Thioridazine interacts with the membrane of mitochondria acquiring antioxidant activity toward apoptosis--potentially implicated mechanisms

Abstract

We evaluated the effects of the phenothiazine derivative thioridazine on mechanisms of mitochondria potentially implicated in apoptosis, such as those involving reactive oxygen species (ROS) and cytochrome c release, as well as the involvement of drug interaction with mitochondrial membrane in these effects. Within the 0 - 100 microM range thioridazine did not reduce the free radical 1,1-diphenyl-2-picryl-hydrazyl (DPPH) nor did it chelate iron. However, at 10 microM thioridazine showed important antioxidant activity on mitochondria, characterized by inhibition of accumulation of mitochondria-generated O2*-, assayed as lucigenin-derived chemiluminescence, inhibition of Fe2+/citrate-mediated lipid peroxidation of the mitochondrial membrane (LPO), assayed as malondialdehyde generation, and inhibition of Ca2+/t-butyl hydroperoxide (t-BOOH)-induced mitochondrial permeability transition (MPT)/protein-thiol oxidation, assayed as mitochondrial swelling. Thioridazine respectively increased and decreased the fluorescence responses of mitochondria labelled with 1-aniline-8-naphthalene sulfonate (ANS) and 1-(4-trimethylammonium phenyl)-6 phenyl 1,3,5-hexatriene (TMA-DPH). The inhibition of LPO and MPT onset correlated well with the inhibition of cytochrome c release from mitochondria. We conclude that thioridazine interacts with the inner membrane of mitochondria, more likely close to its surface, acquiring antioxidant activity toward processes with potential implications in apoptosis such as O2*- accumulation, as well as LPO, MPT and associated release of cytochrome c.

Figure 1

Concentration-response curves for the effects of thioridazine (TR) on state 4 respiration rate (A), state 3 respiration rate (B) and Δψ (C) in isolated rat liver mitochondria. For the respiratory assays, mitochondria (1.5 mg protein) were incubated at 30°C with 5 mM succinate and 2.5 μM rotenone in a standard incubation medium containing 125 mM sucrose, 65 mM KCl and 10 mM HEPES – KOH, pH 7.4, in the presence of 0.5 mM EGTA and 10 mM K₂HPO₄ (respiration medium) in a final volume of 1.5 ml. State 3 respiration was initiated with 0.4 μmol ADP. For the Δψ assays, mitochondria (2 mg protein) incubated in the standard medium plus 2.5 μM rotenone and 0.4 μM rhodamine 123 in a final volume of 2 ml were energized by the addition of 5 mM succinate. The inhibition of the Δψ response of mitochondria was immediate and remained constant for at least 5 min; its extent was calculated 30 s after drug addition by using a calibration curve, as described in Methods. Data are presented as the mean±s.e.mean of three experiments with different mitochondrial preparations.

Figure 2

Effects of 10 μM thioridazine (TR) on mitochondria-generated O₂^•− assayed as lucigenin-derived chemiluminescence. Mitochondria (0.5 mg protein) were incubated with drugs at 30°C in the respiration medium described in the legend to Figure 1, in a final volume of 2 ml. The lucigenin-derived chemiluminesce response was initiated by adding 5 μM lucigenin; data are per cent inhibition of luminescence (integrated area under the curve) in relation to a control in the absence of the drug. The data are presented as the mean±s.e.mean of six experiments with different mitochondrial preparations. Statistical analysis was performed by the Mann – Whitney non-parametric test. ^*Significantly different from control (P<0.05).

Figure 3

Effects of 10 μM thioridazine (TR) on lipid peroxidation assayed as MDA generation (A) and associated mitochondrial swelling (B) and cytochrome c release (C) induced by Fe²⁺/citrate in isolated rat liver mitochondria (Fe²⁺). Mitochondria (1 mg protein) were incubated in the standard medium with 5 mM succinate, 2.5 μM rotenone, 50 μM (NH₄)₂Fe(SO)₄ and 2 mM sodium citrate for 30 min at 37°C (1 ml final volume). See Methods for MDA, mitochondrial swelling and cytochrome c determinations. Data are presented as the mean±s.e.mean of nine (A), nine (B) and six (C) experiments with different mitochondrial preparations. Statistical analysis was performed by Kruskal – Wallis non-parametric analysis of variance (ANOVA) followed by Dunn's multiple comparison test. ^*Significantly different from Fe²⁺ (P<0.05 for A and B; P<0.1 for C).

Figure 4

Effects of 10 μM thioridazine (TR) on mitochondrial swelling (A) and associated oxidation of protein-SH (B) and cytochrome c release (C) induced by 10 μM CaCl₂+0.5 mM t-BOOH in isolated rat liver mitochondria (t-BOOH). Mitochondria (0.4 mg protein) were incubated in the standard medium with 5 mM succinate and 2.5 μM rotenone, at 30°C (1.5 ml final volume). See also Methods for the determinations. Data are presented as the mean±s.e.mean of 10 (A), six (B) and six (C) experiments with different mitochondrial preparations. Statistical analysis was performed by Kruskal – Wallis non-parametric analysis of variance (ANOVA) followed by Dunn's multiple comparison test. ^*Significantly different from t-BOOH (P<0.05).

Figure 5

Concentration-response curves for the effects of thioridazine (TR) on membrane-ANS (A) and membrane-TMA – DPH (B) fluorescence response in isolated rat liver mitochondria incubated in the standard medium, as described in Methods. The relative fluorescence intensity (R.F.I.) was measured after drug addition. Data are presented as the mean±s.e.mean of three experiments with different mitochondrial preparations.