Intracellular Metabolism of α,ß-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.

The major toxicants in cigarette smoke, α,ß-unsaturated aldehydes, such as acrolein (ACR) and crotonaldehyde (CA), and α,ß-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,ß-unsaturated carbonyls produced only their corresponding adducts. These results show that the GSH adducts of α,ß-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,ß-unsaturated ketone, MVK. Such a difference in reactivity to the carbonyl reductase might be related to differences in the cytotoxicity of α,ß-unsaturated aldehydes and ketones.

Methyl vinyl ketone, a toxic ingredient in cigarette smoke extract, modifies glutathione in mouse melanoma cells.

Cigarette smoke contains many harmful chemicals, which contribute to the pathogenesis of smoking-related diseases such as chronic obstructive pulmonary disease, cancer and cardiovascular disease. The cytotoxicity of cigarette smoke is well documented, but the definitive mechanism behind its toxicity remains unknown. Ingredients in cigarette smoke are known to deplete intracellular glutathione (GSH), the most abundant cellular thiol antioxidant, and to cause oxidative stress. In the present study, we investigated the mechanism of cigarette smoke extract (CSE)-induced cytotoxicity in B16-BL6 mouse melanoma (B16-BL6) cells using liquid chromatography-tandem mass spectrometry. CSE and ingredients in cigarette smoke, methyl vinyl ketone (MVK) and crotonaldehyde (CA), reduced cell viability in a concentration-dependent manner. Also, CSE and the ingredients (m/z 70, each) irreversibly reacted with GSH (m/z 308) to form GSH adducts (m/z 378) in cells and considerably decreased cellular GSH levels at concentrations that do not cause cell death. Mass spectral data showed that the major product formed in cells exposed to CSE was the GSH-MVK adduct via Michael-addition and was not the GSH-CA adduct. These results indicate that MVK included in CSE reacts with GSH in cells to form the GSH-MVK adduct, and thus a possible reason for CSE-induced cytotoxicity is a decrease in intracellular GSH levels.

Drug metabolite heterogeneity in cultured single cells profiled by pico-trapping direct mass spectrometry.

AIM: We investigated the heterogeneity of tafluprost metabolism in primary human hepatocytes at a single-cell level by live single-cell mass spectrometry (MS). MATERIALS & METHODS: Picoliter volumes of cytoplasm were analyzed by nano-electrospray ionization MS in order to obtain single-cell metabolite profiles. The subcellular components of a single tafluprost-treated human hepatocyte were isolated and the single-cell metabolite profile was compared with those of traditional bulk hepatocyte analysis. RESULTS: In the bulk hepatocyte analysis, liquid chromatography-MS showed the averaged metabolism of tafluprost to tafluprost acid (TA) and ß-oxidized metabolites. However, live single-cell MS showed that tafluprost metabolism varied among individual cells. In addition, there was significant variation in the quantities of TA and a major metabolite, dinor-TA, among cells, whereas there was no significant variation in 7-ethoxycoumarin metabolism. CONCLUSION: Thus, live single-cell MS successfully detected the heterogeneity of drug metabolism in individual living hepatocytes.

Direct drug metabolism monitoring in a live single hepatic cell by video mass spectrometry.

The metabolism of anti-breast cancer drug, tamoxifen, in a single human hepatocellular carcinoma cell, HepG2, was directly monitored by a video-mass spectroscope. The cytoplasm, a vacuole or nucleus of the cell was directly sucked by a nano-spray tip under a video-microscope, and then was introduced into a mass spectrometer. Unchanged drug molecules were found in cytoplasm and a vacuole, but the metabolites were only found in the cytoplasm. This direct detection of drug metabolites in a live single cell is useful for speedy drug metabolism monitoring.

Live single-cell metabolomics of tryptophan and histidine metabolites in a rat basophil leukemia cell.

A direct and rapid metabolic analysis of a live single cell was performed by live single-cell video-mass spectrometry. The contents of the cytoplasm and a granule were sucked into a nano-electrospray ionization (nano-ESI) tip, and were directly introduced into a Q-TOF mass spectrometer by nano-spray after the addition of an ionization solvent. The metabolic pathways and the locations of tryptophan and histidine metabolites were traced by this method for a cultured rat basophil leukemia cell line (RBL-2H3). The t-values of detected peaks by a t-test between the different location, e.g. cytoplasm and a granule, revealed the molecular localization of each MS peak. A direct and quick metabolomic analysis of a living cell under simultaneous video-microscopic observations was innovated.