Effectsofmethanolonmorphology，viabilityandapoptosis-related genes in cells of arterial bulb and brain cell lines system of Lateolabrax maculatus

【Objective】To investigate the effects of methanol on the morphology and viability on different cell lines of spotted sea bass（Lateolabrax maculatus）at the cellular level，and to lay the foundation for revealing the toxicological effects of microplastics（methanol as solvent）on fish cells.【Method】In vitro，different volume fractions of methanol （1%，3%，5%，7%，9%，11%）were used to treat the cells of arterial bulb and brain cell lines of L. maculatus，and the changes of cell morphology were observed by inverted microscopy，cell viability was detected by MTT，and apoptosisrelated genes like cysteine aspartate protease 3（Caspase3），cysteine aspartate protease 9（Caspase9），Bcl2-associated X protein（Bax）and B-cell lymphoma-2（Bcl-2）were detected by real-time fluoresceence quantitative PCR，and Caspase3 activity was detected by Caspase3 activity assay kit. 【Result】When the volume fraction of methanol was more than 7%， the cells of arterial bulb cell line of L. maculatus became rounded and shrunk，lost the ability to adhere to the wall，and appeared dead cells；the cell viability showed a significant decrease（P<0.05，the same below），the relative expression of Caspase3，Caspase9，Bax and Bcl-2 genes were significantly up-regulated，and Caspase3 activity was highly significantly increased（P<0.01，the same below）. When the volume fraction of methanol was more than 1%，there were gaps between the cells of the brain cell line and the cells tended to retract，the number of adherent cells was greatly reduced，and there were a large number of suspended dead cells；cell viability was significantly reduced，and the relative expression of apoptosisrelated genes were significantly up-regulated，and their relative expression tended to increase with the increase of methanol volume fraction；the effect of methanol on the caspase3 activity of the cells of the brain cell line was concentrationdependent. The caspase3 activity increased with the increase of methanol treatment，and it was highly significantly increased by 5%- 11% methanol treatment.【Conclusion】Methanol significantly up-regulats the expression levels of Caspase3，Caspase9，Bax and Bcl-2 genes and increases Caspase3 activity in cells of arterial bulb and brain cell lines of L. maculatus，indicating that methanol has different toxicity levels to different cell types，and it is especially toxic to brain cells. Therefore，different safe concentrations of methanol should be formulated according to different kinds of cells when choosing methanol as a solvent for microplastics.