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.

Application of in vitro biotransformation data and pharmacokinetic modeling to risk assessment.

The adverse biological effects of toxic substances are dependent upon the exposure concentration and the duration of exposure. Pharmacokinetic models can quantitatively relate the external concentration of a toxicant in the environment to the internal dose of the toxicant in the target tissues of an exposed organism. The exposure concentration of a toxic substance is usually not the same as the concentration of the active form of the toxicant that reaches the target tissues following absorption, distribution, and biotransformation of the parent toxicant. Biotransformation modulates the biological activity of chemicals through bioactivation and detoxication pathways. Many toxicants require biotransformation to exert their adverse biological effects. Considerable species differences in biotransformation and other pharmacokinetic processes can make extrapolation of toxicity data from laboratory animals to humans problematic. Additionally, interindividual differences in biotransformation among human populations with diverse genetics and lifestyles can lead to considerable variability in the bioactivation of toxic chemicals. Compartmental pharmacokinetic models of animals and humans are needed to understand the quantitative relationships between chemical exposure and target tissue dose as well as animal to human differences and interindividual differences in human populations. The data-based compartmental pharmacokinetic models widely used in clinical pharmacology have little utility for human health risk assessment because they cannot extrapolate across dose route or species. Physiologically based pharmacokinetic (PBPK) models allow such extrapolations because they are based on anatomy, physiology, and biochemistry. In PBPK models, the compartments represent organs or groups of organs and the flows between compartments are actual blood flows. The concentration of a toxicant in a target tissue is a function of the solubility of the toxicant in blood and tissues (partition coefficients), blood flow into the tissue, metabolism of the toxicant in the tissue, and blood flow out of the tissue. The appropriate degree of biochemical detail can be added to the PBPK models as needed. Comparison of model simulations with experimental data provides a means of hypothesis testing and model refinement. In vitro biotransformation data from studies with isolated liver cells or subcellular fractions from animals or humans can be extrapolated to the intact organism based upon protein content or cell number. In vitro biotransformation studies with human liver preparations can provide quantitative data on human interindividual differences in chemical bioactivation. These in vitro data must be integrated into physiological models to understand the true impact of interindividual differences in chemical biotransformation on the target organ bioactivation of chemical contaminants in air and drinking water.

Improving cancer dose-response characterization by using physiologically based pharmacokinetic modeling: an analysis of pooled data for acrylonitrile-induced brain tumors to assess cancer potency in the rat.

Historically, U.S. regulators have derived cancer slope factors by using applied dose and tumor response data from a single key bioassay or by averaging the cancer slope factors of several key bioassays. Recent changes in U.S. Environmental Protection Agency (EPA) guidelines for cancer risk assessment have acknowledged the value of better use of mechanistic data and better dose-response characterization. However, agency guidelines may benefit from additional considerations presented in this paper. An exploratory study was conducted by using rat brain tumor data for acrylonitrile (AN) to investigate the use of physiologically based pharmacokinetic (PBPK) modeling along with pooling of dose-response data across routes of exposure as a means for improving carcinogen risk assessment methods. In this study, two contrasting assessments were conducted for AN-induced brain tumors in the rat on the basis of (1) the EPA's approach, the dose-response relationship was characterized by using administered dose/concentration for each of the key studies assessed individually; and (2) an analysis of the pooled data, the dose-response relationship was characterized by using PBPK-derived internal dose measures for a combined database of ten bioassays. The cancer potencies predicted for AN by the contrasting assessments are remarkably different (i.e., risk-specific doses differ by as much as two to four orders of magnitude), with the pooled data assessments yielding lower values. This result suggests that current carcinogen risk assessment practices overestimate AN cancer potency. This methodology should be equally applicable to other data-rich chemicals in identifying (1) a useful dose measure, (2) an appropriate dose-response model, (3) an acceptable point of departure, and (4) an appropriate method of extrapolation from the range of observation to the range of prediction when a chemical's mode of action remains uncertain.

Metabolism of chloroform by cytochrome P450 2E1 is required for induction of toxicity in the liver, kidney, and nose of male mice.

Chloroform is a nongenotoxic-cytotoxic liver and kidney carcinogen and nasal toxicant in some strains and sexes of rodents. Substantial evidence indicates that tumor induction is secondary to events associated with cytolethality and regenerative cell proliferation. Therefore, pathways leading to toxicity, such as metabolic activation, become critical information in mechanism-based risk assessments. The purpose of this study was to determine the degree to which chloroform-induced cytotoxicity is dependent on the cytochromes P450 in general and P450 2E1 in particular. Male B6C3F(1), Sv/129 wild-type (Cyp2e1+/+), and Sv/129 CYP2E1 knockout (Cyp2e1-/- or Cyp2e1-null) mice were exposed 6 h/day for 4 consecutive days to 90 ppm chloroform by inhalation. Parallel control and treated groups, excluding Cyp2e1-null mice, also received an i.p. injection (150 mg/kg) of the irreversible cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) twice on the day before exposures began and 1 h before every exposure. Cells in S-phase were labeled by infusion of BrdU via an implanted osmotic pump for 3.5 days prior to necropsy, and the labeling index was quantified immunohistochemically. B6C3F(1) and Sv/129 wild-type mice exposed to chloroform alone had extensive hepatic and renal necrosis with significant regenerative cell proliferation. These animals had minimal toxicity in the nasal turbinates with focal periosteal cell proliferation. Administration of ABT completely protected against the hepatic, renal, and nasal toxic effects of chloroform. Induced pathological changes and regenerative cell proliferation were absent in these target sites in Cyp2e1-/- mice exposed to 90 ppm chloroform. These findings indicate that metabolism is obligatory for the development of chloroform-induced hepatic, renal, and nasal toxicity and that cytochrome P450 2E1 appears to be the only enzyme responsible for this cytotoxic-related metabolic conversion under these exposure conditions.

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.

Sex-dependent metabolism of xenobiotics.

Sex-dependent differences in xenobiotic metabolism have been most extensively studied in the rat. Because sex-dependent differences are most pronounced in rats, this species quickly became the most popular animal model to study sexual dimorphisms in xenobiotic metabolism. Exaggerated sex-dependent variations in metabolism by rats may be the result of extensive inbreeding and/or differential evolution of isoforms of cytochromes P450 in mammals. For example, species-specific gene duplications and gene conversion events in the CYP2 and CYP3 families have produced different isoforms in rats and humans since the species division over 80 million years ago. This observation can help to explain the fact that CYP2C is not found in humans but is a major subfamily in rats (Table 11). Animal studies are used to help determine the metabolism and toxicity of many chemical agents in an attempt to extrapolate the risk of human exposure to these agents. One of the most important concepts in attempting to use rodent studies to identify sensitive individuals in the human population is that human cytochromes P450 differ from rodent cytochromes P450 in both isoform composition and catalytic activities. Xenobiotic metabolism by male rats can reflect human metabolism when the compound of interest is metabolized by CYP1A or CYP2E because there is strong regulatory conservation of these isoforms between rodents and humans. However, problems can arise when rats are used as animal models to predict the potential for sex-dependent differences in xenobiotic handling in humans. Information from countless studies has shown that the identification of sex-dependent differences in metabolism by rats does not translate across other animal species or humans. The major factor contributing to this observation is that CYP2C, a major subfamily in rats, which is expressed in a sex-specific manner, is not found in humans. To date, sex-specific isoforms of cytochromes P450 have not been identified in humans. The lack of expression of sex-dependent isoforms in humans indicates that the male rat is not an accurate model for the prediction of sex-dependent differences in humans. Differences in xenobiotic metabolism among humans are more likely the consequence of intraindividual variations as a result of genetics or environmental exposures rather than from sex-dependent differences in enzyme composition. A major component of the drug discovery and development process is to identify, at as early a stage as possible, the potential for toxicity in humans. Earlier identification of individual differences in xenobiotic metabolism and the potential for toxicity will be facilitated by improving techniques to make better use of human tissue to prepare accurate in vitro systems such as isolated hepatocytes and liver slices to study xenobiotic metabolism and drug-induced toxicities. Accurate systems should possess an array of bioactivation enzymes similar to the in vivo expression of human liver. In addition, the compound concentrations and exposure times used in these in vitro test systems should mimic those achieved in the target tissues of humans. Consideration of such factors will allow the development of compounds with improved efficacy and low toxicity at a more efficient rate. The development of accurate in vitro systems utilizing human tissue will also aid in the investigation of the molecular mechanisms by which the CYP genes are regulated in humans. Such studies will facilitate the study of the basis for differences in expression of isoforms of CYP450 in humans.

Chloroform-induced cytolethality in freshly isolated male B6C3F1 mouse and F-344 rat hepatocytes.

Chloroform is carcinogenic in rodents but is not mutagenic or DNA reactive. Chloroform-induced hepatocarcinogenesis in rodents is believed to be secondary to events associated with cytotoxicity and cell proliferation. Understanding the mechanisms of chloroform toxicity may provide insights into the mechanisms of carcinogenicity. The goal of these studies was to characterize the cytotoxicity of chloroform in male B6C3F1 mouse and F-344 rat hepatocytes in vitro. We used an in vitro suspension-culture system that reproduced the exposure of the liver to chloroform and the expression of toxicity in vivo. Simulations of a physiologically based dosimetry model for chloroform indicated that the livers of mice and rats were exposed to chloroform concentrations up to 5 mM for 3 h after hepatotoxic doses of chloroform. Freshly isolated male mouse and rat hepatocytes were exposed to chloroform in sealed flasks and then cultured for 24 h as monolayers. Following a 2- or 3-h exposure in suspension, chloroform induced concentration-dependent cytotoxicity (lactate dehydrogenase release) in culture at concentrations higher than 1 mM. Cytolethality was not increased under reduced oxygen tension, indicating that reductive metabolism does not contribute to chloroform-induced toxicity. A threshold of 1 mM chloroform was also found for glutathione (GSH) depletion, with a 50% depletion at 3.8 mM after 2 h. Addition of dithiothreitol, a reducing agent, did not prevent chloroform-induced toxicity, indicating that oxidation of sulfhydryl groups is not critical for toxicity. The lack of protein sulfhydryl group depletion is consistent with this conclusion. Cotreatment with the cytochrome P450 inhibitor 1-phenylimidazole prevented both cytolethality and GSH depletion, indicating that metabolism is necessary for chloroform-induced toxicity. Both species exhibited similar sensitivity toward chloroform toxicity, indicating that toxicity is not sufficient to explain different susceptibility in heptocarcinogenicity. As chloroform metabolism is saturated in the micromolar range, our results indicate that both metabolism and exposure of the liver cells to high concentrations of chloroform are required for toxicity.

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.

Identification of benzene oxide as a product of benzene metabolism by mouse, rat, and human liver microsomes.

Benzene is a ubiquitous environmental pollutant that is known to cause hematotoxicity and leukemia in humans. The initial oxidative metabolite of benzene has long been suspected to be benzene oxide (3,5-cyclohexadiene-1,2-oxide). During in vitro experiments designed to characterize the oxidative metabolism of [14C]benzene, a metabolite was detected by HPLC-radioactivity analysis that did not elute with other known oxidative metabolites. The purpose of our investigation was to prove the hypothesis that this metabolite was benzene oxide. Benzene (1 mM) was incubated with liver microsomes from human donors, male B6C3F1 mice, or male Fischer-344 rats, NADH (1 mM), and NADPH (1 mM) in 0.1 M sodium phosphate buffer (pH 7.4) and then extracted with methylene chloride. Gas chromatography-mass spectrometry analysis of incubation extracts for mice, rats, and humans detected a metabolite whose elution time and mass spectrum matched that of synthetic benzene oxide. The elution time of the benzene oxide peak was approximately 4.1 min, while phenol eluted at approximately 8 min. Benzene oxide also coeluted with the HPLC peak of the previously unidentified metabolite. Based on the 14C activity of this peak, the concentration of benzene oxide was determined to be approximately 18 microM, or 7% of total benzene metabolites, after 18 min of incubation of mouse microsomes with 1 mM benzene. The metabolite was not observed in incubations using heat-inactivated microsomes. This is the first demonstration that benzene oxide is a product of hepatic benzene metabolism in vitro. The level of benzene oxide detected suggests that benzene oxide is sufficiently stable to reach significant levels in the blood of mice, rats, and humans and may be translocated to the bone marrow. Therefore benzene oxide should not be excluded as a possible metabolite involved in benzene-induced leukemogenesis.

Furan-mediated uncoupling of hepatic oxidative phosphorylation in Fischer-344 rats: an early event in cell death.

Furan is a potent rodent hepatotoxicant and carcinogen. The present study was done to examine the effects of furan on hepatic energy metabolism both in vivo and in vitro in male F-344 rats. Furan produced concentration- and incubation time-dependent irreversible reductions in ATP in freshly isolated F-344 rat hepatocytes. Furan-mediated depletion of ATP occurred prior to cell death and was prevented by including 1-phenylimidazole, a cytochrome P450 inhibitor, in the suspensions. Male F-344 rats were treated with furan (0-30 mg/kg, po) and killed 24 hr later to prepare hepatic mitochondria. Furan produced dose-dependent increases in state 4 respiration and ATPase activity. Both of these changes were prevented by 1-phenylimidazole cotreatment. In a separate series of experiments, mitochondria were prepared from isolated rat hepatocytes following incubation with furan (2-100 microM) for 1-4 hr. Furan produced incubation time- and concentration-dependent increases in state 4 respiration and ATPase activity. Furan-mediated mitochondrial changes were prevented by adding 1-phenylimidazole to the hepatocyte suspensions. These results indicate that the ene-dialdehyde metabolite of furan uncouples hepatic oxidative phosphorylation in vivo and in vitro. In vitro studies using an isolated hepatocyte suspension/culture system demonstrated that the concentration response for furan-mediated mitochondrial changes in suspension corresponded with the concentration responses for cell death after 24 hr. Including 1-phenylimidazole or oligomycin plus fructose in hepatocyte suspensions prevented furan-induced cell death after 24 hr in culture. The results of this study indicate that furan-induced uncoupling of oxidative phosphorylation is an early, critical event in cytolethality both in vivo and in vitro.

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.

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.

Refinement and verification of the physiologically based dosimetry description for acrylonitrile in rats.

The physiologically based dosimetry description for acrylonitrile (ACN) and its mutagenic epoxide metabolite 2-cyanoethylene oxide (CEO) in F-344 rats (M. L. Gargas, M. E. Anderson, S.K.O. Teo, R. Batra, T. R. Fennell, and G. L. Kedderis, 1995, Toxicol. Appl. Pharmacol. 134, 185-194) has been refined to include a physiological stomach compartment and the reactions of ACN with tissue glutathione (GSH). The second-order rate constant for reaction of ACN and GSH at pH 7.3 was measured and included in the dosimetry description. Metabolic parameters for ACN and CEO were estimated from oral bolus pharmacokinetic studies and previously obtained iv bolus data (3.4, 47, 55, or 84 mg ACN/kg). Rats were given bolus oral doses of 3, 10, or 30 mg ACN/kg in water, and blood samples were collected at selected time points. ACN and CEO blood concentrations were determined by gas chromatography. The brain and liver concentrations of ACN and CEO were also measured after 10 mg ACN/kg po. ACN elimination from blood was described by saturable P450 epoxidation (Vmax of 5.0 mg/hr/kg and K(M) of 1.5 mg/liter) and first-order GSH conjugation (73 hr(-1)/kg). CEO elimination was described by first-order GSH conjugation (500 hr(-1)/kg). The pharmacokinetic data were well simulated, although CEO blood concentrations after bolus oral dosing were somewhat overestimated. Sensitivity analysis of the dosimetry description indicated that the inhalation exposure route was much more sensitive to changes in metabolic and physiological parameters than either the iv or oral bolus routes. Therefore, inhalation pharmacokinetic data were obtained and compared to simulations of the dosimetry description. Rats were exposed to 186, 254, or 291 ppm ACN for 3 hr. ACN and CEO concentrations were measured in blood, brain, and liver at selected postexposure time points. The dosimetry description accurately simulated the ACN inhalation pharmacokinetic data, providing verification of the parameter estimates. The verified rat dosimetry description for ACN and CEO will be used as the basis for development of a dosimetry description for ACN in people.

Prediction of furan pharmacokinetics from hepatocyte studies: comparison of bioactivation and hepatic dosimetry in rats, mice, and humans.

Furan is a volatile solvent and chemical intermediate that is hepatotoxic and hepatocarcinogenic in rats and mice but is not mutagenic or DNA-reactive. Furan hepatotoxicity requires cytochrome P450 2E1 bioactivation to cis-2-butene-1,4-dial. We have previously shown that furan biotransformation kinetics determined with freshly isolated rat hepatocytes in vitro accurately predict furan pharmacokinetics in vivo [Kedderis et al. (1993) Toxicol. Appl. Pharmacol. 123, 274], suggesting that furan biotransformation kinetics determined with freshly isolated mouse or human hepatocytes can be used to develop species-specific pharmacokinetic models. Hepatocytes from male B6C3F1 mice or human accident victims (n = 3) were incubated with furan vapors to determine the kinetic parameters for furan bioactivation and compared to our previous data for rat hepatocytes. Isolated hepatocytes from all three species rapidly metabolized furan (Vmax of 48 nmol/hr/10(6) mouse hepatocytes, 19-44 nmol/hr/10(6) human hepatocytes, and 18 nmol/hr/10(6) rat hepatocytes) with high affinity (KM ranging from 0.4 to 3.3 microM). The hepatocyte kinetic data and physiological parameters from the literature were used to develop dosimetry models for furan in mice and people. The hepatocyte Vmax values were extrapolated to whole animals assuming 128 x 10(6) hepatocytes/g rodent liver and 137 x 10(6) hepatocytes/g human liver. Simulations of inhalation exposure to 10 ppm furan for 4 hr indicated that the absorbed dose (mg/kg), and consequently the liver dose of cis-2-butene-1,4-dial, was approximately 3- and 10-fold less in humans than in rats or mice, respectively. These results indicate that the target organ concentration, rather than the exposure concentration, is most appropriate for interspecies comparison of dose. The initial rates of furan oxidation in rat, mouse, and human liver were approximately 13-, 24-, and 37-fold greater than the respective rates of blood flow delivery of furan to the liver after 4-hr exposures to < or = 300 ppm. One important consequence of blood flow limitation of furan bioactivation is that the amount of toxic metabolite formed in the liver will be unaffected by increases in Vmax due to the induction of cytochrome P450 2E1. Therefore, the interindividual variations observed in cytochrome P450 2E1 activity among human populations would not be expected to have a significant effect on the extent of furan bioactivation in people. These considerations may be important for human cancer risk assessments of other rapidly metabolized rodent carcinogens.

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.

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.

Conjugation of acrylonitrile and 2-cyanoethylene oxide with hepatic glutathione.

The glutathione (GSH) conjugation of the rat carcinogen acrylonitrile (ACN) and its epoxide metabolite 2-cyanoethylene oxide (CEO) by rat, mouse, and human liver enzymes was characterized in vitro since GSH conjugation is the major disposition pathway for these chemicals in vivo. Mass spectral analyses indicated that S-(2-cyanoethyl)GSH was the product from reaction of GSH and ACN and that S-(cyanohydroxyethyl)GSH reaction products were formed from CEO. Because of the rapid nonenzymic reactions of ACN and CEO with GSH at pH 7.3, the steady-state kinetics of hepatic GSH conjugation were determined at pH 6.5 by HPLC analysis of the products. Hyperbolic kinetics were observed with respect to GSH for the reactions catalyzed by mouse or rat hepatic cytosols at pH 6.5, whereas sigmoidal kinetics were observed with respect to ACN or CEO. This kinetic pattern is consistent with the random sequential kinetic mechanism that has been described for GSH S-transferases. Estimates of the maximal velocities of the reaction at pH 6.5 showed that mouse enzymes had a 4- to 6-fold greater capacity for GSH conjugation of ACN and CEO than rat enzymes. ACN appeared to be conjugated with GSH more efficiently than CEO under these conditions. At physiological pH (7.3), rapid nonenzymic conjugation of GSH (10 mM) with ACN or CEO (5 mM) was observed (approximately 25 and 15 nmol product/min, respectively). Addition of hepatic cytosols or microsomes from rats or mice increased the velocity of GSH conjugation approximately 1.6-fold. A similar velocity enhancement was observed with human liver cytosols for the GSH conjugation of ACN, but not for CEO. Human liver microsomes did not enhance the velocity of GSH conjugation of either substrate. These results suggest that ACN is a better substrate for human liver GSH S-transferases than CEO. Estimation of the initial velocities of the GSH conjugation reactions in intact rodent liver from the in vitro data at pH 7.3 suggests that the enzyme-mediated GSH conjugation of ACN and CEO will be approximately 4-fold greater than the velocity of the direct chemical reaction with GSH.