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
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.
PLoS One. 2016 Dec 22;11(12):e0168721. doi: 10.1371/journal.pone.0168721. eCollection 2016.
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.
在越南，包括致癌物和前致癌物在内的大量有毒物质，每天都从工业和农业活动、食品生产以及医疗服务中释放到环境中。在本研究中，我们报告了基于新型酵母的生物传感器系统的开发，该系统通过与两种质粒共转化来测定遗传毒性致癌物和前致癌物。一种质粒携带人CPR和CYP（CYP3A4、CYP2B6或CYP2D6）基因，而另一种质粒包含RAD54-GFP报告构建体。由此产生的三个同时含有CPR-CYP和RAD54-GFP表达盒的共表达系统分别命名为CYP3A4/CYP2B6/CYP2D6 + RAD54系统，并用于通过响应DNA损伤选择性激活和诱导CPR-CYP和RAD54-GFP表达盒来检测和评估致癌物和前致癌物的遗传毒性潜力。结果表明，前致癌物主要、中度或未被一种CYP酶生物活化，因此可通过特定的共表达系统进行选择性检测。黄曲霉毒素B1和苯并(a)芘主要由CYP3A4 + RAD54系统检测到，而N-亚硝基二甲胺仅中度激活CYP2B6 + RAD54报告系统，且均未被CYP2D6 + RAD54系统识别。相比之下，遗传毒性致癌物甲磺酸甲酯可被所有系统检测到。我们的酵母报告系统可以在384孔微孔板中进行，以提供高效的遗传毒性测试，从而识别各种致癌化合物，并且与现有的96孔遗传毒性生物测定相比，可将化学物质消耗降低约53%。与液体处理机器人相结合，该平台能够以完全自动化和连续的方式快速、经济高效且高通量地筛选大量分析物，而无需用户干预。
