Genotoxic Studies On Ethanol And The Protective Effects Of Procyanidins

Ethanol abuse is one of the most costly health problems in the world. The mechanism by which ethanol exerts its genotoxicity is not well understood. It is generally considered that free radical generation plays an important role in the genotoxicity of ethanol. A better understanding the genotoxic action and the possible mechanism of ethanol could lead to more effective treatment strategies for ethanol abuse. In present study, we investigated genotoxicity of ethanol in vivo and in vitro and revealed its possible molecular mechanisms. Meanwhile, the protective effects of natural antioxidants were also detected.In order to detect the DNA damage in mouse peripheral leukocytes induced by ethanol in vivo, the alkaline single cell gel electrophoresis was used. For understanding the possible mechanism of genotoxic action of ethanol, the urinary 8-OHdG excretions, generation of ROS and total antioxidant capacity of the whole blood were also measured. All the results showed that acute and chronic ethanol administration could induce significant increases in DNA damage in mouse peripheral leukocytes. Meanwhile, 8-OHdG in urine, an oxidative product of DNA, and ROS in the whole blood cells were also increased. The above results firstly provided the clear evidence that ethanol-induced DNA damage in peripheral leukocytes resulted from ethanol-induced oxidative stress in the body.The central nervous system (CNS) is one of the main toxic targets of ethanol. In present study, the DNA damage in brain cells (cerebral cortex, hippocampus, cerebellum, striatum and hypothalamus) induced by ethanol and which brain regions were more vulnerable to the genotoxic action of ethanol were investigated. Furthermore, the concentrations of ethanol and acetaldehyde in brain regions of the mice were detected by using gas chromatography after ethanol administration. The results showed that acute ethanol administration could induce significant DNA damage in cerebellum and hippocampus in mice and the peak increase in DNA damage reached at approx 4 hours post ethanol administration. Chronic ethanol administration induced the DNA damages in cerebellum, hippocampus, hypothalamus and cortex, which could be considered as the targets of chronic ethanol toxicity in the brain. Interestingly, three days after ethanol withdrawal, DNA damage induced by chronic ethanol administration could be almost completely repaired, suggesting that the DNA auto-repairing ability in the brain is quite strong. After ethanol treatment, the concentrations of ethanol and acetaldehyde in brain regions of mice were different and the concentrations of ethanol in cerebellum and hippocampus were higher than the other brain regions. The results suggested that ethanol induced DNA damage in cerebellum and hippocampus was relative to the high concentration of ethanol.Oxygen-derived free radicals have been implicated in many disease processes, including ethanol-induced neurotoxicity. Ethanol exposure has been shown to induce the generation of reactive free radical in vitro and in vivo. Our previous study showed that ethanol-induced DNA damage was resulted from the ethanol-induced oxidative stress. Therefore, it is reasonable to assume that antioxidants might have protective effects on ethanol-induced oxidative DNA damage. In present study, it was clearly demonstrated that the isolated grape seed procyanidin fractions and other antioxidants such as ascorbic acid, vitamin E and resveratrol could markedly decrease the levels of DNA single-strand breaks induced by ethanol. In other words, the grape seed procyanidins and other antioxidants can be used in prevention and treatment of the ailments in central nervous system induced by ethanol abuse.We have demonstrated that cerebellum and hippocampus, especially cerebellum, are the main targets of genotoxicity of ethanol. Further studies were conducted using primary cultures of rat cerebellar granular neurons to investigate the genotoxicity of ethanol in vitro and the releasing of ROS, the HO-1 and OGG1 mRNA expression were simultaneously measured. Whether the grape extracts and red wine extracts have protective effects on ethanol induced DNA damage were also detected. It was found that ethanol could induce significant DNA damage and releasing of ROS in rat cerebellar granular neurons. Ethanol exposed for 3 h and 6 h could up regulate the HO-1 and 0GG1 mRNA expression suggesting a compensatory regulation of the antioxidant defense and DNA repair system. In the present study, grape and red wine extracts could prevent ethanol-induced DNA damage and ROS generation in rat cerebellar granular neurons possibly due to reduction in ethanol-induced oxidative stress. All the results indicated that ethanol could induce oxidative DNA damage in rat cerebellar granular neurons, and grape and red wine extracts could significantly protect the neurons against ethanol-induced genotoxicity. In conclusion, the genotoxicity, especially neurotoxicity, of ethanol resulted from the ethanol-induced oxidative stress was demonstrated. Natural antioxidants, grape and red wine procyanidins, showed protective effects on ethanol-induced genotoxicity, which could be used in prevention and treatment of the ailments in central nervous system induced by ethanol abuse.