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.

Long-term mutagenicity studies with chloroform and dimethylnitrosamine in female lacI transgenic B6C3F1 mice.

The weight of evidence indicates that chloroform induces cancer in the female B6C3F1 mouse liver via a nongenotoxic-cytotoxic mode of action. However, it is probable that DNA damage occurs secondary to events associated with cytolethality and regenerative cell proliferation. The purpose of the present study was to evaluate the potential mutagenic activity of chloroform in the B6C3F1 lacI transgenic mouse liver mutagenesis assay including mutagenic events that might occur secondary to cytolethality. The positive control, dimethylnitrosamine (DMN) is a DNA-reactive mutagen and carcinogen. DMN-induced mutations were anticipated to require only a brief exposure and without further treatment were predicted to remain unchanged over time at those frequencies. Chloroform-induced mutations secondary to toxicity were anticipated to require longer exposure periods and to occur only under conditions that produced sustained cytolethality and regenerative cell proliferation. Female B6C3F1 lacI transgenic mice were treated with daily doses of 2, 4, or 8 mg/kg of DMN by gavage for 4 days and then held until analysis 10, 30, 90, and 180 days postexposure. Livers from DMN-treated mice exhibited a dose-related 2- to 5-fold increase over control mutant frequencies and remained at those levels for 10 through 180 days postexposure. Thus, following the initial induction by DMN no selective mutation amplification or loss was seen for this extended period of time. Female B6C3F1 lacI mice were exposed daily for 6 hr/day 7 days/week to 0, 10, 30, or 90 ppm chloroform by inhalation, representing nonhepatotoxic, borderline, or overtly hepatotoxic chloroform exposures. Timepoints for determination of lacI mutant frequency were 10, 30, 90, and 180 days of exposure. No increase in lacI mutant frequency in the liver was observed at any dose or timepoint with chloroform, indicating a lack of DNA reactivity. DNA alterations secondary to toxicity either did not occur or were of a type not detectable by lacI mutant frequency analysis, such as large deletions.

Patterns of chloroform-induced regenerative cell proliferation in BDF1 mice correlate with organ specificity and dose-response of tumor formation.

It has been reported that chloroform administered to BDF1 mice by inhalation for 2 years at concentrations of 5, 30 or 90 p.p.m. for 6 h/day, 5 days/week induced an increase in renal cell tumors in male but not female mice exposed to the doses of 30 and 90 p.p.m. A small increase in liver tumors was statistically significant in the female mice at 90 p.p.m. if the incidences of carcinomas and adenomas were combined. Because chloroform is not a DNA reactive mutagen, a 13-week time-course and dose-response study was conducted under conditions of the original bioassay to examine whether regenerative cell proliferation was an underlying mechanism of carcinogenesis. Mice were given bromodeoxyuridine via infusion during the last 3.5 days prior to necropsy to label cells in S-phase. Chloroform induced pathology and regenerative cell proliferation, measured as the labeling index (LI, percentage of cells in S-phase), were assessed microscopically and immunohistochemically. Male mice exposed to 30 and 90 p.p.m. exhibited a dose-dependent increase in regenerating tubules within the renal cortex and up to a 31-fold increase in LI. No renal lesions or increased LI were observed in females. Increased centrilobular to midzonal hepatocyte degeneration and vacuolation and a 7-fold increase over controls in the hepatocyte LI were observed in the female mice at 90 p.p.m. at 13 weeks. Males exhibited similar pathology, but the increase in LI was not sustained. The observed correlations between cytolethality and regenerative cell proliferation with tumor formation supports extensive evidence that chloroform induces cancer via a non-genotoxic-cytotoxic mode of action. A concentration of 5 p.p.m. is the no-observed-adverse-effect level for nephrotoxicity, cell proliferation and cancer. An appropriate safety factor applied to this value is a straightforward approach to cancer risk assessment that is consistent with the mode of action of chloroform.

Physiologically based pharmacodynamic modeling of an interaction threshold between trichloroethylene and 1,1-dichloroethylene in Fischer 344 rats.

Physiologically based pharmacokinetic modeling (PBPK) and gas uptake experiments have been used by researchers to demonstrate the competitive inhibition mechanism between trichloroethylene (TCE) and 1,1-dichloroethylene (DCE). Expanding on their work, we showed that this pharmacokinetic interaction was absent at levels of 100 ppm or less of either chemical in gas uptake systems. In this study, we further illustrate the presence of such an interaction threshold at the pharmacodynamic level by examining the interaction effect of either chemical on the other's ability to bind and deplete hepatic glutathione (GSH) in Fischer 344 rats. However, at this end point, the pharmacodynamic interaction is complicated by the ability of the liver to resynthesize GSH in response to its depletion. To quantitatively resolve the interaction effects on GSH content from the resynthesis effects, physiologically based pharmacodynamic (PBPD) modeling is applied. Initially, the PBPD model description of hepatic GSH kinetics was calibrated against previously published data and by gas uptake experiments conducted in our laboratory. Then, the model was used to determine the duration of the gas uptake exposure experiments by identifying the critical time point at which hepatic GSH is at a minimum in response to both chemicals. Subsequently, gas uptake experiments were designed following the PBPK/PD model predictions. In these model-directed experiments, DCE was the only chemical capable of significantly depleting hepatic GSH. The application of TCE to the rats at concentrations higher than 100 ppm obstructed the ability of DCE to deplete hepatic GSH. Since the metabolites of DCE bind to hepatic GSH, this obstruction signaled the presence of metabolic inhibition by TCE. However, TCE, at concentrations less than 100 ppm, was not effective in inhibiting DCE from significantly depleting hepatic GSH. The same observations were made when the ability of DCE to cause hepatic injury, as measured by aspartate aminotransferase serum activity, was assessed. Both conclusions validated the previous findings of the presence of the interaction threshold at the pharmacokinetic level.

1995 STP Young Investigator Award recipient. Increased rate of apoptosis correlates with hepatocellular proliferation in Fischer-344 rats following long-term exposure to a mixture of groundwater contaminants.

Apoptosis was evaluated in the livers of Fischer-344 rats following observations of increased hepatocellular proliferation from exposures, at low parts per million (ppm) levels, to a drinking water mixture of 7 groundwater contaminants during a 6-mo time-course study. The 7 chemicals used are among the most frequently detected contaminants associated with hazardous waste sites: arsenic, benzene, chloroform, chromium, lead, phenol, and trichloroethylene. Significant increases in 5-bromo-2'-deoxyuridine hepatocellular labeling were present in a unique pattern surrounding large hepatic veins (0.5-2.0 mm). This did not appear to be a regenerative response due to cytotoxicity, as assessed by the absence of increased plasma enzyme activity and the absence of hepatocellular lesions. Immunohistochemical staining for apoptosis, using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method showed patterns of labeling in treated animals that directly correlated to areas of increased hepatocyte proliferation. Apoptotic activity was maximum at the 1-mo exposure time point, whereas proliferating hepatocytes reached a maximum rate at the 10-day time point. This may have been triggered as a compensatory response to the increased cell proliferation or as a protective response to remove cells with altered DNA due to chemical mixture exposure. The principal findings of this paper are that (a) apoptosis directly correlated with changes in cell proliferation: (b) observed effects were produced by repeated exposures to a relatively low-level chemical mixture; and (c) the TUNEL method detected apoptotic cells at very early and late stages, potentially increasing the observable time period for apoptosis.

Incorporating Monte Carlo simulation into physiologically based pharmacokinetic models using advanced continuous simulation language (ACSL): a computational method.

Biologically based models with physiological parameters are becoming more popular as a tool to estimate target tissue doses from chemical exposures. However, the majority of current physiologically based pharmacokinetic (PBPK) models do not take into account the uncertainty and/or variability within the various model parameters. Consideration of uncertainty is important to evaluate the predictive ability and complexity of a model as well as identification of parameters which contribute disproportionately to variability in model output. In order to estimate the uncertainty in PBPK model output, a versatile and simple computational method is presented which can be readily incorporated into the majority of PBPK models without extensive additions to model computer code. In this paper, a separate computer program for Monte Carlo simulation is furnished that randomly samples values for model parameters and writes them into a run-time language (command file) format which can then be utilized to execute individual PBPK models. Modifications to the PBPK model allow the desired output to be written to a data file for statistical analysis. The method presented in this paper is applied to a simple PBPK model for benzene disposition.

Physiologically based pharmacokinetic/pharmacodynamic modeling of the toxicologic interaction between carbon tetrachloride and Kepone.

Carbon tetrachloride (CCl4) lethality in Sprague-Dawley rats is greatly amplified by pretreatment of Kepone (decachlorooctahydro-1,3,2-metheno-2H-cyclobuta[cd] pentalen-2-one). The increase in lethality was attributed to the obstruction of liver regenerative processes. These processes are essential for restoring the liver to its full functional capacity following injury by CCl4. Based on the available mechanistic information on Kepone/CCl4 interaction, a physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) model was constructed where the following effects of Kepone on CCl4 toxicity are incorporated: (1) inhibition of mitosis; (2) reduction of repair mechanism of hepatocellular injury; (3) suppression of phagocytosis. The PBPK/PD model provided computer simulation consistent with previously published time-course results of hepatotoxicity (i.e., pyknotic, injured and mitotic cells) of CCl4 with or without Kepone. As a further verification of this model, the computer simulations were also consistent with exhalation kinetic data for rats injected with different intraperitoneal (i.p.) doses of CCl4 in our laboratory. Subsequently, the PBPK/PD model, coupled with Monte Carlo simulation, was used to predict lethalities of rats treated with CCl4 alone and CCl4 in combination with Kepone. The experimental lethality studies performed in our laboratories were as follows: Sprague-Dawley rats were given either control diet or diet containing 10 ppm Kepone for 15 days. On day 16, rats in the Kepone treated group were given i.p. doses of 0, 10, 50, and 100 microliters/kg CCl4 (n = 9) while control rats were exposed to 0, 100, 1000, 3000, and 6000 microliters/kg CCl4 (n = 9). Lethality was observed at the 1000 (1/9), 3000 (4/9), and 6000 (8/9) microliters/kg doses for the control group and at the 50 (4/9) and 100 (8/9) microliters/kg for the treated group. Based on Monte Carlo simulation, which was used to run electronically 1000 lethality experiments for each dosing situation, the LD50 estimates for CCl4 toxicity with and without Kepone pretreatment were 47 and 2890 microliters/kg, respectively. Monte Carlo simulation coupled with the PBPK/PD model produced lethality rates which were not significantly different from the observed mortality, with the exception of CCl4 at very high doses (e.g., 6000 microliters/kg, p = 0.014). Deviation at very high doses of the predicted mortality from the observed may be attributed to extrahepatic systemic toxicities of CCl4, or solvent effects on tissues at high concentrations, which were not presently included in the model. Our modeling and experimental results verified the earlier findings of Mehendale (1990) for the 67-fold amplification of CCl4 lethality in the presence of Kepone. However, much of this amplification of CCl4 lethality with Kepone pretreatment was probably due to pharmacokinetic factors, because when target tissue dose (i.e., model estimated amount of CCl4 metabolites) was used to evaluate lethality, this amplification was reduced to 4-fold.

The use of physiologically-based pharmacokinetic/pharmacodynamic dosimetry models for chemical mixtures.

Human exposure to chemicals is rarely, if ever, limited to a single chemical. Therefore, it is essential that we consider multiple chemical effects and interactions in our risk assessment process. However, with the almost infinitely large number of chemical mixtures in the environment, systematic studies of the toxicology of these chemical mixtures with conventional methodologies and approaches are impossible because of the immense resources and unrealistically long durations required. Thus, the development of 'Predictive and Alternative Toxicology' is imperative. At Colorado State University (CSU), our research effort is entirely devoted to this challenge. In order to have a reasonable chance to deal with the complex issue of toxicology of chemical mixtures, we believe that the following concepts must be considered: (1) the utilization of computer, (2) the exploitation of mathematical/statistical methodologies; (3) developing very focused, mechanistically based, and short-term toxicology studies; (4) coupling computer/mathematical modeling with mechanistically-based toxicology. Our strategy therefore the utilization of physiologically-based pharmacokinetic/pharmacodynamic (PBPK/PD) modeling, coupled with very focused, model-directed toxicology experiments as well as other statistical/mathematical methodologies such as Monte Carlo simulation, isobolographic analysis, and response surface methodology. We believe that 'Predictive and Alternative Toxicology' in terms of tissue dosimetry at the pharmacokinetic and pharmacodynamic levels is achievable with simple and complex but chemically defined mixtures. In this presentation, we describe two ongoing research projects as an illustration of our 'Bottom-Up' and 'Top-Down' approaches for handling the chemical mixtures: (1) PBPK/PD modeling of toxicologic interactions between Kepone and carbon tetrachloride (CCl4) and the coupling of Monte Carlo simulation for the prediction of acute toxicity; (2) the conceptual development of PBPK/PD modeling for a more complex chemical mixture of seven groundwater contaminants from hazardous waste sites and the consideration of subfractionation of this chemical mixture.

The application of physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) modeling for exploring risk assessment approaches of chemical mixtures.

When dealing with health impacts of environmental or occupational exposure such as groundwater contamination from or remediation effort associated with hazardous waste sites, we are obviously not facing individual, single chemicals. Thus, we are immediately confronted with the following questions: (1) Is single chemical risk assessment approach applicable to the multiple chemicals in hazardous waste sites? (2) How do we handle risk assessment of chemical mixtures? Although there were pioneering and commendable efforts from the USEPA to formulate guidelines for risk assessment of chemical mixtures, these guidelines were principally based on additivity concept. As new scientific advances are made, improvement and refinement of risk assessment methodology will be anticipated. At Colorado State University (CSU), our research effort is devoted to the challenges and potential applications of physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) modeling in the risk assessment of chemical mixtures. With the ultimate goal of Predictive Toxicology, 3 specific research projects are described: (1) PBPK/PD modeling of toxicologic interactions between trichloroethylene (TCE) and 1,1-dichloroethylene (1,1-DCE) and the investigation and defining of an 'Interaction Threshold'; (2) PBPK/PD modeling of toxicologic interactions between Kepone and carbon tetrachloride (CCl4) and the coupling of Monte Carlo simulation for the prediction of acute toxicity; (3) PBPK modeling of the inhibition of pharmacokinetics and enzyme kinetics of TCE caused by low-level, repeated dosing of a chemical mixture of 7 groundwater contaminants. Since this paper is meant to be a commentary and the emphasis is on approaches for dealing with chemical mixtures, detailed presentation of data is avoided. These examples illustrate partially our ongoing research activities and the related ideas with respect to possible novel risk assessment applications to chemical mixtures.

A unique pattern of hepatocyte proliferation in F344 rats following long-term exposures to low levels of a chemical mixture of groundwater contaminants.

Most exposures of humans to environmental agents involve mixtures of chemicals, rather than individual chemicals. Some chemicals can cause hepatocellular proliferation and act as neoplastic promoters. Little is known concerning hepatocellular proliferation caused by chemical mixtures such as those found in groundwater at hazardous waste sites. Therefore, a 6 month study was performed to investigate hepatocellular proliferation and histopathological changes in F344 rats after long-term, low-level exposure to a mixture of groundwater contaminants. The seven chemicals used are among the most frequently detected contaminants associated with hazardous waste sites; arsenic, benzene, chloroform, chromium, lead, phenol and trichloroethylene. Male F344 rats were exposed to this mixture, or submixtures of the organic or inorganic chemicals, via drinking water for 6 months. The study design included a time-course experiment (i.e. 3 and 10 days and 1, 3 and 6 months) and a dose-response experiment. Hepatocellular proliferation studies were performed by subcutaneously implanting osmotic mini-pumps to continuously deliver 5-bromo-2'-deoxyuridine for 7 days, which labeled nuclei of proliferating cells. In all groups, there were no differences in weight gain, body weight, liver weight ratios or liver-associated plasma enzymes. Light microscopic evaluation revealed no lesions related to the treatments in any animals. However, significant increases in hepatocellular labeling were observed at the 3 and 10 day and 1 month exposure time points after treatment with the full mixture, as well as the organic or inorganic submixtures. Proliferating hepatocytes expressed a unique labeling pattern surrounding large hepatic veins (0.5-2.0 mm), but not central veins. This did not appear to be a regenerative response due to cytotoxic mechanisms, as assessed by the absence of increased plasma enzyme activity and the absence of hepatocellular lesions.