PCB153 And PCB95 In Combined Exposure Modulate PC12 Cells As Defined By Targeted Lipidomics Analysis

As widespread persistent organic pollutants,polychlorinated biphenyls(PCBs)can cause the damage to biological reproductive system,nervous system and endocrine system et al.on different degrees.These effects are gradually enriched through the food chain,posing a huge threat to the health of humans.The PCBs in the environment tend not to exist in a single form,but in a combined exposure.Therefore,it is of great practical significance to study the combined toxic effects of different PCBs.With the advances of science and technology level,lipidomics arises and provides a new technological thought and method for a more extensive study of toxicity mechanism.The focus of future toxicity tests will shift from overall animal testing to testing strategies based on alternatives such as cells.In this paper,PC12 cells were used to carry out the exposure experiments in single and combined forms of PCB153 and PCB95 and were analyzed by lipidomics in revealing their effects on the endogenous glycerophospholipids.The neurotoxicity induced by PCB153 and PCB95 were discussed at the cellular level.This study first established a detection method of 26 glycerophospholipids in HepG2 cells and PC12 cells.Among these glycerophospholipids,22 are endogenous lipids for these two kind cells.The average recoveries of 4 ectogenic glycerophospholipids in PC12 cells ranged from 73.7%-117.2% with the spiked concentraction of 25 μg/L-1000 μg/L,which indicated the effectiveness of the method.PC12 cells were used for reproducibility test(n=12),and the relative standard deviation(RSD)for each compound was less than 20%,indicating the stability of the method.This method provides a technical basis not only for the detection of glycerophospholipids in cells but also for the subsequent targeted lipidomic studies.This study conducted the cell viability experiments at the dosages of 0.14 μM,1.39 μM and 55.40 μM for PCB153 as well as 0.1 μM,2 μM and 10 μM for PCB95 for 120 h.The cell viabilities ranged from 46.86%-150.74% after the exposure of PCB153 and 64.43%-100.21% of PCB95.The lipidomics results showed that,there were similar increase trends in low and high dosage groups of PCB153 at 120 h,while the trend was opposite in the medium dosage group.When exposed to PCB95 for 120 h,the low and medium dosage groups presented a similar variation trend while the high dosage group changed more dramatically.There were 2,2 and 7 glycerophospholipids up regulated significantly in the low,medium and high dosage group.According to the principles of VIP>1 and t-test P value<0.05,5 potential biomarkers were confirmed after exposure of PCB153 and they were PC(14:0/14:0),PE(16:0/18:1),PE(16:0/18:2),PS(18:0/18:1)and PI(16:0/18:1)respectively.Only PC(14:0/14:0)could be confirmed as a potential biomarker after exposure of PCB95.Based on the high dosages of PCB153 and PCB95,the cell viability experiments in single and combined exposure were conducted for 24 h.It was found that the cell viability in combined exposure group was higher than that in PCB153 group but lower than PCB95 group.The range of glycerophospholipids in the combined exposure group was more extensive.It was analyzed that PC(14:0/14:0)and PS(18:0/18:1)could be confirmed as potential biomarkers after the single and combined exposure.In addition,this study established a screening method based on the fragments of the two fatty acid chains in glycerophospholipids and the parent ions to integrate the MRM information in order to screen unknown glycerophospholipids in PC12 cells.The method screened 62 kinds of glycerophospholipids in PC12 cells primarily.