Individual variability in the detoxification of carcinogenic arylhydroxylamines in human breast.

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.

Combined ascorbate and glutathione deficiency leads to decreased cytochrome b5 expression and impaired reduction of sulfamethoxazole hydroxylamine.

Sulfonamide antimicrobials such as sulfamethoxazole (SMX) have been associated with drug hypersensitivity reactions, particularly in patients with AIDS. A reactive oxidative metabolite, sulfamethoxazole-nitroso (SMX-NO), forms drug-tissue adducts that elicit a T-cell response. Antioxidants such as ascorbic acid (AA) and glutathione (GSH) reduce SMX-NO to the less reactive hydroxylamine metabolite (SMX-HA), which is further reduced to the non-immunogenic parent compound by cytochrome b (5) (b5) and its reductase (b5R). We hypothesized that deficiencies in AA and GSH would enhance drug-tissue adduct formation and immunogenicity toward SMX-NO and that these antioxidant deficiencies might also impair the activity of the b5/b5R pathway. We tested these hypotheses in guinea pigs fed either a normal or AA-restricted diet, followed by buthionine sulfoximine treatment (250 mg/kg SC daily, or vehicle); and SMX-NO (1 mg/kg IP 4 days per week, or vehicle), for 2 weeks. Guinea pigs did not show any biochemical or histopathologic evidence of SMX-NO-related toxicity. Combined AA and GSH deficiency in this model did not significantly increase tissue-drug adduct formation, or splenocyte proliferation in response to SMX-NO. However, combined antioxidant deficiency was associated with decreased mRNA and protein expression of cytochrome b (5), as well as significant decreases in SMX-HA reduction in SMX-NO-treated pigs. These results suggest that SMX-HA detoxification may be down-regulated in combined AA and GSH deficiency. This mechanism could contribute to the higher risk of SMX hypersensitivity in patients with AIDS with antioxidant depletion.