學生姓名:
黃育琳
指導教授:
黃登福
學期:
106上
摘 要:
Since the cyanotoxin saxitoxin (STX) is a neurotoxin and induces ecological changes in aquatic environments, a potential risk to public and environmental health exists. However, data on STX-mediated cytotoxic and genotoxic effects are still scare. The aim of this study is to establish a protocol for isolation and primary culture of neurons from tropical freshwater fish species Hoplias malabaricus for assessment of the effects of STX. Moreover, the cytotoxic and genotoxic potential of STX was examined in two mammalian cell lines. Cells from brain of H. malabaricus was treated with papain for tissue dissociation, and basic fibroblast growth factor (bFGF) supplement nutrition media was added. The dissociated cells were plated with medium and different STX concentrations and the toxic cellular effects such as oxidative stress, neurotoxicity, and genotoxicity were evaluated. Neuro 2A (N2A), a neuroblastoma mouse cell line, and Vero cell line, derived from Vero green monkey kidney cells, were exposed to several concentrations of STX ranging from 0.5 to 64 nM to determine cell viability, induction of apoptosis (DNA fragmentation assay), and formation of micronuclei (cytokinesis-block micronucleus assay; CBMN) following 24 h of incubation. Cultures treated with bFGF showed the greatest adherence, survival and cellular development. STX increased specific activity of glutathione peroxidase and lipoperoxidation levels, which were cytotoxic and genotoxic indicated by the comet assay. The half maximal effective concentration (EC50) values for STX calculated in cell viability tests were 1.01 nM for N2A and 0.82 nM for Vero cells. With increasing STX concentration there was evidence indicating apoptosis induction in Vero cells with a 50% increase in DNA fragmentation compared to control at the highest STX concentration tested (3 nM). The results demonstrated no significant changes in the frequency of micronucleated binucleated cells in N2A and Vero cells exposed to STX, indicating the absence of genotoxicity under these test conditions. In conclusion, the established primary neuronal culture protocol was successfully applied to study in vitro neurotoxicology. There was no apparent cellular necrosis as evidenced by a lack of formation of multinucleated cells. STX causes cytotoxicity and produced death of both cell types tested through an apoptotic process.
10601 seminar abstract.docx