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

Van Ngoc Bui,Huu Duc Nguyen,Hoang Ha Chu,Yvan Bettarel,Thi Yen Hoang,Thi Thuy Linh Trinh,Thi Thu Huyen Nguyen,Vu Thanh Thanh Nguyen,Stefan Wölfl,Nam Hai Truong,Chi Thanh Mai

doi:10.1371/journal.pone.0168721

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.

Highlights

Carcinogens are either genotoxic or nongenotoxic [1]
Cytochrome P450 monooxygenases (CYPs) have been central to the study of toxicology, since they are involved in metabolism of endogenous molecules, detoxification and biotransformation of xenobiotics, drug-drug and drug-food interactions, and bioactivation of potential carcinogens and other pollutants
For cytochrome P450 monooxygenases (CYPs) activity, only microsomes containing properly folded CYPs that were confirmed by reduced carbon monoxide (CO)-difference spectra with a peak at 450 nm were introduced to the activity assay

Summary

Introduction

Carcinogens are either genotoxic or nongenotoxic [1] Genotoxins, such as alkylating agents, can bind to DNA forming DNA adducts and cause damage to the DNA or mutations, which may lead to cancer, while nongenotoxins do not directly cause DNA damage but promote growth or alter the expression or repression of genes by different cellular processes [2, 3]. Procarcinogens, such as polycyclic aromatic hydrocarbon (PAHs), mycotoxins, etc., become carcinogenic only after they are transformed in metabolic processes including bioactivation by cytochrome P450 monooxygenases (CYPs) [4,5,6]. Biosensors will not compete but rather complement official physicochemical methods, with specific benefits in environmental monitoring, food safety and quality control, drug testing and other uses where genotoxicity tests are needed to determine potential genotoxic and mutagenic hazards

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Conclusion