p53和γ-H2AX作为乙醛引起DNA损伤早期生物标记物的实验研究

doi:10.3969/j.issn.1004-616x.2014.02.001

癌变·畸变·突变

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p53和γ-H2AX作为乙醛引起DNA损伤早期生物标记物的实验研究

周学贤，Penny Jones，Paul Carmichael，郝卫东，

1．北京大学公共卫生学院毒理学系，食品安全毒理学研究与评价北京市重点实验室，北京 100191，中国；2．英国联合利华安全与环境保障中心，贝德福德郡 MK44 1LQ，英国

收稿日期:2013-10-10 修回日期:2014-02-10 出版日期:2014-03-30 发布日期:2014-03-30
通讯作者: 郝卫东，E-mail：whao@bjmu.edu.cn
作者简介:周学贤（1987－），女，河北省唐山市人，硕士，研究方向：遗传毒理学。E-mail：zhouxuexian1@163.com

Changes of p53 and γ-H2AX of TK6 cells caused by acetaldehyde

ZHOU Xue-xian， Penny Jones， Paul Carmichael， HAO Wei-dong，

1. Beijing Key Laboratory of Toxicological Research and Risk Assessment for Food Safety, Department of Toxicology, School of Public Health, Peking University, Beijing 100191, China; 2. Unilever, Safety & Environmental Assurance Centre,Bedford Shire, MK44 1LQ, UK

Received:2013-10-10 Revised:2014-02-10 Online:2014-03-30 Published:2014-03-30
Contact: 郝卫东，E-mail：whao@bjmu.edu.cn

摘要/Abstract

摘要：

目的：评价乙醛染毒p53野生型人类淋巴母细胞(TK6)后，细胞中DNA损伤标记物p53、γ-H2AX的表达变化，并与彗星试验中的DNA链断裂指标进行敏感性比较，探讨p53和γ-H2AX 作为乙醛引起DNA损伤的早期生物标记物的可能。方法：乙醛在0.5~20 mmol/L的浓度范围分别染毒TK6细胞20 min、2和12 h后，采用高通量的流式细胞术检测肿瘤抑制蛋白total-p53、磷酸化p53(p-p53)以及磷酸化组蛋白(γ-H2AX)的细胞表达率和表达强度(平均荧光强度)；同时采用碱性彗星试验检测乙醛引起的DNA链断裂指标(细胞拖尾率、尾长、尾部DNA百分含量以及尾矩)的变化。结果：乙醛染毒TK6细胞12 h后，γ-H2AX的表达强度在15及20 mmol/L浓度下显著升高(P<0.05)，p-p53的细胞表达率在0.5~7.5 mmol/L浓度范围内呈现剂量依赖的升高趋势，total-p53的细胞表达率趋势与p-p53相似，但与阴性对照组相比，差异无统计学意义。彗星试验中，细胞拖尾率、尾长、尾部DNA百分含量以及尾矩在5.0~12.5 mmol/L浓度范围内均增加，并呈现剂量依赖性。染毒2 h后，与阴性对照组相比，total-p53和p-p53的细胞表达率在15和20 mmol/L浓度下显著升高(P均<0.05)，细胞拖尾率、尾部DNA百分含量以及尾矩在20 mmol/L浓度升高(P均<0.05)。染毒20 min后，3种生物标志物均未见清晰的变化趋势，4种DNA链断裂指标均未见显著变化(P均＞0.05)。结论：乙醛染毒TK6细胞12 h可诱导p-p53细胞表达率升高、γ-H2AX细胞表达强度升高、 total-p53细胞表达率轻微升高，以及细胞拖尾率、尾长、尾部DNA百分含量、尾矩的升高。p-p53比γ-H2AX和DNA链断裂指标在检测乙醛诱导的DNA损伤方面更加敏感。

关键词: 乙醛, DNA损伤, p53, γ-H2AX, 彗星试验

Abstract:

OBJECTIVE: The aim of this study was to investigate the changes of biomarkers of DNA damage，total p53，phospho-p53 (p-p53) and phospho-H2AX (p-H2AX or γ-H2AX) following exposure of TK6 cells to acetaldehyde and to explore the roles and the sensitivity of p53，γ-H2AX in DNA damage detection by comparison in the indicators of DNA strand breaks (comet assay). METHODS：TK6 cells were exposed to acetaldehyde with different concentrations of 0.5-20 mmol/L for 20 min，2 and 12 h. Flow cytometry was applied to investigate the changes of relevant biomarkers of DNA damage. Alkaline comet assay was used to detect the indicators of DNA strand breaks (percentage of cells with tails，tail length，percentage of tail DNA，tail moment). RESULTS： After exposure to acetaldehyde for 12 h，a significant increase of γ-H2AX expression was observed at acetaldehyde doses of 15 and 20 mmol/L. A significant dose-dependent increase of p-p53 expression rate was found at acetaldehyde dose between 0.5-7.5 mmol/L. In parallel，a similar trend of expression was observed in total p53，but no significant increase was shown. For the comet assay，significant dose-dependent increases of percentage of cells with tails，tail length，percentage of tail DNA，tail moment were obtained after 12 h exposure acetaldehyde dose between 5.0-12.5 mmol/L. 2 h exposure showed that both total p53 and p-p53 expression rates increased significantly at concentrations of 15 and 20 mmol/L of acetaldehyde，and significant increases of percentage of cells with tails，tail DNA，tail moment accurred at the concentration of 20 mmol/L acetaldehyde，but no response was observed in γ-H2AX. For 20 min exposure，no clear trend for all the three biomarkers and no significant change were shown with the indicators of DNA strand breaks. CONCLUSION： Acetaldehyde could induce increase of p-p53 expression rate，γ-H2AX expression amount，percentage of cells with tails，tail length，percentage of tail DNA，tail moment and total p53 expression rate to a less extent. The biomarker p-p53 may be more sensitive reflecting the DNA damage caused by acetaldehyde than γ-H2AX and indicators of DNA strand breaks.

Key words: acetaldehyde, DNA damage, p53, γ-H2AX, comet assay

周学贤，Penny Jones，Paul Carmichael，郝卫东，. p53和γ-H2AX作为乙醛引起DNA损伤早期生物标记物的实验研究[J]. 癌变·畸变·突变, doi: 10.3969/j.issn.1004-616x.2014.02.001.

ZHOU Xue-xian， Penny Jones， Paul Carmichael， HAO Wei-dong，. Changes of p53 and γ-H2AX of TK6 cells caused by acetaldehyde[J]. Carcinogenesis, Teratogenesis & Mutagenesis, doi: 10.3969/j.issn.1004-616x.2014.02.001.

参考文献

[1] International Agency for Research on Cancer(IARC). Monographs on the Evaluation of Carcinogenic Risks to Humans[R]. France：IARC，1999.

[2] International Agency for Research on Cancer(IARC). Monographs on the Evaluation of Carcinogenic Risks to Humans，Alcohol consumption and ethyl carbamate[R]. France：IARC， 2010.

[3] Lambert B，HE SM. DNA and chromosome damage induced by acetaldehyde in human lymphocytes in vitro[J]. Ann NY Acad Sci，1988，534(1)：369-376.

[4] Singh NP，Khan A. Acetaldehyde：genotoxicity and cytotoxicity in human lymphocytes[J]. Mutat Res，1995， 337(1)：9-17.

[5] Lorenti GC，Mechilli M，Proietti DSL，et al. Relationship between DNA lesions，DNA repair and chromosomal damage induced by acetaldehyde[J]. Mutat Res，2009，662(1)：3-9.

[6] Moeller BC，Recio L，Green A，et al. Biomarkers of exposure and effect in human lymphoblastoid TK6 cells following [13C2]-acetaldehyde exposure[J]. Toxicol Sci，2013， 133(1)：1-12.

[7] National Toxicology Program. Report on Carcinogens[R]. USA：NTP，2011.

[8] Grafstr?m RC，Dypbukt JM，Sundqvist K，et al. Pathobiological effects of acetaldehyde in cultured human epithelial cells and fibroblasts[J]. Carcinogenesis，1994， 15(5)：985-990.

[9] Qui?ones A，Rainov NG. Identification of genotoxic stress in human cells by fluorescent monitoring of p53 expression[J]. Mutat Res，2001，494(1)：73-85.

[10] Nelson WG，Kastan MB. DNA strand breaks：the DNA template alterations that trigger p53-dependent DNA damage response pathways[J]. Mol Cell Biol，1994，14(3)：1815-1823.

[11] Levine AJ. p53，the cellular gatekeeper for growth and division[J]. Cell，1997，88(3)：323-331.

[12] Rogakou EP，Pilch DR，Orr AH，et al. DNA double- stranded breaks induce histone H2AX phosphorylation on serine 139[J] . J Biol Chem，1998，273(10)：5858- 5868.

[13] Sedelnikova OA，Rogakou EP，Panyutin IG，et al. Quantitative detection of 125IdU-induced DNA double-strand breaks with γ-H2AX antibody[J]. Radiat Res，2002， 158(4)：486-492.

[14] Mac Phail SH，Banath JP，Yu TY，et al. Expression of phosphorylated histone H2AX in cultured cell lines following exposure to X-rays [J]. Int J Radiat Biol，2003，79(5)：351- 358.

[15] 宾●萍. γ-H2AX与DNA双链断裂关系的研究进展[J]. 卫生研究，2006，36(4)：520-522.

[16] Dickey JS，Redon CE，Nakamura AJ，et al. H2AX： functional roles and potential applications[J]. Chromosoma， 2009，118(6)：683-692.

[17] Yokoyama A，Omori T，Tanaka Y，et al. p53 Protein accumulation，cancer multiplicity，and aldehyde dehydrogenase-2 genotype in Japanese alcoholic men with early esophageal squamous cell carcinoma[J]. Cancer Lett，2007， 247(2)：243-252.

[18] Kang J，Ferguson D，Song H，et al. Functional interaction of H2AX，NBS1，and p53 in ATM-dependent DNA damage responses and tumor suppression[J]. Mol Cell Biol，2005， 25(2)：661-670.

[19] Van AH，Gasser SM. Crosstalk between histone modifications during the DNA damage response[J]. Trends Cell Biol，2009， 19(5)：207-217.

[20] Paull TT，Rogakou EP，Yamazaki V，et al. Acritical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage[J]. Curr Biol，2000，10(15)：886-895.

[21] Dote H，Cerna D，Burgan WE，et al. Enhancement of in vitro and in vivo tumor cell radiosensitivity by the DNA methylation inhibitor zebularine[J]. Clin Cancer Res，2005，11(12)： 4571-4579.

[22] Marietta C，Thompson LH，Lamerdin JE，et al. Acetaldehyde stimulates FANCD2 monoubiquitination，H2AX phosphorylation，and BRCA1 phosphorylation in human cells in vitro ：Implications for alcohol-related carcinogenesis[J]. Mutat Res，2009，664(1)：77-83.

p53和γ-H2AX作为乙醛引起DNA损伤早期生物标记物的实验研究

Changes of p53 and γ-H2AX of TK6 cells caused by acetaldehyde

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