A simplified strategy to assess the cytotoxicity of new psychoactive substances in HepG2 cells using a high content screening assay – Exemplified for nine compounds

Abstract

The study should demonstrate the applicability of a simplified strategy based on a high content screening assay (HCSA) for cytotoxicity testing of new psychoactive substances (NPS) in HepG2 cells. NPS pose a threat for the health care system as usually nothing is known about their (cyto-) toxicity before throwing them onto the drug market. For a straightforward assessment of their cytotoxic potential, a simplified strategy testing six different cytotoxicity indicating parameters within one run by a HCSA was developed. Its applicability should be demonstrated by investigating nine NPS belonging to various chemical classes. Namely, the synthetic cannabinoids 4CN-CUMYL-BINACA and CUMYL-CBMICA, the cathinone derivatives dibutylone, ephylone, and 4-MEAP, the tryptamines 5-MeO-MiPT and 5-MeO-DALT, and the phenylethylamines 25C-NBOMe and 25I-NBOMe. As not only parent compounds may induce cytotoxic effects but also their metabolites, the impact of the CYP-dependent metabolism should also be investigated. HepG2 cells were seeded for 24 h in RPMI medium followed by exposure of the NPS for 48 h in a low and high concentration (7.81 and 125 μM) with or without the unspecific CYP inhibitor 1-amonibenzotriazol (ABT), respectively. Afterwards, cells were loaded for 1 h with a fluorescence dyes cocktail containing 0.8 μM Hoechst 33342, 20 nM TMRM, 1 μM CAL-520, 1 μM TOTO-3. An epifluorescence microscope (BioTek Lionheart FX Automated Microscope) equipped with a 20×/0.45 objective was used for cell plate analysis. The following parameters were monitored in NPS incubations with or without ABT and compared to each other or blank incubations without drug: cell count, nuclear size, nuclear intensity (all Hoechst 33342), mitochondrial membrane potential (TMRM), cytosolic calcium levels (CAL-520), plasma membrane integrity (TOTO-3). A strong cytotoxic potential could be observed for ephylone, CUMYL-CBMICA, and dibutylone, whereas 5-MeO-MiPT showed a moderate potential. 5-MeO-MiPT additionally indicated metabolism-based effects by a decrease of the nuclear size and nuclear intensity at 125 μM in incubations with ABT. As blood concentrations of NPS are often not available and intracellular concentration are usually completely unavailable, the cytotoxicity assessment was based on two different concentrations for the HCSA. However, investigated concentrations were at least in the range of published blood concentrations of dibutylone, ephylone, and 5-MeO-MiPT. Case reports about liver damages have been associated with the intake of the two latter compounds. In turn, the HCSA confirmed the cytotoxic potential of ephylone and 5-MeO-MiPT. Additionally, the cytotoxicity of 5-MeO-MiPT was influenced by its CYP-dependent metabolism. However, it should be mentioned that HepG2 cells are known to have a limited metabolic competence for some CYP enzymes, which may explain that metabolism-based effects could only be observed to a low extend. The simplified HCSA-based strategy allowed to screen NPS from heterogenous chemical classes to get a first assessment on their cytotoxic properties. This information is crucial for a thorough risk assessment of NPS not only for authorities.