title: Evidence of ATP assay as an appropriate alternative of MTT assay for cytotoxicity of secondary effluents from WWTPs.
abstract: Biological tests are effective and comprehensive methods to assess toxicity of environmental pollutants to ensure the safety of reclaimed water. In this study, the canonical MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to evaluate the cytotoxicity of dissolved organic matters (DOMs) of secondary effluents from wastewater treatment plants (WWTPs). It was surprising that most concentrated DOMs treated HepG2 cells yielded much higher signal compared with vehicle control regardless of difference of treatment technologies and seasons. However, there was actually no obvious enhancement of the cell proliferation by microscopy. In order to find out potential reason for the discrepancy, another three assays were performed. The results of ATP assay and flow cytometry showed expected toxicity, which was consistent with microscopy and previous studies, while DNA assay did not exhibit apparent change in treated cells. The possible mechanisms of abnormal MTT signal could be that some materials in secondary effluents isolated by solid extraction with HLB resin directly reacted with MTT and/or enhanced the activity of mitochondrial dehydrogenase. Therefore, the MTT assay is not suitable to assess cytotoxicity of complex mixtures such as secondary effluents, while ATP assay is an optional sensitive method. This study also suggests the importance of choosing both suitable extraction methods and detection assays for toxicity evaluation of component-unknown environmental samples.
authors.affiliation: Environmental Simulation and Pollution Control State Key Joint Laboratory, and State Environmental Protection Key Laboratory of Microorganism Application and Risk Control (MARC), School of Environment, Tsinghua University, Beijing 100084, PR China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Environmental Simulation and Pollution Control State Key Joint Laboratory, and State Environmental Protection Key Laboratory of Microorganism Application and Risk Control (MARC), School of Environment, Tsinghua University, Beijing 100084, PR China; Shenzhen Laboratory of Microorganism Application and Risk Control, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China. Electronic address: luyun@tsinghua.edu.cn.
authors.forename: Yun
authors.initials: Y
authors.lastname: Lu
authors.affiliation: Shenzhen Laboratory of Microorganism Application and Risk Control, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China.
authors.forename: Qian-Yuan
authors.initials: QY
authors.lastname: Wu
authors.affiliation: Environmental Simulation and Pollution Control State Key Joint Laboratory, and State Environmental Protection Key Laboratory of Microorganism Application and Risk Control (MARC), School of Environment, Tsinghua University, Beijing 100084, PR China; Shenzhen Laboratory of Microorganism Application and Risk Control, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China. Electronic address: hyhu@tsinghua.edu.cn.
authors.forename: Hong-Ying
authors.initials: HY
authors.lastname: Hu
authors.affiliation: State Environmental Protection Key Laboratory of Microorganism Application and Risk Control, School of Life Science, Tsinghua University, Beijing 100084, PR China.
authors.forename: Ying-Hua
authors.initials: YH
authors.lastname: Chen
authors.affiliation: School of Life Science, Tsinghua University, Beijing 100084, PR China.
authors.forename: Wan-Li
authors.initials: WL
authors.lastname: Liu
mesh_terms: D000255:Adenosine Triphosphate / Q000032:analysis*; D017209:Apoptosis / Q000187:drug effects; D001681:Biological Assay / Q000379:methods*; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D005434:Flow Cytometry; D056945:Hep G2 Cells; D006801:Humans; D012680:Sensitivity and Specificity; D013778:Tetrazolium Salts / Q000737:chemistry*; D013844:Thiazoles / Q000737:chemistry*; D062065:Wastewater / Q000032:analysis / Q000633:toxicity*; D014874:Water Pollutants, Chemical / Q000032:analysis / Q000633:toxicity*; D018508:Water Purification
chemical_list: D000255:Adenosine Triphosphate; D013778:Tetrazolium Salts; D013844:Thiazoles; D062065:Wastewater; D014874:Water Pollutants, Chemical; C022616:thiazolyl blue
keywords: ATP assay; Cell cycle; Cytotoxicity; MTT assay; Secondary effluent