The Mechanisms Of Embryonic Developmental Toxicity Induced By Perfluorooctane Sulfonate

Perfluorinated compounds (PFCs) represent a large group of chemicals which are characterized by a fully fluorinated hydrophobic linear carbon chain attached to various hydrophilic heads. PFCs are widely used for many industrial purposes and consumer-related applications. This is due to their unique chemical thermal stability, low surface free energy and so on. Among these, perfluorooctane sulfonate (PFOS), its precusors, and related compounds are used in many applications ranging from textiles, carpets, paper, food packing materials. Although PFOS concentrations in environment are very low, for example, PFOS concentration was 5.6 pg/m3 in an exposed urban setting in Japan while lower than 0.1μg/L in surface water, given that general application, stable carbon-fluorine bonds, and the potential to biomagnify in food chains of it, PFOS in the liver was measured to be up to 7,760μg/L wet weight for plaice, and serum level recorded in 2003-2004 was about 20 ng/mL for PFOS.The toxicity and hazard profile for PFOS has been reviewed. PFOS has been found to increase mortality and malformation to zebrafish embryos in dose-and time-dependent manner, regulate various protein levels, and up-regulate p53 and Bax expression. Additionally, liver hypertrophy, lipid vacuolation, and decreased serum cholesterol were detected in rats after subchronic exposure to PFOS. Recently, the authors found that PFOS exposure resulted in spontaneous behavior and habituation, cholinergic system dysfunction, and abnormal proteins expression in neonatal pups. Furthermore, PFOS may induce apoptosis and DNA damage in HepG2 cells, and induce PC-12 cells to differentiate into the acetylcholine phenotype at the expense of the dopamine phenotype in vitro. The potential toxicity of PFOS has not been well characterized, and littile information is known about markers of its toxic action. Although acute toxicity endpoints had provided information after PFOS exposure, whether 2.0 mg/kg·bw PFOS in utero would induce morphological alteration and glia activation following by inflammatory and pathological apoptosis. Furthermore, the potential mechanism of morphological alteration in zebrafish, a vertebrate model, induced by PFOS need to be understood further.Central nervous system (CNS) composes of neurons and non-neuronal cells, the latter including glia and endotheliocytes. Glia cells are predominant as supportive components in CNS and play primary role in disease processes. In this study, pups exposed to PFOS in utero, zebrafish embryos, and microglia cells (N9 cell line) were selected to research potential mechanisms, including activated glia cells may trigger pathological apoptosis and inflammatory, abnormal expression of miRNAs, mRNA levels, and motor neuronal development and proliferation in zebrafish.Section I:Developmental toxicity on liver, lung and brain in nenonatal rats after PFOS exposure in uteroVulnerable periods during the development of the nervous system are sensitive to environmental insults such as trauma, hypoxia-ischemia, and oxidative stress. Injury maybe continuous as unrecoverable lesion in retardant developing brain, although slight repair occurs. In this section, the tissular pathology and potential neurotoxicity in neonatal rats induced by PFOS were performed. Pregnant Sprague-Dawley rats were given 2.0 mg/kg PFOS daily by gavage from gestational day (GD) 2 to GD21. Controls received 0.5% Tween-20 vehicle (4 ml/kg). At parturition, approximately 11.3% of offspring died following by significantly weight loss at 2.0 mg/kg PFOS groups. Tissular immunihistochemistry (IHC) showed focal hemorrhage induced by erythrocytic transudation, blurred hepatic cords and marked hepatomegaly, cytoplasmic vascuolation, meganucleus and multinuclear around with portals in livers. The lung is another important target organ in addition to the liver in rats. In contrast to thinning of the epithelial walls in control pulmonary alveolus, congestion, lung aetlectasis, as well as focal or diffuse thickened epithelial walls were observed in the lungs exposed to PFOS, therefore, the lungs from PFOS-exposed animals looked like immature. Notably, it was the first time to observe the epithelial cells with bigger nucleus abnormal structural ciliated columnar epitheliumon the bronchioles. At the same time, more deeply blue was observed in cerebral nucleus in exposed group.After PFOS exposure the numbers of ED1+, EMAP-Ⅱ+, and nestin+ cells in cortex and hippocampus were similar between two groups, whereas GFAP+ cells in in cortex and hippocampus were significantly higher in PFOS-treated group compared with control. Furthermore, in the PFOS-treated newborns, up-regulation of two proteins, AIF-1 involved in chronic inflammatory processes, NFκB related to inflammatory and apoptosis, were observed, whereas bak, a pro-apoptotic Bcl-2 family protein, showed down-regulation.These finding suggested that PFOS exposure may cause hepatic and respiratory dysfunction, lesion in developing brain by inducing inflammatory and pathological apoptosis.SectionⅡ:Developmental toxicity and the mechanisms of PFOS to zebrafish embryoThe zebrafish embryo is a universal research model for investigating toxicity because it is easy to maintain and handle, with rapid embryogenesis and continuous reproduction. In this study, developmental toxicity and the potential mechanisms of PFOS to zebrafish embryo were studied.Six-hour post-fertilization (hpf) zebrafish embryos were exposed to 1.0 mg/L PFOS until 192 hpf. Control groups received 0.0025% DMSO. Latval mortality as well as malformation ratio was significantly increased after the embryos were exposed until 132 hpf. The fry displaced gross developmental malformations, including pericardiac edema, yolk sac edema, spinal curvature, hypopogmentation, and sidestroke. To test whether developmental malformation was mediated via apoptosis and differential expression of genes in mRNA and protein levels, immunofluorescent staining, acridine orange staining, miRNA microarray screening, and RT-PCR were used.Alpha-tubulin plays a key role in maintaining the functions of motor neurons and can be used as a marker for axon localization, in the brain, the pattern and size of axon tracts can be used as an indicator of malformation. Immunofluorescent staining indicated that alpha-tubulin was highly expressed in the brain and spinal cord of the caudal two thirds at 72 hpf and 120 hpf in control groups. Compared with control groups, exposure to 1.0 mg/L PFOS caused alpha-tubulin in motor neurons to be significantly down-regulated in the same positions, and much thinner and linear expression was observed in the trunk region. However, alpha-tubulin was highly expressed in spinal cord of the caudal two thirds at 72 hpf after PFOS exposure following by similar expression in axon tracts in the brain.PCNA is a cofactor for DNA polymerase and is expressed in all cells during early development and is then down-regulated during morphogenesis and differentiation, being only expressed in late developing prgans and tissues, such as the pectoral fin and the pharyngopalatine arch. The control group showed that PCNA was expressed in a punctiform pattern in branchial arch and pharyngopalatine arch, but it was expressed at lower levels in the telencephalon, diencephalon, cerebellum, and the caudal spinal cord, which was accompanied with linear expression in trunk spinal cord at 72 hpf and 96 hpf. At 120 hpf, PCNA was highly expressed in pharyngopalatine arch, which was accompanied with linear expression in trunk spinal cord and sarcomere. Exposure to 1.0 mg/L PFOS caused PCNA expression in abdomen and the caudak spinal cord to be highly increased compared with control levels at 72 hpf,96 hpf, and 120 hpf. In addition, PCNA was expressed in a punctiform pattern in the brain at 120 hpf following with no expression at 72 hpf and 96 hpf. Given that miRNAs can play essential roles in embryonic development, several brain-specific miRNAs were detected by stem-loop RT-PCR and found that miR-124, miR-128, and miR-153 a/b showed up-regulated whereas miR-181b, miR-125b, and miR-430 family showed down-regulated after PFOS exposure until 120 hpf. Simultaneously, cdk5, a proline-directed serine/threonine kinase, is essential for brain development and function, in this study overexpression of cdk5 inhibited significantly peroxiredoxin-2 (Prx 2) activity (P<0.05). In addition, increasing apoptotic cells around the developing heart were observed after AO staining, and three miRNAs that primarily found in the heart indicated differential expression, for example, significant up-expression were observed in miR-1, miR-138, and down-regulation in miR-133 (P<0.05).These finding suggested that PFOS could induce adverse effects on brain and heart development by disturbing motor neuron development and proliferation and regulating miRNAs and mRNA levels.Section III:Apoptosis and apoptotic pathway induced by PFOS in the murine N9 microglial cell lineApoptosis, or programmed cell death, plays a vital role during both development and the maintenance of tissue homeostasis in CNS, whereas multiple pathological insults could induce non-physilogical apoptosis. Apoptosis can be divided into the extrinsic pathway, initiated through death receptors such as Fas, and the cell intrinsic pathway, initiated through mitochondria. In mammals, mitochondria function as central check-points for many forms of apoptosis. During mitochndrial pathways, the permeabilization of the mitochondrial outer membrane allows the release of cytochrome c, which triggers caspase cascade to orchestrate the death of cells. Although one approach to identify direct developmental neurotoxicity is to use an in vitro model, the molecular mechanisms underlying how PFOS functionally affects to microglia cells remain largely unknown. This study we firstly use murine microglia N9 cells to evaluate apoptosis through mitochondrial pathway after PFOS exposure.In the current study, N9 cells were given 5,10,50,100, and 200μmol/L PFOS for 24 h and 48 h, controls received 0.5% DMSO. Toxicological parameters investigated included: (1) cell viability treated for 24 h and 48 h detected by MTT assay; (2) apoptotic cells shape and ratio were detected by PI and FDA double-staining, Hoechst 33258 staining, and flow cytometry; (3) mitochondrial membrane potential (MMP) measured by Rhodamine 123; (4) p53, Bax, Bcl-2, caspase 3, and caspase 9 mRNA expression detected by RT-PCR, andβ-actin was used as internal reference gene.Herein, decreased viability and increased numbers of apoptotic cells of N9 cells in a dose-dependent manner were observed, and the apoptotic cells ratio was 3.97%,8.32%, 20.81%,33.26%, and 48.72% respectively, and statistically significant increases in ratio were observed in the 50μmol/L and higher dose groups. Additionally, dissipation of MMP, no dose-depedent up-regulation of p53, Bax, caspase 3, and caspase 9 mRNAs, and down-regulation of Bcl-2 mRNA were involved after PFOS exposure. These results suggested that cell apoptosis induced by PFOS mainly was medicated by mitochrondrial pathways.