João Pedro Silva

Cannabis and cannabinoids invite us to reflect on an enduring tension between human intention and biological consequence: the pursuit of relief, pleasure, or new experiences may carry risks that extend beyond what is sought or anticipated. Acute exposure to tetrahydrocannabinol (THC)-containing cannabis can impair attention, memory, coordination, and decision-making, and may cause anxiety, confusion, paranoia, and cardiovascular effects. Frequent cannabis use has also been associated with an increased risk of cannabis use disorder and adverse cognitive and mental health outcomes, although the strength of evidence and causal relationships vary across outcomes (Hoch et al. 2025;National Academies of Sciences, Engineering, and Medicine, 2024).Cannabis use patterns and the availability of cannabis-based products have diversified considerably in recent years, with the market now encompassing products with varying cannabinoid compositions and concentrations, including dried cannabis, extracts and concentrates, edibles, vaping formulations, and products containing individual cannabinoids such as THC and cannabidiol (CBD) (National Academies of Sciences, Engineering, and Medicine, 2024; Tassone et al. 2023). This diversification introduces distinct exposure scenarios, in which formulation, route of administration, dose, frequency of use, and individual susceptibility can influence cannabinoid disposition and effects (Lucas et al. 2018). The importance of formulation and dosing conditions is illustrated by the variability of CBD pharmacokinetics (Moazen-Zadeh et al. 2024).THC-containing edible products pose particular risks because their delayed onset can encourage repeated dosing before effects become apparent, while unintentional ingestion by children can cause severe intoxication (National Academies of Sciences, Engineering, and Medicine, 2024).Despite advances in understanding cannabis toxicology, important gaps remain regarding repeated and long-term exposure, the effects of complex mixtures, and the contribution of contaminants and degradation products to overall toxicity. Addressing these gaps requires research integrating mechanistic toxicology, exposure assessment, and clinical observations to support informed clinical decisions and regulatory risk assessment.The present Research Topic brings together 11 complementary contributions, including original research, reviews, case reports, and a perspective, spanning molecular and cellular mechanisms, pediatric toxicity, cannabinoid hyperemesis, cannabinoid and alcohol co-exposure, inhalation toxicology, product chemistry, and regulatory risk assessment. These contributions are summarized below.This article collection moves beyond a THC-centered view of cannabis toxicity. The review by Teixeira provides an overview of phytocannabinoids and synthetic cannabinoids in the context of recreational use and potential therapeutic applications, highlighting the diversity of their structures, pharmacological targets, and adverse effects. This review provides a conceptual framework for the experimental studies in the collection.Consumer use of CBD has expanded, despite uncertainties concerning repeated exposure and long-term effects (Silva and Carvalho 2022). In this context, Camacho et al. compared purified CBD with a CBD-rich hemp extract at matched CBD concentrations in 0.5 % sesame-oil emulsions, assessing development and reproduction in Caenorhabditis elegans. Juveniles were more sensitive than adults, with reduced body size and developmental delays that persisted after early exposure ended.Continuous exposure beginning at parental reproductive maturity also reduced progeny output. The extract generally produced less pronounced effects than purified CBD.These findings identify hazards in this model; high internal CBD concentrations and interspecies differences preclude direct inference about risk at human consumer or therapeutic doses.Wong et al. compared chemically synthesized and plant-derived CBD, observing similar responses in the in vitro genotoxicity, hepatotoxicity, and cytochrome P450 activity assays examined. In a separate pharmacokinetic study, one synthesized CBD preparation was administered orally to male Sprague-Dawley rats at 26.5 mg/kg/day for 21 days. Repeated dosing increased systemic exposure, with preferential retention in adipose tissue and mesenteric lymph nodes. These findings support comparability for the in vitro endpoints assessed and the importance of repeated-dose toxicokinetics, but do not establish equivalent in vivo behavior or long-term safety across CBD sources.Cannabis toxicology also requires consideration of constituents other than cannabinoids. The mini review by Russo and Whiteley addresses cannabinoid hyperemesis syndrome (CHS), which is associated with prolonged, frequent use of THC-containing cannabis and has also been reported following exposure to other CB1 receptor agonists. The authors discuss proposed mechanisms, diagnostic challenges relative to cyclic vomiting syndrome, and hypotheses concerning pesticide contamination. They also consider non- Masilamani et al. describe a case report from a 7-year-old child who developed asystole lasting more than 15 seconds with apnea approximately seven hours after a reported ingestion of 75 mg of Δ⁸-THC in gummies (approximately 3 mg/kg). The child had already developed lethargy three to four hours after ingestion, despite initially stable vital signs. This case highlights the possibility of delayed severe events following substantial edible exposures, but does not establish a universal observation period.Together, these pediatric contributions support child-resistant packaging, safe storage, clinical recognition, and observation tailored to dose, symptoms, co-exposures, and clinical course.The Perspective by Achuthan et al. provides a regulatory dimension to the collection.Using Canadian data on regulated dried cannabis and cannabis vaping products,...

Neurotoxicity and abuse potential are strongly associated with psychotropic drugs. Although animal models have been used for decades to investigate different forms of toxicity, including neurotoxicity and abuse potential, increasing efforts have been directed toward overcoming their inherent limitations. In this context, the development of New Approach Methodologies (NAMs), aimed at improving chemical safety assessment through more protective and physiologically relevant models, has emerged as a promising strategy to predict the neurotoxic potential of drugs while contributing to the replacement, reduction, and refinement of animal experimentation. Nevertheless, the number of NAMs capable of reliably and efficiently predicting neurotoxicity remains limited. Importantly, psychotropic drugs have been shown to induce epigenetic modifications (e.g., DNA methylation, histone modifications), which are frequently associated with neurotoxic adverse effects. The integration of these alterations into NAMs may therefore represent a major advance for academia, industry, and regulatory agencies in assessing the neurotoxic and abuse potential of such substances. Here, we review the latest evidence on the role of psychotropic drug-induced epigenetic modifications in neurotoxicity and abuse potential. Importantly, we further discuss how integrating these epigenetic alterations into NAMs could advance risk assessment of neurotoxic effects and abuse liability.

Synthetic cannabinoid products are commonly smoked as plant-based mixtures, yet the compounds transferred to or generated in smoke remain poorly characterized. This study qualitatively compared an unheated seized herbal product containing JWH-210 with smoke condensate generated using a vacuum-assisted laboratory apparatus. Cigarette tubes packed with 800 mg of the seized product were combusted for 1 min, and smoke drawn through the collection train was retained on a C18 solid-phase extraction cartridge. The cartridge was sequentially eluted with methanol and dichloromethane, and the extracts were screened by gas chromatography–electron ionization–mass spectrometry. Compound annotations considered mass-spectral library matches, diagnostic ions, signal-to-noise ratios, and retention-index agreement. The representative unheated extracts contained 19 annotated chromatographic peaks representing 13 unique tentative annotations, whereas, the representative smoke-condensate extracts contained 48 peaks representing 43 unique tentative annotations, including 35 observed only after smoke generation. A signal tentatively assigned to JWH-210 was observed before and after smoke generation. Two additional synthetic-cannabinoid-related library matches occurred only in the methanolic smoke extract but remain provisional because authenticated standards, high-resolution mass spectrometry, and matrix-matched controls were unavailable. These findings provide a case-specific qualitative smoke fingerprint relevant to forensic screening but do not establish concentrations, formation pathways, exposure, or toxicological risk and should not be extrapolated directly to human inhalation exposure.

Rimonabant (SR141716A), an inverse agonist of the cannabinoid receptor type 1 (CB ₁ ), once approved for treating obesity and metabolic disorders, was withdrawn shortly after due to psychiatric and psychological adverse events (PPAEs), including depression and suicidality. Although its primary pharmacological mechanism of action is well-characterized, the molecular basis underlying these neuropsychiatric effects remains unclear. Here, we investigated the epigenetic impact of rimonabant exposure, both in vitro and ex vivo, at therapeutically relevant concentrations and doses, with a focus on histone modifications and DNA methylation. In SH-SY5Y human neuroblastoma cells, after 24 and 96 h treatment with 0.01 and 1 µM rimonabant significantly increased global histone H3 and H4 acetylation by 2.7- and 1.4-fold, respectively, without altering global DNA methylation levels. The effects on histone acetylation were partially reversed by a CB ₁ receptor agonist, indicating a role for CB ₁ in the observed epigenetic modulation. Rimonabant also decreased histone deacetylases (HDAC) activity and reduced the levels of H3K4me3 and H3K27me3, marks that have been previously identified in psychiatric perturbations. Moreover, 4-week oral administration of 3 or 15 mg/kg rimonabant to rats produced region- and dose-specific alterations in H3K4me3, H3K27me3, H3K9ac, and 5-methylcytosine levels across the prefrontal cortex, hippocampus, and nucleus accumbens, in line with epigenetic profiles characteristic of depression, anxiety, and schizophrenia. Collectively, these findings demonstrate that rimonabant disrupts key epigenetic regulatory mechanisms in the brain and support the hypothesis that epigenetic dysregulation contributes to its psychiatric liabilities. This work strengthens the value of incorporating epigenetic endpoints into neuropharmacological safety assessments.

Quantitative immunofluorescence is widely used to assess molecular expression and cellular distribution across biological tissues, yet the analysis of large image datasets remains time-consuming and prone to user-dependent variability. To address these limitations, we herein developed a semi-automated workflow that integrates ImageJ/Fiji for image processing, StarDist for nuclear segmentation, and spreadsheet- or Python-based routines for data curation. The pipeline standardizes critical analytical steps, including scale calibration, region-of-interest (ROI) definition, channel selection, and z-stack handling, while preserving essential metadata through a structured file-naming system. Optical density and cell-number metrics are exported automatically in a consistent format, enabling efficient consolidation into a unified dataset. Subsequent curation can be performed either manually in a spreadsheet software or fully automatically through custom Python scripts, allowing extraction of sample identifiers, regions, and markers, as well as calculation of normalized intensity values. Comparison with existing protocols proved that this workflow adheres to widely accepted quantification principles while markedly improving reproducibility, consistency, and analytical throughput. This method offers a straightforward, transparent, and scalable solution for fluorescence-based quantification suitable for laboratories with varying levels of computational expertise.

Mitochondria are essential drivers of neuronal growth, differentiation, and overall brain development. Synthetic cannabinoids (SCs) have been shown to enhance neurite outgrowth in NG108-15 neuroblastoma x glioma cells through CB1 receptor activation, while disrupting mitochondrial function. Here, we demonstrated first-hand the impact of biologically-relevant concentrations (< 1μM) of ADB-FUBINACA (an SC commonly identified in drug seizures) on mitochondrial morphology and dynamics (i.e., fusion, fission and mobility) during the neurodifferentiation of NG108-15 cells. Our findings revealed that, during NG108-15 neurodifferentiation, ADB-FUBINACA reduced the mean mitochondrial area and perimeter by around 10%, while increasing mitochondrial circularity, and decreasing network branching and interconnectivity. Specifically, branch length per mitochondrion and branch junctions declined by 17 and 25% in the neurons’ soma at the end of NG108-15 differentiation (after 72 h). Moreover, 1 nM and 1 µM ADB-FUBINACA markedly decreased the levels of mitochondrial fusion markers (Opa1 and Mfn2) and increased the levels of fission markers Drp1 and Fis1 at the same time point. The percentage of motile mitochondria in neurites also decreased at 72 h, while average speed and total run length per mobile mitochondrion remained unaffected, resulting in an accumulation of stationary mitochondria which may be important, for example, to support neurite extension. Collectively, these findings suggest that while ADB-FUBINACA promotes mitochondrial accumulation in neurites, potentially supporting the energy demands of developing neurites and influencing neurite outgrowth, in the long-term, the fragmentation of the mitochondrial network in the soma may compromise the maintenance of neurites, in terms of energy requirements.

Synthetic cannabinoids (SCs) have been increasingly associated with neurodevelopmental impairment; however, the underlying mechanisms remain poorly understood. In particular, the impact of SCs on mitochondria during neurodifferentiation remains largely unexplored, despite the central role of these organelles in this process. Building upon our previous findings that THJ-2201, a widely used SC, enhances neurite outgrowth in NG108-15 neuroblastoma-glioma cells at biologically relevant concentrations (1 pM–1 μM), we investigated whether this SC influences mitochondrial function, morphology, and dynamics during neurodifferentiation. THJ-2201 exposure caused a 30–40% reduction in intracellular ATP levels in a CB1-dependent manner, along with a 20–30% decrease in TMRE retention during NG108-15 neurodifferentiation. Cells treated with 1 μM THJ-2201 failed to sustain the expected increase in VDAC levels (an indirect marker of mitochondrial mass) during regular differentiation. Concurrently, THJ-2201 elevated PGC-1α levels, a key regulator of mitochondrial biogenesis, by disrupting its translocation to the nucleus. Expression of both fusion (Opa1, Mfn1, and Mfn2) and fission (Drp1 and Fis1) markers exhibited a less pronounced increase between 24 and 72 h in THJ-2201-treated cells. Mitochondrial morphology exhibited alterations in mean area, perimeter, branching, and circularity in the soma after 72 h exposure. Additionally, THJ-2201 reduced mitochondrial mobility in neurites without affecting their average speed or run length and led to a mitochondrial accumulation within neurites, as indicated by decreased Miro1 expression. Overall, these findings suggest that THJ-2201–induced mitochondrial remodelling and redistribution may transiently enhance local energy supply for neurite outgrowth, but at the expense of somatic mitochondrial function, resulting in an overall bioenergetic imbalance.