Transcriptional induction of CYP1A1 by oltipraz in human Caco-2 cells is aryl hydrocarbon receptor- and calcium-dependent

Oltipraz, a synthetic derivative of the cruciferous vegetable product 1,2-dithiole-3-thione, is considered as one of the most potent chemoprotectants. It modulates both cytochrome P-450 (CYP) and glutathione S-transferase expression and activities in rat tissues. Its effects, however, are variable according to the enzyme, tissue, and species. We show here that, as previously found in rat lung and kidney, CYP1A1 is inducible by oltipraz in both rat intestine and Caco-2 cells, a cell line originated from a human colon adenocarcinoma. In these cells, a 50 pm oltipraz treatment increased CYP1A1 mRNA (similar to30-fold), protein and activity. mRNA level was augmented as early as 2 h after the beginning of treatment, suggesting a transcriptional activation, and was maximal between 8 and 12 h. Transient transfection of Caco-2 cells with constructs containing different sizes of the 5'-flanking region of the CYP1A1 gene upstream of the luciferase reporter gene showed an increase in luciferase activity in oltipraz-treated cells, which correlates with the presence of the xenobiotic responsive element (XRE). Furthermore we demonstrated that resveratrol, an antagonist of the aryl hydrocarbon (Ah) receptor, inhibited the induction of both CYP1A1 promoter activity and mRNA by oltipraz, supporting the involvement of the Ah receptor in this induction. In an attempt to further characterize the mechanism of CYP1A1 induction, we showed a rapid increase in intracellular calcium concentration upon treatment of Caco-2 cells with oltipraz. Moreover, the effect of this compound on CY-P1A1 was strongly abolished in the presence of BAPTA-AM, a well known chelator of intracellular calcium, and 2-aminoethyl diphenylborate, an inhibitor of store-operated calcium channels. These results bring the first demonstration that oltipraz activates transcription of the CYP1A1 gene through the Ah receptor-XRE pathway in Caco-2 cells and that CYP1A1 induction relies upon an increase of intracellular calcium concentration.