title: Reductive detoxification of arylhydroxylamine carcinogens by human NADH cytochrome b5 reductase and cytochrome b5.
abstract: 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.
authors.affiliation: Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53706-1102, USA.
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Kurian
authors.forename: Nathaniel A
authors.initials: NA
authors.lastname: Chin
authors.forename: Brett J
authors.initials: BJ
authors.lastname: Longlais
authors.forename: Kristie L
authors.initials: KL
authors.lastname: Hayes
authors.forename: Lauren A
authors.initials: LA
authors.lastname: Trepanier
mesh_terms: D002273:Carcinogens / Q000378:metabolism* / Q000633:toxicity*; D042966:Cytochrome-B(5) Reductase / Q000302:isolation & purification / Q000378:metabolism*; D015786:Cytochromes b5 / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D019811:Hydroxylamine / Q000737:chemistry / Q000378:metabolism; D006898:Hydroxylamines / Q000378:metabolism* / Q000633:toxicity*; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D010084:Oxidation-Reduction; D013379:Substrate Specificity
chemical_list: D002273:Carcinogens; D006898:Hydroxylamines; D019811:Hydroxylamine; D015786:Cytochromes b5; D042966:Cytochrome-B(5) Reductase