Bioenergetics dysfunction, mitochondrial permeability transition pore opening and lipid peroxidation induced by hydrogen sulfide as relevant pathomechanisms underlying the neurological dysfunction characteristic of ethylmalonic encephalopathy

    Bioenergetics dysfunction, mitochondrial permeability transition pore opening and lipid peroxidation induced by hydrogen sulfide as relevant pathomechanisms underlying the neurological dysfunction characteristic of ethylmalonic encephalopathy Gabriela Miranda Fernandez Cardoso, Julia Tauana Pletsch, Belisa Parmeggiani, Mateus Grings, N´ıcolas Manzke Glanzel, Larissa Daniele Bobermin, Alexandre Umpierrez Amaral, Moacir Wajner, Guilhian Leipnitz PII: DOI: Reference:

S0925-4439(17)30202-8 doi:10.1016/j.bbadis.2017.06.007 BBADIS 64790

To appear in:

BBA - Molecular Basis of Disease

Received date: Revised date: Accepted date:

26 January 2017 16 May 2017 10 June 2017

Please cite this article as: Gabriela Miranda Fernandez Cardoso, Julia Tauana Pletsch, Belisa Parmeggiani, Mateus Grings, N´ıcolas Manzke Glanzel, Larissa Daniele Bobermin, Alexandre Umpierrez Amaral, Moacir Wajner, Guilhian Leipnitz, Bioenergetics dysfunction, mitochondrial permeability transition pore opening and lipid peroxidation induced by hydrogen sulfide as relevant pathomechanisms underlying the neurological dysfunction characteristic of ethylmalonic encephalopathy, BBA - Molecular Basis of Disease (2017), doi:10.1016/j.bbadis.2017.06.007

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ACCEPTED MANUSCRIPT Bioenergetics dysfunction, mitochondrial permeability transition pore opening and lipid peroxidation induced by hydrogen sulfide as relevant

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pathomechanisms underlying the neurological dysfunction characteristic of

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ethylmalonic encephalopathy

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Gabriela Miranda Fernandez Cardosoa,e, Julia Tauana Pletscha,e, Belisa Parmeggiania, Mateus Gringsa, Nícolas Manzke Glanzela, Larissa Daniele Bobermina,

Programa de Pós-Graduação em Ciências Biológicas: Bioquímica, Universidade

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a

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Alexandre Umpierrez Amarala,d, Moacir Wajnera,b,c, Guilhian Leipnitza,b,*

Federal do Rio Grande do Sul, Rua Ramiro Barcelos, 2600-Anexo, CEP 90035-003, Porto Alegre, RS, Brazil

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Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde,

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Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos, 2600-Anexo, CEP

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90035-003, Porto Alegre, RS, Brazil Serviço de Genética Médica, Hospital de Clínicas de Porto Alegre, Rua Ramiro

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Barcelos, 2350, CEP 90035-903, Porto Alegre, RS, Brazil Departamento de Ciências Biológicas, Universidade Regional Integrada do Alto

Uruguai e das Missões, Avenida Sete de Setembro, 1621, CEP 99709-910, Erechim, RS, Brazil. e

Co-first authors. Both authors have contributed equally to this work.

*Corresponding Author: Guilhian Leipnitz, Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal de Rio Grande do Sul, Ramiro Barcelos Street, 2600 – Anexo, CEP 90035-003, Porto Alegre, RS – Brazil. Phone: +55 51 3308-5551, fax: +55 51 3308-5540, e-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract Hydrogen sulfide (sulfide) accumulates at high levels in brain of patients with

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ethylmalonic encephalopathy (EE). In the present study, we evaluated whether sulfide

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could disturb energy and redox homeostasis, and induce mitochondrial permeability

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transition (mPT) pore opening in rat brain aiming to better clarify the neuropathophysiology of EE. Sulfide decreased the activities of citrate synthase and aconitase in rat cerebral cortex mitochondria, and of creatine kinase (CK) in rat cerebral

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cortex, striatum and hippocampus supernatants. Glutathione prevented sulfide-induced

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CK activity decrease in cerebral cortex. Sulfide also diminished mitochondrial respiration in cerebral cortex homogenates, and dissipated mitochondrial membrane potential (ΔΨm) and induced swelling in the presence of calcium in brain mitochondria. Alterations in

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ΔΨm and swelling caused by sulfide were prevented by the combination of ADP and cyclosporine A, and by ruthenium red, indicating the involvement of mPT in these effects.

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Furthermore, sulfide increased the levels of malondialdehyde in cerebral cortex supernatants, which was prevented by resveratrol and attenuated by glutathione, and of

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thiol groups in a medium devoid of brain samples. Finally, we verified that sulfide did not alter cell viability and DCFH oxidation in cerebral cortex slices, primary cortical astrocyte cultures and SH-SY5Y cells. Our data provide evidence that bioenergetics disturbance and lipid peroxidation along with mPT pore opening are involved in the pathophysiology of brain damage observed in EE.

Keywords: sulfide; bioenergetics; redox status; mitochondrial permeability transition; rat brain.

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ACCEPTED MANUSCRIPT 1. Introduction Ethylmalonic encephalopathy (EE) is an inherited metabolic disorder of sulfide

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metabolism caused by mutations in the ETHE1 gene, which encodes the sulfur

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dioxygenase ETHE1 (EC 1.13.11.18). ETHE1 is a 30-kDa polypeptide exclusively

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located in the mitochondrial matrix that participates in the catabolic pathway of hydrogen sulfide (sulfide, H2S), oxidizing a sulfur atom extracted from persulfide, converting it to sulfite [1] (Figure 1). Patients affected by EE present developmental delay, progressive

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pyramidal signs, seizures, relapsing petechiae, acrocyanosis and chronic mucoid diarrhea

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[2, 3]. The onset and severity of these symptoms vary from patient to patient but they usually occur early in development and half of the patients die within the first two years of life from metabolic decompensation [4, 5]. Neuropathological findings include

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frontotemporal atrophy, abnormalities in basal ganglia and brainstem gray matter, and malformations in the white matter, dentate nuclei and spinal cord [5-10].

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The biochemical hallmark of EE is the tissue accumulation of sulfide [2]. Sulfide is a biologically active compound that, at micromolar concentrations, strongly inhibits

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cytochrome c oxidase of mitochondrial respiratory chain and short-chain acyl-CoA dehydrogenase [11, 12]. In addition, thiosulfate, generated by the condensation of two molecules of sulfide, and ethylmalonic acid, derived from the carboxylation of the shortchain acyl-CoA dehydrogenase substrate butyrate, are also found at high levels in this disorder [7]. Although the pathogenesis of the neurological dysfunction observed in EE is not yet fully elucidated, data from the literature evidence that toxicity exerted by sulfide accumulation is a key factor. It was shown that this metabolite causes progressive cytochrome c oxidase deficiency due to heme a inhibition and degradation of these complex subunits [11]. Sulfide also inhibits mitochondrial respiration in human-induced

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ACCEPTED MANUSCRIPT pluripotent stem cell-derived cerebrocortical neurons, primary human fibroblasts and COS-7 monkey kidney cells, and increases hydroxyl radical generation and F2-

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isoprostanes in mouse heart and brain [13]. Another study demonstrated that sulfide

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exerts cytotoxicity to rat hepatocytes, which was prevented by cyclosporine and

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trifluoperazine, suggesting that sulfide induces mitochondrial permeability transition (mPT) pore opening in these cells [14]. Moreover, studies performed in fibroblasts of patients reported alterations in the expression of enzymes involved in redox homeostasis,

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as well as decreased levels of reduced glutathione (GSH), Krebs cycle intermediates and

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the redox cofactors NADH and NAD+ [15, 16].

We aimed to better clarify the pathophysiology involved in the neurological damage found in EE by evaluating whether enzymes crucial for cell energy metabolism

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could be sensitive targets to sulfide and whether this metabolite could induce mPT pore opening in brain. The effects of sulfide on redox homeostasis and cell viability were also

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examined.

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Figure 1 Sulfide (H2S) catabolic pathway. 1) In mitochondria, sulfide is fixed to sulfite by sulfide quinone oxidoreductase (SQR) to produce thiosulfate; 2) Thiosulfate sulfur

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transferase (TST) reconstitutes sulfite from thiosulfate with the fixation of a sulfur to reduced glutathione (GSH) forming glutathione persulfide; 3) ETHE1 oxidizes the sulfur

sulfate.

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atom of persulfide generating sulfite; 4) Sulfite oxidase (SUOX) oxidizes sulfite to

2. Material and Methods 2.1 Animals and Reagents Wistar rats (70-100 g) obtained from the Central Animal House of the Department of Biochemistry, ICBS, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil, were used. The animals had free access to water and 20 % (w/w) protein commercial chow (SUPRA, Porto Alegre, RS, Brazil), and were maintained on a 12:12 h light/dark cycle in an air conditioned constant temperature (22 ºC ± 1 ºC) colony 5

ACCEPTED MANUSCRIPT room. The experimental protocol was approved by the Ethics Committee for Animal Research of UFRGS, Porto Alegre, Brazil, and followed the National Institutes of Health

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Guide for the Care and Use of Laboratory Animals (NIH Publications No 80-23, revised

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1978). All efforts were made to minimize the number of animals used and their suffering.

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All chemicals were purchased from Sigma (St. Louis, MO, USA), except for Dulbecco’s Modified Eagle’s Medium (DMEM), Hanks’ balanced salt solution (HBSS), fetal bovine serum (FBS) and other materials for cell cultures that were purchased from

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Gibco/Thermo Scientific (Carlsbad, CA, USA). Sulfide (H2S) was generated by sodium

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hydrosulfide hydrate (NaSH.xH2O), which is commonly used as sulfide donor. Sodium hydrosulfide hydrate solutions were prepared fresh on the day of the experiment. 2.2 Preparation of homogenates and supernatants

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For mitochondrial respiration, rats were euthanized by decapitation, had their brain immediately removed and placed on an ice plate. Cerebral cortex was dissected,

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weighed and homogenized in MiR05 buffer (110 mM sucrose, 60 mM K-lactobionate, 0.5 mM EGTA, 3 mM MgCl2, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES and 0.1%

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BSA, pH 7.1). Homogenates were then exposed to 500 µM sulfide prior to the determination of mitochondrial respiration. For the measurement of oxidative stress parameters and 3(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction, we used cerebral cortex supernatants or slices. Cerebral cortex supernatants were obtained after homogenization of the tissue in 20 mM sodium phosphate buffer, pH 7.4, containing 140 mM KCl, and centrifugation at 750 g for 10 min at 4 ºC. Slices were prepared using a McIlwain tissue chopper. Supernatants or slices were then incubated in the absence or presence of sulfide (1-500 µM) at 37 ºC for 1 h, and used for the determination of malondialdehyde (MDA)

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ACCEPTED MANUSCRIPT levels, carbonyl formation, 2’,7’-dichlorofluorescin (DCFH) oxidation and MTT reduction.

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For creatine kinase (CK) activity determination, cerebral cortex, striatum and

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hippocampus were homogenized (1:10 w/v) in an isosmotic saline solution and

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centrifuged at 800 g for 10 min at 4 ºC. Supernatants were aliquoted and used for the determination of this parameter.

2.3 Preparation of mitochondrial fractions

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Rat cerebral cortex mitochondrial preparations were used for the measurement of citrate synthase (CS), aconitase (ACO) and malate dehydrogenase (MDH) activities,

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while for mitochondrial membrane potential (ΔΨm) and swelling mitochondrial preparations from rat forebrain were used. Forebrain and cerebral cortex mitochondria

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were isolated from rats as previously described [17] with slight modifications [18]. Animals were killed by decapitation, had their brain rapidly removed and put into ice-

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cold isolation buffer containing 225 mM mannitol, 75 mM sucrose, 1 mM EGTA, 0.1 % BSA and 10 mM HEPES, pH 7.2. Forebrain or cerebral cortex was then cut into small

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pieces using surgical scissors, extensively washed to remove blood and homogenized 1:10 in a Dounce homogenizer using both a loose-fitting and a tight-fitting pestle. The homogenate was centrifuged for 3 min at 2,000 g. After centrifugation, the supernatant was again centrifuged for 8 min at 12,000 g. The pellet was suspended in isolation buffer containing 10 μL of 10 % digitonin and centrifuged for 8 min at 12,000 g. The final pellet containing the mitochondria was gently washed and suspended in isolation buffer devoid of EGTA, at an approximate protein concentration of 20 mg.mL-1. Prior to the determination of the parameters, mitochondrial preparations were submitted to a preincubation at 37 ºC for 30 min in the absence or presence of sulfide (100-1,000 µM). Only

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ACCEPTED MANUSCRIPT mitochondrial preparations with respiratory control ratio (RCR) greater than 5 were used in the experiments.

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2.4 Preparation of primary astrocyte cultures

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Primary cortical astrocyte cultures from Wistar rats were prepared essentially as

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previously described [19]. Briefly, cortices of newborn Wistar rats (1-2-day-old) was removed, mechanically dissociated in HBSS and left for decantation for 20 min. Supernatant was collected and centrifuged for 5 min (400 g). Afterwards, the pellet was

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resuspended in the culture medium (DMEM/F12 supplemented with 10 % fetal bovine

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serum, 15 mM HEPES, 14.3 mM NaHCO3, 1 % fungizone and 0.04 % gentamicin, pH 7.4). Cells were plated onto 24-well plates pre-coated with poly-L-lysine at a density of 105 cells/well and cultured at 37 ºC in atmosphere with 5 % of CO2. When the cells

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reached confluence (approximately 14 days in vitro), the culture medium was exchanged with serum-free DMEM/F12, and the cells were incubated with sulfide (500-5,000 µM)

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for 24 hat 37 ºC in atmosphere with 5 % of CO2. After incubation, cells were used to determine DCFH oxidation and MTT reduction.

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2.5 SH-SY5Y cell culture Human neuroblastoma cell line SH-SY5Y, obtained from the American Type Culture Collection (ATCC; USA), was cultured in DMEM/F12, pH 7.4, containing 10% FBS, 15 mM HEPES, 14.3 mM NaHCO3, 1 % amphotericin B and 0.032 % gentamicin, at 37ºC in a humidified atmosphere with 5 % CO2. When cells reached approximately 90 % confluence, they were sub-cultured using 0.05 % trypsin/ethylene-diaminetetracetic acid (EDTA) and seeded in 24-well plates (6x104 cells/well) [20]. SH-SY5Y cells were treated when reached approximately 75% confluence. The culture medium was replaced by DMEM/F12 1% FBS and cells were incubated with sulfide (100-1,000 µM) for 24 h at 37°C in atmosphere with 5% CO2.

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ACCEPTED MANUSCRIPT 2.6 Citric acid cycle (CAC) enzyme activities CS activity was measured according to Shepherd and Garland

[21] by

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determining 5,5’-dithio-bis(2-nitrobenzoic acid) reduction at λ = 412 nm. The activity of

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ACO was measured according to Morrison [22] by following the reduction of NADP+ at

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wavelengths of excitation and emission of 340 and 466 nm. MDH activity was measured according to Kitto [23] by following the reduction of NADH at wavelengths of excitation and emission of 340 and 466 nm, respectively. The activities of the CAC enzymes were

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expressed as nmol.min-1.mg protein-1, mmol.min-1.mg protein-1 or μmol.min-1.mg protein-

2.7 Creatine kinase (CK) activity

CK activity was measured according to the method described by Hughes [24]

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slightly modified by da Silva et al. [25]. The incubation medium consisted of 50 mM Tris buffer, pH 7.5, 7.0 mM phosphocreatine, 7.5 mM MgSO4, and tissue preparations in a

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final volume of 0.1 mL. Sulfide was added to the medium and submitted to a preincubation at 37 °C for 30 min. The reaction was started by the addition of 4.0 mM ADP

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and stopped after 10 min by the addition of 20 L of 50 mM p-hydroxymercuribenzoic acid. The creatine formed was estimated by a colorimetric method in which the color was developed by the addition of 100 L of 20% α-naphthol and 100 L of 20% diacetyl in a final volume of 1.0 mL and read after 20 min at λ = 540 nm. Results were calculated as μmol.min-1.mg protein-1. 2.8 Mitochondrial oxygen consumption The rate of oxygen consumption was measured using an OROBOROS Oxygraph2k (Innsbruck, Austria) in a thermostatically controlled (37 ºC) and magnetically stirred incubation chamber. Sulfide (500 μM) was added to the reaction medium containing cerebral cortex homogenates (1 mg tissue.mL-1) at the beginning of the assay. Respiration

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ACCEPTED MANUSCRIPT of homogenates was then determined using a substrate-uncoupler inhibitor titration protocol with modifications [26]. Pyruvate (5 mM), malate (0.5 mM) and glutamate (10

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mM) (PMG) or succinate (10 mM) were used to determine complex I (CI)- or complex II

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(CII)-linked leak respiration, respectively. ADP was added at 500 µM final concentration

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to obtain oxidative phosphorylation (OXPHOS) capacity. Oligomycin (1 µg/mL) was added to reconstitute convergent CI&II-linked respiration. Titration with the uncoupler carbonyl cyanide m-chlorophenyl hydrazine (CCCP) (0.5-1 µM) was performed to

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determine electron transfer system (ETS) capacity. Rotenone (0.5 µM) was added for determination of CII ETS capacity.

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2.9 Mitochondrial membrane potential (ΔΨm) This parameter was estimated according to the method described by Akerman and

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Wikstrom [27] and Figueira et al. [28], following the fluorescence of the cationic dye safranin O (5 µM) on a spectrofluorometer with magnetic stirring at an excitation and

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emission of 495 and 586 nm, respectively, using 2.5 mM glutamate plus 2.5 mM malate as substrates. Mitochondrial preparations (0.75 mg protein.mL-1) were incubated at 37 ºC

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with sulfide (150 M) in 5 mM HEPES buffer, pH 7.2, containing 150 mM potassium chloride, 5 mM magnesium chloride, 0.015 mM EGTA, 5 mM potassium phosphate, 0.01 % BSA and 1 µg.mL-1 oligomycin A. CaCl2 (30 µM) was added to the reaction medium 50 s after the beginning of the assay. In some experiments, the mitochondrial preparations were incubated with cyclosporin A (CsA; 1 µM), ADP (300 µM) or ruthenium red (RR; 1 µM). In the end of each measurement, maximal depolarization was induced by 1 µM carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP). The results are showed as traces representing FAU. 2.10 Mitochondrial swelling

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ACCEPTED MANUSCRIPT Mitochondrial swelling was assessed by measuring light scattering changes on a spectrofluorometer with magnetic stirring operating at excitation and emission of 540 nm,

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using 2.5 mM glutamate plus 2.5 mM malate as substrates. Mitochondrial preparations

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(0.75 mg protein.mL-1) were incubated at 37 ºC with 150 µM sulfide in 5 mM HEPES

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buffer, pH 7.2, containing 150 mM potassium chloride, 5 mM magnesium chloride, 15 µM EGTA, 5 mM potassium phosphate, 0.01 % BSA and 1 µg.mL-1 oligomycin A. CaCl2 (40 µM) was added to the reaction medium 50 s after the beginning of the assay.

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In the end of each measurement, maximal swelling was induced by the addition of

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alamethicin (40 µg.mL-1), a pore-forming compound. The results are showed as traces representing FAU.

2.11 Malondialdehyde (MDA) levels

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MDA concentrations were measured by the thiobarbituric acid-reactive substances (TBA-RS) method described by Esterbauer and Cheeseman [29], with slight

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modifications. Three hundred microliters of cold 10 % trichloroacetic acid were added to 150 µL of pre-incubated supernatants in the presence of sulfide for 1 h at 37°C and

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centrifuged at 3,000 g for 10 min. Three hundred microliters of the pre-incubated supernatants were then incubated with 300 µL of 0.67 % thiobarbituric acid in 7.1 % sodium sulfate on a boiling water bath for 25 min. The tubes containing the mixture were allowed to cool on running tap water for 5 min. The resulting pink-stained TBA-RS were determined in a spectrophotometer at 532 nm. A calibration curve was performed using 1,1,3,3-tetramethoxypropane, and each curve point was subjected to the same treatment. MDA levels were calculated as nmol MDA.mg protein-1. 2.12 Thiol content Thiol content derived formed from oxidized glutathione (GSSG) was evaluated according to Browne and Armstrong [30] with modifications. Briefly, a commercial

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ACCEPTED MANUSCRIPT solution of GSSG (200 μM) was exposed to sulfide (500 or 1,000 µM) for 60 min in a medium devoid of biological samples and, afterwards, an aliquot (30 µL) of this

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incubation was treated with o-phthaldialdehyde (1 mg/mL). After 15 min, the

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fluorescence was measured using excitation and emission wavelengths of 350 nm and

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420 nm, respectively. Data were expressed as fluorescence arbitrary units (FAU). 2.13 Amplex Red oxidation

Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) is a fluorogenic reagent that

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generates resorufin when oxidized. Briefly, Amplex Red (5.5 µM) was exposed to sulfide

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(500 µM) in a medium containing horseradish peroxidase (2 U.mL-1), and the fluorescence of resorufin was monitored over time in the O2k-Fluorometer (Innsbruck, Austria) [26]. Data were obtained as fluorescence arbitrary units (FAU).

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2.14 2’,7’-Dichlorofluorescin (DCFH) oxidation DCFH oxidation was determined according to the method of LeBel [31], with

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slight modifications, in order to examine reactive oxygen species generation. Cerebral cortex slices were exposed to sulfide (1-1,000 µM) for 1 h at 37 ºC, whereas astrocytes

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and SH-SY5Y cells were exposed to this metabolite for 24 h at 37ºC. Afterwards, tissue slices or cells were incubated with 2’,7’-dichlorofluorescein diacetate (DCF-DA) (5 µM for cortical slices and 10 µM for cells), prepared in 20 mM sodium phosphate buffer, pH 7.4, containing 140 mM KCl, during 30 min at 37 ºC. DCF-DA is permeable to the cell membrane and is deacetylated by esterases to DCFH in the intracellular medium. This nonfluorescent product is converted by reactive species into the highly fluorescent product dichlorofluorescein (DCF). After incubation, fluorescence was measured in a 96well plate using a Fluorescence Microplate Reader at wavelengths of 480 nm (excitation) and 535 nm (emission). The calibration curve was performed with standard DCF (1-100

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ACCEPTED MANUSCRIPT µM). The production of reactive species was calculated as pmol.g tissue-1 (cerebral cortex slices) or fluorescence arbitrary units.mg protein-1 (cells and SH-SY5Y cells).

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2.15 Carbonyl formation

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Carbonyl formation was measured spectrophotometrically according to Reznick

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and Packer [32]. Two hundred microliters of pre-treated supernatants (containing approximately 0.3 mg of protein) were treated with 400 µL of 10 mM 2,4dinitrophenylhydrazine dissolved in 2.5 N HCl or with 2.5 N HCl (blank) and left in the

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dark for 1 h. After that, samples were precipitated with 600 µL of 20% trichloroacetic

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acid and centrifuged for 5 min at 9,000 g. The pellet was then washed with 1 mL ethanol:ethyl acetate (1:1, v/v) and dissolved in 550 µL of 6 M guanidine prepared in 2.5 N HCl at 37°C for 5 min. The calibration curve was performed with BSA (0.5-3 mg/mL).

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Calibration curve and HCl-treated samples (blank) absorbances were measured at 280 nm, and 2,4-dinitrophenylhydrazine-treated samples absorbance was determined at 370

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nm. The results were calculated as nmol of carbonyl groups/mg of protein, using the extinction coefficient of 22,000x106 nmol.mL-1 for aliphatic hydrazones.

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2.16 MTT reduction

Cell viability was examined in cerebral cortex slices exposed to sulfide (100-5,000 µM) for 3 h at 37 ºC, and in cells exposed to this metabolite for 24 h at 37 ºC by measuring the reduction of MTT to a dark violet formazan product [33]. After 1 h, slices and cells were washed twice with 500 µL of Hank´s buffered salt solution (HBSS). MTT reduction assay was performed in plates containing 300 µL HBSS and the reaction was started with the addition of 50 µg.mL-1 MTT. After 45 min incubation at 37 ºC the medium was removed and the slices or astrocytes were submerged in dimethyl sulfoxide in order to extract the colored formazan product. This colored product was measured

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ACCEPTED MANUSCRIPT spectrophotometrically at a wavelength of 570 nm and a reference wavelength of 630 nm. Results were compared to control samples to which 100% viability was attributed.

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2.17 Protein determination

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Protein content was measured by the method of Lowry et al. [34], using bovine

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serum albumin as a standard. 2.18 Statistical analysis

Results are presented as mean ± standard deviation. Assays were performed in

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duplicate or triplicate and the mean was used for statistical calculations. Data were

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analyzed using one-way analysis of variance (ANOVA) followed by the post-hoc Duncan multiple range test when F was significant. ANOVA statistical analysis is reported in the text with the F-statistic, df between (df1), df within (df2), and P value. Only significant F

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values are shown. Differences between groups were considered significant at P < 0.05. All analyses were carried out in a compatible computer using the Statistical Package for

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the Social Sciences software.

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3. Results

3.1 Sulfide decreases CS and ACO activities in cerebral cortex mitochondria Sulfide is a molecule known to react with oxidized thiols, promoting Ssulfhydration on proteins that may lead to modulation or impairment of their function [35-37]. Considering this, we initially investigated the effects of sulfide on the activities of the CAC enzymes CS, ACO and MDH in mitochondrial fractions prepared from cerebral cortex of developing rats. Sulfide significantly decreased the activity of CS [F(3,20)=7.932; P<0.001] and ACO [F(3,20)=364.015; P<0.001], but not of MDH (Figure 2), indicating an impairment of cell bioenergetics.

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Figure 2 Effect of sulfide on the activities of citrate synthase (CS) (A), aconitase (ACO)

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(B) and malate dehydrogense (MDH) (C) in rat cerebral cortex. Tissue supernatants were

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incubated in the presence of sulfide (100-1,000 µM). Values are means ± SD for six independent experiments (animals) performed in triplicate. Controls did not contain the metabolite in the incubation medium. ***P < 0.001, compared to controls (ANOVA

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followed by Duncan multiple range test).

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3.2 Sulfide decreases CK activity in cerebral cortex, striatum and hippocampus supernatants

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The effects of sulfide on the activity of CK, a crucial enzyme for energy transfer and buffer, was also evaluated in different brain structures of rats. It can be observed in Figure 3 that this metabolite decreased the activity of CK in cerebral cortex [F(5,30)=7.110; P<0.01], striatum [F(5,30)=5.724; P<0.01] and hippocampus [F(5,24)=7.116; P<0.01] of rats. We further evaluated whether free radical scavengers could prevent the decrease of CK activity caused by sulfide in cerebral cortex. Figure 4 shows that GSH, but not melatonin, trolox and lipoic acid, fully prevented this effect [F(5,30)=5.798; P<0.01], implying that sulfide decreases CK activity possibly by altering sulfur-containing critical groups in this enzyme structure.

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Figure 3 Effect of sulfide on creatine kinase (CK) activity in rat cerebral cortex (A),

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striatum (B) and hippocampus (C). Tissue supernatants were incubated in the presence of

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sulfide (1-1,000 µM). Values are means ± SD for five to six independent experiments (animals) performed in triplicate. Controls did not contain the metabolite in the incubation

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medium. *P < 0.05, **P < 0.01, compared to controls (ANOVA followed by Duncan

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multiple range test).

Figure 4 Effect of free radical scavengers on sulfide-induced decrease of creatine kinase (CK) activity in rat cerebral cortex. Cerebral cortex supernatants were incubated in the presence of sulfide (500 µM) and reduced glutathione (GSH; 50 µM), trolox (TRO; 5 µM), melatonin (MEL; 1,000 µM) or lipoic acid (LA; 500 µM). Values are means ± SD for six independent experiments (animals) performed in triplicate. Controls did not contain the metabolite in the incubation medium. *P < 0.05, **P < 0.01, compared to 16

ACCEPTED MANUSCRIPT controls; ##P < 0.01, compared to 500 µM sulfide (ANOVA followed by Duncan multiple

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range test).

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3.3 Sulfide decreases mitochondrial respiration in cerebral cortex homogenates

The next step was to evaluate whether sulfide could affect brain mitochondrial

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respiration using PMG or succinate as substrates once sulfide decreased CS and ACO

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activities, and is reported to be a strong cytochrome c oxidase inhibitor [11, 12]. It was verified that sulfide markedly decreased ADP- and CCCP-stimulated respiration, that

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substrates in brain (Figure 5).

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represent OXPHOS and ETS capacity, respectively, supported by CI- and CII-linked

Figure 5 Effect of sulfide (500 µM) on mitochondrial respiration measured by oxygen consumption in cerebral cortex homogenates. Pyruvate (5 mM), malate (0.5 mM) and glutamate (10 mM) (PMG) or succinate (SUC, 10 mM) were used to determine complex I (CI)- or complex II (CII)-linked respiration, respectively. PMG: CI-linked leak respiration; ADP (500 µM): CI-linked oxidative phosphorylation capacity; SUC: CI&IIlinked oxidative phosphorylation capacity; Oligomycin (Oligo, 1 µg/mL): CI&II-linked 17

ACCEPTED MANUSCRIPT leak respiration; CCCP (0.5 or 1 µM): CI&II-linked electron transfer system (ETS) capacity; rotenone (Rot, 0.5 µM): CII-linked ETS capacity. Values are means ± SD for

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three independent experiments performed in triplicate and are expressed as O2 flux

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(ρmol.(s*mg protein)-1). *P < 0.05, **P < 0.01, *** P < 0.001, compared to controls

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(Student´s t test).

presence of Ca2+ in brain mitochondria

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3.4 Sulfide decreases ΔΨm and induces mitochondrial swelling in the

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The effect of sulfide (150 µM) on ΔΨm and mitochondrial swelling was investigated in brain mitochondria in the presence of Ca2+ using glutamate and malate as substrates. Ca2+ was added to evaluate whether sulfide could induce mPT pore opening.

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Figure 6 demonstrates that sulfide decreases ΔΨm [F(3,8)=14.614; P<0.001] (Figure 6A) and induces swelling [F(3,8)=10.529; P<0.05] (Figure 6B), and that these effects were

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blocked by the addition of RR, and the combination of CsA and ADP, suggesting the

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involvement of mPT in these effects.

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Figure 6 Effect of sulfide (150 M) on mitochondrial membrane potential (ΔΨm) (A)

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and swelling (B) using brain mitochondria supported by glutamate plus malate in the presence of Ca2+. (A and B) Sulfide (150 µM) (trace b) was added 50 s after the beginning

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of incubation to the reaction medium containing the mitochondrial preparations (0.75 mg protein.mL-1). Ca2+ (30 µM) was added 100 s afterwards. In some experiments,

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mitochondrial preparations were incubated with sulfide plus cyclosporine A (CsA; 1 µM) and ADP (300 µM) (trace c), or ruthenium red (RR; 1 µM; trace d). Control (trace a) was performed in the presence of Ca2+ (30 µM) and did not contain sulfide. CCCP (1 µM) or alamethicin (Alam; 40 µg.mg of protein-1) was added at the end of the experiments, as indicated. Traces are representative of three independent experiments and express fluorescence arbitrary units (FAU).

3.5 Sulfide induces lipid peroxidation in cerebral cortex supernatants Next the effect of sulfide on MDA levels was evaluated in rat cerebral cortex. Our results show that sulfide increases MDA levels [F(5,24)=10.274; P<0.001] (Figure 7A),

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ACCEPTED MANUSCRIPT indicating lipid peroxidation. It was also verified that resveratrol (Res) totally prevented,

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whereas GSH attenuated this effect [F(3,20)=19.885; P<0.001] (Figure 7B).

Figure 7 Effect of sulfide on malondialdehyde (MDA) levels in rat cerebral cortex.

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Cerebral cortex supernatants were incubated in the presence of sulfide (1-1,000 µM) (A).

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In some experiments tissue supernatants were incubated in the presence of sulfide (1,000

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µM) and reduced glutathione (GSH; 1,000 µM) or resveratrol (Res; 5 M) (B). Values are means ± SD for five to six independent experiments (animals) performed in triplicate. Controls did not contain the metabolite in the incubation medium. ***P < 0.001,

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compared to controls; ###P < 0.001, compared to 1,000 µM sulfide (ANOVA followed by Duncan multiple range test).

3.6 Sulfide increases thiol levels in the presence of oxidized glutathione (GSSG) and reacts with Amplex Red in a medium devoid of biological samples In order to study sulfide reactivity, we exposed a commercial solution of GSSG and a solution of the oxidizable fluorogenic compound Amplex Red to sulfide in a medium devoid of biological samples. We observed that sulfide increased thiol group content when exposing GSSG solution to this metabolite (Figure 8), and that sulfide could oxidize Amplex Red (data not shown). 20

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Figure 8 Effect of sulfide on oxidized glutathione (GSSG) in a medium devoid of biological samples. A commercial solution of oxidized glutathione (GSSG, 200 M) was

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co-incubated with sulfide (500 or 1,000 µM). Values are means ± SD for three

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independent experiments performed in triplicate and are expressed as fluorescence

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arbitrary units (FAU). *** P < 0.001, compared to controls (ANOVA followed by Duncan multiple range test).

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3.7 Sulfide does not alter DCFH oxidation and cell viability in cerebral cortex slices, astrocytes, and SH-SY5Y cells, and carbonyl formation in cerebral cortex supernatants We evaluated reactive oxygen species production by DCFH oxidation and cell viability by MTT reduction in cerebral cortex slices exposed for 3 h, and in primary astrocyte cultures and SH-SY5Y cells exposed for 24 h to sulfide. Tables 1 and 2 show that sulfide did not significantly alter any of these parameters. It was further observed that sulfide did not modify carbonyl formation in cerebral cortex supernatants exposed for 1 h to this metabolite (data not shown).

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Table 1 Effect of sulfide (1-1,000 M) on 2’,7’-dichlorofluorescin (DCFH) oxidation in

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rat cerebral cortex slices and primary astrocyte cultures from rat cerebral cortex, and SH-

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SY5Y cells

1

10

0.74 ± 0.31

0.77 ± 0.38

0.86 ± 0.27

12,355 ± 2,240

-

-

29,437 ± 5,54

-

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Control

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Sulfide (µM)

Cerebral

Cortical astrocytes SH-SY5Y

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cells

500

1,000

0.78 ± 0.23

0.66 ± 0.15

0.58 ± 0.20

-

11,478 ± 1,625

12,543 ± 2,333

32,116 ± 7,067

28,257 ± 5,234

25,580 ± 1,528

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cortex slices

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Data are represented as mean ± SD for four to six independent experiments. DCFH oxidation is expressed as pmol.g tissue-1 (cerebral cortex slices) or fluorescence arbitrary

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units.mg protein-1 (cortical astrocytes and SH-SY5Y cells). No significant alterations

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were observed (ANOVA).

Table 2 Effect of sulfide (100-5,000 M) on [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) reduction in rat cerebral cortex slices and primary astrocyte cultures from rat cerebral cortex, and SH-SY5Y cells Sulfiden (µM) 100

500

1,000

5,000

104 ± 19.6

116 ± 16.2

107 ± 14.9

95.3 ± 16.0

Cortical astrocytes

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109 ± 4.04

109 ± 10.0

116 ± 9.50

SH-SY5Y cells

96 ± 13.8

112 ± 4.36

102 ± 11.0

-

Cerebral cortex slices

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ACCEPTED MANUSCRIPT Data are represented as mean ± SD of three to six independent experiments and expressed

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as percentage of controls. No significant alterations were observed (ANOVA).

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3.8 Cyanide decreases ΔΨm, but does not alter CK activity and MDA levels

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in rat brain

In order to evaluate whether cyanide, which similarly to sulfide is a respiratory chain complex IV inhibitor, could alter bioenergetics and induce lipid peroxidation, we

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investigated its effects on ΔΨm, CK activity and MDA levels. We verified that cyanide

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decreased ΔΨm even in the absence of calcium in brain mitochondrial preparations (Figure 9A). Moreover, cyanide did not alter MDA levels in cerebral cortex (Figure 9B) and CK activity in cerebral cortex, striatum and hippocampus of rats (Figure 9C). These

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differential effects exerted by cyanide as compared to sulfide suggest that sulfide impairs

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bioenergetics and induces lipid peroxidation through hydrosulfide anion generation.

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ACCEPTED MANUSCRIPT Figure 9 Effect of cyanide on mitochondrial membrane potential (ΔΨm) (A), malondialdehyde (MDA) levels (B) and creatine kinase (CK) activity (C) in rat brain.

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Brain mitochondrial preparations were incubated in the presence of cyanide (150 µM)

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(A). Cerebral cortex, striatum or hippocampus supernatants were exposed to cyanide (500

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or 1,000 µM) (B and C). Values are means ± SD for three to four independent experiments (animals) performed in triplicate. Controls did not contain cyanide in the incubation

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medium (ANOVA followed by Duncan multiple range test).

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4. Discussion

EE is a fatal mitochondrial disease clinically characterized by severe neurological symptoms, whose pathophysiology remains to be elucidated. In this regard, previous

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works suggested that sulfide is neurotoxic by inhibiting cytochrome c oxidase and shortchain fatty acid oxidation [11, 12]. Herein we investigated the effects of sulfide on

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bioenergetics, mPT opening and redox status in brain of rats aiming to gain insight into the neurotoxic mechanisms exerted by this metabolite.

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We found that sulfide decreased the activities of the CAC enzymes CS and ACO, which may be caused by alterations on sulfur-containing groups by S-sulfhydration [35, 37, 38]. This assumption is in accordance with our findings demonstrating that sulfide reacts with GSSG leading to an increase of thiol group content. On the other hand, since ACO is an enzyme reported to be a redox sensor of reactive species [39-41], we cannot rule out that reactive species generated via sulfide action also mediate ACO inhibition. Sulfide further decreased CK activity, which was prevented by GSH, suggesting that CKcontaining disulfide groups are also a target for sulfide attack. The mechanism possibly involved in GSH-mediated protection possibly consists in the reaction between this antioxidant and sulfide that is catalyzed by sulfide-coenzyme Q reductase in which GSH

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ACCEPTED MANUSCRIPT accepts the sulfur atom of sulfide forming nontoxic GSSH persulfide [42, 43]. These findings allied to cytochrome c oxidase inhibition [11, 12] may explain the bioenergetics

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dysfunction observed in patients with EE [7, 44, 45].

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Our data also evidenced that sulfide impairs ETS and OXPHOS, corroborating

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previous studies [13, 46-48]. It may be presumed that the decrease of CS and ACO activities, besides cytochrome c oxidase inhibition, elicited by sulfide underlie ETS and OXPHOS impairment. Moreover, sulfide decreased ΔΨm and increased mitochondrial

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swelling in brain mitochondria in the presence of calcium. These alterations were blocked

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by the inhibitors of mPT CsA [49-52] and ADP [53, 54], as well as by the mitochondrial Ca2 + uptake blocker RR [55], indicating induction of mPT pore opening and the involvement of intramitochondrial Ca2 + concentrations in sulfide toxicity. CsA inhibits

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mPT by binding cyclophilin D, a mitochondrial matrix protein that modulates mPT pore through its interaction with the adenine nucleotide translocator (ANT) [49, 51, 56, 57],

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whereas ADP blocks the pore by directly binding to ANT [54, 58]. Taken together our data, it is conceivable that sulfide induces mPT pore opening by disturbing energy

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metabolism. It is further possible that sulfide-induced pore opening occurs via direct interaction of this compound with disulfide groups of proteins that are mPT pore components. Indeed, previous studies showed that the pore is regulated by oxidationreduction state of thiol groups [59-62]. In addition, considering that mPT pore opening leads to cytochrome c release that may trigger apoptosis and that previous findings showed that sulfide is cytotoxic to rat hepatocyte suspensions [14], we examined whether sulfide could alter cell viability in cerebral cortex slices and astrocytes. Although we did not verify alterations on cell viability, it is possible that longer periods of exposure to sulfide or higher concentrations of this metabolite could alter this parameter.

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ACCEPTED MANUSCRIPT Sulfide also induced lipid peroxidation in brain, which was prevented by Res and GSH, suggesting the involvement of reactive species. This is in line with recent findings

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demonstrating that sulfide increases F2-isoprostanes, a final lipid peroxidation product, in

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mouse heart and brain [13]. The fact that GSH prevented sulfide-induced lipid

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peroxidation and also CK activity decrease is in accordance with reports showing that Nacetylcysteine (a GSH precursor) combined with the antibiotic metronidazole prolongs the lifespan of ETHE1-deficient mice and improves clinical conditions of some patients

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[43]. On the other hand, sulfide did not alter DCFH oxidation in cerebral cortex

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supernatants, cortical astrocytes and SH-SY5Y cells, showing that this metabolite does not modify this parameter after short and long periods of exposure. However, we cannot rule out that reactive species not detected by DCFH are generated by sulfide. In this

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regard, we further verified that sulfide per se was able to oxidize Amplex Red, a common probe used to detect hydrogen peroxide production [26], suggesting that sulfide is a very

generation.

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reactive molecule in our experimental conditions and may lead to reactive species

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It is difficult to establish the significance of our results since sulfide concentrations in brain of patients affected by EE are unknown. However, since most physiological effects of sulfide appear to be mediated in the micromolar concentration range [35], it is conceivable that higher concentrations are achieved in brain of ETHE1-deficient individuals. This assumption is reinforced by the fact that thiosulfate, which is considered to reflect the presence of sulfide [1], can reach levels as high as 860 µM in urine [7] and 170 µM in plasma of some patients [43]. On the other hand, the fact that cyanide dissipated ΔΨm without Ca2+ addition, and did not alter CK activity and MDA levels suggests that sulfide-induced toxic effects are specific and possibly mediated by hydrosulfide anion.

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ACCEPTED MANUSCRIPT In conclusion, to our knowledge, this is the first report showing that sulfide decreases the activities of CK and some CAC enzymes, and induces mPT pore opening

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in brain. These findings showing that sulfide causes bioenergetics failure may explain the

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lactic acidosis and mitochondrial microscopic abnormalities found in tissues of patients

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[4, 63-65]. Our data allied to previous reports suggest that antioxidants and/or compounds capable of binding and neutralizing sulfide could be considered as promising clinical

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candidates for treatment of EE.

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ACCEPTED MANUSCRIPT Acknowledgements This work was supported by grants from Conselho Nacional de Desenvolvimento

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Científico e Tecnológico (CNPq), Programa de Apoio a Núcleos de Excelência

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(PRONEX II), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul

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(FAPERGS), Pró-Reitoria de Pesquisa/Universidade Federal do Rio Grande do Sul (PROPESQ/UFRGS), Financiadora de estudos e projetos (FINEP), Rede Instituto Brasileiro de Neurociência (IBN-Net) # 01.06.0842-00 and Instituto Nacional de Ciência

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e Tecnologia em Excitotoxicidade e Neuroproteção (INCT-EN).

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ACCEPTED MANUSCRIPT Highlights Ethylmalonic encephalopathy (EE) is characterized by sulfide accumulation.

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Sulfide decreases creatine kinase, citrate synthase and aconitase activity in brain.

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Sulfide decreases mitochondrial respiration and ΔΨm, and increases swelling.

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Sulfide induces bionergetic dysfunction, lipid peroxidation, and mPT pore opening.

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Sulfide toxicity is involved in the neuropathology of EE.

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