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.

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.

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.

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 calculat%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 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 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.