Scope and Method of Study: We use human Caco-2 and Hep G2 cell lines as model to investigate the MTX effect to human sulfotransferases through enzymatic assay, western blot, and RT-PCR methods. We used MTT assay to detect the cytotoxicity of MTX to these cell lines. Through the reporter gene assay, RNA interference, step wise promoter deletion, site directed promoter mutation, real-time PCR, DNA gel shift assay, and super shift assay, we found several nuclear receptors involved in the transcriptional regulation of hSULT2A1.

Findings and conclusions.: We found MTX can induce human sulfotransferases in a time - and concentration-dependent manner in both human Caco-2 and Hep G2 cell lines. The induction of human sulfotransferases mediated by MTX can be repressed by folic acid in human Hep G2 cell line. The toxicity study indicates the induction of hSULTs was not caused by the cytotoxicity of MTX. Through multiple molecular techniques, we found the IR2 sequence located in -186 to -173 of hSULT2A1 promoter region plays very important function in hSULT2A1 regulation. The nuclear receptor CAR and VDR up-regulate the promoter activity of hSULT2A1, and the PXR down-regulate the promoter activity of hSULT2A1. Obviously, there is cross-talk between different nuclear receptors in hSULT2A1 regulation.