CYP2A13 expressed in human bladder metabolically activates 4-aminobiphenyl

金沢大学大学院医学系研究科機能分子医薬学金沢大学薬学部authorCigarette smoking is the predominant risk factor for bladder cancer. Aromatic amines such as 4-aminobiphenyl (ABP) is the major carcinogens found in tobacco smoke. Although it is generally accepted that ABP is metabolically activated via N-hydroxylation by CYP1A2 in human liver, previous studies using Cyp1a2-null mice indicated the involvement of other enzyme(s). Here we found that CYP2A13 can metabolically activate ABP to show genotoxicity by Umu assay. The Km and Vmax values for ABP N-hydroxylation by recombinant CYP2A13 in E. coli were 38.5 ± 0.6 μM 7.8 ± 0.0 pmol/min/pmol CYP, respectively. The Km and Vmax values by recombinant CYP1A2 were 9.9 ± 0.9 μM and 39.6 ± 0.9 pmol/min/ pmol CYP, respectively, showing 20-fold higher intrinsic clearance than CYP2A13. In human bladder, CYP2A13 mRNA, but not CYP1A2, is expressed at a relatively high level. Human bladder microsomes showed ABP N-hydroxylase activity (K m = 34.9 ± 4.7 μM and Vmax = 57.5 ± 1.9 pmol/min/mg protein), although the intrinsic clearance was 5-fold lower than that in human liver microsomes (Km = 33.2 ± 2.0 μM and Vmax = 293.9 ± 5.8 pmol/min/mg protein). The activity in human bladder microsomes was prominently inhibited by 8-methoxypsoralen, but not by fluvoxamine, anti-CYP1A2 or anti-CYP2A6 antibodies

Association of genetic variation in tamoxifen-metabolizing enzymes with overall survival and recurrence of disease in breast cancer patients

Tamoxifen has been a mainstay of adjuvant therapy for breast cancer for many years. We sought to determine if genetic variability in the tamoxifen metabolic pathway influenced overall survival in breast cancer patients treated with tamoxifen. We examined functional polymorphisms in CYP2D6, the P450 catalyzing the formation of active tamoxifen metabolites, and UGT2B15, a Phase II enzyme facilitating the elimination of active metabolite in a retrospective study of breast cancer patients. We also examined whether the combination of variant alleles in SULT1A1 and UGT2B15 had more of an impact on overall survival in tamoxifen-treated patients than when the genes were examined separately.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44227/1/10549_2004_Article_7751.pd

DNA supercoiling suppresses real-time PCR: a new approach to the quantification of mitochondrial DNA damage and repair

As a gold standard for quantification of starting amounts of nucleic acids, real-time PCR is increasingly used in quantitative analysis of mtDNA copy number in medical research. Using supercoiled plasmid DNA and mtDNA modified both in vitro and in cancer cells, we demonstrated that conformational changes in supercoiled DNA have profound influence on real-time PCR quantification. We showed that real-time PCR signal is a positive function of the relaxed forms (open circular and/or linear) rather than the supercoiled form of DNA, and that the conformation transitions mediated by DNA strand breaks are the main basis for sensitive detection of the relaxed DNA. This new finding was then used for sensitive detection of structure-mediated mtDNA damage and repair in stressed cancer cells, and for accurate quantification of total mtDNA copy number when all supercoiled DNA is converted into the relaxed forms using a prior heat-denaturation step. The new approach revealed a dynamic mtDNA response to oxidative stress in prostate cancer cells, which involves not only early structural damage and repair but also sustained copy number reduction induced by hydrogen peroxide. Finally, the supercoiling effect should raise caution in any DNA quantification using real-time PCR

In vitro bioactivation of N-hydroxy-2-amino-α-carboline

2-Amino-α-carboline (AαC) is a mutagenic and carcinogenic heterocyclic amine present in foods cooked at high temperature and in cigarette smoke. The mutagenic activity of AαC is dependent upon metabolic activation to N-hydroxy-AαC (N-OH-AαC); however, the metabolism of N-OH-AαC has not been studied. We have synthesized 2-nitro-α-carboline and N-OH-AαC and have examined in vitro bioactivation of N-OH-AαC by human and rodent liver cytosolic sulfotransferase(s) and acetyltransferase(s) and by recombinant human N-acetyltransferases, NAT1 and NAT2. The sulfotransferase-dependent bioactivation of N-OH-AαC by human liver cytosol exhibited large interindividual variation (0.5-75, n = 14) and was significantly higher than bioactivation of N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP), Correlation and inhibition studies suggested that the isoform of sulfotransferase primarily responsible for bioactivation of N-OH-AαC in human liver cytosol is SULT1A1. O-Acetyltransferase-dependent bioactivation of N-OH-AαC by human liver cytosol also exhibited large inter-individual variation (16-192, n = 18). In contrast to other N-hydroxy heterocyclic amines, which are primarily substrates only for NAT2, both NAT1 and NAT2 catalyzed bioactivation of N-OH-AαC. The rate of bioactivation of N-OH-AαC by both NAT1 and NAT2 was significantly higher than that for N-OH-PhIP. In rat and mouse liver cytosols, the level of sulfotransferase-dependent bioactivation of N-OH-AαC was similar to the level in the high sulfotransferase activity human liver cytosol. The level of O-acetyltransferase-dependent bioactivation of N-OH-AαC in rat liver cytosol was also comparable with that in the high acetyltransferase activity human liver cytosol, However, the level of O-acetyltransferase-dependent bioactivation of N-OH-AαC in mouse liver cytosol was comparable with that in the low acetyltransferase activity human liver cytosol. In contrast to N-OH-PhIP, bioactivation of N-OH-AαC was not inhibited by glutathione S-transferase activity; however, DNA binding of N-acetoxy-AαC was inhibited 20% in the presence of GSH. These results suggest that bioactivation of N-OH-AαC may be a significant source of DNA damage in human tissues after dietary exposure to AαC and that the relative contribution of each pathway to bioactivation or detoxification of N-OH-AαC differs significantly from other N-hydroxy heterocyclic or aromatic amines