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Permeabilization of the mitochondrial membrane has been extensively associated with necrotic and apoptotic cell death. Similarly to what had been previously observed for B16F10-Nex2 murine melanoma cells, PdC (palladacycle compounds) obtained from the reaction of dmpa (N,N-dimethyl-1-phenethylamine) with the dppe [1,2-ethanebis(diphenylphosphine)] were able to induce apoptosis in HTC (hepatoma, tissue culture) cells, presenting anticancer activity in vitro. To elucidate cell site-specific actions of dmpa:dppe that could respond to the induction of apoptosis in cancer cells in the present study, we investigated the effects of PdC on isolated RLM (rat liver mitochondria). Our results showed that these palladacycles are able to induce a Ca2+-independent mitochondrial swelling that was not inhibited by ADP, Mg2+ and antioxidants. However, the PdC-induced mitochondrial permeabilization was partially prevented by pre-incubation with CsA (cyclosporin A), NEM (N-ethylmaleimide) and bongkreic acid and totally prevented by DTT (dithiothreitol). A decrease in the content of reduced thiol groups of the mitochondrial membrane proteins was also observed, as well as the presence of membrane protein aggregates in SDS/PAGE without lipid and GSH oxidation. FTIR (Fourier-transform IR) analysis of PdC-treated RLM demonstrated the formation of disulfide bonds between critical thiols in mitochondrial membrane proteins. Associated with the mitochondrial permeabilization, PdC also induced the release of cytochrome c, which is sensitive to inhibition by DTT. Besides the contribution to clarify the pro-apoptotic mechanism of PdC, this study shows that the catalysis of specific protein thiol cross-linkage is enough to induce mitochondrial permeabilization and cytochrome c release.

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Organotelluranes exhibit potent antioxidant properties as well as the ability to react with protein thiol groups and, thereby, they are good models to study the mechanism of the mitochondrial permeability transition (MPT). We evaluated the effects of the concentration of organotelluranes, namely RT-03 and RT-04, on rat liver mitochondria. At the concentration range of 0.25-1.0 microM, organotelluranes did not cause any mitochondrial dysfunction. At the concentration range of 5-10 microM, RT-03 and RT-04 caused the Ca2+-dependent opening of the (MPT) pore, regulated by Cyclosporin A. At the concentration range of 15-30 microM the swelling was not inhibited by Cyclosporin A and in the absence of Ca2+, a significant decrease of respiratory control ratio was observed due to concomitant phosphorylation impairment and uncoupling, transmembrane potential disruption, depletion of mitochondrial reduced thiol groups, and alterations in the bilayer fluidity. Above 100 microM, the organotelluranes caused complete inhibition of respiratory chain. Over the whole studied concentration range, RT-03 and RT-04 did not induce mitochondrial oxidative stress assessed by using the reactive oxygen and nitrogen species indicator 2',7'-dichlorodihydrofluorescein diacetate. Further, the organotelluranes also exhibited protective effect against t-butyl hydroperoxide-induced oxidative stress as well as against Fe2+/citrate-induced peroxidation of mitochondrial membranes and PCPECL liposomes. These results point out that MPT pore opening can involve damage exclusively to mitochondrial membrane proteins. The exclusive antioxidant activity observed at nanomolar range is also an interesting new finding described in this work.