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.

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.

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.

Development of a quantitative model incorporating key events in a hepatotoxic mode of action to predict tumor incidence.

Biologically based dose-response (BBDR) modeling of environmental pollutants can be utilized to inform the mode of action (MOA) by which compounds elicit adverse health effects. Chemicals that produce tumors are typically labeled as either genotoxic or nongenotoxic. Though both the genotoxic and the nongenotoxic MOA may be operative as a function of dose, it is important to note that the label informs but does not define a MOA. One commonly proposed MOA for nongenotoxic carcinogens is characterized by the key events cytotoxicity and regenerative proliferation. The increased division rate associated with such proliferation can cause an increase in the probability of mutations, which may result in tumor formation. We included these steps in a generalized computational pharmacodynamic (PD) model incorporating cytotoxicity as a MOA for three carcinogens (chloroform, CHCl(3); carbon tetrachloride, CCL(4); and N,N-dimethylformamide, DMF). For each compound, the BBDR model is composed of a chemical-specific physiologically based pharmacokinetic model linked to a PD model of cytotoxicity and cellular proliferation. The rate of proliferation is then linked to a clonal growth model to predict tumor incidences. Comparisons of the BBDR simulations and parameterizations across chemicals suggested that significant variation among the models for the three chemicals arises in a few parameters expected to be chemical specific (such as metabolism and cellular injury rate constants). Optimization of model parameters to tumor data for CCL(4) and DMF resulted in similar estimates for all parameters related to cytotoxicity and tumor incidences. However, optimization of the CHCl(3) data resulted in a higher estimate for one parameter (BD) related to death of initiated cells. This implies that additional steps beyond cytotoxicity leading to induced cellular proliferation can be quantitatively different among chemicals that share cytotoxicity as a hypothesized carcinogenic MOA.

[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.

Hepatic injury induces contrasting response in liver and kidney to chemicals that are metabolically activated: role of male sex hormone.

Injury to liver, resulting in loss of its normal physiological/biochemical functions, may adversely affect a secondary organ. We examined the response of the liver and kidney to chemical substances that require metabolic activation for their toxicities in mice with a preceding liver injury. Carbon tetrachloride treatment 24 h prior to a challenging dose of carbon tetrachloride or acetaminophen decreased the resulting hepatotoxicity both in male and female mice as determined by histopathological examination and increases in serum enzyme activities. In contrast, the renal toxicity of the challenging toxicants was elevated markedly in male, but not in female mice. Partial hepatectomy also induced similar changes in the hepatotoxicity and nephrotoxicity of a challenging toxicant, suggesting that the contrasting response of male liver and kidney was associated with the reduction of the hepatic metabolizing capacity. Carbon tetrachloride pretreatment or partial hepatectomy decreased the hepatic xenobiotic-metabolizing enzyme activities in both sexes but elevated the renal p-nitrophenol hydroxylase, p-nitroanisole O-demethylase and aminopyrine N-demethylase activities significantly only in male mice. Increases in Cyp2e1 and Cyp2b expression were also evident in male kidney. Castration of males or testosterone administration to females diminished the sex-related differences in the renal response to an acute liver injury. The results indicate that reduction of the hepatic metabolizing capacity induced by liver injury may render secondary target organs susceptible to chemical substances activated in these organs. This effect may be sex-specific. It is also suggested that an integrated approach should be taken for proper assessment of chemical hazards.

Estimation of interindividual pharmacokinetic variability factor for inhaled volatile organic chemicals using a probability-bounds approach.

The derivation of reference concentrations (RfCs) for systemically acting volatile organic chemicals (VOCs) uses a default factor of 10 to account for the interindividual variability in pharmacokinetics (PK) and pharmacodynamics (PD). The magnitude of the PK component of the interindividual variability factor (IVF; also referred to as human kinetic adjustment factor (HKAF)) has previously been estimated using Monte Carlo approaches and physiologically based pharmacokinetic (PBPK) models. Since the RfC derivation considers continuous lifetime human exposure to VOCs in the environment, algorithms to compute steady-state internal dose (SS-ID), such as steady-state arterial blood concentration (Ca) and the steady-state rate of amount metabolized (RAM), can be used to derive IVF-PKs. In this context, probability-bounds (P-bounds) approach is potentially useful for computing an interval of probability distribution of SS-ID from knowledge of population distribution of input parameters. The objective of this study was therefore to compute IVF-PK using the P-bounds approach along with an algorithm for SS-ID in an adult population exposed to VOCs. The existing steady-state algorithms, derived from PBPK models, were rewritten such that SS-ID could be related, without any interdependence, to the following input parameters: alveolar ventilation (Qp), hepatic blood flow (Ql), intrinsic clearance (CL(int)) and blood:air partition coefficient (Pb). The IVF-PK was calculated from the P-bounds of SS-ID corresponding to the 50th and 95th percentiles. Following either specification of probability distribution-free bounds (characterized by minimal, maximal, and mean values) or distribution-defined values (mean, standard deviation and shape of probability distribution where: Qp=lognormal, Ql=lognormal, CL(int)=lognormal and Pb=normal) in RAMAS Risk Calc software version 3.0 (Applied Biomathematics, Setauket, NY), the P-bound estimates of SS-ID for benzene, carbon tetrachloride, chloroform and methyl chloroform were obtained for low level exposures (1ppm). Using probability distribution-defined inputs, the IVF-PK for benzene, carbon tetrachloride, chloroform and methyl chloroform were, respectively, 1.18, 1.28, 1.24, and 1.18 (based on P-bounds for Ca), and 1.31, 1.58, 1.30, and 1.24 (based on P-bounds for RAM). A validation of the P-bounds computation was performed by comparing the results with those obtained using Monte Carlo simulation of the steady-state algorithms. In data-poor situations, when the statistical distributions for all input parameters were not known or available, the P-bounds approach allowed the estimation of IVF-PK. The use of P-bounds method along with steady-state algorithms, as done in this study for the first time, is a practical and scientifically sound way of computing IVF-PKs for systemically acting VOCs.

Calpain released from dying hepatocytes mediates progression of acute liver injury induced by model hepatotoxicants.

Liver injury is known to progress even after the hepatotoxicant is long gone and the mechanisms of progressive injury are not understood. We tested the hypothesis that hydrolytic enzymes such as calpain, released from dying hepatocytes, destroy the surrounding cells causing progression of injury. Calpain inhibitor, N-CBZ-VAL-PHE-methyl ester (CBZ), administered 1 h after a toxic but nonlethal dose of CCl(4) (2 ml/kg, ip) to male Sprague Dawley rats substantially mitigated the progression of liver injury (6 to 48 h) and also led to 75% protection against CCl(4)-induced lethality following a lethal dose (LD75) of CCl(4) (3 ml/kg). Calpain leakage in plasma and in the perinecrotic areas increased until 48 h and decreased from 72 h onward paralleling progression and regression of liver injury, respectively, after CCl(4) treatment. Mitigation of progressive injury was accompanied by substantially low calpain in perinecrotic areas and in plasma after CBZ treatment. Normal hepatocytes incubated with the plasma collected from CCl(4)-treated rats (collected at 12 h when most of the CCl(4) is eliminated) resulted in extensive cell death prevented by CBZ. Cell-impermeable calpain inhibitor E64 also protected against progression of CCl(4)-induced liver injury, thereby confirming the role of released calpain in progression of liver injury. Following CCl(4) treatment, calpain-specific breakdown of alpha-fodrin increased, while it was negligible in rats receiving CBZ after CCl(4). Hepatocyte cell death in incubations containing calpain was completely prevented by CBZ. Eighty percent of Swiss Webster mice receiving a lethal dose (LD80) of acetaminophen (600 mg/kg, ip) survived if CBZ was administered 1 h after acetaminophen, suggesting that calpain-mediated progression of liver injury is neither species nor chemical specific. These findings suggest the role of calpain in progression of liver injury.

A novel method for preparation of animal models of liver damage: liver targeting of carbon tetrachloride in rats.

Animal models prepared by treatment with toxic compounds such as a carbon tetrachloride have been used to examine drug disposition in hepatic diseases. However, it is possible that these compounds accumulate and cause damage to other organs as they are administered systemically. In this study, we used the liver surface application technique to deliver a toxic compound to the liver to prepare an appropriate animal model in which only the liver is significantly damaged. To restrict the absorption area in the liver, a cylindrical diffusion cell was attached to the liver surface of male Wistar rats. Twenty-four hours after direct addition of carbon tetrachloride to the diffusion cell, plasma levels of glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT), and hepatic malondialdehyde (MDA) concentration were increased, while there were no changes in plasma creatinine or renal MDA level. On the other hand, not only GOT, GPT and hepatic MDA, but also creatinine and renal MDA levels were markedly increased by p.o. and i.p. administration of carbon tetrachloride, suggesting renal damage. These results indicated that the animal models of liver damage prepared by utilizing drug delivery techniques to accumulate toxic compounds in the liver would enable us to investigate the precise effects of hepatic disorder on drug disposition.

Use of sensitivity analysis to assess reliability of metabolic and physiological models.

Because ethical considerations often preclude directly determining the human health effects of treatments or interventions by experimentation, such effects are estimated by extrapolating reactions predicted from animal experiments. Under such conditions, it must be demonstrated that the reliability of the extrapolated predictions is not excessively affected by inherent data limitations and other components of model specification. This is especially true of high-level models composed of ad hoc algebraic equations whose parameters do not correspond to specific physical properties or processes. Models based on independent experimental data restricting the numerical space of parameters that do represent actual physical properties can be represented at a more detailed level. Sensitivities of the computed trajectories to parameter variations permit more detailed attribution of uncertainties in the predictions to these low-level properties. S-systems, in which parameters are estimated empirically, and physiological models, whose parameters can be estimated accurately from independent data, are used to illustrate the applicability of trajectory sensitivity analysis to lower-level models.

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).

Mechanism of carbon tetrachloride-induced hepatotoxicity. Hepatocellular damage by reactive carbon tetrachloride metabolites.

CCl4-induced liver damage was modeled in monolayer cultures of rat primary hepatocytes with a focus on involvement of covalent binding of CCl4 metabolites to cell components and/or peroxidative damage as the cause of injury. (1) Covalent binding of 14C-labeled metabolites was detected in hepatocytes immediately after exposure to CCl4. (2) Low oxygen partial pressure increased the reductive metabolism of CCl4 and thus covalent binding. (3) [14C]-CCl4 was bound to lipids and to proteins throughout subcellular fractions. Binding occurred preferentially to triacylglycerols and phospholipids, with phosphatidylcholine containing the highest amount of label. (4) The lipid peroxidation potency of CCl4 revealed subtle differences compared to other peroxidative substances, viz., ADP-Fe3+ and cumol hydroperoxide, respectively. (5) CCl4, but not the other peroxidative substances, decreased the rate of triacylglycerol secretion as very low density lipoproteins. (6) The anti-oxidant vitamin E (alpha-tocopherol) blocked lipid peroxidation, but not covalent binding, and secretion of lipoproteins remained inhibited. (7) The radical scavenger piperonyl butoxide prevented CCl4-induced lipid peroxidation as well as covalent binding of CCl4 metabolites to cell components, and also restored lipoprotein metabolism. The results confirm that covalent binding of the CCl3* radical to cell components initiates the inhibition of lipoprotein secretion and thus steatosis, whereas reaction with oxygen, to form CCl3-OO*, initiates lipid peroxidation. The two processes are independent of each other, and the extent to which either process occurs depends on partial oxygen pressure. The former process may result in adduct formation and, ultimately, cancer initiation, whereas the latter results in loss of calcium homeostasis and, ultimately, apoptosis and cell death.

Attenuation of CCl(4)-induced hepatic fibrosis by GdCl(3) treatment or dietary glycine.

The role of Kupffer cells in CCl(4)-induced fibrosis was investigated in vivo. Male Wistar rats were treated with phenobarbital and CCl(4) for 9 wk, and a group of rats were injected with the Kupffer cell toxicant gadolinium chloride (GdCl(3)) or were fed glycine, which inactivates Kupffer cells. After CCl(4) alone, the fibrosis score was 3.0 +/- 0.1 and collagen protein and mRNA expression were elevated, but GdCl(3) or glycine blunted these parameters. Glycine did not alter cytochrome P-450 2E1, making it unlikely that glycine affects CCl(4) metabolism. Treatment with GdCl(3) or glycine prevented CCl(4)-induced increases in transforming growth factor (TGF)-beta 1 protein levels and expression. CCl(4) treatment increased alpha-smooth muscle actin staining (score 3.0 +/- 0.2), whereas treatment with GdCl(3) and glycine during CCl(4) exposure blocked this effect (1.2 +/- 0.5); there was no staining with glycine treatment. These results support previous in vitro data and demonstrate that treatment of rats with the selective Kupffer cell toxicant GdCl(3) prevents stellate cell activation and the development of fibrosis.

Uptake, tissue distribution, and fate of inhaled carbon tetrachloride: comparison of rat, mouse, and hamster.

Carbon tetrachloride is hepatotoxic in rats, mice, and hamsters. However, rats are less sensitive to the hepatotoxic effects of CCl(4) than the other two species. The purpose of this study was to compare the uptake, tissue distribution, and elimination of CCl(4) by these three rodent species. Groups of 20 F344/Crl BR rats, B6C3F(1) mice, and Syrian hamsters were exposed by nose-only inhalation for 4 h to 20 ppm (14)C-labeled CCl(4). The fate of (14)C was followed in tissues, excreta, and exhaled breath for 48 h after the exposure. At the end of the exposure, concentrations of CCl(4) equivalents (CE) in tissue were highest in liver of rats and mice, but highest in fat for rats. The liver received the highest dose of CCl(4) equivalents with the following species ranking: mouse > hamster > rat. Patterns of CE elimination were species and tissue dependent, with the majority of elimination occurring within 48 h after exposure. Rats eliminated less radioactivity associated with metabolism ((14)CO(2), urine and feces) and more radioactivity associated with parent compound (exhaled activity trapped on charcoal) than did mice or hamsters. The results indicate that ranking of species sensitivity to the hepatotoxic effects of inhaled CCl(4) correlates with CE dose to liver and with the ability to metabolize CCl(4).

Pathogenesis of carbon tetrachloride-induced hepatocyte injury bioactivation of CCI4 by cytochrome P450 and effects on lipid homeostasis.

The CCl4-induced development of liver damage was studied in monolayer cultures of primary rat hepatocytes: (1) CCl4 caused accumulation of triglycerides in hepatocytes following cytochrome P450 induction with beta-naphthoflavone or metyrapone. Ethanol or a high dose of insulin plus triiodothyronine had the same effect. (2) CCl4 increased the synthesis of fatty acids and triglycerides and the rate of lipid esterification. Cholesterol and phospholipid synthesis from acetate was also increased. (3) CCl4 reduced beta-oxidation of fatty acids as assessed by CO2-release and ketone body formation. Hydrolysis of triglycerides was also reduced. (4) The content of unsaturated fatty acids in microsomal lipids was decreased by almost 50% after incubation with CCl4, while saturated fatty acids increased slightly. (5) CCl4 exerted a pronounced inhibitory effect on the exocytosis of macromolecules (albumin), but did not affect secretion of bile acids from hepatocytes.

The utility of PBPK in the safety assessment of chloroform and carbon tetrachloride.

Occupational exposure limits (OELs) for individual substances are established on the basis of the available toxicological information at the time of their promulgation, expert interpretation of these data in light of industrial use, and the framework in which they sit. In the United Kingdom, the establishment of specific OELs includes the application of uncertainty factors to a defined starting point, usually the NOAEL from a suitable animal study. The magnitude of the uncertainty factors is generally determined through expert judgment including a knowledge of workplace conditions and management of exposure. PBPK modeling may help in this process by informing on issues relating to extrapolation between and within species. This study was therefore designed to consider how PBPK modeling could contribute to the establishment of OELs. PBPK models were developed for chloroform (mouse and human) and carbon tetrachloride (rat and human). These substances were chosen for examination because of the extent of their toxicological databases and availability of existing PBPK models. The models were exercised to predict the rate (chloroform) or extent (carbon tetrachloride) of metabolism of these substances, in both rodents and humans. Monte Carlo analysis was used to investigate the influence of variability within the human and animal model populations. The ratio of the rates/extent of metabolism predicted for humans compared to animals was compared to the uncertainty factors involved in setting the OES. Predictions obtained from the PBPK models indicated that average rat and mouse metabolism of carbon tetrachloride and chloroform, respectively, are much greater than that of the average human. Application of Monte Carlo analysis indicated that even those people who have the fastest rates or most extensive amounts of metabolism in the population are unlikely to generate the levels of metabolite of these substances necessary to produce overt toxicity in rodents. This study highlights the value that the use of PBPK modeling may add to help inform and improve toxicological aspects of a regulatory process.

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.