Insights into benzo fury toxicity: 5-(2-aminopropyl)benzofuran and 6-(2-aminopropyl)benzofuran disrupt oxidative, energetic and mitochondrial homeostasis of hepatocytes in vitro

Benzofurans (‘benzo fury’) are synthetic phenethylamines used as alternatives to amphetamines, with a few case reports describing intoxications and barely any information regarding their mechanisms of toxicity. Taking into consideration that the majority of these compounds are orally consumed, the liver stands out as a key target organ for their toxicity, due to the first passage effect. This project was set out to determine the hepatotoxic effects of 6-(aminopropyl)benzofuran (6-APB) and 5-(2-aminopropyl)benzofuran (5-ABP). Human HepaRG and HepG2 cells, and primary rat hepatocytes (PRH) were exposed for 24h to 5-APB (PRH: 0.0377–10.0 mM; HepaRG cells: 0.110–6.500 mM; HepG2 cells: 0.2926–20 mM) and 6-APB (PRH:0.0377–23 mM; HepaRG cells: 1.171–14 mM; HepG2 cells: 0.192–30 mM). PRH were also co-exposed with several cytochrome P450 inhibitors such as metyrapone (CYP2E1), quinidine (CYP2D6), ketoconazole (CYP3A4), and 1-aminobenzotriazole (general P450 inhibitor), to determine the influence of metabolism on benzo fury toxicity. Viability was assessed by the MTT assay and data were fitted to the Logit model. The appraisal of specific cytotoxicity endpoints was carried out in PRH, testing each drug at the respective EC20, EC40, EC50, and EC70 (based on data from MTT assay). For all in vitro models, both benzofurans induced concentration-dependent effects, with PRH displaying the highest sensitivity to 5-APB and 6-APB toxicities. 5-APB was proven to be more toxic (EC50 0.96 mM in PRH; 2.62 mM in HepaRG cells; 3.79 mM in HepG2 cells) than 6-APB (EC50 1.94 mM in PRH; 6.02 mM in HepaRG cells; and 8.18 mM in HepG2 cells). The co-exposure of benzo fury and CYP inhibitors led to a shift to the right of cell death curves, thus indicating lower toxicity when CYP450 metabolism is inhibited. 5-APB and 6-APB led to an increase in the production of reactive oxygen species at the highest tested concentrations, and both drugs elicited concentration-dependent depletion of GSH and ATP, as well as a concentration-dependent increase in LDH leakage, mitochondrial membrane potential, and caspase-3, -9, and -8 activities. Overall, the in vitro hepatotoxicity of 5-APB and 6-APB appears to be triggered by extrinsic and intrinsic apoptotic mechanisms, following disruption of cellular oxidative and energetic homeostasis. The results also point towards a contribution of metabolism in 6-APB and 5-APB toxicity. The data here reported helps to increase the general knowledge regarding novel psychoactive substances, which will contribute to better legislation and better health care regarding these compounds.