A nicotine C-oxidase gene (CYP2A6) polymorphism important for promoter activity.

In humans, several polymorphic variants have been described for the gene encoding the major nicotine C-oxidase, cytochrome P450 2A6 (CYP2A6), which is to a great extent responsible for the large interindividual differences seen at the enzymatic and activity levels. Hitherto, mainly polymorphic variants in the open reading frame have been identified. In the present study, we identified a novel single nucleotide polymorphism (SNP) located in the 5' flanking region of the CYP2A6 gene. Sequencing of 1.4 kb of the 5'-upstream region of the CYP2A6 gene from eight individuals revealed a c.-1013A>G polymorphism defining two new alleles, CYP2A6*1D and CYP2A6*1E, lacking or having also the CYP2A7 3'-UTR. Analysis of genomic DNA from 32 Swedish and 109 Turkish subjects by dynamic allele-specific hybridization (DASH) showed that, in both groups, the variants carrying the c.-1013A>G SNP represent approximately 70% of the total number of alleles. Transfection of HepG2 cells with luciferase reporter constructs containing 1019 bp of the CYP2A6 5'-regulatory sequence showed that the region between c.-1005 and c.-1019 elicited a strong enhancer effect and that the CYP2A6*1D promoter had significantly reduced expression as compared to CYP2A6*1A carrying c.-1013A. Electrophoretic mobility shift assays (EMSA) showed that nuclear proteins from HepG2 and B16A2 cells exhibited a higher binding affinity to the probe harboring c.-1013A as compared to the c.-1013G probe, although the transcription factor(s) responsible for this binding could not be identified. In conclusion, our results indicate the presence of a strong enhancer or promoter responsive element between c.-1005 and c.-1019 in the CYP2A6 gene and that a c.-1013A>G polymorphism in this region affects CYP2A6 transcription.

Polymorphic NF-Y dependent regulation of human nicotine C-oxidase (CYP2A6).

OBJECTIVES: In humans, cytochrome P450 2A6 (CYP2A6) constitutes the principal nicotine C-oxidase. Several different polymorphic CYP2A6 gene variants are known which contribute to the highly variable expression of this enzyme among individuals. In this study we report a novel polymorphism located in the 5' flanking region (-745A > G) of the CYP2A6 gene disrupting a CCAAT box. METHODS AND RESULTS: Electrophoretic mobility shift assays (EMSA) indicated that NF-YA is part of this nuclear protein complex. Chromatin immunoprecipitation revealed that NF-Y recognizes a region of the CYP2A6 5' flanking region located between -932 and -606. EMSA showed that out of the three CCAAT boxes in the CYP2A6 promoter, with CCAAT core sequences located between -839/-835, -748/-744, and -689/-685, only the one at -748/-744 was able to compete with the nuclear protein complex binding to the -748/-744 CCAAT box. Cotransfection experiments indicated that NF-Y acts as a positive regulatory element on CYP2A6 gene regulation. EMSA demonstrated that an NF-Y consensus oligonucleotide but not the -745A > G oligonucleotide competed efficiently with binding of the protein complex to the -748/-744 CCAAT box. Promoter activity of the -745A > G variant was significantly reduced to 78% relative to the wild-type allele in HepG2 cells transfected with luciferase reporter plasmids. Finally, haplotype analysis was carried out comprising the -745A > G variant in combination with all known CYP2A6 3' and 5' flanking single nucleotide polymorphisms: -1013A > G, -48T > G, and the CYP2A6/CYP2A7 3' flank conversion. CONCLUSION: A new haplotype, CYP2A6*1H was identified, with allele frequencies of 3.1% in Swedish and 5.2% in Turkish populations.

Sister chromatid exchanges and micronuclei in peripheral lymphocytes of shoe factory workers exposed to solvents.

We examined sister chromatid exchanges (SCEs) and micronuclei (MN; cytokinesis-block method) in cultured peripheral lymphocytes from 52 female workers of two shoe factories and from 36 unexposed age- and sex-matched referents. The factory workers showed an elevated level of urinary hippuric acid, a biomarker of toluene exposure, and workplace air contained high concentrations of various organic solvents such as toluene, gasoline, acetone, and (in one of the plants only) ethylacetate and methylenediphenyl diisocyanate. The shoe factory workers showed a statistically significant higher frequency of micronucleated binucleate lymphocytes in comparison with the referents. This finding agreed with three preliminary MN determinations (each comprising 27-32 shoe workers and 16-20 controls) performed in one of the plants 2-5 years earlier. The shoe factory workers also had a lower average level of blood hemoglobin than the referents. In contrast, no difference was found between the groups in SCE analysis. Smokers showed significantly higher mean frequencies of SCEs per cell and high frequency cells (HFC) than nonsmokers. Aging was associated with increased MN rates and reduced cell proliferation. Polymorphism of the glutathione S-transferase M1 gene (GSTM1) did not affect the individual level of SCEs; but in smoking shoe workers an effect of the occupational exposure on the frequency of micronucleated cells could be seen only in GSTM1 null subjects. The low prevalence of the glutathione S-transferase T1 (GSTT1) null genotype precluded the evaluation of the influence of GSTT1 polymorphism. Our results show that the shoe factory workers have experienced genotoxic exposure, which is manifest as an increase in the frequency of MN, but not of SCEs, in peripheral lymphocytes. The exposures responsible for the MN induction could not be identified with certainty, but exposure to benzene in gasoline and methylenediphenyl diisocyanate may explain some of the findings.

3'-UTR polymorphism in the human CYP2A6 gene affects mRNA stability and enzyme expression.

Cytochrome P450 2A6 (CYP2A6) is the major nicotine C-oxidase in human and participates in the metabolism of drugs and precarcinogens. The CYP2A6 gene is highly polymorphic and more than 22 different alleles have been described. We here focused on the polymorphism in the 3'-UTR region, in particular the common CYP2A6*1B allele, carrying an unequal crossover element from the pseudogene CYP2A7. Analysis of CYP2A6 expression in a human liver bank (n=46) revealed that the protein level and catalytic activity using coumarin as a substrate were all higher, following a linear gene-dose relationship, in livers carrying one or two copies of CYP2A6*1B, as compared to other CYP2A6 allelic variants. Different variants of the CYP2A6 3'-UTR were cloned into a modified pGL3 plasmid downstream of the luciferase reporter gene. The plasmids, having the proximal promoter of CYP2A6 gene, were transfected into HeLa cells or injected into the tail veins of male CD1 mice. In both systems, the 3'-UTR CYP2A6*1B constructs caused higher reporter gene activity and the CYP2A7 3'-UTR construct lower activity, compared to the CYP2A6*1 3'-UTR constructs. Two SNPs differentiating the 3'-UTR between CYP2A7 and CYP2A6*1B were found to be of importance for the expression in both systems. Analysis of reporter enzyme degradation in HeLa cells showed that luciferase-3'-UTR-CYP2A6*1A had a half-life of approximately 4.9h as compared to 6.3h for luciferase-3'-UTR-CYP2A6*1B. In conclusion, we identified polymorphic motifs in the CYP2A6 3'-UTR of importance for CYP2A6 mRNA stabilization and enzyme expression. Such polymorphism has been described to influence the in vivo rate of nicotine elimination and possibly the cigarette consumption and risk of smoking induced lung cancer.

Transcriptional regulation of the human CYP2A6 gene.

Nicotine C-oxidation is primarily catalyzed by CYP2A6 in humans. This enzymatic activity exhibits a large interindividual variability, which to a great extent is caused by genetic polymorphisms in the CYP2A6 gene. There are large interindividual differences in CYP2A6 mRNA and protein levels, but little is known about the transcriptional regulation of CYP2A6, which can, e.g., explain such differences. Using transient transfections of 5'-deleted CYP2A6 promoter constructs in human hepatoma B16A2 cells, we show that maximal promoter activity was harbored in the sequence spanning from -112 to -61. Putative response elements for the transcription factors hepatocyte nuclear factor-4 (HNF-4)alpha, CCAAT-box/enhancer binding protein (C/EBP)alpha, C/EBPbeta, and octamer transcription factor-1 (Oct-1) were identified in this region, and electrophoretic mobility shift assays showed that these transcription factors bind to the predicted elements. To determine the relevance of these sites, expression vectors for these transcription factors were cotransfected with CYP2A6 promoter constructs in HepG2 cells. HNF-4alpha, C/EBPalpha, and Oct-1 exerted an activating effect, whereas overexpression of C/EBPbeta reduced CYP2A6 promoter activity. To confirm the importance of these sites in vivo, mutated CYP2A6 reporter constructs were injected into mouse liver. Mutation of either HNF-4 or C/EBP-Oct-1 motifs significantly decreased promoter activity, 52 and 26% of wildtype, respectively, whereas when both motifs were mutated the activity in mice decreased to 14% of wild type. In conclusion, the data indicate that the constitutive hepatic expression of CYP2A6 is governed by an interplay between the transcription factors HNF-4alpha, C/EBPalpha, C/EBPbeta, and Oct-1. These results will be important for the identification of new polymorphisms affecting CYP2A6 gene expression.

Genetic polymorphism of CYP1A2 in Ethiopians affecting induction and expression: characterization of novel haplotypes with single-nucleotide polymorphisms in intron 1.

CYP1A2 polymorphism has been well studied in white persons and Asians but not in Africans. We performed CYP1A2 genotype and phenotype analysis using caffeine in Ethiopians living in Ethiopia (n = 100) or in Sweden (n = 73). We sequenced the CYP1A2 gene using genomic DNA from 12 subjects, which revealed a novel intron 1 single-nucleotide polymorphism (SNP), -730C>T. We developed SNP-specific polymerase chain reaction-restriction fragment length polymorphism genotyping and molecular haplotyping methods for the intron 1 SNPs, and four different haplotypes were identified: CYP1A2*1A (wild-type for all SNPs), CYP1A2*1F (-164A), CYP1A2*1J (-740G and -164A), and CYP1A2*1K (-730T, -740G, and -164A), having frequencies of 39.9, 49.6, 7.5, and 3.0%, respectively. The frequency of CYP1A2*1J and CYP1A2*1K among Saudi Arabians (n = 136) was 5.9% and 3.6%, and among Spaniards (n = 117) 1.3% and 0.5%, respectively. Functional significance of the different intron 1 haplotypes was analyzed. Subjects with CYP1A2*1K had significantly decreased CYP1A2 activity in vivo, and reporter constructs with this haplotype had significantly less inducibility with 2,3,7,8-tetrachlorodibenzo-p-dioxin in human B16A2 hepatoma cells. Electrophoretic mobility shift assay using nuclear extracts from B16A2 cells revealed a specific DNA binding protein complex to an Ets element. Efficient competition was obtained using oligonucleotide probes carrying the wt sequence and Ets consensus probe, whereas competition was abolished using probes with the -730C>T SNP alone or in combination with -740T>G (CYP1A2*1K). The results indicate a novel polymorphism in intron 1 of importance for Ets-dependent CYP1A2 expression in vivo and inducibility of the enzyme, which might be of critical importance for determination of interindividual differences in drug metabolism and sensitivity to carcinogens activated by CYP1A2.