Oncology Center, Johns Hopkins Medical Institutions, Baltimore, Maryland 21205.
The substituted 1,2-dithiole-3-thione oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione] protects against the acute and chronic toxicities of many xenobiotics, including aflatoxin B1, in rodents. These protective effects are mediated, in part, through elevation of glutathione S-transferase (GST) activities. Because studies by Coles et al. [Carcinogenesis (Lond.), 6: 693-697, 1985] suggested that the detoxication of aflatoxin through conjugation with glutathione is principally catalyzed by GST homodimer YaYa, we have investigated the regulation of the gene coding for the Ya subunit in the liver of F344 rats following dietary administration of oltipraz. Overall GST activity, as measured by conjugation with 1,2-dichloro-4-nitrobenzene or 1-chloro-2,4-dinitrobenzene, as well as the levels of GST Ya protein, was elevated 1.5-fold by 24 h and maximally (2.7- to 3.5-fold) and persistently after 5 days on a purified diet supplemented with 0.075% oltipraz. Steady state mRNA levels for GST subunit Ya, as quantified by slot blot analysis using rat liver GST complementary DNA clone pGTB38, were also elevated by 24 h, with a maximal elevation of 3-fold observed at 3 days. However, mRNA levels decreased thereafter, despite continued feeding of oltipraz. Northern blot analyses demonstrated that oltipraz did not alter the size of GST mRNA. Transcriptional activity of the GST Ya gene, as determined by nuclear run-off analysis, was increased 2-fold after 24-h feeding of oltipraz, was maximally induced 2.4-fold at 3 days, and returned to near control levels at 7 days, despite sustained feeding of oltipraz. Modulation of GST activity by oltipraz was not accompanied by changes in the methylation pattern at internal sites of the GST Ya gene. These results show that the initial induction of hepatic GST activity during oltipraz exposure correlates with changes in steady state levels of GST mRNA and rates of GST gene transcription; however, the continued elevation of GST enzymatic activities and GST Ya protein levels in the face of declining GST Ya mRNA levels and transcription rates suggests that additional mechanisms may be involved in regulating GST Ya expression by oltipraz.
Department of Pharmacology and Molecular Sciences, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205.
Most chemical carcinogens require activation to reactive electrophilic forms by Phase 1 enzymes (cytochromes P-450) in order to exert their toxic and neoplastic effects. The resultant electrophiles are susceptible to metabolic conjugation and other types of detoxications by Phase 2 enzymes (glutathione transferases, NAD(P)H: quinone reductase, glucuronosyltransferases). The balance between Phase 1 and Phase 2 enzymes is an important determinant of whether exposure to carcinogens will result in toxicity and neoplasia. Measurements of the activity of quinone reductase (QR) provide an efficient method for studying the potency and mechanism of Phase 2 enzyme induction. QR can be measured easily in murine hepatoma cells (Hepa lclc7) grown in microtiter plate wells, and the inductive response of these cells closely parallels the behavior of rodent tissues in vivo. Some inducers (such as large planar aromatics) are bifunctional; they induce both Phase 1 and Phase 2 enzymes and require binding to the Ah receptor and enhanced transcription of the cytochrome P1-450 system. Other inducers (e.g., phenolic antioxidants, 1, 2-dithiole-3-thiones, coumarins, thiocarbamates) are monofunctional and are independent of Ah receptor function. Monofunctional enzyme induction protects against carcinogens. The induction of Phase 2 enzymes by monofunctional inducers depends on the presence, or acquisition by metabolism, of electrophilic centers, and many of these inducers are Michael reaction acceptors. Our search for chemoprotective enzyme inducers for potential use as chemoprotectors in man is currently focused on fumarate derivatives, as well as on the identification of other monofunctional inducers in extracts of vegetables.