Development of human cell biosensor system for genotoxicity detection based on DNA damage-induced gene expression
Background. Human exposure to genotoxic agents in the environment and everyday life represents a serious health threat. Fast and reliable assessment of genotoxicity of chemicals is of main importance in the fields of new chemicals and drug development as well as in environmental monitoring. The tumor suppressor gene p21, the major down-stream target gene of activated p53 which is responsible for cell cycle arrest following DNA damage, has been shown to be specifically up-regulated by genotoxic carcinogens. The aim of our study was to develop a human cell-based biosensor system for simple and fast detection of genotoxic agents.
Methods. Metabolically active HepG2 human hepatoma cells were transfected with plasmid encoding Enhanced Green Fluorescent Protein (EGFP) under the control of the p21 promoter (p21HepG2GFP). DNA damage was induced by genotoxic agents with known mechanisms of action. The increase in fluorescence intensity, due to p21 mediated EGFP expression, was measured with a fluorescence microplate reader. The viability of treated cells was determined by the colorimetric MTS assay.
Results. The directly acting alkylating agent methylmethane sulphonate (MMS) showed significant increase in EGFP production after 48 h at 20 μg/mL. The indirectly acting carcinogen benzo(a)pyren (BaP) and the cross-linking agent cisplatin (CisPt) induced a dose- dependent increase in EGFP fluorescence, which was already significant at concentrations 0.13 μg/mL and 0.41 μg/mL, respectively. Vinblastine (VLB), a spindle poison that does not induce direct DNA damage, induced only a small increase in EGFP fluorescence intensity after 24 h at the lowest concentration (0.1 μg/mL), while exposure to higher concentrations was associated with significantly reduced cell viability.
Conclusions. The results of our study demonstrated that this novel assay based on the stably transformed cell line p21HepG2GFP can be used as a fast and simple biosensor system for detection of genetic damage caused by chemical agents.
Elespuru RK, Agarwal R, Atrakchi AH, Bigger CAH, Heflich RH, Jagannath DR, et al. Current and Future Application of Genetic Toxicity Assays: The Role and Value of In Vitro Mammalian Assays. Toxicol Sci 2009; 109: 172-9.
Sutton MD, Smith BT, Godoy VG, Walker GC. The SOS response: Recent insights into umuDC-dependent mutagenesis and DNA damage tolerance. Ann Rev Genet 2000; 34: 479-97.
Putnam CD, Jaehnig EJ, Kolodner RD. Perspectives on the DNA damage and replication checkpoint responses in Saccharomyces cerevisiae. DNA Repair 2009; 8: 974-82.
Holbrook NJ, Fornace AJ. Response to adversity - molecular control of gene activation following genotoxic stress. New Biologist 1991; 3: 825-33.
Quillardet P, Huisman O, Dari R, Hofnung M. SOS chromotest, a direct assay of induction of an sos function in escherichia-coli k-12 to measure genotoxicity. P Natl Acad Sci USA 1982; 79: 5971-5.
Oda Y, Nakamura S, Oki I, Kató T, Shinagawa H. Evaluation of the new system (umu-test) for the detection of environmental mutagens and carcinogens. Mutat Res 1985; 147: 219-29.
Walmsley RM, Billinton N, Walsh L, Barker MG, Knight AW, Cahill PA. A yeast RAD54-GFP genotoxicity assay, is effective in identifying direct acting mutagens in addition to clastogens not detected by bacterial tests. Toxicol Sci 2003; 72: 1106.
Liu X, Kramer JA, Swaffield JC, Hu Y, Chai G, Wilson AGE. Development of a highthroughput yeast-based assay for detection of metabolically activated genotoxins. Mutat Res-Gen Tox En 2008; 653: 63-9.
Zhou B-BS, Elledge SJ. The DNA damage response: putting checkpoints in perspective. Nature 2000; 408: 433-9.
Sionov RV, Haupt Y. The cellular response to p53: the decision between life and death. Oncogene 1999; 18: 6145-57.
Waldman T, Kinzler KW, Vogelstein B. P21 is necessary for the P53-mediated G1 arrest in human cancer cells. Cancer Res 1995; 55: 5187-90.
Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature 2000; 408: 307-10.
Moldovan G-L, Pfander B, Jentsch S. PCNA, the maestro of the replication fork. Cell 2007; 129: 665-79.
Park SY, Lee SM, Ye SK, Yoon SH, Chung MH, Choi J. Benzo[a]pyrene-induced DNA damage and p53 modulation in human hepatoma HepG2 cells for the identification of potential biomarkers for PAH monitoring and risk assessment. Toxicol Lett 2006; 167: 27-33.
Zegura B, Zajc I, Lah TT, Filipic M. Patterns of microcystin-LR induced alteration of the expression of genes involved in response to DNA damage and apoptosis. Toxicon 2008; 51: 615-23.
Hreljac I, Zajc I, Lah T, Filipic M. Effects of model organophosphorous pesticides on DNA damage and proliferation of HepG2 cells. Environ Mol Mutagen 2008; 49: 360-7.
Ellinger-Ziegelbauer H, Stuart B, Wahle B, Bomann W, Ahr HJ. Comparison of the expression profiles induced by genotoxic and nongenotoxic carcinogens in rat liver. Mutat Res-Gen Tox En 2005; 575: 61-84.
Knasmuller S, Mersch-Sundermann V, Kevekordes S, Darroudi F, Huber WW, Hoelzl C, et al.. Use of human-derived liver cell lines for the detection of environmental and dietary genotoxicants; current state of knowledge. Toxicology 2004; 198: 315-28.
Bressac B, Galvin KM, Liang TJ, Isselbacher KJ, Wands JR, Ozturk M. Abnormal structure and expression of p53 gene in human hepatocellular-carcinoma. P Natl Acad Sci USA 1990; 87: 1973-7.
Mesojednik S, Kamensek U, Cemazar M. Evaluation of shRNA-mediated gene silencing by electroporation in LPB fibrosarcoma cells. Radiol Oncol 2008; 42: 82-92.
Todd MD, Lee MJ, Williams JL, Nalezny JM, Gee P, Benjamin MB, et al. The cat-tox (l) assay - a sensitive and specific measure of stress-induced transcription in transformed human liver-cells. Fund Appl Toxicol 1995; 28: 118-28.
Tanaka H, Arakawa H, Yamaguchi T, Shiraishi K, Fukuda S, Matsui K, et al. A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage. Nature 2000; 404: 42-9.
Guittet O, Hakansson P, Voevodskaya N, Fridd S, Graslund A, Arakawa H, et al. Mammalian p53R2 protein forms an active ribonucleotide reductase in vitro with the R1 protein, which is expressed both in resting cells in response to DNA damage and in proliferating cells. J Biol Chem 2001; 276: 40647-51.
Ohno K, Tanaka-Azuma Y, Yoneda Y, Yamada T. Genotoxicity test system based on p53R2 gene expression in human cells: Examination with 80 chemicals. Mutat Res-Gen Tox En 2005; 588: 47-57.
Ohno K, Ishihata K, Tanaka-Azuma Y, Yamada T. A genotoxicity test system based on p53R2 gene expression in human cells: Assessment of its reactivity to various classes of genotoxic chemicals. Mutat Res-Gen Tox En 2008; 656: 27-35.
Siafakas RA, Richardson DR. Growth arrest and DNA damage-45 alpha (GADD45α). Int J BiochemCell B 2009; 41: 986-9.
Hastwell PW, Chai LL, Roberts KJ, Webster TW, Harvey JS, Rees RW, et al. High-specificity and high-sensitivity genotoxicity assessment in a human cell line: Validation of the GreenScreen HC GADD45a-GFP genotoxicity assay. Mutat Res-Gen Tox En 2006; 607: 160-75.
Birrell L, Cahill P, Hughes C, Tate M, Walmsley RM. GADD45a-GFP GreenScreen HC assay results for the ECVAM recommended lists of genotoxic and non-genotoxic chemicals for assessment of new genotoxicity tests. Mutat Res-Gen Tox En 2010; 695: 87-95.
Zhang R, Niu YJ, Do HR, Cao XW, Shi D, Hao QL, et al. A stable and sensitive testing system for potential carcinogens based on DNA damage-induced gene expression in human HepG2 cell. Toxicol In Vitro 2009; 23: 158-65.
Yang TT, Cheng LZ, Kain SR. Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein. Nucleic Acids Res 1996; 24: 4592-3.
Cormack BP, Valdivia RH, Falkow S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 1996; 173: 33-8.
Lawley PD. Mutagens as carcinogens - development of current concepts. Mutat Res 1989; 213: 3-25.
Beranek DT. Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating-agents. Mutat Res 1990; 231: 11-30.
Jaiswal AS, Narayan S. S(N)2 DNA-alkylating agent-induced phosphorylation of p53 and activation of p21 gene expression. Mutat Res-Fund Mol M 2002; 500: 17-30.
Perlow RA, Kolbanovskii A, Hingerty BE, Geacintov NE, Broyde S, Scicchitano DA. DNA adducts from a tumorigenic metabolite of benzo[a]pyrene block human RNA polymerase II elongation in a sequence- and stereochemistry-dependent manner. J Mol Biol 2002; 321: 29-47.
Wang A, Gu J, Judson-Kremer K, Powell KL, Mistry H, Simhambhatla P, et al. Response of human mammary epithelial cells to DNA damage induced by BPDE: involvement of novel regulatory pathways. Carcinogenesis 2003; 24: 225-34.
Sadikovic B, Rodenhiser DI. Benzopyrene exposure disrupts DNA methylation and growth dynamics in breast cancer cells. Toxicol Appl Pharm 2006; 216: 458-68.
Jagger C, Tate M, Cahill PA, Hughes C, Knight AW, Billinton N, et al. Assessment of the genotoxicity of S9-generated metabolites using the GreenScreen HC GADD45a-GFP assay. Mutagenesis 2009; 24: 35-50.
Corda Y, Job C, Anin MF, Leng M, Job D. Spectrum of DNA platinum adduct recognition by prokaryotic and eukaryotic DNA-dependent RNA-polymerases. Biochemistry 1993; 32: 8582-8.
Pabla N, Huang S, Mi QS, Daniel R, Dong Z. ATR-Chk2 signaling in p53 activation and DNA damage response during cisplatin-induced apoptosis. J Biol Chem 2008; 283: 6572-83.
Marini F, Nardo T, Giannattasio M, Minuzzo M, Stefanini M, Plevani P, et al.. DNA nucleotide excision repair-dependent signaling to checkpoint activation. P Natl Acad Sci USA 2006; 103: 17325-30.
Owellen RJ, Hartke CA, Dickerson RM, Hains FO. Inhibition of tubulin-microtubule polymerization by drugs of vinca alkaloid class. Cancer Res 1976; 36: 1499-502.
Tishler RB, Lamppu DM, Park S, Price BD. Microtubule-active drugs taxol, vinblastine, and nocodazole increase the levels of transcriptionally active P53. Cancer Res 1995; 55: 6021-5.