Study On The Immunotoxicity Of 8:2 Fluorotelomer Alcohol Both In In Vitro And In Vivo Models

8:2 Fluorotelomer alcohol is a kind of widely used surface material in houseware and industrial goods and is ubiquitous in surrounding environment.Because the production and usage of 8:2 FTOH increased by years,more and more researchers interested in the toxicity of 8:2 FTOH.Till now,8:2 FTOH has been associated with hepatotoxicity,nephrotoxicity,reproductive toxicity and endocrine-disrupting effects.However,the immunotoxicity of 8:2 FTOH need further to be studied.In order to fill blank in this filed,murine macrophage cell line RAW 264.7,C57BL/6 mice and murine primary splenocytes were used as in vitro and in vivo models to study the immunotoxicity of 8:2 FTOH.Firstly,murine macrophage cell line RAW 264.7 and murine primary splenocytes were used as the representations of murine innate immune system and adaptive immune system,respectively,to explore the in vitro effects of 8:2 FTOH to murine innate immune system and adaptive immune system,In murine macrophages-related study,hydralazine hydrochloride(Hyd)was used as a kind of reactive carbonyl species(RCS)scavenger.The immunotoxicity of 8:2 FTOH was explored in cell viability and immune function and its possible mechanism was studied.Experiments in cell viability included MTT cell viability assay,Annexin V-FITC /PI staining for cell apoptosis assay,EdU staining for cell proliferation assay,PI staining for cell cycle assay,DCFH-DA staining for intracellular reactive oxygen species(ROS)and RT-qPCR was used to measure the expression of cell proliferationrelated genes.As for immune function,the expressions of pro-inflammatory cytokinesand antigen-presenting-related genes were evaluated by RT-qPCR,and the concentrations of pro-inflammatory cytokines in cell supernatant were measured by ELISA.And FITC-labeled E.coli bioparticles were used to measure the ability of macrophage phagocytosis.Results obtained in the present study showed that 8:2 FTOH exposure caused decrease of cell viability,decline of cell proliferation ability and G1 phase and G2/M phase arrest in RAW 264.7.At the same time,8:2 FTOH exposure not only downregulated the expressions of pro-inflammatory cytokines-and cell cyclerelated genes,but also disturbed the expression of antigen-presenting-related genes.Although call apoptosis,intercell ROS level and the ability of phagocytosis were not changed by the-treatment of 8:2 FTOH at all concentrations used in this study,cotreatment with Hyd partly neutralized the cytotoxicity of 8:2 FTOH.The results in this part illustrated that 8:2 FTOH can induce cell cycle arrest via inhabiting the expression of cell cycle-related genes,and cause the decrease in the cell viability of RAW 264.7 finally.Hyd can partly neutralized the cytotoxicity of 8:2 FTOH and restored the cell viability of RAW 264.7 by eliminating the cell cycle arrest.On the other hand,8:2 FTOH can disturb the immune function of macrophage via inhabiting the expression of pro-inflammatory cytokines-related genes and disturbing the expression of antigenpresenting-related genes.In primary splenocytes-related study,murine primary splenocytes were stimulated by Concanavalin A(Con A)or lipopolysaccharide(LPS)to evaluate the cell viability by MTT assay and the level of IFN-? in cell supernatant was measured by ELISA.As shown in results,the exposure of 8:2 FTOH inhibited the Con A-or LPS-stimulated proliferation of splenocytes.Furthermore,8:2 FTOH inhibited the level of secreted IFN-? in the cell supernatant of Con A-stimulated splenocytes.The results in this part illustrated that 8:2 FTOH can impair the immune function of murine primary splenocytesSecondly,6-week-old male C57BL/6 mice were used as in vivo model to explore the immunotoxicity of 8:2 FTOH.In this study,8:2 FTOH were set to at the concentrations of 0,10,30 and 100 mg/kg/day,and mice were administered with 8:2 FTOH by gavage for 4 weeks.During the whole experimental period,body weights and food consumptions were recorded once a week.After exposure,sera were obtained to measure the levels of pro-inflammatory cytokines by ELISA and the content of GSH and the activities of SOD and CAT were measured by using respective kits.Thymus,liver and spleen were weighted and observed in histopathology after H&E staining.Moreover,the expression of pro-inflammatory genes was evaluated in tissues by RT-qPCR.The level of GSH,and the activities of SOD and CAT were measured in liver by using respective kits.The results showed that exposure to 8:2 FTOH caused decreases of the contents of IL-1? and GSH as well as the activities of SOD and CAT in sera.Increases in liver weights and histological changes were observed in the liver,but no histological change in either the spleen or the thymus was observed after administration of 8:2 FTOH.8:2 FTOH-treatment disordered the expression pro-inflammatory genes in all tissues,and 8:2 FTOH-treatment also decreased the level of GSH as well as the activities of SOD and CAT in liver.The results in this part illustrated that 8:2 FTOH can cause damage in the immune system of mice and the decreases of antioxidant capacity in sera and liver.In summary,8:2 FTOH posed potential immunotoxicity in the immune system of mice.This study reveals the immunotoxicity of 8:2 FTOH and its possible mechanism,as well as provides a theory about regulating the use of FTOHs.