P450 2B1 Research Articles

Metabolism of carcinogenic N-nitroso-N-methylaniline by purified cytochromes P450 2B1 and P450 2B2.

N-Nitroso-N-methylaniline (NMA) is an esophageal carcinogen in the rat. The in vitro enzymatic metabolism of NMA was investigated using cytochromes P450 2B1 and P450 2B2, isolated from liver microsomes of rats pretreated with phenobarbital (PB), reconstituted with NADPH-cytochrome P450 reductase and dilauroylphosphatidylcholine. Formaldehyde is produced by both cytochromes P450 (P450). NMA is a better substrate for P450 2B1 than for P450 2B2. The maximal velocity (Vmax) values are 3.3 and 1.6 nmol HCHO/min per nmol P450 for P450 2B1 and P450 2B2, respectively. Beside formation of formaldehyde, aniline and p-aminophenol (p-AP) are found to be metabolites formed from NMA by both P450 isoenzymes. P450 2B1 also affords phenol, while none was found with the P450 2B2 isoenzyme. Phenol formation presumably arose from direct alpha-C-hydroxylation of NMA via a benzenediazonium ion (BDI) intermediate. The results suggest strongly that P450 2B1 catalyzes both alpha-C-hydroxylation and denitrosation of NMA while P450 2B2 catalyzes only denitrosation. Therefore, the P450 2B1 isoenzyme participates in the activation of NMA to the ultimate carcinogenic BDI.

Evidence for a 1-Electron Oxidation Mechanism in N-Dealkylation of N,N-Dialkylanilines by Cytochrome P450 2B1: KINETIC HYDROGEN ISOTOPE EFFECTS, LINEAR FREE ENERGY RELATIONSHIPS, COMPARISONS WITH HORSERADISH PEROXIDASE, AND STUDIES WITH OXYGEN SURROGATES

Many enzymes catalyze N-dealkylations of alkylamines, including cytochrome P450 (P450) and peroxidase enzymes. Peroxidases, exemplified by horseradish peroxidase (HRP), are generally accepted to catalyze N-dealkylations via 1-electron transfer processes. Several lines of evidence also support a 1-electron mechanism for many P450 reactions, although this view has been questioned in light of reported trends for kinetic hydrogen isotope effects for N-demethylation with a series of 4-substituted N,N-dimethylanilines. No continuous trend for an increase of isotope effects with the electronic parameters of para-substitution was seen for the P450 2B1-catalyzed reactions in this study. The larger value seen with the 4-nitro derivative is consistent with a shift in mechanism due to either a reversible electron transfer step preceding deprotonation or to a hydrogen atom abstraction mechanism. With HRP, the trend is to lower isotope effects with para electron-withdrawing substituents, due to an apparent shift in rate-limiting steps. Biomimetic model high-valent porphyrins showed reduction rates with variously 4-substituted N,N-dialkylanilines that were consistent with a positively charged intermediate; such relationships were not seen for anisole O-demethylation with P450 2B1. In contrast to the case with the NADPH-supported P450 reactions, high deuterium isotope effects ( approximately 7) were seen in the N-dealkylations supported by the oxygen surrogate iodosylbenzene. With iodosylbenzene, colored aminium radicals were observed in the oxidations of aminopyrine, N,N-dimethyl-4-aminothioanisole, and 4-methoxy-N,N-dimethylaniline. With the latter compound, a substantial intermolecular deuterium isotope effect was observed for N-demethylation. In the N-dealkylation of N-ethyl,N-methylaniline by P450 2B1 (NADPH-supported), the ratio of N-demethylation to N-deethylation was 16. Although it is probably possible for P450s to catalyze amine N-dealkylations via hydrogen atom abstraction when such a course is electronically or sterically favored, we interpret the evidence to favor a 1-electron pathway with N,N-dialkylamines with P450 2B1 as well as HRP and several biomimetic models.

Secobarbital-mediated Inactivation of Cytochrome P450 2B1 and Its Active Site Mutants: PARTITIONING BETWEEN HEME AND PROTEIN ALKYLATION AND EPOXIDATION

Secobarbital (SB) is a relatively selective mechanism-based inactivator of cytochrome P450 2B1, that partitions between epoxidation and heme and protein modification during its enzyme inactivation. The SB-2B1 heme adduct formed in situ in a functionally reconstituted system has been spectrally documented and structurally characterized as N-(5-(2-hydroxypropyl)-5-(1-methylbutyl)barbituric acid)protoporphyrin IX. The SB-protein modification has been localized to 2B1 peptide 277-323 corresponding to the active site helix I of cytochrome P450 101. The targeting of heme and this active site peptide suggests that the 2B1 active site topology could influence the course of its inactivation. To explore this possibility, the individual SB epoxidation, heme and protein modification, and corresponding molar partition ratios of the wild type and seven structural 2B1 mutants, site-directed at specific substrate recognition sites, and known to influence 2B1 catalysis were examined after Escherichia coli expression. These studies reveal that Thr-302 is critical for SB-mediated heme N-alkylation, whereas Val-367 is a critical determinant of 2B1 protein modification, and Val-363 is important for SB epoxidation. SB docking into a refined 2B1 homology model coupled with molecular dynamics analyses provide a logical rationale for these findings.

Interactions of 8-anilino-1-naphthalenesulfonic acid (ANS) and cytochrome P450 2B1: role of ANS as an effector as well as a reporter group.

Interactions of cytochrome P450 2B1 were probed using 8-anilino-1-naphthalenesulfonic acid (ANS), a well known and frequently used reporter group for hydrophobic interactions. Titration of cytochrome P450 2B1 with ANS revealed 6.6 +/- 0.2 ANS binding sites per molecule of P450 2B1 with a Kd value of 42 +/- 2 microM. In our evaluation of the consequences of the binding of ANS to cytochrome P450 2B1, we found that the binding of ANS to P450 2B1 increased benzphetamine demethylation activity by 1.5-fold, indicating a role for ANS as an effector in addition to its role as a reporter group. Kinetic analysis of the effects of ANS on P450 2B1-dependent demethylation activity revealed that ANS increased both the Vmax and K(m) of the benzphetamine demethylation reaction. ANS stimulation of activity appears not to be due to the replacement or augmentation of the role of lipid since studies of binding and catalytic activities in the presence and absence of added lipid gave the same array of effects. These results demonstrate that ANS can bind to cytochrome P450 2B1 as would be expected of a reporter group probe but show in addition that this probe also acts as an effector molecule stimulating catalytic activity. Thus, results of ANS studies should be viewed with caution since the molecule may play more than one role in its reaction with a protein.

Inductive effect of telazol ® on hepatic expression of cytochrome p450 2B in rats

Telazol ®, a 1:1 combination of tiletamine HCl and zolazepam HCl, is an anesthetic and immobilizing agent that has been in use in veterinary medicine and animal field studies for more than a decade. No information is available, however, regarding the effects of Telazol ®, or its constituents, on hepatic cytochromes P450. The purpose of the present study was to assess the effect of Telazol ® on the rat hepatic cytochrome P450 system. Adult male rats were given a single intraperitoneal injection of Telazol ® at a dose of 20, 40, 80, or 120 mg/kg body weight (six rats/dose), while control rats received the vehicle only. Animals were killed 24 hr later, and hepatic microsomes were prepared. Treatment with Telazol ® resulted in dose-dependent increases in benzyloxyresorufin O-dealkylase and testosterone 16β-hydroxylase activities. Ethoxyresorufin O-deethylase, P-nitrophenol hydroxylase, and testosterone 6β- and 7α-hydroxylase activities were essentially unaltered at all doses of the drug. Densitometric quantitation of immunoblots probed with polyclonal antibody against cytochrome P450 2B1 indicated a 17-fold increase in the hepatic level of cytochrome P450 2B1 for rats treated with the highest dose of Telazol ®. In contrast, the level of cytochrome P450 2B2 was increased slightly but not significantly. In the presence of 0.5 mg of anti-cytochrome P450 2B1 IgG/nmol P450, benzyloxyresorufin O-dealkylase activity was inhibited by 92% in hepatic microsomes prepared from a rat treated with Telazol ® at a dose of 120 mg/kg compared with only 25% inhibition in hepatic microsomes from a control rat. In summary, the results demonstrate that Telazol ® specifically induced expression of the cytochrome P450 2B isozymes in rats.

Phenobarbital Induction Mediated by a Distal CYP2B2 Sequence in Rat Liver Transiently Transfected in Situ

The promoter activities of the genes for cytochrome P450 2B1 (CYP2B1) and cytochrome P450 2C1 (CYP2C1) have been assayed by direct injection of promoter-luciferase chimeric genes into rat liver. Activities of minimal promoters for CYP2C1 and CYP2B1 were detectable in untreated animals but were not increased by treatment of the animals with phenobarbital. After insertion to the 5' side of the minimal promoters of one to three copies of the CYP2B2 sequence from -2318 to -2155, a phenobarbital-responsive element in primary hepatocyte cultures (Trottier, E., Belzil, A., Stoltz, C., and Anderson, A. (1995) Gene (Amst.) 158, 263-268), phenobarbital treatment induced the activity of the CYP2C1 promoter by 5-15-fold and the CYP2B1 promoter by 2.5-5-fold. Mutation of a basal transcription element-like motif and a CCAAT/enhancer binding protein element in the CYP2B1 proximal promoter region reduced expression, but 3-4-fold induction by phenobarbital was retained. Mutation of the "Barbie box," a putative phenobarbital-responsive element (He, J.-S., and Fulco, A. J. (1991) J. Biol. Chem. 266, 7864-7869) in the CYP2B1 proximal promoter did not reduce the relative response to phenobarbital. These results demonstrate that direct injection of DNA into rat liver may be used to assay phenobarbital responsiveness of cytochrome P450 genes. In this system, a distal CYP2B2 element mediates a response to phenobarbital, and proximal elements, including the Barbie box, are not required for the induction.

Involvement of a cytochrome P450 system in microsomal debromination of alpha-(bromisovaleryl)urea.

The reductive debromination of a hypnotic, (alpha-bromisovaleryl)urea to (3-methylbutyryl)urea by rat liver microsomes was studied. Pretreatment of rats with cytochrome P450 inducers such as phenobarbitone, 3-methylcholanthrene, acetone and pregnenolone-16 alpha-carbonitrile enhanced the debromination of (alpha-bromisovaleryl)urea by liver microsomes. Microsomal debromination was inhibited by cytochrome P450 inhibitors such as metyrapone, alpha-naphthoflavone, SKF 525-A and carbon monoxide. Microsomal debromination was enhanced by addition of NADPH-cytochrome P450 reductase and inhibited by addition of an antibody against the flavo enzyme to the liver microsomes. A reconstituted cytochrome P450 system containing NADPH-cytochrome P450 reductase, and cytochrome P450 1AI or P450 2BI exhibited debrominating activity toward the hypnotic. These results indicated that a cytochrome P450 system plays an essential role in the microsomal debromination of (alpha-bromisovaleryl)urea.

The CYP2B1 proximal promoter contains a functional C/EBP regulatory element.

Cytochromes P450 2B1 and 2B2 (CYP2B1 and CYP2B2) are well-known phenobarbital-inducible genes in rat liver. Potential transcriptional regulatory elements in the proximal promoter regions of rat CYP2B genes were analyzed by transfection in HepG2 hepatoma cells and by binding of nuclear proteins. Deletion of sequences from -1,400 to -110 had modest effects on promoter activity, but further deletion to -57 decreased the transcriptional activity by more than 90%, suggesting the presence of strong cis-acting elements in this region. Sequences similar to a basal transcription element (BTE) in CYP1A1 and a proposed phenobarbital responsive element (Barbie box) are present from -89 to -67. However, no protection was detected in these regions by DNase I footprinting assay. Instead, a region (FP1) from -64 to -45 was protected by liver nuclear extracts. Mutation of either the BTE or FP1 sequences of CYP2B1, or both, reduced transcriptional activity by 70-80% in HepG2 cells. FP1 was identified as a functional C/EBP site by co-transfection of C/EBP expression vectors and supershift assays with C/EBP antisera. Binding of liver nuclear proteins to sequences within the -110 to +1 region was not detectably altered by pretreatment of rats with phenobarbital.

Cytochrome P450-mediated activation of phenanthrene in genetically engineered V79 Chinese hamster cells

V79 Chinese hamster cells genetically engineered for rat cytochromes P450 1A1, 1A2, 2B1 and human cytochromes P450 1A1, 1A2, 2A6, 2E1, and 3A4 are being applied in metabolism studies on polycyclic aromatic hydrocarbons. This study presents the results on phenanthrene as the prototypic polycyclic aromatic hydrocarbon possessing a bay region. Phenanthrene is of less importance regarding cytotoxicity and carcinogenicity as compared to e.g. benzo[ a]pyrene or 7,12-dimethylbenz[ a]anthracene. However, phenanthrene is more readily converted to metabolites which are exreted in higher amounts than those from any other polycyclic aromatic hydrocarbon. Therefore, its metabolites are of diagnostic value in epidemiological and occupational exposure studies. For this reason, it is worthwhile to understand the metabolism of phenanthrene in detail, e.g. allocating metabolites and cytochromes P450s. In accordance to previous observations cytochromes P450 1A1 and 1A2 were the most active forms towards phenanthrene. However, metabolite profiles differed between rat and human homologues of cytochromes P450, in particular for cytochrome P450 1A2. The predominant metabolite formed by rat cytochrome P450 1A2 was the K region trans-9,10-dihydrodiol, whereas human cytochrome P450 1A2 produced similar amounts of the trans-1,2-, trans-3,4- and trans-9,10-dihydrodiol. High amounts of trans-1,2-dihydrodiol, the metabolic precursor of the bay-region dihydrodiol epoxide, were also formed by human cytochrome P450 1A1 compared to its rat homologue. Unexpectedly, human cytochrome P450 2E1 showed a remarkable catalytic activity to metabolize phenanthrene to its trans-9,10-dihydrodiol. Utilizing recombinant CYPs in live V79 cells appears to be a valuable too yielding results important for the evaluation of exposure data and risk assessment for humans.

Transcriptional regulation of the CYP2B1 and CYP2B2 genes by C/EBP-related proteins

Cytochrome P450 (CYP) 2B1 and 2B2 are encoded by two closely related genes, CYP2B1 and CYP2B2, that are expressed at low levels in adult rat liver but are induced markedly by the administration of the drug phenobarbital (PB) or other structurally unrelated hydrophobic compounds to animals. Very little is understood about the molecular mechanisms that control both basal and induced transcription of these genes. We have identified two liver specific DNase I hypersensitive sites associated with the CYP2B1 and CYP2B2 ( CYP2B) genes. One site, which maps to a region in the 5′-flanking region between −2.2 and −2.3 kb, became more resistant to DNase I cleavage in nuclei from PB-treated rats; the converse was true of the other hypersensitive site, which maps to the proximal promoter region between −0.05 and −0.15 kb. DNase 1 footprint analysis revealed three prominent and one weak footprinted regions in the promoter region in the vicinity of the proximal hypersensitive site. Using competitor oligonucleotides, we determined that one footprinted region (FT2), between −42 and −66 bp, is likely to represent a binding site for CCAATT enhancer binding protein (C/EBP) family members. Indeed, bacterial expressed recombinant C/EBPα bound at this site and formed a footprint pattern identical to the pattern observed with liver nuclear extract. In vitro transcription assays demonstrated that the FT2 site contributed strongly to promoter activity, since its mutation reduced transcription by 80%. Two other sites identified by footprint analysis (FT1 and FT3) are also required to maintain high basal transcription of CYP2B2 promoter constructs in an in vitro transcription assay. Transient transfection experiments confirmed the expectation that C/EBPα could activate the 1.4 kb CYP2B promoter constructs, with mutation of the FT2 site impairing both basal transcription and transactivation by exogenous C/EBPα.

Differential Effects of Nonhydroxylated Flavonoids as Inducers of Cytochrome P450 1A and 2B Isozymes in Rat Liver

Flavanone, flavone, and tangeretin differentially affected the activities of cytochrome P540 1A and 2B isozymes in rat liver. Flavone and, to a lesser extent, tangeretin, increased activities of ethoxyresorufinO-deethylase, methoxyresorufinO-demethylase, and pentoxyresorufinO-dealkylase (PROD), whereas flavanone mainly enhanced PROD activity. Immunoblot analysis indicated that flavone and tangeretin increased cytochrome P450 1A1, 1A2, and 2B1,2 forms, whereas flavanone only enhanced the cytochrome P450 2B isozymes. Northern blot study showed that flavone and tangeretin increased the level of the cytochrome P450 1A2 mRNAs. The concentration of the other mRNAs were slightly or not affected by flavonoids. These results suggest that the induction of P450 1A2 by flavone and tangeretin might involve a transcriptional and/or post-transcriptional mechanism.

Modulation of constitutive and inducible hepatic cytochrome(s) P-450 by interferon β in mice

Aims/Methods: Interferon β is used as a therapeutic agent, but its effects on the hepatic cytochrome P-450-dependent drug metabolizing system have not yet been characterized. We investigated the effect of interferon β on cytochrome P-450 in mice. Results: Interferon β (2×10 5 units/mouse) significantly reduced total hepatic cytochrome P-450 (20%) and the activity of NADPH cytochrome C reductase (12%) 24 h after administration; lower doses had no such effect. Various monooxygenase activities were slightly reduced, the one most affected being 7-ethoxycoumarin O-deethylase (29%). In phenobarbital-treated mice, interferon β reduced the induction of total cytochrome P-450 (22%), the activities of pentoxyresorufin O-dealkylase (38%), benzyloxyresorufin O-dealkylase (30%), erythromycin N-demethylase (30%), 7-ethoxycoumarin O-deethylase (16%) and cytochrome P-450 2B1 (33%) and 3A (45%) proteins. In β-naphthoflavone-treated mice, interferon β lowered the induction of total cytochrome P-450 (18%), the activities of ethoxyresorufin O-deethylase (31%) and of 7-ethoxycoumarin O-deethylase (25%) and of cytochrome P-450 1A1 protein (31%). Conclusions: Thus it appears that induced cytochrome(s) P-450 were susceptible to interferon β, this effect not being influenced by the type of inducer. Since various members of the same cytochrome P-450 subfamilies catalyze oxidation of drugs in humans, our findings have potential significance as regards the fate of drugs or exogenous compounds given to patients receiving interferon β.

Identification of the heme adduct and an active site peptide modified during mechanism-based inactivation of rat liver cytochrome P450 2B1 by secobarbital.

The olefinic barbiturate secobarbital (SB) is a sedative hypnotic known to be a relatively selective mechanism-based inactivator of rat liver cytochrome P450 2B1. Previous studies have demonstrated that such inactivation results in prosthetic heme destruction and irreversible drug-induced protein modification, events most likely triggered by P450 2B1-dependent oxidative activation of the olefinic pi-bond. However, the precise structure of the SB-modified heme and/or the protein site targeted for attack remained to be elucidated. We have now isolated the SB-heme adduct from P450 2B1 inactivated by [14C]SB in a functionally reconstituted system and structurally characterized it by electronic absorption spectroscopy and tandem collision-induced dissociation (CID), matrix-assisted laser desorption ionization on time of flight (MALDI-TOF), and liquid secondary ion mass spectrometry in the positive mode (+ LSIMS) as the N-(5-(2-hydroxypropyl)-5-(1-methylbutyl)barbituric acid)protoporphyrin IX adduct. The [14C]SB-modified 2B1 protein has also been isolated from similar inactivation systems and subjected to lysyl endopeptidase C (Lys-C) digestion and HPLC-peptide mapping. A [14C]SB-modified 2B1 peptide was thus isolated, purified, electrotransferred onto a poly-(vinylidene) membrane, and identified by micro Edman degradation of its first N-terminal 17 residues (S277NH(H)TEFH(H)ENLMISLL293) as the Lys-C peptide domain comprised of amino acids 277-323. This peptide thus includes the peptide domain corresponding to the distal helix I of P450 101, a region highly conserved through evolution, and which is known not only to flank the heme moiety but also to intimately contact the substrates. This finding thus suggests that SB-induced protein modification of P450 2B1 also occurs at the active site and, together with heme N-alkylation, contributes to the SB-induced mechanism-based inactivation of P450 2B1.

Inhibition of rat liver cytochrome P450 isozymes by isothiocyanates and their conjugates: a structure-activity relationship study.

A series of arylalkyl and alkyl isothiocyanates, and their glutathione, cysteine, and N-acetylcysteine conjugates were used to study their inhibitory activity toward the dealkylation of ethoxyresorufin (EROD), pentoxyresorufin (PROD), and methoxyresorufin (MROD) in liver microsomes obtained from the 3-methylcholanthrene or phenobarbital-treated rats. These reactions are predominantly mediated by cytochrome P450 (P450) isozymes 1A1 and 1A2, 2B1 and 1A2, respectively. All isothiocyanates inhibited PROD more readily than EROD. Increases in the alkyl chain length of arylalkyl isothiocyanates to C6 resulted in an increased inhibitory potency in these assays; at longer alkyl chain lengths (C8-C10) the inhibitory potency declined. The IC50s for phenethyl isothiocyanate (PEITC) were 47, 46 and 1.8 microM for EROD, MROD and PROD, respectively. Substitution of an additional phenyl group on PEITC also increased the inhibitory potency; the IC50s for 1,2-diphenylethyl isothiocyanate (1,2-DPEITC) and 2,2-diphenylethyl isothiocyanate (2,2-DPEITC) were 0.9 and 0.26 microM for EROD, and 0.045 and 0.13 microM for PROD, respectively. The relative inhibitory potency of PEITC and its conjugates was N-acetylcysteine-PEITC (PEITC-NAC) < glutathione-PEITC (PEITC-GSH) < cysteine-PEITC (PEITC-CYS) < PEITC. The observations that the parent isothiocyanates were more potent inhibitors than the conjugates suggest that dissociation of the conjugate is required for activity. Naturally occurring alkyl isothiocyanates, sulforaphane (SFO) and allyl isothiocyanate (AITC), were very weak inhibitors in the assays. These results suggest the potential of isothiocyanates as structural probes for studying P450 isozymes. In addition, the inhibitory activity of isothiocyanates for PROD correlated with the previously demonstrated tumor inhibitory potency in (4-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induced A/J mouse lung tumor bioassays, which supports earlier findings that P450 2B1 is one of the major isozymes involved in NNK activation and that inhibition of this isozyme is an important mechanism for the chemopreventive activity of isothiocyanates.

Role of cytochrome P450 in DNA damage induced by N-nitrosodialkylamines in cultured rat hepatocytes.

The involvement of cytochrome P450 in the cytotoxicity and DNA damage-repair induced by N-nitrosodipropylamine (NDPA), N-nitroso-n-butyl-n-propylamine (NBPA), and N-nitrosodibutylamine (NDBA) was investigated in cultured hepatocytes isolated from untreated, phenobarbital (PB)- and pyridine (PYR)-pretreated rats. Pretreatment of rats with PB caused a 10-fold increase in the sensitivity of hepatocytes to the cytotoxic actions of NDPA, NBPA and NDBA as measured by trypan blue exclusion, whereas PYR pretreatment increased the sensitivity of hepatocytes to NDPA and NBPA, but not to NDBA. This elevated sensitivity correlated well with increased 7-pentoxyresorufin depentylase activity catalyzed by P450 2B1 in cultures from PB-pretreated rats and enhanced p-nitrophenol hydroxylase activity of P450 2E1 in cultures of hepatocytes from PYR-pretreated rats. Unscheduled DNA synthesis showed that DNA damage-repair was significantly increased in freshly isolated hepatocytes from PB- and PYR-pretreated rats. With increasing time in culture, however, there was a marked reduction in the DNA damage repair response, concomitant with a decrease in the cytotoxicity of NDPA, NBPA and NDBA in primary cultures of hepatocytes. Coincident with this, a rapid loss in the specific activities of P450 2B1 and 2E1 was detected during the first 48 h in all primary cultures. Although N-nitrosodimethylamine (NDMA), used as a positive control, produced high nuclear grain counts in cultures from PYR-pretreated rats, the toxic effect of NDMA in rat hepatocytes was much weaker than that observed with NDPA, NBPA and NDBA. This result suggests that the type of DNA damage or repair efficiently induced by NDPA, NDBA or NDBA might differ from that due to NDMA.

Cytochrome P450-dependent transformations of 15R- and 15S-hydroperoxyeicosatetraenoic acids: stereoselective formation of epoxy alcohol products.

Although there are many reports of epoxy alcohol synthesis from lipoxygenase products (fatty acid hydroperoxides) in mammalian tissues, there are no well-defined examples of the stereoselective synthesis of individual epoxy alcohol diastereomers. An earlier report on the metabolism of 15S-hydroperoxyeicosatetraenoic acid (15S-HPETE) in rat liver microsomes suggested such a specific reaction [Weiss, R. H., et al. (1987) Arch. Biochem. Biophys. 252, 334-338]. To characterize this reaction further, we set out to determine the precise structures and mechanism of biosynthesis of the epoxy alcohol products. We compared the products formed from 15R- and 15S-HPETE by hematin (a nonenzymatic reaction), by liver microsomes isolated from control and phenobarbital-treated rats, and by purified cytochrome P450 2B1. Eight epoxy alcohol isomers were identified by mass spectrometry and 1H NMR. In the hematin reaction, the major products are four epoxy alcohols with the epoxide in the trans configuration, diastereomers are formed in similar amounts, and the 15-HPETE enantiomers give indistinguishable patterns of products. By contrast, the liver microsomes and P450 2B1 enzyme form predominantly single diastereomers, and the configuration of the epoxide is dependent on the stereochemistry of the substrate. The main product formed from 15S-HPETE is 11S-hydroxy-14S,15S-trans-epoxyeicosa-5Z,8Z,12E- trienoic acid, and the amounts increase upon phenobarbital induction. The main products from 15R-HPETE are 11-hydroxy-14S,15R-epoxyeicosa-5Z,8Z,12E-t rienoic acid from microsomes from control rats and 13-hydroxy-14S,15R-cis-epoxyeicosa-5,8,11-trienoic acid in microsomes from phenobarbital-induced rats. The P450 2B1 enzyme gave products similar to those from the phenobarbital-induced microsomes. Analysis of an incubation using the 18O-labeled 15S-HPETE substrate demonstrated 97.6% retention of both hydroperoxy oxygens in the major product with progressively lower 18O retentions in the minor products (74-32%), possibly reflecting degrees of enzymatic control of these reactions. These results establish a precedent for the stereoselective synthesis of epoxy alcohols by mammalian cytochrome P450s.

The effects of dietary ellagic acid on rat hepatic and esophageal mucosal cytochromes P450 and phase II enzymes.

Ellagic acid (EA), a naturally occurring plant polyphenol possesses broad chemoprotective properties. Dietary EA has been shown to reduce the incidence of N-2-fluorenylacetamide-induced hepatocarcinogenesis in rats and N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumors. In this study changes in the expression and activities of specific rat hepatic and esophageal mucosal cytochromes P450 (P450) and phase II enzymes following dietary EA treatment were investigated. Liver and esophageal mucosal microsomes and cytosol were prepared from three groups of Fisher 344 rats which were fed an AIN-76 diet containing no EA or 0.4 or 4.0 g/kg EA for 23 days. In the liver total P450 content decreased by up to 25% and P450 2E1-catalyzed p-nitrophenol hydroxylation decreased by 15%. No changes were observed in P450 1A1, 2B1 or 3A1/2 expression or activities or cytochrome b5 activity. P450 reductase activity decreased by up to 28%. Microsomal epoxide hydrolase (mEH) expression decreased by up to 85% after EA treatment, but mEH activities did not change. The hepatic phase II enzymes glutathione S-transferase (GST), NAD(P)H:quinone reductase [NAD-(P)H:QR] and UDP glucuronosyltransferase (UDPGT) activities increased by up to 26, 17 and 75% respectively. Assays for specific forms of GST indicated marked increases in the activities of isozymes 2-2 (190%), 4-4 (150%) and 5-5 (82%). In the rat esophageal mucosa only P450 1A1 could be detected by Western blot analysis and androstendione was the only P450 metabolite of testosterone detectable. However, there were no differences in the expression of P450 1A1, the formation of androstendione or NAD(P)H:QR activities between control and EA-fed rats in the esophagus. Although there was no significant decrease in overall GST activity, as measured with 1-chloro-2,4-dinitrobenzene (CDNB), there was a significant decrease in the activity of the 2-2 isozyme (66% of control). In vitro incubations showed that EA at a concentration of 100 microM inhibited P450 2E1, 1A1 and 2B1 activities by 87, 55 and 18% respectively, but did not affect 3A1/2 activity. Using standard steady-state kinetic analyses, EA was shown to be a potent non-competitive inhibitor of both liver microsomal ethoxyresorufin O-deethylase and p-nitrophenol hydroxylase activities, with apparent Ki values of approximately 55 and 14 microM respectively. In conclusion, these results demonstrate that EA causes a decrease in total hepatic P450 with a significant effect on hepatic P450 2E1, increases some hepatic phase II enzyme activities [GST, NAD-(P)H:QR and UDPGT] and decreases hepatic mEH expression. It also inhibits the catalytic activity of some P450 isozymes in vitro. Thus the chemoprotective effect of EA against various chemically induced cancers may involve decreases in the rates of metabolism of these carcinogens by phase I enzymes, due to both direct inhibition of catalytic activity and modulation of gene expression, in addition to effects on the expression of phase II enzymes, thereby enhancing the ability of the target tissues to detoxify the reactive intermediates.

Mechanisms of myricetin mutagenicity in V79 cells: involvement of radicalar species.

Myricetin is a flavonol that is widely distributed in edible plants and although it has been proved to be genotoxic in bacteria and to induce significant concentration-dependent nuclear DNA degradation concurrent with lipid peroxidation, very little is known about its mechanisms of genotoxicity. In this work we tried to evaluate the role of rat cytochromes P450 in the genotoxicity of myricetin and to study the role that radicalar species may have in its mutagenicity. The results obtained show that the genotoxicity of myricetin as assessed by the induction of chromosomal aberrations is not different in V79 cells lines genetically engineered for the expression of rat cytochromes P450 1A1, 1A2, and 2B1, compared to parental cell lines. We have also been able to show that reactive oxygen species resulting from the autooxidation of myricetin at pH values above neutrality have an important role in its mutagenicity. Therefore, under some conditions, myricetin can act as a prooxidant.

Identification of the cytochrome P450 isozymes involved in the metabolism of N-nitrosodipropyl-,N-nitrosodibutyl- and N-nitroso-n-butyl-n-propylamine.

The metabolism of N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA) and N-nitroso-n-butyl-n-propylamine (NBPA) was investigated in vitro using liver microsomes and purified isoforms of cytochrome P450 in a reconstituted system. Liver microsomes were prepared from rats pretreated with phenobarbital (PB), pyridine (PYR), beta-naphthoflavone (BNF), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), clofibrate (CLO) or from untreated rats. The purified cytochrome P450s used in the reconstituted system were rat 1A1 and 2B1 and rabbit 2E1. The rates of metabolism and the product profiles for NDPA, NDBA and NBPA changed significantly depending on the pretreatment of the rats or the identity of the purified cytochrome P450 isoforms. Induction by PB dramatically increased cleavage of NDPA, NDBA and NBPA at C-N bonds, leading to substantial increases in formation of the respective aldehydes and the overall metabolic rates. Microsomes from PYR-pretreated rats exhibited increased activities for formation of formaldehyde and propionaldehyde from NDPA and NBPA. Microsomes from BHT-pretreated rats showed a slight increase in activity for N-dealkylation of NDBA and BNPA. Treatment with BHA decreased the overall metabolism of NDBA, but slightly increased N-dealkylation of NBPA. Microsomal metabolism of NDPA, NDBA and NBPA was decreased by pretreatment with BNF and CLO. Results from studies using the reconstituted system with purified cytochrome P450 isoforms demonstrated that cytochrome P450 2B1 specifically catalyzed alpha-hydroxylation of these three long chain nitrosamines with high activity. Cytochrome P450 2E1 catalyzed formation of formaldehyde and propionaldehyde from NDPA and NBPA, but did not catalyze formation of acetaldehyde or butyraldehyde. Cytochrome P450 1A1 exhibited no activity for metabolism of NDPA, NDBA and NBPA. The contributions of cytochrome P450 2B1 and 2E1 to N-dealkylation reactions were determined using inhibitory monoclonal antibodies (mAb). With microsomes from PB-pretreated rats, inhibition by mAb-2B1 indicated a 62% contribution by cytochrome P450 2B1 to debutylation of NDBA and 65% to depropylation of NDPA. In microsomes from PYR-pretreated rats inhibition by mAbs also showed a role for cytochrome P450 2E1 in depropylation of NDPA. These studies provide a better understanding of the role of various forms of cytochrome P450 in metabolic activation of these long chain N-nitrosodialkylamines to potentially toxic, mutagenic and carcinogenic intermediates.

Oxidation of 9-alkylanthracenes by cytochrome P450 2B1, horseradish peroxidase, and iron tetraphenylporphine/iodosylbenzene systems: anaerobic and aerobic mechanisms.

Variously substituted alkylanthracenes were studied as models for polycyclic hydrocarbon oxidations. 9-Methylanthracene was oxidized to 9-(hydroxymethyl)anthracene, 10-methyl-10-hydroxy-9-anthrone, and anthraquinone in several systems, including (i) NADPH- and O2-fortified rat liver microsomes, (ii) cytochrome P450 (P450) 2B1 Supported by either iodosylbenzene (PhIO) or a mixture of NADPH-P450 reductase, NADPH, and O2, (iii) horseradish peroxidase and either H2O2 or ethyl hydroperoxide, and (iv) a mixture of iron tetraphenylporphine (FeTPP) and PhIO (in anhydrous CH2Cl2/MeOH). The microsomal system also formed dihydrodiols from 9-methyl- and 9-ethylanthracenes. The formation of the three oxidized products by the P450/NADPH/O2 system was dependent upon O2 label from 18O2 was incorporated into the products, and no label from H2(18O) was incorporated. No label from 18O2 was incorporated into the three products in the FeTPP/PhIO system. In the horseradish peroxidase/H2O2 system, the formation of the three products was decreased when O2 was omitted, and label from both H2(18)O and 18O2 was incorporated into all three products. The results are interpreted in terms of three mechanisms. One is used by the FeTPP and P450 systems, with all oxygen transfers coming from an FeO entity. The other two pathways are utilized by horseradish peroxidase and begin with formation of a radical cation, which can undergo reactions either with H2O or with O2 to form the products detected here. The involvement of a 9-methylanthracene radical cation in the P450 and FeTPP pathways is a possibility, but rapid rearrangement and oxygen rebound must be invoked. Comparisons of products from various 9-alkylanthracenes do not provide evidence that one-electron oxidation is an integral part of the epoxidation process with these compounds. The significance of the lack of trapping of radical (by H2(18O) in the P450 reactions to DNA adduct formation is considered.