Cumulative Deleterious Effects of Tetrahydrocannabinoid (THC) and Ethanol on Mitochondrial Respiration and Reactive Oxygen Species Production Are Enhanced in Old Isolated Cardiac Mitochondria.

Delta 9 tetrahydrocannabinol (THC), the main component of cannabis, has adverse effects on the cardiovascular system, but whether concomitant ethanol (EtOH) and aging modulate its toxicity is unknown. We investigated dose responses of THC and its vehicle, EtOH, on mitochondrial respiration and reactive oxygen production in both young and old rat cardiac mitochondria (12 and 90 weeks). THC dose-dependently impaired mitochondrial respiration in both groups, and such impairment was enhanced in aged rats (-97.5 ± 1.4% vs. -75.6 ± 4.0% at 2 × 10-5 M, and IC50: 0.7 ± 0.05 vs. 1.3 ± 0.1 × 10-5 M, p < 0.01, for old and young rats, respectively). The EtOH-induced decrease in mitochondrial respiration was greater in old rats (-50.1 ± 2.4% vs. -19.8 ± 4.4% at 0.9 × 10-5 M, p < 0.0001). Further, mitochondrial hydrogen peroxide (H2O2) production was enhanced in old rats after THC injection (+46.6 ± 5.3 vs. + 17.9 ± 7.8%, p < 0.01, at 2 × 10-5 M). In conclusion, the deleterious cardiac effects of THC were enhanced with concomitant EtOH, particularly in old cardiac mitochondria, showing greater mitochondrial respiration impairment and ROS production. These data improve our knowledge of the mechanisms potentially involved in cannabis toxicity, and likely support additional caution when THC is used by elderly people who consume alcohol.

Complete investigations (autopsy, toxicology, and histology) in a death due to apixaban overdose.

The dead body of a 54-year-old man was found at home by his partner. He was off work due to depression. A letter with suicidal intention was present on the scene. He was known to be a heavy drinker, and near the body, an empty bottle of whisky was found. In addition, 2 empty blisters of Eliquis (apixaban) 5 mg, corresponding to 40 tablets, were identified. Apixaban is an oral anticoagulant, acting as a factor Xa inhibitor. Autopsy findings were mostly unremarkable, except numerous bruises and some superficial self-inflected wounds. Histology showed hematomas of calyces and renal pelvis and in the liver, several areas of perivenular haemorrhagic necrosis. Others organs were congestive. Femoral venous blood alcohol was 0.11 g/L. In femoral venous blood, a toxic concentration of apixaban was measured at 1184 ng/mL using LC-MS/MS. Other drugs found at therapeutic concentrations included diazepam (99 ng/mL), nordiazepam (171 ng/mL), flecainide (447 ng/mL), and mianserine (65 ng/mL). Using liquid chromatography coupled to high-resolution mass spectrometry, 2 metabolites were identified, O-desmethyl-apixaban (61.8% of the apixaban response) and hydroxyl-apixaban (4.5% of the apixaban response). Long-term therapy was confirmed by a concentration of 10390 pg/mg in pubic hair.

Thermal bone injuries: postmortem computed tomography findings in 25 cases.

INTRODUCTION: Fire death investigations attempt to determine whether a subject was alive or dead before the fire started. Therefore, it is essential to assess if the bone damage is traumatic or the result of exposure to heat. This observational study aims to expose the specific CT semiology of thermal bone lesions to allow the forensic radiologist to identify and distinguish them from traumatic lesions that would have preceded death. MATERIALS AND METHODS: We retrospectively reviewed the CT findings of 25 bodies with thermal bone lesions for which a postmortem computed tomography (PMCT) was performed prior to an autopsy. Imaging findings were correlated to the autopsy data to identify the specific features of thermal bone lesions. RESULTS: Specific signs of thermal injuries to bone were identified on PMCT on all deceased cases. Thermal damages predominated in areas directly exposed to flames (rib cage, distal extremities) with less soft tissue coverage ("soft tissue shielding"). The mottled appearance of bone marrow was a constant sign of burned bones. Heat fractures such as trans-diploic fractures of flat bones and beveled ("flute-mouthpiece") fractures of extremities seemed specifically related to thermal mechanism. In addition, we provided a better description of superficial cortical fissures of flat bones ("ancient Chinese porcelain") and observed a "stair step" fracture of a long bone until now undescribed in radiological literature. CONCLUSION: Thermal bone lesions have specific CT findings, different on several points from traumatic injuries. Their knowledge is essential for radiologists and forensic physicians to provide an accurate report of injury and conclusions.

The use of multiple keratinous matrices (head hair, axillary hair, and toenail clippings) can help narrowing a period of drug exposure: experience with a criminal case involving 25I-NBOMe and 4-MMC.

The objective of this publication is to present the interest of collecting several keratinous specimens in order to document possible drug impairment at the time of the assault, when knowledge solely occurred 7 months after. A subject committed a murder and within minutes after the crime self-inflicted serious wounds. He was charged to the hospital where he slowly recovered. After several weeks, he was sent to prison. During this period, intelligence indicated possible drug impairment at the time of the assault after using 25I-NBOMe and 4-MMC. Head hair (4 cm), axillary hair, and toenails were collected 7 months after the crime. New psychoactive substances were tested in each specimen using LC-MS/MS, which revealed the presence of 25I-NBOMe and 4-MMC in axillary hair (2 and 6 pg/mg) and toenails (1 and 5 pg/mg). However, the perpetrator claimed that the positive findings were due to contamination in prison. Therefore, the head hair was also tested and results returned negative (LOQ at 1 pg/mg), demonstrating absence of contamination during the last 4 months before collection. Combining the window of drug detection in axillary hair (about 4 to 8 months) and the one of toenail clippings (up to 8 months), and excluding drug exposure during the previous 4 months as well as external contamination as the head hair results were negative, allowed us to conclude that the positive findings in axillary hair and toenails are more likely than not consistent with consumption of both 25I-NBOMe and 4-MMC at the time of the crime.

Be careful with lentils! About a forensic observation.

The discovery of exogenous particles in the broncho-pulmonary tree is frequently described in forensic literature, especially in lung samples, in the context of aspirated gastric content during the death agony period or during resuscitation. We report an original observation of a multi-visceral dispersion of exogenous particles detected, in an 8-year-old boy, who allegedly fell from a 2-m high brick-wall. The autopsy found major liver fracture and diaphragm rupture with massive internal hemorrhage without gastric wall rupture. The histological analyses have identified round to oval bodies in the lung bronchi, alveoli, and, rarely, in vascular sections, and also on the surface of several samples. These particles stained strongly pink by the periodic acid Schiff method, evoking dried vegetables. Two hypotheses were invoked: aspirated vegetable particles into the bronchial tree or parasitic infection, like pinworm larva. In order to characterize the nature of these particles, different legumes were cooked, embedded in paraffin wax, and examined under light microscope. Simultaneously, morphological comparison between the gastric content and pinworm larva and lentils was made and a PCR analysis was performed on gastric fluid sample. The DNA sequencing showed a Fabaceae plant family, Lens culinaris. The possibility of a hematogenous dissemination of the starch grains during a perimortem aspiration of gastric content seems unlikely, and a contamination from the gastric content of the organs samples during the autopsy or the pathologic macroscopic and microscopic processes seems to be the principal hypothesis. The formal identification of such particles is important to avoid the misdiagnosis of a potential parasitic infection. The risk of confusion can be detrimental in some circumstances.

Perspectives in Evaluating Selective Androgen Receptor Modulators in Human Hair: A Short Communication.

BACKGROUND: As hair testing increases the window of drug detection and permits the differentiation of long-term use from a single exposure when performing segmental analyses (which also allows establishing the pattern of use), this matrix should be considered as a suitable complement to standard investigations in clinical, forensic, and sport toxicology. The authors were recently involved in 3 cases where hair analysis was used to demonstrate the use of selective androgen receptor modulators (SARMs), including ligandrol (LGD-4033), andarine (S-4), and ostarine (S-22). SARMs are increasingly being abused as "safe" alternatives to steroids. METHODS: After decontamination using dichloromethane, hair specimens were segmented, cut into very small segments (<1 mm), incubated overnight in a buffer, and extracted using a mixture of organic solvents. Drugs were tested using liquid chromatography-tandem mass spectrometry and confirmed using liquid chromatography/HRMS. RESULTS: The determined concentrations were as follows: ligandrol, 14-42 pg/mg; andarine, 0.1-0.7 pg/mg; and ostarine, 3-21 pg/mg. CONCLUSIONS: To enhance performance, SARMs must be used on a long-term basis, which can have serious clinical consequences, including liver damage, myocardial infarction, and blood clots. Hair testing for SARMs has additional benefits versus urine analysis as it can detect the parent compound and numerous metabolites.

The Importance of Autopsy in a Case of Digestive Perforation Undetected by Postmortem Computed Tomography.

ABSTRACT: Cases of foreign body ingestion in the forensic literature are mainly described in children or psychiatric patients. Postmortem imaging can detect most foreign bodies, but its sensitivity depends, among other things, on the type of item and its location. In some cases, the ingestion of foreign bodies can remain unnoticed and have serious consequences for the patient. We describe the case of a patient who died in a psychiatric seclusion room with no obvious cause and for whom a forensic autopsy was requested. Further investigations showed the existence of a subdural hematoma associated with a midline shift, secondary to a skull fracture that was considered to be the cause of death. Toxicological analyses identified in blood several drugs, including diazepam (24 ng/mL) and its major metabolite nordazepam (24 ng/mL), propranolol (57 ng/mL), paliperidone (9 ng/mL), and loxapine (620 ng/mL). The forensic autopsy revealed the existence of a gastrointestinal perforation after the ingestion of a plastic teaspoon, which the postmortem CT scan had failed to detect. Although technological advances continue to assist the forensic pathologist in his diagnosis, autopsy still has a leading role in forensic investigations and does not yet seem to be replaceable by imaging techniques alone.

Murdered while under the influence of 3-MeO-PCP.

The abuse of new psychoactive substances (NPS) has been dramatically increasing all around the world since the late 2000s. The availability of hundreds of NPS in the past decade is challenging for both public health and global drug policies. A 39-year-old woman, known as a multidrug addict, was murdered by her partner by ligature strangulation. A comprehensive toxicological screening by gas chromatography and ultra-high performance liquid chromatography with tandem mass spectrometry revealed the simultaneous presence of ethanol (1.37 g/L), diazepam (157 ng/mL) and nordiazepam (204 ng/mL), cocaine (25 ng/mL) and benzoylecgonine (544 ng/mL), and (3-methoxy-(1-(1-phenylcyclohexyl)piperidine) or 3-MeO-PCP, a dissociative hallucinogen anesthetic drug. Concentrations of 3-MeO-PCP were 63, 64, and 94 ng/mL in femoral blood, bile, and urine, respectively. Hair tested also positive for 3-MeO-PCP on 3 × 2-cm segments at 731, 893, and 846 pg/mg, indicating long-term abuse of the drug. This seems to be the first ever reported hair concentrations. Major impairment of the victim, including visual hallucinations and alteration of behavior, was attributed to the mixture of all the drugs, with a major contribution of 3-MeO-PCP. The toxicological findings were compared to the few reports available in the medical literature.

Assessment of Pregabalin Use by Hair Testing.

BACKGROUND: The detection and analysis of drugs in hair has progressively emerged as a consequence of the enhanced sensitivity of analytical techniques used in forensic toxicology; a greater advantage in using this matrix respect to classical ones (i.e., urine and blood) is an easier and noninvasive sample collection, even when the careful supervision of law-enforcement officers is required to avoid the risk that the sample may be adulterated or replaced. Moreover, according to the length of the hair, the history of drug exposure can be retrospectively monitored from few weeks up to months or years since sample collection. OBJECTIVE: Given the potential negative effects of pregabalin, an antiepileptic and analgesic drug with a high risk of misuse and abuse, the laboratory was asked to test for the drug in hair. METHOD: A new ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry was developed. The method involves incubation of 25 mg of cut hair in acetonitrile for 2 h in an ultrasonic bath and separation on an Acquity HSS C18 column (150 × 2.1 mm × 1.8 µm) maintained at 50°C in a thermostatically controlled oven. A gradient elution was performed. RESULTS: The method was fully validated according to international standards. The limit of quantitation of the test was 10 pg/mg. Five authentic cases of pregabalin in hair segments were tested using the method and the results were in the range 17-1487 pg/mg. CONCLUSION: This new method was found suitable to monitor both patients under pregabalin therapy and dependent subjects.

Postmortem Hyperthermia: Two Case Reports and a Review of the Literature.

In this daily practice, the forensic pathologist is rarely confronted with postmortem hyperthermia associated with the rapid onset of rigor mortis. We report 2 similar cases where the rectal temperature value taken during the on-scene investigations by the forensic pathologist was greater than 40°C (104°F) in both cases, and rigor mortis was complete within less than 6 hours postmortem. The first case was due to a deadly intoxication by ecstasy and the second one to the deadly association of methadone and a possible neuroleptic malignant syndrome. Infection-related deaths were eliminated. Thus, the association of postmortem hyperthermia and rapid-onset rigor mortis would suggest in the first hypothesis a toxic death, particularly 3,4-methylenedioxymethamphetamine. However, an autopsy and toxicological analysis are necessary to confirm the cause of death.

Bone and soft tissue histology: a new approach to determine characteristics of offending instrument in sharp force injuries.

This paper describes a new approach to determine characteristics of the implement used to inflict trauma which involves the histological analysis of exogenous particles. Based on Locard's principle "every contact leaves a trace," we decided to assess whether histological examination of bone and soft tissue around a penetrating injury (sharp force trauma) could provide evidence of the offending implement. Case reports and experimental studies have demonstrated the potential of cut mark features in bone to identify the causative implement and potentially assist in identifying the perpetrator. Scanning electron microscopy in combination with energy-dispersive X-ray spectrometry (SEM/EDS) have previously been reported to identify exogenous particles from various implements. In medical research, histological techniques are used to study the impact of foreign particles in tissues originating from implants. However, the routine use of histology in forensic medicine focuses on understanding type and timing of injuries. Based on three forensic cases, the results presented in this paper demonstrate that histology offers a cost-efficient and reliable means to detect foreign particles related to offending implement and/or to the environment where the victim was located. The interpretation of histological results was performed in conjunction with the macroscopic autopsy findings and anthropological analysis of bone samples.

Update on injury mechanisms in abusive head trauma--shaken baby syndrome.

Violently shaking a baby leads to clinical presentations ranging from seizures to cardiopulmonary arrest. The main injuries sustained are retinal hemorrhages, subdural hemorrhages, and sometimes fractures and spine injury. It is important to have a global view of the injuries sustained by the infant to correctly discuss the biomechanical aspects of abusive head trauma. Recent works based on finite element models have shown that whiplash-shaking alone is enough to generate vitreo-retinal traction leading to retinal hemorrhage and to cause the rupture of bridging veins leading to subdural hemorrhage. We will review the main papers dealing with the mechanisms of shaken baby syndrome and present the most relevant hypothesis concerning the biomechanical aspects of injuries related to shaken baby syndrome.

Evidence of exposure to flecainide in a newborn by keratinous matrices testing and interpretation of the findings.

Pediatric poisoning represents a serious problem all around the world. Abuse or neglect of children by adults must be highlighted in children exposed to drugs to which they would not normally have access. Usually, segmental hair analysis would allow in these contexts to determine whether the exposure was unique or repetitive. Hair and nail samples from a 9-month-old girl were received in our laboratory for analysis, after the child was hospitalized due to severe dehydration caused by her mother's neglect. At the admission, flecainide, an antiarrhythmic never prescribed to the child, was identified in the daughter urine. Using an LC-MS/MS method, flecainide tested positive in the child's hair at the following concentrations: 66 pg/mg (root to 1 cm), 61 pg/mg (1-2 cm), and 125 pg/mg (2-3 cm). Traces below the limit of quantification (1 pg/mg) were also present in the nail clippings. These concentrations are much lower than those obtained in adults under daily treatment. Given the different pharmacokinetic and dynamic parameters in children, the different rate of hair growth, and the greater porosity of the hair, which makes it more prone to external contamination, the interpretation of hair findings in children remains very complicated. In this case, it can be assumed that the presence of the drug in the urine indicates systemic incorporation and that administration had occurred for some months (three positive segments). The interpretation of hair tests from young children needs a global review of all the findings, as a positive result cannot stand alone to claim repetitive exposures.

Testing for clomiphene in keratinous matrices using LC-MS/MS in doping purpose: Is a single intake of clomiphene detectable in hair and nail clippings?

Clomiphene is a selective estrogen receptor modulator. It is indicated for the treatment of female infertility issues but in sport, it can be misused to stimulate endogenous testosterone secretion in men. Therefore, it has been prohibited at all times by the World Anti-doping Agency. The aim of this study was to get data to be able to interpret concentrations in athletes. A healthy volunteer (male, 62 years-old) ingested a single therapeutic dose of clomiphene (Clomid™, 50 mg). Strands of hair (blond, 4 cm) were collected one month after the ingestion. Body hair (beard, axillary, pubic and chest hair), and finger and toenails were collected over 4-5 months. A previous method was modified to identify and quantify clomiphene in keratinous matrices. 30 mg of specimen were sonicated and incubated in 1 mL of methanol, in presence of 200 pg of clomiphene-D5 (internal standard). After centrifugation and evaporation of the organic phase, the samples were analyzed using LC-MS/MS. Linearity was verified in hair and nail clippings between 1 and 500 pg/mg. The limits of detection and quantification were determined at 0.3 and 1 pg/mg respectively. The study demonstrated that clomiphene tested positive in all the analyzed specimens at 9 pg/mg in head hair, from 28 to 486 pg/mg (body hair) and from 4 to 57 pg/mg (nails). Clomiphene was identified for the first time in multiple keratinous matrices. This study demonstrated that a single oral therapeutic dose is detectable in keratinous matrices over a long period of time.

In vitro characterization of protonitazene metabolites, using human liver microsomes, and first application to two urines collected from death cases.

Protonitazene, or N,N-diethyl-5-nitro-2-[(4-propoxyphenyl)methyl]-1H-benzimidazole-1-ethanamine, is a novel synthetic opioid, which belongs to the nitazene family. Over the last four years, nitazenes have re-emerged on the new psychoactive substances market and have been reported in several fatal intoxication cases. The metabolism of several nitazene analogues have already been studied, but to date, no data exists regarding protonitazene. The aim of the study was the detection of protonitazene and its metabolites in authentic human urine collected in two fatal intoxication cases, comparing the data after in vitro incubation with human liver microsomes, and subsequent analysis by ultra-performance liquid chromatography-tandem mass spectrometry and ultra-performance liquid chromatography-high-resolution mass spectrometry. Protonitazene metabolites, including N-desethyl-protonitazene, 5-amino-protonitazene and 4-hydroxy-nitazene, were characterized in vitro and were identified in the urine of both cases. The ratios between metabolites and parent protonitazene, higher than 1, were calculated to estimate the proportionality of metabolites. The results suggest that testing protonitazene metabolites should increase the window detection of exposure to protonitazene.

Testing for Kratom alkaloids in fingernail clippings - not only mitragynine.

Kratom (Mitragyna speciosa) is a species of large tree that grows in Southeast Asia and is part of the Rubiaceae family. Its fresh leaves are harvested for their medicinal properties and used for their psychoactive effects. Kratom contains many biologically active alkaloids, including mitragynine and 7-OH-mitragynine, which are considered the two most important psychoactive components and constitute approximately 66% and 2% of the total alkaloid content. Other alkaloids are present in the plant, such as speciogynine, speciociliatine and paynantheine, but have less psychoactive activity. Over the past decade, the sale of kratom powder has increased on the Internet. This led to a significant increase in forensic cases. Given the lack of data existing in the literature, and the total absence of data in nails, the authors report a study to determine the best target alkaloids for documenting kratom consumption in this matrix. Fingernail clippings from a supposed kratom powder user were analyzed after liquid-liquid extraction, chromatography separation using a HSS C18 column and performed on an ultra-high performance liquid chromatography coupled to a tandem mass spectrometer. In the specimen, mitragynine was quantified at 229 pg/mg, speciogynine and paynantheine were both quantified at 2 pg/mg, and speciociliatine was quantified at 19 pg/mg. 7-OH-mitragynine was not detected. The interpretation of these concentrations is complex, since there is currently no reference in the literature, as this is the first identification of mitragynine and other kratom alkaloids in nails. Nevertheless, in view of the high concentration of mitragynine, the subject seems to be a repetitive user of kratom. According to the measured concentrations, it seems that mitragynine remains the best target to document kratom consumption, but the identification of the other alkaloids would enhance the specificity of the test.

Is it really possible to kill with insulin without leaving traces? From lifesaver to killer, the issues surrounding the analytical characterization of postmortem insulin illustrated by an exemplary case.

Evidence of an insulin overdose is very complicated in the medico-legal field. The analysis and subsequent interpretation of results is complex, especially when treating postmortem blood samples. The instability of insulin, the special pre-analytical conditions and the absence of specific analytical methods has led most laboratories not to analyze insulin in their routine with a consequent underestimation of cases. This paper aims to assess the difficulties associated with the analytical characterization of insulin by describing a case that typically represents most of the inconveniences encountered following a suspected insulin overdose. The case concerns a man found dead at home by his brother. After an external examination, which did not reveal a specific cause of death, toxicological analysis was requested which did not reveal any substance of toxicological interest. Only 9 months later, it was reported to the toxicologist that the subject was diabetic, on insulin lispro treatment and that three empty syringes were found next to his body. Following analysis by LC-high-resolution mass spectrometry, the presence of insulin lispro at a concentration of 1.1 ng/mL, a therapeutic concentration, was evidenced. Despite the low concentration found, overdose cannot be excluded and this paper will describe the criteria evaluated to reach this conclusion. This case highlights that the interpretation of a postmortem insulin concentration is very complex and requires the evaluation of various elements including the circumstances of death, the subject's medical history, the interval between death and sampling and the sample storage.

Cortisone in hair of elementary school girls and its relationship with childhood stress.

UNLABELLED: Children may be exposed to stressful situations with adverse effects on their physiological and psychological health. As cortisone may be a useful additional biomarker for stress research and as it has been shown to be detectable in human hair, this study measured physiological concentrations of hair cortisone in 223 elementary school girls and explored its relationship with child-reported estimates of stress, more specifically questionnaires on major life events (i.e., Coddington Life Events Scale for Children), emotions (i.e., anger, anxiety, sadness, and happiness), and coping strategies (i.e., emotion- versus problem-focused coping). Cortisone concentrations were positively correlated with the overall life event score for the past 6 months (rho = 0.223, p = 0.004), as well as with the negative event score for this period (rho = 0.227, p = 0.003; N = 165). Cortisone did not correlate with emotions or coping styles reported by the children. CONCLUSION: Despite its exploratory nature, this study may suggest elevated hair cortisone concentrations under psychosocial stress in young children. Although the observed findings should be interpreted with prudence, this study may encourage further research elucidating the potential importance and relevance of hair cortisone analysis as an additional or substituting stress biomarker for hair cortisol.

First identification of dapagliflozin in human hair: Development of a new liquid chromatography with tandem mass spectrometry method.

Sodium-glucose cotransporter 1 inhibitors are a new class of drugs used for the treatment of type II diabetes. Due to their diuretic capabilities and the glycosuria they induce, these molecules cause effective weight loss that could attract the interest of a wider public than diabetics with all the health consequences knowing the adverse effects of these substances. In order to reveal a past exposure to these substances, hair analysis can be very useful especially in the medicolegal context. There are no data in the literature about gliflozin testing in hair. In this study, a method was developed for the analysis of three molecules belonging to the gliflozin family (dapagliflozin, empagliflozin and canagliflozin) using a liquid chromatography system coupled to tandem mass spectrometry. After decontamination with dichloromethane, gliflozins were extracted from hair following incubation in methanol in the presence of dapagliflozin-d5. Validation showed acceptable linearity for all compounds between 10 and 10,000 pg/mg, with limit of detection and limit of quantification at 5 and 10 pg/mg, respectively. Repeatability and reproducibility were below 20% at three concentrations for all analytes. The method was subsequently applied to the hair of two diabetic subjects under dapagliflozin treatment. In one of the two cases, the result was negative, while in the second case, the concentration was 12 pg/mg. Due to the absence of data, it is difficult to explain the absence of dapagliflozin in the hair of the first case. Physico-chemical characteristics of dapagliflozin could explain its bad incorporation in hair, making detection difficult even after daily treatment.