Selective Destruction of Cytochrome P-450d and Associated Monooxygenase Activity by Carbon Tetrachloride in the Rat

1. The effects of acute treatment of 3-methylcholanthrene (MC)-induced rats with carbon tetrachloride (CCI₄) on the content and activity of the polycyclic aromatic hydrocarbon-inducible forms of cytochrome P-450 (P-450c and P-450d) in liver and kidney have been determined.

2. Post-treatment of MC-induced rats with CCl₄ in vivo decreased the specific content of total, spectrally determined, P-450 in both hepatic and renal microsomes, by 60% and 40%, respectively. CCl₄ treatment destroyed almost all of the hepatic P-450d (specific content after 6h, <2% of control), but had no effect on P-450c, which increased slightly over the 6h, to 30% above control values.

3. Immunocytochemical measurements demonstrated greater loss of P-450d from the centrilobular and midzonal than from periportal regions of the liver.

4. Hepatic phenacetin O-deethylase, an activity catalysed specifically by P-450d in this tissue, was dramatically decreased following administration of CCl₄ to MC-induced rats. Loss of monooxygenase activity was highly correlated with the decrease in P-450d content (r=0.947, P<0.001). Aryl hydrocarbon hydroxylase activity of liver, catalysed almost entirely by P-450c, was unchanged and neither activity was affected in kidney.

5. Treatment of MC-induced rats with CCl₄ causes a selective loss of hepatic P-450d and associated monooxygenase activities. Phenacetin O-deethylation is catalysed specifically by P-450d in liver, but not in kidney. The mechanism for this destruction of P-450d may be suicide activation of CCl₄, but the rate of such activation appears to be much lower than with P-450b. Alternatively, P-450d may be particularly sensitive, and P-450c particularly resistant, to the active metabolite of CCl₄ diffusing from a distant site of formation.     

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.     

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.     

Amplification of CCl4 toxicity by chlordecone: destruction of rat hepatic microsomal cytochrome P-450 subpopulation.

Previous work has established marked amplification of CCl4 hepatotoxicity by prior exposure to chlordecone (CD). Since CCl4 is toxic by virtue of its bioactivation by the hepatomicrosomal cytochrome P-450 (cyt P-450) system, which is in turn destroyed, our first interest was to determine if cyt P-450 isozymes were selectively destroyed in this interaction. CoCl2 also decreased hepatic P-450 contents, so our other interest was to observe whether CoCl2 selectively decreased or spared CCl4 metabolizing P-450 enzymes. Solubilized hepatic microsomes from variously treated rats were used. The treatment protocol was dietary CD (10 ppm, for 15 d), and CCl4 (100 microliters/kg, ip). The treatments were CD alone, CCl4 alone, CD + CCl4 and with or without CoCl2 (60 mg/kg/d, sc for 2 d) treatment on d 13 and 14 of the dietary protocol. The control group received normal diet and corn oil vehicle. The key mixed-function oxidase (MFO) parameters measured were microsomal protein, cyt P-450 content, and aminopyrine demethylase (APD). Decrease of P-450 levels ranged from 2.2-fold (CD + CCl4) to 1.3-fold (CD + CoCl2). APD activity decreased by 48 and 26.6% in CD + CCl4 and CD + CoCl2 treatments, respectively. Using an anion-exchange high-performance liquid chromatography (HPLC) column, solubilized microsomal hemoproteins were resolved into five peaks. The P-450 content associated with each peak was determined. In CD rats there was slight increase in peak heights, whereas peak heights in CCl4 and control treatments were similar. CoCl2 decreased all peaks, the decrease of peak I being maximal. In CD + CCl4 treatment, absence of peaks II and III was noted. Microsomal proteins stained for heme showed decreased staining intensity of hemo-protein bands, particularly band 4 (MW 52,000), which was absent in CD + CCl4 interaction. These findings suggest that (1) CoCl2 does not selectively decrease or spare any P-450 isozymes and (2) CD + CCl4 interaction does destroy specific P-450 isozymes.     

Structural comparison of monoclonal antibody immunopurified pulmonary and hepatic cytochrome P-450 from 3-methylcholanthrene-treated rats

A pulmonary cytochrome P-450 was purified from lung microsomes of 3-methylcholanthrene (MC)-treated rats by immunoaffinity chromatography using a monoclonal antibody to MC-induced rat liver cytochrome P-450. The isolated pulmonary cytochrome P-450 was MC-inducible and had an apparent molecular weight of 57 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight, as well as the NH2-terminal sequence of the first nine amino acids of the pulmonary cytochrome P-450, was identical to that of an epitopically related MC-induced rat liver cytochrome P-450. In addition, partial proteolysis of both cytochromes P-450 yielded indistinguishable peptide patterns on SDS-Page. Treatment of rats with MC, therefore, induces a pulmonary cytochrome P-450 which is structurally identical to the MC-induced hepatic enzyme by several criteria.     

Effects of starvation on microsomal cytochrome P-450 and laurate-omega-hydroxylation of rat kidney and liver

Cytochrome P-450 (P-450) content and laurate-omega-oxidation activity in rat kidney and liver microsomes were investigated following starvation. Multiple forms of P-450 were analyzed by one dimensional separation using peroxidase stained SDS-continuous gradient polyacrylamide gel electrophoresis. Gels of the hepatic microsomes treated with phenobarbital showed three P-450 bands, and the renal microsomes showed one sharp band, which was induced remarkably by starvation and coincided with the middle molecular form of P-450 from the hepatic microsomes. Since laurate-omega-oxidation activity was induced specifically by starvation but not by drug treatment, in both the kidney and the liver microsomes, the middle molecular form of P-450 might catalyze laurate-omega-oxidation. It seemed, therefore, that a special P-450 subunit catalyzing laurate-omega-oxidation has a greater function in the renal rather than hepatic microsomes because the specific laurate-omega-oxidation activity per starvation induced P-450 content was relatively similar in both the kidney and the liver.     

Role of P-450c in the formation of a reactive intermediate of chlorotrianisene (TACE) by hepatic microsomes from methylcholanthrene-treated rats

A previous study has shown that chlorotrianisene is metabolized by hepatic microsomal cytochrome P-450 monooxygenase(s) to a reactive intermediate that binds covalently to microsomal proteins [Juedes, Bulger, and Kupfer: Drug Metab. Dispos. 15, 786 (1987)]. Covalent binding of chlorotrianisene in hepatic microsomes is dramatically stimulated by treatment of rats with methylcholanthrene (MC), which is known to induce two major P-450 isozymes, P-450c (IA1) and P-450d (IA2). To determine whether P-450c and/or P-450d are involved in catalysis of covalent binding of chlorotrianisene, antibodies to P-450c and P-450d were used. Incubations of chlorotrianisene were conducted with liver microsomes from MC-treated rats (MC microsomes) and a monoclonal antibody (mAb) raised to the major MC-induced isozyme P450c, mAb 1-7-1, or a polyclonal monospecific antibody (pAb) to P-450d, pAb anti-d (-c). At a 5:1 ratio of antibody to microsomal protein, mAb 1-7-1 inhibited covalent binding by 67%, whereas pAb anti d (-c) showed a 10% inhibition. Maximal inhibition by mAb 1-7-1 was 89% at a 100:1 ratio of antibody to microsomal protein. From these findings it was concluded that P-450c is the major isozyme responsible for the metabolism of chlorotrianisene to the covalently binding reactive intermediate in MC microsomes. Additionally, it was observed that potentiation of covalent binding occurred with the noninhibitory mAbs used in these incubations. Substituting bovine serum albumin (BSA) for antibodies showed that this increase in binding is probably due to an increase in acceptor sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Co-induction of cytochrome P-450 isozymes in rat liver by 2,4,5,2',4',5'-hexachlorobiphenyl or 3-methoxy-4-aminoazobenzene

A multitude of xenobiotics have been demonstrated to co-induce either cytochromes P-450c and P-450d or cytochromes P-450b and P-450e in rat hepatic microsomes. Recently, the compounds 2,4,5,2',4',5'-hexachlorobiphenyl (HCB) and 3-methoxy-4-aminoazobenzene (3-MeO-AAB) have been suggested as selective inducers of cytochrome P-450b (Eur. J. Biochem. 151:67 (1985)) and P-450d (Biochem. Biophys. Res. Commun. 133:1072 (1985)), respectively. Since the identification of inducers with such unique characteristics would have implications with regard to the mechanism of induction of all four isozymes, we have examined the induction of cytochromes P-450b and P-450e by HCB and cytochromes P-450c and P-450d by 3-MeO-AAB in liver microsomes from adult male rats. Immunoblot analysis with monoclonal antibodies directed against cytochromes P-450b and P-450e indicate that HCB induces both isozymic species at the three dosage levels examined (10, 90, and 180 mg/kg). Similarly, 3-MeO-AAB does not appear to represent a unique inducer. Immunoblots of hepatic microsomes from animals treated with three different dosage regimens of 3-MeO-AAB demonstrate that, even at the lowest dosage level (50 mg/kg), both cytochromes P-450c and P-450d are induced. Moreover, immunoinhibition of 7-ethoxyresorufin O-deethylase (EROD) activity by monospecific antibody against either cytochrome P-450c or P-450d confirms this result. 3-MeO-AAB increases this enzyme activity 10-fold; approximately one-third of this induced activity is inhibited with monospecific anti-P-450c, while two-thirds is inhibited with monospecific anti-P-450d. This study also demonstrates that hepatic EROD activity is not an accurate estimate of cytochrome P-450c content since the majority of this enzyme activity in control and 3-MeO-AAB-treated rats is inhibited with monospecific anti-P-450d but not with monospecific anti-P-450c.     

In vitro inhibition of hepatic drug oxidation by thioridazine. Kinetic analysis of the inhibition of cytochrome P-450 isoform-specific reactions

The phenothiazine tranquilizer thioridazine has been associated with drug interactions in man. This study investigated the capacity of the drug to inhibit hepatic drug oxidations mediated by cytochromes P-450 (P-450) in microsomes in vitro. Thioridazine was a potent linear mixed-type inhibitor of P-450b-dependent 7-pentoxyresorufin O-depentylase activity in phenobarbital-induced rat liver. The kinetic analysis revealed the enzyme-substrate dissociation constant (Ks) to be 1.6 microM whereas the dissociation constant of the enzyme-inhibitor complex (Ki) was 0.11 microM. In contrast, 7-ethoxyresorufin O-deethylase activity (mediated by P-450c) in beta-naphthoflavone-induced rat hepatic microsomes was inhibited to a lesser extent (Ki = 2.4 microM) in relation to the Ks value (0.5 microM). Spectral studies indicated that the efficiency of thioridazine binding in phenobarbital-induced microsomes was about 25-fold greater than in microsomes from beta-naphthoflavone-induced rat liver. This finding is consistent with the relative capacity of thioridazine to inhibit oxidase activities catalyzed by P-450b and P-450c. Mixed-function oxidase activities catalysed by other P-450s were also inhibited by thioridazine, although to a lesser extent than those catalysed by forms b and c. Thus, the 6 beta- and 16 beta-hydroxylations of androst-4-ene-3,17-dione in hepatic microsomes from untreated rats were inhibited to a similar extent (I50S = 52 and 43 microM, respectively). The 7 alpha- and 16 alpha-hydroxylase pathways were approximately only half as susceptible to inhibition by thioridazine. These findings demonstrate the capacity of thioridazine to inhibit a range of P-450-dependent drug oxidations, with those catalysed by forms b and c most susceptible. The present study strongly suggests that drug interactions elicited by thioridazine are most likely a consequence of inhibitory interactions with P-450 enzymes.     

Carbon tetrachloride changes the activity of cytochrome P450 system in the liver of male rats: role of antioxidants.

The cytochrome P-450 enzymes are responsible for the oxidation of xenobiotic chemicals including drugs, pesticides, and carcinogens. These enzymes include cytochrome P450, cytochrome b(5), arylhydrocarbon (benzo[a]pyrene) hydroxylase (AHH), NADPH-cytochrome C reductase and dimethylnitrosamine N-demethylase I (DMN-dI). Changes in the activities of the above mentioned enzymes were studied in the liver microsomes of rats treated with antioxidants (ascorbic acid (AA), DL-a-tocopherol (vitamin E, VE), garlic) as single- and repeated doses prior to the administration of a single dose of CCl(4). Pretreatment of rats with single doses of AA, VE, or garlic prior to the administration of CCl(4) was found to decrease the hepatic content of cytochrome P450, and the activities of DMN-dI and AHH. On the other hand, these treatments induced the hepatic content of cytochrome b(5) and the activity of NADPH-cytochrome c reductase. Pretreatment of rats with repeated doses of AA, VE, or garlic for 12 consecutive days prior to the administration of CCl(4) as single dose was potentially decreased the activities of cytochrome P450, DMN-dI and NADPH-cytochrome c reductase. Also, the activity of AHH decreased after treatments of rats with repeated doses of garlic prior to the administration of CCl(4). It was noted that repeated doses of antioxidants are more effective than single dose in decreasing the activity of drug-metabolizing enzymes. It is concluded that repeated doses of antioxidants or garlic could reduce the toxic effects exerted by CCl(4) upon the liver, and probably other organs, through inhibition of cytochrome P450 system that activates CCl(4) into its active metabolite, trichloromethyl radical. Moreover, inhibition of cytochrome P450 system could also reduce the toxic and carcinogenic effects of chemical carcinogens such as benzo(a)pyrene and dimethylnitrosamine. The mechanisms of antioxidant protection were discussed in the text.     

Variation in inducibility of cytochrome P-450c and aryl hydrocarbon hydroxylase in rat liver, lung, kidney, pancreas and nasopharynx

The effect of 3-methylcholanthrene (MC) treatment on the cytochrome P-450c content of various rat tissues was examined by measuring the level of immunodetectable P-450c in conjunction with its aryl hydrocarbon hydroxylase (AHH) activity. Immunoblots revealed that P-450d was induced in the nasopharynx and pancreas in addition to its previously reported induction in the liver, lung and kidney. In contrast to P-450c, induction of the immunorelated P-450d was observed only in the liver. The specific content of immunodetected P-450c in the tissue homogenates decreased in the order: liver, nasopharynx, pancreas, lung, kidney. The corresponding AHH activities decreased in the order: liver, kidney, lung, nasopharynx, pancreas. The ratio of AHH activity to P-450c content varied widely among tissues: ratios of 37.2:1.7:0.47:0.04:0.02 were obtained for the kidney, liver, lung, nasopharynx and pancreas, respectively. The absence of a direct relationship between the levels of AHH and P-450c indicates that the extrahepatic activity may partially derive from P-450 forms other than P-450c and/or the specific activity of P-450c varies among different tissues.     

Regulation of renal cytochrome P-450. Effects of two-thirds hepatectomy, cholestasis, biliary cirrhosis and post-necrotic cirrhosis on hepatic and renal microsomal enzymes

The possibility of a relationship between hepatic and renal cytochrome P-450 contents was assessed in rats with liver disease. In rats killed 3 days after two-thirds hepatectomy (a model for hepatocellular insufficiency), the total microsomal cytochrome P-450 content of the whole liver was decreased by 60% as compared to that in control rats; renal cytochrome P-450 was increased by 30% while the 7-ethoxycoumarin deethylase activity of kidney microsomes was increased by 80%. In rats killed 7 days after bile duct ligation (a model for cholestasis) or 35 days after bile duct ligation (a model for biliary cirrhosis), hepatic cytochrome P-450 was decreased by 60% and 45%, respectively, while renal cytochrome P-450 content was increased by 50% and 150%, respectively. In contrast, in rats killed 15 days after the last dose of carbon tetrachloride, 1.3 ml/kg twice weekly for 3 months (a model for post-necrotic cirrhosis), both hepatic and renal cytochrome P-450 contents remained unchanged. Phenobarbital (80 mg/kg daily for 3 days) was a poor inducer of renal cytochrome P-450 in sham-operated rats but became a potent inducer of renal cytochrome P-450 in rats with two-thirds hepatectomy. We conclude that renal cytochrome P-450 is increased in three models in which hepatic cytochrome P-450 contents are decreased (two-thirds hepatectomy, cholestasis and biliary cirrhosis), but remains unchanged in a model of severe liver pathology, in which hepatic cytochrome P-450 content is not modified (late, post-necrotic cirrhosis). The hypothetical role of endogenous inducer(s) is discussed.     

Drug metabolizing activity in rats with chronic liver injury induced by carbon tetrachloride: relationship with the content of hydroxyproline in the liver

The drug metabolizing activity of rat liver during long-term administration of carbon tetrachloride (CCl4), and its relationship with the content of hydroxyproline (Hyp) in the liver were examined. The contents of cytochrome P-450 (P-450) and cytochrome b5 (b5) and the metabolization of aniline, aminopyrine, 7-ethoxycoumarin (7-EC) and benzo(a)pyrene (B(a)P) in the microsomal fraction were examined five days after the final administration of CCl4. The contents of P-450 and b5 and the activity to metabolize the four substrates were gradually reduced as the Hyp content in the liver increased. However, aminopyrine N-demethylation and B(a)P hydroxylation, particularly the latter, was more reduced than aniline hydroxylation and 7-EC O-deethylation in the early stage of hepatic fibrosis. Such differences may be due mainly to the different P-450 subtypes affected by CCl4.     

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.     

Induction of drug-metabolizing enzymes in Gunn rat liver. Effect of polycyclic aromatic hydrocarbons on cytochrome P-450 regulation

Response of congenitally jaundiced rats (Gunn rats) to administration of polycyclic aromatic hydrocarbons (PAH) was investigated and compared to that of Wistar rats. Unlike Wistar, Gunn males did not exhibit changes in the overall cytochrome P-450 content of hepatic microsomes. The first step in the induction process (i.e. presence of cytosolic receptors for PAH) was found present and functionally similar (number of sites, Kd) to that of Wistar rats from which the Gunn strain is derived. An increase in monooxygenase activities related to P-450c and P-450d isoenzymes specifically induced by PAH was noticed, whereas no effect could be detected on the glucuronidation rate of either 4-nitrophenol, testosterone or estrone. As determined by immunoquantification after Western blotting, the isoenzymatic profile of P-450 from PAH-treated male Gunn rats showed an increase of P-450c and P-450d accompanied by an equivalent decrease in P-4502c (major male-specific isoenzyme). The balance between increase in P-450c and P-450d and decrease in P-4502c may explain the absence of increase in the total P-450 in PAH-treated male Gunn rats. Such a response was not observed in PAH-treated male Wistar rats or in female rats of both strains. In contrast, the response of male Gunn rats to PB treatment was similar to that observed in Wistar rats, i.e. increase in overall cytochrome P-450 content of hepatic microsomes and of specific isoenzyme P-450b/e. A possible regulation of P-450 isoenzyme synthesis by the intracellular haem pool might be involved.     

EFFECTS OF MOTORCYCLE EXHAUST ON CYTOCHROME P-450-DEPENDENT MONOOXYGENASES AND GLUTATHIONE S-TRANSFERASE IN RAT TISSUES

The effects of motorcycle exhaust (ME) on cytochrome P-450 (P-450) -dependent monooxygenases were determined using rats exposed to the exhaust by either inhalation, intratracheal, or intraperitoneal administration. A 4-wk ME inhalation significantly increased benzo[a]pyrene hydroxylation, 7-ethoxyresorufin O-deethylation, and NADPH-cytochrome c reductase activities in liver, kidney, and lung microsomes. Intratracheal instillation of organic extracts of ME particulate (MEP) caused a dose- and time-dependent significant increase of monooxygenase activity. Intratracheal treatment with 0.1 g MEP extract/ kg markedly elevated benzo[a]pyrene hydroxylation and 7- ethoxyresorufin O-deethylation activities in the rat tissues 24 h following treatment. Intraperitoneal treatment with 0.5 g MEP extract/ kg/d for 4d resulted in significant increases of P-450 and cytochrome b contents and NADPH-cytochrome c reductase 5 activity in liver microsomes. The intraperitoneal treatment also markedly increased monooxygenases activities toward methoxyresorufin, aniline, benzphetamine, and erythromycin in liver and benzo[a]pyrene and 7-ethoxyresorufin in liver, kidney, and lung. Immunoblotting analyses of microsomal proteins using a mouse monoclonal antibody (Mab) 1-12-3 against rat P-450 1A1 revealed that ME inhalation, MEP intratracheal, or MEP intraperitoneal treatment increased a P-450 1A protein in the hepatic and extrahepatic tissues. Protein blots analyzed using antibodies to P-450 enzymes showed that MEP intraperitoneal treatment caused increases of P-450 2B, 2E, and 3A subfamily proteins in the liver. The ME inhalation, MEP intratracheal, or MEP intraperitoneal treatment resulted in significant increases in glutathione S -transferase activity in liver cytosols. The present study shows that ME and MEP extract contain substances that can induce multiple forms of P-450 and glutathione S-transferase activity in the rat.     

Specific binding of polyhalogenated aromatic hydrocarbon inducers of cytochrome P-450d to the cytochrome and inhibition of its estradiol 2-hydroxylase activity

Treatment of male Sprague-Dawley rats with 3,4,5,3',4',5'-hexabromobiphenyl (HBB) at 10 mumol/kg followed by purification of hepatic microsomal cytochrome P-450d revealed that HBB remained specifically bound to P-450d throughout purification. Binding was noncovalent since HBB was removed by extraction with dichloromethane. Although HBB induced both cytochrome P-450c and P-450d, specific immunoprecipitation of these isozymes from HBB-treated rats showed that HBB was associated only with cytochrome P-450d. Quantitation of HBB and cytochrome P-450d in microsomes from HBB-treated rats suggested a 0.9:1 ratio of HBB to cytochrome P-450d. Five other halogenated aromatic hydrocarbon inducers of cytochrome P-450d, bearing steric similarity to HBB (including 2,3,7,8-tetrachlorodibenzo-p-dioxin), were associated with cytochrome P-450d when used to induce cytochrome P-450d in rats. HBB inhibited estradiol 2-hydroxylase activity of purified cytochrome P-450d in a noncompetitive manner with an I50 of 38 nM for 50 nM P-450d whereas its noncoplanar isomer, 2,4,5,2',4',5'-hexabromobiphenyl, had an I50 over 700-fold higher. Thus certain polyhalogenated aromatic hydrocarbons, with the capacity to induce cytochrome P-450d also bind to the cytochrome when used as inducing agents and inhibit catalytic activity of the cytochrome.     

Induction of rat cytochrome P-450 3 and its mRNA by 3,4,5,3',4',5'-hexachlorobiphenyl

Rat cytochrome P-450 3 (P-450 3) is a constitutive hepatic steroid hormone 7 alpha-hydroxylase which is relatively unresponsive to a number of monooxygenase-inducing agents. The present study demonstrates that a polyhalogenated aromatic hydrocarbon inducer, 3,4,5,3',4',5'-hexachlorobiphenyl (HCB), induces P-450 3 in livers of adult male rats, and that the increase is the result of an increase in the mRNA for this enzyme. Cytochrome P-450 3 and its mRNA were increased more slowly and to a lesser extent than cytochrome P-450c (P-450c) and its mRNA, indicating that these enzymes are not regulated coordinately in liver. The maximum increase in P-450 3 and P-450 3-dependent androstenedione 7 alpha-hydroxylase activity (2- to 3-fold) occurred 7 days after administration of HCB, in contrast to the increase in P-450c (greater than 200-fold) which was maximal by 3-5 days. The rate of induction of P-450 3 mRNA was also slower [maximum increase (9-fold) at 5 days after HCB administration] than that of P-450c mRNA [maximum increase (30-fold) at 2-3 days]. Moreover, a higher dose of HCB was required to produce maximum induction of P-450 3 (50 mg/kg) than that required to produce maximum induction of P-450c (10 mg/kg). P-450 3 was not detected on Western blots of lung, kidney, or prostate microsomes isolated from control or HCB-treated rats (less than or equal to 2% of that found in livers of HCB-treated rats). Moreover, P-450 3-dependent steroid 7 alpha-hydroxylase activity was not detected in these extrahepatic tissues of control or HCB-treated rats (less than or equal to 1% of that found in the corresponding liver microsomes of untreated or HCB-treated rats). In contrast, P-450c was increased dramatically by HCB in lung, kidney, and prostate tissues, indicating differential expression of P-450c and P-450 3 in extrahepatic tissues.     

High affinity phenacetin O-deethylase is catalysed specifically by cytochrome P450d (P450IA2) in the liver of the rat

Phenacetin is metabolized primarily by O-deethylation to paracetamol (POD activity), a reaction catalysed by cytochrome P450. The high affinity component of POD activity is inducible in rat liver by treatment of the animals with polycyclic aromatic hydrocarbons. Following treatment with hydrocarbons such as 3-methylcholanthrene (MC) and isosafrole (ISF) both cytochromes P450c (P450IA1) and P450d (P450IA2) are also induced in rat liver. Studies with the reconstituted enzymes have shown that both forms of P450 catalyse phenacetin O-deethylation at rates that exceeded that of the high affinity component of activity of hepatic microsomal preparations from 3-methylcholanthrene-treated rats (at 4 microM phenacetin: P450c, 440 +/- 40 pmol/nmol/min; P450d, 1030 +/- 10 pmol/nmol/min; microsomal fraction, 163 pmol/mg/min). Specific inhibitory antibodies (both monoclonal and monospecific polyclonal) were used to define the specificity of microsomal POD activity. These studies have shown that hepatic high affinity POD activity is exclusively catalysed by cytochrome P450d in both untreated rats and in rats pretreated with MC.