Cellular Kinetics of Induction by Oltipraz and Its Keto Derivative of Detoxication Enzymes in Human Colon Adenocarcinoma Cells1

Oltipraz [5-(2.pyrazinyl)-4-methyl-1,2-dithiole-3-thionej is a synthetic dithiolethione with chemopreventive activity against carcinogen-induced neoplasia of liver, lung, and colon in several animal model systems. Protection from tumor formation is associated with elevation of Phase H enzymes, including glutathione (GSH) transferase and NAD(P)H:quinone oxidoreductase (DT-diaphorase) in experimental carcinogenesis models in vivo. To investigate the time and dose relationships of the pharmacological action of oltipraz and to develop a model for its investigation, a human colon adenocarcinoma HT29 cell line was primarily used In this cell line, oltipraz resulted in increased activity of both GSH transferase and DT-diaphorase. At the maximum effective concentration (100 ELM), the elevation of GSH transferase was 3-fold and that of DT-diaphorase was 2-fold. The optimal duration of oltipraz exposure to HT29 cells was 24 h, following which the peak in enzyme activity was 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 period of drug exposure and remained elevated, even as catalytic activities declined to control levels, suggesting additional mechanisms for control of the activity of this enzyme. More prolonged drug exposure was associated with less induction of the detoxication enzymes, prompting an investigation of the possible toxicity of oltipraz to these cells. Although the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide assay revealed inhibition of proliferation (IC50, 100 LM oltipraz), a clonogenic assay demonstrated no loss of clonogenicity. Oltipraz is known to be extensively metabolized in many species; two Received 1 1/26/96; revised 2/7/97; accepted 2/10/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I Supported in part by Grants CA-06972, CA-49820, and CA-56036 from the National Cancer Institute, and an appropriation from the Commonwealth of Pennsylvania. 2 To whom requests for reprints should be addressed, at Kimmel Cancer Center, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107. Fax: (215) 503-3155; E-mail: [email protected]. major metabolites include a 3-ketone (metabolite 2, M2) and a molecular rearrangement to a pyrrolopyrazine derivative (metabolite 3, M3), numerous conjugates of which are formed in vivo. To investigate the potential cause of the lag in response, we synthesized two major oltipraz metabolites (M2 and M3) and tested their efficacy in enzyme induction. The activity ofDT-diaphorase was induced similarly by both oltipraz and M2 (2.6versus 2.8-fold baseline) at 100 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-state mRNA peaked at 24 h as with the parent compound. Thus, the oxidative desulfuration of oltipraz results in the formation of an active metabolite, but this process is not rate limiting for the induction of detoxicating 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 active as the parent compound and may be considered for clinical development in its own right.