Universidade Federal do Pampa, Uruguaiana, Rio Grande do Sul, Brazil.
Toxicol Sci. 2010 Sep;117(1):133-43. doi: 10.1093/toxsci/kfq185. Epub 2010 Jun 23.
Ebselen (Ebs) and diphenyl diselenide [(PhSe)(2)] readily oxidize thiol groups. Here we studied mitochondrial swelling changes in mitochondrial potential (Deltapsim), NAD(P)H oxidation, reactive oxygen species production, protein aggregate formation, and oxygen consumption as ending points of their in vitro toxicity. Specifically, we tested the hypothesis that organochalchogens toxicity could be associated with mitochondrial dysfunction via oxidation of vicinal thiol groups that are known to be involved in the regulation of mitochondrial permeability (Petronilli et al. J. Biol. Chem., 269; 16638; 1994). Furthermore, we investigated the possible mechanism(s) by which these organochalchogens could disrupt liver mitochondrial function. Ebs and (PhSe)(2) caused mitochondrial depolarization and swelling in a concentration-dependent manner. Furthermore, both organochalchogens caused rapid oxidation of the mitochondrial pyridine nucleotides (NAD(P)H) pool, likely reflecting the consequence and not the cause of increased mitochondrial permeability (Costantini, P., Chernyak, B. V., Petronilli, V., and Bernardi, P. (1996). Modulation of the mitochondrial permeability transition pore (PTP) by pyridine nucleotides and dithiol oxidation at two separate sites. J. Biol. Chem. 271, 6746-6751). The organochalchogens-induced mitochondrial dysfunction was prevented by the reducing agent dithiothreitol (DTT). Ebs- and (PhSe)(2)-induced mitochondrial depolarization and swelling were unchanged by ruthenium red (4microM), butylated hydroxytoluene (2.5microM), or cyclosporine A (1microM). N-ethylmaleimide enhanced the organochalchogens-induced mitochondrial depolarization, without affecting the magnitude of the swelling response. In contrast, iodoacetic acid did not modify the effects of Ebs or (PhSe)(2) on the mitochondria. Additionally, Ebs and (PhSe)(2) decreased the basal 2' 7' dichlorofluorescin diacetate (H(2)-DCFDA) oxidation and oxygen consumption rate in state 3 and increased it during the state 4 of oxidative phosphorylation and induced the formation of protein aggregates, which were prevented by DTT. However, DTT failed to reverse the formation of protein aggregates, when it was added after a preincubation of liver mitochondria with Ebs or (PhSe)(2). Similarly, DTT did not reverse the Ebs- or (PhSe)(2)-induced Deltapsim collapse or swelling, when it was added after a preincubation period of mitochondria with chalcogenides. These results show that Ebs and (PhSe)(2) can effectively induce mitochondrial dysfunction and suggest that effects of these compounds are associated with mitochondrial thiol groups oxidation. The inability of cyclosporine A to reverse the Ebs- and (PhSe)(2)-induced mitochondrial effects suggests that the redox-regulated mitochondrial permeability transition (MPT) pore was mechanistically regulated in a manner that is distinct from the classical MPT pore.
依布硒啉（Ebs）和二苯二硒醚[(PhSe)2]很容易氧化硫醇基团。在这里，我们研究了线粒体肿胀变化在线粒体膜电位（Δψm）、NAD(P)H 氧化、活性氧物质产生、蛋白质聚集体形成和耗氧量方面作为它们体外毒性的终点。具体来说，我们测试了这样一个假设，即有机硒化合物的毒性可能与线粒体功能障碍有关，通过氧化已知参与调节线粒体通透性的毗邻硫醇基团（Petronilli 等人，J. Biol. Chem.，269;16638;1994）。此外，我们还研究了这些有机硒化合物可能破坏肝线粒体功能的可能机制。Ebs 和 (PhSe)2 以浓度依赖的方式引起线粒体去极化和肿胀。此外，这两种有机硒化合物都导致线粒体吡啶核苷酸（NAD(P)H）池的快速氧化，这可能反映了线粒体通透性增加的后果，而不是原因（Costantini，P.，Chernyak，B.V.，Petronilli，V.和 Bernardi，P.（1996）。通过在两个不同的位点上的吡啶核苷酸和二硫醇氧化来调节线粒体通透性转换孔（MPTP）。J. Biol. Chem.，271，6746-6751）。还原剂二硫苏糖醇（DTT）可以防止有机硒化合物引起的线粒体功能障碍。Ebs-和（PhSe）2-诱导的线粒体去极化和肿胀不受钌红（4μM）、叔丁基对苯二酚（2.5μM）或环孢菌素 A（1μM）的影响。N-乙基马来酰亚胺增强了有机硒化合物引起的线粒体去极化，而不影响肿胀反应的幅度。相比之下，碘乙酸并没有改变 Ebs 或（PhSe）2 对线粒体的影响。此外，Ebs 和（PhSe）2 降低了基础 2'7'二氯荧光素二乙酸（H（2）-DCFDA）氧化和氧消耗率在状态 3 并在氧化磷酸化的状态 4 增加它，并诱导蛋白质聚集体的形成，这是由 DTT 预防的。然而，当 DTT 在与 Ebs 或（PhSe）2 预孵育后添加到肝线粒体中时，它未能逆转蛋白质聚集体的形成。同样，当 DTT 在与硫属化合物预孵育后添加到线粒体中时，它也未能逆转 Ebs 或（PhSe）2 诱导的Δψm 崩溃或肿胀。这些结果表明，Ebs 和（PhSe）2 可以有效地诱导线粒体功能障碍，并表明这些化合物的作用与线粒体硫醇基团的氧化有关。环孢菌素 A 不能逆转 Ebs 和（PhSe）2 诱导的线粒体作用表明，氧化还原调节的线粒体通透性转换（MPT）孔以与经典 MPT 孔不同的方式在机制上受到调节。
