Date of Award

2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Pharmacology

First Advisor

Trevor M. Penning

Abstract

3-Nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen detected in diesel exhaust and air pollution. It requires metabolic activation via nitroreduction to form DNA adducts and promote mutagenesis. NAD(P)H:quinone oxidoreductase 1 (NQO1) was identified as the major nitroreductase responsible for 3-NBA activation in liver, but this may not model inhalation exposures. I sought to determine whether human aldo-keto reductase (AKR) enzymes could display novel nitroreductase activity towards 3-NBA because they are widely expressed in the lung and AKR1C3 bioactivates nitroaromatic prodrug PR-104A. Using discontinuous enzymatic assays monitored by UV-HPLC, I determined that AKR1C1-1C3 catalyze three successive 2-electron nitroreductions towards 3-NBA to form the reduced product, 3-aminobenzanthrone. Evidence of the intermediates was obtained by UPLC-HRMS. AKR1C1-1C3 and NQO1 have equivalent kcat/Km values and contribute equally to the nitroreduction of 3-NBA in lung epithelial cell lines. Notably, AKR1C1-1C3 and NQO1 are induced by NF-E2 p45-related factor 2 (Nrf2) which raises the prospect that Nrf2-targeted chemopreventive agents may exacerbate 3-NBA toxification. I examined 3-NBA bioactivation in adenocarcinomic human alveolar basal epithelial (A549) cells which possess constitutively active Nrf2 signaling. To evaluate the role of Nrf2 signaling on this process, Nrf2 signaling was modulated by heterozygous (Nrf2-Het) and homozygous Nrf2 knockout (Nrf2-KO). Pharmacological Nrf2 activators (sulforaphane, synthetic triterpenoids) were evaluated in human bronchial epithelial cells (HBEC3-KT) which possess functional Nrf2 signaling. Changes in AKR1C1-1C3 and NQO1 expression by Nrf2 knockout or use of Nrf2 activators were confirmed by qPCR, immunoblots, and enzyme activity assays. I observed a reduction of 3-NBA bioactivation in the A549 Nrf2 KO cell lines (53% reduction in A549 Nrf2-Het cells and 82% reduction in A549 Nrf2-KO cells) and 40-60% increases in 3-NBA bioactivation due to Nrf2 inducers in HBEC3-KT cells. Enhanced 3-NBA metabolic activation due to Nrf2 activity may increase DNA adducts and promote mutagenesis. Nrf2 activation has been used as a chemopreventive strategy despite its increasingly recognized potential to promote oncogenesis and chemotherapy resistance. Our data suggest that Nrf2 activation may also be deleterious in preventing cancer initiation for certain carcinogen exposures (i.e. diesel exhaust) and should be utilized with caution.

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Available to all on Monday, September 26, 2022

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