ALTERNATIVE TOXICOLOGICAL METHODS
ALTERNATIVE TOXICOLOGICAL METHODS
Edited by
Harry Salem Sidney A. Katz
CRC PR E ...
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ALTERNATIVE TOXICOLOGICAL METHODS
ALTERNATIVE TOXICOLOGICAL METHODS
Edited by
Harry Salem Sidney A. Katz
CRC PR E S S Boca Raton London New York Washington, D.C.
This edition published in the Taylor & Francis e-Library, 2005. “To purchase your own copy of this or any of Taylor & Francis or Routledge’s collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.”
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Historical Developments in the Humane Care and Use of Research Animals: The First 4000 Years +DUU\6DOHPDQG6LGQH\$.DW]
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Table 1.1 Chronology for the Enactment of Animal Welfare Legislation Year
State
Year
State
Year
State
1828 1835 1838 1938 1842 1845 1848 1851 1851 1852 1854 1856 1857 1858 1859 1859 1860 1861
New York Massachusetts Connneticut Wisconsin New Hampshire Missouri Virginia Iowa Minnesota Kentucky Vermont Texas Rhode Island Tennessee Kansas Washington Pennsylvania Nevada
1864 1864 1867 1868 1868 1869 1871 1871 1871 1872 1873 1873 1873 1875 1879 1879 1880
Idaho Oregon New Jersey California West Virginia Illinois District of Columbia Michigan Montana Colorado Delaware Indiana Nebraska Georgia Arkansas Louisiana Mississippi
1880 1881 1881 1883 1883 1884 1887 1887 1889 1890 1891 1893 1895 1898 1913 1913 1921
Ohio North Carolina South Carolina Alabama Maine Hawaii New Mexico South Dakota Florida Maryland North Dakota Oklahoma Wyoming Utah Alaska Arizona Virgin Islands
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THE FIRST 4000 YEARS
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THE FIRST 4000 YEARS
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REFERENCES $QLPDO:HOIDUH,QVWLWXWH $QLPDOVDQG7KHLU/HJDO5LJKWV $6XUYH\RI$PHULFDQ/DZV IURPWR $QLPDO:HOIDUH,QVWLWXWH:DVKLQJWRQ'&S )\H:% 3URÀOHVLQFDUGLRORJ\ 0DUVKDOO+DOO&OLQ&DUGLRO ² 5XVVHOO:06DQG%XUFK5/ 7KH3ULQFLSOHVRI+XPDQH([SHULPHQWDO7HFKQLTXHV 0HWKXHQ/RQGRQ
CHAPTER
2
A History of Interagency Approaches to Alternatives and Establishment of the Interagency Regulatory Alternatives Group 6LGQH\*UHHQ
CONTENTS ,QWURGXFWLRQ (VWDEOLVKPHQWRI,5$* ,5$*:RUNVKRSV (YROXWLRQRI,&&9$0 5HIHUHQFHV
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The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM): Recent Progress in the Evaluation of Alternative Toxicity Testing Methods :LOOLDP66WRNHV
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ALTERNATIVE TOXICOLOGICAL METHODS
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BACKGROUND AND HISTORY OF THE ICCVAM 7KH1DWLRQDO,QVWLWXWHVRI+HDOWK 1,+ 5HYLWDOL]DWLRQ$FWRI 3XEOLF/DZ GLUHFWHGWKH1DWLRQDO,QVWLWXWHRI(QYLURQPHQWDO+HDOWK6FLHQFHV 1,(+6 WRHVWDEOLVKFULWHULDIRUWKHYDOLGDWLRQDQGUHJXODWRU\DFFHSWDQFHRIDOWHUQDWLYHWHVWLQJ PHWKRGVDQGWRGHYHORSDSURFHVVE\ZKLFKVFLHQWLÀ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,&&9$0 ,QGHYHORSLQJLWVUHSRUWWKHFRPPLWWHHVRXJKWEURDGLQSXW DQGSDUWLFLSDWLRQIURPLQWHUHVWHGVWDNHKROGHUVLQFOXGLQJLQGXVWU\DFDGHPLDDQLPDO ZHOIDUH RUJDQL]DWLRQV DQG WKH LQWHUQDWLRQDO FRPPXQLW\ 7KH DG KRF ,&&9$0 Table 3.1 Member Agencies Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) Consumer Product Safety Commission Department of Defense Department of Energy Department of Health and Human Services Agency for Toxic Substances and Disease Registry Food and Drug Administration National Institute for Occupational Safety and Health National Institutes of Health, Office of the Director National Cancer Institute National Institute of Environmental Health Sciences National Library of Medicine Department of the Interior Department of Labor Occupational Safety and Health Administration Department of Transportation Research and Special Programs Administration Department of Agriculture Environmental Protection Agency Table 3.2 Test Method Validation and Acceptance Criteriaa Validation Criteria Clear statement of proposed use Biological basis/relationship to effect of interest provided Formal detailed protocol Reliability assessed Relevance assessed Limitations described All data available for review Data quality: Ideally good laboratory practices (GLPs) Independent scientific peer review Acceptance Criteria Fits into the regulatory testing structure Adequately predicts the toxic endpoint of interest Generates data useful for risk assessment Adequate data available for specified uses Robust and transferable Time and cost effective Adequate animal welfare consideration (3 Rs) a
These are shortened versions of the adopted criteria. For the full text see: National Institute of Environmental Health Sciences (NIEHS), Validation and Regulatory Acceptance of Toxicological Test Methods: A Report of the ad hoc Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), NIH publication 97-3981, Research Triangle Park, NC, 1997.
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ICCVAM Authorization Act (U.S. Code, 2000).
ICCVAM EVALUATION OF ALTERNATIVE METHODS
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Table 3.4 The Duties of the ICCVAMa Consider petitions from the public for review and evaluation of new and revised test methods for which there is evidence of scientific validity Coordinate the technical review and evaluation of new and revised test methods of interagency interest Submit ICCVAM test recommendations to each appropriate federal agency Facilitate and provide guidance on validation criteria and processes Facilitate: Interagency and international harmonization of test protocols that encourage the reduction, refinement, and replacement of animal test methods Acceptance of scientifically valid test methods and awareness of accepted methods Make ICCVAM final test recommendations and agency responses available to the public Prepare reports on the progress of this act and make these available to the public a
ICCVAM Authorization Act (U.S. Code, 2000).
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Test Sponsor Submission of Test Method
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Figure 3.1
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1. Experimental
2. Modeling
3. Validation (literature data)
in vitro data on kinetics
kinetic modeling
kinetics in vivo
in vitro data on dynamics
prediction of target tissue
(e.g., CNC, EC20, EC50)
45
concentrations (e.g., NOEL, LOEL) prediction of dynamics
in vivo systemic
prediction of systemic
Figure 5.1
toxic doses
toxic doses
(e.g., NOED, LOED,
(e.g., NOED, LOED,
LD50)
LD50)
Building blocks of the ECITTS scheme.
Compounds and Test Battery (LJKW ZHOOFKDUDFWHUL]HG WHVW FRPSRXQGV ZKLFK SRVVHVV DFWLYLW\ LQ WKH FHQWUDO RU SHULSKHUDO QHUYRXV V\VWHPV ZHUH VHOHFWHG 7KHVH ZHUH DFU\ODPLGH FDIIHLQH GLD]HSDP QKH[DQHKH[DQHGLRQH OLQGDQH SDUDWKLRQSDUDR[RQ SKHQ\WRLQ DQG WROXHQH $Q LQ YLWUR WHVW EDWWHU\ ZLWK JHQHUDO DQG QHXURVSHFLÀF HQGSRLQWV ZDV GHVLJQHGLQRUGHUWRFRYHUHQGSRLQWVIRUEDVDOF\WRWR[LFLW\PRUSKRORJ\SK\VLRORJ\ DQGQHXURFKHPLVWU\ :DOXPHWDO )RUVE\HWDO 7KH KXPDQ QHXUREODVWRPD FHOO OLQH 6+6<< KDV EHHQ XVHG H[WHQVLYHO\ DV D PRGHOIRUV\PSDWKHWLFJDQJOLRQFHOOV 5RVVHWDO DQGZDVFKRVHQIRUWKHVH VWXGLHV7KHFHOOVFDQEHIXUWKHUGLIIHUHQWLDWHGZKHQFXOWXUHGLQVHUXPIUHHPHGLXP ZLWKUHWLQRLFDFLG 15$ ZKLFKLQGXFHVWKHFHOOVWRGHYHORSDQH[WHQVLYHQHWZRUN RI QHXULWHV 3nKOPDQ HW DO )LJXUH )XUWKHUPRUH WKH H[FLWDELOLW\ DQG
a Figure 5.2
b
(a) Native and (b) retinoic acid-differentiated SH-SY5Y cells.
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ALTERNATIVE TOXICOLOGICAL METHODS
Table 5.1 Endpoints Studied in the in Vitro Neurotoxicity Test Battery for the ECITTS Project Endpoint Cytotoxicity/Inhibition of cell growth Neurite degeneration (ND) Protein synthesis rate (PSR) Basal intracellular free Ca2+ concentration (basal Ca2+) Voltage operated Ca2+ channels (VOCC) Phospholipase C-coupled acetylcholine receptor signal transduction (mAChR peak) Acetylcholine-induced capacitive Ca2+ entry (mAChR plateau)
Assay
Toxicity level
Total cellular protein content Number of neurites per cell [3H] leucine incorporation in proteins during 2 hr Fura-2/Ca2+, fluorescence
Basal cytotoxicity Morphology Physiology
High potassium-induced Ca2+ flux, fluorescence Carbachol-activated, immediate transient Ca2+ peak, fluorescence Carbachol-activated, secondary Ca2+ plateau, fluorescence
Neurochemistry
Physiology
Neurochemistry
Neurochemistry
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Table 5.2 Effects of the Test Compounds, Determined in Differentiated Human Neuroblastoma SH-SY5Y Cells, on in Vitro Endpoints as Presented in Table 5.1 Compound Acrylamide Acrylamide Caffeine Diazepam Lindane Lindane Lindane Parathion/paraoxon Phenytoin
In vitro effect
Concentration QM or QM/hr) (Q
20% cytotoxicity CNC; neurite degeneration/time CNC; inhibition of VOCC CNC; inhibition of VOCC CNC; inhibition of VOCC 20% increased basal [Ca2+]i 50% cytotoxicity CNC; neurite degeneration/time CNC; inhibition of protein synthesis
920 11 10 49 3.4 35 150 0.5 87
CNC, critical cellular neurotoxic concentration; VOCC, voltage operated calcium channels.
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ALTERNATIVE TOXICOLOGICAL METHODS
Estimated doses (mg/kg) or (mg/kg/day)
100.0
10.0
10
1
0.1
0.01
0.001 0.001
0.01
0.1
1
10
100
1000
Experimental doses (mg/kg) or (mg/kg/day) Lindane (CNC: inhibition of VOCC vs. LOED: learning; s.c.) Lindane (EC20: increased basal Ca2+ vs. LOED: convulsions; s.c.) Lindane (50% cytotoxicity vs. lowest LD50; a.) Lindane (50% cytotoxicity vs. highest LD50; a.) Acrylamide (20% cytotoxicity vs. LOED: gait; a.) Acrylamide (CNC: ND vs. LOED: startle response; 10 days) Acrylamide (CNC: ND vs. LOED: startle response; 30 days) Acrylamide (CNC: ND vs. LOED: startle response; 90 days) Caffeine (CNC: inhibited VOCC vs. LOED: anti-nociception; a.) Diazepam (CNC: inhibited VOCC vs. LOED: time to emerge; a.) Phenytoin (CNC: inhibition of PSR vs. operant learning; a.) Parathion/paraoxon (CNC of paraoxon: ND vs. LOED: tail-pinch response of parathion; a.)
Figure 5.3
Estimated versus experimental doses after (a) acute and/or (s.c.) subchronic exposure. See Table 5.1 for endpoint definitions. The line represents the identity.
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Summary of the OECD’s New Guidance Document on the Recognition, Assessment, and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation (UURO=HLJHU
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Table 6.1 Common Conditions and Clinical Signs Abdominal rigidity Abortion Agalactia Anemia Analgesia Anuria Apathy Ataxia/incoordination Bleeding Blepharospasm Blood in feces or urine Blood around nose, eyes Boarded abdomen Body temperature, abnormal Body weight loss or emaciation Breathing difficulties (Dyspnea) Cachexia Chewing, persistent Chromodachryorrhea Circling Comatose Compulsive behavior Constipation Convulsions Corneal ulceration Coughing/sneezing Cyanosis Dehydration Diarrhea
Discharge, abnormal Dyspnea (difficult breathing) Epistaxis (nasal bleeding) Excitable Eyelid closure Eyes fixed/sunken Fractured bone Gasping Grooming—failure to do Hunched/stiff posture Hyperreflexia Immobile/inactive Jaundice (icterus) Joints swollen Kyphosis Lateral position Limping/lameness Locomotory behavior Lordosis Loss of condition, body muscle Mammary gland abnormalities Moribund Motor excitation Not eating/drinking Oedema Pale mucous membranes Paralysis Paresis Piloerection
Pinna reflex Prostrate Pruritis Pupillary constriction/dilation Rales, pulmonary Rectal prolapse Recumbency, prolonged Red eye(s)/nose Reflexes Retention of feces Righting reflex Salivation, excessive or abnormal Seizures Self-mutilation Skin bruising/color/crepitus Spasm Staggering Sunken flanks Suppuration Swellings Tenesmus Tetany Tremor Urine retention Vaginal prolapse Vocalization Vomiting
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Table 7.1 Models and Areas of Research and Specific Techniques That Cause Distress Research Models or Areas Non-Pain-Induced Distress Aggression Anxiety Cancer Depression Diabetes Drug addiction and withdrawal Environmental stress Fear Immunological research Infectious disease Motion sickness Nutrition research Panic Pharmacology (some) Psychopathology Radiation research Stress (psychological) Toxicology (induced effects) Transgenic research
(e.g., Vogel conflict-drinking model) (tumor burden, cachexia, carcinogenicity testing) (e.g., learned helplessness, forced swimming, maternal deprivation)
(e.g., hot, cold) (e.g., vaccine potency testing)
(e.g., nutrient deprivation) (e.g., tumor necrosis factor, capsaicin research) (other than anxiety, depression, fear, etc., mentioned above)
Pain-Induced Distress Arthritis Burn research Cancer research Chronic pain studies1 Dental studies Inflammation studies Experimental surgery Muricide Orthopedic studies Trauma research
(tumor pain)
(e.g., organ transplantation/rejection) (as a model of aggression, neophobia, etc.)
Specific Techniques Non-Pain-Induced and Pain-Induced Distress Anesthesia aftereffects Antibody production Aversive stimuli Bleeding techniques Complete Freund’s Adjuvant Control group Deprivation Dosing techniques Granuloma techniques Gut loop studies
(polyclonal and monoclonal) (e.g., electric shock) (including retro-orbital bleeding) (animals denied experimental treatments) (e.g., water, food, sleep, or social partners/experiences) (e.g., gavage)
(continued)
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ALTERNATIVE TOXICOLOGICAL METHODS
Table 7.1 (continued) Models and Areas of Research and Specific Techniques That Cause Distress Knockout technology Restraint Surgery sequelae 1
Acute pain should not be a problem if the guidelines of the International Association for the Study of Pain (IASP, 1979) are followed.
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Table 7.2 The USDA’s Current Pain and Distress Categories and a Proposed Modification, as Well as Related Features of the Two Systems A. Current Scheme USDA Category C (63%)1 D (29%) E (8%)
Pain and/or Distress Little or None Yes or No2 Yes
Anesthesia/ Analgesia
Full ACUC Review
Alternative Literature Search
No Yes No
Yes Yes Yes
No Yes Yes
B. Proposed Scheme Category I II III IV 1 2
Pain and/or Distress
Anesthesia/ Analgesia
Full IACUC Review
Alternative Literature Search
Minor or None Minor or None Moderate Severe
No Yes Yes or No Yes or No
No Perhaps Yes Yes
No Perhaps Yes Yes
Numbers in parentheses are USDA figures for 2000. Animals listed in USDA category D were given pain- or distress-relieving drugs, but these drugs may not have been sufficient to relieve all pain and distress throughout the experiment.
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Table 7.3 State to State Variation in Reporting Animal Use in Column E (Unalleviated Pain or Distress) for States Using Greater Than 20,000 Animals
State Nationwide California Delaware Georgia Illinois Indiana Iowa Kansas Maryland Massachusetts Michigan Minnesota
Percentage of Animals in Column E 8 4 15 2 3 18 28 16 12 1 15 3
Percentage of Animals in Column E
State Missouri Nebraska New Jersey New York North Carolina Ohio Pennsylvania Texas Virginia Washington Wisconsin Federal Agencies
22 15 6 11 6 3 8 3 0 20 9 7
Note: USDA data from 2000. Table 7.4 States That Reported Less Than 1% of Animal Use in Column E between 1995 and 1997 Alaska Arizona Hawaii Kentucky Louisiana Maine
(300) (5,000) (500) (5,300) (16,800) (800)
Mississippi Nevada Oklahoma Oregon Rhode Island S. Carolina
(2,000) (3,000) (4,300) (4,700) (2,100) (6,100)
Tennessee Utah Vermont Virginia West Virginia Wyoming
(10,900) (4,600) (1,100) (19,200) (1,700) (300)
Data from the USDA. Figures in parentheses indicate the average number of animals used across all pain categories.
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Table 7.5 Selected Elements of Institutional Policies on Monoclonal Antibody Production Available on the World Wide Web Penn State Monitoring subj. with solid tumors Priming Number of taps Monitoring postinoculation Replacement fluid after ascites harvest Anesthesia during tap
Stanford
U Iowa
U Minnesota
Not specified
3v/week
Not specified
3v/week
As low as 0.1 ml pristane Max 3 taps, last terminal Daily
Not specified
0.2 ml max pristane 2 taps, last after euthanasia Daily
0.5 ml max pristane Not specified
1–2 ml of saline subcutaneous
Not specified
Not specified
Not specified
Not specified
Anesthesia can be used
Not specified 3v/week for first week, then daily Not specified
Anesthesia used for new personnel
Daily
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CHAPTER
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Meeting Industry and Regulatory Needs for Alternative Test Methods to the Draize Rabbit Eye Irritation Test 6KHUU\/:DUG
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The Ocular Surface: Barrier Function and Mechanisms of Injury and Repair 0DUWLQ5HLP
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ELECTROLYTES AND METABOLITES IN CORNEAL STROMA AND EPITHELIUM (OHFWURO\WHV IRU H[DPSOH VRGLXP 1D SRWDVVLXP . FKORULGH &O FDO FLXP &D DQGVXOIDWH 62 IRUPLPSRUWDQWSDUWVRIWKHLRQLFPLOLHXIRUOLYLQJ FHOOV 6FKUDJHHWDO *OXFRVHLVNQRZQWREHDPDMRUQXWULHQWPHWDEROLWH RIWKHFRUQHDOWLVVXHV 5HLP E /DFWDWHLVWKHSURGXFWRIWKHDQDHURELFHQHUJ\ SURGXFLQJ PHWDEROLVP 7DEOH ,Q WKH FRUQHD OLNH LQ PDQ\ RWKHU WLVVXHV DGH QRVLQHWULSKRVSKDWH $73 DQGDGHQRVLQHGLSKRVSKDWH $'3 DUHPDMRUFDUULHUVRI FHOOXODUHQHUJ\7KHUHIRUH$73OHYHOVDUHFRQVLGHUHGWRLQGLFDWHWKHYLWDOLW\RIWLVVXHV DQG FHOOV 5HLP HW DO +HQQLJKDXVHQ HW DO 6LQFH$73 OHYHOV Table 9.1 Stroma of rabbit cornea, mean ± SD, hydration = 3.2 ± 0.4 QMol/g H2O (n = 20) Na 125 ± 42 Cl 123 ± 25 K 24 ± 4 S 105 ± 21 P 12 ± 4
QMol/g net weight (n = 15) Lactate 9.66 ± 1.2 Glucose 3.63 ± 0.8 ATP 0.20 ± 0.05 ADP 0.07 ± 0.03 ASC 4.72 ± 1.44
Note: ASC = ascorbate. Levels of some metabolites and electrolytes in the corneal stroma. Adenosine triphosphate (ATP) and adenosine diphosphate (ADP) are strictly intracellular metabolites. Since the corneal stroma contains keratocytes only in about 10% of its volume, their levels are much lower than in the epithelium (see Table 9.2). Lactate is present in the extracellular as well as in the intracellular space and consequently shows levels close to those of the epithelium (Reim et al., 1970, 1978; Fischern, 1996).
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Table 9.2 Metabolite levels of rabbit corneal epithelium QMol/g wet weight, m ± SEM ATP ADP ATP/ADP AMP Glucose Lactate GSH GSSG ASC
3.03 ± 0.10 (n = 14) 0.26 ± 0.02 (n = 14) 12.23 ± 0.89 (n = 14) 0.93 ± 0.08 (n = 14) 2.02 ± 0.22 (n = 14) 9.89 ± 1.02 (n = 14) 3.03 ± 0.43 (n = 21) 0.28 ± 0.05 (n = 21) 11.55 ± 1.05 (n = 24)
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Table 9.3 Grading of Eye Burns I
II
III
IV
Immediate signs Erosion Hyperemia
Large erosion Ischemia 1/3 Chemosis
Surface defect Ischemia >1/2 Rose chemosis Corneal opacity
Epithelia destroyed Deep ischemia >3/4 Dense corneal opacity Conjunctival necroses Sclera porcelain white Discoloration and atrophy of iris Fibrin exudate
Later signs Regeneration
Recirculation Regeneration
Persistent erosion Ulceration Vascularization Scars
Proliferation Large ulcerations Melting of cataract Glaucoma Scarification
Note: Grading of eye burns according to clinical signs. The upper part lists immediate damage visible, the lower one later secondary events. The classification of signs was developed from various authors and by clinical experiences (Hughes, 1946; Roper-Hall, 1965; Thoft, 1978; Reim and Kuckelkorn, 1995)
Figure 9.1
Eye with a mild alkali burn stage I (Table 9.3). The corneal epithelium was completely lost, but the stroma remained undamaged and clear. The conjunctiva showed hyperemia, but no swelling or ischemia. The damage healed within a few days.
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Figure 9.2
ALTERNATIVE TOXICOLOGICAL METHODS
Lime burn stage II (Table 9.3). The whole corneal and some conjunctival epithelium was destroyed. The corneal stroma exhibited little superficial turbidities. The lower conjunctiva is demonstrated by upgaze. It was swollen (chemosis). Superficially in the conjunctiva, ischemia is recognized by the interrupted blood columns. With the lit lamp microscope, bloodstream could not be detected. Underneath the otherwise pale conjunctiva, intact sclera appeared with a faint rose background.
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Figure 9.3
Clinical appearance of a severe chemical injury grade IV. The inner margin of the upper lid showed a white line of necrosis. The conjunctiva appeared flat and white, also from necrosis, which presumably included visible parts of the sclera. In the upper left region, some hemorrhages were deposited in necrotic conjunctiva. Ischemia was evident. The cornea was completely turbid. The outlines of iris and pupil could be hardly identified.
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Figure 9.4
95
Melting of the anterior eye segment, nine days after most severe burn from liquid metal. There were extended necroses of all conjunctival, subconjunctival, and scleral tissues, appearing homogeneously white and slippery. Only in the right upper region, some hemorrhages in necrotic tissues showed red color. The cornea was opaque in the upper marginal parts. The lower and central cornea was melted away and the iris and lens exposed. Since at that time (1977) corneal donor material was not available, the eye was lost and had to be removed. In the melting tissues of the anterior eye segment, high activities of N-acetylglucose aminidase (NAcGA, E.C.3.2.1.50) and cathepsin-D (E.C.3.4.23.5) were found (Reim, 1982a).
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96
ALTERNATIVE TOXICOLOGICAL METHODS
Cornea and conjunctiva Severe lesion, severe response PGE2α, Interleukins, LT 4, Subst-P, VIP, CGRP Mild lesion weak response
IL-1, IL-6 IL-8, TNF
PMNs
PMNs, macrophages
T-lymphocytes B-lymphocytes Plasma cells
_
+
O2 OH -radicals lysosomal enzymes
Cellular and humoral antibodies Restitution
Inflammation Figure 9.5
Scars
Ulceration
Flow diagram of inflammatory cascade following chemical and thermal injuries of the eye. The inflammatory response is a quantitative process produced by the affected tissues and the leucocytes involved (Ghattacherjee et al., 1979; Reim et al., 1980, 1993, 1997; Reim, 1982a; Rochels et al., 1982; Kulkarni and Srinivasan, 1983; Becker et al., 1991, 1995; Reim and Leber, 1992; Reim and Becker, 1995).
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Table 9.4 Cytokines in Tears EGF p regeneration of epithelium TGFbeta 2 p inhibits proliferation TNFalpha, in inflammation Many others, but presumably released from damaged surface epithelia Note: Cytokines and growth factors in tears influencing the corneal epithelium (Mishima et al., 1991; Kruse and Tseng, 1994; Sotozono and Kinshita, 1998).
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100
10
1 Keratitis Inflammat.Cone Ulceration
Figure 9.6
Decompensation Dystrophy, Scars Keratoconus
Interleukin-1F (IL-1) and Interleukin-6 (IL-6) in human corneal buttons from keratoplasty. Total number of cases: 127. The logarithmic ordinate shows the concentrations found in pg/mg extractable protein. The symbols represent the median, squares stand for IL-1, rhombs for IL-6. The error bars demonstrate the 75% percentiles. In the abscissa, the diagnoses of the cases were indicated corresponding to the position of the symbols (Becker et al., 1995). Inflamed corneas revealed very high levels of IL-1 and IL-6. The levels in the uninflamed, quiete corneas were lower by an order of magnitude.
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N-Acetylglucose aminidase in human tears wmol/min/ml 10
Atopic conjunctivitis
1
Range of 8 samples from human burns Normal 0.24 s 0.09 (9)
0.1 Figure 9.7
Activity of N-acetylglucose aminidase (NAcGA, E.C.3.2.1.50) in human tears collected from nine human cases with eye burns stage I and II and an atopic patient. Please note that the ordinate is in logarithmic scale! The enzyme activity (QMol/min/ml) increased considerably in surface diseases.
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ALTERNATIVE TOXICOLOGICAL METHODS
Table 9.5 Stroma of Rabbit Cornea, QMol/g H2O, mean ± SD
EDXA Na Cl S P Ca
Normal (n = 20) 125 123 105 12 3
± ± ± ± ±
Alkali Burn, Denuded, Rinsed for 16 Days, 4v daily with 0.9% NaCl (n = 8)
42 25 21 4 3
90 65 24 22 1
± ± ± ± ±
11 15 4 22 3
Note: Changes of the levels of some electrolytes in the corneal stroma after alkali burn. The denuded stroma was rinsed with saline, four times daily for 16 days. Na, Cl, and especially S were decreased, P increased (Fischern et al., 1998).
Calcification and Contamination 0RUHRYHU ZKHQ H\HV GHQXGHG IURP HSLWKHOLXP ZHUH ULQVHG ZLWK SKRVSKDWH EXIIHURULIWKH\ZHUHWUHDWHGZLWKH\HGURSVFRQWDLQLQJSKRVSKDWHEXIIHUDVVROYHQW RUSKRVSKDWHVDOWVRIGUXJVVXFKDVSUHGQLVRORQHSKRVSKDWHUDSLGFRUQHDOFDOFLÀ FDWLRQZDVREVHUYHGLQFOLQLFDOSDWLHQWVDQGLQPRGHOH[SHULPHQWV 7DEOH )LJXUHV ² 5HLPHWDO 6FKUDJHHWDO ,QDGGLWLRQKXPDQFRUQHDOEXWWRQV REWDLQHGIURPEXUQWH\HVRQNHUDWRSODVW\DVZHOODVFRQMXQFWLYDOVDPSOHVUHYHDOHG D UHPDUNDEO\ KLJK FRQWDPLQDWLRQ E\ YDULRXV PHWDOV DQG PLQHUDOV 6FKUDJH DW HO 6FKLUQHU HW DO 7KHVH FRQWDPLQDWLRQV ZHUH H[SODLQHG RQ RQH KDQGE\LPSXULWLHVLQWKHWHVWPDWHULDOVDQGRQWKHRWKHUKDQG E\VHFRQGDU\LQWUR GXFWLRQRISDUWLFOHVZLWKRSKWKDOPLFVROXWLRQVXVHGLQWRSLFDOWKHUDS\RQWKHGHQXGHG VWURPDVXUIDFH 5HLPHWDO Scarring ,QPDQ\FDVHVWKHXOFHUDWLRQVGLGQRWSHUIRUDWH7KHQSUROLIHUDWLRQWLVVXHFRY HUHG WKH ZKROH DQWHULRU H\H VHJPHQW DQG WUDQVIRUPHG LW LQWR VKULQNLQJ VFDUV7KLV LQGXFHGV\PEOHSKDUDWKDWWKHPRWLOLW\RIWKHJOREHZDVUHVWULFWHG 5HLPDQG6DULF 5HLP D RUWKHJOREHZDVWRWDOO\LPPRELOL]HG+HDOLQJRIWKHVHFDVHV Table 9.6 Stroma of Rabbit Cornea, QMol/g H2O, Mean ± SD
EDXA Na Cl S P Ca
Normal (n = 20) 125 123 105 12 3
± ± ± ± ±
42 25 21 4 3
Alkali Burn, Denuded, Rinsed for 16 Days, 4v Daily with Phosphate Buffer (n = 8) 105 88 28 623 435
± ± ± ± ±
22 33 4 307 198
Note: Changes of the levels of some electrolytes in the corneal stroma after alkali burn and rinsing with isotonic phosphate buffer, four times daily for 16 days. Na, Cl, and especially S were decreased, but P and Ca were largely increased. Clinically, calcification of the cornea was observed (Schrage, 1997; Fischern et al., 1998; Haller, 2001).
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Figure 9.8
101
Left eye of a 16-year-old boy six months after a most severe chemical injury. In this accident, a highly alkaline etching fluid used to work on electronic parts spilled into both eyes of the patient. In this case, a severe inflammatory response had developed and remained for years. The conjunctiva-like proliferation tissue surrounding the cornea was swollen and very hyperemic. The cornea was devoid of epithelium. It showed extended ulceration especially in its marginal parts and was generally thinned. The upper right cornea showed white calcification. To save the eye from melting, a keratoplasty was performed. The excised cornea was examined with electron dispersive x-ray analysis method (EDXA) (see Figures 9.9 and 9.10).
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Figure 9.9
Scanning electron microscopy (SEM) on a cross section of the cornea seen in Figure 9.8. Magnification v200. The upper part shows calcification, the lower one parallel corneal lamellae (Schrage et al., 1988, 1993, 1996).
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ALTERNATIVE TOXICOLOGICAL METHODS
Figure 9.10 Electron dispersive x-ray analysis (EDXA) of the calcified cornea as demonstrated in Figures 9.8 and 9.9. The spectra of the x-rays backscattered at scanning electron microscopy (SEM) showed as expected high peaks for calcium (Ca) and phosphorus (P). But the most prominent peak from this sample was emitted from silicon (Si). Thus, EDXA revealed an unexpected high contamination of the cornea by silicone, which might have explained the severe and longstanding inflammatory response in this case (Schrage et al., 1988, 1993, 1996).
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Figure 9.11 Eye of a 42-year-old male 2 years after severe lime burn. Heavy scar formation could not be prohibited. The cornea was covered with thick highly vascularized proliferation tissue. The conjunctiva developed strong scars between the globe and the lids, reducing eye motility. The conjunctival scars also deformed the lid margins. The hyperemic, red scar tissue showed that the inflammatory response had not subsided after 2 years. The eye was practically blind and had bad prognoses for surgical rehabilitation.
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CHAPTER
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Evaluation and Refinement of the Bovine Cornea Opacity and Permeability Assay -RKQ/8EHOV-DPHV'3DDXZDQG3KLOOLS/&DVWHUWRQ
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111
Stainless Steel Ring
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O-Ring Top Holder Cornea
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Figure 10.1 Cross-section of the new cornea holder. In contrast to the old holder, the new holder clamps onto sclera rather than cornea. Also, the shape of the chamber fits the normal curvature of the cornea in contrast to the flat chamber of the current BCOP holder.
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112
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Table 10.1 Absorbance at 570 nm (A570) of Bovine Corneas Exposed to Various Treatments and Incubated in MEM for 3 h Treatment
None
Intact, 35ºC, MEM w/o Epi, 35ºC, MEM w/o Epi/Endo, 4ºC, H2O Isopropanol Acetone 30% TCA 1% NaOH 30% SLS
0.05 ± 0.03 0.11 ± 0.03 0.67 ± 0.13 — — — — —
Exposure 10 min 1 min — — — 0.59 ± 1.38 ± 1.43 ± 1.69 ± 0.095 ±
0.08 0.22 0.08 0.22 0.03
30 sec
— — — 0.23 ± 0.04 1.07 ± 0.21 2.28 ± 0.20 1.28 ± 0.29 0.48 ± 0.28
— — — 0.24 ± 0.07 0.87 ± 0.25 1.96 ± 0.06 0.36 ± 0.19 0.21 ± 0.11
Note: Intact is untreated cornea incubated in MEM. w/o Epi is cornea with epithelium removed, not exposed to chemical, and incubated in MEM for 3 h. w/o Epi/Endo is cornea with both epithelium and endothelium removed, not exposed to chemical, and incubated in H2O at 4ºC. Values are mean ±SD, n = 5–10. (Data cited from Ubels et al. [2000]. With permission of Elsevier Science.)
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30 sec 1 min
mg H20 / mg cornea
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Figure 10.2 Corneal hydration (mg H2O/mg cornea) following exposure to test substances for 30 sec, 1 min, or 10 min and incubation in MEM for 3 h. Intact is untreated cornea incubated in MEM. w/o Epi is cornea with epithelium removed, not exposed to chemical, and incubated in MEM for 3 h. w/o Epi-Endo is cornea with both epithelium and endothelium removed, not exposed to chemical, incubated in H2O at 4rC. Mean ±SD, n = 5. Values for intact, without Epi, and without Epi-Endo were all significantly different from each other. 30-sec values and unmarked 1min values are not different than intact value. # Significantly different than 30-sec and 1-min values within group. * Significantly different than 30-sec and 10-min values within group. (ANOVA and Dunnett’s test, P e0.05). (Data cited from Ubels et al. [2000]. With permission of Elsevier Science.)
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Figure 10.3 Corneal endothelial cell layers stained with Alizarin Red S and trypan blue. Twenty percent of the endothelial layer is damaged after mounting in the old corneal holder (left), and none of the endothelial layer is damaged after mounting in the new holder (right). The streaks of damaged cells exhibited after mounting in the old holder are characteristic of wrinkling caused by the holder. Magnification 35v. (Reproduced from Ubels et al. [2002]. With permission of Elsevier Science.)
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CHAPTER
11
Corneal Organ Culture for Ocular Toxicity Test of Commercial Hair Care Products )X6KLQ;<XDQG.H3LQJ;X
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117
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Culture Treatment 7KUHH KDLU FDUH SURGXFWV WHUPHG *$ *% DQG *& ZHUH DSSOLHG GLUHFWO\ RU GLOXWHG ZLWK 0(0 ÀUVW DQG WKHQ DSSOLHG GURSZLVH RQWR WKH FHQWHU VXUIDFH RI WKH FRUQHD$IWHUGLVFUHWHWLPHLQWHUYDOVWKHWUHDWHGFRUQHDVZHUHZDVKHG²WLPHVE\ GLSSLQJLQWR3%6VROXWLRQDQGWKHQSURFHVVHGIRUHLWKHUFHOOVXUIDFHELRWLQODEHOLQJ RUHSLWKHOLDOQXFOHDUH[WUDFWLRQ Surface Biotinylation-Tight Junction Permeability Assay &XOWXUHGFRUQHDVZHUHZHWWHGZLWKIUHVKO\PDGHPJPOVXOIR1+6/&ELRWLQ 3LHUFH&KHPLFDOV5RFNIRUG,/ LQ+DQN·VEDODQFHGVDOWVROXWLRQFRQWDLQLQJP0 &D&ODQGP00J&O &KHQHWDO $IWHUDPLQLQFXEDWLRQWKHVXUIDFH ODEHOHGFRUQHDVZHUHULQVHGZLWK3%6HPEHGGHGLQ2&7IUR]HQLQOLTXLGQLWURJHQ DQGVHFWLRQHGRQDFU\RVWDWDWDWKLFNQHVVRIQP7KHVHFWLRQVZHUHÀ[HGZLWKLFH FROG DFHWRQH DQG ODEHOHG ZLWK UKRGDPLQHDYLGLQ ' LQ 3%6 FRQWDLQLQJ ERYLQH VHUXPDOEXPLQ %6$ IRUK7KHVOLGHVZHUHPRXQWHGDQGH[DPLQHGXQGHUD1LNRQ (FOLSVH(ÁXRUHVFHQFHPLFURVFRSHHTXLSSHGZLWKD6SRWGLJLWDOFDPHUD Electrophoretic Mobility Shift Assay (EMSA) 7R SUHSDUHG FHOO H[WUDFWV IRU (06$ WKH HSLWKHOLDO FHOOV ZHUH UHPRYHG IURP FXOWXUHGERYLQHFRUQHDVH[SRVHGWRKDLUFDUHSURGXFWVXQGHUDGLVVHFWLRQPLFURVFRSH ZLWKDVFDOSHOEODGH&HOOVIURPHDFKFRUQHDZHUHUHVXVSHQGHGLQQORIH[WUDFWLQJ EXIIHU P0+HSHV.2+DWS+ P0GLWKLRWKUHLWRO>'77@P0HWK\OHQH GLDPLQHWHWUDDFHWLFDFLG>('7$@P0.&OJO\FHUROP0SKHQ\OPH WK\OVXOIRQ\OÁXRULGH>306)@13 DQGLQFXEDWHGZLWKSHULRGLFDJLWDWLRQDW r&IRUK3URWHLQFRQFHQWUDWLRQZDVGHWHUPLQHGXVLQJPLFUR%&$SURWHLQDVVD\ UHDJHQWNLW 3LHUFH&KHPLFDO&R 7KHROLJRQXFOHRWLGH&*&77*$7*$*7&$*& &**$$IRU$3ELQGLQJDQGWKHROLJRQXFOHRWLGH$*&77&$*$****$777& &*$*$**IRU1)O%ELQGLQJZHUHODEHOHGXVLQJ7SRO\QXFOHRWLGHNLQDVHDQG >K3@$73)RUGHWHFWLRQRI$3DQG1)O%'1$ELQGLQJDFWLYLWLHV 3ODEHOHG ROLJRQXFOHRWLGHVZHUHLQFXEDWHGZLWKDQJRIHSLWKHOLDOH[WUDFWHGSURWHLQVDQGWKH UHDFWLRQPL[WXUHZDVVXEMHFWHGWR(06$DVVD\DVGHVFULEHGE\;XHWDO
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Figure 11.1 Biotin surface labeling to visualize epithelial barrier. Cultured bovine cornea was incubated with sulfo-NHS-LC-biotin for 30 min and then embedded in OCT, snapfrozen, and sectioned (6 Qm). Cryostat sections (8 Qm) were (A) stained directly with hematoxylin to reveal corneal morphology (B) or incubated with rhodamineavidin D to visualize the bound biotin. The rhodamine staining represents biotinylation of accessible surface of normal bovine cornea and linear staining at the corneal surface indicates functional epithelial TJ barrier in cultured corneas. Ep, epithelium; BM, basement membrane; St, stroma that consists of fibroblasts. A and B are mirror orientations of the same corneal sections.
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Figure 11.2 Tight junction permeability assay of cultured bovine corneas in response to challenge of three hair care products. Corneas in culture were treated with 100%, 50% (not shown), and 25% chemicals, and TJ permeability of corneal epithelium was assessed by surface biotinylation as described in Figure 11.1. Inserts: corneal sections stained directly with hematoxylin to reveal corneal morphology. Note: extended biotinylation of the corneal surface caused by GA and GB exposure in a concentration dependent manner. However, no disruption of TJ was observed in GC treated cornea.
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Figure 12.1 Confocal image of a corneal equivalent with surrounding sclera containing chick embryonic dorsal root ganglion (not shown). Neurites, labeled with nerve-specific antineurofilament antibody are seen traveling through the sclera (S) parallel to the corneal periphery and branching into the cornea (C). White dashed line, corneal periphery; arrows, parallel neurites; arrowheads, branching neurites.
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Additional exposure times (min)
90% <90% but > 30% <30%
60, 240 5, 60 1, 5
THE EPIOCULAR PREDICTION MODEL
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136
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Calculation of Effective Time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etermination of Prediction Model 'UDL]H GDWD ZHUH DYDLODEOH IRU ZDWHUVROXEOH PDWHULDOV LQ WKH (&(72& GDWDEDVH %DJOH\HWDO $OVR'UDL]HGDWDIRUDQDGGLWLRQDOFRVPHWLFRU SHUVRQDOFDUHSURGXFWVLQJUHGLHQWVZHUHPDGHDYDLODEOHWR0DW7HNE\FRPPHUFLDO FRPSDQLHV LQWHUHVWHG LQ WKH GHYHORSPHQW RI D GDWDVHW IRU (SL2FXODU $OO PDWHULDOVDUHOLVWHGLQ7DEOH$Q(7IRUWKH PDWHULDOVZDVGHWHUPLQHG XVLQJ WKH SURFHGXUHV GHVFULEHG DERYH )RU PDWHULDOV ZLWK 'UDL]H VFRUHV DW D VSHFLÀHG FRQFHQWUDWLRQ D VROXWLRQ RI WKH VSHFLÀHG FRQFHQWUDWLRQ ZDV PDGH XS SULRUWRGHWHUPLQDWLRQRIWKH6*DQGWKHVROXWLRQZDVXVHGQHDWRUGLOXWHGWR GHSHQGLQJRQWKH6*$OOPDWHULDOVZHUHWHVWHGDWOHDVWWZLFHXVLQJWZRGLIIHUHQW ORWVRI(SL2FXODUWLVVXH $QDYHUDJH(7IRUWKHYDULRXVPDWHULDOVZDVGHWHUPLQHGDQGWKH'UDL]HVFRUH ZDVSORWWHGYHUVXVORJ (7 /LQHDUUHJUHVVLRQURXWLQHVLQWKHJUDSKLFVSURJUDP 6OLGHZULWH9HUVLRQ $GYDQFH *UDSKLFV 6RIWZDUH (QFLQLWDV &$ ZHUH XVHG WR ÀQGWKHEHVWÀWWRWKHVHGDWDWRGHWHUPLQHDSUHGLFWLRQHTXDWLRQ Testing of Prediction Model 'UDL]H VFRUHV IRU YDULRXV FRQVXPHU DQG SHUVRQDO FDUH SURGXFWV LQFOXGLQJ ÀYH VKDPSRRVVL[KDQGIDFHERG\VRDSVÀYHODXQGU\GHWHUJHQWVIRXUGLVKZDVKLQJOLT XLGV DQG IRXU VNLQ ORWLRQV ZHUH PDGH DYDLODEOH WR 0DW7HN &RUSRUDWLRQ E\ 0% 5HVHDUFK/DERUDWRULHV 6SLQQHUVWRZQ3$ $OOPDWHULDOVZHUHRIIWKHVKHOISURGXFWV DYDLODEOHDWUHWDLOVWRUHV(DFK(7ZDVGHWHUPLQHGDVRXWOLQHGDERYHLQDWOHDVW WZR VHSDUDWH ORWV RI (SL2FXODU 8VLQJ HDFK (7 SUHGLFWHG 'UDL]H VFRUHV ZHUH FDOFXODWHGDQGDQDYHUDJHSUHGLFWHG'UDL]HVFRUHZDVFRPSXWHGIRUHDFKPDWHULDO 6XEVHTXHQWO\WKHLQYLWURSUHGLFWHG'UDL]HVFRUHZDVSORWWHGYHUVXVWKHDFWXDO'UDL]H VFRUHVWKHGDWDZHUHÀWWHGWRWKHOLQHSUHGLFWHG'UDL]H DFWXDO'UDL]H \ [ DQG DFRUUHODWLRQFRHIÀFLHQWUZDVGHWHUPLQHG
THE EPIOCULAR PREDICTION MODEL
137
Table 13.2 In Vitro and In Vivo Data Used to Generate the Prediction Model; In Vitro Data from EpiOcular ET-50 Determinations; In vivo Data from ECETOC Database or Commercial Sources Conc. tested
# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
Benzalkonium chloride (10%) Benzalkonium chloride (5%) Benzalkonium chloride (1%) Cetyl pyridinium bromide (10%) Cetyl pyridinium bromide (1%) Cetyl pyridinium bromide (0.1%) Glycerol Sodium hydroxide (10%) Sodium hydroxide (1%) Propylene glycol Sodium dodecyl sulfate (30%) Sodium dodecyl sulfate (15%) Sodium dodecyl sulfate (3%) Trichloro acetic acid (30%) Trichloro acetic acid (3%) Triton X-100 (10%) Triton X-100 (5%) Triton X-100 (1%) Tween 20 (100%) Body/hand wash Body/hand wash Eye gel/colorant Eye gel/colorant Face/body wash Face/body wash Face/body wash Hand/body lotion Hand/body lotion Hand/body lotion Shampoo—baby Shampoo—baby Conditioner Shampoo—regular Shampoo—regular Na2-ricinoleadmido MEA sulfosuccinate Sodium trideceth sulfate Cetrimonium chloride Stearalkonium chloride Cocamide DEA Disodium cocoamphodipropionate Surfactant blend Surfactant blend Final formulation shampoo Final formulation shampoo Final formulation shampoo Final formulation shampoo Final formulation shampoo
2.0% 1.0% 0.2% 2.0% 0.2% 0.02% 20.0% 2.0% 0.2% 20.0% 6.0% 3.0% 0.6% 6.0% 0.6% 2.0% 1.0% 0.2% 20.0% 20% 20% 20% 20% 20% 20% 20% 20% 20% 20% 20% 20% 20% 20% 20% 20% 20% 20% 20% 20% 20% 20% 20% 20% 20% 20% 20% 20%
ET-50 (min)
Draize (MMAS)
1.07 1.0 5.9 9.0 30.1 240.0 240.0 1.0 2.3 240.0 2.1 5.1 9.0 1.0 155.1 2.5 5.3 36.7 240.0 9.1 14.8 240 240 240 6.5 10.2 240 240 240 30.8 25.7 240 6.0 8.7 108.9
108.0 83.8 45.3 89.7 36.0 2.7 1.7 108.0 25.8 1.3 60.5 59.2 16.0 106.0 6.7 68.7 33.1 1.7 4.0 32 35 0 2 2 25 40 2 2 3 10 18 2 30 35 0
2.5 116.9 240 240 11.2 19.2 40.4 26.1 29.1 4.2 9.3 9.0
33 6.67 14 0 15.3 6.0 2.67 4.0 12.5 32.7 31.6 34.4 (continued)
138
ALTERNATIVE TOXICOLOGICAL METHODS
Table 13.2 (continued) In Vitro and In Vivo Data Used to Generate the Prediction Model; In Vitro Data from EpiOcular ET-50 Determinations; In vivo Data from ECETOC Database or Commercial Sources Conc. tested
# 48 49 50 51 52 53 54 55 56 57 58 59
Final formulation shampoo Final formulation shampoo Final formulation shampoo Final formulation shampoo Final formulation shampoo Hydro-alcohol (hair spray) solution 10% fatty alcohol ethoxylate Eye makeup remover (surfactant sol.) Lactic acid (3% solution) Oleic acid Skin care emulsion Body spray
20% 20% 20% 20% 20% 20% 20% 20% 20% 20% 20% 20%
ET-50 (min)
Draize (MMAS)
31.0 63.1 47.1 29.4 42.1 84.1 189 240 240 240 240 240
3.9 3.5 8.3 6.57 4.8 6.0 3.5 0 0 2 0 0
Quality Control Testing (DFKZHHNO\ORWRI(SL2FXODUWLVVXHLVWHVWHGXVLQJWKHFRPPRQVXUIDFWDQW7ULWRQ ; 6LJPD &KHPLFDO &RPSDQ\ 6W /RXLV 02 2QH KXQGUHG PLFUROLWHUV RI D VROXWLRQRI7ULWRQ;LQXOWUDSXUHZDWHU WR02KP DUHDSSOLHGWR GXSOLFDWH(SL2FXODUWLVVXHV 1 IRUDQGPLQ LQDGGLWLRQ Q/RI XOWUDSXUHZDWHUDUHDSSOLHGWRWULSOLFDWHWLVVXHVIRUPLQWRVHUYHDVWKHQHJDWLYH FRQWURO7KHLQWUDORWYDULDELOLW\RIWKHWLVVXHZDVDVVHVVHGE\GHWHUPLQLQJDQDYHUDJH VWDQGDUGGHYLDWLRQDQGFRHIÀFLHQWRIYDULDWLRQ &9 vVWDQGDUGGHYLDWLRQDYHU DJH IRU HDFK RI WKHVH WLPHG DSSOLFDWLRQV LQ DGGLWLRQ WKH &9V ZHUH DYHUDJHG WR FRPSXWHDQDYHUDJH&9IRUWKHHQWLUHORW7KHLQWHUORWYDULDELOLW\ZDVDVVHVVHGE\ FRPSXWLQJ WKH (7 IRU HDFK WLVVXH ORW DQG WKHQ E\ FRPSDULQJ HDFK (7 IRU 7ULWRQ ; IURP DOO (SL2FXODU WLVVXH ORWV SURGXFHG GXULQJ WKH FDOHQGDU \HDUV ²)LQDOO\WKHDYHUDJH0772'IRUWKHQHJDWLYHFRQWUROWLVVXHVDQGWKH UHVXOWDQW&9ZHUHGHWHUPLQHG Interlaboratory Testing 7LVVXHVIURPDVLQJOHSURGXFWLRQORWZHUHSDFNDJHGDQGVKLSSHGWRWKH3URFWHU DQG *DPEOH &RPSDQ\ &LQFLQQDWL 2+ DQG WKH ,QVWLWXWH IRU ,Q 9LWUR 6FLHQFHV *DLWKHUVEXUJ0' ,QDGGLWLRQWLVVXHIURPWKHVDPH(SL2FXODUORWZDVUHWDLQHG DW 0DW7HN DQG VWRUHG DW r& IRU KU SULRU WR WHVWLQJ 5HVXOWV IURP WKH WZR ODERUDWRULHVDQGIURP0DW7HN·VODERUDWRU\ZHUHFRPSDUHGIRUWKHSRVLWLYHFRQWURO 7ULWRQ ; DQG IRU WZR DGGLWLRQDO PDWHULDOV IURP WKH (&(72& GDWDEDVH %DJOH\HWDO D VRGLXPGRGHF\OVXOIDWH 6'6 DQG E EHQ]DO NRQLXPFKORULGH %$. 1RWH $VSHUWKHSURFHGXUHVGHVFULEHGDERYH6'6DQG %$. ZHUH WKH DFWXDO FRQFHQWUDWLRQV WHVWHG LQ WKH WKUHH ODEV 'RVH UHVSRQVH FXUYHVZHUHFRQVWUXFWHGXVLQJ1 WLVVXHVDQGDQ(7ZDVGHWHUPLQHGLQHDFK ODEIRUHDFKRIWKHWKUHHPDWHULDOV$VZLWKWKHGDWDEDVHWHVWLQJUHSURGXFLELOLW\RI
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WKH WHVWLQJ UHVXOWV ZDV DVVHVVHG E\ FDOFXODWLQJ DYHUDJHV VWDQGDUG GHYLDWLRQV DQG FRHIÀFLHQWVRIYDULDWLRQIRUHDFKRIWKHPDWHULDOVWHVWHG,QDGGLWLRQDFRHIÀFLHQWRI YDULDWLRQZDVGHWHUPLQHGIRUHDFKRIWKHWLPHSRLQWVWHVWHGLQHDFKODERUDWRU\DQG DQDYHUDJH&9ZDVFDOFXODWHGIRUWKHODERUDWRU\)LQDOO\WKH(7IRUHDFKPDWHULDO IURPWKHWKUHHODEVZDVFRPSDUHG Testing of Ultramild Materials 7KHPHWKRGXVHGWRWHVWYHU\PLOGPDWHULDOVLHWKRVHWRZKLFKWKH'UDL]HWHVW ZDVLQVHQVLWLYH 00$6 GLIIHUHGIURPWKHPHWKRGGHVFULEHGDERYHLQWZRNH\ DVSHFWV D WKHPDWHULDOVZHUHDSSOLHGQHDWUHJDUGOHVVRIWKH 6*DQG E H[SRVXUH WLPHVZHUHH[WHQGHGXSWRKU7KH(7ZDVGHWHUPLQHGDVSUHYLRXVO\GHVFULEHG XVLQJWKH077WLVVXHYLDELOLW\DVVD\
RESULTS Histological Characterization of the EpiOcular Tissue Model &URVVVHFWLRQVRI+ (VWDLQHG(SL2FXODUWLVVXHDQGDUDEELWFRUQHDDUHVKRZQ LQ)LJXUH7KHVWUXFWXUHVDUHRIHTXDOWKLFNQHVVDQGFRQVLVWRIDFXERLGDOOD\HU RI EDVDO FHOOV ZLWK DGGLWLRQDO OD\HUV RI ÁDWWHQHG FHOOV 7KHUH LV QR HYLGHQFH RI D JUDQXODURUFRUQLÀHGOD\HUDVZRXOGEHVHHQZLWKIXOO\GLIIHUHQWLDWHGNHUDWLQRF\WHV FXOWXUHGDWWKHDLU²OLTXLGLQWHUIDFH 5HJQLHUHWDO 0DNHWDO %RHOVPD HW DO &HOOV RQ WKH DSLFDO VXUIDFH RI ERWK WLVVXHV DUH VTXDPRXV DQG VKRZ HORQJDWHGQXFOHL
A
B
Figure 13.1 Hematoxylin and Eosin (H&E) stained histological cross sections of (A) EpiOcular tissue model and (B) rabbit cornea epithelium and underlying stroma. Tissues were fixed in 10% formalin, embedded in paraffin, and stained with H&E. Final magnification 360v.
140
ALTERNATIVE TOXICOLOGICAL METHODS
OCL-200 Prediction Equation Draize Score (MMAS)
150 r = 0.90
125
59 materials
100 75 50 25 0 1
10
100
300
ET-50 (min) Prediction Equation: Draize (MMAS) = –4.74 + 101.7/
(ET-50) 95% Prediction Intervals: Draize (MMAS) = –30.54 + 100.4/
(ET-50) Draize (MMAS) = 21.07 + 102.9/
(ET-50) Figure 13.2 Graphical depiction of in vivo and in vitro data used to derive the prediction equation. All materials tested that had specific gravity >0.95 were diluted to 20% in ultrapure water. If actual ET-50 exceeded 240 min, ET-50 was set equal to 240 min. If ET was less than 1 min, ET-50 was set to 1 min.
Determination of Prediction Model 7KH FKHPLFDO QDPH RU SURGXFW FDWHJRU\ WKH UHVSHFWLYH (7 DQG WKH 'UDL]H VFRUHIRUWKHPDWHULDOVXVHGWRFRQVWUXFWWKHSUHGLFWLRQHTXDWLRQDUHJLYHQLQ7DEOH (7 DQG 'UDL]H VFRUHV DUH SORWWHG LQ )LJXUH 7KH RSWLPDO ÀW IRU WKH SUHGLFWLRQHTXDWLRQZDVGHWHUPLQHGWREH 'UDL]H 00$6 ²(7 ZKHUHWKH(7LVH[SUHVVHGDVe(7ePLQ 7KH FRQÀGHQFHLQWHUYDOVIRUWKHSUHGLFWLRQHTXDWLRQ SUHGLFWLRQLQWHU YDO DUH 8SSHUOLPLW 'UDL]H00$6 (7 /RZHUOLPLW 'UDL]H00$6 ²(7 Testing of Prediction Model 7DEOH VKRZV SUHGLFWHG DQG DFWXDO 'UDL]H VFRUHV IRU WKH ÀQDO IRUPXODWLRQ FRVPHWLFSHUVRQDOFDUHSURGXFWVXVHGWRWHVWWKHXWLOLW\RIWKHSUHGLFWLRQPRGHO$ JUDSKLFDOFRPSDULVRQRIWKHDFWXDO'UDL]HDQGSUHGLFWHG'UDL]HVFRUHVLVVKRZQLQ )LJXUH7KHDELOLW\WRFRUUHFWO\SUHGLFWWKHLQYLYR'UDL]HVFRUHVZDVDVVHVVHG
THE EPIOCULAR PREDICTION MODEL
141
Table 13.3 EpiOcular Prediction Model Testing: Comparison between In Vivo and In Vitro Predicted Draize Code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Predicted Draize
Product
10599 10599 10599 10599 10599 10599 10599 10599 10599 10599 10599 10599 10599 10599 10599 10599 10599 10599 10599 10599 10599 10599 10599 10599
A B C D E F G H I J K L M N O P Q R S T U V W X
Body wash Body wash Dishwashing liquid Hand soap liquid Dishwashing liquid Facial soap Dishwashing liquid Dishwashing liquid Laundry detergent Laundry detergent Laundry detergent Dishwashing liquid Shampoo Shampoo Shampoo Hand soap liquid Skin lotion Shampoo Skin lotion Shampoo Body wash Laundry detergent Laundry detergent Skin lotion
Actual Draize (MMAS)
14.6 31.3 51.3 28.0 25.2 15.6 50.8 60.5 37.3 1.8 33.4 96.9 6.5 32.9 8.4 18.1 1.8 46.9 1.8 39.3 41.2 1.8 39.9 1.8
16.7 44.7 38.3 24.7 39.3 9.3 39.0 50.3 37.3 0.7 37.7 37.7 4.0 41.7 3.3 13.3 0.7 33.7 0.0 37.7 33.0 0.7 44.0 0.7
7HVWLQJRI2&/3UHGLFWLRQ0RGHO ,Q9LYR 00$6 YV3UHGLFWHG'UDL]H
3UHGLFWHG'UDL]H
FRQVXPHUSURGXFWVWHVWHG U PDWHULDOV U PDWHULDOV
,Q9LYR'UDL]H 00$6 Figure 13.3 Comparison of predicted and actual Draize scores for consumer products including: shampoos (5), face/body soap (6), dishwashing liquids (4), laundry detergents (5), and skin lotions (4). The predicted Draize scores were calculated based on the ET-50 using the prediction equation shown in Figure 13.2.
142
ALTERNATIVE TOXICOLOGICAL METHODS
Table 13.4 EpiOcular QC Testing Results for Positive (0.3% Triton X-100) and Negative (ultrapure water) Controls Calendar Year
Tissue Lots
Triton ET-50 (min)
Neg. Control (OD)
Avg. Lot CV (%)
2000 1999 1998 1997 1996
60 84 85 81 47
23.1s6.0 22.6s5.0 25.2s5.6 22.9s4.7 24.9s6.3
1.441 1.433 1.354 1.343 1.274
5.5 5.6 5.5 5.4 5.2
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ET-50 (mins): BAK SDS 5.75 6.39 5.97 6.04 0.32 5.36
3.12 3.30 3.72 3.38 0.31 9.19
Triton 25.40 26.59 29.75 27.24 2.25 8.26
Intralaboratory Reproducibility Avg. CV (%) 7.75 7.14 9.56
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Table 13.6 ET-50 for Ultramild Materials for Which the Draize Test Is Insensitive In Vivo Concentration
Draize (MMAS)
ET-50 (min)
Benzalkonium chloride (BAK) 10.00% 5.00% 1.00% 0.30% 0.10% 0.03%
108.0 83.8 45.3 8.67 0 0
1.1 1.0 5.9 28.9 212.7 2053.0
Sodium dodecyl sulfate (SDS) 30.00% 15.00% 3.00% 1.00% 0.30% 0.10%
60.5 59.2 16.0 0.67 0 0
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CHAPTER
14
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RESULTS Histologic Characteristics of the Three-Dimensional Corneal Epithelial Constructs +LVWRORJLFDOO\WKHWKUHHGLPHQVLRQDOFRQVWUXFWVRI,+&(&UHVHPEOHGWKHVWUDWLÀHG FHOOXODURUJDQL]DWLRQRIKXPDQFRUQHDOHSLWKHOLXP )LJXUH 7KHQRQNHUDWLQL]HG VXSHUÀFLDOFHOOVZHUHÁDWWHQHGLQWKHSODQHRIWKHFRQVWUXFW7KHRXWHUPHPEUDQHVRI WKHVH FHOOV ZHUH WKURZQ LQWR PLFURYLOOL D IHZ PLFURQV LQ OHQJWK &HOOV LQ DOO OD\HUV H[KLELWHGDZHOOGHYHORSHGF\WRVNHOHWDOQHWZRUNDQGZHUHDWWDFKHGWRDGMDFHQWFHOOV E\ GHVPRVRPHV )LJXUHV DQG &HOOV RI WKH LQWHUPHGLDWH OD\HU GLVSOD\HG PRUHODWHUDOF\WRSODVPLFH[WHQVLRQVWKDQWKRVHLQWKHEDVDOOD\HUVLPLODUWRZLQJFHOOV LQKXPDQFRUQHDOHSLWKHOLXP0HDQWKLFNQHVVRIWKHFRQVWUXFWVZDVQP QP 6( Q VLPLODUWRWKDWRIQRUPDOKXPDQFRUQHDOHSLWKHOLXP
Figure 14.1 Light micrograph of a plastic section from a construct after 24 hr equilibration at 37°C. The tissue organization reveals a stratified appearance with columnar basal cells, defined wing cells, and flattened superficial cells. Toluidine blue and basic fuchsin, original magnification 160v.
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Figure 14.2 Transmission electron micrograph of the construct after 24 hr equilibration at 37°C. The superficial cells give rise to numerous villus processes. The nucleus is oriented parallel to the surface. Some small, spot-like junction arrangements are seen between cells at the surface. Desmosomes are present between adjacent cells. Original magnification 12,600v.
Figure 14.3 Transmission electron micrograph of basal cells of the three-dimensional construct. The nucleus of the basal cell is upright. Within the cell, there is an extensive cytoskeletal network. Mitochondria surround the nucleus and desmosomes join adjacent cells. Original magnification 10,000v.
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Figure 14.4 High-power transmission electron micrograph of the basal membrane of a basal cell revealing numerous mature hemidesmosomes. Also visible are the components of the hemidesmosomes: the intracellular membrane placode, filaments radiating through the membrane, and the typical band in what would be the lamina lucida. The amorphous band at the bottom is the polycarbonate support membrane of the construct. Original magnification 75,000v.
7KH EDVDO FHOO OD\HU FRQVLVWHG RI D VLQJOH OD\HU RI FROXPQDU FHOOV ZKRVH EDVDO PHPEUDQHVUHVWHGRQWKHLQVHUW )LJXUHVDQG $WWKHXOWUDVWUXFWXUDOOHYHO WKHEDVDOPHPEUDQHVUHYHDOHGPDWXUHKHPLGHVPRVRPHVZLWKLQWHUQDOSODFRGHVDQG ÀODPHQWVUDGLDWLQJWKURXJKWKHFHOOPHPEUDQH )LJXUH 7KHF\WRSODVPRIWKH FROXPQDU FHOOV ZDV ÀOOHG ZLWK PDWV RI ÀODPHQWV DQG VSDUVH PLWRFKRQGULD ZHUH ORFDWHGDURXQGWKHQXFOHXV Biochemical Characteristics of the Three-Dimensional Corneal Epithelial Constructs 7KH WKUHHGLPHQVLRQDO FRQVWUXFWV ZHUH DVVHVVHG IRU LPPXQRUHDFWLYLW\ WR WKH HSLWKHOLDO FHOOVSHFLÀF NHUDWLQV D JURXS RI ZDWHULQVROXEOH SURWHLQV WKDW IRUP WKH PDMRUFRPSRQHQWVRIWKHF\WRVNHOHWDOFRPSOH[.HUDWLQVDUHGLYLGHGLQWRDFLGLFDQG EDVLF VXEIDPLOLHV DQG DUH IRXQG WRJHWKHU LQ SDLUV7KH FKDUDFWHUL]DWLRQ RI VSHFLÀ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ÀFNHUDWLQ )LJXUH
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Figure 14.5 Western blots of the acidic (AE1) and the basic (AE3) keratin family in the threedimensional construct: A. The reactivity pattern for AE1 in the three-dimensional construct (Lane 1) is similar to that of immortalized human corneal epithelial cells (IHCEC) cultured for 4 weeks (Lane 2). B. The reactivity pattern for AE3 (Lane 1) is similar to that of cultured IHCEC at 1 week (Lane 2) and 2 weeks (Lane 3) and human donor corneal epithelial cells obtained within 24 hr after death (Lane 4). The molecular weights of several cytokeratin isoforms recognized by the respective antibodies are indicated below under AE1 and AE3, respectively.
Figure 14.6 Western blot of the 64 kDa cornea-specific keratin AE5 in the three-dimensional construct. Lanes show the construct on Day 0 (immediately upon arrival), Day 1 (24 hr equilibration at 37°C), and Day 2 (48 hr at 37°C), as well as the fresh human donor corneal epithelial cell positive control (+) and the rabbit lacrimal gland negative control (–).
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Figure 14.7 Western blot of laminin in the three-dimensional construct. One band is seen at 220 kDa and another at 440 kDa. Lane 1, three-dimensional construct; Lane 2, fresh human corneal donor epithelial cells from cadaver eyes; Lane 3, immortalized human corneal epithelial cells cultured for 4 weeks.
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Figure 14.9 Top: Western blot analysis for phosphorylated (active) p42/p44 mitogen-activated protein kinase (MAPK) after treatment with benzalkonium chloride. Positive control (Ctrl +), epidermal growth factor-stimulated A431 cells. Negative control (Ctrl –), unstimulated A431 cells. Bottom: The histogram shows the increase in intensity level (activity) of p42/p44 with increasing concentrations of benzalkonium chloride (BAK). The absence of activity with 0.1% benzalkonium chloride is likely due to cell death resulting from toxic insult.
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The Human Corneal Epithelial HCE-T TEP Assay for Eye Irritation: Scientific Relevance and Summary of Prevalidation Study Results 6KHUU\/:DUG0D[LPR*DFXOD-UDQG+HQU\)(GHOKDXVHU
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Corneal epithelium
Ocular surface (cornea, conjunctiva and iris)
Rabbit (in vivo)
Test Tissue
Human (in vitro)
Test Species
Superficial to eye
Superficial to cell cultures
Exposure Route
Until washed out by blinking and tearing (<5 min)
5 min
Exposure Time 5 concentrations with highest equal to that evaluated in Draize test Concentration defined by toxicologist (neat or diluted)
Concentration Tested Concentration of test material causing 15% of fluorescein to penetrate through the corneal epithelium (FR85) Cornea: area of damage and opacity Conjunctiva: chemosis, redness and discharge Iris: degree of effect
Endpoint Measured
Area of corneal damage is related to epithelial cytotoxicity and junctional disruption; opacity is related to penetration of test material into stroma. Conjunctiva damage similar to cornea—epithelial toxicity and penetration. Iris—material has penetrated to cause more severe damage.
Cytotoxicity and junctional disruption in the corneal epithelium
Mechanism of Tissue Damage
Table 15.1 Similarities and Differences in the Endpoint Measured in the HCE-T TEP Assay and in the Draize Eye Irritation Test
THE HUMAN CORNEAL HCE-T TEP ASSAY 167
168
ALTERNATIVE TOXICOLOGICAL METHODS
tear film
tight junction
desmosomal junction stroma Figure 15.2 Cross-sectional diagram of the human corneal epithelium. A drawing illustrating the multiple cell layers, the apical tight junctions, and the intercellular desmosomal junctions of the corneal epithelium.
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Figure 15.3 Cross sections of stratified HCE-T cultures visualized by transmission electron microscopy following 5-min superficial exposures to (A) cell culture medium (KGM) (5000v); and (B) 0.01% benzalkonium chloride (BAC) (6000v). Ap = apical surface of the culture; M = collagen membrane. [Photos contributed by S.D. Dimitrijevich.]
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Figure 15.4 Cross sections of stratified HCE-T cultures, visualized by transmission electron microscopy, which were fixed in a special buffer to retain the mucin layer. The dark-stained surface material is the mucin produced by the corneal epithelial cells. (A) A continuous layer of mucin is found on the surface of a differentiated culture (maintained at the air–liquid interface in serum-free medium containing 1.15 mM calcium) in which the TER is high (tight junctions are intact); and (B) mucin is found between the cells in this non-differentiated culture (maintained submerged in serum-free medium containing 0.15 mM calcium) in which the TER is low (tight junctions not intact).
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Day 3
% of Control
100 80 60 40 20 0 0.00
TEP MTT TER
0.02
0.04
SDS(%)
0.06
0.00
0.02
0.04
0.06
SDS(%)
Figure 15.5 Three assays were used to evaluate the dose-dependent effects of sodium dodecyl sulfate (SDS) on HCE-T cultures on the day of treatment (day 1), and 48 hr later (day 3). TEP, transepithelial permeability to fluorescein; TER, transepithelial electrical resistance; MTT, cell viability assay using the MTT dye [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]. Each dose is represented by triplicate cultures, and the error bars are the standard deviations.
172
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30.0 25.0 20.0 15.0 10.0 5.0
–20.000
0.0 0.000
20.000
40.000
60.000
80.000
100.000
120.000
24 Hr Fluorescein Figure 15.6 Fluorescein staining data from rabbit eyes following surfactant-containing formulation exposure correlates well with Draize tissue score data (modified maximum average score, MMAS and modified maximum average corneal score, MMCS) from the same animals. The error bars are the standard deviations; n = 5–6 rabbits evaluated per Draize test; and n = 2–4 rabbits evaluated for fluorescein staining at 24 hr. [Data from Gettings et al., 1996.]
174
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120
MMAS R2 = 0.632 MMCS R2 = 0.7057
100
24 Hr R2 = 0.7361 Fluorescein
Draize Data
80
60
40
20
-0.8
-0.6
-0.4
-0.2
0 0
0.2
0.4
0.6
-20
logFR85 Figure 15.7 The relationship between the in vivo 24-hour fluorescein staining data and the in vitro log FR85 values for the subset of the CTFA formulations that are represented in the HCE-T TEP assay Prediction Model. The error bars are the standard deviations; n = 2 TEP assays; n = 5–6 rabbits per Draize test; and n = 2–4 rabbits per fluorescein data.
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sodium laureth sulfate cocamidopropyl betaine
Surfactant Ingredients
5.0 0.5 12.0 2.0 0.4 0.15
25.0 15.0
1.14 1.0 0.67 0.44 15.0 6.0 1.5 1.5
25.0 5.0
Percent Formula (w/w)
anionic nonionic — —
anionic anionic nonionic nonionic
cationic nonionic cationic nonionic amphoteric anionic cationic nonionic
anionic amphoteric
Surfactant Class
MAS 57.4 CS 38.0 COS 2.0
MAS 37.8 CS 20.0 COS 1.0
MAS 22.0 CS 10.0 COS 0.5
MAS 4.8 CS 0.0 COS 0.0 MAS 14.2 CS 0.0 COS 0.0
Draize Scoresb
Concentration used in Draize test, and initial concentration for dilution in TEP assay. MAS is the maximum average score; CS is the corneal score; COS is the corneal opacity score. Federal Hazardous Substances Act (FHSA) classifications: –, negative; +, positive; –/+ repeat test (FHSA, 1979).
100%
Hair conditioner
a
2.5%
Bubble bath
Product Type
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Table 15.2 Prevalidation Study Test Materials and Draize Data
+
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–/+
–
–
FHSA Classc
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LogFR85 Figure 15.8 Overlay of the prevalidation study test results for five test materials in the MAS Prediction Model (PM). The solid line represents the nonlinear regression curve of the MAS PM, and the dashed lines are the 95% confidence intervals. (A) Overlay of the 60 log FR85 values in the MAS PM. There are 12 values for each of the five test materials on the plot, but, due to overlap in the data, all 12 points may not be distinct. (B) Fit of the average log FR85 value for each of the five test materials in the MAS PM. The result for each test material is the average of 12 assays.
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Table 15.3 The Nonirritant/Irritant Classification of the Five Test Materials as Determined by the Draize Test and by the HCE-T TEP Assay Test Material
Draize Score Classificationa
TEP Assay MAS PMb
TEP Assay CS PMb
TEP Assay COS PMb
Bubble bath Hair conditioner Cleansing gel Shampoo 1 Shampoo 2
1 1 2 2 2
1 1 2 2 2
1 1 2 2 2
1 1 1 2 2
a
Draize classification was the same across the MAS, CS, and COS scores, except for the cleansing gel which was an irritant by the MAS and CS, but a nonirritant by the COS. b Draize scores for the classifications: nonirritant MAS e 15; nonirritant CS < 5; nonirritant COS < 0.667. Note: Draize maximum average score, MAS; corneal score, CS; corneal opacity score, COS. Table 15.4 Average HCE-T TEP Assay Results for Five Test Materials in Three Laboratories; The Predicted MAS and Class from the TEP assay Are Compared to the Draize MAS and Class for Each Test Result Test Material Bubble bath Hair conditionerc Cleansing gel Shampoo 1 Shampoo 2 a b
c
Draize MASa
Draize Classb
Predicted MASa
Predicted Classb
4.8 14.2 22.0 37.8 57.4
1 1 2 3 4
4.20 1.82 16.15 34.62 41.18
1 1 2 3 3
MAS, maximum average score. The four classification cutoffs for MAS are based on the scheme proposed by Kay and Calandra (1962): MAS 0–15, minimal (class 1); MAS 15.1–25, mild (class 2); MAS 25.1–55, moderate (class 3); MAS > 55, severe (class 4). Hair conditioner was less water soluble than other test materials, which may account for its underprediction when it was diluted.
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Table 15.5 Variability in the Draize MAS Compared to the Predicted MAS for the HCE-T TEP Assay Prevalidation Study Data Test Material
Intralab CV (%) for Draize MASa
Bubble bath
22.82
Hair conditionerd
19.67
Cleansing gel
53.24
Shampoo 1
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Shampoo 2
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Interlab CV (%) for Predicted MASc 6.03
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12.49
2.83
a
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CHAPTER
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Allergic Contact Hypersensitivity: Mechanisms and Methods* 'HQLVH06DLOVWDG
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ICCVAM
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REGULATORY ACTION Figure 17.1 ICCVAM review process: the local lymph node assay. (Modified from Sailstad, 2000.)
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BACKGROUND Contact Hypersensitivity and Allergic Contact Dermatitis Test Methods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ÀFDWLRQ RI &+ SRWHQWLDO RI FKHPLFDOV7KH UHTXHVW DQG LQWHQGHG XVH RI D QHZ PHWKRG PXVW EH FOHDU DQG LQ WKLV FDVH WKH VSRQVRUV ZHUHQRWUHTXHVWLQJHOLPLQDWLRQRIH[LVWLQJWHVWVEXWDFFHSWDQFHRIWKH//1$DV DQDOWHUQDWLYHPHWKRGWKDWFRXOGEHXVHGLQOLHXRIWKHWUDGLWLRQDO*3WHVWLIGHVLUHG DQGDSSURSULDWH Background: An Example of the Dilemma of GP Tests ,Q ZRUNZDVLQLWLDWHGLQWKH([SHULPHQWDO7R[LFRORJ\'LYLVLRQ (7' RI 7KH (QYLURQPHQWDO 3URWHFWLRQ $JHQF\ (3$ DV DQ LQWHUDJHQF\ DJUHHPHQW ZLWK WKH$UP\ WR HYDOXDWH WKH DOOHUJLF SRWHQWLDO RI WKUHH G\HV 6DLOVWDG HW DO 7KHVHG\HVZHUHXVHGLQ0FRORUHGVPRNHJUHQDGHV7KHUHZDVFRQFHUQ IRUWKHLQGLYLGXDOVUHSHDWHGO\H[SRVHGWRWKHVHG\HVLQWKHPDQXIDFWXULQJIDFLO LWLHVDQGWKHLUSRWHQWLDOWRGHYHORSDOOHUJLHVRUK\SHUVHQVLWLYLW\WRWKHFKHPLFDOV 7KH QDWXUH RI WKHVH G\HV DFFHOHUDWHG WKH LQYHVWLJDWLRQ LQ RXU ODERUDWRU\ IRU DYDLODELOLW\ HIIHFWLYHQHVV DQG SUDFWLFDOLW\ RI PHWKRGV WR DVVHVV WKH VHQVLWL]LQJ SRWHQWLDORIFKHPLFDOV +\SHUVHQVLWLYLW\ LV GHÀQHG DV H[FHVVLYH KXPRUDO RU FHOOXODU UHVSRQVH WR DQ DQWLJHQ ZKLFK FDQ OHDG WR WLVVXH GDPDJH 7KH $UP\ '\H 3URMHFW UHTXLUHG WKH HYDOXDWLRQRIWKHDVWKPDOLNH7\SH,K\SHUVHQVLWLYLW\DQGWKH7\SH,9FRQWDFWK\SHU VHQVLWLYLW\UHVSRQVHV ,QWHUDFWLRQEHWZHHQSXOPRQDU\DQGLPPXQRWR[LFRORJLVWVLQ(7'SURYLGHGQHF HVVDU\ H[SHULHQFH WR HYDOXDWH WKH SXOPRQDU\ UHVSRQVHV EURQFKRFRQVWULFWLRQ DQG F\WRSKLOLFDQWLERG\IRUPDWLRQIURPDUHSHDWHG G\H LQKDODWLRQH[SRVXUHDQGFKDO OHQJHV\VWHPWKDWZDVSUHYLRXVO\HVWDEOLVKHG +RZHYHUWKHODFNRIH[SHUWLVHLQWKH&+DQG$&'DUHDUHTXLUHGDQH[WHQVLYH UHYLHZRIH[LVWLQJPRGHOVDQGPRGLÀ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Mechanisms: Contact Hypersensitivity/Allergic Contact Dermatitis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nduction Phase 7KH ÀUVW SKDVH VHQVLWL]DWLRQ LQGXFWLRQ SKDVH RULJLQDWHV XSRQ HSLFXWDQHRXV DSSOLFDWLRQ RI D KDSWHQ ,W LV EHOLHYHG WKDW KDSWHQV FRXSOH WR FDUULHU SURWHLQV RQ GHUPDODQGHSLGHUPDOFHOOVWREHFRPHIXOO\LPPXQRJHQLF7KHGHQGULWLFFHOOVZLWKLQ VNLQ/DQJHUKDQVFHOOV /& WDNHXSDQGSURFHVVWKHDQWLJHQ KDSWHQ DQGPLJUDWH WRWKHUHJLRQDOGUDLQLQJO\PSKQRGHZKHUHWKH\SUHVHQWWKHDQWLJHQWRO\PSKRF\WHV
INDUCTION PHASE
ELICITATION PHASE
CHEMICAL ALLERGEN
EDEMA AND ERYTHEMA
LANGERHANS CELL (LC)
INITIAL NONSPECIFIC INFLAMMATORY MEDIATORS MIGRATION TO LOCAL LYMPH NODE
LC AND LYMPHOCYTE INTERACTION
“PRIMED” LYMPHOCYTES
CELLULAR INFLUX CYTOKINES, COSTIMULATORY, ADHESION MOLECULES INCREASE
SPECIFIC RESPONSE
LYMPHOCYTE PROLIFERATION
Figure 17.2 Illustration of contact hypersensitivity (CH) mechanisms: induction and elicitation phase. During the induction phase of CH, immature dendritic cells of the skin, called “Langerhans cells” (LC), effectively uptake and process the allergen. Simultaneously, epidermal keratinocytes and the LCs themselves release cytokine mediators which assist the LCs in the migration to the draining lymph node and maturation into effective antigen-presenting cells. In the lymph node, LC to T lymphocyte interactions occur, which is followed by lymphocyte proliferation. The proliferation results in “primed” effector lymphocytes which maintain a memory for the specific allergen. The elicitation phase of CH appears to involve two mechanisms of action. Initially, allergens cause direct cellular action releasing a series of nonspecific inflammatory mediators. These mediators are responsible for some of the cellular influx into the site of allergen challenge. Additionally, the “primed” lymphocytes are called into the area in a very specific response. These responses and the cytokines, costimulatory factors, and adhesion molecules act to produce the end results of erythema and edema. (Modified from Selgrade et al., 2001.)
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TEST METHODS Traditional Contact Hypersensitivity Tests—Guinea Pig 7KH *3 KDV EHHQ LGHQWLÀHG DV DQ DQLPDO PRGHO IRU &+ IRU PRUH WKDQ \U 7KHUHDUHVHYHUDOJXLQHDSLJWHVWVIRUWKHDVVHVVPHQWRI&+7KHVHLQFOXGHWKHJXLQHD SLJPD[LPL]DWLRQWHVW *307 WKH%XHKOHUDVVD\ %$ )UHXQG·VFRPSOHWHDGMXYDQW WHVWVSOLWDGMXYDQWRSHQHSLGHUPDODQG'UDL]H7KHWHVWVPRVWFRPPRQO\DFFHSWHG DQGXVHGE\UHJXODWRU\DJHQFLHVDUHWKH*307DQG%$ .OHFDN :KLOH WKHUH DUH YDULDWLRQV LQ WKH *3 WHVW PHWKRGV WKH *307 DQG WKH %$ DUH WKH PRVW FRPPRQ )LJXUH 6DLOVWDG %RWK RI WKHVH WHVWV UHTXLUH DQLPDOVSHUWHVWJURXS7KH\UHO\RQWKHVHQVLWL]DWLRQDQGFKDOOHQJHSKDVHVUHTXLULQJ DERXW PRQWK WR SHUIRUP DQG WKH YLVXDO DVVHVVPHQW RI WKH HU\WKHPD UHVSRQVH .OHFDN 7KH*307GHSHQGVRQWKHLQGXFWLRQDQGHOLFLWDWLRQSKDVHRI&+UHTXLULQJWKH UHSHDWHGGHUPDOVHQVLWL]DWLRQRIJXLQHDSLJVDQGWKHQWKHHYDOXDWLRQRIWKHFKDOOHQJH RUHOLFLWDWLRQUHVSRQVH7KLVWHVWLVQRWHGIRULWVDELOLW\WRGHWHFWZHDNVHQVLWL]HUV,W KDVDOVREHHQFULWLFL]HGIRUJLYLQJIDOVHSRVLWLYHUHVSRQVHV7KLVPHWKRGDOVRUHTXLUHV WKHXVHRISRWHQWLDOO\LUULWDWLQJ)UHXQG·VFRPSOHWHDGMXYDQWYLDLQWUDGHUPDOLQMHFWLRQV 2Q GD\ WKHUH LV D FKDOOHQJH H[SRVXUH ZKLFK LV YLVXDOO\ DVVHVVHG IRU HU\WKHPD KU ODWHU7KH DVVHVVPHQW RI WKH FKDOOHQJH VLWH LV JUDGHG QR YLVXDO FKDQJH SDWFK\ HU\WKHPD FRQÁXHQW HU\WKHPD FRQÁ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
200
ALTERNATIVE TOXICOLOGICAL METHODS
Guinea Pig Maximization Test
Buehler Assay 20 animals/group
Topical antigen application: ID injection w/ or w/o FCA
Induction
Topical antigen application: closed patch
Days 5-8
Day 20–22 topical application
48, 72 h after challenge
Days 0, 6–8, and 13–15
Challenge
Day 27–28 topical application (untreated flank for 6 h)
Endpoint Analysis
21, 24, 48 h after removing patch
erythema
Figure 17.3 Guinea pig models. (Modified from Sailstad, 2002.)
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ALLERGIC CONTACT HYPERSENSITIVITY: MECHANISMS AND METHODS
Days 1, 2, 3
201
Agent applied to ears
IV tail injection of isotope
Day 6
Day 6: 5 h post IV injection
Lymph nodes removed
Proliferation measured: Isotope incorporation expressed as disintegration per minute (dpm) Figure 17.4 The local lymph node assay. (Modified from Sailstad, 2002.)
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34.1 32.3 34.9 31.1 22.1 15.1
212.0 204.9 216.9 218.5 190.4
2571.9 2175.7 2322.7 2260.1 2261.4 1788.9
0.5 1 2 4 10 24
0.5 1 2 4 10
0.5 1 2 4 10 24
285.9 405.4 355.2 502.7 435.5 627.2
24.2 21.7 23.7 21.0 28.2
3.0 3.2 4.1 4.9 5.6 8.1
0.2 0.2 0.2 0.2 0.3 0.4
Cover
— — — — — —
— — — — —
— — — — — —
0.1 0.1 0.2 0.3 0.4 0.7
Carbon Filter
74.2 95.2 98.9 93.3 88.3 86.0
9.2 8.7 13.3 8.7 10.8
1.4 1.3 1.2 1.2 1.2 0.9
0.1 0.2 0.2 0.2 0.3 0.4
0.3 0.3 0.1 0.1 0.2 47.9
0.01 0.01 0.01 0.01 0.1
270 Qg/cm2 3.40 0.01 3.21 0.02 4.91 0.02 3.21 0.7 3.99 3.5 2934 Qg/cm2 2.53 0.1 3.24 0.2 3.37 0.5 2.18 2.3 3.01 15.2 2.92 97.1
0.001 0.001 0.005 0.003 0.3 2.1
0.001 0.001 0.001 0.001 0.01 0.2
Feces
42 Qg/cm2 3.18 0.004 2.99 0.02 2.78 0.02 2.81 0.2 2.66 2.1 1.83 5.5
0.01 0.01 0.01 0.02 0.2 0.5
Urine
3 Qg/cm2 4.00 6.00 7.17 7.93 9.63 12.53
Skin Qg/cm2 %
Absorbed is sum of urine, feces, cage wash, and carcass. Source: Lythgoe, 1990a.
a
0.22 2.2 2.0 1.8 1.2 0.9
Wash
0.5 1 2 4 10 24
Exposure (hr)
0.4 0.2 0.6 1.1 1.6 8.4
0.04 0.05 0.05 0.06 0.56
0.01 0.01 0.3 0.01 0.4 0.6
0.001 0.001 0.001 0.01 0.01 0.02
Cage Wash
40.3 150.9 98.5 74.8 114.3 216.7
12.9 15.1 8.1 12.3 21.1
1.7 2.5 1.8 3.6 7.0 4.4
0.1 0.1 0.2 0.3 0.4 0.2
Carcass
40.9 153.04 99.6 78.3 131.3 369.9
12.9 15.2 8.2 13.0 25.2
1.7 2.5 1.9 3.9 9.8 12.6
0.1 0.1 0.2 0.3 0.6 0.9
1.39 5.22 3.39 2.67 4.47 12.61
4.79 5.63 3.03 4.83 17.52
3.95 5.98 4.38 9.24 23.06 29.64
3.79 4.28 7.32 9.93 19.55 31.37
Absorbed Qg/cm2 %
Table 18.1 Acetochlor In Vivo (Mean dose distribution as Qg equivalents of acetochlor per cm2. Mean of four male rats.)
214 ALTERNATIVE TOXICOLOGICAL METHODS
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Table 18.2 Acetochlor In Vitro (Mean dose distribution in vitro dermal absorption in rat skin. Mean of four to seven skin samples. Results presented as Qg/cm2.) Exposure (hr)
Absorbed Qg/cm2 %
Wash
Skin Qg/cm2 %
Donor
Loop
3.60 3.77 3.84 2.98 2.52 2.75
0.08 0.04 0.03 0.05 0.01 0.02
0.55 0.44 0.50 0.43 0.59 0.41
2.30 2.47 3.09 2.28 2.11 1.73
1.41 0.97 0.57 0.58 0.78 0.14
1.03 1.02 1.25 1.38 0.98 1.21
4.06 5.22 8.84 5.09 7.11 4.53
10.5 14.3 19.4 4.41 5.59 5.52
13.4 16.7 8.25 11.6 11.6 13.3
Total Recovered
3.02 Qg/cm2 0.05 1 2 4 10 24
0.41 0.82 1.14 1.37 2.15 2.12
13.68 27.12 37.78 45.30 71.19 70.13
1.73 1.32 0.95 0.80 0.17 0.13
0.11 0.11 0.12 0.09 0.08 0.08
2.89 2.73 2.73 2.75 2.99 2.77
47.3 Qg/cm2 0.5 1 2 4 10 24
2.02 3.90 10.3 16.0 20.7 28.7
4.27 8.25 21.78 33.83 43.76 60.68
25.6 28.2 18.7 13.6 7.1 2.9
1.09 1.17 1.46 1.08 1.00 0.79
31.1 35.3 32.2 32.7 30.6 33.7
318 Qg/cm2 0.5 1 2 4 10 24
6.58 21.4 33.4 80.9 182 246
2.07 6.73 10.50 25.44 57.23 77.36
283 383 261 262 158 67
12.9 16.6 28.1 16.2 22.6 14.4
325 452 350 375 379 347
3095 Qg/cm2 1 2 4 10 24
17 104 137 264 757
0.55 3.36 4.43 8.53 24.46
2411 3146 2103 2344 1598
143 187 111 196 202
4.62 6.04 3.59 6.33 6.53
311 313 316 294 182
118 138 161 156 127
2999 3858 2828 3253 2866
Note: The 0.5-hr exposure was not performed at the high dose. Source: Clowes and Scott, 1990a.
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ALTERNATIVE TOXICOLOGICAL METHODS
Table 18.3 Acetochlor In Vitro (Mean dose distribution in vitro dermal absorption in human skin. Mean of six skin samples. Results expressed as Qg/cm2.) Exposure (hr)
Absorbed Qg/cm2 %
Wash
Skin Qg/cm2 %
Donor
Loop
Total Recovered
3.02 Qg/cm2 2 10 24
0.015 0.074 0.261
0.486 2.44 8.63
2.36 2.61 1.54
0.273 0.271 0.092
9.04 9.98 3.03
0.035 0.136 0.098
0.359 0.194 0.244
3.12 3.86 2.80
2.61 2.31 3.09
3.04 3.28 2.24
47.3 Qg/cm2 2 10 24
0.140 1.70 3.63
0.296 3.59 7.68
34.2 30.4 26.2
1.89 4.43 1.63
3.99 9.37 3.45
41.9 42.7 37.4
318 Qg/cm2 2 10 24
0.816 5.69 43.6
0.256 1.79 13.7
239 221 245
18.9 12.1 18.2
5.94 3.82 5.72
70.4 91.8 62.3
22.4 15.8 15.8
352 347 384
3095 Qg/cm2 2 10 24
4.57 22.4 32.5
0.148 0.722 1.05
2796 2057 1669
51.5 80.5 38.4
1.67 2.60 1.24
772 896 671
260 259 217
3884 3314 2628
Source: Clowes and Scott, 1990b.
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217
Table 18.4 Comparison of the Percent Absorbed In Vitro and In Vivo in Rat Skin; Data Are from Tables 18.1 and 18.2 Dose Qg/cm2) (Q
0.5
1.0
2.0
4.0
10.0
24.0
Exposure Duration (hr) 3.0 in vivo 3.02 in vitro 42.5 in vivo 47.3 in vitro 270 in vivo 318 in vitro 2934 in vivo 3095 in vitro
3.8 13.7 4.0 4.3 4.8 2.1 1.4 —
4.3 27.1 6.0 8.2 5.6 6.7 5.2 0.6
7.3 37.8 4.4 21.8 3.0 10.5 3.4 3.4
9.9 45.3 9.2 33.8 4.8 25.4 2.7 4.4
19.5 71.2 23.1 43.8 9.3 57.2 4.5 8.5
31.4 70.1 29.6 60.7 17.5 77.4 12.6 24.5
4.6 3.7 5.3 1.6
3.7 1.9 6.2 1.9
2.2 2.1 4.4 1.9
Ratio in Vitro/in Vivo 3.02/3.0 47.3/42.4 318/270 3095/2934
3.6 1.1 0.4 —
6.3 1.4 1.2 0.1
Dose 1 Dose 2
5.2 5.0 3.5 1.0
Dose 3 Dose 4
7 6
Ratio vitro/vivo
5 4 3 2 1 0 0
5
10
15
20
25
Exposure (h)
Figure 18.1 The ratio of the in vitro absorption to the in vivo absorption with exposure duration in the rat. Dose ratios 1, 2, 3, and 4 are in order of low dose ratio to high dose ratio. Data are from Table 18.4.
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218
ALTERNATIVE TOXICOLOGICAL METHODS
Table 18.5 Comparison of the Percent Absorbed In Vitro in Rat and Human Epidermal Membrane Preparations; Data Are from Tables 18.2 and 18.3 Dose Qg/cm2) (Q
2.0
10.0
24.0
Exposure Duration (hr) 3.03 rat human 47.3 rat human 318 rat human 3095 rat human
37.8 0.49 21.8 0.30 10.5 0.26 3.4 0.15
3.03 47.3 318 3095
77.1 72.7 40.4 22.7
71.2 2.44 43.8 3.59 57.2 1.79 8.5 0.72
70.1 8.63 60.7 7.68 77.4 13.7 24.5 1.05
Ratio Rat/Human 29.2 12.2 32.0 11.8
8.1 7.9 5.6 23.3
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Ratio rat/human
60 50 40 30 20 10 0 0
5
10
15
20
25
Duration of Exposure (h)
Figure 18.2 The ratio of the rat in vitro absorption to the human in vitro absorption with exposure duration. Dose ratios 1, 2, 3, and 4 are in order of low dose ratio to high dose ratio. Data are from Table 18.5.
VALIDATING IN VITRO DERMAL ABSORPTION STUDIES
219
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CHAPTER
19
A Molecular Diagnostic Approach to Irritant or Allergic Patch Testing Using the DermPatch 0HOLVVD0)LW]JHUDOG9HUD%0RUKHQQ6WDFH\%+XPSKUH\ 1LUPDOD-D\DNXPDUDQG/DZUHQFH$5KHLQV
CONTENTS %DFNJURXQG 0DWHULDOVDQG0HWKRGV 7DSH6WULSSLQJDQG([WUDFWLRQRI7RWDO51$ 5LERQXFOHDVH3URWHFWLRQ$VVD\ ,QGXFWLRQRI(U\WKHPDWRXV5HDFWLRQVRQWKH6NLQ 5HVXOWV 'LVFXVVLRQ 5HIHUHQFHV
BACKGROUND (OXFLGDWLRQRIWKHPROHFXODUHYHQWVWKDWOHDGWRLUULWDQWFRQWDFWGHUPDWLWLV ,&' RU DOOHUJLF FRQWDFW GHUPDWLWLV $&' LQ KXPDQ VNLQ ZLOO EH KHOSIXO LQ GHYHORSLQJ PHFKDQLVWLFWR[LFRORJLFDODVVD\VWKDWSHUPLWWKHUHGXFWLRQUHÀQHPHQWRUUHSODFH PHQW RI DQLPDOV LQ SURGXFW VDIHW\ WHVWLQJ 7KH ÀUVW DOWHUQDWLYH WR[LFRORJLFDO WHVW DSSURYHGE\,&&9$0ZDVDPHFKDQLVWLFWHVWWKHPXULQHORFDOO\PSKQRGHDVVD\ 0//1$ ZKLFKUHSODFHGWKHJXLQHDSLJPD[LPL]DWLRQWHVWLQ 7KH0//1$ PHDVXUHVO\PSKRF\WHSUROLIHUDWLRQWKURXJK+PHWK\OWK\PLGLQHXSWDNHLQWKHGUDLQ LQJ O\PSK QRGHV RI DQLPDOV WRSLFDOO\ H[SRVHG WR WKH WHVW DUWLFOH 6LQFH WKH WHVW LV EDVHGRQHYHQWVRFFXUULQJGXULQJWKHLQGXFWLRQSKDVHRIDOOHUJLFFRQWDFWGHUPDWLWLV
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221
222
ALTERNATIVE TOXICOLOGICAL METHODS
Table 19.1 Skin Cytokins (adapted from Gerberick et al., 1998) Cytokines IL-1E IL-1F IL-3 IL-6 IL-7 IL-8 IL-10 IL-12 IL-15 G-CSF M-CSF GM-CSF TGF-E TGF-F TNF-E MIP-1E MIP-1F IP-10
Constitutive or inducible expression in Langerhans cells Keratinocytes Fibroblasts – + – + – – – –
+ – + + + + +
– – – – + – + + –
+ + + + + + – + +
+ + + – + – + + + – + – + – – – +
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IRRITANT OR ALLERGIC PATCH TESTING USING THE DERMPATCH
223
Table 19.2 mRNA Cytokine Profiles from Human Skin Biopsy or Human Cell Samples
TNF-E IFN-K IL-2 GM-CSF IL-1E (human cells) IL-1F (human cells) IL-4 IL-6 IL-10 IL-12 p35 (human cells) IL-12 p40 (human cells)
ACD
ICD
increased increased increased increased dependent on allergen increased increased increased increased no change increased
increased increased increased increased increased increased not determined not determined no change no change no change
Source: Adapted from Wakem and Gaspari, 2000.
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Allergic
Irritant
low molecular weight, lipid soluble less critical ++++ necessary interaction of antigen with primed T cells ++++ early ++++ ++ increased ++
acids, alkalies, surfactants, solvents, oxidants, enzymes more critical ++ not necessary damage to keratinocytes
Source: Adapted from Marks and DeLeo.
+++ later ++++ ++ decreased ++
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ALTERNATIVE TOXICOLOGICAL METHODS
Table 19.4 Clinical and Histological Aspects of Contact Dermatitis Feature
Allergic
Irritant
Itch Pain, burning Erythema Vesicles Pustules Hyperkeratosis Fissuring Sharp demarcation Reaction delay after contact Spongiosis Dermal edema Necrotic keratinocytes Ballooning degeneration Lymphocytic infiltrate Neurotrophilic infiltrate
++++ (early) ++ ++++ ++++ + ++ ++ yes days ++++ ++++ ++++ + ++++ +
+++ (late) ++++ (early) ++++ + +++ +++ ++++ yes minutes to hours ++++ ++++ ++++ +++ ++++ +++
Source: Adapted from Marks and DeLeo.
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Tissue Culture Well Culture Insert ALI Tissue Membrane Medium
Figure 20.1 Schematic representation of the air–liquid interface (ALI) tissue culture technique. Table 20.1 Epithelial Tissues Successfully Cultured at the ALI Skin equivalentsa Keratinocytes only Keratinocytes plus fibroblastsb Keratinocytes plus melanocytes Keratinocytes plus Langerhans cells Ocular corneal epitheliuma Tracheal/bronchiala epithelium Tracheal/bronchial submucosal glands Vaginal epitheliuma,b Gingival epitheliuma,b a b
Cultured at MatTek Corp. In development.
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Figure 20.2 (A) Histological cross section of H&E stained EpiDerm-200. Magnification = 440v. (B) Transmission electron micrograph of ruthenium tetroxide stained intercellular lamellar lipid sheets (150,000v).
Figure 20.3 Top macroscopic view of MelanoDerm tissues containing normal human melanocytes derived from Black donor skin (400v magnification).
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• Asian (A) • Black (B) • Caucasian (C) Figure 20.4 Development of pigmentation in MelanoDerm tissue produced with melanocytes derived from various skin phototypes. The figure shows the top macroscopic view of tissue inserts. Day 0 indicates the day of shipment of a fully differentiated tissue. Pigmentation develops during additional culture of the fully differentiated tissue by the end user.
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10-7M MSH, 3 ng/ml FGF 5x10-8 MSH, 1.5 ng/ml FGF 10-8 MSH, 0.3 ng/ml FGF No MSH or FGF added Figure 20.5 Pigmentation of MelanoDerm tissue (Black melanocytes) induced by E-MSH and F-FGF as shown by top macroscopic view of tissue inserts. Tissues were cultured in media containing the indicated concentrations of growth factors for up to 20 days following shipment of commercial MelanoDerm tissue.
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Day:
3
7
10
14
Kojic Acid—topical
Control
Figure 20.6 Lightening effect of topical kojic acid on MelanoDerm tissue. Tissues containing Black melanocytes were treated topically with 1% kojic acid for the indicated number of days (25 Ql applied topically every other day). The top macroscopic view of the tissues reveals the visually observable lightening effect.
Table 20.2 Lightening of MelanoDerm Tissue by Topical Treatment with 1% Kojic Acid Treatment H2O 1% kojic acid
Qg/tissue) Melanin content (Q Day 10 Day 14 19.9 11.4
35.3 18.3
Figure 20.7 EpiDerm-FT tissue contains normal human fibroblasts cultured within a collagen type I dermal matrix. A fully differentiated epidermis derived from normal human keratinocytes is cultured on the top of the dermis.
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Figure 20.8 Immunohistochemical staining of HLA-DR on human Langerhans cells within (A) ImmunoDerm tissue and (B) excised human skin.
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MTT (% untreated control)
120
Chloropromazine
100 80
-UVA +UVA 42%
60 68% 40 87%
74%
20
-2%
0 0.001
0.003
0.010
0.032
0.100
[Concentration %] Figure 20.9 Phototoxicity of chlorpromazine. EpiDerm tissues were topically treated with the indicated concentrations of chlorpromazine and incubated overnight. On the following day tissues were either irradiated with 6 J/cm2 of UVA or kept in the dark. Tissues were then rinsed, fed with fresh medium, and incubated overnight. Finally, tissue viability was determined by the MTT assay. A decrease in tissue viability of 30% or greater between the irradiated and nonirradiated tissues indicates a phototoxic effect.
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Table 20.3 EpiDerm-200 Triton X-100 ET50 Database Summary Year 2000 1999 1998 1997 1996 1995 a
a
EPI-200 Triton ET-50 6.76 6.75 7.24 6.78 6.74 6.65
EPI-200 Triton C.V.
Lots
Avg. C.V.
16.4 18.2 17.9 15.9 14.6 77.8
89 146 175 228 184 112
6.2 5.7 9.2 9.9 9.6 4.9
Through September 2000.
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Figure 20.10 Changes in cancer-related gene expression following UVB-irradiation of EpiDerm tissue. AtlasTM human cancer cDNA expression array analysis of gene expression changes induced in EpiDerm tissues 6 hr after irradiation with 175 mJ/cm2 UVB.
Table 20.4 UVB-Irradiation of EpiDerm Tissue WAF-1 (+) MAP Kinase p38 (+) Growth-arrest-specific protein (+) c-Myc binding protein MM-1 (+) TRAF-interacting protein (+) Caspases (+) Death-associated protein kinase (DAP kinase 1, +) p53-induced protein (+) GADD45 (+) DNA excision repair protein ERCC1 (+) DNA-repair protein XRCC1 (+) Placenta growth factors 1+2 (+) TIMP-1 (+) T-plasminogen activator (+) Rho GDP dissociation inhibitor 1 (+) Endothelin 2 (–) IL-6 (–) Leukocyte interferon-inducible peptide (–) 60S ribosomal protein (–)
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– +
– +
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Cycles Figure 20.11 Induction of PlGF expression following UVB-irradiation of EpiDerm tissue. Agarose gel electrophoresis of products obtained by RT-PCR of total RNA isolated from EpiDerm tissue 20 hours following UVB-irradiation. (–) no irradiation. (+) UVB-irradiated. The expected PlGF PCR product is 273 bp.
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Figure 20.12 EpiDerm tissues cultured in 24 well (EpiDerm-224) or 96 well (EpiDerm-296) high throughput ALI formats. Each tissue has its own individual media reservoir to avoid cross-contamination of samples.
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Figure 20.13 Total RNA-96 isolation kit designed for high throughput total RNA isolation from EpiDerm-296.
Figure 20.14 Agarose gel electrophoresis of total RNA isolated from EpiDerm-296 with the Total RNA-96 isolation kit. Average yield is approximately 10 Qg DNA-free total RNA/EpiDerm-296 tissue.
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Cellular Resistance of Tetrahymena to Sulfur Mustard 3HWHU/3ODWWHERU]H-DPHV(6LVWUXQNDQG:LOOLDP-6PLWK
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INTRODUCTION 6XOIXUPXVWDUG 60ELV FKORURHWK\O VXOÀGH LVDSRWHQWDON\ODWLQJDJHQWWKDW FRQWLQXHVWREHDWKUHDWDVDFKHPLFDOZHDSRQ5HVHDUFKKDVVKRZQWKDW60LVKLJKO\ UHDFWLYHWRPDQ\FHOOXODUQXFOHRSKLOHVZLWK'1$DSSHDULQJWREHDFULWLFDOWDUJHW 7KHVH DON\ODWLRQ HYHQWV LQYROYH '1$ VWUDQG EUHDNV DV ZHOO DV WKH IRUPDWLRQ RI LQWUDVWUDQG RU LQWHUVWUDQG FURVVOLQNV 3DSLUPHLVWHU 7KH ODWWHU HYHQWV DUH ODUJHO\WKRXJKWWRFDXVHWKHREVHUYHG60LQGXFHGUHGXFWLRQLQ'1$V\QWKHVLVDQG VXEVHTXHQWLQWHUUXSWLRQRIWKHQRUPDOFHOOF\FOH (PLVVRQDQG6PLWK ,QKXPDQVH[SRVHGWR60WKHÀUVWWDUJHWRILQFDSDFLWDWLRQLVWKHH\HVIROORZHG E\DODWHQWSHULRGLQZKLFKYHVLFDWLRQRFFXUV'HVSLWHRFXODULQMXULHVKDYLQJEHHQ UHSRUWHG VLQFH :RUOG :DU , WKH FXUUHQW OHDG WKHUDSLHV SURYLGH DW EHVW WUDQVLHQW V\PSWRPDWLFUHOLHI$EDWWHU\RIFDQGLGDWHWKHUDSHXWLFFRPSRXQGVH[LVWVEXWKDVQRW EHHQDGHTXDWHO\WHVWHGDVWKHFXUUHQWWHVWPRGHOVWDQGDUG'UDL]HWHVW 'UDL]HHWDO 251
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MATERIALS AND METHODS Cell Culture and SM Exposures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ÀQDOFRQFHQWUDWLRQRIRUP07KLVZDVIROORZHG E\D GLOXWLRQDQGVXEVHTXHQWVHULDOGLOXWLRQVWRWKHGHVLUHGFRQFHQWUDWLRQ8SRQ GLOXWLRQ 60 ZDV LPPHGLDWHO\ DGGHG WR D KHDOWK\ 7HWUDK\PHQD FXOWXUH PL[HG IRU aVHFRQDYRUWH[DFWLRQPL[HUDQGSODFHGRQDURFNLQJSODWIRUP7KHQHJDWLYH FRQWURO ZDV WUHDWHG LGHQWLFDOO\ EXW 60 ZDV DEVHQW IURP WKH FXOWXUH PHGLXP DQG FHOOV$WVSHFLÀFWLPHSRLQWVDEDFWHULRORJLFORRSIXORIWKLVPL[WXUH aPO ZDV SODFHG RQ D PLFURVFRSH FRYHUJODVV DQG LQYHUWHG LQWR D GHSUHVVLRQ VOLGH DQG FHOOV ZHUH H[DPLQHG IRU PRWLOLW\ DW v PDJQLÀFDWLRQ7KH PRWLOLW\ SDWWHUQ RI WKH 7HW UDK\PHQD H[FOXGLQJVSHHGRIPRYHPHQW ZDVREVHUYHGHYDOXDWHGDQGTXDQWLÀHG (DFK ORZSRZHU PLFURVFRSLF ÀHOG FRQWDLQHG DSSUR[LPDWHO\ WR RUJDQLVPV 0RWLOLW\ZDVHVWLPDWHGE\PDNLQJDQDSSUR[LPDWHFRXQWRIWKHWRWDOQXPEHURIFHOOV LQWKHPLFURVFRSLFVDPSOHDQGWKHQXPEHURIFHOOVPRYLQJQRUPDOO\&HOOVPRYLQJ VORZHURUIDVWHUWKDQWKHFRQWUROVZHUHFRQVLGHUHGWREHPRYLQJQRUPDOO\ Coulter Counter Studies &HOOV ZHUH H[DPLQHG ZLWK D &RXOWHU +LDOHDK )/ =0 SDUWLFOH FRXQWHU$Q DOLTXRW PO RI FHOO VXVSHQVLRQ ZDV PL[HG ZLWK PO RI &RXOWHU ,VRWRQ LQ DQ $FFXYHWWHDQGUHDGRQWKHFRXQWHU&HOOQXPEHUDQGDYHUDJHVL]HZHUHUHFRUGHG
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aDWP0aDWP0DQGaDWP0$WP0 FHOOV KDG GHFUHDVHG LQ VL]H EXW WKLV FHOO SRSXODWLRQ ZDV GHWHUPLQHG WR QRW EH DOLYH %\KUSRVWH[SRVXUHYLDEOHFHOOVKDGQHDUO\UHJDLQHGWKHLULQLWLDOVL]H
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CHO and HeLa PBL HEK Tetrahymena Yeast
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0 10 -8
10 -7
10 -6
10 -5
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SM Concentration [M] Figure 21.1 Cell viability vs. [SM]. Viability of six different cell lines was analyzed over a range of SM concentrations. Symbols are represented within the graph.
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ALTERNATIVE TOXICOLOGICAL METHODS
60 8.0 mM 4.0 mM 2.0 mM
50
0.5 - 1.0 mM 0.3 mM & Positive Control
Cell Number
40
30
20
10
0 0
10
20
30
40
50
60
70
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Time (h) Figure 21.2 Effects of SM on cell number over time. The number of Tetrahymena was determined after exposure to a range of SM concentrations and over a period of 72 hr. Symbols are represented within the graph.
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Studies of Cellular Biochemical Changes Induced in Human Cells by Sulfur Mustard :LOOLDP-6PLWK2IÀH(&ODUN,,,)UHG0&RZDQ0DXUD('H-RVHSK &KDULVVH'DYHQSRUWDQG&ODUN/*URVV
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INTRODUCTION 7KH PLVVLRQ RI WKH 86 $UP\ 0HGLFDO 5HVHDUFK ,QVWLWXWH RI &KHPLFDO 'HIHQVH 05,&' LQFOXGHV EDVLF UHVHDUFK LQWR WKH PHFKDQLVPV RI DFWLRQ RI FKHPLFDOWKUHDWDJHQWV7KHUHVXOWVREWDLQHGIURPVXFKVWXGLHVDUHWKHQXVHGWR GHÀQHSKDUPDFRORJLFDOLQWHUYHQWLRQVWUDWHJLHVWRSURWHFWZDUÀJKWHUVDQGFLYLO LDQV DJDLQVW LQMXU\ E\ WKHVH DJHQWV 7KH FHOOXODU SKDUPDFRORJ\ WHDP DW WKH 05,&'IRFXVHVLWVUHVHDUFKRQWKHDFWLYHFHOOXODUDQGWLVVXHSDWKRJHQLFPHFK DQLVPV DFWLYH IROORZLQJ H[SRVXUH WR VXOIXU PXVWDUG dGLFKORURHWK\O VXOÀGH $UP\GHVLJQDWLRQ+' 7KLVUHSRUWSUHVHQWVVRPHRIWKHUHVXOWVRIWKRVHVWXGLHV DQGUDLVHVTXHVWLRQVSHUWDLQLQJWRPRGHOVDQGSURFHGXUHVHPSOR\HG
MATERIALS AND METHODS Cell Cultures HEK 3ULPDU\FXOWXUHVRIDGXOWQRUPDOKXPDQHSLGHUPDONHUDWLQRF\WHV +(.V ZHUH REWDLQHG IURP &ORQHWLFV &RUSRUDWLRQ 6DQ 'LHJR &$ 7KH FHOOV ZHUH SDVVDJHG LQWRRUFPWLVVXHFXOWXUHÁDVNVPDLQWDLQHGLQ.HUDWLQRF\WHJURZWKPHGLD .*0&ORQHWLFV DQGJURZQWRYDULRXVFRQÁXHQFLHV+(.VZHUHH[SRVHGWRHLWKHU RU Q0 +' DORQJ ZLWK XQWUHDWHG FRQWUROV 3RVWH[SRVXUH LQFXEDWLRQ WLPHV ZHUH IURP WR KU DIWHU ZKLFK FHOOV ZHUH KDUYHVWHG E\ 7U\SVLQ('7$ &ORQHWLFV&RUSRUDWLRQ HeLa +H/DFXOWXUHVZHUHPDLQWDLQHGLQFPFXOWXUHÁDVNV &RUQLQJ*ODVV:RUNV &RUQLQJ1< LQPLQLPDOHVVHQWLDOPHGLXP 0(0 ZLWKIHWDOFDOIVHUXP )&6 DWr&DQG&2 :KHQ WKH FXOWXUHV ZHUH WR FRQÁXHQW WKH PHGLXP ZDV UHPRYHG DQG UHSODFHG ZLWK D PHGLXP FRQWDLQLQJ +' WR Q0 DQG WKH FHOOV ZHUH LQFXEDWHG IRU PLQ LQ DQ DSSURYHG IXPH KRRG DW URRP WHPSHUDWXUH WR UHPRYH DQ\ KD]DUGRXV YDSRUV $IWHU KU WKH PHGLXP ZDV UHPRYHG DQG UHSODFHG ZLWK IUHVK 0(0 ZLWKRXW +' WR LQFXEDWH DQ DGGLWLRQDO WR KU 7KH FHOOV ZHUH KDUYHVWHGE\UHPRYLQJWKHPHGLXPDQGDGGLQJSKRVSKDWHEXIIHUHGVDOLQHFRQWDLQ LQJWU\SVLQDQG('7$WRWKHÁDVN7KHGHWDFKHGFHOOVZHUHSLSHWWHGLQWRDFRQLFDO FHQWULIXJH WXEH FRQWDLQLQJ 0(0 ZLWK )&6 DQG 7U\SVLQ LQKLELWRU VROXWLRQ &ORQHWLFV6DQ'LHJR&$ 7KHFHOOVZHUHKDUYHVWHGYLDFHQWULIXJDWLRQDWvJ IRU PLQ7KH VXSHUQDWH ZDV UHPRYHG DQG WKH FHOOV VXVSHQGHG LQ PO RI P07ULVEXIIHU S+ DQGP00J&O7KHFXOWXUHVZHUHPDLQWDLQHGRQLFH XQWLOWKHDVVD\ZDVLQLWLDWHG
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RESULTS 2XU LQLWLDO DWWHPSWV DW SXOVHGÀHOG JHO HOHFWURSKRUHVLV 3)*( RI +(. '1$ IROORZLQJH[SRVXUHWR+'XVHGWKHSURFHGXUHGHVFULEHGE\%HQWOH\HWDO LQ ZKLFKWUHDWHGFHOOVDUHSODFHGLQDJDURVHSOXJVH[SRVHGWRO\VLQJEXIIHUVDQGWKHQ WUDQVIHUUHG WR WKH JHOV 7KLV VWHS SUHVXPDEO\ ZRXOG VLJQLÀFDQWO\ UHGXFH WKH WLPH DQGKDQGOLQJIRURSWLPL]HGDQDO\VLVEXWZHFRXOGQRWGHWHFW'1$PLJUDWLRQWKURXJK WKH3)*(7RHQVXUHWKDW+(.'1$H[KLELWHGQRUPDOPLJUDWLRQSDWWHUQVWKURXJK RXU 3)*( ZH XVHG D FRPPHUFLDO '1$ LVRODWLRQ NLW IURP %RHKULQJHU0DQQKHLP GHVLJQHGIRUPDPPDOLDQFHOOVDQGWLVVXHV$OWKRXJKZHREWDLQHGORZ\LHOGVRI'1$ WKHSXULW\RIWKHLVRODWHVZDVLQVXIÀFLHQWDVMXGJHGE\WKH QPUDWLR PXVW EH! 7KHVHUHVXOWVDORQJZLWKWKRVHRIRXURWKHULVRODWLRQDWWHPSWVDUHOLVWHGLQ 7DEOH 7R YDOLGDWH WKH DELOLW\ RI WKH NLWV WR LVRODWH KXPDQ '1$ ZH XVHG KXPDQ SHULSKHUDOEORRGOHXNRF\WHV 3%/ DQGREWDLQHGJRRG\LHOGUHFRYHULHVZLWKDGHTXDWH SXULW\8VLQJSKHQROFKORURIRUPH[WUDFWLRQRI'1$ZHDOVRIDLOHGWRJHQHUDWHGHFHQW \LHOGVRISXUH'1$IURPFRQWURO+(.:HPRGLÀHGWKHNLWSURFHGXUHDVGHVFULEHG LQWKHPHWKRGVDQGXOWLPDWHO\\LHOGHGVXIÀFLHQWTXDQWLWLHVRISXUH'1$IURPFRQWURO +(.7KLVPRGLÀHGH[WUDFWLRQSURFHGXUHZDVWKHQXVHGRQ+'H[SRVHGFHOOVDQG 7DEOHVKRZVWKHKLJK\LHOGVDQGSXULWLHVREWDLQHG Table 22.1 Problems Seen with DNA Isolation from HEK
Procedure
Date
Sample
Purity Level of DNA (260/280 Ratio)
Purification of HEK DNA using Boehringer Mannheim Kit for mammalian cells Purification of lymphocyte DNA using Boehringer Mannheim Kit for mammalian blood Phenol/chloroform extraction of HEK DNA
8/4/98 8/18/98 8/24/98 9/2/98 9/8/98 9/29/98 10/1/98 10/7/98 10/13/98 10/19/98 10/27/98 11/5/98
Control Control Control Control Control Control Control Control Control Control Control Control
1.35 1.65 1.35 1.82 1.81 1.93 1.22 1.35 1.47 1.90 1.86 1.86
Purification of HEK DNA using Boehringer Mannheim Kit for mammalian cells (modified procedure)
DNA Yield (mg/ml) 60 45 25 189 185 194 60 33 67 94 93 98
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Table 22.2 Purification of HEK DNA using Boehringer Mannheim Kit for Mammalian Cells Exposed to Sulfur Mustard (HD) Date 11/19/98
11/26/98
12/8/98
Sample Control 50 mM HD 100 mM HD 300 mM HD Control 50 mM HD 100 mM HD 300 mM HD Control 50 mM HD 100 mM HD 300 mM HD
Purity Level of DNA (260/280 Ratio)
DNA Yield
1.81 2.10 1.89 1.82 1.85 1.99 1.86 1.81 1.98 1.96 1.91 1.86
122 163 150 199 194 213 198 172 183 179 176 199
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Figure 22.1 Time-dependent response of PARP activity following HD exposure of (top) HeLa cells and (bottom) HEK cells. Two concentrations of HD were used, 10 and 100 QM, and PARP activities are presented as percent maximal response.
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200
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Hrs post HD C1q
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– ++ +++
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+ = weak; ++ = moderate; +++ = intense; NT = not tested. Grading of staining was judged visually by fluorescence microscope and HEK controls not exposed to HD were negative for fluorescence.
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Figure 22.3 Concentration-dependent increase in the secretion of IL-8 from HEK exposed to HD.
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CHAPTER
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Table 23.1 HEK Housekeeping Gene Transcripts at 16 Hr Following Sulfur Mustard Gene Ratioa Gene Transcript 14-3-3 zeta protein 23 kDa Highly Basic Protein E-Tubulin F-Actin Glyceraldehyde 3-phosphate dehydrogenase HLA class I histocompatibility antigen C-4 alpha chain Hypoxanthine-guanine phosphoribosyltransferase Ribosomal Protein S9 Ubiquitin a
b
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25 QM SM
200 QM SM
317 1689 307 236 612 487
0.7 2.0 0.57 1.2 0.44 0.94
0.44 2.3 1.4 1.2 1.1 1.0
59
0.9
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2.1 0.73
Induction ratio from phosphorimage densitometeric measurements of listed gene transcript normalized to the sum total of the detected transcript intensity/pixel density. Control intensity (grayscale pixel density) adjusted from background. Background was set at the median intensity of the blank space between the six array panels. The subtracted background intensity ranged from 19 to 43.
Table 23.2 HEK Inflammation-Associated Transcripts at 16 Hr Following Sulfur Mustard Gene Ratioa Gene Transcript CD40 Interleukin 1 E Interleukin 1 F Interleukin 2 receptor E subunit Interleukin 6 Interleukin 7 receptor E subunit Interleukin 8 Interleukin 13 Interleukin 15 Macrophage inflammatory protein 2E S19 ribosomal protein Tumor necrosis factor E a
b
c
Control Intensityb
25 QM SM
200 QM SM
86 67 101 148 494 131 16 349 215 11 886 50
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Induction ratio from phosphorimage densitometeric measurements of listed gene transcript normalized to the sum total of the detected transcript density. Control intensity (grayscale pixel density) adjusted from background. Background was set at the median intensity of the blank space between the six array panels. The subtracted background intensity ranged from 19 to 43. Transcript not detected above background.
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Figure 23.1 Atlas cDNA array nylon blot image. HEK were exposed to 200 QM sulfur mustard for 16 h and mRNA isolated and 32P labeled as cDNA. The array contains doubledot blots of cDNA from 588 transcriptionally regulated genes and 9 housekeeping genes at the bottom of the array (see 3 housekeeping genes in rectangular box). Boxes 1 and 2 show expression of macrophage inflammatory protein 2E and interleukin 8, respectively. These inflammatory transcripts were at very low expression levels in control HEK.
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Figure 24.1 HEKs were exposed to the indicated HD concentrations and then incubated for 24 hr at 37SC. Viabilities were determined by using these dyes as described in the Experimental section.
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CHAPTER
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Comparison of Spectrophotometric and Fluorometric Assays of Proteolysis in Cultured Human Cells Exposed to Sulfur Mustard 6XVDQ$.HOO\&KDUOHQH0&RUXQ0DULDQ51HOVRQ&ODUN/*URVV DQG:LOOLDP-6PLWK
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INTRODUCTION 6XOIXUPXVWDUG dGLFKORURGLHWK\OVXOÀGH+' LVDSRWHQWYHVLFDWLQJDJHQW ZKRVHPHFKDQLVPRIEOLVWHUIRUPDWLRQLVSRRUO\XQGHUVWRRG0LFURVFRSLFH[DPL QDWLRQ RI WKH OHVLRQ LQWLPDWHV WKH LQYROYHPHQW RI SURWHRO\VLV LQ WKH FDVFDGH RI HYHQWV OHDGLQJ WR WKH HSLGHUPDO²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ÀFDF\7KHVFUHHQLQJSURFHVVGHPDQGVWKHGHYHORSPHQWRIERWKVHQVLWLYH DQG UHSURGXFLEOH LQ YLWUR DVVD\V WKDW FDQ EH XVHG WR TXDQWLWDWH WKH ELRPDUNHUV IRUPHG DV WKH UHVXOW RI +' H[SRVXUH LQ WDUJHW WLVVXHV:H KDYH LQYHVWLJDWHG WKH XVH RI FRPPHUFLDO SURWHDVH VXEVWUDWHV ZLWK HLWKHU FKURPRJHQLF RU ÁXRURJHQLF SURSHUWLHVWRPHDVXUHWKHELRPDUNHUSURWHRO\WLFDFWLYLW\LQERWK3%/VDQG+(.V WKDW KDYH EHHQ H[SRVHG WR +' 7KH PHDVXUHPHQW RI SURWHRO\WLF DFWLYLW\ ZDV TXDQWLWDWHG E\ ERWK VSHFWURSKRWRPHWULF DQG ÁXRURPHWULF PHWKRGV 6XLWDELOLW\ RI WKHVH DVVD\V IRU WKH VFUHHQLQJ RI SRWHQWLDO PHGLFDO FRXQWHUPHDVXUH HIÀFDF\ ZLOO EHDVVHVVHG
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Keratinocyte Growth 1RUPDOKXPDQHSLGHUPDONHUDWLQRF\WHVZHUHSXUFKDVHGDVVHFRQGSDVVDJHFHOOV IURP&ORQHWLFV7KHFHOOVZHUHJURZQVXEFXOWXUHGDQGWKHQGLVSHQVHGLQWRWLVVXH FXOWXUHSODWHVDWWKHDSSURSULDWHGHQVLW\IRUH[SHULPHQWDWLRQ 6PLWKHWDO HD Exposure +'ZDVGLOXWHGLQWRHLWKHU.*0 IRUNHUDWLQRF\WHV RU530, IRUO\PSKRF\WHV DQGDGGHGWRWKHDSSURSULDWHFHOOFXOWXUHSODWHVLQDFKHPLFDO VXUHW\KRRGWR\LHOG ÀQDO+'FRQFHQWUDWLRQVUDQJLQJIURPWRQ0$IWHUKUDWURRPWHPSHUDWXUH WR DOORZ K\GURO\VLV RI DJHQW WKH WLVVXH FXOWXUH SODWHV ZHUH WUDQVIHUUHG WR D S& LQFXEDWRUXQGHUDKXPLGLÀHG&2DWPRVSKHUHIRUWKHGXUDWLRQRIWKHSRVWH[SR VXUHLQFXEDWLRQSHULRG Chromozym® Assay (24 hr Postexposure to HD) Keratinocytes 7ZHQW\PLFUROLWHUVRIWKHDSSURSULDWH&KURPR]\PVXEVWUDWHZDVDGGHGWRHDFK ZHOORIWKHZHOOSODWH7KHSODWHVZHUHLQFXEDWHGIRUKUDWS&)LIW\PLFUROLWHUV RIVXSHUQDWHZDVUHPRYHGIURPHDFKVDPSOHDQGSODFHGLQWRD ZHOOSODWH7KH ZHOO SODWH ZDV UHWXUQHG WR WKH LQFXEDWRU 7KH ZHOO SODWH ZDV SODFHG LQ WKH PLFURSODWH VFDQQLQJ VSHFWURSKRWRPHWHU DQG WKH DEVRUEDQFH DW QP ZDV GHWHU PLQHG7KHODVWWZRVWHSVZHUHUHSHDWHGDJDLQDWKU Lymphocytes 6XSHUQDWHQOZDVUHPRYHGDQGGLVFDUGHGIURPHDFKZHOO$QDOLTXRWRI QO RI v ² 0 &KURPR]\P 7+ ZDV DGGHG WR HDFK ZHOO IRU D ÀQDO YROXPHRIQO7KHSODWHZDVLQFXEDWHGDWS&IRUKU$QDOLTXRWRIQO RIVXSHUQDWHZDVUHPRYHGDQGDGGHGWRDFOHDQ ZHOOSODWH7KH ZHOOSODWH ZDVSODFHGLQWKHPLFURSODWHVFDQQLQJVSHFWURSKRWRPHWHUDQGWKHDEVRUEDQFHDW QPZDVGHWHUPLQHG Intracellular Protease Activity $W KU SRVWH[SRVXUH WR +' QO RI HDFK &HOO3UREH HQ]\PH VXEVWUDWH ZDV DGGHGWRWKHDSSURSULDWHZHOOVRIWKHZHOOSODWH7KHSODWHVZHUHWKHQLQFXEDWHG IRU KU DW S& )OXRUHVFHQFH ZDV WKHQ PHDVXUHG XVLQJ WKH &\WR)OXRU PXOWLZHOO SODWHUHDGHUZLWKH[FLWDWLRQVHWDWQPDQGHPLVVLRQUHDGDWQP
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Chromozym® Substrates Chromozym® TH Tosyl-glycyl-prolyl-arginine-4-nitranilide acetate N-Methylsulfonyl-D-Phe-Gly-Arg-4-nitranilide acetate Chromozym® t-PA Chromozym® TRY Carbobenzoxy- valyl-glycyl-arginine-4-nitrilanilide acetate Benzoyl-β-alanyl-glycyl-arginine-4-nitranilide acetate Chromozym® U CellProbe® Enzyme Substrates: Nonfluorescent (aa)x -Fl Substrate-Dye complex ➾➾➾
Enzyme AAPV- Elastase (AAPV) D-aminopeptidase A (DAA) K-Aminopeptidase B (KAB) G-Aminopeptidase (GA) L- Aminopeptidase (LA) A- Aminopeptidase M (AAM) R- Aminopeptidase B (RAB)
Fluorescent Dye + Nonfluorescent Substrate leaving group Substrate Dl-Alanyl-Alanyl-Prolyl-Rho 110 Asp-Asp-Rho 110 Lysine-Lysine-Rho 110 Gly-Gly-Rho 100 Leu-Leu-Rho Alanine-Alanine-Rho 110 Arginine-Arginine-Rho 110
Figure 25.1 Substrate descriptions.
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Figure 25.2 Protease activity was measured in two donors as a function of cell number. Increasing the cell concentration above 2 v 106/well did not increase protease activity and this concentration was used in all subsequent studies.
Figure 25.3 PBL from five different donors were exposed to 250 QM HD and then incubated for 24 hr. Protease activity using the Chromozym® TH substrate was measured as described in Material and Methods. Significant protease activity occurred reproducibly. All assays were run in triplicate and all donors were evaluated multiple times on different days.
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Figure 25.4 HEK were exposed to indicated HD concentrations and incubated for 20 hr. Chromogenic protease assays were performed as described in the Materials and Methods. The protease activity was only evident at the highest concentration of HD, and only the Chromozym® U and Chromozym® t-PA substrates were significantly hydrolyzed.
Figure 25.5 HEK were exposed to HD at the indicated concentrations, and protease activity was measured fluorometrically 24 hr later as described in the Material and Methods section. KAB and GA substrates were significantly hydrolyzed above the control values while elastase (AAPV) and DAA were decreased.
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Effects of Low Dose Sulfur Mustard on Growth and DNA Damage in Human Cells in Culture :LOOLDP-6PLWK%HWKDQ\67ROLYHU2IÀH(&ODUN,,,-DQHW0RVHU (ULF:1HDOOH\-XDQLWD-*X]PDQDQG&ODUN/*URVV
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Figure 26.1 PBLs were exposed to buffer or to buffer plus HD. Four hours following exposure, cells were harvested and treated with 0.001% H2O2 or buffer. HD concentrations of 5 QM or greater inhibited expression of SSB in the comet assay. Points represent mean ±SEM from two experiments. 40
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Figure 26.2 HEKs were exposed to buffer or to buffer plus HD. Four hours following exposure, cells were harvested and treated with 0.002% H2O2 or buffer. Results are similar to those seen with PBLs. Points represent mean ±SEM from two experiments.
EFFECTS OF LOW DOSE SULFUR MUSTARD ON HUMAN CELLS
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Figure 26.3 PBLs were exposed to buffer or to buffer plus CEES. Four hours following exposure, cells were harvested and treated with 0.001% H2O2 or buffer. CEES did not inhibit expression of H2O2-induced SSB. Points represent mean ±SEM from two experiments.
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Figure 26.4 HEKs were exposed to buffer or to buffer plus CEES. Four hours following exposure, cells were harvested and treated with 0.002% H2O2 or buffer. Results are similar to those seen with PBLs. Points represent mean ±SEM from two experiments.
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Figure 26.5 HEKs were exposed to buffer or to buffer plus HD. Eighteen hours following exposure, cells were harvested and treated with 0.002% H2O2 or buffer. Points represent mean ±SEM from two experiments.
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Figure 26.6 HEKs were exposed to buffer or to buffer plus HD. Twenty-four hours following exposure, cells were harvested and treated with 0.002% H2O2 or buffer. Points represent mean ±SEM from two experiments.
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Imaging Sulfur Mustard Lesions in Basal Cells and Human Epidermal Tissues by Confocal and Multiphoton Laser Scanning Microscopy 5REHUW-:HUUOHLQDQG-DQQD60DGUHQ:KDOOH\
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Figure 27.1 Basal cell adhesion complex. A microvesicle (lower left panel) showing details of the dermal–epidermal separations characteristic of a sulfur mustard blister. Hemidesmosomes (arrowhead), at the roof of the blister, are well displaced from the basement membrane (bm) and lamina densa (ld). An expanded model of the intact adhesion complex (circumscribed area) includes the intracellular keratin filaments K5 and K14 and their facilitated attachments to the transmembrane E6F4 integrin receptors. The exodomains of E6F4 are shown linked by laminin 5 to the basement membrane zone.
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Figure 27.2 Keratin 5 images from control cultures of HEK recorded by multiphoton imaging (A), showed the elaborate cytoskeletal matrix and distribution of these filaments within the cell cytoplasm. Image intensity and K5 concentration were greatest around the nucleus of each cell, and a lacy network of delicate filaments projected out toward the cell extremities. Analysis of confocal images from K5 controls (B) and HD-exposed populations (C) showed a statistically significant (p < 0.01) 29.2% decrease in intensity at 1 hr postexposure to sulfur mustard.
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Figure 27.3 Multiphoton keratin 14 images from control HEK cultures (A) showed elaborate cytoskeletal distribution comparable to that of keratin 5. Image intensity and K14 concentration were greatest around the nucleus. Lacy networks of filaments projected out to the cell extremities where they interfaced closely with those of adjacent cells (arrow). At 1 hr postexposure to sulfur mustard, K14 images (B) indicated a disruption of organization, resulting in withdrawal of filaments from the plasma membrane margins, appearance of punctate nodules (arrows), and a substantial loss of cytoskeletal definition.
Figure 27.4 Analysis of K14 confocal images from replicate cultures of HEK sham-treated controls (A and C) and HD-exposed populations (B and D) showed a statistically significant (p < 0.01) 30.14% decrease in image intensity (K14 expression) at 1 hr postexposure and a nearly complete loss of expression (79% decrease in image intensity) at 2 hr.
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Figure 27.5 A multiphoton montage showing the organization of E6 integrins on an explant of human epidermis. Serial slices (A and B) illustrate the receptor outline on successive cross sections through the epidermal rete pegs. The three-dimensional reconstruction (C) and the stereo image (D) are from the same Z-series of serial slices. Together, they show the topographic complexity of ventral epidermis plus the circular shape and extensive distribution of E6F4-integrin receptors.
Figure 27.6 A multiphoton image (slice) of human epidermis showing in cross section the distribution and circular shape of F4 integrins (arrows) on the basal cell surface.
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Figure 27.7 Multiphoton images of E6F4 receptors in human epidermis exposed to sulfur mustard indicated unraveling and loss of circular shape at 1 hr postexposure (A) and an almost total loss of E6F4 expression at the basal cell surface by 2 hr postexposure (B, C). At 2 hr postexposure, only a basolateral pattern of residual fluorescence remained to outline the constituent basal cells. Loss of E6 and F4 integrin also occurred spontaneously in epidermal tissues following dermal–epidermal separation; therefore, the effects may not be strictly related to sulfur mustard exposure.
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Figure 27.8 Analysis of confocal images from HEK in replicate control and HD-exposed cultures indicated a statistically significant (p < 0.01) decrease of 27.3% and 26.3% in image intensity of E6 and F4 integrins, respectively, at 1 hr postexposure. The decrease was characterized by a loss of fluorescence from the surface of attached basal cells, resulting in a honeycomb pattern of residual, basolateral fluorescence. Postexposure image patterns from cultures were very similar to those recorded from intact epidermal tissues (see Figures 27.7B,C).
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Suppression of Sulfur Mustard-Increased IL-8 in Human Keratinocyte Cell Cultures by Serine Protease Inhibitors: Implications for Toxicity and Medical Countermeasures* )UHG0&RZDQ&ODUHQFH$%URRPÀHOGDQG:LOOLDP-6PLWK
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METHODS Reagents 6XOIXU PXVWDUG +' dGLFKORURGLHWK\O VXOÀGH ZLWK D SXULW\ RI ! ZDV REWDLQHG IURP WKH 86 $UP\ (GJHZRRG &KHPLFDO %LRORJLFDO &HQWHU $EHUGHHQ 3URYLQJ *URXQG 0' +XPDQ HSLGHUPDO NHUDWLQRF\WHV +(.V DQG NHUDWLQRF\WH JURZWKVHUXPIUHHPHGLD .*0 ZHUHSXUFKDVHGIURP&DPEUH[:DONHUVYLOOH0' 17RV\O/O\VLQHFKORURPHWK\ONHWRQH 7/&. ZDVREWDLQHGIURP6LJPD6W/RXLV 02(WK\OSJXDQLGLQREHQ]RDWHK\GURFKORULGH FRPSRXQG ZDVREWDLQHGIURP LQKRXVHDQWLYHVLFDQWVFUHHQLQJSURJUDP 86$UP\0HGLFDO5HVHDUFK,QVWLWXWHRI &KHPLFDO'HIHQVH$EHUGHHQ3URYLQJ*URXQG0'
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315
Table 28.1 Candidate Antivesicant Drug Screening: Statistically Positive Reduction of at Least 50% in Edema or Histopathology in the Mouse Ear or Hairless Mouse Edema
Histopathology
Anti-Inflammatory Drugs Fluphenazine dihydrochloride Indomethacin Olvanil Hydrocortisone Olvanil (saturated) Retro olvanil Olvanil (urea analog) Octyl homovanillamide Dexamethasone
63 53 65 62 65
50 96 91 71 53 84 81 100 72
Scavenger Drugs 2-Mercaptopyridine-1-oxide 6-Methyl-2-Mercaptopyridine-1-oxide 4-Methyl-2-Mercaptopyridine-1-oxide Dimercaprol
57 43
65 56 94 92
Protease Inhibitor 1-(4-aminophenyl)-3-(4-chlorophenyl)urea HCl N-(OP)-L-Ala-L-Ala-benzy ester hydrate 1(G-T)-4-(4-methyl phenylsemithiocarbazide)
54 63 50
PADPRP Inhibitor 3-(4d-Bromophenyl)ureidobenzamide Benzoylene urea
74 54 Other
Hydrogen peroxide gel, 3%
58
Data generated by the U.S. Army Medical Research Institute of Chemical Defense Anti-vesicant Drug Screen.
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Percent of IL-8
100 80 60 40 20 0 Control 0.0
62.5
125.0 250.0
500.0 1000.0
TLCK (µM)
Figure 28.1 Percent IL-8 of HD-exposed HEK following treatment with TLCK.
MUSTARD-INDUCED IL-8
317
120
Percent of IL-8
100 80 60 40 20 0 Control 0.0
31.25 62.5 125.0 250.0 500.0 1000.0
1579 (µM) Figure 28.2 Percent IL-8 of HD-exposed HEK following treatment with 1579.
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CHAPTER
29
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Proposed Mechanism of HD Action Proteins Inflammatory Cells HD Membranes Ca++
DNA
Strand Breaks
PARP NAD + depletion
Inflam Metabolic Disruption
Protease release
Inhibition of glycolysis
CELLULAR TISSUE
Toxicity
Breakdown of epithelial attachment
Memb Ca++ Epidermal-dermal separation
Inflam
Hyd VESICATION
Figure 29.1 The cellular and tissue alterations induced by HD that are proposed to result in blister formation. HD can have many direct effects such as alkylation of proteins and membrane components (Memb), as well as activation of inflammatory cells. One of the main macromolecular targets is DNA, with subsequent activation of poly(ADP-ribose) polymerase (PARP). Activation of PARP can initiate a series of metabolic changes culminating in protease activation. Within the tissue, the penultimate event is the epidermal–dermal separation that occurs in the lamina lucida of the basement membrane zone. Accompanied by a major inflammatory response and changes in the tissue hydrodynamics (Hyd), fluid fills the cavity formed at this cleavage plane and presents as a blister. Table 29.1 Strategies for Pharmacologic Intervention of the HD Lesion Biochemical Event DNA alkylation DNA strand breaks PARP activation Disruption of calcium Proteolytic activation Inflammation a
Pharmacologic Strategy Intracellular scavengers Cell cycle inhibitors PARP inhibitors Calcium modulators Protease inhibitors Antiinflammatories
Example N-acetyl cysteine Mimosine Niacinamide BAPTAa AEBSFa Indomethacin; Olvanil
BAPTA is a calcium chelator; AEBSF is a sulfonyl fluoride compound.
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Table 29.2 Candidate Countermeasures with Greater Than 50% Efficacy in Mouse Ear Model Percentage Reduction in Pathology Anti-Inflammatory Drugs Fluphenazine dihydrochloride Indomethacin Olvanil Olvanil (saturated) Retro olvanil Olvanil (urea analog) Octyl homovanillamide
50 96 91 53 84 81 100
Scavenger Drugs 2-Mercaptopyridine-1-oxide 6-Methyl-2-mercaptopyridine-1-oxide 4-Methyl-2-mercaptopyridine-1-oxide Dimercaprol Na 3-sulfonatopropyl glutathionyl disulfide Hydrogen peroxide gel, 3%
66 56 94 78 64 58
Protease Inhibitors 1-(4-aminophenyl)-3-(4-chlorophenyl) urea N-(OP)-L-Ala-L-Ala-benzy ester hydrate Ethyl p-guanidino benzoate hydrochloride
54 62 62
PARP Inhibitors 3-(4d-Bromophenyl)ureidobenzamide Benzoylene urea 4-Amino-1-naphthol hydrochloride tech Total number of positive compounds = 19
74 54 80
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CHAPTER
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Molecular Neurotoxicology of 6-Hydroxydopamine and Methamphetamine: Lessons Derived from Transgenic Models -HDQ/XG&DGHW
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Figure 30.1 Molecular neurotoxicology of methamphetamine.
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CHAPTER
31
A Microassay Method Using a Neuroblastoma Cell Line to Examine Neurotoxicity of Organophosphate Mixtures /DXULH(5RV]HOO%HQMDPLQ&.UDPHUDQG*OHQ-/HDFK
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ÀFDQWO\ LQKLELWHG S DW SDUDR[RQ FRQFHQWUDWLRQV JUHDWHUWKDQv²Q07KHKLJKHVWFRQFHQWUDWLRQRISDUDR[RQ²Q0UHVXOWHG LQ QHDUO\ LQKLELWLRQ RI $&K( DFWLYLW\ 'LFKORUYRV VLJQLÀFDQWO\ LQKLELWHG $&K(DFWLYLW\DWFRQFHQWUDWLRQVDERYHv²Q07KHKLJKHVWFRQFHQWUDWLRQRI GLFKORUYRVQ0UHVXOWHGLQDQLQKLELWLRQRIDSSUR[LPDWHO\&RQFHQWUDWLRQV DERYHWKLVUHVXOWHGLQGHFUHDVHGFHOOXODUYLDELOLW\ GDWDQRWVKRZQ 7KHDSSDUHQW (&IRUSDUDR[RQZDVv²Q0ZKLOHWKHDSSDUHQW(&IRUGLFKORUYRV ZDVQ07KHVHYDOXHVDUHLQDJUHHPHQWZLWKWKRVHRI(KULFKHWDO LQ ZKLFK VLPLODU H[SHULPHQWV ZHUH SHUIRUPHG ZLWK WHUPLQDOO\ GLIIHUHQWLDWHG 6+ 6<<QHXUREODVWRPDV 1.400
Absorbance (405 nm)
1.200 1.000
500,000
0.800
250,000 125,000
0.600
62,500
0.400
31,000
0.200
16,000 8,000
0.000 -0.200
0
15
30
45
60
Time (min) Figure 31.1 Determination of optimal cell number per well and reaction time for AChE activity.
344
ALTERNATIVE TOXICOLOGICAL METHODS
Concentration Dichlorvos (µM)
Absorbance (405 nm)
Control
1.E-02
5.E-02
1.E-01
5.E-01
1.E+00
5.E+00
1.E+01
1.60
1.60
1.40
1.40
1.20
1.20
*
1.00
1.00
* *
0.80 0.60
0.60
*
*
0.40
0.80
*
*
0.20
0.40
*
*
0.20
0.00
0.00 Control 1.E-07 1.E-06 1.E-05 5.E-05 1.E-04 5.E-04 1.E-03 5.E-03 1.E-02 5.E-02 1.E-01
Concentration Paraoxon (µM) Paraoxon
Dichlorvos
Figure 31.2 Effect of dichlorvos or paraoxon on AChE activity of SH-SY5Y neuroblastomas. *p < 0.05.
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10-2 D/10-5 Px
10-2 D/10-4 Px
10-2 D/10-3 Px
10-1 D/10-5 Px
10-1 D/10-4 Px
10-1 D/10-3 Px
1.0 D/10-5 Px
1.0 D/10-4 Px
1.0 D/10-3 Px
10-5 Px
10-4 Px
10-3 Px
10-2 D
10-1 D
0.000
1.0 D
0.200 control
Absorbance (405 nm)
1.600
µM Dichlorvos (D) or Paraoxon (Px) Figure 31.3 Effect of dichlorvos (D) or paraoxon (Px) alone or in combination on AChE activity of SH-SY5Y neuroblastomas. *p < 0.05.
IN VITRO ASSESSMENT OF ORGANOPHOSPHATE NEUROTOXICITY
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Table 31.1 Effect of Paraoxon (Px) or Dichlorvos (D) on AChE Activity of SH-SY5Y Neuroblastomas, Significance Level 0.05 OP concentration QM) (Q control 10–2 D 10–1 D 1.0 D 10–5 Px 10–4 Px 10–3 Px 10–2 D/10–5 Px 10–2 D/10–4 Px 10–2 D/10–3 Px 10–1 D/10–5 Px 10–1 D/10–4 Px 10–1 D/10–3 Px 1.0 D/10–5 Px 1.0 D/10–4 Px 1.0 D/10–3 Px
Absorbance at 405 nm ± SEM 1.269 1.373 1.198 0.831 1.352 0.811 0.362 1.332 0.630 0.418 1.370 0.647 0.453 0.432 0.590 0.339
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.031 0.091 0.033 0.023 0.081 0.086 0.024 0.042 0.077 0.017 0.167 0.104 0.027 0.151 0.156 0.052
Measured inhibition (%) –8 6 34 –7 36 72 –5 50 67 –8 49 64 66 54 73
Predicted inhibition (%)
Significant vs. control?
x x x –15 28 63 –1 42 77 28 71 106
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Figure 32.1 Integrin heterodimers transduce signals both into and out of the cell via interactions with extracellular ligands and cytoplasmic accessory or regulatory proteins, the cytoskeleton, and signal transduction proteins. Abbreviations: arginine-glycineaspartic acid peptide (RGD), calreticulin (Cal), focal adhesion kinase (Fak), paxillin (Pax), phosphatidylinositol-3 kinase (PI3), vinculin (Vin).
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INTEGRIN +
+ Intracellular Accessory Protein
Extracellular Ligand
Receptor Activation/Clustering
Cell Migration
Tissue Organization
Re-Organization of Cytoskeletal Proteins
Neurite Elongation
Activation of Signal Transduction Pathways
Cell - Cell Communication
Cell Cycle
Gene Expression
Cell Survival
Figure 32.2 Integrin-mediated cell functions.
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Protein (Westerns) Expression Levels mRNA (Northerns, RPA)
Identify Integrin Subunits
Cell Adhesion Assay Function Proliferation/ Differentiation Neuronal (PC12, N2A) Cell Lines Glial (C6)
Develop In Vitro Test Systems
Isolated Cells (Astrocytes, Oligoden) Tissue Culture Primary Cultures/ Co-Cultures
Test Known Agents
Validate Known Agents
Test New Compounds
Neuropathic
TMT, MeHg
Axonpathic
Acrylamide
Synaptopathic
MDMA
In Vivo
TMT, MeHg
In Vitro
Unknown Mechanisms
In Vivo
Suspected Neurotoxicants
Figure 32.3 Approach to validation of integrin subunit expression levels as molecular biomarkers of neurotoxicity. Abbreviations: trimethyltin (TMT), methylmercury (MeHg), 3,4-methylenedioxymethamphetamine (MDMA), RNAse protection assay (RPA).
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CHAPTER
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Two-Photon Fluorescence Microscopy: A Review of Recent Advances in Deep-Tissue Imaging &KHQ<XDQ'RQJ/LO\+VX.L+HDQ.LP&KULVWRI%XHKOHU3HWHU'.DSODQ 7KRPDV'+DQFHZLF]DQG3HWHU7&6R
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Figure 33.1 Jablonski diagrams for (a) one-photon and (b) two-photon excitation. One-photon excitation occurs through the absorption of a single photon. The two-photon process occurs through the simultaneous absorption of two lower energy photons. After either excitation process, the fluorophore relaxes to the lowest energy level of the first excited electronic state. The subsequent fluorescence emission process is independent of the mode of excitation.
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Figure 33.2 A schematic of two-photon fluorescence microscope design.
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Figure 33.3 Two-photon autofluorescence images of human skin. Top left: stratum corneum. Top right: basal layer. Bottom: fibrous dermal layer (Zeiss Fluar 100v oil).
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Figure 33.4 Image restoration of two-photon images of ex vivo human skin using maximum likelihood approach. Autofluorescence (left) and blind deconvoluted (right) images of human basal layer. Top: lateral view. Bottom: axial section (Zeiss Fluar 40v oil).
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Figure 33.5 Two independent spectral components isolated in ex vivo human skin based on SMCR: (a) a spectral component corresponds to elastin fibers in the dermis and (b) a spectral component corresponds to melanin (or a fluorophore that colocalizes with melanin) in the epidermal–dermal junction. For (a) and (b): (left) a twodimensional image of the concentration distribution of the spectral component; (right, top) the spectrum of this component; (right, bottom) the depth distribution profile of this component.
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The Application of Genomics and Proteomics to Toxicological Sciences 6WHSKDQ&KHYDOLHU:LOOLDP'3HQQLHDQG,DQ.LPEHU
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Protein expression and posttranslational modification Mass spectrometry Protein database search mRNA expressed
Protein identity
Research lead
Toxicological marker
Toxicant signature
Figure 34.1 Schematic of genomic and proteomic technologies: The first step in toxicogenomics (right) is the construction of a microarray that involves the amplification by PCR and the immobilization of known DNA sequences (either cDNA or oligonucleotides) on a solid support. The mRNA prepared from a biological model can be labeled and hybridized to the microarray and visualized using phosphorimager scanning. Subsequent bioinformatic analyses using appropriate software allows determination of the extent of hybridization of the labeled probes to the corresponding arrayed cDNA spots, and a comparison of control with test samples permits quantitative assessment of changes in gene expression associated with treatment. Total protein content from a biological model treated with a toxicant is separated on two-dimensional gel electrophoresis according to isoelectric point (first dimension) and molecular weight (second dimension), allowing bioinformatic analysis of differences in protein expression of treated versus untreated samples. Therefore, there is no preliminary work such as array construction for proteomic studies (left), but proteins with altered expression have to be identified subsequently. Individual proteins of interest are excised from two-dimensional-gels, digested with trypsin, and applied to a mass spectrometer. Identification of these proteins is obtained by searching protein databases with mass spectrometry data.
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Table 35.1 IARC Class I or 2A Human Carcinogen, or NTP Reasonably Anticipated Human Carcinogen Tg.AC Topical
TgrasH2
p53+/–
XPA–/–/p53+/–
+ + – + – – nd
+ nd + nd +/– + +
+ + + + + – nd
nd* + nd +** nd – nd
+ + +# + +
+/– + – nd nd
+ + +/– –
+ + + nd nd
Genotoxic Benzene Benzo(a)pyrene Cyclophosphamide 7,12-Dimethylbenzanthracene Melphalan Phenacetin Procarbazine Nongenotoxic Cyclosporin A Diethylstilbestrol 17-F-estradiol (or ethinyl estradiol#) Oxymetholone 2,3,7,8-TCDD
* nd: no adequate data available on the performance of the compound in that model. **Positive in 6 month XPA–/– and positive in 6 month p53+/–; not tested in XPA–/–/P53+/– bitransgenic. Table 35.2 Genotoxic Trans-Species Rodent Carcinogens
p-Cresidine 2,4-Diaminotoluene Diethylnitrosamine Dimethylnitrosamine N-Ethylnitrosourea Glycidol N-Methylnitrosourea Phenolphthalein Thiotepa Urethane 4-Vinyl-1-cyclohexene-diepoxide*
Tg.AC Topical
TgrasH2
P53+/–
XPA–/–/P53+/–
– + nd nd nd – nd nd nd nd –
+ nd + + + + + – + + +
+ – nd + + – + + nd + +
+ nd nd nd nd nd nd nd nd nd nd
* Applied dermally to each model tested.
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Table 35.3 Nongenotoxic Rodent Carcinogens and Human Carcinogenicity Unlikely or Uncertain Tg.AC Topical
TgrasH2
P53+/–
XPA–/–/P53+/–
nd + nd – nd nd nd + – – – –
– + nd + – nd – nd – – – +
nd – – +/– – – – – – – – –
nd nd nd – – nd nd nd – – – +*
Chlorpromazine Clofibrate Dieldrin Diethylhexylphthalate Haloperidol D-Limonene Metaproterenol Pentachlorophenol Phenobarbital Reserpine Sulfamethoxazole WY-14643
* Positive in 6 month XPA–/–, not tested in XPA–/–/p53+/– bitransgenic. Table 35.4 Rodent Noncarcinogens
Genotoxic p-Anisidine 2-Chloroethanol 1-Chloro-2-propanol 2,6-Diaminotoluene 8-Hydroxy-quinoline Nongenotoxic Ampicillin Benzethonium chloride D-Mannitol Oleic acid diethanolamine Phenol Resorcinol Rotenone Sulfisoxazole
Tg.AC Topical
TgrasH2
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XPA–/–/P53+/–
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– nd – – –
nd nd nd nd nd
nd – nd – – + – –
– nd – nd nd – – –
nd nd nd – nd – – nd
nd nd – nd nd nd nd nd
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Figure 36.1 Rat toxicology U34 gene array. This gene expression display was created by GeneSpring® (Silicon Genetics) gene array analysis software from our data (rat brain RNA 1 h postexposure to CPF) read from an Affymetrix Rat Toxicology U34 GeneChip®. In the original display (depicted in gray tones here), red and purple blocks represented up-regulated genes and the blue represented down-regulated genes. Gray blocks represented genes whose expression is essentially the same as in the control animal.
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RESULTS Selection of Genes to Measure ,QRUGHUWRPRVWHIÀFLHQWO\IRFXVRXU´ÀUVWSDVVµ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Analysis of Gene Expression Patterns ,QRUGHUWRPRUHREMHFWLYHO\FODVVLI\WKHXSDQGGRZQUHJXODWLRQRIJHQHH[SUHV VLRQRIWLPHZHJURXSHGWKHJHQHVLQWRWKHVL[PRVWFRPPRQSDWWHUQVREVHUYHGLQ WKHGDWD1HDUO\DOORIWKHJHQHH[SUHVVLRQGDWDIURPWKH'1$PLFURDUUD\VÀWLQWR RQHRIVL[FDWHJRULHV )LJXUH Quantification of Gene Expression Patterns in the Brain ,Q RUGHU WR TXDQWLI\ WKH W\SHV RI DOWHUDWLRQV LQ JHQH H[SUHVVLRQ LQGXFHG E\ H[SRVXUH WR &3) ZH DVVLJQHG QHDUO\ DOO RI WKH JHQHV IRU ZKLFK ZH KDG VXIÀFLHQW GDWDWRRQHRIWKHVL[PRVWFRPPRQSDWWHUQVREVHUYHGLQWKHGDWD7KHDQDO\VLVRI WKHEUDLQJHQHH[SUHVVLRQOHYHOVLQWKH&3)H[SRVHGUDWXVLQJWKH5DW1HXURELRORJ\ 8*HQH&KLSUHYHDOVWKDWJHQHVDQG(67V RXWRIWKHJHQHVDQG (67VRQWKHFKLSIDOOLQWRWKH$SDWWHUQJHQHVDQG(67V IDOOLQWRWKH% SDWWHUQJHQHVDQG(67V ÀWLQWRWKH&SDWWHUQ JHQHVDQG(67V ÀWLQWRWKH'SDWWHUQJHQHVDQG(67V ÀWLQWRWKH(SDWWHUQDQGJHQHV DQG(67V ÀWLQWRWKH)SDWWHUQ7KHUHPDLQLQJRIJHQHVDQG(67VKDYH EHHQH[FOXGHGIURPDQDO\VLVGXHWRLQFRPSOHWHGDWDRUGXHWRRXWOLHUVWDWXVRIRQH RUPRUHRIWKHGDWDSRLQWV
A.
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D.
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Figure 36.2 Illustrations of the six expression pattern categories. The numerals under the xaxis represent hours after CPF exposure. Y-axis represents relative gene expression level. (A) No alteration of gene expression; (B) initial up-regulation, then return to normal by 24 h; (C) initial up-regulation, then return to normal by 4 h; (D) delayed up-regulation by 4 h, then return to normal by 24 h; (E) delayed upregulation by 24 h; (F) rapid down-regulation, then return to normal by 4 h.
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Figure 36.3 The relative percentages of the six patterns of gene expression detected in the CPF-exposed rats.
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ALTERNATIVE TOXICOLOGICAL METHODS
Figure 36.4 Scatterplot of data from rat toxicology U34 GeneChip.
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BuChe Activity (O.D. at 412 nm)
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Hours Post-Exposure Figure 36.5 In vivo effects of CPF on BuChE activity.
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Figure 37.1 Comparison of toxicological features of HCN and nerves gases, such as sarin. The larger arrow represents inhaled HCN. The smaller arrow represents the effective dosage, after detoxification and loss of the 30% of inhaled HCN that is exhaled (Moore and Gates, 1946), leaving 0.7 retained. HCN concentrations up to 30 mg/m3 are normally neutralized by human detoxification systems before effects become observable (Prentiss, 1937).
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Figure 37.2 Contrasting plots of data from the same rat exposures to HCN (after Ballantyne, 1987). One plot (left) reflects use of exposures with fixed duration and varied HCN concentrations. The steep initial slope shows that HCN must be very concentrated for a small number of breaths to deliver a lethal dosage. The second plot presents the data results as customarily displayed, given a fixed toxic gas concentration and varied exposure durations. LCt50 values tend to increase as more time becomes available for detoxification.
Figure 37.3 Comparison of responses from different strains of rats under similar HCN exposure conditions (after Levin et al., 1985). Log–log plots of the data reveal differences of effective HCN concentrations and probit slopes of lines but do not show which strain better represents the human race.
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Figure 37.4 Relationships of anatomical and pharmacological factors leading to lethality for mammals that inhale HCN. Nonionized HCN is rapidly absorbed into blood to move the short distance from alveoli to the sensor. The sensor response (or lack of a signal) communicates a need for oxygen to respiratory neurones that initiate hyperventilation. Increased minute volumes multiply HCN intake until respiratory center poisoning leads to apnea.
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Table 37.1 Hydrogen Cyanide Concentrations and Effects Associated with Various HCN Exposure Conditions in Animals and Man Mg/m3 a
30,000 9,300b 7,740b 4,000a 3,200b 2,230 a b
Effects Rat, inhalation LC50 Men, 11/11 hyperventilate Men, “most hyperventilate” Rat, inhalation LC50 Men, “50% hyperventilate” Pig, LC50 within 2 min of inhalation
Ref. Levin et al., 1985 Bodansky and Helm, 1944 Cope and Abramovitz, 1959 Ballantyne, 1987 Wexler et al., 1947 Stemler et al., 1994
Lethal concentration for 50% of subject rats, 0.1 min inhalation exposure. Equivalent HCN mg/m3 dosage calculated for intravenous sodium cyanide solution.
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Figure 37.5 Observed breathing rates and blood cyanide concentrations of three miniature pigs exposed to HCN for 2 min (after Stemler et al., 1994). Values for blood concentrations of cyanide, at the top and right side of Figure 37.5, are presented with the corresponding respiratory rate values. Although data acquired after the onset of HCN exposure for 2 min are very limited, they are consistent with other indicators that an aortic blood HCN concentration of 4 mg/m3 at 5 min is a threshold value for a lethal outcome.
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Figure 37.6 Average whole blood cyanide levels in dogs after four continuous intravenous slow infusion trials (1 mg/kg/min) with NaCN solution at 4.0 mg/ml (after Vick et al., 2000). RA indicates respiratory arrest and cessation of NaCN infusion. Methemoglobin formation by 20 mg/ml hydroxylamine hydrochloride solution was initiated 30 sec after RA. Although 3.6 Qg/kg was the average value observed at RA, it appears that survival is dependent upon avoidance of blood cyanide concentrations above 4 Qg/kg.
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Figure 37.7 Triaxial depiction that was hand drawn to integrate (1) time course information reported hourly or by the day; (2) mustard vapor dosage information; and (3) levels of human effect severity for the given time and dosage. The dashed line represents data from one particularly relevant experiment (Unde and Dunphy, 1944) among more than 100 experimental reports that were considered.
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Table 37.2 Estimates of Percent of Military Personnel with Indicated Degree of Performance-Degradation per Day after Onset of Gastrointestinal Symptoms/Signs from Infection with Enterotoxigenic Escherichia coli Bacteriaa Day of Onsetb 1 2 3 4 5 6 7 8 9 10 11 12 13 14 a
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Percentage of cases with degraded military performance % New Recoveredg Casesc No.d (25%)c No.e (50%)c No.f (100%)c (%)c (14.6) (19.0) (19.3) (17.1) (9.8) (6.1) (4.1) (2.9) (2.0) (1.5) (1.3) (1.1) (0.7) (0.5)
9 (47.4) 7 (36.8) 4 (21.1) 4 (21.1) 2 (10.5) 2 (10.5) 4 (21.1) 4 (21.1) 3 (15.8) 3 (15.8) 2 (10.5) 1 (5.3) 0.5 (2.6) (0)
7 (36.8) 7 (36.8) 10 (52.6) 8 (42.1) 8 (42.2) 6 (31.6) 3 (15.8) 2 (10.5) 1 (5.3) (0) (17) (18) (18.5) (19)
3 (15.5) 3 (15.8) (0) (7) (9) (11) (12) (13) (15) (16) (89.5) (94.7) (97.4) (100.0)
0 (0) 2 (10.5) 5 (26.3) (36.8) (47.4) (57.9) (63.2) (68.4) (78.9) (84.2)
Percentages based upon data from 19 proven cases of enterotoxigenic Escherichia coli infection acquired in Mexico City (Merson et al., 1976). Onset day 1 is day 3 from exposure to infection. Calculated from daily rate curve (Fischer and Mershon, 1994). Cases (9) with uncomplicated traveler’s diarrhea, performance 25% degraded on day 1. Cases (7) with changed activities, performance 50% degraded during onset day 1. Cases (3) with recovery time in bed; performance 100% degraded during onset day 1. Cases (19) all had some degree of incapacitation during onset day 1.
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Figure 37.9 This cartoon is included to suggest that modeling represents a combination of art and science. The art of modeling methodology is applied when conventional testing methods are not applicable for collection of experimental results. In each case, previously collected data were reacquired and analyzed to provide a best possible estimate of reality.
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Field Data _a sh 85064594 _a RC582.17 _a Immunotoxicology _a Immunologic toxicology _a Immunotoxicity _w g _a Immunopathology _w g _a Toxicology _a Immunopharmacology
Explanations Record Number Class Number Subject Heading UF (Use For Term) UF (Use For Term) BT (Broader Term) BT (Broader Term) RT (Related Term)
Figure 38.1 LC authority record for Immunotoxicology, supplemented with explanatory notations.
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Field Data 01037pam__2200325_a_4500 996520 9910520155750.7 900824sl991____njua_____b____001_0_eng_c _9 (DLC) 90011279 _a 7 _b cbc _c orignew _d 1 _e ocip _f 19 _g y-gencatlg _a CIP ver. ea10 to SL 05-13-91 _a 90011279 _z 0849388153 _a DNLM/DLC _c DLC _d DLC _a RA1224.3 _b .G457 1991 _a QH 465.C5 G328 _a 615.9/02 _2 20 _a Genetic toxicology / _c editors, Albert P. Li, Robert H. Heflich. _a Boca Raton : _b CRC Press, _c cl991. _a x, 493 p. : _b ill. ; _c 24 cm. _a Includes bibliographical references. _a Includes index. _a Genetic toxicology. _a Carcinogens. _a Chromosome Abnormalities _x chemically induced. _a Mutagens _x adverse effects. _a Mutation. _a Li, A. P. _a Heflich, Robert H., _d 1946_b c-GenColl _h RA1224.3 _i .G457 1991 _t Copy 1 _w
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LC Classification: NLM Class No.: Dewey Class No.:
90011279 Genetic toxicology / editors, Albert P. Li, Robert H. Heflich Boca Raton : CRC Press, c1991. Li, A. P. Heflich, Robert H., 1946x, 493 p.,: ill.; 24 cm. 0849388153 Includes index. Includes bibliographical references. Genetic toxicology. Carcinogens. Chromosome Abnormalities--chemically induced. Mutagens--adverse effects. Mutation. RA1224.3 .G457 1991 QH465.C5 G328 615.9/02 20
Figure 38.4 LC OPAC Full Record display for Genetic Toxicology, AP Li, and RH Heflich, editors, 1991.
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IN SILICO APPROACHES FOR PBPK MODEL PARAMETERS In Silico Approaches for Tissue:Air Partition Coefficients 7LVVXH DLU3&VGHVFULEHWKHUHODWLYHFRQFHQWUDWLRQVRIYRODWLOHRUJDQLFFKHPLFDOV 92&V LQWLVVXHVDQGDLUDWVWHDG\VWDWH%RWK/)(W\SH46$5PRGHOVDQGELRORJ LFDOO\EDVHGDOJRULWKPVKDYHEHHQGHYHORSHGIRUSUHGLFWLQJWLVVXH DLUSDUWLWLRQFRHI ÀFLHQWVRIDYDULHW\RIFKHPLFDOV 7DEOH ,QGHYHORSLQJD/)(W\SH46$5IRUKXPDQWLVVXH DLU3&V$EUDKDPDQG:HDWK HUVE\ REVHUYHGWKDWQRQSRODUVROXWHVRQO\QHHGHGKH[DGHFDQH DLUSDUWLWLRQLQJ + DK\GURSKRELFGHVFULSWRU ZKHUHDVDQHOHFWURVWDWLFGHVFULSWRU T ZDVLPSRUWDQW IRUIXQFWLRQDOO\VXEVWLWXWHGFRPSRXQGVVXFKDSURSDQRO$EUDKDPDQG:HDWKHUVE\ FRUUHFWO\ XQGHUVFRUH WKH LPSRUWDQFH RI OLPLWLQJ WKH XVH RI WKHVH HTXDWLRQV
Inert Gases; LMWVOCs Inert Gases; LMWVOCs Inert Gases; LMWVOCs Inert Gases; LMWVOCs
H H H H
log Pkidney:air = –1.084 + 0.417R2 + 0.226 T + + 3.624 E + + 2.926 F + + 0.534 log Phe:a
0.0983 [ FY + 2.213
log Padipose:air = (0.7341xv) – (0.029 [ VY ) – (1.57(1/1x)) – (0.559(1/1xv)) –
Steric descriptors
log Pmuscle:air = –1.14 + 0.544R2 + 0.216 T + + 3.4714 E + + 2.924 F + + 0.578 log Phe:a
log Plung:air = –1.300 + 0.667R2 + 0.680 T + + 3.539 E + + 3.35 F + + 0.458 log Phe:a
log Pliver:air = –1.031 + 0.059R2 + 0.774 T + + 0.593 E + + 1.049 F + + 0.654 log Phe:a
Haloalkanes
Inert Gases; LMWVOCs
H
log Pheart:air = –1.208 + 0.128R2 + 0.987 T + + 0.643 E + + 1.783 F + + 0.597 log Phe:a
R
Inert Gases; LMWVOCs
log Pbrain:air = –1.074 + 0.427R2 + 0.286 T + + 2.781 E + + 2.787 F + + 0.609 log Phe:a
log Padipose:air = –0.294 – 0.172R2 + 0.729 T + + 1.7474 E + + 0.219 F + + 0.895 log Phe:a H
Chemical Classc
Inert Gases; LMWVOCs
QSARs: LFE-type equations
Speciesb
H
Electrostatic descriptors
Approacha
Table 40.1 In Silico Approaches for Estimating the Tissue:Air Partition Coefficients (P) of Chemicals
(continued)
Gargas et al. (1988)
Abraham and Weathersby (1994)
Abraham and Weathersby (1994)
Abraham and Weathersby (1994)
Abraham and Weathersby (1994)
Abraham and Weathersby (1994)
Abraham and Weathersby (1994)
Abraham and Weathersby (1994)
Reference
IN SILICO APPROACHES FOR PBPK MODELING 485
log(Padipose:water – Vwt) = 0.9Po:w + 0.31 log(Pkidney:water – Vwt) = 0.72Po:w – 0.56 log(Pliver:water – Vwt) = 1.06Po:w – 1.43 log(Pmuscle:water – Vwt) = 0.63Po:w – 0.60 ln Padipose:air = 0.032Tb – 5.456
Hydrophobic descriptors
log Pmuscle:air = (0.9951xv) – (0.018(1/ [ VY )) – (0.4244 [ FY ) – (0.559(1/1xv) + (0.602(1/1xv)) – 1.334
– 0.217
Y – 0.194NF log Pmuscle:air = 0.3991xv – (0.007(1/ [ VY )) + 0.295QH + 0.2594 [ SF
log Pmuscle:air = 0.379QH – 0.278NCl + 0.536NBr – 0.190NF + 0.169NCl – 0.439
log Pliver:air = 1.0721Xv – 0.021/ [ VY + 0.647/1Xv – 0.3044 [ FY – 1.212
log Pliver:air = –0.6851xv – (0.020(1/ [ )) + 0.232QH + (0.298(1/1xv)) + 0.104NCl – 0.726
Y V
log Pliver:air = (1.0721xv) – (0.021(1/ [ VY )) + (0.647(1/1xv)) – (0.3044 [ FY ) – 1.212 log Pliver:air = 0.366NCl – 0.588NBr + 0.345QH – 0.179NF – 0.007
log Padipose:air = 1.0371xv – (0.007(1/ [ VY )) + 0.022QH – 0.1773 [ FY – 0.199NF – 0.0036
log Padipose:air = 0.7341Xv – 0.0291 [ VY – 1.570/1Xv – 0.5591Xv – 0.0984 [ FY + 2.213 log Padipose:air = 0.563NCl + 1.028NBr + 0.467NC + 0.270QH – 0.199NF – 0.097
Approacha
Haloalkanes Haloalkanes Haloalkanes
R R R
F F F F H
R
Chloroethanes; Benzene Chloroethanes; Benzene Chloroethanes; Benzene Chloroethanes; Benzene Haloalkanes
Haloalkanes
Haloalkanes
Haloalkanes
R
R
Haloalkanes
R
Haloalkanes
Haloalkanes
R
R
Haloalkanes
Chemical Classc
R
Speciesb Reference
Bertelsen et al. (1988) Bertelsen et al. (1988) Bertelsen et al. (1988) Bertelsen et al. (1988) Csanady and Laib (1990)
Gargas et al. (1988)
Gargas et al. (1988)
Gargas et al. (1988)
Csanady and Laib (1990)
Gargas et al. (1988) Gargas et al. (1988)
Gargas et al. (1988)
Gargas et al. (1988)
Gargas et al. (1988)
Csanady and Laib (1990)
Table 40.1 (continued) In Silico Approaches for Estimating the Tissue:Air Partition Coefficients (P) of Chemicals
486 ALTERNATIVE TOXICOLOGICAL METHODS
Pkidney:air = –0.920 + 0.764 log Po:a Pliver:air = –0.388 + 0.502 log Pw:a + 0.497 log Po:a Pliver:air = 0.432 + 1.064 log Pw:a Pliver:air = 0.746 log Po:a + 0.178 log Pw:a – 0.767 Pliver:air = 0.871 log Po:a – 1.044
Pliver:air Plung:air Plung:air Plung:air
log log log log log
log log log log
–0.875 + 0.773 log Po:a –0.21 log Po:a + 0.91 log Pw:a + 0.41 –0.057 + 0.870 log Pw:a + 0.146 log Po:a 0.057 + 0.978 log Pw:a
Pbrain:air = –0.850 + 0.773 log Po:a Pbrain:air = –3.692 + 1.253RG Pkidney:air = –0.18 log Po:a + 0.82 log Pw:a + 0.53 Pkidney:air = 0.277 + 1.111 log Pw:a Pkidney:air = 0.466 log Po:a + 0.379 log Pw:a – 0.332 Pkidney:air = 0.700 log Po:a – 0.877
log log log log log log
= = = =
H H H H H H
Padipose:air = 0.174 + 0.910 log Po:a Pbrain:air = –0.16 log Po:a + 0.82 log Pw:a + 0.47 Pbrain:air = 0.274 + 0.537 log Pw:a + 0.444 log Po:a Pbrain:air = 0.394 + 1.096 log Pw:a Pbrain:air = 0.471 log Po:a + 0.630 log Pw:a – 0.305 Pbrain:air = 0.844 log Po:a – 1.124
log log log log log log
H H H H
H H H H H
H H H H H H
H H H H H
ln Pliver:air = 0.022Tb – 4.638 log Padipose:air = 0.209 + 0.0628 log Pw:a + 0.8868 log Po:a log Padipose:air = 0.21 log Po:a + 0.24 log Pw:a log Padipose:air = 0.782 log Po:a + 0.201 log Pw:a + 0.432 log Padipose:air = 0.901 log Po:a + 0.150
Inert gases; LMWVOCs Hydrophilic VOCs Inert gases; LMWVOCs Inert gases; LMWVOCs
Inert gases; LMWVOCs Inert gases; LMWVOCs Inert gases; LMWVOCs Hydrophobic VOCs LMWVOCs
Inert gases; LMWVOCs Inert gases; LMWVOCs Hydrophilic VOCs Inert gases; LMWVOCs Hydrophobic VOCs LMWVOCs
Inert gases; LMWVOCs Hydrophilic VOCs Inert gases; LMWVOCs Inert gases; LMWVOCs Hydrophobic VOCs LMWVOCs
Haloalkanes Inert gases; LMWVOCs Hydrophilic VOCs Hydrophobic VOCs LMWVOCs
Csanady and Laib (1990) Abraham et al. (1985) Tichy (1991b) Tichy (1991a) Fiserova-Bergerova et al. (1984) Abraham et al. (1985) Tichy (1991b) Abraham et al. (1985) Abraham et al. (1985) Tichy (1991a) Fiserova-Bergerova et al. (1984) Abraham et al. (1985) Abraham et al. (1985) Tichy (1991b) Abraham et al. (1985) Tichy (1991a) Fiserova-Bergerova et al. (1984) Abraham et al. (1985) Abraham et al. (1985) Abraham et al. (1985) Tichy (1991a) Fiserova-Bergerova et al. (1984) Abraham et al. (1985) Tichy (1991b) Abraham et al. (1985) Abraham et al. (1985) (continued)
IN SILICO APPROACHES FOR PBPK MODELING 487
H H H H H H
log log log log log log H H H H H H H H H H H R R R R
log Pmuscle:air = –0.852 + 0.768 log Po:a log Pmuscle:air = –3.247 + 0.965RG Padipose:air = 0.447Po:a + 0.075Pw:a + 6.59
Pbrain:air = (0.026So + 0.51Sw)/Sa Pbrain:air = 0.020Po:a + 0.380Pw:a + 0.94
Pkidney:air = (0.014So + 0.51Sw)/Sa Pkidney:air = 0.011Po:a + 0.400Pw:a + 0.69
Pkidney:air = –0.391 + 0.550 log Pw:a + 0.440 log Po:a Pliver:air = (0.028So + 0.51Sw)/Sa Pliver:air = 0.028Po:a + 0.79
Pmuscle:air = 0.014Po:a + 0.384Pw:a + 0.94
ln Padipose:air = 0.032Tb – 5.456 ln Pliver:air = 0.022Tb – 4.638 log Padipose:air = 0.920 log Po:a + 0.136 log Padipose:air = 0.927 log Po:a – 0.032 log Pw:a + 0.120
Plung:air = –0.833 + 0.911 log Po:a Pmuscle:air = –0.19 log Po:a + 0.82 log Pw:a + 0.54 Pmuscle:air = 0.49 log Po:a + 0.39 log Pw:a – 0.31 Pmuscle:air = –0.263 + 0.575 log Pw:a + 0.423 log Po:a Pmuscle:air = 0.351 + 1.108 log Pw:a Pmuscle:air = 0.652 log Po:a – 0.702
H H
Speciesb
log Plung:air = 0.373 log Po:a + 0.416 log Pw:a – 0.216 log Plung:air = 0.644 log Po:a – 0.815
Approacha
LMWVOCs LMWVOCs LMWVOCs LMWVOCs
LMWVOCs; CFCs
Inert gases; LMWVOCs LMWVOCs LMWVOCs; CFCs
LMWVOCs LMWVOCs; CFCs
LMWVOCs LMWVOCs; CFCs
Inert gases; LMWVOCs Inert gases; LMWVOCs LMWVOCs; CFCs
Inert gases; LMWVOCs Hydrophilic VOCs Hydrophobic VOCs Inert gases; LMWVOCs Inert gases; LMWVOCs LMWVOCs
Hydrophobic VOCs LMWVOCs
Chemical Classc Reference Tichy (1991a) Fiserova-Bergerova et al. (1984) Abraham et al. (1985) Tichy (1991b) Tichy (1991b) Abraham et al. (1985) Abraham et al. (1985) Fiserova-Bergerova et al. (1984) Abraham et al. (1985) Abraham et al. (1985) Meulenberg and Vijverberg (2000) Paterson and Mackay (1989) Meulenberg and Vijverberg (2000) Paterson and Mackay (1989) Meulenberg and Vijverberg (2000) Abraham et al. (1985) Paterson and Mackay (1989) Meulenberg and Vijverberg (2000) Meulenberg and Vijverberg (2000) Csanady and Laib (1990) Csanady and Laib (1990) Gargas et al. (1989) Gargas et al. (1989)
Table 40.1 (continued) In Silico Approaches for Estimating the Tissue:Air Partition Coefficients (P) of Chemicals
488 ALTERNATIVE TOXICOLOGICAL METHODS
R R R
Pkidney:air = 0.097Po:a + 0.826Pw:a
Pliver:air = 0.026Po:a + 0.878Pw:a + 2.36
Pmuscle:air = 0.010Po:a + 0.772Pw:a + 0.29
c
b
a
LMWVOCs; CFCs
LMWVOCs; CFCs
LMWVOCs; CFCs
LMWVOCs; CFCs
R, H R, H
LMWVOCs LMWVOCs
Poulin and Krishnan (1996a) Poulin and Krishnan (1996c)
Gargas et al. (1988) Gargas et al. (1988) Gargas et al. (1989) Gargas et al. (1988) Gargas et al. (1989) Meulenberg and Vijverberg (2000) Meulenberg and Vijverberg (2000) Meulenberg and Vijverberg (2000) Meulenberg and Vijverberg (2000) Meulenberg and Vijverberg (2000)
T + = dipolarity/polarizability, E + = overall hydrogen-bond acidity, F + = overall hydrogen-bond basicity, 1Xv, [ VY , 1X, 4 [ FY , 3 [ FY , 4Xvpc = connectivity indices, NBr = number of bromide atoms in the molecule, NC = number of carbon atoms in the molecule, NCl = number of chloride atoms in the molecule, NF = number of fluoride atoms in the molecule, Phe:a = hexadecane:air partition coefficient, Po:a = n-octanol:air partition coefficient (or vegetable oil:air), Po:w = n-octanol:water partition coefficient (or vegetable oil:water), Pw:a = water:air partition coefficient, QH = variable dependant on the polarity of the molecule due to the presence of hydrogen atoms, R2 = Excess molar refraction, Rg = average solubility in entire set of solvent systems, Sa = solubility in air, So = solubility in n-octanol (or vegetable oil), Ss = solubility in saline, Sv = solubility in vegetable oil, Sw = solubility in water, Tb = boiling point, Vnt = volume fraction of neutral lipids in tissues, Vpt = volume fraction of phospholipids in tissues, and Vwt = volume fraction of water in tissues. F = fish, H = human, and R = rats. CFCs = chlorofluorocarbons, LMWVOCs = low molecular weight volatile organic chemicals, and VOCs = volatile organic chemicals.
Ptissue:air = (SsVwt + SvVnt + 0.7SsVpt + 0.3SvVpt)/Sa Ptissue:air = Po:wPw:a(Vnt + 0.3Vpt) + Pw:a(Vwt + 0.7Vpt)
R
Pbrain:air = 0.054Po:a + 0.832Pw:a
Haloalkanes Haloalkanes LMWVOCs Haloalkanes LMWVOCs LMWVOCs; CFCs
Biologically based algorithms
R R R R R R
log Padipose:air = 1.027 log Po:a – 0.046 log Pw:a – 0.119 log Pliver:air = 0.574 log Po:a + 0.302 log Pw:a – 0.278 log Pliver:air = 0.730 log Po:a + 0.128 log Pw:a – 0.550 log Pmuscle:air = 0.477 log Po:a + 0.365 log Pw:a – 0.374 log Pmuscle:air = 0.644 log Po:a + 0.180 log Pw:a – 0.725 Padipose:air = 0.594Po:a + 0.085Pw:a + 9.40
IN SILICO APPROACHES FOR PBPK MODELING 489
490
ALTERNATIVE TOXICOLOGICAL METHODS
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log Pplasma:air = –1.48 + 0.490R2 + 2.04 T + + 3.5074 E + + 3.911 F + + 0.157 log Phe:a
log (Pblood:water – Vwb) = 0.7Po:w – 0.75 ln Pblood:air = 0.038Tb – 13.3 log Pblood:air = 0.0109Tb – 2.584 log Pblood:air = –0.14 log Po:a + 0.86 log Pw:a + 0.47 log Pblood:air = 0.685 log Po:a – 0.6565 log Pblood:air = 0.45 log Pw:a + 1.21 log Pblood:air = –0.003 log Pw:a + 1.47 log pblood:air = –0.074 + 0.802 log Pw:a + 0.218 log Po:a log Pblood:air = –0.07 log Sw + 1.21
Hydrophobic descriptors
log Pblood:air = 0.0072MW + 0.197 log Pblood:air = 0.321NBr + 1.06 Pblood:air = 0.07MW + 5.59 log Pblood:air = 0.443QH – 0.303NF + 0.225NCl + 0.510NBR + 0.155NC – 0.104
Steric descriptors
Speciesb
F H H H H H H H H
Chloroethanes; benzene Aliphatic hydrocarbons Trihalomethanes Hydrophilic VOCs Trihalomethanes VOCs VOCs Inert gases; LMWVOCs VOCs
Trihalomethanes Trihalomethanes Aliphatic hydrocarbons Haloalkanes
Inert Gases; LMWVOCs
H
H H H R
Inert Gases; LMWVOCs
Chemical Classc
H
QSARs: LFE-type equations
log Pblood:air = –1.269 + 0.612R2 + 0.916 T + + 3.614 E + + 3.381 F + + 0.362 log Phe:a
Electrostatic descriptors
Approacha
Table 40.2 In Silico Approaches for Estimating the Blood:Air Partition Coefficients (P) of Chemicals
Bertelsen et al. (1998) Csanady and Laib (1990) Batterman et al. (2002) Tichy (1991b) Batterman et al. (2002) Laass (1987) Laass (1987) Abraham et al. (1985) Laass (1987) (continued)
Batterman et al. (2002) Batterman et al. (2002) Perbellini et al. (1985) Gargas et al. (1988)
Abraham and Weathersby (1994)
Abraham and Weathersby (1994)
Reference
IN SILICO APPROACHES FOR PBPK MODELING 491
Pblood:air Pblood:air Pblood:air Pblood:air Pblood:air Pblood:air
Pblood:air Pblood:air Pblood:air Pblood:air Pblood:air Pblood:air Pblood:air Pblood:air Pblood:air Pblood:air Pblood:air Pblood:air Pblood:air Pblood:air Pblood:air Pblood:air Pblood:air Pblood:air Pblood:air Pblood:air Pblood:air
log log log log log log
log log log log log log log log log log log log log log log log log log log log log
= = = = = = = = = = = = = = = = = = = = =
= = = = = =
–0.820 + 0.754 log Po:a 0.09 log Sw + 8.25 log Vo – 11.09 0.11 log Sw + 1.91 0.180 log Po:a + 0.889 log Pw:a + 0.054 0.20 log Sw + 1.29 0.22 log Pw:a + 0.67 log Po:a – 0.98 0.22 log Sw + 10.78 log Vw – 40.99 0.262 + 0.996 log Pw:a 0.27 log 1000/P + 5.10 log Vo – 6.67 0.31 log Sw + 3.90 log Vo – 4.53 0.35 log 1000/P + 1.01 0.35 log Sw + 0.79 log 1000/P + 1.34 log Vo – 2.23 0.37 log Sw + 10.09 log Vw – 38.40 0.38 log Sw + 0.91 log 1000/P – 0.45 0.45 log Sw + 0.81 log 1000/P – 0.40 0.48 log Sw + 0.75 log 1000/P + 1.67 log Vo – 2.77 0.51 log 1000/P + 0.37 0.581 log Po:a + 0.332 log Pw:a – 0.599 0.63 log 1000/P + 0.38 0.65 log Po:a – 0.84 0.851 log Sw + 1.78
–0.09 log Po:a + 2.45 –0.102 + 0.675 log Pw:a + 0.315 log Po:a –0.295 + 0.588 log Pw:a + 0.411 log Po:a –0.338 log Po:a + 3.121 –0.6737 + 0.5319 log Po:a log Pw:a 0.695 log Po:a – 1.076
Approacha
H H H H H H H H H H H H H H H H H H H H H
H H H H H H
Speciesb
Inert gases; LMWVOCs VOCs VOCs Hydrophobic VOCs VOCs VOCs VOCs Inert gases; LMWVOCs VOCs VOCs VOCs VOCs VOCs VOCs VOCs VOCs VOCs LMWVOCs VOCs VOCs VOCs
VOCs Inert gases; LMWVOCs Inert gases; LMWVOCs Halogenated hydrocarbons VOCs LMWVOCs
Chemical Classc
Table 40.2 (continued) In Silico Approaches for Estimating the Blood:Air Partition Coefficients (P) of Chemicals
Laass (1987) Abraham et al. (1985) Abraham et al. (1985) Tichy et al. (1984) Sato and Nakajima (1979) Fiserova-Bergerova et al. (1984) Abraham et al. (1985) Laass (1987) Laass (1987) Tichy (1991a) Laass (1987) Laass (1987) Laass (1987) Abraham et al. (1985) Laass (1987) Laass (1987) Laass (1987) Laass (1987) Laass (1987) Laass (1987) Laass (1987) Laass (1987) Laass (1987) Gargas et al. (1989) Laass (1987) Laass (1987) Laass (1987)
Reference
492 ALTERNATIVE TOXICOLOGICAL METHODS
= = = = = = =
0.08e0.0308Tb 0.00442Po:a 0.88Pw:a + 0.012 0.89Pw:a + 0.011Po:a 0.90 log Pw:a – 461 Pw:a + (Po:a/100) Sw(1 + 0.0035Po:w)/Sa
log Pblood:air = Pw:a[VlbPo:w0.85 + Vprb(86.2/Po:w + 3.70)] + Vwb log Pblood:air = 0.426 log Po:a + 0.515 log Pw:a – 0.070 log Pblood:air = 0.553 log Po:a + 0.351Pw:a – 0.286 Pblood:air = 0.0054Po:a + 0.931Pw:a + 1.16
Pblood:air Pblood:air Pblood:air Pblood:air Pblood:air Pblood:air Pblood:air
log Pblood:air = 0.984 log Pw:a + 0.053 log Pblood:air = 1.07 log Pw:a + 0.27 log Po:a – 0.79 log Pblood:air = 1.21 log Vo – 0.17 log Pblood:air = 3.05 – 0.34Po:n log Pblood:air = –3.922 + 1.369RG log Pblood:air = 5.89 log Vw – 21.43 log Pblood:air = 7.86 log Vo – 10.40 log Pblood:air = 8.90 log Vw – 33.40 log Pmilk:air = 0.900 log Po:a – 1.095 log Pplasma:air = –0.079 + 0.896 log Pw:a + 0.149 log Po:a log Pplasma:air = –0.082 + 0.894 log Pw:a + 0.152 log Po:a log Pplasma:air = –0.848 + 0.890 log Po:a log Pplasma:air = –3.696 + 1.208RG log Pplasma:air = 0.038 + 1.019 log Pw:a Pblood:air = 0.0072Po:a + 0.898Pw:a + 0.03
LMWVOCs Haloalkanes LMWVOCs LMWVOCs; CFCs
Aliphatic hydrocarbons Aliphatic hydrocarbons VOCs LMWVOCs Esters; alcohols Anaesthetics LMWVOCs
H H H H H H H H, R R R R
Ketones; ethers; gases VOCs VOCs Ketones Inert gases; LMWVOCs VOCs VOCs VOCs Trihalomethanes Inert gases; LMWVOCs Inert gases; LMWVOCs Inert gases; LMWVOCs Inert gases; LMWVOCs Inert gases; LMWVOCs LMWVOCs; CFCs
H H H H H H H H H H H H H H H
Tichy et al. (1984) Laass (1987) Laass (1987) Cabala et al. (1992) Abraham et al. (1985) Laass (1987) Laass (1987) Laass (1987) Batterman et al. (2002) Abraham et al. (1985) Abraham et al. (1985) Abraham et al. (1985) Abraham et al. (1985) Abraham et al. (1985) Meulenberg and Vijverberg (2000) Perbellini et al. (1985) Perbellini et al. (1985) Feingold (1976) Tichy et al. (1984) Kaneko et al. (1994) Eger and Larson (1964) Paterson and Mackay (1989) Connell et al. (1993) Gargas et al. (1988) Gargas et al. (1989) Meulenberg and Vijverberg (2000) (continued)
IN SILICO APPROACHES FOR PBPK MODELING 493
c
b
Chloroethanes Chloroethanes Chloroethanes
R
Chemical Classc
F H
LMWVOCs LMWVOCs
Poulin and Krishnan (1996c) Poulin and Krishnan (1996b)
Fouchécourt et al. (2000) Fouchécourt and Krishnan (2000) Fouchécourt and Krishnan (2000)
Reference
T + = dipolarity/polarizability, E + = overall hydrogen-bond acidity, F + = overall hydrogen-bond basicity, BS = Basic structure, fe = fraction of erythrocytes in blood, fp = fraction of plasma in blood, MW = molecular weight, NBr = number of bromide atoms in the molecule, NC = number of carbon atoms in the molecule, NCl = number of chloride atoms in the molecule, nCL = number of CL fragments, nCL2 = number of CL2 fragments, nCL3 = number of CL3 fragments, NF = number of fluoride atoms in the molecule, nH3 = number of H3 fragments, P = vapor pressure, Phe:a = hexadecane:air partition coefficient, Po:a = n-octanol:air partition coefficient (or vegetable oil:air), Po:n = vegetable oil:nitrogen partition coefficient, Po:w = n-octanol:water partition coefficient (or vegetable oil:water), Pw:a = water:air partition coefficient, QH = variable dependant on the polarity of the molecule due to the presence of hydrogen atoms, R2 = excess molar refraction, Rg = parameters relative to the solvent, Sa = solubility in air, Ss = solubility in saline, Sv = solubility in vegetable oil, Sw = solubility in water, Tb = boiling point, Vlb = volume fraction of lipids in blood, Vnb = volume fraction of neutral lipids in blood, Vne = volume fraction of neutral lipids in erythrocytes, Vnp = volume fraction of neutral lipids in plasma, Vo = surface tension, Vpb = volume fraction of phospholipids in blood, Vpe = volume fraction of phospholipids in erythrocytes, Vpp = volume fraction of phospholipids in plasma, Vprb = volume fraction of proteins in blood, Vw = heat released due to evaporation of the substance at boiling temperature, Vwb = volume fraction of water in blood, Vwe = volume fraction of water in erythrocytes, and Vwp = volume fraction of water in plasma. F = fish, H = human, and R = rats. CFCs = chlorofluorocarbons, LMWVOCs = low molecular weight volatile organic chemicals, and VOCs = volatile organic chemicals.
R, H R, H
Biologically based algorithms
Pblood:air = Po:wPw:a(Vnb + 0.3Vpb) + Pw:a(Vwb + 0.7Vpb) Pblood:air = [fe(SsVwe + SvVne + 0.7SsVpe + 0.3SvVpe) + fp(SsVwp + SvVnp + 0.7SsVpp + 0.3SvVpp)]/Sa
a
Speciesb QSARs: Free–Wilson-type equations
Pblood:water = BS (C-C) (28.4) + nCL2(–12.9) + nCL3(12.9) Pblood:air = BS (C-C) (26.2) + nH3(–34.9) + nCL(–4.51) + nCL2(29.4) + nCL3(11.5) Pblood:air = BS(C-C) (45.6) + nH3(–51.5) + nCL(–8.86) + nCL2(36.4) + nCL3(11.1)
Approacha
Table 40.2 (continued) In Silico Approaches for Estimating the Blood:Air Partition Coefficients (P) of Chemicals
494 ALTERNATIVE TOXICOLOGICAL METHODS
IN SILICO APPROACHES FOR PBPK MODELING
495
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Inert gases; HMWOCs; LMWVOCs H2-R antagonists Inert gases; LMWVOCs; CFCs Inert gases; LMWVOCs; CFCs Inert gases; LMWVOCs; CFCs Inert gases; LMWVOCs; CFCs Inert gases; LMWVOCs; CFCs PCBs H2-R antagonists
H H H H H H R
log Pheart:blood = –0.346 + 0.204 T + – 2.150 E + – 0.853 F + + 0.931Vx
log Pkidney:blood = –0.188 + 0.226R2 – 0.559 T + – 0.433 F + + 0.832Vx
log Pliver:blood = –0.270 + 0.233R2 – 0.375 T + – 1.004 E + – 1.118 F + + 0.832Vx
log Plung:blood = –0.150 – 0.195 T + + 0.389Vx
log Pmuscle:blood = –0.222 – 0.479 T + – 0.517 F + + 0.999Vx
Padipose:plasma = 1.9988 – 0.5004UNS + 0.1793NPL + 0.05931DIFF2
0.7567 F
+
+ 1.189Vx
log Pbrain:blood = 0.088 + 0.264R2 – 0.966 T
+
– 0.7057 E +
–
log Pbrain:blood = –0.166 + 0.239R2 – 0.626 T + – 0.368 E + – 0.615 F + + 1.072Vx log Pbrain:blood = –0.0148PSA + 0.152 log Po:w + 0.139 log Pbrain:blood = 1.359 + 0.338 log Pcyh – 0.00618Vm H H
Inert gases; LMWVOCs; CFCs
Chemical Classc
Inert gases; LMWVOCs; CFCs
H
QSARs: LFE-type equations
Speciesb
H
3.267 F + + 2.275Vx
log Padipose:blood = 0.168 + 0.198R2 + 0.130 T + – 1.211 E + –
Steric descriptors
Approacha
Table 40.3 In Silico Approaches for Estimating the Tissue:Blood Partition Coefficients (P) of Chemicals
(continued)
Abraham and Weathersby (1994) Abraham and Weathersby (1994) (48) Norinder and Haeberlein (2002)
Clark (1999) Kalizan and Markuszewski (1996) Abraham and Weathersby (1994) Abraham and Weathersby (1994) Abraham and Weathersby (1994)
Abraham and Weathersby (1994)
Abraham and Weathersby (1994)
Reference
IN SILICO APPROACHES FOR PBPK MODELING 497
H2-R antagonists; Inert gases; SOMs
R R R
log Pbrain:blood = –0.021PSA – 0.003MV + 1.643 log Pbrain:blood = –0.0322DPSA + 1.33
$ $ Pliver:blood = ?(Vlt 3R Z +Vwt)/(Vlb 3R Z + Vwb)A+ B
$ $ Pkidney:blood = ?(Vlt 3R Z + Vwt)/(Vlb 3R Z + Vwb)A+ B
$ $ Pbrain:blood = ?(Vlt 3R Z + Vwt)/(Vlb 3R Z + Vwb)A+ B
$ $ Padipose:blood = ?(Vlt 3R Z + Vwt)/(Vlb 3R Z + Vwb)A+ B
log Pbrain:blood = 0.39 log Po:w + 0.68 log Pbrain:blood = 0.054Go + 0.43
LMWVOCs LMWVOCs LMWVOCs
H, R H, R H, R
H2-R antagonists
Drugs, hormones H2-R antagonists; LMWVOCs LMWVOCs
R
log Pbrain:blood = 1.296 + 0.309 log Pcyh – 0.00570MW
H2-R antagonists
HMWOCs
Drug-like molecules
H H H, R
R
log Pbrain:blood = 0.476 + 0.541 log Po:w – 0.00794MW
Hydrophobic descriptors
R
0.6987 F + + 0.995Vx log Pbrain:blood = –0.218(NN + NO) + 0.235 log Po:w – 0.027
log Pbrain:blood = –0.038 + 0.198R2 – 0.687 T + – 0.7157 E + –
Inert gases; HMWOCs; LMWVOCs HMWOCs
R
log Pbrain:blood = –0.01Vm + 0.35 log Po:w + 0.99I3 + 1.25
Inert gases; volatile hydrocarbons
R
log Pbrain:blood = 0.00116MW + 0.272 log Po:w – 0.088
H2-R antagonists
Chemical Classc
R
Speciesb
log Pbrain:blood = –0.088 + 0.272 log Po:w – 0.00116MW
Approacha Reference
DeJongh et al. (1997)
DeJongh et al. (1997)
DeJongh et al. (1997)
Seydel and Schaper (1982) Lombardo et al. (1996) DeJongh et al. (1997)
Norinder and Haeberlein (2002) Kalizan and Markuszewski (1996) Kalizan and Markuszewski (1996)
Kalizan and Markuszewski (1996) Norinder and Haeberlein (2002) Norinder and Haeberlein (2002) Clark (1999) Norinder and Haeberlein (2002) Norinder and Haeberlein (2002)
Table 40.3 (continued) In Silico Approaches for Estimating the Tissue:Blood Partition Coefficients (P) of Chemicals
498 ALTERNATIVE TOXICOLOGICAL METHODS
Basic drugs Basic drugs Basic drugs Basic drugs Basic drugs Basic drugs Basic drugs Basic drugs Basic drugs Basic drugs
Rb Rb Rb Rb Rb Rb Rb Rb Rb Rb
Pkidney:plasma = 0.075 3R Z
Pheart:plasma = 0.032 3R Z
Pheart:blood = 1.678 3R Z
Pgut:plasma = 0.058 3R Z
Pgut:blood = 3.002 3R Z
Pbrain:plasma = 0.062 3R Z
Pbrain:blood = 3.157 3R Z
Pbone:plasma = 0.036 3R Z
Pbone marrow:blood = 1.975 3R Z
Padipose:plasma = 0.016 3R Z
Padipose:blood = 0.915 3R Z
H2-R antagonists HMWOCs Basic drugs
R R Rb
log Pbrain:plasma = –0.48 (log Poct-cyc + 0.89 ln Padipose:blood = 0.05§i + 0.021
Drugs; LMWVOCs; anaesthetics H2-R antagonists
R R
log Pbrain:blood = 0.4275 – 0.3873nacc,solv + 0.1092 log Po:w – 0.0017Apol log Pbrain:blood = 1.979 + 0.373 log Pcyh – 0.00275Vwav
HMWOCs HMWOCs HMWOCs H2-R antagonists; LMWVOCs
LMWVOCs
R R R R
H, R
Ln Pkidney:blood = 0.0065§o Ln Pliver:blood = 0.025§i Ln Pmuscle:blood = 0.0069§i log Pbrain:blood = 0.035(Gsolv + 0.259
$ $ Pmuscle:blood = ?(Vlt 3R Z + Vwt)/(Vlb 3R Z + Vwb)A+ B
(continued)
Yokogawa et al. (2002)
Yokogawa et al. (2002)
Yokogawa et al. (1990)
Yokogawa et al. (2002)
Yokogawa et al. (1990)
Yokogawa et al. (2002)
Yokogawa et al. (1990)
Yokogawa et al. (2002)
Yokogawa et al. (1990)
Yokogawa et al. (2002)
Yamaguchi et al. (1996) Yamaguchi et al. (1996) Yamaguchi et al. (1996) Norinder and Haeberlein (2002) Feher et al. (2000) Kalizan and Markuszewski (1996) Testa et al. (2000) Yamaguchi et al. (1996) Yokogawa et al. (1990)
DeJongh et al. (1997)
IN SILICO APPROACHES FOR PBPK MODELING 499
Basic drugs Basic drugs Basic drugs Basic drugs Basic drugs Basic drugs Basic drugs Basic drugs
Rb Rb Rb Rb Rb Rb Rb
Chemical Classc
Rb
Speciesb
Chloroethanes Chloroethanes Chloroethanes Chloroethanes Chloroethanes Chloroethanes Chloroethanes Chloroethanes
F F F H H H R R
QSARs: Free–Wilson-type equations
Padipose:blood = BS(C-C)(94.5) + nCL2(–29.2) + nCL3(29.2) Pliver:blood = BS(C-C)(2.93) + nCL2(–0.238) + nCL3(0.238) Pmuscle:blood = BS(C-C)(3.02) + nCL2(–0.175) + nCL3(0.175) Padipose:blood = BS(C-C)(49.2) + nH3(–0.440) + nCL(–14.54) + nCL2(–6.65) + nCL3(26.5) Pliver:blood = BS(C-C)(2.64) + nH3(–0.61) + nCL(–0.66) + nCL2(–0.18) + nCL3(1.68) Pmuscle:blood = BS(C-C)(1.11) + nH3(0.08) + nCL(–0.02) + nCL2(–0.21) + nCL3(0.15) Padipose:blood = BS(C-C)(30.1) + nH3(–9.88) + nCL(–6.02) + nCL2(–3.90) + nCL3(17.3) Pliver:blood = BS(C-C)(1.79) + nH3(–0.9) + nCL(–0.38) + nCL2(–0.21) + nCL3(1.27)
Pspleen:blood = 3.002 3R Z
Pskin:plasma = 0.058 3R Z
Pskin:blood = 2.997 3R Z
Pmuscle:plasma = 0.099 3R Z
Pmuscle:blood = 4.928 3R Z
Plung:plasma = 0.031 3R Z
Plung:blood = 1.158 3R Z
Pliver:plasma = 0.064 3R Z
Approacha Reference
Fouchécourt Fouchécourt Fouchécourt Fouchécourt (2000) Fouchécourt (2000) Fouchécourt (2000) Fouchécourt (2000) Fouchécourt (2000)
and Krishnan
and Krishnan
and Krishnan
and Krishnan
et al. (2000) et al. (2000) et al. (2000) and Krishnan
Yokogawa et al. (1990)
Yokogawa et al. (2002)
Yokogawa et al. (1990)
Yokogawa et al. (2002)
Yokogawa et al. (1990)
Yokogawa et al. (2002)
Yokogawa et al. (1990)
Yokogawa et al. (2002)
Table 40.3 (continued) In Silico Approaches for Estimating the Tissue:Blood Partition Coefficients (P) of Chemicals
500 ALTERNATIVE TOXICOLOGICAL METHODS
c
b
Chloroethanes
LMWVOCs
Ketones; Alcohols; Esters
R R, H
LMWVOCs
H
Poulin and Krishnan (1996b)
Poulin and Krishnan (1995b)
Poulin and Krishnan (1995a)
Fouchécourt and Krishnan (2000)
T + = dipolarity/polarizability, E + = overall hydrogen-bond acidity, F + = overall hydrogen-bond basicity, (Gsolv = free energy of solvation in hexadecane, 7i = molecular structure Fujita value, 7o = molecular structure Fujita value, A1, A2 = Collander-type coefficient, Apol = polar surface area, B = correction factor, BS = basic structure, DIFF = variable dependant on the number of chloride atoms in the aromatic cycle, DPSA = dynamic polar surface area, fe = fraction of erythrocytes in blood, fp = fraction of plasma in blood, I3 = variable dependant on the presence of an amino nitrogen or carboxyl group, MV = molecular volume, MW = molecular weight, nacc,solv = number of solvated hydrogen-bond acceptors, nCL = number of CL fragments, nCL2 = number of CL2 fragments, nCL3 = number of CL3 fragments, nH3 = number of H3 fragments, NN = number of nitrogens, NO = number of oxygens, NPL = variable dependant on the number of chloride atoms in the molecule in ortho position, oG = Gibbs free energy related to the solvation of the substance in water, Pcyh = cyclohexane:water partition coefficient, Po:w = n-octanol:water partition coefficient (or vegetable oil:water), Poct-cyc = octanol-cyclohexane, PSA = polar surface area, R2 = Excess molar refraction, So = solubility in n-octanol (or vegetable oil), Sw = solubility in water, UNS = variable dependant on the number of atoms in the molecule that are not chlorides, Vlb = volume fraction of lipids in blood, Vlt = volume fraction of lipids in tissue, Vm = molar volume, Vnb = volume fraction of neutral lipids in blood, Vne = volume fraction of neutral lipids in erythrocytes, Vnp = volume fraction of neutral lipids in plasma, Vnt = volume fraction of neutral lipids in tissues, Vpb = volume fraction of phospholipids in blood, Vpe = volume fraction of phospholipids in erythrocytes, Vpp = volume fraction of phospholipids in plasma, Vpt = volume fraction of phospholipids in tissues, Vwav = volume of water needed in order to solubilize the substance, Vwb = volume fraction of water in blood, Vwb = volume fraction of water in blood, Vwe = volume fraction of water in erythrocytes, Vwp = volume fraction of water in plasma, Vwt = volume fraction of water in tissue, Vwt = volume fraction of water in tissues, and Vx = McGowan characteristic volume. F = fish, H = human, and R = rats. CFCs = chlorofluorocarbons, HMWOCs = high molecular weight organic chemicals, LMWVOCs = low molecular weight volatile organic chemicals, PCBs = polychlorobiphenyls, and VOCs = volatile organic chemicals.
Ptissue:blood = (SoVnt + Sw0.7Vpt + So0.3Vpt + SwVwt)/(SoVnb + Sw0.7Vpb + So0.3Vpb + SwVwb) Ptissue:blood = (Po:wVnt + Vwt + Po:w0.3Vpt + 0.7Vpt)/[fe(Po:wVne + Vwe + Po:w0.3Vpe + 0.7Vpe) + fp(Po:wVnp + Vwp + Po:w0.3Vpp + 0.7Vpp)] Ptissue:blood = [Po:w(Vnt + 0.3Vpt) + (Vwt + 0.7Vpt)]/[Po:w(Vnb + 0.3Vpb) + (Vwb + 0.7Vpe)]
a
R
Biologically based algorithms
Pmuscle:blood = BS(C-C)(0.69) + nH3(–0.12) + nCL(0.04) + nCL2(–0.12) + nCL3(0.17)
IN SILICO APPROACHES FOR PBPK MODELING 501
502
ALTERNATIVE TOXICOLOGICAL METHODS
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log log log log log log log log log log log log log log log log log log log log log log
(1/fu(plasma) – 1) = 0.994 log Po:w – 1.10 (1/fu(plasma) – 1) = 0.994 log Po:w – 1.10 2 + 13 log P (1/Ka(plasma)) = –3.91 log Po:w o:w – 13.7 (1 – fu(brain)) = 0.36 log Po:w – 1.07 (1 – fu(plasma)) = 0.276 log Po:w + 1.2 (1 – fu(plasma)) = 0.30 log Po:w – 1.03 (1 – fu(plasma)) = 0.33 log Po:w + 1.94 1/Ka(albumin binding) = –0.85 log Po:w + 2.73 1/Ka(albumin binding) = –0.97 log Po:w + 3.24 1/Ka(albumin binding) = –0.97 log Po:w – 0.70I + 3.24 1/Ka(albumin binding) = –0.99 log Po:w + 2.49 Kalbumin binding = 0.89 log Po:w + 1.47 Kalbumin binding = 1.15 log Po:w + 1.23 Kalbumin binding = 1.23 log Po:w – 0.056 Kalbumin binding = 1.32 log Po:w + 0.37 Kalbumin binding = 1.39 log Po:w – 1.19 Kalbumin binding = 1.65 log Po:w – 2.57 Kplasma protein binding = 0.73(Rmui + 1.46 Ka(blood protein binding) = 0.5047T – 0.665 (1/fu(plasma) – 1) = 1.011 log Po:w – 1.745 (1 – fu)/fu(adipose) = log 0.750 + 0.936 log Po:w (1 – fu)/fu(brain) = log 0.073 + 0.860 log Po:w
Hydrophobic descriptors
Approachb Chemical Class
H H H H H H H H H H H H H H H H H H H R R R
Aromatic acids Organic acids Cephalosporins Barbiturates Penicillins Barbiturates Tetracyclines Sulfapyrimidines; sulfapyridines Sulfapyridines Sulfapyrimidines; sulfapyridines Sulfapyrimidines Sulfapyrimidines Sulfonamides Steroid bisguanylhydrazones Penicillins Cardenolides Steroid hormones Sulfapyridines Penicillins Organic acids Barbituric acids Barbituric acids
QSARs: LFE-type equations
Speciesc
Table 40.4 In Silico Approaches for Estimating Protein Binding of Chemicalsa
Testa et al. (2000) Laznicek et al. (1987) Testa et al. (2000) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982) Bird and Marshall (1967) Laznicek et al. (1987) Nesterov et al. (1998) Nesterov et al. (1998) (continued)
Reference
IN SILICO APPROACHES FOR PBPK MODELING 503
c
b
a
(1 – fu)/fu(gut) = log 0.099 + 0.824 log Po:w (1 – fu)/fu(heart) = log 0.135 + 0.780 log Po:w (1 – fu)/fu(kidney) = log 0.676 + 0.619 log Po:w (1 – fu)/fu(liver) = log 1.775 + 0.504 log Po:w (1 – fu)/fu(lung) = log 0.164 + 0.841 log Po:w (1 – fu)/fu(muscle) = log 0.080 + 0.835 log Po:w (1 – fu)/fu(pancreas) = log 0.022 + 1.095 log Po:w (1 – fu)/fu(plasma) = log 0.016 + 0.975 log Po:w (1 – fu)/fu(red blood cell) = log 0.178 + 0.677 log Po:w (1 – fu)/fu(skin) = log 0.271 + 0.736 log Po:w (1 – fu)/fu(spleen) = log 0.126 + 0.841 log Po:w (1 – fu)/fu(stomach) = log 0.058 + 0.939 log Po:w (1 – fu)/fu(testis) = log 0.120 + 0.747 log Po:w Kplasma protein binding = 0.33(Rmui – 0.53I + 4.08 (1/fu(plasma) – 1) = 1.016 log Po:w – 1.275
R R R R R R R R R R R R R R Rb
Speciesc Barbituric acids Barbituric acids Barbituric acids Barbituric acids Barbituric acids Barbituric acids Barbituric acids Barbituric acids Barbituric acids Barbituric acids Barbituric acids Barbituric acids Barbituric acids Sulfapyridines Organic acids
Chemical Class Nesterov et al. (1998) Nesterov et al. (1998) Nesterov et al. (1998) Nesterov et al. (1998) Nesterov et al. (1998) Nesterov et al. (1998) Nesterov et al. (1998) Nesterov et al. (1998) Nesterov et al. (1998) Nesterov et al. (1998) Nesterov et al. (1998) Nesterov et al. (1998) Nesterov et al. (1998) Seydel and Schaper (1982) Laznicek et al. (1987)
Reference
K = protein affinity constant (Freundlich isotherm), Ka = protein affinity constant (Scatchard isotherm) and fu = unbound fraction. Po:w = octanol:water partition coefficient, T = molecular hydrophobicity constant, I = family indicator variable, (Rmui = variable dependant on the resistance constant due to diffusion of the nonionized form in the lipid membrane. H = humans, Rb = rabbit, and R = rat.
log log log log log log log log log log log log log log log
Approachb
Table 40.4 (continued) In Silico Approaches for Estimating Protein Binding of Chemicalsa
504 ALTERNATIVE TOXICOLOGICAL METHODS
IN SILICO APPROACHES FOR PBPK MODELING
505
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506
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H H R
log CL(renal) = –0.5 log Po:w + 3 log CL(renal) = –0.5 log Po:w + 13 log CL(hepatic) = –0.54(Rmui – 0.51
) log CL(renal) = –log(0.35 + 0.013 3R Z
H H H
R
H
H
H H H H H R
CL (intrinsic; hepatic) = 0.0555 Po:w1.05 log CL(renal) = –0.24(log Po:w)2 – 0.04 log Po:w + 0.58
Hydrophobic descriptors
1/log CL(intrinsic; hepatic) = 3.58 – 0.058S_sCl – 0.57S_aaO – 0.47Shadow Z length – 0.75CIC 1/log CL(intrinsic; hepatic) = –3.11 – 0.10Dipole – mag + 13.25Jurs – RPCG + 0.57Jurs – RPCS + 0.00013Apol CL(intrinsic; hepatic) = 25Steric + 44Electrostatic + 20LUMO + 11HINT
Steric descriptors
Speciesb QSARs: LFE-type equations
log CL(hepatic) = 0.64 log Po:w – 0.98IP + 9.33 log CL(hepatic) = 0.055Energy – 0.95IP – 0.53HBD + 10.63 log CL(hepatic) = 0.067Energy – 1.01IP – 0.34HBD – 0.43(E + 14.66 log CL(hepatic) = 0.094Energy – 1.18IP – 0.74(E + 18.65 log CL(hepatic) = 0.65 log Po:w – 0.40IP – 0.37HBD + 0.0025Hf + 3.63 Metabolic ratio = 2.72Q6 + 1.96EH + 0.014SN + 6.43
Electrostatic descriptors
Approacha
Table 40.5 In Silico Approaches for Estimating Clearances (CL) of Chemicals
NSAID ß-blockers Sulfonamides
Basic drugs Probenecid analogs Probenecid analogs
Commercially available drugs Commercially available drugs Haloalkanes
Benzodiazepines Benzodiazepines Benzodiazepines Benzodiazepines Benzodiazepines Dichlorobiphenyls
Chemical Classc
(2000) (2000) (2000) (2000) (2000) and Dickins (2002)
Smith et al. (1996) Smith et al. (1996) Seydel and Schaper (1982) (continued)
Yokogawa et al. (2002) Seydel and Schaper (1982) Seydel and Schaper (1982)
Waller et al. (1996)
Ekins and Obach (2000)
Ekins and Obach (2000)
Lewis Lewis Lewis Lewis Lewis Lewis
Reference
IN SILICO APPROACHES FOR PBPK MODELING 507
CL(renal) = –0.41(Rmui – 0.80 CL(renal) = –0.51 log Po:w – 0.33 CL(renal) = –log [0.048 + 6.98 v 10–4(107T)1.394] CL(total) = –0.74(Rmui + 0.22pKa – 1.73 E(hepatic) = 0.045 log Po:w – 0.32
c
b
a
Basic drugs Basic drugs
Rb Rb
Seydel and Schaper (1982) Seydel and Schaper (1982)
Ishizaki et al. (1997)
Ishizaki et al. (1997)
Seydel and Schaper (1982) Yamaguchi et al. (1996) Seydel and Schaper (1982) Seydel and Schaper (1982) Yamaguchi et al. (1996) Ishizaki et al. (1997)
Reference
(E = variable related to molecular orbitals, (Rmui = variable dependant on the resistance constant due to diffusion of the nonionized form in the lipid membrane, T = molecular hydrophobicity constant, Apol = polar surface area, CIC = complementary information content, Dipole-mag = dipole moment, EH = HOMO energy, Electrostatic = Coulombic interaction energy, Energy = minimum internal energy, HBD = potential hydrogen bond donor atoms in the molecule, Hf = enthalpy of formation, HINT = hydrophobic field energy, IP = ionization potential, Jurs-RPCG = relative positive charge, Jurs-RPCS = relative positive charge surface area, LUMO = lowest unoccupied molecular orbital energy, Po:w = n-octanol:water partition coefficient (or vegetable oil:water), Po:w, app = apparent octanol:water partition coefficient, Q6 = net atomic charge on carbon atom at biphenyl ring position, R2(CH3) = methyl fragment at R2 position, R2(C2H5) = ethyl fragment at R2 position, R1(OC3H7) = propyl ether fragment at R1 position, S_aaO = E-state indices for oxygen atoms with two aromatic bonds, S_sCl = E-state indice for chlorine atoms with a single bond, Shadow Z length = length of the molecule in Z dimension, SN = total nucleophillic superdelocalizability, and Steric = Van der Waals interaction energy. F = fish, H = human, and R = rats. CFCs = chlorofluorocarbons, HMWOCs = high molecular weight organic chemicals, LMWVOCs = low molecular weight volatile organic chemicals, and VOCs = volatile organic chemicals.
R R
Xylidines Xylidines
Sulfonamides Sulfapyridines Xylidines Sulfonamides HMWOCs Basic drugs
Chemical Classc
R R R R R Rb
Speciesb
QSARS: Free–Wilson-type equations
log CL(renal) = 0.417R2(CH3) – 0.744R1(OC3H7) + 1.33 log CL(total) = 0.49R2(CH3) + 0.57 R2(C2H5) + 0.25R1(OC3H7) + 1.76
CL(intrinsic; hepatic) = 0.248 3R ZDSS
CL(intrinsic; hepatic) = 0.0875 3R Z
CL(intrinsic; hepatic) = 3.828 3R Z
log log log log log
Approacha
Table 40.5 (continued) In Silico Approaches for Estimating Clearances (CL) of Chemicals
508 ALTERNATIVE TOXICOLOGICAL METHODS
Y log Vmax (oxidation) = –1.6764 [ FY + 0.4243 [ FY – 0.134 [ SF + 1.622
log kcat/Km(demethylation) = 0.53 log Po:w + 3.47 log V = 0.55 log Po:w
Hydrophobic descriptors
V(n-demethylation) = 0.005SA – 0.52 V(n-demethylation) = 0.038SA – 0.00001SA2 – 25.64
Speciesc
R R
X-C6H4N(CH3)2 Barbiturates
Amines Amines
Haloalkanes
R R R
Toluenes Ethylamines Ethylamines PCBs
Nitriles Toluenes Halothanes Barbiturates Anilines X-C6H4N(CH3)2
Chemical Classd
H H H R
H H H H H R
QSARs: LFE-type equations
log kcat/Km(Oxidation) = 0.0347SA – 2.29(E + 1.92 log Vmax (n-demethylation) = 0.18 Length – 1.94 log Vmax (n-demethylation) = 3.50 Length – 0.13 Length2 – 23.9 log V(oxidation) = 2.4861 – 0.1364NPL * NSIDE + 0.5694UNS – 0.2433NOM * NMC + 0.001227MW * NUNSTOT + 0.8242IND – 1.1493MOD
Steric descriptors
log V(oxidation) = 0.894 – 0.111diameter – 0.007(E log kcat (oxidation) = 19.97 – 0.024(H{ – 0.95IP log V(oxidation) = 26.90 – 2.58IP log kcat (oxidation) = 0.024Vol – 0.23Q – 1.14 log kcat (oxidation) = 1.33 – 0.15Q log kcat (demethylation) = –0.68 W– + 1.06
Electrostatic descriptors
Approachb
Table 40.6 In Silico Approaches for Estimating Reaction Rates of Chemicalsa
Hansch and Leo (1995) Hansch and Leo (1995) (continued)
Lewis and Dickins (2002) Lewis and Dickins (2002)
Gargas et al. (1988)
Lewis and Dickins (2002) Lewis (2001) Lewis (2001) Parham and Portier (1998)
Lewis and Dickins (2002) Lewis and Dickins (2002) Lewis and Dickins (2002) Lewis and Dickins (2002) Lewis and Dickins (2002) Hansch and Leo (1995)
Reference
IN SILICO APPROACHES FOR PBPK MODELING 509
d
c
b
a
Speciesc
Chloroethanes
Chemical Classd
Fouchécourt and Krishnan (2000)
Reference
kcat = catalytic rate, Km = enzyme affinity constant, V = metabolic rate, Vmax = maximal velocity of metabolism, and Vmaxc = body weight normalized maximal velocity of metabolism. Y Y Y (E = LUMO energy – HOMO energy, (H{ = hydrogen abstraction energy, Q = dipolar moment of the molecule, 4 [ F , 3 [ F , [ SF = connectivity indices, BS = basic structure, diameter = diameter of the molecule, IND = variable dependant on experimental data used, IP = ionization potential, Length = length of the molecule, MOD = variable dependant on experimental data used, MW = molecular weight, nCL = number of CL fragments, nCL2 = number of CL2 fragments, nCL3 = number of CL3 fragments, nH3 = number of H3 fragments, NMC = number of meta chlorines, NOM = number of adjacent unsubstituted ortho-meta carbon pairs, NPL = variable dependant on the number of chloride atoms in the molecule in ortho position, NSIDE = variable dependant on the number of chloride atoms in the molecule in meta position, NUNSTOT = variable dependant on the number of chloride atoms in the molecule, Po:w = n-octanol:water partition coefficient (or vegetable oil:water), SA = surface area, UNS = variable dependant on the number of atoms in the molecule that are not chloride, Vol = volume of the molecule, and W– = Hammet constant. F = fish, H = human, and R = rats. PCBs = polychlorobiphenyls.
R, H
QSARS: Free–Wilson-type equations
Vmaxc = BS(C-C)(51.6) + nH3(14.6) + nCL(–4.84) + nCL2(10.2) + nCL3(–16.9)
Approachb
Table 40.6 (continued) In Silico Approaches for Estimating Reaction Rates of Chemicalsa
510 ALTERNATIVE TOXICOLOGICAL METHODS
2 – 0.50 log 1/Km(oxidation) = 1.39 log Po:w – 0.22 log Po:w log 1/Km(sulfation) = 2.93F2 + 1.16T2 + 0.91T3 + 0.82MR2 – 0.59IFOH + 1.29IET + 2.59 –log Km(oxidation) = 43.27 – 4.03(E – 0.60 log Po:w log 1/Km(glucoronidation) = 0.83 log Po:w + 1.37 log 1/Km(hydrolysis) = 0.056Z1H2O + 0.051Z2H2O + 0.026Z3H2O + 0.04Z4H2O + 4.616 log 1/Km(hydrolysis) = 0.066Z1H2O + 4.259 log 1/Km(hydrolysis) = 0.44T+ 4.08 log 1/Km(hydrolysis) = 0.40T+ 4.40 log 1/Km(NADP-oxidation) = 0.69 log Po:w + 2.90 log Km(n-demethylation) = –0.55 log Po:w + 2.67 log Km(oxidation) = 0.61 log Po:w + 2.23 log Km(oxidation) = 1.02 log Po:w + 2.98
Hydrophobic descriptors
Speciesb
H H H R R R R R R R R R
H H H R R R Rb
QSARs: LFE-type equations
log 1/Km(demethylation) = 0.46 log Po:w + 0.63W – + 2.62 log 1/Km(sulfation) = 0.92 log Po:w – 1.48MR4 – 0.64MR3 + 1.04MR2 + 0.67W – + 4.01 log Km(acetylation) = –0.42 log Pui + 0.14pKa – 2.89 Km(oxidation) = [(Qiad(T)i)/|IPi – b|] + c log Km(acetylation) = 0.17pKa – 0.69 log Km(acetylation) = –0.42(Rmu:i + 0.15pKa – 1.39 log Km(acetylation) = 0.07pKa + 0.31 log Po:w – 0.33
Electrostatic descriptors
Approacha
Barbiturates Phenols Toluenes Phenols Phenylhippurates Phenylhippurates Phenylhippurates Phenylhippurates Drugs Morphines Carbamates Pyrazoles
X-C6H4N(CH3)2 Phenols Sulfonamides Alkenes Sulfonamides Sulfonamides Sulfonamides
Chemical Classc
Table 40.7 In Silico Approaches for Estimating the Michaelis–Menten Affinity Constant (Km) of Chemicals
Lewis and Dickins (2002) Hansch and Leo (1995) Lewis and Dickins (2002) Hansch and Leo (1995) Kim (1993) Kim (1993) Kim (1993) Kim (1993) Seydel and Schaper (1982) Hansch and Leo (1995) Hansch and Leo (1995) Hansch and Leo (1995) (continued)
Hansch and Leo (1995) Hansch and Leo (1995) Seydel and Schaper (1982) Csanady et al. (1995) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982)
Reference
IN SILICO APPROACHES FOR PBPK MODELING 511
R Rb
log Km(oxidation) = 0.79 log Po:w + 1.46 log Km(oxidation) = 1.04 log Po:w + 1.10
c
b
a
R, H
Chloroethanes
4-nitrophenyl alkyl ethers Alkylbenzenes Toluenes
Chemical Classc
Fouchécourt and Krishnan (2000)
Hansch and Leo (1995) Hansch and Leo (1995)
Hansch and Leo (1995)
Reference
(E = LUMO energy – HOMO energy, W– = Hammet constant, Q = dipolar moment of the molecule, T,T2,T3 = molecular hydrophobicity constants, (Rmui = variable dependant on the resistance constant due to diffusion of the nonionized form in the lipid membrane, a = orbital availability, b = LUMO energy, BS = basic structure, c = variable dependant on the molecular size, d(T) = normalized electron density, F2 = variable dependant on the electrical field induced by ortho positioned atoms, lFOH = variable dependant on the number of F OH groups in the molecule, lET = variable dependant on the family of the substance, IP = ionization potential, MR2,3,4 = molar refractivity indices, nCL = number of CL fragments, nCL2 = number of CL2 fragments, nCL3 = number of CL3 fragments, nH3 = number of H3 fragments, pKa = log dissociation constant of an acid in water, Po:w = n-octanol:water partition coefficient (or vegetable oil:water), Pui = n-octanol:water partition coefficient for the nonionized form, and Z1, 2, 3, 4H2O = variables corresponding to the potential energy for the interaction between the molecule and water. F = fish, H = human, and R = rats. PCBs = polychlorobiphenyls.
Km = BS(C-C)(3.8) + nH3(–2.59) + nCL(–0.37) + nCL2(0.79) + nCL3(0.19)
QSARs: Free–Wilson-type equations
R
Speciesb
log Km(oxidation) = 1.05 log Po:w + 1.22
Approacha
Table 40.7 (continued) In Silico Approaches for Estimating the Michaelis–Menten Affinity Constant (Km) of Chemicals
512 ALTERNATIVE TOXICOLOGICAL METHODS
IN SILICO APPROACHES FOR PBPK MODELING
513
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ZKHUH3YR Z YHJHWDEOHRLO ZDWHU3&3S Z SURWHLQ ZDWHU3&IRUVWUDWXPFRUQHXP 'O FRHIÀFLHQWIRUGLIIXVLRQLQWRWKHOLSLGIUDFWLRQRIVWUDWXP FRUQHXPDQG'S FRHIÀFLHQWIRUGLIIXVLRQLQWRWKHSURWHLQIUDFWLRQRIVWUDWXPFRUQHXP ,Q WKH DERYH DOJRULWKP WKH FRHIÀFLHQWV DQG DUH VSHFLHVVSHFLÀFDQGUHIHUWRWKHIUDFWLRQDOFRQWHQWRIOLSLGLQVNLQWKHSDWKOHQJWK IRU D K\SRWKHWLFDO WRUWXRXV GLIIXVLRQ SDWKZD\ LQ VWUDWXP FRUQHXP WKH VXP RI WKH IUDFWLRQDOFRQWHQWRIZDWHUDQGSURWHLQLQVNLQDQGWKHSDWKOHQJWKIRUDK\SRWKHWLFDO WUDQVFHOOXODUGLIIXVLRQSDWKZD\DFURVVWKHFRUQHRF\WHVRIVWUDWXPFRUQHXPUHVSHF WLYHO\7KHUHPDLQLQJWHUPVDUHFKHPLFDOVSHFLÀFDQGFDQEHGHULYHGIURPPROHFXODU VWUXFWXUH 3S Z LQ VWUDWXP FRUQHXP KDV EHHQ UHODWHG WR 3R Z WKURXJK DQ HPSLULFDO UHODWLRQVKLS 3RXOLQ DQG .ULVKQDQ 9DOLGDWLRQ RI WKLV DOJRULWKP ZDV
LMWVOCs; HMWOCs LMWVOCs; HMWOCs Alcohols, steroids
H H H
log Kp = 0.44R2 – 0.49 T + – 1.487 E + – 3.447 F + + 1.94Vx – 5.13
log Kp = –0.59 T + – 0.637 E + – 3.487 F + + 1.79Vx – 5.05
log Kp = –5.33 – 0.62 T + – 0.387 E + – 3.347 F + + 1.85Vx
LMWVOCs; HMWOCs LMWVOCs; HMWOCs Alcohols, steroids Alcohols, steroids Alcohols LMWVOCs; HMWOCs Hydrocorticone esters Dermal drugs; LMWVOCs; HMWOCs LMWVOCs; HMWOCs Alcohols, steroids Steroids LMWVOCs; HMWOCs
H H H H H H H H H H H
Kp = (b1 + b2Po:w)e(b3MW) log Kp = –5.14 – 0.477Ca + 0.237Cd + 0.038Pol log Kp = –6.14 – 0.427Ca + 0.237Cd + 0.21L – 0.11W log Kp = –7.29 + 0.15Pol log Kp = b1 + b2 log Po:w + b3MW0.5
log Kp = 0.652 log Po:w – 0.00603MW – 0.623ABSQon – 0.313SsssCH – 2.3
log Kp = 0.77 log Po:w – 0.0103MW – 2.33
2
V
log Kp = –0.786OT + 0.252 O– 1.617 T – 5.767 log Kp = 0.82 log Po:w – 0.0093Vm – 0.039MPt – 2.36 log Kp = 0.84 log Po:w – 0.07(log Po:w)2 – 0.27Hb – 1.84 log MW + 4.39
log Kp = –0.428H– 4.80 ; 93& + 28.06
E –1 Kp = (b1 + 0.0025/(b2 + b3 + 3R Z )) MWb5
H
Steric descriptors
LMWVOCs; HMWOCs
Chemical Classc
H
QSARs: LFE-type equations
Speciesb
log Kp = –0.6267Ca – 23.87(Q+)/E 0.289SsssCH – 0.0357SsOH – 0.482IB + 0.405BR + 0.834
Electrostatic descriptors
Approacha
Table 40.8 In Silico Approaches for Estimating the Skin Permeability Coefficient (Kp) of Chemicals
Moss et al. (2002) Moss et al. (2002)
Moss et al. (2002) Ghafourian and Fooladi (2001)
Patel et al. (2002)
Moss et al. (2002) Raevsky and Schaper (1998) Raevsky and Schaper (1998) Raevsky and Schaper (1998) Moss et al. (2002) Ghafourian and Fooladi (2001)
Moss et al. (2002)
Ghafourian and Fooladi (2001)
Moss et al. (2002)
Moss et al. (2002)
Moss et al. (2002)
Reference
514 ALTERNATIVE TOXICOLOGICAL METHODS
H
log Kp = 3.99 log TA + 4.53 T V – 0.762OT – 11.364
Steroids Phenols
H H
log Kp = –0.207 log 3R Z + 1.49 log Po:w – 5.42
+ 2.39 log Po:w – 8.71 log Kp = –0.37 log 3R Z
Kp Kp Kp Kp
= = = =
0.544 log Po:w – 2.88 0.80 log Po:w – 8.883 –1.46 (log Po:w + 0.29 log Po:w – 3.75 –4.36 – 0.387Ca + 0.247Cd
/(b3 + 3 ))
c
b
+
H
Acids; Alcohols; Hydrocarbons
Mechanistically based equations Poulin and Krishnan (2001)
Seydel and Schaper (1982) Ghafourian and Fooladi (2001) Testa et al. (2000) Raevsky and Schaper (1998)
Testa et al. (2000)
Seydel and Schaper (1982)
Moss et al. (2002)
Moss et al. (2002)
Ghafourian and Fooladi (2001)
Ghafourian and Fooladi (2001)
a
+
+
H = solubility parameter, T = dipolarity/polarizability, E = overall hydrogen-bond acidity, F = overall hydrogen-bond basicity, 7Ca = hydrogen bond acceptor free energy in the molecule, 7Cd = hydrogen bond donor in the molecule, 2O = molecular shape index, ; 93& = connectivity indices, ABSQon = sum of absolute charges on oxygen and nitrogen atoms, b1, b2, b3, b4, b5 = regression coefficients without any assigned role, BR = number of rotatable bonds, Dl = coefficient for diffusion into the lipid fraction of stratum corneum, Dp = coefficient for diffusion into the protein fraction of stratum corneum, Hb = number of hydrogen bonds formed by the substance, IB = Balaban index, L = molecular length, MPt = melting point, MW = molecular weight, OT = number of hydrogen bonding heteroatoms, Po:w = n-octanol:water partition coefficient (or vegetable oil:water), Pol = describes bulk or volume related effects, Pp:w = protein:water partition coefficient for stratum corneum, Pvo:w = vegetable oil:water partition coefficient, q– = the most negative charge on the hydrogen bond accepting heteroatoms, Q+/E = positive charge per unit volume, T V = sum of atomic charges on hydrogen bonding heteroatoms, T V = sum of atomic charges on hydrogen bonding hydrogens, R2 = excess molar refraction, SsOH = sum of E-state indices for all hydroxy groups, SsssCH = sum of E-state indices for all methyl groups, TA = total solvant accessible surface, Vm = molar volume, Vx = McGowan characteristic volume, and W = molecular width. F = fish, H = human, and R = rats. CFCs = chlorofluorocarbons, HMWOCs = high molecular weight organic chemicals, LMWVOCs = low molecular weight volatile organic chemicals, and VOCs = volatile organic chemicals.
* 0.028Dl /0.0340) + (Pp:w * 0.88D p/0.0018) Kp = (Pvo:w
log log log log
K p = b1( 3
Aliphatic alcohols Hydrocorticone esters Alcohols, steroids Steroids
HMWOCs
E R Z
H H H H
Pharmaceuticals
E R Z
Kp = 1.17 v 10–7 3R Z + 2.73 v 10–8
H
Barbiturates; Isoquinoline; Salicyclic acid Alcohols, Steroids
H
Hydrophobic descriptors
H
log Kp = 28.4q– + 0.018Vm + 2.824
IN SILICO APPROACHES FOR PBPK MODELING 515
516
ALTERNATIVE TOXICOLOGICAL METHODS
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n Silico Approaches for Oral Absorption Constants 7KH UDWH FRQVWDQW IRU RUDO DEVRUSWLRQ RI FKHPLFDOV PD\ EH D VLQJOH QXPEHU UHÁHFWLQJ WKH VXP WRWDO RI DOO SURFHVVHV LQYROYHG RU LW PD\ EH D VHW RI FRQVWDQWV HDFK RQH RI ZKLFK PD\ GHVFULEH WKH UDWH RI DEVRUSWLRQ DORQJ WKH JDVWURLQWHVWLQDO WUDFW7KHVLPSOHVWGHVFULSWLRQRIRUDODEVRUSWLRQLQ3%3.PRGHOVXVHVDÀUVWRUGHU UDWHFRQVWDQW .D RUDO WRDSSUR[LPDWHWKLVSURFHVV$QGWKHYDOXHRIWKLVSDUDPHWHU LVIUHTXHQWO\REWDLQHGE\ÀWWLQJWKHPRGHOWRVHULDOEORRGFRQFHQWUDWLRQGDWDREWDLQHG IROORZLQJRUDOGRVLQJ ,Q VLOLFR HIIRUWV LQ PRGHOLQJ DEVRUSWLRQ KDYH PDLQO\ IRFXVHG RQ GHYHORSLQJ VRIWZDUH SDFNDJHV WKDW DUH FDSDEOH RI SUHGLFWLQJ WKH IUDFWLRQ DEVRUEHG LQ KXPDQV 5DHYVN\DQG6FKDSHU %DOLPDQHHWDO $JRUDPHWDO 5DHYVN\ HW DO $PRQJ WKHVH WKH$&$7 DQG WKH L'($ PRGHOV DUH FRPPRQO\ EHLQJXVHGIRUSUHFOLQLFDODQDO\VLVE\WKHSKDUPDFHXWLFDOLQGXVWU\ $JRUDPHWDO *UDVVDQG6LQNR 7KHVHPRGHOVGRQRWSURYLGHVHSDUDWHHVWLPDWHVRI DEVRUSWLRQUDWHFRQVWDQWVVLQFHWKH\DOVRLQWHJUDWHÀ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ÀUVWSDVVHIIHFWLQDGGLWLRQ WR WKH UDWH RI DEVRUSWLRQ UHPDLQV XQFOHDU VHH 7DEOHV WR &XUUHQWO\ QR PHFKDQLVWLF DOJRULWKPV DUH DYDLODEOH WR SURYLGH SUHGLFWLRQV RI .D RUDO YDOXHV RI FKHPLFDOVSULRUWRWHVWLQJ 7KH LQ VLOLFR DSSURDFKHV GHVFULEHG DERYH FDQ EH LQFRUSRUDWHG ZLWKLQ 3%3. PRGHOVWRREWDLQVLPXODWLRQVRIEORRGDQGWLVVXHFRQFHQWUDWLRQVIRUQHZFKHPLFDOV SULRUWRWHVWLQJDVGHVFULEHGLQWKHIROORZLQJVHFWLRQ
INTEGRATING IN SILICO APPROACHES INTO RISK ASSESSMENT 3%3.PRGHOVIDFLOLWDWHWKHLQFRUSRUDWLRQRIWKHHVWLPDWHVRIWKHYDULRXVFKHP LFDOVSHFLÀF SDUDPHWHUV ZLWK WKRVH RI SK\VLRORJLFDO SDUDPHWHUV WR VLPXODWH WKH
Speciesb
R R R R R R R R R R R R R R R
F
Chemical Class
Sulfonamides Sulfonamides Pharmaceuticals Organic acids Sulfonamides Xanthenes Carbamates Antihistamines Sulfonylureas Carbamates Sulfonylureas Sulfonamides Sulfonamides Phenols; Anilines; Esters Organic anions
Barbiturates
QSARs: LFE-type equations
Ka(absorption) = km[(1/Rf) – 1]n’/(Q + [(1/Rf) – 1]n’) log Ka(absorption) = –0.04 (log Po:w)2 + 0.22 log Po:w + 0.04 log Ka(absorption) = 0.067 + log Po:w – log(1.4 + Po:w) log Ka(absorption) = –0.082(log Po:w)2 + 0.268 log Po:w + 3.96 log Ka(absorption) = –0.09 (log Po:w)2 + 0.44 log Po:w – 0.396 log Ka(absorption) = 0.09 log Po:w + 0.83 log Ka(absorption) = 0.18 log Po:w + 0.23 log Ka(absorption) = 0.24 log Po:w – 1.37 log Ka(absorption) = 0.30 log Po:w – 0.07(log Po:w)2 – 2.38 log Ka(absorption) = 0.3 log Po:w – 0.57 log (0.34 Po:w + 1) – 0.15I – 0.74 log Ka(absorption) = 0.46 log Po:w – 0.36 log (0.60Po:w + 1) – 0.23 log Ka(absorption) = 0.5 log Po:w –0.61 log (0.07Po:w + 1) – 0.39 log Ka(absorption) = 0.502 log Po:w – log (0.053Po:w0.0862 + 1) – 0.384 log Ka(absorption) = 0.56 log Po:w – (0.04Po:w0.84 + 1) – 0.63 log Ka(absorption) = 1.36 log Po:w + 0.36
Hydrophobic descriptors
log Ka(absorption) = –0.58 log Po:w + 0.35pKa – 1.77
Electrostatic descriptors
Approacha
Table 40.9 In Silico Approaches for Estimating the Oral Absorption Constant (Ka) of Chemicals
Testa et al. (2000) Seydel and Schaper (1982) Yamaguchi et al. (1996) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982) Seydel and Schaper (1982) (continued)
Seydel and Schaper (1982)
Reference
IN SILICO APPROACHES FOR PBPK MODELING 517
b
a
Speciesb
Sulfonylureas
Chemical Class
Seydel and Schaper (1982)
Reference
allyl = allyl group, Br = bromide, BS = basic structure, C2H5 = ethyl group, C6H5 = aromatic ring group, CH3 = methyl group, Cl = chloride group, cyC6H11 = cyclohexyl group, H = hydrogen group, I = family indicator variable, i-C3H7 = isopropyl group, i-C4H9 = isobutyl group, km = rate constant for transfer out of the membrane, n = group occurrence in molecule, n’ = constant specific to the equation without any given role, n-C3H7 = n-propyl group, n-C4H9 = n-butyl group, NO2 = nitrooxide group, OCH3 = methyl ether group, pKa = log dissociation constant of an acid in water, Po:w = n-octanol:water partition coefficient (or vegetable oil:water), Q = constant specific to the equation without any given role, Rf = reverse-phase TLC lipophilicity parameter, and t-C4H9 = tert-butyl group. F = fish and R = rats.
R
QSARs: Free–Wilson type equations
log Ka(absorption) = BS(BEN-SO2-NHCONH)(–2.272) + nH (0) + n2-CH3 (0.088) + n4CH3 (0.074) + n4-C2H5 (0.163) + n4-OCH3 (–0.229) + n2-NO2 (–0.324) + n3-NO2 (–0.207) + n4-NO2 (–0.323) + n4-Cl (0.198) + n4-Br(0.122) + nnC4H9(0) + nCH3 (–0.638) + nC2H5 (–0.361) + nn-C3H7 (–0.145) + ni-C3H7 (–0.244) + ni-C4H9(–0.035) + nt-C4H9 (0.149) + ncy-C6H11(0.135) + nallyl (–0.419) + nC6H5 (–0.088)
Approacha
Table 40.9 (continued) In Silico Approaches for Estimating the Oral Absorption Constant (Ka) of Chemicals
518 ALTERNATIVE TOXICOLOGICAL METHODS
IN SILICO APPROACHES FOR PBPK MODELING
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BS = basic structure (C-C).
BSa
H3
Cl
Cl2
Cl3
1 1 1 1 1 1 1 1 1
1 1 0 1 0 0 0 0 0
1 0 2 0 1 1 0 0 0
0 1 0 0 1 0 2 1 0
0 0 0 1 0 1 0 1 2
520
ALTERNATIVE TOXICOLOGICAL METHODS
1,1,1-trichloroethane
1,1,2,2-tetrachloroethane
Cl Cl
Cl
H
Cl
Cl Cl
H
Cl
H
H
H
Basic structure: (-C-C-)
Molecular fragments: 1 x (-C-C-) 1 x H3 1 x Cl3
Molecular fragments: 1 x (-C-C-) 2 x Cl2H
Figure 40.1 Chemical description methodology used in this study. The chemicals are represented as a basic structure (C-C) with substituents on the two carbons. Examples of the description of 1,1,1 trichloroethane and 1,1,2,2 tetrachloroethane are presented.
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Table 40.11 Contributionsa of Chloroethane Structural Features to Rat Partition Coefficientsb and Metabolic Constantsc
a
b
c
Fragments
Pb
Pl
Ps
Pf
Vmaxc
Km
BS Cl2 Cl3 Cl H3 r2
56.8 42.7 7.00 –9.60 –50.1 0.98
2.02 –0.319 1.60 –0.506 –0.653 0.91
0.746 –0.0181 0.233 0.00710 –0.0770 0.99
28.9 –1.16 14.1 –7.22 –8.56 0.96
52.7 9.40 –15.3 –7.22 12.9 0.82
3.75 0.863 –0.0932 –0.234 –1.65 0.88
Contributions were obtained by multiple linear regression from experimental data on chloroethane, 1,1-dichloroethane, 1,2-dichloroethane, 1,1,2-trichloroethane, 1,1,1,2-tetrachloroethane, 1,1,2,2-tetrachloroethane, pentachloroethane, and hexachloroethane. BS = basic structure (C-C). Pb, Pl,, Ps, and Pf refer to blood:air, liver:blood, slowly perfused tissue:blood and fat:blood partition coefficients, respectively. Vmaxc (Qmol/hr/kg) and Km (QM) refer to maximal velocity of metabolism and affinity constant, respectively.
Table 40.12 Contributionsa of Chloroethane Structural Features to Human Partition Coefficientsb
a
b
Fragments
Pb
Pl
Ps
Pf
BS Cl2 Cl3 Cl H3 r2
37.4 29.6 7.53 –8.92 –39.3 0.83
2.72 –0.365 2.16 –0.446 –0.699 0.98
1.099 –0.163 0.166 0.0510 0.0450 0.91
38.9 0.105 12.2 –10.6 –1.05 0.94
Contributions were obtained by multiple linear regression from experimental data on chloroethane, 1,1-dichloroethane, 1,2-dichloroethane, 1,1,2-trichloroethane, 1,1,1,2-tetrachloroethane, 1,1,2,2-tetrachloroethane, and hexachloroethane. BS = basic structure (C-C). Pb, Pl, Ps, and Pf refer to blood:air, liver:blood, slowly perfused tissue:blood and fat:blood partition coefficients, respectively.
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1000
Estimated parameter value
y = 1.0089x R2 = 0.9159
100
10
1
0.1 0.1
1
10
100
1000
Experimental parameter value
Figure 40.2 Comparison of rat experimental and predicted parameter values. Experimental values from Gargas et al. (1988, 1989).
Estimated parameter value
1000
y = 0.8984x R2 = 0.8557
100
10
1
0.1 0.1
1
10
100
1000
Experimental parameter value
Figure 40.3 Comparison of human experimental and predicted parameter values. Experimental values were derived from Gargas et al. (1988, 1989).
IN SILICO APPROACHES FOR PBPK MODELING
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Table 40.13 Comparison of Experimentala (Exp) and QSAR-Estimated (Est) Values of Rat Partition Coefficientsb and Metabolic Constantsc for 1,1,1-Trichloroethane
a b
c
Parameter
Exp
Est
Pb Pl Ps Pf Vmaxc Km
5.67 1.52 0.56 46.4 43.1 3.14
13.7 2.97 0.90 34.5 50.3 2.01
Experimental data from Gargas et al. (1988, 1989). Pb, Pl,, Ps, and Pf refer to blood:air, liver:blood, slowly perfused tissue:blood and fat:blood partition coefficients, respectively. Vmaxc (Qmol/hr/kg) and Km (QM) refer to maximal velocity of metabolism and affinity constant, respectively.
Table 40.14 Comparison of Experimentala (Exp) and QSAREstimated (Est) Values of Human Partition Coefficientsb for 1,1,1-Trichloroethane
a b
Parameter
Exp
Est
Pb Pl Ps Pf
2.53 3.40 1.25 104
5.56 4.18 1.31 50.1
Data derived from Gargas et al. (1989). Pb, Pl, Ps, and Pf refer to blood:air, liver:blood, slowly perfused tissue:blood and fat:blood partition coefficients, respectively.
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Figure 40.4 Quantitative structure-activity relationship (QSAR) physiologically based pharmacokinetic (PBPK) modeling framework. User input consists of the exposure scenario and chemical structure information such as the number of fragments constituting the molecule. This information is fed to the program that contains the model constants, the Free–Wilson type SPR, the contribution values of each molecular fragment (Cs) and of the basic structure (BS) to the model parameters (P), and the simulation algorithms. The model can then simulate the pharmacokinetics of the chemical in biota and then provide its profile as output. The example of 1,1,1-trichloroethane is shown.
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Tissue concentrations obtained using conventional-type PBPK model (mg/L)
526
ALTERNATIVE TOXICOLOGICAL METHODS
10
1 y = 1.2184x R2 = 0.9746 0.1
0.01
0.001
0.0001 0.0001
0.001
0.01
0.1
1
10
Tissue concentrations obtained using QSPR-PBPK type model (mg/L)
Figure 40.5 Comparison of steady-state blood and tissue concentrations of chloroethanes in rats exposed to 1 ppm, as simulated by conventional and QSAR PBPK models. Qg/L) of 1,1,1Table 40.15 Steady-State Tissue Concentrations (Q Trichloroethane in Rat and Humans Estimated Using the Conventional (PBPK) and QSAR-Based (QSAR) Physiologic Model Following a Continuous Exposure to 1 ppm Rat
Human
Tissue
QSAR
PBPK
QSAR
PBPK
Blood Liver Slowly perfused Fat Richly perfused
22.9 6.77 20.7 790 68.1
16.6 4.22 9.31 770 25.2
12.8 6.23 16.7 502 53.4
8.5 5.61 10.7 472 28.9
Table 40.16 Steady-State Arterial Blood Concentration (Cass) Obtained Using the Conventional (PBPK) and QSAR-Based (QSAR) Physiological Model in Rats Exposed to the NOAEL of 1,1,1-Trichloroethane (875 ppm) and the Corresponding Environmental Concentration (Ci) in Humans Derived Using the Human Conventional (PBPK) and QSAR-Based (QSAR) Physiological Models Endpoint Rat Cass (mg/L) Human Ci (ppm)a a
QSAR
PBPK
59.3 6342
24.9 4252
Calculated using the QSAR-derived Cass (59.3 mg/L).
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Figure 42.1 Frame sequence showing the evolution of the action potential in an atrial tissue using the Nygren model for the electrophysiology of the tissue. The times of the frames going clockwise are (a) 50 ms, (b) 100 ms, (c) 150 ms, and (d) 200 ms. The stimulus consisted of a current pulse of 40 Q%cm2 delivered to the leftmost column of atrial cells. The stimulus lasted for 2 ms and VM in the legend is the voltage in mV. (continued)
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Figure 42.1 (continued) Frame sequence showing the evolution of the action potential in an atrial tissue using the Nygren model for the electrophysiology of the tissue. The times of the frames going clockwise are (a) 50 ms, (b) 100 ms, (c) 150 ms, and (d) 200 ms. The stimulus consisted of a current pulse of 40 Q%cm2 delivered to the leftmost column of atrial cells. The stimulus lasted for 2 ms and VM in the legend is the voltage in mV.
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Figure 42.2 Action potential, entering from the left and encountering a region of high potassium concentration region at t = 40 and 250 ms, respectively. The hyperkalemic region is along the tissue central axis, in the center taking up 1 cm 2 of the 3 cm v 3 cm tissue.
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Figure 42.3 This figure contrasts the effect of potassium current blockers on the action potential. The left-hand panel shows the voltage, that is, the action potential in the tissue without modulation of the currents. The effect of reducing the iKr and iKs, such as is effected by newer antiarrhythmic drugs such as azimilide, is shown in the right-hand panel. Modulation of the wave distortion and erratic behavior should be noted.
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Submillimeter-Wave Frequency Studies of the Vibrational Modes of Deoxyribonucleic Acid: A Metric for Mutagenicity? 75*OREXV%/*HOPRQW0%\NKRYVNDLD-+HVOHU'/:RRODUG $&6DPXHOV-2-HQVHQ+6DOHPDQG:5/RHURS
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RESULTS AND DISCUSSION Computational Studies 7\SLFDOFRPSXWDWLRQDOUHVXOWVIRUDUHSUHVHQWDWLYHROLJRPHUWKDWZDVVWXGLHGDV D KHOLFDO GRXEOHVWUDQGHG '1$ SRO\PHU DQDORJ DUH VKRZQ LQ )LJXUH IRU WZR YDOXHVRIWKHOLQHZLGWKSDUDPHWHU$QRWDEOHIHDWXUHRIWKHSUHGLFWHGVSHFWUDLVWKH ÀQHVWUXFWXUHZKLFKLVSUHGLFWHGRQWKHEDVLVRIORZIUHTXHQF\YLEUDWLRQDOPRGHV
Absorption
Poly(dA)Poly(dT)
0
100
200
300
400
γ = 2 cm -1
500
γ = 7 cm
0
100
200
300
400
500
Poly(dAdT)Poly(dTdA)
0
100
200
300
400
500
100
200
300
400
500
-1
0
Frequency (cm-1) Figure 43.1 Calculated spectra of Poly(dA)Poly(dT) and of Poly(dAdT)Poly(dTdA) at two linewidths: 2-wavenumber and 7-wavenumber.
562
ALTERNATIVE TOXICOLOGICAL METHODS
Table 43.1 Comparison of Predicted Vibrational Band Positions (in Wavenumbers) of Poly(dA)Poly(dT) with Values Observed by Experiments in the Literature Calculations
Experiment (Powell, 1987)
Calculations
Experiment (Powell, 1987)
20 43 57 71 94 106 131 160
Unknown Unknown 62 80 95 106 136 —
172 199 211 — 270 348 400 459
170 200 214 238 — — — —
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III
Relative Absorbance
I
3500 3250 3000 2750 2500 2250 2000 1750 1500 1250 1000 750 500 250
0
-1
Wavenumber (cm ) Figure 43.2 Experimental absorbance spectra of herring DNA in the mid-infrared through the very far infrared region.
FREQUENCY STUDIES OF THE VIBRATIONAL MODES OF DNA
0.60
Herring DNA Salmon DNA
0.55
Transmission
563
0.50 0.45 0.40 0.35 0.30 0.25 24
22
20
18
16
14
12
10
Wavenumber (cm-1) Figure 43.3 Fine structure in the absorbance spectra of herring and salmon DNA in the very far infrared region.
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Scattering Parameter, S12
0.9
SalmonDNA HerringDNA 0.8
0.7
0.6 75.0
80.0
85.0
90.0
95.0
100.0
105.0
110.0
Frequency, GHz Figure 43.4 Transmission scattering parameter (S12) measurement of herring and salmon DNA in the microwave (W-band) region.
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CHAPTER
44
Trophoblast Toxicity Assay (TTA): A Gestational Toxicity Test Using Human Placental Trophoblasts +DUYH\-.OLPDQ
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567
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ALTERNATIVE TOXICOLOGICAL METHODS
Cytotrophoblast
cAMP hCG
TGFß LIF
Phorbol Esters
HJK
Villous Syncytiotrophoblast
Anchoring Trophoblasts
Invading Trophoblasts
hCG
TUN
PAI-1
Figure 44.1 Pathways of trophoblast differentiation. Just as the basal layer of the skin gives rise to keratinocytes, the cytotrophoblast — the stem cell of the placenta — gives rise to the differentiated forms of trophoblasts. (Left) Within the chorionic villi, cytotrophoblasts fuse to form the overlying syncytiotrophoblast. The villous syncytiotrophoblast makes the majority of the placental hormones, the most studied being hCG. cAMP, EGF, and even hCG itself have been implicated as stimulators of this differentiation pathway. In addition to upregulating hCG secretion, cAMP has also been shown to down-regulate trophouteronectin (TUN) synthesis. (Center) At the point where chorionic villi make contact with external extracellular matrix (decidual stromal ECM in the case of intrauterine pregnancies), a population of trophoblasts proliferates from the cytotrophoblast layer to form the second type of trophoblast — the junctional trophoblast. These cells form the anchoring cell columns that can be seen at the junction of the placenta and endometrium throughout gestation. Similar trophoblasts can be seen at the junction of the chorion layer of the external membranes and the decidua. The junctional trophoblasts make a unique fibronectin — trophouteronectin — that appears to mediate the attachment of the placenta to the uterus. TGFF and LIF have been shown to induce cultured trophoblasts to secrete increased levels of trophouteronectin, while down-regulating hCG secretion. (Right) Finally, a third type of trophoblast differentiates toward an invasive phenotype and leaves the placenta entirely — the invasive intermediate trophoblast. In addition to making human placental lactogen, these cells also make urokinase and plasminogen activator inhibitor-1 (PAI-1). Phorbol esters have been shown to increase trophoblast invasiveness in in vitro model systems and to upregulate PAI-1 in cultured trophoblasts. The general theme that comes from these observations is that specific factors are capable of shifting the differentiation pathway of the cytotrophoblast toward one of the above directions while turning off differentiation toward the other pathways. See text for details.
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Figure 44.2 Purification of cytotrophoblasts from term placenta. A term placenta is minced and digested with trypsin and DNAse. The supernatant is passed through calf serum to inactivate the digestive enzymes; then these pellets are pooled and placed on a Percoll gradient to separate out the cytotrophoblasts. (From Kliman et al. (1986) Endocrinology, 118(4), 1567–1582. With permission.)
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Figure 44.3 In vitro morphologic differentiation of cytotrophoblasts. After purification, the cytotrophoblasts are dispersed as individual cells (left). When plated in culture media containing serum, these cells flatten out and begin to move toward each other. After 24 hr in culture, aggregates begin to appear, with some evidence of cell fusion (center). After 72 hr in culture, most of the trophoblasts have fused and formed large, multinucleated syncytiotrophoblasts. (From Kliman et al. (1986) Endocrinology, 118(4), 1567–1582. With permission.)
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Figure 44.4 hCG secretion by trophoblasts in culture. Percoll-gradient purified cytotrophoblasts were cultured in DMEM media for four days. Media was changed daily and assayed for hCG by radioimmunoassay. hCG was not detectable at the time of initial plating. (From Kliman et al. (1986) Endocrinology, 118(4), 1567–1582. With permission.)
Table 44.1 Regulation of Trophoblast hCG Secretion Factor
Trophoblasts (Trimester)
Effect on hCG Secretion
CAMP HCG GnRH
Term Term Term
Stimulates Stimulates Stimulates
GnRH F-Adrenergic agonists Dexamethasone Inhibin
First, Term First Term Term
Not clear Stimulates Stimulates Inhibits
Activin
Term
Activin EGF Thyroid hormone Thyroid stimulating hormone Interleukin-1 Interleukin-6 Basement membrane
First First, Term First, Term Term
Potentiates GnRH stimulation of hCG secretion Stimulates Stimulates Stimulates Inhibits
First First First
Stimulates Stimulates Stimulates
Decidual protein
Term
Inhibits
Prolactin
Term
Inhibits
References (Feinman et al., 1986) (Shi et al., 1993) (Belisle et al., 1989; Szilagyi et al., 1992) (Kelly et al., 1991) (Oike et al., 1990) (Ringler et al., 1989a) (Petraglia et al., 1987, 1989, 1991) (Petraglia et al., 1991)
(Steele et al., 1993) (Maruo et al., 1987) (Maruo et al., 1991) (Beckmann et al., 1992) (Yagel et al., 1989b) (Nishino et al., 1990) (Truman and Ford, 1986) (Ren and Braunstein, 1991) (Yuen et al., 1986)
574
ALTERNATIVE TOXICOLOGICAL METHODS
Figure 44.5 Effects of 8-bromo-cAMP on hCG and progesterone secretion by cultured cytotrophoblasts. Percoll-gradient purified cytotrophoblasts were cultured for 48 hr in the absence (䉱) or presence (䢇) of 8-bromo-cAMP. hCG (A) and progesterone (B) were quantitated in the medium at 24-hr intervals. Values presented are the mean ± SE from six separate experiments. At each time point, 8-bromo-cAMP-treated cultures secreted significantly more (p < 0.014, by the Wilcoxon signed rank test) progesterone and hCG than did control cultures. (From Feinman et al. (1986) J. Clin. Endocrinol. Metab., 63(5), 1211. With permission.)
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