Neurotoxicity Of Chronic Exposure To Perfluorooctane Sulfonate And Itsmechanisms,in Vitroand In Vivo

As a kind of organic fluorine material, perfluorooctane sulphonate (PFOS) is widely used in clothing, carpets, textiles, upholstery, paper, packaging and cleaning products. Recent studies show PFOS were checked in river, lake and even bottled water. Moreover, measurable levels were detected in the general population in many countries. These show that PFOS is persistent, bioaccumulative and toxic, its biological effects and potential health hazards receives more and more attention and concern. The nervous system is accumulative and sensitive taget for PFOS. However, neurotoxic effects of PFOS and its mechanism are still lack of systematic research. In this study, we aim to evaluate neurotoxicity induced by chronic PFOS exposure and to explore the potential mechanisms using nematode Caenorhabditis elegans, mice and human neuroblastoma cells SH-SY5Y, in vivo and in vitro. It provides new evidences to further reveal neurotoxic effects of PFOS.Wide type N2Caenorhabditis elegans was exposed at different concentrations of PFOS (0.2,2,20and200μ M) for48h from L1stage.The endpoints of growth,locomotor behavior and chemotaxis learning behavior were checked. GFP labelled transgenic C. elegans strains were used to investigate effects of PFOS on relevant neurons. Adult mice were intraperitoneally exposed to PFOS with dosage of0,5,20or40mg/kg for6weeks.We mainly assayed escape lantency in Morris water maze, and the morphology of hippocampal neurons by HE staining. SH-SY5Ycells were exposed with5,25or50μM PFOS for48h. Using MTT methord to estimate Cell viability. And Cell apoptosis was estimated by Annexin-V/PI staining assay. Then reactive oxygen species (ROS) level, maleic dialdehyde (MDA) content, superoxide dismutase (SOD) activity and glutathione peroxidase (GPX) activity were respectively checked.Main results are as follows:(1) It shows the obvious lethal effects of PFOS in C. elegans.Its LC50 values are respectively3.484,2.028and0.842mM for24h,48h and72h exposure. Exposure to sublethal dosage,200μM PFOS, obiviously inhibited growth of nematodes. Compared to the control, body length and body width significantly decreased after PFOS exposure. In addition, the significant decreases of forward movement frequency, body bends and head thrashes were observed in20μM PFOS exposure group, in comparison with the control.(2)20μM PFOS exposure induced a significant increase of chemotaxis index for towards NaCl in comparison with the control, which shows the obvious decrease of learning ability in the exposed nematodes. Assays in GFP labelled transgenic C. elegans strains showed that treatments with PFOS influenced ASE sensory neurons but not the cholinergic neurons or dopamine neurons.(3) After6-week exposed to PFOS, mice showed lesser activities and slower responses in comparison with the control animals. Morris water mazem test showed that the escape latency of20mg/kg PFOS group was significantly raised compared to the control group in the first training day. While, in the first, second and fifth day, the escape latency all significantly delayed after chronic exposure to PFOS in the dose of40mg/kg. It shows that PFOS exposure caused the decline of spatial learning and memory ability. Statistical analysis for morphology in hippocampus showed that declined neuron density and smaller cell body with dark stained nucleus in CA1region of mice at40mg/kg PFOS group, and neuron density declined in CA3region as well.(4) Cell viability and apoptosis experiments showed PFOS caused obvious cytotoxicity in the SH-SY5Y cells. PFOS inhibited cell viability, meanwile,25μM and50μM PFOS induced respectively8.30%and11.23%cell apoptosis. While the antioxidantNAC, rescued cells from PFOS-induced apoptosis via blocking ROS.(5) PFOS induced oxidative stress in SH-SY5Y cells.25μM and50pM PFOS increased ROS production with a level of1.6-fold and2.4-fold of the control, which could be inhibited by antioxidant NAC. PFOS caused significant increase of MDA content and SOD activity in dose dependent manner, but significant decrease of GPX activity.These results indicate:(1) Sublethal doses of PFOS exposure caused the decline of forward movement, body bend and head thrash in nematodes, and motor activity in mice. It indicates that chronic exposure of low dosage PFOS can induce chronic neurotoxicity, which influences locomotor behaviors of animals.(2) PFOS caused defects of chemotaxis learning behavior in nematodes, and the decline of spatial learning and memory in mice. It indicates that chronic exposure to sublethal PFOS affects learning and memory function of animals.(3) We found PFOS exposure significantly affect the function of sensory neurons ASE, while cholinergic neurons and dopamine neurons did not be affected. And selective neuron degeneration of the hippocampus in CA1and CA3region are closely related to PFOS-induced learning and memory disorders in mice. These indicate that neurotoxicity of chronic exposure to PFOS may be associated with selective toxicity on certain neurons.(4) PFOS induced apoptotic death, and increased ROS level and SOD activity in dose-dependent manner, but actually decreased GPX activity. These results suggest that PFOS induced neurotoxicity may closely associated with oxidative stress and cell apoptosis by disturbing the balance of intracellular oxidative and antioxidant system, which mediated cell damages and apoptosis death.For the first time, this study providesthe detailed description about neurotoxic effects of chronic exposure to PFOS, combining with in vivo and in vitro assays. As a persistent environment contaminant, PFOS should be received particular concerns, about its ecologic safety and health risk of chronic exposure to PFOS. Our study suggests that PFOS-induced chronic neurotoxicity including specific nerve cells damage, degeneration and apoptosis, may be associated with degenerative neurological diseases. In addition, cell oxidative damages via disturbing oxidative and antioxidant balance paly an important role in PFOS-induced neurotoxicity. It hints that preventive protection from oxidative damage may reduce neurotoxic effects of PFOS exposure.