Novel Psychoactive Substances (NPS) Footprint by Cytokine Release and Volatilome Profile: Advances in Clinical Toxicology

The rising widespread availability of NPS has heightened interest in clinical toxicology. NPS are substances that mimic the effects of traditional drugs but whose pharmacological and side effects are poorly known, giving birth to NPS monitoring centres (Early Warning System) aimed to rapidly detect, assess, and respond to public social-health threats. NPS exhibit mostly neurotoxic effects on monoamine and glutamatergic systems. Studies show they affect cytokine and chemokine (CKs) expression and significantly altering cell metabolism. Our aim is to investigate the possible effects of selected NPS on in vitro CKs release, as marker of inflammation, and on volatilome profile, as specific metabolite signature. Whole blood from 8 healthy volunteers (1:1 F/M) were cultured for 48h in presence and absence of selected NPS (35μM): PCA, α- PHP, 3-CMC, Butyrylfentanyl, 5F-AKB-48. CKs levels were detected in the medium via Luminex technology. Volatile metabolites were characterized using an HS-SPME-GC×GC mass spectrometry. Coupled t-test shows PCA, an amphetamine-like stimulant, decreases IP10 levels (P=0.011), suggesting a downregulation of the inflammatory response IFNγ-mediated. Conversely, αPHP, a synthetic cathinone, significantly increases IL8 (P=0.001), IP10 (P=0.008) and MCP1 (P=0.031) levels, potentially triggering excessive inflammation and tissue damage. Accordingly, the cathinone 3CMC increases IL8 levels (P=0.048). Butyrylfentanyl, unlike other opioids, increases IL8 levels (P=0.02). As previously reported, the cannabinoid 5FAKB48 downregulates the anti-inflammatory cytokine IL10 (P=0.041). Finally, to trace a possible blood-volatile NPS-footprint, the high-separation and identification capabilities of the GC×GC-TOF MS system allowed us to detect a profile of more than 700 specific metabolites changing after treatment. Our results suggest NPS impact CKs release and volatilome profile, raising awareness in NPS toxicology. In particular, volatilome assessment approach represents a promising tool for studying the in vivo NPS effects on cell biology and improving toxicological screening. It also enables personalized multi-OMIC analyses to assess individual dose-dependent and time-dependent response, also considering the different genomic and epigenomic backgrounds.