Emerging club drugs: 5-(2-aminopropyl)benzofuran (5-APB) is more toxic than its isomer 6-(2-aminopropyl)benzofuran (6-APB) in hepatocyte cellular models

Abstract

New phenylethylamine derivatives are among the most commonly abused new psychoactive substances. They are synthesized and marketed in lieu of classical amphetaminic stimulants, with no previous safety testing. Our study aimed to determine the in vitro hepatotoxicity of two benzofurans [6-(2-aminopropyl)benzofuran (6-APB) and 5-(2-aminopropyl)benzofuran (5-APB)] that have been misused as ‘legal highs’. Cellular viability was assessed through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, following 24-h drug exposure of human hepatoma HepaRG cells (EC₅₀ 2.62 mM 5-APB; 6.02 mM 6-APB), HepG2 cells (EC₅₀ 3.79 mM 5-APB; 8.18 mM 6-APB) and primary rat hepatocytes (EC₅₀ 964 μM 5-APB; 1.94 mM 6-APB). Co-incubation of primary hepatocytes, the most sensitive in vitro model, with CYP450 inhibitors revealed a role of metabolism, in particular by CYP3A4, in the toxic effects of both benzofurans. Also, 6-APB and 5-APB concentration-dependently enhanced oxidative stress (significantly increased reactive species and oxidized glutathione, and decreased reduced glutathione levels) and unsettled mitochondrial homeostasis, with disruption of mitochondrial membrane potential and decline of intracellular ATP. Evaluation of cell death mechanisms showed increased caspase-8, -9, and -3 activation, and nuclear morphological changes consistent with apoptosis; at concentrations higher than 2 mM, however, necrosis prevailed. Concentration-dependent formation of acidic vesicular organelles typical of autophagy was also observed for both drugs. Overall, 5-APB displayed higher hepatotoxicity than its 6-isomer. Our findings provide new insights into the potential hepatotoxicity of these so-called ‘safe drugs’ and highlight the putative risks associated with their use as psychostimulants.

Key points

• The isomer 5-APB is more hepatotoxic than 6-APB.

• At biologically relevant concentrations, CYP2D6 and 3A4 contribute to the toxification of psychoactive 5-APB and 6-APB. CYP2E1 seems also implicated in 5-APB bioactivation.

• Both benzofury concentration-dependently increase oxidative species, disturb mitochondrial potential homeostasis and energetic levels.

• 5-APB and 6-APB stimulate autophagy and apoptosis at low in vitro concentrations.