Just Accepted by Toxicology Mechanisms and Methods Toxicity of Brominated Flame Retardants, BDE-47 and BDE-99 stems from impaired mitochondrial bioenergetics. Murilo Pazin, Lilian Cristina Pereira, Daniel Junqueira Dorta doi: 10.3109/15376516.2014.974233
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Abstract Polybrominated diphenyl ethers (PBDEs)are used as flame retardants, and they have been detected in human blood, adipose tissue and breast milk, a consequence of their physicochemical and bioaccumulative properties, as well as their high environmental persistence. Many studies reports liver toxicity related to exposure to PBDEs. In the present study, we investigated the toxicity of BDE-47 and BDE-99 at concentrations ranging from 0.1 to 50 µM in isolated rat liver mitochondria. We evaluated how incubation of a mitochondrial suspension with the PBDEs affected the mitochondrial inner membrane, membrane potential, oxygen consumption, calcium release, mitochondrial swelling, and ATP levels to find out whether the tested compound interfered with the bioenergetics of this organelle. Both PBDEs were toxic to mitochondria: BDE-47 and BDE-99 concentrations equal to or higher than 25 and 50 µM, respectively, modified all the parameters used to assess mitochondrial bioenergetics, which culminated in ATP depletion. These effects stemmed from the ability of both PBDEs to cause Membrane Permeability Transition (MPT) in mitochondria, which impaired mitochondrial bioenergetics. In particular, BDE-47, which has fewer bromine atoms in the molecule, can easily overcome biological membranes what would be responsible for the major negative effects exerted by this congener when compared with BDE-99.
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Toxicity of Brominated Flame Retardants, BDE-47 and BDE-99 stems from impaired mitochondrial bioenergetics.
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Murilo Pazina, Lilian Cristina Pereiraa, Daniel Junqueira Dortab
a
Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Análises
Clínicas, Toxicológicas e Bromatológicas, Universidade de São Paulo, Av. Bandeirantes, 3900, CEP:14040901, Ribeirão Preto – São Paulo – Brazil b
Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Departamento de Química,
Universidade de São Paulo, Av. Bandeirantes, 3900, CEP:14040901, Ribeirão Preto – São Paulo – Brazil
Corresponding author: Daniel Junqueira Dorta Phone/Fax: +55–16–36020544 Email: [email protected]
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ABSTRACT Polybrominated diphenyl ethers (PBDEs)are used as flame retardants, and they have been
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detected in human blood, adipose tissue and breast milk, a consequence of their physicochemical and bioaccumulative properties, as well as their high environmental persistence. Many studies reports liver toxicity related to exposure to PBDEs. In the present study, we investigated the toxicity of BDE-47 and BDE-99 at concentrations ranging from 0.1 to 50 µM in isolated rat liver mitochondria. We evaluated how incubation of a mitochondrial suspension with the PBDEs affected the mitochondrial inner membrane, membrane potential, oxygen consumption, calcium release, mitochondrial swelling, and ATP levels to find out whether the tested compound interfered with the bioenergetics of this organelle. Both PBDEs were toxic to mitochondria: BDE-47 and BDE-99 concentrations equal to or higher than 25 and 50 µM, respectively, modified all the parameters used to assess mitochondrial bioenergetics, which culminated in ATP depletion. These effects stemmed from the ability of both PBDEs to cause Membrane Permeability Transition (MPT) in mitochondria, which impaired mitochondrial bioenergetics. In particular, BDE-47, which has fewer bromine atoms in the molecule, can easily overcome biological membranes what would be responsible for the major negative effects exerted by this congener when compared with BDE-99. Key-words: PBDEs, mitochondria, ATP depletion, bioenergetics
INTRODUCTION
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Flame retardants are applied as a safety measure to prevent fires. These compounds have been used in the electronics, textile, and automotive industries, among others; they are also added to materials such as wood, plastic, paper, cooking utensils, just to mention a few examples (Chevrier et al., 2010; Angioni et al., 2013).
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The demand for flame retardants has increased: for instance, between 2000 and 2011 application of these substances has risen from 310,000 to 2 million ton/year, representing approximately 640% growth (Frol, 2013). According to this last survey, brominated flame retardants constituted about 20% (400,000 ton/year) of all the flame retardants used worldwide (Frol, 2013). Nowadays, over 175 chemicals are classified as flame retardants, being the largest groups the halogenated (brominated and chlorinated) and non-halogenated (derivatives of phosphorus, nitrogen, melamines) compounds (Ravichandran et al., 2011). Polybrominated diphenyls ethers (PBDEs) are one of the most often employed – they effectively prevent of flame from spreading and are inexpensive (Pestana et al., 2008). Unfortunately, PBDEs persist in the environment because they can bioaccumulate and biomagnify, which characterize them as Persistent Organic Pollutants (POPs) (She et al., 2013). Apart from this persistence, several studies have reported that they are potentially toxic, indeed, they underlie hepatotoxicity (Nash, Szabo, Carey, 2013), immunological changes (Fair et al., 2012), neurotoxicity (Slotkin et al., 2013) and endocrine disruption (Yu et al., 2011). On the basis of literature reports on PBDEs hepatotoxicity, mitochondria isolated from rat liver constitute an excellent experimental model (Pereira et al., 2012). Mitochondria recognizably are cellular energy-producing organelles; they also play a key role in the maintenance of many cellular functions. (Otera, Ishihara, Mihara, 2013).
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BDE-47 and BDE-99 are the most common PBDEs in nature. Additionally, they have high toxic potential. Indeed, several studies with cell cultures have concluded that BDE-47 and BDE99 can induce apoptotic cell death, suggesting mitochondria are a primary target of cell toxicity (Souza et al., 2013; Hu, Hu, Xu, 2009).
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A large number of publications have shown that some mitochondrial disorders are closely associated with the toxicity of various compounds (Rana, Rera, Walker, 2013; Pereira, Souza, Dorta, 2013; Huang et al., 2011), and that many of these effects are correlated with defects in the respiratory chain functionality. Toxic compounds may reduce ATP production, generate free radicals and alter the mechanisms regulating intracellular calcium. These cellular events may induce DNA, proteins, and lipids oxidation as well as, opening of permeability transition pores, all of which underlie the mechanisms of cell death by apoptosis (Lemasters, 1999). Therefore, this study aimed to evaluate the toxic mechanisms of action of BDE-47 (tetraBDE) and BDE-99 (penta-BDE) on the bioenergetics of mitochondria using mitochondria isolated from rat liver.
MATERIAL AND METHODS Standard solutions of congeners of PBDEs, BDE-47 and BDE-99 (Accustandard, USA), were used. BDE-47 and BDE-99 doses between 0.1 and 50 µM were employed during the tests. The toxic potential of these compounds was evaluated using mitochondria isolated from rat liver cells.
Chemicals
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The reagents rotenone; carbonyl cyanide 3-chlorophenylhydrazone (CCCP); succinate; glutamate; malate; adenosine 5-diphosphate (sodium salt) (ADP); safranin-ο; 1-anilino-8naphthalene sulfonate salt (ANS); diphenylhexatriene (DPH); 1-(4-(trimethylamine)phenyl)-6phenylhexa-1,3,5 triene (TMA-DPH); ethylene glycol bis(b-aminoethyl ether)-N,N,N,N-
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tetraacetic acid (EGTA); Ruthenium Red; dimethyl sulfoxide (DMSO); tert-butyl hydroperoxide solution (t-BOOH), and Kit firefly luciferin–luciferase assay system for ATP-Assay were purchased from Sigma-Aldrich Chemical Co (St Louis, MO, USA). The reagents 2,7dichlorodihydrofluorescein diacetate (H2DCFDA) and Calcium Green 5N were acquired by Molecular Probes (OR, USA). Both PBDEs were obtained from Sigma-Aldrich Chemical Co (St Louis, MO, USA). All the other reagents were of the highest commercial purity degree. The amounts of DMSO required to dissolve both PBDEs did not affect the assays (data not show). All the stock solutions were prepared using glass-distilled deionized water.
Animal Care Male Wistar rats weighing approximately 180-200 g were used in this study. A maximum of four rats per cage were maintained under standard laboratory conditions; water and food were provided ad libitum. The experimental protocols were approved by the Ethics Committee for the Use of Laboratory Animals of the University of São Paulo – Ribeirão Preto campus under Protocol number: 12.1.227.53.0.
Mitochondria Isolation
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The animals were euthanized, and their livers (10–15 g) were immediately removed; sliced into 50 mL of buffer containing sucrose 250 mM, EGTA 1 mM and HEPES–KOH 10 mM, pH 7.2; and homogenized three times at 1 min intervals in a Potter–Elvehjem homogenizer. Rat liver mitochondria were isolated by standard differential centrifugation (Pedersen et al., 1978). The
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homogenates were centrifuged at 580 × g for 5 min; the supernatant was further centrifuged at 10 300 × g for 10 min. The pellets were suspended in 10 mL of buffer containing 250 mM sucrose, EGTA 0.3 mM, and HEPES–KOH 10 mM, pH 7.2, and centrifuged at 3400 × g for 15 min. The final mitochondrial pellet was suspended in 1 mL of buffer containing sucrose 250 mM and HEPES–KOH 10 mM, pH 7.2, and used within 3 h. The mitochondrial protein content was determined by the biuret reaction. During all the procedures, the mitochondria were maintained at 4 ºC.
Evaluation of how BDE-47 and BDE-99 interact with the mitochondrial membrane DPH, TMA-DPH, and ANS insertion into membranes elicits a fluorescence response (F), the Stern–Volmer equation describes its static quenching as: F0/F = 1 + KSV (Q), where F0 and F are fluorescence intensities in the absence and presence of the quencher, respectively; and KSV is the Stern–Volmer constant. To evaluate interaction of PBDEs with the mitochondrial membrane, the mitochondria (1 mg protein.mL-1) were incubated with DPH 0.5 µM, ANS 75 µM, and TMA-DPH 1.04 µM in the standard reaction buffer (sucrose 125 mM, KCl 65 mM, HEPES-KOH 10 mM, pH 7.2) at 30 ºC, followed by addition of varying amounts of PBDEs in a final volume of 2 mL. The fluorescence was measured on an F-4500 spectrofluorometer (Hitachi, Tokyo, Japan) at excitation and emission wavelengths of 360/430 nm, 380/485 nm, and 362/432 for DPH, ANS, and TMA-DPH, respectively (Rodrigues et al., 2002).
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Continuous-monitoring mitochondrial assays Mitochondrial respiration was polarographically monitored using an oxygraph (Hansatech, Norfolk, England) equipped with a Clark-type oxygen electrode (Chance, Willians, 1956). The
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mitochondria (1 mg protein.mL-1) were incubated in 1 mL of the standard reaction buffer containing EGTA 0.5 mM, and K2HPO4 10 mM, pH 7.2 at 30 ºC, using glutamate and malate 5 mM and potassium succinate 5 mM (and rotenone 2.5 µM) as the oxidizable substrates for complexes I and II, respectively. State III respiration was initiated with 400 nmoles of ADP, and PBDEs were added at the time of testing (Cain, Skilleter, 1987).
The Mitochondrial Membrane Potential (ΔΨ) was spectrofluorometrically monitored using 1 mg of protein mL-1 and safranin-o 10 µM as the probe. An F-4500 spectrofluorometer (Hitachi, Tokyo, Japan) with the 495/586 nm excitation/emission wavelength pair (Akerman, Wikstrom, 1976), was used for this purpose. A standard reaction buffer and K2HPO4 10 mM, pH 7.2, was employed at the time of testing. After membrane potential was generated, aliquots of the PBDEs congeners at different concentrations were added to evaluate their effect on the kinetics of mitochondrial membrane potential. At the end of the incubation period, the membrane potential was completely dissipated using CCCP (1 µM).
Mitochondrial swelling was estimated from the decrease in the apparent absorbance at 540 nm in the presence of 0.4 mg of protein mL-1 using a Model DU-70 spectrophotometer (Beckman, Coulter Inc., Fullerton, CA, U.S.A.). Along with the mitochondria, different concentrations of BDE-47 and BDE-99 were added to analyze whether they interfered with
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mitochondrial swelling. To understand the mechanisms that trigger the process better, Cyclosporine A (CsA) 1 µM, Ruthenium Red (RR) 0.5 µM, and N-ethylmaleimide (NEM) 25 µM were used as modulators of the process. The PBDEs were added to the standard reaction
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buffer together, with K2HPO4 10 mM, pH 7.2 at the time of testing (Lemasters et al., 1987)
The Mitochondrial Ca2+ efflux was spectrofluorimetrically monitored using Calcium Green 5N 150 nM as probe, 506/531 nm excitation/emission wavelength pair, and 1 mg of protein mL1
. The PBDEs in the standard reaction buffer plus and K2HPO4 10 mM, pH 7.2, were added at the
time of testing. The total calcium efflux was performed using CCCP (1 μM). When the mitochondria capture Calcium Green 5N linked to Ca2+ the fluorescence detected is diminished, than if the fluorescence increases again, means that mitochondria cannot handle the calcium (Rajdev, Reynolds, 1993).
Determination of ATP levels Mitochondrial ATP levels were determined using the firefly luciferin–luciferase assay system (Thore, 1979). The mitochondrial suspension (1 mg protein.mL-1) was centrifuged at 9000 × g for 5 min, at 4 °C, and the pellet was treated with 1 mL of ice-cold 1 M HClO4. After centrifugation at 14 000 × g for 5 min at 4 °C, aliquots (100 μL) of the supernatants were neutralized with 70 μL of KOH 2 M suspended in Tris–HCl 100 mM, pH 7.8 (final volume = 1 mL) and centrifuged again. The bioluminescence was measured in the supernatant using a Sigma–Aldrich assay kit according to the manufacturer’s instructions, on an AutoLumat LB953 Luminescence photometer (Perkin Elmer Life Sciences, Wildbad, Germany). Statistical Analysis
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The experimental data were evaluated by analysis of variance (ANOVA), followed by a post-hoc analysis, including the use of Dunnett’s test, to compare the several treated groups with respective control. The software GraphPrism version 5.0 for Windows was employed. Results
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with p < 0.05 were considered statistically significant.
RESULTS The two tested congeners, BDE-47 and BDE-99, significantly impacted the fluorescence of the three probes used to assess these PBDEs ability to insert in the mitochondrial membrane (Figure 1). The results suggested that both congeners were able to interact with either the hydrophobic or hydrophilic region of the mitochondrial inner membrane, thereby interfering with the activity of proteins and other molecules present in these regions. The tested compounds affected the hydrophobic probe DPH the most: at 50 µM, BDE-47 and BDE-99 reduced the fluorescence of this probe by approximately 20% as compared with the control. To assess mitochondrial respiration, it is necessary to evaluate whether the PBDEs present in the inner mitochondrial membrane can alter the electron transport chain. More specifically, we conducted an assay to confirm the ability of the target PBDEs to insert and interact with complexes I and II of the respiratory chain. Table 1 shows how much BDE-47 and BDE-99 affected oxygen consumption by mitochondria isolated from rat liver using glutamate and malate as oxidizable substrates for site I, and succinate as oxidizable substrate for complex II. In the latter case, addition of rotenone inhibited complex I, to prevent reverse flow of electrons. The respiratory control ratio (RCR), values revealed decreased oxygen consumption at BDE-47 concentrations of 25 and 50 µM. BDE-99 elicited the same effects, at the higher concentration only.
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The interaction of both PBDEs with the inner mitochondrial membrane directly correlated with dissipation of the mitochondrial membrane potential. Figure 2 shows a significantly different effect after addition of BDE-47 (25, and 50 µM) (Fig. 2A) and BDE-99 (50 µM) (Fig. 2B). .
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Evaluation of mitochondrial swelling demonstrated the ability of PBDEs to modified the permeability of the mitochondrial membrane and consequently its integrity and selectivity. Figure 3 evidenced statistically significant differences (p < 0.05) between the treated and the control group (without addition of PBDE). BDE-47 (Fig. 3A) exerted a significant effect at the two highest concentrations (25 and 50 µM); for BDE-99 (Fig. 3B), this effect occurred only at 50 µM. Modulators partially inhibited this swelling, showing that this effect might depend on calcium. Because calcium homeostasis in the mitochondria is essential for proper operation, we examined whether the loss of permeability by these organelles was associated with calcium release. The presence of BDE-47 (25 and 50 µM) and BDE-99 (50 µM), prompted significant release of calcium ion. This was in line with the other analyzed parameters and indicated that BDE-47 and BDE-99 directly participated in dysregulation of mitochondrial calcium (Fig. 4). Finally, investigation of mitochondrial ATP levels after incubation of the mitochondria with both PBDEs (Fig. 5) revealed significantly lower mitochondrial ATP levels upon interaction with BDE-47 (10, 25, and 50 µM) and BDE-99 (25 and 50 µM). Hence, mitochondrial respiratory chain disruption can lead to reduced ATP levels. DISCUSSION The liver is the major site of foreign substances metabolism and therefore an constitutes important target for damaging effects (Jaeschke et al., 2002). Mitochondrial dysfunction is a
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relevant mechanism in the pathogenesis of many toxicants in mammals, especially hepatotoxicity (Amacher, 2005). Mitochondria play a central role in cell life and death. At the mitochondrial level, several potential compounds can cause toxicity. The results of this work have demonstrated that PBDEs
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are some of such compounds. Like many other compounds, description of the complete toxicological profile of PBDEs is also lacking in the literature. Our results indicated that BDE-47 (tetra-BDE) and BDE-99 (penta-BDE) interacted with the mitochondrial membrane and localized in the inner mitochondrial membrane, possibly lowering mitochondrial membrane fluidity and impairing energy processes in the mitochondria. Indeed, these processes are sensitive to membrane organization (Dorta et al., 2005). These results were in line with the ability of PBDEs to reduce oxygen consumption by the mitochondria. Analysis of mitochondrial respiration revealed that both BDE-47 and BDE-99 decreased oxygen consumption by the analyzed electron transport chain complexes. This effect stemmed from inhibition of the respiratory chain – results indicated diminished state III of respiration which is the moment when mitochondria convert ADP to ATP. Nakagawa and colleagues (2007) observed the same effect when they evaluated the toxic effects of another compound also classified as POPs – Tetrabromo Bisphenol A; ATP depletion occurred and the membrane potential decreased, indicating changes in the inner mitochondrial membrane. In this work, the toxicity of BDE-47 and BDE-99 directly related to their ability to interact with the inner mitochondrial membrane, which interfered with mitochondrial bioenergetics. The changes in mitochondrial respiration also referred to the loss of membrane potential elicited by both PBDEs. This close relationship between respiration/membrane potential could be interpreted as oxidation of substrates in the mitochondrial matrix and outside the inner
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membrane mediated by the electron transport chain, which could also be defined as a system for transmembrane electron transport. On the other hand, this transmembrane system should promote proton transfer from inside to outside the mitochondrion, to generate a membrane potential. From the energy standpoint, this membrane electrochemical potential would be
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necessary for ATP synthesis (La Piana et al., 1998). In addition, some toxic agents could affect the energy balance in the cells by increasing ATP consumption and/or reducing ATP synthesis. We analyzed this phenomenon by measuring ATP depletion, to find out whether the tested compounds affected this important mitochondrial mechanism. According to our results, the studied PBDEs significantly reduced the amount of ATP. Pereira, Souza and Dorta (2013) described the same outcome for BDE-100: its interaction with the mitochondrial membrane depleted ATP levels and altered respiratory parameters. Labeling with the probe calcium green 5N, to assess calcium release from mitochondrion, showed that the tested congeners also affected the mitochondrial calcium homeostasis: BDE-47 at 25 and 50 µM and BDE-99 at 50 µM interfered with this parameter and lead to excessive calcium release during the assay. Our result was similar to that found by Coburn, Currás-Collazo and Kodavanti (2008). These authors verified that BDE-47 also influenced calcium homeostasis in mitochondria isolated from nerve cells, which affected the cell signaling system. Hence, this class of compounds affects calcium homeostasis in both liver tissue and cells of the central nervous system. The impact of PBDEs on calcium homeostasis probably originated from mitochondrial swelling induced by BDE-47 and BDE-99. In other words, changes in calcium efflux and mitochondrial swelling were directly related to loss of mitochondrial membrane permeability. In fact, as indicated by the test involving swelling modulators, such modulators completely
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suppressed the effect of PBDEs. This outcome pointed to possible mechanisms of action for BDE-47 and BDE-99. In the presence of CsA, these PBDEs did not induce swelling. Therefore, it was possible to infer that in the absence of CsA these flame retardants may have caused swelling via opening of permeability transition pores (PTP).Therefore, CsA prevented
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cyclophilin D (CypD) from binding to ANT in the inner membrane which inhibited mitochondrial swelling to occur (Rao, Carlson, Yan, 2014). The test that employed RR as inhibitor showed that neither BDE-47 nor BDE-99 caused swelling. This result corroborated those obtained in the presence of CsA: PTP opening usually depends on calcium. As observed by Wang and colleagues (2005), even RR 0.1 M was able to inhibit the uniporter calcium transport channel. Blockage of this channel hindered calcium into entry the mitochondria and the consequent opening of PTP. According to the literature, the mitochondrial permeability transition (MPT) involves sudden increase in inner mitochondrial membrane permeability to solutes with molecular masses up to 1500 Da, due to opening of a highly conductive channel that is sensitive to Cyclosporin A and is Ca2+-dependent (Rasola, Bernardi, 2007). Several physiological and pathogenic processes could trigger MTP in both intact cells and isolated mitochondria (Hajnoczky, Davies, Madesh, 2003). In addition, numerous stressful responses (calcium dysregulation, reactive oxygen species, genotoxic stress, activation of proteases) could also trigger MPT, suggesting that mitochondria act as a universal stress sensor (Roberts, Goping, Bleackley, 2003). Our results suggested that MPT generated PTP, which in turn depleted mitochondrial ATP levels; PTP opening modified ATP levels due to an electrochemical imbalance. As already reported in the literature, ATP depletion is one of the key events induced by toxic compounds as a consequence of suppressed electron transport/oxidative phosphorylation, mitochondrial
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membrane potential dissipation, and calcium homeostasis dysfunction (Mingatto et al., 2002). In summary, our results have demonstrated that exposure to the PBDE congeners (BDE-47 and BDE-99) evaluated in this study modified bioenergetic parameters and damaged the mitochondria. BDE-47 had more potent toxic effects as compared with BDE-99; indeed, for all
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the tested bioenergetic parameters BDE-47 was able to exert adverse effects at lower concentrations than BDE-99. This different toxicity may be linked to the quantity and position of bromine atoms, in the PBDE. Models used to study the structure-activity relationship of PBDE have previously demonstrated that less brominated PBDEs with bromine atoms in the ortho position and no bromine atoms in the meta position were generally more toxic (Harju et al., 2007). In our case, BDE-47 has fewer bromine atoms and these atoms are in positions that made this congener more toxic than BDE-99 due to its facility to overcome biological membranes. It is important to notice that the effects observed here appears on higher levels that the ones still being found in humans or animals tissue. However, even though, they were listed as persistent organic pollutants (POP) by the Stockholm Convention in 2009, and banned from Europe since 2004, because of their physical chemical characteristics, there is also strong evidence that the levels of some of these flame retardants are increasing, doubling every 3 to 5 years (Richardson, 2008). These facts demonstrates that PBDEs are substances that are still in the process of accumulating in the environment and biota with biomagnifaction through the top of the food chain.
CONCLUSION This work provides relevant additional information about the toxicodynamics of two of the main representatives of brominated flame retardants, BDE-47 and BDE-99. We have concluded
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that both PBDEs display high toxic potential at their highest tested concentrations, thereby affecting mitochondrial bioenergetics. The PBDEs act on the inner mitochondrial membrane, interfering in parameters such as oxygen consumption, mitochondrial membrane potential, calcium homeostasis, and energy production. As judged from the bioenergetics results, BDE-47,
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a tetra-BDE, exhibits the highest toxicity as compared with BDE-99, a penta-BDE. The more potent toxic effects originate from the lower amount and position of bromine atoms in the structure of tetra-BDE. Elucidation of the structure-activity relationship of these compounds will facilitate understanding the mechanisms through which PBDEs exert their toxic effects.
ACKNOWLEDGEMENTS The authors thank Professor Carlos Curti, PhD, Professor Luciane Carla Alberici, PhD, and the Biochemistry Laboratory of the Faculty of Pharmaceutical Sciences of Ribeirão Preto - USP / Brazil for technical support. The authors thank for the financial support – grant #2009/06912-6 and #2012/04542-0 São Paulo Research Foundation (FAPESP).
DECLARATION OF INTEREST The authors report no declarations of interest
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Lemasters JJ, Digiuseppi J, Nieminem AL, Herman B. (1987). Blebbing. free calcium and mitochondrial membrane potential preceding cell death hepatocytes. Nature, 325, 78-81. Mingatto FE, Rodrigues T, Pigoso AA, Uyemura SA, Curti C, Santos AC. (2002). The critical role of mitochondrial energetic impairment in the toxicity of nimesulide to hepatocytes. J Pharmacol Exp Ther, 303, 601-607.
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Nakagawa Y, Suzuki T, Ishii H, Ogata A. (2007). Biotransformation and cytotoxicity of a brominated flame retardant, tetrabromobisphenol A, and its analogues in rat hepatocytes. Xenobiotica, 37, 693-708. Nash JT, Szabo DT, Carey GB. (2013). Polybrominated diphenyl ethers alter hepatic phosphoenolpyruvate carboxykinase enzyme kinetics in male Wistar rats: implications for lipid and glucose metabolism. J Toxicol Environ Health A, 76, 142-56. Otera H, Ishihara N, Mihara K. (2013). New insights into the function and regulation of mitochondrial fission. Biochim Biophys Acta, 1833, 1256-68. Pedersen PL, Greenawalt JW, Reynafarje B, Hullihen J, Decker GL, Soper JW, Bustamente E. (1978). Preparation and characterization of mitochondria and submitochondrial particles of rat liver and liver-derived tissues. Methods Cell Biol, 20, 411-81. Pereira LC, Souza AO, Dorta DJ. (2013). Polybrominated Diphenyl Ether Congener (BDE-100) Induces Mitochondrial Impairment. Basic Clin Pharmacol Toxicol, 112, 418-24. Pereira LC, Souza AO, Pazin M, Dorta DJ. (2012). Mitocôndria como alvo para avaliação de toxicidade de xenobiótico. Revista Brasileira de Toxicologia, 25, 1-14. Pestana CR, Borges KB, Fonseca P, Oliveira DP. (2008). Risco ambiental da aplicação de éteres de difenilaspolibromadas como retardantes de chama. Rev Bras Toxicol, 21, 41-8. Rajdev S, Reynolds IJ. (1993). Calcium green-5N, a novel fluorescent probe for monitoring high intracellular free Ca2+ concentrations associated with glutamate excitotoxicity in cultured rat brain neurons. Neurosci Lett., 162, 149-52. Rana A, Rera M, Walker DW. (2013). Parkin overexpression during aging reduces proteotoxicity, alters mitochondrial dynamics, and extends lifespan. Proc Natl Acad Sci U S A, 110, 8638-43. Rao VK, Carlson EA, Yan SS. (2014). Mitochondrial permeability transition pore is a potential drug target for neurodegeneration. Biochim Biophys Acta, 1842, 1267-72. Rasola A, Bernardi P. (2007). The mitochondrial permeability transition pore and its involvement in cell death and in disease pathogenesis. Apoptosis, 12, 815-833. Ravichandran S, Bouldin RM, Kumar J, Nagarajan R. (2011). A renewable waste material for the synthesis of a novel non-halogenated flame retardant polymer. J Cleaner Product, 19, 454-58. Richardson, S.D. (2008). Environmental Mass Spectrometry: Emerging Contaminants and Current Issues. Analytical Chemistry, 80, 4373–4402. Roberts DL, Goping IS, Bleackley RC. (2003). Mitochondrial at the heart of the cytotoxic attack. Biochem Biophys Res Commun, 304, 513-18.
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Rodrigues T, Santos AC, Pigoso AA, Mingatto FE, Uyemura SA, Curti C. (2002). Thioridazine interacts with the membrane of mitochondria acquiring antioxidant activity toward apoptosis-potentially implicated mechanisms. Br J Pharmacol, 136, 136–42.
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She YZ, Wu JP, Zhang Y, Peng Y, Mo L, Luo XJ, Mai BX. (2013). Bioaccumulation of polybrominated diphenyl ethers and several alternative halogenated flame retardants in a small herbivorous food chain. Environ Pollut, 174, 164-70. Slotkin TA, Card J, Infante A, Seidler FJ. (2013). BDE99 (2,2',4,4',5-pentabromodiphenyl ether) suppresses differentiation into neurotransmitter phenotypes in PC12 cells. Neurotoxicol Teratol, 37, 13-7. Souza AO, Pereira LC, Oliveira DP, Dorta DJ. (2013). BDE-99 congener induces cell death by apoptosis of human hepatoblastoma cell line - HepG2. Toxicol In Vitro, 27, 580-87. Thore, A. (1979). Technical aspects of bioluminescent firefly luciferase assay of ATP. Sci Tools, 26, 30-4. Wang C, Lin Z, Dong Q, Lin Z, Lin K, Wang J, Huang J, Huang X, He Y, Huang C, Yang D, Huang C. (2012). Polybrominated diphenyl ethers (PBDEs) in human serum from Southeast China. Ecotoxicol Environ Saf, 78, 206-11. Yu L, Lam JCW, Guo Y, Wu RSS, Lam PKS, Zhou B. (2011). Parental transfer of polybrominated diphenyl ethers (pbdes) and thyroid endocrine disruption in zebrafish. Environ Sci Technol, 45, 10652-9.
FIGURE LEGENDS Figure 1. Interaction of BDE-47 (A) and BDE-99 (B) with the inner mitochondrial membrane as measured by the probes ANS, TMA-DPH, and DPH, as described in materials and methods. The data are presented as the means ± SEM of a series of three experiments.
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Figure 2. A) Concentration-response effect of BDE-47 (A) and BDE-99 (B) on dissipation of the mitochondrial membrane potential in mitochondria isolated from rat liver (1.0 mg protein mL-1) as evaluated using Safranin-o as probe, incubated as described in materials and methods. Data represent the mean ± SEM of three determinations with different mitochondrial preparations,
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relative to the control in the absence of the compound. *Statistically significant results (p < 0.05) as compared with the control.
Figure 3. Effect of BDE-47 (A) and BDE-99 (B) on mitochondrial swelling in mitochondria isolated from rat liver (0.4 mg protein mL-1), incubated as described in materials and methods. The maximum swelling was observed as induced by inorganic phosphate (Pi) 1 µM. The figure on the right shows how modulators Cyclosporin A (CsA),N-ethylmaleimide (NEM), and Ruthenium Red (RR) affect the action of PBDEs. Data represent the mean ± SEM of three determinations with different mitochondrial preparations, relative to the control in the absence of the compound. *Statistically significant results (p < 0.05) as compared with the control.
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Figure 4. Concentration-response effect of BDE-47 (A) and BDE-99 (B) on the efflux of calcium from mitochondria isolated from rat liver (1.0 mg protein mL-1) as measured with Calcium Green 5N probe, incubated as described in materials and methods. Points represent the mean ± SEM of three determinations with different mitochondrial preparations as compared with the control. *Statistically significant results (p < 0.05) as compared with the control. The total release of calcium induced by CCCP was 1 µM.
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Figure 5. Concentration-response effect of BDE-47 (A) and BDE-99 (B) on ATP levels in mitochondria isolated from rat liver (1.0 mg protein mL-1) as evaluated using the luminescence obtained from the reaction of luciferin/luciferase, incubated as described in materials and methods. Data represent the mean ± SEM of three determinations with different mitochondrial preparations, relative to the control in the absence of the compound. *Statistically significant results (p < 0.05) as compared with the control. The fall in ATP levels induced by CCCP was 1 µM.
Table 1. Respiratory parameters: state 3 (V3), state 4 (V4) and respiratory control ratio (RCR) using glutamate and malate for complex I, and succinate for complex II in mitochondria isolated 21
from rat liver, under the influence of BDE-47 and BDE-99. Data represent the mean ± SEM of three determinations with different mitochondrial preparations. *Means different from the
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control (no addition of PBDEs) in accordance with Dunett test (p
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Abstract Polybrominated diphenyl ethers (PBDEs)are used as flame retardants, and they have been detected in human blood, adipose tissue and breast milk, a consequence of their physicochemical and bioaccumulative properties, as well as their high environmental persistence. Many studies reports liver toxicity related to exposure to PBDEs. In the present study, we investigated the toxicity of BDE-47 and BDE-99 at concentrations ranging from 0.1 to 50 µM in isolated rat liver mitochondria. We evaluated how incubation of a mitochondrial suspension with the PBDEs affected the mitochondrial inner membrane, membrane potential, oxygen consumption, calcium release, mitochondrial swelling, and ATP levels to find out whether the tested compound interfered with the bioenergetics of this organelle. Both PBDEs were toxic to mitochondria: BDE-47 and BDE-99 concentrations equal to or higher than 25 and 50 µM, respectively, modified all the parameters used to assess mitochondrial bioenergetics, which culminated in ATP depletion. These effects stemmed from the ability of both PBDEs to cause Membrane Permeability Transition (MPT) in mitochondria, which impaired mitochondrial bioenergetics. In particular, BDE-47, which has fewer bromine atoms in the molecule, can easily overcome biological membranes what would be responsible for the major negative effects exerted by this congener when compared with BDE-99.
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Toxicity of Brominated Flame Retardants, BDE-47 and BDE-99 stems from impaired mitochondrial bioenergetics.
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Murilo Pazina, Lilian Cristina Pereiraa, Daniel Junqueira Dortab
a
Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Análises
Clínicas, Toxicológicas e Bromatológicas, Universidade de São Paulo, Av. Bandeirantes, 3900, CEP:14040901, Ribeirão Preto – São Paulo – Brazil b
Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Departamento de Química,
Universidade de São Paulo, Av. Bandeirantes, 3900, CEP:14040901, Ribeirão Preto – São Paulo – Brazil
Corresponding author: Daniel Junqueira Dorta Phone/Fax: +55–16–36020544 Email: [email protected]
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ABSTRACT Polybrominated diphenyl ethers (PBDEs)are used as flame retardants, and they have been
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detected in human blood, adipose tissue and breast milk, a consequence of their physicochemical and bioaccumulative properties, as well as their high environmental persistence. Many studies reports liver toxicity related to exposure to PBDEs. In the present study, we investigated the toxicity of BDE-47 and BDE-99 at concentrations ranging from 0.1 to 50 µM in isolated rat liver mitochondria. We evaluated how incubation of a mitochondrial suspension with the PBDEs affected the mitochondrial inner membrane, membrane potential, oxygen consumption, calcium release, mitochondrial swelling, and ATP levels to find out whether the tested compound interfered with the bioenergetics of this organelle. Both PBDEs were toxic to mitochondria: BDE-47 and BDE-99 concentrations equal to or higher than 25 and 50 µM, respectively, modified all the parameters used to assess mitochondrial bioenergetics, which culminated in ATP depletion. These effects stemmed from the ability of both PBDEs to cause Membrane Permeability Transition (MPT) in mitochondria, which impaired mitochondrial bioenergetics. In particular, BDE-47, which has fewer bromine atoms in the molecule, can easily overcome biological membranes what would be responsible for the major negative effects exerted by this congener when compared with BDE-99. Key-words: PBDEs, mitochondria, ATP depletion, bioenergetics
INTRODUCTION
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Flame retardants are applied as a safety measure to prevent fires. These compounds have been used in the electronics, textile, and automotive industries, among others; they are also added to materials such as wood, plastic, paper, cooking utensils, just to mention a few examples (Chevrier et al., 2010; Angioni et al., 2013).
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The demand for flame retardants has increased: for instance, between 2000 and 2011 application of these substances has risen from 310,000 to 2 million ton/year, representing approximately 640% growth (Frol, 2013). According to this last survey, brominated flame retardants constituted about 20% (400,000 ton/year) of all the flame retardants used worldwide (Frol, 2013). Nowadays, over 175 chemicals are classified as flame retardants, being the largest groups the halogenated (brominated and chlorinated) and non-halogenated (derivatives of phosphorus, nitrogen, melamines) compounds (Ravichandran et al., 2011). Polybrominated diphenyls ethers (PBDEs) are one of the most often employed – they effectively prevent of flame from spreading and are inexpensive (Pestana et al., 2008). Unfortunately, PBDEs persist in the environment because they can bioaccumulate and biomagnify, which characterize them as Persistent Organic Pollutants (POPs) (She et al., 2013). Apart from this persistence, several studies have reported that they are potentially toxic, indeed, they underlie hepatotoxicity (Nash, Szabo, Carey, 2013), immunological changes (Fair et al., 2012), neurotoxicity (Slotkin et al., 2013) and endocrine disruption (Yu et al., 2011). On the basis of literature reports on PBDEs hepatotoxicity, mitochondria isolated from rat liver constitute an excellent experimental model (Pereira et al., 2012). Mitochondria recognizably are cellular energy-producing organelles; they also play a key role in the maintenance of many cellular functions. (Otera, Ishihara, Mihara, 2013).
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BDE-47 and BDE-99 are the most common PBDEs in nature. Additionally, they have high toxic potential. Indeed, several studies with cell cultures have concluded that BDE-47 and BDE99 can induce apoptotic cell death, suggesting mitochondria are a primary target of cell toxicity (Souza et al., 2013; Hu, Hu, Xu, 2009).
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A large number of publications have shown that some mitochondrial disorders are closely associated with the toxicity of various compounds (Rana, Rera, Walker, 2013; Pereira, Souza, Dorta, 2013; Huang et al., 2011), and that many of these effects are correlated with defects in the respiratory chain functionality. Toxic compounds may reduce ATP production, generate free radicals and alter the mechanisms regulating intracellular calcium. These cellular events may induce DNA, proteins, and lipids oxidation as well as, opening of permeability transition pores, all of which underlie the mechanisms of cell death by apoptosis (Lemasters, 1999). Therefore, this study aimed to evaluate the toxic mechanisms of action of BDE-47 (tetraBDE) and BDE-99 (penta-BDE) on the bioenergetics of mitochondria using mitochondria isolated from rat liver.
MATERIAL AND METHODS Standard solutions of congeners of PBDEs, BDE-47 and BDE-99 (Accustandard, USA), were used. BDE-47 and BDE-99 doses between 0.1 and 50 µM were employed during the tests. The toxic potential of these compounds was evaluated using mitochondria isolated from rat liver cells.
Chemicals
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The reagents rotenone; carbonyl cyanide 3-chlorophenylhydrazone (CCCP); succinate; glutamate; malate; adenosine 5-diphosphate (sodium salt) (ADP); safranin-ο; 1-anilino-8naphthalene sulfonate salt (ANS); diphenylhexatriene (DPH); 1-(4-(trimethylamine)phenyl)-6phenylhexa-1,3,5 triene (TMA-DPH); ethylene glycol bis(b-aminoethyl ether)-N,N,N,N-
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tetraacetic acid (EGTA); Ruthenium Red; dimethyl sulfoxide (DMSO); tert-butyl hydroperoxide solution (t-BOOH), and Kit firefly luciferin–luciferase assay system for ATP-Assay were purchased from Sigma-Aldrich Chemical Co (St Louis, MO, USA). The reagents 2,7dichlorodihydrofluorescein diacetate (H2DCFDA) and Calcium Green 5N were acquired by Molecular Probes (OR, USA). Both PBDEs were obtained from Sigma-Aldrich Chemical Co (St Louis, MO, USA). All the other reagents were of the highest commercial purity degree. The amounts of DMSO required to dissolve both PBDEs did not affect the assays (data not show). All the stock solutions were prepared using glass-distilled deionized water.
Animal Care Male Wistar rats weighing approximately 180-200 g were used in this study. A maximum of four rats per cage were maintained under standard laboratory conditions; water and food were provided ad libitum. The experimental protocols were approved by the Ethics Committee for the Use of Laboratory Animals of the University of São Paulo – Ribeirão Preto campus under Protocol number: 12.1.227.53.0.
Mitochondria Isolation
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The animals were euthanized, and their livers (10–15 g) were immediately removed; sliced into 50 mL of buffer containing sucrose 250 mM, EGTA 1 mM and HEPES–KOH 10 mM, pH 7.2; and homogenized three times at 1 min intervals in a Potter–Elvehjem homogenizer. Rat liver mitochondria were isolated by standard differential centrifugation (Pedersen et al., 1978). The
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homogenates were centrifuged at 580 × g for 5 min; the supernatant was further centrifuged at 10 300 × g for 10 min. The pellets were suspended in 10 mL of buffer containing 250 mM sucrose, EGTA 0.3 mM, and HEPES–KOH 10 mM, pH 7.2, and centrifuged at 3400 × g for 15 min. The final mitochondrial pellet was suspended in 1 mL of buffer containing sucrose 250 mM and HEPES–KOH 10 mM, pH 7.2, and used within 3 h. The mitochondrial protein content was determined by the biuret reaction. During all the procedures, the mitochondria were maintained at 4 ºC.
Evaluation of how BDE-47 and BDE-99 interact with the mitochondrial membrane DPH, TMA-DPH, and ANS insertion into membranes elicits a fluorescence response (F), the Stern–Volmer equation describes its static quenching as: F0/F = 1 + KSV (Q), where F0 and F are fluorescence intensities in the absence and presence of the quencher, respectively; and KSV is the Stern–Volmer constant. To evaluate interaction of PBDEs with the mitochondrial membrane, the mitochondria (1 mg protein.mL-1) were incubated with DPH 0.5 µM, ANS 75 µM, and TMA-DPH 1.04 µM in the standard reaction buffer (sucrose 125 mM, KCl 65 mM, HEPES-KOH 10 mM, pH 7.2) at 30 ºC, followed by addition of varying amounts of PBDEs in a final volume of 2 mL. The fluorescence was measured on an F-4500 spectrofluorometer (Hitachi, Tokyo, Japan) at excitation and emission wavelengths of 360/430 nm, 380/485 nm, and 362/432 for DPH, ANS, and TMA-DPH, respectively (Rodrigues et al., 2002).
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Continuous-monitoring mitochondrial assays Mitochondrial respiration was polarographically monitored using an oxygraph (Hansatech, Norfolk, England) equipped with a Clark-type oxygen electrode (Chance, Willians, 1956). The
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mitochondria (1 mg protein.mL-1) were incubated in 1 mL of the standard reaction buffer containing EGTA 0.5 mM, and K2HPO4 10 mM, pH 7.2 at 30 ºC, using glutamate and malate 5 mM and potassium succinate 5 mM (and rotenone 2.5 µM) as the oxidizable substrates for complexes I and II, respectively. State III respiration was initiated with 400 nmoles of ADP, and PBDEs were added at the time of testing (Cain, Skilleter, 1987).
The Mitochondrial Membrane Potential (ΔΨ) was spectrofluorometrically monitored using 1 mg of protein mL-1 and safranin-o 10 µM as the probe. An F-4500 spectrofluorometer (Hitachi, Tokyo, Japan) with the 495/586 nm excitation/emission wavelength pair (Akerman, Wikstrom, 1976), was used for this purpose. A standard reaction buffer and K2HPO4 10 mM, pH 7.2, was employed at the time of testing. After membrane potential was generated, aliquots of the PBDEs congeners at different concentrations were added to evaluate their effect on the kinetics of mitochondrial membrane potential. At the end of the incubation period, the membrane potential was completely dissipated using CCCP (1 µM).
Mitochondrial swelling was estimated from the decrease in the apparent absorbance at 540 nm in the presence of 0.4 mg of protein mL-1 using a Model DU-70 spectrophotometer (Beckman, Coulter Inc., Fullerton, CA, U.S.A.). Along with the mitochondria, different concentrations of BDE-47 and BDE-99 were added to analyze whether they interfered with
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mitochondrial swelling. To understand the mechanisms that trigger the process better, Cyclosporine A (CsA) 1 µM, Ruthenium Red (RR) 0.5 µM, and N-ethylmaleimide (NEM) 25 µM were used as modulators of the process. The PBDEs were added to the standard reaction
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buffer together, with K2HPO4 10 mM, pH 7.2 at the time of testing (Lemasters et al., 1987)
The Mitochondrial Ca2+ efflux was spectrofluorimetrically monitored using Calcium Green 5N 150 nM as probe, 506/531 nm excitation/emission wavelength pair, and 1 mg of protein mL1
. The PBDEs in the standard reaction buffer plus and K2HPO4 10 mM, pH 7.2, were added at the
time of testing. The total calcium efflux was performed using CCCP (1 μM). When the mitochondria capture Calcium Green 5N linked to Ca2+ the fluorescence detected is diminished, than if the fluorescence increases again, means that mitochondria cannot handle the calcium (Rajdev, Reynolds, 1993).
Determination of ATP levels Mitochondrial ATP levels were determined using the firefly luciferin–luciferase assay system (Thore, 1979). The mitochondrial suspension (1 mg protein.mL-1) was centrifuged at 9000 × g for 5 min, at 4 °C, and the pellet was treated with 1 mL of ice-cold 1 M HClO4. After centrifugation at 14 000 × g for 5 min at 4 °C, aliquots (100 μL) of the supernatants were neutralized with 70 μL of KOH 2 M suspended in Tris–HCl 100 mM, pH 7.8 (final volume = 1 mL) and centrifuged again. The bioluminescence was measured in the supernatant using a Sigma–Aldrich assay kit according to the manufacturer’s instructions, on an AutoLumat LB953 Luminescence photometer (Perkin Elmer Life Sciences, Wildbad, Germany). Statistical Analysis
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The experimental data were evaluated by analysis of variance (ANOVA), followed by a post-hoc analysis, including the use of Dunnett’s test, to compare the several treated groups with respective control. The software GraphPrism version 5.0 for Windows was employed. Results
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with p < 0.05 were considered statistically significant.
RESULTS The two tested congeners, BDE-47 and BDE-99, significantly impacted the fluorescence of the three probes used to assess these PBDEs ability to insert in the mitochondrial membrane (Figure 1). The results suggested that both congeners were able to interact with either the hydrophobic or hydrophilic region of the mitochondrial inner membrane, thereby interfering with the activity of proteins and other molecules present in these regions. The tested compounds affected the hydrophobic probe DPH the most: at 50 µM, BDE-47 and BDE-99 reduced the fluorescence of this probe by approximately 20% as compared with the control. To assess mitochondrial respiration, it is necessary to evaluate whether the PBDEs present in the inner mitochondrial membrane can alter the electron transport chain. More specifically, we conducted an assay to confirm the ability of the target PBDEs to insert and interact with complexes I and II of the respiratory chain. Table 1 shows how much BDE-47 and BDE-99 affected oxygen consumption by mitochondria isolated from rat liver using glutamate and malate as oxidizable substrates for site I, and succinate as oxidizable substrate for complex II. In the latter case, addition of rotenone inhibited complex I, to prevent reverse flow of electrons. The respiratory control ratio (RCR), values revealed decreased oxygen consumption at BDE-47 concentrations of 25 and 50 µM. BDE-99 elicited the same effects, at the higher concentration only.
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The interaction of both PBDEs with the inner mitochondrial membrane directly correlated with dissipation of the mitochondrial membrane potential. Figure 2 shows a significantly different effect after addition of BDE-47 (25, and 50 µM) (Fig. 2A) and BDE-99 (50 µM) (Fig. 2B). .
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Evaluation of mitochondrial swelling demonstrated the ability of PBDEs to modified the permeability of the mitochondrial membrane and consequently its integrity and selectivity. Figure 3 evidenced statistically significant differences (p < 0.05) between the treated and the control group (without addition of PBDE). BDE-47 (Fig. 3A) exerted a significant effect at the two highest concentrations (25 and 50 µM); for BDE-99 (Fig. 3B), this effect occurred only at 50 µM. Modulators partially inhibited this swelling, showing that this effect might depend on calcium. Because calcium homeostasis in the mitochondria is essential for proper operation, we examined whether the loss of permeability by these organelles was associated with calcium release. The presence of BDE-47 (25 and 50 µM) and BDE-99 (50 µM), prompted significant release of calcium ion. This was in line with the other analyzed parameters and indicated that BDE-47 and BDE-99 directly participated in dysregulation of mitochondrial calcium (Fig. 4). Finally, investigation of mitochondrial ATP levels after incubation of the mitochondria with both PBDEs (Fig. 5) revealed significantly lower mitochondrial ATP levels upon interaction with BDE-47 (10, 25, and 50 µM) and BDE-99 (25 and 50 µM). Hence, mitochondrial respiratory chain disruption can lead to reduced ATP levels. DISCUSSION The liver is the major site of foreign substances metabolism and therefore an constitutes important target for damaging effects (Jaeschke et al., 2002). Mitochondrial dysfunction is a
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relevant mechanism in the pathogenesis of many toxicants in mammals, especially hepatotoxicity (Amacher, 2005). Mitochondria play a central role in cell life and death. At the mitochondrial level, several potential compounds can cause toxicity. The results of this work have demonstrated that PBDEs
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are some of such compounds. Like many other compounds, description of the complete toxicological profile of PBDEs is also lacking in the literature. Our results indicated that BDE-47 (tetra-BDE) and BDE-99 (penta-BDE) interacted with the mitochondrial membrane and localized in the inner mitochondrial membrane, possibly lowering mitochondrial membrane fluidity and impairing energy processes in the mitochondria. Indeed, these processes are sensitive to membrane organization (Dorta et al., 2005). These results were in line with the ability of PBDEs to reduce oxygen consumption by the mitochondria. Analysis of mitochondrial respiration revealed that both BDE-47 and BDE-99 decreased oxygen consumption by the analyzed electron transport chain complexes. This effect stemmed from inhibition of the respiratory chain – results indicated diminished state III of respiration which is the moment when mitochondria convert ADP to ATP. Nakagawa and colleagues (2007) observed the same effect when they evaluated the toxic effects of another compound also classified as POPs – Tetrabromo Bisphenol A; ATP depletion occurred and the membrane potential decreased, indicating changes in the inner mitochondrial membrane. In this work, the toxicity of BDE-47 and BDE-99 directly related to their ability to interact with the inner mitochondrial membrane, which interfered with mitochondrial bioenergetics. The changes in mitochondrial respiration also referred to the loss of membrane potential elicited by both PBDEs. This close relationship between respiration/membrane potential could be interpreted as oxidation of substrates in the mitochondrial matrix and outside the inner
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membrane mediated by the electron transport chain, which could also be defined as a system for transmembrane electron transport. On the other hand, this transmembrane system should promote proton transfer from inside to outside the mitochondrion, to generate a membrane potential. From the energy standpoint, this membrane electrochemical potential would be
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necessary for ATP synthesis (La Piana et al., 1998). In addition, some toxic agents could affect the energy balance in the cells by increasing ATP consumption and/or reducing ATP synthesis. We analyzed this phenomenon by measuring ATP depletion, to find out whether the tested compounds affected this important mitochondrial mechanism. According to our results, the studied PBDEs significantly reduced the amount of ATP. Pereira, Souza and Dorta (2013) described the same outcome for BDE-100: its interaction with the mitochondrial membrane depleted ATP levels and altered respiratory parameters. Labeling with the probe calcium green 5N, to assess calcium release from mitochondrion, showed that the tested congeners also affected the mitochondrial calcium homeostasis: BDE-47 at 25 and 50 µM and BDE-99 at 50 µM interfered with this parameter and lead to excessive calcium release during the assay. Our result was similar to that found by Coburn, Currás-Collazo and Kodavanti (2008). These authors verified that BDE-47 also influenced calcium homeostasis in mitochondria isolated from nerve cells, which affected the cell signaling system. Hence, this class of compounds affects calcium homeostasis in both liver tissue and cells of the central nervous system. The impact of PBDEs on calcium homeostasis probably originated from mitochondrial swelling induced by BDE-47 and BDE-99. In other words, changes in calcium efflux and mitochondrial swelling were directly related to loss of mitochondrial membrane permeability. In fact, as indicated by the test involving swelling modulators, such modulators completely
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suppressed the effect of PBDEs. This outcome pointed to possible mechanisms of action for BDE-47 and BDE-99. In the presence of CsA, these PBDEs did not induce swelling. Therefore, it was possible to infer that in the absence of CsA these flame retardants may have caused swelling via opening of permeability transition pores (PTP).Therefore, CsA prevented
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cyclophilin D (CypD) from binding to ANT in the inner membrane which inhibited mitochondrial swelling to occur (Rao, Carlson, Yan, 2014). The test that employed RR as inhibitor showed that neither BDE-47 nor BDE-99 caused swelling. This result corroborated those obtained in the presence of CsA: PTP opening usually depends on calcium. As observed by Wang and colleagues (2005), even RR 0.1 M was able to inhibit the uniporter calcium transport channel. Blockage of this channel hindered calcium into entry the mitochondria and the consequent opening of PTP. According to the literature, the mitochondrial permeability transition (MPT) involves sudden increase in inner mitochondrial membrane permeability to solutes with molecular masses up to 1500 Da, due to opening of a highly conductive channel that is sensitive to Cyclosporin A and is Ca2+-dependent (Rasola, Bernardi, 2007). Several physiological and pathogenic processes could trigger MTP in both intact cells and isolated mitochondria (Hajnoczky, Davies, Madesh, 2003). In addition, numerous stressful responses (calcium dysregulation, reactive oxygen species, genotoxic stress, activation of proteases) could also trigger MPT, suggesting that mitochondria act as a universal stress sensor (Roberts, Goping, Bleackley, 2003). Our results suggested that MPT generated PTP, which in turn depleted mitochondrial ATP levels; PTP opening modified ATP levels due to an electrochemical imbalance. As already reported in the literature, ATP depletion is one of the key events induced by toxic compounds as a consequence of suppressed electron transport/oxidative phosphorylation, mitochondrial
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membrane potential dissipation, and calcium homeostasis dysfunction (Mingatto et al., 2002). In summary, our results have demonstrated that exposure to the PBDE congeners (BDE-47 and BDE-99) evaluated in this study modified bioenergetic parameters and damaged the mitochondria. BDE-47 had more potent toxic effects as compared with BDE-99; indeed, for all
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the tested bioenergetic parameters BDE-47 was able to exert adverse effects at lower concentrations than BDE-99. This different toxicity may be linked to the quantity and position of bromine atoms, in the PBDE. Models used to study the structure-activity relationship of PBDE have previously demonstrated that less brominated PBDEs with bromine atoms in the ortho position and no bromine atoms in the meta position were generally more toxic (Harju et al., 2007). In our case, BDE-47 has fewer bromine atoms and these atoms are in positions that made this congener more toxic than BDE-99 due to its facility to overcome biological membranes. It is important to notice that the effects observed here appears on higher levels that the ones still being found in humans or animals tissue. However, even though, they were listed as persistent organic pollutants (POP) by the Stockholm Convention in 2009, and banned from Europe since 2004, because of their physical chemical characteristics, there is also strong evidence that the levels of some of these flame retardants are increasing, doubling every 3 to 5 years (Richardson, 2008). These facts demonstrates that PBDEs are substances that are still in the process of accumulating in the environment and biota with biomagnifaction through the top of the food chain.
CONCLUSION This work provides relevant additional information about the toxicodynamics of two of the main representatives of brominated flame retardants, BDE-47 and BDE-99. We have concluded
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that both PBDEs display high toxic potential at their highest tested concentrations, thereby affecting mitochondrial bioenergetics. The PBDEs act on the inner mitochondrial membrane, interfering in parameters such as oxygen consumption, mitochondrial membrane potential, calcium homeostasis, and energy production. As judged from the bioenergetics results, BDE-47,
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a tetra-BDE, exhibits the highest toxicity as compared with BDE-99, a penta-BDE. The more potent toxic effects originate from the lower amount and position of bromine atoms in the structure of tetra-BDE. Elucidation of the structure-activity relationship of these compounds will facilitate understanding the mechanisms through which PBDEs exert their toxic effects.
ACKNOWLEDGEMENTS The authors thank Professor Carlos Curti, PhD, Professor Luciane Carla Alberici, PhD, and the Biochemistry Laboratory of the Faculty of Pharmaceutical Sciences of Ribeirão Preto - USP / Brazil for technical support. The authors thank for the financial support – grant #2009/06912-6 and #2012/04542-0 São Paulo Research Foundation (FAPESP).
DECLARATION OF INTEREST The authors report no declarations of interest
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FIGURE LEGENDS Figure 1. Interaction of BDE-47 (A) and BDE-99 (B) with the inner mitochondrial membrane as measured by the probes ANS, TMA-DPH, and DPH, as described in materials and methods. The data are presented as the means ± SEM of a series of three experiments.
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Figure 2. A) Concentration-response effect of BDE-47 (A) and BDE-99 (B) on dissipation of the mitochondrial membrane potential in mitochondria isolated from rat liver (1.0 mg protein mL-1) as evaluated using Safranin-o as probe, incubated as described in materials and methods. Data represent the mean ± SEM of three determinations with different mitochondrial preparations,
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relative to the control in the absence of the compound. *Statistically significant results (p < 0.05) as compared with the control.
Figure 3. Effect of BDE-47 (A) and BDE-99 (B) on mitochondrial swelling in mitochondria isolated from rat liver (0.4 mg protein mL-1), incubated as described in materials and methods. The maximum swelling was observed as induced by inorganic phosphate (Pi) 1 µM. The figure on the right shows how modulators Cyclosporin A (CsA),N-ethylmaleimide (NEM), and Ruthenium Red (RR) affect the action of PBDEs. Data represent the mean ± SEM of three determinations with different mitochondrial preparations, relative to the control in the absence of the compound. *Statistically significant results (p < 0.05) as compared with the control.
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Figure 4. Concentration-response effect of BDE-47 (A) and BDE-99 (B) on the efflux of calcium from mitochondria isolated from rat liver (1.0 mg protein mL-1) as measured with Calcium Green 5N probe, incubated as described in materials and methods. Points represent the mean ± SEM of three determinations with different mitochondrial preparations as compared with the control. *Statistically significant results (p < 0.05) as compared with the control. The total release of calcium induced by CCCP was 1 µM.
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Figure 5. Concentration-response effect of BDE-47 (A) and BDE-99 (B) on ATP levels in mitochondria isolated from rat liver (1.0 mg protein mL-1) as evaluated using the luminescence obtained from the reaction of luciferin/luciferase, incubated as described in materials and methods. Data represent the mean ± SEM of three determinations with different mitochondrial preparations, relative to the control in the absence of the compound. *Statistically significant results (p < 0.05) as compared with the control. The fall in ATP levels induced by CCCP was 1 µM.
Table 1. Respiratory parameters: state 3 (V3), state 4 (V4) and respiratory control ratio (RCR) using glutamate and malate for complex I, and succinate for complex II in mitochondria isolated 21
from rat liver, under the influence of BDE-47 and BDE-99. Data represent the mean ± SEM of three determinations with different mitochondrial preparations. *Means different from the
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control (no addition of PBDEs) in accordance with Dunett test (p
