Comparison of Tacrine-Induced Cytotoxicity in Primary Cultures of Rat,Mouse, Monkey,Dog, Rabbit,and Human Hepatocytes

ABSTRACT

Tacrine is the first drug approved for the treatment of Alzheimer's disease. Approximately 50% of patients treated with tacrine develop elevated serum aminotransferase levels, an indication of potential hepatotoxicity. the mechanism of human hepatoxicity has been difficult to study, because of the absence of an animal model. Therefore, this study compared the cytotoxicity induced by tacrine in primary rat, mouse, monkey, dog, rabbit and human hepatocytes to determine differences in response to tacrine between species in vitro Cytotoxicity was assessed by determination of extra-and intracellular lactate dehydrogenase. the ratio of intracellular enzyme to total enzyme (i.e intracellular and extracellular) was used to represent the viabilities of the cultures. Concentration-dependent cytotoxicity occurred after four and 24-hour exposure over a tacrine concentration range of 0 to 380 µg/ml. Cytotoxic potency of tacrine in hepatocytes from human, dog, mouse and rat was not significantly different; monkey hepatocytes appeared slightly more sensitive, while rabbit hepatocytes appeared slightly less sensitive than human hepatocytes. Increased time of exposure to tacrine decreased the concentration necessary to induce a cytotoxic response This in vitro model suggests only minimal differences in sensitivity to tacrine-induce cytotoxicity; therefore, cytotoxicity in primary cultures of hepatocytes from various species would appear to be related to common metabolite(s) and/or mechanism of cellular injury.     

The Metabolism of Tolamolol* in the Mouse,Rat, Guinea-pig,Rabbit and Dog

1. [³H, ¹⁴C]Tolamolol was well absorbed after oral administration to mice, rats, guinea-pigs, rabbits and dogs.

2. The major route for excretion of. radioactivity by mice, rats and guineapigs was the faeces; in rabbits the major route was the urine. Dogs excreted similar amounts of radioactivity by both routes. Biliary excretion of radioactivity by the rat and guineapig was demonstrated.

3. Tolamolol was extensively metabolized by all five species. The major metabolite in mice, rats, guinea-pigs and rabbits was the product of hydroxylation of the tolyl ring, which was excreted as such and as the glucuronide and sulphate conjugates.

4. In the dog the major metabolite was the acid resulting from hydrolysis of the carbamoyl group. This acid was also excreted by the rabbit, but was only a minor metabolite in the other species studied.     

Human,Rat, and Mouse Metabolism of Resveratrol

Purpose. Resveratrol, a phenolic phytoalexin occurring in grapes, wine, peanuts, and cranberries, has been reported to have anticarcinogenic, antioxidative, phytoestrogenic, and cardioprotective activities. Because little is known about the metabolism of this potentially important compound, the in vitro and in vivo metabolism of trans-resveratrol were investigated. Methods. The in vitro experiments included incubation with human liver microsomes, human hepatocytes, and rat hepatocytes and the in vivo studies included oral or intraperitoneal administration of resveratrol to rats and mice. Methanol extracts of rat urine, mouse serum, human hepatocytes, rat hepatocytes, and human liver microsomes were analyzed for resveratrol metabolites using reversed-phase high-performance liquid chromatography with on-line ultraviolet-photodiode array detection and mass spectrometric detection (LC-DAD-MS and LC-UV-MS-MS). UV-photodiode array analysis facilitated the identification of cis- and trans-isomers of resveratrol and its metabolites. Negative ion electrospray mass spectrometric analysis provided molecular weight confirmation of resveratrol metabolites and tandem mass spectrometry allowed structural information to be obtained. Results. No resveratrol metabolites were detected in the microsomal incubations, and no phase I metabolites, such as oxidations, reductions, or hydrolyzes, were observed in any samples. However, abundant trans-resveratrol-3-O-glucuronide and trans-resveratrol-3-sulfate were identified in rat urine, mouse serum, and incubations with rat and human hepatocytes. Incubation with -glucuronidase and sulfatase to release free resveratrol was used to confirm the structures of these conjugates. Only trace amounts of cis-resveratrol were detected, indicating that isomerization was not an important factor in the metabolism and elimination of resveratrol. Conclusion. Our results indicate that trans-resveratrol-3-O-glucuronide and trans-resveratrol-3-sulfate are the most abundant metabolites of resveratrol. Virtually no unconjugated resveratrol was detected in urine or serum samples, which might have implications regarding the significance of in vitro studies that used only unconjugated resveratrol.     

Permeability Characteristics of Various Intestinal Regions of Rabbit,Dog, and Monkey

The in vitro permeability of a series of both hydrophilic and lipophilic compounds, as defined by the octanol/water partition coefficient, was measured in four segments of rabbit, monkey, and dog intestine using a side-by-side diffusion cell. A linear relationship was established for tissue resistance to hydrophilic compound diffusion in jejunum and colon among rabbit, monkey, and dog. The results suggest that rabbit jejunum is twice as permeable as monkey and dog jejunum. The colonic tissues of monkey, rabbit, and dog demonstrate similar permeabilities. Measuring the permeabilities of different tissues with compounds of similar physicochemical properties allows comparison of tissue restriction to transport. Thus, in vitro permeability measurements may be used to investigate physiological differences of various intestinal tissue segments that influence tissue permeability. Investigating the permeability of different intestinal segments from various species could allow the identification of an appropriate in vitro intestinal permeability model that will lead to the prediction of intestinal absorption in humans, eliminating the need for extensive and often misleading in vivo animal testing.     

Ethylmorphine Metabolism in Isolated Rat Hepatocytes

The metabolism of ethylmorphine has been studied in suspensions of isolated rat hepatocytes. Early during incubation, the two major metabolic intermediates detected were morphine and norethylmorphine following N- and O-dealkylation of ethylmorphine, respectively. During subsequent incubation the concentration of the second metabolic intermediate, normorphine increased, before the concentration peaked at approximately 20 μM (100 μM ethylmorphine). Both morphine and normorphine were glucuronidated to form morphine-3-glucuronide and normorphine-3-glucuronide, respectively, which appeared to be the major metabolic end products. The percentage of ethylmorphine metabolized to morphine-3-glucuronide was found to be dependent on the initial concentration of ethylmorphine. With increasing initial ethylmorphine concentration the relative formation of morphine-3-glucuronide was reduced (29±10% at 5 μM, 18 ± 5% at 20 μM, and 15 ± 4% at 100 μM mean ± S.D., n=10). The concentrations of ethylmorphine and its metabolites were found to be higher in liver cells than in medium. Thus the ratios between the intra-/extra-cellular concentrations of ethylmorphine increased somewhat from an initial value of 4 during the period for which ethylmorphine could be detected intracellularly. The drug metabolites all exhibited ratios above 10 for the initial 100 min. of incubation. With time these ratios showed a decline, but even for prolonged incubation the ratios were 5 or higher for the end products. Thus considerable drug concentration gradients existed across the cell membrane of isolated rat hepatocytes.     

Excretion and Biotransformation of Carboxymethyl-cysteine in Rat,Dog, Monkey and Man

1. Following an oral dose of S-carboxymethyl[³⁵S]cysteine, monkey (rhesus and African green), rat, dog, and man excreted 77, 88, 95, and 100% respectively of the ³⁵S radioactivity in urine and 7·0, 2·5, 0·7, and 0·3% in faeces during a 3 to 4 day period.

2. The principal drug-related components excreted were unchanged carboxymethylcysteine, dicarboxymethyl sulphide and inorganic sulphate.

3. Rat, dog, and man excreted primarily dicarboxymethyl sulphide and unchanged carboxymethylcysteine and no inorganic sulphate (rat, 7%).

4. Monkey excreted largely inorganic sulphate, moderate amounts of dicarboxymethyl sulphide and a trace of unchanged drug.     

The Metabolism of Talampicillin in Rat,Dog and Man

1. After administration of [phthalidyl-¹⁴]talampicillin (Talpen® to rat. dog and man, radioactivity was excreted mainly in the urine (90%, 86% and 98% in rat, dog and man respectively).

2. After administration of [ampicillin-¹⁴C]talampicillin, radioactivity was excreted in the urine of rats and dogs to a lesser extent (35% in both species) and only a small proportion of the dose was excreted in the bile (6% in rats, less than 0·1% in dogs).

3. The pattern of radiometaboletes was very similar in extracts of the urines of rat, dog and man dosed orally with [phthalidyl-14C]talampicillin. The major metabolite was 2-hydroxymethylbenzoic acid.

4. Unchanged talampicillin was present in the hepatic portal vein blood of dog and thus reached the liver, whereas in rat, no parent compound could be detected in portal vein blood. This result may help to explain differences in toxicity of the compound in rat and dog.

5. Studies in vitro showed that the intestinal wall is an important site of hydrolysis of talampicillin in rat and dog.     

Ethanol Inhibits Acetate Metabolism in Rat Hepatocytes

Abstract: The metabolism of acetate at concentrations of 1, 2.5, 5 and 10 mM was investigated in freshly isolated hepatocytes from 48 hr fasted, female rats in the absence and presence of 10 mM ethanol. The maximal capacity for acetate metabolism was 0.85 μmol/(10⁸ cells · min). Ethanol caused a 20% decrease in the apparent V_max for acetate metabolism and an increase in the apparent Km for acetate from 3.0 to 4.6 mM. At physiological concentration of acetate (~ 1 mM) and in the absence of an inhibitory effect of ethanol, the capacity for acetate metabolism was 15–20% of the rate of acetate formation from ethanol and the inhibitory effect of ethanol further reduced it to 10–15%. The results thus explain the well-known but hitherto not understood fact that only a small fraction of acetate produced in the liver during ethanol oxidation is further metabolized by the liver, while the majority is exported for oxidation in other tissues. Finally, a new method for calculation of liver acetate uptake in the presence of ethanol is presented.     

Metabolism of Debrisoquine Sulphate in Rat,Dog and Man

1. The metabolism of debrisoquine sulphate in the dog has been studied and is similar to that in rat and man.

2. Two acidic urinary metabolites, shown to be present in rat, dog and man, have been isolated from rat urine. After derivatization they were characterized by n.m.r. and mass spectroscopy as methyl 2-[2-(4,6-dimethylpyrimidylamino)-methyl]-phenylacetate and 2-[2-(4,6-dimethylpyrimidylamino)-ethyl]benzoate.     

The Metabolism of Ftorafur in the Beagle Dog and Rhesus Monkey

1. Ftorafur, a fluorinated pyrimidine nucleoside antimetabolite, is metabolized by the beagle dog and rhesus monkey to 5-fluorouracil, which is subsequently biotransformed to the corresponding nucleosides, to α-fluoro-β-ureidopropionic acid, to urea and to CO₂.

2. In the dog, urea was the primary urinary metabolite while in the monkey, α-fluoro-β-ureidopropionic acid predominated.

3. The dog and monkey excrete about 35% of the recovered dose as CO₂.

4. The possibility that ftorafur is a relatively inactive transport form of 5-fluorouracil is discussed.     

The Metabolism of Terbutaline in Dog and Rat

Abstract

1. The metabolic fate of [³H]terbutaline has been studied in dog after oral, intravenous and subcutaneous administration and in rat after oral and intravenous administration. In 3–4 days the dog excreted 75% of the dose in the urine after oral administration and more than 90% after intravenous or subcutaneous administration; the remainder was in the faeces. The rat in 24 h excreted about 13% in the urine and 61% in the faeces after oral administration and 48% in the urine and 35% in the faeces after intravenous administration.

2. After oral administration of [³H]terbutaline, the time course of radioactivity concentration was the same in lung, heart and serum; low levels of unchanged drug were found in all tissues. After intravenous administration, the concentration of unchanged drug was higher in lung and heart than in serum.

3. In dog, 1·7% of an intravenous dose was excreted into bile in 6 h. In rat, about 37% of the dose was recovered in the bile during 12 h.

4. Enzymic hydrolysis of urine showed that terbutaline is metabolized by conjugation, forming a glucuronide in rat but probably a sulphate in dog.     

The Metabolism of Tinidazole in the Rat and Dog

Abstract

1. [³⁵S]Tinidazole was administered to dogs and rats by oral and intravenous routes. Methods for the determination of tinidazole and total nitroimidazoles are described.

2. The main route for excretion of radioactivity was the urine in both species.

3. In the dog the serum contained predominantly unchanged drug.

4. In both species unchanged drug accounted for about half the urinary radioactivity; the remaining radioactivity was due to four metabolites.

5. One metabolite is the product of hydroxylation of the 2-methyl group and another is its O-glucuronide. The other two metabolites have not been identified, but they do not appear to be simple nitroimidazoles.

6. It is concluded that the metabolic fate of tinidazole is similar in dogs and rats.     

The Metabolism of Isopropyl Oxitol in Rat and Dog

1. An intraperitoneal dose of [¹⁴C]isopropyl Oxitol is rapidly metabolized in the rat.

2. The major routes of excretion of radioactivity are the urine (73% dose) and in the expired air as [¹⁴C]carbon dioxide (14%).

3. The major urinary metabolites were characterized as isopropoxyacetic acid (30% of the urinary radioactivity), N-isopropoxyacetyl glycine (46%) and ethanediol (13%).

4. The metabolism of the compound in the dog is similar to that in the rat.     

The Metabolism of 5-(4-Acetamidophenyl)Pyrazin-2(1H)-One in Rat,Dog and Cynomolgus Monkey

1. The metabolism and disposition of ¹⁴C-acetamidophenyl pyrazinone has been studied in rat, dog and cynomolgus monkey. The compound was well absorbed and rapidly excreted in urine and faeces by all three species.

2. Distribution of ¹⁴C-pyrazinone was rapid and extensive with the exception of the central nervous system where concentrations were at, or below, the limit of detection.

3. Whereas, in in vitro studies, metabolites (but not the parent compound) weakly inhibited some activities of the cytochrome P-450 system, there was evidence from in vivo studies in the rat that the compound and/or its metabolite(s) are weak selective inducers of cytochrome P-450.

4. Metabolite patterns were similar in all three species. The major route of metabolism was glucuronidation at the oxygen of the pyrazinone ring. Other metabolites originated from metabolism by gut microflora with subsequent hepatic metabolism.     

Comparative Toxicology of Temelastine: A Novel H1 Antagonist in Dog, Rat, and Monkey

Comparative Toxicology of Temelastine: A Novel H, Antagonistin Dog, Rat, and Monkey. POOLE, A., BETTON, G. R., SALMON, G.,SUTTON, T., AND ATTERWILL, C. K. (1990). Fundam. Appl. Toxwol.14, 71–83. The toxicity of temelastine 2-[4-(5-bromo-3-methylpyrid-2-yl)butyl-amino]-5-[(6-methylpyTid-3-yl)methyl]-4-pyrimidone a potent, selective, competitive hista-mineHrreceptor antagonist was examined in dogs and rats. The majortoxicologjcal response seen in the dog was marked, but intermittentand reversible, increases in the plasma activity of a numberof liver-associated enzymes, viz alanine aminotransferase (ALT),glutamate dehydro-genase (GLDH), and alkaline phosphatase (ALP).The increases first seen in two male dogs treated for 30 consecutivedays at a dose of 300 mg/kg became apparent at lower doses,i.e., 100 and 33.3 mg/kg/day, in 6- and 12-month studies. Althoughthe increases were suggestive of hepatotoxicity, the only histologicalchanges were increases in hepatocellular lipofuscin pigmentand foci of macrophages seen in dogs treated at 300 mg/kg for12 months. Rats treated for up to 12 months at doses as highas 300 mg/kg/day showed no treatment-related increases in plasmaenzymes although increases in liver weights and hepatocellularlipofuscin pigment together with centrilobular hypertrophy wereseen in the 300 mg/kg/day treatment group. To investigate differencesin hepatic responsiveness between species dogs, rats, and monkeyswere exposed to high concentrations of temelastine by continuous24-hr intravenous infusion. The results of the study showedthe dog to be most sensitive to the hepatic effects of temelastine.The major toxicological effect of temelastine in the rat wasa histopathological lesion of the thyroid gland characterizedby agglomeration and depletion of colloid, follicular epithelialhypertrophy and reduced follicular size. The no-effect dosefor this lesion was between 10 and 33.3 mg/kg/day. These histopathologicalchanges, characteristic of a "TSH-driven" thyroid gland, werenot seen in the thyroid glands of dogs.     

Drug Metabolism Activities of Isolated Rat Hepatocytes in Monolayer Culture

Abstract: The levels of cytochrome P-450 in hepatocytes cultured as monolayers for 22 hrs in Dulbecco's modified Eagle medium supplemented with serum and insulin was reduced to approximately 40% of initial values of freshly isolated hepatocytes. In correspondance with this the activities of the cytochrome P-450 monooxygenases aryl hydrocarbon (benzo(a)pyrene) hydroxylase (AHH) and ethylmorphine (EM) N-demethylase were reduced to 40 and 22% of their initial activities, respectively. Modifying the culture medium through omission of cysteine and cystine, and adding dexamethazone and delta-amino levulinic acid, increased the content of cytochrome P-450 to 59 % and EM N-demethylase to 46 % of initial values, but was without effect on AHH activity. However, further modifications by adding high concentrations of asparagine and leucine increased AHH activity to 62% of initial values, but did not further enhance the total content of cytochrome P-450 or the EM N-demethylase activity. The activities of cytochrome P-450 reductase, flavin containing monooxygenase, epoxide hydrolase and glutathione S-transferase decreased less (to about 70–80% of initial values) than cytochrome P-450 associated monooxygenase activities, whereas UDP-glucuronyl transferase decreased to about 50% of initial values. In contrast to what was observed regarding cytochrome P-450 and associated monooxygenase activities, modification of the incubation conditions did not affect the non-cytochrome P-450 enzymatic activities.     

In Vitro and in Vivo Metabolism of the Anti-Cancer Agent CI-1040, a MEK Inhibitor, in Rat, Monkey, and Human

PURPOSE: The use of in vitro and in vivo models using both rodent and non-rodent species plays an important role with regard to metabolism during the drug development process. In this study, we compared the metabolism of a MEK inhibitor (CI-1040) using in vitro and in vivo models with that observed in a cancer patient. METHODS: Radiolabeled CI-1040 was assessed for metabolism using rat and monkey liver microsomes and hepatocytes, as well as in Wistar rats and cynomolgus monkeys via oral administration. Human bile and plasma samples were obtained immediately after administration of CI-1040 to a patient with advanced colon cancer. A combination of HPLC-radiochromatography (HPLC-RAM), LC/MS and LC/MS/MS experiments were used to analyze all resulting metabolites. Unlabeled CI-1040 was administered (100 mg/day, QD) for 15 days to a patient suffering from colon cancer. Bile was collected by the insertion of a T-tube directly into the bile duct over a 14-h period. Metabolites were also monitored in the patient's plasma. RESULTS: Analysis of the metabolites in all species using in vitro and animal models demonstrated that CI-1040 undergoes extensive oxidative metabolism (14 metabolites identified) with subsequent glucuronidation of the hydroxylated metabolites. Metabolites were predominantly excreted through the bile in the animal models. CONCLUSIONS: Overall, the in vitro and animal models in combination provided comprehensive coverage for all metabolites observed in human bile and plasma. In conclusion, the results obtained in this study demonstrate the utility of conducting investigations across species in order to gain complete coverage for successfully predicting human metabolites of new compounds in development.     

Simulating Intravitreal Injections in Anatomically Accurate Models for Rabbit, Monkey, and Human Eyes

Purpose

To develop models for rabbit, monkey, and human that enable prediction of the clearance after intravitreal (IVT) injections in one species from experimental results obtained in another species.

Methods

Anatomically accurate geometric models were constructed for rabbit, monkey, and human that enabled computational fluid dynamic simulation of clearance of an IVT injected bolus. Models were constructed with and without the retrozonular space of Petit. Literature data on clearance after IVT injection of substances spanning a range of molecular weight up to 157?kDa were used to validate the rabbit model.

Results

The space of Petit had a significant increase on the clearance of slowly diffusing substances cleared by the anterior pathway by reducing the bottleneck for drug efflux. Models that did not include this zone could not accurately predict the clearance of slowly diffusing substances whose clearance was accelerated by intraocular pressure-driven convection.

Conclusions

The ocular anatomy must be carefully reconstructed in the model to enable accurate predictions of clearance. This method offers an alternative means for scaling experimental data from one species to another that may be more appropriate than other simple approaches based entirely upon scaling of compartment volumes and flow rates.     

Modulation of Glucose Metabolism in Isolated Rat Hepatocytes by 1,1,1,2-Tetrafluoroethane

Modulation of Glucose Metabolism in Isolated Rat Hepatocytesby 1,1,1,2-Tetrafluoroethane. OLSON, M.J., REIDY,C. A., ANDJOHNSON, J.T. (1990). Fundam Appl Toxicol. 15,270–280.The thennodynamic behavior and lack of ozone-depleting potentialof 1,1,1,2-tetrafluoro-ethane (R-134a) suggest it as a likelyreplacement for dichlorodifluoromethane (R-12), now used asthe refrigerant in many air-conditioning systems. To furtherthe presently incomplete toxicological analysis of R- 134a,the effects of R-134a on cell viability and functional competenceof glucose metabolism were evaluated in suspension culturesof hepatocytes derived from fed or fasted rats. R-134a concentrationsup to and including 75% (750,000 ppm) in the gas phase of sealedculture flasks did not produce evidence of cytolethality (LDHleakage) following 2 hr of exposure; in contrast, halothane(l,l,l-trifluoro-2-bromo-2-chloroethane) caused cell death ata gas phase concentration of only 1250 ppm. In hepatocytes isolatedfrom fed rats, R-134a at concentrations of 12.5 to 75% increasedglycolysis (production of lactate + pyruvate) in a concentration-dependentmanner, no effect was observed at 5%. At 25%, R-12 and 1,1,2,2-tetrafluoro-l,2-dichloroethane(R-l14) were of equal potency to R-134a in stimulating glycolysis;l,l,l,2,2-pentafluoro-2-chloroethane (R-115) depressed glycolysisslightly. Halothane, at concentrations as low as 300 ppm, markedlyincreased rates of glycolysis. Glucose production by hepatocytesof fed rats was decreased by R-134a, R-12, and R-114 only atconcentrations of 25% or more. On the other hand, halothane(>300 ppm) potently decreased glucose production by hepatocytes.In cells isolated from livers of fasted rats, R-134a exposureinhibited gluconeo-genesis in a concentration-dependent manneralthough this effect was not significant until R-134a concentrationsreached 12.5%. Comparative potency studies showed that R-l 34a,R-12, or R-l 14 (25% gas phase) inhibited gluconeogenesis aboutequally while as little as 300 ppm halothane was effective andR-115 (25%) was without effect. Considering that the thresholdfor alteration of the rate of glucose metabolism in this invitro paradigm is about 12.5% R-134a, we conclude that toxicologicallysignificant alteration of glucose-linked bioenergetics is unlikelyat the levels of R-l34a exposure anticipated in workplace orenvironment     

The Metabolism of Acetonitrile to Cyanide by Isolated Rat Hepatocytes

The Metabolism of Acetonitrile to Cyanide by Isolated Rat Hepatocytes.FREEMAN, J. J., and HAYES, E. P. (1987). Fundam. Appl. Toxicol.8, 263–271. The metabolism of saturated nitriles, includingacetonitrile, has been assumed to occur by a cytochrome P-450-dependentoxidation at the -carbon, yielding a cyanohydrin intermediatewhich may spontaneously degrade to hydrogen cyanide and an aldehyde.However, results of studies in our laboratory suggest that formaldehydeis not a metabolite of acetonitrile. Since acetonitrile is structurallysimilar to iodomethane, a substrate for glutathione (GSH) S-transferases,we hypothesized that the metabolism of acetonitrile to cyanidemight also occur by a nucleophilic substitution reaction involvingGSH. The present studies were conducted to investigate thesehypotheses and to further our study of the effects of acetoneon acetonitrile metabolism. Female Sprague–Dawley ratswere pretreated with buthionine sulfoximine BSO (4 mmol/kg ip,at –4 and –2 hr), cobalt heme (90 µmol/kgsc, at –48 hr), acetone (1960 mg/kg po, at –24 hr),or vehicle, and hepatocytes were isolated after collagenaseperfusion of the liver. BSO reduced the cellular GSH contentby >80%, but did not appear to affect the metabolism of acetonitrile:the liberation of cyanide correlated with cytochrome P-450,and not GSH, concentrations. Cobalt heme depleted hepatocellularcytochrome P-450 (–45%) content, decreased cell yieldand viability, and resulted in a marked reduction in the metabolismof acetonitrile to cyanide. Cobalt heme did not affect the recoveryof sodium cyanide from hepatocyte suspensions. Pretreatmentof rats with acetone resulted in a twofold increase in the metabolismof acetonitrile to cyanide. Addition of acetone in vitro inhibitedacetonitrile metabolism, with an IC50 of 319 µM. Theseresults suggest that the metabolism of acetonitrile to cyanideoccurs by a cytochrome P-40-dependent pathway, and not by anucleophilic substitution reaction with GSH.