Hepatic And Cardiac Developmental Toxicity Of PFOS Prenatal Exposure

Perfluorooctane sulfonate (PFOS), a widespread environmental pollutant, is a breakdown product of related perfluorooctanesulfonamides, which were used in many industrial and commercial applications. Due to the extremely stable and accumulative nature of PFOS, it has been considered as a persistent organic pollutant and has been found in high concentrations in serum and liver in wildlife and humans. Increased incidences of hepatotoxicity and developmental toxicity have been reported to be the main toxicity and hazard profile of PFOS. However, the mechanism underlying hepatic effects and developmental toxicity observed in the PFOS-treated mammalians was not well known. Notably, it is proven that PFOS can cross the placental barrier and cause toxicity in developmental mammalians.As we known, early life stage exposure to toxicants would increase the risk of adverse effects. In the past decade, increasing evidence has been reported to support the associations between exposures during the intrauterine period and health outcomes later in life. So it is possible to find predictive biomarkers for increased incidence of liver toxicity and cardiac developmental toxicity induced by PFOS.To test the hypothesis whether prenatal exposure of PFOS to the livers of postnatal rat, may related with the alteration of genomic DNA methylation level and promoter region methylation of individual genes; and to investigate whether prenatal exposure of PFOS to the hearts of postnatal rat, may related with the alteration of cardiac mitochondrial dysfunction and structure damage. Pregnant Sprague-Dawley (SD) rats were exposed to perfluorooctane sulfonate (PFOS) and then livers and hearts of weaned (Postnatal Day 21) offspring rats were investigated through epigenetic effects and mitochondrial injury, respectively. Part I:Prenatal exposure to PFOS altered DNA methylation levels in postnatal SD rat liversObjectives:To study whether prenatal exposure of PFOS to the livers of postnatal rat, may related with the alteration of DNA methylation level. Methods:After sperm positive was observed, the pregnant rats gavage exposed to different doses of PFOS (control,0.1 mg/kg/d,0.6 mg/kg/d,2.0 mg/kg/d) during the gestational day 2-21, then the dams was allowed to give birth and the livers of postnatal day 21 were collected, and the global DNA methylation level, the methylation status of LINE-1 and the methylation status of CpG islands were evaluated with the global DNA methylation kit, BSP combined cloned sequencing, and AP-PCR, respectively. Results:The global DNA methylation level at the highest dose group decreased significantly (p<0.05); but there were no significant differences of methylation levels of LINE-1 among the groups (p>0.05); The AP-PCR method showed significant difference of CpG islands methylation in three dosed group compared to the control (p<0.05); in addition, the expression of DNMT3a at the highest dose group was significantly increased (p<0.05). Conclusion:In livers of postnatal rats, the global DNA hypomethylation level and the decrease of CpG islands methylation were related to prenatal exposure to PFOS.Part II:Prenatal exposure to PFOS altered individual genes methylation levels of livers from postnatal SD ratObjectives:The adverse environmental exposure in early life may have deleterious effects on animals through epigenetic aspects. The current study examined the possibility of early epigenetic alteration in PFOS-exposed rat liver. Methods:Pregnant Sprague-Dawley (SD) rats were exposed to Perfluorooctane sulfonate (PFOS) at doses of 0.1,0.6 and 2.0 mg/kg/d and 0.05% Tween 80 as control by gavage from gestation days 2 to 21. The dams were allowed to give birth and liver samples from weaned (Postnatal Day 21) offspring rats were analyzed for individual genes such as tumor suppressor gene glutathione S-transferase pi (GSTP) and p16 promoter methylation level, as well as related genes expression level. Results:In PFOS exposed weaned rats, compared to the control, methylation of critical CpG sites (+79,81,84) in GSTP promoter was found up to 30% methylated in the livers of treated rats, while p16 promoter methylation was not affected. In addition, the up-regulated expression of GSTP was observed and this increase was associated with its main pathway of transcription regulation:Keapl-Nrf2/MafK. Conclusion:early induced hypermethylation in critical cytosines within the GSTP gene promoter region may be a significant biomarker of hepatic PFOS burden, though their direct role in PFOS induced-hepatotoxicity, including its potential carcinogenic action, needs further research.Part III:Prenatal exposure to PFOS injured cardiac mitochondrial inner membrane of postnatal SD ratObjectives:Xenobiotics exposure in early life may have adverse effects on animals' development through mitochondrial damage or dysfunction. The current study demonstrated the possibility of early cardiac mitochondrial damage in PFOS-exposed rat heart. Methods:Pregnant Sprague-Dawley (SD) rats were exposed to Perfluorooctane sulfonate (PFOS) at doses of 0.1,0.6 and 2.0 mg/kg/d and 0.05% Tween 80 as control by gavage from gestation days 2 to 21. The dams were allowed to give birth and heart tissues from weaned (Postnatal Day 21) offspring rats were analyzed for mitochondrial damage through histology observation, ultramicrostructure by electron microscope, global gene expression profile by microarray, related mRNA and proteins or enzymes expression levels by quantitative PCR and western blot. Results:The mitochondria was damaged at 2.0 mg/kg/d dosage group; there were significant vacuolization and inner membrane injury appeared at the hig dosage group; The global gene expression profile showed significant difference in level of some mRNA expression related with mitochondrial structure and function at 2.0 mg/kg/d dosage group, compared to the control; in addition, the expression of several gene at the highest dose group was indeed significantly increased (p<0.05) through quantitative PCR and western blot analysis. Finally, the changes among groups were almost dose-dependent. Conclusion:In hearts of postnatal rats, the mitochondrial injury level and the status of their function were related to prenatal exposure to PFOS. Thus, early induced changes in cardiac mitochondrial structure and function may be a significant phenomenon in rat development after PFOS exposure, though their direct role in PFOS induced developmental toxicity, including its contribution to development of heart or the whole organism, needs further research.