The quantification of total PPARgamma ligand activity in serum samples and the association with adverse metabolic health endpoints
Edwards, Lariah Marie
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It is well recognized that the global increase in obesity and metabolic diseases over the last several decades cannot be solely attributed to aging and modern lifestyle trends (i.e., excess caloric intake and lack of physical activity). Metabolism disrupting chemicals (MDCs) are environmental and consumer product chemicals that act at the molecular level in multiple organs to affect systemic metabolic homeostasis and are hypothesized to increase the risk of obesity and metabolic diseases. The nuclear receptor peroxisome proliferator activated receptor γ (PPARγ) regulates insulin sensitivity, adipogenesis, and mature adipocyte maintenance, function and survival, which make it beneficial as a therapeutic target. However, PPARγ is also a target for structurally diverse MDCs that may not induce its health-promoting biological effects. One such PPARγ agonist is triphenyl phosphate (TPHP), an organophosphate ester commonly used in consumer products. Accumulating evidence from animal and in vitro studies demonstrates that MDCs act on multiple organs (e.g., liver, adipose, pancreas) to disrupt glucose and lipid homeostasis. Epidemiological studies have characterized human exposure to complex mixtures of MDCs and associated that exposure to obesity and metabolic diseases. However, not all suspected MDCs have been investigated in human epidemiological studies. Furthermore, these studies are complicated given the role of complex chemical mixtures. The objectives of this dissertation were: 1) to investigate the role of TPHP, a suspected MDC in animal studies, as a metabolic disruptor in humans, 2) to develop an improved biomarker of exposure to mixtures of environmental PPARγ ligands, the Serum PPARγ Activity Assay (SPAA), and 3) to investigate environmental exposures in a human cohort using the SPAA. In the first aim, we used publicly available data from the 2013-2014 National Health and Nutrition Examination Survey to investigate the association between urinary diphenyl phosphate (DPHP), a metabolite of TPHP and biomarker of exposure, and measures of body adiposity as well as markers of type 2 diabetes risk. In adjusted multivariate linear regression models, urinary DPHP was positively associated with increased waist circumference, body mass index, and sagittal abdominal diameter in adults aged 20-50 years, but not in older adults aged 51-79 years. In all adults, urinary DPHP was not associated with any marker of type 2 diabetes risk (fasting plasma glucose, hemoglobin A1c, homeostatic model assessment of insulin resistance, 2-hour oral glucose). In the second aim, we developed and optimized the SPAA as a tool to measure cumulative PPARγ ligand activity in human serum samples and then used the assay to assess exposure to PPARγ ligands in a human cohort. SPAA generates robust PPRE transcriptional activity using Cos-7 cells transfected with a human PPARγ1 expression vector along with a PPRE (DR1)-driven luciferase vector. With small volumes of serum, the SPAA reliably detected high PPRE transcriptional activity levels induced by rosiglitazone, a potent and efficacious therapeutic PPARγ agonist, in serum from experimentally exposed mice. The SPAA also detected significant differences in the PPRE transcriptional activity induced by U.S. based human commercial serum samples. The abrogation of the activity by a PPARγ antagonist, T0070907, confirmed the receptor-specificity of the human serum-induced activity. Finally, we investigated PPARγ agonist exposure in serum samples from a population of Danish women with extensive environmental chemical biomarker data using SPAA and used an additivity model to estimate the contribution of a subset of the chemicals towards measured activity. The serum samples from the Danish cohort induced PPRE transcriptional activity in the SPAA, but with an overall lower efficacy than a U.S. based serum samples. Modeling of the PPRE transcriptional activity induced by the perfluoroalkyl substances and a polybrominated diphenyl ether at concentrations measured in the Danish serum with effect summation demonstrated that these chemicals are unlikely to be the source of the serum activity. Together, these findings demonstrate that TPHP exposure is associated with metabolic disruption, specifically adiposity, in people, which supports toxicological research, and lay the foundation for future work using the SPAA as an exposure biomarker in human populations.