Study On The Developmental Toxicity Induced By Exposure Of Hexafluoropropylene Oxide Dimer(HFPO-DA)in Chicken Embryos

Objective: The aims of this study were to investigate potential developmental toxicities following exposure to hexafluoropropylene oxide dimer(HFPO-DA)in chicken embryos,to determine the toxic doses of HFPO-DA,and to explore the underlying molecular mechanisms.So as to further supplement the toxicological information of HFPO-DA and to provide new evidences for health risk assessment of population exposed to HFPO-DA.Methods: Fertilized eggs(embryonic day zero,ED0)were evenly allocated to six treatment groups according to egg weight: vehicle control group(sunflower oil),positive control group(2 mg/kg egg weight PFOA),1,2,4 and 8 mg/kg egg weight HFPO-DA.Prior to incubation(Embryonic day zero,ED0),the eggs were exposed to PFOA or HFPO-DA via air cell injection and then placed in the incubator.After hatching,the hatchling chickens were anesthetized,subjected to electrocardiography,and sacrificed.Serum,heart and liver tissue samples were collected.The tissue samples were then utilized to investigate potential developmental cardiotoxicity and hepatotoxicity induced by HFPO-DA exposure.The serum concentrations of HFPO-DA in developmentally exposed hatchling chickens were determined with quadrupole time of flight liquid chromatograph-mass spectrometer(QTOF LC/MS).Electrocardiography results were analyzed for cardiac functional changes evaluation.Hematoxylin& eosin staining(HE staining)and oil red O staining were conducted on paraffin sections of heart tissues and frozen sections of liver tissues respectively to assess the pathological changes induced by HFPO-DA exposure.After demonstration of HFPO-DA exposure induced developmental toxicities in chicken embryos,in ovo gene silencing via lentivirus(Lentivirus transfection)was conducted to investigate the role of peroxisome proliferator-activated receptor alpha(PPARα)in the observed effects.The fertilized eggs were evenly divided into five treatment groups by egg weight: vehicle control group(C),control lentivirus group(CV),PPARα silencing lentivirus group(PV),HFPO-DA exposure group(H4)and HFPO-DA exposure + PPARα silencing lentivirus co-treatment group(H4PV).On ED0,4 mg/kg egg weight HFPO-DA was air-cell injected to H4 and H4 PV group eggs,while other groups received sunflower oil(vehicle control),then the incubation was initiated.On ED2,PPARα silencing lentivirus was microinjected into PV and H4 PV group embryos,control lentivirus was injected into CV group embryos,and sterile saline was injected into embryos in other groups,and then embryos were put back to incubators and allowed to hatch.The hatchling chickens were anesthetized,subjected to echocardiography,and sacrificed.Serum,heart and liver tissue samples were collected.Western blotting was performed to confirm the silencing efficacy of PPARα silencing lentivirus in the heart and liver tissues of hatchling chickens.The cardiac functional changes were evaluated by analyzing the electrocardiography data,and the cardiac and hepatic morphological changes were assessed with HE and oil red O staining,respectively.Western blotting was used to detect the protein expression levels of cluster of differentiation 36(CD36)and enoyl-Co A hydratase and 3-hydroxyacyl Co A dehydrogenase(EHHADH),downstream target genes of PPARα,in heart and liver tissues of hatchling chickens.Results:On ED0,chicken embryos were exposed to 0,1,2,4 and 8 mg/kg egg weight HFPODA,resulting in hatchling serum concentrations of 0,35.7,131.53,242.45 and 242.75 ng/m L,respectively,as indicated by Q-TOF LC/MS.At lower doses(1 and 2 mg/kg egg weight)HFPO-DA,no significant changes in cardiac function,cardiac morphology or liver morphology were observed.On the other hand,higher dose HFPO-DA exposure(4 and 8 mg/kg egg weight)induced developmental cardiotoxicity in chicken embryos,mainly manifested as increased heart rate and thinner right ventricular wall.At the same time,developmental hepatotoxicity of chicken embryo,mainly manifested by the significant increase of lipid droplets deposition(steatosis)in the liver tissue.These effects are consistent with the known toxicity following exposure to 2 mg/kg egg weight PFOA in chicken embryos.The PPARα gene was silenced effectively by in ovo gene silencing via lentivirus transfection.Western blotting results indicated that the silencing efficiency of PPARα was 52% in the chicken heart tissues,and 40% in the chicken liver tissues.PPARα silencing reversed the significant thinning of right ventricular wall induced by HFPO-DA exposure at 4 mg/kg egg weight,and effectively alleviated the increase in heart rate and increase of lipid droplets in liver tissue,but the alleviating effect did not fully abolish the toxicological effects.These results suggest that PPARα plays an important role in the development of cardiac and liver toxicity induced by HFPO-DA exposure in chicken embryos,but is not the sole target of HFPO-DA.In addition,protein expression levels of CD36 and EHHADH,downstream genes of PPARα,were significantly increased in HFPO-DA exposed chicken heart tissues,and decreased to normal levels after PPARα silencing,suggesting roles of PPARα-regulated fatty acid metabolism-related and peroxisome proliferator-related genes in HFPO-DA-induced developmental cardiotoxicity.However,only CD36 protein expression was significantly increased in liver tissue,while EHHADH protein expression was not significantly changed,suggesting partially different molecular mechanisms between HFPO-DA-induced developmental cardiotoxicity and hepatotoxicity.Conclusion:(1)Exposure to HFPO-DA during chicken embryo development induced developmental cardiotoxicity in the form of increased heart rate,thinning of right ventricular wall,and developmental hepatotoxicity mainly manifested by increased steatosis.(2)The same dose of HFPO-DA and PFOA exposure resulted in a large difference in serum concentration,suggesting differences in the toxicokinetics of HFPO-DA and PFOA.Comparing to PFOA,HFPO-DA seems to have less biological accumulation in chicken embryo.(3)The toxic effects of HFPO-DA exposure on the developmental heart and liver were similar to those of PFOA,but to a lesser extent comparing to PFOA.(4)PPARα still plays an important role in the developmental cardiotoxicity and hepatotoxicity induced by HFPO-DA exposure,and is related to its regulation of fatty acid metabolism-related and peroxisome proliferator-related genes,which is similar to that of PFOA.