TY - JOUR T1 - Regulation of thiol environment of the N-demethylation and ring hydroxylation of N,N-dimethyl-4-aminoazobenzene (DAB) by rat liver microsomes. JF - Drug Metabolism and Disposition JO - Drug Metab Dispos SP - 13 LP - 18 VL - 14 IS - 1 AU - W G Levine Y1 - 1986/01/01 UR - http://dmd.aspetjournals.org/content/14/1/13.abstract N2 - A study was conducted on the regulation by thiol environment of microsomal metabolism of the azo dye hepatocarcinogen, N,N-dimethyl-4-aminoazobenzene (DAB). Physiological concentrations of glutathione (GSH) stimulated N-demethylation and ring hydroxylation of the dye in normal and phenobarbital (PB)-treated microsomes. However, little effect of GSH was seen with microsomes from beta-naphthoflavone (BNF)-treated rats. The synthetic thiol, dithiothreitol (DTT), stimulated ring-hydroxylation of DAB but inhibited N-demethylation at all concentrations in control nd PB-induced microsomes. A biphasic response to DTT was obtained with BNF microsomes; inhibition of N-demethylation was seen only at low concentrations (0.1 mM) and a return to control values occurred at higher concentrations. DTT inhibition was shown to be specific for the first N-demethylation step, whereas the second was slightly stimulated at concentrations greater than 3.0 mM. Agents which alkylate [N-ethylmaleimide (NEM), p-hydroxymercuribenzoate] or oxidize [5,5'-dithiobis(nitrobenzoic acid) or Ellman's reagent] protein SH groups inhibited DAB metabolism. Inhibition of microsomal NADPH-cytochrome c reductase activity by p-hydroxymercuribenzoate required an order of magnitude more inhibitor than was needed to block DAB metabolism. This suggests that DAB metabolism requires viable SH groups other than those involved in NADPH-cytochrome c reductase activity. NEM, in contrast, inhibited the N-demethylation of DAB and NADPH-cytochrome c reductase at approximately the same concentrations. Ring-hydroxylation was stimulated by high (greater than 1 mM) concentrations of NEM, implying a different enzymic mechanism for this pathway.(ABSTRACT TRUNCATED AT 250 WORDS) ER -