Cytotoxicity and transcriptional activation of stress genes in human liver carcinoma cells (HepG2) exposed to cadmium chloride.

Epidemiological studies have shown that there exists some correlation between cadmium exposure and human cancers. The evidence that cadmium and cadmium compounds are probable human carcinogens is also supported by experimental studies reporting induction of malignant tumors formation in multiple species of laboratory animals exposed to these compounds. In vitro studies with mammalian cells have also shown that cadmium is clastogenic, but its mutagenic potential is rather weak. In this research, we performed the MTT assay for cell viability to assess the cytotoxicity of cadmium chloride (CdCl2), and the CAT-Tox (L) assay to measure the induction of stress genes in thirteen different recombinant cell lines generated from human liver carcinoma cells (HepG2), by creating stable transfectants of different mammalian promoter-chloramphenicol acetyltransferase (CAT) gene fusions. Cytotoxicity experiments with the parental cell line yielded a LC50 of 6.1 +/- 0.8 microg/mL, upon 48 h of exposure. Four (metallothionein--HMTIIA, 70-kDa heat shock protein--HSP70, xenobitic response element--XRE, and cyclic adenosine monophosphate response element--CRE) out of the 13 constructs evaluated showed statistically significant inductions (p < 0.05). The induction of these genes was concentration-dependent. Marginal inductions were also recorded for the c-fos, and 153-kDa growth arrest DNA damage (GADD153) promoters, indicating a potential for CdCl2 to damage DNA. However, no significant inductions (p > 0.05) of gene expression were recorded for cytochrome P4501A1--CYP1A1, glutathion-S-transferase Ya subunit--GST Ya, nuclear factor kappa (B site) response element--NFkappaBRE, tumor suppressor protein response element--p53RE, 45-kDa growth arrest DNA damage--GADD45, 78-kDa glucose regulated protein--GRP78, and retinoic acid response element--RARE. As expected, these results indicate that metallothioneins and heat shock proteins appear to be excellent candidates for biomarkers for detecting cadmium-induced proteotoxic effects at the molecular and cellular levels. Induction of XRE indicates the potential involvement of CdCl2 in the biotransformation process in the liver, while activation of CRE indicates stimulation of cellular signaling through the protein kinases pathway.

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. However, similar investigations with atrazine did not show any significant effects of this chemical on HepG2 cells, even at its maximum solubility of 100 microg/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 (HepG2), by creating stable transfectants of different mammalian promoter-chloramphenicol acetyltransferase (CAT) gene fusions. Cytotoxicity experiments yielded LC50 values of 11.9 +/- 2.6 microg/mL for arsenic trioxide in de-ionized water, and 3.6 +/- 0.4 microg/mL for arsenic trioxide in 100 microg/mL atrazine; indicating a 3 fold increase in arsenic toxicity associated with the atrazine exposure. Co-exposure of HepG2 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, metallothioneinIIa--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--NFkappaBRE 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.

Transcriptional activation of stress genes and cytotoxicity in human liver carcinoma cells (HepG2) exposed to 2,4,6-trinitrotoluene, 2,4-dinitrotoluene, and 2,6-dinitrotoluene.

The CAT-Tox (L) assay has recently been developed and validated for detecting and quantifying the specific molecular mechanisms that underlie toxicity of various xenobotic chemicals. We performed this assay to measure the transcriptional responses associated with 2,4,6-trinitrotoluene (TNT) and 2 of its byproducts [2,4 and 2,6-dinitotoluenes (DNTs)] to 13 different recombinant cell lines generated from human liver carcinoma cells (HepG2) by creating stable transfectants of mammalian promoter chloramphenicol acetyltransferase (CAT) gene fusions. Cytoxicity test with the parental HepG2 cells, using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]-based assay for cell viability, yielded LC50 values of 105 +/- 6 mg/mL for TNT in 1% dimethyl sulfoxide (DMSO), and > 300 mg/mL for DNTs, upon 48 h of exposure. TNT appeared to be more toxic than 2,4-DNT, which also showed a higher toxicity compared to 2,6-DNT. Of the 13 recombinant constructs evaluated, 8 (CYP 1A1, GST Ya, XRE, HMTIIA, c-fos, HSP70, GADD153, and GADD45), 5 (c-fos, HSP70, GADD153, GADD45, and GRP78), and none showed inductions to significant levels (p < 0.05), for TNT, 2,4-DNT, and 2,6-DNT, respectively. For most constructs, the induction of stress genes was concentration-dependent. These results show the potential for TNT and 2,4-DNT to cause protein damage and/or perturbations of protein biosynthesis (HSP70 and GRP78), alterations in DNA sequence or its helical structure (c-fos, GADD153, GADD45), and the potential involvement of TNT in the biotransformation process (CYP 1A1, GST Ya, XRE), and in the toxicokinetics of metal ions (HMTIIA). Within the range of concentrations tested (0-300 mg TNT or DNT/mL in 1% DMSO), no significant inductions (p > 0.05) of NFKBRE, p53RE, CRE, and RARE were found.