Effect of CYP2D6, 2C19, and 3A4 Phenoconversion in Drug-Related Deaths.

Molecular autopsy is a very important tool in forensic toxicology. However, many determinants, such as co-medication and physiological parameters, should be considered for optimal results. These determinants could cause phenoconversion (PC), a discrepancy between the real metabolic profile after phenoconversion and the phenotype determined by the genotype. This study's objective was to assess the PC of drug-metabolizing enzymes, namely CYP2D6, 2C19, and 3A4, in 45 post-mortem cases where medications that are substrates, inducers, or inhibitors of these enzymes were detected. It also intended to evaluate how PC affected the drug's metabolic ratio (MR) in four cases. Blood samples from 45 cases of drug-related deaths were analyzed to detect and determine drug and metabolite concentrations. Moreover, all the samples underwent genotyping utilizing the HaloPlex Target Enrichment System for CYP2D6, 2C19, and 3A4. The results of the present study revealed a statistically significant rate of PC for the three investigated enzymes, with a higher frequency of poor metabolizers after PC. A compatibility was seen between the results of the genomic evaluation after PC and the observed MRs of venlafaxine, citalopram, and fentanyl. This leads us to focus on the determinants causing PC that may be mainly induced by drug interactions. This complex phenomenon can have a significant impact on the analysis, interpretation of genotypes, and accurate conclusions in forensic toxicology. Nevertheless, more research with more cases in the future is needed to confirm these results.

Metabolomics Provides Novel Insights into the Potential Toxicity Associated with Heated Tobacco Products, Electronic Cigarettes, and Tobacco Cigarettes on Human Bronchial Epithelial BEAS-2B Cells.

Smoking is an established risk factor for various pathologies including lung cancer. Electronic cigarettes (e-cigs) and heated tobacco products (HTPs) have appeared on the market in recent years, but their safety or, conversely, their toxicity has not yet been demonstrated. This study aimed to compare the metabolome of human lung epithelial cells exposed to emissions of e-cigs, HTPs, or 3R4F cigarettes in order to highlight potential early markers of toxicity. BEAS-2B cells were cultured at the air-liquid interface and exposed to short-term emissions from e-cigs set up at low or medium power, HTPs, or 3R4F cigarettes. Untargeted metabolomic analyses were performed using liquid chromatography coupled with mass spectrometry. Compared to unexposed cells, both 3R4F cigarette and HTP emissions affected the profiles of exogenous compounds, one of which is carcinogenic, as well as those of endogenous metabolites from various pathways including oxidative stress, energy metabolism, and lipid metabolism. However, these effects were observed at lower doses for cigarettes (2 and 4 puffs) than for HTPs (60 and 120 puffs). No difference was observed after e-cig exposure, regardless of the power conditions. These results suggest a lower acute toxicity of e-cig emissions compared to cigarettes and HTPs in BEAS-2B cells. The pathways deregulated by HTP emissions are also described to be altered in respiratory diseases, emphasizing that the toxicity of HTPs should not be underestimated.

Integrating rare genetic variants into DPYD pharmacogenetic testing may help preventing fluoropyrimidine-induced toxicity.

Variability in genes involved in drug pharmacokinetics or drug response can be responsible for suboptimal treatment efficacy or predispose to adverse drug reactions. In addition to common genetic variations, large-scale sequencing studies have uncovered multiple rare genetic variants predicted to cause functional alterations in genes encoding proteins implicated in drug metabolism, transport and response. To understand the functional importance of rare genetic variants in DPYD, a pharmacogene whose alterations can cause severe toxicity in patients exposed to fluoropyrimidine-based regimens, massively parallel sequencing of the exonic regions and flanking splice junctions of the DPYD gene was performed in a series of nearly 3000 patients categorized according to pre-emptive DPD enzyme activity using the dihydrouracil/uracil ([UH2]/[U]) plasma ratio as a surrogate marker of DPD activity. Our results underscore the importance of integrating next-generation sequencing-based pharmacogenomic interpretation into clinical decision making to minimize fluoropyrimidine-based chemotherapy toxicity without altering treatment efficacy.

Extracellular vesicles derived from nasopharyngeal carcinoma induce the emergence of mature regulatory dendritic cells using a galectin-9 dependent mechanism.

Nasopharyngeal carcinoma-derived small extracellular vesicles (NPCSEVs) have an immunosuppressive impact on the tumour microenvironment. In this study, we investigated their influence on the generation of tolerogenic dendritic cells and the potential involvement of the galectin-9 (Gal9) they carry in this process. We analysed the phenotype and immunosuppressive properties of NPCSEVs and explored the ability of DCs exposed to NPCSEVs (NPCSEV-DCs) to regulate T cell proliferation. To assess their impact at the pathophysiological level, we performed real-time fluorescent chemoattraction assays. Finally, we analysed phenotype and immunosuppressive functions of NPCSEV-DCs using a proprietary anti-Gal9 neutralising antibody to assess the role of Gal9 in this effect. We described that NPCSEV-DCs were able to inhibit T cell proliferation despite their mature phenotype. These mature regulatory DCs (mregDCs) have a specific oxidative metabolism and secrete high levels of IL-4. Chemoattraction assays revealed that NPCSEVs could preferentially recruit NPCSEV-DCs. Finally, and very interestingly, the reduction of the immunosuppressive function of NPCSEV-DCs using an anti-Gal9 antibody clearly suggested an important role for vesicular Gal9 in the induction of mregDCs. These results revealed for the first time that NPCSEVs promote the emergence of mregDCs using a galectin-9 dependent mechanism and open new perspectives for antitumour immunotherapy targeting NPCSEVs.

Impact of Electronic Cigarettes, Heated Tobacco Products and Conventional Cigarettes on the Generation of Oxidative Stress and Genetic and Epigenetic Lesions in Human Bronchial Epithelial BEAS-2B Cells.

Electronic cigarettes (e-cig) and heated tobacco products (HTP) are often used as smoking cessation aids, while the harm reduction effects of these alternatives to cigarettes are still the subject of controversial debate, in particular regarding their carcinogenic potential. The objective of this study is to compare the effects of e-cig, HTP and conventional cigarette emissions on the generation of oxidative stress and genetic and epigenetic lesions in human bronchial epithelial BEAS-2B cells. Our results show that HTP were less cytotoxic than conventional cigarettes while e-cig were not substantially cytotoxic in BEAS-2B cells. E-cig had no significant effect on the Nrf2 pathway, whereas HTP and cigarettes increased the binding activity of Nrf2 to antioxidant response elements and the expression of its downstream targets HMOX1 and NQO1. Concordantly, only HTP and cigarettes induced oxidative DNA damage and significantly increased DNA strand breaks and chromosomal aberrations. Neither histone modulations nor global DNA methylation changes were found after acute exposure, regardless of the type of emissions. In conclusion, this study reveals that HTP, unlike e-cig, elicit a biological response very similar to that of cigarettes, but only after a more intensive exposure: both tobacco products induce cytotoxicity, Nrf2-dependent oxidative stress and genetic lesions in human epithelial pulmonary cells. Therefore, the health risk of HTP should not be underestimated and animal studies are required in order to determine the tumorigenic potential of these emerging products.

Dalbavancin plasma concentrations in 133 patients: a PK/PD observational study.

OBJECTIVES: Limited pharmacokinetics data support dalbavancin long-term use in off-label indications and the optimal dosing regimen is debated. We aimed to describe dalbavancin concentrations in an observational retrospective multicentre study. METHODS: Patients from 13 French hospitals, treated with 1500 mg doses of dalbavancin and for whom therapeutic drug monitoring was performed from June 2018 to March 2021 were included. Dalbavancin plasma concentrations were described at peak and 1, 2, 3, 4, 6 and 8 weeks after the last 1500 mg dose. Concentrations in patients weighing more or less than 75 kg and with a GFR greater or less than 60 mL/min were compared. Microbiological data were collected and dalbavancin MIC was measured when possible. RESULTS: One hundred and thirty-three patients were included (69% treated for bone and joint infections, 16% for endocarditis). Thirty-five patients received a single dose of dalbavancin and 98 received several administrations. Two, 3 and 4 weeks after the last dose, median plasma concentrations were respectively 25.00, 14.80 and 9.24 mg/L for the first doses and 34.55, 22.60 and 19.20 mg/L for the second or subsequent doses. Weight and renal function had an impact on pharmacokinetics. Infection was documented in 105 patients (Staphylococcus spp. in 68% of cases). Staphylococcus aureus was isolated in 32.5% of cases (median MIC: 0.047 mg/L) and Staphylococcus epidermidis in 27% of cases (median MIC of 0.047 mg/L). CONCLUSIONS: Plasma concentrations of dalbavancin were consistent with those described in clinical trials and those sought during the industrial development of the molecule.

A current overview of the pharmacological composition of "La Chimique" consumed in Mayotte: Preliminary results of the CHASSE-MAREE protocol.

Mayotte Island, a French department located in the Mozambique Channel, has for several years been faced with the consumption of "La Chimique" (LC), reputed (but extremely poorly documented) to be a mixture of tobacco and synthetic cannabinoid receptor agonists (SCRAs). One of the objectives of the CHASSE-MAREE protocol is to assess the composition and heterogeneity of LC products through successive LC sample collection campaigns among users. Currently underway, we present here the first analytical results (samples collected in 2022). Between September and December 2022, 80 samples were collected throughout the island over three periods: 70 in the usual form of LC (small folded papers containing a plant-like sample, mostly tobacco), 6 powders, and 4 cigarettes. Analysis was performed using liquid chromatography with high-resolution mass spectrometry. The detected substances (number of detections) included SCRAs (MDMB-4en-PINACA [35], ADB-FUBIATA [25], MDMB-INACA [16], ADB-BUTINACA [15], AFUBIATA [11], 4F-MDMD-BICA [7], CH-PIATA [14], 5C-APINACA [3], BZO-HEXOXIZID [2], and 4F-ABINACA [1]), nicotine (68), tetrahydrocannabinol (THC), cannabinol (CBN), and cannabidiol (CBD) (2), medications (amantadine [11], cyamemazine [6], and acetaminophen [3]), and a designer benzodiazepine (bromazolam [4]). The SCRAs currently in use are varied, and the market for "cooks" (those who prepare LC) is dispersed according to where and when samples are collected. These preliminary results will be supplemented by analysis of samples collected in the first half of 2023 and by an improved description of the current panorama of consumption of LC in Mayotte (mapping, effects felt and dependence, etc.).

Lead, Mercury, and Cadmium Concentrations in Blood Products Transfused to Neonates: Elimination Not Just Mitigation.

Lead (Pb), mercury (Hg), and cadmium (Cd) are identified as potent developmental neurotoxicants. Neonates are the main group receiving multiple blood transfusions. The exposure of neonates to these heavy metals (HMs) can occur through blood transfusions. This study aimed to determine the concentrations of lead (Pb), mercury (Hg), and cadmium (Cd) in various blood products (plasma, platelets, packed red blood cells (pRBCs), and whole blood (WB)) to explore the probability of concurrent exposure of these HMs and to identify the metal load per transfusion with risk assessment. Residual bloods from blood bank bags were collected after neonatal transfusion. Pb, Hg, and Cd concentrations were determined in 120 samples of blood products by inductively coupled plasma mass spectrometry (ICP-MS). Pb and Cd levels were over the normal levels in 19.2 and 5.9% of all blood units, respectively. In 35 and 0.8% of blood units, the Pb and Cd concentrations, respectively, were higher than that recommended for transfusions in premature neonates. The anticipated safe value was surpassed by 2.5% for Cd of all transfusions, primarily because of WB. However, Hg was detected only in 5.8% of all samples and their concentrations were within the normal range. The concurrent neonatal exposure to Pb, Hg, and Cd was statistically significant. Hazard quotients of Hg and Cr were >1 and Pb cancer risk was 2.41 × 10-4. To the best of our knowledge, this study is the first report examining Pb, Hg, and Cd in blood products other than WB and pRBCs using ICP-MS. This study demonstrated the exposure of neonates to Pb, Hg, and Cd during transfusion with a considerable amount of Pb. It confirms the significant concurrent exposure to the three HMs, which maximize their potential developmental neurotoxicity with a high probability of developing non-carcinogenic and carcinogenic health effects.

Metabolic ratios and SNPs implicated in tramadol-related deaths.

Tramadol (TR) metabolism is performed by polymorphic enzymes that are influenced by genetic polymorphisms. Within this scope, the study presented here aimed to describe 41 genetic variants within CYP2D6, CYP2B6, and CYP3A4 genes in 48 cases of TR-related death that may be involved in the response to TR and to assess whether there is a correlation between these genetic variants and metabolic ratios (MRs). Blood samples from 48 victims of a TR-related death were analyzed to determine the concentrations of TR and its metabolites [O-desmethyltramadol (M1) & N-desmethyltramadol (M2)] using a LC-MS/MS method. All the samples were also genotyped for 41 common CYP2D6, CYP2B6, and CYP3A4 single nucleotide polymorphisms (SNPs) using the HaloPlex Target Enrichment system. Cases with the T/- genotype (rs35742686 in CYP2D6) had significantly higher M2/M1 ratio than cases with T/T genotype and cases with the G/A genotype (rs35599367 in CYP3A4) had significantly higher MR2 (TR/M2) ratio than cases with G/G genotype. The frequency of tested SNPs which belong to CYP2D6, CYP2B6, and CYP3A4 revealed the over-presentation of 2 SNPs (rs1058172 in CYP2D6 and rs4803419 in CYP2B6) in TR overdose group, which could have toxicological implications. These results indicate these polymorphisms in CYP2D6, CYP2B6, and CYP3A4 might influence the function and could increase the risk of toxicity. However, these findings should be supported in future studies with larger groups of cases.

Insights into the regulation of cellular Mn2+ homeostasis via TMEM165.

Golgi cation homeostasis is known to be crucial for many cellular processes including vesicular fusion events, protein secretion, as well as for the activity of Golgi glycosyltransferases and glycosidases. TMEM165 was identified in 2012 as the first cation transporter related to human glycosylation diseases, namely the Congenital Disorders of Glycosylation (CDG). Interestingly, divalent manganese (Mn) supplementation has been shown to suppress the observed glycosylation defects in TMEM165-deficient cell lines, thus suggesting that TMEM165 is involved in cellular Mn homeostasis. This paper demonstrates that the origin of the Golgi glycosylation defects arises from impaired Golgi Mn homeostasis in TMEM165-depleted cells. We show that Mn supplementation fully rescues the Mn content in the secretory pathway/organelles of TMEM165-depleted cells and hence the glycosylation process. Strong cytosolic and organellar Mn accumulations can also be observed in TMEM165- and SPCA1-depleted cells upon incubation with increasing Mn concentrations, thus demonstrating the crucial involvement of these two proteins in cellular Mn homeostasis. Interestingly, our results show that the cellular Mn homeostasis maintenance in control cells is correlated with the presence of TMEM165 and that the Mn-detoxifying capacities of cells, through the activity of SPCA1, rely on the Mn-induced degradation mechanism of TMEM165. Finally, this paper highlights that TMEM165 is essential in secretory pathway/organelles Mn homeostasis maintenance to ensure both Golgi glycosylation enzyme activities and cytosolic Mn detoxification.

Interpretation of hair and nails findings in an infant death case related to maternal addiction to tramadol.

An 11-month-old boy was found dead. Autopsy findings (cyanosis and polyvisceral congestion) and blood tramadol (TR) concentration of 6240 µg/L were consistent with an acute TR intoxication. In this poisoning situation, owing to the mother's statements (TR addiction leading to daily TR-orange juice mixture preparation accidentally used for the baby bottle preparation by the mother's partner), and the question of possible previous TR administrations to the infant, hair and/or nails (infant, mother, partner, 6-year-old sister) analysis was performed. Hair (2-cm-long hair segments from proximal [S1] to distal [S3]) and nails concentrations (pg/mg; nd: not detected) were as follows: Infant (hair: TR 1420 [S1], 1622 [S2], 2736 [S3]; O-DMT 16-38; N-DMT 34-100 [TR in significant quantities in the hair decontamination bath]-toenails: TR 584; O-DMT 8; N-DMT 15), mother (hair: TR 2340 [S1], 2150 [S2], 2500 [S3]; O-DMT 704-1170; N-DMT 827-1360), mother's partner (fingernails: TR 72; O-DMT nd; N-DMT nd) and sister (hair: TR 261 [S1], 524 [S2]; O-DMT 15 [S1], 16 [S2]; N-DMT 20 [S1], 38 [S2]). Metabolite ratio (infant and sister hair) was comparable to those observed in hair of pharmaceutical industry employees manufacturing tramadol. TR in washing baths, low observed nail concentrations (infant and partner) confirm (i) TR-related mother's addiction and (ii) external contamination issues (TR in sweat of the child at the time of death and in living environment) to explain the infant's keratinized samples results. This case report illustrates the interest of analyzing keratinized matrices of the whole family in such a situation.

In the case of extensively putrefied bodies, the analysis of entomological samples may support and complement the toxicological results obtained with other alternative matrices.

Entomotoxicological analysis is not part of routine toxicological analysis. This work aims to present two cases to illustrate the potential of entomological samples as complementary matrices to identify substances in cases of advanced putrefaction. (Case#1) A woman wasexhumed after 14 months to ascertain the exact cause of death. She died after six weeks of hospitalization because of intestinal ischemia followed by multiorgan failure. (Case#2) The corpse of a woman, known to have a psychiatric disorder, was discovered in her apartment. The state of decomposition of the body was consistent with a post-mortem period of several weeks (approximately 6 weeks). Toxicological investigations were performed in the biological and entomological samples of case#1 (hair, adipocere, brain, and pupae) and of case#2 (hair, bone, flies, and pupae) using liquid chromatography with high-resolution mass spectrometry and tandem mass spectrometry detection methods. In case#1, several drugs and metabolites were detected. In particular, the pupae analyses allowed the objectification of morphine administration, whereas morphine was only found in adipocere, but not in hair nor in brain. In case#2, the pupae analyses allowed the detection of three metabolites of quetiapine, and the flies analyses allowed the detection of valpromide, which was only detected in hair. In conclusion, the pupae and flies analyses in these two cases complemented the results obtained in the other alternative biological samples, which may guide hypotheses about the possible causes of death. Nevertheless, additional data and case reports would be of benefit to assess the value of entomotoxicology in routine forensic investigations.

Prediction of a Large-Scale Database of Collision Cross-Section and Retention Time Using Machine Learning to Reduce False Positive Annotations in Untargeted Metabolomics.

Metabolite identification in untargeted metabolomics is complex, with the risk of false positive annotations. This work aims to use machine learning to successively predict the retention time (Rt) and the collision cross-section (CCS) of an open-access database to accelerate the interpretation of metabolomic results. Standards of metabolites were tested using liquid chromatography coupled with high-resolution mass spectrometry. In CCSBase and QSRR predictor machine learning models, experimental results were used to generate predicted CCS and Rt of the Human Metabolome Database. From 542 standards, 266 and 301 compounds were detected in positive and negative electrospray ionization mode, respectively, corresponding to 380 different metabolites. CCS and Rt were then predicted using machine learning tools for almost 114,000 metabolites. R2 score of the linear regression between predicted and measured data achieved 0.938 and 0.898 for CCS and Rt, respectively, demonstrating the models' reliability. A CCS and Rt index filter of mean error ± 2 standard deviations could remove most misidentifications. Its application to data generated from a toxicology study on tobacco cigarettes reduced hits by 76%. Regarding the volume of data produced by metabolomics, the practical workflow provided allows for the implementation of valuable large-scale databases to improve the biological interpretation of metabolomics data.

Four-year follow-up of surface contamination by antineoplastic drugs in a compounding unit.

OBJECTIVES: This study aimed to monitor the contamination by antineoplastic drugs on work surfaces in a compounding unit 4 years after its implementation. METHODS: This descriptive study was done in a unit performing on average 45 000 preparations per year. Surface sampling points (N=23) were monitored monthly in the frame of routine activity from the opening of an anticancer drug compounding unit. Contamination with nine antineoplastic drugs (cyclophosphamide, ifosfamide, dacarbazine, 5-fluorouracil, methotrexate, gemcitabine, cytarabine, irinotecan and doxorubicin) was assessed on wipes with a local liquid chromatography coupled with a tandem mass spectrometer analysis. The contamination rate (CR, %) was prospectively monitored every month during the entire study period. The occurrence of critical incidents was also registered. The effect of each safety measure implemented during this period was also analysed. RESULTS: Based on the 1104 samples collected between March 2016 and March 2020, the CR was 18.5%. If three different critical incidents among a vial breakage that occurred were individually considered, this CR was slightly lower than that in the literature. Eight months after opening and taking different corrective actions, the overall CR dropped from 42.39% to 11.52% (p<0.001). Contamination was limited to the area that includes the compounding room and, more precisely, the welder and the QC-Prep+ sampling points. CONCLUSIONS: From the beginning of the study and from month to month, surface contamination was limited to the nearest sampling points to the compounding unit. This 4-year monitoring study allowed us to determine the intravenous conventional antineoplastic drugs and sampling points to be focused on.

2-MeOPP positive hair result in case of urapidil treatment.

The metabolism of urapidil to 2-MeOPP induces the risk of detection of 2-MeOPP in biological samples (blood, urine and hair) in case of urapidil treatment. This is supported by two case reports and an in vitro study of urapidil metabolism.

Therapeutic Drug Monitoring and Pharmacogenetic Testing as Guides to Psychotropic Drug Dose Adjustment: An Observational Study.

To avoid the failures in therapy with psychotropic drugs, treatments can be personalized by applying the results of therapeutic drug monitoring and pharmacogenetic testing. The objective of the present single-center observational study was to describe the changes in psychotropic drug management prompted by therapeutic drug monitoring and pharmacogenetic testing, and to compare the effective drug concentration based on metabolic status with the dose predicted using an in silico decision tool for drug-drug interactions. The study was conducted in psychiatry wards at Lille University Hospital (Lille, France) between 2016 and 2020. Patients with data for at least one therapeutic drug monitoring session or pharmacogenetic test were included. Blood tests were performed for 490 inpatients (mainly indicated by treatment monitoring or failure) and mainly concerned clozapine (21.4%) and quetiapine (13.7%). Of the 617 initial therapeutic drug monitoring tests, 245 (40%) complied with good sampling practice. Of the patients, 51% had a drug concentration within the therapeutic range. Regardless of the drug concentration, the drug management did not change in 83% of cases. Thirty patients underwent pharmacogenetic testing (twenty-seven had also undergone therapeutic drug monitoring) for treatment failure; the plasma drug concentration was outside the reference range in 93% of cases. The patient's metabolic status explained the treatment failure in 12 cases (40%), and prompted a switch to a drug metabolized by another CYP450 pathway in 5 cases (42%). Of the six tests that could be analyzed with the in silico decision tool, all of the drug concentrations after adjustment were included in the range estimated by the tool. Knowledge of a patient's drug concentration and metabolic status (for CYD2D6 and CYP2C19) can help clinicians to optimize psychotropic drug adjustment. Drug management can be optimized with good sampling practice, support from a multidisciplinary team (a physician, a geneticist, and clinical pharmacist), and decision support tools.