Possible metabolic pathways of ethanol responsible for oxidative DNA damage in human peripheral lymphocytes.
BACKGROUND
Ethanol abuse, especially binge drinking, can be toxic to human organs. However, there have been few studies on the genotoxicity induced by ethanol in human peripheral lymphocytes under binge drinking conditions. The purpose of this study was to investigate the oxidative DNA damage induced by ethanol in human peripheral lymphocytes in vitro and the possible mechanism associated with ethanol metabolism. The concentrations of ethanol investigated in this study were 50 and 100 mM, which are equal to the ethanol concentrations in blood after binge drinking. The maximum concentration we used was 150 mM although it is not the typical blood ethanol concentration seen during binge drinking, and most people may die at such a high concentration. The purpose of using this maximum concentration was to obtain more detailed evidence about the genotoxicity induced by ethanol. The DNA repair process was also studied.


METHODS
Peripheral lymphocytes were isolated from donors who were nonsmokers and not ethanol drinkers. Oxidative DNA damage, possible metabolic pathways of ethanol in human peripheral lymphocytes, and the repair system involved in the DNA auto-repair process were examined by comet assay, flow cytometry, time-of-flight mass spectrometry (TOF-MS), reverse transcription-polymerase chain reaction (RT-PCR), and western blotting.


RESULTS
The results showed that ethanol at the concentrations of 50, 100, and 150 mM significantly induced the oxidative DNA damage in human peripheral lymphocytes in vitro, which was accompanied by a parallel increase in the generation of 8-OHdG, intracellular hydroxyl radical, and reactive oxygen species (iROS). The DNA damage induced by ethanol could be attenuated by alcohol dehydrogenase 1B (ADH1B) or acetaldehyde dehydrogenase 2 (ALDH2) inhibitor, and the mRNA expression levels of ADH1B and ALDH2 were increased markedly by ethanol. The inhibitor of cytochrome P450 2E1 (CYP2E1) had no effect on ethanol-induced DNA damage, and CYP2E1 mRNA expression was not affected by ethanol. Furthermore, ethanol-induced DNA damage could be auto-repaired by lymphocytes. The expression of 8-oxoguanine DNA glycosylase 1 (OGG1) and the X-ray repair cross-complementation group 1 (XRCC1), 2 core enzymes in the base excision repair (BER) system, were increased in both of transcriptional and protein levels after ethanol treatment.


CONCLUSIONS
This study provides direct evidence that ethanol can induce oxidative DNA damage in human peripheral lymphocytes in vitro, and its mechanism may be associated with the metabolism of ethanol by the ADH1B/ALDH2 pathway. Moreover, ethanol-induced DNA damage can be auto-repaired by human peripheral lymphocytes possibly mediated by the BER system.
Yan
Yu
Y
Department of Pharmacology, Shenyang Pharmaceutical University, China.
Yang
Jing-Yu
JY
Mou
Yan-Hua
YH
Wang
Li-Hui
LH
Zhang
Hong
H
Wu
Chun-Fu
CF
D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002492:Central Nervous System Depressants / Q000378:metabolism / Q000633:toxicity*; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D045647:DNA Glycosylases / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000187:drug effects*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D000431:Ethanol / Q000378:metabolism* / Q000633:toxicity*; D005260:Female; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D053858:Metabolic Networks and Pathways; D018384:Oxidative Stress / Q000187:drug effects* / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D000076105:X-ray Repair Cross Complementing Protein 1
D002492:Central Nervous System Depressants; D004268:DNA-Binding Proteins; D017382:Reactive Oxygen Species; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; D000431:Ethanol; D000426:Alcohol Dehydrogenase; D019392:Cytochrome P-450 CYP2E1; D045647:DNA Glycosylases; C479058:oxoguanine glycosylase 1, human