Effect of dietary butylated hydroxytoluene on nuclear envelope cytochrome P-450 during the initiation and promotion stages of hepatocarcinogenesis.

The anticarcinogenic effect of the dietary antioxidant butylated hydroxytoluene (BHT) correlates with a preservation of nuclear envelope (NE) cytochrome P-450 in rats undergoing chemically induced hepatocarcinogenesis. This effect of BHT on NE cytochrome P-450 was observed during both the initiation and the promotion stages of hepatocarcinogenesis. Complex interactions between the two different mechanisms of action of BHT (i.e., enzyme induction and antioxidant activity) may account for some of the differences between the patterns of response to BHT observed during initiation and promotion.     

Effects of ethylene glycol on hepatic microsomal cytochrome P-450

The effect of ethylene glycol on rat hepatic microsomal cytochrome P-450 was studied in vitro and in vivo. The destruction of cytochrome P-450 was not seen in vitro. The addition of 1 mM NADPH also did not change. When ethylene glycol was added to drinking water at a concentration of 1.0% for 7 days, there was no change in the contents of microsomal protein, cytochrome P-450, b5 and heme. While NADPH-cytochrome C reductase activity of the exposed group did not change, NADH-ferricyanide reductase activity increased significantly.     

Ethanol-induced increase in NADH-dependent monooxygenase activities of hepatic microsomal cytochrome P-450

NADH-dependent hepatic microsomal monooxygenase activities were measured in the presence and absence of NADPH in material from adult male rats given ethanol in a liquid diet. Controls were given a liquid control diet (control group; lipid as substitute for ethanol) or rat chow (untreated group). Ethanol feeding elevated microsomal aniline hydroxylase activity and did not change ethylmorphine N-demethylase activity. NADH supported 21-24% of the NADPH-driven aniline hydroxylase activity in ethanol, control and untreated microsomes, but only about 6% of ethylmorphine N-demethylase activity. In the presence of NADPH, NADH gave 13-14% increase in aniline hydroxylase activity in microsomes from control and untreated rats, but only 3% in ethanol microsomes. In contrast, the presence of NADPH increased many times the effect of NADH on ethylmorphine N-demethylation with no striking difference between the groups. In another series of experiments, demethylation of 4-nitroanisole was elevated after ethanol feeding (4-fold with NADPH; 5-fold with NADH) and phenobarbital treatment (8-fold with NADPH, 2-fold with NADH). In the ethanol-induced activity, NADH and NADPH were less than additive. In the control and untreated and the phenobarbital-induced activities, NADH and NADPH were additive or possibly synergistic in driving the activity. Both ethanol and phenobarbital elevated cytochrome P-450; ethanol also elevated cytochrome b5 measured as NADH-reducible cytochrome.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of nuclear receptors in cytochrome P-450 induction by xenochemicals

This review summarizes recent findings indicating that members of the orphan nuclear receptor superfamily regulate the synthesis of their CYP genes which code CYP enzymes involved in metabolism of endogenous and exogenous compounds. The foreign compounds metabolism and the role played by individual cytochrome P450 (CYP) enzymes in the activation and detoxification of xenochemicals prevalent in the environment are important areas of molecular pharmacology and toxicology. The advances in our understanding of the mechanisms through which foreign chemicals impact on these CYP-dependent metabolic processes have been made during the past years. Role for three "orphan" nuclear receptor superfamily members, designated CAR (constitutive androstane receptor), PXR/SXR (pregnelone X receptor) and PPAR (peroxisome proliferator activated receptor), in respectively mediating the induction of hepatic CYPs belonging to families CYP2, CYP3, and CYP4 has now been established. The CYP gene products such as CYP3A, CYP2B and PPAR are essential for metabolism of endogenous steroid hormones, fatty acids and various xenobiotics including drugs. Unexpectedly, it has been shown that SXR, which regulates CYP3A, can also regulate CYP2B via recognition of the phenobarbital response element (PBRE). In a type of functionally symmetry, orphan receptor CAR was found to activate CYP3A through SXR/PXR response element. Indeed, SXR/PXR binds to inverted (IR-6) and direct (DR-4) response element localized to regulatory DNA regions of human CYP3A4 and rat CYP3A23 genes, respectively. These observations provide a rational explanation for the activation of multiple CYP gene classes by certain xenobiotics as well as the propensity for drug-drug interactions. In addition, both endogenous and exogenous ligands which act as activators of nuclear receptors can result in disruption of cellular homeostasis.     

Hepatic cytochrome P-450 and microsomal heme oxygenase in copper-deficient rats

Copper deficient and control rats were pair-fed from weaning a milk-based diet. Each animal received 25 mg of iron parenterally. After 6 weeks of dietary manipulations the animals were killed. As expected, rats fed the copper-deficient diet were anemic. Copper-dependent enzyme activities and copper content of plasma and liver confirmed that copper deficiency was present in these rats. Hepatic microsomal cytochrome P-450 specific contents were similar in control and copper-deficient animals in the uninduced state and following induction of cytochrome P-450 by treatment with phenobarbital. However, microsomal heme oxygenase was increased in copper-deficient animals compared with controls whether they were treated with phenobarbital or not. No differences were observed in splenic microsomal heme oxygenase between dietary groups. These studies suggest that hepatic heme catabolism is enhanced in copper deficiency. The explanation for this enhancement may be related to increased hepatic iron accumulation in copper-deficient animals or to the associated defect of selenium metabolism in copper deficiency.     

Effects of retinoic acid on hepatic cytochrome P-450 dependent enzymes in rats under different vitamin A status

The temporal effects of retinoic acid supplementation on hepatic cytochrome P-450-dependent enzymes were studied on the rat. Four groups of male weanling rats were fed semi synthetic diets: two groups containing 0 or 4.4 mg retinol equivalents per kg diet as retinyl palmitate (A- RA- and A+ RA- groups) and two similar groups supplemented with all trans retinoic acid (12 mg/kg diet) (A- RA+ and A+ RA+ groups). After five or ten weeks of feeding, the rats were killed, liver microsomes were prepared and assayed for aniline hydroxylase, aminopyrine N demethylase activities and cytochrome P-450 levels. Whereas no change was observed between the four groups after 5 weeks, the following modifications appeared after 10 weeks: Vitamin A deficiency decreased hepatic drug metabolism by phase I enzymes (hydroxylase and N demethylase) but only when liver storage pool was not detectable. Vitamin A concentration as low as 4 micrograms/g is sufficient to avoid any perturbation of these enzymes. Parallel to a sparing effect on liver reserves of vitamin A, retinoic acid maintained a normal activity of enzymes of xenobiotic metabolism. However, retinoic acid treatment produced an alteration of phase I enzymes in vitamin A supplemented group (A+ RA+). As this was accompanied by a doubling of vitamin A liver reserves, compared to A+ RA- group, it is suggested that this might result from a liver vitamin A overloading, leading to membrane damage perturbing microsomal enzymes. These results indicate the need for a more careful use of retinoids as a therapeutic agent.     

Potentiation of ethanol-induced hepatic vitamin A depletion by phenobarbital and butylated hydroxytoluene

Administration of ethanol, phenobarbital or butylated hydroxytoluene (BHT) each resulted in significantly lower hepatic vitamin A than in untreated controls. When ethanol was combined with either phenobarbital or BHT, a striking potentiation of the depletion was observed, resulting in hepatic vitamin A values of less than 5% of normal, whether expressed per gram of liver or per 100 g body wt. These effects were observed for both retinol and retinyl esters, measured by high pressure liquid chromatography. By contrast, lung levels of retinol and retinyl esters were significantly higher in rats treated with ethanol, but remained unchanged in rats treated with phenobarbital or butylated hydroxytoluene compared with control animals.     

Paradoxical effects of cobaltous chloride treatment on 2-methylnaphthalene disposition and hepatic cytochrome P-450 content in carp

Effects of cobaltous chloride (CoCl₂) treatment on the disposition of 2-methylnaphthalene (2-MeNap)-derived radioactivity was studied in adult carp. The fish were treated with CoCl₂ (60 mg/kg, ip) or an equivalent volume of saline (control) 36 and 12 hr before 2-MeNap administration (5 mg/kg, ip). Carp were killed 12, 24, 48 and 72 hr later. CoCl₂-treated carp had less 2-MeNap-derived radioactivity in gallbladder bile and a greater amount in the liver at all of these times. Solvent extraction of bile revealed that virtually all of the radioactivity was due to polar 2-MeNap metabolites in both groups. Solvent extraction of liver showed that the greater total amount of 2-MeNap-derived radioactivity in the CoCl₂ treatment group was due to a greater amount of parent compound being present; amount of polar 2-MeNap metabolites in the liver of CoCl₂-treated carp and control carp was similar. Inasmuch as 2-MeNap metabolism in carp is thought to involve monooxygenation in the liver, our results suggest that the altered hepatobiliary disposition of 2-MeNap in CoCl₂-treated carp may be due to depressed 2-MeNap metabolism by liver microsomal enzymes. However, total hepatic cytochrome P-450 content and activity of an associated monooxygenase enzyme (7-ethoxycoumarin-O-deethylase) were similar in CoCl₂-treated and control carp throughout the study. To resolve this paradox (apparent depression of hepatic 2-MeNap metabolism by CoCl₂ but lack of effect on hepatic cytochrome P-450 content and 7-ethoxycoumarin-O-deethylase activity), it is suggested that a minor form of cytochrome P-450 may be responsible for hepatic 2-MeNap metabolism in carp and be selectively inhibited or degraded by CoCl₂. Inherent in this postulate is the assumption that this minor form of cytochrome P-450 does not contribute significantly to total cytochrome P-450 content of carp liver or 7-ethoxycoumarin-O-deethylase activity.Supported by a grant from the University of Wisconsin Sea Grant College Program, USPHS Grant ES 01332, and in part by NIEHS Aquatic Biomedical Center Grant ES 01985, Preliminary results were presented at the Eighteenth Annual Meeting of the Society of Toxicology, New Orleans, Louisiana, March 1979. A portion of this research was conducted at the University of Wisconsin-Madison Biotron, a controlled environmental research facility supported by the University of Wisconsin.     

Effects of ethylene oxide on hepatic microsomal cytochrome P-450: an in vitro study

The effect of ethylene oxide on rat hepatic microsomal cytochrome P-450 was studied in vitro. Cytochrome P-450, but not b5, was decreased by ethanol. When hepatic microsome was bubbled with ethylene oxide gas, cytochrome P-450 content remained unchanged when compared with CO2 bubbling. The addition of NADPH 1 mM also did not change cytochrome P-450 significantly. The excessive high exposure to 500 mM ethylene oxide also did not affect microsomal cytochrome P-450. These results indicate that ethylene oxide does not destroy directly the hepatic microsomal cytochrome P-450 under these conditions.     

Studies on hepatic mitochondrial cytochrome P-450 during Plasmodium yoelii infection and pyrimethamine treatment in mice

Hepatic mitochondrial cytochrome P-450 and b(5) activities were significantly depressed, whereas heme and hemozoin were increased during Plasmodium yoelii infection. Type II, aniline-HCl binding efficacy and polyacrylamide gel electrophoretic profile also depicted impairment of cytochrome P-450 during infection. However, the above alterations were more pronounced in the infected hepatic mitochondria, compared to microsomes. Oral treatment of pyrimethamine (10 mg/kg body weightx4 days) to P. yoelii-infected mice brought the altered levels of mitochondrial and microsomal cytochrome P-450 and b(5) to almost normal, whereas heme and hemozoin levels remained unchanged.     

Effects of lead on drug metabolizing enzymes, cytochrome P-450 and hemeoxygenase in rats

The effects of lead on the drug metabolizing system in liver microsomes and porphyrin metabolism in the bone marrow were studied using male Wistar rats (about 250 g in weight). To study the acute effects of lead, rats were given lead injection intraperitoneally once a day for three consecutive days at a dose of 0 (control), 0.1, 1.0, 10 or 50 mg/kg of lead in the form of lead acetate in a 5% glucose solution. In the 2nd experiment, the chronic effects of lead were studied by administering lead at a dose of 0 (control), 5 and 20 mg/kg once a week for 9 wk for a total of 10 administrations. After the last injection, each rat was fasted for 22 h in a metabolic cage to prevent the animal from eating bed chips or feces and was then sacrificed by decapitation. The rat liver microsome enzymes were used to evaluate the effects of lead on the hepatic functions. In the acute stage, lead decreased the activities of drug metabolizing enzymes, such as aniline hydroxylase and aminopyrine N-demethylase, and decreased the contents of microsomal cytochrome P-450 and cytochrome b5. In the chronic stage, lead decreased the cytochrome P-450 and cytochrome b5 contents and induced hypertrophic liver, but did not affect the activity of aniline hydroxylase. These findings suggest that the rat gradually gained resistance against lead toxicity in the chronic stage. In a supplementary experiment, lead was found to decrease the contents of heme in the microsome and to increase the activity of hemeoxygenase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ornithine alpha-ketoglutarate counteracts the decrease of liver cytochrome P-450 content in burned rats.

The effect of ornithine alpha-ketoglutarate (OKG) on cytochrome P-450 enzyme activities was studied in a well-defined model of injury (burn followed by fasting then subsequent hypocaloric diet) administered to young rats for 3 d. Hepatic microsomes were prepared by ultracentrifugation and levels of cytochromes P-450 were determined spectrophotometrically. The activities of ethoxy-resorufin-O-deethylase (EROD), benzyloxy-resorufin-O-dealkylase (BROD), and erythromycin demethylase were measured as markers of P-450 1A, 2A, and 3A isotypes respectively. The level of total hepatic microsomal proteins (8 mg/mL) remained constant. The level of cytochrome P-450 (1.14+/-0.08 nmol/mg microsomal proteins) was decreased by a hypocaloric diet (23%, P = 0.003) and burn further enhanced this phenomenon (15%, P = 0.03). Both healthy and burned rats receiving OKG showed the same level of cytochrome P-450 as the rats fed ad libitum. OKG supplementation counteracted the enhancement (40%) of EROD activity induced by hypocaloric diet but did not influence BROD and erythromycin demethylase activities. OKG sustained cytochrome P-450 levels in rats fed a hypocaloric diet, even after burning. These findings indicate that OKG may favor drug metabolism in this injured population.     

Preventive effects of phylloquinone on hemorrhagic death induced by butylated hydroxytoluene in male rats.

The effects of vitamin K on hemorrhagic death induced by dietary butylated hydroxytoluene (BHT) were studied. Male Sprague-Dawley rats were given BHT or two phenolic antioxidants (2,4,6-tri-tert-butylphenol and 2,5-di-tert-butylhydroquinone) in combination with a 24% casein basal diet. The levels of the phenols were chosen to nearly equal LD50 (40 days). Hemorrhagic death, hemorrhage and a decrease in prothrombin index caused by 1.20% BHT were prevented by simultaneously adding phylloquinone (0.68 mumole/kg/day). Phylloquinone also inhibited the effect of the related phenolic antioxidants. Ten nanomoles of phylloquinone injected into the femoral vein on day 3 of feeding 1.2% BHT increased the prothrombin concentration from 28% of normal to 100% of normal within 18 to 24 hours. Phylloquinone oxide also prevented hypoprothrombinemia due to BHT. These results suggest that BHT-induced hemorrhagic death may be caused by direct and/or indirect vitamin K deficiency, and its mechanism may be different from those of warfarin.     

Microsomal cytochrome P-450 degradation by in vitro lipid peroxidation

In this study, the influence of in vivo lipid peroxidation (LPO) on cytochrome P-450 (P-450) degradation was investigated using rat liver microsomes. To identify the nature of P-450 degradation, three different perturbant LPO-initiation systems were employed: NADPH/ADP-Fe, cumene hydroperoxide (CHP), and 2,2'-azobis (2-amidino- propane) hydrochloride (AAPH). The results show that each of these systems readily induced P-450 degradation during in vitro LPO and that the progression and extent of the degradation increased with incubation time. However, attempts to elicit P-450 degradation by the use of hydrogen peroxide, superoxide, or hexanal failed to induce damage. Interestingly, the addition of several well-known radical scavengers and radical scavenging enzymes, including superoxide dismutase and catalase, into the incubation media provided little protection against P-450 degradation or malondialdehyde (MDA) formation. It was found, however, that sulfhydryl compounds, including GSH and substrates of P-450-dependent monooxygenases, provided varying degrees of protection. Based on the specificity of protective action, it was concluded that the structural stability of P-450 to defend against LPO requires reduced thiols and/or substrate binding. This suggests that P-450 degradation by LPO is closely related to the oxidation of certain essential thiol groups located at the substrate binding site of the P-450 molecule during LPO reaction.