Whole-genome sequencing-based antimicrobial resistance and shedding dynamics of Escherichia coli isolated from calves before and after antimicrobial group treatments.

The fattening of calves is often associated with high antimicrobial use and the selection of antimicrobial resistance (AMR). The objective of this observational longitudinal study was to describe the AMR and strain dynamics, using whole-genome sequencing (WGS), of fecal Escherichia coli in a cohort of 22 calves. All calves received antimicrobial group treatments on Day (D) 1 (oxytetracycline, intramuscularly) and on D4 through D12 (doxycycline, in-feed). Additionally, eight calves received individual parenteral treatments between D7 and D59, including florfenicol, amoxicillin, marbofloxacin, and gamithromycin. Rectal swabs were collected from all calves on D1 (prior to treatment), D2, D9, and D82. The swabs were spread onto Enterobacterales-selective agar, and three E. coli colonies per plate were subjected to WGS. Out of 264 isolates across all calves and sampling times, 80 unique strains were identified, a majority of which harbored genes conferring resistance to tetracyclines, streptomycin, and sulfonamides. The diversity of strains decreased during the in-feed antimicrobial group treatment of the calves. On D82, 90% of isolates were strains that were not isolated at previous sampling times, and the median number per strain of AMR determinants to tetracyclines, florfenicol, ß-lactams, quinolones, or macrolides decreased compared to D9. Additionally, clonal dissemination of some strains represented the main transmission route of AMR determinants. In this study, WGS revealed important variations in strain diversity and genotypic AMR of fecal E. coli over time in calves subjected to group antimicrobial treatments. IMPORTANCE: The continued emergence and spread of antimicrobial resistance (AMR) determinants are serious global concerns. The dynamics of AMR spread and persistence in bacterial and animal host populations are complex and not solely driven by antimicrobial selection pressure. In calf fattening, both antimicrobial use and carriage prevalence of antimicrobial-resistant bacteria are generally recognized as high. This study provides new insights into the short-term, within-farm dynamics and transmission of AMR determinants in Escherichia coli from the dominant fecal flora of calves subjected to antimicrobial group treatments during the rearing period. The diversity of E. coli strains decreased over time, although, in contrast to previous observations in extended-spectrum ß-lactamase-producing Enterobacterales, the predominance of a few clones was not observed. The spread of AMR determinants occurred through the dissemination of clonal strains among calves. The median number per strain of AMR determinants conferring resistance to selected antimicrobials decreased toward the end of the rearing period.

Identification, synthesis and quantification of eutylone consumption markers in a chemsex context.

Eutylone is a cathinone-derived synthetic amphetamine scheduled by the World Health Organization and European Monitoring Centre for Drugs and Drug Addiction since 2022 due to its growing consumption. We report here an eutylone intoxication involving a 38-year-old man and a 29-year-old woman in a chemsex context. A bag containing a white crystalline powder labelled as a research product was found in their vehicle. Nuclear magnetic resonance and liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analyses identified the powder as eutylone and confirmed purity superior to 99%. LC-HRMS data analysis using molecular networking allowed to propose new eutylone metabolites in blood samples in a graphical manner. We described 16 phase I (e.g. hydroxylated or demethylated) and phase II metabolites (glucuroconjugates and sulfoconjugates). The same metabolites were found both in male and female blood samples. Toxicological analyses measured eutylone concentration in blood samples at 1374 ng/mL and 1536 ng/mL for the man and the woman, respectively. A keto-reduced metabolite (m/z 238.144) was synthesized to permit its quantification at 67 ng/mL and 54 ng/mL in male and female blood samples, respectively. Overall, the identification of these metabolites will increase the knowledge of potential drug consumption markers and allow to implement mass spectrometry databases to better monitor future drug abuse or consumption.

In silico and in vitro metabolism studies of the new synthetic opiate AP-237 (bucinnazine) using bioinformatics tools.

The recent emergence of new synthetic opioids (NSOs) compounds in the illicit market is increasingly related to fatal cases. Identification and medical care of NSO intoxication cases are challenging, particularly due to high frequency of new products and extensive metabolism. As the study of NSO metabolism is crucial for the identification of these drugs in cases of intoxication, we aimed to investigate the metabolism of the piperazine NSO AP-237 (= bucinnazine). Two complementary approaches (in silico and in vitro) were used to identify putative AP-237 metabolites which could be used as consumption markers. In silico metabolism studies were realized by combining four open access softwares (MetaTrans, SyGMa, Glory X, Biotransformer 3.0). In vitro experiments were performed by incubating AP-237 (20 µM) in differentiated HepaRG cells during 0 h, 8 h, 24 h or 48 h. Cell supernatant were extracted and analyzed by liquid chromatography coupled to high-resolution mass spectrometry and data were reprocessed using three strategies (MetGem, GNPS or Compound Discoverer®). A total of 28 phase I and six phase II metabolites was predicted in silico. Molecular networking identified seven putative phase I metabolites (m/z 203.154, m/z 247.180, m/z 271.180, two m/z 289.191 isomers, m/z 305.186, m/z 329.222), including four previously unknown metabolites. Overall, this cross-disciplinary approach with molecular networking on data acquired in vitro and in silico prediction enabled to propose relevant candidate as AP-237 consumption markers that could be added to mass spectrometry libraries to help diagnose intoxication.

Severe 25E-NBOH intoxication associated with MDPHP intake: a case report, metabolism study, and literature review.

N-Benzylphenethylamine derivatives are 5-HT2A receptor agonists with hallucinogenic properties, including NBOMe (N-(2-methoxybenzyl)-2-(3,4,5-trimethoxyphenyl)ethan-1-amine) and NBOH (2-(((2,5-dimethoxyphenethyl)amino)methyl)phenol). We reported here the case of a 23-year-old man who presented a serotoninergic syndrome and a loss of consciousness following the consumption of a powder labelled as 25I-NBOH. Toxicological analyses of biological samples were carried out using a liquid chromatography high-resolution mass spectrometry. Two new psychoactive substances were identified and confirmed with certified reference materials: 25E-NBOH (2-(((4-ethyl-2,5-dimethoxyphenethyl)amino)methyl)phenol) and MDPHP (1-(benzo[d][1,3]dioxol-5-yl)-2-(pyrrolidin-1-yl)hexan-1-one). Pharmaceuticals administered to the patient during his medical care were found in plasma and urine. 25E-NBOH and MDPHP concentrations were respectively at 2.3 ng/mL and 3.4 ng/mL in plasma, and 25.7 ng/mL and 30.5 ng/mL in urine. 25I-NBOH (2-(((4-iodo-2,5-dimethoxyphenethyl)amino)methyl)phenol) was specifically searched in both samples and was not detected. These results are discussed along with a literature review on human cases of exposure to N-benzylphenethylamine derivatives. Using molecular networking approach, we propose the first 25E-NBOH metabolism study using authentic biological samples (plasma and urine). We described seven metabolites (M1 to M7), including two phase I (m/z 330.172; m/z 288.160) and five phase II metabolites (m/z 464.191, m/z 478.207, m/z 492.223, m/z 508.218; m/z 396.156). The M6 (m/z 492.223) was the most intense ion detected in plasma and urine and could be proposed as a relevant 25E-NBOH consumption marker. Overall, we described an original case of 25E-NBOH poisoning and identified metabolites that could potentially be used as consumption markers to detect 25E-NBOH intoxications with a higher confidence level and probably a longer detection window.

Estimation of carcass chemical composition in beef-on-dairy cattle using dual-energy X-ray absorptiometry (DXA) scans of cold half-carcass or 11th rib cut.

The aim of the present study was to estimate the chemical composition (water, lipid, protein, mineral, and energy contents) of carcasses measured postmortem using dual-energy X-ray absorptiometry (DXA) scans of cold half-carcass or 11th rib cut. One hundred and twenty beef-on-dairy (dam: Swiss Brown, sire: Angus, Limousin, or Simmental) bulls (n = 66), heifers (n = 42), and steers (n = 12) were included in the study. The reference carcass composition measured after grinding, homogenization, and chemical analyses was estimated from DXA variables using simple or multiple linear regressions with model training on 70% (n = 84) and validation on 30% (n = 36) of the observations. In the validation step, the estimates of water and protein masses from the half-carcass (R2 = 0.998 and 0.997; root mean square error of prediction [RMSEP], 1.0 and 0.5 kg, respectively) and 11th rib DXA scans (R2 = 0.997 and 0.996; RMSEP, 1.5 and 0.5 kg, respectively) were precise. Lipid mass was estimated precisely from the half-carcass DXA scan (R2 = 0.990; RMSEP = 1.0 kg) with a slightly lower precision from the 11th rib DXA scan (R2 = 0.968; RMSEP = 1.7 kg). Mineral mass was estimated from half-carcass (R²â€…= 0.975 and RMSEP = 0.3 kg) and 11th rib DXA scans (R2 = 0.947 and RMSEP = 0.4 kg). For the energy content, the R2 values ranged from 0.989 (11th rib DXA scan) to 0.996 (half-carcass DXA scan), and the RMSEP ranged from 36 (half-carcass) to 55 MJ (11th rib). The proportions of water, lipids, and energy in the carcasses were also precisely estimated (R2 ≥ 0.882) using either the half-carcass (RMSEP ≤ 1.0%) or 11th rib-cut DXA scans (RMSEP ≤ 1.3%). Precision was lower for the protein and mineral proportions (R2 ≤ 0.794, RMSEP ≤ 0.5%). The cattle category (sex and breed of sire) effect was observed only in some estimative models for proportions from the 11th rib cut. In conclusion, DXA imaging of either a cold half-carcass or 11th rib cut is a precise method for estimating the chemical composition of carcasses from beef-on-dairy cattle.

Assessment of the water, lipid, protein, mineral, and energy contents of beef carcass allows for an understanding of the bovine growth physiology and is key to determining the carcass's commercial value at the slaughterhouse. Direct measurement of the carcass chemical composition requires postmortem grinding and homogenization of a half-carcass to perform chemical analyses. This reference method is expensive, time-consuming, and destructive of edible meat. The aim of the present study was to develop an alternative and nondestructive method to determine carcass chemical composition based on image scans obtained using dual-energy X-ray absorptiometry (DXA). Equations were calibrated to estimate the carcass composition based on the DXA scans of a whole half-carcass or a single-rib cut in an accurate, precise, fast, and reproducible way. These were established for seven types of beef-on-dairy cattle of different sexes and breeds of sire, which are among the most commonly used in specialized beef-on-dairy fattening production systems worldwide.

Use of innovative, cross-disciplinary in vitro, in silico and in vivo approaches to characterize the metabolism of chloro-alpha-pyrrolidinovalerophenone (4-Cl-PVP).

Synthetic cathinones constitute a family of new psychoactive substances, the consumption of which is increasingly worldwide. A lack of metabolic knowledge limits the detection of these compounds in cases of intoxication. Here, we used an innovative cross-disciplinary approach to study the metabolism of the newly emerging cathinone chloro-alpha-pyrrolidinovalerophenone (4-Cl-PVP). Three complementary approaches (in silico, in vitro, and in vivo) were used to identify putative 4-Cl-PVP metabolites that could be used as additional consumption markers. The in silico approach used predictive software packages. Molecular networking was used as an innovative bioinformatics approach for re-processing high-resolution tandem mass spectrometry data acquired with both in vitro and in vivo samples. In vitro experiments were performed by incubating 4-Cl-PVP (20 µM) for four different durations with a metabolically competent human hepatic cell model (differentiated HepaRG cells). In vivo samples (blood and urine) were obtained from a patient known to have consumed 4-Cl-PVP. The in silico software predicted 17 putative metabolites, and molecular networking identified 10 metabolites in vitro. On admission to the intensive care unit, the patient's plasma and urine 4-Cl-PVP concentrations were, respectively, 34.4 and 1018.6 µg/L. An in vivo analysis identified the presence of five additional glucuronoconjugated 4-Cl-PVP derivatives in the urine. Our combination of a cross-disciplinary approach with molecular networking enabled the detection of 15 4-Cl-PVP metabolites, 10 of them had not previously been reported in the literature. Two metabolites appeared to be particular relevant candidate as 4-Cl-PVP consumption markers in cases of intoxication: hydroxy-4-Cl-PVP (m/z 282.1254) and dihydroxy-4-Cl-PVP (m/z 298.1204).

A Transversal Approach Combining In Silico, In Vitro and In Vivo Models to Describe the Metabolism of the Receptor Interacting Protein 1 Kinase Inhibitor Sibiriline.

Sibiriline is a novel drug inhibiting receptor-interacting protein 1 kinase (RIPK1) and necroptosis, a regulated form of cell death involved in several disease models. In this study, we aimed to investigate the metabolic fate of sibiriline in a cross-sectional manner using an in silico prediction, coupled with in vitro and in vivo experiments. In silico predictions were performed using GLORYx and Biotransformer 3.0 freeware; in vitro incubation was performed on differentiated human HepaRG cells, and in vivo experiments including a pharmacokinetic study were performed on mice treated with sibiriline. HepaRG culture supernatants and mice plasma samples were analyzed with ultra-high-performance liquid chromatography, coupled with tandem mass spectrometry (LC-HRMS/MS). The molecular networking bioinformatics tool applied to LC-HRMS/MS data allowed us to visualize the sibiriline metabolism kinetics. Overall, 14 metabolites, mostly produced by Phase II transformations (glucuronidation and sulfation) were identified. These data provide initial reassurance regarding the toxicology of this new RIPK1 inhibitor, although further studies are required.

Arylcyclohexylamine Derivatives: Pharmacokinetic, Pharmacodynamic, Clinical and Forensic Aspects.

Since the 2000s, an increasing number of new psychoactive substances (NPS) have appeared on the drug market. Arylcyclohexylamine (ACH) compounds such as ketamine, phencyclidine and eticyclidine derivatives are of particular concern, given their rapidly increasing use and the absence of detailed toxicity data. First used mainly for their pharmacological properties in anesthesia, their recreational use is increasing. ACH derivatives have an antagonistic activity against the N-methyl-D-aspartate receptor, which leads to dissociative effects (dissociation of body and mind). Synthetic ketamine derivatives produced in Asia are now arriving in Europe, where most are not listed as narcotics and are, thus, legal. These structural derivatives have pharmacokinetic and pharmacodynamic properties that are sometimes very different from ketamine. Here, we describe the pharmacology, epidemiology, chemistry and metabolism of ACH derivatives, and we review the case reports on intoxication.

Carbofuran self-poisoning: forensic and analytic investigations in twins and literature review.

Carbofuran is a pesticide widely used in agricultural context to kill insects, mites, and flies by ingestion or contact. Along with literature review, we aimed to (i) present the clinical, autopsy, and toxicological findings of carbofuran self-poisonings in two 69-year-old twins, resulting in the death of one of them and (ii) assess carbofuran metabolite distribution using molecular networking. Quantitative analysis of carbofuran and its main metabolites (3-hydroxycarbofuran and 3-ketocarbofuran) was carried out using an original liquid chromatography-tandem mass spectrometry method on biological samples (cardiac or peripheral blood, urine, bile, and gastric contents). Toxicological analysis of post-mortem samples (twin 1) highlighted high concentrations of carbofuran and its metabolites in cardiac blood, bile, and gastric contents. These compounds were also quantified in blood and/or urine samples of the living brother (twin 2), confirming poisoning. Using molecular networking approach to facilitate visualization of mass spectrometry datasets and sample-to-sample comparisons, we detected two more metabolites (7-phenol-carbofuran and 3-hydroxycarbofuran glucuronide) in bile (twin 1) and urine (twin 2). These results highlight the value of (i) these compounds as carbofuran consumption markers and (ii) bile samples in post-mortem analysis to confirm poisoning. From an analytical point of view, molecular networking allowed the detection and interpretation of carbofuran metabolite ammonium adducts which helped to confirm their identification annotations, as well as their structural data. From a clinical point of view, the different outcomes between the two brothers are discussed. Overall, these cases provide novel information regarding the distribution of carbofuran and its metabolites in poisoning context.

Mise en évidence d'intoxications par ingestion de champignons supérieurs : expérience au CHU de Rennes.

L'α- et la ß-amanitine sont de puissantes toxines de champignons supérieurs responsables de cytolyses hépatiques graves pouvant menacer le pronostic vital. En France, les données des centres antipoison rapportent un nombre croissant d'intoxications aux champignons depuis 2016, justifiant le besoin de méthodes de diagnostic biologique robustes. En laboratoire de toxicologie hospitalière, l'objectivation d'une intoxication par les amanitines à partir de prélèvements sanguins ou urinaires constitue ainsi un élément important dans la prise en charge du patient intoxiqué. L'objectif de ce travail consiste à réaliser une mini-revue de la littérature sur le dosage des amanitines dans les fluides biologiques pour le diagnostic des intoxications aux amanitines. Les caractéristiques des amanitines, les méthodes analytiques, les données d'interprétation, les applications pratiques ainsi que les perspectives d'utilisation des techniques de dosage y sont présentées. À travers une comparaison de deux techniques analytiques de chromatographie liquide couplée à de la spectrométrie de masse en tandem utilisées au Centre hospitalier universitaire de Rennes (Waters Xevo TQ XSTM et Thermo Scientific Q ExactiveTM), cet article présente également le retour d'expérience de biologistes médicaux dans l'amélioration continue des méthodes de dosages des amanitines.

Comparison of Illicit Drug Seizures Products of Natural Origin Using a Molecular Networking Approach.

Drug powder composition analysis is of particular interest in forensic investigations to identify illicit substance content, cutting agents and impurities. Powder profiling is difficult to implement due to multiple analytical methods requirement and remains a challenge for forensic toxicology laboratories. Furthermore, visualization tools allowing seizure products identification appear to be under-used to date. The aim of this study is to present the utility of molecular networking for the composition establishment of natural origin drugs. A powder suspected to contain heroin and three powders suspected to contain cocaine obtained from law enforcement agency seizures were analyzed using untargeted screening by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS/MS). Molecular networking and metabolite annotation applied to suspected heroin sample allowed rapid confirmation of its illicit content (heroin), the identification of structurally related major impurities (6-monoacetylmorphine, 6-monoacetylcodeine, noscapine, and papaverine), as well as cutting agents (acetaminophen and caffeine). The cocaine powder profiling allowed the comparison of its constituents in a semi-quantitative manner (cocaine, benzoylecgonine, trans/cis-cinnamoylcocaine, trimethoxycocaine, hexanoylecgonine methylester, caffeine, hydroxyzine, levamisole, and phenacetin), bringing additional information for their identification, including geographically sourcing of natural product and their putative place in the supply chain. Although this approach does not replace the profiling techniques used by forensic laboratories, the use of molecular networks provides a visual overview of structurally related constituents which aids the comparison and investigation of seizure powders. Molecular networks offers here an ideal way to depict structurally related and unrelated compounds in these often complex mixtures of chemicals.

New psychoactive substance cocktail in an intensive care intoxication case elucidated by molecular networking.

BACKGROUND: The recreational use of new psychoactive substances (NPS) is increasing worldwide. Among them, the arylcyclohexylamine family including phencyclidine (PCP) and ketamine derivatives is increasing. We report a non-fatal intoxication mainly due to arylcyclohexylamine compounds illustrated by molecular networking (MN). CASE DETAILS: A 37-year-old man with a history of drug abuse was discovered with several bags labeled as research chemicals around him and traces of powder on his nose. He was rehydrated, intubated, and admitted to the intensive care unit (ICU). Urine and drug were analyzed by liquid chromatography-mass spectrometry for NPS identification. Several NPS were quantified in urine: 3-OH-PCP at 12,085 mg/L, 3-MeO-PCP at 1100 mg/L, 2F-DCK at 147 mg/L, N-ethylhexedrone at 165 mg/L and CMC at 48 mg/L. Using a bioinformatic approach, a molecular network was built to confirm the consumption of powders contained in the bags by comparison with patient's urine. DISCUSSION: This case illustrates the interest of MN to (i) perform sample-to-sample comparison, (ii) target quantification methods, and (iii) allow proper management to confirm the relevance of the treatment.

Metabolite elucidation of 2-fluoro-deschloroketamine (2F-DCK) using molecular networking across three complementary in vitro and in vivo models.

This work first aims to investigate metabolites of 2-fluoro-deschloroketamine (2F-DCK), a new arylcyclohexylamine derivatives (a group of dissociative ketamine-based substances) using two in vitro experimental approaches, and to compare obtained results by means of molecular networking. Metabolites of 2F-DCK were investigated using both human liver microsomes (HLMs) and hepatic (HepaRG) cell line incubates using molecular networking approach: 2F-DCK pure substance was incubated with HLMs for up to 1 h at two concentrations (100 and 500 µM) and with HepaRG cells for two time periods (8 and 24 h) at one concentration (20 µM). In vitro obtained results were subsequently applied to a 2F-DCK-related fatality case. In vitro-produced metabolites were investigated using high-resolution accurate mass spectrometry using Orbitrap mass analyzer technology. Thirteen metabolites were in vitro produced and several metabolic pathways can be postulated. Seven additional metabolites were found in post-mortem samples (bile and urine) of the case, comprising three Phase II metabolites, which appear to be minor in vivo metabolites. HLMs and HepaRG cell models appear to be complementary and obtained data allowed the identification of several specific 2F-DCK metabolites in biological samples. In practical terms, observed metabolic ratios suggested that nor-2F-DCK (208.1137 m/z) and a hydrogenated metabolite (224.1443 m/z) could be proposed as reliable metabolites to be recorded in HRMS libraries in order to improve detection of 2F-DCK use.

Alcohol and drug consumption among motor vehicle drivers in the Brittany region of France: A 9-year cross-sectional population study.

The primary objective of the present study was to evaluate the frequency of positive tests for alcohol and drugs during roadside testing or after road accidents among drivers in the Brittany region of France. The study's secondary objective was to describe the blood concentrations of the substances found during these tests, in order to provide a scientific basis for the establishment or modification of legislative threshold values for road injuries prevention. We performed a cross-sectional study of a database compiled by Rennes University Hospital's toxicology laboratory in the Brittany region of France between 2010 and 2018. Driver's age, sex, and test status (positive or negative), and blood levels of ethanol, 9-tetrahydrocannabinol (THC), methylene dioxymethamphetamine (MDMA), amphetamine, benzoylecgonine and 6-monoacetylmorphine (6-MAM) were collected. Twelve thousand four hundred and ninety-seven drivers (males: 86.1%; median (range) age: 29 (15-94)) have provided roadside blood samples, giving a total of 25,998 test results. Among the 10,996 drivers with at least one positive test, the median blood concentrations of ethanol, THC, MDMA, amphetamine, benzoylecgonine, and 6-MAM were respectively 1.82 g/L, 2.41 ng/mL, 138.4 ng/mL, 67.7 ng/mL, 173.3 ng/mL, and 0.97 ng/mL. 1159 (10.54%) of the 10,996 drivers tested positive for two or more substances, and 151 (1.4%) tested positive for three or more substances. With the exception of heroin, the currently recommended threshold values appear to be appropriate for road injuries prevention with regard to the concentrations observed in offenders.

Soybean Meal Can Be Replaced by Faba Beans, Pumpkin Seed Cake, Spirulina or Be Completely Omitted in a Forage-Based Diet for Fattening Bulls to Achieve Comparable Performance, Carcass and Meat Quality.

The aim of the study was to investigate the complete substitution of imported soybean meal in beef cattle diets and the consequences on performance, meat, and adipose tissue quality. Thirty growing crossbred Limousin bulls, with an initial bodyweight of 164 ± 13 kg and 4.3 ± 0.3 months of age, were fed a grass/maize-silage based diet with little additional concentrate (0.5:0.3:0.2). Concentrates contained either soybean meal (positive control), faba beans, pumpkin seed cake, or spirulina (Arthrospira platensis), resulting in about 226 g crude protein (CP)/kg concentrate dry matter (DM) and 158 g CP/kg total diet DM. A grain-based concentrate providing just 135 g CP/kg concentrate DM and 139 g CP/total diet DM served as a negative control. Bulls of all groups had comparable average daily gains (1.43 ± 0.1 kg) and feed intakes (6.92 ± 0.37 kg). Carcass and meat quality did not differ among groups. The fatty acid profile of meat lipids was hardly affected. These results indicate that soybean meal can be replaced by any of the tested protein sources without impairing performance or meat quality. Importantly, bulls fed the negative control achieved a fattening and slaughter performance comparable to that of the protein-supplemented groups without affecting meat and adipose tissue quality. Thus, the present findings suggest that feeding crossbred bulls a grass/maize-silage based diet does not require additional protein supplementation.

In vivo and in vitro α-amanitin metabolism studies using molecular networking.

Amanitin poisonings are among the most life-threatening mushroom poisonings, and are mainly caused by the genus Amanita. Hepatotoxicity is the hallmark of amanitins, powerful toxins contained in these mushrooms, and can require liver transplant. Among amatoxins, α-amanitin is the most studied. However, the hypothesis of a possible metabolism of amanitins is still controversial in this pathophysiology. Therefore, there is a need of clarification using cutting-edge tools allowing metabolism study. Molecular network has emerged as powerful tool allowing metabolism study through organization and representation of untargeted tandem mass spectrometry (MS/MS) data in a graphical form. The aim of this study is to investigate amanitin metabolism using molecular networking. In vivo (four positive amanitin urine samples) and in vitro (differentiated HepaRG cells supernatant incubated with α-amanitin 2 µM for 24 h) samples were extracted and analyzed by LC-HRMS/MS using a Q Exactive™ Orbitrap mass spectrometer. Using molecular networking on both in vitro and in vivo, we have demonstrated that α-amanitin does not undergo metabolism in human. Thus, we provide solid evidence that a possible production of amanitin metabolites cannot be involved in its toxicity pathways. These findings can help to settle the debate on amanitin metabolism and toxicity.

Average transfer factors are not enough: The influence of growing cattle physiology on the transfer rate of polychlorinated biphenyls from feed to adipose.

Food of animal origin accounts for >90% of the overall human exposure to polychlorinated biphenyls (PCBs). Food regulatory maximum levels help to control this exposure, but bovine meat has been found to be prone to exceed those occasionally. In order to ensure the chemical safety of bovine meat, the aim was to explore the dependency of the bioconcentration (BCF) and biotransfer (BTF) factor, and assimilation efficiency (AE) of PCBs on carcass lipid proportion and growth rate of beef cattle. Eleven bulls were fattened for 293 days with three different diets (7.0, 7.4, 7.5 MJ net energy for growth kg-1 dry matter) at PCB background levels, until slaughter at 530 or 600 kg body weight. Feed and perirenal adipose tissue were sampled for PCB analyses via GC/HRMS and carcass lipid proportion was estimated by the 11th rib dissection technique. For all tested PCBs, BCF (ranging from 0.7 to 18.4) and BTF (ranging from 0.1 to 2.7) decreased at least 1.5 up to 10.6-fold when the carcass lipid proportion increased by 4%, resulting from a typical dilution process. For a faster growth rate of 0.18 kg d-1 however, only a non-significant increasing trend in transfer factors (1.1 to 2.1-fold) was seen. Besides, the transfer factors increased with PCB chlorination degree, non-ortho substitution and lipophilicity. These results underpin the complex interaction between animal physiology and PCB physicochemical properties, making it challenging to interpret average transfer factors to support chemical risk assessment and management.

Molecular Networking for Drug Toxicities Studies: The Case of Hydroxychloroquine in COVID-19 Patients.

Using drugs to treat COVID-19 symptoms may induce adverse effects and modify patient outcomes. These adverse events may be further aggravated in obese patients, who often present different illnesses such as metabolic-associated fatty liver disease. In Rennes University Hospital, several drug such as hydroxychloroquine (HCQ) have been used in the clinical trial HARMONICOV to treat COVID-19 patients, including obese patients. The aim of this study is to determine whether HCQ metabolism and hepatotoxicity are worsened in obese patients using an in vivo/in vitro approach. Liquid chromatography high resolution mass spectrometry in combination with untargeted screening and molecular networking were employed to study drug metabolism in vivo (patient's plasma) and in vitro (HepaRG cells and RPTEC cells). In addition, HepaRG cells model were used to reproduce pathophysiological features of obese patient metabolism, i.e., in the condition of hepatic steatosis. The metabolic signature of HCQ was modified in HepaRG cells cultured under a steatosis condition and a new metabolite was detected (carboxychloroquine). The RPTEC model was found to produce only one metabolite. A higher cytotoxicity of HCQ was observed in HepaRG cells exposed to exogenous fatty acids, while neutral lipid accumulation (steatosis) was further enhanced in these cells. These in vitro data were compared with the biological parameters of 17 COVID-19 patients treated with HCQ included in the HARMONICOV cohort. Overall, our data suggest that steatosis may be a risk factor for altered drug metabolism and possibly toxicity of HCQ.

New insights into quetiapine metabolism using molecular networking.

Metabolism is involved in both pharmacology and toxicology of most xenobiotics including drugs. Yet, visualization tools facilitating metabolism exploration are still underused, despite the availibility of pertinent bioinformatics solutions. Since molecular networking appears as a suitable tool to explore structurally related molecules, we aimed to investigate its interest in in vitro metabolism exploration. Quetiapine, a widely prescribed antipsychotic drug, undergoes well-described extensive metabolism, and is therefore an ideal candidate for such a proof of concept. Quetiapine was incubated in metabolically competent human liver cell models (HepaRG) for different times (0 h, 3 h, 8 h, 24 h) with or without cytochrom P450 (CYP) inhibitor (ketoconazole as CYP3A4/5 inhibitor and quinidine as CYP2D6 inhibitor), in order to study its metabolism kinetic and pathways. HepaRG culture supernatants were analyzed on an ultra-high performance liquid chromatography coupled with tandem mass spectrometry (LC-HRMS/MS). Molecular networking approach on LC-HRMS/MS data allowed to quickly visualize the quetiapine metabolism kinetics and determine the major metabolic pathways (CYP3A4/5 and/or CYP2D6) involved in metabolite formation. In addition, two unknown putative metabolites have been detected. In vitro metabolite findings were confirmed in blood sample from a patient treated with quetiapine. This is the first report using LC-HRMS/MS untargeted screening and molecular networking to explore in vitro drug metabolism. Our data provide new evidences of the interest of molecular networking in drug metabolism exploration and allow our in vitro model consistency assessment.

Self-inflicted neck wounds under influence of lysergic acid diethylamide: A case report and literature review.

RATIONALE: Lysergic acid diethylamide (LSD) is a highly potent psychedelic drug derived from ergot alkaloids. The available literature data derived from controlled studies or usage in a medical setting seem reassuring; however the literature contains very rare cases of fatal self-inflicted injuries associated with LSD exposure. The behavioral disorder that created the conditions conducive to death is a maladaptive or irrational response to the psychiatric manifestations induced by the substance. PATIENT CONCERN: Here, we report the case of a 26-year-old man found dead with large neck wounds in a locked house. No medical history other than recreational use of alcohol and narcotics was reported as well as any history of psychotic disease. The entirety of the other investigations carried out did not demonstrate the presence of a third party at the place of death and a dropper bottle containing LSD was found near the body. DIAGNOSIS: We report the first case of fatal self-inflicted neck wounds with a cutting instrument in the context of acute exposure to LSD in a patient with no psychiatric history and without suicidal symptoms at the time of the self-aggressive act. INTERVENTION AND OUTCOMES: In the present work, we used a validated method using liquid chromatography coupled with mass spectrometry for simultaneous quantification of LSD and its metabolites (O-H-LSD and Nor-LSD) in whole blood and urine samples. LSD and O-H-LSD were respectively found at 1460 and 182 pg/mL in blood. In the urine, the concentrations of LSD, nor-LSD, O-H-LSD were, respectively, 3670, 201, and 4890 ng/L. LESSONS: This observation is particularly relevant in view of the resurgence of interest in the therapeutic use of LSD, notwithstanding the fact that the literature has not demonstrated a link between suicidal risk and acute or chronic exposure to LSD.