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SHRNA-MEDIATED SILENCING OF AS3MT EXPRESSION MODULATES THE CAPACITY OF HEPG2 CELLS TO METHYLATE INORGANIC ARSENIC
Citation:
DROBNA, Z., D. J. THOMAS, AND M. STYBLO. SHRNA-MEDIATED SILENCING OF AS3MT EXPRESSION MODULATES THE CAPACITY OF HEPG2 CELLS TO METHYLATE INORGANIC ARSENIC. Presented at 45th Annual Society of Toxicology Meeting 2006, San Diego, CA, March 05 - 09, 2006.
Description:
Several methyltransferases have been linked to the oxidative methylation of inorganic arsenic (iAs) in mammalian cells. However, the relative contributions of these enzymes to the overall capacity of cells to methylate iAs have not been characterized. Arsenic (+3 oxidation state) methyltransferase (AS3MT) that is expressed in rat and human hepatocytes catalyzes all steps in this pathway, yielding mono-, di-, and trimethylated metabolites that contain arsenic in either +3 or +5 oxidation states. Here we used post-transcriptional suppression of AS3MT expression by a short hairpin RNA (shRNA) to examine its role in the metabolic transformation of iAs in human hepatic cells. Three target 21-nucleotide sequences were selected for human AS3MT mRNA (NCBI, NM_020682), using a BLOCK-iT RNAi Designer (Invitrogen). shRNA oligonucleotides designed for each of the target sequences (both sense and antisense) were cloned into a retroviral pSIREN-RetroQ expression vector (Clontech) that contains a puromycin resistance gene and the human U6 promoter. The three shRNA expression vectors, as well as an empty pSIREN-RetroQ vector were used to transduce human hepatocellular carcinoma (HepG2) cells. Four clonal HepG2 cell lines were established, including HepG2/shRNA/A, HepG2/shRNA/B, HepG2/shRNA/C, and HepG2/shRNA/empty. AS3MT mRNA and protein levels were analyzed in each cell lines using quantitative PCR and immunoblot analysis, respectively. Capacity to methylate iAs was examined in cells exposed to 73As-arsenite for 66 hours. The PCR profiles show that the silencing of AS3MT expression was most effective in HepG2/shRNA/A cells. The rate of iAs methylation was suppressed by 95% in clonal HepG2/shRNA/A cells as compared to parental HepG2 cells. These results suggest that AS3MT is the key enzyme in the pathway for methylation of iAs in human hepatic cells. (This abstract does not reflect U.S. EPA policy.)