Toxic effects of chemical warfare agent mixtures on the mussel Mytilus trossulus in the Baltic Sea: A laboratory exposure study.

Baltic blue mussels (Mytilus trossulus) were implemented to assess potential toxicity, health impairments and bioaccumulation of dumped chemical warfare agents on marine benthic organisms. Mussels were collected from a pristine cultivation side and exposed under laboratory conditions to different mixtures of chemical warfare agents (CWAs) related phenyl arsenic compounds, Clark I and Adamsite as well as chloroacetophenone. Using a multi-biomarker approach, mussels were assessed thereafter for effects at different organisational levels ranging from geno-to cytotoxic effects, differences in enzyme kinetics and immunological responses. In an integrated approach, chemical analysis of water and tissue of the test organisms was performed in parallel. The results show clearly that exposed mussels bioaccumulate the oxidized forms of chemical warfare agents Clark I, Adamsite (DAox and DMox) and, to a certain extent, also chloroacetophenone into their tissues. Adverse effects in the test organisms at subcellular and functional level, including cytotoxic, immunotoxic and oxidative stress effects were visible. These acute effects occurred even at the lowest test concentration.

Comparison of in vitro metabolism and cytotoxicity of capsaicin and dihydrocapsaicin.

Capsaicin and dihydrocapsaicin are the major active components in pepper spray products, which are widely used for law enforcement and self-protection. The use of pepper sprays, due to their irreversible and other health effects has been under a strong debate. In this study, we compared metabolism and cytotoxicity of capsaicin and dihydrocapsaicin using human and pig liver cell fractions and human lung carcinoma cell line (A549) in vitro. Metabolites were screened and identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Using liver cell fractions, a novel aliphatic hydroxylated metabolite (m/z 322) was detected to dihydrocapsaicin but no structure was found corresponding to capsaicin. Instead, a novel phase I metabolite of capsaicin, corresponding to the structure of aliphatic demethylation and dehydrogenation (m/z 294) was identified. In addition, two novel conjugates, glycine conjugates (m/z 363 and m/z 365) and bi-glutathione (GSH) conjugates (m/z 902 and m/z 904), were identified for both capsaicin and dihydrocapsaicin. The medium of the exposed A549 cells contained ω-hydroxylated (m/z 322) and alkyl dehydrogenated (m/z 304) forms, as well as a glycine conjugate of capsaicin. As to dihydrocapsaicin, an alkyl dehydrogenated (m/z 306) form, a novel alkyl hydroxylated form, and a novel glycine conjugate were found. In A549 cells, dihydrocapsaicin evoked vacuolization and decreased cell viability more efficiently than capsaicin. Furthermore, both compounds induced p53 protein and G1 phase cell cycle arrest. Usefulness of the found metabolites as biomarkers for capsaicinoid exposures will need further investigations with additional toxicity endpoints.

Applying human and pig hepatic in vitro experiments for sulfur mustard study: screening and identification of metabolites by liquid chromatography/tandem mass spectrometry.

RATIONALE: Sulfur mustard is a chemical warfare agent (CWA) with high toxicity and complex metabolism. This study aimed at identification of new metabolic biomarkers for sulfur mustard using in in vitro exposures and various mass spectrometric techniques. METHODS: Human and pig liver subcellular fractions were used as biocatalysts. Metabolites were screened by liquid chromatography and tandem mass spectrometry (LC/MS/MS) using positive electrospray ionization (ESI). For structural identification, product ion scans (MS/MS, MS(3) ) and accurate mass measurements using liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) were acquired. RESULTS: Sulfur mustard is metabolized in vitro by S-oxidation and glutathione (GSH) conjugations. One S-oxidized metabolite, bis(2-chloroethyl) sulfoxide (m/z 175), was formed in both species only when liver microsomes were present in incubations, and it was the main metabolite if GSH was not added into the reaction mixture. However, conjugation with GSH was found to be a spontaneous reaction in physiological pH and buffered solution. Three GSH conjugates of sulfur mustard were detected and identified, among which two were novel; 2-((2-(S-glutathionyl)ethyl)thio)ethanol (m/z 412) and 2-((2-(S-glutathionyl)ethyl)thio)ethyl phosphate (m/z 492). CONCLUSIONS: To our knowledge, this was the first time that S-oxidized metabolites and GSH conjugates of sulfur mustard have been detected and identified from human samples in vitro by LC/MS/MS. The usefulness of the GSH conjugates to serve as biomarkers for sulfur mustard exposure in human samples requires further studies.

Analysis of nitromethane from samples exposed in vitro to chloropicrin by stable isotope dilution headspace gas chromatography with mass spectrometry.

Chloropicrin (trichloronitromethane) is a widely used soil fumigant and an old chemical warfare agent. The metabolism of chloropicrin is not well known in mammals but nitromethane has been shown to be one of its main metabolites. Here, a fast and simple headspace gas chromatography with mass spectrometry method was applied for the measurement of nitromethane from aqueous samples. The analytical method was validated using stable isotope labeled internal standard and a small sample volume of 260 µL. No conventional sample preparation steps were needed. The method was accurate (relative standard deviations ≤1.5%) and linear (R(2) = 0.9996) within the concentration range of 0.1-6.0 µg/mL. This method was used to measure nitromethane in in vitro incubations with human and pig liver cell fractions containing enzymes for xenobiotic metabolism, exposed to chloropicrin. The results indicate that the presence of glutathione is necessary for the formation of nitromethane from chloropicrin. Also, nitromethane was formed mostly in liver cytosol fractions, but not in microsomal fractions after the incubation with chloropicrin. Our results suggest that although nitromethane is not the unequivocal biomarker of chloropicrin exposure, this method could be applied for screening the elevated levels in humans after chloropicrin exposure.

Verification and quantification of saxitoxin from algal samples using fast and validated hydrophilic interaction liquid chromatography-tandem mass spectrometry method.

Hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method was validated with algal samples for verification and quantification of saxitoxin (STX), a potent neurotoxin which is listed in the Chemical Weapons Convention (CWC) in Schedule 1A. Isocratic elution, conventional bore HILIC column and high flow rate together with accurate post-column splitter provided detection of STX in 6.5 min with total analysis time of 9 min per sample. STX analogue, gonyautoxin 1 (GTX 1) was used as an internal standard. Sample preparation of freeze-dried algae included liquid extraction and centrifugal filtering with mean recovery of 99.9% at concentration level of 10 ng/ml (n=3). Retention times for STX and GTX 1 were 6.47±0.03 min and 4.44±0.01 min (n=45), respectively. Four diagnostic product ions were used for reliable verification of saxitoxin. Method was found to be precise and linear (R(2)=0.9714 and R(2)=0.9768) in concentration ranges of 5-50 ng/ml and 25-200 ng/ml, respectively. For saxitoxin, calculated LOD was 3 ng/ml and LLOQ 11 ng/ml. Validation was conducted using spiked algal matrix since this method is not only needed for verification analysis for the CWC but also for safety analysis of other environmental samples for presence of STX. Identification criteria for verification of STX with HILIC-MS/MS method are discussed.

Development and validation of efficient stable isotope dilution LC-HESI-MS/MS method for the verification of ß-lyase metabolites in human urine after sulfur mustard exposure.

As a part of the project for screening unequivocal biomarkers after sulfur mustard exposure, a quantitative method for the determination of ß-lyase metabolites 1,1'-sulfonylbis-[2-(methylsulfinyl)ethane] (SBMSE) and 1-methylsulfinyl-2-[2-(methylthio)ethylsulfonyl]ethane (MSMTESE) was validated. Full validation was conducted according to the FDA guidelines for method validation using pooled human urine as a sample matrix. The metabolites were extracted from urine with an optimized sample preparation procedure using ENV+ solid phase extraction cartridge with reduced volume of sample and solvents. Metabolites were detected by improved and faster liquid chromatography-heated electrospray ionization-tandem mass spectrometry (LC-HESI-MS/MS) method with two transitions of each chemical using non-buffered eluents, post-column splitter and higher flow-rate. These provided over five times faster analysis than previously published method providing 4.5 min/sample cycle time, to achieve up to 200 samples per day (24 h). Quantification was performed using deuterium labelled internal standard of SBMSE. The method was linear over the concentration range of 5-200 ng/ml (average correlation coefficients were R(2)=0.997 and R(2)=0.989) for both ß-lyase metabolites, SBMSE and MSMTESE, respectively. The average retention times for SBMSE and MSMTESE were 1.96±0.01 min and 3.24±0.03 min (n=54). Calculated limits of detection were 4 ng/ml for both SBMSE and MSMTESE, respectively. Lower limits of quantification were 10 ng/ml and 11 ng/ml for SBMSE and MSMTESE, respectively. This validated method was successfully used in the First Confidence Building Exercise on Biomedical Samples Analysis organized by the Organisation for the Prohibition of Chemical Weapons (OPCW). Identification criteria for analysing unequivocal biomarkers of sulfur mustard with LC-MS/MS after alleged use is discussed and proposed based on the validation and exercise results.

Capsaicinoids, chloropicrin and sulfur mustard: possibilities for exposure biomarkers.

Incapacitating and irritating agents produce temporary disability persisting for hours to days after the exposure. One can be exposed to these agents occupationally in industrial or other working environments. Also general public can be exposed in special circumstances, like industrial accidents or riots. Incapacitating and irritating agents discussed in this review are chloropicrin and capsaicinoids. In addition, we include sulfur mustard, which is an old chemical warfare agent and known to cause severe long-lasting injuries or even death. Chloropicrin that was used as a warfare agent in the World War I is currently used mainly as a pesticide. Capsaicinoids, components of hot pepper plants, are used by police and other law enforcement personnel as riot control agents. Toxicity of these chemicals is associated particularly with the respiratory tract, eyes, and skin. Their acute effects are relatively well known but the knowledge of putative long-term effects is almost non-existent. Also, mechanisms of effects at cellular level are not fully understood. There is a need for further research to get better idea of health risks, particularly of long-term and low-level exposures to these chemicals. For this, exposure biomarkers are essential. Validated exposure biomarkers for capsaicinoids, chloropicrin, and sulfur mustard do not exist so far. Metabolites and macromolecular adducts have been suggested biomarkers for sulfur mustard and these can already be measured qualitatively, but quantitative biomarkers await further development and validation. The purpose of this review is, based on the existing mechanistic and toxicokinetic information, to shed light on the possibilities for developing biomarkers for exposure biomonitoring of these compounds. It is also of interest to find ideas for early effect biomarkers considering the need for studies on subchronic and chronic toxicity.