| Objective:To study the effects of oxidative stress and antioxidative stress in neuron damgae indunced by BDE-153, and to provide a scientific basis for the possible mechanism of developmental neurotoxicity of BDE-153. Methods:Part I:(1)In vivo, male puppy Sprague Dawley rats at postnatal day 10(PND 10) were randomly divided into four groups depended on their litters and weights, 10 puppy rats per group. Puppy rats were administrated with different concentrtions of BDE-153 solution(olive oil, 1 mg/kg, 5 mg/kg and 10 mg/kg BDE-153) by intraperitoneal injection once based on 10 ml/kg body weight. After two months, hippocampus mophorlogy and ultrastructure were observed under optical and electronic microscope, nerve cell apoptosis rate was assayed by TUNEL staining and flow cytometry, and lactate dehydrogenase leakage rate was detected by spectrophotometry.(2)In vitro, primary rat neurons were cultured and identified using neuron specific cytoskeletal protein β-tubulin Ш. Comparable well-grown confluent neurons out of the same batch were classified and treated with medium(blank control), DMSO, 10, 20 and 40 μM BDE-153 for 48 h. Neurons’ morphological changes were observed under an inverted optical microscope. The cell viability was assayed by CCK-8 kit and the LDH activity was detected by spectrophotometry. Using Hoechst 33258 staining, AO/EB double staining and flow cytometry to detect nerve cell apoptosis.Part II: The treatment of animal and cell were mentioned above(See Part I). AfterBDE-153 treatment, ROS were detected with flow cytometry, and MDA, NO and NOS were detected using the special kits. n NOS1 m RNA and protein expressions were detected using real-time quantitative PCR, immunofluorescence, Weston Blot, and Enzyme-linked immuno sorbent assay(ELISA).Part III: The treatment of animal and cell were mentioned above(See Part II). After treated with BDE-153, SOD activity and GSH content were measured by the special kits. Prx1 and Prx2 m RNA and protein levels were assayed using real-time quantitative PCR, immunofluorescence, Weston Blot, and ELISA. Results:Part I:(1)Results in vivo, in the controls’ hippocampus CA1 region, under an optical microscope, the controls’ hippocampus cells arrayed structurally with tight cell-connections and had regular shapes and numbers. However, with increasing concentrations of BDE-153, the cells were observed to be in disarray and a reduction in the number was noted. In addition, the karyoplasmic gap had widened. Under a transmission electron microscope, a large and round nucleus with evenly distributed chromatin inside was located in the center and surrounded by abundant organelles, including the endoplasmic reticulum(ER), ribosome, and mitochondria. In the treated groups, the nucleus was smaller and was observed to be slightly broken; the chromatin condensed and gathered to the nuclear membrane. The organelles, in particular the endoplasmic reticulum, were swollen, dissolved, had vacuolar degeneration, and in some cases even disappeared.With the increasing BDE-153 dose, the LDH activity was gradually increased the proportion of TUNEL positive cells was significantly increased, and IHS scores were significantly increased in the 1, 5, and 10 mg/kg BDE-153 groups(P < 0.05). Further, the cell apoptosis rates detected by flow cytometry were significantly increased in the 5 and 10 mg/kg BDE-153 groups(P < 0.05).(2)Results in vitro, after treated with BDE-153, the nerve cells viability were gradually decreased and the LDH leakage rates were increased. In addition, neurons’ morphology was changed, as BDE-153 dose increased, there was a reduction in neurons and they tended to cluster, there were fewer and looser cell-connections, and the cell body was smaller with reduced and shortened axons and dendrites. Hoechst 33258 staining, AO/EB double staining and flow cytometry detection showed that with the BDE-153 dose, cell apoptosis rates were increased; Compared to the controls, the apoptosis rates of 20 and 40 μM BDE-153 groups were significantly increased(P < 0.05).Part II:(1)Results in vivo: with the increasing BDE-153 dose, the contents of lipid peroxidation products MDA and ROS were increased, Compared with the control group, the MDA contents in 1, 5 and 10 mg/kg BDE-153 groups were significantly increased and the effects were dose dependent(P < 0.01); The content of ROS in the 10 mg/kg BDE-153 group was significantly higher than the controls(P < 0.01). In addition, the level of t NOS in 1, 5 and 10 mg/kg BDE-153 groups were significantly higher than the controls(P < 0.01); The level of NO in the 10 mg/kg BDE-153 group was significantly higher than the control and 1 mg/kg BDE-153 groups(P < 0.01); The expression levels of n NOS1 m RNA and protein in hippocampal cells were gradually increased; The percentage of n NOS1 positive cells and immunohistochemistry(IHS) scores were both increased, and the 10 mg/kg BDE-153 groups were significantly higher than the controls(P < 0.01);The expression levels of n NOS1 m RNA and protein in the 5 and 10 mg/kg BDE-153 groups were significantly higher than the controls(P < 0.01).(2)Results in vitro, compared with the control group, the contents of MDA in the 10, 20 and 40 μM BDE-153 groups were significantly increased(P < 0.01). With the BDE-153 dose, the content of NO and the activity of n NOS in the neurons were gradually increased. Compared to the control group, the NO contents in the 20 and 40 μM BDE-153 groups were significantly increased(P < 0.01); And the n NOS1 activities in the DMSO group and the treated groups were significantly increased(P < 0.01). Moreover, the ROS contents of DMSO, 10, 20 and 40 μM BDE-153 groups were significantly increased compared to the controls(P < 0.05).Part III:(1)Results in vivo,with the increasing BDE-153 dose, the contents of antioxidant substances SOD and GSH were reduced. Compared to the controls, the SOD activities of 1, 5 and 10 mg/kg BDE-153 groups were significantly decreased(P < 0.01); But the GSH contents of those groups were significantly increased(P < 0.01). Moreover, with the BDE-153 dose, the Prx1 and Prx2 mRNA and protein expressions were significantly decreased; The Prx1 m RNA and protein expressions in 1, 5 and 10 mg/kg BDE-153 groups were significantly decreased compared to the controls(P < 0.01 or P < 0.05); The Prx2 m RNA expressions in the 5 and 10 mg/kg BDE-153 groups were significantly lower than control groups(P < 0.01).(2)Results in vitro, compared to the controls, the SOD activity and GSH content in treated groups were significantly decreased, and had a dose-response relationship. We also assayed the expressions of Prx1 and Prx2 m RNA and protein, we found that the expression levels were gradually decreased with the increasing BDE-153 dose. The hippocampal m RNA and protein of Prx1 and Prx2 in the 10, 20 and 40 μM BDE-153 groups were significantly decreased compared to the controls(P < 0.05).Conclusions:(1) BDE-153 induced neuron damage, showing decreased cell vitality, increased LDH leakage and cell apoptosis.(2) The increased oxidative stress and decreased antioxidative stress is probably one of the main mechanism of neuron damage induced by BDE-153. |
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