PT - JOURNAL ARTICLE AU - Vijaya M. Lakshmi AU - Terry V. Zenser AU - Bernard B. Davis TI - Rat Liver Cytochrome P450 Metabolism of<em>N</em>-Acetylbenzidine and <em>N</em>,<em>N</em>′-Diacetylbenzidine DP - 1997 Apr 01 TA - Drug Metabolism and Disposition PG - 481--488 VI - 25 IP - 4 4099 - http://dmd.aspetjournals.org/content/25/4/481.short 4100 - http://dmd.aspetjournals.org/content/25/4/481.full SO - Drug Metab Dispos1997 Apr 01; 25 AB - To provide the information necessary for assessing risk and preventing tumorigenesis, the metabolism ofN-acetylbenzidine andN,N′-diacetylbenzidine was assessed with rat liver microsomes from control and β-naphthoflavone-treated rats. The oxidation of [3H]N-acetylbenzidine to [3H]N′-hydroxy-N-acetylbenzidine (N′HA), [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 β-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 N′HA was at the limit of detection. With control microsomes, N′HA was a major metabolite with more N′HA (49 ± 6 pmol/mg protein/min) produced than NHA (38 ± 5). Whereas the oxidation ofN,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 β-naphthoflavone-treated microsomes. Metabolism of [3H]N-acetylbenzidine and [3H]N,N′-diacetylbenzidine by β-naphthoflavone-treated microsomes was completely inhibited by the specific cytochrome P4501A1/1A2 inhibitors α-naphthoflavone and ellipticine at 10 μM. Except for the &lt;30% inhibition observed with the cytochrome P4502E1 inhibitor (disulfiram), inhibitors of cytochrome P4503A1/3A2 (troleandomycin) and P4502C6 (sulfinpyrazone) were not effective at 10 μM. N′HA 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 N′HA formation. The nonspecific cytochrome P450 inhibitor SKF-525A (10 μM) exhibited a partial dose–response inhibition (maximum 41% of complete reaction mixture) of N′HA formation, but did not alter NHA formation. In contrast, the nonspecific cytochrome P450 inhibitor, 2,4-dichloro-6-phenylphenoxyethylamine prevented formation of both N′HA and NHA. β-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 notN,N′-diacetylbenzidine. More adduct was detected with control than β-naphthoflavone-treated microsomes. Results are consistent with cytochrome P4501A1/1A2 playing the major role in N-acetylbenzidine andN,N′-diacetylbenzidine metabolism by liver microsomes from control and β-naphthoflavone-treated rats. The formation of N′HA by control, but not by β-naphthoflavone-treated, rats and its insensitivity to inhibition by cytochrome P4501A1/1A2 inhibitors were unexpected. The American Society for Pharmacology and Experimental Therapeutics