Rulten S L, Hodder E, Ripley T L, Stephens D N, Mayne L V
Trafford Centre for Medical Research, University of Sussex, Falmer, Brighton, BN1 9RY, United Kingdom.
Alcohol Clin Exp Res. 2008 Jul;32(7):1186-96. doi: 10.1111/j.1530-0277.2008.00673.x.
The largest cause of neurological damage to children is prenatal exposure to alcohol and chronic alcohol use in adults is associated with neurodegeneration, dementia and long-term behavioral changes. Microarray analysis identified the DNA damage response (DDR) gene, Fanconi anemia (Fanc) D2, to be robustly upregulated in mouse midbrain following 24-hour in vivo exposure to ethanol. In this study, we investigate the ability of ethanol to generate DNA strand breaks, predicted substrates for the Fanc pathway and the potential role of FANCD2 in the DDR to ethanol in brain.
The effect of ethanol on FANCD2 mRNA levels was measured by quantitative real time PCR using mouse brain and human neuronal cells. FANCD2 protein levels and ubiquitination were measured by Western blotting and immunocytochemistry. DNA damage induction by ethanol/acetaldehyde was measured using the Comet assay and gamma H2AX immunocytochemistry. Levels of DNA and RNA synthesis were measured in cell strains using (3)H-thymidine or (3)H-uridine up-take.
Chronic exposure to ethanol induced FANCD2 in mouse midbrain in vivo and in the nucleus of human neuronal cells in culture. However, there was no concomitant increase in the amount of ubiquitinated FANCD2. Acetaldehyde also induced nonubiquitinated FANCD2 protein, and we were able to demonstrate the ability of acetaldehyde to generate DNA double strand breaks, lesions which normally induce ubiquitination of FANCD2. Ethanol also inhibited both RNA and DNA synthesis in proliferating cells consistent with effects on transcription and replication.
In contrast to other DNA damaging agents, ethanol/acetaldehyde generated DNA strand breaks without inducing ubiquitination of FANCD2, despite increasing protein levels in the nucleus. These data are consistent with recent reports that suggest the Fanconi anemia pathway plays an important role in the adult brain in response to DNA damage. Further work is required to establish what this role is, in particular the potential function of nonubiquitinated FANCD2 and its role in the DNA damage response in postmitotic neurons and neural precursor cells.
儿童神经损伤的最大原因是产前接触酒精，而成人长期饮酒与神经退行性变、痴呆及长期行为改变有关。微阵列分析表明，在体内暴露于乙醇24小时后，小鼠中脑中DNA损伤反应（DDR）基因范可尼贫血（Fanc）D2显著上调。在本研究中，我们调查了乙醇产生DNA链断裂的能力、Fanc途径的预测底物以及FANCD2在脑中对乙醇的DDR中的潜在作用。
使用小鼠脑和人神经元细胞，通过定量实时PCR检测乙醇对FANCD2 mRNA水平的影响。通过蛋白质印迹法和免疫细胞化学检测FANCD2蛋白水平和泛素化。使用彗星试验和γH2AX免疫细胞化学检测乙醇/乙醛诱导的DNA损伤。使用（3）H-胸腺嘧啶核苷或（3）H-尿苷摄取法测量细胞株中DNA和RNA的合成水平。
长期暴露于乙醇可在体内诱导小鼠中脑及培养的人神经元细胞核中的FANCD2。然而，泛素化的FANCD2数量并未随之增加。乙醛也可诱导非泛素化的FANCD2蛋白，并且我们能够证明乙醛产生DNA双链断裂的能力，这种损伤通常会诱导FANCD2的泛素化。乙醇还抑制增殖细胞中的RNA和DNA合成，这与对转录和复制的影响一致。
与其他DNA损伤剂不同，乙醇/乙醛产生DNA链断裂但不诱导FANCD2的泛素化，尽管细胞核中的蛋白水平有所增加。这些数据与最近的报道一致，即范可尼贫血途径在成人大脑中对DNA损伤的反应中起重要作用。需要进一步开展工作以确定这一作用是什么，特别是非泛素化FANCD2的潜在功能及其在有丝分裂后神经元和神经前体细胞的DNA损伤反应中的作用。
