Discovery and characterization of a cytochrome b5 variant in humans with impaired hydroxylamine reduction capacity.

OBJECTIVES: We have shown that cytochrome b5 (cyt b5), along with its reductase, NADH cytochrome b5 reductase (b5R), is capable of direct xenobiotic biotransformation. We hypothesized that functionally significant genetic variability in cyt b5 could be found in healthy individuals. BASIC METHODS: Cyt b5 cDNAs were prepared from peripheral blood mononuclear cells from 63 individuals. MAIN RESULTS: One individual was heterozygous for a sequence variant in cyt b5 (A178G), with a predicted amino acid substitution of T60A. This variant, when expressed in Escherichia. coli, maintained a similar Vmax for the hydroxylamines of sulfamethoxazole, 4-aminobiphenyl, and 2-amino-l-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP), compared with wild type cyt b5, with a modestly increased Km (2 to 3.5-fold) for each substrate. When expressed in a mammalian system (HeLa cells), however, T60A was associated with a 70% reduction in cyt b5 protein expression compared with wild type. mRNA expression for both isoforms were comparable in HeLa cells, and translation of these mRNAs in a rabbit reticulocyte lysate system with inhibited proteasomal machinery were also similar. Incubation of these translated enzymes with uninhibited rabbit reticulocyte lysate, however, indicated greater susceptibility of T60A to proteasomal degradation. CONCLUSIONS: These data indicate that a naturally occurring variant in cyt b5, T60A, leads to modestly altered affinity for hydroxylamine substrates and dramatically reduced cyt b5 expression. Work is underway to determine the prevalence of this and other variants in cyt b5 or b5R in a larger population, and to determine the association of such variants with differences in hydroxylamine reduction in vivo.

Reductive detoxification of arylhydroxylamine carcinogens by human NADH cytochrome b5 reductase and cytochrome b5.

Heterocyclic and aromatic amine carcinogens are thought to lead to tumor initiation via the formation of DNA adducts, and bioactivation to arylhydroxylamine metabolites is necessary for reactivity with DNA. Carcinogenic arylhydroxylamine metabolites are cleared by a microsomal, NADH-dependent, oxygen-insensitive reduction pathway in humans, which may be a source of interindividual variability in response to aromatic amine carcinogens. The purpose of this study was to characterize the identity of this reduction pathway in human liver. On the basis of our findings with structurally similar arylhydroxylamine metabolites of therapeutic drugs, we hypothesized that the reductive detoxification of arylhydroxylamine carcinogens was catalyzed by NADH cytochrome b5 reductase (b5R) and cytochrome b5 (cyt b5). We found that reduction of the carcinogenic hydroxylamines of the aromatic amine 4-aminobiphenyl (4-ABP; found in cigarette smoke) and the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP; found in grilled meats) was indeed catalyzed by a purified system containing only human b5R and cyt b5. Specific activities were 56-346-fold higher in the purified system as compared to human liver microsomes (HLM), with similar Michaelis-Menten constants (K(m) values) in both systems. The stoichiometry for b5R and cyt b5 that yielded the highest activity in the purified system was also similar to that found in native HLM ( approximately 1:8 to 1:10). Polyclonal antisera to either b5R or cyt b5 significantly inhibited N-hydroxy-4-aminobiphenyl (NHOH-4-ABP) reduction by 95 and 89%, respectively, and immunoreactive cyt b5 protein content in individual HLM was significantly correlated with individual reduction of both NHOH-4-ABP and N-hydroxy-PhIP (NHOH-PhIP). Finally, titration of HLM into the purified b5R/cyt b5 system did not enhance the efficiency of reduction activity. We conclude that b5R and cyt b5 are together solely capable of the reduction of arylhydroxylamine carcinogens, and we further hypothesize that this pathway may be a source of individual variability with respect to cancer susceptibility following 4-ABP or PhIP exposure.