| In May 2011, a food scandal which was called“Plastic unfantastic”burst in Taiwan,making the main crime of this event, the chemical substance, di (2-ethylhexyl)phthalate (DEHP), get an unprecedented attention. DEHP is used widely in industry toenhance the plasticity of plastics, and it is mainly used in polyvinyl chloride (PVC)plastic products. The plastics that contain DEHP are used in food wrapper, foodpackage materials, toys and medical devices. In this event, DEHP was added tobeverages by unscrupulous businessmen to substitute the legal food addictive,beverage cloud. This scandal was the most severe food safety event in recent years inTaiwan. Besides, DEHP may migrate to food and water from food package materialsmade with PVC which contains DEHP.There were abundant research referring to the toxicity of DEHP to both animals andhuman body. Except the effects of DEHP to reproductive and developmental systems,there were a mass of experiments implying that DEHP caused toxicity to different celllines through oxidative damage in vitro. Oxidative damage is the consequence ofunbalance of oxidation and anti-oxidation, which is an important mechanism thatinduces apoptosis to tissues and cells.There are high levels of substances related to oxidation, and low levels ofantioxidant enzymes and non-enzymic free radical scavenging agents in human brains,which makes the brains extremely fragile to oxidative damage. Death of neurons inbrain is related to various neurodegenerative diseases. Hippocampus is the mostimportant place of learning and memory. The damage to hippocampal neurons isclosely related to Alzheimer’s disease (AD). This research is focused on the toxiceffect of DEHP to hippocampal neurons, thus to explore its effect to the centralnervous system (CNS).Aim: to research the toxic effect of DEHP to hippocampal neurons and CNS atcellular level, thus to provide data support to safety assessment of DEHP.Methods: Culture the hippocampal neurons from Wistar fetal rats withserum-free culture method. The growing of hippocampal neurons and the effect ofDEHP to morphology of neurons was observed via a converted fluorescencemicroscope. The concentration of DEHP used in this study was figured out and triedaccording to the concentration mentioned in other studies referring to DEHP toxicity in vitro. Effect of DEHP to cellular viability was detected with MTT method. Weobserved the morphological changes of cellular nucleus via Hoechst 33258fluorescent staining, and tested the activity of Caspase-3, the typical index ofapoptosis, to research the effect of DEHP to neuronal apoptosis. At last we tested thelevel of ROS through DCFH-DA fluorescent staining and a laser scanning confocalmicroscope to analyze the fluorescence intensity. We tested the activity of SOD, theantioxidase, and the expression level of GSH to study the oxidative damage of DEHPto hippocampal neurons.Results: Serum-free culture gained hippocampal neurons with higher puritycompared with neurons cultured with fetal bovine serum (FBS). DEHP concentrationused in the experiments was finally settled as 0.25mM, 0.5mM, 1mM, 2mM, 3mMafter trying. DEHP induced morphological change and decreased cellular viability ofhippocampal neurons, and this effect has a dose-effect relationship, with cellularviability decreased to 52% of control when expose to DEHP with a concentration of3mM. After fluorescence staining with Hoechst 33258, nuclei shrinkage and apoptoticbodies were observed, with a significant upregulation of Caspase-3, implyingneuronal apoptosis. DEHP damage resulted in mass production of ROS, decreasing ofSOD activity and lower expression of GSH. DEHP caused toxicity to hippocampalneurons through oxidative stress. |
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