 50 mL polypropylene conical tube(s) m a salt/ice bath (-5°C). 2. Add PMSF to 0.2 m&fand somcate (-50% power, 6 x 20 s bursts, allowing time for the sample to cool between bursts) The suspension should noticeably change from opaque to partially translucent whrle turning darker m color 3 Centrifuge at 12OOg for 15 min to remove cell debris. It may be necessary to resonicate the pellet m fresh KMDG buffer to improve recovery. 4. Centrifuge supematant at 150,OOOg for 60 min at 4°C discard the resulting supematant (cytosol) and resuspend the pellet (membranes) m cold 0.25 M sucrose using a Teflon homogenizer. Aliquot the resuspended membranes, determine cytochrome P450 content (see Subheading 3.3. and Notes 14-17), freeze in liquid nitrogen, and store at -7O’C.

3.6. Purification of His-Tagged Cytochromes P450 from E. coli 3.6.1. Triton X- 774 Solubiliza tion

and Phase Separation (see Notes 19-21) 1. Add 3 mL of KP, buffer A per gram of spheroplasts (P450 substrate, DTT, DNase, and PMSF should be added mrmediately before use), resuspending and homogenizmg the pellet with a Teflon homogenizer. Note: It 1s essential to completely remove all of the TES buffer from the previous step or the phase separation will be less than optrmal. 2. Transfer to a glass beaker or flask and add Triton X-l 14 dropwise to a final concentration of 0.7% while stirring moderately wrth a magnetic stirrer Stir for an additional 30 min. 3. Centrifuge at 100,OOOg for 30 mm. A dark reddish-brown detergent-rich phase should be present near the top of each centrifuge tube. Separate this layer from the detergent-poor phase using a plastic Pasteur pipet. Recentrifugation may be necessary to more completely remove the detergent-poor phase. 4. Gradually dilute the cloudy detergent-rich phase with a IO-fold excess of KP, buffer B The solution will become transparent withm a few mm. 5. Determme cytochrome P450 content spectrophotometrrcally (see Subheading 3.3. and Notes 14-17)

3.6.2. NP2 NTA Agarose Chromatography 1, Chill a column (with appropriate fittings and tubmg for gravity-flow chromatography) at 4’C. 2. Pour a suspension of Nif2 NTA agarose resin (4°C) into the column and allow to stand for 10 min. Use approx 1 mL of resin for every 50-100 nmol of cytochrome P450 (see Note 22). Open outlet valve and drain the excess hquid from the column until the hquid level reaches the top of the settled resin bed. Do not allow the

Jenkins, Pikuleva, and Waterman

188

column to run dry at any point. Usmg a pipet, slowly add 1 column volume of KP, Buffer B (wtthout dtsturbmg the column bed) and allow it to dram through the column. Next, add enough KP, Buffer B to prevent the resin bed from runnmg dry (during gravity-flow chromatography) and equilibrate the column with 5-10 column volumes of KP, Buffer B. 3. Apply the diluted detergent-rich phase to the column at a rate of 0.5-l .OmL/mm (see Note 23) 4 Wash the column with approx 5-l 0 column volumes of KP, Buffer C 5 Connect the outlet tubing to a fraction collector and slowly elute with KP, Buffer C containing 30 mM histidine Read the absorbance of the collected fractions (418 nm), pool those contammg P450, and quantify the total amount of cytochrome P450 as described in Subheading 3.3. and Notes 14-17. At least 50% of the applied cytochrome P450 should be recovered after Ni2+ NTA agarose chromatography (see Notes 24 and 25).

3.6.3. Hydroxylapatite

(HA) Chromatography

1. Prepare approx 1 mL of hydrated HA resin per 20-40 nmol of P450. One gram of dry resin swells to 2-3 mL. To hydrate, one part of HA should be added to six parts water (at 4”C), gently swirled, and allowed to settle for 10 min. Decant water and tine particles (which will reduce the flow rate) Repeat twice 2. Pour and equilibrate the HA column in a manner similar to that for the Ni*+ NTA column except for the use of KPr Buffer D as the equilibration buffer Gradually dilute (l:lO, v/v) the pooled fractions from the Ni*+ NTA column with KP, Buffer D 3 Apply the diluted Ni2+ NTA column eluate to the HA column (flow rate = 0 20.5 mL/mm) and wash the column extensively to remove Triton X-100 and substrate (if present) with a potassium phosphate buffer of at least 10 mMcontammg 20% (v/v) glycerol, 0.2% (w/v) sodium cholate, and 0.1 mMDTT. Higher phosphate concentratrons can be used, depending on the P450. 4. Attach the outlet tubing to a fraction collector and slowly elute the protein with 400 mA4potassmm phosphate, pH 7 4, containing 20% (v/v) glycerol, 0.4% (w/v) sodium cholate and 0.1 mM DTT. Recovery of cytochrome P450 after HA chromatography should be 50-90% (see Note 26). 5 Dialyze fractions containing cytochrome P450 against 50 mM potassium phosphate, pH 7.4, containing 20% (v/v) glycerol, 0 1 mM DTT, 0 1 n-J4 EDTA and 0.1% (w/v) sodium cholate (see Notes 27 and 28). 6. Quantify cytochrome P450 as described m Subheading 3.3. and Notes 14-17 Ahquot mto 1.5-mL microfuge tubes, rapidly freeze in liqutd nitrogen, and store at -7OY!.

3.7. Concluding

Remarks

In thts chapter, we have outlined basic strategtes and conditions for success-

ful high-level expression of bovine P45Osccand P45Ocl7 in E. coli, which, m theory, should be universally applicable to any eukaryotic P450. In reality,

Cyfochrome P45U in E. colt

189

however, application of these and other techniques to other cytochromes P450 has demonstrated that, with our current level of understanding, cytochrome P450 expression in E. coli is unpredictable, even when comparmg very closely related cytochromes P450. To date, only a few cytochromes P450 can be classified as high-level expressors (>500 nmol/L of culture), whereas many are at least intermediate level expressors (100-500 nmol/L). Those that give comparatively lower yields (cl00 nmol P45O/L) or no yield at all (e.g., P45Oc18, P450arom, and P45Oc21) might be inherently unstable at the mRNA and/or protein level in E. colz, although specific characteristics which might account for this instability are not yet known. This should not in any way discourage attempts to express cytochromes P450 in E. colz. In fact, direct comparison of bovine P45Oc17 expression in COS cells, yeast, Sfs cells and E. colz (16) has demonstrated E. coli to be a very favorable system for expression of this P450. Ultimately, variations m expression plasmid, host strain and growth conditions will most likely need to be made by trial and error to maximize expression of each cytochrome P450 in E. colz. 4. Notes 1. For microsomalcytochromesP450,direct substitutionof the first eight codonsof a given CYP cDNA with the sequenceencoding MALLLAVF(L) (Fig. 1) does not always result in the most highly expressingconstruct(17,lJ). In those cases, partial deletron ofthe membraneanchorwas required (followed by attachmentof MALLLAVF(L)) to produce a more favorable result Note that the N-terminus of P45Oscc does not contam this sequence, although substitution of an alanine at the second posttton was found to be important (12). 2. Introduction of a His tag greatly simplifies purification of a cytochrome P450 from E. colz The high affinity (Kd = lo-i3 M> of His-tagged proteins allows chromatography on Ni+2 NTA, a powerful first step in the purification. We have

found His-tagged P45Oc17 to be normal spectrally and catalytically. P45Osccis routinely purified from E coli by chromatography on DEAE-cellulose, hydroxylapatite, and adrenodoxm-Sepharose (12) Because this procedure has been developed specifically for purification of bovine P45Osccand not mitochondrial cytochromes P450 in general, it will not be described in detail here Cytochromes P450 have been purified from E. colz by several different strategies, which are described m the literature (e g., refs. 14,18-20); however, a thorough description

of theseproceduresis beyond the scopeof this chapter. 3. It is almost impossible to predict a priori which expression plasmid will work

best for microsomal cytochromesP450. Although the pCWOri+ vector hasbeen the most widely used, other vectors listed in the Methods section have produced good yields as well (19,20). If the pCWOri+ vector is not mnnediately available, pSP19glOL is probably the best alternative for expression of microsomal cytochromes P450 (10). It should be noted that pTrc99A is the only vector that has

given high-level expressionof mitochondrial cytochromesP450 to date.

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4 For unknown reasons, cytochrome P450 expression in E co& m several cases,

5 6 7

8

9

10.

11. 12

13. 14.

15.

has been observed to be strain dependent. Strains that have been most amenable to high-level expression are JM109, TOPP 3, DHSa, and XL-l Blue. Successful high-level expression of a given cytochrome P450 m E co11appears to be largely dependent on a relatrvely narrow set of growth conditrons. TB medmm has been wrdely used for expression of cytochromes P450 m E. colz We have observed that cells that have been recently transformed and plated out produce the best yields of cytochrome P450 relative to freezer stocks of transformed cells or those that have been stored on LB-Amp plates for several days at 4°C. The time of induction by IPTG is critrcal. If the cells are Induced during late instead of early logarithmic growth, the level of cytochrome P450 produced ~111 be substantially reduced. Addrtron of &ALA has been shown to improve the expression of several cytochromes P450, often those expressed at relatively low to moderate levels (IO200 nmol P45O/L of culture). Presumably, &ALA, a heme precursor, Increases cytochrome P450 expressron by enhancing heme brosynthesrs P45Oscc levels are Increased approx twofold (from 250 to 500 nmol P45O/L of culture) by the presence of &ALA. &ALA does not appreciably affect bovine P45Oc 17 expression (normally 300-600 nmol P45O/L of culture). Trace elements also can be included in TB medmm, although then exact effect on the cytochrome P450 expression levels IS unknown A 4000X stock (100 mL) of trace elements 1sprepared from the followmg compounds. 2.7 g FeCl, * 6Hz0, 0.2 g ZnClz * 4Hz0,0.2 g CoClz * 6H20, 0 2 g Na,MoO, 2Hz0, 0 1 g CaClz 2H,O, 0.1 g CuClz, and 0.05 g H3B03 dissolved m 1.2 MHCl (20). Although 48 h is a typical growth period for maximal cytochrome P450 expression, other trme pomts (between 24 and 72 h) should be checked for each P450 Incubatron temperature has been shown to be an important factor (9) For most eukaryotrc cytochromes P450 that have been expressed m E ~011, optimal temperatures have ranged between 28-32°C. Different shaking speeds (between 125-250 rpm) should be investigated to maxrmize expressron. The medium/flask volume ratio has also been found to be important (9). Generally, the greater the aeratron of the culture (i.e., the lower the medium/flask volume ratio), the faster the culture ~111 reach a maximum level of expression. Cells expressmg P45Oscc are typically grown m 0.5 L medmm/2.8 L flask, whereas for P45Oc 17 this ratio is 1 .O L medmm/2.8 L flask. Complete reduction (by sodmm drthronite) and CO binding by P45Oc17 wrthin whole E. colt cells requires at least 15 mm For P45Oscc, this process requrres 20-30 mm. Values obtained from measuring whole cell spectra should be considered only as estimates. This 1s especially true rf the level of expression is low, because interference from endogenous bacterral proteins will be more pronounced.

Cytochrome P450 in E. coli 16 Although E. colz is not known to contain endogenous cytochromes P450, it does possess a membrane-bound hemoprotein complex, cytochrome o, whmh possesses an absorbance maximum at 416 nm and a trough between 428-436 nm m its reduced-CO/reduced difference spectrum (22). Denatured hemoprotem cytochrome P450 exhibits a peak around 420 nm (referred to as P420) which, if present in whole cells, will overlap the cytochrome o peak at 4 16 nm 17. When determining reduced-CO/reduced difference spectra of E co/z membranes or detergent-solubihzed proteins, sample dilution may be necessary For P45Oscc, a phosphate buffer (50 mA4 potassium phosphate, pH 7 4,20% (v/v) glycerol) is typically used. For P45Oc 17, the presence of detergent and substrate m KP, buffer B (without PMSF) help to stabilize the enzyme. 18 Less sodium dtthtonite (~1 mg) should be used when reducing E. COEE membrane or detergent-solubilized protein samples. A large excess of sodium dtthtomte will acidify the buffer and promote cytochrome P450 denaturatton 19. Substrate or ligand (tf known) should be added to KP, Buffer A at a reasonable concentration to help stabilize the cytochrome P450 upon solubtlization. 20. After addition of Triton X-l 14 to spheroplasts, the resulting suspension will become very viscous as a result of released chromosomal DNA. The presence of DNase should reduce the vtscosity within 10 mm. 21. This procedure typically solubilizes 50-80% of the spectrally detectable cytochrome P450 in whole cell suspensions. 22. If the cytochrome P450 content of the diluted detergent-rich phase (to be applied to the Nt2+ NTA resin) is low (cl00 nmol/L of startmg culture), more resin than indicated may be required to improve recovery 23 Resuspension of the top of the column (using a stirring rod) durmg loadmg may be necessary if the flow rate becomes significantly reduced 24 A considerable amount of the Ni2+ may dissociate from the column during elution, although this does not appear to denature the majority of the P450. Under these conditions, there may be an initial shoulder (absorbmg at 418 nm) which appears to be a combmatton of bacterial protein contaminants and partially denatured P450. This should be discarded and the main peak contammg the majority of cytochrome P450 should be further purified on hydroxylapatite, 25. If Nr2” leaching occurs durmg elution, the resin should be regenerated accordmg to the manufacturer’s recommendations before re-equtllbrating and applymg another sample. 26. Cytochrome P450 bound to HA should be washed and eluted in less than 12 h. Recovery from HA will be stgmficantly reduced if the cytochrome P450 IS m contact with the resin for longer times. 27. Concentration of the HA column fractions, if necessary, should be done before dialysis. Centriflo CFSOA ultrafiltration membrane cones (Amtcon, Danvers, MA) work well for this purpose. 28. Depending on the P450, it may be possible to omit the detergent from the dialyses buffer without causing the protein to precipitate.

192

Jenkins, hkuleva,

and Waterman

Acknowledgments Support for investigation of expression of P45Oc17 and P45Oscc in E. colz has been provided by USPHS Grants GM37942 and ES07028.

References 1, Zuber, M. X., Simpson, E. R., and Waterman, M. R. (1986) Expression of bovine 17a-hydroxylase cytochrome P-450 cDNA in nonsteroidogemc (COS 1) cells Science 234, 1258-1261 2 Asseffa, A , Smith, S J , Nagata, K , Gillette, J , Gelbom, H V , and Gonzalez, F. J. (1989) Novel exogenous heme-dependent expression of mammalian cytochrome P450 usmg baculovuus. Arch &o&em Brophys 274,481-490 3 Oeda, K , Sahaki, T , and Ohkawa, H. (1985) Expression of rat liver cytochrome P450MC cDNA m Saccharomyces cerevwae DNA 4,203-2 10 4 Barnes, H. J., Arlotto, M. P , and Waterman, M. R. (1991) Expression and enzymatic acitivity of recombinant cytochrome P450 17a-hydroxylase m Escherlchla COIL Proc Nat1 Acad Set USA 88,5597-5601. 5 Sambrook, J , Fritsch, E. F., and Matuatis, T (1989) Molecular Clonmg. A Laboratory Manual, 2nd ed. (Ford, N., Nolan, C , and Ferguson, M., eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 6 Muchmore, D C., McIntosh, L. P., Russell, C. B , Anderson, D E , and Dahlquist, F W. (1989) Expression and mtrogen- 15 labeling of proteins for proton and mtrogen- 15 nuclear magnetic resonance Methods Enzymol 177,4&73 7. Bordier, C. (198 1) Phase separation of integral membrane proteins m Triton X1 14 solution J B1o1 Chem. 256, 1604-1607 8. Stormo, G. D , Schneider, T D., and Gold, L. (1982) Characterization of the translational imtiation sites m E. toll. Nucleic Acids Res. 10,297 l-2996 9. Barnes, H J. (1992) Heterologous Expression of Bovine Cytochrome P45017a Hydroxylase Ph D. Thesis, Umversity of Texas Southwestern Medical Center at Dallas IO. Barnes, H. J (1996) Maximizing expression of eukaryotic cytochrome P45Os m Escherlchra ~011.Methods Enzymol 272,3-14. Il. Wada, A., Mathew, P. A., Barnes, H J., Sanders, D , Estabrook, R W., and Waterman, M. R. (1991) Expression of functional bovine cholesterol side chain cleavage cytochrome P450 (P45Oscc) in Escherzchla COELArch Blochem Blophys 290,376380. 12. Wada, A. and Waterman, M R (1992) Identification by site-directed mutagenesis of two lysme residues m cholesterol side chain cleavage cytochrome P450 that are essential for adrenodoxm bmdmg. J, Btol Chem. 267,22,877-22,882. 13. Pikuleva, I. BJorkhem, I. and Waterman, M. R. (1997) Expression, purification, and enzymatic properties of recombinant human cytochrome, P45Oc27 (CYP27), Arch Blochem Blophys 343,123-130 14. Richardson, T H , Jung, F., Griffin, K. J., Wester, M , Rauncy, J L., Kemper, B , Borngeim, L. M , Hassett, C., Omlecinski, C J , and Johnson, E F. (1995) A universal approach to the expression of human and rabbit cytochrome P45Os of the 2C subfamily m Eschenchla cob. Arch Blochem Blophys 323, 87-96

Cytochrome P450 in E. coli

193

15 Omura, T. and Sato, R (1964) The carbon monoxtde-binding pigment of liver microsomes. J Blol Chem 239,2379--2385. 16 Barnes, H. J., Jenkins, C. M , and Waterman, M. R. (1994) Baculovirus expression of bovine cytochrome P45Oc 17 m Sf9 cells and comparison with expression in yeast, mammalian cells, and E colz. Arch Blochem Biophys 315,489-494. 17. Sandhu, P., Baba, T., and Guengerich, F. P. (1993) Expression of modified cytochrome P450 2ClO (2C9) m Escherichia coli, purification, and reconstitutton of catalytic activity. Arch. Blochem. Biophys. 306,443-450 18 Sandhu, P., Guo, Z., Baba, T., Martm, M. V , Tukey, R. H., and Guengerich, F.P (1994) Expression of modified human cytochrome P450 lA2 m Escherichia cob. stabilization, purification, spectral characterization, and catalytic activities of the enzyme. Arch. Blochem Biophys 309,168-l 77. 19. John, G. H., Hasler, J A, He, Y , and Halpert, J. R (1994) Escherichza coli expression and characterization of cytochromes P450 2B11, 2B 1, and 2B5 Arch Biochem Biophys. 309,367-375 20 Halkier, B A , Nielsen, H L., Koch, B., and Moller, B L. (1995) Purtflcation and characterization of recombinant cytochrome P450TyR expressed at high levels m Escherichta coli. Arch Blochem Blophys 322,369-377. 2 1 Bauer, S. and Shtloach, J. (1974) Maximal exponenttal growth and yield of E colz obtainable in a bench-scale fermentor. Blotechnol Bloeng 16,933-941 22. Poole, R. K. and Ingledew, W. J. (1987) Pathways of electrons to oxygen, m Escherzchia colt and Salmonella typhlmunum. Cellular and Molecular Biology, (vol. I) (Netdhardt, F. C , Ingraham, J. L , Low, K B , Magasamk, B , Schaechter, M., and Umbarger, H E., eds.), American Society for Microbiology, Washington, DC, pp. 170-200.

23 Expression in Yeast

of Mammalian

Cytochromes

P450

Yoshiyasu Yabusaki 1. Introduction Recent extensive research on cytochrome P450 (P450) has clarified that P450 constrtutes a superfamrly of hemoprotems, which are beheved to have evolved from a common ancestor. The versatility of P450 is mainly owing to the existence of a large number of P450 species, each of which shows a broad but somewhat unique substrate specificity. Mammalian P450 species are found m subcellular membranes, such as mitochondria and endoplasmic retrculum, and have several common properties: molecular size, structure, and immunological and brochemical characteristics. The existence of closely related P450 species has made it rather difficult to isolate a given P450 from others by classical brochemical-puriticatton procedures. However, molecular biology has provided a powerful tool to reveal the ammo-acid sequence of a specific P450 species from analysrs of the nucleotide sequence of its cloned cDNA. When a cDNA for a particular P450 species with an unknown function is isolated, it is prerequtstte for the characterrzatron of the P450 species to establish a suitable expression system for the cDNA. The expression system IS also useful to investigate the structure-function relationship of the enzyme. Mammalian P450 species are bound to mtcrosomal or mitochondrral membranes, and require electron transfer enzymes, ferredoxm and nicotinamide ademne dinucleotide [phosphate] reduced form (NAD[P]H)-ferredoxm reductase for mrtochondrial P450, and NADPH-P450 reductase for mtcrosomal P450, to exhibit their activities. Thus, it IS desirable that the host cells contain such intracellular compartments and electron-transfer enzymes. Based on these criterra, yeast is one of the best host cells for expression of P45Os.Although yeast From Methods In Molecular Sto/ogy, Vol 107 Cytochrome P450 Protocols Edlied by I R PhIllips and E A Shephard 0 Humana Press Inc , Totowa, NJ

195

Yabusaki

196

has its own P450 and NADPH-P450 reductase responsible for ergosterol biosynthesis, the cellular content of this P450 species is very low in yeast under the culture conditions descrrbed below, and can be negligible as compared with heterologously expressed P450. Besides yeast, several expression systems including bacteria (Escherichza coli), and cultured insect (baculovirus), mammalian and plant cells are also used to express P450 cDNA. Some of these are described in other chapters (see Chapters 22,24, and 25)

2. Materials 2.1. Cultivation

of Yeast

1 Yeast strain: Saccharomyces cerevwae AH22 (MATa, led-3, leu2-112, hu4519, canl, [czr+], [rho”J) was used for the present study This strain was deposited as No. 38626 to American Type Culture Collection and can be obtained therefrom. Other yeast strains, reported m the hterature, have also been used as a transformation host 2. YPD medium. 2% (w/v) Bactopeptone (Difco; Detroit, MI), 1% (w/v) yeast extract (Difco), and 2% (w/v) glucose. Autoclaved before use Stable for several months at room temperature. 3. SD medium. 0 67% (w/v) yeast mtrogen base wtthout ammo acids (Difco) and 2% (w/v) glucose. Autoclaved before use. Stable for several months For the cultivation of transformed yeast, SD medium is supplemented with histidme (20 mg/L). Histidine solution (2 mg/mL) is autoclaved separately and stored at room temperature

2.2. Transformation

of Yeast

1. Lithium chloride: 1 M solutton m dtsttlled water Autoclaved before use Stable for several months at room temperature 2. Polyethylene glycol. 70% (w/v) PEG4000 in distilled water. Autoclaved before use Stable for several months at room temperature. 3. SD-agar plate. To autoclaved SD-medium containing 2% (w/v) agar (Difco), histidine solution (2 mg/mL) is added to a final concentration of 20 &/mL. The mixture is then poured mto plates.

2.3. Construction

of Expression

Plasmid

1 P450 and NADPH-P450 reductase cDNAs. A full-length cDNA coding for rat ltver P450 (CYPl Al) is isolated as described (1) Clonmg of yeast NADPHP450 reductase gene is described elsewhere (2) 2. Yeast vector: The yeast expression vector pAAH was used in the present study This vector (a gift from B. Hall, University of Washington, Seattle, WA) harbors yeast alcohol dehydrogenase I (ADH) gene promoter and termmator sequences, and a leu2 gene for selection of yeast transformants by leucine complementation. pAAH also contains a yeast 2 pm ori, for replication in yeast, and pBR322-

197

Mammalian P450 in Yeast

derived ori and amplclllin-resistance gene for replication and selection, respectively, in E cok 3. Restriction enzymes and other DNA modifying enzymes. These enzymes for recombinant DNA techniques can be obtained from several suppliers Restriction digestion, ligation, and blunt-ending are performed under conditions recommended by the supphers

2.4. Determination

of P450 Content and Monooxygenase

Activity

1. Washing buffer. 100 &potassium phosphate (KP1) buffer, pH 7.0 Prepare 1 M each of KH,PO, and K,HP04, and mix equal volumes of each solution to make 1 h4 stock KP1 buffer, pH 7.0. 2. Sodium dlthiomte 3. 7-Ethoxycoumarm* 20 mM solution m 50% (v/v) ethanol 4. NADPH: 100 mM solution

2.5. Preparation of Microsomal Fraction and Sodium Dodecyl Sulfate (SDS)-Polyacrylamide Gel Electrophoresis (PA GE) 1. Zymolyase- 1OOT (Selkagaku, Tokyo, Japan) This enzyme preparation isolated from Arthrobacter Euteus contains /3-1,6-glucanase activity (see Note 1). 2 Zymolyase buffer 10 mM Tns-HCl, pH 7.5, contammg 2 M sorbltol, 0 1 mM dlthlothreltol (DTT) and 0 1 mM ethylene dlamme tetra-acetic acid (EDTA) 3 Somcatlon buffer 10 mMTns-HCl, pH 7 5, containing 0.65 M sorbitol, 0 1 mA4 DTT, and 0.1 mM EDTA 4 Phenylmethylsulfonyl tluorlde (PMSF). 200 mM solution m ethanol 5 Solubllizatlon buffer: 50 n-u!4 Tris-HCl, pH 6.8, containing 40% (v/v) glycerol, 1% (w/v) SDS, 10% (v/v) 2-mercaptoethanol, 1 mM PMSF, and 0 02% (w/v) Coomassle Bromophenol Blue 6 10% polyacrylamide gel containing SDS.

3. Methods 3.1. Construction

of Yeast-Expression

Plasmid

1 The expression plasmid pAMC 1 for rat CYP 1A 1 was constructed as described m detail (ref. 3, also see Note 2). 2 In order to utilize a unique Hind111 site between the ADH promoter and termmator of pAAH5, HIndI sites were created both before and after the CYPlAlcoding sequence of pAU157. 3. For expression of rat CYPl Al and overexpression of yeast NADPH-P450 reductase m yeast, an expression plasmid pAMR2 was constructed, the details of which are described elsewhere (ref. 4, see Notes 3 and 4) 4. Expression units for CYPl Al, under the control of ADH promoter and terminator, and for yeast P450 reductase under the control of its own promoter and termnator, were both mserted into a single plasmid.

198

Yabusakl

5. For expression of a fusion enzyme between rat CYPlA 1 and yeast NADPH-P450 reductase m yeast, an expression plasmid pAFCR1 was constructed. The fusion enzyme consists of the entire sequence of CYP 1A 1 and a partial sequence for the amino-terminal truncated soluble form of yeast P450 reductase (see Notes 5 and 6). A detailed protocol for the construction of pAFCR1 has been described elsewhere (5)

3.2. Transformation of Yeast Cells with Constructed Expression Plasmids 1. Cultivate a single colony of S cerevrszae AH22 strain m 5 mL YPD medium m a sterile test tube for 16 h (or overnight) at 30°C 2. Place 1 mL of the culture m an Eppendorf tube and collect the yeast cells by centrifugation at 3000g for 2 mm 3 Wash the pelleted cells with 0 2 A4 LiCl and suspend m 20 pL 1 h4 LiCl 4. Add approx 1 pg of the expression plasmtd (10 pL) and 30 pL 70% PEG, and mix by vortexmg 5. Stand mixture for 60 mm at 30°C 6 Add 140 pL of distilled water and mix by vortexmg 7. Plate 100 pL onto each of two SD-agar plates. 8. Incubate the plates for 2-3 d at 30°C (see Notes 7 and 8)

3.3. Cultivation of Transformants and Determination of P450 Expression 1. Pick several colonies by sterile toothpick or wire mto 5-mL SD medium in a sterile test tube for cultivatton for 16 h (or overnight) at 30°C Mark the mdtvidual colonies on plates or streak onto another SD-plate for storage (see Note 9) 2. After saturation of growth, transfer the 5-mL culture into 250 mL SD medium in a sterile flask (500 mL) and continue the cultivatton for a further 16 h (or overnight) at 30°C 3 Measure the optical density (OD) at 660 nm and calculate the cell density (see Note 10) 4. Collect yeast cells from the culture by centrtfugatton at 2000g for 5 mm, wash

5 6 7 8. 9.

them with 10 mL of washing buffer, and finally suspend m 2 mL of washing buffer. Pour 1 mL of the suspended cells mto each of two quartz cuvets Place both cuvets m a dual-beam spectrophotometer (Hitachi 557) and correct the baseline between 400 and 500 nm. Bubble CO gas mto the sample cuvet for about 1 mm Add 2-3 mg of sodium dithiomte to both sample and reference cuvets. Cover each cuvet with Paratilm and invert to mix contents Monitor the difference spectrum between 450 and 490 nm automattcally and calculate P450 concentratton based on a molar extinction coefficient of 9 1 mA@ cm-’

Mammalian P450 m Yeast 3.4. Preparation

of Microsomal

Fraction

1. Collect yeast cells (about 2 x log cells) from the culture by centnfugatlon, wash them with somcation buffer and suspend them m 3 mL of Zymolyase buffer, containing 10 mg of Zymolyase-1OOT. 2 Incubate the mixture with gentle shaking for 1 h at 30°C to obtam spheroplasts. Measure the decrease m ODsoo to monitor the lysls of yeast cell walls 3 Wash the spheroplasts twice with Zymolyase buffer and finally suspend them m 10 mL of sonication buffer. 4 Disrupt the spheroplasts by somcation at 60W for 5 mm on Ice (see Note 11) 5 Centrifuge the lysate at 3000g for 10 min at 4°C to remove undisrupted cells and cell debris. 6. Centrifuge the supernatant at 148,000g for 80 mm at 4°C to precipitate the microsomal fraction. 7. Suspend the pelleted mlcrosomal fraction in a small volume (50 &-1 mL) of sonication buffer

3.5. SDS-PAGE 1. Collect yeast cells (3-5 x lo7 cells) in an Eppendorf tube by centrifugation, wash with 0.8 mL of Zymolyase buffer, and suspend them in 0 3 mL of the same buffer containmg 500 pg of Zymolyase- 1OOT. 2 Incubate the mixture for 1 h at 30°C. Collect the spheroplasts by centrlfugatlon at 3000g for 2 min and wash them twice with Zymolyase buffer. 3. Add 50 pL of solubihzatlon buffer to the pelleted spheroplasts and heat at 1OO’C for 3 min prior to loadmg onto an SDS-polyacrylamide gel 4 After electrophoresls, stain the gel with Coomassle Brllhant Blue (see Note 12).

3.6. Measurement

of Monooxygenase

Activity

1. For measurement of monooxygenase activity of intact yeast cells, cultivate yeast cells to a density of 1.3 x lo7 cells/ml m SD medium 2. Add 7-ethoxycoumarin m 50% methanol to the culture to a final concentration of 0.5 mM, and continue the cultivation. 3. After several hours Incubation, remove a 1 0-mL ahquot from the culture and centrifuge it at 3000g for 2 mm. 4 Measure 7-hydroxycoumarm formed m the culture supernatant by fluorlmetry (see Note 13) 5. For measurement of monooxygenase activity in the mlcrosomal fraction, prepare a reaction mixture (1.0 mL) containing 100 mM KPl buffer, pH 7.0, 0 5 mM NADPH, and an appropriate volume of the microsomal fraction. 6. Preincubate the reactlon mixture for 5 min at 37°C and start the reaction by addltlon of 7-ethoxycoumarm to a concentration of 0 5 m1!4. 7 After incubation for 5 mm at 37Y!, add 31.25 pL of 30% TCA and 1 mL of chloroform to terminate the reaction

200

Yabusaki

8. Extract the chloroform layer (0 3 mL) with 3 mL of 10 mMNaOH, 0.1 MNaCl 9 Measure 7-hydroxycoumarin formed, by fluortmetry at an excttation of 550 nm and an emission of 588 nm.

4. Notes 1 We have used Zymolyase- 1OOT as a lytic enzyme for yeast However, other lyttc enzymes may be used to digest yeast cell walls (7). 2 For construction of the expression plasmtd for rat CYPl Al, we used only the coding sequence for CYPlAl and deleted its 5’ and 3’ noncodmg sequences. In addition, the upstream sequence between the ADH promoter and the ATG translational initiation codon was found to be very important for efficient expression of heterologous genes m yeast. We employed a short (AT)-rich sequence before the ATG codon For expression of bovine adrenal CYP17 cDNA, this modtfication resulted m the production of a twofold greater amount of P450 hemoprotem (8) 3 Although yeast cells contain endogenous NADPH-P450 reductase activtty, co-expression (overexpresston) of P450 reductase with P450 enhances the P450dependent monooxygenase acttvtty in yeast. 4 We have compared the effect of overproduction of rat and yeast P450 reductase m yeast, leading to the conclusion that yeast P450 reductase is better than rat P450 reductase with respect to the increase in monooxygenase acttvtty (4,9). Thts may be related to the greater stabihty in yeast cells of the yeast reductase compared to the rat reductase However, overexpresston of human P450 reductase m yeast has been reported to enhance monooxygenase activity (10,ll). 5. The ammo-terminal hydrophobtc regton of P450 is important for correct localization of the newly synthesized polypeptide into yeast microsomal membranes, as well as for tts enzymatic activity. On the other hand, the amino-terminal hydrophobic region of P450 reductase 1srequired only for anchoring the enzyme to the mtcrosomal membranes and is not necessary for its acttvtty Thus, the ammo-terminal hydrophobic region of P450 reductase was omitted m the construction of the P450/reductase fusion. More detailed analysis on the effects of the hinge region between P450 and P450 reductase domams was descrtbed m my review article (6) and references cited therein. 6. For the constructton of acttve P450ireductase fusion protein, the P450 domam should be placed at the ammo-terminus of the fuston (12). This structural feature seems to be important for efficient intramolecular electron transfer from the reductase domain to the P450 domain. 7. For transformation of yeast, thus simple alkaline metal method 1s recommended The plasmid DNA can be prepared by a standard munprep method and used for transformation wtthout further purification The transformation efficiency is about 102-1 03/pg of plasmid DNA. 8. Yeast transformatton can also be performed by a spheroplast method as described m our earlier publmatton (J), although this procedure is more complicated and time-consuming.

Mammalian P450 in Yeast

201

9. This 1s very important because yeast colonies differ from each other m then growth and level of expression of P450 Select the best colony among a few transfonnants by measuring the expresston of P450. 10. We have plotted a correlation curve between OD at 660 nm and cell density, and calculated the cell density based on this plot. The relation between OD at 660 nm and actual cell number is dependent on the spectrophotometer used 11. Avoid a temperature increase by keeping the spheroplast suspension on ice. 12. If an antiserum for the P450 species is available, it can be used to measure the expression of the P450 in yeast by immunoblotting After electrophoresis proteins can be transferred from the gel to a blottmg membrane and detected by using the antiserum and a labeled secondary antibody (see Chapter 43) 13 It is easy to measure P450-dependent monooxygenase activity in intact yeast cells. However, caution should be exercised when calculating specific activity (or turnover) based on cell number or P450 amount, because during cultivation the yeast cells multiply and cellular P450 content varies

References 1 Yabusaki, Y., Shtmizu, M , Murakami, H , Nakamura, K , Oeda, K., and Ohkawa, H. (1984) Nucleotide sequence of a full-length cDNA coding for 3-methylcholanthrene-mduced rat liver cytochrome P-450MC Nucleic Aczds Res. 12,2929-2938. 2 Yabusaki, Y , Murakami, H., and Ohkawa, H (1988) Primary structure of Saccharomyces cereviszae NADPH-cytochrome P450 reductase deduced from nucleotide sequence of its cloned gene. J Bzochem. 103, 1004-1010. 3. Oeda, K., Sakaki, T., and Ohkawa, H. (1985) Expression of rat liver cytochrome P-450MC cDNA m Saccharomyces cereviszae. DNA 4,203-2 10. 4. Murakami, H., Yabusaki, Y , Sakaki, T., Shibata, M , and Ohkawa, H (1990) Expression of cloned yeast NADPH-cytochrome P450 reductase gene in Saccharomyces cerevtslae. J Blochem 108, 859-865 5. Sakaki, T., Kominami, S., Takemori, S., Ohkawa, H., Akiyoshi-Shlbata, M., and Yabusaki, Y. (1994) Kinetic studies on a genetically engmeered fused enzyme between rat cytochrome P450IAl and yeast NADPH-P450 reductase Btochemistry 33,49334939. 6. Yabusaki, Y (1995) Artificial P450/reductase fusion enzymes: what can we learn from their structures? Blochtmle 77, 594-603. 7. Guengerich, F. P., Brian, W. R , Sari, M. A., and Ross, J T (1991) Expression of mammalian cytochrome P450 enzymes usmg yeast-based vectors Methods Enzymol. 206, 130-145. 8 Sakaki, T., Shibata, M., Yabusaki, Y , Murakami, H , and Ohkawa, H. (1989) Expression of bovine cytochrome P45Oc 17 cDNA m Saccharomyces cerevwae DNA 8,409-418 9 Murakaml, H , Yabusaki, Y , and Ohkawa, H. (1986) Expression of rat NADPHcytochrome P-450 reductase cDNA in Saccharomyces cerevwae. DNA 5, l-10

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Yabusaki

10. Eugster, H. P., Bartsch, S., Wurgler, F. E , and Sengstag, C (1992) Functional co-expression of human oxrdoreductase and cytochrome P450 1Al in Saccharomyces cerewszae results in increased EROD actlvtty. Biochem. Bzophys. Res. Commun. 185,641-647

11. Urban, P., Truan, G., Galtier, J. C., and Pompon, D. (1993) Xenobrotrc metabolism in humamzed yeast: Engineered yeast cells producing human NADPH-cytochrome P450 reductase, cytochrome b5, epoxide hydrolase and P45Os. Blochem. Sot. Trans 21, 1028-1034. 12. Sakaki, T., Shibata, M , Yabusakt, Y , Murakami, H., and Ohkawa, H. (1990) Expression of bovine cytochrome P45Oc21 and Its fused enzyme with yeast NADPH-cytochrome P450 reductase m Saccharomyces cerevzszae DNA Cell Blol 9,603-614

24 Expression of Cytochromes in a Baculovirus System

P450

Steven R. Hood, Girish Shah, and Peter Jones 1. Introduction The baculovirus expression system is a highly effective system for the largescale productron of recombinant proteins m insect cells. The major attraction of baculovn-us as an expression vector system IS the virus-encoded polyhedrin andpI genes. These genes produce large amounts of polyhedrin and p 10 proteins in virus-infected insect cells m the later stages of the virus-replication cycle. The polyhedrin protein, controlled by the polh promoter, IS required m the normal infection cycle to package virus partrcles wrthm occlusron bodies or polyhedra, which protect the virus particles from the environment during the period between mfectton of susceptrble hosts. The function of the p 10 protein is not clear as yet but it is thought to be involved m polyhedra formation and IS controlled by the plO promoter. Polyhedrin and p10 are not required to maintain an infection m cultured cells in vitro. The polyhedrm and pl0 genes can be replaced with foreign gene sequences thus enabling expression of recombinant protein from the polyhedrin (polh) andpZ0 promoters. The baculoviru-insect cell expression system provides a eukaryotrc envtronment that is generally conducive to the proper folding, drsulfide bond formatron, ohgomerizatron, and/or other posttranslation modifications required for the biological activity of some eukaryotic proteins. For instance, glycosylations are performed using insect cell-derived sugars and thus result m the production of near authentic versions of the original proteins. Furthermore, microsomal membrane fractions prepared from infected cells can be utrhzed in standard drug metabolism assaysin a similar way to liver microsomes. The baculovims system has been used for the expression of several rsoforms of P450 and a list of these is shown in Table 1 (I-16). The availability of two From Methods m Molecular &o/ogy, Vol 107 Cytochrome P450 Protocols Edited by I R PhIllIps and E A Shephard 0 Humana Press Inc , Totowa, NJ

203

204 Table 1 P450 lsoforms in Baculovirus

Hood, Shah, and Jones that have been Expressed Infected Insect Cells

Isoform 1Al 2Al 2D6 2Cll 2C8 2C9 2El 2J2 3A4 3A7 17A 19A (Aromatase)

Reference Buters et al (I); Grant et al (2) Grogan et al (3) Paine et al (4) Btagmt and Ceher (5) Zeldm et al (6) Grogan et al (3) Grogan et al (3) Patten and Kock (7) Wu et al (8) Buters et al (9). Lee et al (10) Sakuma et al (II) 4A 11 Imaoka et al (12) Barnes et al (13) Lahde et al (14), Sigle et al (IS), Amarnah and Simpson (16)

strong promoters (polh andpl0) in the same vnus allows the coexpression of a cytochrome P450 and the NADPH-cytochrome P450 reductase (P450 reductase). This coexpression results in significantly higher catalytic activity than m a system in which the expressed P450 is reconstituted with purified P450 reductase (10). In our laboratories, the cDNAs for the human cytochrome P450 enzymes are cloned mto baculovnuses by replacing the polyhedrm gene. Further, the cDNA for the P450 reductase is cloned m the P450 baculovnuses by replacing the ~20 gene. The recombinant baculoviruses are propagated m Spodoptera frugiperdu (S’) cells to generate high-titer virus stocks. Trichoplusia ni (T. ni) cells are used for protein expression and scale-up. Smallscale (0.1-5 L) expression is carried out in spinner- and/or shaker-flask cultures, whereas large scale (10-50 L) expression is carried out in agitated, sparged bioreactors. Infected cells are harvested by centrtfugatton (small-scale preparations) or tangential-flow filtration (large-scale preparations) and these are processed to isolate microsomes, which are assayed for P450 activity. 2. Materials 2.1. DNA Cloning and Packaging

Kits

1. There are a wide range of commerctally available krts for the cloning and packaging of DNA into baculovirus We have used the Baculogold (Pharmmgen, San Diego, CA) and Bat-to-Bat (Life Technologies, Bethesda, MD) protocols m our laboratones 2 5-bromod-chloro-3-mdolyl-S-o-galactosrde (Xgal) 3 Isopropylthio-P-o-galactoside (IPTG).

205

P450 Expression by Baculovirus 2.2. Cell Lines

1, SfP-cell line derived from Spodoptera fmgiperda, the fall army worm, is available from the American Type Culture Collection (ATCC, Rockville, MD) 2. T nz-cell line derived from Tr~hoplusw ni, the cabbage looper worm, IS avadable from Invttrogen (San Diego, CA).

2.3. Propagatjon For small-scale

and Maintenance work, commercially

of Cell Lines available

media are used.

I. 5” cells are routinely cultured in TC 100 medium (purchased from Ltfe Technologies as both liquid and powder), supplemented wrth 5% fetal bovine serum (purchased from Tissue Culture Services, and batch tested for Insect cell growth) The medium 1s also supplemented with penictllm-streptomycin (200 U/mL and 200 pg/mL, respectively), gentamicin (50 pg/mL), amphotericm B (2 5 pg/mL), and Pluromc F-68 (0 2% w/v) (all purchased from Life Technologtes). To prepare 1 L of TC 100 medium from powder. a Heat 850 mL of Mini-Q (Milhpore, Watford, UK) water (or deiomzed-dtstilled water) to 15 to 30°C (room temperature) b. Add powdered medium to the water with gentle sttrrmg. Rmse original package wtth a small volume of water to remove all traces of powder and add to the solution, Star the solutton until all lumps have dtssolved. (NB, do not heat the solutton to >3O”C) , c Add 0.35 g sodium bicarbonate (NaHCOs) and allow to dissolve. d. Make the volume up to 900 mL with Mini-Q (or deionized-distilled) water. e Adjust to pH 6.1 using 10 Mpotassmm hydroxtde. Add in small altquots (l-2 mL at a time). Upon each addition, a small precipitate forms that clears within seconds. (NB, the pH must not be allowed to rise above pH 6 3, as an insoluble precipitate will form.) f. Add fetal bovine serum, penicillin-streptomycin, gentamicin, amphotericm B, and Pluromc F-68 at the required concentrations. Adjust volume to 1 L g. Sterilize the solution by membrane filtration (Sartorms, Goettmgen, Germany or Millipore, Bedford, MA) usmg 0.2~pm pore size membranes. h Store medtum at 4°C m the dark and use withm 4 wk of preparation 2. T nz cells are routinely cultured m EX401 medium (purchased from Ttssue Culture Services [Botolph Claydon, Bucks, UK] as both liquid and powder), supplemented with penictllin-streptomycin, gentamicm and amphoterrcm B (concentrations as for TCIOO medium) and Pluromc F-68 (0 1% w/v) Prepare 1 L of EX40 1 medium from powder as described above in item 1, steps a-h 3. Culture vessels: a. Small-scale cultures Both cell lines are maintamed as suspension cultures in a variety of vessels ranging from 500-mL Erlenmeyer flasks (culture volume C200 mL), 2-L Fernbach shaker flasks (culture volume 500 mL-1.5 L) (Nalgene), glass spinner flasks (culture volume 1-6 L) (Bellco)

Hood, Shah, and Jones

206

4 5 6. 7. 8 9. 10 11.

b Large-scale cultures. For large-scale culture of T nz cells we use 15-L (Btolaffitte), 36-L Bellco glass spmners, and 75-L (Chemap) microbial vessels The control schemes (stu-rer speed, head space gassing, and sparge au pressure) are designed m house to maintam a preset dissolved oxygen level. pH IS not controlled. The 36-L spinners are fitted wtth a bottom sparge tube (>lO p bubble size), a sampling tube, and DO, and pH electrodes The shaker flask and spinner cultures are maintained at 125-150 rpm on an orbital shaker (purchased from Forma and New Brunswtck) Flask cultures have foam closures allowing for exchange of gases, whereas the spinner cultures are continuously gassed mto the headspace with au The magnettc stirrer tables (purchased from Bellco) for the spinner cultures and the shaker platforms (Innova and Forma) are housed in Bellco roller-bottle cabmets maintamed at 28°C. Trypan blue 0.4% (w/v) solutton: dtlute 1.5 with complete culture medium and sterrhze by filtratton (0.2 pm) before use m cell-vtabthty studtes Dimethyl sulfoxtde (DMSO): tissue culture grade Mycoplasma detection ktt* Gen Probe mycoplasma detection ktt (Labs Implex, UK) 2% (v/v) Hycolm disinfectant solution 0 5% (w/v) neutral red 4% (w/v) agarose solution (Life Technologies). Silicone 1520 anttfoam (Dow-Cornmg, Cornmg, NY) Equipment mcludes benchtop cooler: StrataCooler Cryo Benchtop Cooler (Stratagene, La Jolla, CA).

2.4. Expression

of Cytochromes

f450

1. Hemin chloride: Prepare a 2 mg/mL stock solution of hemm chloride in ethanol/ 1 N NaOH solution (1.1 v/v) and sterthze by filtratton through a 0 45+m filter Store solution at 4°C m the dark Hemm chloride is used at a final concentratton of 2 &mL. 2 6-ammo-levuhmc actd (5-6 ALA). Prepare a 50 mA4 stock solutton in EX401 medium and sterilize usmg 0.2 pm filter Store frozen at-20°C 5-6 ala 1sused at a final concentration of 100 @4 3. Ferric citrate (Fe Ctt): Prepare a 41 rnA4 stock solutton m boilmg water and sterilize (when cool) using 0.2 pm filter Store at 4°C in the dark. Fe tit 1sused at a final concentration of 100 @4

2.5. Harvesting of Cells and Preparation of Microsomal Membranes 1. Phosphate buffered saline (PBS) 2 Resuspension buffer: 0.1 A4 Potassium phosphate buffer (pH 7.4) made up m Milh-Q water and supplemented with 20% (v/v) glycerol and 1 mMEDTA. 3. Homogenizer and centrifuges for microsome preparation a. Potter homogenizer (Brown) with a 60-mL teflon pestle and glass mortar

P450 Expression by Baculovirus

207

b. Cells aresedimentedin aSorvall-DuPont(Sorvall, Stevenage,Hens, UK) RC3C floor-standmg centrifuge using 1-L polycarbonatebottleswith sealedlids. c The 9000g S9 centrifugation is performed in a Sorvall RCSC floor-standing centrifuge using SLA 1500or SLA3000 rotors andpolycarbonate bottles d. Ultracentrifugation is performed in a BeckmanEX 90 (Fullerton, CA) centrifuge using a 45 Ti rotor and 96-mL polycarbonatecentrifuge bottles

3. Methods 3.1. Cloning and Packaging of P450 cDNAs into Baculovirus The large srzeof the baculovirus genome (128 kb) prohibits the direct insertion of cDNAs by routine cloning techniques. Therefore, it IS necessary to clone the cDNA into a shuttle plasmid that is capable of recombinmg with the genome to give a functional recombinant virus. A wide range of vectors and packaging kits are commercially available, but these can be drvided mto two types according to the method of selection of recombinants. The basis of each protocol is outlined in Subheadings 3.1.1. and 3.1.2., but the manufacturer’s mstructions should be followed when using commercial kits. 3.1.7. Recombinant Selection in Insect Cells (e.g., the Baculogold Sys tern) The cDNAs for the P450 and P450 reductase are cloned into a transfer vector (e.g., pAcUWSl), downstream of the appropriate promoter. A sample of the plasmid IS cotransfected with linear copies of the AcMNPV baculovirus genome mto Sfl cells. Recombination between homologous sequences on the vector and the genome occur at a low frequency to form recombinant baculoviral genomes containing the P450 and P450-reductase genes. The original AcMNPV genome contains the La& gene m the polyhedrin site, which is replaced m the recombination event. By growing the host cells m a medium containing Xgal and IPTG, the nonrecombmant plaques are blue, whereas the recombinant plaques are colorless. The identification of these plaques requires some experience. Such a clonmg strategy is detarled m Lee et al (10). 3.1.2. Recombinant Selection in Bacteria (e.g., the Bat-to-Bat System) This system was first described by Luckow et al. (17) and forms the basis of the Bat-to-Bat system (Gibco-BRL). As with the system described in Subheading 3.1.1., the cDNA to be expressed is cloned into a transfer vector (pFASTBAC) downstream of the polh promoter. The recombinant plasmid IS then transformed into an E. colz cell lme containing a circular baculovirus genome (bacmid), and a homologous recombmation event occurs. The bacmrd

Hood, Shah, and Jones

208

contain elements of the 1acZ gene that allow the recombinant bacmid to be selected by blue/white screening. In the presence of X-gal and

and the plasmtd

IPTG, recombinant bacmrds produce blue colomes on a white background. The cells containing the recombinant bacmid are then scaled up and the DNA extracted. This DNA is then transfected into Sfs cells by lipofection. We have modified the pFASTBAC vector to include the cDNA for the P450 reductase under the control of the pl0 promoter. This new vector 1s used as a standard shuttle vector for a range of human P450 cDNAs.

We have found it easier to identify the blue recombmant colonies produced by the Bat-to-Bat system than the clear recombinant plaques produced by the Baculogold system. 3.2. Propagation

and Maintenance

Both S’ and T. nz cells are routinely TClOO and EX401 media, respectively

of Cell Lines mamtamed m suspension culture in (both supplemented as described m

Subheading 2.3.), m a variety of shaker and spinner culture flasks. 1. Seed cells at an mittal density of 2-4 x IO5 cells/ml. 2. Count cells and determine viability (trypan blue exclusion) dally (see Note 1) 3 Passage stock cultures every 2-3 d (see Note 2) For expansion of cell cultures, transfer cells from an exponentially growing culture mto a fresh vessel contammg warm medium (28°C) to give an inittal cell density of 24 x 105/mL It IS important that the cultures are not left to overgrow, as this often presents problems with growth of subsequent subculture.

3.3. Establishing Frozen Stocks of Cells The following procedure is used to establish frozen stocks of Sj?2and T ni cells. Routinely,

a frozen bank of 50 x 0.9-mL veals of cells is established

(see

Note 3). 1. From an exponentially growing culture of S’ or T ni cells, remove a volume of culture containing at least 5 x lo8 cells. Centrifuge at 300g for 10 mm at 4°C. 2 Discard the supernatant and resuspend the cell pellet m 45 mL of the appropriate complete culture medium (see Subheading 2.3.). Aspirate with a pipet to break up clumps. 3 Add 5 mL of DMSO to the cell suspension and mix well. 4. Dispense 0.9-mL aliquots of this solution into labeled cryovials 5. Place vials m a precooled (4°C overnight) Stratacooler benchtop cooler and leave at 4OC for 2 h. 6. Transfer the cooler containing the vials to a -80°C freezer and leave at this temperature overmght 7. Remove the vials from the cooler and transfer to a -135°C freezer or a ltqutd nitrogen cryostore

209

P450 Expression by Baculovirus

3.4. Establishing

Cell Culture from a Frozen Stock

To establish a cell culture from a frozen bank, the following procedure is used. 1 Warm 50 mL of the approprtate complete medium (see Subheading 2.3.) to approx 28’C and drspense into a 50-mL centrifuge tube 2 Place a vial of frozen cells into a beaker containing water at approx 37°C. 3. Transfer the contents of the thawed vial aseptically into the warm medmm in the centrifuge tube and centrifuge at 3009 for 10 min at room temperature 4. Discard the supematant and resuspend the cells in 50 mL of the appropriate complete medium (see Subheading 2.3,) (approx 28’C) and place m a 500-mL Erlenmeyer flask. 5. Incubate the flask of cells at 28’C on a shaker platform (125-150 rpm) 6. The culture is ready for further expansion after 4-6 d.

3.5. Mycoplasma Check We routinely (once every 2 mo) check for the presence of mycoplasma m insect cell cultures with the Gen Probe Mycoplasma TC Detection system. All stocks to be frozen and cells that have been in culture longer than 2 mo are checked for mycoplasma. A prerequisite for the mycoplasma test is that all cells to be tested are washed free of antrbrotlcs and cultured in antrbiottc-free medium prior to testing. Media to be tested should have been exposed to cells for at least 3 d.

3.6. Scale-Up of Baculovirus Stock To prepare high-ttter

worktng virus stock, the following

procedure

IS used.

1 Centrifuge S$?Jcells from an exponentially growing culture at 300g for 10 mm at room temperature. 2. Resuspend the cell pellet in fresh growth medmm at a density of approx 1 x 1O6 cells/ml in an appropriate spinner flask. 3. Add recombmant baculovnus to grve an effective multtphcrty of infection (MOI) of0 1. 4. Culture the cells at 28°C with the spinner speed kept m the range of 125-150 rpm. Take samples daily for cell density/viability evaluatton. 5. When the viabtlity drops below 50%, harvest the cells by centrtfugatron at 1OOOg for 10 min at 4°C usmg either sterile 250-mL comcal centrtfuge tubes or 1-L centrifuge bottles. Collect the supernatant aseptically and store in the dark at 4°C. 6. Remove a small sample (100 mL) and store separately at -80°C. Thts frozen stock IS the master stock and is not normally touched unless all other stocks have lost viral actrvtty or become contaminated. 7. Remove another small sample, store separately, and use to determine the virus titer (see Subheading 3.7.).

210

Hood, Shah, and Jones

8. If the virus stock 1s of high activity (i.e , >107 plaque forming units/ml j&ii/ mL]) , it is used to prepare viral stocks for use m large-scale expression work. We do not use recombinant baculoviruses that have been passaged more than five times.

3.7. Determination of Viral Titer by Plaque Assay Plaque assays are carried out to determine virus titer in p&/ml followmg procedure (see Note 4).

using the

1 Seed S’ cells (from an exponentially growmg culture) mto 35-mm Petri dishes m fully supplementedTClOO medium at a densityof 1 5 x lo6 cells/ 1 5-2 0 mL 2 Allow cells to attach for 2 h at 28’C 3. Remove the medium by plpet and add, m a dropwlse manner to the center of the dish, 0.4 mL containing an appropriate vuus dilution (serial log dilutions of the vn-us to be tltered, prepared in the growth medium) (see Note 4). Do this m duplicate. 4. Leave the dishes at room temperature for approx 1 h, on a level surface, to allow the virus to adsorb to the cells. Rock the dishes gently at 20 mm intervals to

ensure even coverageof the cells with the virus. 5 Remove the vuus moculum and discard into Hycolm dismfectant. 6. Add 2 mL of agarose/growth medium solution (1 1, v/v) and allow to set at room temperature for 20 mm (see Note 4). 7 Add 1 mL of growth medmm on top of the agarose overlay. 8. Incubate the dishes at 28°C for 4 d 9. Remove the medium from the overlay and add to the overlay 1 mL of 0 5% (w/v) neutral red solution. Leave for 2-4 h Decant the excess stain mto dismfectant Invert the dishes and store in the dark overnight. 10. Examine the plates The dilution that gives 20-50 plaques/well 1sused for determmmg the titer. For example, if there are an average of 35 plaques per dish for the 1od dilution, the vuus titer is calculated as 35 x 2.5 x lo6 p&/ml (the factor of 2.5 1sused because the virus is added in a volume of 0.4 mL).

3.8. Expression of Cytochrome P450 Enzymes For each P450 enzyme, small-scale (1-L) experiments using T. ni cells are set up to determine the optimum conditions for expression. These mclude the amount of baculovlrus added, time course of harvest, and the concentration and range of precursors used (see Note 5). These conditions are then apphed to fermenter cultures. 3.8. I. Small-Scale Expression The following procedure is used for small scale expression studies. 1. Resuspend exponentially growing T. nl cells in fresh EX401 medium supplemented with 5% fetal bovine serum, antibiotics, amphoterlcm B, and Pluromc F-68 (as described m Subheading 2.3.), at a density of approx 1 x lo6 cells/ml in either a 2-L Fernbach shaker flask or a 3-L spinner flask (see Note 6).

P450 Expression by Baculovirus

Fig. 1. Cell count and viability for a 1-L fermentation performed in a shaker flask. The virus was added on Day 0 at an MO1 of 2. 2. Add recombinant baculovirus (usually at an MO1 of 2). Heme precursors (e.g., hemin chloride, ferric citrate, etc.) are also added at this stage (see Subheading 2.4., steps l-3). 3. Incubate cells at 28”C, with the rotating speed of the shaker/spinner set at 125-150 tpm. 4. Remove samples daily for cell density/viability and P450 activity measurements. For the P450 activity determination, centrifuge 250 mL of culture at 1OOOgfor 10 min at 4°C. Discard the supematant and isolate microsomes from the cell pellet (see Subheading 3.10.).

Figure 1 illustrates

a typical

growth profile

for a 1-L culture grown in a

shaker flask.

3.8.2. Large-Scale

Expression

The following procedure is used to set up fermenter expression (see Notes 6 and 7).

cultures for large-scale

1. Fill the fermenter with EX401 medium supplemented with 5% fetal bovine serum. 2. Warm the medium in the fermenter to 28’C. 3. Add an inoculum of exponentially growing T. ni cells to the fermenter to give an initial cell density of l-3 x lO?mL. Also add appmx 5 mL of silicone 1520 antifoam. 4. Take samples daily to monitor cell density and viability, and pH. When the cell density reaches l-2 x lO’?mL, add baculovirus at an MO1 of 2. Also add precur-

Hood, Shah, and Jones

Fig. 2. Cell count and viability for a 36-L fermentation performed in a stirred fermenter. The virus was addedon Day 3 at an MO1 of 2. sors at the time of virus addition (see Subheading harvestedafter 3 d of virus infection.

2.4.). The cells are usually

The growth profile for a typical 36 L fermentation is shown in Fig. 2. 3.8.3. Large-Scale 3.8.3.1.

Fermentation

GROWING INSECT CELL CULTURE IN MICROBIAL BIOREACTORS

For growing insect cells we use Biolafitte 15 L, and Chemap 75 L microbial fermenters. Damaging shear is minimized in the fermenters by the design and use of a dissolved oxygen (D02) control scheme dependent on DO2 set point error to control the agitation rate, the airflow rate, and the vesselhead pressure. Maximum permitted shear rates are estimated based on previous work carried out in shaker flasks. Minimum agitation rates are estimated to prevent sedimentation. 3.8.3.2.

IN~CULUM PORTS

The inoculation procedure (steaming connections in sifu) is modified to minimize the time between inoculum preparation and vessel inoculation (the viability of the cells decreases rapidly if they are left unstirred and unaerated for any length of time).

P450 Expression by Baculovirus

213

Fig. 3. Cell count and viability for a 10-L fermentation performed in a microbial bioreactor. The virus was added on Day 3 at an MO1 of 2.

3.8.3.3. STEAMTRACES Local hot-spots are minimized by steaming sample ports for 10 min only at the time of sampling. Typical growth profiles for a 10-L and a 50-L microbial bioreactor are shown in Figs. 3 and 4, respectively. 3.9. Harvesting of cell Pellets At the end of each fermentation, the cells are harvested and the resulting cell pellets processed to yield microsomes (see Subheading 3.10.). The method of harvesting is determined by the culture volume. Cultures up to 10 L are harvested by centrifugation, whereas larger volumes are processed by tangential flow filtration. (28,19). 3.9.1. Harvesting by Centrifugation 1. Sediment the cells from the fermentation by centrifugation at 1162g for 10 mitt at 4°C. In our laboratories, this is performed in 1-L polycarbonate bottles with sealed caps (Beckman) in a Sorvall DuPont RA3C. 2. At the end of the first centrifirgation, discard the supematant. Pour fresh culture into the same bottles and repeat the centrifugation. 3. Once all the culture has been centrifuged, resuspend the pellets in a small volume of PBS and pool into one bottle.

214

Hood, Shah, and Jones

Fig. 4. Cell count and viability for a 50-L fermentation performed in a microbial bioreactor. The virus was addedon Day 3 at an MO1 of 2. 4. Centrifuge the cells again andresuspendthe pellet in fresh PBSin order to remove tracesof the culture medium. 5. After a final centrifugation, processthe pellet to yield microsomes or store at -8O‘C until required. 3.92. Large-Scale Harvesting Large-scale harvesting is performed by membrane filtration (Z&19). 1. To maximizerecovery, flush the systemwith a volume of PBSequal to the holdup of the systemto recover any material that is held in the systemor adhered to the membrane.The recovery cycle is caked out with the permeatevalve closed and pump off. 2. Repeatuntil the volume of concentraterecovered is 6 L. 3. Sedimentthe concentrateas described in Subheading 3.9.1. 4. Freezethe cells from a 36-L culture in onepellet, and those from a 50-L fermentation in two aliquots. 3.10. Preparation of Microsomes The following protocol is for the preparation of microsomes from the pellet of cells obtained from a 36-L fermentation (or from half the pellet obtained from a 50-L fermentation). Homogenize the pellets and dispense the microsomes in a cold room (4OC) or on ice.

P450 Expression by Baculovirus

215

1 Resuspend the cell pellet in an appropriate volume of resuspension buffer (see Note 8). If the pellet was frozen after harvesting, defrost slowly by adding 200300 mL of warm buffer (<‘25”C), incubate m a warm bath (37’C), and then collect the melted mixture Repeat until the pellet is thawed (take care not to exceed the total resuspension volume). 2 Pour a 60-80-mL ahquot of the pellet/buffer mix mto the homogenizer vessel and disrupt the cells with 10 slow strokes of the pestle Pour the homogenate mto a 500-mL centrifuge bottle. Repeat the procedure until all the pellet mixture has been homogenized or until two bottles have been filled 3. Centrifuge the homogenate at 9000g for 30 min at 4°C (7300 rpm m an SLA3000 rotor m a Sorvall RA3C), and pour the supematant (the S9) into a beaker Take care not to disturb the pellet, and leave some of the supematant m the tube to avoid carry over of cell debris 4. Pipet the supernatant into 72-mL polycarbonate ultracentrifuge bottles. Balance the bottles against each other to a tolerance of 10 mg. (Take the balance with the Mlest tube and then make the rest up with buffer. NB, it IS important to balance the tubes with resuspension buffer because the 20% (v/v) glycerol content negates the use of water) 5. Centrifuge the remaining homogenate while the tubes are bemg balanced and the pestle and mortar are being rinsed with water and then resuspension buffer. 6. Transfer the centrifuge tubes into the rotor and place into the ultracentrifuge Run the centrifuge at 100,OOOg (average) for 70 min at 4°C (36,000 rpm in a 45Ti rotor). During this run, repeat step 5 for the second batch of S9. 7 At the end of the ultracentrlfuge run, remove the tubes from the rotor and keep on Ice. Replace with the balanced tubes from the second batch and repeat the centrifugatlon run (see Note 9). 8. Decant the supematant from the microsomal pellets. The pellet may have a soft upper layer and care should be taken not to pour this off with the supematant. 9 Add 30 mt. of resuspension buffer to the first tube and resuspend the pellet by washing and scraping with a 50-r& pipet. Vortex the solution and transfer mto the next tube. Transfer the buffer/pellet mix from tube to tube until all the pellets have been resuspended. Then transfer the pooled resuspension into the homogenizer vessel. 10. Add a further 20 mL of resuspension buffer to the first tube and rinse by vortexmg. Pass this rinse from tube to tube and finally combine with the resuspended mlcrosomes in the homogenizer Homogenize the pellet with 10 slow strokes of the pestle and pour the microsomes into a fresh 250-mL beaker on ice. 11 Repeat steps 8-10 with the second set of tubes and pool the resultmg microsomes with the first batch (this is best done by putting both batches in the homogenizer vessel and mixing with the pestle on slow speed to avoid spillages). 12. Aliquot the mlcrosomes into prelabeled cryovlals and snap freeze m liquid mtrogen (see Note 10). Store the majority of the mlcrosomes m 1-mL aliquots, but take some O-25-mL samples for the determination of protein concentration and P450 content. Once frozen, store the cryovials at -80°C until use and do not refreeze once thawed.

216

Hood, Shah, and Jones

Table 2 Suggested Homogenization and Final Microsomal Resuspension Volumes for Various Sizes of Fermentations Culture size/L 1 2

5 10 36 50

Number of batches

Total buffer volume (mL)

Ultracentrlfuge tubes per batch

1 1 1 1 1 2

60 120 280 575 1700 (2X) 1700

1 2 4 8 24 (2X) 24

Microsomal VOlhL 5

10 15 30 100 (2X) 100

4. Notes 1 Trypan blue cell viability test is performed by mlxmg 50 pL of cell sample with 50 @, of 1.5 diluted trypan blue. 2 Routinely, cell cultures are not passaged beyond 2 mo and are checked for mycoplasma contammation on a regular basis (4-8 wk) Mycoplasma-contaminated cultures are destroyed by autoclaving. 3 Frozen stocks of Sj?3and T. nz cells are routinely established in growth medium supplemented with 10% (v/v) DMSO at a density of 1 x 1O7cells/ml It is important that the cells used for freezing are in an exponential phase of growth 4 In the plaque assay, it IS important that the cell monolayer ts not allowed to dry out at any stage, especially durmg virus addition and removal The temperature of the agarose/medmm mix 1scrltical We routinely incubate the solution at 37°C and, before addition to the 35-mm assay plate, the solution ts cooled at room temperature until it barely feels warm to the hand. It 1sthen quickly added on top of the monolayer 5. Heme precursors are added to prevent the depletion of the intracellular heme pool becoming a rate llmitmg factor for the production of functional P450 protein. It is important to optimize the type and amount of precursors for each cell type and lsoform 6. Headspace area in both shaker and spinner flasks 1scritical and culture volume exceeding headspace volume results in poor growth 7 Cell clumping 1s usually caused by Inadequate stirring speed of the culture vessel. Both ,S’fsand T nz will tolerate speeds up to 150-200 rpm and the addition of Pluronic F-68 1s essential to mimmlze shear damage and mamtam a high cell vlabihty 8. There is no hard formula for the amount of buffer used for resuspension of the pellet We have found that this 1s dictated by the volume of S9 that could be processed to mlcrosomes in a day. We do not recommend the storage of insect cell S9 overnight as this seems to have a detrimental effect on the final P450 yield. The buffer volumes used in our laboratory are shown in Table 2.

P450 Express/on by Baculowrus

217

9. In this protocol, we use two Beckman 45Ti rotors (capacity 6 x 72 mL) runnmg m Beckman Ex 90 ultracentrifuges By performing two batch runs m each rotor a total of 1700 mL of S9 can be processed to microsomes in a 4-h period. 10. This method of microsomal preparation routinely yields 100 mL of mtcrosomes at a protein content of 15-20 mg/mL from a 3 6-L culture The final protein concentration varies depending on the final cell count and viabihty at the time of harvest. In our hands, typical P450 yields range from 50 to 250 pmol P450lmg microsomal protein, as determined by CO difference spectral analysis

References 1. Buters, J T , Shou, M., Hardwick, J. P , Korzekwa, K R , and Gonzalez, F. J. (1995) cDNA-directed expression of human cytochrome P450 CYP 1A 1 using baculovnus Drug Met Dlspos 23,696-701 2 Grant, D. F., Greene, J. F. , Pmot, F., Borhan, B., Moghaddam, M. F , Hammock, B. D., Ohkawa, H., Luo, G., and Guenthner, T. M. (1996) Development of an in situ toxicology assay system using recombinant baculovnuses. Bzochem. Pharmacol 51,503-5 15 3 Grogan, J , Shou, M , Andrusiak, E A , Tamura, S., Buters, J T , Gonzalez, F J , and Korzekwa, K (1995) Cytochrome P4502A 1,2El and 2C9 cDNA-expression by insect cells and partial purification usmg hydrophobic chromatography Blochem Pharmacol 50,1509-l 5 15 4 Paine, M. J. I., Gilham, D., Roberts, G. C. K., and Wolf, C. R (1996) Functional high level expression of cytochrome P450 CYP2D6 using baculovnus expression systems. Arch Blochem Bzophys 338, 143-150 5. Biagmi, C. and Celier, C. (1996) cDNA directed expression of two allehc variants of cytochrome P450 2Cll using COS and SF2 1 insect cells Arch Bzochem Blophys

326,298-305.

6 Zeldin, D. C , DuBois, R. N., Falck, J. R., and Capdevtla, J H (1995) Molecular cloning, expression and characterisation of an endogenous human cytochrome P450 arachadonic acid epoxygenase isoform Arch Blochem Bzophys 232, 76-86 7. Patter-t, C J. and Koch, P (1995) Baculovnus expression of human P450 2E 1 and cytochrome b5 Arch Blochem. Biophys 238, 143-150 8. Wu. S., Moomaw, C R., Tomer, K. B., Falck, J. R., and Zeldm D C. (1996) Molecular clonmg and expression of CYP2J2, a human cytochrome P450 arachadonic acid epoxygenase highly expressed m heart. J Blol. Chem 271(7), 3460-3468. 9 Buters, J. T. M., Korzekwa, K. R., Kunze, K. L., Omata, Y., Hardwick, J. P , and Gonzalez F J (1994) cDNA-directed expression of human cytochrome P450 CYP3A4 using baculovnus Drug Met Dispos 22,688492. 10 Lee, C. A ., Kadwell, S H., Kost, T A., and Serabjit-Singh, C. J. (1995) CYP3A4 expressed m insect cells Infected with a recombinant baculovuus containing both CYP3A4 and human NADPH-Cytochrome P450 reductase is catalytically similar to human liver microsomal CYP3A4 Arch Bzochem Bzophys 319, 157-167.

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and Jones

11 Sakuma, T., Kttamura, R., Yokio, T., and Kamataki, T (1995) Efficient complementary DNA-directed expression of human fetal liver cytochrome P450 (CYP3A7) m insect cells using baculovtrus. Bzochem. Mol. Bzol Int 35, 447-455.

12. Imaoka, S., Ogawa, H , Kimura, S., and Gonzalez, F J.(1993) Complete cDNA sequence and cDNA-directed expression of CYP4A11, a fatty acid omegahydroxylase expressed m human kidney. DNA Cell Bzol 12,893-899. 13. Barnes, H. J., Jenkins, C. M., and Waterman, M. R. (1994) Baculovu-us expression of bovine cytochrome P45Oc17 m Sf9 cells Arch Bzochem Bzophys 315, 489-494.

14. Lahde, M., Raumo, H., Pelkonen, O., Karp, M., and Oker-Blom, C (1993) Expression of human placental cytochrome P450 aromatase (CYP19) cDNA m insect cells using a luciferase based baculovirus vector. Bzochem. Bzophys. Res. Commun 197,151 I-1517 15. Sigle, R. O., Titus, M. A. Harada, N , and Nelson, S. D. (1994) Baculovuus-mediated high level expression of human placental aromatase (CYP19Al). Bzochem Biophys

Res Commun. 201,69&700

16. Amarne H. B. and Simpson E R (1995) Expression of a recombinant derivative of human aromatase P450 m insect cells utilising the baculovuus vector system Mol Cell Endocrznol.

109, l-5

17. Luckow, V A , Lee, S C., Barry, G F , and Olins, P. 0 (1993) Efficient generation of infectious recombinant baculoviruses by site-specific transposon-medtated insertion of foreign genes into a baculovirus genome propagated m Escherzchza coli J Vzrol 67,4566-4579

18 Radlett, P. J (1972) The concentration of mammalian cells in a tangential flow filtration unit. J Appl Chem. Bzotechnol 22,495-499 19 Tom, R. L., Caron, A W., Massie, B., and Kamen, A A. (1995) Scale up of recombinant vuus and protein production m stirred tank reactors Methods Mol Biol 39,203-224

25 Expression of Cytochromes in Mammalian Cells

P450

Els M. de Groene 1. Introduction In the past few years, much research effort has been directed toward the expression of human cytochrome P450 cDNAs in mammalian cell Imes. The use of such cell lines allows the investigation of the metabohsm of xenobiotics by mdivtdual cytochromes P450 (1,Z). Stable expression of cytochrome P450 cDNAs can be achieved by using vectors derived from Epstein Barr virus (3), SV40-based vectors (4), and by retrovnal infection (S-7). In this chapter, stable expression of human cytochrome P450 cDNAs in NIH/ 3T3 cells using retroviral infection will be described (see Fig. 1). The retroviral vector-part of the plasmid contains two long-terminal repeats (LTRs; necessary for integration, promotion, and polyadenylation), a packaging sequence psi (w), and the foreign cytochrome P450 cDNA under the control of the cytomegalovirus promoter. This retroviral vector is transfected mto a “helper” cell lme (~2) which is ecotropic. The virus particles produced by an ecotropic helper cell lme are only capable of infecting rodent cells, whereas amphotroptc virus particles can infect rodent, human, monkey, and dog cells. The helper cell line produces pivotal viral proteins being the env protein (core of the vnus particle), the gag protein (antigen determination), and the pol protem (reverse transcriptase). When RNA of the retroviral vector is expressed in the ~2 cells, these proteins recognize the packaging sequence and this results m the production and budding of virus particles containing retroviral vector RNA. Such virus particles are able to infect ecotropic cells where the RNA IS released in the cytoplasm. The viral RNA is transcribed by reverse transcriptase to DNA which migrates to the nucleus of the cell. This strategy results m efficient integration From Methods m Molecular Biology, Voi 107 Cytochrome P450 Protocols EdIted by I R Phillrps and E A Shephard 0 Humana Press Inc , Totowa,

219

NJ

de Groene

220

pst n

transfection

virus particle

(3

0

infection

Fig. 1. Pathway of replication-defective retroviral virus infection. Fulllength cytochrome P450 cDNA is inserted in the Hind111 site of the Moloney murine leukemia virus-based vector LNCX. Abbreviations: LTR (long-terminal repeat); psi (w = packaging sequence); NE0 (neomycin-resistance gene); CMV (cytomegalovirus promoter); G (gag gene); P (pol gene = reverse transcriptase); E (env gene). of the viral DNA into the host genome. No spreading infection can occur because the virus particles are replication-deficient (8). Many retroviral vectors are available; the vector used in this protocol, LNCX, also contains the cDNA for the neomycin-resistance gene (see Fig. 1).

Cytochromes P450 in Mammalian Cells

221

This is used to select the cells that have successfully integrated the retroviral DNA into the genome of the host cell. Individual colonies of cells resistant to G418 (aneomycin analog) are selected and cultured separately. The level of expression of cytochrome P450 mRNA in each clone is determined. Those clones with the highest expression are further examined for their cytochrome P450 activity, using substrates specific for the different cytochrome P45Os.All the cytochrome P450-expressing cell lines are compared with a vector-infected cell line containing no cytochrome P450 cDNA. This chapter focuses on the methods for transfection, infection, and RNA analysis necessary to develop genetically engineered cell lines. In addition, a method for analysis of the cytochrome P450-mediated catalytic activity of such cell lines will be briefly described. 2. Materials 2.1. Plasmid DNA Preparation All cytochrome P450 cDNAs were cloned in the Hind111site of the Moloney murine leukemia virus-based vector LNCX (7,9) by standard clonmg techniques and stored as bacterial stocks at -70°C (10). For DNA Isolation the following materials are used: 1. Lurla Bertam (LB) medium: 10 g/L NaCl, 10 g/L peptone, 5 g/L yeastextract Dissolve m water and autoclave(store at 4°C). 2 Qlaprep megaplasmld isolation kit (Qlagen GmbH, Hllden, Germany). 3. Isopropanol. 4 Ethanol (70%) in water. 2.2. Cell Culture Cells are cultured in Dulbecco’s modified Eagle’s medmm (DMEM) supplemented with 10% fetal calf serum (FCS), 0.375% sodium bicarbonate, 100 U/mL penicillin, 100 mg/mL streptomycin and 2 mM L-glutamine, at 37°C and 5% C02. The followmg stock solutions are used: 10X DMEM (Gibco BRL, Gaithersburg, MD). 7.5% sodium bicarbonate (Gibco BRL). 100X Penicillin/ streptomycin (Gibco BRL). 200 mML-glutamine (dissolve in water, filter-sterilize (0.22 p) and store at -2OOC). 1OX Trypsin solution (Gibco BRL) diluted to 1X trypsin in phosphate-buffered saline (PBS). PBS: 137 mA4 NaCl, 2.7 mA4 KCI, 0.8 mMNaH2P04, 1.5 W K2HP04, dissolve m water and autoclave. 2.3. Transfection and Infection 1. 1MCaC12,dissolvein water andfilter-sterilize(0.22 pm) (storeat4’C). 2. 2X HBS: 280 mMNaC1,50 mMHEPES, 1.5mMNa2HP04, dissolve m water and adjustto pH 7.1 + 0.05 (very important),filter-sterilize (0 22 m) (storeat -20°C).

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3 Polybrene (20 pg/mL) (Sigma, St LOUIS, MO), dissolve in water, and filter-sterlllze (0 22 pm) (store at 4OC) 4 G418 (20 g/L) (Glbco BRL), dissolve in DMEM, and filter-stenhze (0 22 w) (store at 4°C).

2.4. RNA Analysis There are many commercially available kits for RNA Isolation. The two kits we have tested have both proven to be efficient and each produced a high yield

of intact RNA. 1. RNA Insta-pure System (Eurogentec S. A., Seramg, Belgium) or TRIzol (Gibco BRL) 2. Chloroform 3 Isopropanol 4, Ethanol (70%) m water. 5 0 1% sodium dodecyl sulfate (SDS) m sterile water

3. Methods 3.1. Preparation

of DNA for Transfection

1. 2. 3 4. 5 6

Inoculate 10 & bacterial stock m 500 mL LB. Incubate at 37”C, ovemtght. Centrifuge the bacterial culture for 15 min at 5OOOgand remove the medium Isolate plasmld DNA according to the Qlaprep handbook (see Note 1) The DNA 1spreclpltated with 0 7 volumes of isopropanol Centrifuge for 30 min at 20,OOOg at 4°C and remove supematant Wash the DNA pellet with 5 mL cold 70% ethanol and centrifuge again for 15 min as described m item 5. 7. Dissolve the DNA pellet in 0.5 mL water. Determme the concentration and purity from the absorbance at 260 nm and 280 nm (1 ODZbO= 50 pg/mL; A260/A280ratio for pure DNA = 1.6-l 8). To sterilize the solution

of plasmld

DNA m water extract with 1 volume

of

chloroform/lsoamylalcohol (24: 1;v/v), vortex and centrifuge 10 mm at 13,000g. Collect the upper aqueous layer, containing the DNA, in a sterile tube. The DNA solution is now sufficiently sterile to be transfected into cells. Store at -2O’C. 3.2. Cell Culture

The murine fibroblast NIH/3T3 cells (ATCC CRL 1658) and the “helper” cell line, ~2, which 1sderived from NIH/3T3 cells (21) are cultured in 75 cm2 culture flasks containing 10-15 mL complete DMEM medium (hereafter DMEM refers to complete DMEM contammg all supplements). The cells are trypsimzed when they have reached 90% confluency (for NIH/3T3 cells twice a week [see Note 21).

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223

1. Remove the medium and wash the cells with 2 mL PBS. 2 Incubate the cells with 2 mL trypsin (1X) until they detach (within 5 mm [see Note 31). 3. Suspend the cells m the trypsm and transfer 0.2 mL mto a fresh culture flask containing 10-15 mL DMEM.

3.3. Transfection and Infection 3.3.1. Transfection The retrovlral vectors are transfected mto ~2 cells accordmg to the calcium phosphate coprecipitation method (12) with some minor alterations (see Note 4). 1. Take a 75 cm* culture flask containing ~2 cells that are not more than 80% confluent. 2. Trypsinize the cells m 2 mL trypsin and add 2 mL DMEM. 3. Take a small sample from the cell suspension and count the cells usmg a hemocytometer 4. Collect 2 x lo6 cells in a separate tube and store on ice. 5. Dilute 15-20 pg plasmid DNA to a final volume of 375 $ with sterile water 6 Add 125 p.L 1 MCaC12 to the DNA solutton. 7 Pipet 0 5 mL 2X HBS into another tube 8 Slowly add the DNA/CaC12 solution to the 2X HBS while contmuously shaking the mixing tube; a “blue mist” will appear (see Note 5) 9 Incubate this mixture for 15 mm at room temperature. 10. During the incubation, centrifuge the ~2 cells for 5 mm at 200g and remove the supernatant. 11. Resuspend the cell pellet m the DNA precipitation mixture and incubate for 15 min at room temperature. 12. Add DMEM to a final volume of 7 mL and transfer to a 75 cm2 culture flask 13. Incubate for 6 h at 37°C and 5% CO*. 14. Remove the culture medium and wash the cells twice with 5 mL PBS Add 10 mL DMEM and return cells to the incubator. 15. After 2 d the culture medium (containing the replication-deficient virus particles) can be collected and filtered through a 0.45-pm filter in order to remove any ~2 cells that might have detached. This filtrate can be stored at -7O’C up to 6 mo and IS used to infect NIH/3T3 cells (see Note 6).

3.3.2. Infection 1 Plate 1 x lo6 NIH/3T3 cells m a 75 cm2 culture flask and culture overnight. 2. Remove the medium and incubate the cells for 2 h with the filtrate containing the replication defective vnus particles (see Subheading 3.3., item 15) supplemented with polybrene 8 pg/mL 3. Replace the virus-containing medium with DMEM and culture the cells for 2 d.

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4. Trypsimze the cells and plate them at different cell dilutrons (1:50, 1: 10, 1:5, and so on) m 75 cm2 culture flasks containmg DMEM supplemented with G418 (1 g/L) 5. Use untreated NIH/3T3 cells as a control for infection 6. DMEM supplemented with G418 has to be replaced every 2 or 3 d dependmg on the number of dead cells m the culture. 7. After 2 wk individual cell colonies can be observed and ptcked (see Note 7). 8, For picking colomes, use Pasteur-plpets wtth a bent end (hold m gas flame and let the tip of the pipet bend 90”, sterilize by autoclavmg) or, tf Petri-dishes are used instead of culture flasks, use an Eppendorf prpet trp. 9. Place a microscope in the lammar-flow cabmet 10. Fill a 24-well plate with DMEM supplemented with G418 (0 5 g/L). 11 Pick the colonies while looking through the microscope and transfer to the 24-well plate for culturing 12. When the cells reach confluency m the wells, wash them and trypsimze with a drop of trypsm 13. Resuspend the cells using a I-mL Eppendorf pipet and place m a 25 cm2 culture flask.

3.4. RNA Analysis Cells that are growing m a monolayer in a 3.5-cm Petri dish can be lysed drrectly usmg 1 mL RNA Insta-pure (for frozen cell pellets (-70°C) use 0.2 mL for 1~10~ cells). 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Solubilize the lysate by prpeting rt up and down a few times Add 0.1 mL chloroform per 1 mL lysate, vortex for 15 s, and place on ice for 5 min. Centrifugate for 15 mm at 12,000g (4OC). The aqueous phase (upper layer), now approx half of Its initial volume, is transferred to a fresh tube. Add an equal volume of tsopropanol, vortex, and place the samples for 15 min at 4°C Centrifuge for 15 mm at 12,000g (4°C). Wash the RNA pellet with 1 mL 70% ethanol and centrifuge again as m step 6. Remove the supernatant and let the pellet briefly dry (not too long because very dry pellets are more dtffcult to drssolve) Dissolve the pellet m 100 p.L water contammg 0.1% SDS (see Notes 8 and 9). Determine the concentratton by the absorbance at 260 nm and 280 nm (1 ODZ6a = 40 pg RNA/mL; AZ6,jA2s0 ratio for pure RNA = 1 8-2.0) RNA dot blots are made on Hybond N+ and are hybridized with [32P]dCTP labeled cytochrome P450 cDNA according to standard procedures (10)

3.5. Activity

Analysis

Culture 3 x lo6 cells in a 75 cm2 culture flask in DMEM. Incubate the cells the followmg day for 3, 6, or 18 h in 5 mL DMEM contaming the substrate spectfic for the particular human cytochrome P450 being

Cytochromes F450 In Mammalian Cells

225

expressed (see Notes 1042). Serum-free medium (Ultraculture, BloWhittaker, Walkerville, MA) is used when metabolites are analyzed by high-pressure liquid chromatography (HPLC). 4. Notes 1. DNA can also be isolated by standard techmques m whtch phenol/chloroform purification steps are used, but this is not directly suitable for transfectton. The DNA should be purified by equilibrium centrifugation m cestum chlortde/ ethidium bromide gradients (10). 2 NIW3T3 are Iibroblasts which possesscontact inhibition, when the cells are grown to 100% confluency, they stop growing. However, tf they are not trypsmtzed at the appropriate time they lose their contact inhibition and can transform 3 Some cell lines need a longer period of time to detach; thts ttme can be decreased by incubating them at 37°C. For some cell lmes, trypsin 1s toxic. Then do not Incubate the cells with 2 mL trypsm, but “wash” the cells wtth trypsm and after detaching suspend them in DMEM 4 Here the transfectton IS performed using the calcmmphosphate prectpttatton procedure Another method is transfection with a lipid/DNA complex For this method, several kits are commercially available (e.g., Lipofectm, Gtbco-BRL; DOTAP, Boehrmger Mannhetm). 5. When a real precipitate 1sobserved instead of a “blue mist,” the pH of 2X HBS ts most likely not correct. Another possibility IS that the DNA is not pure enough 6. Although hamster cells are not suscepttble to retrovtral mfectton, genettcally engineered hamster cell lines can still be produced using retroviral vectors via transfection alone. 7. If no colonies are observed after two weeks and all the cells are dead, check that the DNA used for transfection was mainly m the supercotled form It 1s also possible that the ~2 cells have lost most of their expresston of viral protems and thereby then “helper” function. The latter can be avoided by culturmg the ~2 cells under hypoxanthme, ammopterm, thymtdine (HAT) selection 8. An mcubatlon at 60°C may be required for complete drssolvmg of RNA. 9. Handle RNA with care. autoclaved or diethylpyrocarbonate (DEPC)-treated solutions should be used. 10. Start activity studtes with 18 h incubations, then try shorter mcubatton times. 11 The amount of catalytic activity can be increased by co-expresston of NADPHcytochrome P450 oxtdoreductase cDNA (13, Gentest, Woburn, MA). 12. Examples of specific substrates used to test catalytic acttvity m cell lmes expressing human cytochromes P450 (3-7,13,14). CYPlAI, ethoxyresorufin, CYPlA2, caffeine, ethoxyresorufin; CYP2A6, coumarm, CYP2B6, ethoxytrifluoro-methylcoumarm, CYP2C8/9/10, tolbutamide, diclofenac, paclitaxel, CYP2C 19, mephenytom; CYP2D6, debnsoqum, dextromethorphan, bufuralol, CYP2E, amlm, p-nitrophenol, CYP3A4, testosterone.

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References 1 Gonzalez, F J., Crespt, C L., and Gelboin, H V. (199 1) cDNA-expressed human cytochrome P45Os a new age of molecular toxtcology and human risk assessment. Mutation Res 247, 113-127. 2 Langenbach, R , Smith, P. B., and Crespi, C L (1993) Recombmant DNA approaches for the development of metabolic systems used in m vttro toxicology Mutation Res 277,25 L-275. 3. Crespt, C. L. (1993) Human cell lines, derived from AHH-1 TK+/- human lymphoblasts, genetically engineered for expression of cytochromes P450. Tox~ology 82,89-104 4. Doehmer, J. (1993) V79 Chinese hamster cells genetically engineered for cytochrome P450 and then use m mutagenicity and metabolism studies ToxzcoEogy 82, 105-I 18. 5. Nouso, K., Thorgeisson, S. S., and Battula, N. (1992) Stable expresston of human cytochrome P4502EI in mammalian cells. metabohc acttvatton of nitrosoamme and formatton of adducts wtth cellular DNA. Cancer Res 52, 1796-1800. 6 Salompaa, P., Hakkala, J , Pasanen, M., Pelkonen, 0 , Vahakas, K , Battula, N., Nouso, K., and Raunio, H. (1993) Retrovuus-mediated stable expression of human CYP2A6 m mammalian cells. Eur J. Pharmacol. 248,95-102. 7. de Groene, E. M., Hassing, I. G. A. M., Blom, M. J , Semen, W., Fink-Gremmels, J , and Horbach, G J. (1996) Development of human cytochrome P450-expressmg cell lines application m mutagenicity testmg of ochratoxin A Cancer Res 56,299-304. 8 Varmus, H E. (1982) Form and function of retrovtral provn-uses. Sczence 216, 8 12-820. 9 Miller, A. D., and Rosman, G. J. (1989) Improved retrovtral vectors for gene transfer and expression Bzotechnzques 7,980-990. 10 Sambrook, J , Fritsch, E. F , and Maniatis, T (1989) Molecular Clonzng* A laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 11. Mann, R., Mulligen, R. C , and Baltimore, D. (1983) Construction of a retrovuus packaging mutant and tts use to produce helper-free defective retrovuus Celi 33, 153-159 12 Graham, F. L., and Van der Eb, A. J. (1973) A new technique for the assay of mfecttvity of human adenovirus 5 DNA. Vwology 29,241-264. 13. Schneider, A., Schmalix, W. A., Suuguri, V , de Groene, E. M., Horbach, G J., Kleingeist, B., Lang, D , Backer, R., Belloc, C , Beaunne, P , Gretm, H , and Doehmer, J. (1996) Arch. Bzochem Bzophys. 332,295-304 14. Sonmchsen, D S., Lm, Q., Schuetz, E. G., Schuetz, J D., Pappo, A , and Relhng, M. V. (1995) Variabtlity in human cytochrome P450 pa&axe1 metabohsm J Pharmacol Expt. Ther. 275,566-575.

Monoclonal

Antibodies

to Cytochromes

P450

Harry V. Gelboin, Magang Shou, lnna Goldfarb, Tian J. Yang, and Kristopher Krausz 1. Introduction 7.1. Monoclonal

Antibodies:

Properties

and Production

Monoclonal antibodies (MAbs) are produced by the “hybridoma” technology developed by Kohler and Mllstem m 1975 (I), which is a major and definmg achievement of modern biology (2). The hybrldoma methodology producmg MAbs was derived from the clonal selection theory of Bumet and colleagues (3), which stated that each B-lymphocyte antlbody-forming cell and its progeny is committed to the production of a single, unique type of antibody molecule. This chemically defined MAb molecule spectkally recognizes a single antigenic determinant or “epltope” on the protein antigen, which, m our studies, is a specific individual cytochrome P450 (P450). This specificity of the MAb continues for the lifetime of the hybrldoma cell, which can be frozen or grown m culture indefinitely, and with its progeny, 1sa immortal source of an epitope-specific MAb. The MAbs are chemically defined, pure, epitope specific, and reproducible for limitless generations of cells. 1.2. Production of MAbs MAbs are produced by the fusion of myeloma tumor cells with isolated dissociated B lymphocytes of the spleen from mice nnmumzed with a particular immunogen. The fused cells or “hybndomas” combine the characteristics of the B lymphocytes, which are committed to the contmuous and stable production of a specific antibody, with those of the myeloma tumor cells, which impart the stable and immortal properties. The individual hybridomas are cloned and screened for the desired MAb with various assays such as binding enzymelinked immunosorbent assays (ELISA) immunoblotting, and P450 enzymeFrom: Methods m Molecular Bology, Vol 107 Cytochrome P450 Protocols E&ted by I R Phillips and E. A Shepherd 0 Humana Press Inc , Totowa, NJ

227

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Gelboin et al.

mhibitory activity. The selected hybridoma clone is usually subcloned three times to ensure monoclonality. The hybrldomas can be grown in cell culture in a defined medium or larger amounts of MAbs can be obtained by injecting the hybridomas into the peritoneal cavity of mice where the ascites fluid contammg large quantities of the specific MAb can be collected. MAbs are directed to conformational or sequential epitopes of the P450 protein. Conformatlonal determinants depend on the native spatial conformation of the P450, whereas sequential epitopes depend only on the sequence of ammo-acid residues. In each case, the antlgenic determinants are topographic, i.e., composed of structures on the protein surface. MAbs to epltopes of different types and position may help elucidate the topography of the P450 antigen in relation to the mlcrosomal membrane, identify sites that are immunodommant, and help characterize the active site of the enzyme. Two major uses of the MAbs are the detection and quantification of specific P45Osand the determination of the role of each P450 in the metabolism of a particular substrate (4,.5). 1.3. MAbs and P45Os (6) MAbs probes are reagents par excellence for the highly specific detection and quantification of proteins. They are ideal for the study of the multrple isoforms of P45Os,which collectively catalyze the oxldatron of a multitude of xenoblotlcs and endoblotlcs which include drugs, carcinogens, toxins, and some endogenous metabohtes. Some of the P45Osare polymorphic m the human population and m certain casesare found to be defective m a percentage of the population (7). These individuals are especially sensitive to the toxic effects of drugs that would normally be metabolized by the P450 that was defective. An example of this is the debrlsoquine polymorphism, m which debrisoqume presents a toxic hazard to the patient (8) The MAbs can measure the protein concentration and the contribution of each individual P450 to specific substrate metabolism in a tissue contaming multiple forms of P450 (4,5). This ability would permit a phenotyping of individuals in respect to their complement of the individual P45Os contributing to the metabolism of administered drugs, carcinogens, or toxic materials. This information in turn can be used to identify the appropriate drug and its dosing level and also as an aid in drug development. 1.4. MAbs and Polycional Antibodies (PAbs) MAbs are far superior reagents than PAbs for most analytic purposes. Immunogens have many different determinants and thus the exposed B-cells collectively produce a large number of different antibodies that recognize different epitopes on the antigen. PAbs are thus impure heterogeneous reagents that bind multiple epltopes and, therefore, are likely to exhibit considerable undesired cross-reactivity. Further, repeated immunizations are required for new antl-

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229

body production and each nnmunization produces a different quality of antibody preparation. A useful manual (9) for the production and characterization of MAbs and PAbs has been published. 2. Materials 2.1. lmmuniza tion 1. Freund’s complete adjuvant 2. Freund’s incomplete adjuvant.

2.2. Production of Hybridomas The myeloma cell line, NS- 1, supplied by the Fredrick Cancer Research and Development Center (FCRDC), resistant to 8-azaguamne, was used as the myeloma fusion partner. 1. RPM1 1640 medmm-Complete (supplemented with 2 r& L-glutamine, 10% fetal bovine serum (FBS), and antibiotics/antimycotlcs, If desired. 2. HAT media-Complete as (RPMI)-Complete except that it contains 20% FBS and is supplemented with 10 fl hypoxanthme, 0.4 @4 ammopterm, and 16 @4 thymldme, and 10% Hybridoma Cloning Factor (IGEN) 3. HT media-Complete as (RPMI)-Complete except that it contains 20% FBS and 1s supplemented with 10 flhypoxanthme, 16 flthymldine and 5% hybndoma cloning factor. 4. Hank’s Balanced Salt Solution (HBSS). 5. Sterile polyethylene glycol (PEG- 1500): 50% (w/v) in 75 mM HEPES, pH 7.0. 6. RPM1 1640 without FBS, not supplemented. 7. Equipment includes sterile microscope slides (with frosted ends), sterile &well tissue-culture plates or 60 x 15 mm style plates, and a tissue-culture hood.

2.3. Culturjng

and CYoning Hybridomas

1. RPM1 1640 medium-Complete, supplemented with 10% or with 20% FBS, 2 mM L-glutamine and, if desired, antibiotics/amtimycotlcs. 2. 10% hybndoma cloning factor: HCF; (IGEN). 3. Freezing medium: 50% RPM1 1640,40% FBS, 10% dlmethylsulfoxlde (DMSO). 4. IX coating solution (KPI Labs).

5. Equipment includes hemocytometer,96-well tissue-cultureplates,multichannel pipettor, and Beckman immunoassay plates.

2.4. Hydroxyapatite

Column Chromatography

1 Hydroxyapatite (HAP GradeOBlo-Rad, Hercules, CA). 2. Equilibration buffer. 0.01 M sodium phosphate, pH 6.8, 0.02% (w/v) sodium azide. 3. Elution buffers: (a) 0.01 Msodmm phosphate, pH 6.8. (b) 0.3 Msodlum phosphate, pH 6.8.

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230

4. Regeneration buffers: a. 0.5 A4 sodium phosphate, pH 6.8. b. 1 MNaCl. c. 0.01 A4 sodium phosphate, pH 6 8,O 02% (w/v) sodium azlde

3. Methods 3.1. Immunization We have successfully used a 3-wk hyperimmunlzation schedule. A typical schedule is outlined below. All tnjections are given rntraperrtoneally (i/p), using a 23-gage needle. Three to SIX 4-6-wk-old Balb/c mice are used. 1. Inject mice i/p with 30 ug of immunogen diluted 1:l (v/v) with Complete Freund’s adjuvant (d 0) (see Notes 1 and 2) 2 One week later (d 7), inject mice again but mix lmmunogen with 1 volume Incomplete Freund’s adjuvant 3. Take test bleed to determine if an unmune response has occurred (d 12-13) 4. Inject mice a third time using Incomplete Freund’s adjuvant (d 14). 5. Three days after the last injection (i.e., on d 17) kill mice for hybridization (fusion) (see Note 2)

3.2. Hybridoma

Production

All reagents should be prepared the day before fusion and, where indtcated, warmed to 37°C for at least 1 h prior to fusion. Sterile conditions and a trssueculture hood are required. NS-1 myelomas are grown in RPM1 1640-complete medium (see Notes 3 and 4). I

2. 3.

4. 5

6. 7

Place 3 x lo7 myeloma cells in a 50-mL conical centrifuge tube and centrifuge at 500g for 10 min, remove supernatant, and resuspend m 40 mL HBSS (37’C) This is considered one complete wash. Repeat step 1 and keep the myeloma cells in the tube at room temperature Kill an immunized mouse , then remove its spleen, and place m a sterile culture dish with 6 mL HBSS (37°C). Cover the dish. Remove the mouse and the dissecting instruments, and wipe the hood with 70% ethanol. Move spleen to another hood and continue with the fusion. Squeeze the spleen between the frosted ends of two microscope slides to obtain splenocytes. Repeat unttl only the connective tissue of the spleen remains. Discard spleen Transfer splenocytes by ptpet mto the tube with the myeloma cells Centrifuge at SOOgfor 10 mm. Remove the supernatant, and gently resuspend m 30 mL HBSS (This is the second wash). Repeat for a third wash. However, do not resuspend the pellet, and allow one drop of the HBSS to remam m the tube when removmg the supematant. Gently tap the tube to loosen the cell pellet and place into a 37°C water bath for 3 mm. Remove tube, and, under sterile conditions, while rotating the tube,

MAbs to Cytochromes P&Xl

8.

9.

10.

11. 12

231

SLOWLY add 1 ml of prewarmed (37°C) PEG solution dropwise during a 1-min period. Cells will aggregate into clumps owing to the PEG Contmue to rotate the tube for 1 min after the addition of PEG. Add, extremely gently and slowly, 10 mL prewarmed (37°C) RPM1 1640 (no FBS; no supplements) to the tube while continuing to rotate the tube Gently add 30 mL of RPM1 1640 to the tube to ddute the PEG. It 1snot necessary to continue to rotate the tube at this point. Allow to stand at room temperature for 10 min Centrifuge at 300g for 10 mm. Remove the supernatant Carefully add HATComplete medium (up to 35 mL) to the pellet, pipetmg down the wall of the tube to loosen the pellet DO NOT FORCEFULLY resuspend the pellet. It should dissolve with time. Allow the cells to remain in the tube for 1 h at 37°C m a CO, incubator (CO2 at 5%) Plate the cells into 96-well tissue-culture plates at a density of 1 x 104-1 x lo5 cells/well, based on Initial myeloma concentration in 0.2 mL HAT-Complete medium. Hybridoma growth should be evident under the microscope m 5-6 d and to the naked eye m 7-10 d. Replace HAT-Complete medium 5-6 d after plating, then switch to HT-Complete medium 12-14 d after initial plating Begin to screen the mdivldual wells for antibody production when they are at least 50% confluent with cell growth (see Subheading 3.6.).

3.3. Cuhuring Hybridomas (see Note 6) 1. Hybridomas grow best at a cell concentration of between 1 x 104/mL to 1 x 106/mL Do not allow the cells to reach a concentration of 1.2 x 106/mL, or cell death ~111 occur. In general, hybrldomas will double every 24-36 h. This will depend on the type of myeloma used Although hybrldomas usually grow in suspension, they may adhere shghtly to plastic tissue cultureware. If this happens, dislodge the cells by lightly tapping the sides of the flask or by usmg a stream of medium from the plpet. 2. Count the cells and dislodge them from the plastic Calculate the cell concentration based on dilutions. 3. Dilute cells by adding the appropriate amount of RPM1 1640-Complete medium to obtain the desired concentration. You may want to split equal volumes of cells from one flask into two or more flasks.

3.4. Freezing and Storage of Hybridomas (see Note 6) Freeze hybridomas in 1-mL aliquots at a cell density of 5 x 1O6to 5 x 107/ vial using the freezing medium (at 4°C). Resuspend the cells in the medium, pipet into cryovials, and place into a -8OOC freezer. The vials need to cool at a slow rate. This can be accomphshed by packing the vials tightly m a Styrofoam box, or wrapping the vials in several layers of paper towels, or, for a more uniform and consistent approach, use an insulated freezing container (available from NALGENE, cat. no. 5100-0001).

Gelboin et al. After the vials have been at -80°C for at least 5 h, they should be transferred to a liquid nitrogen freezer (vapor phase) for long-term storage. 3.5. Cloning of Hybridomas A single hybrtdoma will produce antibodies directed to a single epttope. When a population of hybridomas originate from the same cell, the cells represent a clone, and the antibodies they produce are monoclonal. The method described here can be employed because the cells are plated at a low cell density after fusion, and are thus “almost cloned.” Nevertheless, cloning should be performed at least three times to ensure monoclonality. 1. Count the cells to be cloned usmg a hemocytometer.Calculate the number of cells needed (i e ,3 plates at 96 wells/plate x 0 5 cells/well = 144 cells needed for one hybridoma lme). 2 Pipet cells mto medium (0 2 mL/well x 96 wells/plate x 3 plates = 57 6 mL medmm) 3. Using a multichannel prpettor, pipet 0 2 mL of the cell/medmm mixture into each well of the 96-well plates. Incubate at 37”C, 5% C02. Clones appear in 7-10 d. Screen the supernatant at 50% confluency (see Subheading 3.6.) Pick 5-10 positive clones and contmue to expand, freeze, clone, or begin large volume production of the desired monoclonal.

3.6. Screening for the Production of Antibodies As the hybrids begin to grow, each individual well on the 96-well plates that has cell growth IS assigned a number and spent medium from these wells 1s screened for the presence of antibody 1. Coat Beckman immunoassay plates with the desired antigen m 1X coating solution at 1 5 pmol/well (100 pL) (see Note 7) 2 Spent medium 1sscreened by ELISA performed by standard methods using alkaline phosphatase-coqugated goat F(ab’2) fragment to mouse IgG(H -I- L), mouse IgG (Fc specific), and mouse IgM (p-chain specific). 3. Each positive well rescreened and then cloned using complete medium contammg 10% HCF 4. Hybrids of interest are cloned at least three times

3.7. Ascites Production Ascites (or ascitic fluid) is an intraperitoneal fluid from a mouse that has developed a peritoneal tumor. The tumor IS induced by injectmg a mouse i/p with hybridomas. The peritoneal cavity serves as an excellent growth chamber for the hybridomas, which can grow to a very high density and produce a solution of high-titer antibodies. However, the ascites will contam other proteins as well. Typical concentration of protein in ascites is 20-40 mg/mL; and antibody

MAbs to Cytochromes P450

233

concentration can range from l-l 0 mg/mL. One mouse can yield from 2-l 0 mL of ascites fluid, if tapped more than once, Before injection of the hybridomas, mice should be primed by irritating the perttoneum, whrch will cause secretion of nutrients and lymphoid cells into the peritoneal cavity. 1. Adult Balb/c mice (6 wk-old) are primed with 0.2 mL pristane (2,6,10,14-tetramethyldecanotc acid) or incomplete Freund’s adjuvant i/p. 2. One to two weeks later, mlect i/p 1 x lo6 to 5 x lo6 hybridoma cells m 0.5 mL phosphate buffered saline (PBS) or HBSS. 3. One to two weeks after mlecting the cells, ascites should begm to bmld up When the abdomen is noticeably larger, but before the mouse has difficulty moving, remove fluid 4 Using a 19-gage needle, with a 5-mL syringe, withdraw as much flutd as possible. Return the mouse to its cage. Many mice ~111 produce more ascites fluid and can be tapped again in a few days then killed 5. Incubate ascites at 37°C for 1 h Transfer to 48°C and mcubate for between 4 h to overnight. 6. Centrifuge the ascites at 3000g for 15 min Remove the oil layer if present, and carefully remove the supernatant from the cell pellet. If debris IS still present, centrifuge again. 7. Ascites can be stored at -20°C for up to 1 yr. For long-term storage, -80°C is recommended. Prepare several aliquots of the ascites so that repeated freeze/thaw cycles are avoided.

3.8. Hydroxyapa fife Purification of MA 6s from Mouse Ascites Fluid There are several methods for MAb purification (9,IO). The followmg 1s one that we have used. 1. Hydrate hydroxyapatite in equthbration buffer and pack into a 2 5 x 15 cm col-

umn (bed volume of approx 74 mL). 2. Wash column with at least 3 bed volumes of equihbration buffer to allow column to equilibrate. 3. Up to 35 mL of ascites fluid can be applied to column Clarify 30 + 5 mg protein/ mL ascites fluid by centrifugation at 20,OOOg for 30 min at 4°C and dilute 1.5 before loading on column 4. Elute bound proteins with 500 mL of a 0.01 A4 to 0.3 M linear phosphate gradient, pH 6.8, at room temperature, with a flow rate of 1 mL/mm. Collect 4-5 mL fractions. 5. Albumm and other serum proteins elute first. Antibody will eiute m the phosphate gradient between approx 0.17-0.23 Molar. Of the approx 1.O g of total ascrtes protein on the column 2-10% (20-100 mg) are MAbs. 6. Columns can be regenerated by washing with 3 bed volumes 0 5 A4 sodium phosphate, 3 bed volumes of 1 MNaCl; then with 6 bed volumes of 0.01 A4 sodium phosphate, pH 6.8,O 02% (w/v) sodmm azide.

234

Gelboin et al.

3.9. lsotyping of MAbs Murine antibodies are divided mto five classes based on the type of heavy chain m the Ig molecule. They are IgG, IgM, IgA, IgE, and IgD. The heavy chains of IgG molecules can be further divided into four subclasses: IgG,, IgG2,, IgGZb, and IgGs, IgG, and IgM are the predommatant type found in the blood, therefore most hybridomas produce one or the other. Hybridomas producing IgA are rare, and ones producing IgE or IgD are extremely rare. If a particular class or subclass of MAb is desired, then Initial screening can be done after fusion and cloning. Using an anti-Ig specific second antibody would eliminate the need for this procedure. If class and/or subclass are unimportant, the initial screening does not need to be performed using a second antibody specific for all mouse antibodies. Determination of class and subclass of the MAb would then be necessary. This can be achieved by methods such as Ouchterlony double diffusion or antibody-capture ELISA (9). 3.10. Preparation of P450 Immunogens Numerous P45Os have been successfully purified from animal tissues (II) and used as nnmunogens for MAb production (6). However, this purification is laborious and difficult, especially for P45Os that are present m low abundance. Recently, a large number of rodent and human P450 cDNAs have been cloned and many of these have been inserted mto various vectors that enable the individual P450 protein to be expressed in heterologous host cells such as bacteria, yeast, COS cells, insect cells, and human AHH-1 lymphoblastoid cells (12). The expressed P45Os can be purified, characterized, and used as an immunogen for MAb production. 3.11. Use of Inhibitory MAbs Indrvidual expressed P45Oscan be characterized for substrate and product specificity, but this does not enable dtermination of the quantitative contribution of an individual P450 to the metabolism of a substrate in situ. This is largely owing to the inability to quantify both the amount and enzyme activity of individual P45Os in the presence of multiple P45Os. MAbs that inhibit the enzyme activity of specific P45Os can be used to determine the quantitative contribution of a specific P450 to any reaction measured m a tissue preparation containing multiple P45Os.Table l(4) shows the inhibitory effects of MAb 329-9 on the metabolism of seven substrates catalyzed by human cytochrome P450 3A4. The inhibition ranges from 80-95% for six substrates and is 60% for the seventh. The addition of MAb 3-29-9 to human liver microsomes (HLM) inhibited the metabolism of the seven substratesfrom O-86%, each value detinmg the contribution of P450 3A4 to the metabolism of each substrate.The MAb

235

MAbs to Cytochromes P450 Table 1 Mab 3-29-9 Inhibition of Metabolism by Human Cytochrome P450 3A4 Substrates p-Nitroanisole (0-demethylatton) Phenanthrene {aromatic epoxzdatlon) Diazepam (alzphatzc hydroxylatlon) Diazepam (N-demethylatlon) Testosterone (6P-hydroxylatzon) Taxol (aromatic hydroxylatlon) Cyclosporine (oxzdatzon)

of Different

Substrates

% Inhtbmon by MAb 3-29-9 HLMn RLMb P450 3A4 60

0

0

94

32

0

93

13

10

88

28

10

95

86

20

91

84

0

70-80

-

-

OHLM = Human her mlcrosomes, &LM = Rat her muxosomes

did not inhibit metabolism catalyzed by rat liver mrcrosomes (RLM) and thus does not cross-react. Table 2 (5) shows a similar study of the effect of MAb l73-18, which is inhibitory to human P450 2El activity. Thus, determinatton of the extent of inhibition of an enzymic activity by a MAb is useful for defining the role of an mdivrdual P450 m xenobiotrc and endoblotic metabolism. 4. Notes 1 The amount of immunogen and period of unmuntzatton can vary greatly (9) and the various methods may affect the yield of different MAb subtypes We have successfully used 10-100 pg per immunization The amount of tmmunogen depends on its purity, availability, the number of mice immunized and its antigenie potency. We generally use 30 pg in 100 pL/mjection/mouse Smaller volumes can be used, but the maxtmum volume including adJuvant should not exceed 0.5 mL for i/p injections. The Intervals of immunization vary greatly among laboratories. Freund’s adjuvant is the most commonly used for nnmun~zation. There are two types: complete and mcomplete. Complete adjuvant contains inactive Mycobacterium tuberculosrs, which induces a strong immune response and is used primarily for the initial injectton Incomplete Freund’s does not contam M tuberculosis, and is generally used for subsequent injections One part adjuvant is used/one part nnmunogen, to a maximum of 0.5 mL/r/p injection

236

Gelboin et al. Table 2 MAb 1-73-18 inhibition by Human Cytochrome Substrates p-Nitroanisole (0-demethylatlon) Phenanthrene (aromatic epoxldatlon) 4-Methylamsole (cresol formation) 4-Methylamsole (alcoholformatlon) Toluene (benzyl alcohol) Chlorzoxazone (6-hydroxylatron)

of Metabolism P450 2El

of Different

Substrates

% Inhtbmon by MAb l-73- 18 P450 2E 1 HLMa RLMb 92

35

0

85

23

2

60

25

0

80

12

0

90

40

0

90

72

10

“HLM = Human liver mlcrosomes, bRLM = Rat liver mtcrosomes 2. An alternative, 3-mo, munun~zation schedule can be used This mvolves mlecttons once a month for three months, taking a test bleed 1 wk after the second mlection. An mrmune response may not always be apparent with the 3-wk schedule, and longer mrmunizatton periods wrll often decrease the number of different antibodies formed, but increase the maturation of the antibody affimttes 3. Hybridomas are formed by the union of a myeloma, an immortal tumor cell lme, with an antibody secreting cell (from the spleen of muuumzed mice) that 1scommitted to the production of a single antibody. The resulting hybridoma secretes an abundant and constant amount of a specific and unique antibody. There are several myeloma cells lines generated from Balb/c mice; NS-1 and Sp2/0 are the most commonly used. We have successfully used NS-1 Unfused myelomas do not survive m HAT-supplemented medium. Splenocytes are resistant to HAT, but do not survive in tissue culture unless hybridized with myeloma cells. Surviving cells are the result of the hybridization of a myeloma cell and a splenocyte, and are called hybridomas. 4 NS- 1 cells grow best at a concentration of between 1 x lo4 and 8 x lo5 cells/ml The concentration should not exceed 1.2 x lo6 cells/ml NS-1 cells usually double m approx 24 h Count and dilute as necessary For fnston, 3 x 1O7myeloma cells are used for every 1 x lo* splenocytes. One mouse spleen typically yields 5 x 107-2 x lo8 cells A maximum of 1 x lo8 splenocytes are used for one fusion 5 Established hybridomas grow well m a broad range of standard tissue-culture media. The two most commonly used are RPM1 1640 and Dulbecco’s modified

MAbs to Cytochromes P450

237

Eagle medium (DMEM) The best cell growth is obtained usmg FBS as a supplement, although sera from other animals (horse, calf) are also adequate No other growth supplements are necessary except when cells are at a very low density (1 x 104/mL or less), as when clonmg. Medium supplemented with FBS has very little contamination of foreign IgG, but if this presents a problem, there are defined serum-free media avarlable commercially that can be used 6 As hybridomas are the product of much labor and are invaluable resources, freezing hybridoma cell lines properly is quite important Freezmg the cells at a low passage helps to ensure a stable population. 7 We have used baculovirus-expressed P450 3A4 as the antigen Owing to the low yield of highly purified expressed protein, we used partially purified baculovnusexpressed P450 3A4 for purposes of screening hybrids. To check for specificity of the antibodies, we used lysate from cells infected with wild-type baculovirus that had gone through the same partial purification scheme.

References 1 Kohler, G and Milstem, C (1975) Continous cultures of fused cells secreting antibody of predefined specificity. Nature (Lond ) 256,495497 2 Yelton, D. E. and Scharff, M. D (198 1) Monoclonal antibodies* a powerful new tool in biology and medicme Annu. Rev. Blochem 50,657-680. 3. Burnet, M. (1959) The Clonal Selection Theory ofAcqulred Immunity. Vanderbilt University Press, Nashville, TN. 4 Gelbom, H V., Krausz, K W , Goldfarb, I., Buters, J. T M , Yang, S K., Gonzalez, F. J , Korzekwa, K. R , and Shou, M. (1995) An mhibitory monoclonal antibody to human cytochrome P450 3A4. Blochem Pharmacol 50,184 l-l 850 5 Gelboin, H. V , Goldfarb, I , Krausz, K W , Grogan, J , Korzekwa, K R , Gonzalez, F J., and Shou, M (1996) Inhibitory and non-inhibitory monoclonal antibodies to human cytochrome P450 2El Chem Res Toxzcol 9, 1023-1030 6 Gelboin, H. V (1993) Monoclonal antibodies and cytochrome P450. Pharmacol Rev. 45,413-453.

7. Gonzalez, F. J and Idle, J R.(l994) Pharmacogenetic phenotypmg and genotyping. present status and future potential Cluucal Pharmacohnet 26, 59-70. 8. Dahl, M. L. and Bertilsson, L (1993) Genetically variable metabolism of antidepressants and neuroleptic drugs m man. Pharmacogentzcs 3,61-70 9. Harlow, E. and Lane, D. (1988) Antzbodles A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 10. Stanker, L.H., Vanderlaan, M. And Juarez-Salmas, H (1985) One-step purification of mouse monoclonal antibodies from ascites fluid by hydroxylapatite chromatography. J Immunological Methods 76, 157-169. 11. Waterman, M. R. and Johnson, E. F. (1991) Methods in Enzymol 12 Gonzalez, F J. and Korzekwa, K. R (1995) Cytochrome P450 Expression Systems. Annu Rev. Pharmacol. Toxicol. 35,369-390.

27 Targeting Antipeptide Towards Cytochrome

Antibodies P450 Enzymes

Robert J. Edwards 1. Introduction Antibodies agamst cytochrome P450 enzymes can be produced by immunizing animals with synthetic peptides that mimtc small regions of the apoprotein. As the amino-acid sequencesof a large number of cytochrome P450 enzymes are known (I), this approach to ratsing antibodies can be applied to most of the major cytochrome P450 enzymes in human, rat and other species. This 1sa relattvely simple, rapid and effective method of producmg antibodies. The anttpeptide approach to antibody production overcomes the need to use purified cytochrome P450 enzymes as mununogens (2). Cytochrome P450 purification is an arduous procedure and is especially difficult in ttssues that have a low cytochrome P450 content. Also, purtfication of human cytochrome P450 enzymes is often hmited by the availability of suitable quantities of tissue. Further, both polyclonal and monoclonal antibodies (PAbs/MAbs) raised against purified cytochrome P450 may cross-react with other cytochrome P450 enzymes (3-S). In contrast, a high degree of binding specificity can be achieved by directing antipepttde antibodies towards umque regions of the target antigen. Successfully produced antipeptide antibodies recognize both native and denatured cytochrome P450, hence these antibodies are particularly suited to immunoblotting (6,7) and immunocytochemistry using formalin-fixed secttons (8,9). Unlike antibodies produced by other techniques, the epitope of an antrpeptide antibody IS predetermined, hence antibodies can be directed towards specific surface regions of cytochrome P450. The structure and function of cytochrome P450 enzymescan be studied using such antibodies through immunoinhibition of enzyme activity (1 O-12). From Methods m Molecular Elrology, Vol 107 Cytochrome P450 Protocols Edrted by I R Phtlllps and E A Shephard 0 Humana Press Inc , Totowa, NJ

239

240

Edwards

In this chapter, methods for the production immunoblotting studies will be described.

of antibodies

suitable

for

2. Materials 1 Lyophlhzed peptlde (see Notes l-8). 2 Keyhole limpet hemocyanin (KLH) may be purchased as a partially purified product in 65% saturated ammonium sulfate (Calbiochem, Nottingham, UK). Dlssolve 250 mg of KLH m 20 mL of water and dialyze five times agamst 1 L of 0 1 M sodium phosphate buffer, pH 7 2. Measure the protem concentration of the solution and store frozen at -20°C in 5-mL aliquots. 3 Freshly prepared 0 0 1 M 5,5’-dlthlobls(2-nitrobenzolc acid) (DTNB) dissolved in 0.1 A4 potassium phosphate buffer, 1 mM ethylene diamine tetra-acetlc acid (EDTA), pH 8 0 4. Freshly prepared 0.15 M m-malelmidobenzoyl-Nhydroxysuccmlmlde ester (MBS) dissolved m dlmethyl formamlde. 5. Freund’s complete and Freund’s mcomplete adjuvants. Stable at 4°C for >l yr 6 Sephadese G-25 column (25 x 2 2 cm) 7. Phosphate buffered saline (PBS). 1.5 mM KH2P04 8.1 mM Na2HP04, 2 7 mA4 KCl, 137 mMNaC1, pH 7 5

3. Methods 3.7. Conjugation

of Peptide to Carrier Protein

1 Weigh about 10 pm01 of thlopeptlde (see Notes l-8) and dissolve m 2 mL of 0.05 M sodium phosphate buffer, pH 6.0 (see Note 9). 2. Remove 25 pL and dilute to 2.5 mL m 0 1 Mpotassium phosphate buffer, 1 n-&J EDTA, pH 8.0. Prepare a series of standard solutions between O-100 p&! 2-mercaptoethanol in 0.1 A4 potassmm phosphate buffer, 1 mM EDTA, pH 8.0. To 1-mL ahquots of diluted thlopeptlde and standard solutions of 2-mercaptoethanol, add 0.05 mL of DTNB solution and mix. The presence of thlol 1s indicated by production of a yellow colored solution. Measure the absorbance of samples at 4 12 nm and quantify the amount of thiopeptlde present by comparison with the standards 3. In preparation for conJugatlon to the thlopeptlde, derivatize KLH with MBS (see Notes 10 and 11) Typically 20 mg of KLH dissolved in 5 mL of 0.1 A4 sodium phosphate buffer, pH 7.2 is used. While mixing, add, over a period of 3-5 mm, 95 pL of MBS solution (i.e., 0.22 mg MBS/mg KLH) and allow to react for 30 min 4 Purify derlvatlzed KLH by gel tiltratIon using a Sephadex G-25 column, eqmhbrated m 0.05 Msodmm phosphate buffer, pH 6 0, at a flow rate of 2.5 mL/mm. Monitor the absorbance of the eluent at 280 nm and collect 5 mL fractions MBSderivatlzed KLH 1s eluted over 10-15 mL in the void volume of the column 5 Mix thlopeptlde and MBS-denvatlzed KLH at a ratio of 1 ~01 thlopeptlde to 2 4 mg KLH. AdJust the mixture to pH 7.5 using 1 M sodmm hydroxide. Mix for 2 h at room temperature and then dialyze three times agamst 500 mL PBS (see Notes 12 and 13)

Antibodies Against Cytochrome 450 3.2. Immunization

5 6 7 8 9 10 11

241

(see Notes 14 and 15)

Collect a preimmune blood sample from a marginal ear vein of a 3 kg male New Zealand White rabbit Allow the blood to clot and centrifuge at 9508 for 15 mm Collect the serum and store at -20°C. Dilute KLH-peptide conjugate to 0.4 mL in PBS. Place 0 75 mL of Freund’s complete adjuvant m a 5 mL vial and, while vortexmixing, gradually add 0.75 mL of KLH-peptide conjugate solution This should produce a thick emulsion, Inject 1 mL of the mixture of Freund’s adjuvant and peptide conjugate (200 pg) mto the rabbit subcutaneously at four sites (50 pg mL per site) All subsequent injections are prepared as described m item 4, except that Freund’s incomplete adjuvant is used After 2 wk, inject 200 pg of pepttde conjugate in Freund’s adjuvant subcutaneously at four sites (50 pg per site) After a further 2 wk, administer 200 pg of the pepttde conjugate m Freund’s adjuvant mtramuscularly at two sttes (100 pg per site). One week later, collect a blood sample from a marginal ear vein and prepare the antiserum as descrtbed in item 2 Test antiserum for bmdmg to cytochrome P450 enzymes (see Notes 16-18) Admimster booster injections intramuscularly at approx 1 mo Intervals, and collect blood 1 wk later on each occasion

4. Notes 4.1. Selection

of Peptide

1. The Directory of Cytochrome P450-Containing Systems, which can be found at http://www.icgeb.trieste.ttip450/, contains up-to-date mformation on cytochrome P450 sequences 2 A number of factors need to be considered when selecting a peptide for immumzation. If an antibody is required for tmmunoblotting and/or immunocytochemistry then the choice can be quote stratghtforward. Antibodies directed towards the C-termmus of cytochrome P450 are the simplest to produce and are the most reliable for reacting with the target cytochrome P450 enzyme (7) This IS because, by couplmg through its N-termmus, the orientation of the peptide is the same as the C-termmus of the cytochrome P450 enzyme and resultant antibodies recognize both equally well. Anttbodies that bind to most of the major forms of cytochrom P450 in humans and rats have been produced m this way (several examples are shown m Figs. 1 and 2) Alternatively, antibodies may be directed towards internal hydrophilic regions of the protein A simple computer program may be used to identify such regions (15) It is also useful to consider the predicted secondary structure of the protem, and direct antibodies to loop regions, I.e., nonalpha helical, nonbeta sheet regions (11). These regions have an inherent greater atomic mobihty and such flexibihty 1sthought to be advantageous for the

242

Edwards

Anti-CYPlAl

Anti-CYP

1A2

Anti-CYP2B1/2

Anti-CYPZD

1

Anti-CY P2E 1

Anti-CY P.3A I

Fig. 1. A series of immunoblots demonstrating the selectivity of binding of various antipeptide antibodies targeted against rat P450 enzymes. Antibodies were raised against the C-terminus of rat CYPIAI (QHLQA), CYPZDI (REQGL), CYP2El (VIPRS), and CYP3AI (IITGS), and against internal sequences of CYPlA2 (TGALFKHSENYK, residues 283-294) and to a region common to CYP2Bl and CYP2B2 (IDTYLLRMEKEK, residues 265-276 of CYP2Bl and CYP2B2). Liver microsomal fraction was prepared from rats which were either untreated (UT) or treated with 3-methylcholanthrene (MC), sodium phenobarbital (PB), streptozotocin (STZ), or pregnenolone 16a-carbonitrile (PCN), as described previously (13,24). To each lane, 10 pg of microsomal protein was applied, except for immunoblots developed with antXYPZB1/2 and antXYP3Al antibodies where 5 pg of microsomal protein was used. After electrotransfer onto nitrocellulose filters, each blot was developed with antiserum diluted 1:4000, except for the anti-CYP3Al antiserum, which was used at a dilution of 1: 16,000. Only the central section of blots corresponding to the molecular-weight range of 50-60 kDa is shown. No other immunoreactive bands were detected.

Antibodies Against Cytochrome 450

Anti-CYPlAl

243

-

Anti-CYP2El 1 Anti-CYP3A4

Fig. 2. A series of immunoblots demonstrating the selectivity of binding of various antipeptide antibodies targeted against human cytochrome P450 enzymes. Antibodies were raised against the C-terminus of human CYPlAl (MQLRS), CYPlBl (KETCQ), CYP2El (VIPRS), and CYP3A4 (TVSGA), and to an internal sequence of CYPlA2 (TGALFKHSKKGPR, residues 284-296). The binding of antibodies to insect cell microsomal fractions containing recombinant human CYPl Al (rCYPlA1, 2 pg) or CYPlB 1 (rCYP 1B 1, 10 pg), and lymphoblastoid cell microsomal fraction containing recombinant human CYPlA2 (rCYPlA2, 10 pg), CYP2El (rCYP2E1, 5 pg), or CYP3A4 (rCYP3A4, 10 pg) was compared with binding to a pool of 6 samples of human liver microsomal fraction (HLM, 25 pg). Microsomal fractions containing recombinant enzymes were obtained from Gentest (Woburn, MA). After electrotransfer onto nitrocellulose filters, each blot was developed with antiserum diluted 1:4000. Only the central section of blots corresponding to the molecular-weight range of 50-60 kDa is shown. No other immunoreactive bands were detected.

cross-reaction of antipeptide antibodies with protein antigen. Good success with this approach has been found, but it should be recognized that although antibodies that bind to peptide are almost always produced, their cross-reaction with protein is not certain.

Edwards 3 One of the advantages of using the antipeptide approach is that antibodies can be directed to predetermined sites on the surface of cytochrome P450. This allows studies of the function of cytochrome P450 enzymes by directing to regions on the surface of the cytochrome P450 enzymes anttbodies that interfere wtth catalytic activity (1042). To raise such anttbodies, it is useful to consider models of the three-dtmenaonal structure of eukaryotic cytochrome P450 enzymes (1617). 4. Whatever use is intended for the antibody, an important issue 1s hkely to be the specificity of binding. Thus property can be predetermined by directing antibodies to sequences that are unique to one form of cytochrome P450. Although in many cases the choice may be clear, it is possible that similar, but not identical sequences occur m other cytochrome P450 enzymes. It is difficult to devise gurdelmes to deal with this gray area. However, in the case of C-termmally directed antibodies, the C-termmal residue 1soften critical for anttbody binding, and alteration of this amino acid alone can greatly reduce antibody bmdmg (7). The sequence of the selected peptide should also be compared with the sequences of proteins other than cytochrome P450. A complete search of sequences m protein sequence databases can be performed using computer programs such as FASTA or BLAST (18). 5. The size of the peptide used to raise antibodies should also be considered. When raising antibodies agamst the C-terminus of cytochrome P450 enzymes, the author has mostly used pepttdes representing the last five ammo acid residues of the protein with the addition of cysteine to the N-termmus for the purpose of conjugation to carrier protein However, for several cytochrome P450 enzymes m the CYP2B, CYP2C, and CYP2D subfamilies, cysteine 1s already present as the fifth residue from the C-terminus. In these cases, the naturally occurrmg cysteme was used for coupling to the carrier protein. This effectively reduced the mnnuruzing sequence to a four-amino-acid residue pepttde. Nevertheless, antibodies raised against these peptides also bound successfully to their target cytochrome P450 enzymes In human CYPlBl, the C-terminus contains cysteme m the penultimate position. This problem was overcome by synthesizing a hexapeptide containing two cysteme residues with different side-chain protectmg groups. At the N-termmus, a trttyl cysteme was used (as m most other syntheses), but the cysteine in the penultimate position was protected with a t-butylthio group. Deprotectron and cleavage of the peptide from the solid-phase resin was performed for 2 h m 95% trifluoroacetic acid, 2 5% water, and 2 5% triisopropylsilane. Under these condttions, the thiol of the cysteme residue at the N-termmus was unmasked, whereas the t-butylthio-protected cysteme residue remained protected. After couplmg of the peptide to KLH, the t-butylthio group was removed by treatment with 0.2 M 2-mercaptoethanol. The conjugate was then dialyzed against 0 05 A4 sodium phosphate, 0 1 M sodium chloride, 1 mM EDTA, 1 mM dithrothreitol (DTT), pH 7.4. Immumzation with this conjugate resulted in antibodies that bound to human CYPl B 1 (see Note 18). 6. As regards targeting internal sequences of cytochrome P450 enzymes, peptrdes of between 7 and 12 amino-acid residues have been used successfully Although it 1stempting to synthesize peptides as large as possible, this 1soften restricted by

Antibodies Against Cytochrome 450 considerations of similarity with other cytochrome P450 enzymes. Thus, a longer peptide may have a better chance of producing antibodies that bmd to the target cytochrome P450, but tf the peptide contains regions that occur in other cytochrome P450 enzymes, then the resultant antibodies may also bind to these forms

4.2. Peptide Synthesis 7. Details of the methods for the synthesis of peptides has been described in a previous volume of this series and elsewhere (19,201. We have employed N-a-9fluorenylmethoxycarbonyl (Fmoc) chemistry usmg a semiautomated pepttde synthesizer to make peptides on solid phase resms. Both kieselguhr and Polyhipe supports contammg polydimethylacrylamide functionahzed with ethylenediamme, norleucine and trifluoroacetic acid-labile 4-hydroxymethylphenoxyacetlc acid have been used, Alternatively, manual synthesis of peptides was accomplished using 4-benzyloxybenzyl alcohol resm (Wang resin) For convenience, these resins were purchased with the first (C-termmal) ammo acid already coupled. Fmoc-protected ammo acids were activated using benzotriazole-l-yloxy-tris-pyrrolidinophosphomum hexafluorophosphate; alternatively, preformed activated pentafluorophenyl esters were used, except for serine and threonme, which were coupled as 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazme esters Typically, cysteine was added to the N-terminus of peptides for the purpose of conjugatton to protems; therefore, peptides were cleaved from the resin, and sidechain protectmg groups removed from amino acids, by treatment for 2 h with 95% trifluoroacetic acid and 5% ethanedithiol. This mixture is effective m scavenging trityl groups liberated from cysteine and preserving the thiol group After removal of trifluoroacetic acid by rotary evaporation the peptide was precipitated by addition of ice-cold diethyl ether. The peptide was sedimented by centrifugation (25Og for 10 min), the supernatant removed and the precipitated peptide washed a further three times in a similar manner Finally, the peptide was dried under a stream of nitrogen and dissolved m 5-l 0 mL of 0 5 A4 acetic acid, before being lyophihzed. Occasionally, a peptide may be msoluble in this solution If so, the suspension was lyophilized and small portions of the peptide were tested for solubihty m other solvents, e.g., glacial acetic acid or dimethylformamide Often, once dissolved, the solution may be successfully diluted mto 0.5 A4acetic acid. If it IS necessary to use neutral or alkaline conditions to dissolve the peptide, then precautions should be undertaken to avoid oxidation of the thiol group, e.g by addition of EDTA and degassing of solutions 8 Peptides were purified by gel filtration using a Sephadex G-l 5 coltmrn (30 x 1.5 cm) in 0.5 A4 acetic acid using a flow rate of 1 mL/min Fractions of 4 mL were collected and analyzed for the presence of thtopeptide using DTNB. The purity of each thiol-contammg fraction was assessed by high-pressure liquid chromatography (HPLC). HPLC was performed on a Nucleosil C 18 10 pm column (Jones Chromatography, Hengoed, UK) with constant momtormg of the eluent at 210 nm. The gradient employed was typically O-25% (v/v) acetomtrile (contammg 0.1% [v/v] trifluoroacetic acid) over 10 mm at a flow rate of 2 mL/mm, but

Edwards this gradient can be extended for more hydrophobic peptides. Typically, products of >90% purity were obtamed Most commonly, lmpurltles are peptlde with side-chain protecting groups attached (either because they were not removed or reattached during the deprotection reaction). Owing to the strong ultraviolet (UV) absorbance of such groups, the amount of lmpurlty 1soften overestimated Frequently, lmpurlties can be removed or reduced to an acceptable level by the gelfiltration chromatography step, probably as a result of increased hydrophobic interaction of the Impurity. Thus, impurities are usually retarded during the chromatography. Fractions contammg thlopeptide with the greatest purity were pooled and lyophllrzed. Further purification of these small hydrophlhc peptides was rarely necessary The composltlon of each peptide was confirmed by electrospray mass spectrometry.

4.3. Conjugation 9 If solublllty problems were encountered during the synthesis, then it 1s possible that the peptlde will not dissolve m 0 05 A4 sodium phosphate buffer, pH 6 0 Attempts should be made to dissolve the peptlde as described m Note 7. If this proves ineffective, then It 1s still possible to measure the thiol content of the suspension, provided the peptide is soluble in the alkaline buffer used for this purpose. Often, such peptldes can still be successfully coupled 10. In order to ensure a good immunological reaction, It 1susually necessary to conjugate peptldes to a carrier protein. KLH is the carrier protein of choice for Immunizations. This protein 1s strongly immunogenic and antibodies against KLH do not cross-react with mammalian proteins The preparations available from Calbiochem are soluble in 0 1 Mphosphate buffer; however, KLH obtained from other suppliers should be tested for solubility, because some have been found to contam very little soluble material. Also, note that in low ionic strength buffers, KLH will salt out of solution 11 The use of MBS for coupling thiopeptlde to carrier protein results m a complex with peptide coupled m a specific orientation. This also avoids masking ofpotentlally antlgemc groups m the peptlde. Other cross-lmkmg reagents such as glutaraldehyde and carbodumide have been used successfully, but these reagents often cross-link through the reactive side-chains of ammo acids, i.e., the E-ammo group of lysme or the carboxyhc acid group of aspartate and glutamate residues 12 In some cases, the resulting peptlde-KLH conjugate may become cloudy as a result of some preclpltatlon Such conjugates appear to be equally effective unmunogens as soluble conjugates. 13. The degree of conjugation of peptlde to KLH can be assessed by comparing the amino acid analysis of conjugated and unconjugated KLH. Typically, a ratio of 0.2 pm01 peptide/mg carrier protein is achieved. Although not quantltatlve, it 1s more convenient to assess the success of the conjugation reaction by slmultaneously coupling peptlde to lysozyme under the same conditions used to couple peptide to KLH. Because lysozyme has a relatively low mol wt, the result of the coupling reaction can be easily assessed by sodium dodecyl sulfate-polyacryla-

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mide gel electrophoresis (SDS-PAGE) using 15% (w/v) polyacrylamrde gels Successful conjugation is evident as an increase m the size of lysozyme, usually as a series of bands of increasing mol wt.

4.4. Immunization 14. When antibodies are raised against peptrdes representing the C-termmus of cytochrome P450 enzymes, antibodies of the required specificity and bindmg strength are usually produced m the first bleed, and subsequent booster injections have little effect on these characterrstics. However, antibodtes raised agamst peptides that represent internal sequences of cytochrome P450 enzymes tend to be more variable m their binding to cytochrome P450 enzymes Often antibodies with the desired bmdmg characteristics may be produced m the first blood sample, but It is not uncommon for the best antibodies to be produced after one or more booster injections Occasionally, the converse may occur and antibodies of the desired characteristics are lost with booster injections. It is important, therefore, to assess antisera as soon as possible after collection and decide whether to continue or terminate the nnmun~zatron procedure 15 Most of this work has been performed using rabbits: however, other spectes may also be used for the production of antipeptide antibodies. Sheep have been successfully used, although the binding strength of the antibodies tends to be a little lower than those produced m the rabbit.

4.5. Assessment by lmmunoblotting 16. Immunoblot analysis is particularly useful in determining the specificity of binding of the antibodies produced. We have used SDS-PAGE using 9% gels to separate microsomal proteins and electroblottmg to transfer the protems onto nitrocellulose filters (21,22). Nonspecific binding sites on the filter are blocked by mcubation m 3% (w/v) bovine serum albumin (BSA) m PBS for 1 h at room temperature or 4°C overmght. After rinsing in 50 mL PBS, filters are mcubated for 1 h at room temperature man appropriate dilution of antiserum (e.g., 1 l,OOO1: 16,000) m 20 mL of 0 1% (w/v) BSA in PBS The filter is washed five times with about 25 mL washing solution (PBS containing 0.05% (w/v) BSA and 0.05% (v/v) Tween-20) each time and then incubated for 1 h at room temperature m 20 mL goat antirabbit IgG-peroxidase conjugate diluted 1*25,000 m 0.1% (w/v) BSA in PBS. After this, the filter is washed five ttmes as previously described. Jmmunoreactivity is detected using enhanced chemilummescence followmg the mstructtons supplred by the manufacturer (Amersham plc, Little Chalfont, UK) 17 For antirat cytochrome P450 antibodies, specificity can be demonstrated by comparing the bmdmg to liver microsomal membrane proteins isolated from rats treated with various cytochrome P450-mducmg compounds (Fig. 1). Thus, the anti-CYP 1A 1 antibody bound to a single band m microsomal membrane proteins isolated from rats treated with 3-methylcholanthrene, but not to mlcrosomal membrane proteins isolated from any of the other rats. The anti-CYPlA2 antibody bound to a single band in microsomal proteins isolated from all the rats, and the

Edwards intensity of the immunoreactive band was greatly increased m those treated with 3-methylcholanthrene. The antt-CYP2B l/2 anttbody bound only to mtcrosomal proteins from rats treated with sodium phenobarbital, where two bands, corresponding to CYP2Bl and CYP2B2, were detected. The anti-CYP2Dl antibody bound to a single band with similar intensity m all rats, regardless of their treatment. The anti-CYP2El antibody bound to a single band m microsomal proteins from all rats, and the intensity of the immunoreacttve band was mcreased in those treated with streptozotocin. Finally, the anti-CYP3Al antibody bound to a single band in microsomal proteins from all rats, and the intensity of the immunoreactrve band was very strongly Increased after treatment with pregnenolone 1~CXcarbomtrile These results are consistent with the known pattern of expression of these cytochrome P450 enzymes upon exposure of rats to the inducing agents. 18. For the antihuman cytochrome P450 antibodies, specificity can be assessed by examinmg the bmding to recombinant human cytochrome P450 enzymes (Fig. 2). In the examples shown, each of the antihuman cytochrome P450 antibodies bound only to their respective recombinant cytochrome P450 antigens Antibodies targeted agamst CYPlA2, CYP2E1, and CYP3A4, but not CYPlAl or CYPlBl, bound to human liver mlcrosomal membrane proteins, consistent with the expression of these cytochrome P450 enzymes m normal human liver

References 1 Nelson, D. R., Koymans, L , Kamataki, T , Stegeman, J. J., Feyeretsen, R , Waxman, D. J., Waterman, M. R , Gotoh, O., Coon, M. J., Estabrook, R. W , Gunsalus, I. C., and Nebert, D. W (1996) P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetzcs 6, l-42 2 Parkinson, A. and Gemzlk, B. (1991) Production and purtticatron of anttbodies against rat hver P450 enzymes. Methods Enzymol. 56,233-245. 3. Thomas, P. E., Bandiera, S., Reik, L. M., Mames, S. L , Ryan, D. E., and Levm, W. (1987) Polyclonal and monoclonal antibodies as probes of rat hepatic cytochrome P-450 isozymes. Fed Proc. 46,2563-2566 4. Wrighton, S. A., Vandenbranden, M., Becker, G, W., Black, S D., and Thomas, P. E. (1992) Two monoclonal antrbodres recognizing different epitopes on rat cytochrome IIB 1 react with human IIE 1. A402 Pharmacol 41,76-82. 5. Goldfarb, I., Korzekwa, K., Krausz, K. W , Gonzalez, F., and Gelbom, H V. (1993) Cross-reactivity of thirteen monoclonal anttbodies with ten vaccima cDNA expressed rat, mouse and human cytochrome P45Os Biochem Pharmacol 46,787-790. 6. Edwards, R. J., Murray, B. P., Singleton, A. M., Murray, S., Davies, D S., and Boobis, A R. (1993) Identification of the epttope of an anti-peptide antibody which binds to CYPlA2 m many specres including man Biochem Pharmacol 46,2 13-220 7. Edwards, R. J., Smgleton, A. M , Murray, B P., Davies, D. S , and Boobts, A. R (1995) Short synthetic peptides exploited for reliable and specific targeting of antibodies to the C-termmi of cytochrome P450 enzymes. Biochem Pharmacol. 49,39-47.

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8. Anilkumar, T. V., Goidmg, M., Edwards, R. J., Lalani, E., Sarraf, C. E , and Alison, M, R. (1995) The resistant hepatocyte model of carcmogenesrs m the rat the apparent Independent development of oval cell proliferation and early nodules. Carcinogenesis U&845-853. 9. Goldmg, M., Sarraf, C. E., Lalam, E., Anilkumar, T V., Edwards, R J , Nagy, P , Thorgeirsson, S. S., and Ahson, M R. (1995) Oval cell differentiation mto hepatocytes in the acetylaminofluorene-treated regenerating rat liver. Hepatologv 22, 1243-1253. 10. Edwards, R. J , Singleton, A. M., Murray, B. P., Murray, S., Boobts, A R , and Davies, D. S. (199 1) Identification of a functionally conserved surface region of rat cytochromes P450IA. Biochem. J 278,749-757 11. Edwards, R. I., Singleton, A M., Murray, B. P., Sesardic, D., Rich, K J , Davies, D. S., and Boobis, A. R (1990) An anti-peptide antibody targeted to a specrtic region of rat cytochrome P450IA2 inhibits enzyme activity. Blochem J. 266,497-504. 12. Adams, D. A., Edwards, R. J., Davies, D S., and Boobis, A R. (1997) Specific inhibition of human CYPlA2 using a targeted antibody. Blochem. Pharm 54, 189-197 13. Rich, K J , Sesardic, D , Foster, J R , Davies, D. S., and Boobis, A. R. (1989) Immunohlstochemical localization of cytochrome P450b/e m hepatlc and extrahepatic tissues of the rat. Blochem Pharmacol. 38,3305-3322. 14. Debra, K., Boobis, A. R., Davies, D. S , and Edwards, R J (1995) Dlstributton and mduction of CYP3Al and CYP3A2 in rat liver and extra-hepattc tissues Biochem Pharmacot

50,2047-2056.

15. Edwards, R. J., Murray, B. P , and Boobis, A R. (1991) Anti-pepttde antibodies in studies of cytochromes P450IA. Methods Enzymol 206,220--233 16. Edwards, R. J., Murray, B. P., Boobis, A. R., and Davies, D. S (1989) Identification and location of alpha-hehces in mammalian cytochromes P450. Blochemzstry 28,3762-3770

17. Edwards, R. J , Murray, B. P., Singleton, A. M., and Boobis, A. R (1991) Orientation of cytochromes P450 in the endoplasmic reticulum. Blochemwtry 30, 7 l-76. 18. Pearson, W. R. and Miller, W. (1992) Dynamic programming algorithms for biological sequence comparison Methods Enzymol. 210,575-601. 19 Pennmgton, M W and Dunn, B. M. (1994) Peptlde Syntheses Protocols Humana, Totowa. 20. Atherton, E. and Sheppard, R C. (1989) Solid phase pepttde synthesis-a practical approach. IRL Press, Oxford University, Oxford, UK. 21. Edwards, R. J., Singleton, A. M., Sesardic, D., Boobis, A R., and Davies, D. S (1988) Antibodies to a synthetic peptide that react specrfically with a common surface region on to hydrocarbon-inducible isoenzymes of cytochrome P-450 Blochem. Pharmacol

37,3735-3741

I

22. Edwards, R. J., Murray, B. P , Murray, S , Schulz, T., Neubert, D., Gant, T W , Thorgeirsson, S. S., Boobis, A. R., and Davies, D S (1994) Contribution of CYPl Al and CYPlA2 to the activation of heterocychc ammes in monkeys and human. Carclnogenesis 15,82!9-836.

28 Signals for Retention of Cytochrome P450 in the Endoplasmic Reticulum Elzbieta Skorupa and Byron Kemper 1. Introduction According to the current model for the subcellular compartmentalizatron of proteins, any protein entering the secretory pathway will be transported by bulk flow to the cell surface unless a signal medrating its organelle-specific retention IS present. Endoplasmic reticulum (ER) membrane protems, such as cytochromes P450, must have a sequence or structural features that prevent transport from the ER. For some ER membrane proteins, such signals have been identified and are encoded by a short ammo acid sequence such as a double lysme motif at the C-termmus of the cytoplasmic tall or positively charged residues at the N-terminus (reviewed m ref. I). ER retention signals may mediate either the direct retention (exclusion from further transport) or retrieval from a post-ER compartment (I). Microsomal P45Os do not contain any of the known ER retention signals and appear to be excluded from the retrieval pathway (2,3). However, there have been reports that some P45Os are present in either the Golgi or plasma membrane (415). P45Osare inserted into the ER membrane by the N-terminal signal/anchor sequence, which also encodes an ER-retentton function (3,6). The cytoplasmic domain of microsomal P45Os,minus the N-termmal29 ammoacid residue signal/anchor sequence, also plays a role m retention m the ER (7). Although this domain is localized on the cytoplasmic side of the ER membrane, its potential association with the membranes via additional sequences has been suggested (8). Most studies defining cellular targeting have utilized chimeric proteins. The basic rationale behind this strategy is that the fusion of a sequence encoding a potential localization signal to a reporter protein with a different cellular locaFrom Methods m Molecular Bology, Vol 107 Cytochrome P450 Protocols Edrted by I R Phillips and E A Shephard 0 Humana Press Inc , Totowa, NJ

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tron should direct the chrmerrc protein to the cellular location specified by the signal. Thus, identification of a particular sequence of P450 as an ER locahzatlon (retentron) signal generally requires two types of evidence: first, thus sequence should localize a chimeric protem to the ER and second, mutatron or deletion of thus sequence alters the cellular location of the protein (either the P450 or a chimera). The first kmd of evidence, i.e., imposing ER retention on a chlmerrc protein, relies on the assumption that the location of a protein IS determined not by a positive transport signal, but rather a retention signal and that a reporter protem by Itself IS not retamed in the ER. Although early transport experrments with simple peptides, devoid of signal sequences,were interpreted as evidence for transport from the ER as the default pathway, upon re-evaluation, the kinetlcs of transport are more consrstent wrth the presence of positive transport srgnals in secreted proteins (1,9). This further suggests the need for caution in selectmg the appropriate reporter. Nonetheless, proteins wrthout a posrtlve transport signal are likely to be transported out of the ER at a slower rate by bulk flow unless some property or sequence of the protein prevents export. This followmg chapter describes methods that are used to map the ER retention signal(s) of mlcrosomal P45Os,which utrhze chlmerrc proteins. 2. Materials (see Note 7) 2.1. Chime& Protein Construction 2.1.1, Cytochrome P450 Microsomal P450 can be divided mto 2 mam domams. the N-termmal srgnal/anchor sequence of about 25 amino-acid resrdues and the remammg cytoplasmtc domain of about 470 residues. Some P45Os contain also a short sequence preceding the membrane anchor. These are the mam domains that have been analyzed for the presence of the ER locahzation/ retentron function and/or membrane association. To obtain the DNA fragments encoding one of these domains, for the constructron of chimerrc protems, either natural restrictton sites are used or appropriate restrrction sites are engineered by silent mutations introduced by site-directed mutagenesrs or polymerase chain reaction (PCR). The N-termmal signal/anchor sequence is defined as a 17-25 stretch of hydrophobic ammo-acid residues, usually bordered by negatrvely charged residues at the N-terminus and positively charged residues at the C-terminus. When studying only the cytoplasmrc domain, rt may be Important to retain it m the enzymatically active form to avoid the retention of misfolded proteins by the quality control systemof the ER. In thuscase, it may be important to include m the chlmeric protein construction a linker region of P450 (ammo-acid residues between the membrane anchor and strongly conserved pro-pro-gly-pro [PPGP] sequence) followmg the surrogate signal for membrane insertion (7).

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2.1.2. Reporter Protein An ideal reporter protein should have the following

features.

1. Normally targeted to a cellular location other than the ER. For example, a cytoplasmlc protein that 1s known to be compatible with transport to the cell surface when attached to an appropriate signal sequence is a good candidate for studies involving the membrane insertion signal of P450 smce the remainder of P450 IS on the cytoplasmic side of the ER membrane On the other hand, a Golgi or plasma membrane specific protein (or its domain encoding the localization signal) is a feasible reporter for the studies on the cytoplasmic domain of P450. However, m this case rt is Important that such a reporter does not have specific requirements for transport out of the ER, such as ohgomerization or bmdmg with another protein/subunit, which might be deficient m a fusion protem. 2 Undergoes organelle-specific modification, for example phosphorylatlon or glycosylation, durmg the transport through the secretory pathway used to monitor the localization of a chimera. A typical modlficatlon is N-glycosylatlon, which allows the use of endoglycosldase H (endo H) sensitivity to distinguish ER locahzed glycoprotems (endo H sensitive) from those that were transported out of the ER at least to the medial Golgl (endo H resistant) (see Note 2) 3 A standard method for detecting the reporter. The examples of such methods include a Immunological detection with an antibody against the reporter protem or its selected domain. This IS particularly important m studies mvolvmg only a small P450 domain for which there is no antI- P450 antibody (see Note 3). b. Enzyme activity assay This method can be used, for example, with reporters such as /3-galactosldase or alkaline phosphatase fused to the P450 N-termmal slgnallanchor c. Intrinsic fluorescence This method of detection can be applied with green fluorescent protein (GFP) as a reporter In studies involving the N-terminal P450 signal/anchor sequence, a cytoplasmic or a secretory protein devoid of its own signal sequence can be used as a reporter, because the P450 signal functions also m targeting the chimerlc molecule to the ER membrane. However, when studies mvolve only the cytoplasmic domain(s) of P450, any potential reporter will also need a signal sequence and membrane anchor without ER-retention properties. In this case, a Golgi or plasma-membrane protein that contains a signal sequence can be used. The GFP is a promising reporter for studies on subcellular localization. Its use eliminates the need for any posttranslational modifications or for specific antibodies. It is a small cytoplasmlc protein that seems to be compatible with transport to any subcellular compartment when fused to a targeting signal and the location of the hybrid can be directly observed using fluorescent mlcroscopy (10). Our preliminary studies suggest that this reporter can be used directly in studies involving the N-terminal domain of P450, which targets it to the ER. Furthermore, fusion of GFP to the C-terminus of a full-length P450

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2C2 does not interfere with the ER localization or folding, because the chimera remains enzymatically active (unpublished). When only the cytoplasmrc domain of P450 is studied, a signal sequence without ER-retention function will be needed. GFP vectors can be obtained from Clontech and allow for both, N- and Ctermmal fusions in any desirable frame. These vectors can be used in many different cell-lures and work particularly well rn COS 1 cells (see Note 4).

2.1.3. Expression Vectors Transfections in COS cells work partrcularly well with expression vectors containmg SV40 origins of replicatron, which are amplified in the cells and result in relatively high levels of expression. The authors have been usmg pCMV4 and pCMV5 vectors (2,6,7) which contain all the features important for expression in COS cells. 1 The SV40 orrgm of rephcatron, 2 The strong human cytomegalovnus (CMV) transcrrption promoter, 3. The human growth hormone (hGH) transcription termination and polyadenylatron signal, 4 The bacterrophage fl origin of replication for the productron of smglestranded DNA, 5 A prokaryottc origin of rephcatton, and 6. An ampicillin-resistance gene. Multiple-clonmg sites m these vectors contam a series of unique restrrctron sites for clonmg cDNA downstream from the CMV promoter.

2.2. Cell-Culture and Transfection 2.2.1. Cell-Culture Media and Supplementary

Materials

1. Cells. COSl cells can be purchased from ATCC (Rockvlle, MD) * Grow as a monolayer m Dulbecco’s modrtied Eagle medmm (DMEM), hrgh glucose formula (4 5 g/ L), contammg 10% calf serum m a humrdrtied CO2 incubator with 5% CO, 2 DMEM. To prepare 1 L of DMEM, dissolve one pouch of powdered medium (Gibco BRL, Garthersburg, MD) m about 900 mL of autoclaved water, add 3.7 g of NaHC03, and adjust the pH of the medmm to 7.2 usmg erther 1 MHC1 or 1 M NaOH. After brmgmg the final volume to 1 L with water, the solutron 1s sterrlized by passing through 0.2~pm filters (Nalgene, Milwaukee, WI). Sterrlrzed medium may be stored at 4°C for several months. 3 Complete growth medium. To obtain complete medium, add calf serum (Sigma, St Louis, MO) to the DMEM to a final concentratron of 10% and 0.0 1 volume of a 100X stock solutron of pemcillin and streptomycm (Stgma) to produce final concentrations of pemcrllm and streptomycin of 100 U/mL and 100 g/mL, respectively. Store at 4°C and use withm 2 mo. 4. Dulbecco’s phosphate-buffered saline (PBS)* To make 1 L of PBS, dissolve in about 700 mL of distilled water: 8 g NaCl, 0.2 g KCl, 1 15 g Na2HP04, and 0.2 g

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KH,PO,. AdJust the pH to 7.4 with 0.1 NNaOH, add water to 1 pL and sterilize by autoclavmg. Alternatively, to obtain 1 L of PBS, one pouch of powdered mixture (Gibco) is dissolved m about 950 mL water After adJustmg the pH to 7 4 and the final volume to 1 L the solution IS sterilized by autoclavmg 5. 10% (v/v) dimethylsulfoxide (DMSO) m DMEM. Incubation of cells with 10% (v/v) DMSO “shocks” the cells and increases transfection efficiency. 10% v/v DMSO m sterile DMEM should be made just before use 6. DMEM lacking methionine and cysteme: This medium is used for radiolabelmg transfected cells. To a 100 mL bottle of DMEM lacking methionme, cysteine, and glutamine (ICN, Costa Mesa, CA) add 1 mL of 200 ML-glutamme (Gibco). Store at 4°C. Just before radiolabeling, mix the destred volume of this medium with 35STranslabel (ICN) to a final radioactrvrty of 200-500 pCi/mL

2.2.2. Solutions for Transfection with DEAE-Dextran 1 Diethylaminoethyl DEAE-dextran: To prepare a stock solution of 10 mg/mL DEAE-dextran, 200 mg of DEAE- dextran M W. 500,000 (Pharmacia, Uppsala, Sweden, or Sigma) is dissolved m PBS, the volume IS adJusted to 20 mL and the solution is autoclaved for 20 min This solution may be stored at -2O’C for several mo. 2 Chloroqume: To prepare a 250 mM stock solution, dissolve 515 9 mg of chloroqume (diphosphate salt) (Sigma) m water. After adJustmg the volume to 4 mL, filter-sterilize the solution using a 0.45 unr Mrlhpore filter. The sterile solution can be stored at -20°C for several mo if wrapped m aluminum foil to protect from light 3. DDC solution. DDC is a mixture of DEAE-dextran (10 mg/mL) in PBS and 2.5 mM chloroqume. Add 100 pL of 250 mM chloroquine to 10 mL of DEAEdextran (IO mg/mL) Store as 1-mL ahquots at -20°C. Make fresh every 3-4 mo 4. Medium for transfection. DMEM contammg 10% (v/v) NuSerum (Collaborative Research).

2.3. Analysis of the Subcellular Localization of Expressed Proteins 2.3.1. Radtolabeling of Transfected Cells, lmmunoprecipitation and Endo H Sensitivity Assay 1. 35S Translabel (ICN). 2. Radioimmunoprecrpitation (RIPA) buffer. RIPA buffer is used to lyse cells and contains. 10 mMTris-HCI, pH 7.4, 150 mMNaC1, 1.O% (w/v) sodrum deoxycholate, 1% (v/v) Triton X-100, 0.1% (w/v) sodium dodecylsulfate (SDS), 1 mM phenylmethylsultonyl fluoride (PMSF) (added just before use from a 100 mM stock solution m absolute ethanol). 3. Antibody solution Polyclonal antnsera or monoclonal antibodies (MAbs) against P450 or a reporter protein may be used. 4. TENN buffer: 50 mMTris-HCI, pH 7.5, 5 mA4ethylenediamme tetra-acetic acid (EDTA), 150 mMNaC1,0.5% (v/v) Nomdet P-40.

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5. Protetn A-Sepharose. To prepare 5 mL of protem A-Sepharose slurry (Pharmacia), add 0 2 g of protein A-Sepharose to 40 mL of TENN buffer, mix, and leave on the bench until the beads settle m the bottom of the tube. Centrifuge for 5 mm and remove the supernatant. Repeat the wash once more, and, after pelletmg the beads, add 4 mL of TENN buffer. Store at 4°C 6. TSA buffer: 10 mMTris-HCl, pH 7.4, 150 mMNaC1. 7. 50 mM Trrs-HCI, pH 6.8 8. Releasing buffer This buffer is used to release the antigen/antibody complex from protein A-Sepharose and contams 50 m&f Trrs-HCl, pH 6.8, 0.4% (w/v) SDS, 25 mA4 2-mercaptoethanol. 9 2X endo H buffer 300 mA4 sodium acetate, pH 5 5, 1% (v/v) Triton X-l 00 10. Endoglycosidase H, 1 mU/pL (Boehrmger Mannhelm) 11. 5X gel loadmg buffer. 250 mM Tns-HCl, pH 6.8, 10% (w/v) SDS, 0.5% (w/v) bromophenol blue, 50% (v/v) glycerol, 25% (v/v) 2-mercaptoethanol

2.3.2. Subcellular Fractionation of Transfected Cells 1, Sucrose solutions: 2.0 M, 1.3 I@ 1 0 M, 0 6 M, 0.25 M, all made m 5 mA4HEPESKOH, pH 6.8 2 2X RIPA This is a twofold concentration of the solution described in Subheading 2.3.1.

2.3.3. Endoplasmic Reticulum (NADPH-Dependent Cytochrome c Reductase) and Golgj (Galactosyltransferase) Marker Assays 1. 2. 3. 4 5 6. 7 8 9 10. 11.

0.6 Mpotassium phosphate buffer, pH 7 7. 20 mM EDTA. 5 mA4 cytochrome c. Dissolved m water and stored frozen at -20°C. 25 WNADPH (Sigma) Dissolved m water, allquoted into 50 pI.. samples, and stored at -70°C Dtscard when color turns yellow 1 mMUDP-galactose (Sigma). Dissolved m water and stored at -2O’C 70 mg/mL ovalbumm (Sigma) Dissolved m water and stored at -20°C 1 MMnC12. 20% (v/v) Triton X- 100 1 M Tris-HCl, pH 7.5 UDP [3H-Gal] 11 32 Ci/mmol (NEN, Boston, MA) 10% (w/v) and 5% (w/v) trrchloroacetic acid (TCA)

2.3.4. lmmunofluorescence

of Transfected Cells

1 10X PBS. Dissolve one pouch of powdered PBS (Gibco) m about 90 mL water and after adjusting to pH 7.4, bring the volume to 100 mL with water Sterilize by autoclavmg and store at room temperature 2. 2 5% (w/v) paraformaldehyde: Resuspend 1 g of paraformaldehyde m about 20 mL of water, add 4 drops of 1 NNaOH, and dissolve by heating at 60°C until the solution clarifies Add 4 mL of 1OX PBS, adJust the pH to 7.2 with 1 N acetic acid, and brmg the final volume to 40 mL with water. Make fresh just before use

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3 2% (v/v) Tnton X-100 in PBS. This solution IS used for solubihzatlon ofthe cells. 4. 4% (w/v) gelatin: Dissolve 4 g of gelatin in a final volume of 100 mL water, sterilize by autoclavmg and store at 4°C. 5. PBS/gelatin: For 1 L of this solution, mix 100 mL of 10X PBS, 100 mL of 4% (w/v) gelatm, and adJust the volume to 1 L with water. 6 Primary antibody solution: Prepare a dllutlon of the primary antlbody m PBS/ gelatin solution assuming that 30 & of diluted mixture ~111 be needed for each cover slip. Centrifuge for 5 min in a microfuge (12,000g) Just before use and transfer the supematant to a fresh tube. Store on Ice. ‘7. Fluorochrome-conjugated secondary antibody solution’ Resuspend 2 mg of pure antibody in 1.5 mL water and centrifuge in a microfuge (12,OOOg) for 5 min Transfer the supernatant to a new tube. Store 10-a ahquots m a box wrapped in aluminum foil at -70°C. Just before use, prepare enough of the desired dllution in PBS/gelatin to have 30 clr, per cover slip 8. Cover shp mounting medium: To prepare 10 mL of p-phenylenediamine-based mounting medium, mix 10 mg of p-phenylenediamme, 1 mL of 1OX PBS and 9 mL of glycerol Star until dissolved, ahquot 0 5 mL into small vials, and store protected from light at -20°C Discard when color turns yellowish after several mo and prepare a fresh stock 9. Cover slip sealer. Either the commercially available preparation Pro-Texx (Lerner Laboratories, Pittsburgh, PA) or clear nail polish can be used However, nail polish should not be used for the GFP studies.

3. Methods 3.7. Preparation

of DNA Encoding

the Chimeric Protein

Once the reporter protein has been selected, the DNA fragment for the desired region is ligated to the DNA encoding the P450 domam of interest and the entire recombmant DNA is inserted mto the pCMV expressron vector by standard recombinant DNA techmques. The clone of the desired hybrid protein IS identified by restrlction digestion and DNA sequencing and a larger amount of the DNA to be used in the transfectlon is purified. For the DEAEdextran-mediated transfection of COSl cells, it is not necessary to purify the plasmid DNA by CsCl gradient centnfugation. Mini-prep DNA or preparations purified by Qiaprep spin columns (Qiagen, Chatsworth, CA) or Qlagen tip 100 columns (Qiagen) are of sufficient purity (see Note 5). To obtain 100-200 E of plasmid DNA, a single colony of the desired clone is inoculated into I mL of LB-broth contammg an appropriate antibiotic and mcubated with shaking at 37°C for 6-12 h. This culture IS used to inoculate 100 mL of medium (LB or 2xTY) containing the antlblotlc, which IS then incubated at 37OC for 12-l 8 h. Pelleted cells are processed accordmg to the Qiagen protocol for tip 100 column purification (Qiagen). Typically, about 100-200 ~18of supercoiled DNA free of cbromosomal DNA andRNA is obtainedusing this protocol.

Skorupa and Kemper

258 3.2. Transfection

of COS7 Cefls (see Notes 6 and 7)

1. One d before the transfection, actively growmg COSl cells are harvested and added to new plates to give 4060% confluency the followmg day Usually, a confluent 25 cm2 flask of cells is resuspended m 25 mL of the complete medtum, and 2 mL of this suspension 1s plated mto each well of a 6-well plate or each 35-mm (diameter) plate One mL of complete medium IS added to each dish (to bring the volume to 3 mL) and the cells are returned to the CO2 mcubator. 2 The followmg day the transfection mixes are prepared For each 35-mm plate or one well of a 6-well plate, place 1 mL of prewarmed DMEM/lO% (v/v) NuSerum m a sterile polystyrene tube (Falcon, Los Angeles, CA) and add 40 pI. of DDC Mix well by vortexing. Add 1-2 pg of the appropriate DNA (add water to the mock sample) and mix again by vortexmg (see Notes 8 and 9) 3 Remove the medium from the cells and wash the monolayers with PBS prewarmed at 37°C. Add transfection mix to each dish and contmue the mcubation at 37°C for 34 h 4. Remove the transfection mix and add 1 mL of 10% DMSO in DMEM to each plate Incubate for 2 mm at room temperature, remove the mtxture, and gently wash the cells with 2 mL of PBS. 5. Add 3 mL of complete medium to each dish and return the plates to the CO2 incubator (see Notes 10 and 11)

3.3. Metabolic

Radiolabeling

Because the maximum

of Transfected

expression

Cells

of the transfected

DNA

is observed

between 4&96 h after the transfectlon, this IS usually the optimal time for radlolabelmg of the cells. At this time, cells should be almost confluent. 1. Aspirate the medium from the plates, and wash the cells twice with PBS prewarmed at 37°C 2. Add 1 mL of prewarmed DMEM lacking methionme and cysteine to each plate and incubate for 20 mm in the CO, mcubator to deplete the endogenous pools of these amino acids. 3 Prepare labeling medium by mixing prewarmed DMEM lacking cysteme and methionme with 35STranslabel to make 300 pL per 35 mm plate (or one well) at 200-500 @YmL (see Note 12) 4 Aspirate the medium from the plates and add 300 pL of radiolabelmg medmm Gently rock the plates every 15-20 min to prevent drying of the cells Continue incubation for l-3 h if only labeling (not pulse-chase) is needed and contmue as described in step 6. If a pulse-chase IS to be performed, as in the endo H assay, a 30-min Incubation with radioisotope is followed by the chase (step 5) 5. For the chase incubation, remove the labeling medium (discard in radtoactive waste), wash the cells twice with prewarmed complete medium and add 3 mL of complete medium, supplemented with 2 mM nonradioactive methionine, to each dish Continue the incubatton for 3-4 h (see Note 13).

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6. Put the plates on Ice and remove the medium (discard m radioactive waste). Wash the cells twice with ice-cold PBS. Add 0.5 mL of ice-cold RIPA buffer containing 1 rmVPMSF to each plate (well) and leave on ice for 5-10 mm Transfer the cellular lysates to microfuge tubes and vortex briefly. Centrifuge m a mlcrofuge (12,000g) for 10 min at 4°C and transfer the supernatants to fresh tubes Use unrnedlately for lmmunopreclpltation or freeze the supernatants at -7O’C.

3.4. lmmunoprecipitation

and Endoglycosidase

H Assay

1 Add l-5 @, of the appropriate antibody solution (usually l-5 pL of polyclonal antiserum or 0.5-I pL of purified monoclonal antlbody [MAb]) to each lysate and leave on a rotating platform at 4°C for 2-4 h (see Note 15). 2. Add 100 clr, of protein A-Sepharose slurry to each sample and continue the incubation on a rotating platform for an additional 1 h (see Note 16). 3 Centrifuge the mixtures in a microfuge (12,OOOg) for 2 mm and gently aspirate the supernatant so that the Sepharose pellet IS not disturbed Add 1 mL of icecold RIPA buffer to each pellet, mix well by vortexmg, and centrifuge as previously noted. Remove the supernatant and repeat the wash with RIPA buffer 4 Wash the pellets with 1 mL of ice-cold TSA buffer and then with 1 mL of 50 mM Tns-HCl, pH 6.8. Carefully remove the washing buffer 5 If the samples are to be directly analyzed by autoradiography, add 30 ,uL of the SDS gel loading buffer to each Sepharose pellet and boil the samples for 3 mm. For the endo H assay, do step 6. 6. Add 20 & of the releasing buffer to each pellet and boil the samples for 5 mm. Centrifuge m a mlcrofuge (12,000g) for 2 min and transfer a lo-& aliquot from each sample to each of two new tubes. 7 Add 10 pL of 2X endo H buffer to each ahquot and either l-2 pL of water (controls) or l-2 $ of endo H (l-2 mu). Incubate the samples at 37°C overnight. 8. Add 5 ,LLLof 5X SDS gel loading buffer to each sample and boll for 3 mm Analyze by SDS-polyacrylamlde gel electrophoresls (PAGE) and autoradiography

3.5. Subcellular

Fractionation

1. Plate the cells for the transfection as described m Subheading 3.2. except on loo-mm (diameter) plates (from the 25 cm2 flask of confluent cells resuspended m 25 mL of the medmm, plate 10 mL per 100~mm plate) The next day, transfect the cells using the DEAE-dextran method, scaling everythmg up fivefold as compared to 35-mm plates. Thus, each loo-mm plate will need 5 mL of the transfection mix containing 200 pL DDC solution and 5-10 pg of DNA 2. Radiolabeling of transfected cells. Forty-eight hours after transfectlon, wash the transfected cells twice with prewarmed PBS and premcubate with 2 mL of DMEM lacking cysteme and methiomne for 30 mm Remove the premcubation medium and add 1.5 mL of the labeling medium containing 200-500 pCl/mL of 35S-Translabel and incubate for 1 h. Remove the labeling medmm, wash the cells twice with prewarmed PBS and add 10 mL of the complete medmm supplemented with 2 rruV methlonme to each plate. Continue the chase mcubatlon for 4-5 h

260

Skorupa and Kemper

3 Put the plates on ice and aspirate the medium. Wash the cells twice with ice-cold PBS as before (Subheading 3.3., item 6). Add I mL of 0 25 M sucrose solutton to each plate and scrape the cells from the surface using a rubber policeman or cell scraper. Transfer the cell suspension to microfuge tubes and centrifuge for 5 min at 1700g m a cold room Discard the supernatant, and wash the pelleted cells with 1 mL of 0.25 M sucrose. Centrifuge as before, remove the supernatant, and resuspend the cells in 1 4 mL of 0 25 M sucrose Proceed to the next step or freeze the cells m -70°C 4 Prepare the sucrose gradients (11) m mtrocellulose tubes (Beckman, Fullerton, CA) by gently layering wtth a plastic ptpet the following volumes of sucrose soluttons. 1 mL of 2 M sucrose, 3.4 mL of 1.3 M sucrose, 3 4 mL of 1 0 M sucrose, and 2 75 mL of 0 6 M sucrose. It is best to prepare the gradrents using 4°C solutions Just before homogenizing the cells. 5 Transfer the cell suspension to the tube of a Dounce homogenizer chilled on ice and homogemze the cells with 32 strokes using a B pestle (all done on ice) Load the lysate on top of the sucrose gradient (see Note 17). 6 Centrimge for 3 h at 260,OOOg m an SW.41 rotor (Beckman). 7. Collect 1 mL fracttons and use all or half of each (0 5 mL) for nnmunoprecipitatron by mixmg with an equal volume of me-cold 2x RIPA buffer. Follow the immunoprecipitatton protocol, as described m Subheading 3.4. After the final wash of the immunoprecipttates with 50 mMTris-HCl, pH 6 8, add 30-50 pL of 1X SDS gel-loading buffer to each pellet andboil the samplesfor 3 min Analyze the samples by SDS-PAGE and autoradtography

3.6. Assays for Enzyme Markers for Subcellular (see Notes 18and 19)

Organelles

In order to assay for the activity of ER and Golgi-membrane marker enzymes, one or two 100~mm plates of nontransfected COS 1 cells are used for subcellular fractionation as described m Subheading 3.5. An aliquot of each 1-mL fraction 1sthen used for assays. 3.6.1. Assay for the Endoplasmic- Reticulum Marker Enzyme, NADPH-Cytochrome c Reductase The actrvity of NADPH-cytochrome c reductase is assayed by measuring the initial rate of cytochrome c reduction (12). For each sample, mix m a microfuge tube: 44 pL of gradient fraction, 50 pL of 0.6 M potassium phosphate buffer, pH 7.7,0.5 I& of 20 mM EDTA, 5 uL of 5 rU4 cytochrome c. At this step, prewarm the sample for 1 mm at 37”C, transfer it to a cuvet, add 1 & of 25 WNADPH, and mix. Measure the absorbance at 550 nm every 30 s for about 3 mm. Determine the activity by calculating the slope.

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261

3.6.2, Assay for the Golgi Marker Enzyme, Galactosyltransferase This assay IS based on the protocol of Bole et al. (IIjS For each sample to be assayed, 34 & of the protein sample is mixed with 16 pL of the premix containing: 5 p,L of 1 mMUDP-galactose, 5 & of ovalbumin (70 mg/mL), 1 &L of 1MMnC&, 1.5 & of 20% (v/v) Trlton X-100,2.5 & of 1MTns-HCl, pH 7.5 and 1 pL of UDP-[3H-GaI]. Incubate the reactlons for 1 h at 37°C. To measure the background activity, 34 pL of 0.6 M sucrose is used instead of the protein fraction. After the incubation, spot 30 @ of each sample on Whatman 3MM filter paper and measure TCA-precipitable radioactivity. To measure TCAprecipitable radioactivity, the filters are air-dried for about 10 mm, then placed in ice-cold 10% (w/v) TCA for 10 min, washed three times m 5% (w/v) TCA and once in methanol, and then air-dried. Radioactivity is determined by liquid-scintillation counting. 3.7. lmmunofluorescence of Transfected Cells 3.7.7. Plating Cells for lmmunofluorescence 1. To prepareclean and sterilecover slips,wash them three timesm acetoneusing a somcator(each time using fresh acetone) and, after air-drying, sterilize by autoclavmg. 2. Using forceps dipped in alcohol and flamed, place five coverslips m each 35-mm plate and add 2 mL of sterile 0.1% (w/v) gelatin. Keep in refrigerator for 15 mm, then remove gelatm. 3 Plate cells, transfected one day earlier as described m Subheading 3.2., mto the cover slip-containing dishes. Make sure that the cover slips stick to the bottom of the plate and do not float. Return the plates to the COZ Incubator and contmue the mcubatlon for one more day

3.7.2. Processing the Transfected Cells for lmmunostaining (see Note 20) Cells are used 48 h after transfection and should be almost confluent. All subsequent steps can be done at room temperature. 1, Wash the cells with PBS three times. 2. Fix the cells by adding 2-3 mL of freshly made 2.5% (w/v) p-formaldehyde to each plate and incubate the plates for 20-30 mm at room temperature. 3. Wash the cells twice with PBS/gelatin 4. If cells have to be permeablhzed, add 2-3 mL of 0.1% (v/v) Triton-X 1OO/PBS to each plate and incubate the plates for 5 min (see Note 21). 5. Wash the cells twice with PBSlgelatm. 6. Drain the cover slips well and transfer them to individual wells of a multlwell plate. 7. Place on each cover slip 30 & ofprimary antibody solution. Incubate for 30-40 min.

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8. Add about 2 mL of PBS/gelatin to each well, aspirate, and replace with fresh PBS/gelatin for 10 mm. Repeat this wash procedure four times. 9 Layer 30 pL of secondary antibody solution on each cover shp, cover the plate with alummum foil and incubate for 30-40 min. 10 Wash with PBS/gelatin as after the primary antibody mcubatlon (see step 8). Avold allowmg the cover slips to dry and keep the plate covered with foil and away from the light. 11. Dram the cover slips carefully and place each on a drop of mounting solution on a microscope slide. Dram the excess solution usmg a Pasteur plpet attached to a suction pump and seal with either Pro-Texx or nail polish. 12. Observe cells under the microscope. Store the slides, protected from light, m the refrigerator. 4. Notes

4.1. Construction of Chimeric Proteins and Reporter Selection In most cases, there are multtple suppliers of materials. Those listed are the suppliers that we use but, unless expllcltly stated, it IS not essential that materials be obtained from the stated suppliers If a selected reporter has all the advantages (lack of positive transport signals, compatlblhty with transport through the secretory pathway when attached to the transport signal) except for an easily traceable modlflcatlon, such as a glycosylatlon signal, a signal for the glycosylatlon may be mtroduced mto the hybrid protein by mutagenesis It 1svery important, when selecting the site for this modlficatlon, to consider the optimal context of the glycosylatlon recogmtlon signal and its distance from the membrane (13,14) In addition, some P45Os are naturally glycosylated m the N-terminal peptlde preceding the signal sequence (14) and this can be exploited m the studies on the ER retention mediated by the N-terminal domain of P450 Alternatively, sequences containing potential glycosylation &es present m the cytoplasmrc domain of some P45Os have been inserted m front of the signal/anchor peptlde (2). Immunological detection of a chlmeric protein may often be slmpllfied by attaching a c-myc epltope tag to it. This 1sa very short peptlde for which MAbs are widely available. Presence of this tag at the N-terminus of P450 2C2 does not interfere with the ER localization or folding of the protein (C. Chen and B Kemper, unpublished). The use of the GFP as a reporter may circumvent many possible problems encountered with other reporters The excellent resolution allows even an unexperienced researcher to dlstmgursh the localization of the fluorescing protein m the ER from that of Golgl or plasma membrane.

4.2. DNA Preparation 5. Although m most cases DNA purified with Qlagen 1sof sufficient punty for transfection, sometimes it may be necessary to use CsCl gradient purification. Very often, more crude preparations of DNA perform very well and good transfection

Retention of Cytochrome P450 can be obtamed using mint-prep DNA. The authors often obtain small amounts (10-20 pg) of suffictently pure DNA from 2-mL cultures processed on Qtaprep columns (Qiagen) This procedure takes about 20 mm 6. Although transient transfection m COS cells IS a good model system for studies on the localization stgnals, it has several potential problems of which the users of this system should be aware: a. Aggregation of the overexpressed protein High level expression of some P45Os has been shown to induce colocahzatton of large amounts of nnmunodetectable P450 in a nuclear fraction as a result of aggregation (5) b. A rapid turnover of the expressed protems. Although this IS not a malor problem for localization studies, turnover is much faster in COSl cells than m liver cells m VIVO, which might affect mterpretation of localization studies. c. Abnormal folding. Under the condittons of strong overexpresston, a fractton of the expressed protein may be misfolded, which hampers enzymattc activity assays and may affect localization.

4.3. Transfection 7. The most important factors in efficient transfection using the DDC method are* exponenttally growing cells plated for transfectton, fresh transfectmg medium (with pH of 7 3-7 4) and a good quality DDC mixture DDC IS toxic to the cells and it IS important to watch the cells durmg the transfectton for signs of toxtctty, m which case the transfection should be stopped after a shorter time. 8 It 1s very important to mix the DDC and medium well before addmg DNA, to avoid the formation of a precipttate on the cells When formation of a precipitate 1srepeatedly observed, new medium and DDC may be needed 9. If, after placing the transfection mtxture on the cells, a precipitate 1s observed, it 1s stall worth contmumg with the experiment in most cases. Although m such cases the efficiency of transfection will be decreased, sigmficant expression of the transfected protein will be observed However, It is safer to stop the transfection after a shorter time (3 h, rather than 4 h) and proceed with extra caution when washing the cells. In thts case, it 1s advantageous to passage the cells to fresh plates the followmg day. 10, Many transfection protocols call for replating the transfected cells mto new dashes 24 h after the transfection to mcrease the level of expression However, in most cases, this step can be omitted without a stgmticant decrease in the expression level of P450 in COSl cells 11 When GFP is used as a reporter, a strong signal is easily detected even 20 h after the transfection, shortening by half the usual time needed for significant expression in transient transfection

4.4. Radiolabeling,

Immunoprecipitation,

and Endo H Assay

12 For short (C30 min) pulse labelmg or when working with proteins with a short life time, tt may be helpful to increase the activity of radiolabeled amino acids to 800-1000 pCi/mL. On the other hand, to obtain a steady-state locahzatton of the

264

13.

14. 15

16

Skorupa

and Kemper

hybrid protein studied, a longer time, I e , overmght labelmg can be used In this case, the volume of the labelmg medium should be increased to 1.5 mL and 2% (v/v) calf serum should be included Because the kinetics of transport out of the ER is different for different proteins, the actual time for chase mcubation for the endo H assay should be established expertmentally In most cases, a significant fraction of glycoprotems not retamed m the ER acqutres endo H resistance after less than 1 h of chase incubation and 3-4 h chase is suffictent for all the pulse-labeled protem to move out of the ER. To prevent degradation of protems, additional protease mhibitors may be included m the RIPA buffer. Leupeptm, pepstatm, and aprotmm are most commonly used. If the primary antibody gives a htgh background (brings down many nonspecific protems), tt may be helpful to preclear the cellular lysate wtth a 30-min preincubation with nommmune serum, followed by protein A-Sepharose mcubatton and removal of precipltable maternal with the Sepharose by centrtfugatton Alternatively, nommmune serum may be omitted and premcubatton done with only protem A-Sepharose Although most antibodies bind to protein A-Sepharose wtth high affinity, for some IgG subclasses, protein G has much higher affinity, and m those cases protem G- Sepharose should be used

4.5. Subcellular

Fractionation

17. In some cases, subcellular fracttonation on a dtscontmuous sucrose gradient may give better resolutton of organelles if the cellular lysate IS first centrifuged at low speed (SOOg) for 5 mm and the supernatant, devotd of the nuclear pellet, used for fractionation. However, m thts case a significant loss of ER-associated proteins may be observed. In the authors’ studies, good separation of ER and Golgt membranes could be achieved with the total lysate.

4.6. Assays for Marker Enzymes 18. When the chimeric protein under study seems to localize m compartments other than ER, for example the Golgt, tt wtll be necessary to check for its presence in the plasma membrane also. In addition to immunofluorescence (see Note 21), the evidence for that can be obtained by comparing its localization wtth the plasma membrane marker, 5’ nucleotidase (6,16). 19 The mam problem with the ER and Golgi marker assays derives from having insufficient material to detect the respecttve enzyme activittes. In most cases, one or two 100~mm plates of confluent cells should yield a sufficient amount of membrane proteins for those assays. If not, tt is possible that a significant number of cells remained intact owing to msuflicrent homogenization, usually the result of a pestle that IS too loose.

4.7. lmmunofluorescent

Analysis

20. During the initial round of experiments, when the specifictty and the purity of the antibodies used is not well known, it wtll be helpful to include several controls*

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265

a Mock-transfected cells incubated with the antigen-spectfic antibody and respective secondary antibody, b. DNA-transfected cells mcubated with pretmmune serum and then the secondary antibody, and c. DNA-transfected cells incubated with the secondary antibody only. 21. If a particular chimeric protem IS transported to the cell surface and has a domain with the anttgenic epitope exposed on the extracellular side of the plasma membrane, n.nmunofluorescent stammg of nonpermeabthzed cells should be performed.

References 1 Rothman, J. and Wieland, F. T. (1996) Protein sortmg by transport vesicles Sczence 272,227- 234 2. Szczesna-Skorupa, E. and Kemper, B. (1993) An N-terminal glycosylatton signal on cytochrome P450 is restricted to the endoplasmtc reticulum in a lummal ortentation. J. Biol Chem. 268, 1757-1762. 3. Murakami, K , Mihara, K., and Omura, T. (1994) The transmembrane region of microsomal cytochrome P450 identified as the endoplasmic reticulum retention signal J Blochem 116,164175 4. Robin, M A , Maratrat, M , Loeper, J., Durandschneider, A M , Tmel, M , Ballet, F., Beaune, P., Feldman, G , and Pessayre, D (1995) Cytochrome P450 2B follows a vesicular route to the plasma membrane m cultured rat hepatocytes. GastroenteroZogy 108, 1110-l 123 5. Neve, E P A., Ehasson, E , Pronzato, M. A , Albano, E , Marmari, U , and Ingelman-Sundberg, M. (1996) Enzyme-specific transport of rat liver cytochrome P450 to the Golgi apparatus Arch Blochem Biophys 333,459-465 6. Ahn, K., Szczesna-Skorupa, E. and Kemper, B. (1993) The ammo-terminal 29 amino acids of cytochrome P450 2C 1 are sufficient for retention m the endoplasmic retmulum. J Biol. Chem 268, 1872618733. 7. Szczesna-Skorupa, E , Ahn, K., Chen, C. D., Doray, B , and Kemper, B. (1995) The cytoplasmm and N-terminal transmembrane domains of cytochrome P450 contam independent signals for retentton m the endoplasmic reticulum J Brol Chem 270,24,327-24,333 8 Uvarov, V Y., Sotmchenko, A I , Vodovozova, E L , Molotkovsky, J G , Kolesanova, E. F., Lyulkm, Y A , Stier, A., Krueger, V , and Archakov, A I (1994) Determination of membrane- bound fragments of cytochrome P450 2B4. Eur. J Biochem. 222,483-489. 9. Schekman, R. and Orci, L. (1996) Coat protems and vesicle budding Sczence 271, 1526- 1533.

10 Kain, S. R., Adams, M , Kondepudi, A., Yang, T.-T , Ward, W. W., and Kitts, P. (1995) Green fluorescent protein as a reporter of gene expresston and protein localization. BioTechnzques 19, 650-655 11. Bole, D. G., Hendershot, L M., and Kearney, J. F (1985) Posttranslational association of mnnunoglobulm heavy chain bmdmg protein with nascent heavy chams in nonsecretmg and secreting hybridomas. J, Cell Biol 102, 1558-1566.

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12. Omura, T. and Takesue, S (1970) A new method for simultaneous purtfication of cytochrome b, and NADPH-cytochrome c reductase from rat liver microsomes. J Blochem 67,249-257.

13 Shakin-Eshleman, S. H., Sprtalmk, S. L., and Kasturr, L. (1996) The ammo acid at the X position of an Asn-X-Ser sequeon IS an important determinant of N-linked core-glycosylatron efficiency J B~ol Chem 271,6363-6366 14. Shrmozawa, O., Sakaguchr, M , Ogawa, H., Harada, N., Mihara, K., and Omura, T. (1993) Core glycosylatron of cytochrome P--450(arom). J Bzol Chem. 268, 21,399-21,402 15. Clark, B. J. and Waterman, M. R. (1991) The hydrophobrc ammo-terminal sequence of bovine 17-hydroxylase is required for the expression of a functional hemoprotem m COS 1 cells. J. Brol Chem. 266,5898-5904. 16. Schmrrnel, S. D , Kent, C., Brschoff, R., and Vagelos, R. R. (1973) Plasma membranes from cultured muscle cells: rsolatton procedure and separatron of putative plasma membrane marker enzymes Proc. Nat1 Acad Scl USA 70,3 195-3 199.

29 Quantification of Cytochrome P450 Gene Expression by RNase Protection Analysis Colin N. A. Palmer 1. Introduction Many attempts to describe the regulation of mdivldual cytochrome P450 (CYP) gene products m animal tissues have been frustrated by the lack of sensitivity and specificity of the assays used. Most enzymatic assays are hampered by the overlapping substrate specificity of these enzymes, and the generation of high-affinity antibodies that are specific for individual isoforms is not a trivial task. Many CYP isoforms have close relatives, some of which can have as much as 98% identity at the RNA nucleotlde-sequence level, and filter-hybridization techniques using labeled cDNA probes are unable to dlscrlminate between such mRNAs. Although noncoding portions of the cDNAs may be used as probes, as they are more likely to offer regions of divergence within subfamllles, this 1snot often possible because 1 many lsoforms retain a high degree of slmllarity well Into their 3’ noncoding region, 2. in many cases very little 3’ noncoding sequence has been determined, and 3. these regions may contarn repetitwe elements These factors severely limit the use of 3’ noncoding cDNA probes for the quantification of CYP mRNA expression

The use of end-labeled ohgonucleotides as hybridization probes to dlscnmlnate between distinct but highly related sequences is not sufficiently sensitive for the accurate quantification of low-abundance CYP mRNAs. The polymerase chain reaction (PCR) has increasingly been used for the quantification of mRNAs over the past few years, and the initial ease of operation IS indeed very tempting; however, it can be very laborious to obtain rigorous quantltative and accurate results using PCR methodology. From Methods m Molecular Biology, Vo/ 107 Cytochrome P450 Protocols Edlted by I R Phllhps and E A Shephard 0 Humana Press Inc , Totowa, NJ

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To overcome these limitations, we have utrlized a RNase protection assay. This assay involves the preparation of radiolabeled antisense RNA probes, hybridization of these probes to total RNA in solutron, and subsequent removal of unhybrrdized RNA by RNase digestion (I) The advantage of solutron hybrrdizatlon over filter hybridization 1sthat a small hybrrdizatron volume can be used, thus increasing the efficiency of hybridization. Also, as there are no steps between RNA preparation and hybridization, loss of the target mRNA species is prevented. The use of a single-stranded RNA probe allows the use of

high concentratrons of probe without obtaining the probe/probe hybrtdizatron, which may occur when double-stranded cDNA probes are used. The RNase digestion eliminates all illegitimately hybridized probe, owing to the ability of the enzyme to cut at mismatches and single-stranded loops. This results m specificity beyond that obtamed by differential melting of illegitimate hybrids. RNase protection has been used successfully to quantify the expression of mdi-

vtdual members of the CYP2B (2), CYP2C (3), CYP3A and CYP4A (4,s) subfamilies.

The specificity

of this assay is illustrated

by its ability to discrimmate

between the mRNAs encoding CYP2B 1 and CYP2B2 (2) and between the members of the complex rabbit CYP4A subfamily (6). RNaseprotection 1salso proving invaluable in the quantification of mRNAs encoding xenobiotic-activated transcnption factors that regulate the expression of cytochrome P450 genes (7).

2. Materials All materials should be “RNase diethylpyrocarbonate (DEPC)-treated

2.1. Riboprobe

Free”. All chemicals H,O.

Template Preparation

are dissolved

in

and Transcription

1 pBluescript plasmid contammg a fragment of the approprrate cDNA. For the transcription reactions a high-quality DNA template IS required for optimal performance. Plasmid DNA should be prepared from a suitable Escherzchm colz strain (e.g , DH5a or XL-l Blue) by alkaline lysis followed by CsCl density gradient centrifugation or Qiagen ion-exchange chromatography. 2 Restriction endonucleases from standard suppliers may be used to perform lmearization reactions 3 Protemase K (Boehringer Mannheim, Indianapolis, IN): Prepare a 10 mg/mL stock m water and store m single-use aliquots at -2O’C. 4. 10% (w/v) sodium dodecyl sulfate (SDS). 5. Phenol/chloroform: prepare a mixture of 1 vol phenol to 1 vol chloroform. 6. 3 M sodium acetate. 7. Ethanol. 8 70% (v/v) ethanol. 9. DEPC-treated water.

P450 Gene Expression

269

10. 5X transcription buffer; 0.75 M dlthlothreltol (DTT), solutions of nucleoslde triphosphates (NTP mix) (2.5 &each of UTP, ATP, GTP, and 0.125 mMCTP); and T7 and T3 RNA polymerases are conveniently obtained as components of a transcrlptlon kit from Stratagene (La Jolla, CA) 11. RNase inhibitor II (Stratagene). 12. [a-32P] CTP (800 Cl/mmol) (Amersham International Buckinghanshlre, UK) 13 0.5 M Na2HP0,. 14. DE81 filter paper (Whatman, Clifton, NJ). 15 RNase-free DNase (Stratagene). 16 Formamide (“Ultra-Pure,” Gibco/BRL, Gaithersburg, MD): Store at -20°C For use m the preparation of RNA load buffer and RNase protection hybndization buffer 17. RNA load buffer: 97% (v/v) formamide, 1% (w/v) Flcoll, 0.05% (w/v) bromopheno1 blue, 0 05% (w/v) xylene cyanol. 18. Urea (“Ultra-Pure,” Gibco/BRL). 19 40% (w/v) Acrylamide, 2.1% Bisacrylamlde 20 8 A4 urea/6% polyacrylamide gel prepared from items 18 and 19. 21 X-Ray film (KODAK-XAR) 22 RNA elutlon buffer: 0.5 M ammonium acetate, 1 mM ethylene dlamme tetraacetic acid (EDTA), 1% (w/v) SDS. 23. tRNA (Boehrmger Mannhelm): 10 mg/mL stock 24. RNase protectlon hybrldizatlon buffer. 80% (v/v) formamide, 40 mMPIPES, pH 6 4,0 4 A4 NaCl, 1 mlcp EDTA. 25. Pellets of frozen CO2 26 Scmtlllatlon fluid (Aquasol or equivalent). 27. Saran wrap

2.2. /?Nase Profecfion

Assay

1 tRNA (Boehrmger Mannhelm): 10 mg/mL stock 2. RNase A ( Boehrmger Mannhelm): Prepare a 10 mg/mL stock solution m water and store at -20°C. 3 RNase A digestlon solution 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 200 mM NaCl, 100 mMLiC1, RNase A (40 pg/mL). Store at 4°C. Boil for 10 mm and then chill on ice nmnedlately before use. 4. 10% (w/v) SDS. 5. Proteinase K (Boehringer Mannheim). see Subheading 24 item 3 6. Phenol/chloroform. see Subheading 2.1., item 5 7 Ethanol 8. RNA load buffer. see Subheading 2.1., item 17. 9. 8 MUrea/ 6% polyacrylamide gel: see Subheading 2.1., items 18-20 10. Gel fixing solution: 10% (v/v) methanol, 10% (v/v) acetic acid. Il. X-Ray film (KODAK-XAR). 12 Pellets of frozen CO2

Palmer

270 2.3. TCA Precipitation

of Protected

RNA

1 Yeast total RNA: Prepare a 10 mg/mL stock solution. Store m aliqouts at -20°C. 2 10% (w/v) trichloroacettc actd (TCA)

3 5% (w/v) TCA. 4 Glass-fiber filters 5 Scintillatton fluid (Aquasol or equivalent)

3. Methods 3.7. Template Linearization 1. Dtgest the recombinant plasmid template with a restriction endonuclease that cuts the construct once at a position 5’ of the cDNA msert (see Notes 1 and 2) It is important not to use an enzyme that produces a 3’ overhang as this may lead to mapproprtate transcrtption 2 Monitor dtgestion by agarose-gel electrophorests of an aliquot of the reaction mixture Do not proceed unttl -95% digestion has occurred, as circular DNA will be preferentially transcribed The products of such “run round” transcription are useless for the RNase protection assay 3 Upon complete digestion, add Protemase K to a final concentration of 50 pg/mL and SDS to a final concentration of 0 5% (w/v) Incubate at 37’C for 20 mm. 4 Extract once with an equal volume of phenol/chloroform To the aqueous phase, add sodmm acetate to a final concentration of 0 3 M and 3 volumes of ethanol. Freeze on pellets of frozen CO* for 10 min and then centrifuge in a microcentrifuge at full speed for 10 mm 5 Wash the pellet with 70% ethanol and then allow the pellet to an-dry 6. Resuspend the lmeartzed DNA in DEPC-treated water.

3.2. Transcription of Template and Purification of RNA Transcript To obtain an “antisense” transcript, constructs should be transcribed from the promoter that lies at the 3’ end of the cDNA, T7 or T3, dependmg on the orientation of the insert in pBluescnpt. The transcript should be gel-purified m order to obtain clean RNase protections. 1 To a RNase free 0.5-mL microcentrifuge tube add. 5 p.L 5 x Transcriptton buffer 5 @ NTP mix 1 p.L 0.75 MDTT. 2 5 pL [a-32P] CTP (50 pCt> O-8 5 pL DEPCtreated HZ0 (to make final reactton volume 25 pL). l-9 pL linearized DNA template (0.2 pg). 1 pL RNase inhibitor II (40 units) 1 l.iL T3 or T7 RNA Polymerase (50 units) Incubate the reaction mix for 30 min at 37°C 2. The progress of the reaction may be monitored by spotting 0 5 pL of the reaction mix onto a small piece of Whatman DE8 1 chromatography paper. Leave to atrdry for 5 mm and wash in 0.5 MNa2HP04 for 5 mm. The radtoacttvtty remaining on the filter corresponds to CTP incorporated into the probe. Incorporation of

P450 Gene Express/on

3. 4. 5 6. 7

8 9.

10. Il. 12. 13.

271

30-60% of the total radioactivity m 30 mm 1sideal for the productton of a htghquality riboprobe. To the transcription reaction mtx add 1 pL (10 units) of RNase-free DNase and continue the mcubatton for a further 15 mm Precipitate the transcript by the addition of l/10 volume of 3M sodium acetate and 3 volumes of ethanol, and place tube on pellets of frozen CO, for 30 mm Centrifuge for 5 min at 15,OOOg,remove the supernatant and resuspend the pellet in 4 p,L of RNA load buffer. Heat the sample at 80°C for 3 mm. Load onto a 0.5~mm-thick urea/polyacrylamtde gel (see Subheading 2.1., item 20) mini vertical-slab gel (e.g., Mighty Small from Hoeffer or Mini-protean from Bio-Rad, Hercules, CA), and subject to electrophoresis for 30 mm at 6W Wrap the gel m Saran wrap and expose the gel to X-Ray film for 3 mm Mark the position of the gel on the X-ray film usmg a marker pen and develop the film. Cut a wmdow m the autoradtogram that corresponds to the position of the fulllength transcript and, using the pen marks to positton the gel on the developed autoradiogram, excise the portion of gel contammg full-length transcrtpt Place the gel fragment into 0.3 mL of RNA elutton buffer Incubate at 37°C for 2 h m an orbital shaking incubator to elute RNA Ethanol precipitate the eluted RNA in the presence of 30 pg tRNA carrier, and resuspend in 200 pL of RNase protection hybridizatton buffer Determme the yield by liquid scmttllation spectrometry. Dilute the probe to 1 x lo4 cpm/pL to prevent excessive mtermolecular radiolysts and thus prolong Its “shelf hfe ” Store the probe in aliquots at -20°C

3.3. Hybridization 1. Concentrate the total RNA by lyophtlization, or dilute to give the reqmred amount of RNA in 5-25 pL of hybridization buffer. Do not use more than 40 pg of total RNA as we have found that the response 1snot linear m assays contammg htgher concentrattons of total RNA 2 Perform all hybridizations m a volume of 30 pL m 0.5 mL microcentrtfuge tubes, Add between 4 x 1O4and 1 x 1O5cpm of probe per hybridization reaction Add tRNA to bring the total amount of RNA present m the hybrtdtzation mixture to 30 pg. Always mclude one hybridization mtxture that contains tRNA alone. This serves as a negattve control 3. Heat the hybridtzation mixture at 80°C for 10 min and then Incubate at 45°C overmght.

3.4. RNase Treatment 1. Centrifuge tubes briefly to collect hybridization mixture at the bottom of the tube 2. Add 350 pL of RNase A digestion solution. Incubate at 30°C for 30 min 3. Stop the RNA digestton by addition of 10 pL of 10% SDS and 10 pL of proteinase K and incubate at 37°C for 20 mm.

272

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4 Transfer contents to a 1 5-mL microcentrifuge tube and extract the digest with an equal volume of phenol/chloroform 5. To the aqueous phase, add 3 volumes of ethanol and leave on pellets of frozen CO, for 30 min Centrlfige at top speed m a mlcrocentflfuge for 10 mm and remove the ethanol, taking care not to disturb the pellet Centrifbge the tube briefly to collect the remainder of the ethanol from the sides of the tube and remove by careful plpetmg. It ts important to remove all excess ethanol, but not to allow the pellet to dry out. Excess ethanol may cause the sample to float out of the wells of the gel, and drying of the pellet will inhibit solubilization of the sample 6 Resuspend the pellet m 4 pL of RNA load buffer (vortex thoroughly)

3.5. Elecfrophoretic

Analysis of Protected RNA (see Note 3)

1 Heat sample at 80°C for 3 mm. 2 Load onto a prerun 1.5 mm-thick urea/polyacrylamlde gel (see Subheading Z.Z., item 9). Also load radlolabeled size markers in one of the wells (see Note 4). Electrophorese at 6W/mimgel until the xylene cyan01 is 1 cm from the bottom of the gel. 3. After electrophoresls, wash the gel for 1 h m gel fixing solution m order to fix the RNA in the gel and remove the urea 4. Rinse the gel m water. 5 Dry gel at 80°C for 1 h m a gel dryer. 6. Expose gel to X-ray film.

3.6. TCA Precipitation

of Protected

RNA

An alternative to electrophoretic analysis 1s the direct measurement of TCAprecipltable RNA. This is suitable only for the quantification of moderate to highly abundant gene products that do not have closely related lsoforms. 1 After stopping RNA digestion (see Subheading 3.4., step 3), add 100 pg of yeast total RNA and 300 pL of 10% (w/v) TCA Incubate on ice for 30 mm. 2 Apply the sample to a glass-fiber filter using a vacuum manifold, and rmse with approx 3 mL of 5% (w/v) TCA 3. Dry the filter under an Infrared lamp 4. Place the dry filter m 5 mL of scmtlllation fluid and determine the radioactivity bound to the filter by hquld-scintlllatlon spectrometry.

3.7. Calculation

of Specific Radioactivity

of Probe (see Notes 5-7)

The specific radloactlvlty of the probe 1s determined by the concentration of CTP and radionuclide m the transcription reaction. In the reaction conditions described m Subheading 3.2., 1 & of reaction mix contains 25 pmol of CTP and 5 x IO6 cpm. The number of moles of probe In 5 x lo6 cpm of TCAprecipltable radioactlvlty, may be calculated by dividing 25 pmol by the number of C residues present m the hybridizing section of probe. (Do not include

any residues that are present in the transcribed polylinker sequence.)

P450Gene

Express/on

273

Sample calculation: For a CYPZA6 antisense probe of 169 nucleotides contaming 42 C residues: 5 x 1O6cpm = 25 pmol CTP, therefore 5 x 1O6cpm = 25/ 42 pmol or 0.6 pmol of radiolabeled probe. 0 6 pmol = 0.6 x 10-l* mol x 6.023 x 1O-23molecules/mol = 3.6 x 10” molecules of probe, therefore 5 x 1O6cpm of radlolabeled probe = 3.6 x 10’ 1molecules, a specific radioactivity of 5 x lo6 cpm/3.6 x 10” molecules = 1.39 x lo-5 cpm/molecule. This specific radioactivity can be used to calculate the number of probe molecules protected in a RNase protection assay. For example, if a protected signal correspondmg to 500 cpm (by comparison with a standard curve) was obtained in a RNase protection assay containing 40 pg total RNA prepared from human liver and the CYP2A6 riboprobe described above the number of molecules protected can be calculated as follows: 5 x lo2 cprn/1.39 x 10B5cpm/molecule = 3.6 x lo7 molecules. The concentration of the mRNA 1sthus 3.6 x lo7 molecules/40 pg = 8 x 1O5 molecules/pg of total RNA. This value may be related to the amount of tissue used m the assay by assuming a 5 pg yield of RNA from each hepatocyte (8). In this assay 40 pg of RNA was used, which corresponds to 40 x 10” g/ 5 x 10-l * g/cell = 8 x lo6 cells. The concentration of CYP2A6 mRNA in this human liver sample is therefore 3.6 x 1O7molecules/8 x 1O6cells = 4.5 molecules/cell. 4. Notes 1 It is of vital importance for the success of the experiment that the template region that is to be used should be carefully consldered. With the CYP genes, It 1slmportant that a region of the cDNA 1s chosen that contains as many mismatches as possible with respect to other members of the same subfamily This should allow complete digestion of lllegltimate hybrids. It is also important that regions that are known to be subject to allelic variation are not chosen for use as probes, because thrs may result m unwanted cleavage of the hybrids. The size of the template LSalso important as it is easier to produce high quahty small transcripts, and small transcripts are less prone to “breathmg” artifacts in the protection assay. It is easiest to use probes from 100 to 400 nucleotldes long, but probes of other sizes can be used. 2. Several companies now sell RNase protectlon probe templates and one of these, Amblon, also sells RNase protection kits This kit has the advantage of being a single-tube assay that does not require protemase K digestion or phenol extraction It produces good results, but is rather expensive if you plan to do many assays. Amblon also markets nonisotoplc RNase protection assay kits, but these have not yet been utihzed m the quantification of CYP mRNAs

274

Palmer

3. It is possible to detect several CYP mRNAs m a single hybridizatton assay if probes of different lengths are used. The use of long sequencing gels would permit the analysis of more than 20 CYP tsoforms m one assay. Tins approach IS excellent for “mass screening” of CYP expression; however, it may not be suitable for accurate quantification because the stgnal to noise ratio in assays with so many probes will be reduced. This IS parttcularly true when isoforms of greatly dlffering abundance are bemg analyzed In these cases, the background signal from an abundant tsoform may mask the signal obtained for a low-abundance isoform 4. A suttable set of markers for sizing the protected fragments 1s the 1 kb ladder from Grbco/BRL+ Thts can be end-labeled with [IX-~~S] dATP usmg Klenow DNA polymerase and standard reaction protocols A typical RNase protection expenment is shown m Fig. 1 If single base pair accuracy 1s required then the RNase protecttons may be electrophoresed through a sequencing gel, with a sequencing reaction serving as an ideal size marker. 5. In order to determine the amount of radioactivity present m the protected fragments, we have to compare the signals obtained with our samples with signals obtamed from known amounts of radioactive probe. In order to do this, various drlutlons of undigested, full-length probe should be electrophoresed m an identtcal manner to the RNase digestions. These should be vtsualtzed along with the experimental samples. The most accurate and convenient method to generate the standard curve IS by phosphorimager analysis The linear range of most phosphorimage scans extends more than four orders of magnitude. however, care must stall be taken to avoid usmg overexposures. The easiest quahty-assurance step to avoid overexposure is to take a one-dtmensional line plot of the strongest signals. Overexposure is apparent when the stgnal peak has a plateau or flat top Densltometry of autoradiograms may also be used. This is more laborious and must be performed wtth great care and many different exposures as the linearity of each exposure 1s less than one order of magnitude In all cases, the signal obtained with a negattve control RNase digestion (e.g , one containmg tRNA alone) should be used to determine nonspecific radioactivtty, and the value obtained should be subtracted from the experimental samples. Do not subtract this value from the samples used to generate the standard curve, subtract the background determined from a blank lane. The signal obtained with various amounts of radioactivity may be plotted graphically and a linear regression obtained. The equatton of this line may be used to interpolate the values obtained with the experimental samples In practice, a standard curve of 15-20 samples between 10 cpm and 5000 cpm should cover the range of signals obtained with most probes for cytochrome P450 mRNAs Signals above 5000 cpm should be treated with caution because a significant amount of the probe wtll have been protected. In these cases, the assay should be repeated with a lower amount of total RNA. Figure 2 shows the quantitative nature of the RNase protection assay 6. Subheading 3.7. describes the calculation of expression of mRNA in terms of “molecules per cell.” This relies entirely on the quanttfication of the input RNA by spectrophotometrtc means and using an assumptton of yield of RNA per cell

275

P450 Gene Expression HlJb$a;NL&/ER MPUNDIGESTED PROBE

12 34567

8

L

Fig. 1. Analysis of human liver cytochrome b5 mRNA by RNase protection. A 379nucleotide riboprobe was transcribed from pBShB5300 and used to assay 10 pg of total RNA from eight different human liver samples (tracks l-g). A track containing 3sS-labeled 1 kb ladder as a molecular weight marker is included (M). Also included are tracks containing undigested (P) and completely digested (-) probe. The mobility of the 379-nucleotide riboprobe (UNDIGESTED PROBE) and the 300-nucleotide species resulting from protection by hybridization to cytochrome bs mRNA (PROTECTED PROBE) is indicated..

t Input sense RNA (pg)

Fig. 2. RNase protection analysis of known amounts of sense RNA. Sense RNA was transcribed from a CYP2A6 cDNA in the presence of low specific activity 35S-labeled CTP and the yield of transcript was determined by scintillation spectrometry. Known amounts of sense RNA were included in RNase protection assays with a 32P-labeled CYP2A6 antisense riboprobe. The radioactivity present in the protected fragments was determined by comparison with a standard curve as described in Note 5 and the amount of protected RNA was calculated based on the specific radioactivity of the probe as described in Subheading 3.7. The signal may be normalized to the number of cells actually used in the preparation by calculating the yield of a DNA preparation from the same tissue. This would allow the mRNA abundance to be expressed as molecules per mg of DNA.

Palmer It may be more desirable to express the mRNA abundance as a ratio of the mRNA of some fixed “housekeeping” gene This approach is limited by the fact that some commonly used “housekeeping” genes may be regulated in the systems that you may be investigating. We have utilized P450 reductase as a normahzation probe m human tissues because we found that its abundance varied less than threefold among 20 human livers and was expressed m simrlar amounts in all tissues studied (9). Other studies have confirmed the lack of variability of P450 reductase expresston m human tissues (8). This may not be suitable for all systems because P450 reductase 1s known to be regulated m rodent liver by xenobiotics and we have found that fetal tissues contain very little P450 reductase mRNA Other commonly used normalizatton probes Include those encodmg p-actin, ribosomal protems, or glyceraldehyde-phosphate dehydrogenase However, these gene products are known to be hormonally controlled or to vary m abundance from tissue to tissue (I/X11) Perhaps the best choice for a normalizatton probe ts one agamst either 18s or 28s rRNA, because the abundance of the target would be a reflection of the amount of total RNA present. In general, one should be wary of the method of normalization and be vigilant for systematic errors in analysts that may be introduced by the normahzatton procedure 7 One of the most common problems encountered m RNase protection is mabthty to produce an mtact rtboprobe. There are several reasons that may account for a poor-quality transcrtpt. These include RNase contammation, excess template, and faulty reagents It is important to use high-quality DNA and reagents, and the RNase mhtbttor stock should be renewed frequently (every few months) High levels of incorporation (8O-100%) durmg transcription may Indicate that too much template is present, which may lead to htgh levels of transcripttonal mitiation that cannot be supported by the concentration of nucleotides m the reaction mixture. This will result in high levels of premature termination In order to obtain full-length transcripts, the amount of template should be reduced to produce between 30 and 60% mcorporation during the reactton period It 1s generally easier to produce high-quality probes if their size 1s relatively small. For this reason, it is best to use probes between 100 and 400 nucleotides in length. Another common problem 1s mcomplete digestion wtth RNase. Incomplete digestion 1s indicated by obtammg a signal m the negative control. This may be seen as a fragment corresponding to the full-length probe rather than one derived from legmmate protection by the target mRNA. This could be owing to template contammation and can be rectified by preparing fresh probe using a minimal amount of template and extensive DNase treatment. This will always be a problem if the probe is not gel-purified Incomplete digestion can also be produced by the intrinsic structure of the RNA probe The only solution to this problem 1sto design a new transcriptton template from a different section of the cDNA

P450 Gene Expression

277

References 1 Myers, R M , Latin, Z , and Martians, T. (1985) Detection of smgle base pair mismatches by RNase protectton. Sczence 230, 1242-1246 2 Akrawi, M , Rogters, V , Vandenberghe, Y , Palmer, C. N , Vercruysse, A., Shephard, E A., and Phtllips, I. R (1993) Maintenance and mductton m co-cultured rat hepatocytes of components of the cytochrome P450-mediated monooxygenase. Blochem Pharmacol 45, 1583-l 59 1 3. Tapner, M., Liddle, C., Goodwin, B., George, J , and Farrell, G C (1996) Interferon gamma down-regulates cytochrome P450 3A genes m primary cultures of well-differentiated rat hepatocytes. Hepatology 24,367-373 4 Palmer, C N , Richardson, T H , Griffin, K J , Hsu, M H , Muerhoff, A S , Clark, J. E , and Johnson, E F (1993) Characterization of a cDNA encoding a human kidney, cytochrome P-450 4A fatty acid omega-hydroxylase and the cognate enzyme expressed m Eschertchia colt Blochlm Blophys Acta 1172, 161-166. 5. Corrm, C., Feletou, M , Canet, E., and Vanhoutte, P. M (1996) Inhtbnors of the cytochrome P450-mono-oxygenase and endothelmm-dependent hyperpolartzations m the guinea-pig isolated carotid artery Br J Pharmacol 117, 607-610 6 Roman, L J , Palmer, C. N , Clark, J. E , Muerhoff, A S., Griffin, K J , Johnson, E F , and Masters, B. S. (1993) Expression of rabbit cytochromes P4504A which catalyze the omega-hydroxylatton of arachtdomc acid, fatty actds, and prostaglandins. Arch Blochem Bzophys 307,57-65 7. Palmer, C N A., Hsu, M. H., Griffin, K J , Raucy, J L., and Johnson, E F., (1998) Peroxisome proliferator activated receptor- a expression in human hver Mol. Pharmacol.

53, 14-22.

8 Czerwmski, M , McLemore, T L., Gelboin, H V , and Gonzalez, F. J (1994) Quanttfication of CYP2B7, CYP4B1, and CYPOR messenger RNAs in normal human lung and lung tumors Cancer Res. 54, 1085-l 09 1 9. Shephard, E A., Palmer, C. N., Segall, H J , and Phillips, I. R (1992) Quanttflcation of cytochrome P450 reductase gene expression m human tissues Arch Blochem. Blophys. 294, 168-l 72 10 Rolland, V., Dugan, I., Le, L. X., and Lavau, M. (1995) Evtdence of increased glyceraldehyde-3-phosphate dehydrogenase and fatty acid synthetase promoter acttvittes m transiently transfected adipocytes from genetically obese rats. J Blol Chem 270,1102-l 106 11. Kawaguchi, H., Yavart, R., Stover, M. L., Rowe, D W., Ratsz, L G., and Pilbeam, C C. (1994) Measurement of interleukin- 1 sttmulated constituttve prostaglandm G/H synthase (cyclooxygenase) mRNA levels m osteoblastic MC3T3-El ceils using competttive reverse transcriptase polymerase chain reaction. Endocr Res 20,219233

Hepatocyte Cultures in Drug Metabolism and Toxicological Research and Testing Vera Rogiers and Antoine Vercruysse 1. General Background to the Use of Hepatocyte-Based In Vitro Models The safety for man of new pharmaceuticals is ensured by in VIVOtestmg, at a first stage by using experimental animals and later, m clmrcal trials, by administration

of the drugs to human volunteers (I). On a global scale, ammal-

based in vivo testing mvolves a large number of vertebrates, partrcularly rodents (2). As the safety criteria for new drugs progressrvely increase, so does the use of experimental animals. However, this is no longer ethically, economtcally, or scientifically acceptable. The solution to this problem may lie, at least m part, in the consistent use of in vitro models in drug metabolism and toxicological research and testing. The liver IS the major organ with respect to drug metabolism.

Because drug

metabolism and toxicity are inherently linked and the liver is known to be one of the most commonly affected target organs in preclinical toxictty studies, several liver-based in vitro models have been developed, including purified enzymes, subcellular fractions, isolated hepatocytes m suspensron and in culture, mrmortalized

liver cell lines, recombinant

liver cell lines, liver slices, and

even perfused whole organs (3). Major problems are associated wtth the use of such liver-based in vitro models, however, owing to either limited

viability

tant differentiated

functions. Consequently, the practmal

applicability

liver-specific

of isolated hepatocytes

or the reduction or loss of rmpor-

m suspension (4-5 h) and in short-term

primary monolayer cultures (2-3 d) is limited. Nevertheless, they have been used to investigate drug metabolism, cytotoxicrty, hepatotoxrcity, genotoxrcity, From Methods m Molecular B/o/ogy, Vol 107 Cytochrome P450 Protocols E&ted by I R PhIllIps and E A Shephard 0 Humana Press Inc , Totowa, NJ

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and for mechanistic studies of biologrcal and/or toxtcological actton, btokinetic studies, and species selection (4). Some toxicological problems, however, can be addressed only by longer exposure of cells to the drugs under investigation. These include studtes of the inducing capacities of new molecules, some drug mteractrons, long-term effects of endogenous and exogenous factors on drugmetabolism patterns, and subchronic/chronic toxrcrty (5,6). Consequently, the development, characterization, and validation of long-term cultures of hepatocytes as m vitro experimental models represents an important goal in toxicology, drug development, and risk assessment. To be of value for drug development, such models should be able to express key phase 1 and phase 2 drug-metabolizmg enzymes in amounts comparable with those m VIVOand for longer time perrods (2-3 wk) than m short-term conventronal hepatocyte monolayer cultures. At present, such an tdeal culture system does not exist, although it IS clear that the more sophisticated hepatocyte culture systemsdisplay promising properties (7,s). These models take into account a number of elements of the micro-environment including cell-medium interactions, soluble medium components, cell-cell interactions, cell-matrix interactions, extra-cellular matrix components, cell shape, and polarity (78). 2. Metabolic Competence of Short-Term vs Long-Term 2.1. Short- Term Cultures

Hepatocyte

Cultures

Short-term cultures or conventional monolayer cultures of hepatocytes are kept for 2 to 3 d and during that limited period they maintain both phase 1 and phase 2 drug-metabohzmg enzymes at an acceptable level m comparrson with in vivo (9). Thus, they are reasonably efficient models for establishing the biotransformatron pattern of new drugs m different species, including man (4), and for this reason have been widely used as an in vitro model for drugmetabolism studies and for toxicological research and testing. They provide a good model for studying the dnect effects of hepatotoxins on the cells and the toxic events caused by active metabolites formed. However, as a function of culture time hepatocytes undergo both morphologrcal changes (they flatten and spread out), and phenotyprc changes, and consequently, after 3 d or even earlier, no longer accurately reflect xenobrotic metabolism in VIVO(6). Of particular concern IS the reduction m the concentratrons of several of the cytochromes P450 and the loss of response to some wellknown inducers of these enzymes (10-12). A similar problem with respect to phase 2 drug-metabolizing enzymes (13-16) and noncytochrome P450-dependent phase 1 metabolism (I 7) occurs.

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2.2. Long-Term Cultures From the recent literature, it is clear that the maintenance of the differentiated state of cultured hepatocytes as a function of time IS strongly dependent on a complex environment in which exogenous factors, cell-matrix and cell-cell interactions play key roles ($6). The more sophisticated culture models, including 1 coculturesof hepatocyteswith helper cells, 2. collagen gel cultures,and 3. liver spheroids, are thus better candidates to study long-term effects of drugs. 2.2.7. Cocultures of Hepatocytes with Helper Cells (see Chapter 33 for Methods) In this system, hepatocytes are cultured together with nonparenchymal cells m order to mimic better the in vivo situation in which hepatocytes are m contact with nonparenchymal cells via the space of Disse. Several continuous cell lines from different tissues have been used as helper cells (18,19), but the most successful coculture system was obtained with ratliver epithelial cells of primitive biliary origin (20). The hepatocytes not only display a longer viability but also retain to a greater extent the morphological and biochemical characteristics of cells in vivo (6). This is true for hepatocytes from various species, including humans, and from adults and fetuses (21-23). Recent work with cocultured rat hepatocytes at the enzymatic, protein, and mRNA levels revealed a steady-state situation for at least 10 d in which phase 2 enzymes are qualitatively maintained (13,14,16,24-26), the cytochrome P450 content is partly preserved (10), cytochrome P450 forms are selectively maintamed (l&24,25,27) and the noncytochrome P450-dependent flavin-containmg monooxygenase (FMO) is well-expressed (17). It could also be shown that the hormonal regulation of glutathione S-transferase (GST) and FM0 (28) is preserved throughout the culture time, which is not the case in conventionally cultured hepatocytes (17). The mRNAs encoding members of the CYP2B subfamily, CYP2Bl and CYP2B2, are maintained in cocultured rat hepatocytes and they remam inducible by phenobarbital and sodium valproate (VP) to an extent comparable to that observed in vivo (11,29,30). Also quantitatively expressed are the genes coding for NADPH cytochrome P450 reductase, cytochrome bg, and cytochrome P450 4A (11,12). The early production and deposition of extracellular matrix components (31,32) has been suggested to play a key role m the maintenance of both high levels of gene transcription (33) and cell-cell communication via gap junctions

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(34). It has also been shown that plasma-membrane constituents of rat liver epithelial cells used in cocultures are responsible for the stabihzation of gapjunctional mtercellular communication between parenchymal cells (35), and a liver regulatory protein (LRP), present on both hepatocyte and eptthehal cell membranes, has been identified (36). Coculture conditions have also been associated with less oxidative stressthan observed m conventional culture conditions (37,367. In addition, cocultured rat hepatocytes can deal better with oxtdattve stressfrom exogenous molecules than conventionally cultured cells (39), pointing to then importance m toxicologtcal studies concerned with this type of problem. Disadvantages of the coculture technique are related to the fact that the isolatton of the bihary epithehal cells is time-consummg and not well-standardized. Also, the second cell type contributes to the DNA and protem contents, which are both used to normalize most biochemical and metabolic parameters 2.2.2. Collagen-Gel

Cultures (see Chapter 32 for Methods)

Two models seemto be of particular importance: collagen-gel sandwich configuration cultures and immobilization gel cultures of hepatocytes. In the former model, hepatocytes are sandwiched between two layers of rat-tail tendon collagen type I (M-42). In a first step, hepatocytes are seeded onto a collagen layer and allowed to attach. A second layer of collagen ISthen spread over the attached hepatocytes 1 d after seeding. The second layer of collagen prevents flattening and spreading of the hepatocytes. A simple variation of this model is the collagen-gel mnnobihzation culture of hepatocytes. The cells are allowed to settle within a flutd collagen preparation prior to gelatmation, which results m the entrapment of the cells withm the gel matrix (43). In both systems, the cells retam their polarity and cuboidal shape and an architecture similar to that found m vtvo is retained (4&46). A dtstribution of actin filaments resembling that present m vivo is maintained throughout the enttre penod of culture, which 1sm sharp contrast to the stressfibers that appear m the cytoplasm of conventionally cultured hepatocytes (41,47). Liver-specific functions, including the secretion of urea, albumm, transfer&, fibrinogen, and bile salts, are maintained for up to 6 wk (44) and it has been claimed that continued synthesis of collagen by the hepatocytes may be critical for hepatocyte function (48). However, results on the charactertzation of this model with regard to drug metabolism and toxicity studies are rather scarce. Stable CYP3A expression could be shown for 2 wk in sandwich cultures of human and rat hepatocytes as judged by the biotransformation patterns of urapidil, an a-blocking drug (49), and two mununosuppressants, tacrolimus and sirohmus (50). The maintenance for 3 wk of the O-demethylation and subsequent O-glucuronidatton pathway

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of pimobendan was reported by Pahermk et al. (51) using immobihzation cultures of human hepatocytes. CYP 1A 1 expression remained present and inducible by 3,3’,4,4’-tetrachlorobiphenyl for at least 2 wk in a sandwich culture of rat hepatocytes. This was concluded from measurement of the conversion rate of ethoxyresorufin to resorufin (52). The a, n and o class GST isoenzymes are stably expressed for at least 3 wk m both collagen-gel culture models (53), but changes in the subunit pattern occur (54). The inducibility of the GSTs by several inducers, either of a more general nature or specific for one of the GST classes, is well-maintained (Beken, personal communication). In human hepatocyte immobilization-gel cultures, it was also shown that the conjugation patterns for acetammophen were stable for 2 wk (43). The expression and regulation of P45Os have not yet been investigated m depth. In human hepatocyte immobilization-gel cultures, the CYPlAl-dependent O-demethylation of p-mtroanisole could be maintained for 3 wk (43). From our own results in rat hepatocytes, it appears that a steady-state maintenance of the ethoxycoumarinO-deethylase activity (CUP1 Al- and CYP2Bldependent) around 25% of the value found for freshly isolated hepatocytes, is obtained in both sandwich and immobilization gel cultures for at least 2 wk (55). A 2- to 3-fold induction is observed after exposure of rat hepatocytes to phenobarbital (3.2 mM) m sandwich cultures. In immobihzation gels, a more moderate effect was seen (55). Other data on phase 1xenobiotic metabolism can be summarized as follows. Epoxide hydrase activity, a noncytochrome P450-dependent phase 1 enzyme activity, is stably expressed m both culture systems. A steady-state of about 30% of the initial activity observed m freshly isolated rat hepatocytes has been observed. L-proline addition to the culture medium had no effect. In contrast, phenobarbital exposure provoked an induction of 2- to 2.5 fold (55). Practical constraints of both culture models are associated with the presence of collagen. In order to gain accessto the hepatocytes for metabolrc and toxicity studies, the collagen usually has to be enzymatically digested (53). In addition, drugs or substrates added must be able to penetrate the collagen layer in order to reach the cells. Consequently, then physicochemical properties are of importance (43). Another problem, which occurs particularly m immobihzanon gels, is the presence of dead and dying cells that become entrapped during initiation of the cultures. They cannot be removed as in conventional cultures and cocultures but remain m the cultures. Thus, leaked enzymes can damage neighboring cells and their protein and DNA contents can cause erroneous results in normalization of biochemical and metabohc parameters (53).

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2.2.3. Lwer Spheroids In this model, the attachment of hepatocytes to a solid support IS inhibited, they aggregate, and form floating three-dimensional structures. One method is based on the culture of hepatocytes on nontissue-culture plastic on a rotary shaker (56). Other methods involve the culture of hepatocytes with nonadherent substrata, includmg poly (2-hydroxy-ethylmethacrylate) (p.HEMA), hverderived proteoglycans, and a collagen-conjugated thermoresponsive polymer (57-59). A combination of both the rotary shaker and coating with p.HEMA can be used resulting in a faster and more efficient technique (60). In spheroids, hepatocytes maintain not only m vrvo morphological characteristics, but also liver-specific functions including albumin, transferrin and urea secretion, and bile-acid formation. With respect to drug metabolism, it is known that 7-ethoxycoumarm O-deethylase, GST, and UDP-glucuronyltransferase activities are expressed in spheroids (8). In addition, normal lidocame metabolism and peroxisomal proliferation, associated with hepatocarcinogemcity m rodents, have been reported (61). A disadvantage of the use of spheroids, however, is that larger sizes(200~urn diameter and more) contain inner cells that degenerate because of dehydration. A diffusion gradient of nutrients, oxygen, and also of test compounds across the spheroids will limit the exposure of the inner cells. Furthermore, the functionality of liver spheroids strongly depends on the culture conditions used, m particular on the type of medium (8). 3. Toxicity Studies with Hepatocyte Cultures 3.1. Screening and Ranking of Toxic Compounds As already mentioned, freshly isolated hepatocyte suspensions and shortterm cultures of hepatocytes represent the most popular in vitro models for comparing and ranking the relative toxicity of different classes of chemicals (8,9). Ranking is done by determmation of the I& value of each compound, which is the concentration of the test compound causing a 50% inhibition of the toxicity parameter measured. Toxicity parameters can be subdivided either into hver-specific and nonliver-specific toxicity markers or into basic cytotoxicity and metabolic-competence markers (Table 1). However, a serious drawback of this method IS the wide range of concentrations that need to be tested, with maxima far m excessof real plasma and tissue levels, This could be owing to either a low metabolic rate or a short exposure time m vitro. Thus in vitro systems that allow exposure times and metabolic rates more comparable to those observed in vivo (long-term culture models of hepatocytes) seem more promising in vitro models for future work ($62).

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Table 1 Liver-Specific and Nonspecific Criteria as Quantitative Indicators of Toxicity (8,63,74) Liver-specific criteria Specific plasma protem synthesis: albumin, transferrm

Ureogenesis Apohpoprotem syntheses Peroxisome proliferation Cytochrome P-450 induction/inhibition Btle actds uptake, coqugatlon, secretion Gluconeogenesis Glycogen synthesis Nonliver-specific criteria Plasma membrane integrity. Dye uptake (e.g., trypan blue) Intracellular enzyme leakage (e.g., lactate dehydrogenase) Intracellular ion leakage (e.g., K+ release) Intracellular accumulation of external molecules (e.g., succinate) Lysosomal integrity: Neutral red uptake Levels of acid phosphatase Mitochondrial activity. ATP content/nucleotide ratios Oxygen consumption Reduction of a formozan salt (MTT test) DNA damage Metabolic competence* Total protein synthesis Glutathione content Lipid peroxidatton Covalent binding to macromolecules

The quality of the results obtained will depend not only on the choice of the culture model but also on: 1. the choice of the toxictty marker(s); 2. the biotransformation pattern and metabolic rate in vitro of the compound(s) under mvestigatron (additional factors include age, sex, and species of cell source), 3. the standardization and validation of the protocols used (e.g., culture media composition, culture atmosphere, and substrates added); 4. the physicochemical properties of the compound(s) under study (e g., volatility and lipid solubihty);

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5 availablhty of in vwo data on the compound(s) tested (e.g , histological, biochemical, and toxicological) Generally, the measurement of metabolic competence parameters is a more sensitive method than the use of basic cytotoxicity markers (63,64). A typical example from our own research is the finding of a concentration-dependent inhibition of gluconeogenesis in rat hepatocyte suspensions caused by exposure to the anti-epileptic compound sodium VP and its unsaturated 4-ene metabome, and the lack of measurable lactate dehydrogenase leakage mto the mcubation medium (65). Exposure of rat hepatocyte cultures to milacemide, another anti-epileptic molecule, causeda significant inhibition of several phase- 1 drugmetabolizing enzymes and also important morphological changes, whereas the lactate dehydrogenase leakage index again remained unchanged (66). However, there 1ssome evidence that markers of metabolic competence are not always more sensitive than basic cytotoxicity endpoints m detecting the toxicity of a compound (8). It should also be mentioned that the suitability of hepatocyte-based m vitro systemsas models for screening compounds early m drug development is more convmcmg when the compounds to be ranked are structurally related. 3.2. Studies of Genotoxic

Compounds

Rat hepatocyte short-term cultures have been used for some years to help to assessthe genotoxic potential of chemicals and drugs (67,68). They are wellsuited for quantification of DNA fragmentation and DNA repair after exposure to the genotoxic substances.These assaysare considered valid models for predicting potentially genotoxlc effects in humans (69-71). However, for some compounds, differences m response between rodent and human cells were noticed, highlighting the importance of species differences (72). These differences are sometimes quite subtle. Benzo(a)pyrene and 2-acetylammofluorene, for example, form the same DNA adducts in humans and rat, but quantitative differences occur in that a greater number of carcmogemc DNA adducts are present m human hepatocytes than m those of rats (73). It was therefore suggested that whenever information on a human carcmogemc hazard must be obtained, human hepatocyte cultures would provide more useful mformation. 3.3. Mechanistic Studies Hepatocyte suspensions and short-term cultures are again the most popular in vitro models to obtain valuable information on the nature of the hepatotoxic mechanism of a compound. Mechanisms to be studied may be a solvent effect on the cellular membrane, affecting its physicochemical properties; the production of reactive oxygen species and free radicals, provoking lipid peroxi-

Hepatocyte Cultures dation of the cellular membranes or glutathrone depletion; and covalent binding to macromolecules (74). In such assayshepatocytes of different species, including human, may be used. Examples of successesachieved through the use of short-term cultures of human hepatocytes are the elucidation of the direct potentiation of the hepatotoxic effect of opioids by ethanol (7.9 and of the mechanism of the hepatotoxic effect of acetaminophen (76). However, when the biotransformation of the compound under investigation is a key factor and long-term exposure is necessary to induce hepatotoxicity, long-term culture may represent a far better in vitro model. In this respect, the use of cocultures of hepatocytes with primitive biliary cells may be of importance. The suitability of this model has been shown m the elucidation of the hepatotoxic properties of the anti-epileptic drug sodium VP (30). Indeed, m rats VP is metabolized by CYP2Bl to 4-ene VP and 2,4-dtene VP (77), both known to be strong inducers of microvestcular steatosis and potent inhibitors of fatty acid P-oxidatton. In cocultures, VP was found to be a potent inducer of CYP2Bl and the same results could also be reproduced in vivo (30). Thus VP

induces both in vivo and in vitro its own metabolism, leading to an overexposure of the liver cells to toxic unsaturated VP metabolites. Several other examples have been reported showing similar results in vitro as in vwo (78,79).

4. Conclusions Numerous mvestrgations have demonstrated the usefulness of hepatocytebased m vitro models for drug metabohsm and toxicity studies. In particular, hepatocyte suspensions and short-term cultures of hepatocytes have been applied with successm: 1. establishing the biotransformation

pathways of new drugs in the liver,

2. ranking of compounds within a particular chemical class;

3. elucidation of the molecular mechanism of hepatotoxicity; of species differences

and

4. mvestigatlon

However, the phenotypic changes and rapid lossesof xenobiotic-metabolizmg enzymes of the hepatocytes may, in some cases, lead to erroneous results. Many potential ways of overcoming this problem have been investigated including the use of different culture media, additives, culturmg on a substratum of components of the extracellular matrix or on microcarrrers, and coculturing with other cell types (SO). However, each of these methods has achieved only limited success.Consequently, the development and use of longterm cultures of functionally stable hepatocytes remains a major goal. The protocols used for the isolation of hepatocytes and their short-term culture differ considerably among laboratories and validated protocols do not yet exist (81) (see Chapter 3 1 for methods for the isolation of rat hepatocytes). A

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first step in this direction has been taken by the European Centre for the Valtdation of Alternative Methods (ECVAM) by organizing a workshop on the practical applicability of hepatocyte cultures m routme testing (d), and further efforts are underway. At present, only the use of short-term hepatocyte cultures m unscheduled DNA-synthesis assaysto detect genotoxic substanceshas been officially accepted (68), but it is clear that more can be accomplished. It seems quite feasible to expand the actual m vitro acute toxicity testmg of compounds (by hepatocyte suspension and short-term cultures) to in vitro chronic toxicny testing by improving the already existing more sophisticated long-term culture models described in Subheading 2.2. When animal models are used (m vtvo and m vitro) to demonstrate the safety of a potential drug, the critical question is whether or not the model used truly reflects the human situation. To answer this, in vitro metabolism studies on human hepatocytes can be of great help (82) (see Chapter 36 for methods for the preparation and culture of human hepatocytes). As the availability of human liver is very limited and the preparation of hepatocytes from this material is often problematic (83,84) it is very useful to be able to cryopreserve human hepatocytes whenever available. However, procedures for this have not yet been standardized and are themselves problematic (85). Therefore, it is often more practical to select a species that has a pattern of metabolism similar or close to humans. The use of hepatocyte suspensions and their cultures can thus help to identify species suitable for further investigation in vivo as well as m vitro. However, it remains a challenge to develop better protocols for the cryopreservation of human hepatocytes and those of dogs and monkeys, which are often used in toxicology as the second nonrodent species. References 1 Roglers, V , Blaauboer,B , Maurel, P , Phillips, I., and Shephard,E. (1995) Hepatocyte-basedzn vztromodelsandtheir application in pharmaco-toxicology.Toxzc m Vitro 9,685-694.

2. Anonymous (1994) Animal experimentation in Canadaand in the Netherlands. AT’A 22,310-313

Gibson, C. G and Skett,P. (1994) in Introduction to Drug Metabohm, 2nd ed, Chapmanand Hall, London, UK. 4. Blaauboer, B J., Boobis, A. R., Castell, J V., Coecke, S., Groothuls, G. M. M., Gulllouzo, A., Hall, T. J , Hawksworth, G M , Lorenzon,G., Mlltenburger, H. G., Rogiers, V , Skett,P.,Villa, P , andWiebel, F. J (1994) The practical applicability of hepatocytecultures in routine testing.ATLA 22, 23 1-24 1.

3

5 Gu~llouzo, A, Morel, F., Ratanasavanh, D , Chesnk, C., and Guguen-Gulllouzo,

C

(1990) Long-term culture of functional hepatocytes.TOXK zn Vztro 4,4151127. 6. Rogiers, V., and Vercruysse,A (1993) Rat hepatocytecultures andcoculturesin biotransformation studiesof xenoblotics.Tox~ology 82, 193-208

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7 Rogiers, V. (1993) Cultures of Human Hepatocytes n-rIn Vitro Pharmaco-Toxrcology, m Human Cells m In Vitro Pharmaco-Toxicology (Rogrers, V , Sonck, W., Shephard, E., and Vercruysse, A., eds), VUB, Brussels, Belgium, pp. 77-l 15 8. George, E., Ham&on, G., and Westmoreland, C. (1996) The use of zn vitro models m hepatotoxicrty testmg. TEN 3, 142-152 9 Gurllouzo, A. (1986) Use of cultured hepatocytes for xenobionc metabolism and cytotoxlclty studies, in Isolated and CulturedHepatocytes (Gutllouzo, A., and Guguen-Guillouzo, C., eds.), Les Editions Inserm, John Libbey-Eurotext, Paris, France, pp. 3 13-332 10. Rogiers, V., Vandenberghe, Y. Callaerts, A., Verleye, G , Cornet, M , Met-tens, K , Sonck, W., and Vercruysse, A. (1990) Phase I and phase II xenobtotrc bto transformatton in cultures and cocultures of adult rat hepatocytes Bzochem Pharmacol 40,170 l-l 706. 11 Akrawr, M , Rogters, V , Vandenberghe, Y , Palmer, C. N A., Vercruysse, A , Shephard, E. A., and Phillips, I. R. (1993) Maintenance and mductton m cocultured rat hepatocytes of components of the cytochrome P-450 mediated monooxygenase. Blochem. Pharmacol. 45,1583-1591. 12 Akrawt, M., Shephard, E A., Phrlhps, I R., Vercruysse, A, and Rogters, V (1993) Effects of phenobarbital and valproate on the expression of cytochrome P-450 in cocultured rat hepatocytes Toxx. m Vitro 7,477-480 13 Vandenberghe, Y., Glatse, D., Meyer, D., Gutllouzo, A., and Ketterer, B. (1988) Glutathione transferase isoenzymes in cultured rat hepatocytes Biochem Pharmacol

37,2482-2485

14 Vandenberghe, Y., Ratanasavanh, D., Glaise, D , and Guillouzo, A (1988) Influence of medium composition and culture conditions on glutathrone S-transferase activity in adult rat hepatocytes during culture. In Vitro Cell. Dev. Blol 24,281-288. 15 Vandenberghe, Y., Morel, F , Forcers, A , Ketterer, B., Vercruysse, A , Gurllouzo, A., and Rogters, V. (1989) Effect of phenobarbital on the expression of glutatrone S-transferase tsoenzymes m cultured rat hepatocytes. FEBS Lett. 251,5%64 16 Vandenberghe, Y., Morel, F , Pemble, S., Taylor, J. B., Rogiers, V., Ratanasavanh, D., Vercruysse, A , Ketterer, B , and Guillouzo, A. (1990) Changes m expression of mRNA coding for glutathione S-transferase subunits l-2 and 7 m cultured rat hepatocytes. Mel Pharmacol 37,372-376. 17 Coecke, S , Mertens, K , Segaert, A., Callaerts, A, Vercruysse, A., and Rogiers, V. (1992) Spectrophotometnc measurement of flavin-contammg monooxygenase activity in freshly isolated rat hepatocytes and then cultures. Anal Blochem. 205,285-288 18 Kuri-Harcuch, W. and Mendoza-Figueroa, T (1989) Cultrvation of adult rat hepatocytes on 3T3 cells * expression of various liver differentiated functtons. Dzfferentiation 41, 148-157 19. Donato, M. J., Castell, J. V , and Gomez-Lechon, M. J. (1994) Cytochrome P450 activities in pure and cocultured rat hepatocytes. Effects of model mducers. In Vitro Cell Dev Biol 30A, 825-832 20. Beg&, J., Guguen-Guillouzo, C., Pasdeloup,

N., and Gutllouzo, A (1984) Prolonged maintenance of active cytochrome P-450 in adult rat hepatocytes cocultured with another liver cell type. Hepatology 4, 839-842

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21 Guguen-Gmllouzo, C., Clement, B , Lescoat, G., Glaise, D , and Guillouzo, A (1984) Modulatton of human fetal hepatocyte survival and differentiation by interactions with a rat liver epithehal cell lme. Dev Bzol 105,21 L-220. 22. Clement, B , Guguen-Gmllouzo, C., Campion, J. P , Glaise,D., Bourel, M , and Guillouzo, A. (1984) Long-term cocultures of adult human hepatocytes with rat liver epithehal cells . modulation of active albumin secretion and accumulation of extracellular material HepatoZogy 3,373-380 23. Lescoat, G., Thtze, N., Clement, B , Guillouzo A, and Guguen-Guillouzo, C (1985) Control of albumm and a fetoprotem secretion by fetal and neonatal rat hepatocytes maintained m coculture. Cell Diff l&259-268 24. Niemann, C , Gauthier, J. C., Richer%, L., Ivanov, M A., Melcion, C., and Cordier, A (1991) Rat adult hepatocytes in primary pure and mixed monolayer culture Blochem Pharmacol 42,373-379. 25. Utesch, D , and Oesch, F. (1992) Dependency of the in vitro stabilrzation of differentiated functions in parenchymal cells on the type of cell lme used for coculture. In Vztro Cell Dev. Blol 28A, 193-198. 26 Vons, C , Pegorier, J P., Guard, J., Kohl, C , Ivanov, M. A., and France, D. (1991) Regulation of fatty-acid metabolism by pancreatic hormones m cultured human hepatocytes. Hepatologv 13, 1126-l 130. 27. Maier, P (1988) Development of m vitro toxicity tests with cultures of freshly isolated rat hepatocytes Experientla 44,807-8 17. 28 Coecke, S (1994) Hormonal regulation of flavm-containing monooxygenase and glutathione S-transferase m rat liver an in vztro approach. Doctoral thesis VriJe Urnversrtert Brussel, Department of Toxicology, Brussels, Belgium, pp. l-190 29. Rogiers, V , Callaerts, A , Vercruysse, A., Akrawi, M., Shephard, E , and Phillips, I. (1992) Effects of valproate m xenobiotic biotransformation m rat hvers zn vzvo and in vztro experiments Pharm. Weekbl Scz 14, 127-13 1 30 Rogiers, V , Akrawi, M., Vercruysse, A., Phillips, I R., and Shephard, E A (1995) Effects of the anticonvulsant, valproate, on the expression of components of cytochrome-P-450-mediated monooxygenase system and glutathione S-transferases. Eur J. Blochem 231,337-343. 3 I. Clement, B., Guguen-Guillouzo, C , Grimaud, J. A , Rrssel, M., and Guillouzo, A (1988) Effect of hydrocortisone on deposition of types I and IV collagen m cultured rat hepatocytes Cell Mol Biol 34,449A60. 32. Clement, B., Resean, P. Y., Baffet, G., Loreal, O., Lehry, D., Campion, J. P , and Guillouzo, A. (1988) Hepatocytes may produce lammm in fibrotic liver and primary culture. Hepatology 8,794--803 33. Frashn, J. M., Kneip, B., Vaulont, S , Glaise, D., Munmch, A., and Guguen-Guillouzo, C. (1985) Dependence of hepatocyte specific gene expression on cell-cell interactions in primary culture. EMBO J 4,2487-249 1. 34. Mesnil, M , Fraslm , J. M , Piccoh, C., Yamasaki, H., and Guguen-Guillouzo, C (1987) Cell contact but not junctional communication (dye coupling) with biliary epithelial cells is required for hepatocytes to mamtam differentiated functions Exp. Cell Res. 173,524-533.

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35 Diener, B., Beer, N., Durk, H., Trarser, M., Utesch, D., Wieser, R. J , and Oesch, F. (1994) Gap junctional mtercellular communication of cultured rat liver parenchymal cells IS stabthzed by epithelial cells and then isolated plasma membranes. Experlentra 50, 12 1-126. 36. Corlu, A., Kneip, B., Lhadi, C., Leray, G., Glaise, D., Baffet, G., Bourel, D., and GuguenGutllouzo, C. (1991) A plasma membrane protein involved m cell contact-mediated regulation of tissue specific genes in adult hepatocytes. J Cell Bzol. 115,505-5 15. 37. Mertens, K., Rogters, V., and Vercruysse, A. (1993) Glutathlone dependent detoxicatron m adult rat hepatocytes under various culture condmons. Arch. Toxzc. 67,680-685. 38. Mertens, K., Roglers, V., and Vercruysse, A. (1993) Measurement

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of malondialdehyde m cultures of adult rat hepatocytes. Toxw. En Vatro 7, S439-441 Mertens, K., Kaufman, S., Waterschoot, S., Vercruysse , A , and Roglers, V. (1996) Effect of tertiary butylhydroperoxide-induced oxldattve stress on glutathione content and thiobarbrturtc acid reactive substances production in cultures and cocultures of adult rat hepatocytes. Toxzc zn Vztro 10, 507-511. Dunn, J. C. Y., Yarmush, M. L , Koebe, H G., and Tompkins, R G (1989) Hepatocyte functron and extracellular matrix geometry long-term culture in a sandwich contiguratton FASEB J 3,174-177 Dunn, J. C. Y., Tompkins, R. G , and Yarn-rush, M L. (1991) Long-term m vitro function of adult hepatocytes m a collagen sandwich configuratron Bzotechnol Progr. 7,237-245. Dunn, J. C. Y., Tompkins, R G., and Yarmush, M. L (1992) Hepatocytes in collagen sandwich* evidence for transcriptional and translational regulation J Cell Blol. 116, 1043-l 053 Koebe, H. G , Pahernik, S , Eyer, P , and Schildberg, F W. (1994) Collagen gel nnmobilizatton : a useful cell culture technique for long-term metabolism studies on human hepatocytes. Xenobzottca 24,95-107. Yarmush, M. L., Toner, M., Dunn, J , Rotem, A, Hubel, A , and Tompkins, R G. (1992) Hepatic tissue engineering. Development of critical technologres. Ann NY Acad Sci. 665,238-252. Knop, E., Bader, A., Boker, K., Pichlmayr, R., and Sewing, K. F (1995) Ultrastructural and functronal differentiation of hepatocytes under long-term culture conditions. Anat Ret 242,337-349. Lecluyse, E., Audus, K. L., and Hochman, J. H. (1994) Formation of extensive canalicular networks by rat hepatocytes cultured m collagen-sandwich configuration. Am. J Physzol. 266, C1764-C1774. Ezzell, R. M., Toner, M., Hendricks, K., Dunn, J. C. Y., Tompkins, R. G., and Yarmush, M. L. (1993) Effect of collagen gel configuratton on the cytoskeleton in cultured rat hepatocytes. Exp Cell Res 208,442-452. Lee, J., Morgan, J. R., Tompkins, R. G and Yarmush, M. L. (1993) Prolme-mediated enhancement of hepatocyte function in a collagen gel sandwich culture configuration. FASEB J 7,586-59 1,

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49 Bader, A , Zech, K , Crome, O., Chrtsttans, U , Ptchlmayr, R., and Sewmg, K. F (1994) Use of organotypical cultures of prtmary hepatocytes to analyse drug biotransformation m man and animals. Xenobiotlca 24, 623-633 50. Bader, A , Knop, E , Boker, K. H W., Crome, O., Fruhauf, N., Gonschior, A. K , Christians, U., Esselmann, H., Pichlmayr, R., and Sewing, K F. (1996) Tacrohmus (FK506) biotransformation in primary rat hepatocytes depends on extracellular matrtx geometry. Naunyn-Schmzedeberg’s Arch Pharmacol 353,461+73. 5 1 Pahernik, S A , Schmid, J , Sauter, T , Schddberg, F W , and Koebe, H G. (1995) Metabolism of pimobendan in long-term human hepatocyte culture ry1vivo-ln wtro comparison. Xenobiotxa 25, 8 1I-823. 52. Rotem, A., Matthew, H. W. T., Hstao, P H., Toner, M., Tompkms, R. G , and Yarmush, M. L. (1995) The activtty of cytochrome P450 1Al m stable cultured rat hepatocytes. Toxzc in titro 9, 139-149. 53 Beken, S , Pauwels, M , Pahemik, S , Koebe, H G , Vercruysse, A, and Roglers, V (1996) Glutathione S-transferase activtty in collagen gel sandwtch and tmmobthsation cultures of rat hepatocytes. Toxzc in Vztro 11(6), 741-752 54 Beken, S., Pahermk, S., Koebe, H. G , Vercruysse, A., and Rogiers, V (1997) Cell morphology, albumin secretton and glutathione S-transferase expressron in collagen gel sandwtch and immobtlisatton cultures of rat hepatocytes Toxzc zn Vztro 11(5), 409-4 16 55. De Smet, K., Callaerts, A., Vercruysse, A., and Rogiers, V. (1997) Effect of pheuobarbnal ouf-ethoxycoumarin 0-deethylase and mtcrosomal epoxide hydrase activities m collagen gel cultures of rat hepatocytes Toxic. zn Vitro 11(5), 455-463 56 Li, A P., Colbum, S. M , and Beck, D J. (1992) A stmphfied method for the culturing of primary adult rat and human hepatocytes as multicellular spheroids In Vitro Cell Dev Blol 28A, 673-677 57 Landry, J , Bermer, D., Quellet, C., Goyette, R., and Marceau, N. (1985) Spheroidal aggregate culture of rat liver cells : htstotyplc reorgamzatton, biomatrix deposition, and maintenance of functional acttvtties. J Cell Blol. 101,914-923 58. Kotde, N , Sakaguchi, K., Koide, Y., Asano, K., Kawaguchi, M L., Matsushlma, H., Takenami, T., ShmJi, T , Mot-t, M , and Tsujt, T (1990) Formation of multtcellular spheroids composed of adult rat hepatocytes in dishes with positively charged surfaces and under other nonadherent envtronments. Exp Cell Res 186,227-235 59. Ueno, K, Miyashita, A., Endoh, K, Takezawa, T., Yamazakt, M., Mori, Y , and Satoh, T (1992) Formatton of multicellular spheroids composed of rat hepatocytes. Res Comm Chem Path01 Pharmacol 77, 107-120. 60. Hamilton, G., Fox, R., Atterwill, C. K., and Gedorge, E. (1995) Liver spheroids as a long term model for hver toxicity m vitro. Hum Exp Toxzc. 15, 153 6 1 Roberts, R. A., and Soames, A R. (1993) Hepatocyte spheroids. prolonged hepatocyte viability for m vitro modehng of nongenotoxtc carcmogenesis. Fundam Appl Toxzc 21, 149-158.

62. Ratanasavanh, D., Beaune, P , Baffet, G., Rtssel, M., Kremers, P , Guengertch, F. P., and Guillouzo, A. (1986) Immunochemtcal evidence for the maintenance of cytochrome P-450 isozymes, NADPH cytochrome C reductase, and epoxide

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hydrolase in pure and mixed primary cultures of adult human hepatocytes. J. Htstochem. Cytochem. 34,521--533. Guillouzo, A. (1992) Hepatotoxtcity, in In Vitro Toxzczty Testzng (Frazier, J M., ed.), Marcel Dekker, NY, pp. 45-83. Goethals, F , Krack, G., Deboyser, D , Vossen, P., and Roberfrotd, M (1984) Critical biochemical functions of isolated hepatocytes as sensitive indicators of chemical toxicity. Fundam. Appl. Toxzc. 4,441-450 Rogiers, V , Vandenberghe, Y., and Vercruysse A. (1985) Inhibition of gluconeogenesis by sodium valproate and its metabolites m isolated rat hepatocytes. Xenobiotlca 15,759-765. Rogiers, V , Vandenberghe, Y., Vanhaecke, T., Geerts, A , Callaerts, A., Carleer, J., Roba, J., and Vercruysse, A. (1997) Observation of hepatotoxic effects of 2-npentylaminoacetamte (Milacemide) m rat liver by a combined m vzvo/zn vztro approach. Arch Toxzc 71,271-282 Butterworth, B. E , Smith-Oliver, T., Earle, L., Lowry, D. J., White, R. D., Doohttle, D. J., Working, P K., Cattley, R. C., Jirtle, R., Michalopoulos, G., and Strom, S. (1989) Use of primary cultures of human hepatocytes in toxicology studres. Cancer Res. 49, 1075-l 084. Swterenga, S. H. H., Bradlaw, J. A., Brrllmger, R. L., Gilman, J. P. W., Nestmann, E. R., and San, R C (1991) Recommended protocols based on a survey of current practice in genotoxtcity testing laboratories. I* unscheduled DNA synthesis assay in rat hepatocyte cultures Mutat Res. 246,235-253. Butterworth, B. E., Earle, L. L., Strom, S., Jutle, R., and Michalopoulos, G. (1983) Induction of DNA repair m human and rat hepatocytes by 1,6-dimtropyrene. Mutat Res 122,73-80. Strom, S. C , Jirtle, R. L , and Michalopoulos, G (1983) Genotoxic effects of 2-acetylammofluorene on rat and human hepatocytes. Environ Health Perspect 49,165-170. Martelh, A., Robbiano, L., Gmliano, L., Pino, A., Angehm, G , and Brambilla, G. (1985) DNA fragmentation by N-nitrosodimethylamme and methylmethanesulfonate in human hepatocyte primary cultures. Mutat Res 144,209-2 11 Martelli, A., Allavena, A , Robbtano, L., Mattioli, F., and Brambilla, G (1990) Comparison of the sensitivity of human and rat hepatocytes to the genotoxic effects of metronidazole Pharmacol TOXK 66, 329-334 Monteith, D. K. and Gupta, R. C. (1992) Carcinogen-DNA adducts in cultures of rat and human hepatocytes. Cancer Lett. 62, 87-93. Guillouzo, A. (1995) Hepatotoxicite zn vitro, in Toxzcologze Cellulazre In Vztro Mthodes et Applzcatlons (Adolphe, M., Gutllouzo, A., and Marano, F , eds.), Les Editions Inserm, Paris, France, pp. 69-120. Jover, R., Ponsoda, X., Gomez-Lechon, M. L , and Castell, J. V. (1992) Potentiatton of heroin and methadone hepatotoxictty by ethanol: an zn vztro study using cultured human hepatocytes. Xenobzotzca 22,471-478 Birge, R. B., Bartolone, J B., Hart, S. G. E., Nishaman, E. V., Tyson, C. A., Khairallah, E A , and Cohen, S D (1990) Acetominophen hepatotoxicity * corre-

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31 Isolation of Rat Hepatocytes Karen De Smet, Sonja Beken, Tamara Vanhaecke, Marleen Pauwels, Antoine Vercruysse, and Vera Rogiers 1. Introduction Because the liver is the main organ involved in the metabolism and toxicity of xenobiotics, isolated hepatocytes from various species, mcludmg humans, and their cultures, constitute attractive in vitro models for pharmaco-toxtcological studies (see Chapter 30). Initial attempts to isolate adult parenchymal cells by mechanical methods were not very successful, and were concerned more with cell yield than with cell Integrity, viability, or functionality (I). Major progress was made by the mtroductlon of enzymes as dlssoclatmg agents, e.g. collagenase and hyaluronidase. In 1969, Berry and Friend (2) established an in situ collagenase perfusion technique for rat liver to obtain a high yield of viable hepatocytes. An important modification, introduced by Seglen (3,4) was the supplementation of the collagenase solution with Ca2+m a second perfusion step. This two-step collagenase procedure is still used today with only slight modifications. Usually, a perfusion with Ca2+-free buffer is carried out first to attack the calcium-containing bridges between the cells, followed by a second perfusron with Ca2+-containing buffer with collagenase. Ca2+is necessaryto activate the collagenase, which digests the collagen matrix. This technique can be applied to the liver of various species,including humans, and usually a cell population of hepatocytes with less than 5% nonparenchyma1cells is obtained. 2. Materials 1 60 mg/mL Sodium pentobarbital. Stablefor months in refrigerated bottle 2 5000 IU/mL Sodturnheparin Stablefor months in refrigerated bottle. From Methods m Molecular Bology, Vol 107 Cytochrome P450 Protocols Edlted by I R PhWps and E A Shephard 0 Humana Press Inc , Totowa, NJ

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8

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De Smet et al. Collagenase, type I (Clostridiopeptidase A). Stable for months tf refrigerated dry, Warm up to room temperature before opening the bottle Trypan blue: 0.4% (w/v) in 0 85% (w/v) NaCl. Alcohol, 70% (v/v) Ca2+-free Krebs-Henseleit buffer (KHB), pH 7 4 2 10 mL of 0.154 M NaHCO,, saturated for 60 mm with carbogen (95% 0, and 5% CO,), 1000 mL of 0 154 A4 NaCl,40mLofO.l54MKCl, 10mLofO 154MKH2P0,,and10mLof0.154M MgSO,, all in double-distilled water. The buffer is sterthzed by passmg through a 0.22~pm filter and can be stored at 4°C for 6 mo Ca2+-containing KHB, pH 7.4, 1270 mL of Ca2+-free Krebs-Henseleit buffer and 30 mL of 0.110 A4 CaCl, m double-distdled water The buffer is sterrhzed by passing through a 0 22-pm filter and can be stored at 4°C for 6 mo HEPES (N-2-hydroxyethylpiperazme-N’-2-ethanolsulfomc acid) buffer, pH 7 65. 0.80% (w/v) NaCl, 0.01% (w/v) Na,HPO, 12H20, 0.02% KCl, 0 038% HEPES in Milhpore-quality water The buffer is sterdlzed by passing through a 0 22+un filter and can be stored at 4°C for 6 mo. Leibovitz medmm* 1 47% (w/v) Letbovitz and 0 20% (w/v) bovine serum albumm (BSA) m Milhpore-quality water. The medium is sterilized by passing through a 0.22~pm filter and can be stored at 4°C for 6 mo. Standard medium : Dulbecco’s Modified Eagle’s Medium (DMEM) containmg 4 5 mg/mL glucose and 0 584 mg/mL L-glutamine . This sterile medium can be stored for 6 mo at 4°C. Wash medium: Standard medmm supplemented with 7 3 IU/mL benzyl pemcillm, 50 pg/mL streptomycin sulfate, 50 pg/mL kanamycm monosulfate, and 10 pg/mL sodium ampicillm This medium is sterile and can be stored for 7 d at 4°C Phosphate-buffered saline (PBS), pH 7 65.0.28% (w/v) NaCl, 0 02% (w/v) KCl, 0 3 1% (w/v) Na2HP04 * 12H20, and 0.02% (w/v) KH2P04 in Mtlhpore-quality water The buffer is sterihzed by passing through a 0 22-pm filter and can be stored at 4°C for 9 mo Equipment mcludes a 63-pm perlon filter, a 0.22~pm filter, sterile volumetric pipets, a glass cannula, a bile cannula (inner diam 0.28 mm, outer diam 0.061 mm), a hemocytometer, two 50-mL sterile centrifuge tubes, a perfusion apparatus (Fig. l), a laminar flow cabmet, an inverse-phase light microscope, a perfusion pump, a centrifuge, and a thermostated bath.

3. Methods 3.1. Sterilization

of the Perfusion

Apparatus

1. Sterilize the perfusion apparatus with 400 mL of alcohol (70%) and circulate for 15 mm 2 Rinse the apparatus three times with sterile Millipore-quality water (10 min/ circulation).

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/

Pump

i:

-

Hz0 37°C

Hz0 37°C stainless steel weight WsW

device

Fig 1 Schematic representation of the perfusion apparatus

3.2. Isolation

of the Rat Liver (see Note 1)

1 Hepatocytes are isolated from male Sprague Dawley rats (f200 g), that have access to water and food ad llbrtum (see Note 2A). 2. Anesthetize the rat by injecting sodmm pentobarbrtal solution (0.1 mL/lOO g body weight) mtraperitoneally 4. Shave the rat’s abdomen, disinfect with alcohol, and unmobrhze the rat on the surgery table. 5. Open the abdomen with a U-shaped mcision. 6. Ligate the bile duct twice, close the lower ligature, insert the bile cannula, and fix it by closing the upper ltgature. 7. Ligate the vena cava once and the vena porta twice 8. Inject 1 mL of freshly prepared heparin solution (0.1 mL heparin [5OOOIU/mL]/2.4 mL physrological saline) m the vena saphena medialis Final concentration of heparin is 2OOIU/mL 9 Close the lower ligature of the vena porta, msert the glass cannula m the vena porta, and close the upper ligature 10. Close the ligature of the vena cava. 11. Remove the liver from the rat and rinse it with Ca2+-free IU-IB

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3.3. Two-Step

Perfusion

of the Rat Liver (see Notes 2b, 3, and 4)

1 Circulate 250 mL Ca2+-free KHB m the perfusion apparatus, thermostated at 42°C (to obtain 37°C m the liver). Gas it with 5% CO2 and 95% 0, 2. Transfer the excised rat liver to the perfusion plate and connect the glass cannula, already mserted in the vena porta (perfusion rate: 40-50 mL/min, temperature* 37°C). The connectmg tubes have to be completely free of air bubbles 3. First perfusion step Remove the blood from the liver with 100 mL Ca2+-free KHB and circulate the remammg 150 mL Ca2+-free KHB for 15 mm. 4. Second perfusion step: Dtssolve 18,400 digestion umts of collagenase, type I, m 10 mL of gassed Ca2+-containing KHB Sterihze the solution by passing through a 0 22-pm filter and add tt to the 150 mL of Ca2+-free KHB m the perfusion apparatus. The final concentration of collagenase is 115 collagenase digestion units/ml Circulate for 25 mm 5 Disconnect the perfused rat liver from the perfnston apparatus and transfer It to a Petri dish tilled with Letbovitz medium.

3.4. Purification 1. 2. 3 4. 5 6. 7. 8. 9. 10.

of Rat Hepatocytes

Open the Glisson’s capsule and suspend the cells m Leibovitz medmm. Filter the resulting cell suspenston through a sterile perlon filter (63 pm). Allow the cells to sediment for 15 mm Remove the supernatant, wash the cells with 40 mL of HEPES buffer and drspense the cell suspension mto two 50 mL sterile centrifuge tubes Centrifuge the cell suspension for 1 min at 63g Remove the supernatant and wash the cell pellet with 20 mL of HEPES buffer. Centrifuge the cell suspension for 1 mm at 63g. Remove the supernatant and wash the cell pellet with wash medium (20 mL per tube). Centrifuge the cell suspension for 1 min at 63g. Remove the supematant and resuspend the cell pellet m 100 mL of wash medium

3.5. Determination

of the Cell Viability (see Note 5)

1. Mtx 600 pL of Ca2+-containing KHB with 200 pL of trypan blue solution and 400 pL of cell suspension. 2. Count the viable (white) and dead (blue) cells in a hemocytometer under a light microscope (10 x 20 Usually 4 fields are counted per chamber 3 Calculate the mean number of viable and dead cells viability (in %) = (# viable cells x lOO)/[(# viable cells) + (# dead cells)] cell concentration (in cells/ml) = [(# viable cells) x 3 x lOOO]/O 1 3 = dilution factor of the cell suspension 1000 = conversion factor from mm3 to mL 0.1 = volume contained by 1 field of the hemocytometer 4. Only cell suspenstons with a viability of 80% or more are used for in vrtro purposes

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4. Notes 1. An important source of variation between the results obtained from different laboratories, when using isolated rat hepatocytes and then cultures for pharmacotoxicological purposes, IS the way in which the cell isolation is performed. A validated standardized procedure does not yet exist However, some recommendations for the lsolatlon of rat hepatocytes have been made m the report of ECVAM Workshop 1 (5). 2. From our own experience, it appears that the followmg variables may be of importance. Once chosen, they should be rigorously kept constant throughout the experiments a. With respect to the cell source: - strain, age, gender and health condltlon of the rats. - food composition, feeding schedule, type of bedding, light/dark cycle, and environmental stress factors. b. With respect to the two-step perfusion technique. - heparin admmlstration before cannulation - presence of Ca*+-chelating agents m the first perfusate, If they are used, their concentration and type. - Ca2+-concentration m the second perfusate - length of the perfusion times (constant time length or visual stop) - type, quality, activity, and storage conditions of the collagenase used. Collagenase shows a great deal of variation in enzyme activity and specificity when purchased from different vendors or even among different batches from the same vendor Therefore several different batches of collagenase should be tested to determine which one best preserves the function to be assessed m the isolated hepatocytes - presence of other dissociating enzymes, e g , hyaluronidase, trypsm - composltlon of perfusion buffers, their pH, level of oxygenation, temperature, and osmolarity. - use of either carbogen gassing (95% O2 and 5% C02) m the case of HC03 buffers or HEPES to maintain the buffer pH at 7 4-7.5. The pH can be finally adjusted with 0 1 N NaOH. - type of tubmg, cleaning and sterilizing methods for the perfusion apparatus and the tubing - temperature, pH, and composition of the dispersing medium for the freshly isolated hepatocytes (e.g., presence, concentration and type of fetal calf serum [FCS] and BSA). - washing and purification procedures of the cell suspension (length of centrifugation time, g-value, number of washings and composltlon, pH, osmolarity, and volume of washing buffers) 3. During liver perfusion, some general problems may occur such as poor perfusion of the liver, mcomplete digestion of the liver, or poor recovery of the cells Causes and solutions are taken up m Table 1 (6)

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Table 1 Troubleshooting Problem Poor perfusion

of the Two-Step

Collagenase

Cause Air bubbles m liver

Particulates in liver Liver blood clots

Incomplete digestion of the liver

Poor recovery of cells

Poor perfusion Insufficient enzyme concentration No Ca*+ m buffer Large portions of nonperfused liver Incomplete digestion Low number of viable cells

Cell clumping Gross microbial contamination

Perfusion

of Liver

Solution Avoid bubbles during portal vem cannulation and transfer to the perfusion apparatus A bubble trap is necessary Filter buffers and enzymes Use heparm, flush liver rapidly and completely to remove endogenous blood. See poor perfusion Increase amount or specific activity of collagenase Add Ca*+ to 5 mmol/L See poor perfusion See incomplete digestion of the liver Try different types or batches of collagenase, decrease amount of collagenase; during perfusion, keep liver moist Try addition of DNase I Clean perfusion apparatus, prepare fresh sterile buffers

4 Liver perfusion can be carried out both wrth a nonrecirculatmg or a recirculatmg apparatus Although the former technique 1snot influenced by the release of metabolic products from the liver (which continuously change the composmon of the perfksate), this system is mconvement and expensive because large amounts of digesting enzyme are used. Therefore, a recirculating system (see Fig. 1) IS most commonly applied. 5 It is highly recommended that the suspensions of freshly isolated rat hepatocytes should be used only if a mmimum viability of at least 80% (as measured by excluston of trypan blue) and a mmimum attachment yield of 80% are obtained immedtately followmg isolation (5).

References 1. Anderson, N. G. (1953) The mass isolation of whole cells from rat liver. Sczence 117,627-628

2 Berry, M. N. and Friend, D S (1969) High yield preparation of isolated rat liver parenchymal cells. J Cell Blol 43, 506-520.

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3. Seglen, P. 0. (1972) Preparation of rat liver cells. I Effect of Ca2’ on enzymatic dispersion of isolated, perfused liver. Exp Cell Res 74,45&454 4. Seglen, P. 0 (1976) Preparation of isolated rat liver cells. Methods Cell Bzol 13, 29-83 5 Blaauboer, B J., Boobis, A. R., Castell, J V , Coecke, S , Groothms, G M M., Guillouzo, A., Hall, T J., Hawksworth, G. M., Lorenzon, G., Miltenburger, H. G , Rogiers, V., Skett, P , Villa, P., and Wiebel, F. J. (1994) The practical applicability of hepatocyte cultures m routme testing. A TU 22, 23 l-24 1 6. Alpmt, G., Philips, J. 0 , Vroman, B., and La Russo, N F (1994) Recent advances m the isolation of liver cells Hepatology 20,494-5 14.

32 Collagen-Gel

Cultures of Rat Hepatocytes:

Collagen-Gel Sandwich and Immobilization

Cultures

Sonja Beken, Tamara Vanhaecke, Karen De Smet, Marleen Pauwels, Antoine Vercruysse, and Vera Rogiers 1. Introduction Collagen-gel cultures are relatively new m vitro models for long-term culture of functional hepatocytes. In these models, the cells are cultured between two layers of the extracellular matrix protein, hydrated collagen type I. Numerous studies have demonstrated the importance of the presence of extracellular matrix during the culture of liver cells. Indeed, extracellular matrix plays a role m the modulation of several aspects of cellular function, includmg cell adhesion, migration, spreading, differentiation, growth regulation, and liver-specific gene expression and its regulation. Thus it is believed that the culture of hepatocytes in these new models better represents the situation in viva . It has been clearly shown that the cellular polarity of the hepatocytes is restored m these cultures and a nearly physiological secretion of albumin, transferrin, fibrinogen, urea, and bile salts, maintained for more than 6 wk, has been reported (1-3). However, with respect to the drug-metabolizing capacity of the cultured cells, nearly no data are available (see Chapter 30, Subheading 2.2.). Nevertheless great confidence exists that these culture systemscould represent a major breakthrough in the development of a long-term model for functional hepatocytes. Such an m vitro model is greatly needed for the in vitro study of the expression and regulation of the various key phase I and phase II drugmetabolizing pathways. A collagen-gel sandwich culture model and an easy-to-apply modtfication, the immobilization culture, are described in detail in this Chapter. The former technique is based on the work of Dunn et al. (3), the latter on that of From Methods m Molecular Biology, Vol 107 Cytochrome P450 Protocols Edlted by I R PhMps and E A Shephard 0 Humana Press Inc , Totowa, NJ

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304 Koebe et al. (4). The collagen-rsolation of Elsdale and Bard (5).

2. Materials 2.1. Preparation 1, 2. 3 4. 5 6. 7. 8 9.

method 1sa modrfication

of Rat-Tail Collagen

of the method

(Type I)

1% (w/v) NaCl m Millipore-quality water 3% (v/v) CHsCOOH in Millipore-quality water. 30% (w/v) NaCl in Milhpore-quahty water. 5% (w/v) NaC1/0.6% (v/v) CHsCOOH m Mllhpore-quality water 0.6% (v/v) CHsCOOH m Mlllipore-quality water 1 mA4 HCl in Mtllipore-quality water Chloroform Liquid antiseptic soap. 4% (w/v) chlorhexidm digluconate. Equipment includes 2 forceps, 4 Petri dishes (diam 15 cm), 4 (5 x 5 cm) sterile compresses, a 500~mL beaker with seal, a 500-mL stertle glass bottle, 6 centrifuge bottles of 250 mL, dialysis membranes (MWCO: 12-14,000, diam : 29 mm), and a cooled centrifuge

2.2. Collagen-Gel

Sandwich

Culture

1. Standard medmm: Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 mg/mL glucose and 0.584 mg/mL L-glutamme. This medium is sterile and can be stored for 6 mo at 4°C. 2. TO medium Standard medium supplemented with 10% fetal bovine serum (FBS), 7.3 IU/mL benzyl pernctllm, 50 pg/mL streptomycm sulfate, 50 pg/mL kanamytin monosulfate, 10 pg/mL sodmm ampicillin, 0 5 U/mL insulin (from bovine pancreas, cell-culture tested), and 0.007 ug/mL glucagon (from porcme pancreas, cell-culture tested). This medmm is stertle and can be stored for 7 d at 4°C 3 T4 medmm. Standard medium supplemented with 0 02 pg/mL eptdermal growth factor, 7 5 pg/mL hydrocortisone hemtsuccinate, and anttbtottcs, insulin and glucagon as descrtbed for the TO medtum This medium is sterile and can be stored for 7 d at 4°C. 4 Wash medmm. Standard medium supplemented wtth antibiotics as described for the TO medmm. This medium is sterile and can be stored for 7 d at 4°C. 5. Thtoglycollate medium* 5 g/L yeast extract, 15 g/L tryptone, 5.5 g/L glucose, 0.5 g/L sodium thloglycollate, 2.5 g/L NaCl, 0 5 g/L L-cystine, 0.001 g/L resazurme and 0.5 g/L agar 6. Rat-tail collagen-gel (type I) (1.1 mg/mL). Prepare the required concentration from the collagen-gel (type I) stock solution (see Subheading 3.1.) by dtlutmg with a sterile 1 rnA4 HCl solutton. Prepare freshly before use. 7. 10 times concentrated DMEM contammg 3.7% (w/v) NaHCO,. This medium 1s sterilized by passing through a 0.22~pm filter and can be stored for 3 mo at 4°C. 8. Equipment includes stertle plastic Petri dishes of 6, 10, and 15 cm diam., 15 mL sterile centrifuge tubes, sterile Pasteur plpets and volumetric ptpets, a lammar-

Collagen-Gel

Cultures and Hepatocytes

flow cabinet, an incubator (water Jacketed, 37°C humtdified containing 5% CO,), and a thermostated bath (37°C).

2.3. Collagen-Gel

immobilization

305 atmosphere of an

Culture

1. Standard medium DMEM contammg 4.5 mg/mL glucose and 0 584 mg/mL L-glutamme. This medium is sterile and can be stored for 6 mo at 4“C. 2. TO medium. Standard medium supplemented with 5% FBS and antibiotics, insulin, and glucagon as described for the TO medium m Subheading 2.2. This medium is sterile and can be stored for 7 d at 4°C. 3. T30’ medium: Standard medium supplemented with 2 5% FBS and antibiotics, msulm and glucagon as described for the TO medium in Subheading 2.2. This medium is sterile and can be stored for 7 d at 4’C. 4. T4 medium: as described m Subheading 2.2. 5. Thioglycollate medium as described m Subheading 2.2. 6. Rat-tail collagen-gel (type I) (1.63 mg/mL). Prepare the required concentration as described m Subheading 2.2. 7. 10 times concentrated DMEM contammg 3.7% (w/v) sodmm bicarbonate This medmm 1s stertltzed by passmg through a 0.22~pm filter and can be stored for 3 mo at 4°C 8. Equipment asdescribed in Subheading 2.2. plus a cryostat and a cooling plate (15°C).

3. Methods 3.1. Preparation of Rat-Tail Collagen (Type I) 3.1-I. Collection of the Collagen Fibers 1 Collect the tails from male Sprague-Dawley rats (usually after isolation of the liver) and store them at -30°C. 2. Scrub the rat tails with the liquid antiseptic soap. 3. Dissect the collagen fibers from a number of rat tails (usually nine tails are used) 4 Collect these in 1% (w/v) NaCl, wash them once in 1% (w/v) NaCl, and twice m double-distilled water. 5. Stir the collagen fibers m 3% (v/v) CH$OOH (approx 200 mL/tail) overnight at 4°C.

3.1.2. Purification 1. Carefully filter the collagen through four layers of 5 x 5 cm sterile compresses. 2. Centrifuge the collagen at 11,OOOgfor 2 h at 4°C. 3 Measure the volume of the supematant, and add dropwise to the supematant, while stirring at 4°C 0 2 volume of 30% (w/v) NaCl. 4. Leave the collagen solution, without stirring, for at least 1 h at 4°C (or overnight) 5. Centrifuge the collagen at 175Og for 30 min at 4°C. 6. Wash the pellet twice with 5% (w/v) NaCl/O 6% (v/v) CH,COOH (200 mL/centrifuge bottle).

Beken et al.

306 7 After each wash, centrifuge for 30 min at 1750 g at 4’C 8 Dtssolve the pellet m 0.6% (v/v) CHsCOOH (approx 50 n&/tall). 9 Stir the collagen solutton overnight at 4°C.

3.1.3. Dialysis 1. Transfer the collagen solutton to dtalysrs membranes 2. Dialyze five times for at least 4 h against 10 volumes of 1 mM HCl at 4°C 3 Centrifuge the collagen solutton at 11,OOOg for 2 h at 4°C

3.1.4. Sterilization 1. Measure the volume of the supematant and add 0 003 volumes of chloroform. 2. Transfer the collagen solution to a 500-mL beaker with seal 3, Stir for 48 h at 4°C.

3.7.5. Lyophilizatlon 1 Transfer the collagen solutton to a sterile 500~mL bottle and store at 4°C. 2. Lyophlhze two 5-mL samples of the obtained collagen solutton in order to determine the concentratton Note Usually a collagen concentratton of about 3 mg/mL 1s obtained

3.2. Collagen-Gel

Sandwich

Culture (see Notes 1-5)

1 Isolate rat hepatocytes as described m Chapter 3 1 2 The sterthty of the medta IS checked by adding 1 mL of the prepared medium to 25 mL of autoclaved thioglycollate medmm and mcubatmg thts mixture at 37°C. After 2 d the eventual contammatton of the thtoglycollate medmm is mvesttgated 3 Precoatmg of the culture dishes. a. Place the rat-tall collagen-gel (type I) (1.1 mg/mL), the 10 times concentrated DMEM, and a 15-mL sterile centrifuge tube on ice in the laminar-flow cabtnet b. Mix, m a 15-mL sterile centrifuge tube on ice, 1 part of 10 times concentrated DMEM with 10 parts of collagen-gel (1.1 mg/mL) c. Disperse 1.00, 2 75, or 6.25 mL of thts mixture over 6, 10 or 15 cm dram. plastic Petri dishes, respectively The mixture has to be completely dispersed over the surface of the Petri dishes. d. Transfer the precoated Petri dishes to the incubator Precoated Petri dishes can be stored m the incubator for up to 5 d. 4. Seeding of the rat hepatocytes: a. Warm the TO and T4 media for about 30 min in a thermostated bath (37’C) before use b Seed the rat hepatocytes at a density of 0.4 x 1O6cells/ml of TO medium in 4, 11, or 25 mL in 6, 10 or 15 cm diam. Petri dishes, respectively c. 4 h after seeding, renew the medium with 4, 11, or 25 mL T4 medmm in 6, 10, or 15 cm diam. Petri dishes, respectively.

Collagen-Gel

Cultures and Hepatocytes

307

5. Adding the second layer of rat-tall collagen gel (type I), completmg the sandwich configuration, 24 h after cell seeding: a. Warm the wash and T4 media for about 30 mm m a thermostated bath (37°C) before use. b. Place the rat-tail collagen-gel (type I) (1.1 mg/mL), the 10 times concentrated DMEM, and a 15-mL sterile centrifuge tube on ice m the laminar-flow cabinet. c. Wash the cells present m the 6, 10, or 15 cm dram. Petri dishes twice with 2, 5, or 10 mL wash medium, respectively. Do this very carefully d. Mix, m a 15-mL centrifuge tube on ice, 1 part of 10 times concentrated DMEM with 10 parts of collagen-gel (1.1 mg/mL). e. Disperse 1.00, 2.75 or 6.25 mL of this mixture into 6, 10, or 15 cm diam plastic Petri dishes, respectively. The mixture has to be completely dispersed over the surface of the Petri dishes. Place the completed collagen-gel sandwich cultures m the incubator for about f 45 mm (see Note 1). g. Add 4, 11, or 25 mL of T4 medium to each of the 6, 10, or 15 cm diam Petri dishes, respectively. 6 Renew the medium every day thereafter with the same volume of T4 medium

3.3. Collagen-Gel 1 2. 3. 4 5. 6.

7 8 9 10. 11.

lmmobilizafion

Culture (see Notes I-5)

Isolate rat hepatocytes as described in Chapter 3 1. The sterility of the media is checked as described in Subheading 3.2. Cool the Petri dishes required on a cooling plate at 15°C Place a suspension of 1.6 x lo6 cells/ml, the rat-tail collagen-gel (type I) (1.63 mg/mL), the 10 times concentrated DMEM, and a 15-mL sterile centrifuge tube on ice in the lammar-flow cabinet Mix, in a 15-mL sterile centrifuge tube on ice, 1 part of 10 times concentrated DMEM with 10 parts of collagen-gel (1 63 mg/mL) Mix, on me, 1.OO,2.75, or 6.25 mL of thus mixture with 1.OO,2.75, or 6.25 mL of cell suspension (1.6 x IO6 cells/ml), respectively, and disperse into 6, 10, or 15 cm diam. cooled plastic Petri dishes, respectively. Leave the Petri dishes on the coolmg plate (15’C) for 15 mm. Place the collagen-gel rmmobrlrzation cultures in the incubator for about 30 mm (see Note 1). Add 4, 11, or 25 mL of T30’ medium to 6, 10, or 15 cm diam. Petri dishes contaming the collagen-gel immobilization cultures, respectively 4 h after seeding renew the medium with 4, 11, or 25 mL of T4 medium for the 6, 10, or 15 cm diam. Petri dishes, respectively. Renew the medmm every day thereafter with the same volume of T4 medium

4. Notes 1. The collagen type I gel used for immobiltzation and sandwich cultures is a hydrogel. The process of gel formation is a crucial step m order to obtain a solid gel. Therefore, care must be taken not to disturb this process while transferring

Beken et al.

2

3.

4

5.

freshly coated sandwich cultures or freshly prepared immobdlzation cultures to the incubator. Moreover, further manipulation of these cultures has to be done with care, because any mechanical stress will cause the gel to detach. Because only few publications have appeared regarding the drug metabohsm capacity of collagen-gel cultures of hepatocytes (see Chapter 30, Subheading 2.2.2.), the inter- and mtra-laboratory reproducibihties of the results obtained m these culture models are not yet known The variables mentioned in Chapter 33, Subheading 4., Note 2. probably also hold for collagen-gel cultures of hepatocytes. To date, however, supportmg data are not available. From our own experience (1,2) some practical constraints can be identified a. To obtain access to the hepatocytes for metabohc or toxicrty studies, enzymatic digestion of the collagen-gel is often necessary b The normahzatton of biochemtcal and metabolic parameters may be problematic: The expression of enzymatic activities vs cytosohc protein concentration is not possible without enzymatic digestion of the collagen present The collagen entraps culture-medium proteins and these cannot be completely washed out In contrast, the expression of results vs microsomal protein concentration poses no problems. The expression of enzymatic activtties vs the DNA content of the cells is not valid. The DNA remains entrapped m the collagen-gel and dying or dead cells have the same DNA content as viable cells c During the mitiation of cultures (in particular for immobihzation cultures) and as a function of culture time (for both culture models), dying or dead cells can become entrapped m the collagen-gel. Then protein and DNA content can not only cause erroneous results, but also the leaked enzymes can damage neighboring cells. d Coculture of hepatocytes with rat-liver eptthelial cells of primitive biliary origin (as described in Chapter 33, Subheadings 2.2. and 3.2.) m both collagengel models IS not successful, because confluent layers cannot be obtained and the collagen environment seems to be deleterious for the rat-liver epithelial cells. The exposure of hepatocytes to drugs or substrates IS limited by the rate of penetration of these substances through the collagen-gel On this particular topic, vntually no data are available m the literature (4).

References 1. Beken, S., Pauwels, M., Pahermk, S , Koebe, H.-G., Vercruysse, A. and Rogters, V. (1997) Collagen gel sandwich and immobihzation cultures of rat hepatocytes. problems encountered m expressing glutathione S-transferase activities Toxzcol In Vitro l&741-752. 2 Beken, S , Pahermk, S , Koebe, H -G., Vercruysse, A., and Rogiers, V (1997) Cell morphology, albumin secretton, and glutathione S-transferase expression m collagen gel sandwich and immobilization cultures of rat hepatocytes Toxzcol In Vztro 11,409-416.

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309

3. Dunn, J C. Y , Tompkms, R. G., and Yarmush, M.L. (1991) Long-term zn vztro functron of adult hepatocytes in a collagen sandwich contiguratton. Brotechnol. Prog 7,237245.

4 Koebe, H.-G , Pahermk, S , Eyer, P., and Schtldberg, F.-W (1994) Collagen gel mnnobihzation a useful cell culture technique for long-term metabolic studies m human hepatocytes Xenobzotzca 24,95-107. 5. Eisdale, T. and Bard, J (1972) Collagen substrata for studies on cell behavior. J Cell Blol. 54,62&-637.

33 Rat Hepatocyte

Cultures

Conventional Monolayer Cultures and Cocultures with Rat liver Epithelial Cells Tamara Vanhaecke, Karen De Smet, Sonja Beken, Marleen Pauwels, Antoine Vercruysse, and Vera Rogiers 1. Introduction To survive for more than a few hours, hepatocytes must attach to a support When cultured under conventional conditions they form a monolayer that usually survrves for about 1 week. However, the hepatocytes undergo phenotypic changes, including dedifferentiation and the selective loss of some key pathways of xenobiotic metabolism (see Chapter 30). The use of more sophtsttcated culture techniques, such as coculture of rat hepatocytes with rat liver epithelial cells (RLEC) of primitive biliary origin, that more closely mimic the situation in viva results in a longer survival time and better maintenance of drug-metabolizing capacity (see Chapter 30). Both culture techniques are described here in detail. They are based on the work of Guguen-Guillouzo (I), Guillouzo (2), and Wtlhams et al. (5). 2. Materials 2.1. Conventional Monolayer Cultures 1 Standardmedium, pH 5 2: 75% Minimum EssentialMedium, 25% Medium 199, 1mg/mL bovine serumalbnnun(BSA) (fraction V, mmlmum 96%) and 10 clg/mL Insulin (from bovine pancreas,prepared from starting material with activity 27.8 IU/mg). This medium 1ssterilizedby passingthrough a 0.22~pmfilter and can be stored for 3 mo at -20°C 2. TO-medium:Standardmedium supplementedwith 10%(v/v) fetal bovine serum; 2.2 mg/mL NaHC03, 7 3 IU/mL benzyl penicillin, 50 pg/mL streptomycmsulFrom Methods m Molecular Broiogy, Voi 107 Cytochrome P450 Protocols Edlted by I Pi Phllltps and E A. Shephard 0 Humana Press Inc , Totowa, NJ

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fate, 50 pg/mL kanamycm monosulfate, and 10 pg/mL sodmm amprcrllm. Thus medium is sterilely prepared and can be stored for 7 d at 4’C 3 Thioglycollate medium: 5 g/L yeast extract, 15 g/L tryptone, 5 5 g/L glucose, 0 5 g/L sodmm thtoglycollate, 2 5 g/L NaCl, 0.5 g/L L-cystme, 0.001 g/L resazurme, and 0.5 g/L agar 4. Equipment includes sterile plastrc Petri dishes of 15, 10 or 6 cm diam, sterile volumetrrc pipets, sterile Pasteur prpets, a laminar-arrflow cabmet, an incubator (37’C, water jacketed, humrdified atmosphere of 95% air and 5% CO,), a thermostated bath (37’C), and a phase-contrast inverse light mrcroscope

2.2. Cocultures with RLEC 2.2.1. Isolation of RLEC 1 Equipment includes two sterile 100~mL bottles, two sterile nylon filters (80 um) in funnels, a sterile glass Petri dish of 10 cm dram., a sterrle magnet, sterile plastic 50-mL centrifuge tubes, sterile plastic Petri dishes of 4 cm dram., a fine platrnum-needle, sterile volumetrrc prpets, sterile Pasteur ptpets, laminar-airflow cabmet, an incubator (37’C, water jacketed, humidified atmosphere of 95% au and 5% CO,), a thermostated bath (37”C), a centrifuge, and a phase-contrast inverse light microscope 2. Stenle phosphate-buffered saline (PBS), pH 7 65, (1 L): 0.28% (w/v) NaCl, 0 3 1% (w/v) Na2HP04 12H20, 0.02% (w/v) KCl, 0.02% (w/v) KH2P04 m Mtlliporequality water sterrhzed by passing through a 0.22~pm filter 3 Trypsm solution: 0 25% trypsm (from porcine pancreas, 12,700 U/mg protem) in PBS, sterilized by passing through a 0.22~pm filter (approx 67 mL/g liver). 4 Wrlham’s medmm E, can be stored for 6 mo at 4°C. 5. 200 mL William’s medium E for RLEC (WM) supplemented with antrbrotrcs (as described for TO-medium m Subheading 2.1.) and 0.1 mg/mL L-glutamme. This medium is sterilely prepared and can be stored for 7 d at 4°C. 6 500 mL WM supplemented with 10% (v/v) (FBS) (WMF) This medium IS sterilely prepared and can be stored for 7 d at 4°C.

2.2.2. Culture of RLEC 1. Equipment includes 75 cm2 sterile flasks treated for tissue culture, sterile volumetric ptpets, sterile Pasteur prpets, an incubator (37°C water jacketed, humtdrfled atmosphere of 95% air and 5% C02), a thermostated bath (37”C), lammar-airflow cabmet, and a phase-contrast inverse light microscope 2 Sterile PBS (see Subheading 2.2.1., item 2). 3 Sterile trypsin-ethylenedramme tetra-acetic acid (EDTA). 4 WMF (see Subheading 2.2.1., item 6).

2.2.3. Cryopreserva tion of RL EC 1. Equipment includes liquid nitrogen, sterile 2-mL Nunc-tubes, sterile plastrc 15-mL centrifuge tubes, sterile volumetric ptpets, sterile Pasteur prpets, laminar-airflow cabmet, an incubator (37’C, water jacketed, humidified atmosphere of 95% air

Rat Hepa tocyte Cultures

2. 3. 4 5.

313

and 5% CO,), a thermostated bath inverse light microscope. Sterile PBS (see Subheading 2.2.1., Sterile trypsm-EDTA. WMF (see Subheading 2.24 item WMF supplemented with 20% (v/v)

(37”C), a centrifuge, and a phase-contrast item 2). 6) dimethyl sulfoxide (DMSO).

2.2.4. Coculture 1. The same materials as for the conventional monolayer cultures (see Subheading 2.1.) 2 Sufficient confluent layers of RLEC (from not later than the 30th passage) for the coculture experiment planned.

3. Methods 3.1. Conventional Monolayer (see Notes 1 and 2)

Cultures of Rat Hepatocytes

1 Prepare the media, check their sterthty by adding 1 mL of the medium to 25 mL autoclaved throglycollate medium and incubating this mixture at 37°C. After 2 d the thioglycollate medium IS investigated for contammation Discard if cloudy. 2 Before use, warm the media for about 30 mm in a thermostated bath (37°C) 3 Isolate rat hepatocytes as described in Chapter 3 1 4. Seed the hepatocytes at a density of 1.6 x lo6 cells/283 cm2 Petrt dish, 4.4 x lo61 78.5 cm2 Petri dish or 1 x lo7 cells/177 cm2 Petri dish m 4, 1 I, and 25 mL TO-medium, respectively. 5. Allow cell attachment by incubation of the hepatocyte cultures at 37°C m a humidtfled atmosphere of 95% air and 5% CO, 6 4 h after cell seeding, renew the medium with 4 mL (28 3 cm2 Petri dishes), 11 mL (78.5 cm2 Petri dishes) or 25 mL (177 cm2 Petri dishes) TO-medium (37°C) supplemented with 7 x l&* M hydrocortisone hemisuccinate. 7. Renew the medmm every day thereafter with the same amounts of serum-free TO-medium (37°C) supplemented with 7 x 10” Mhydrocortisone hemtsuccmate and 0.25 pg/mL amphotericm B.

3.2. Cocultures of Rat Hepatocytes 3.2.1. Isolation of RLEC 1. 2. 3. 4. 5

with RLEC

Weigh a sterile, 100-mL bottle with lid containing 50 mL sterile PBS Decapitate 10 Sprague-Dawley rats of 8-10 d old Open the abdominal walls of the animals and remove the hver Collect the livers m the 50 mL PBS Reweigh the bottle, the difference between this weight and that determined m item 1 IS the mass of collected liver. 6 Remove the PBS and transfer the livers to a sterile glass Petri dish of 10 cm diam. 7. Add 8 mL sterile PBS and cut the livers into small pieces.

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8 Transfer these to the 0 25% (w/v) trypsin solutton (20 mL/g collected hver) in a lOO-mL sterile bottle, add a sterile magnet, seal the bottle and let stir for 10 min on a magnetic stirrer 9. Leave for a few min 10 Remove the trypsin solution carefully with a IO-mL sterile plpet. 11. Add 20 mL trypsm solutton/g collected liver and stir for 15 mm 12. Leave for a few mm and filter the supernatant through an 80-pm nylon filter mto a 50-mL plastic centrifuge tube (save the material that does not pass through the filter). 13 Centrifuge the filtrate for 5 min at 62Og. 14. Remove the supernatant, leaving approx 10 mL above the pellet. 15. Dilute the pellet to 35 mL with WM and resuspend 16. Centrifuge for 5 mm at 620g 17. Remove the supernatant completely and resuspend the pellet m 1.66 mL WM per g collected liver (=A) 18. To the liver material that did not pass through the filter (step 12), add 60 mL trypsin solution and stir for 15 mm 19. Repeat steps 12 to 17 (=B). 20 Combme A and B together. If an aggregate forms, refilter 2 1 Prepare cultures of four different dilutions. For example, for 3 g collected liver m a total volume of 10 mL WM (= A+B), place 0 25,0.50,0.75 and 1 00 mL A+B mixture mto four different 4 cm dram. Petri dishes Dilute each to a total volume of 4 00 mL with WMF 22 Incubate the cultures for 20 mm (= first series) 23. Transfer the medium of the first series of cultures into new Petri dishes, make up to 4 mL with WMF, and incubate for 20 mm (= second series). 24 Renew the medium of the first series with 4 mL WMF and incubate these for 24 h 25. After 20 mm, transfer the medium of the second series into new Petri dishes, dilute to 4 mL with WMF and incubate for 20 mm (= thud series) 26. Renew the medium of the second senes with 4 mL WMF and incubate these for 24 h 27 After 2 h, transfer the medium of the thnd series mto new Petri dishes, make up to 4 mL with WMF, and incubate for 24 h (= fourth series) 28. Renew the medium of the thud series with 4 mL WMF and place these m the incubator for 24 h. 29. After 24 h, renew the medium of all Petri dishes with WMF 30. Thereafter, renew the medium every 48 h until colonies of RLEC are visible under the microscope (after a few wk). 31

Scan the Petri dishes every day for fibroblasts. When fibroblasts are present, scrape these off using a heated platinum-needle 32 When the colonies are large enough (1 e >lOO cells/colony), transfer the RLEC

mto new 4 cm diam Petri dishes and let these form new colonies by repeating steps 29 and 30. 33. After 2-3 mo, a pure population of good divtdmg RLEC is obtained. They are further subcultured m 75 cm* culture flasks.

315

Rat Hepatocyte Cultures 3.2.2, Culture of RLEC

1. Add to a 75 cm* culture flask 10 mL WMF at 37°C. 2. Transfer the RLEC from a single Petri dish (as obtained m Subheading 2.2.1., step 33) into a culture flask (= replication number 1). 3. Incubate the cells for 24 h 4 Renew with 10 mL WMF at 37°C. 5. Renew medium thereafter every 2 d with 10 mL WMF until a confluent layer of RLEC IS obtained (approx 8 d). 6. Remove the medium 7. Add 5 mL sterile PBS (37’C) per flask, shake gently, and remove the PBS 8. Add 5 mL sterile trypsm-EDTA (37°C) per flask, shake gently and leave the flask sealed for 5 mm at room temperature 9. Remove the trypsm-EDTA, reseal the flask, and place tt for approx 15 min in the mcubator. 10. Detach the RLEC by tapping the flask. Confirm that the cells have detached by examinatton under the mtcroscope (i.e., are in floating suspension) 11. Add 20 mL WMF at 37’C per flask and transfer 10 mL cell suspension mto two new sterile culture flasks (= rephcatron number 2). Mix gently to spread the cell suspension over the flask. 12. Repeat steps 3-11 until enough confluent layers of RLEC are obtained to set up the coculture experiment planned or cryopreserve the RLEC m Nunc-tubes

3.2.3. Cryopreservation

of RLEC (see Note 3)

1. Remove the medium from a confluent layer of RLEC. 2. Wash cells with 5 mL stertle PBS (37°C) per flask 3. Add 5 mL sterile trypsin-EDTA (37°C) per flask, shake gently, and leave the flask closed for 5 min at room temperature. 4 Remove the trypsm-EDTA, seal the flask, and place it in the incubator for approx 15 mm. 5. Detach the RLEC by tappmg the flask. Check under the microscope that the cells have detached (i.e. are floating in suspension) 6. Add 10 mL WMF per flask. 7. Transfer the cell suspenston into sterile plasttc 15-mL centrrfuge tubes 8 Centrifuge for 5 min at 620g 9. Resuspend the pellet in 4 mL WMF 10. Homogenize well (by ptpeting up and down) and divide the suspension between four stertle 2-mL Nunc-tubes (1 mL suspension per tube). 11. Add to each tube 1 mL WMF supplemented with 20% (v/v) DMSO. 12. Seal the tubes and cryopreserve in hqutd nitrogen (write the rephcatton number on the tube).

3.2.4. Coculture (see Notes 2 and 3) 1. Follow steps l-5 of Subheading 3.1. for plating the hepatocytes.

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2 After attachment of the hepatocytes (-3.5 h): a Detach the confluent layers of RLEC from the bottom of the flasks as described in Subheading 3.2.3., steps l-5. b. Prepare the cocultures as follows: Add a quarter, half, or the total amount of the cells derived from a 75 cm2 flask of confluent RLEC suspended in 4, A 1, or a4 mL renewing medium, to Petrr dishes of 28.3,78.5, or 177 cm2, respectively. This consrsts of TO-medium (37°C) supplemented with 7 x l@ A4 hydrocorttsone hemrsuccmate 3. Every day thereafter, renew the medium with either 4, 11, or 25 mL serumfree TO-medium (37°C) supplemented with 7 x 10” M hydrocortrsone hemtsuccurate and 0 25 pg/mL amphotericin B, according to the size of the Petri dishes

4. Notes 1 A high degree of variability has been observed between the results of drug metabolism studies obtained either from different cultures of rat hepatocytes within the same laboratory or from stmrlar cultures m different laboratones. Some of these drscrepancres can be explained by the many variables described m Chapter 30 (see Subheading 4.) 2. From the hterature (2) and our own experience (3,4) addmonal factors can be summarized as follows. Of importance are: - the composmon of the culture media, in particular the serum concentratron, the inclusion of hormones, growth factors, differentiation-inducing agents such as DMSO, inducers, and other soluble factors; - the pH and osmolarity of the culture media, - the type of plastic culture dishes or flasks used, their coating and whether or not they underwent a special treatment for cell culture, - cell density and extent of cell-cell mteractrons, - the oxygen supply during culture; - the presence of phenol red m the culture media and its interference with some assays; - the duratron of exposure to substrates; - the presence of organic solvents m which water insoluble substances are added and their effect on the endpoints being measured, - the use of appropriate control cultures; - the lack of standardized protocols used to study drug metabolism in cultured cells at the functional enzyme activity, protein, and mRNA levels 3. Problems specrfic for cocultures of rat hepatocytes with RLEC are. - poor characterrzatron of the helper cells, - poor standardrzation of the isolation method of RLEC; - use of RLEC with a drfferent rephcatron number m different experiments; - contribution of the RLEC to the DNA and protein contents, which are used to normalize most biochemical and metabohc parameters.

Rat Hepatocyte

Cultures

317

References 1. Guguen-Guillouzo, C., Clement, B., Baffet, G., Beaumont, C., Morel-Channy, E., Glaise, D., and Guillouzo, A (1983) Maintenance and reversibility of active albumin secretion by adult rat hepatocytes co-cultured with another liver epithelial cell type. Exp Cell Res 143,47--M. 2. Guillouzo, A. (1986) Use of isolated and cultured hepatocytes for xenobtotrc metabolism and cytotoxictty studies, m Isolated and Cultured Hepatocytes (Guillouzo, A. and Guguen-Gmllouzo, C., eds.), John Lrbbey, London, UK, pp. 313-332 3. Rogiers, V (1993) Cultures of human hepatocytes, m In Vitro Pharmaco-Toxicolony (Rogiers V., Sonck, W., Shephard, E., and Vercruysse, A, eds), VUB, Brussels, Belgium, pp 77-l 15 4. Rogiers, V. and Vercruysse, A. (1993) Rat hepatocyte cultures and co-cultures m biotransformation studies of xenobiotics. Toxzcology 82, 193-208 5 Williams, G M., Weisburger, E. K., and Weisburger, J. H (197 1) Isolation of and long-term cell culture of epithehal-like cells from rat liver Exp Cell Res 69, 106112

34 Isolation of Periportal and Pericentral Hepatocytes Rolf Gebhardt 1. Introduction Liver parenchyma shows a remarkable heterogeneity of the hepatocytes along the porto-central axis, particularly with respect to the expression of cytochromes P450 and other enzymes involved in the biotransformation and conjugation of xenobiotics (I and references therein). This mlcrodiversity IS best revealed by immunohlstochemical techniques or by in sztu hybridization on liver sections. However, when the focus IS on metabolic activltles, stralghtforward access to heterogeneity is via isolation of hepatocyte subpopulations. Because of the considerable variability of enzyme distribution, it is impossible to design a separation technique that accounts for every kind of metabolic zonation. However, the rough separation of the hepatocytes mto two subpopulations, namely the perlportal and the pericentral populations, provides a suitable approach to studying many aspects of hepatocyte heterogeneity m drug metabolism. Several techniques have been established for the separation of perlportal and pericentral hepatocytes. The most suitable separation technique leading to consistent results is the so-called digitonin-collagenase perfusion method developed independently by Quistorff (2) and Lindros and coworkers (3). Based on the dlstrtbutlon of clearly zonated enzymes,such as glutamme synthetase, a reasonable entlchment of both cell populations could be demonstrated (4). 2. Materials 2.1. Isolation of Hepatocyte Subpopulations 1. PerfusIon media (see Note 1): a. Medium A: 137 mA4sodium chloride, 5.4 mM potassiumchloride, 1 2 rruI4 magnesium sulfate, 0.15 mM potassium dlhydrogenphosphate, 0.79 mh4 From Methods m Molecular Bfology, Vol 107 Cytochrome P450 Protocols Edlted by I R Phllllps and E A Shephard 0 Humana Press Inc , Totowa, NJ

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Gebhardt

320

2

3. 4. 5. 6.

dlsodmm hydrogenphosphate, 1 mM calcium chloride, 10 mM HEPES, and 0. I % glucose, adjusted to pH 7.4 b Medium B 145 mM sodium chloride, 5 4 mA4 potassmm chloride, 0 77 mA4 magnesium sulfate, 0.93 mM magnesium chloride, 0 44 rnA4 potassmm dihydrogenphosphate, 0.34 mM dlsodium hydrogenphosphate, and 10 mM HEPES, adjusted to pH 7.4. c. Medmm C: 145 mM sodium chloride, 5 4 mM potassmm chloride, 0 77 mM magnesium sulfate, 0 93 mM magnesmm chloride, 0 44 mA4 potassmm dlhydrogenphosphate, 0.34 mM dlsodmm hydrogenphosphate, and 10 mA4 HEPES, 0 2% glucose and 0 2% bovine serum albumin (BSA) (defatted), adjusted to pH 7.4. Dlgltonin. 7 mM in medium A Dissolve by boiling Cool to 37°C and stenllze by filtration mto a sterile syringe for the infuser immediately before use Caution Do not use If not completely dissolved Collagenase (units determined with z-glycyl-L-propyl-glycyl-glycyl-L-propyl-~alanme). Can be stored at -20°C for up to one year if kept dry (see Note 2) PercoIl@ (Pharmacla, Uppsala, Sweden) Mix Percoll with 1O-fold concentrated medium A to obtain a solution with a density of 1.08 g/mL. 0.5% Trypan blue (m salme) Stable for 3 wk when refrigerated If solution 1s clumpy, clear by filtration. Equipment Usual equipment for the lsolatlon of hepatocytes (see Chapter 3 1). In addition: roller pump with low pulsation, mfusor (Braun Melsungen, Braunschwelg, Germany); sterile syrmge for mfisor

2.2. Evaluation of Hepatocyte 2.2 1. Enzyme Assays

Separation

2 2.1.1. GLUTAMINE SYNTHETASE 1. 2. 3. 4. 5 6. 7.

250 m&f Imldazole. Adjust to pH 6.8. Stable for weeks at 4°C. 250 mM L-glutamine. Adjust to pH 6 8. Keep frozen at -20°C 250 WHydroxylamme-HCI. Adjust to pH 6.8. Stable for weeks at 4°C. 1 6 mM ADP* Adjust to pH 6.8 Keep frozen at -20°C 250 rnA4 Sodium arsenate: Adjust to pH 6 8 Keep frozen at -20°C 10 mM Manganese chloride: Adjust to pH 6 8 Stable for months at 4°C Stop solution* 0 37 M FeCl, in 0 2 A4 trichloroacetic acid and 0.67 M HCl Keep cold. 8. Equipment includes suitably sized test tubes, water bath, and spectrophotometer

2.2.1.2. ALANINE AMINOTRANSFERASE 1, 1 MAlanme in 0.1 Mphosphate buffer, pH 7.4: Stable for 6 mo at 4’C. Add a few drops of chloroform to prevent growth of microorganisms. 2. 13 mMNADH in 10 mA4 sodium bicarbonate: Stable for 4 wk at 4°C 3. 0.5 mg/mL lactate dehydrogenase m glycerol: For stablhty see manufacturer.

321

Isolation of Hepa tocytes 4 0 66 A4 a-Oxoglutarate Stable for 4 wk under refrigeration 5. Equipment mcludes suitably sized cuvets and spectrophotometer

2.2.1.3. 1

2 3. 4. 5. 6 7 8.

3-HYDROXY-%METHYLGLUTARYL

CoA

REDUCTASE

(HMGCoA REDUCTASE): Solution A:50 mMImidazole, 90 mMethylenediammetetra-acetic acid (EDTA), 1 mM ethyleneglycotetra-acetic acid (EGTA), 150 mA4 potassium fluoride, 100 mMglucose-6-phosphate, 1 mMNADP, 7 mA4dithiothrettol (DTT), and 1 mMHMGCoA. Adjust to pH 7 2 3-hydroxy-3-methyl[3-i4C]glutaryl-coenzyme A. Solution B: dissolve 30 ng digitonin in 100 J.IL solution A and add 1 U glucose6-phosphate dehydrogenase. Concentrated HCl. Acetone and benzene, analytical grade. Iodine vapor. Scintillation cocktail Equipment: silica gel thin layer plates (Whatman K5D), water bath, and scmtlllation counter.

2.2.2. lmmunocytochemistry 1. William’s medium E supplemented with 2 mM glutamme. Stable when stertle and refrigerated. 2 Phosphate-buffered saline (PBS). 150 mM NaCl and 10 mA4 phosphate (sodmm salts) at pH 7.4. Stable for 2 mo when refrigerated. 3. 3.5% paraformaldehyde in PBS: Dissolve by heating to 90°C. Cool before use. Stable for one day only (refrigeration) Cautzun: This reagent is toxic 4. 10% Goat serum m PBS: Keep frozen. Stable for two years, tf frequent thawing and freezing is avoided. 5. Primary antibody: Use antiserum or monoclonal antrbodtes (MAbs) directed against a highly zonated enzyme, e g., anti-(glutamine synthetase) serum (IgG) prepared in rabbit Dilute with PBS. Use dilution suggested by the suppher or test for optimal dilution 6 Secondary antibody: This antiserum must be directed against the tsotype of the prtmary antiserum or antibody (e.g., antirabbit IgG prepared m goat). Dtlute 1.50 with PBS. 7. Peroxtdase-anttperoxidase (PAP) complex: This complex must be prepared from the same species as the prtmary antibody (e.g., rabbit) Dilute according to the data provided by the supplier. 8. 100 mA4Tris-HCl buffer: Adjust to pH 7.6. Stable for 3 mo when refrtgerated 9. Staining solution: 0.5 mg/mL Dtammobenzidme, 2 mg/mL aminotrtazole, in 100 mM Tris-HCl buffer. Add H,Oz (to a final concentration of 0.0 1%) immedlately before use. Prepare fresh for each staining Caution: These reagents are toxic. 10 Equipment. Collagen-coated cover slips (20 x 20 mm), mtcroscope slides, ttght sealing moist incubation chamber.

Gebhardt

322 3. Methods 3.1. Isolation of Hepatocyte Subpopulations 3.1.1. Liver Perfusion (see Note 3)

1 Anesthetize rat by mtraperitoneal mlection of sodmm pentobarbital (60 mg/kg body weight) 2 Open the peritoneal cavity, ligate the vena cava inferior, and cannulate the portal vem by standard technique (see Note 4) 3. Flush the liver free of blood with medium A at a rate of 35 mL/mm (see Note 5). 4. Contmue perfusion until a second cannula is placed at the vena cava superior. 5. Switch quickly from infusion of medium A to the digitonin solution Note that the direction of mfusion is dependent on the subpopulation to be isolated. For periportal cells, mfusion is retrograde through the vena cava superior, for pericentral cells, mfusion 1s orthograde through the portal vem 6 Infuse the digitonm solution at a constant rate of 2 n&/mm by means of an infuser 7 Carefully check the pattern of decolorizatton becommg visible on the surface of the liver. The pattern should be homogeneous all over the liver (see Notes 6 and 7) 8 Stop the infusion of digitonin when the typical pattern for orthograde or retrograde perfusion (Fig. 1) is reached. 9 Switch immediately to mfusion of medium B m the direction opposite to that of the digitonin infusion 10 Continue to infuse medium B until at least 950 mL are used up Then switch to orthograde perfusion, if not already the case 11 While leaving the liver VI sztu, connect the cannula m the vena cava superior with the reservoir of a recirculation perfusion device 12 Switch to recirculating perfusion (orthograde) with medium C contammg collagenase (160 U/100 mL) Perfusion should be performed preferably at a constant flow of 35 mL/mm. 13 After 2 mm add calcium chloride to recirculating medium C (final concentration 5w 14. When the liver becomes soft (usually after 15-20 mm) or shows some small

ruptures, stop the perfusion. Perfuse for no longer than 25 min. 15. Carefully take each lobe at its base with a pan of tweezers, cut it free, and trans-

fer to a beaker containing cold medium A. 16. Disrupt the liver capsule by cutting it several times. Take the lobes and shake

them until the cells are dispersed. Discard the remaming (white) vascular tree 17. Filter the cell suspension through three layers of cheese cloth and finally through

100 nm gauze

3.1.2. Purification of Hepatocytes 1. Place 50 mL of hepatocyte suspension m a suitably sized tube. Centrifuge in a swinging bucket rotor at 50g for 3.5 mm. Discard supernatant. 2. Refill tube with medium A and centrifuge agam. Repeat twice

323

Isolation of Hepatocytes

A

BoAoAoAo*o*o*

“9A0,0,0~0~0 O~O~OAOAOAO~ Ao~o,o~o,o~o o.o.o.o.opo. Fig. 1. Schematic illustration of the staining pattern of a liver characteristic for the isolation of (A) periportal and (B) perlcentral hepatocytes Black area, normal-colored liver tissue; white area, tissue decolored by dlgltonm, triangles, portal tracts; small white circles, central veins Note that the periportal region (contmuous network) IS always larger than the pericentral (circular) region

3. Produce a Percoll gradient by centrifuging 35 mL of a Percoll solution of a density of 1.08 g/mL at 20,000-30,OOOg for 1O-l 5 mm (see Note 8) 4. Place 2-3 x 10’ hepatocytes m 10 mL of medium A on top of 35 mL of the Percoll gradient. Centrifuge in a swinging bucket rotor at 15Og for 15 mm. 5 Collect 2-mL fractions from bottom to top Dilute each fraction fivefold with 0 9% NaCl Centrifuge twice at 50g for 2 min

3.1.3. Determination of Cell Yield and Viability Carefully suspend the isolated hepatocytes in cold medium A. Gently mix an aliquot of 250 @ with 100 @, of Trypan blue solution Transfer an aliquot to a hemocytometer and count numbers of cells excludmg or staining with Trypan blue. Viability IS expressed as the percentage of cells that exclude the dye (see Notes 9 and 10). 3.2. Evaluation of Hepatocyte Separation 3.2.7. Giutamine Synthetase Assay

(see Notes 11-14)

1. Prepare assay mix immediately before use. Add 2 vol of lmidazole, 2 vol of L-glutamine, 1 vol of hydroxylamme, 1 vol of ADP, 1 vol of sodium arsenate,

and 1 vol of manganesechloride exactly in this sequence.Warm to 37°C (see Note 11).

2. Preparea suitablehomogenateof the hepatocytesin 50 nMTns-HCl, pH 6.8, at a concentration of l-2 x 1O6cells/ml using a Dounce homogenizer or a somcator. 3. Mix 100 pL, cell homogenate and 400 pL assay mix and incubate at 37°C for 15 to 30 min. Run blanks with homogenate, but wlthout ADP and sodium arsenate m the assay mix.

Gebhardt

324

4 Terminate the mcubatlon by addmg 1 mL of stop solution Keep samples on ice Centrifuge at 3000g. 5. Measure absorbance m a spectrophotometer at 535 nm 6 Calculate enzyme actlvlty. A (mU/mL) = E (mOD) 34.7/t (min)

3.2.2. Alanine Aminotransferase

(1)

Assay

1 Assay mix: add 30 vol of alanme/phosphate buffer, 0 5 vol NADH, and 0.5 vol lactate dehydrogenase Stable for 12 h at room temperature 2. Add 3 vol of assay mix to 0.5 vol of cell homogenate (see Subheading 3.2.1., item 2) m a cuvet and incubate at 37°C for 3 mm 3 Place into spectrophotometer and add 0 1 vol of a-oxoglutarate. MIX well and record the decrease of absorbance at 340 nm (see Note 12) 4. Calculate activity from the slope of the curve using ENADH= 6.6 cm*/ptnole.

3.2.3. HMGCoA Reductase Assay 1 Add 2 kBq 3-hydroxy-3-methyl[3-‘4C]glutaryl-coenzyme A to 50 & of solution B 2. Add 100 pL of hepatocyte suspension (see Subheading 3.2.1., item 2) to 50 pL preheated (37°C) solution B containing the radiotracer, mix, and incubate at 37OC 3. Terminate assay after 10 min by adding 20 & HCl. Blanks are terminated at zero time (see Note 13) 4. Incubate at 37°C for at least 30 mm to convert the mevalonate formed mto mevalonolactone. 5 Chromatograph ahquots on silica gel thin-layer plates m acetone.benzene (1 1, v/v), vlsuallze with iodine vapor, scrape into scmtlllatlon vials, add scmtlllatlon cocktail, and measure radloactlvlty

3.2.4. lmmunocytochemistry 1. Incubate hepatocytes suspended m culture medmm on collagen-coated cover slips in a Petri dish Let cells attach for at least 2 h (see Note 15) 2. Discard culture medium and nonattached cells. Wash once with PBS. Fix m icecold 3 5% paraformaldehyde for 20 min 3. Wash twice with PBS 4. Place a drop (SO-100 pL) of 10% goat serum on a microscope slide. Remove cover slip with cells from the Petri dish and place it upside down on the drop Be careful to avoid formation of air bubbles. 5. Incubate m a tight-sealing moist chamber (e g., large Petri dish) at room temperature for 30 mm 6. Remove cover slip and dip m PBS. Wash twice by dipping m fresh PBS 7 Repeat steps 4-6 with 80-100 pL of primary antibody diluted with PBS (see Notes 16 and 17) Extend the incubation period to l-2 h (see Note 18) Wash extensively m fresh PBS.

325

isolation of Hepatocytes

8. Repeat steps 4-6 with 80-100 pL of secondary antibody diluted appropriately m PBS (see Note 16) Incubate for 30 min to 1 h. Wash extensively with PBS 9 Repeat steps 4-6 with 80-100 pL of the PAP complex Incubate for 30 mm to 1 h. Wash extensively with PBS 10. Transfer cover shp to 100 MTris-HCl, pH 7.6. 11. Place cover slip right side up m an approprtate chamber (e.g., small Petri dish) and add freshly prepared staining solution (about 1 mL) Incubate for not longer than 30 mm. If background staining is too Intense (check under microscope), remove cover slip and wash with Tris-HCl (see Note 19) 12. Put cover slip m PBS and view under microscope (see Notes 20 and 21) Other methods for assessing the quality of hepatocyte separation and the cultivation of cell subpopulations are described m Notes 22-27

4. Notes 4.1. Isolation

of Hepatocyte

Subpopulations

1. Although successful isolation achieved by perfusion wtth bicarbonate-buffered media and 5% COZ + 95% O2 for equilibration has been described in the hterature, we found it better to use HEPES-buffered media and 100% 0, 2. Because pure collagenase is msufficient, the choice of collagenase is of utmost importance for successful isolation of the hepatocytes; the collagenase must be contaminated with other (unknown) proteolytic activities It is recommended that different batches from various suppliers be tested and compared. A large quantity of an optimal batch can then be ordered and stored in appropriate aliquots at -20°C. 3 In thts protocol the two-step isolation procedure described by Seglen (5) is used with slight modifications 4. Preferably, the liver should be perfused zn situ m order to avoid any disturbance of blood flow that might result from twisting and other manipulations necessary for removing the liver when perfusing it m the isolated state. Perfusion zn sztu helps to ensure an even and simultaneous perfusion of all sinusoids. 5. Normal perfusion should be done with constant flow at 35 mL/mm, preferably with a pump with low pulsation. Alternatively, a constant pressure can be obtained through the use of a column of water of about 80 cm 6. The zonal destructron of the hepatocytes can be Judged from the white-colored pattern appearing at the surface of the hepatocytes when the digitonin solution is infused. For isolating periportal cells, the pattern should consist of white dots marking the center of the lobules (Fig. 1). Stop the perfusion when the diameter of the dots is as large as the distance between dots. For isolating pericentral cells the inverse pattern (white network) should occur. Perfusion is stopped when the network has completely fused and the remaining central brown dots have approximately the same diameter as the white dots m the aforementioned case. 7. If the blood flow IS not disturbed, the respective white pattern should be homogeneous over the entire surface of the liver. In case of gross mhomogenemes, the

326

8.

9

10

11.

12. 13.

14.

15.

16. 17.

18. 19.

20.

Gebhardt liver should be disposed of. If inadequate perfusion is restrtcted to distinct lobes such lobes can be entirely removed after the digestion with collagenase. Sometimes only small peripheral areas are not well-perfused These pieces of tissue should be cut off after collagenase digestion before the lobes are taken out Viability can be improved by percoll gradient sedimentation. This is suggested particularly when hepatocyte subpopulations are used. However, a high viabthty determined by Trypan blue staining does not necessarily ensure a high plating efficiency during subsequent cultivation of the hepatocyte subpopulattons Yield of isolated cells per liver is usually only about 50% and 35% for periportal and pericentral hepatocytes, respectively, compared with the yield of a normal hepatocyte preparation This is somewhat less than one would expect from the respective fraction of these cell populations. Vtability is also not as high as for a total hepatocyte preparation; usually between 82 and 90% only. It should be noted that hepatocytes isolated by this technique may show increased leakage of small molecules up to the size of glutathione The assay is performed as described (6). When preparing the assay mix, it is important to add the reagents m the correct sequence If the mix gets turbid (white), discard! Absorbance is linear up to about 600 mOD. If the recorded decrease m absorbance is not linear, extend premcubahon period or check cuvet for contaminating dust. The assay IS performed accordmg to Geelen et al (7) with slight modificatrons. The enzyme assay is lmear up to 5 mm and with protein concentrations m the range 0.06 to 0.6 mg of cell protein per assay. [3H]mevalonolactone can be used as an internal standard To ensure that a reasonable separation of periportal and pericentral hepatocytes has been achieved, the periportal/pericentral ratios of enzyme activities should be 1.6, and >2 5 for glutamine synthetase, alanme aminotransferase, and HMGCoA reductase, respectively (4). Instead of mcubation for attaching the hepatocytes, a cytofuge can be used In this case isolated cells can be fixed without a delay that may result in continuous loss of zonated properties. Tween-20 (0 05-O 5%) can be added to the antiserum-containing solutions m order to avoid nonspecific binding of antibodies The selection of an appropriate target for the primary antiserum 1s important, because only a few molecules show a zonal distribution clear enough to be useful for the distmction of pertportal and pericentral hepatocytes Incubations, particularly with the primary antiserum, can be performed at 4°C overnight. For estimatmg the mtensny of background staining, omit the primary antiserum and/or the PAP complex If background stammg is too high, use greater dilutions of the PAP complex or reduce the concentration of H,OZ. Alternatively, cover slips could be dehydrated by taking through a graded series of ethanol, immersed m xylene, and mounted usmg conventtonal mounting such as DePeX (Bioproducts, Ingelheim, Germany)

Isolation of Hepatocytes

327

21 For better comparison, two different markers can be visualized using different antisera and substrates (8) 22. Hepatocytes In situ can be prelabeled with dyes such as acrldme orange via perfusion m orthograde and retrograde direction (9). The relative fraction of pertportal and pencentral hepatocytes can then be estimated from fluorescence-activated cell sorter (FACS) analysis of the hepatocyte populations obtamed 23 The different metabolic performance of the hepatocyte subpopulatlons can be used to obtain a rough indication of the quality of the separation (IO,IZ) 24 Northern-blot analysts could also be used for assessing the quality of separation provided cDNAs for highly zonated enzymes or other molecules are available Examples of this approach have been published by Twisk et al (22) 25. Hepatocyte subpopulattons can be cultured using techniques and media suitable for the cultivation of total hepatocytes. It 1s advantageous, however, to use culture conditions similar to the conditions to which these cell populations are exposed m vlvo (c-f ref. 13) 26. The cultured subpopulations of hepatocytes show a remarkable metabohc stability with respect to certain functions (4,10), whereas other mitially highly zonated functions eqmhbrate rapidly This IS the case for mevalonate synthesis and gluconeogenesis. 27. With respect to drug metabolism, the loss of many phase I reactions IS more pronounced m pencentral than m penportal hepatocytes leading to comparably low activities after I or 2 d m culture (24,15). Differences m phase II reactions are retained for longer periods of time (15).

References 1. Gebhardt, R. (1992) Metabolic zonation of the liver. regulation and implications for liver function. Pharmacol Ther 53,275-354. 2 Qmstorff, B. (1985) Gluconeogenesis m penportal and penvenous hepatocytes of rat liver, isolated by a new high-yield digitonm-collagenase perfusion technique. Blochem J 229,22 1-226. 3. Lindros, K. 0. and Penttila, K. E. (1985) Digrtonm-collagenase perfusion for efficient separation of periportal or penvenous hepatocytes. Blochem. J 228,757-760. 4. Burger, H.-J., Gebhardt, R., Mayer, C., and Mecke, D (1989) Different capacities for ammo acid transport m periportal and perivenous hepatocytes isolated by digitonin/collagenase perfusion. Hepatologv 9,22-28 5 Seglen, P. 0 (1976) Preparation of isolated rat liver cells. Methods Cell Bzol 13, 29-83 6. Gebhardt, R. and Williams, G. M. (1986) Amino acid transport m established adult rat liver epithelial cell lmes. Cell Bzol. Toxlcol 2,9-12. 7 Geelen, M. J H., Papiez, J. S , Girgrs, K , and Gibson, D M. (1991) IN SITU measurement of HMG-CoA reductase activity in dtgitomn-permeabilized hepatocytes. Biochem Bzophys Res Commun 180,525-530 8. Gebhardt, R., Lindros, K , Lamers, W. H., and Moorman, A F. M (1991) Hepatocellular heterogeneity in ammonia metabolism* demonstration of limited

328

9

10.

11.

12.

13

14.

15.

Gebhardt colocahzatton of carbamoylphosphate synthetase and glutamine synthetase. Eur J Cell Blol 56,464-467. Gumucio, J J , May, M , Dvorcak, C , Chianale, J., and Massey, V. (1986) The isolation of functionally heterogeneous hepatocytes from the proximal and distal half of the liver acmus m the rat. Hepatoloa 6,932-944 Quistorff, B , Dich, J , and Grunnet, N. (1986) Perlportal and pertvenous hepatocytes retam then zonal characteristics m primary culture Blochem Blophys Res Commun 139, 1055-1061 Tosh, D , Alberti, G. M , and Agms, L (1988) Glucagon regulation of gluconeogenesis and ketogenesls m pertportal and pertvenous hepatocytes Bzochem J 256, 197-204. Twlsk, J., Hoekman, M. F M., Mager, W. H., Moorman, A F. M , de Boer, P A J , Prmcen, H. M G., and Gebhardt, R. (1995) Heterogeneous distribution of cholesterol 7a-hydroxylase and sterol 27-hydroxylase m the rat liver acmus IS regulated at the mRNA and translational level. J Urn Invest. 95, 1235-1242 Probst, I., Schwartz, P., and Jungermann, K. (1982) Induction m primary culture of gluconeogemc and glycolyttc hepatocytes resemblmg periportal and pertvenous cells. Eur J Bzochem 126,271-278. Suolinna, E. M , Pentttla, K E., Wmell, B. M., Sjoholm, A. C., and Lmdros, K 0. (1989) Drug metabolism by perlportal and perivenous rat hepatocytes Comparison of phase I and phase II reactions and their induclbility during culture Bzochem Pharmacol. 38,1329-l 334 Gebhardt, R., Alber, J., Wegner, H., and Mecke, D (1994) Different drug metabolizmg capacities m cultured periportal and permentral hepatocytes. Blochem Pharmacol 48,76 l-766

35 Perifusion

Culture of Hepatocytes

Rolf Gebhardt 1. Introduction Cultivation of cells in Petri dishes or culture flasks is usually performed by changing the medium after certain periods of time, e.g., 24 h (stationary cultivation). Clearly, the concentration of nutrients and other components as well as of added hormones will decrease over this period because of the metabolic activity

of the cultured cells Conversely,

metabolic

end products such as lac-

tate, ammonium ions and even bile salts will accumulate. In order to replace the sequential changes of the culture medium by a continuous supply, several systems for perifusion or circumfusion of the cells with medium have been designed. Usage of one such approach, reported for cultivation of hepatocytes (I), indicated that perifusion was beneficial to the cultured hepatocytes, resulting in a prolonged life-span and an enhanced metabolic performance In particular, it was found that the hormonal response to dexamethasone and glucagon was increased (2), partly because steady-state concentrations of hormones are established even at low initial concentrations. Since these early studies, the technique for perifusion has improved and it has been found that oxygen tension within the culture medium is a very critical parameter; surprismgly, m contrast to previous assumptions that oxygen supply in normal stationary cultures is msufficient (3), higher oxygen tensions, in conjunction with elevated flow rates, proved to be cytotoxic under perifusion conditions. If this and other special features and requirements are considered, perifusion cultivation has considerable advantages over conventional culture techniques, particularly for studies on biotransformation and drug toxicity (4,5). As most commercially available perifusion systemsare either limited m their usage or are not suitable for the cultivation of hepatocytes, the basic requireFrom Methods m Molecular Ebology, Vol 107 Cytochrome P450 Protocols Edlted by I R Phllhps and E A Shephard 0 Humana Press Inc , Totowa, NJ

329

330

Gebhardt _____

c-

(closed)

___---.

Fig. 1 SchematIc illustration of the components of a penfuslon system. B, bubble trap; C, culture chamber; I, mfusor for labile compounds, LHC, lower heat control, M, medium reservoir; N, needle; OX, oxygenator; P, pump; UHC, upper heat control, S, switch for flow direction The flow direction of the medium m the open and closed perlfuslon mode 1salso indicated The lllustratlon is not to scale.

ments for an easy-to-build penfusIon system are described herem m addition to the basic principles of perifusion cultivation.

2. Materials 2.7. Perifusion System (see Notes 7 and 2) 2.1.1. Peripheral Components 1 Medium reservoir (Fig. 1): The medium reservoir may consist of any bottle, flask, or other container that can be sterilized and sealed m a way that prevents contammatlon of the medium. It must be equipped with an mlet and outlet connection 2. Medium pump (Fig. 1): Any peristaltic pump with low pulsation Supported flow range 0 5-20 mL/h 3 Bubble trap. A simple glass bubble trap with a capacity of 10 mL is sufficient (Fig. 1) (see Note 3)

4 The oxygenator (Fig. 1): It should allow the easy saturation of the culture medmm with oxygen and carbon dloxlde, while contaming as little medium as possible. It may consist of 10-15 bundles of tmy gas-permeable (silicone) tubes (length 100 mm; outer diameter: 0.7 mm, wall thickness. 0 05 mm) that are glued together and sit within a larger gas-impermeable tube (glass) through which the gas-mlxture streams m a countercurrent to the culture medium (see Notes 4 and 5) 5. Tubing should consist of chemically mert and autoclavable material, e g., sillcone or polytetrafluorethylene (PTFE) (see Note 6)

6. Injection needles,madeof refined steel,that have anopening at the side of the tip (Hamilton). Use one for the inlet and two for the outlet 7 Temperature control is very important In principle, the penfusion system can be operated m any thermal incubator Alternatively, two independently operating thermal plates for warming the top and bottom of the culture chamber are suffi-

331

Hepatocyte Perifuslon Culture A

dutlet

Inlet

__.__ it

130

~-i

Fig. 2. Details of the culture chamber. The tllustratton shows (A) the lid, (B) the cover, (C) a vertical and longttudinal section, and (D) a view, from above, of the bottom part of the culture chamber. CS, culture supports (76 x 26 x 1 mm), e g., mtcroscope slides; SR, silicone ring; the holes for inlet and outlet (34 mm m diameter) are stoppered wtth s&cone rubber (dashed area). All measurements are m mm. The tllustration is not to scale cient (Fig. 1). Preferably, the temperature of the upper heat control should be 1°C higher than that of the lower one.

2.1.2. Central Components: Culture Chambers and Cell Supports 1. The culture chamber must fulfil several requtrements: a. It should be made of a material that is chemically inert and can be autoclaved, such as polycarbonate (MakrolonR). b. It should consist of a cover (Fig. 2B) and a bottom part (containing a trough) separated by a silicone rmg (Fig. 2C,D). The sillcone ring should tighten the chamber when the cover and the bottom part are squeezed agamst each other by slight mechanical pressure from the chamber holder or the heat control c. The size and shape of the trough should be chosen so as to enable the establishment of an almost lammar flow of medium (Fig. 2D).

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Gebhardt

d. The total area of the trough should allow the cultivatton of sufficrent cells for the planned experrments. e. The height of the trough should not be less than 8 mm, m order to accommodate the culture supports (1 mm), the layer of culture medium (1 mm), and leave sufficient space (6 mm) to prevent the medium from reaching the upper margin, in case the chamber IS shghtly trlted The front and rear of the chamber should be equipped with three holes of 4 mm diameter, sealed wtth sihcone rubber (Fig. 2C,D) (see Notes 7 and 8) f The cover IS srmply a plate with a groove for the srhcone ring (Fig. 2B) g Preferably, the cover and the bottom part should be translucent, in order to allow visual mspectlon of the cultures h. For use m stattonary culture, a hd (Fig. 2A) fittmg to the bottom part of the culture chamber IS required that IS autoclavable, translucent (Macrolon), and does not fully seal the chamber, thus allowmg for exchange of gas (like the cover of a Petrr dash) 2 The chamber holder. a. The chamber holder should allow the tight sealmg of the culture chamber by any simple mechamsm squeezing the cover and the bottom part together. b. Preferably, the chamber holder IS attached to the thermal plate constltutmg the lower heat control for the culture chamber. 3 The culture supports: a Culture supports can be made either of autoclavable material or of other biocompatible plastic that can be sterilized by irradiatron (see Note 9) b The size of the supports should match the dimensions of the trough of the culture chamber. Preferably, up to five dtfferent supports should completely cover the bottom of the trough (Fig. 2D). c The supports should be precoated with components of the extracellular matrix (such as collagen). 4. Reagents. a Collagen b 0.1% (v/v) glacial acetic acid c Hank’s buffered salt solutron.

2.2. Perifusion Cultivation (see Note 10) 1 Seeding medium: Wrlllams’ medium E supplemented with 10% newborn calf serum, 2 mA4 L-glutamine, peniclllm (50 U/mL), streptomycm (50 ng/mL), and 0.1 pA4 dexamethasone (see Note 11). 2 Short-term perifusion medium. Williams’ medium E supplemented with 2 n-&I L-glutamme, penicillin (50 U/mL), streptomycm (50 pg/mL), and 0.1 @Idexamethasone. 3 Long-term penfusion medium. Williams medium E supplemented with 2.5% newborn calf serum, 2 mA4 L-glutamine, penicillin (50 U/mL), streptomycm (50 pg/mL), 0.1 @4 msulm, 0.1 @4 glucagon, and 0.1 pA4 dexamethasone.

333

Hepatocyte Perifusion Culture

4 The gas-mixture recommended here is composed of 13% oxygen, 5% CO,, and 82% nitrogen (see Note 12). 5 Isolated primary rat hepatocytes with a viability of more than 90%, suspended in seeding medium. 6. Rat liver eptthehal cells (LEC) isolated from juvemle livers (10 d postpartum) according to Wtlhams (6)

3. Methods 3.1. Preparation of the Perifusion 3.1.1. Sterilization and Coating

System

1 Sterilize the complete system by autoclaving. It 1spreferable to separate the system into the culture chamber, which is sterrhzed separately, and the other components, which can be sterilized when fully connected. The latter should be separated from each other only if free access of steam IS otherwise blocked or hampered. All open endings should be loosely wrapped with alummum foil Sterilization of the culture chamber is preferably performed with culture supports already in place on the bottom of the trough Make sure that steam has free access to the Interior of the chamber. If necessary, small parts of the perifusion system and tubmg can alternatively be sterilized by immersion m 70% ethanol followed by careful drying m a lammar flow cabinet. 2. After the culture chamber has been autoclaved and allowed to cool to room temperature, cover culture supports wtth a collagen matrtx. For a lo-mL chamber, add approx 15 mL of collagen m 0 1% (v/v) glacial acetic acid and incubate m a humidified atmosphere for at least 24 h. Discard the solutton and wash the plates twice with Hank’s buffered salt solution The chamber should be kept humid until seeding of the hepatocytes

3.1.2. Assembly and Filling of the System 1 Assemble the complete perifusion system, except for the culture chamber, under a laminar air flow in a safety hood accordmg to Fig. 1 2 Add perifusion medium to the reservoir and close it tightly. For short-term perrfusion m the closed system (up to 48 h), the system should contain 5x the volume of medium m the culture chamber. For long-term perifusion (up to 7 d, the system should contain 15x the volume of medium in the culture chamber 3 In order to minimize the time required to fill the tubing and the other components with medium, a sterile additional tube connected with a syringe should be attached to the injection needle (the end of the tubing) and the medium should be sucked through the tubing. Before the medium reaches the end of the tubing, clamp the tubing, m order to avoid leakage or backward flow of medium. Fillmg should be performed just before connecting the components to the culture chamber (1 e , after step 4 of Subheading 3.1.3.)

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3.1.3. Loading of the Chambers with Hepatocytes 1 Place sterile culture supports on the bottom of the culture chamber (if not already done). 2. Add 10 mL of cell suspension (e.g., 1.25 x lo6 hepatocytes per mL of seeding medium) Ensure an equal distribution of the inoculated cells. 3, Cover the bottom part of the culture chamber with the hd and place the complete chamber mto a normal CO2 incubator (37°C 95% humid@ to allow for the proper attachment of the cells (usually 2 h) 4 Remove unattached cells together with the medmm and replace with the appropriate per&ion medmm

3.1 4. Establishment of Cocultures (see Notes 13 and 14) 1 If cocultures are to be estabhshed, the concentration of the hepatocyte suspension used above (see Subheading 3.1.3., step 2) should be reduced by 50% (1 e , to 0.625 x lo6 hepatocytes per mL of seeding medium). 2. When the hepatocytes have attached to the culture supports (see Subheading 3.1.3., step 3), gently shake the chamber to remove nonattached cells and discard the medium. 3. Add 10 mL of a suspension of liver epithehal cells (1 x lo6 cells per mL of seeding medium). 4. Place the covered chamber back mto the incubator and keep it there for 6-8 h. 5 Remove unattached cells together with the medium and replace with the appropriate perifusion medmm

3.2. Perifusion Cultivation (see Notes 15 and 16) 3.2.1. Preparation for Perifusion 1 Place the culture chamber under a laminar au flow and replace the lid with the sterile cover of the chamber. 2 Fix the complete chamber in the chamber holder (avoid tilting). Close the chamber holder, pressing down the cover part of the chamber for tight sealmg. Note that the pressure may rise withm the chamber. 3. Connect the chamber to the needles for inlet and outlet according to Fig. 2 4. Switch on the pump and monitor the system until a contmuous flow of medmm is established

3.2.2. Closed Perifusion (see Note 17) 1. For the closed perifusion mode connect the outlet with the reservoir (Fig. 1) 2 Adjust the position of the culture chamber such that the level of the medmrn wtthm the chamber matches the level of the medium within the reservoir Note that this condition is essential for optimal flow of medium (see Note 18). 3 Make sure that long-term cultivation with the closed perifusion system is performed only with long-term perifusion medium (see Notes 19 and 20). 4. Adjust the flow rate to the mode and duration of cultivation (see Note 21). a. Less than 24 h. 5-20 mL/h. b More than 24 h* l-5 mL/h.

335

Hepatocyte Perifuslon Culture 3.2.3. Open Perifusion

1. For the open-penfusion mode, let the medium drain from the chamber (Fig. 1). 2. The level of the medium within the culture chamber need not be the same as that m the reservoir 3 If the open perifusion mode is operated for less than 24 h, use serum-free medium only. Make sure that long-term cultivation with the open-penfusion system IS performed exclusively with long-term perifusion medium. 4 Adjust the flow rate to the mode and duration of cultivation: a. Less than 6 h: up to 50 mL/h. b Between 6 and 24 h. up to 20 mL/h.

3.2.4. Steady State Kinetic Measurements I

2 3.

4. 5

(see Note 22)

For steady-state kmetic measurements of drug metabolism, use a special culture chamber holdmg only one culture support, preferably perifused m longitudmal orientation. Operate in open perifusion mode only. Infuse the compound to be tested directly before the inlet needle to the chamber (see Fig. l), usmg a separate pump or mfusor Add a marker substance to the mfusion, in order to determine the lag-phase for the marker to dram from the chamber. Use half of this lag-phase as the average reference time “zero” for the kinetic measurements. Collect the medium immediately after draining from the chamber. A fraction collector may be used The flow rate has to be adjusted to allow the rapid establishment of a steady-state condition as well as the detection of the parent compound and the expected metabolites (see Note 23).

3.2.5. Enzyme Induction 1 For determmations of the induction of enzymes involved m the metabolism of xenobiotlcs, the penfusion system usually has to be operated for a period of 24-72 h 2. If toxic metabolites of the mducmg agent are expected, the open-perifusion mode should be used. 3. The inducing agent may be added to the medium within the reservoir or infused directly before the inlet needle, depending on its stability (see Note 24) 4. Individual culture supports may be removed from the chamber during the course of the perifusion or after termmation for measuring the activity or the amount of the respective enzyme(s) Note. All manipulations that could lead to contammation of the system should be performed m a laminar air flow cabinet. 5. Cell homogenates can be obtamed by rinsing the culture supports with ice-cold saline or the appropriate buffer, scraping the cells mto a small amount of buffer, and sonicating the sample. 6. Aliquots may be used for enzyme-activity assays, western blots, and protein determmations

Gebhardt 3.3. Other Perifusion Systems Several perifusron systemsare commercially available. Most of them allow the perifusion of small amounts of cells sufficrent only for observations of the influence of culture media (including factors or compounds to be tested) on cell viability and morphology. A surtable system for brotransformation studres IS the “modulculttvator” (4), which is produced by Urtz GmbH & Co. KG (8955 1 Konigsbronn, Germany). Because of its modular design, this system is especially suited for comparative studies of different drugs or different concentrations of drugs and inducers. 4. Notes I The perifuston system descrtbed here is a slightly modified version of that descrtbed by Gebhardt and Mecke (1) Information on a commercially available version can be obtained from the author 2. Basic requirements for the materials of the culture chamber and pertpheral parts of the perifusion system include: a. Sterilizable m an autoclave Other methods for sterihzation may be apphcable, but usually are not as convenient (Beware of using gas sterilization, because even after extensive venttlation, remains of the toxic gas may diffuse out of plastic for many days) b. Noncytotoxic plastic. If there are doubts, a simple test can be performed as follows: immerse the respecttve part(s) in a small amount of culture medium, leave for 1 d m the refrigerator or at room temperature, remove the medium and add tt to normal hepatocyte cultures. Apply any of the conventional viability assays for determination of cytotoxictty c Translucent material. If the material of the culture chamber does not allow the visual mspection of the cultures under an inverted mtcroscope, the culture supports may be removed aseptically and placed in an appropriate Petri dish 3. The bubble trap may also be used to adpt the temperature of the culture medium to 37% This is necessary because saturation with gas is temperature-dependent 4 The device for oxygenation is of utmost importance. It must ensure that oxygen tension in the medium stays within a narrow range between 30 to 70 mm Hg (4090 @4) and that the medmm is in equthbrmm with sufficient CO* to establish the correct pH of 7.4 with the bicarbonate concentratton of Wrlltams’ medium E For the silicone-tube oxygenator suggested here, the gas mixture described m Subheading 2.2., item 4, is optimal However, if other devices are used for gas exchange, the gas mixture has to be adapted accordingly In any case, it is recommended that oxygen saturation and pH of the medium are measured before it enters the culture chamber 5. The distance between the outlet of the oxygenator and the inlet of the culture chamber should be as short as possrble and the tubmg should be almost gasimpermeable, m order to lose as little oxygen (or C02) as possible.

Hepatocyte Perifwon Culture

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6. The tubing connectmg the culture chamber with the medium reservoir (for the closed mode) should not be too narrow (approx 3 mm dtameter), in order to allow easy back-flow of medium and to prevent occasional air bubbles. Always use two outlet needles Note that back-flow is pressure-driven; pumping is not recommended. 7 The culture chamber should not be completely filled with culture medium, but should have room (not too much!) for a gas atmosphere above the l-mm-thick layer of culture medium. This ensures that there is no gradient in gas saturation across the culture chamber. Make sure that the culture medium does not touch the cover of the chamber 8 The flow of medmm within the culture chamber should be homogeneous (lamtnar). This condition IS difficult to sattsfy because mfuston of the medmm through one needle creates some inhomogenetty right from the beginning If there is any doubt as to whether all parts of the culture area are pertfused almost equally, the flow characteristics should be visualized. This can be done by fillmg the chamber wtth a clear solution prior to the ml&on of a colored (Trypan blue) solution of the same composition and osmolartty. 9 The culture supports should consist of (biocompatible) plastic or glass slides If glass is used it must be sibcomzed by immersion in silicone oil (Sigma, St. Louis, MO) and heating at 100°C for 1 h. All materials should be coated with collagen, matrtgel or other extracellular matrix components (EMC) It is essential that the EMC does not detach during cultivation, particularly if long-term cultivation is performed 10 In the system described here, bicarbonate-contammg media are used because these are best suited for the cultivation of hepatocytes. However, if gas exchange with COZ is not feasible, a bicarbonate-free verston of Williams’ medium E can be used as the basic medium The various supplements can be used accordingly. 11 Williams’ medium E can be replaced by a special modification of Waymouth medium as described (I), 12 The basic components of the gas mixture are oxygen, CO% and nitrogen The relative concentrattons are dependent upon the properties of the oxygenator and the content of sodium btcarbonate m the culture medium 13 Cocultivation wtth LEC certamly prolongs hepatocyte survival and supports the performance of hepatocellular functions in stationary cultures (7) as well as m perifusion (4). However, it should be noted that the LEC are much more sensitive than hepatocytes to changes in pH, oxygen concentration, and osmolanty Thus, if these parameters are not controlled carefully, the LEC may selecttvely detach and die. 14 Alternattvely (or in combination) to coculttvatton, the hepatocyte monolayer may be covered with a second layer of collagen matrix (sandwich culture) as described by Dunn et al. (8). Pertfusion for long periods of time may result in proteolyttc breakdown of the collagen layer If this occurs, the addition of trypsm inhibitors is recommended

Gebhardt 15 PenfUsion, m general, has been found to improve hepatocyte survival and the metabohc performance of these cells (1,2,4,9). Because of the many problems that may occur m a self-made system like the one described here, frequent momtoring of the oxygen tension and pH of the medium before tt enters, and after it leaves, the culture chamber 1s recommended. Likewise, the viability, metabohc performance and waste-product formatton should be assayed at appropriate intervals. Assays should mclude the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolmm bromtde (MTT) assay, enzyme mductron (e g., tyrosme ammotransferase by dexamethasone), and measurements of lactate accumulation m the culture medium 16. By cocultivation with LEC, an even better phenotypic stabilization of the hepatocytes can be obtained (3) With respect to biotransformation of drugs, however, cocultivation may result m altered phase II reactions (test with pure LEC) and may hamper easy determmatton of hepatocyte-related metaboltc rates. 17 The closed perifusion mode is chosen for cultivation for studies on enzyme mduction, for producing large amounts of drug metabohtes, and for studies on proliferation (9) The open perifuston mode 1s chosen for kinetic studies (drug metabolism), the production of mtermediates, and for cytotoxicity studies (4) 18. Switching between the closed and the open mode (and vice versa) is possible, provided the right medium 1schosen. 19. Short-term perifusion medium can be serum-free and may even lack some components of the basic medium, provided that the cells survtve for the necessary period of time when exposed to such artificial condttions 20. Long-term perifusion medium should at least contain the supplements listed m Subheading 2.2. When longer periods of cultivation are intended (>l wk), the addition of small amounts of serum IS unavoidable. 21 Flow rates should not exceed the limits given, particularly for long-term penfusion, otherwise, cells might detach or even die. 22 For steady-state kmetic measurements it ts important that the ratio between the number of cells and the volume of medium is as high as possible and that samples are taken immediately after the medium has flowed over the cells. By reducing the diameter of the tubing that drams the medium a long lag-phase can be avoided 23. The flow rate should be adjusted to the expected metabolic rates, in order rapidly to establish a steady-state condition and to facilitate determmation of metabolic products 24 For studies on mductton of enzymes it is important to take mto account that inducers may be active m the pertfusion system at concentrations much lower than those found wrth conventtonal cultivation (4,9)

References 1. Gebhardt, R. and Mecke, D. (1979) Perifused monolayer cultures of rat hepatocytes as an improved m vitro system for studies on ureogenesis. Exp Cell Res 124,349-359

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2. Gebhardt, R. and Mecke, D. (1979) Permissive effect of dexamethasone on glucagon induction of urea-cycle enzymes in perifused monolayer cultures of rat hepatocytes. Eur. J. Blochem 97,29-35. 3. Wallach, D. F. and Sherwood, P. (1976) Diffusion m tissue cultures on gas-permeable and impermeable supports. J Theor. Bzol. 56,443-458 4. Gebhardt, R., Wegner, H., and Alber, J (1996) Penfusion of co-cultured hepatocytes: optimization of studies on drug metabolism and cytotoxicity m vitro Cell Biol. Toxlcol. 12,57-68 5. Gebhardt, R (1994) Improved drug metabohzmg capacity of hepatocytes co-cultured with epithelial cells and maintained m a perifusion system, m Alternatzves to Anzmal Testing (Remhard C , ed.), Verlag Chemie, Wemheim, Germany, pp. 141-146. 6. Williams, G. M. (1976) Primary and long-term culture of rat hver epithelial cells Methods Cell Biol. 14,357-364. 7 Guguen-Gmllouzo, C., Clement, B., Baffet, G., Beaumont, C Morel-Chany, E , Glaise, D., and Guillouzo, A. (1983) Maintenance and reversibility of active albumin secretion by adult rat hepatocytes co-cultured with another liver epithehal cell type Exp Cell Res 143,47-54. 8 Dunn, J C , Yarmush, M. L , Koebe, H G., and Tompkms, R G. (1989) Hepatocyte function and extracellular matrix geometry* long-term culture m a sandwich configuration. FASEB J 3, 174-177. 9. Gebhardt, R and Fischer, S (1995) Enhanced sensitivity of perifused primary rat hepatocytes to mitogens and growth modulation by carcinogens Toxic ln Vitro 9, 445-45 1.

Human Hepatocyte

Culture

Jean Bernard Ferrini, Jean-Claude Ourlin, Lydiane Pichard, Gerard Fabre, and Patrick Maurel 1. Introduction Primary

culture of hepatocytes

IS an in vitro model widely used to mvestt-

gate various aspects of liver physiology and pathology (1). In particular, such cultures have been extensively used for assessing the expression and function of drug-metabolizing enzymes including cytochromes P450 (CUP), drug metabolism, drug-drug interactions, and the mechanisms of cytotoxtcity and

genotoxrcrty. Most of these studies have been carried out with rodent hepatocytes. However, because of species-specificity m both the regulation and activity of drug-metabolizing enzymes, extrapolation from animals to humans is not generally possible. For this reason, several groups have developed human hepatocyte culture systems (2-s). The technique used to isolate human hepatocytes is based on the two-step collagenase perfusion first mtroduced by Berry and Friend (9) and modified by Seglen (10). Originally performed in sztu for obtaining hepatocytes from the adult rat, this technique of perfusion has been adapted to the ex vivo treatment of human liver tissue. The aim of this chapter is to describe the authors’ experience in the rsolatlon of hepatocytes from human liver tissue and the preparation of short- and long-term cultures

1.7. Human Liver Samples 1. The use of human hver samples for hepatocyte preparation for sclentrfic purposes has to be approved by National Ethics Cornmrttees or by other regulatory authorities 2 Because of the extensive use of donor livers for transplantanon, the avallablhty of whole human liver has dramatically decreased in the last few years Some donor livers are considered by surgeons to be unsuitable for transplantation (owing to a From Methods m Molecular Bology, Vol 107 Cytochrome P450 Protocols Edlted by I R PhIllIps and E A Shephard 0 Humana Press Inc , Totowa, NJ

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et al

high level of steatosts or cholestasts for example); however, these are generally not suitable for hepatocyte isolation either (see Note 1) 3 Our main source of human liver tissue therefore consists of hepattc lobes or smaller fragments resected for medical purposes. In general, the pathologies requnmg such resections include primary tumor, metastasis, adenoma, angioma, or hydatid cyst. This kmd of sample has many advantages. a. as it would otherwise be discarded, the impact of ethical considerations is greatly reduced; b it is generally used within 4 h of removal (and m many occasions withm 1 h), the cold ischenna period thus being reduced with respect to whole donor livers; c anatomo-pathologtcal data are available; d a sufficient amount of histologically normal tissue 1sgenerally available 7.2. Requirements 1, The quality of the perfusion is crttical for hepatocyte isolation. To this end, several requirements concerning the liver sample itself must be fulfilled 2. Mmimum leakage must occur during perfusion For this purpose, the sample must be encapsulated m Ghsson’s capsula on all areas except, obviously, the edge left by the surgeon. If a tumor 1spresent wtthm the tissue to be perfused, it should be removed at the end of the perfusion and not before. Indeed, tumor resection before sample condmonmg would strongly compromise the quality of the perfusion. Thus point requires good coordmatton with the surgeon and the anatomopathologist (see Note 2) 3 There must be several veins apparent on the cut edge, these will be used for perfusion Usually, these will have been sutured by the surgeon during the operation to avoid excessive bleeding and must be reopened for perfusion (see Note 3) 4 The mass of the liver sample is another critical parameter. It should be between approx 50 and 400 g The problem with small samples is the difficulty of finding good vascular entries to obtain satisfactory perfusion The problem with samples that exceed 400 g is that the ratio of the mass of tissue to the mass of collagenase is too high Thts results m a decrease in the yield and quality of hepatocytes. When a whole liver is used, it is better to split it mto two or three smaller pieces each meeting the requirements of items 2 and 3.

1.3. Conditioning When the tissue is collected m a distant surgery department (transport duration within 3-4 h) rt 1s washed m the operating theater wtth 24 L of Euro Collins buffer at 4OC, depending on the size of the sample. Washing is considered to be sufficrent when the effluent shows limited blood coloratron. The sample ts then placed in a sterile plastic bag, in the presence of a sufficient amount of Euro Collins buffer to overlay it, and 1s transported on me. When the tissue 1s collected locally (transport duration less than 1 h), the Euro Collins washing step is omrtted.

343

Human Hepatocyte Culture 2. Materials 2.1. Buffers and Solutions Buffers and solutions are prepared in deionized through 0.22~pm filters, and stored in refrigerated

water, sterilized by passing stoppered bottles.

I Euro-Collms buffer. 2.05 g/L KH,P04, 7.4g/L K,HPO,, 1 12 g/L KCl, 0 84 g/L NaHCOs, and 35 g/L glucose, pH 7.33. 2. N-[2-hydroxyethyl]piperazme-w-[2-ethanesulfomc acid] (HEPES) buffer 20 mM HEPES, 120 mMNaC1, 5 mA4KC1, 0.5% glucose, pH 7.4. 3 [Ethylene-bis(oxyethylenenitrilo)]tetraacettc acid (EGTA) solution* 0 5 mM EGTA, 58.4 n&J NaCl, 5.4 mA4 KCl, 0.44 mM KH2P04, 0 34 mA4 NaHPO,, 25 mMN-trzs[hydroxymethyl]methylglycme, pH 7 2. 4. Antibiotic solution* 10 000 U/mL pemcillm, 10 mg/mL streptomycm Add 10 mL/L to HEPES buffer and to the EGTA solution 5. Fungtzone solutton 250 pg/mL fungtzone. Add 3 mL/L to HEPES buffer and EGTA solution 6. Antioxidant solution. 100 mM manmtol, 100 mM sorbitol, 100 mM glutathtone. Add 1 mL/L to HEPES buffer before washing the tissue 7. 70 mM CaC12 solutton. Add to HEPES buffer for collagenase solutton 8 Glutamme solution. 8.75 g glutamine are added to 250 mL water; heat at 37°C for a few mm m a thermostated water bath to solubtlize. (For supplementatton of Ham-F12-Wtlham’s E [H/W] medium). 9. BSA solution for hepatocyte isolation* dtssolve 5 g of BSA (fraction V)/L of HEPES buffer. Sterilize by passing through a 0 22+an filter. Supplement wtth antibiottcs and fungizone. 10. BSA solution for supplementation of William’s medium E (W) dissolve 2.5 g BSA m a final volume of 100 mL of W Sterthze by passing through a 0.22-p filter. 11 PurseptR (Merz and Co, Frankfurt, Germany) for hqutd and instrument decontamination A 3% solution of this product inactivates viruses, including hepatms B virus and HIV, m less than 12 h.

2.2. Culture Media We use two different chemically and hormonally defined culture media for short (approx 1 wk) or long term (approx 1 mo) cultures.

2.2.1. H/W Medium (Short-Term

Cultures) (11)

1. Ham-F 12 medium: Dissolve the amount of powdered medium required for 5 L in approx 4.5 L of deionized water. Add 5.88 g NaHCOs and, after 15 mm bubblmg with a mixture of 95% O2 and 5% COZ, adjust to pH 7.4. Adjust volume to 5 L 2. William’s medium E. Dissolve the amount of powdered medium required for 5 L in approx 4.5 L deionized water. Add 11 g NaHCO, and, after 15 mm bubbling wrth a mixture of 95% O2 and 5% COZ, adJust to pH 7.4. Adjust volume to 5 L

Ferrini et al.

344

3. Combme H and W media and sterilize by passing through a 0 22-pm filter The mixture 1s kept refrigerated in the dark m 1-L stoppered bottles H and W media may also be purchased (but at a higher price) as liquid media. 4 MIX of additives for Ham-F12/Wtlham’s E medium (Mix H/W). Preparation for supplementing 25 L of culture medium The Mix H/W 1s prepared by combming three submixes, 1,2, and 3, that are mdividually prepared as follows (see Note 4). Submix 1 3 1.5 g glucose, 2.5 x lo6 units pemctllm and 2.5 g streptomycm are first dissolved in 500 mL deionized water and combined with 250 mL glutamme solution and 75 mL fungtzone solutton. Submrx 2: 1.1 g sodium pyruvate, 1 mg dexamethasone (dissolved m 500 pL ethanol), and 1.25 g transferrm are drssolved m a final volume of 75 mL with deionized water Submtx 3: 100 pL ethanolamme, 50 mg msulm (dissolved m 10 mL water containing 100 pL glacial acetic acid), 5 mg glucagon (dissolved in 10 mL water containing 100 pL of 1 A4 NaOH), and 37 5 mg lmoleic acid are dissolved m a final volume of 25 mL with deionized water The three submixes are mdivtdually sterilized by passing through 0.22~pm filters and combined to form the MIX H/W (total volume 925 mL). Mix H/W IS allquoted in 37-mL fractions, the volume necessary for 1 L of H/W medium, m 50-mL Falcon tubes and stored at -20°C until use 5. Vitamin C solution: 50 mg m 2 mL of detomzed water. Sterilized by passing through a 0 22-pm filter. Prepare just before use. 6 The final chemrcally and hormonally defined H/W medium is prepared just before use by supplementmg 1 L H/W medium with 37 mL Mix H/W and 2 mL vitamin C solution

2.2.2. William’s Medium E (long-Term

Cultures) (12)

1 Dissolve two 5 L-doses powdered medium in approx 8 L deionized water. Add 23 83 g HEPES and 22 g NaHCO,, and, after 30 mm bubbling with a mixture of 95% O2 and 5% CO*, adjust to pH 7 2 Adjust volume to 10 L 2. Sterilize by passing through a 0 22-pm filter and keep refrigerated m the dark m 1-L stoppered bottles 3. The submtx for additives is prepared by adding m a final volume of 35 mL of W medmm 50 mg insulin, 25 mg linoletc acid, 25 mg transferrm, 500 ug prolactm, 5 mg somatotropm, 1.8 1 mg hydrocorttsone, 64 pg selenium acetate, 10 pg cholera toxin, 0 3 pg ethanolamme, 10 mg glucagon, 250 pg epldermal growth factor, and 100 pg liver growth factor. Add each of these as a concentrated solution (1000 times the final concentration) either m water (insulin m the presence of 0.02% acetic acid; linoletc acid, transferrin, prolactin, somatotropm, selenium, cholera toxin, glucagon in the presence of 10 nnI4 hydrochloric acid, eptdermal growth factor and liver growth factor m the presence of 0 1 M acetic acid) or m dimethylsulfoxtde (DMSO) (hydrocortisone and ethanolamme). Note that, with respect to the ortgmal formulatton of Lanford et al. (12), the amounts of eptder-

Human Hepatocyte Culture

345

ma1 growth factor and of glucagon are halved, and thyrotropin releasing factor is omitted The submix is sterilized by passing through a 0.22~pm filter. 4. Mix of additives for W medium (Mix W). Preparation for supplementing 5 L culture medium. The Mix W is prepared by combmmg 100 mL BSA solution, 35 mL of the additive submix, 50 mL of antibiotic solution and 15 mL fungizone solution. Mix W is aliquoted in 40-mL fractions (volume necessary for 1 L) m 50-mL Falcon tubes and stored at -80°C until use. 5. The final chemically and hormonally defined W medium IS prepared Just before use by supplementmg 1 L of medium with 40 mL of Mix W.

2.3. Collagenase

Solution

1. Prepare 1 L of HEPES buffer supplemented with anttbrottcs, fungizone, and 10 mL of 70 r&4 CaClz and divide it into two parts of 250 mL and 750 mL 2. Dissolve 500 mg Type IV collagenase (Sigma, St. Louis, MO) m the 250-mL aliquot of this buffer and sterilize by passing through 0.45- and 0.22~pm filters if necessary (see Note 5). Because of the cost of collagenase, this solution should be prepared only when perfusion of the tissue has been shown to proceed correctly (see Subheading 3.2., step 3). 3. Add to the 750-mL abquot of HEPES buffer This solution of collagenase will be used to dissociate the liver tissue.

2.4. Cell Culture Materials 1. 2 3. 4. 5 6. 7. 8. 9. 10. 11.

Type I collagen-coated dishes (60 or 100 mm diameter) from Iwalo Glass (Iwaki, Japan) Fetal calf serum (FCS) tested for hepatocyte cultures Lammar-flow mtcrobiology safety cabmet Nylon filter (250 mesh) sterilized by autoclavmg. Perfusion vessel (Pyrex or stainless steel), rubber tubing (hoses), teflon terminal tip, and stoppers that can be sterilized by autoclavmg Thermostated water bath for buffers and solutions Heater for perfusion vessel Pump for tissue perfusion with flux adjustment between 10 and 500 mL/mm. Vacuum liquid-aspiration device (for removal of liquid waste) Waste collectors for tissue, liquids (blood, perfusion effluents) and other solid materials (gloves, Whatman paper, alummum foil, and so on). Decontamination reservotr (50 L) for dissection instruments, perfusion vessel, tubing, and other reusable materials Standard apparatus for cell culture: incubators, low speed centrifuge, optical microscope, rotary agitator, and so on.

3. Methods 3.1. Safety Conditions 1. Virological analysis of the patient from whom the liver sample has been resected must be carried out shortly before or at the time of the operation. The serologies

Fermi et al.

2

3. 4

5

include hepatitis A, B, and C viruses, and HIV All laboratory staff should be vaccinated against hepatitis B virus and clearly informed of the possible risks of infection. Even when the vnologtcal analysis 1s negative, all expertmentation with human tissue samples must conform to the safety pohcies regarding the protectton of staff and the containment standard of the equipment and of the laboratory rooms m which tissue processmg, isolation of, and expertmentatton on, cell cultures are to be performed (European standard containment laboratory type L2) In cases where donor tissue 1s infected with a hepatotrophtc vuus, tsolatton and culture must be performed m a containment laboratory type L3 (see Note 6) All steps of hepatocyte tsolatton and culture are carried out m a lammar vertlcalflow microbrology safety hood, to protect not only the staff from contammation, but also the liver sample. Staff must wear sterile gloves, glasses, masks, and disposable coats and boots. All materials and llqutd wastes must be decontaminated prior to discarding or restertllzation by autoclavmg (for recycled materials). Instruments and materials to be reused are decontaminated by mrmersion m 3% Pursept (final concentration) for 24 h Prepare 50 L of this solutton in an appropriate reservoir shortly before hepatocyte rsolatlon. Liquid wastes are stored in an appropriate reservoir in the presence of 3% Pursept (final concentration) for 24 h. Other materials such as used culture dishes are decontammed by autoclaving before being discarded

3.2. Perfusions 1 Upon arrival m the laboratory, the liver sample is placed in the perfusion vessel and the edge is carefully examined m order to locate the various veins that will be used for perfusion (see Note 3). The volumes indicated below for buffers and solutions are adequate for a sample of approx 300 g, for smaller or larger samples, these should be modified accordmgly. 2. All soluttons and buffers are kept at 37’C. Do not oxygenate soluttons and buffers before perfusion, because this will generate oxtdative stress m the tissue. 3. The tissue 1s first washed with 2 L HEPES buffer supplemented with anttoxtdants, antibiotics, and fungizone, at a rate of approx 1 ml/mm/g of tissue with no recirculation. Anttoxidants are expected to reduce the concentration of oxidized species produced upon reoxygenation of the tissue owing to the reperfuston after the cold tschemia In this step, blood 1swashed away (tf this has not previously been carried out with Euro-Collins buffer m the operating theater), the tissue 1s warmed to 37°C and it can be ensured that perfusion 1sproceeding correctly (see Note 7). During this and further perfusion steps, the cannula is inserted successively m all veins present on the edge for approx 30 s each (one vem at a time). Care must be taken not to mlure the veins during this operation. 4. If perfusion is proceedmg normally, preparation of collagenase solutton should be started at this point (see Subheading 2.3., item 2).

347

Human Hepatocyte Culture

5. The tissue is then perfused with 1 L of supplemented EGTA solution (see Subheading 2.1., item 4), under the same conditions as described above, with no recirculation 6 The tissue is perfused with 1 L supplemented HEPES buffer (see Subheading 2.1., item 4) to remove EGTA, under the same conditions as previously described At the end of this step, the reservou of the perfuston vessel is emptied and washed several times with HEPES buffer. 7 The tissue is then perfused with the concentrated collagenase solution under the conditions previously described, except that here the solution is recirculated and that the rate of perfusion is reduced to 100 mL/min The duration of this step varies from one sample to another, but generally lasts for a maximum of 20 mm. During this step, softening of the tissue gradually appears as well as marbling, indicating that dissociation is proceeding efficiently (see Note 8)

3.3. Hepatocyte

Isolation

and Washing

1. At the end of the collagenase perfusion, the liver sample is transferred mto a new stainless steel vessel, the Ghsson’s capsula is opened m several places, and the tumor or metastasis, if present, is (are) removed and sent to anatomopathology (see Note 2). 2. The tissue is gently disrupted with scissors. 3 The homogenate is complemented with 5-10 volumes of 0 5% BSA m HEPES buffer 4 The homogenate is filtered through a nylon filter (250 mesh) and the filtrate is distributed into 150-mL centrifuge tubes The filter is washed twice with approx 100 mL of HEPES buffer to collect the hepatocytes that are trapped m the undissociated tissue homogenate. 5 Tubes are centrifuged for 3 mm at 50g. 6. The supematant is discarded and the pellet, representing the hepatocytes, is gently resuspended in 100 mL of H/W culture medium per tube by 5 successive up and down runs with a pipet (see Note 9). 7. Steps 5 and 6, are repeated twice At the end of the last centrifugation, the yield of the preparation may be roughly estimated by measurmg the volume of the pellet: 1 mL of pellet represents approx 1 x lo8 cells For more precise counting of cells, see next steps. 8 At the end of the last washing, the pellet is resuspended m an equal volume of H/W culture medmm and homogemzed gently with a pipet as described m step 6 9. 500 pL of hepatocyte suspension is dispersed m 9 5 mL of H/W culture medium. 250 pL of this suspension are placed m a polystyrene tube and supplemented with 50 pL of a 1% Trypan blue solution, After 2 mm at room temperature, a lo-pL aliquot of this suspension is placed in the compartment of a hemocytometer cell for counting. 10. Yield and viabihty of cells are classically evaluated by exammauon under a mtcroscope using the Trypan-blue exclusion test In our hands, the yield and viabihty are, on average, 7 x lo6 cells per gram of liver tissue and 85%, respectively (see Note 10).

Ferrini et al. 3.4. Hepafocyfe

Plating and Culfure

1, After evaluation of yield and vlablhty, an appropriate amount of H/W culture medium is complemented with FCS (5% in volume) 2. The hepatocyte suspension is diluted m this medium to 3.5 or 10 x lo6 viable cells/ml for plating m 60 or 100 mm dlam culture dish, respectively 3. Culture dishes are distributed on stainless steel trays (7 dishes of 100 mm or 18 dishes of 60 mm per tray) and 2 or 7 mL of culture medium are added per 60 or 100 mm dish, respectively. 4 Then, 1 mL of an appropnately diluted suspension of cells (3 5 or 10 x lo6 viable cells) is added per dish. This number of cells per dish corresponds to a confluent monolayer (see Note 11). Care must be taken to resuspend cells frequently by gentle circular agitation whilst dlstrlbutmg to the culture dish. 5 Cells are evenly distributed on the dish by gentle agitation (see Note 11) 6 Culture dishes are then placed m an incubator, m a humid atmosphere of air 5% CO2 at 37’C. 7. After 4 h, the serum-supplemented medium 1s discarded and replaced with 3 or 8 mL of new serum-free medium per 60 or 100 mm dish, respectively (see Note 12).

8 The culture medium 1s then renewed every 24 or 48 h, for short- or long-term cultures, respectively (see Note 13).

4. Notes 1. With high levels of steatosis or cholestasis, we generally found either poor yield, poor viability (<65%), and poor attachment of cells to culture dishes, or no dlssociation at all 2 The tumor IS generally not “perfused” by collagenase, this 1s apparent from the fact that the tumor does not become warm during perfusion. It IS dlssected out after collagenase perfuslon and sent to the anatomopathologlst m an appropriate storage medium. 3 The most convenient manner of proceeding here is to make a new edge (by cutting a 0.5- to l-cm thick slice), parallel to the one left by the surgeon This reveals various vascular entries and, at the same time, allows collection of some fresh tissue samples that will be used for comparisons in further studies 4. Because of the numerous additives required to supplement the basal culture medium, it 1spreferable to prepare a concentrated solution of all the additives, so that only one addrtlon to the medium 1snecessary; however, because of the dlfficulty of filtration for sterihzatlon, we had to split this concentrated solution mto three submixes. 5 We only use batches of collagenase with a specific actlvlty greater than 400 Sigma U/mg. Some batches appear to be contaminated by micro-organisms This is assessedby microsocopic examination of an aliquot of culture medium supplemented with 1 mg of collagenase after 24-48 h mcubatlon at 37°C under normal

Human Hepatocyte Culture

6

7

8

9.

10.

11.

12.

349

culture condmons. In such cases, the collagenase solution has to be sterrhzed either directly by passmg through a 0.22+m filter or, to prevent the filter from becoming clogged, first through a 0 45pm filter prtor to a second filtratton, through a 0.22~pm filter We have already prepared and cultured hepatocytes from a patrent who was infected with hepatrtrs C vu-us. Yield, vrabrhty, and culture aspect were not significantly different from those observed with normal liver. This is checked by touching the tissue upon perfusion and verrfymg the homogeneous warming of the liver sample. Should a part of the sample remain cold durmg this step, rt means that the vein irrigating this area either has not been found (and it would be necessary to search for it to improve the yield of preparation) or it 1s obstructed by a clot. In the latter case, a reperfusron at a higher rate may be tried (care must be taken not to damage the tissue by excessive pressure) It is important to monitor this step by direct touching of the tissue Correct perfusion and dissoctatron 1srevealed by swellmg of the various areas of the sample when moving from one vein to another The pellet IS generally formed by one “tight” part in the bottom and one “soft” part above The “soft” part contams damaged cells and erythrocytes that should be removed. This 1sperformed by slowly trltmg the tube. The “soft” pellet may represent up to 30% of material at the first washing step Its volume gradually decreases thereafter to become negligible at the third washing Removal of the “soft” pellet is crmcal m obtaining a preparation with a viability greater than 80%, and a high purity. As a general rule, we have observed that the quicker the procedure for the tsolatton of hepatocytes, the better the quality of cells and subsequent cultures Therefore, isolation must be performed as rapidly as possible Indeed, here, time 1s quality! Nevertheless, the yield and vrabrhty are widely variable from one sample to another and range between 1 and 10 x 1Ogcells, and 70 and 90%, respectively The reasons for this variability are difficult to identity, parameters such as duration of cold ischemia, health status of the liver, pathology, formatron of intratissue clots, or quality of perfusion (number and state of vessels used for perfusing), could possibly be involved The hepatm drfferenttated phenotype is maintained m confluent but not m subconfluent cultures (13) Establishment of a homogeneous confluent-cell monolayer throughout the dish 1stherefore crrtrcal. Once the dishes present on one tray are supplemented with culture medium and cells, the tray is held horrzontally and gently agitated from left to right (1 cycle/s for 10 s), and back and forth (1 cycle/s for 10 s) with a pause of 5 s in between, This allows the suspension to be spread homogeneously on the dash and avoids rotational movement of the suspensron, which would result in preferential distribution of cells towards the periphery of the dish wrth low density in the center. The culture medium must be aspirated and poured gently to avoid detachment of cells. Medium changes ~111be facilitated by placing the trays on an mclmed plane.

Ferrm et al.

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13. Phenotype characterization Several markers of hepatic phenotype have been investigated and shown to be maintained m our cultures for at least a week (shortterm cultures, STC) and 5 wk (long-term cultures, LTC). These include: a the mductton of CYPlAs and CYP3A4 in response to dioxin and rtfampicm, respectively (STC and LTC) (1422); b. the mductton of glucuronosyltransferases (STC, not tested in LTC) (23,24); c the oxtdative metabolism of drugs and reconstitution of drug-drug mteracttons (STC and LTC) (25,26); d. the production of albumin (STC and LTC), al-antitrypsm, fibrinogen and plasmmogen (LTC), e the production of apohpoprotems, ApoA, ApoB, lpa (STC and LTC) (13), f. the production of acute phase proteins (ceruloplasmm, fibrinogen, ferritm) m response to cytokmes II-l, 11-6, tumor necrosis factor (STC) (27), g the mitotic response of cells to spectfic hepatotrophic factors including epiderma1 growth factor, transforming growth factor a, hepatocyte growth factor and serum from patients with liver failure (STC, under low cell density) (21%30), h vnus delta and vnus C mfectton and replication (31)

Acknowledgments This work has been supported in part by Synthelabo-Recherche ChtllyMazarin, France (JB Ferrim), and Glaxo-France (JC Ourlin). The authors wish to thank Professors Jacques Domergue, and Jean Michel Fabre (CHR Saint Eloi, Montpellier France), Henri Joyeux (Centre Anttcancereux Val d’Aurelle, Montpellrer France), Gtlles Fourtamer (CHR Ranguerl, Toulouse France), Patrice Le Treut (CHR la Conception, Marseille France), Jean Baulleux (CHR La Croix Rousse, Lyon France), for providing human liver tissue, and Dr. Cohn Young for editorial assistance.

References 1 Gebhardt, R (1996) Special issue on international congress on hepatocytes* applications in cell biology, toxicology and medicme. Cell BzoE Tox~col. pp. l-65 2 Maurel, P. (1996) The use of adult human hepatocytes m primary culture and other in vitro systems to investigate drug metabolism m man Adv Drug Del Rev 22,105132 3. Daujat, M., Pichard, L., Pmeau, T., Fabre, G., Bonfils, C , and Maurel, P (1991) Induction protocols for cytochromes P450IIIA m vwo and m primary cultures of animal and human hepatocytes Methods Enzymol. 206,345-353 4 Morel, F , Beaune, P , Ratanasavanh, D , Flmols, J P , Yang, C S , Guengench, F P., and Guillouzo, A (1990) Expression of cytochrome P-450 enzymes m cultured human hepatocytes. Eur J Blochem 91,437-444.

5 Gomez-Lechon, M. J., Castell, J , Gmllen, I., O’Connor, E., Nakamura, T., Fabra, R , and Trullenque, R. (1995) Effects of hepatocyte growth factor on the growth

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and metabolism of human hepatocytes in primary culture. Hepatology 21, 1248-1254. 6. Ismail, T , Howl, J , Wheatley, M., McMaster, P , Neuberger, J M , and Stram, A J (1992) Growth of normal human hepatocytes m primary cultures Effect of hormones and growth factors on DNA synthesis. Hepatology 14, 1076-1082 7 Kostrubsky, V E , Strom, S. C , Wood, S G , Wrighton, S A , Sinclair, P R , and Sinclair, J. F (1995) Ethanol and isopentanol increase CYP3A and CYP2E m primary cultures of human hepatocytes Arch Blochem Bzophys 322,5 16-520 8. Li, A. P., Rasmussen, A , Xu, L , and Kammski, D. L. (1995) Rifampicm mducnon of hdocame metabolism in cultured human hepatocytes J Pharmacol Exp Ther 274,673-677 9. Berry, M. N and Friend, D S (1969) High yield preparation of isolated rat liver parenchymal cells. J Cell Biol 43,506-520 10. Seglen, P. 0 (1976) Preparation of isolated rat liver cells Methods Cell Bzol 18, 29-83

11 Isom, H. C. and Georgoff, I (1984) Quantitative assay for albumin-producing liver cells after Simian vn~.~s40 transformation of rat hepatocytes mamtamed m chemically defined medium. Proc. Nat1 Acad. Scz USA. 81,6378-6382 12 Lanford, R. E., Carey, K. D , Estlack, L. E., Smith, G C , and Hay, R. V. (1989) Analysis of plasma protein and hpoprotem synthesis m long-term primary cultures of baboon hepatocytes maintained m serum-free medmm. In Vitro Cell Dev Blol 25, 174-l 82 13. Greuet, J , Pichard, L., Ourlin, J. C., Bonfils, C., Domergue, J , Le Treut, P., and Maurel, P. (1997) Effect of cell density and epidermal growth factor on the mducible expression of CY3A and CYPlA genes in human hepatocytes in primary culture HepatoEogy 25, 1166-l 175 14. Daujat, M., Fabre, I., Diaz, D , Pichard, L., Fabre, G., Fabre, J M , Saint Aubert, B., and Maurel, P. (1990) Inducibtlity and expression of class IA and IIIA cytochromes P450 m primary cultures of adult human hepatocytes. Bzochem Pharmacol (Life Scl Adv.) 9,3 15-326. 15. Diaz, D , Fabre, I., DauJat, M., Samt Aubert, B., Bones, P , Michel, H , and Maurel, P (1990) Omeprazole is an aryl hydrocarbon-hke Inducer of human hepatic cytochrome P450. Gastroenterology 99,737-747 16. Dalet-Beluche, I , Boulenc, X., Fabre, G , Maurel, P , and Bonfils, C. (1992) Purification of two cytochrome P450 isozymes related to CYP2A and CYP3A gene families from monkey (baboon, Papio papio) liver microsomes. cross-reactivity with human forms. Eur. J. Biochem. 204,64 l-648. 17. Pichard, L., Fabre, I., DauJat, M., Domergue, J , Joyeux, H., and Maurel, P. (1992) Effect of corticosteroids on the expression of cytochromes P450 and on cyclosporin A oxidase activity m primary cultures of human hepatocytes A401 Pharmacol. 41, 1047-1055. 18. Curi-Pedrosa, R., Daujat, M., Pichard, L., Ourlin, J. C., Clair, P., Gervot, L , Lesca, P , Domergue, J., Joyeux, H., Fourtamer, G., and Maurel, P (1994) Omeprazole

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19.

20 21

22.

23

24

25

26

27

28.

29.

30.

3 1.

Fermi et al and lansoprazole are mixed mducers of CYPlA and CYP3A m human hepatocytes m primary culture. J Pharmacol Exp Ther 269,384-392 Park, G. R., Pichard, L , Tmel, M , Larroque, C , Elston, A , Domergue, J., Detxonne, B., and Maurel, P. (1994) What changes drug metabolism m crtttcally ill pattents? Two prehmmary studies m isolated human hepatocytes. Anaesthesza 49,18&191 Maurel, P (1996) The CYP3A famtly, m Cytochromes P450 Metabolic and Toxzcologzcal Aspects (Ioanmdes, C , ed.), CRC, Boca Raton, FL, pp 241-270 Wright, M. C., Maurel, P , and Pame, A. J. (1996) Induction of cytochrome P450 3A by metyrapone in human hepatocyte in culture Human Exp Toxxol 15, 203,204 Greuet, J , Pichard, L , Bonfils, C , Domergue, J , and Maurel, P. (1996) The fetal specific gene CYP3A7 1s inducible by rifamptcm m adult human hepatocytes m primary culture. Blochem Biophys Res Commun 225,689--694 Fabre, G., Bourrie, M., Marti, E., Fabre, I., Roque, C., Saint Aubert, B , Joyeux, H , Maurel, P , Berger, Y., and Cano, J. P (1990) Inductbtlity of both cytochrome P450 and UDP-glucuronosyl-transferase isozymes m primary cultures of human hepatocytes. Blochem Pharmacol (LEfe Scl. Adv) 9,79-91 Abid, A , Sabolovm, N., Batt, A. M., Siest, G., Maurel, P., Gmllemm, F., and Magdalou, J (1994) Induction of UDP-glucuronosyltransferases and cytochromes P450 in human hepatocytes. Cell. Pharmacol. 1,257-262. Ptchard, L , Fabre, I, Fabre, G , Domergue, J , Saint Aubert, B., Mourad, G , and Maurel, P (1990) Cyclosporm A drug interactions. screening for mducers and mhtbitors of cytochrome P-450 (cyclosporm A oxtdase) m primary cultures of human hepatocytes and m liver microsomes. Drug Metab Dzspos l&595-606 Maurice, M., Pichard, L., DauJat, M., Fabre, I , Joyeux, H., Domergue, J , and Maurel, P (1992) Effects of tmidazole dertvatives on cytochromes P450 from human hepatocytes m primary culture FASEB J 6, 752-758 Muntane-Relat, J , Ourlm, J C , Domergue, J., and Maurel, P. (1995) Differential effects of cytokmes on the inducible expression of CYPlAl, CYPlA2, and CYP3A4 m human hepatocytes m primary culture. Hepatology 22, 1143-1153 Blanc, P., Etienne, H., DauJat, M., Fabre, I., Pichard, L., Domergue, J., Joyeux, H , Fourtamer, G., and Maurel, P (1991) Antiproliferative effect of FK506 and cyclosporm on adult human hepatocytes m culture. Transplant Proc 23, 2821-2824 Blanc, P., Etienne, H., DauJat, M., Fabre, I., Zindy, F , Domergue, J , Astre, C., Samt Aubert, B , Michel, H , and Maurel, P (1992) Mitotic responsiveness of cultured adult human hepatocytes to epidermal growth factor, transforming growth factor a, and human serum. Gastroenterology 102, 1340-l 350. Blanc, P., Liautard, J., Greuet, J., Daures, J. P., Fabre, J M., Larrey, D , Mtchel, H., and Maurel, P. (1995) Effect of anti-ulcer drugs on DNA synthesis in adult normal human hepatocytes m culture. Hepatologv 22,s 14-8 19. Ferrmt, J. B , Pichard, L., Domergue, J., and Maurel, P. (1997) Long-term primary cultures of adult human hepatocytes. Chemzco-Bzol Inter 107,3 l-45

37 Reconstruction Marie-Cbcile

of Human Epidermis

In Vitro

Lenoir-Viale

1. Introduction Bell et al. (I) showed that a dermal equivalent can be obtained by mixing living fibroblasts with a solution of collagen that will gel and further contract mto a firm lattice by extrusion of water. Epidermal keratmocytes can be seeded on top of these lattices, giving rise to a simplified skin (2,3). Another source of keratmocytes

is the outer root sheath of the hair follicle,

which is one of the specialized appendages of human skm involved m epiderma1repair (4). Plucking hair follicles is a nonmvasive method that requires no medical qualification. Because the hair follicle will regrow, it can be considered as an infinite source of keratmocytes. A well-stratified and differentiated epidermis, which closely resembles human skin epidermis, has been obtained by implanting hair follicles vertically mto freshly cast dermal equivalents, thereafter raising the cultures to the air-liquid interface, where they are mamtamed for 2-3 wk (5). The reconstructed human epidermis has been shown to possess enzymes that catalyze the sequential transformation of drugs and endogenous compounds (6). Therefore such a system has the potential to serve as a model to study drug metabolism in vitro. In this chapter, methods are described for the preparation of dermal eqmvalents and the reconstruction of human epidermis (epidermalization). 2. Materials 2.1. Preparation

of Dermal Equivalents

1. Minimal Essential Medium (MEM) with Earle’s salts containing 25 mMHEPES, but lacking L-glutamine and NaHC03 (MEM-HEPES).

From Methods m Molecular Bology, Vol 107 Cytochrome P450 Protocols Edlted by I R PhIllIps and E A Shephard 0 Humana Press Inc , Totowa, NJ

353

354

Lenoir- Via/e

2 3 4. 5 6. 7 8 9. 10. 11 12. 13. 14 15

Fetal calf serum (FCS) (see Note 1) 200 mA4 L-glutamine (L-Glu). 100 mM sodium pyruvate Nonessential ammo actds (NEAA) Pemcillm (10,000 IV) - streptomycm (10,000 pg/mL) (PS) Fungtzone or amphothertcin B (250 pg/mL) (F) Gentamicin sulfate (50 mg/mL) (G). MEM 1OX concentrate 7.5% (w/v) NaHCO,. 0.1 NNaOH 0.5 N acetic acid: use for first dialysis of collagen solution. 0 1% (v/v) (0 017 N> Acetic acid (AcOH). Prepare from glacial acetic acid Double-dtsttlled water (DDW) Collagen type I* must be unpepsmized and can be obtained from calf skm (commercial source: Jacques Boy, Reams, France or Gattefosse, Gennevtlhers, France) (see Notes 2 and 3). Dialysis tubing SPECTRAPOR (Spectrum Medical Industries, Los Angeles, CA), cat. no 132655,32mm diameter, molecular weight cut off 6000-8000 Culture medium MEM-HEPES, 10% (v/v) FCS, 0 1% (v/v) PS, 1% (v/v) sodium pyruvate, 1% (v/v) NEAA, 1% (v/v) L-Glu, F (0.125 pg/mL). Human dermal fibroblasts. can be obtained either from skm or from a commercially available source (see Note 4) MEM 1 76X concentrate: To prepare 100 mL, mix 17 6 mL MEM 10X concentrate, 5 16 mL 7 5% NaHCOs, 1 76 mL L-Glu, 1.76 mL NEAA, 1 76 mL sodmm pyruvate, 0 176 mL PS, 0 088 mL F, 0.176 mL G, 71 52 mL DDW Store all solutions at 4°C and prepare all media under sterile conditions.

16. 17. 18. 19

2.2. Reconstruction

of Human Epidermis

(Epidermalization)

1 MEM with Earle’s salts lacking L-glutamme, but containing NaHCOs (2 2 g/L) (MEM). 2 Hydrocorttsone (HC) (Sigma, St. Louts, MO., cat. no. H4001) Prepare a stock solution (500 pg/rnL) by dissolvmg 5 mg in 1 mL ethanol and then dilute li 10 m MEM Store in 0 4 mL aliquots at -20°C 3. Cholera toxin (CTX) (Sigma, cat no C3012) Prepare a stock solution (1 mg/mL) in sterile DDW Store at 4°C 4. Epidermal growth factor (EGF) Prepare a stock solution (10 pg/mL) m phosphate-buffered salme (PBS). Store 0.5~mL ahquots at -20°C. 5 Lattice culture medium* MEM, 10% (v/v) FCS, 1% (v/v) L-Glu, 1% (v/v) sodium pyruvate, 1% (v/v) NEAA, 0 1% (v/v) PS, HC (0 4 pg/mL), EGF (10 ng/mL), CTX (0.01 pg/mL, l&i0 M) 6. Stamless-steel grids

Human Epidermis Reconstruction In Vitro

355

3. Methods 3.1. Preparation of Dermal Equivalents 3.7.7. Dialysis of Commercial Collagen Solution Dialyze this solution (using dialysis tubing previously boiled in DDW) for 1 x 48 h agamst 0.5 Nacetic acid, then for three times 12 h agamst successive changes of AcOH (see Subheading 2.1,, item 13), in a 5-L Erlenmeyer flask, at 4”C, with magnetic stirrmg, under sterile conditions. 3.1.2. Preparation of Dermal Equivalents (see Note 5) 1. Calculate the volume of each solution needed according to the number of dermal equivalents to be prepared. For one 10 mL volume lattice, mix’ 4.6 mL MEM 1.76X concentrate, 0.9 mL FCS, 0 5 mL 0.1 N NaOH, 0.9 mL AcOH, 4 x lo5 fibroblasts m 1 mL culture medium. The volume of the lattices can be increased according to the desired size and thickness of the dermal equivalents. 2 In a large Erlenmeyer flask containmg a magnetic stirring bar, pool the solutions listed m item 1, then add the cells at the last moment, maintaining a gentle magnetic stirring 3. Ahquot the pooled solutions into 25-mL Erlenmeyer flasks (7.9 mL/ flask). 4. Slowly and carefully add 2.2 mL collagen solution along the inner surface of the Erlenmeyer flask. The volume of collagen depends on the concentration of the solution. If the collagen solution is too dilute, increase the volume of collagen solution and decrease the volume of AcOH added (see item 1) m order to keep the final total volume at 10 mL. 5. MIX the pooled solutions vigorously (rotary shaking by hand). Prepare one Erlenmeyer flask at a time, two at most. 6. Pour each pooled solution (10 mL) into a 60-mm diameter bacterlologlcal Petri dish and place it immediately into an incubator at 37’C with an atmosphere of 5% coz. 7. Leave in the incubator for 10-l 5 min for the lattices to gel.

3.2. Reconstruction of Human Epidermis (Epidermalization) 3.2.1. Preparation of the Hair Follicle Explants 1. Prepare two 35-mm diameter culture dishes containing 1.5 mL of the culture medium (see Subheading 2.1., item 17). 2. Pluck hair follicles from several areas of the scalp of normal volunteers (see Note 6). Use only follicles in the anagen phase, i.e. those having a visible bulb and sheath. 3 Cut the hair shafts 2 mm above the sheath and immerse the hair follicles m culture medium contained in one of the two dishes. 4. Remove the bulbs with scissors, because their soft end would hamper the implantation of the explant into the collagen gel

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Lenoir- Via/e

Fig. 1. Plucked anagen hair follicle, with the bulb (B), hair shaft (HS), inner-root sheath (IRS) and outer-root sheath (ORS). Arrows indicate where hair follicle is sectioned for implantation. Open arrowhead locates the opening of the sebaceous gland duct. Dotted lines represent the portions that are actually inserted into the collagen lattice. Bar represents 500 pm. Adapted from ref. 5 with permission granted by Academic Press. 5. Cut each follicle into two pieces and immerse each piece in the culture medium contained in the second dish. Five half-follicles are required for each 60-mm diameter lattice. A plucked anagen hair follicle is shown in Fig. 1.

3.2.2. Reconstruction

of Human Epidermis (Epidermalization)

1. Plant the explants in an upright position in the freshly cast collagen gel. Plant five half-follicles in a 60-mm diameter dish or 25 in a 120 x 120~mm square dish. Incubate at 37’C in an atmosphere containing 5% CO*. The gel contracts within a few hours. Check for detachment from the edges and the bottom of the dish. 2. On d 6, after contraction of the lattices, raise the cultures onto stainless-steel grids. Add lattice culture medium so that the dermal equivalents are in contact with the medium but the hair follicles are exposed to air (see Note 7). 3. Change the medium twice a week. 4. On d 12, using two tweezers, remove the hair follicles from the lattices, taking care not to damage the growing epithelium. By d 15, the lattices should be covered completely by keratinocytes (see Fig. 2). Complete differentiation is normally obtained after 3 or 4 wk (see Figs. 3 and 4) (see Note 8).

4. Notes 1. Batches of FCS are not equivalent and it is very important to test each batch to obtain an optimal differentiation of the reconstructed epidermis. 2. The batch of collagen must be tested because time of gelling varies with batches and implantation must be done when the dermal equivalent becomes solid (i.e., when it becomes opaque). Moreover the quality and concentration of the collagen solution is important for the contraction of the dermal equivalent, which should have a final size of about 15-18 mm diameter from a 60-mm diameter

Human Epidermis Reconstruction

In Vitro

357

Fig. 2. Progress of outgrowth of epidermal sheet after hair follicles were implanted into a dermal equivalent. Hair follicles of two different donors were cultured for indicated periods (in d) before staining with 1% rhodamin B followed with Nile blue sulfate (l/10 000 dilution). Adapted from ref. 5 with permission granted by Academic Press.

initial size. This optimal final size allows a good thickness of the dermal equivalent so that it is firm enough to be easily handled when it is raised onto the grid. 3. Alternatively, collagen can be prepared from rat tails: a. Kill the rats, preferably young (150475 g) Sprague Dawley, by cervical dislocation and remove the tails. b. Wash freshly obtained tails with cold tapwater and soak in 70% (v/v) ethanol for 20 min. c. Remove the skin and pull out the tendons with forceps, one segment at a time, starting from the thin end. Never allow tendons to become dry. Place tendons in sterile distilled water and weigh them.

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Lenoir- Viale

Fig. 3. Morphology of reconstructed epidermis obtained after 28 d of culture. (SB, stratum basale, SS, stratum spinosum, SG, stratum granulosum, SC, stratum corneum). Bar represents 50 pm. Adapted from ref. 5 with permission granted by Academic Press.

4.

5.

6. 7.

8.

d. Mince the tendons into small pieces with scissors and place the pieces into AcOH (40 n&/g of tendon). Keep the mixture at 4°C for 2 d, stirring occasionally by hand. e. Centrifuge the mixture at 4’C for 60 min at 10,OOOg. f. Recover the supematant, which corresponds to a collagen solution (2-3 mg/mL) that is ready to use (should be clear). Fibroblasts from different donors do not grow in the same manner and they do not have the same capacity to contract the dermal equivalent. This capacity should be tested and the number of fibroblasts used to prepare one lattice should be adjusted as necessary to obtain the optimal final size and thickness. Although dermal equivalents are prepared in bacteriological Petri dishes, tibroblasts tend to attach to the edges and the bottom of the dish. For this reason, it is important to prepare the dermal equivalents in the morning and to check the detachment l-2 h later and also in the evening and the following day. A good outgrowth was observed in about 80% of the cultures obtained from the hair follicles of the 30 donors tested (SJ, so it is important to verify the capacity of the follicles to give rise to epithelium. The quantity of medium under the grid is critical; the grid must be flat so that the medium reaches the grid homogeneously in order to obtain a homogeneous differentiation of the reconstructed epidermis. Epidermalization can be done also by inserting skin punch-biopsies into a freshly cast dermal equivalent (7).

Human Epidermis Reconstruction

In Vitro

Fig. 4. Distribution of differentiation markers in the reconstructed epidermis: 67-kDa keratin (A), membrane transglutaminase (IS), involucrin (C), and filaggrin (D). Dotted line represents the dermal-epidermal junction. Bar represents 25 pm. Adapted from ref. 5 with permission granted by Academic Press.

References 1. Bell, E., Ivarsson, B., and Merrill, C. (1979) Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc. Natl. Acad. Sci. USA 76, 1274-1278. 2. Bell, E., Ehrlich, H. P., Buttle, D., and Nakatsuji, T. (198 1) Living tissue formed in vitro and accepted as a skin-equivalent tissue of full thickness. Science 211, 1052-1054.

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3 Asselmeau, D., Bernard, B. A , Bailly, C., Darmon, M., and Prumeras, M (1986) Human epidermis reconstructed by culture: 1slt normal? J Invest Dermatol 86, 181-185. 4. Irvin, T.T. (1981) Prlnczples andpractrce of wound healmg Year Book Medical, Cambridge, UK 5 Lenoir, M C , Bernard, B A., Pautrat, G., Darmon, M , and Shroot, B (1988) Outer root sheath cells of human hair follicle are able to regenerate a fully dlfferentrated epidermis m vitro. Dev Biol 130,6 10420. 6 Pham, M. A , Magdalou, J , Siest, G , Lenoir, M C , Bernard, B A., Jamoulle, J C , and Shroot, B (1990) Reconstrtuted epidernns. a novel model for the study of drug metabolism m human epidermis J Invest Dermatol 94,74%752 7. Coulomb, B., Saiag, P., Bell, E., Brertburd, F., Lebreton, C., Haslan, M , and Dubertret, L. (1986) A new method for studying eptdermahzatron m vitro. Br J Dermatol 114,91-101.

Gene Transfer and Expression Frank Gaunitz and Monika Papke 1. Jntroduction

DNA transfectton has become an important technique in molecular biology. The fusron of putative gene-regulatory sequences to reporter genes and their mtroduction into a eukaryotic cell can be used for a detailed analysis of cisacting DNA regulatory sequences (1). DNA regulatory elements such as those responsive to CAMP (2) or to glucocorticoids and antiglucocortrcoids (3) as well as to drugs and xenobiotics such as phenobarbital (4,s) can be identified through the use of approprtate reporter-gene constructs. Cotransfection experiments with vectors expressing specific transcrtption factors can be used to identify the role of these transcription factors in the expression of specific genes (6) and m addition, the developmental activation of a promoter can be examined (7). The expression of functional protein m transfected cells can be useful for studies on genetic toxicology (8) or biochemical pathways (9). By the mtroduction of mutated DNAs that produce modified protems m the transfected cell, the functional significance of protein domains (10) and individual ammoacid residues (11,12) can be elucidated, and the transfection of cDNAs isolated from patients has already proved useful for the analysis of molecular defects that result m genetic disorders (13). For the elucidation of regulatory processes,cell lmes are frequently used for transfection experiments becausethey can be easily transfected by several techniques. In contrast, primary cells, especially cultured hepatocytesalthough they should resemble the situation in vivo more closely-are less frequently used because they are more difficult to transfect. The low transfection efficiencies and cytotoxic effects with different protocols are a common problem. From Methods m Molecular Brology, Vol 107 Cytochrome P450 Protocols Edlted by I R Phllhps and E A Shephard 0 Humana Press Inc , Totowa, NJ

361

Gaunitz and Papke In order to overcome these obstacles, we have developed improved methods for transfection of primary cultured hepatocytes. A CaP04-based protocol (14) and a method using the catiomc lipid MaxifectmTM that are routmely used m our laboratory will be described m this chapter. The chorce of the method to be used depends on the question to be addressed and the number of transfectrons that need to be performed. Both methods are equally reproducible, but lipofection with Maxifectin yields higher transfectlon efficiency and 1s less cumbersome to perform. Although the protocols described were specifically developed for the transfection of primary cultured hepatocytes, they can be used for the transfection of prrmary cells derived from various tissues and also of cell lines, and result m equal or even greater transfectton efficiencies, especially with cell lmes. In addition to the transfectron protocols, we will describe three highly sensitive luminometric reporter-gene assays that we routinely use m our laboratory for the analysis of DNA regulatory elements. 1.1. Cotransfection of Reporter of Transfection Experiments

Genes and Analysis

Several approaches designed to elucidate mechanisms involved in transcrrptronal regulation require the delivery of more than one gene to the cells. If, for example, the role of a specific transcription factor in the expression of a reporter gene is to be Investigated, by use of a plasmid expressmg the factor m the target cell, it is necessary that the expression plasmid enters the same cell as the appropriate reporter gene. Both methods described m thrs chapter can be used to transfect more than one gene mto a single cell tf the plasmids are mixed prior to incubation with the transfection agent. In fact, rt is strongly recommended that a control plasmrd 1scotransfected together with the reporter-gene construct m order to normalize for different transfection efficiencies. In our laboratory we usually cotransfect a reporter gene expressing /3-galactosidasein order to correct for transfection efficiency. Figure 1 presents data from several transfection experiments done with two plasmids. One expresses a P-galactosidase activrty, the other a beetle luciferase actrvrty. Each data point representsthe two measured activities obtained from a single transfection Although the activities obtained m each transfection experiment differed owing to dtfferent transfection efficienctes, luciferase and P-galactosrdaseacttvrty always correlate as can be seen from the linear fit. If a high background of endogenous P-galactosrdase is a problem, a control plasmrd expressing Renilla lucrferase can be used. The relative level of expression from a specific reporter gene can be determined as shown in Fig. 2.

363

Gene Transfer and Expression 1600000

,

3z B 12000001400000

f 100000049, 800000'2

600000

-

$0

400000

-

$z

200000

-

01 0

.,.,',.,',.,.,l 20000

40000

60000

luctferme

80000

activity

100000

120000

140000

(rlu)

Fig 1 Comparison of luclferase and P-galactosldase expression from two cotransfected plasmids m transfectlon experiments with different efficiencies Rlu, relative luminescence units

2. Materials 2.1. Cell Culture Conditions 1. William’s medium E supplemented with 10% fetal calf serum (FCS), 2 mM glutamme, 10eg A4 msulm, 10m7M dexamethasone, 500 U/mL penicillin, and 40 U/mL streptomycm (see Note 1). 2. Incubator at 37”C, 90% humidity, 5% CO;, and 95% air.

2.2. Transfection

Using Calcium Phosphate

1. 1.O A4 CaCl*: Filter-sterdlze and store at -20°C (see Note 2). 2. 2xHeBS: 0 28 A4 NaCl, 10 mMKC1, 1.5 mM NaH2P04, 2% glucose, 42 mM N-2-Hydroxyethylplperazine-N’-2-ethanesulfomc acid (HEPES), pH 7 10 (see Note 3). 3 HeBS-glycerol. 15% glycerol, 1xHeBS (prepared from the 2xHeBS stock). 4. TE: 10 mMTris-HCl, pH 7.6, 1 mA4EDTA. 5 Supercoiled plasmid DNA: 1 ctg/pL m TE (see Note 4). 6. Polystyrene conical tubes (Falcon@’ 2095, 17 x 120 mm) (see Note 5).

2.3. Transfection

Using Maxifectin

Reagent

1. Maxifectm kit (Surovoy, Rottenburg, Germany) mcluding Enhancer solution. 2 Binding buffer 0.9% NaCl, 10 mM HEPES, pH 7 4

Unifectm

and

Gaunitz and Papke

364 EAl(RG)

-EAl(Buffcr)

-

EAl(0) - EAl(Buffcr)

WW

relative level of expression =

WV

EA2(CP) - EA2(Buffer)

I

EA2(0) - EA2(Buffer)

VW

P(O)

if the endogenous activity of the enzymes measured is low enough (EAl(0) -EAl(Buffer) andEA2(0) - EAZ(Buffer)) this formula can be simplified to: EAl(RG) relative

-EAl(Buffer)

level of expression =

EA2(CP) - EA2(Buffer) EAl(RG): LU(CP): EXl(Buffer): &\2(Buffer):

Enq me-AC tit tty from tbe Enzyme-Activity from the Background activtty from (e.g. autolummesccnce) Background achvrty from

P(RG):

Amount

EAl(0):

Assay1 performedwth cells not transfectedwith plasmid (e.g. endogenous IJ-galactostdase activity) Awnyl performed with cells not trrnsfected with pbrmid Amount of protein in ljsrte of untr*asfWcd cells

EA2(0): P(0):

of protein

reporter gene control plssmrd substrate of assay1 substrate

of nury2

ia tysate of traasfected

cells

Fig 2 Parameters and calculation used m the interpretation of a transfectron expelvnent 3. TE* 10 mMTris-HCl, pH 7.6, 1 miMEDTA. 4 Supercooled plasmld DNA: 1 pg/& m TE (see Note 4)

2.4. Harvest

of Cells

1. Hank’s solution: 137 miI4 NaCl, 5 4 mM KCl, 0 4 mM MgS04, 0.5 mM MgC12 6H2O, 0.35 mA4Na2HP04, 0 44 mM KHPPOJ, 2 mM HEPES, pH 7 4 2. Lysrs buffer: 77 mM K2HP04, 23 mM KH2P04, 0 2% Trtton X-100, 1 rmI4 dithrothreltol (DTT; to be added Just before use), pH 7 8 (see Note 6)

2.5. In Situ Detection

of pGa/actosidase

Activity

1. 1 A4 Sodium Phosphate Buffer-Stock (SPB-Stock). 770 nnI4 Na2HP04, 230 mM NaH2P04, pH 7.3. 2. Paraformaldehyde solutron: Drssolve 8 g of paraformaldehyde m 150 mL 0.1 M SPB (preheated to 60°C prepared from the SPB-Stock with sterile water) Add one drop of 10 M NaOH to clear the solution (see Note 7). Store the solutton at 4°C for not more than 4 wk

Gene Transfer and Expression

365

3. Fixative: Dilute the paraformaldehyde solution twofold with 0 1 M SPB (prepared from the SPB-Stock) and add 8 pL of a 25 % glutaraldehyde solutlon/mL of fixative 4. Potassium ferricyanide-stock: 50 mMK,Fe(CN),. Store at 4°C m the dark for not more than 3 mo 5. Potassium ferrocyamde-stock. 50 mA4 K,Fe(CN),. Store at 4°C m the dark for not more than 3 mo. 6. X-Gal stock: Dissolve 20 mg X-Gal (5-Bromo-4-chloro-3-mdolyl-p-o-galactopyranoside) in 1 mL of NJ-dimethyl formanude. Store m a glass contamer at -20°C. 7. Staining solution: 100 mM SPB, 1.3 mA4 MgCl, (from a 1 A4 stock), 3 mM K,Fe(CN&, 3 mM K,Fe(CN),, X-Gal (1 mg/mL) Prepare from stock solutions Just before use and filter through a 0.2~pm disposable filtration unit 8 Phosphate-buffered saline (PBS) without Ca2+ and Mg2+* 2.7 mM KCl, 1.5 n-J4 KH,PO,, 140 mMNaC1,6.5 mMNa2HP0,, pH 7 4

2.6. /S-Galactosidase

Assay

1 Galacto-LightTM Chemllummescent Reporter Assay Kit (Troplx, Bedford, MA), m&ding Galacton, Galacton Reaction Buffer Dlluent and Light EmIsslon Accelerator. 2 Lummometer

2.7. Beetle Luciferase

Assay

1. Beetle Luciferase Assay Reagent: 20 mMTncme, 1.07 mM(MgCO& Mg(OH), 5H20, 2.67 mM MgS04, 0 1 mM ethylenedlamine tetra-acetic acid (EDTA), 33 3 mM DTT, 270 @4 Coenzyme A, 470 fl Luciferin, 530 pA4, ATP, pH 7 8, store in ahquots at -70°C (see Note 8). 2 Luminometer 2.8. Renilla

Luciferase

Assay

1. Dual Luclferase Reporter Assay System (Promega, Madison, WI), including Stop & GloTM Buffer, Stop & GloTM Substrate and Stop & Glo Substrate Solvent 2. Luminometer.

3. Methods 3.1. Transfection

Using Calcium Phosphate

1. Four h before the start of transfection, remove the medium from the cells and add 1 5 mL of fresh medium per well of a &well plate (see Note 9) 2. Start the CaP04/DNA precipitate preparation 25-30 mm before the solution IS added to the cells Dilute 1 MCaCl, to 250 mA4wrth sterile dlstllled water Then supplement 500 & of this solution with 12 ~18supercolled plasmld DNA and add It dropwlse to 500 $ 2xHeBS m a polystyrene conical tube. Addition should take 60 s and is performed using a glass Pasteur plpet. Durmg the addition, bubble air from the bottom of the tube by use of a second glass Pasteur plpet attached to

366

Gaunitz and Papke

a mechanical pipetor. After addition, mix the solution by vortexmg for 2-3 s at maximum speed and incubate at room temperature for 25-30 mm. 3 Before addition of the CaPO,/DNA precipitate to the cells resuspend It by carefully plpetmg it twice through a 1000~clr, plpet tip (see Note 10) 4. 200 pL of the mixed preclprtate should then be added dropwlse to the cells m one well of a Macroplate m 1 5 mL medium During the addition, gently swirl the dish (see Note 11) 5. After an 8-h exposure to the precipitate, wash the cells twice with Hank’s solution Then add 0.5 mL HeBS-glycerol solution to each well and expose the cells to this solution for exactly 2 min at room temperature. Aspirate the glycerol, then rinse the cells with Hank’s solution followed by a rinse with fresh medium (see Note 12).

6. After the addition of fresh growth medmm, the cells are incubated until lysls of the cells.

3.2. Transfection

Using Maxifectin

Reagent

1. Dilute 1 pg DNA mto 100 pL bmdmg buffer. 2 Add 2 pL enhancer solution and Incubate at room temperature for 10 mm (see Note 13)

3 MIX 7 5 pL Umfectm solution with 100 pL of bmdmg buffer m a separate tube (see Note 14)

4 Combme the DNA/enhancer solution with the Umfectin solution by gentle mixing and incubate at room temperature for 10-l 5 mm 5. Remove the cells from the incubator and add 50-200 pL of the transfection mixture to the cells m 1.5 mL of culture medium (see Notes 9 and 15). 6 After 4 h, aspirate the medium, add fresh growth medium, and incubate the cells until lysls (see Note 16)

3.3. Harvest

of Cells

At an appropriate

time after transfectlon,

the cells can be harvested

(see

Note 17). 1. Rinse the cells twice with Hank’s solution. 2 Add between 100 pL and 1 mL lysls buffer to the cells (see Note 18) 3 Remove the cells from the wells with a rubber policeman and transfer the lysate to a mlcrocentrlfuge tube (see Note 19). 4. To remove cell debris, centrifuge the lysate for 2 min at 12,OOOgand transfer the cleared lysate to a fresh tube prior to reporter-enzyme analysis (see Note 20).

3.4. In Situ Staining for p-Galactosidase 1 2. 3. 4. 5

Activity

Aspirate the medmm from the cells. Rinse the cells twice with PBS at room temperature. Add 1 mL fixative per well of a Macroplate and mcubate for 5 mm on ice Aspirate the fixative and rinse cells twice with PBS Add 1 mL staining solution per well of a Macroplate and incubate the plate at 37°C for at least 30 mm (see Note 21).

Gene Transfer and Expression 3.5. pGa/actosidase

367

Assay

1. Transfer 2 pL cell lysate or an appropriate dilution of it to a luminometer sample tube (see Note 22) 2. Dilute Galacton loo-fold m Galacto-Light Reaction Buffer (as much as needed for the number of assays) 3. Add 67 pL of diluted Galacton to the sample m the lummometer sample tube. 4. After exactly 60 mm of incubation at room temperature, add 100 & Light Emlssion Accelerator and measure enzyme activity in a luminometer (see Note 23)

3.6. Beetle Luciferase

Assay

1. Transfer 10 pL cell lysate to a lummometer sample tube (see Note 22). 2. Add 100 $.. Beetle Luclferase Assay Reagent and measure luminescence after a delay of 2 s.

3.7. Renilla Luciferase

Assay

1. Transfer 4 pL cell lysate to a luminometer sample tube (see Notes 22 and 23). 2 Add 20 pL beetle luciferase assay reagent and measure lummescence after a delay of 2 s (see Note 24) 3. Add 20 pL Stop & Glo Substrate diluted 50-fold in Stop & Glo Substrate Solvent and measure lummescence after a delay of 2 s

4. Notes 1. The transfectlon protocols presented have been optimized for the transfection of primary cultures of hepatocytes from different species, including rat and human. For these primary cultures, William’s E medium supplemented m the way described is well-suited for cultivation from lsolatlon throughout transfectlon. Other cells used m our laboratory are usually kept m growth medium appropriate for them and are transferred to William’s E medium 4 h prior to transfection Although highest transfectlon efficiencies are obtained with the supplements listed, they can be omitted If they are known to mfluence the outcome of the desired experiment Primary cultured hepatocytes should be cultivated for at least 12 h in medium supplemented with FCS It should also be noted that with certain systems of lipofection antibiotics should be omitted from the medium 2 We obtained the best results with CaC12 * 4H,O Suprapur@ (Merck, Rahway, NJ) 3 Because the pH of the solution is a very critical parameter we recommend that several stocks of 2xHeBS (e.g., 5 stocks, 500 mL each) from pH 7.0 to 7.2 are tested to discover which 1sbest for optimal transfectlon efficiency The best stock can be filter-stenhzed and stored m convenient aliquots at -20°C 4. Only high-purity supercooled DNA should be used for transfectlon. In our laboratory we purify plasmld from XL-l blue bacteria by affinity chromatography on Qiagen@ (Chatsworth, CA) columns. We usually determine the quality of the DNA by spectrophotometry and estimate the amount of nicked plasmld m the preparation by electrophoresls through a 0.8% agarose gel contammg ethldmm

368

Gaunitz and Papke

bromide (0 4 ug/mL). DNA with more than 5% of relaxed plasmtd strongly reduces transfection efficiency 5. In our experience, other tubes, even if they are made from identical material, may not work. 6 This lysis buffer has proved useful for cells transfected with beetle luciferaseand P-galactosidase-vectors. However, if Rendla luciferase plasmids with weak promoters are used, the passive lysis buffer supplied m the Dual Luciferase Reporter Assay System (Promega) is recommended, because Triton X- 100 may greatly intensify autoluminescence of coelenterazme, which is the substrate for Renilla luciferase 7 Prepare the paraformaldehyde solution m a fume hood and wear a mask and gloves when handling paraformaldehyde. Usually only one drop of 10 MNaOH is sufficient to clear the solution. If not, add more drops at a rate of 1 drop/min 8. The composmon of this reagent is accordmg to a protocol of Promega (Technical Bulletin No. 16 1) It can be purchased ready-made from Promega and is included m the Dual Luctferase Reporter Assay System. 9. Freshly isolated hepatocytes are usually plated at a density of 0 5 x lo6 cells per 35mm dish of a 6-well plate (Macroplate, TC, Gremer, Nuertmgen, Germany) Fresh medium is added 2 h after seeding. In our experience, primary cultured hepatocytes as well as trypsimzed cells should not be transfected withm the first 20 h after tsolatton or treatment with trypsin, because several promoters seem to be regulated m a very unpredictable manner during this time, probably by the activation of transcription factors induced by the stressof isolation or trypsinization. 10 Only use high-quality sterile pipet tips that have a very smooth surface e g , Multi@ tips. 11. In order to prevent changes m pH and temperature that may be critical for transfectton eftictency at this step, tt is tmportant that the cells are not removed from the incubator for more than 6 mm 12. Depending on the viabthty of the cells used, the glycerol step may be omitted, because glycerol is toxic for the cells and weak hepatocytes may suffer from this treatment. However, the calcium phosphate/DNA precipitate should be removed by two washes wtth Hank’s solution, followed by a wash with fresh medium. 13. The use of enhancer solution supplied wrth the Maxtfectm ktt enhances transfection efficiency for most types of cells we use m our laboratory. However, there are reports that for certain types of cells, Umfectm alone works equally well or even better 14. The ratto of DNA to Umfectm and enhancer solution described m this protocol IS optimal for the transfection of hepatocytes and many cell lines used m our laboratory. However, because the optimal amount of Umfectm m relation to the amount of DNA used is dependent on the cell type to be transfected some optimazation can be done by varying the amounts used For optimtzatton we recommend the amount of Umfectm be varied between 5 clr, and 20 p.L. 15. Transfection efficiency with Maxifectm seems to be strongly dependent on cell density In our laboratory, most cells tested showed higher transfection efficiency the lower the cell density at time of transfection. Depending on the problem to be

Gene Transfer and Expression

16.

17.

18

19 20

21.

22

23.

24

369

addressed, it must be considered whether a high overall number of cells transfected or a high ratio of transfected to untransfected cells 1smore important Although tt IS not m any case necessary to remove the transfectton medium, a better viability of transfected primary cultured hepatocytes can be obtained tf the solution is removed 4 h after the start of transfection The optimal ttme for harvest of cells depends on the type of cells and the transfectton protocol used. With CaP04 transfectton of prtmary cultured hepatocytes, expression from different reporter genes 1s highest between 10 and 20 h after transfectton (14). However, new data obtained from HepG2 cells transfected wtth Maxifectm (unpublished) mdicates highest expression close to 75 h after transfection. Therefore we strongly recommend that an imttal transfectton experiment be performed to find the best time for harvest of cells. The amount of lysis buffer to be used is dependent on the expected level of expression. For 0 5 x lo6 cells m a well of a Macroplate, use 100-200 pL of the descrtbed lysis buffer and 1 mL if the passive lysts buffer (Dual LuciferaseTM Reporter Assay System) is used. If the passive lysts buffer (Dual Luciferase Reporter Assay System) IS used simply rock the culture plates at room temperature for 15 mm and then transfer the lysate to a mtcrocentrifuge tube For the enzyme assays described, it is usually unnecessary to clear the lysate by centrifugation. However, we recommend that the lysate be cleared if subsequent protein determmations are to be performed Blue-stamed /3-galactosidase posittve cells are usually observed 30 min after the addition of stammg solution Stammg can be prolonged up to 24 h, but care should be taken that the wells do not dry out. If we use plasmids wtth very strong enhancers and promoters, e.g., cytomegalovirus-elements, for the determination of transfection efficiency, we usually measure a lysate dilution equtvalent to between 250 and 1000 cells (see also Note 23). When performing the enzyme assays described in these protocols care has to be taken that determinations are done m the linear range of the assays, because we reduced the amount of substrates with regard to the suggesttons of the vendors. Espectally if strong enhancers or promoters are used, substrate may be depleted very quickly and/or the linear range of the luminometer used may be exceeded Although measurement ts, of course, not necessary if no plasmid with beetle luctferase activity has been cotransfected, it is necessary to add the beetle luciferase-assay reagent for the determination of Renilla luciferase activity

References 1. O’Leary, K. A , McQuiddy, P , and Kasper, C. B. (1996) Transcrtpttonal regulation of the TATA-less NADPH cytochrome P-450 oxidoreductase gene Arch Blochem Biophys 330,271-280 2 Nason, T. F., Han, X.-G., and Hall, P. F. (1992) Cychc AMP regulates expression of the rat gene for steroid 17a-hydroxylase/C17-20 lyase P-450 (CYP17) in rat Leydig cells. Bzochzm Bzophys. Acta 117 1, 73-80.

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3 Burger, H -J., Schuetz, J. D., Schuetz, E. G., and Guzelian, P S. (1992) Paradoxtcal transcriptional activation of rat hver cytochrome P-450 3Al by dexamethasane and the antlglucocorttcotd pregnenolone 16a-carbomtrtle: analysis by transient transfection into primary monolayer cultures of adult rat hepatocytes. Proc Nat1 Acad Set USA 89,2145-2149

4. Shaw, P. M., Adesmk, M., Weiss, M C , and Corcos, L (1993) The phenobarbital-induced transcriptional activation of cytochrome P-450 genes IS blocked by the glucocorticoid-progesterone antagonist RU486. Mel Pharmacol 44,775-783 5. Hahn, C. N., Hansen, A. J., and May, B K. (1991) Transcrrptlonal regulation of the chicken CYP2Hl gene. J Biol Chem 266, 17,031-17,039. 6. Legraverend, C., Eguchi, H., Strom, A., Lahuna, 0 , Mode, A , Tollet, P , Westm, S., and Gustafsson, J.-A. (1994) Transactivation of the rat CYP2C13 gene promoter involves HNF- 1, HNF-3, and members of the orphan receptor subfamily Blochemlstry

33,9889-9897.

7. Yamamoto, T., Chapman, B. M., Clemens, J. W., Richards, J. S , and Soares, M J. (1995) Analysis of cytochrome P-450 side-chain cleavage gene promoter activation during trophoblast cell differentiation. Mel Cell Endocrinol 113, 183-194. 8. Crespi, C. L., Penman, B. W., Gonzalez, F. J., Gelboin, H. V., Galvin, M , and Langenbach, R. (1993) Genetic toxicology using human cell lines expressing human P-450. Bzochem. Sot. Trans. 21, 1023-1028. 9 Guo, Y -D , Strugnell, S., Back, D. W., and Jones, G. (1993) Transfected human liver cytochrome P-450 hydroxylates vltamm D analogs at different side-chain positions. Proc Nat1 Acad Scz USA 90, 8668-8672 10. Clark, B. J., and Waterman, M R. (1991) The hydrophobic ammo-terminal sequences of bovine 17a-hydroxylase IS required for the expression of a functional hemoprotem m COS 1 cells. J Biol Chem. 266, 5898-5904. 11 Kltamura, M., Buczko, E., and Dufau, M. L. (199 1) Dlssoclatton of hydroxylase and lyase acttvlttes by site-directed mutagenesis of the rat P450t7, A401 Endocrmol

$1373-1380

12. Kadohama, N , Zhou, D., Chen, S., and Osawa, Y. (1993) Catalytic efficiency of expressed aromatase following site-directed mutagenesis. Biochim Blophys. Acta 1163, 195-200 13. Yanase, T., Waterman, M. R., Zachmann, M., Winter, J. S. D., Simpson, E. R , and Kagimoto, M (1992) Molecular basis of apparent isolated 17,20-lyase deficiency’ compound heterozygous mutations in the C-termmal region (Arg(496) + Cys, Gln(461) + Stop) actually cause combined 17a-hydroxylase/17,20-lyase defictency Blochlm Bzophys Acta 1139,275-279. 14. Gaumtz, F., Papke, M., and Gebhardt, R. (1996) Transient transfectlon of primary cultured hepatocytes using CaP04/DNA preclpttatton. BzoTechnzques 20, 8264332

39 Lipid-Mediated Gene Transfer into Normal Adult Human Hepatocytes in Primary Culture Jean-Claude Marie-Cbcile

Ourlin, Marie-Jose Vilarem, Martine Daujat, Harricane, and Patrick Maurel

1. Introduction Functional analysis of the 5’4lankmg region of a gene has become a routme procedure for identifying the c&acting DNA elements involved m the control of the transcriptional activity of the gene (I). This experimental approach requires the transfection of reporter plasmids, harboring various fragments of the 5’-flanking region of the gene, into appropriate cultured cells and the subsequent measurement of the activity of the reporter gene, either constitutively or in response to regulatory stimuli. Continuous cell lines derived from mahgnant tissues have been extensively used for this purpose because they can be easily cultured and transfected. However, these cell lines have two major drawbacks: they are dedifferentiated with respect to the normal tissue from which they originate; that is, the tissue-specific transcription factors are generally expressed at a low level, if at all; and their culture requires the use of serum, the chemical and hormonal composition of which is not fully defined. In fact, it would be preferable to transfect normal cells in a primary culture in which the phenotype of the tissue of origin is maintained. The problem with this approach is that the methods currently used to transfect cell lines, including electroporation and calcmm phosphate- or diethylaminoethyl (DEAE) dextran-DNA coprecipitation, are generally not convenient for primary cultures because of their toxicity (2-4). Recently, Felgner and collaborators introduced the use of cationic liposomes for the efficient transfer of genes into mammalian cells (5-7). The basis of this method depends on hposomes that contam two species of lipid, a catiomc From Methods In Molecular Btology, Vol 107 Cytochrome P450 Protocols Edlted by I R PhMps and E A Shephard 0 Humana Press Inc , Totowa, NJ

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amphiphlhc lipid and a neutral phospholipid, which together form a complex with the polynucleotlde (DNA or RNA); this complex 1s stabilized by electrostatic interactions. The complex interacts through Its residual positive charges with the negative charges at the surface of the cellular membrane and penetrates the cell either by endocytosis or by direct fusion, dependmg on the nature of the hposome. The polynucleotlde is then delivered mto the cytosol and ultimately targeted to the nucleus through a mechanism that 1s not completely understood. This method is being used more and more frequently to transfect cells previously considered as resistant to transfection by classical methods, notably human keratmocytes and vascular smooth-muscle cells, and rat hepatocytes (8-10). The aim of this chapter 1sto describe our experience of lipid-mediated gene transfer into normal adult human hepatocytes m primary culture (II), using an inexpensive catlonic hposome 3P[N-(N:N’-dlmethylaminoethane)-carbamoyl] cholesterol (DC-chol) (12).

2. Materials 1. Hepatocytes are isolated as described m Chapter 36 m the present issue. 2. Cells are plated on plastic dishes, precoated or not with collagen, at 1, 2, or 4 x lo6 cells per 60-mm diameter dish, as described m Chapter 36 (see Note 1) 3 Culture condltlons are as described Chapter 36 m serum-free Ham-FlYWllllam’s E medium as used for short-term cultures 4 Lipids* LlpofectlnR (Glbco BRL, Eragny, France); dloleylphosphatydylethanolamme (DOPE) solution (20 mg/mL or 26.88 mM) from Fluka (Buchs, Switzerland); DC-chol prepared as described (12) (see Subheading 3.1. and Note 2). 5. Chloroform, cholesteryl chloroformate and NJ-dlmethylethylendlamine from Sigma Aldrich-Chimle (Saint Quentm Fallavier, France). 6. N-[2-hydroxyethyllplperazme-N’-[2-ethanesulfomc acid] (HEPES) (Sigma, Saint Louis, MO) buffer 20 mM, pH 7 8. 7 Sterile 5-mL polypropylene tubes (for somcatlon of llposomes) and sterile 5-mL polystyrene tubes (for llposome-plasmld complex formation) (Falcon, Los Angeles, CA) 8 Somcator (1 OOW maximum) with a probe adapted for volumes of l-2 mL 9 Plasmids. pSV2-(chloramphemcol acetyltransferase [CAT] as the reporter gene) and pbl-TK-CAT (m which the CAT reporter gene 1scontrolled by the thymidine kinase promoter) were kindly provided by Dr. A. Le Cam (INSERM, Montpelher, France) and pSV-@GAL (/3-galactosidase as reporter gene) was purchased from Promega (Madison, WI). In both pSV2-CAT and pSV-PGAL plasmids, the reporter gene is under the control of the SV40 promoter. pbl-l Al-TK-CAT contains a fragment of the CYP 1A 1 5’-flanking region, extending from -1560 to -900, placed upstream of the thymldme kmase promoter and the CAT reporter gene (13). 10 Plasmids are prepared using either the Qiagen Kit (Chatsworth, CA) or Nucleobond cartrldges (Machery-Nagel, D&en, Germany) Plasmid solutions are

Lipid- Media ted Gene Transfer

11 12. 13. 14. 15.

16. 17.

18. 19.

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prepared m sterile water at 1 ug/pL. Stock solutions are frozen at -20°C Workmg solutions are kept at 4OC Optimem medium (Gibco BRL). Store at 4’C. Bicinchoninic assay kit for measurement of protein concentration (Pierce, Rockford, IL). Phosphate buffered saline (PBS) Cell-fixatton solution: 2% formaldehyde, 0 2% glutaraldehyde m water. Prepare enough for 1 mL per culture dish Histologtcal reactton mixture for P-GAL assay. 4-chloro-5-bromo-3-mdoyl+ galactopyranoside (1 mg/mL), 5 mM potassium ferrmyamde, 5 mA4 potassium ferrocyamde, 2 mM magnesium chloride, in PBS. Prepare enough for 1 mL per culture dish. Cell lysts solution. 250 mMTrts-HCl, pH 7.8,0.5% Triton X-100. CAT assay reagents. Chloramphenicol, [3H]-acetyl Coenzyme A (200 mCi/ mmol) from NEN (Les Ulis, France), Econofluor scintillation cocktail (Packard, Menden, CT). Solution of epidermal growth factor (EGF) from Sigma at 2 pg/mL in the culture medium. Store m ahquots at -20°C until use. CYPlA inducer solutions. 1 fl2,3,7,8-tetrachlorodibenzo-p-dloxm (TCDD) from BCP Instruments (Lyon, France) and 25 mM P-naphthoflavone (BNF) m culture-grade dimethylsulfoxide (DMSO) from Sigma. Store m ahquots at -20°C until use.

3. Methods 3.1. Preparation DC-chol

of D&ho/

1s prepared as follows, as prevrously

described (12).

1. Solution 1: 2.25 g cholesteryl chloroformate in 5 mL of dry chloroform 2 Solution 2. 2 mL N,N-dtmethylethylendlamme m 3 mL dry chloroform 3. Solution 1 is added dropwtse to solution 2 at 0°C. The reactton is complete at this temperature and leads to the formation of DC-chol 4 The excess NJ-dimethylethylendiamme is removed by evaporation of chloroform under vacuum in a rotary evaporator, and redissolving the powder m a mimmum volume of absolute ethanol The DC-chol 1srecrystallized at 4°C and dried. This step is repeated twice more. The yield is approx 20% (0.5 g). 5 The DC-chol is analyzed by mass spectroscopy A umque peak at 500 must be obtained. The powder should be stored in a destccator at room temperature Under these conditions, DC-chol is stable for more than a year

3.2. Preparation of DC-chol Liposomes It is essential that the liposomes be prepared under sterile conditions. 1. Prepare a solution of DC-chol m chloroform at 5 mg/mL or 10 mA4 2. For standard DC-chol liposome (molar ratio DC-chol to DOPE: 1.2/0.8, at a final hptd concentration of 1.2 mg/mL), mtx 120 pL of DC-chol solutton with 30 pL

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of DOPE solution (see Subheading 2.4.). Evaporate off the chloroform under nitrogen at room temperature Add 1 mL of HEPES buffer and keep at 4°C overnight for rehydration of the lipid mixture. 3. Sterihze a sonicator probe m ethanol Sonicate the lipid mixture at 0°C at 8OW, for SIX 20-s bursts, each separated by 1-min intervals. The suspension obtained should not be turbid Stored at 4”C, the DC-chol hposome suspension is stable for at least 2 wk (see Note 3)

3.3. Electron Microscopy

of Liposome

Preparations

1 Coat grids with formvar as described in an electron microscopy handbook (14) 2 Prepare freshly sonicated liposomes in 200 mM HEPES buffer, pH 7.8, at a final lipid concentration of 1.2 pg/p.L 3 Just before application to the grid, dilute the liposome suspension IO-fold with buffer 4 While holding the grid with tine forceps, place a small drop (20 pL) of diluted liposome suspension on the grid and leave for 1 mm. 5 Take the grid with the absorbed specimen and place onto a drop of buffer on parafilm for 5 s Merely touch the drop with the specimen side of the grid. Repeat this step once. 6. Place the grid with the absorbed specimen onto a drop of 2% aqueous uranyl acetate on parafilm for 30 s Repeat this step once 7 Finally, remove excess stain by touching the edge of the grid with a piece of filter paper. The specimen IS then an-dried (see Note 4). 8. Liposomes are examined with a Jeol2000 EX electron microscope at an accelerating voltage of 80KV with an objective aperture of 20 pm.

3.4. Standard

Transfection

Conditions

1. Four hours after plating of hepatocytes at 1 x lo6 cells per 60 mm diameter collagen-coated dish, the Ham-F 12/William’s E culture medium supplemented with 5% fetal calf serum (FCS) IS removed and the monolayer of cells is washed once with serum-free medium (see Chapter 36) (see Notes 5 to 7). 2. Two mL of fresh serum-free medium are added per dish. 3. Appropriate ahquots of DC-chol hposome suspension (25 pg lipid/l x lo6 cells) or of hpofectm (30 pg lipid/l x lo6 cells, prepared as recommended by the supplier) and plasmid solution (5 pg/l x lo6 cells, the same amount is used for transfection with DC-chol or with lipofectm) are mixed m a final volume of 200 pL of optrmem medium The mixture IS Incubated for 10 mm at room temperature (see Note 8). 4. The liposome-plasmid mixture is added to the 2 mL of culture medium and the culture dishes are placed in the adCOz incubator at 37°C for 6 h (DC-chol llposomes) or 8 h (hpofectmR hposomes) (see Note 9). 5 In control expernnents, cells are treated similarly except that the plasmid is omitted 6. Transfection is stopped by aspiration of the liposome-plasmid-supplemented medium

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7 Three mL of fresh medium are then added per culture dish and the culture is continued until cells are harvested for measurement of reporter-gene acttvity (see Notes 10 to 12)

3.5. Treatments of Cells After Transfection (see Note 13) 3.5.1. Treatment to Stimulate Entry into S Phase 1. Four h after plating of hepatocytes at 1 x lo6 cells per 60 mm diameter collagencoated dish, the medium is renewed m the presence of 30 pL of EGF solution/ 3 mL of medium. Final concentration of EGF is 20 ng/mL (see Note 14) 2 The culture medium is subsequently renewed every 24 h, in the presence of EGF 3. Transfection of cells may be carried out at 4, 28, 52, 76, or 100 h after platmg, under standard conditions 4. Cells are harvested and analyzed for reporter gene activity In control experiments cells are cultured m the absence of EGF (see Note 15)

3.5.2. Treatment to Stimulate CYPl Al Gene Expression 1 Cells are transfected 4 h after plating, under standard condltlons (see Subheading 3.4.), and the medium 1s subsequently renewed every 24 h for 96 h

2. Immediately after transfectton, and 24,48, or 72 h later, cells are treated for 24 h with the CYPlAl inducer dissolved m 3 pL of DMSO and added in 3 mL of medium. Control cells are-treated with the vehicle alone 3 After 24 h of treatment, cells are harvested and analyzed for reporter gene activity (see Note 16)

3.6. p-GAL Assay 1. This assay allows direct visualization dishes per experimental pomt.

of transfected cells (blue cells) Use two

2. Cell monolayers are washed twrce with 2 mL PBS/culture dish

3 Cells are fixed for 5 min at 4°C with 1 mL fixation solution/dish. 4. After washing twice with 2 mL PBS/dish, cell monolayers are overlaid 1 mL of histological reaction solution and incubated for 18 h at 37’C.

with

5. Culture dishes are examined by phase-contrast microscopy and intensely blue cells (tranfected cells) are counted m a field covering 60% of the culture dish (see Note 17).

3.7. CAT Assay 1. Cell monolayers are washed twice with 2 mL PBS/culture dish, and cells are harvested by scrapmg with a rubber policeman in 1 mL of PBS per dish. Use two dishes per experimental point. 2 Cells obtained after a brief centrifugation at 2000g are lysed by incubation for 15 mm at room femperature in approx 2 volumes of lysts solution, with Intermittent vortexmg.

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3. The cell homogenate is centrifuged at 10,OOOgfor 8 mm at 4°C the supernatant (cell lysate) IS collected and the protein concentration measured. 4. The cell lysate is heated at 60°C for 10 mm to mactivate endogenous acetylases and deacetylases 5. 50 pg of cell lysate protem are mixed with 1 mA4 chloramphemcol and 30 pM [3H] acetyl Coenzyme A (0.5 pCi) m a final volume of 100 pL of 250 mA4 TrisHCl, pH 7.8,s dethylenediamine tetra-acetic acid (EDTA), and the reaction is allowed to proceed for 90 min at 37°C (see Note 18). 6. 90 pL of the reaction mixture are added to 10 mL of scintillation cocktail and the mixture is vortexed for 1 mm before being placed m a scintillation counter 7 The radioactivity is measured over a period of a few mm to 3 h, depending on the efficiency of transfection It reflects the amount of radioactivity transferred from the aqueous to the organic phase and hence incorporated into acetyl chloramphemcol(1). 8 The CAT activity is expressed m cpm/mg protein/h (see Note 19)

4. Notes 1. Although we recommend the culturing of hepatocytes at confluence (4 x 1O6 cells per 60-mm diameter dish ) on collagen-coated dishes for optimum mamtenance of the hepatic phenotype (Chapter 36), the search for condtttons allowing efficient transfection may require the use of different culture conditions (see also Note 5). 2. A major drawback of hpofectm and other commercial hposomes is their excessive price In contrast, DC-chol allows the preparation of inexpensive hposomes that are as efficient as hpofectm for the transfection of human hepatocytes (11) 3 For sonication, several observations have been made a. Bath somcators were inefficient in our hands Liposomes prepared m this way were not active m transfectmg cells, m the electron microscope they appeared as large vesicles instead of rounded hposomes. b. A probe somcator must be used. A 3-mm diameter probe, used with roundbottomed polypropylene tubes of l-cm diameter containing 2 mL suspension gave satisfactory results. c. A series of 20-s sonication periods, with 1 min m between, was found to be more efficient than continuous somcation, presumably because this allows appropriate cooling of the suspension, thus favoring the formation of small liposomes d Different batches of hposomes can be pooled to avoid variability from one preparation to another 4. Because grids are generally not uniform, it is necessary to make two specimens for each sample and to search for the best stammg on each grid. If staining is uneven, first check that the grid wetting is appropriate (sometimes an improvised treatment might be necessary to make the grid surface more or less hydrophilic) and then dilute the spectmen.

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5 Conditions given m this section are those giving the most efficient transfectton (as concluded from data obtained with 10 different human hepatocyte cultures) and are referred to as our standard transfection condmons. For any mvestigation, experimental condmons for transfection should be adapted to the conditions m which the gene under consideration is expressed. For example, investigating the transcriptional activity of constructs of a gene Involved in the regulation of the cell cycle will require a subconfluent culture and the treatment of cells with growth factors (I.5,16). On the other hand, investigating a gene whose expression is part of the specific hepatic phenotype will require a confluent culture (17). The density of cells m the monolayer is an essential parameter for transfection. Detailed mvestigatron demonstrated that the efficiency of transfection decreases by approx 50% as the cell density mcreases from 3.5 x lo4 to 14 x lo4 cell/cm*, the last value correspondmg to a confluent monolayer. A similar pattern was obtained with either lipofectm or DC-chol In contrast, the presence or absence of collagen as a substratum did not affect the efficiency of transfection (II). The age of the culture is another essential parameter. The efficiency of transfecnon decreases as the age of the culture mcreases from 4 h to 60 h. This decrease is observed with both lipofectin and DC-chol (11) For hposome-plasmid preparations, use 5-r& polystyrene tubes because the complex has a tendency to adhere to polypropylene tubes. Do not use a vortex, instead, gently mix by hand. The range of values tested for the DC-chol/DOPE ratio was: 2:0, 1 5.0 5, 1 2:0.8,0.5: 1 5, and 0:2. For a constant amount of ltptd (either 20 or 30 pg, at the ratio 1.2:0.8 for DC-chol/DOPE), the efficiency of transfection increased with increasmg amount of plasmid to reach a maximum (at 5 pg plasmid DNA) then decreased as the amount of plasmid increased to 10 pg per assay For a constant amount of plasmid (5 or 10 pg), the efficiency of transfection increased, with increasing amounts of lipid, to reach a maximum (at 25 or 30 pg with DC-chol or bpofectm, respectively) and decreased as the amount of lipid increased further to 50 pg per assay (11). 9 The duration of exposure of cells to the lipid-plasmid mixture varied from 4-l 6 h. The efficiency of transfection was found to be roughly constant over this penod (11). 10. Under the conditions reported here, neither lipofectm nor DC-chol are toxic to the cells, as assessed by measurement of de nova protein synthesis In addition, the activity of the reporter gene (P-GAL or CAT) remains constant for at least 96 h (11). Il. On average, the efficiency of transfection is not sigmficantly different with lipofectm or DC-chol: 0.35% + 0.66 (n = 6) or 0.23% + 0.29 (n = 3), respectively However, the variabihty from one culture to another is quite large: from 0.04 to 2.0% for lipofectin and from 0.0 to 0.7% for DC-chol, as assessed by the P-GAL assay. The reason for this variability is not known but it appears not to be related to the viability of hepatocytes after isolation (II). 12. By comparison, the efficiency of transfection is much greater when human cell lines are tested. Thus, for example, under our standard condmons the percent of cells transfected through the use of hpofectm and DC-chol, respectively, was

378

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14.

15

16.

17.

18.

19.

Our/m et al. 3.0 + 0 8 and 0 5 + 0.1 for HepG2, 8.0 + 2.0 and 20.0 + 4 0 for Caco-2; 6 0 f 1 5 and 2.0 & 0.4 for WRL68 (llJ3). Depending on the gene under mvestigatton and on the nature of the stirnull that control its expression, it may be necessary to submit the cells to various treatments after transfection Two examples are described here. EGF, transforming growth factor a, and hepatocyte growth factor are all able to stimulate the entry into S-phase of cultured human hepatocytes (15,16) The use of EGF may be preferred for its low cost in comparison to the others, unless a growth factor-specific gene activation is to be analyzed The efficiency of transfection appears to be greatly increased when cells are treated with EGF In particular, the effictency of transfection parallels the rate of DNA synthesis in the cells, which, under our culture conditions, reaches a maximum after 48 h of treatment with EGF For example, if 48-h-old cultures are transfected, the efficiency of transfection is increased at least fivefold with respect to cells cultured in the absence of EGF (11) Under these condmons, the transcriptional activity of the CYPl Al-CAT construct 1s inducible by both TCDD and BNF as expected (21). Thts mductbtlrty seems to be greater m cells transfected usmg hpofectin as compared to DC-chol, whereas the presence of collagen as a substratum and the cell density (between 1 and 4 x 1O6cells per 60-mm dish) have no effect. Treatment of cells with CYP 1A inducers does not affect the efficiency of transfection as assessed by P-GAL reporter-gene activity Some eukaryotrc cells express a P-galactostdase-like enzyme that is able to blotransform 4-chloro-5-bromo-3-mdoyl-P-galactopyranoside This leads to a blue-colored background of variable Intensity, depending on the cell type This activity is located in the acidic compartments of the cell m contrast to the noncompartmentatron of the transfected enzyme. Thus, only mtense blue staming of the cell is characteristtc of efficient transfection Because the background increases wtth time, the length of incubation to reveal enzyme activity must not exceed 18 h. This problem can be overcome by usmg a reporter gene containing a nuclear localization signal Because of the variability m the efficiency of transfection from one experiment to another, the conditions for measuring CAT activity (duration of mcubatton and amount of cell lysate) have to be adapted for each series of experiments, m order to obtam a signal m the appropriate range of sensitivity The efficiency of transfection by hposomes varies widely with the type of cell to be transfected and the nature of the lipids used to prepare liposomes. Therefore, the present protocol should be restricted to primary cultures of human hepatocytes and to the hposomes used here

Acknowledgments This work has been supported in part by Glaxo-France (JC Ourlin). The authors wish to thank Professors Jacques Domergue, and Jean Michel Fabre (CHR Saint Elol, Montpellier, France), Henri Joyeux (Centre AnticancCreux

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Val d’Aurelle, Montpelller, France), Gllles Fourtanler (CHR Ranguell, Toulouse, France), and Jean Baulleux (CHR La Croix Rousse, Lyon, France), for providing human liver tissue, and Cohn Young for editorial assistance. References 1. Kmgston, E. and Sheen, J. (1994) Uses of fusion genes m mammalian transfectlon m Current Protocols In Molecular Biology, vol 1 (Ausubel, F M , Brent, R , Kingston, R. E., Moore, D. D., Seidman, J G., Smith, J A., and Struhl, K., eds.), John Wiley and Sons, NY, pp 961-969 2. Neumann, E., Schaefer-Ridder, M., Wang, Y , and Hofschneider, P H (1982) Gene transfer mto mouse myeloma cells by electroporation m high electric field EMBO J 1,841-845. 3 Chen, C. and Okayama, H. (1987) High-efficiency transformation of mammalian cells by plasmld DNA. Mel Cell. Blol 7,2745-2752 4. Takai, T and Ohmon, H (1990) DNA transfectlon of mouse lymphoid cells by the combmation of DEAE-dextran-mediated DNA uptake and osmotic shock procedure. Bzochzm. Bzophys. Acta 1048, 105-109 5. Feigner,

P. L., Gadek,

T. R , Holm,

M , Roman,

R , Chan, H

W , Wenz,

M ,

Northrop, J. P., Rmglod, G. M., and Danielsen, M (1987) Lipofection: a highly efficient, hpld-medlated DNA-transfectlon procedure. Proc Nat1 Acad. Scz USA 84,7413-7417 6 Felgner, P L. and Rmgold, G. M (1989) Catiomc liposome-medlated transfectlon. Nature 337,387,388. 7 Felgner, J H., Kumar, R., Sridhar, C. N., Wheeler, C J , Tsar, Y J , Border, R , Ramsey, P., Martm, M., and Felgner, P. L. (1994) Enhanced gene dellvery and mechamsm studies with a novel series of catlomc lipid formulations J B~of Chem. 269,2550-2561.

8 Jiang, C K., Connolly, D., and Blumenberg, M (1991) Comparison of methods for transfection of human epidermal keratmocytes. J Invest Dermatol 97,96%973. 9. Pickering, J. G., Jekanowskl, J., Weir, L., Takeshita, S., Lesordo, D W , and Isner, J. M. (1994) Liposome-mediated gene transfer into human vascular smooth muscle cells. Czrculatzon 89, 13-21. 10. Jarnagm, W. R., Debs, R. J., Wang, S. S., and Bissell, D. M. (1992) Catlomc hpldmediated transfection of liver cells in primary culture. Nuclezc Aczds Res 20, 4205-4211 11. Ourlm, J. C., Vilarem, M. J., DauJat,M., Harricane, M. C., Domergue,J., Joyeux, H., Baulieux, J., and Maurel, P. (1997) Lipid-medlated transfection of normal adult human hepatocytes in primary culture. Anal Biochem 247,34-44 12. Gao, X. and Huang, L. (1991) A novel catlonic liposome reagent for efficient transfection of mammalian cells. Biochem. Blophys. Res Commun 179,280--285. 13. Daujat, M., Charrasse, S., Fabre, I., Lesca, P., Jounaidl, Y , Larroque, C , Poellinger, L., and Maurel, P. (1996) Induction of CYPl Al gene by benzlmldazole derivatives during Caco-2 cell differentiation. Evidence for an aryl-hydrocarbon receptor-mediated mechanism. Eur J Blochem. 237,642-652.

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14. Hayat, M. A. (1989) Prmciples and technzques of electron microscopy. bzologzcal appllcatlons CRC, Boca Raton, FL, pp. 52-54 15. Blanc, P , Etienne, H , Daqat, M., Fabre, I , Zindy, F , Domergue, J , Astre, C , Saint Aubert, B., Mlchel, H , and Maurel, P. (1992) Mitotic responsiveness of cultured adult human hepatocytes to epidermal growth factor, transforming growth factor a, and human serum Gastroenterology 102,1340-1350 16. Strain, A. J., Ismail, T , Tsubouchl, H , Arakakl, N., Hishida, T , Kitamura, N , Daikuhara, Y., and Nakamura, T. (199 1) Native and recombinant human hepatocyte growth factors are highly potent promoters of DNA synthesis in both human and rat hepatocytes J Clrn Invest 87, 1853-1857. 17 Greuet, J., Pichard, L., Ourlin, J C , Bonfils, C., Domergue, J., Le Treut, P., and Maurel, P. (1997) Effect of cell density and epidermal growth factor on the mducible expression of CY3A and CYP 1A genes m human hepatocytes m primary culture. Hepatology 25, 1166-l 175

40 Identification of Regulatory Protein-Binding in Cytochrome P450 Genes by Means of the Gel-Retardation Assay

Sites

Helen Dell, Siew Cheng Wong, Ian R. Phillips, and Elizabeth A. Shephard 1. Introduction The gel-retardatton, band-shift, or electromobtltty shift assay 1s used to investigate protein-DNA mteractions. Nuclear protein from the desired source is incubated with a radiolabeled DNA fragment. The products of the reaction are then analyzed by electrophoresls through a nondenaturmg polyacrylamtde gel. Free DNA will migrate rapidly through the gel, but DNA bound to protein will move more slowly, i.e., the migration of bound DNA ts retarded. The protein-DNA complexes formed can be detected by autoradtography. Transcription of several cytochrome P450 (P450) genes IS increased in response to foreign chemtcals. The gel-retardation assayhas played an tmportant role in the tdentification of 5’ flanking sequences that bind transcriptton factors in response to treatment of cells or animals with various xenobtottcs. Such studies have been carried out in concert with DNase I footprmtmg experiments (Chapter 41) and cell-transfection assaysof P450 gene constructs (Chapters 38 and 39). In this way, regulatory sequences and transcription factors have been identified that play an important role in the induction of CYP 1A 1 by polycyclic aromatic hydrocarbons (I) and members of the CYP4A subfamily by peroxisome proliferators (2). The identification of DNA sequencesthat regulate the phenobarbital-induced expression of genes encoding members of the CYP2B, 2C, and 3A subfamilies has proved more difficult. Until recently, condtttons for maintaining the phenobarbital response m hepatocyte cultures had not been established, and it was thus impossible to delineate DNA regulatory sequences through the use of P450-reporter gene From Methods m Molecular Bology, Vol 107 Cytochrome P450 Protocols E&ted by I R Ph!lllps and E A Shephard 0 Humana Press Inc , Totowa, NJ

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constructs in transfection experiments. However, in the absence of a suitable phenobarbital-responsive cell line, gel retardation assayshave proved invaluable for the identification of CYP2B regulatory sequencesthrough their abrhty to bmd truns-acting regulatory protems. Usmg the gel-retardation assay, the region between -66 and -42 of the CYPZBl and 2B2 genes has been shown to bind members of the c/EBP family of transcription factors (3-5). A srmrlar sequence between -45 and -64 of the Cyp2blO gene also binds c/EBP family members (6). Using a quantitative gelretardation assay, the DNA-protein complex formed wrth this region of CYP2B2 1s fourfold more abundant with extracts from phenobarbital-treated rats (3). A member of the octamer class of transcrtptron factors forms a protem-DNA complex with the -199 to -183 region of the CYP2B2 gene that is more abundant with liver nuclear extracts isolated from rats treated with phenobarbital (3). Recently, Roe et al., (7) have shown protem-binding to an API site in the -144 1 region of the CYP2B2 gene that 1salso increased m response to phenobarbital. The base sequence required for protein binding can be confirmed usmg a mutated sequence as the probe or by mcludmg heterologous or homologous competrtor DNA m the assay. If a transcription factor is suspected of binding to a parttcular sequence, then the assay can be carried out m the presence of antibodies to the candidate protem. If the antibody binds to a protein present in the DNA-protein complex, the size of the complex will increase, thus further retarding its mrgration through the gel. This is the so-called supershift effect. Alternatively, if the antibody recognizes the DNA-bindmg region of the protein, then no complex will form. DNA-protein mteractrons will vary according to the source of nuclear protem and the DNA fragment used. In this chapter, we describe a general protocol for the gel-retardation assay.The parameters that can be varied to optimize the for-matronof a particular DNA-protein complex are noted in Subheading 4. 2. Materials

2.1. Preparation of DNA for Radiolabeling DNA molecules for use as probes in gel-retardation assays can be derived from cloned DNAs by restrtctron-enzyme digestion or amplification by the polymerase chain reaction (PCR), or can be produced as synthetic doublestranded ohgonucleottdes (see Notes 1 and 2). 2.1. I. DNA Molecules Produced via Restriction-Enzyme Digestion or PCR Amplification I Restriction endonucleases:available from several suppliers, e.g., Pharmacia Biotech (St. Albans, Herts, UK) or New England Biolabs (Beverly, MA)

Gel-Retardation

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2. Agarose: ukraPURE low-meltmg-point agarose (Gibco-BRL, Gaithersburg, MD) 3 50X TAE buffer. 2 MTrizma@ base (Tris[hydroxymethyl]aminomethane), 5 71% (v/v) glacial acetic acid, 0.05 A4 EDTA Dissolve 242 g Trizma@ base m distilled water, then add 57 1 mL glacial acetic acid and 100 mL 0.5 M EDTA, pH 8 0. Make solution up to 1 L with distilled water, autoclave, and store at room temperature 4. Spm-X centrifuge filter unit (0.22 pm cellulose acetate, COSTAR, Cambndge, MA) 5 DNA extraction buffer: 20 mA4 Tris-HCl, pH 8 0, 1 mM EDTA, pH 8 0 Autoclave and store at room temperature. 6. Phenol/chloroform (1 1, v/v). mix equal volumes of phenol and chloroform Wrap container in aluminum foil Store at 4°C 7 DNA precipitating agents. a 3 M sodium acetate, pH 5 2 b 10 A4 ammonium acetate. Autoclave and store at room temperature. 8 Ethanol* absolute and 70% (v/v)

2.1.2. Synthetic Double-Stranded

Ol/gonucleotides

1 10X Annealing buffer. 0.1 MTris-HCl, pH 8 0, 0 1 M MgC12, 0.5 MNaCl Sterilize by passing through a 0 22 pm Millex@-GP filter unit (Mtlllpore, Bedford, MA.) and store at -20°C.

2.2. Radiolabeling

DNA Molecules

2.2.1. DNA Molecules with a 5’-Overhang 1. Radioisotope. [a-32P]dCTP (3000 Cmnmol, 10 pCi/pL) (NEN Research Products, Boston, MA) (see Note 3) 2. Ultrapure dNTP Set (2’-deoxynucleoside 5’-trtphosphates) (Pharmacta). 100 mM stocks of dATP, dGTP, dCTP, and dTTP. Dilute each to 10 nut4 with sterile distilled water and store at -20°C. 3. E. coli DNA polymerase I, Klenow fragment, cloned, FPLCpureB (10 II/$, Pharmacia). 4. 10X One-Phor-All buffer plus 100 nut4 Trts-acetate, pH 7 5, 100 mM magnesium acetate and 500 mA4 potassium acetate (Pharmacia)

2.2.2. DNA Molecules with Blunt Ends 1. Radioisotope: [y2P]ATP (3000 Ct/mmol, 10 pCI/pL) (NEN Research Products) 2 Ready-to-goTM T4 polynucleottde kmase kit (Pharmacta) Contains, when reconstituted in a volume of 50 pL, 8-10 U FPLCpureB T4 polynucleottde kmase, 50 mA4 Trts-HCl, pH 7 6, 10 mA4 MgCl,, 5 mA4 dithiothrettol (DTT), 0 1 mM spermidme, 0 1 mA4 EDTA, pH 8 0,O 2 w ATP

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2.3. Recovery of DNA Probe and Measurement of Incorporated

Radioactivity

1. Purlficatlon columns (see Note 4). a NucTrap@ probe purification column (Stratagene, Cambridge, UK) b Chroma SpinTM Column (Clontech Laboratories, Palo Alto, CA). 2 STE buffer. 100 mMNaC1, 20 mM Tns-HCl, pH 7 5, 10 mA4 EDTA. Autoclave and store at room temperature. 3. DE81 ion-exchange paper (Whatman Scientific, Maidstone, Kent, UK). 4. Scmtlllatlon fluid. Ecoscmt A (Natlonal Diagnostics, Atlanta, GA) or equivalent. 5 Plastic scmtlllatlon vials (National Diagnostics) 6 0 5 M Na2HP04 (AnalaR grade, BDH Laboratory Supplies, Poole, UK) autoclave and store at room temperature 7 Ethanol. 95% (v/v) 8. Equipment includes a bench-top centrifuge with swinging bucket rotor (e g , Centaur II, MSE, Therm0 FI, Crawley, UK) and a scintillation counter.

2.4. Gel-Retardation

Assay

1OX TBE buffer. 0 89 M Trlzma base, 0.89 A4 boric acid, 20 mM EDTA. Weigh out 108 g Trlzma base and 55 g boric acid. Dissolve m distilled water and add 40 mL 0.5 MEDTA, pH 8.0. Make solution up to 1 L with distilled water Autoclave and store at room temperature 5X Binding buffer: 60 mA4N-2-hydroxyethylplperazine-N’-2-ethanesulfomc acid (HEPES), pH 7.9,20 mMTris-HCl, pH 7 9,300 mMKCl,l50 mA4NaC1,25 mM MgC&, 25 mA4 DTT, 0.5 mM EDTA, pH 8.0, 62 5% (v/v) glycerol (8) (see Note 5) Sterilize by filtration through a 0.22-w Mlllex@-GP filter unit and store in ahquots at -20°C Poly (dI.dC) poly (d1 dC) DNA copolymer (double strand, sodium salt, Pharmacla Blotech). prepare a 5 mg/mL stock solution m sterile 5 mM NaCl. Store in aliquots at -20°C. N,N,N:N’-tetramethylethylenedlamme (TEMED) (Bio-Rad, Hercules, CA). 10% (w/v) ammonium persulfate (BDH Laboratory Supplies, Lutterworth, UK): prepare fresh m sterile distilled water Protogel (National Diagnostics). 30% (w/v) acrylamide, 0 8% (w/v) blsacrylamlde, gas stabilized (see Note 6). For example, to make a 6% polyacrylamlde gel, mix 625 @ 10X TBE, 5 mL Protogel, 19.1 mL water. Add 250 p.L 10% (w/v) ammonium persulfate and 25 pL TEMED Pour the gel immediately Dilution buffer 25 mA4 HEPES, pH 7.6, 0 1 mM EDTA, pH 8 0, 40 mM KCl, 10% (v/v) glycerol, 1 mMDTT Sterilize by filtration through a 0 22-q MillexGP filter unit and store m ahquots at -20°C. Proteinase K (Boehrmger Mannhein, Mannhelm, Germany). Prepare a 25-mg/mL solution m 50 rml4 Tris-HCl, pH 8.0, 1 mA4 CaCl*. Incubate for 5 mm at 37°C. Store in aliquots at -20°C.

Gel-Retardabon

Assay

385

9 Gel-loading buffers. a Loadmg buffer A 30% (v/v) glycerol, 0.2% (w/v) bromophenol blue. b. Loading buffer B* 30% (v/v) glycerol, 0.2% (w/v) bromophenol blue, 0.25% (w/v) xylene cyan01 Sterilize by filtration through a 0 22-pm Millex-GP filter unit and store m aliquots at -20°C (see Note 7) 10. Nuclear protein extract prepare according to chosen method (see Note 8) Determme protein concentration by the Lowry method (9) (see Note 9). Store m ahquots at -70°C (see NotelO). 11, Equipment includes a PROTEAN II xi cell-electrophoresis apparatus (or equivalent), a power pack, and gel-dryer. 2.4.1.

Competitive Gel-Retardation

Assays

DNA molecules used as competitors can be derived from cloned DNAs by restriction-enzyme drgestion or PCR amplificatron, or can be synthetic doublestranded olrgonucleotides.

2.4.2. Use of Antibodies to identify DNA-Binding

Proteins

Antibody to the protem of interest (Santa Cruz Biotechnology, CA; or Upstate Brotechnology, Lake Placid, NY).

Santa Cruz,

3. Methods 3.1. Preparation of DNA for Radiolabeling 3.1.1. DNA Molecules Produced via Restriction Enzyme Digestion or PCR Amplification 1 Electrophorese the DNA through a low melting pomt agarose gel m 1X TAE buffer and excise the DNA fragment of interest. 2 Purify the DNA fragment from the agarose using either a Spin-X filter umt (according to the manufacturer’s mstructrons) or by phenol/chloroform extraction (10) (see Note 11). 3. Precipitate the DNA wtth 2 vol of absolute ethanol and 0.2 vol of either 3 A4 sodium acetate, pH 5.2, or 10 A4 ammonium acetate at -70°C for at least 1 h or preferably overnight. 4. Pellet the DNA by centnfugation for 20 min at ml1 speed in an Eppendorfcentnfuge. 5. Wash the pellet with 70% (v/v) ethanol and dry under vacuum. 6. Dissolve the pellet m an appropriate volume of sterile water and determine the DNA concentration (see Note 12). Store at -20°C.

3.1.2. Synthetic Double-Stranded

Oligonucleotides

1. In a 1.5-mL Eppendorf tube, mix equimolar amounts of the two single-stranded complementary oligonucleotides with 10 pL of 10X annealmg buffer and add sterile water to a total volume of 100 uL

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2. Incubate at 65°C for 10 mm to allow strands to anneal

3. Allow to cool to room temperature. 4. Store at -20°C

3.2. Radiolabeling

DNA Probes

3.2.1, DNA Molecules with a 5’ Overhang 1 In a 1S-mL Eppendorf tube, mix 100-300 ng of the DNA to be labeled, 2 pL 1OX One-Phor-All buffer PLUS, 2 pL 10 mMdATP, 2 pL 10 mMdGTP, 2 pL 10 mM dTTP, and sterile water to make up the volume to 17 pL. 2 Add 2 uL [cL-32P]dCTP and 1 pL of E coli DNA polymerase I, Klenow fragment 3. Flick the tube to mix contents, and collect the reaction mixture at the bottom of the tube by centrifuging briefly m an Eppendorf centrifuge 4. Incubate at 37’C for 45 min 5. Recover probe as described m Subheading 3.3.

3.2.2. DNA Molecules with Blunt Ends We found the ready-to-go

T4 polynucleotide

kmase kit (Pharmacia)

easy to

use. The kit contains, in a single tube, all components except the DNA to be labeled, and the radioactive isotope. Radlolabel DNA according to the manufacturer’s instructions. 3.3. Recovery of DNA Probe and Measurement of Incorporated

Radioactivity

1 Radiolabeled DNA molecules are separated from unincorporated deoxynucleotldes by using either a Nuctrap push column or a Chroma SpmTM Column according to the respective manufacturer’s instructions 2 Before loading onto the column apply 1 pL of radiolabelmg reaction mixture onto each of two separate pieces of DE8 1 ion exchange paper. 3 Apply 1 pL of the DNA fraction eluted from the column onto each of two separate pieces of DE8 1 paper. 4. Wash one piece of the DE81 paper (from before and after column purification) with 0 5 MNa2HP04 to remove unincorporated nucleotides The other piece of each pair should not be washed 5 Place each of the four pieces of DE8 1 paper into separate scmtillatlon vials and add 5 mL of scintillation fluid. 6 Determine radroactivlty by liquid-scintillatton spectrometry For use as a probe in gel-retardation assays, the DNA should have a specific radioactivity of ~1 x 1O4cpm/ng.

3.4. Gel-Retardation

Assay

1. Pour the polyacrylamide (see Note 14).

gel (see Note 13) and leave to polymerize overnight

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387

2. Rinse the wells with TBE buffer using a syringe. Prerun the gel at 10-15 V/cm for 30 min m TBE buffer (see Note 15). 3. To a 1 5-mL Eppendorf tube on ice, add 5 pg nuclear protein (see Note 16), 4 & 5X binding buffer, 2.5 pg poly dI.dC (see Note 17) and sterile water to make up the volume to 19 & Nuclear proteins should be diluted to an appropriate concentration in dilution buffer and should comprise no more than a fifth of the total volume of the reaction mixture. 4. Flick each tube to mix contents, and collect the reaction mixture at the bottom of the tube by centrifuging briefly m an Eppendorf centrifuge. 5. Incubate reaction mixtures on ice for 10 mm. 6. Add 1 pL DNA probe (0.5-l ng) (see Note 18), mix, and centrifuge briefly in an Eppendorf centrifuge. 7. Incubate on Ice for 30 min. 8 Add 2 p.l of gel-loading buffer (see Note 7) 9. Before loading the sample onto the gel, recirculate the runmng buffer, and rmse the wells again. 10. Load samples on gel and electrophorese at 10-15 V/cm until the free probe has almost reached the end of the gel (e.g., on a 6% polyacrylamlde gel a 60-bp probe ~111comlgrate with the bromophenol blue). Recirculate the running buffer either contmuously with a pump or manually every 30 mm 11 Transfer the gel onto 3MM chromatography paper (Whatman Scientific), cover with Saran wrap and dry using a slab gel dryer at 80°C 12. Autoradiograph overnight at -70°C with intensifying screens (see Note 19).

3.5. Pro teinase K Digestion Set up the reaction mixture as described in Subheading 3.4., step 3. After the 30-min binding incubation, add 5 pg protemase K and incubate the mixture at 37°C for 15 min before loading onto the gel (see Note 20).

3.6. Competitive Gel-Retardation Assays These are usually performed concurrently with standard gel-retardation assays. 1 To a 1.5-mL Eppendorf tube on ice, add 4 p.L 5X binding buffer, 2.5 pg poly dI.dC (see Note 17), nonradiolabeled DNA to be used as competitor (see Note 21), 1 pL DNA probe (0.5-l ng) and sterile water to make up the volume to 15 p.L. 2. Mix and add 5 pg of nuclear protein extract. 3. Mix again by flicking the tube and centrifuge briefly in an Eppendorf centrifuge. 4. Processsampleas describedin Subheading 3.4., steps 7-12.

3.7, Use of Antibodies to identify DNA-Binding Proteins 1. 2. 3. 4.

In a 1.5~mL Eppendorf tube, set up reaction mix as m Subheading Add l-2 pg of antibody to the tube and mix (see Note 22). Incubate on ice for 10 min. Process sample as described in Subheading 3.4., steps 6-12.

3.4., step 3

Dell et al. 4. Notes 1. The DNA molecules used as probes can be from 16 to 400 bp long, although it has been reported that oligonucleotides of less than 20 bp have a reduced affinity for the bmdmg protein, even though they encompass the same bmdmg site as longer DNA molecules (11) 2. Smgle-stranded oligonucleotides can be designed so that when the complementary strands are annealed they form double-stranded ohgonucleotides with either a blunt end or 5’-ends with a 4-base overhang. The former can be end-labeled by means of polynucleotide kmase, and the latter via a fill-m reaction using E cob DNA polymerase. 3 The use of radiolabeled dCTP in the fill-in reaction may not always be appropnate as the 5’ overhang may not contain a guanine Also, we have found that the Klenow fragment of E colr DNA polymerase 1snot very effective at filling-m the two outermost bases of any 5’-overhang Thus fill-m labeling of a HzndIII restnctlon fragment (AJAGCTT) will be more efficient with radlolabeled dATP than with radiolabeled dCTP 4 Several types of column are suitable for use. The followmg are easy to use and yield probes of high purity a NucTrap probe purification columns have the advantage of requiring no centrifugatlon step and can separate a wide range of DNA sizes, from 17 to 50,000 bp b Chroma spm columns are available m a variety of matrix pore sizes and column volumes In our lab, the Chroma Spin- 10 column (cat. no. K1300-2) has been successfully used for the purification of DNA probes ranging m size from 16 to 200 bp 5 The composition of the binding buffer may have to be adjusted to obtain optimal bmdmg a. Initially a titration of KCl/NaCl concentration over the range 50-500 mM should be carrled out. b Ca*+ or Mg*+ can be added to a concentration of 50 mA4 c. When using cytoplasmic fractions or high-salt nuclear extracts, the inclusion m the reaction mix of nomonic or zwitteriomc detergents such as Nomdet P-40 or CHAPS was found to increase protein-DNA binding (12). 6. Gels of 6 or 8% polyacrylamide are the most commonly used. However, if the protein-DNA complex fails to enter the gel or if it is suspected that more than one type of protein-DNA complex is present, the percentage of polyacrylamlde can be decreased to 4% The latter is often the case with DNA fragments >lOO bp, which may contain several different protem-binding sites 7 For DNA probes of less than 80 bp, we use loading-buffer A and for those longer than 80 bp, we use loading-buffer B. The mclusion of xylene cyanol, which migrates more slowly than does bromophenol blue, enables the mlgratlon of larger DNAs to be monitored more easily. For example, for a 200-bp DNA on a 4% gel, electrophoresis should be stopped when the xylene cyan01 is 4 cm from the bottom of the gel

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Fig. 1. Gel-retardation assays of nuclear proteins extracted by different methods. A radiolabeled DNA fragment extending from -34 to -86 of the CYP2B2 gene was incubated with 5 pg of nuclear proteins extracted from rat liver by the methods of Sierra (13) (protocol 1, tracks 1 and 2), Jose-Estanyol et al. (IS) (protocol 2, tracks 3 and 4), Lavery and Schibler (14) (protocol 3, tracks 5 and 6), and Deryckere and Gannon (16) (protocol 4, tracks 7 and 8). The binding reactions were carried out at 0°C in the presence of 2 pg poly dI.dC. The reaction mixture loaded in track 9 (-) contained no nuclear protein. Reaction mixtures were electrophoresed through a 6% polyacrylamide gel, which was then autoradiographed. 8. Many methods for the extraction of fractions containing DNA-binding proteins have been reported in the literature. Figure 1 shows a comparison of results obtained with protein extracts isolated according to the methods of Sierra (13), Lavery and Schibler (14), Jose-Estanyol et al. (15), and the 1 h mini-prep method of Deryckere and Gannon (16). The method of Sierra (13), although relatively complicated, gave the greatest amount of protein-DNA complexes per pg of nuclear protein. The 1 h mini-prep method was the easiest, but at least 20 pg of protein extract had to be used to obtain any detectable protein-DNA complex. 9. Because of the viscosity of the nuclear protein extract, aliquots may not contain equivalent concentrations of protein. Therefore, it is better to determine the concentration of protein in each aliquot rather than to rely on a concentration determined on the whole extract. 10. Nuclear protein extracts should be stored in small aliquots at -7OOC and should not be frozen and thawed more than a few times. Extracts that have been stored for more than a year can produce smearing along the gel tracks in a gel-retardation assay. We have also observed a slight variation in results obtained with dif-

Dell et al.

11. 12

13.

14.

15

16

17.

18.

ferent preparations of nuclear proteins It is useful to test a new batch against previous batches m a gel retardation assay For small amounts of DNA, the Spin-X filter method is more efficient. As the amount of DNA 1soften small, rather than measuring absorbance at 260 nm the DNA concentration can be determined by compartson with known amounts of ethidium bromide-stained DNA spotted onto Saran wrap or electrophoresed through an agarose gel. Longer gels give better separation of DNA-protein complexes. We routinely use 1 5-mm thick 16 x 16 cm gels. The use of a 1O-well comb with this size gel yields much sharper and distinct bands than a 20-well comb It 1s possible to pour the gel on the same day as it is used, but it is important to allow sufficient time for complete polymertzation (90-120 mm). Use of a gel that has not polymerized completely will result m smearing along the tracks and/ or u-regular migration. Other runnmg buffers, such as TE (10 mA4 Tris-HCl, pH 8 0, 1 mA4 EDTA, pH 8 0), Tn.+glycme (50 mMTru+glycine, pH 9 4) or TAE (see Subheading 2.1.1.), can be used The concentration of TBE buffer can be varied according to the probe and the source of nuclear protein used (8,11). Low ionic-strength buffers are preferred because too high a salt concentration causes DNA-protem complexes to aggregate, and this retards their migration through the gel. We routinely use TBE buffer at 0 25X or 0.5X concentration Various amounts of nuclear protein extract can be used m gel-retardation assays We find that 5 1.18of nuclear protein extracted by the method of Sierra (13) is a good starting amount. Less may be required if the nuclear protems have been partially purified or enriched Poly d1 dC acts as a nonspecific competitor DNA to reduce nonspecific mteractions between nuclear proteins and the DNA probe. The amount of poly d1 dC added depends on the probe and the amount of nuclear protein Too little can result in the smearing of retarded bands or formation of multiple nonspecific complexes Some complexes may even fail to enter the gel Too much will reduce or prevent the formation of complexes. 0.5 pg of poly d1 dC per 1 pg nuclear protein extract works well in our hands. For partially purified or enriched nuclear proteins, the poly dI.dC concentration has to be substantially reduced for the detection of specific complexes (17). The optimal poly d1 dC concentration required for each DNA probe can be determined by performing a series of assays with increasing amounts of poly d1 dC for a fixed amount of nuclear protein The DNA probe should be added in excess m order to bind all specific nuclear proteins Usually, 2 x lo4 cpm of the radiolabeled probe per assay will be well in excess. The amount of probe can be mcreased if more nuclear proteins are added. In our studies, it is particularly important to add excess probe because treatment of the animals with phenobarbttal can increase the abundance of nuclear protein(s) that bind to particular regions of the CYPZBI promoter (Fig. 2, tracks 1 and 2).

Gel-Retardation

Assay

391

Fig. 2. Competitive gel-retardation assays: A radiolabeled DNA fragment extending from -348 to -45 1 of the CYP2Bl gene was incubated, at 0°C in the presence of 2.5 pg poly dl.dC, with 5 pg of nuclear proteins extracted from the livers of rats treated with phenobarbital (PB) or of untreated animals (U). Assays contained no competitor (-) or a loo-fold molar excess of unlabeled competitor, either the -348 to -45 1 DNA fragment or a double-stranded oligonucleotide containing a &BP consensus binding site, as indicated. The reaction mix in track 5 was incubated with 5 pg proteinase K. Reaction mixtures were electrophoresed through a 4% polyacrylamide gel, which was then autoradiographed. 19. To obtain sharper bands, the gel can be autoradiographed at room temperature for several days without intensifying screens. This is particularly useful when using a probe that forms many different proteiwDNA complexes. 20. The addition of proteinase K to one of the gel-retardation assay mixtures can confirm that the retarded bands represent proteipDNA complexes. 21. The competition assay is used to investigate the specificity of a particular protein-DNA interaction. The inclusion in the assay of a large molar excess of nonradiolabeled DNA probe can determine whether the protein-DNA interaction is sequence-specific. The use as competitors of synthetic oligonucleotides

Dell et al.

Fig. 3. Gel retardation assays in the presence of antibodies to DNA-binding proteins. (A) A radiolabeled DNA fragment extending from -44 to -67 of the CYP2B2 gene was incubated with 2.5 pg of rat liver nuclear protein extract in the presence (+) or absence (-) of 1 pg of antibodies specific for c/EBPa, j3, or 6, as indicated. Reaction mixtures contained 2.5 pg poly dI.dC. Numbers indicate the protein-DNA complexes supershifted by antibodies to c/EBPa (track 1) and c/EBPP (track 2), respectively. (B) A radiolabeled double-stranded oligonucleotide corresponding to the -183 to -199 region of the CYP2B2 gene was incubated with 5 pg of nuclear protein extract from the liver of phenobarbital-treated rats in the presence of either an antibody that recognizes the DNA-binding domain of Ott 1 and Ott 2 proteins (OctV2) or one that does not recognize these proteins (control), or in the absence of antibody (-). Reaction mixtures contained 2.5 l.tg poly dI.dC. Reaction mixtures were electrophoresed through a 6% (A) or a 4% (B) polyacrylamide gel, which was then autoradiographed.

corresponding to consensus protein-binding sites or to short sections of the DNA probe can, respectively, aid identification of the protein that is bound, and delineation of the DNA-binding site (Fig. 2). Unlabeled competitor DNA should be added at a molar excess of between 50- to 500-fold depending on the affinity of the protein for the DNA probe. 22. The inclusion in a gel-retardation assay mix of an antibody specific for a particular DNA-binding protein can reveal whether the protein is present in the DNAprotein complex formed. If the antibody interacts with the DNA-binding protein, it can result either in a supershift of the complex, because of the presence of the antibody, or an inhibition of complex formation, if the DNA-binding domain of the protein is masked by interaction with the antibody (Fig. 3).

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Acknowledgments We thank Shaun Thomas, Department of Hematology, University College London, for the Oct1/2 antibody. H. D. was a reclplent of a Wellcome Trust Toxicology Studentshlp. S. C. W. is a recipient of a EDB/Glaxo (Singapore) Scholarship. The work was supported by grants from the Cancer Research Campaign and the Wellcome Trust (ref. no 042495). References 1. Whitlock, J. P., Okino, S. T., Dong, L., Ko, H. P., Clarke-Katzenberg, R , Ma, Q., and Li, H. (1996) Induction of cytochrome P450 1A 1’ a model for analysing mammalian gene transcription FASEB J. 10,809-8 18 2. Johnson, E. F , Palmer, C. N. A., Griffin, K. J , and Hsu, M -H (1996) Role of the peroxisome proliferator-activated receptor m cytochrome P450 4A gene regulation FASEBJ. 10,1241-1248. 3. Shephard, E A., Forrest, L. A , Shervington, A., Fernandez, L. M , Claramella, G , and Phllllps, I R. (1994) Interaction of proteins with a cytochrome P450 2B2 gene promoter: ldentlfication of two DNA sequences that bmd protems that are enriched or activated m response to phenobarbital DNA Cell Brol 13, 793-804 4 Luc, P V., Adesnik, M , Ganguly, S., and Shaw, P M. (1996) Transcrlptlonal regulation of the CYP2BI and CYP2B2 genes by UEBP-related proteins Biochem Pharmacol 51,345-356. 5 Sommer, K , Ramsden, R , Sldhu, J., Costa, P., and Omlecmskl, C J (1996) Promoter region analysis of the rat CYP2Bl and CYP2B2 genes. Pharmacogenetxs 6,369-374 6. Honkakoskl, P., Moore, R., Gynther, J., and Negishl, M (1996) Characterlzatlon of phenobarbital-inducible mouse CypZblO gene transcription m primary hepatocytes. J Biol Chem 271,9746-9753. 7. Roe, A L., Bloum, R. A , and Howard, G V (1996) In vlvo phenobarbital treatment increases protein-binding to a putative AP-1 site in the CYP2B2 promoter. Blochem Biophys Res Comm. 228, 1 I@-1 14. 8 Rosette, C. and Karm, M. (1995) Cytoskeletal control of gene expression: depolymerisatlon of mlcrotubules activates NF-kB. J. Cell Blol 128, 111 l-l 119. 9 Lowry, O., Rosebrough, N., Farr, A., and Randall, R. (1951) Protein measurement with the Folm phenol reagent. J Blol Chem 193,265-275. 10. Sambrook, J., Fntsch, E. F., and Mamatis, T. (1989) Molecular Clonmg, A Laboratory Manual, vol. 3,2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11. Scott, V., Clark, A. R., and Docherty, K. (1993) The gel retardatzon assay. Methods m Molecular Biology, vol 31. Protocols for Gene Analysis (Harwood, A. J., ed ), Humana, Totowa, NJ, pp. 339-347. 12 Hassanain, H. H., Dal, W., and Gupta, S. L (1993) Enhanced gel mobility shift assay for DNA-binding factors Anal. Biochem 213, 162-167 13. Sierra, F. (1990) Laboratory Gwde to In Vitro Transcnptzon. BloMethods, vol 2. Birkhauser Verlag, Basel.

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Dell et al.

14. Lavery, D. J., and Schtbler, U. (1993) Circadian transcrtptron of the cholesterol‘i’a-hydroxylase gene may involve the liver-enriched b-ZIP protem DBP Genes Dev. 7,1871-1884 15 Jose-Estanyol, M , Pollard, A , Foiret, D., and Danan, J.-L (1989) A common liver-spectfic factor binds to the rat albumin and a-foetoprotein promoters m vitro and acts as a positive trans-acting factor in vivo. Eur J Biochem. 181,761-766 16. Deryckere, F. and Gannon, F. (1994) A one-hour mmtpreparation technique for the extraction of DNA-binding proteins from animal tissues BzoTechniques 16,405. 17. Larouche, K., Bergeron, M.-J., Leclerc, S., and Guerm, S (1996) Optimization of competitor poly(dI-dC). poly(dI-dC) levels is advised in DNA-protein interaction studies mvolvmg enrtched nuclear proteins. BtoTechnzques 20,43%-444

41 Cytochrome P450 Gene Regulation Analysis of Protein-DNA Interactions In Situ Steven T. Okino and James P. Whitlock, Jr. 1. Introduction Most mechanistic analyses of gene regulation are performed m contexts different from the physiological conditions of the intact cell. For example, transcnption factors are often studied according to their ability to modulate the expression of plasmid-based reporter genes rather than endogenous chromosomal genes; DNA regulatory elements are usually analyzed as oligonucleotides m vitro or as components of recombmant plasmids, rather than m their native chromosomal setting. Such studies, though mformative, may not falthfully reflect gene regulation in vivo. In addition, many studies ignore the regulatory effects of chromatin structure on gene expression. In studies that do address chromatin structure, reconstituted chromatin is often analyzed m vitro; m such situations, the stoichtometric relationship between DNA and transcription factors may not resemble those found m vivo, and other important regulatory macromolecules may be inactive or missing. Therefore, it is important to develop approaches for studying gene regulation under conditions similar to those that exist in VIVO. In viva footprinting using dimethyl sulfate is a powerful techmque that detects protein-DNA mteractions on endogenous genes in intact cells (1,Z). Studies using this technique have helped to determine mechanisms by which native genes are regulated in vivo (1,3). However, dimethyl-sulfate footprmtmg is hmited in that it cannot detect protein-DNA interactions at sites that lack a guanine residue (such as the TATA box) nor those that occur on the sugarphosphate backbone or in the minor DNA groove. In addition, this technique has limited usefulness m analyzing chromatin structure. Here, we descrtbe a From Methods 1r1 Molecular Biology, Vol Edited by I R Phllllps and E A Shephard

395

107

Cytochrome

0 Humana

P450

Protocols

Press Inc , Totowa,

NJ

396

Okino and Whitlock, Jr.

technique that can be used for analyzing chromosomal protein-DNA interactions and chromatm structure znsitu. The authors have developed these procedures during our analyses of the activation of the cytochrome P4501Al gene (Cyplal) by the environmental contaminant 2,3,7,8-tetrachlorod~benzo-pdioxin (TCDD). We have shown that induction of Cyplal gene transcription by TCDD is associated with disruption of nucleosomes, increased accesslblhty of enhancer and promoter DNA, and recruitment of general transcription factors to the promoter (4,5). These findings provide insight mto the mechanisms by which xenoblotlcs regulate the expression of specific mammalian cytochrome P450 genes. 2. Materials 2.7. Isolation

of Nuclei and Treatment,

In Situ, with DNase I

1 Cells: Four lo-cm plates of cells per sample (about 1 x 10’ cells/plate). Best results are obtained rf cells are ~80% confluent and growing exponentially. 2. DNase I dilution buffer: 20 n&f Tris-HCl, pH 7 6, 50 mM NaCl, 1 mM dlthlothreltol (DTT), bovine serum albumin (BSA), (100 pg/mL) 50% (v/v) glycerol Store at -20°C in 1-mL ahquots 3. DNase I: Add 10 mL of DNase I dilution buffer to a 100 mg bottle of DNase I (Boehringer Mannhelm, Mannhelm, Germany, 2000 umts/mg, cat. no. 104-l 59) to produce 10 mg/mL DNase I stock solutlon. Store at -20°C m 1-mL ahquots 4. Buffer A: 300 mA4 Sucrose, 60 mA4 KCl, 60 mA4 Tns-HCl, pH 8 0, 2 mM ethylenedlamme tetra-acetic acid (EDTA). Make fresh; 34 ml/sample are required. 5. Buffer A + 0 5% (v/v) Nonidet P-40 (NP-40, Sigma, St Louis, MO, cat. no N-6507). Make fresh; 4 ml/sample are reqmred 6. Buffer B: 150 mMSucrose, 80 mMKCl,35 mA4HEPES, pH 7 4,5 mMK2HP04, 5 mA4 MgCl*, 2 mM CaC&. Make fresh; 1 mL per sample is required 7. Buffer B + DNase I (20 pg/mL). Make fresh and add DNase I Just prior to use, 1 ml/sample 1srequired 8 Proteinase K (Sigma, cat. no P4914). Prepare as a 20 mg/mL stock in water Store at -20°C in 1-mL aliquots. 9. Buffer C: 20 mM Tns-HCl, pH 8.0, 20 mM NaCl, 20 mM EDTA, 1% (w/v) sodium dodecyl sulfate (SDS), protemase K (600 pg/mL). Make fresh and add protemase K just before use; 2 ml/sample are required 10. 15 mL Polypropylene centrifuge tubes (Applied Scientific, [South San Francisco, CA] cat. no AS2022).

2.2. Purification

of Genomic DNA

1. Buffer-saturated phenol and phenol:chloroform (1: 1, v/v ) (Gibco-BRL, Galthersburg, MD, cat. no. 155 13 and 15593 respectively). 2 RNase A (Sigma, cat no. R9009): Prepare as a 10 mg/mL stock in 10 mA4TnsHCl, pH 7 5, 15 mM NaCl; boll for 15 min, then allow to slowly cool to room temperature. Store at -20°C in 1-mL aliquots.

Cytochrome P450 Gene Regulation

397

3 10X RNase A buffer. 500 mM Tns-HCl, pH 7.5, 100 mM MgCl,, 50 mMDTT Store at -20°C in 1-mL aliquots. 4 3 M Sodmm acetate, pH 5 2 5. 100% Ethanol. 6 TE: 10 mA4Tris-HCI, pH 7.5, 1 mMEDTA 7 1.5-mL Microcentrifuge tubes (Umted Scientific Products, cat. no MCT-175-A)

2.3. DNase I Digestion of Genomic DNA In Vitro 10X in vitro DNase I buffer: 500 mA4 Tris-HCl, pH 7.5, 100 mA4 MgC12, BSA (500 pg/mL). Store at -2OOC in I-mL aliquots. 2.4.5’ End Labeling

of Primer 3 Kmase buffer: 700 mMTris-HCI, pH 7.6, 100 mMMgCl*,

1 10X Polynucleotide 5 mM DTT. Store at -2O’C m 1-mL ahquots. 2. [Y-~~P] ATP (3000 Ci/mmol, 10 mCi/mL) (Amersham, Arlmgton Herghts, IL, cat. no. AA0068) Store at 4°C. 3. T4 Polynucleotide kmase (New England BioLabs, Beverly, MA, 10,000 units/ml, cat. no 201L). Store at -20°C 4. NucTrap probe purification columns (Stratagene, La Jolla, CA, cat no 400701)

2.5. Ligation-Mediated

Polymerase

Chain Reaction (PCR)

1 Oligonucleotides: All ohgonucleotides must be purified on a 20% (w/v) polyacrylamide gel Resuspend the DNA m TE and quantify by measurrng absorbance in a spectrophotometer at 260 nm usmg the formula (pmol/pL) = (A2&0.01 x N)

(1) Where N represents the length of the oligonucleotide m bases. Keep a concentrated stock of each ohgonucleotide (about 100 pmol/pL) and drlute an ahquot of each oligonucleotide in TE for use as a working stock. Store all ohgonucleotides at -2O’C in 20-pL aliquots 2. Preparation of linkers. Synthesize and gel-purify the following ohgonucleotrdes. a. Long-linker oligonucleottde (25-mer). 5’-GCGGTGACCCGGGAGATCTGAATTC-3’ b. Short-linker oligonucleotide (11 mer): 5’-GAATTCAGATC-3’ c. Mrx the short- and long-linker oligonucleottdes to a final concentratron of 20 pmol/pL each, m 250 mMTris-HCl, pH 7 7 Heat at 95’C for 5 mm Transfer to a 72’C heat block and slowly cool to 22°C. Then transfer the heat block to the cold room and allow to cool to 4°C overmght The annealed linkers can reman-r at 4°C for several weeks. 3 Preparation and design of prrmers: Synthesize and gel-purify the followmg oligonucleotides a Linker primer: The linker primer is the long oligonucleottde used m making the linker, it is a 25-mer containing 15 GC residues Lurker-primer working stock is 25 pmol/pL.

4 5

6 7

8

9. 10 11 12 13 14 15 16

b Primer 1. Should be a 20-22-mer contammg about 50% GC content. Primer 1 working stock is 0.3 pmol/pL. c. Primer 2. Should be a 25mer contammg about 15 GC residues (similar to the linker primer) Primer 2 working stock is 25 pmol/pL. Primer 2 must be 3’ to primer 1. It can overlap primer 1 by up to 12 bases If primer 2 does not overlap primer 1, they should not be separated by more than 50 bases d. Primer 3: Should be a 27-28-mer containing about 17 GC residues. Primer 3 working stock is 4 pmol/pL. Primer 3 must be 3’ to primer 2 It can overlap prtmer 2 by up to 15 bases. If primer 3 does not overlap primer 2, they should not be separated by more than 200 bases The area to be analyzed should be between 25 and 250 bases away from the 3’ end of primer 3. It is possible to use the same primer 1 and primer 2 and multiple primer 3’s spaced apart from each other to analyze different regions of a gene. 5X First-strand buffer. 200 mMNaC1, 50 mMTris-HCl, pH 8.9,25 mA4MgS04, 0.05% (w/v) Gelatin. Store at -20°C m 1-mL altquots 5X Amplification buffer: 200 mM NaCl, 100 mM Trts-HCl, pH 8 9, 25 mM MgSO,, 0.05% (w/v) Gelatin, 0 5% (v/v) Triton X- 100. Store at -20°C in 1-mL aliquots tRNA (Sigma, cat no. R0128) Prepare as a 10 mg/mL stock m TE Store at-20°C in 1-mL aliquots Stop solution. 10 mM Tris- HCl, pH 7.5,4 mMEDTA, 260 mM sodmm acetate, pH 7.0,67 pg/mL tRNA. Thts solution can be made as a stock wtthout tRNA and stored at room temperature Add tRNA Just before use 20 mM dNTPs Mix together equal volumes of the four 100 mM dNTP stocks (Pharmacta, Piscataway, NJ, cat no. 27-2035-01) and water Store at -20°C m 50-pL ahquots 2 mM dNTPs: Make a 1: 10 dilution of the 20 mM dNTP stock with water Store at -20°C in 75-pL aliquots Vent DNA Polymerase (New England BioLabs, 2000 units/ml, cat. no 254L) Store at -20°C 1 M Tris-HCI, pH 7.5 1 MM&l,. 1 M DTT. Store at -20°C m I-mL ahquots. 100 mMATP (Pharmacta, cat. no 27-2056-01) Store at-20°C m 20-pL ahquots T4 DNA hgase (Promega, Madison, WI, 3000 umts/mL, cat. no. Ml 802). Store at -20°C. 0.5-mL Thermocycler reaction tubes (Perkin Elmer, Norwalk, CT, cat. no. N801-0280)

2.6. Polyacrylamide-Gel

Electrophoresis

1 10X TBE: Dissolve 108 g of tris base and 55 g of boric acid m 800 mL of water Add 40 mL of 0.5 M EDTA, pH 8 0, and water to give a final volume of 1 L 2. 20% Acrylamide/urea: Mix together 2 1 g of urea, 9.66 g ofacrylamtde, 0.33 g of bisacrylamtde, and 5 mL. of 10X TBE. Add water to give a final volume of 50 mL.

Cytochrome P450 Gene Regulation

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3. 8% Acrylamldelurea: Mix together 210 g of urea, 38.66 g of acrylamlde, 1 33 g of bisacrylamlde, and 50 mL of 1OX TBE Add water to make a final volume of 500 mL. 4. Loading Buffer: deionized formamide (Gibco-BRL, ultraPURE) containing 0.3% (w/v) xylene cyanol, 0.3% (w/v) bromophenol blue, and 1 mA4EDTA, pH 8.0. 5. Fix solution: 15% (v/v) methanol, 5% (v/v) acetic acid. 6. Whatman 3MM filter paper 7. Hyperfilm-MP (Amersham).

3. Methods 3.1. Isolation

of Nuclei and Treatment

with DNase I (see Note 1)

1. Cool buffer A and buffer A + NP-40 on ice. Buffers B and C should be at room temperature. 2 Aspirate medium from plates. Rinse each plate with 5 mL Ice cold buffer A Add 2.5 mL buffer A to each plate and scrape, using a rubber policeman, to dtslodge cells. Usmg a 1-mL plpet, transfer the cells from all plates mto a 15-mL centnfuge tube on ice. 3. Pellet cells by centlfugation

at 5OOg for 5 min at 4T

Aspirate

supematant

Resuspend cells m 4 mL of buffer A. Add 4 mL of buffer A + NP-40, mix gently, and incubate on Ice for 5 min. 4. Pellet nuclei by centlfugatlon at 500g for 5 mm at 4°C (see Note 2). Asprrate supematant. Resuspend nuclei in 1 mL of buffer B. Add 1 mL of buffer B + DNase I, mix gently, and mcubate at room temperature for 90 s (see Note 3) Stop the reactions by adding 2 mL of buffer C (see Note 4) Incubate at 37°C for 3 h.

3.2. Purification

of Genomic DNA

1. Extract the vfscous solution with an equal volume of phenol. Transfer the aqueous phase to a new 15-mL centrifuge tube and re-extract with an equal volume of phenol-chloroform (1.1, v/v). Transfer the aqueous phase to a new 15-mL centrlfuge tube and add 400 pL of 3 Msodium acetate and 10 mL of ethanol to preclpitate the DNA. Store at -20°C overnight 2. Pellet the DNA by centrifugation at 5,000g for 30 min at 4°C. Aspirate the supernatant and air-dry the DNA pellet. Resuspend the DNA in 355 pL water, transfer to a 1.5-mL microcentrifuge tube. Add 40 pL of 10 X RNase A buffer and 5 pL of RNase A. Incubate at 37°C for 1 h. 3. Extract the DNA with an equal volume of phenol-chloroform (1: 1, v/v). Transfer the aqueous phase to a 1.5-mL microcentrifuge tube contammg 1 mL of ethanol and 40 @., of 3 M sodmm acetate. Cool on dry Ice for 30 mm or at -20°C for at least 2 h. 4. Pellet the DNA by centrifugatlon in a mlcrocentrlfuge at 16,OOOg for 15 min at 4°C. Aspirate the supematant and air-dry the DNA pellet. Resuspend the DNA m 200 & of TE Determme the DNA concentration by measuring absorbance m a spectrophotometer at 260 nm (see Note 5). Dilute the DNA to 1 mg/mL rn TE. Store at -2O’C.

3.3. DNase I Digestion

of Purified

Genomic DNA

1 Isolate genomtc DNA as indicated m Subheadings 3.1. and 3.2., but do not treat the nuclei with DNase I (i.e , m step 4 of Subheading 3.1. resuspend the nuclei pellet m 2 mL of buffer B, then add 2 mL of buffer C and incubate at 37°C for 3 h) 2 Dilute DNase I in DNase I dilution buffer to obtam final concentrations of 2 0, 1 5, 1.0,0.5, 0 25, 0 1, and 0 pg DNase I/mL. Store on ice 3. In each of seven 1.5-mL microcentrifuge tubes, mix 50 pL of genomic DNA, 40 pL of 10X in vitro DNase I buffer and 270 p.L water, warm to 37°C. 4. Add 40 pL of a DNase I dtlutton to each tube. Mix gently and incubate at 37°C for 5 min 5. Extract the DNA with an equal volume of phenol-chloroform (1 1, v/v) Transfer the aqueous phase to a 1 S-mL microcentrifuge tube containing 1 mL ethanol and 40 uL of 3 M sodium acetate Cool on dry ice for 30 mm or at -20°C for at least 2 h 6. Pellet the DNA by centrifugation m a microcentrtfuge at 16,000g for 15 mm at 4°C. Aspirate the supernatant and au-dry the DNA pellet. Resuspend the DNA m 40 pL of TE Determine the DNA concentration by measurmg absorbance m a spectrophotometer at 260 nm. Dilute the DNA to 1 mg/mL in TE 7 Check the extent of DNase I digestion by agarose-gel electrophoresis. Appropriately digested DNA should range between 200 bp and 3 kb m size. Store DNA at -2O’C.

3.4. 5’ End Labeling

of Primer 3

1 Label 2 pmol of primer 3 (0.5 pL of primer 3 working stock) per DNA sample to be analyzed. If analyzmg more than 20 DNA samples, do two separate labeling reactions 2. In a 1.5-n& microcentrifuge tube mix primer 3 and water to make a volume of 10 $ Add 3 pL of 1OX polynucleottde kmase buffer, 15 pI.. of [Y-~~P] ATP and 2 pL of T4 polynucleotide kmase. Incubate at 37°C for 1 h. 3. Remove unmcorporated [Y-~~P] ATP. We use NucTrap Probe purification columns accordmg to the manufacturer’s directions. 4. Add 2.5 volumes ethanol and 0.1 volume 3 Msodmm acetate Cool on dry ice for 30 min or at -20°C for at least 2 h. 5. Pellet the DNA by centrifugation in a microcentrifuge at 16,000g for 15 mm at 4°C. Aspirate the supernatant and au-dry the DNA pellet

3.5. Ligation-Mediated

PCR

1 First strand reaction. a Add 4 pL (4 ug) of purified genomic DNA to a 0.5-mL thermocycler tube. Place on ice. b Using Table 1, assemble the first strand reaction mix on ice, addmg Vent DNA polymerase last (all volumes are m pL)

Cytochrome P450 Gene Regulation Table 1 First-Strand

Reaction

Mix

# of samples

1

5X 1st strand buffer 2 rmI4 dNTPs Primer 1 (0.3 pmol/&) Vent (2 umts/&) Water

6 3.6 1

Table 2 Linker Ligation # of samples

401

2

4

6

8

10

12

12 24 36 48 60 72 7.2 14.4 21 6 28 8 36 43.2 2 4 6 8 10 12

05 1 15 30

2 60

3 90

4 120

5 6 150 180

4

6

8

10

132 22 5 366 9 45 30 9 178.5

176 30 488 12 6 40 12 238

22 375 61 15 75 50 15 297 5

14

16

18

84 96 108 50+4 57.6 64.8 14 16 18

20 120 72 20

7 210

8 240

9 270

10 300

14

16

18

20

Mix 1

2

1 MTns-HCl, pH 7.5 2 2 4 4 8.8 BSA (1 mg/mL) 375 75 15 1 M MgClz 0.61 122 244 1MDTT 1.5 3 6 100 mMATP 075 15 3 Lmkers 5 10 20 Llgase(3 umts/pL) 1.5 3 6 Water 29.75 595 119

12

264 308 45 52 5 732 854 18 21 9 105 60 70 18 21 357 416.5

352 39.6 60 67 5 976 1098 24 27 12 135 80 90 24 27 476 535.5

44 75 122 30 15 100 30 595

c Add 26 & of the first-strand reaction mix to each DNA sample MIX gently and centrifuge briefly (10 s) to get all liquid to the bottom of the tube Do not overlay these samples with mineral 011 d. Place samples into a thermocycler preheated to 95°C (we use a DNA Thermal Cycler 480 from Perkm Elmer Cetus) Incubate at 95°C for 5 mm, then at 42°C for 30 mm and 76’C for 10 mm Cool to 4°C Remove samples from thermocycler and place on ice 2. Linker ligation. a. Using Table 2, assemble the linker ligation mix on Ice, adding the lmkers and the T4 DNA hgase last (all volumes are m pL), b. To each sample from step 1, item d, add 45 $ of the linker ligation mix Mix gently, then incubate at 17°C overmght. 3. Amplification reaction. a. To each sample from step 2, item b, add 8.3 pL, of 3 M sodium acetate, 1 pL tRNA (10 pg) and 225 Ils, ethanol. Cool on dry ice for 30 mm or at -20°C for at least 2 h b Pellet the DNA by centrlfugation in a mlcrocentrlfuge at 16,000g for 15 mm at 4°C Aspirate the supematant and air-dry the DNA pellet Resuspend the DNA in 70 & of water and place on ice

402 Table 3 Amplification # of samples 5X Amplification Buffer 20 mMdNTPs Primer 2 (25 pmoW4 Linker primer (25 PmoWJ Vent (2 units/&) Water

Okino and Whitlock, Jr. Mix 1

2

4

6

8

20

40

80

120

160

10

12

14

16

18

20

200 240

280

320

360

400

1 0.4

2 0.8

4 1.6

6 2.4

8 32

10 4

12 48

14 56

16 64

18 7.2

20 8

0.4

0.8

1.6

2.4

3.2

4

4.8

5.6

6.4

72

8

3 6 9 13 4 26 8 40.2

12 53.6

15 67

18 80.4

15 6.7

21 24 93 8 107

27 120

30 134

Table 4 Labeling Mix # of samples

1

2

4

6

8

10

12

14

16

18

20

5XAmphficatlon Buffer 20 mA4dNTPs Vent (2 umts/pL) Water

1

2

4

6

8

10

12

14

16

18

20

05 0.5 3

1 1 6

2 2 12

3 3 18

4 4 24

5 5 30

6 6 36

7 7 42

8 8 48

9 9 54

10 10 60

c. Using Table 3, assemble the amplification mix on ice, adding Vent DNA polymerase last (all volumes are in &) d. To each sample from step 3, item b, add 30 $ of the amplification mix. Overlay with 50 pL of mineral 011.Mix gently and centrifuge briefly (10 s) to get all liquid to the bottom of the tube. e. Place samples into a thermocycler preheated to 95°C. Incubate as follows 1 Cycle. 95°C for 3 mm, 60°C for 2 mm, 76“C for 3 mm. 15 Cycles: 95°C for 1 min, 60°C for 2 min, 76“C for 3 min + 5 s/cycle Cool to 4OC Remove samples from thermocycler and place on ice. 4 Labeling reaction a. Using Table 4, assemble the labeling mix on Ice, adding Vent DNA polymerase last (all volumes are in $) b. Resuspend the primer 3 pellet from, Subheading 3.4., step 5 m 5 & of labeling mix/sample c. Add 5 pL of the primer 3/labelmg mix to each sample from step 3, item e, pipeting through the mineral 011 overlay to the bottom of the tube. Mix gently.

Cyfochrome P450 Gene Regulation

403

d. Place samples into a thermocycler preheated to 95°C. Incubate as follows* 2 cycles: 95°C for 3 min, 66°C for 2 min, 76°C for 10 mm Cool to 4°C. Remove samples from thermocycler and place on ice. 5. Isolation of labeled DNA a. Pipet the DNA samples (leave behind the mmeral oil) mto 1.5-mL microcentrifuge tubes contaming 400 pL of phenol/chloroform (1: 1, v/v) and 295 pL of stop solution. b. Vortex vigorously, centrtfuge m a microcentrifuge at 16,OOOg for 5 mm at 22’C and transfer the aqueous phase to a microcentrifuge tube contammg I mL ethanol Cool on dry ice for 30 mm or at -2O’C for at least 2 h c Pellet the DNA by centrrfirgation in a microcentrifuge at 16,OOOgfor 15 mm at 4°C. Aspirate the supernatant and an-dry the DNA pellet. d Resuspend the DNA pellet m 10 $ of loading buffer. The sample is now ready for analysis by polyacrylamide-gel electrophoresis. Store at -20°C.

3.6. Polyacrylamide-Gel

Electrophoresis

1 2. 3 4

Pour an 8% acrylamide/urea gel in an apparatus suitable for DNA sequencing. Pre-run the gel m 1X TBE for 30 min. Heat the DNA samples to 95’C for 3 mm. Load 3 p.L of the DNA samples into individual wells on the acrylamide gel. Electrophorese until the xylene cyanol dye is about 314 of the way to the bottom of the gel. 5 Soak the gel m fix solution for 20 mm, transfer to Whatman 3MM filter paper, and dry on a gel dryer 6 Expose the dried gel to autoradiography film (we use Hyperfilm-MP) overnight at -20°C using intensifying screens (see Notes 6 and 7) 7. This procedure can detect changes in the accessibllty of genomic DNA to DNase I likely reflecting a change in the chromatin (nucleosomal) structure and constitutive and mducible protein-DNA mteractions on an endogenous gene. a. When comparing samples, a significant and reproducible difference m banding intensity may reflect a difference m the chromatm structure (for example, see refs. 4 and 5) b. To detect protein-DNA interactions, it is sometimes necessary to equalize the banding mtensity by dilutmg some samples with loading buffer as indicated Protein-DNA interations are indicated by a region of DNA that is protected from DNase I digestion when compared with naked genomic DNA digested with DNase I in vitro (for example, see refs. 4 and 5)

4. Notes 4.7. Isolation

of Nuclei and Treatment

with DNase I

1. The optimal amount of DNase I to use should be determined empirically In our hands, 10 pg DNase I/mL works best for the mouse hepatoma cell lme Hepa lclc7

404

Okino and Whitlock, Jr

2. The cell pellet should be white The nuclear pellet may be considerably smaller than the cell pellet (about l/2-1/4 of the size) and more translucent. If the nuclear pellet IS not visibly different from the cell pellet, a high concentration of NP-40 should be used If there is little or no nuclear pellet, a lower concentration of NP-40 should be used 3. When preparing multiple samples simultaneously, stagger the addition of DNase I by 15 s so that all of the reactions can be stopped at exactly 90 s. 4. The solution, which contams nuclei, should become vtscous immediately after the addition of buffer C.

4.2. Purification

of Genomic DNA

5. The expected yield IS about 300 pg of genomic DNA per sample.

4.3. Polyacrylamide-Gel

Electrophoresis

6 If no bands are present on the initial autoradiogram, the annealmg may be too high Try lowermg the annealing temperature of the reaction and/or the labeling reaction m 3’C mcrements. 7 If the lanes are smeary with no distmct banding pattern, the annealing may be too low, Try increasing the annealing temperature of the reaction and/or the labeling reaction in 3°C increments.

temperatures amplification temperatures amplification

References 1. Mueller, P. R. and Wold, B. (1989) In vivo footprmting of a muscle specific enhancer by ligation mediated PCR Science 246, 780-786. 2. Wu, L., Okino, S T., and Whitlock, Jr, J. P. (1994) In viva protein-DNA mteracttons associated with gene transcription. Methods Mol Genet 5,53-64 3. Wu, L. and Whitlock, Jr., J. P (1992) Mechamsm of dtoxm action. Ah receptormediated increase m promoter accessibihty m vivo Proc. Nat1 Acad Scl USA 89,4811-4815 4. Okino, S. T. and Whitlock, Jr., J. P. (1995) Dioxin induces localized, graded changes in chromatm structure* implications for Cyp 1Al gene transcription Mol Cell Blol l&3714-3721. 5. Ko, H. P., Okino, S T., Ma, Q., and Whitlock, Jr., J. P. (1996) Dioxm-Induced CYPlAl transcrtptton m vtvo: the aromatic hydrocarbon receptor mediates transactivation, enhancer-promoter communication, and changes in chromatin structure. Mol Cell Blol. 16,430-436.

42 Analysis of Cytochrome P450 Polymorphisms Ann K. Daly, Sophia C. Monkman, Joanne Smart, Annette Steward, and Suzanne Cholerton 1. Introduction A number of common functtonally significant genetic polymorphisms in genes encoding certain cytochromes P450 have been described and characterized (for review, see refs. I and 2). Individuals may lack certain enzyme activities or have higher or lower than normal activities owing to the presence of certain variant alleles. These mdividuals may be at altered risk of developmg adverse drug reactions or drseases associated wtth xenobiotrc exposure, mcludmg cancer. Variant alleles can be detected by the direct approach of genotyping where the individual’s DNA is directly examined usually by an assayinvolving use of the polymerase chain reactton (PCR), or by phenotypmg, where the individual’s pattern of metabolites produced from a probe drug is examined. This chapter describes the detection of common polymorphisms m cytochrome P450 genes using both genotyping and phenotypmg approaches. Genotyping is now generally preferred to phenotyping because it is more convenient, requiring only a single blood sample, which can be taken at any time. It can also be used on stored blood or other tissue samples. Phenotypmg, however, is useful when investigating drug mteractrons m viva and in situations where the molecular basis of a polymorphism is unclear. In the sections of this chapter covering genotyping, we describe general methods for PCR and analysts of PCR products that have been used to genotype for pharmacogenetic polymorphisms, together with the specific conditions used to detect a range of known polymorphisms in cytochrome P450 genes. Methods for phenotypic detection of polymorphisms in CYP2D6 and CYP2A6 are also described.

From Methods !n Molecular Srology, Vol 107 Cytochrome P450 Protocols Edited by I R PhIllIps and E A Shephard 0 Humana Press Inc , Totowa, NJ

405

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2. Materials 2. I. Genotyping 2.1.1. Preparation of DNA from Human Blood 1. Human blood l-5 mL 1scollected usmg etther ethylenediaminetetra-acetic acid (EDTA) or citrate as antlcoagulant If required, the blood can be stored at -20°C for up to 1 yr or at -80°C for longer periods prior to DNA extraction. 2. Lysls buffer: 320 mM sucrose, 5 mM MgCl*, 1% (v/v) Triton X-100, 10 mM Tns-HCl, pH 8.0. The buffer is made up omlttmg the Trlton X-100 and autoclaved. Triton X- 100 is added to the autoclaved solution while still warm and the solution 1simmediately mixed vigorously to ensure uniform mixing. Store at 4°C 3 Solution B 150 mA4 NaCl, 60 mM EDTA, 1% (w/v) sodium dodecyl sulfate (SDS), 400 mM Tris-HCl, pH 8 0 The buffer is made up omitting the SDS and autoclaved. After coolmg, the SDS is added and the solution is stored at room temperature 4. 5 A4 sodium perchlorate. Store at room temperature 5 Chloroform, precooled to -2O’C 6. Ethanol, precooled to -20°C 7 70% (v/v) ethanol. 8. 10 mM Tris-HCl, 1 mM EDTA, pH 7.4 Autoclave. Store at room temperature.

2.1.2. PCR 1 Nucleotlde mix (2 mM each of dATP, dGTP, dCTP, dTTP). Stock solutions of each of the four nucleotides are purchased from a commercial supplier (such as Boehrmger Mannheim, Lewes, UK) at a concentration of 100 mM, pH 7 0. To prepare 1 0 mL of 2 tinucleotide mix, add 20 $., of each 100 mMnucleotlde stock to 920 & of stenle water. Mix well. Dispense mto 50 & aliquots Store at -20°C 2. Thermostable DNA polymerase. Thermostable DNA polymerases are available from a variety of suppliers. For most genotypmg assays, Blotaq DNA polymerase from Blolme 1sused (London, UK), but Tbr DNA polymerase 1salso used (NBL Gene Sciences, Cramlington, Northumberland, UK) and the Expand PCR system (Boehrmger-Mannhelm, Lewes, UK) in certain assays (see Note 1) 3. 10X PCR buffer: 500 mMKC1, 15 mMMgCl,, 100 mMTris-HCl, pH 8.8, 1% (v/v) Triton X- 100. The buffer should be autoclaved before addition of the Trlton X-100 Store in small aliquots at -20°C 4 Thermocycler. 5 PCR primers: The particular ollgonucleotide primers required for each assay are listed m the Methods section. Primers are normally diluted to a concentration of 25 w in water and stored in small aliquots at -20°C. Satisfactory results are obtained from unpurified oligonucleotide preparations.

2.7.3. Gel Electrophoresis

of PCR Products

1. 1OX Tris-Borate EDTA buffer (TBE)* 0.9 M Tns-borate, 20 mM EDTA. To prepare 1 L, dissolve 108 g Trlzma base and 55 g Boric acid in 900 mL water Add

Cytochrome P450 Polymorphisms

2.

3.

4. 5.

6.

407

40 mL 0.5 M EDTA, pH 8.0. Make up volume to 1 L wtth water. Autoclave Store at room temperature. Agarose* Depending on the precise requirements of the assay, either standard or high-resolution agarose gels may be run. Standard agarose gels may be prepared using molecular biology-grade agarose, which is available from a variety of suppliers. For high-resolution gels, we use FMC Nu-sieve 3:1 agarose (Flowgen, Staffordshrre, UK). 30% acrylamide solution: Dissolve 29 g acrylamide and 1 g N,N’-methylenebisacrylamlde to a final volume of 100 mL m water. Cm&on. Acrylamlde 1s a neurotoxin and should be handled with great care, especially when weighing Acrylamide blsacrylamide solutions are available from a variety of commercial supplters and may be a more convenient and safe alternative to preparing the stock solution in the laboratory 10% (w/v) ammomum persulfate. Ethidium bromide (EB) solution: Dissolve 0.1 g sohd ethidlum bromide m 10 mL water. EB is a mutagen and must be handled with care The solution is stored at 4°C m a foil-wrapped universal container. Molecular-weight standards* Where the products of a PCR reaction or restriction digest are of 1000 bp or less, a 100 bp DNA ladder is used (Gibco-BRL, Paisley, UK), which consists of DNA molecules m the range 100 to 1500 bp and gives bands every 100 bp If the products of the PCR reaction are longer than 1000 bp, we use h DNA digested with the restriction enzyme HzndIII, which gives bands of 23 kb, 9.4 kb, 6.6 kb, 4.4 kb, 2.3 kb, 2.0 kb, 564 bp, and 125 bp This material is avadable commercially from a range of suppliers

2.2. Phenotyping 2.2.1. Debrisoquine

Phenotyping

1 Clear glass bijou vials (16 mL) with open-top caps and polytetrafluoroethylene (PTFE)-lmed seals (Tuff-Bond discs, Pierce & Warrmer, Chester, UK) 2 Heating block, used m fume cupboard 3 Debrlsoquine capsules for oral ingestion, contammg 10 mg debrlsoquine (Declinax, Roche Products, Herts, UK) 4. Urine collection containers and 25 mL universal storage tubes 5 Debrlsoquine (available from Sigma [Poole, UK] and ICN [Thame, UK]), 4-hydroxydebrlsoqume (Ultrafine Chemicals, Manchester, UK) and 7-methoxyguanoxan (see Note 2), each 1 g/L in methanol:water (1.49, v/v) (see Note 3). 6. Drug-free urine. 7. 1 M Sodium bicarbonate. 8 Hexafluoroacetylacetone (HFAA) (Aldrich, Dorset, UK) (see Note 4). 9. Toluene, dlstol grade, used from a glass PTFE dispenser 10 3 A4 Sodium hydroxide (see Note 5). 11. Gas chromatograph with electron-capture detector suitable for capillary chromatography (see Note 6) 12. Capillary gas chromatography column 40 m x 0.25 mm Id x 0.25 w DB 1 (see Note 7).

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2.2.2. Coumarin Phenotyping 1. Coumarin capsules for oral ingestion containing 2 mg of coumarm (see Note 8). 2 Urme-collection containers and 25mL universal storage tubes 3. 7-hydroxycoumarm (1 mg/mL) in 10% (v/v) disttlled water in ethanol Stable at 4°C for up to 1 mo. 4 P-glucuromdase (Type H-2 from HeEzxpomatia, Sigma, Poole, UK). 5. Chloroform: high-pressure liquid chromatography (HPLC) grade. 6 Ethyl acetate: HPLC grade. 7. The lower phase of chloroform-distilled water-ethyl acetate-acetic actd (24.12*6:0 5, v/v) Make fresh as required. 8 Thin-layer chromatography (TLC) plates Precoated high performance thm-layer chromatographic glass-backed s&a gel 60 F254, 10 x 20 cm, (Merck, Darmstadt, Germany) 9 Automatic sample applicator for TLC plates, such as the Lmomat IV (Camag, Muttenz, Switzerland). 10 Hortzontal developing chamber for TLC plates. 11, Densitometer capable of scanning fluorescent TLC plates together with dataanalysis system. We use a Camag TLC scanner II connected to a Camag SP4290 TLC integrator.

3. Methods 3.1. Geno typing 3.1.1. Preparation of DNA from Human Blood 1 Nuclei are prepared by addition of the blood to 35 mL lysts buffer m a 50-mL conical polypropylene centrifuge tube and, after gentle mixing, centrtfugation at 2000g for 10 min at 4°C 2. The pellet is resuspended in 2 mL solution B and transferred to a 15-mL polypropylene centrifuge tube To this, 0 5 mL 5 M sodium perchlorate is added The suspension is rotary mixed at room temperature for 15 mm and then incubated at 65°C for 30 min. Two mL chloroform is added and the mixture rotary mixed at room temperature for 10 min, followed by centrifugatlon at 14OOg for 10 mm to separate the phases. 3. The aqueous DNA-containing upper phase is transferred to a fresh tube and 2 volumes of ethanol added The tube is inverted gently to precipitate DNA and the DNA spooled onto a disposable plastic loop The spooled DNA IS briefly washed m 70% ethanol and allowed to dry at room temperature for 20 min. 4. The DNA is then resuspended rn 200 pL 10 mMTris-HCl, 1 mA4EDTA, pH 7 4, by incubation at 60°C for 8-16 h and stored at 4°C until required 5. The yield, concentratton, and purity of the DNA is assessed spectrophotometritally. The absorbance at 260 nm and 280 nm for typically a 1 in 50 dtlutton of the DNA is determined. The AZbOis used to calculate the DNA concentration and both AZ6c and A,,, as a measure of purity (see Note 9).

Cytochrome P450 Polymorphisms

409

3.1.2. General PCR procedures 1 A master-mix consisting of PCR buffer, nucleotrdes, prrmers, enzyme, and water is prepared. Depending on the precise assay, either 25 pL or 50 pL is allowed for each assay. To prepare 1 mL of our regular master-mix, to a 1S-mL microfuge tube add 100 pL 10X PCR buffer, 100 ,L& nucleotides stock, 10 pL of each of primer 1 and primer 2 (assummg a 25 j,1J4stock), 595 pL (if necessary, adJust volume to ensure final volume of 1 mL) sterile water and, finally, 25 units (usually 5 pL) Tuq polymerase. All pipeting should be done using automatic pipets reserved for this purpose (see Note 10). Mix gently and spin briefly m a microcentrifuge. 2. Pipet the appropriate volume (25 or 50 pL) mto 0.5-mL microcentrifuge tubes Add genomic DNA (100 ng-1 pg) for amplification (see Note 11) At least one tube should be used as a “no DNA” blank and appropriate controls of known genotype should also be included (see Note 10). Centrifuge briefly and overlay with 100 pL mineral oil (see Note 12). 3. Place the tubes m a thermocycler programmed for the recommended temperatures and numbers of cycles (see individual assays for details)

3.1.3. Analysis of PCR Products by Electrophoresis 3.1.3.1. USEOF ELECTROPHORESIS 1. On completion of the PCR reaction, rt 1soften useful before additional analysis is performed to determine, by gel electrophoresis, whether the amphfication has been successful In the case of allele-specific PCR assays, electrophoretic analySIS of the products of PCR is the final step of the procedure. 2. The precise type of gel and electrophoresis conditions will depend on a number of factors, including size of fragments to be analyzed and whether separation of a number of fragments of similar molecular weight is required. 3 It 1s essential that molecular-weight standards be run on all gels. For most purposes, we use 100 bp DNA ladder markers and in these protocols it is assumed that this is the recommended molecular weight marker unless otherwise stated. 3.1.3.2.

AGAROSE-GEL

ELECTROPHORESIS

1. Agarose mmigels consisting of either standard or high-resolution agarose of dimensions 7.6 cm x 10 cm are run using the GNA- 100 apparatus from Pharmacia (Milton Keynes, UK) and are prepared by dissolvmg the required weight of agarose in 50 mL TBE buffer m a comcal flask. 2. Dissolution is achieved by heating in a microwave oven or on a Bunsen burner with regular mixing 3, The solution is allowed to cool to approx 60°C and EB is added to a final concentration of 0 5 pg/mL. 4. The solution is then poured into the gel mold, two combs are added to form the sample wells, and the gel IS allowed to set 5 The gel is transferred to an electrophoresis tank and completely covered with TBE buffer.

Daly et al.

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6. Up to 8 pL gel sample is mixed with 2 & gel loading buffer and applied to the gel using an automattc pipet. 7 Electrophoresls 1s carried out at 70 V for 3@-60 mm until the tracker dyes are well-separated. 8 The DNA bands are visualized on a translllummator 3.1.3.3.

POLYACRYLAMIDE-GEL ELECTROPHORESIS

1. For analysis of PCR products by polyacrylamlde-gel electrophoresls, we run gels of dimensions 15 cm x 17 cm using the Model VI 5 17 vertical gel-electrophoreSIS system from Glbco-BRL. 2. A 10% polyacrylamlde gel 1sprepared by mixing 16.7 mL 30% acrylamlde solution, 5 mL 10X TBE buffer and water up to 50 mL m a measuring cylmder To this, 0.5 mL 10% ammonium persulfate and 50 @ TEMED is added and, after rapid mlxmg, the gel solution 1spoured between sealed gel plates and the wellforming comb applied to the top of the gel. 3 The gel IS allowed to polymerize for 30 mm It is then placed m the electrophoreSIS tank and an appropriate volume of TBE added Up to 20 p.L sample 1smixed with 5 & gel-loading buffer and apphed to the wells using an automatic plpet 4. Electrophoresls 1scarried out at 150 V (constant voltage) until the bromophenol blue marker has reached the bottom of the gel 5 The gel 1sstained m 300 mL EB solution (0 5 @mL) for 15 min and, after brief destaining m water, 1s viewed on a ultraviolet (UV) transillummator. 3.7.4.

Digestion of PCR Products with Restriction Enzymes

A number of the genotypmg assays described m Subheadings 3.1.5.-3.1.10. involve restriction digests. These are normally carried out m PCR buffer (see Note 13) at the temperature recommended by the supplier. Normally either 10 or 20 & PCR product 1s digested with l-10 units of enzyme. 3.7.5. CY P2D6 Genofyp’ng A large number of variant CYP2D6 alleles have been described (3). However, at least 95% of those homozygous or heterozygous for inactivating mutations can be detected by genotypmg for the four alleles described m Subheadings 3.1.5.1,3.1.5.3. (see Note 14). 3 1.5.1

CYf2D6*3

AND CYP2D6*#

ALLELES

1. The forward primer is Gl (5’-TGCCGCCTTCGCCAACCACT-3’) and the reverse 1s Bl (5’-GGCTGGGTCCCAGGTCATAC-3’) (see Note 15) Satlsfactory results are obtained only with Tbr polymerase and each PCR reaction is performed m 50 @ of master mix containing Tbr polymerase and 3% dimethylsulfoxlde (DMSO) but otherwise prepared as described m Subheading 3.1.2. 0 5-l B genomlc DNA is used for each assay.

Cytochrome P450 Polyf77orphisms 380

wtlwt

W”4 -

280 -

l&)-

wtm -

411 WV’*3 *3/*4 *3/*3 -

-m--

-

-

160 130--

-

100 -

-w--

--

-----

Fig. 1. Detectton of CYP2D6*3 and CYP2D6*4 alleles by PCR Schematic representation of a typical polyacrylamide gel showing a range of band patterns. Bands smaller than 100 bp are not shown because they stain poorly with ethidmm bromide. The band sizes are shown m base pans. wt = wild-type by this assay. 2 After mmal mcubatton for 1 mm at 93°C 35 cycles of mcubatton for 1 mm at 93°C 1.5 min at 63°C and 5 mm at 70°C followed by 1 cycle of 10 mm at 70°C are carried out 3 A PCR product of 826 bp is produced by the reaction. 4 20 pL of product 1s digested with 1 pL BstNI (at 10 U/pL) and 1 @, BsaAI (at 5 U/l&), at 5O’C for 3 h. To control for digestion by BsaAI, 3 pL of a 50 pL PCR reaction of a region of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, can be added to the digest (see Note 16). The GAPDH gene amphtication product (591 bp) contains a single BsaAI site, and 1sdigested to give two bands of 500 bp and 91 bp. A similar control for BstNI digestion IS not necessary because there are several sites for the enzyme m the amphfied CYP2D6 fragment, m addition to the polymorphic site. 5. The digestion products are analyzed on a 10% polyacrylamide gel (see Subheading 3.1.3.3.). The various band patterns observed with different genotypes are shown schematically m Fig. 1. All samples except those from SubJects homozygous for the CYP2D6*.5 allele should contain invariant bands of 139 bp, 63 bp, 28 bp, 25 bp, and 10 bp Subjects with at least one allele other than CYP2D6*3 and CYP2D6*4 should show additional bands of 280, 181, 100, and 20 bp. Subjects positive for CYP2D6*4 will show additional bands of 380, 18 1, and 20 bp Subjects positive for CYP2D6*3 will show additional bands of 280, 16 1, and 100 bp. Bands of less than 100 bp may not be clearly visible on an EB-stained gel (see Note 17). 3.152 CYP2D6*5 ALLELE 1. The forward primer 1sCYP- 13 (5’-ACCGGGCACCTGTACTCCTCA-3’) and the reverse primer CYP-24 (5’-GCATGAGCTAAGGCACCCAGAC-3’) Each amplification 1sperformed in a total volume of 25 pL using enzyme and buffer 1 from the PCR-Expand system and 0.2-0.5 pg genomic DNA.

Daly et al.

412 2 The PCR reaction conditions

are 1 cycle of 1 mm at 93”C, followed by 30 cycles of 1 min at 93”C, 2 mm at 65”C, and 10 mm at 68”C, then 1 cycle of 10 mm at 70°C 3. The PCR products are analyzed on a 1.0% agarose gel with HzndIII-digested h DNA as molecular-weight standard. Samples positive for the CYP2D6*5 allele (either heterozygous or homozygous) give a product of 3 5 kb. If both alleles are CYP2D6*5-negative, no PCR product is produced (see Note 17) All samples are assayed m duplicate (see Note 18). 3.1.5.3.

CYPZD6*6

ALLELE

1 Two allele-specific PCR reactions (reaction 1 and reactlon 2) are carried out m parallel with the forward primer 2G (5’-CTCGGTCTCTCGCTCCGCAC-3’) m both cases and the reverse primers 9G (5’-CAAGAAGTCGCTGGAGCTGT-3’) m reactlon 1 and 10G (5’-CAAGAAGTCGCTGGAGCTGG-3’) in reaction 2 Satisfactory results are obtained only with Tbr polymerase and each PCR reaction 1s performed m 25 pL of master mix containing Tbr polymerase and 3% DMSO together with the other components described m Subheading 3.1.2. 0 2-0.5 pg genomlc DNA is used for each assay. 2. The temperature conditions are 30 cycles of 1 mm at 95’C, 1.5 mm at 54”C, and 3 mm at 70°C 3. The products are analyzed by electrophoresis through a 1% agarose gel Subjects negative for CYP2D6*6 should give a band of 174 bp for reaction 1 only, subjects heterozygous should give bands of 174 bp for both reactions, and SubJects homozygous for CYP2D6*6 should give a band of 174 bp for reaction 2 only 3.1.6. CYP2C19

Genotyping

Two PCR assays are carried out in parallel to detect the CYP2CZ9*2 (ml) and CYP2C19*3 (m2) alleles, respectively. Together these assays should allow identification of at least 80% of all individuals defective in CYP2C 19 activity (see Note 19). 3.1.6.1.

CYPZC~~*ZALLELE

1. The forward primer 1sMEP- 1(5’-ATTGAATGAAAACATCAGGATTG-3’) and the reverse MEP-2 (5’-GTAAGTCAGCTGCAGTGATTA-3’). Each amphficatlon is carried out m a volume of 50 pL and 0.5-l 0 pg genomlc DNA is used 2. The PCR conditions are 40 cycles of 1 mm at 93”C, 1 75 min at 55°C and 2 mm at 7O”C, followed by 1 cycle of 10 mm at 7O’C. 3 A PCR product of 169 bp is produced by the reaction. 4 10 pL of PCR product 1sincubated with 2 U of SmaI for 3 h at 37’C. The dlgestlon products are analyzed on a 2 8% high-resolution agarose gel. A homozygous wild-type sample will show bands of 120 bp and 49 bp. The 169-bp product remains undigested m a sample homozygous for CYP2C19*2. A heterozygous sample ~111 show bands of 169 bp, 120 bp and 49 bp

Cytochrome P450 Polymorphisms

413

3.1.6.2. CYP2C79*3 ALLELE 1 The forward primer 1s MEP-32 (5’-TATTATCTGTTAACTAATATGA-3’) and the reverse MEP-4 (5’-ACTTCAGGGCTTGGTCAATA-3’) Each amphfication is canted out in a volume of 50 pL with 0.5-l pg genomtc DNA 2 The condittons are 30 cycles of 1 mm at 93’C, 1 5 mm at 52“C, and 2 mm at 70°C 3. A product of 271 bp is obtained. 4 20 & of the product is digested with 15 units BarnHI at 37’C for 3 h and the digest analyzed by electrophorests through a 4% high-resolution agarose gel. The presence of the CYP2C19*3 allele is indicated by loss of the single BamHI site. A homozygous wild-type sample will show bands of 175 bp and 96 bp The 27 1-bp product remains undigested m a sample homozygous for CYPZCI 9*3 A heterozygous sample will show bands of 271 bp, 175 bp, and 96 bp 3.7.7.

CYP2El

There are two well-characterized polymorphisms in the CYPZEl gene that occur at 1019 bp upstream of the transcription start site (detectable with RsaI) and at 7668 bp in intron 6 (detectable with DraI), respectively (1). 3.1.7.1.

RSAI POLYMORPHISM

1. The forward primer is RSI (5’-TTCATTCTGTCTTCTAACTGG-3’) and the reverse, RS2 (5’-CCAGTCGAGTCTACATTGTCA-3’) Each amphticatton is carried out in a volume of 50 clr, with 0.5-l pg genomic DNA 2. The PCR conditions are 35 cycles of 1 mm at 93”C, 2 min at 48’C and 2 mm at 7O”C, followed by 1 cycle of 10 min at 70°C. 3. A PCR product of 4 10 bp is produced by the reaction. 4. 10 $ of PCR product 1sincubated with 2 U of RsaI for 3 h at 37°C. The dlgestlon products are analyzed on a 2.8% high-resolution agarose gel A homozygous wild-type sample will show bands of 360 bp and 50 bp The 410-bp product remains undigested m a homozygous mutant sample. A heterozygous sample will show bands of 410 bp, 360 bp, and 50 bp. 3.1.7.2. L&AI POLYMORPHISM 1 The forward primer is DR3 (5’-TTCATTCTGTCTTCTAACTGG-3’) and the reverse, DR4 (5’-CCAGTCGAGTCTACATTGTCA-3’) Each amphticatton is carried out m a volume of 50 pL using the regular assay mix and 0.5-1.0 pg genomtc DNA. 2. The PCR conditions are 35 cycles of 1 mm at 93”C, 2 min at 55’C and 2 min at 7O”C, followed by 1 cycle of 10 mm at 70°C. 3. A PCR product of 995 bp 1sproduced by the reactton. 4 10 pL of PCR product is incubated with 2 U of DruI for 3 h at 37°C The dlgesnon products are analyzed on a 1.0% agarose gel. A homozygous wild-type sample will show bands of 572 bp, 302 bp, and 121 bp A homozygous mutant

samplewill show bandsof 874 bp and 121bp. A heterozygoussamplewill show bands of 874 bp, 572 bp, 302 bp, and 121 bp

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3.1.8. CYP2C9 There are two separate CYP2C9 polymorphisms known. Each results m an ammo-acid substitution. Based on recent recommendations, the three alleles are now named CYP2C9*1 (wild-type), CYP2C9*2 (R&Z) and CYP2C9*3

(3). 3.1.8.1 CYP2C9*2 ALLELE(R,.& (1359L)

POLYMORPHISM)

1. The forward primer IS C9a (S-GGATATGAAGCAGTGAAGGAA-3’) and the reverse, C9b (5’-GGCCTTGGTTTTTCTCAACTC-3’) Each amplification IS carried out in a volume of 50 pL usmg the regular assay mix and 0 5-l .O pg genomic DNA. 2 The PCR conditions are 35 cycles of 1 mm at 93”C, 1.5 mm at 6O”C, and 2 mm at 70°C 3 A PCR product of 420 bp IS produced by the reactlon. 4 20 @L of the product 1sdigested with AvaII at 37°C for 3 h and the digest analyzed by electrophoresis through a 4% high-resolution agarose gel The presence of the mutation is indicated by loss of the single AvuII site A homozygous mutant sample will show a single band of 420 bp A homozygous wild-type sample will show bands of 363 and 57 bp. A heterozygous sample ~111 show bands of 420 bp, 363 bp, and 57 bp

3.1.8.2. CYP~C~*~ALLELE

(1359LPOLYMORPHISM)

1. The forward primer 1s C9c (5’-TGCACGAGGTCCAGAGATGC-3’) and the reverse, C9d (5’-AAACATGGAGTTGCAGTGTA-3’) The forward primer contains a single base pair mismatch which generates a new NsiI site in individuals with the wild-type CYP2C9*1 sequence present Each amplification 1s carried out m a volume of 50 pL using the regular master-mix and 0.5-l 0 pg genomic DNA. 2. The PCR condltlons are 35 cycles of 1 mm at 93’C, 2 mm at 59”C, and 2 mm at 70°C. 3 A product of 13 1 bp IS obtained 4 20 pL of the product 1s digested with the enzyme NszI for 3 h at 37°C and the digest analyzed by electrophoresis through a 4% high-resolution agarose gel The presence of the CYP2C9*3 allele 1s indicated by the loss of the single NszI site created by the PCRpnmer. Individuals homozygous for CYP2C9*3 will give a single band of 13 1 bp, whereas heterozygotes will gave bands of 13 1, 110, and 2 1 bp and those negative for CYP2C9*3 will give bands of 110 and 21 bp

3.7.9. CYP2A6 Two CYP2A6 polymorphisms have been described. Both affect exon 3 of the gene with CYp2A6*2 (CYP2A6vZ) resultmg in an amino-acid substitution and CYP2A6*3 (CYP2A6v2) a series of amino-acid substitutions (4). Both alleles can be detected using a long PCR assay, followed by reampllfication of exon 3 and digestion of this product with two chagnostic restriction enzymes in parallel.

Cytochrome P450 Polymorphisms

415

1. The forward primer IS F4 (S-CCTCCCmGCTGGCTGTGTCCCAAGCTAGGC-3’) and the reverse R4 (5’-CGCCCCTTCCTTTCCGCCATCCTGCCCCCAG-3’). Each amplification 1s carried out in a volume of 25 pL using the Expand-PCR system and 0.5-l .O pg genomic DNA. To avoid mlspnmmg, all components are kept on ice before and after mixing. 2. The thermocycler is preheated to 93“C and the samples added Followmg addltion of all samples, the temperature IS held at 93°C for 1 mm, followed by 30 cycles of 1 mm at 93”C, 1 mm at 66°C and 5 mm at 68”C, with a final extension of 10 min at 68°C 3. A PCR product of 7 8 kb is produced by the reaction. 4 After the long PCR reaction, 1 pL product is reamplified in a nested reaction The primers are E3F (5’-GCGTGGTATTCAGCAACGGG-3’) and E3R (5’-TCGTCCTGGGTCTTTTCCTTC-3’). A regular master-mix 1sused m this case 5. For the reamplificatlon, the PCR conditions are 35 cycles of 30 s at 93”C, 30 s at 64’C, and 60 s at 7O’C. 6. A PCR product of 201 bp is produced by the reaction. 7 Two lo-pL aliquots of the product are separately digested with 1 unit of&m1 (to detect CYP2A6*2) and 1 unit of D&I (to detect CYp2A6*3). Both digests are performed at 37°C for 3 h 8 The digestion products are analyzed on a 2.8% high-resolution agarose gel The presence of the CYP2A6*2 allele is indicated by digestion with XcmI to yield bands of 141 bp and 60 bp and the CYP2A6*3 allele by digestion with DdeI to yield bands of 142 bp and 59 bp Heterozygotes will show various combmatlons of these band patterns. 3.7.70.

CYPl Al

Two polymorphisms, both resulting in ammo-acid substitutions, are known to occur in exon 7 of CYPlA I. The variant alleles have been tentatively termed CYPlAl*2B and CYPlAl*4 (see Note 20) and result m amino-acid substitutions of Ile462Val and Thq6,Asn, respectively. The assay used for detection of CYPlAI*2B also detects CYPlA1*4 and, to differentiate between these two alleles, it IS necessary to carry out a second PCR assaythat specifically detects CYPlAl”4. 1. The forward primer is 1AlX (5’-GAACTGCCACTTCAGCTGTCT-3’) and the reverse, 1AlY (5’-CCAGGAAGAGAAAGACCTCCCAGCGGGCCA-3’) (see Note 21). Each ampllficatlon 1scarried out m a volume of 50 pL using the regular master-mix and 0.5-l 0 pg genomic DNA. 2. PCR conditions are 35 cycles of 1 min at 94”C, 1 mm at 68”C, and 1 mm at 70°C 3. A PCR product of 195 bp IS obtained. 4. The product is digested with NcoI and the digests analyzed by electrophoresis through a 2.8% high-resolution agarose gel. The presence of either the CYPlAl*2B or CYPlAl”4 alleles is indicated by a single band of 195 bp owing to loss of the single NcoI site For other alleles, two bands of 163 and 32 bp, are obtained.

Daly et al

416

5 For subjects that are either homozygous for CYPlAl*2B/CYPIAi *4 or heterozygous, an additional assay that discrtmmates between CYPlAl*2B and CYPlAl*4 1s carried out 6. The forward primer is IA17FP (5’-CCACTCACTTGACACTTCTG-3’) and the reverse, lA17RP (5’-TAGACAGAGTCTAGGCCTCA-3’). Each ampllfication is carried out m a volume of 50 pL using the regular master-mix and 0 5-l .O pg genomic DNA 7. The PCR condttions are 35 cycles of 1 mm at 93°C 1.5 mm at 55°C and 2 min at 70°C 8. A product of 38 1 bp is obtained. 9. 20 pL product IS digested with BsaI for 5 h at 50°C and the digests analyzed by electrophoresis through a 1.8% agarose gel. The presence ofthe CYPIAI *4 allele is indicated by the absence of a BsaI site and a single band of 38 1 bp. The presence of a BsaI site that gives two bands of 201 bp and 180 bp indicates that the positive result with NcoI 1sowing to the presence of the CYPlAI *2B allele (see Note 20)

3.2. Phenotyping 3.2.1. CYP2D6 Phenotyping with Debrlsoquine 3 2.1 1. DEBRISOQUINE STANDARDS 1 Dilute debrisoqume and 4-hydroxydebnsoqume mto a lomt aqueous stock solution of approx 200 mg/L. 2. Dtlute the stock solutton mto drug-free urine to produce standard solutions of 0.0,0.5, 1.0, 2.5, 5.0, and 10.0 mg/L of both compounds. 3 Assay the standards vs quality controls, separately produced as described in Subheading 3.2.1.1., steps 1 and 2, and, if satisfactory, store m aliquots (approx 1 mL) at -2O’C until required. 4 Dilute the internal standard into water to produce a workmg solutton of approx 10 mg/L (see Note 22). Store at 4°C. 3.2.1 2. URINE COLLECTION (SEE NOTE 23) 1. Immediately after emptying bladder, sublect takes a single 10 mg capsule of debrtsoquine. 2. All urme passed over the next 8 h is collected m a urine container Note final volume (see Note 24) Gently agitate container to mtx urine 3. Take approx 15 mL aliquot and store m universal tube at -2O“C until required 3.2.1.3

ANALYSIS OF URINE

Standards, duplicate.

samples,

and quality

controls

(in samples)

are analyzed

m

1 To urine sample or standard (400 pL) add internal standard (50 pL) and sodium bicarbonate (100 pL), in a blJou vial. 2. In a fume cupboard, add HFAA (50 &) and toluene (1 mL) (see Note 25). Cap vials with PTFE face of seals to inside.

Cytochrome P450 Polymorphisms

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3 Agitate vials to mix contents and place m heating block m a fume cupboard. Heat (1 OO”C, 60 min) and then cool to room temperature before proceeding. 4. Remove caps (see Note 26) Add sodium hydroxide (5 mL) and more toluene (2 mL). Replace caps. 5. Vortex thoroughly (10 s exactly), centrifuge (2000g for 5 mm) and transfer supernatant to new tube or autosampler vial for injection onto gas chromatograph 3.2.1.4. GAS CHROMATOGRAPHY 1. The samples are applied to a gas chromatograph using the following conditrons. a. Temperatures* Injection port, 300°C; Electron Capture Detector, 300°C. b. Split ratio: 100.1, 1 pL injected. c. The oven temperature program depends on whether hydrogen or helium IS used as carrier gas. For hydrogen (15 psi) the program is: initial temperature 190°C for 5.5 mm, a ramp of 20Wmin to 260°C and final time 2 mm at 260°C. For helium (25 psi), imtral temperature 90°C for 0.5 min, ramp 1 30°C min to 210°C ramp 2 O.S”C/min to 215°C ramp 3 IOWmin to 240°C and final time 5 mm at 240°C. 2 Debrisoqume elutes first followed by 4-hydroxydebrtsoqume and the Internal standard, but precise elution times will depend on the column and the carrier gas (see Note 27). The signal from the detector IS monitored usmg an integrator (see Note 28) and peak area ratios for both debrtsoquine and 4-hydroxydebrisoquine relative to the mtemal standard are calculated. 3 Standard curves of peak area ratio vs concentration for both debrisoqume and 4-hydroxydebrrsoqume are prepared and used to calculate concentrations m the unknown urine samples. 4. The concentrations of debnsoqume and 4-hydroxydebrrsoqume are used to calculate a metabolic ratio of debrisoquine/4-hydroxydebrisoquine (see Note 29).

3.2.2. CYP2A6

Phenotyping with Coumarin

3.2.2.1. URINE COLLECTION 1. Immediately after emptying bladder, subject takes a single capsule contammg 2 mg of coumarin. 2. All urine produced in subsequent 8-h period is collected, pooled, and the total volume recorded. 3. An aliquot (20 mL) is stored frozen (-2O’C) until required for analysis 3.2.2.2.

ANALYSIS OF URINE FOR UNCONJUGATED 7-HYDFIOXYCOUMARIN

1. Add chloroform (3.5 mL) to urine (2 mL) or standard solution of 7-hydroxycoumarin (2 mL; 5-100 ng/mL) (see Notes 30 and 31) 2. Vortex-mix (0.5 min), mechanically shake (20 mm), and centrifuge (2OOOg for 5 min). 3. Remove and discard aqueous layer. 4 Transfer a portion (-2 mL) of the organic layer to a fresh tube and evaporate to dryness under vacuum at room temperature

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3.2.2.3. ANALYSIS OF URINE FOR CONJUGATED 7-HYDROXYCOUMARIN 1 Incubate urme (0.5 mL) with P-glucuronidase (4000 U) for 16 h at 37’C 2. Remove an aliquot (100-200 l.tL) and make up to 2 mL with dlsttlled water 3 Add chloroform (3.5 mL) and proceed as m Subheading 3.2.2.2. 3.2.2.4. I 2 3 4. 5

6

CHROMATOGRAPHIC PROCEDURES AND DENSITOMETRIC ANALYSIS

Reconstitute dry residues from the procedures in Subheadings 3.2.2.2. and 3.2.2.3. (see Note 32). Apply a portion (10 pL) to a TLC plate using a Lmomat IV automatic sample applicator (see Note 33) Place plate in Camag horizontal developing chamber m the saturation configuration with mobile phase When separation is complete, remove plate and dry at room temperature Scan plate usmg a Camag TLC scanner II connected to a Camag SP4290 TLC Integrator at 3 13 nm using the mercury lamp in the reflection mode in the presence of a K400 secondary filter. The integrator converts the stgnal from the scanner to both peak areas and peak heights Standard curves of peak areas for the standard soluttons of 7-hydroxycoumarm vs concentratrons are prepared and the concentrations of 7-hydroxycoumarm m urme samples are calculated

4. Notes 1. There are a variety of thermostable DNA polymerases available For many of the assaysTaq DNA polymerase from a variety of suppliers will give satisfactory results However, for certam assays, use of Tbr DNA polymerase, which has a longer halflife at 96°C and a lower error rate than Tag polymerase is recommended For amphIications of templates m excess of 2 kb, use of a kit mtended for long-range PCR is recommended These kits are available from several supphers and generally consist of a mixture of a DNA polymerase without proofreadmg 3’ to 5’ exonuclease activity, such as Taq DNA polymerase, and a proofreading enzyme such as Pwo DNA polymerase. For addmonal mformation on thermostable polymerases see ref. 5 2. Guanoxan is a possible substitute 3. Concentrations are free-base equivalent: weight of any salt must be taken into consideratton 4 Also known as l,l, 1,5,5,5-hexafluoro-2,4-pentanedtone Purchase as approx 15-mL solutton and altquot m approx 1-mL portions mto amber glass vials with PTFE-faced seals. Store at 4°C. 5. Prepare m fume cupboard because it may heat and give off vapor durmg preparation, 6 The essential ingredients are capillary chromatography with split mJection, electron-capture detectton, and an appropriate recorder, integrator, or computer for recordmg data 7 A standard analytical column with a nonpolar polysiloxane phase Within reason, a shorter or longer column could be used, likewise a thicker or thinner stationaryphase film

Cytochrome P450 Polymorphisms

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8. Capsules contaming 5 mg of coumarin have also been used for phenotypmg procedures (6). 9. Yield and purity of genomlc DNA IS determined by measuring absorbance at 260 nm and 280 nm. On the basis of the AzbOreading, DNA concentration 1scalculated, an absorbance of 1 being equivalent to a DNA concentration of 50 pg/mL If the A,,,jA,s, ratio ~1.6, the sample may be contaminated with protein, resulting in difficulties with amphficatlon by PCR. Further purification may be necessary. If only small quantities of starting material are available, it may not be possible to quantify the amount of DNA present but it is still possible to obtain a PCR product. Information on obtaining DNA from archival samples, such as pathological specimens, is provided in ref. I. 10. PCR is a sensitive techruque and there 1s a risk that incorrect results may be obtained owing to contammatlon of samples with small amounts of PCR product from previous amplifications The risk of this occurring can be greatly reduced by using separate areas of the laboratory for DNA preparation, for setting up PCR reactlons, and for analyzing PCR products. It is particularly important that different pipets are used for each of these processes. It 1s also important that controls for contammatlon detection be included in each set of assays. These should include a “no DNA” blank tube with all other components present and at least one sample of known genotype. It is also valuable to genotype samples on two separate occasions. More mformatlon on avoiding contammatlon and decontamination methods 1sprovided m ref. 5 11. The authors routinely add 0 5 E DNA to a 50 pL reaction but have obtained PCR products from 100 ng starting material when necessary 12 To avoid evaporation of the reaction, it 1s essential that It be overlaid with light mineral 011.This is not detrimental to the reaction or to subsequent sample processmg. Alternatively, some thermocyclers have heated lids to prevent evaporation. 13. Most restriction enzymes show optimum activity at a defined pH and salt concentratton. However, the majority of enzymes show sufficient activity in PCR buffer to allow direct digestion of PCR products followmg amphficatlon. Further information is provided in ref. 5. 14 At least nine different defective CYP2D6 alleles have been described (3). However, current evidence (A. K. Daly, unpublished data) suggests that, for most purposes, genotyping for the four most common defective alleles (CYP2D6*3, CYP2D6*4, CYP2D6*5, and CYP2D6*6) will identify in excess of 95% of poor metabolizers. The other alleles tend to be very rare and assaymg for them will not greatly improve sensitlvlty of poor metabohzer detection. 15. The reverse primer has a single base-pair mismatch that results m a BsaAI site being generated if the AZb3, deletion characteristic of the CYPZD6*3 allele is present. 16. Including a positive control for BsaAI digestion can be useful BstNI dlgestion is already internally controlled owing to the presence of several invariant sites m the PCR product. For BsaAI, we add to the digestion a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR product, which contams a BsaAI site. The product 1s

420

17

18

19

20

21 22. 23. 24.

25. 26 27.

28.

Daly et al. prepared using the forward primer R4 (5’-AGAAACAGGAGGTCCCTACT-3’) and the reverse primer R5 (5’-GTCGGGTCAACGCTAGGCTG-3’) and 35 cycles of 1 mm at 93°C 1 5 mm at 58°C and 2 min at 70°C followed by 1 cycle of 10 mm at 70°C. A PCR product of 591 bp IS produced by the reaction and yields digestlon products of 500 bp and 9 1 bp when drgested with BsaAI. CYP2D6 poor metabolizers will have two defective alleles present Genotype should be assigned on the basis of the data from the three assays descrtbed m Subheading 3.1.5. Individuals who fall to give a product for both the CYP2D6*3/ “4 and the CYPZD6*6 assays but are positive for CYP2D6*5 are CYP2D6*5 homozygotes. There has been no linkage observed between the mactivatmg mutations characteristtc of CYP2D6*3, *4, and *6 (3), and therefore subjects who are heterozygous for two of these alleles are assumed to have two defective alleles present and to be poor metabohzers. Because no PCR product is obtained m mdivrduals lackmg the CYP2D6*5 allele, an internal posrtive control would be useful Attempts to design such a control have not yet been successful and it is therefore suggested that the CYP2D6*5 assay be carrred out in duphcate on two separate occasions and that positive and negattve controls should always be included Allele nomenclature 1s in accordance with recent recommendatrons (3) The defecttve alleles were formerly referred to as CYP2CZ9ml and CYP2C19m2 (8,9). The CYP2CI9*3 allele is very uncommon among Europeans but occurs in approx 20% of Oriental subjects lacking CYP2C 19 activity Nomenclature is as suggested in a recent publicanon (IO). The CYPlAl*2B may be associated wtth an increased rusk of lung cancer development, possibly owing to increased enzyme activity, but the significance of CYPIAI *4 is still unclear (10) The reverse prtmer contains a single base-pair mismatch that introduces a NcoI site into the wild-type sequence (21) Adjust the concentration as necessary to suit the sensitivtty of the electron capture detector. This procedure IS generally carried out overnight, following a normal day If the container 1saccurately marked, total urine volume can be noted, an aliquot taken by the mdrvidual bemg phenotyped, and the remainder discarded. Otherwise total collection will have to be measured in the laboratory before takmg an aliquot for analysis. Volumes of solvent and sample can be altered, but the ratio of aqueous:orgamc phases should not be greater than I .2. Take care to identify mdtvidual seals and carefully replace as some crystals may have formed on them Typical retention times usmg hydrogen as a carrier gas are debrisoqume, 3 22 mm, 4-hydroxydebrisoquine, 4 46 mm; and 7-methylguanoxan, 7.22 mm; and using helium as a carrier gas are debrisoqume, 12.3 mm, 4-hydroxydebrisoqume, 15 3 mm; and 7-methoxyguanoxan, 20 9 mm. Wrth a Hewlett-Packard 3396 Integrator, the run parameters used are’ attenuatron = -1, chart speed = 0.5, threshold = -4 and peak wtdth = 0.04.

Cytochrome P450 Polymorphisms

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29 Individuals lacking CYP2D6 activity (poor metabolizers) are normally defined as those showing a metabolic ratio m excess of 12.6, although lower hmits mcluding 5 4 and 10.0 have also been suggested. 30. The concentration of 7-hydroxycoumarm in urine is calculated by comparison of unknowns with caltbration curves constructed using solutions contammg known amounts of authentic 7-hydroxycoumarm taken through the procedures described m Subheading 3.2.2. 3 1. It is advisable to analyze urine samples in duplicate. 32. Owing to the volatility of chloroform, it is advisable to reconstitute the samples one at a time, apply the sample to the plate, and then reconstitute the next 33. If a 10 x 20 cm plate is used and samples are applied to both sides of the plate m 6 mm bands with a lane separation of 4 mm, each side of the plate can accommodate six urine samples m duplicate and stx standards in smglicate.

References 1. Daly, A. K (1995) Molecular basis of polymorphic

drug metabolism J MoI Med 73,539-553. 2 Evans, D A P (1993) Genetic Factors zn Drug Therapy Clznzcal and Molecular Pharmacogenetzcs Cambridge Umverstty Press, Cambridge, UK. 3. Daly, A K , Brockmoller, 3 , Broly, F , Eichelbaum, M , Evans, W. E., Gonzalez, F. J., Huang, J.-D , Idle, J. R , Ingelman-Sundberg, M , Ishizaki, T , JacqzAlgram, E., Meyer, U. A., Nebert, D W , Steen, V. M., Wolf, C R., and Zanger, U. M. (1996) Nomenclature for human CYP2D6 alleles Pharmacogenetlcs 6, 193-20 1. 4 Fernandez-Salguero, P , Hoffman, S. M. G , Cholerton, S., Mohrenweiser, H , Raunio, H., Pelkonen, O., Huang, J., Evans, W. E., Idle, J R , and Gonzalez, F. J. (1995) A genetic polymorphism in coumarin 7-hydroxylatton: sequence of the human CYP2A genes and identification of variant CYP2A6 alleles Am J. Hum Genet 57,65 I-660. 5. Newton, C. R. (1995) PCR Essentzal Data John Wiley, Chichester, UK 6 Rautio, A., Kraul, A., KOJO, A , Salmela, E , and Pelkonen, 0 (1992) Interindivtdual variability of coumarm 7-hydroxylation m healthy volunteers. Pharmacogenetics 2,227-233

7. Jackson, D. P , Hayden, J. D., and Qutrke, P (1991) Extraction of nucleic acid from fresh and archival material, m PCR, A Practxal Approach (McPherson M. J., Quirke P., and Taylor G R., eds.), IRL,, Oxford, UK, pp. 29-50 8. de Morals, S. M. F., Wtlkinson, G. R., Blaisdell, J., Nakamura, K., Meyer, U. A , and Goldstein, J A ( 1994) The major genetic defect responsible for the polymorphism of S-mephenytoin metabolism in humans. J. Bzol Chem 269, 15,419-15,422. 9. de Morals, S. M F., Wilkinson, G. R., Blaisdell, J , Meyer, U. A., Nakamura, K , and Goldstein, J. A (1994) Identification of a new genetic defect responsible for the polymorphtsm of (S)-mephenytom metabolism in Japanese. Mel Pharmacol 46,594-598

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10. Cascorbr, I., Brockmoller, J., and Roots, I. (1996) A C4887A polymorphism m exon 7 of human CYPIAI-populatton frequency, mutation linkages and impact on lung cancer susceptibility. Cancer Res 56,4965-4969. 11. Shields, P. G., Bowman, E D., Harrington, A M , Doan, V. T. and Weston, A. (1993) Polycyclic aromatic hydrocarbon-DNA adducts m human lung and cancer susceptibility genes. Cancer Res. 53,3486-3492.

43 Induction of Cytochrome P450 1A (CYPl A) in Fish A Biomarker for Environmental

Pollution

Bente M. Nilsen, Karin Berg, and Anders Goksaryr 1. Introduction Inducible proteins are attractive candidates for biomarkers-i.e. biological responses that reflect exposure to, or effects of, environmental pollutants in an organism (I). Blomarkers are increasingly regarded as powerful and mformatlve tools m ecotoxlcology and environmental management (1). The inductlon of cytochrome P450 IA (CUP1 A) in fish has been evaluated as a sensitive, convenient, “early warning” signal of orgamc xenoblotlcs m the aquatic environment (2-6). In a number of field and laboratory studies, the response of this enzyme system in fish to widespread environmental pollutants such as polyaromatic hydrocarbons (PAH), polychlorinated biphenyls (PCBs), dioxms, oil compounds, pesticides, and so on, has been demonstrated and validated (2-6). A common structural feature of the inducing compounds has been identified as a planar, aromatic structure resulting in bmdmg to the Ah receptor, thereby initiating the induction process leading to increased amounts of CYPlA mRNA, protem, and catalytic activity (see ref. 5). This induction process is routinely measured by catalytic assays using benzo(a)pyrene or 7-ethoxyresorufin as substrates m aryl hydrocarbon hydroxylase (AHH) or 7-ethoxyresorufin 0-deethylase (EROD) assays,respectively. As specific antibodies against fish CYPl A proteins have become avallable, immunochemical techniques such as Western blotting, enzyme-lmked immunosorbent assay (ELISA) and immunohlstochemlstry have gained in popularity (4,7,8). With cDNA sequences from a number of fish species available (see ref. 9), analytical tools for mRNA measurements have also been applied in such studies. From Methods In Molecular Bfology, Vol Edited by I R Phllllps and E A Shephard

423

107 Cytochrome 0 Humana

Press

P450 Protocols Inc.

Totowa,

NJ

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Nilsen, Berg, and Goksoyr

In catalytic assays, sample preservation is a critical part of quaky assurance. Suboptimal sampling or storage may lead to loss of catalytic activity, and high levels of inducing compounds or xenobiottc antagonists may affect the measured activtty. Immunochemical techniques, where the amount of CYPlA protein binding to a specific antibody is measured, are usually not dependent on btologtcally active samples. Furthermore, ELISA methods can be developed into rapid, time-saving techniques where large numbers of samples can be simultaneously assayed. In monitoring the aquatic environment, it has been suggested that both catalytic and immunochemical techniques be applied as a quality control (IO). In this chapter, protocols for sampling and sample preparation of fish tissue are presented, and the EROD assay is described m detail. This method has been adopted m many national

and international

monitoring

programs

(IO-

12). Also, immunochemtcal assays for CYPlA detection m fish tissue using ELBA or Western blotting are presented. mRNA measurements are very sensitive to sample degradation, and appear to be less amenable to large field sampling programs. Methods referring to CYPl A mRNA analyses will not be described here.

2. Materials 2.1. Sampbng Equipment includes dissection equipment (scissors, forceps, scalpel), and a liquid nitrogen container or -8OOC freezer. 2.2. Preparation of Postmitochondrial Microsomes from Fish Tissue

Supernatant

(PMS) and

1. Homogenizatron buffer for PMS: 0 1 M sodmm phosphate, pH 7.4, 0.15 M KCl, 1 mA4 ethylenedramme tetra-acetic acid (EDTA), 1 mM dithiothreitol (DTT), 10% (v/v) glycerol. Dissolve 13.8 g NaH2P04 * HZ0 and 11.2 g KC1 m 500 mL dlstrlled water. Add 0.372 g EDTA, 0.154 g DTT and 115 mL 87% (v/v) glycerol. Adjust to pH 7 4 with NaOH and add distilled water to 1000 mL. 2 Homogenizatron buffer for mrcrosomal fractrons: 0.1 A4 sodium phosphate, pH 7.4,O 15 MKCl, 1 mMEDTA, 1 mA4DTT. Prepare as homogenization buffer for PMS (see item 1) but without the addition of glycerol 3. Resuspension buffer for mrcrosomal fractions: 0 1 A4 sodmm phosphate, pH 7.4, 0.15 M KCl, 1 mM EDTA, 1 m&I DTT and 20% (v/v) glycerol. Prepare as buffer described m item 1, but with 230 mL 87% (v/v) glycerol. 4. Equipment includes a Potter-Elvehjem teflon-glass homogemzer and a liquid nitrogen container or -80°C freezer.

C YP 1A Induction in Fish

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2.3. EROD Analysis 1. EROD-buffers are selected to give the best reaction rate for CYPlA from the specific fish species to be tested. Usually 0.1 Msodium phosphate or 0.1 MTrtsHCl buffers wrth pH 7 O-g.5 are used The optimum buffers for some selected fish specres are given below (8,13): a. Atlantic salmon (Saimo s&r): 0.1 M sodmm phosphate, pH 7 6 b. Atlantic cod (Gadus morhua): 0.1 Msodium phosphate, pH 7.8 c. Plaice (Pleuronectes platessa): 0.1 M sodium phosphate, pH 7.0. 2 Ethoxyresorufin solution, 0.4 mM 7-ethoxyresorufin m dimethylsulfoxrde (DMSO) Dissolve 1 mg 7-ethoxyresorufin in 10 mL DMSO. Determine the exact concentration by making a 1.100 dilutton m EROD-buffer and reading the absorbance at 482 mu (extinction coefficient = 22.5 mA@lcm-’ f14J. Store at -2O’C in light-proof bottles. 3. Nigotinamide ademme dinucleotide phosphate (NADPH) stock solution 10 mM NADPH Drssolve 8.3 mg NADPH m 1.O mL distilled water May be stored for 1 wk at -20°C. 4 Resorufin standard solution* 15-20 pA4 resorufin m DMSO. Prepare stock solution by drssolvmg 1 mg resorutin m 50 mL DMSO. The stock solution can be stored at -2O*C protected from light Dilute the stock solution 1.3 m DMSO, read absorbance at 572 nm, and calculate the exact concentration by using the extinctron coefticrent 73 2/rnW’lcm-1 (15). Ahquot and store at-20°C protected from light. 5. Fluorescence spectrophotometer. 6. Recordmg device for fluorrmetrlc output

2.4. ELBA 1 Coating buffer 50 mMcarbonate/bicarbonate, pH 9 6 Dissolve I 59 g Na,COs and 2.93 g NaHCOs in 1000 mL distilled water. Store at 4°C. 2. Phosphate-buffered saline (PBS), pH 7 3 Dtssolve 1.15 g Na,HP04, 0 2 g KH2P04, 8.0 g NaCl, and 0.2 g KC1 in 800 mL distilled water. Adjust to pH 7.3, and adjust volume to 1000 mL with distilled water. Store at 4°C 3 TPBS: 0.05% (v/v) Tween-20 m PBS Dissolve 1 mL Tween-20 m 2000 mL PBS. Store at 4°C. 4. Blockmg solution: 2% (w/v) bovine serum albumm (BSA) m PBS. May be stored for a few d at 4°C. 5. Monoclonal or polyclonal antibodies (MAbs/PAbs) against fish CYPlA (primary antibody). A limited number of such antibodies have been prepared and characterized (16Zl), (for revrews, see refs. 2,3), and some are commercially avarlable (e.g., monoclonal mouse anticod CYPIA, NP-7 from Brosense Laboratories AS, Bergen, Norway). Many of these antibodies cross-react with CYPlA from a variety of fish species and may thus be used to detect CYPlA from more than one species of fish.

Nilsen, Berg, and Gokwyr

426

6. Secondary antlbody against mouse or rabbit IgG (depending on the orlgm of the primary antibody) conjugated with horseradish peroxldase (HRP). A large number of such secondary antIbodIes are commercially available from a variety of manufacturers 7. Developing solution* 0.04% (w/v) o-phenylenediamine dlhydrochloride (OPD), 0.012% (v/v) H,O,. Dissolve 15 mg OPD m 37 5 mL distilled water and add 15 pL 30% H,O, Make fresh as required. Caution: Beware of the toxic nature of OPD (OPD m tablet form 1s avadable from some compames). 8. Stop solution: 4 NH,SO,. Dissolve 53 mL H,SO, (95-97%) m 500 mL dlstllled water. Cut&on: Beware of the hazardous nature of sulfurrc acid. 9. Equipment includes 96-well microplates (Nunc Maxisorp, Roskllde, Denmark), microplate washer (optlonal), and microplate reader

2.5. Western Blotting 1. Blotting buffer: 25 mM Tris, 192 mA4 glycine, 20% (v/v) methanol. Mix 3 03 g Trls base, 14.4 g glycme, and 200 mL methanol. Make up to a final volume of 1000 mL with dlstilled water Store at 4°C 2. Tris-buffered saline (TBS), pH 7.5 Dissolve 4 8 g Tris base and 58.4 g NaCl m approx 1800 mL distilled water. Adjust to pH 7.5 with 5 M HCl, then adjust volume to 2000 mL with distilled water. Store at 4OC. 3. TTBS: TBS containing 0.05% (v/v) Tween-20. Dissolve 1 mL Tween-20 m 2000 mL TBS. Store at 4°C. 4. Blocking solution: 3% (w/v) gelatin in TTBS. Dissolve 3 g gelatin in 100 mL TTBS by carefully heating the solution. The solution may be stored at 4°C for a few days, and must then be reheated before use. 5 Developing solution. (A) Dissolve 30 mg 4-chloro-1 -naphthol in 10 mL methanol, (B) Add 30 & cold 30% Hz02 to 50 mL TBS Protect solutions against light. Mix (A) and (B) Just prtor to use and pour immediately over the membrane 6. MAbs or PAbs against fish CYPIA. (see Subheading 2.4., item 5) 7. Secondary antlbody against primary antrbody labeled with HRP (see Subheading 2.4., item 6). 8. Electroblotting equipment. There are numerous types on the market The procedure described here 1s for use with a wet blotting apparatus. 9 Nltrocellulose membrane and filter paper (Whatman 3MM or eqmvalent)

3. Methods 3.1. Sampling Throughout

(see Note 1) this procedure keep tissue and tissue fractions cold (on ice).

1. Kill the fish as quickly as possible Carefully dissect out tissues, avoiding rupturmg the gall bladder (bile may contam monooxygenase inhlbltors) (see Note 2) 2, Remove tissue immediately and transfer mto a beaker on Ice. Remove excess blood from the tissue fractions by washing in homogenization buffer. 3. If fish are killed m the field without access to centrifuges, small pieces of ttssue can be frozen in liquid nitrogen until further processmg m the laboratory.

CYPlA induction in Fish

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3.2. Preparation of PMS and Microsomes from Fish Tissue (see Notes 3 and 4) Throughout this procedure keep tissue and tissue fractions at 0-4°C. 1 Homogenization: a. Weigh the fresh or frozen liver sample (commonly 0.5-Z g is used), and allow frozen tissue to thaw on ice Add 4 mL cold homogemzation buffer per g liver and mince the tissue with scissors. Use homogemzation buffer for PMS (with 10% (v/v) glycerol) rf only PMS is being prepared, and homogenization buffer for microsome fractions if microsome fractions are being prepared. b. Transfer the tissue to a prechilled tissue homogenizer (Potter-Elvehjem type) and homogenize with five to seven passes with a motor driven teflon pestle 2. Preparation of PMS fractions: a. Transfer the crude homogenate to Eppendorf tubes (or other centrifuge tubes) and centrifuge at 12,000g for 20 min at 4°C. b. Collect the supernatant with a pipet, takmg care to avoid the pellet and the floating lipid layer The 12,000g supernatant (PMS) produced m this step may be analyzed directly if mlcrosomes are not required. 3. Preparation of microsome fractions: a. Microsome fractions are prepared by further centrifugation of the PMS. Transfer the cold PMS to ultracentrifuge tubes and centrifuge at 100,OOOg for 60 mm at 4’C. b. Carefully remove the supernatant (cytosol fraction) by aspiration to leave the mtcrosomal pellet. If a transparent glycogen pellet is present beneath the microsome pellet, carefully flush the microsomes loose from the glycogen with resuspension buffer using a Pasteur prpet. c. Using a teflon-glass tissue homogenizer, resuspend the mtcrosomal pellet in cold resuspension buffer (0.5-2 mL) to a protein concentratron of approximately 5-l 5 mg protein/ml (normally a 1: 1 ratio between buffer volume and tissue weight). 4. Storage. The PMS or microsome fraction may be kept on ice if enzyme analysis is being immediately performed (or within a few h). The fractrons can also be stored m ahquots at -80°C for later analysis.

3.3. EROD (see Notes 5-7) 1. Set the excitation and emissron monochromators of the spectrofluorimeter to 535 nm and 585 nm, respectively. 2. Place PMS or microsome fractions, freshly prepared or thawed from -80°C for the first time, on ice until analysis (see Note 8). 3. Perform the assay m temperature controlled cuvets or at room temperature.

4. Add 1.96mL EROD buffer, 10$7-ethoxyresorufin substratesolution (see Note 9) and 20 pL PMS or microsome fraction to the cuvet. 5. Mix well, either by continuous stirring using a small magnetrc flea m the cuvet, or by inverting the cuvet 2 or 3 times.

Nilsen, Berg, and Goksaryr 6. Place the cuvet m the spectrofluorimeter, and start the spectrofluorimeter to record the base line 7. Add 10 $ NADPH solution to initiate the reaction and mix well as noted (see item 5) (see Note 9) 8. Measure resort& productton over 2-3 min by recording the change in fluorescence (see Note 10). 9. Add 10 p.L resorufin solution to the cuvet as internal standard Mix well and record the increase in fluorescence (see Note 11). 10. Determine the protein content of the tissue fractions, using, e.g., the Lowry assay (22) or the Bradford assay (23) with BSA as standard (see Note 12) 11 Calculate the specific activity of the enzyme (pmol/mm/mg protein) m the sample by using the followmg formula (see Fig. 1): pmol resorufinlminlmg

protem = Fs/mm x R/F, x lNs x l/Cs

(1)

Fs/mm = Increase m sample fluorescence per min, R = Amount of resorutin added as internal standard (pmol), F, = Increase in fluorescence owing to resorutin standard, V, = Sample volume (mL), Cs = Protein concentration of sample (mg/mL)

3.4. ELlSA (see Notes 13 and 14) 3.4. I. Coating 1 Dilute the samples m coating buffer to a concentration of 10-100 pg total protem/mL (see Note 15). Optimum coatmg concentratton must be determined for each application (see Note 16) Mix well and keep on ice. 2 Make a diagram of a 96-well microplate, avord the wells along the edges, and mark three wells for each sample (triplicates) Add 100 & sample to each well accordmg to the diagram Add coating buffer to all empty wells (see Note 18) 3 Cover the plate and incubate at 4°C overnight. This will allow the proteins m the sample to adsorb to the well surface 4 Wash the wells three times with 200 pL TPBS/well. Leave the last washing solution in the wells for 3-5 min as a soaking step (see Note 17).

3.4.2. Blocking 1 Add 200 $ blockmg solution to all wells, mcludmg one blank row along the edge (see Note 19). 2. Incubate at room temperature for 45-60 min. This step will block all remaining protein-binding sites m the well. 3. Wash the wells three times in TPBS as above (see Subheading 3.4.1., item 4)

3.4.3. Primary Antibody 1. Dilute the MAb or PAb agamst fish CYPlA in 1% BSA m PBS Optimum dilution of the primary antibody must be determined for each antibody and for each fish species to be tested (see Note 20). 2. Add 100 pL primary antibody solution to each well.

CYPIA Induction in Fish

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of

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Adh,O” of resorufin

Time (min)

Fig. 1. Schematic drawing of the fluorescent output during 7-ethoxyresorufm O-deethylase (EROD) assay. NADPH and the internal standard resort& are added to the sample at the times Indicated The increase in fluorescence after addition of NADPH (Fs) and after addition of resorutin (FR) IS measured as indicated, and the enzymatic activity IS calculated using the formula given in Subheading 3.3., item 11. 3 Cover the plate and incubate at 37’C for 1 h or overnight at 4°C (see Note 21) 4. Wash the wells three times m TPBS as above (see Subheading 3.4.1., item 4)

3.4.4. Secondary Antibody 1. Dilute the HRP-labeled secondary antibody according to the manufacturer’s instructions in 1% BSA in PBS. If the primary antibody (see Subheading 3.4.3.) is of mouse origin, a labeled secondary antibody against mouse IgG must be used. Similarly, if the primary antibody is of rabbit origin, a labeled secondary antibody against rabbit IgG must be used. 2 Add 100 l.L secondary antibody solution to each well 3. Cover the plate and incubate at room temperature for 1 h 4. Wash the wells five times m TPBS.

430

Nilsen, Berg, and Gokwyr

3.4.5. Developing 1. Prepare the developing solutron Just before use and add 100 $ to each well 2. Let the color reaction develop for 5-30 mm (depending on the intensity of the reactron). 3. Stop the reactron by addmg 50 $4 N H2S04. 4. Read the absorbance in each well at 492 nm using a mlcroplate reader Subtract the absorbance value of blank wells (usmg the mean value of all blank wells).

3.5. Western Blotting 3.5.1. Slotting 1. Equilibrate the prevrously electrophoresed sodmm dodecyl sulfate (SDS)-polyacrylamide gel (see Note 22), containing separated proteins from mrcrosome fractrons (10 pg total protem loaded per well), or PMS (40 pg total protein per well), in blotting buffer for at least 10 mm (see Notes 23 and 24) 2. Cut the mtrocellulose membrane to the size of the gel, wet rt m drstilled water, and equilibrate the membrane m blotting buffer for at least 10 mm Wear gloves at all times when handling the membrane and assembling the membrane-gel sandwich 3. Using a gel holder with sponge pads, supplied with the electroblottmg apparatus, assemble a sandwich as follows: Wet one sponge pad and place it on the cathode side of the gel holder Cut two sheets of filter paper to the size of the gel, wet them m blottmg buffer, and place one sheet on top of the sponge pad. Place the pre-equilibrated gel on top of the filter paper and place the nitrocellulose membrane on top of the gel Roll wrth a clean glass tube to carefully squeeze out any air bubbles trapped between the gel and the membrane Place the second wetted filter paper on top of the membrane and roll again to remove au bubbles. Complete the sandwich by placmg another sponge pad on top of the filter paper and closmg the gel cassette. 4 Place the cassette in the blotting tank filled with blotting buffer. Connect to a power supply and transfer for 1 h at a constant voltage of 100 V (approprrate for 0 75-mm thrck gels, thicker gels may need longer transfer trmes) (see Note 25)

3.5.2. Blocking 1. Remove the membrane from the sandwich assembly, and equilibrate it in TTBS for 5 mm 2 Place the membrane in blocking solutron for 30-45 min at room temperature on a gentle shaker (see Note 26). This step will block sites on the membrane not occupied by sample protein 3. Wash the membrane 2 x 5 mm m TTBS

3.5.3. Primary Antibody 1. Dilute the primary MAb or PAb against fish CYPlA in TTBS with 1% gelatin. Optimum drlution of the primary antibody must be determined for each antibody and for each fish species to be tested (see Note 27).

CYPlA Induction in Fish

431

2. Add the solution to the membrane, using a volume Just sufficient to cover the membrane. 3. Incubate overnight at room temperature on a gentle shaker (see Note 28) 4. Wash the membrane 2 x 5 min in TTBS.

3.5.4. Secondary Antibody 1 Dilute the HRP-labeled secondary antlbody according to the manufacturer’s instructions in TTBS with 1% gelatin If the primary antibody (see Subheading 3.5.3.) is of mouse ongin, a labeled secondary antibody agamst mouse immunoglobulms must be used. Similarly, If the primary antlbody is of rabbit ongin, a labeled secondary antlbody against rabbtt immunoglobulins must be used (see Note 29). 2 Incubate 3 h at room temperature on a gentle shaker. 3 Wash the membrane 2 x 5 min m TTBS and 1 x 5 mm m dlstilled water

3.5.5. Developing 1. Prepare developing solution just prior to use Mix (A) and (B) and nnmedlately pour over the membrane (see Note 30). 2 After 5-30 mm bands should appear. If the staining IS weak, transfer to dlstilled water and leave the membrane in the tray protected from light. More bands may appear and weak bands may get stronger several hours later If the staimng 1sstrong, wash at least 2 x 5 min with distilled water to stop further darkening of the bands 3 The developed membranes may be stored for months in distilled water at 4”C, or an-dried at room temperature. To obtain a permanent record of the result, the membrane should be photographed, preferably when still wet

4. Notes 4.1. Sampling 1. In laboratory studies, groups of control fish are normally included m the expenmental deugn. When sampling feral fish, it 1sequally Important to mclude pnstine reference sampling sites with slmllar habltat properties as the suspected contaminated sites All sites should be as similar as possible m terms of size and sex of species caught, water salinity and temperature, as well as depth and sedlment type when sampling bottom-dwelling fish species (5). An alternative to this strategy 1sto use cagmg of fish from a genetically homogeneous group, to ensure that the fish are exposed m the experimental area for the prescribed period of time (see, e.g., 24,25). 2. Blliary metabolltes may mhibit EROD actlvlty and care should be taken to avoid contammatlon with bile when dissecting out the liver.

4.2. Preparation

of PMS and Microsomes

from Fish Tissue

3. The post-mltochondnal supernatant (PMS, sometimes called the S-9 or S-12 fraction) is the supernatant generated by centrifugatlon of a tissue homogenate at 900& 12,000g. The microsomes are precipitated by a 100,OOOgcentritigatlon of the PMS

432

Nilsen, Berg, and Gokmyr

4. The liver is the richest source of CYPlA m the fish, and this tissue is most wtdely used for testing CYPlA induction m fish It has been shown, however, that CYPIA is also found m other tissues (see, e.g., 26), and tt may be of interest to test other tissues such as kidney, heart, or gill during a sampling program In this case, optimized procedures for preparatton of subcellular fractions from the various trssues should be consulted or developed.

4.3. EROD Analysis 5 In this protocol, the catalytic activity of CYPlA is measured by a fluorrmetrtc EROD assay. CYP 1A catalyzes the O-deethylation of 7-ethoxyresorufin givmg the product resorufin, which is measured fluorimetrically using an excitation wavelength of 535 nm and an emission wavelength of 585 nm (27,28) The resort& formation is dependent on the presence of enzyme, substrate, oxygen, and the cofactor NADPH. An alternattve method is to follow the formatton of resorufin production spectrophotometrically at 572 nm (15) This method, however, appears to be less sensttrve than the fluortmetrtc alternatrve. 6. The EROD reaction is temperature-dependent and the assay 1s performed at a defined temperature, usually 20°C. Because of adaptatton of fish to ambient temperature, EROD activity is dependent not only on assay temperature, but also on environmental temperature at the sate of fish samplmg (see, e g., 29) Thus, m order to be able to compare samples from fish caught at different locations, care should be taken to avoid large differences in the water temperature at the different sites. 7. With each batch of samples tested, positive and negative control samples with known EROD actrvtty should be mcluded as a quahty control of the assay and the mstrumentatlon. In our laboratory, stocks of P-naphthoflavone(BNF)-Induced and nonmduced cod mrcrosomes are used as positive and negative controls, respectively. The control mrcrosomes are alrquoted into small volumes and stored at -80% and each alrquot is thawed and used only once for EROD measurements. 8. The enzymatic acttvtty of CYPlA is easily destroyed by protein denaturatton during field samplmg, processing of samples, or suboptimal storage conditions. Storage of ttssue or tissue fractions at -8O*C or m liqutd nitrogen, and processmg of samples at 0-4”C is therefore essential to minimtze protein denaturatton m samples for EROD analysts Repeated freezing and thawing of samples also stgmficantly reduce the enzymattc activity ofCYP1A. Frozen samples should therefore not be thawed more than once before EROD analysis 9. The substrate and the cofactor NADPH are added to the EROD reactton mrx m excess. Ideally the concentrations of substrate and NADPH should be determmed by titration to find the concentrations that give maximal activity in the assay. A substrate concentration of at least 2-4x the KM value should be used. The K, value of CYPl A from the liver of different fish species varies greatly, but the substrate concentration described in this protocol (2 pA4in the final reaction mix) has been successfully used for fish CYPlAs with observed KM values ranging from 50 nA4 to 400 r&f.

CYPlA induction in Fish

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10. In order to measure true reaction rates, incubation mixtures should not exceed a resorufm production of 0.2-O 3 nmol/mm. Higher reactlon rates may exhlblt nonlinear kinetics and result in underestimation of the true reaction rate. Dllutlon of the PMS or microsome fraction m homogenization buffer may be necessary to obtain an appropriate reactlon rate in such cases. If, on the other hand, a low EROD activity is determmed, the volume of the sample may be Increased to 50100 & (the volume of buffer should be reduced accordmgly). 11 The addition of a known amount of resorufin as internal standard 1s necessary because of the quenching effect of variable amounts of protem in the samples We typically use 150-200 pmol resorufin, but the amount may vary depending on the mstmment settings required for any particular sample. 12 Enzyme activity determined by EROD is usually normalized to total protein in the sample tested Normal&g the activity to the wet weight of tissue used for the reaction is also possible, but the argument against this approach is that in certain situations, especially when perfomung EROD measurements at sea, it is not possible to accurately weigh small pieces of tissue. When normahzmg the EROD values to total protein the method of protein determination is critical. Stagg and Addison (28) have shown that protein determination was a major source of differences m the EROD values reported from different laboratories. Usual methods for protein determmatlon are the Lowry assay (22) or the Bradford assay (23). The latter method is often preferred because it 1squicker and can be performed m a 96-well plate. A modified Lowry assay, which 1s rapid, can be performed in a 96-well plate and has a broader linear range than the Bradford method, has recently been introduced (DC protein assay from BloRad Laboratories, Hercules, CA: BCA protein assay from Pierce, Rockford, IL). It has been suggested that this method, with BSA as standard, should become the standard protem assay for these analyses (30).

4.4. ELBA 13 This procedure describes an indirect ELISA where the antigen 1sunmoblllzed on a microtiter plate, a primary antibody specific for the antigen 1s allowed to bind to the antigen, and a secondary antibody ConJugated to the enzyme HRP IS allowed to bmd to the primary antibody. The immunocomplex 1s detected by the addition of a substrate, which is cleaved by the ConJugated HRP to generate a colored reaction product that is spectrophotometrlcally detected. 14. The ELISA procedure described is a semiquantitative assay, giving a relative measure of CYPlA m different samples. Although it is theoretically possible to develop a quantitative assay to determine the absolute amount of CYPlA m microsome or PMS samples by preparing a standard curve with purified CYPl A, such an approach is difficult because purified CYPl A 1s available from only a very limited number of fish and, more importantly, because of the nature of the CYPlA protein CYPlA is a hydrophobic protein that 1s bound within membranes in microsome and PMS fractions. Experiments where known amounts of purified CYPlA have been added to mlcrosome fractions have shown that com-

434

15.

16

17. 18.

19. 20.

Nilsen, Berg, and Gokssyr ponents of the mtcrosome preparation affect binding between CYPlA and anttbody in such a way that a standard curve prepared wtth purtfied protein will not give an accurate measure of the amount of CYPlA m a microsome fraction (Gokseyr, unpublished results). The antigen used for coating may be either a microsome fraction or a PMS fraction. A microsome fraction is, however, a richer source of CYPlA than a PMS, and there may be cases where only a microsome fraction will give a signal m ELISA If the preparation of a microsome fraction IS impossible, a PMS fraction may be further enriched in CYPlA content by precipitation with CaCl,. To this end, 0.32 MCaCl, is added to the PMS fraction to a final concentratton of 12 mM CaCl*, the solution is centrifuged at 15,OOOgm an Eppendorf centrrfuge, and the pellet 1s resuspended by somcatron in resuspension buffer for mlcrosomal fractions (see Subheading 2.2., item 3). To allow comparison of different samples, the total amount of protein added to the microplate wells should be the same for each sample A coating concentration of 10-100 pg total protein per mL has been successfully used for different fish species and different primary antibodies m our laboratory. The optimal amountlconcentratlon of anttgen to be used m the coating step must, however, be determined for each fish species and antibody combination. To this end, coat the plates with a serial dilution of the positive control sample (see Note 7) and perform the ELISA with both primary antibody and secondary antibody m excess. Plot the values and select the lowest coating concentration that will yield a strong signal. In some cases, where all the field samples contain much less CYPlA than the positive control sample, it may be useful to use a higher antigen concentration for coating m order to be able to discriminate between samples with low, but different contents of CYPlA The optimal coating concentratton in such cases may be best determined by preparing a serial dilutron of one of the field samples Instead of the positive control sample Coated plates may be stored with coating buffer at 4°C for several days without any detectable adverse effects. Addition of detergents durmg the coating step should be avoided because tt does not sigmticantly improve the ELISA signal, and concentrations of more than 0.001-0.01% of some detergents may result in reduced ELISA signal (7). The 2% (w/v) BSA blockmg solution may be substituted with 0.2% (v/v) Tween-20 in PBS or 3% (w/v) nonfat dry milk in TPBS. The primary antibodies used for detection of CYPl A in fish are usually crossreacting antibodies that specifically bind to CYPlA from a variety of different fish species. Owing to the large number of different fish species and the difficulties in preparing purified CYPl A, it is not practical to prepare specific antibodies against CYPlA from all kinds of fish. Instead, cross-reacting antibodies are prepared by taking advantage of the significant degree of sequence simtlartty between CYPlA from different fish species, which means that antibodies raised against CYPlA from one species will often cross-react with CYPlA from a variety of other species (3,21). The affinity of the antibody for CYP 1A from different

CYPIA Induction in Fish fish may, however, vary greatly. Optimum dilution of the primary antibody must therefore be determined for each combmation of antibody and fish species. To this end, plates coated with the optimum antigen concentratron, detem-nned as described m Note 16, are incubated wrth a serial dilution of primary antibody, followed by the secondary antibody m excess. Plot the values and select a concentration of antibody that IS within the linear part of the curve Maximum range 1s obtained by choosmg a point near the saturatton level. 21. Incubation of the primary antibody for 1 h at 37°C has been successfully used with a variety of MAbs and PAbs in our laboratory Wtth some antibodies, however, increased sensitivity may be obtained if the prtmary antibody is incubated overnight at 4°C.

4.5. Western Blotting 22. Western blotting combines gel electrophoresis to separate the proteins in the microsome or PMS fractions with the specificity of mnnunological detection. The gel electrophoresis step is not described here, but we routinely run our samples in SDS-polyacrylamide gels, using P-12% polyacrylamide and the buffer system of Laemmli (31). The samples are denatured by boiling in the presence of SDS and 2-mercaptoethanol (31). 23. The amount of total protem needed to be able to detect the CYPl A band depends upon whether a microsome or PMS fraction is used and the aftimty of the primary antibody. 10 l.tg total protein for mrcrosome fractions and 40 pg total protem for PMS fractions have been successfully used m our laboratory with a variety of antibodies agamst CYPlA. If the band 1s weak, e g , m cases with weakly induced samples or a cross-reacting antibody with weak affinity for CYPl A from the particular species tested, tt may be useful to increase the amount of total protein loaded on the gel. To allow for compartson of different samples, the total amount of protein should be the same for each sample. 24. The gels are equtlibrated m blottmg buffer to remove electrophoresrs buffer salts and detergents. This step also allows the gel to adjust to its final size before blotting, since some gels (<12% polyacrylamide) may shrink in the methanol blotting buffer used. Equilibration may result m diffusion and broadening of bands, and the gels should therefore not be left m blotting buffer longer than necessary. Ten min 1ssufficient for thin (< 1 mm) gels, thicker gels may need a longer time. 25. Blotting ttmes may need to be optimized for the particular gel and blotting system used. Thicker gels need longer transfer times. One h 1s sufficient for our system with 0.75-l mm gels. 26. The blocking solution (3% gelatin in TTBS) may be substituted with 5% nonfat dry milk m TTBS. The dilution buffer for the primary and secondary antibody should then also be substituted with 5% nonfat dry milk m TTBS. 27. Optimum dilution of the primary antibody must be determined for each antibody and fish-specres combmatton. As already noted above (Note 20), antibodies against fish CYP 1A are usually cross-reacting antibodies that bind CYP 1A from a variety of fish species, but the afftntty for CYP 1A may vary greatly between

436

Nilsen, Berg, and Gokmyr

species, It is therefore not uncommon that the optimum dilution of the antibody for different fish species may vary 1O-fold or more. 28. Incubation with the primary-antibody solution overnight at room temperature has been successfully used with a variety of different anti-CYP 1A antibodies m our laboratory. Shorter incubation ttmes (not less than 2 h at room temperature) may be suffictent for some antibodies. 29. Incubatton with the secondary-antibody solution for 3 h at room temperature has been found to give the best results with our primary antibody/secondary antibody combinations. Shorter mcubation times (1 h at room temperature or at 37°C) may be sufficient m some cases. 30. In the procedure described here, a chromogenic substrate is used to develop the bands In cases where the signal IS weak, lummescent substrates may be used instead to Increase the sensitivity of detection. Enhanced chemilummescence (ECL) substrates are available from many manufacturers and should be used according to the manufacturer’s mstructions. The membrane (after the final wash) is incubated with the luminescent substrate for 30 s to a few mm, then sealed m a thm plastic membrane and exposed to X-ray film, usually for a few s or min.

References 1. Peakall, D. B. (1994) Biomarkers: the way forward m envtronmental assessment. Toxxol Ecotox~ol. News 1, S-60 2. Gokseyr, A. and Fdrlm, L (1992) The cytochrome P450 system m fish, aquattc toxicology, and environmental monitoring. Aquat Toxxof 22,287-312. 3 Stegeman, J. J. and Hahn, M E. (1994) Biochemistry and molecular biology of monooxygenases: current perspectives on forms, functions and regulation of Cytochrome P450 in aquatic species, m Aquatzc Tox~ology Molecular, Biochemzcal, and Cellular Perspectzves (Maims, D. C. and Ostrander, G K., eds ), Lewis Publishers, Boca Raton, FL, pp 87-204. 4. Fdrlin, L , Goksnryr, A , and Hussy, A -M. (1994) Cytochrome P450 monooxygenase as mdicator of PCB/dioxin like compounds m fish, m Bzologzcal Momtorzng of Estuanne and Coastal Waters (Kramer, K , ed.) CRC, Boca Raton, FL, pp. 135-150. 5. Goksoyr, A. (1995) Use of cytochrome P450 1A (CYPlA) in fish as a bromarker of aquatic pollution. Arch Toxzcology (Supp. 17), 8@-95 6 Bucheli, T D. and Fent, K. (1995) Induction of cytochrome P450 as a biomarker for environmental contammation in aquatic ecosystems Crzt Rev Envzron Sci Technol 25,201-268 7 Goksoyr, A (1991) A semi-quantitative cytochrome P450IAl ELISA. A simple method for studying the monooxygenase mduction response m environmental monitoring and ecotoxicological testing of fish. Scr Tot Envlr. 101,255-262 8 Goksoyr, A , Andersson, T., Buhier, D R., Stegeman, J J., Williams, D E., and Fbrlin, L. (1991) Immunochemical cross-reactivity of P-naphtoflavone-mducible cytochrome P-450 (P4501A) m liver microsomes from different fish species and rat. Fzsh Physlol Bzochem 9, 1-13.

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9. Morrison, H., Oleksiak, M. F., Cornell, N. W., Sogm, M. L , and Stegeman, J J (1995) Identification of Cytochrome P450 1A genes from two teleost fish, toadfish (Opsanus tau) and scup (Stenotomus chrysops), and phylogenettc analysis of CYPlA genes. Btochem. J. 308,97-104. 10 Collier, T. K., Anulaction, B. F , Stein, J. E., Goksoyr, A., and Varanast, U (1995) A field evaluation of cytochrome P450 1A as a btomarker of contaminant exposure in three spectes of flattish Envtron Toxicol. Chem 14, 143-152. 11. Burgeot, T., Bocquene, G., Pmgray, G., Godefroy, D., Legrand, J., Dimeet, J., Marco, F., Vincent, F., Henocque, Y., Jeanneret, H. O., and Galgani, F (1994) Monitoring biological effects of contammatton in marine fish along French Coasts by measurement of ethoxyresorufin-o-deethylase acttvity. Ecotoxtcol Envzron SaJ 29, 13 l-147. 12. North Sea Task Force (1993) North Sea Qua&y Status Report 1993 Oslo and Paris Commisstons, London. Olsen & Olsen, Fredensborg, Denmark, pp 132+vt. 13 Goksoyr, A. and Larsen, H E. (199 1) The cytochrome P450 system of Atlantic salmon (Salmo salar) I Basal properties and induction of P450 1Al m liver of immature and mature fish. Fish Phystol. Bzochem. 9,339-349. 14. Prough,

R. A , Burke,

M

D , and Mayer,

R T (1978)

Direct

fluorometrlc

methods for measurmg mtxed-function oxtdase activity. Meth Enzymol. 52, 372-377 15. Klotz, A. V., Stegeman, J. J , and Walsh, C (1984) An alternattve 7-ethoxyresorufin 0-deethylase acttvtty assay. a continuous visible spectrophotometric method for measurement of cytochrome P-450 monooxygenase acttvlty Anal Biochem 140, 138-145. 16. Williams, D. E., and Buhler, D. R. (1984) Benzo(a)pyrene-hydroxylase catalyzed by purified isozymes of cytochrome P-450 from j3-naphthoflavone-fed rambow trout. Btochem Pharmacol 33,3743-3753. 17 Goksoyr, A. (1985) Purification of hepatic mrcrosomal cytochromes P-450 from P-naphthoflavone-treated Atlantic cod (Gadus morhua), a marme teleost fish. Biochim Biophys Acta 840,409-417

18. Park, S. S., Miller, H Gelboin, H. V (1986) P-450E of the marme 3-methylcholanthrene

, Klotz, A. V., Kloepper-Sams, P. J , Stegeman, .I J , and Monoclonal antibodies to liver microsomal cytochrome fish Stenotomus chrysops (scup). cross reactivity with induced rat cytochrome P-450. Arch Biochem Btophys

249,339-350

19. Zhang, Y. S., Goksoyr, A., Andersson, T , and Forlm, L. (1991) Inmal purtfication and characterization of hepatic microsomal cytochrome P-450 from BNFtreated perch (Percafluvtattlts). Comp. Btochem Physiol 98B, 97-103. 20. Myers, C. R., Sutherland, L A., Haasch, M. L., and Lech, J. J (1993) Antibodtes to a synthetic peptide that react specifically with rainbow trout hepatic cytochrome P450 1Al. Environ Toxtcol. Chem 12, 1619-1616. 21. Husary, A -M., Strtetkvern, K O., Maaseide, N P , Olsen, S. O., and Gokseyr, A (1996) Polyclonal and monoclonal antibodies agamst immunoaffimty purified Atlantic cod (Gadus morhua L.) CYPlA Mel Mar Btol Biotech 5,84-92.

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22 Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) Protein measurement with the folin phenol reagent. J Bzol Chem. 193,265-275 23 Bradford, M M. (1976) A rapid and sensitive method for the quantltatlon of microgram quantities of protein utlhzmg the principle of protein-dye-bmdmg Analyt Biochem. 72,248-254

24. Beyer, J., Sandvik, M , Hylland, K., Fjeld, E., Egaas, E , Aas, E , SkBre, J. U , and Goksayr, A (1996) Contammant accumulation and blomarker responses m flounder (Platzchthysjlesus L.) and Atlantic cod (Gadus morhua L.) exposed by cagmg to polluted sediments m Snrrfjorden, Norway. Aquatzc Toxlcol. 36,75-98. 25. Gadagbul, B K.-M. and Gokseyr, A. (1996) CYPlA and other blomarker responses to effluents from a textile ml11 m the Volta River (Ghana) using caged tilapla (Oreochromls nllotxus) and sediment-exposed mudflsh (Clarlas anguzllaris).

Bzomarkers

1,252-26

1.

26 Hussy, A.-M., Myers, M S , Wllhs, M. L , Collier, T K , Celander, M , and Goksaryr, A. (1994) Immunohlstochemical localization of CYPlA and CYP3Ahke lsozymes m hepatlc and extrahepatlc tissues of Atlantic cod Toxzcol Appl Pharmacoi

129,294-308

27. Burke, M. D and Mayer, R. T. (1974) Ethoxyresorufin direct fluorometrlc assay of a mlcrosomal 0-dealkylation which IS preferentially mduclble by 3-methylcholanthrene. Drug Metab Dzsp. 2,583-588. 28. Stagg, R. M. and Addison, R F. (1995) An inter-laboratory comparison of measurements of ethoxyresorufin 0-deethylase actlvlty m dab (Llmanda llmanda) hver. Marwe Environ Res 40,93-l 08 29. Sleiderink, H M., Beyer, J , Everaarts, J M , and Boon, J. P (1995) Influence of temperature on cytochrome P450 1A in dab (Limanda llmanda) from the southern North Sea* results from field surveys and a laboratory study. Marzne Environ Res 39, 67-71 30 Stagg, R and McIntosh, A. (1997) Determination of monooxygenase activity in the liver of the dab (Lzmanda hmanda) by the fluorlmetnc measurement of 7ethoxyresorufin-0-deethylase (EROD) activity ICES Techn. w Marzne Envrron sci. 22, p. 17 3 1. LaemmLl, U. K. (1970) Cleavage of structural protems during the assembly of the head of bacteriophage T4. Nature 227,680-685.

44 Generation of Allelic Series in Mice by Gene Targeting Maureen K. Bunger and Christopher

A. Bradfield

1. Introduction The ability to manipulate the murme genome via the use of gene targeting m pluripotent embryonic stem cells is arguably one of the most important scientific achievements of the last decade (1-4j. This technology holds the potential to produce murme strains and cell-culture systems that harbor an allehc series of mutations at almost any given locus, As a result, models can now be generated m complex eukaryotic systems in which a gene of interest IS no longer expressed (“null” allele) or m which some aspect of tts function or expresston has been altered. The techniques commonly used m the generation of null alleles have been the focus of many excellent review artrcles, books, and laboratory manuals (5-9). Therefore, our goal m preparing thts chapter was to draw attention to gene-targeting strategies that provide mechanistic insight when null alleles provide incomplete mformatton. Additionally, we discuss some examples of how allelic series can sometimes yield surprising results that arise when different laboratories use different approaches to create a null mutation at the same locus. We would like to discuss how gene targeting has proven to be a useful techmque for pharmacologists. However, owing to the nascent stage of the technology, few examples are available in this area. Therefore, our presentation will be more general in nature. First, we provide an overvrew of the most popular gene-targeting approaches in use today. Second, we describe gene-targeting experiments at the CFTR locus, arguably one of the largest allelic series to date. Thud, we provide a brief review of targeting expertments at loci encoding cytochrome P450 structural genes and their regulators. We hope that the chapter will help the reader appreciate how gene targeting can be From Methods in Molecular Biology, Vol 107 Cytochrome P450 Protocols Edlted by I R Ph&ps and E A Shephard 0 Humana Press Inc , Totowa, NJ

439

Bunger and Bradfield used to generate novel mouse models that are useful m drug design and for the investigation of metabohc pathways and the mechanism of action of toxicants. One method to generate a null allele at a given locus is to introduce a targeted mutation via homologous recombmation into the genome of pluripotent mouse embryonic stem (ES) cells (2-4). When injected into blastocysts, ES cells mix with the inner cell mass of the blastocyst giving rise to a number of cell lineages to produce chimeric progeny. When the targeted ES cells give rise to the germ line m the chimera, standard breeding schedules can be used to generate murine lines that are either heterozygous or homozygous for the targeted mutations (10). Most often, either replacing or disrupting exons required for function of a gene product or regulatory elements required for tts expression generates null alleles. Thus, null alleles may be the result of a block in transcrrption of the gene, decreased stability or activity of the gene product, or a combination of any of these. One common strategy is to generate null alleles through the use of a targeting construct m which the ammoglycoside phosphotransferase gene (Neo) is flanked by genomtc fragments derived from a region of the gene to be disrupted. Expression of the Neo-selectable marker 1s often driven by the phosphoglycerate kmase (PGK) promoter, which is highly active in murine ES cells. When the construct integrates via homologous recombination at the target locus, the ES cells attain resistance to toxic aminoglycoside drugs such as G4 18-sulfate. Targeting constructs are typically designed in such a way that homologous recombmation will disrupt or delete a cnttcal sequence within the gene of interest, as well as provide a marker to allow selection for this event (Fig. 1) (2-4). Integration of vector DNA into the ES-cell genome can occur by both homologous and nonhomologous mechanisms. Using classical targeting constructs, the ratio of homologous to nonhomologous integration events can be highly variable, typically ranging from l/20 to l/40,000 m recombinant cells that survive positive selectron (11,12). Thus, simple targeting constructs that only take advantage of a positive-selection step can yield large numbers of colonies that contain mtegration events at nontarget sites (via nonhomologous integration). To overcome this potential problem, targeting constructs often include, in addition to a positive-selectable marker, a negative-selectable marker that flanks one or both of the genomic arms of the construct. In one popular strategy known as positive-negative selection, the herpes simplex vu-us thymidine kinase gene (HSV-tk), which encodes a viral enzyme that produces toxic metabolites in the presence of ganctclovn (GANC), is placed at one or both ends of the targeting construct (Fig. 1) (13). In the event of homologous recombmation the HSV-tk doesnot integrate, rendering cells resistant to GANC. Ideally, since heterologous integration of the entire construct leads to insertion of HSV-tk mto the genome, only cells that have integrated the

441

Generation of Allelic Series A Homologous Recombination

,,,eo HSV-tk

Targetingconstruct Wild-typeallele

G418’,Ganciclovir’ 1 Homolgous recombination

B Non-homologousrecombination HSV-tk

Targetingvector Non-homologous site

1

G418’,GancicloviP HSV-fk

Random integration

Fig. 1. Positive-negative gene targeting: (A) A positive-selection marker such as Neo is inserted into the coding region of the targeting construct flanked by genomic sequence.A negative-selection marker, HSV-tk, is cloned onto either or both ends. Homologous recombination results in insertion of Neo in the genome and loss of HSV-fk. Resulting ES cells are G418- and GANC-resistant. (B) The targeting construct insertsby nonbomologousrecombination resulting in insertion of both Neo and HSV-tk randomly into the genome.Cells are G418-resistant,but are GANC-sensitive. vector-derived genomic fragments by homologous recombination will survive in media containing both G418 and GANC. In addition to Neo and HSV-tk, specially designed Hprt minigenes have also been successfully used in positive-negative gene targeting and are especially versatile since Hprt can substitute for either Neo or HSV-tk. Sufficient expression of the Hprt gene product, hypoxanthine phosphoribosyl transferase (HPRT), in cells confers both resistance to hypoxanthine-aminopterin thymidine (HAT) and sensitivity to 6-thioguanine (TG). The versatility of Hprt as both a positive- and negative-selection marker can simplify the generation of gene-targeting constructs where both the negative and positive selectable markers are required in tandem (see Subheadings 2.1.-2.3.) (14,IJ). However, HPRT is endogenously expressed in ES cells, therefore the use of Hprt as a selectable marker is limited to ES-cell lines that are HPRT- (16,17). It is important to point out that in practice, survival of recombinant ES cells using any type of negative selection is a function of both

Bunger and Bradfield the efficiency of homologous recombination and the spontaneous loss-of-function of the negative-selectable gene. Consequently, efficiency of selectton for homologous recombmatton can vary stgmficantly for each targeting construct and targeted locus and the utility of posmve-negative selection for any parttcular locus can only be determined empirrcally. Whereas this chapter places considerable emphasis on the use of Neo, HSV-tk, and Hprt as selectable markers, tt should be noted that Hyg and Pur are examples of addtttonal posttive-selectable markers and Diphtheria toxin-A IS a example of an additional negattveselectable marker for use in targeting-vector construction (18-20). Although partrcular emphasis IS often placed on the generation of whole-animal models, it is important to point out that the parental ES cells constitute a powerful experimental system. Targeting the second allele with an addmonal targeting construct or the use of high G418 concentrations for selection of nonreciprocal crossing-over events can give rtse to “double knockout” clones (“homozygous null”) (19,21,22). Double-knockout cell lines have great experimental utility since ES cells can be mampulated in culture to differentiate down a number of different cell lineages allowing the role of a partrcular gene product on a specific differentiation process to be studied in vitro (23,24). Furthermore, ES cells can form teratomas when injected subcutaneously in nude mice (25). ES cells therefore have the potential not only to facthtate targeted alterations m the genome of the whole mouse, but also can provide excellent models to study the role of a given gene on tumor progression and cellular differentiation. Although null alleles generated through deletions or inserttons have provided great insights into the function of numerous gene products, tt may be more mformattve to generate mutant alleles encodmg normally expressed gene products displaying altered function or mutant alleles that are expressed only m certam cell lineages or at certain developmental time points (conditional null mutations). Null alleles may be inappropriate when one is attemptmg to generate murine models of human disease states related to alterations in protein function, not necessartly loss of protem expresston (26-28). Furthermore, murme strains harboring condittonal mutant alleles can be partrcularly useful when examming the role of a gene product on the development or physiology of a partmular tissue type or when null alleles lead to embryonic lethality and prevent study later m development (2%31). Generation of condttronal null mutations IS only briefly menttoned here, but should be recognized as a valuable method to study trssue and developmental-specrfc effects of null mutations (29,31). Finally, the ability to generate and examme the consequences of novel mutations within the murine genome that result m hypomorphic phenotypes rather than null phenotypes 1sa powerful way to deduce the role of specific protein subdomains on complex biological pathways.

Generation of Allelic Series

443

2. Strategies With the advent of ES-cell technologres came the ability to readrly create informative allelic servesin mice by generating targeted mutations at specific sites in any gene product. Allelic variations can be generated through gene targeting by the introduction of mutations such as amino acid substrtutions, insertions and deletions, alterations m regulatory elements, and complete gene replacement. Important principles applicable to any gene-targetmg experiment should be constdered when discussing gene-targeting techniques. Fnst, multiple passagmg of ES cells can potenttally compromrse the pluripotency of the cells, therefore protocols designed to minimize the time ES cells spend m culture are preferred. Second, successful cotransfer of selectable marker and a nearby mutation 1s inversely proportional to the distance between the two (32,33). Therefore, careful attention must be paid to the distance between mutation and marker gene in the design of constructs used for any strategy that relies on cotransfer of the mutation with a marker gene. Third, efficiency of homologous recombinatton with a DNA construct that is tsogemc to the ES-cell line is 20-25 times greater than wrth nonisogenic DNA (34,35) Therefore rt is important to be aware of the length and fidelity of homology that exists between the construct and the target locus (12,36). Fourth, careful attention must be paid to the choice of markers to be mcluded and to provide a means to screen for the correct targeting event via the rdentification, mtroductron, or removal of an mformative restriction site, or by polymerase chain reaction (PCR) (3 7-39). Our primary goal in this chapter is to describe gene-targetmg techniques that result in “clean” transfer of the mutation to the locus leaving mmlmal traces of exogenous DNA sequence. In many gene-targeting strategies, the selectable marker 1sused to disrupt genes in order to generate null alleles and often remains Inserted m the locus. However, m several cases,insertion of the selectable marker gene into certain locr has resulted m unexpected cis effects on the regulation of nerghbormg genes. These effects appear to be the result of the presence of enhancers and promoters required for expression of selectable marker (40,41). During the past 6 yr, four techniques have emerged based on the positive-negative selectron strategy and have each proven successful in systematically introducing targeted mutatrons mto the mouse germ lure. All of the techniques described here are either inherently designed to generate clean mutations, or can be adapted to generate nearly clean mutations by concomitant use of the Cre-LoxP recombination system (42,43). A generic discussion of these four targeting techniques and the use of the Cre-LoxP recombinatron system follows, along with an example we feel demonstrates the power of using gene targeting to generate allelic series in mice.

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2.1. In and Out1 Hit and Run The “in-out” or “hit and run” approach borrows technology used in generatmg targeted mutations in yeast (44,45). This technique requires construction of an insertional-type targeting vector containing both positive- and negativeselection markers, like iVeo and HW-tk, m tandem and a single long stretch of homologous sequence containing the desrred mutation. Use of the Hprt minigene as both the positive and negative selectable marker has also been used successfully in thus procedure (46). In the hit-and-run approach, the targeting construct is lmearized within the homologous region and electroporated mto ES cells. It is thought that the cell employs double-strand gap repair mechanisms to insert the targeting construct mto the genome m reverse orientation at the region of homology (O-type recombination), leading to a duplication of homologous sequences separated by vector-derived marker genes (Fig. 2) (47,48). A two-step selection process is then employed, In the first step, cells are grown m medium containing G4 18 (for Neo-containing vector) or HAT (for Hprt-containmg vector), which selects for insertion of the vector mto the genome. Subsequently, cells are grown in medium containmg GANC (for HSV&-containing vector) or TG (for Hprt-containing vector). This second step selects for a smgle reciprocal recombination event that results from a looping out of one or the other homologous region, resolution of the duplication, and subsequent loss of vector-derived sequences (49). An advantage of hit and run is that one can screen for events in which all extraneous vector DNA sequence has been eliminated, thereby generating an allele in which no exogenous DNA sequence remains. One potential complication of the hit and run technique is high background resultmg after negative-selection stemming from the spontaneous loss of expression of the negative-selection marker gene. Since during this process both marker genes are lost, it is often advantageous to screen clones for reversion to sensitivity in positive-selection medium m addition to resistance m negative-selection medium m order to reduce background (SO). Furthermore, the prolonged time m culture required to complete the two-step selection process may compromise the ability of the ES cells to contribute to the germ line. Hit and run targeting has been used successfully to introduce clean mutations into murine genes encoding apolipoprotem B, Hox2.6, and the cystic fibrosis transmembrane conductance regulator (45,46,51).

2.2. Tag and Exchange and Double Replacement “Tag and exchange” and double-replacement strategies (50,52) require two replacement-type targeting vectors (R-type recombination) with homology to the region of interest (Figs. 3 and 4). The first vector contains both positive-

445

Generation of Allelic Series A

Hit/in Neo l-/Wtk \

I

G418’, GancicloviP HSV-tk Neo : rfm-1

B

Tj71

Run/Out

* L

m

I

lntrachromosomal

recombrnation

G418”, Ganciclovir’ Tj:

I

Mutant allele

Fig. 2 Hit and run/m and out: (A) A mutation is introduced mto the codmg regton flanked on both sides by genomic sequence. The targetmg construct contains both postttve- and negative-selection markers m tandem The construct is lmearized wtthin homologous sequence Recombmation at the correct site results in duplication of the genomtc region and insertion of both markers Cells are G418-reststant and GANCsensitive. (B) A single reciprocal mtrachromosomal recombinatton excises one or the other duplicate along with the marker genes Resultant cells are G4 1&sensitive and GANC-resistant. Clones must be screened to determine which duplicate was excised.

and negative-selection markers such as Neo and HSV-tk m tandem or Hprt alone, with flanking homologous sequences from the target gene. An mrtial homologous recombination event with this vector serves to “tag” the site of interest with concomitant gain of resistance to G4 18 (or HAT). The second targeting vector contains the mutation of interest flanked by homologous sequence in the same region of the genome that has been tagged. This construct does not require a selectable marker, only the presence of the mutation of interest. Homologous recombination serves to “exchange” the tagged site with the

Bunger and Bradfield

446 A

Neo

Tag

HSV-fk

1^1

fl

\I

A

Tr: 1

w

First targeting vector

l-n---l

Wild-type allele

G418’, Ganciclovif Tagged allele

B

Exchange

Second targeting vector Tagged allele

t

1

G4181, Ganciclovir’ Mutant allele

Fig. 3. Tag and exchange: (A) The allele is tagged by insertion of tandem positiveand negative-selection markers inserted into a neutral genomic region near the site to be modified. Homologous recombination results in cells that are G418-resistant and GANC-sensitive. (B) A second targeting construct contains the mutation of interest within genomic sequence homologous to the tagged site. Homologous recombination results in cells that are G4 1&sensitive and GANC-resistant. Recombinant cells must be screened for the specific mutation.

sequence containing the mutation and subsequent loss of both marker genes. Clones are selected for loss of the negative-selection marker and survivors are screened for the presence of the mutation. The difference between tag and exchange and double replacement is in the initial targeting step. Tag and exchange inserts marker genes into neutral sequence adjacent to the area to be mutated, whereas double replacement uses the marker genes to replace genomic sequence. The advantage of replacing the sequences that are to be mutated in the second step is that it increases the range over which new mutations can be introduced. Using tag and exchange, the probability that cross-over events will result in replacement of both the marker genes and incorporation of the desired insertion or mutation is inversely proportional to the distance between the selection cassettes and the mutation (32,331. Therefore, tag and exchange is most valuable when one is attempting

447

Generation of Allelic Series A First replacement

q-l

L

e

r-77

.*.*-*

. ..* . . . . -....

*...

‘. ,5-J

First targeting vector m

El

Wild-type allele

G41IP,Ganciciovif I Neo HSV-fk w I

Tagged allele

B Second replacement Second targeting vector a

Neo HSV-fk

Tagged allele

G41Ip, Ganciclovir’ Mutant allele

Fig. 4. Double replacement: (A) The allele is partially replaced in the targeted region by tandem positive and negative markers flanked by homologous sequence. Homologous recombination results in cells that are G418-resistant and GANC-sensitive. (B) A second targeting construct contains the mutation within replaced sequence and sequence homologous to the regions flanking the deletion. Homologous recombination replaces the markers with the genomic sequence containing the mutation, resulting in cells that are G4 18-sensitive and GANC-resistant.

to generate an allelic series at a shorter genomic region such as a promoter or within a single exon. However, when it is desirable to create mutations along the entire length of a locus that spans larger regions of the genome, multiple tags must be generated. With double replacement, deletions of 20 kb have been achieved in the first step such that new mutations could be easily introduced anywhere along that region in the second step (52). The advantage of both the tag-and-exchange and double-replacement strategies is that once the locus is tagged, mutational diversity may be achieved utilizing a series of markerless targeting vectors. Another advantage of both these techniques, as with the hit

448

Bunger and Bradfield

and run method, is that vector sequence can be entirely replaced by homologous sequence, ehmmating the mtroduction of any exogenous sequence mto the locus. 2.3. Plug and Socket “Plug and socket” also requires two targeting vectors (53) (Fig. 5). However, in this technique the initial targeting construct, the “socket,” consists of two different positive-selection cassettes,one of which is made nonfunctional by a mutation. The second targeting construct consists of homology containing the desired mutation and the same nonfunctional positive-selection cassette, the “plug,” containing a distmct mutation that complements the one introduced in the first step. In practice, the mutated selection markers should contain deletions at either end, such that spontaneous reversion is unlikely and only recombination within the coding sequence of the marker re-establishes function of the second positively selectable gene. Useful positive-selection markers can be any combmation of, but are not ltmited to, Neo, hyg,pur, and Hprt. As with the tag and exchange method, the clone containing the socket can be used to introduce many different mutations into the same site with subsequently derived plug vectors. This method has been successfully used m repeated plugand-socket targeting to generate an allellc series at the murme adult P-globm gene locus (4653). The advantage of this method versus tag and exchange is in the marked reduction of background that results from random mtegration of the targeting vector. This background reduction is attributed to the fact that only cells that have undergone homologous recombination within the second marker gene will survive positive selection in the second step. Unfortunately, this requires that the second marker cassette remains inserted m the genome. To overcome this potential problem and as depicted m Fig. 5, carefully placed 1oxP sites can be included m the targeting vector, which allows the employment of the bacteriophage Pl Cre recombinase gene product to excise the marker gene (see Subheading 2.5.). This serves to eliminate any potential czs effects the remaining marker gene may confer from the ES-cell genome prior to blastocyst injection (54). 2.4. Single-Step Cotransfer One of the simplest techniques described is single-step cotransfer (Fig. 6) (32). This method employs a single replacement-type targeting vector in which a positive-selection marker, such as Neo or Hprt , is inserted within neutral homologous sequence (such as an mtron) near the desired mutation (55). Homologous recombination will ideally replace endogenous sequence with both the marker and the desired mutation. Much like tag and exchange, the

449

Generation of Allelic Series A

Socket

First targeting construct 1

,T, 1

G418’ Socket

B mug

Mutant allele

b

= LOXP site

Fig. 5. Plug and socket: (A) Two tandem positive-selection markers are inserted into neutral genomic sequence. The second marker contains a mutation that renders it nonfunctional, such as a 5’ deletion (HprtA,), to create the “socket.” Homologous recombination results in cells that are G4 18-resistant, but HAT-sensitive. (B) A second targeting construct is generated that contains a complementary 3’ deletion of the second positive-selection marker (@MA,). Only homologous recombination in which crossover points are in the genome and the second positive-selection marker result in cells that are HAT-resistant and G418-sensitive. LoxP sites can be inserted to facilitate removal of the second marker before injection of cells into the blastocyst.

probability of cotransfer of marker and mutation decreases with the distance between them. However, distances of as much as 3 kb have been shown to cotransfer with a rate of 50% (32). Recombinants are selected in a single selec-

Bunger and Bradfield

450

Targeting construct

G418’, Ganciclovir’

+ Cre recombinase

1

G41b

l

Mutant allele

Fig. 6. Single-stepcotransfer: A positive-selection marker is inserted into neutral region adjacent to the site of the mutation and flanked by homologous genomic sequence.HSV-tk can be placed at either end. Homologous recombination results in cells that are G418- and GANC-resistant and recombinant cells must be screenedfor the mutation. LoxP sitescan be inserted where depicted to facilitate removal of the marker gene before injection of cells into the blastocyst. tion step and can be screened for the presence of the mutation. The advantages of this method are: It requires generation of only a single targeting construct, and it requires only a single selection step reducing the length of time the ES cells spend in culture. However, as with the plug-and-socket method, the selection marker remains integrated into the genome. Therefore, as shown in Fig. 6, it may be desirable to include ZoxP sites flanking iVeo in the targeting construct to facilitate the excision of Neo using Cre before injection of cells into the blastocyst. 2.5. Cfe-LoxP The bacteriophage P 1 Cre recombinase catalyzes site-specific recombination between two 34-bp repeats called ZoxP sites. Recombination between two direct-repeat ZoxP sites occurs such that any intervening sequence is excised (42,43). Thus, any vector-derived sequence that is flanked by ZoxP sites can be excised at both the cellular and organismal level by coexpression of Cre (Fig. 7). The reverse reaction is also possible, whereby the Cre-ZoxP system can be used to introduce sequence (“knock-in”) in a site-specific manner. However, this

Generation of Allelic Series

451

Excision using Cre-/oxP

b = LoxP site

Fig. 7. Cre-LoxP System:BacteriophagePI Cre-recombinaseprotein directs sitespecific recombination betweentwo unidirectional 1oxPsitesresulting in excision of intervening sequence.A single 1oxPsite remains.

type of recombination occurs at a much lower frequency (56). Introduction of ZoxP sites by homologous recombination allows the ability to generate large deletions in a single step, including deletion of the marker genes introduced during targeting (41). For example, Cre-ZoxP -mediated gene targeting has been used successfully to generate very informative deletions in the IgH gene locus in mice where ZoxP sites were placed to flank both genomic sequence and the marker genes. Transient transfection of a Cre-recombinase expression construct into the ES-cell clones resulted in generation of the deletion plus concomitant removal of the marker genes (57). Furthermore, the Cre-ZoxP system can be used to generate conditional null mutations by designing targeting vectors to introduce ZoxP sites surrounding key regulator sequences,protein subdomains, or even entire genes. The resultant mouse can then be bred with a mouse containing the Cre recombinase as a transgene under the regulation of a tissue- or cell-specific promoter. Excision of sequence between the ZoxP sites occurs in vivo in the resultant offspring, leading to tissue-specific knockouts of the targeted gene product (30,31,58). One important application of the Cre-ZoxP system is that it can be used to excise marker genes and their associated promoters, enhancers, and polyadenylation signals that may interfere with regulation of neighboring genes. As noted above, use of simple insertion or replacement-type targeting vectors can lead to permanent insertion of the marker gene into the genome. To overcome this problem, investigators have exploited the use of the Cre-ZoxP recombination system. By flanking the marker genes of the targeting vector with

452

Bunger and Bradfield

1oxP sites, transient transfection of a Cre expression vector mto recombmant

ES cells will regenerate a locus that is free of the marker sequence leaving behind only a single 1oxP site. A major limitation associated with using this approach is transient expression of Cre can be inefficient. Therefore, including a negative-selection marker, such as HSV-tk or Hprt, within the region flanked by the ZoxP sites can facilitate selection for cells that underwent an exctston event. The efficiency of Cre-mediated recombination m ES cells by transient transfection of Cre followed by a negative selection is upwards of 99%, obviatmg the need for addtttonal screening by Southern blot or PCR (54). However, this additional selection step can increase the extent of passagmg the ES cells must endure prior to blastocyst injection. The Cre-ZoxP technology also allows “recycling” of selection markers, facihtatmg multiple targeting events at different loci m the same ES-cell lme and offering the potential for retargetmg of the same locus (54). For example, it is sometimes desirable to generate ES cells that are homozygous nulls at the targeted allele for use in in vitro differentiation or studies involving teratoma formation (23). Previously, one way that this was achieved was through the use of a second targeting vector containing a different set of selectable markers (19). This process has limited versatility because of the relatively small pool of ES cell-compatible selection markers that can be used. Therefore, it is advantageous to be able to rid the genome of the selection markers followmg targeting events such that these selection markers can be used repeatedly in the same ES-cell line. Moreover, use of the Cre-EoxP system allows versatlhty in creatmg targeting vectors that can perform multiple functions in the generation of both null alleles and subtle mutations of those alleles. For example, as outlined m Fig. 8, creation of a targeting vector for use in tag-and-exchange methodology can be altered to include 1oxP sites flanking the tandem copies of Neo and HSV-tk, or Hprt alone. This entire sequence is then used to disrupt coding sequence by homologous recombmation in ES cells and subsequent transient expression of Cre creates the null allele that 1smissing marker genes, Prior to transient transfection with Cre, recombmant ES cells can be frozen and saved for future use as a tagged allele for tag and exchange to generate a series of mutations at that particular site. Alternatively, the ES cells containing the null allele after excision of the marker gene with Cre can be retargeted at a different locus usmg the same selection marker m a new targeting construct or retargeted at the second allele using the same targeting construct. It is important to note, however, that any targeting event that results in msertion of HSV-tk mto the genome will not be valuable for generation of a mouse lme containing the desired mutation owing to the fact that the presence of HSV-TK can cause sterility in male mice (59).

Generation of Allelic Series A

453 B

Tag

Null Allele MO

HSV-fk Ganciclovir’

+ Cre I G418’, Ganciclovilq

Null Allele

G41@, Ganciclovir’

Fig. 8. Knock in/knock out using tag and exchange and Cre-LoxP: (A) Tag-andexchange technique can be used to tag an allele with markers that are flanked by ZoxP sites (see text) while at the same time disrupting the coding sequence, resulting in cells that are G418-resistant and GANC-sensitive. (B) Cells containing the tagged allele can be transiently transfected with Cre recombinase expression vector to create a null mutation that is free of marker gene sequence, resulting in cells that are GANC-resistant and G418-sensitive. (C) Cells containing the tagged allele can also be retargeted with multiple constructs containing missense mutations, resulting in an allelic series.

3. CFTR: An Example The emergence of the ability to systematically generate allelic series at a single locus by gene targeting in ES cells has yet to be fully exploited as a way to facilitate research into the complex mechanisms of gene products in vivo. However, in several cases,multiple alleles of certain gene products have been generated by different laboratories and have resulted in crucial observations alluding to both the power of gene targeting as well as the variability that can result among different targeting events at a single gene locus. One such case emerges from targeting at the cystic fibrosis transmembrane-conductance regulator (CFTR), a CAMP-induced chloride channel located on the apical membrane of secretory epithelial cells (60.61). In humans, defects in the CFTR lead to cystic fibrosis (CF), an autosomal recessive disorder characterized by

Bunger and Bradfield

454 Table 1 Classes of Mutations and Their Affected Processes at Human Cftr Locus Class Class Class Class Class Class

Affected process I II III IV V

Biosynthesis Protein processing Channel regulation Channel conductance Reduced synthesis

chronic lung obstruction, pancreatic-enzyme deficiency, and neonatal mtestinal blockage. CF is one of the most common genetic disorders in humans, affecting roughly 1 m 2500 newborns (28,62). Clmical analysis shows that 100% of CF patients exhibit lung pathology, whereas 85-90% show additional pancreatic-enzyme deficiency, and 10% also exhibit neonatal mtestmal blockage (28,62). Analysis of the human Cftr locus has led to the observation that more than 500 different alleles exist, including large gene deletions, single ammo acid deletions, aberrant splicing, and nonsense and missense mutations. Many of these distinct mutations lead to some form of clmical cystic fibrosis (62). In humans, mutations at the Cftr locus have been divided into five general classes based on their molecular consequences and are described m Table 1 (62). It is estimated that 50% of the mutations leading to cystic fibrosis result m the expression of an aberrant gene product rather than giving rise to a null allele (28). In an effort to generate models to aid m the design of therapeutic strategies to lrmtt the progression of CF, a number of targeted mutations at the Cft locus have been introduced mto the murine germ line. To date, there have been seven such mouse models for CF generated by different targeting strategies at the Cftr locus (.51,55,63-6 7). Common to all of these mouse models IS a reduction or absence of chloride-channel conductance m isolated epithehal cells. Despite similar experimental

ObJectwes, each targeting

strategy has resulted m a mouse

model displaying significantly differing mcidences of intestinal obstruction, pancreatic pathology, and perinatal lethality (Table 2). Four gross disruptions of the Cftr locus, denoted cftF1cAM, cftFIEDN, cfttr”*BAy, and cftrm’uNc accordmg to the originatmg institution, have been generated by either targeted msertion or replacement at the Cftr locus (63,65,66,68). In all four cases, the marker gene remained inserted in the locus. Ratcliff et al. (68) and Snouwaert et al.

Table 2 Mouse Models

of Cystic Fibrosis

Mouse model

Generated

by Gene Targeting

Mutational strategy

in ES Cells Phenotype

Class I/Nulls cftP'UNC

Replacement-type

cfttr"'EDN

Insertion-type

cftP'cAM

Replacement-type

cfttr"lBAY

Insertion-type

gene targeting, exon 10

gene targeting, exon 10 gene targeting, exon 10

gene targeting, exon 3

Mild respuatory tract, pancreas, and gall bladder pathology; intestinal obstruction; permatal mortahty (90% by PND30) (65) Defective chlorrde-ion transport, mrld respiratory-tract and mtestmal pathology; no permatal mortal@ (63) Defective chloride-ion transport; pancreatic and respiratory-tract pathology; intestmal obstructton; permatal mortahty (80% by PNDS) (64). Defective chloride-ion transport; pancreatrc and respiratory-tract pathology; intestmal obstruction; perinatal mortality (40% by PND7) (66)

Class II @AFSOSCAM

Single-step cotransfer, AF508

+AFSOSNET

Hit and run, AF508

Class III cj-tP5’D

Single-step cotransfer, G55 1D

PND = post natal day

Defective chloride-ton transport; pancreatic and respiratory-tract pathology; intestinal obstructron, pennatal mortality (35% by PND 16) (67). Reduced chloride-ton transport, mild respiratory-tract pathology, mcomplete mtestmal obstruction, no permatal mortality (51) Defective chloride-ion transport, no respiratory-tract pathology, mtestmal obstructton, permatal mortahty (35% by PND 35) (55)

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(65) disrupted exon 10 using a replacement-type gene targeting usmg Hprt or Neo markers, respectively. Dorm et al. (63) and O’Neal et al. (66) used msertional-type gene targeting to introduce stop codons in exons 10 and 3, respectively. The molecular basis for the phenotypic differences that resulted from these independent targeting events is not entirely understood, although alternative splicing around the Neo marker of the cftrEDN allele has been shown to produce a low level of CFTR protein capable of wlldtype activity and may explain why this mouse model displayed the mildest phenotype of the four (69). Introduction of subtle mutations at Cftr may provide a more appropriate model of human CF. Almost 70% of CF in humans is the result of a deletion at residue 508 (AF508) of the CFTR protein, The AF508 mutation is classified as a class II mutation because it results m abnormal cellular processmg of CFTR and degradation of the protein before it reaches its final destination on the apical plasma membrane surface. Two groups have introduced specific mutations to mimic the human AF508 through two of the techniques described above (Table 2) (51,67). The AF508 mutation has been shown to yield a temperature sensitive protein product that is capable of functionmg normally when cells are grown at the permissive temperature, 27°C. Fmdmg ways to mimic the phenotype associated with the permissive temperature in vivo could be a therapeutic breakthrough. In order to generate mouse models to enable the design of therapies to overcome the AF508 mutation phenotype, van Doommck et al. (51) and Colledge et al. (67) used the hit-and-run technique and single step cotransfer technique, respectively, to introduce the AF508 mutation mto the mouse genome. The resultant mice contain a deletion of the phenylalanme residue at position 508, but produce normal-length transcripts from the Cftr locus. Furthermore, isolated tracheal epithelia cells from both of these mice exhibited the abihty to express functional CFTR when grown at 27OC (67,70). Despite the similarity of these two targeted alleles, subtle differences m the phenotypes of the two mouse models remain. The cj?@jo8 mouse generated by single-step cotransfer technique also resulted in an msertion of the Hprt mmigene into the mtron downstream of exon 10. The resultant mice displayed a phenotype close to that of mice with disrupted Cftr alleles described above, including reduced CFTR mRNA amounts, a high rate of intestinal blockage, and perinatal mortality. The cftf1508 mouse generated by hit and run resulted in normal transcript levels and absence of the gross intestinal defects and perinatal lethality seen in the other CF mice described. The mechanism for these differences has not yet been explained. A second common mutation represented in roughly 3% of CF patients is the G55 1D mutation, a class III mutation that allows normal processmg of the gene product, but results m a chloride channel that is nonresponsive to CAMP

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induction (62). Compared with patients that carry the AF508 mutation, G55 1D patients show a threefold reduction in mcidence of neonatal mtestmal obstruction. A mouse model of the G55 1D mutation would be useful as a tool m the design of therapeutic strategies and to address questions regarding genotype/ phenotype correlations. Delaney et al. have generated such a mouse using the single-step cotransfer method where two Neo selection markers were Inserted mto intronic sequence near the site of the G551D mutation (55). These mice exhibited reduced amounts of CFTR mRNA, a high incidence of perinatal lethality due to intestinal obstruction, and absence of pathology m the lungs and pancreas. Interestmgly, this allele produces a full-length transcript that is expressed and processed correctly and the protein product properly traffics to the plasma membrane. The value of generating the cfiF508 and the cftrGS51D mouse models of CF m addition to Cftr nulls is not only to provide mouse models that may facilitate drug design, but also serves to provide a measure of reproducibility for different targeting strategies. Finally, although targeting strategy is an important contrtbutor to differential phenotypes m related murine models, tt IS important to point out that the differences in the phenotypes are not solely caused by different mutant alleles. Genetic background of the mice and their housmg environments are important factors to be considered when addressing the genotype/phenotype correlation for each mutation. Furthermore, the validity of therapeutic strategies resulting from use of these CF mouse models relies on resolution of the mechanism underlying the phenotypic differences among them. 4. Examples from the P450 Field Many of the observations made m the studies of the Cftr locus are reiterated by studies of null alleles of proteins important for cytochrome P450 functton and expression, One inference that is drawn from multiple-targeting experiments at the Cftr locus is that null alleles developed in different laboratories often have surprisingly different phenotypes. It is important to reemphasize that these differences may be allelic m nature, may result from cu-effects caused by the introduction of a marker gene into the locus, may be because of different genetic background, or may be caused by environmental factors. However, it is also important to emphasize that these apparent discrepancies do not necessarily represent experimental error and that it may be useful to think of the resultant phenotypic dtfferences as opportunities to understand the underlying biology. Several examples from the recent literature demonstrate that the influences of genetic background can play an important role in determining phenotypes of null alleles (71-73). Examination of the influences of genetic background by determining correlations between genetic polymorphisms and particular phenotypes of a null allele provides a powerful tool for the identifi-

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cation of interacting loci. Genettc background constderations are parttcularly relevant for the P450 field because of the role P45Osplay in the bioactivation of toxins and procarcmogens, to which susceptibthty among mouse strains 1s highly polymorphtc. Moreover, multiple null alleles of P450-encoding loci can provide direct evidence to the contribution of and functional redundancy among P450 isoforms in relationship to normal phystology and xenobiotic metabolism. The CYPlA2 and CYP2El cytochrome P450 enzymes have been impltcated in the metabohsm of many commonly used drugs and ubiquitous envlronmental toxtcants. The CYPlA2 isoform has a wide spectrum of catalytic activities, ranging from the hydroxylation of the environmental carcmogens benzo(a)pyrene and aflatoxin-Bl to the oxidatton of endogenous estrogens (74,75). The CYPlA2 lsoform is constrtutively expressed in a number of tissues durmg development but is also inducible by xenobiotics that activate the aryl hydrocarbon receptor (AHR) (75,76). Two null alleles at the Cypla2 locus have been generated by independent laboratortes and these alleles display markedly different phenotypes. One Cypla2 null allele was generated by a positive-negative-selection approach that used Neo to disrupt the first codmg exon of the Cypla2 structural gene. This allele resulted in neonatal lethality, usually within 15-45 min after birth, because of respiratory distress (77). The lethal phenotype, however, was not completely penetrant as a small number of the Cypla2c-/-) survivors displayed no apparent abnormalities. Interestingly, another Cypla2 null allele that replaced exons 3, 4, and 5 m addition to disrupting exon 2 with an Hprt mimgene, resulted m a null line in which homozygous nulls developed normally (78). The reason for the difference m phenotype between these two mutant alleles is not known, but the incomplete penetrance of the lethal phenotype in one of the nulls implies the mvolvement of other 10~1.As mentioned throughout this chapter, these phenotyptc discrepancies could also be the result of true allehc differences resulting from unique targetmg strategies or regulatory effects of the marker genes on neighboring alleles. Additional differences m housing condttions, mcludmg pathogens present m mdtvtdual housmg facihttes, cannot be ruled out. Understanding the contrtbunon of these variables to the dissimilar Cypla2 null phenotypes may shed light on the biology of the CypIa2 locus. The CYP2E 1 enzyme is inducible upon ethanol treatment or in response to starvation and is capable of metabohzmg substrates such as acetaminophen, dimethyl sulfoxide, vinyl chloride, and ethylene glycol (79). Addtttonally, evidence exists to suggest that CYP2El is also capable of metabolizmg endogenous ketone bodies (SO). A null allele of Cyp2el was generated by positive-negative selection in ES cells using a construct that replaced exon 2 in the Cyp2el structural gene (81). The resultant homozygous null mice developed normally but dtsplayed altered metabohsm toward CYP2El substrates

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such as acetaminophen and benzene (81,82). Although no surprismg developmental phenotypes resulted from generating the null allele of Cypdel, the resulting mouse model has utility m the study of the biological effects and interactions of drugs that mduce CYP2El or are metabolized by CYP2EImedlated mechanisms. The AHR and the aryl hydrocarbon receptor nuclear translocator (ARNT) proteins are members of the PAS superfamily of protems. In response to exogenous hgands such as dloxms and polychlorinated blphenyls, the activated AHIUARNT heterodimer regulates the expression of a number of xenoblotlcmetabohzmg enzymes, including CYPl Al, CYPlBl, and CYPlA2 (reviewed in ref. 83). Genetic evidence in naturally occurring murine strains polymorphlc at the Ahr locus demonstrates that the AHR mediates most, If not all, toxic effects of dioxins and related compounds (84). Cell culture, yeast and in vitro models of AHIUARNT signaling have been crucial to our understandmg of the transcriptional responses to dioxin exposure (reviewed in ref. 85). However, because many aspectsof dioxin toxicity appear to occur only m whole animals, simpler model systems have afforded only a limited understanding of the biology of the AHR and ARNT and their respecttve roles m the plelotropic effects of dioxins In addition, cell culture and m vitro models are of hmited utlllty m our attempts to understand whether the AHR or ARNT play Important roles in mammalian development. In an attempt to extend our understanding of this topic, null alleles have been generated at both the Ahr and Arnt 10~1.The phenotypes resulting from null alleles at Ahr included a 25 to 40% decrease in relative liver weight as well as a subtle portal fibrosis. These results suggest that the AHR plays an important role m normal liver function and development and provide some of the most compelling evidence yet that the AHR possessesphysiological Importance that extends beyond its role in an adaptive response to xenobiotlc exposure. In addition, this biological relationship may have uncovered a potential target pathway for dioxin-mduced toxicity. Finally, although experimental evidence from AHR null animals treated with 2,3,7,8 tetrachlorodlbenzo-pdioxin (TCDD) restates the importance of AHR-mediated signaling in toxicity, these studies may have uncovered a novel AHR-independent lung toxlclty that occurs at very high doses of TCDD (86,87). Results from targeting at the Ahr locus also repeat a familiar theme that null alleles generated by different strategies do not always generate identical phenotypes (88,89). The null allele generated by positive-negative selection at exon 1 (designated Ahr A1’A1)displayed a high incidence of neonatal lethality because of defects m the immune system, including decreased splenic and peripheral lymphocytes, durmg the first 10-12 wk of life. These mice also displayed pronounced hepatlc fibrosis that may have compromised normal liver

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functron (86,88,90). In contrast, the Ahr null allele generated by positive-negative selection to replace exon 2 (destgnatedAhP2’A2), which codes for the basic region of the AHR, dtsplayed a temporal hepatocyte mtcrovestcular steatosts in the first 3 wk postpartum that completely resolved by 3 mo (89). Other than a reduced relative liver weight that persisted throughout development, the Ahp21A2mice developed normally, displayed no defictts m lymphocyte populations, and no lethality was associated with the null allele. As an explanation of these disparate phenotypes, differences m environment and c&effects resulting from the targeting event should be considered. One of the most interesting variables that may explain these different phenotypes is genetic background. Although both null alleles were generated in ES cells derived from 129 strain mice, the tndtvtdual cell lines, designated Jl and Rl, used for the generation of the Ah#“A’and Ah#2/A2alleles, respectively, are genetically different (91). Independent segregation of alleles resulting from crosses of the chimeras with C57BL/6 mice and the subsequent brother-sister matings to generate the homozygous null will generate a number of founders, all of which will be genetically drstmct at a number of loct other than Ahr. The most common way to eliminate this vartabthty is to backcross onto a defined genetic background for at least 10 generations to produce a congenic line, or to perform multigeneration brother-sister matings to develop a novel rsogenic line. Grven these limitations, early characterization of any null allele is compromised by this genetic variability. This variability 1sexacerbated when mdividual laboratories back-cross nulls onto different genetic backgrounds and in different environments. Until these mice are directly compared on similar genetic backgrounds in the same environment, tt will be difficult to determine the exact nature of the phenotypic differences between the Ahr null alleles. More importantly, if appropriate genetic experiments demonstrate that genetic background is a factor, this will suggest that addittonal loci are mvolved m mediating AHR signaling and provide a genetic approach to identify those genes. Indeed, early genetic studies involving aryl hydrocarbon hydroxylase response to 3-methylcholanthrene suggest that other loci are crucial for thts signaling pathway (92). A null allele at the Aunt locus has also provided significant insights into the biology of the encoded protem. The ARNT protein is crucial to hgand-induced signaling through the AHR. As the Arnt null allele was being generated, a series of experiments indicated that the ARNT protein also forms a transcripttonally active heterodimer with hypoxia inducible factor 1a (HIF 1a) and that this complex is upregulated m response to hypoxia (93,941 The ARNT/HIFl a heterodtmer has been shown to upregulate genes encoding a number of factors important m cellular and organismal responses to low oxygen, such as vascular endothehal growth factor (VEGF) and erythropoietm (95). Null alleles at the

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Aunt locus display an embryonic lethal phenotype resulting from aberrant vasculogenesis of the yolk-sac, with all embryos dying between embryonic day 9.5 and 10.5. This phenotype is similar to that observed m Vefl/-, and TFcd+ mice (96-99). Although the Arnt(-/-) phenotype has provrded valuable insights mto the role of ARNT in hypoxia signaling and fetal development, the lethal phenotype severely limits the utility of these mice to discern physiological roles of ARNT later in development or the role that ARNT plays in AHR signaling. Thus, the ARNT story provides an excellent example in which generation of an inducible null allele or tissue-specific null alleles of Arnt via the Cre-LoxP system will be necessary to facihtate studies into the nature of ARNT signaling in xenoblotic metabolism and mammahan development. In addition, targeting at the Arnt locus points to the versatility of using double-knockout ES cells. Experiments in ARNT(-/-) ES cells generated by the step-up procedure revealed new roles for ARNT in modulating glycolytic pathways m addition to its known role in transducing hypoxia and dioxin-responsive signaling (99). 5. Conclusions The generation of targeted mutations m pluripotent embryonic stem cells is a powerful tool in creating allelic variants of critical gene products. It is hoped that such alleles will allow the study of mutant gene products in vivo and provide a pathway to establish mouse models of human disease.Before the advent of gene targeting in ES cells, generation of allelic variations m mice relied on spontaneous or chemically induced mutations. The use of gene targeting can complement these earlier approaches by allowing generation and study of predetermined mutations m the mouse genome. The design of the targeting construct and choice of strategy can be viewed as the most critical steps m generating genetically engineered animal models by the methods described above. The use of marker genes as a way of enriching populations of cells containmg the correctly targeted mutation has emerged as a powerful tool but also the source of a host of potential complicatrons when interpreting genotype/phenotype correlatrons. Thus, many of the approaches described here are designed to result in a marker-free, mutant locus, alleviating many of the phenotypic ambiguities resulting from the presence of marker genes and them cis elements within the genome. The Cre-loxP recombination system is but one of the many strategies that can provide a route to a nearly clean targeting event and allow the recycling of selection markers m the generation of double-knockout ES cells. The power of these techniques to generate informative allelic series at individual loci has implications not only to provide in viva models for deciphering mechanistic properties of complex biological pathways, but also to provide models for testing therapeutic strategies relating to human disease and metabolism.

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Acknowledgments We thank Jennifer Schmidt, John Petrim, and members of the Bradfield Lab for technical insight and review of the manuscript. This work was supported by grants from the National Institutes of Health (ES 06883, ES07015, and CA07175) and The Burroughs Wellcome Foundation. References 1. Smtthtes, O., Gregg, R G , Boggs, S S , Koralewski, M. A., and Kucherlapati, R S (1985) Insertion of DNA sequences mto the human chromosomal beta-globm locus by homologous recombinatton Nature 317,230-234. 2. Hooper, M., Hardy, K., Handyside, A , Hunter, S., and Monk, M (1987) HPRTdeficient (Lesch-Nyhan) mouse embryos derived from germlme colomzation by cultured cells. Nature 326,292-295 3 Capeccht, M. (1989) Altering the genqme by homologous recombmation. Sczence 244, 1288-1292. 4 Keller, B. H , Hagemann, L J , Doetschman, T , Hagaman, J R , Huang, S , Wilhams, P J., Fnst, N L , Maeda, N., and Smithies, 0. (1989) Germ-line transmission of a planned alteratton made m a hypoxanthme phosphoribosyltransferase gene by homologous recombination m embryomc stem cells. Proc Nat1 Acad Scr USA 86,8927-893 1. 5. Sedivy, J M and Joyner, A M (1992) Germline gene targeting m mice, m Gene Targeting. W. H Freeman, New York, pp. 146-149. 6. Hogan, B., Beddmgton, R , Constantmt, F , and Lacy, E (1994) Manipulatmg the Mouse Embryo: A Laboratory Manual, 2nd ed Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 7. Wasserman, P. M and DePamphths, M. L. (1993) Guide to techniques in mouse development, m Methods Enzymol , vol 225 (Abelson, J. N. and Simon, M I , eds.), Academic, New York, pp. 855-877. 8 Hooper, M. L (1992) Embryonal Stem Cells. Introducing Planned Changes into the Animal Germllne Hardwood Academic Publishers, Chur, Swttzerland. 9. Galli-Taliadoros, L A., Sedgwick, J D., Wood, S. A, and Korner, H. (1995) Gene knock-out technology. a methodological overvtew for the interested novice. J. Immunol. Methods 181, 1-15 10. Bradley, A., Evans, M., Kaufman, M H , and Robertson, E. (1984) Formation of germ-lme chimaeras from embryo-derived teratocarcmoma cell lines Nature 309,255-256. 11 Ramirez-Sohs, R., Davis, A. C., and Bradley, A. (1993) Gene targeting m embryonic stem cells.Methods Enzymol 225, 855-878. 12. Hasty, P., Rivera-Perez, J , Chang, C , and Bradley, A (1991) Target frequency

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