| Part 1:Prenatal PFOS Exposure Induces Oxidative Stress and Apoptosis in the Lung of Rat Off-springObjective:The main purpose of this study was to investigate the ability of prenatal PFOS exposure to induce oxidation and apoptosis in rat offspring lungs.Methods:Pregnant rats were dosed orally with PFOS (0,0.1 and 2.0 mg/kg/day) from gestation days (GD) 1 to 21. Lung samples from postnatal day (PND) 0 and 21 pups were analyzed for the toxic effects of PFOS. The content of PFOS in serum and lung was analyzed using LC/MS/MS, H&E staining, TUNEL, Real time PCR and western blot were used to detect the histopathological changes, cell apoptosis, the expression of proapoptotic and antiapoptotic molecules mRNA, respectively. In addition, the release of cytochrome c (Cyt c) from mitochondria to cytoplasm, the activities of caspase 3,8,9 and SOD, levels of MDA, GSH were also detected.Results:The concentrations of PFOS in sera and lung were also increased in a dose-dependent manner. Compared to the controls, significant differences in body weight from PND 0 to PND 21and postnatal mortality within PND 3 were observed in the high dose group. It was shown that dams that received 2.0 mg/kg/day caused sever histopathological changes along with marked oxidative injuries and increased cell apoptosis in neonatal lungs, and further examinations showed that the ratio of Bax to Bcl-2, release of cyt c from mitochondria, expressions of proapoptotic genes (Fas and Fas-L), and activities of caspase-3,-8 and -9 were up-regulated correspondingly. There was no significant histopathological change and cell apoptosis in neonatal lungs at the low dose group, even though the level of MDA and bax mRNA of PND 0 lungs, and the expression of Fas mRNA of PND 21 lungs, were increased slightly.Conclution:In summary, the present study demonstrated that prenatal PFOS exposure could overwhelm the homeostasis of antioxidative systems to result in oxidative stress and activate both caspase-dependent death pathways in rat offspring lungs.Part 2:PFOS Induced Apoptosis via ROS-Mediated Mitochondrial Dysfunction in A549 CellsObjective:The main purpose of this study was to investigate the direct effects of PFOS on A549 lung epithelial cells, since the study in part 1 could not distinguish whether the pulmonary toxicity induced by prenatal PFOS exposure represented direct lung toxic mechanisms as any of the innumerable possibilities from cryptic and overt systemic toxicity.Methods:After A549 cells were exposed to various concentration of PFOS (0-200μM), MTT, flow cytometry, DCFH-DA, and JC-1 were used to detect cell viability, cell apoptosis rate, ROS level and mitochondrial membrane potential (MMP), respectively. Levels of MDA and GSH, activities of SOD, caspase 3,8, and 9 were also evaluated. In addition, we further investigated the protective effects of NAC against PFOS-induced cell apoptosis, and the different responses of A549 to PFOS under cultures supplemented with different concentration sera. Results:Compared to controls, after 24 h,48 h or 72 h of PFOS exposure, the cell viabilities were clearly decreased in concentration and time-dependent manners. Furthermore, cell apoptosis rate, levels of ROS, MDA, activities of SOD, caspase 3 and 9, were increased, while levels of GSH and MMP were inclined significantly, after 24 h of PFOS exposure. Pre-treatment with NAC for 2 h obviously suppressed PFOS-induced increase of intracellular ROS levels, cell viability loss and apoptosis. In addition, it was also found that the addition of sera protected the cells from the detrimental effects of PFOS.Conclusion:PFOS has the obvious toxic effects on A549 cells, and the intrinsic cell death pathway, activated by ROS-mediated mitochondrial dysfunction, takes part in the PFOS-induced A549 cell apoptosis. In light of the protective effect of sera, the fact that the fetus and newborn possess lower content of these proteins suggested that greater lung toxicity may occur at blood levels of PFOS that are nontoxic to adults.
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