Ring- and N-Hydroxylation of 2-acetylaminofluorene by reconstituted mouse liver microsomal cytochrome P-450 enzyme system

The N- and ring-hydroxylation of 2-acetylaminofluorene (AAF) are examined with a reconstituted cytochrome P-450 enzyme system from liver microsomal fractions from both control and 3-methylcholanthrene (MC)-pretreated mice. Partial purification of cytochrome P-450 fraction is achieved by bacterial protease treatment of microsomes followed by Triton X-100 solubilization and ammonium sulfate precipitation. Both cytochrome P-450 and NADPH-cytochrome c reductase fractions are required for optimum oxidative activity. Hydroxylation activity is determined by the source of cytochrome P-450 fraction; cytochrome P-450 fraction from MC-pretreated mice is several fold more active than that from controls.     

Metabolism of lidocaine by purified rat liver microsomal cytochrome P-450 isozymes

The metabolism of lidocaine was studied using rat liver microsomes or a reconstituted lidocaine monooxygenase system with one of eight forms of cytochrome P-450 purified from liver microsomes from untreated- (P450 UT-2 and UT-5), phenobarbital- (P450 PB-1, PB-2, PB-4, and PB-5) or 3-methylcholanthrene- (P450 MC-1 and MC-5) treated rats. A reverse phase high-performance liquid chromatography system capable of simultaneously assaying four major lidocaine metabolites, namely, monoethylglycinexylidide (MEGX), 3-hydroxylidocaine (3-OH LID), methylhydroxylidocaine (Me-OH LID) and glycinexylidide (GX), was employed to determine the rate of formation of each metabolite. Untreated microsomes generated MEGX, Me-OH LID, and 3-OH LID, but the formation of GX was not detected. In male rat liver microsomes, MEGX was the major metabolite of lidocaine when a concentration of 1 mM was employed. The formation of MEGX and Me-OH LID was increased significantly (P less than 0.01) by microsomes from phenobarbital-treated rats, and the formation of 3-OH LID was increased with 3-methylcholanthrene. The study with the reconstituted system with purified cytochrome P-450 isozymes revealed that all eight forms of cytochrome P-450 used have an ability to N-deethylate lidocaine to form MEGX. Among these isozymes, cytochrome P450 PB-4 and P450 UT-2 showed a higher turnover number for the formation of MEGX. Me-OH LID was formed exclusively by P450 PB-5, and 3-OH LID exclusively by P450 MC-1. Selectivity of cytochrome P450 PB-5 for aromatic methyl hydroxylation of lidocaine was confirmed by an inhibition study; formation of Me-OH LID by microsomes of rats treated with phenobarbital was inhibited completely by antibody against P450 PB-5. It was concluded that different cytochrome P-450 isozymes metabolize lidocaine with a different rate and different position selectivities. Since a specific substrate of cytochrome P450 PB-5 (P-450e) is not known, lidocaine may be a useful substrate for the identification of P450 PB-5.     

Induction of liver microsomal NADPH cytochrome C reductase and cytochrome P-450 by some new synthetic pyrethroids

Newly developed synthetic pyrethroids were evaluated for their ability to alter microsomal cytochrome P-450 and NADPH cytochrome c reductase in rats. Permethrin (80:20 cis-trans), 50 mg/kg/day po, increased cytochrome P-450 after 4, 8, or 12 days of administration and NADPH cytochrome c reductase after 8 or 12 days. A mixture containing less of the cis form (40:60 cis-trans) did not alter either cytochrome P-450 or NADPH cytochrome c reductase after 4 days of administration but increased both after 8 or 12 days. α-Cyano analogs of permethrin (40:60 cis-trans and 97:3 cis-trans) did not induce either cytochrome P-450 or NADPH cytochrome c reductase. None of the preparations altered body weight gain. Permethrin, but not its α-cyano analog, appears to be a weak inducer of the mixed function oxidase system.     

Induction of cytochrome P-450 isozymes by chromanamine derivatives in rat liver

1. Pretreatment of rats with 6-(3-picolyl)amino-2,2,5,8-tetramethylchromane (PATC) for 7 days resulted in a significant increase in the activities of benzphetamine N-demethylase, p-nitroanisole O-demethylase and aniline hydroxylase in liver microsomes prepared 24?h after the last treatment.

2. Analysis by Western blot showed that PATC induces cytochrome P-450 b, P-450 c and P-450 d, which are the major forms of cytochrome P-450 in liver microsomes of rats when pretreated with phenobarbital and 3-methylcholanthrene.

3. Exposure of liver sections to the antibodies to cytochrome P-450 b and P-450 c resulted in intense immunostaining within the centrilobular regions, but produced staining of considerably weaker intensity in the perilobular region. Semiquantitative immunochemical analysis, by image analyser, of cytochrome P-450 b and P-450 c showed that centrilobular hepatocytes were stained more intensively than perilobular hepatocytes.

4. These results indicate that PATC induces cytochromes P-450 b and P-450 c, in the centrilobular hepatocytes to a greater degree than those in the perilobular hepatocytes.

5. Co-administration of PATC with pentobarbital caused a significant increase in pentobarbital sleeping time. Furthermore, PATC was found to cause a decrease in the activity of benzphetamine N-demethylase in liver microsomes prepared 30?min after treatment with the drug.     

Induction of cytochrome P-450 isozymes by chromanamine derivatives in rat liver

1. Pretreatment of rats with 6-(3-picolyl)amino-2,2,5,8-tetramethylchromane (PATC) for 7 days resulted in a significant increase in the activities of benzphetamine N-demethylase, p-nitroanisole O-demethylase and aniline hydroxylase in liver microsomes prepared 24 h after the last treatment. 2. Analysis by Western blot showed that PATC induces cytochrome P-450 b, P-450 c and P-450 d, which are the major forms of cytochrome P-450 in liver microsomes of rats when pretreated with phenobarbital and 3-methylcholanthrene. 3. Exposure of liver sections to the antibodies to cytochrome P-450 b and P-450 c resulted in intense immunostaining within the centrilobular regions, but produced staining of considerably weaker intensity in the perilobular region. Semiquantitative immunochemical analysis, by image analyser, of cytochrome P-450 b and P-450 c showed that centrilobular hepatocytes were stained more intensively than perilobular hepatocytes. 4. These results indicate that PATC induces cytochromes P-450 b and P-450 c, in the centrilobular hepatocytes to a greater degree than those in the perilobular hepatocytes. 5. Co-administration of PATC with pentobarbital caused a significant increase in pentobarbital sleeping time. Furthermore, PATC was found to cause a decrease in the activity of benzphetamine N-demethylase in liver microsomes prepared 30 min after treatment with the drug.     

Induction of liver microsomal cytochrome P-450 2B1 by dimethyl diphenyl bicarboxylate in rats.

Dimethyl diphenyl bicarboxylate (dimethyl-4,4'-dimethyloxy-5,6,5',6'-dimethylene-dioxy-di phe nyl-2,2'- bicarboxylate, DDB), a synthetic mimic of the natural product schizandrin C, is used in China as a hepatoprotective agent to improve the liver functions of patients with hepatitis or under cancer chemotherapy. In this study, we investigated the effects of DDB on liver microsomal drug-metabolizing enzymes. When male Sprague-Dawley rats were treated with a daily intragastric dose of DDB (200 mg.kg-1) for 3 d, the microsomal pentoxyresorufin dealkylase activity and P-450 2B1 protein levels were markedly increased. The fold increase was lower than that by phenobarbital (75 mg.kg-1, ip once daily x 3 d). The level of P-450 2B1 mRNA was elevated by DDB but the magnitude of the elevation was much less than that caused by phenobarbital. DDB also increased the rates of testosterone hydroxylation at positions 16 beta, 16 alpha, 6 beta, and 2 beta as well as the rate of ethoxyresorufin dealkylation, suggesting moderate increases in the levels of P-450 3A and P-450 1A1 in addition to the huge increase in P-450 2B1. The level of glutathione S-transferase was also slightly increased, but the levels of P-450 2E1 and NAD(P)H: quinone oxidoreductase were not changed. The results indicate that DDB is an inducer of P-450 2B1.     

Induction of hepatic microsomal cytochrome P-450 by mirex and kepone.

The chlorinated insecticides, mirex and Kepone, pose a threat to human health as a consequence of their pollution of the environment. We investigated their potential to affect synergistically the toxicity of other xenobiotics and the pharmacological function of drugs by induction of hepatic microsomal enzymes. Male rats were induced by ip injection of mirex (50 or 5 mg/kg/day for 5 days) or Kepone (10 or 1 mg/kg/day for 5 days). Metabolic activity was tested with warfarin and biphenyl using high-performance liquid chromatographic assays. The high doses of both compounds induced cytochrome P-450 with absorbance bands (reduced, CO complex) at 449 nm. Cytochrome concentrations were enhanced twofold relative to controls. Mirex resembled 3-methylcholanthrene and benzo[a]pyrene by inducing formation of 6-hydroxywarfarin but differed in not inducing 8-hydroxywarfarin. Kepone resembled phenobarbital in inducing 7-hydroxywarfarin but differed in its effects on the other metabolites. The low dose of mirex induced higher amounts of 4′-hydroxywarfarin than did the high dose. The metabolite profiles with high and low doses of Kepone also showed marked variations from one another. Mirex and Kepone are carcinogenic in rats and mice but, in contrast to the polycyclic aromatic carcinogens, do not markedly enhance the activity of microsomal biphenyl 2-hydroxylase relative to biphenyl 4-hydroxylase. We conclude that mirex and Kepone induce hepatic mixed-function oxidase profiles which differ from one another and from the classical inducers, phenobarbital and 3-methylcholanthrene. Mirex apparently only induces one of the enzymes induced by 3-methylcholanthrene. The enzyme profiles arising from the insecticides are dose dependent and will thus potentiate qualitatively differing effects depending on the level of ingestion.     

Induction of rat liver microsomal cytochrome P-450 isozymes and epoxide hydrolase by a series of 4'-substituted-2,3,4,5-tetrachlorobiphenyls

We have shown previously that 2,3,4,5-tetrachlorobiphenyl is ineffective as an inducer of rat liver microsomal cytochrome P-450. Addition of a single para-chloro substituent in the otherwise unsubstituted phenyl ring, to give 2,3,4,4',5-pentachlorobiphenyl, produces a potent cytochrome P-450 inducer with both phenobarbital- and 3-methylcholanthrene-type characteristics. In the present study, 2,3,4,5-tetrachlorobiphenyl was substituted in the para(4') position with 12 other functional groups. The 4'-X-C12H5Cl4 derivatives were tested as inducers of cytochromes P-450a--P-450e and epoxide hydrolase, by immunochemical analysis of liver microsomes prepared 4 days after a single treatment (500 mumol/kg) of 1-month-old male Long Evans rats. When the para' substituent was a halogen (F, Cl, Br or I), the derivative induced both cytochromes P-450b and P-450e, and cytochromes P-450c and P-450d, which are the major phenobarbital- and 3-methylcholanthrene-inducible isozymes, respectively. A similar type of induction was observed with a second group of derivatives substituted with CN, NO2 or CF3. However, a derivative containing CH3CO--(which is also a meta-directing, ring-activating substituent) failed to induce cytochromes P-450a-P-450e at the dosage and time tested. Members of a third group of derivatives, which contained an ortho/para-directing, ring-activating substituent) were either ineffective inducers (OH, CH3, CH3O--), or were inducers of cytochromes P-450c and P-450d (isopropyl or t-butyl). Hence, 4'-substitution with a bulky lipophilic substituent conferred 3-methylcholanthrene- but not phenobarbital-type characteristics on 2,3,4,5-tetrachlorobiphenyl. Some of the derivatives tested, namely those substituted with Cl, Br, I and CF3, were remarkably effective inducers of cytochrome P-450a, causing a 10-11-fold induction of this isozyme. Data on the induction of cytochrome P-450c were analyzed by multiparameter linear regression in an attempt to correlate the biological activity of the 4'-X-C12H5Cl4 derivatives with the physiochemical properties of the various substituents. From these results, and those reported recently, we propose that binding of the 4'-X-C12H5Cl4 derivatives to the rat cytosolic Ah receptor is favored by increasing the electronegativity, lipophilicity and hydrogen bonding characteristics of the 4' substituent, whereas enzyme induction (both in vivo and in cultured rat hepatoma cells) is also governed by a fourth characteristic, the STERIMOL factor, which gives a measure of the width of the substituent.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of rat liver microsomal cytochrome P-450 steroid hydroxylase reactions by imidazole antimycotic agents

The imidazole antimycotic agents ketoconazole, miconazole and clotrimazole were tested for their abilities to inhibit the reactions involved in the oxidative metabolism of androst-4-ene-3,17-dione by rat liver microsomal cytochromes P-450. All three compounds were found to function as potent inhibitors of steroid hydroxylase reactions, producing 50% inhibition of 6 beta-, 16 beta-, and 16 alpha-hydroxylase activities at concentrations between 10(-7) and 10(-5) M. The antimycotic agents, when added to liver microsomes, bound to cytochrome P-450 with high affinity to produce a "type II" spectral complex. These agents showed differential inhibition of the various steroid hydroxylases and were found not to affect the activities of the liver microsomal steroid 5 alpha-reductase or the androst-4-ene-3,17-dione 17-oxidoreductase. The results presented demonstrate an interaction of these imidazole antimycotic agents with the various cytochromes P-450 of liver microsomes, resulting in selective inhibition of monooxygenase activity.     

Interspecies homology of liver microsomal cytochrome P-450. A form of dog cytochrome P-450 (P-450-D1) crossreactive with antibodies to rat P-450-male

P-450-male is a male specific form of cytochrome P-450 in rat liver microsomes. Cytochrome P-450 crossreactive with anti-P-450-male antibodies was purified to an electrophoretical homogeneity from liver microsomes of male beagle dogs. The specific content of the purified cytochrome P-450 (P-450-D1) was 16.9 nmol/mg protein. The apparent monomeric molecular weight of P-450-D1 was 48,000, which was smaller than P-450-male (51,000). P-450-D1 showed similarities in spectral properties, N-terminal amino acid sequence, and catalytic activities with some limited exceptions: P-450-D1 did not catalyze 2 alpha-hydroxylation of testosterone and progesterone and catalyzed 21-hydroxylation of progesterone. Based on these results, we propose that P-450-D1 is a form of cytochrome P-450 in the same gene subfamily as P-450-male.     

Cimetidine: an inhibitor and an inducer of rat liver microsomal cytochrome P-450

1. Cimetidine pretreatment of male Sprague-Dawley rats caused a significant increase in the specific content of total hepatic cytochrome P-450, supporting the hypothesis that this H2-receptor antagonist has monooxygenase induction effects. 2. Quantitative ultrastructural studies of liver of cimetidine-pretreated animals also supported this hypothesis in showing a significant proliferation of smooth endoplasmic reticulum. These ultrastructural changes were qualitatively similar to those produced by treatment of rats with phenobarbital, a well-characterized monooxygenase-inducing agent whose effects were studied for comparative purposes. 3. Competitive inhibition of metoprolol alpha-hydroxylation by cimetidine in liver microsomes prepared from untreated animals (Ki = 18.8 microM) was also demonstrated. 4. These results allowed testing of the hypothesis (Burnet et al. 1986) that inhibition of a defined monooxygenase should lead to induction of the synthesis of the relevant cytochrome P-450 isozyme. 5. The finding that metoprolol alpha-hydroxylase activity of liver microsomes was lowered, not elevated, by pretreatment of animals with cimetidine argues against the concept of a causal link between monooxygenase inhibition and induction.     

Purification and characterization of human liver microsomal cytochrome P-450 2A6

Cytochrome P-450 (P-450) 2A6 was purified by chromatography of human liver microsomes. The final preparation was electrophoretically homogeneous and contained 16 nmol of P-450/mg of protein. The amino-terminal amino acid sequence of the protein (first 13 residues) matched that of the reported cDNA exactly. The UV-visible spectrum indicated that the isolated hemoprotein was in the low-spin form. The protein was recognized by rabbit antibodies raised against rat P-450 2A1, and a rabbit antiserum against the P-450 2A6 preparation was also prepared. With these antibodies, it was estimated that P-450 2A6 accounted for a maximum of 1% of the total P-450 present in the human liver microsomes; the level varied greater than 100-fold among the 20 samples examined. Purified P-450 2A6 catalyzed coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation at rates similar to those measured in the human liver sample used to prepare P-450 2A6, and these two microsomal activities were strongly inhibited by the antibodies. The purified P-450 2A6 enzyme also catalyzed low levels of 4,4'-methylene-bis(2-chloroaniline) (MOCA) N-oxidation and activation of aflatoxin B1, 6-aminochrysene, 2-amino-3-methylimidazo[4,5-f]quinoline, and 2-amino-3,5-dimethylimidazo [4,5-f]quinoline to genotoxic products; the antibody inhibited the activity of purified P-450 2A6 towards aflatoxin B1 and 6-aminochrysene but did not inhibit these reactions in human liver microsomes (MOCA N-oxidation was inhibited approximately 20%). Human P-450 2A6 did not catalyze testosterone 7 alpha-hydroxylation, a characteristic activity of the related rat P-450 2A1 protein. These results emphasize the need to characterize individual P-450 enzymes in order to understand their functions in the context of more complex systems.     

Metabolism of diamantane by rat liver microsomal cytochromes P-450

1. Diamantane binds to liver microsomes from phenobarbital-treated rats with an apparent Ks value of 5.2 x 10(-7) mol/l. This value being lower than that obtained for perhydrophenanthrene indicates that diamantane is very strongly bound to microsomal cytochrome P-450. 2. Metabolic studies show that liver microsomes from phenobarbital-treated rats readily metabolize diamantane to mono-, di- and possibly tri-hydroxy derivatives, whereas liver microsomes from beta-naphthoflavone-induced rats do not bind this hydrocarbon or metabolize it. 3. Reconstituted cytochromes P-450 b and e were more efficient in the hydroxylation of diamantane than liver microsomes; metabolites formed by the reconstituted system do not include all the products formed by microsomes, which indicates the involvement of forms of cytochrome P-450 other than the isozymes b and e.     

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.     

Induction of rat hepatic microsomal cytochrome P-450 by 2,3',4,4',5,5'-hexachlorobiphenyl

The following evidence suggests that 2,3',4,4',5,5'-hexachlorobiphenyl resembles isosafrole as an inducer of hepatic microsomal cytochrome P-450d in the immature male Wistar rat. First, the major hepatic microsomal polypeptide (Mr = 52,000), intensified after treatment of rats with 2,3',4,4',5,5'-hexachlorobiphenyl, comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with cytochrome P-450d (i.e. the major isosafrole-inducible polypeptide) but had an electrophoretic mobility intermediate between cytochrome P-450b (Mr approximately equal to 51,500) and cytochrome P-450c (Mr = 56,000) (i.e. the major phenobarbital- and 3-methylcholanthrene-inducible polypeptides respectively). Second, when pairs of various xenobiotics were coadministered to rats at doses effecting maximal induction of hepatic microsomal cytochrome P-450, the inductive effects of 2,3',4,4',5,5'-hexachlorobiphenyl were additive with those of phenobarbital, 3-methylcholanthrene and pregnenolone-16 alpha-carbonitrile but not with those of isosafrole. The inductive effects of phenobarbital, 3-methylcholanthrene, pregnenolone-16 alpha-carbonitrile and isosafrole were all expressed additively with each other. Third, in contrast to phenobarbital and pregnenolone-16 alpha-carbonitrile treatment, treatment of rats with 2,3',4,4',5,5'-hexachlorobiphenyl, isosafrole or 3-methylcholanthrene failed to increase markedly the proportion of total cytochrome P-450 capable of forming a 446 nm-absorbing complex with metyrapone. Fourth, the in vitro metabolism of isosafrole, catalyzed by hepatic microsomes from rats treated with 2,3',4,4',5,5'-hexachlorobiphenyl, isosafrole or 3-methylcholanthrene, produced complexes between ferrous cytochrome P-450 and a methylenedioxyphenyl metabolite, the spectra of which were between 400 and 500 nm and were similar to each other but which were readily distinguishable from the spectra of the product adducts formed during the metabolism of isosafrole by hepatic microsomes from rats treated with corn oil (control), phenobarbital, or pregnenolone-16 alpha-carbonitrile.