Uncovering 4-Cl-α-PPP toxicity and mitigation strategies using Caenorhabditis elegans as a translational model

Background: Synthetic cathinones, commonly referred to as “bath salts”, are a class of New Psychoactive Substances designed to mimic the effects of traditional drugs of abuse. These compounds exert amphetamine-like effects by interacting with dopamine, serotonin, and noradrenaline transporters, leading to a range of adverse outcomes, including neurotoxicity [1, 2]. However, for several emerging synthetic cathinones, such as 4'-chloro-alpha-pyrrolidinopropiophenone (4-Cl-α-PPP), their toxicological profile remains poorly characterized, highlighting the need for further investigation. Additionally, exploring mitigation strategies to counteract their harmful effects may represent a promising approach. Objective: Using C. elegans as a discovery platform, this study aims to characterize the effects of 4-Cl-α-PPP on animal development, lifespan, reproductive behavior, and potential heritable toxicological signatures, as well as to identify novel strategies to mitigate its toxicity. Methods: Synchronized L1-stage animals of the DC19 [bus-5(br19)] strain (~200 per condition) were exposed in liquid medium to increasing concentrations of 4-Cl-α-PPP. After 72 h of incubation in M9 buffer supplemented with OP50 bacteria as a food source [3], survival was assessed by counting live and dead worms [2]. Further experiments were conducted to evaluate the protective effect of N-acetyl-cysteine (NAC; 1 mM) against 4-Cl-α-PPP-induced reductions in survival. Results: Exposure to increasing concentrations of 4-Cl-α-PPP resulted in a concentration-dependent decrease in animal survival. While no significant effects were observed at lower concentrations (≤0.05 mM), a marked reduction in survival was detected at 0.75 mM [survival percentage (mean ± standard deviation): control (0 mM) = 98.47 ± 1.66; 0.75 mM = 18.78 ± 17.97, p<0.01], becoming more pronounced at higher concentrations (≥1.0 mM), with complete lethality observed at 2.5 mM [survival percentage (mean ± standard deviation): 1.0 mM = 0.29 ± 0.87, p<0.0001; 2.5 mM = 0.00 ± 0.00, p<0.0001]. Notably, co-incubation with NAC (1 mM) attenuated the decrease in survival induced by 4-Cl-α-PPP. Additional studies addressing developmental, lifespan, reproductive, and heritable effects are currently ongoing. Conclusions: These findings show that 4-Cl-α-PPP causes a marked, concentration-dependent decrease in animal survival, partially prevented by NAC, indicating that oxidative stress plays a major role in 4-Cl-α-PPP toxicity.