Atrazine potentiation of arsenic trioxide-induced cytotoxicity and gene expression in human liver carcinoma cells (HepG2)

Recent studies in our laboratory indicated that arsenic trioxide has the ability to cause significant cytotoxicity, and induction of a significant number of stress genes in human liver carcinoma cells, HepG2 [1]. However, similar investigations with atrazine did not show any significant effects of this chemical on HepG2 cells, even at its maximum solubility of 100 mug/mL in 1% dimethyl sulfoxide (DMSO). Further cytogenetic studies were therefore carried out to investigate the combined effects of arsenic trioxide and atrazine on cell viability and gene expression in immortalized human hepatocytes. Cytotoxicity was evaluated using the MTT-assay for cell viability, while the CAT-Tox (L) assay was performed to measure the induction of stress genes in thirteen different recombinant cell lines generated from human liver carcinoma cells (HepG(2)), by creating stable transfectants of different mammalian promoter - chloramphenicol acetyltransferase (CAT) gene fusions. Cytotoxicity experiments yielded LC50 values of 11.9 +/- 2.6 mug/mL for arsenic trioxide in de-ionized water, and 3.6 +/- 0.4 mug/mL for arsenic trioxide in 100 mug/mL atrazine; indicating a 3 fold increase in arsenic toxicity associated with the atrazine exposure. Co-exposure of HepG(2) cells to atrazine also resulted in a significant increase in the potency of arsenic trioxide to upregulate a number of stress genes including those of the glutathione-S-transferase Ya subunit-GST Ya, metallothionein(IIa)-HMTIIA, 70-kDa heat shock protein-HSP70, c-fos, 153-kDa growth arrest and DNA damage (GADD153), 45-kDa growth arrest and DNA damage (GADD45), and 78-kDa glucose regulated protein-GRP78 promoters, as well as the xenobiotic response element-XRE, tumor suppressor protein response element-p53RE, cyclic adenosine monophosphate response element-CRE, and retinoic acid response element-RARE. No significant changes were observed with respect to the influence of atrazine on the modulation of cytochrome P450 1A1-CYP 1A1, and nuclear factor kappa (B site) response element-NF kappa BRE by arsenic trioxide. These results indicate that co-exposure to atrazine strongly potentiates arsenic trioxide-induced cytotoxicity and transcriptional activation of stress genes in transformed human hepatocytes.