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Study of the Intervention Effect of Melatonin on ACR-Induced Neurobehavioral Damage in Rats

Author: ZhuLanLan
Tutor: YanHong
School: Huazhong University of Science and Technology
Course: Health Toxicology
Keywords: melatonin acrylamide neurobehavioral damage oxidative damage DNA damage
CLC: R114
Type: Master's thesis
Year: 2011
Downloads: 37
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Abstract

Acrylamide (acrylamide) is a white crystalline substance and is precursor substance widely used in the production of polyacrylamide. It is mainly used for water purification treatment, pulp processing and so on. Starchy foods are prone to produce acrylamide at high temperatures cooking (>120℃). Studies have shown that human exposure to acrylamide through the digestive tract, respiratory tract, skin and mucous membrane and other ways.Acrylamide is detected in some fried and grilled starchy foods such as French fries, etc. The content is 500 times higher than the World Health Organization (WHO) allowed in drinking water (0.5μg / L).A large number of animal studies shows that acrylamide mainly cause neurotoxicity, reproductive and developmental toxicity and only neurotoxicity are proved to human.Neurotoxicity mainly are degenerative changes in peripheral nerve and central nervous system.The acute or subacute poisoning mainly shows central nervous system disorder, the chronic poisoning mainly shows peripheral neuropathy disorder,but the mechanism is still not fully elucidated.Melatonin (melatonin, MT) is the main hormone secreted by the pineal gland, it can regulate mood, sleeping, immunity, sex, reproduction and other physiological functions,and plays an important role in aging, tumor growth and the occurrence and development of some elderly patients. Studies reported that MT had antioxidant activity as an efficient free radical scavenger, and has a strong water-soluble and fat-soluble to pass through a variety of biological barriers in the body in 20th century 90 years. MT could effectively scavenge hydroxyl radicals, peroxy alkyl radicals, superoxide anions, and plays an important role in preventing oxidative damage and protecting the vitality of antioxidant enzymes in the body. MT has a good prospect in the prevention of neurodegenerative disease such as Alzheimer’s disease (AD),parkinson’s disease (PD) and Huntington’s disease.In the presently study,we explore the intervention of MT on the basis of ACR results in nervous system damage(neurological damage,central nervous system oxidative damage and DNA injury), Subacute toxicity of acrylamide poisoning characteristics and possible mechanism were also explored,providing the scientific basis for the prevention and treatment of acrylamide poisoning.PartⅠNeurobehavioral toxicity induced by ACR alone and ACR with MT intervention in ratsObjective: To explore the neurobehavioral toxicity induced by ACR alone and ACR with MT intervention in rats.Methods: SD male rats were divided randomly into 4 groups (9 per group):control group, ACR group (40 mg/kg), MT group (5 mg/kg) and MT+ACR group (40 mg/kg ACR+ 5 mg/kg MT ). Control group was treated with equivalent volume of NS, ACR group was intragastric administration with 40 mg/kg ACR solution, MT group was intraperitonealed with 5 mg/kg MT, MT + ACR group was intragastric administration with 40 mg/kg ACR solution and intraperitonealed with 5 mg/kg MT for 12 days.The body weights,gait scores,tail-flick time and expand spacing hindlimb were determined every three days. 24h after the last treatment, rats were decapitated, cerebral cortex, cerebellum and sciatic nerve were separated on ice and preserved in freshly prepared formalin fixative preparing for HE staining.Results: (1) Compared with control group, the body weight of ACR group decreased 9.4% (P <0.05), 12.2% (P <0.01), 16.3% (P <0.01), 23.8% (P <0.01) on the third day, 6th day, 9th days, 12th day.The body weight of MT+ACR group decreased 8.9% (P <0.05) on the 6th day, 8.8%(P <0.05)on the 9th day,15.8%(P <0.01)on the 12th day. Compared with ACR group, the body weight of MT+ACR group increased 8.9% on the 9th day (P <0.05) and growed 10.6% on the 12th day (P <0.05).(2) Compared with control group, the gait score of ACR group and MT+ACR group were significantly increased since the third day(P <0.01). Compared with ACR group, the gait score of MT+ACR group reduced 25.1%(P <0.01)on the 6th day and decreased 20%(P <0.01)on the 9th day. (3) Compared with control group, on the 9th and 12th day, the tail flick time of ACR group reduced 26.4% (P <0.01), 55%(P <0.01), MT + ACR group decreased 26.4%(P <0.01),33.3%(P <0.01). Compared with ACR group, tail flick time of MT + ACR group prolonged 48.1% (P <0.01) on the 12th day. (4) With the exposure time, the distance between hind legs of ACR group was distinctly on the increase, MT + ACR group showed a slight increasing trend. Compared with control group, ACR group were significantly increased (P <0.01) from the 6th day, MT + ACR group began to widen significantly from the 9th day (P <0.01). Compared with ACR group, MT + ACR group shortened limbs spacing 19.6% (P <0.01) on the 6th day. (5) HE staining showed that cerebral cortex and sciatic nerve was normal in each group,but Purkinje cell nuclear condensation was observed in cerebellum of ACR group, No abnormal lesions can be seen in MT + ACR group.Conclusion: ACR sub-acute exposure can lead to neurological damage in rats, such as weight loss, high gait score,wide hind legs spacing,short tail flick time and others, Purkinje cell nuclear condensation was observed in cerebellum from the morphology. MT intervention can alleviate the symptoms of ACR poisoning, neurological and cerebellar structural damage. PartⅡBrain oxidative damage induced by ACR alone and ACR with MT intervention in ratsObjective: To explore brain oxidative damage induced by ACR alone and ACR with MT intervention in rats.Methods: Animal groups and treatments were the same as the first part. Malondialdehyde (MDA), the total superoxide dismutase (T-SOD),glutathione (GSH) and glutathione peroxidase (GSH-Px) in cerebral cortex and cerebellum were assayed by almighty microplate assay.Results:(1)The results in Cerebral cortex: compared with control group, T-SOD activity of ACR group was significantly reduced by 9.94%(P <0.05)and GSH content decreased by 19.90% (P <0.01); Compared with ACR group, T-SOD activity of MT + ACR Group increased by 15.67% (P <0.01).(2)The results in erebellum: compared with control group, MDA content of ACR Group increased by 20.36% (P <0.05),T-SOD activity, GSH content decreased by 18.33% (P <0.05), 20.67% (P <0.01). Compared with ACR group, MDA content of MT + ACR Group decreased 20.51% (P <0.01).Conclusion: ACR sub-acute exposure can cause oxidative damage in rat brain tissue, Lipid peroxidation in cerebellum shows significant change, but not obvious change in cerebral cortex MT simultaneous intervention can prevent oxidative damage in rats exposed to ACR. Oxidative stress may mediate the neurotoxic effect of ACR.PartⅢBrain DNA damage induced by ACR alone and ACR with MT intervention in ratsObjective: To explore brain DNA damage induced by ACR alone and ACR with MT intervention in rats.Methods: Animal groups and treatments were the same as the first part. Cerebral cortex and cerebellum were prepared for single cell suspension, adjusting the cell concentration of 1×106 / ml. DNA damage of cells in cerebral cortex and cerebellum were assayed by single cell gel electrophoresis determination.Results(:1)Comet tail length, tail DNA% and Olive tail moment percentage were not statistically different (P > 0.05) between each group in cerebral cortex. (2)Compared with control group, tail length, tail DNA% and Olive tail moment percentage of ACR group and MT + ACR group in cerebellum were significantly increased (P <0.01); Compared with ACR group, tail length of MT + ACR group reduced by 39.1% (P <0.01), tail DNA% decreased 52.1% (P <0.01), Olive tail moment decreased 56.8% (P <0.01).Conclusion: ACR sub-acute exposure can cause DNA damage in cerebellum cells, but no obvious change in cerebral cortex cells. MT simultaneous intervention can alleviate the DNA damage in cerebellum of rats exposed to ACR. DNA damage may be due to a large amount of free radicals or reactive oxygen species induced by ACR. Lipid peroxidation damage in cerebellum may lead to DNA damage.In summary, ACR sub-acute exposure mainly shows neurobehavioral damage and the structure of cerebellar lesions. Neurological injury is more significant than the structural damage. Biological macromolecules shows cerebellar DNA damage and lipid peroxidation damage, lipid peroxidation damage is not obvious and DNA damage is not observed in the cerebral cortex. MT intervention can alleviate the damages induced by ACR .Oxidative stress may mediate the neurotoxic effect of ACR. Lipid peroxidation damage in cerebellum may be related to structural damage or high free radicals or reactive oxygen species. DNA damage may be due to a large amount of free radicals or reactive oxygen species induced by ACR, and free radicals or reactive oxygen species directly attack DNA molecules. Lipid peroxidation damage in cerebellum may lead to nerve cell membrane permeability transition and DNA damage, the specific mechanism needs further study.

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