Metabolism Of Carbon Tetrachloride Research Articles

Inhibition of CYP2E1 Activity does not Abolish Pulsatile Urine Alcohol Concentrations During Chronic Alcohol Infusions

Continuous, long-term intragastric infusions of ethanol leads to a two-step induction of hepatic cytochrome P 450 2E1 (CYP2E1) that is correlated with blood ethanol concentrations (BECs) and urine alkohol concentrations (UECs). In addition, long-term and continuous ethanol infusion does not produce a steady-state BEC, but results in pulsatile BECs and UECs having peak-peak duration of approximately 6 days and ethanol concentrations ranging from near zero to over 500 mg/dl. In the present study, rats were treated with ethanol (levels reaching 13 g · kg−1· day−1) for 38 days in the presence of compounds reported to block CYP2E1 activity or expression, to study the possible involvement of CYP2E1 in the pulsatile BECs. The inhibitors used were chlormethiazole (CMZ); diallysulfide (DAS); phenethyl isothiocyanate (PET) and dihydrocapsacin (DHC). Hepatic microsomal metabolism of carbon tetrachloride and p-nitrophenol, as well as mean CYP2E1 apoprotein levels, were significantly greater (P≤0.05) in ethanol-treated rats than in control rats, whereas rats treated with DAS, CMZ or PET had significantly (P≤0.05) reduced p-nitrophenol and carbon tetrachloride metabolism and lower CYP2E1 apoprotein levels compared to those of ethanol controls. UECs were variable in all ethanol-treated groups and there was a typical pulsatile pattern that had a mean interpulse interval (the number of days between the peaks of two consecutive pulses) ranging over 5.4±0.3–6.0±0.7 days and a mean amplitude (nadir to peak UEC) of 415 ± 39–337 ± 33 mg/dl. None of the putative CYP2E1 blockers altered the pulsatile nature of ethanol in urine. Our results suggest that pulsatile UECs are not the result of variations in the amount of CYP2E1.

Postulated Carbon Tetrachloride Mode of Action: A Review

Under the 2005 U.S. EPA Guidelines for Carcinogen Risk Assessment (1), evaluations of carcinogens rely on mode of action data to better inform dose response assessments. A reassessment of carbon tetrachloride, a model hepatotoxicant and carcinogen, provides an opportunity to incorporate into the assessment biologically relevant mode of action data on its carcinogenesis. Mechanistic studies provide evidence that metabolism of carbon tetrachloride via CYP2E1 to highly reactive free radical metabolites plays a critical role in the postulated mode of action. The primary metabolites, trichloromethyl and trichloromethyl peroxy free radicals, are highly reactive and are capable of covalently binding locally to cellular macromolecules, with preference for fatty acids from membrane phospholipids. The free radicals initiate lipid peroxidation by attacking polyunsaturated fatty acids in membranes, setting off a free radical chain reaction sequence. Lipid peroxidation is known to cause membrane disruption, resulting in the loss of membrane integrity and leakage of microsomal enzymes. By-products of lipid peroxidation include reactive aldehydes that can form protein and DNA adducts and may contribute to hepatotoxicity and carcinogenicity, respectively. Natural antioxidants, including glutathione, are capable of quenching the lipid peroxidation reaction. When glutathione and other antioxidants are depleted, however, opportunities for lipid peroxidation are enhanced. Weakened cellular membranes allow sufficient leakage of calcium into the cytosol to disrupt intracellular calcium homeostasis. High calcium levels in the cytosol activate calcium-dependent proteases and phospholipases that further increase the breakdown of the membranes. Similarly, the increase in intracellular calcium can activate endonucleases that can cause chromosomal damage and also contribute to cell death. Sustained cell regeneration and proliferation following cell death may increase the likelihood of unrepaired spontaneous, lipid peroxidation- or endonuclease-derived mutations that can lead to cancer. Based on this body of scientific evidence, doses that do not cause sustained cytotoxicity and regenerative cell proliferation would subsequently be protective of liver tumors if this is the primary mode of action. To fulfill the mode of action framework, additional research may be necessary to determine alternative mode(s) of action for liver tumors formed via carbon tetrachloride exposure.

Comparative effects of dimethylsulfoxide on metabolism and toxicity of carbon tetrachloride and dichloromethane

The effects of dimethylsulfoxide (DMSO) on the metabolism and toxicity of chlorinated methanes were examined. Male mice were treated with DMSO (1, 2.5 or 5 ml kg(-1), i.p.) prior to challenge with dichloromethane (CH(2)Cl(2)) or carbon tetrachloride (CCl(4)). Blood carboxyhemoglobin elevation resulting from metabolic conversion of CH(2)Cl(2) to carbon monoxide was inhibited dose-dependently by DMSO pretreatment. The elevation of serum aspartate aminotransferase, alanine aminotransferase and sorbitol dehydrogenase activities induced by CCl(4) (0.1 mmol kg(-1)) was not changed in mice pretreated with DMSO at 1 ml kg(-1), but depressed significantly at a greater dose of DMSO. However, DMSO failed to alter the hepatotoxicity of CCl(4) injected at a dose of 0.2 mmol kg(-1). DMSO induced the microsomal p-nitrophenol hydroxylase and p-nitroanisole O-demethylase activities as early as 2 h following the treatment. Microsomal disposition of CH(2)Cl(2) and CCl(4) was measured using a vial equilibration technique. The disappearance of CH(2)Cl(2) was inhibited competitively by addition of DMSO. But DMSO did not affect the metabolic degradation of CCl(4). The results indicate that DMSO has multiple effects on metabolism and toxicity of xenobiotics. DMSO induces the hepatic metabolizing activity mediated by CYP2E1, but the presence of this solvent in the enzyme site may inhibit directly the enzymatic interaction with a substrate. The toxicological significance of DMSO-induced effects on such an interaction may be variable depending on the properties of each substrate. The invulnerability of CCl(4) metabolism to the effects of DMSO appears to be related to its high affinity for the lipophilic CYP enzyme site.

Protective role of heme oxygenase-1 induction in carbon tetrachloride-induced hepatotoxicity

Reductive metabolism of carbon tetrachloride (CCl 4) is thought to cause lipid peroxidation which results in hepatic injury. Heme oxygenase-1 (HO-1) (EC 1.14.99.3), the rate-limiting enzyme in heme catabolism, is known to be induced by oxidative stress and to confer protection against oxidative tissue injuries. In this study, we examined the role of HO-1 induction in a rat model of CCl 4-induced acute liver injury. CCl 4 treatment (1 mL/kg, intraperitoneally) produced severe hepatic injury in rats as revealed by significant increases in serum alanine transaminase (ALT) (EC 2.6.1.2) activity and hepatic malondialdehyde (MDA) content, severe liver cell injury, and increases in hepatic tumor necrosis factor-α (TNF-α) mRNA expression and DNA binding activity of nuclear factor-κB (NF-κB). Following CCl 4 treatment, hepatic HO-1 expression was markedly increased both at transcriptional and protein levels in hepatocytes, especially around the central vein. HO-1 induction was mediated in part through a rapid increase in microsomal free heme concentration presumably derived from hepatic cytochrome P450. Inhibition of HO activity by tin-mesoporphyrin (Sn-MP), which resulted in a sustained increase in microsomal free heme concentration, exacerbated liver injury, as judged by the sustained increase in serum ALT activity, extensive hepatocytes injuries, a more pronounced expression of hepatic TNF-α mRNA and an enhanced NF-κB activation. These findings indicate that induction of HO-1 is an adaptive response to CCl 4 treatment, and it may be critical in the recovery of hepatocytes from injury. Our findings also suggest that HO-1 induction may play an important role in conferring protection on hepatocytes from oxidative damage caused by free heme.

Increased cytotoxicity of carbon tetrachloride in a human hepatoma cell line overexpressing cytochrome P450 2E1.

Cytotoxic free radicals generated during the metabolism of carbon tetrachloride by cytochrome P450 2E1 (CYP2E1) are thought to cause hepatotoxicity. Here, the cytotoxic effects of carbon tetrachloride in a liver cell line expressing CYP2E1 (HLE/2E1) are compared with those in the mother cell line (HLE). The effects of carbon tetrachloride on the gene expression of HSP70, a potential marker of oxidative stress, were also examined. The viability of HLE/2E1 cells after exposure to carbon tetrachloride was significantly decreased compared with that of HLE cells. Northern blot analysis revealed that the HSP70 mRNA level was significantly increased after carbon tetrachloride treatment in both cell lines, while the magnitude of its increase was much greater in HLE/2E1 cells than in HLE cells. These results suggest that the oxidative stress induced by CYP2E1 plays an important role in the increase in cytotoxicity of carbon tetrachloride in CYP2E1-overexpressing cells.

Mechanism of aerobic transformation of carbon tetrachloride by poplar cells.

The biochemical mechanism of carbon tetrachloride transformation by poplar cells was investigated using an axenic poplar cell culture. After one-day incubations of poplar cells under aerobic conditions, about 1.5% of dosed carbon tetrachloride was transformed to carbon dioxide, about 0.001% to chloroform and about 3% of the carbon was bound to insoluble poplar cellular materials. The production of carbon dioxide increased under aerobic conditions while the formation of chloroform and cell binding of carbon tetrachloride-carbon was enhanced under anaerobic conditions. Both carbon dioxide production and cell binding were significantly inhibited by a general inhibitor of cytochrome P-450 activity (carbon monoxide) and by specific P-450 2E1 inhibitors (chlorzoxazone, isoniazid, 4-methylpyrazole and 1-phenylimidazole). However, no inhibitory effects were observed when the cells were incubated in the presence of lignin peroxidase inhibitors (NaVO3 and 3-amino-1,2,4-triazole). These results suggest that an enzyme similar to mammalian cytochrome P450-2E1 is involved in the metabolism of carbon tetrachloride by poplar cells. This study demonstrates an environmental biodegradative process for carbon tetrachloride that operates under aerobic conditions.

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 K m (affinity) and V max (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 V max to K m 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 ( 14CO 2 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 V max and K m, respectively. Normalizing the rate of metabolism ( V ma / K m) by milligrams liver protein, the rate of metabolism of CCl4 differs across species, withxhamster > mouse > rat > human.

Cytochrome P450 2E1 is the primary enzyme responsible for low-dose carbon tetrachloride metabolism in human liver microsomes

We examined which human CYP450 forms contribute to carbon tetrachloride (CCl 4) bioactivation using hepatic microsomes, heterologously expressed enzymes, inhibitory antibodies and selective chemical inhibitors. CCl 4 metabolism was determined by measuring chloroform formation under anaerobic conditions. Pooled human microsomes metabolized CCl 4 with a K m of 57 μM and a V max of 2.3 nmol CHCl 3/min/mg protein. Expressed CYP2E1 metabolized CCl 4 with a K m of 1.9 μM and a V max of 8.9 nmol CHCl 3/min/nmol CYP2E1. At 17 μM CCl 4, a monoclonal CYP2E1 antibody inhibited 64, 74 and 83% of the total CCl 4 metabolism in three separate human microsomal samples, indicating that at low CCl 4 concentrations, CYP2E1 was the primary enzyme responsible for CCl 4 metabolism. At 530 μM CCl 4, anti-CYP2E1 inhibited 36, 51 and 75% of the total CCl 4 metabolism, suggesting that other CYP450s may have a significant role in CCl 4 metabolism at this concentration. Tests with expressed CYP2B6 and inhibitory CYP2B6 antibodies suggested that this form did not contribute significantly to CCl 4 metabolism. Effects of the CYP450 inhibitors α-naphthoflavone (CYP1A), sulfaphenazole (CYP2C9) and clotrimazole (CYP3A) were examined in the liver microsome sample that was inhibited only 36% by anti-CYP2E1 at 530 μM CCl 4. Clotrimazole inhibited CCl 4 metabolism by 23% but the other chemical inhibitors were without significant effect. Overall, these data suggest that CYP2E1 is the major human enzyme responsible for CCl 4 bioactivation at lower, environmentally relevant levels. At higher CCl 4 levels, CYP3A and possibly other CYP450 forms may contribute to CCl 4 metabolism.

P-644 - Stimulation of Carbon Tetrachloride Metabolism by Alcohols in Guinea-Pig Liver Microsomes

P-644 - Stimulation of Carbon Tetrachloride Metabolism by Alcohols in Guinea-Pig Liver Microsomes

Clarification of the Relationship Between Free Radical Spin Trapping and Carbon Tetrachloride Metabolism in Microsomal Systems

It has been proposed that the C-phenyl- N-tert-butylnitrone/trichloromethyl radical adduct (PBN/ •CCl 3) is metabolized to either the C-phenyl- N-tert-butylnitrone/carbon dioxide anion radical adduct (PBN/ •CO 2 −) or the glutathione (GSH) and CCl 4-dependent PBN radical adduct (PBN/[GSH- •CCl 3]). Inclusion of PBN/ •CCl 3 in microsomal incubations containing GSH, nicotinamide adenine dinucleotide phosphate (NADPH), or GSH plus NADPH produced no electron spin resonance (ESR) spectral data indicative of the formation of either the PBN/[GSH- •CCl 3] or PBN/ •CO 2 − radical adducts. Microsomes alone or with GSH had no effect on the PBN/ •CCl 3 radical adduct. Addition of NADPH to a microsomal system containing PBN/ •CCl 3 presumably reduced the radical adduct to its ESR-silent hydroxylamine because no ESR signal was observed. The Folch extract of this system produced an ESR spectrum that was a composite of two radicals, one of which had hyperfine coupling constants identical to those of PBN/ •CCl 3. We conclude that PBN/ •CCl 3 is not metabolized into either PBN/[GSH- •CCl 3] or PBN/ •CO 2 − in microsomal systems.

P-274 - Stimulation of Carbon Tetrachloride Metabolism by Anesthetics in Guinea-Pig Liver Microsomes

P-274 - Stimulation of Carbon Tetrachloride Metabolism by Anesthetics in Guinea-Pig Liver Microsomes

A pharmacokinetic model of anaerobic in vitro carbon tetrachloride metabolism

Carbon tetrachloride (CCl 4) 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 (CHCl 3) formation from CCl 4 is nonlinear with dose. Dose is traditionally expressed as the amount of CCl 4 added to the vial. In this study, a pharmacokinetic model has been developed to calculate the concentration of CCl 4 in the microsomal suspension. Hepatic microsomes prepared from fed and fasted animals were incubated with CCl 4 under anaerobic conditions and formation of CHCl 3 over a 5-min incubation period was monitored by headspace gas chromatography. Dose-response curves, based on total amount of CCl 4 added to the microsomes, revealed a nonlinear, biphasic appearance of CHCl 3, with fasting slightly increasing CHCl 3 production in microsomes prepared from fasted rats. Microsomes were also pretreated with the CYP2E1 inhibitor, diallyl sulfone (DAS), before addition of CCl 4. 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 CCl 4 concentrations. Following DAS pretreatment, the saturable portion of the dose-response curve was inhibited more than the linear phase with the biphasic CHCl 3 production becoming more linear. DAS inhibition eliminated the effect of fasting on CHCl 3 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 K rmm of 2.3 mg/l (15 μM). The linear phase rate constant ( k f) was determined to be 0.046 h −1 ( k fc = 0.03 h −1). In conclusion, a pharmacokinetic model has been developed for anaerobic in vitro metabolism of CCl 4 to CHCl 3 that estimates metabolic rates based on CHCl 3 formation and actual CCl 4 concentration in the microsomal suspension.

Tandem mass spectrometry study of C-phenyl- N-tert-butyl nitrone spin adducts from in vitro rat liver microsomal metabolism of bromotrichloromethane and carbon tetrachloride

Electron ionization and thermospray were used in conjunction with tandem mass spectrometry methods to identify trichloromethyl/C-phenyl-N-tert-butyl nitrone (PBN) spin adducts produced in rat liver microsomal dispersions that had been treated with reduced nicotinamide adenine dinucleotide phosphate (NADPH)-generating system and BrCCl3 (or CCl4). In the identification of PBN spin adducts, a scan of precursors of m / z 57 was utilized to confirm the presence of PBN spin adducts, because PBN spin adducts produce m / z 57 from tert-butyl as a characteristic fragment. Use of deuterated PBN (PBN-d9 deuterated at tert-butyl; PBN-d 14 deuterated at both phenyl and tert-butyl) improved the recognition of PBN adducts in mixtures by precursor ion scans, because m / z 66 (which corresponds to the deuterated tert-butyl group) is characteristic and, unlike m / z 57, it is not a common fragment for any other compounds. Two new PBN spin adducts that were not detected before by electron paramagnetic resonance or mass spectrometry were identified by these methods for the first time.

Inhibition of CYP2E1 activity does not abolish pulsatile urine alcohol concentrations during chronic alcohol infusions.

Continuous, long-term intragastric infusions of ethanol leads to a two-step induction of hepatic cytochrome P450 2E1 (CYP2E1) that is correlated with blood ethanol concentrations (BECs) and urine alcohol concentrations (UECs). In addition, long-term and continuous ethanol infusion does not produce a steady-state BEC, but results in pulsatile BECs and UECs having peak-peak duration of approximately 6 days and ethanol concentrations ranging from near zero to over 500 mg/dl. In the present study, rats were treated with ethanol (levels reaching 13 g.kg-1.day-1) for 38 days in the presence of compounds reported to block CYP2E1 activity or expression, to study the possible involvement of CYP2E1 in the pulsatile BECs. The inhibitors used were chlormethiazole (CMZ); diallysulfide (DAS); phenethyl isothiocyanate (PET) and dihydrocapsacin (DHC). Hepatic microsomal metabolism of carbon tetrachloride and p-nitrophenol, as well as mean CYP2E1 apoprotein levels, were significantly greater (P < or = 0.05) in ethanol-treated rats than in control rats, whereas rats treated with DAS, CMZ or PET had significantly (P < or = 0.05) reduced p-nitrophenol and carbon tetrachloride metabolism and lower CYP2E1 apoprotein levels compared to those of ethanol controls. UECs were variable in all ethanol-treated groups and there was a typical pulsatile pattern that had a mean interpulse interval (the number of days between the peaks of two consecutive pulses) ranging over 5.4 +/- 0.3-6.0 +/- 0.7 days and a mean amplitude (nadir to peak UEC) of 415 +/- 39-337 +/- 33 mg/dl. None of the putative CYP2E1 blockers altered the pulsatile nature of ethanol in urine. Our results suggest that pulsatile UECs are not the result of variations in the amount of CYP2E1.

Study of the isolation and stability of α-trichloromethylbenzyl(tert-butyl)aminoxyl, the trichloromethyl radical adduct of α-phenyl-tert-butylnitrone (PBN)

The aminoxyl produced from trapping trichloromethyl radicals by α-phenyl-tert-butylnitrone (PBN) has been isolated. Isolation has been achieved from the metabolism of carbon tetrachloride by rat liver microsomal dispersions followed by extraction or by photolysis of bromo(trichloro)methane in benzene. Chromatographic techniques were used to purify the isolated aminoxyl. The stability of this spin adduct aminoxyl has been evaluated in various solvents and at different pH in buffer solutions. The intrinsic stability of this spin adduct is remarkable. Solutions of this aminoxyl survive in aromatic solvents almost unchanged for over 90 days. In aqueous media the spin adduct is stable for over 30 days. Light and low pH cause decay but the rates are moderate. The spin adduct is difficult to oxidize and slow to reduce. The outstanding stability of this alkyl spin adduct must be associated in some manner with the proximity of the trichloromethyl group.

Reductive Activation of Carbon Tetrachloride by Human Haemoglobin

The reductive metabolism of carbon tetrachloride (CC14) by human haemoglobin (Hb) was observed in vitro by absolute absorption spectra recorded under anaerobic conditions. The following results were obtained: 1) a decrease of the 430nm peak typical of free reduced Hb (Hb2+); 2) the formation of a shoulder of absorbance, attributable to the production of a complex between Hb2+and a metabolite of CC14carbon monoxide (Hb-CO); and 3) the oxidation of some Hb2+to methaemoglobin (Hb3+). The concentration of these three forms — Hb2+, Hb-CO and Hb3+— during anaerobic incubation of Hb with CC14was calculated algebraically from the absolute spectra. CO production was then calculated from the concentration of Hb-CO, using a suitable calibration curve. Interestingly, under identical experimental conditions, a substrate-dependent loss of Hb-derived haem, but not of Hb itself nor of haem-derived porphyrin fluorescence, was measured. Preliminary HPLC studies to clarify the discrepancy and, in particular, the role and fate of the haem group, showed two substrate-dependent modified haem products. The results indicate that human Hb is able to catalyse the reductive activation of CCl4, and suggest that, during the process, its prosthetic group haem may be modified by CC14metabolites to products which maintain a tetrapyrrolic structure but are unable to react with pyridine.

The anomally of pyridine nucleotide synergism in carbon tetrachloride metabolism

Carbon tetrachloride metabolism was examined in hepatic microsomes isolated from control and phenobarbital-treated Sprague-Dawley rats to determine the mechanism of pyridine nucleotide synergism. An NADPH generator increased metabolism two fold as determined by lipid peroxidation. Addition of NADP to the reaction system did not alter the maximum velocity, but did decrease the Km for NADPH from 61 μM to 7.6 μM in control and from 21 μM to 6.3 mM PB microsomes. Addition of NAD + produced an increase in metabolism similar to NADH. Substrates and competitive inhibitors of nucleotide pyrophosphatase also enhanced CCl 4 metabolism. A high correlation ( r=0.947) was indicated between the percent inhibition of nucleotide pyrophosphatase and the percent synergism of NADPH-catalyzed CCl 4 metabolism. Thus, pyridine nucleotiode synergism in CCl 4 metabolism appears to result from the increased availability of NADPH produced by a decreased degradation of the NADPH by the nucleotide pyrophosphatase.

Microsomal metabolism of carbon tetrachloride: Participation of pyridine nucleotide synergism

The role of pyridine nucleotide synergism in CCl4 metabolism was evaluated for its potential contribution to enhanced lipid peroxidation. Male Sprague-Dawley rats receiving either no treatment (control) or treatment with phenobarbital (PB) were used to prepare hepatic microsomes. Metabolism was evaluated in the presence and absence of an NADPH generator system and in the presence and absence of NADH. The generator system produced a greater extent of metabolism for both control and PB microsomes. NADH-catalyzed CCl4 metabolism occurred to a similar extent in control and PB microsomes, amounting to 9-10% and 5-6% of the NADPH rate in control and PB microsomes, respectively. Synergism by NADH occurred at the lowest concentrations of NADPH, apparently decreasing the Km for NADPH and having little effect on the Vmax. Addition of NAD+ produced synergism, as did the addition of 5' AMP, an inhibitor of nucleotide pyrophosphatase. Thus, the synergistic increase in CCl4 metabolism produced by NADH may occur in part from an increased availability of NADPH, as a result of decreased degradation, rather than by electron donation from NADH.

Carbon tetrachloride metabolism in partially hepatectomized and sham‐operated rats pre‐exposed to chlordecone (kepone)

The potentiation of CCl4 toxicity by pre-exposure to chlordecone (CD) is well established. Chlordecone-induced metabolism of CCl4 and suppressed hepatocellular repair have been offered as possible mechanisms for this potentiation. Recent work using the partially hepatectomized (PH) rat as a model for an actively regenerating liver has provided supportive evidence for the latter hypothesis. The present study was initiated to determine if metabolism and disposition of 14CC14 is altered in the PH rat, and if this is a contributing factor to the reported protective effect afforded by the PH procedure. Male Sprague-Dawley rats (150-175 g) maintained on dietary CD (10 ppm) for 15 days were partially hepatectomized or sham-operated (SH) on day 15. Another group of CD-pretreated rats received 0.9% CoCl2 (60 mg/kg, sc, qd for 2 days) in lieu of the surgical procedure. On day 16 the rats were challenged with a single dose of CCl4 (100 microL/kg, ip) containing 20 muCi 14CCl4. A radiolabel inventory consisting of exhaled 14CCl4, 14CO2 production, total hepatic 14C, free 14CCl4 and covalently bound 14C was taken over a 6-hr time period. Lipid peroxidation and serum enzyme activities [aspartate aminotransferase (AST) and alanine aminotransferase (ALT)] were measured in indices of toxicity. Neither CD pretreatment alone nor CoCl2 treatment alone produced significant alterations in metabolism of low dose (100 microliters/kg) CCl4. No significant difference in 14CCl4 recovery or 14CO2 production was detected for PH versus SH rats. Hepatic 14CCl4-derived 14C (per gram tissue) was greater in PH rats. Values for free 14CCl4, covalently bound 14C, and lipid peroxidation were similar for SH and PH rats.(ABSTRACT TRUNCATED AT 250 WORDS)