Hepatoprotective effects of Tagetes lucida root extract in carbon tetrachloride-induced hepatotoxicity in Wistar albino rats through amelioration of oxidative stress.

CONTEXT: The roots of Tagetes lucida Cav. (Asteraceae) have antioxidant and antimicrobial properties. OBJECTIVE: This study aimed to examine the hepatoprotective effects of T. lucida roots ethanol extract (TLRE) using carbon tetrachloride (CCl4)-induced hepatotoxicity in rats. MATERIALS AND METHODS: The active ingredients of TLRE were identified by high-performance liquid chromatography, infra-red spectrum, and mass spectrometric procedures. Ninety rats were distributed into four main groups: positive, therapeutic, protective, and negative group. The therapeutic group was implemented using CCl4 (a single dose of 2 mL/kg) before TLRE or silymarin administration. Meanwhile, the protective group was implemented by administering CCl4 (a single dose of 2 mL/kg) after force-feeding TLRE or silymarin. Each therapeutic and protective group was divided into three subgroups: force-fed with saline, TLRE (500 mg/kg), and silymarin (25 mg/kg). The positive group was split into two subgroups that were force-fed TLRE and silymarin. Positive, therapeutic, and protective groups were compared to the negative group (untreated rats). CCl4, TLRE, and silymarin were orally administrated using a gastric tube. RESULTS: In the therapeutic and protective groups, TLRE significantly reduced liver enzymes, i.e., aspartate aminotransferase (12.47 and 6.29%), alanine aminotransferase (30.48 and 11.39%), alkaline phosphatase (17.28 and 15.90%), and cytochrome P450-2E1 (39.04 and 48.24%), and tumour necrosis factor-α (53.72 and 53.72%) in comparison with CCl4-induced hepatotoxicity controls. CONCLUSIONS: TLRE has a potent hepatoprotective effect with a good safety margin. After a repeated study on another type of small experimental animal, their offspring, and an experiment with a large animal, this study may lead to clinical trials.

CM-101: Type I Collagen-targeted MR Imaging Probe for Detection of Liver Fibrosis.

Purpose To evaluate the biodistribution, metabolism, and pharmacokinetics of a new type I collagen-targeted magnetic resonance (MR) probe, CM-101, and to assess its ability to help quantify liver fibrosis in animal models. Materials and Methods Biodistribution, pharmacokinetics, and stability of CM-101 in rats were measured with mass spectrometry. Bile duct-ligated (BDL) and sham-treated rats were imaged 19 days after the procedure by using a 1.5-T clinical MR imaging unit. Mice were treated with carbon tetrachloride (CCl4) or with vehicle two times a week for 10 weeks and were imaged with a 7.0-T preclinical MR imaging unit at baseline and 1 week after the last CCl4 treatment. Animals were imaged before and after injection of 10 µmol/kg CM-101. Change in contrast-to-noise ratio (ΔCNR) between liver and muscle tissue after CM-101 injection was used to quantify liver fibrosis. Liver tissue was analyzed for Sirius Red staining and hydroxyproline content. The institutional subcommittee for research animal care approved all in vivo procedures. Results CM-101 demonstrated rapid blood clearance (half-life = 6.8 minutes ± 2.4) and predominately renal elimination in rats. Biodistribution showed low tissue gadolinium levels at 24 hours (<3.9% injected dose [ID]/g ± 0.6) and 10-fold lower levels at 14 days (<0.33% ID/g ± 12) after CM-101 injection with negligible accumulation in bone (0.07% ID/g ± 0.02 and 0.010% ID/g ± 0.004 at 1 and 14 days, respectively). ΔCNR was significantly (P < .001) higher in BDL rats (13.6 ± 3.2) than in sham-treated rats (5.7 ± 4.2) and in the CCl4-treated mice (18.3 ± 6.5) compared with baseline values (5.2 ± 1.0). Conclusion CM-101 demonstrated fast blood clearance and whole-body elimination, negligible accumulation of gadolinium in bone or tissue, and robust detection of fibrosis in rat BDL and mouse CCl4 models of liver fibrosis. © RSNA, 2017 Online supplemental material is available for this article.

Putative mechanisms of environmental chemical-induced steatosis.

Liver disease is a major health issue characterized by several pathological changes, with steatosis (fatty liver) representing a common initial step in its pathogenesis. Steatosis is of critical importance because prevention of fatty liver can obviate downstream pathologies of liver disease (eg, fibrosis). Recent studies have shown a strong correlation between chemical exposure and steatosis. The work described here identifies chemicals on the US Environmental Protection Agency's Integrated Risk Information System (IRIS) that induce steatosis and investigates putative mechanisms by which these chemicals may contribute to this pathological condition. Mitochondrial impairment, insulin resistance, impaired hepatic lipid secretion, and enhanced cytokine production were identified as potential mechanisms that could contribute to steatosis. Taken together, this work is significant because it identifies multiple mechanisms by which environmental chemicals may cause fatty liver and expands our knowledge of the possible role of environmental chemical exposure in the induction and progression of liver disease.

Period1 mediates rhythmic metabolism of toxins by interacting with CYP2E1.

The biological clock is an endogenous biological timing system, which controls metabolic functions in almost all organs. Nutrient metabolism, substrate processing, and detoxification are circadian controlled in livers. However, how the clock genes respond to toxins and influence toxicity keeps unclear. We identified the clock gene Per1 was specifically elevated in mice exposed to toxins such as carbon tetrachloride (CCl4). Mice lacking Per1 slowed down the metabolic rate of toxins including CCl4, capsaicin, and acetaminophen, exhibiting relatively more residues in the plasma. Liver injury and fibrosis induced by acute and chronic CCl4 exposure were markedly alleviated in Per1-deficient mice. These processes involved the binding of PER1 protein and hepatocyte nuclear factor-1alpha (HNF-1α), which enhances the recruitment of HNF-1α to cytochrome P450 2E1 (Cyp2e1) promoter and increases Cyp2e1 expression, thereby promoting metabolism for toxins in the livers. These results indicate that PER1 mediates the metabolism of toxins and appropriate suppression of Per1 response is a potential therapeutic target for toxin-induced hepatotoxicity.

A single pretreatment with clofibric acid attenuates carbon tetrachloride-induced necrosis, but not steatosis, in rat liver.

The present study investigated whether a single pretreatment with clofibric acid suppresses liver injury in rats after CCl4 intoxication. Rats received a single pretreatment with clofibric acid (100 mg/kg, i.p.) 1 h prior to a CCl4 (1 mL/kg, p.o.) challenge, and were euthanized 24 h after the CCl4 administration. A single pretreatment with clofibric acid effectively suppressed increases in the serum aminotransferase activities and the severity of necrosis following the CCl4 challenge, whereas the pretreatment did not protect against CCl4-induced fatty liver. The clofibric acid pretreatment did not affect blood concentrations of CCl4 in the early stage after CCl4 dosing, or the level of the CCl4 reaching the liver 1 h after the CCl4 challenge. Moreover, the clofibric acid pretreatment did not affect the intensity of the covalent binding of the [14C]CCl4 metabolite to microsomal proteins and lipids. The clofibric acid pretreatment did not alter microsomal cytochrome P450 2E1 activity. Based on these results, we conclude that protection against CCl4-induced hepatocellular necrosis by a clofibric acid pretreatment does not require its repeated administration, and that a single and brief pre-exposure to clofibric acid prior to CCl4 dosing markedly suppresses necrosis without affecting the development and progression of steatosis.

[Changes in the toxicity and immunotoxity of tetrachloromethane and carbophos under the action of 2,4,6-triphenyl-4H-selenopyran and their connection with cytochrome P-450 dependent monooxygenase system].

It is established in experiments on noninbred rats that 2,4,6-triphenyl-4H-selenopyrane (peroral administration in a dose of 0.8 mg/kg during 3 days) induces cytochrome P450, thus increasing the toxicity and immunotoxicity of carbon tetrachloride (metabolized via "lethal synthesis"), and reduces the analogous effects of carbophos, the biotransformation of which proceeds via the formation of low-toxicity and nontoxic metabolites.

Extrahepatic metabolism by CYP2E1 in PBPK modeling of lipophilic volatile organic chemicals: impacts on metabolic parameter estimation and prediction of dose metrics.

Physiologically based pharmacokinetic (PBPK) models are increasingly available for environmental chemicals and applied in risk assessments. Volatile organic compounds (VOCs) are important pollutants in air, soil, and water. CYP2E1 metabolically activates many VOCs in animals and humans. Despite its presence in extrahepatic tissues, the metabolism by CYP2E1 is often described as restricted to the liver in PBPK models, unless target tissue dose metrics in extrahepatic tissues are needed for the model application, including risk assessment. The impact of accounting for extrahepatic metabolism by CYP2E1 on the estimation of metabolic parameters and the prediction of dose metrics was evaluated for three lipophilic VOCs: vinyl chloride, trichloroethylene, and carbon tetrachloride. Metabolic parameters estimated from fitting gas uptake data with and without extrahepatic metabolism were similar. The impact of extrahepatic metabolism on PBPK predictions was evaluated using inhalation exposure scenarios relevant for animal toxicity studies and human risk assessment. Although small, the relative role of extrahepatic metabolism and the differences in the predicted dose metrics were greater at low exposure concentrations. The impact was species dependent and influenced by Km for CYP2E1. The current study indicates that inhalation modeling for several representative VOCs that are CYP2E1 substrates is not affected by the inclusion of extrahepatic metabolism, implying that liver-only metabolism may be a reasonable simplification for PBPK modeling of lipophilic VOCs. The PBPK predictions using this assumption can be applied confidently for risk assessment, but this conclusion should not necessarily be applied to VOCs that are metabolized by other enzymes.

Carbon tetrachloride hepatotoxicity as a function of age in female Fischer 344 rats.

Severity of liver damage 24 h after intraperitoneal administration of carbon tetrachloride (0.2 ml/kg) was evaluated in female Fischer 344 rats aged 5, 14 and 28 months, i.e. in young adulthood, middle age and old age. Carbon tetrachloride-induced hepatotoxicity, as judged by the leakage of hepatic enzymes into the bloodstream and the disappearance of hepatic microsomal cytochrome P450, was much less severe in old rats than in young-adult rats. For example, serum sorbitol dehydrogenase (SDH) activity following carbon tetrachloride administration was 680 mumol/min/l in old rats compared with 1710 mumol/min/l in young-adult rats, and the loss of hepatic cytochrome P450 was 25% of the total amount in old rats compared with 50% of the total in young-adult rats. Spin trapping and electron spin resonance (ESR) spectroscopy were utilized to measure the conversion of carbon tetrachloride to trichloromethyl radicals in vivo. This primary bioactivation step occurred at similar rates in female rats aged 5, 14 and 28 months. In addition, the total nonheme iron contents in livers of rats in the three age groups were similar. Thus, the age associated attenuation of carbon tetrachloride-induced hepatotoxicity was not explained on the basis of decreased bioactivation to reactive species or decreased availability of iron for promotion of lipid peroxidation. The results suggest that other factors are important determinants of age-associated changes in sensitivity to toxic chemicals.

Biotransformation of carbon tetrachloride and lipid peroxidation promotion by liver nuclear preparations from different animal species.

Liver nuclear preparations from male Syrian Golden hamster (SG); C3H mice and Sprague-Dawley (SD) rats were able to biotransform CCl4 to CHCl3. That ability was not NADPH dependent and proceeded to an equal extent under N2 or air. Studies in more detail with C3H mice preparations revealed that only one of the processes was of an enzymatic nature and that it was inhibited by 1 mM EDTA. There was a correlation between liver nuclear ability to biotransform CCl4 to CHCl3 in the species tested and their liver carcinogenic response to CCl4. That correlation was not observed when biotransformation was studied using liver slices instead of liver nuclei. Liver nuclear preparations from the 3 species were able to promote a lipid peroxidation (LP) process in the presence of CCl4. The process was fully NADPH dependent in the case of SG and SD preparations but not in C3H mice. Study of the process in detail in the case of C3H mice shows that in that case LP was heat and EDTA sensitive, particularly in the absence of NADPH. There was no correlation between the intensity of CCl4 promoted LP either in liver nuclear or liver slices preparations in the 3 species tested and their carcino genic response to CCl4. Results might suggest that LP does not determine or rate limit the process of cancer development by CCl4 but do not exclude its participation in a given stage of the overall process.

Effects of electron acceptors and donors on transformation of tetrachloromethane by Shewanella putrefaciens MR-1.

Transformation of chlorinated aliphatic compounds was examined in Shewanella putrefaciens strain MR-1, an obligately respiring facultative anaerobe. Under anaerobic conditions, MR-1 has been shown to transform tetrachloromethane to trichloromethane (24%), CO2 (7%), cell-bound material (50%) and unidentified nonvolatile products (4%). The highest rate and extent of transformation were observed with MR-1 cells grown under iron(III)-respiring conditions. Lactate, formate and hydrogen were the most effective electron donors. Tetrachloromethane was not degraded in the presence of oxygen. Transformation of other chlorinated methanes and ethenes was not observed.

Optimization of the dose and route of injection, and characterisation of the time course of carbon tetrachloride-induced hepatotoxicity in the rat.

INTRODUCTION: The purpose of this study was to optimize carbon tetrachloride-induced hepatotoxicity in the rat with respect to dose, route of injection, and time course. METHODS: Male Wistar albino rats, 4 to 6 weeks old and weighing 130-180 g were used. Hepatotoxicity was evaluated by measuring the activity of serum enzymes (alkaline phosphatase [ALP], alanine aminotransferase [ALT], and aspartate aminotransferase [AST]) as well as serum total bilirubin level. RESULTS: Intraperitoneal injection of carbon tetrachloride (CCl(4)) increased the activity of ALP (from 64.9 to 137.3 U/l), ALT (from 106.6 to 693.1 U/l), and AST (from 113.8 to 693.9 U/l). Plasma bilirubin level increased (from 0.119 to 0.42 mg/dl). In contrast, subcutaneous injection of CCl(4) had no effect on these variables. The optimum intraperitoneal dose of CCl(4) was found to be 2 ml/kg body weight (dissolved in an equal volume of olive oil), and this increased the level of bilirubin and the activity of the three enzymes significantly, without causing death of the animals. Hepatotoxicity was observed within 2 h of intraperitoneal injection of CCl(4) and reached a peak after 24 h. Bilirubin level and serum enzyme activities declined gradually to normal levels by 3 days after CCl(4) injection. CONCLUSION: It is possible to reliably evoke reversible hepatotoxicity in rats by intraperitoneal injection of 2 ml/kg CCl(4).

A pharmacokinetic model describing pulsatile uptake of orally-administered carbon tetrachloride.

Many rodent bioassays have been conducted using oral gavage for delivery of test chemicals. Highly lipophilic compounds are generally administered to rodents dissolved in corn oil, a dosing vehicle shown to influence xenobiotic toxicity, carcinogenicity and pharmacokinetics by altering chemical absorption processes. In this paper, we present a multi-compartmental description of the gastrointestinal (GI) tract linked to a physiologically based pharmacokinetic (PB-PK) model to describe the complex oral uptake of carbon tetrachloride (CCl4) administered in corn oil and 0.25% Emulphor. The GI submodel was described using a series of subcompartments, each subcompartment described with an absorption constant (Ka, 1/h), a bioavailability term (A, unitless), and a compartment emptying time (T, h). The model was parameterized by fitting multi-peak blood and exhaled breath chamber concentration-time profiles following oral gavage of CCl4 in corn oil and aqueous vehicles to male Fischer 344 rats. Successful fitting of experimental data was accomplished by varying values of Ka, A, and T until adequate fits were obtained. Values of Ka and A required to fit data from aqueous gavage were greater than corn oil. Utilization of the multi-compartmental GI tract submodel provided increased precision in fitting complex oral uptake profiles compared to previously used one- and two-compartment oral uptake models. This model provides estimates of absorption rate constants and bioavailabilities as well as providing a framework for generation of more complete, physiologically-realistic descriptions of oral absorption.

Physiologically based pharmacokinetic/pharmacodynamic modeling of chemical mixtures and possible applications in risk assessment.

Human exposure to chemicals, be it environmental or occupational, 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 method is imperative. 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 exploitation of recent advances in computational technology; (2) the utilization of mathematical/statistical modeling; (3) coupling computer modeling with very focused, mechanistically based, and short-term toxicology studies. Our approach is, therefore, the utilization of physiologically based pharmacokinetic/pharmacodynamic (PB-PK/PD) modeling, coupled with very focused, model-directed toxicology experiments as well as other statistical/mathematical modeling such as isobolographic analysis and response surface methodology. Tissue dosimetry at the pharmacokinetic and pharmacodynamic levels is achievable with simple and complex, but chemically defined, mixtures. Our long-term goal is to formulate innovative risk assessment methodologies for chemical mixtures. In this presentation, one of our specific research projects is described: PB-PK/PD modeling of toxicologic interactions between Kepone and carbon tetrachloride (CCl4) and the coupling of Monte Carlo simulation for the prediction of acute toxicity.

Induction of serum-borne immunomodulatory factors in B6C3F1 mice by carbon tetrachloride. I. Carbon tetrachloride-induced suppression of helper T-lymphocyte function is mediated by a serum borne factor.

Following carbon tetrachloride-induced liver injury, hepatotrophic factors are synthesized and released into the serum to facilitate the regeneration of damaged hepatic tissue. We investigated the possibility that immunosuppression could be mediated through induction of a serum factor(s) because in vivo exposure of B6C3F1 mice to carbon tetrachloride selectively inhibits T-cell-dependent immune responses. Addition of mouse serum (5% by volume) obtained from mice treated with carbon tetrachloride (250 or 500 mg/kg/day for 7 days) to naive spleen cell cultures markedly suppressed the sheep red blood cell antibody-forming cell response compared to controls (P < 0.01). Immunosuppression was observed in mice sensitized with sheep red blood cells 48 h, but not 24 or 72 h, following one dose of carbon tetrachloride (1000 mg/kg). Only serum isolated from mice 48 h following exposure to a single dose of carbon tetrachloride (1000 mg/kg) suppressed the antibody-forming cell response when added in vitro to spleen cell cultures. Biodistribution studies using [14C]-labelled carbon tetrachloride demonstrated that accumulation of the [14C]-label was primarily associated with excretory organs (liver, kidneys and lungs) but not with the serum, red blood cells, or spleen. Surprisingly, 24 and 48 h following exposure to [14C]-labelled carbon tetrachloride, an increase in radioactivity was detected in the thymus. The distinct profile of immunosuppressive activity associated with serum isolated from carbon tetrachloride-treated mice and the biodistribution studies clearly demonstrating a negligible amount of carbon tetrachloride or metabolites in the serum strongly implicate the role of a carbon tetrachloride-induced serum borne immunosuppressive factor.

The role of metabolism in carbon tetrachloride-mediated immunosuppression. In vitro studies.

In vitro studies were performed to determine the role of metabolic bioactivation in mediating immunosuppression by CCl4. Direct addition of CCl4 to naive spleen cell cultures sensitized with either sheep erythrocytes, DNP-Ficoll or lipopolysaccharide (LPS) resulted in a marked inhibition of the antibody forming cell (AFC) response to all three of the selected antigens at 3.0 mM concentration in culture. However, this inhibition was primarily due to the direct cytotoxic effects of CCl4 on spleen cells following 3-5 days of culture in the presence of the chemical as evidenced by a decrease in cell number and viability and by the absence of selective effects on T-cell dependent humoral responses which is contradictory to the effects observed in vivo. Co-incubation of splenocytes for 1 h with primary hepatocytes, but not with subcellular metabolic activation systems, such as S9 or microsomes, enhanced the immunotoxic effects of CCl4 in vitro. Interestingly, a 3-h co-incubation of spleen cells with metabolically active hepatocytes in primary culture resulted in an even greater potentiation of the immunotoxic effects of CCl4 as determined by the T-cell dependent IgM AFC response. Conversely, under identical conditions, CCl4 did not suppress humoral responses to the polyclonal B-cell activator LPS which is in agreement with the effects produced by in vivo exposure to CCl4. It is important to emphasize that for the metabolic activation studies (i.e. co-incubation with either S9, microsomes or hepatocytes), spleen cells were washed free of CCl4 immediately following the co-incubation period. Control splenocyte cultures (i.e. no metabolic activation system) incubated in the presence of CCl4 alone at 3.0 mM over a 3-h time-period, had no effect on spleen cell function, number or viability. In agreement with our previous findings which indicate that pretreatment of mice with inducers and inhibitors of the mixed function oxygenase system prior to CCl4 administration modulated the immunotoxic effects of CCl4 in vivo, these results lead us to conclude that immunotoxicity by CCl4 requires metabolic activation.

A pharmacokinetic model of anaerobic in vitro carbon tetrachloride metabolism.

Carbon tetrachloride (CCl4) is a potent hepatotoxic agent whose toxicity is mediated through cytochome P450-dependent metabolism. Results from anaerobic in vitro experiments with hepatic microsomes isolated from male F-344 rats indicate that chlorofom (CHCl3) formation from CCl4 is nonlinear with dose. Dose is traditionally expressed as the amount of CCl4 added to the vial. In this study, a pharmacokinetic model has been developed to calculate the concentration of CCl4 in the microsomal suspension. Hepatic microsomes prepared from fed and fasted animals were incubated with CCl4 under anaerobic conditions and formation of CHCl3 over a 5-min incubation period was monitored by headspace gas chromatography. Dose-response curves, based on total amount of CCl4 added to the microsomes, revealed a nonlinear, biphasic appearance of CHCl3, with fasting slightly increasing CHCl3 production in microsomes prepared from fasted rats. Microsomes were also pretreated with the CYP2E1 inhibitor, diallyl sulfone (DAS), before addition of CCl4. In uninhibited microsomes, there appeared to be a high-affinity saturable phase of metabolism occurring at lower concentrations followed by a linear phase at higher CCl4 concentrations. Following DAS pretreatment, the saturable portion of the dose-response curve was inhibited more than the linear phase with the biphasic CHCl3 production becoming more linear. DAS inhibition eliminated the effect of fasting on CHCl3 formation. The best fit kinetic constants for the saturable phase resulted in an estimate of V(max) of 0.017 mg/h/mg protein (V(maxc) = 7.61 mg/h/kg) and Km of 2.3 mg/l (15 microM). The linear phase rate constant (kf) was determined to be 0.046 h-1) (kfc = 0.03 h-1). In conclusion, a pharmacokinetic model has been developed for anaerobic in vitro metabolism of CCl4 to CHCl3 that estimates metabolic rates based on CHCl3 formation and actual CCl4 concentration in the microsomal suspension.

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.

Application of physiologically based pharmacokinetic modelling to combination toxicology.

Non-additive toxicity has been demonstrated in laboratory animals for a large number of temporally separated or concurrent multiple chemical exposures. These exposures are typically at concentrations higher than those found in the environment, leading to the question of the applicability of the results to the human situation. Physiologically based pharmacokinetic (PBPK) modelling has been applied successfully to single chemicals; its utility for extrapolation across species and dose has been demonstrated. Use of PBPK modelling in the study of chemical mixtures is increasing although still limited. The use of PBPK modelling by various investigators in the field of combination toxicology is reviewed. PBPK modelling has been used to examine: the role of increased metabolism in non-additive toxicity resulting from temporally separated exposures; the influence of the time interval separating two chemical exposures; and the role of inhibition of metabolism in concurrent exposure to two chemicals. In summary, development of a PBPK or PBPK/pharmacodynamic model for a combined exposure provides a basis for extrapolation across species, route and dose, and a useful tool for risk assessment.

Comparative metabolism of carbon tetrachloride in rats, mice, and hamsters using gas uptake and PBPK modeling.

No study has comprehensively compared the rate of metabolism of carbon tetrachloride (CCl4) across species. Therefore, the in vivo metabolism of CCl4 was evaluated using groups of male animals (F344 rats, B6C3F1 mice, and Syrian hamsters) exposed to 40-1800 ppm CCl4 in a closed, recirculating gas-uptake system. For each species, an optimal fit of the family of uptake curves was obtained by adjusting Michaelis-Menten metabolic constants Km (affinity) and Vmax (capacity) using a physiologically based pharmacokinetic (PBPK) model. The results show that the mouse has a slightly higher capacity and lower affinity for metabolizing CCl4 compared to the rat, while the hamster has a higher capacity and lower affinity than either rat or mouse. A comparison of the Vmax to Km ratio, normalized for milligrams of liver protein (L/h/mg) across species, indicates that hamsters metabolize more CCl4 than either rats or mice, and should be more susceptible to CCl4-induced hepatotoxicity. These species comparisons were evaluated against toxicokinetic studies conducted in animals exposed by nose-only inhalation to 20 ppm 14C-labeled CCl4 for 4 h. The toxicokinetic study results are consistent with the in vivo rates of metabolism, with rats eliminating less radioactivity associated with metabolism (14CO2 and urine/feces) and more radioactivity associated with the parent compound (radioactivity trapped on charcoal) compared to either hamsters or mice. The in vivo metabolic constants determined here, together with in vitro constants determined using rat, mouse, hamster, and human liver microsomes, were used to estimate human in vivo metabolic rates of 1.49 mg/h/kg body weight and 0.25 mg/L for Vmax and Km, respectively. Normalizing the rate of metabolism (Vmax/Km) by milligrams liver protein, the rate of metabolism of CCl4 differs across species, with hamster > mouse > rat > human.

Reactive oxygen species in the progression of CCl4-induced liver injury.

Pretreatment of rats with large doses of vitamin A (retinol) dramatically increased the hepatotoxicity of carbon tetrachloride (CCl4). Experiments were performed to elucidate the mechanism of this potentiation. Hypervitaminosis A was produced by oral administration of retinol, 250,000 IU/kg for seven days. CCl4 was then administered at a dose of 0.15 ml/kg, ip. This large dose of vitamin A did not enhance the biotransformation of CCl4, but did produce a 4-fold increase in CCl4-induced lipid peroxidation, as assessed by ethane exhalation. Because vitamin A has been shown to activate macrophages, it was hypothesized that this increased lipid peroxidation and liver injury resulted from the release of reactive oxygen species from activated Kupffer cells. By using a chemiluminescence assay, an enhanced release of free radicals was detected in Kupffer cells isolated from vitamin A pretreated rats. In addition, Kupffer cells from vitamin A pretreated rats displayed enhanced phagocytic activity in vitro, towards sheep red blood cells. In vivo, vitamin A pretreated rats cleared carbon particles from the blood 2-3 times faster than non-pretreated rats. In vivo administration of superoxide dismutase (SOD) 2 hr after CCl4 exposure did not influence CCl4 toxicity in control rats but did block the enhanced ethane exhalation and also the potentiation of CCl4 liver injury in vitamin A treated rats. Administration of methyl palmitate, an inhibitor of Kupffer cell function, did not inhibit CCl4 toxicity in control rats, but did effectively block enhanced ethane exhalation and potentiation of CCl4 injury in vitamin A treated rats. We conclude that potentiation of CCl4 hepatotoxicity by hypervitaminosis A is mediated in part by reactive oxygen species released from activated Kupffer cells.