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BMC Biotechnol. 2015 Nov 26;15:107. doi: 10.1186/s12896-015-0224-y.

A novel quantification method for the total demethylation potential of aquatic sample extracts from Bohai Bay using the EGFP reporter gene.

Qian Y1, Wang X2, Lv Z3, Guo C4, Han M5, Wu J6,7, Yang Y8, Yang Y9, Jiang Y10, Wei Y11, Nie J12, Liang B13,14, Zhang J15, Wang X16,17.

Author information

1
State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China. qianyan@craes.org.cn.
2
College of Life Science and Bioengineering, Beijing University of Technology, Beijing, 100124, China. wangxiaoli@bjut.edu.cn.
3
State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China. lvzhanlu1015@163.com.
4
State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China. chenpreteen@sina.com.
5
State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China. hanmei@craes.org.cn.
6
State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China. wujiabing159@sina.com.
7
School of Public Health, Anhui Medical University, Hefei, 230032, China. wujiabing159@sina.com.
8
School of Public Health, Anhui Medical University, Hefei, 230032, China. yyj580719@163.com.
9
College of Life Science and Bioengineering, Beijing University of Technology, Beijing, 100124, China. yishu-y@bjut.edu.cn.
10
State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China. lr07@sohu.com.
11
State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China. weiyj@craes.org.cn.
12
State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China. niejing@craes.org.cn.
13
State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China. baobao45@126.com.
14
School of Public Health, Anhui Medical University, Hefei, 230032, China. baobao45@126.com.
15
State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China. zhangjl@craes.org.cn.
16
State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing, 100012, China. Xlwang@craes.org.cn.
17
Institute of Environmental Health and Related Product Safety, Chinese Center for Disease Control and Prevention, Beijing, 100021, China. Xlwang@craes.org.cn.

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.

PMID:
26610601
PMCID:
PMC4660669
DOI:
10.1186/s12896-015-0224-y
[Indexed for MEDLINE]
Free PMC Article

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