Demetrio Raldúa

Octodrine (2-amino-6-methylheptane; DMHA) is a sympathomimetic stimulant detected in sports-performance and weight-loss supplements despite limited toxicological characterization and regulatory concerns. Here, we used 7-day-post-fertilization zebrafish larvae to characterize the acute cardiac, neurobehavioral, and transcriptional effects of octodrine after 2 h of exposure. Cardiac responses were biphasic: 5 µM octodrine significantly increased atrial and ventricular rates, whereas concentrations ≥ 150 µM produced progressive cardiac depression and higher concentrations disrupted atrioventricular conduction. At 450 µM, all larvae showed complete absence of detectable cardiac activity; mortality was not independently assessed. Modelling of the inhibitory phase yielded IC₅₀ values of 195.6 µM for atrial rate and 188.8 µM for ventricular rate. At substantially lower concentrations, octodrine significantly reduced spontaneous locomotor activity. After Holm correction across all 24 planned time-by-treatment comparisons, a transient effect remained detectable at 50 nM, with more sustained hypoactivity at 0.5 and 5 µM across most of the 2 h exposure period. At the transcriptional level, 3.5 µM octodrine significantly reduced fosab and egr1 expression, whereas fkbp5 was induced at selected concentrations; npas4a showed a significant overall treatment effect but no dose-specific comparison remained significant after blocked analysis and multiplicity correction. nr4a1 was significantly downregulated at 350 µM, whereas th1 and kcnh2a expression was not significantly altered. Together, these findings show that neurobehavioral/transcriptional responses and pronounced cardiac dysfunction occurred over different concentration ranges under the conditions tested. The results provide an initial in vivo hazard characterization of octodrine and support further investigation of its neuropharmacological mechanisms, internal exposure, and cardiovascular safety.

Octodrine (2-amino-6-methylheptane; DMHA) is a sympathomimetic stimulant detected in sports-performance and weight-loss supplements despite limited toxicological characterization and regulatory concerns. Here, we used 7-day-post-fertilization zebrafish larvae to characterize the acute cardiac, neurobehavioral, and transcriptional effects of octodrine after 2 h of exposure. Cardiac responses were biphasic: 5 µM octodrine significantly increased atrial and ventricular rates, whereas concentrations ≥150 µM produced progressive cardiac depression and higher concentrations disrupted atrioventricular conduction. Exposure to 450 µM resulted in 100% lethality. Nonlinear concentration–response analysis yielded EC50 values of 194.8 µM for atrial rate and 187.9 µM for ventricular rate. At substantially lower concentrations, octodrine significantly reduced spontaneous locomotor activity, with effects detectable at 50 nM and more sustained hypoactivity at 0.5 and 5 µM across most of the 2 h exposure period. At the transcriptional level, exposure to 3.5 µM suppressed several neuronal activity–responsive genes, including fosab, egr1, and npas4a, while nr4a1 was downregulated at higher concentrations. The stress-responsive glucocorticoid target fkbp5 was induced at selected concentrations, whereas th1 and kcnh2a expression was not significantly altered. Together, these findings reveal a marked separation between low-concentration neurobehavioral and transcriptional effects and overt cardiac toxicity at substantially higher concentrations. The results provide an initial in vivo toxicological profile of octodrine and support further investigation of its neuropharmacological mechanisms, internal exposure, and cardiovascular safety.

Background/Objectives: Synthetic cathinones are a rapidly evolving class of new psychoactive substances whose structural diversity complicates toxicological risk assessment. Methylenedioxy cathinones occupy a pharmacological space between MDMA-like entactogens and more dopaminergic stimulant cathinones, but their direct cardiac liabilities remain poorly characterized. This study aimed to compare the cardiotoxic and neurobehavioral profiles of methylone, butylone, pentylone and their N,N-dimethyl analogues, and to determine how alkyl-chain extension and terminal amine substitution shape functional toxicity. Methods: Wild-type short-fin zebrafish (Danio rerio) embryos were used as a multiparametric New Approach Methodology. Cardiac rhythmicity was assessed in 3 days post-fertilization embryos after acute exposure to methylone, butylone, pentylone, dimethylone, dibutylone, dipentylone, dihexylone and diheptylone by high-speed video microscopy and dynamic pixel-based analysis, focusing on atrial chronotropy and atrioventricular conduction. Basal locomotor activity was evaluated in 5 days post-fertilization eleutheroembryos over 120 min using automated video tracking. Results: Negative chronotropy increased with alkyl-chain extension, with the monoalkyl subset following the rank order methylone < butylone < pentylone. Among dialkyl analogues, dihexylone and, especially, diheptylone produced the strongest atrial-rate inhibition. AV conduction impairment was more heterogeneous but became prominent among higher-liability analogues, with diheptylone showing the lowest AV-block midpoint descriptor and complete lethality at 1000 µM. Locomotor profiling revealed predominantly hypoactive phenotypes, with sustained late-phase inhibition especially for dipentylone, dihexylone and diheptylone. Conclusions: Alkyl-chain extension and terminal amine substitution shaped cardiac and neurobehavioral toxicity in a structure-dependent manner. The zebrafish workflow provides a structure-oriented framework for prioritizing emerging methylenedioxy cathinones with comparatively higher functional cardiac liability.

Synthetic cathinones are a class of new psychoactive substances (NPS) whose structural diversity and rapid evolution complicate toxicological risk assessment. Methylenedioxy cathinones, including methylone, butylone, pentylone and their N,N-dimethyl analogues, occupy a pharmacological space between MDMA-like entactogens and more dopaminergic stimulant cathinones. Although their monoamine transporter profiles and psychostimulant effects have been partially characterized, their direct cardiac liabilities remain poorly understood. Here, we used zebrafish embryos as a multiparametric New Approach Methodology to compare the cardiotoxic and neurobehavioral profiles of methylone, butylone, pentylone, dimethylone, dibutylone, dipentylone, dihexylone and diheptylone. Cardiac rhythmicity was assessed after acute exposure by high-speed video microscopy and dynamic pixel-based analysis, focusing on atrial chronotropy and atrioventricular (AV) conduction. Negative chronotropy increased with alkyl-chain extension, with the monoalkyl subset following the rank order methylone < butylone < pentylone. Among dialkyl analogues, dihexylone and especially diheptylone produced the strongest atrial rate inhibition. AV conduction impairment was more heterogeneous but became prominent among the higher-liability analogues, with diheptylone showing the lowest AV-block EC50 and complete lethality at 1000 µM. Time-resolved locomotor profiling revealed predominantly hypoactive phenotypes, with the most sustained late-phase inhibition observed for dipentylone, dihexylone and diheptylone. Integration of locomotor and cardiac endpoints showed that locomotor effects generally occurred below concentrations producing severe AV conduction impairment, although endpoint separation varied across analogues. Overall, this study provides a structure-oriented zebrafish framework for prioritizing emerging methylenedioxy cathinones with comparatively higher functional cardiac liability.

The rapid emergence of New Psychoactive Substances (NPS), particularly pyrrolidinophenone derivatives, poses a significant challenge for public health and forensic toxicology. While their neuropharmacological profiles as dopamine transporter inhibitors are well-documented, their cardiac toxicity remains poorly understood. This study employs a multiparametric New Approach Methodology (NAM) using zebrafish embryos to integrate neurobehavioral and cardiotoxic endpoints for comparative hazard prioritization. We evaluated nine pyrrolidine-containing cathinones, including α-PVP, MDPV, α-PiHP, MDPiHP, α-D2PV, 3-Cl-, 4-Cl-, and 3,4-Cl-α-PVP, and 4-F-3-Me-α-PVP, on locomotor activity and cardiac rhythmicity using high-speed video microscopy and dynamic pixel analysis. Across the series, compounds induced concentration-dependent negative chronotropy and, in most cases, locomotor suppression. Crucially, we identified a functional dissociation between atrial rate control and atrioventricular (AV) conduction. The 3,4-dichloro substitution (3,4-Cl-α-PVP) was the most potent inducer of negative chronotropy (EC₅₀ = 52.6 μM), whereas 4-Cl-α-PVP exhibited a distinct pro-arrhythmic liability, increasing the incidence of 2:1 AV block. Time-course locomotor profiling indicated that α-PVP and chlorinated analogs were among the most potent behavioral modifiers. Using a Functional Safety Index (AV block EC₅₀/locomotor EC₅₀-like), we show that most compounds exhibit wide separations between neurobehavioral inhibition and severe conduction impairment, while specific substitutions, particularly para-chlorination, are associated with comparatively reduced functional separation between these endpoints within the assay. Overall, these data demonstrate that subtle structural changes within the pyrrolidinophenone scaffold can shape distinct arrhythmic phenotypes and functional safety profiles, supporting zebrafish-based integrated screening as a rapid platform for prioritizing emerging synthetic cathinones with comparatively higher cardiac liability within this experimental framework.

The rapid emergence of New Psychoactive Substances (NPS), particularly pyrrolidinophenone derivatives, poses a significant challenge for public health and forensic toxicology. While their neuropharmacological profiles as dopamine transporter inhibitors are well-documented, their cardiac toxicity remains poorly understood. This study employs a high-resolution New Approach Methodology (NAM) using zebrafish embryos to integrate neurobehavioral and cardiotoxic endpoints for comparative hazard prioritization. We evaluated nine pyrrolidine-containing cathinones, including α-PVP, MDPV, α-PiHP, MDPiHP, α-D2PV, 3-Cl-, 4-Cl-, and 3,4-Cl-α-PVP, as well as 4-F-3-Me-α-PVP, on locomotor activity and cardiac rhythmicity in zebrafish embryos using high-speed video microscopy and dynamic pixel analysis.. Across the series, compounds induced concentration-dependent negative chronotropy and, in most cases, locomotor suppression. Crucially, we identified a functional dissociation between atrial rate control and atrioventricular (AV) conduction. The 3,4-dichloro substitution (3,4-Cl-α-PVP) was the most potent inducer of negative chronotropy (EC₅₀ = 52.6 μM), whereas 4-Cl-α-PVP exhibited a distinct pro-arrhythmic liability, increasing the incidence of 2:1 AV block. Time-course locomotor profiling indicated that α-PVP and chlorinated analogs were among the most potent behavioral modifiers. Using a Functional Safety Index (AV block EC₅₀ / locomotor EC₅₀-like), we show that most compounds exhibit wide separations between neurobehavioral inhibition and severe conduction impairment, while specific substitutions—particularly para-chlorination—are associated with comparatively reduced safety margins. Overall, these data demonstrate that subtle structural changes within the pyrrolidinophenone scaffold can shape distinct arrhythmic phenotypes and functional safety profiles, supporting zebrafish-based integrated screening as a rapid platform for prioritizing emerging synthetic cathinones with disproportionate cardiac risk.

Environmental exposure to endocrine disruptors such as Tributyltin (TBT) has been established as a serious threat causing neurological impairment, reproduction dysfunction and lipid dysregulation. However, the molecular mechanisms underlying TBT-induced toxicity, particularly its capacity to disrupt epigenetic programming during critical developmental windows, remain poorly understood. State-of-the-art multi-omic approaches have emerged to consider the complexity of the different omic systems, crucial for a better characterization of the mechanisms of action of pollutants. In the present study, a novel multi-omic analysis integrating epigenomics, transcriptomics, and metabolomics was conducted on zebrafish eleutheroembryos exposed from 2 to 5 days post-fertilization to two non-toxic TBT concentrations (3 and 30 nM). To capture not only the toxic mechanisms but also multifaceted molecular interactions from gene to phenotype, multi-omics results were complemented by behavioural and neurotransmitter content analyses at the end of the exposures and after a 3-day clearance period. TBT induced the appearance of 5 280 differentially methylated regions, with significant enrichment in intergenic, CpG islands, and promoter regions. Our results also revealed that altered differentially methylated regions were involved in key biological processes, including nervous system development, synaptic signaling, and muscle structure development. Multi-omic analysis identified significant alterations in neurotransmitter signaling pathways, which were then confirmed through neurotransmitter content analysis. TBT also affected responses at the organismal level, particularly impairing cognitive performance. Altogether, the integration of multi-omic strategies demonstrated the potential to revolutionize the future of toxicology by enhancing the understanding of environmental pollutant effects, paving the way for more refined risk assessment and regulatory frameworks.