Kinetics of N- and C-hydroxylations of 2-acetylaminofluorene in male Sprague-Dawley rat liver microsomes: implications for carcinogenesis.
The metabolism of 2-acetylaminofluorene (AAF) has been studied in male Sprague-Dawley rat liver microsomes over a concentration range of 0.02 to 300 microM, and kinetic parameters have been determined for five oxidative pathways. The N-hydroxylation of AAF was best described by a single enzyme system with a mean Km of 0.033 microM and a mean Vmax of 3.63 pmol/mg/min. Pretreatment of animals with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) caused a marked induction of N-hydroxylase activity while phenobarbital had no effect. Biphasic kinetics for the 7-hydroxylation of AAF were observed in both control and TCDD- and phenobarbital-induced microsomes. The high-affinity Km [0.051 +/- 0.015 (S.E.) microM; n = 3] in control microsomes was 3 orders of magnitude lower than the low-affinity Km (103 +/- 16 microM; n = 3) indicating that each isoenzyme predominated at vastly different substrate concentrations. The mean Vmax values for the low- and high-affinity enzymes were 3.5 and 1351 pmol/mg/min, respectively. TCDD pretreatment markedly induced the activity of the low-capacity enzyme and reduced the activity of the high-capacity enzyme. Phenobarbital caused a significant induction of both enzyme pathways. Biphasic kinetics were also observed for the 5-, 3-, and 1-hydroxylations of AAF in control and phenobarbital-induced microsomes, but in TCDD-pretreated microsomes only 1-hydroxylation exhibited biphasic kinetics. TCDD caused a marked induction of these metabolic pathways while phenobarbital had no effect. Nonclassical kinetics were observed for the 9-hydroxylation of AAF, and at high substrate concentrations detoxification via this pathway and 7-hydroxylation predominated. However, at low concentrations, metabolic activation of AAF via N-hydroxylation was a major pathway. These data indicate that multiple forms of cytochrome P-450 are involved in AAF metabolism and that the balance between metabolic activation and detoxification of this substrate is dependent on both concentration and previous exposure to inducers.
D015073:2-Acetylaminofluorene / Q000378:metabolism*; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D006900:Hydroxylation; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains / Q000378:metabolism*
D003577:Cytochrome P-450 Enzyme System; D015073:2-Acetylaminofluorene; D010634:Phenobarbital