6alpha-hydroxylation of taurochenodeoxycholic acid and lithocholic acid by CYP3A4 in human liver microsomes

The aim of the present study was to identify the enzymes in human liver catalyzing hydroxylations of bile acids. Fourteen recombinant expressed cytochrome P450 (CYP) enzymes, human liver microsomes from different donors, and selective cytochrome P450 inhibitors were used to study the hydroxylation of taurochenodeoxycholic acid and lithocholic acid. Recombinant expressed CYP3A4 was the only enzyme that was active towards these bile acids and the enzyme catalyzed an efficient 6alpha-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid. The Vmax for 6alpha-hydroxylation of taurochenodeoxycholic acid by CYP3A4 was 18.2 nmol/nmol P450/min and the apparent Km was 90 microM. Cytochrome b5 was required for maximal activity. Human liver microsomes from 10 different donors, in which different P450 marker activities had been determined, were separately incubated with taurochenodeoxycholic acid and lithocholic acid. A strong correlation was found between 6alpha-hydroxylation of taurochenodeoxycholic acid, CYP3A levels (r2=0.97) and testosterone 6beta-hydroxylation (r2=0.9). There was also a strong correlation between 6alpha-hydroxylation of lithocholic acid, CYP3A levels and testosterone 6beta-hydroxylation (r2=0.7). Troleandomycin, a selective inhibitor of CYP3A enzymes, inhibited 6alpha-hydroxylation of taurochenodeoxycholic acid almost completely at a 10 microM concentration. Other inhibitors, such as alpha-naphthoflavone, sulfaphenazole and tranylcypromine had very little or no effect on the activity. The apparent Km for 6alpha-hydroxylation of taurochenodeoxycholic by human liver microsomes was high (716 microM). This might give an explanation for the limited formation of 6alpha-hydroxylated bile acids in healthy humans. From the present results, it can be concluded that CYP3A4 is active in the 6alpha-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid in human liver.     

The effect of a 2-h exposure to cigarette smoke on the metabolic activation of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in A/J mice.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, induces lung adenomas in A/J mice following a single intraperitoneal (i.p.) injection. However, inhalation of mainstream cigarette smoke does not induce or promote NNK-induced lung tumors in this mouse strain purported to be sensitive to chemically-induced lung tumorigenesis. The critical events for NNK-induced lung tumorigenesis in A/J mice is thought to involve O(6)-methylguanine (O(6)MeG) adduct formation, GC-->AT transitional mispairing, and activation of the K-ras proto-oncogene. The objective of this study was to test the hypothesis that a smoke-induced shift in NNK metabolism led to the observed decrease in O(6)MeG adducts in the lung and liver of A/J mice co-administered NNK with a concomitant 2-h exposure to cigarette smoke as observed in previous studies. Following 2 h nose-only exposure to mainstream cigarette smoke (600 mg total suspended particulates/m(3) of air), mice (n=12) were administered 7.5 micromol NNK (10 microCi [5-3H]NNK) by i.p. injection. A control group of 12 mice was sham-exposed to HEPA-filtered air for 2 h prior to i.p. administration of 7.5 micromol NNK (10 microCi [5-3H]NNK). Exposure to mainstream cigarette smoke had no effect on total excretion of NNK metabolites in 24 h urine; however, the metabolite pattern was significantly changed. Mice exposed to mainstream cigarette smoke excreted 25% more 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) than control mice, a statistically significant increase (P<0.0001). Cigarette smoke exposure significantly reduced alpha-hydroxylation of NNK to potential methylating species; this is based on the 15% reduction in excretion of the 4-(3-pyridyl)-4-hydroxybutanoic acid and 42% reduction in excretion of 4-(3-pyridyl)-4-oxobutanoic acid versus control. Detoxication of NNK and NNAL by pyridine-N-oxidation, and glucuronidation of NNAL were not significantly different in the two groups of mice. The observed reduction in alpha-hydroxylation of NNK to potential methylating species in mainstream cigarette smoke-exposed A/J mice provides further mechanistic support for earlier studies demonstrating that concurrent inhalation of mainstream cigarette smoke results in a significant reduction of NNK-induced O(6)MeG adduct formation in lung and liver of A/J mice compared to mice treated only with NNK.     

Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by inducible and constitutive cytochrome P450 enzymes in rats.

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces tumor formation in the liver, lung, nasal cavity, and pancreas of rats. Metabolic activation is required for the tumorigenicity of this compound. The involvement of cytochrome P450 enzymes in NNK bioactivation was investigated in rats by studies with chemical inducers and antibodies against P450s. Liver microsomal enzymes catalyzed the formation of 4-oxo-1-(3-pyridyl)-1-butanone (keto aldehyde), 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol), 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone (NNK-N-oxide), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) from NNK. When the activity was expressed on a per nanomole P450 basis, treatments of rats with 3-methylcholanthrene (MC), phenobarbital (PB), pregnenolone 16-alpha-carbonitrile (PCN), Aroclor 1254 (AR), safrole (SA), and isosafrole (ISA) increased the keto aldehyde formation in liver microsomes 2.0-, 2.4-, 3.8-, 2.5-, 2.1-, and 1.8-fold, respectively; PB, AR, SA, and ISA increased the keto alcohol formation 1.7-, 1.3-, 2.0-, and 1.3-fold, respectively. The extents of induction were more pronounced when expressed on a per milligram protein basis, due to the higher microsomal P450 contents in the induced microsomes. The formation of NNK-N-oxide was markedly increased by PB and PCN and slightly increased by AR, SA, and ISA. However, the formation of NNAL, the major metabolite due to carbonyl reduction, was not increased by the treatments but was decreased by AR, ISA, and acetone (AC). The kinetic parameters of NNK metabolism by control, MC-, PB-, and PCN-induced liver microsomes were obtained. A panel of monoclonal (anti-1A1, -2B1, -2C11, and -2E1) and polyclonal (anti-1A2, -2A1, and -3A) antibodies were used to assess the involvement of constitutive hepatic P450 enzymes in NNK metabolism. Keto aldehyde formation was inhibited by anti-1A2 and anti-3A (about 15%) but not by others; the formation of keto alcohol was inhibited by anti-1A2, anti-2A1, and anti-3A (by 13-26%). In incubations with lung microsomes, the formation of keto aldehyde, keto alcohol, NNK-N-oxide, and NNAL were observed. With nasal mucosa microsomes, however, only keto aldehyde and keto alcohol formation were appreciable. SA and AC significantly decreased NNK metabolism in lung and nasal mucosa microsomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of vitamins and common drugs on reduction of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in rat microsomes.

4-(Methylnitrosamino)-1-(3-pyridyl)-butanone (NNK) is a tobacco-specific nitrosamino that requires metabolic activation by cytochrome P450 enzymes. The activation of NNK by cytochrome P450 enzymes leads to the formation of different metabolites. Detoxification of NNK usually occurs via carbonyl reduction to its hydroxyl product, 4-(methylnitrosamino)-1-(3-pyridyl)-butanol (NNAL). In the present study, the influences of common vitamins and P450 modulators on the reduction of NNK by rat microsomes were studied. The formation of NNAL but not other metabolites was detected by the described HPLC method. Among the vitamins tested, vitamins E, A (retinol), B6 and B5 were found to be marginal effective upon reduction of NNK while vitamins A (cis-acid), A (trans-acid), D2, D3, K1, K3, B1 and A (crocetin) increased the formation of NNAL from 3 to 21%. The effect of vitamin C-palmitate (<10 microM) was most pronounced followed by crocetin upon reduction of NNK. Clonidine, tolbutamide and atropine slightly increased the reduction of NNK while cimetidine showed no effects. The modulation of NNK reduction could reduce the carcinogenic potential of NNK, since the main detoxification pathway of NNK involves carbonyl reduction.     

Nicotine and 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Binding and Access Channel in Human Cytochrome P450 2A6 and 2A13 Enzymes

Cytochromes P450 (CYP) from the 2A subfamily are known for their roles in the metabolism of nicotine, the addictive agent in tobacco, and activation of the tobacco procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Although both the hepatic CYP2A6 and respiratory CYP2A13 enzymes metabolize these compounds, CYP2A13 does so with much higher catalytic efficiency, but the structural basis for this has been unclear. X-ray structures of nicotine complexes with CYP2A13 (2.5 Å) and CYP2A6 (2.3 Å) yield a structural rationale for the preferential binding of nicotine to CYP2A13. Additional structures of CYP2A13 with NNK reveal either a single NNK molecule in the active site with orientations corresponding to metabolites known to form DNA adducts and initiate lung cancer (2.35 Å) or with two molecules of NNK bound (2.1 Å): one in the active site and one in a more distal staging site. Finally, in contrast to prior CYP2A structures with enclosed active sites, CYP2A13 conformations were solved that adopt both open and intermediate conformations resulting from an ∼2.5 Å movement of the F to G helices. This channel occurs in the same region where the second, distal NNK molecule is bound, suggesting that the channel may be used for ligand entry and/or exit from the active site. Altogether these structures provide multiple new snapshots of CYP2A13 conformations that assist in understanding the binding and activation of an important human carcinogen, as well as critical comparisons in the binding of nicotine, one of the most widely used and highly addictive drugs in human use.     

Characterization of testosterone 16 alpha-hydroxylase (I-P-450(16) alpha) induced by phenobarbital in mice

Cytochrome P-450 (I-P-450(16) alpha), which is associated with phenobarbital-induced testosterone 16 alpha-hydroxylation activity, was purified from livers of phenobarbital-treated female 129/J mice on the basis of the specific hydroxylation activity in fractions eluted from columns of octylamino-Sepharose 4B, hydroxylapatite, DEAE-Bio-Gel A, and isobutyl-Sepharose 4B. The specific cytochrome P-450 content of the purified I-P-450(16) alpha fraction was 12.4 nmol/mg of protein, and it had an apparent molecular weight of 54K. The specific activity of reconstituted testosterone 16 alpha-hydroxylation activity with the purified I-P-450(16) alpha fraction was 6-8 nmol min-1 (nmol of cytochrome P-450)-1. Rabbit antibody raised against the purified I-P-450(16) alpha fraction inhibited nearly 100% of the 16 alpha-hydroxylation activity in liver microsomes of phenobarbital-treated female 129/J mice but did not affect hepatic microsomal 16 alpha-hydroxylation activity of untreated male and female 129/J mice at all. In hepatic microsomes of phenobarbital-treated male 129/J mice, 70% of the 16 alpha-hydroxylation activity, at most, was catalyzed by I-P-450(16) alpha, and the residual 30% of the activity was catalyzed by C-P-450(16) alpha. The increase of I-P-450(16) alpha by phenobarbital was due to de novo synthesis of I-P-450(16) alpha, and this induction was not sexually regulated in 129/J mice. Anti-C-P-450(16) alpha [Harada, N., & Negishi, M. (1984) J. Biol. Chem. 259, 12285-12290] did not inhibit the 16 alpha-hydroxylation catalyzed by I-P-450(16) alpha; thus, I-P-450(16) alpha and C-P-450(16) alpha are immunochemically distinct isozymes of testosterone 16 alpha-hydroxylase.     

Functional characterization of medaka CYP3A38 and CYP3A40: kinetics and catalysis by expression in a recombinant baculovirus system

Phylogenic analysis of the teleost genomic lineages has demonstrated the precedent for multiple genome duplications. Among many of the genes duplicated, cytochrome P450 genes have undergone independent diversification, which can be traced to a single ancestral gene. In teleosts, cytochrome P450s, from all major families, have been identified. Among these, the CYP3A family has been cloned in several teleost species and demonstrated to contain multiple paralogs differing in gene expression patterns and tissue distribution. Herein we characterized the catalytic and kinetic activities of two medaka CYP3A paralogs (CYP3A38 and CYP3A40) with benzyloxyresorufin (BFC), a fluorescent 3A-selective substrate, and testosterone, a known metabolic substrate for CYP3A enzymes. Recombinant CYP3A was produced using the baculovirus expression vector system in Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn5) insect cells and accounted for up to 24% of total cellular protein. Following addition of a heme-albumin conjugate to log phase cells, spectral P450 content reached a maximum of 560 and 2350 pmol/mg microsomal protein for CYP3A38 and CYP3A40, respectively. Incubations containing recombinant CYP3A, human NADPH-cytochrome P-450 oxidoreductase reductase, human cytochrome b5, and a NADPH generation system catalyzed the dealkylation of BFC and hydroxylation of testosterone with a high degree of stereoselectivity. However, efficiencies and specificities were significantly different between the two isoforms. Km and Vmax activities based on BFC-catalysis were 0.116 and 0.363 muM, and 7.95 and 7.77 nmol/min/nmol P450 for CYP3A38 and CYP3A40, respectively. CYP3A38 preferentially catalyzed testosterone hydroxylation at the 6beta-, 2beta- and 16beta-positions with minor hydroxylation at other positions within the steroid nucleus. Testosterone catalysis with CYP3A40 was limited predominantly to the 6beta- and 2beta-positions. Putative identification of CYP3A substrate recognition sites (SRS) 1-6 indicates that 12 of the 49 amino acid differences between CYP3A38 and CYP3A40 OFRs occur in SRS regions previously known to be associated with steroid hydroxylation. We suggest that differences in kinetics and catalytic activities are a result of amino acid substitutions in SRS regions 1, 3 and 5 within the CYP3A38 and CYP3A40 protein sequence.     

A high-level expression of CYP2A in the lung of the suncus (Suncus murinus) and its role in the activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

Northern blot analysis of mRNA prepared from the lung of Suncus murinus (suncus), which was classified as an ancestor of primates, revealed that the expression level of cytochrome P450 2A (CYP2A) mRNA was about 100-fold higher than in the lung from rats and mice. To confirm that the pulmonary CYP2A of the suncus had a catalytic activity, the metabolism of a specific substrate for CYP2A6, (+)-cis-3,5-dimethyl-2-(3-pyridyl) thiazolidin-4-one hydrochloride (SM-12502), was determined. The intrinsic clearance for SM-12502 S-oxidation by the suncus lung microsomes was calculated to be 99-fold higher than that by rat liver microsomes. The mutagen-producing activity of a 9,000 g supernatant fraction prepared from suncus lung was examined using 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as a promutagen. The results showed that the suncus lung possessed 82-fold higher mutagen-producing activity than the rat lung, indicating that NNK was efficiently activated by the CYP2A isoform expressed in the suncus lung and that the suncus was a sensitive animal species to the genotoxicity of NNK contained in tobacco smoke.     

The profile of urinary metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in rats is determined by its pulmonary metabolism.

Metabolism of the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in rats was compared to metabolism in primary lung and liver cells. Untreated rats and rats pretreated with phenobarbital, acetone or phenethyl isothiocyanate (PEITC) were used for all experiments. Also the influence of [-]-1-methyl-2-[3-pyridyl]-pyrrolidine (nicotine) administered concomitantly with NNK, or incubated with isolated cells, upon NNK metabolism was investigated and found to be only marginal upon alpha-hydroxylation and pyridine N-oxidation in vivo. In hepatocytes nicotine inhibited NNK pyridine N-oxidation, alpha-hydroxylation and glucuronidation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), whereas in lung cells the influence of nicotine was not as pronounced. In vivo phenobarbital induced alpha-hydroxylation and pyridine N-oxidation. In vitro the effects of the modulators were most pronounced upon hepatocytes, where phenobarbital greatly induced pyridine N-oxidation and PEITC inhibited alpha-hydroxylation. NNAL was conjugated to its beta-glucuronide in lung cells at four times higher rates than in hepatocytes. The ratios of the sum of N-oxides to the sum of alpha-hydroxylation products in vivo were similar to those in lung cells, especially at low NNK concentrations (1 microM), while in hepatocytes alpha-hydroxylation was more pronounced. The same correlation of metabolism in isolated lung cells with whole rats was observed if oxidative NNAL metabolism was related to oxidative NNK metabolism. Here hepatocytes showed a much higher formation of NNAL oxidation products than either lung cells formed, or rats excreted in urine. This was true despite a lower rate of metabolism in the lung than in liver if based on cell number, the rate based on mg protein was four times higher in lung than liver. Only after phenobarbital treatment was the contribution of hepatic metabolism to excreted metabolites important. In conclusion the lung which is also the target of NNK carcinogenesis, and not the liver, is the organ with the most important contribution to NNK and NNAL metabolism at concentrations relevant to human exposure.     

NADPH- and hydroperoxide-supported 17beta-estradiol hydroxylation catalyzed by a variant form (432L, 453S) of human cytochrome P450 1B1

Human cytochrome P450 1B1 (CYP1B1) catalyzes the hydroxylation of 17beta-estradiol (E(2)) at C-4, with a lesser activity at C-2. The E(2) 4-hydroxylase activity of human CYP1B1 was first observed in studies of MCF-7 breast cancer cells. Sequencing of polymerase chain reaction products revealed that CYP1B1 expressed in MCF-7 cells was not the previously characterized enzyme but a polymorphic form with leucine substituted for valine at position 432 and serine substituted for asparagine at position 453. To investigate the NADPH- and organic hydroperoxide-supported E(2) hydroxylase activities of the 432L, 453S form of human CYP1B1, the MCF-7 CYP1B1 cDNA was cloned and the enzyme was expressed in Sf9 insect cells. In microsomal assays supplemented with human NADPH:cytochrome P450 oxidoreductase, the expressed 432L, 453S form catalyzed NADPH-supported E(2) hydroxylation with a similar preference for 4-hydroxylation as the 432V, 453N form, with maximal rates of 1.97 and 0.37 nmol (min)(-1)(nmol cytochrome P450)(-1) for 4- and 2-hydroxylation, respectively. Cumeme hydroperoxide efficiently supported E(2) hydroxylation by both the 432V, 453N and 432L, 453S forms at several-fold higher rates than the NADPH-supported activities and with a lesser preference for E(2) 4- versus 2-hydroxylation (2:1). The hydroperoxide-supported activities of both forms were potently inhibited by the CYP1B1 inhibitor, 3,3',4, 4',5,5'-hexachlorobiphenyl. These results indicate that the 432V, 453N and 432L, 453S forms of CYP1B1 have similar catalytic properties for E(2) hydroxylation, and that human CYP1B1 is very efficient in catalyzing the hydroperoxide-dependent formation of catecholestrogens.     

Cloning and expression of a pig liver taurochenodeoxycholic acid 6alpha-hydroxylase (CYP4A21): a novel member of the CYP4A subfamily

A cytochrome P450 expressed in pig liver was cloned by polymerase chain reaction using oligonucleotide primers based on amino acid sequences of the purified taurochenodeoxycholic acid 6alpha-hydroxylase. This enzyme catalyzes a 6alpha-hydroxylation of chenodeoxycholic acid, and the product hyocholic acid is considered to be a primary bile acid specific for the pig. The cDNA encodes a protein of 504 amino acids. The primary structure of the porcine taurochenodeoxycholic acid 6alpha-hydroxylase, designated CYP4A21, shows about 75% identity with known members of the CYP4A subfamily in rabbit and man. Transfection of the cDNA for CYP4A21 into COS cells resulted in the synthesis of an enzyme that was recognized by antibodies raised against the purified pig liver enzyme and catalyzed 6alpha-hydroxylation of taurochenodeoxycholic acid. The hitherto known CYP4A enzymes catalyze hydroxylation of fatty acids and prostaglandins and have frequently been referred to as fatty acid hydroxylases. A change in substrate specificity from fatty acids or prostaglandins to a steroid nucleus among CYP4A enzymes is notable. The results of mutagenesis experiments indicate that three amino acid substitutions in a region around position 315 which is highly conserved in all previously known CYP4A and CYP4B enzymes could be involved in the altered catalytic activity of CYP4A21.     

Characterization of human liver leukotriene B(4) omega-hydroxylase P450 (CYP4F2)

We previously reported the cloning of a human liver leukotriene B(4) (LTB(4)) omega-hydroxylase P450 designated CYP 4F2 [Kikuta et al. (1994) FEBS Lett. 348, 70-74]. However, the properties of CYP 4F2 remain poorly defined. The preparation solubilized using n-octyl-beta-D-glucopyranoside from microsomes of CYP 4F2-expressing yeast cells catalyzes v- hydroxylation of LTB(4), 6-trans-LTB(4), lipoxin A(4), 8-hydroxyeicosatetraenoate, 12-hydroxyeicosatetraenoate, and 12-hydroxystearate in the presence of rabbit liver NADPH-P450 reductase. In addition, the enzyme shows ethoxycoumarin O-deethylase and p-nitroanisole O-demethylase activities. The enzyme was purified to apparent electrophoretic homogeneity from yeast cells by sequential chromatography of solubilized microsomes through amino-n-hexyl-Sepharose 4B, DEAE-HPLC, and hydroxylapatite HPLC columns. The final preparation showed a specific content of 11.1 nmol of P450/mg of protein, with an apparent molecular mass of 56.3 kDa. CYP 4F2 was distinguished from the closely homologous CYP 4F3 (human neutrophil LTB(4) omega-hydroxylase) by its much higher K(m) for LTB(4), inability to omega-hydroxylate lipoxin B(4), and extreme instability.     

Metabolism of 1,8-cineole by human cytochrome P450 enzymes: identification of a new hydroxylated metabolite

Human metabolism of the monoterpene cyclic ether 1,8-cineole was investigated in vitro and in vivo. In vitro, the biotransformation of 1,8-cineole was investigated by human liver microsomes and by recombinant cytochrome P450 enzymes coexpressed with human CYP-reductase in Escherichia coli cells. Besides the already described metabolite 2alpha-hydroxy-1,8-cineole we found another metabolite produced at high rates. The structure was identified by a comparison of its mass spectrum and retention time with the reference compounds as 3alpha-hydroxy-1,8-cineole. There was a clear correlation between the concentration of the metabolites, incubation time and enzyme content, respectively. CYP3A4/5 antibody significantly inhibited the 2alpha- and 3alpha-hydroxylation catalyzed by pooled human liver microsomes. Further kinetic analysis revealed that the Michaelis-Menten K(m) and V(max) for oxidation of 1,8-cineole in position three were 19 microM and 64.5 nmol/min/nmol P450 for cytochrome P450 3A4, and 141 microM and 10.9 nmol/min/nmol P450 for cytochrome P450 3A5, respectively. To our knowledge, this is the first time that 3alpha-hydroxy-1,8-cineole is described as a human metabolite of 1,8-cineole. We confirmed these in vitro results by the investigation of human urine after the oral administration of cold medication containing 1,8-cineole. In human urine we found by GC-MS analysis the described metabolites, 2alpha-hydroxy-1,8-cineole and 3alpha-hydroxy-1,8-cineole.     

Increased expression of cytochrome P450 IIIA2 in male rat liver after dietary vitamin A supplementation

In this study dietary vitamin A supplementation (25 IU/g diet) was assessed for its effect on hepatic microsomal P450 content and on P450 enzyme-specific drug oxidase activities in rats. Intake of the supplemented diet by male rats over a 15-week period resulted in a fivefold increase in hepatic vitamin A stores over those measured in control liver from rats that received a balanced diet without vitamin A supplementation. Serum retinol was unchanged and there was no evidence of hepatocellular injury in any of the animals. There was a 26% increase in P450 content in vitamin A-supplemented rat liver and regioselective androst-4-ene-3,17-dione (androstenedione) and progesterone hydroxylation revealed changes in several P450 pathways. Thus, androstenedione 16 alpha-hydroxylation (P450 IIC11-mediated) and progesterone 21-hydroxylation (P450 IIC6-mediated) were decreased slightly to 80 and 74% of respective control activities while P450 IIA1/2-dependent androstenedione 7 alpha-hydroxylation was slightly increased. In contrast, the 6 beta-hydroxylations of androstenedione and progesterone were increased to 169 and 152% of control following dietary supplementation. Kinetic analysis of androstenedione 6 beta-hydroxylation revealed an increase in maximal reaction velocity (Vmax 4.00 +/- 0.47 vs 2.20 +/- 0.10 nmol/min/mg protein) but the Km was unchanged, suggesting an increase in enzyme concentration. Consistent with this assertion, immunoquantitation of the steroid 6 beta-hydroxylase, P450 IIIA2, revealed a 158% increase in the microsomal expression of this enzyme (9.8 +/- 2.7 vs 6.2 +/- 1.3 ng/micrograms microsomal protein). From these studies it now seems clear that vitamin A, as a dietary additive in nontoxic doses, has the capacity to alter the activity of hepatic microsomal drug oxidases by modulating the expression of P450 enzymes.     

A conspicuous down-regulating effect of morphine on essential steroid hydroxylation reactions and certain drug N-demethylations.

This study was conducted to explore the potency of morphine to induce reductions of specific cytochrome P450 isoenzyme functions. Male Sprague-Dawley rats were treated with escalating doses (20-125 mg/kg per day) of morphine for 2 weeks in order to study the effects on the following cytochrome P450 catalyzed reactions: 16 alpha-hydroxylation of dehydroepienderosterone (DHA) and progesterone; 17 alpha- and 21-hydroxylation of progesterone; N-demethylation of ethymorphine, codeine and morphine as well as O-dealkylation of ethylmorphine and codeine. 16 alpha-Hydroxylation of DHA and progesterone and 17 alpha-hydroxylation of progesterone decreased to 18, 12 and 10% of control activities, respectively. The N-demethylation of ethylmorphine and codeine decreased to 34 and 43% of control activities, respectively. Morphine treatment had no effect on the 21-hydroxylation reactions or the O-dealkylation of ethylmorphine or codeine. A monoclonal antibody (Mab) against rat liver cytochrome P450 2 c/RLM 5 exerted a 66-73% inhibition of the N-demethylation of ethylmorphine and codeine, respectively, whereas the O-dealkylation reactions were not affected. This Mab inhibited the 16 alpha- and 17 alpha-hydroxylation of DHA and progesterone, whereas the 21-hydroxylation reactions were unaffected. The steroid hydroxylation reactions in rat adrenals were not altered upon morphine treatment. Our data suggest that a major part of the 16 alpha- and 17 alpha-steroid hydroxylations are catalyzed by the same (or closely related) cytochrome(s) P450 as the opioid N-demethylation reactions.     

The N-terminus of the Qcr7 protein of the cytochrome bc(1) complex in S. cerevisiae may be involved in facilitating stability of the subcomplex with the Qcr8 protein and cytochrome b

The role of the hydrophobic membrane-binding segments of NADPH-cytochrome P450 reductase (CPR) and cytochrome b(5) remain undefined. We have expressed four different recombinant flavocytochromes containing b(5) linked to CPR with different hydrophobic segments as linkers. These fusion proteins have been expressed in Escherichia coli and purified and some of their physical properties and electron transfer activities described in the accompanying paper. Of interest is the presence of internal "membrane-binding" hydrophobic segments in these flavocytochromes. This paper describes the ability of these flavocytochromes to reconstitute in vitro two P450 activities that have been reported to be stimulated by the addition of b(5) (the 17,20-lyase activity of CYP17A and the 6 beta hydroxylation of testosterone catalyzed by CYP3A4) and two P450 reactions that do not respond to the presence of b(5) (the 17 alpha-hydroxylation of progesterone catalyzed by CYP17A and the omega hydroxylation of lauric acid catalyzed by CYP4A1). The present study shows that a hydrophobic "membrane-binding" segment must be present in the artificial flavocytochromes in order to successfully reconstitute in vitro hydroxylation activities with P450s. Differences in the effectiveness of the different flavocytochromes to reconstitute enzymatic activities depends on the P450 tested and the nature of the hydrophobic linker segment present in the purified recombinant flavocytochromes. The hypothesis is proposed that differences in the surface topology of a P450 may dictate differences in their docking with the CPR or b(5) component of a fusion protein, resulting in differences in the rates of electron transfer to the P450.     

Biotransformation of (-)-verbenone by human liver microsomes

The biotransformation of (-)-verbenone was investigated with human liver microsomes by using GC-MS. Regioselective biotransformation was observed when (-)-verbenone was incubated with the liver microsomes. (-)-10-Hydroxyverbenone was formed from (-)-verbenone of kinetic analysis showed that the Km and Vmax values for the hydroxylation of (-)-verbenone by liver microsomes from three human samples, HG-70, HG-56 and HG-23, were 1.1 mM and 4.8 nmol/min/nmol P450, 0.6 mM and 2.1 nmol/min/nmol P450, and 2.8 mM and 4.6 nmol/min/nmol P450, respectively.     

Structure of the human lung cytochrome P450 2A13

The human lung cytochrome P450 2A13 (CYP2A13) activates the nicotine-derived procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) into DNA-altering compounds that cause lung cancer. Another cytochrome P450, CYP2A6, is also present in human lung, but at much lower levels. Although these two enzymes are 93.5% identical, CYP2A13 metabolizes NNK with much lower K(m) values than does CYP2A6. To investigate the structural differences between these two enzymes the structure of CYP2A13 was determined to 2.35A by x-ray crystallography and compared with structures of CYP2A6. As expected, the overall CYP2A13 and CYP2A6 structures are very similar with an average root mean square deviation of 0.5A for the Calpha atoms. Like CYP2A6, the CYP2A13 active site cavity is small and highly hydrophobic with a cluster of Phe residues composing the active site roof. Active site residue Asn(297) is positioned to hydrogen bond with an adventitious ligand, identified as indole. Amino acid differences between CYP2A6 and CYP2A13 at positions 117, 300, 301, and 208 relate to different orientations of the ligand plane in the two protein structures and may underlie the significant variations observed in binding and catalysis of many CYP2A ligands. In addition, docking studies suggest that residues 365 and 366 may also contribute to differences in NNK metabolism.     

Species differences in 5 alpha-androstane-3 beta,17 beta-diol hydroxylation by rat, monkey, and human prostate microsomes.

The 6 alpha-, 7 alpha-, and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by rat prostate microsomes appears to be catalyzed by a single, high-affinity cytochrome P450 enzyme. In the present study we have examined the hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by prostate microsomes from cynomolgus monkeys and from normal subjects and patients with benign prostatic hyperplasia. Our results suggest that although rat, monkey, and human prostate microsomes catalyze the 6 alpha-, 7 alpha-, and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol, these pathways of oxidation in monkeys and humans are not catalyzed by a single cytochrome P450 enzyme. The ratio of the three metabolites was not uniform among prostate microsomal samples from individual humans or monkeys. The 6 alpha-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol varied independently of both the 7 alpha- and 7 beta-hydroxylation, which varied in unison. The 6 alpha-, 7 alpha-, and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by monkey prostate microsomes appeared to be differentially affected by in vivo treatment of monkeys with beta-naphthoflavone or dexamethasone. Treatment of a monkey with dexamethasone appeared to cause a 2.5-fold increase in both the 7 alpha- and the 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol without increasing the 6 alpha-hydroxylation. The 7 alpha- and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by human and monkey prostate microsomes, but not the 6 alpha-hydroxylation, was inhibited by antibody against rat liver NADPH-cytochrome P450 reductase. Similarly, the 7 alpha- and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by human prostate microsomes, but not the 6 alpha-hydroxylation, was markedly inhibited (greater than 85%) by equimolar concentrations of the imidazole-containing antimycotic drugs ketoconazole, clotrimazole, and miconazole. These results suggest that the 7 alpha- and 7 beta-hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by monkey and human prostate microsomes is catalyzed by a cytochrome P450 enzyme, whereas the 6 alpha-hydroxylation is catalyzed by a different enzyme which may or may not be a cytochrome P450 monooxygenase. The hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol by prostate microsomes from normal human subjects was quantitatively and qualitatively similar to its hydroxylation by prostate microsomes from patients with benign prostatic hyperplasia.(ABSTRACT TRUNCATED AT 400 WORDS)     

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone modulation of cytokine release in U937 human macrophages

 The nicotine-derived N-nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is one of the most abundant and potent carcinogens found in tobacco smoke. NNK induces lung tumors in rodents and is most likely involved in lung carcinogenesis in humans. Studies on the metabolism and carcinogenicity of NNK have been extensive. However, its effects on the immune system have not been investigated thoroughly. Considering that tobacco smoking partially suppresses the immune response in humans, and that immune surveillance plays a critical role in cancer development, we examined the effects of NNK on the production of selected cytokines. In a previous study, we observed an inhibition of NK cell activity and IgM secretory cell number in NNK-treated A/J mice [Rioux and Castonguay (1997) J Natl Cancer Inst 89: 874]. In this study, we demonstrate that U937 human macrophages activate NNK to alkylating intermediates by α-carbon hydroxylation and detoxify NNK by N-oxidation. We observed that NNK, following activation, induces the release of soluble tumor necrosis factor (TNF), but inhibits interleukin(IL)-10 synthesis. We also report that 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone, and nitroso(acetoxymethyl)methylamine, which generate the same alkylating intermediates as NNK, have similar effects on TNF and IL-10. This suggests that pyridyloxobutylating and methylating intermediates generated from NNK are potent modulators of the immune response. The levels of IL-6, granulocyte/macrophage-colony-stimulating factor and macrophage chemotactic protein 1 were also decreased in supernatants of NNK-treated U937 macrophages. In contrast, IL-2 synthesis in Jurkat cells was inhibited by NNK treatment. This is the first study demonstrating that NNK, via its alkylating intermediates, alters the cytokine synthesis profile in human cells. Modulation of cytokine synthesis by NNK might partially explain the immunosuppresion observed in smokers. Inhibition of immune functions, resulting from NNK activation to alkylating agents, may facilitate lung tumor development. Received: 3 February 2000 / Accepted: 15 September 2000