Mechanism(s) of Toxic Action of Zn and Selenite: A Study on AS-30D Hepatoma Cells and Isolated Mitochondria

Abstract

Mitochondria of AS-30D rat ascites hepatoma cells are found to be the main target for Zn(2+) and sodium selenite (Na(2)SeO(3)). High [mu]M concentrations of Zn(2+) or selenite were strongly cytotoxic, killing the AS-30D cells by both apoptotic and necrotic ways. Both Zn(2+) and selenite produced strong changes in intracellular generation of reactive oxygen species (ROS) and the mitochondrial dysfunction via the mitochondrial electron transport chain (mtETC) disturbance, the membrane potential dissipation, and the mitochondrial permeability transition pore opening. The significant distinctions in toxic action of Zn(2+) and selenite on AS-30D cells were found. Selenite induced a much higher intracellular ROS level (the early event) compared to Zn(2+) but a lower membrane potential loss and a lower decrease of the uncoupled respiration rate of the cells, whereas the mtETC disturbance was the early and critical event in the mechanism of Zn(2+) cytotoxicity. Sequences of events manifested in the mitochondrial dysfunction produced by the metal/metalloid under test are compared with those obtained earlier for Cd(2+), Hg(2+), and Cu(2+) on the same model system.

Figure 1

Time- and dose-dependent action of Zn²⁺ and selenite on AS-30D cell viability assayed by the trypan blue exclusion test. Time of incubation used was 3, 24, and 48 h. The results are expressed in % to corresponding control and presented as mean values of four independent experiments ±SE. *P < 0.05.

Figure 2

Induction of apoptosis in AS-30D cells by different concentrations of Zn²⁺ and selenite. The cells were cultivated in RPMI 1640 medium without (control) or with the indicated concentration (in μM) of Zn²⁺ and selenite for 24 h, stained with propidium iodide, and analysed by flow cytometry. Percentage of the sub-G₁ fraction, characteristic for apoptotic cells, is indicated in the upper left corner of each panel. A typical experiment out of at least three independent ones for each compound is shown.

Figure 3

Action of different concentrations of Zn²⁺ and selenite on the mitochondrial transmembrane potential after 24 h incubation with AS-30D cells. JC-1 is a cell-penetrating dye that accumulates within mitochondria maintaining high ΔΨ_mito and changes its emission fluorescence from green to red that can be followed by flow cytometry. The percentage of cells with red (R2) and green (R3) JC-1 fluorescence, reflecting high and low ΔΨ_mito, respectively, was estimated; R2 is indicated in the upper left corner of each panel. The protonophoric uncoupler CCCP plus the potassium ionophore valinomycin, acting together to completely collapse ΔΨ_mito, have always been used as the “positive” control. A typical experiment out of at least three independent ones is shown.

Figure 4

Action of cyclosporine A on changes in intracellular ROS formation produced by high Zn²⁺ and selenite in AS-30D cells. (a) Se: 50 μM; 3 h; (b) Zn: 250 μM; 24 h. The cells were cultivated in RPMI 1640 medium without (control) or with the indicated concentration (in μM) of selenite and Zn²⁺ for 3 h and 24 h, respectively, in the absence or presence of 1 μM CsA. The overall ROS production was calculated as the geometric mean of total green fluorescence of the oxidation product of DCFH₂ and is indicated in the upper right corner of each panel; for other details, see Section 2. A typical experiment out of at least three independent ones for each compound is shown.

Figure 5

Simultaneous recordings of four mitochondrial parameters (respiration, ΔΨ_mito, K⁺, and Ca²⁺ fluxes) with the help of O₂, TPP⁺, K⁺, and Ca²⁺-selective electrodes after treatment of isolated RLM with Zn²⁺ in the absence (a) or presence of CsA (b). Mitochondria (1 mg protein/mL) were incubated at room temperature in a medium containing 120 mM NaCl, 2 mM NaH₂PO₄, 10 mM HEPES (pH 7.4), 5 mM Glu, and 5 mM Mal. The additions of Zn²⁺ (5 μM), K⁺(100 μM), Ca²⁺(200 μM), and dithionite are indicated by arrows. [CsA] was 1 μM. 20 μM of Ca²⁺were present in the assay medium from the beginning of experiment. Downward deflection indicates decrease in [O₂], [TPP⁺], [K⁺ _free], and [Ca²⁺ _free] in the medium. The results are representative for a series of two independent experiments.

Figure 6

Simultaneous recordings of four mitochondrial parameters (respiration, ΔΨ_mito, K⁺, and Ca²⁺ fluxes) with the help of O₂, TPP⁺, K⁺, and Ca²⁺-selective electrodes after treatment of isolated RLM with sodium selenite in the absence (a) or presence of CsA (b). The additions of Na₂SeO₃ (5 μM) are indicated by arrows. The remainder is as in Figure 5.

Figure 7

Simultaneous recordings of four mitochondrial parameters (respiration, ΔΨ_mito, K⁺, and Ca²⁺ fluxes) with the help of O₂, TPP⁺, K⁺, and Ca²⁺-selective electrodes after treatment of isolated RLM with Ca²⁺ in the absence (a) or presence of CsA (b). The additions of Ca²⁺ (50 μM) are indicated by arrows. The remainder is as in Figure 5.

Figure 8

Simultaneous recordings of four mitochondrial parameters (respiration, ΔΨ_mito, K⁺, and Ca²⁺ fluxes) with the help of O₂, TPP⁺, K⁺, and Ca²⁺-selective electrodes after treatment of isolated RLM with Cd²⁺ in the absence (a) or presence of CsA (b). The additions of Cd²⁺ (5 μM) are indicated by arrows. The remainder is as in Figure 5.