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.     

Increase of a form of UDP-glucuronyltransferase glucuronizing various phenolic xenobiotics and the corresponding translatable mRNA in 3-methylcholanthrene-treated rat liver

Induction of hepatic microsomal UDP-glucuronyltransferase activity toward various phenolic xenobiotics by 3-methylcholanthrene treatment of rats was observed, and the process of the induction was studied. We had previously purified a form of UDP-glucuronyltransferase (called GT-1) having a catalytic activity toward phenolic xenobiotics from liver microsomes of 3-methylcholanthrene-treated rats. The antibodies against GT-1 inhibited the enzyme activity toward those xenobiotics in liver microsomes, and bound to a single protein having a molecular weight of about 54,000 Da (same value as that of GT-1) among microsomal proteins on immunoblotting analysis. The amount of GT-1 protein in hepatic microsomes was found to be increased in close correspondence with the activity increase by 3-methylcholanthrene treatment, by immunoblotting analysis using an uninducible cytochrome P-450 reductase as a negative standard. It was shown by in vitro translation assays that the protein increase described above resulted from the enhancement of the level of translatable mRNA encoding for GT-1. Increases in the amount of the protein immunochemically corresponding to GT-1 in the microsomes from liver of phenobarbital-treated rats and from extrahepatic organs, such as kidney, small intestine, and lung, of phenobarbital- or 3-methylcholanthrene-treated rats were also observed.     

Metabolism of benzo[a]pyrene VI. Stereoselective metabolism of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol to diol epoxides

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-1) and (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-2) are highly mutagenic diol epoxide diastereomers that are formed during metabolism of the carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Remarkable stereoselectivity has been observed on metabolism of the optically pure (+)- and (?)-enantiomers of the dihydrodiol which are obtained by separation of the diastereomeric diesters with (?)-α-methoxy-α-trifluoromethylphenylacetic acid. The high stereoselectivity in the formation of diol epoxide-1 relative to diol epoxide-2 was observed with liver microsomes from 3-methylcholanthrene-treated rats and with a purified cytochrome P-448-containing monoxygenase system where the (?)-enantiomer produced a diol epoxide-2 to diol epoxide-1 ratio of 6 : 1 and the (+)-enantiomer produced a ratio of 1 : 22. Microsomes from control and phenobarbital-treated rats were less stereospecific in the metabolism of enantiomers of BP 7,8-dihydrodiol. The ratio of diol epoxide-2 to diol epoxide-1 formed from the (?)- and (+)-enantiomers with microsomes from control rats was 2 : 1 and 1 : 6, respectively. Both enantiomers of BP 7,8-dihydrodiol were also metabolized to a phenolic derivative, tentatively identified as 6,7,8-trihydroxy-7,8-dihydrobenzo[a]pyrene, which accounted for ～30% of the total metabolites formed by microsomes from control and phenobarbital-pretreated rats whereas this metabolite represents ～5% of the total metabolites with microsomes from 3-methylcholanthrene-treated rats. With benzo[a]pyrene as substrate, liver microsomes produced the 4,5-, 7,8- and 9,10-dihydrodiol with high optical purity (>85%), and diol epoxides were also formed. Most of the optical activity in the BP 7,8-dihydrodiol was due to metabolism by the monoxygenase system rather than by epoxide hydrase, since hydration of (±)-benzo[a]pyrene 7,8-oxide by liver microsomes produced dihydrodiol which was only 8% optically pure. Thus, the stereospecificity of both the monoxygenase system and, to a lesser extent, epoxide hydrase plays important roles in the metabolic activation of benzo[a]pyrene to carcinogens and mutagens.     

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.     

Cleavage of nascent UDP glucuronosyltransferase from rat liver by dog pancreatic microsomes

Antibody to mouse UDP glucuronosyltransferase, previously shown to cross-react with rat transferase [1], immunoadsorbed 3 electrophoretically distinct transferase forms from the microsomes of untreated and phenobarbital-treated rats and 4 forms from 3-methylcholanthrene treated animals. The forms from phenobarbital-treated or control animals ranged in molecular weights from 49,000 to 52,000 daltons, and those from 3-methylcholanthrene-treated rats ranged from 51,000 to 57,000 daltons. The intensity of the electrophoretic bands indicated that the levels of at least two forms were increased by the administration of either compound.In contrast, only a 52,000-dalton electrophoretic band was observed after immunoadsorption of in vitro translated products using poly(A) RNA isolated from either control, phenobarbital-, or 3-methylcholanthrene-treated rats. When dog pancreatic microsomes were included in the in vitro translation assay for either of the poly(A) RNA preparations, part of the 52,000-dalton band remained and a new band of about 50,000 daltons was generated. This processed transferase form(s) appeared to be inserted into or sequestered by the microsomes. These results indicate that some of the electrophoretic variants of rat liver transferase arise by posttranslational modifications and that at least one rat transferase form undergoes proteolytic cleavage of an approximate 2,000-dalton peptide fragment during insertion into the membrane.     

Strain differences of the ability to hydroxylate methotrexate in rats

Converting activity of methotrexate (MTX) to 7-hydroxymethotrexate (7-OH-MTX) was examined using eight strains of rats. Marked variability of the activity was found in liver cytosols from the rats. The highest activity was observed with Sea:SD rats, followed by LEW/Sea and Jcl:Wistar rats. The lowest activity was observed with WKA/Sea rats. The difference in the activity between Sea:SD and WKA/Sea strains was 104-fold. The variation was correlated to the strain difference of benzaldehyde oxidase activity in the rats. The cytosolic 7-hydroxylase activities in other tissues of Sea:SD rats were much higher than those of WKA/Sea, similarly to the case in liver. The liver microsomes of Sea:SD rats exhibited no 7-hydroxylase activity toward MTX even in the presence of NADPH. The cytosolic 7-hydroxylating activity of the livers of Sea:SD rats was inhibited by menadione, β-estradiol, chlorpromazine and disulfiram, inhibitors of aldehyde oxidase, but not oxypurinol, an inhibitor of xanthine oxidase. The purified aldehyde oxidase from the livers of Sea:SD rats exhibited a significant 7-hydroxylating activity toward MTX. However, xanthine oxidase had no ability to hydroxylate MTX. These facts suggest that MTX hydroxylating activity in rats is predominantly due to aldehyde oxidase, and the strain differences are due to the variations of the flavoenzyme level.     

Cytochrome P-455 nm complex formation in the metabolism of phenylalkylamines. IX. A comparative study with enantiomeric 2-nitroso-1-phenylpropane in microsomes and a reconstituted cytochrome P-450 system from rat liver

Formation of cytochrome P-455 nm complexes was investigated with enantiomeric 2-nitroso-1-phenylpropane--the C-nitroso analogue of amphetamine--and optically active N-hydroxyamphetamine, in the presence of NADPH. For comparative reasons, three different drug-metabolizing enzyme systems were used, namely microsomes from control and phenobarbital-treated rats, and a reconstituted system containing the main phenobarbital-induced form of cytochrome P-450 from rat liver. In microsomes obtained from phenobarbital-treated rats, pronounced differences in the kinetics of complex formation were observed between the enantiomeric C-nitroso compounds, but not between the isomers of N-hydroxyamphetamine. In the reconstituted enzyme system the S-nitroso compound formed the P-455 nm chromophore at the highest initial rate, while the R analogue was devoid of complexing activity. The rates of complex formation from the N-hydroxylamine enantiomers were high and equal.     

Aldrin epoxidation catalyzed by purified rat-liver cytochromes P-450 and P-448. High selectivity for cytochrome P-450

Aldrin epoxidation was studied in monooxygenase systems reconstituted from purified rat liver microsomal cytochrome P-450 or P-448, NADPH-cytochrome c reductase, dilauroylphosphatidylcholine and sodium cholate. Cytochrome P-450, purified from hepatic microsomes of phenobarbital-treated rats, exhibited a high rate of dieldrin formation. The low enzyme activity observed in the absence of the lipid and sodium cholate was increased threefold by addition of dilauroylphosphatidylcholine and was further stimulated twofold by addition of sodium cholate. The apparent Km for aldrin in the complete system was 7 +/- 2 microM. SKF 525-A, at a concentration of 250 microM, inhibited aldrin epoxidation by 65%, whereas 7,8-benzoflavone had no inhibitory effect at concentrations up to 250 microM. Addition of ethanol markedly increased epoxidase activity. The increase was threefold in the presence of 5% ethanol. When cytochrome P-448 purified from hepatic microsomes of 3-methylcholanthrene-treated rats was used, a very low rate of epoxidation was observed which was less than 3% of the activity mediated by cytochrome P-450 under similar assay conditions. Enzyme activity was independent of the lipid factor dilauroylphosphatidylcholine. The apparent Km for aldrin was 27 +/- 7 microM. The modifiers of monooxygenase reactions, 7,8-benzoflavone, SKF 525-A and ethanol, inhibited the activity mediated by cytochrome P-448. The I50 was 0.05, 0.2 and 800 mM, respectively. These results indicate that aldrin is a highly selective substrate for cytochrome P-450 species present in microsomes of phenobarbital-treated animals and is a poor substrate for cytochrome P-448. The two forms of aldrin epoxidase can be characterised by their turnover number, their apparent Km and their sensitivity to modifiers, like 7,8-benzoflavone and ethanol.     

Differential oxidase activity of hepatic and pulmonary microsomal cytochrome P-450 isozymes after treatment with cytochrome P-450 inducers.

Phenobarbital, 3-methylcholanthrene, acetone and pyrazole were used as inducers of cytochrome P450 and the NADPH-dependent oxidase activity (O-2 production) of pulmonary and hepatic microsomes was determined. Oxidase activity of microsomes from 3-methylcholanthrene-treated rats was significantly decreased as compared to that of controls when expressed on the basis of cytochrome P450 content (30% decrease for liver, 60% decrease for lung). The oxidase activity of liver microsomes from pyrazole-treated rats showed a significant increase, whereas phenobarbital treated microsomes had average superoxide-generating activity. The contribution of cytochromes CYP 1A, CYP 2B and CYP 2E1 to superoxide-generating activity was investigated using monoclonal antibodies. Monoclonal antibody 1-91-3 against CYP 2E1 inhibited superoxide generation by 58% in liver microsomes from pyrazole-treated rats. Monoclonal antibodies 1-7-1 and 2-66-3 against CYP 1A and CYP2B, respectively, had no effect on superoxide generation. These results indicate that different cytochrome P450 isoforms are mainly responsible for differential superoxide generating activities of microsomes and complement the reconstitution study of Morehouse and Aust. Furthermore, our study indicates that CYP 1A1, induced by 3-MC, demonstrates an unusually low oxidase activity.     

Intralobular distribution of UDP-glucuronosyltransferase in livers from untreated, 3-methylcholanthrene- and phenobarbital-treated rats

A 3-methylcholanthrene-inducible enzyme form of UDP-glucuronosyltransferase has been localized within the liver lobule both immunohistochemically and enzymatically in microdissected centrilobular and periportal liver tissue. Livers of untreated, 3-methylcholanthrene- and phenobarbital-treated rats have been compared. The enzyme was detected in hepatocytes throughout the liver. However both immunohistochemical determination of the enzyme level and biochemical determination of its activity towards 1-naphthol revealed a heterogeneous distribution of the enzyme. In untreated controls and 3-methylcholanthrene-treated rats both enzyme activity and histochemical staining was highest in centrilobular hepatocytes. However, after phenobarbital-treatment enzyme staining and activity was highest in periportal hepatocytes, suggesting that the differentially inducible enzyme activities may be localized in different zones of the liver lobule. The results demonstrate that the 3-methylcholanthrene-inducible UDP-glucuronosyltransferase is preferentially expressed in centrilobular hepatocytes.     

Production of superoxide radical in reductive metabolism of a synthetic food-coloring agent, indigocarmine, and related compounds

Indigocarmine, which is widely used as a synthetic colouring agent for foods and cosmetics in many countries, was reduced to its leuco form and decolorized by rat liver microsomes with NADPH under anaerobic conditions. The reductase activity was enhanced in liver microsomes of phenobarbital-treated rats, and inhibited by diphenyliodonium chloride, a NADPH-cytochrome P450 reductase (P450 reductase) inhibitor, but was not inhibited by SKF 525-A or carbon monoxide. Indigocarmine reductase activity was exhibited by purified rat P450 reductase. In contrast, when indigocarmine was incubated with rat liver microsomes and NADPH under aerobic conditions, superoxide radical was produced and its production was inhibited by superoxide dismutase and diphenyliodonium chloride. When indigocarmine was incubated with purified rat P450 reductase in the presence of NADPH, superoxide radical production was enhanced 17.7-fold (similar to the enhancement of indigocarmine-reducing ability) as compared with that of rat liver microsomes. A decrease of one molecule of NADPH was accompanied with formation of about two molecules of superoxide radical. P450 reductase exhibited little reductase activity towards indigo and tetrabromoindigo, which also afforded little superoxide radical under aerobic conditions. These results indicate that indigocarmine is reduced by P450 reductase to its leuco form, and superoxide radical is produced by autoxidation of the leuco form, through a mechanism known as futile redox cycling.     

Anaerobic dehalogenation of halothane by reconstituted liver microsomal cytochrome P-450 enzyme system

Cytochrome P-450 from liver microsomes of phenobarbital-treated rabbits catalyzed anaerobic dehalogenation of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) when combined with NADPH and NADPH-cytochrome P-450 reductase. Cytochromes P-450B₁ and P-448 from liver microsomes of untreated rabbits were less active. Triton X-100 accelerated the reaction. Unlike anaerobic dehalogenation of halothane in microsomes, the major product was 2-chloro-1,1,1-trifluoroethane and 2-chloro-1,1-difluoroethylene was negligible. These products were not detected under aerobic conditions, and dehalogenation activity was inhibited by carbon monoxide, phenyl isocyanide and metyrapone.     

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.     

The effect of the bay-region 12-methyl group on the stereoselective metabolism at the K-region of 7,12-dimethylbenz[a]anthracene by rat liver microsomes.

The enantiomers of a trans-5,6-dihydrodiol formed in the metabolism of 7,12-dimethylbenz[a]anthracene by rat liver microsomes (microsomal fractions) were resolved by chiral stationary-phase high-performance liquid chromatography. The major 7,12-dimethylbenz[a]anthracene trans-5,6-dihydrodiol enantiomer and its hydrogenation product 5,6,8,9,10,11-hexahydro-trans-5,6-diol were found to have 5S,6S absolute configurations by the exciton chirality c.d. method. The R,R/S,S enantiomer ratios of 7,12-dimethylbenz[a]anthracene trans-5,6-dihydrodiol formed in the metabolism of 7,12-dimethylbenz[a]anthracene by liver microsomes from untreated, 3-methylcholanthrene-treated and phenobarbital-treated male Sprague-Dawley rats were found to be 11:89, 6:94, and 5:95 respectively. These findings and those reported previously on the metabolic formations of trans-5,6-dihydrodiols from 7-methylbenz[a]anthracene and 12-methylbenz[a]anthracene suggest that the 12-methyl group in 7,12-dimethylbenz[a]anthracene plays an important role in determining the stereoselective metabolism at the K-region 5,6-double bond. Furthermore, the finding that formation of 5S,6S-dihydrodiol as the predominant enantiomer was not significantly affected by the isoenzymic composition of cytochrome P-450 present in microsomes prepared from the livers of the rats pretreated with the different inducing agents indicates that the stereoselectivity depends on the substrate metabolized rather than on the precise nature of the metabolizing-enzyme system.     

The proximate carcinogen trans-3,4-dihydroxy-3,4-dihydro-dibenz[c,h]acridine is oxidized stereoselectively and regioselectively by cytochrome 1A1, epoxide hydrolase and hepatic microsomes from 3-methylcholanthrene-treated rats.

Metabolism of the proximate carcinogen trans-3,4-dihydroxy-3,4-dihydrodibenz[c,h]acridine has been examined with rat liver enzymes. The dihydrodiol is metabolized at a rate of 2.4 nmol/nmol of cytochrome P450 1A1/min with microsomes from 3-methylcholanthrene-treated rats, a rate more than 10-fold higher than that observed with microsomes from control or phenobarbital-treated rats. Major metabolises consisted of a diastereomeric pair of bis-dihydrodiols (68-83%), where the new dihydrodiol group has been introduced at the 8,9-position, tetraols derived from bay region 3,4-diol-1,2-epoxides (15-23%), and a small amount of a phenolic dihydrodiol(s) where the new hydroxy group is at the 8,9-position of the substrate. A highly purified monooxygenase system reconstituted with cytochrome P450 1A1 and epoxide hydrolase (17 nmol of metabolites/nmol of cytochrome P450 1A1/min) gave a metabolite profile very similar to that observed with liver microsomes from 3-methylcholanthrene-treated rats. Study of the stereoselectivity of these microsomes established that the (+)-(3S,4S)-dihydrodiol gave mainly the diol epoxide-1 diastereomer, in which the benzylic 4-hydroxyl group and epoxide oxygen are cis. The (-)-(3R,4R)-dihydrodiol gave mainly diol epoxide-2 where these same groups are trans. The major enantiomers of the diastereomeric bis-dihydrodiols are shown to have the same absolute configuration at the 8,9-position. Correlations of circular dichroism spectra suggest this configuration to be (8R,9R). The (8R,9S)-oxide may be their common precursor.     

Reduction of tertiary amine N-oxides by liver preparations: function of aldehyde oxidase as a major N-oxide reductase

Reduction of tertiary amine N-oxides to the corresponding amines by liver preparations was investigated with imipramine N-oxide and cyclobenzaprine N-oxide under anaerobic conditions. Rabbit liver cytosol in the presence of an electron donor of aldehyde oxidase exhibited a significant N-oxide reductase activity which is comparable to the activity of the liver microsomes supplemented with NADPH. Rabbit liver aldehyde oxidase also exhibited the N-oxide reductase activity in the presence of its electron donor, indicating that the activity observed in the liver cytosol is due to this cytosolic enzyme. Furthermore, the tertiary amine N-oxide reductase activity of liver cytosols from rats, mice, hamsters and hogs was demonstrated by comparison with that of liver microsomes from these mammalian species.     

Studies on the microsomal mixed function oxidase system: redox properties of detergent-solubilized NADPH-cytochrome P-450 reductase.

Hepatic microsomal NADPH-cytochrome P-450 reductase was solubilized from rabbit liver microsomes in the presence of detergents and purified to homogeneity by column chromatography. The purified reductase had a molecular weight of 78 000 and contained 1 mol each of FAD and FMN per mol of enzyme. On reduction with NADPH in the presence of molecular oxygen, an 02-stable semiquinone containing one flavin free radical per two flavins was formed, in agreement with previous work on purified trypsin-solubilized reductase. The reduction of oxidized enzyme by NADPH, and autoxidation of NADPH-reduced enzyme by air, proceeded by both one-electron equivalent and two-electron equivalent mechanisms. The reductase reduced cytochrome P-450 (from phenobarbital-treated rabbits) and cytochrome P-448 (from 3-methylcholanthrene-treated rabbits). The rate of reduction of cytochrome P-450 increased in the presence of a substrate, benzphetamine, but that of cytochrome P-448 did not.     

Immunochemical characterization of some monooxygenase activities in liver microsomes from untreated and phenobarbital-treated rats

Two major forms of liver microsomal cytochrome P 450, one from untreated rats (P 450 A₂NI) and the other from phenobarbital-treated rats (P 450 B₂PB), were partially purified. Reconstitution of monooxygenase activities of purified enzymes and inhibition patterns of these activities by antibodies in microsomes gave the following results: 1) aniline hydroxylase activity is mainly supported by cytochrome P 450 A₂NI. This form is the major one in microsomes from control rats, but is also found at minute amounts in microsomes from phenobarbital-treated rats. It behaves as a constitutive form. 2) 4-nitroanisole-and benzphetamine-demethylase activities are mainly supported by cytochrome P 450 B₂PB which is predominant in phenobarbital-treated rats but is also present in control microsomes at low levels. 3) 4-nitroanisole-O-demethylase activity is less specific than benzphetamine-N-demethylase activity towards cytochrome P 450 B₂PB.     

Preparation and characterization of monoclonal antibodies against a form of hamster liver cytochrome P-450 highly specific to aflatoxin B1

Monoclonal antibodies were prepared against a form of cytochrome P-450 (designated as cytochrome P-450-I) purified from 3-methylcholanthrene-treated hamster livers which is highly specific to aflatoxin B1. The cytochrome P-450-I was detected in ELISA and Western blots in liver microsomes from 3-methylcholanthrene-treated hamsters and also from non-treated and phenobarbital-treated hamsters in smaller amounts. However, none of the liver microsomes from 3-methylcholanthrene-treated rat, rabbit, guinea pig and Suncus murinus contained the cytochrome P-450-I. These results suggest that cytochrome P-450-I is specific to hamster and is induced mainly by 3-methylcholanthrene.     

Hepatic catecholestrogen synthases: differential effect of sex, inducers of cytochromes P-450 and of antibody to the glucocorticoid inducible cytochrome P-450 on NADPH-dependent estrogen-2-hydroxylase and on organic hydroperoxide-dependent estrogen-2/4-hydroxylase activity of rat hepatic microsomes

Formation of catecholestrogens (CE) by rat hepatic microsomes was re-examined because as recently shown; (1) CE formation can be catalyzed by an NADPH-dependent estrogen-4-hydroxylase (E-4-H(NADPH)) and by a peroxidatic, organic hydroperoxide-dependent estrogen-2/4-hydroxylase (E-2/4-H(OHP)), in addition to the established NADPH-dependent estrogen 2-hydroxylase (E-2-H(NADPH)); and (2) the indirect radiometric and the COMT-coupled radioenzymatic assays, used in many previous studies, may fail to provide an accurate measure, in particular, of 4-OH-CE. Using a direct product isolation assay, hepatic microsomes of both male and female rats were shown to express E-2/4-H(OHP) activity with properties similar to those of peroxidatic activity in other tissues. The activities of E-2/4-H(OHP) and E-2-H(NADPH) were affected differently by 5 out of 7 inducers of cytochromes P-450 administered in vivo. Phenobarbital and dexamethasone caused a 4- and 2-3-fold increase in E-2-H(NADPH) activity, respectively, but only a 38 and 20% increase in E-2/4-H(OHP) activity. Ketoconazol and beta-naphtoflavone caused a modest increase in E-2-H(NADPH) activity but a decrease in OHP-dependent activity. Clofibrate decreased peroxidatic activity by 50% and NADPH-dependent activity by approximately 20%. Both activities were increased by ethanol but decreased by isoniazide, an agent which induces the same form of cytochromes P-450 as ethanol. Polyclonal antibody against P-450p, a form of P-450 induced by glucocorticoids, inhibited E-2-H(NADPH) but not E-2/4-H(OHP) activity of untreated and of dexamethasone- and phenobarbital-treated rats. This study establishes that CE formation may occur in liver via the peroxidatic pathway and indicates that this pathway depends on forms of P-450 different from those mediating E-2-H(NADPH) activity. It also confirms and extends previous observations of the involvement of multiple, constitutive and induced forms of cytochrome P-450 in NADPH-dependent 2-hydroxylation in liver.