Effects Of Dichloro-diphenyl-trichloroethane (DDT) On The Oxidization-antioxidization System And The Interventional Effects Of TBHQ

Dichloro-diphenyl-trichloroethane (DDT) is one of the organochlorine pesticides, and it has been widely used as pesticide in China from 1950's to 1980's. It's undoubtedly that DDT caused significant pollution to the environment because of its chemical stable, easy residual property. Although DDT has already been banned in many countries, but the potential adverse effects to human health should not be disregarded because of its characteristics mentioned above. Moreover, researchers owed the increasing incidence of the neurodegenerative diseases to the aggravating environment pollution. For example, the incidence of PD (Parkinson's Disease) showed distinct dose-effect correlation with the sale quantity of insecticides and the distribution of places for lumbering and planting vegetables. By using a case-control study, Semchuk found that the usage of herbicides and insecticides was the risk factor, and there was a dose-response relationship between the incidence of PD and the length of labouring time in the field, but insecticides were found as the only hazardous factor for PD when the confounding risk factors were under control. Cao and his colleges also indicated that the usage of pesticides can be considered as a risk factor for the development of PD.Oxidative stress occupies an important place in the mechanisms of neurotoxicity study. During the past few years, particular attentions have been directed to oxidative stress because it could well interpret the mechanism of delayed neurotoxicity and accumulated neurotoxicity. Now, more and more researches indicate that oxidative stress is associated with many neurodegenerative diseases, such as Parkinson's disease, Huntington's disease, Alzheimer's disease and multiple sclerosis, and it may also play a key role in the process of these diseases. In the past few decades, researches about DDT mainly focused on the liver toxicity,reproductive toxicity and endocrine disruption effects, the study of nervous system was still comparatively rare, a majority of them always centered about electrophysiology experiment in vitro and the Substantial Nigra and Striatum dopaminergic pathways. As for the effects of DDT on the oxidization-antioxidization system of nerve tissue, little is know about it. Therefore, the current study select the cerebral cortex, hippocampus and cerebellum of Sprague-Dawley male rats as subjects investigated and study the associated index of the oxidization-antioxidization system. In additon, we also studied the effects of DDT on oxidization-antioxidization system in PC12 cells and employed tBHQ to observe its antagonistic effects on oxidative stress induced by DDT, so as to provide new insights for the prevention and therapy in DDT poisoning.PartⅠOxidative Stress induced by DDT in different brain tissues Objective: To study the oxidative stress induced by DDT in nerve tissue. Methods: Sprague-Dawley male rats were divided into four groups including solvent control groups and three DDT-treated groups (20,40,80mg/kg). Test substances were administered intragastric and rats were sacrificed 7 days later after treatment. Malonyldiadehyde (MDA), total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-PX), glutathione reductase (GR) and glutathione-S- transferases (GST) in cerebral cortex, hippocampus and cerebellum were examined. Results: Compared with the solvent control:①With the dose increaseing, the content of MDA in cerebral cortex , hippocampus and cerebellum significantly increased while T-SOD activity decreased significantly.②GSH-PX activity of cerebral cortex significantly increased at low dose group,but significantly decreased at moderate dose and high dose group (P <0.05). In hippocampus and cerebellum, With the dose increaseing, GSH-PX activity decreased significantly.③GR activity of hippocampus decreased significantly at moderate dose and high dose groups (P<0.05). In cerebellum, it only decreased at high dose group (P <0.05). In cerebral cortex,the alteration of GR activity at different dose groups was not observed.④GST activity of cerebral cortex significantly increased only at low,moderate dose group. In cerebellum, it significantly increased at moderate dose group but decreased at high dose groups (P<0.05). In hippocampus, it significantly decreased at all dose groups (P<0.05).Conclusions: DDT can induce oxidative stress in dose-response manner in nerve tissue. The tissue oxidative damage might be one of the primary mechanisms of neurotoxicity of DDT.PartⅡEffects of DDT on the ROS levels in different nerve tissueObjective: On the basis of known oxidative damage induced by DDT, investigate the effects of DDT on ROS levels in the cerebral cortex,hippocampus and cerebellum of rats so as to understand the production mechanism of ROS.Methods: Sprague-Dawley male rats were divided into four groups including solvent control groups and three DDT-treated groups (20,40,80mg/kg). Test substances were administered intragastric and rats were sacrificed 7 days later after treatment. ROS levels were examined by fluorophotometry. Results: Compared with the solvent control, in cerebral cortex, the levels of ROS significantly increased in all the DDT groups, levels of ROS in 80mg/kg group singnificantly increased 37% when compared with the 40mg/kg group, but it had no significant difference when compared with the 20mg/kg group. In hippocampus, with the dose increaseing, the ROS levels in all the dose groups showed an increasing tendency, but only in 80mg/kg group, it showed significant difference when compared with the control and the 20mg/kg group. In cerebellum, the levels of ROS significantly increased in all the DDT groups when compared with the solvent control, but there was no significant difference among the three dose groups.Conclusions: DDT can induce the ROS levels increase in cerebral cortex,hippocampus and cerebellum of rats, but an obvious dose-response relationship is not observed. Furthermore, this increase differed from the clinical symptoms. It is suggested that the increasing ROS is not determinative in the pathogenesis of DDT, the increasing ROS may contribute to mediate certain signal transduction pathway.PartⅢEffects of DDT on the ROS levels in vitro of PC12 cell and the antagonistic effects of antioxidant tBHQ Objective: To investigate the effects of DDT on ROS levels in PC12 cells and the antagonistic effects of tBHQ.Methods: 40μmol/L tBHQ was pre-incubated with PC12 cells for 16h. Cells were divided into 6 groups: control group,1×10-9 mg/ml DDT group,1×10-8 mg/ml DDT group,tBHQ group,tBHQ plus 1×10-9 mg/ml DDT group and tBHQ plus 1×10-8 mg/ml DDT group. After the administration of DDT for 2h,6h,12h and 24h, fluorophotometry was used to examined the ROS levels in PC12 cells.Results: Both the two DDT treatment group significantly increased the ROS levels in PC12 cells at each time point, but the dose-response relationship was not obvious. In the time-response relationship of DDT treatment group, ROS levels decreased firstly and then increased with the passage of time in 1×10-9 mg/ml DDT group, but this time-response relationship is not remarkable in 1×10-8 mg/ml DDT group. Besides, the tBHQ pretreatment group does not influence the ROS levels in PC12 cells, DDT treatment cooperated with tBHQ significantly decreased the ROS levels in PC12 cells when compared with the DDT group.Conclusions: DDT can induce the increase of ROS levels in PC12 cells and this result is in conformity with the experiments in vivo, tBHQ can efficiently antagonise the toxic effects of DDT at gived dosage in this experiment.PartⅣEffects of DDT on the oxidization-antioxidization system invitro of PC12 cell and the antagonistic effects of antioxidant tBHQ Objective: To investigate the effects of DDT on the oxidization-antioxidization system in vitro of PC12 cell line and the antagonistic effects of tBHQ.Methods: 40μmol/L tBHQ was pre-incubated with PC12 cells for 16h. Cells were divided into 6 groups: control group,1×10-9 mg/ml DDT group,1×10-8 mg/ml DDT group,tBHQ group,tBHQ plus 1×10-9 mg/ml DDT group and tBHQ plus 1×10-8 mg/ml DDT group. After the administration of DDT for 2h,6h,12h and 24h, spectrophotometry was used to examined the MDA,T-SOD,GSH-PX,GR and GST, the content of GSH andγ-GC was detected by HPLC.Results: In vitro, tBHQ could noticeably antagonise the oxidative stress induced by DDT to some extend; little influene on SOD induced by tBHQ was observed; Among those antioxidases induced by tBHQ, the elevation of GR activity occurred before the phaseⅡdetoxification enzyme. Conclusions: The antagonistic effects of tBHQ is in time-dependent and dose-dependent manner, over time or dose will weaken the effects. It is only working with SOD that the GSH dependent antioxidization system can efficiently scavenge the free radicals and transport the xenobiotic compounds outside the cells. The rapid elevation of GR activity may be an adaptive response mechanism to the external stimuli which universally exists in nature.