CELLULAR KINETICS OF INDUCTION BY OLTIPRAZ AND ITS KETO DERIVATIVE OFDETOXICATION ENZYMES IN HUMAN COLON ADENOCARCINOMA CELLS

Oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione] is a synthet ic dithiolethione with chemopreventive activity against carcinogen-ind uced neoplasia of liver, lung, and colon in several animal model syste ms, Protection from tumor formation is associated with elevation of Ph ase II enzymes, including glutathione (GSH) transferase and NAD(P)H:qu inone oxidoreductase (DT-diaphorase) in experimental carcinogenesis mo dels in vivo. To investigate the time and dose relationships of the ph armacological action of oltipraz and to develop a model for its invest igation, a human colon adenocarcinoma HT29 cell line was primarily use d, In this cell line, oltipraz resulted in increased activity of both GSH transferase and DT-diaphorase, At the maximum effective concentrat ion (100 mu M), the elevation of GSH transferase was 3-fold and that o f DT-diaphorase was 2-fold, The optimal duration of oltipraz exposure to HT29 cells was 24 h, following which the peak in enzyme activity wa s observed at 24 h after removal of the drug, and activity had almost returned to control levels after 72 h in drug-free media, Steady-state mRNA levels for DT-diaphorase were observed to increase during the pe riod of drug exposure and remained elevated, even as catalytic activit ies declined to control levels, suggesting additional mechanisms for c ontrol of the activity of this enzyme, More prolonged drug exposure wa s associated with less induction of the detoxication enzymes, promptin g an investigation of the possible toxicity of oltipraz to these cells , Although the (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium brom ide assay revealed inhibition of proliferation (IC50, 100 mu M oltipra z), a clonogenic assay demonstrated no loss of clonogenicity, Oltipraz is known to be extensively metabolized in many species; two major met abolites include a 3-ketone (metabolite 2, M2) and a molecular rearran gement to a pyrrolopyrazine derivative (metabolite 3, M3), numerous co njugates of which are formed in vivo, To investigate the potential cau se of the lag in response, we synthesized two major oltipraz metabolit es (M2 and M3) and tested their efficacy in enzyme induction, The acti vity of DT-diaphorase was induced similarly by both oltipraz end M2 (2 .6- versus 2.8-fold baseline) at 100 mu M, whereas M3 was inactive at all concentrations, M2 also resulted in a 5.8-fold elevation of steady -state DT-diaphorase mRNA levels, Both enzyme activity and steady-stat e mRNA peaked at 24 h as with the parent compound, Thus, the oxidative desulfuration of oltipraz results in the formation of an active metab olite, but this process is not rate limiting for the induction of deto xicating enzymes, These data support the use of intermittent schedules in oltipraz in clinical trials of chemoprevention because of evidence of attenuation of response, The metabolite M2, but not M3, is as acti ve as the parent compound and may be considered for clinical developme nt in its own right.