Date of Award
2024
Degree Type
Dissertation
Degree Name
Doctor of Philosophy in Pharmaceutical Sciences
Specialization
Pharmacology and Toxicology
Department
Biomedical and Pharmaceutical Sciences
First Advisor
Angela Slitt
Abstract
Per- and polyfluoroalkyl substances (PFAS) are a unique class of over 15,000 manmade chemicals that are still widely used in manufacturing and are resistant to degradation. Accumulation in the environment and general population has been associated with an array of adverse health outcomes. Perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), and perfluorohexanesulfonic acid (PFHxS) specifically have been associated with markers of liver injury, high serum cholesterol, thyroid disease, low fetal birth weight, suppressed vaccine response, and cancer. Exposure to PFAS is commonly investigated individually, however, humans are exposed to a multitude of PFAS daily. Additionally, with the pervasive nature of these chemical, people of all ages are affected by their toxic effects. This dissertation focuses on both a global and targeted approach to elucidating mechanisms of PFAS liver toxicity, both individually and as a mixture, in young and adult mice. The studies herein introduce novel approaches, including offspring liver omics’ analyses of perinatal PFAS mixture exposures and PFOS exposures in targeted protein murine knockout mouse models, to understand and evaluate the underlying mechanisms of PFAS liver toxicity in both neonatal and adult mice.
In manuscript 1, livers from pups gestationally and lactationally exposed to individual PFOA, PFOS, PFHxS, or a PFAS mixture were investigated for proteomic alterations at postnatal day (PND) 21. Herein, sequential window acquisition of all theoretical mass spectra - liquid chromatography-mass spectrometry (SWATH-LC/MS) and Ingenuity Pathway Analysis (IPA) software were utilized to investigate global proteomic changes in the exposed offsprings’ liver. Individual and commonly observed proteins between treatments and diets were explored to tease out the differing proteomic signatures. Specific proteins involved in pathways concerning lipid transport, storage, synthesis, and catabolism, xenobiotic metabolism, and inflammation were investigated.
In manuscript 2, neonatal livers indirectly exposed to PFOA, PFOS, PFHxS, or a PFAS mixture through gestation and lactation were investigated for transcriptomic alterations at postnatal day (PND) 21. Herein, the QuantSeq 3' mRNA-Seq Library Prep Kit, BlueBee genomics platform, and IPA software were utilized to investigate global transcriptomic changes in the exposed pup livers. Treatment and diet-specific genes that were individually or commonly observed were investigated to tease out the unique transcriptomic signatures. Selected genes involved in pathways regarding xenobiotic metabolism, inflammation, and lipid transport, storage, synthesis, and catabolism were further investigated.
In manuscript 3, a knockout (KO) mouse model with a specific targeted deletion of organic anion transporting polypeptide 2b1 (Oatp2b1) was utilized to investigate the contribution of this supposed PFOS uptake transporter in vivo. A single 1 mg/kg-bw dose of PFOS was administered to C57BL/6J Oatp2b1-/- male and female mice. Plasma was collected at 30 min, 1, 3, 8, 12, 24, 48 hours, 5, 7, and 14 days; females were euthanized on day 5 and males on day 14. LC/MS was utilized to evaluate plasma, liver, kidney, and brain PFOS concentrations.
In manuscript 4, the contribution of albumin to the uptake and distribution of PFOS in vivo was investigated utilizing a KO murine model with a specific targeted deletion of albumin from the blood. Albumin-/- KO C57BL/6J male mice were dosed with either 0.5 mg/kg-bw/day or 10 mg/kg-bw/day for 7 days. PFOS concentrations in plasma, liver, intestines, kidneys, and urine were determined using LC/MS. Plasma alanine aminotransferase (ALT), triglycerides (TG), and free fatty acids (FFA) were investigated and liver lipids were isolated. SWATH-LC/MS was utilized to investigate the liver proteome in the 0.5 mg/kg-bw/day study and quantitative reverse transcription polymerase chain reaction (RT-qPCR) examined specific genes commonly dysregulated with PFOS exposure in both studies.
Altogether, manuscripts 1 and 2 revealed the importance of investigating the impact of PFAS exposure in sensitive populations, such as the developing fetus. Maternal exposure to individual PFAS and/or a PFAS mixture was shown to cause significant adverse alterations to the offspring liver proteome and transcriptome, showing compelling concordance between datasets. Results suggest individual PFAS and mixtures need to further be evaluated for differing behaviors seen in vivo. Manuscripts 3 and 4 utilized protein specific murine KO models to delve into a targeted mechanistic approach of PFAS liver toxicity. OATP2B1 was found to have minimal involvement in PFOS uptake, opposing previous in vitro findings for this uptake protein. Albumin had a large contribution to altered PFOS distribution in tissues, but was found to not play a major role in liver enlargement. This research highlights potential mechanisms underlying PFAS liver toxicity in both neonatal and adult mice, and provides future mechanistic studies a jumping off point to aid in the understanding of how PFAS exert their adverse effects to help inform future regulations for these chemicals.
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.
Recommended Citation
Kaye, Emily, "MECHANISTIC APPROACH TO UNDERSTANDING ADVERSE LIVER OUTCOMES RELATED TO PFAS EXPOSURE IN NEONATAL AND ADULT MICE" (2024). Open Access Dissertations. Paper 1641.
https://digitalcommons.uri.edu/oa_diss/1641