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.

Genotoxic Assays for Measuring P450 Activation of Chemical Mutagens

“…The RadarScreen assay uses a RAD54 β-galactosidase reporter construct in which β-galactosidase cleaves the substrate into galactose and luciferin [70]. HUG1 encodes an inhibitor of ribonucleotide reductase, while RNR3 encodes the large subunit of ribonucleotide reductase that is specifically induced because of DNA damage [71]. The fluorescent markers can be enhanced using yEGFP, engineered for yeast codon bias.…”

“…To mitigate this deficiency, human CYPs have been introduced into the strains for both plate assays and reporter assays. For example, Bui et al [71] expressed CYP1A2, CYP2C9, CYP3A4, and CYP2D6 in a strain that monitors RAD54-GFP. Sengstag et al [50] and Fasullo et al [64,67] have expressed CYP1A1, CYP1A2, and CYP3A4 in strains that monitor translocations, mutations, and unequal SCE.…”

“…The agents tested include polyaromatic hydrocarbons (BaP-DHD), mycotoxins (AFB1), and heterocyclic aromatic amines (2-amino-3,8-dimethylimidazo-[4,5-f ]quinoxaline (MeIQx), 2-amino-3, 4-dimethylimidazo-[4,5-f ] quinoline (MeIQ ), and 2-amino-3-methylimidazo-[4,5-f ]quinoline (IQ )). Bui et al [71] introduced human CYPs into strains to measure induction of GFP using the reporter RAD54-GFP. Li et al [52] used a sensitive fluorimetric assay to measure inhibition of secreted dextranase; the assay consists of strains expressing Lipomyces kononenkoae amylase, CYP3A4, and hOR [52].…”

Overview of Piezoelectric Biosensors, Immunosensors and DNA Sensors and Their Applications

“…Genetic information from a pathogen and human tissue or blood sample are common analytes for these biosensors. However, carcinogens interacting with immobilized chromosomes or double stranded DNA chains can be mentioned as a possible pair: analyte and the biorecognition part of the biosensor [ 50 , 51 , 52 , 53 ].…”

Yeast-Based Biosensors: Current Applications and New Developments

“…In addition to carcinogens, numerous chemicals such as polycyclic aromatic hydrocarbon and mycotoxins are pro-carcinogens: i.e., they become carcinogenic only after they have been bio-activated by cellular metabolic processes. Ngoc Bui and colleagues designed a yeast-based biosensor able to determine and evaluate pro-carcinogens presents in environmental samples [37].…”