Search

Microorganism-based genotoxicity assessments are vital for evaluating potential chemical-induced DNA damage. In this study, we developed both chromosomally integrated and single-copy plasmid–based reporter assays in budding yeast using a RNR3 promoter–driven luciferase gene. These assays were designed to compare the response to genotoxic chemicals with a pre-established multicopy plasmid–based assay. Despite exhibiting the lowest luciferase activity, the chromosomally integrated reporter assay showed the highest fold induction (i.e., the ratio of luciferase activity in the presence and absence of the chemical) compared with the established plasmid-based assay. Using CRISPR/Cas9 technology, we generated mutants with single- or double-gene deletions, affecting major DNA repair pathways or cell permeability. This enabled us to evaluate reporter gene responses to genotoxicants in a single-copy plasmid–based assay. Elevated background activities were observed in several mutants, such as mag1Δ cells, even without exposure to chemicals. However, substantial luciferase induction was detected in single-deletion mutants following exposure to specific chemicals, including mag1Δ, mms2Δ, and rad59Δ cells treated with methyl methanesulfonate; rad59Δ cells exposed to camptothecin; and mms2Δ and rad10Δ cells treated with mitomycin C (MMC) and cisplatin (CDDP). Notably, mms2Δ/rad10Δ cells treated with MMC or CDDP exhibited significantly enhanced luciferase induction compared with the parent single-deletion mutants, suggesting that postreplication and for nucleotide excision repair processes predominantly contribute to repairing DNA crosslinks. Overall, our findings demonstrate the utility of yeast-based reporter assays employing strains with multiple-deletion mutations in DNA repair genes. These assays serve as valuable tools for investigating DNA repair mechanisms and assessing chemical-induced DNA damage. Key points: • Responses to genotoxic chemicals were investigated in three types of reporter yeast. • Yeast strains with single- and double-deletions of DNA repair genes were tested. • Two DNA repair pathways predominantly contributed to DNA crosslink repair in yeast. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

By damaging DNA molecules, genotoxicants cause genetic mutations and also increase human susceptibility to cancers and genetic diseases. Over the past four decades, several assays have been developed in the budding yeast Saccharomyces cerevisiae to screen potential genotoxic substances and provide alternatives to animal-based genotoxicity tests. These yeast-based genotoxicity tests are either DNA alteration-based or DNA stress-response reporter-based. The former, which came first, were developed from the genetic studies conducted on various types of DNA alterations in yeast cells. Despite their limited throughput capabilities, some of these tests have been used as short-term genotoxicity tests in addition to bacteria- or mammalian cell-based tests. In contrast, the latter tests are based on the emergent transcriptional induction of DNA repair-related genes via activation of the DNA damage checkpoint kinase cascade triggered by DNA damage. Some of these reporter assays have been linked to DNA damage-responsive promoters to assess chemical carcinogenicity and ecotoxicity in environmental samples. Yeast-mediated genotoxicity tests are being continuously improved by increasing the permeability of yeast cell walls, by the ectopic expression of mammalian cytochrome P450 systems, by the use of DNA repair-deficient host strains, and by integrating them into high-throughput formats or microfluidic devices. Notably, yeast-based reporter assays linked with the newer toxicogenomic approaches are becoming powerful short-term genotoxicity tests for large numbers of compounds. These tests can also be used to detect polluted environmental samples, and as effective screening tools during anticancer drug development.By damaging DNA molecules, genotoxicants cause genetic mutations and also increase human susceptibility to cancers and genetic diseases. Over the past four decades, several assays have been developed in the budding yeast Saccharomyces cerevisiae to screen potential genotoxic substances and provide alternatives to animal-based genotoxicity tests. These yeast-based genotoxicity tests are either DNA alteration-based or DNA stress-response reporter-based. The former, which came first, were developed from the genetic studies conducted on various types of DNA alterations in yeast cells. Despite their limited throughput capabilities, some of these tests have been used as short-term genotoxicity tests in addition to bacteria- or mammalian cell-based tests. In contrast, the latter tests are based on the emergent transcriptional induction of DNA repair-related genes via activation of the DNA damage checkpoint kinase cascade triggered by DNA damage. Some of these reporter assays have been linked to DNA damage-responsive promoters to assess chemical carcinogenicity and ecotoxicity in environmental samples. Yeast-mediated genotoxicity tests are being continuously improved by increasing the permeability of yeast cell walls, by the ectopic expression of mammalian cytochrome P450 systems, by the use of DNA repair-deficient host strains, and by integrating them into high-throughput formats or microfluidic devices. Notably, yeast-based reporter assays linked with the newer toxicogenomic approaches are becoming powerful short-term genotoxicity tests for large numbers of compounds. These tests can also be used to detect polluted environmental samples, and as effective screening tools during anticancer drug development.By damaging DNA molecules, genotoxicants cause genetic mutations and also increase human susceptibility to cancers and genetic diseases. Over the past four decades, several assays have been developed in the budding yeast Saccharomyces cerevisiae to screen potential genotoxic substances and provide alternatives to animal-based genotoxicity tests. These yeast-based genotoxicity tests are either DNA alteration-based or DNA stress-response reporter-based. The former, which came first, were developed from the genetic studies conducted on various types of DNA alterations in yeast cells. Despite their limited throughput capabilities, some of these tests have been used as short-term genotoxicity tests in addition to bacteria- or mammalian cell-based tests. In contrast, the latter tests are based on the emergent transcriptional induction of DNA repair-related genes via activation of the DNA damage checkpoint kinase cascade triggered by DNA damage. Some of these reporter assays have been linked to DNA damage-responsive promoters to assess chemical carcinogenicity and ecotoxicity in environmental samples. Yeast-mediated genotoxicity tests are being continuously improved by increasing the permeability of yeast cell walls, by the ectopic expression of mammalian cytochrome P450 systems, by the use of DNA repair-deficient host strains, and by integrating them into high-throughput formats or microfluidic devices. Notably, yeast-based reporter assays linked with the newer toxicogenomic approaches are becoming powerful short-term genotoxicity tests for large numbers of compounds. These tests can also be used to detect polluted environmental samples, and as effective screening tools during anticancer drug development. [ABSTRACT FROM AUTHOR]