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.

Fig 1

Activity test of recombinant human CPR (A) and recombinant human CYP3A4, CYP2B6, and CYP2D6 (B). 3A4+, 2B6+, and 2D6+ are microsomes of clones coexpressing both CPR and CYP genes; 3A4–, 2B6–, and 2D6– or CPR–are microsomes of clones expressing only one gene, the CYPs or CPR, respectively; NC (negative control) are microsomes of clones bearing the control pESC-URA plasmid. The standard deviation values of the measurements of fluorescence microplate assay (B) were all less than 5% of the calculated values and are thus not presented here.

Fig 2. Fluorescence induction in yeast cells transformed with different gene constructs in response to DNA damage.

Yeast-based biosensors were either nontreated (NT, control) or exposed to increasing concentrations of AFB1 (A), BaP (B), NDMA (C), and MMS (positive genotoxin, D). CYP3A4 + RAD54: strain transformed with both CPR-CYP3A4 and RAD54-GFP constructs; RAD54: strain transformed with only the RAD54-GFP construct; NCs (negative control) system: strain transformed with two control pESC-URA and pUMGP5 plasmids. The GFP fluorescence intensity of measurements was compared within linear range of GFP signals by calculation of GFP fold induction. The horizontal dashed line at 1.3 fold GFP induction is used as cutoff or genotoxicity threshold. Other negative (untransformed yeast cells) and process (medium) controls are not presented here.