Effects of arsenic, cadmium, chromium, and lead on gene expression regulated by a battery of 13 different promoters in recombinant HepG2 cells

Toxic metals occur naturally at low concentrations throughout the environment, but are found in higher concentrations at many of the hazardous waste sites on the EPA Superfund list. As part of the Agency for Toxic Substances and Disease Registry (ATSDR) mandate to evaluate the toxicity of metals and mixtures, we chose four of the high-priority metal pollutants from ATSDR's HAZ-DAT list, including arsenic, cadmium, chromium, and lead, to test in a commercially developed assay system, CAT-Tox(L) (Xenometrix). This assay employs a battery of recombinant HepG2 cell lines to test the transcriptional activation capacity of xenobiotics in any of 13 different signal transduction pathways. Our specific aims were to identify metal-responsive promoters and determine whether the pattern of gene expression changed with a mixture of metals. Humic acid was used in all assays as a carrier to help solubilize the metals and, in all cases, the cells were exposed to the humic acid-metal mixture for 48 h. Humic acid alone, at 50-100 muM, showed moderate activation of the XRE promoter, but little other notable activity. As(V), at doses of 50-250 muM, produced a complex profile of activity showing significant dose-dependent induction of the hMTIIA, GST Ya, HSP70, FOS, XRE, NF kappa BRE, GADD153, p53RE, and CRE promoters. Pb(II) showed dose-related induction of the GST Ya, XRE, hMTIIA, GRP78, and CYP IA1 promoters at doses in the range of 12-100 muM. Cd(II), at 1.25-15 muM, yielded significant dose-dependent induction of hMTIIA, XRE, CYP IA1, GST Ya, HSP70, NF kappa BRE, and FOS. Whereas Cr(III) yielded small, though significant inductions of the CRE, FOS, GADD153, and XRE promoters only at the highest dose (750 muM), Cr(VI) produced significant dose-related inductions of the p53RE, FOS, NF kappa BRE, XRE, GADD45, HSP70, and CRE promoters at much lower doses, in the range of 5-10 muM. Assays testing serial dilutions of a mixture comprising 7.5 muM Cd(II), 750 muM Cr(III), and 100 muM Pb(II) (the combination of metals most frequently found at National Priority List sites) showed significant dose-dependent induction of the hMTIIA promoter, but failed to show dose-related induction of any other promoter and showed no evidence of synergistic activation of gene expression by the metals in this mixture. Our results thus show metal activation of gene expression through several previously unreported signal transduction pathways, including As(V) induction of GST Ya, FOS, XRE, NFkBRE, GADD153, p53RE, and CRE; Pb(II) induction of GST Ya, XRE, Cyp IA1, and GADD153; Cd(II) induction of NFkBRE, Cyp IA1, XRE, and GST Ya; and Cr(VI) induction of p53RE, XRE, GADD45, HSP70, and CRE promoters, and thus suggest new insights into the biochemical mechanisms of toxicity and carcinogenicity of metals. It is also an important finding that no evidence of synergistic activity was detected with the mixture of Cd(II), Cr(III), and Pb(II) tested in these assays.