Mitochondrial dysfunction induced by different organochalchogens is mediated by thiol oxidation and is not dependent of the classical mitochondrial permeability transition pore opening

Abstract

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.

FIG. 1.

Glutathione peroxidase cycle of ebselen. GSH represent a glutathione molecule. Different thiol groups (including that of mitochondrial membranes) could replace GSH in this figure.

FIG. 2.

Thiol oxidase futile cycle of diorganochalcogens (X = Se or Te). RSH can represent a low molecular endogenous molecule (glutathione, cysteine, etc.) or thiol-containing proteins (δ-ALA-D, Na⁺, K⁺-ATPase, mitochondrial proteins).

FIG. 3.

Effects of organochalchogens on ΔΨm and mitochondrial swelling. Isolated rat liver mitochondria (0.5 mg) were incubated in standard medium (see composition in the Materials and Methods), and the ΔΨm or swelling was monitored as described in the Material and Methods. (A) Effect of Ebs (1–5μM) on ΔΨm and (B) effect of Ebs (1–5μM) on mitochondrial swelling. (C) Effect of (PhSe)₂ (10–40μM) on ΔΨm and (D) effect of (PhSe)₂ (10–40μM) on mitochondrial swelling. The mitochondria (0.5 mg/ml), organochalchogens, or 2,4 DNP (100μM) were added where indicated by arrows. The traces are representative of three to five independent experiments.

FIG. 4.

Effect of organochalchogens on mitochondrial NAD(P)H oxidation. Isolated rat liver mitochondria (0.5 mg) were incubated in standard medium, and the mitochondrial NAD(P)H oxidation was monitored as described in the Material and Methods. The Ebs, (PhSe)₂, or Ca²⁺/Pi were added where indicated by the arrow. The traces are representative of three independent experiments.

FIG. 5.

Effect of DTT on organochalchogens-induced mitochondrial dysfunction. Effect of DTT (10μM) on Ebs (5μM)-induced mitochondrial depolarization (A) or swelling (B). Effect of DTT (80μM) on (PhSe)₂ (20μM)-induced mitochondrial depolarization (C) or swelling (D). The mitochondria (0.5 mg/ml), organochalchogens, or 2,4 DNP (100μM) were added where indicated by arrows in medium containing DTT. The traces are representative of three independent experiments.

FIG. 6.

Effect of organochalchogens on ROS generation. Isolated rat liver mitochondria (0.5 mg) were incubated in standard medium (see standard medium in the Materials and Methods) containing 1μM H₂-DCFDA. Ebs (5μM), (PhSe)₂ (20μM), or 2,4 DNP (100μM) were added, and the H₂-DCFDA fluorescence was monitored during 10 min. The delta of fluorescence (final fluorescence – initial fluorescence) was used to do the calculations. The data are expressed as percent of control. Data represent the mean ± SE of three separate determinations performed in duplicates. *p < 0.05 compared with control by Duncan's multiple range test.

FIG. 7.

Effect of organochalchogens on oxygen consumption. Isolated rat liver mitochondria (0.15 mg/ml) were incubated in standard medium, and the oxygen consumption was measured as described in the Materials and Methods. Effect of Ebs (5μM) (A) and (PhSe)₂ (20μM) (B) on mitochondrial oxygen consumption. The arrows indicate sequential additions of Mito, 0.15 mg/ml mitochondria; Subst, 10mM succinate; Ebs, 5μM ebselen (A); 20μM (PhSe)₂ (B); ADP, 0.2mM ADP; and FCCP, 5μM carbonylcyanide p-trifluoromethoxyphenylhydrazone. Similar results were obtained with at least two independent mitochondrial preparations.

FIG. 8.

Effect of organochalchogens on protein aggregates formation. Representative SDS-polyacrylamide slab gel electrophoresis of membrane protein from rat liver mitochondria. In each lane, samples of 40 μg of protein were applied to a 12% acrylamide running gel after 1 h of incubation under conditions described in the Material and Methods in the absence (A) or presence (B) of DTT. (C) The mitochondria were incubated with organochalchogens in conditions described in (A) and after DTT was added for 10 min (10μM for Ebs and 80μM for (PhSe)₂). Lane 1, molecular standard weight; Lane 2, control (DMSO); Lane 3, (PhSe)₂ (20μM); and Lane 4, Ebs (5μM). Gels are representative from three to four different experiments.