Metabolic activation of nitroarenes and Nrf2-Keap1

Nitroarenes are carcinogens found in car exhaust and prominent in diesel exhaust. They pollute the air we breathe, contribute to air pollution which is rising due to climate change. Nitroarenes of concern are 1-nitropyrene (1-NP), 1,8-dinitropyrene (1,8-DNP), 3-nitrobenzanthrone (3-NBA) and 6-nitrochyrsene (6-NC). Nitroarenes undergo metabolic activation by nitroreduction via a nitroso- and hydroxylamino- intermediate and amine product and numerous enzymes have been implicated. In the last funding period, we found that prominent nitroreductases involved in the metabolic activation of 1-NP, 1,8-DNP and 3-NBA in human lung cells were the aldo-keto reductases AKR1C1-AKR1C3; furthermore, the genes encoding these enzymes were induced by the NRF2-KEAP1 stress response pathway and genetic knock out of this pathway eliminated nitroarene activation. These findings were among the first to demonstrate that activation of the NRF2-KEAP1 pathway may be harmful in the context of nitroarene exposure and could initiate the carcinogenesis process rather than prevent it. In this renewal we hypothesize that AKR1C enzymes induced by the NRF2-KEAP1 pathway activate nitroarenes to intermediates that adduct proteins and DNA, with a resultant increase in mutation, that ultimately leads to increased carcinogenesis. This hypothesis will be tested by using the following human lung cell lines: A549 (human adenocarcinoma cells with constitutive expression of NRF2 due to hypermethylation and mutation of KEAP1); A549 cells with NFE2L2/NRF2 heterozygous and homozygous knockout generated by CRISPR/Cas9, and HBEC3-KT cells (immortalized human bronchial epithelial cells with inducible NRF2). In Aim 1: We will determine whether human AKRs metabolically activate 6-nitrochrysene (6-NC) in the human lung cells described using both genetic and pharmacological approaches. 6-NC is unique in that it can be activated by monooxygenation and nitroreduction. We will determine whether human AKR1C enzymes reduce the nitro group of 6-NC-1,2-DHD to form 6-aminochrysene-1,2-DHD (6-AC-1,2-DHD) on route to DNA adducts or whether their dihydrodiol dehydrogenase activity will yield a highly reactive 6-nitrochysene-1,2-dione. In Aim 2: We will use a targeted proteomics approach to determine whether nitrosoarenes generated by AKR1C enzymes adduct proteins that control redox-state including KEAP1 itself in HBEC3-KT cells. Detection of sulfinamide and sulfonamide adducts in human lung cells would demonstrate that nitrosoarenes may modify the proteome and affect tumorigenesis. In Aim 3: We will determine whether the adductome resulting from nitroarene exposure in A549 and HBEC3-KT cells is NRF2 dependent. Covalent and oxidatively damaged DNA adducts will be measured by stable-isotope dilution liquid chromatography mass spectrometry. In Aim 4: We will determine for the first time if AKR1C enzymes increase the mutagenicity of nitroarenes on the HPRT gene. We will determine the mutation frequency, potency, pattern, spectrum, and signature and whether the distribution of single base substitutions is similar to COSMIC (catalog of somatic mutations in cancer) observed in human cancers.