Potential role of circulating microRNAs as early markers of preeclampsia

Abstract Objective To identify microRNAs (miRNAs) differentially expressed at early stages of gestation (12–14 weeks) in the serum of pregnant women, who later developed severe preeclampsia (sPE) in the third trimester of pregnancy ( n = 24) compared to women with normal pregnancy ( n = 24). Materials and Methods Sera from 12–14-week-gestation whole blood were subjected to microarray analysis with TaqMan Low Density Array chips (human microRNA panel V3.0), and to quantitative real-time polymerase chain reaction. Results By using the TaqMan Low Density Array chip technology, 19 mature miRNAs appeared differentially expressed in the group of women who later developed sPE as compared to normal women. The expression of four miRNAs (miR-1233, miR-520, miR-210, miR-144) was validated by quantitative real-time polymerase chain reaction analysis. MiR-1233 was the most overexpressed in the serum of women who later developed sPE. Conclusion Circulating miRNAs deserve further investigation in order to explore their potential role in the pathogenesis of preeclampsia. In particular, miR-1233 might represent a potential marker of early sPE

Acute DOB and PMA Administration Impairs Motor and Sensorimotor Responses in Mice and Causes Hallucinogenic Effects in Adult Zebrafish

The drastic increase in hallucinogenic compounds in illicit drug markets of new psychoactive substances (NPS) is a worldwide threat. Among these, 2, 5-dimetoxy-4-bromo-amphetamine (DOB) and paramethoxyamphetamine (PMA; marketed as "ecstasy") are frequently purchased on the dark web and consumed for recreational purposes during rave/dance parties. In fact, these two substances seem to induce the same effects as MDMA, which could be due to their structural similarities. According to users, DOB and PMA share the same euphoric effects: increasing of the mental state, increasing sociability and empathy. Users also experienced loss of memory, temporal distortion, and paranoia following the repetition of the same thought. The aim of this study was to investigate the effect of the acute systemic administration of DOB and PMA (0.01-30 mg/kg; i.p.) on motor, sensorimotor (visual, acoustic, and tactile), and startle/PPI responses in CD-1 male mice. Moreover, the pro-psychedelic effect of DOB (0.075-2 mg/kg) and PMA (0.0005-0.5 mg/kg) was investigated by using zebrafish as a model. DOB and PMA administration affected spontaneous locomotion and impaired behaviors and startle/PPI responses in mice. In addition, the two compounds promoted hallucinatory states in zebrafish by reducing the hallucinatory score and swimming activity in hallucinogen-like states

Untargeted Metabolic Profiling of 4-Fluoro-Furanylfentanyl and Isobutyrylfentanyl in Mouse Hepatocytes and Urine by Means of LC-HRMS

The diffusion of new psychoactive substances (NPS) is highly dynamic and the available substances change over time, resulting in forensic laboratories becoming highly engaged in NPS control. In order to manage NPS diffusion, efficient and innovative legal responses have been provided by several nations. Metabolic profiling is also part of the analytical fight against NPS, since it allows to identify the biomarkers of drug intake which are needed for the development of suitable analytical methods in biological samples. We have recently reported the characterization of two new analogs of fentanyl, i.e., 4-fluoro-furanylfentanyl (4F-FUF) and isobutyrylfentanyl (iBF), which were found for the first time in Italy in 2019; 4F-FUF was identified for the first time in Europe and was notified to the European Early Warning System. The goal of this study was the characterization of the main metabolites of both drugs by in vitro and in vivo experiments. To this end, incubation with mouse hepatocytes and intraperitoneal administration to mice were carried out. Samples were analyzed by means of liquid chromatography-high resolution mass spectrometry (LC–HRMS), followed by untargeted data evaluation using Compound Discoverer software with a specific workflow, designed for the identification of the whole metabolic pattern, including unexpected metabolites. Twenty metabolites were putatively annotated for 4F-FUF, with the dihydrodiol derivative appearing as the most abundant, whereas 22 metabolites were found for iBF, which was mainly excreted as nor-isobutyrylfentanyl. N-dealkylation of 4F-FUF dihydrodiol and oxidation to carbonyl metabolites for iBF were also major biotransformations. Despite some differences, in general there was a good agreement between in vitro and in vivo samples

New insights into methoxetamine mechanisms of action: Focus on serotonergic 5-HT 2 receptors in pharmacological and behavioral effects in the rat

Methoxetamine (MXE) is a dissociative substance of the arylcyclohexylamine class that has been present on the designer drug market as a ketamine-substitute since 2010. We have previously shown that MXE (i) possesses ketamine-like discriminative and positive rewarding effects in rats, (ii) affects brain processing involved in cognition and emotional responses, (iii) causes long-lasting behavioral abnormalities and neurotoxicity in rats and (iv) induces neurological, sensorimotor and cardiorespiratory alterations in mice. To shed light on the mechanisms through which MXE exerts its effects, we conducted a multidisciplinary study to evaluate the various neurotransmitter systems presumably involved in its actions on the brain. In vivo microdialysis study first showed that a single administration of MXE (0.25 and 0.5 mg/kg, i.v.) is able to significantly alter serotonin levels in the rat medial prefrontal cortex (mPFC) and nucleus accumbens. Then, we observed that blockade of the serotonin 5-HT2 receptors through two selective antagonists, ketanserin (0.1 mg/kg, i.p.) and MDL 100907 (0.03 mg/kg, i.p.), at doses not affecting animals behavior per se, attenuated the facilitatory motor effect and the inhibition on visual sensory responses induced by MXE (3 mg/kg, i.p.) and ketamine (3 mg/kg, i.p.), and prevented MXE-induced reduction of the prepulse inhibition in rats, pointing to the 5-HT2 receptors as a key target for the recently described MXE-induced sensorimotor effects. Finally, in-vitro electrophysiological studies revealed that the GABAergic and glutamatergic systems are also likely involved in the mechanisms through which MXE exerts its central effects since MXE inhibits, in a concentration-dependent manner, NMDA-mediated field postsynaptic potentials and GABA-mediated spontaneous currents. Conversely, MXE failed to alter both the AMPA component of field potentials and presynaptic glutamate release, and seems not to interfere with the endocannabinoid-mediated effects on mPFC GABAergic synapses. Altogether, our results support the notion of MXE as a NMDA receptor antagonist and shed further lights into the central mechanisms of action of this ketamine-substitute by pointing to serotonin 5-HT2 receptors as crucial players in the expression of its sensorimotor altering effects and to the NMDA and GABA receptors as potential further important targets of action

Potential of the zebrafish model for the forensic toxicology screening of NPS: A comparative study of the effects of APINAC and methiopropamine on the behavior of zebrafish larvae and mice

Abstract The increased diffusion of the so-called novel psychoactive substances (NPS) and their continuous change in structure andconceivably activity has led to the need of a rapid screening method to detect their biological effects as early as possible after their appearance in the market. This problem is very felt in forensic pathology and toxicology, so the preclinical study is fundamental in the approach to clinical and autopsy cases of difficult interpretation intoxication. Zebrafish is a high-throughput suitable model to rapidly hypothesize potential aversive or beneficial effects of novel molecules. In the present study, we measured and compared the behavioral responses to two novel neuroactive drugs, namely APINAC, a new cannabimimetic drug, and methiopropamine (MPA), a methamphetamine-like compound, on zebrafish larvae (ZL) and adult mice. By using an innovative statistical approach (general additive models), it was found that the spontaneous locomotor activity was impaired by the two drugs in both species: the disruption extent varied in a dose-dependent and time-dependent manner. Sensorimotor function was also altered: i) the visual object response was reduced in mice treated with APINAC, whereas it was not after exposure to MPA; ii) the visual placing responses were reduced after treatment with both NPS in mice. Furthermore, the visual motor response detected in ZL showed a reduction after treatment with APINAC during light-dark and dark-light transition. The same pattern was found in the MPA exposed groups only at the dark-light transition, while at the transition from light to dark, the individuals showed an increased response. In conclusion, the present study highlighted the impairment of spontaneous motor and sensorimotor behavior induced by MPA and APINAC administration in both species, thus confirming the usefulness of ZL as a model for a rapid behavioural-based drug screening

MAM-2201 acute administration impairs motor, sensorimotor, prepulse inhibition, and memory functions in mice: a comparison with its analogue AM-2201

Rationale1-[(5-fluoropentyl)-1H-indol-3-yl](4-methyl-1-naphthalenyl) methanone (MAM-2201) is a potent synthetic cannabinoid receptor agonist illegally marketed in "spice" products and as "synthacaine" for its psychoactive effects. It is a naphthoyl-indole derivative which differs from its analogue 1-[(5-Fluoropentyl)-1H-indol-3-yl](1-naphthylenyl) methanone (AM-2201) by the presence of a methyl substituent on carbon 4 (C-4) of the naphthoyl moiety. Multiple cases of intoxication and impaired driving have been linked to AM-2201 and MAM-2201 consumption.ObjectivesThis study aims to investigate the in vitro (murine and human cannabinoid receptors) and in vivo (CD-1 male mice) pharmacodynamic activity of MAM-2201 and compare its effects with those induced by its desmethylated analogue, AM-2201.ResultsIn vitro competition binding studies confirmed that MAM-2201 and AM-2201 possess nanomolar affinity for both CD-1 murine and human CB1 and CB2 receptors, with preference for the CB1 receptor. In agreement with the in vitro binding data, in vivo studies showed that MAM-2201 induces visual, acoustic, and tactile impairments that were fully prevented by pretreatment with CB1 receptor antagonist/partial agonist AM-251, indicating a CB1 receptor mediated mechanism of action. Administration of MAM-2201 also altered locomotor activity and PPI responses of mice, pointing out its detrimental effect on motor and sensory gating functions and confirming its potential use liability. MAM-2201 and AM-2201 also caused deficits in short- and long-term working memory.ConclusionThese findings point to the potential public health burden that these synthetic cannabinoids may pose, with particular emphasis on impaired driving and workplace performance

Neuronal dysfunction associated with cholesterol deregulation

Cholesterol metabolism is crucial for cells and, in particular, its biosynthesis in the central nervous system occurs in situ, and its deregulation involves morphological changes that cause functional variations and trigger programmed cell death. The pathogenesis of rare diseases, such as Mevalonate Kinase Deficiency or Smith–Lemli–Opitz Syndrome, arises due to enzymatic defects in the cholesterol metabolic pathways, resulting in a shortage of downstream products. The most severe clinical manifestations of these diseases appear as neurological defects. Expanding the knowledge of this biological mechanism will be useful for identifying potential targets and preventing neuronal damage. Several studies have demonstrated that deregulation of the cholesterol pathway induces mitochondrial dysfunction as the result of respiratory chain damage. We set out to determine whether mitochondrial damage may be prevented by using protective mitochondria-targeted compounds, such as MitoQ, in a neuronal cell line treated with a statin to induce a biochemical block of the cholesterol pathway. Evidence from the literature suggests that mitochondria play a crucial role in the apoptotic mechanism secondary to blocking the cholesterol pathway. Our study shows that MitoQ, administered as a preventive agent, could counteract the cell damage induced by statins in the early stages, but its protective role fades over time

Worsening of the Toxic Effects of (±) Cis -4,4′-DMAR Following Its Co-Administration with (±) Trans -4,4′-DMAR: Neuro-Behavioural, Physiological, Immunohistochemical and Metabolic Studies in Mice

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/4.0/).4,4’-Dimethylaminorex (4,4’-DMAR) is a new synthetic stimulant, and only a little information has been made available so far regarding its pharmaco-toxicological effects. The aim of this study was to investigate the effects of the systemic administration of both the single (±)cis (0.1–60 mg/kg) and (±)trans (30 and 60 mg/kg) stereoisomers and their co-administration (e.g., (±)cis at 1, 10 or 60 mg/kg + (±)trans at 30 mg/kg) in mice. Moreover, we investigated the effect of 4,4′-DMAR on the expression of markers of oxidative/nitrosative stress (8-OHdG, iNOS, NT and NOX2), apoptosis (Smac/DIABLO and NF-κB), and heat shock proteins (HSP27, HSP70, HSP90) in the cerebral cortex. Our study demonstrated that the (±)cis stereoisomer dose-dependently induced psychomotor agitation, sweating, salivation, hyperthermia, stimulated aggression, convulsions and death. Conversely, the (±)trans stereoisomer was ineffective whilst the stereoisomers’ co-administration resulted in a worsening of the toxic (±)cis stereoisomer effects. This trend of responses was confirmed by immunohistochemical analysis on the cortex. Finally, we investigated the potentially toxic effects of stereoisomer co-administration by studying urinary excretion. The excretion study showed that the (±)trans stereoisomer reduced the metabolism of the (±)cis form and increased its amount in the urine, possibly reflecting its increased plasma levels and, therefore, the worsening of its toxicity.Peer reviewedFinal Published versio

Pharmaco-toxicological effects of the novel tryptamine hallucinogen 5-MeO-MiPT on motor, sensorimotor, physiological, and cardiorespiratory parameters in mice—from a human poisoning case to the preclinical evidence

Rationale: The 5-methoxy-N-methyl-N-isopropyltryptamine (5-MeO-MiPT, known online as “Moxy”) is a new psychedelic tryptamine first identified on Italian national territory in 2014. Its hallucinogen effects are broadly well-known; however, only few information is available regarding its pharmaco-toxicological effects. Objectives: Following the seizure of this new psychoactive substances by the Arm of Carabinieri and the occurrence of a human intoxication case, in the current study we had the aim to characterize the in vivo acute effects of systemic administration of 5-MeO-MiPT (0.01–30 mg/kg i.p.) on sensorimotor (visual, acoustic, and overall tactile) responses, thermoregulation, and stimulated motor activity (drag and accelerod test) in CD-1 male mice. We also evaluated variation on sensory gating (PPI, prepulse inhibition; 0.01–10 mg/kg i.p.) and on cardiorespiratory parameters (MouseOx and BP-2000; 30 mg/kg i.p.). Lastly, we investigated the in silico ADMET (absorption, distribution, metabolism, excretion, toxicity) profile of 5-MeO-MiPT compared to 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) and N,N-dimethyltryptamine (DMT). Results: This study demonstrates that 5-MeO-MiPT dose-dependently inhibits sensorimotor and PPI responses and, at high doses, induces impairment of the stimulated motor activity and cardiorespiratory changes in mice. In silico prediction shows that the 5-MeO-MiPT toxicokinetic profile shares similarities with 5-MeO-DIPT and DMT and highlights a cytochrome risk associated with this compound. Conclusions: Consumption of 5-MeO-MiPT can affect the ability to perform activities and pose a risk to human health status, as the correspondence between the effects induced in mice and the symptoms occurred in the intoxication case suggests. However, our findings suggest that 5-MeO-MiPT should not be excluded from research in the psychiatric therapy field

In vitro and in vivo pharmaco-toxicological characterization of Novel Psychoactive Substances (NPS): focus on Novel Synthetic Opioids (NSOs)

The rapid emergence of novel psychoactive substances (NPS) in the global drug market poses a significant threat to public health and presents a challenge to drug policy. Often, there is insufficient information about the adverse clinical effects and social hazards linked to NPS, hindering the development of effective preventive measures and treatment strategies. The widespread dissemination and diversity of New Synthetic Opioids (NSOs) have introduced complexity into recreational NPS markets globally. Given the limited understanding of the pharmaco-toxicological properties of NSOs, my research project seeks to address the gaps in this area. It is a component of a multicenter research project affiliated with the Anti-Drug Policy Department (DPA) of the Presidency of the Council of Ministers. The University of Ferrara's Forensic Toxicology and Translational Medicine Laboratory is a national coordinator for this initiative. The aim of this multidisciplinary project is to investigate the effects of the emerging NSOs in a large spectrum calling different disciplinaries. In particular, the characterization of NSOs in this project involved in silico (Absorption, Distribution, Metabolism, Excretion and Toxicity-ADMET prediction), in vitro (receptor binding and Gi/β-arrestin 2 interaction tests, genotoxicity tests), in vivo (mouse model, zebrafish model) and ex-vivo (histological analysis) approaches. Sex differences in the pharmaco-toxicology of some NSOs was also evaluated. The in silico ADMET and zebrafish model were used to find rapid alternative methods for NSOs screening. As a last approach of my research project is the development of potential therapeutic interventions, I applied Naloxone (opioid receptor antagonist) alone or with Antalarmin (CRF-1 receptor antagonist) to reverse the toxic effects of the studied opioids (particular focus on cardiorespiratory alteration) and evaluate the potential role played by stress in their toxic action. The extensive results obtained through the multidisciplinary approach applied in my PhD project have unveiled substantial pharmaco-toxicological insights and novel mechanisms attributed to NSOs toxicity. Moreover, these findings suggest new rapid screening tools of NSOs. A part of these findings is published in significant scientific journals. Disseminating this data aims to increase awareness of the risks linked to NSOs and facilitate the development of more effective therapeutic strategies for managing NSO-related intoxications.La rapida comparsa di nuove sostanze psicoattive (NSP) nel mercato globale della droga rappresenta una minaccia significativa per la salute pubblica e rappresenta una sfida per le politiche anti-droga. Spesso le informazioni sugli effetti clinici avversi e sui rischi sociali legati alle NSP sono insufficienti, il che ostacola lo sviluppo di misure preventive e strategie di trattamento efficaci. L’ampia diffusione e la diversità dei nuovi oppioidi sintetici (NOS) hanno introdotto una complessità nei mercati delle NSP a livello globale. Data la limitata comprensione delle proprietà farmaco-tossicologiche degli NOS, il mio progetto di ricerca si figura di colmare le lacune in questo settore. Questo studio di Dottorato si inserisce in un progetto di ricerca multicentrico istituito dal Dipartimento Politiche Antidroga (DPA) della Presidenza del Consiglio dei Ministri. Il Laboratorio di Tossicologia Forense del Dipartimento di Medicina Traslazionale dell'Università degli Studi di Ferrara è coordinatore nazionale di questo progetto. Lo scopo di questo studio multidisciplinare è quello di indagare con più tecniche complementari gli effetti dei NOS emergenti sul territorio nazionale ed internazionale. In particolare, la caratterizzazione farmaco-tossicologica dei NOS è stata eseguita mediante studi in silico (previsione di assorbimento, distribuzione, metabolismo, escrezione e tossicità-ADMET), in vitro (binding recettoriale ed interazione Gi/β-arrestina 2, test di genotossicità), in vivo (modello murino, modello di larva di zebrafish) ed approcci ex-vivo (analisi istologica). Sono state anche valutate le differenze farmaco-tossicologiche di alcuni NOS in funzione del sesso del soggetto. Il modello ADMET in silico e il modello zebrafish sono stati utilizzati per trovare metodi sperimentali alternativi rapidi per lo screening dei NOS. Come ultima fase del mio progetto di ricerca è stato studiato un potenziale intervento terapeutico nelle intossicazioni acute da NOS. In particolare, ho studiato gli effetti del Naloxone (antagonista dei recettori oppioidi) da solo e con la co-somministrazione di Antalarmina (antagonista recettore CRF-1) per invertire gli effetti tossici acuti indotti dagli oppioidi studiati (con particolare attenzione all'alterazione cardiorespiratoria) e valutare il potenziale ruolo esercitato dallo stress nella loro azione tossica. Gli estesi risultati ottenuti attraverso l'approccio multidisciplinare applicato nel mio progetto di dottorato hanno permesso di identificare importanti effetti farmaco-tossicologiche e nuovi meccanismi di azione alla base della tossicità dei NOS. Inoltre, questo studio ha permesso di identificare nuovi strumenti di screening rapido per i NOS. Una parte di questi risultati è stata pubblicata su importanti riviste scientifiche. La diffusione di questi dati mira ad aumentare la consapevolezza dei rischi legati ai NOS e a facilitare lo sviluppo di strategie terapeutiche più efficaci per la gestione delle intossicazioni dei NOS