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Development and evaluation of yeast-based GFP and luciferase reporter assays for chemical-induced genotoxicity and oxidative damage

  • Applied genetics and molecular biotechnology
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Abstract

We aimed to develop the bioassays for genotixicity and/or oxidative damage using the recombinant yeast. A genotoxicity assay was developed using recombinant Saccharomyces cerevisiae strain BY4741 with a green fluorescent protein (GFP) reporter plasmid, driven by the DNA damage-responsive RNR3 promoter. Enhanced fluorescence induction was observed in DNA repair-deficient strains treated with methyl methanesulfonate, but not with hydrogen peroxide. A GFP reporter yeast strain driven by the oxidative stress-responsive TRX2 promoter was newly developed to assess oxidative damage, but fluorescence was poorly induced by oxidants. In place of GFP, yeast strains with luciferase gene reporter plasmids (luc2 and luc2CP, encoding stable and unstable luciferase, respectively) were prepared. Transient induction of luciferase activity was clearly detected only in a TRX2 promoter-driven luc2CP reporter strain within 90 min of oxidant exposure. However, luciferase was strongly induced by hydroxyurea in the RNR3 promoter-driven luc2 and GFP reporter strains over 8 h after the exposure, suggesting that the RNR3 promoter is continuously upregulated by DNA damage, whereas the TRX2 promoter is transiently activated by oxidative agents. Luciferase activity levels were also increased in a TRX2-promoter-driven luc2CP reporter strain treated with tert-butyl hydroperoxide and menadione and weakly induced with diamide and diethyl maleate. Weakly enhanced luciferase activity induction was detected in the sod1Δ, sod2Δ, and rad27Δ strains treated with hydrogen peroxide compared with that in the wild-type strain. In conclusion, tests using GFP and stable luciferase reporters are useful for genotoxicity, and oxidative damage can be clearly detected by assay with an unstable luciferase reporter.

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References

  • Afanassiev V, Sefton M, Anantachaiyong T, Barker G, Walmsley R, Wölfl S (2000) Application of yeast cells transformed with GFP expression constructs containing the RAD54 or RNR2 promoter as a test for the genotoxic potential of chemical substances. Mutat Res 464:297–308

    Article  CAS  PubMed  Google Scholar 

  • Ames BN, Durston WE, Yamasaki E, Lee FD (1973) Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc Natl Acad Sci U S A 70:2281–2285

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ames BN, Shigenaga MK, Hagen TM (1993) Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci U S A 90:7915–7922

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Benton MG, Glasser NR, Palecek SP (2008) Deletion of MAG1 and MRE11 enhances the sensitivity of the Saccharomyces cerevisiae HUG1P-GFP promoter-reporter construct to genotoxicity. Biosens Bioelectron 24:736–741. doi:10.1016/j.bios.2008.06.033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Berry DB, Gasch AP (2008) Stress-activated genomic expression changes serve a preparative role for impending stress in yeast. Mol Biol Cell 19:4580–4587. doi:10.1091/mbc.E07-07-0680

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bianchi V, Pontis E, Reichard P (1986) Changes of deoxyribonucleoside triphosphate pools induced by hydroxyurea and their relation to DNA synthesis. J Biol Chem 261:16037–16042

    CAS  PubMed  Google Scholar 

  • Boronat S, Piña B (2006) Development of RNR3- and RAD54-GUS reporters for testing genotoxicity in Saccharomyces cerevisiae. Anal Bioanal Chem 386:1625–1632. doi:10.1007/s00216-006-0751-4

    Article  CAS  PubMed  Google Scholar 

  • Boulton SJ, Jackson SP (1996) Saccharomyces cerevisiae Ku70 potentiates illegitimate DNA double-strand break repair and serves as a barrier to error-prone DNA repair pathways. EMBO J 15:5093–5103

    CAS  PubMed  PubMed Central  Google Scholar 

  • Brendel M, Bonatto D, Strauss M, Revers LF, Pungartnik C, Saffi J, Henriques JA (2003) Role of PSO genes in repair of DNA damage of Saccharomyces cerevisiae. Mutat Res 544:179–193

    Article  CAS  PubMed  Google Scholar 

  • Broomfield S, Chow BL, Xiao W (1998) MMS2, encoding a ubiquitin-conjugating-enzyme-like protein, is a member of the yeast error-free postreplication repair pathway. Proc Natl Acad Sci U S A 95:5678–5683

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Brown JA, Sherlock G, Myers CL, Burrows NM, Deng C, Wu HI, McCann KE, Troyanskaya OG, Brown JM (2006) Global analysis of gene function in yeast by quantitative phenotypic profiling. Mol Syst Biol 2(2006):0001. doi:10.1038/msb4100043

    CAS  PubMed  Google Scholar 

  • Bui VN, Nguyen TT, Bettarel Y, Nguyen TH, Pham TL, Hoang TY, Nguyen VT, Nghiem NM, Wölfl S (2015) Genotoxicity of chemical compounds identification and assessment by yeast cells transformed with GFP reporter constructs regulated by the PLM2 or DIN7 promoter. Int J Toxicol 34:31–43. doi:10.1177/1091581814566870

    Article  CAS  PubMed  Google Scholar 

  • Carmel-Harel O, Stearman R, Gasch AP, Botstein D, Brown PO, Storz G (2001) Role of thioredoxin reductase in the Yap1p-dependent response to oxidative stress in Saccharomyces cerevisiae. Mol Microbiol 39:595–605

    Article  CAS  PubMed  Google Scholar 

  • Chang EC, Crawford BF, Hong Z, Bilinski T, Kosman DJ (1991) Genetic and biochemical characterization of Cu,Zn superoxide dismutase mutants in Saccharomyces cerevisiae. J Biol Chem 266:4417–4424

    CAS  PubMed  Google Scholar 

  • Cooke MS, Evans MD, Dizdaroglu M, Lunec J (2003) Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J 17:1195–1214. doi:10.1096/fj.02-0752rev

    Article  CAS  PubMed  Google Scholar 

  • Coyle MB, Strauss B (1970) Cell killing and the accumulation of breaks in the DNA of HEp-2 cells incubated in the presence of hydroxyurea. Cancer Res 30:2314–2319

    CAS  PubMed  Google Scholar 

  • Dolz-Edo L, Rienzo A, Poveda-Huertes D, Pascual-Ahuir A, Proft M (2013) Deciphering dynamic dose responses of natural promoters and single cis elements upon osmotic and oxidative stress in yeast. Mol Cell Biol 33:2228–2240. doi:10.1128/Mcb.00240-13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dunham M, Gartenberg MR, Brown GW (2015) Methods in yeast genetics and genomics. A Cold Spring Harbor Laboratory course manual, 2015 edition. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Eide L, Bjørås M, Pirovano M, Alseth I, Berdal KG, Seeberg E (1996) Base excision of oxidative purine and pyrimidine DNA damage in Saccharomyces cerevisiae by a DNA glycosylase with sequence similarity to endonuclease III from Escherichia coli. Proc Natl Acad Sci U S A 93:10735–10740

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Elledge SJ, Zhou Z, Allen JB, Navas TA (1993) DNA damage and cell cycle regulation of ribonucleotide reductase. BioEssays 15:333–339

    Article  CAS  PubMed  Google Scholar 

  • Friedberg EC, Walker GC, Siede W, Wood RD, Schultz RA, Ellenberger T (2005) DNA repair and mutagenesis, 2nd edn. American Society for Microbiology Press, Washington DC

    Google Scholar 

  • Gasch AP, Werner-Washburne M (2002) The genomics of yeast responses to environmental stress and starvation. Funct Integr Genomics 2:181–192

    Article  CAS  PubMed  Google Scholar 

  • Gasch AP, Spellman PT, Kao CM, Carmel-Harel O, Eisen MB, Storz G, Botstein D, Brown PO (2000) Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell 11:4241–4257

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gietz D, St Jean A, Woods RA, Schiestl RH (1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20:1425

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Herrero E, Ros J, Belli G, Cabiscol E (2008) Redox control and oxidative stress in yeast cells. Biochim Biophys Acta 1780:1217–1235. doi:10.1016/j.bbagen.2007.12.004

    Article  CAS  PubMed  Google Scholar 

  • Ichikawa K, Eki T (2006) A novel yeast-based reporter assay system for the sensitive detection of genotoxic agents mediated by a DNA damage-inducible LexA-GAL4 protein. J Biochem 139:105–112. doi:10.1093/jb/mvj011

    Article  CAS  PubMed  Google Scholar 

  • Jamieson DJ (1998) Oxidative stress responses of the yeast Saccharomyces cerevisiae. Yeast 14:1511–1527. doi:10.1002/(SICI)1097-0061(199812)14:16<1511::AID-YEA356>3.0.CO;2-S

    Article  CAS  PubMed  Google Scholar 

  • Jayaraman M, Radhika V, Bamne MN, Ramos R, Briggs R, Dhanasekaran DN (2005) Engineered Saccharomyces cerevisiae strain BioS-OS1/2, for the detection of oxidative stress. Biotechnol Prog 21:1373–1379. doi:10.1021/bp050104j

    Article  CAS  PubMed  Google Scholar 

  • Jia X, Xiao W (2003) Compromised DNA repair enhances sensitivity of the yeast RNR3-lacZ genotoxicity testing system. Toxicol Sci 75:82–88

    Article  CAS  PubMed  Google Scholar 

  • Jia X, Zhu Y, Xiao W (2002) A stable and sensitive genotoxic testing system based on DNA damage induced gene expression in Saccharomyces cerevisiae. Mutat Res 519:83–92

    Article  CAS  PubMed  Google Scholar 

  • Kasai H (1997) Analysis of a form of oxidative DNA damage, 8-hydroxy-2′-deoxyguanosine, as a marker of cellular oxidative stress during carcinogenesis. Mutat Res 387:147–163

    Article  CAS  PubMed  Google Scholar 

  • Klaus V, Hartmann T, Gambini J, Graf P, Stahl W, Hartwig A, Klotz LO (2010) 1,4-Naphthoquinones as inducers of oxidative damage and stress signaling in HaCaT human keratinocytes. Arch Biochem Biophys 496:93–100. doi:10.1016/j.abb.2010.02.002

    Article  CAS  PubMed  Google Scholar 

  • Kosower NS, Kosower EM, Wertheim B, Correa WS (1969) Diamide, a new reagent for the intracellular oxidation of glutathione to the disulfide. Biochem Biophys Res Commun 37:593–596

    Article  CAS  PubMed  Google Scholar 

  • Kuge S, Jones N (1994) YAP1 dependent activation of TRX2 is essential for the response of Saccharomyces cerevisiae to oxidative stress by hydroperoxides. EMBO J 13:655–664

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kuge S, Arita M, Murayama A, Maeta K, Izawa S, Inoue Y, Nomoto A (2001) Regulation of the yeast Yap1p nuclear export signal is mediated by redox signal-induced reversible disulfide bond formation. Mol Cell Biol 21:6139–6150. doi:10.1128/Mcb.21.18.6139-6150.2001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lee J, Godon C, Lagniel G, Spector D, Garin J, Labarre J, Toledano MB (1999) Yap1 and Skn7 control two specialized oxidative stress response regulons in yeast. J Biol Chem 274:16040–16046

    Article  CAS  PubMed  Google Scholar 

  • Lewinska A, Grzelak A, Bartosz G (2008) Application of a YHB1-GFP reporter to detect nitrosative stress in yeast. Redox Rep 13:161–171. doi:10.1179/135100008X259268

    Article  CAS  PubMed  Google Scholar 

  • Lichtenberg-Fraté H, Schmitt M, Gellert G, Ludwig J (2003) A yeast-based method for the detection of cyto and genotoxicity. Toxicol in Vitro 17:709–716

    Article  PubMed  Google Scholar 

  • Liu X, Kramer JA, Swaffield JC, Hu Y, Chai G, Wilson AG (2008) Development of a highthroughput yeast-based assay for detection of metabolically activated genotoxins. Mutat Res 653:63–69. doi:10.1016/j.mrgentox.2008.03.006

    Article  CAS  PubMed  Google Scholar 

  • Molina-Navarro MM, Castells-Roca L, Belli G, García-Martínez J, Marín-Navarro J, Moreno J, Pérez-Ortín JE, Herrero E (2008) Comprehensive transcriptional analysis of the oxidative response in yeast. J Biol Chem 283:17908–17918. doi:10.1074/jbc.M800295200

    Article  CAS  PubMed  Google Scholar 

  • Morano KA, Grant CM, Moye-Rowley WS (2012) The response to heat shock and oxidative stress in Saccharomyces cerevisiae. Genetics 190:1157–1195. doi:10.1534/genetics.111.128033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ochi Y, Sugawara H, Iwami M, Tanaka M, Eki T (2011) Sensitive detection of chemical-induced genotoxicity by the Cypridina secretory luciferase reporter assay, using DNA repair-deficient strains of Saccharomyces cerevisiae. Yeast 28:265–278. doi:10.1002/yea.1837

    Article  CAS  PubMed  Google Scholar 

  • Oda Y, Nakamura S, Oki I, Kato T, Shinagawa H (1985) Evaluation of the new system (umu-test) for the detection of environmental mutagens and carcinogens. Mutat Res 147:219–229

    Article  CAS  PubMed  Google Scholar 

  • O’Doherty PJ, Lyons V, Higgins VJ, Rogers PJ, Bailey TD, Wu MJ (2013) Transcriptomic insights into the molecular response of Saccharomyces cerevisiae to linoleic acid hydroperoxide. Free Radic Res 47:1054–1065. doi:10.3109/10715762.2013.849344

    Article  PubMed  Google Scholar 

  • Outten CE, Falk RL, Culotta VC (2005) Cellular factors required for protection from hyperoxia toxicity in Saccharomyces cerevisiae. Biochem J 388:93–101. doi:10.1042/Bj20041914

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Prolla TA, Christie DM, Liskay RM (1994) Dual requirement in yeast DNA mismatch repair for MLH1 and PMS1, two homologs of the bacterial mutL gene. Mol Cell Biol 14:407–415

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Putnam CD, Jaehnig EJ, Kolodner RD (2009) Perspectives on the DNA damage and replication checkpoint responses in Saccharomyces cerevisiae. DNA Repair (Amst) 8:974–982. doi:10.1016/j.dnarep.2009.04.021

    Article  CAS  Google Scholar 

  • Quillardet P, Huisman O, D’Ari R, Hofnung M (1982) SOS chromotest, a direct assay of induction of an SOS function in Escherichia coli K-12 to measure genotoxicity. Proc Natl Acad Sci U S A 79:5971–5975

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ramotar D, Popoff SC, Gralla EB, Demple B (1991) Cellular role of yeast Apn1 apurinic endonuclease/3′-diesterase: repair of oxidative and alkylation DNA damage and control of spontaneous mutation. Mol Cell Biol 11:4537–4544

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Reagan MS, Pittenger C, Siede W, Friedberg EC (1995) Characterization of a mutant strain of Saccharomyces cerevisiae with a deletion of the RAD27 gene, a structural homolog of the RAD2 nucleotide excision repair gene. J Bacteriol 177:364–371

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Robertson JB, Stowers CC, Boczko E, Johnson CH (2008) Real-time luminescence monitoring of cell-cycle and respiratory oscillations in yeast. Proc Natl Acad Sci U S A 105:17988–17993. doi:10.1073/pnas.0809482105

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S (2004) Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 73:39–85. doi:10.1146/annurev.biochem.73.011303.073723

    Article  CAS  PubMed  Google Scholar 

  • Schiestl RH, Prakash S (1990) RAD10, an excision repair gene of Saccharomyces cerevisiae, is involved in the RAD1 pathway of mitotic recombination. Mol Cell Biol 10:2485–2491

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sha W, Martins AM, Laubenbacher R, Mendes P, Shulaev V (2013) The genome-wide early temporal response of Saccharomyces cerevisiae to oxidative stress induced by cumene hydroperoxide. PLoS One 8:e74939. doi:10.1371/journal.pone.0074939

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Symington LS (2002) Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. Microbiol Mol Biol Rev 66:630–670

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Trotter EW, Grant CM (2002) Thioredoxins are required for protection against a reductive stress in the yeast Saccharomyces cerevisiae. Mol Microbiol 46:869–878

    Article  CAS  PubMed  Google Scholar 

  • van Loon AP, Pesold-Hurt B, Schatz G (1986) A yeast mutant lacking mitochondrial manganese-superoxide dismutase is hypersensitive to oxygen. Proc Natl Acad Sci U S A 83:3820–3824

    Article  PubMed  PubMed Central  Google Scholar 

  • Walmsley RM, Billinton N, Heyer WD (1997) Green fluorescent protein as a reporter for the DNA damage-induced gene RAD54 in Saccharomyces cerevisiae. Yeast 13:1535–1545

    Article  CAS  PubMed  Google Scholar 

  • Walsh L, Hastwell PW, Keenan PO, Knight AW, Billinton N, Walmsley RM (2005) Genetic modification and variations in solvent increase the sensitivity of the yeast RAD54-GFP genotoxicity assay. Mutagenesis 20:317–327. doi:10.1093/mutage/gei044

    Article  CAS  PubMed  Google Scholar 

  • Wei T, Zhang C, Xu X, Hanna M, Zhang X, Wang Y, Dai H, Xiao W (2013) Construction and evaluation of two biosensors based on yeast transcriptional response to genotoxic chemicals. Biosens Bioelectron 44:138–145. doi:10.1016/j.bios.2013.01.029

    Article  PubMed  Google Scholar 

  • Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, Andre B, Bangham R, Benito R, Boeke JD, Bussey H, Chu AM, Connelly C, Davis K, Dietrich F, Dow SW, El Bakkoury M, Foury F, Friend SH, Gentalen E, Giaever G, Hegemann JH, Jones T, Laub M, Liao H, Liebundguth N, Lockhart DJ, Lucau-Danila A, Lussier M, M’Rabet N, Menard P, Mittmann M, Pai C, Rebischung C, Revuelta JL, Riles L, Roberts CJ, Ross-MacDonald P, Scherens B, Snyder M, Sookhai-Mahadeo S, Storms RK, Véronneau S, Voet M, Volckaert G, Ward TR, Wysocki R, Yen GS, Yu K, Zimmermann K, Philippsen P, Johnston M, Davis RW (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285:901–906

    Article  CAS  PubMed  Google Scholar 

  • Xiao W, Chow BL (1998) Synergism between yeast nucleotide and base excision repair pathways in the protection against DNA methylation damage. Curr Genet 33:92–99

    Article  CAS  PubMed  Google Scholar 

  • Xiao W, Chow BL, Hanna M, Doetsch PW (2001) Deletion of the MAG1 DNA glycosylase gene suppresses alkylation-induced killing and mutagenesis in yeast cells lacking AP endonucleases. Mutat Res 487:137–147

    Article  CAS  PubMed  Google Scholar 

  • Zhang M, Hanna M, Li J, Butcher S, Dai H, Xiao W (2010) Creation of a hyperpermeable yeast strain to genotoxic agents through combined inactivation of PDR and CWP genes. Toxicol Sci 113:401–411. doi:10.1093/toxsci/kfp267

    Article  CAS  PubMed  Google Scholar 

  • Zhang M, Zhang C, Li J, Hanna M, Zhang X, Dai H, Xiao W (2011) Inactivation of YAP1 enhances sensitivity of the yeast RNR3-lacZ genotoxicity testing system to a broad range of DNA-damaging agents. Toxicol Sci 120:310–321. doi:10.1093/toxsci/kfq391

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The authors thank Kazuho Inomata, Shogo Matsunobu, Yuki Takahashi, Ayumu Tanabe, Chiaki Hamaya, Koei Yachi, and other members of the laboratory for technical support and discussions. This work was supported in part by grants from the Smoking Research Foundation, the Hibi Science Foundation, and a Grant-in-Aid for Scientific Research in Innovative Areas “Plasma Medical Innovation” (24108005) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (to T.E.).

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  1. Molecular Genetics Laboratory, Division of Bioscience and Biotechnology, Department of Environmental and Life Sciences, Toyohashi University of Technology, 1-1 Hibarigaoka, Tempaku-cho, Toyohashi, Aichi, 441-8580, Japan

    Hajime Suzuki, Takahiro Sakabe, Yuu Hirose & Toshihiko Eki

  2. The Electronics-Inspired Interdisciplinary Research Institute (EIIRIS), Toyohashi University of Technology, 1-1 Hibarigaoka, Tempaku-cho, Toyohashi, Aichi, 441-8580, Japan

    Yuu Hirose

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  1. Hajime Suzuki
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  4. Toshihiko Eki
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Correspondence to Toshihiko Eki.

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This study was partially funded by the Smoking Research Foundation, the Hibi Science Foundation, and a Grant-in-Aid for Scientific Research in Innovative Areas “Plasma Medical Innovation” (24108005) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (to T.E.).

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Suzuki, H., Sakabe, T., Hirose, Y. et al. Development and evaluation of yeast-based GFP and luciferase reporter assays for chemical-induced genotoxicity and oxidative damage. Appl Microbiol Biotechnol 101, 659–671 (2017). https://doi.org/10.1007/s00253-016-7911-z

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