Fig 1.
Structure, photophysical properties and intracellular accumulation of 4'I-MPP+.
(A) Structures of MPP+ and 4'I-MPP+; (B) UV-Vis spectra of 4'I-MPP+ (5 μM) in PBS, methanol, and acetonitrile; (C) Excitation and emission spectra of 4'I-MPP+ (5 μM) in PBS, methanol, and acetonitrile); (D) Light and Fluorescence (Ex/Em 340/470-550 nm) images of 4'I-MPP+ (50 μM) treated differentiated MN9D cells. (a) light image; (b) florescence image at zero time after 4'I-MPP+ treatment; (c) florescence image after 2 h after 4'I-MPP+ treatment of the same cell population; (E) The time courses of the cellular uptakes of 100 μM MPP+ and 4'I-MPP+ which were determined either by HPLC-UV [16] or by measuring the intrinsic fluorescence of intracellular 4'I-MPP+.
Fig 2.
Comparison of the toxicological characteristics of 4'I-MPP+ with respect to MPP+.
(A) 4'I-MPP+ is selectively toxic to dopaminergic MN9D (reference to HepG2) cells similar to MPP+ as determined by the MTT assay. Data are presented as mean ± SD (n = 6). ٭p<0.005 and #p<0.05 versus no toxin controls; (B) Both MPP+ and 4'I-MPP+ (200 μM) inhibit the ubiquinone dependent NADH oxidation activity of rat brain mitochondrial complex-I. The complex-I inhibition potency of 4'I-MPP+ is greater than that of MPP+ at 200 μM concentrations; (C) Both MPP+ and 4'I-MPP+ deplete intracellular ATP levels. Intracellular ATP levels of MPP+ or 4'I-MPP+ (100 μM) treated MN9D and HepG2 cells (for 6 h at 37 °C) were quantified as detailed in Materials and Methods; The data are presented as mean ±± S.D (n = 3). ٭p< 0.0025 versus the controls with no toxin.
Fig 3.
Increase of intracellular ROS levels by 4'I-MPP+ and MPP+ leading to specific apoptotic cell death.
(A) The intracellular ROS levels were determined using the non-specific ROS sensitive probe DCFH-DA in MPP+ or 4'I-MPP+ treated (100 μM, 1 h at 37 °C) MN9D and HepG2 cells as detailed in Materials and Methods. The data are present as mean ± S.D (n = 3). ٭p<0.005 and #p<0.02 compared with controls with no toxins; (B) 4'I-MPP+-mediated ROS production in MN9D cells is localized to the mitochondria. The DCF fluorescence (Ex/Em 488/524 nm) images of 4'I-MPP+ treated (250 μM for 4 h at 37 °C) MN9D cells were recorded as detailed in Materials and Methods; (C) Both MPP+ and 4'I-MPP+ cause apoptotic MN9D cell death. Apoptotic chromatin condensation was visualized by observing the increase in nuclear DAPI fluorescence (Ex/Em 358/461 nm) in MPP+ or 4'I-MPP+ (250 μM) treated (for 12 h) MN9D cells.
Fig 4.
Mitochondrial accumulation and membrane depolarization by 4'I-MPP+.
(A) Intracellular localization of cytosolic 4'I-MPP+. Differentiated and undifferentiated MN9D and HepG2 cells were loaded with 100 μM 4'I-MPP+ and 200 nM MitoTracker Green FM and dual fluorescence (4'I-MPP+ and MitoTracker Green FM) images were recorded. (B) MPP+ and 4'I-MPP+ uptake into isolated MN9D cell mitochondria. The uptakes of MPP+ or 4'I-MPP+ (400 μM; for 45 min at 37 °C) were determined as detailed in Materials and Methods. Data are presented as mean ± S.D (n = 3). ٭p< 0.005 versus the zero-time controls; (C and D) Mitochondrial membrane potential depolarization by MPP+ and 4'I-MPP+. MPP+ or 4'I-MPP+-mediated MN9D or HepG2 cell mitochondrial membrane depolarizations were monitored by observing the decay of intracellular TMRM fluorescence.
Fig 5.
The effect of plasma and mitochondrial membrane potentials on the uptake and cellular distribution of MPP+ and 4'I-MPP+ in MN9D and HepG2 cells.
(A) The effect of FCCP on MPP+ and 4'I-MPP+ uptakes into MN9D and HepG2 cells. Cells were pre-incubated with FCCP (5 μM for 30 min) and then with MPP+ or 4'I-MPP+ (50 μM, for 45 min at 37 °C) and the intracellular MPP+ or 4'I-MPP+ levels were quantified as detailed in Materials and Methods. The data are presented as mean ± SD (n = 3). ٭p<0.005 and #p<0.025 versus respective controls without FCCP. (B) The effect of FCCP on the intracellular distribution of 4'I-MPP+ in differentiated MN9D and HepG2 cells. Cells were pre-incubated with 5 μM FCCP 30 min and then with 100 μM 4'I-MPP+ for 1 h at 37 °C. 4'I-MPP+ fluorescence images (Ex/Em 340/470-550 nm) were recorded. Controls were carried out in an identical manner, except that FCCP was omitted from the incubation media.
Fig 6.
Characteristics of MN9D mitochondrial uptake of 4'I-MPP+.
(A) Extracellular Ca2+ inhibit the mitochondrial uptake of 4'I-MPP+. The uptake of 4'I-MPP+ (200 μM; for 45 min at 37 °C) into isolated intact MN9D mitochondria were determined as described in Materials and Methods in the presence or absence of extracellular Ca2+. ٭p<0.005 versus zero Ca2+ controls. (B) Effects of NCX inhibitors on the MN9D mitochondrial uptake of 4'I-MPP+. The effects of CGP37157, 2APB, and KBR7943 (50 μM) on the mitochondrial uptake of 4'I-MPP+ (200 μM; for 45 min at 37 °C) were determined as detailed in Materials and Methods. The data are represented as mean ± S.D (n = 3). ٭p<0.005 versus controls. (C) Concentration dependence of the inhibition of MN9D mitochondrial uptake of 4'I-MPP+ by CGP37157. The experiments were carried out using a similar protocol as above with varying concentrations of CGP37157 (10–50 μM). The data are presented as mean ± S.D (n = 3). ٭p<0.005 versus controls. (D) The effect of CGP37157, on the mitochondrial accumulation of 4'I-MPP+ in live HepG2 cells. The effect of CGP37157 (50 μM, preincubated for 30 min at 37 °C) on the mitochondrial uptake of 4'I-MPP+ (100 μM, for 1 h at 37 °C) was monitored by dual fluorescence imaging using the mitochondrial probe MitoTracker Green and 4'I-MPP+. Controls were treated under similar conditions, except that CGP37157 was excluded from the incubation medium. (E) The effect of CGP37157 and KBR7943 on the 4'I-MPP+-mediated mitochondrial depolarization of HepG2 cells. HepG2 cells were incubated with either 50 μM CGP37157 or KBR7943 for 30 min and then with 50 nM TMRM for 45 min at 37 °C, and finally with 4'I-MPP+ (100 μM final concentration). The mitochondrial depolarization was followed by monitoring the intracellular TMRM fluorescence (Ex/Em 543/573 nm) as a function of time. Controls were treated identically except that CGP37157 and KBR7943 were excluded from the initial incubation media. (F) The effect of extracellular Ca2+ on MPP+ and 4'I-MPP+ cellular uptake. Cells were incubated with 50 μM MPP+ or 4'I-MPP+ in KRB-HEPES or EGTA-HEPES (1.3 mM EGTA with no Ca2+) for 45 min, at 37 °C. MPP+ and 4'I-MPP+ uptakes were measured by HPLC-UV. The data are presented as mean ± S.D (n = 3). ٭p<0.003 versus KRB-HEPES controls. (G) The effect of CGP37157 on the 4'I-MPP+ mediated mitochondrial membrane depolarization under longer incubation conditions. HepG2 cells were incubated with or without 10 μM CGP37157 followed by 50 nM TMRM, and finally with 4'I-MPP+ (100 μM, 0 or 4h) and the intracellular TMRM fluorescence was recorded. Controls were carried out using an identical protocol except that either CGP37157 or 4'I-MPP+ were omitted from the incubation media.
Fig 7.
CGP37157 protect MN9D cells from MPP+ and 4'I-MPP+ toxicities.
Cells were pre-incubated with 0–50 μM CGP37157 for 30 min followed by 80 μM MPP+ or 25 μM 4'I-MPP+ for 8 h. The relative cell viabilities were determiner using MTT assay, as detailed above. Data are presented as mean ± SD (n = 6). ٭p<0.0001 and #p<0.005 versus no CGP37157 controls.
Table 1.
The relative elution times and hydrophobicities of 4'-subtituted MPP+ derivatives.