Forensic Toxicology最新文献

Purpose: Methamphetamine (MA) is widely abused worldwide and has long been a major social concern. To provide basic information for comparing MA toxicokinetics in humans, we analyzed changes in the serum and urinary concentrations of MA and its metabolites in mice and compared the toxicokinetic profiles at the toxic dose with those at the therapeutic dose.

Methods: Mice were administered therapeutic (1.5 mg/kg) or toxic (15 mg/kg) doses and blood and urine samples were collected. The serum concentrations of MA and its metabolite amphetamine (AMP), as well as the urinary concentrations of MA and its metabolites-AMP, p-hydroxymethamphetamine (OHMA), p-hydroxyamphetamine (OHAMP), and norephedrine (NEP) -were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Results: The serum AUC_0-24 values for MA and AMP were approximately 16 and 41 times higher, respectively, in the toxic dose group than those in the therapeutic dose group. The urinary AMP and OHAMP excretion levels were approximately 3.6 and 2.2 times higher, respectively, in the toxic dose group. The urinary [AMP] / [MA] ratio at all collected points and [AMP] / [OHMA] ratio in the 0-24 h sample were significantly higher in the toxic than in the therapeutic dose group.

Conclusions: The results obtained from this study suggest that the metabolism of AMP to OHAMP and NEP was saturated during intoxication. Furthermore, the determination of whether a toxic dose had been administered within 24 h or after 24 h would be possible through a joint evaluation of the urinary [AMP] / [MA] and [AMP] / [OHMA] ratios.

Purpose: The cellular mechanisms underlying tolerance development to psychostimulant-induced neurotoxicity remain poorly understood. This study investigated these mechanisms using pyrrolidinophenone derivatives (PPs), potent amphetamine-type stimulants with strong dopaminergic activity and high cytotoxicity, aiming to establish a neuronal tolerance model and to explore adaptive processes relevant to substance use disorder.

Methods: Human SK-N-SH neuronal cells were chronically exposed to α-pyrrolidinooctanophenone (α-POP) to prepare the drug-resistant cell line, SH/POP. Transcriptomic profiling was performed to identify gene expression alterations associated with tolerance development.

Results: Cell sensitivity assay showed that SH/POP cells can survive at lethal concentrations (> 40 μM) of α-POP. RNA sequence analysis of the resistant cells identified alterations in 1,298 differentially expressed genes and the gene ontology analysis surmised an upregulation of calcium-binding-related genes. The development of α-POP resistance reduced the basal Ca²⁺ concentration and suppressed the caspase-3 activation elicited by the drug. Additionally, the development down-regulated the phosphorylation of a transcription factor cAMP response element-binding protein (CREB) and expressions of CREB-target genes, Fos proto-oncogene AP-1 transcription factor subunit and neurotensin. Furthermore, constitutive activation of endoplasmic reticulum stress responses and selective enhancement of trypsin-like proteasome activity in SH/POP cells were detected.

Conclusions: Resistance development of neuronal cells to PPs is ascribable to suppression of calcium-dependent apoptosis and CREB signaling, and constitutive activation of endoplasmic reticulum stress responses. The SH/POP cell line represents a novel in vitro model to study molecular adaptations to psychostimulant toxicity and provides insights into neuroadaptive mechanisms underlying substance use disorder.

Purpose: Orexin receptor antagonists (ORAs) are a novel class of medications used in the treatment of insomnia. With increasing restrictions on benzodiazepine prescriptions, a rise in ORA overdose is expected. To enable the prediction of clinical severity and formulation of appropriate treatment strategies, we developed a rapid analytical method for detecting ORAs in plasma samples using a Monolithic solid-phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS).

Method: Extraction was performed using MonoTip C18. All steps from pretreatment to elution were conducted using centrifugation. Quantification was carried out using GC-MS with temperature-programmed analysis by positive ion electron ionization, using suvorexant-d₆ as the internal standard. The method was applied to plasma samples from actual ORA overdose cases to evaluate its practical applicability.

Result: The calibration curves demonstrated excellent linearity over the range of 10-2,000 ng/mL, with correlation coefficients of at least 0.9999. Reproducibility showed a coefficient of variation (CV) between 0.6% and 6.9%, and recovery rates were over 83%. ORA concentrations in overdose patient samples were successfully quantified using this method.

Conclusion: MonoTip C18 utilizes a reduced amount of solvent, thereby eliminating the need for evaporation-to-dryness steps. As a result, the entire procedure, encompassing SPE to GC-MS detection, can be completed within 40 min. This single protocol is applicable to all ORAs currently available in Japan and is suitable for both clinical and forensic toxicological applications.

Purpose: Some toxic plants have strong morphological similarities with edible wild plants. Therefore, poisoning often occurs due to accidental ingestion. It is important to identify poisonous plants in edible plant mixtures, even if the samples have been digested. In the present study, we developed a method for genus and species identification in mixed samples using massive parallel sequencing (MPS).

Methods: Veratrum oxysepalum and Colchicum autumnale are the most common causative plants of such accidents and their morphological features are similar to those of the edible Hosta sieboldiana. In this study, we used V. oxysepalum and C. autumnale as the target poisonous plant species. We developed and optimized an MPS analysis method for trnL and rbcL regions that are commonly used in plant species identification. Initially, DNA from poisonous plants (V. oxysepalum and C. autumnale) and edible plants (H. sieboldiana) were mixed in various ratios and analyzed using MPS. Next, we prepared cooked materials and simulated gastric contents from V. oxysepalum, C. autumnale, and H. sieboldiana and analyzed them using MPS.

Results: We detected both poisonous and edible plant DNA when mixed in equal amounts. Poisonous plants were also detected in cooked or simulated gastric acid content. These results suggest that our method can be used to identify the genera or species of plants present in cooked materials and simulated gastric contents.

Conclusions: These results indicate that MPS techniques are useful for the forensic analysis of plant materials.

Purpose: 3,4-Methylenedioxypyrovalerone (MDPV) is a potent psychostimulant substance endowed with addictive properties. As mammalian target of rapamycin (mTOR) mediates neuroadaptive changes responsible for development of addiction, the current study evaluated whether rapamycin, a potent and selective inhibitor of mTOR, prevents induction and expression of behavioral sensitization in mice treated with MDPV.

Methods: Locomotor sensitization was used as an animal model of early phase of addiction. C57BL/6JRj mice were treated with rapamycin before administration of MDPV during the induction phase of sensitization, or during the final 5 days of the withdrawal. Sensitization was assessed based on the measurement of locomotor activity after treatment with MDPV.

Results: Rapamycin administered on days 1-7 inhibited induction of sensitization characterized by increased horizontal and vertical locomotor activity on day 7 compared to day 1. Additionally, when given during the withdrawal from MDPV, rapamycin blocked expression of sensitization, defined as augmented response to MDPV on day 21 compared to day 1.

Conclusions: Abolishment of locomotor sensitization to MDPV by rapamycin suggests that neuroadaptive changes underlying this phenomenon are dependent on the mTOR signaling and warrants further research on possible application of mTOR inhibitors in treatment of addiction.

Purpose: Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are direct metabolites of ethanol (EtOH) and sensitive biomarkers of alcohol consumption. However, despite extensive studies in Western populations, data on Japanese individuals are limited. This study characterized the pharmacokinetics of EtG and EtS in Japanese adults and evaluated the influence of dose, genetic polymorphisms, and habitual alcohol use.

Methods: Twenty-eight healthy Japanese adults received either 1.0 or 0.2 g/kg of pure EtOH (high or low dose). Whole blood and urine samples were collected for 24 h, and EtG and EtS were quantified using validated liquid chromatography-tandem mass spectrometry. Pharmacokinetic parameters were analyzed. The urinary excretion and recovery of EtG and EtS were estimated. Associations between genetic polymorphisms in alcohol-metabolizing and conjugation-related enzymes and Alcohol Use Disorders Identification Test (AUDIT) scores were also evaluated.

Results: EtG and EtS persisted longer than EtOH in both blood and urine. Both metabolites were excreted in the urine in a dose-dependent manner. Individuals carrying ALDH2*1/*2 showed a significantly higher urinary EtG formation rate than those carrying ALDH2*1/*1 (wild type). The AUDIT score showed a modest positive association with the urinary formation rate of EtS but not with EtG. The 24 h urine from high-dose participants exceeded international cutoffs, whereas that from low-dose participants was below the quantification limits.

Conclusions: EtG and EtS showed sustained detectability, and their urinary excretion was dose-dependent, indicating their utility as biomarkers of recent alcohol intake. These findings support their potential forensic applications of EtG and EtS in Japan.

Clinical trial registration: Japan Registry of Clinical Trials (jRCT), jRCT1070240083 (registered 2024-12-10).

Purpose: A powder found at a fatality scene, labeled as the synthetic cathinone 3',4'-methylenedioxy-α-pyrrolidinohexiophenone (MDPHP) and most likely smoked using a crack pipe, was analyzed. The powder was identified as very potent synthetic cannabinoid ADB-BUTINACA/ADB-BINACA with a high purity (> 98%). This case highlights the risks associated with mislabeled novel psychoactive substances (NPS), particularly those purchased online.

Methods: The powder was analyzed using liquid chromatography-high resolution mass spectrometry (LC-HRMS), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and nuclear magnetic resonance (NMR) spectroscopy. Comprehensive toxicological screening, including NPS, was performed in urine and blood. ADB-BUTINACA was quantified using a standard addition method on various post-mortem matrices: femoral and heart blood, urine, stomach content, bile fluid and liver tissue. Scalp hair was analyzed via external calibration to assess potential long-term exposure.

Results: ADB-BUTINACA concentrations were 34.5 ng/mL in femoral blood, 101 ng/mL in heart blood and 3.1 ng/mL in urine. Traces of MDMB-BUTINACA were found in the powder and hair, but not in other biological matrices. ADB-BUTINACA metabolites were detected in all biological matrices. MDPHP was found at low concentrations (< 2 ng/mL) in blood and urine but not in the powder, indicating prior cathinone use. A toxicological significance score (TSS) of 3 was assigned for this monointoxication with ADB-BUTINACA.

Conclusions: This case demonstrates fatal poisoning due to extremely high ADB-BUTINACA concentrations in post-mortem blood samples, emphasizing the severe risks associated with mislabeled substances. It underscores the importance of drug checking services to prevent poisonings and overdoses caused by highly potent NPS.