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.

中文翻译：

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新兴俱乐部药物：在肝细胞模型中，5-（2-氨基丙基）苯并呋喃（5-APB）比其异构体6-（2-氨基丙基）苯并呋喃（6-APB）更具毒性。

新的苯乙胺衍生物是最常滥用的新型精神活性物质。它们是合成的，可以代替经典的两性兴奋剂上市销售，没有经过先前的安全性测试。我们的研究旨在确定被滥用为“合法高位”的两种苯并呋喃[6-（2-氨基丙基）苯并呋喃（6-APB）和5-（2-氨基丙基）苯并呋喃（5-APB）]的体外肝毒性。 。在人类肝癌HepaRG细胞暴露24小时后，通过3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化物（MTT）还原试验评估细胞活力（EC50 2.62 mM 5-APB ； 6.02 mM 6-APB），HepG2细胞（EC50 3.79 mM 5-APB； 8.18 mM 6-APB）和原代大鼠肝细胞（EC50 964μM5-APB； 1.94 mM 6-APB）。最敏感的体外模型，原代肝细胞的共孵育，CYP450抑制剂与CYP3A4共同发挥代谢作用，尤其是通过CYP3A4代谢时，对两种苯并呋喃均具有毒性作用。此外，6-APB和5-APB浓度依赖性地增加了氧化应激（显着增加了反应性物种和氧化型谷胱甘肽，并降低了降低的谷胱甘肽水平）和不稳定的线粒体稳态，从而破坏了线粒体膜电位并降低了细胞内ATP。对细胞死亡机制的评估显示，caspase-8，-9和-3的激活增加，并且核形态学变化与凋亡一致。在高于2 mM的浓度下，坏死占了上风。两种药物都观察到典型的自噬的酸性囊泡细胞器的浓度依赖性形成。总体而言，5-APB的肝毒性比其6-异构体更高。