title: Procarcinogens - Determination and Evaluation by Yeast-Based Biosensor Transformed with Plasmids Incorporating RAD54 Reporter Construct and Cytochrome P450 Genes.
abstract: 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.
authors.affiliation: National Key Laboratory of Gene Technology, Institute of Biotechnology (IBT), Vietnam Academy of Science and Technology (VAST), Hanoi, Vietnam.|Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Heidelberg, Germany.
authors.forename: Van Ngoc
authors.initials: VN
authors.lastname: Bui
authors.affiliation: Thai Nguyen University of Sciences, Thai Nguyen University, Thai Nguyen, Vietnam.
authors.forename: Thi Thu Huyen
authors.initials: TT
authors.lastname: Nguyen
authors.affiliation: National Key Laboratory of Gene Technology, Institute of Biotechnology (IBT), Vietnam Academy of Science and Technology (VAST), Hanoi, Vietnam.
authors.forename: Chi Thanh
authors.initials: CT
authors.lastname: Mai
authors.affiliation: Institute of Research for Development (IRD), UMR MARBEC, Montpellier, France.
authors.forename: Yvan
authors.initials: Y
authors.lastname: Bettarel
authors.affiliation: National Key Laboratory of Gene Technology, Institute of Biotechnology (IBT), Vietnam Academy of Science and Technology (VAST), Hanoi, Vietnam.
authors.forename: Thi Yen
authors.initials: TY
authors.lastname: Hoang
authors.affiliation: National Key Laboratory of Gene Technology, Institute of Biotechnology (IBT), Vietnam Academy of Science and Technology (VAST), Hanoi, Vietnam.
authors.forename: Thi Thuy Linh
authors.initials: TT
authors.lastname: Trinh
authors.affiliation: National Key Laboratory of Gene Technology, Institute of Biotechnology (IBT), Vietnam Academy of Science and Technology (VAST), Hanoi, Vietnam.
authors.forename: Nam Hai
authors.initials: NH
authors.lastname: Truong
authors.affiliation: National Key Laboratory of Gene Technology, Institute of Biotechnology (IBT), Vietnam Academy of Science and Technology (VAST), Hanoi, Vietnam.
authors.forename: Hoang Ha
authors.initials: HH
authors.lastname: Chu
authors.affiliation: Thai Nguyen University of Sciences, Thai Nguyen University, Thai Nguyen, Vietnam.
authors.forename: Vu Thanh Thanh
authors.initials: VT
authors.lastname: Nguyen
authors.affiliation: Vietnam National University of Agriculture, Hanoi, Vietnam.
authors.forename: Huu Duc
authors.initials: HD
authors.lastname: Nguyen
authors.affiliation: Institute of Pharmacy and Molecular Biotechnology (IPMB), Heidelberg University, Heidelberg, Germany.
authors.forename: Stefan
authors.identifier: http://orcid.org/0000-0002-9977-174X
authors.initials: S
authors.lastname: Wölfl
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D002273:Carcinogens / Q000494:pharmacology*; D065702:Cytochrome P-450 CYP2B6 / Q000235:genetics; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics; D051544:Cytochrome P-450 CYP3A / Q000235:genetics; D004249:DNA Damage / Q000187:drug effects; D004265:DNA Helicases / Q000235:genetics; D045643:DNA Repair Enzymes / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects*; D017930:Genes, Reporter / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009152:Mutagenicity Tests; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*
chemical_list: D002273:Carcinogens; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D065702:Cytochrome P-450 CYP2B6; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; C075468:RAD54 protein, S cerevisiae; D004265:DNA Helicases; D045643:DNA Repair Enzymes