0090-9556/97/2504-0481-0488$02.00/0
DRUG METABOLISM AND DISPOSITION
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
Vol. 25, No. 4

Rat Liver Cytochrome P450 Metabolism of N-Acetylbenzidine and N,N-Diacetylbenzidine

Vijaya M. Lakshmi, Terry V. Zenser, and Bernard B. Davis

Veterans Administration Medical Center, and Department of Biochemistry and Division of Geriatric Medicine, St. Louis University School of Medicine

To provide the information necessary for assessing risk and preventing tumorigenesis, the metabolism of N-acetylbenzidine and N,N-diacetylbenzidine was assessed with rat liver microsomes from control and -naphthoflavone-treated rats. The oxidation of [³H]N-acetylbenzidine to [³H]N-hydroxy-N-acetylbenzidine (NHA), [³H]N-hydroxy-N-acetylbenzidine (NHA), and ³H-ring oxidation products was assessed. For [³H]N,N-diacetylbenzidine, the formation of [³H]N-hydroxy-N,N-diacetylbenzidine (NHDA) and the ³H-ring oxidation product was assessed. With -naphthoflavone-treated microsomes, the rate of NHA formation was 8-fold more than observed with control. Although significant formation of ring-oxidation products was demonstrated, the formation of NHA was at the limit of detection. With control microsomes, NHA was a major metabolite with more NHA (49 ± 6 pmol/mg protein/min) produced than NHA (38 ± 5). Whereas the oxidation of N,N-diacetylbenzidine was not observed with control microsomes, significant formation of NHDA (421 ± 49 pmol/mg protein/min) and ring-oxidation (182 ± 28) product was observed with -naphthoflavone-treated microsomes. Metabolism of [³H]N-acetylbenzidine and [³H]N,N-diacetylbenzidine by -naphthoflavone-treated microsomes was completely inhibited by the specific cytochrome P4501A1/1A2 inhibitors -naphthoflavone and ellipticine at 10 µM. Except for the <30% inhibition observed with the cytochrome P4502E1 inhibitor (disulfiram), inhibitors of cytochrome P4503A1/3A2 (troleandomycin) and P4502C6 (sulfinpyrazone) were not effective at 10 µM. NHA formation by control microsomes was not prevented by any of these inhibitors. Conditions that inhibit flavin-dependent monooxygenase metabolism, methimazole (1 mM), and heat treatment (37°C for 60 min) were also ineffective in preventing NHA formation. The nonspecific cytochrome P450 inhibitor SKF-525A (10 µM) exhibited a partial dose-response inhibition (maximum 41% of complete reaction mixture) of NHA formation, but did not alter NHA formation. In contrast, the nonspecific cytochrome P450 inhibitor, 2,4-dichloro-6-phenylphenoxyethylamine prevented formation of both NHA and NHA. -Naphthoflavone treatment increased [³H]N-acetylbenzidine binding to DNA, but not [³H]N,N-diacetylbenzidine. Binding of both compounds to DNA was inhibited by ellipticine. N-(3-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine was detected by ³²P-postlabeling in microsomal incubations with N-acetylbenzidine, but not N,N-diacetylbenzidine. More adduct was detected with control than -naphthoflavone-treated microsomes. Results are consistent with cytochrome P4501A1/1A2 playing the major role in N-acetylbenzidine and N,N-diacetylbenzidine metabolism by liver microsomes from control and -naphthoflavone-treated rats. The formation of NHA by control, but not by -naphthoflavone-treated, rats and its insensitivity to inhibition by cytochrome P4501A1/1A2 inhibitors were unexpected.