Glutathione S-transferase-mediated chlorothalonil metabolism in liver and gill subcellular fractions of channel catfish.

Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile) is a broad spectrum fungicide that is a potent acute toxicant to fish. Therefore, the metabolism of chlorothalonil was investigated in liver and gill cytosolic and microsomal fractions from channel catfish (Ictalurus punctatus) using HPLC. All fractions catalyzed the metabolism of chlorothalonil to polar metabolites. Chlorothalonil metabolism by cytosolic fractions was reduced markedly when glutathione (GSH) was omitted from the reaction mixtures. The lack of microsomal metabolism in the presence of either NADPH or an NADPH-regenerating system indicated direct glutathione S-transferase (GST)-catalyzed conjugation with GSH without prior oxidation by cytochrome P450. Cytosolic and microsomal GSTs from both tissues were also active toward 1-chloro-2,4-dinitrobenzene (CDNB), a commonly employed reference substrate. In summary, channel catfish detoxified chlorothalonil in vitro by GST-catalyzed GSH conjugation in the liver and gill. The present report is the first to confirm microsomal GST activity toward CDNB in gill and toward chlorothalonil in liver, and also of gill cytosolic GST activity towards chlorothalonil, in an aquatic species.

Kinetics of conjugation and oxidation of nitrobenzyl alcohols by rat hepatic enzymes.

Previous work has suggested that quantitative differences in the in vitro and in vivo metabolism of mononitrotoluene isomers are a result of differences in the hepatic conjugation and oxidation of the first metabolic intermediates, the mononitrobenzyl alcohols. We have determined the steady-state kinetic parameters, Vmax, Km and V/K, for the metabolism of the nitrobenzyl alcohols by rat hepatic alcohol dehydrogenase, glucuronyltransferase, and sulfotransferase. 3-Nitrobenzyl alcohol was the best substrate for cytosolic alcohol dehydrogenase (Vmax = 1.48 nmoles/min/mg protein, V/K = 3.15 X 10(-3) nmoles/min/mg protein/microM, Km = 503 microM). Vmax and Km values for 4-nitrobenzyl alcohol were similar, but V/K was about 60% of that for 3-nitrobenzyl alcohol. 2-Nitrobenzyl alcohol was not metabolized by the alcohol dehydrogenase preparation used here, but it was metabolized to 2-nitrobenzoic acid by a rat liver mitochondrial preparation. 2-Nitrobenzyl alcohol was the best substrate for microsomal glucuronyltransferase (Vmax = 3.59 nmoles/min/mg protein, V/K = 11.28 X 10(-3) nmoles/min/mg protein/microM, Km = 373 microM). The Vmax for 3-nitrobenzyl alcohol was similar, but the V/K was about half and the Km was about twice that for 2-nitrobenzyl alcohol. The Vmax for 4-nitrobenzyl alcohol was about 40% and the V/K was about half that for 2-nitrobenzyl alcohol. The best substrate for cytosolic sulfotransferase was 4-nitrobenzyl alcohol (Vmax = 1.69 nmoles/min/mg protein, V/K = 37.21 X 10(-3) nmoles/min/mg protein/microM, Km = 48 microM). The Vmax values for the other two benzyl alcohols were similar, but the V/K and Km values were about 11 and 400%, respectively, of those for 4-nitrobenzyl alcohol. These data are in qualitative agreement with results obtained when the nitrobenzyl alcohols were incubated with isolated hepatocytes, but they do not allow quantitative modeling of the data from hepatocytes.

In vitro human tissue models in risk assessment: report of a consensus-building workshop.

Advances in the technology of human cell and tissue culture and the increasing availability of human tissue for laboratory studies have led to the increased use of in vitro human tissue models in toxicology and pharmacodynamics studies and in quantitative modeling of metabolism, pharmacokinetic behavior, and transport. In recognition of the potential importance of such models in toxicological risk assessment, the Society of Toxicology sponsored a workshop to evaluate the current status of human cell and tissue models and to develop consensus recommendations on the use of such models to improve the scientific basis of risk assessment. This report summarizes the evaluation by invited experts and workshop attendees of the current status of such models for prediction of human metabolism and identification of drug-drug interactions, prediction of human toxicities, and quantitative modeling of pharmacokinetic and pharmaco-toxicodynamic behavior. Consensus recommendations for the application and improvement of current models are presented.

Furan-induced liver cell proliferation and apoptosis in female B6C3F1 mice.

Furan is a potent rodent hepatocarcinogen that probably acts through non-genotoxic mechanisms involving hepatotoxicity and regenerative hepatocyte proliferation. In addition to inducing necrosis, cytotoxicants like furan may also induce cytolethality through apoptosis which has been suggested to play a key role in carcinogenesis. Hepatocyte proliferation and apoptosis were studied in female B6C3F1 mice exposed to furan by oral gavage for 3 weeks at National Toxicology Program (NTP) bioassay doses (8 and 15 mg/kg body weight) and lower (4 mg/kg). Furan treatment led to a 2- to 3-fold significant increase in liver-related enzymes and bile acids in blood serum as compared to the control group. These changes were accompanied by minor subcapsular inflammation and minimal necrosis at 8 and 15 mg furan/kg. A dose-related increase in bromodeoxyuridine-labeling index (1.4- to 1.7-fold) and hematoxylin- and eosin-defined apoptotic index (6- to 15-fold) was observed at 8 and 15 mg/kg. Co-treatment of mice with aminobenzotriazole, an irreversible inhibitor of cytochromes P-450, prevented the observed hepatotoxic effects induced by furan. These results indicate that furan elicits hepatotoxicity in a dose-related manner through a toxic metabolite and, furthermore, suggest that apoptosis is an important form of cell death at hepatocarinogenic doses under short-term conditions.

Extrapolation of in vitro enzyme induction data to humans in vivo.

Enzyme induction generally increases the rate and extent of xenobiotic metabolism in vitro, but physiological constraints can dampen these effects in vivo. Biotransformation kinetics determined in hepatocytes in vitro can be extrapolated to whole animals based on the hepatocellularity of the liver, since the initial velocity of an enzyme-catalyzed reaction is directly proportional to the total enzyme present in the cell. The biotransformation kinetics of various xenobiotics determined with isolated hepatocytes in vitro have been shown to accurately predict pharmacokinetics in whole animals. Analysis of the kinetic data, using physiologically based pharmacokinetics, allows extrapolation of xenobiotic biotransformation across dose routes and species in a biologically realistic context. Several fold variations were observed in the bioactivation of the hepatotoxicant furan by isolated human hepatocytes, due to induction of cytochrome P450 2E1. Extrapolation of these data to humans in vivo showed that furan bioactivation was limited by hepatic blood flow delivery of the substrate. One important consequence of hepatic blood flow limitation is that the amount of metabolite formed in the liver is unaffected by increases in Vmax due to enzyme induction. Therefore, interindividual variations in cytochrome P450 2E1 among human populations would not affect the bioactivation of many rapidly metabolized hazardous chemical air pollutants. The hepatic blood flow limitation of biotransformation is also observed after oral bolus dosing of rapidly metabolized compounds. More slowly metabolized xenobiotics, such as therapeutic agents, are only partially limited by hepatic blood flow and other processes.

Hepatic macromolecular covalent binding of mononitrotoluenes in Fischer-344 rats.

The mononitrotoluenes are important industrial chemicals which display isomeric specificity in their ability to induce hepatic DNA excision repair in Fischer-344 rats. Covalent binding of the structurally related hepatocarcinogen, 2,6-dinitrotoluene, to hepatic DNA is markedly decreased by prior administration of the sulfotransferase inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP). The objectives of this study were to determine whether hepatic macromolecular covalent binding of the mononitrotoluene isomers differed and to determine whether covalent binding of the mononitrotoluenes to hepatic DNA in vivo was decreased by inhibitors of sulfotransferase. Male Fischer-344 rats were given a single oral dose of [ring-U-14C]-2-, 3- or 4-nitrotoluene (2-, 3- or 4-NT) and killed at various times thereafter. Livers were removed and analyzed for total and covalently bound radiolabel. Maximal concentrations of total radiolabel were observed between 3 and 12 h after the dose, and there were no large differences among the 3 isomers in peak concentrations achieved. Covalent binding to hepatic macromolecules was maximal 12 h after administration for all three isomers. Thereafter, concentrations of covalently bound 2-NT-derived material were always 2-6 times higher than those of 3- or 4-NT-derived material. When DNA was isolated from livers of rats given the mononitrotoluenes 12 h previously, only 2-NT was observed to covalently bind at concentrations above the limits of detection of the assay. The covalent binding of 2-NT, but not that of 3- or 4-NT, to both total hepatic macromolecules and DNA was markedly decreased by prior administration of either PCP or DCNP. Covalent binding to hepatic DNA was decreased by greater than 96%. The results of this study correlate well with studies which have demonstrated that 2-NT, but not 3- or 4-NT, induces DNA excision repair. Furthermore, they suggest that 2-NT, like the hepatocarcinogen 2,6-dinitrotoluene, requires the action of sulfotransferase for its conversion to a species capable of covalently binding to hepatic DNA.

Present status of the application of cryopreserved hepatocytes in the evaluation of xenobiotics: consensus of an international expert panel.

Successful cryopreservation of freshly isolated hepatocytes would significantly decrease the need for freshly-procured livers for the preparation of hepatocytes for experimentation. Hepatocytes can be prepared, cryopreserved, and used for experimentation as needed at different times after isolation. Cryopreservation is especially important for research with human hepatocytes because of the limited availability of fresh human livers. Based on the cumulative experience of this international expert panel, a consensus was reached on the various aspects of hepatocyte cryopreservation, including cryopreservation and thawingprocedures and applications of the cryopreserved hepatocytes. Key to successful cryopreservation includes slow addition of cryopreservants, controlled-rate freezing with adjustment for the heat of crystallization, storage at -150 degrees C, and rapid thawing. There is a general consensus that cryopreserved hepatocytes are useful for short-term xenobiotic metabolism and cytotoxicity evaluation.

Vehicle-dependent oral absorption and target tissue dosimetry of chloroform in male rats and female mice.

Chloroform-induced toxicity in rodents depends on oral dose regimen. We evaluated the absorption and tissue dosimetry of chloroform after gavage administration in various vehicles to male Fischer 344 rats and female B6C3F1 mice. Animals received a single dose of chloroform in corn oil, water, or aqueous 2% emulphor at doses (15-180 and 70-477 mg/kg for rats and mice) and dose volumes (2 and 10 ml/kg for rats and mice) used in previously reported toxicity studies. Blood, liver, and kidney chloroform concentration-time courses were determined. Gavage vehicle had minimal effects on chloroform dosimetry in rats. In mice, however, tissue chloroform concentrations were consistently greater for aqueous versus corn oil vehicle. At the low dose volume used for rats (2 ml/kg) gavage vehicle may not play a significant role in chloroform absorption and tissue dosimetry, at the higher dose volume used for mice (10 ml/kg), vehicle may be a critical factor.

Rodent tissue distribution of 2-cyanoethylene oxide, the epoxide metabolite of acrylonitrile.

The direct acting mutagen 2-cyanoethylene oxide (CEO), formed in the liver by oxidation of acrylonitrile (ACN), is thought to mediate the extrahepatic carcinogenic effects of ACN in rats. This study determined the tissue distribution of CEO (3 mg/kg p.o.) in F-344 rats and B6C3F1 mice. Radioactivity from [2,3-14C]CEO was widely distributed in the major organs of rodents by 2 h and decreased by 71% to 90% within 24 h, demonstrating that there was no preferential tissue uptake or retention of CEO. CEO was detected in rodent blood and brain 5-10 min after an oral dose of ACN (10 mg/kg), demonstrating that this mutagenic epoxide metabolite circulates to extrahepatic target organs following ACN administration.

The role of regenerative cell proliferation in chloroform-induced cancer.

Chloroform produces cancer by a nongenotoxic-cytotoxic mode of action, with no increased cancer risk expected at noncytotoxic doses. The default risk assessment for inhaled chloroform relies on liver tumor incidence from a gavage study with female B6C3F1 mice and estimates a virtually safe dose (VSD) at an airborne concentration of 0.000008 ppm of chloroform. In contrast, a 1000-fold safety factor applied to the NOAEL for liver cytotoxicity from inhalation studies yields a VSD of 0.01 ppm. This estimate relies on inhalation data and is more consistent with the mode of action of chloroform.

Biochemical basis of hepatocellular injury.

The hepatotoxic response elicited by a chemical agent depends on the concentration of the toxicant (parent compound or metabolite) delivered to the hepatocytes across the liver acinus via blood flow. Hepatotoxicants produce characteristic patterns of cytolethality in specific zones of the acinus due to the differential expression of enzymes and the concentration gradients of cofactors and toxicant in blood across the acinus. Most hepatotoxic chemicals produce necrosis, characterized by swelling in contiguous tracts of cells and inflammation. This process has been contrasted with apoptosis, where cells and organelles condense in an orderly manner under genetic control. Biotransformation can activate a chemical to a toxic metabolite or decrease toxicity. Quantitative or qualitative species differences in biotransformation pathways can lead to significant species differences in hepatotoxicity. Fasted rodents are more susceptible to the hepatotoxic effects of many chemicals due to glutathione depletion and cytochrome P-450 induction. Freshly isolated hepatocytes are the most widely used in vitro system to study mechanisms of cell death. Hepatotoxicants can interact directly with cell macromolecules or via a reactive metabolite. The reactive metabolite can alkylate critical cellular macromolecules or induce oxidative stress. These interactions generally lead to a loss of calcium homeostasis prior to plasma membrane lysis. Mitochondria have been shown to be important cellular targets for many hepatotoxicants. Decreasing hepatocellular adenosine triphosphate concentrations compromise the plasma membrane calcium pump, leading to increased cellular calcium concentrations. Calcium-dependent endonucleases produce double-strand breaks in DNA before cell lysis. These biochemical pathways induced by necrosis-causing toxicants are similar to the biochemical pathways involved in apoptosis, suggesting that apoptosis and necrosis differ in intracellular and extracellular control points rather than in the biochemistry involved in cell death.

Steady state kinetics of chloroperoxidase-catalyzed N-demethylation reactions.

The kinetic mechanism of the chloroperoxidase-catalyzed N-demethylation of N,N-dimethylaniline supported by ethyl hydroperoxide was determined from initial rate and inhibition studies. When the concentration of N,N-dimethylaniline was varied in the presence of several different fixed concentrations of ethyl hydroperoxide, a double reciprocal plot of the data gave a series of parallel lines. Parallel lines were also observed when the data were plotted as a function of the concentration of ethyl hydroperoxide. A linear double reciprocal plot was obtained when the concentrations of both substrates were varied in a constant ratio. Competitive substrate inhibition was observed for N,N-dimethylaniline with a KI of 4.78 mM. Competitive substrate inhibition was also observed for ethyl hydroperoxide but the secondary replot of the slopes of the double reciprocal lines versus the concentrations of ethyl hydroperoxide intercepted the origin, indicating that no abortive binary complex between ethyl hydroperoxide and the enzyme was formed. These results suggested that the inhibition of the demethylation reaction by ethyl hydroperoxide was due to the reaction of ethyl hydroperoxide with chloroperoxidase compound I to evolve oxygen. N,N-Dimethylaniline inhibited the chloroperoxidase-catalyzed evolution of oxygen from ethyl hydroperoxide with a KI (0.111 mM) essentially identical with its KM for demethylation (0.122 mM). The inhibition of the demethylation reaction by 2,5-dimethylfuran was competitive with respect to N,N-dimethylaniline and uncompetitive with respect to ethyl hydroperoxide, consistent with dead-end inhibition in a ping-pong system. The results of the initial rate and inhibition studies are consistent with a Ping Pong Bi Bi mechanism as the minimal kinetic model for chloroperoxidase-catalyzed N-demethylation reactions. In this model, ethyl hydroperoxide reacts with chloroperoxidase to form the oxidized enzyme intermediate compound I with the concomitant release of ethanol. N,N-Dimethylaniline then binds to compound I and is oxidized, resulting in the formation of N-methylaniline and formaldehyde and the regeneration of the native peroxidase.

Inhibition of the microsomal N-hydroxylation of 2-amino-6-nitrotoluene by a metabolite of methimazole.

Inhibition studies were used to investigate the identity of the microsomal enzyme(s) responsible for the NADPH-dependent N-hydroxylation of 2-amino-6-nitrotoluene. The N-hydroxylation reaction was inhibited by several cytochrome P-450 inhibitors as well as by methimazole, a substrate for flavin-containing monooxygenase. Heat inactivation of flavin-containing monooxygenase had no effect on the rate of the reaction but abolished the inhibition by methimazole. These results indicate that the flavin-containing monooxygenase-mediated metabolism of methimazole produced an inhibitor of the cytochrome P-450-catalyzed N-hydroxylation reaction. When glutathione was included in the incubation the inhibition by methimazole was abolished, presumably due to the reduction of oxygenated metabolites of methimazole. These results show that methimazole inhibition does not necessarily implicate flavin-containing monooxygenase in microsomal N-hydroxylation reactions.

Loss of rat liver microsomal cytochrome P-450 during methimazole metabolism. Role of flavin-containing monooxygenase.

The role of flavin-containing monooxygenase (FMO) in the decrease in cytochrome P-450 content during the microsomal metabolism of methimazole (N-methyl-2-mercaptoimidazole) was investigated by heat inactivation of FMO. Incubation of liver microsomes from untreated Fischer 344 rats with NADPH and methimazole resulted in a 25% loss of cytochrome P-450 detectable as its ferrous-carbon monoxide complex. The same extent of cytochrome P-450 loss was observed with 1 and 20 mM methimazole, suggesting saturation of the process. There was no significant loss of cytochrome P-450 when microsomal FMO was heat-inactivated prior to incubation with NADPH and methimazole. Heat pretreatment of the microsomes did not affect cytochrome P-450 concentrations and cytochrome P-420 was not observed. These results indicate that FMO-catalyzed metabolism of methimazole is necessary for the loss of cytochrome P-450 in microsomes from untreated rats. Sulfite and N-methylimidazole, the ultimate products of methimazole metabolism, did not cause a significant loss of cytochrome P-450. There was no loss of cytochrome P-450 when glutathione was included in the incubation with methimazole, suggesting that cytochrome P-450 loss was due to an interaction with oxygenated metabolites of methimazole formed by FMO. Losses of cytochrome P-450 were also observed after incubation of microsomes from phenobarbital- (31%) of beta-naphthoflavone-pretreated rats (44%) with NADPH and methimazole. In contrast to microsomes from untreated rats, heat inactivation of FMO did not prevent the loss of cytochrome P-450 in microsomes from the pretreated rats. These results indicate that both phenobarbital and beta-naphthoflavone induce isozymes of cytochrome P-450 capable of directly activating methimazole.

Peroxidase-catalyzed N-demethylation reactions. Substrate deuterium isotope effects.

Deuterium isotope effects on the kinetic parameters for the hydroperoxide-supported N-demethylation of N,N-dimethylaniline catalyzed by chloroperoxidase and horseradish peroxidase were determined using N,N-di-(trideuteromethyl)aniline. The isotope effect on the Vmax for the chloroperoxidase-catalyzed demethylation reaction supported by ethyl hydroperoxide was 1.42 +/- 0.31. The isotope effects on the Vmax for the horseradish peroxidase-catalyzed reaction supported by ethyl hydroperoxide and hydrogen peroxide were 1.99 +/- 0.39 and 4.09 +/- 0.27, respectively. Isotope effects ranging from 1.76 to 5.10 were observed on the Vmax/Km for the hydroperoxide substrate (i.e. the second order rate constant for the reaction of the hydroperoxide with the peroxidase to form compound I) in both enzyme systems when the N-methyl groups of N,N-dimethylaniline were deuterated. These results are not predicted by the simple ping-pong kinetic model for peroxidase-catalyzed N-demethylation reactions. The data are most simply explained by a mechanism involving the transfer of deuterium (or hydrogen) from N,N-dimethylaniline to the enzyme during catalysis. The deuterium must subsequently be displaced from the enzyme by the hydroperoxide, causing the observed isotope effects.

pH kinetic studies of the N-demethylation of N,N-dimethylaniline catalyzed by chloroperoxidase.

The effect of pH on the kinetic parameters for the chloroperoxidase-catalyzed N-demethylation of N,N-dimethylaniline supported by ethyl hydroperoxide was investigated from pH 3.0 to 7.0. Chloroperoxidase was found to be stable throughout the pH range studied. Initial rate conditions were determined throughout the pH range. The Vmax for the demethylation reaction exhibited a pH optimum at approximately 4.5. The Km for N,N-dimethylaniline increased with decreasing pH, while the Km for ethyl hydroperoxide varied in a manner paralleling Vmax. Comparison of the Vmax/Km values for N,N-dimethylaniline and ethyl hydroperoxide indicated that the interaction of N,N-dimethylaniline with chloroperoxidase compound I was rate-limiting below pH 4.5, while compound I formation was rate-limiting above pH 4.5. The log of the Vmax/Km for ethyl hydroperoxide was independent of pH, indicating that chloroperoxidase compound I formation is not affected by ionizations in this pH range. The plot of the log of the Vmax/Km for N,N-dimethylaniline versus pH indicated an ionization on compound I with a pK of approximately 6.8. The plot of the log of the Vmax versus pH indicated an ionization on the compound I-N,N-dimethylaniline complex, with a pK of approximately 3.1. The results show that chloroperoxidase can demethylate both the protonated and neutral forms of N,N-dimethylaniline (pK approximately 5.0), suggesting that hydrophobic binding of the arylamine substrate is more important in catalysis than ionic bonding of the amine moiety. For optimal catalysis, a residue in the chloroperoxidase compound I-N,N-dimethylaniline complex with a pK of approximately 3.1 must be deprotonated, while a residue in compound I with a pK of approximately 6.8 must be protonated.