Date of Award

2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmaceutical Sciences

First Advisor

Bingfang Yan

Abstract

Carboxylesterases (CES) catalyze both hydrolytic and synthetic reactions and play important roles in drug metabolism and lipid mobilization. Many factors such as age and dietary supplements have been shown to regulate the CES expression. The expression of CES1 shows the developmental regulation and is highly induced by antioxidants. The goals of this project were to determine whether CES2, like CES1, is developmentally regulated and to investigate how antioxidants induced the expression of CES1 at the molecular level. The ontogenic studies showed the mRNA levels of CES2 exhibited a postnatal surge (1-31 days versus 35-70 days) in both liver and duodenum. The levels of CES2 protein increased with age as well. However, individual donor multi-sampling CES2 expression studies showed the significant correlation between the duodenum and jejunum but insignificant correlation between the liver and duodenum. Moreover, the metabolic enzyme cytochrome P450 3A4 (CYP3A4) which share substrates with CES2 in many case have a comparable age related expression pattern but the mRNA level of CYP3A4 in the duodenum showed otherwise. The mechanistic studies on CES1 induction used the dissected regulatory sequence of the CES1A1 gene to locate the element supporting the transactivation. A novel element was identified and designated as sensitizing/antioxidant response element (S/ARE). Comparing with the known antioxidant element ARE4, the novel element supported whereas the ARE4 element repressed the transactivation. The Electrophoretic mobility shift assay and Chromatin immunoprecipitation experiments demonstrated that the S/ARE element serves as a major site to interact with the CES1A1 gene and both elements were bound by nuclear factor-E2 related factor-2 (Nrf2). In conclusion, CES2 and CYP3A4 are expressed under developmental regulation and whether the regulation occurs in a gene-dependent or an organ-dependent manner. Both positive and negative Nrf2 response elements exist even within the same gene. The identification of ARE4 supported Nrf2 repression, given the fact that Nrf2 is generally considered to confer transactivation activity.

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