Role of Human Aldo-Keto Reductases in the Metabolic Activation of the Carcinogenic Air Pollutant 3-Nitrobenzanthrone

Abstract

3-Nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen detected in diesel exhaust particulate and ambient air pollution. It requires metabolic activation via nitroreduction to promote DNA adduct formation and tumorigenesis. NAD(P)H:quinone oxidoreductase 1 (NQO1) has been previously implicated as the major nitroreductase responsible for 3-NBA activation, but it has recently been reported that human aldo-keto reductase 1C3 (AKR1C3) displays nitroreductase activity toward the chemotherapeutic agent PR-104A. We sought to determine whether AKR1C isoforms could display nitroreductase activity toward other nitrated compounds and bioactivate 3-NBA. Using discontinuous enzymatic assays monitored by UV-HPLC, we determined that AKR1C1−1C3 catalyze three successive two-electron nitroreductions toward 3-NBA to form the reduced product 3-aminobenzanthrone (3-ABA). Evidence of the nitroso- and hydroxylamino- intermediates were obtained by UPLC-HRMS. Km, kcat, and kcat/Km values were determined for recombinant AKR1C and NQO1 and compared. We found that AKR1C1, AKR1C3, and NQO1 have very similar apparent catalytic efficiencies (8 vs 7 min−1 mM−1) despite the higher kcat of NQO1 (0.058 vs 0.012 min−1). AKR1C1−1C3 possess a Km much lower than that of NQO1, which suggests that they may be more important than NQO1 at the low concentrations of 3-NBA to which humans are exposed. Given that inhalation represents the primary source of 3-NBA exposure, we chose to evaluate the relative importance of AKR1C1−1C3 and NQO1 in human lung epithelial cell lines. Our data suggest that the combined activities of AKR1C1−1C3 and NQO1 contribute equally to the reduction of 3-NBA in A549 and HBEC3-KT cell lines and together represent approximately 50% of the intracellular nitroreductase activity toward 3-NBA. These findings have significant implications for the metabolism of nitrated polycyclic aromatic hydrocarbons and suggest that the hitherto unrecognized nitroreductase activity of AKR1C enzymes should be further investigated.

Induction of the Antioxidant Response by the Transcription Factor NRF2 Increases Bioactivation of the Mutagenic Air Pollutant 3-Nitrobenzanthrone in Human Lung Cells

“…Among the human genes regulated by NRF2, AKR1C1–1C3 and NQO1 are consistently the most overexpressed in response to pharmacological activators of NRF2 and by activation of NRF2 by KEAP1 knockdown. , The metabolic activation of 3-NBA requires nitroreduction by these enzymes in human lung cells where 34% of 3-NBA nitroreduction is attributed to AKR1C1–1C3 and up to 40% is attributed to NQO1 . Given the importance of AKR1C1–1C3 and NQO1 nitroreductase activity, we sought to determine the role of NRF2-KEAP1 signaling on 3-NBA toxication in vitro as measured by 3-ABA formation, cell viability, and cytotoxicity in two human lung cell lines: A549 cells that harbor somatic mutations in KEAP1 and epigenetic silencing of its promoter which lead to high consititutive NRF2 expression and HBEC3-KT cells with wildtype KEAP1 that are sensitive to NRF2 activators.…”

“…The fluorescence of 3-ABA was used to measure the nitroreduction of nonfluorescent 3-NBA in cell culture as published previously . To assess the contribution of NRF2 signaling in the metabolic activation of 3-NBA, experiments were conducted in various A549 cell lines (A549 wt, A549 NRF2-Het, and A549 NRF2-KO).…”

“…We recently demonstrated that aldo-keto reductase 1C1, 1C2, and 1C3 (AKR1C1–1C3) display 3-NBA nitroreductase activity (Figure ). This is the second recorded observation that AKR1C isoforms, which are primilarily recognized as ketosteroid reductases and/or hydroxysteroid dehydrogenases, exhibit reductase activity toward nitroaromatic compounds in an oxygen-independent manner. , In both of these cases, AKR1C family members could compete with the widely recognized nitroreductase NADPH:quinone oxidoreductase 1 (NQO1). We found that the catalytic efficiencies of NQO1, AKR1C1, and AKR1C3 are equivalent for the reduction of 3-NBA and that these enzymes contribute equally in the reduction of 3-NBA in human bronchial epithelial cells in vitro .…”

“…Metabolic activation pathways of 3-NBA via AKR1C1–1C3 and NQO1 lead to DNA adduct formation. Metabolic activation of 3-NBA involves a six-electron reduction of the nitro-group involving sequential formation of the nitroso-, hydroxylamino-, and amine products catalyzed by NQO1 and AKR1C1–1C3, modified from ref . The hydroxylamino intermediate, N -hydroxy-3-aminobenzanthrone ( N -OH-3-ABA) is intercepted by sulfonation or acetylation which creates an excellent leaving group for attack of the intermediate nitrenium or carbenium ion by DNA bases.…”

Nitroreduction: A Critical Metabolic Pathway for Drugs, Environmental Pollutants, and Explosives

“…Studies on the metabolic activation of the chemotherapeutic agent PR-104A using transfected cDNAs showed that surprisingly AKR1C3 and not NQO1 was the major NTR involved in its bioactivation . The NTR activity of AKR1C3 has since been expanded to include the nitroarenes, 3-nitrobenzanthrone and 1-NP . The AKRs catalyze an ordered bi−bi kinetic mechanism in which the NADPH is the first substrate to bind and NADP + is last to leave. , The final products of these reactions are amines, and the formation of the intermediate nitroso- and hydroxylamino compounds is difficult to observe and may be related to their rapid propensity to undergo further reduction.…”