Date of Award

2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Pharmacology

First Advisor

Trevor M. Penning

Second Advisor

Harry . Ischiropoulos

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are pervasive environmental and dietary toxicants generated from the incomplete combustion of organic material, including fossil fuels, food, and tobacco. To exert carcinogenicity PAHs require metabolic activation to electrophilic metabolites. Metabolism involves cytochrome P450s (P450s) and epoxide hydrolase to yield more water-soluble hydroxy-PAHs (phenols, dihydrodiols and tetraols), while aldo-keto reductases (AKRs) convert PAH trans-dihydrodiols into PAH ortho (o)-quinones. PAHs can also act as xenoestrogens. However, the role of PAHs in endometrial cancer, a cancer predominantly associated with unopposed estrogen action is unknown. In this dissertation I characterize the status of estrogen receptors (ERs) and the aryl hydrocarbon receptor (AhR) in the Ishikawa endometrial cell line and assess whether the representative PAH, benzo[a]pyrene (B[a]P), can be metabolized to estrogenic compounds in the presence and absence of induction of P450s by 2,3,7,8-tetrachlorodibenzo[b,e][1,4]-dioxin (TCDD). Using stable-isotope dilution high performance liquid chromatography and APCI tandem mass spectrometry in the selected reaction monitoring mode, I analyzed B[a]P metabolism in Ishikawa cells. Estrogenic activity of B[a]P metabolites was determined by the endogenous estrogen inducible alkaline phosphatase reporter gene and an exogenous estrogen response element (ERE) luciferase reporter construct. I also assessed whether PAHs can induce a proliferative phenotype via ER- and non-ER-regulated pathways. I demonstrate that, 5 µM B[a]P can be metabolized in human endometrial cells into 3-OH-B[a]P and B[a]P-7,8-dione in sufficient amounts to activate ER and that induction of P450 activity by 10 nM TCDD increases the amount of estrogenic metabolites formed. I also show that of the estrogenic B[a]P metabolites, only B[a]P-7,8-dione induces endometrial cell proliferation, which is unlikely regulated by ERs instead by the epidermal growth factor receptor (EGFR) and activation of the mitogen-activated protein kinase (MAPK) pathway. This work indicates that human endometrial cells can metabolize PAHs into estrogenic metabolites, which may play a role in the disruption of ER signaling in the endometrium and also induce cell proliferation through non-ER-regulated pathways.

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