Inorganic phosphate stimulates the toxic effects of Tl+ in rat liver mitochondria

We studied action of inorganic phosphate (P(i)) on toxic effects of Tl+ in isolated rat liver mitochondria. This is a convenient model to study the toxicity of heavy metals. P(i) markedly retarded contraction of energized mitochondria swollen in the TlNO3 medium and even stronger stimulated swelling and state 4 of succinate-energized mitochondria in the TlNO3 medium. A valinomycin-induced decrease of K+-diffusion potential was also accelerated by Tl+ in the presence of P(i). The mitochondrial permeability transition pore in the medium containing Ca2+, TlNO3, and nitrates of univalent cations was distinctly stimulated by P(i). However, P(i) did not affect both the Tl+-stimulated swelling of nonenergized mitochondria in the TlNO3 medium and swelling of energized mitochondria in the Tl acetate medium. Respiration stimulated by 2,4-dinitrophenol and monoamine oxidase activity of energized mitochondria were not affected by Tl+ regardless of the presence of P(i). We suggested that stimulation by P(i) of toxic action of Tl+ in mitochondria and cells could be due to even greater enhancement of uncoupling of mitochondria as shown by an additional increase of swelling and state 4, and in the greater probability of opening of MPTP in the presence of P(i) and Ca2+.     

Mersalyl prevents the Tl+-induced permeability transition pore opening in the inner membrane of Ca2+-loaded rat liver mitochondria

It was earlier shown that the calcium load of rat liver mitochondria in medium containing TlNO₃ and KNO₃ resulted in the Tl⁺-induced mitochondrial permeability transition pore (MPTP) opening in the inner membrane. This opening was accompanied by an increase in swelling and membrane potential dissipation and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration. This respiratory decrease was markedly leveled by mersalyl (MSL), the phosphate symporter (PiC) inhibitor which poorly stimulated the calcium-induced swelling, but further increased the potential dissipation. All of these effects of Ca²⁺ and MSL were visibly reduced in the presence of the MPTP inhibitors (ADP, N-ethylmaleimide, and cyclosporine A). High MSL concentrations attenuated the ability of ADP to inhibit the MPTP. Our data suggest that the PiC can participate in the Tl⁺-induced MPTP opening in the inner membrane of Ca²⁺-loaded rat liver mitochondria.     

Effects of Tl<Superscript>+</Superscript> on ion permeability,membrane potential and respiration of isolated rat liver mitochondria

It is known that permeability of the inner mitochondrial membrane is low to most univalent cations (K⁺, Na⁺, H⁺) but high to Tl⁺. Swelling, state 4, state 3, and 2,4-dinitrophenol (DNP)-stimulated respiration as well as the membrane potential (ΔΨ_mito) of rat liver mitochondria were studied in media containing 0–75 mM TlNO₃ either with 250 mM sucrose or with 125 mM nitrate salts of other monovalent cations (KNO₃, or NaNO₃, or NH₄NO₃). Tl⁺ increased permeability of the inner mitochondrial membrane to K⁺, Na⁺, and H⁺, that was manifested as stimulation of the swelling of nonenergized and energized mitochondria as well as via an increase of state 4 and dissipation of ΔΨ_mito. These effects of Tl⁺ increased in the order of sucrose <K⁺ <Na⁺ ≤ NH₄⁺. They were stimulated by inorganic phosphate and decreased by ADP, Mg²⁺, and cyclosporine A. Contraction of energized mitochondria, swollen in the nitrate media, was markedly inhibited by quinine. It suggests participation of the mitochondrial K⁺/H⁺ exchanger in extruding of Tl⁺-induced excess of univalent cations from the mitochondrial matrix. It is discussed that Tl⁺ (like Cd²⁺ and other heavy metals) increases the ion permeability of the inner membrane of mitochondria regardless of their energization and stimulates the mitochondrial permeability transition pore in low conductance state. The observed decrease of state 3 and DNP-stimulated respiration in the nitrate media resulted from the mitochondrial swelling rather than from an inhibition of respiratory enzymes as is the case with the bivalent heavy metals.     

A thiol oxidation interpretation of the Cu2+ effects on rat liver mitochondria

A comparative study of the Cu2+ effects, binding and reduction, has been performed on rat liver mitochondria. In the first minutes, Cu2+ (less than or equal to 50 micron) is massively bound and reduced to the extent of 70%-80% while a simultaneous activation of respiration takes place. Then the remaining 20% or so of Cu2+ are progressively bound and reduced while respiratory inhibition, Ca2+ and Mg2+ effluxes, and swelling are observed. EDTA, used as a copper chelator, prevents or reduces the copper effects and removes part of the bound copper, according to the time of introduction in the incubation medium after Cu2+. The results suggest that the two steps of the copper binding and the effects following involve mainly first the outer (cytosol side) proteins of the inner membrane and then those of the inner membrane. 100 microM dithiothreitol and 100 microM glutathione used as antioxidant thiol reagents prevent, as does EDTA, but do not reverse the 25 microM copper effects. They also decrease the copper binding; however, no relationship between binding and preventive action is observed. It is shown that glutathione and dithiothreitol have a specific potent ability to reduce Cu2+, which explains that in presence of these reagents copper may react with mitochondria partly or entirely in the form of Cu+. These findings suggest that Cu2+ in its Cu+ form has no mitochondrial effect. A mechanism of copper action involving oxidation of some membrane thiol groups is discussed.     

Selective toxicity of chrysin on mitochondria isolated from liver of a HCC rat model

Flavonoids are natural compounds that show various biological effects, such as the anti-cancer effect. Chrysin is a flavonoid compound found in honey and propolis. Studies have shown that chrysin has anti-cancer activity due to induction of apoptosis signaling. In the present study, we examined the cytotoxic effect of chrysin against liver mitochondria obtained from the hepatocellular carcinoma (HCC) rat model. Diethylnitrosamine (DEN) and 2-acetylaminofluorene (2-AAF) was used for induction of HCC. Mitochondria were isolated from liver hepatocytes using differential centrifugation. Then, hepatocytes and mitochondria markers related to apoptosis signaling were investigated. Our finding indicated an increase in mitochondrial reactive oxygen species (ROS) generation, collapse in the mitochondrial membrane potential (MMP), swelling in mitochondria, and cytochrome c release (about 1.6 fold) after exposure of mitochondria obtained from the HCC rats group with chrysin (10, 20, and 40 µM) compared to the normal rats group. Furthermore, Chrysin was able to increase caspase-3 activity in the HCC rats group (about 2.4 fold) compared to the normal rats group. According to the results, we proposed that chrysin could be considered as a promising complementary therapeutic candidate for the treatment of HCC, but it requires a further in vivo and clinical studies.