Ouwais Aljabasini

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.

Zebrafish behavioral profiling is increasingly used in environmentally oriented New Approach Methodologies (NAMs) for chemical hazard assessment, including neuroactive chemicals. However, interpreting acoustic/vibrational startle habituation requires understanding how different quantification strategies influence toxicological readouts. In this study, we performed a controlled cross-platform comparison of two common habituation metrics in 5-dpf zebrafish eleutheroembryos following acute chemical exposure. Using embryos from the same spawning and shared stock solutions, we evaluated: (i) a centroid-based workflow (30 fps), quantifying post-stimulus displacement as area under the curve (AUC), and (ii) a high-speed kinematic workflow (1000 fps), classifying individual startle events to calculate %habituation.Eight neuroactive reference chemicals were tested to benchmark both approaches and to improve interpretation of an environmentally relevant neurobehavioral screening endpoint. The high-speed kinematic workflow showed high directional concordance with expected modulation patterns for 6/8 compounds and provided detailed resolution of C-start mechanics. Meanwhile, the centroid-based workflow, a robust and widely accessible standard in the field, reproduced expected directions for 3/8 compounds, capturing robust modulations for specific neuroactive profiles while showing lower sensitivity to other chemicals, such as ketamine, despite their marked effect on habituation kinetics. Notably, results for compounds such as nimodipine showed that both assays are essential for identifying complete loss of the motor response, thereby avoiding misinterpretation of performance deficits as altered plasticity.Overall, our findings show that while both platforms capture behavioral shifts, readout choice substantially influences the sensitivity and interpretation of chemical effects. This study supports fit-for-purpose endpoint selection, strengthening environmental hazard assessment and prioritization.

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.

Synthetic cathinones such as 3,4-methylenedioxypyrovalerone (MDPV) are potent psychostimulants with high abuse potential, yet their systemic toxicity and neurobehavioral effects remain poorly characterized during early development. Using Danio rerio (zebrafish) embryos and larvae, we performed an integrated assessment of the cardiotoxic, behavioral, and molecular effects of MDPV. Acute exposure of 3 days post-fertilization (dpf) embryos produced a marked, concentration-dependent bradycardia and atrioventricular (AV) conduction block, leading to reduced ventricular activity and complete AV dissociation at the highest concentrations (EC₅₀ = 228 µM). Quantitative analysis of ventricular motion revealed a significant decrease in cardiac output (CO) at all tested concentrations and a reduction in ejection fraction (EF) only at 480 µM, while fractional shortening (FS) and stroke volume (SV) remained unchanged, indicating predominant chronotropic and conduction effects with secondary contractile impairment. In 5 dpf larvae, MDPV caused a sustained, concentration-dependent decrease in basal locomotor activity (EC₅₀ = 2.51 µM) but did not affect prepulse inhibition (PPI) of the acoustic startle response (ASR), unlike dextroamphetamine, which enhanced PPI via dopaminergic D₂ receptor activation. Short-term (2 h) exposure of 3 dpf embryos to 0.4–400 µM MDPV induced transcriptional changes in dopaminergic and stress-responsive genes, whereas expression of major repolarizing potassium channel genes (kcnh6a and kcnq1) remained unaltered. Collectively, these results demonstrate that MDPV exerts potent negative chronotropic effects likely through direct functional interference with cardiac repolarization, while neurobehavioral effects occur at concentrations nearly two orders of magnitude lower than cardiotoxic thresholds, supporting zebrafish as a predictive model for the integrative assessment of psychostimulant toxicity.

Synthetic cathinones such as 3,4-methylenedioxypyrovalerone (MDPV) are potent psychostimulants with high abuse potential, yet their systemic toxicity and neurobehavioral effects remain poorly characterized during early development. Using Danio rerio (zebrafish) embryos and larvae, we performed an integrated assessment of the cardiotoxic, behavioral, and molecular effects of MDPV. Acute exposure of 3 days post-fertilization (dpf) embryos produced a marked, concentration-dependent bradycardia and atrioventricular (AV) conduction block, leading to reduced ventricular activity and complete AV dissociation at the highest concentrations (EC₅₀ = 228 µM). Quantitative analysis of ventricular motion revealed a significant decrease in cardiac output (CO) at all tested concentrations and a reduction in ejection fraction (EF) only at 480 µM, while fractional shortening (FS) and stroke volume (SV) remained unchanged, indicating predominant chronotropic and conduction effects with secondary contractile impairment. In 5 dpf larvae, MDPV caused a sustained, concentration-dependent decrease in basal locomotor activity (EC₅₀ = 2.51 µM) but did not affect prepulse inhibition (PPI) of the acoustic startle response (ASR), unlike dextroamphetamine, which enhanced PPI via dopaminergic D₂ receptor activation. Short-term (2 h) exposure of 3 dpf embryos to 0.4–400 µM MDPV induced transcriptional changes in dopaminergic and stress-responsive genes, whereas expression of major repolarizing potassium channel genes (kcnh6a and kcnq1) remained unaltered. Collectively, these results demonstrate that MDPV exerts potent negative chronotropic effects likely through direct functional interference with cardiac repolarization, while neurobehavioral effects occur at concentrations nearly two orders of magnitude lower than cardiotoxic thresholds, supporting zebrafish as a predictive model for the integrative assessment of psychostimulant toxicity.