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We examined the 4'-hydroxylation of flurbiprofen in rat hepatocytes and liver microsomes in order to know whether the metabolism of flurbiprofen is changed on its administration to experimental animals after overnight fasting, because starvation and fasting change both the composition of cytochrome P450s (CYPs) and metabolic activity. CYPs involved in the hydroxylation were determined by various CYP inhibitors and inhibitory antibodies against rat CYP2C11 and CYP2E1 using the microsomes in fasting and feeding. The results provided a possibiliy that the 4'-hydroxylation might be regulated by CYP2C11, but not by CYP2E1, at fasting rather than feeding.

It was demonstrated that trans-stilbene was metabolically activated to the estrogenic compound by rat liver microsomes (Sugihara et al., Toxicol. Appl. Pharmacol., 167, 46-54 (2000)). In this study, determination of the isoforms of cytochrome P450 involved in the oxidation of the proestrogen, trans-stilbene, to its hydroxylated metabolites was examined. When trans-stilbene was incubated with rat liver microsomes in the presence of NADPH, estrogenic compounds, trans4-hydroxystilbene and trans-4,4'-dihydroxystilbene were formed. Comparison of the oxidase activity among liver microsomes of untreated, 3-methylcholanthrene-treated, acetone-treated, clofibrate-treated, dexamethasone-treated and phenobarbital-treated rats toward trans-stilbene showed that those from 3-methylcholanthrene-treated rats exhibited the highest activity. Human liver microsomes also catalyzed the oxidation in varying degrees. Variation in trans-stilbene oxidase activity was closely correlated to that of phenacetin O-deethylase activity. The oxidase activity was inhibited by alpha-naphthoflavone; however, in this case trans-4,4'-dihydroxystilbene was not detected. The oxidase activity toward trans-stilbene was exhibited by recombinant human cytochrome P450 1A1 and 1A2 expressed in a human B lymphoblastoid cell line.

The effect of 4-(4-chlorobenzyl)pyridine (4-CBP) on rat hepatic microsomal cytochrome P450 (P450) and its molecular species (CYP2B1, 2E1, 3A2, 2C11, and 2C12), and on drug-metabolizing enzyme activities were examined in vivo and in vitro. Treatment of rats with 4-CBP resulted in the induction of P450 and drug-metabolizing enzymes in a dose-dependent manner, but it was markedly inhibitory at higher dose levels. Immunoblot analyses revealed that 4-CBP induces both CYP2B1 and 2E1; however, both were decreased by increasing the dose of 4-CBP. The in vitro inhibitory experiment revealed that 4-CBP strongly inhibited benzphetamine N-demethylase activity, but not dimethylnitrosamine N-demethylase activity. The present findings provide information on the induction and inhibition effect of chlorinated benzylpyridine on hepatic microsomal P450s and drug-metabolizing enzymes in vivo and in vitro.

The formation of 7-oxo-delta8-tetrahydrocannabinol (7-oxo-delta8-THC) from 7beta-hydroxy-delta8-THC was found in hepatic microsomes of rats. The activity was stereoselective and about 3-fold higher than that from 7alpha-hydroxy-delta8-THC. The oxidative activity of 7alpha- and 7beta-hydroxy-delta8-THC to 7-oxo-delta8-THC was significantly higher in male than in female, and significantly enhanced by both dexamethasone and phenobarbital, and then inhibited up to about 20% of the control value by antibody against P450GPF-B, presumably a member of the 3A subfamily, a major enzyme responsible for the formation of 7-oxo-delta8-THC in guinea pigs. This antibody also inhibited the formation of 7alpha- and 7beta-hydroxy-delta8-THC, and 7-oxo-delta8-THC from delta8-THC by hepatic microsomes of rats. These results indicate that there is a sex-related difference in the oxidation of 7-hydroxy-delta8-THC to 7-oxo-delta8-THC and the reaction is mainly catalyzed by P450 enzyme(s) belonging to the 3A subfamily as major enzyme(s) of microsomal alcohol oxygenase in rats.

Imipramine (IMI) N-oxidase activity in brain microsomes from rats of both sexes was determined by high performance liquid chromatography, and compared with the results in rat liver microsomes. Brain and liver microsomal IMI N-oxidation was sensitive to thermal inactivation and had an optimal pH at around 9.0. IMI N-oxidase activity (15.54 pmol/min/mg protein) in brain microsomes was about one-hundredth that of liver microsomes (2.08 nmol/min/mg protein) at a substrate concentration of 5 mM. IMI N-oxidase activities in both brain and liver microsomes displayed biphasic kinetics that associated a low Km-low Vmax element with a high Km-high Vmax component. Furthermore, a significant sex difference was observed in Vmax values (male>female) in both phases, but Km values were similar between male and female rats, resulting in a significant sex difference (male>female) in intrinsic clearance values (Vmax/Km) of the low-Km and the high-Km phases.

The stereoselectivity in 4-hydroxylation of bunitrolol (BTL), a beta-adrenoreceptor blocking agent, was examined in liver microsomes from monkeys (marmosets and Japanese monkeys) and compared with the results of human liver microsomes. The formation of (+)-4-OH-BTL from (+)-BTL from (+)-BTL was significantly higher than that of (-)-4-OH-BTL from (-)-BTL in the liver microsomal fractions from the two kinds of monkeys. The 4-OH-BTL-forming activity from racemic BTL was significantly lower than from enantiomeric BTL, indicating a possible metabolic interaction between BTL enantiomers. The in vitro profiles observed in the monkeys were very similar to those in humans, but the stereoselectivity in BTL metabolism [(+)-BTL > (-)-BTL] in the primates was found to be reverse to that in rats [S. Narimatsu et al., Anal. Biochem., 222, 256-261 (1994)]. The 4-OH-BTL-forming activity from BTL enantiomers was significantly suppressed by quinidine and quinine, while the former was more potent than the latter, and also by alpha-naphthoflavone. Furthermore, the activity was also suppressed by antisera against rat cytochromes P450-2D2 and -1A2 in concentration-dependent manners. However, kinetics showed that enantiomeric BTL 4-hydroxylation was monophasic in liver microsomes from marmosets of both genders and from male Japanese monkeys. These results suggest that cytochrome P450-2D and -1A enzymes with similar Km values are involved in BTL 4-hydroxylation in monkey liver microsomes.

Antioxidative inhibition by protoporphyrin (PP) of peroxidative damage in lysosomes, mitochondria and microsomes of rat liver was investigated at 24 h after an intravenous administration of PP. Using a lysosome-containing (3500 x g) fraction, the release of lysosomal marker enzymes, acid phosphatase and aryl sulfatase, from lysosome which had been stimulated by L-ascorbic acid (AsA), was decreased dose-dependently, as was the inhibition of lipid peroxidation by PP in the fraction. Swelling of mitochondria induced by Fe 2+ and AsA was also inhibited in the PP-injected rat. In microsomes, lipid peroxidation stimulated by AsA caused a decrease in activity of a microsomal marker enzyme, glucose 6-phosphatase, and in P450 content. The extent of the decrease by AsA, both in activity and content, was diminished in PP-administered rat liver microsomes. These results indicate that PP protects those subcellular fractions from deterioration by lipid peroxidation.

We isolated a form of cytochrome P450 (P450) from hepatic microsomes of untreated doguera baboons. The final preparation (referred to as P450 BLa) was apparently homogenous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The estimated minimum molecular weight of P450 BLa was 50 kDa. The N-terminal amino acid sequence of P450 BLa (identified 10 residues) was identical with that of P450 3A8 purified from cynomolgus monkeys. This protein was cross-reactive with antibodies raised against P450 3A4 and P450 CMLc which were P450 3A enzymes purified from hepatic microsomes of humans and cynomolgus monkeys, respectively. P450 BLa was capable of catalyzing testosterone 6 beta-hydroxylation and zonisamide reduction. P450 BLa antibody inhibited the activity of testosterone 6 beta-hydroxylase, but not the activities of testosterone 16 alpha- and 16 beta-hydroxylases in liver microsomes of doguera baboons. From these lines of evidence we conclude that P450 BLa can be classified as part of the P450 3A subfamily and acts as a constitutive testosterone 6 beta-hydroxylase in hepatic microsomes of doguera baboons.

The inductive effects of pretreatment with 10 synthetic chloroflavone isomers and congeners (80 mg/kg/d for 3 d, i.p.) on hepatic microsomal monooxygenases were examined with male Wistar rats. All chloroflavone congeners, except 3'-chloroflavone (CF), significantly increased (1.2-to 2-fold) the content of total cytochrome P-450 (P-450s) in microsomes. The effects of these congeners were evaluated based on the activities of microsomal aminopyrine N-demethylase, aniline hydroxylase and scoparone (6, 7-dimethoxycoumarin) O-demethylase, and the CO difference spectrum and sodium dodecyl sulfate-gel electrophoretogram of cytochrome P-450s. The results were compared with those of phenobarbital (PB), 3-methylcholanthrene (MC) and PB plus MC. Based mainly on effects on the CO difference spectrum and ratio of the two O-demethylase activities of scoparone, 2'-CF and 2', 4'-dichloroflavone (DCF) were categorized as the PB-type, 4'-CF and 6, 8-DCF as the MC-type, 3', 6-DCF and 3', 5', 6-trichloroflavone as weak MC-type, and 6-CF and 2', 6-DCF as mixed (PB plus MC)-type inducers. A comparison of chloroflavone isomers and congeners indicated that (1) the presence of the 2'-chloro substituent of the flavone system is a minimal requirement for exhibiting the PB-type effect, (2) the absence of the 2'-chloro substituent is possibly that for exhibiting the MC-type effect and (3) the induction potencies by individual chloroflavone congeners may not be related to the degrees of chlorination.

The effect of glutathione depletor diethylmaleate on rat hepatic glutathione S-transferase and glutathione peroxidase was studied in vivo and in vitro. When diethylmaleate (600 mg/kg) was given i.p. to rats, liver glutathione was depleted within 2 h and recovered to the control level 5 h after diethylmaleate treatment. Both glutathione S-transferase and peroxidase activities in microsomes, not in cytosol, were markedly increased during glutathione depletion and only glutathione S-transferase activity remained at high levels after recovery of the glutathione content. The increase in microsomal glutathione S-transferase and peroxidase activities with concomitant exhaustion of glutathione was also observed by perfusion of the isolated liver with diethylmaleate (10 mM). When liver microsomes were incubated with diethylmaleate in vitro at 37 degrees C, glutathione S-transferase, but not peroxidase, activity was increased; the increase was not reversed by dithiothreitol. These results indicate that diethylmaleate activates microsomal glutathione S-transferase by direct reaction to the enzyme during glutathione depletion and suggest that glutathione S-transferase activity and glutathione peroxidase activity in the microsomal enzyme may be differently regulated.

The apparent in vitro kinetic constants of uridine diphosphate-glucuronyltransferase (UDP-GT) activities towards E6080, 1-naphthol (1-N) and 4-hydroxybiphenyl (4-HB) were determined using microsomes, to assess the effect of inducing agents and evaluate species and tissue differences. In rats, the 3-methylcholanthrene and beta-naphthoflavone treatments increased the Vmax app/KM app values for E6080, 13- and 8-fold, and those for 1-N, 1.9- and 1.7-fold, respectively, but did not affect those for 4-HB. Phenobarbital was ineffective on the UDP-GT activity toward E6080. In rats and rhesus monkeys, the intestinal mucosa had higher specific UDP-GT activity toward E6080 than did the liver, and the rank order of the Vmax app/KM app value for E6080 in the intestinal mucosa was rhesus monkey > rat > guinea pig > beagle. In guinea pig, the Vmax app/KM app value for E6080 in the liver was 4 times higher than that in the intestinal mucosa. Both the Vmax app/KM app values for 1-N and 4-HB in the liver of all species studied were higher than those in the intestinal mucosa, and guinea pig liver showed the highest values, while, about the Vmax app for 1-N and 4-HB, beagle liver had the highest values. In the beagle intestinal mucosa, the Vmax app/KM app values for all 3 substrates studied were extremely low.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of two polychlorinated biphenyls, 3, 4, 3', 4'- and 2, 5, 2', 5'-tetrachlorobiphenyl (TCB), was studied using rat liver microsomes and the four forms of cytochrome P-450 (P-450), P-450b, P-450e, P-450c and P-450d. At first, effects of various inducers of P-450 such as phenobarbital (PB), 3-methylcholanthrene (MC), isosafrole (ISF) and pregnenolone 16α-carbonitrile on the formation of metabolites of these TCBs by liver microsomes were compared. 3, 4, 3', 4'-TCB was significantly metabolized by liver microsomes from MC-treated rats to form two previously reported metabolites, 4-hydroxy-3, 5, 3', 4'-TCB and 5-hydroxy-3, 4, 3', 4'-TCB with a relative ratio of 2.5 : 1. Incubation with microsomes from untreated or PB-treated rats produced none of the metabolites. On the other hand, 2, 5, 2', 5'-TCB was metabolized to 3-hydroxy-2, 5, 2', 5'-TCB most easily by liver microsomes from PB-treated rats and at a moderate rate by liver microsomes from ISF-treated rats. Activities of microsomes from untreated or MC-treated rats to hydroxylate 2, 5, 2', 5'-TCB were low or undetectable. When these TCB hydroxylase activities were examined with a reconstituted system consisting of each P-450, reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase, dilauroylphosphatidylcholine and NADPH-generating system, only P-450c catalyzed both the 4-and 5-hydroxylations of 3, 4, 3', 4'-TCB at a ratio of 2.2 : 1. On the contrary, the hydroxylation of 2, 5, 2', 5'-TCB proceeded efficiently with P-450b and P-450e, being more efficient with the former. P-450d did not show any catalytic activity toward 3, 4, 3', 4'-TCB and 2, 5, 2', 5'-TCB. These results indicated that P-450c and P-450b play a major role in the metabolism of 3, 4, 3', 4'-TCB in MC-treated microsomes and of 2, 5, 2', 5'-TCB in PB-treated microsomes, respectively. The inhibition studies using antibodies against P-450c and P-450b further supported the above conclusions.

The metabolism of strychnine was studied with hepatic microsomes of rats, mice, guinea pigs, rabbits and dogs, using high-performance liquid chromatography. Eight metabolites were found by the incubation with rabbit liver microsomes. Five of them were identified as strychnine N-oxide (St N-oxide), 2-hydroxystrychnine (2-OH St), strychnine 21,22-epoxide, 16-hydroxystrychnine (16-OH St) and 18-oxostrychnine by comparing the chromatographic and spectral data to those of authentic samples. Significant differences in metabolic profiles of strychnine were observed among the above species. The main metabolite in rats and mice was 16-OH St, while in guinea pigs and rabbits, and in dogs, it was 2-OH St, and St N-oxide, respectively. The metabolic activity in guinea pig liver microsomes was much higher than those of other species. There seems to be a fairly good inverse correlation between the metabolic activity and strychnine toxicity in guinea pigs and other animal species.

Androst-4-ene-3,6-dione derivatives 2-4 and 3 alpha-methoxy-4-en-6-one steroid 7 were prepared and tested for their ability to inhibit aromatase in human placental microsomes. The 16 alpha-bromide 2, the 16 alpha-alcohol 3, and the 3 alpha-methoxide 7 of this series were effective competitive inhibitors of aromatase with apparent Ki's of 150 nM, 1.18 microM, and 700 nM. Compound 2 caused a time-dependent, biphasic loss of aromatase activity in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) while compound 7 caused a time-dependent, pseudo-first order inactivation of the activity, with kinact's of 0.417 and 0.036 min-1 for compounds 2 and 7. NADPH and oxygen were required for the time-dependent inactivation and the substrate, androst-4-ene-3,17-dione, prevented it in each case.

The hepatic microsomal metabolism of cocaine and the pharmacological effects of some metabolites were studied in experimental animals. Hepatic microsomes from mice, rats and guinea pigs catalyzed the oxidation of cocaine to m- and p-hydroxycocaines. Only trace amounts of m-hydroxycocaine were detected in the extract of the incubation mixture with rabbit hepatic microsomes. The total amount of the two hydroxycocaines was less than 12% that of norcocaine which was the most predominant microsomal metabolite in all the animals species examined. The administration of p-hydroxycocaine (20 mg/kg i.p.) to mice significantly increased locomotor activity (total distance and number of rearing movements). The effect of p-hydroxycocaine was more active or comparable with that of cocaine, indicating that this metabolite is an active metabolite of cocaine.

The influence of aging on the reductase activity of acetohexamide, an oral antidiabetic drug with a ketone group, was examined in liver microsomes and cytosol of male rats. Acetohexamide reductase activities in liver microsomes of male rats at 26 and 31 months of age were much lower than that in liver microsomes of male rats at 9 weeks of age. Testectomy markedly decreased acetohexamide reductase activity in liver microsomes of the 9-week old rats and the decreased enzyme activity was significantly increased by testosterone administration. These results indicate, at least in part, that aging decreases the enzyme activity by decreasing the secretion of testosterone from the testes. On the other hand, aging (26 months of age) did not affect acetohexamide reductase activity in liver cytosol of male rats, although the enzyme activity at 31 months of age was slightly but significantly lower than that in liver cytosol of male rats at 9 weeks of age. Testectomy or testosterone administration had no effect on the enzyme activity in liver cytosol of 9-week old male rats.

The tissue and subcellular distributions of 14C-2,3,4,7,8-pentachlorodibenzofuran (PenCDF), one of the most important causal agents of yusho, were studied using rats. More than 60% of the radioactivity given orally was accumulated in the liver after 5 d and this high percentage persisted over a period of 3 weeks. Subcellular fractionation of the liver homogenate showed unusual separation by PenCDF-pretreatment, but the distribution of radioactivity was just parallel to those of cytochrome P-450 content and glucose-6-phosphatase (EC3.1.3.9) activity. Gas chromatographic analysis provided evidence that the extracts from the liver and its subcellular fractionations contained only unchanged PenCDF. Those results strongly suggest that PenCDF has some affinity to endoplasmic reticulum of rat liver.

A simple assay method for testosterone hydroxylase activity by thin-layer chromatographyultraviolet spectrophotometry was developed. By using this method, the effects of various H2-receptor antagonists (cimetidine, CIM ; metiamide, MET ; ranitidine, RAN ; famotidine, FAM) or imidazole (IMZ) on the hydroxylations of testosterone by mouse liver microsomal enzymes were studied in vitro. CIM and MET inhibited the 6β-, 7α-and 16α-hydroxylations in a dose-dependent manner. RAN and FAM had little inhibitory effect on any hydroxylation. IMZ inhibited the 6β-hydroxylation to the same degree as CIM and MET, while the effects on the 7α-and 16α-hydroxylations were very weak. The kinetic data indicated that these drugs inhibited the three hydroxylation reactions competitively and the affinities for each hydroxylase varied from drug to drug. The results suggest that the inhibitory actions of CIM or MET on the hydroxylations of testosterone are due to the imidazole ring structure, and the side chain structures may play a role in the enhancement of affinity to the enzymes, particularly the 7α-and 16α-hydroxylases. On the other hand, RAN and FAM containing a ring structure different from imidazole had little effect on any testosterone hydroxylase.

Renal Microsomal N-nitrosodimethylamine (NDMA) N-demethylation was measured in several species by a sensitive radiometric method using [14C] NDMA as a substrate and 2, 4-dinitrophenylhydrazine (DNPH) as a trapping agent of the [14C] formaldehyde formed. The activities were the highest in mice and lowest in hamsters. The activities in rats could not be detected. Among several strains of mice studied the DDY strain was the highest in its activities. Although nicotinamide adenine dinucleotide supported NDMA demethylation by about 32% of nicotinamide adenine dinucleotide phosphate in kidney and only 16% in liver, other properties (pH profiles, km values, and effects of inhibitors) exhibited almost similar results in liver as compared to kidney.

The effect of protoporphyrin IX (PP) on lipid peroxidation was investigated in rat liver homogenates, in hepatic subcellular organelles such as mitochondria and microsomes, and in a mixture of unsaturated fatty acids. PP inhibited both the Fe2+ and ascorbic acid (AsA)-stimulated lipid peroxidation and the concomitant increase of oxygen consumption in the homogenates. PP failed to inhibit auto-oxidation in the mixture of unsaturated fatty acids. PP inhibited the lipid peroxidation both in mitochondria stimulated by AsA and in microsomes stimulated by AsA or a reduced nicotinamide adenine dinucleotide phosphate-generating system. Its antioxidative effects were less pronounced in the heat-denatured subcellular fractions. Soluble fraction (SF) enhanced the antioxidative action of PP on the lipid peroxidation in boiled microsomes, but boilde SF lessened the action of PP.These findings indicate that PP has an antioxidative effect on lipid peroxidation in liver, and that the antioxidative effect of PP is mediated by factors present in liver, some of which are heat-labile and some heat-stable.

When estradiol 17-sulfate was incubated with rat liver microsomes in the presence of a reduced nicotinamide adenine dinucleotide phosphate (NADPH)-generating system, 4-hydroxyestradiol 17-sulfate was obtained as a minor product accompanying the major metabolite, 2-hydroxyestradiol 17-sulfate. This 4-hydroxylation was shown to occur without cleavage of the conjugate group. At the substrate concentration of 200 μM, which is about twice the Km value of estradiol 17-sulfate 2-hydroxylase, the amounts of 2-and 4-hydroxylated products formed by liver microsomes from male rats were 15.03±1.43 and 0.23±0.01%, respectively. Analogous results were obtained with microsomes from female rats under the same conditions : the yields of 2-and 4-hydroxylated metabolites were 8.75±0.97 and 0.14±0.02%, respectively. Thus, the relative ratios of 2-and 4-hydroxylation of estradiol 17-sulfate were approximately 60 : 1 in both sexes. A synthesis of the authentic conjugate, 4-hydroxyestradiol 17-sulfate, and a method for its assay by high-performance liquid chromatography with an electrochemical detector are also described.

The β-glucosidation and β-glucuronidation of pantothenic acid (PaA) in dog liver microsome were compared with those of p-nitrophenol. PaA underwent β-glucosidation in the presence of uridine diphosphate glucose (UDPG). However, p-nitrophenol did not conjugate β-glucose but underwent β-glucuronidation in the presence of uridine diphosphate glucuronic acid (UDPGA). The UDP-glucuronyltransferase effect on p-nitrophenol was 1000 times larger than the UDP-glucosyltransferase effect on PaA. But no glucuronidation of PaA occurred. The optimum pH for the glucosidation of PaA was 8.5. These results indicate that PaA has a high specificity for β-glucosidation, and that β-glucosidation is an essential pathway in the metabolism of PaA in dogs.

The mechanism of species difference in N-hydroxyphenacetin mutagenicity was investigated by using the Salmonella/microsome mutagenicity test and high-performance liquid chromatographic (HPLC) analysis. Mutagenicity of N-hydroxyphenacetin in Salmonella typhimurium TA100 was about 10 times more efficiently detected with a liver 9000g supernatant fraction (S9) from hamsters than with the corresponding fraction from rats in the absence of a reduced nicotinamide adenine dinucleotide phosphate-generating system. Paraoxon and sodium fluoride, both inhibitors of microsomal amidase, not only inhibited the mutagenicity of N-hydroxyphenacetin, but also retarded the formation of the deacetylation product, p-nitrosophenetole, a strong intrinsic mutagen. The N-hydroxyphenacetin-deacetylation activity was about 20 times higher in liver microsomes from the hamster than in those from the rat.

The acetohexamide reducing activity in hepatic 10,000 X g supernatant was significantly higher in male than in female rats. Evidence obtained in this study suggests that the microsomal carbonyl reductase may contribute to the sex difference in the reductive metabolism of acetohexamide in rats.