title: A novel quantification method for the total demethylation potential of aquatic sample extracts from Bohai Bay using the EGFP reporter gene.
abstract: BACKGROUND
  The demethylation potential of environmental pollutants is possibly an innate part of their comprehensive health risk. This paper develops a novel method called TDQ to quantify the demethylation epigenetic toxicity, termed the 5-AZA-CdR demethylation toxic equivalency, of aquatic samples from the heavily polluted Bohai Bay using Hep G2 cell lines transiently transfected with the pEGFP-C3 plasmid containing a methylated promoter of the EGFP reporter gene inserted artificially in vitro.
  
  
  RESULTS
  If the aquatic sample extract has strong total demethylation potential to the promoter, its methylation level will decrease, and increased green fluorescence will be observed under microscopy after TDQ co-incubation. The 5-AZA-CdR was selected as a representative demethylation agent to validate the principle of the TDQ method on three levels: significant dose-response relationships between the concentration of 5-AZA-CdR and the methylation level of promoters, mRNA expression level of the EGFP gene, and the fluorescence intensity of EGFP proteins. Twenty extracts from aquatic samples are successfully quantified with the TDQ test. Eight of them return meaningful results ranging from 0.00004 to 0.20053 μM 5-AZA-CdR toxicity equivalents.
  
  
  CONCLUSIONS
  The TDQ method is a reliable and rapid assay for the quantification of the DNA demethylation potential of aquatic sample extracts, which may shed light on the safety evaluation of food material.
authors.affiliation: State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China. qianyan@craes.org.cn.
authors.forename: Yan
authors.initials: Y
authors.lastname: Qian
authors.affiliation: College of Life Science and Bioengineering, Beijing University of Technology, Beijing, 100124, China. wangxiaoli@bjut.edu.cn.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China. lvzhanlu1015@163.com.
authors.forename: Zhanlu
authors.initials: Z
authors.lastname: Lv
authors.affiliation: State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China. chenpreteen@sina.com.
authors.forename: Chen
authors.initials: C
authors.lastname: Guo
authors.affiliation: State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China. hanmei@craes.org.cn.
authors.forename: Mei
authors.initials: M
authors.lastname: Han
authors.affiliation: State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China. wujiabing159@sina.com.|School of Public Health, Anhui Medical University, Hefei, 230032, China. wujiabing159@sina.com.
authors.forename: Jiabing
authors.initials: J
authors.lastname: Wu
authors.affiliation: School of Public Health, Anhui Medical University, Hefei, 230032, China. yyj580719@163.com.
authors.forename: Yongjian
authors.initials: Y
authors.lastname: Yang
authors.affiliation: College of Life Science and Bioengineering, Beijing University of Technology, Beijing, 100124, China. yishu-y@bjut.edu.cn.
authors.forename: Yishu
authors.initials: Y
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China. lr07@sohu.com.
authors.forename: Yan
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China. weiyj@craes.org.cn.
authors.forename: Yongjie
authors.initials: Y
authors.lastname: Wei
authors.affiliation: State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China. niejing@craes.org.cn.
authors.forename: Jing
authors.initials: J
authors.lastname: Nie
authors.affiliation: State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China. baobao45@126.com.|School of Public Health, Anhui Medical University, Hefei, 230032, China. baobao45@126.com.
authors.forename: Bao
authors.initials: B
authors.lastname: Liang
authors.affiliation: State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China. zhangjl@craes.org.cn.
authors.forename: Jinliang
authors.initials: J
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China. Xlwang@craes.org.cn.|Institute of Environmental Health and Related Product Safety, Chinese Center for Disease Control and Prevention, Beijing, 100021, China. Xlwang@craes.org.cn.
authors.forename: Xianliang
authors.initials: X
authors.lastname: Wang
mesh_terms: D001374:Azacitidine / Q000031:analogs & derivatives* / Q000633:toxicity; D001483:Base Sequence; D019175:DNA Methylation / Q000187:drug effects*; D000077209:Decitabine; D044127:Epigenesis, Genetic / Q000187:drug effects*; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D008969:Molecular Sequence Data; D009792:Oceans and Seas; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D018675:Toxicity Tests; D014874:Water Pollutants, Chemical / Q000633:toxicity*
chemical_list: D014874:Water Pollutants, Chemical; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000077209:Decitabine; D001374:Azacitidine