The kinetic study of the inhibition of human cholinesterases by demeton-S-methyl shows that cholinesterase-based titration methods are not suitable for this organophosphate.

The organophosphorus insecticide, demeton-S-methyl (DSM), is considered as a good surrogate of the highly toxic nerve agent VX for skin absorption studies due to similar physico-chemical properties and in vitro percutaneous penetration profile. But, when skin distribution was estimated by measuring inhibition of cholinesterase activity, the results were poorly reproducible. The various grades of commercial DSM solutions were suspected to be the origin of the discrepancies. This hypothesis was tested by measuring inhibition of human acetyl- and butyrylcholinesterase by two commercial DSM solutions. The inhibition rate was independent on the enzyme concentration confirming pseudo-first order conditions. But complete inhibition of butyrylcholinesterase activity was achieved only when the DSM concentration was at least 1500-fold higher than the enzyme concentration. Besides, complete inhibition of acetylcholinesterase was never achieved. Mass spectrometry analysis of the inhibited butyrylcholinesterase adducts identified monomethoxyphosphorylated-serine, the aged product of inhibition by DSM or a derivative with a modified leaving group. Neither spontaneous reactivation nor aging of the dimethoxyphosphorylated-serine could account for the inhibition kinetics observed, suggesting an overly complicated kinetic scheme not compatible with the requirement of a titration experiment. In conclusion, cholinesterase-based analytical methods should be avoided for DSM titration in skin penetration studies.

Methodological contributions towards LC-MS/MS quantification of free VX in plasma: An innovative approach.

A novel analytical method has been developed to detect and quantify VX (O-ethyl S-(2(diisopropylamino) ethyl) (methylphosphonothioate)) in plasma using an LC-MS/MS technique. VX detection and quantification in plasma following percutaneous exposure represent a formidable challenge and it is an important part of the ongoing struggle against chemical warfare agents. Liquid-liquid extraction of VX from plasma was performed and it generated a recovery rate of approximately 65% followed by an LC-MS/MS analysis in a 100% organic phase. An Allure biphenyl column (Restek) was tested with detection limit at 0.5 pg/mL (5 µL injected). Initial application was focused on human skin grafted on nude mice as an experimental model with proper adjustments done for very small quantities of plasma.

High-resolution image cytometry of rat sperm nuclear shape, size and chromatin status. Experimental validation with the reproductive toxicant vinclozolin.

Recent studies have shown that the complex inter-related processes of sperm chromatin organization and nuclear morphogenesis, both of which are important fertility determinants, may be disrupted by chemicals. A high-resolution image cytometry method has been developed, using the fluorescent dye bisbenzimide, for the measurement of 20 features of the sperm nucleus related to size, form and chromatin status in the rat. For the complete set of features measured and from a total of 150 spermatozoa assessed per sample, the overall coefficient of reproducibility was 5%. Then, an experimental validation of the method was carried out in rats chronically exposed to the antiandrogenic reproductive toxicant vinclozolin and control animals. Univariate statistics revealed significant vinclozolin-induced changes for 19 out of 20 morphometric and chromatin features. Stepwise linear discriminant analysis classified correctly 84.3% of the sperm nuclei with only four features selected. The accuracy and reproducibility of the cytometry assessment of the sperm nuclei together with the results of the experimental validation suggest this method may be a new powerful tool for use in reproductive toxicology.

Child overdose: the value of the French pharmacovigilance causality assessment method.

BACKGROUND: In France, the regional pharmacovigilance centres manage drug overdose as adverse drug reactions (ADRs) using the French ADR causality assessment method, and some poison control centres (e.g. in Paris) do likewise for the most serious cases. AIM: The aim of the study was to analyse and compare the chronological and semiological scores calculated with this method, in cases of drug overdose and ADRs in children recorded in the French pharmacovigilance ADR database. RESULTS: In total, 7963 cases were analysed. The distribution of semiological criteria in drug overdose and ADR cases differed significantly (G-test), with a similar result for chronological criteria (but to a lesser degree). CONCLUSION: The distinction of two types of criteria in the French ADR causality assessment method appears useful for analysing cases of drug overdose in children. This finding triggers a renewed interest in the use of this method of causality assessment of drug overdose in children, and possibly in adults.