Browsing by Subject "Per- and polyfluoroalkyl substances (PFAS)"

At this moment in human history, there is almost a one hundred percent chance that the readers of this dissertation have detectable levels of per- and polyfluoroalkyl substances (PFAS) in their bodies. Their “nonstick” and long-lasting properties have made PFAS attractive for widescale use in industries and commercial products. Large scale manufacturing applications combined with lack of regulatory efforts have led to ubiquitous human exposure for almost eight decades. Whereas the United States (U.S.) Environmental Protection Agency (EPA) estimates that at least 600 PFAS are in commercial use in the U.S. today, much of the existing toxicological research focuses on two “legacy” PFAS, perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA). It is well established that human exposure to PFOS and PFOA can lead to numerous adverse health outcomes, including liver and immune toxicity, thyroid disease, kidney and testicular cancers, cardiovascular disease, and reproductive and developmental effects like maternal hypertensive disorders, increased risk of miscarriage, and fetal growth restriction. Unfortunately, very little is known about the toxicity of “replacement” PFAS, including perfluorobutane sulfonic acid (PFBS), which is increasingly detected in the environment and within humans. Another troubling gap in the scientific literature is lack of understanding regarding the toxicity of PFAS mixtures, considering human exposure to PFAS does not occur in isolation. Concerningly, much of the available research has focused on toxicity of individual PFAS although humans are exposed to mixtures of PFAS in their daily lives. These deficiencies understanding the scope and mechanisms of human toxicity to alternative PFAS and PFAS mixtures poses challenges for adequate risk assessment to protect public health. This dissertation aims to improve knowledge regarding how maternal exposure to an understudied PFAS and to a PFAS mixture impacts maternal and fetal health. These endpoints are of specific interest because PFAS have been associated with adverse maternal and fetal health outcomes, including endocrine disruption, preeclampsia, preterm birth, and fetal growth restriction. Since it is widely accepted that dysregulated placentation underlies a number of adverse pregnancy outcomes for mother and offspring, the hypothesis of this dissertation is that exposure to replacement PFAS and environmental PFAS mixtures pose significant risks to maternal and fetal health during pregnancy through their effects on the development and function of the placenta. Effects of PFBS are described in chapters 2 – 4, whereas effects of an environmental PFAS mixture are described in chapter 5 – 6. In chapter 2, the transcriptomic effects of PFBS exposure are investigated on three placental trophoblastic cell types. RNA sequencing (RNA-seq) was performed on PFBS-dosed extravillous trophoblast (EVT), cytotrophoblast (CTB), and syncytiotrophoblast (STB) cells using two cellular models. Investigation of EVTs used the immortalized HTR8/SVneo line, while CTBs and STBs were examined using a novel human trophoblastic stem cell line. RNA-seq identified 75 significantly dysregulated genes in PFBS-dosed EVTs, 16 genes of which are associated with placentation and preeclampsia pathogenesis. RNA-seq identified 14 significantly dysregulated genes in PFBS-dosed STBs, in which one gene is involved in angiogenesis and has been implicated in preeclampsia pathogenesis, and five genes are involved with mitochondrial function. Interestingly, no significantly dysregulated genes were detected in PFBS-dosed CTBs. Overall, these experiments identified dysregulated expression of genes involved with cell-specific functions in both EVTs and STBs, presenting a mechanistic link between PFBS exposure, dysregulated placentation, and development of pregnancy complications like preeclampsia. While chapter two presents in vitro evidence that PFBS can disrupt placentation, chapters 3 and 4 describe the effects of PFBS exposure on maternal and fetal health, respectively, using an in vivo model of pregnancy. This approach involved a New Zealand White rabbit (Oryctolagus cuniculus) model of pregnancy, which was selected for study due to rabbit hemodynamics resembling humans during pregnancy and the structural similarities between human and rabbit placentas. Additionally, this model provided the ability to obtain blood pressure measurements and multiple biological fluid and tissue collections, in amounts sufficient for multiple assays. Dams were exposed to control, PFBS-low dose, or PFBS-high dose drinking water. One week after drinking water exposure began, dams were bred, and pregnancy confirmed via ultrasound on gestational day (GD) 15. On GD 25, dams were sacrificed, and maternal and fetal organs were evaluated and measured. Maternal health effects, investigated in chapter 3, included maternal blood pressure, weights and measures, histopathology, clinical chemistry panels, and thyroid hormone levels. At the high dose of PFBS exposure, dams exhibited significant changes in pulse pressure and renal resistive index measure, calculated from blood pressure measurements, which is suggestive of changes in arterial structure and kidney function that may result in hypertension and renal diseases. Adverse structural changes in kidney histopathology provided additional evidence of kidney toxicity from PFBS exposure. Fetal health and placental effects are described in chapter 4, which includes fetal viability, body weights and measures, histopathology, placental weight and morphology, and placental RNA sequencing. Significant changes in fetal crown-rump distance were detected in fetuses from dams receiving the high PFBS dose. Utilization of a mixed model statistical approach identified a significant interaction term between PFBS high dose and fetal sex when evaluating placental weight, suggesting a sex-specific effect on placental weight with PFBS exposure. Additionally, the fetal body weight: placental weight ratio was decreased in the PFBS high group and had a significant sex by exposure interaction term. As this measure is a common proxy for placental insufficiency, PFBS exposure may decrease placental functions that play an important role in achieving optimal fetal development. Together, these two observations demonstrate that PFBS can alter clinically relevant fetoplacental endpoints, some of which are sex-specific. Further investigation of the placenta via RNA sequencing identified one significantly dysregulated gene, AGT, in PFBS high dose placentas as compared to controls. AGT is well-characterized for its role in placentation and preeclampsia. Overall, chapters 3 and 4 present data demonstrating significant maternal and fetal outcomes, respectively, from maternal PFBS exposure in an in vivo experimental model of pregnancy. The results presented in these chapters support the hypothesis that PFBS exposure during gestation leads to adverse health outcomes, seen through maternal effects, like renal injury and hypertension, and fetal effects, like decreased growth parameters and adverse placenta function. Over the course of our lives, including those of pregnant women, PFBS exposure does not occur in isolation. Thus, chapters 5 and 6 investigate the effects of exposure to an environmentally relevant PFAS mixture. The mixture was formulated to mimic the levels of PFAS measured in the tap water of a central North Carolina community (Pittsboro, NC). This mixture includes PFBS along with nine other PFAS. The same rabbit model of pregnancy used for the work presented in chapters 3 and 4 was used to investigate maternal and fetal health outcomes from gestational exposure to this PFAS mixture. Maternal endpoints investigated in chapter 5 revealed significant increased body weight, increased kidney and liver weights, adverse kidney histopathology, and a marker of kidney dysfunction in the clinical chemistry panel in PFAS-exposed dams. Although increased blood pressure and dysregulated thyroid hormone levels observed in the PFAS-exposed group did not reach statistical significance, trends observed with these outcomes require consideration and further investigation in both future in vivo and human population studies. Chapter 6 describes adverse placental outcomes, but no observed adverse fetal health endpoints, resulting from maternal exposure to the environmentally relevant PFAS mixture. Statistical analysis via a mixed model detected a significant interaction term between PFAS exposure and sex when evaluating body weight: placental weight ratio, suggesting that female placental efficiency is disrupted by exposure to a PFAS mixture. Interestingly, a significantly higher number of placentas with abnormal gross morphology were observed in PFAS exposed dams as compared to controls, with a higher incidence in females than males in the PFAS exposed group. Finally, RNA sequencing identified 14 differentially expressed genes between control and PFAS-exposed placenta samples, nine of which have an established functional relevance to pregnancy outcomes. Overall, chapters 5 and 6 support the hypothesis that maternal exposure to an environmentally relevant mixture of PFAS leads to adverse effects on maternal and placental outcomes, although fetal effects were not detected using the measures employed. In summary, this dissertation provides fundamental evidence that PFBS and an environmentally relevant PFAS mixture can elicit adverse health outcomes on both maternal and fetal health. Many of these outcomes may be underlaid by a dysregulated placenta, but other avenues of toxicity were made apparent, including renal injury and hypertension. This research adds to the emergent literature that replacement PFAS and mixtures pose a significant concern for maternal and fetal health.