南京大学学报(自然科学版) ›› 2020, Vol. 56 ›› Issue (5): 719–728.doi: 10.13232/j.cnki.jnju.2020.05.011

• • 上一篇    

砷和二氯乙酰胺对HepG2细胞的联合暴露毒性效应

王智志,陈玲,张徐祥,吴兵()   

  1. 污染控制与资源化研究国家重点实验室，南京大学环境学院，南京，210023
  • 收稿日期:2020-06-29 出版日期:2020-09-30 发布日期:2020-09-29
  • 通讯作者: 吴兵 E-mail:bwu@nju.edu.cn
  • 基金资助:
    江苏省科技支撑计划社会发展项目(BE2017709);国家重点研究发展计划(2016YFA0203103)

Combined toxic effects of arsenic and dichloroacetamide in human HepG2 cells

Zhizhi Wang,Ling Chen,Xuxiang Zhang,Bing Wu()   

  1. State Key Laboratory of Pollution Control and Resource Reuse，School of the Environment，Nanjing University，Nanjing，210023，China
  • Received:2020-06-29 Online:2020-09-30 Published:2020-09-29
  • Contact: Bing Wu E-mail:bwu@nju.edu.cn

RichHTML

5

PDF (PC)

582

摘要：

饮用水中污染物间的联合毒性效应对评估饮用水健康风险具有重要意义.选择饮用水中备受关注的砷（arsenic，As）和消毒副产物二氯乙酰胺（dichloroacetamide，DCAcAm）为研究对象，分析人肝癌细胞HepG2暴露于As（0.375和0.75 mg·L-1）和DCAcAm（0.1，1和10 mg·L-1）24 h后的联合毒性效应.结果表明，DCAcAm单独暴露未降低HepG2细胞活力，但与As联合暴露时可增加As的细胞毒性.从化学检测和生物分子调控通路水平分析了联合毒性机制：DCAcAm提高了HepG2细胞对As的富集水平，并通过提高S?腺苷甲硫氨酸（S?adenosine methionine,SAM）含量增加As的甲基化水平；DCAcAm还增强了As对氧化应激和DNA损伤等相关基因表达和代谢物水平的影响.上述研究结果表明，在进行饮用水中As毒性效应评估及其风险评价时，DCAcAm等消毒副产物的影响值得关注.

关键词: 砷, 二氯乙酰胺, 联合毒性, 基因表达, 代谢组学

Abstract:

Arsenic (As) and disinfection byproducts are of widespread concern in drinking water due to their high health risks. However，there is little report about the effect of co?exposure on their toxicological behaviors. In this paper，the dichloroacetamide (DCAcAm) was chosen as the representative disinfection byproduct. The human HepG2 cells were used to explore the combined toxicity of As (0.375 and 0.75 mg·L-1) and DCAcAm (0.1，1 and 10 mg·L-1). Experimental results showed that DCAcAm could significantly increase the cytotoxic effects of As with a certain dose?effect relationship. Underlying mechanisms of the combined toxicity were furtherly analyzed. First，the concentrations of total As and As species in the HepG2 cells were analyzed using ICP?MS and HPLC?ICP?MS，respectively. The concentrations of methyl donor，S?adenosine methionine (SAM)，were determined by ELISA. The results showed that DCAcAm increased intracellular As accumulation in HepG2 cells，and increased the methylation level of As by increasing the SAM contents，which might be an important reason that DCAcAm increased As toxicity. Furthermore，the high?throughput PCR and NMR?based metabolomics studies were used to analyze the biological processes after combined exposure of As and DCAcAm. Results showed that DCAcAm enhanced the changes in expressions of genes related to intracellular oxidative stress and DNA damage，such as EGFR，ABCC2，APEX1 and ERCC2. Metabonomics analysis indicated that combined exposure of As and DCAcAm significantly altered the amino acid metabolism of HepG2 cells，decreased the levels of valine，leucine and isoleucine，and inhibited the process of aminoacyl?tRNA biosynthesis，which were similar with the effects of As alone exposure.The above results provide a mechanistic explanation for the synergistic toxic effects of As and DCAcAm. The results of this paper also show that during the evaluation of the toxic effects and health risks of As in drinking water，the influences of low concentration DCAcAm and other disinfection byproducts deserve our high attention.

Key words: arsenic, DCAcAm, combined toxicity, gene expression, metabolomics

中图分类号: 

  • X172

表1

检测指标及所用仪器"

检测指标细胞培养容器检测仪器
总As含量75 cm2细胞培养瓶ICP?MS
As形态75 cm2细胞培养瓶HPLC?ICP?MS
SAM6孔细胞培养板ELISA试剂盒，酶标仪

甲基化

基因表达

6孔细胞培养板

高通量qPCR仪

384孔模块

细胞活力96孔细胞培养板CCK?8试剂盒，酶标仪
基因表达6孔细胞培养板高通量qPCR仪，OpenArray芯片
代谢组学75 cm2细胞培养瓶NMR核磁共振

图1

As和DCAcAm联合暴露对HepG2细胞活力的影响（a）DCAcAm；（b）0.375 mg·L-1 As与DCAcAm联合暴露；（c）0.75 mg·L-1 As与DCAcAm联合暴露；n=18，三个生物学重复，每个生物学重复包括六个技术平行；*p<0.05"

图2

As和DCAcAm联合暴露下HepG2细胞内总As浓度（a）0.375 mg·L-1 As与DCAcAm联合暴露；（b）0.75 mg·L-1 As与DCAcAm联合暴露；n=3，*p<0.05"

表2

As和DCAcAm联合暴露下HepG2细胞中As形态浓度百分比（n=3，*p<0.05）"

AsⅢDMA+MMAAsⅤ
0.375 mg·L-1 As57.06%14.14%28.80%

0.375 mg·L-1 As+

0.1 mg·L-1 DCAcAm

55.15%18.04% *26.81%

0.375 mg·L-1 As+

1 mg·L-1 DCAcAm

56.20%15.12%28.68%

0.375 mg·L-1 As+

10 mg·L-1 DCAcAm

52.79%16.28% *30.93%
0.75 mg·L-1 As60.95%18.53%20.52%

0.75 mg·L-1 As+

0.1 mg·L-1 DCAcAm

59.68%21.32% *19.01%

0.75 mg·L-1 As+

1 mg·L-1 DCAcAm

61.58%18.84%19.58%

0.75 mg·L-1 As+

10 mg·L-1 DCAcAm

60.06%20.04% *19.90%

图3

As和DCAcAm联合暴露对HepG2细胞内SAM含量的影响（a）DCAcAm；（b）0.375 mg·L-1 As与DCAcAm联合暴露；（c）0.75 mg·L-1 As与DCAcAm联合暴露；n=3，*p<0.05"

图4

As和DCAcAm联合暴露对HepG2细胞基因表达的影响（a）细胞基因表达变化的热图分析；（b）差异表达基因数量venn图；（c~f）差异表达基因富集分析"

图5

As和DCAcAm联合暴露对HepG2细胞代谢的影响（a，b）代谢产物的PLS?DA；（c，d）差异代谢产物数量venn图；（e，f）差异代谢产物热图"

表3

As和DCAcAm联合暴露组代谢产物的KEGG通路注释"

Pathway Name

0.375

mg·L-1 As

0.375 mg·L-1 As+0.1 mg·L-1 DCAcAm0.375 mg·L-1 As+1 mg·L-1 DCAcAm0.375 mg·L-1 As+10 mg·L-1 DCAcAm

0.75

mg·L-1 As

0.75 mg·L-1 As+0.1 mg·L-1 DCAcAm0.75 mg·L-1 As+1 mg·L-1 DCAcAm0.75 mg·L-1 As+10 mg·L-1 DCAcAm
氨酰基?tRNA生物合成9 （1.1E-8）8 （4.4E-8）9 （4.4E-9）10 （1.1E-9）7（7.3E-6）8 （3.3E-7）9 （2.5E-8）9 （7.13E-9）
精氨酸生物合成3 （0.0011）3 （9.3E-4）
苯丙氨酸、酪氨酸和色氨酸的生物合成2 （0.0014）2 （9.4E-4）2 （0.0011）2 （0.0016）2（0.0015）2 （0.0014）
D?谷氨酰胺和D?谷氨酸代谢2 （0.0033）2 （0.0023）2 （0.0028）2 （0.0038）2（0.0038）2 （0.0036）2 （0.0038）2 （0.003）
缬氨酸、亮氨酸和异亮氨酸的生物合成2 （0.0061）3 （1.9E-4）3 （2.2E-4）
丙氨酸、天冬氨酸和谷氨酸代谢3 （0.0083）4 （3.4E-4）3 （0.0065）3 （0.01）4 （9.7E-4）3 （0.0073）
苯丙氨酸代谢2 （0.0096）2 （0.0067）2 （0.0081）3（4.6E-4）3 （4.1E-4）
1 Andra S S，Makris K C，Botsaris G，et al. Evidence of arsenic release promoted by disinfection by?products within drinking?water distribution systems. Science of the Total Environment，2014，472：1145-1151.
2 Guo H M，Wen D G，Liu Z Y，et al. A review of high arsenic groundwater in mainland and taiwan，China：distribution，characteristics and geochemical processes. Applied Geochemistry，2014，41：196-217.
3 Jochem M，Ende L，Isasa M，et al. Targeted degradation of glucose transporters protects against arsenic toxicity. Molecular and Cellular Biology，2019，39(10)：e00559-18.
4 Wang Y B，Ezemaduka A，Li Z H，et al. Joint toxicity of arsenic，copper and glyphosate on behavior，reproduction and heat shock protein response in Caenorhabditis elegans. Bulletin of Environmental Contamination and Toxicology，2017，98(4)：465-471.
5 孙贵范. 饮用水中砷浓度最高允许限值研究进展. 中华流行病学杂志，2009，28：6-8.
6 Liu S，Guo X C，Zhang X X，et al. Impact of iron precipitant on toxicity of arsenic in water：a combined in vivo and in vitro study. Environmental Science & Technology，2013，47(7)：3432-3438.
7 高乃云. 饮用水消毒副产物形成与控制研究. 北京：中国建筑工业出版社，2011，236.
8 曹中琦，盖世博. 活性炭吸附去除水中高毒性N?DBPs卤代乙酰胺. 净水技术，2019(7)：78-84.
Cao Z Q，Gai S B. Adsorption of haloacetamides as highly toxic N?DBPs removal in water by activated carbon. Water Purificatin Technology，2019(7)：78-84.
9 曹文平，赵鑫，王国祥等. 消毒副产物二氯乙酰胺的去除特性及对斑马鱼的毒性. 中国环境科学，2017，37(3)：1073-1081.
Cao W P，Zhao X，Wang G X，et al. Removal property and toxicity research on zebrafish of the disinfection?byproduct?dichloroacetamide. China Environmental Science，2017，37(3)：1073-1081.
10 Yu S L，Lin T，Chen W，et al. The toxicity of a new disinfection by?product，2,2?dichloroacetamide (DCAcAm)，on adult zebrafish (danio rerio) and its occurrence in the chlorinated drinking water. Chemosphere，2015，139：40-46.
11 Wang Y M，Zhu G C，Engel B，et al. Probabilistic human health risk assessment of arsenic under uncertainty in drinking water sources in jiangsu province，China. Environmental Geochemistry and Health，2020，42(7)：2023-2037.
12 Yin X，Jiang B L，Feng Z X，et al. Comprehensive evaluation of shallow groundwater quality in central and southern jiangsu province，China. Environmental Earth Sciences，2017，76(11)：400.
13 Nieuwenhuijsen M J，Toledano M B，Eaton N E，et al. Chlorination disinfection byproducts in water and their association with adverse reproductive outcomes：a review. Occupational and Environmental Medicine，2000，57(2)：73-85.
14 Yang F，Zhang J，Chu W H，et al. Haloactamides versus halomethanes formation and toxicity in chloraminated drinking water. Journal of Hazardous Materials，2014，274：156-163.
15 Stalter D，O'Malley E，Von Gunten U，et al. Fingerprinting the reactive toxicity pathways of 50 drinking water disinfection by?products. Water Research，2016，91：19-30.
16 States J C. Disruption of mitotic progression by arsenic. Biological Trace Element Research，2015，166(1)：34-40.
17 Zhou X X，Sun X，Mobarak C，et al. Differential binding of monomethylarsonous acid compared to arsenite and arsenic trioxide with zinc finger peptides and proteins. Chemical Research in Toxicology，2014，27(4)：690-698.
18 Yang P，Zhou B，Cao W C，et al. Prenatal exposure to drinking water disinfection by?products and DNA methylation in cord blood. Science of the Total Environment，2017，586：313-318.
19 Plewa M J，Muellner M G，Richardson S D，et al. Occurrence，synthesis and mammalian cell cytotoxicity and genotoxicity of haloacetamides：an emerging class of nitrogenous drinking water disinfection byproducts. Environmental Science & Technology，2007，42(3)：955-961.
20 Koch I，McPherson K，Smith P，et al. Arsenic bioaccessibility and speciation in clams and seaweed from a contaminated marine environment. Marine Pollution Bulletin，2007，54(5)：586-594.
21 李景岩，张爱君. 砷代谢与砷毒性作用机制的关系. 中国地方病防治杂志，2011，26(5)：345-347.
22 Dopp E，Hartmann L M，Von Recklinghausen U，et al. Forced uptake of trivalent and pentavalent methylated and inorganic arsenic and its cyto?/genotoxicity in fibroblasts and hepatoma cells. Toxicological Sciences，2005，87(1)：46-56.
23 李响利. 砷甲基化过程中S?腺苷甲硫氨酸和砷与人砷甲基化酶结合位点的研究. 博士学位论文. 南京：南京大学，2014.
Li X L. Investigation of the binding sites of S?adenosylmethionine and arsenic in human arsenic (Ⅲ) methytransferase during arsenic methyltion Ph.D Dissertation. Nanjing：Nanjing University，2014.
24 Chikkanna A，Mehan L，Sarath P，et al. exposures Arsenic ，poisoning，and threat to human health：arsenic affecting human health∥Environmental exposures and human health challenges. Hershey：IGI Global，2019：86-105.
25 Prakash C，Soni M，Kumar V. Mitochondrial oxidative stress and dysfunction in arsenic neurotoxicity：a review. Journal of Applied Toxicology，2016，36(2)：179-188.
26 Ma L，Li J，Zhan Z B，et al. Specific histone modification responds to arsenic?induced oxidative stress. Toxicology and Applied Pharmacology，2016，302：52-61.
27 周勤华，周盛梅. 氨酰?tRNA合成酶的研究进展. 中国生物工程杂志，2009，29(6)：130-134.
Zhou Q H，Zhou S M. Research progress on aminoacyl?tRNA synthetase. China Biotechnology，2009，29(6)：130-134.
28 Wei N，Shi Y，Truong L N，et al. Oxidative stress diverts tRNA synthetase to nucleus for protection against DNA damage. Molecular Cell，2014，56(2)：323-332.
29 Park S G，Ewalt K L，Kim S. Functional expansion of aminoacyl?tRNA synthetases and their interacting factors：new perspectives on housekeepers. Trends in Biochemical Sciences，2005，30(10)：569-574.
30 邢晓丹，张木子，黎明等. 牛磺酸和甲硫氨酸亚砜亚胺对高血氨症大弹涂鱼氧化应激和炎症反应相关基因影响的比较研究. 海洋与湖沼，2018，49(3)：646-652.
Xing X D，Zhang M Z，Li M，et al. Comparison effects of taurine and methionine sulfoximine on genes involved in oxidative stress and inflammation of hyperammonemia mudskipper. Oceanologia et Limnologia Sinica，2018，49(3)：646-652.
[1] 韩永和，贾梦茹，傅景威，向　萍，史孝霞，崔昕毅，罗　军，陈焱山*. 不同浓度砷酸盐胁迫对蜈蚣草根际微生物群落功能多样性特征的影响[J]. 南京大学学报(自然科学版), 2017, 53(2): 275-.
[2] 王少华，徐兆文，陆现彩，王汝成，王浩，陈，朱翔. 南京大平山铜矿砷的赋存状态[J]. 南京大学学报(自然科学版), 2014, 50(2): 181-.
[3] 张礼;刘学军;. 一种基于Gamma模型的RNA-seq数据分析方法[J]. 南京大学学报(自然科学版), 2013, 49(4): 466-474.
[4]  孙曙光，尹颖，郭红岩**
.  大气CO2浓度升高对砷污染水体生态风险的影响*[J]. 南京大学学报(自然科学版), 2013, 49(3): 387-393.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!

版权所有 © 2021 《南京大学学报(自然科学)》

地址：江苏省南京市鼓楼区汉口路22号，南京大学学报编辑部，210093

电话：025-83592704 , 传真：025-83592704        技术支持：北京玛格泰克科技发展有限公司