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Kinetics of testosterone 6beta-hydroxylation were determined using a reconstituted system that consisted of CYP3A4, cytochrome b5 and NADPH-cytochrome P450 oxidoreductase (OR) with similar ratios as those seen in human liver microsomes and compared with those determined using human liver microsomes. Two reconstituted systems were constructed in accordance with two human liver microsomal samples that showed extremely high and low ratios of OR/CYP3A4. The Km values of testosterone 6beta-hydroxylation obtained from the reconstituted systems with high and low OR/CYP3A4 ratios were 29.3 and 35.2 microM, respectively, which were similar to that of the corresponding human liver microsomal samples (23.2 and 40.0 microM, respectively). However, Vmax values obtained from the reconstituted systems (3.7 and 0.8 pmol/min/pmol CYP3A4) were much lower than those from the human liver microsomes (44.2 and 31.1 pmol/min/pmol CYP3A4). The results suggest that the interaction between substrate and CYP3A4 in the reconstituted systems appear to be similar to human liver microsomes but that the velocity of the substrate metabolism in the reconstituted systems is different from that in human liver microsomes. In conclusion, our reconstituted systems could be used for the determination of affinity but not for the determination of the maximum velocity of substrate metabolism. Further studies on the protein-protein interactions between CYP3A4, OR, cytochrome b5 and/or a specific lipid environment are required to establish a reconstituted system showing similar kinetic properties to those of human liver microsomes.

Aims: To determine the cytochrome P450 (CYP) isoforms involved in the oxidation of propofol by human liver microsomes., Methods: The rate constant calculated from the disappearance of propofol in an incubation mixture with human liver microsomes and recombinant human CYP isoforms was used as a measure of the rate of metabolism of propofol. The correlation of these rate constants with rates of metabolism of CYP isoform-selective substrates by liver microsomes, the effect of CYP isoform-selective chemical inhibitors and monoclonal antibodies on propofol metabolism by liver microsomes, and its metabolism by recombinant human CYP isoforms were examined., Results: The mean rate constant of propofol metabolism by liver microsomes obtained from six individuals was 4.2 (95% confidence intervals 2.7, 5.7) nmol min(-1) mg(-1) protein. The rate constants of propofol by microsomes were significantly correlated with S-mephenytoin N-demethylation, a marker of CYP2B6 (r = 0.93, P < 0.0001), but not with the metabolic activities of other CYP isoform-selective substrates. Of the chemical inhibitors of CYP isoforms tested, orphenadrine, a CYP2B6 inhibitor, reduced the rate constant of propofol by liver microsomes by 38% (P < 0.05), while other CYP isoform-selective inhibitors had no effects. Of the recombinant CYP isoforms screened, CYP2B6 produced the highest rate constant for propofol metabolism (197 nmol min-1 nmol P450-1). An antibody against CYP2B6 inhibited the disappearance of propofol in liver microsomes by 74%. Antibodies raised against other CYP isoforms had no effect on the metabolism of propofol., Conclusions: CYP2B6 is predominantly involved in the oxidation of propofol by human liver microsomes.

Background/aims: Although the activity of the liver in metabolizing and eliminating various drugs decreases in endotoxemia, the mechanism remains to be elucidated. The generation of nitric oxide by the inducible type of nitric oxide synthase increases in endotoxemia. Nitric oxide readily reacts with heme proteins such as cytochrome P450 that metabolize various compounds, including steroids and eicosanoids. The purpose of this study was to determine the effect of nitric oxide on the function of hepatic cytochrome P450 in endotoxemic rats., Methods: To determine the dynamic aspects of nitric oxide metabolism, hepatic levels of the inducible type of nitric oxide synthase and heme-iron nitrosyl complexes, and plasma levels of nitrite and nitrate were determined in rats before and after intravenous administration of lipopolysaccharide. Changes in the levels of P450 isoforms and testosterone hydroxylation activity in hepatic microsomes were also determined. To evaluate in vivo CYP3A2 activity, midazolam sleep time was measured., Results: When lipopolysaccharide increased the hepatic inducible type of nitric oxide synthase and plasma levels of nitric oxide metabolites, the intensity of low-spin signal of electron spin resonance responsible for the ferric form of P450 decreased with a concomitant increase in heme-iron nitrosyl complexes in the liver. Lipopolysaccharide-related nitric oxide generation is followed by an early decrease in the levels of cytochrome P450 and of testosterone hydroxylation activity in liver microsomes. Midazolam sleep time was prolonged by lipopolysaccharide. All these early changes were prevented by the inhibitor of nitric oxide synthase, N(G)-iminoethyl-L-ornithine. Moreover, lipopolysaccharide suppressed the gene expression of CYP2C11 and CYP3A2. Decreases in levels of cytochrome P450 and their mRNAs were more pronounced at 24 h after LPS administration, but apparently they are NO-independent., Conclusions: These results suggest that lipopolysaccharide-induced modulation of cytochrome P450 may occur via the interplay of two different mechanisms and that, especially in the early phase, nitric oxide-dependent inhibition is more important.

To investigate the effect of central inflammation due to bacterial infection, such as meningitis, on the activities of hepatic cytochromes P450 (CYPs), rats were injected intracerebroventricularly (i.c.v.) with 0.1 microg of bacterial lipopolysaccharide (LPS). The LPS i.c.v. injection significantly decreased the total P450 contents (by 30% of the levels of control rats treated with saline i.c.v.), the contents of CYP1A (48%), 2B (54%), 2C11 (37%) and 3A (40%) and related drug metabolizing activities, 7-ethoxycoumarin O-deethylation (36%), imipramine N-demethylation (41%) and erythromycin N-demethylation (33%) in liver microsomes 24 h after the treatment. In contrast, intraperitoneal (i.p.) injection of LPS at the same dose as i.c.v. (0.1 microg) did not significantly affect the hepatic microsomal contents of total P450 or the content of each individual CYP isozyme and its activity. CYP2D1 protein and the activity of imipramine 2-hydroxylase were not significantly decreased by LPS injection regardless of the route of administration. The inhibitory effects of 0.1 microg i.c.v. LPS on the activities of these CYPs were almost equal to those of 10 microg i.p. LPS, and 0.01 microg of i.c.v. LPS significantly decreased the activity of imipramine N-demethylase only. Therefore, the LPS i.c.v. injection resulted in CYP isozyme-selective inhibition at an ineffective dose when injected i.p.. It is suggested that a central inflammation, such as meningitis, differentially decreases the levels of hepatic CYP isozymes. A possible involvement is discussed of the central nervous system in this down-regulation.

We observed significant strain differences [Dark Agouti (DA) > Wistar] in 1,3,7-trimethyluric acid formation (C-8 hydroxylation) during caffeine metabolism, though not in N-demethylations, in adult male DA and Wistar rats. In contrast, adult female and immature male rats of both DA and Wistar strains did not show significant differences in activity levels of C-8 hydroxylation. Kinetic studies using liver microsomes revealed that adult male DA rats have a larger Vmax for C-8 hydroxylation than do Wistar rats. Troleandomycin (TAO), known as a cytochrome P450 (CYP) 3A inhibitor, and an anti-rat CYP3A2 polyclonal antibody effectively reduced C-8 hydroxylation by rat liver microsomes in a concentration-dependent manner, suggesting that C-8 hydroxylation in rats is mediated largely by an isoform(s) of the CYP3A subfamily. Troleandomycin and the antibody did not inhibit the N-demethylations of caffeine by rat liver microsomes. Treatment of rats with CYP3A inducers caused a marked increase in C-8 hydroxylase activity. These results indicate that the rat CYP3A subfamily is capable of catalyzing C-8 hydroxylation of caffeine as is the case for human CYP3A4. The results of western blotting analysis using anti CYP3A antiserum showed that the staining intensity of the protein band in DA rat liver microsomes was higher than that in Wistar rat liver microsomes. We concluded that marked sex-dependent strain differences in C-8 hydroxylation of caffeine between Wistar and DA rats are due to the differences in the levels of expression of CYP3A in these strains of rats.

Cytochrome P450 enzymes belonging to the CYP2D subfamily have been shown to be one of determinants of the polymorphic drug oxidations in the human and the rat. Debrisoquine 4-hydroxylation is a typical reaction catalyzed by these enzymes. However, various strains of mice were observed to have much lower debrisoquine 4-hydroxylase activity than Wistar rats, whereas other monooxygenase activities in mice toward bunitrolol, propranolol, imipramine and amitriptyline, which are mediated by the CYP2D enzymes in the rat, were comparable to those of the rats. Immunoblot analysis of mouse liver microsomes with an antibody raised against a rat CYP2D enzyme indicated that the mouse liver contained a P450 enzyme(s) immunochemically related to the rat CYP2D enzyme. The antibody inhibited propranolol ring-hydroxylase and imipramine 2-hydroxylase activities, as well as testosterone 16alpha-hydroxylase activity, a typical reaction of mouse CYP2D9, but not debrisoquine 4-hydroxylase activity in mouse liver microsomes. We partially purified a P450 enzyme (designated P450 ML2d) from livers of male ddY mice by monitoring the cross-reactivity with the antibody. The partially purified enzyme was indicated to belong to the CYP2D subfamily from its N-terminal amino acid sequence, but the homology of the sequence to other CYP2D enzymes of the mouse (CYP2D9-11) was 62%, suggesting that P450 ML2d is a novel P450 enzyme. P450 ML2d had the oxidation activities for the rat CYP2D-substrates, such as propranolol 4-hydroxylation and imipramine 2-hydroxylation, in higher rates than those of the microsomes, but did not exhibit debrisoquine 4-hydroxylase activity. Our result is the first finding that a mouse CYP2D enzyme also metabolizes substrates for the rat CYP2D enzyme, in addition to steroids, but the enzyme had a limited specificity for the substrates of the CYP2D enzymes of the rat and the human.

Acetaldehyde was oxidized by rat and human hepatic microsomes in the presence of NADPH. We designated this NADPH-dependent oxidation system MAOS (microsomal acetaldehyde-oxidizing system), to distinguish it from the NAD-dependent acetaldehyde oxidation system of acetaldehyde dehydrogenase in mitochondria and cytosol. This activity was increased 2.3-fold by giving rats ethanol. Judging from the Vmax/Km values, the metabolic capacity of rat hepatic microsomes for MAOS activity was increased 24-fold by ethanol. The acetaldehyde oxidation activity of eight forms of purified rat cytochrome P450 was investigated in a reconstituted system. CYP2E1 had the highest level, followed by CYP1A2 and 4A2. Immunoinhibition studies showed that an anti-CYP2E1 antibody inhibited 90% of the MAOS activity in rats given ethanol. NADPH-dependent acetate formation was 12% or 33.6% of the NAD-dependent acetate formation in liver homogenates of control rats and those treated with ethanol, respectively. We investigated human MAOS activity further. Among the 10 forms of human cytochrome P450 expressed in yeast, CYP2E1 had especially high acetaldehyde oxidation activity. The correlation of MAOS activity with the levels of immunoreactive CYP2E1 in individual human microsomes was highly significant (r2 = 0.88, P < .01). These results indicate that hepatic CYP2E1 mainly contributes to MAOS in rats and humans, the pathway of which may play an alternative role against acetaldehyde in the liver after alcohol consumption together with acetaldehyde dehydrogenase in the metabolism of acetaldehyde.

To assess the contribution of cytochrome P450 (P450) to the microsomal ethanol oxidation system (MEOS) in humans, we examined ethanol oxidization activity in human hepatic microsomes and multiple forms of human hepatic P450s expressed in B-lymphoblastoid cells. Acetaldehyde produced by the MEOS was converted into a fluorescent derivate with cyclohexane-1,3-dione and analyzed by high-performance liquid chromatography using a fluorescence detector. The ethanol oxidation activity of seven forms of human P450s was investigated. P450s 2E1 and 1A2 formed acetaldehyde at high rates. In immunoinhibition studies, anti-P450 2E1 antibody inhibited the ethanol oxidation activity of human hepatic microsomes by 54%, and anti-P450 1A2 antibody inhibited ethanol oxidation activity by 21%. 7,8-Benzoflavone, an inhibitor of P450 1A forms, also inhibited this activity by 38%. The correlation of ethanol oxidation activity with the levels of immunoreactive P450 2E1 in individual human microsomes was highly significant (r = 0.91, P < .001), and the correlation of ethanol oxidation activity with the levels of P450 1A2 was also highly significant (r = 0.87, P < .001). The Km values of P450s 2E1 and 1A2 for ethanol oxidation were 16.5 and 23.6 mM, respectively. These results indicated that P450 2E1 was a major contributor to the MEOS in humans; however, P450 1A2 was considered to play an important role in the MEOS.

NADPH-dependent activity of acetaldehyde oxidation was investigated in microsomes by assaying [14C]acetic acid produced from [14C]acetaldehyde with ion-exchange column. Rat hepatic microsomes exhibited acetaldehyde oxidation activity in the presence of NADPH. This activity was induced 2-fold by the treatment of rats with ethanol. We designated this NADPH-dependent oxidation system as microsomal acetaldehyde-oxidizing system (MAOS), to distinguish from the NAD-dependent acetaldehyde oxidation system by acetaldehyde in mitochondria and cytsol. We further investigated essential enzymes contributing to MAOS activity. Acetaldehyde oxidation activity was investigated in eight forms of purified P-450 in a reconstituted system. Cytochrome P-450 (CYP) 2E1 had the highest oxidation activity and CYP1A2 and CYP4A2 had the next highest activity. Other forms had low activity. To assess the contribution of these forms to MAOS activity, immunoblot was done. CYP2E1 was induced 2-fold by ethanol treatment, but CYP1A2 and CYP4A2 were not reflecting the MAOS activity increased by ethanol treatment. These results suggest that CYP2E1 is the essential enzyme in the MAOS of rats.

The effects of 4-phenylalkylpyridines (chain length of 0-5, 7, 9 and 11 carbon atoms) on the induction of hepatic microsomal P450 and its multiple forms (1A1/2 (1A1, 1A2), 3A2, 2B1/2, 2C6 and 2E1) in the male and female rat have been investigated. P450 induction gradually declined with increasing chain length of the 4-phenylalkylpyridines. Immunoblot analysis revealed that three pyridine compounds having methylene units of 0, 1 and 2 only induced P4501A2, whereas those having 4, 5 or 7 methylene units only induced 1A1 in the male rat. In the female rat, however, we could not observe such a chain length-dependent differential induction of the P4501A subfamily. Induced levels of P4503A2 and 2E1 were dependent on total P450 specific content, but 2C6 was increased in a chain length-dependent manner in both sexes. These results provide new information on the differential effects of pyridine-containing compounds on P450 induction in the rat.

Alprenolol 4-hydroxylation and N-desisopropylation in liver microsomes from male Wistar rats were kinetically analyzed to be biphasic. In the 4-hydroxylation at a low substrate concentration (5 microM), significant strain [Wistar > Dark Agouti (DA)] and sex (male > female) differences were observed, and the differences decreased at a high substrate concentration (1 mM). In the N-desisopropylation, only a strain difference (Wistar > DA) was observed at the low substrate concentration. Cytochrome P450BTL (P450BTL, corresponding to CYP2D2) in a reconstituted system with 5 microM alprenolol had high 4-hydroxylase activity, which was about 10 times that of P450ml corresponding to CYP2C11, and N-desisopropylase activity at a similar extent to P450ml. The two microsomal activities at 5 microM alprenolol were efficiently decreased by antibodies against P450BTL and by sparteine, a typical substrate of the CYP2D subfamily. Polyclonal antibodies against P450ml and P450PB-1 (corresponding to CYP3A2) partially suppressed only N-desalkylation at 5 microM, whereas they reduced the two activities at 1 mM. P450ml showed a high N-desisopropylase activity at a substrate concentration of 1 mM, where the sex difference was not observed. Furthermore, P450PB-2 corresponding to CYP2C6, which is one of the major P450 isozymes in female rats, also had 4-hydroxylase and N-desalkylase activities. These results suggest that a CYP2D isozyme(s) is the primary enzyme in alprenolol 4-hydroxylation and N-desisopropylation in a lower substrate concentration range, and that the involvement of some male-specific P450 isozyme(s) other than CYP2C11 or CYP3A2 may cause the sex difference in the 4-hydroxylation. In a higher substrate concentration range, CYP2C11 is thought to play a major role particularly in N-desisopropylation in male rats.(ABSTRACT TRUNCATED AT 250 WORDS)

We have characterized a chemically reactive propranolol (PL) metabolite which binds to proteins in rat liver microsomes. During incubation with rat liver microsomes (1 mg of protein) fortified with an NADPH-generating system, 4-hydroxypropranolol (4-OH-PL) quickly disappeared from the reaction medium, but none of the possible metabolite peaks was detected under the high-performance liquid chromatographic conditions used. The consumption of 4-OH-PL depended on microsomes and NADPH. The reaction was not affected by inhibitors of cytochrome P450 or FAD monooxygenase, but was markedly diminished in the presence of cytosol and ascorbic acid. The effect of cytosol was inhibited by potassium cyanide but not by sodium benzoate or dimethyl sulfoxide, and was also not affected by heating at 60 degrees C for 30 min, suggesting that superoxide (SO) ion was involved in the reaction and that it was blocked by superoxide dismutase (SOD) present in the cytosol. Cu,Zn-SOD, purified from cytosol, effectively mimicked the suppressive effect of cytosol. Incubation of 4-OH-PL in an SO-generating system of xanthine and xanthine oxidase generated 1,4-naphthoquinone (1,4-NQ), which was identified by TLC, HPLC, and GC/MS. 1,4-NQ was also formed in microsomal incubates containing NADPH and small amounts of microsomes (below 0.1 mg of protein). These results indicate that 4-OH-PL is converted by SO, or some reactive oxygen species derived from it, to 1,4-NQ which binds to proteins and is one of the reactive metabolites of PL.

A form of P450 [termed P450(b-1)] was purified from male bear liver microsomes. The specific content of the final P450(b-1) preparation was 11.26 nmol/mg protein, and recovery was 0.20% of the microsomal P450. The apparent molecular weight of P450(b-1) was 54,000. The absorption spectrum of P450(b-1) indicated that this protein was a low- and high-spin mixed type P450 in the oxidized form. The carbon monoxide complex of reduced P450(b-1) showed an absorption peak at 450.5 nm. The reconstituted system containing P450(b-1) catalyzed the metabolism of aminopyrine, benzo[a]pyrene, 7-ethoxycoumarin, imipramine and propranolol, of which P450(b-1) most strongly catalyzed aminopyrine N-demethylation and imipramine N-demethylation. The N-terminal amino acid sequence of P450(b-1) was highly homologous to that of P450-D1 from liver microsomes of male beagle dogs. P450(b-1) showed similarities in spectral properties, N-terminal amino acid sequence, and catalytic activities to rat P450 2C11. P450(b-1) was immunochemically cross-reactive with anti-P450 2C11 antibody and very weakly cross-reactive with anti-P450 2E1 antibody, but did not react with anti P450 1A1 or 2B1 antibodies. On the basis of these results, we suggest that P450(b-1) belongs to the P450 2C subfamily.

Background: Ropivacaine is a local anesthetic with a long duration of action. Although it is less toxic than bupivacaine, local anesthetic toxicity is possible when the plasma concentration is increased. Because ropivacaine is an amide-type local anesthetic, it is metabolized by cytochrome P450 (P450) in the liver, and its elimination and plasma concentration can be dependent on the level of P450. The purpose of this investigation was to elucidate the metabolism of ropivacaine by human hepatic P450., Methods: The metabolism of ropivacaine was compared using recombinant human and purified rat hepatic P450 isozymes. An inhibition study using antibodies against rat P450 was performed using hepatic microsomes from human and rat to identify which P450s are involved in ropivacaine metabolism., Results: Ropivacaine was metabolized to 2',6'-pipecoloxylidide (PPX), 3'-hydroxyropivacaine (3'-OH Rop), and 4'-hydroxyropivacaine (4'-OH Rop) by hepatic microsomes from human and rat. PPX was a major metabolite of both human and rat hepatic microsomes. In a reconstituted system with rat P450. PPX was produced by CYP2C11 and 3A2, 4'-OH Rop by CYP1A2, and 3'-OH Rop by CYP1A2 and 2D1. Formation of PPX in rat hepatic microsomes was inhibited by anti CYP3A2, but not by CYP2C11 antibody, and formation of 3'-OH Rop was inhibited by CYP1A2 and 2D1 antibodies. Anti CYP3A2 and 1A2 antibodies inhibited the formation of PPX and 3'-OH Rop in human hepatic microsomes, respectively. Recombinant human P450s expressed in lymphoblast cells were used for further study. CYP3A4 and 1A2 formed the most PPX and 3'-OH Rop, respectively. Ropivacaine N-dealkylation and 3'-hydroxylation activities correlated well with the level of CYP3A4 and 1A2 in human hepatic microsomes, respectively., Conclusions: Ropivacaine was metabolized to PPX, 3'-OH Rop, and 4'-OH Rop by hepatic P450. PPX was a major metabolite in human hepatic microsomes. CYP3A4 was involved in producing PPX. CYP1A2 was involved in the formation of 3'-OH Rop in human hepatic microsomes.

1. The changes in the activity of drug-metabolizing enzymes and in the content of P450 isozymes in renal and hepatic microsomes after treatment of the male Sprague-Dawley rat with cis-diamminedichloroplatinum (Cisplatin, CDDP) were examined. 2. NADPH-P450 reductase activity in renal microsomes was significantly increased by treatment with CDDP, but lauric acid omega- and (omega-1)-hydroxylation activities of renal microsomes were not increased. 3. The level of P4502C23 was increased significantly and levels of P4504A2 and 4A8 tended to increase in renal microsomes. 4. In hepatic microsomes, lauric acid omega-hydroxylation activity was increased, but (omega-1)-hydroxylation activity was not. Levels of P4502C11 and 3A2, which are male-specific forms, were decreased, whereas levels of P4502A1, 2C7 and 2E1 were increased in hepatic microsomes. The levels of P4504A2 and 4A3 were increased by CDDP and the level of P4504A1 was not changed. Changes in the protein levels of P450 by CDDP were consistent with those in the mRNA levels reported previously (LeBlanc et al. 1992). 5. Male-specific forms in rat liver such as P4502C11 were decreased by CDDP, but those in the kidney such as P4504A2 was not. Therefore, CDDP has different influences on the regulation of hepatic and renal P450s.

The metabolism of trimethadione (TMO), a useful indicator of hepatic drug-oxidizing capacity in rats and humans, was studied using 14 different forms of rat cytochrome P450 (CYP1A1, 1A2, 2A1, 2A2, 2B1, 2B2, 2C6, 2C7, 2C11, 2C12, 2C13, 2E1, 3A2 and 4A2) and three forms of human cytochrome P450 (CYP1A2, 2C and 3A4). TMO N-demethylation was increased by treating rats with phenobarbital. CYP2C11 and 2B1 had high TMO N-demethylase activity, but 1A1 and 1A2 had low activity. Antibodies raised to CYP2C11 and 2B1/2 inhibited TMO N-demethylation in hepatic microsomes of untreated and phenobarbital-treated rats, respectively. In a reconstituted system, human CYP3A4 and 2C produced efficiently dimethadione (DMO), but CYP1A2 did not catalyse TMO N-demethylation. Antibodies raised to CYP3A2 and 2C11 inhibited TMO N-demethylation in human hepatic microsomes. These results indicated that the N-demethylation of TMO is catalysed mainly by CYP2C11 and 2B1 in rat hepatic microsomes, and that human CYP3A4 and an unspecified isoform of the 2C subfamilies contribute to TMO N-demethylation in human liver.

Osteogenic Disorder Shionogi (ODS) rats, which cannot synthesize ascorbic acid due to a deficiency of L-gulonolactone oxidase, become scorbutic when not supplied with dietary ascorbic acid. We used the deficient rats to study the effects of ascorbic acid on the amount of cytochrome P450 enzymes in liver microsomes. The total amount of hepatic cytochrome P450 in ODS rats deprived of ascorbic acid was lower by approximately 40%, whereas ODS rats fed with ascorbic acid and the wild strain had the same level of total hepatic cytochrome P450. Western blot analysis for various forms of cytochrome P450 in liver microsomes indicated that the amount of CYP1A2 was significantly higher in ascorbic acid deficient rats. On the other hand, amounts of CYP2B2 and 3A were lower, and those of CYP2E1 and CYP2C6/11 were unaffected. In accordance with the higher amount of CYP1A2, Northern blot analysis showed increased expression of CYP1A2 mRNA. The capacity of microsomes to produce mutagens from 2-amino-6-methyl-dipyrido[1,2-a:3',2'-d]imidazole acetate (Glu-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole acetate (Trp-P-2) was higher in scorbutic ODS rats by the Ames test. These results indicate that the effects of ascorbic acid deficiency on the expression of cytochrome P450 in ODS rat livers are form-specific and that the increased CYP1A2 is associated with increased metabolic activation of promutagens in the scorbutic state.

Two P-450s with debrisoquine 4-hydroxylation activity, designated P-450 UT-7 and UT-7b, were purified and partially purified, respectively, from hepatic microsomes of untreated male rats. Both purified P-450s with an apparent molecular weight of 49,000, were associated with another protein with an apparent molecular weight of 29,000 which was designated 29 k-protein. The CO-reduced spectra of both P-450 UT-7 and UT-7b showed a peak at 448 nm. The NH2-terminal amino acid sequences of P-450 UT-7 and UT-7b were the same as the amino acid sequences of CYP2D1 and CYP2D2 deduced from the cDNA, respectively, except for the lack of a terminal methionine for P-450 UT-7b. In a reconstituted systems, P-450 UT-7 and UT-7b catalyzed lidocaine 3-hydroxylation and N-deethylation in the presence of the 29 k-protein. The Km and Vmax values for lidocaine 3-hydroxylation were 3.6 microM and 0.50 nmol/min/nmol of P-450 for P-450 UT-7, and 3.6 microM and 0.93 nmol/min/nmol of P-450 for P-450 UT-7b, respectively. Antibody against P-450 UT-7, which also cross-reacted with P-450 UT-7b, inhibited lidocaine 3-hydroxylation in liver microsomes from untreated male rats, but had little effect on lidocaine N-deethylation. These findings suggested that lidocaine 3-hydroxylation in hepatic microsomes from untreated male rats was catalyzed by P-450 UT-7 and/or UT-7b.P-450 UT-7 not containing 29 k-protein was obtained as the non-absorbed fraction from hydroxylapatite HPLC. The activities of debrisoquine 4-hydroxylation as well as lidocaine 3-hydroxylation and N-deethylation in a reconstituted system with P-450 UT-7 without 29 k-protein were one-fifth of those of P-450 UT-7 containing 29 k-protein at the same substrate concentration. These findings suggested that the 29 k-protein was essential to express the maximal metabolic activities. However, the lidocaine metabolic activity in a reconstituted system with P-450 UT-7 containing 29 k-protein and in hepatic microsomes were not inhibited by 29 k-protein antibody.

Mitochondrial NADPH-linked aquacobalamin reductase was purified and characterized to clarify its enzymatic properties. The enzyme was purified about 360-fold over rat liver mitochondrial membranes in a yield of 7.5%. The purified enzyme was homogenous in SDS-PAGE. The molecular mass (M(r)) of the enzyme was calculated to be 65 kDa by SDS-PAGE and by Toyopearl HW55 gel filtration, indicating that the enzyme is a monomeric polypeptide with M(r) of 65 kDa. The enzyme was a flavoprotein containing 1 mol of FAD and FMN per mole of the enzyme. The enzyme was specific for NADPH as electron donor and had the ability to reduce cytochrome c (15.4 mumol.min-1 x mg protein-1), potassium ferricyanide (4.9 mumol.min-1 x mg protein-1) and 2,6-dichlorophenolindophenol (16.8 mumol.min-1.mg protein-1) as well as aquacobalamin (6.4 mumol.min-1 x mg protein-1). Although the enzyme immunoreacted with an antibody against NADPH-cytochrome P-450 reductase, which had the activity of the NADPH-linked aquacobalamin reductase in rat liver microsomes, the mitochondrial enzyme and the microsomal enzyme had different enzymological properties.

Microsomal aldehyde oxygenase (MALDO) activity for 9-anthraldehyde (9-AA) was significantly higher in the male than in the female adult rat liver. 9-AA MALDO activity was also significantly enhanced by pretreatment with dexamethasone and phenobarbital, whereas it was not significantly changed by 3-methylcholanthrene or acetone. Several cytochrome P450 isozymes purified from rat hepatic microsomes were able to catalyze the oxidation of 9-AA to 9-anthracene carboxylic acid (9-ACA) in the presence of NADPH, NADPH-cytochrome P450 reductase and dilauroylphosphatidylcholine. Under the ordinary conditions of the reconstituted system, the catalytic activities (nmol/min/nmol P450) of cytochrome P450s, 2A1, 2B2, 2C6, 2C11 and 3A2 were 1.53 (1.37 in the presence of cytochrome b5), 1.20 (2.06), 4.87 (7.75), 18.0 (21.6) and 0.90 (1.17), respectively. Cytochrome P450 2C11 (CYP 2C11) showed the highest catalytic activity of the cytochromes examined. In the reconstituted system using the lipids extracted from microsomes, CYP 3A2 more effectively catalyzed the oxidation of 9-AA to 9-ACA, and its catalytic activity (nmol/min/nmol P450) was 3.33 or 6.61 in the absence or presence of cytochrome b5, respectively. The antibody against CYP 2C11 inhibited by 90% the hepatic microsomal oxidation of 9-AA MALDO activity in adult male rats, but the activity was not inhibited by antibody against CYP 3A2. These results show that the individual forms of cytochrome P450 have a catalytic activity for the oxidation of 9-AA to 9-ACA, and that CYP 2C11 is the major constitutive catalyst of 9-AA MALDO activity in untreated adult male rat liver.

A form of P450 [termed P450(h-1)] was purified from the liver microsomes of a male horse to electrophoretic homogeneity. The specific content of the final P450(h-1) preparation was 14.8 nmol/mg of protein and the recovery was 0.38% of the microsomal P450. The apparent molecular weight of P450(h-1) was 52,000 Da. The absorption spectra of P450(h-1) indicated that P450(h-1) was a low- and high-spin mixed type P450 in the oxidized form. The reconstituted system containing P450(h-1) could catalyze benzphetamine N-demethylation, 7-ethoxycoumarin O-deethylation, and testosterone 16 alpha-hydroxylation. In the horse hepatic microsomes, aniline p-hydroxylation and testosterone 6 beta-hydroxylation, in addition to the above reactions, were detected. The N-terminal amino acid sequence of P450(h-1) was highly homologous to that of rat P450 2C11. Western blot analysis using anti-P450(h-1) antibody revealed that this antibody most strongly recognized P450 2C13 among ten rat P450s belonging to eight different subfamilies involved in hepatic drug metabolism. This anti-P450(h-1) antibody inhibited the testosterone 16 alpha-hydroxylase activity in horse liver microsomes. These results suggest that P450(h-1) belongs to the P450 2C subfamily and contributes to the testosterone 16 alpha-hydroxylation in horse liver microsomes.

Hepatic microsomal cytochrome P450s, which are involved in the metabolism of drugs, hormones, prostaglandins and fatty acids, change when animals develop diabetes. We studied changes in cytochrome P450 isozymes in both hepatic and renal microsomes of rats with diabetes caused by streptozocin, and compared the results with changes in catalytic activities in the microsomes. In hepatic microsomes of diabetic rats, the amount of cytochrome P450 2E1, an acetone-inducible isozyme, was two and a half times that of control rats, and that of P450 4A2, a major renal isozyme, was three times that in the controls. The amounts of cytochrome P450s 2A1, 2C6, 2C7, 3A2 and 4A3 increased in hepatic microsomes of diabetic rats, and P450 2C11 decreased. Treatment with insulin restored these to the levels in the controls. The catalytic activities of aniline hydroxylation, 7-ethoxycoumarin O-dealkylation, testosterone 2 beta, 6 beta, 7 alpha, and 16 beta-hydroxylation, and omega-, (omega-1)-hydroxylation of lauric acid were high in the hepatic microsomes of diabetic rats, and testosterone 2 alpha and 16 alpha-hydroxylation activities were low. In renal microsomes of diabetic rats, cytochrome P450s 2E1, 4A2 and K-4 were induced, and omega- and (omega-1)-hydroxylation activities were high. These changes were reversed by insulin treatment. The induction and suppression of cytochrome P450 isozymes in diabetic rats were consistent with the changes in the catalytic activities. In both hepatic and renal microsomes, P450s 2E1 and 4A2 were induced, altered metabolism of ketones and fatty acids in diabetes may contribute to these changes.

1. The effect of cyclosporin A, an immunosuppressive drug, on the levels of individual cytochromes P450 of rat liver was studied by immunoblotting with specific antibodies. Changes in the metabolic activities of the microsomal enzymes were similarly studied. 2. Total P450 was decreased by treatment with cyclosporin A for 17 days. NADPH-P450 reductase activity was also decreased by treatment with cyclosporin A for 10 days and decreased further after 17 days. 3. Cytochromes 2B2 and 2C6 concentrations were decreased by treatment with cyclosporin A for 17 and 10 days, respectively. Administration of cyclosporin A for 17 days also decreased the levels of the male specific cytochromes 2C11 and 3A2, and consequently decreased testosterone 2 alpha-, 16 alpha-, 2 beta- and 6 beta-hydroxylation activities. Shorter periods (7 and 10 days) of cyclosporin A treatment decreased the level of cytochrome 3A2, which metabolizes cyclosporin A. In contrast, the level of cytochrome 2C13 was unchanged.

Many kinds of cytochrome P450s were purified from rat hepatic microsomes, and their role in the metabolization of nicotine in a reconstituted system examined. Of four phenobarbital-inducible P450s, P450 2B1 had the highest nicotine oxidation activity and P450 2B2 showed a low rate of nicotine oxidation, whereas P450 2C6 and 3A2 had no detectable activity toward nicotine. Among eleven other purified cytochrome P450s tested, P450 2C11 had high nicotine oxidation activity and P450 1A2 and 2D1 showed low catalytic activity toward nicotine. The other cytochrome P450s, P450 1A1, 2A1, 2A2, 2C7, 2C12, 2C13, 2E1 and 4A1, had no detectable nicotine oxidation activity. Based on these results, participation of cytochrome P450s in nicotine metabolism in human and animal livers is discussed.

The suppression by cannabidiol (CBD) of the liver microsomal drug-metabolizing enzyme activities in female rats was demonstrated and its mechanism was examined. Pretreatment of rats with CBD (10 mg/kg, i.p.) caused temporary decreases in contents of cytochrome P450 (P450) and b5 and NADPH-cytochrome c (P450) reductase activity compared with values from the vehicle control group. p-Nitroanisole O-demethylase, aniline hydroxylase, d-benzphetamine N-demethylase and delta 9-tetrahydrocannabinol 11-hydroxylase were also decreased by the CBD pretreatment. The latter two activities took a longer time to return to control levels than the former two. However, the CBD pretreatment, which reduced the protein level of P450 UT-2 (CYP2C11) in adult male rats, did not decrease the protein level of P450 F-1 (CYP2C6) or F-2 (CYP2C12) in liver microsomes from female rats. These results suggest that the mechanisms by which CBD suppresses liver microsomal drug-metabolizing enzyme activities are different in male and female rats.

ESR measurements were performed for the reaction systems composed of t- or n-butylhydroperoxide and cytochrome P-450 (UT-2), purified from rat liver microsomes. On addition of n-butylhydroperoxide to the P-450 at pH 7.4, ESR signal due to a ferric low-spin species (g1 = 2.29, g2 = 2.24 and g3 = 1.96) was recorded. The observed g-parameters agreed well with those of a model thiolate-heme-iron(III)-peroxide complex, Fe(III)TPP(-S-TGE) (-OO-t-butyl) (g1 = 2.285, g2 = 2.198 and g3 = 1.959). In terms of the g-parameters, the new P-450 complex was concluded to be a P-450-butyl peroxide adduct, in which a butyl peroxide anion ligates at the sixth position of heme iron(III).

The role of several P450 enzymes in the metabolism of diazepam (DZ) has been investigated. Hepatic microsomes of adult male rats were pretreated with antisera raised against the P450s CYP3A2, 2B1, 2C6, 2C11, 2D1 and 2E1, and their influence on the subsequent metabolism of DZ was determined by simultaneously measuring the changes in the relative rates of formation of its metabolites. Several forms of P450 were found to be positively involved in DZ metabolism. Antisera of the "male-specific" P450 enzyme CYP2C11 partially inhibited both DZ N-demethylase and C3 hydroxylase activities (60%) which resulted in decreased formations of N-desmethyl-DZ (NDZ) and 3-hydroxy-DZ (3HDZ), respectively. In a reconstitution experiment with the purified enzyme, CYP2C11 predominantly catalysed the formation of NDZ from DZ. Antisera of a further male-specific P450 CYP3A2 strongly inhibited (95%) the C3 hydroxylase of DZ and thus 3HDZ formation. A corresponding reconstitution experiment with this same P450 enzyme gave 3HDZ as principal product. CYP2D1 antisera inhibited the aromatic hydroxylation of DZ (98%) and subsequent formation of 4'-hydroxy-DZ (4'HDZ). This enzyme was also observed to inhibit DZ N-demethylase activity (60%). A reconstitution experiment with pure CYP2D1 catalysed the formation of both 4'HDZ and NDZ.

The genotoxic and mutagenic activation of aflatoxin B1 (AFB1) by hepatic, renal, and pulmonary microsomes and purified cytochrome P450s was investigated in Salmonella typhimurium TA1535/pSK1002 cells in which an umu response shows DNA damage. The activity of the hepatic microsomes was greatest. Pulmonary microsomes had moderate activity and renal microsomes had low activity. P450 2C11, 2B1, 3A2, 4A2, 4B1, K-2, and K-4 were assayed in a reconstituted system with dilauroylphosphatidylcholine (DLPC). P450 2C11 (a major hepatic cytochrome P450 in male rats) had high activity. P450 2B1 (a major form as well as P450 4B1 in pulmonary microsomes) and K-2 (a minor form in renal microsomes) had moderate activity. P450 4A2 (a major form in renal microsomes), P450 K-4 (a renal form), and P450 4B1 had low activity. P450 3A2 did not have high activity in these conditions but it had high activity toward AFB1 in a modified reconstituted system with a lipid mixture and sodium cholate instead of DLPC only. The activities of other forms were not enhanced by the modification of reconstituted system. Anti-P450 2C11 or 3A2 antibodies inhibited the bioactivation of AFB1 by hepatic microsomes to 50%. These results suggest that the greater ability of hepatic microsomes as compared with pulmonary and renal microsomes to metabolize AFB1 to mutagenic products is a function of the relative proportions of the highly active cytochrome P450s, P450 2C11 and 3A2, in the liver.

The proportions of different hepatic microsomal cytochrome P450s expressed in uremic rats were studied with specific antibodies and with a steroid hydroxylase assay. In male uremic rats, the hepatic levels of P450 2C11, a male-specific form, and 3A2, a male-dominant form, were decreased to about 30% at 5 weeks after the induction of uremia. These changes were paralleled by decreases in the activities of testosterone 2 alpha-, 16 alpha-, and 6 beta-hydroxylation. The level of P450 2A1, abundant in immature rats, was increased 2-fold by uremia and accompanied by an increase in testosterone 7 alpha-hydroxylation activity. The levels of P450 2C6 and 2E1 were not changed by uremia. The levels of male-specific and male-dominant forms such as P450 2C11 and 3A2 are affected by the serum level of testosterone, which was decreased in the male rats with uremia. Therefore, castrated rats were prepared to compare the effects of testosterone on hepatic cytochrome P450s in uremic rats with those in castrated rats. When testosterone was administered to the castrated rats, the decreased levels of both P450 2C11 and 3A2 returned to normal. However, the administration of testosterone to the uremic rats did not prevent the decrease in the levels of these P450s. In female rats, changes in the levels of cytochrome P450s were not as great during uremia as those in male uremic rats. The level of P450 2C12, a female-specific form, was not changed; the level of P450 2A1 was increased by 50%, that of 3A2 which is barely detected in female rats was increased by 60%, and that of 2E1 was increased by 25%. These results suggested that the changes in the hepatic levels of cytochrome P450s were affected by factors other than testosterone in uremic rats.

The possible roles of cytochrome P450 (P450) enzymes in the metabolic activation of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) by rat liver microsomes have been examined in a system containing the bacterial tester strain Salmonella typhimurium NM2009, a newly developed strain showing high O-acetyltransfer activities. The DNA-damaging activity could be determined by measuring expression of the umu gene in a plasmid containing the fused umuC-lacZ gene construct in the bacteria. The following lines of evidence support the view that both NDMA and NDEA are principally oxidized to reactive products by P450 2E1 in rat liver microsomes. First, NDMA and NDEA were activated by rat liver microsomes in a protein- and substrate-dependent manner and the former chemical was more active than the latter; both activities were induced in rats treated with P450 2E1 inducers such as ethanol, acetone and isoniazid and by starvation. Second, activation of NDMA and NDEA were both inhibited significantly by antibodies raised against rat P450 2E1 and by P450 2E1 inhibitors such as diethyldithiocarbamate and 4-methylpyrazole in rat liver microsomes. Finally, in reconstituted monooxygenase systems containing purified rat P450 enzymes, P450 2E1 gave the highest rates of the activation of both NDMA and NDEA; the addition of rabbit cytochrome b5 to the system caused about a 1.5-fold increase in both reactions. In separate experiments we also found that N-nitrosomethylacethoxymethylamine, a compound that reacts with DNA after ester cleavage, is more genotoxic in S.typhimurium NM2009 than in S.typhimurium NM2000, a strain that is defective in O-acetyltransferase activity. Part of the pathway involved in the activation of nitrosamines is suggested to be acetylation of alkyldiazohydroxides formed by P450 or acetylesterase, because the genotoxic activity of N-nitrosomethylacethoxymethylamine in S.typhimurium NM2009 could be inhibited by the O-acetyltransferase inhibitor pentachlorophenol. These results indicate that NDMA and NDEA are oxidized to gentoxoic products by rat liver microsomes and that a P450 2E1 enzyme plays a major role in the activation of these two potent carcinogens. The activation pathway of N-nitrosodialkylamines through acetylation by O-acetyltransferase has been proposed. This simple bacterial system for measuring genotoxicity should facilitate studies on the activation of N-nitroso alkylamines.

The effects of fasting on hepatic cytochrome P450 in the mature male house musk shrew, Suncus murinus (suncus), were studied by Western blot analyses and enzyme assays. The content of P450IIE protein was decreased, by fasting, to 24% of the control level in contrast to the results with rats, in which P450IIE protein was increased to 172% by fasting. These changes reflected on catalytic activities, such as aniline hydroxylase and N-nitrosodimethylamine demethylase activities, which were decreased to about 45% and 28%, respectively, of the control levels by fasting, while in fasting rats, the catalytic activities of these enzymes were 2-3-fold higher than in controls.

A cytochrome P-450 isozyme, P-450 bunitrolol (BTL), catalyzing bunitrolol 4-hydroxylation was partially purified from liver microsomes of adult male Sprague-Dawley rats by hydrophobic affinity chromatographic (omega-aminooctyl-Sepharose 4B) and high-performance liquid chromatographic (anion-exchange diethylaminoethyl-5PW) techniques. The specific content of the final preparation was 5.02 nmol/mg protein, which was 7.8-fold that of microsomes. It showed two protein bands of 49 and 32 kDa in sodium dodecylsulfate-polyacrylamide gel electrophoresis. N-Terminal 20 amino acid sequence of the protein of a higher molecular mass (49 kDa) isolated by an electroblotting technique is 94% homologous with that of CYP2D2. In a reconstituted system including NADPH-cytochrome P-450 reductase and an NADPH-generating system, the final preparation had the highest activity toward BTL and debrisoquine 4-hydroxylation among 12 isozymes of cytochrome P-450 examined. Kinetic parameters, KM and Vmax values, of P-450 BTL calculated for BTL 4-hydroxylation were 10.7 microM and 19.68 nmol/min/nmol P-450, respectively, whereas those values (mean +/- SE) of rat liver microsomes were 0.84 +/- 0.05 microM and 2.05 +/- 0.11 nmol/min/nmol P-450. When preincubated with rat liver microsomes, the antibody against the final P-450 BTL preparation suppressed bunitrolol and debrisoquine 4-hydroxylase activities dose-dependently and almost completely. These results suggest that cytochrome P-450 BTL and its immunochemically related P-450 isozyme(s) play a major role in debrisoquine 4-hydroxylation as well as in BTL 4-hydroxylation in rat liver microsomes.

The content of cytochrome P-450 in liver microsomes from guinea pigs was decreased by ascorbic acid-deficiency. Since ascorbic acid is an antioxidant in vivo, the possible involvement of lipid peroxidation in this phenomenon was investigated. In fact, the level of lipid peroxides in liver homogenates of guinea pigs was increased by ascorbic acid deficiency. The level was significantly decreased when the animals were given tocopherol acetate (25 mg/kg/day, s.c.) with an ascorbic acid-free diet. The activities of aminopyrine N-demethylase, aniline hydroxylase, p-nitroanisole O-demethylase and 7-ethoxycoumarin O-deethylase, and the content of cytochrome P-450 spectrally determined did not restore the control level by the administration of tocopherol acetate to the ascorbic acid-deficient animals. Western blot analysis of liver microsomes with antibodies to rat P-450IA2 (P-448-H), P-450IIB1 (P-450b) and human P-450IIIA4 (P-450NF) showed that ascorbic acid-deficiency resulted in a decrease in the amount of cytochrome P-450 immunochemically related to P-450IA2, but not the amounts of the forms of cytochrome P-450 cross-reactive with antibodies to P-450IIB1 and P-450IIIA4. The reduced amounts of cytochrome P-450 cross-reactive with antibodies to rat P-450IA2 in liver microsomes of ascorbic acid-deficient animals remained unchanged even when lipid peroxidation was inhibited by tocopherol acetate, suggesting that there is a mechanism(s) other than lipid peroxidation involved in the reduction of amounts of cytochrome P-450 by ascorbic acid deficiency.

Oxidative metabolism of delta 9-tetrahydrocannabinol (THC) by liver microsomes was studied in female rats. Delta 9-THC was mainly biotransformed to 11-hydroxy-delta 9-THC (11-OH-delta 9-THC) and 9 alpha,10 alpha-epoxy-hexahydrocannabinol (EHHC) by liver microsomal fraction of adult female rat. Two isozymes of cytochrome P-450 (P-450) [F-1 (IIC6) and F-2 (IIC12)] were purified from liver microsomes of female rats and oxidation activities toward delta 9-THC were assessed in the reconstituted system containing NADH-P-450 reductase and cytochrome b5. P-450 F-1 showed considerable activity toward 11-OH-delta 9-THC formation (10.62 nmol/min/nmol of P-450), whereas P-450 F-2 did not show any activity toward delta 9-THC oxidation under the conditions used. Preincubation of microsomes with antiserum against P-450 F-1 obtained from rabbits caused a marked decrease in 11-OH-delta 9-THC formation, whereas antiserum against P-450 F-2 did not exhibit any inhibitory effect on the oxidation of delta 9-THC by liver microsomes of adult female rats. Further, antiserum against P-450 F-1 or F-2 did not affect the microsomal formation of 9 alpha,10 alpha-EHHC from delta 9-THC. These results indicate that P-450 F-1 and its immunochemically related P-450 isozyme(s) play important roles in the formation of an active metabolite, 11-OH-delta 9-THC, from delta 9-THC by liver microsomes of adult female rats.

Age-related changes in the levels of multiple forms of cytochrome P-450 as well as in the testosterone hydroxylation activities of hepatic microsomes of male and female rats of different ages from 1 week to 104 weeks (24 months) were investigated. The total cytochrome P-450 measured photometrically did not change much with age in either male and female rats. Testosterone 2 alpha-, 2 beta-, 6 beta-, 15 alpha-, 16 beta-hydroxylation activities of male rats were much higher than those in female rats and were induced developmentally. These activities in male rats declined with aging to the very low level in female rats by 104 weeks of age. Testosterone 7 alpha-hydroxylation activity was maximum at 3 weeks of age in rats of both sexes. The levels of individual cytochrome P-450s were measured by immunoblotting. P450IA1 and IA2 (3-methylcholanthrene-inducible forms) and P450IIB1 and IIB2 (phenobarbital-inducible forms) were detected at low levels in rats of both sexes at all ages. P450IIA2, IIC11 and IVA2 were detected in male rats only and were induced developmentally. These male-specific forms disappeared in male rat liver at 104 weeks of age. P450IIC12, a typical female-specific form, was induced developmentally in female rats and was also detected in male rats at 3 and 104 weeks of age. P450IIIA2 (testosterone 6 beta-hydroxylase) was induced developmentally in male rats, but disappeared when the rats were 104 weeks of age. In female rats, P450IIIA2 was detected only at 1 and 3 weeks of age. P450IIA1, IIC6, IIE1 and IVA3 were detected in rats of both sexes at any age. P450IIC6 and IVA3 were induced developmentally and detected at a similar level in rats of both sexes. The level of P450IIA1 was maximum at 3 weeks of age in rats of both sexes. The changes in the level of P450IIE1 during aging were small compared with the changes in other cytochrome P-450s used in this study. These observations provide concrete evidence to our earlier hypothesis that each of the forms of cytochrome P-450 in male rats alter with aging in different patterns resulting in a practical feminization of over-all cytochrome P-450 composition at old age.

Cytochrome P450 isozymes purified from rat hepatic microsomes were able to catalyse the oxidation of 11-oxo-delta 8-tetrahydrocannabinol (11-oxo-delta 8-THC) to delta 8-THC-11-oic acid in the presence of NADPH, cytochrome P450 reductase and dilauroylphosphatidylcholine. The catalytic activities (nmol/min/nmol P450) of cytochrome P450s, UT-2 (IIC11), UT-4 (IIA2), UT-5 (IIC13), PB-1, PB-2 (IIC6), PB-4 (IIB1), MC-1 (IA2), MC-5 (IA1) and IF-3 (IIA1), were 0.69, 0.08, 0.07, 0.23, 0.46, 0.02, 0.06, 0.07 and 0.34, respectively, whereas the activities of cytochrome P450s, PB-5 (IIB2) and DM (IIE1), were less than 0.02 nmol/min/nmol P450. Cytochrome P450 IIC11 showed the highest catalytic activity of the cytochromes examined. The mechanism for the oxidation of 11-oxo-delta 8-THC to delta 8-THC-11-oic acid by cytochrome P450 IIC11 was established as being an oxygenation since one atom of oxygen-18 was exclusively incorporated into the carboxylic acid formed under 18O2. The antibody raised to cytochrome P450 IIC11 inhibited by 60% the hepatic microsomal oxidation of 11-oxo-delta 8-THC to delta 8-THC-11-oic acid in male rats. These results indicate that cytochrome P450 IIC11 is a major form of the cytochrome to catalyse the oxidation of 11-oxo-delta 8-THC to delta 8-THC-11-oic acid in the hepatic microsomes of male rats and that the oxidation of aldehyde to carboxylic acid is a catalytic activity common to most isozymes of P450.

The effect of lipid peroxidation in vitro on the amounts of several forms of cytochrome P-450 in liver microsomes from guinea-pigs was investigated. Lipid peroxide formation in liver microsomes from ascorbic acid (VC)-deficient animals was much higher than that observed in control animals. The antibodies to rat P-450IA2 (P-448-H), P-450IIB1 (P-450b) and human P-450IIIA4 (P-450NF) recognized one or two forms of cytochrome P-450 in liver microsomes of guinea-pigs. Neither cytochrome P-450 cross-reactive with anti-P-450IIB1 antibodies nor cytochrome P-450 cross-reactive with antibodies to P-450IIIA4 was virtually affected by microsomal lipid peroxidation induced by NADPH in vitro. In contrast, the forms of cytochrome P-450 immunochemically related to P-450IA2 were decreased with the increased level of lipid peroxide formation. The form-specific degradation of cytochrome P-450 due to lipid peroxidation was in agreement with our previous observation that the amounts of cytochrome P-450 cross-reactive with antibodies to P-450IA2 but not with antibodies to P-450IIIA (P-450PB-1) were predominantly decreased in VC-deficient guinea-pigs compared to control animals in vitro.

For identification of microsomal cytochrome P-450 (P-450) enzymes which catalyze 2- or 4-hydroxylations of estrogens in the rat liver, estradiol (E2) and estradiol 17-sulfate (E2-17-S) were selected as the substrates and incubated with various kinds of purified P-450 enzymes: PB-1, PB-2, PB-4 and PB-5 obtained from phenobarbital-treated male rats (Sprague-Dawley); MC-1 and MC-5 from 3-methylcholanthrene-treated male rats; and UT-1, UT-2, UT-4 and UT-5 from untreated animals. The reactions were carried out under the P-450-reconstructed system, and the resulting products were determined by HPLC using electrochemical detection. All the enzymes tested were shown to have varying degrees of catalytic activities for 2-hydroxylation of the two substrates; UT-1 and UT-2 had the highest activity. Of the induced P-450 enzymes, PB-2 and MC-1 showed fairly high catalytic activity for 4-hydroxylation of E2. The P-450 enzymes obtained from the untreated male rats, especially UT-4, showed the highest catalytic activity for 4-hydroxylation of the two substrates. From these results and also from kinetic experiments, the P-450 enzymes which catalyze 2- and 4-hydroxylations of estrogen were considered to be different species. A part of E2 was converted to such metabolites as estrone and those having a hydroxyl group at positions 6 beta, 15 alpha or 16 alpha, each production of which was estimated to be catalyzed by single or multiple P-450s.

The differences in the levels of cytochrome P-450s in hepatic and renal microsomes between spontaneously hypertensive rats (SHR) and normotensive control rats (Wistar Kyoto rats, WKY) were investigated by Western blotting with a specific antibody. Differences in the metabolic activity of the microsomes were also studied. In hepatic microsomes, the content of P450 PB-1 (IIIA2) was 140% higher in SHR than in WKY and the content of P450 IF-3 (IIA1) in SHR was one-seventh that in WKY. The differences reflected the increase in testosterone 6 beta-hydroxylation activity and decrease in testosterone 7 alpha-hydroxylation activity in hepatic microsomes of SHR. The level of P450 K-5 (IVA2) in hepatic microsomes of SHR was 4-times that in microsomes of WKY. The levels of other cytochrome P-450s in SHR were not very different from those in WKY. In renal microsomes, the levels of three renal cytochrome P-450s, P450 K-2, K-4, and K-5, were measured. The level of P450 K-5 (fatty acid omega-hydroxylase) in SHR was 50% higher than that in WKY and the difference reflected the increase in lauric acid omega- and (omega-1)-hydroxylation activities of the renal microsomes of SHR. The levels of P450 K-2 and K-4 did not differ in both rats.

Ascorbic acid (VC) deficiency resulted in a decrease in the activities of aminopyrine N-demethylase, aniline hydroxylase, and p-nitroanisole O-demethylase and in the content of cytochrome P-450, as spectrally determined, whereas it caused an increase in the activities of 6 beta-hydroxylases for testosterone and progesterone in liver microsomes of guinea pigs. Western blot analysis of liver microsomes with antibodies to rat P-448-H (P-4501A2), P-450j (P-450IIE), P-450 PB-1 (P-450IIIA), and P-450b (P-450IIB1) showed that VC deficiency decreased the amount of cytochrome P-450 immunochemically related to P-450IA2 and P-450IIE but did not change the amount of the form that was cross-reactive with antibodies to P-450IIB1 and tended to slightly increase (not statistically significantly) the amount of the form of the cytochrome immunochemically related to P-450IIIA. The larger decrease by VC deficiency in the amount of cytochrome P-450 that was cross-reactive to the rat P-450IA2 resulted in a lower capacity of liver microsomes to activate promutagens, such as 2-amino-3-methyl-imidazo(4,5-f)quinoline and aflatoxin B1. These results indicate that VC deficiency in guinea pigs differentially affects the content of individual forms of cytochrome P-450.

The potent hepatocarcinogen 3-methoxy-4-aminoazobenzene (3-MeO-AAB) has been reported to be bioactivated to mutagenic intermediates by rat liver microsomal cytochrome P450 (P450) and to be a selective inducer of rat P450IA2. In this study we have further investigated the roles of individual rat and human P450 enzymes in the bioactivation of this hepatocarcinogen in a Salmonella typhimurium TA1535/pSK1002 system where umu response is indicative of DNA damage. 3-MeO-AAB was found to be bioactivated by liver microsomal enzymes from rats and humans in this assay system. The liver microsomal activities are increased by pretreatment of rats with various P450 inducers such as phenobarbital (PB), beta-naphthoflavone (BNF), dexamethasone (DEX), acetone, ethanol, isoniazid (INH), diphenylhydantoin and valproic acid, and can be inhibited considerably by SKF-525A and metyrapone. alpha-Naphthoflavone (ANF) is also an inhibitor for the reaction catalyzed in BNF-treated rats, but stimulated the microsomal activity in DEX-treated rats. Evidence has also been obtained that specific antibodies raised against P450IIB1, P450IA1 or IA2, P450IIE1, and P450IIIA2 inhibited the activation in liver microsomes from rats pretreated with PB, BNF, INH and DEX respectively, suggesting the possible roles of several P450 enzymes in the bioactivation of 3-MeO-AAB. The results obtained with reconstituted monooxygenase systems containing various rat P450 enzymes are highly supportive of this conclusion. Human liver microsomal activation of 3-MeO-AAB was also inhibited to various extents by antibodies raised against P450IA2, P450MP, P450IIE1 and P450IIIA4. In a reconstituted system containing purified forms of human P450, P450IA2 was the most active in catalyzing 3-MeO-AAB, followed by P450IIIA4 and P450MP. ANF, a known activator of P450IIIA-catalyzed reactions, caused an increase in activation of 3-MeO-AAB in human liver microsomal and P450IIIA4- and P450MP-containing reconstituted systems. From these results it is concluded that multiple P450 enzymes in rat and human liver microsomes are involved in the bioactivation of 3-MeO-AAB, regardless of its selective induction of the rat P450IA2 gene.

The metabolism of lidocaine by human hepatic microsomes and purified human cytochrome P-450s, P-450NF (P450IIIA4), P-450MP (a P450IIC form) and P-450PA (P450IA2) was examined and their metabolism was compared with that by rat hepatic cytochrome P-450s. Human hepatic microsomes produced monoethylglycinexylidide (MEGX) and 3-hydroxylidocaine (3-OH-LID) from lidocaine. In a reconstituted system with dilauroylphosphatidylcholine, P-450NF efficiently produced MEGX. P-450PA was not efficient in lidocaine N-deethylation (formation of MEGX) but produced 3-OH-LID. P-450NF and P-450MP did not produce 3-OH-LID. Lidocaine N-deethylation activity of P-450NF was enhanced in a modified reconstituted system with a phospholipid mixture and sodium cholate. P-450NF appears to be an ortholog to rat P450 PB-1 (P450IIIA2). Anti-P450 PB-1 antibody cross-reacted with P-450NF and efficiently inhibited lidocaine N-deethylation in human hepatic microsomes. The correlation of lidocaine N-deethylation activity with the concentration of P-450NF determined immunochemically with anti-P450 PB-1 antibody was good (r = 0.81). In addition, correlation between P-450NF content estimated with anti-P450 PB-1 and anti-P-450NF preparations was good (r = 0.96). These results suggest that rat P450 PB-1 and human P-450NF have closely related properties and P-450NF is the major enzyme involved in lidocaine N-deethylation in human hepatic microsomes.

delta 9-Tetrahydrocannabinol (THC) was incubated with a reconstituted system consisting of dilauroylphosphatidylcholine, NADPH-cytochrome c reductase, cytochrome b5, and cytochrome P-450 (P-450) isozyme UT-2, UT-4, or UT-5, which was purified from liver microsomes of adult male rats. It was biotransformed by UT-2 to 11-OH-delta 9-THC and 3'-OH-delta 9-THC, and by UT-4 to 8 beta-OH-delta 9-THC and 11-OH-delta 9-THC. UT-5, however, showed only a little activity for 11-OH-delta 9-THC formation. Activity of the isozyme UT-2 for 11-OH-delta 9-THC formation from delta 9-THC was calculated to be 4.07 nmol/min/nmol P-450 while those of UT-2 for the formations of 16 alpha-OH-testosterone (16 alpha-OH-T), 2 alpha-OH-T, and androstenedione from testosterone were 14.7, 6.6, and 2.2 nmol/min/nmol P-450, respectively. Anti-P-450 UT-2 IgG fraction obtained from rabbit serum dose-dependently suppressed formations of 16 alpha-OH-T, 2 alpha-OH-T, and androstenedione from testosterone with liver microsomes of adult male rats. The antibody, in the amount that inhibited above 90% of 16 alpha-OH-T and 2 alpha-OH-T formations from testosterone, also reduced 80% of the microsomal formations of 11-OH-delta 9-THC and 3'-OH-delta 9-THC from delta 9-THC, as compared with control experiments using preimmune IgG fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanism of inhibitory effect of cannabidiol (CBD) on the hepatic drug-metabolizing enzyme system was studied in adult male rats in vivo. Time course studies revealed that microsomal d-benzphetamine N-demethylation and testosterone 2 alpha-, 16 alpha- and 17-oxidation were markedly suppressed 6 to 48 h after the single administration of CBD (10 mg/kg, intraperitoneally). Decreases in activities of aniline hydroxylation and p-nitroanisole O-demethylation and in content of total cytochrome P450 were intermittent and moderate. On the other hand, no change was observed in reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome c reductase activity or cytochrome b5 content in the hepatic microsomes of the CBD-treated rats. Western blotting analysis showed a marked decrease in the male-specific cytochrome P450 UT-2 in the hepatic microsomes, especially 24 to 48 h after pretreatment with CBD. It is possible that CBD given 6 to 12 h before the sacrifice might interact with cytochrome P450 as a substrate, resulting in inhibition of the drug-metabolizing enzyme activities in the earlier stages. In the later stages from 24 to 48 h after CBD treatment, the reduction in content of the male-specific cytochrome P450 UT-2 may play a major role in the inhibitory effect of CBD on the hepatic drug-metabolizing enzyme system in the adult male rat in vivo.

The metabolism of arachidonic acid, lauric acid and prostaglandin A1 by rat hepatic microsomes and multiple forms of cytochrome P-450 purified from rat hepatic microsomes was studied. Arachidonic acid was hydroxylated by hepatic microsomes of male rats by omega- and (omega-1)-hydroxylation. Phenobarbital treatment of rats decreased the hydroxylation activity slightly, but 3-methylcholanthrene treatment increased the hydroxylation activity 2-fold. However, lauric acid and prostaglandin A1 omega- and omega-1)-hydroxylation activities decreased after treatment with phenobarbital and 3-methylcholanthrene. Arachidonic acid and lauric acid were metabolized with similar ratios of omega- and (omega-1)-hydroxylation, but prostaglandin A1 was efficiently metabolized at the omega-position by hepatic microsomes of untreated male rats. In a reconstituted system with purified cytochromes P-450, P450 UT-1, UT-2 (P-450h), MC-1 (P-450d) and MC-5 (P-450c) effectively hydroxylated arachidonic acid at both the omega- and (omega-1)-position. P450 UT-8 hydroxylated arachidonic acid only at the omega-position. P450 DM (P-450j) hydroxylated arachidonic acid at the (omega-1)-position efficiently. Lauric acid was also hydroxylated by P450 UT-1, UT-2, PB-1, PB-2, MC-1, IF-3 (P-450a) and DM, at the (omega - 1)-position only. Only P450 UT-8 could hydroxylate laruic acid at the omega-position. Prostaglandin A1 was efficiently and specifically metabolized by P450 UT-8 with omega-hydroxylation. P450 UT-2 and PB-1 could hydroxylate prostaglandin A1 by (omega-1)-hydroxylation, but with low activity.

The effects of starvation on the composition of 12 different cytochrome P450s in rat hepatic microsomes were studied with a specific antibody. Changes in the metabolic activity of the microsomes were studied at the same time. P450 DM (P450j) was induced 2.5-fold by a 48-h starvation and its increase reflected the increase of metabolic activity of hepatic microsomes toward aniline, 7-ethoxycoumarin, and N-nitrosodimethylamine. P450 K-5, the major renal cytochrome P450 in untreated male rat, was also induced 2.5-fold by a 48-h starvation. P450 UT-2 (P450h) and P450 UT-5 (P450g), typical male-specific forms, decreased with starvation. P450 UT-2 had high testosterone 2 alpha- and 16 alpha-hydroxylation activities. These activities of hepatic microsomes were reduced with the decrease in P450 UT-2. P450 PB-1, testosterone 6 beta-hydroxylase, was increased time-dependently by starvation. P450 UT-4 (RLM2), a minor male-specific form, was not changed by starvation. P450 PB-2 (P450k), present in both sexes, was changed little by starvation. P450 PB-4 (P450b) and P450 PB-5 (P450e) are strongly induced in rat liver by phenobarbital in coordinate fashion. Starvation increased P450 PB-4 12-fold but reduced P450 PB-5 to 22% of the control level. P450 MC-1 (P450d) was decreased by starvation. P450 MC-5 (P450c) was barely detected in control rats and was not changed by starvation. P450 IF-3 (P450a), rich in immature rats, was increased by starvation, accompanied by an increase in testosterone 7 alpha-hydroxylation activity in the hepatic microsomes. We further investigated whether new cytochrome P450s appeared upon starvation by comparison of chromatographic profiles of cytochrome P450 from starved rats with those of cytochrome P450 from control rats using HPLC. Three new cytochrome P450s were detected in the starved rats. These cytochrome P450s were purified to homogeneity. One of them was P450 DM, judging from spectral properties, catalytic activity, and the NH2-terminal sequence. The two other forms were designated P450 3b and 4b. The minimum molecular weights of P450 3b and 4b were 53,000 and 52,000, respectively, and their CO-reduced absorption maxima were at 449 and 452 nm, respectively. P450 3b metabolized aminopyrine, N-nitrosodimethylamine, 7-ethoxycoumarin, and lauric acid, but with low activity. P450 4b was efficient in lauric acid omega- and (omega-1)-hydroxylation only. The spectral properties, catalytic activity, peptide map, and NH2-terminal sequence of P450 4b agreed with those of P450 K-5. P450 3b was a new cytochrome P450, judged by these criteria.

Six rat hepatic cytochromes P-450, named P-450IF-1-6, were purified from hepatic microsomes of immature female rats by high-performance liquid chromatography with anion-exchange, cation-exchange, and hydroxylapatite columns. The purified forms, except for P-450IF-4, gave a single protein-staining band on SDS-polyacrylamide gel electrophoresis, with a minimum molecular weight of 50,000 for P-450IF-1, 49,000 for P-450IF-2, 47,000 for P-450IF-3, 53,500 for P-450IF-5, and 54,000 for P-450IF-6. The CO-reduced spectral maximum of these forms was at 450 nm for P-450IF-1, 448 nm for P-450IF-2, 451 nm for P-450IF-3, 449 nm for P-450IF-4, 449 nm for P-450IF-5, and 450 nm for P-450IF-6. All of these cytochromes had the low-spin state of heme in the oxidized form. P-450IF-4 had high metabolic activity for both benzphetamine and 7-ethoxycoumarin. P-450IF-5 had moderate activity toward 7-ethoxycoumarin. P-450IF-3 catalyzed the hydroxylation of testosterone at the 7 alpha-position effectively, but the other forms did not hydroxylate testosterone. Analysis of the NH2-terminal sequence showed that P-450IF-1, 2, 3, 5, and 6 differed structurally from each other. The sequences of P-450IF-1 and IF-2 were somewhat homologous, but the NH2-terminal sequences of the other forms were all different. Based on these results, we concluded that P-450IF-1 corresponded to one of the phenobarbital-inducible forms in male rat liver. P-450IF-2 was a female-specific form and its concentration was low.(ABSTRACT TRUNCATED AT 250 WORDS)

A diabetes-inducible form of cytochrome P-450, termed P-450DM, was purified to electrophoretical homogeneity (MW 51,000) by high-performance liquid chromatography from liver microsomes of diabetic rats induced with streptozotocin. The CO-reduced absorption maximum of P-450DM was at 452 nm and the oxidized heme iron appeared to be predominately in the high-spin state as deduced from the Soret maximum at 395 nm. P-450DM was active in aniline hydroxylation and N-nitrosodimethylamine demethylation. The dealkylation activity toward 7-ethoxycoumarin by P-450DM was much enhanced by the addition of cytochrome b5.

The mechanism of the suppression of an ethanol-inducible cytochrome P-450 (P-450DM/j) by pituitary hormone has been studied in rats. The hepatic content of P-450DM/j protein quantitated by Western blots was low but was 2-fold higher in male than female untreated rats (75 and 34 pmol/mg of protein, respectively). The content was increased 2.6-fold (male) and 5.6-fold (female) by hypophysectomy and the sex-related difference was abolished. Treatment of hypophysectomized rats with human growth hormone (hGH), but not with prolactin, reversed the increased amounts of P-450DM/j protein. The hGH-induced suppression was more effective with the continuous infusion than intermittent injection. The hepatic level of P-450DM/j mRNA, determined by the use of a 23-mer oligonucleotide probe, was also changed by hypophysectomy and/or hGH-treatment, largely in parallel with the changes in the content of P-450DM/j protein and microsomal p-nitrophenol and aniline hydroxylations. These results suggest that growth hormone exerts the suppressive effect on P-450DM/j through a somatogenic receptor-mediated process. In another growth hormone-depleted condition, diabetes, the hepatic level of P-450DM/j mRNA was also increased to a level similar to that in hypophysectomized rats, but the protein content was 2- to 3-fold higher in diabetic than hypophysectomized rats. These results indicate, in addition to the reduction of serum growth hormone level, the presence of another stimulatory factor, which acts translationally or posttranslationally in livers of diabetic rats. On the other hand, coordinate changes in the level of P-450DM/j protein and the mRNA in hypophysectomized rats indicate that growth hormone acts rather directly and suppresses the level of P-450DM/j mainly at a pretranslational step in rat livers.