Carbon tetrachloride-induced changes in the activity of phase II drug-metabolizing enzyme in the liver of male rats: role of antioxidants

Glutathione S-transferases and glutathione play an important role in the detoxification of most toxic agents. In the present study, the protective effects of some antioxidants (L-ascorbic acid (AA), vitamin E (VE) or garlic) on carbon tetrachloride-induced changes in the activity of alanine amino transferase (ALT), aspartate amino transferase (AST), glutathione S-transferase (GST), and the level of glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) were studied. The activities of ALT, and AST were assayed in plasma, whereas the activity of GST and the levels of GSH and TBARS were determined in the livers of rats. The current study included two experiments. In the first experiment, animals received single oral dose of CCl4 (400 mg/kg body weight) after administration of AA (100 mg/kg b.w.), VE (100 mg/kg b.w.) or garlic (800 mg/kg b.w.) as single oral doses. In the second experiment, rats received repeated oral doses of antioxidants for 12 consecutive days followed by a single oral dose of CCl4 on the 13th day and killed after that by 24 h. Treatment of male rats with CCl4 significantly increased the activity of ALT and AST in plasma, and the levels of both GSH and TBARS in the liver. On the other hand, CCl4 inhibited the activity of GST after single dose treatment. Single-dose treatments of rats with AA, VE or garlic prior to the administration of CCl4 could not restore the alterations in the activity of ALT and AST caused by CCl4 to the normal control level. However, repeated dose treatments with these agents restored such alterations to the normal level. We observed that VE is more effective than AA and garlic in restoring the inhibition of GST activity caused by CCl4 to the normal level after single dose treatments. On the other hand, AA and VE are more effective than garlic in restoring the induced TBARS level caused by CCl4 to the normal control level after repeated dose treatments. It has been observed that the tested antioxidants were able to antagonize the toxic effects of CCl4, and such counteracting effects were more pronounced when they were administered as repeated doses prior to administration of CCl4.     

Effect of some hypoglycemic herbs on the activity of phase I and II drug-metabolizing enzymes in alloxan-induced diabetic rats

Polycyclic aromatic hydrocarbons (PAHs) and N-nitrosamines (NNA) are mainly activated by cytochrome P450s, and their associated enzyme activities such as aryl hydrocarbon (benzo(a)pyrene) hydroxylase (AHH), N-nitrosdimethylamine N-demethylase I (NDMA-dI), NADPH-cytochrome C reductase, and detoxified by glutathione S-transferase (GST) and glutathione (GSH). The present study shows the influence of Cymbopogon proximus (Halfa barr), Zygophyllum coccineum L. (Kammun quaramany), Lupinus albus (Termis) as herbs capable of inducing hypoglycemia on the activity of the above mentioned enzymes in the liver of diabetic rats. Alloxan was administered as a single dose (120 mg/kg body weight) to induce diabetes and the herbs were administered to diabetic rats as repeated doses for 4 weeks. Alloxan-induced diabetes significantly increased the blood glucose level by 93% compared to the control level. On the other hand, repeated-dose treatments of diabetic rats with Cymbopogon proximus and Lupinus albus are more effective than Zygophyllum coccineum in restoring the elevated blood glucose level to the normal level. Alloxan treatment increased the hepatic activity of cytochrome P450, NADPH-cytochrome C reductase, AHH, NDMA-dI, GST and GSH by 112, 122, 82, 99, 64 and 26%, respectively. These herbs decreased the activity of above mentioned enzymes in the liver of diabetic rats compared to alloxan-treated rats. We conclude that alloxan increased the activity of cytochrome P450 system and that such herbs reduced these activities. The toxic effects of PAHs (e.g. benzo(a)pyrene) and NNA (e.g. N-nitrosdimethylamine) could be increased in the liver of diabetic rats through induction of their corresponding bioactivating enzymes. On the other hand, hypoglycemic herbs could alleviate the deleterious effects of these carcinogens in the liver of diabetic rats since these herbs reduced the hepatic content of cytochrome P450 and other associated enzyme activities compared to the diabetic group. Such alterations in the activity of phase I and II drug-metabolizing enzymes should be considered when therapeutic drugs are administered to diabetic patients since most of drugs are metabolized mainly by the cytochrome P450 system.     

Narcotic drugs change the expression of cytochrome P450 2E1 and 2C6 and other activities of carcinogen-metabolizing enzymes in the liver of male mice

Drug-metabolizing enzymes play a great role in the bioactivation and also detoxification of zenobiotics and carcinogens such as N-nitrosamines and polycyclic aromatic hydrocarbons (PAHs). Therefore, the present study was undertaken to investigate the effect of narcotic drugs such as cannabis (hashish) and diacetylmorphine (heroin) on the activity of N-nitrosodimethylamine N-demethylase I [NDMA-dI], arylhydrocarbon [benzo(a)pyerne] hydroxylase [AHH], cytochrome P450 (CYP), cytochrome b(5), NADPH-cytochrome c reductase, glutathione-S-transferase, and levels of glutathione and thiobarbituric acid-reactive substances (TBARS). In addition, the present study showed the influence of hashish and heroin after single (24 h) and repeated-dose treatments (4 consecutive days) on the expression of cytochrome P450 2E1 (CYP 2E1) and cytochrome P450 2C6 (CYP 2C6). The expression of CYP 2E1 was slightly induced after single-dose and markedly induced after repeated dose-treatments of mice with hashish (10 mg kg(-1) body weight). Contrarily, heroin markedly induced the expression of CYP 2C6 after single-dose and potentially reduced this expression after repeated-dose treatments. It is believed that N-nitrosamines are activated principally by CYP 2E1 and in support of this, the activity of NDMA-dI was found to be increased after single- and repeated-dose treatments of mice with hashish by 23 and 41%, respectively. In addition, single- and repeated-dose treatments of mice with hashish increased: (1) the total hepatic content of CYP by 112 and 206%, respectively; (2) AHH activity by 110 and 165%, respectively; (3) NADPH-cytochrome c reductase activity by 21 and 98%, respectively; (4) and glutathione level by 81 and 173%, respectively. Also, single-dose treatments of mice with heroin increased the total hepatic content of CYP, AHH, NADPH-cytochrome c reductase, and glutathione level by 126, 72, 39, 205%, respectively. However, repeated dose-treatments of mice with heroin did not change such activities except cytochrome c reductase activity increased by 20%. Interestingly, the level of free radicals, TBARS, was potentially decreased after single or repeated-dose treatments with either hashish or heroin. It is clear from this study that the effects of hashish are different from those of heroin on the above mentioned enzymes particularly after repeated dose treatments. It is concluded that hashish induced the expression of CYP 2E1 and other carcinogen-metabolizing enzymes activities, and this induction could potentiate the deleterious effects of N-nitrosamines and aromatic hydrocarbons, e.g. benzo(a)pyrene, upon the liver and probably other organs. Such alterations may also change the therapeutic actions of other drugs, which are primarily metabolized by the P450 system, when administered to peoples using hashish or heroin.     

Effects of saikosaponin-d on enhanced CCl4-hepatotoxicity by phenobarbitone

The effects of saikosaponin-d extracted from the roots of Buplerum falcatum L. on increased toxicity of CCl4 and increased activities of microsomal enzymes induced by phenobarbitone have been examined. Saikosaponin-d showed protection against the CCl4-hepatotoxicity enhanced by phenobarbitone. It also inhibited increases in the content of cytochrome P450 and NADPH-cytochrome c reductase activity, which are induced by the phenobarbitone treatment, but the spectral characteristics of P450 were not altered. The rate of microsomal lipid peroxidation by NADPH and CCl4 was significantly lowered in-vitro in rats pretreated with phenobarbitone and saikosaponin-d compared with those pretreated with phenobarbitone alone.     

Relationship between survival times of rats exposed to lethal level of nitrogen dioxide and arylhydrocarbon hydroxylase activity in lungs

A previous paper has shown that survival times of rats exposed to lethal levels of NO2 were prolonged by administration of enzyme-inducing agents prior to NO2 exposure, and that the survival time of female rats was longer than that of male rats. In order to investigate a mechanism on prolongation of survival times by administration of enzyme-inducing agents and on sex-difference, a relationship between survival times and microsomal enzyme activity in lungs of rats administered enzyme-inducing agents was examined. The survival times of rats exposed to 65 ppm NO2 were prolonged with increase of 3MC doses, and AHH activity in lungs of rats rose with increase of 3MC doses. A significant correlation between the survival time and AHH activity was observed. Induction of AHH activity of female rats administered 3MC was higher than that of male rats. NADPH-dependent cytochrome c reductase activity in lungs of male and female rats did not change with 3MC administration, and a significant relationship between the survival time and NADPH-dependent cytochrome c reductase activity was not observed. These results suggest that cytochrome P1-450 system in lung microsomes may have protective action against the toxicity of NO2.     

Modulation of carbon tetrachloride hepatotoxicity and xenobiotic-metabolizing enzymes by corticosterone pretreatment, adrenalectomy and sham surgery

These investigations sought to determine the role of physiological concentrations of natural glucocorticoids in modulating chemical toxicity, and to ascertain if effects on toxicity may be due to alterations of chemical metabolizing enzymes by glucocorticoids. The hepatotoxic response to carbon tetrachloride (CCl4) in adrenalectomized or naive Long Evans rats treated with corticosterone was assessed. Alterations of hepatic cytochrome P-450 concentration, mono-oxygenase activities, NADPH-cytochrome (P-450)c reductase activity, and glutathione S-transferase activity were examined. Adrenalectomy and to a lesser extent sham surgery were protective, but corticosterone administration increased CCl4 hepatotoxicity. Corticosterone administration to adrenalectomized or sham-operated rats reduced the protective effect of these treatments. Correlating with the in vivo response, mono-oxygenase activities decreased after adrenalectomy and sham surgery, but increased with glucocorticoid administration. These studies suggest that basal, stress-associated, and pharmacological concentrations of a natural glucocorticoid can modify chemical toxicity and alter hepatic enzymes important to chemical metabolism.     

Induction of hepatic cytochrome P-450 and xenobiotic metabolizing enzymes in rats gavaged with an Alberta crude oil

Changes in body weight gain and in biochemical parameters of blood and liver were assessed in Sprague-Dawley rats after multiple oral administration of three test doses of an Alberta crude oil (ACO). Rats treated with ACO (1.25-5 ml/kg) did not show statistically significant (p greater than .05) differences from control, corn-oil treated (5 ml/kg) rats, in body weight gains, liver weight, and blood biochemical indicators of liver (alanine aminotransferase, gamma glutamyltransferase), kidney (blood urea nitrogen, creatinine), and erythrocyte (adenosine 5'-triphosphate, 2,3-diphosphoglyceric acid, reduced glutathione) cytotoxicity. Treatment with ACO, however, caused statistically significant (p less than .05) and dose-related increases from control in (1) microsomal protein and cytochrome P-450 content, and NADPH-cytochrome c reductase, aryl hydrocarbon hydroxylase (AHH), and 7-ethoxycoumarin-O-deethylase (7-ECOD) activities, and (2) cytosolic glutathione transferase activity of liver. The induction of hepatic cytochrome P-450 and xenobiotic-metabolizing enzymes in microsomes of ACO-treated rats was probably associated with dose-related changes in isozymic forms of cytochrome P-450, as evidenced by (1) appearance of a 448-nm spectral peak in microsomes of ACO-treated rats and (2) differences in the inhibition pattern of AHH and 7-ECOD activities in microsomes of control and ACO-treated rats upon treatment with metyrapone and 7,8-benzoflavone.     

Modification of cytochrome P-450, NADPH-cytochrome c reductase and aryl hydrocarbon hydroxylase activities by schistosomicidal drugs

This study was planned to investigate the modification of the mouse microsomal monooxygenase enzymes using various schistosomicidal drugs. Enzymes investigated were cytochrome P-450, NADPH-cytochrome c reductase and aryl hydrocarbon hydroxylase (AHH). Administration of oxamniquine and niridazole increased, whereas praziquantel and hycanthone lowered the cytochrome P-450 content. An apparent increase in the activity of NADPH-cytochrome c reductase was only observed with oxamniquine. The in vivo and in vitro effects of schistosomicidal drugs on the activity of AHH were investigated using benzo(a)pyrene (BP) as substrate. Oxamniquine and niridazole significantly increased the AHH activity in vivo and in vitro, while the antimonial drugs enhanced the enzyme activity only in vivo. On the other hand, praziquantel and hycanthone lowered the AHH activity only in vivo. Metrifonate did not show any effect either in vivo or in vitro. The mechanisms by which these drugs modify the AHH activity are discussed in the text.     

Alterations in hepatic Phase I and Phase II biotransformation enzymes by garlic oil in rats

Studies were conducted to examine the effect of a single and repeated administrations of garlic oil (diallyl sulfide) on Phase I and Phase II biotransformation enzymes in rats. Adult, male Sprague-Dawley rats treated with a single dose of garlic oil (500 mg/kg i.p.) showed a significant depression of hepatic cytochrome P-450, aminopyrine Ndemethylase and aniline hydroxylase while microsomal protein content, cytochrome b₅, NADPH-cytochrome c reducase, benzphetamine N-demethylase and cytosolic glutathione, S-transferase remained unaffected 24 h following the treatment. Although certain microsomal enzymes were depressed, there was no liver damage caused by garlic oil as judged by the putative serum enzyme test. On the other hand, daily administration of garlic oil (50 mg/kg) i.p. for 5 days) produced a significant increase in hepatic cytochrome P-450, aminopyrine N-demethylase and benzphetamine N-demethylase activities, but not in the rest of the aforementioned parameters of biotransformation reactions. These data indicate that the effect of garlic oil on the hepatic drug-metabolizing enzyme system is dose-dependent.     

Microsomal electron transport and xenobiotic monooxygenase activities during the embryonic period of development in the killifish,Fundulus heteroclitus

The developmental patterns of aryl hydrocarbon hydroxylase (AHH) activity and NADPH-cytochrome c reductase activity were followed during embryonic development in Fundulus. AHH activity was localized in microsomal fractions prepared from whole Fundulus embryos and eleutheroembryos. On the basis of this subcellular localization, the requirements of O₂ and NADPH for activity, and sensitivity to carbon monoxide and cytochrome c inhibition, the AHH activity in Fundulus embryos and eleutheroembryos appeared to be cytochrome P-450 dependent. AHH activity was measurable in stages prior to the appearance of the liver rudiment, and during subsequent embryonic development the extrahepatic tissues were likely to have contributed substantially to the AHH activity measured. At all stages assayed before hatching, microsomal AHH specific activity remained uniformly low, but within 24 hr of hatching, AHH specific activity increased about ninefold. This posthatching increase in AHH activity was not age dependent, nor developmental stage dependent, but rather required hatching, and was not due to the presence of endogenous inhibitors in prehatching stages. The levels of NADPH-cytochrome c reductase activity and AHH activity were not closely correlated in whole embryo and eleutheroembryo microsomes, but the AHH activity in these preparations apparently was not limited by the levels of the NADPH-cytochrome c (P-450) reductase. The presence of AHH activity in Fundulus embryos during the period of active organogenesis, prior to hatching, indicates that this species is likely to be susceptible to a variety of teratogens requiring metabolic activation, and this may be the case for other species of fish as well.     

Isopropanol enhancement of cytochrome P-450-dependent monooxygenase activities and its effects on carbon tetrachloride intoxication

Acute or chronic treatment of rats with isopropanol caused a significant increase in hepatic cytochrome P-450 content and a two- to threefold increase in aniline hydroxylase and 7-ethoxycoumarin O-deethylase activities, but no significant change in ethylmorphine N-demethylase or benzo(a)pyrene hydroxylase activity. In rats treated with isopropanol and challenged with CCl4, liver toxicity of CCl4 was characteristically potentiated, as assessed by elevation of serum glutamic-pyruvic transaminase (SGPT) levels. Isopropanol pretreatment also potentiated CCl4-induced damage to the hepatic monooxygenase system. In addition to a decrease in cytochrome P-450, rats treated with isopropanol and challenged with CCl4 showed a nonspecific decrease not only in aniline hydroxylase and 7-ethoxycoumarin O-deethylase activities, but also in ethylmorphine N-demethylase, benzo(a)pyrene hydroxylase, and NADPH-cytochrome c reductase activities. These results were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized microsomes. The electrophoretic results showed that isopropanol pretreatment markedly potentiated the CCl4-caused destruction of cytochrome P-450 hemeproteins. The data strongly suggest that isopropanol increases one or more forms of cytochrome P-450 which selectively enhance the metabolism of CCl4 to an active metabolite. This active metabolite then causes a nonselective damage to the microsomal mixed-function oxidase system.     

Induction of cytochrome P-450 related metabolic activities in the rat ventral prostate

Rats were treated with β-naphthoflavone (BNF) or phenobarbital (PB), $\text{80}\text{mg}\text{kg}$ i.p. for 4 days and microsomes prepared from the ventral prostate were assayed for aryl hydrocarbon hydroxylase (AHH), 7-ethoxyresorufin-O-deethylase (7-EOD) and NADPH-cytochrome c reductase activities. The relative increase after BNF treatment was approx. 1000 times for AHH, and 800 times for 7-EOD, while the NADPH-cytochrome c reductase activity was not significantly altered. PB treatment caused no significant effects. Treatment with BNF led to an increased in vitro formation of all measured benzo(a)pyrene (B(a)P) metabolites, especially phenols. Carbon monoxide (CO) and α-naphthoflavone (α-NF) inhibited 7-EOD- and AHH-activities. The rat ventral prostate contains inducible cytochrome P-450-dependent enzymes, a circumstance of potential importance in the etiology of prostatic carcinoma.     

Effect of ethylene glycol monomethyl ether and diethylene glycol monomethyl ether on hepatic metabolizing enzymes

Glycol ethers have been extensively used in industry over the past 40-50 years. Numerous studies on the toxicity of glycol ethers have been performed, however, the effects of glycol ethers on the hepatic drug metabolizing enzymes are still unknown. We studied the changes of the putative metabolic enzymes, that is, the hepatic microsomal mixed function oxidase system and cytosolic alcohol dehydrogenase, by the oral administration of diEGME and EGME. Adult male Wistar rats were used. DiEGME was administered orally; 500, 1000, 2000 mg/kg for 1, 2, 5 or 20 days and EGME was 100, 300 mg/kg for 1, 2, 5 or 20 days. Decreases in liver weights were produced by highest doses of diEGME (2000 mg/kg body wt/day for 20 days) and EGME (300 mg/kg body wt/day for 20 days). DiEGME increased hepatic microsomal protein contents and induced cytochrome P-450, but not cytochrome b5 or NADPH-cytochrome c reductase. The activity of cytosolic ADH was not affected by diEGME administration. On the other hand, EGME did not change cytochrome P-450, cytochrome b5 or NADPH-cytochrome c reductase. The activity of cytosolic ADH was increased by repeated EGME treatment. Therefore it is suspected that the enzyme which takes part in the metabolism of diEGME is different from that of EGME, although diEGME is a structural homologue of EGME.     

Chronic treatment with the endocannabinoid anandamide increases cytochrome P450 metabolizing system in the rat

The aim of this work was to investigate the effects of single and repeated administration of the endogenous cannabinoid anandamide (20 mg/kg i.p.) on cytochrome P450-mediated biotransformation in the rat. In liver microsomes from chronically treated rats, an increase in cytochrome P450 content and in the activity and immunoreactivity of cytochrome P450 reductase was detected. Immunoblot analysis of the hepatic microsomal proteins revealed an increase in the relative level of cytochrome P450 2B1/2 and 3A2. The activity of monooxygenase enzymes linked to specific cytochrome P450 isoforms was significantly enhanced. This increase in the content and activity of the cytochrome P450 system was also seen in liver microsomes from acutely treated rats; however, these increases were smaller than those seen after prolonged treatment. After acute treatment, the brain cytochrome P450 and b(5) content was increased, whereas after chronic treatment, only that of b(5) was enhanced. Cytochrome P450 reductase activity and its relative abundance were increased only in the brains of chronically treated rats. The present findings demonstrate that anandamide administration increased the metabolic activity of the cytochrome P450 system in rat liver and brain.     

Comparison of effects of xenobiotics on extrahepatic and hepatic microsomal drug-metabolizing enzymes in mice

The content of microsomal protein is the same in both kidneys and small intestine, corresponding to 57% of the control value expressed as 100% in the untreated liver. The contents of P450 and cytochrome b(5), and the activity of NADPH-cytochrome c reductase in the kidney were higher than those in the small intestine, which were 17%, 22% and 41% of controls, respectively, in the former and 5%, 11% and 22% of controls in the latter. As compared with similar measurements made in the liver, the activities of substrate-metabolizing enzymes in these extrahepatic organs were very low. The activities of renal aniline hydroxylase, aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase, 7-methoxycoumarin O-demethylase and benzo(a)pyrene hydroxylase were 6%, 5%, 3%, 0.6% and 0.2% of controls, respectively. The activities of these enzymes in the small intestine were lower than those in the kidney or below the limits of detection. These results suggested that isoforms or their contents of P450 responsible for these substrate biotransformations are different among liver, kidneys and small intestine. Meantime, this study showed similar significant inductions by phenobarbital and rifampin of small intestinal and hepatic microsomal drug-metabolizing enzymes. In contrast, neither phenobarbital nor rifampin was capable of increasing renal microsomal enzymes, with the exception of benzo(a)pyrene hydroxylase which was induced by rifampin. These findings indicated that both liver and small intestine, but not kidneys contain the same phenobarbital- and rifampin-inducible P450 isoforms, cytochrome b(5) and NADPH-cytochrome c reductase. In addition, CCl(4) could be bioactivated by CYP2E1 to free radicals in the kidney which caused destruction of microsomal enzymes. In mice pretreated with phenobarbital, CCl(4) also attenuated the increase in content of P450 in the small intestine, which appeared to be a result of induction by phenobarbital of CYP2E1.     

Influence of liver disease and environmental factors on hepatic monooxygenase activity in vitro

Summary The effects of liver disease and environmental factors on hepatic microsomal cytochrome P-450 content, NADPH-cytochrome c reductase (reductase) activity and aryl hydrocarbon hydroxylase (AHH) activity have been simultaneously investigated in 70 patients undergoing diagnostic liver biopsy. The activity of reductase was not significantly affected by the presence of liver disease or any of the environmental factors studied. Cytochrome P-450 content decreased with increasing severity of liver disease whereas AHH activity was only significantly reduced in biopsies showing hepatocellular destruction. None of the parameters of monooxygenase activity varied significantly with the age or sex of the patients. Alcohol excess was associated with decreased cytochrome P-450 content and AHH activity and this effect was independent of the histological status of the biopsy. Both high caffeine intake and cigarette smoking increased AHH activity in the absence of any change in cytochrome P-450 content. There was a positive correlation between the number of meat meals eaten per week and cytochrome P-450 content. Chronic treatment with enzyme-inducing anticonvulsants appeared to increase both cytochrome P-450 content and AHH activity. Despite differential effects of liver disease and environmental influences on cytochrome P-450 content and AHH activity there was a highly significant correlation between the two parameters. The results of the present study correlate well with the known effects of disease and environment on drug metabolism in vivo.     

Medroxyprogesterone acetate improvement of the hepatic drug-metabolizing enzyme system in rats after chemical liver injury

Medroxyprogesterone acetate (MPA) has an inducing effect on the hepatic drug-metabolizing enzyme system in the rat. The effect of MPA on the liver metabolism was further evaluated here by investigating the restoration of hepatic function after chemical liver injury in female rats. The hepatic injury was induced by pretreating the animals with CCl4 and dimethylnitrosamine for 4 weeks, after which rats treated with MPA for a week were compared with rats showing spontaneous regeneration upon treatment with the MPA vehicle only. Changes in various parameters of the drug-metabolizing enzyme system were used as indices of hepatic function together with liver protein content. The results showed that MPA therapy increased the cytochrome P-450 content and the activity of NADPH-cytochrome c reductase, the monooxygenase enzymes benzo[a]pyrene hydroxylase and aminopyrine N-demethylase, epoxide hydrolase and glutathione S-transferase. MPA increased the relative values in the rats with liver injury almost equally to, or even more than, that seen in the intact animals in comparison to the corresponding vehicle-treated rats. MPA seemed to enhance protein synthesis during liver regeneration, as indicated by changes in total liver protein and in the gel electrophoresis pattern of the microsomal proteins. The hepatic enzyme induction and enhancement of protein synthesis achieved by MPA after liver injury may be of value in the treatment of liver diseases.     

Effect of o-benzyl-p-chlorophenol on drug-metabolizing enzymes in rats

o-Benzyl-p-chlorophenol (BCP) is widely used as a broad spectrum disinfectant. Treatment of male Fischer 344 rats with BCP resulted in an increase in cytochrome P-450 content and an accompanying decrease in aryl hydrocarbon hydroxylase (AHH) activity in both liver and kidney microsomes. Several other drug-metabolizing enzymes were not affected by BCP treatment. However, in kidney, BCP induced NADPH-cytochrome c reductase and uridine diphosphate glucuronyl transferase activities and caused a small increase in total cytochrome P-450 content and glutathione concentration. The cytochrome P-450 isozymes induced by BCP were fractionated by high pressure liquid chromatography (HPLC). The HPLC profile following BCP treatment most closely resembled that seen after phenobarbital. Using an immunoblotting procedure and a radioimmunoassay, it was shown that the increase in cytochrome P-450 content in the liver after BCP treatment was, in part, due to an increase in the phenobarbital-inducible isozymes, P-450b + e. In the kidney, the increase in total cytochrome P-450 content after BCP exposure was not due to an increase in P-450b + e. The decrease in AHH activity appeared to be caused by noncompetitive inhibition of constitutive AHH activity by BCP. BCP also inhibited benzphetamine demethylation, although to a lesser extent. The failure to observe an increase in benzphetamine demethylase activity in vivo, despite the induction of P-450b, was probably due to the concomitant induction and inhibition of drug-metabolizing enzymes by BCP.     

One-electron reductive bioactivation of 2,3,5,6-tetramethylbenzoquinone by cytochrome P450.

Bioreductive activation of quinones in mammalian liver has generally been attributed to NADPH-cytochrome P450 reductase. However, in view of the 20-30-fold molar excess of cytochrome P450 over NADPH-cytochrome P450 reductase on the endoplasmic reticulum of the rat liver cell and the capability of cytochrome P450 to bind and reduce xenobiotics, it was considered of interest to investigate the possible role of cytochrome P450 in the bioreduction of quinones. In the present study, 2,3,5,6-tetramethyl-1,4-benzoquinone (TMQ) was chosen as a model quinone. First, TMQ was found to bind at the metabolic active site of phenobarbital (PB)-inducible cytochrome P450s of rat liver microsomes, indicating that TMQ is a potential substrate for cytochrome P450-mediated biotransformation. Second, with electron spin resonance, one-electron reduction of TMQ to a semiquinone free radical (TMSQ) was found to occur in these microsomal fractions. SK&F 525-A, a well-known inhibitor of cytochrome P450, strongly inhibited TMSQ formation in these subcellular fractions without affecting NADPH-cytochrome P450 reductase activity. One-electron reductive bioactivation of TMQ was further investigated with purified NADPH-cytochrome P450 reductase alone and in reconstituted systems of purified cytochrome P450-IIB1 and NADPH-cytochrome P450 reductase. As measured by ESR, purified cytochrome P450-IIB1 in the presence of NADPH-cytochrome P450 reductase was able to reduce TMQ to TMSQ at a much greater rate than in the presence of NADPH-cytochrome P450 reductase alone. Reduction of TMQ was also investigated by measuring the initial rate of NADPH oxidation by TMQ under anaerobic conditions. Inhibitors of cytochrome P450, namely SK&F 525-A and antibodies against PB-inducible cytochrome P450s, caused a substantial decrease in reductive metabolism in PB-treated microsomes. These antibodies were also effective in the inhibition of TMQ-induced NADPH oxidation in a complete reconstituted system of equimolar concentrations of cytochrome P450-IIB1 and NADPH-cytochrome P450 reductase, indicating that the reaction was specific for cytochrome P450-IIB1. Finally, initial rates of NADPH oxidation were determined in reconstituted systems containing varying amounts of NADPH-cytochrome P450 reductase and cytochrome P450-IIB1 to determine the contribution of either enzyme in the reduction of TMQ. As expected, NADPH-cytochrome P450 reductase was able to reduce TMQ to a small extent. However, reconstitution in the presence of increasing amounts of cytochrome P450-IIB1 (relative to NADPH-cytochrome P450 reductase) resulted in increasing rates of TMQ-induced NADPH oxidation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Changes in cytochrome P450 molecular species in rat liver in chloroform intoxication

The effect of CHCl3 on the composition of hepatic microsomal cytochrome P450 species was compared with that of CCl4 in rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (3MC). The administration of CHCl3 hardly affected cytochrome P450 content in non-treated rat liver, but caused a similar degree of depletion in the content as observed after CCl4 administration in PB-pretreated rats. In the pretreatment with 3MC, the administration of CHCl3 brought about a marked decrease in the content to 24% of control after 12 hr, while CCl4 reduced the content only to one-half of control. It was demonstrated by SDS-polyacrylamide gel electrophoresis and Whatman DE-52 anion-exchange chromatography that 3MC-induced P450 species decreased with CHCl3, while it was affected little by CCl4 treatment. The activity of benzo[a]pyrene hydroxylase was altered together with the change in the content of cytochrome P450 species. The administration of CHCl3 to PB-pretreated rats caused the depletion in PB-induced P450. These findings indicate that cytochrome P450 species induced with 3MC as well as PB are highly susceptible to CHCl3 intoxication, whereas the administration of CCl4 depletes the PB-induced species without affecting the 3MC-induced species.