Intracellular Metabolism of α,β-Unsaturated Carbonyl Compounds, Acrolein, Crotonaldehyde and Methyl Vinyl Ketone, Active Toxicants in Cigarette Smoke: Participation of Glutathione Conjugation Ability and Aldehyde–Ketone Sensitive Reductase Activity

Abstract

The major toxicants in cigarette smoke, α,β-unsaturated aldehydes, such as acrolein (ACR) and crotonaldehyde (CA), and α,β-unsaturated ketone, methyl vinyl ketone (MVK), are known to form Michael-type adducts with glutathione (GSH) and consequently cause intracellular GSH depletion, which is involved in cigarette smoke-induced cytotoxicity. We have previously clarified that exposure to cigarette smoke extract (CSE) of a mouse melanoma cell culture medium causes rapid reduction of intracellular GSH levels, and that the GSH–MVK adduct can be detected by LC/MS analysis while the GSH–CA adduct is hardly detected. In the present study, to clarify why the GSH–CA adduct is difficult to detect in the cell medium, we conducted detailed investigation of the structures of the reaction products of ACR, CA, MVK and CSE in the GSH solution or the cell culture medium. The mass spectra indicated that in the presence of the cells, the GSH–CA and GSH–ACR adducts were almost not detected while their corresponding alcohols were detected. On the other hand, both the GSH–MVK adducts and their reduced products were detected. In the absence of the cells, the reaction of GSH with all α,β-unsaturated carbonyls produced only their corresponding adducts. These results show that the GSH adducts of α,β-unsaturated aldehydes, CA and ACR, are quickly reduced by certain intracellular carbonyl reductase(s) and excreted from the cells, unlike the GSH adduct of α,β-unsaturated ketone, MVK. Such a difference in reactivity to the carbonyl reductase might be related to differences in the cytotoxicity of α,β-unsaturated aldehydes and ketones.

Cigarette smoking is a major risk factor for cardiovascular disease, cancer and chronic obstructive pulmonary disease.¹⁾ However, the exact mechanism behind such smoking-related human diseases is not well understood. The cytotoxic effects of cigarette smoke have been shown to be due to the presence of many oxidants and reactive aldehydes.^2–4) Among them, the α,β-unsaturated aldehydes acrolein (ACR) and crotonaldehyde (CA), abundantly present in cigarette smoke, have been often reported to strongly react with protein and peptide thiol groups.^5–10) In particular, glutathione (GSH), a tripeptide containing a thiol group, is an important antioxidant and essential cofactor for antioxidant enzyme glutathione peroxidase. ACR and CA can chemically or enzymatically react with GSH to form their corresponding Michael adducts. Reddy et al.¹¹⁾ reported that exposure of GSH solutions to the gas phase of cigarette smoke results in rapid GSH depletion by reaction with ACR and CA, while GSH–aldehyde adducts are only slightly detected in cells exposed to cigarette smoke. Colombo et al.¹²⁾ demonstrated by MS analyses that exposure of human gingival fibroblasts to cigarette smoke rapidly depletes intracellular GSH, and also that the total GSH consumption is due to the release of GSH–ACR and GSH–CA adducts from the cells. Furthermore, Van der Toorn et al.⁷⁾ demonstrated by direct-infusion MS combined with enzymatic assays that a substantial amount of GSH in epithelial cells is irreversibly modified to GSH–ACR and GSH–CA derivatives, thereby depleting the total available GSH pool. Consequently, α,β-unsaturated aldehydes as well as oxidants are thought to mediate oxidative stress, which has been implicated in the pathogenesis of smoking-related diseases.^5,6,13)

Using GC/MS, we previously identified methyl vinyl ketone (MVK), an α,β-unsaturated ketone, as another active ingredient in cigarette smoke and demonstrated that MVK is abundantly present in cigarette smoke extract (CSE).¹⁴⁾ Furthermore, we clarified using LC/MS/MS and NMR that MVK could easily react with tyrosine at 37°C, a chemically reactive amino acid, to form N-(3-oxobutyl)-Tyr via Michael addition reaction.¹⁴⁾ We also found that GSH levels in mouse melanoma cells are rapidly reduced by exposure to CSE, and that MVK, ACR and CA irreversibly react with intracellular GSH to form the corresponding GSH adducts. GSH–CA and GSH–ACR adducts are barely detected, whereas the GSH–MVK adduct is clearly detected.^3,4) In this study, we chose B16–BL6 mouse melanoma cells, which are of a black and highly metastatic cell line,¹⁵⁾ in order to examine the effect of CSE and its active ingredients on the metastatic ability of these cancer cells.

The purpose of this study was to use LC/MS/MS to find why the GSH–CA and GSH–ACR adducts are hardly detected in mouse melanoma cells. First, we analyzed in detail the structure of reaction products from a GSH solution with MVK, CA or ACR solution. Second, we examined the modification of GSH in mouse melanoma cells exposed to CSE. Finally, we speculated on how α,β-unsaturated carbonyl compounds could be detoxified in the cells.

Experimental

Materials

Caster Frontier One (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). Fetal bovine serum (FBS) was from BioWest Co. (Nuaille, France). Ethylenediaminetetraacetic acid (EDTA) trypsin solution (EDTA: 2.2 mM, trypsin: 0.25%) was from Mediatech, Inc. (Manassas, VA, U.S.A.). Penicillin/streptomycin solution (penicillin: 50000 U/mL, streptomycin: 50 mg/mL) was from Cosmo Bio Co., Ltd. (Tokyo, Japan). Dulbecco’s modified Eagle’s medium (DMEM) with L-glutamine was from Invitrogen Corp. (Carlsbad, CA, U.S.A.). Dulbecco’s phosphate-buffered saline without calcium and magnesium [DPBS(−)] was from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). LC/MS grade H₂O and CH₃OH were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and LC/MS grade formic acid and an octadecyl silyl (ODS) column (Cosmosil 5C₁₈-AR-II 4.6 mm×150 mm) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan).

Preparation of CSE

CSE was prepared by modification of a technique described in a previous report for the preparation of new standards.¹⁴⁾ Briefly, CSE was prepared by bubbling into phosphate-buffered saline [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 that stuck to a Cambridge filter was 150 mg.¹⁶⁾ 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.

Cell Culture

The highly metastatic B16–BL6 mouse melanoma cell line was kindly provided by Dr. Futoshi Okada of Tottori University (Yonago, Japan). The cells were cultured in DMEM containing 10% FBS and 0.1% penicillin/streptomycin solution at 37°C in humidified 5% CO₂–95% air. Viable cells were trypsinized and enumerated using a Coulter Counter (Coulter Electronics Inc., Hialeah, FL, U.S.A.).

In Vitro Reaction of GSH with MVK, CA and ACR

To identify the major reaction products of intracellular GSH with CSE, a 2 mM solution of GSH in DPBS(−) was added to an equal volume of 20% CSE together with 20 µM solution of MVK, CA and ACR in DPBS(−). After incubation for 30 min at 37°C, the chemical structures of the GSH-related products in each reaction solution were analyzed and identified by LC/MS and LC/MS/MS.

Exposure of Mouse Melanoma Cells to CSE, MVK, CA and ACR

B16–BL6 mouse melanoma cells (5×10⁶ cells) in 10 mL cell culture medium were exposed to 10% CSE and 10 µL of 100 µM MVK, CA and ACR, for 30 min at 37°C. The cells were harvested with EDTA trypsin solution and subsequently resuspended in 1 mL of DPBS(−). Assay samples were prepared with a modification of the technique described in previous reports. Briefly, the cells were washed with 1 mL DPBS(−) by centrifugation at 200×g for 5 min.¹⁷⁾ They were then washed 3 more times under the same conditions. Next, the cell pellets were collected and lysed with 50 µL of 70% methanol.¹⁸⁾ The cell lysates were centrifuged at 17400×g for 5 min at 4°C, and the resulting supernatants were collected and analyzed by LC/MS and LC/MS/MS. Cell culture medium that had been exposed to CSE was directly used for the analysis of GSH metabolites.

Triple-Quadrupole Mass Spectrometry and HPLC Conditions

A 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 scan and MS/MS analysis coupled to an Alliance HT 2795 Separations Module (Waters Co., Milford, MA, U.S.A.). The optimized conditions were as follows: source temperature, 120°C; desolvation temperature, 350°C; flow rate of cone nitrogen, 100 L/h and flow rate of desolvation nitrogen, 1000 L/h. Capillary and cone voltages were 3.0 kV and 20 V, respectively. The flow rate of argon collision gas for fragmentation in the product ion mode was 0.3 mL/min (3.37–3.39×10⁻³ mbar) by which the collisional energy was optimized for the fragment ions of GSH (5–25 eV). All chromatographic separations were performed using a Cosmosil 5C₁₈-AR-II column (4.6 mm×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. For the main gradient analysis, the initial elution solvent consisted of 1% solvent B until t=2 min, and using a linear gradient, it was raised to 40% solvent B at t=12 min. This condition was maintained for 3 min until t=15 min, then using a linear gradient, it was raised further to 95% solvent B at t=18 min. This condition was maintained for 3 min until t=21 min. Finally, the gradient solvent was lowered to 1% solvent B by 22 min and maintained for 2 min. The LC oven temperature was set at 27°C and the injection volume was 5 µL.

High-Resolution (HR)-MS and HPLC Conditions

A high-resolution mass spectrometer Orbitrap Velos Pro (Thermo Fisher Scientiffic K. K., Yokohama, Japan) was used for elemental analysis. It was equipped with Prominence UFLC (LC-20AD) (Shimadzu Co., Kyoto, Japan). The spray voltage was set at 3500 V. The resolution of the equipment was set at 100000, and the capillary temperature was set at 250°C. The selected range of measurement was from m/z 100 to 650. The HPLC conditions are the same as described above.

Results

Reaction Products of GSH with MVK, CA, ACR or CSE in Vitro or in Cells

To clarify the structure of the products obtained in the presence or absence of the cells, we first compared the reaction products of GSH with α,β-unsaturated carbonyl compounds unsaturated (MVK, CA, ACR) and CSE, in vitro or in the cells.

When the MVK, CA or ACR solution was directly added to the GSH solution for 30 min at 37°C, peaks with m/z 378 at t_R 16.2 min (Fig. 1a), m/z 378 at t_R 17.2 min (Fig. 2a) and m/z 364 at t_R 14.6 min (Fig. 3a) were detected, respectively. On the other hand, when the MVK, CA or ACR solution was added to the mouse melanoma cell culture medium, the peaks at m/z 378 (t_R 16.2 min) and m/z 380 (t_R 16.9 min) for MVK (Fig. 1b), the peak at m/z 380 (t_R 17.1 min) for CA (Fig. 2b) and the peak at m/z 366 (t_R 14.9 min) for ACR (Fig. 3b) were detected. The same results were obtained when a GSH solution (Fig. 4a) or the cell culture medium (Fig. 4b) was treated with CSE. However, the protonated molecule peaks at m/z 380 and m/z 366 were obtained only in the presence of cells (Fig. 4b). These data indicate that the proposed molecular weights masses of 379 and 365 Da obtained in the presence of cells are larger by 2 Da than the 377 and 363 Da obtained by the reaction of GSH with the α,β-unsaturated carbonyl compounds in the absence of cells. Intracellular GSH may react rapidly with α,β-unsaturated carbonyl compounds via Michael addition to generate their corresponding adducts (GSH–MVK, GSH–CA and GSH–ACR). As for the increase of 2 Da, it is supposed that these GSH adducts are reduced by certain intracellular carbonyl reductase(s). The product ion spectrum of the alcohol form of GSH is shown in Fig. 5, for comparison with GSH and GSH–MVK. The product ions m/z 251 and m/z 305 (Fig. 5a) increased by 2 Da from the product ions of GSH–MVK, m/z 249 and m/z 303 (Fig. 5b). These results indicate that an increase of 2 Da was caused by the SH conjugated compound.

Fig. 1. Total Ion Current Chromatogram (TICC) and Extracted Ion Chromatograms of m/z 378 and m/z 380 Obtained from Reaction Products of (a) 1 mM GSH Solution Directly Treated with 10 µM MVK and (b) B16–BL6 Mouse Melanoma Cells (5×10⁶ Cells) Treated with 10 µM MVK at 37°C for 30 min

Protonated molecule peak of m/z 378 appeared at (a) and (b); m/z 380 only appeared in the case of (b). This indicates that GSH conjugated MVK ([M+H]⁺: m/z 378) was translated into m/z 380 in the cells.

Fig. 2. TICC and Extracted Ion Chromatograms of m/z 378 and m/z 380 Obtained from Reaction Products of (a) 1 mM GSH Solution Directly Treated with 10 µM CA and (b) B16–BL6 Mouse Melanoma Cells (5×10⁶ Cells) Treated with 10 µM CA at 37°C for 30 min

Protonated molecule peak of m/z 378 appeared mainly at (a); m/z 380 only appeared in the case of (b). This indicates that GSH conjugated CA ([M+H]⁺: m/z 378) was almost completely translated into m/z 380 in the cells.

Fig. 3. TICC and Extracted Ion Chromatograms of m/z 364 and m/z 366 Obtained from Reaction Products of (a) 1 mM GSH Solution Directly Treated with 10 µM ACR and (b) B16–BL6 Mouse Melanoma Cells (5×10⁶ Cells) Treated with 10 µM ACR at 37°C for 30 min

Protonated molecule peak of m/z 364 appeared mainly at (a); m/z 366 only appeared in the case of (b). This indicates that GSH conjugated CAR ([M+H]⁺: m/z 364) was almost completely translated into m/z 366 in the cells.

Fig. 4. TICC and Extracted Ion Chromatograms of m/z 308, m/z 364, m/z 366, m/z 378 and m/z 380 Obtained from Reaction Products of (a) 1 mM GSH Solution Directly Treated with 10% CSE and (b) B16–BL6 Mouse Melanoma Cells (5×10⁶ Cells) Treated with 10% CSE at 37°C for 30 min

Protonated molecule peaks of m/z 366 and m/z 380 appeared at (b). This indicates that GSH conjugated MVK, CA ([M+H]⁺: m/z 378) and ACR ([M+H]⁺: m/z 364) were almost completely translated into m/z 380 and 366 in the cells.

Fig. 5. Product Ion Mass Spectra of (a) Alcohol Form of GSH–MVK (GSH–MVK–OH, m/z 380) That Was Obtained from B16–BL6 Mouse Melanoma Cells (5×10⁶ Cells) Exposed to 10 µM MVK at 37°C for 30 min, (b) GSH–MVK (m/z 378) That Was Obtained from 1 mM GSH Exposed to 10 µM MVK at 37°C for 30 min and (c) GSH That Was Obtained from Unexposed Control of the Cells (5×10⁶ Cells)

Sample (a) and (c) were analyzed after being deproteinized with 70% methanol/DPBS(−). The collisional energy used was 10–15 eV.

In the presence of cells, we confirmed that the protonated molecule [M+H]⁺ at m/z 380 (t_R 17.1 min) and at m/z 366 (t_R 14.8 min) correspond to the reduced products of GSH–CA and GSH–ACR adducts, respectively, and the protonated molecule [M+H]⁺ at m/z 378 (t_R 16.1 min) and at m/z 380 (t_R 16.9 min) correspond to the GSH–MVK adduct and its reduced product, respectively (Fig. 4b). These results indicate that in the presence of cells, the GSH adducts of α,β-unsaturated aldehydes (CA and ACR) are immediately reduced to each alcohol product and the GSH adduct of the α,β-unsaturated ketone (MVK) is reduced relatively slowly to its alcohol product.

HR-MS Analysis of the Reduced Products of GSH Adducts

We analyzed the precise molecular formulae of the alcohol compounds, which were 2 Da more than the GSH adducts of the α,β-unsaturated carbonyl compounds obtained in cells, using high-resolution mass spectrometer. In the cells treated with CSE, peaks were obtained at m/z 380 (t_R 16.5, 16.8 min) and at m/z 366 (t_R 14.4 min), and their measured exact mass of the protonated molecules appeared at m/z 380.14780 and m/z 380.14734 and at m/z 366.13165, respectively. Similarly, in the cells treated with MVK, CA and ACR, peaks of [M+H]⁺ appeared at m/z 380 (t_R 16.5 min), m/z 380 (t_R 16.8 min) and m/z 366 (t_R 14.4 min), respectively, and their values of measured exact mass of protonated molecules appeared at m/z 380.14742 (t_R 16.5 min), m/z 380.14755 (t_R 16.8 min) and m/z 366.13198 (t_R 14.2 min), respectively. From the measured exact mass, it is proposed molecular formulas are C₁₄H₂₆O₇SN₃ for [M+H]⁺ at m/z 380 and C₁₃H₂₄O₇SN₃ for [M+H]⁺ at m/z 366. These measured and calculated values of exact mass and error are summarized in Table 1. On the other hand, the GSH–MVK and GSH–CA adducts and the GSH–ACR adduct which were formed by Michael addition in a cell-free medium, appeared at the peaks of m/z 378 and m/z 364, respectively. The measured exact masses of these compounds appeared at m/z 378.13214 (t_R 15.6 min) and m/z 364.11658 (t_R 14.3 min) and the proposed molecular formulae and error of these product compounds were C₁₄H₂₄O₇SN₃ for [M+H]⁺ at 378.13295 with error 2.14 ppm and C₁₃H₂₂O₇SN₃ for [M+H]⁺ at 364.11730 with error 1.97 ppm. In addition, the molecular formulae of [M+H]⁺ at m/z 380 (C₁₄H₂₆O₇SN₃) and [M+H]⁺ at m/z 366 (C₁₃H₂₄O₇SN₃) were those in which two atomic hydrogen (2H) atoms were added to the molecular formulas of [M+H]⁺ at m/z 378 (C₁₄H₂₄O₇SN₃) and [M+H]⁺ at m/z 364 (C₁₃H₂₂O₇SN₃), respectively.

Table 1. The Measured Exact Mass and Error of the Protonated Molecule Peaks (m/z 380, 366) in the Cells (B16–BL6) Exposed to CSE, MVK, CA and ACR, and Calculated Exact Mass of Proposed Molecular Formula

	m/z	tR (min)	Measured	Proposed elemental composition (MH+)	Calculated	Error (ppm)
CSE	380	16.5	380.14780	C14H26O7SN3	380.14860	2.10
	380	16.8	380.14734	C14H26O7SN3	380.14860	3.31
	366	14.4	366.13165	C13H24O7SN3	366.13295	3.55
MVK	380	16.5	380.14742	C14H26O7SN3	380.14860	3.10
CA	380	16.8	380.14755	C14H26O7SN3	380.14860	2.76
ACR	366	14.2	366.13198	C13H24O7SN3	366.13295	2.64

The results obtained above indicate that the peaks of m/z 380 and m/z 366 correspond to the 2H reduction compounds of GSH–MVK (m/z 378) and GSH–CA (m/z 378) adducts and that of the GSH–ACR (m/z 364) adduct, respectively; namely, the alcohol product of each GSH adduct.

Analysis of GSH Metabolites Formed in Cells Exposed to CSE

In order to obtain more information about the GSH metabolites, which formed in the cell, we analyzed the cell and culture medium obtained on a time course basis. In the total ion current chromatogram (TICC) of deproteinized B16–BL6 cells, the peaks corresponding to the GSH–ACR adduct (m/z 364), and its alcohol product (m/z 366), the GHS–MVK adduct (m/z 378) and its alcohol product (m/z 380) and the alcohol product of the GSH–CA adduct (m/z 380) were observed 5 min after addition of CSE into the mouse melanoma cell culture medium (Fig. 6a). The peaks at m/z 366, 378 and 380 were observed in the culture medium even 30 min after their reactions (Fig. 6b), but the peak at m/z 364 was rarely observed. These results show that the GSH–CA and GSH–ACR adducts formed in the cell are rapidly metabolized to become their stable alcohol products.

Fig. 6. TICC of Selected Ion Monitoring of (a) Deproteinized B16–BL6 Mouse Melanoma Cells (5×10⁶ Cells) and (b) Their Culture Medium

The chromatogram was obtained from control sample, and after treated with CSE for 5 and 30 min. The peaks of GSH–ACR [M+H]⁺ m/z 364, GSH–ACR–OH [M+H]⁺ m/z 366, GSH–MVK [M+H]⁺ m/z 378, GSH–MVK–OH [M+H]⁺ m/z 380, GSH–CA–OH [M+H]⁺ m/z 380 were observed at 5 min after in the cell. On the other hand, the peak of GSH–ACR–OH [M+H]⁺ m/z 366, GSH–MVK [M+H]⁺ m/z 378, GSH–MVK–OH [M+H]⁺ m/z 380, GSH–CA–OH [M+H]⁺ m/z 380 were observed at 30 min after in the culture medium.

These results indicate that in the cell, MVK rapidly forms the GSH–MVK adduct, which is quickly transported out of the cell. In contrast, ACR and CA similarly form their corresponding GSH adducts, but these adducts are immediately reduced to their alcohol products, which then are quickly transported out of the cell. These metabolites of the extracellular fluid (e.g. blood plasma) might serve as markers for oxidative stress due to cigarette smoking.

Discussion

Recent studies have shown that α,β-unsaturated carbonyl compounds, CA, ACR and MVK, present in cigarette smoke primarily conjugate with nucleophilic amino acid residues via Michael addition reaction.^19–21) These carbonyl compounds rapidly react with intracellular GSH and form their corresponding adducts, thereby depleting cellular GSH^4,22) and leading to cell damage.^4,22,23) In addition, it has been reported that a substantial amount of GSH in epithelial cells exposed to the gaseous phase of cigarette smoke is irreversibly modified to the GSH–ACR adduct (m/z 364) and GSH–CA adduct (m/z 378) by direct infusion mass spectra of the cell lysate, but the GSH–MVK adduct (m/z 378) and GSH–CA adduct are not separated.⁷⁾ Furthermore, it has been reported that the GSH–ACR and GSH–CA adducts can be detected in airway epithelial cells exposed to cigarette smoke, although the relative yields were limited.¹¹⁾

Our results clearly showed that GSH adducts of α,β-unsaturated carbonyls (ACR, CA and MVK) are promptly formed by reaction of GSH and CSE, and in the presence of mouse melanoma cells, the aldehyde compounds (CA and ACR) rapidly react with intracellular GSH to form each adduct, which is considerably reduced to each alcohol product, while the ketone compound MVK also rapidly forms its corresponding adduct but with slower reduction to its alcohol product. Such alcohol production did not occur in the absence of the cells. Our results also confirmed by HR-MS that, in the cells, the peak at m/z 378 is the GSH–MVK adduct, the peaks at m/z 380 are the alcohol products of the GSH–MVK and GSH–CA adducts, and the peak at m/z 366 is the alcohol product of the GSH–ACR adduct. These results suggest that an enzyme reducing the carbonyl groups of the GSH adducts of CA, ACR and MVK exists in the cells.

Aldose reductase is a cytosolic reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent aldo–keto reductase that catalyzes the reduction of a variety of aldehydes and carbonyls. This enzyme catalyzes the reduction of GSH conjugates of α,β-unsaturated aldehydes with higher catalytic efficiency than free aldehydes.^24,25) The aldo–keto reductase family 1 member B10 is an important protein protecting the host cell against α,β-unsaturated carbonyls and their GSH-conjugates.²²⁾ Shen et al.²⁶⁾ have reported that aldo–keto reductase family 1 member B1 efficiently reduces GSH-conjugated carbonyl compounds and plays an important role in the detoxification of their compounds. Since such a reductase is ubiquitously present in tissues/organs, we surmise that GSH adducts of CA, ACR and MVK are reduced to their alcohol products in the cells, although there are differences in the reaction rate. In a previous study, we revealed that MVK has a fairly stronger cytotoxicity than CA in mouse melanoma cells.⁴⁾ This phenomenon can be explained by the fact that aldo–keto reductases in the cells are more sensitive to the GSH–CA adduct than the GSH–MVK adduct, although further study is needed to elucidate such differences in the cytotoxicity of α,β-unsaturated carbonyls in cigarette smoke.

The LC/MS/MS method for the determination of GSH conjugates of α,β-unsaturated carbonyls and their polar metabolites may be useful for assessing the oxidative damage of cells since these carbonyl compounds are intracellularly produced during the metabolism of lipids, carbohydrates, amino acid, vitamins and steroids²⁶⁾ as well as ubiquitous environmental pollutants from industrial waste and cigarette smoke and vehicular exhaust.^19,27,28)

Conclusion

Using mass spectrometric analyses, we have elucidated the metabolic pathway in mouse melanoma cells of α,β-unsaturated carbonyl compounds (aldehydes CA and ACR, and ketone MVK) present in cigarette smoke extract (CSE). Intracellular GSH reacts rapidly with CA, ACR and MVK to generate their corresponding GSH–MVK, GSH–CA and GSH–ACR adducts, after which the GSH–CA and GSH–ACR adducts are reduced rapidly by aldose reductase in cells to produce their alcohol products but the GSH–MVK adduct is reduced more slowly. Furthermore, using HR-MS, we determined the molecular formula of the alcohol products of the GSH adducts formed in the cell culture medium by addition of CSE, MVK, CA and ACR. These results show that the α,β-unsaturated carbonyl compounds in CSE penetrate the cell membrane easily and react with the intracellular GSH to form their conjugates, which are reduced to each alcohol product by carbonyl reductases, after which the alcohol products are excreted into the extracellular fluid. Our findings suggest the possibility of using the alcohols of GSH adducts of α,β-unsaturated carbonyl compounds as biomarkers of oxidative stress in the blood after cigarette smoking.

Acknowledgment

This study was supported in part a Grant from the Smoking Research Foundation, Japan.

Conflict of Interest

The authors declare no conflict of interest.

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