Roles Of P25/CDKk5-p53Signaling Pathway In BaP-induced Neuronal Apoptosis

[Objective] To investigate the role of p25/CDK5-p53signaling pathway in BaP-induced cortical neuronal apoptosis.[Methods]1. The neurons from cerebral cortex of0-3day’s neonatal rats were incubated for5days at a density of1000cells/mm. In the next experiments, the primary cortical neuronal cells were exposed to different concentrations of BaP (0,10,20,40μmol/L) for different time (Oh,6h,12h,24h,48h).2.①Neuronal apoptosis rates were detected by neuronal cell morphological changes and flow cytometry. Neuronal cell viability was detected by MTT assay.②DNA damage was evaluated by the Olive Tail Moments (OTM) and the BPDE-DNA adducts in neurons.③The levels of Reactive oxygen species were detected by flow cytometry.④The levels of protein of the p25/CDK5pathways expression were detected by Western-blot.⑤The levels of mRNA expression of the p25/CDK5pathways were detected by RT-PCR.[Results]1. After treated with BaP for48h, the viability of cells exposed to BaP decreased13.9%,19.0%and22.5%with the increased dose of BaP at10,20,40μmol/L, respectively. With increase of BaP dose and exposure time, cell processes retracted and intercellular junction reduced; many nuclei appeared shrunken with irregular shapes and sizes, which represent hyper-condensed and extensive chromatin fragment. AO-EB staining showed that the normal neuronal cells displayed uniformly green stained and were alive. After BaP treatment, the neurons appeared to be orange colored apoptotic cells. Factorial design analysis of variance shows that neuronal cells apoptosis rate was dependent on the dose and time of BaP exposure, and there was an interaction between the dose and time (P<0.001). After treated with BaP for48h, compared with the blank control group, neuronal apoptosis rates were increased0.66-,1.90-and2.14-times in the low, medium and high dose groups respectively.2. The OTM increased significantly in a dose-and time-dependent manner compared with the blank control group; and there was an interaction between the dose and time (P<0.001). After treated with BaP for48h, compared with the blank control group, the OTM was increased0.33-,0.77-and1.68-times in the low, medium and high dose groups respectively. The quantity of BPDE adducts was dependent on the dose and time of BaP exposure, and there was an interaction between the dose and time (P0.001). After treated with BaP for48h, compared with the blank control group, the quantity of BPDE adducts were increased1.02-,1.27-and1.59-times in the low, medium and high dose groups respectively.3. The reactive oxygen species was dependent on the dose and time of BaP exposure, and there was an interaction between the dose and time (P<0.001). After treated with BaP48h, compared with the blank control group, the reactive oxygen species were increased0.75-,0.97-and1.64-times in the low, medium and high dose groups respectively.4. The levels of α-spectrin, p35/25, CDK5, p53and p-p53Ser15significantly increased in a dose-and time-dependent manner compared with the blank control group, and there was an interaction between the does and time (P<0.001). After treated with BaP for48h, compared with the blank control group, the levels of a-spectrin, p35/25, CDK5, p53and p-p53Ser15were increased1.71-,1.95-and2.40-times,0.94-,1.19-and1.69-times,0.91-,2.00-and3.03-times,1.40-,1.78-and3.10-times,0.45-,0.51-and0.73-times,1.25-,1.36-, and1.96-times in the low, medium and high dose groups respectively.5. Neuronal apoptosis rates, the protein expression patterns of a-spectrin, p35/25, CDK5, p53and p53Ser15all decreased compared with the cells without cotreatment with MDL28170, and with those treated with BaP for48h, they decreased0.47-,0.68-and0.71-fold,0.46-,0.47-and0.50-fold,0.42-,0.47-and0.48-fold,0.54-,0.57-and0.70-fold,0.29-,0.34-and0.37-fold,0.48-,0.45-and0.53-fold, respectively.6. There was a down-regulation of apoptosis rates, p53and p53Ser15in cultures cotreated with the CDK5inhibitor roscovitine. And there was no significant difference at48h after exposure in cells exposed to different concentrations of BaP, and after treated with BaP for48h, they decreased0.37-,0.42-and0.47-fold,0.37-,0.42-and0.60-fold;0.38-,0.30-and0.37-fold;0.53-,0.50-and0.58-fold.7. After treatment with BaP for48h, the levels of calpain, p35, CDK5mRNA expression increased in a dose dependent manner compared with the blank control group of BaP exposure, and increased8.02-,2.96-,1.94-fold, respectively.[Conclusion]1. BaP could induce oxidative stress and DNA damage of neural cells in a dose-and time-dependent manner.2. BaP could induce neural cells apoptosis in a dose-and time-dependent manner. As far as time series, BaP might result in cell apoptosis in the way of DNA damage and oxidative stress.3. p25/CDK5-p53signaling pathway may be an important factor evoked by DNA damage and oxidative stress in BaP-induced neuronal apoptosis.