In Vivo Studies On Apoptosis In Gobiocypris Rarus Induced By BaP

Apoptosis is critically important for normal development and homeostasis of the organism, and also occurs as a defense mechanism such as in immune reactions or when cells are damaged by disease or noxious agents. To investigate the carcinogenic mechanism and apoptosis induced by BaP, Chinese rare minnow (Gobiocypris rarus) was exposed to waterbrone BaP for28d at variety concentrations (1.0μg/L、5.0μg/L、20.0μg/L、80.0μg/L). Then the apoptosis and histopathologyical changes of the liver, intestines and gills were studied. Real-time PCR was used to quantitatively determined the mRNA levels of some key genes involved in mitochondria pathway of apoptosis. Major achievements are as follows:1Cloning and sequence analysis of genes involved in mitochondria pathway of apoptosis in Gobiocypris rarusThe partial sequences of bcl-2、apaf-1、caspase-3and caspase-9were cloned and sequenced from the liver of Gobiocypris rasus by RT-PCR. Sequence analysis showed that the caspase-3and caspase-9genes shared the highest sequence affinity with Tanichthys albonubes, while bcl-2and apqf-1genes showed the highest sequence affinity with Gobio gobio and Cyprinus carpio, respectively. Phylogenetic trees constructed based on nucleic acids of other species showed that Gobiocypris rarus has the closest genetic relationship with Tanichthys albonubes and Danio rerio.2Histopathological changes of livers, intestines and gills in Gobiocypris rarus after28d exposure to BaPHE (hematoxylin-eosin staing) method was used to investigate the histopathological changes of livers, intestines and gills. The result revealed that the hisgological structure in liver, intestines and gills of Chinese rare minnow were damaged to some extents. For example, liver cells turgescence, karyopyknosis and karyorrhexis; goblet cell of intestinal mucosa manifolded; epithetial hyperplasia of gills, amalagamation of gill lamellae.3Apoptosis of Gobiocypris rarus induced by BaP after28d exposureTUNEL (TdT-mediated dUTP Nick-End Labeling) method was employed to study the apoptosis of hepatocyte induced by BaP. TUNEL results showed that BaP can induce cell apoptosis in the liver, not in the intestines and gills of Gobiocypris rarus. A dose-related increase in apoptotic index was observed at these concentrations. The apoptotic index at20μg/Land80μg/L was significant different from the control.4Quantitation of some key genes involved in mitochondria pathway of apoptosis in the liver of Gobiocypris rarus by Real-time PCRThe expression of cyp1a1, p53, bax, bcl-2, caspase-9, caspase-3, and apaf-1in the livers of Gobiocypris rarus were determined after28d exposure. The mRNA levels of cyplal, p53, bax, caspase-9genes were up-regulated corresponding to the concentrations of BaP. No significant was observed at the concentration of1.0μg/L (p>0.05), however, the mRNA levels of these genes increased significantly compared with control at the levels of5.0,20.0and80.0μg/L BaP (p<0.05). The mRNA levels of bcl-2increased2.07-fold in the groups compared with control by treating with5.0μg/L BaP (p<0.05), no significance was observed at the concentration of1.0、20.0、80.0μg/L compared with control (p>0.05). On the contrary, the mRNA levels of apaf-1were significantly down-regulated at all concentrations, with decrease of0.46-,0.42-,0.39-and0.47-fold compared with the control group, respectively. However, caspase-3transcription pattern was not significantly influenced after BaP exposure (p>0.05). The results showed that different mechanisms involved in apoptosis following exposure to BaP, rather than exclusively caspases. BaP induced apoptosis via activation of some genes involved in mitochondria pathway, including cyplal, p53, bax and caspase-9.