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J Biol Chem
J Biol Chem
The Journal of Biological Chemistry
0021-9258
1083-351X
American Society for Biochemistry and Molecular Biology

S0021-9258(24)02127-6
10.1016/j.jbc.2024.107626
107626
JBC Communication
Nicotine inhalation and metabolism triggers AOX-mediated superoxide generation with oxidative lung injury
Zweier Jay L. Jay.Zweier@osumc.edu
1∗
Kundu Tapan 1
Eid Mahmoud S. 1
Hemann Craig 1
Leimkühler Silke 2
El-Mahdy Mohamed A. 1
1 Division of Cardiovascular Medicine, and the EPR Center, Department of Internal Medicine, Davis Heart and Lung Research Institute, College of Medicine, The Ohio State University, Columbus, Ohio, USA
2 Department of Molecular Enzymology, Institut für Biochemie und Biologie, Universität Potsdam, Potsdam, Germany
∗ For correspondence: Jay L. Zweier Jay.Zweier@osumc.edu
02 8 2024
9 2024
02 8 2024
300 9 10762612 2 2024
22 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
With the increasing use of vaping devices that deliver high levels of nicotine (NIC) to the lungs, sporadic lung injury has been observed. Commercial vaping solutions can contain high NIC concentrations of 150 mM or more. With high NIC levels, its metabolic products may induce toxicity. NIC is primarily metabolized to form NIC iminium (NICI) which is further metabolized by aldehyde oxidase (AOX) to cotinine. We determine that NICI in the presence of AOX is a potent trigger of superoxide generation. NICI stimulated superoxide generation from AOX with Km = 2.7 μM and Vmax = 794 nmol/min/mg measured by cytochrome-c reduction. EPR spin-trapping confirmed that NICI in the presence of AOX is a potent source of superoxide. AOX is expressed in the lungs and chronic e-cigarette exposure in mice greatly increased AOX expression. NICI or NIC stimulated superoxide production in the lungs of control mice with an even greater increase after chronic e-cigarette exposure. This superoxide production was quenched by AOX inhibition. Furthermore, e-cigarette-mediated NIC delivery triggered oxidative lung damage that was blocked by AOX inhibition. Thus, NIC metabolism triggers AOX-mediated superoxide generation that can cause lung injury. Therefore, high uncontrolled levels of NIC inhalation, as occur with e-cigarette use, can induce oxidative lung damage.

Keywords

aldehyde oxidase
electronic cigarettes
superoxide
free radicals
EPR spin-trapping
Abbreviations

AOX aldehyde oxidase

EC electronic cigarettes

NIC nicotine

NICI NIC iminium

RLX raloxifene

ROS reactive oxygen species

Reviewed by members of the JBC Editorial Board. Edited by Ursula Jakob
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pmcNicotine (NIC) is the active and addicting element of tobacco cigarettes and electronic cigarettes (EC). Given the harmful effects of tobacco cigarettes, EC were first proposed as a safer alternative; however, a number of recent studies have shown that EC can also cause a variety of toxicities (1, 2, 3, 4). Chronic NIC exposure can play a role in the induction and progression of cardiovascular and pulmonary disorders (5, 6, 7). While chronic exposure to high concentrations of NIC upregulates numerous vasoconstrictors and inflammatory mediators that have a critical role in inducing disease (8, 9), it is not clear how this occurs and how NIC or its metabolism triggers this process.

NIC metabolism in humans occurs in two major steps involving the enzymes P450 (CYP2A6) and aldehyde oxidase (AOX) (Fig. 1A). In the first step NIC is metabolized to the nicotine Δ1′(5′)-iminium ion (NICI), and in the second step NICI is further metabolized to the major NIC metabolic product cotinine (10, 11, 12). AOX is a molybdo-flavoenzyme similar to xanthine oxidase (13, 14, 15). It catalyzes the oxidation of aldehydes and N-heterocyclic compounds with electron transfer to O2 (14, 15, 16, 17). While this process would be predicted to generate reactive oxygen species (ROS) including superoxide (O2•−) and hydrogen peroxide (18, 19), the magnitude and process of ROS formation has not been characterized and its potential pathophysiological significance is unknown.Figure 1 Nicotine (NIC) metabolism forms NIC iminium (NICI) that in the presence of human aldehyde oxidase-1 (hAOX1) is a potent source of O2•−.A, in the major pathway of NIC metabolism, NIC is first oxidized by Cytochrome P450 2A6 (CYP2A6)-catalyzed 5′-oxidation to nicotine Δ1′(5′)-iminium ion, followed by the hAOX1 catalyzed oxidation of nicotine Δ1′(5′)-iminium ion (NICI) to cotinine with the concomitant production of superoxide (O2•−) and hydrogen peroxide (H2O2). B, SDS-PAGE of purified recombinant hAOX1: Lane 1, molecular weight (MW) markers; lane 2, purified hAOX1, showing a single major band at ∼148 kDa. C, measurement of O2•− generation by cytochrome-c (Cyt-c) reduction from recombinant hAOX1 and NICI in air-saturated 50 mM phosphate buffer, pH 7.8, containing 0.1 mM EDTA, 100 μM Cyt-c, 100 U/ml catalase, and 40 nM hAOX1. The reaction was initiated by adding NICI in varying concentrations as labeled for each time course curve. D, kinetic analysis of the O2•− generation rate for a given NICI concentration. Data were fit to the Michaelis-Menten equation by non-linear regression with Km and Vmax values determined as shown. E, effects of the hAOX1 inhibitor raloxifene (RLX) and of SOD1. Values shown are mean ± SD, n = 4. F, EPR spin-trapping of O2•− generation from hAOX1 and NICI performed with 25 nM hAOX1, 10 μM NICI, 100 U/ml catalase, and 10 mM DIPPMPO in chelexed phosphate-buffered saline with 0.1 mM DTPA, pH 7.4. No O2•−–derived signal was detected with 0 μM NICI, but with 10 μM NICI, a large signal was seen, best simulated as a combination of DIPPMPO-OOH (84%) and DIPPMPO-OH (16%) adducts (Sim), with EPR parameters: DIPPMPO-OOH aP ∼ 49.83 G, aN ∼ 13.14 G, aHβ ∼ 11.12 G, aHƔ ∼ 0.90 G, and DIPPMPO-OH aP ∼ 47.92 G, aN ∼ 12.90 G, aHβ ∼ 13.64 G. G, time course of the increase in the EPR signal of the O2•−-derived DIPPMPO–OOH adduct, associated with the spectrum in F with the spectrum shown after the signal plateaued.

Therefore, we measure and characterize the process of ROS generation from human AOX in the presence of NICI. Using the isolated enzyme, we measure the kinetics of this process determining its rates of O2•− generation and substrate concentration-dependence. In a murine EC-exposure model, we observe that NICI or NIC stimulate AOX-mediated O2•− generation in the lungs. This is exacerbated by chronic EC vaping exposure, where induction of high AOX levels leads to enhanced ROS production that causes oxidative lung injury, while EC vehicle without NIC had much less effect.

Results

Human recombinant AOX1 (hAOX1) was purified and characterized by SDS gel electrophoresis with single major band seen at ∼150 kDa molecular weight with purity of >95% (Fig. 1B). UV-Vis absorption spectra showed the typical profile of hAOX1 with absorbance bands centered at 350 and 450 nm and a shoulder at 550 nm (20). The hAOX1 had high enzyme activity of ∼1150 nmol/min/mg enzyme with phenanthridine substrate as reported (21).

In order to measure the production of O2•− from hAOX1 in the presence of the major NIC metabolite NICI, cytochrome-c (Cyt-c) reduction assays were performed as a function of NICI concentration from 1 to 50 μM. With substrate concentrations as low as 1 μM in the presence of hAOX1 (40 nM) strong O2•− generation was seen that further increased with increasing NICI concentrations (Fig. 1C). The rate of O2•− generation as a function of NICI concentration exhibited Michaelis-Menton kinetics with a Km of 2.7 ± 0.4 μM with a Vmax of 794 ± 22 nmol/min/mg of hAOX1 (Fig. 1D). Since recombinant hAOX1 preparations including that assayed have only partial occupation of the molybdenum cofactor binding site of ∼60% (21), one can estimate the Vmax for the holoenzyme as ∼1323 nmol/min/mg. Thus, hAOX1 metabolizes NICI with high affinity and very high rate of O2•− production.

To further validate the formation of O2•− from hAOX1 and NICI, experiments were performed in the presence of SOD1 (0.1 μM). SOD1 abolished the Cyt-c reduction (Fig. 1E). Furthermore, the AOX inhibitor raloxifene (RLX; 0.5 μM) also quenched Cyt-c reduction by >95%. No significant O2•− formation was seen in the absence of either NICI or hAOX1 (Fig. 1E).

EPR spin-trapping was used to directly confirm the process of O2•− generation. While no signal was seen with hAOX1 (25 nM) alone, following the addition of 10 μM NICI a large signal of trapped O2•− (DIPPMPO-OOH) appeared (Fig. 1F). Computer simulations showed that 84% of the observed signal was present as the –OOH adduct of directly trapped O2•− with 16% as –OH adduct. In kinetic measurements, the levels of the -OOH adduct increased rapidly over the first 180 s and then plateaued (Fig. 1G).

Thus, it is clear that μM levels of NICI in the presence of hAOX1 are sufficient to produce a high rate of O2•− and ROS generation. EC liquids typically contain 12 to 36 mg/ml of NIC that corresponds to 74 to 222 mM NIC. In human EC users, NIC levels of ∼ 50 μM were measured in sputum 30 min after vaping and it was extrapolated that initial NIC levels in the lung could be much higher in the mM range (22).

In order to determine if chronic exposure to the process of NICI-induced O2•− generation from AOX can cause lung toxicity and oxidative injury, experiments were performed in a chronic mouse model of EC-exposure designed to mimic exposure levels that occur in humans (6, 23). This mouse model enables dose-controlled EC exposure with assessment of organ-specific toxicity, providing lung tissue that cannot be similarly obtained in humans. Of note, while there is only one AOX isoform in humans, mice have 4 AOX isoforms with AOX1 as the major isoform expressed in the lungs along with AOX3 (24). The mouse AOX1 has 83% homology to hAOX1. Both human and mouse AOXs are structurally and functionally similar as dimers of two identical 150 kD subunits with N-terminal domain containing two [2Fe-2S] clusters, a central FAD-binding domain and a C-terminal molybdenum cofactor containing the substrate binding domain (13, 24).

In the mouse EC exposure model, we measured the levels of AOX1 present with or without exposure for 16 weeks to aerosol generated from EC-liquid containing 24 mg/ml NIC (ECN). Immunoblotting of lung tissue demonstrated that AOX1 was constitutively expressed in the lungs of control mice and that EC-exposure increased lung AOX1 expression by over 6-fold (Fig. 2, A and B). Immunohistology confirmed the presence of AOX1 in the lungs and the increase following ECN-exposure with very high expression of AOX1 in the bronchiolar epithelium with increased expression also present in the alveoli (Fig. 2, C and D).Figure 2 AOX1 is expressed in the lungs with a marked increase following EC exposure leading to enhanced NICI or NIC–derived O2•−generation. Lung sections and lung tissue homogenates from mice exposed for 16 weeks to air (Air) or EC aerosol generated from EC liquid containing 24 mg/ml NIC (ECN) were studied. A, immunoblotting (IB) of lung homogenates for AOX1 and for GAPDH loading control. B, quantitation of AOX1 expression from IB. C, immunofluorescence (IF) of lung sections incubated with primary antibody (Ab) against AOX1 followed by fluorescent-labeled secondary Ab (green). DAPI was used as a nuclear stain (blue). D, quantitative analysis of AOX1 from IF images. E, fluorescence detection of O2•− in lung sections incubated with dihydroethidium (DHE; 10 μM) for 30 min alone (E1) or together with: 10 μM NICI (E2); 10 μM NICI + 100 μM SOD mimetic (SODm) MnTBAP (E3); 10 μM NICI + 100 μM AOX inhibitor raloxifene (RLX) (E4); or 100 μM NIC (E5). Red fluorescence arises from the O2•−-mediated oxidation of DHE. Images in C and E were obtained by confocal microscopy with the white scale bar corresponding to 50 μm. Bar graphs in E show quantitative analysis of O2•− generation in arbitrary units (AU). Graphs show means ± SD of data from 3 mice. Statistical analysis was done by unpaired t test in panels B & D and two-way ANOVA followed by Tukey's multiple comparison test in E. For B, D & E, p values are marked for comparison of ECN to Air. In E, # significant from DHE for matched Air or ECN treatments. For comparison across Air and ECN groups 1 to 5, p values are: for Air- 2 versus 1 (<0.0001), 3 versus 1 (0.3700), 4 versus 1 (0.9865), 5 versus 1 (<0.0001): for ECN- 2 versus 1 (p < 0.0001), 3 versus 1 (<0.0006), 3 versus 1 (0.0006), 4 versus 1 (0.0023), 5 versus 1 (p < 0.0001); $ significant from DHE/NICI for matched Air or ECN treatment. For comparison across Air and ECN groups 1 to 5, p values are: for Air- 3 versus 2 (<0.0001), 4 versus 2 (<0.0001), 5 versus 2 (0.0936); for ECN- 3 versus 2 (<0.0001), 4 versus 2 (<0.0001), 5 versus 2 (0.0724); for ECN 3 versus 2 (<0.0001), 4 versus 2 (<0.001), 5 versus 2 (0.0724).

O2•− generation in the lungs of these mice was assessed using the fluorescence probe DHE which is oxidized by O2•− to 5-hydroxy-ethidium that exhibits red fluorescence (25, 26). It was observed that paralleling the rise in AOX1 expression, ECN-exposure increased the levels of O2•− generated in the lungs of these mice compared to that seen with control air-exposure (Fig. 2E1). Addition of NICI (10 μM) stimulated O2•− generation in the lungs of air-exposed mice, while even higher levels were seen with ECN-exposure (Fig. 2E2). The observed fluorescence was confirmed to be derived from O2•− as it was quenched by addition of a SOD mimetic (SODm) (Fig. 2E3). The elevated O2•− generation was shown to be largely derived from AOX-mediated metabolism as it was blocked by AOX inhibition with RLX (Fig. 2E4). It was also observed that addition of NIC (100 μM) resulted in similar stimulation of O2•− generation as with NICI (Fig. 2E5). Thus, the process of NIC and NICI metabolism to cotinine results in AOX-mediated O2•− generation.

In order to determine if this NIC-stimulated ROS generation was associated with oxidative lung damage, we assayed oxidative protein and DNA modifications with comparative measurements performed following 16 weeks of exposure in 3 groups of mice: (1) air-control; (2) EC vehicle (ECV); and (3) EC vehicle with 24 mg/ml of NIC (ECN). ECN-exposure increased protein and DNA oxidation, detected by ELISA, with ∼5-fold higher levels of protein carbonylation and >3-fold increase in the level of oxidized guanine species compared to lungs of matched ECV-exposed mice with only trace levels in air-control mice (Fig. 3, A and B). Immunohistology also showed much higher levels of protein tyrosine nitration in the bronchial epithelium and alveoli of lungs from ECN-exposed mice than in lungs of ECV-mice with only trace levels in lungs of air-control mice (Fig. 3, C and D). DNA oxidation with increased 8-hydroxy-2′-deoxyguanosine was also seen in the lungs of ECN-exposed mice compared with ECV-exposed or air-control mice (Fig. 3, E and F). Thus, ECN inhalation was associated with oxidative lung injury well above that seen with NIC-free vehicle.Figure 3 Protein carbonylation, tyrosine nitration, and DNA oxidative damage. Measurements were performed in lung homogenates and sections from mice exposed for 16 weeks to air (Air) or electronic cigarette (EC) aerosol generated from EC liquid containing 0 mg/ml NIC (ECV) or 24 mg/ml NIC (ECN). A, ELISA quantitation of protein carbonyls. B, ELISA for oxidized guanine species. C, immunofluorescence (IF) of lung sections incubated with primary antibody (Ab) to nitrotyrosine (NT) followed by corresponding fluorescent-labeled secondary Ab (red). DAPI (blue) was used as a nuclear stain. D, quantitation of the NT-derived IF in C, expressed as arbitrary units (AU). E, IF of lung sections incubated with primary Ab to 8-hydroxy-2′-deoxyguanosine (8-OHdG) followed by corresponding fluorescent-labeled secondary Ab (green). F, quantitative analysis of 8-OHdG-derived IF. Images in (C) & (E) were obtained by confocal microscopy with the white bar corresponding to 50 μm. A, B, D, and F show means ± SD of data from 5 mice. Statistical analysis was done by one-way ANOVA with Tukey's multiple comparison test. p values for intergroup comparisons are as shown. EC exposure with inhalation of aerosol containing NIC triggered much greater protein and DNA oxidative damage than matched exposure without NIC.

Further experiments were performed to directly evaluate the role of AOX in the oxidative lung injury seen with ECN-exposure. Mice were subjected to a high intensity protocol of 6 h/day of ECN exposure for 14 consecutive days with and without daily pretreatment with the potent AOX inhibitor raloxifene (RLX). Following this exposure, measurements of oxidative lung injury were performed. With RLX treatment compared to ECN alone, protein carbonyl levels in lung tissue were decreased 2.5-fold to levels similar to those in air-control mice, and malondialdehyde protein adduct levels were similarly decreased (Fig. 4, A and B). Immunohistology also showed that RLX-treatment greatly decreased nitrotyrosine and 8-hydroxy-2′-deoxyguanosine levels (Fig. 4, C and D). Thus, AOX inhibition prevented the ECN exposure-induced increase in oxidative lung injury.Figure 4 AOX inhibition prevents EC-induced oxidative lung injury. Measurements were performed in lung homogenates and sections from mice exposed for 14 days to either fresh air (Air) or EC aerosol generated from EC liquid containing 24 mg/ml NIC (ECN) with daily intraperitoneal injection of vehicle or raloxifene (RLX) (25 mg/kg) (ECN + RLX). A, ELISA quantitation of protein carbonyls. B, ELISA for malondialdehyde (MDA)-protein adducts. C, immunofluorescence (IF) of lung sections incubated with primary antibody (Ab) against nitrotyrosine (NT) followed by corresponding fluorescent-labeled secondary Ab (red). DAPI (blue) was used as a nuclear stain. D, quantitation of the NT-derived IF in (C), expressed as arbitrary units (AU). E, IF of lung sections incubated with primary Ab against 8-hydroxy-2′-deoxyguanosine (8-OHdG) followed by corresponding fluorescent-labeled secondary Ab (green). F, quantitative analysis of 8-OHdG-derived IF. Images in C & E were obtained by confocal microscopy with the white bar corresponding to 50 μm. Data in (A), (B), (D), and (F) are means ± SD from 5 mice. Statistical analysis was done as in Figure 3. p values for intergroup comparisons are as shown. AOX inhibition prevented EC-induced oxidative lung damage with a decrease in protein carbonylation, lipid peroxidation, tyrosine nitration and DNA oxidative damage.

Discussion

In view of the well-established toxicity of tobacco cigarette smoking, electronic cigarettes were developed and proposed as a safe form of inhaled NIC delivery. However, with EC use, sporadic lung injury has been reported with lung toxicity seen in humans and animal models (2, 3, 4, 5, 6). As NIC delivery from EC products is poorly controlled with high levels of NIC present in EC liquids, there is the potential for high levels of NIC to be delivered to the lungs. In view of this, there is a need to better understand the ramifications of EC-mediated NIC delivery and metabolism within the lungs.

We observed that the process of NIC metabolism intrinsically results in O2•− generation through the reaction of the NIC metabolite NICI with AOX. NICI is a unique high affinity substrate for AOX1 (16, 17). We show that it generates O2•− with Vmax estimated for the holoenzyme of 1323 nmol/min/mg, corresponding to a turnover number, kcat, of 199 min−1. Since the Km value is measured as 2.7 μM, the catalytic efficiency (kcat/Km) is 74 min−1 μM−1. Thus, this reaction proceeds with a high rate even with low μM levels of NICI so that it can result in high levels of ROS generation.

In our chronic mouse EC-exposure model, designed to mimic human EC aerosol exposure (6, 23), we make the novel observation that EC-exposure greatly increases AOX1 expression in the lungs leading to increased O2•− generation that is greatly stimulated by NICI or NIC but blocked by AOX inhibition. In comparative studies of mice chronically exposed to EC aerosol with NIC versus EC vehicle alone, it was clearly seen that oxidative damage to proteins and nucleic acids was greatly increased in the presence of NIC. While significant increases in oxidative damage occurred even in the absence of NIC, in the presence of NIC oxidative damage was greatly increased. Furthermore, in experiments with AOX inhibition oxidative lung injury was greatly decreased.

Thus, with EC exposure that contains high levels of NIC, marked induction of AOX1 expression in the lungs occurs, which in the presence of NIC and its NICI metabolite leads to high levels of O2•− and secondary ROS generation. This in turn was shown to trigger cellular injury with oxidative modification of proteins and DNA. Therefore, the high levels of NIC delivered to the lungs by EC inhalation can result in oxidative damage that could contribute to the lung injury and inflammation observed in some EC users. The severity of this process would depend on the concentrations of NIC in the EC liquid as well as the amounts of aerosol inhaled, which are a function of device power and puff duration. To decrease the high levels of NIC-mediated oxidant stress in the lungs, the levels of NIC present in the EC aerosol and the amounts delivered should be limited. Our observations indicate that the process of high-level NIC delivery to the lungs as can occur with EC use can be intrinsically harmful.

Experimental procedures

Materials

Chemicals, reagents, and other materials were from Millipore-Sigma, unless noted otherwise. S(−)-Nicotine-Δ1′(5′)-iminium diperchlorate was provided by Dr R.S. Obach (Pfizer). 5-(Diisopropoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DIPPMPO) was from Enzo-Alexis.

Expression and purification of hAOX1

Cloning, expression and purification of hAOX1 was as reported (20).

Cytochrome c (Cyt-c) reduction

O2• − formation was detected at 24 °C by measuring the rate of the SOD-inhibitable reduction of ferric-Cyt-c using an Agilent 8453 diode array spectrophotometer. Initial rates were calculated using the extinction coefficient of ferrous-Cyt-c at 550 nm of 21,000 M−1 cm−1 (27).

Electron paramagnetic resonance (EPR) spectroscopy

EPR spectra were recorded on a Bruker EMX spectrometer with HS resonator as reported (28). The sample was placed in a flat cell and measurements performed at ambient temperature using DIPPMPO (10 mM) spin trap in chelexed phosphate buffered saline (PBS) pH 7.4. EPR parameters were: microwave frequency, 9.78 GHz; microwave power, 20 mW; modulation amplitude, 0.5 G; time constant, 82 ms; scan time, 42 s. EPR spectral simulations were performed as described (29, 30).

Animals and EC-Exposure

Studies were approved by the Institutional Laboratory Animal Care and Use Committee (IACUC) and conformed to NIH Guidelines for the Care and Use of Laboratory Animals. Sixteen-week-old male C57BL/6J mice from Jackson Labs, were acclimatized for 2 weeks, then exposed to EC aerosol containing 0 mg/ml NIC (ECV) or 24 mg/ml NIC (ECN) in polyethylene glycol and glycerol (1:1 v/v). Exposure to EC aerosol was performed using our computer-controlled high-throughput vaping exposure system (31). The aerosol was generated by an EC Mod (JoyeTech, eVic Basic) with 0.2 Ω Kanthal wire coil and 25 W power. To determine if chronic exposure causes oxidative lung injury, mice were exposed to ECV or ECN for 2 h/day, 5 days/wk for 16 weeks. To evaluate the effect of AOX inhibition on EC-induced lung injury, mice were intraperitoneally injected daily with 20 mg/kg RLX in 7% aqueous DMSO or vehicle alone and exposed to ECN 6 h/day for 14 days, with exposures divided into two 3-h blocks separated by a 60-min interval during which mice had access to fresh air, food and water.

Immunoblotting

Lung tissue was homogenized in lysis buffer containing 62 mM Tris (pH 6.8), 10% glycerol, 2% sodium dodecyl sulfate (SDS) supplemented with protease/phosphatase inhibitor and centrifuged at 12,000g for 30 min at 4 °C. Protein concentration was determined by Bio-Rad DC protein assay (Bio-Rad). Supernatant was mixed with SDS sample loading buffer, heated at 70 °C for 5 min, separated on a gradient (4–20%) polyacrylamide gel (Bio-Rad) and electroblotted on a PVDF membrane (Bio-Rad). Membranes were incubated for 1 h at 37 °C in Tris-buffered saline-0.1% Tween 20 (TBST) containing 5% nonfat dry milk and washed ×3 with TBST, then incubated overnight at 4 °C in TBST-1% milk containing primary rabbit polyclonal antibody (Ab) with high affinity for human and mouse AOX1 (immunogen sequence 571–616 of hAOX1 that has 83% homology to mAOX1 with much lower homology to mAOX3 (Invitrogen) (1:500 dilution)) or GAPDH (Cell Signaling Technology) (1:2000 dilution), then washed ×3 with TBST, followed by addition of horseradish peroxidase-linked secondary anti-rabbit Ab in TBST (1:2000 dilution) (Cell Signaling Technology) for 1 h at 37 °C. Membranes were washed in TBST, then developed using immunoblotting detection reagent (Clarity Max Western ECL). Protein bands were captured and analyzed using a high-resolution KwikQuant Imager and Software (Kindle Biosciences). Protein bands were normalized to GAPDH. Each Ab used was validated for specificity versus the purified target as documented by the manufacturer’s data sheets and confirmed in our studies.

Immunofluorescence

Lungs were dissected, inflated, placed in optimal cutting temperature (OCT) embedding compound (Sakura Finetek), and cryosectioned. 5 μm sections were placed on chilled super-frosted slides, permeabilized using 0.5% Triton-X, fixed with 2% paraformaldehyde for 10 min, and blocked by 10% goat serum and 1% BSA in PBS-0.5% tween 20 (PBST). Sections were incubated overnight with primary Abs at 4 °C, washed with PBST, and incubated for 1 h at 37 °C in PBST containing fluorescent secondary Abs. Anti-fade mounting medium (SouthernBiotech) containing 1 μM nuclear stain 4,6-diamidino-2-phenylindole (DAPI) was applied and coverslips placed on the sections. Primary rabbit polyclonal Ab, as above, against AOX1 (1:100 dilution), NT (Cell Signaling Technology) (1:100 dilution), or 8-OHdG (Santa Cruz Biotechnology, Dallas, TX) (1:100 dilution) were used with secondary anti-rabbit or anti-mouse Alexa Fluor 594 and Alexa Fluor 488 Abs (Invitrogen) at 1:1000 dilutions. Sections were imaged by confocal microscopy (FluoView 3000; Olympus America) and images analyzed using FluoView 3000 software. At least 3 sections per mouse, with at least 4 fields per section and 3 to 5 mice/group were examined. Each Ab was validated for specificity versus the purified target on immunoblotting and also tested in cells with high target expression as provided by the manufacturer’s data sheets and confirmed in our studies.

Assay of O2•− generation in lung

Dihydroethidium (DHE)-derived fluorescence was used to detect O2•− generation in tissue sections as described (23). Sections were incubated with 10 μM DHE (Molecular Probes, Inc., OR) in the dark for 30 min, either alone or with addition of 10 μM NICI alone or together with AOX1 inhibitor RLX 100 μM, superoxide dismutase mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP; SODm) 100 μM, or NIC 100 μM. Sections were washed with PBST, anti-fade mounting medium containing 1 μM DAPI applied, coverslips placed, and imaged by confocal microscopy as above. DHE-derived fluorescence was detected with excitation and emission wavelengths of 490 nm and 555 nm (32).

Assay of protein carbonyls and malondialdehyde

Protein carbonyls were assayed from lung homogenate by colorimetric ELISA kits (Abcam, Boston, MA & Cell Biolabs) per manufacturer’s recommendations. Protein carbonyls are derivatized to dinitrophenyl hydrazone (DNPH). Primary Ab against DNPH is used, followed by HRP-conjugated secondary. Protein carbonyls were quantitated by comparing absorbance at 450 nm to that of a known oxidized BSA standards. Malondialdehyde-protein adducts were assayed by colorimetric ELISA (Cell Biolabs) per manufacturer’s recommendations.

Assay of nucleic acid oxidation

Oxidized guanine species including 8-hydroxy-2′-deoxyguanosine (8-OHdG), 8-hydroxyguanine (8-OHG), and 8-hydroxyguanosine, were measured by ELISA kit (Cayman, Ann Arbor, MI) per manufacturer’s recommendations from DNA extracted from lung tissue homogenized in 0.1 M phosphate buffer, pH 7.4 with 1 mM EDTA. DNA was extracted using a genomic DNA extraction kit (Invitrogen), then digested with nuclease P1, and incubated for 10 min with alkaline phosphatase at 37 °C.

Data availability

Data will be shared upon request. Contact Jay L. Zweier, 420 West 12th Ave, TMRF 116A, Columbus, Ohio 43210, email: jay.zweier@osumc.edu.

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Acknowledgments

We are grateful to Dr R.S. Obach of Pfizer who provided the S(−)-Nicotine-Δ1′(5′)-iminium diperchlorate used.

Author contributions

J. L. Z., C. H., S. L., T. K., M. S. E., and M. A. E-M writing–review & editing; J. L. Z. writing–original draft; J. L. Z. supervision; J. L. Z. project administration; J. L. Z. funding acquisition; J. L. Z. conceptualization. C. H., T. K., M. S. E., and M. A. E-M visualization, C. H., T. K., M. S. E., and M. A. E-M investigation; S. L. resources. M. S. E. Writing – review & editing, Visualization, Investigation.

Funding and additional information

This work was supported by 10.13039/100000054 National Cancer Institute , 10.13039/100000002 National Institutes of Health , and 10.13039/100002590 American Lung Association Grants CA270608 , R01HL135648 , R01HL131941 , and GRT00060635 to J.L.Z. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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