Studies On The Endocrine-disrupting Effects Of PFOA And PFOS

Perfluorinated compounds (PFCs) are synthetic fully fluorinated organiccompounds that have been widely used as surfactants in industrial and commercialapplications. They are stable chemicals that is both lipid-and water-repellent.Moreover, PFCs have been listed as persistent organic pollutants (POPs). Thewidespread distribution and persistence of PFCs in humans and the environment havegenerated great concern about potential health impacts.There is growing evidence that PFCs may disrupt the endocrine system.Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonate (PFOS) are therepresentative and commonly used PFCs. Further characterization of theendocrine-disrupting effects of PFOA and PFOS is required for assessing potentialhealth risks. Methods developed by U.S.EPA involve a battery of in vitro and in vivoscreening assays to identify substances that interact with endocrine systems. Toinvestigate the endocrine disruption potencies of PFOA and PFOS, a combination of in vitro and in vivo assays was employed in the present study. Reporter gene assayswere used to detect receptor mediated (anti-)estrogenic,(anti-)androgenic,(anti-)thyroid hormone activities and PPAR activation of PFOA/PFOS. Thesteroidogenic effects of PFOA/PFOS were measured at both the hormone and genelevels in the human adrenocortical carcinoma cell line H295R. Additionally, azebrafish-based short-term screening method and female pubertal rat assay weredeveloped to detect the potential effect of PFOA/PFOS on endocrine function in vivo.Our research focused on a detailed characterization of PFOA/PFOS in an attempt todetermine the endocrine-disrupting effects of these chemicals.Part I Assessing the endocrine-disrupting effects of PFOAand PFOS by in vitro assaysChapter I Assessing hormone receptor activities of PFOAand PFOS in reporter gene assaysObjectiveThe major way by which endocrine disrupting chemicals (EDCs) disrupt thephysiological process is through interaction with nuclear hormone receptors. Reportergene assay has been established as a powerful tool for testing receptor agonists andantagonists among chemicals. Hormone receptor mediated reporter gene assays wereused to detect (anti-)estrogenic (anti-)androgenic,(anti-)thyroid hormone activitiesand PPAR activation of PFOA and PFOS.MethodsIn ER mediated reporter gene assay, CV-1cell line was transfected with ER expression vector rER/pCI and the reporter plasmid pERE-TATA-Luc+. In TR mediated reporter gene assay, CV-1cell line was transfected with TR expressionvector pGal4-L-TR and the reporter plasmid pUAS-tk-luc. In PPAR mediatedreporter gene assay, CV-1cell line was transfected with PPAR expression vectorPG-PPAR and the reporter plasmid PBR-PPRE. Cells were treated with PFOA orPFOS alone to investigate the agonistic activities of these chemicals. For antagonisticactivity tests, cells were treated with PFOA/PFOS with E2/T3. MDA-kb2cell linehad been stably transfected with the pMMTV.neo.luc reporter gene plasmid. Theywere treated with various concentrations of tested chemicals with or without DHT toinvestigate the agonistic and antagonistic activities.ResultsPFOA and PFOS acted alone had no effect on reporter gene expression ascompared with vehicle control. PFOA and PFOS co-treatment with1×10-9M E2could increase the luciferase expression at the tested concentrations (3×10-9-3×10-7M). None of the tested groups showed androgenic and antiandrogenic activities in ARreporter gene assay. PFOA and PFOS did not have TR agonistic activity in TRreporter gene assay. PFOA (1×10-8-3×10-7M) displayed anti-thyroid hormone effectswith5×10-9M T3in the medium. While PFOS co-treated with T3could also suppressthe expression of luciferase at concentrations of1×10-7M and3×10-7M. In PPAR reporter gene assay, PFOA and PFOS activated PPAR at all concentrations.ConclusionResults here demonstrated that PFOA and PFOS has additive estrogenic activity,anti-thyroid hormone activity and PPAR activation in vitro. They could have effectson the endocrine system through interfering with nuclear receptor. Chapter II The steroidogenic effects of PFOA and PFOSin H295R steroidgenesis AssayObjectiveChemicals can exert endocrine-disrupting effects via non-receptor mediatedpathways. The human adrenocortical carcinoma cell line H295R has been shown tobe a potential in vitro model for screening adverse effects of chemicals onsteroidogenesis. In this study, H295R cells were used to determine the effects ofPFOA and PFOS on steroidogenesis.MethodsIn this study, the steroidogenic effects of PFOA and PFOS were measured inH295R cell. The hormone levels of estradiol (E2) and testosterone (T), the expressionof major steroidogenic genes and the key steroidogenic gene regulator steroidogenicfactors1(SF-1) were measured after PFOA and PFOS exposure in H295Rsteroidogenesis assay.ResultsPFOA and PFOS could induce E2production and reduce T production in aconcentration-dependent manner. E2level were significantly increased by PFOA andPFOS at1×10-8M and3×10-8M, respectively. While T level were decreased byPFOA and PFOS at3×10-8M and1×10-7M, respectively. The expression patterns ofthe10steroidogenic genes were altered by PFOA and PFOS exposure. PFOAsignificantly induced the expression of17βHSD1,3βHSD2, CYP11B2, CYP19.Expressions of CYP17,17βHSD4, CYP11A were decreased by PFOA exposure. Genelevels of HMGR, StAR, CYP11A, CYP19,3βHSD2, CYP11B2were elevated by PFOS.For CYP17,17βHSD1and CYP21, the relative responses were suppressed after PFOStreatment. For the key steroidogenic gene regulator SF-1, gene expression wasdown-regulated within the tested concentration range. SF-1protein level gradually decreased in H295R cells as PFOA exposure concentration increased.ConclusionPFOA and PFOS could alter steroid hormone synthesis and the majorsteroidogenic genes expression in H295R steroidogenesis assay. Results heredemonstrated that PFOA and PFOS have the ability to alter steroidogenesis. Part II Assessing the endocrine-disrupting effects of PFOAand PFOS by in vivo assaysChapter I Effects of PFOA and PFOS on development andendocrine function in zebrafishObjectiveA short-term zebrafish embryo exposure assay was developed to detect thepotential effects of PFOA and PFOS on endocrine system development and functionin vivo.MethodsZebrafish embryos from4h post fertilization (hpf) to120hpf were exposed to100μg/L,200μg/L, and500μg/L PFOA or PFOS. The embryos’ survivals and thestage of embryonic development were recorded daily. Genes related to estrogenreceptor production (esr1and esr2b), early thyroid development (hhex and pax8), andsteroidogenic enzyme synthesis (cyp17, cyp19a, and cyp19b) were quantified byQ-RT-PCR. To identify genes and pathways involved in the endocrine-disruptingeffects, mRNA microarray analysis were conducted. ResultsExposure to PFOA and PFOS did not affect hatching and larval survival rates inzebrafish embryos. No significant lethal and developmental abnormality wasobserved during the whole exposure time. PFOA cause a significantly increase ofesr1at500μg/L and no changes for esr2b. Exposure to PFOA up-regulated hhex andpax8expression levels at200and500μg/L. No significant changes of cyp17, cyp19a,and cyp19b were observed. Exposure to PFOS (500μg/L) led to an increase in theexpression level of esr1but a decrease in the expression of esr2b. An apparentconcentration-dependent increase of hhex and pax8expression was observed afterPFOS treatment. Suppression of cyp17, cyp19a, and cyp19b was found at200and500μg/L PFOS exposure. Microarray analysis revealed several classes ofsignificantly regulated genes which could be related to cellular, metabolic,development and biological regulation processes in zebrafish.ConclusionIn short-term zebrafish embryo exposure assay, PFOA and PFOS could alter theexpressions of estrogen receptor, early thyroid development and steroid synthesisrelated genes. Further large-scale genetic study using microarrays provided moreinformation on exploring the mechanisms of PFCs-mediated endocrine-related effectsin zebrafish. Chapter II Effects of PFOA and PFOS on pubertydevelopmentObjectiveLimited information about effects of PFOA and PFOS on puberty developmentis available. We evaluated the effects and explored the neuroendocrine mechanism of neonatal and juvenile PFOA/PFOS exposure on female puberty maturation. We alsoevaluated the effects of PFOA/PFOS on liver.MethodsFemale rats were injected with PFOA or PFOS at0.1,1and10mg/kg/dayduring postnatal day (PND)1-5or26-30. Body weight, anogenital distance (AGD),vaginal opening (VO), estrous cyclicity and other development indexes were recorded.Serum levels, ovaries histology and follicles numbers were evaluated. Geneexpressions of Kiss1, Kiss1r, ERα, ERβ and immunohistochemistry of kisspeptin inthe hypothalamic anteroventral periventricular (AVPV) and arcuate (ARC) nucleiwere quantified. Liver weight were measured. Fatty acids in serum were determinedby GC/MS. Analysis of biomarkers for hepatic metabolism were also assessed.ResultsVO and first estrus were significantly advanced in10mg/kg PFOA,1and10mg/kg PFOS groups after neonatal and juvenile exposure. Neonatal PFOA/PFOSexposure increased body weight and AGD in a non-dose-dependent manner. Estradioland luteinizing hormone (LH) levels were increased after neonatal and juveniletreatments. Irregular estrous cycles occurred more frequently in0.1and1mg/kgPFOA/PFOS groups. Although no altered ovarian morphology was observed, folliclesnumbers were reduced in neonatal groups. Kiss1, Kiss1r, ERα mRNA expressions inAVPV and ARC were decreased after two periods’ exposure. Suppression ofkisspeptin fiber intensities were also evoked by PFOA/PFOS exposure especially atthe high dose in AVPV and ARC. Results also showed that liver weight weresignificantly increased in the highest PFOA/PFOS treated groups.Changes in serumfatty acids composition and metabolic disturbance in liver by both PFOA/PFOS wereobserved. ConclusionNeonatal and juvenile exposure to environmental levels of PFOA/PFOS couldaffect the regulation of hypothalamic-pituitary-gonadal (HPG) axis leading toadvancing puberty onset. Kisspeptin/GPR54system played a key role in the effects.Neonatal exposure to low-dose of PFOA/PFOS could affect serum fatty acidmetabolism and other metabolic processes in liver.