LC-MS/MS quantification of olanzapine in hair after alkaline digestion.

Olanzapine (OLZ), a second-generation antipsychotic drug, is effective in the treatment of acute psychosis, schizophrenia, agitation, bipolar mania, and other psychiatric problems. Antipsychotics are prescribed drugs, which lead the drug abuser to illegal methods of access. This behavior also demonstrates the association of OLZ with criminal involvement, commonly observed at forensic crime scenes. The acute toxicity and even death resulting from OLZ exposure have been highlighted in numerous studies. Therefore, developing analytical techniques to detect OLZ is essential for forensic toxicology. This study aimed to develop a specific and reliable LC-MS/MS method for OLZ detection and quantification in hair samples. The method was validated in terms of selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), trueness, precision, and uncertainty. The range of linearity was between 0.1-100 ng/mg, with LOD and LOQ values established at 0.036 ng/mg and 0.1 ng/mg, respectively. All validation results are within acceptable parameters. The validated method has been applied to authentic hair samples. The variation of OLZ concentrations in 12 hair segments (2 from Case 1 and 10 from Case 2) from two drug-positive patients, ranging from 0.131 to 0.460 ng/mg, is presented in this study. Although several studies have been conducted to determine OLZ in hair samples using segmental analysis via hair solubilization, this study is the first to determine OLZ in hair samples after "digestion" with comparative parameters prior to chromatographic analysis.

Sex differences in the neural and behavioral effects of acute high-dose edible cannabis consumption in rats.

The consumption of D9-tetrahydrocannabinol (THC)- or cannabis-containing edibles has increased in recent years; however, the behavioral and neural circuit effects of such consumption remain unknown, especially in the context of ingestion of higher doses resulting in cannabis intoxication. We examined the neural and behavioral effects of acute high-dose edible cannabis consumption (AHDECC). Sprague-Dawley rats (6 males, 7 females) were implanted with electrodes in the prefrontal cortex (PFC), dorsal hippocampus (dHipp), cingulate cortex (Cg), and nucleus accumbens (NAc). Rats were provided access to a mixture of Nutella (6 g/kg) and THC-containing cannabis oil (20 mg/kg) for 10 minutes, during which they voluntarily consumed all of the provided Nutella and THC mixture. Cannabis tetrad and neural oscillations were examined 2, 4, 8, and 24-h after exposure. In another cohort (16 males, 15 females), we examined the effects of AHDECC on learning and prepulse inhibition, and serum and brain THC and 11-hydroxy-THC concentrations. AHDECC resulted in higher brain and serum THC and 11-hydroxy-THC levels in female rats over 24 h. AHDECC also produced: 1) Cg, dHipp, and NAc gamma power suppression, with the suppression being greater in female rats, in a time-dependent manner; 2) hypolocomotion, hypothermia, and anti-nociception in a time-dependent manner; and 3) learning and prepulse inhibition impairments. Additionally, most neural activity and behavior changes appear 2 h post-ingestion, suggesting that interventions around this time might be effective in reversing/reducing the effects of AHDECC. Significance Statement The effects of high-dose edible cannabis on behaviour and neural circuitry are poorly understood. We found that the effects of acute high-dose edible cannabis consumption, which include decreased gamma power, hypothermia, hypolocomotion, analgesia, and learning and information processing impairments, are time- and sex-dependent. Moreover, these effects begin 2 h after AHDECC and last for at least 24 h, suggesting that treatments should target this time window in order to be effective.

Postmortem Diffusion of Aconitum Alkaloids and Their Metabolites in Rabbits.

OBJECTIVES: To explore the postmortem diffusion rule of Aconitum alkaloids and their metabolites in poisoned rabbits, and to provide a reference for identifying the antemortem poisoning or postmortem poisoning of Aconitum alkaloids. METHODS: Twenty-four rabbits were sacrificed by tracheal clamps. After 1 hour, the rabbits were administered with aconitine LD50 in decocting aconite root powder by intragastric administration. Then, they were placed supine and stored at 25 ℃. The biological samples from 3 randomly selected rabbits were collected including heart blood, peripheral blood, urine, heart, liver, spleen, lung and kidney tissues at 0 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h and 96 h after intragastric administration, respectively. Aconitum alkaloids and their metabolites in the biological samples were analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). RESULTS: At 4 h after intragastric administration, Aconitum alkaloids and their metabolites could be detected in heart blood, peripheral blood and major organs, and the contents of them changed dynamically with the preservation time. The contents of Aconitum alkaloids and their metabolites were higher in the spleen, liver and lung, especially in the spleen which was closer to the stomach. The average mass fraction of benzoylmesaconine metabolized in rabbit spleen was the highest at 48 h after intragastric administration. In contrast, the contents of Aconitum alkaloids and their metabolites in kidney were all lower. Aconitum alkaloids and their metabolites were not detected in urine. CONCLUSIONS: Aconitum alkaloids and their metabolites have postmortem diffusion in poisoned rabbits, diffusing from high-content organs (stomach) to other major organs and tissues as well as the heart blood. The main mechanism is the dispersion along the concentration gradient, while urine is not affected by postmortem diffusion, which can be used as the basis for the identification of antemortem and postmortem Aconitum alkaloids poisoning.

Characterizing Freshwater Ecotoxicity of More Than 9000 Chemicals by Combining Different Levels of Available Measured Test Data with In Silico Predictions.

Ecotoxicological impacts of chemicals released into the environment are characterized by combining fate, exposure, and effects. For characterizing effects, species sensitivity distributions (SSDs) estimate toxic pressures of chemicals as the potentially affected fraction of species. Life cycle assessment (LCA) uses SSDs to identify products with lowest ecotoxicological impacts. To reflect ambient concentrations, the Global Life Cycle Impact Assessment Method (GLAM) ecotoxicity task force recently recommended deriving SSDs for LCA based on chronic EC10s (10% effect concentration, for a life-history trait) and using the 20th percentile of an EC10-based SSD as a working point. However, because we lacked measured effect concentrations, impacts of only few chemicals were assessed, underlining data limitations for decision support. The aims of this paper were therefore to derive and validate freshwater SSDs by combining measured effect concentrations with in silico methods. Freshwater effect factors (EFs) and uncertainty estimates for use in GLAM-consistent life cycle impact assessment were then derived by combining three elements: (1) using intraspecies extrapolating effect data to estimate EC10s, (2) using interspecies quantitative structure-activity relationships, or (3) assuming a constant slope of 0.7 to derive SSDs. Species sensitivity distributions, associated EFs, and EF confidence intervals for 9862 chemicals, including data-poor ones, were estimated based on these elements. Intraspecies extrapolations and the fixed slope approach were most often applied. The resulting EFs were consistent with EFs derived from SSD-EC50 models, implying a similar chemical ecotoxicity rank order and method robustness. Our approach is an important step toward considering the potential ecotoxic impacts of chemicals currently neglected in assessment frameworks due to limited test data. Environ Toxicol Chem 2024;00:1-14. © 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Effects of Metabolic Disruption on Lipid Metabolism and Yolk Retention in Zebrafish Embryos.

A subgroup of endocrine-disrupting chemicals have the ability to disrupt metabolism. These metabolism-disrupting chemicals (MDCs) can end up in aquatic environments and lead to adverse outcomes in fish. Although molecular and physiological effects of MDCs have been studied in adult fish, few studies have investigated the consequences of metabolic disruption in fish during the earliest life stages. To investigate the processes affected by metabolic disruption, zebrafish embryos were exposed to peroxisome proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone, the PPARγ antagonist T0070907, and the well-known environmentally relevant MDC bisphenol A. Decreased apolipoprotein Ea transcript levels indicated disrupted lipid transport, which was likely related to the observed dose-dependent increases in yolk size across all compounds. Increased yolk size and decreased swimming activity indicate decreased energy usage, which could lead to adverse outcomes because the availability of energy reserves is essential for embryo survival and growth. Exposure to T0070907 resulted in a darkened yolk. This was likely related to reduced transcript levels of genes involved in lipid transport and fatty acid oxidation, a combination of responses that was specific to exposure to this compound, possibly leading to lipid accumulation and cell death in the yolk. Paraoxonase 1 (Pon1) transcript levels were increased by rosiglitazone and T0070907, but this was not reflected in PON1 enzyme activities. The present study shows how exposure to MDCs can influence biochemical and molecular processes involved in early lipid metabolism and may lead to adverse outcomes in the earliest life stages of fish. Environ Toxicol Chem 2024;00:1-14. © 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

The effect of cannabis edibles on driving and blood THC.

BACKGROUND: Cannabis has been shown to impact driving due to changes produced by delta-9-tetrahydrocannabinol (THC), the psychoactive component of cannabis. Current legal thresholds for blood THC while driving are based predominantly on evidence utilizing smoked cannabis. It is known that levels of THC in blood are lower after eating cannabis as compared to smoking yet the impact of edibles on driving and associated blood THC has never been studied. METHODS: Participants drove a driving simulator before and after ingesting their preferred legally purchased cannabis edible. In a counterbalanced control session, participants did not consume any THC or cannabidiol (CBD). Blood was collected for measurement of THC and metabolites as well as CBD. Subjective experience was also assessed. RESULTS: Participants consumed edibles with, on average, 7.3 mg of THC, which is less than the maximum amount available in a single retail package in Ontario, providing an ecologically valid test of cannabis edibles. Compared to control, cannabis edibles produced a decrease in mean speed 2 h after consumption but not at 4 and 6 h. Under dual task conditions in which participants completed a secondary task while driving, changes in speed were not significant after the correction for multiple comparison. No changes in standard deviation of lateral position (SDLP; 'weaving'), maximum speed, standard deviation of speed or reaction time were found at any time point or under either standard or dual task conditions. Mean THC levels were significantly increased, relative to control, after consuming the edible but remained relatively low at approximately 2.8 ng/mL 2 h after consumption. Driving impairment was not correlated with blood THC. Subjective experience was altered for 7 h and participants were less willing/able to drive for up to 6 h, suggesting that the edible was intoxicating. INTERPRETATION: This is the first study of the impact of cannabis edibles on simulated driving. Edibles were intoxicating as revealed by the results of subjective assessments (VAS), and there was some impact on driving. Detection of driving impairment after the use of cannabis edibles may be difficult.

Cannabis Use in Parkinson's Disease: Patient Access to Medical Cannabis and Physician Perspective on Product Safety.

The rate of medical cannabis use has increased in parallel with the number of states legalizing its use. Parkinson's disease (PD) patients are of particular concern due to their higher cannabis use rate than in the general US population (25-40% PD patient cannabis users vs. ~18% in the general population), as well as their susceptibility to environmental contaminants in cannabis, including pesticides, toxic elements, solvents, microbes, and mycotoxins. In order to address the complex nature of this industry, we examined the changes in PD-related qualifying conditions in the U.S. from 2019 to 2023. We also conducted an online survey to gain insight into the knowledge, risk perceptions, and opinions regarding medical cannabis and contamination issues from physicians who treated PD patients. The number of states including PD-related qualifying conditions increased over the past 5 years from 28 to 36 states. These conditions included PD (increasing from 14 to 16 states), muscle spasms (14 to 24), anxiety (1 to 5), and pain (17 to 35). State-by-state comparisons revealed high variability in the language used to describe the different qualifying conditions. Online surveys were sent out to 45 neurologists and movement disorder specialists who primarily treated PD patients. The response rate was 44% from nine states (AZ, CA, FL, MA, MN, WI, PA, IL, and NM). When asked if they were aware of any contaminants in cannabis products, we found that 65% of the physicians were unaware of any contaminants commonly found in cannabis and only 25%, 15%, and 15% of them were aware of pesticide, toxic element, and solvent contaminants, respectively. In their free-text opinion response on the health impact of cannabis-borne contaminants, "long-term effect" (35%) and "comorbidities and PD prognosis" (40%) were identified as the two most common themes. These results point to the need for further regulatory deliberation regarding risks and susceptibility to cannabis contaminants. Additionally, education is needed to inform physicians on cannabis safety issues. Further research will identify the implementation strategies to reduce contaminant exposure and protect patient health.

Rapamycin and Starvation Mitigate Indomethacin -Induced Intestinal Damage through Preservation of Lysosomal Vacuolar ATPase Integrity.

Nonsteroidal anti-inflammatory drugs (NSAIDs) possess anti-inflammatory, antipyretic, and analgesic properties and are among the most commonly used drugs. Although the cause of NSAID-induced gastric ulcers is well-understood, the mechanism behind small intestinal ulcers remains elusive. In this study, we examined the mechanism through which indomethacin (IM), a prominent NSAID, induces small intestinal ulcers, both in vitro and in vivo In IEC6 cells, a small intestinal epithelial cell line, IM treatment elevated levels of LC3-Ⅱ and p62. These expression levels remained unaltered after treatment with chloroquine or bafilomycin, which are vacuolar ATPase (V-ATPase) inhibitors. IM treatment reduced the activity of cathepsin B, a lysosomal protein hydrolytic enzyme, and increased the lysosomal pH. There was a notable increase in subcellular co-localization of LC3 with Lamp2, a lysosome marker, post-IM treatment. The increased lysosomal pH and decreased cathepsin B activity were reversed by pretreatment with rapamycin (Rapa) or glucose starvation, both of which stabilize V-ATPase assembly. To validate the in vitro findings in vivo, we established an IM-induced small intestine ulcer mouse model. In this model, we observed multiple ulcerations and heightened inflammation following IM administration. However, pretreatment with Rapa or fasting, which stabilize V-ATPase assembly, mitigated the IM-induced small intestinal ulcers in mice. Co-immunoprecipitation studies demonstrated that IM binds to V-ATPase in vitro and in vivo These findings suggest that IM induces small intestinal injury through lysosomal dysfunction, likely due to the disassembly of lysosomal V-ATPase caused by direct binding. Moreover, Rapa or starvation can prevent this injury by stabilizing the assembly. Significance Statement This study elucidates the largely unknown mechanisms behind small intestinal ulceration induced by indomethacin and reveals the involvement of lysosomal dysfunction via V-ATPase disassembly. The significance lies in identifying potential preventative interventions, such as rapamycin treatment or glucose starvation, offering pivotal insights that extend beyond NSAID-induced ulcers to broader gastrointestinal pathologies and treatments, thereby providing a foundation for novel therapeutic strategies aimed at a wide array of gastrointestinal disorders.

American College of Medical Toxicology Research Agenda 2024-2030.

ACMT recognizes the pivotal role of high-quality research in advancing medical science. As such, the establishment of a formal research agenda for ACMT is a leap forward in communicating the priorities of the College, its members, and the patient populations we serve. This thoughtfully crafted agenda will serve as a strategic compass for ACMT, guiding our pursuit of scientific discovery, fostering innovation, and enhancing outcomes for patients and communities affected by poisonings and exposures.

The influence of cosmetic treatments in the uptake of in vitro cocaine contamination in hair.

Hair analysis is a powerful tool to assess drug use, yet the challenge of external contamination complicates its interpretation. Understanding the influence of cosmetic hair treatments is pivotal as their presence may affect this phenomenon. This study investigated the effects of four cosmetic treatments (bleach, henna, gel, and dry shampoo) on the external in vitro contamination of cocaine and its primary metabolite, benzoylecgonine (BE). Hair samples were divided into four groups: A-hair treated with cosmetics then contaminated; B-hair contaminated then subjected to cosmetic treatment; and C-hair solely contaminated (control group). Negative hair samples (n = 24) were immersed in a cocaine and BE aqueous solution of 1 µg/mL for 24 h. All hair samples were analyzed by a LC-MSMS procedure successfully validated according to ANSI/ASB Standard 036 guidelines (limit of quantification at 10 pg/mg). Henna in Group A (n = 13) resulted in the most substantial reduction for cocaine (92%), while bleach in Group B (n = 15) showed an 80% decrease. For BE, Group A henna (n = 13) exhibited a 50% reduction, and Group B bleach (n = 15) demonstrated a 71% decrease, all compared to Group C (n = 24). The study found no significant differences concerning hair color (black (n = 3), brown (n = 10), red (n = 5) and blond (n = 6)) or shape (straight (n = 6), wavy (n = 16), curly (n = 1), and coily (n = 1)). All analysis were performed in triplicate with variations below 20%. These findings emphasize that cosmetic treatments do affect cocaine/BE concentrations in hair when exposed to external contamination, highlighting the importance of considering an individual's cosmetic history prior to interpretation.

Intoxications involving methoxyacetylfentanyl and U-47700: a study of 3 polydrug fatalities.

Novel synthetic opioids (NSOs) represent an emerging group of novel psychoactive substances, acting as agonists at the opioid receptors. NSOs include fentanyl-related compounds, e.g. methoxyacetylfentanyl (MeACF), and non-fentanyl analogs, e.g. "U compounds" including U-47700. Here we present three cases of death involving MeACF and U-47700, with particular reference to preliminary data on pharmacokinetics and tissue distribution.After a complete post-mortem examination, general unknown screenings and analysis of drugs of abuse were performed on postmortem samples by immunoassays, gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. To quantify the analytes of interest in post-mortem blood and tissues, the standard addition method was used. A toxicological significance score (TSS), weighing the role of the NSO in each death case, was assigned.Case 1 died at the hospital after consumption of U-47700, methadone (serum levels: 2,600 ng/ml and 37 ng/ml), tilidine and benzodiazepines. In case 2, U-47700 (204 ng/ml) together with methadone (290 ng/ml), flubromazepam (480 ng/ml) and diazepam (300 ng/ml) were detected in peripheral blood. In case 3, methoxyacetylfentanyl (266 ng/ml), furanylfentanyl (4.3 ng/ml) 4-ANPP (15 ng/ml) and alprazolam (69 ng/ml) were quantified in femoral blood. In all cases, the NSO likely contributed to the death (TSS = 3).NSOs appear to be often consumed in the setting of polydrug intoxications, especially in combination with other opioids and benzodiazepines, which often exert synergistic effects. The standard addition method remains the most reliable in post-mortem analysis and toxicological results should always be evaluated together with circumstantial and autopsy data.

Analyzing high-throughput assay data to advance the rapid screening of environmental chemicals for human reproductive toxicity.

While high-throughput (HTP) assays have been proposed as platforms to rapidly assess reproductive toxicity, there is currently a lack of established assays that specifically address germline development/function and fertility. We assessed the applicability domains of yeast (S. cerevisiae) and nematode (C. elegans) HTP assays in toxicity screening of 124 environmental chemicals, determining their agreement in identifying toxicants and their concordance with reproductive toxicity in vivo. We integrated data generated in the two models and compared results using a streamlined, semi-automated benchmark dose (BMD) modeling approach. We then extracted and modeled relevant mammalian in vivo data available for the matching chemicals included in the Toxicological Reference Database (ToxRefDB). We ranked potencies of common compounds using the BMD and evaluated correlation between the datasets using Pearson and Spearman correlation coefficients. We found moderate to good correlation across the three data sets, with r = 0.48 (95% CI: 0.28-1.00, p<0.001) and rs = 0.40 (p=0.002) for the parametric and rank order correlations between the HTP BMDs; r = 0.95 (95% CI: 0.76-1.00, p=0.0005) and rs = 0.89 (p=0.006) between the yeast assay and ToxRefDB BMDs; and r = 0.81 (95% CI: 0.28-1.00, p=0.014) and rs = 0.75 (p=0.033) between the worm assay and ToxRefDB BMDs. Our findings underscore the potential of these HTP assays to identify environmental chemicals that exhibit reproductive toxicity. Integrating these HTP datasets into mammalian in vivo prediction models using machine learning methods could further enhance the predictive value of these assays in future rapid screening efforts.

Neuroinflammation is dependent on sex and ovarian hormone presence following acute woodsmoke exposure.

Woodsmoke (WS) exposure is associated with significant health-related sequelae. Different populations can potentially exhibit varying susceptibility, based on endocrine phenotypes, to WS and investigating neurological impacts following inhaled WS is a growing area of research. In this study, a whole-body inhalation chamber was used to expose both male and female C57BL/6 mice (n = 8 per group) to either control filtered air (FA) or acute WS (0.861 ± 0.210 mg/m3) for 4 h/d for 2 days. Neuroinflammatory and lipid-based biological markers were then assessed. In a second set of studies, female mice were divided into two groups: one group was ovariectomized (OVX) to simulate an ovarian hormone-deficient state (surgical menopause), and the other underwent Sham surgery as controls, to mechanistically assess the impact of ovarian hormone presence on neuroinflammation following FA and acute WS exposure to simulate an acute wildfire episode. There was a statistically significant impact of sex (P ≤ 0.05) and statistically significant interactions between sex and treatment in IL-1ß, CXCL-1, TGF-ß, and IL-6 brain relative gene expression. Hippocampal and cortex genes also exhibited significant changes in acute WS-exposed Sham and OVX mice, particularly in TGF-ß (hippocampus) and CCL-2 and CXCL-1 (cortex). Cortex GFAP optical density (OD) showed a notable elevation in male mice exposed to acute WS, compared to the control FA. Sham and OVX females demonstrated differential GFAP expression, depending on brain region. Overall, targeted lipidomics in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) serum and brain lipids demonstrated more significant changes between control FA and acute WS exposure in female mice, compared to males. In summary, male and female mice show distinct neuroinflammatory markers in response to acute WS exposure. Furthermore, ovarian hormone deficiency may impact the neuroinflammatory response following an acute WS event.

Current status of keratinized matrices in Toxicology: Comparison of hair and nails.

Nails are a keratinized matrix that has been proposed as an alternative to hair to evaluate long-term and retrospective consumption of drugs of abuse and pharmaceuticals. This matrix has been gaining interest in recent years, with new studies focusing on the analysis of fingernails and/or toenails for different substances. However, nails and hair present differences in structure, growth, and incorporation pathways that may affect drug incorporation and analysis and complicate the interpretation of the results. To better understand the results in nail samples, a comparison of concentrations found in hair, fingernails, and toenails has been described in the literature for some drugs. This review unifies the results found in the literature, with special interest on studies that report paired samples from the same individuals. Differences between fingernail and toenail samples, as well as proposed cut-offs in nails, are also discussed. Definite conclusions can be reached for some drugs, but, in general, more standardized studies are needed to better understand nail results.

Status of single-cell RNA sequencing for reproductive toxicology in zebrafish and the transcriptomic trade-off.

The utilization of transcriptomic studies identifying profiles of gene expression, especially in toxicogenomics, has catapulted next-generation sequencing to the forefront of reproductive toxicology. An innovative yet underutilized RNA sequencing technique emerging into this field is single-cell RNA sequencing (scRNA-seq), which provides sequencing at the individual cellular level of gonad tissue. ScRNA-seq provides a novel and unique perspective for identifying distinct cellular profiles, including identification of rare cell subtypes. The specificity of scRNA-seq is a powerful tool for reproductive toxicity research, especially for translational animal models including zebrafish. Studies to date not only have focused on 'tissue atlassing' or characterizing what cell types make up different tissues but have also begun to include toxicant exposure as a factor that this review aims to explore. Future scRNA-seq studies will contribute to understanding exposure-induced outcomes; however, the trade-offs with traditional methods need to be considered.

FetoML: Interpretable predictions of the fetotoxicity of drugs based on machine learning approaches.

Pregnant females may use medications to manage health problems that develop during pregnancy or that they had prior to pregnancy. However, using medications during pregnancy has a potential risk to the fetus. Assessing the fetotoxicity of drugs is essential to ensure safe treatments, but the current process is challenged by ethical issues, time, and cost. Therefore, the need for in silico models to efficiently assess the fetotoxicity of drugs has recently emerged. Previous studies have proposed successful machine learning models for fetotoxicity prediction and even suggest molecular substructures that are possibly associated with fetotoxicity risks or protective effects. However, the interpretation of the decisions of the models on fetotoxicity prediction for each drug is still insufficient. This study constructed machine learning-based models that can predict the fetotoxicity of drugs while providing explanations for the decisions. For this, permutation feature importance was used to identify the general features that the model made significant in predicting the fetotoxicity of drugs. In addition, features associated with fetotoxicity for each drug were analyzed using the attention mechanism. The predictive performance of all the constructed models was significantly high (AUROC: 0.854-0.974, AUPR: 0.890-0.975). Furthermore, we conducted literature reviews on the predicted important features and found that they were highly associated with fetotoxicity. We expect that our model will benefit fetotoxicity research by providing an evaluation of fetotoxicity risks for drugs or drug candidates, along with an interpretation of that prediction.

Difficulties associated with the interpretation of post-mortem toxicology.

While post-mortem (PM) toxicology results provide valuable information towards ascertaining both the cause and manner of death in coronial cases, there are also significant difficulties associated with the interpretation of PM drug levels. Such difficulties are influenced by several pharmacokinetic and pharmacodynamic factors including PM redistribution, diffusion, site-to-site variability in drug levels, different drug properties and metabolism, bacterial activity, genetic polymorphisms, tolerance, resuscitation efforts, underlying conditions and the toxicity profile of cases (i.e. single- or mixed-drug toxicity). A large body of research has been dedicated to better understanding and even quantifying the influence of these factors on PM drug levels. For example, several investigative matrices have been developed as potential indicators of PM redistribution, but they have limited practical value. Reference tables of clinically relevant therapeutic, toxic and potentially fatal drug concentrations have also been compiled, but these unfortunately do not provide reliable reference values for PM toxicology. More recent research has focussed on developing databases of peripheral PM drug levels for a variety of case-types to increase transferability to real-life cases and improve interpretations. Changes to drug levels after death are inevitable and unavoidable. As such, guidelines and practices will continue to evolve as we further our understanding of such phenomena.

CarcSeq detection of lorcaserin-induced clonal expansion of Pik3ca H1047R mutants in rat mammary tissue.

Lorcaserin is a 5-hydroxytryptamine 2C (serotonin) receptor agonist and a non-genotoxic rat carcinogen, which induced mammary tumors in male and female rats in a two-year bioassay. Female Sprague Dawley rats were treated by gavage daily with 0, 30, or 100 mg/kg lorcaserin, replicating bioassay dosing but for shorter duration, 12 or 24 weeks. To characterize exposure and eliminate possible confounding by a potentially genotoxic degradation product, lorcaserin and N-nitroso-lorcaserin were quantified in dosing solutions, terminal plasma, mammary and liver samples using ultra high-performance liquid chromatography-electrospray tandem mass spectrometry. N-nitroso-lorcaserin was not detected, supporting lorcaserin classification as non-genotoxic carcinogen. Mammary DNA samples (n = 6/dose/timepoint) were used to synthesize PCR products from gene segments encompassing hotspot cancer driver mutations (CDMs), namely regions of Apc, Braf, Egfr, Hras, Kras, Nfe2l2, Pik3ca, Setbp1, Stk11, and Tp53. Mutant fractions (MFs) in the amplicons were quantified by CarcSeq, an error corrected next-generation sequencing approach. Considering all recovered mutants, no significant differences between lorcaserin dose groups were observed. However, significant dose-responsive increases in Pik3ca H1047R mutation were observed at both timepoints (ANOVA, p < 0.05), with greater numbers of mutants and mutants with greater MFs observed at 24 weeks as compared to 12 weeks. These observations suggest lorcaserin promotes outgrowth of spontaneously occurring Pik3ca H1047R mutant clones leading to mammary carcinogenesis. Importantly, this work reports approaches to analyze clonal expansion and demonstrates CarcSeq detection of the carcinogenic impact (selective Pik3ca H0147R mutant expansion) of a non-genotoxic carcinogen using a treatment duration as short as 3 months.