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. Author manuscript; available in PMC: 2024 Feb 20.
Published in final edited form as: Chem Res Toxicol. 2023 Jan 24;36(2):270–280. doi: 10.1021/acs.chemrestox.2c00337

Role of human aldo-keto reductases and nuclear factor erythroid 2-related factor 2 in the metabolic activation of 1-nitropyrene via nitroreduction in human lung cells

Anthony L Su †, Trevor M Penning †,‡,*
PMCID: PMC9974908  NIHMSID: NIHMS1871388  PMID: 36693016

Abstract

1-Nitropyrene (1-NP) is a constituent of diesel exhaust and classified as a group 2A probable human carcinogen. The metabolic activation of 1-NP by nitroreduction generates electrophiles that can covalently bind DNA to form mutations to contribute to cancer causation. NADPH-dependent P450 oxidoreductase (POR), xanthine oxidase (XO), aldehyde oxidase (AOX), and NAD(P)H:quinone oxidoreductase 1 (NQO1) may catalyze 1-NP nitroreduction. We recently found that human recombinant aldo-keto reductases (AKRs) 1C1-1C3 catalyze 1-NP nitroreduction. NQO1 and AKR1C1-1C3 are genes induced by nuclear factor erythroid 2-related factor 2 (NRF2). Despite this knowledge, the relative importance of these enzymes and NRF2 to 1-NP nitroreduction is unknown. We used a combination of pharmacological and genetic approaches to assess the relative importance of these enzymes and NRF2 in the aerobic nitroreduction of 1-NP in human bronchial epithelial cells, A549 and HBEC3-KT. 1-NP nitroreduction was assessed by the measurement of 1-aminopyrene (1-AP), the six-electron reduced metabolite of 1-NP, based on its intrinsic fluorescence properties (λex and λem). We found that co-treatment of 1-NP with salicylic acid, an AKR1C1 inhibitor, or ursodeoxycholate, an AKR1C2 inhibitor, for 48 h decreased 1-AP production relative to 1-NP treatment alone (control) in both cell lines. R-sulforaphane (SFN) or 1-(2-Cyano-3,12,28-trioxooleana-1,9(11)-dien-28-yl)-1H-imidazole (CDDO-Im), two NRF2 activators, each increased 1-AP production relative to control only in HBEC3-KT cells, which have inducible NRF2. Inhibitors of POR, NQO1, and XO failed to modify 1-AP production relative to control in both cell lines. Importantly, wild-type A549 cells with constitutively active NRF2 produced more 1-AP than A549 cells with heterozygous expression of NFE2L2/NRF2, which were able to produce more 1-AP than A549 cells with homozygous knockout of NFE2L2/NRF2. Together, these data show dependence of 1-NP metabolic activation on AKR1Cs and NRF2 in human lung cells. This is the second example whereby NFE2L2/NRF2 is implicated in the carcinogenicity of diesel exhaust constituents.

Keywords: 1-Aminopyrene, fluorescence, CRISPR/Cas-9, reactive oxygen species, nitroreduction, rotenone, A549 cells, HBEC3-KT cells

Graphical Abstract

graphic file with name nihms-1871388-f0001.jpg

Introduction

Ambient air pollution was estimated to contribute to 14.1% of lung cancer deaths in 2017.1,2 Diesel exhaust is a constituent of air pollution and ranks as a group 1 known human carcinogen by the International Agency for Research on Cancer (IARC).3,4 In a pooled analysis of 11 case-control studies, diesel exhaust exposure was associated with lung cancer, even after controlling for occupation.5 In mice, diesel exhaust exposure also causes pulmonary fibrosis,6 which is a known risk factor for lung cancer.7 Despite these findings, mechanisms by which diesel exhaust exposure causes lung cancer is incomplete. In prior studies, we showed that the metabolic activation of 3-nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one, 3-NBA), one of the most mutagenic constituents of diesel exhaust,8-10 was metabolically activated by nitroreduction mediated by human aldo-keto reductases (AKRs) and NAD(P)H:quinone oxidoreductase 1 (NQO1)11 and was dependent on nuclear factor erythroid 2-related factor 2 (NRF2),12 encoded by NFE2L2. However, it is unknown if this pathway is implicated in the metabolic activation of other nitroarenes found in diesel exhaust.

1-Nitropyrene (1-NP) is a constituent of diesel exhaust and a group 2A probable human carcinogen by IARC.3,4 1-NP is mutagenic at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in the presence of rat liver S9 in Chinese hamster ovary (CHO) cells13 and induces 6-thioguanine-resistant mutants in human hepatoma HepG2 cells.14 The metabolic activation of 1-NP by nitroreduction contributes to the mutagenicity of the compound. Nitroreduction can occur aerobically via three successive two-electron reductions or anaerobically via six successive one-electron reductions.15,16 Common metabolites in both mechanisms of nitroreduction of 1-NP include 1-nitrosopyrene (1-NOP, two-electron reduced), then N-hydroxyl-1-aminopyrene (N-OH-1-AP, four-electron reduced), and then 1-aminopyrene (1-AP, six-electron reduced).17,18 1-Aminopyrene can be N-acetylated to form N-acetyl-1-aminopyrene.19,20 The N-OH-1-AP intermediate is of interest because it can become N,O-acetylated to form N-acetoxy-1-aminopyrene to result in a good leaving group so that the tautomeric nitrenium and carbenium ions form to produce covalent adducts with DNA (e.g., N-(deoxyguanosin-8-yl)-1-aminopyrene).17,18,21,22 Significantly, under aerobic conditions, the nitroso, and hydroxylamino products in nitroreduction may become oxidized to generate reactive oxygen species (ROS).23 1-NP exposure itself generates ROS,24 which may form oxidative DNA lesions, such as the mutagenic lesion 8-oxo-2’-deoxyguanosine,25,26 and represents another mechanism for 1-NP mutagenicity.

We recently showed that, in parallel with 3-NBA,11 1-NP was also metabolically activated by the nitroreductase activity catalyzed by AKRs27 but the relative contribution of these enzymes in human lung cells is unknown. Other nitroreductases for 1-NP include NADPH-dependent P450 oxidoreductase (POR), xanthine oxidase (XO), aldehyde oxidase (AOX), and NQO1.28-33 However, POR, XO, and AOX are anaerobic nitroreductases, and studies involving POR or NQO1 did not determine kinetic parameters for the nitroreduction of 1-NP. Notably, NQO1 and AKR1C1-1C3 are downstream target genes of the NRF2/Kelch-like ECH-associated protein 1 (KEAP1) system.34-37 1-NP generates electrophilic and oxidative stress,38,39 which would modify reactive and redox-sensitive cysteines on KEAP1 and thereby activate NRF240-42 to stimulate the transcription of genes that contain an antioxidant response element (ARE), including NQO1 and AKR1C1-1C3.34-37 Therefore, NRF2/KEAP1 could be a modulator of and responder to 1-NP nitroreduction.

We now investigate the extent to which the nitroreduction of 1-NP is dependent on AKRs, NQO1, other nitroreductases, and its regulation by NRF2 in human lung cells, A549 and HBEC3-KT. Whereas HBEC3-KT cells have inducible NRF2,12,43 A549 cells have a hypermethylated and mutated KEAP1 to lead to constitutively active NRF2.44-47 Wild-type A549 cells that have two functional copies of NRF2/NFE2L2, A549 cells with heterozygous expression of NRF2/NFE2L2 by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9, and A549 cells with homozygous knockout of NRF2/NFE2L2 by CRISPR/Cas9 were used. A549 cells with gene-edited NRF2/NFE2L2 have corresponding less expression of AKR1C1-1C3 and NQO1.12 Assessment of 1-NP nitroreduction was performed through in-cell fluorescence measurements utilizing fluorescence properties specific to 1-AP. We hypothesized that the nitroreduction of 1-NP will be dependent on AKR1C1-1C3 and NRF2 and that NRF2 activators and inhibitors will increase and decrease 1-NP nitroreduction, respectively.

Materials and Methods

Chemicals and Reagents

Diphenyleneiodonium sulfate (DPI) was from Toronto Research Chemicals (Toronto, ON). 1-(2-Cyano-3,12,28-trioxooleana-1,9(11)-dien-28-yl)-1H-imidazole (CDDO-Im) was from Tocris Bioscience (Bristol, UK). 1-Nitropyrene (1-NP), dimethyl sulfoxide (DMSO), 2-chloroethyl ethyl sulfide (CEES), menadione (MD), allopurinol (ALP), all-trans retinoic acid (ATRA), and n-octylamine (OCA) were from Sigma-Aldrich (St. Louis, MO). Rotenone (ROT) and R-sulforaphane (SFN) were from Cayman Chemical (Ann Arbor, MI). Flufenamic acid (FA), ursodeoxycholate (UD), and indomethacin (INDO) were from ICN Biomedicals, Inc. (Costa Mesa, CA). Salicylic acid (SA) and dicoumarol (DIC) were from Acros Organics (Geel, Belgium). N-[4-[2,3-dihydro-1-(2-methylbenzoyl)-1H-indol-5-yl]-5-methyl-2-thiazolyl]-1,3-benzodioxole-5-acetamide (ML385) was from Selleck Chemicals (Berlin, Germany). 1-Aminopyrene (1-AP) was from TCI America (Portland, OR). (4-(2-Hydroxy-2-methylpropyl)piperidin-1-yl)(5-methoxy-1H-indol-2-yl)methanone (ASP9521) and 3-4-trifluoromethyl-phenylamino-benzoic acid (BMT4-159) were synthesized in-house and confirmed to have over 95% purity by high-performance liquid chromatography (HPLC). All compounds used in the current study and their biological activities are listed in Table S1. A549 cells with heterozygous (+/−) and homozygous (−/−) knockout of NFE2L2, which encodes NRF2, were generated using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)912,48,49 and were gifts from John D. Hayes at the University of Dundee, who also provided A549 wild-type control cells that were generated by transfection with an empty pLentiCRISPRv2 vector and survived puromycin treatment.12,48,49

Cell Culture

A549 wild-type cells were purchased from American Type Culture Collection (ATCC; Manassas, VA; ATCC #CCL-185). A549 cells were grown in Kaighn’s Modification of Ham’s F-12 Medium (F12-K) (Gibco, Waltham, MA) supplemented with 10% v/v fetal bovine serum (FBS) (Hyclone, Characterized) (FisherScientific, Waltham, MA) and penicillin-streptomycin at 100 U/mL and 100 μg/mL, respectively (Gibco, Waltham, MA). Human bronchial epithelial cells (HBEC3-KT) were a gift from Dr. John Minna (University of Texas Southwestern Medical Center). HBEC3-KT cells were grown in Keratinocyte Serum-Free Medium (Gibco, Waltham, MA) supplemented with human recombinant Epidermal Growth Factor and Bovine Pituitary Extract (Gibco, Waltham, MA). All cells were grown to 70-80% confluency prior to subculture. Trypsin-EDTA (0.25%) (Gibco, Waltham, MA) was used to detach cells during subculture subsequent to a wash with Dulbecco’s phosphate buffered saline (DPBS) (Gibco, Waltham, MA). All cells are maintained in a humidified incubator at 37°C with 95% ambient air and 5% CO2.

Detection of 1-AP in Cell Culture

Cells were plated at 2.0 x 104 cells/well (200 μL/well) in 96-well black opaque plates (Product 3916, Corning, Corning, NY). A549 cells were treated 24 h after plating. HBEC3-KT cells were treated 48 h after plating. Cell densities at the time of treatment were 3.4 x 104 cells/well for A549 cells and 3.8 x 104 cells/well for HBEC3-KT cells. Cells were treated in medium equivalent to the growth medium for the cells, and 0.1% (v/v) DMSO was always used as the vehicle control. We measured 1-AP according to the fluorescence excitation and emission wavelength properties of 1-AP (λex = 360 nm, λem = 426 nm) (Figures S1 and S2). To ensure negligible fluorescence of all the media at our wavelengths of interest for 1-AP, calibration curves of fluorescence versus 1-AP quantity were generated using each medium in the absence of cells and checked to indicate low fluorescence background (e.g., negligible y-intercept value corresponding to absence of 1-AP) (Figure S3). 1-NP was not found to fluorescence at the λex = 360 nm, λem = 426 nm wavelengths of interest in each medium (Figures S1-S3); the same was seen for all other chemicals used in the current study (data not shown), except for the case of menadione in the medium used for HBEC3-KT cells; hence, that data are uninterpretable and not presented. The calibration curves for fluorescence versus 1-AP for each medium was used to convert a fluorescence reading into a 1-AP quantity, with correction for cell auto-fluorescence (cells were plated each experiment for this specific purpose to modify the y-intercept of the calibration curve). Measurements were made using the Synergy 2 multi-detection microplate reader (Biotek Instruments Inc., Winooski, VT). Each independent experiment was performed in triplicate.

In the experiments where fluorescence was measured at one time point, protein quantity was determined from the same exact cells to normalize 1-AP production. To determine protein quantity, after the determination of the fluorescence readings, medium was immediately aspirated from cells and cells were washed with DPBS. Cells were then lysed with Pierce RIPA lysis buffer (ThermoFisher Scientific, Waltham, MA). Lysates were analyzed for protein quantity using the bicinchoninic acid (BCA) assay (ThermoFisher Scientific, Pierce, Waltham, MA) as per the instructions of the manufacturer.

Statistical Analysis

Data were analyzed by one-way ANOVA followed by Tukey’s post-hoc multiple comparisons of means, which compares each experimental group to all other experimental groups within one graph. In the case of two factors within the experiment (e.g., varied NFE2L2/NRF2 status and treatment status), data were analyzed by two-way ANOVA followed by Tukey’s post-hoc multiple comparisons of means. All data were analyzed using GraphPad Prism version 9 (La Jolla, CA). P < 0.05 was considered statistically significant.

Results

Time and concentration-dependence of 1-AP production from A549 and HBEC3-KT cells exposed to 1-NP

Wild-type A549 cells were able to reduce 1-NP into 1-AP in a time and concentration dependent manner (Figure 1A). For example, when A549 cells were incubated with 10 μM 1-NP, they generated 144.2 ± 16.07 (mean ± SEM) pmol 1-AP and 364.9 ± 37.81 pmol 1-AP at the 24-h and 96-h time points, respectively, showing time dependence. Also, at the 48-h time point, A549 cells receiving 1 μM 1-NP and 5 μM 1-NP were able to generate 41.38 ± 3.222 pmol 1-AP and 154.8 ± 11.31 pmol 1-AP, respectively, showing concentration dependence. Similar time and concentration dependence of 1-AP formation was observed in HBEC3-KT cells (Figure 1B). However, A549 cells had a higher capacity than HBEC3-KT cells to generate 1-AP. For example, following incubation with 5 μM 1-NP, A549 cells generated 243.9 ± 21.40 pmol 1-AP whereas HBEC3-KT cells generated 96.08 ± 11.29 pmol 1-AP at the 96-h time point. It was noted that the rate of 1-AP production seemed to plateau in A549 cells at the latter time points while the rate of 1-AP production was non-linear and seemed to increase over time for HBEC3-KT cells, especially at higher 1-NP concentrations (Figures 1A and 1B).

Figure 1:

Figure 1:

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Time and concentration-dependence of 1-AP generation in (A) A549 cells and (B) HBEC3-KT cells. N = 6 independent experiments performed in triplicate for each for (A) and n = 3 independent experiments performed in triplicate for each for (B). All error bars represent mean ± SEM.

Effect of AKR1C inhibitors on the ability of A549 and HBEC3-KT cells to perform 1-NP nitroreduction

We examined the effect of AKR1C isoform specific inhibitors to attenuate 1-NP nitroreduction in wild-type A549 cells and HBEC3-KT cells. Treatment with salicylic acid50,51 or ursodeoxycholate52,53 as inhibitors of AKR1C1 or AKR1C2, respectively, decreased the amount of 1-AP produced by A549 cells by 51.1% and 47.2%, respectively, relative to 1-NP treatment only (control) (p = 0.0149 and = 0.0253, respectively) (Figure 2A). Treatment with flufenamic acid54-57 as a pan-AKR1C inhibitor resulted in the formation of 61.4% of 1-AP compared to control (p = 0.0784, NS) (Figure 2A). Of the AKR1C3 inhibitors (ASP9521,58 BMT4-159,55 and indomethacin54,59) investigated, indomethacin decreased 1-AP production relative to control by 33.5% (p = 0.0385); ASP9521 or BMT4-159 did not produce a statistical difference in 1-AP formation compared to the control (p = 0.7300 and = 0.3881, respectively) (Figure 3A). In HBEC3-KT cells, application of flufenamic acid, salicylic acid, or ursodeoxycholate decreased the amount of 1-AP produced in culture by 95.2%, 80.5%, and 72.7%, respectively, relative to control (p < 0.0001, < 0.0001, and < 0.0001, respectively) (Figure 2B). Indomethacin decreased 1-AP production by 32.8% relative to control (p = 0.0385), and the effect of ASP9521 or BMT4-159 were not statistically significant (p = 0.7489 and = 0.1625, respectively) (Figure 3B). These data implied that in HBEC3-KT cells the nitroreduction of 1-NP was solely dependent on AKRs but that other enzymes may contribute to nitroreduction of 1-NP in A549 cells.

Figure 2:

Figure 2:

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Effect of AKR1C inhibitors on the ability of (A) A549 cells and (B) HBEC3-KT cells to produce 1-AP following 1-NP exposure. N = 5 independent experiments performed in triplicate for each for (A) and n = 4 independent experiments performed in triplicate for each for (B). Each individual data point represents the average of technical replicates within one experiment. Statistical analysis was performed with one-way ANOVA followed by Tukey’s post-hoc multiple comparison of means. Groups with non-shared superscript letters are significantly different (p < 0.05); shared superscript letters show no statistical significance between compared groups. Error bars represent mean ± SEM. Treatment durations were 48 h for (A-B). Abbreviations: 1-NP, 1-nitropyrene; FA, flufenamic acid; SA, salicylic acid; UD, ursodeoxycholate.

Figure 3:

Figure 3:

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Effect of AKR1C3-specific inhibitors on the ability of (A) A549 cells and (B) HBEC3-KT cells to produce 1-AP following 1-NP exposure. N = 4 independent experiments performed in triplicate for each for (A) and (B). Each individual data point represents the average of technical replicates within one experiment. Statistical analysis was performed with one-way ANOVA followed by Tukey’s post-hoc multiple comparison of means. Groups with non-shared superscript letters are significantly different (p < 0.05); shared superscript letters show no statistical significance between compared groups. Error bars represent mean ± SEM. Treatment durations were 48 h for (A-B). Abbreviations: 1-NP, 1-nitropyrene; ASP9521, (4-(2-hydroxy-2-methylpropyl)piperidin-1-yl)(5-methoxy-1H-indol-2-yl)methanone; BMT4-159, 3-4-trifluoromethyl-phenylamino-benzoic acid; INDO, indomethacin.

Effect of NRF2 activators and inhibitors on the ability of A549 and HBEC3-KT cells to perform 1-NP nitroreduction

In wild-type A549 cells, application of SFN60 or CDDO-Im61 as NRF2 activators did not modify 1-AP production relative to control (p = 0.9597 and = 0.6419, respectively) (Figure 4A). Application of ML38562 or ATRA63,64 as NRF2 inhibitors decreased 1-AP production by 30.9% and 42.6%, respectively, relative to control (p = 0.0478 and = 0.0034, respectively) (Figure 4A). In HBEC3-KT cells, SFN or CDDO-Im increased 1-AP production by 3.63-fold and 4.89-fold, respectively, relative to control (p < 0.0001 and < 0.0001, respectively) (Figure 4B). ML385 or ATRA did not change 1-AP production to a statistically significant amount (p = 0.6138 and = 0.9287, respectively) (Figure 4B). However, when ML385 or ATRA were applied as co-treatments with CDDO-Im, 1-AP formation was decreased relative to the CDDO-Im-treated group lacking ML385 or ATRA by 24.3% and 43.5%, respectively (p < 0.0001 and < 0.0001, respectively) (Figure 4C). These data implied that the metabolic activation of 1-NP was induced by NRF2 activators only in HBEC3-KT cells.

Figure 4:

Figure 4:

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Effect of NRF2 modulators on the ability of (A) A549 cells and (B-C) HBEC3-KT cells to produce 1-AP following 1-NP exposure. N = 6 independent experiments performed in triplicate for each for (A) and n = 4 independent experiments performed in triplicate for each for (B) and (C). Each individual data point represents the average of technical replicates within one experiment. Statistical analysis was performed with one-way ANOVA followed by Tukey’s post-hoc multiple comparison of means. Groups with non-shared superscript letters are significantly different (p < 0.05); shared superscript letters show no statistical significance between compared groups. Error bars represent mean ± SEM. Treatment durations were 48 h for (A-C). Abbreviations: 1-NP, 1-nitropyrene; SFN, R-sulforaphane; CDDO-Im, 1-(2-cyano-3,12,28-trioxooleana-1,9(11)-dien-28-yl)-1H-imidazole; ML385, N-[4-[2,3-dihydro-1-(2-methylbenzoyl)-1H-indol-5-yl]-5-methyl-2-thiazolyl]-1,3-benzodioxole-5-acetamide; ATRA, all-trans retinoic acid.

Effect of inhibitors of other nitroreductases on the ability of A549 and HBEC3-KT cells to perform 1-NP nitroreduction

To investigate the role of other nitroreductases in 1-NP activation in wild-type A549 cells, a number of other enzyme inhibitors were used. Neither CEES (a POR and thioredoxin reductase inhibitor),65,66 menadione (an aldehyde oxidase inhibitor),67-70 dicoumarol (a NQO1 inhibitor),71 nor allopurinol (a xanthine oxidase inhibitor)68,70,72,73 decreased 1-AP production relative to control (Figure 5A). By contrast, rotenone, a mitochondrial transport chain complex I inhibitor of NADH-ubiquinone reductases,74,75 decreased 1-AP production by 46.3% relative to control (p = 0.0083) (Figure 5A). In HBEC3-KT cells, neither CEES, dicoumarol, allopurinol, nor rotenone decreased 1-AP production relative to control (Figure 5B). However, HBEC3-KT cells treated with 1-NP, CDDO-Im, and rotenone had decreased 1-AP production relative to cells treated with just 1-NP and CDDO-Im alone by 41.7% (p = 0.0012) (Figure 5C), suggesting that activated NRF2 was necessary for rotenone-suppression of 1-AP production. Menadione was not used in the experiments with HBEC3-KT cells because it fluoresced in the medium for HBEC3-KT cells at the wavelengths of interest for 1-AP detection.

Figure 5:

Figure 5:

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Effect of inhibitors of additional nitroreductases on the ability of (A) A549 cells and (B-C) HBEC3-KT cells to produce 1-AP following 1-NP exposure. N = 6 independent experiments performed in triplicate for each for (A), n = 4 independent experiments performed in triplicate for each for (B), and n = 5 independent experiments performed in triplicate for each for (C). Each individual data point represents the average of technical replicates within one experiment. Statistical analysis was performed with one-way ANOVA followed by Tukey’s post-hoc multiple comparison of means. Groups with non-shared superscript letters are significantly different (p < 0.05); shared superscript letters show no statistical significance between compared groups. Error bars represent mean ± SEM. Treatment durations were 48 h for (A-C). MD was not included for HBEC3-KT cells because a false positive signal developed with MD in the HBEC3-KT medium. Abbreviations: 1-NP, 1-nitropyrene; CEES, 2-chloroethyl ethyl sulfide; MD, menadione; DIC, dicoumarol; ALP, allopurinol; ROT, rotenone; CDDO-Im, 1-(2-cyano-3,12,28-trioxooleana-1,9(11)-dien-28-yl)-1H-imidazole.

Because CEES is a non-selective inhibitor of POR and thioredoxin reductase,65,66 we investigated the effect of additional POR inhibitors on 1-AP formation. In wild-type A549 cells, neither DPI76,77 nor OCA78,79 used as POR inhibitors altered 1-AP production relative to control (Figure 6A). In HBEC3-KT cells, 1-AP production was not altered by DPI or OCA relative to control (Figure 6B).

Figure 6:

Figure 6:

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Effect of POR inhibitors on the ability of (A) A549 cells and (B) HBEC3-KT cells to produce 1-AP following 1-NP exposure. N = 4 independent experiments performed in triplicate for each for (A-B). Each individual data point represents the average of technical replicates within one experiment. Statistical analysis was performed with one-way ANOVA followed by Tukey’s post-hoc multiple comparison of means. Groups with non-shared superscript letters are significantly different (p < 0.05); shared superscript letters show no statistical significance between compared groups. Error bars represent mean ± SEM. Treatment durations were 48 h for (A-B). Abbreviations: 1-NP, 1-nitropyrene; DPI, diphenyleneiodonium sulfate; OCA, n-octylamine.

Effect of NFE2L2 gene-deletion on ability of A549 cells to perform 1-NP nitroreduction

A549 wild-type cells containing two functional copies of NFE2L2, A549 cells with heterozygous expression of NFE2L2, and A549 cells with homozygous knockout of NFE2L2, were each evaluated in the context of 1-NP exposure, 1-NP and menadione exposure, and 1-NP and rotenone exposure (Figure 7). A549 cells with heterozygous expression of NFE2L2 and A549 cells with homozygous knockout of NFE2L2 had reduced 1-AP production compared to A549 wild-type cells with two functional copies of the NFE2L2 gene by 59.6% and 96.0%, respectively (p < 0.0001 and < 0.0001, respectively) when treated with 1-NP. With cells treated with 1-NP and menadione, A549 cells with heterozygous NFE2L2 expression and A549 cells with homozygous NFE2L2 knockout had reduced 1-AP production compared to A549 wild-type cells by 57.9% and 92.0%, respectively (p < 0.0001 and < 0.0001, respectively). With cells treated with 1-NP and rotenone, A549 cells with heterozygous NFE2L2 expression and A549 cells with homozygous NFE2L2 knockout had reduced 1-AP production compared to A549 wild-type cells by 58.2% and 94.6%, respectively (p < 0.0001 and < 0.0001, respectively). Within wild-type A549 cells, only rotenone decreased 1-AP production relative to control (p = 0.0370). Within A549 cells with heterozygous NFE2L2 expression, neither menadione nor rotenone decreased 1-AP production relative to control (p = 0.9988 and = 0.9558, respectively). Within A549 cells with homozygous NFE2L2 knockout, neither menadione nor rotenone decreased 1-AP production relative to control (p > 0.9999 and > 0.9999, respectively). In two-way ANOVA, the effect of A549 NFE2L2 status and the effect of inhibitor treatment were both statistically significant (p < 0.0001 and = 0.0404, respectively), but the interaction between the two was not statistically significant (p = 0.1788). These data imply that NRF2 is necessary for the inhibitory effect of rotenone and the effect of menadione, which also trended towards inhibition but was not statistically significant.

Figure 7:

Figure 7:

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Effect of NFE2L2/NRF2 gene editing on the ability of A549 cells to produce 1-AP following 1-NP exposure. N = 6 independent experiments performed in triplicate for each. Each individual data point represents the average of technical replicates within one experiment. Statistical analysis was performed with two-way ANOVA followed by Tukey’s post-hoc multiple comparison of means. Groups with non-shared superscript letters are significantly different (p < 0.05); shared superscript letters show no statistical significance between compared groups. Error bars represent mean ± SEM. Treatment durations were 48 h. Abbreviations: 1-NP, 1-nitropyrene; MD, menadione; ROT, rotenone.

Discussion

We used pharmacological approaches to evaluate the contribution of a variety of nitroreductases (POR, XO, AOX, NQO1, and AKR1C1-1C3) and pharmacological and genetic approaches to evaluate the contribution of NRF2 to the nitroreduction of 1-NP in human lung cells. POR, XO, and AOX are anaerobic nitroreductases and catalyze nitroreduction by successive one-electron steps.28-33,80 Our study showed that these enzymes did not play a role in the conversion of 1-NP to 1-AP. By contrast, NQO1 and AKR1C1-1C3 catalyze nitroreduction by successive two-electron steps.11,80 Our study shows that AKR1C1-AKR1C3 play a prominent role in the conversion of 1-NP to 1-AP. The properties of wild-type A549 cells with constitutively active NRF244-47 and of HBEC3-KT cells with inducible NRF212,43 permitted different pharmacological and genetic NFE2L2/NRF2 modulation to assess the NRF2 dependence of 1-NP nitroreduction.

Although both A549 wild-type and HBEC3-KT cells were able to generate 1-AP in a time- and concentration-dependent manner when exposed to 1-NP, the time course of 1-AP formation between the two cell lines differed. Whereas the rate of 1-AP production in A549 cells plateaued, the rate of 1-AP production in HBEC3-KT cells increased in a non-linear fashion especially at the higher concentrations of 1-NP. This may reflect the activation of NRF2 by 1-NP in HBECT3-KT cells at later time points to increase AKR1C1-1C3 expression and enhance 1-NP nitroreduction. 1-NP has been previously shown to activate NRF2.38,39 This could occur via modification of cysteine residues in KEAP1 by the formation of nitrosothiols or by their modification by ROS produced during the reoxidation of intermediates of nitroreduction. A549 cells were able to generate more 1-AP than HBEC3-KT cells regardless of time point with equivalent 1-NP concentrations. This likely reflects the expression of constitutively active NRF2 in A549 cells. Oncogenic properties of A549 cells could also contribute to increased nitroreduction. For example, differences in IL-1β and IL-6,81,82 which regulate AKR1C1-1C2 and AKR1C3, respectively,83 may also be determinants of differences in nitroreduction but their involvement are beyond the scope of the current study.

AKR1C inhibitors showed that AKR1Cs were the major nitroreductases for 1-NP in HBEC3-KT cells. For example, flufenamic acid and salicylic acid reduced 95.2% and 80.5% of 1-AP production in these cells, respectively. By contrast, salicylic acid reduced 51.1% of 1-AP production in A549 wild-type cells treated with 1-NP, and the effect of flufenamic acid was not statistically significant. The differences observed in these cell types may reflect the presence of additional 1-NP nitroreductase(s) in A549 cells. The effect of AKR1C3 inhibitors on 1-AP production was more similar between the cell types. In both cell types, we achieved up to about 35% inhibition of 1-AP production with indomethacin. By contrast, the effects of ASP9521 and BMT4-159 were less pronounced. The differential effects of these inhibitors could be related to the specificity of different organic anion transporters expressed in lung cells, such as SLC22A7 or OAT2,84-86 which would govern inhibitor uptake. It is noted that indomethacin is carboxylic acid, BMT4-159 is a nitro-naphthylaminobenzoate with a powerful electron-withdrawing group, and ASP9521 is not an anion. Additionally, unlike salicylic acid and ursodeoxycholate, flufenamic acid contains a trifluoromethyl group. The trifluoromethyl group is a bulky electron-withdrawing substituent that could provide steric hinderance and limit the uptake of flufenamic acid by organic anionic transporters, such as SLC22A7 or OAT2,84-86 affecting bioavailability and efficacy. Our findings show that AKR1C3 may not be as important as a 1-NP nitroreductase in HBEC3-KT cells where AKR1C1-1C2 inhibitors provided approximately 70-80% inhibition of 1-NP reduction.

We find that 1-NP nitroreduction is dependent on NRF2 in A549 wild-type and HBEC3-KT cells using pharmacological activation or inhibition. A549 wild-type cells did not show increased nitroreduction in response to NRF2 activators, SFN60 and CDDO-Im,61 but had decreased nitroreduction in response to NRF2 inhibitors, ML38562 and ATRA (metabolite of vitamin A)63,87. ML385 directly binds to Neh1 of NRF262 and ATRA forms a complex with retinoic acid receptor alpha to bind Neh7 of NRF2.63,64 The absence of an effect of NRF2 activators was expected in A549 cells in which NRF2 is constitutively active.12 Our study is the first to document the efficacy of NRF2 inhibitors to reduce 1-AP production in A549 wild-type cells and indicates that NRF2 pharmacological inhibition can be used to mitigate 1-NP nitroreduction. In HBEC3-KT cells, NRF2 activators, SFN and CDDO-Im, increased nitroreduction but the effect of NRF2 inhibitors, ML385 or ATRA by themselves, was not statistically significant. HBEC3-KT cells induced with CDDO-Im, however, were susceptible to ML385 or ATRA inhibition of 1-AP production, implying that NRF2 has to be activated before the NRF2 inhibitors will have an effect.

We also used inhibitors of other nitroreductases to identify the additional enzymes that may reduce 1-NP in wild-type A549 cells. To ensure cell-type specificity, we performed these experiments in both A549 wild-type and HBEC3-KT cells. The only statistically significant effect by inhibitor alone was observed with rotenone, which decreased 1-AP formation in a A549-cell specific manner. This effect may be partially attributable to rotenone-stimulated ROS generation,88,89 whereby the sequential reduction of oxygen to hydrogen peroxide would intercept superoxide anion as a reductant for 1-NP, a process that may be dependent on mitochondrial superoxide dismutase (mtSOD). As mtSOD is predicted to be constitutively active in the presence of constitutively active NRF2,90 this property would decrease the available superoxide anion to act as a reductant for 1-NP nitroreduction. Our finding that rotenone suppression of nitroreduction was observed in HBEC3-KT cells treated with CDDO-Im to activate NRF2, but not in HBEC3-KT cells in the absence of CDDO-Im, is consistent with the concept that high levels of NRF2 and mtSOD are required for rotenone to decrease nitroreduction. The prospect that rotenone activates NRF2 in HBEC3-KT cells is unlikely because this phenomenon would have increased AKR1C1-1C3 expression and increase nitroreduction in these cells to a greater extent than observed. The possibility also exists that complex I NADH-ubiquinone reductase is a nitroreductase itself, perhaps aided by semiquinone as a reductant, to render the rotenone inhibition effective. Menadione also generates superoxide anion and hydrogen peroxide;91,92 however, its effect on 1-AP production did not reach statistical significance in A549 cells. The lack of a significant effect of menadione implies that its target(s) (i.e., AOX) do not act as an aerobic 1-NP nitroreductase.

The use of A549 cells with heterozygous and homozygous NFE2L2/NRF2 knockdown supports the premise that 1-NP nitroreduction is NRF2-dependent and provides insight into the modulation of nitroreduction by rotenone and possibly menadione. A549 wild-type cells had a greater capacity to reduce 1-NP by nitroreduction than A549 cells with heterozygous NFE2L2 knockout, which in turn produced more 1-AP than A549 cells with homozygous NFE2L2 knockout. The homozygous knockout led to an almost complete loss of 1-NP reduction, showing the process is NRF2-dependent. Menadione or rotenone did not produce further inhibition of 1-NP nitroreduction in the A549 cells with gene-edited NFE2L2, suggesting that any effects of these agents in wild-type A549 cells were also mediated by NRF2. In support of this observation, NADH-ubiquinone reductase, the target of rotenone,74,75 may be regulated indirectly by NRF2 because NRF2 in fibroblasts was necessary to maintain the mitochondrial NADH supply93 necessary for NADH-ubiquinone reductase activity. Inactive NADH-ubiquinone reductase would prevent a rotenone effect on nitroreduction. Although the effect of menadione in A549 wild-type cells is not statistically significant, the AOX1 gene is downstream of NRF2,94 consistent with the expectation that cells with compromised NRF2 may not be sensitive to AOX1 inhibition. Because A549 cells with homozygous NFE2L2 knockout have no expression of AKR1C1-1C312 and have lost their ability to perform 1-NP nitroreduction that is not further altered by rotenone or menadione, we conclude that NRF2-regulated AKR enzymes are the major culprits responsible for 1-NP reduction. Genetic knockdown of NFE2L2/NRF2 in A549 cells was previously shown to eliminate the nitroreduction of 3-NBA, another component of diesel exhaust, demonstrating its dependence on NRF2 for its metabolic activation, as well.12 Our data support the premise that NRF2 activation may be required for the metabolic activation of all nitroarenes.

With the exception of indomethacin, which was used at 10 μM to avoid fluorescence interference, the concentrations of the activators and inhibitors used in the current study were identical to those used in respective studies on 3-NBA11,12 so that this study can be compared to the other two. The concentrations of the inhibitors used are expected to be saturating based on IC50 values for the respective enzymes.50,53-56,58,59,62-66,68,71,73,74,77,79 CEES, OCA, and DPI were used at just below cytotoxic concentrations (data not shown). The concentrations of the two activators used, SFN and CDDO-Im, were shown to have about equal effects on nuclear NRF2 translocation based on western blots.12

The detection of 1-AP formation implies that metabolism of 1-NP has proceeded through upstream metabolites, such as N-OH-1-AP, the hydroxylamino metabolite needed for formation of the N-(deoxyguanosin-8-yl)-1-aminopyrene DNA adduct. Whether the differences in 1-AP production seen with NRF2 modulation correlates to a difference in formation of DNA adducts or mutations derived from 1-NP exposure remains to be determined.

In summary, we demonstrate a clear role for AKR1C1-1C3 and NRF2 in the nitroreduction of 1-NP in human lung cells, A549 and HBEC3-KT (Figure 8). Additional modulators of 1-NP nitroreduction in A549 cells may include rotenone-targeted inhibition of NADH-ubiquinone reductase and ROS. Our study shows a dark side of NRF2 activation because its induction of AKR1C1-1C3 can increase the metabolic activation of 1-NP and 3-NBA, suggesting that NRF2 activators should be used with caution in trials aimed to reduce risk of lung cancer from air pollution.

Figure 8:

Figure 8:

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Scheme of the dependence of nitroreduction of 1-NP on NRF2 and AKR1C1-1C3 in a system of constitutive NRF2 versus inducible NRF2. We found that AKR1C1-1C3 inhibitors reduce 1-AP production in each cell type, and effects of flufenamic acid, salicylic acid, and ursodeoxycholate were particularly potent in HBEC3-KT cells, each causing over 70% inhibition to support that AKR1Cs are the major 1-NP nitroreductases in HBEC3-KT cells. Whereas NRF2 activators were effective at increasing 1-AP production in HBEC3-KT cells that have inducible NRF2, NRF2 inhibitors were effective at decreasing 1-AP production in A549 cells that have constitutively active NRF2. By contrast, we did not find POR inhibitors, a NQO1 inhibitor, or a XO inhibitor to reduce 1-AP production in either cell type. Our results with rotenone imply a role of ROS or mitochondrial nitroreductases in the nitroreduction of 1-NP, particularly for A549 cells where AKR1C inhibition could not explain all contribution to 1-NP nitroreduction.

Supplementary Material

Supporting Infromation

Table S1: Compounds used in the study

Figure S1: Fluorescence excitation spectra of (A) 100% MeOH blank, (B) 21.3 nM 1-AP in 100% MeOH, and (C) 1.33 μM 1-NP in 100% MeOH

Figure S2: Fluorescence emission spectra of (A) 100% MeOH blank, (B) 21.3 nM 1-AP in 100% MeOH, and (C) 1.33 μM 1-NP in 100% MeOH

Figure S3: Fluorescence calibration curves for 1-AP and 1-NP at 360 nm excitation and 426 nm emission

Acknowledgements

The authors thank Dr. John D. Hayes from the University of Dundee for provision of the A549 cells with heterozygous NFE2L2 (+/−), A549 cells with homozygous NFE2L2 (−/−), and the respective control cells. The authors thank members of the Penning laboratory for helpful scientific discussions.

Funding Information

This project was supported National Institutes of Health (NIH) grants P30-ES013508 (TMP) and R01-ES029294 (TMP) and by Training Grant T32-ES019851 (ALS) awarded by the National Institute of Environmental Health Sciences (NIEHS). The contents expressed in this publication are solely the responsibility of the authors and do not necessarily represent the views of the NIH or NIEHS.

Footnotes

Conflict of Interest

TMP is a member of the Expert Panel for the Research Institute for Fragrance Materials, the founder of Penzymes, and is a consultant for Propella Therapeutics.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Infromation

Table S1: Compounds used in the study

Figure S1: Fluorescence excitation spectra of (A) 100% MeOH blank, (B) 21.3 nM 1-AP in 100% MeOH, and (C) 1.33 μM 1-NP in 100% MeOH

Figure S2: Fluorescence emission spectra of (A) 100% MeOH blank, (B) 21.3 nM 1-AP in 100% MeOH, and (C) 1.33 μM 1-NP in 100% MeOH

Figure S3: Fluorescence calibration curves for 1-AP and 1-NP at 360 nm excitation and 426 nm emission

NIHMS1871388-supplement-Supporting_Infromation.docx (176.4KB, docx)

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