Interests and limits of bone marrow analysis in forensic toxicology : contribution to quantitative interpretation of bone marrow concentrations

L'objectif de cette thèse était de faire le point sur la place de l'analyse de la moelle osseuse (MO) en tant que matrice alternative au sang en toxicologie médicolégale. Une méthode analytique a été développée et validée pour la quantification du citalopram, du diazepam et ses métabolites (nordazepam, temazepam, oxazepam) dans la MO et 10 autres matrices d'intérêt médicolégal. Cette procédure a été appliquée avec succès dans des cas réels pour l'analyse de matrices dégradées et a permis l'établissement d'une cinétique tissulaire chez l'animal au cours d'une étude pharmacocinétique. Cette cinétique animale a été intégrée dans une modélisation PBPK afin de prédire chez l'homme la distribution tissulaire du citalopram, du diazepam et son métabolite principal, le nordazepam, après administration orale thérapeutique. Ces simulations donnent des clefs intéressantes pour l'interprétation quantitative des concentrations tissulaires en toxicologie médicolégale. Une étude a été conduite pour déterminer l'influence du site de prélèvement sur la détermination des concentrations médullaires de caféine et sur la corrélation de ces concentrations avec les dosages sanguins. Elle montre que le site de prélèvement de MO est un paramètre important à prendre en considération dans l'interprétation quantitative des analyses de MO. L'ensemble de ce travail confirme l'intérêt de la MO en toxicologie médicolégale. Des études expérimentales ont permis d'approfondir les connaissances de cette matrice autour des problématiques du prélèvement, de l'analyse et de la distribution ante mortem afin de contribuer à l'interprétation qualitative et quantitative des analyses réalisées sur la MOThe aim of this work was to evaluate the interest of bone marrow (BM) analysis in forensic toxicology, as an alternative matrix to blood. An analytical method was developed and validated for the quantification of citalopram, diazepam, and its main metabolites (nordazepam, temazepam, oxazepam) in BM and 10 others matrices of forensic interest. This procedure was successfully applied to real cases for putrified sample analyses and to establish a tissue kinetic in rabbit samples for a pharmacokinetic study. These animals kinetics were implemented in PBPK modeling to predict in human tissue distribution of citalopram, diazepam, and its metabolite, nordazepam, after oral therapeutic administration. These predictions gave some clues to interpret quantitatively tissue concentrations in forensic toxicology. A study was also performed to examine whether BM sample location may influence post mortem BM quantification and correlation between BM and blood concentrations. Caffeine was used as test compound. Sample location was found to be an important parameter to consider in quantitative interpretation of BM analyses. This work confirmed the interest of BM in forensic toxicology. Experimental studies improved our knowledge on this matrix about the problematic of sample location, analytical procedure and ante mortem distribution to contribute to qualitative and quantitative interpretation of BM analyse

Intérêts et limites de l'analyse de la moelle osseuse en toxicologie médicolégale : contribution à l'interprétation quantitative des concentrations médullaires

The aim of this work was to evaluate the interest of bone marrow (BM) analysis in forensic toxicology, as an alternative matrix to blood. An analytical method was developed and validated for the quantification of citalopram, diazepam, and its main metabolites (nordazepam, temazepam, oxazepam) in BM and 10 others matrices of forensic interest. This procedure was successfully applied to real cases for putrified sample analyses and to establish a tissue kinetic in rabbit samples for a pharmacokinetic study. These animals kinetics were implemented in PBPK modeling to predict in human tissue distribution of citalopram, diazepam, and its metabolite, nordazepam, after oral therapeutic administration. These predictions gave some clues to interpret quantitatively tissue concentrations in forensic toxicology. A study was also performed to examine whether BM sample location may influence post mortem BM quantification and correlation between BM and blood concentrations. Caffeine was used as test compound. Sample location was found to be an important parameter to consider in quantitative interpretation of BM analyses. This work confirmed the interest of BM in forensic toxicology. Experimental studies improved our knowledge on this matrix about the problematic of sample location, analytical procedure and ante mortem distribution to contribute to qualitative and quantitative interpretation of BM analysesL'objectif de cette thèse était de faire le point sur la place de l'analyse de la moelle osseuse (MO) en tant que matrice alternative au sang en toxicologie médicolégale. Une méthode analytique a été développée et validée pour la quantification du citalopram, du diazepam et ses métabolites (nordazepam, temazepam, oxazepam) dans la MO et 10 autres matrices d'intérêt médicolégal. Cette procédure a été appliquée avec succès dans des cas réels pour l'analyse de matrices dégradées et a permis l'établissement d'une cinétique tissulaire chez l'animal au cours d'une étude pharmacocinétique. Cette cinétique animale a été intégrée dans une modélisation PBPK afin de prédire chez l'homme la distribution tissulaire du citalopram, du diazepam et son métabolite principal, le nordazepam, après administration orale thérapeutique. Ces simulations donnent des clefs intéressantes pour l'interprétation quantitative des concentrations tissulaires en toxicologie médicolégale. Une étude a été conduite pour déterminer l'influence du site de prélèvement sur la détermination des concentrations médullaires de caféine et sur la corrélation de ces concentrations avec les dosages sanguins. Elle montre que le site de prélèvement de MO est un paramètre important à prendre en considération dans l'interprétation quantitative des analyses de MO. L'ensemble de ce travail confirme l'intérêt de la MO en toxicologie médicolégale. Des études expérimentales ont permis d'approfondir les connaissances de cette matrice autour des problématiques du prélèvement, de l'analyse et de la distribution ante mortem afin de contribuer à l'interprétation qualitative et quantitative des analyses réalisées sur la M

Analyse de la moelle osseuse en toxicologie médicolégale

LYON1-BU Santé (693882101) / SudocSudocFranceF

α-Methyltryptamine (α-MT) Metabolite Profiling in Human Hepatocyte Incubations and Postmortem Urine and Blood

: α-MT is a hallucinogenic and stimulant tryptamine that was involved in several overdose fatalities in the United States and Europe. Analytical toxicology, and particularly the identification of metabolite biomarkers in biological samples, often is the only way to prove tryptamine use in clinical and forensic caseworks. We aimed to identify optimal α-MT metabolite biomarkers of consumption in humans. We identified α-MT metabolites in 10-donor-pooled human hepatocyte incubations and postmortem urine and blood from an α-MT overdose case using in silico metabolite predictions, liquid chromatography high-resolution-tandem mass spectrometry (LC-HRMS/MS), and software-assisted data mining. Nine metabolites were identified in vitro and eight additional metabolites were found in urine; five metabolites were found in blood. Metabolic transformations were hydroxylation, O-sulfation, O-glucuronidation, N-glucuronidation, and N-acetylation, consistent with the metabolism of structural analogues. The findings in hepatocyte incubations and postmortem samples were consistent, proving the in vitro model suitability. We suggest α-MT, hydroxy-α-MT glucuronide, and two hydroxy-α-MT sulfates as biomarkers of α-MT use in non-hydrolyzed urine; we suggest α-MT, two hydroxy-α-MT sulfates and N-acetyl-α-MT as biomarkers of α-MT use in blood. Further studies on α-MT clinical and forensic caseworks with different doses and routes of administration are necessary to better explore α-MT metabolism

α-Methyltryptamine (α-MT) Metabolite Profiling in Human Hepatocyte Incubations and Postmortem Urine and Blood

α-MT is a hallucinogenic and stimulant tryptamine that was involved in several overdose fatalities in the United States and Europe. Analytical toxicology, and particularly the identification of metabolite biomarkers in biological samples, often is the only way to prove tryptamine use in clinical and forensic caseworks. We aimed to identify optimal α-MT metabolite biomarkers of consumption in humans. We identified α-MT metabolites in 10-donor-pooled human hepatocyte incubations and postmortem urine and blood from an α-MT overdose case using in silico metabolite predictions, liquid chromatography high-resolution-tandem mass spectrometry (LC-HRMS/MS), and software-assisted data mining. Nine metabolites were identified in vitro and eight additional metabolites were found in urine; five metabolites were found in blood. Metabolic transformations were hydroxylation, O-sulfation, O-glucuronidation, N-glucuronidation, and N-acetylation, consistent with the metabolism of structural analogues. The findings in hepatocyte incubations and postmortem samples were consistent, proving the in vitro model suitability. We suggest α-MT, hydroxy-α-MT glucuronide, and two hydroxy-α-MT sulfates as biomarkers of α-MT use in non-hydrolyzed urine; we suggest α-MT, two hydroxy-α-MT sulfates and N-acetyl-α-MT as biomarkers of α-MT use in blood. Further studies on α-MT clinical and forensic caseworks with different doses and routes of administration are necessary to better explore α-MT metabolism

Elimination des résidus médicamenteux dans les eaux résiduaires urbaines par charbon actif en poudre - Etude du procédé CarboPlus

National audienc

Treatment of pharmaceuticals by PAC: case study of a full-scale pilot at Seine Centre WWTP.

International audienceThe micropollutants fate in the environment has become an increasing topic of interest last decades, especially in heavily urbanized area. In addition to priority pollutants (WFD), a large number of molecules such as pesticides, personal care products, pharmaceuticals, flame retardants, artificial sweeteners, etc. is detected in the environment and also represents a potential threat for it (Rogers 1996, Jørgensen and Halling-Sørensen 2000, Heberer 2002). Furthermore, the fate of pollutants within the wastewater treatment plants (WWTP) is today well studied and WWTP effluents are generally considered as an important source of contamination for a long time, especially in the case of urban areas (Heberer 2002). Tertiary treatments are nowadays developed because some molecules are persistent to primary and biological treatments. Different kinds of technologies already exist and are mainly imported from drinking water industry. Adsorption on activated carbon seems to be one of the most interesting of these processes regarding its simplicity, efficiency and functioning costs. Some recent papers have already shown its efficiency over persistent micropollutants (Margot et al. 2013; Boehler et al. 2012). In this context, the LEESU (Water, Environment and Urban Systems Laboratory), through the framework of the OPUR (Observatory of Urban Pollutants) research programme and in close partnership with the Parisian public company (SIAAP) which manages wastewaters of the 8 million inhabitant conurbation, launched this study on the tertiary treatments. Thus, a full-scale activated carbon adsorption pilot (50 m3/h) has been set up at the Seine Centre WWTP (capacity of 250 000 m3/day) and is currently studied in order to i) characterize the efficiency of the process for priority and emerging pollutants removal and ii) characterize the sorption mechanism and the different parameters influencing it (organic matter competition, operating parameters, activated carbon structure and properties, etc.). The process consists in a fluidized bed of powdered activated carbon (PAC) reactor. As coagulant and flocculant are added to the system, the bed stays steady and no filtration system is needed to separate water and PAC. In addition, a new type of activated carbon, developed for the process, will also be tested. These micro-grains have an intermediary size (300 - 400 µm) compared to powder ( 1 mm), what should allow a high efficiency together with a higher ease of operating for the process as no chemicals are needed and solid retention time can be higher. Furthermore, the micro-grain selected for this study is regenerated and can easily be recycled, what is consistent with an environmental application. In fact, a recycling by the supplier system can be implemented, potentially resulting in cost reductions and in avoiding the waste carbon handling issue. Considering this, the selection of the process configuration (PAC or micro-grain) will then be possible depending on each site situation and treatment requirements. The study is organized in 3 phases. The first phase consists in an optimization of the pilot, where different configurations (PAC dose, hydraulic retention time and nature of carbon) are tested, while the second phase will operate at steady state. Finally, the third phase will allow testing the micro-grain configuration. A strategy of intensive (every 2 weeks during 2 years) and large (> 130 molecules) screening campaigns has been built to get a very large database and range of information, by measuring inlet and outlet concentrations of pharmaceuticals, pesticides, priority substances and metals during a total of 30 campaigns. Preliminary results tend to highlight a removal of 15-50% of dissolved organic carbon (DOC) and a slight decrease of nitrogenous pollution (10-30%) whereas TSS are not eliminated. Regarding micropollutants, only pharmaceuticals results are currently available. A significant removal (40-90%) of these molecules seems possible even for the most hydrophilic (log KOW < 4) ones such as antibiotics and beta-blockers. Moreover, a clear influence of the operating parameters can be highlighted. This is in good accordance with recent and similar works in Switzerland and Germany (Margot et al. 2013; Boehler et al. 2012). In addition, a significant relation between micropollutant, DOC and UV-254 signal removals seems to exist, and the potential of using measure of UV-254 as a control tool of the efficiency of the process will be evaluated. This presentation aims at presenting results from the two PAC phases and the first feedbacks from the micro-grain phase. A focus will be done on micropollutants fate, but classical wastewater treatment parameters will also be presented to have a precise and complete overview of the potential impact of the addition of a tertiary treatment to the urban areas WWTPs

Elimination des résidus médicamenteux dans les eaux résiduaires urbaines par charbon actif en poudre - Etude du procédé CarboPlus

National audienc

Removal of emerging micropollutants from WWTP discharges: is activated carbon suitable for wastewater application?

International audienc