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.

Mass Spectrometric Approaches to the Identification of Potential Ingredients in Cigarette Smoke Causing Cytotoxicity

“…Cytotoxic effects of CSE have been reported in various cells. 21,22) We clarified that CSE reduces cell viability in a concentration-dependent manner in B16-BL6 cells. In the presence of 1% CSE, cell viability was significantly reduced to about 45.3% at 24 h of incubation.…”

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

“…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.…”

“…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][7][8] Fur-thermore, 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.…”

Using an <i>Escherichia Coli</i> K-12/<i>recA-gfpmut2</i> Microbial Biosensor to Assess the Impact of Cyclophosphamide and L-ascorbic Acid Residues on Living Bacteria Cells

“…Free radicals could prompt peroxidation of lipid in membranes and interfere with cell components, causing disruption of normal functions. Disturbance mediated by oxidative stress results in stimulation of signaling pathways and thus increased production of proinflammatory cytokines and apoptosis [12][13][14][15][16][17][18][19][20][21].…”