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.
ABSTRACT: 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.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.(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.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.
Project description: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.
Project description:BACKGROUND:The aim of this study was to evaluate the association between CYP2A13 polymorphisms and lung cancer susceptibility using the HapMap database. METHODS:A case-control analysis of 532 subjects with lung cancer and 614 controls with no personal history of the disease was performed. The tag SNPs rs1645690 and rs8192789 for CYP2A13 were selected, and the genetic polymorphisms were confirmed experimentally through real-time PCR, cloning, and sequencing assay. RESULTS:SNP frequency in this study was consistent with the HapMap Project database of Han-Chinese and lung cancer risk was associated with CYP2A13 polymorphisms in non-smokers. CYP2A13 shares a 93.5% identity with CYP2A6 in the amino acid sequence and the homologous sequences may interfere with the study of SNPs of CYP2A13. CONCLUSIONS:CYP2A13 may be a potential key metabolic enzyme gene in the carcinogenesis of lung cancer in non-smokers. The common polymorphisms of CYP2A13 may be candidate biomarkers for lung cancer susceptibility in Han-Chinese.
Project description:CYP2A13 is a human cytochrome P450 (P450) enzyme important in the bioactivation of the tobacco-specific lung procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). CYP2A13 expression levels vary dramatically among lung biopsy samples from patients, presumably owing in part to a suppression of CYP2A13 expression by disease-associated inflammation. Here, we determined whether CYP2A13 expression in the lungs of CYP2A13-humanized mice is suppressed by the presence of lung tumors. Tissues from an NNK lung tumor bioassay were examined. CYP2A13-humanized mice (95-100%) had multiple lung tumors at 16 weeks after NNK (30 or 50 mg/kg) treatment; whereas only ?9% of saline-treated CYP2A13-humanized mice had lung tumor (?1/lung). Mice with lung tumors, from the NNK-treated groups, were used for dissecting adjacent tumor-free lung tissues; whereas mice without visible lung tumors, from the saline-treated group, were used as controls. Compared with the controls, the levels of CYP2A13 protein and mRNA were both reduced significantly (by ?50%) in the NNK-treated groups. The levels of mouse CYP2B10 and CYP2F2 mRNAs were also significantly lower in the dissected normal lung tissues from tumor-bearing mice than in lungs from the control mice. Pulmonary tissue levels of three proinflammatory cytokines, tumor necrosis factor alpha, interferon gamma, and interleukin-6, were significantly higher in the tumor-bearing mice than in the controls, indicating occurrence of low-grade lung inflammation at the time of necropsy. Taken together, these findings support the hypothesis that CYP2A13 levels in human lungs can be suppressed by disease-associated inflammation in tissue donors, a scenario causing underestimation of CYP2A13 levels in healthy lungs.
Project description:The potential carcinogenicity of naphthalene (NA), a ubiquitous environmental pollutant, in human respiratory tract is a subject of intense debate. Chief among the uncertainties in risk assessment for NA is whether human lung CYP2A13 and CYP2F1 can mediate NA's respiratory tract toxicity.We aimed to assess the in vivo function of CYP2A13 and CYP2F1 in NA bioactivation and NA-induced respiratory tract toxicity in mouse models.Rates of microsomal NA bioactivation and the effects of an anti-CYP2A antibody were determined for lung and nasal olfactory mucosa (OM) from Cyp2abfgs-null, CYP2A13-humanized, and CYP2A13/2F1-humanized mice. The extent of NA respiratory toxicity was compared among wild-type, Cyp2abfgs-null, and CYP2A13/2F1-humanized mice following inhalation exposure at an occupationally relevant dose (10 ppm for 4 hr).In vitro studies indicated that the NA bioactivation activities in OM and lung of the CYP2A13/2F1-humanized mice were primarily contributed by, respectively, CYP2A13 and CYP2F1. CYP2A13/2F1-humanized mice showed greater sensitivity to NA than Cyp2abfgs-null mice, with greater depletion of nonprotein sulfhydryl and occurrence of cytotoxicity (observable by routine histology) in the OM, at 2 or 20 hr after termination of NA exposure, in humanized mice. Focal, rather than gross, lung toxicity was observed in Cyp2abfgs-null and CYP2A13/2F1-humanized mice; however, the extent of NA-induced lung injury (shown as volume fraction of damaged cells) was significantly greater in the terminal bronchioles of CYP2A13/2F1-humanized mice than in Cyp2abfgs-null mice.CYP2F1 is an active enzyme. Both CYP2A13 and CYP2F1 are active toward NA in the CYP2A13/2F1-humanized mice, where they play significant roles in NA-induced respiratory tract toxicity. https://doi.org/10.1289/EHP844.
Project description:Nasopharyngeal carcinoma (NPC) is characterized by a high prevalence in Southern China, especially among Cantonese individuals of the Guangdong Province. Epidemiological studies have suggested that frequent exposure to high levels of nitrosamine from preserved foods such as salted fish could be a risk factor for NPC. Cytochrome P450 encompasses a family of enzymes that metabolize carcinogens and CYP2A13, a member of this family, is expressed predominantly in the respiratory tract with the highest levels in the nasal mucosa. In an effort to test whether a correlation exists between CYP2A13 genetic polymorphism and the risk of developing NPC, we sequenced all nine exons and the exon-intron junctions of the CYP2A13 gene in 45 NPC patients. We identified a total of 21 single nucleotide polymorphism (SNPs), including 7 novel SNPs. The most frequent functional variant allele was 74A-1757G-3375T-7233G with a haplotype frequency of 7.8% in the 45 NPC cases. In addition, a stop codon mutation was detected in one case. We then selected the 3 most frequent SNPs and one stop codon mutation to expand our study to a case-control analysis within the Cantonese population. A novel haplotype consisting 8 SNPs in introns, and four additional novel SNPs were identified; but no correlation between CYP2A13 genetic polymorphism and individual susceptibility to NPC was observed.
Project description:It has been proven that cytochrome P450 enzyme 2A13 (CYP2A13) played an important role in the association between single nucleotide polymorphisms (SNP) and human diseases. Cytochrome P450 enzymes are a group of isoenzymes, whose sequence homology may interfere with the study for SNP. The aim of this study is to explore the interference on the SNP study of CYP2A13 caused by homologous sequences.Taqman probe was applied to detect distribution of rs8192789 sites in 573 subjects, and BLAST method was used to analyze the amplified sequences. Partial sequences of CYP2A13 were emplified by PCR from 60 cases. The emplified sequences were TA cloned and sequenced.For rs8192789 loci in 573 cases, only 3 cases were TT, while the rest were CT heterozygotes, which was caused by homologous sequences. There are a large number of overlapping peaks in identical sequences of 60 cases, and the SNP of 101 amino acid site reported in the SNP database is not found. The cloned sequences are 247 bp, 235 bp fragments.The homologous sequences may interfere the study for SNP of CYP2A13, and some SNP may not exist.
Project description:CYP2A13, CYP2B6, and CYP2F1 are neighboring cytochrome P450 genes on human chromosome 19, and the enzymes that they encode overlap in substrate specificity. A CYP2A13/2B6/2F1-transgenic mouse, in which CYP2A13 and 2F1 are both expressed in the respiratory tract and CYP2B6 is expressed in the liver, was recently generated. We generated a CYP2A13 (only) transgenic mouse so that the specific activity of CYP2A13 can be determined. The CYP2B6 and CYP2F1 genes in the CYP2A13/2B6/2F1 genomic clone were inactivated via genetic manipulations, and CYP2A13 was kept intact. A CYP2A13 (only) transgenic (2A13-TG) mouse was generated using the engineered construct and then characterized to confirm transgene integrity and determine copy numbers. The 2A13-TG mice were normal in gross morphology, development, and fertility. As in the CYP2A13/2B6/2F1-transgenic mouse, CYP2A13 expression in the 2A13-TG mouse was limited to the respiratory tract; in contrast, CYP2B6 and 2F1 proteins were not detected. Additional studies using the CYP2A13-humanized (2A13-TG/Cyp2abfgs-null) mouse produced by intercrossing between 2A13-TG and Cyp2abfgs-null mice confirmed that the transgenic CYP2A13 is active in the bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a lung procarcinogen. The 2A13-TG mouse should be valuable for assessing specific roles of human CYP2A13 in xenobiotic toxicity in the respiratory tract.
Project description:Cytochrome P450 3A4 (CYP3A4) metabolizes ?50% of all clinically used drugs. Although CYP3A4 expression varies widely between individuals, the contribution of genetic factors remains uncertain. In this study, we measured allelic CYP3A4 heteronuclear RNA (hnRNA) and mRNA expression in 76 human liver samples heterozygous for at least one of eight marker SNPs and found marked allelic expression imbalance (1.6-6.3-fold) in 10/76 liver samples (13%). This was fully accounted for by an intron 6 SNP (rs35599367, C>T), which also affected mRNA expression in cell culture on minigene transfections. CYP3A4 mRNA level and enzyme activity in livers with CC genotype were 1.7- and 2.5-fold, respectively, greater than in CT and TT carriers. In 235 patients taking stable doses of atorvastatin, simvastatin, or lovastatin for lipid control, carriers of the T allele required significantly lower statin doses (0.2-0.6-fold, P=0.019) than non-T carriers for optimal lipid control. These results indicate that intron 6 SNP rs35599367 markedly affects expression of CYP3A4 and could serve as a biomarker for predicting response to CYP3A4-metabolized drugs.
Project description:1.?1-Chloropyrene, one of the major chlorinated polycyclic aromatic hydrocarbon contaminants, was incubated with human cytochrome P450 (P450 or CYP) enzymes including CYP1A1, 1A2, 1B1, 2A6, 2A13, 2B6, 2C9, 2D6, 2E1, 3A4 and 3A5. Catalytic differences in 1-chloropyrene oxidation by polymorphic two CYP1B1 and five CYP2A13 allelic variants were also examined. 2.?CYP1A1 oxidized 1-chloropyrene at the 6- and 8-positions more actively than at the 3-position, while both CYP1B1.1 and 1B1.3 preferentially catalyzed 6-hydroxylation. 3.?Five CYP2A13 allelic variants oxidized 8-hydroxylation much more than 6- and 3-hydroxylation, and the variant CYP2A13.3 was found to slowly catalyze these reactions with a lower kcat value than other CYP2A13.1 variants. 4.?CYP2A6 catalyzed 1-chloropyrene 6-hydroxylation at a higher rate than the CYP2A13 enzymes, but the rate was lower than the CYP1A1 and 1B1 variants. Other human P450 enzymes had low activities towards 1-chloropyrene. 5.?Molecular docking analysis suggested differences in the interaction of 1-chloropyrene with active sites of CYP1 and 2?A enzymes. In addition, a naturally occurring Thr134 insertion in CYP2A13.3 was found to affect the orientation of Asn297 in the I-helix in interacting with 1-chloropyrene (and also 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK) and caused changes in the active site of CYP2A13.3 as compared with CYP2A13.1.
Project description:Excision repair cross-complementation group 5 (ERCC5) gene plays an important role in nucleotide excision repair, and dysregulation of ERCC5 is associated with increased lung cancer risk. Haplotype and diplotype analyses were conducted in normal bronchial epithelial cells (NBEC) to better understand mechanisms responsible for interindividual variation in transcript abundance regulation of ERCC5 We determined genotypes at putative ERCC5 cis-regulatory SNPs (cis-rSNP) rs751402 and rs2296147, and marker SNPs rs1047768 and rs17655. ERCC5 allele-specific transcript abundance was assessed by a recently developed targeted sequencing method. Syntenic relationships among alleles at rs751402, rs2296147, and rs1047768 were assessed by allele-specific PCR followed by Sanger sequencing. We then assessed association of ERCC5 allele-specific expression at rs1047768 with haplotype and diplotype structure at cis-rSNPs rs751402 and rs2296147. Genotype analysis revealed significantly (P < 0.005) higher interindividual variation in allelic ratios in cDNA samples relative to matched gDNA samples at both rs1047768 and rs17655. By diplotype analysis, mean expression was higher at the rs1047768 alleles syntenic with rs2296147 T allele compared with rs2296147 C allele. Furthermore, mean expression was lower at rs17655 C allele, which is syntenic with G allele at a linked SNP rs873601 (D' = 0.95). These data support the conclusions that in NBEC, T allele at SNP rs2296147 upregulates ERCC5, variation at rs751402 does not alter ERCC5 regulation, and that C allele at SNP rs17655 downregulates ERCC5 Variation in ERCC5 transcript abundance associated with allelic variation at these SNPs could result in variation in NER function in NBEC and lung cancer risk.