Db. Tully et al., Effects of arsenic, cadmium, chromium, and lead on gene expression regulated by a battery of 13 different promoters in recombinant HepG2 cells, TOX APPL PH, 168(2), 2000, pp. 79-90
Toxic metals occur naturally at low concentrations throughout the environme
nt, but are found in higher concentrations at many of the hazardous waste s
ites 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 e
mploys a battery of recombinant HepG2 cell lines to test the transcriptiona
l activation capacity of xenobiotics in any of 13 different signal transduc
tion pathways. Our specific aims were to identify metal-responsive promoter
s and determine whether the pattern of gene expression changed with a mixtu
re of metals. Humic acid was used in all assays as a carrier to help solubi
lize 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 a
ctivation of the XRE promoter, but little other notable activity. As(V), at
doses of 50-250 muM, produced a complex profile of activity showing signif
icant dose-dependent induction of the hMTIIA, GST Ya, HSP70, FOS, XRE, NF k
appa BRE, GADD153, p53RE, and CRE promoters. Pb(II) showed dose-related ind
uction 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, a
nd FOS. Whereas Cr(III) yielded small, though significant inductions of the
CRE, FOS, GADD153, and XRE promoters only at the highest dose (750 muM), C
r(VI) produced significant dose-related inductions of the p53RE, FOS, NF ka
ppa 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 met
als most frequently found at National Priority List sites) showed significa
nt dose-dependent induction of the hMTIIA promoter, but failed to show dose
-related induction of any other promoter and showed no evidence of synergis
tic activation of gene expression by the metals in this mixture. Our result
s thus show metal activation of gene expression through several previously
unreported signal transduction pathways, including As(V) induction of GST Y
a, FOS, XRE, NFkBRE, GADD153, p53RE, and CRE; Pb(II) induction of GST Ya, X
RE, 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 a
nd carcinogenicity of metals. It is also an important finding that no evide
nce of synergistic activity was detected with the mixture of Cd(II), Cr(III
), and Pb(II) tested in these assays.