Effects of Trichlorotelluro-dypnones on Mitochondrial Bioenergetics

Article pubs.acs.org/crt

Effects of Trichlorotelluro-dypnones on Mitochondrial Bioenergetics and Their Relationship to the Reactivity with Protein Thiols César H. Yokomizo,† Felipe S. Pessoto,‡ Tatiana Prieto,‡ Rodrigo L. O. R. Cunha,‡ and Iseli L. Nantes*,‡ †

Departamento de Biologia Molecular, Universidade Federal de São Paulo, R. Botucatu, 740, São Paulo, SP Brazil Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Av. dos Estados, 5001, Santo André, SP Brazil

‡

ABSTRACT: The effect of four trichlorotelluro-dypnones, named compounds 1, 2, 3, and 4, on the bioenergetics of isolated rat liver mitochondria (RLM) and cells was investigated. In a dose-dependent manner, the studied organotelluranes promoted Ca2+dependent mitochondrial swelling inhibited by cyclosporine A and were associated with a decrease of the total mitochondrial protein thiol content. These effects characterize the opening of the classical mitochondrial permeability transition pore. Despite the reactivity with mitochondrial protein thiol groups, these compounds did not promote significant glutathione depletion. In the absence of Ca2+, the organotelluranes promoted mitochondrial loss of ΔΨ in RLM concomitant with respiratory control decrease due to an increase of the state 4 respiration rate. In these conditions, mitochondrial swelling was absent, and thiol content was higher than that in the presence of Ca2+. The differentiated effects observed in the presence and absence of Ca2+ are probably related to the effects of that ion on membrane structure, with repercussions for the exposure of specific reactive protein thiol groups. In smooth muscle cells, these compounds promoted the loss of mitochondrial ΔΨ and apoptosis. The loss of ΔΨ was not preceded by a decrease of cell viability that is consistent with mitochondria as the primary targets for the action of these organotelluranes.

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INTRODUCTION In the past, tellurium and selenium derivatives were considered highly toxic compounds. However, the identification of selenium cysteine as the 21st proteic amino acid present in a diverse set of prokaryotic and eukaryotic enzymes1−7 raised questions about the relativity of chalcogen toxicity. Tellurium is a chalcogen element that is able to form inorganic and organic derivatives.8−10 Telluranes belong to the class of organochalcogen compounds and have been intensively studied.8 The organotellurium compounds are divided into two distinct groups: the divalent derivatives comprising tellurols, diorganotellurides, and diorganoditellurides, and the hypervalent derivatives comprising organotellurium trihalides, diorganotellurium dihalides, organotellurium oxides, organotellurates, and organopertelluranes.8 The comprehension and manipulation of the biological effects of organotelluranes remain a challenge. Considering the diversity of potential therapeutic applications for organotelluranes, it is important to perform parallel studies about their toxicity.11,12 There are few reports about the toxicity of © XXXX American Chemical Society

organotelluranes, and most of these are limited to their relative toxicity in animals, the effects in tissues, and the capacity to inhibit cellular proliferation.8,13−15 Studies addressing the structure−activity relationships (SARs) and biochemical mechanisms of organotelluranes toxicity are scarce.16−18 The studies focused on the biological targets for organotelluranes have suggested that many of the biological effects observed for these compounds are related to a peculiar chemistry with thiol. The reactivity of organotelluranes with thiol groups of biomolecules are involved both in the beneficial antioxidant and deleterious properties of organotelluranes.8,16 The thiol-reactive forms of organoselenium and organotellurium compounds depend on the diorganyldichalcogenyl compounds (Nox = −1) and the organochalcogenuranes (Nox = +4). Organochalcogenides (Nox = −2) are thiol reactive when a leaving group is bonded to the chalcogen atom, whereas when the leaving group is absent, the thiol reactive form should Received: December 14, 2014

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DOI: 10.1021/tx5005166 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX

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Chemical Research in Toxicology

MPTP opening by CsA is Pi-independent, and the participation of PiC in MPTP could not be corroborated by knockout studies.31,32 More recently, the participation of F1Fo ATP synthase has been postulated, and Alavian et al. proposed that its c subunit ring could act as the pore per se.33,34 However, the liposome-reconstituted c subunit of F1Fo ATP synthase was insensitive to Ca2+ and CsA and required higher concentrations of ATP and ADP than those previously described as necessary to inhibit MPTP opening. Thus, in a recent article, Halestrap proposed that the formation of an ATP synthasome, composed of interacting ANT, PiC, and F1Fo ATP synthase (as previously described by the group of Pedersen and other research groups), could be imperative for MPTP formation.31,35−38 Therefore, despite the advances concerning the participation of proteins in MPTP, only the role of SH oxidation has been conclusively established. Conditions of oxidative stress can promote SH oxidation and the formation of disulfide bonds, as well as thiol reagents such as phenyl arsine oxide (PAO), eosine, maleimide, and organotelluranes. In this scenario, the studies of the biological effects of organotelluranes are relevant for a better comprehension of the participation of protein thiol groups in MPTP. In the present study, we described the biological effects of four structurally related trichlorotelluro-dypnones, named compounds 1, 2, 3, and 4 (Chart 2). The structure of these compounds enables the

be the product of its oxidation, a chalcogenoxide (Nox = +4).8,19,20 Previously, we had described the peculiar biological effects of two telluranes, RT-03 and RT-04, which were compatible with the characteristics of thiol reagents (Chart 1).16 Chart 1. Molecular Structures of Organotelluranes RT-03 and RT-04

The reactivity of RT-03 and RT-04 with thiol groups of proteins was confirmed by their inhibitory action on the cysteine proteases cathepsins and by the titration of cysteine thiol groups.21,22 RT-03 and RT-04 were able to induce cyclosporine A (CsA)-sensitive and CsA-insensitive mitochondrial permeability transition (MPT) in a dose-dependent manner.16 Furthermore, the regulated and CsA-insensitive mitochondrial permeability transition pore (MPTP) openings triggered by RT-03 and RT-04 were shown to be related to their reactivity with thiol groups. These compounds promoted the cross-linkage of mitochondrial membrane proteins that had been prevented by DTT and NEM. RT-03 and RT-04 are hydrophobic and can be expected to be preferentially partitioned into membranes. Therefore, at high organotellurane concentrations, the attack to thiol groups of membrane proteins in association with significant changes in the membrane fluidity should be enough to induce cyclophilin D-independent opening of the pore.16 These results suggest that, at high concentrations, the effect of organotelluranes on membrane organization could have significant influence on mitochondrial function. A peculiar and interesting characteristic exhibited by the organotelluranes RT-03 and RT-04 is their antioxidant activity, which provides an unusual condition in which the pore is formed concomitantly with efficient antioxidant protection. This characteristic suggests that the lipid damage observed under conditions in which MPTP opening was promoted by oxidative stress is not decisive for the event and constitutes only a side effect.16,21 In recent years, numerous studies have characterized the conditions and agents involved in the activation and inhibition of MPTP opening; however, the participation of specific mitochondrial proteins for this event remained an unanswered and controversial question. First, adenine nucleotide translocase (ANT) and cyclophilin D were proposed as obligatory components of the MPT. The proposed participation of ANT in the MPTP led to studies into the mechanism responsible for the Ca2+-stimulated MPTP opening.23,24 In addition, the voltage-dependent anion channel (VDAC) and hexokinase have also been assigned as components of the MPTP.25−27 Although a regulatory role for ANT in MPT had already been confirmed, further studies by Kokoszka et al., Krauskopf et al., and Baines et al. demonstrated the nonessential role of the ADP/ATP translocator and the voltage-dependent anion channels for the MPTP.28−30 The phosphate carrier (PiC) has also been postulated as the cyclophilin D binding component of the pore and related to the capacity of phosphate to promote activation of the MPTP opening. However, the inhibition of

Chart 2. Molecular Structures of Trichlorotelluro-dypnones Used in the Present Study

occurrence of the formation of Te−O bonds and a pseudooctahedral structure. The peculiar structures of compounds 1−4 resemble the immunomodulator AS101 and enable one to gain new insights into the SAR of organotelluranas.11 Thefore, these compounds are promising for therapeutic applications, but as they are newly designed molecular structures, it is important to characterize dosedependent toxic effects on normal cells that have been envisaged in the present study.

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MATERIALS AND METHODS

Chemicals. All reagents were commercial products of the highest purity grade available from Sigma-Aldrich (St. Louis, MO, USA) or Molecular Probes (Eugene, OR, USA). Trichlorotelluro-dypnones (1−4) were synthesized as described by Huang et al. for acetone but substituting acetophenone, ortho-methoxyacetophenone, para-methoxyacetophenone, and para-ethoxyacetophenone.39 Isolation of Rat Liver Mitochondria. Liver mitochondria were isolated by conventional differential centrifugation from male Wistar rats.40 The livers were homogenized in 250 mM sucrose, 1 mM EGTA, and 10 mM HEPES buffer (pH 7.4). The mitochondrial suspension was washed twice in the same medium containing 0.1 mM B


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cells were maintained in fluorescence media at 1 × 104 cells/ experiment in the presence of studied organotelluranes (25 μM), after preincubation with fluorophores (Dioc 20 μM/mL and DAPI 10 20 μM/mL) as already mentioned. Cell viability was tested by neutral red assay after cell incubation with telluranes, which was done in the presence of 2% FCS (fetal calf serum). To avoid tellurane compound aggregation, the cells were washed 2× in PBS solution, and 0.1 mL of 50 μg of NR/mL prepared in DMEM free FCS was added to each well (the NR solution was filtered to remove fine precipitates of dye crystals), and the plate was incubated for 3 h under the same cell culture conditions. Thereafter, the medium was removed, the cells were washed 1× with PBS before 0.1 mL fixation solution (3% formaldehyde, 1% CaCl2) was rapidly applied followed by the addition of 0.1 mL of extraction solution (1% glacial acid acetic on ethanol). After an additional 10 min of incubation at room temperature and brief agitation on a microtiter-plate shaker, the absorbance of the plate wells was measured at 540 nm. The viability percentage (means of three experiments) was based on control cells incubated in the same conditions in the absence of trichlorotelluro-dypnones.47,48 Cytomorphology. Microscopy images of the cells were obtained using a widefield Leica DMI 6000B microscope (Leica Microsystems, Germany) with a HCX PL APO 40×/0.85 objective coupled to an ultrafast digital camera (Leica DFC365 FX, Leica Microsystems, Germany). The nuclei in the same visual field were stained with DAPI (10 μg/mL), and energized mitochondria were stained by the retention of the dye DiOC6(3) incubated at 37 °C for 30 min. Statistical Analyses. The statistical analyses were performed using GraphPad software available at the following site: http://statpages. org/anova1sm.html. The posthoc tests (Bonferroni correction) were also done by GraphPad software at http://graphpad.com/quickcalcs/ posttest1.cfm. Computational Analyses of the Temporal Loss of the Mitochondrial Membrane Potential. The progressive loss of transmembrane potential in each condition, as evidenced by the loss of DiOC6(3) fluorescence, was recorded by video. For all videos, the frames were individually extracted as images in “ppm” extension. In the images, each of the three subsequent bytes represents one pixel of the color components red, green, and blue (RGB). Each component, or byte, is a numeric value of a minimum of 0 and a maximum of 255 and corresponds to a color intensity value in the digital images. Therefore, by defining the contribution of coupled mitochondria (brightness of green light) in each frame as the sum of all green components of pixel values, it is possible to determine the temporal changes in the mitochondria transmembrane potential (i.e., the kinetics of the loss of mitochondrial potential). This process was automated with a script for UNIX platforms, which requires the open-source software ffmpeg available at https://www.ffmpeg.org/about.html and a C language compiler.

EGTA, and the final pellet was resuspended in 250 mM sucrose to a final protein concentration of 80−100 mg/mL. Standard Medium for Assays with Mitochondria. The reactions were performed in medium containing 125 mM sucrose, 65 mM KCl, 10 mM HEPES-KOH, pH 7.4, 5 mM potassium succinate (+ 2.5 μM rotenone), and 10 μM CaCl2 or 10 μM EGTA, when indicated. Mitochondrial Swelling. Mitochondria (0.4 mg of protein) were incubated in 1.5 mL of a medium containing 125 mM sucrose, 65 mM KCl, 10 mM HEPES-KOH, pH 7.4, 5 mM potassium succinate (+ 2.5 μM rotenone), and 10 μM CaCl2 at 30 °C for 15 min. Mitochondrial swelling was estimated from the decrease in absorbance at 540 nm measured by a Hitachi U-2000 Spectrophotometer (Tokyo, Japan).41 Measurements of Mitochondrial Transmembrane Electrical Potential (ΔΨm). Mitochondrial ΔΨ was estimated through fluorescence changes of rhodamine 123 (0.4 μM) recorded on a model F-2500 spectrofluorometer (Hitachi Ltd., Tokyo, Japan) operating at λexcitation and λemission wavelengths of 505 and 525 nm respectively, with a slit width of 5 nm.42,43 Determination of Total Thiol Content. After 15 min of incubation under swelling conditions, mitochondria were treated with trichloroacetic acid (5% final concentration) and centrifuged at 4500g for 10 min. The pellet was suspended with 1 mL of 0.5 M potassium phosphate buffer, pH 7.6, and, after the addition of 0.1 mM DTNB, absorbance was determined at 412 nm. The amount of thiol groups was calculated from ε = 13.600 M−1cm−1.44 Determination of Mitochondrial Reduced Glutathione and Other Nonprotein Thiol Content. After 15 min of incubation under swelling conditions, the mitochondrial suspension was treated with 0.5 mL of 13% trichloroacetic acid and centrifuged at 900g for 3 min. Aliquots (100 μL) of the supernatant were mixed with 2 mL of 100 mM NaH2PO4 buffer, pH 8.0, containing 5 mM EGTA. One hundred microliters of o-phthalaldehyde (1 mg/mL) was added, and the fluorescence was measured 15 min later using the 350/420 nm excitation/emission wavelength pair in an F-2500 fluorescence spectrophotometer (Hitachi, Ltd., Tokyo, Japan).45 This method is also sensitive to other nonprotein thiol content. Mitochondrial Respiration. Mitochondria (1 mg protein/mL) were incubated in a medium containing 125 mM sucrose, 65 mM KCl, 0.5 mM EGTA, 10 mM K2HPO4, and 10 mM HEPES-KOH, pH 7.2, at 30 °C. Mitochondrial respiration was monitored polarographically by an anoxygraph equipped with a Clark-type oxygen electrode (Gilson Medical Electronics, Middleton, WI). State 4 respiration was initiated by the addition of 5 mM potassium succinate (2.5 μM rotenone), and state 3 respiration was initiated by the addition of 400 nmol ADP. Respiratory control ratios ± standard deviation (RCR ± SD) were determined according to Chance and Willians.46 The values of RCR for compound 2 were presented as percentages, and the absolute values are shown in Table 1.

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Table 1. Absolute RCR Values Obtained for Compound 2 [compd 2] (μM) 0 0.5 2.5 5 7.5 10 12.5 15

RESULTS Effect of Trichlorotellurium(IV)-dypnones (1−4) on Mitochondrial Permeability, Transmembrane Potential (ΔΨ), and Total Content of Protein Thiol Groups in the Presence and Absence of Ca2+. Four trichlorotellurium(IV)-dypnones (1−4), were synthesized from the corresponding acetophenones and tellurium tetrachloride. The compounds differed according to the substituents at the aromatic rings. The dypnones were probed for their effects on mitochondrial bioenergetics. Rat liver mitochondria were incubated with dypnones in standard medium in the presence of 10 μM Ca2+ during 15 min at 30 °C. In Figure 1, the results obtained with compounds 2 and 4 are representative of the effects of the studied dypnones on mitochondrial bioenergetics that are characterized by swelling (Figure 1A, lines b and b′), loss of transmembrane potential ΔΨ (Figure 1B, lines b and b′), and decrease of RCR (Figure 1C). The inset of Figure 1A shows the

RCR ± SD 6.7 6.3 4.5 4.1 3.9 3.2 3.5 3.4

± ± ± ± ± ± ± ±

0.1 0.15 0.9 1.1 0.5 0.07 0.3 0.2

Cell Culture and Experiments. Rabbit aortic smooth muscle cells (SMCs) isolated from tunica intima and media of normal male New Zealand rabbit aorta (Abbott Laboratories, North Chicago, IL) were grown in high glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen, Carlsbad, Ca, USA) at 37 °C/ 5% CO2 (Sanyo MCO-20AIC, Japan). For microscopy experiments, C


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EC50 curves obtained for compounds 2 and 4. Similar results were obtained for compounds 1 and 3 (not shown). The mitochondrial permeability promoted by Te(IV)dypnones 1, 2, 3, and 4 was concentration dependent, and the maximal effects were observed in the following concentrations: 50, 15, 25, and 25 μM, respectively. The mitochondrial permeabilization promoted by Te(IV)-dypnones was sensitive to CsA (Figure 2A, lines c and c′), prevented by the Ca2+

Figure 2. Control left and right panels show, respectively, SMC labeled with DiOC6(3) immediately after and 6 h after incubation. The RT-03 left and right panels show, respectively, SMC immediately after and 6 h after incubation with 20 μmol/L RT-03 (positive control). The other panels correspond, respectively, to the effect of 25 μmol/L of compounds 1, 2, 3, and 4 on the mitochondrial transmembrane potential of SMC, immediately after and 6 h after treatment. Bellow, H represents the time-dependent SMC viability incubated for 2 h with compounds 1, 2, 3, and 4 and represented, respectively, by blue, magenta, green, and royal blue lines and symbols. The SMC viability was determined by neutral red as described in Materials and Methods.

Figure 1. (A) Swelling of rat liver mitochondria (RLM) induced by compounds 2 and 4. Lines a and a′ represent the control experiment of swelling, and lines b and b′ represent the condition of mitochondrial permeabilization induced by 15 and 25 μmol/L of compounds 2 and 4, respectively. Line c and c′ show the effect of CsA and line d the effect of 15 μM EGTA on RLM incubated with compound 2. The inset represents the EC50 curve obtained for RLM incubated with different concentrations of compounds 2 and 4. (B) Temporal variation of ΔΨ in the absence (line a) and presence (line b) of 15 and 25 μmol/L of compounds 2 and 4, respectively. (C) The effect of compound 2 on the RCR of RLM performed in medium without CaCl2. Compound 2 was used in the following concentrations: 0.5, 2.5, 5, 7.5, 10, 12.5, and 15 μmol/L. (D) The effect of dypnones on the mitochondrial membranethiol (light gray) and reduced gluthatione (dark gray) content in the absence (−) and presence (+) of 10 μmol/ L Ca2+; the first column represents the negative control experiment and the second column the positive control experiment in the presence of 0.6 mM t-BuOOH. In panels C and D, the results are the average ± SD of three experiments carried out with different mitochondrial preparations.

chelator EGTA (Figure 1A, line d and d′) and associated with an increase of state 4 respiration rate (uncoupling) (Figure, 1C). The mitochondrial permeabilization promoted by Te(IV)dypnones was also associated with a decrease of mitochondrial membranethiol content, which was also Ca2+-sensitive (Figure 1D). Considering that the RCR decrease promoted by Te(IV)dypnones is unrelated to respiration inhibition (Figure 1C), it is probable that proteins of the respiratory chain were not the targets for the chemical attack of these compounds. Considering the well-known reactivity of telluranes with thiols, we evaluated the effect of Te(IV)-dypnones on the SH and GSH content of RLM. Despite the significant capacity to deplete mitochondrial membrane SH content, the same concentrations of Te(IV)-dypnones did not cause significant depletion of total reduced GSH content compared with the results obtained by t-BuOOH (0.6 mM) (Figure 1D). The D


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normal with round and homogeneous nuclei. Green fluorescence in the majority of cells indicates that the mitochondria are coupled. Figure 2B shows a significant loss of mitochondrial ΔΨ in the SMC cells at approximately 3 min, the time necessary to adjust microscopy for image capture, after the addition of 20 μmol/L of RT-03 (left panel). The mitochondrial ΔΨ was completely abolished after 6 h of incubation with RT-03 (right panel). Figure 2C shows the snap shots of SMC taken ∼3 min and 12 h after incubation with 25 μmol/L of compound 1. In comparison with the control conditions, compound 1 led to significant loss of SMC ΔΨ around 3 min after the addition to the cell suspension. Figure 2D, E, and F shows that compounds 2, 3, and 4 have similar effects on SMC bioenergetics. Around 3 min after the addition of 25 μmol/L of compounds 2, 3, and 4 in suspensions of SMC labeled with DiOC6(3), significant amounts of mitochondria remained coupled. However, the ΔΨ of SMC cells was equally abolished after 6 h of incubation with compounds 2, 3, and 4. During the 6 h of incubation of SMC cells under the different conditions mentioned above, the cell images were captured in video, and the kinetics of the mitochondrial membrane potential was computationally determined (Material and Methods section). Figure 2G shows that, relative to the negative control, compound 1 and RT-03 lead SMC cells to lose almost 50% of ΔΨ at early incubation times. Compounds 2, 3, and 4 did not promote early loss of ΔΨ. The results obtained for SMC incubated with Te (IV)-dypnones show that cell membranes are not a barrier for their action in cells. Considering that except for RT-03 and compound 1, significant loss of ΔΨ loss has been observed 1−2 h after the addition of trichlorotelluro-dypnones, a question is raised: is the mitochondrial transmembrane potential changing due to the alteration of mitochondrial bioenergetics via the trichlorotelluro-dypnones, or are the compounds toxic to the cells? To address this question, we performed experiments to determine the effect of organotelluranes on the SMC viability by neutral red during the first 2 h of incubation (Figure 2H). This incubation time is not enough to observe a significant loss of ΔΨ for SMC treated with the four investigated trichlorotellurodypnones. The neutral red assay is based on the capacity of viable cells to take up the dye neutral red that is then incorporated into lysosomes. Therefore, the neutral red assay is an efficient test of cytotoxicity that allows the estimation of the number of cultured viable cells submitted to different conditions. Considering that the procedure is more sensitive than other cytotoxicity tests such as tetrazolium salts, enzyme leakage, or protein content, we chose this assay to more specifically determine the effects of organotelluranes in cells.47 SMC were incubated for a total time of 2 h in the absence and in the presence of the four trichlorotelluro-dypnones in a medium containing neutral red. The loss of SMC viability treated with trichlorotelluro-dypnones was around 10−20% after 2 h of incubation with organotelluranes, and the values are close to the control values (Figure 2H). Considering that a significant loss of mitochondrial transmembrane potential was observed for SMC cells at the early times of incubation with organotelluranes and that it was almost immediate for compound 1 (Figure 2 G), we concluded that the loss of the mitochondrial transmembrane potential occurs due to the alteration of mitochondrial bioenergetics by the action of the organotelluranes. The viability of SMC cells with impaired aerobic ATP production is consistent with previously published data showing that the inhibition of SMC respiratory chain

dypnones studied here were also innocuous relative to the effect of t-BuOOH on GSH content (Figure 1D). The depletion of mitochondrial GSH content by t-BuOOH was related to the reduction of the peroxide by GPx at the expense of GSH. Taken together, these results are consistent with a preferential partition of dypnones into the mitochondrial membranes. Considering that experiments assessing ΔΨ and RCR are performed in the presence of EGTA, the dissipation of transmembrane potential and the uncoupling promoted by Te(IV)-dypnones could be the result of a permeabilization unrelated to MPTP formation. The EC50 values of RCR obtained for the Te(IV)-dypnones were consistently different from those obtained for the swelling and SH depletion promoted by these compounds (Table 2). It is important to Table 2. EC50 Values for the Effects of Te(IV)-Dypnones on RLM compds

SH EC50 (μM)

swelling EC50 (μM)

1 2 3 4

9.9 ± 0.6 7.0 ± 1.8 12.4 ± 0.5 8.3 ± 0.4

9.6 ± 1.0 7.4 ± 0.4 9.8 ± 1.0 15.6 ± 1.5

RCR EC50 (μM) 6.3 1.7 6.9 3.0

± ± ± ±

0.75 0.2 1.2 0.3

note that Ca2+ affects membrane structure and protein SH exposure.16 Therefore, the EC50 values obtained in the presence and absence of Ca2+ probably result from the reaction of the dypnones with different mitochondrial membrane proteins. Table 2 presents the osmotic swelling (mitochondrial permeability), SH oxidation, and RCR EC50 values for the three studied Te (IV)-dypnones. Table 2 shows that for compounds 1 and 2, almost identical EC50 values were obtained for swelling and membrane SH decrease. These results suggest that the SH groups that were chemically attacked by the Te(IV)-dypnones are related to the structure of proteins involved in the formation of MPTP. Differently, the SH depletion EC50 values obtained for compounds 3 and 4 were different from those obtained for swelling. For compound 3, the SH depletion EC50 value was 20% higher than that obtained for mitochondrial swelling. However, for compound 4 the difference is more significant because the SH depletion EC50 value was ∼50% lower than that obtained for mitochondrial swelling. This result is consistent with compound 4 being a less specific reactant for the proteins that are assembled in the MPTP structure. Effect of Te(IV)-dypnones on the Bioenergetics of Cells. Considering that the effects of trichlorotelluro-dypnones on the bioenergetics of isolated RLM could be impaired in cells by the barrier of the plasmatic membrane, the Te(IV)dypnones were also probed in cultured smooth muscle cells (SMC). Figure 2 shows the comparative effects of RT-03 (positive control) and compounds 1, 2, 3, and 4 on the mitochondrial transmembrane potential of SMC relative to the negative control (untreated SMC). The left and right images in Figure 2A show the SMC immediately after and 6 h after incubation in the absence of dypnones, respectively. The mitochondrial membrane potential was accessed by fluorescence microscopy of cells labeled with the dye DiOC6(3). DiOC6(3) is a cell-permeant, green-fluorescent, lipophilic dye which, in low concentrations, selectively stains the mitochondria of live cells.42,49−53 In the absence of any Te(IV) compounds (Figure 2A, left and right panels), SMC appeared E


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Chemical Research in Toxicology Scheme 1. Postulated Mechanism for the Reaction of Trichlorotelluro-dypnones with Thiolsa

a

A represents the associative mechanism, and B represents the dissociative mechanism. Compound 1 was chosen to represent the four trichlorotelluro-dypnones.

4 did not induce GSH depletion nor exhibit antioxidant activity, both effects that were observed for RT-03 and RT-04.16 The predominant attack on the thiol content of RLM membranes suggests that compounds 1, 2, 3, and 4 had partitioned in the lipid bilayers of mitochondria, where they reacted with the membrane proteins. Although accumulating inside the mitochondrial membranes, leading to MPTP opening, the telluranes studied here did not promote extensive aggregation of proteins when compared to the effects of RT-03 and RT-04. These results are suggestive of a more specific attack of compounds 1, 2, 3, and 4 on the proteins involved in the formation of the MPTP.55 The effects of RT-03 and compounds 1, 2, 3, and 4 that were observed in isolated rat liver mitochondria were also observed in cultured cells. These results demonstrated that the cell plasma membrane is not a barrier for the telluranes, which can enter the mitochondria leading to ΔΨ loss. The kinetics of ΔΨ loss exhibited by the trichlorotelluro-dypnones studied here are similar; however, only compound 1 led to immediate loss of ΔΨ when added to a cell suspension at the concentration of 25 μmol/L. Consistently, compound 1 was the unique dypnone that promoted the highest depletion of the mitochondrial protein thiol content. The biological effects of organotelluranes are related to the reactivity with thiols. The chemistry of tellurium has been studied by several authors, and particularly, the chemistry of different Te(IV) compounds, including a trichlorotellurane, AS101, with protein thiols was very well studied by Albeck et al. regarding kinetics, stability, and reaction mechanism.11,56,57 It is known that Te(IV) compounds react with nucleophiles such as carboxylates, alcohols, and thiols and produces (nucleophile)4Te products. In aqueous solution, the (nucleophile)4Te compounds may exchange ligands by interacting with other nucleophiles or hydrolyze to TeO2. However, exclusively

(complexes I and II) promoted by NO production by the synergistic action of interferon-γ plus tumor necrose factor-α (IFN-γ + TNF-α) does not affect trypan blue exclusion.54 In that study, the increase of lactate production by IFN-γ + TNFα-treated SMC corroborated that these cells can survive by changing aerobic metabolism to glucose fermentation.

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DISCUSSION Organochalcogen compounds of selenium and tellurium occur in several classes deriving from the oxidation states of these elements, which varies from −2 to +6.8 The thiol-reactive forms of organoselenium and organotellurium compounds depend on the diorganyldichalcogenyl compound (Nox = −1) and the organochalcogenurane (Nox = +4). Previously, the effects of RT-03 and RT-04 on isolated rat liver mitochondria were characterized. The structural characteristics of RT-03 and RT04 are compatible with the thiol reagents. Consistently, RT-03 and RT-04 were able to induce CsA-sensitive and CsAinsensitive MPT in a dose-dependent manner. Furthermore, the regulated and CsA-insensitive MPTP openings triggered by RT-03 and RT-04 were shown to be related to their reactivity with thiol groups. These compounds promoted the crosslinkage of mitochondrial membrane proteins that had been prevented by DTT and NEM. At high concentrations, the hydrophobicity of RT-03 and RT-04 was responsible for changes to the membrane organization, with repercussions on mitochondria function. A peculiar and interesting characteristic exhibited by the organotelluranes RT-03 and RT-04 was their antioxidant activity. Therefore, these telluranes led to an unusual condition in which the MPTP is formed concomitantly with efficient antioxidant protection. Similar to RT-03 and RT04, compounds 1, 2, 3, and 4 promoted dose-dependent mitochondrial swelling associated with depletion of the thiol content of RLM membranes. However, compounds 1, 2, 3, and F


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Chemical Research in Toxicology Funding

Te(SR)4 may disproportionate to Te(SR)2 and RSSR (eq 1).58,59 Te(SR)4 → Te(SR)2 + RSSR

We thank FAPESP (2010/10349-2, 2012/07456-7), CNPq (Conselho Nacional de Desenvolvimento Cienti fí co e Tecnológico), CAPES (Coordenaçaõ de Aperfeiçoamento de ́ Pessoal de Nivel Superior), and Núcleo de Bioquimica e Biotecnologia/Universidade Federal do ABC for financial support of this work.

(1)

The reaction type described by eq 1 was corroborated to occur with the catalytic thiol group of two cysteine proteases, papain, and cathepsin B and the thrichlorotellurane AS101 leading to the inhibition of the enzymes in a time- and concentration-dependent manner.11 For these enzymes, the covalent inhibition was supported by the persistence of inhibition after gel permeation chromatography. Further, AS101-inactiveted papain could be reactivated by a thiol exchange reaction with low molecular thiols such as cysteine. The reaction mechanism of Te(IV) with nucleophiles is not well understood; however, by assuming a similarity with the chemistry of phosphorus, it was previously proposed that the reaction mechanism of Te(IV) toward nucleophiles could occur by mechanisms that are similar to SN2-type and SN1-type reactions (named associative and dissociative mechanisms, respectively, Scheme 1A and B).11 The base of the associative mechanism is that Te(IV) compounds are formally pentavalent considering the four ligands and the equatorial lone pair electrons. This structural characteristic makes feasible the addition of another ligand in the equatorial plane. In a dissociative mechanism, a Te(IV) compound undergoes first the dissociation of an equatorial ligand generating a positive charge that is compensated by the equatorial lone pair electrons. The crystal and molecular structures have been determined for compounds 1, 2, and 3 (manuscript in preparation). All the three structures present intramolecular bonding between the carbonyl oxygen donor resident on the dypnone structure and the tellurium(IV) center. These compounds have pseudo-octahedral geometries and are characterized as the monomeric group of organotellurium(IV) trihalides. Therefore, similarly to that was proposed for AS101, it is more probable that a dissociative mechanism for the reaction of the trichlorotelluro-dypnones toward thiols occurs. In this structure, while the equatorial lone pair electrons favor the loss of a ligand, the octahedral structure impairs the associative mechanism (Scheme 1A). All of the trichlorotellurodypnones studied here exhibit a Te−O bond that is absent in the RT-03 structure. The differences in tellurium moieties could be related to the reactivity with proteins and the antioxidant activity for lipids. Also, it should be noted that the different effects of trichlorotelluro-dypnones on mitochondria and cultured cells were modulated by the presence and position of the methoxy groups appended to the phenyl rings. The Ca2+dependence for the depletion of the protein thiol groups suggests that the process is related to the exposure of the lateral chains of cysteine residues, facilitating their chemical attack by Te.60 The absence of methoxy groups in compound 1 does not produce the steric hindrance that might prevent access of the Te moiety to the cysteine lateral chains of mitochondrial proteins. However, the structural differences relative to the RT03 might lower the reactivity of the dypnones but are sufficient for accessing those proteins involved in MPTP assembly.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to David da Mata for technical assistance and to Ricardo Nantes Liang, B.Sc. for the software development that determined the curves of ΔΨ decay from the analysis of the images.

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ABBREVIATIONS MPTP, mitochondrial permeability transition pore; CsA, cyclosporine A; SMC, smooth muscle cells; RLM, rat liver mitochondria; ADP, adenine diphosphate; RCR, respiratory control ratio; SAR, structure−activity relationship; DTT, dithiothreitol; NEM, n-ethylmaleimide; ANT, adenine nucleotide translocase; VDAC, voltage-dependent anion channel; PAO, phenyl arsine oxide; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); EGTA, ethylene glycol tetraacetic acid; KCl, potassium chloride; DAPI, 4′,6-diamidino-2-phenylindole; DIOC6(3), 3,3′-dihexyloxacarbocyanine iodide; EC50, half maximal effective concentration; NR, neutral red

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Effects of Trichlorotelluro-dypnones on Mitochondrial Bioenergetics

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