Biochemical and genotypic characterization of 4-aminobiphenyl-hydroxlamine reduction in human breast

Carcinogens such as 4-aminobiphenyl (4-ABP), in tobacco, and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP), found in grilled meats, are known rodent mammary carcinogens. These two compounds are not directly genotoxic; however once oxidized to their respective arlhydroxylamine metabolites, they can undergo further bioactivation to form a reactive nitrenium ion thought to initiate cancer by forming DNA adducts. Therefore, the reduction of arlhydroxlamines back to their parent compounds by cytochrome b5 (b5) and NADH cytochrome b5 reductase (b5R) is considered a detoxification mechanism.;Although PhIP and 4-ABP lead to mammary cancer in animal models, epidemiological studies examining the relationship between PhIP or 4-ABP exposure (via well-done meat intake or smoking) and breast cancer risk have yielded mixed results. In addition, there is wide variability in DNA adduct formation after in vitro exposure of breast epithelial cells from individual women to standardized amounts of arylhydroxylamine carcinogens. This may be due, in part, to local differences in arylhydroxylamine disposition. We hypothesize that b5 and b5R catalyze arylhydroxylamine detoxification locally in the breast, and that polymorphic variation in the genes that encode b5 (CYB5A ) and b5R (CYB5R3) have an important influence on arylhydroxylamine detoxification capacity.;This thesis describes the characterization of 4-aminobiphenyl-hydroxlamine (4-ABP-NHOH) reduction by h5 and h5R in human breast. The apparent K m in breast microsomes was similar to that found in the recombinant system and in liver microsomes, with a Vmax about one-fourth of that in liver. A remarkable amount of individual variability was observed in reduction activities (79-fold) and b5 and b5R protein expression (> 20-fold). Reduction activities in individual breast microsomes correlated with expression of both immunoreactive b5 and b5R, and h5 antisera inhibited 4-ABP-NHOH reduction by more than 70%. Interestingly, b5 and b5R protein expression were also highly correlated, suggesting possible co-regulation. Most of this variability could not be explained by single nucleotide polymorphisms (SNPs) in the promoter, coding, and 3'UTR regions of CYB5A and CYB5R3 These findings suggest the need to develop a better understanding of the transcriptional regulation of CYB5A and CYB5R3, and its effects on individual variability in this detoxification pathway in breast and other cancer target tissues.