Clinical Toxicology - Vol 48, issue 7, Aug 2010

Clinical Toxicology (2010) 48, 675–694 Copyright © Informa UK, Ltd. ISSN: 1556-3650 print / 1556-9519 online DOI: 10.3109/15563650.2010.516752

REVIEW LCLT

The clinical toxicology of metamfetamine LEO J. SCHEP, ROBIN J. SLAUGHTER, and D. MICHAEL G. BEASLEY Metamfetamine – clinical toxicology

National Poisons Centre, Department of Preventive and Social Medicine, University of Otago, Dunedin, New Zealand

Introduction. Metamfetamine is a highly addictive amfetamine analog that acts primarily as a central nervous system (CNS) stimulant. The escalating abuse of this drug in recent years has lead to an increasing burden upon health care providers. An understanding of the drug’s toxic effects and their medical treatment is therefore essential for the successful management of patients suffering this form of intoxication. Aim. The aim of this review is to summarize all main aspects of metamfetamine poisoning including epidemiology, mechanisms of toxicity, toxicokinetics, clinical features, diagnosis, and management. Methods. A summary of the literature on metamfetamine was compiled by systematically searching OVID MEDLINE and ISI Web of Science. Further information was obtained from book chapters, relevant news reports, and web material. Epidemiology. Following its use in the Second World War, metamfetamine gained popularity as an illicit drug in Japan and later the United States. Its manufacture and use has now spread to include East and South-East Asia, North America, Mexico, and Australasia, and its world-wide usage, when combined with amfetamine, exceeds that of all other drugs of abuse except cannabis. Mechanisms of toxicity. Metamfetamine acts principally by stimulating the enhanced release of catecholamines from sympathetic nerve terminals, particularly of dopamine in the mesolimbic, mesocortical, and nigrostriatal pathways. The consequent elevation of intra-synaptic monoamines results in an increased activation of central and peripheral a- and b-adrenergic postsynaptic receptors. This can cause detrimental neuropsychological, cardiovascular, and other systemic effects, and, following long-term abuse, neuronal apoptosis and nerve terminal degeneration. Toxicokinetics. Metamfetamine is rapidly absorbed and well distributed throughout the body, with extensive distribution across high lipid content tissues such as the blood-brain barrier. In humans the major metabolic pathways are aromatic hydroxylation producing 4-hydroxymetamfetamine and N-demethylation to form amfetamine. Metamfetamine is excreted predominantly in the urine and to a lesser extent by sweating and fecal excretion, with reported terminal half-lives ranging from ∼5 to 30 h. Clinical features. The clinical effects of metamfetamine poisoning can vary widely, depending on dose, route, duration, and frequency of use. They are predominantly characteristic of an acute sympathomimetic toxidrome. Common features reported include tachycardia, hypertension, chest pain, various cardiac dysrhythmias, vasculitis, headache, cerebral hemorrhage, hyperthermia, tachypnea, and violent and aggressive behaviour. Management. Emergency stabilization of vital functions and supportive care is essential. Benzodiazepines alone may adequately relieve agitation, hypertension, tachycardia, psychosis, and seizure, though other specific therapies can also be required for sympathomimetic effects and their associated complications. Conclusion. Metamfetamine may cause severe sympathomimetic effects in the intoxicated patient. However, with appropriate, symptom-directed supportive care, patients can be expected to make a full recovery. Keywords Metamfetamine; Methamphetamine; Drug abuse; Sympathomimetic toxidrome; 4-Hydroxymetamfetamine; Amfetamine; Amphetamine; Hypertension; Tachycardia; Necrotizing vasculitis; Psychosis

Introduction Metamfetamine, known also as methamphetamine, methylamfetamine, N-methylamfetamine, desoxyephedrine, phenylisopropylmethylamine, and N,a-dimethylphenethylamine, is a methylated analogue of amfetamine. It has similar pharmacological properties to amfetamine, acting primarily as a central nervous system stimulant, but differs in having more pronounced effects due to its greater lipophilicity. Although metamfetamine has been used therapeutically to treat exogenous obesity and attention deficit disorder,1 it is

Received 23 April 2010; accepted 16 August 2010. Address correspondence to Leo J. Schep, National Poisons Centre, Department of Preventive and Social Medicine, University of Otago, PO Box 913, Dunedin, New Zealand. E-mail: [email protected]

more often associated with clandestine manufacture and distribution, arising from its increasing illicit abuse as a central nervous system (CNS) stimulant. High purity metamfetamine, commonly known as “Speed” (although this may also describe amfetamine and other common stimulant drugs of abuse), is easily manufactured in clandestine laboratories by amateur chemists without any formal training in synthetic chemistry. The ease and low cost of this illicit manufacturing has resulted in metamfetamine becoming widely available and commonly abused. In many countries around the world it has become a significant drug of abuse and public health problem. Isolated or repetitive use of metamfetamine can be hazardous, and because of its popularity, intoxication has become a relatively common clinical scenario, producing a variety of potentially lethal effects. An understanding of these toxic effects and medical treatment is therefore essential for the

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successful management of patients suffering this form of intoxication.

Methods An extensive literature review was performed by systematically searching OVID MEDLINE (January 1950–March 2010) and ISI Web of Science (1900–March 2010). Bibliographies of identified articles were screened for additional relevant studies including non-indexed reports. Non-peerreviewed sources were also included: books, relevant newspaper reports, and applicable Web material. This review identified 1,459 papers, excluding duplicates. This list was screened for those associated with metamfetamine toxicity in humans and those that succinctly described the mechanism of action, clinical features, and treatment protocols. Articles employed in this review included case reports, case series, animal studies and review articles that were considered relevant.

Physico-chemical properties Metamfetamine (CAS 537-46-2) belongs to a class of sympathomimetic drugs called phenylethylamines.2 Essentially, they consist of a phenylethylamine structure (an aromatic ring with a two-carbon side chain leading to a terminal amine group) with attached groups to the amine, the alpha or beta carbons or on the aromatic ring (Fig. 1). Both amfetamine and metamfetamine possess a methyl derivative on the alpha carbon of the ethylamine side chain; metamfetamine has an additional methyl derivative on the amine (Fig. 1). Its chemical formula is C10H15N and the molecular mass is 149.24. The hydrochloride salt of metamfetamine forms as white to translucent crystals,3 whereas the free base forms a dark liquid. The hydrochloride salt is relatively pure and, in contrast to amfetamine sulfate, is sufficiently volatile to allow vaporization and inhalation.3 A more detailed summary of its properties is presented in Table 1. There are therefore two isomeric forms of metamfetamine: (+)-metamfetamine and (−)-metamfetamine (Fig. 1) and their pharmacological profiles are distinct. The former enantiomer is the dominant CNS stimulant and is five times more biologically active than the (−) enantiomer,3 which has greater peripheral sympathomimetic activity. The (−) enantiomer is also formed as a metabolite of selegiline,8 an antiParkinsonian drug.

Epidemiology Amfetamine-like drugs have been part of human history for many centuries. Traditional Chinese medicine includes ephedrine and pseudoephedrine extracts from the stems of Ephedra sinica Stapf (ma huang) and other species of this genera,

Fig. 1. Structures of phenylethylamine and the stereoisomers of amfetamine and metamfetamine.

Table 1. Physicochemical properties of metamfetamine4–7 Property Form Boiling point Melting point Density pKa Log p Solubility

Description Colorless liquid; hydrochloride salt is a white powder or translucent crystal 212°C Crystals: 170–175°C Not available ∼9.9 2.07 500 mg/mL water, soluble in diethyl ether and ethanol; hydrochloride salt is readily soluble in water

to treat asthma and other bronchial disorders.9 Amfetamine was first synthesized in Germany in 1887, and metamfetamine in 1893 in Japan.10 The first reported misuse of amfetamine was in 1937 when it was used by students in Minnesota to avoid sleep during examination periods.11 Thereafter both amfetamine and metamfetamine were widely used both clinically and illicitly during the Second World War by the Americans, Germans, and Japanese and became a serious problem in post-war Japan.10,12 Increasing popularity of metamfetamine as a drug of abuse within the United States led to its illicit production in San Francisco by 1962.13 In later years this was expanded by Mexican traffickers with

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Metamfetamine – clinical toxicology increased distribution to the West, Southwest, and Midwest States.13,14 The total number of present metamfetamine users worldwide is uncertain. In 2007, the United Nations Office on Drugs and Crime (UNODC) estimated that between 16 and 51 million people aged 15–64 consumed amfetamines, of whom 54–59% were metamfetamine users.15 Although this estimate is imprecise, metamfetamine consumption is clearly at epidemic proportions. Indeed, an earlier report from the UNODC suggests the combined consumption of metamfetamine and amfetamine exceeds all other drugs of abuse except cannabis.16 Production and consumption of metamfetamine is currently most concentrated in East and Southeast Asia and North America, but there are increasing reports of similar activity in Latin America and Australasia.17 Statistical data on drug seizures for 2007 indicated that most occurred in East and Southeast Asia (56%), followed by North America,17 whereas Europe’s contribution was only 2%. Operations in China, Myanmar, and the Philippines account for most of the recent productions in East and Southeast Asia.15 Within Europe, ecstasy is the main amfetamine of abuse, although there is growing evidence of increased manufacturing of metamfetamine in Lithuania, increased use in Slovakia, and an outward movement of its use beyond the Czech Republic, where it has long been established.17,18

Mechanisms of toxicity Metamfetamine acts in a manner similar to amfetamine, but with the addition of the methyl group to the chemical structure. It is more lipophilic (Log p value 2.07, compared with 1.76 for amfetamine),4 thereby enabling rapid and extensive transport across the blood–brain barrier.19 Metamfetamine causes an increased release of key endogenous monoamines,20 principally dopamine, from the sympathetic nerve terminals. Dopaminergic neurons are involved in three major systems in the brain: the mesolimbic, mesocortical, and nigrostriatal pathways, influencing emotional and motivation responses, reward systems, and motor control.21 Under normal physiological conditions, catecholamines, including dopamine, are synthesized in the presynaptic terminal and are found in both the cytoplasm and the presynaptic vesicles. They are released by exocytosis into the neuronal synapse in response to presynaptic action potentials, and act at postsynaptic receptors to promulgate the action potential.22 Released monoamines will be taken back into the presynaptic terminals by the respective uptake transporters, and metabolized.23 Metamfetamine facilitates the increased release of the key monoamine neurotransmitters by several molecular processes (see Fig. 2). Metamfetamine enters the presynaptic terminals by passive diffusion across the lipid membrane and through the membrane-bound catecholamine-uptake transporters (dopamine, norepinephrine, and serotonin transporters).24

677 Within the cytosol, metamfetamine enters the presynaptic vesicle through membrane-bound vesicular monoamine transporter-2 (VMAT-2) and facilitates the redistribution of the monoamines into the cytosol by disrupting the pH gradient whose presence is essential for driving the accumulation of the monoamines into the vesicles.24,25 Increased monoamine concentrations within the cytosol leads to their increased movement into the synapse through the respective catecholamine transporters; this occurs in the opposite direction to the usual reuptake mechanism.26 Catecholamines at elevated concentrations within the synapse compete with, and are partially blocked by, metamfetamine for reuptake through the catecholamine transporters, thereby promoting prolonged neuronal activity.27 Metamfetamine also decreases the expression of catecholamine transporters on the neuronal cell surface,28 inhibits intracellular monoamine oxidase activity,29 and promotes the intracellular expression of tyrosine hydroxylase, which leads to increased dopamine synthesis.30 Enhanced cytosol concentrations of dopamine can also lead to its increased oxidation in the neuronal cytoplasm to form dopamine quinine.25 This compound, in association with transition metals, superoxide radicals, and hydrogen peroxide, undergoes redox recycling, leading to oxidative stress, mitochondrial injury, neuronal apoptosis, and nerve terminal degeneration.31 The accumulated neuronal injury from chronic metamfetamine abuse is evidenced by losses of striatal dopamine32,33 and serotonin transporters,34 and subsequent decreases in dopamine and serotonin concentrations.35 Animal studies of chronic metamfetamine exposure have also shown evidence of long-term injuries to presynaptic dopaminergic and serotonergic terminals.36,37

Toxicokinetics Absorption Following ingestion, metamfetamine is readily absorbed across the gastrointestinal tract. Controlled studies with therapeutic formulations have indicated tmax values ranging from 3.13 to 6.3 h post-ingestion.38–40 Following intranasal administration of the powder, peak plasma concentrations similarly do not occur until approximately 3–4 h post-exposure.41,42 With inhalation of the vapor, metamfetamine rapidly appears in the plasma but plasma concentrations increase slowly, peak concentrations being reached at 2.5 (±0.5) h,43 possibly because of the ongoing availability of the drug, initially trapped in the mucosa of the upper respiratory tract. In contrast to these delays in maximum concentrations, subjects experienced peak subjective effects as early as a few minutes after inhalation.43 The estimated bioavailability from smoke inhalation has ranged from 67% to 90.3 + 10.4%,41,44 with the differences in part depending on smoking technique and the temperature of the flame.41 Following ingestion, one estimate was 67.2 ±

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Fig. 2. Schematic diagram summarizing the mechanisms whereby metamfetamine facilitates the release of dopamine and other catecholamines from neuronal terminals into the synapse. Metamfetamine (▲) enters the cell by passive diffusion (i) or via membranebound dopamine reuptake transporters (ii). Metamfetamine causes the redistribution of dopamine (●) from presynaptic vesicles into the neuronal cytosol (iii) and promotes the activity and expression of tyrosine hydroxylase (iv) thereby contributing to elevated dopamine concentrations within the cytosol, leading to increased movement into the synapse via the dopamine transporter (ii). Metamfetamine also prolongs monoamine neuronal activities by blocking their presynaptic re-uptake (v), decreasing the expression of transporters at the cell surface and inhibiting monoamine oxidase activity (vi).

3.1%38 whereas the percentage of unmetabolized drug absorbed systematically following intranasal insufflations was greater at 79%.41

venous use.48 Because of metamfetamine’s low molecular weight and high lipid solubility, there is also considerable transfer from maternal to fetal blood.49–51

Distribution

Metabolism

Metamfetamine is distributed to most parts of the body. Reported volumes of distribution in one study of habitual abusers were 3.73 ± 0.94 and 3.80 ± 1.05 L/kg following doses of 0.25 mg/kg and 0.5 mg/kg, respectively.45 One study has suggested that in the presence of ethanol, the volume of distribution of metamfetamine decreases, which may be due to ethanol displacing metamfetamine from peripheral binding sites.45 There is limited information on whether metamfetamine significantly binds to plasma proteins; some binding has been demonstrated for amfetamine (∼20%).46 As metamfetamine has a relatively high lipophilicity (Table 1), it would be expected to distribute extensively across high lipid-content tissues such as the blood–brain barrier.47 Unsurprisingly, metamfetamine is also distributed into breast milk,48 appearing in the milk within minutes of intra-

The predominant site of metamfetamine metabolism is the liver, mainly involving the cytochrome isoenzyme, CYP2D6. In humans, the major metabolic pathways are aromatic hydroxylation producing 4-hydroxymetamfetamine and N-demethylation to form amfetamine.52,53 Other minor metabolites include norephedrine, 4-hydroxyamfetamine, 4-hydroxynorephedrine, and possibly benzyl methyl ketoxime and benzoic acid.52,54 A summary of these pathways is presented in Fig. 3. Glucuronide and sulfate conjugates of 4-hydroxymetamfetamine are also formed.55 Interindividual differences in metabolism may be largely due to CYP2D6 variability.53 Metabolism appears to be suppressed by ethanol consumption,56,57 although, paradoxically, chronic ingestion of ethanol may increase the rate of metamfetamine hydroxylation.56

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Fig. 3. A summary of the metabolic pathways of metamfetamine52,54: (i) Aromatic hydroxylation, (ii) N-demethylation, (iii) β-hydroxylation, (iv) oxidative deamination, and (v) side chain oxidation. In humans, the major metabolites are 4-OH metamfetamine and amfetamine.

Although metamfetamine is metabolized to amfetamine (Fig. 3), investigations with therapeutic doses of metamfetamine suggest that the contribution of amfetamine to observed subjective effects is negligible.43,44 Peak concentrations of amfetamine in volunteers were substantially lower than those of the parent drug throughout the investigated time interval. In addition, the time to achieve peak plasma concentrations of amfetamine was delayed. Studies found that the amfetamine peak can be delayed from 10 to 24 h following inhalation43,44 and 17 + 3.3 h following intravenous injection.44 Although it is expected there would be at least some delay before the concentrations of the metabolite peaked, the authors of these studies did not hypothesize why it was so long. However, the clinical pharmacokinetics of high doses of illicit metamfetamine remain to be described adequately and theoretically higher doses may produce greater clinical effects attributable to the amfetamine metabolite. Elimination Metamfetamine is excreted predominantly in the urine, and to a lesser extent by sweating and fecal excretion.52,58 One study found that about 90% of a 14C-labelled dose was excreted in urine over the first 4 days following ingestion, with the majority of the drug appearing in the first 48 h.54 Studies investigating the percentage of renal elimination of the parent drug compared with metabolites have produced varied

results, with values for the former ranging from 18% to 55%, versus up to 15% as the 4-hydroxymetamfetamine metabolite, and 2% to 10% as amfetamine.38,44,54,59,60 However, such differences are not surprising, given the likely considerable interindividual variability in metabolism, and possible changes in individual metabolic efficiency with more regular use. Other factors affecting urinary elimination include dose, urine flow, and urinary pH.38 As metamfetamine is a weak base (pKa ∼9.9),61 acidification of the urine can markedly enhance its excretion.59 Under these conditions it remains predominantly in the ionized state, resulting in significantly decreased renal tubular reabsorption, leading to 55–70% excretion of the unchanged drug. In contrast, as little as 1–2% is eliminated unchanged in highly alkaline urine.59 The renal clearance of metamfetamine averaged 214 ± 120 mL/min following a 10-mg dose and 120 ± 33 mL/min following a 20-mg dose.60 In a second study, following the ingestion of 10 or 20 mg doses, clearance averaged 159 ± 18 mL/min.38 As renal clearance values have been reported in excess of glomerular filtration rates, it is postulated that elimination could occur partly by active transport, which may become saturated.60 Reported terminal half-lives for metamfetamine have varied considerably from 9.1 ± 4.0 h to 25.2 ± 6.0 h,38,39,44,60,62– 64 although most of the reports cited mean values between 9 and 12 h.38,39,44,62,64 The route of drug administration does not appear to significantly alter the half-life.41,44

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Clinical features Exposure to metamfetamine can occur through a variety of routes, including ingestion,131,179 injection,147 nasal insufflation,113 and inhalation (smoking)70; less common routes are urethral,180 rectal,67,82 and vaginal.72 Effects following metamfetamine exposure can vary widely, depending on dose, route, duration (acute and/or chronic), and pattern (e.g., frequency) of use. A comprehensive summary of signs and symptoms reported in published case reports and case series is presented in Table 2. The reviewed literature indicates a predominance of adrenergic effects, characteristic of a sympathomimetic toxidrome, although with significant variations between patients. At relatively low doses of between 5 and 50 mg, the major effects include euphoria, positive mood, heightened general arousal, and decreased fatigue, with acute improvement in sustained attention and a reduced appetite, as well as evidence of moderate tachycardia and hypertension.41–44,62,64,181–191 A summary of dose–responses with key cardiovascular parameters are presented in Table 3. Subjective effects and measures of cardiovascular function seem to increase with dose,42 although there is limited information pertaining to specific effects at higher doses. One clinical study involved 16 inpatients in a psychiatric unit, all of whom had previously been admitted for amfetaminerelated psychosis, were administered escalating intravenous doses of metamfetamine to replicate their previous condition.192 The drug precipitated clinical psychosis in 11 patients with doses ranging from 55 to 240 mg; another patient required 640 mg to induce this clinical condition. Reported effects included thirst, diaphoresis, paresthesia, hypertension, throbbing headaches, paranoia, hallucinations, and aggressive thoughts or behaviors.192 It is predicted that doses greater than 150 mg would cause toxic effects in an infrequent user.3 Overall, reported clinical effects following higher doses of metamfetamine in adults may include tachycardia, hypertension, palpitations, tachypnea, chest pain, gastrointestinal upset, mydriasis, diaphoresis, hyperthermia, and hyperreflexia, along with CNS effects of anxiety, agitation, delirium, and psychosis.72,73,81,83,89,94,114 A variety of cardiovascular and cerebrovascular injuries may develop following exposure to metamfetamine. Effects include ventricular dysrhythmias,77 myocardial dysfunction and ischemia with or without infarction,78,83,106,113 cardiomyopathy,70,123 and aortic and other vascular dissections.105,145,148 A major factor is vasoconstriction and/or vasospasm, which can occur because of excess catecholamine activity upon a1-adrenoreceptors. This may result in myocardial and other tissue ischemia and, along with the hyperdynamic circulation, is a major cause of hypertension. Inadvertent arterial injections of sympathomimetic drugs may additionally cause localized vasospasm,193 although this does not appear to have been reported for metamfetamine. Traditional or magnetic resonance angiography of cerebral vessels may demonstrate narrowing or frank occlusion of small- and medium-sized arteries, or beading (regions of

L.J. Schep et al. blood vessels where segments of stenosis alternate with normal or dilated intervening sections).75,76,135–137,143,194 Pathological assessment has shown evidence of necrotizing vasculitis (an inflammatory reaction of blood vessels, resulting in fibrinoid necrosis of tissue and associated leukocytic infiltration of the blood vessel walls) that is similar to periarteritis nodosa.195 Ensuing disorders may include cerebral ischemia152 with ischemic stroke143 and/or hemorrhagic stroke, which may arise from intracerebral104,194 or subarachnoid hemorrhage.152,194 Cerebral edema,153 acute lung injury,171 and ischemic colitis have also been reported.131,133 Vasculitis can also result in impaired limb perfusion,140 visual compromise,174 and/or renal and hepatic failure.93,108 Hepatic damage may also arise from any of a variety of causes, including direct toxicity, the secondary effects of hypotension, hypoxia, hyperthermia or lipid peroxidation, or as a complication of viral hepatitis or other infection caused by the use of contaminated needles.71,95,111 Repetitive movement disorders such as choreoathetosis, although uncommon, can develop in both adults and children.85,96,118 Serotonin toxicity, although not reported specifically for metamfetamine, has been reported with dexamfetamine in combination with venlafaxine and citalopram.196 As metamfetamine both increases presynaptic release and prevents reuptake of serotonin,197 there is a theoretical risk of serotonin toxicity following its use in association with other serotonergic agents. Patients with agitation, excessive muscular activity, and hyperthermia are at risk of developing rhabdomyolysis65,81,96,118,178 and subsequent renal failure.89,101,111,178 Hyperkalemia can occur secondary to renal impairment93,94 or as a result of rhabdomyolysis. Profuse sweating and tachypnea may lead to significant fluid and electrolyte depletion.94 Metabolic acidosis and coagulopathy can develop secondary to dehydration, inadequate peripheral perfusion, seizures, or hyperthermia.66,68,79,81 However, hyponatremia secondary to increased secretion of antidiuretic hormone and/or increased fluid intake, as described with 3,4-methylenedioxymetamfetamine (ecstasy) intoxication,198 has not been reported following exposure to metamfetamine. Ocular effects can include not only mydriasis,79,94 but also keratitis,175 corneal ulceration,175,176 decreased visual acuity or transient loss of vision,173,174 and retinal hemorrhage.173 Most of these ocular effects are thought to be due to the sympathomimetic effects of vasoconstriction, vasospasm, or complicating necrotizing vasculitis.173,174 Fatal outcomes are well recognized, typically arising from cardiac dysrhythmias, myocardial infarction, cardiorespiratory arrest, intractable seizures, hypoxic brain damage, hyperthermia, or intracerebral bleed.68,70,73,79,89,92,109,150,153,199

Chronic misuse In addition to these effects, the long-term use of metamfetamine in high doses may lead to non-ischemic cardiomyopathy,112 congestive heart failure,70,112,117 pulmonary hypertension,122

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Table 2. Anticipated signs and symptoms following acute and chronic exposure to metamfetamine Class Cardiac

Vasculature

Neurological

Signs and symptom

Case reports (references)

Tachycardia Hypertension Bradycardia Hypotension Cardiomegaly Congestive heart failure Chest Pain Coronary vasospasm Myocardial infarction/Ischemia Elevated troponin ECG abnormalities Abnormal Q Abnormal QRS Poor R wave progression Abnormal QT T changes ST changes Unspecified dysrhythmia Sinus tachycardia Sinus bradycardia Ventricular tachycardia Ventricular fibrillation Left or right bundle branch block Heart block Myocardial hypertrophy Ventricular dysfunction Cardiomyopathy Myocarditis Myocardial necrosis Contraction band necrosis Ischemic colitis Vasculitis (angiitis) Carotid artery aneurysm Vascular dissection Vascular beading Paresthesia Numbness Hyperreflexia Tremor Rapid and rolling eye movements Drowsiness Headache (including occipital)

65–93 65, 67, 69, 72, 75, 77, 84, 90–93, 101–103 106 66, 70, 74, 79, 106–108 91 111 66, 77, 83, 106, 113 106 70, 78, 83, 101, 106, 113 65, 77, 82, 106, 111

Cerebral ischemia Cerebral infarcts/stroke Cerebral hemorrhage Intracerebral/intracranial/ intraventricular Subarachnoid Hemiparesis or hemiplegia

116 107 77, 103, 118 77, 78, 101 77, 83, 101, 106, 107, 113 75 70, 72, 89, 101, 116

78, 121 106 122–124 70, 77, 122, 123 70, 77, 107, 123 111 101 101, 129, 130 131–134 75, 76, 93, 103, 108, 135–144 145 105, 109, 116, 145–148 75, 76, 135–138, 143 67, 81 136, 143 72, 137, 138, 147 65, 73, 74, 92 85, 88 66, 73, 75, 76, 80, 102, 107, 135, 147 66, 75, 80, 81, 87, 101–103, 135, 136, 138, 142–144, 146, 147, 149–151 101, 143 102, 136, 143, 146 75, 76, 80, 87, 135, 137–139, 141, 142, 144, 147, 149–151, 153–156 80, 138, 139, 147, 149 87, 91, 102, 135, 137, 142–144, 146, 147, 151, 154

Case series (references) 94–100 97, 99, 100, 104, 105 94 109, 110 112 105, 112, 114 99, 110, 115 100

115, 117 117 117 115, 117 99, 115, 117 117 117 117, 119 119, 120

110, 117, 125, 126 110, 112, 117 112, 127, 128

109, 110, 112, 126 104

105

98

104, 114 152 104, 152 104, 109, 127, 152 104, 109, 110, 125, 152 104 (Continued)

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Table 2. (Continued) Class

Signs and symptom Aphasia Ataxia Seizure Coma Cerebral edema Death

Psychiatric

Agitation

Respiratory

Anxiety Crying Aggressive behavior Confusion/disorientation Delirium/hallucinations Paranoia Psychosis Choreoathetosis Tachypnea Dyspnea Apnea Aspiration pneumonia Pulmonary edema

Hepatic Ocular

Renal

Metabolic

Pulmonary hypertension Abnormal liver function tests Acute liver failure Mydriasis Intraretinal hemorrhage Decreased/blurred vision Amaurosis fugax (transient loss of vision) Retinal vasculitis Keratitis/corneal ulceration Crystalline retinopathy Elevated serum creatinine Elevated blood urea nitrogen Urinary retention Hematuria Hyperuricemia Oliguria Renal insufficiency/failure Hyperkalemia Hypokalemia Hypocalcemia Hyperglycemia Hypoglycemia Elevated lactate dehydrogenase

Case reports (references) 102, 143, 144, 146 150 72, 73, 75, 76, 87, 89, 92, 139, 147, 153 72, 75, 79, 92, 101, 139, 151, 153, 154, 158 76, 79, 101, 141, 149, 153, 156, 159, 160 68, 70, 73, 75, 79, 89, 92, 101, 108, 120, 124, 139, 141, 145, 149, 151, 153, 156, 158–163 65, 67, 69, 72, 81, 82, 85, 88, 111, 147, 163 86 66, 71, 74, 84, 89, 150 73, 76, 80, 87, 89, 111, 138, 151 68, 71, 74, 85, 89, 165–168 85, 165, 167, 168 71, 85, 165–167 85, 86, 88, 118 66, 67, 70, 74, 75, 79, 84–86, 88, 91, 107, 111 66, 70, 77, 107 72, 92 72 68, 70, 73, 75, 79, 91, 103, 107, 111, 123, 129, 130, 141, 156, 158–162, 171 122 71, 111, 153 71, 79, 89, 111 65, 69, 72–75, 79, 82, 84, 90, 150, 154 173 80, 103, 136, 173 86, 174

Case series (references) 104 98 97, 98, 157 94, 157 127 104, 105, 110, 127

94–100, 114 99, 100 96, 98 99 104, 114, 164 94, 95, 99, 114, 164, 169 164 95, 164, 169, 170 96

112

110, 127 172

94

75, 174 175, 176 177 66, 74, 89, 93, 101, 103, 111 101, 103 90 90, 107, 138 81 79, 81, 90 79, 89, 91, 93, 101, 108 79, 93, 101 107 81 66, 72, 121 85 71, 84, 91, 118

157 157

157 94, 100, 105, 157 94

(Continued)

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Table 2. (Continued) Class

Signs and symptom Metabolic acidosis Hyperthermia

Hematological

Musculoskeletal

Gastrointestinal

Thrombocytopenia Leukocytosis Disseminated intravascular coagulation Muscle rigidity Elevated creatinine kinase Myalgia Rhabdomyolysis Nausea/vomiting Diarrhea Abdominal pain Hematemesis Hematochezia Diaphoresis Flushed face

Dermal

Case reports (references)

Case series (references)

66, 68, 71, 72, 74, 84, 107, 121 71, 74, 75, 79, 81, 89, 91, 101, 108, 111, 163 111, 153 81, 85, 90, 108, 133 81, 94, 111 74 65, 71, 72, 74, 78, 81–84, 89, 101, 111, 118, 121 81 65, 71, 72, 74, 81, 101, 111, 118 77, 80, 81, 93, 103, 107, 108, 135, 142, 144, 147, 149, 160 81, 133 65, 81, 90, 103, 106, 108, 131–134, 158 89, 138 89, 132 66, 74, 81, 85, 89, 113 76

and cognitive impairment.200 Irreversible neuronal 201,202 likely caused in part by long-term dopamine changes, depletion32 may develop, and isolated reports suggests that some syndromes such as parkinsonism may be causally linked in some patients with a previous history of metamfetamine abuse.203 The adverse psychiatric effects following irreversible neuronal changes can include a lasting psychosis, similar to

66, 97, 100, 157 94, 96–98, 100, 104, 125, 157 110 94

94, 96, 97, 100, 178

94, 98, 100, 157, 178 95, 98, 104

95, 105, 114

94, 99

paranoid schizophrenia.164,204 This is typically manifested by hallucinations, delusions, and paranoia;164,167 behavior can become bizarre, destructive, and violent.205 Self-mutilation without suicidal ideation has been reported.206 Changes in the physical appearance of chronic abusers often occur, as an aging effect is commonly produced.207 These physical changes usually result from associated malnutrition,

Table 3. Mean changes in key cardiovascular parameters of heart rate, diastolic and systolic blood pressure (relative to their respective baseline values) following exposure to metamfetamine by various routes Dose (mg) 12* 15 15* 15.5 17.5* 25* 30 30 30 30 30 30 30* 30* 35* 40 50 50*

Route

Heart rate (BPM)

Diastolic BP (mmHg)

Systolic BP (mmHg)

Reference

Intranasal Intravenous Oral Intravenous Intravenous Intranasal Inhalation Inhalation Intravenous Intravenous Intravenous Intravenous Oral Oral Intravenous Inhalation Intranasal Intranasal

1 9 9 25 18 13 32 30 21 18 28 13 8 – 14 30 21 19

7 12 10 6 14 9 17 17 12 9 14 14 20 14 20 10 10 17

4.9 11 13 23 22 16 18 20 21 18 18 20 30 28 35 16 20 24

42 181 185 44 62 42 44 43 189 186 182 181 185 64 62 41 41 42

*Doses were based on the weight of a 70-kg adult.

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684 weight loss, and poor hygiene.207 A further physical complication is “meth mouth” (significant caries of the teeth), which is thought to be due mainly to neglect and poor oral hygiene or poor diet. Additional contributing factors may also include xerostoma and/or bruxism.208,209 Excessive tooth decay has been erroneously attributed to contaminants of illicitly manufactured metamfetamine; contaminants appear not to contribute in any significant manner to meth mouth.209 Tolerance to the effects of metamfetamine has been reported in chronic users.92,210 Reports describe long-term addicts taking up to 15 g per day in divided doses without evidence of serious acute morbidity.211 Dependence can also develop with chronic use, so that a period of abstinence typically results in a withdrawal syndrome, marked more by psychological than physical complaints.212 Initial symptoms are thought to be due to depletion of CNS neurotransmitters and may consist of exhaustion, depression, agitation, and anxiety; this early phase is typically labeled “the crash.”212–214 Following this initial phase, further withdrawal effects may occur, including symptoms of prolonged depression with anhedonia, as well as insomnia or hypersomnia, anxiety, irritability, paranoia, aggression, and craving for the drug.212,214,215 The severity appears related to the dosage and duration of previous metamfetamine use.216 The majority of withdrawal symptoms tend to regress linearly over time,213 typically persisting for 5 days to 3 weeks,215,216 although fatigue and depression may continue for up to 12 months.212 Severe depression during withdrawal may lead to suicidal ideation requiring inpatient psychiatric management.212 Although there are presently no pharmaceutical agents available that may assist in minimizing metamfetamine dependence, recent clinical trials suggest some medications and two agonist replacements show some promise.217 Effects during pregnancy Metamfetamine use during pregnancy may have adverse effects upon the growth and development of the fetus. It is thought complications can occur by either direct transplacental transfer of the drug itself or secondary to its placental or maternal effects, leading to changes in the in utero environment. Such secondary changes may result from the vasoconstrictive effects of the drug; ovine models have demonstrated maternal hypertension and decreased uteroplacental blood flow, accompanied by fetal hypertension, hypoxia, and acidosis.218,219 Chronic metamfetamine use may also lead to poor maternal nutrition, thereby contributing to adverse fetal effects.220 The most common effect noted in newborns appears to be decreased weight, length, and head circumference.220–222 Metamfetamine abuse during pregnancy may also lead to placental insufficiency and/or abruption;49,220 premature delivery, fetal death, and maternal deaths have also been reported.153 Congenital anomalies including clefting, cardiac anomalies, cranial abnormalities, and abnormal brain development have additionally been reported with metamfetamine and

L.J. Schep et al. other amfetamines.223–226 However, the overall validity of these reports may be limited due to small sample sizes, reporting bias, reliance on maternal reporting of drug use, multiple drug exposures, or other nondrug factors. A mild withdrawal syndrome has been noted in newborns.220,227 There is only limited data, but one series of reports following children exposed to amfetamines in utero reported increased behavioral problems at 4 years old (especially if the mother was still addicted),228 aggression and hyperactivity at 8 years,229,230 and difficulties with physical fitness activities at 14 years.231 The work was, however, limited by small sample size, prenatal polydrug use, the lack of a control group, and the possible contribution of poor parental skills associated with the continued abuse of metamfetamine.

Effects upon children In children, the effects of direct exposure most commonly consist of agitation, irritability, crying, tachycardia, and vomiting. Less common effects include hyperthermia, ataxia, roving eye movements, rhabdomyolysis, and seizures.69,86,88,96–98,232 However, severe toxicity is not often reported in children following exploratory ingestions; in one series of 22 cases, seizures only occurred in 2 (9%) of 22 patients.97

Management Diagnosis The diagnosis of metamfetamine intoxication is typically made on the basis of the patient’s history along with clinical features of sympathomimetic poisoning. This diagnosis should be considered for any clinical presentation involving hyperthermia, excess sympathetic tone, or hallucinations.233 Confusion can arise particularly with pediatric admissions, for example, in which intoxicated patients have been misdiagnosed as with Centruroides sculpturatus envenoming.88,98 Qualitative analytical tests have been developed and a variety of immunoassays are available for the detection of metamfetamine and other related amfetamines. Unfortunately, these urine immunoassays are not highly sensitive or specific, and can lead to false-positive results.234–236 Additionally, a true-positive result does not necessarily indicate that the presenting illness is due to metamfetamine toxicity. If required, confirmation of metamfetamine exposure can typically be obtained using gas chromatography-mass spectrometry,237 liquid chromatography-mass spectrometry, or thinlayered chromatography. However, the lack of correlation between serum or urine concentrations and clinical effects means such tests are of limited value in acute management of the patient and should not be considered a routine component of assessment procedures. Additionally, as the diagnosis is usually made based on clinical effects, neither is identification of the stimulant often required.

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Stabilization

Supportive care

Patients may present to an emergency department in a critical condition with seizures, cardiac arrest, or stroke (or other acute consequences of arterial spasm or rupture). Those ingesting packaged metamfetamine (body packers/stuffers) may suffer overwhelming poisoning if packages leak their contents.73,158,159 Agitation and combativeness can make initial treatment of these patients difficult, and emergency intervention is often the priority. Significant hypertension, agitation, neurological, or respiratory compromise, or hyperthermia requires prompt, aggressive treatment. Appropriate airway management including airway control, oxygenation, and ventilation are required in obtunded patients.

Cardiovascular Hypertension is common, often severe, and presents a risk for intracranial hemorrhage. In mild cases, a benzodiazepine should suffice, along with providing a calming environment. If blood pressure does not settle, further control should be attempted with short-acting titratable antihypertensive agents such as nitroprusside246 0.5–1.5 μg/kg/min initially by intravenous infusion, then increased in steps of 500 ng/kg/min every 5 min within the range 0.5–8 μg/kg/min247 or an α1-adrenergic receptor antagonists such as phentolamine248 2–5 mg intravenously, repeated if necessary.247 Beta blockers are contraindicated as the blockade of β2-receptors could theoretically lead to increases in blood pressure because of unopposed α1-adrenergic receptor stimulation.246 Labetalol, 50–200 mg by intravenous infusion at a rate of 2 mg/min,247 has been recommended as a third-line agent in patients with refractory hypertension given its mixed α- and β-receptor antagonist properties,246 but caution is still warranted as its β-antagonist effects exceed its α-antagonist effect.249 Acute aortic dissection secondary to hypertensive crisis should be treated with both α- and β-blockers in combination.105 Periodic clinical and radiographic follow-up is recommended to help identify any secondary aneurysmal dilation that may occur.105 Mild sinus tachycardia may also respond to a benzodiazepine and the provision of a non-stimulating environment. In the absence of circulatory compromise, further intervention may not be required, but hemodynamically significant sinus tachycardia may increase the risk of myocardial ischemia and infarction and/or ventricular dysrhythmias.246 Therefore, if refractory to benzodiazepine treatment, very cautious use of a nonselective β-blocking agent to control heart rate may be considered.246 Serial ECGs should be monitored for onset of dysrhythmias. Supraventricular and ventricular dysrhythmias should be managed following standard advanced cardiac life support.233 Correction of hypoxia, acidosis, and metabolic abnormalities will minimize the risk of dysrhythmia.250 Elevated serial cardiac enzymes in association with ECG features of ischemia and chest pains may suggest the occurrence of an acute coronary syndrome (a collection of clinical effects consistent with myocardial ischemia), which can progress to acute myocardial infarction.112,113 These parameters should therefore be monitored closely. Additionally, coronary artery angiography may be required for diagnostic, prognostic, and therapeutic reasons. Echocardiography may be indicated and is especially recommended if there is cause to suspect cardiomyopathy (more associated with chronic metamfetamine use).70,123 Treatment recommendations for stimulant-induced acute coronary syndromes are mainly derived from those for cocaine intoxication,246 which recommend a benzodiazepine initially and a calming environment. Pharmacological treatments for unstable angina and myocardial infarction generally follow standard protocols. Nitroglycerin in either sublingual, oral, topical, or intravenous

Decontamination There are limited data on the benefits or otherwise of decontamination following oral ingestion; however, it is unlikely to be of significant benefit in the majority, because of the drug’s rapid absorption, and patients often present late and are less than cooperative. Nevertheless, although the efficacy of activated charcoal has not been assessed formally in human metamfetamine poisoning, its prompt use may assist in minimizing the adverse effects from a large recent ingestion. In a mouse model, activated charcoal given contemporaneously with metamfetamine has been shown to delay the onset of toxicity and decrease early mortality.238 However, charcoal should only be considered in patients who are alert, stable, and cooperative, and who have ingested a potentially toxic amount up to 1 h previously.239 Charcoal 50–100 g should be administered cautiously, because of the risk of impending seizures and/or loss of airway protective reflexes.233 In the case of significantly symptomatic patients, the risk of administration likely outweighs any benefit74 and general supportive measures should take precedence. Gastric lavage is unlikely to be of benefit233 and the induction of emesis is not recommended.240 Patients suspected of body packing158,241 or stuffing242 should undergo abdominal imaging using CT, although this may not be conclusive.100 Gastrointestinal decontamination may be of benefit in these patients. The role of activated charcoal in this situation has not been well defined, but it can be considered in those at risk of packet rupture.243 Whole bowel irrigation with polyethylene glycol solution can also be considered for the removal of ingested packets of illicit drugs.244 This is best administered through a nasogastric tube with suggested dosing regimens of 500 mL/h in children 9 months to 6 years, 1000 mL/h in children 6–12 years, and 1500–2000 mL/ h in adolescents and adults.244 It should be continued until the rectal effluent is clear or until there is no evidence of packages in the gastrointestinal tract.244 In situations of symptomatic body packers who are expelling degenerating or leaking packages, or if complete mechanical bowel obstruction occurs, monitoring in an intensive care unit and surgical removal of packages through laparotomy may be required.243,245

686 form has been used and titrated to effect in patients suffering from cocaine intoxication.251,252 In cases of unstable angina and myocardial infarction refractory to benzodiazepine and nitrate therapy, a potent α1-receptor antagonist such as phentolamine246,253 2–5 mg intravenously may be considered.247 Angioplasty may be indicated in cases of coronary artery stenosis.113 Hypotension is a rare finding, usually occurring late in the course of poisoning secondary to other factors, and appears to be associated with a poor prognosis.73,89,94 Milder cases are treated initially with intravenous fluids such as plasma expanders or crystalloids; a poor response or progression to shock requires vasopressors233 and admission to an intensive care unit with central cardiovascular monitoring. Neurological/psychiatric Severe agitation, psychosis, and/or choreoathetoid movements may be prominent following metamfetamine intoxication. Patients with only minor agitation may be managed without pharmaceutical intervention by providing a quiet, nonstimulating environment. More severely agitated patients may require physical restraints to prevent self harm; pharmacological sedation should then be instituted immediately as resistance to restraints can lead to continued heat production and rhabdomyolysis. The intravenous route of drug administration may be difficult or unsafe in agitated patients, although it has the advantage of enabling titration of the medication to effect.254 In these circumstances, initial intramuscular administration can be used until intravenous access is gained.255 With repeat intramuscular administration there is the risk that under- or over-sedation may occur.254,256 There is controversy concerning the role of benzodiazepines versus antipsychotics for controlling agitation.254,257 Benzodiazepines, antipsychotics, or both appear to be commonly used in emergency departments for treating agitation of uncertain etiology,255,258 but there are few studies systematically comparing the use of these agents,258 and this limitation also applies to agitation induced specifically by metamfetamine. However, initial control of agitation should be undertaken with benzodiazepines rather than antipsychotic medications.233,257,259–261 Initially, in an adult diazepam 5–10 mg intravenous or lorazepam 1–2 mg intravenous should be given; doses can be repeated until the patient is sedated.261 Midazolam 1–4 mg can be administered intramuscularly in patients without intravenous access.233 High doses may be required;262 one author describes total doses exceeding 100 mg of diazepam or its equivalent being required to achieve adequate sedation.260 Antipsychotics such as droperidol, olanzapine, and haloperidol can induce cardiac dysrhythmia or hypotension, interfere with thermoregulation, or precipitate extrapyramidal side-effects including dystonic reactions.257,259,263 Consequently, they have not generally been recommended as first-line agents for the control of agitation.257 Nevertheless,

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L.J. Schep et al. droperidol has been used successfully to sedate patients with metamfetamine toxicity.84,264 In an open-label study264 involving a subset of metamfetamine-intoxicated patients presenting to an emergency department,265 it was shown to act more quickly and require fewer repeat doses than lorazepam to achieve adequate sedation.264 However, use of these compounds as second-line management should only be considered in patients without cardiovascular abnormalities or elevated temperature.250 They may antagonize some of the effects of amfetamines by dopamine blockade,248 and therefore may be helpful for symptoms arising from stimulation of dopaminergic receptors, such as choreoathetosis.96 Intracerebral and subarachnoid hemorrhages are recognized complications of intoxication, and are thought to be due to acute hypertension (secondary to vasoconstriction or vasculitis, and/or increased cardiac output), leading to vessel wall stress and rupture.80,102,135,138,143,152,194,266 In addition, vasoconstriction may possibly lead to cerebral ischemia101,152 with risk of ischemic stroke.143 Any patient complaining of a severe headache, or displaying evidence of hemiparesis, hemiplegia, or reduced consciousness, requires intracranial CT imaging; this, however, may not detect all abnormalities. Susceptibility may be increased in patients with arteriovenous malformations.80 Significantly symptomatic or comatose patients may develop intracranial hematomas requiring surgical evacuation.102,135,142,147,154,267 Although corticosteroids have been used in the management of vasculitis and intracerebral hemorrhage,76,104,108,137,153 only slow improvement of neurological symptoms has been reported, generally in alert patients with evidence of only minor injuries.76 Seizures typically appear to be short-lived and respond to benzodiazepines.233 However, if prolonged, they may contribute to hyperthermia, metabolic acidosis, and rhabdomyolysis. Initial pharmacological treatment should be with a benzodiazepine: lorazepam 4 mg by slow intravenous injection (into a large vein) in an adult (in a child under 12 years 100 μg/kg; max. 4 mg), repeated once after 10 min if necessary; alternatively, diazepam 10 mg intravenously in an adult at a rate of 5 mg/min (in a child under 12 years 300–400 μg/kg), repeated once after 10 min if necessary.247 If, however, seizures are refractory, phenobarbital (10 mg/kg, infused at a rate of not more than 100 mg/min)247 may be necessary as second-line therapy. If adequate control is still not achieved, muscle paralysis with assisted ventilation (general anesthesia) may be required.233 Fluid and electrolytes Significant body fluid depletion can arise from various factors, including tachypnea and profuse sweating, which may be due in part to hyperthermia generated by the increased muscular and metabolic activity. Decreased fluid intakes may also exacerbate dehydration. Acid–base status, serum electrolytes,

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Metamfetamine – clinical toxicology and fluid balance should be closely monitored. Intravenous rehydration may be required, guided with invasive hemodynamic monitoring, if necessary.94 Severe hyperkalemia should be treated with hemodialysis94 if it does not respond to dextrose and insulin. Metabolic Severe hyperthermia may develop as a result of disturbance of central thermoregulatory systems or muscle hyperactivity because of agitation or seizures.263 As outlined above, benzodiazepines should be used to control agitation and reduce muscle activity, whereas fluid replacement is required to correct dehydration. External cooling measures should also be employed; these include water mist and fans, ice packs, and baths.250,263 If unsuccessful, neuromuscular paralysis with ventilatory support is recommended.263 Metabolic acidosis, which may arise secondary to seizures and/or inadequate tissue perfusion,250 is associated with a poor outcome,94 and should be managed with adequate administration of sodium bicarbonate and fluid replacement.72,74 Refractory hyperthermia could also suggest serotonin toxicity; particularly at risk are those taking other therapeutic or recreational serotonergic drugs. As noted, benzodiazepines are useful for agitation, and experimental animal studies suggest that they may also have a nonspecific serotonin agonist effect.268 Although there have been no randomized controlled trials, cyproheptadine and chlorpromazine may be useful if a diagnosis of serotonin toxicity is made.269,270 Cyproheptadine is only available in oral form and should be given by mouth or through a nasogastric tube in unconscious patients. The suggested initial dose is 12 mg, followed by 2 mg 2 h later, then titrating against response until improvement in autonomic and neurological abnormalities is achieved.269,271 A recommended maintenance dose regimen is 8 mg every 6 h.269 Chlorpromazine can be administered parenterally; the initial suggested dose is 12.5 mg, with a maximum of 1 mg/kg.272 Rhabdomyolysis Those suffering severe agitation, excessive muscular activity, or hyperthermia are at risk of developing rhabdomyolysis.94,118,263 Any patient who presents to hospital with severe agitation should have their creatine kinase activity measured. 273 Serum or urine myoglobin concentrations may also be of use. 274 If present, rhabdomyolysis should be treated aggressively with intravenous fluids to ensure good renal output, thus reducing urine myoglobin concentrations and the attendant risk of renal damage.275 Although urinary alkalinization might also help minimize risk of myoglobin-induced renal damage, 97,275 care should be taken as alkalinization has the effect of increasing retention of metamfetamine, slowing its excretion.59

687 Renal Metamfetamine may cause contraction of the bladder sphincter leading to urinary retention.90 If the patient has a distended bladder or is suffering suprapubic pain, they require a physical examination and ultrasonography. If there is evidence of a contracted bladder sphincter, catheterization should be undertaken. Acute renal failure can develop subsequent to rhabdomyolysis, vasculitis, hyperthermia, circulatory collapse, or a combination of these factors.79,84,93,273 Careful monitoring to detect evidence of early renal failure is required. This includes monitoring urine output and serum creatinine concentrations. Acute renal failure should be treated urgently with hemodialysis, hemodialfiltration, or hemofiltation. Gastrointestinal Any patient presenting with abdominal pain or a history of recent bloody diarrhea and having a history of drug abuse suggests the possibility of ischemic colitis, and therefore requires a CT scan or sonography. Low-grade ischemia typically heals within a short period of time, whereas full thickness ischemia may require prompt resection.133 Enhanced elimination As metamfetamine is a weak base (pKa ∼9.9),61 acidification of the urine can enhance its excretion59 because in more acidic urine it becomes mostly ionized, and thus renal tubular reabsorption is minimized.84 An early case report advised urinary acidification;84 however, this is no longer recommended clinically as there are limited data on its effectiveness, and the attendant risk of causing or aggravating acidosis, seizures, or rhabdomyolysis would outweigh any potential benefits.276 Hemodialysis, hemoperfusion, hemofiltration, and other techniques to enhance removal of metamfetamine are unlikely to be effective, because of its high volume of distribution, and are not recommended. Multiple doses of activated charcoal are not thought to be of significant benefit. In a rat model, it did not enhance the elimination of metamfetamine.277

Conclusions Metamfetamine, an N-methylated analog of amfetamine, is a widely abused drug that acts as a stimulant by causing enhanced release of catecholamines from sympathetic nerve terminals, particularly those in the mesolimbic, mesocortical, and nigrostriatal pathways. The resulting elevated concentrations can lead to detrimental psychological, cardiovascular, and other systemic effects, and, following long-term abuse, neuronal apoptosis, and nerve terminal degeneration. Patients with metamfetamine poisoning are expected to make a full recovery, provided they receive prompt supportive care.

688

Acknowledgments We thank AJ Barnes (New Zealand National Poison Centre) for drawing Fig. 2.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.

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L.J. Schep et al. 200. Simon SL, Domier C, Carnell J, Brethen P, Rawson R, Ling W. Cognitive impairment in individuals currently using methamphetamine. Am J Addict 2000; 9:222–2231. 201. Thompson PM, Hayashi KM, Simon SL, Geaga JA, Hong MS, Sui Y, Lee JY, Toga AW, Ling W, London ED. Structural abnormalities in the brains of human subjects who use methamphetamine. J Neurosci 2004; 24:6028–6036. 202. Ernst T, Chang L, Leonido-Yee M, Speck O. Evidence for long-term neurotoxicity associated with methamphetamine abuse: a 1H MRS study. Neurology 2000; 54:1344–1349. 203. Rudnicki SA, Archer RL, Labib BT. Motor neuron disease in methamphetamine abusers. Amyotroph Lateral Scler 2007; 8:126–127. 204. Iwanami A, Kato N, Nakatani Y. P300 in methamphetamine psychosis. Biol Psychiatry 1991; 30:726–730. 205. Zweben JE, Cohen JB, Christian D, Galloway GP, Salinardi M, Parent D, Iguchi M. Psychiatric symptoms in methamphetamine users. Am J Addict 2004; 13:181–190. 206. Israel JA, Lee K. Amphetamine usage and genital self-mutilation. Addiction 2002; 97:1215–1218. 207. Winslow BT, Voorhees KI, Pehl KA. Methamphetamine abuse. Am Fam Physician 2007; 76:1169–1174. 208. Hamamoto DT, Rhodus NL. Methamphetamine abuse and dentistry. Oral Dis 2009; 15:27–37. 209. Shaner JW, Kimmes N, Saini T, Edwards P. “Meth mouth:” rampant caries in methamphetamine abusers. AIDS Patient Care STDS 2006; 20:146–150. 210. Comer SD, Hart CL, Ward AS, Haney M, Foltin RW, Fischman MW. Effects of repeated oral methamphetamine administration in humans. Psychopharmacology (Berl) 2001; 155:397–404. 211. Kramer JC, Fischman VS, Littlefield DC. Amphetamine abuse. Pattern and effects of high doses taken intravenously. JAMA 1967; 201:305–309. 212. Meredith CW, Jaffe C, Ang-Lee K, Saxon AJ. Implications of chronic methamphetamine use: a literature review. Harv Rev Psychiatry 2005; 13:141–154. 213. Barr AM, Markou A, Phillips AG. A “crash” course on psychostimulant withdrawal as a model of depression. Trends Pharmacol Sci 2002; 23:475–482. 214. Gawin FH, Ellinwood EH. Cocaine and other stimulants – actions, abuse, and treatment. N Engl J Med 1988; 318:1173–1182. 215. Newton TF, Kalechstein AD, Duran S, Vansluis N, Ling W. Methamphetamine abstinence syndrome: preliminary findings. Am J Addict 2004; 13:248–255. 216. McGregor C, Srisurapanont M, Jittiwutikarn J, Laobhripatr S, Wongtan T, White JM. The nature, time course and severity of methamphetamine withdrawal. Addiction 2005; 100:1320–1329. 217. Karila L, Weinstein A, Aubin HJ, Benyamina A, Reynaud M, Batki SL. Pharmacological approaches to methamphetamine dependence: a focused review. Br J Clin Pharmacol 2010; 69:578–592. 218. Stek AM, Baker S, Fisher BK, Lang U, Clark KE. Fetal responses to maternal and fetal methamphetamine administration in sheep. Am J Obstet Gynecol 1995; 173:1592–1598. 219. Burchfield DJ, Lucas VW, Abrams RM, Miller RL, Devane CL. Disposition and pharmacodynamics of methamphetamine in pregnant sheep. JAMA 1991; 265:1968–1973. 220. Smith L, Yonekura ML, Wallace T, Berman N, Kuo J, Berkowitz C. Effects of prenatal methamphetamine exposure on fetal growth and drug withdrawal symptoms in infants born at term. J Dev Behav Pediatr 2003; 24:17–23. 221. Little BB, Snell LM, Gilstrap LC III. Methamphetamine abuse during pregnancy: outcome and fetal effects. Obstet Gynecol 1988; 72:541–544. 222. Smith LM, LaGasse LL, Derauf C, Grant P, Shah R, Arria A, Huestis M, Haning W, Strauss A, Della Grotta S, Liu J, Lester BM. The infant development, environment, and lifestyle study: effects of prenatal methamphetamine exposure, polydrug exposure, and poverty on intrauterine growth. Pediatrics 2006; 118:1149–1156.

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Metamfetamine – clinical toxicology 223. Arria AM, Derauf C, Lagasse LL, Grant P, Shah R, Smith L, Haning W, Huestis M, Strauss A, Della Grotta S, Liu J, Lester B. Methamphetamine and other substance use during pregnancy: preliminary estimates from the Infant Development, Environment, and Lifestyle (IDEAL). Matern Child Health J 2006; 10:293–302. 224. Dixon SD, Bejar R. Echoencephalographic findings in neonates associated with maternal cocaine and methamphetamine use: incidence and clinical correlates. J Pediatr 1989; 115:770–778. 225. Cloak CC, Ernst T, Fujii L, Hedemark B, Chang L. Lower diffusion in white matter of children with prenatal methamphetamine exposure. Neurology 2009; 72:2068–2075. 226. Chang L, Smith LM, LoPresti C, Yonekura ML, Kuo J, Walot I, Ernst T. Smaller subcortical volumes and cognitive deficits in children with prenatal methamphetamine exposure. Psychiatry Res 2004; 132:95–106. 227. Oro AS, Dixon SD. Perinatal cocaine and methamphetamine exposure: maternal and neonatal correlates. J Pediatr 1987; 111:571–578. 228. Billing L, Eriksson M, Steneroth G, Zetterström R. Pre-school children of amphetamine-addicted mothers. I. Somatic and psychomotor development. Acta Paediatr Scand 1985; 74:179–184. 229. Billing L, Eriksson M, Jonsson B, Steneroth G, Zetterstrom R. The Influence of environmental factors on behavioral-problems in 8-yearold children exposed to amphetamine during fetal life. Child Abuse Negl 1994; 18:3–9. 230. Eriksson M, Billing L, Steneroth G, Zetterström R. Health and development of 8-year-old children whose mothers abused amphetamine during pregnancy. Acta Paediatr Scand 1989; 78:944–949. 231. Cernerud L, Eriksson M, Jonsson B, Steneroth G, Zetterström R. Amphetamine addiction during pregnancy: 14-year follow-up of growth and school performance. Acta Paediatr 1996; 85:204–208. 232. Suchard JR, Curry SC. Methamphetamine toxicity. Pediatr Emerg Care 1999; 15:306. 233. McKinney PE, Palmer RB. Amphetamines and derivatives. In: Brent J, Wallace KL, Burkhart KK, Phillips SD, Donovan JW, eds. Critical Care Toxicology: Diagnosis and Management of the Critically Poisoned Patient. Philadelphia PA: Elsevier Mosby; 2005:761–766. 234. D’Nicuola J, Jones R, Levine B, Smith ML. Evaluation of six commercial amphetamine and methamphetamine immunoassays for crossreactivity to phenylpropanolamine and ephedrine in urine. J Anal Toxicol 1992; 16:211–213. 235. Roberge RJ, Luellen JR, Reed S. False-positive amphetamine screen following a trazodone overdose. J Toxicol Clin Toxicol 2001; 39:181–182. 236. Grinstead GF. Ranitidine and high-concentrations of phenylpropanolamine cross react in the emit monoclonal amphetamine methamphetamine assay. Clin Chem 1989; 35:1998–1999. 237. Valentine JL, Middleton R. GC-MS identification of sympathomimetic amine drugs in urine: rapid methodology applicable for emergency clinical toxicology. J Anal Toxicol 2000; 24:211–222. 238. McKinney PE, Tomaszewski C, Phillips S, Brent J, Kulig K. Methamphetamine toxicity prevented by activated charcoal in a mouse model. Ann Emerg Med 1994; 24:220–223. 239. Chyka PA, Seger D, Krenzelok EP, Vale JA. Position paper: singledose activated charcoal. Clin Toxicol (Phila) 2005; 43:61–87. 240. Seger D, Muelenbelt J. Position paper: ipecac syrup. J Toxicol Clin Toxicol 2004; 42:133–143. 241. Wetli CV, Mittleman RE. The body packer syndrome – toxicity following ingestion of illicit drugs packaged for transportation. J Forensic Sci 1981; 26:492–500. 242. Roberts JR, Price D, Goldfrank L, Hartnett L. The bodystuffer syndrome – a clandestine form of drug overdose. Am J Emerg Med 1986; 4:24–27. 243. Booker RJ, Smith JE, Rodger MP. Packers, pushers and stuffers-managing patients with concealed drugs in UK emergency departments: a clinical and medicolegal review. Emerg Med J 2009; 26:316–320. 244. Position paper: whole bowel irrigation. J Toxicol Clin Toxicol 2004; 42:843–854.

693 245. Marc B, Baud FJ, Maisonblanche P, Leporc P, Garnier M, Gherardi R. Cardiac monitoring during medical-management of cocaine body packers. J Toxicol Clin Toxicol 1992; 30:387–397. 246. Guidelines 2000 for Cardiopulmonary Resuscitation and Emergency Cardiovascular Care. Part 8: advanced challenges in resuscitation: section 2: toxicology in ECC. The American Heart Association in collaboration with the International Liaison Committee on Resuscitation. Circulation 2000; 102:1223–1228. 247. Joint Formulary Committee. British National Formulary 59. London: Pharmaceutical Press; 2010. 248. Derlet RW, Albertson TE, Rice P. Antagonism of cocaine, amphetamine, and methamphetamine toxicity. Pharmacol Biochem Behav 1990; 36:745–749. 249. Richards DA, Tuckman J, Prichard BN. Assessment of alpha-adrenoceptor and beta-adrenoceptor blocking actions of labetalol. Br J Clin Pharmacol 1976; 3:849–855. 250. Greene SL, Kerr F, Braitberg G. Review article: amphetamines and related drugs of abuse. Emerg Med Australas 2008; 20:391–402. 251. Brogan WC, Lange RA, Kim AS, Moliterno DJ, Hillis LD. Alleviation of cocaine-induced coronary vasoconstriction by nitroglycerin. J Am Coll Cardiol 1991; 18:581–586. 252. Hollander JE, Hoffman RS, Gennis P, Fairweather P, Disano MJ, Schumb DA, Feldman JA, Fish SS, Dyer S, Wax P, Whelan C, Schwarzwald E. Nitroglycerin in the treatment of cocaine-associated chest pain – clinical safety and efficacy. J Toxicol Clin Toxicol 1994; 32:243–256. 253. Albertson TE, Dawson A, de Latorre F, Hoffman RS, Hollander JE, Jaeger A, Kerns W, Martin TG, Ross MP. TOX-ACLS: toxicologicoriented advanced cardiac life support. Ann Emerg Med 2001; 37:S78–S90. 254. Knott JC, Isbister GK. Sedation of agitated patients in the emergency department. Emerg Med Australas 2008; 20:97–100. 255. Downes MA, Healy P, Page CB, Bryant JL, Isbister GK. Structured team approach to the agitated patient in the emergency department. Emerg Med Australas 2009; 21:196–202. 256. Spain D, Crilly J, Whyte I, Jenner L, Carr V, Baker A. Safety and effectiveness of high-dose midazolam for severe behavioural disturbance in an emergency department with suspected psychostimuantaffected patients. Emerg Med Australas 2008; 20:112–120. 257. Whelan KR, Dargan PI, Jones AL, O’Connor N. Atypical antipsychotics not recommended for control of agitation in the emergency department. Emerg Med J 2004; 21:649. 258. Yildiz A, Sachs GS, Turgay A. Pharmacological management of agitation in emergency settings. Emerg Med J 2003; 20:339–346. 259. Dubin WR, Weiss KJ, Dorn JM. Pharmacotherapy of psychiatric emergencies. J Clin Psychopharmacol 1986; 6:210–222. 260. Chiang WK. Amphetamines. In: Flomenbaum NE, Goldfrank LR, Hoffman RS, Howland M, Lewin NA, Nelson LS, eds. Goldfrank’s Toxicologic Emergencies. 8th ed. New York: McGraw-Hill; 2006:1118–1132. 261. Henry JA. Amphetamines. In: Ford MD, Delaney KA, Ling LJ, Erickson T, eds. Clinical Toxicology. Philadelphia, PA: Saunders Company; 2001:620–626. 262. Gray SD, Fatovich DM, McCoubrie DL, Daly FF. Amphetaminerelated presentations to an inner-city tertiary emergency department: a prospective evaluation. Med J Aust 2007; 186:336–339. 263. Callaway CW, Clark RF. Hyperthermia in psychostimulant overdose. Ann Emerg Med 1994; 24:68–76. 264. Richards Jr, Derlet RW, Duncan DR. Methamphetamine toxicity: treatment with a benzodiazepine versus a butyrophenone. Eur J Emerg Med 1997; 4:130–135. 265. Richards Jr, Derlet RW, Duncan DR. Chemical restraint for the agitated patient in the emergency department: lorazepam versus droperidol. J Emerg Med 1998; 16:567–573. 266. Bostwick DG. Amphetamine induced cerebral vasculitis. Hum Pathol 1981; 21:1030–1033. 267. Kessler JT, Jortner BS, Adapon BD. Cerebral vasculitis in a drug abuser. J Clin Psychiatry 1978; 39:559–564.

694 268. Nisijima K, Shioda K, Yoshino T, Takano K, Kato S. Diazepam and chlormethiazole attenuate the development of hyperthermia in an animal model of the serotonin syndrome. Neurochem Int 2003; 43:155–164. 269. Boyer EW, Shannon M. The serotonin syndrome. N Engl J Med 2005; 352:1112–1120. 270. Isbister GK, Buckley NA, Whyte IM. Serotonin toxicity: a practical approach to diagnosis and treatment. Med J Aust 2007; 187:361–365. 271. Isbister GK. Comment: serotonin syndrome, mydriasis, and cyproheptadine. Ann Pharmacother 2001; 35:1672–1673. 272. Chan BS, Graudins A, Whyte IM, Dawson AH, Braitberg G, Duggin GG. Serotonin syndrome resulting from drug interactions. Med J Aust 1998; 169:523–525.

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L.J. Schep et al. 273. Richards JR, Bretz SW, Johnson EB, Turnipseed SD, Brofeldt BT, Derlet RW. Methamphetamine abuse and emergency department utilization. West J Med 1999; 170:198–202. 274. Huerta-Alardin AL, Varon J, Marik PE. Bench-to-bedside review: rhabdomyolysis – an overview for clinicians. Crit Care 2005; 9:158–169. 275. Bosch X, Poch E, Grau JM. Rabdomyolysis and acute kidney injury. N Engl J Med 2009; 361:62–72. 276. Albertson TE, Derlet RW, Van Hoozen BE. Methamphetamine and the expanding complications of amphetamine. West J Med 1999; 170:214–219. 277. Hutchaleelaha A, Mayersohn M. Influence of activated charcoal on the disposition kinetics of methamphetamine enantiomers in the rat following intravenous dosing. J Pharm Sci 1996; 85:541–545.

Clinical Toxicology (2010) 48, 695–708 Copyright © Informa UK, Ltd. ISSN: 1556-3650 print / 1556-9519 online DOI: 10.3109/15563650.2010.516263

REVIEW LCLT

Cocaine, metamfetamine, and MDMA abuse: the role and clinical importance of neuroadaptation DONNA SEGER Neuroadaptation

Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA

Introduction. This article reviews the role and clinical importance of specific neuroadaptations that may occur following use of cocaine, metamfetamine, and 3,4,methylenedioxymetamfetamine (MDMA). Methods. A literature search was performed using OVID MEDLINE and PubMed for all years to the present date, which identified 250 papers of which 154 were considered relevant. Mechanisms of action of cocaine and metamfetamine. Cocaine and metamfetamine increase central nervous system synaptic dopamine primarily by increasing the release of dopamine into the synapse and binding to the dopamine reuptake transporter, which prevents the reuptake of dopamine from the synapse back into the nerve cell. Synaptic dopamine then stimulates post synaptic receptors. The continued release of dopamine and prevention of reuptake results in a supraphysiological concentration of dopamine, which causes euphoria or a “high.” The greater the concentration of dopamine, the greater the high. Continued supraphysiological concentrations of dopamine and postsynaptic receptor stimulation may cause physiological and anatomical changes (neuroadaptations) in the central nervous system (CNS) synapse that attempt to maintain homeostasis. An example of a dopaminergic neuroadaptation is the decrease in number of post synaptic D2 receptors that occurs when synaptic dopamine concentrations remain supraphysiological. This neuroadaptation attempts to maintain homeostasis, that is, the decreased number of D2 receptors provides fewer receptors to be constantly stimulated by increased synaptic dopamine. Although metamfetamine also increases synaptic dopamine similarly to cocaine, metamfetamine also increases cytoplasmic dopamine, which causes CNS oxidative stress and neurotoxicity. The clinical impact of the oxidative stress is unknown. Mechanisms of action of MDMA. MDMA increases concentrations of synaptic serotonin by increasing the release of serotonin and binding to the serotonin reuptake transporter, preventing the reuptake of serotonin from the synapse back into the nerve cell. An example of a serotonergic neuroadaptation is a decrease in the number of serotonin presynaptic autoreceptors (one of the regulators of synaptic serotonin concentration) to maintain homeostasis. MDMA also causes a decrease in serotonergic biochemical markers and neuronal axotomy in rats and nonhuman primates. Abstinence may allow reinnervation, but the axonal regrowth pattern is abnormal. Whether axotomy and reinnervation also occur in humans is unknown. Pharmacogenomics may play a role in the varied response of the individual to MDMA. Conclusions. Neuroadaptations may be transient or permanent. The duration of drug use or drug concentration needed to cause neuroadaptations is unknown, but some neuroadaptations begin shortly after initiation of drug use and are dependent on variables such as genetics and age at the initiation of use. Understanding the concept of neuroadaptation and some specific neuroadaptations that occur will allow clinicians to better understand the interindividual variability in response to drugs of abuse. Keywords

Synapse; Serotonin; Dopamine; Neurotoxicity; Receptors

Introduction Constant stimulation causes cells to make compensatory physiological and/or anatomical changes to maintain homeostasis (equilibrium). Compensatory changes in the central nervous system (CNS) that are caused by drugs of abuse are called neuroadaptations and these oppose the acute reinforcing effects of the drugs. If drug use ceases, the neuroadaptive changes may reverse over days to weeks. However, as

Received 9 July 2010; accepted 13 August 2010. Address correspondence to Donna Seger, Department of Medicine, Vanderbilt University Medical Center, 501 Oxford House, Nashville, TN 373232-4632, USA. E-mail: [email protected]

repeated drug use remodels neural circuits and synapses, the adaptive changes may become permanent.1–5 This review will address specific neuroadaptations that occur following use of the psychostimulants cocaine, metamfetamine (methamphetamine), and 3,4,methylenedioxymetamfetamine (MDMA; ecstasy). Neuroadaptations in the dopaminergic system following the use of cocaine and metamfetamine will be discussed as well as neuroadaptations in the serotonergic system following the use of MDMA. The review does not attempt to address comprehensively the neuroadaptive changes caused by these psychostimulants, only to explain the basis of a few specific adaptive processes. It will not address tolerance (reduction of acute drug effects with repeated exposure) or withdrawal (unmasked adaptation causing symptoms) as much has been written about these

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neuroadaptations.2,4 The neurotoxicity of the drugs is described to distinguish the toxicity from the adaptive changes.

Methodology A comprehensive literature search was performed using OVID MEDLINE and PubMed. Bibliographies of identified articles were reviewed for additional references and cross reference. All articles were from peer-reviewed journals. Search terms included neuroadaptation, serotonin, dopamine, cocaine, metamfetamine, methamphetamine, ecstasy, MDMA, synapse, receptor, drugs of abuse, plasticity, and neurotoxicity. This search identified 250 references of which 152 were considered relevant.

The synapse The CNS synapse is where neurons communicate with one another, not by direct continuity but by means of chemical substances called neurotransmitters. These neurotransmitters are synthesized in the neuron and moved into vesicles through vesicular transporters. The acidic pH of the vesicle protects the neurotransmitters from degradation in the more basic neuronal cytoplasm. Depolarization of the nerve causes the vesicle to fuse with the plasma nerve membrane and release the neurotransmitter into the synaptic cleft, the small gap between the two neurons. The amount of neurotransmitter released is determined by the synaptic and vesicular concentration of the neurotransmitter and the degree of stimulation of the autoreceptor (determined by concentration of the synaptic neurotransmitter) on the presynaptic nerve cell membrane. Autoreceptor stimulation inhibits neurotransmitter synthesis and release into the synapse.6–8 The neurotransmitter diffuses through the synaptic cleft and produces effects by binding to a receptor (protein) on the postsynaptic nerve cell membrane. Neurotransmitter effects are terminated by a plasma membrane reuptake transporter, which moves the synaptic neurotransmitter back into the neuron (Fig. 1).9

Neuroadaptation of synaptic receptors – a change in number Drugs of abuse may be receptor agonists or may induce the release of neurotransmitters that stimulate the postsynaptic receptor. During development, each neuron is programmed to respond to a certain level of input from each neurotransmitter. If a neuron receives too many impulses from increased synaptic neurotransmitter (induced by drugs of abuse), the neuron responds by decreasing the number of receptors (downregulation) for this neurotransmitter. Conversely, if a

Fig. 1. The Synapse.

neuron receives too many impulses from a certain neurotransmitter, the neuron responds by increasing the number of receptors (upregulation). Other receptor neuroadaptations include changes in the receptor function or post-receptor mechanisms that oppose the effect of the drug.10–12

Dopamine Dopamine synthesis and metabolism In the neuronal soma, the amino acid tyrosine is hydroxylated by tyrosine hydroxylase to L-dopa. Tyrosine hydroxylase is the rate-limiting enzyme and is sensitive to negative feedback. L-Dopa undergoes decarboxylation by the L-amino acid decarboxylase to dopamine. Synaptic dopamine that is not removed by the reuptake transporter is enzymatically broken down by catechol-O-methyl transferase and monoamine oxidase.13 Dopaminergic pathways Of the four major dopamine pathways in the brain (mesocortical, mesolimbic, nigrostriatal, and hypothalamic), the mesolimbic and mesocortical pathways play a critical role in mediating the hedonistic and addictive properties of drugs of abuse. The majority of dopaminergic neurons lie in the ventral tegmental area of the midbrain. Axons extending from the soma to the cerebral cortex, especially the frontal lobes, comprise the mesocortical pathway, which is involved in the conscious experience of drug use, drug expectation, and craving. Axons projecting to the nucleus accumbens (in the limbus) comprise the mesolimbic pathway, which is involved in memory, motivating behavior, acute reinforcing effects of drugs, and conditioned responses linked to craving. The mesolimbic and mesocortical pathways operate in parallel, yet they interact with one another and with other pathways.11,14,15

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Neuroadaptation Dopamine transporters control the concentration of synaptic and intracytoplasmic dopamine Two transporters regulate the concentration of dopamine in the cytoplasm and in the synapse. Vesicular monoamine transporters (VMAT) are classified as VMAT-1, which is found in peripheral neuroendocrine cells, and VMAT-2, which is found in neurons. VMAT-2 transports dopamine (synthesized in the neuronal soma) from the neuronal cytoplasm into the vesicles for storage.16 Once dopamine has been released into the synapse, the dopamine reuptake transporter transfers dopamine back into the cytoplasm of the presynaptic nerve terminal11,17 (see Fig. 1). Dopamine receptors In the CNS there are at least five types of dopamine receptors that fall into two families based on whether they increase (D1) or decrease (D2) the synthesis of the second messenger, cyclic adenosine monophosphate. Postsynaptic D1 receptors are located primarily on striatal spiny neurons that project to the internal part of the globus pallidus/substantia nigra pars reticularis. Postsynaptic D2 receptors are located on spiny neurons projecting to the external part of the globus pallidus.2 D2 receptors are also located on the presynaptic neuronal membrane and act as autoreceptors that help to regulate the concentration of dopamine released into the synapse.13,18–22 The pleasure principle and synaptic dopamine The behavioral and clinical effects of drugs of abuse are caused by multiple neurotransmitters acting in multiple brain areas, but the most important neurotransmitter is dopamine. Hedonistic activity and euphoria (from either natural rewards or drugs of abuse) occur primarily as a result of firing of dopamine neurons that causes dopamine release into the synapse of the limbus11,23 and stimulation of the D2 receptor.2,24 With repeated activity, dopaminergic neuronal firing begins when stimuli (or cues) predict the activity that will follow. Drug-induced neuronal firing increases synaptic dopamine to supraphysiological concentrations.5,25–29 The greater the concentration of synaptic dopamine, the greater the “high.” As drug use continues, the synaptic concentration (set point) at which pleasure is achieved is changed, and there is a blunting of mechanisms that mediate positive reinforcement.4,11 In contrast, natural reinforcers lose their ability to increase synaptic dopamine and supraphysiological synaptic concentrations are not attained.5,25–30 Although most studies have examined the role of dopamine during acute or chronic drug use, dopamine interacts with other important neurotransmitters such as glutamate (involved in molecular changes associated with learning) and g-amino butyric acid (which modulates the magnitude of the dopamine response). With long-term drug use, neural adaptations also occur in the glutamatergic and GABAergic pathways,

697 and these adaptations influence the dopamine response. As these drugs are used repetitively, neuroadaptive molecular changes are induced in the brain.31,32

Cocaine and dopamine Cocaine is a psychostimulant drug that acts at dopaminergic, noradrenergic, and serotonergic synapses, but its behavioral effects are primarily because of its action at the dopaminergic synapse where it increases synaptic dopamine.33 Cocaine increases synaptic dopamine in two ways. Firstly, cocaine increases the release of dopamine into the synapse. The elevated concentrations of synaptic dopamine activate both D1 and D2 receptors. Secondly, cocaine binds to the dopamine reuptake transporter preventing the reuptake of synaptic dopamine34 and prolonging the effects of dopamine at its receptors. D2 receptors play an important role in mediating the acute effects of cocaine. D1 receptor pathways are activated with prolonged use of cocaine through immediate early genes, which are genes that can be induced transiently and rapidly (within hours), and are important mediators of neuroadaptation.35–41 Neuroadaptation of the D2 receptor Genetic determination of D2 receptor number Positron emission tomography (PET) scans record the uptake and washout of radioactive markers that compete with endogenous neurotransmitters and can therefore measure receptor densities. PET scans have demonstrated decreased D2 receptor density (number) in people who abuse cocaine or metamfetamine. The question is whether the D2 receptor number is determined genetically (making those with fewer D2 receptors vulnerable to drug abuse) and/or whether the number of D2 receptors decreases during drug use (causing continued drug use). Subjects without psychiatric or neurological diseases or drug abuse have significant inter-subject variability in D2 receptor number on PET scans indicating that genetics is one determinant of the number of D2 receptors.42–47 D2 receptor number determines the response to drugs of abuse One of the determinants of an individual’s response to drugs of abuse is the number of D2 receptors. Fewer D2 receptors increase vulnerability for drug abuse as people with decreased D2 receptors find supraphysiological concentrations of synaptic dopamine much more pleasurable than those with a greater number of dopamine receptors. In a human volunteer study, those who reported the administration of the stimulant methylphenidate (an inhibitor of the dopamine transporter) as pleasurable had significantly fewer D2 receptors than those who reported the administration of the stimulant as unpleasant.43,48

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Animal experiments also support the concept that the number of D2 receptors determines the response to drugs of abuse. When rats are trained to self-administer alcohol, and D2 receptors are injected into the nucleus accumbens through an adenoviral receptor, there is an initial decrease in alcohol intake (more receptors available to be stimulated so less alcohol is required for the sensation of pleasure). However as D2 receptors decrease, drug intake increases again.49 The importance of D2 receptor number in the initiation of drug use or maintenance of addiction is unclear.50 Neuroadaptation – the change in D2 receptor number When monkeys are trained to self-administer food with a lever, and cocaine is subsequently substituted for food, the continued self-administration of cocaine causes a decrease in the number of D2 receptors in the dorsal and ventral striatum33,51 (see Fig. 251). D2 receptor numbers can also be changed by environmental influences. When monkeys are housed together, D2 receptors increase in monkeys that become dominant. The dominant monkeys are able to control resources and that ability may induce an increase in D2 receptors. The density of D2 receptors in individually housed monkeys (before housing together) does not predict social

rank, that is, the number of D2 receptors is not a trait variable that influences dominant hierarchies. When both dominant and subordinate monkeys are allowed subsequently to selfadminister cocaine, subordinate monkeys (with fewer D2 receptors) self-administer significantly more cocaine than dominant monkeys. Subordinate monkeys will even choose cocaine over food. The environmentally induced change in D2 receptors (a neuroadaptation) and subsequent drug use, as determined by the number of D2 receptors, demonstrates that environmental variables that change the number of D2 receptors increase vulnerability to future cocaine use.51 There are other clinical consequences of a decrease in the number of D2 receptors. Human imaging studies of addicts during withdrawal or protracted abstinence reveal that a decrease in D2 receptors is associated with reduced cerebral metabolism in the prefrontal cortex (hypofrontality). These same frontal regions become hypermetabolic during cue-induced craving. Cocaine-dependent subjects with hypofrontality demonstrate poor impulse control and compulsive drug use and poor performance on neuropsychatric tests that assess prefrontal cortical function. Subjects with the lowest number of D2 receptors had the lowest metabolic values. D2 receptor number may indirectly regulate cerebral metabolism.23,52–54

2 monkeys Baseline

6 months

R-1241

R-1249

Fig. 2. PET imaging of D2.51 D2 receptors (light areas) decrease with continued cocaine administration.

12 months

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Neuroadaptation Recovery of D2 receptor number during abstinence In a single monkey who self-administered cocaine for 3 years, D2 receptors were still decreased following 7 months of abstinence.55 In a different set of experiments, three monkeys who used cocaine for 1 week had a normal number of D2 receptors following 3 weeks of abstinence: three of the five monkeys who self-administered cocaine for 12 months had complete recovery of D2 receptor numbers following 3 months of abstinence: and two of the five monkeys had no recovery in D2 receptor number following 1 year of abstinence.51 The rate of recovery was not related to total cocaine intake.51 Recovery of D2 receptors following abstinence is variable and related more to the duration of drug use than dose of drug.54,55 Conclusions The number of D2 receptors is genetically determined. People with fewer D2 receptors are more susceptible to drug abuse. Chronic cocaine use decreases the number of D2 receptors, which promotes drug craving. Both drugs and the environment can change D2 receptor number, and recovery of D2 receptors during abstinence is variable. A decrease in the number of D2 receptors reflects a neuroadaptation to maintain homeostasis during chronic cocaine use.33

699 dopamine which causes neurotoxicity.16,56 Minor mechanisms by which metamfetamine increases cytoplasmic dopamine include increasing the activity of tyrosine hydoxylase (which increases production of dopamine) and inhibiting monamine oxidase (which metabolizes dopamine). However, the major mechanism is the action of metamfetamine on the dopamine transporter, VMAT-2. The resulting concentration of cytoplasmic and synaptic dopamine is the result of the combined actions of metamfetamine17,56–64 (see Fig. 3). Increased synaptic dopamine – dopamine reuptake transporter malfunction Dopamine reuptake transporter In addition to binding to the dopamine reuptake transporter and preventing reuptake from the synapse, metamfetamine also reverses the direction of dopamine transport causing the transporter to move dopamine from the cytoplasm into the synapse. The mechanism by which this occurs is unknown.16,64,65 Metamfetamine decreases the function of the dopamine reuptake transporter within 1 h of ingestion. Following the ingestion of a single dose of metamfetamine, dopamine transport normalizes within 24 h, but following the ingestion of multiple high doses of metamfetamine, dopamine transport only normalizes partially.16

Neuroadaptation of the D1 receptor D1 receptors play a major role in mediating behavioral responses to cocaine as well as permanent modification of neuronal circuits that are involved in reward-related learning and memory. Cocaine-induced stimulation of the D1 receptor induces immediate early genes that control the transcription factors (proteins that bind to regulatory regions) of other genes and act on intracellular messengers thereby changing cellular gene expression.5,35 Changing cellular gene expression may cause a reorganization of neuronal circuits, changes of neuronal activity and changes in cellular functions such as intracellular signaling and synaptic modification. The more times the altered circuitry is stimulated (by repeated cocaine exposure), the more permanent it becomes.36 The altered circuitry reflects a neuroadaptation in reward-related learning and memory processes in the mesocortocolimbic dopamine system.36–41

4 1 vMAT MAO 5 TYROSINE DOPA DAT

2

3 DAT

Metamfetamine and dopamine Like cocaine, metamfetamine induces euphoria by increasing synaptic dopamine. Metamfetamine is a lipophilic weak base and enters nerve terminals readily by diffusing across the plasma membrane. Once inside the terminal, metamfetamine causes the release of dopamine into the synapse and binds to the dopamine reuptake transporter to prevent reuptake. In contrast to cocaine, metamfetamine also increases cytoplasmic

Fig. 3. Mechanisms by which Meth increases synaptic and cytosolic dopamine (DA). 1. redistributes DA from synaptic vesicles to cytosol; 2. reverses transport of DA through plasma membrane transporters; 3. decreases DA transporters at cell surface; 4. inhibit MAO; 5. increases expression of tyrosine hydroxylase.57

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Neuroadaptation of the dopamine reuptake transporter Postmortem and PET studies have demonstrated that chronic metamfetamine use decreases dopamine reuptake transporter density in certain brain regions that have been associated with motor and cognitive impairment.66–68 However, dopamine reuptake transporter density may return to normal slowly during prolonged drug abstinence, implying that the initial decrease in transporter density is a neuroadaptive response to the increased synaptic dopamine. However, even if dopamine reuptake transporter density normalizes following abstinence, cognitive deficits may still persist.69,70 Neuroadaptation of D2 receptors Not surprisingly PET studies in human metamfetamine users reveal decreased D2 receptors that may represent downregulation from exposure to increased synaptic dopamine concentrations.71

the vesicle interior becomes more alkaline. The alkalinization causes the vesicle to collapse pushing dopamine into the cytoplasm.59–62 Decreased vesicular dopamine causes a decrease in dopamine released into the synapse following depolarization. However, the actions of metamfetamine on the dopamine reuptake transporter also play a role in the overall concentration of synaptic dopamine.16 Neurotoxicity In neuronal cell cultures, metamfetamine produces selective degeneration of dopamine neuron terminals without cell body loss. Acidotropic uptake of metamfetamine causes osmotic swelling and vacuoles. Hyperthermia and oxidative stress may be the initial event in metamfetamine neurotoxicity.72 Oxidative stress because of increased cytoplasmic dopamine

Increased cytoplasmic dopamine VMAT-2 transporter malfunction Metamfetamine causes a redistribution of the VMAT-2 dopamine transporter within the nerve terminal, which makes the transporter less available to the dopamine molecule, decreasing the ability to move cytoplasmic dopamine into the protective vesicle (see Fig. 4).65 In addition to decreasing the movement of cytoplasmic dopamine into the vesicle, metamfetamine pushes vesicular dopamine into the cytoplasm by two methods. Firstly, metamfetamine binds to VMAT-2, which results in vesicular dopamine efflux into the cytoplasm. Secondly, the weak base metamfetamine moves across the vesicle membrane in its uncharged form and accumulates in the acidic vesicle in its charged form (now less able to permeate the vesicle membrane). The acidic pH gradient in the vesicles provides the energy that allows vesicular metamfetamine accumulation against its concentration gradient. As the basic metamfetamine molecule continues to accumulate,

“Drug-free’’

“DA releasers’’ (eg., amphetamine analogs)

vesicles

DA

(eg., cocaine, methylphenidate)

DA

DA DA

“DA reuptake inhibitors’’

Metamfetamine collapses acidic organelles such as synaptic vesicles (as well as lysosomes and endosomes) moving dopamine into the cytoplasm. There, dopamine reacts with molecular oxygen to form reactive oxygen species (ROS) such as superoxide- and hydroxyl-free radicals and hydrogen peroxide. The process is called intracellular oxidative stress.17,58,73,74 Oxidative stress is an imbalance between biochemical processes leading to the production and removal of ROS, which damage all cellular biomacromolecules (lipids, sugars, proteins, polynucleotides) and can lead to secondary products that also cause damage. The CNS is vulnerable to the oxidative insult because of a high rate of oxygen utilization, poor concentration of antioxidants, high concentration of both polyunsaturated lipids (most susceptible to oxidation), and redox-active transition metals (capable of catalytic generation of ROS). Oxidative stress is necessary for the neurotoxic effects seen following metamfetamine administration in animals.75 Of interest, cocaine increases vesicular dopamine uptake (so cytoplasmic dopamine is not increased) and is not known to cause the formation of ROS. Agents that sequester dopamine within synaptic vesicles may be neuroprotective in neurodegenerative diseases.16 It is difficult to interpret the interaction of the neuroadaptive changes and the toxic changes. Even less is known about the clinical implications of either of these.

DA DA

DA DA DA

Unable to move Nonmembrane location of DA back into vesiclesvesicles, cytoplasmic DA low cytoplasmic DA forms ROS

Fig. 4. VMAT-2 distribution (follows vesicles).16

MDMA and serotonin Serotonin Serotonin synthesis and metabolism Serotonin is synthesized from the amino acid tryptophan in the CNS neurons and in enterochromaffin cells in the gastrointestinal tract. Tryptophan is transported actively into the

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Neuroadaptation

701 H COOH C C NH2 H H

L-TRYPTOPHAN N H O2 tetrahydropteridine HO L-5-HYDROXYTRYPTOPHAN

tryptophan hydroxylase

N H

vitamin B6 HO 5-HYDROXYStored in vessicles** TRYPTAMAINE (SEROTONIN, 5-HT)

** (Rate-limiting)

H COOH C C NH2 H H L-aromatic amino acid decarboxylase

N H

H H C C NH2 H H

5-HT N-acetylase

MAO

HO 5-HYDROXYINDOLE ACETALDEHYDE aldehyde dehydrogenase

HO N

NAD H O C C OH H

H (Major metabolite) ** 5-HYDROXYINDOLE ACETIC ACID (5-HIAA)

N N H

H O C C H H NADH

aldehyde reductase

HO N

H H C C OH H H

H 5-HYDROXYTRYPTOPHOL

**Biochemical markers unique to serotonergic neurons

Fig. 5. Serotonin synthesis and metabolism.

CNS where it is metabolized by tryptophan hydroxylase, the rate-limiting enzyme in the synthetic pathway. The major metabolite in this metabolic pathway is 5-hydroxyindoleacetic acid (HIAA). As tryptophan hydroxylase is not regulated by end-product inhibition nor saturated with substrate, the concentration of brain tryptophan hydroxylase influences the synthesis of serotonin. Once synthesized, serotonin is stored immediately in cytoplasmic vesicles to protect it from degradation76 (see Fig. 5).

neurotransmitter is terminated by a single serotonin reuptake transporter (SERT), which removes serotonin from the synaptic cleft. Serotonin that escapes neuronal reuptake and storage is inactivated by monoamine oxidase localized in postsynaptic elements and surrounding cells. In addition to being a neurotransmitter and released into discrete synapses, serotonin is also a neuromodulator as it is released from axonal varicosities into the extraneuronal space and diffuses to outlaying targets.76–78

Serotonergic pathways Of the numerous midline nuclei in the CNS, nine raphe nuclei contain paired neurons distributed along the entire length of the brain stem and are the primary source of serotonin. Consistent with the many actions attributed to serotonin, such as mood, affect, appetite, temperature regulation, and sexual activity, the axons extend to many areas of the brain.

The serotonin reuptake transporter The transporter regulates many aspects of serotonin homeostasis such as serotonin brain concentration, serotonin receptor function, and the firing rate of serotonergic neurons in the different raphe nuclei.77–83 MDMA is a CNS psychostimulant related structurally to metamfetamine. “Ecstasy” is the common street name for this illicit drug with mixed stimulant and mild hallucinogenic actions. It is known to cause feelings such as closeness to others and empathy. MDMA is a potent releaser and/or reuptake inhibitor of presynaptic serotonin, dopamine, and norepinephrine. The acute psychological effects of MDMA are

The serotonin synapse Depolarization causes the release of serotonin into the synapse, which stimulates the postsynaptic serotonin receptors that mediate the effects of serotonin. Action of this

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702 thought to be primarily because of the acute increase in synaptic serotonin.84 MDMA administration to animals causes a rapid release of serotonin into the synapse, prevents reuptake by binding to the serotonin transporter, and causes a decrease in the biochemical markers (serotonin, 5 HIAA, and tryptophan hydroxylase) unique to serotonergic neurons.85–87 Much has been written about MDMA-induced neurotoxicity in animals, such as axotomy (destruction of serotonin axons and axon terminals) and subsequent abnormal reinnervation.88 Little has been written about the neuoradaptations, which may be caused by MDMA, including irreversible binding to tryptophan hydroxylase, reversible binding to the serotonin transporter, and downregulation of postsynaptic serotonin receptors. A brief review of neurotoxicity is presented to clarify how it differs from neuroadaptation. The following evidence is from rodents and nonhuman primates, unless otherwise stated. Neurotoxicity Reductions in biochemical markers indicate axotomy MDMA causes a decrease in the three biochemical markers unique to serotonin neurons [serotonin, 5 HIAA (metabolite), and tryptophan hydroxylase (enzyme)]. Decreased reuptake of synaptic serotonin decreases the concentration of neuronal serotonin and subsequently HIAA. The irreversible inhibition of tryptophan hydroxylase (which lasts for days) causes longlasting reductions in the biochemical markers.89 It has been assumed that the loss of biochemical markers is because of axotomy, that is, the destruction of serotonin axons and axon terminals90,91 in the forebrain with sparing of serotonin cell bodies in the brain stem, as demonstrated by immunohistochemical findings.92,93 Immunohistochemical findings support axotomy Immunohistochemical stains demonstrating fragmented axons following MDMA exposure is presumed evidence that MDMA causes destruction of serotonin axons and axonal terminals.90–96 Loss of fine axons projecting from dorsal raphe nuclei with sparing of thick axons projecting from median raphe nuclei is also seen.97 These studies support the theory (initiated by loss of biochemical markers) that MDMA causes axotomy.98,99 The lack of damage to other neurotransmitter systems in the rat or primate suggests that MDMA is a selective serotonergic neurotoxin.88 Abnormal reinnervation following axotomy As MDMA does not affect the nerve cell bodies, there is potential for regeneration (sprouting) of serotonin axons following axotomy.100–102 Although regeneration occurs, the reinnervation patterns are highly abnormal, demonstrating either increased or decreased densities of axons compared with controls.90,93 The axonal sprouting patterns may be species dependent100,103,104 with altered reinnervation patterns

D. Seger occurring more frequently in primates than in rodents.88 Abnormal reinnervation patterns may be permanent as evidenced by the persistence of abnormal patterns 7 years following MDMA exposure in squirrel monkeys. Factors that appear to influence axonal recovery include the distance of the damaged terminal field from its nerve cell body of origin; the size and severity of the initial lesion; the proximity of the injured axons to myelinated fiber tracts;105 and species differences such as the length of serotonin axonal projections and degree of axon myelination.88 Conflicting studies regarding axotomy Other animal studies demonstrate no axonal destruction and suggest that serotonin and other biochemical markers may be sequestered and unavailable to the stain or test. Intact nerve terminals possessing less serotonin could produce misleading results if the stain could not stain neurons with lesser concentration of serotonin.106 In addition, other validated markers of neurotoxicity do not consistently demonstrate axotomy. For example, MDMA does not increase glial fibrillary acidic protein, a structural protein that detects neuronal degeneration in astroglia.106–109 The problem is that although this protein may be useful for detecting many forms of neuronal injury, its sensitivity for detecting small lesions of fine unmyelinated serotonin axons has not been determined.110 To add to the confusion, differences in dosage, route of administration, species, and methodology may be the source of the disparate conclusions. The confounder of hyperthermia Another confounder is that MDMA induces hyperthermia and hyperthermia enhances neurotoxicity.88,111–113 Hyperthermia is not dose-related114 and may be related to a deficiency of the enzyme CYP2D6 (see subsequent section on pharmacogenomics).106,115 Applicability of animal studies to humans Animal studies that assess whether MDMA induces axotomy and abnormal reinnervation are inconclusive. An additional question is whether repeated high doses of MDMA that induce neurotoxicity in rats and primates relate to typical human patterns of MDMA use. Neuroadaptations Downregulation of serotonin receptors In rats MDMA causes a downregulation of postsynaptic 5-HT2a receptors, similar to the downregulation of dopamine receptors caused by cocaine. Receptor density then increases after MDMA treatment is discontinued.116 In humans, single photon emission computerized tomography (SPECT) studies reveal decreased receptor density in recent MDMA users compared with controls, but receptor density increases to normal in ex-MDMA users.117 There appears to be a neuroadaptive compensatory upregulation

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Neuroadaptation of these receptors after depletion of synaptic serotonin118,119 or depletion of tryptophan.120 All of these receptor neuroadaptations attempt to maintain homeostasis. MDMA binds to tryptophan hydroxylase MDMA binds to tryptophan hydroxylase and irreversibly inhibits the enzyme. Although the onset of inhibition does not occur for several hours, the inhibition lasts for days until new enzyme is synthesized. Enzyme inactivation is increased by hyperthermia.89 Tryptophan hydroxylase activity is regulated by the serotonin reuptake transporter as subsequently described.79 MDMA binds to the serotonin reuptake transporter causing metabolic neuroadaptations Although MDMA also causes the release of other neurotransmitters (such as dopamine and norepinephrine) and binds to their respective reuptake transporters, MDMA has the highest affinity for SERT.85–87 When MDMA binds to the SERT, not only is reuptake of serotonin from the synapse prevented but the movement of serotonin into the synapse is increased. The result is supraphysiological concentrations of synaptic serotonin. As the serotonin transporter becomes unavailable and serotonin accumulates in the synapse, the serotonin presynaptic autoreceptors are downregulated and tryptophan hydroxylase activity increases in an attempt to increase the concentration of serotonin. Therefore changes in the density of SERT sites cause changes in serotonin metabolism.121 SERT knockout animals have markedly reduced concentrations of serotonin and 5-HIAA but normal amounts of tryptophan oxidase demonstrating the neuroadaptive response of tryptophan hydroxylase to a lower density of transporter sites.79 In humans, PET and SPECT scans, which use radioligands that label the transporter, have demonstrated decreases in the SERT concentration in MDMA users.122 A SPECT study revealed no difference in SERT concentration among past MDMA users and drug naive subjects.123 Other brain-imaging and post mortem findings also indicate that a decrease in SERT concentration may reverse following abstinence124–126 further suggesting that the decrease in transporter concentration is a short-term functional neuroadaptation rather than neurotoxicity. As data are conflicting, methodological questions such as reliability and validity in assessment of transporter availability must be raised.122,127 There may be functional inactivation of the transporter, not loss of the serotonin transporter. Tests using antiSERT antibodies in rats do not demonstrate depletion of the transporter.106 Regulation of serotonin transporter-binding sites may change following exposure to other drugs (such as cocaine and tobacco) independently of any changes in nerve terminals.128,129 Questions include whether estrogen, gender, and polymorphism affect the transporter130–133 or if preexisting conditions caused decreased number of CNS serotonin neurons. Unfortunately, most imaging studies did not confirm drug use with analytical testing.129 How these findings in the serotonergic system impact later psychiatric morbidity is unknown.

703 In summary, studies indicate that decreased serotonin reuptake transporter concentration causes a downregulation of the serotonin autoreceptors and increases tryptophan oxidase activity. These neuroadaptations attempt to regulate presynaptic homeostasis by regulating the amount of serotonin in the brain.79,134 The extent to which MDMA causes these neuroadaptations is unknown, but we do know that the concentration of SERT on the cell membrane can be changed rapidly by MDMA binding.121,135–137 Confounders in human neurotoxicity and neuroadaptation Human recreational MDMA users demonstrate poor verbal and visual memory,84 and poor executive cognitive functioning compared with controls.138–142 These deficits are consistent with altered functioning in the serotonergic system given the central role of serotonin in memory and executive cognitive functioning. Also, pre-drug use deficiencies in serotonergic functioning may predispose to drug use.143 MDMA users are notoriously polydrug abusers; cannabis is often a concurrent substance of abuse,143–145 and the history of drug use is often not confirmed analytically. How the neurotoxic effects seen in animals apply to humans is unknown. How the neuroadaptations demonstrated in humans impact cognitive effects is also unknown. Pharmacogenomics of MDMA Pharmacogenomics may play an important role in determining the individual response to MDMA. Polymorphisms (variants) in genes cause variable (interindividual) response to drugs. Polymorphism of receptors and cytochrome enzymes offer one explanation for the varying interindividual susceptibility to drug dependence and addiction.146 CYP2D6 catalyzes hydroxylation or demethylation of more than 20% of drugs and more than 80 distinct phenotypes (polymorphism). The phenotype (allelic variants) determines the rate of drug metabolism. A person with homozygous alleles containing inactivating mutations at CYP2D6 are poor drug metabolizers, those with one or two functional alleles exhibit intermediate or extensive metabolism, and those with duplicated genes experience ultrarapid metabolism.91,146 In humans, the main metabolic pathway of MDMA is mediated through the cytochrome CYP2D6 enzyme. Lack of functional activity of this enzyme occurs in approximately 10% of Caucasians who are classified as poor metabolizers. These individuals might be more susceptible to acute toxicity.147–149 CYP2D6 is also inhibited by MDMA. When a second dose of MDMA is administered 24 h after the first dose, the MDMA plasma concentration is 30% greater than it was following the first dose. The exposure of CYP2D6 to the first dose impairs disposition of the second dose and leads to accumulation of MDMA. Impaired elimination of MDMA may lead to higher and sustained concentrations of the parent

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704 drug and increased acute toxicity in individuals with this genetic disposition.150,151

Conclusions Cocaine, metamfetamine, and MDMA cause dysfunction of CNS intracytoplasmic and plasma membrane transporters that move neurotransmitters in and out of intracellular vesicles, neurons, and synapses. Supraphysiological concentrations of synaptic neurotransmitters is one result. Neuroadaptations occur to maintain homeostasis and may be transient or permanent. The length of time of use or the total amount of drug required to cause these neuroadaptations is unknown. These neuroadaptations help explain subsequent responses to acute and chronic drug use and addiction.

Acknowledgment Special thanks to Suzanne Brock for help with preparation of this manuscript.

Declaration of interest The author report no conflicts of interest. The author alone is responsible for the content and writing of this paper.

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Clinical Toxicology (2010) 48, 709–717 Copyright © Informa UK, Ltd. ISSN: 1556-3650 print / 1556-9519 online DOI: 10.3109/15563650.2010.505197

ARTICLE LCLT

Cobinamide is superior to other treatments in a mouse model of cyanide poisoning ADRIANO CHAN1,2, MAHESWARI BALASUBRAMANIAN2, WILLIAM BLACKLEDGE2, OTHMAN M. MOHAMMAD2, LUIS ALVAREZ1,2, GERRY R. BOSS2, and TIMOTHY D. BIGBY1,2 Cobinamide for cyanide poisoning

1 2

Medicine Service, VA San Diego Healthcare, San Diego, CA 92161 Department of Medicine, University of California, San Diego, CA 92093, USA

Context. Cyanide is a rapidly acting cellular poison, primarily targeting cytochrome c oxidase, and is a common occupational and residential toxin, mostly via smoke inhalation. Cyanide is also a potential weapon of mass destruction, with recent credible threats of attacks focusing the need for better treatments, as current cyanide antidotes are limited and impractical for rapid deployment in mass casualty settings. Objective. We have used mouse models of cyanide poisoning to compare the efficacy of cobinamide (Cbi), the precursor to cobalamin (vitamin B12), to currently approved cyanide antidotes. Cbi has extremely high affinity for cyanide and substantial solubility in water. Materials and Methods. We studied Cbi in both an inhaled and intraperitoneal model of cyanide poisoning in mice. Results. We found Cbi more effective than hydroxocobalamin, sodium thiosulfate, soldium nitrite, and the combination of sodium thiosulfate–sodium nitrite in treating cyanide poisoning. Compared to hydroxocobalamin, Cbi was 3 and 11 times more potent in the intraperitoneal and inhalation models, respectively. Cobinamide sulfite (Cbi-SO3) was rapidly absorbed after intramuscular injection, and mice recovered from a lethal dose of cyanide even when given at a time when they had been apneic for over 2 min. In range-finding studies, Cbi-SO3 at doses up to 2000 mg/kg exhibited no clinical toxicity. Discussion and Conclusion. These studies demonstrate that Cbi is a highly effective cyanide antidote in mouse models, and suggest it could be used in a mass casualty setting, because it can be given rapidly as an intramuscular injection when administered as Cbi-SO3. Based on these animal data Cbi-SO3 appears to be an antidote worthy of further testing as a therapy for mass casualties. Keywords

Antidote; Poisoning management; Poisoning; Hydroxocobalamin

Introduction Cyanide is an extremely potent and rapidly acting cellular poison. Cytochrome c oxidase appears to be its primary intracellular target, although cyanide binds to other metalloenzymes.1 Hydrogen cyanide (HCN) gas, the cyanide form present under physiological conditions, reacts with purified cytochrome c oxidase in two steps: 1) relatively rapid formation of an enzyme–HCN intermediate; and 2) slow conversion of the intermediate to a stable product, possibly an enzyme–cyanide ion complex that blocks mitochondrial electron transport.2,3 The lethal dose (LD)50 of potassium cyanide (KCN) for animals is in the range of 2–8 mg/kg, with as little as 50 mg fatal to humans.2

Received 27 March 2010; accepted 28 June 2010. Address correspondence to Timothy D. Bigby, Department of Medicine, University of California, Medicine Service, VA San Diego Healthcare, 3350 La Jolla Village Drive, San Diego, CA 92161, USA. E-mail: [email protected]

Cyanide appears to have been used as a weapon dating back to ancient Rome.4 Because it is easy and inexpensive to make, it is a potential weapon of mass destruction, either as HCN gas in an enclosed space or as potassium or sodium cyanide added to water or food supplies. It was used in the Nazi concentration camps during the Holocaust as Zyklon B, a stabilized form of cyanide. The Jonestown Massacre in 1978 is the most recent mass cyanide poisoning, and, in 2003, United States intelligence authorities learned of a credible al-Qaeda plot to use cyanide in the New York subway system.5 Current treatments for cyanide poisoning are hydroxocobalamin (OHC), sodium nitrite (NaNO2), and sodium thiosulfate (Na2S2O3), all of which must be given by intravenous injection. 6–8 We have shown that cobinamide (Cbi) is superior to OHC as a cyanide antidote in cultured cells, Drosophila melanogaster,9 and in a sublethal rabbit model.10 When combined with sodium sulfite, intramuscular Cbi rapidly and effectively reverses cyanide toxicity in the rabbit model.11 We now show that Cbi is superior to the current treatments for cyanide poisoning in two lethal mouse models, and is highly effective by intramuscular injection when used with sodium sulfite.

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Materials and methods Materials Male C57BL/6J mice, 6–12 weeks old, were from Jackson Laboratories (Bar Harbor, ME, USA), and were fed Teklad 7001 standard diet from Harlan Laboratories (Madison, WI, USA) ad libitum. All studies were performed according to NIH Guidelines for the Care and Use of Laboratory Animals, and approved by the Institutional Animal Care and Use Committee of the Veterans Administration San Diego Healthcare System. KCN (Fisher Scientific Inc., Waltham, MA, USA) was dissolved immediately before use in 0.1 M NaOH for the inhalation model, and in 10 mM Na2CO3 for the intraperitoneal injection model; the pKa of HCN is 9.3, and thus in these alkaline solutions cyanide is present as a nonvolatile salt. A 4.3 L gas chamber constructed of acrylic glass (Plexiglas®) was maintained at 30°C using a heated-air circulation system regulated by a feedback loop controller (Watlow, Winona, MN, USA) (please see full details and Fig. 1 in the supplement available at http://informahealthcare.com/doi/suppl/ 10.3109/15563650.2010.505197). Aquohydroxocobinamide (Cbi), referred to as cobinamide throughout the text, was prepared from OHC (Wockhardt LTD, Mumbai, India)

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Choice of the animal model and study conditions Mice were chosen for these studies because they are the smallest mammal in which the proposed work could be conducted. C57BL/6 mice were used because they are a wellcharacterized, in-bred mouse strain used in prior studies of cyanide toxicity. Cyanide treatment is classified as a USDA Pain and Distress Category E condition, and the IACUC of the VA San Diego and the investigators deemed the study acceptable only if the mice were anesthetized. The investigators realized this might have impacted the outcome, but concluded that without the use of anesthesia the proposed work was inhumane. Exposure of mice to inhaled cyanide

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under mild alkaline conditions using cerium hydroxide.12 The Cbi product was isolated on a weak cation-exchange column eluted with a NaCl gradient, and was desalted on a C18 reversed-phase column. The final product was concentrated on a rotary evaporator and by lyophilization. By high-performance liquid chromatography analysis, the Cbi used in these studies was >95% pure, with the major contaminant being OHC carried through unhydrolyzed. All other chemical reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA) and were of the highest purity available.

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Fig. 1. Comparison of available cyanide antidotes against inhaled cyanide. Maximal clinical doses of currently available cyanide antidotes were compared to cobinamide (0.2 mmol/kg; 200 mg/kg) in C57BL/6 mice exposed to 534, 801, and 908 ppm of cyanide gas for 30 min (LC100). These included OHC (0.2 mmol/kg; 200 mg/kg), sodium thiosulfate (2.6 mmol/kg), and sodium nitrite (1.3 mmol/kg) (n = 5–6 per condition). No OHC or sodium nitrate animals survived at the lowest dose of cyanide gas. All cobinamide-treated animals survived, whereas 80%, 60%, and none of sodium thiosulfate-treated animals survived when treated with 534, 801, and 908 ppm, respectively. The combination of sodium thiosulfate and sodium nitrite was fully effective except when 908 ppm cyanide was administered, in which only 20% of animals survived (n = 5). The data were plotted and log transformed for nonlinear regression analysis. We determined LC50 inhaled cyanide of 803 and 901 ppm for sodium thiosulfate and the combination of sodium thiosulfate/sodium nitrite, respectively. For cobinamide, the LC50 was significantly in excess of 908 ppm. (Cbi = cobinamide; Na2S2O3 = sodium thiosulfate; NaNO2 = sodium nitrite; OHC = hydroxocobalamin.).

Mice were anesthetized with isoflurane (Baxter Healthcare Co., Deerfield, IL, USA) in the airtight gas chamber described above using an amount of liquid isoflurane calculated to deliver 2% when fully evaporated. This led to a surgical plane of anesthesia within 5 min, which was maintained throughout the experiment. Once the mice were anesthetized, HCN gas was generated in the chamber by injecting 100 mM KCN into a glass beaker containing 10 mL of 1 M sulfuric acid. Mice were exposed to the gas for 30 min, and were observed for the onset of respiratory arrest. The HCN concentration in the chamber was stable over the duration of exposure, when measured in gas samples using previously described methods.9,13 Mice were given cyanide antidotes or saline solution by intraperitoneal injection 15 min prior to being placed into the cyanide gas chamber. They were observed for survival during the 30 min interval of exposure and for the following 3 days. In all cases, at least five animals were studied per condition. Intraperitoneal injection of mice with cyanide Mice were anesthetized with 3% isoflurane in an induction chamber, and maintained at 2% isoflurane using a nose cone; core temperature was kept at 36.5°C using a temperaturecontrolled warming table. The mice were then administered antidotes or saline solution intravenously via lateral tail vein in a volume of 100 μL. Immediately following antidote administration, 20 mM KCN was injected into the peritoneal

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Cobinamide for cyanide poisoning cavity in 200 μL. The antidotes and cyanide were given via different routes to avoid possible direct interaction prior to systemic delivery to the animal. Animals were observed for 1 h for the onset of death, defined as apnea without further respiratory effort or movement, or palpable cardiac pulsation. In all circumstances, at least five animals were studied per condition. Measurement of red blood cell cyanide concentration Cyanide in blood is bound almost exclusively to methemoglobin in red blood cells (RBCs); thus, blood cyanide can be measured by separating RBCs from plasma, and acidifying the RBCs to release cyanide as HCN gas.14 Heparinized whole blood was collected by intracardiac puncture at the time of sacrifice. It was centrifuged and the pelleted RBCs were lysed in ice-cold water. The lysates were placed into glass tubes sealed with stoppers holding plastic center wells (Kontes Glass Co., Vineland, NJ, USA) containing 0.1 M NaOH. A volume of 10% trichloroacetic acid equal to the lysate was added through the septum of the stopper, and the tubes were shaken at 37°C for 60 min. After cooling to room temperature, cyanide trapped in the NaOH was measured in a spectrophotometric assay following its reaction with p-nitrobenzaldehyde and o-dinitrobenzene at 560 nm.13,15 Concentrations were determined from standard curves using freshly prepared KCN dissolved in 0.1 M NaOH. Measurement of mouse plasma and urinary thiocyanate concentrations Plasma was obtained as described above, and urine was collected after sacrifice by intravesical puncture. Samples were placed into the stoppered tubes containing plastic center wells as described above, and thiocyanate was oxidized to cyanide at 37°C using acidified potassium permanganate.16 Ethanol was injected through the stopper after 3–5 min to quench the reaction. The resultant HCN gas trapped in the NaOH in the center wells was measured as described above for measuring RBC cyanide.

711 Measurement of plasma Cbi concentration Blood samples were heparinized and plasma was separated by centrifugation. Cbi in the plasma was converted to dicyanocobinamide by adding KCN to a final concentration of 5 mM. Protein in the plasma was denatured by heating the samples to 80°C for 15 min in a chemical fume hood, followed by adding an equal volume of acetonitrile. The samples were vortexed for 5 min, and centrifuged at 8, 900 × g forg 15 min at 4°C. The supernatants were dried by rotary vacuum, re-constituted in 0.2 mL water, and clarified through a 0.20 μm filter. The samples were analyzed on a high-performance liquid chromatography system using a C18 reversed-phase column eluted with a gradient from 20 mM potassium phosphate, pH 4.6 containing 0.16 mM KCN (solvent A) to 60% methanol/water (solvent B; 1 min to 40% B, 11 min to 50% B, and 1 min to 100% B; flow rate 1 mL/min). Dicyanocobinamide eluted at 16 min, and was detected by spectral absorption at 366 nm; it was quantified by comparison to authentic dicyanocobinamide (Sigma-Aldrich) standards over a 60-fold concentration range. Data analysis Survival curves were analyzed by the log-rank (Mantel–Cox) test. Dose–response curves were analyzed by log transformation of the dose followed by nonlinear regression analysis with reporting of the LD50 or effective dose (ED)50 and the 95% confidence interval (CI). Studies measuring cyanide or thiocyanate concentrations were analyzed by repeated measures analysis of variance with a Bonferroni post hoc test for multiple comparisons. These data are reported as the mean ± standard error of the mean. Simple means (two samples) were analyzed using an unpaired Student’s t-test. All analyses were performed using Prism software, version 5 (GraphPad Software, San Diego, CA, USA). Differences were considered significant when p £ 0.05.

Results Determination of lethal and sublethal doses of inhaled and injected cyanide

Intramuscular injection of Cbi We administered 0.2 mmol/kg (200 mg/kg) of Cbi or cobinamide sulfite (Cbi-SO3; Cbi mixed with equimolar sodium sulfite) in 50 μL into the gastrocnemius muscle of mice. To examine the kinetics of Cbi, these animals were rapidly sacrificed at 2.5, 5, 10, 60, and 360 min after the intramuscular injection. To examine the efficacy of these preparations when administered intramuscularly, the intramuscular injection was preceded 3 min by an intraperitoneal injection of 0.16 mmol/kg of KCN (a lethal dose) and the animals were observed for death as an end point. In all cases, at least five animals were studied per condition.

To establish the lethal concentration (LC)50 and LC100 of inhaled cyanide gas during a 30 min exposure or the LD50 and LD100 of intraperitoneal administration of KCN, the upand-down procedure for acute toxicity testing was used.17 In response to 534 ppm HCN (LC100) mice would become apneic and die within 30 min. The LC50 was found to be 451 ppm (95% CI, 424–480; n = 5) (Supplemental Fig. 2), which is higher than previously reported in the literature.2,18 However, these earlier studies were performed in a different mouse strain, and without general anesthesia. General anesthesia appears to decrease toxicity by preventing the hyperventilation that occurs in awake animals in response to

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Fig. 2. Dose–response to cobinamide and OHC in the inhaled model. Mice were injected with vehicle, cobinamide (Cbi), or OHC 15 min before being placed in an exposure chamber and exposed to cyanide gas at 534 ppm for 30 min. Data were plotted in dose– survival curves and the dose log transformed for nonlinear regression analysis. The ED50 regression analysis was 0.029 (29 mg/kg) (95% CI, 0.025–0.033) and 0.301 mmol/kg (416 mg/kg) for cobinamide and OHC, respectively (n = 5 per condition).

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Cyanide distribution and metabolism To study cyanide distribution and clearance, mice were exposed to 260 ppm HCN gas for 30 min, and then allowed to recover. RBC cyanide, and plasma and urine thiocyanate were measured prior to cyanide exposure, immediately after exposure, and at 2 and 6 h post exposure (Supplemental Fig. 4). The RBC cyanide concentration peaked immediately after exposure and then decayed over the ensuing 6 h. The urine thiocyanate concentration increased as the RBC cyanide decreased. No change was observed in the plasma thiocyanate concentration, which remained low throughout the study, indicating that thiocyanate was freely excreted in the urine. Efficacy of antidotes in inhaled model of cyanide poisoning Cyanide antidotes available in the United States were compared to Cbi at the LC100 of inhaled cyanide. OHC, Na2S2O3, and NaNO2 were used at doses of 0.2, 2.6, and 1.3 mmol/kg, respectively.2,4 These doses are the maximal recommended clinical doses when calculated on a milligrams per kilogram basis, and exceed the recommended human dose when calculated on an mg/m2 basis.4 Cbi was used at the same molar dose as OHC. No animals treated with OHC or Na2NO2 survived the lowest concentration of cyanide gas, 534 ppm. When used at these doses, only Cbi

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inhaled cyanide.19 Intraperitoneal injection of KCN at 0.16 mmol/kg induced apnea and death within 5–9 min. The observed LD50 was 0.144 mmol/kg (10 mg/kg) (95% CI, 0.090–0.232; n = 5) (Supplemental Fig. 3), which is similar to prior studies.2,20

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Fig. 3. Measurement of RBC cyanide, and plasma and urine thiocyanate. Mice were injected in the peritoneal cavity with 0.2 mmol/kg of OHC, cobinamide (Cbi), or buffer. Fifteen minutes later they were anesthetized in the exposure chamber and exposed to 260 ppm of cyanide gas for 30 min, and then sacrificed immediately (time zero), or at 2 or 6 h after removal from the chamber. Mice not exposed to cyanide were sacrificed at the time other animals were placed in the chamber (shown as control). Red blood cell cyanide (panel A), and plasma (panel B) and urine thiocyanate (panel C) were measured as described in Methods section (n = 3 per condition).

and Na2S2O3 resulted in survival (Fig. 1) (p < 0.0001). To further compare the efficacy of these two agents, the cyanide concentration was increased to 801 ppm; all Cbitreated animals survived, whereas only 60% of Na2S2O3treated animals survived (p = ns). However, at this cyanide concentration, the combination of Na2S2O3 and NaNO2, a recommended clinical treatment, was fully effective. To

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Fig. 4. Dose–response to cobinamide and OHC in the intraperitoneal model. Mice were given varying doses of cobinamide (Cbi) or OHC intravenously immediately before receiving an intraperitoneal injection of 0.24 mmol/kg of KCN. Data were plotted as dose–response curves and the ED50 calculated by log transformation of the dose and nonlinear regression analysis of the data. The calculated ED50 was 0.054 (95% CI, 0.041–0.072) (54 mg/kg) and 0.175 mmol/kg (95% CI, 0.162 ± 0.189) (242 mg/kg) for cobinamide and OHC, respectively (n = 5 for each condition).

compare Cbi to the combination of Na2S2O3 and NaNO2, animals were challenged with a cyanide dose of 908 ppm. All Cbi-treated animals survived, whereas only 20% of the Na2S2O3 and NaNO2-treated animals survived (p < 0.015). Plotting survival against the inhaled cyanide concentration increased the LC50 for cyanide to 803, 901, and significantly greater than 908 ppm for Na2S2O3, combination of Na2S2O3 and NaNO2, and Cbi, respectively (Fig. 1) when the data were log transformed and analyzed by nonlinear regression. Thus, Cbi was superior to these established treatments for cyanide poisoning. To more accurately compare the efficacy of Cbi to OHC, we administered a range of doses of the two compounds. A dose as low as 0.1 mmol/kg (100 mg/kg) of Cbi was fully effective, with 100% survival (Fig. 2) (p < 0.0002). OHC was significantly less effective with a dose 0.4 mmol/kg (553 mg/kg) required to obtain 100% survival (Fig. 2) (p < 0.03). The dose that produced 50% survival (ED50) was 0.029 (29 mg/kg) (95% CI of 0.025– 0.033) and 0.301 mmol/kg (416 mg/kg) for Cbi and OHC, respectively. Therefore, Cbi was about 10-fold more potent than OHC in this inhaled model of cyanide poisoning. To compare biochemical evidence of efficacy of Cbi and OHC, we measured RBC cyanide, as well as plasma and urine thiocyanate levels immediately, and 2 and 6 h after a sub-LD (260 ppm) of inhaled cyanide. We found that RBC cyanide increased 50-fold immediately in response to inhaled cyanide (24.9 ± 6.2 vs. 0.5 ± 0.03 μg/g protein; HCN alone vs. control), but did not differ significantly from controls in Cbi-treated animals at any time (3.0 ± 0.4 vs. 0.5 ± 0.03 μg/g protein; Cbi vs. control) (Fig. 3A). Plasma thiocyanate was reduced significantly at

2 h in Cbi-treated animals (15.9 ± 3.3 vs. 23.5 ± 5.0 μg/g protein; Cbi vs. HCN alone), whereas it was not reduced significantly in OHC-treated animals compared to those treated with HCN alone (26.8 ± 1.0 vs. 23.5 ± 5.0 μg/g protein; OHC vs. HCN alone) (Fig. 3B). Urine thiocyanate slowly increased in all HCN-treated animals, but in both Cbi- and OHC-treated animals it never rose to levels observed in those treated with HCN alone (477.7 ± 93.8 vs. 151.0 ± 24.4 vs. 237.8 ± 41.1 μg/g: HCN alone vs. OHC vs. Cbi) (Fig. 3C). Thus, the increased potency of Cbi compared to OHC as a cyanide antidote was reflected in cyanide and thiocyanate blood concentrations. Comparison of Cbi and OHC in an intraperitoneal model of cyanide poisoning We next compared Cbi and OHC in a parenteral cyanide model. Mice were given either Cbi or OHC intravenously at various doses immediately before they were given an intraperitoneal injection of 0.24 mmol/kg (16 mg/kg) of KCN (about 6 μmol per mouse). Survival was the observed endpoint (Fig. 4). With Cbi, 100% survival was observed at 0.16 mmol/kg (160 mg/kg), whereas 0.32 mmol/kg (442 mg/kg) of OHC was required for 100% survival. The data were plotted as a dose–response curve, and the dose log transformed. The data were then subjected to nonlinear regression analysis. The calculated ED50 was 0.054 (95% CI, 0.041–0.072) (54 mg/kg) and 0.175 mmol/kg (95% CI, 0.162 ± 0.189) (242 mg/kg) for Cbi and OHC, respectively (Fig. 4). Thus, Cbi was again more potent than OHC, and the difference in relative potencies of the two compounds between the inhaled and injection models is considered in the Discussion. Intramuscular injection of Cbi Cyanide is an extremely rapid metabolic poison, and cyanide-poisoned victims may be unconscious, hypotensive, and die rapidly if untreated. Therefore, intramuscular injection may be the most expeditious, viable route, especially in a setting of mass casualties. We found that Cbi was absorbed slowly, and we noticed that some of the animals injected with Cbi developed paresis of the injected limb. Cbi reacts with nitric oxide (NO)21,22 and we hypothesized that Cbi was inducing localized ischemia by consuming NO, thereby retarding its own absorption. To prevent this possible sequence of events, we added sodium sulfite to Cbi. Sulfite binds to cobalamin with a reasonably high affinity.23 We showed that Cbi-SO3 was absorbed more effectively than Cbi (Fig. 5A) with peak absorption occurring at 5 and 60 min, respectively. Cbi-SO3 had a half-life of 32.3 min (95% CI, 27.2–39.6; n = 5) using a one-phase decay model. Cbi measurements did not adequately fit this model because of its slow absorption phase, but the half-life was estimated to be greater than 6 h. We compared these

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Fig. 5. Intramuscular cobinamide. (A) Kinetics of intramuscular absorption of cobinamide. Cobinamide in a volume of 50 μL was injected intramuscularly into the gastrocnemius muscle. Solutions included cobinamide and cobinamide sulfite. Animals were sacrificed quickly at noted times, and blood was obtained by intracardiac puncture. Cobinamide in plasma was measured as described in Methods section by high-performance liquid chromatography. Cobinamide sulfite had a half-life of 32.3 min (95% CI, 27.2–39.6; n = 5) using a one-phase decay model. Cobinamide measurements did not adequately fit this model because of its slow absorption phase, but the half-life was estimated to be greater than 6 h (n = 3). (B) Effectiveness of cobinamide intramuscular preparations. Cobinamide or cobinamide sulfite (0.2 mmol/kg; 200 mg/kg) was injected intramuscularly 3 min after mice received a lethal intraperitoneal injection of KCN (0.16 mmol/ kg). A Kaplan–Meier survival curve is shown (n = 5 for each condition).

Range-finding toxicity studies

two preparations in their ability to prevent death in the intraperitoneal KCN injection model (Fig. 5B). In both cases, 0.2 mmol/kg (200 mg/kg) of Cbi was administered intramuscularly. Only Cbi-SO3 resulted in 100% survival.

We performed general range-finding toxicity studies of Cbi in mice, assessing clinical parameters and survival. We administered Cbi at increasing amounts by intraperitoneal injection, and the animals were observed for up to 7 days for adverse effects. Animals injected with 0.2 mmol/kg (200 mg/kg) tolerated this dose without observable abnormalities. However, 0.4 mmol/kg (400 mg/kg) reduced spontaneous activity, which was followed by respiratory distress, hunched posture, piloerection, and ultimately death by 36 h (Fig. 7). Log transformation of the dose and linear regression analysis revealed an LD50 of 0.32 mmol/kg (320 mg/kg). We postulated that this adverse effect could be from Cbi binding endogenous NO, and, therefore, tested Cbi-SO3. At doses up to 2.0 mmol/ kg (2000 mg/kg), Cbi-SO3 induced no clinical signs of toxicity with animals surviving for at least 7 days (Fig. 7; only doses up to 0.8 mmol/kg (800 mg/kg) are shown in the figure for clarity). We did not try doses higher than 2000 mg/kg because the United States Food and Drug Administration considers 2000 mg/kg an appropriate limit dose in rodent toxicity studies.24

Efficacy of Cbi sulfite post poisoning

Discussion

Pre-poisoning treatment models are not always useful in predicting efficacy of antidotes. Therefore, we further examined the effectiveness of Cbi-SO3 relative to the timing of KCN poisoning. Mice were given intramuscular Cbi-SO3, either before or up to 4 min after a LD of intraperitoneal KCN (Fig. 6). The Cbi-SO3 was 100% effective up to 3 min post cyanide

In the United States, three cyanide antidotes are available: nitrites (e.g., NaNO2 and amyl nitrite), Na2S2O3, and OHC (vitamin B12a).25 Nitrites generate met(ferric)hemoglobin, which has a high affinity for cyanide, but can no longer bind oxygen; thus, nitrites can exacerbate the carbon mon-

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Fig. 7. Range-finding toxicity studies of cobinamide and cobinamide sulfite. General range-finding toxicity studies of intraperitoneal cobinamide (Cbi) or cobinamide sulfite (Cbi-SO3) were performed. Animals were observed for up to 7 days. Data were plotted as dose–response curves and the LD50 by log transformation of the dose and nonlinear regression analysis. The LD50 for cobinamide was 0.32 mmol/kg (320 mg/kg). Although only doses up to 0.8 mmol/kg (800 mg/kg) are shown, no mortality or adverse effect was observed with any dose of cobinamide sulfite up to and including a dose of 2 mmol/kg (2000 mg/kg) (n = 5 for each dose).

oxide-induced reduction in oxygen-carrying capacity in smoke-inhalation victims. Moreover, nitrites can induce vasodilatation, causing hypotension.2 Na2S2O3 acts as a sulfur donor for the enzyme rhodanese, which detoxifies cyanide by converting it to thiocyanate, but rhodanese is limited both in cellular amount and tissue distribution. OHC binds cyanide with a relatively high affinity (KA ∼1012 M−1),26 but 5–10 g are required for cyanide poisoning. We have shown previously that Cbi is effective as a cyanide scavenger in cultured cells,9 a fly model,27 and nitroprusside-induced cyanide toxicity in mice.28 We now show that Cbi is effective in two lethal mouse models of cyanide poisoning, and demonstrate it is superior, in our models, to currently available treatments. Although Cbi was absorbed poorly, Cbi-SO3 was rapidly absorbed from an intramuscular site, and protected mice from cyanide-induced death, even when administered after cyanide. Evans previously showed that Cbi neutralizes cyanide in mice and rabbits, but he administered it by intravenous injection and did not strictly compare it to other cyanide antidotes.29 With the exception of amyl nitrite, currently approved drugs for cyanide poisoning are available only as intravenous preparations, limiting their usefulness in a mass casualty setting. The time required to start intravenous lines and administer relatively large fluid volumes would be prohibitively long in treating many cyanide-poisoned persons in the field. Therefore, an intramuscular preparation that is rapidly absorbed would be highly desirable. To develop a model of cyanide inhalation, we needed to construct a suitable exposure chamber. Cyanide gas (HCN) is not commercially available, and, therefore, a flowthrough exposure system with accurately controlled cyanide

concentrations is not feasible. Requirements of a sealed chamber are that gases must equilibrate rapidly, and the chamber must be maintained above the boiling point of HCN (26°C). We found that our chamber generated reproducible, stable concentrations of cyanide with a sustained level of anesthetic gas throughout the exposure period (at least 30 min). The lethal LC50 we observed for cyanide (451 ppm for 30 min) was higher than that previously reported in mice.18,30 Three factors may contribute to this difference: 1) mouse strains vary in their sensitivity to cyanide and C57BL/6 mice are relatively resistant; 2) previous reports used measured concentrations of cyanide gas that tend to underestimate the concentration of cyanide because of gas loss or condensation at room temperature; and 3) the earlier studies were performed in awake mice that likely hyperventilated on initial cyanide exposure,18,30 whereas our studies were performed with anesthetized mice. Cbi is the penultimate compound in cobalamin biosynthesis, lacking the dimethylbenzimidazole nucleotide tail coordinated to the cobalt atom in the lower axial position.21 Whereas cobalamin has only a free upper ligand binding site, Cbi has free upper and lower binding sites; moreover, the dimethylbenzimidazole group has a negative trans effect on the upper binding site, thereby reducing cobalamin’s affinity for ligands.22 The net effect is that Cbi binds two cyanide ions, and has a greater affinity for cyanide than OHC, with a KA overall of ∼1022 M−1.26 In addition, Cbi is at least five times more water-soluble than OHC. These three chemical differences translate into smaller volumes of administration of Cbi than OHC, and we calculate that 5 mL of a 200 mM Cbi solution should neutralize one human LD50 of cyanide (5 mL can be given intramuscularly in the gluteal region). We found that Cbi is considerably more effective than OHC, and that the difference is more pronounced in the inhaled than the parenteral model of cyanide poisoning. Thus, Cbi is 11 times more effective in the inhaled model and 3 times more effective in the parenteral model. Although there are several possible explanations for these differences in efficacy, the most plausible are the kinetics of the two models. Because of the time needed to absorb cyanide gas into the circulation, the inhaled model leads to a slower onset of toxicity, whereas, in the intraperitoneal model, cyanide is absorbed rapidly and distributes into the vascular system at a rate approaching that of intravenous injection. Other small molecules administered to mice by intraperitoneal injection are rapidly absorbed.31 A difference between two compounds is, therefore, more likely to be seen in the inhaled model where these compounds have a longer time to act. We should note that the inhaled model is more representative of real-life circumstances, in which people are likely to be exposed to cyanide gas. We have shown previously that Cbi has high affinity for nitric oxide (NO).21,22 Binding NO in vivo may lead to systemic hypertension as occurs with intravenous OHC,32 and to localized vasoconstriction when given by intramuscular injection. Initial studies with intramuscular Cbi supported this impression, because post-mortem examination of

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716 injected animals demonstrated significant quantities of residual Cbi at the injection site (TDB, unpublished observations). We found that Cbi-SO3 does not bind NO in vitro, and that it is rapidly absorbed and highly effective. Moreover, it exhibited no clinical toxicity, even at a dose of 2000 mg/kg (2.0 mmol/kg). Although the precise LD50 for cyanide is not known in humans, lethal poisoning has occurred with as little as 50 mg (∼0.5–1 mg/kg). Mice are more resistant to the effects of cyanide than humans with the LD50 for KCN reported to be between 2 and 8 mg/kg, depending on the strain and mode of cyanide exposure.20,33 We found that C57BL/6 mice are particularly resistant, with an LD50 for cyanide of 9.75 mg/kg (0.15 mmol/kg). Thus, the amounts of Cbi required to rescue humans from a lethal cyanide dose is likely to be considerably less than that required in this study. The current study has several limitations. First, anesthetized animals do not approximate real-life exposures. However, we felt that studies in awake animals would be inhumane, and current animal care guidelines strongly discourage and limit the use of awake animals in studies of toxins such as cyanide.34,35 Anesthetics might bias our data by inducing hypotension, which could increase the susceptibility of the animal to the cardio-depressant effects of cyanide. Alternatively, in the inhaled model, anesthetics could protect animals through decreased minute ventilation or by preventing hyperventilation in response to cyanide gas.19 Second, in the parenteral model, the onset of death was very rapid, leaving only a narrow window for intervention. Third, the studies were not conducted in a randomized fashion. However, all animals were the same in-bred strain from the same supplier, and the observed effects were highly reproducible. And, fourth, the studies were not blinded because of the complexity of doing this, and the nature of the antidotes (Cbi and OHC are both intensely colored).

Conclusion Cyanide, in practical terms, cannot be regulated. It is used in countless industrial applications, is cheap to make, and is abundant. Hundreds of thousands tons of cyanide are manufactured each year in the United States.4 The National Institutes of Health and the Department of Defense have both emphasized the need for new, more-effective, and less-toxic treatments for cyanide poisoning that can be deployed rapidly in a mass casualty setting.4,36 We conclude that Cbi may be an agent that satisfies these requirements.

Declaration of interest This work was supported in part by the Department of Veterans Affairs (TDB) and the National Institutes of Health Counter ACT U01 NS058030 (GRB, TDB).

A. Chan et al.

References 1. Scheffler IE. Mitochondria make a come back. Adv Drug Deliv Rev 2001; 49(1–2):3–26. 2. Salkowski AA, Penney DG. Cyanide poisoning in animals and humans: a review. Vet Hum Toxicol 1994; 36(5):455–466. 3. Way JL. Cyanide intoxication and its mechanism of antagonism. Annu Rev Pharmacol Toxicol 1984; 24:451–481. 4. Baskin SI, Brewer TG. Chapter 10: Cyanide poisoning. In: Zajtchuk R, Bellamy RF, eds. Medical Aspects of Chemical and Biological Warfare. Washington, DC: Office of The Surgeon General, Borden Institute, Walter Reed Army Medical Center; 1997. 5. Suskind R. The One Percent Doctrine: Deep Inside America’s Pursuit of Its Enemies Since 9/11. New York, NY: Simon & Schuster; 2006. 6. Alcorta R. Smoke inhalation & acute cyanide poisoning. Hydrogen cyanide poisoning proves increasingly common in smoke-inhalation victims. Jems 2004; 29(8):Suppl 6–15; quiz Suppl 6–7. 7. Alarie Y. Toxicity of fire smoke. Crit Rev Toxicol 2002; 32(4):259–289. 8. Silverman SH, Purdue GF, Hunt JL, Bost RO. Cyanide toxicity in burned patients. J Trauma 1988;28(2): 171–176. 9. Broderick KE, Potluri P, Zhuang S, Scheffler IE, Sharma VS, Pilz RB, Boss GR. Cyanide detoxification by the cobalamin precursor cobinamide. Exp Biol Med (Maywood) 2006; 231(5):641–649. 10. Brenner M, Mahon SB, Lee J, Kim J, Mukai D, Goodman S, Kreuter KA, Ahdout R, Mohammad O, Sharma VS, Blackledge W, Boss GR. Comparison of cobinamide to hydroxocobalamin in reversing cyanide physiologic effects in rabbits using diffuse optical spectroscopy monitoring. J Biomed Opt 2010; 15(1):017001. 11. Brenner M, Kim JG, Mahon SB, Lee J, Kreuter KA, Blackledge W, Mukai D, Patterson S, Mohammad O, Sharma VS, Boss GR. Intramuscular cobinamide sulfite in a Rabbit model of sublethal cyanide toxicity. Ann Emerg Med 2009; 55: 352–363. 12. Renz P. Some intermediates in the biosynthesis of vitamin B12. Method Enzymol 1971; 18(Part 3):82–92. 13. Guilbault GG, Kramer DN. Ultra sensitive, specific method for cyanide using p-nitrobenzaldehyde and o-dinitrobenzene. Anal Chem 1966; 38(7):834–836. 14. Lundquist P, Rosling H, Sorbo B. Determination of cyanide in whole blood, erythrocytes, and plasma. Clin Chem 1985; 31(4):591–595. 15. Gewitz HS, Pistorius EK, Voss H, Vennesland B. Cyanide formation in preparations from Chlorella rulgaris Beijerinck: effect of sonication and amygdalin addition. Planta (Berl) 1976; 131:145–148. 16. Boxer GE, Rickards JC. Determination of thiocyanate in body fluids. Arch Biochem Biophys 1952; 39:292–300. 17. Bruce RD. An up-and-down procedure for acute toxicity testing. Fundam Appl Toxicol 1985; 5(1):151–157. 18. Esposito FM, Alarie Y. Inhalation toxicity of carbon monoxide and hydrogen cyanide gases released during the thermal decomposition of polymers. J Fire Sci 1988; 6:195–242. 19. Doi M, Ikeda K. Postanesthetic respiratory depression in humans: a comparison of sevoflurane, isoflurane and halothane. J Anesth 1987; 1(2):137–142. 20. Moore SJ, Ho IK, Hume AS. Severe hypoxia produced by concomitant intoxication with sublethal doses of carbon monoxide and cyanide. Toxicol Appl Pharmacol 1991; 109(3):412–420. 21. Broderick KE, Singh V, Zhuang S, Kambo A, Chen JC, Sharma VS, Pilz RB, Boss GR. Nitric oxide scavenging by the cobalamin precursor cobinamide. J Biol Chem 2005; 280(10):8678–8685. 22. Sharma VS, Pilz RB, Boss GR, Magde D. Reactions of nitric oxide with vitamin B12 and its precursor, cobinamide. Biochemistry 2003; 42(29):8900–8908. 23. Dolphin D. B12. New York, NY: Wiley-Interscience; 1981. 24. Guidance on Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals. M3 (R2). 2008; 15 July 2008. http://www.fda.gov/downloads/Regulatory Information/Guidances/ucm129524.pdf. Accessed 26 March 2010.

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Cobinamide for cyanide poisoning 25. Gracia R, Shepherd G. Cyanide poisoning and its treatment. Pharmacotherapy 2004; 24(10):1358–1365. 26. Hayward GC, Hill HA, Pratt JM, Vanston NJ, Williams RJ. The chemistry of vitamin B12. IV. The thermodynamic trans-effect. J Chem Soc [Perkin 1] 1965; September:6485–6493. 27. Broderick KE, Chan A, Balasubramanian M, Feala J, Reed SL, Panda M, VS, Pilz RB, Bigby TD, Boss GR. Cyanide produced by human isolates of Pseudomonas aeruginosa contributes to lethality in Drosophila melanogaster. J Infect Dis 2008; 197(3):457–464. 28. Broderick KE, Balasubramanian M, Chan A, Potluri P, Feala J, Belke DD, McCulloch A, Sharma VS, Pilz RB, Bigby TD, Boss GR. The cobalamin precursor cobinamide detoxifies nitroprusside-generated cyanide. Exp Biol Med (Maywood) 2007; 232(6):789–798. 29. Evans CL. Cobalt compounds as antidotes for hydrocyanic acid. Br J Pharmacol Chemother 1964; 23:455–475. 30. Matijak-Schaper M, Alarie Y. Toxicity of carbon monoxide, hydrogen cyanide and low oxygen. J Combust Toxicol 1982; 9:21.

717 31. Gentry RT, Rappaport MS, Dole VP. Serial determination of plasma ethanol concentrations in mice. Physiol Behav 1983; 31(4):529–532. 32. Borron SW, Baud FJ, Barriot P, Imbert M, Bismuth C. Prospective study of hydroxocobalamin for acute cyanide poisoning in smoke inhalation. Ann Emerg Med 2007; 49(6):794–801, e1–2. 33. Norris JC, Moore SJ, Hume AS. Synergistic lethality induced by the combination of carbon monoxide and cyanide. Toxicology 1986; 40(2):121–129. 34. OPRR/ARENA IACUC Guidebook. 2002. 26 March 2009. http:// grants.nih.gov/grants/olaw/olaw.htm. Accessed 26 March 2010. 35. United States Public Health Service Policy on Humane Care and Use of Laboratory Animals, 2002. . http://grants.nih.gov/grants/olaw/references/ phspol.htm. Accessed 26 March 2010. 36. NIH CounterACT Program: Countermeasures Against Chemical Threats. 2009. http://www.ninds.nih.gov/research/counterterrorism/ counterACT_home.htm. Accessed 26 March 2010.

Supplemental methods and data for LCLT

Cobinamide is superior to other treatments in a mouse model of cyanide poisoning Methods Inhaled Cyanide Exposure Chamber. A custom 4.3 L gas exposure chamber was constructed to maintain a constant concentration of cyanide gas under a controlled temperature and rapid circulation system. It was composed of Plexiglas® and sealed airtight by an O-ring under the lid held in place by six screw fittings (Supplemental Figure 1; Panel A is a photograph of the chamber, and Panel B is a schematic). Section A of the chamber is the animal compartment that holds four mice comfortably. It is separated from Section B by a fine plastic grate. Near the rear of the box along the grate is a circulating fan powered by a power supply in Section C. The fan has a metal Peltier device heated by power delivered from Section C. A thermistor in Section B monitors temperature continuously, adjusting power delivery to the Peltier device

(A)

providing fine temperature control to 30 ± 0.5oC. Section C is a sealed separate container that houses all electronics. Two ports in the lid over Section B are for injecting liquid isoflurane and KCN solution into a glass beaker containing 1M H2SO4. A port on the far right side of Section A is for sampling chamber gas. Cyanide Gas Inhalation Model. Mice (C57BL/6) were placed in the cyanide exposure chamber described in Figure 1. They were anesthetized over 5 min with isoflurane at a steady-state concentration of 2%. Doses of KCN in 0.1 M NaOH, pH 12 were added to 10 ml of 1 M H2SO4 to generate HCN gas, and the mice were kept in the chamber for 30 min. The parts per million (ppm) concentration was calculated and confirmed by direct measurement. The latter was performed by drawing 24 ml of gas from the chamber into a gas tight syringe containing 1 ml of 0.1 M NaOH, and measuring the cyanide concentration as described in the Methods of the primary manuscript.

KCN Intraperitoneal Injection Model. Mice were anesthetized with 2% isoflurane on a temperature controlled warming table. When fully anesthetized, they were injected intraperitoneally with various volumes of 20 mM KCN in 10 mM Na2CO3, pH 9.5. The animals were observed for onset of apnea and death. Anesthetic Injection Port

C KCN Gas Generator

Fan

Electronics

(B)

Animal Exposure Chamber

B Separation Grate

Sample Port

A

Measurement of RBC Cyanide, and Plasma and Urine Thiocyanate. Mice were exposed for 30 min to 260 ppm of inhaled cyanide in the exposure chamber. They were sacrificed immediately after removal from the chamber (time zero), or at 2 or 6 h after removal from the chamber. Mice not exposed to cyanide were sacrificed at the time other animals were placed in the chamber (shown as Control). Red blood cell cyanide, and plasma and urine thiocyanate were measured as described in Methods of the primary manuscript.

Cyanide Injection Port

Results Supplemental Figure 1. Inhaled Cyanide Exposure Chamber. A) Photograph of the Plexiglas® chamber. B) Schematic of the chamber.

Cyanide Gas Inhalation Model. Mice exposed for 30 min to 313 ppm of cyanide all survived (Supplemental Figure 2A).

100

75

75 Survival (%)

Survival (%)

100

50 25

50

25

0 0

100 200 300 400 500 Inhaled Cyanide Gas (ppm)

0 0.00

600

Supplemental Figure 2. Cyanide Gas Inhalation Model. Mice (C57BL/6) were placed in the cyanide exposure chamber, anesthetized with isoflurane, and exposed to increasing ppm of HCN gas. The dose was log transformed and a non-linear regression analysis was performed (n = 6 per condition).

Mortality increased with increasing ppm of HCN, with 534 ppm being 100% lethal (LC100). These data were replotted in concentration vs. survival plot and the LC50 estimated by non-linear regression analysis to be 451 ppm (Supplemental Figure 2B).

0.05

0.10 KCN (mmol/kg)

0.15

Supplemental Figure 3. KCN Intraperitoneal Injection Model. Mice were anesthetized with 2% isoflurane and then injected intraperitoneally with various volumes of 20 mM KCN in 10 mM Na2CO3, pH 9.5. Apnea consistently occurred within 50 sec in all animals. The data were plotted as shown. A log transformation of dose, followed by non-linear regression analysis was performed to measure LD50 (n = 6 per condition).

RBC-CN

250

Serum-SCN

400

Urine-SCN

200

300

150 100

100 50 0

0 Control

Cyanide and Thiocyanate Measurements. Measurement of red blood cell cyanide in mice exposed to 260 ppm (a sublethal dose) demonstrated that the cyanide concentration peaked immediately after exposure and quickly decayed thereafter (Supplemental Figure 4). Plasma thiocyanate did not change significantly during the time course. However, urine thiocyanate increased up to 6 h post-exposure.

500

Thiocyanate (µg/ml)

KCN Intraperitoneal Injection Model. Mice injected with 0.10 mmol/kg of KCN all survived (Supplemental Figure 3). Mortality increased with increasing doses of KCN with 100% morality (LD100) observed at a concentration of 0.16 mmol/kg. These data were plotted demonstrating a very steep dose-response (Supplemental Figure 3). The observed LD50 was 0.144 mmol/kg (10 mg/kg)(95% CI of 0.090 to 0.232; n = 5).

Cyanide (µg/gm RBC protein)

600 300

0.0 2.0 Time (h)

6.0

Supplemental Figure 4. Measurement of RBC Cyanide and Plasma and Urine Thiocyanate. Mice exposed for 30 min to 260 ppm of inhaled cyanide were sacrificed immediately (time zero), or at 2 or 6 h after exposure. Mice not exposed to cyanide were sacrificed at the time other animals were placed in the chamber (shown as Control). Red blood cell cyanide, and plasma and urine thiocyanate were measured as described in Methods (n = 3 per condition).

Clinical Toxicology (2010) 48, 718–724 Copyright © Informa UK, Ltd. ISSN: 1556-3650 print / 1556-9519 online DOI: 10.3109/15563650.2010.488640

ARTICLE LCLT

Predicting acute complicated glyphosate intoxication in the emergency department JEONG MI MOON and BYEONG JO CHUN Glyphosate herbicide intoxication

Department of Emergency Medicine, Chonnam National University Hospital, Gwangju, Republic of Korea

Background. Glyphosate herbicide intoxication results in a range of mortality and morbidity, depending on patients’ factors. Predicting which patient will need intensive medical treatment might help reduce mortality by providing prompt treatment, as well as triage those patients not likely to develop complications. Thus, we sought to identify independent factors that could predict which patient will develop subsequent medical complications. Methods. Seventy-six patients presenting with acute glyphosate herbicide ingestion at Chonnam National University Hospital were enrolled in this retrospective study. To identify the predictive factors for complications, objective variables easily assessed at presentation including previously reported predictive factors for mortality, such as age, vital signs, X-ray abnormalities, and laboratory findings, were analyzed by univariate and multivariate stepwise logistic regression analyses. Results. Of the 76 patients, 32 (42.1%) had medical complications and 2 (2.6%) died. Metabolic acidosis was the most common medical complication. Whereas metabolic acidosis, respiratory failure, hypotension, acute kidney injury, hyperkalemia, and seizures developed within 24 h, acute pancreatitis occurred a few days after the ingestion. The univariate analysis showed that an advanced age, amount ingested >100 mL, X-ray abnormalities, elevated amylase, alanine aminotransferase (ALT), and blood nitrogen urea were significant factors. However, the multivariate analysis showed that advanced age, elevated ALT, and X-ray abnormalities were independent factors associated with serious complications and the need for intensive medical treatment. Conclusions. The results of this study showed that age > 50 years, X-ray abnormalities, and ALT > 40 U/L were significant predictive factors for complications in patients with glyphosate surfactant herbicide poisoning; patients with these findings might require admission to the intensive care unit. Keywords Poisoning; Glyphosate; Herbicide; Complication

Introduction Glyphosate, marketed as a combination of polyoxyethylene amine surfactant and glyphosate as an isopropylammonium salt, is now the most common post-emergent nonselective herbicide used in agriculture in the United States and worldwide.1,2 Glyphosate is absorbed into the plant in a process facilitated by the surfactant, and the glyphosate targets the synthesis of chlorophyll-related molecules by competitive inhibition of the enzyme enolpyruvylshikimate phosphate synthase, present in plants but not in animals.3 This herbicide has been sold under many names: Roundup, Zero Weed Killer, and others since 1974.4,5 In Korea, glyphosate surfactant herbicides have been marketed under several names with 16.2–41% concentrations of glyphosate, and polyoxyethylene amine as a surfactant. Because of its easy access, glyphosate herbicide exposure is not uncommon. The American Association of Poison Control Centers reported that glyphosate

Received 27 October 2009; accepted 22 April 2010. Address correspondence to Byeong Jo Chun, Department of Emergency Medicine, Chonnam National University Hospital, Gwangju, Republic of Korea. E-mail: [email protected]

herbicide was the most common herbicide with human exposure in the United States, accounting for 4,268 cases in 2008.6 However, the reported mortality of glyphosate ingestion varies by country. In the United States, only 2 among the 4,268 patients developed major toxicity and 5 patients died. Only 1.0% intentionally ingested glyphosate.6 Mortality has been reported to be from 8.0 to 29.3% in Taiwan and 16.1% in Japan.7–11 A recent Taiwan study enrolling 74.6% of patients with suicidal ingestion of glyphosate herbicide reported serious medical complications in 4.9% of patients and death in 7.2% of patients.12 This wide variation in mortality and morbidity may be explained by patient characteristics such as age, or the intent of the exposure. Independent factors that are readily available in the emergency department (ED) and that can predict which patients with glyphosate herbicide ingestion are at risk for critical complications might reduce both the duration of hospitalization and mortality by rapid intensive treatment. In addition, such factors can help triage patients not likely to develop medical complications. Several previous studies have identified clinical and laboratory parameters that could serve as independent predictive factors associated with mortality, including age > 40 years, amount ingested, hypotensive shock, heart rate > 100/min,

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Glyphosate herbicide intoxication pulmonary infiltration, acidosis, potassium > 5.5 mmol/L, suicide attempt, and creatinine > 1.4 mg/dL (123.76 μmol/ L).7,8,10,11 However, these studies are limited by the outcome endpoint used, which was only mortality. In addition, some had low statistical power. Three studies reported that the predictors associated with the severity of glyphosate herbicide poisoning include an older age, larger amount ingested, suicidal attempt, receipt of atropine therapy, a longer elapsed time to presentation, and esophageal and laryngeal injury.12–14 However, some parameters may not be immediately available in the ED, such as the evaluation for the presence of esophageal injury, or may not be exact, such as elapsed time, and some variables suggested to be important have not had the cutoff levels defined. No prior study has been conducted to identify objective variables that can be easily assessed in the ED and used to predict which patients with glyphosate ingestion are at risk for the development of serious subsequent medical complications including death. Therefore, we sought to 1) describe the clinical features of patients with acute glyphosate poisoning and 2) identify independent factors that could be used to predict which patients will develop subsequent serious medical complications requiring intensive medical therapy.

Methods This study was a retrospective cohort study conducted at Chonnam National University Hospital, an academic tertiary care center in the Republic of Korea. The annual ED census is about 30,000 patients. This study was approved by our hospital institutional review board. We used the hospital electronic medical record system to obtain the medical record of patients who were candidates for this study. The patient selection criteria included patients presenting to the ED with glyphosate herbicide intoxication between January 1998 and January 2009 and who were older than 18 years of age at presentation. A total of 129 patients were identified. However, 28 patients who ingested glyphosate with another drug were excluded; in addition, 10 patients with missing data, 7 patients with clinical manifestations of a cholinesterase inhibitor intoxication (including decreased RBC cholinesterase activity on serial testing in 5 patients), 5 patients poisoned with glyphosate through inhalation or other nonoral routes, and 3 patients who died on arrival were excluded from the analysis. Thus, 76 patients were enrolled. Past medical history included hypertension in three, diabetes mellitus in six, and ischemic heart disease in one patient. The medical records were reviewed and the following data were collected: 1) variables that were readily available at presentation in the ED: age, gender, vital signs, laboratory test results, mental status, electrocardiogram (ECG), and the interpretation of chest X-ray by a radiologist; 2) variables related to the ingestion event: the amount of glyphosate

719 ingested, co-ingestion with alcohol, intent of the exposure, and elapsed time from ingestion to arrival at the ED; and 3) variables related to treatment and outcome: use of gastric lavage or activated charcoal, medical complications, length of hospitalization, and cause of death. The estimated amount of ingestion was defined as follows: a spoon as 5 mL, a mouthful as 25 mL, a cup as 100 mL, and a bottle as 300 mL. The corrected QT interval (QTc) in the ECGs was calculated using Bazett’s formula (QTc = QT/√RR).15 Prolongation of the QTc interval was defined as a QTc interval greater than 440 ms.15 Medical complications were defined as follows: acute kidney injury, respiratory failure, hypotension (the need for pressor support to maintain blood pressure after admission), metabolic acidosis (pH < 7.35 and HCO3− < 20 mmol/L), hyperkalemia (potassium > 6.0 mEq/L), acute pancreatitis [lipase (reference range 7–60 U/L) > 180 U/L and C-reactive protein (reference range 0.2–0.5 mg/dL) > 15 mg/dL accompanied by abdominal pain that was not explained by other causes], hepatic damage [alanine aminotransferase (ALT) > 400 U/L and bilirubin > 4 mg/dL], seizures, or cardiac arrest. Acute kidney injury was defined as an absolute increase in the serum creatinine (Cr) of ≥0.3 mg/dL (26.52 μmol/L) or a percentage increase in the serum Cr ≥ 50% from baseline.16 Respiratory failure was defined as an arterial oxygen pressure less than 60 mmHg while breathing room air or the need for mechanical ventilation.17 In addition, any other conditions encountered during the review of the patient medical records that were deemed to be serious and clinically significant by the investigator were rated as medical complications.

Data analysis Baseline data and outcomes were summarized by frequency tabulation for categorical variables or medians for continuous variables with the exception of age, amount of ingestion, and duration of hospitalization, which are expressed as means. Patients were divided into two groups according to the development of complications. Patients with a medical complication during hospitalization were assigned to the complicated group. To identify the predictive factors associated with a high risk for complications, objective variables easily assessed at presentation were evaluated, including previously suggested predictors of mortality, such as age > 50 (years), heart rate > 100/min, amount of ingested > 100 mL, intentional ingestion, X-ray abnormalities, and laboratory results, by univariate analysis. However, the variables that were used to define complications such as initial hypotension, low pH, and HCO3−, and elevated Cr and potassium were excluded because patients with these deteriorations at presentation were placed in the complicated group. The results of the laboratory tests were categorized based on values for the upper limit of normal at our hospital. Univariate analysis of the association between each covariate and outcome was performed. Covariates with p-values less than 0.05 were considered

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J.M. Moon and B.J. Chun

sufficient for the inclusion of the variable in the multivariate stepwise logistic regression analysis, with the significance level set at p > 0.05. Estimated odds ratios and confidence intervals were calculated for all significant variables. All statistical analyses were performed using the Statistical Package for the Social Sciences version 15.0.

Results Patient characteristics We enrolled 76 patients and 32 (42.1%) among them showed at least one complication during hospitalization. The gender ratio did not differ between the two groups (complicated 71.0% vs. noncomplicated 68.9%, p = 0.729). The initial mean systolic blood pressure of the patients was 120.9 ± 31.6 mmHg and the mean heart rate at presentation was 81.5 ± 19.7/min. Pneumonic infiltration in 7 patients and pulmonary edema in 10 patients was present on the initial chest X-ray (Table 1). Fifty-eight (76.3%) out of 76 patients initially had Table 1. The characteristics of patients with glyphosate herbicide intoxication Variables

Total (n = 76)

Age (years) Male Exposure Elapsed timea (min) Intentional ingestion Co-ingestion with alcohol Amount ingested (mL) Clinical manifestation Altered mental state Sore throat Nausea/vomiting Abdominal pain Diarrhea Chest pain X-ray abnormalities ECG abnormalities at presentation QTc prolongation Sinus tachycardia First-degree AV block ST-T abnormality Sinus bradycardia Wide QRS tachycardia Treatment Gastric lavage within 2 h Amount of gastric lavage (L) Administration of charcoal Hemodialysis Outcome Duration of hospitalization (h) Death

55.1 ± 16.3 53 (69.7%)

a

180 (20–720) 62 (81.6%) 15 (22.1%) 161.5 ± 125.7 25 (32.9%) 25 (32.9%) 16 (21.1%) 15 (19.7%) 3 (3.9%) 2 (2.6%) 17 (22.4%) 30/58 (51.7%) 8/58 (13.8%) 6/58 (10.3%) 6/58 (10.3%) 3/58 (5.2%) 1/58 (1.7%) 34 (48.6%) 10 (1–17) 15 (19.7%) 3 (3.9%) 152.8 ± 214.7 2 (2.6%)

Time interval from ingestion to arrival at the hospital.

an ECG on admission. The most common abnormality on the ECG was QTc interval prolongation, followed by sinus tachycardia. These abnormal findings did not require any specific treatment and disappeared prior to discharge. The elapsed time from ingestion to arrival to the hospital varied from 20 to 720 min and was similar in the two groups (complicated 260.7 ± 337.8 min vs. noncomplicated 247.8 ± 211.1 min, p = 0.729). Gastrointestinal decontamination of gastric lavage and administration of charcoal were similar in the two groups. Hemodialysis was used in three patients who were discharged without any sequelae. The indications for hemodialysis were metabolic acidosis in two patients and hyperkalemia with acute renal failure in one patient.

Outcome The duration of hospitalization after ingestion was significantly longer in the complicated group (complicated 232.9 ± 250.2 h vs. noncomplicated 91.8 ± 161.0 h, p = 0.008) (Table 1). The most frequent complication of glyphosate herbicide ingestion was metabolic acidosis (36.8%), followed by respiratory failure (27.6%) (Table 2). Most of the complications developed within 24 h after ingestion, whereas acute pancreatitis developed a few days after glyphosate herbicide ingestion. Out of 32 patients with complications, 17 (53.1%) had at least two complications. In 27 out of 28 patients with metabolic acidosis, the acidosis resolved with fluid administration, sodium bicarbonate replacement, or hemodialysis. However, metabolic acidosis was refractory to fluid and sodium bicarbonate administration in one patient. Out of 28 patients with metabolic acidosis, 22 (78.6%) demonstrated anion gap higher than 16 mmol/L. In contrast, the presence of urinary ketones was assessed in 25 out of 28 patients and only 6 (24.0%) patients were found to have urinary ketones. Three patients with hypoxia (PaO2 < 60 mmHg at room air) responded to oxygen administration via oxygen mask. Five patients required intubation because of an altered mental state in addition to hypoxia. Thirteen (61.9%) of 21 patients with respiratory failure needed mechanical ventilation. Extubation in 18 patients was performed between 1 and 34 days after ingestion. Acute kidney injury was detected between 1 and 21 h after ingestion in nine patients with acute kidney injury; the highest median level of Cr was 3.2 (range 1.5–8.2) mg/dL [282.9 (range 132.6–724.9) μmol/L] at 21 h (median) after ingestion. Serum Cr normalized after fluid administration in seven patients and hemodialysis in one patient. The median potassium increased to 7.1 (6.5–7.4) mEq/L in four patients and returned to normal at 10 h (median) after ingestion with the administration of insulin/glucose and calcium gluconate, or hemodialysis. All of the patients with hyperkalemia had a metabolic acidosis. Acute kidney injury was present in three out of four patients with hyperkalemia.

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Glyphosate herbicide intoxication

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Table 2. Complications of patients with glyphosate herbicide intoxication Total

Onset timea (h)

Lasting durationb (h)

Progression to refractory one (%)

28 (36.8%) 21 (27.6%) 14 (18.4%) 9 (11.8%) 9 (11.8%) 4 (5.3%) 4 (5.3%) 1 (1.3%)

3.0 (1.0–25.5) 6.0 (1.0–54.8) 2.6 (1.0–25.9) 197.4 (169.3–225.5) 2.8 (1.0–21.8) 12.1 (2.0–210.0) 1.2 (1.0–21.8) 19.1

11.0 (5.8–104.5) 72.0 (1.3–830.0) 46.5 (4.5–52.7) 294.4 (191.3–397.5) 41.5 (2.6–169.3)

1 (3.6%) 1 (4.8%) 1 (7.1%) 0 (0%) 1 (11.1%) 2 (50.0%) 0 (0%) 0 (0%)

Complication Metabolic acidosis Respiratory failure Hypotension Acute pancreatitis Acute kidney injury Cardiac arrest Hyperkalemia Seizure a

39.0 (2.6–169.3)

Period from arrival at hospital to appearance of complications (median and range). Period from appearance to resolution of complications (median and range).

b

Nine patients (11.8%) had acute pancreatitis, and at 8 (7–9) days after glyphosate herbicide ingestion the peak median level of lipase was 624 (range 190–1,758) U/L. It returned to below 100 U/L at 12 (8–16) days with nothing per oral and parenteral nutrition support after the ingestion without associated complications. One patient showed a generalized tonic clonic seizure that was controlled by intravenous benzodiazepine 19 h after ingestion. Cardiac arrest developed within 24 h after ingestion in three cases and 210 h after ingestion in one case during hospitalization, and two patients out of the four patients were successfully resuscitated. One patient died because of refractory hypotension and metabolic acidosis 22 h after ingestion, despite the administration of vasopressor and sodium bicarbonate. The other patient died 10 days after ingestion because of acute respiratory distress syndrome combined with acute kidney injury. All surviving patients with complications were discharged without chronic sequelae.

Risk stratification The patients in the complicated group were significantly older than patients in noncomplicated group (complicated 61.1 ± 14.3 years vs. noncomplicated 51.0 ± 16.5 years, p = 0.007). The complicated group ingested significantly larger amounts of

glyphosate herbicide (complicated 197.6 ± 120.4 mL vs. noncomplicated 136.0 ± 124.5 mL, p = 0.042). Univariate analysis revealed significant differences in age > 50 years, amount ingested > 100 mL, X-ray abnormalities, and elevated ALT, blood urea nitrogen, and amylase between the two groups (Table 3). In the multivariate analysis, age > 50 years (54.5% of specificity), X-ray abnormalities (88.6% of specificity), and ALT > 40 U/L (95.5% of specificity) were independent factors for the prediction of serious medical complications that required intensive treatment (Table 4).

Discussion The purpose of this study was to identify the early predictive factors of patients at risk for critical complications after glyphosate herbicide ingestion. The fatality rate of patients with acute glyphosate herbicide ingestion was 2.6%. Compared to another study that included high ratio of intentional ingestion, the fatality rate was low. This may be explained by the fact that we excluded three patients who died on arrival to the hospital after glyphosate herbicide ingestion. If we included these patients, the fatality rate would be 6.3%, which is similar to the reports of Chen et al.12 The successful

Table 3. Univariate analysis of the outcome of patients with glyphosate herbicide ingestion

Age > 50 (years) Intentional ingestion Amount ingested > 100 (mL) Tachycardia (HR > 100/min) X-ray abnormalities BUN > 20 (mg/dL) Sodium > 145 (mEq/L) ALT > 40 (U/L) Glucose > 120 (mg/dL) Amylase > 100 (U/L)

Total (n = 76)

Noncomplicated group (n = 44)

Complicated group (n = 32)

p-Value

44 (57.9%) 62 (81.6%) 49 (64.5%) 7 (9.2%) 17 (22.4%) 16 (21.1%) 6 (7.9%) 9 (11.8%) 42 (55.3%) 30 (39.5%)

20 (45.5%) 35 (79.5%) 24 (54.5%) 2 (4.5%) 5 (11.4%) 5 (11.4%) 3 (6.8%) 2 (4.5%) 25 (56.8%) 13 (29.5%)

24 (75.0%) 27 (84.4%) 25 (78.1%) 5 (15.6%) 12 (37.5%) 11 (34.4%) 3 (9.4%) 7 (21.9%) 17 (53.1%) 17 (53.1%)

0.010 0.841 0.034 0.099 0.007 0.015 0.683 0.021 0.749 0.038

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J.M. Moon and B.J. Chun Table 4. Independent predictors identified by multivariate analysis Variables Age > 50 (years) X-ray abnormalities ALT > 40 (U/L)

b

Odds ratios

p-Value

95% confidence intervals

−1.322 −1.281 −2.365

0.267 0.278 0.094

0.027 0.049 0.012

0.083–0.861 0.078–0.994 0.015–0.595

use of hemodialysis to treat metabolic acidosis or hyperkalemia in three patients who survived also contributed to decreased mortality rate, in contrast to other reports.7 The complications of glyphosate herbicide intoxication include cardiovascular instability, respiratory distress, metabolic acidosis, and hepatorenal dysfunction. Some have found metabolic acidosis as the most common complication, and others have reported that shock and respiratory failure are common.7,8,12 This discrepancy may be explained by different definitions of metabolic acidosis. Several studies have reported metabolic acidosis in glyphosate herbicide intoxication; however, the underlying cause of the metabolic acidosis has not been clearly defined.4,7,8 In our study, 78% of patients with metabolic acidosis had a high anion gap. Serum lactate was evaluated in 16 patients and it significantly correlated with pH, with a Spearman’s rank correlation coefficient of −0.589 (p = 0.016). Systemic factors, including hypotension, hypoxia, cardiac failure, administration of catecholamine, and hepatic damage, can contribute to the development of lactic acidosis in glyphosate herbicide intoxication. Out of eight patients with acute kidney injury, seven had metabolic acidosis. A high anion gap metabolic acidosis might be attributed to acute renal injury and/or lactate acidosis. Further study is needed to determine the cause of the high anion gap metabolic acidosis to understand the pathophysiology of glyphosate herbicide toxicity for improved treatment and better patient outcome. The surfactant in glyphosate herbicide has been suggested to contribute to hypotension through myocardial depression.18 In this study, hypotension might not be due to hypovolemia because of the finding of normal central venous pressure and normal hemoglobin. In addition, we found that glyphosate induced several ECG abnormalities such as sinus tachycardia, and first degree atrioventricular block. This is the first report to show a link between the severity of glyphosate herbicide ingestion and QTc prolongation. The QTc interval was more prolonged in the complicated group at admission (complicated 470.8 ± 48.9 ms vs. noncomplicated 438.0 ± 37.3 ms, p = 0.010). Glyphosate itself or the surfactant might affect the repolarization of the conduction system in cardiac ventricles by direct or secondary effects, because the QTc prolongation was observed on admission and resolved at discharge without specific treatment. Acute renal tubular necrosis was found on postmortem examination in suicidal glyphosate herbicide ingestion.11 Tissue concentration in the kidneys was higher than that in brain, liver, and blood.19 In this study, six patients with acute kidney injury simultaneously had hypotension and seven

patients had respiratory failure, as well as acute kidney injury. Hypotension or hypoxia may cause or contribute to the development of acute kidney injury in addition to the direct toxicity of the glyphosate herbicide. Patients with acute pancreatitis had at least one of other complications that developed earlier such as hypotension or respiratory failure. The development of acute pancreatitis may be attributed to tissue hypoperfusion, or hypoxia induced by early developed complications. In contrast to the other complications induced by glyphosate herbicide ingestion, acute pancreatitis has been described in only one prior case report.20 However, pancreatitis induced by organophosphate or carbamate ingestion has been more commonly reported.21,22 The acute pancreatitis induced by organophosphate intoxication is thought to be due to acetylcholine release from the pancreatic nerves and prolonged hyperstimulation of pancreatic acinar cells.23 This is the first study to investigate the development of acute pancreatitis in patients with glyphosate herbicide ingestion. Pancreatitis might have been overlooked previously, considering the high frequency of 11.8% in this study. When patients report abdominal pain 24 h after glyphosate herbicide ingestion, physicians should consider the possibility of pancreatitis. The advanced age, X-ray abnormalities, and elevated ALT were selected in the predictive model to identify patients at risk for developing serious complications, including death. In the elderly, a higher risk of serious complication could be explained by a poorer physiological condition, altered toxicokinetics and toxicodynamics, as well as the presence of additional morbidities and a higher risk for aspiration.14 X-ray abnormalities indicate pulmonary toxicity, such as pulmonary edema or aspiration pneumonia, one of the suggested causes of mortality or morbidity in prior reports and confirmed by the findings of this study.7,14 The value of ALT corresponding to the upper 95% of the normal population was 37.5 U/L.24 Elevation of ALT (ALT > 40 U/L) at presentation may be indicative of underlying liver abnormalities caused by other diseases such as hepatitis B or alcohol. In particular, chronic alcohol consumption induces functional and morphological changes in the mitochondria, oxidative stress, and impairment of the antioxidant defense of the liver.25 Patients with these underlying liver diseases may be more vulnerable to drugs that cause mitochondrial dysfunction and oxidative injury, such as glyphosate herbicide.26,27 Because mitochondria are essential for the regulation of intracellular aerobic energy production, the reduced energy as well as oxidative stress may contribute to complications.28 In

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Glyphosate herbicide intoxication addition, elevated ALT might serve as a warning sign of subclinical hypoperfusion, or hypoxia at presentation. Four out of seven patients with elevated ALT, in the complicated group, had hypotension or respiratory failure from 3 to 13.5 h after ingestion in this study. We suggest that the initial serum ALT, advanced age, and pulmonary infiltration could be used to identify patients with glyphosate herbicide ingestion who appear well at admission but develop subsequent complications. This may maximize the chances of identifying patients who are most likely to benefit from rapid admission and intensive treatment.

723 the presence of medical complications at presentation such as hypotension and metabolic acidosis may be useful for identifying patients with glyphosate poisoning at risk for complications. This finding may improve the opportunity for identifying patients most likely to benefit from more rapid triage and increased levels of care.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.

Limitations The limitations of this study include the following. First, this was a retrospective study. As a result, the elapsed time from ingestion to arrival to the ED may be under- or overestimated. However, this variable was not included in the univariate analysis. Second, we could not completely exclude a selection bias because we excluded seven patients with missing laboratory data. This might be explained by the absence of significant symptoms following accidental small ingestion of glyphosate herbicide. All of these cases were discharged within 48 h after presentation without sequelae. The selection bias caused by the exclusion of these patients was likely low. Third, there might be a referral bias. The number of patients with mild or no symptoms after glyphosate herbicide ingestion was small; that was most likely because the study was conducted at a tertiary care hospital where patients with severe symptoms tended to be transferred. Fourth, all patients had a history of exposure to glyphosate, but the diagnosis was not confirmed by laboratory testing. Gas chromatography can be used to detect serum glyphosate, but this testing is not usually available in the ED.29 Instead, only patients that brought to the hospital the bottle from which they drank with the word “glyphosate” on the package label were included in this study. Fifth, the severity of acute pancreatitis was not determined by radiologic study. A serum lipase more than 3 times the normal limit was significantly correlated with severe pancreatic morphological changes.30 A C-reactive protein more than 15 mg/dL was a sensitive predictor of the progression of severity from moderate to severe.31 Sixth, we did not completely exclude the effects of gastric lavage on the respiratory complications that developed after admission. However, a patient with an altered mental status received gastric lavage or drainage in state with intubation; this would lower the risk for gastric lavage causing the respiratory complications. Seventh, the significance of the intentional ingestion might have been undervalued because of the potential denial of coverage by healthcare insurance.

Conclusion The results of this study suggest that advanced age, elevation of ALT, and pulmonary infiltration at presentation as well as

References 1. Jauhiainen A, Rasanen K, Sarantila R, Nuutinen J, Kangas J. Occupational exposure of forest workers to glyphosate during brush saw work. Am Ind Hyg Assoc J 1991; 52:61–64. 2. Lavy T, Cowell J, Steinmetz J, Massey J. Conifer seedling nursery worker exposure to glyphosate. Arch Environ Contam Toxicol 1992; 22:6–13. 3. Williams GM, Kroes R, Munro IC. Safety evaluation and risk assessment of the herbicide roundup and its active ingredient, glyphosate, for humans. Regul Toxicol Pharmacol 2000; 31:117–165. 4. Stella J, Ryan M. Glyphosate herbicide formulation: a potentially lethal ingestion. Emerg Med Australas 2004; 16:235–239. 5. Wu JJ, Chang SS, Tseng CP, Deng JF, Lee CC. Parenteral glyphosate surfactant herbicide intoxication. Am J Emerg Med 2006; 24:504–506. 6. Bronstein AC, Spyker DA, Cantilena LR Jr., Green JL, Rumack BH, Giffin SL. 2008 Annual report of the American Association of Poison Control Centers’ national poison data system: 26th annual report. Clin Toxicol 2009; 47:911–1084. 7. Lee HL, Chen KW, Chi CH, Huang JJ, Tsai LM. Clinical presentations and prognostic factors of a glyphosate surfactant herbicide intoxication: a review of 131 cases. Acad Emerg Med 2000; 7:906–910. 8. Lee CH, Shih CP, Hsu KH, Hung DZ, Lin CC. The early prognostic factors of glyphosate surfactant intoxication. Am J Emerg Med 2008; 26:275–281. 9. Sawada Y, Nagai Y, Ueyma M, Yamamoto I. Probable toxicity of surface active agent in commercial herbicide containing glyphosate. Lancet 1988; 1:299. 10. Talbot AR, Shiaw MH, Huang JS, Yang SF, Goo TS, Wang SH, Chen CL, Sanford TR. Acute poisoning with a glyphosate surfactant herbicide: a review of 93 cases. Hum Exp Toxicol 1991; 10:1–8. 11. Tominack RL, Yang GY, Tsai WJ, Chung HM, Deng JF. Taiwan National Poisoning Center of glyphosate surfactant herbicide ingestions. J Toxicol Clin Toxicol 1991; 29:91–109. 12. Chen YJ, Wu ML, Deng JF, Yang CC. The epidemiology of glyphosate surfactant herbicide poisoning in Taiwan, 1986–2007: a poison center study. Clin Toxicol 2009; 47:670–677. 13. Chang CY, Peng YC, Hung DZ, Hu WH, Yang DY, Lin TJ. Clinical impact of upper gastrointestinal tract injuries in glyphosate-surfactant oral intoxication. Hum Exp Toxicol 1999; 18:475–478. 14. Hung DZ, Deng JF, Wu TC. Laryngeal survey in glyphosate intoxication: a pathophysiological investigation. Hum Exp Toxicol 1997; 16:596–599. 15. Chan A, Isbister GK, Kirkpatrick CMJ, Dufful SB. Drug induced QT prolongation and torsade de pointes: evaluation of a QT nomogram. QJM 2007; 100:609–615. 16. Mehta RL, Kellum JA, Shah SV, Molitoris BA, Ronco C, Warnock DG, Levin A, Acute Kidney Injury Network. Acute kidney injury network:

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report of an initiative to improve outcome in acute kidney injury. Crit Care 2007; 11:R31–R38. Talcott JA, Finberg R, Mayer RJ, Goldman L. The medical course of cancer patients with fever and neutropenia. Clinical identification of a low risk subgroup at presentation. Arch Intern Med 1988; 148:2561–2568. Tai T, Yamashita M, Wakimori H. Hemodynamic effects of Roundup, glyphosate and surfactant in dogs. Jpn J Toxicol 1990; 3:63–68. Menkes DB, Temple WA, Edwards IR. Intentional self poisoning with glyphosate containing herbicides. Hum Exp Toxicol 1991; 10:103–107. Hsiao CT, Lin LJ, Hsiao KY, Chou MH, Hsiao SH. Acute pancreatitis caused by severe glyphosate surfactant oral intoxication. Am J Emerg Med 2008; 26:384.e3–384.e5. Brahmi N, Blel Y, Kouraichi N, Abidi N, Thabet H, Amamou M. Acute pancreatitis subsequent to voluntary methomyl and dichlorvos intoxication. Pancreas 2005; 31:424–427. Harputluoglu MM, Demirel U, Alan H, Ates F, Aladag M, Karincaoglu M, Hilmioglu F. Pancreatic pseudocyst development due to organophosphate poisoning. Turk J Gastroenterol 2007; 18:122–125. Chang CB, Chang CC. Refractory cardiopulmonary failure after glyphosate surfactant intoxication: a case report. J Occup Med Toxicol 2009; 4:2. Ozer JS, Chetty R, Kenna G, Palandra J, Zhang Y, Lanevschi A, Koppiker N, Souberbielle BE, Ramaiah SK. Enhancing the utility of

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alanine aminotransferase as a reference standard biomarker for drug induced liver injury. Regul Toxicol Pharmacol 2010; 56:237–246. Reddy VD, Padmavathi P, Varadacharyulu NCh. Emblica officinalis protects against alcohol-induced liver mitochondrial dysfunction in rats. J Med Food 2009; 12(2):327–333. Peixoto F. Comparative effects of the Roundup and glyphosate on mitochondrial oxidative phosphorylation. Chemosphere 2005; 61:1115–1122. Malatesta M, Perdoni F, Santin G, Battistelli S, Muller S, Biggiogera M. Hepatoma tissue culture cells as a model for investigating the effects of low concentrations of herbicide on cell structure and function. Toxicol In Vitro 2008; 22:1853–1860. Hussaini SH, Farrington EA. Idiosyncratic drug induced liver injury: an overview. Expert Opin Drug Saf 2007; 6:673–874. Motojyuku M, Saito T, Akieda K, Otsuka H, Yamamoto I, Inokuchi S. Determination of glyphosate, glyphosate metabolites, and glufosinate in human serum by gas chromatography mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2008; 875:509–514. Lankisch PG, Petersen M, Gottesleben F. High, not low, amylase and lipase levels indicate severe acute pancreatitis! Z Gastroenterol 1994; 32(4):213–215. Pezzilli R, Billi P, Miniero R, Fiocchi M, Cappelletti O, MorselliLabate AM, Barakat B, Sprovieri G, Miglioli M. Serum interleukin-6, interleukin-8, and beta 2-microglobulin in early assessment of severity of acute pancreatitis. Comparison with serum C-reactive protein. Dig Dis Sci 1995; 40:2341–2348.

Clinical Toxicology (2010) 48, 725–729 Copyright © Informa UK, Ltd. ISSN: 1556-3650 print / 1556-9519 online DOI: 10.3109/15563650.2010.498790

ARTICLE LCLT

Dimethylformamide metabolism following self-harm using a veterinary euthanasia product PHILIPPE HANTSON1,2, ANTOINE VILLA3, ANNE-CÉCILE GALLOY1, SARA NEGRI4, GIULIA ESABON4, FABIEN LAMBIOTTE5, VINCENT HAUFROID2, and ROBERT GARNIER3 Dimethylformamide metabolism and NAC

1

Intensive Care, Cliniques Saint-Luc, Brussels, Belgium Louvain Centre for Toxicology and Applied Pharmacology, Université catholique de Louvain, Brussels, Belgium 3 Poison Center, Fernand Widal Hospital, Paris, France 4 Research Laboratory on Airborne Pollutants, Fondazione Salvatore Maugeri, Pavia, Italy 5 Intensive Care, Centre Hospitalier de Sambre-Avesnois, Maubeuge, France 2

Background. A veterinary euthanasia drug containing embutramide, mebezonium, tetracaine, and dimethylformamide (DMF; T-61® or Tanax®) may cause serious manifestations or even fatalities after self-poisoning. Immediate toxicity is mainly due to a general anesthetic and due to a neuromuscular blocking agent, while delayed hepatotoxicity seems related to the solvent DMF. The protective role of N-acetylcysteine (NAC) administration remains debatable. Material and methods. Two male veterinarians (50- and 44-year-old) attempted suicide by injecting T-61 in the precordial area for the first one, and by ingesting 50 mL for the second. Both received NAC (for 14 days in the first case and only for 20 h in the second). Urine was collected for the serial determination of DMF, N-methylformamide (NMF), and N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC). Results. Both patients developed only mild signs of liver injury. The metabolite of DMF, NMF, appeared rapidly in the urine, while a further delay was necessary for AMCC excretion. The kinetics of elimination of DMF and DMF metabolites were slightly slower than those reported in exposed workers. Conclusions. While both patients had a favorable outcome, there is no clear evidence that NAC could directly influence NMF and AMCC excretion. Further investigations of NMF and AMCC excretion, with and without NAC, would be indicated. Keywords

Acute poisoning; Hepatotoxicity; N-acetylcysteine

Introduction T-61® is a veterinary euthanasia drug containing embutramide, a general anesthetic, mebezonium, a neuromuscular blocking agent, tetracaine, a local anesthetic, and dimethylformamide (DMF) as a solvent. Each milliliter of the solution contains 200 mg embutramide, 50 mg mebezonium, 5 mg tetracaine, and 0.6 mL DMF in aqueous solution. A few cases of T-61 poisoning through oral, intramuscular, or intracardiac administration have been reported. Immediate toxicity is mainly due to the anesthetics and mebezonium that are responsible for coma and respiratory failure. In survivors, delayed hepatotoxicity has been observed and is attributable to DMF metabolites. The exact mechanism of DMF-induced hepatotoxicity and the effectiveness of N-acetylcysteine (NAC)

Received 16 March 2010; accepted 2 June 2010. Address correspondence to Philippe Hantson, Intensive Care, Cliniques Saint-Luc, Avenue Hippocrate 10, Brussels 1200, Belgium; Louvain Centre for Toxicology and Applied Pharmacology, Université catholique de Louvain, Brussels 1200, Belgium. E-mail: [email protected]

treatment are controversial. We report two new cases of T61 poisoning with toxicokinetic data treated with NAC.

Patient 1 A 50-year-old veterinarian was found unconscious by his assistant after the injection of an unspecified amount of T-61 in the precordial area. It was unclear to what extent the route of administration was intracardiac or subcutaneous. Two bottles of T-61 (50 mL each) were found nearby. This patient had a past medical history of severe ischemic cardiopathy and coronary artery bypass graft surgery. He was a smoker and obese (75 kg, 165 cm, BMI 28), with dyslipidemia, depression, and chronic ethanol abuse. Forty minutes later, the first medical rescuers noted that he was still unconscious (Glasgow Coma Scale was 6/15: E1, V1, M4), but breathing spontaneously (oxygen saturation 87%); blood pressure was 102/58 mmHg. The electrocardiogram (ECG) showed sinus tachycardia (120 bpm) with ST segment depression in posterior leads and V5–V6. After orotracheal intubation for mechanical ventilation, the patient was referred to an intensive care unit (ICU).

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One hour later, he was still comatose with temperature 34.7°C; hemodynamic instability required fluid repletion and norepinephrine administration. Serum troponin-I was 0.19 ng/mL (N: <0.14 ng/mL) ; creatinine kinase (CK) 866 IU/L (N: <171 IU/L); and blood ethanol 2.87 g/L. Intravenous administration of NAC was started, using the same protocol as for paracetamol poisoning (150 mg/kg over 30 min as loading dose, followed by 50 mg/kg over 4 h, and 100 mg/kg over 16 h). The treatment was then continued for 14 days by the oral route (70 mg/kg every 4 h). The patient regained consciousness within a few hours and improved hemodynamically. The ECG was unchanged. The serum troponin-I level peaked at 0.56 ng/mL 18 h after admission and normalized on day 3; CK normalized within 4 days. Echocardiography revealed an ischemic dilated cardiopathy (ejection fraction 25%). Liver function tests (Table 1) revealed hepatitic injury from day 3. No biological signs of hepatic failure were observed. Serum amylase and lipase levels remained normal. There was also no impairment of renal function. The patient made a complete recovery. He was discharged from the ICU on day 6 to a psychiatry unit. Urine was collected daily from the 3rd to the 16th day for the quantification of excretion of DMF and its metabolites,

N-methylformamide (NMF) and N-acetyl-S-(N-methylcarbamoyl) cysteine (AMCC).

Table 1. Patient 1: Follow-up of liver function tests

Methods of urine DMF and DMF metabolites measurement

Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 13

Total bilirubin (mg/L)

Direct bilirubin (mg/L)

N: 3–12

N: 0–2

13 12 17 20 19 14 14 12 4

2 1 3 4 3 3 4 4 <1

AST (IU/L)

ALT (IU/L)

N: 6–33 N: 14–63 19 18 49 64 86 76 123 146 18

23 20 21 91 182 174 236 296 67

Patient 2 A 44-year-old veterinarian (75 kg, 185 cm, BMI 22) was admitted with coma and respiratory failure requiring mechanical ventilation 80 min after having intentionally ingested the content of a bottle (50 mL) of T-61. The patient had no history of previous medical history and was a nondrinker. He had vomited once before the arrival of the first medical rescuers. In addition to altered consciousness, fasciculations were also noted at this time. On admission to the ICU, the patient received 30 g of activated charcoal via nasogastric tube. Intravenous NAC was started within 4 h of T-61 ingestion, using the same protocol as for paracetamol poisoning. Laboratory investigations are shown in Table 2. There was a prompt neurological recovery and extubation was performed less than 6 h after hospital admission. The patient had an uneventful clinical course and was discharged home on day 8. Serial urine samples were obtained for the determination of DMF, NMF, and AMCC.

DMF and NMF were measured in urine using gas chromatography with nitrogen phosphorus detector (GC-NPD) in the laboratory of the Industrial and Environmental Toxicology Unit (Brussels, Belgium) using a validated method.1 Briefly, urine samples were diluted with a methanol solution containing the internal standard (N,N-dimethylacetamide) and the supernatant was injected on a CP WAX52 CB (25 m, 0.32 mm, 1.2 μm) column. Using this gas chromatographic method, the intermediate metabolite N-hydroxymethyl-N-methylformamide (HMMF) is also quantified as NMF due to its spontaneous degradation into NMF in the heated injector. The analytical

Table 2. Patient 2: Follow-up of liver function tests Patient 1 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 12 Day 13 Day 14 Day 15 Day 16

DMF (mg/24 h)

NMF (mg/24 h)

AMCC (mg/24 h)

Patient 2

DMF (mg/6 h)

NMF (mg/6 h)

AMCC (mg/6 h)

7.85 2.04 1.02 0.69 0.71 0.35 0 0 0 0 0 0

807.47 58.31 24.82 8.58 1.24 0.53 0 0 0 0 0 0

272.23 47.52 44.99 114.7 17.01 20.23 10.01 5.30 4.39 3.08 3.83 5.04

Day 1 (18:30) Day 2 (0:30) Day 2 (6:30) Day 2 (12:30) Day 2 (18:30) Day 3 (0:30) Day 3 (6:30) Day 3 (12:30)

88.2 169.3 69.5 93.0 46.2 17.4 2.5 2.0

15.1 74.1 167.5 222.5 242.1 203.2 179.7 113.1

0.0 0.0 0.0 4.0 4.9 22.0 129.2 256.2

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performance of the method is confirmed by its inclusion (certification for NMF analysis) in the German External Quality Assessment Scheme (G-EQUAS) for toxicological analyses in biological materials. AMCC was measured in urine using a high-performance liquid chromatographic method with ultraviolet detection as described elsewhere.2 AMCC determinations were performed by the Salvatore Maugeri Foundation (Pavia, Italy). Limits of quantification (LOQ) in urine were respectively 0.5, 1, and 2 mg/L, and limit of detection (LOD) 0.2, 0.3, and 0.9 mg/L for DMF, NMF, and AMCC.

Results of the determination of DMF metabolites excretion The results of urinary excretion of DMF, NMF, and AMCC for patients 1 and 2 are shown in Table 3. The maximal urine concentration for DMF, NMF, and AMCC was respectively 2.1, 215, and 72.8 mg/L for patient 1, and 225.7, 478.6, and 366 mg/L for patient 2. Patient 1: NMF (t1/2:0.49 days from day 3 to day 7) and AMCC (t1/2: 2.35 days from day 3 to day 16). Patient 2: DMF (t1/2: 3.92 h from day 2 [12:30] to day 3 [12:30]). In patient 1, the urinary elimination half-life (calculated from the absolute amount excreted) was 0.49 days for NMF (from day 3 to day 7) and 2.35 days for AMCC (from day 3 to day 16). In patient 2, the urinary elimination half-life was 3.92 h for DMF (from day 2 [12:30] to day 3 [12:30]).

Discussion In T-61 poisoning, acute toxicity is primarily related to the anesthetic and neuromuscular blocking properties of the Table 3. Patients 1 and 2: Quantitative urinary excretion of DMF, NMF, and AMCC Total bilirubin

Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14

(mg/L)

Direct bilirubin (mg/L)

AST (IU/L)

ALT (IU/L)

N: 1–14

N: 0.5–3

N: <35

N: <45

53 40 182 774 507 188 47 32 27 27 21 18 20

30 24 93 439 495 352 176 135 106 84 68 53 47

13 25 17 9 7 7 8

6 11 8 5 4 3 4

9 8 7

3 3 2

LOQ in urine was respectively 0.5, 1, and 2 mg/L for DMF, NMF, and AMCC; and LOD was respectively 0.2, 0.3, and 0.9 mg/L for DMF, NMF, and AMCC. The determination of NMF also includes HMMF.

actives resulting in coma and respiratory failure.3 Delayed hepatotoxicity has been observed in suicide cases that recovered from the initial manifestations and can be attributed to the solvent, DMF, or its metabolites.3,4 From 1979, 15 publications have reported a total of 32 cases of T-61 poisoning. As in our cases, the patients were mainly veterinarians, veterinary assistants, or members of their families. The victims were mostly men (66%). Their median age was 37 years (6 to 58 years). Poisoning generally resulted from a suicidal attempt; T-61 was ingested in 10 cases, or was injected intravenously, intramuscularly, or subcutaneously in 8, 6, and 6 cases, respectively; two patients attempted an intracardiac administration. Six fatalities were recorded in the pre-hospital setting and were likely due to respiratory depression. Biological signs consistent with hepatocellular injury were noted in 14 cases. In at least six cases, the severity of liver injury was assessed by a prolonged prothrombin time, a drop in factor V concentration, or the development of hyperammonemic encephalopathy. The smallest amounts reported having caused delayed hepatotoxicity were: 5 mL for the subcutaneous route, 12 mL for the intramuscular, 12 mL for the intracardiac, 15 mL for the oral, and 30 mL for the intravenous. The delay from exposure to hepatotoxicity varied from 2 to 22 days. One patient died on day 9 from hepatic failure; he had ingested 50 mL of T-61 and did not receive NAC.4 Another patient recovered from severe hepatic failure after having received reduced glutathione (GSH) from day 5 to 27.5 Four patients were treated with NAC for a duration ranging from 2 to 7 days. All recovered, after having had hepatocellular injury, mild in one case, and a more severe in the other three.3,6–8 DMF-induced hepatotoxicity is mostly observed after professional exposure in man and has been assessed by experimental studies in animals. The severity of the liver injury is directly related to the dose; however, exposure to DMF can cause symptoms even at low concentrations.9 DMF is primarily metabolized in the liver (by P450 2E1) to HMMF, which is further transformed to NMF. NMF has been shown to be further metabolized by two metabolic pathways: hydroxylation of the N-methyl group to form N(hydroxymethyl)formamide and formation of AMCC via a reactive metabolic intermediate, methyl isocyanate (MIC)10–12 (Fig. 1). The formation of AMCC results from the conjugation of MIC with GSH.13 There is a difference between the metabolism of DMF in humans and rodents. Biotransformation of DMF to AMCC is an important pathway in humans but not in rodents. The pathway leading to MIC has been implicated in NMF-induced liver toxicity. The role of GSH depletion in MIC-induced hepatotoxicity is not fully established. In a rodent model, there was a lack of increase in acetaminophen-induced hepatotoxicity by DMF.14 To the best of our knowledge, this is the first time that AMCC has been measured following acute intoxication with DMF. As illustrated in Table 3, in patient 2, NMF excretion seems to be the highest on day 2 when AMCC urinary excretion

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P. Hantson et al.

Fig. 1. Hypothetical mechanisms of DMF metabolism.

increases. Unfortunately, in patient 2, further urine samples were not available and the urinary AMCC excretion peak was probably not reached by day 3 (12:30 am). In patient 1, urinary concentrations of NMF and AMCC were measured only on day 3, when they were maximal. Mraz studied absorption, metabolism, and elimination of N,N-dimethylformamide in human volunteers.15 The excretion curves of the particular compounds reached their maximum 6–8 h (DMF), 6–8 h (HMMF), 8–14 h (NMF), and 24–34 h (AMCC) after exposure. The half-lives of excretion were approximately 2, 4, 7, and 23 h respectively.15 Other authors reported median halflives of 5.1 h and 22.1 h for NMF and AMCC, respectively.16 In our patients, the elimination kinetics of DMF and DMF metabolites were slightly slower than those reported in exposed workers. The rationale to give NAC is that MIC should cause depletion of cellular GSH, alteration of protein sulfhydryls, and impairment of signaling pathways probably due to oxidative stress within the hepatocyte. The hepatoprotective effect of NAC was investigated in a few experiments conducted in rats.8,17,18 Using serum sorbitol dehydrogenase (SDH) as a marker of DMFinduced hepatotoxicity, Buylaert et al. found a lower increase in SDH in NAC-treated animals.8 In another experiment, Lu et al. investigated the effects of the treatment with sulfhydryl compounds on the changes of superoxide dismutase (SOD) and

xanthine oxidase (XOS) induced by DMF toxicity.17 They found that, compared to the poisoning group, the activity of SOD in liver homogenate was significantly reduced in the group treated with NAC. Finally, NAC offered protection in Balb/c-mice against NMF, which is an hepatotoxicant in rodents.18 Our two patients were exposed to very significant amounts of NMF and AMCC, when compared to the biological exposure indices (BEIs) proposed by the American Conference of Governmental Industrial Hygienists (ACGIH) for workers occupationally exposed to the threshold limit value (TLV) of DMF in breathable air. However, only mild features of delayed liver injury occurred, confirming previous observations. This has been explained by the fact that DMF-induced hepatotoxicity is due to a toxic metabolite and that with high DMF exposure auto-inhibition of DMF of the formation of the metabolite occurs.8,19 Together with our kinetic data, this time course would also support a longer duration of NAC administration (if indicated) till AMCC is no more detectable. The protective effect of NAC cannot be affirmed from these two single cases. If hepatoxicity is related to GSH depletion, the first patient may have been at higher risk due to obesity and chronic ethanol abuse; however, acute ethanol intake may also have some protective effects by inhibition of CYP 2E1.20 In future cases, it would be interesting to extend the measurement of urinary AMCC, with and without NAC

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Dimethylformamide metabolism and NAC use, in order to better investigate the effect of NAC administration on the rate of AMCC excretion.

729

9.

Declaration of interest 10.

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.

References 1. Nomiyama T, Haufroid V, Buchet JP, Miyauchi H, Tanaka S, Yamauchi T, Imamiya S, Seki Y, Omae K, Lison D. Insertion polymorphism of CYP2E1 and urinary N-methylformamide after N,N-dimethylformamide exposure in Japanese workers. Int Arch Occup Environ Health 2001; 74(7):519–522. 2. Imbriani M, Negri S, Ghittori S, Maestri L. Measurement of urinary N-acetyl-S-(N-methylcarbamoyl)cysteine by high-performance liquid chromatography with direct ultraviolet detection. J Chromatogr B Analyt Technol Biomed Life Sci 2002; 778(1–2):231–236. 3. Hantson P, Semaan C, Jouret JC, Rahier J, Lauwerys R, Brunain JP, Mahieu P. Intracardiac injection of T-61, a veterinary euthanasia drug. J Toxicol Clin Toxicol 1996; 34(2):235–239. 4. Nicolas F, Rodineau P, Rouzioux JM, Tack I, Chabac S, Meram D. Fulminant hepatic failure in poisoning due to ingestion of T61, a veterinary euthanasia drug. Crit Care Med 1990; 18(5):573–575. 5. Trevisani F, Tame MR, Bernardi M, Tovoli C, Gasbarrini A, Panarelli M, Gasbarrini G. Severe hepatic failure occurring with T61 ingestion in an attempted suicide. Early recovery with the use of intravenous infusion of reduced glutathione. Dig Dis Sci 1993; 38(4):752–756. 6. Fabre M, Bruel F, Cabot C, Navarro P, Fanjaud G, Burgat-Sacaze V, Virenque C. Une intoxication volontaire grave par ingestion de 75 mL de T-61 euthanasiant veterinaire (abstract). XXXIème Congrès de la société de Toxicologie Clinique; Nancy, France; 1993. 7. Juguet F, Moirand R, Caubet A, Guyader D, Brissot P, Curtes JP. Hépatite aigue après ingestion de T-61, traitée précocement par N-Acetyl Cystéine (abstract). XXXIème Congrès de la société de Toxicologie Clinique; Nancy, France; 1993. 8. Buylaert W, Calle P, De Paepe P, Verstraete A, Samyn N, Vogelaers D, Vandenbulcke M, Belpaire F. Hepatotoxicity of N,N-dimethylformamide

11.

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20.

(DMF) in acute poisoning with the veterinary euthanasia drug T-61. Hum Exp Toxicol 1996; 15(8):607–611. Hamada M, Abe M, Tokumoto Y, Miyake T, Murikami H, Hiasa Y, Matsuura B, Sati K, Onji M. Occupational liver injury due to N,NDimethylformamide in the synthetics industry. Intern Med 2009; 48:1647–1650. Mraz J, Turecek F. Identification of N-acetyl-S-(N-methylcarbamoyl) cysteine, a human metabolite of N,N-dimethylformamide and N-methylformamide. J Chromatogr 1987; 414(2):399–404. Mraz J, Cross H, Gescher A, Threadgill MD, Flek J. Differences between rodents and humans in the metabolic toxification of N,Ndimethylformamide. Toxicol Appl Pharmacol 1989; 98(3):507–516. Kafferlein HU, Angerer J. N-methylcarbamoylated valine of hemoglobin in humans after exposure to N,N-dimethylformamide: evidence for the formation of methyl isocyanate? Chem Res Toxicol 2001; 14(7):833–840. Haufroid V, Lison D. Mercapturic acids revisited as biomarkers of exposure to reactive chemicals in occupational toxicology: a minireview. Int Arch Occup Environ Health 2005; 78(5):343–354. Kim TH, Kim YW, Shin SM, Kim CW, Yu IJ, Kim SG. Synergistic hepatotoxicity of N,N-dimethylformamide with carbon tetrachloride in association with endoplasmic reticulum stress. Chem Biol Interact 2010; 184(3):492–501 Mraz J, Nohova H. Absorption, metabolism and elimination of N,Ndimethylformamide in humans. Int Arch Occup Environ Health 1992; 64(2):85–92. Lauwerys R, Hoet P. Industrial Chemical Exposure; Guidelines for Biological Monitoring. 3rd ed. Boca Raton, FL: Lewis Publishers; 2001:459–466. Lu ZQ, Qiu QM, Miao XJ, Hu GX. Experimental study on detoxication effect of sulfhydryl compounds in acute poisoning of dimethylformamide. Zhongguo Wei Zhong Bing Ji Jiu Yi Xue 2007; 19(4):233–235. Pearson PG, Gescher A, Harpur ES. Hepatotoxicity of N-methylformamide in mice. I. Relationship to glutathione status. Biochem Pharmacol 1987; 36(3):381–384. Mraz J, Jheeta P, Gescher A, Hyland R, Thummel K, Threadgill MD. Investigation of the mechanistic basis of N,N-dimethylformamide toxicity. Metabolism of N,N-dimethylformamide and its deuterated isotopomers by cytochrome P450 2E1. Chem Res Toxicol 1993; 6(2):197–207. Wrbitzky R. Liver function in workers exposed to N,N-dimethylformamide during the production of synthetic textiles. Int Arch Occup Environ Health 1999; 72(1):19–25.

Clinical Toxicology (2010) 48, 730–736 Copyright © Informa UK, Ltd. ISSN: 1556-3650 print / 1556-9519 online DOI: 10.3109/15563650.2010.504187

ARTICLE LCLT

Utility of serum lactate to predict drug-overdose fatality ALEX F. MANINI1, ASHISH KUMAR2, DEAN OLSEN3, DAVID VLAHOV4, and ROBERT S. HOFFMAN5 Utility of lactate

1

Mount Sinai School of Medicine, Emergency Medicine, New York, NY, USA Mount Sinai School of Medicine, Emergency Medicine Residency Program, New York, NY, USA 3 Saint Barnabas Hospital, Emergency Medicine, Bronx, NY, USA 4 New York Academy of Medicine, New York, NY, USA 5 New York University School of Medicine, Emergency Medicine, New York, NY, USA 2

Context. Poisoning is the second leading cause of injury-related fatality in the United States. An elevated serum lactate concentration identifies medical and surgical patients at risk for death; however, its utility in predicting death in drug overdose is controversial and unclear. Objective. We aimed to evaluate the prognostic utility of serum lactate concentration for fatality in emergency department (ED) patients with acute drug overdose. Materials and Methods. This was a case–control study at two urban university teaching hospitals affiliated with a regional poison control center. Data were obtained from electronic medical records, poison center data, and the office of the chief medical examiner. Controls were consecutive acute drug overdoses over a 1-year period surviving to hospital discharge. Cases were subjects over a 7-year period with fatality because of drug overdose. Serum lactate concentration was obtained from the initial blood draw in the ED and correlated with fatality. Results. During the study period, 873 subjects were screened with 50 cases and 100 controls included. Drug exposures and baseline characteristics were similar between groups. Mean lactate concentration (mmol/L) was 9.88 ± 6.7 for cases and 2.76 ± 2.9 for controls (p < 0.001). The receiver operating characteristic area under the curve for prediction of fatality was 0.87 (95% CI: 0.81–0.94). The optimal lactate cutpoint was 3.0 mmol/L (84% sensitivity, 75% specificity), which conferred a 15.8-fold increase in odds of fatality (p < 0.001). Conclusion. In this derivation study, serum lactate concentration had excellent prognostic utility to predict drug-overdose fatality. Prospective validation in the ED evaluation of drug overdoses is warranted.

Keywords Overdose; Fatality; Acute poisoning

Introduction Each year, there are over 1.5 million drug-related emergency department (ED) visits and over 2.4 million poison exposures reported to the American Association of Poison Control Centers in the United States.1–2 Poisoning is defined as injury resultant from exposure to drugs, chemicals, or natural toxins and is currently the second leading cause of injuryrelated fatality in the United States.3 Although poisoning is an infrequent cause of cardiac arrest in elderly patients, it is the leading cause of cardiac arrest in victims <40 years of age.2–4 The prognostic and diagnostic utility of a serum lactate concentration in the initial evaluation of drug overdose is historically controversial.5 Lactate concentration is a useful prognostic indicator for mortality in both medical and surgical patients including those with sepsis,6–9 trauma,10–11 myocardial

Received 7 May 2010; accepted 22 June 2010. Address correspondence to Alex F. Manini, Mount Sinai School of Medicine, Emergency Medicine, One Gustave L. Levy Place, Box 1620, New York, NY 10029, USA. E-mail: [email protected]

infarction with cardiogenic shock,12 and in undifferentiated intensive care unit (ICU) patients.13 Current guidelines for the initial approach to management of the patient with a drug overdose do not include routine evaluation of serum for a lactate concentration.14–16 However, lactate concentration is an established prognostic marker for the evaluation of patients with elevated anion gap metabolic acidosis,17 as well as selected drug overdoses (e.g., metformin, acetaminophen),18–19 selected chronic drug toxicities (e.g., stadivudine),20 and chemical poisoning (e.g., cyanide).21 Despite this evidence, specific indications to obtain a serum lactate concentration in drug-overdose patients remain unclear and studies to clarify the role of serum lactate concentration in the evaluation of patients with drug-overdose emergencies are needed. We designed a case–control study to explore the prognostic significance of serum lactate concentration in acute drug-overdose emergencies. We aimed to calculate the diagnostic test characteristics of serum lactate concentration for drug-overdose fatality and to assign an optimal cutpoint for prediction of mortality. We hypothesized that an elevated serum lactate concentration following acute drug overdose would have excellent prognostic utility for drugoverdose fatality.

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Utility of lactate

Materials and methods Study design This was a case–control study to evaluate the prognostic utility of serum lactate concentration for acute drug-overdose emergencies. Cases were identified retrospectively and controls were enrolled prospectively. Setting EDs from two urban, tertiary-care hospitals were used for enrollment of controls. Both EDs have annual visit volumes in excess of 50,000 and are staffed 24 h per day with boardcertified emergency physicians. The regional Poison Control Center (PCC) used for enrollment of cases in the study has an annual referral volume of 70,000. The study protocol was approved by the Institutional Review Board (IRB) for all participating institutions. Case adjudication Patients with a fatal drug overdose reported to the regional PCC over a 7-year period (2000–2006) were eligible for inclusion as cases. All human deaths from a single large metropolitan city that were attributed to poisoning were analyzed regardless of age. To be included, deaths were adjudicated as either poison-related fatality (PRF) or not by an adjudication committee composed of three medical toxicologists with a range of clinical experience (3–25 years practicing clinical toxicology, all board certified by either the American Board of Emergency Medicine or the American Osteopathic Board of Emergency Medicine). After review of the medical record, each adjudicator was asked to determine the relationship between poisoning and the cause of death according to one of the following five categories: 1) probable/definite, 2) possible, 3) unclear, 4) improbable, or 5) definitely unrelated. For each case, these five determinations were then dichotomized into PRF (1 or 2) versus non-PRF (3, 4, or 5) in accordance with validated adjudication methods in the epidemiology literature.22 Dichotomized data from two adjudicators were initially used to decide if the death was drug-overdose related. Disagreements were settled by a third adjudicator. Adjudicators were blinded to the current study purpose and hypothesis. Exclusion criteria were the following: no serum lactate concentration obtained in the ED, alternative diagnosis (per adjudication), chronic presentation (i.e., not acute), nondrug overdose (e.g., plant), dermal or inhalational exposures only, age <18 years, anaphylaxis, and subjects with incomplete data. Selection of controls Control data were prospectively collected over a 1-year period (April 1, 2007–March 31, 2008) with IRB approval.

731 Control patients were selected from consecutive ED patients presenting with acute drug overdose who were severe enough to warrant bedside consultation with the medical toxicology service but were not complicated by in-hospital fatality. Exclusion criteria were exactly the same as that for cases (see above) with the additional exclusion of in-hospital fatality (five subjects). Fatalities excluded as controls were not included as cases. Control patients were referred from the EDs of two urban, university teaching hospitals. All controls underwent consultation from the PCC-affiliated medical toxicology service consisting of at least one clinical fellow supported by a board-certified medical toxicologist. All control patients had evaluation for drug-overdose emergencies including bedside history, physical examination, and screening laboratory tests while in the ED. Data collection and processing Electronic and paper medical records were provided for all cases and controls with IRB approval. Standardized data collection was performed by a single blinded abstractor and data were stored in a de-identified electronic database. Sources of data included hospital medical records (primary source), PCC electronic records, death reports or cause of death data from the Office of the Chief Medical Examiner (when available), and any additional paper notes (e.g., toxicology consultation service note). Using a standardized data collection instrument, demographics (age, gender), history of exposure (intent, timing, medications, etc.), and drug exposure information (using data from both history and toxicology testing) were recorded. Drug exposures were categorized into standard classes based on mechanism of action (e.g., benzodiazepines, sympathomimetics) to facilitate subgroup analysis. In addition to confirmation of exposure by history, laboratory confirmation of exposures using routine toxicology screens was recorded, if available. A blood gas (either arterial or venous) was performed on patients as was clinically necessary per the clinicians’ judgment. Serum toxicology (acetaminophen, salicylate, ethanol, and rarely, selected drug concentrations on an individual basis per clinicians’ judgment) and urine toxicology screens (most commonly included amphetamines, opioids, benzodiazepines, cocaine metabolite, barbiturates, phencyclidine, tetrahydrocannabinol, tricyclics) were performed per clinicians’ judgment. Methods of measurement Venous serum lactate concentration was drawn at the bedside for all control patients. The decision to measure serum lactate was made at the discretion of the treating physician as part of clinical care, and results were readily available to the clinicians in real time. Only the initial ED lactate was used in the analysis for both cases and controls; as such, subsequent lactates even if changed or abnormal were not included in the

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732 analysis. Serum was analyzed using amperometric electrodes with enzymatic membranes, and run using Radiometer ABLTM 700 analyzers. According to the manufacturer, the range of normal values for venous serum lactate concentration is 1.0–2.5 mmol/L.

Primary data analysis Categorical and continuous variables were assessed using the chi-squared and t-test, respectively, with two-tailed alpha equal to 0.05. Odds ratios with 95% confidence intervals were calculated to evaluate associations between individual drug classes and hyperlactatemia. Receiver operating characteristic (ROC) curves were created to determine the diagnostic test characteristics and the optimal cutpoint for serum lactate concentration. The optimal cutpoint was defined as the serum lactate concentration which maximized the sum of sensitivity plus specificity rounded to the nearest integer. Computer analysis was performed using SPSS version 17 software (SPSS, Inc., Chicago, IL, USA).

A.F. Manini et al. insufficient data (n = 6), anaphylaxis (n = 1). In total, 359 subjects were excluded, leaving 100 controls for data analysis. Baseline characteristics Demographics (age, gender) and comorbidities (hypertension, diabetes, congestive heart failure, chronic obstructive pulmonary disease, coronary disease) were similarly distributed between cases and controls (p = NS). Table 1 summarizes baseline characteristics of all subjects in the study. Drug exposures Proof of at least one drug exposure (either serum drug concentrations or urine toxicology screens) was obtained in 80 and 83% of cases and controls, respectively. Exposure information comparing cases and controls is summarized in Table 2. Aside from ethanol co-ingestion (n = 40), the most common drug exposures in all 150 subjects were acetaminophen (n = 39), opioids (n = 26), and benzodiazepines (n = 26). No drug exposure categories were significantly associated with increased risk of hyperlactatemia (defined as a serum

Sample size and power With 50 cases available to be analyzed in the PCC electronic database, and assuming that the control group would have a mean serum lactate concentration of 2 mmol/L, we estimated the need to analyze data from 100 consecutive control subjects. This would yield 90% power to detect a twofold difference (i.e., clinically meaningful) in mean serum lactate concentration using the t-test.

Results Subject enrollment During the study period, a total of 873 subjects were screened. Application of inclusion and exclusion criteria resulted in analysis of 50 cases (acute drug-overdose fatalities) and 100 controls (acute drug-overdose survivors). For cases, mortality occurred at a median of hospital day 3 (mean 5.6; range day 1–71). PCC referral fatalities over the study period (n = 414) were eligible if deemed to be drug-related deaths by toxicologist adjudication (n = 227), at which point application of exclusion criteria [no lactate drawn (n = 94), insufficient data (n = 23), nondrug overdose (n = 18), age <18 (n = 15), chronic presentation (n = 14), dermal/inhalational (n = 6), alternative diagnosis (n = 6), anaphylaxis (n = 1)] yielded 50 cases for analysis. Eligible controls (n = 459) over the study period were excluded if any of the following criteria were met: deaths (n = 5), no lactate obtained (n = 187), chronic presentation (n = 57), nondrug overdose (n = 37), age <18 (n = 33), alternative diagnosis (n = 21), dermal/inhalational exposure (n = 12),

Table 1. Baseline characteristics of 50 cases and 100 control subjects

Baseline characteristic Demographics Age Males Comorbidities Hypertension Diabetes mellitus Coronary artery disease Congestive heart failure COPD Permanent pacemaker

Cases (N = 50) % or mean ± SD

Controls (N = 100) % or mean ± SD

41.6 ± 17 50

37.9 ± 15.8 63

16 10 6 2 2 2

7 3 3 1 2 0

COPD, chronic obstructive pulmonary disease; N, number of subjects; SD, standard deviation.

Table 2. Exposure information comparing cases and controls

Exposure information Number of exposures Single drug overdose Multiple drug overdose Unknown drug exposure* Proof of at least one exposure

Cases (N = 50) % or mean ± SD

Controls (N = 100)% or mean ± SD

2.3 ± 1.6 38 60 2 80

2.2 ± 1.3 35 61 5 83

N, number of subjects; SD, standard deviation. *At least one of the drugs to which the patient was exposed was an unknown drug.

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Table 3. Most common drug exposures in study subjects and risk of hyperlactatemia by exposure class

Ethanol (co-ingestion) Acetaminophen Benzodiazepines Opioids Sympathomimetics Antipsychotics Antidepressants Anticonvulsants Cardiovascular Medications Salicylates Total

40 39 26 26 23 22 17 11 8 8 150

53 62 31 58 48 50 47 73 38 75 51

1.1 1.8 0.37 1.4 0.87 0.97 0.85 2.8 0.57 3.1 –

25

95% CI 0.54–2.2 0.86–3.8 0.15–0.91 0.6–3.3 0.36–2.1 0.39–2.4 0.31–2.3 0.71–10.9 0.13–2.5 0.6–15.8 –

Exposure class listed in descending order of frequency of ingestion among all 150 subjects. Statistically significant OR listed in bold. CI, confidence interval; HL, hyperlactatemia; OR, odds ratio. *Hyperlactatemia defined as above the upper normal limit of venous lactate concentration, >2.5 mmol/L.

lactate concentration >2.5 mmol/L per assay). Benzodiazepines were the only category significantly associated with the decreased risk of hyperlactatemia (OR 0.37; CI 0.15–0.91). Drug exposure categories were similar between groups and the most common drug exposures with odds of hyperlactatemia are summarized in Table 3. Lactate concentration and blood gas data Mean serum lactate concentration (mmol/L) was 9.88 ± 6.7 for cases and 2.76 ± 2.9 for controls (p < 0.001). Raw lactate data comparing cases to controls are demonstrated in Fig. 1. Elements of the blood gas (pH, PCO2, HCO3) were available in 45 (90%) cases and 97 (97%) controls. Elements of the blood gas were highly associated with fatality (all p < 0.001 using t-test) and are each summarized in Table 4. Sensitivity analysis was performed by removing all 39 subjects with acetaminophen (APAP) co-ingestion from the analysis (because of prior data associating lactate with fatality in APAP overdose);19 in the remaining 111 subjects, mean serum lactate remained significantly associated with fatality (p < 0.001). Lactate cutpoint The ROC curve for ability of a serum lactate concentration to predict fatality is demonstrated in Fig. 2. The c-statistic for area under the ROC curve was 0.87 (95% CI, 0.81–0.94) and was statistically significant (p < 0.0001). The optimal serum lactate concentration integer cutpoints that maximized the sum of sensitivity and specificity were 3.0 mmol/L (84% sensitivity, 75% specificity) and 5.0 mmol/L (68% sensitivity, 93% specificity), respectively. A lactate cutpoint of 3.0 mmol/ L conferred 15.8-fold increased odds of fatality (OR 15.8; CI

Lactate (mmol/L)

Exposure class

Number of total subjects HL* (%) OR

30

20 15 10 5 0 Controls

Cases

Fig. 1. Lactate data comparing cases with controls. This figure demonstrates a box-plot of lactate concentration for cases (fatalities) and control (survivors) subjects. The box-plot represents the median, 25th and 75th quartiles, outliers, and extreme outliers by a line, a box, open circles (ο) and asterisks (*), respectively. Table 4. Lactate and selected blood gas data comparison Laboratory test Lactate (mmol/L) pH† PCO2 (mmHg) HCO3‡ (mmol/L) Total

Cases: mean (SD) 9.88 (6.7) 7.14 (0.20) 29.6 (11) 10.5 (5.4) n = 45

Controls: mean (SD)

p-Value

2.77 (2.9) 7.38 (0.09) 41.9 (10.1) 23.7 (3.5) n = 97

<0.001 <0.001 <0.001 <0.001 –

*p-Values calculated using the t-test for continuous variables. † Venous and arterial pH values used unchanged for cases; venous pH for al controls. ‡ Calculated using formula: pH = 6.1 + log HCO3/0.0306 × PCO2. Notes: HCO3, bicarbonate; mmHg, millimeters mercury; mmol/L, milli moles per liter; N, number; PCO2, partial pressure of carbon dioxide; SD standard deviation.

6.5–38). Diagnostic test characteristics of selected serum lactate concentration cutpoints are summarized in Table 5.

Discussion In this derivation study, serum lactate concentration had excellent prognostic utility to predict fatality in drug-overdose emergencies. Using ROC analysis, initial venous lactate concentrations obtained in the ED had outstanding diagnostic test characteristics. By maximizing the sum of sensitivity and specificity, selection of optimal integer cutpoints for lactate concentration occurred at 3.0 and 5.0 mmol/L. Hyperlactatemia in the setting of overdose may occur from many mechanisms, all of which suggest major metabolic insult and systemic compromise. Mechanisms to account for hyperlactatemia from specific drug overdoses are myriad and

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1.0

Sensitivity

0.8 0.6 0.4 0.2 0.0 0.0

0.2

0.4 0.6 1 – Specificity

0.8

1.0

Fig. 2. ROC curve for prediction of overdose fatality using initial serum lactate. This figure demonstrates the ROC curve of initial serum lactate concentration to predict mortality. The area under the curve of 0.87 was statistically significant. The cutpoints that maximized the sum of sensitivity and specificity were 3.0 and 5.0 mmol/L, respectively. ROC, receiver operating characteristics.

Table 5. Diagnostic test characteristics of selected lactate cutpoints Lactate Sensitivity % cutpoint (CI) HL* >3.0 >4.0 >5.0

90 (82–98) 84 (74–94) 72 (60–85) 68 (55–81)

Specificity % (CI)

NPV % (CI)

PPV % (CI)

66 (57–75) 75 (66–84) 88 (82–95) 93 (88–98)

93 (87–99) 90 (84–97) 86 (79–93) 85 (78–92)

57 (46–68) 63 (51–75) 75 (63–87) 83 (71–95)

CI, confidence intervals; HL, hyperlactatemia; NPV, negative predictive value; PPV, positive predictive value. *Hyperlactatemia from assay defined per manufacturer as lactate concentration >2.5 mmol/L.

include the following: hypoperfusion because of vasoconstriction (e.g., ergots)23 or hypotension (e.g., beta blockers);24 muscle activity because of seizures (e.g., cocaine)25 or myoclonus (e.g., serotonin syndrome);26 altered metabolism of lactate because of increased production (e.g., propylene glycol)27 or decreased clearance (e.g., metformin);28 duration of unconsciousness;5 mitochondrial DNA changes (e.g., nucleoside inhibitors);29 and failure of cellular respiration because of poisoning of glycolysis (e.g., arsenic),30 the Kreb’s cycle (e.g., monofluoroacetate),31 electron transport (e.g., carbon monoxide),32 or uncoupling of oxidative phosphorylation (e.g., salicylism).33 The association of hyperlactatemia with mortality has been extensively studied in medical and surgical patients.6,7,10–11,34 It also correlates with mortality in ED patients with sepsis.7 The Surviving Sepsis Campaign uses a serum lactate concentration cutpoint as one trigger to begin early goal-directed therapy regardless of blood pressure,35 as hyperlactatemia correlates with mortality when studied in patients with severe sepsis even while excluding organ failure and shock.8 Organ failure and mortality during trauma also show a positive

correlation to admission lactate concentrations.10 Similarly, hyperlactatemia is associated with assessing the severity of various poisonings such as with cyanide and acetaminopheninduced fulminant hepatic failure.36,37 Hyperlactatemia is now being studied in the pre-hospital setting and has shown promising results in identifying patients early in their presentation who are at increased risk for morbidity and mortality.38 Our data build upon the above prior evidence to include patients with acute drug overdose. We utilized a validated technique of adjudication22 to categorize overdoses resulting in PRF, and then correlated these results with observed hyperlactatemia. Cases were selected from the PCC database, rather than from the same two hospitals used to select the controls, because the study would not have otherwise been feasible given the rarity of PRF. Selection of controls from a 1-year period, rather than from the 7-year time period of case ascertainment, yielded enough patients to adequately power the statistical analysis; thus, evaluation of a longer time period was not necessary. We cannot exclude the possibility that changes in overdose epidemiology or differential use of lactate measurement between these two time periods may have biased our results; however, these possibilities are unlikely to have biased away from the null and are thus unlikely to have impacted the results meaningfully. The implications of these results may be to help risk stratify patients early in the course of the ED presentation. Identification of those at risk for drug-overdose fatality may help prevent in-hospital adverse events and aid ICU triage. Risk stratification by serum lactate concentration may also help lead to expedite interventions such as life-saving antidotal therapy, when indicated. Prospective validation in the ED evaluation of drug overdoses is necessary to confirm these derivation data. Strategies that incorporate lactate concentration into ICU triage from the ED in drug-overdose emergencies may be warranted. Interestingly, benzodiazepines were the only drugs that demonstrated a significantly decreased risk of hyperlactatemia. When benzodiazepines were co-ingested in overdose, there was 63% lower odds of hyperlactatemia (p < 0.05). This finding is novel but can be explained plausibly by CNS depression, muscle relaxation, and possible suppression of seizure activity, all of which contribute to lowering the serum lactate concentration.

Limitations Our study has several limitations including those common to all retrospective case–control studies, namely the inability to calculate incidence or prevalence of hyperlactatemia or fatality. A large subset of subjects were excluded because of absence of ED serum lactate, which may have biased the lactate cutpoint data; however, this would probably bias toward the null hypothesis as clinicians are typically more likely to draw lactate in more severely ill patients. Another consideration is the study setting, as the study was performed in an urban tertiary referral center, and results might not be applicable to all

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Utility of lactate centers; however, our subject population was highly diverse and likely represents a general ED population. The presence of a bedside medical toxicology consultation may have improved outcomes for controls as opposed to cases; however, this would have biased toward the null as sicker control subjects with theoretically high lactates would presumably have better prognosis. Cases were poison center referrals at the discretion of the ED physician, which may have biased our case population. Additionally, because of limited patients for subgroup analysis we could not identify drug combinations that were particularly prone to produce hyperlactatemia or fatality as an additive effect; however, in future larger studies we plan to examine particularly toxic drug combinations (type, dose, etc.). And finally, time to obtaining serum lactate concentration, as well as whether the source was arterial or venous, may also limit interpretation of our data. However, our data represent real-world scenario, which adds credence to the overall concept of generalizability.

735

9. 10.

11.

12.

13.

14.

15. 16.

Conclusions In this derivation study, serum lactate concentration had excellent prognostic utility to predict drug-overdose fatality. Prospective validation in the ED evaluation of patients with drug overdose is warranted. Identification of those at risk for fatality may prevent adverse events and aid ICU triage.

17.

18.

19.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.

References 1. Substance Abuse and Mental Health Services Administration. Drug Abuse Warning Network, May 2005. http://druginfo.samhsa.gov/. Accessed on 2 June 2008. 2. Bronstein AC, Spyker DA, Cantilena LR, Green J, Rumack BH, Heard SE. 2007 Annual Report of the American Association of Poison Control Centers’ National Poison Data System (NPDS): 25th Annual Report. Clin Toxicol (Phila) 2008; 46:927–1057. 3. Paulozzi L, Crosby A, Ryan G. Increases in age-group-specific injury mortality – United States, 1999–2004. MMWR 2007; 56:1281–1284. 4. McCaig LF, Burt CW. Poisoning-related visits to emergency departments in the United States, 1993–1996. J Toxicol Clin Toxicol 1999; 37:817–826. 5. Schuster HP, Kapp S, Prellwitz W, Schuster CJ, Weilemann LS. Significance of hyperlactatemia in acute hypnotic drug poisoning. J Mol Med 1981; 59:599–605. 6. Fall PJ, Szerlip HM. Lactic acidosis: from sour milk to septic shock. J Intensive Care Med 2005; 20:255–271. 7. Shapiro NI, Howell MD, Talmor D, Nathanson LA, Lisbon A, Wolfe RE, Weiss JW. Serum lactate as a predictor of mortality in emergency department patients with infection. Ann Emerg Med 2005; 45:524–528. 8. Mikkelsen ME, Miltiades AN, Gaieski DF, Goyal M, Fuchs BD, Shah CV, Bellamy SL, Christie JD. Serum lactate is associated with mortality

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in severe sepsis independent of organ failure or shock. Crit Care Med 2009; 37:1670–1677. Jones AE, Puskarich MA. Is lactate the “Holy Grail” of biomarkers for sepsis prognosis? Crit Care Med 2009; 37:1812–1813. Mankis P, Jankowski S, Zhang H, Kahn RJ, Vincent JL. Correlation of serial blood lactate levels to organ failure and mortality after trauma. Am J Emerg Med 1995; 13:619–622. Callaway DW, Shapiro NI, Donnino MW, Baker C, Rosen CL. Serum lactate and base deficit as predictors of mortality in normotensive elderly blunt trauma patients. J Trauma 2009; 66:1040–1044. Valente S, Lazzeri C, Vecchio S, Giglioli C, Margheri M, Bernardo P, Comeglio M, Chiocchini S, Gensini GF. Predictors of in-hospital mortality after percutaneous coronary intervention for cardiogenic shock. Int J Cardiol 2007; 114:176–182. Khosravani H, Shahpori R, Stelfox HT, Kirkpatrick AW, Laupland KB. Occurrence and adverse effect on outcome of hyperlactatemia in the critically ill. Crit Care 2009; 13:R90. Epub 2009 June 12. Erickson TB, Thompson TM, Lu JJ. The approach to the patient with an unknown overdose. Emerg Med Clin North Am 2007; 25:249–281; abstract vii. Burns MJ, Schwartzstein RM. General approach to drug poisoning in adults. In: Basow DS, ed. UpToDate, Waltham, MA; 2010. Wolf SJ, Heard K, Sloan EP, Jagoda AS; American College of Emergency Physicians. Clinical policy: critical issues in the management of patients presenting to the emergency department with acetaminophen overdose. Ann Emerg Med 2007; 50:292–313. Manini AF, Hoffman RS, McMartin KE, Nelson LS. Relationship between serum glycolate and falsely elevated lactate in severe ethylene glycol poisoning. J Anal Toxicol 2009; 33:174–176. Dell’aglio DM, Perino LJ, Kazzi Z, Abramson J, Schwartz MD, Morgan BW. Acute metformin overdose: examining serum pH, lactate level, and metformin concentrations in survivors versus nonsurvivors: a systematic review of the literature. Ann Emerg Med 2009; 54:818–823. Bernal W, Donaldson N, Wyncoll D, Wendon J. Blood lactate as an early predictor of outcome in paracetamol-induced acute liver failure: a cohort study. Lancet 2002; 359:558–563. Miller KD, Cameron M, Wood LV, Dalakas MC, Ko-vacs LA. Lactic acidosis and hepatic steatosis associated with use of stavudine: report of four cases. Ann Int Med 2000; 133:192–195. Baud FJ, Borron SW, Mégarbane B, Trout H, Lapostolle F, Vicaut E, Debray M, Bismuth C. Value of lactic acidosis in the assessment of the severity of acute cyanide poisoning. Crit Care Med 2002; 30:2044–2050. Curb JD, McTiernan A, Heckbert SR, Kooperberg C, Stanford J, Nevitt M, Johnson KC, Proulx-Burns L, Pastore L, Criqui M, Daugherty S; WHI Morbidity and Mortality Committee. Outcomes ascertainment and adjudication in the Women’s Health Initiative. Ann Epidemiol 2003; 13:S122–S128. Kuhn FE, Johnson MN, Gillis RA, Visner MS, Schaer GL. Effect of cocaine on the coronary circulation and systemic hemodynamics in dogs. J Am Coll Cardiol 1990; 16:1481–1491. Love JN, Howell JM, Litovitz TL, Klein-Schwartz W. Acute beta blocker overdose: factors associated with the development of cardiovascular morbidity. J Toxicol Clin Toxicol 2000; 38:275–281. Hulme J, Sherwood N. Severe lactic acidosis following alcohol related generalized seizures. Anaesthesia 2004; 59:1228–1230. Boyer EW, Shannon M. The serotonin syndrome. N Engl J Med 2005; 352:1112–1120. Jorens PG, Demey HE, Schepens PJ, Coucke V, Verpooten GA, Couttenye MM, Van Hoof V. Unusual D-lactic acid acidosis from propylene glycol metabolism in overdose. J Toxicol Clin Toxicol 2004; 42:163–169. Stumvoll M, Nurjhan N, Perriello G, Dailey G, Gerich JE. Metabolic effects of metformin in non-insulin-dependent diabetes mellitus. N Engl J Med 1995; 333:550–554. Cote HCF, Brumme ZL, Kevin JP, Craib J, Alexander CS, Wynhoven B, Ting L, Wong H, Harris M, Harrigan PR, O’Shaughnessy MV, Montaner JSG. Changes in mitochondrial DNA as a matter of nucleoside toxicity in HIV-infected patients. N Engl J Med 2002; 346:811–820.

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736 30. Pal S, Chatterjee AK. Protective effect of methionine supplementation on arsenic-induced alteration of glucose homeostasis. Food Chem Toxicol 2004; 42:737–742. 31. Chi CH, Chen KW, Chan SH, Wu MH, Huang JJ. Clinical presentation and prognostic factors in sodium monofluoroacetate intoxication. J Toxicol Clin Toxicol 1996; 34:707–712. 32. Benaissa ML, Mégarbane B, Borron SW, Baud FJ. Is elevated plasma lactate a useful marker in the evaluation of pure carbon monoxide poisoning? Intensive Care Med 2003; 29:1372–1375. 33. Stolbach AI, Hoffman RS, Nelson LS. Mechanical ventilation was associated with acidemia in a case series of salicylate-poisoned patients. Acad Emerg Med 2008; 15:866–869. 34. Smith I, Kumar P, Molloy S, Rhodes A, Newman PJ, Grounds RM, Bennett ED. Base excess and lactate as prognostic indicators for

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patients admitted to intensive care. Intensive Care Med 2001; 27:74–83. Rivers EP, Coba V, Visbal A, Whitmill M, Amponsah D. Management of sepsis with early resuscitation. Clin Chest Med 2008; 29:689–704. Baud FJ, Borron SW, Megarbane B, Trout H, Lapostolle F, Vicaut E, Debray M, Bismuth C. Value of lactic acidosis in the assessment of the severity of acute cyanide poisoning. Crit Care Med 2002; 30:2044–2050. Schmidt LE, Larsen FS. Prognostic implications of hyperlactemia, multiple organ failure and systemic inflammatory response syndrome in patients with acetaminophen-induced acute liver disease. Crit Care Med 2006; 34:337–343. Jansen TC, van Bommel J, Mulder PG. The prognostic value of blood lactate levels relative to that of vital signs in the pre-hospital setting: a pilot study. Crit Care 2008; 12:R160.

Clinical Toxicology (2010) 48, 737–744 Copyright © Informa UK, Ltd. ISSN: 1556-3650 print / 1556-9519 online DOI: 10.3109/15563650.2010.507548

ARTICLE LCLT

Acute illnesses associated with exposure to fipronil—surveillance data from 11 states in the United States, 2001–2007 SOO-JEONG LEE1, PRAKASH MULAY2, BRIENNE DIEBOLT-BROWN3, MICHELLE J. LACKOVIC4, LOUISE N. MEHLER5, JOHN BECKMAN6, JUSTIN WALTZ7, JOANNE B. PRADO8, YVETTE A. MITCHELL9, SHEILA A. HIGGINS10, ABBY SCHWARTZ11, and GEOFFREY M. CALVERT1 Acute illnesses associated with fipronil exposure

1

Division of Surveillance, Hazard Evaluations, and Field Studies, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, Cincinnati, OH, USA 2 Bureau of Environmental Public Health Medicine, Florida Department of Health, Tallahassee, FL, USA 3 Environmental and Injury Epidemiology and Toxicology Branch, Texas Department of State Health Services, Austin, TX, USA 4 Office of Public Health, Louisiana Department of Health and Hospitals, New Orleans, LA, USA 5 Department of Pesticide Regulation, California Environmental Protection Agency, Sacramento, CA, USA 6 Public Health Institute, Oakland, CA, USA 7 Office of Environmental Public Health, Oregon Public Health Division, Oregon Department of Human Services, Portland, OR, USA 8 Office of Environmental Assessments, Washington State Department of Health, Olympia, WA, USA 9 Bureau of Occupational Health, New York State Department of Health, Troy, NY, USA 10 North Carolina Department of Health and Human Services, Raleigh, NC, USA 11 Division of Environmental Health, Michigan Department of Community Health, Lansing, MI, USA

Introduction. Fipronil is a broad-spectrum phenylpyrazole insecticide widely used to control residential pests and is also commonly used for flea and tick treatment on pets. It is a relatively new insecticide and few human toxicity data exist on fipronil. Objective. This paper describes the magnitude and characteristics of acute illnesses associated with fipronil exposure. Methods. Illness cases associated with exposure to fipronil-containing products from 2001 to 2007 were identified from the Sentinel Event Notification System for Occupational Risks (SENSOR)-Pesticides Program and the California Department of Pesticide Regulation. Results. A total of 103 cases were identified in 11 states. Annual case counts increased from 5 in 2001 to 30 in 2007. Of the cases, 55% were female, the median age was 37 years, and 11% were <15 years old. The majority (76%) had exposure in a private residence, 37% involved the use of pet-care products, and 26% had work-related exposures. Most cases (89%) had mild, temporary health effects. Neurological symptoms (50%) such as headache, dizziness, and paresthesia were the most common, followed by ocular (44%), gastrointestinal (28%), respiratory (27%), and dermal (21%) symptoms/ signs. Exposures usually occurred from inadvertent spray/splash/spill of products or inadequate ventilation of the treated area before re-entry. Conclusions. Our findings indicate that exposure to fipronil can pose a risk for mild, temporary health effects in various body systems. Precautionary actions should be reinforced to prevent fipronil exposure to product users. Keywords

Fipronil; Pesticides; Poisoning; Surveillance; Phenylpyrazole

Introduction Every year, several new pesticide active ingredients are introduced into the market in the United States.1 To ensure that new pesticide products, particularly those containing new active ingredients, do not pose unreasonable risks to human health, post-marketing surveillance efforts are needed to identify any adverse health effects associated with these products.

Received 26 April 2010; accepted 6 July 2010. Address correspondence to Geoffrey M. Calvert, National Institute for Occupational Safety and Health, 4676 Columbia Parkway, R-17, Cincinnati, OH 45226, USA. E-mail: [email protected]

Fipronil is a relatively new insecticide that belongs to the phenylpyrazole family and was first registered by the US Environmental Protection Agency (EPA) in 1996.2 As a broad-spectrum insecticide, fipronil is widely used to control various residential, veterinary, and agricultural pests such as ants, beetles, cockroaches, fleas, ticks, termites, and weevils.2 Fipronil disrupts g-aminobutyric acid (GABA) receptors in the central nervous system thereby blocking GABA-gated chloride channels, resulting in excessive neuronal stimulation and death of the target insect.2 Fipronil has higher affinity for GABA receptors in insects than in mammals and thus produces greater toxicity in insects.2 Additional selective toxicity of fipronil to insects is produced by blockage of neuronal glutamate-gated chloride channels, which are found only in invertebrates.3

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Over the last decade, the usage of fipronil has increased considerably and fipronil residues can now be found in 40% of American homes.4 Meanwhile, information on human health effects from fipronil poisoning is very limited and only a few reports are available in the literature.5–8 Reported symptoms include conjunctivitis, headache, dizziness, nausea, vomiting, abdominal pain, oropharyngeal pain, cough, sweating, sensory impairment, weakness, drowsiness, agitation, and seizure. Recently, the US EPA intensified scrutiny of the spot-on insecticides due to an apparent increase in the number of adverse reaction reports among treated pets.9 This prompted the present evaluation of the human toxicity associated with fipronil exposure, including spot-on fipronil-containing insecticides for pets. Multi-state surveillance data on pesticide illness in the United States were used. This paper describes the magnitude and characteristics of acute illnesses associated with fipronil exposure among humans identified from 2001 to 2007. This paper also presents three case reports from 2008 and 2009 to illustrate different patterns of fipronil exposure and to provide evidence that problems with fipronil persist.

in state health departments. Currently, 12 states participate in the SENSOR-Pesticides program: Arizona, California, Florida, Iowa, Louisiana, Michigan, New Mexico, New York, North Carolina, Oregon, Texas, and Washington. Cases exposed between January 1, 2001 and December 31, 2007 were included. Few cases were identified before 2001. The year 2007 is the most recent year for which complete surveillance data were available. Most SENSOR-Pesticides states provided data for the entire study period. However, three states joined the SENSOR-Pesticides program after 2001 and contributed data for fewer years (Iowa, 2006–2007; New Mexico, 2005–2007; and North Carolina, 2007). Arizona identifies very few cases of acute pesticide poisoning overall, and data from this state were excluded from analyses. CDPR is an agency under the California EPA and operates its own pesticide illness surveillance program. The state surveillance programs collect information on pesticide poisoning cases identified from various sources (e.g., poison control centers, workers’ compensation systems, state agencies responsible for pesticide regulation, and physician reports) and classify cases based on the strength of evidence for pesticide exposure, health effects, and toxicological evidence supporting the association between exposure and health effects.10 Table 1 provides case definitions used by the SENSOR-Pesticides program and CDPR. The SENSOR-Pesticides program and CDPR use slightly different case definitions and categories. Definite, probable, possible, and suspicious cases from SENSOR-Pesticides and definite, probable, and possible cases from CDPR were included in this study. Fipronil cases included persons who were exposed to a single fipronilcontaining product only or to at least one fipronil-containing product when exposure involved multiple pesticide products.

Methods Cases that reported acute illness or injury associated with exposure to fipronil were identified from two data sources: the Sentinel Event Notification System for Occupational Risks (SENSOR)-Pesticides program and the California Department of Pesticide Regulation (CDPR). The SENSORPesticides program is run by the National Institute for Occupational Safety and Health (NIOSH) and collects pesticide illness surveillance data annually from state programs residing

Table 1. Case classification matrix for fipronil-related illnesses by the SENSOR-Pesticides program Classification category Classification criteriaa Exposure Health effects Causal relationship

Definite 1 1 1

Probable 1 2 1

2 1 1

Possible

Suspicious

2 2 1

1 or 2 1 or 2 4

Source: CDC. Case definition for acute pesticide-related illness and injury cases reportable to the national public health surveillance system. Available at http://www.cdc.gov/niosh/topics/pesticides/pdfs/casedef2003_revAPR2005.pdf. a Cases are classified as definite, probable, possible, or suspicious based on scores for exposure, health effects, and causal relationship. Exposure scores: 1 = laboratory, clinical, or environmental evidence for exposure; 2 = evidence of exposure based solely on written or verbal report from the patient, a witness, or applicator. Health effects scores: 1 = two or more new post-exposure signs or laboratory findings reported by a licensed health professional; 2 = two or more post-exposure symptoms reported by the patient. Causal relationship scores: 1 = the observed health effects are consistent with the known toxicology of the pesticide; 4 = insufficient toxicological information available to determine the causal relationship. Note: Case classifications are slightly different between the SENSOR-Pesticides program and CDPR’s Pesticide Illness Surveillance Program. CDPR classifies cases as definite, probable, and possible based on the relationship between exposure and health effects: definite = both physical and medical evidence document exposure and consequent health effects; probable = limited or circumstantial evidence supports a relationship to pesticide exposure; possible = evidence neither supports nor contradicts a relationship. More information is available at http:// www.cdpr.ca.gov/docs/whs/pisp/brochure.pdf.

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Acute illnesses associated with fipronil exposure Variables of interest in the analysis included year of exposure, source of the case report, age, gender, location of exposure, work-relatedness, type of fipronil product (i.e., pet-care product versus other), health effects, illness severity, type of activity at the time of exposure, and factors contributing to the exposure. Work-related exposures referred to exposures that occurred while at work. Illness severity was categorized into low, moderate, and high using standard criteria.10 Low severity refers to mild illnesses that generally resolve without treatment and with minimal lost work time (<3 days). Moderate severity refers to illnesses that are generally systemic and require medical treatment. They may require hospitalization (≤3 days) and lost work time (≤5 days). High severity refers to life-threatening or serious health effects which can result in permanent impairment or disability and may require hospitalization (>3 days) and substantial lost work time (>5 days). Contributing factors to fipronil exposure were coded by a retrospective review of available information. This study was exempt from consideration by the federal Human Subjects Review Board because only surveillance data were analyzed and each state removes any personal identifiers from the data prior to submission to NIOSH. Data analysis Data analysis was performed with SAS v 9.1. Descriptive statistics were used to characterize cases, and cases were stratified by the type of fipronil product. To eliminate duplicate cases that may have been identified by both the SENSORPesticides and CDPR programs, California cases from each program were compared on date of exposure, age, sex, pesticide active ingredients, and county of exposure (Personal identifiers were not available). Three cases were identified by both programs and these cases were counted only once in the data analyses.

Results From 2001 through 2007, a total of 103 acute illness cases associated with fipronil exposure were identified in 11 states; 92 cases by SENSOR-Pesticides and 11 cases by CDPR. Florida, Texas, and Louisiana accounted for 58% (n = 60) of all cases. Iowa and New Mexico reported no cases. For the 7-year period, reported cases increased from 5 in 2001 to 30 in 2007 (Fig. 1). Most cases (n = 72; 70%) were identified through poison control centers. A total of 86 (83%) cases were exposed to a single fipronil-containing product, and 17 (17%) cases were exposed to multiple pesticide products, at least one of which was a fipronil-containing product. The fipronil products associated with these illnesses are provided in Table 2. Pet-care products (Frontline®) were responsible for 38 (37%) cases. Of cases exposed to pet-care products, 33 were by spot-on treatment products and 5 were by spray products. Table 3 provides selected characteristics of the cases. Fifty-seven (55%) cases were female. The median age of

739

Fig. 1. Acute illnesses related to fipronil exposure by year and the type of product—11 states, 2001–2007 (n = 103).

affected persons was 37 years (range: <1–86 years), and 11 (11%) cases were <15 years old. A total of 78 (76%) cases had exposure in a private residence and work-related exposures accounted for 27 (26%) cases. Neurological symptoms predominated (50%), followed by ocular (44%), gastrointestinal (28%), and respiratory (27%) symptoms. Detailed health effects are presented in Table 4. Ninety-two (89%) cases were classified as having low-severity illness and 9 (9%) had moderate-severity illness. There were two cases (2%) of high-severity illness, both of whom were pest control operators. One case with high-severity illness had a brief episode of seizure, blurred vision, and dizziness (previous medical history is not known) while applying Maxforce® Roach Gel and Termidor® (EPA Registration numbers were unidentified). During the application, this case used only chemicalresistant gloves and no other personal protective equipment (PPE). The other case with high-severity illness developed dyspnea, diaphoresis, tremor, paresthesia, and slurred speech while applying Termidor® 80 WG (EPA Registration No. 264-569), which required hospitalization for 7 days. Information on PPE use was not available for this case. Factors contributing to fipronil exposures are presented in Table 5. The most common factors included inadvertent splash/spray/spill (e.g., due to human error or unexpected pet movement), inadequate ventilation of the treated area before re-entry, failure to leave the treated area during application, required PPE not worn, pesticide products stored or used within reach of children, and contact with residue (e.g., handling pet before applied product dried). Case reports The following three cases illustrate different patterns of exposure to fipronil products.

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S.J. Lee et al. Table 2. Fipronil products used in fipronil-related illness cases—11 states, 2001–2007 (n = 103) Product name Fiprogard SC Fiprogard 80 WG Frontline Plus For Dogs Frontline Plus For Cats Frontline Spray Treatment Frontline Top Spot For Dogs Frontline Top Spot For Cats Frontlinea Maxforce ABF4 Maxforce Ant Bait F1 Maxforce IBH10 Maxforce Roach Gel Maxforcea Product: RBF5 Termidor SC Insecticide Termidor 80 WG Insecticide Termidora Unknown

Registration No.

Toxicityc

Restricted use

No. of cases

432-901b 432-900b 65331-5 65331-4 65331-1 65331-3 65331-2 Not available 432-1264, 64248-21b 64248-10b 64248-19b Not available Not available 432-1259, 64248-14b 7969-210, 264-568b 7969-209, 264-569b Not available Not available

3 2 3 3 3 3 3 3 3 3 3 3 3 3 3 2 — —

(Canceled) (Canceled) No No No No No No No (Canceled) (Canceled) No No No Yes Yes — —

10 2 19 6 5 5 1 2 2 1 1 1 1 3 23 13 2 8

a

Detailed product names are not available. No longer active. The product was canceled or transferred to a different company. c Toxicity categories of pesticide products are based on established criteria of the Environmental Protection Agency (40 CFR part 156). Category 1 is given for pesticides with the greatest toxicity and category 4 for pesticides with the least toxicity. The signal word for each category is as follows: 1 (danger), 2 (warning), and 3 (caution). b

Case 1. In September 2009, a 38-year-old pest control technician in Texas, who had worked in termite control for over a year and had never worn PPE, developed dizziness, a shaky feeling, hand stiffness and tingling, abdominal pain, diarrhea, and tachycardia after spraying Termidor® SC (EPA Registration No. 7969-210). He was taken to the Emergency Department by ambulance and underwent decontamination. He was discharged about 6 h later when symptoms resolved. He continued to feel slightly irritable and weak, and was not able to work for 2 days. His illness was classified as moderate severity and was placed in the case definition classification category of “definite”. Case 2. In April 2008, a 33-year-old woman in Washington State developed sore throat, headache, and difficulty in breathing after returning to her apartment after it had been sprayed with Termidor® SC (EPA Registration No. 7969210) for ants. The pest control company had sprayed her living room, kitchen, and bathroom, and told her to stay out for 1.5 h. She returned home 3.5 h later and became symptomatic. Her symptoms resolved after ventilating the apartment. Her illness was classified as low severity and was placed in the case definition classification category of “possible”. Case 3. In March 2009, a 65-year-old woman in New York State developed a pruritic rash on her neck, scalp, arms, face, ears, and chest after playing with a dog treated with Frontline® Top Spot® for Dogs (EPA Registration No. 65331-3). Unaware that her husband had treated their dog, she played with the dog within hours of treatment. She sought medical treatment 4 days after symptom onset. The illness was classified as low

severity and was placed in the case definition classification category of “possible”.

Discussion Fipronil is one of the most commonly used insecticides in American homes. However, limited data are available on the toxicity of this pesticide in humans. Analyzing pesticide illness surveillance data from 2001 to 2007, we identified 103 acute illness cases associated with fipronil exposure in 11 states and the annual number of reported cases was shown to increase over time. The findings showed that reporting of acute illness related to fipronil exposure was relatively uncommon and most cases were related to residential exposures. However, it should be noted that pesticide-related illnesses, especially nonoccupational exposure cases, are substantially underreported.11 The cases identified in this report should serve as sentinels to warn of the need to reinforce the importance of precautionary measures to prevent fipronil exposure and subsequent adverse health effects. Our findings showed that the vast majority of cases had low-severity illness, indicating that fipronil exposure, in general, poses a low risk of mild, temporary health effects. Similarly, a report by the Paris Poison Center in France documented that most cases presented with no or mild symptoms probably because these cases experienced relatively low exposures.5 Consistent with the fact that the central nervous

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Table 3. Acute illnesses related to fipronil exposure by selected characteristics—11 states, 2001–2007 Characteristics State (years contributing data) Florida (2001–2007) Texas (2001–2007) Louisiana (2001–2007) California (2001–2007) Oregon (2001–2007) Washington (2001–2007) New York (2001–2007) North Carolina (2007) Michigan (2001–2007) Iowa (2006–2007) New Mexico (2005–2007) Status Definite Probable Possible Suspicious Sex Male Female Age <15 15–24 25–34 35–44 45–54 55–64 65+ Unknown Location of exposure Private residence Commercial facility Institution (e.g., hospital) Unknown Work-relatedness Yes No Unknown Type of activity at the time of exposure Routine indoor living activity Applying/Handling pesticide Routine work activity Routine outdoor living activity Unknown Severity of illness High Moderate Low

Total (n = 103)

Pet-care product (n = 38)

Other product (n = 65)

23 19 18 13 12 8 5 4 1 0 0

6 2 9 0 10 5 3 3 0 0 0

17 17 9 13 2 3 2 1 1 0 0

2 15 79 7

2 5 29 2

0 10 50 5

46 57

11 27

35 30

11 8 20 17 14 13 6 14

7 2 9 9 0 4 1 5

4 6 11 8 14 9 5 6

78 8 4 13

36 1 0 1

42 7 4 12

27 71 5

1 37 0

26 34 5

39 38 7 5 14

12 20 0 1 5

27 18 7 4 9

2 9 92

0 3 35

2 6 57

system is the primary target of fipronil, neurological symptoms were the most commonly observed health effects. Ocular symptoms were also commonly reported, which were usually related to inadvertent splash/spray to the eyes. Although it was rare, high-severity illnesses requiring hospitalization or presenting with seizure were also identified in

our study. The two cases with high-severity illness were both pest control operators who were exposed on-the-job while applying fipronil products. Although most fipronil-related illnesses identified in this report arose from non-occupational exposures, our findings suggest that occupational exposures to fipronil, which can involve repetitive exposure to products

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S.J. Lee et al. Table 4. Clinical manifestations of fipronil-related illness—11 states, 2001–2007 (n = 103) Health effects

No.

Neurological Headache Dizziness Paresthesia Muscle weakness Confusion Ocular Irritation, pain, inflammation, lacrimation Conjunctivitis Gastrointestinal Nausea Vomiting Respiratory Upper respiratory pain/irritation Dyspnea Cough Wheezing or exacerbation of asthma Dermatologic Irritation, pain, rash, erythema Pruritus, swelling, hives Cardiovascular Tachycardia, palpitation Other Fatigue

51 24 14 10 7 7 45 38 11 29 22 14 28 16 9 8 5 22 15 12 4 2 11 9

Note: Presented are health effects reported by at least 5 cases, except for cardiovascular effects. The sum of health effects exceeds 103 because some cases had more than one health effect.

with higher concentrations, can pose a risk of more severe health effects. Thus, strict compliance with required PPE including chemical-resistant gloves, long-sleeved shirt, long pants, socks, and shoes, and protective eyewear such as goggles, a faceshield or safety glasses with front, brow, and temple protection should be reinforced among professional users.

This study also found that pet-acare products (Frontline®) were related to more than one-third of cases and accounted for the majority of childhood cases (64%). This finding suggests the need for special attention by parents to prevent exposure among children. In the United States, 39% of households own at least one dog and 33% own at least one cat12 and many of these pets are treated for fleas and ticks. Pet owners who use fipronil should remember that these petcare products are pesticides with inherent toxicity. As such, when handling these products, appropriate precautions should be taken to avoid contact with skin, eyes, or clothing during the application and also to prevent exposure to residue on the treated pets by avoiding contact with the treated areas of the pet until dry as instructed by the product label. Additionally, users may need to avoid such contact at least until bathing or shampooing treated pets, which the label recommends not be undertaken until at least 48 h after application. An experimental study showed that for up to four weeks after spot-on treatment of dogs with Frontline®, fipronil residues were detected on gloves worn while petting the dogs for 5 min.13 The effect of Frontline® is reported to last for a month and monthly reapplications are usually recommended. Thus, repetitive and/or chronic exposure to low doses may occur if precautionary actions are not taken. Fipronil product users should be aware of the potential presence of residue after using the product. Employing good hygiene such as handwashing after contacting treated pets would help to minimize exposure. Moreover, a recent study showed that pet owners had an increased risk for the presence of fipronil residue in their homes.14 The study measured fipronil and its degradates in 24 Texas residences and found that the median concentration of total fipronil was 15 times greater in indoor dust than in outdoor dust, and the concentration of fipronil sulfide in indoor dust was four times greater in pet-owners’ residences than non-pet-owners’ residences. Although these residue exposures to fipronil and its metabolites may not produce acute toxicity, the effects of chronic, low-level exposure are

Table 5. Factors that contributed to fipronil exposure—11 states, 2001–2007 (n = 103) Factorsa Inadvertent spray/splash/spill (due to human error, pet behavior, package design, etc.) Inadequate ventilation of treated area before re-entry or early re-entry Required personal protective equipment not worn or inadequate People were in the treated area during application Pesticide stored or used within reach of child Contact with treated pets Inappropriate use (excessive application, outdoor product used indoors) Notification/posting absent or ineffective Off-site movement of pesticides Applicator not properly trained or supervised Other Unknown a

Cases can have more than one contributing factors.

No. of cases 23 16 7 7 7 6 6 4 3 3 7 25

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Acute illnesses associated with fipronil exposure unknown. Furthermore, some fipronil degradates, such as fipronil desulfinyl (the primary photodegradate), are more potent in blocking mammalian chloride channels than fipronil,2 which raises additional concern about the chronic health effects from fipronil residue exposure. Animal studies have demonstrated that fipronil can produce chronic neurological, developmental, reproductive, and endocrine toxicity (summarized in Ref. 2).2 Furthermore, EPA classifies fipronil as a possible human carcinogen based on an increased incidence of thyroid tumors in rats.15 Because chronic health effects are not typically captured by acute pesticide poisoning surveillance systems and are absent from our report, and because little is known about these as related to fipronil, further research is needed to better evaluate chronic health effects of fipronil products. In addition, because the residues of several pesticides may be found in many homes (e.g., permethrin is found in up to 89% of American homes),4 consideration should be given to study the chronic health effects that may arise from exposure to these potentially synergistic pesticide mixtures. We found that exposures often occurred from inadequate ventilation in the treated space before re-entry. This factor contributed to the illness of 16 individuals, and is illustrated in Case 2. Although Case 2 stayed away from her treated apartment even longer than she was told from the pest control operator, she still developed symptoms when she re-entered her home. The label for Termidor® SC, a restricted use pesticide, has the following instruction “DO NOT allow residents, children, other persons or pets into the immediate area during application. DO NOT allow residents, children, other persons or pets into treated area until sprays have dried.”16 A restricted entry interval (REI), which is the time interval after a pesticide application when re-entry should be avoided to prevent exposure to hazardous residues, is provided for some restricted use and over-the-counter pesticides (e.g., total release foggers). Providing recommended re-entry intervals would be more informative for users of fipronil-containing restricted use products, compared to a recommendation to wait for sprays to dry. Also, given that cases became ill after staying in the treated area during the application, precautionary information can be strengthened by instructing residents to leave the treated home, apartment, or structure during the application and until expiration of the REI. The findings in this report are subject to several limitations. First, the number of cases identified by passive surveillance systems likely underrepresents the true magnitude of fipronil-associated illnesses. Additionally, the number of cases is not comparable across states and years because the presented data are case counts, not rates, and also because data from three states were available for only a part of the study period. Second, the surveillance data are limited to acute health effects with a short latency. Third, the data may include false-positive cases because clinical findings of fipronil poisoning are nonspecific and diagnostic tests for fipronil overexposure are not routinely available. Fourth, cases exposed to multiple products may have had some symptoms erroneously attributed to fipronil. Likewise,

743 some health symptoms may have been caused by the solvents and adjuvants present in the fipronil products. Lastly, most cases were identified through poison control centers and information for these cases largely rely on self-reports. However, for the vast majority of these cases, the surveillance systems conduct additional follow-up to embellish the amount of information known, which increases data reliability and quality.

Conclusion In conclusion, exposure to fipronil can pose a risk for mild, temporary health effects in humans. Those using fipronil products should take all necessary precautions to prevent exposure to fipronil. Fipronil users should comply with all label instructions including use of PPE such as eye protection and chemical-resistant gloves. To prevent exposure to post-application surface/air residue, product labels can be improved by adding more detailed precautionary information such as warning about the potential for exposure to humans from contact with treated pets, the length of time the potential for fipronil exposure from treated pets exists, and the duration of REIs before entering treated spaces. Finally, the public, particularly pet-owners, should be aware of the potential presence of residential fipronil residues and employ good hygiene to minimize exposure. Disclaimer: The findings and conclusions in this paper are those of the authors and do not necessarily represent the views of the National Institute for Occupational Safety and Health or each author’s state agency.

Declaration of interest Funding to support this study was provided by NIOSH, EPA, and the state agencies that contributed data. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.

References 1. US Environmental Protection Agency. Fact Sheets on New Active Ingredients, 2010. http://www.epa.gov/opprd001/factsheets. Accessed 1 August 2010. 2. National Pesticide Information Center. Fipronil: Technical Fact Sheet, 2009. http://npic.orst.edu/factsheets/fiptech.pdf. Accessed 1 August 2009. 3. Zhao X, Yeh JZ, Salgado VL, Narahashi T. Fipronil is a potent open channel blocker of glutamate-activated chloride channels in cockroach neurons. J Pharmacol Exp Ther 2004;310:192–201. 4. Stout DM 2nd, Bradham KD, Egeghy PP, Jones PA, Croghan CW, Ashley PA, Pinzer E, Friedman W, Brinkman MC, Nishioka MG, Cox DC. American Healthy Homes Survey: a national study of residential

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5.

6. 7.

8.

9.

10.

pesticides measured from floor wipes. Environ Sci Technol 2009;43:4294–4300. Gasmi A, Chataigner D, Garnier R, Lagier G. Toxicity of fipronilcontaining insecticides. Report of 81 cases from the Paris Poison Center. Vet Hum Toxicol 2001;43:247. Chodorowski Z, Anand JS. Accidental dermal and inhalation exposure with fipronil—a case report. J Toxicol Clin Toxicol 2004;42:189–190. Fung HT, Chan KK, Ching WM, Kam CW. A case of accidental ingestion of ant bait containing fipronil. J Toxicol Clin Toxicol 2003;41:245–248. Mohamed F, Senarathna L, Percy A, Abeyewardene M, Eaglesham G, Cheng R, Azher S, Hittarage A, Dissanayake W, Sheriff MHR, Davies W, Buckley NA, Eddleston M. Acute human self-poisoning with the N-phenylpyrazole insecticide fipronil—a GABAA-gated chloride channel blocker. J Toxicol Clin Toxicol 2004;42:955–963. US Environmental Protection Agency. EPA Evaluation of Pet Spot-On Products: Analysis and Mitigation Plan, 2010. http://www.epa.gov/ pesticides/health/petproductseval.html. Accessed 26 July 2010 Calvert GM, Mehler LN, Alsop J, De Vries AL, Besbelli N. Surveillance of pesticide-related illness and injury in humans. In: Krieger RI, ed.

S.J. Lee et al.

11.

12.

13.

14.

15.

16.

Hayes’ Handbook of Pesticide Toxicology. 3rd ed. New York: Elsevier; 2010:1313–1369. Mehler LN, Schenker MB, Romano PS, Samuels SJ. California surveillance for pesticide-related illness and injury: coverage, bias, and limitations. J Agromedicine 2006;11:67–79. The Humane Society of the United States. U.S. pet ownership statistics, 2009. http://www.humanesociety.org/issues/pet_overpopulation/facts/ pet_ownership_statistics.html. Accessed 3 March 2010. Jennings KA, Canerdy TD, Keller RJ, Atieh BH, Doss RB, Gupta RC. Human exposure to fipronil from dogs treated with frontline. Vet Hum Toxicol 2002;44:301–303. Mahler BJ, Van Metre PC, Wilson JT, Musgrove M, Zaugg SD, Burkhardt MR. Fipronil and its degradates in indoor and outdoor dust. Environ Sci Technol 2009;43:5665–5670. US Environmental Protection Agency. Fipronil; Pesticide Tolerance, 1998. http://www.epa.gov/EPA-PEST/1998/July/Day-17/p18987.htm. Accessed 26 July 2010. BASF Corporation. Termidor SC Termiticide/Insecticide, 2008. http:// www.americanpest.net/docs/msds/termidor-sc-label.pdf. Accessed 26 July 2010.

Clinical Toxicology (2010) 48, 745–749 Copyright © Informa UK, Ltd. ISSN: 1556-3650 print / 1556-9519 online DOI: 10.3109/15563650.2010.502122

ARTICLE LCLT

Potential for erroneous interpretation of poisoning outcomes due to changes in National Poison Data System reporting BRUCE ANDERSON1, XUEHUA KE2, and WENDY KLEIN-SCHWARTZ1 Potential problems with NPDS reporting changes

1

Maryland Poison Center, University of Maryland School of Pharmacy, Baltimore, MD, USA Department of Pharmaceutical Health Services Research, University of Maryland School of Pharmacy, Baltimore, MD, USA

2

Background/Objective. In 2006, the annual report of poison centers in the United States changed the method of reporting profiles for generic substance categories from all exposures to single-substance exposures only. The objective of this study is to describe the potential impact of this reporting change on longitudinal analysis of outcomes. Methods. Generic substance categories with data available for all years of the study were manually extracted from Table 22 of the National Poison Data System (NPDS) annual reports for 2002–2007. For each generic substance category, the following data were extracted for each of the 6 years: total number of substance mentions (2002–2005) or single-substance exposures (2006–2007), reason (unintentional or intentional), pediatric exposures (children age <6 years), and outcomes of major effect and death. Data were compared using descriptive analysis (Wilcoxon signed-rank test) and negative binomial regression. Results. There were 65 generic substance categories (30 drug categories and 35 nondrug categories) that had data in all study years. For drug categories the average annual number of reported deaths by substance category decreased by 80.8%, from 2,229 in year 2002–2005 to 428 after the 2006 reporting change (p < 0.0001). The average annual number of reported major outcomes by substance category dropped by 76.0% (p < 0.0001). The impact on nondrug categories was similar: the annual average number of deaths and major effects by substance category decreased by about 50% from 394 and 4,639 per year during 2002–2005 to 198 deaths (p < 0.0001) and 2,357 major effects (p ≤ 0.0001) during 2006–2007. After controlling for potential covariates, multivariate regression showed that there were significant decreases in average rates of reported deaths (61.7 and 35.9%) and major effects (36.3 and 11.2%) for drug categories and nondrug categories, respectively (p < 0.01 for all). Conclusions. Overall rates of major outcomes and deaths reported to poison control centers from 2002 to 2007 have remained constant. The new method of describing demographic data in Table 22 results in outcomes that are different from those reported in previous NPDS annual reports. Comparing NPDS generic substance outcome data before and after the reporting change in 2006 will yield inaccurate results if the change in reporting methodology is not taken into account. Keywords Poison control centers; Toxicology; Reproducibility of results; Fatality; Epidemiology

Introduction Poison centers in the United States receive calls from the public and health professionals regarding potentially toxic exposures. For each call, data such as patient demographics, toxic effects, treatments, and medical outcomes are documented in online programs and are submitted in real time to the American Association of Poison Control Centers (AAPCC) National Poison Data System (NPDS). An annual report documenting the collective experience of all poison centers in the United States is published annually. From 1983 to 2007, 45,964,981 human exposures have been reported to poison centers.1 These annual reports are frequently cited in medical literature and by various governmental agencies and are viewed as a unique and important source of information Received 15 February 2010; accepted 4 June 2010. Address correspondence to Bruce Anderson, Maryland Poison Center, University of Maryland School of Pharmacy, 220 Arch St, Baltimore, MD 21201, USA. E-mail: [email protected]

on poisoning in the United States. Between 1986 and 2006, the annual reports of the AAPCC were cited in other references a total of 2,574 times. Since the first AAPCC annual report in 1983, several tables report outcomes including Table 11 (“Outcome by age”), Table 12 (“Outcome by reason”), and Table 22, a comprehensive table documenting the demographic profile of exposure cases by generic category of substances and products for non-pharmaceuticals and pharmaceutics. Prior to 2006, the data in Table 22 included all substance mentions (i.e., total exposures), regardless of whether more than one substance was involved. This meant that if a patient died after exposure to three different substances, death outcomes would be noted for each of those generic substance categories in Table 22. Starting in 2006, the table was changed so that data on age categories, reason, health-care facility treatment, and outcomes included single-substance exposures only, although data for all implicated substances continued to be collected (see Images 1 and 2). Tables 11 and 12 are reports of outcomes by case, not by substance. Reporting methods for these tables

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B. Anderson et al.

Image 1. Page 646 of the 2004 annual report of the American Association of Poison Control Centers’ Toxic Exposure Surveillance System.4

Image 2. Page 1020 from the 2007 annual report of the American Association of Poison Control Centers’ National Poison Data System (NPDS).1

did not change over time. Rates of major outcomes and deaths reported in Tables 11 and 12 from 2002 to 2007 remained constant. Changes in data reporting and construction of important tables in the AAPCC NPDS annual report can lead to erroneous conclusions regarding trends in a number of cases and medical outcomes. The purpose of this study was to document the impact of this reporting change on serious outcomes (major effects, death) by comparing the 2006 and 2007 annual reports with earlier reports on poison center data.

Methods The data sources for this study are the published AAPCC annual reports for 2002–2007.1–6 For each generic category, the following data were extracted from Table 22 (“Demographic profile of exposure cases by generic category”) for each of the 6 years: total number of substance mentions (2002–2005) or single-substance exposures (2006–2007),

reason (unintentional or intentional), pediatric exposures (children age < 6 years), and outcomes of major effect and death. As the formatting of the names for some of the generic substance categories in the published annual reports has changed over time, only those generic substance categories that had data available for all 6 study years were analyzed. Descriptive statistics, including Wilcoxon signed-rank test, were used to compare data pre-reporting change (e.g., 2002– 2005) and post-reporting change (e.g., 2006–2007). We calculated average annual numbers of each characteristic (such as death, major effects, total number of exposures, reason, and pediatric exposures) for each generic drug and nondrug category in both pre-change and post-change periods. Wilcoxon signed-rank test was used because the distributions of each characteristic among generic substance categories in drug or nondrug categories were not normally distributed. Negative binomial regression examined whether or not there were differences in the average rates of reporting outcomes of major effect or death pre-2006 versus 2006–2007. The regression adjusted for other factors such as pediatric

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exposures (children age < 6 years), intentional versus unintentional exposures, total annual numbers of exposures, and category types (drug or nondrug categories) to confirm that the observed changes in rates of reporting outcomes of major effect or death were not mainly due to these variables. Negative binomial regression was used because the distributions of major effects and deaths are highly right-skewed and the variances of the variables are much larger than their means. Generalized estimating equations were used to adjust between-category and within-category correlations because of repeated measures on the same categories. We hypothesize that within drug or nondrug substance categories, there is a significant change in the rate of reported deaths or major effects before and after the reporting rule changed. SAS 9.1 (SAS Institute Inc., Cary, NC, USA) was used for all data analyses. The study was submitted to the University of MarylandInstitutional Review Board and determined to not require the board review as it does not involve participation or information from human subjects.

Results Descriptive statistics are presented in Tables 1 and 2. There were a total of 65 generic categories in the analysis (30 drug substance categories and 35 nondrug substance categories). For drug categories the average annual number of reported deaths decreased significantly by 80.8%, from 2,229 during

2002–2005 to 428 after the 2006 reporting change (p < 0.0001; Wilcoxon signed-rank test). The average annual number of major outcomes dropped significantly by 76.0% from 26,865 to 6,460 in the same period (p < 0.0001). From Table 2, for nondrug substance categories, the average annual number of deaths and major outcomes reported decreased significantly from 394 and 4,639 in pre-change period to 198 (p < 0.0001) and 2,357 (p < 0.0001) in the post-change period, respectively. These each represent a decrease of approximately 50%. Differences between the pre- and postchange period were also significant for total number of exposures, reason, and pediatric exposures for both drug substance categories and nondrug substance categories (p < 0.05). Regression results for death counts show that there was no significant difference in average rates of reported deaths across years for nondrug categories before year 2006 (p = 0.2185), while in 2006–2007 the average rate of reported deaths by substance category was significantly less than that in the prechange period by 35.9% (p = 0.0027), when holding other factors fixed (pediatric exposures, intentional exposures, unintentional exposures, total exposures). For drug categories, before the reporting change, the average rates of reported deaths by substance category were not significant (p = 0.1003) across years. Starting in 2006, the average rate of reported deaths in drug categories decreased significantly by 61.7% (p < 0.0001) when compared to that before the reporting change. Regression results for major outcomes demonstrate that after adjusting other covariates such as annual total number of exposures, annual number of cases in those aged less than

Table 1. Descriptive statistics for characteristics of drug substance categories from NPDS’ Table 22 before and after reporting change (n = 30) Characteristics Deaths Major effects Exposures Pediatric exposures (age < 6 years) Intentional exposures Unintentional exposures

Average annual count 2002–2005

Average annual count 2006–2007

p-Value*

2,229 26,865 1,354,883 570,541 395,817 887,371

428 6,460 1,416,767 521,396 160,739 758,860

<0.0001 <0.0001 0.0004 0.0007 <0.0001 0.0002

NPDS, National Poison Data System. *Wilcoxon signed-rank tests were used to compare data pre-change (2002–2005) and post-change (2006–2007).

Table 2. Descriptive statistics for characteristics of nondrug substance categories from NPDS’ Table 22 before and after reporting change (n = 35) Characteristics Deaths Major effects Exposures Pediatric exposures (age < 6 years) Intentional exposures Unintentional exposures

Average annual count 2002–2005

Average annual count 2006–2007

p-Value*

394 4,639 1,381,694 714,508 72,364 1,271,044

198 2,357 1,339,685 672,718 36,110 1,156,788

<0.0001 <0.0001 0.0439 0.0072 0.0024 ≤0.0001

NPDS, National Poison Data System. *Wilcoxon signed-rank tests were used to compare data pre-change (2002–2005) and post-change (2006–2007).

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Discussion Changes in reporting are a natural evolution of a data collection system. The reporting system for the AAPCC has evolved several times over 25 years. The purpose of this report is to illustrate how a specific change in reporting can markedly impact the interpretation of poison center data and trends. Changes in the reporting of NPDS Table 22 (“Demographic profile of exposure cases by generic category”) that occurred effectively in 2006 can lead to the erroneous conclusion that there has been a reduction in the reported number of deaths and major effects reported to U.S. poison centers over time. This study evaluated 65 generic substance categories and found significant decreases in average annual number of reported major effects and deaths in Table 22 in 2006 and 2007 compared to 2002–2005. These differences persisted after controlling for covariates. Therefore, these data show that the “apparent” drop in death counts and major outcome counts can be primarily attributed to the change in reporting methodology. The reporting change was instituted to allow for a more precise evaluation of the association between substances and outcome. When more than one substance is involved, it is difficult to elucidate the extent to which each substance contributed to outcome, especially in a summary table. Certain substances such as ethanol might be associated with major effects and deaths as a co-ingestant in more serious intentional exposures. The change in reporting was intended to eliminate this uncertainty regarding relative contribution of multiple substances to outcome. Unfortunately, the reporting change may be unappreciated by many users of these data. The NPDS annual reports document that data are presented differently in Table 22 in 2006 and 2007 compared to previous reports. In both of these reports running into well over 100 pages, there is a warning immediately following the abstract that “Comparison of exposure or outcome data from previous AAPCC Annual Reports is problematic. . . . Table 22 (Exposure cases by generic category) restricts the breakdown including deaths to single substance cases to improve precision and avoid misrepresentation.” This statement is close to 55 pages prior to

the start of Table 22. Although, the table title states “single substance,” because of the fact that the first data column is “number of case mentions” (i.e., includes cases with more than one substance), one might misinterpret the fact that the remaining columns (age, reason, health-care facility treatment, outcome) are single substance only. It is easy to envision how health professionals, public health agencies, industry, laypersons, and the media, who are looking for trends in poison center data over time, may not fully appreciate the impact of the reporting change. This reporting change could have profound research, public health, and public reporting implications if not taken into account. To illustrate the potential problems, the impact of this reporting change on oxycodone can be seen in Fig. 1, which plots the change in total numbers of exposures, major effects, and deaths over time for oxycodone from 2002 to 2007. The annual average number of cases of oxycodone deaths reported to U.S. poison centers prior to this reporting change was 44.8. After the reporting change, the annual average number of oxycodone deaths drops to 8.0. Without an appreciation for the change in reporting, these apparent improved outcomes may be erroneously attributed to advances in poisoning patient management or to public health programs to distribute naloxone in communities with major opioid addiction issues. The pharmaceutical industry could use these data to support a position that the magnitude of the problem of serious oxycodone abuse/misuse has diminished such that further regulation is unnecessary. The reporting change could lead to “unexplained” discrepancies between poison center data and other monitoring systems such as the Drug Abuse Warning Network, especially when patterns of prescription opioid abuse/misuse are studied. Policy analysts may erroneously conclude that public health efforts undertaken to address prescription opioid abuse have resulted in improved outcomes. Misinterpreting these findings could lead to inaccurate assessment of the impact of prevention efforts.

10,000

1,000 Log of count

6 years, annual number of intentional cases, and annual number of unintentional cases, for nondrug categories, before the year 2006, there was no significant difference in average rates of reported major outcomes by substance category across years (p = 0.4992). Starting in 2006, the rate of reported major effects by substance category dropped significantly by 11.2% (p = 0.0002) compared with that in pre-change period. For drug categories, before the reporting change, the average rates of reported major outcomes decreased by approximately 6.8% (p < 0.0001) per year. Starting in 2006, the average rate for reported major effects in drug categories decreased significantly further by 36.2% (p < 0.0001) when compared to that in pre-change period.

B. Anderson et al.

Total

100

Major Death

10

1

2002

2003

2004

2005

2006

2007

Fig. 1. Changes in outcome over time for oxycodone.

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Potential problems with NPDS reporting changes Potential solutions to the problems associated with the reporting change include reverting to the original reporting method or devising a mechanism to present both the singlesubstance and multi-substance exposure data in Table 22. Given the length of the annual report the latter may be difficult to achieve in the printed publication. However, the annual report is available on the AAPCC Web site and could include an addendum with the second version of Table 22. Simply having a second version will alert users of the data to the fact that they need to be vigilant about how they use and interpret the data.

Conclusion The overall rate of outcomes and deaths has not changed over time. However, definitions for reporting outcomes in Table 22 of the AAPCC NPDS annual report changed between 2005 and 2006. The new reporting method means that outcomes are reported only for single-substance exposures. For substances that are commonly co-ingested with other substances, comparing previous data may yield inaccurate results if the change in reporting is not appreciated. The result of this reporting change is that researchers and public policy analysts who rely on the published NPDS annual reports will not be able to perform accurate longitudinal outcome assessments by substance category that cover pre- and post-reporting change periods.

749

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.

References 1. Bronstein AC, Spyker DA, Cantilena LR Jr, Green JL, Rumack BH, Heard SE. 2007 annual report of the American Association of Poison Control Centers’ National Poison Data System (NPDS): 25th annual report. Clin Toxicol 2008; 46(10):927–1057. 2. Watson WA, Litovitz TL, Rodgers GC, Klein-Schwartz W, Youniss J, Rose RS, Borys D, May M. 2002 annual report of the American Association of Poison Control Centers Toxic Exposure Surveillance System. Am J Emerg Med 2003; 21:353–421. 3. Watson WA, Litovitz TL, Rodgers GC, Klein-Schwartz W, Youniss J, Reid N, Rouse WG, Rembert RS, Borys D. 2003 annual report of the American Association of Poison Control Centers Toxic Exposure Surveillance System. Am J Emerg Med 2005; 22:335–404. 4. Watson WA, Litovitz TL, Rodgers GC, Klein-Schwartz W, Reid N, Youniss J, Flanagan A, Wruk KM. 2004 annual report of the American Association of Poison Control Centers Toxic Exposure Surveillance System. Am J Emerg Med 2005; 23:335–404. 5. Lai MW, Klein-Schwartz W, Rodgers GC, Abrams JY, Haber DA, Bronstein AC, Wruk KM. 2005 annual report of the American Association of Poison Control Centers National Poisoning and Exposure Database. Clin Toxicol 2006; 44:803–932. 6. Bronstein AC, Spyker DA, Cantilea LR Jr, Green J, Rumack BH, Heard SE. 2006 annual report of the American Association of Poison Control Centers National Poison Data System (NPDS). Clin Toxicol 2007; 45:815–917.

Clinical Toxicology (2010) 48, 750–751 Copyright © Informa UK, Ltd. ISSN: 1556-3650 print / 1556-9519 online DOI: 10.3109/15563650.2010.489902

BRIEF COMMUNICATION LCLT

Dextrose 50% as a better substitute for saturated salt solution in mothball float test KA YUEN TANG1, CHI KEUNG CHAN2, and FEI LUNG LAU2 Dextrose 50% in mothball float test

1

Accident and Emergency Department, Caritas Medical Centre, Hong Kong, China Hong Kong Poison Information Centre, Hong Kong, China

2

We describe the use of dextrose 50% solution to differentiate naphthalene and paradichlorobenzene in the mothball float test. Its advantages over saturated salt solution are discussed. Keywords Mothballs; Naphthalenes; Paradichlorobenzenes; Poisoning

Children may present to hospitals following accidental ingestion of mothballs of three types: camphor, naphthalene, and paradichlorobenzene. These are commonly available in most countries. It is important to identify the mothball ingredients as the toxicities, diagnostic evaluations, and treatments are different. The mothball float test first described by Koyama can help differentiate the three types of mothballs.1,2 Naphthalene mothballs float in saturated salt solution but sink in water. Paradichlorobenzene mothballs sink in both water and saturated salt solution. Camphor mothballs float in both water and saturated salt solution. The test depends on their specific gravities, and they are listed in ascending order as follows: camphor 0.980, water 1.000, naphthalene 1.100, saturated salt solution 1.197, and paradichlorobenzene 1.437.1,2 Among the various medical solutions, we noted that the specific gravity of dextrose 50% is around 1.180,3 which lies between naphthalene and paradichlorobenzene, so naphthalene should float in dextrose 50% whereas paradichlorobenzene should sink in it. As dextrose 50% is more readily available and more quickly prepared in hospitals than saturated salt solution, we decide to test whether dextrose 50% can differentiate naphthalene mothballs from paradichlorobenzene mothballs. The specific gravities of other medical solutions such as mannitol 20% (1.099), gelofusine (1.017), hypertonic saline 5.85% sodium chloride (1.041), and dextrose 10% (1.034) should preclude their use in the mothball float test, yet they were also tested in this study. Ten naphthalene mothballs and ten paradichlorobenzene mothballs, all made from different manufacturers, were tested

Received 13 April 2010; accepted 26 April 2010. Address correspondence to Ka Yuen Tang, Accident and Emergency Department, Caritas Medical Centre, 111 Wing Hong Street, Sham Shui Po, Hong Kong, China. E-mail: [email protected]

in this study. The weights of the mothballs tested ranged from 3 to 40 g. They were either in the form of “ball,” “tablet,” “cake,” or “triangular bar.” We put each mothball into the following solutions: dextrose 50%, saturated salt solution, mannitol 20%, gelofusine, hypertonic saline 5.85%, and dextrose 10% to see whether it sank or floated. Saturated salt solution was prepared according to Koyama by putting three

Fig. 1. The naphthalene mothball (arrow) floated while the paradichlorobenzene mothball (arrowhead) sank in dextrose 50%. The weights of both mothballs were the same (40 g).

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Dextrose 50% in mothball float test

751

Fig. 2. The naphthalene mothball sank in a salt solution with inadequate stirring for 10 s. Noted the presence of undissolved salt in the solution (left). The naphthalene mothball started to float in the salt solution after stirring for 20 s (middle). The naphthalene mothball floated freely in the salt solution after stirring for 45 s (right).

tablespoonfuls of table salt (45 g) into half a glass of water (100 mL) and stirring it vigorously for 45 s, or until the salt does not dissolve any more.1 All the 10 naphthalene mothballs floated and all the 10 paradichlorobenzene mothballs sank in saturated salt solution and dextrose 50% (Fig. 1). Both dextrose 50% and saturated salt solution were 100% accurate in differentiation between naphthalene mothballs and dichlorobenzene mothballs in this study (p < 0.001). Other medical solutions including mannitol 20%, gelofusine, hypertonic saline 5.85%, and dextrose 10% failed to differentiate naphthalene mothballs from paradichlorobenzene mothballs. Although both dextrose 50% and saturated salt solution performed equally well in this study, there are several advantages of using dextrose 50% over saturated salt solution in mothball float test. First, if the salt solution is not saturated, naphthalene mothballs may sink, leading to a false interpretation.1 We noted in this study that it happened especially when not enough salt was used or not enough time was taken to stir the solution (Fig. 2). On the other hand, the quality control of dextrose 50% is usually well prepared by the suppliers and can thus give a more consistent result. The mothball float test was originally developed for household use, whereas in hospitals or clinics dextrose 50% solutions were more readily available. Third, 100 mL of dextrose 50% is more quickly prepared than saturated salt solution because you do not need to take time to stir the solution. Fourth, dextrose 50% is cheap and costs only HK $7.5 (US $1) for 100 mL. We suggest that dextrose 50% should be used as a

substitute of saturated salt solution in mothball float test in hospitals, whereas saturated salt solution should be applied in household use.

Acknowledgment I am grateful to B. Braun Medical Ltd. for providing information on specific gravities of its products.

Declaration of interest The authors report no declarations of interest. The authors alone are responsible for the content and writing of this paper.

References 1. Koyama K, Yamashita M, Ogura Y, Ando Y, Fukuda T, Matsuzaki Y. A simple test for mothball component differentiation using water and a saturated solution of table salt: its utilization for poison information service. Vet Hum Toxicol 1991; 33:425–427. 2. Fukuda T, Koyama K, Yamashita M, Koichi N, Takeda M. Differentiation of naphthalene and paradichlorobenzene mothballs based on their difference in specific gravity. Vet Hum Toxicol 1991; 33(4):313–314. 3. Sparks DR, Chimbayo A, Cripe J, Smith R, Boring K, Najafi N. Preventing medication infusion errors & venous air embolisms using a micromachined specific gravity sensor. Drug Deliv Technol 2004; 4(4). http:// www.mems-issys.com/pdf/issystech16.pdf. Accessed 8 April 2010.

Clinical Toxicology (2010) 48, 752–754 Copyright © Informa UK, Ltd. ISSN: 1556-3650 print / 1556-9519 online DOI: 10.3109/15563650.2010.498379

BRIEF COMMUNICATION LCLT

Hepatorenal injury induced by cutaneous application of Atractylis gummifera L. ASMA BOUZIRI, ASMA HAMDI, KHALED MENIF, and NEJLA BEN JABALLAH Poisoning by Atractylis gummifera L.

Pediatric Intensive Care Unit, Children’s Hospital of Tunis, Tunis, Tunisia

Introduction. In Mediterranean countries, intoxication by Atractylis gummifera L. is frequent and characterized principally by hepatorenal injury, often fatal. Its toxicity after a cutaneous application is unknown. We report a case of poisoning by A. gummifera L. induced by repeated cutaneous application. Case report. A 30-month-old boy was admitted in our pediatric intensive care unit in coma (Glasgow Coma Scale 8). Investigations showed hepatic cellular injury, cholestasis, decreased prothrombin level, and increased creatinine. History from the parents revealed repeated and occlusive cutaneous application of A. gummifera L. on a skin burn. Qualitative analysis of urine confirmed the diagnosis of A. gummifera poisoning. The child was discharged after 16 days of hospitalization with residual renal insufficiency. Discussion. Poisoning by A. gummifera L. after cutaneous application has not previously been reported in the literature. The prevention of this poisoning, particularly frequent in Mediterranean countries, is mainly based on the education of the public concerning the dangers of this plant. Keywords Acute poisoning; Hepatotoxicity; Herbs; Acute renal failure

Introduction Atractylis gummifera L. (bird-lime or blue thistle) is an herbaceous and thorny plant of the family of Asteraceae widely spread in Mediterranean countries. Published reports indicate oral ingestion results in life-threatening poisoning. The plant is a public health concern for children in developing countries where it grows.1–3 The potential for toxicity after cutaneous application is unknown. We report a case of poisoning by A. gummifera L. in a 30-month-old boy induced by repeated cutaneous application.

Case report A previously healthy 30-month-old Tunisian boy was admitted to our pediatric intensive care unit in coma with a 2 days history of fever and vomiting. On physical examination, the child was comatose (Glascow Coma Scale 8), with pulse of 183 beats per minute, blood pressure of 94/43 mmHg, respiratory rate of 41 breaths per minute, dextrostix of 0.5 g/L, temperature of 36.4°C, and decrease in urine output (0.3 mL/kg/h during the first day of hospitalization) without focal neurological signs or convulsions. He had icteric sclera and a deep Received 31 March 2010; accepted 31 May 2010. Address correspondence to Asma Bouziri, Pediatric Intensive Care Unit, Children’s Hospital of Tunis, Tunis, Tunisia. E-mail: [email protected]

second-degree burn of the left arm (burn cutaneous surface estimated to 2–3% of the body surface). The hemodynamic and hydration state of the child were normal. He was intubated and ventilated for coma. History from the parents revealed the occurrence, 6 days previously, of a boiling water burn of the left arm which was treated by repeated local application of occlusive bandage containing cooked A. gummifera. The plant was prepared by boiling crude cut root in 1 L of water for approximately 30 min. The bandage was changed every 12 h. We eliminated also, questioning, the possibility of an accidental ingestion of the plant by the child. Lumbar puncture was normal. The initial biological results were the following: blood urea, 58.4 mmol/L (normal 3.5–6.5 mmol/L); blood creatinine, 482 μmol/L (normal 60–120 μmol/L); sodium, 134 μmol/L; potassium, 7.4 mmol/L; glucose, 3.32 mmol/L; calcium, 2 μmol/L; AST 1,830 IU/L (normal <20 IU/L); ALT, 4,170 IU/L (normal <45 IU/L); total bilirubin, 9.2 mg/dL (reference range 0–1.1 mg/dL); direct bilirubin, 7.1 mg/dL (reference range 0–0.3 mg/dL); alkaline phosphatase, 320 IU/L (normal range 44–147 IU/L); INR, 5.06; hemoglobin, 12.5 g/dL; white blood cell count, 21,200/mm3; platelet count, 236,000/mm3. The research for schizocytes on the blood smear was negative. The arterial blood gas was normal. Viral hepatitis A (IgM anti-VHA), B (antigen HBs), and C serology was negative. Paracetamol was undetectable. Several causes of hepatorenal injury were then considered such as hepatorenal damage after burn, hepatorenal syndrome after sepsis, Reye syndrome, A. gummifera systemic poisoning, and hepatobiliary duct injuries.

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Poisoning by Atractylis gummifera L. A toxicological qualitative analysis of urine and gastric samples, taken at admission, was performed using a thin layer chromatography on silica gel. The presence of atractyloside (ATR) was revealed by a color-developing agent (para-dimethylaminobenzaldehyde) giving a purple spot and a characteristic smell of valerianic acid after passage in the steam room. This analysis was negative in the gastric liquid and positive in the urine confirming the diagnosis of A. gummifera poisoning. A few hours after admission, the child developed ventricular tachycardia with hypotension, which was immediately treated by external cardioversion. This dysrhythmia was attributed to severe hyperkalemia, and peritoneal dialysis was performed urgently. The clinical course was of progressive restoration of consciousness allowing the extubation of the child after 4 days. The evolution of the biological parameters was marked by the improvement of the hepatic and renal function (INR, 1.6; ALT, 278 IU/L; blood urea, 32 mmol/L; and creatinine, 250 μmol/L before discharge). The child was transferred to a pediatric general ward after 6 days of hospitalization in the pediatric intensive care unit. He was discharged from the hospital after 10 days with residual kidney insufficiency requiring conservative treatment.

Discussion Atractylis gummifera L. is a rural plant easily accessible in Mediterranean countries. The poisoning by this plant is mostly accidental and occurred mainly after oral ingestion of the whitish substance secreted by the plant which looks like chewing gum.2,4 Rarely, the poisoning can be caused by the use of A. gummifera L. as medicinal plant because of its antipyretic, diuretic, abortive, purgative, and emetic properties.5,6 In the dried state, this plant is also used in folk medicine in local application for its healing properties (e.g., for skin, abscesses).6 In our patient, the most likely source for toxicity is dermal and not oral because the area of the skin burn was dressed each time and the child did not have access to the plant. Review of the PubMed literature suggests no previous reports of poisoning by A. gummifera L. after dermal exposure. The misunderstanding of the toxicity of this plant explains the occurrence of accidents by excessive uses such as that observed in our patient. All the parts of the plant are toxic, with toxicity lessening from root to leaves.2 The major toxic components of the A. gummifera L. are diterpenoid glucosides: ATR (atractylate of potassium) and carboxyatractyloside (gummiferrine). Several factors, including the climate, the composition of the soil, the time of harvest, and genetic factors, influence the content of diterpenoid glucosides in the rhizome of A. gummifera.7 ATR, isolated mainly from the roots of this plant, is a powerful inhibitor of oxidative phosphorylation and the Krebs cycle in mitochondria. This action is exerted especially in cells rich in mitochondria such as hepatocytes and in proximal tubular epithelial cells, which contain carriers that

753 allow ATR to cross the cell membrane. ATR interacts with the adenine nucleotide translocator, a mitochondrial protein contained in the inner membrane. This protein is responsible for the antiport of ATP and ADP, an important system for oxidative phosphorylation and constitutes a part of the permeability transition pore complex involved in mitochondrial membrane permeabilization. The selective binding of ATR to this protein has two consequences. First, ATR inhibits ADP transport, thus blocking oxidative phosphorylation and Krebs cycle reactions. Second, ATR induces opening of the mitochondrial permeability leading to release of soluble intermembrane proteins, including cytochrome c. The translocation of cytochrome c from mitochondria to the cytosol is a crucial step in Fas-induced apoptosis. The inhibition of mitochondrial phosphorylation leads to hepatic necrosis and renal failure in animals and humans.7 It is also the cause of increase in glucose utilization with exhaustion of the hepatic and muscular stocks of glycogen and inhibition of the glucuneogenesis explaining hypoglycemia, a characteristic feature of ATR poisoning, that can be present in the absence of liver failure.1,7,8 The other typical symptoms of A. gummifera poisoning are gastrointestinal problems including nausea, vomiting, epigastric and abdominal pain, and diarrhea.8 Some reports also describe general anxiety, headache, drowsiness, arrhythmia, and convulsions.2 In several cases, these symptoms are followed by coma. The laboratory findings may indicate severe hepatocellular damage (marked increased in AST, ALT, and bilirubin) and acute renal failure.7,8 Our patient developed a severe form of A. gummifera poisoning with gastrointestinal upset, coma, hepatic failure, acute renal failure, and arrhythmia. The ventricular tachycardia observed in our patient can be attributed either to a direct cardiovascular toxicity of the plant or to severe hyperkalemia. The diagnosis of A. gummifera poisoning is based on the clinical history, the symptoms, and the identification of the toxins of the plant in the urine or in the gastric liquid.1,2,9 Simple methods for the detection of ATR poisoning are at present restricted to thin layer chromatography in urine and gastric liquid and are useful only in the case of severe poisoning such as in our case. Immunoassays, high-performance liquid chromatography, nuclear magnetic resonance, and a recently developed high-performance liquid chromatography/mass spectrometry method have yet to be applied to clinical diagnoses.10 No specific pharmacological treatment is currently available to treat A. gummifera intoxications. All therapeutic approaches, including fluid and electrolyte replacement, cardiovascular and respiratory support, seizure control, and conventional therapeutic methods for severe hepatic and renal failure, are symptomatic.2,7,9 The prognosis in severe forms is often poor with a fatal outcome.2 It is mainly determined by fulminant hepatitis and acute renal insufficiency.2,9 In spite of the severity of symptoms presented by our patient, he recovered with only renal sequelae. This report illustrates an original case of A. gummifera poisoning induced by repeated and occlusive cutaneous

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754 application of this plant on a skin burn leading to a severe form of toxicity with coma, hepatic failure, acute renal failure, and arrhythmia. The prevention of this life-threatening poisoning is mainly based on the education of the public concerning the dangers of this plant.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper. Informed consent was obtained from parents before the submission of this article.

References 1. Skalli S, Alaoui I, Pineau A, Zaid A, Soulaymani R. Atractylis gummifera L. poisoning: a case report. Bull Soc Pathol Exot 2002; 95:284–286.

A. Bouziri et al. 2. Hamouda C, Hédhili A, Ben Salah N, Zhioua M, Amamou M. A review of acute poisoning from Atractylis gummifera L. Vet Hum Toxicol 2004; 46:144–146. 3. Eddelston M, Persson H. Acute plant poisoning and antitoxin antibodies. J Toxicol Clin Toxicol 2003; 41:309–315. 4. Megueddem M, Djafer R. Intoxication au chardon à glu. Faculté de médecine d’Annaba, Algérie. Toxicol Clin 2002; 18:5–10. 5. Peyrin-Biroulet L, Barraud H, Petit-Laurent F, Ancel D, Watelet J, Chone L, Hudziak H, Bigard MA, Bronowicki JP. Hépatotoxicité de la phytothérapie: données cliniques, biologiques, histologiques et mécanismes en cause pour quelques exemples caractéristiques. Gastroenterol Clin Biol 2004; 28:540–550. 6. Chardon G, Viala A, Vignais P, Stanislas A. L’intoxication par le chardon à glu. Thérapie 1964; 19:1313–1322. 7. Daniele C, Dahamna S, Firuzi O, Sekfali N, Saso L, Mazzanti G. Atractylis gummifera L. poisoning: an ethnopharmacological review. J Ethnopharmacol 2005; 97:175–181. 8. Georgiou M, Sianidou L. Hepatotoxicity due to Atractylis gummifera L. Clin Toxicol 1988; 26:487–493. 9. Madani N, Sbaï H, Harandou M, Boujraf S, Achour S, Khatouf M, Kanjaa N. Atractylis gummifera poisoning in a pregnant woman. Presse Med 2006; 35:1828–1830. 10. Stewart MJ, Steenkamp V. The biochemistry and toxicity of atractyloside: a review. Ther Drug Monit 2000; 22:641–649.

Clinical Toxicology (2010) 48, 755–756 Copyright © Informa UK, Ltd. ISSN: 1556-3650 print / 1556-9519 online DOI: 10.3109/15563650.2010.508044

BRIEF COMMUNICATION LCLT

Hydrogen sulfide toxicity in a thermal spring: a fatal outcome HALE DALDAL, BAYRAM BEDER, SIMAY SERIN, and HULYA SUNGURTEKIN Hydrogen sulfide toxicity in a thermal spring

Department of Anaesthesiology and Reanimation, Pamukkale University School of Medicine, Kinikli, Denizli Turkey

Introduction. Hydrogen sulfide (H2S) is a toxic gas with the smells of “rotten egg”; its toxic effects are due to the blocking of cellular respiratory enzymes leading to cell anoxia and cell damage. Case presentation. We report two cases with acute H2S intoxication caused by inhalation of H2S evaporated from the water of a thermal spring. Two victims were found in a hotel room were they could take a thermal bath. A 26-year-old male was found unconscious; he was resuscitated, received supportive treatment and survived. A 25-year-old female was found dead. Autopsy showed diffuse edema and pulmonary congestion. Toxicological blood analysis of the female revealed the following concentrations: 0.68 mg/L sulfide and 0.21 mmol/L thiosulfate. The urine thiosulfate concentration was normal. Forensic investigation established that the thermal water was coming from the hotel’s own illegal well. The hotel was closed. Conclusion. This report highlights the danger of H2S toxicity not only for reservoir and sewer cleaners, but also for individuals bathing in thermal springs. Keywords

Inhalation exposure; Acute toxicity; Hydrogen sulfide

Introduction Hydrogen sulfide (H2S) is a flammable, colorless gas with a characteristic odor of rotten egg. It is also present in sewers in a high concentration and occurs naturally in natural gas, volcanic gases, and near thermal springs.1 The major route of entry into the body is through the lungs. The mechanism of the toxicity is related primarily to inhibition of oxidative phosphorylation in the oxidative processes of cells.2 We report two cases of acute H2S toxicity which was occurred by inhalation of H2S in Pamukkale thermal springs.

Case presentation A 26-year-old male and a 25-year-old female checked in at a thermal hotel in Pamukkale thermal springs at 02.00 am and requested a wake-up call at 08.00 am. When they did not respond to the wake-up service, the hotel staff entered the room and the two individuals were found unconscious. Emergency services were called. The female patient was found dead near the bathroom on the floor, the male patient was found unconscious in bed, some distance from the bathroom when paramedics arrived. He was rapidly transferred to the Pamukkale University Hospital, approximately 20 min by ambulance. He was breathing spontaneously and received O2 therapy during transport. Emergency responders reported a Received 19 May 2010; accepted 9 July 2010. Address correspondence to Hale Daldal, Department of Anaesthesiology and Reanimation, Pamukkale University School of Medicine, Kinikli, Denizli, Turkey. E-mail: [email protected]

very strong, pungent odor in the hotel room, and the bathroom taps were found open. At presentation the man was unconscious, his Glasgow coma scale was 5/15, respiratory rate was 28/min, blood pressure was 116/85 mmHg, pulse was 135 beats/min, and peripheral oxygen saturation was 74% in the emergency department. He was transferred to the intensive care unit. The result of blood gas analysis was pH 7.09, PO2 52 mmHg, PCO2 72 mmHg, bicarbonate and lactate concentrations were normal. He had sinus tachycardia on ECG and pulmonary edema on chest X-ray. Total WBC count was 29.700 cells/μL. His serum chemistry showed mildly elevated liver enzymes (AST 56 U/L, ALT 47 U/L), normal renal function tests, and electrolytes. Alcohol and drugs of abuse were not detected on screening. He was rapidly intubated with propofol IV 2 mg/kg and high frequency mechanical ventilation therapy was administered. Diuretic therapy was started for the treatment of noncardiogenic pulmonary edema. He received ventilation therapy for 16 h. His consciousness level improved and he was extubated after 18 h. He was discharged well on the third day of hospitalization. He has had no sequela and cognitive function is normal. The deceased woman’s autopsy was performed 3 h after death and revealed findings compatible with H2S toxicity. On making the main incision, a pungent characteristic rotten-egg odor spread in the autopsy room and present in the thermal spring room supported our suspicion of H2S toxicity. Macroscopic findings were similar to findings of general asphyxia. Petechia hemorrages were noted on serous membranes; the left lung revealed diffuse subpleural bleeding on its surface. There was diffuse edema and pulmonary congestion. Blood

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756 and urine samples and lung, liver, kidney and cardiac muscle tissue samples were taken for toxicological analysis. Gas chromatography/mass spectrometry analysis showed 0.68 mg/L sulfide and 0.21 mmol/L thiosulfate in the blood sample. The concentration of thiosulfate in the urine sample was normal. There were no other toxic agents found in toxicological analysis.

Discussion H2S is a colorless gas heavier than air with a characteristic odor of rotten eggs. Odor is an unreliable guide to high concentrations of the gas because it causes rapidly olfactory tolerance.2 H2S exposures occur almost exclusively by the inhalational route. H2S occurs naturally in crude petroleum, natural gas, volcanic gases, and thermal springs.3 Several cases of H2S toxicities had been reported in the literature. Most of these cases were chronic toxicity; however, acute toxicities are also reported in industrial workers, sewers, and the individuals who committed to suicide with H2S content of chemicals.1,3–7 Normally H2S exposures, such as in reservoir cleaning, are due to organic materials (i.e., mud or sewage) which are known to contain H2S.8 The interesting feature of this case is the H2S exposure in a thermal spring. Ordinarily, thermal water is collected in a control center and is distributed through the networks to the hotels in Pamukkale after toxicological and microbiological analyses. The forensic investigation revealed that the hotel in which the victims stayed was not using the thermal water through the centralized network. Thermal water used in this hotel was from an illegal artesian well. Thus the use of unprocessed thermal water through the individual artesian directly is believed to be the cause of the accumulation of H2S. H2S in the body is detoxified rapidly by oxidation into sulfide or thiosulfate. Kage et al. reported that thiosulfate in

H. Daldal et al. urine is the only indicator to prove H2S toxicity in non-fatal cases, while the analysis of sulfide in fatal cases should be accompanied by the measurement of thiosulfate in blood.4 The concentrations of sulfide and thiosulfate in blood samples from the female victim were 0.68 mg/L and 0.21 mmol/L, respectively. The concentrations were at least 14 and 7 times higher than the levels in healthy persons. In contrast to this high level in the blood, thiosulfate was not detected in the urine sample (below 0.003 mmol/L), perhaps suggesting rapid death. In conclusion, this report highlights the danger of H 2S toxicity, not only in reservoir and sewer cleaners in the thermal springs, but also the individuals who bath with thermal water in thermal springs.

References 1. Nikkanen HE, Burns MM. Severe hydrogen sulfide exposure in a working adolescent. Pediatrics 2004; 113:927–929. 2. Kerns W. Cyanide and hydrogen sulfide. In: Goldfrank LR, Flomenbaum NE, Lewin NA, eds. Goldfrank’s Toxicologic Emergencies. NewYork: McGraw-Hill, 2002:1498–1510. 3. Lee EC, Kwan J, Leem JH. Hydrogen sulfide intoxication with dilated cardiomyopathy. J Occup Health 2009; 51:522–525. 4. Kage S, Ikeda H, Ikeda N, Tsujita A, Kudo K. Fatal hydrogen sulfide poisoning at a dye works. Leg Med 2004; 6:182–186. 5. Knight LD, Presnell SE. Death by sewer gas: case report of a double fatality and review of the literature. Am J Forensic Med Pathol 2005; 26:181–185. 6. Gerasimon G, Bennett S, Musser J, Rinard J. Acute hydrogen sulfide poisoning in a dairy farmer. Clin Toxicol (Phila) 2007; 45:420–423. 7. Kobayashi K, Fukushima H. Suicidal poisoning due to hydrogen sulfide produced by mixing a liquid bath essence containing sulfur and a toilet bowl cleaner containing hydrochloric acid. Chudoku Kenkyu 2008; 21:183–188. 8. Kage S, Ito S, Kishida T, Kudo K, Ikeda N. A fatal case of hydrogen sulfide poisoning in a geothermal power plant. J Forensic Sci 1998; 43:908–910.

Clinical Toxicology (2010) 48, 757–761 Copyright © Informa UK, Ltd. ISSN: 1556-3650 print / 1556-9519 online DOI: 10.3109/15563651003749290

ARTICLE LCLT

Lead poisoning from use of bronze drinking vessels during the late Chinese Shang dynasty: an in vitro experiment ALAN D. WOOLF1, TERENCE LAW2, HOI-YING ELSIE YU2, NICHOLAS WOOLF3, and MARK KELLOGG2 Lead poisoning in Shang dynasty bronzes

1

Department of Medicine, Children’s Hospital Boston, Boston, MA, USA Department of Laboratory Medicine, Children’s Hospital Boston, Boston, MA, USA 3 Connecticut College, New London, CT, USA 2

Introduction. Bronze drinking vessels famous for their intricate carvings and used by the aristocracy in the Chinese Shang dynasty (1555– 1145 BCE) are known to have been fabricated with alloys containing soft metallic lead. The contribution of lead leaching from such vessels into the fermented grain wines drunk by the Chinese nobility in ancient times has not been previously estimated. Methods. Three bronze vessels containing 8% lead by weight were fabricated to resemble the late Shang bronze goblets. Shaoxing drinking rice wine was purchased locally and placed in the vessels, using a white grape wine and water as comparisons. Sampling was performed at baseline, 2 min, and then at days 1, 2, 4, and 7. Lead concentrations in the liquid matrix were measured using atomic absorption spectroscopy. Results. Significant amounts of lead leached into the liquid within one day: 13,900 μg/L in water, 45,900 μg/L in rice wine, and 116,000 μg/L in white wine. Lead continued to leach into both the grape and rice wines with the passage of time. Discussion. Significant lead contamination of Shaoxing rice wine was detected when it was left in bronze goblets fabricated to resemble the Shang dynasty vessels. If a liter of contaminated wine was drunk daily, the daily intake of lead could have been as high as 85 mg. Such a high degree of contamination could cause chronic lead poisoning, affecting the health of the Shang nobility who used bronze beverage containers, before lead was excluded from the manufacture of bronze. Keywords

Lead poisoning; Poisoning; Toxicity; Intoxication

Lead was one of the first metals used by civilizations dating back thousands of years. Lead pieces have been found in excavations in Troy (3000–2500 BCE), predynastic Egypt (2500 BCE), and Assyria (2000 BCE).1 The low melting point of lead and its malleability were ideal characteristics for its use in the making of alloys by the early metallurgists. One of the earliest societies of Chinese people, the Shang dynasty (1555–1145 BCE) left extensive burial mounds discovered in urban districts along the Yellow River in Hebei, Henan, and Shanxi provinces of northern China including Anyang and other sites where a trove of artifacts, including numerous pottery shards or intact pieces and bronze objects, has been recovered.2–6 These magnificent bronzes represent what has been called the “bronze age” of Chinese history and include weaponry, utensils, religious ornaments, cooking vessels, cauldrons, goblets, and beverage vessels, all fashioned from mined and alloyed tin, copper, and lead. Early bronzing techniques included metallic lead either as a natural contaminant or as an intentionally added ingredient. Bronze

Received 1 March 2010; accepted 3 March 2010. Address correspondence to Alan D. Woolf, Department of Medicine, Children’s Hospital Boston, 300 Longwood Avenue, Boston, MA 02115, USA. E-mail: [email protected]

drinking vessels unearthed in tombs of royalty from the late Shang period have been found to contain lead. Linduff previously reported an average 7.2% lead content in 30 Shang bronze vessels tested in the Freer Gallery.7 She observed that the percentage of lead composition of the late Shang and Western Chou (ca. 1050–950 BCE) bronze pieces ranged from 4.2% to as high as 21.54%. Lead contamination of food and beverage containers has been a known environmental source of lead poisoning in humans since antiquity. Lead contaminated the wine, water, and foods of ancient Roman aristocrats, and they made their wine in leaden vessels, even adding lead as a preservative or to improve taste.8 Gilfillan observed that the chief sources of lead poisoning in upper class Romans were wine, grape syrup, and preserved fruits.9 Lead has been detected in the skeletal remains of the early Peruvians.10 Lead-contaminated food and drink affected the health of people living in diverse cultures from ancient Greeks, Italians, and Iraqis to more recent examples including American colonists and slaves living in Barbados.11 The Chinese aristocrats of the Shang dynasty were known to drink wine as an everyday beverage as well as during ritualized ceremonial rites. Evidence of fermented millet- and rice-based grain wines, with an alcohol content of 10–15% by weight, has been found in pottery sherds of 16 vessels found

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758 in an archeological dig of the neolithic period in the Henan province, as well as in well-preserved liquids sealed in the Shang dynasty bronze vessels found at Anyang.6 The Shang believed in after-life and anticipated that the dead would need food and drink, and so placed hundreds of bronze vessels in the burial mounds of their dead emperors and royalty, viewing ceremonial use of wine as a means of communicating with the dead.12 Acquisition of bronze objects denoted the accumulation of wealth and distinction; these aromatic fermented wines were only drunk by the Shang nobility. Because they were reportedly heavy drinkers, they might have also been at a disproportionately high risk of chronic lead poisoning, as opposed to the other classes in the Shang society. The quantitative measurement of how much metallic lead could be leached from these vessels has not been previously attempted.

Materials and methods Materials Bronze vessels The three bronze containers used in this study were replicas modeled after the Shang dynasty specimens of museum-quality. They were manufactured by Paul Cavanagh at the Paul King Foundry in Johnston, Rhode Island, USA, in July 2009 (Fig. 1). The metal composition of these replicas was 78% copper, 14% tin, and 8% metallic lead; these percentages were specifically chosen based on data presented in the report by Linduff.7 The process used was an alloy melted and poured into molds using the “lost wax molding” methodology. Sand molding in sectional casting, which was the method probably used during the Shang period, could not be employed. All three vessels were fabricated to hold approximately 600 mL of liquid, and their dimensions were as follows: height 11 cm, diameter (top) 12 cm, and diameter (bottom) 6 cm.

Fig. 1. Bronze drinking vessels fabricated to resemble ancient Chinese late Shang dynasty bronze wine goblets.

A.D. Woolf et al. Wine For this study, Shaoxing drinking wine was used as the test liquid. This rice-based wine is a modern-day version of the fermented rice-based alcoholic beverages consumed during the late Shang dynasty. The wine was purchased in July 2009 at Ming’s Supermarket in Boston, MA, USA. Two different brands were purchased: Hua Diao and Jia Fan labels, each containing 15% alcohol by volume. A 50% mix of each brand was used for the rice wine experiment. Water and white wine (Chenin blanc, containing 13% alcohol by volume) were used as comparisons in the experiment.

Methods Experimental procedure On day 0 of the study, 500 mL of rice wine, white wine, or water were poured into one of the three bronze replicas. Ten milliliters of liquids was removed for lead measurement at 2 min and then 1, 2, 4, and 7 days after the initial day of the experiment. Additional samplings took place on day 14, 28, and 44 in the second experiment. All incubations were at room temperature. At the end of the first experiment, all vessels were rinsed three times with de-ionized water before the second round of experiments using fresh wine or water. Lead level testing Lead concentration was measured by atomic absorption spectroscopy (AAS) (atomic absorption spectrophotometer model A analyst 600 and model A analyst 800 equipped with graphite furnace, Zeeman background correction system, and lead hollow cathode lamp; Perkin Elmer, Norwalk, CT, USA). Six microliters of the sample mixture prepared with matrix modifier (see below) was heated in a graphite furnace to 2,450°C. During the process, the atomized lead sample was excited for absorbance at l = 283.3 nm for 2 s by a lead hollow cathode lamp. Matrix-specific standards containing 0, 10, 50, 250, and 500 μg/L of lead were prepared by spiking aqueous lead standard solution of 1,000 μg/mL (Perkin Elmer) into water, rice wine, and white wine, respectively. One hundred microliters of standard was mixed with 200 μL lead matrix modifier (2 g ammonium phosphate monobasic in 10 mL 10% Triton-X QS 200 mL ddH2O) prior to heating in the atomic absorption spectrophotometer. Each set of standards (0, 10, 50, 250, and 500 μg/L) was used to calibrate for the respective matrix (e.g., standards made in water were used to generate the standard curve for water specimens). Three levels of QC prepared for routine blood lead measurements (two purchased from BioRad, Hercules, CA, USA; one made inhouse) were run after calibrations to verify calibrations. The precision (coefficients of variation) for these quality control levels at 80, 300, and 400 μg/L were 4.4, 3.3, and 3.7% (n = 200 each), respectively. Experimental specimens (water, rice wine, and white wine) taken at different time points were diluted with the respective blank solution and mixed with lead

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matrix modifier before lead measurement to assure the measured concentrations were within the reportable range of our assay (0–500 μg/L). The laboratory at Children’s Hospital, Boston, MA, USA, meets the requirements of the Centers for Disease Control and Prevention (CDC) lead and multielement proficiency and the College of American Pathologists proficiency testing for blood lead. Vessel leaching standardization The three vessel replicas were tested to determine whether similar amounts of lead would be leached from the vessel using the CDC standardized protocol for lead contamination assessment.13 Each vessel was rinsed with de-ionized water three times before the addition of 500 mL of acetic acid (4%, v/v). After 24 h, lead content was measured using a similar method as outlined above except that standards were prepared using acetic acid to generate the calibration curve.

Results None of the three liquids had detectable lead contamination at baseline before they were decanted into the bronze vessels. The results of the first experiment are shown in Table 1 and Fig. 2. A significant amount of lead was leached into the liquid immediately after filling (397–2,590 μg/L). The time to peak concentrations of lead in the three liquids differed: dispersal of lead into the water peaked at 4 days, whereas the peak in white

wine occurred at day 1, and that of Shaoxing rice wine occurred at day 7. The results of the second experiment are shown in Table 1 and Fig. 2. Again there was measurable leaching of lead into all three solutions, although amounts were lower. Figure 2 p portrays the rate of lead leaching in the two experiments, with incremental accumulations over time. A difference in lead content between the three vessels would also explain the differences in leaching into the liquids. Following the CDC protocol described in the methods section using 4% (v/v) acetic acid, we determined that the amount of lead leached from all three vessels was similar: vessel A − 222,000 μg/L, vessel B − 193,000 μg/L, and vessel C − 207,000 μg/L.

Discussion Our findings support the contention that the Shang nobility could have suffered lead poisoning from the habitual drinking of fermented wines stored in bronze vessels of this period. Rice wine stored in the replicas used in this study reached concentrations as high as 85,000 μg/L after 7 days of storage, and in a used, washed vessel reached as high as 32,800 μg/L at 7 days, and 49,200 μg/L by 60 days. By comparison, previous studies have estimated that lead concentrations in ancient Roman wines fell within the range of 200–1,500 μg/L.14 An analysis of uncorked English port wines made between 1770 and 1820 reported lead levels of 320–1,900 μg/L.15 Much higher lead content could theoretically be found in the ancient Chinese wine

Table 1. Measurement of lead accumulation in test solutions in three bronze vessels First experiment Time (in bronze vessel) Predecanting 2 min 1 day 2 days 4 days 7 days

Water pH 6.0 (μg/L)

Chenin blanc (white wine) pH 3.3 (μg/L)

Shaoxing rice wine pH 4.1 (μg/L)

0 397 13,900 23,200 25,600 13,400

0 2,480 116,000 103,000 95,000 113,000

0 2,590 45,900 76,700 80,000 85,000

Second experiment Time (in bronze vessel) Predecanting 2 min 1 day 2 days 4 days 7 days 14 days 28 days 44 days 60 days

Water pH 6.0 (μg/L)

White grape wine pH 3.3 (μg/L)

Shaoxing rice wine pH 4.1 (μg/L)

0 1,480 3,590 4,310 7,900 12,300 15,500 13,300 12,600 17,500

0 5,280 33,400 36,300 43,600 50,200 61,300 70,600 71,200 83,600

0 4,570 26,200 29,100 30,800 32,800 36,600 39,800 42,800 49,200

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A.D. Woolf et al.

(A)

(B)

80,000

100,000 80,000 60,000

Water Rice wine White wine

40,000 20,000 0

0

2 4 6 Length of liquid storage in wine vessel (days)

8

Lead concentration (µg/L)

Lead concentration (µg/L)

120,000

70,000 60,000 50,000 40,000 Water Rice wine White wine

30,000 20,000 10,000 0

0

10 20 30 40 Length of liquid storage in wine vessel (days)

50

Fig. 2. Lead measurement in liquid stored in the fabricated ancient bronze vessel. Shiny precipitates were observed in water for both (A) first experiment and (B) second experiment.

in light of our experiments and this could have caused lead poisoning in those Shang dynasty aristocrats who drank it habitually. Overt lead poisoning (abdominal colic, neuropathy, anemia, and a blood lead level of 130 μg/dL) has been described in a 63-year-old man who chronically drank homemade wine, stored in a contaminated container, containing 61,000 μg/L lead.16 The extent of leaching depends on the composition of the vessel, the nature of the lead (as a salt vs. metallic), the internal dimensions and surface area of the vessel, the duration of contact between liquid and vessel, and the temperature, pH, and miscibility of the solution. Because we did not prewash the vessels, residual surface powder from manufacture could have accounted for the higher rates of lead contamination seen in the first versus the second experiment. We chose to store the wine at room temperature; if the Shang warmed their wine, it could have increased the extent of lead leaching. An experiment by Hofmann, in which he boiled various wines in leaden vessels according to ancient Roman instructions, found levels of 390–781 mg lead per liter of wine.17 The rate of lead leaching into a liquid has previously been shown to be enhanced with increasing acidity.18 Differences in the rates of lead leaching by the three liquids in this study could be explained in part by their pH, because white grape wine (pH 3.3) had the highest lead concentrations, rice wine (pH 4.1) had an intermediate amount of lead leaching, whereas water (pH 6.0) had the lowest amount. The practice of storing wine in bronze containers for variable periods of time before its consumption would increase the risk of lead poisoning. This study showed that stored wine continued to leach increasing amounts of lead over time, at least for the first 60 days. Thus, although newly poured wine into bronze goblets would be contaminated with significant amounts of lead within minutes, the use of stored rice wine from contaminated vessels would greatly magnify the hazard by increasing the concentration of lead in the wine many-fold. Several limitations to this study may restrict the conclusions to be drawn from its results. The Shaoxing rice wine

produced commercially today is likely different in many aspects from the fermented rice and millet beverages produced in the Shang period of Chinese history. There are no published studies which give details of the composition, alcohol concentration, and pH of the ancient wines, although traces have been detectable in some vessels unearthed in the Anyang district. Likewise, the contemporaneous casting method to produce the bronze drinking vessels for this experiment was different from that used by metallurgists in the Shang period and we could not account for differences in materials, variations in interior dimensions, or degenerative changes of the authentic vessels because of weathering over time. We chose one set of dimensions as typical, but this does not cover the hundreds of variations in the volume capacities and surface areas of the Shang bronzes. Toxic levels of lead in the wine would still have been produced despite these variations in vessel casting, weathering, and size, and the handling of the wine, given the extreme amounts of lead leaching found in our experiments.

Conclusions This study confirms the hypothesis that significant quantities of lead could have been leached into acidic fermented wine decanted into the Shang bronze drinking and beverage storage vessels. If the Shang nobility drank a liter of wine daily, their lead intake could have been 85 mg or higher, a dose that would have threatened their health.

Acknowledgments The authors gratefully acknowledge the contributions of Ms. Emma Hawley and Christina Bellamy in facilitating this project and to the support and resources provided by the Granada Media Group, ITV Productions, and Anglia

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Lead poisoning in Shang dynasty bronzes Factual Productions, all in London, Great Britain, without which the project could not be accomplished. Funding for the fabrication of the vessels was provided by Anglia Factual Productions. The authors would like to thank Ms. Alison Clapp, the librarian at the Children’s Hospital, and Ms. Julia Whelan, reference librarian at the Countway Library at Harvard Medical School, for their invaluable help in researching previous publications. Mr. Paul Cavanagh of the Paul King Foundry in Johnston, Rhode Island, kindly fabricated the bronze vessels. We also thank Amy Kellogg for her thorough editing and suggestions to the manuscript.

Declaration of interest This work was supported in part by a grant from the Agency for Toxic Substances and Disease Registry with additional support from the U.S. Environmental Protection Agency, administered through the Association of Occupational and Environmental Clinics (AOEC), Washington, D.C. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.

References 1. Waldron HA. Lead poisoning in the ancient world. Med Hist 1973; 17(4):391–399. 2. Chinnery J. Treasures of China: The Glories of the Kingdom of the Dragon. London, UK: Duncan Baird Publishers; 2008.

761 3. Needham J. Science and Civilization in China. Volume 1: Introductory Orientations. London, UK: Cambridge University Press; 1961. 4. Needham J. Clerks and Craftsman in China and the West. London, UK: Cambridge University Press; 1970. 5. Chang KC. Art, Myth, and Ritual. Cambridge, MA: Harvard University Press; 1983. 6. McGovern PE, Zhang J, Tang J, Zhang Z, Hall GR, Moreau RA, Nuñez A, Butrym ED, Richards MP, Wang CS, Cheng G, Zhao Z, Wang C. Fermented beverages of pre- and proto-historic China. Proc Natl Acad Sci U S A 2004; 101(51):17593–17598. 7. Linduff KM. The incidence of lead in late Shang and early Chou ritual vessels. Expedition 1977; 19(3):7–16. 8. Steinbock RT. Lead ingestion in history. N Engl J Med 1979; 301(5):277. 9. Gilfillan SC. Lead poisoning and the fall of Rome. J Occup Med 1965; 7:53–60. 10. Ericson JE, Shirahata H, Patterson CC. Skeletal concentrations of lead in ancient Peruvians. N Engl J Med 1979; 300(17):946–951. 11. Wittmers L, Aufderheide A, Rapp G, Alich A. Archaeological contributions of skeletal lead analysis. Acc Chem Res 2002; 35(8):669–675. 12. Paper J. The Spirits Are Drunk. Albany, NY: State University of New York; 1995. 13. Parsons PJ, Chisolm JJ. Centers for Disease Control & Prevention, Atlanta, GA; 1997. http://www.cdc.gov/nceh/lead/publications/1997/ pdf/c1.pdf. Accessed 27 February 2010. 14. Nriagu JO. Saturnine gout among Roman aristocrats: did lead poisoning contribute to the fall of the empire? N Engl J Med 1983; 308(11):660–663. 15. Ball GV. Two epidemics of gout. Bull Hist Med 1971; 45:401–408. 16. Antonini G, Ferracuti S, Pennisi E, Monarca B. Wine poisoning as a source of lead intoxication. Am J Med 1989; 87(2):238–239. 17. Hofmann KB. Die Getranke der Griechen und Romer vom hygienische Standpunkte. Arch Gesch Med 1883; 6:26–40. 18. Lin SW, Vargas-Galarza Z, Felix-Navarro RM. Optimizing the conditions for leaching lead from solid waste produced by purometallurgical process of recycling automobile used batteries. J Mex Chem Soc 2006; 50(2):64–70.

Clinical Toxicology (2010) 48, 762–763 Copyright © Informa UK, Ltd. ISSN: 1556-3650 print / 1556-9519 online DOI: 10.3109/15563650.2010.484394

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Postmortem changes in carbon monoxide poisoning RICARDO JORGE DINIS-OLIVEIRA1,2,3, FÉLIX CARVALHO3, TERESA MAGALHÃES1,4,5, and AGOSTINHO SANTOS1,5 Carbon monoxide poisonings

1

Faculty of Medicine, University of Porto, Porto, Portugal Department of Clinical Analysis and Public Health, Center of Research in Health Technologies (CITS)-IPSN-CESPU, CRL, Vila Nova de Famalicão, Portugal 3 REQUIMTE, Department of Toxicology, Faculty of Pharmacy, University of Porto, Porto, Portugal 4 Biomedical Sciences Institute Abel Salazar, University of Porto, Porto, Portugal 5 National Institute of Legal Medicine I.P., Faculty of Medicine, University of Porto, Porto, Portugal 2

Carbon monoxide (CO) is produced when organic matter is burned in an inadequate supply of oxygen. Death from CO intoxication may occur either from deliberate self-harm or in the course of unintentional poisoning following fires.1,2 CO binds to hemoglobin, much more strongly than oxygen does, leading to the formation of carboxyhemoglobin (COHb), resulting in hypoxia and subsequently to anoxia.1,2 A COHb concentration about 50–60% is usually considered fatal. In patients with concomitant morbidity, in particular cardiovascular and lung disease, interpretation of lower COHb concentrations may be more difficult. Moreover, establishing the cause of death in a victim who has been in a house fire may be problematic if samples are not obtained early after the event. Indeed, if the CO-intoxicated victim survives for several hours, postmortem blood samples may fail to show the intoxication through the measurement of COHb concentrations. In these circumstances, blood taken at the time of admission to the hospital is of particular value, in addition to scene investigation and postmortem changes. Particularly, autopsy findings in CO deaths are fairly characteristic and can be important forensic clues to the cause of death. We illustrate these findings in the case of CO poisoning associated with smoke inhalation in the accompanying photographs. Lividity can exhibit a cherry-red or bright-pink color (Fig. 1) in Caucasians, as a consequence of COHb formation. As mentioned above, this discoloration may be lost if COHb levels fall during resuscitation. Noteworthy, similar lividity will develop in case of death because of intense cold (hypothermia), cyanide poisoning, following the morgue’s refrigeration of a recently deceased body, and also if the area of the cadaver was covered by wet clothing. In victims

Fig. 1. (A) Lividity of cherry-red or bright-pink color in Caucasians, as a consequence of COHb formation. (B) Smoke in the face, nostrils, and mouth is suggestive of CO poisoning but does not prove that the person died from CO inhalation. It only confirms that smoke covered these structures.

Received 29 March 2010; accepted 7 April 2010. Address correspondence to Ricardo Jorge Dinis-Oliveira, Institute of Legal Medicine, Faculty of Medicine, University of Porto, Jardim Carrilho Videira, Porto 4050-167, Portugal. E-mail: [email protected]; [email protected]

with dark skin, the discoloration is prominent in the conjunctivae, nail beds, and mucosa of the lips. If the deceased is burned, the pathologist must attempt to determine whether the victim was alive or already dead when the fire occurred. For a victim found at the fire scene, the saturation ratio of COHb can be an indicator for judging, but the results can be difficult to interpret. In the absence of

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763 however, does not necessarily mean that the individual was dead prior to the start of the fire. Finally, although smoke in the face, nostrils, and mouth is suggestive of CO poisoning, it does not prove that the person died from CO inhalation but only that smoke covered these structures. Internally, the musculature, the internal viscera, and blood collected will have a bright cherry-red coloration (Fig. 2).

Acknowledgment Ricardo Dinis-Oliveira acknowledges FCT for his Post-Doc grant (SFRH/BPD/36865/2007).

Declaration of interest Fig. 2. Smoke soot covering the larynx, trachea, and bronchi suggests that the victim was alive when the fire occurred and was intoxicated by CO.

breathing during fire, COHb levels in nonsmokers can reach 3% and those in smokers may be up to 10%.3 Values above 10%, therefore, can be useful to indicate that the individual has been exposed to CO during life, but the converse is not necessarily true. Examination of individuals overcome by smoke inhalation will usually reveal soot in the nostrils and mouth (Fig. 1A) as well as coating the larynx, trachea, and bronchi (Fig. 2). Absence of soot,

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.

References 1. Mayes RW. ACP Broadsheet No 142: November 1993. Measurement of carbon monoxide and cyanide in blood. J Clin Pathol 1993; 46:982–988. 2. Prockop LD, Chichkova RI. Carbon monoxide intoxication: an updated review. J Neurol Sci 2007; 262:122–130. 3. Yeoh MJ, Braitberg G. Carbon monoxide and cyanide poisoning in fire related deaths in Victoria, Australia. J Toxicol 2004; 42:855–863.

Clinical Toxicology (2010) 48, 764–765 Copyright © Informa UK, Ltd. ISSN: 1556-3650 print / 1556-9519 online DOI: 10.3109/15563650.2010.502123

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Cutaneous loxoscelism caused by Loxosceles anomala FÁBIO BUCARETCHI1, EDUARDO MELLO DE CAPITANI1, STEPHEN HYSLOP1, RAFAEL SUTTI1, THOMAZ A.A. ROCHA-E-SILVA2, and ROGERIO BERTANI3 Cutaneous loxoscelism caused by Loxosceles anomala

1

Faculty of Medical Sciences, State University of Campinas, Campinas Poison Control Center, Campinas, Brazil Department of Physiological Sciences, Faculty of Medical Sciences, Santa Casa de Sao Paulo, Sao Paulo, Brazil 3 Butantan Institute, Sao Paulo, Brazil 2

A previously healthy 35-year-old female was bitten on the anterior right thigh by a brown spider while dressing her trousers; the spider was stored and later identified as an adult female Loxosceles anomala. Clinical evolution involved a relatively painless bite with mild itching, followed by local, indurated swelling and a transient, generalized erythrodermic rash at 24 h post-bite. The local discomfort was progressive, and involved changes in the lesion pattern, with pain of increasing intensity. The patient was admitted 60 h post-bite, showing an irregular blue plaque surrounded by an erythematous halo lesion, located over an area of indurated swelling. Considering the presumptive diagnosis of cutaneous loxoscelism, she was treated with five vials of anti-arachnidic antivenom i.v. without adverse effects. There was progressive improvement, with no dermonecrosis or hemolysis; complete lesion healing was observed by Day 55. The clinical features and outcome were compatible with cutaneous loxoscelism and similar to those reported for other Loxosceles species. Keywords Brown spider; Cutaneous loxoscelism; Loxosceles anomala

A previously healthy 35-year-old female was bitten on the anterior right thigh by a brown spider while dressing her trousers (1 p.m., time zero); the spider was killed and stored at the time of the bite for identification. Clinical evolution involved a relatively painless bite with mild itching. At 24 h post-bite the patient noted progressive local swelling with induration, associated with a transient, generalized erythrodermic rash that was most intense on the face and trunk. By 48 h post-bite, there was progressive local discomfort with pain of increasing intensity (stinging burning sensation) and changes in the lesion pattern. Photos of the lesion e-mailed by the patient to the Campinas Poison Control Center 57 h post-bite revealed an irregular blue plaque surrounded by an erythematous halo. There was no complaint of fever, pallor, jaundice, or change in urine color; a presumptive diagnosis of cutaneous loxoscelism was then made. The patient was admitted to the university hospital 3 h later and had an irregular lesion (6 cm × 4 cm) with the characteristics described above, located over an area (20 cm × 12 cm) of indurated swelling. In view of the progressive signs of local envenoming, the patient was treated with five vials of anti-arachnidic antivenom i.v. [AV, Instituto Butantan, Brazil; 5 mL/vial containing Received 6 May 2010; accepted 15 June 2010. Address correspondence to Fábio Bucaretchi, Centro de Controle de Intoxicacoes HC/UNICAMP, Campinas, CEP 13081-970, Brazil. E-mail: [email protected]

F(ab´)2 antibodies against Loxosceles gaucho, Phoneutria nigriventer, and Tityus serrulatus venoms] without adverse effects. Approximately 18 h after AV infusion there was a reduction in the pain and a qualitative clinical assessment that the lesion progression had been stopped; at this point, the patient was discharged. Sequential hemograms revealed no indication of hemolysis. The patient did not develop dermonecrosis and complete lesion healing was observed by Day 55 post-bite. Fig. 1(A) and (B) shows the lesion aspect 6 and 55 days post-bite, respectively. On the fifth day post-bite, the patient brought the dead spider that had caused the bite, subsequently identified by an expert arachnologist (co-author RB) as a female adult of Loxosceles anomala.1 The genus Loxosceles (Heineken and Lowe, 1832) has a worldwide distribution in temperate and tropical regions and comprises at least 100 species.2–4 Envenoming by these spiders generally results in local dermonecrosis with gravitational spreading, whereas severe systemic complications, such as intravascular hemolysis and acute renal failure, are unusual.3,4 The diagnosis of loxoscelism is difficult and rarely based on the identification of the spider, but on epidemiological data, historical findings, and clinical signs and symptoms.3,4 In general, less than 15% of bitten patients bring the spider for identification3; in addition, as the clinical signs and symptoms are not particularly pronounced during the first few hours after a bite, the patients usually present for evaluation only 24–48 h after being bitten.3,4

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765 (B)

Fig. 1. Aspect of the local lesion 6 (A) and 55 (B) days post-bite, respectively. (A) Irregular blue plaque surrounded by an erythematous halo, resembling local vasculitis. (B) Complete lesion healing 55 days post-bite.

Most bites of Loxosceles in Brazil are caused by L. intermedia, L. laeta, and L. gaucho.3,4 Loxosceles anomala is an uncommon species,1 and no confirmed cases of bites by this species have previously been reported. However, the circumstance, clinical features, and outcome described here were compatible with cutaneous loxoscelism, and were similar to those reported for other Loxosceles species.3,4 AV has been used to treat clinical loxoscelism in Brazil since the 1960s and shows good cross-reactivity in neutralizing the dermonecrotic and lethal activities of several Loxosceles venoms in rabbits.5 Those who advocate the use of AV based on anecdotal and extensive clinical experience stress that AV therapy can be beneficial in decreasing the lesion size and cure time, and in attenuating the systemic effects.5 In conclusion, the clinical findings described here indicate that L. anomala venom probably shares similar activities with those of other Loxosceles species.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.

References 1. Álvares ESS, Rodrigues T, De Maria M. On Loxosceles anomala (Mello Leitão) (Araneae: Sicariidae). Rev Ibérica Aracnol 2004; 10:293–295. 2. Platnick NI. The World Spider Catalog, Version 10.5. American Museum of Natural History. http://research.amnh.org/iz/spiders/catalog/ SICARIIDAE.html. Accessed 30 March 2010. 3. Hogan CJ, Barbaro KC, Winkel K. Loxoscelism: old obstacles, new directions. Ann Emerg Med 2004; 44:608–624. 4. Da Silva PH, da Silveira RB, Appel MH, Mangili OC, Gremski W, Veiga SS. Brown spiders and loxoscelism. Toxicon 2004; 44:693–709. 5. Pauli I, Puka J, Gubert IC, Minozzo JC. The efficacy of antivenom in loxoscelism treatment. Toxicon 2006; 48:123–137.

Clinical Toxicology (2010) 48, 766–767 Copyright © Informa UK, Ltd. ISSN: 1556-3650 print / 1556-9519 online DOI: 10.3109/15563650.2010.503657

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Methemoglobinemia induced by indoxacarb intoxication YA-JU WU1, YU-LI LIN1, HAN-YU HUANG2, and BANG-GEE HSU3 Indoxacarb poisoning causes methemoglobinemia

1

Department of Internal Medicine, Buddhist Tzu Chi General Hospital, Hualien, Taiwan Division of Medical Intensive Care Unit, Buddhist Tzu Chi General Hospital, Hualien, Taiwan 3 Division of Nephrology, Department of Internal Medicine, Buddhist Tzu Chi General Hospital, Hualien, Taiwan 2

Indoxacarb is a recently introduced insecticide whose mode of action is blockage of voltage-gated sodium channels. There are limited data on human ingestion. A case of 68-year-old healthy male who presented with general cyanosis because of methemoglobinemia following the ingestion of indoxacarb is presented. After receiving a methylene blue injection, the patient recovered without sequelae. Keywords Methemoglobinemia; Indoxacarb

Introduction Indoxacarb (methyl-(S)-N-[7-chloro-2,3,4a,5-tetrahydro-4a(methoxycarbonyl) indeno[1,2-e][1,3,4]oxadiazin-2-ylcarbonyl]-4′-(trifluoromethoxy)carbanilate) is an oxadiazine insecticide that blocks neuronal voltage-dependent sodium channels, resulting in impaired nerve function, feeding cessation, paralysis, and death of insect.1,2 Once indoxacarb is absorbed or ingested, feeding cessation occurs almost immediately even though it may take several days for insects to die.1 Indoxacarb has low mammalian toxicity and there are limited data with regard to effects after human ingestion.1,2 Here, we report a case of acquired methemoglobinemia occurring after indoxacarb ingestion.

Case report A 68-year-old man had previously been in good health and did not have a glucose 6-phosphate dehydrogenase deficiency history. He intentionally ingested 50 mL of indoxacarb insecticide (Avatar®; DuPont Crop Protection, Taipei, Taiwan; 14.5% indoxacarb; 79.5% inert ingredients; 6% other components-related isomers and impurities) in a suicide attempt. He ingested no other drugs before admission. He was sent to the emergency department 2 h later with the bottle of indoxacarb (methyl-(S)-N-[7-chloro-2,3,4a,5-tetrahydro-4a-(methoxycarbonyl) indeno[1,2-e][1,3,4]oxadiazin-2-ylcarbonyl]-4′-(trifluoromethoxy)carbanilate). His vital signs were body temperature

Received 12 April 2010; accepted 20 June 2010. Address correspondence to Bang-Gee Hsu, Division of Nephrology, Department of Internal Medicine, Buddhist Tzu Chi General Hospital, Hualien, Taiwan. E-mail: [email protected]

36.3°C, heart rate of 110 beats per minute, respiratory rate of 18 breaths per minute, and blood pressure of 112/70 mmHg. His consciousness was clear (Glasgow Coma Scale: E4V5M6) but he reported dizziness, headache, and fatigue at presentation to the hospital. His laboratory data showed white blood cell count of 13,600/μL, hemoglobin 15.0 g/dL, platelets 318,000/μL, alanine aminotransferase 25 IU/L, blood urea nitrogen 13 mg/dL, creatinine 1.0 mg/dL, sodium 134 mEq/L, and potassium 3.9 mEq/L. Arterial blood gas data on 25% oxygen therapy revealed pH of 7.38, PaCO2 35.4 mmHg, PaO2 187.7 mmHg, HCO3- 20.9 mmol/L, and arterial oxyhemoglobin saturation (SaO2) was 99.6%. There was no active lung lesion on chest X-ray. However, cyanotic lips and nails were noticed (Fig. 1A and B). The pulse oxymeter was only 82%. Blood methemoglobin (MetHb; Cobas b221, Roche Diagnostics, Indianapolis, IN, USA) level was 63.5%. Intravenous methylene blue 2 mg/kg was administered immediately and 4 h later, the second dose because cyanotic lips were persistent. Eight hours later, the blood MetHb was 3.5%. Hemolytic anemia did not occur during hospitalization. Three days later, the patient was discharged in stable condition.

Discussion Methemoglobin is an altered state of hemoglobin in which the ferrous (Fe2+) ions of heme are oxidized to the ferric (Fe3+) state.3,4 The symptoms of methemoglobinemia are often vague and nonspecific, but as methemoglobin levels increase, symptoms typically increase too. Levels of 20–50% will cause symptoms such as respiratory distress, dizziness, headache, and fatigue. Lethargy and stupor develop at levels around 50% and death may occur around 70%.4 There are a number of methods to detect methemoglobin, such as co-oximeters,

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Indoxacarb poisoning causes methemoglobinemia (A)

(B)

767 anemia.8 Metabolism in rats after oral dosing noted that most of the dose was excreted within 96 h. In urine, metabolites were cleaved products (indane or trifluoromethoxyphenyl ring products), whereas in feces, major metabolites retained both these moieties. Major metabolic reactions included hydroxylation of the indane ring, hydrolysis of the carboxymethyl group from the amino nitrogen, and opening of the oxadiazine ring, which gave rise to cleaved products.8 Indoxacarb had aromatic metabolites that can biotransform into active intermediates that produce methemoglobin.3 There are limited data with regard to human ingestion. The literature provides only two case reports of indoxacarb ingestion with methemoglobinemia. One case was treated by methylene blue with ascorbic acid in India and the other was treated only by methylene blue in Korea.9,10 Our patient was treated by methylene blue because he had the clinical symptoms of methemoglobinemia. In summary, methemoglobinemia is one of the syndromes which occur after indoxacarb ingestion. Here we have described a 68-year-old healthy male who presented with general cyanosis because of methemoglobinemia following the ingestion of indoxacarb and was successfully treated with methylene blue injection.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.

Fig. 1. Cyanotic lips (A) and nails (B) were found after indoxacarb ingestion.

simple bedside testing of chocolate brown blood on filter paper, semiquantitative bedside testing, and spectrophotometer-based readings.4–6 The most laboratory diagnosis of methemoglobinemia in clinical was used co-oximeters.4 In our case, cyanotic lips and nails were noticed with dizziness, headache, and fatigue at emergency department and methemoglobinemia diagnosed. Rebound methemoglobinemia as high as 60% has been reported up to 18 h after methylene blue administration, because of prolonged absorption of the implicated agent from topical or enteric sites.7 Indoxacarb is an oxadiazine insecticide that acts on insect neuronal voltage-dependent sodium channels and is now registered for use on apples and pears with low mammalian toxicity.1,2 High single oral doses of indoxacarb caused gait abnormalities, incoordination, hypoactivity, convulsions, tremors, hypothermia, hair loss, labored respiration, discharge, hemolytic anemia, and vocalization in rats.8 Human toxicity includes eye irritation, blurred vision, skin sensitization with allergic rashes, alteration in blood cell counts, and/or

References 1. Lapied B, Grolleau F, Sattelle DB. Indoxacarb, an oxadiazine insecticide, blocks insect neuronal sodium channels. Br J Pharmacol 2001; 132:587–595. 2. Song W, Liu Z, Dong K. Molecular basis of differential sensitivity of insect sodium channels to DCJW, a bioactive metabolite of the oxadiazine insecticide indoxacarb. Neurotoxicology 2006; 27:237–244. 3. Bradberry SM. Occupational methaemoglobinaemia. Mechanisms of production, features, diagnosis and management including the use of methylene blue. Toxicol Rev 2003; 22:13–27. 4. Camp NE. Methemoglobinemia. J Emerg Nurs 2007; 33:172–174. 5. Henretig FM, Gribetz B, Kearney T, Lacouture P, Lovejoy FH. Interpretation of color change in blood with varying degree of methemoglobinemia. J Toxicol Clin Toxicol 1988; 26:293–301. 6. Shihana F, Dissanayake DM, Buckley NA, Dawson AH. A simple quantitative bedside test to determine methemoglobin. Ann Emerg Med 2010; 55:184–189. 7. Guay J. Methemoglobinemia related to local anesthetics: a summary of 242 episodes. Anesth Analg 2009; 108:837–845. 8. TOXNET – Databases on toxicology, hazardous chemicals, environmental health, and toxic releases. http://toxnet.nlm.nih.gov. Accessed 3 June 2010. 9. Prasanna L, Rao SM, Singh V, Kujur R, Gowrishankar. Indoxacarb poisoning: an unusual presentation as methemoglobinemia. Indian J Crit Care Med 2008; 12:198–200. 10. Park S, Lee J, Park J. Indoxacarb pesticide poisoning with methemoglobinemia. Toxicol Lett 2008; 180(Suppl. 1):S172.

Clinical Toxicology (2010) 48, 768–769 Copyright © Informa UK, Ltd. ISSN: 1556-3650 print / 1556-9519 online DOI: 10.3109/15563650.2010.497763

LETTERS TO THE EDITOR 1556-3650 LCLT Clinical 1556-9519 Toxicology Toxicology, Vol. 1, No. 1, Sep 2010: pp. 0–0

Chronic renal failure associated with heavy metal contamination of drinking water: A clinical report from a small village in Maharashtra

Table 1. Heavy metal content in blood and in the water sources of two patients measured by ICP-AES Type of sample

Lead (μg/dL)

Arsenic (μg/dL)

Copper (μg/dL)

Cadmium (μg/dL)

100 100

BDL BDL

BDL BDL

29 68

119 BDL BDL BDL 43

BDL BDL BDL BDL BDL

BDL 23 1 0.03 BDL

BDL 4 2 0.10 BDL

5

3

1

5

To the Editor: LetterBawaskar H.S. to the editor et al.

Cadmium and lead are divalent cations which in excess damage the proximal renal tubules leading to irreversible and progressive renal damage.1,2 Exposure to low environmental cadmium and lead may accelerate progressive renal insufficiency.3 In India, drinking water is the main source of lead and cadmium contamination, believed to be responsible for renal failure.4 Here we report our recent experience in a rural Indian community suggesting the need for improved environmental and health monitoring in such areas. We detected an index case of chronic renal failure in a routine health camp. We confirmed 23 deaths due to renal failure (age 17–70, mean 47.3 years) from hospital records, with a further 18 cases with signs and symptoms suggestive of chronic renal failure during the survey of 2053 individuals in a village in Buldana district of Maharashtra, India. Laboratory support is weak in this area, and full biochemical data are not readily available. Hospital records and clinical findings in 18 cases revealed that all reported puffiness, anemia, and a raised serum creatinine 2–4 mg/dL (mean 2.40) (normal < 1.4 mg/dL). Additional features were proteinuria in 13 (72%), anorexia in 11 (61%), hypertension in 8 (44%), renal glycosuria and hematuria in 6 (33%), constipation in 4 (22%), and recurrent renal calculi in 3 (16%). One case had a high serum uric acid (9.30 mg/dL) with small irregular kidneys on ultrasonography. The cases were seen by six physicians and nephrologists and were receiving regular diuretics, antihypertensives, and low-salt diet. Three patients were undergoing regular weekly dialysis. As a result of progressive decline in health and regular death due to chronic renal failure, the villagers felt that their illness has no medical remedy and avoided free hospitalization, investigations, and renal biopsy. They see death due to kidney disease as a fact of life in this region. With much difficulty, we obtained written consent of two cases for blood analyses and collected water from drinking sources for analysis. Results in Table 1 suggest heavy metal contamination from the water supply as a potential cause of renal injury. The character of the local soil is clay and the main occupation of affected villagers is farming. Cadmium-contaminated phosphate manures and lead-containing pesticides are common sources of contamination of drinking water in this clay soil.4 We plan to undertake a large-scale epidemiological studies in this and other regions to quantify the public health hazard of heavy metal contamination and to better define the clinical syndrome, so that corrective public health measures can be started, including regulation of heavy metal concentrations in commonly used fertilizers and pesticides. This case series illustrates the problems of identifying and managing environmental toxicity in poor rural situations.

Received 27 May 2010; accepted 28 May 2010. Address correspondence to Himmatrao Saluba Bawaskar, Bawaskar Hospital and Research Center, Mahad, Raigad 402301, Maharashtra, India. E-mail: [email protected]

Blood Patient-1 Patient-2 Water Bore well-1 Bore well-2 Bore well-3 Bore well-4 Open well-1 Limit of detection by ICP-AES

BDL, below detectable limit. Method: ICP-AES (inductively coupled plasmaatomic emission spectrophotometry). Limit: permission limit (as per iS-10500-1983); lead: <10 μg/dL; arsenic: <5 μg/dL; copper: <5 μg/dL; and cadmium: <1 μg/dL.

Himmatrao Saluba Bawaskar, Pramodini Himmatrao Bawaskar, and Parag Himmatrao Bawaskar Bawaskar Hospital and Research Center, Mahad, Raigad, Maharashtra, India

References 1. Hotz P, Buchet JP, Bernard A, Lison D, Lauwerys R. Renal effects of low level environmental cadmium exposure: 5-year follow-up of a subcohort from the cadmium study. Lancet 1999; 254:1508–1513. 2. Gonick HC. Nephrotoxicity of cadmium and lead (Review article). Indian J Med Res 2008; 128:335–352. 3. Lin JL, Lin-Tan DT, Hsu KH, Yu CC, Environmental lead exposure and progression of chronic renal disease in patients without diabetes. N Engl J Med 2003; 348:277–286. 4. Khwaja AR, Singh R, Raju M, Tandon SN. The geo-environmental cycle of cadmium: a case study. Environmentalist 1997; 17:103–108. 1556-3650 LCLT Clinical 1556-9519 Toxicology Toxicology, Vol. 1, No. 1, Aug 2010: pp. 0–0

Response to “A multicenter comparison of the safety of oral versus intravenous acetylcysteine for treatment of acetaminophen overdose” Authors’ M. Punja reply et al.

To the Editor: We read with great interest the study by Heard et al. that compared the safety of oral and intravenous administration of N-acetylcysteine (NAC) for acetaminophen overdose.1 Although many studies report overall adverse

Received 19 July 2010; accepted 30 July 2010. Address correspondence to David H. Jang, Department of Medical Toxicology, New York University, 455 First Avenue, Room 123, NY 10016, USA. E-mail: [email protected]

Clinical Toxicology vol. 48 no. 7 2010

Letters to the editor

769

event rates of 30–40% and rates of anaphylactoid reaction around 3–6%, which is consistent with this study, we feel that the true rate is undetermined given methodological flaws in similar retrospective chart reviews.2 We are concerned with the assignment of hypotension and tachycardia as “not related” adverse effects in the group receiving intravenous (IV) administration. The authors fail to provide any description as to how the unblinded abstractors determined which adverse events were considered related or unrelated to the ongoing administration of NAC. It is also striking that there were a high number of deaths in patients who received IV NAC given the lack of evidence to support that they were more severely poisoned prior to treatment or had a substantially longer delay to initiation of the antidote. Although in our experience, and in the literature,3–5 the administration of IV NAC involves a low incidence of serious anaphylactoid reaction and rarely, death, and has proven efficacy in the delayed and prolonged treatment of patients with acetaminophen-induced fulminant hepatic failure, we request that the authors attempt to address this significant discrepancy. Mohan Punja Department of Emergency Medicine, Beth Israel Medical Center, New York, NY, USA David H. Jang Department of Medical Toxicology, New York University, New York, NY, USA Robert S. Hoffman NYC Poison Center, New York, NY, USA

References 1. Heard K. A multicenter comparison of the safety of oral versus intravenous acetylcysteine for treatment of acetaminophen overdose. Clin Toxicol 2010; 48(5):424–430. 2. Schmidt LE, Dalhoff K. Risk factors in the development of adverse reactions to N-acetylcysteine in patients with paracetamol poisoning. Br J Clin Pharmacol 2001; 51(1):87–91. 3. Zyoud SH, Syed Sulaiman SA, Sweileh WM, Awang R, Al-Jabi SW. Incidence of adverse drug reactions induced by N-acetylcysteine in patients with acetaminophen overdose. Human Exp Toxicol 2010; 29(3):153–160. 4. Cassidy N, Tracey JA, Drew SA. Cardiac arrest following therapeutic administration of N-acetylcysteine for paracetamol overdose. Clin Toxicol 2008; 46(9):921. 5. Appelboam AV, Dargan PI, Knighton J. Fatal anaphylactoid reaction to N-acetylcysteine: caution in patients with asthma. Emerg Med J 2002; 19:594–595.

the adverse events as related or not during the abstraction. They coded an event as related if it was temporally related to acetylcysteine administration and if the investigator could not identify another cause. We considered more rigorous definitions but felt that we could not reasonably develop an exhaustive list of rules and exceptions. We therefore decided to rely on the clinical assessment made using the best information available during a retrospective chart review. We recognize that a prospective study or a different definition may have produced different results, but we feel that our method would detect significant adverse events related to acetylcysteine. A second concern was the “high number of deaths” in patients who received IV acetylcysteine. The overall mortality in the IV group was approximately 6%. Although this is higher than the 1–2% mortality reported in the other studies of acetylcysteine,1 it is similar to the 7% reported in a community hospital2 and it is lower than the 28% reported in a referral center for liver transplant.3 As several of our sites were liver transplant centers, we believe that the mortality is not unexpected for this population. We strongly disagree with the suggestion that the IV group was not more severely poisoned than the oral group. Although the median ALT and creatinine of the groups were similar between the oral and IV groups, the 90th percentile for the baseline ALT for the IV group was 4414 IU/L compared to 951 IU/L in the oral group and the 90th percentile for the creatinine in the IV group was 2.8 mg/dL compared to 1.3 mg/dL in the oral group. These differences show that although the measures of acuity were similar in the overall populations, the “sickest” patients in the IV group had a more severe injury at baseline than the “sickest” patients in the oral group. We believe that this difference in baseline severity accounts for the majority in the observed differences in outcome. This project was supported by the 2004 American College of Clinical Toxicology Multi-Center Research Grant and an unrestricted research grant from Cumberland Pharmaceuticals. Rocky Mountain Poison and Drug Center (RMPDC) was the coordinating center for this multicenter study. All funds were paid out to the participating centers and RMPDC received no support for this project. RMPDC has clinical, consulting, and research contracts with Cumberland Pharmaceuticals (a manufacturer of IV acetylcysteine) and McNeil Consumer Healthcare (a manufacturer of acetaminophen products). The investigators were responsible for the design, performance, and analysis of the study and for the drafting and revisions of this article. The sponsors had no role in the study design and performance and did not review the article prior to acceptance. Dr. Heard was supported by an Award Number K08DA020573 from the National Institute on Drug Abuse. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute On Drug Abuse or the National Institutes of Health.

1556-9519 1556-3650 LCLT Clinical Toxicology Toxicology, Vol. 1, No. 1, Aug 2010: pp. 0–0

Author’s reply to response to “A multicenter comparison of the safety of oral versus intravenous acetylcysteine for treatment of acetaminophen overdose.”

Kennon Heard Rocky Mountain Poison Center, Denver, CO, USA On behalf of the Toxicology Investigators Network Authorship Group

Author’s K. Heard reply

To the Editor:

References We thank the authors for their comments. Their first concern is that we fail to provide any description as to how the investigators determined if adverse events were related to acetylcysteine. As noted in our methods (3rd paragraph, 2nd sentence), we asked the investigators to categorize

Received 29 July 2010; accepted 30 July 2010. Address correspondence to Kennon Heard, Rocky Mountain Poison Center, 990 Bannock Street, Denver, CO 80262, USA. E-mail: [email protected]

1. Smilkstein MJ, Knapp GL, Kulig KW, Rumack BH. Efficacy of oral N-acetylcysteine in the treatment of acetaminophen overdose. Analysis of the national multicenter study (1976 to 1985). N Engl J Med, 1988; 319(24):1557–1562. 2. Schiodt FV, Rochling FA, Casey DL, Lee WM. Acetaminophen toxicity in an urban county hospital. N Engl J Med, 1997; 337(16):1112–1117. 3. Pakravan N, Simpson KJ, Waring WS, Bates CM, Bateman DN. Renal injury at first presentation as a predictor for poor outcome in severe paracetamol poisoning referred to a liver transplant unit. Eur J Clin Pharmacol, 2009; 65(2):163–168.

Clinical Toxicology (2010) 48, 770 Copyright © Informa UK, Ltd. ISSN: 1556-3650 print / 1556-9519 online DOI: 10.3109/15563650.2010.517385

Errata LCLT

p16 promoter methylation in Pb2+-exposed individuals Kovatsi Leda, Georgiou Elisavet, Ioannou Antrea, Haitoglou Costas, Tzimagiorgis George, Tsoukali Helen, and Kouidou Sofia, Volume 48, Issue 2, pp 124–128.

Toxicology Investigator Network Authorship Group Wilford Hall Medical Center: Vikhyat S. Bebarta.

The publishers would like to apologise as the above authors have been printed with their names in the wrong order. They have been listed with surname first, followed by first name. This has been corrected in the online version. The list should read as follows:

UCSD Medical Center: Richard F. Clark.

Leda Kovatsi, Elisavet Georgiou, Antrea Ioannou, Costas Haitoglou, George Tzimagiorgis, Helen Tsoukali and Sofia Kouidou.

New York City Poison Center: Oladapo Odujebe.

xxxxx

A multicenter comparison of the safety of oral versus intravenous acetylcysteine for treatment of acetaminophen overdose*, Kennon Heard, Volume 48, Issue 5, pp 424–430. xxxxx

The publishers would like to apologise as Kennon Heard was not the only author of this article. The correct list of authors is as follows:

Indiana University School of Medicine: Louise Kao, Blake Froberg.

Carolinas Medical Center: Eric Lavonas, Ming Qi. Hartford Hospital: Joao Delgado, John McDonagh. LSU Health Sciences Center: Tom Arnold.

Albert Einstein Medical Center: Gerry O’Malley, Claudia Lares, Elizabeth Aguilera. Denver Health-Rocky Mountain Poison Center. Richard Dart MD, PhD, Kennon Heard MD, Chriss Stanford MA, Jamie Kokko MPH, Greg Bogdan PhD, Carrie Mendoza MD, Sara Mlynarchek MPH, Sean Rhyee MD, Jason Hoppe DO, William Haur MD, Hock Heng Tan MD, Nguyen Nguyen Tran MD, Shawn Varney MD, Amy Zosel MD, Jennifer Buchanan MD, Mohammed Al-Helial MD.