Abstract
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 μg/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 CdC12 to damage DNA. However, no significant inductions (p > 0.05) of gene expression were recorded for cytochrome P450 1A1 – CYP1A1, glutathion-S-transferase Ya subunit – GST Ya, nuclear factor kappa (B site) response element – NFkBRE, 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 CdC12 in the biotransformation process in the liver, while activation of CRE indicates stimulation of cellular signaling through the protein kinases pathway.
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References
Chettle DR, Ellis KJ: Further scientific issues in determining an occupational standard for cadmium. Am J Ind 22: 117–124, 1992
Mason HJ, Davison AG, Wright AL, Guthrie CJG, Fayers PM, Venables KM, Smith NJ, Chettle DR, Franklin DM, Scott MC, Holden H, Gompertz D, Newman-Taylor AJ: Relations between liver cadmium, cumulative exposure, and renal function in cadmium alloy workers. Br J Ind Med 45: 793–805, 1988
Drasch GA: An increase of cadmium body burden for this century: An investigation on human tissues. Sci Total Environ 26: 111–120, 1983
Voors AW, Shuman MS, Johnson WD: Additive statistical effects of cadmium and lead on heart related disease in a North Carolina USA autopsy series. Arch Environ Health 37: 98–102, 1982
Ferm VH, Layton WM Jr.: Teratogenic and mutagenic effects of cadmium. In: J.O. Nriagu (ed). Cadmium in the Environment. Part 2, Health Effects. John Wiley, New York, 1981, pp 743–756
EPA: Ambient Water Quality Criteria for Cadmium. Rep. 440/5-80-025. US Environmental Protection Agency, Washington, DC, 1980, 183 pp
Nomiyama K: Carcinogenicity of cadmium. Jpn J Indust Health 24: 13–23, 1982
International Agency for Research on Cancer: Cadmium and cadmium compounds. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans: Supplement 7. WHO, Lyons, France, 1987, pp 140–142
International Agency for Research on Cancer: IARC Monographs on the Evaluation of Carcinogenic Risks to Humans: Beryllium, Cadmium Mercury and Exposures in The Glass Industry. IARC Scientific Publications no. 58. IARC, Lyons, France, 1993, pp 119–238
Elinder CG, Kjellstrom T, Hogstedt C, Andersson K, Spang G: Cancer mortality of cadmium workers. Br J Ind Med 42: 651–655, 1985
Occupational Safety and Health Administration, US Department of Labor: Occupational exposure to cadmium; final rules. Fed Reg 57: 42102–42463, 1992
Kazantzis G, Lam TH, Sullivan KR: Mortality of cadmium-exposed workers: A five-year update. Scand J Work Environ Health 14: 220–223, 1988
Oberdorster G: Airborne cadmium and carcinogenesis of the respiratory tract. Scand J Work Environ Health 12: 523–537, 1986
Sullivan K, Waterman L: Cadmium and cancer: The current position. Report of an international meeting in London, September 1988. Ann Occup Hyg 32: 557–560, 1988
Thun MJ, Elinder CG, Friberg L: Scientific basis for an occupational standard for cadmium. Am J Ind Med 20: 629–642, 1991
Waalkes MP, Oberdorster G: Cadmium Carcinogenesis. Biological Effects of Heavy Metals, Vol. II. CRC Press, Boca Raton, FL, 1990
Waalkes MP, Coogan TP, Barter RA: Toxicological principles of metal carcinogenesis with special emphasis on cadmium. Crit Rev Toxicol 22: 175–201, 1994
de Kort WLAM, Verschoor MA, Wibowo AAE, van Hemmen JJ: Am J Ind Med 11: 145–156, 1987
Elinder CG: Other toxic effects. In: K.J. Ellis (ed). Cadmium and Health, Vol. 2. CRC Press, Boca Raton, FL, 1986, pp 160–197
Bucio L, Souza V, Albores A, Sierra A, Chavez E, Carabez A, Gutierrez-Ruiz MC: Cadmium and mercury toxicity in a human fetal hepatic cell line (WRL-68 cells). Toxicology 102: 285–299, 1995
Bhattacharyya MH, Wilson AK, Rajan SS, Jonah M: Biochemical pathways in cadmium toxicity. In: R.K. Zalups, J. Koropatnick (eds). Molecular Biology and Toxicology of Metals. Taylor and Francis, New York, 2000, pp 34–74
Mosmann T: Rapid colorimetric assay for cellular growth and survival: Applications to proliferation and cytotoxicity assays. J Immunol Meth 65: 55–63, 1983
Todd MD, Lee MJ, Williams JL, Nalenzy JM, Gee P, Benjamin MB, Farr SB: The CAT-Tox assay: A sensitive and specific measure of stress induced transcription in transformed human liver cells. Fundament Appl Toxicol 28: 118–128, 1995
Tchounwou PB, Wilson BA, Schneider J, Ishaque A: Cytogenetic assessment of arsenic trioxide toxicity in the Mutatox, Ames II, and CATTox assays. Metal Ions Biol Med 6: 89–91, 2000
Chang LW: An introduction to metal effects on other organ systems. In: L.W. Chang, L. Mgs, T. Suzuki (eds). Toxicology of Metals. CRC Lewis Publishers, Boca Raton, FL, 1996, pp 885–899
Habeebu SS, Liu J, Klaassen CD: Cadmium-induced apoptosis in mouse liver. Toxicol Appl Pharmacol 149: 203–209, 1998
Cherian MG, Howell SB, Imura N, Klaassen CD, Koropatnick J, Lazo JS, Waalkes MP: Contemporary issues in toxicology: Role of metallothionein in carcinogenesis. Toxicol Appl Pharmacol 126: 1–5, 1994
Richards RI, Heguy A, Karin M: Structural and functional analysis. The human metallothionein-I gene: Differential induction by metal ions and glucocorticoids. Cell 7: 263–272, 1984
Leber AP, Miya TS: A mechanism for cadmium-and zinc-induced tolerance to cadmium toxicity: Involvement of metallothionein. Toxicol Appl Pharmacol 37: 403–414, 1976
Goering PL, Klaassen CD: Tolerance to cadmium-induced hepatotoxicity following cadmium pretreatment. Toxicol Appl Pharmacol 74: 308–313, 1984
Wong KL, Cachia R, Klaassen CD: Comparison of the toxicity and tissue distribution of cadmium in newborn and adult rats after repeated administration. Toxicol Appl Pharmacol 56: 317–325, 1980
Goering PL, Klaassemn CD: Resistance to cadmium-induced hepatotoxicity in immature rats. Toxicol Appl Pharmacol 74: 321–329, 1984
Nordberg GF, Goyer R, Nordberg M: Comparative toxicity of cadmium chloride on mouse kidney. Arch Pathol 99: 192–197, 1975
Dudley RE, Gammal LM, Klaassen CD: Cadmium-induced hepatic and renal injury in chronically exposed rats: Likely role of hepatic cadmium-metallothionein in nephrotoxicity. Toxicol Appl Pharmacol 77: 414–426, 1985
Goering PL, Waalkes MP, Klaassen CD: Toxicology of cadmium. In: R.A. Goyer, M.G. Cherian (eds). Handbook of Experimental Pharmacology, Vol. 115. Springer-Verlag, Heidelberg, New York, 1995, pp 189–213
Petering DH, Fowler BA: Roles of metallothionein and related proteins in metal metabolism and toxicity: Problems and perspectives. Environ Health Perspect 65: 217–224, 1986
Cherian MG: Metallothionein and its interaction with metals. In: R.A. Goyer, M.G. Cherian (eds). Handbook of Experimental Pharmacology, Vol. 115. Springer-Verlag, Heidelberg, New York, 1995, pp 121–137
Yao EG, Denison MS: DNA sequence determinants for binding of transformed Ah receptor to a dixion-responsive enhancer. Biochemistry 31: 5060–5067, 1992
Pimental RA, Liang B, Yee GK, Wilhelmsson A, Poellinger L, Paulson KE: Dioxin receptor and C/EBP regulate the function of the glutathione-S-transferase Ya gene xenobiotic element. Mol Cell Biol 13: 4265–4373, 1993
Safe S: Polychlorinated biphenyls (BCBs), dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and related compounds: Environmental and mechanistic considerations, which support the development of toxic equivalency factors (TEFs). Crit Rev Toxicol 21: 51–88, 1990
Poland A, Knutson JC: 2,3,7,8-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: Examination of the mechanism of toxicity. Annu Rev Pharmacol Toxicol 22: 517–542, 1982
Schmidt JV, Bradfield CA: Ah receptor signaling pathways. Annu Rev Cell Dev Biol 12: 55–89, 1996
Safe S: Comparative toxicology and mechanism of action of polychlorinated dibenzo-p-dioxins and dibenzofurans. Annu Rev Pharmacol Toxicol 26: 371–399, 1986
Sadek CM, Allen-Hoffman BL: Suspension-mediated induction of hepa 1c1c7 CYP1a-1 expression is dependent on the Ah receptor signal transduction pathway. J Biol Chem 269: 31505–31509, 1994
Weiss C, Kolluri SK, Kiefer F, Gottlicher M: Complementation of Ah receptor deficiency in hepatoma cells: Negative feedback regulation and cell cycle control by the Ah receptor. Exp Cell Res 226: 154–163, 1996
Ma Q, Whitlock JP Jr: The aromatic hydrocarbon receptor modulates the Hepa 1c1c7 cell cycle and differentiated state independently of dioxin. Mol Cell Biol 16: 2144–2150, 1996
Fujii-Kuriyama Y, Imataka H, Sogawa K, Yasumoto KI, Kikuchi Y: Regulation of CYP1A1 expression. FASEB J 6: 706–710, 1992
Gonzales GJ, Nebert DW: Autoregulation plus upstream positive and negative control regions associated with transcriptional activation of the mouse P1 (450) gene. Nucleic Acids Res 13: 7269–7288, 1985
Hankinson O: The aryl hydrocarbon receptor complex. Annu Rev Pharmacol Toxicol 35: 307–340, 1995
Whitlock JP Jr, Okino S, Dong L, Ko H, Clarke-Ktzenberg R, Ma Q, Li H: Induction of cytochrome P4501A1: A model for analyzing mammalian gene transcription. FASEB J 10: 809–818, 1996
Tchouwnou PB, Wilson BA, Schneider J, Ishaque AB: Cytogenetic assessment of arsenic trioxide in the Mutatox, Ames II and CAT-Tox (L) assays. In: J.A. Centeno, Ph. Collery, G. Vernet, R.B. Finkelman, H. Gibb, J.C. Etienne (eds). Metal Ions in Biology and Medicine, Vol. 6. Paris, France, 2000, pp 89–91
Morimoto RI, Tissieres A, Georgopoulos C (eds): Stress Proteins in Biology and Medicine. Cold Spring Harbor Laboratory Press, New York, 1990
Mosser DD, Theodorakis NG, Morimoto RI: Coordinate changes in heat shock element-binding activity and HSP70 gene transcription rates in human cells. Mol Cell Biol 8: 4736–4744, 1988
Morimoto RI, Sarge KD, Abravaya K: Transcriptional regulation of heat shock genes: A paradigm for inducible genomic responses. J Biol Chem 267: 2987–21990, 1992
Sarge KD, Murphy SP, Morimoto RI: Activation of heat shock gene transcription by heat shock factor1 involves oligomerization, acquisition of DNA-binding activity, and nuclear localization and can occur in the absence of stress. Mol Cell Biol 13: 1392–1407, 1993
Hatayama T, Asai Y, Wakatsuki T, Kitamura T, Imahara H: Regulation of hsp 70 synthesis induced by cupric sulfate and zinc sulfate in thermotolerant HeLa cells. J Biochem (Tokyo) 114: 592–597, 1993
Abe T, Konishi T, Katoh T, Hirano H, Matsukuma K, Kashimura M, Higashi K: Inductiion of heat shock 70 mRNA by cadmium is mediated by gluthathione suppressive and non-suppressive triggers. Biochem Biophys Acta 201: 29–36, 1994
Borrelli E, Montmayeur JP, Foulkes NS, Sassone-Corsi P: Signal transduction and gene control: The camp pathway. Crit Rev Oncol 3: 321–338, 1992
Lee KAW, Masson N: Transcriptional regulation by CREB and its relatives. Biochim Biophys Acta 1174: 221–233, 1993
Smith DR, Toft DO: Steroid receptors and their associated proteins. Mol Endocrinol 7: 4–11, 1993
Gorski J, Furlow JD, Murdoch FE, Fritsch M, Kanenko K, Ying C, Malayer JR: Perturbations in the model of estrogen receptor regulation of gene expression. Biol Reprod 48: 8–14, 1993
Ignar-Trowbridge DM, Pimentel M, Parker MG, McLachlan JA, Korach KS: Peptide growth factor cross-talk with the estrogen requires the A/B domain and occurs independently of protein kinase C or estradiol. Endocrinol 137: 1735–1744, 1996
Ignar-Trowbridge DM, Nelson KG, Bidwell MC, Curtis SW, Washborn TF, McLachlan JA, Korach KS: Coupling of dual signaling pathways: Epidermal growth factor action involves the estrogen receptor. Proc Natl Acad Sci 89: 4658–4662, 1992
Cho H, Katzenellenbogen BS: Synergistic activation of estrogen receptor-mediated transcription by estradiol and protein kinase activators. Mol Endocrinol 7: 441–452, 1993
Tang N, Enger MD: Cd2+-induced c-myc mRNA accumulation in NRK-49F cells is blocked by the protein kinase inhibitor H7 but not HA 1004, indicating that protein kinase C is a mediator of the response. Toxicology 81: 155–164, 1993
Luethy JD, Holbrook NJ: The pathway regulating GADD153 induction in response to DNA damage is independent of protein kinase C and tyrosine kinases. Cancer Res 54: 1902–1906, 1994
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Tchounwou, P.B., Ishaque, A.B. & Schneider, J. Cytotoxicity and transcriptional activation of stress genes in human liver carcinoma cells (HepG2) exposed to cadmium chloride. Mol Cell Biochem 222, 21–28 (2001). https://doi.org/10.1023/A:1017922114201
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DOI: https://doi.org/10.1023/A:1017922114201