Metabolomics perspectives of the ecotoxicological risks of polycyclic aromatic hydrocarbons: A scoping review.

Polycyclic Aromatic Hydrocarbons (PAHs) represent persistent environmental pollutants ubiquitously distributed in the environment. Their presence alongside various other contaminants gives rise to intricate interactions, culminating in profound deleterious consequences. The combination effects of different PAH mixtures on biota remains a relatively unexplored domain. Recent studies have harnessed the exceptional sensitivity of metabolomic techniques to unveil the significant ecotoxicological perils of PAH pollution confronting both human populations and ecosystems. This article furnishes a comprehensive overview of current literature focused on the metabolic repercussions stemming from exposure to complex mixtures of PAHs or PAH-pollution sources using metabolomics approaches. These insights are obtained through a wide range of models, including in vitro assessments, animal studies, investigations on human subjects, botanical specimens, and soil environments. The findings underscore that PAH mixtures induce cellular stress responses and systemic effects, leading to metabolic dysregulations in amino acids, carbohydrates, lipids, and other key metabolites (e.g., organic acids, purines), with specific variations observed based on the organism and PAH compounds involved. Additionally, the ecological consequences of PAH pollutants on plant and soil microbial responses are emphasized, revealing significant changes in stress-related metabolites and nutrient cycling in soil ecosystems. The complex interplay of various PAHs and their metabolic effects on several models, as elucidated through metabolomics, highlight the urgency of further research and the need for comprehensive strategies to mitigate the risks posed by these widespread environmental pollutants.

Toxicity of pesticides widely applied on soybean cultivation: Synergistic effects of fipronil, glyphosate and imidacloprid in HepG2 cells.

The transgenic soy monoculture demands supplementation with pesticides. The aim of this study was to evaluate the individual and mixture effects of fipronil, glyphosate and imidacloprid in human HepG2 cells. Cytotoxicity was evaluated after 48-h incubations through MTT reduction and neutral red uptake assays. Free radicals production, mitochondrial membrane potential, DNA damage, and release of liver enzymes were also evaluated. Data obtained for individual agents were used to compute the additivity expectations for two mixtures of definite composition (one equipotent mixture, based in the EC50 values achieved in the MTT assay; the other one based in the acceptable daily intake of each pesticide), using the models of concentration addition and independent action. The EC50 values for fipronil, glyphosate and imidacloprid were 37.59, 41.13, and 663.66 mg/L, respectively. The mixtures of pesticides elicited significant synergistic effects (p < 0.05), which were greater than the expected by both addictive predictions. Decreased in mitochondrial membrane potential and increased in the transaminases enzymatic activities were observed. As they occur simultaneously, interactions between pesticides, even at non-effective single levels, can reverberate in significant deleterious effects, justifying the need for a more realistic approach in safety evaluations to better predict the effects to human health.

The burden of injury in Central, Eastern, and Western European sub-region: a systematic analysis from the Global Burden of Disease 2019 Study.

BACKGROUND: Injury remains a major concern to public health in the European region. Previous iterations of the Global Burden of Disease (GBD) study showed wide variation in injury death and disability adjusted life year (DALY) rates across Europe, indicating injury inequality gaps between sub-regions and countries. The objectives of this study were to: 1) compare GBD 2019 estimates on injury mortality and DALYs across European sub-regions and countries by cause-of-injury category and sex; 2) examine changes in injury DALY rates over a 20 year-period by cause-of-injury category, sub-region and country; and 3) assess inequalities in injury mortality and DALY rates across the countries. METHODS: We performed a secondary database descriptive study using the GBD 2019 results on injuries in 44 European countries from 2000 to 2019. Inequality in DALY rates between these countries was assessed by calculating the DALY rate ratio between the highest-ranking country and lowest-ranking country in each year. RESULTS: In 2019, in Eastern Europe 80 [95% uncertainty interval (UI): 71 to 89] people per 100,000 died from injuries; twice as high compared to Central Europe (38 injury deaths per 100,000; 95% UI 34 to 42) and three times as high compared to Western Europe (27 injury deaths per 100,000; 95%UI 25 to 28). The injury DALY rates showed less pronounced differences between Eastern (5129 DALYs per 100,000; 95% UI: 4547 to 5864), Central (2940 DALYs per 100,000; 95% UI: 2452 to 3546) and Western Europe (1782 DALYs per 100,000; 95% UI: 1523 to 2115). Injury DALY rate was lowest in Italy (1489 DALYs per 100,000) and highest in Ukraine (5553 DALYs per 100,000). The difference in injury DALY rates by country was larger for males compared to females. The DALY rate ratio was highest in 2005, with DALY rate in the lowest-ranking country (Russian Federation) 6.0 times higher compared to the highest-ranking country (Malta). After 2005, the DALY rate ratio between the lowest- and the highest-ranking country gradually decreased to 3.7 in 2019. CONCLUSIONS: Injury mortality and DALY rates were highest in Eastern Europe and lowest in Western Europe, although differences in injury DALY rates declined rapidly, particularly in the past decade. The injury DALY rate ratio of highest- and lowest-ranking country declined from 2005 onwards, indicating declining inequalities in injuries between European countries.

Cocaine: An Updated Overview on Chemistry, Detection, Biokinetics, and Pharmacotoxicological Aspects including Abuse Pattern.

Cocaine is one of the most consumed stimulants throughout the world, as official sources report. It is a naturally occurring sympathomimetic tropane alkaloid derived from the leaves of Erythroxylon coca, which has been used by South American locals for millennia. Cocaine can usually be found in two forms, cocaine hydrochloride, a white powder, or 'crack' cocaine, the free base. While the first is commonly administered by insufflation ('snorting') or intravenously, the second is adapted for inhalation (smoking). Cocaine can exert local anaesthetic action by inhibiting voltage-gated sodium channels, thus halting electrical impulse propagation; cocaine also impacts neurotransmission by hindering monoamine reuptake, particularly dopamine, from the synaptic cleft. The excess of available dopamine for postsynaptic activation mediates the pleasurable effects reported by users and contributes to the addictive potential and toxic effects of the drug. Cocaine is metabolised (mostly hepatically) into two main metabolites, ecgonine methyl ester and benzoylecgonine. Other metabolites include, for example, norcocaine and cocaethylene, both displaying pharmacological action, and the last one constituting a biomarker for co-consumption of cocaine with alcohol. This review provides a brief overview of cocaine's prevalence and patterns of use, its physical-chemical properties and methods for analysis, pharmacokinetics, pharmacodynamics, and multi-level toxicity.

4-Fluoromethamphetamine (4-FMA) induces in vitro hepatotoxicity mediated by CYP2E1, CYP2D6, and CYP3A4 metabolism.

4-Fluoromethamphetamine (4-FMA) is an amphetamine-like psychoactive substance with recognized entactogenic and stimulant effects, but hitherto unclear toxicological mechanisms. Taking into consideration that the vast majority of 4-FMA users consume this substance through oral route, the liver is expected to be highly exposed. The aim of this work was to determine the hepatotoxic potential of 4-FMA using in vitro hepatocellular models: primary rat hepatocytes (PRH), human hepatoma cell lines HepaRG and HepG2, and resorting to concentrations ranging from 37 µM to 30 mM, during a 24-h exposure. EC50 values, estimated from the MTT viability assay data, were 2.21 mM, 5.59 mM and 9.57 mM, for each model, respectively. The most sensitive model, PRH, was then co-exposed to 4-FMA and cytochrome P450 (CYP) inhibitors to investigate the influence of metabolism on the toxicity of 4-FMA. Results show that CYP2E1, CYP3A4 and CYP2D6 have major roles in 4-FMA cytotoxicity. Inhibition of CYP2D6 and CYP3A4 led to left-geared shifts in the concentration-response curves of 4-FMA, hinting at a role of these metabolic enzymes for detoxifying 4-FMA, while CYP2E1 inhibition pointed towards a toxifying role of this enzyme in 4-FMA metabolism at physiologically-relevant concentrations. The drug also destabilised mitochondrial membrane potential and decreased ATP levels, increased the production of reactive oxygen and nitrogen species and compromised thiol antioxidant defences. 4-FMA further affected PRH integrity by interfering with the machinery of apoptosis and necrosis, increasing the activity of initiator and effector caspases, and causing loss of cell membrane integrity. Potential for autophagy was also observed. This research contributes to the growing body of evidence regarding the toxicity of new psychoactive substances, in particular regarding their hepatotoxic effects; the apparent influence of metabolism over the resulting cytotoxicity of 4-FMA shows that there is a substantial degree of unpredictability of the consequences for users that could be independent of the dose.

Biosynthetic versatility of marine-derived fungi on the delivery of novel antibacterial agents against priority pathogens.

Despite the increasing number of novel marine natural products being reported from fungi in the last three decades, to date only the broad-spectrum cephalosporin C can be tracked back as marine fungal-derived drug. Cephalosporins were isolated in the early 1940s from a strain of Acremonium chrysogenum obtained in a sample collected in sewage water in the Sardinian coast, preliminary findings allowing the discovery of cephalosporin C. Since then, bioprospection of marine fungi has been enabling the identification of several metabolites with antibacterial effects, many of which proving to be active against multi-drug resistant strains, available data suggesting also that some might fuel the pharmaceutical firepower towards some of the bacterial pathogens classified as a priority by the World Health Organization. Considering the success of their terrestrial counterparts on the discovery and development of several antibiotics that are nowadays used in the clinical setting, marine fungi obviously come into mind as producers of new prototypes to counteract antibiotic-resistant bacteria that are no longer responding to available treatments. We mainly aim to provide a snapshot on those metabolites that are likely to proceed to advanced preclinical development, not only based on their antibacterial potency, but also considering their targets and modes of action, and activity against priority pathogens.

Overview of Synthetic Cannabinoids ADB-FUBINACA and AMB-FUBINACA: Clinical, Analytical, and Forensic Implications.

ADB-FUBINACA and AMB-FUBINACA are two synthetic indazole-derived cannabinoid receptor agonists, up to 140- and 85-fold more potent, respectively, than trans-∆9-tetrahydrocannabinol (∆9-THC), the main psychoactive compound of cannabis. Synthesised in 2009 as a pharmaceutical drug candidate, the recreational use of ADB-FUBINACA was first reported in 2013 in Japan, with fatal cases being described in 2015. ADB-FUBINACA is one of the most apprehended and consumed synthetic cannabinoid (SC), following AMB-FUBINACA, which emerged in 2014 as a drug of abuse and has since been responsible for several intoxication and death outbreaks. Here, we critically review the physicochemical properties, detection methods, prevalence, biological effects, pharmacodynamics and pharmacokinetics of both drugs. When smoked, these SCs produce almost immediate effects (about 10 to 15 s after use) that last up to 60 min. They are rapidly and extensively metabolised, being the O-demethylated metabolite of AMB-FUBINACA, 2-(1-(4-fluorobenzyl)-1H-indazole-3-carboxamide)-3-methylbutanoic acid, the main excreted in urine, while for ADB-FUBINACA the main biomarkers are the hydroxdimethylpropyl ADB-FUBINACA, hydroxydehydrodimethylpropyl ADB-FUBINACA and hydroxylindazole ADB-FUBINACA. ADB-FUBINACA and AMB-FUBINACA display full agonism of the CB1 receptor, this being responsible for their cardiovascular and neurological effects (e.g., altered perception, agitation, anxiety, paranoia, hallucinations, loss of consciousness and memory, chest pain, hypertension, tachycardia, seizures). This review highlights the urgent requirement for additional studies on the toxicokinetic properties of AMB-FUBINACA and ADB-FUBINACA, as this is imperative to improve the methods for detecting and quantifying these drugs and to determine the best exposure markers in the various biological matrices. Furthermore, it stresses the need for clinicians and pathologists involved in the management of these intoxications to describe their findings in the scientific literature, thus assisting in the risk assessment and treatment of the harmful effects of these drugs in future medical and forensic investigations.

From street to lab: in vitro hepatotoxicity of buphedrone, butylone and 3,4-DMMC.

Synthetic cathinones are among the most popular new psychoactive substances, being abused for their stimulant properties, which are similar to those of amphetamine and 3,4-methylenedioxymethamphetamine (MDMA). Considering that the liver is a likely target for cathinones-induced toxicity, and for their metabolic activation/detoxification, we aimed to determine the hepatotoxicity of three commonly abused synthetic cathinones: butylone, α-methylamino-butyrophenone (buphedrone) and 3,4-dimethylmethcathinone (3,4-DMMC). We characterized their cytotoxic profile in primary rat hepatocytes (PRH) and in the HepaRG and HepG2 cell lines. PRH was the most sensitive cell model, showing the lowest EC50 values for all three substances (0.158 mM for 3,4-DMMC; 1.21 mM for butylone; 1.57 mM for buphedrone). Co-exposure of PRH to the synthetic cathinones and CYP450 inhibitors (selective and non-selective) proved that hepatic metabolism reduced the toxicity of buphedrone but increased that of butylone and 3,4-DMMC. All compounds were able to increase oxidative stress, disrupting mitochondrial homeostasis and inducing apoptotic and necrotic features, while also increasing the occurrence of acidic vesicular organelles in PRH, compatible with autophagic activation. In conclusion, butylone, buphedrone and 3,4-DMMC have hepatotoxic potential, and their toxicity lies in the interference with a number of homeostatic processes, while being influenced by their metabolic fate.

Pharmacokinetics and Pharmacodynamics of Salvinorin A and Salvia divinorum: Clinical and Forensic Aspects.

Salvia divinorum Epling and Játiva is a perennial mint from the Lamiaceae family, endemic to Mexico, predominantly from the state of Oaxaca. Due to its psychoactive properties, S. divinorum had been used for centuries by Mazatecans for divinatory, religious, and medicinal purposes. In recent years, its use for recreational purposes, especially among adolescents and young adults, has progressively increased. The main bioactive compound underlying the hallucinogenic effects, salvinorin A, is a non-nitrogenous diterpenoid with high affinity and selectivity for the k-opioid receptor. The aim of this work is to comprehensively review and discuss the toxicokinetics and toxicodynamics of S. divinorum and salvinorin A, highlighting their psychological, physiological, and toxic effects. Potential therapeutic applications and forensic aspects are also covered in this review. The leaves of S. divinorum can be chewed, drunk as an infusion, smoked, or vaporised. Absorption of salvinorin A occurs through the oral mucosa or the respiratory tract, being rapidly broken down in the gastrointestinal system to its major inactive metabolite, salvinorin B, when swallowed. Salvinorin A is rapidly distributed, with accumulation in the brain, and quickly eliminated. Its pharmacokinetic parameters parallel well with the short-lived psychoactive and physiological effects. No reports on toxicity or serious adverse outcomes were found. A variety of therapeutic applications have been proposed for S. divinorum which includes the treatment of chronic pain, gastrointestinal and mood disorders, neurological diseases, and treatment of drug dependence. Notwithstanding, there is still limited knowledge regarding the pharmacology and toxicology features of S. divinorum and salvinorin A, and this is needed due to its widespread use. Additionally, the clinical acceptance of salvinorin A has been hampered, especially due to the psychotropic side effects and misuse, turning the scientific community to the development of analogues with better pharmacological profiles.

Cellular uptake and toxicity of gold nanoparticles on two distinct hepatic cell models.

Gold nanoparticles (AuNPs) have huge potential for various biomedical applications, but their successful use depends on their uptake and possible toxicity in the liver, their main site for accumulation. Therefore, in this work we compared the cytotoxic effects induced by AuNPs with different size (~ 15 nm and 60 nm), shape (nanospheres and nanostars) and capping [citrate- or 11-mercaptoundecanoic acid (MUA)], in human HepaRG cells or primary rat hepatocytes (PRH) cultivated with serum-free or Foetal Bovine Serum (FBS)-supplemented media. The safety assessment of the AuNPs demonstrated that overall they present low toxicity towards hepatic cells. Among all the tested AuNPs, the smaller 15 nm spheres displayed the highest toxicity. The toxicological effect was capping, size and cell-type dependent with citrate-capping more toxic than MUA (PRH with FBS), the 15 nm AuNPs more toxic than 60 nm counterparts and PRH more sensitive, as compared to the HepaRG cells. The incubation with FBS-free media produced aggregation of AuNPs while its presence greatly influenced the toxicity outcomes. The cellular uptake of AuNPs was shape, size and capping dependent in PRH cultivated in FBS-supplemented media, and significantly different between the two types of cells with extensively higher internalization of AuNPs in PRH, as compared to the HepaRG cells. These data show that the physical-chemical properties of AuNPs, including size and shape, as well as the type of cellular model, greatly influence the interaction of the AuNPs with the biological environment and consequently, their toxicological effects.

Toxicokinetics and Toxicodynamics of Ayahuasca Alkaloids N,N-Dimethyltryptamine (DMT), Harmine, Harmaline and Tetrahydroharmine: Clinical and Forensic Impact.

Ayahuasca is a hallucinogenic botanical beverage originally used by indigenous Amazonian tribes in religious ceremonies and therapeutic practices. While ethnobotanical surveys still indicate its spiritual and medicinal uses, consumption of ayahuasca has been progressively related with a recreational purpose, particularly in Western societies. The ayahuasca aqueous concoction is typically prepared from the leaves of the N,N-dimethyltryptamine (DMT)-containing Psychotria viridis, and the stem and bark of Banisteriopsis caapi, the plant source of harmala alkaloids. Herein, the toxicokinetics and toxicodynamics of the psychoactive DMT and harmala alkaloids harmine, harmaline and tetrahydroharmine, are comprehensively covered, particularly emphasizing the psychological, physiological, and toxic effects deriving from their concomitant intake. Potential therapeutic utility, particularly in mental and psychiatric disorders, and forensic aspects of DMT and ayahuasca are also reviewed and discussed. Following administration of ayahuasca, DMT is rapidly absorbed and distributed. Harmala alkaloids act as potent inhibitors of monoamine oxidase A (MAO-A), preventing extensive first-pass degradation of DMT into 3-indole-acetic acid (3-IAA), and enabling sufficient amounts of DMT to reach the brain. DMT has affinity for a variety of serotonergic and non-serotonergic receptors, though its psychotropic effects are mainly related with the activation of serotonin receptors type 2A (5-HT2A). Mildly to rarely severe psychedelic adverse effects are reported for ayahuasca or its alkaloids individually, but abuse does not lead to dependence or tolerance. For a long time, the evidence has pointed to potential psychotherapeutic benefits in the treatment of depression, anxiety, and substance abuse disorders; and although misuse of ayahuasca has been diverting attention away from such clinical potential, research onto its therapeutic effects has now strongly resurged.

Epigenetics and the endocannabinoid system signaling: An intricate interplay modulating neurodevelopment.

The endocannabinoid (eCB) system is a complex system comprising endogenous cannabinoids (eCBs), their synthesis and degradation enzymes, and cannabinoid receptors. These elements crucially regulate several biological processes during neurodevelopment, such as proliferation, differentiation, and migration. Recently, eCBs were also reported to have an epigenetic action on genes that play key functions in the neurotransmitter signaling, consequently regulating their expression. In turn, epigenetic modifications (e.g. DNA methylation, histone modifications) may also modulate the function of eCB system's elements. For example, the expression of the cnr gene in the central nervous system may be epigenetically regulated (e.g. DNA methylation, histone modifications), thus altering the function of the cannabinoid receptor type-1 (CB1R). Considering the importance of the eCB system during neurodevelopment, it is thus reasonable to expect that alterations in this interaction between the eCB system and epigenetic modifications may give rise to neurodevelopmental disorders. Here, we review key concepts related to the regulation of neuronal function by the eCB system and the different types of epigenetic modifications. In particular, we focus on the mechanisms involved in the intricate interplay between both signaling systems and how they control cell fate during neurodevelopment. Noteworthy, such mechanistic understanding assumes high relevance considering the implications of the dysregulation of key neurogenic processes towards the onset of neurodevelopment-related disorders. Moreover, considering the increasing popularity of cannabis and its synthetic derivatives among young adults, it becomes of utmost importance to understand how exogenous cannabinoids may epigenetically impact neurodevelopment.

The Synthetic Cannabinoids THJ-2201 and 5F-PB22 Enhance In Vitro CB1 Receptor-Mediated Neuronal Differentiation at Biologically Relevant Concentrations.

Recreational use of synthetic cannabinoids (SCs) before and during pregnancy poses a major public health risk, due to the potential onset of neurodevelopmental disorders in the offspring. Herein, we report the assessment of the neurotoxic potential of two commonly abused SCs, THJ-2201 and 5F-PB22, particularly focusing on how they affect neuronal differentiation in vitro. Differentiation ratios, total neurite length, and neuronal marker expression were assessed in NG108-15 neuroblastoma x glioma cells exposed to the SCs at non-toxic, biologically relevant concentrations (≤1 µM), either in acute or repeated exposure settings. Both SCs enhanced differentiation ratios and total neurite length of NG108-15 cells near two-fold compared to vehicle-treated cells, in a CB1R activation-dependent way, as the CB1R blockade with a specific antagonist (SR141718) abrogated SC-induced effects. Interestingly, repeated 5F-PB22 exposure was required to reach effects similar to a single THJ-2201 dose. Cell viability and proliferation, mitochondrial membrane potential, and intracellular ATP levels were also determined. The tested SCs increased mitochondrial tetramethyl rhodamine ethyl ester (TMRE) accumulation after 24 h at biologically relevant concentrations but did not affect any of the other toxicological parameters. Overall, we report firsthand the CB1R-mediated enhancement of neurodifferentiation by 5F-PB22 and THJ-2201 at biologically relevant concentrations.

Drinking to death: Hyponatraemia induced by synthetic phenethylamines.

Synthetic phenethylamines are widely abused drugs, comprising new psychoactive substances such as synthetic cathinones, but also well-known amphetamines such as methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA, ecstasy). Cathinones and amphetamines share many toxicodynamic mechanisms. One of their potentially life-threatening consequences, particularly of MDMA, is serotonin-mediated hyponatraemia. Herein, we review the state of the art on phenethylamine-induced hyponatremia; discuss the mechanisms involved; and present the preventive and therapeutic measures. Hyponatraemia mediated by phenethylamines results from increased secretion of antidiuretic hormone (ADH) and consequent kidney water reabsorption, additionally involving diaphoresis and polydipsia. Data for MDMA suggest that acute hyponatraemia elicited by cathinones may also be a consequence of metabolic activation. The literature often reveals hyponatraemia-associated complications such as cerebral oedema, cerebellar tonsillar herniation and coma that may evolve to a fatal outcome, particularly in women. Ready availability of fluids and the recommendation to drink copiously at the rave scene to counteract hyperthermia, often precipitate water intoxication. Users should be advised about the importance of controlling fluid intake while using phenethylamines. At early signs of adverse effects, medical assistance should be promptly sought. Severe hyponatraemia (<130 mmol sodium/L plasma) may be corrected with hypertonic saline or suppression of fluid intake. Also, clinicians should be made aware of the hyponatraemic potential of these drugs and encouraged to report future cases of toxicity to increase knowledge on this potentially lethal outcome.

Diet aid or aid to die: an update on 2,4-dinitrophenol (2,4-DNP) use as a weight-loss product.

During the last decades, we have witnessed unparalleled changes in human eating habits and lifestyle, intensely influenced by cultural and social pressures. Sports practice became strongly implemented in daily routines, and visits to the gym peaked, driven by the indulgence in intensive 'weight-loss programs'. The pledge of boasting a healthy and beautiful body instigates the use of very attractive 'fat burners', which are purportedly advertised as safe products, easily available in the market and expected to quickly reduce body weight. In this context, the slimming properties of 2,4-dinitrophenol (2,4-DNP) galvanised its use as a weight-loss product, despite the drug ban for human consumption in many countries since 1938, due to its adverse effects. The main symptoms associated with 2,4-DNP intoxication, including hyperthermia, tachycardia, decreased blood pressure, and acute renal failure, motivated a worldwide warning, issued by the Interpol Anti-Doping Unit in 2015, reinforcing its hazard. Information on the effects of 2,4-DNP mainly derive from the intoxication cases reported by emergency care units, for which there is no specific antidote or treatment. This review provides a comprehensive update on 2,4-DNP use, legislation and epidemiology, chemistry and analytical methodologies for drug determination in commercial products and biological samples, pharmacokinetics and pharmacodynamics, toxicological effects, and intoxication diagnosis and management.

Emerging club drugs: 5-(2-aminopropyl)benzofuran (5-APB) is more toxic than its isomer 6-(2-aminopropyl)benzofuran (6-APB) in hepatocyte cellular models.

New phenylethylamine derivatives are among the most commonly abused new psychoactive substances. They are synthesized and marketed in lieu of classical amphetaminic stimulants, with no previous safety testing. Our study aimed to determine the in vitro hepatotoxicity of two benzofurans [6-(2-aminopropyl)benzofuran (6-APB) and 5-(2-aminopropyl)benzofuran (5-APB)] that have been misused as 'legal highs'. Cellular viability was assessed through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, following 24-h drug exposure of human hepatoma HepaRG cells (EC50 2.62 mM 5-APB; 6.02 mM 6-APB), HepG2 cells (EC50 3.79 mM 5-APB; 8.18 mM 6-APB) and primary rat hepatocytes (EC50 964 µM 5-APB; 1.94 mM 6-APB). Co-incubation of primary hepatocytes, the most sensitive in vitro model, with CYP450 inhibitors revealed a role of metabolism, in particular by CYP3A4, in the toxic effects of both benzofurans. Also, 6-APB and 5-APB concentration-dependently enhanced oxidative stress (significantly increased reactive species and oxidized glutathione, and decreased reduced glutathione levels) and unsettled mitochondrial homeostasis, with disruption of mitochondrial membrane potential and decline of intracellular ATP. Evaluation of cell death mechanisms showed increased caspase-8, -9, and -3 activation, and nuclear morphological changes consistent with apoptosis; at concentrations higher than 2 mM, however, necrosis prevailed. Concentration-dependent formation of acidic vesicular organelles typical of autophagy was also observed for both drugs. Overall, 5-APB displayed higher hepatotoxicity than its 6-isomer. Our findings provide new insights into the potential hepatotoxicity of these so-called 'safe drugs' and highlight the putative risks associated with their use as psychostimulants.

Biodistribution and metabolic profile of 3,4-dimethylmethcathinone (3,4-DMMC) in Wistar rats through gas chromatography-mass spectrometry (GC-MS) analysis.

3,4-Dimethylmethcathinone (3,4-DMMC) is a new psychoactive substance whose recreational use and trade have recently increased. Given the absence of information on the toxicokinetics of 3,4-DMMC, the present work aimed at validating a GC-MS methodology for the drug quantification in biological matrices, and further characterizing its biodistribution in Wistar rats. The method was validated based on the evaluation of the drug stability, limit of detection and quantification, linearity, selectivity, precision, accuracy and recovery. To characterize biodistribution, Wistar rats were administered with 20 or 40 mg/Kg of 3,4-DMMC i.p.. After 1 h or 24 h, rats were anaesthetized, euthanized and blood, brain, liver, heart, kidneys, lungs, spleen, urine (only at 24 h), and a portion of gut, muscle and adipose tissue were collected for analysis. After 1 h, 3,4-DMMC was present in all analysed matrices, and the presence of two metabolites was further detected in all of them. The drug accumulation was higher in kidneys, lungs, spleen and brain. After 24 h, 3,4-DMMC was only present in urine, along with five metabolites. All metabolites were tentatively identified. Through elucidation of the most appropriate analytical matrices and the metabolites that may have the largest detection windows, these data are expected to assist in future clinical and forensic investigations.

The new psychoactive substance 3-methylmethcathinone (3-MMC or metaphedrone) induces oxidative stress, apoptosis, and autophagy in primary rat hepatocytes at human-relevant concentrations.

3-Methylmethcathinone (3-MMC or metaphedrone) has become one of the most popular recreational drugs worldwide after the ban of mephedrone, and was recently deemed responsible for several intoxications and deaths. This study aimed at assessing the hepatotoxicity of 3-MMC. For this purpose, Wistar rat hepatocytes were isolated by collagenase perfusion, cultured and exposed for 24 h at a concentration range varying from 31 nM to 10 mM 3-MMC. The modulatory effects of cytochrome P450 (CYP) inhibitors on 3-MMC hepatotoxicity were evaluated. 3-MMC-induced toxicity was perceived at the lysosome at lower concentrations (NOEC 312.5 µM), compared to mitochondria (NOEC 379.5 µM) and cytoplasmic membrane (NOEC 1.04 mM). Inhibition of CYP2D6 and CYP2E1 diminished 3-MMC cytotoxicity, yet for CYP2E1 inhibition this effect was only observed for concentrations up to 1.3 mM. A significant concentration-dependent increase of intracellular reactive species was observed from 10 µM 3-MMC on; a concentration-dependent decrease in antioxidant glutathione defences was also observed. At 10 µM, caspase-3, caspase-8, and caspase-9 activities were significantly elevated, corroborating the activation of both intrinsic and extrinsic apoptosis pathways. Nuclear morphology and formation of cytoplasmic acidic vacuoles suggest prevalence of necrosis and autophagy at concentrations higher than 10 µM. No significant alterations were observed in the mitochondrial membrane potential, but intracellular ATP significantly decreased at 100 µM. Our data point to a role of metabolism in the hepatotoxicity of 3-MMC, which seems to be triggered both by autophagic and apoptotic/necrotic mechanisms. This work is the first approach to better understand 3-MMC toxicology.

A multiparametric study of gold nanoparticles cytotoxicity, internalization and permeability using an in vitro model of blood-brain barrier. Influence of size, shape and capping agent.

Gold nanoparticles (AuNPs) have biomedical application on imaging and due to increased optical performance, star-shaped AuNPs are of special interest. Because shape, size and capping greatly influence their toxicokinetics and toxicodynamics, a systematic multiparametric comparative study of the influence of these parameters on the cytotoxicity, internalization, and in vitro permeability was conducted in human Cerebral Microvascular Endothelial Cell line (hCMEC/D3), an in vitro model of the human blood-brain barrier (BBB). AuNPs of different size (14 nm and ∼50 nm), shape (spheres and stars), and coating (11-mercaptoundecanoic acid or MUA and sodium citrate) were synthesized and fully characterized. The time- and concentration-dependent cytotoxic profile of the tested AuNPs differed for the different AuNPs. Generally, toxicity was greater for stars relative to sphere-shaped AuNPs, and citrate coating was more toxic than MUA. Regarding the influence of size, smaller-sized AuNPs were more cytotoxic when compared at the same Au concentration. However, when the concentration of AuNPs was expressed as the number of AuNPs/mL, a higher degree of cytotoxicity was noted for the larger ̴50 nm AuNPs. To understand the influence of size, shape and capping, a systematic study design, in which only one of the variables changes, is determinant for correct data interpretation. Considering the results herein presented, for the sake of comparison of differently-sized AuNPs, it is preferable to design the study based upon the number of nanoparticles, since at a fixed Au concentration the number of particles available to promote effect is higher for smaller-sized AuNPs. Cellular internalization also differed among the tested AuNPs; although all were unable to cross the in vitro BBB, the intracellularly accumulated AuNPs can induce cell damage and later compromise BBB integrity and permeability.

Benzo fury: A new trend in the drug misuse scene.

Benzofurans, also known by users as benzo fury or benzofury, are synthetic phenethylamines and constitute the third most prominent group of new psychoactive substances (NPS). As the use of these substances has been spread as an alternative to the classic illicit psychostimulants, such as amphetamines, their legal status was reviewed, resulting in an utter prohibition of these NPS in many countries worldwide. Herein, the prevalence of abuse, chemistry, biological effects, metabolism, and the potential harms and risky behaviors associated with the abuse of benzofurans are reviewed. The congeners of this group are mainly consumed recreationally at electronic dance music parties, in polydrug abuse settings. Benzofurans preferentially act by disturbing the functioning of serotonergic circuits, which induces their entactogenic and stimulant effects and is the reason behind the considerable number of recent benzo fury-related deaths. The slight interaction of these drugs with the dopaminergic system justifies the rewarding effects of these drugs. To date, published evidence on the mechanisms of toxicity of benzo fury is very limited but a body of research is now beginning to emerge revealing an alarming public health threat regarding the abuse of these NPS.