Individual Variability in the Detoxification of Carcinogenic Arylhydroxylamines in Human Breast

Abstract

Cytochrome b(5) (b5) and NADH cytochrome b(5) reductase (b5R) detoxify reactive hydroxylamine (NHOH) metabolites of known arylamine and heterocyclic amine mammary carcinogens. The aim of this study was to determine whether NHOH reduction for the prototypic arylamine 4-aminobiphenyl (4-ABP) was present in human breast and to determine whether variability in activity was associated with single nucleotide polymorphisms (SNPs) in the coding, promoter, and 3'untranslated region (UTR) regions of the genes encoding b5 (CYB5A) and b5R (CYB5R3). 4-ABP-NHOH reduction was readily detected in pooled human breast microsomes, with a K(m) (280μM) similar to that found with recombinant b5 and b5R, and a V(max) of 1.12 ± 0.19 nmol/min/mg protein 4-ABP-NHOH reduction varied 75-fold across 70 individual breast samples and correlated significantly with both b5 (80-fold variability) and b5R (14-fold) immunoreactive protein. In addition, wide variability in b5 protein expression was significantly associated with variability in CYB5A transcript levels, with a trend toward the same association between b5R and CYB5R3. Although a sample with a novel coding SNP in CYB5A, His22Arg, was found with low reduction and b5 expression, no other SNPs in either gene were associated with outlier activity or protein expression. We conclude that b5 and b5R catalyze the reduction of 4-ABP-NHOH in breast tissue, with very low activity, protein, and messenger RNA expression in some samples, which cannot be attributed to promoter, coding, or 3'UTR SNPs. Further studies are underway to characterize the transcriptional regulation of CYB5A and CYB5R3 and begin to understand the mechanisms of individual variability in this detoxification pathway.

The Involvement of Mitochondrial Amidoxime Reducing Components 1 and 2 and Mitochondrial Cytochrome b5 in N-Reductive Metabolism in Human Cells

“…Because mARC was identified in the OMM (2,11,16), it is likely that the mitochondrial isoform of CYB5 is the physiological partner of mARC. However, reduction studies of a two-component microsomal system consisting of CYB5A and its reductase have been described (21)(22)(23)(24). By siRNA-mediated down-regulation of CYB5A or CYB5B in HEK-293 and by analyzing CYB5A knock-out mice, we demonstrate that only the mitochondrial heme protein is involved in the N-reductive metabolic pathway.…”

Polymorphisms in the carcinogen detoxification genes CYB5A and CYB5R3 and breast cancer risk in African American women

“…SNPs were selected based on the following criteria: 1) a variant previously observed in breast or liver samples with low protein expression or arylhydroxylamine reduction activities [35,36]; 2) a reported non-synonymous cSNP (NCBI SNP database; www.ncbi.nlm.nih.gov/); or 3) a reported SNP in a region predicted to affect protein function, splicing, or transcription factor or miRNA binding, even if apparently rare. For the latter predictions, the possible effects of coding polymorphisms on protein function were evaluated using PMut (mmb.pcb.ub.es/PMut/), SIFT (sift.jcvi.org/), and PolyPhen-2 (genetics.bwh.harvard.edu/pph2/ index.shtml) software.…”

“…SNPs that failed the Taqman assay were evaluated by pyrosequencing, using the PSQ TM 96MA System (Biotage AB, Uppsala, Sweden), at the University of Wisconsin Carbone Comprehensive Cancer Center. Both SNP screening techniques were validated by running positive and negative genomic DNA controls from liver or breast samples, where available, in which the allele of interest had been previously established by direct sequencing [35,36]. A total of 144 ancestry informative markers (AIMs) were also genotyped to estimate African and European ancestry [41].…”

“…We have found individual differences in cytochrome b 5 and NADH cytochrome b 5 reductase protein expression and arylhydroxylamine detoxification activities in both human liver and breast samples, with more than 75-fold variability in activities in 70 breast samples from women undergoing reduction mammoplasty or lumpectomy [35,36]. We further found polymorphisms in the genes CYB5A and CYB5R3 , which encode b5 and b5R, in tissues with outlier low protein expression and activities [35,36].…”

“…We further found polymorphisms in the genes CYB5A and CYB5R3 , which encode b5 and b5R, in tissues with outlier low protein expression and activities [35,36]. Many of these variants were found only or predominantly in African American samples.…”