trans-2-Pentenal, an Active Compound in Cigarette Smoke, Identified via Its Ability to Form Adducts with Glutathione

Abstract

α,β-Unsaturated carbonyl compounds readily form adducts with SH or NH₂ residues, which are nucleophilic agents, by Michael addition. Glutathione (GSH) is a tripeptide that contains an SH residue and functions as an antioxidant. We demonstrated previously that acrolein (ACR), crotonaldehyde (CA), and methyl vinyl ketone (MVK) are present in nicotine- and tar-removed cigarette smoke extract (CSE) and reacted with GSH in B16-BL6 mouse melanoma cells to form GSH-ACR, GSH-CA, and GSH-MVK adducts, suggesting a possible mechanism for CSE-induced cytotoxicity. In this study, we searched for novel α,β-unsaturated carbonyl compounds other than ACR, CA, and MVK. We selected candidate compounds in CSE based on accurate mass values generated using LC/MS analysis of products formed between CSE and GSH, and identified these using GC/MS analysis and library screening. As a result, we isolated trans-2-methyl-2-butenal, 2-methyl-2-cyclopenten-1-one, 3-methyl-2-cyclopenten-1-one, and furfural, which were poorly reactive with GSH and only very weakly inhibited growth of Colon-26 mouse carcinoma cells and BALB/3T3 clone A31 mouse normal cells. We also isolated 2-cyclopenten-1-one, trans-2-pentenal, 3-methyl-2-butenal and ethyl vinyl ketone, which were highly reactive with GSH and significantly inhibited the growth of both cell lines. Our data suggest that the reactivity of compounds in CSE with GSH may be positively correlated with the effect on inhibiting cell growth. Notably, trans-2-pentenal showed marked inhibition of carcinoma cells growth, whereas this compound exhibited little inhibitory effect on normal cells. trans-2-Pentenal may be a potent candidate or seed for antitumor agents.

Introduction

Cigarette smoking is a high-risk factor for cardiovascular diseases, chronic obstructive pulmonary disease (COPD), and cancers.^1–4) More than 4800 chemical compounds in cigarette mainstream smoke have been identified.⁵⁾ Cigarette mainstream smoke is composed of gaseous and particulate phases, with the gaseous phase arbitrarily defined as the portion of the smoke aerosol that passes through a Cambridge filter. The particulate phase is the portion trapped on the Cambridge filter. The particle sizes range from 0.1 to 1 µm in diameter, and the particulate phase contains components such as nicotine and tar. Many researchers have been interested in the particulate phase, especially tar and nicotine, with the aim of clarifying the effects of tar and nicotine on human beings. Tar is the major carcinogen, and nicotine is the major addictive agent in tobacco smoke. Nicotine also gives rise to highly carcinogenic nitrosamines that induce cancers of the lung, upper aerodigestive tract, and pancreas.³⁾

We instead focused on the gaseous phase rather than the particulate components, and used nicotine- and tar-removed cigarette smoke extract (CSE) in our experiments. We previously detected three α,β-unsaturated carbonyl compounds (acrolein [ACR], crotonaldehyde [CA], and methyl vinyl ketone [MVK]) as active components in CSE. These compounds irreversibly modified glutathione (GSH) by the Michael addition reaction, inducing intracellular GSH depletion, and thereby inhibiting the growth ratio of mouse melanoma cells.^6–8) Furthermore, we investigated the anti-metastatic action of CSE on highly metastatic mouse Colon-26 carcinoma cells using syngeneic BALB/c mice. Colon-26 cells were injected into the spleen of mice, where the cells grew as the primary lesion, and some cells metastasized from the spleen to liver and established metastatic lesions. CSE (10, 30, and 100%) was administered intraperitoneally daily to mice for 14 d after tumor inoculation. As a result, the relative spleen weights of CSE-administered mice did not differ significantly from those of control mice. However, the relative liver weights of mice administered 30% CSE was significantly decreased compared with control mice. Namely, 30% CSE significantly inhibited the metastasis of Colon-26 cells from the spleen to the liver. In order to identify the active component in CSE, we examined the action of MVK on the invasiveness of Colon-26 cells, and we confirmed that MVK significantly reduced the invasiveness of cells.⁹⁾

In the present study, we searched for novel α,β-unsaturated carbonyl compounds in CSE to examine the anticancer action by utilizing the reactivity with GSH by the Michael addition. First, CSE was mixed with GSH and the reaction solution was analyzed using a high-resolution mass spectrometer to determine the elemental composition of newly formed peaks, and then we selected candidate compounds. Second, these candidate compounds were identified using GC/MS spectral data and library screening, and the reactivity of these compounds with GSH was analyzed using LC/MS. Finally, we investigated the effects of eight α,β-unsaturated carbonyl compounds in CSE on the proliferation of Colon-26 mouse colorectal carcinoma cells and BALB/3T3 clone A31 mouse embryo normal cells.

Experiments

Materials

Winston XS Caster FR One Box cigarettes were purchased from Japan Tobacco, Inc. (Tokyo, Japan). Cambridge filters (Heinr. Borgwaldt GmbH, Hamburg, Germany) were used to remove almost all particles and nicotine from the cigarette smoke. Fetal bovine serum (FBS), minimum essential medium (MEM), and Roswell Park Memorial Institute (RPMI)-1640 medium were from Thermo Fisher Scientific, Inc. (Waltham, MA, U.S.A.). Ethylenediaminetetraacetic acid (EDTA) trypsin solution (EDTA: 2.2 mM, trypsin: 0.25%) was from Mediatech, Inc. (Manassas, VA, U.S.A.). Dulbecco’s phosphate-buffered saline without calcium and magnesium [DPBS (−)] was from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). 2-Cyclopenten-1-one, trans-2-pentenal, trans-2-methyl-2-butenal, 3-methyl-2-butenal, 2,4-hexadienal, and furfural were from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 2-Ethylacrylaldehyde and ethyl vinyl ketone were obtained from Alfa Aesar Co., Inc. (Heysham, U.K.). 2-Methyl-2-cyclopenten-1-one and N-acetyl-L-cysteine (NAC) were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). 3-Methyl-2-cyclopenten-1-one was obtained from Combi-Blocks, Inc. (San Diego, CA, U.S.A.). LC/MS grade H₂O and CH₃OH were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), and LC/MS grade formic acid and an octa decyl silyl (ODS) column (Cosmosil 5C18-AR-II 4.6 × 150 mm) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan).

Preparation of CSE

Cigarettes were continuously combusted with reduced pressure generated using an aspiration pump (Nippon Rikagaku Kikai Co., Ltd., Tokyo, Japan), with the flow rate set at 1.0 L/min. The main stream of the cigarette smoke was passed through a Cambridge filter to remove the tar phase and nicotine, and subsequently bubbled into 15 mL of DPBS (−). The combustion of the cigarette was repeated, unless otherwise specified, until the dry weight of the tar phase trapped on the Cambridge filter reached 150 mg.¹⁰⁾ This solution was regarded as 100% CSE. The CSE was immediately filtered through a 0.22-µm filter, stored at −80°C, and diluted to the required concentration with DPBS (−) when necessary. The final concentrations of these solutions are expressed as a percentage of the initial content.

Search for Effective α,β-Unsaturated Carbonyl Compounds in CSE

The mixed reaction solution of GSH and CSE was analyzed using a high-resolution mass spectrometer. From the results of the elemental composition of new peaks in a chromatogram of the solution, some candidate compounds were estimated, and the corresponding authentic samples were purchased. Each candidate compound (1 µL) was mixed with GSH solution (1 mM) and reacted for 30 min at 37°C, and then the reaction products were analyzed using LC/MS with electrospray ionization (ESI) in positive ion mode. CSE, the gas phase of CSE, and purchased candidate compounds were analyzed using GC/MS. Newly identified α,β-unsaturated carbonyl compounds with high peak intensities in GC mass chromatograms were quantified by GC/MS.

High Resolution Triple-Quadrupole MS Conditions

A high-resolution mass spectrometer, Orbitrap Velos Pro (Thermo Fisher Scientific K. K., Yokohama, Japan), was used for elemental analysis. It was equipped with a 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.

Triple-Quadrupole MS Conditions

A Quattro Premier triple-quadrupole LC/MS (Micromass, Manchester, U.K.) with an ESI source was used for positive ion mode Q1 scanning and analysis with MS/MS coupled to an Alliance HT 2795 Separations Module (Waters Co., Milford, MA, U.S.A.). The optimized conditions were described in a previous report.⁸⁾

HPLC Conditions

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 described in the previous report were used for elution.⁸⁾

GC/MS Conditions

A mass spectrometer (Automass SUN, JEOL Ltd., Tokyo, Japan) equipped with GC (6890N, Agilent Technology Inc., Santa Clara, CA, U.S.A.) was used for analysis of active compounds that are α,β-unsaturated carbonyl compounds in CSE. GC/MS conditions were as follows: ionization energy, 70 eV; current, 300 µA; PM voltage, 500 V; source temperature, 250°C; interface temperature, 250°C; inlet temperature, 250°C; He gas, 1.0 mL/min (at constant flow); splitless mode. Total ion current (TIC) GC/MS was used to identify and quantify the search compounds. Chromatographic separations were performed using a Zebron capillary GC column ZB-WAX (Phenomenex Inc., Torrance, CA, U.S.A.), 30 m long × 0.25 mm I.D. × 1.00-µm film thickness, phase: 100% polyethylene glycol. GC conditions to separate active compounds in CSE were as follows: initial oven temperature, 40°C; fold time, 2 min; rate temperature 4°C/min rise to 100°C; fold time, 0 min; rate temperature 40°C/min rise to 220°C, hold time, 0 min; rate temperature 40°C/min down to 40°C, hold time, 1 min. The α,β-unsaturated carbonyl compound was quantified from the calibration curve of a standard sample using the peak area of the extracted ion chromatogram of the molecular ion. Extracted ion chromatogram of m/z 82 for 2-cyclopenten-1-one, m/z 84 for trans-2-pentenal and ethyl vinyl ketone, and m/z 96 for 2-methyl-2-cyclopenten-1-one and furfural were used for quantification.

Cells

The BALB/c-mouse-derived colon carcinoma cell line (Colon-26) and embryonic semi-normal cell line (BALB/3T3 clone A31) were provided by Riken BioResource Research Center (Ibaraki, Japan). Colon-26 cells were grown in RPMI 1640 medium containing 10% heat-inactivated FBS. BALB/3T3 clone A31 cells were grown in MEM containing 10% heat-inactivated FBS. Both cell lines were cultured in an incubator (a humidified atmosphere consisting of 5% CO₂ at 37°C).

Proliferation Assay

Cells were seeded at 1 × 10⁵ cells/2 mL in each well of a 12-well culture plate. Cells were treated with 100 µM 2-cyclopenten-1-one, trans-2-pentenal, trans-2-methyl-2-butenal, 3-methyl-2-butenal, ethyl vinyl ketone, 2-methyl-2-cyclopenten-1-one, 3-methyl-2-cyclopenten-1-one, and furfural, and incubated for 72 h in a CO₂ incubator at 37°C. As necessary, NAC (50 µM) was added just before treating cells with 100 µM 2-cyclopenten-1-one, 10 µM trans-2-pentenal, 100 µM 3-methyl-2-butenal, and 10 µM ethyl vinyl ketone. Viable cells were enumerated using a Coulter counter (Coulter Z1, Beckman Coulter, Tokyo, Japan).

Statistical Analysis

Results are expressed as the mean ± standard error (S.E.). Statistical analyses were performed with Tukey tests using the Graphpad Prism 4 software package (Graphpad Software, Inc., San Diego, CA, U.S.A.). Differences were considered significant at p < 0.05.

Results

Searches for Novel α,β-Unsaturated Carbonyl Compounds in CSE

We attempted to detect active unsaturated carbonyl compounds other than ACR, CA, and MVK in CSE based on the reactivity with GSH by the Michael addition reaction. First, a GSH solution was added to CSE and reacted at 37°C for 30 min, and then the reacted products were analyzed using high-resolution MS coupled with HPLC. Based on the measured accurate mass value, the molecular composition of the compounds reacted with GSH were calculated and the candidate compounds were estimated. The measured accurate mass value, molecular weight of the estimated unsaturated carbonyl compound, and its element composition are shown in Table 1. From the results of the element composition, we purchased commercially available standard products. The purchased compounds are shown in Table 2 and their structures are shown in Fig. 1.

Table 1. Composition Analyses of Products after the Reaction of CSE with GSH

Retention time (min)	[M + H]+	Measured accurate mass	Estimated elemental composition	Calculated exact mass	Difference (mDa)	Increased mass value from GSH	Elemental composition of the candidate compound
16.4	390	390.13298	C15H24O7N3S	390.13295	0.03	82	C5H6O
18.5	392	392.14790	C15H26O7N3S	392.14860	−0.70	84	C5H8O
15 (14)	404	404.11176	C15H22O8N3S	404.11221	−0.45	96	C5H4O2
18–19 (16–17)	404	404.14851	C16H26O7N3S	404.14860	−0.09	96	C6H8O

Table 2. Candidate Compounds Utilized as Standards

Mr 82 (C5H6O)	2-Cyclopenten-1-one
Mr 84 (C5H8O)	trans-2-Pentenal
	trans-2-Methyl-2-butenal
	3-Methyl-2-butenal
	2-Ethylacrylaldehyde
	Ethyl vinyl ketone
Mr 96 (C6H8O)	2,4-Hexadienal
	2-Methyl-2-cyclopenten-1-one
	3-Methyl-2-cyclopenten-1-one
Mr 96 (C5H4O2)	Furfural

Fig. 1. Structures of Candidate Effective Compound in CSE

Identification and Quantification of Highly Reactive α,β-Unsaturated Carbonyl Compounds in CSE

GC/MS analysis was used to identify target compounds present in CSE. The gas and liquid phases of CSE and the purchased standard compounds were analyzed by GC/MS. The retention time and MS of each was compared. As a result, 2-cyclopenten-1-one, trans-2-pentenal, trans-2-methyl-2-butenal, 3-methyl-2-butenal, ethyl vinyl ketone, 2-methyl-2-cyclopenten-1-one, 3-methyl-2-cyclopenten-1-one, and furfural were identified as effective compounds in CSE and 2,4-hexadienal was not included in CSE. However, 2-ethylacrylaldehyde was unclear. Extracted ion chromatograms are shown in Table 3.

Table 3. Retention Times in GC Mass Chromatograms of CSE, and Commercially Available α,β-Unsaturated Carbonyl Compounds That Showed the Same Retention Times

M+·	Retention time (min)	Commercially available α,β-unsaturated carbonyl compounds
82	18:38	2-Cyclopenten-1-one
84	6:58	2-Ethylacrylaldehyde
	7:35	—
	8:11	—
	8:28	Ethyl vinyl ketone
	11:00	trans-2-Methyl-2-butenal
	12:08	trans-2-Pentenal
	14:24	—
	14:50	3-Methyl-2-butenal
96	18:52	2-Methyl-2-cyclopenten-1-one
	19:23	—
	19:40	Furfural
	20:09	3-Methyl-2-cyclopenten-1-one

Compounds that have high peak intensity in GC mass spectra, 2-cyclopenten-1-one, trans-2-pentenal, ethyl vinyl ketone, 2-methyl-2-cyclopenten-1-one and furfural were quantified by GC/MS. ACR, CA, and MVK, which are well known as α,β-unsaturated carbonyl compounds present in CSE, were also quantified simultaneously. These results indicated that these compounds were present in CSE in approximately equivalent amounts to ACR, CA, and MVK, which have high reactivity to GSH (Fig. 2).

Fig. 2. Concentrations of the Main α,β-Unsaturated Carbonyl Compounds in CSE

Investigation of Reactivity of α,β-Unsaturated Carbonyl Compound Found to Be Present in CSE with GSH

The reactivity of eight compounds (2-cyclopenten-1-one, trans-2-pentenal, trans-2-methyl-2-butenal, 3-methyl-2-butenal, ethyl vinyl ketone, 2-methyl-2-cyclopenten-1-one, 3-methyl-2-cyclopenten-1-one, and furfural) with GSH was investigated. They were reacted with GSH and the mixed solution was analyzed by LC/MS. The extracted ion chromatograms of [M + H]⁺, GSH adducts of these compounds, are shown in Fig. 3. Some spectra had multiple peaks and the product was assumed to be stereoisomer. However, we could not purchase the standard samples for these GSH adducts, their configuration could not be assigned. 2-Cyclopenten-1-one (m/z 390), trans-2-pentenal (m/z 392), 3-methyl-2-butenal (m/z 392), and ethyl vinyl ketone (m/z 392) showed high peak intensities. However, trans-2-methyl-2-butenal (m/z 392), 2-methyl-2-cyclopenten-1-one (m/z 404), and 3-methyl-2-cyclopenten-1-one (m/z 404) showed low peak intensities. Furfural (m/z 404) showed no peak intensity. Accordingly, the reactivity of 2-cyclopenten-1-one, trans-2-pentenal, 3-methyl-2-butenal, and ethyl vinyl ketone with GSH were higher than those of trans-2-methyl-2-butenal, 2-methyl-2-cyclopenten-1-one, 3-methyl-2-cyclopenten-1-one, and furfural. Among these compounds, ethyl vinyl ketone was presumed to be highly reactive due to the terminally located olefin.

Fig. 3. Extracted Ion Chromatograms of Reaction Products of GSH with α,β-Unsaturated Carbonyl Compounds

1.0 × 10⁸ in (a)–(e) and 5.0 × 10⁶ in (f)–(h) indicated the absolute intensity of ion detection, and each value indicates 100% relative intensity.

Effects of Eight α,β-Unsaturated Carbonyl Compounds on the Growth of Colon-26 Cells and BALB/3T3 Clone A31 Cells

One hundred micromoles of 2-cyclopenten-1-one, trans-2-pentenal, 3-methyl-2-butenal, and ethyl vinyl ketone inhibited the growth of Colon-26 cells at 72 h by 58.4, 97.5, 33.6, and 98.0%, respectively. On the other hand, trans-2-methyl-2-butenal, 2-methyl-2-cyclopenten-1-one, 3-methyl-2-cyclopenten-1-one, and furfural only very weakly inhibited the growth of Colon-26 cells (Fig. 4). One hundred micromoles of 2-cyclopenten-1-one, trans-2-pentenal, 3-methyl-2-butenal, and ethyl vinyl ketone decreased the growth of BALB/3T3 clone A31 cells by 39.6, 48.9, 17.9, and 95.9%, respectively. However, 2-cyclopenten-1-one, trans-2-pentenal, and ethyl vinyl ketone had weaker inhibitory effects on the growth of BALB/3T3 clone A31 cells compared with Colon-26 cells (Fig. 4).

Fig. 4. Effects of the Eight Identified Compounds on the Growth of Colon-26 Cells and BALB/3T3 Clone A31 Cells

Both cell types were treated with each compound at 100 µM concentration for 72 h. Cells were enumerated using a Coulter counter. Data are mean ± S.E. (n = 3).

Furthermore, the inhibitory actions of 2-cyclopenten-1-one, trans-2-pentenal, 3-methyl-2-butenal, and ethyl vinyl ketone on the growth of Colon-26 cells were significantly antagonized by NAC, a GSH precursor (Fig. 5).

Fig. 5. Antagonistic Effects of 50 µM N-Acetyl-L-cysteine (NAC) on Colon-26 Cell Growth Inhibited by (a) 100 µM 2-Cyclopenten-1-one, (b) 10 µM trans-2-Pentenal, (c) 100 µM 3-Methyl-2-butenal, or (d) 10 µM Ethyl Vinyl Ketone

Data are mean ± S.E. (n = 3). * p < 0.05.

Discussion

Many researchers have reported effective uses of gaseous ingredients in cigarette smoke. Facchinetti et al. showed that ACR and CA, two α,β-unsaturated aldehydes, were contained in CSE at micromolar concentrations by GC/MS and mimicked CSE in evoking the release of the neutrophil chemoattractant interleukin (IL)-8 and of the pleiotropic inflammatory cytokine tumor necrosis factor (TNF)-α from the human macrophagic cell line U937 using an enzyme-linked immunosorbent assay (ELISA) kit. In addition, ACR (10–30 µM) also induced the released IL-8 from cultured human alveolar macrophages and THP-1 macrophagic cells. On the other hand, a saturated aldehyde, acetaldehyde, was ineffective. Furthermore, CSE-evoked IL-8 release was markedly (>80%) inhibited by NAC (0.1–3 mM) or glutathione monoethyl ester (1–3 mM).¹¹⁾ Noya et al. reported that ACR and MVK were responsible for the acute cytotoxicity of CSE through protein kinase C (PKC)/reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)-dependent and -independent mechanisms, whereas 2-cyclopenten-1-one was responsible for the delayed cytotoxicity of CSE through a PKC/NOX-independent mechanism.¹²⁾ Most recently, Hatai et al. reported that MVK inhibited the invasiveness of mouse rectal carcinoma Colon-26 cells.⁹⁾

In this study, we searched for effective and unidentified α,β-unsaturated aldehydes other than ACR, CA, and MVK. It is well-known that α,β-unsaturated carbonyl compounds readily form adducts with GSH by Michael addition. GSH is a tripeptide that contains an SH residue, and it has maintenance functions in cell homeostasis as an antioxidant. In addition, Horiyama et al. demonstrated that MVK included in CSE reacted with GSH in B16-BL6 mouse melanoma cells to form GSH-MVK adducts, and thus a possible reason for CSE-induced cytotoxicity was a decrease in intracellular GSH levels.⁷⁾ Therefore, we selected candidate compounds for unidentified α,β-unsaturated carbonyl compounds in CSE from the measured accurate mass values of LC/MS analysis of the reaction products of CSE with GSH, and identified these using GC/MS analysis and library screening.

As a result, we identified trans-2-methyl-2-butenal, 2-methyl-2-cyclopenten-1-one, 3-methyl-2-cyclopenten-1-one, and furfural, which were poorly reactive with GSH and barely inhibited the growth of Colon-26 cells and BALB/3T3 clone A31 cells. On the other hand, 2-cyclopenten-1-one, trans-2-pentenal, 3-methyl-2-butenal, and ethyl vinyl ketone, which were highly reactive with GSH, significantly inhibited the growth of Colon-26 cells and BALB/3T3 clone A31 cells. Among four reactive compounds, 10 µM trans-2-pentenal and ethyl vinyl ketone inhibited Colon-26 cells growth moderately, and 50 µM NAC antagonized their inhibitory actions completely. Furthermore, 100 µM 2-cyclopenten-1-one and 3-methyl-2-butenal inhibited Colon-26 cells growth moderately, and 50 µM NAC antagonized their inhibitory actions significantly, but the antagonistic effect of NAC was incomplete. Our data suggested that the reactivity of compounds in CSE with GSH may be positively correlated with the effect of inhibiting the growth of Colon-26 cells and BALB/3T3 clone A31 cells. Among the eight α,β-unsaturated carbonyl compounds identified here, it was notable that trans-2-pentenal showed marked inhibition of the growth of Colon-26 carcinoma cells, whereas this compound exhibited little inhibitory effect on BALB/3T3 clone A31 normal cells. We speculated that the GSH concentration in Colon-26 cell was higher than in BALB/3T3 cells, and trans-2-pentenal formed adducts with its target GSH with higher affinity than the other compounds.

Carretero et al. demonstrated that B16 mouse melanoma cells with a high GSH content showed higher metastatic activity from the spleen to liver.¹³⁾ Huang et al. also indicated that the GSH level was increased during liver growth as a result of the up-regulation of γ-glutamylcysteine synthetase heavy subunit and GSH synthetase. This increase, in turn, facilitated cell growth.¹⁴⁾ In accordance with their reports, we suggest that highly active α,β-unsaturated carbonyl compounds form adducts with GSH, thereby decreasing the GSH content in cells and showing an inhibitory action on cell growth.

In conclusion, we identified 2-cyclopenten-1-one, ethyl vinyl ketone, trans-2-pentenal, and 3-methyl-2-butenal as novel α,β-unsaturated carbonyl compounds in CSE. Furthermore, we demonstrated that these new compounds were present at almost same concentration as ACR, CA and MVK, which are well-known reactive compounds, using GC/MS. Most notably, the cytotoxicity of trans-2-pentenal was specific to tumor cells rather than normal cells, and trans-2-pentenal may be a potent candidate or seed for antitumor agents. The results of this study may lead us to the discovery of new α,β-unsaturated carbonyl compounds contained in CSE and candidate compounds for antitumor agents in them.

Acknowledgment

This study was supported by a Special Grant from the Smoking Research Foundation for Biomedical Research of Japan.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

References

• 1) Smith C. J., Fischer T. H., Atherosclerosis, 158, 257–267 (2001).
• 2) Ezzati M., Lopez A. D., Lancet, 362, 847–852 (2003).
• 3) Hoffmann D., Hoffmann I., El-Bayoumy K., Chem. Res. Toxicol., 14, 767–790 (2001).
• 4) IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, IARC Monogr. Eval. Carcinog. Risks Hum., 83, 1–1438 (2004).
• 5) Green C. R., Rodgman A., Recent Adv. Tobacco Sci, 22, 131–304 (1996).
• 6) Takahashi Y., Horiyama S., Honda C., Suwa K., Nakamura K., Kunitomo M., Shimma S., Toyoda M., Sato H., Shizuma M., Takayama M., Chem. Pharm. Bull., 61, 85–89 (2013).
• 7) Horiyama S., Takahashi Y., Hatai M., Honda C., Suwa K., Ichikawa A., Yoshikawa N., Nakamura K., Kunitomo M., Date S., Masujima T., Takayama M., Chem. Pharm. Bull., 62, 772–778 (2014).
• 8) Horiyama S., Hatai M., Takahashi Y., Date S., Masujima T., Honda C., Ichikawa A., Yoshikawa N., Nakamura K., Kunitomo M., Takayama M., Chem. Pharm. Bull., 64, 585–593 (2016).
• 9) Hatai M., Yoshikawa N., Kinoshita E., Horiyama S., Kagota S., Shinozuka K., Nakamura K., In Vivo, 32, 493–497 (2018).
• 10) Higashi T., Mai Y., Noya Y., Horinouchi T., Terada K., Hoshi A., Nepal P., Harada T., Horiguchi M., Hatate C., Kuge Y., Miwa S., PLOS ONE, 9, e107856 (2014).
• 11) Facchinetti F., Amadei F., Geppetti P., Tarantini F., Di Serio C., Dragotto A., Gigli P. M., Catinella S., Civelli M., Patacchini R., Am. J. Respir. Cell Mol. Biol., 37, 617–623 (2007).
• 12) Noya Y., Seki K., Asano H., Mai Y., Horinouchi T., Higashi T., Terada K., Hatate C., Hoshi A., Nepal P., Horiguchi M., Kuge Y., Miwa S., Toxicology, 314, 1–10 (2013).
• 13) Carretero J., Obrador E., Anasagasti M. J., Martin J. J., Vidal-Vanaclocha F., Estrela J. M., Clin. Exp. Metastasis, 17, 567–574 (1999).
• 14) Huang Z. Z., Chen C., Zeng Z., Yang H., Oh J., Chen L., Lu S. C., FASEB J., 15, 19–21 (2001).