Procarcinogens - Determination and Evaluation by Yeast-Based Biosensor Transformed with Plasmids Incorporating RAD54 Reporter Construct and Cytochrome P450 Genes

• Published in: PloS one 2016-12, Vol.11 (12), p.e0168721-e0168721
• Main Authors: Bui, Van Ngoc, Nguyen, Thi Thu Huyen, Mai, Chi Thanh, Bettarel, Yvan, Hoang, Thi Yen, Trinh, Thi Thuy Linh, Truong, Nam Hai, Chu, Hoang Ha, Nguyen, Vu Thanh Thanh, Nguyen, Huu Duc, Wölfl, Stefan
• Format: Article
• Language: English
• Subjects: Aflatoxin B1; Aflatoxins; Agricultural economics; Benzo(a)pyrene; Bioassays; Biocompatibility; Biology and life sciences; Biosensing Techniques - methods; Biosensors; Biotechnology; Carcinogens; Carcinogens - pharmacology; Cassettes; Chemical compounds; Cost analysis; CYP2D6 protein; Cytochrome; Cytochrome P-450; Cytochrome P-450 CYP2B6 - genetics; Cytochrome P-450 CYP2D6 - genetics; Cytochrome P-450 CYP3A - genetics; Cytochrome P450; Deoxyribonucleic acid; DNA; DNA damage; DNA Damage - drug effects; DNA Helicases - genetics; DNA Repair Enzymes - genetics; Engineering and Technology; Environmental Engineering; Environmental Sciences; Food contamination & poisoning; Food production; Gene expression; Gene Expression Regulation, Fungal - drug effects; Genes; Genes, Reporter - genetics; Genetic aspects; Genotoxicity; Genotoxicity testing; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Health care; High-throughput screening; Humans; Laboratory equipment; Life Sciences; Medicine and Health Sciences; Metabolites; Methyl methanesulfonate; Mutagenicity Tests; N-Nitrosodimethylamine; Plasmids; Plasmids - genetics; Pyrene; Research and analysis methods; Saccharomyces cerevisiae - drug effects; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins - genetics; Science; Toxic substances; Yeast; Yeasts
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0168721

In Vietnam, a great number of toxic substances, including carcinogens and procarcinogens, from industrial and agricultural activities, food production, and healthcare services are daily released into the environment. In the present study, we report the development of novel yeast-based biosensor systems to determine both genotoxic carcinogens and procarcinogens by cotransformation with two plasmids. One plasmid is carrying human CPR and CYP (CYP3A4, CYP2B6, or CYP2D6) genes, while the other contains the RAD54-GFP reporter construct. The three resulting coexpression systems bearing both CPR-CYP and RAD54-GFP expression cassettes were designated as CYP3A4/CYP2B6/CYP2D6 + RAD54 systems, respectively and used to detect and evaluate the genotoxic potential of carcinogens and procarcinogens by selective activation and induction of both CPR-CYP and RAD54-GFP expression cassettes in response to DNA damage. Procarcinogens were shown to be predominantly, moderately or not bioactivated by one of the CYP enzymes and thus selectively detected by the specific coexpression system. Aflatoxin B1 and benzo(a)pyrene were predominantly detected by the CYP3A4 + RAD54 system, while N-nitrosodimethylamine only moderately activated the CYP2B6 + RAD54 reporter system and none of them was identified by the CYP2D6 + RAD54 system. In contrast, the genotoxic carcinogen, methyl methanesulfonate, was detected by all systems. Our yeast-reporter system can be performed in 384-well microplates to provide efficient genotoxicity testing to identify various carcinogenic compounds and reduce chemical consumption to about 53% as compared with existing 96-well genotoxicity bioassays. In association with a liquid handling robot, this platform enables rapid, cost-effective, and high-throughput screening of numerous analytes in a fully automated and continuous manner without the need for user interaction.