Stereoselectivity of cytochrome P-450c in the formation of naphthalene and anthracene 1,2-oxides

Absolute configurations of the arene 1,2-oxides formed from napththalene and anthracene by cytochrome P-450c, the predominant isozyme of cytochrome P-450 found in the livers of rats treated with 3-methylcholanthrene, were determined via two different approaches. The first consisted of trapping the arene oxides with N-acetyl-L-cysteine to form S-conjugates, methylation of the conjugates with diazomethane, and separation of the resulting diastereomeric esters by reversed phase high performance liquid chromatography. Analysis by this procedure of the arene oxides formed from radioactive naphthalene and anthracene by a highly purified and reconstituted monooxygenase system containing cytochrome P-450c indicated that 73 and greater than or equal to 95%, respectively, of the metabolically formed arene oxides consisted of the (+)-(1R,2S)-enantiomer. In the second approach, each hydrocarbon was incubated with a reconstituted system containing both cytochrome P-450c and epoxide hydrolase. Under these conditions, the predominant metabolites are trans-1,2-dihydrodiols formed by epoxide hydrolase catalyzed trans-addition of water to the arene oxide intermediates. In both cases, the (-)-(1R,2R)-dihydrodiols predominated; 92% for naphthalene and 99% for anthracene. Enzyme-catalyzed addition of water to (+)- and (-)-anthracene 1,2-oxide and (+)-napthalene 1,2-oxide occurred exclusively (greater than 99%) at the allylic 2-position. The (-)-(1S,2R)-naphthalene 1,2-oxide, however, is converted to a 40:60 mixture of the (-)-(1R,2R)- and (+)-(1S,2S)-dihydrodiols by benzylic and allylic attack, respectively, resulting in increased enantiomeric purity of the dihydrodiol relative to the oxide. Thus, qualitatively and quantitatively both approaches indicate that the (+)-arene (1R,2S)-oxides predominate. The results are discussed in terms of the steric constraints of a proposed model for the catalytic binding site of cytochrome P-450c.     

Differential stereoselectivity on metabolism of triphenylene by cytochromes P-450 in liver microsomes from 3-methylcholanthrene- and phenobarbital-treated rats

Metabolism of triphenylene by liver microsomes from control, phenobarbital(PB)-treated rats and 3-methylcholanthrene(MC)-treated rats as well as by a purified system reconstituted with cytochrome P-450c in the absence or presence of purified microsomal epoxide hydrolase was examined. Control microsomes metabolized triphenylene at a rate of 1.2 nmol/nmol of cytochrome P-450/min. Treatment of rats with PB or MC resulted in a 40% reduction and a 3-fold enhancement in the rate of metabolism, respectively. Metabolites consisted of the trans-1,2-dihydrodiol as well as 1-hydroxytriphenylene, and to a lesser extent 2-hydroxytriphenylene. The (-)-1R,2R-enantiomer of the dihydrodiol predominated (70 to 92%) under all incubation conditions. Incubation of racemic triphenylene 1,2-oxide with microsomal epoxide hydrolase produced dihydrodiol which was highly enriched (80%) in the (-)-1R,2R-enantiomer. Experiments with 18O-enriched water showed that attack of water was exclusively at the allylic 2-position of the arene oxide, indicating that the 1R,2S-enantiomer of the oxide was preferentially hydrated by epoxide hydrolase. Thiol trapping experiments indicated that liver microsomes from MC-treated rats produced almost exclusively (greater than 90%) the 1R,2S-enantiomer of triphenylene 1,2-oxide whereas liver microsomes from PB-treated rats formed racemic oxide. The optically active oxide has a half-life for racemization of only approximately 20 s under the incubation conditions. This study may represent the first attempt to address stereochemical consequences of a rapidly racemizing intermediary metabolite.     

Stereoselective formation of benz[a]anthracene (+)-(5S,6R)-oxide and (+)-(8R,9S)-oxide by a highly purified and reconstituted system containing cytochrome P-450c

The principal oxidative metabolites formed from benz[a]anthracene (BA) by the rat liver microsomal monooxygenase system are the 5,6- and 8,9-arene oxides. In order to determine the enantiomeric composition and absolute configuration of these metabolically formed arene oxides, an HPLC procedure has been developed to separate the six isomeric glutathione conjugates obtained synthetically from the individual enantiomeric arene oxides. Both (+)- and (?)-BA 5,6-oxide gave the two possible positional isomers, but only one positional isomer was formed in each case from (+)- and (?)-BA 8,9-oxide. When [¹⁴C]-BA was incubated with a highly purified and reconstituted monooxygenase system containing cytochrome P-450c, and glutathione was allowed to react with the arene oxides formed, radio-active adducts were formed predominantly (>97%) from the (+)-(5S,6R) and (+)-(8R,9S) enantiomers. The present results are in accord with theoretical predictions of the steric requirements of the catalytic binding site of cytochrome P-450c.     

Selectivity in the binding of hydroxylated benzo[a]pyrene derivatives to purified cytochrome P-450c

The interaction of rat liver microsomal cytochrome P-450c with potential benzo[a]pyrene (BP) metabolites has been compared with the binding of BP by optical and fluorescence spectroscopy. Fluorescence quenching of the phenolic derivatives of BP derives from 1:1 complex formation with P-450c, is a function of the position of the hydroxyl substituent, and correlates with the concomitant increase in high-spin cytochrome observed in parallel optical titrations. The proportion of high-spin cytochrome seen when P-450c was reconstituted in dilauroylphosphatidylcholine vesicles (60 micrograms/mL) ranged from about 7% for the 3- and 7-phenols to 75% for 11- and 12-phenols. BP and all 12 methyl-BP derivatives have comparable high affinities for P-450c (50-70% high spin). Kd determinations with purified P-450c indicated very strong binding of BP phenols that induce high-spin complexes (4-, 5-, 9-, 10-, 11-, and 12-phenols; Kd = 3-25 nM). Inhibition of n-octylamine binding by the 3- and 7-phenols indicated weak interactions (Kd = 80-90 nM), even though low-spin complexes were formed. Inhibition of BP metabolism catalyzed by P-450c with BP phenols correlated with their respective dissociation constants. These results suggest that phenolic substitution at certain positions on BP (1, 2, 3, 7, or 8) interferes with binding to the active site while substitutions at the other positions either enhance or have no effect on binding. BP dihydrodiols [including the (+)- and (-)-BP 7,8-dihydrodiols] were relatively ineffective in forming high-spin complexes (approximately 20%), and fluorescence quenching of dihydrodiols by P-450c also saturated at low levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereoselective metabolism of the (+)-(S,S)- and (-)-(R,R)-enantiomers of trans-3,4-dihydroxy-3,4-dihydrobenzo[c]-phenanthrene by rat and mouse liver microsomes and by a purified and reconstituted cytochrome P-450 system

Metabolism of (+)-, (-)-, and (+/-)-trans-3,4-dihydroxy-3, 4-dihydrobenzo[c]phenanthrenes by liver microsomes from rats and mice and by a purified monooxygenase system reconstituted with cytochrome P-450c has been examined. Bay-region 3,4-diol 1,2-epoxides are minor metabolites of both enantiomers of the 3,4-dihydrodiol with liver microsomes from 3-methylcholanthrene-treated rats or with the reconstituted system (less than 10% of total metabolites). Microsomes from control and phenobarbital-treated rats and from control mice form higher percentages of these diol epoxides (13-36% of total metabolites). Microsomes from 3-methylcholanthrene-treated rats and cytochrome P-450c in the reconstituted system form exclusively the diol expoxide-1 diastereomer, in which the benzylic hydroxyl group and oxirane oxygen are cis to each other, from the (+)-(3S,4S)-dihydrodiol. The same enzymes selectively form the diol expoxide-2 diastereomer, with its oxirane oxygen and benzylic hydroxyl groups trans to each other, from the (-)-(3R,4R)-dihydrodiol (77% of the total diol epoxides). Liver microsomes from control rats show similar stereoselectivity whereas liver microsomes from phenobarbital-treated rats and from control mice are less stereoselective. Three bis-dihydrodiols and three phenolic dihydrodiols are also formed from the enantiomeric 3,4-dihydrodiols of benzo[c]phenanthrene. A single diastereomer of one of these bis-dihydrodiols with the newly introduced dihydrodiol group at the 7,8-position accounts for 79-88% of the total metabolites of the (-)-(3R,4R)-dihydrodiol formed by liver microsomes from 3-methylcholanthrene-treated rats or by the reconstituted system containing epoxide hydrolase. In contrast, the (+)-(3S,4S)-dihydrodiol is metabolized to two diastereomers of this bis-dihydrodiol, a third bis-dihydrodiol, and two phenolic dihydrodiols.     

Metabolism of benzo[c]phenanthrene by rat liver microsomes and by a purified monooxygenase system reconstituted with different isozymes of cytochrome P-450

Metabolism of the environmental pollutant and weak carcinogen benzo[c]-phenanthrene (B[c]Ph) by rat liver microsomes and by a purified and reconstituted cytochrome P-450 system is examined. B[c]Ph proved to be one of the best polycyclic aromatic hydrocarbon substrates for rat liver microsomes. It is metabolized by microsomes from control rats and by rats treated with phenobarbital or 3-methylcholanthrene at 3.9, 4.2 and 7.8 nmol/nmol cytochrome P-450/min, respectively. Principal metabolites are dihydrodiols along with small amounts (less than 10%) of phenols. The K-region 5,6-dihydrodiol is the major metabolite and accounts for 77-89% of the total metabolites. The 3,4-dihydrodiol with a bay-region 1,2-double bond is formed in much smaller amounts and accounts for only 6-17% of the total metabolites, the highest percentage being formed by microsomes from control rats. Highly purified monooxygenase systems reconstituted with cytochrome P-450a, P-450b and P-450c and epoxide hydrolase form predominantly the 5,6-dihydrodiol (95-97% of total metabolites) and only a small percentage of the 3,4-dihydrodiol (3-5% of total metabolites). The 3,4-dihydrodiol is formed with higher enantiomeric purity by microsomes from 3-methylcholanthrene-treated rats (88%) than by microsomes from control rats (78%) or phenobarbital-treated rats (60%). In each case the (3R,4R)-enantiomer predominates. B[c]Ph 5,6-dihydrodiol formed by all three microsomal preparations is nearly racemic.     

Novel stereoselectivity of rat liver cytochrome(s) P450 toward enantiomers of the trans-1,2-dihydrodiol of triphenylene

Metabolism of 3H-labeled (+)-(S,S)- and (-)-(R,R)-1,2-dihydrodiols of triphenylene by rat liver microsomes and 11 purified isozymes of cytochrome P450 in a reconstituted monooxygenase system has been examined. Although both enantiomers were metabolized at comparable rates, the distribution of metabolites between phenolic dihydrodiols and bay-region, 1,2-diol 3,4-epoxide diastereomers varied substantially with the different systems. Treatment of rats with phenobarbital (PB) or 3-methylcholanthrene (MC) caused a slight reduction or less than a twofold increase, respectively, in the rate of total metabolism (per nanomole of cytochrome P450) of the enantiomeric dihydrodiols compared to microsomes from control rats. Among the 11 isozymes of cytochrome P450 tested, only cytochromes P450c (P450IA1) and P450d (P450IA2) had significant catalytic activity. With either enantiomer of triphenylene 1,2-dihydrodiol, both purified cytochrome P450c (P450IA1) and liver microsomes from MC-treated rats formed diol epoxides and phenolic dihydrodiols in approximately equal amounts. Purifed cytochrome P450d (P450IA2), however, formed bay-region diol epoxides and phenolic dihydrodiols in an 80:20 ratio. Interestingly, liver microsomes from control or PB-treated rats produced only diol epoxides and little or no phenolic dihydrodiols. The diol epoxide diastereomers differ in that the epoxide oxygen is either cis (diol epoxide-1) or trans (diol epoxide-2) to the benzylic 1-hydroxyl group. With either purified cytochromes P450 (isozymes c or d) or liver microsomes from MC-treated rats, diol epoxide-2 is favored over diol epoxide-1 by at least 4:1 when the (-)-enantiomer is the substrate, while diol epoxide-1 is favored by at least 5:1 when the (+)- enantiomer is the substrate. In contrast, with liver microsomes from control or PB-treated rats, formation of diol epoxide-1 relative to diol epoxide-2 was favored by at least 2:1 regardless of the substrate enantiomer metabolized. This is the first instance where the ratio of diol epoxide-1/diol epoxide-2 metabolites is independent of the dihydrodiol enantiomer metabolized. Experiments with antibodies indicate that a large percentage of the metabolism by microsomes from control and PB-treated rats is catalyzed by cytochrome P450p (P450IIIA1), resulting in the altered stereoselectivity of these microsomes compared to that of the liver microsomes from MC-treated rats.     

Regulation of three forms of cytochrome P-450 and epoxide hydrolase in rat liver microsomes. Effects of age, sex, and induction

A procedure for the preparation of monospecific antibody directed against rat liver microsomal cytochrome P-45-a is described. This antibody, together with monospecific antibodies to cytochromes P-450b and P-450c, has been used to show that these three forms of cytochrome P-450 are distinct and share no common antigenic determinants. These antibodies (a) give single immunoprecipitin bands with detergent-solubilized microsomes; (b) do not cross-react with the purified heterologous antigens in Ouchterlony double diffusion analyses; (c) have no effect on catalytic activity of the heterologous antigens but completely inhibit the enzymatic activity of the homologous antigens; and (d) remove only the homologous antigen from detergent-solubilized microsomes when covalently bound to a solid support. With radial immunodiffusion assay, we have quantitated these three forms of cytochrome P-450 in liver microsomes after treatment of rats with seven different inducers of cytochrome P-450. The levels of these cytochrome P-450 isozymes vary independently and are also regulated by the age and sex of the animal. The antibodies have also been used to assess the contribution of cytochromes P-450a, P-450b, and P-450c in the metabolism of xenobiotics by rat liver microsomes. A large proportion of benzo(a)pyrene metabolism and testosterone 16 alpha-hydroxylation in microsomes from untreated rats is not catalyzed by cytochromes P-450a, P-450b, and P-450c. Epoxide hydrolase, another microsomal enzyme involved in the metabolism of xenobiotics, was also quantitated by radial immunodiffusion after prior treatment of rats with microsomal enzyme inducers. The inductions of epoxide hydrolase varies independently of the induction of cytochromes P-450a, P-450b, and P-450c.     

Electron-transport cytochrome P-450 system is involved in the microsomal metabolism of the carcinogen chromate

The kinetics of chromate reduction by liver microsomes isolated from rats pretreated with phenobarbital or 3-methylcholanthrene with NADPH or NADH cofactor have been followed. Induction of cytochrome P-450 and NADPH-cytochrome P-450 reductase activity in microsomes by phenobarbital pretreatment caused a decrease in the apparent chromate-enzyme dissociation constant, Km, and an increase in the apparent second-order rate constant, kcat/Km, but did not affect the kcat of NADPH-mediated microsomal metabolism of chromate. Induction of cytochrome P-448 in microsomes by 3-methylcholanthrene pretreatment did not affect the kinetics of NADPH-mediated reduction of chromate by microsomes. The kinetics of NADH-mediated microsomal chromate reduction were unaffected by the drug treatments. The effects of specific enzyme inhibitors on the kinetics of microsomal chromate reduction have been determined. 2'-AMP and 3-pyridinealdehyde-NAD, inhibitors of NADPH-cytochrome P-450 reductase and NADH-cytochrome b5 reductase, inhibited the rate of microsomal reduction of chromate with NADPH and NADH. Metyrapone and carbon monoxide, specific inhibitors of cytochrome P-450, inhibited the rate of NADPH-mediated microsomal reduction of chromate, whereas high concentrations of dimethyl-sulfoxide (0.5 M) enhanced the rate. These results suggest that the electron-transport cytochrome P-450 system is involved in the reduction of chromate by microsomal systems. The NADPH and NADH cofactors supply reducing equivalents ultimately to cytochrome P-450 which functions as a reductase in chromate metabolism. The lower oxidation state(s) produced upon chromate reduction may represent the ultimate carcinogenic form(s) of chromium. These studies provide evidence for the role of cytochrome P-450 in the activation of inorganic carcinogens.     

Metabolism of dichlorobiphenyls by highly purified isozymes of rat liver cytochrome P-450

Hepatic mixed-function oxidase metabolism of the ubiquitous pollutant polychlorinated biphenyls (PCBs) is implicated in their toxification and detoxification. We used dichlorobiphenyls (DCBs) as models to investigate the effect of the chloro substituent sites on this metabolism experimentally and by molecular orbital calculations. Reconstituted, purified cytochrome P-450 PB-B and BNF-B, the major terminal oxidase isozymes of this system, from phenobarbital (PB)- and beta-naphthoflavone (BNF)-induced rats were used to investigate this metabolism. Both isozymes are also induced by PCBs. High-performance liquid chromatography (HPLC) was used to detect, quantify, and isolate metabolites. Metabolite structures were identified by mass spectrometry, dechlorination to identifiable hydroxybiphenyls, and HPLC retention times. All DCBs yielded 3- and 4- but no 2-monohydroxylated metabolites (3,3'-DCB also yielded a dihydroxy metabolite). Di-o-chloro-substituted DCBs were metabolized primarily by cytochrome P-450 PB-B, mono-o-chloro substituted DCBs by both isozymes approximately equivalently, and DCBs without o-chloro substituents by BNF-B primarily. Thus PB-B preferentially metabolizes noncoplanar DCBs and BNF-B coplanar DCBs. The cytochrome isozymes exhibited differing regioselectivities for DCB metabolism - PB-B hydroxylated unchlorinated phenyl rings and BNF-B chlorinated rings. Incorporation of epoxide hydrolase yielded DCB dihydrodiols, and hydroxy metabolite patterns were consistent with those calculated from ring-opened arene oxide intermediates. Thus the rates and regioselectivities of metabolism and thus possibly the toxicity and carcinogenicity of DCBs are dependent on the cytochrome P-450 isozymes induced.     

Oxidative metabolism of the carcinogen 6-fluorobenzo[c]phenanthrene. Effect of a K-region fluoro substituent on the regioselectivity of cytochromes P-450 in liver microsomes from control and induced rats

Oxidative metabolism of the carcinogen 6-fluorobenzo[c]phenanthrene (6-FB[c]Ph) was compared with that of benzo[c]phenanthrene (B[c]Ph) to elucidate the enhancement of carcinogenicity of B[c]Ph by the 6-fluoro substituent. Liver microsomes from untreated (control), phenobarbital-treated, and 3-methylcholanthrene-treated rats metabolized 6-FB[c]Ph at rates of 3.5, 1.5, and 7.7 nmol of products/nmol of cytochrome P-450/min, respectively. The rates of metabolism of B[c]Ph by the same microsomes were 2.9, 1.6, and 5.5 nmol of products/nmol of cytochrome P-450/min, respectively. Whereas the K-region 5,6-dihydrodiol was the major metabolite of B[c]Ph, the major metabolite of 6-FB[c]Ph was the K-region 7,8-oxide, which underwent slow rearrangement to an oxepin. Thus, the 6-fluoro substituent blocks oxidation at the 5,6-double bond and inhibits hydration of the K-region 7,8-oxide by epoxide hydrolase. Substitution with fluorine at C-6 caused an almost 2.5-fold increase in the percentages of the putative proximate carcinogens, i.e. benzo-ring dihydrodiols with bay-region double bonds, when liver microsomes from 3-methylcholanthrene-treated rats were used. Little or no increase was observed in their formation by liver microsomes from control or phenobarbital-treated rats. Interestingly, liver microsomes from control rats formed almost 3-fold as much 3,4-dihydrodiol as isosteric 9,10-dihydrodiol. The R,R-enantiomers of the 3,4- and 9,10-dihydrodiols and the S,S-enantiomer of the 7,8-dihydrodiol were predominantly formed by all three microsomal preparations.     

Effects of a 6-fluoro substituent on the metabolism of benzo(a)pyrene 7,8-dihydrodiol to bay-region diol epoxides by rat liver enzymes

Metabolism of trans-7,8-dihydroxy-7,8-dihydro-6-fluorobenzo(a)pyrene by liver microsomes from 3-methylcholanthrene-treated rats and by a highly purified monooxygenase system, reconstituted with cytochrome P-450c, has been examined. Although both the fluorinated and unfluorinated 7,8-dihydrodiol formed from benzo(a)pyrene by liver microsomes share (R,R)-absolute configuration, the fluorinated dihydrodiol prefers the conformation in which the hydroxyl groups are pseudodiaxial due to the proximate fluorine. The fluorinated 4,5- and 9,10-dihydrodiols are also greater than 97% the (R,R)-enantiomers. For benzo(a)pyrene, metabolism of the (7R,8R)-dihydrodiol to a bay-region 7,8-diol-9,10-epoxide in which the benzylic hydroxyl group and epoxide oxygen are trans constitutes the only known pathway to an ultimate carcinogen. With the microsomal and the purified monooxygenase system, this pathway accounts for 76-82% of the total metabolites from the 7,8-dihydrodiol. In contrast, only 32-49% of the corresponding diol epoxide is obtained from the fluorinated dihydrodiol and this fluorinated diol epoxide has altered conformation in that its hydroxyl groups prefer to be pseudodiaxial. Much smaller amounts of the diastereomeric 7,8-diol-9,10-epoxides in which the benzylic hydroxyl groups and the epoxide oxygen are cis are formed from both dihydrodiols. As the fluorinated diol epoxides are weaker mutagens toward bacteria and mammalian cells relative to the unfluorinated diol epoxides, conformation appears to be an important determinant in modulating the biological activity of diol epoxides. One of the more interesting metabolites of 6-fluorinated 7,8-dihydrodiol was a relatively stable arene oxide, probably the 4,5-oxide, which is resistant to the action of epoxide hydrolase.     

Differential time course of induction of rat liver microsomal cytochrome P-450 isozymes and epoxide hydrolase by Aroclor 1254

The time course of induction of rat liver microsomal cytochromes P-450a, P-450b + P-450e, P-450c, and P-450d and epoxide hydrolase has been determined in immature male rats administered a single large dose [1500 mumol (500 mg)/kg body wt] of the polychlorinated biphenyl mixture Aroclor 1254. Differential regulation of these xenobiotic-metabolizing enzymes was indicated by their characteristic patterns of induction. The rate of induction of cytochrome P-450a and epoxide hydrolase was relatively slow, and steady-state levels of these enzymes were maintained from approximately Days 9 to 15 after Aroclor 1254 treatment. In contrast, cytochrome P-450c was maximally induced 2 days after Aroclor 1254 treatment and remained at a constant level through Day 15. Steady-state levels of cytochrome P-450d, beginning 1 week after Aroclor 1254 treatment, were preceded by a fairly rapid rate of induction and possibly by a small decline from maximal levels observed around Days 4 to 5. Like those of the other cytochrome P-450 isozymes and epoxide hydrolase, the levels of cytochromes P-450b + P-450e were constant from Day 9 to 15 after Aroclor 1254 treatment. However, an unexpected but reproducible decline (approximately 25%) in total cytochrome P-450 content observed between Days 4 and 9 after Aroclor 1254 treatment principally reflected a dramatic and totally unanticipated decrease (approximately 45%) in the level of cytochromes P-450b + P-450e. This transient decline in the level of cytochromes P-450b + P-450e was not due to an unusual effect of a mixture of polychlorinated biphenyls, since identical results were obtained with two individual congeners, namely 2,3,4,5,4'-penta- and 2,3,4,5,3',4'-hexachlorobiphenyl, that induced the same isozymes as Aroclor 1254. In contrast, when rats were treated with 2,4,5,2',4',5'-hexachlorobiphenyl, which induces cytochromes P-450a and P-450b + P-450e and epoxide hydrolase but not cytochromes P-450c or P-450d, maximal levels of cytochromes P-450b + P-450e were attained on Day 4 and no decrease was observed over the next 11 days. These results suggest that there may be an interaction in the regulation of induction of certain individual cytochrome P-450 isozymes.     

Isoenzyme-specific phosphorylation of cytochromes P-450 and other drug metabolizing enzymes

A series of fourteen cytochrome P-450 isoenzymes was treated with three different protein kinases and found to divide into isoenzymes phosphorylated by both the cyclic AMP-dependent kinase and the calcium-phospholipid-dependent kinase (P-450 PB 3a and PB 2e), by none of these kinases (P-450 PB 1b, MC 1b, UT 1, and thromboxane synthase), and by either the cyclic AMP-dependent kinase (P-450 LM 2, PB 2d, and PB 3b) or the calcium-phospholipid-dependent kinase (P-450 PB 1a, PB 2a, MC 1a, LM 3c, and LM 4). Other components of the monooxygenase system, cytochrome P-450 reductase, cytochrome b5, cytochrome b5 reductase as well as microsomal epoxide hydrolase, were poor substrates for the kinases employed. On the other hand, glutathione transferases 1-2 and 4-4, but not 3-3, were relatively good substrates for the calcium-phospholipid-dependent kinase.     

NADPH-cytochrome P-450 reductase from rat liver: purification by affinity chromatography and characterization.

(NADPH)-cytochrome P-450 reductase was purified to apparent homogeneity by a procedure utilizing nicotinamide adenine dinucleotide phosphate (NADP)-Sepharose affinity column chromatography. The purified flavoprotein has a molecular weight of 79 700 and catalyzes cytochrome P-450 dependent drug metabolism, as well as reduction of exogenous electron acceptors. Aerobic titration of cytochrome P-450 reductase with NADPH indicates that an air-stable reduced form of the enzyme is generated by the addition of 0.5 mol of NADPH per mole of flavin, as judged by spectral characteristics. Further addition of NADPH causes no other changes in the absorbance spectrum. A Km value for NADPH of 5 micron was observed when either cytochrome P-450 or cytochrome c was employed as electron acceptor. A Km value of 8 +/- 2 micron was determined for cytochrome c and a Km of 0.09 +/- 0.01 micron was estimated for cytochrome P-450.     

Role of electrostatic interactions in the reaction of NADPH-cytochrome P-450 reductase with cytochromes P-450

Chemical modification of cytochrome P-450 reductase was used to determine the involvement of charged amino acids in the interaction between the reductase and two forms of cytochrome P-450. Acetylation of 11 lysine residues of the reductase with acetic anhydride yielded a 20-40% decrease in the apparent Km of the reductase for cytochrome P-450b or cytochrome P-450c using either 7-ethoxycoumarin or benzphetamine as substrates. A 20-45% decrease in the Vmax was observed except for cytochrome P-450b with 7-ethoxycoumarin as substrate, where there was a 27% increase. Modification of carboxyl groups on the reductase with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) and methylamine, glycine methyl ester, or taurine as nucleophiles inhibited the interaction with the cytochromes P-450. We were able to modify 4.0, 7.9, and 5.9 carboxyl groups using methylamine, glycine methyl ester, or taurine, respectively. The apparent Km for cytochrome P-450c or cytochrome P-450b was increased 1.3- to 5.2-fold in a reconstituted monooxygenase assay with 7-ethoxycoumarin or benzphetamine as substrate. There were varied effects on the Vmax. There was no significant change in the conformation of the reductase upon chemical modification with either acetic anhydride or EDC. These results strongly suggest that electrostatic interactions as well as steric constraints play a role in the binding and electron transfer step(s) between the reductase and cytochrome P-450.     

Induction of different isozymes of cytochrome P-450 and of microsomal epoxide hydrolase in rat liver by 2-acetylaminofluorene and structurally related compounds

The amounts of five different forms of cytochrome P-450 and of microsomal epoxide hydrolase were determined immunochemically in rat liver microsomes before and after treatment of the animals with 2-acetylaminofluorene and 15 structurally related compounds. The amount of cytochrome P-450c was found to be increased about 60-fold after treatment with 2-aminofluorene and 3-aminofluorene. Administration of 1-aminofluorene, 4-aminofluorene, 2-acetylaminofluorene and nitrofluorene increased this isozyme about 15-19 times. 2-Aminofluorene was found to inhibit the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin to a cytoplasmic receptor 50% at a concentration of 3.12 microM, while no such inhibition could be detected with 2-acetylaminofluorene. Induction of ethoxyresorufin O-deethylase activity was found to be highly correlated (+0.95) with the induction of cytochrome P-450c. Also correlated with the induction of this form was the amount of cytochrome P-450d (+0.84), which could be maximally increased about fourfold. Cytochromes P-450b + e were induced by 2-acetylaminofluorene, 4-acetylaminofluorene and fluorene (about tenfold), while 4-aminofluorene and 4-acetylaminofluorene were found to elevate cytochrome P-450PB/PCN-E about threefold. Microsomal epoxide hydrolase was induced by many of the compounds tested, with 2,7-diaminofluorene, 2,7-diacetylaminofluorene, 2-acetylaminofluorene and 2-(N-hydroxy)acetylaminofluorene being the most potent. No correlation of the induction of this enzyme with the induction of any isozyme of cytochrome P-450 was observed.     

Regio- and stereo-selective metabolism of 4-methylbenz[a]anthracene by the fungus Cunninghamella elegans.

Metabolism of 4-methylbenz[a]anthracene by the fungus Cunninghamella elegans was studied. C. elegans metabolized 4-methylbenz[a]anthracene primarily at the methyl group, this being followed by further metabolism at the 8,9- and 10,11-positions to form trans-8,9-dihydro-8,9-dihydroxy-4-hydroxymethylbenz[a]anthracene and trans-10,11-dihydro-10,11-dihydroxy-4-hydroxymethylbenz[a]anthracene. There was no detectable trans-dihydrodiol formed at the methyl-substituted double bond (3,4-positions) or at the 'K' region (5,6-positions). The metabolites were isolated by reversed-phase high-pressure liquid chromatography and characterized by the application of u.v.-visible-absorption-, 1H-n.m.r.- and mass-spectral techniques. The 4-hydroxymethylbenz[a]anthracene trans-8,9- and -10,11-dihydrodiols were optically active. Comparison of the c.d. spectra of the trans-dihydrodiols formed from 4-methylbenz[a]anthracene by C. elegans with those of the corresponding benz[a]anthracene trans-dihydrodiols formed by rat liver microsomal fraction indicated that the major enantiomers of the 4-hydroxymethylbenz[a]anthracene trans-8,9-dihydrodiol and trans- 10,11-dihydrodiol formed by C. elegans have S,S absolute stereochemistries, which are opposite to those of the predominantly 8R,9R- and 10R,11R-dihydrodiols formed by the microsomal fraction. Incubation of C. elegans with 4-methylbenz[a]anthracene under 18O2 and subsequent mass-spectral analysis of the metabolites indicated that hydroxylation of the methyl group and the formation of trans-dihydrodiols are catalysed by cytochrome P-450 mono-oxygenase and epoxide hydrolase enzyme systems. The results indicate that the fungal mono-oxygenase-epoxide hydrolase enzyme systems are highly stereo- and regio-selective in the metabolism of 4-methylbenz[a]anthracene.     

Immunocytochemical evidence for the maintenance of cytochrome P-450 isozymes, NADPH cytochrome C reductase, and epoxide hydrolase in pure and mixed primary cultures of adult human hepatocytes

Specific polyclonal antibodies were used to investigate the distribution of two cytochrome P-450 isozymes (5 and 8), NADPH cytochrome c reductase, and epoxide hydrolase in adult human hepatocytes cultured alone or co-cultured with rat liver epithelial cells. The enzymes were localized by the indirect immunoperoxidase technique following fixation with a paraformaldehyde-glutaraldehyde mixture and membrane permeabilization with saponin. The pattern of distribution of the four enzymes after 24 hr in culture was similar to that found in vivo. Virtually all the hepatocytes exhibited nearly homogeneous positive staining for cytochrome P-450-8, whereas only 60-80% were positive for cytochrome P-450-5. Nearly homogeneous staining was also observed in all hepatocytes for NADPH cytochrome c reductase and epoxide hydrolase. During the first 12 days in pure culture, the intensity of staining, as well as the number of positively stained cells, decreased slightly except for epoxide hydrolase, which did not show any obvious change. In contrast, even after 15 days in co-culture the extent of staining for all the enzymes decreased less than in pure culture. These results indicate that adult human hepatocytes continue to express specific drug-metabolizing enzymes for several days in culture and provide further evidence that those cells are more stable than rodent hepatocytes in primary culture.     

Specificity in the activation and inhibition by flavonoids of benzo[a]pyrene hydroxylation by cytochrome P-450 isozymes from rabbit liver microsomes

The effect of flavone and 7,8-benzoflavone on the metabolism of benzo[a]pyrene to fluorescent phenols by five cytochrome P-450 isozymes obtained from rabbit liver microsomes was determined. Benzo[a]pyrene metabolism was stimulated more than 5-fold by the addition of 600 microM flavone to a reconstituted monooxygenase system consisting of NADPH, cytochrome P-450 reductase, dilauroylphosphatidylcholine, and cytochrome P-450LM3c or cytochrome P-450LM4. In contrast, an inhibitory effect of flavone on benzo[a]pyrene metabolism was observed when cytochrome P-450LM2, cytochrome P-450LM3b, or cytochrome P-450LM6 was used in the reconstituted system. 7,8-Benzoflavone (50-100 microM) stimulated benzo[a]pyrene metabolism by the reconstituted monooxygenase system about 10-fold when cytochrome P-450LM3c was used, but benzo[a]pyrene hydroxylation was strongly inhibited when 7,8-benzoflavone was added to the cytochrome P-450LM6-dependent system. Smaller effects of 7,8-benzoflavone were observed on the metabolism of benzo[a]pyrene by the cytochrome P-450LM2-, cytochrome P-450LM3b-, and cytochrome P-450LM4-dependent monooxygenase systems. These results demonstrate that the activating and inhibiting effects of flavone and 7,8-benzoflavone on benzo[a]pyrene metabolism depend on the type of cytochrome P-450 used in the reconstituted monooxygenase system.