2018 Volume 66 Issue 7 Pages 721-726
Highly reactive α,β-unsaturated carbonyl compounds, such as acrolein (ACR), crotonaldehyde (CA) and methyl vinyl ketone (MVK), are environmental pollutants present in high concentrations in cigarette smoke. We have previously found that these carbonyl compounds in cigarette smoke extract (CSE) react with intracellular glutathione (GSH) to produce the corresponding GSH-ACR, GSH-CA and GSH-MVK adducts via Michael addition reaction. These adducts are then further reduced to the corresponding alcohol forms by intracellular aldo-keto reductases in highly metastatic mouse melanoma (B16-BL6) cells and then excreted into the extracellular fluid. This time, we conducted a similar study using sheep erythrocytes and found analogous changes in the sheep erythrocytes after exposure to CSE as those with B16-BL6 cells. This indicates similarity of the detoxification pathways of the α,β-unsaturated carbonyl compounds in sheep blood cells and B16-BL6 cells. Also, we found that the GSH-MVK adduct was reduced by aldose reductase in a cell-free solution to generate its alcohol form, and its reduction reaction was completely suppressed by pretreatment with epalrestat, an aldose reductase inhibitor, a member of the aldo-keto reductase family. In the presence of sheep blood cells, however, reduction of the GSH-MVK adduct was partially inhibited by epalrestat. This revealed that some member of the aldo-keto reductase superfamily other than aldose reductase is involved in reduction of the GSH-MVK adduct in sheep blood. These results suggest that blood cells, mainly erythrocytes are involved in reducing the inhalation toxicity of cigarette smoke via an aldo-keto reductase pathway other than that of aldose reductase.
Cigarette smoking is a serious risk factor for cardiovascular disease, lung cancer and chronic obstructive pulmonary disease.1) The cytotoxic effects of cigarette smoke have been shown to be due to a complex interaction of oxidants and reactive α,β-unsaturated carbonyl compounds,2–4) such as acrolein (ACR), crotonaldehyde (CA) and methyl vinyl ketone (MVK). The cytotoxicity of these α,β-unsaturated carbonyl compounds is due to their reaction with intracellular nucleophilic amino acid residues.5–9) In particular, the formation of Michael adducts from their reaction with glutathione (GSH) is considered to deplete intracellular GSH and lead to cell damage.3,10,11) Thus, it is important to elucidate the metabolic pathways of these toxic carbonyl compounds.
In a previous study, using LC/MS analysis, we revealed that ACR, CA and MVK chemically react with reduced GSH (the thiol form) to form the respective Michael adducts.4) Additionally, we determined the corresponding GSH adducts of ACR, CA and MVK in B16-BL6 mouse melanoma cells exposed to cigarette smoke extract (CSE). We revealed that the GSH-CA and GSH-ACR adducts are produced as intermediates and then are promptly reduced in the cells. Their reduced forms (alcohol forms) are detected in large amounts in both the cells and extracellular medium, while the GSH-MVK adduct is produced as an intermediate in the cells but is only partially reduced.12) However, it is not known whether metabolic changes of α,β-unsaturated carbonyl compounds found in the cancer cells also occur in mammalian normal cells (e.g., erythrocytes).
The purpose of this study was to investigate whether the metabolic pathway of α,β-unsaturated carbonyl compounds observed in melanoma cancer cells can be similarly detected in sheep blood cells, and also to clarify the mechanism of the reduction reaction of the GSH-MVK adducts by using aldose reductase, which is a member of the aldo-keto reductase family and its inhibitor epalrestat.13,14)
Winston XS Caster FR One Box (cigarette brand name) cigarettes were purchased from Japan Tobacco Inc., (Tokyo, Japan), and Cambridge filters were purchased from Hiener Borgwaldt KC (Hamburg, Germany). ACR, CA and MVK were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Sheep blood in Alsever’s solution was purchased from Nippon Bio-Test Laboratories Inc. (Saitama, Japan) and used within 2 weeks of expiration. It contains equal amounts of sheep blood and Alsever’s solution, which serves as an anticoagulant consisting of 2.05% glucose, 0.8% sodium citrate, 0.055% citric acid, and 0.42% sodium chloride. Dulbecco’s phosphate-buffered saline without calcium and magnesium [DPBS (−)] was from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). Aldose reductase (human recombinant) and β-nicotinamide-adenine dinucleotide phosphate reduced form tetrasodium salt (NADPH) were purchased from BioVision Inc. (Milpitas, CA, U.S.A.) and Wako Pure Chemical Industries, Ltd. (Osaka, Japan), respectively. Epalrestat, LC/MS grade H2O and CH3OH were obtained from Wako Pure Chemical Industries, Ltd., and LC/MS grade formic acid and an octa decyl silyl (ODS) column (Cosmosil 5C18-AR-II 4.6 mm×150 mm) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan).
Preparation of CSECSE was prepared by modification of a technique described in a previous report15) for the preparation of new standards.16) Briefly, CSE was prepared by bubbling into DPBS (−) the mainstream of smoke (gas phase) from which the particulate phase, including tars and nicotine, had been almost completely removed by passage through a Cambridge filter using an aspiration pump. The pump flow rate was kept constant (1 L/min). Smoke was bubbled until the weight of the tar adhering to the Cambridge filter was 150 mg. The CSE solution was immediately filtered through a 0.22-µm filter. The resulting solution, designated 100% CSE, was stored at −80°C and diluted to various concentrations with DPBS (−) before use. The final concentrations of these solutions are expressed as percent values.
Exposure of CSE to Sheep Blood in Alsever’s SolutionSheep blood in Alsever’s solution (97% volume) was mixed with CSE (3% volume) for 1, 5, 15, 30, 60 and 120 min at 37°C. The concentrations of ACR, CA and MVK present in this sheep blood were in the range of 10 to 15 µM.4,17,18) The mixtures were centrifuged at 2690×g for 10 min at room temperature and separated into blood cell components and supernatants. Both fractions were deproteinized by adding 9 times the volume of 75% acetonitrile and then were centrifuged at 2690×g for 10 min at room temperature. The metabolites of ACR, CA and MVK in the supernatants of both fractions were analyzed by LC/MS/MS.
Synthesis of the GSH-MVK AdductThe GSH-MVK adduct (L-γ-glutamyl-S-4-(2-oxobutyl)-L-cysteinyl-glycine) was synthesized as a substrate for measuring aldose reductase activity. An aliquot of a 1 mM solution of GSH/DPBS (−) was added to an equal volume of 1 mM MVK/DPBS (–) and kept for about 24 h at room temperature. Evidence for the formation of the GSH-MVK adduct came from the appearance of m/z 378 ([M+H]+, M=C14H23N3O7S) and the retention time of this peak on the LC/MS spectrum. The 1H- and 13C-NMR characteristics of the GSH-MVK adduct (L-γ-glutamyl-S-4-(2-oxobutyl)-L-cysteinyl-glycine) in d6-DMSO at 500 MHz (JNM-ECA500, JEOL, Tokyo, Japan) were: δ (1H, ppm) 8.52 (1 H), 8.28 (1 H), 4.42 (1 H), 3.70 (2 H), 3.29 (1 H), 2.90 (1 H), 2.64 (1 H), 2.70 (2 H), 2.66 (2 H), 2.32 (2 H), 2.09 (3 H), 1.90 (2 H); δ (13C, ppm) 206.97, 171.97, 170.81, 170.67, 169.98, 53.29, 52.44, 42.92, 41.22, 33.54, 31.63, 29.68, 26.90, 25.43. On the other hand, we noted the disappearance of the peaks of the double bond position of MVK in 1H- and 13C-NMR spectra, 5.99 ppm (1 H), 137.2 ppm; 6.27 ppm (1 H) and 6.26 (1 H), 129.7 ppm.
Measurements of Reductase Activity in Sheep Blood CellsTo clarify the type of reductase in sheep blood cells, we examined the enzyme activity using aldose reductase and its enzyme activity inhibitor, epalrestat, and its substrate, the GSH-MVK adduct.
Before measuring the reductase activity in sheep blood cells, we investigated the effects of epalerstat on the reduction reaction of the GSH-MVK adduct by aldose reductase in the absence of cells. The aldose reductase (1 mg/mL, 10 µL) with NADPH (2 mM/DPBS (−), 240 µL) was mixed for 5 min at 37°C, and then 10 µL of epalrestat (1 mM) or DPBS (−) was added as a control to 85 µL of this reductase solution and the reaction was allowed to continue at 37°C for 10 min. The concentration of epalrestat at this time was 0.1 mM. Next, the substrate GSH-MVK adduct (Mr 377, 0.5 mM, 5 µL) was added to both solutions and reacted at 37°C for 30–35 min. After that, it was used as a sample for LC/MS measurement, and data were obtained at regular time intervals utilizing the LC/MS acquisition time function.
Next, we examined the effects of epalerstat on the reduction reaction of the GSH-MVK adduct by aldose reductase in the presence of sheep blood cells. Epalerstat (1 mM, 20 µL) or DPBS (–) (20 µL) as a control was added to the sheep blood in Alsever’s solution (180 µL) at 37°C. After 10 min, MVK (1 mM, 2 µL), as the substrate, was added to both solutions. The concentrations of epalrestat and MVK were 0.1 mM and approximately 10 µM, respectively. After 1, 5, 15, 30 and 60 min, sheep blood cells in each reaction solution were collected by centrifugation and then deproteinized by adding 9 volumes of cold 75% CH3CN/H2O and further centrifuged at 2690×g for 10–15 min. Each supernatant was used as a sample for LC/MS analysis.
Triple-Quadrupole MS and HPLC ConditionsA Quattro Premier triple-quadrupole LC/MS (Micromass, Manchester, U.K.) with an electrospray ionization (ESI) source was used for positive and negative ion mode Q1 scanning and MS/MS analysis coupled to an Alliance HT 2795 Separations Module (Waters Co., Milford, MA, U.S.A.). The optimized conditions were described in a previous report. All chromatographic separations were performed using a Cosmosil 5C18-AR-II column (4.6×150 mm). The mobile phase was composed of water containing 0.05% formic acid as solvent A and methanol as solvent B, and the flow rate was set at 0.3 mL/min. A linear gradient analysis was used for LC conditions in the separation. The same conditions as those of the previous report were used for the elution conditions.12)
When CSE (3% volume) was added to the sheep blood in Alsever’s solution (97% volume), MVK, CA and ACR in CSE reacted rapidly (within 1 min) with intracellular GSH and produced the corresponding GSH-MVK (C14H23N3O7S), GSH-CA C14H23N3O7S) and GSH-ACR (C13H21N3O7S) adducts. These GSH conjugated compounds were analyzed by using selected reaction monitoring (SRM) transition of m/z 378>231 (retention time tR 16.2 min) m/z 378>231 (tR 17.2 min) and m/z 364>217, respectively. For the product ion used for SRM transition ion, y1 ion (m/z 251, 237) was used for the alcohol form of this compound, and the dehydrated ion from y1 (m/z 231, 217) ion was used in the case of aldehyde and ketone (shown in the supplementary materials). Only slight SRM transition of the protonated molecular peak of the GSH-MVK adduct (m/z 378>231) was detected in the blood cells (Fig. 1a) and the extracellular fluid (Fig. 1b), while no SRM transition of protonated molecular peaks of GSH-CA and GSH-ACR adducts (m/z 378>231 and m/z 364>217, respectively) was detected at all. The corresponding alcohol forms of GSH-MVK, GSH-CA and GSH-ACR adducts (SRM transition of m/z 380>251 [tR 16.90 min], m/z 380>251 [tR 17.14 min] and m/z 366>237, respectively) were detected in the blood cells (Figs. 1c, 2a, 3a, respectively). Also, in the extracellular fluid, the three alcohol forms began to increase gradually with the passage of time (Figs. 1d, 2b, 3b). The metabolic pathway of α,β-unsaturated carbonyl compounds with Michael addition involved in GSH and subsequent enzymatic reduction reaction is presented in Fig. 4. In this experiment, no hemolysis of blood cells was observed.
(a) In the blood cells and (b) in the extracellular fluid, after exposure of sheep blood cells to CSE. Peak area of GSH-MVK-OH (alcohol form of GSH-MVK adduct) (SRM transition of m/z 380>251) (c) in the sheep blood cells and (d) in the extracellular fluid, after exposure of sheep blood cells to CSE.
(a) In the sheep blood cells and (b) in the extracellular fluid, after exposure of sheep blood cells to CSE.
(a) In the sheep blood cells and (b) in the extracellular fluid, after exposure of sheep blood cells to CSE.
GSH-MVK adduct is thought to be reduced to the alcohol form (GSH-MVK-OH) by reductase present in the cell. We employed cell-free conditions using synthesized GSH-MVK, as a substrate, in order to identify the intracellular aldo-keto reductases involved in the reduction of the GSH adducts of α,β-unsaturated carbonyl compounds. In the absence of sheep blood cells, we confirmed that a commercially available aldose reductase (AKR 1B1), one of the aldo ketoreductase, can almost reduce the GSH-MVK adduct to produce its alcohol form in the presence of NADPH, based on the findings that the peak of SRM transition of m/z 378>231 was reduced while the peak of the SRM transition of m/z 380>251 appeared on the MS of the reaction products. The results of the enzyme activity of aldose reductase are shown in Fig. 5a. The SRM peak area ratio between the transition of m/z 380>251 and m/z 378>231 (addition of DPBS (–), as a control) increased with time. After that, we examined the effect of epalrestat, an aldose reductase inhibitor, on the reduction reaction of GSH-MVK adduct. Epalrestat was added to the medium containing aldose reductase in the presence of NADPH and the substrate GSH-MVK adduct was added. On the mass spectra of the reaction compound, the SRM peak of the GSH-MVK adduct (SRM transition of m/z 378>231) showed almost no change and the SRM ion peak of GSH-MVK-OH (SRM transition of m/z 380>251) appeared very slowly and weakly. In Fig. 5b, the enzyme inhibitory effect of epalrestat was shown by the SRM peak area ratio between the transition of m/z 380>251 and m/z 378>231. Epalrestat completely inhibited aldose reductase activity.
However, when epalrestat was added to sheep blood and then MVK was added, both SRM ion peaks of the GSH-MVK adduct (transition of m/z 378>231) and GSH-MVK-OH (transition of m/z 380>251) appeared to the same extent, and the SRM ion peak of GSH-MVK-OH (transition of m/z 380>251) decreased with time in the MS of the deproteinated reaction supernatant (Fig. 6). This result means that epalrestat can hardly inhibit the activity of reductase in sheep blood cells. Further experiments are required to identify the active reductase in sheep blood cells, as the aldo-keto reductase superfamily is composed of many isoforms.
α,β-Unsaturated carbonyl compounds, including ACR, CA and MVK, are involved in cigarette smoke-produced reactive oxidants.2–4,15) Intracellular GSH plays an important role in antioxidant defense.19–21) The present study shows that the α,β-unsaturated carbonyl compounds in CSE are rapidly taken up into sheep blood cells and react with intracellular GSH to produce the respective Michael adducts, which are promptly reduced to the corresponding alcohol forms and excreted into the extracellular fluid. In our previous paper, very similar results were reported on the metabolism of α,β-unsaturated carbonyl compounds in mouse melanoma (B16-BL6) cells exposed to CSE, although aldo-keto reductase activity in mouse melanoma cells was weaker than that in the blood cells.12) These findings indicate that mammalian blood cells as well as cancer cells can detoxify the α,β-unsaturated carbonyl compounds in cigarette smoke. Therefore, it is speculated that the blood plays an important role in protecting the body from toxic chemicals such as α,β-unsaturated carbonyl compounds in cigarette smoke during human smoking. Reduced forms of GSH adducts of α,β-unsaturated carbonyl compounds in the blood may become oxidative stress biomarkers after cigarette smoking. In this study, we used sheep blood cells as normal mammalian blood cells because of their commercial availability at a reasonable price.
Aldose reductase is a member of the NADPH-dependent aldo-keto reductase superfamily.22,23) Aldo-ketoreductases, such as aldose reductase (AKR1B1) and carbonyl reductase 1 (CBR1), are known to be involved in the detoxification of unsaturated carbonyl compounds derived from biogenic and medical chemicals.24–26) Previous papers have reported that AKR1B126) reduces glutathione conjugates of unsaturated carbonyl compounds and CBR124) decreases anthracycline compounds. Therefore, we assumed that in the cells, aldose reductase reduces the GSH-MVK adduct to its alcohol type (GSH-MVK-OH) and planned an experiment using this enzyme. The GSH-MVK adduct was reduced by aldose reductase in a cell-free solution to generate its hydroxide, and its reduction reaction was completely suppressed by pretreatment with the aldose reductase inhibitor epalrestat, but no such inhibition by epalrestat was observed in the sheep blood solution. This revealed that members of the aldo-keto reductase superfamily other than aldose reductase seem to be involved in the reduction of the GSH-MVK adduct in sheep blood.
In conclusion, the toxic α,β-unsaturated carbonyl compounds in CSE are efficiently metabolized and detoxified by sheep blood cells, mainly erythrocytes. Therefore, when a human smokes, α,β-unsaturated carbonyl compounds may be absorbed from the pulmonary alveoli and detoxified in the blood cells.
This study was supported in part by a Grant from the Smoking Research Foundation, Japan.
The authors declare no conflict of interest.
The online version of this article contains supplementary materials.
