NADPH-dependent oxidation of benzidine by rat liver.
This study used liver microsomes from control an naphthoflavone-treated rats to evaluate NADPH-dependent oxidation of benzidine. With microsomes from beta-naphthoflavone-treated rats, the rates of formation of aqueous soluble metabolite (HPLC analysis) and protein and DNA binding were 835 +/- 81, 14.5 +/- 1.8 and 0.71 +/- 0.14 pmol/mg/min respectively. beta-Naphthoflavone treatment elicited 12.3-, 1.8- and 14.2-fold increases in benzidine metabolism compared with controls as judged by HPLC and protein and DNA binding respectively. For microsomes from treated animals, Km and Vmax values were 47 +/- 6 micromol and 1.13 +/- 0.16 nmol/mg protein/min respectively. All of the metabolic parameters were inhibited to varying degrees by glutathione (1 or 10 mM), N-acetylmethionine (10 mM) and ascorbic acid (10 mM). Following glutathione addition, at least two new metabolite peaks were observed, representing -6% of the total radioactivity recovered by HPLC. Neither metabolite was 3-(glutathion-S-yl)benzidine. Cytochrome P450 inhibitors (10 micro) specific for different members of cytochrome gene families 1-3 indicated that benzidine was metabolized by cytochrome P450 1A1/1A2. Ellipticine and alpha-naphthoflavone, specific 1A1/1A2 inhibitors, elicited 50% inhibition at -0.2 and 0.5 micro respectively. Electron impact and negative ion chemical ionization mass spectro- metry identified the aqueous soluble metabolite as 3-hydroxybenzidine. The lability of 3-hydroxybenzidine observed at pH > 7.0 was prevented by ascorbic acid. Thus, cytochrome P450 1A1/1A2 NADPH-dependent metabolism of benzidine to 3-hydroxybenzidine was demonstrated.
Lakshmi
V M
VM
VA Medical Center and Department of Biochemistry and Division of Geriatric Medicine, St Louis University School of Medicine, MO 63125, USA.
Zenser
N T
NT
Hsu
F F
FF
Mattammal
M B
MB
Zenser
T V
TV
Davis
B B
BB
D000818:Animals; D001560:Benzidines / Q000378:metabolism*; D001711:Biotransformation; D002851:Chromatography, High Pressure Liquid; D018736:DNA Adducts / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D014316:Tritium; D019324:beta-Naphthoflavone / Q000494:pharmacology*
D001560:Benzidines; D018736:DNA Adducts; D014316:Tritium; C029876:benzidine; D009249:NADP; D019324:beta-Naphthoflavone