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.

A chemical approach to searching for bioactive ingredients in cigarette smoke.

Cigarette smoke, a collection of many toxic chemicals, contributes to the pathogenesis of smoking-related diseases such as chronic obstructive pulmonary disease and cancer. Much work has been done on the chemical analysis of ingredients in cigarette smoke, but there are few reports on the active ingredients that can modify biomolecules. We used a sensitive liquid chromatography-mass spectrometry (LC/MS) and LC/MS/MS method to show that L-tyrosine (Tyr), an amino acid with a highly reactive hydroxyl group, readily reacts with cigarette smoke extract (CSE) at body temperature (37°C) to form various Tyr derivatives. Among these derivatives were N-(3-oxobutyl)-Tyr and two acetylated compounds, N-acetyl-Tyr and O-acetyl-Tyr, which were synthesized by reaction of Tyr with methyl vinyl ketone and acetic anhydride, respectively, at 37°C. The presence of methyl vinyl ketone and acetic anhydride in CSE was confirmed by gas chromatography-mass spectrometry (GC/MS). These results indicate that Tyr can easily react with active ingredients in CSE. The present analytical methods should aid the search for active ingredients in cigarette smoke.

Negative and positive ion mode LC/MS/MS for simple, sensitive analysis of sorbic acid.

Sorbic acid (SA: CH(3)-CH=CH-CH=CH-COOH) is one of the widely used food preservatives, although there have been some reports of its toxic activity, for example, on DNA and skin cells. In order to examine the effects of SA on mammalian tissues, we have developed a highly sensitive analytical method using LC/MS/MS with positive and negative ion mode electrospray ionization (ESI). In a previous study, we found that a nonacidic eluent offers better ionization efficiency than acids or their ammoniun salts. However, optimal results could not be obtained because the anion form of SA is poorly retained on a conventional reversed phase column. To resolve this problem, we chose a new type of column and used high-resolution mass spectrometry and positive ion mode analysis. There have only been a few reports using these methods in the positive mode, for example derivatized SA, because acid compounds such as SA are usually used in the negative ion mode. However, a new type of low-carbon-content and polar-endcapped C18 phase column was developed for better separation of SA from the matrix. High-resolution selected reaction monitoring (SRM) gave the best signal to noise ratio in normal-resolution SRM. In the positive ion mode, the CH(3)OH-0.05% HCOOH/0.1% CH(3)COOH eluent system yielded the best ionization efficiency. We propose a highly sensitive and simple analysis using a two-ion-mode ESI SRM method. Such systems should allow quantification of the amount of SA in or around the cells, without the need for pretreatment such as solid phase extraction.

Sensitive and simple analysis of sorbic acid using liquid chromatography with electrospray ionization tandem mass spectrometry.

Sorbic acid (SA: CH(3)-CH=CH-CH=CH-COOH) and its salts are widely used as preservatives in foodstuff because of their growth inhibitory effects on mold, yeast and a wide range of bacteria. However, it is still unclear whether SA and its salts are actually incorporated in these organisms and a higher organisms like mammalian cells. Acidic compounds such as SA are usually analyzed by HPLC with eluents containing acetic acid, formic acid and their ammonium acetates, but such acidic buffers may suppress the ionization efficiency of the acidic compounds in negative-mode electrospray ionization (ESI). In this study, we present a sensitive and simple method for analysis of SA by HPLC with non-acidic solvents such as CH(3)CN/CH(3)OH-H(2)O by negative ion mode ESI-LC/MS. As a result, SA at less as 30 fmol was selectively determined by the selected reaction monitoring (SRM) mode. It was defined as the peak area with a signal-to-noise ratio (S/N) of 3. Good linearity was obtained in the range from 55 fmol (S/N 3) to 500 fmol (r(2)=0.9968) for SA by using LC/MS with the SRM mode. We also show that the method is useful to analyze SA level in the cytosol of mastocytoma cells, which were pretreated with SA. These results suggest the applicability of this method for the highly sensitive determination of SA in the mammalian tissues and cells.

Solvents inducing oxidation of hydroxylamines.

Hydroxylamines gradually undergo oxidation to their oximes on being dissolved in organic solvent (e.g. methanol). This phenomenon was followed by (1)H-NMR and liquid chromatography-mass spectrometry (LC-MS). The oxidation rate was estimated from the peak area observed on the mass chromatogram at the protonated molecule or fragment ion on LC-atmospheric pressure chemical ionization (APCI)-MS. The results showed that the oxidation rate of hydroxylamines depended on the solvent type.

Relative population of S-form and F-form conformers of bryonolic acid and its derivatives in equilibrium in CdCl3 solutions.

Relative populations of S-form (D-E rings: boat-boat form) and F-form (D-E rings: chair-chair form) conformers, in equilibrium in CDCl3 solutions, of 20 derivatives (2-21) of bryonolic acid (D:C-friedoolean-8-en-3beta-ol-29-oic acid) (1) were calculated from NMR spectral data (J-values and chemical shifts), with the aid of molecular mechanic calculation using a MM2/CONFLEX program system. The principal deciding factor of the population ratio was found to be whether the functionality at C-29 is trigonal or tetrahedral; the S-form: F-form was 0:100-32:68 for the "trigonal" type and 48:52-100:0 for the "tetrahedral." The reliability of the results is discussed.