Heme Modification Contributes to the Mechanism-Based Inactivation of Human Cytochrome P450 2J2 by Two Terminal Acetylenic Compounds.

The mechanism-based inactivation of human CYP2J2 by three terminal acetylenic compounds: N-(methylsulfonyl)-6-(2-propargyloxyphenyl)hexanamide (MS), 17-octadecynoic acid (OD), and danazol (DZ) was investigated. The loss of hydroxyebastine (OHEB) carboxylation activity in a reconstituted system was time- and concentration-dependent and required NADPH for MS and OD, but not DZ. The kinetic constants for the mechanism-based inactivation of OHEB carboxylation activity were: KI of 6.1 µM and kinact of 0.22 min-1 for MS and KI of 2.5 µM and kinact of 0.05 min-1 for OD. The partition ratios for MS and OD were ∼10 and ∼20, respectively. Inactivation of CYP2J2 by MS or OD resulted in a loss of the native heme spectrum and a similar decrease in the reduced CO difference spectrum. A heme adduct was observed in the MS-inactivated CYP2J2. The possible reactive metabolite which covalently modified the prosthetic heme was characterized by analysis of the glutathione conjugates formed by MS or OD following oxygenation of the ethynyl moiety. Liquid chromatography-mass spectrometry showed that inactivation by MS or OD did not lead to modification of apoprotein. Interaction of CYP2J2 with DZ produced a type II binding spectrum with a Ks of 2.8 µM and the IC50 for loss of OHEB carboxylation activity was 0.18 µM. In conclusion, heme modification by MS and OD was responsible for the mechanism-based inactivation of CYP2J2. The results suggest that the ethynyl moiety of MS and OD faces the heme iron, whereas the isoxazole ring of DZ is preferentially oriented toward the heme iron of CYP2J2.

Mechanism-Based Inactivation of Human Cytochrome P450 2B6 by Chlorpyrifos.

Chlorpyrifos (CPS) is a commonly used pesticide which is metabolized by P450s into the toxic metabolite chlorpyrifos-oxon (CPO). Metabolism also results in the release of sulfur, which has been suggested to be involved in mechanism-based inactivation (MBI) of P450s. CYP2B6 was previously determined to have the greatest catalytic efficiency for CPO formation in vitro. Therefore, we characterized the MBI of CYP2B6 by CPS. CPS inactivated CYP2B6 in a time- and concentration-dependent manner with a kinact of 1.97 min(-1), a KI of 0.47 µM, and a partition ratio of 17.7. We further evaluated the ability of other organophosphate pesticides including chorpyrifos-methyl, diazinon, parathion-methyl, and azinophos-methyl to inactivate CYP2B6. These organophosphate pesticides were also potent MBIs of CYP2B6 characterized by similar kinact and KI values. The inactivation of CYP2B6 by CPS was accompanied by the loss of P450 detectable in the CO reduced spectrum and loss of detectable heme. High molecular weight aggregates were observed when inactivated CYP2B6 was run on SDS-PAGE gels indicating protein aggregation. Interestingly, we found that the rat homologue of CYP2B6, CYP2B1, was not inactivated by CPS despite forming CPO to a similar extent. On the basis of the locations of the Cys residues in the two proteins which could react with released sulfur during the metabolism of CPS, we investigated whether the C475 in CYP2B6, which is not conserved in CYP2B1, was the critical residue for inactivation by mutating it to a Ser. CYP2B6 C475S was inactivated to a similar extent as wild type CYP2B6 indicating that C475 is not likely the key difference between CYP2B1 and CYP2B6 with respect to inactivation. These results indicate that CPS and other organophosphate pesticides are potent MBIs of CYP2B6 which may have implications for the toxicity of these pesticides as well as the potential for pesticide-drug interactions.

Essential role of the cytochrome P450 enzyme CYP2A5 in olfactory mucosal toxicity of naphthalene.

Naphthalene (NA), a ubiquitous environmental pollutant that can cause pulmonary and nasal toxicity in laboratory animals, requires cytochrome P450 (P450)-mediated metabolic activation to cause toxicity. Our recent study using a Cyp2f2-null mouse showed that CYP2F2 plays an essential role in NA-induced lung toxicity, but not in NA-induced nasal toxicity. The aim of this study was to determine whether mouse CYP2A5, abundantly expressed in nasal olfactory mucosa (OM) and the liver, but less in the lung, plays a major role in the bioactivation and toxicity of NA in the OM. We found, by comparing Cyp2a5-null and wild-type (WT) mice, that the loss of CYP2A5 expression led to substantial decreases in rates of NA metabolic activation by OM microsomes. The loss of CYP2A5 did not cause changes in systemic clearance of NA (at 200 mg/kg, i.p.). However, the Cyp2a5-null mice were much more resistant than were WT mice to NA-induced nasal toxicity (although not lung toxicity), when examined at 24 hours after NA dosing (at 200 mg/kg, i.p.), or to NA-induced depletion of total nonprotein sulfhydryl in the OM (although not in the lung), examined at 2 hours after dosing. Thus, mouse CYP2A5 plays an essential role in the bioactivation and toxicity of NA in the OM, but not in the lung. Our findings further illustrate the tissue-specific nature of the role of individual P450 enzymes in xenobiotic toxicity, and provide the basis for a more reliable assessment of the potential risks of NA nasal toxicity in humans.

The effect of ritonavir on human CYP2B6 catalytic activity: heme modification contributes to the mechanism-based inactivation of CYP2B6 and CYP3A4 by ritonavir.

The mechanism-based inactivation of human CYP2B6 by ritonavir (RTV) in a reconstituted system was investigated. The inactivation is time, concentration, and NADPH dependent and exhibits a K(I) of 0.9 µM, a k(inact) of 0.05 min⁻¹, and a partition ratio of approximately 3. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that the protonated molecular ion of RTV exhibits an m/z at 721 and its two major metabolites are an oxidation product with MH⁺ at m/z 737 and a deacylated product with MH⁺ at m/z 580. Inactivation of CYP2B6 by incubation with 10 µM RTV for 10 min resulted in an approximately 50% loss of catalytic activity and native heme, but no modification of the apoprotein was observed. RTV was found to be a potent mixed-type reversible inhibitor (K(i) = 0.33 µM) and a type II ligand (spectral dissociation constant-K(s) = 0.85 µM) of CYP2B6. Although previous studies have demonstrated that RTV is a potent mechanism-based inactivator of CYP3A4, the molecular mechanism responsible for the inactivation has not been determined. Here, we provide evidence that RTV inactivation of CYP3A4 is due to heme destruction with the formation of a heme-protein adduct. Similar to CYP2B6, there is no significant modification of the apoprotein. Furthermore, LC-MS/MS analysis revealed that both CYP3A4 and human liver microsomes form an RTV-glutathione conjugate having a MH⁺ at m/z 858 during metabolism of RTV, suggesting the formation of an isocyanate intermediate leading to formation of the conjugate.

Bioactivation of the nasal toxicant 2,6-dichlorobenzonitrile: an assessment of metabolic activity in human nasal mucosa and identification of indicators of exposure and potential toxicity.

The herbicide 2,6-dichlorobenzonitrile (DCBN) is a potent nasal toxicant in rodents; however, it is not known whether DCBN causes similar nasal toxicity in humans. The tissue-selective toxicity of DCBN in mouse nasal mucosa is largely dependent on target tissue bioactivation by CYP2A5. The human orthologues of CYP2A5, CYP2A6 and CYP2A13, are both expressed in nasal mucosa and are capable of activating DCBN. In this study, we directly determined the ability of human nasal mucosa to bioactivate DCBN. We also tested the suitability of a glutathione conjugate of DCBN (GS-DCBN) or its derivatives as biomarkers of DCBN exposure and nasal toxicity in mouse models. We found that human fetal nasal mucosa microsomes catalyze the formation of GS-DCBN, with a Km value comparable to that of adult mouse nasal mucosa microsomes. The activity of the human nasal mucosa microsomes was inhibited by 8-methoxypsoralen, a known CYP2A inhibitor. GS-DCBN and its metabolites were detected in the nasal mucosa and nasal-wash fluid obtained from DCBN-treated mice, in amounts that increased with escalations in DCBN dose, and they were all still detectable at 24 h after a DCBN treatment (at 10 mg/kg). Further studies in Cyp2a5-null mice indicated that GS-DCBN and its metabolites in nasal-wash fluid were generated in the nasal mucosa, rather than in other organs. Thus, our data indicate for the first time that the human nasal mucosa is capable of bioactivating DCBN and that GS-DCBN and its metabolites in nasal-wash fluid may collectively serve as indicators of DCBN exposure and potential nasal toxicity in humans.

Bioactivation of the cancer chemopreventive agent tamoxifen to quinone methides by cytochrome P4502B6 and identification of the modified residue on the apoprotein.

The nonsteroidal antiestrogen tamoxifen was introduced as a treatment for breast cancer 3 decades ago. It has also been approved as a chemopreventive agent and is prescribed to women at high risk for this disease. However, several studies have shown that use of tamoxifen leads to increased risk of endometrial cancer in humans. One potential pathway of tamoxifen toxicity could involve metabolism via hydroxylation to give 4-hydroxytamoxifen (4OHtam), which may be further oxidized to form a quinone methide. CYP2B6 is a highly polymorphic drug-metabolizing enzyme, and it metabolizes a number of clinically important drugs. Earlier studies from our laboratory have shown that tamoxifen is a mechanism-based inactivator of CYP2B6. The aim of the current study was to investigate the possible formation of reactive intermediates through detection of protein covalent binding and glutathione ethyl ester adduct (GSHEE) formation. The incubation of tamoxifen with 2B6 gave rise to an adduct of 4OHtam with glutathione, which was characterized as the 4OHtam quinone methide + GSHEE with an m/z value of 719, and the structure was characterized by liquid chromatography-tandem mass spectrometry. The metabolic activation of tamoxifen in the CYP2B6 reconstituted system also resulted in the formation of an adduct to the P4502B6 apoprotein, which was identified using liquid chromatography mass spectrometry. The site responsible for the inactivation of CYP2B6 was determined by proteolytic digestion and identification of the labeled peptide. This revealed a tryptic peptide ¹88FHYQDQE¹94 with the site of adduct formation localized to Gln193 as the site modified by the reactive metabolite formed during tamoxifen metabolism.

Potential biological functions of cytochrome P450 reductase-dependent enzymes in small intestine: novel link to expression of major histocompatibility complex class II genes.

NADPH-cytochrome P450 reductase (POR) is essential for the functioning of microsomal cytochrome P450 (P450) monooxygenases and heme oxygenases. The biological roles of the POR-dependent enzymes in the intestine have not been defined, despite the wealth of knowledge on the biochemical properties of the various oxygenases. In this study, cDNA microarray analysis revealed significant changes in gene expression in enterocytes isolated from the small intestine of intestinal epithelium-specific Por knock-out (named IE-Cpr-null) mice compared with that observed in wild-type (WT) littermates. Gene ontology analyses revealed significant changes in terms related to P450s, transporters, cholesterol biosynthesis, and, unexpectedly, antigen presentation/processing. The genomic changes were confirmed at either mRNA or protein level for selected genes, including those of the major histocompatibility complex class II (MHC II). Cholesterol biosynthetic activity was greatly reduced in the enterocytes of the IE-Cpr-null mice, as evidenced by the accumulation of the lanosterol metabolite, 24-dihydrolanosterol. However, no differences in either circulating or enterocyte cholesterol levels were observed between IE-Cpr-null and WT mice. Interestingly, the levels of the cholesterol precursor farnesyl pyrophosphate and its derivative geranylgeranyl pyrophosphate were also increased in the enterocytes of the IE-Cpr-null mice. Furthermore, the expression of STAT1 (signal transducer and activator of transcription 1), a downstream target of geranylgeranyl pyrophosphate signaling, was enhanced. STAT1 is an activator of CIITA, the class II transactivator for MHC II expression; CIITA expression was concomitantly increased in IE-Cpr-null mice. Overall, these findings provide a novel and mechanistic link between POR-dependent enzymes and the expression of MHC II genes in the small intestine.

Generation and characterization of a CYP2A13/2B6/2F1-transgenic mouse model.

CYP2A13, CYP2B6, and CYP2F1, which are encoded by neighboring cytochrome P450 genes on human chromosome 19, are active in the metabolic activation of many drugs, respiratory toxicants, and chemical carcinogens. To facilitate studies on the regulation and function of these human genes, we have generated a CYP2A13/2B6/2F1-transgenic (TG) mouse model (all *1 alleles). Homozygous transgenic mice are normal with respect to gross morphological features, development, and fertility. The tissue distribution of transgenic mRNA expression agreed well with the known respiratory tract-selective expression of CYP2A13 and CYP2F1 and hepatic expression of CYP2B6 in humans. CYP2A13 protein was detected through immunoblot analyses in the nasal mucosa (NM) (∼100 pmol/mg of microsomal protein; similar to the level of mouse CYP2A5) and the lung (∼0.2 pmol/mg of microsomal protein) but not in the liver of the TG mice. CYP2F1 protein, which could not be separated from mouse CYP2F2 in immunoblot analyses, was readily detected in the NM and lung but not the liver of TG/Cyp2f2-null mice, at levels 10- and 40-fold, respectively, lower than that of mouse CYP2F2 in the TG mice. CYP2B6 protein was detected in the liver (∼0.2 pmol/mg of microsomal protein) but not the NM or lung (with a detection limit of 0.04 pmol/mg of microsomal protein) of the TG mice. At least one transgenic protein (CYP2A13) seems to be active, because the NM of the TG mice had greater in vitro and in vivo activities in bioactivation of a CYP2A13 substrate, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (a lung carcinogen), than did the NM of wild-type mice.

Respective roles of CYP2A5 and CYP2F2 in the bioactivation of 3-methylindole in mouse olfactory mucosa and lung: studies using Cyp2a5-null and Cyp2f2-null mouse models.

The aim of this study was to determine whether mouse CYP2A5 and CYP2F2 play critical roles in the bioactivation of 3-methylindole (3MI), a tissue-selective toxicant, in the target tissues, the nasal olfactory mucosa (OM) and lung. Five metabolites of 3MI were identified in NADPH- and GSH-fortified microsomal reactions, including 3-glutathionyl-S-methylindole (GS-A1), 3-methyl-2-glutathionyl-S-indole (GS-A2), 3-hydroxy-3-methyleneindolenine (HMI), indole-3-carbinol (I-3-C), and 3-methyloxindole (MOI). The metabolite profiles and enzyme kinetics of the reactions were compared between OM and lung, and among wild-type, Cyp2a5-null, and Cyp2f2-null mice. In lung reactions, GS-A1, GS-A2, and HMI were detected as major products, and I-3-C and MOI, as minor metabolites. In OM reactions, all five metabolites were detected in ample amounts. The loss of CYP2F2 affected formation of all 3MI metabolites in the lung and formation of HMI, GS-A1, and GS-A2 in the OM. In contrast, loss of CYP2A5 did not affect formation of 3MI metabolites in the lung but caused substantial decreases in I-3-C and MOI formation in the OM. Thus, whereas CYP2F2 plays a critical role in the 3MI metabolism in the lung, both CYP2A5 and CYP2F2 play important roles in 3MI metabolism in the OM. Furthermore, the fate of the reactive metabolites produced by the two enzymes through common dehydrogenation and epoxidation pathways seemed to differ with CYP2A5 supporting direct conversion to stable metabolites and CYP2F2 supporting further formation of reactive iminium ions. These results provide the basis for understanding the respective roles of CYP2A5 and CYP2F2 in 3MI's toxicity in the respiratory tract.

Role of CYP2A5 in the bioactivation of the lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in mice.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen. Previously, we have demonstrated that NNK-induced lung tumorigenesis in mice depends on target-tissue bioactivation by pulmonary cytochrome P450 (P450) enzymes. The present study was designed to test the hypothesis that mouse CYP2A5 plays an essential role in NNK bioactivation in mouse lung. The role of CYP2A5 in NNK bioactivation was studied both in vitro and in vivo, by comparing the kinetic parameters of microsomal NNK metabolism and tissue levels of O(6)-methylguanine (O(6)-mG) (the DNA adduct highly correlated with lung tumorigenesis) between wild-type (WT) and Cyp2a5-null mice. In both liver and lung microsomes, the loss of CYP2A5 resulted in significant increases in the apparent K(m) values for the formation of 4-oxo-4-(3-pyridyl)butanone, which represents the reactive intermediate that produces O(6)-mG in vivo. The loss of CYP2A5 did not change circulating levels of NNK or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in mice treated intraperitoneally with NNK at either 20 or 100 mg/kg. However, the levels of lung O(6)-mG were significantly lower in Cyp2a5-null than in WT mice; the extent of the reduction was greater at the 20 mg/kg dose (∼40%) than at the 100 mg/kg dose (∼20%). These results indicate that CYP2A5 is the low-K(m) enzyme for NNK bioactivation in mouse lung. It is noteworthy that the remaining NNK bioactivation activities in the Cyp2a5-null mice could be inhibited by 8-methoxypsoralen, a P450 inhibitor used previously to demonstrate the role of CYP2A5 in NNK-induced lung tumorigenesis. Thus, P450 enzymes other than CYP2A5 probably also contribute to NNK-induced lung tumorigenesis in mice.

Role of intestinal cytochrome P450 (P450) in modulating the bioavailability of oral lovastatin: insights from studies on the intestinal epithelium-specific P450 reductase knockout mouse.

The extents to which small intestinal (SI) cytochrome P450 (P450) enzymes control the bioavailability of oral drugs are not well defined, particularly for drugs that are substrates for both P450 and the P-glycoprotein (P-gp). In this study, we have determined the role of SI P450 in the clearance of orally administered lovastatin (LVS), an anti-hypercholesterolemia drug, using an intestinal epithelium (IE)-specific P450 reductase knockout (IE-Cpr-null) mouse model. In the IE-Cpr-null mouse, which has little P450 activities in the IE, the oral bioavailability of LVS was substantially higher than that in wild-type (WT) mice (15 and 5%, respectively). In control experiments, the clearance rates were not different between the two strains, either for intraperitoneally dosed LVS, which bypasses SI metabolism, or for orally administered pravastatin, which is known to be poorly metabolized by P450. Thus, our results demonstrate a predominant role of SI P450 enzymes in the first-pass clearance of oral LVS. The absence of IE P450 activities in the IE-Cpr-null mice also facilitated the identification of the molecular targets for orally administered grapefruit juice (GFJ), which is known to inhibit LVS clearance in humans. We found that pretreatment of mice with oral GFJ enhanced the systemic exposure of LVS in WT, but not in IE-Cpr-null mice, a result suggesting that the main target of GFJ action in the small intestine is P450, but not P-gp.

Potent mutagenicity of 3-methylindole requires pulmonary cytochrome P450-mediated bioactivation: a comparison to the prototype cigarette smoke mutagens B(a)P and NNK.

3-Methylindole (3MI) is a preferential pneumotoxicant found in cigarette smoke. A number of lung-expressed human cytochrome P450 enzymes, including 1A1, 2F1, and 2A13, catalyze the metabolism of 3MI to reactive intermediates that fragment DNA, measured with the Comet assay to assess DNA damage, in a cytochrome P450-dependent manner in primary normal human lung cells in culture, but the mutagenesis of 3MI has been controversial. In the present study, the mutagenic potential of 3MI was compared to the prototypical cigarette smoke carcinogens benzo(a)pyrene (B(a)P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). 3MI, B(a)P, and NNK were incubated with the Salmonella typhimurium strain TA98, which is known to detect the most common subtype of cigarette smoke-induced mutagenicity, frameshift mutations in DNA, and with Salmonella typhimurium strain TA100, which detects base pair substitution mutants, with five sources of P450-mediated bioactivation: rat liver S9, human lung microsomes, recombinant CYP2A13, purified CYP2F3, and recombinant CYP1A1. Only B(a)P was mutagenic in TA100, and it was bioactivated by human lung microsomes and rat liver S9 sources of P450s. However, with the TA98 strain, CYP1A1, CYP2A13, CYP2F3, and human lung microsomes bioactivated 3MI to highly mutagenic intermediates, whereas neither human nor rat liver S9 subcellular fractions formed mutagenic intermediates from 3MI. Quantitative Western blot analysis verified that all three respiratory enzymes were present in human lung microsomes in widely varying amounts. These results indicate that metabolism of 3MI by human lung-expressed cytochrome P450 enzymes but not hepatic P450s elicits equivalent or higher mutagenicity than the prototype cigarette smoke mutagens B(a)P and NNK and indicates that 3MI is a likely human pulmonary carcinogen.

The pneumotoxin 3-methylindole is a substrate and a mechanism-based inactivator of CYP2A13, a human cytochrome P450 enzyme preferentially expressed in the respiratory tract.

3-Methylindole (3MI), a respiratory tract toxicant, can be metabolized by a number of cytochromes P450 (P450), primarily through either dehydrogenation or epoxidation of the indole. In the present study, we assessed the bioactivation of 3MI by recombinant CYP2A13, a human P450 predominantly expressed in the respiratory tract. Four metabolites were detected, and the two principal ones were identified as indole-3-carbinol (I-3-C) and 3-methyloxindole (MOI). Bioactivation of 3MI by CYP2A13 was verified by the observation of three glutathione (GSH) adducts designated as GS-A1 (glutathione adduct 1), GS-A2 (glutathione adduct 2), and GS-A3 (glutathione adduct 3) in a NADPH- and GSH-fortified reaction system. GS-A1 and GS-A2 gave the same molecular ion at m/z 437, an increase of 305 Da over 3MI. Their structures are assigned to be 3-glutathionyl-S-methylindole and 3-methyl-2-glutathionyl-S-indole, respectively, on the basis of the mass fragmentation data obtained by high-resolution mass spectrometry. Kinetic parameters were determined for the formation of I-3-C (V(max) = 1.5 nmol/min/nmol of P450; K(m) = 14 muM), MOI (V(max) = 1.9 nmol/min/nmol of P450; K(m) = 15 muM) and 3-glutathionyl-S-methylindole (V(max) = 0.7 nmol/min/nmol of P450; K(m) = 13 muM). The structure of GS-A3, a minor adduct with a protonated molecular ion at m/z 453, is proposed to be 3-glutathionyl-S-3-methyloxindole. We also discovered that 3MI is a mechanism-based inactivator of CYP2A13, given that it produced a time-, cofactor-, and 3MI concentration-dependent loss of activity toward 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, with a relatively low K(I) value of approximately 10 muM and a k(inact) of 0.046 min(-1). Thus, CYP2A13 metabolizes 3MI through multiple bioactivation pathways, and the process can lead to a suicide inactivation of CYP2A13.

Mechanisms of differential expression of the CYP2A13 7520C and 7520G alleles in human lung: allelic expression analysis for CYP2A13 heterogeneous nuclear RNA, and evidence for the involvement of multiple cis-regulatory single nucleotide polymorphisms.

OBJECTIVE: To identify the mechanisms underlying the decreased allelic expression of a common CYP2A13 allele (7520C>G) in the human lung; CYP2A13 is expressed selectively in the respiratory tract, and is highly efficient in the metabolic activation of several chemical carcinogens. METHODS: The 7520C/G alleles were compared for mRNA stability in cells and relative heterogeneous nuclear RNA (hnRNA) levels in human lungs. Promoter region single nucleotide polymorphisms (SNPs) were identified and analyzed through in-vitro reporter gene assays and gel-shift assays, to uncover the causative SNPs responsible for the decreased allelic expression. RESULTS: (i) The 7520C>G SNP does not influence CYP2A13 mRNA stability in CYP2A13-transfected human lung or nasal epithelial cells; (ii) levels of the 7520G hnRNA were consistently lower (<10%) than the levels of the 7520C hnRNA in lung samples from nine heterozygous individuals; (iii) three SNPs (-1479T>C, -3101T>G, and -7756G>A) in linkage disequilibrium with the 7520C>G variation were found to cause altered interaction with DNA-binding proteins and decreases in promoter activity; (iv) the suppressive effects of the -1479T>C, -3101T>G, and -7756G>A SNPs on the CYP2A13 promoter were additive, whereas the negative effects of the -1479T>C SNP were enhanced by methylation of -1479C. CONCLUSION: The decrease in the expression of 7520G allele was because of the cumulative suppressive effects of multiple SNPs, with each by itself having a relatively small effect on CYP2A13 transcription.

Characterization of CYP2A13*2, a variant cytochrome P450 allele previously found to be associated with decreased incidences of lung adenocarcinoma in smokers.

CYP2A13, a human cytochrome P450 enzyme expressed mainly in the respiratory tract, is believed to play an important role in the initiation of smoking-induced lung cancer. CYP2A13.1 has high efficiency in the metabolic activation of a major tobacco-specific carcinogenic nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). CYP2A13(*)2, a variant allele, was previously found to be associated with decreased incidence of lung adenocarcinoma in smokers. The aim of the present study was to determine whether the CYP2A13.2 protein has decreased enzyme activity and/or expression levels in the lung, compared with CYP2A13.1. CYP2A13.2 has two sequence variations from CYP2A13.1: R25Q and R257C. We compared the activities of heterologously expressed CYP2A13.1 and CYP2A13.2 toward several known CYP2A13.1 substrates: NNK, N-nitrosomethylphenylamine, N,N-dimethylaniline, 2'-methoxyacetophenone, and hexamethylphosphoramide. Our results indicated that CYP2A13.2 was 20 to 40% less active than CYP2A13.1 with the substrates tested. We also determined the levels of the CYP2A13(*)2 mRNA, relative to the level of the CYP2A13(*)1 mRNA, in the lung tissue from (*)1/(*)2 heterozygotes. We found that the CYP2A13(*)2 allele was associated with a level of allelic expression approximately 40% lower than that of the CYP2A13(*)1 allele. Sequence analysis of the promoter region of the CYP2A13(*)2 allele identified a 26-nucleotide deletion. Functional analysis of a 2-kilobase pair CYP2A13-luciferase promoter construct indicated that the 26-nucleotide deletion causes decreases in CYP2A13 promoter activity in the A549 human lung cell line. These findings suggest that the reported association of the CYP2A13(*)2 allele with decreased incidences of lung adenocarcinoma in smokers can be at least partly explained by a decrease in CYP2A13 function.

CYP2A13: variable expression and role in human lung microsomal metabolic activation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

CYP2A13 is the most efficient cytochrome P450 enzyme in the metabolic activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific lung carcinogen. The aims of this study were to determine the levels of CYP2A13 protein in human lung microsomes and to ascertain whether CYP2A13 plays any role in lung microsomal NNK metabolic activation. The expression of CYP2A6 and CYP2A13 was examined using a high-resolution immunoblotting method, following immunopurification with an anti-CYP2A5 antibody. We found that, of 116 human lung microsomal samples analyzed, approximately 90% had detectable CYP2A6, whereas only 12% had detectable CYP2A13 with a detection limit of approximately 2 fmol of CYP2A/mg protein. For the majority of microsomal samples analyzed, the level of CYP2A13 was found to be lower than the level of CYP2A6; overall, the highest level of CYP2A13 found ( approximately 20 fmol/mg protein) was approximately 10-fold lower than the highest level of CYP2A6 detected. Quantitative RNA-polymerase chain reaction analysis confirmed that the highly variable expression of the CYP2A proteins was consistent with variations in the levels of the corresponding CYP2A mRNAs in the same tissue samples. It is noteworthy that the level of CYP2A13, but not CYP2A6, was correlated with lung microsomal NNK metabolic activation activity. Furthermore, the addition of 8-methoxypsoralen, a CYP2A inhibitor, led to greater inhibition of NNK metabolic activation in microsomes containing relatively high levels of CYP2A13 than in samples containing no detectable CYP2A13. Taken together, these data indicate that human lung microsomal CYP2A13 is active in NNK metabolic activation. Therefore, individuals having relatively high levels of CYP2A13 expression will likely have an increased risk of developing smoking-related lung cancer.

Generation and characterization of a transgenic mouse model with hepatic expression of human CYP2A6.

The aim of this study was to prepare and characterize a transgenic mouse model in which CYP2A6, a human P450 enzyme, is expressed specifically in the liver. CYP2A6, which is mainly expressed in human liver, is active toward many xenobiotics. Our transgene construct contained the mouse transthyretin promoter/enhancer, a full-length CYP2A6 cDNA, and a downstream neomycin-resistance gene for positive selection in embryonic stem cells. Hepatic expression of the CYP2A6 transgene was demonstrated by immunoblotting, whereas tissue specificity of CYP2A6 expression was confirmed by RNA-PCR. The transgenic mouse was further characterized after being backcrossed to the B6 strain for six generations. Hepatic microsomes from homozygous transgenic mice had activities significantly higher than those of B6 mice toward coumarin. The in vivo activity of transgenic CYP2A6 was also determined. Systemic clearance of coumarin was significantly higher in the transgenic mice than in B6 controls, consistent with the predicted role of CYP2A6 as the major coumarin hydroxylase in human liver. The CYP2A6-transgenic mouse model should be valuable for studying the in vivo function of this polymorphic human enzyme in drug metabolism and chemical toxicity.