Genotoxicity Evaluation of The Novel Psychoactive Substance MTTA.

MTTA, also known as mephtetramine, is a stimulant novel psychoactive substance characterized by a simil-cathinonic structure. To date, little has been studied on its pharmaco-toxicological profile, and its genotoxic potential has never been assessed. In order to fill this gap, the aim of the present work was to evaluate its genotoxicity on TK6 cells in terms of its ability to induce structural and numerical chromosomal aberrations by means of a cytofluorimetric protocol of the "In Vitro Mammalian Cell Micronucleus (MN) test". To consider the in vitro effects of both the parental compound and the related metabolites, TK6 cells were treated with MTTA in the absence or presence of an exogenous metabolic activation system (S9 mix) for a short-term time (3 h) followed by a recovery period (23 h). No statistically significant increase in the MNi frequency was detected. Specifically, in the presence of S9 mix, only a slight increasing trend was observable at all tested concentrations, whereas, without S9 mix, at 75 µM, almost a doubling of the negative control was reached. For the purposes of comprehensive evaluation, a long-term treatment (26 h) was also included. In this case, a statistically significant enhancement in the MNi frequency was observed at 50 µM.

Acute Cardiovascular and Cardiorespiratory Effects of JWH-018 in Awake and Freely Moving Mice: Mechanism of Action and Possible Antidotal Interventions?

JWH-018 is the most known compound among synthetic cannabinoids (SCs) used for their psychoactive effects. SCs-based products are responsible for several intoxications in humans. Cardiac toxicity is among the main side effects observed in emergency departments: SCs intake induces harmful effects such as hypertension, tachycardia, chest pain, arrhythmias, myocardial infarction, breathing impairment, and dyspnea. This study aims to investigate how cardio-respiratory and vascular JWH-018 (6 mg/kg) responses can be modulated by antidotes already in clinical use. The tested antidotes are amiodarone (5 mg/kg), atropine (5 mg/kg), nifedipine (1 mg/kg), and propranolol (2 mg/kg). The detection of heart rate, breath rate, arterial oxygen saturation (SpO2), and pulse distention are provided by a non-invasive apparatus (Mouse Ox Plus) in awake and freely moving CD-1 male mice. Tachyarrhythmia events are also evaluated. Results show that while all tested antidotes reduce tachycardia and tachyarrhythmic events and improve breathing functions, only atropine completely reverts the heart rate and pulse distension. These data may suggest that cardiorespiratory mechanisms of JWH-018-induced tachyarrhythmia involve sympathetic, cholinergic, and ion channel modulation. Current findings also provide valuable impetus to identify potential antidotal intervention to support physicians in the treatment of intoxicated patients in emergency clinical settings.

The Old and the New: Cardiovascular and Respiratory Alterations Induced by Acute JWH-018 Administration Compared to Δ9-THC-A Preclinical Study in Mice.

Several new psychoactive substances (NPS) are responsible for intoxication involving the cardiovascular and respiratory systems. Among NPS, synthetic cannabinoids (SCs) provoked side effects in humans characterized by tachycardia, arrhythmias, hypertension, breathing difficulty, apnoea, myocardial infarction, and cardiac arrest. Therefore, the present study investigated the cardio-respiratory (MouseOx Plus; EMKA electrocardiogram (ECG) and plethysmography TUNNEL systems) and vascular (BP-2000 systems) effects induced by 1-naphthalenyl (1-pentyl-1H-indol-3-yl)-methanone (JWH-018; 0.3-3-6 mg/kg) and Δ9-tetrahydrocannabinol (Δ9-THC; 0.3-3-6 mg/kg), administered in awake CD-1 male mice. The results showed that higher doses of JWH-018 (3-6 mg/kg) induced deep and long-lasting bradycardia, alternated with bradyarrhythmia, spaced out by sudden episodes of tachyarrhythmias (6 mg/kg), and characterized by ECG electrical parameters changes, sustained bradypnea, and systolic and transient diastolic hypertension. Otherwise, Δ9-THC provoked delayed bradycardia (minor intensity tachyarrhythmias episodes) and bradypnea, also causing a transient and mild hypertensive effect at the tested dose range. These effects were prevented by both treatment with selective CB1 (AM 251, 6 mg/kg) and CB2 (AM 630, 6 mg/kg) receptor antagonists and with the mixture of the antagonists AM 251 and AM 630, even if in a different manner. Cardio-respiratory and vascular symptoms could be induced by peripheral and central CB1 and CB2 receptors stimulation, which could lead to both sympathetic and parasympathetic systems activation. These findings may represent a starting point for necessary future studies aimed at exploring the proper antidotal therapy to be used in SCs-intoxicated patient management.

Behavioral and Pharmacokinetics Studies of N-Methyl-2-Aminoindane (NM2AI) in Mice: An Aminoindane Briefly Used in the Illicit Drug Market.

Drug forums are considered as the main platform sources that have contributed to the increase in NPS popularity, especially for those not yet known to law enforcement and therefore not yet illegal. An example is the new synthetic stimulant NM2AI, which has a very short history of human use and abuse. Little is known regarding this compound, but some information from internet forums and the scientific literature indicates NM2AI as a structural derivate of MDAI, which is known for its entactogenic activity. Indeed, the purpose of this study is to evaluate, for the first time, the in vivo acute effect induced by the intraperitoneal injection of NM2AI (1-10-30-100 mg/kg) in mice. We demonstrate the sensory (by visual placing and object tests) and physiological (core temperature measurement) function variations, nociceptor (by tail pinch test) and strength (grip test) alterations, and sensorimotor (time on rod and mobility) decrease. Moreover, we verify the mild hallucinogenic effect of NM2AI (by startle/prepulse inhibition test). Lastly, we perform a pharmacokinetic study on mice blood samples, highlighting that the main active metabolite of NM2AI is 2-aminoindane (2AI). Taken together, our data confirm the suspected entactogenic activity of NM2AI; however, these in vivo effects appear atypical and less intense with respect to those induced by the classic stimulants, in surprising analogy with what is reported by networked users.

Genotoxicological Characterization of (±)cis-4,4'-DMAR and (±)trans-4,4'-DMAR and Their Association.

The novel psychoactive substance (NPS) 4-Methyl-5-(4-methylphenyl)-4,5-dihydroxazol-2-amine (4,4'-DMAR) shows psychostimulant activity. Data on the acute toxicity of 4,4'-DMAR are becoming increasingly available, yet the long-term effects are still almost unknown. In particular, no data on genotoxicity are available. Therefore, the aim of the present study was to evaluate its genotoxic potential using the "In Vitro Mammalian Cell Micronucleus Test" (MNvit) on (±)cis-4,4'-DMAR and (±)trans-4,4'-DMAR and their associations. The analyses were conducted in vitro on human TK6 cells. To select suitable concentrations for MNvit, we preliminarily evaluated cytotoxicity and apoptosis. All endpoints were analysed by flow cytometry. The results reveal the two racemates' opposite behaviours: (±)cis-4,4'-DMAR shows a statistically significant increase in micronuclei (MNi) frequency that (±)trans-4,4'-DMAR is completely incapable of. This contrast confirms the well-known possibility of observing opposite biological effects of the cis- and trans- isomers of a compound, and it highlights the importance of testing single NPSs that show even small differences in structure or conformation. The genotoxic capacity demonstrated stresses an additional alarming toxicological concern related to this NPS. Moreover, the co-treatments indicate that consuming both racemates will magnify the genotoxic effect, an aspect to consider given the unpredictability of illicit drug composition.

The Genotoxicity of Acrylfentanyl, Ocfentanyl and Furanylfentanyl Raises the Concern of Long-Term Consequences.

Three fentanyl analogues Acrylfentanyl, Ocfentanyl and Furanylfentanyl are potent, rapid-acting synthetic analgesics that recently appeared on the illicit market of new psychoactive substances (NPS) under the class of new synthetic opioids (NSO). Pharmacotoxicological data on these three non-pharmaceutical fentanyl analogues are limited and studies on their genotoxicity are not yet available. Therefore, the aim of the present study was to investigate this property. The ability to induce structural and numerical chromosomal aberrations in human lymphoblastoid TK6 cells was evaluated by employing the flow cytometric protocol of the in vitro mammalian cell micronucleus test. Our study demonstrated the non-genotoxicity of Fentanyl, i.e., the pharmaceutical progenitor of the class, while its illicit non-pharmaceutical analogues were found to be genotoxic. In particular, Acrylfentanyl led to a statistically significant increase in the MNi frequency at the highest concentration tested (75 µM), while Ocfentanyl and Furanylfentnyl each did so at both concentrations tested (150, 200 µM and 25, 50 µM, respectively). The study ended by investigating reactive oxygen species (ROS) induction as a possible mechanism linked to the proved genotoxic effect. The results showed a non-statistically significant increase in ROS levels in the cultures treated with all molecules under study. Overall, the proved genotoxicity raises concern about the possibility of serious long-term consequences.

In Vivo Bio-Activation of JWH-175 to JWH-018: Pharmacodynamic and Pharmacokinetic Studies in Mice.

3-(1-Naphthalenylmethyl)-1-pentyl-1H-indole (JWH-175) is a synthetic cannabinoid illegally marketed for its psychoactive cannabis-like effects. This study aimed to investigate and compare in vitro and in vivo pharmacodynamic activity of JWH-175 with that of 1-naphthalenyl (1-pentyl-1H-indol-3-yl)-methanone (JWH-018), as well as evaluate the in vitro (human liver microsomes) and in vivo (urine and plasma of CD-1 male mice) metabolic profile of JWH-175. In vitro binding studies showed that JWH-175 is a cannabinoid receptor agonist less potent than JWH-018 on mouse and human CB1 and CB2 receptors. In agreement with in vitro data, JWH-175 reduced the fESPS in brain hippocampal slices of mice less effectively than JWH-018. Similarly, in vivo behavioral studies showed that JWH-175 impaired sensorimotor responses, reduced breath rate and motor activity, and increased pain threshold to mechanical stimuli less potently than JWH-018. Metabolic studies demonstrated that JWH-175 is rapidly bioactivated to JWH-018 in mice blood, suggesting that in vivo effects of JWH-175 are also due to JWH-018 formation. The pharmaco-toxicological profile of JWH-175 was characterized for the first time, proving its in vivo bio-activation to the more potent agonist JWH-018. Thus, it highlighted the great importance of investigating the in vivo metabolism of synthetic cannabinoids for both clinical toxicology and forensic purposes.

Cytokine storm and histopathological findings in 60 cases of COVID-19-related death: from viral load research to immunohistochemical quantification of major players IL-1ß, IL-6, IL-15 and TNF-α.

This study involves the histological analysis of samples taken during autopsies in cases of COVID-19 related death to evaluate the inflammatory cytokine response and the tissue localization of the virus in various organs. In all the selected cases, SARS-CoV-2 RT-PCR on swabs collected from the upper (nasopharynx and oropharynx) and/or the lower respiratory (trachea and primary bronchi) tracts were positive. Tissue localization of SARS-CoV-2 was detected using antibodies against the nucleoprotein and the spike protein. Overall, we tested the hypothesis that the overexpression of proinflammatory cytokines plays an important role in the development of COVID-19-associated pneumonia by estimating the expression of multiple cytokines (IL-1ß, IL-6, IL-10, IL-15, TNF-α, and MCP-1), inflammatory cells (CD4, CD8, CD20, and CD45), and fibrinogen. Immunohistochemical staining showed that endothelial cells expressed IL-1ß in lung samples obtained from the COVID-19 group (p < 0.001). Similarly, alveolar capillary endothelial cells showed strong and diffuse immunoreactivity for IL-6 and IL-15 in the COVID-19 group (p < 0.001). TNF-α showed a higher immunoreactivity in the COVID-19 group than in the control group (p < 0.001). CD8 + T cells where more numerous in the lung samples obtained from the COVID-19 group (p < 0.001). Current evidence suggests that a cytokine storm is the major cause of acute respiratory distress syndrome (ARDS) and multiple organ failure and is consistently linked with fatal outcomes.

Evaluation of Cytotoxic and Mutagenic Effects of the Synthetic Cathinones Mexedrone, α-PVP and α-PHP.

Mexedrone, α-PVP and α-PHP are synthetic cathinones. They can be considered amphetamine-like substances with a stimulating effect. Actually, studies showing their impact on DNA are totally absent. Therefore, in order to fill this gap, aim of the present work was to evaluate their mutagenicity on TK6 cells. On the basis of cytotoxicity and cytostasis results, we selected the concentrations (35-100 µM) to be used in the further analysis. We used the micronucleus (MN) as indicator of genetic damage and analyzed the MNi frequency fold increase by flow cytometry. Mexedrone demonstrated its mutagenic potential contrary to the other two compounds; we then proceeded by repeating the analyzes in the presence of extrinsic metabolic activation in order to check if it was possible to totally exclude the mutagenic capacity for α-PVP and α-PHP. The results demonstrated instead the mutagenicity of their metabolites. We then evaluated reactive oxygen species (ROS) induction as a possible mechanism at the basis of the highlighted effects but the results did not show a statistically significant increase in ROS levels for any of the tested substances. Anyway, our outcomes emphasize the importance of mutagenicity evaluation for a complete assessment of the risk associated with synthetic cathinones exposure.

Single Exposure to the Cathinones MDPV and α-PVP Alters Molecular Markers of Neuroplasticity in the Adult Mouse Brain.

Synthetic cathinones have gained popularity among young drug users and are widely used in the clandestine market. While the cathinone-induced behavioral profile has been extensively investigated, information on their neuroplastic effects is still rather fragmentary. Accordingly, we have exposed male mice to a single injection of MDPV and α-PVP and sacrificed the animals at different time points (i.e., 30 min, 2 h, and 24 h) to have a rapid readout of the effect of these psychostimulants on neuroplasticity in the frontal lobe and hippocampus, two reward-related brain regions. We found that a single, low dose of MDPV or α-PVP is sufficient to alter the expression of neuroplastic markers in the adult mouse brain. In particular, we found increased expression of the transcription factor Npas4, increased ratio between the vesicular GABA transporter and the vesicular glutamate transporter together with changes in the expression of the neurotrophin Bdnf, confirming the widespread impact of these cathinones on brain plasticity. To sum up, exposure to low dose of cathinones can impair cortical and hippocampal homeostasis, suggesting that abuse of these cathinones at much higher doses, as it occurs in humans, could have an even more profound impact on neuroplasticity.