Molecular and biochemical evidences on the protective effects of triiodothyronine against phosphine-induced cardiac and mitochondrial toxicity

    Molecular and biochemical evidences on the protective effects of Triiodothyronine against phosphine-induced cardiac and mitochondrial toxicity Amir Hossein Abdolghaffari, Amir Baghaei, Reza Solgi, Maziar Gooshe, Maryam Baeeri, Mona Navaei-Nigjeh, Shokoufeh Hassani, Abbas Jafari, Seyed Mehdi Rezayat, Ahmad Reza Dehpour, Shahram Ejtemaei Mehr, Mohammad Abdollahi PII: DOI: Reference:

S0024-3205(15)00389-6 doi: 10.1016/j.lfs.2015.07.026 LFS 14462

To appear in:

Life Sciences

Received date: Revised date: Accepted date:

15 April 2015 12 July 2015 26 July 2015

Please cite this article as: Abdolghaffari Amir Hossein, Baghaei Amir, Solgi Reza, Gooshe Maziar, Baeeri Maryam, Navaei-Nigjeh Mona, Hassani Shokoufeh, Jafari Abbas, Rezayat Seyed Mehdi, Dehpour Ahmad Reza, Mehr Shahram Ejtemaei, Abdollahi Mohammad, Molecular and biochemical evidences on the protective effects of Triiodothyronine against phosphine-induced cardiac and mitochondrial toxicity, Life Sciences (2015), doi: 10.1016/j.lfs.2015.07.026

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ACCEPTED MANUSCRIPT Molecular and biochemical evidences on the protective effects of Triiodothyronine against phosphine-induced cardiac and mitochondrial toxicity Amir Hossein Abdolghaffaria,b,c,d, Amir Baghaeid, Reza Solgid, Maziar Gooshee,f, Maryam

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Baeerid, Mona Navaei-Nigjehd, Shokoufeh Hassanid, Abbas Jafarid, Seyed Mehdi Rezayatc,g,

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a

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Ahmad Reza Dehpourc,e, Shahram Ejtemaei Mehrc, Mohammad Abdollahid,h,i*

Department of Pharmacology, School of Medicine, International Campus, Tehran University of

Medical Sciences, (TUMS- IC), 1417653861,Tehran, Iran b

Pharmacology and Applied Medicine, Department of Medicinal Plants Research Center, Institute

c

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of Medicinal Plants, ACECR, 141554364, Karaj, Iran

Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences,

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1417614411, Tehran, Iran d

Department of Toxicology and Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences

Research Center, Tehran University of Medical Sciences, Tehran 1417614411, Iran e

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Experimental Medicine Research Center, Tehran University of Medical Sciences, 1417614411,

f

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Tehran, Iran

Students’ Scientific Research Center (SSRC), Tehran University of Medical Sciences,

g

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1417614411, Tehran, Iran

Department of Pharmacology and Toxicology, Pharmaceutical Sciences Branch & Pharmaceutical

Sciences Research Center, Islamic Azad University (IAUPS), 194193311, Tehran, Iran i

International Campus, Tehran University of Medical Sciences, Tehran1417614411, Iran.

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h

Endocrinology & Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences

Institute, Tehran University of Medical Sciences, Tehran

Correspondence to: Prof. Mohammad Abdollahi, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 1417614411, Iran; Email: [email protected] or [email protected] Telephone and fax: +98-21-66959104

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ACCEPTED MANUSCRIPT Abstract Aim: Aluminum phosphide (AlP) is a widely used fumigant and rodenticide. While AlP ingestion leads to high mortality, its exact mechanism of action is unclear. There are ample evidences suggesting cardioprotective effects of triiodothyronine (T3). In this study, we aimed to examine the

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potential of T3 in the protection of a rat model of AlP induced cardiotoxicity.

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Main methods: In order to induce AlP intoxication animals were intoxicated with AlP (12 mg/kg;

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LD50) by gavage. In treatment groups, T3 (1, 2 and 3 µg/kg) was administered intra-peritoneally 30 min after AlP administration. Animals were connected to the electronic cardiovascular monitoring device simultaneously after T3 administration. Then, electrocardiogram (ECG), blood pressure (BP), and heart rate (HR) were monitored for 180 minutes. Additionally, 24 h after AlP

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intoxication, rats were deceased and the hearts were dissected out for evaluation of oxidative stress, cardiac mitochondrial function (Complex I, II and IV), ATP/ADP ratio, caspase 3 & 9, and

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apoptosis by flow cytometry.

Key findings: The results demonstrated that AlP intoxication causes cardiac toxicity presenting with changes in ECG patterns such as decrement of HR, BP and abnormal QRS complexes, QTc and ST

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height. T3 at a dose of 3µg/kg significantly improved ECG and also oxidative stress parameters.

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Furthermore, T3 administration could increase mitochondrial function and ATP levels within the cardiac cells. In addition, administration of T3 showed a reduction in apoptosis through diminishing

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the caspases activities and improving cell viability. Significance: Overall, the present data demonstrate the beneficial effects of T3 in cardiotoxicity of

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AlP.

Keywords: Triiodothyronine, Aluminum phosphide, Phosphine gas (PH3), Oxidative stress, Mitochondrial toxicity, Apoptosis

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ACCEPTED MANUSCRIPT Introduction Aluminum phosphide (AlP) is a commonly used insecticide, rodenticide and fumigant. Poisoning by deliberate self-ingestion of AlP is a common cause of death and socioeconomic loss worldwide, especially in developing countries [2,13,26]. AlP is sold as pallet, tablet, porous blister pack,

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sachets, and as dusts [43].

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While the exact mechanism of AlP toxicity is still unclear, several studies suggest that Phosphine

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gas (PH3) is a key player in AlP toxicity. PH3 is a highly reactive radical, which can freely diffuse into intracellular compartments. PH3 is released from AlP upon contact with water, moisture or hydrochloric acid of the stomach [43]. There are ample evidences suggesting that PH3 can initiate a nucleophilic attack and reduce vital enzymes [3].

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AlP intoxication is mostly fatal by causing multiorgan damage through denaturation of cell membranes [53,56,57]. While AlP can cause a wide range of clinical manifestations, circulatory

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failure is the most common cause of mortality and morbidity in AlP ingested patients [5,60]. Ventricular arrhythmias or dysfunction is a primary outcome of AlP induced cardiac toxicity. Virtually any type of arrhythmia and conduction abnormality such as tachycardia and bradycardia

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following AlP poisoning [8,12].

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can occur. On the other hand, there are several studies reporting reduced left ventricular ejection

Evidences for the mechanism of PH3 demonstrated that it disrupts mitochondrial activity through

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inhibition of cytochrome c oxidase (complex IV), decrement of complex I and II activity and also impairment in ATP synthesis [59,20]. Consequently, inhibition of the electron transport chain (ETC) could lead to a raise of reactive oxygen species (ROS) production [3]. In addition, PH3 can

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exacerbate oxidative stress through inhibition of catalase, peroxidase and increasing the superoxide dismutase (SOD) activity [9]. Most patients with established AlP poisoning die despite intensive care. There are several choices used for the management of AlP poisoning, including gastric lavage with potassium permanganate solution and oral sodium bicarbonate (Bicarb) with activated charcoal. In addition, oral coconut oil and intravenous magnesium have been proposed to be effective [56,49]. Despite many efforts conducted in order to overcome this challenge, the management of AlP poisoning is still a big obstacle in the world. Besides, there is no specific antidote and the major part of management remains supportive only [3]. Thyroid hormones (THs) are inotropic hormones, which could be potentially used to support hemodynamics [21,22,33,47,48]. Subsequently, several clinical studies appeared using triiodothyronine (T3) or thyroxine (T4) to treat heart failure. Short-term intravenous administration of T3 to patients with advanced congestive heart failure was reported to improve cardiac output and decrease arterial vascular resistance [28]. Page 3 of 30

ACCEPTED MANUSCRIPT On the basis of the evidence obtained from cells, animals, and even humans, it seems likely that timely treatments targeting the TH signaling may promote endogenous regeneration of the damaged myocardium [39,54,50]. THs are well-known regulators of mitochondrial biogenesis and function [65,27]. THs have been shown to limit cell apoptosis under stress conditions in several models

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[24,39]. A previous study demonstrated that THs are potent regulators of cardiac muscle

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contractility [10]. In the present study, we aimed to investigate the potential of T3 in the

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cardioprotection of a rat model of AlP-poisoning through examining electrocardiographic and biochemical parameters of toxicity.

Materials and Methods

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Ethics

The procedures implemented throughout the study were approved by the Ethics Committee of

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Tehran University of Medical Sciences in accordance with the Standards for the Care and Use of Laboratory Animals with code number 89-03-33-11232.

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Chemicals

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The following compounds were used throughout the study: AlP (95% purity) was purchased from Samiran Pesticide Formulating Co. (Tehran, Iran). Triiodothyronine (T3) (Sandoz Co.). The

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mitochondria isolation kit was obtained from Bio-Chain Ins. (Newark, New Jersey, USA). Annexin V-FITC/PI was obtained from Beijing Biosea Biotechnology Co, Ltd (Beijing, China). Adenosine diphosphatesodium

salt

(ADP),

adenosine

triphosphate

disodium

salt

(ATP),

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tetrabutylammoniumhydroxide (TBAHS), methanol (HPLC grade), column (SUPELCOSIL™ LC18-T)from Supelco (Antrim, UK), acetic acid, FeCl3-6H2O, sodium sulfate,trichloroacetic acid (TCA), potassium dihydrogen phosphate anhydrous (KH2PO4, analytical grade), 2,4,6-tripyridyl-striazine (TPTZ), 2-thiobarbituric acid (TBA), rotenone, 2,6-dichloroindophenol (DCIP), antimycin A, collagenase. All other chemicals were of the highest purity available and were purchased from Aldrich Chemical Co. Sigma Chemical or Co. (St Louis, Missouri, USA).

Animals Sixty male Albino Wistar rats weighing 200-250 g were used in this study. The animals were housed in standard polycarbonate cages in groups of 4–5 and kept in a temperature-controlled room (22° C) with a 12 h light/12 h dark cycle. Animals were acclimated at least 2 days before experiments with free access to food and water. The experiments were conducted between 09:00 and 13:00. All procedures were carried out in accordance with institutional guidelines for animal Page 4 of 30

ACCEPTED MANUSCRIPT care and use. The groups consisted of at least twelve animals and each animal was used only once. Additionally, efforts were made to reduce animal suffering and to use only the number of animals necessary to produce reliable scientific data.

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Induction of AlP intoxication

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AlP (4-16 mg/kg; dissolved in 2 ml of almond oil) was orally administered (gavage, in almond oil)

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to different groups of rats. The mortality rate was recorded for 24 h. For this purpose, the oral LD50 of AlP was calculated according to Probit analysis (12 mg/kg) and was used for induction of cardiac toxicity and related events [6,32].

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Experimental design

A total number of 60 animals were assigned randomly to 5 groups, each comprising 12 rats. Rats

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were treated according to the following schemas:

In the first step of this study, in order to evaluate the electrocardiogram (ECG) parameters, rats were received 12 mg/kg AlP orally (LD50) in all groups except control group which received almond oil.

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Then the animals were assigned randomly to 5 groups, including 1) Control group received almond

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oil alone; 2) AlP group received aluminum phosphide (12 mg/kg); 3) AlP+T3-1group received AlP (12 mg/kg)+T3 (1 µg/kg); 4) AlP+T3-2 group received AlP (12 mg/kg)+T3 (2 µg/kg); 5) AlP+T3-3

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group received AlP (12 mg/kg)+T3 (3 µg/kg). T3 was administered by intra-peritoneal injection (i.p) 30 min after AlP administration by gavage in the groups which received T3. In order to evaluate the biochemical parameters, rats were assigned randomly to 5 groups that all

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groups except the control group received 0.25 LD50 of AlP including 1) Control group received almond oil alone; 2) AlP group received aluminum phosphide (0.25 LD50); 3) AlP+T3-1 group received AlP (0.25 LD50)+T3 (1 µg/kg); 4) AlP+T3-2 group received AlP (0.25 LD50)+T3 (2 µg/kg); 5) AlP+T3-3 group received AlP (0.25 LD50)+T3 (3 µg/kg). The doses of T3, as well as the time interval between drug injection and AlP intoxication (30 min) were chosen according to literature review [16,24,32].

Determination of electrocardiogram (ECG) parameters Thirty minutes after AlP gavage, animals were anesthetized by intra-peritoneal injection of (30 mg/kg) thiopental sodium and rapidly connected to the PowerLab device (PowerLab 4/35 Data Acquisition Systems, AD Instruments, Australia) for complete monitoring of ECG. For maintaining full general anesthesia, 30 mg/kg thiopental sodium was repeated after 40 minutes and 2.5 hours until the end of the experiment. ECG needle electrodes of the Powerlab™ were inserted under the skin of the right hand and both legs of the anesthetized rat (position II) and continuous ECG data Page 5 of 30

ACCEPTED MANUSCRIPT were achieved for 3 h. For each ECG tracing, QRS complexes and the segments of QT, and ST were measured. ECGs were analyzed by PowerLab system software. The heart rate (HR) and systolic blood pressure (BP) were recorded every 3 minutes by using the tail cuff which was

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connected to the anesthetized rat tail [6,32].

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Collection of samples

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Twenty-four hours after administration of 0.25 LD50 AlP and treatment with T3 in all treatment groups, rats were sacrificed and the hearts were dissected out and washed in ice-cold saline (4°C) to remove the blood, 100-150 mg of fresh cardiac tissue was collected according to mitochondria isolation kit protocol and then remained tissues were immediately frozen and stored at -80°C for

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various assays. For analysis of oxidative stress parameters, the heart was homogenized in a suitable buffer using a tissue homogenizer at 4°C. The homogenates were centrifuged at 3500 g for 20min

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and the supernatant was used for oxidative stress analyses [6].

Thyroid hormone analyses

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Three hours, post administration of an LD50 dose of AlP and treatment with different doses of T3,

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1 ml blood samples were obtained from the tail vein and kept on ice until centrifuged at 16000 g for 15 minutes at 4°C. Serum samples were collected to measure T3 and T4 concentrations using a

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chemiluminescence assay [29].

Assessment of oxidative stress

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Determination of Ferric Reducing/Antioxidant Power (FRAP) The FRAP test is performed on the basis of the antioxidant power of plasma to deoxidizeFe 3+‫ ‏‬to Fe2+‫‏‬. The reagents included 300 mM acetate buffer (pH 3.6) with 16 ml acetic acid per liter of buffer solution, 10 mM TPTZ in 40 mM HCl and 20 mM FeCl3.Working FRAP reagent was prepared as required by mixing 25 ml acetate buffer, 2.5 ml TPTZ solution and 2.5 ml FeCl 3 solution. Ten micro liters of H2O diluted sample was then freshly added to 300 ml reagent warmed at 37C. The complex between TPTZ‫‏‬and Fe2+gives a blue color with absorbance at 593 nm [1].

Lipid peroxidation assay One of the end products ofthe oxidation of polyunsaturated fatty acids is Malonedialdehyde (MDA) that reacts with thiobarbituric acid (TBA) to produce a complex that can be determined by spectrophotometer, named as TBA reactive substances (TBARS).Sampleswere diluted with buffered saline (1:5), aliquot (400 ml) +TCA (28%w/v, 800 ml); and centrifuged at 3000 × g (30 min, 4°C).Thensupernatant (600 ml)+ TBA (1% w/v, 150 ml); incubated (15 min, 95C) + nPage 6 of 30

ACCEPTED MANUSCRIPT butanol (4 ml), then the solution were centrifuged and absorption of the supernatant measured at 532 nm in a BioTek® spectrophotometer. The method was calibrated with tetraethoxypropanestandard solutions [61].

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Superoxide dismutase (SOD) assay

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The basis of an assay of SOD is the reaction of SOD and the indicator molecule, NBT. The rate of

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increase at 560 nm over a 5-min time period indicates the reduction of NBT by superoxide. Varying amounts of total protein were added to the reaction until maximal inhibition was obtained as determined by spectrophotometry. Total SOD activity was determined as the amount of protein necessary for half-maximal inhibition of the NBT reaction. The method is set up in the lab and the

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procedure was described previously [55].

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Measurement of reactive oxygen species (ROS)

To measure reactive oxygen species (ROS) generation, a fluorometric assay using intracellular oxidation of 2,7-dichlorofluoroscein diacetate (DCFH-DA) was performed, that was set up in our

Measurement of total thiol

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laboratory and was described previously in detail [30].

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To measure total thiol (SH), a volume of sample (0.2 mL) was mixed with 0.6 mL of Tris–EDTA buffer (Tris base [0.25 M], EDTA [20 mM], pH 8.2) in a 10-mL test tube and then mixed with 40 mL of DTNB (10 mM) in methanol. The final volume was made up to 4.0 mL by adding 3.16 mL

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of methanol. The test tube, after capping, was centrifuged at 3000 g for 10 minutes at an ambient temperature. After 15-20 minutes, the color appeared. The absorbance of the supernatant was measured at 412 nm [44].

Determination of cardiac mitochondrial function Complex I (NADH –ubiquinone Oxidoreductase) activity assay Complex I activity assay was determined spectrophotometrically (340 nm) by monitoring the oxidation sensitive to rotenone of NADH to NAD+in the presence and absence rotenone.The decrease in absorbance of NADHat 340 nm, was recorded as the total activity of complex I, for 3 min by a spectrophotometer from BioTek® instruments, Inc. (Winooski, USA). The enzyme activity was calculated using an extinction coefficient of 6.22 mM-1cm-1 at 340 nm and was reported as µM NADH/min/mg mitochondrial protein [58].

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ACCEPTED MANUSCRIPT Mitochondrial Complex II activity assay The 2,6-dichlorophenolindophenol (DCPIP) reduction shows the Complex II (succinate–ubiquinone oxidoreductase)specific activity, which was determined by Spectrophotometric analysis at 600 nm. The mitochondria were preincubated in potassium phosphate buffer, MgCl 2, and succinate and then

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after adding antimycin A, rotenone, KCN, and DCPIP, the baseline was recorded for 3 min. The

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reaction was started with ubiquinone and the enzyme-dependent reduction of DCPIP was measured

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for 3–5 min at 600 nm. The complex II activity was calculated using a DCPIP standard curve and was reported as µM DCIP/min/mg of mitochondrial protein [17].

Complex IV (cytochrome c oxidase) activity assay

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First of all, the cytochrome c was reduced by adding enough sodium hydrosulfite that, mitochondrial protein and lubrol-PX in potassium phosphate buffer were then added to the reduced

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cytochrome C to start the reaction. According to the previously established spectrophotometric method, the decrease in optical absorption at 550 nm was measured for 3–6 min. Data were presented as the natural logarithm of the absorbance divided by time and reported as the first-order

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rate constant (k) min/ mg of mitochondrial protein [14].

Determination of ADP/ATP ratio

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In order to calculate the ADP/ATP ratio, 300 mg heart tissue of each rat was sonicated in 250 µL of TCA (6%) and then centrifuged at 12,000 g for 10 min at 4ºC. The supernatant was removed and neutralized with potassium hydroxide (KOH; 4 M). The HPLC system (Waters Chromatography

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Division, Milford, MA, USA) was performed with a 510-pump and solvent delivery system, column (SUPELCOSIL1 LC-18-T, Supelco, Inc., Bellefonte, USA) with a guard column, and 486 UV-Visible Detector all from Waters (Milford, Massachusetts, USA). Isocratic elution (flow: 1 ml/min; 254nm) with tetrabutylammonium hydrogen sulfate (4 mM) in potassium phosphate buffer (0.1 M; pH =5.5) and methanol (85:15 v/v) was used according to the protocol provided. The levels of ATP and ADP were determined with standard curve and then the ratio was calculated [31].

Caspase 3 and 9 assays Caspase 3 and 9 activities were measured by colorimetric assays based on the identity of specific amino acid sequences by these caspases. The tetrapeptide substrates were labeled with the chromophore r-nitroaniline (rNA). rNA is released from the substrate upon cleavage by caspase and produces a yellow color that is monitored by an ELISA reader at 405 nm. The amount of caspase activity present in the sample is proportional to the amount of yellow color produced upon cleavage. Page 8 of 30

ACCEPTED MANUSCRIPT Briefly, the pretreated islets were lysed in the supplied lysis buffer and were incubated on ice for10 min. The whole cell lysates were incubated in caspase buffer (100 mM HEPES, pH 7.4, 20% glycerol, 0.5 mM EDTA, 5 mM dithiothreitol) containing 100 mM of caspase-3 and -9 specific substrate (N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVDpNA:), N-acetyl-Leu-Glu-His-Asp-

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p-nitroanilide (Ac-LEHD-pNA), respectively) for 4 h at 37°C. Then, absorbance was measured at

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405 nm. Caspase activity was defined as nanomoles per rNA released per hour per milligram of

Apoptosis and necrosis analysis by flowcytometry

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protein (nanomoles per hours milligrams of protein) by a rNA calibration curve [36].

Measurement of apoptosis and necrosis by flow cytometery needs isolated single cells, for this

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purpose, the rats were grouped in five (n= 3 in each group) and received AlP and T3 in the same manner as described in the study design. The rats were anesthetized, then the left ventricles of heart

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were rapidly removed in each rat and used the collagenase digestion method that described in references [23,52,63]. The cells were washed with phosphate-buffered saline (PBS) at room temperature and stained with annexin V-FITC and propidium iodide (PI) according to the kit

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instructions. The stained cells were incubated in binding buffer and cell death was analyzed by

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flowcytometry (Apogee, UK). The samples were run on the flow cytometer (at least 2×104 events for each sample); afterwards, data acquisition was performed and analyzed with the apogee

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histogram software. Density plots of flowcytometry are included; the percentage of alive cells (lower left quadrant; annexin V; PI), the percentage of cells in apoptosis (lower right quadrant; annexin V; PI), late apoptosis (upper right quadrant; annexin V; PI), and necrosis (upper left

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quadrant; annexin V; PI) [38,64].

Statistical analysis

Results have been presented as means±SEM. Also data were given as the median and 95% confidence interval. All statistical analyses were performed using Stats Direct version 3.0.146. Assays were performed in triplicate and the mean was used for the statistical analyses. Statistical significance was determined using the one-way ANOVA test, followed by the post-hoc Tukey test. P<0.05 was considered to be statistically significant.

Results Electrocardiogram parameters As shown in Table 1, examination of ECG from AlP administered rats revealed significant and serious changes. After administration of AlP, HR was significantly decreased in all groups in comparison to control group. Treatment with T3 at doses of 2 and 3 µg/kg caused a significant Page 9 of 30

ACCEPTED MANUSCRIPT increase in HR in comparison to AlP group (P<0.05). Increasing of HR was observed in T3-2 µg/kg over 150-180 min and in T3-3 µg/kg after 120-150 min (Table 2). Administration of AlP caused a decrease of BP in all groups in comparison to control group. Treatment with T3 at doses of 2 and 3 µg/kg demonstrated a remarkable increment in BP after 120-180 min (P<0.05), however, at a dose

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of 1 µg/kg could not increase this parameter significantly (Table 3). Furthermore, abnormal QRS

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complexes, QTc and ST height were observed in the AlP group in comparison to control group.

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QRS interval increased during 30-60 min in AlP group in comparison to control group (P<0.05), although treatment with T3 at all doses decreased this parameter in comparison with the AlP group (P<0.05) (Table 1). AlP gavage caused a significant depression in ST height in comparison to control group (P<0.05), however remarkable rise in ST height was observed in groups that received

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T3 at all doses (P<0.05) (Table 1). Hence, according to the ECG results, T3 revealed protective

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effects against AlP induced cardiac damage (Figure 1).

Thyroid hormone assay

As shown in Table 4, serum T3 concentration in AlP group showed no significant changes

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compared to the control group. Although, treatment with T3 at all doses significantly increased

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level of serum T3 concentration in comparison with control and AlP group (P<0.05). Data

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represented that serum T4 concentration showed no significant changes in all groups.

Measurement of heart tissue oxidative stress FRAP

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As shown in Figure 2A, antioxidant power was decreased in the AlP group 93.56 (95% Confidence intervals (CI) 89.24-97.90) as compared with control group 124.57 (95% CI 119.37-129.77) (P<0.001). FRAP value significantly increased in T3 groups in a dose of 2 μg/kg 110.57 (95% CI 102.94-118.19) and 3 μg/kg 114.12 (95% CI 110.63-117.62) in comparison to AlP group 93.56 (95% CI 89.24-97.90) (P<0.001). Although improvement of FRAP during treatment with 3 μg/kg were significantly higher than 2 μg/kg in comparison to AlP group.

Lipid proxidation (LPO) As shown in Figure 2B, LPO was increased in the AlP group 206.3 (95% CI 175.94-236.65) as compared with control group 120.36 (95% CI 69.270-171.45) (P<0.001). Administration of T3 at doses of 2 μg/kg 148.42 (95% CI137.89-158.95) (P<0.01) and 3 μg/kg 127.26 (95% CI 102.25152.26) (P<0.001) significantly reduced LPO level in heart tissue in comparison to AlP group, although this decrement is lower in 3 μg/kg than 2 μg/kg. Page 10 of 30

ACCEPTED MANUSCRIPT Superoxide dismutase (SOD) As shown in Figure 2C, SOD significantly increased in the AlP group 0.31 (95% CI 0.025-0.036) in comparison to control group 0.018 (95% CI 0.010-0.025) (P<0.01). Treatment with T3 with all three doses decreased SOD in comparison to AlP group, but T3 with 3 µg/kg 0.017 (95% CI 0.013-

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0.020) significantly decreased SOD in comparison to AlP group 0.031 (95% CI 0.024-0.036)

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(P<0.01).

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Reactive Oxygen Species (ROS)

As shown in Figure 2D, AlP 2.523 (95% CI 1.931-3.114) significantly increased ROS levels in comparison to control group 1.653 (95% CI 1.031-2.274) (P<0.01). ROS levels decreased in all

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treatment groups with T3 in comparison to AlP group. Treatment with T3 at a dose of 2 μg/kg 1.885 (95% CI 1.709-2.061) could significantly decrease this level compared to other group (P<0.05).

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Furthermore, administration of T3 at a dose of 3 μg/kg 1.705 (95% CI 1.437-1.973) could decrease levels of ROS in comparison to AlP group 2.523 (95% CI 1.931-3.114) (P<0.01).

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Thiol molecules

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As shown in Figure 2E, AlP 156.04 (95% CI 154.9-157.17) could significantly decrease soluble thiol level in comparison to control group 199.98 (95% CI 183.56-216.14) (P<0.001). Treatment

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with T3 at a dose of 3 μg/kg 181.34 (95% CI 172.2-190.49) could improve and elevate level of thiol in comparison to AlP group 156.04 (95% CI 154.9-157.17) (P<0.001).

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Mitochondrial cardiac activity

With the aim of analyzing the cardiac mitochondrial function, activities of respiratory chain in each complex were measured individually (separately). Complex I activity was shown in Figure 3A, which revealed no significant changes in all groups that received AlP in comparison to control group, even in groups that treated with T3 in all doses. Furthermore, complex II and IV showed decreased activity after administration of AlP (complex II: 149.13 (95% CI145.43-152.84), complex IV: 2.87 (95% CI 1.85-3.88)] in comparison to control group (complex II: 185.29 (95% CI 166.95-203.63), complex IV: 7.25 (95% CI 6.15-8.35)] (P<0.05). Administration of T3 in all doses could increase activity in complex II and IV over 24 hours. Although administration of T3 at a dose of 3μg/kg could significantly increase the activity of complex II 175.35 (95% CI 167.48-183.23) and complex IV 5.9 (95% CI 5.56-6.24) in comparison to AlP group (Figures 3B and 3C) (P<0.05).

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ACCEPTED MANUSCRIPT Measurement of cardiac energy as ADP/ATP As shown in Figure 3D, administration of AlP 4.52 (95% CI 4.48-4.56) reduced cellular ATP level (increase in the ADP/ATP ratio) in comparison to control group 2.45 (95% CI2.38-2.52) (p<0.05). Treatment with T3 (at three doses) could increase cellular ATP levels in a dose dependent manner

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in comparison to AlP group. However, treatment with T3 in comparison to other treatment groups

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in a dose of 3 μg/kg 2.8 (95% CI 2.75-2.86) could significantly increase ATP levels in heart tissue

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cells in comparison to the AlP group 4.52 (95% CI 4.48-4.56) (P<0.001). Caspase 3 and 9

AlP caused a significant increase in the cardiac activities of caspase 3,165.14 (95% CI 113.25-

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217.03) and caspase 9, 139.67 (95% CI 129.49-149.85) when compared to control group [Caspase3: 100 (95% CI 79.427-120.57), Caspase 9: 95.71 (95% CI 89.48-101.95) (P<0.01). Treatment with

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T3 at doses of 1, 2 and 3μg/kg demonstrated a significant and dose dependent reduction in the cardiac activities of caspase 3 and caspase 9 in comparison to AlP group. Administration of T3 at a dose of 3 μg/kg significantly decreased caspase 3, 102.75 (95% CI 86.75-118.75) in comparison to

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AlP group 165.14 (95% CI 113.25-217.03) (P<0.01), furthermore treatment with T3 at the same

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dose caused a significant decrease of caspase 9, 101.04 (95% CI 82.92-119.14) in comparison to

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AlP group 139.67 (95% CI 129.49-149.85) (P<0.001) (Figure 4B).

Apoptosis and necrosis by flowcytometry The aim of the flow cytometry in our study was to evaluate the cell death using annexin-V-FITC

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and PI, which stains Phosphatidylserine and DNA residues. As shown in the Figure 4A the percentage of cell viability was significantly decreased in AlP group, 75% (95% CI 69-81) in comparison to control group, 96% (95% CI 88.32-103.68) (P<0.05). The cell viability of all treated groups with T3 was higher than AlP group, although only the group treated with T3 at a dose of 3 μg/kg 96% (95% CI 88.32-103.68) was significantly higher than AlP group75% (95% CI 69-81) (P<0.05). Furthermore, the percentage of necrotic cells, in AlP group, 6% (95% CI5.52-6.48) significantly increased in comparison to control group, 1% (95% CI 0.92-1.08) (P<0.001). Also treatment with T3 at all doses could significantly decrease necrotic cells in comparison to the AlP group6% (95% CI 5.52-6.48) (P<0.001).

Discussion In this study, we demonstrated cardio-protective effects of T3 in acute AlP toxicity model in rats by investigating mitochondrial complexes, ATP levels, apoptotic and oxidative stress biomarkers. Our Page 12 of 30

ACCEPTED MANUSCRIPT findings demonstrated that T3 administration significantly increases mitochondrial complexes activities and ATP levels. Furthermore, apoptotic and oxidative biomarkers were dramatically reduced following T3 treatment. Interestingly, our biochemical findings were consistent with a significant improvement in electrocardiographic parameters.

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Despite the fact that AlP poisoning is a leading cause of death and socioeconomic loss worldwide;

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there is limited knowledge about the underlying mechanisms. The main cause of mortality and

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morbidity in AlP poisoning is cardiac dysfunction. Phosphine gas released as a result of AlP reaction with water, moisture or hydrochloric acid of the stomach, mediates the cardiotoxic effect of AlP. Ample evidences suggest that mitochondrial impairment and oxidative stress are two crucial modulators of AlP induced cell loss and contractile dysfunction in myocardium [41,4].

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While heart failure and refractory hypotension are major clinical manifestations of cardiac toxicity in AlP poisoning, several reports addressed further signs, including tachycardia, bradycardia, atrial

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flutter and fibrillation, ST and T wave changes[41]. Besides, autopsy examinations revealed focal necrosis, neutrophil and eosinophil infiltration, congestion, separation and fragmentation of myocardial fibers, and nonspecific vacuolation of myocytes [5].

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There are mounting evidence suggesting that T3 administration has an acute ameliorative effect on

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cardiac contractility that improve cardiac function in several animal models of cardiac dysfunction, including ischemia-reperfusion injury and heart failure [18,34,37,46]. Previous studies have

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indicated that THs have positive inotropic properties that could potentially be used in clinical practice [7,25,29,51]. Furthermore, Dillman et al demonstrated that administration of T3 can improve cardiac myosine ATPase activity and restored cardiac myosine v1 predominancy [19].

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According to the results of our study, administration of T3 could alleviate AlP-induced hypotension and bradycardia after 120-150 min. Furthermore, this study revealed that administration of T3 could normalize QRS, QTc and ST height in comparison to AlP group. Evolving data indicated that PH3 induced electron transport chain (ECT) impairment through decreasing complex I, II and IV (cytochrome C). Inhibition of ETC can cause ATP reduction and ROS production through slowing down of electron flow with resultant electron leakage. On the other hand, PH3 can cause oxidative tissue damage through inhibition of antioxidant enzymes (ROS scavengers). Impairment of mitochondrial activity and oxidative stress balance can consequently activate necrotic and apoptotic pathways. These processes can play pivotal role in the pathophysiology of AlP induced multi-organ dysfunction, though most of the patients do not demonstrate these clinical manifestations because of the preceding cardiac dysfunction and arrhythmias and resultant sudden death [8,35]. Indeed, in a series of experiments, we have addressed the contribution of mitochondrial impairment and oxidative stress in the pathogenesis of AlP induced cardiac toxicity in rat [6,41]. Based on the results of our study, phosphine toxicity led Page 13 of 30

ACCEPTED MANUSCRIPT to 70% drop in mitochondrial complex IVactivity, which in turn resulted in significant surge in ROS production through transfer of ETC electrons to molecular oxygen. In addition, phosphineinduced inhibition of antioxidant enzymes aggravates this situation. In contrast, by administration of T3, activities of cytochrome c oxidase and levels of antioxidant enzymes both returned to normal

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state which is confirmed by declining level of ROS compared to the control group.

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Concluding remarks raise the possibility of involvement of cardiac mitochondrial damage, ATP

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depletion and oxidative stress in the pathogenesis of AlP induced cardiac toxicity. In this regard, our findings indicated that oral administration of AlP resulted in cardiac dysfunction as revealed by increasing cell death via apoptotic/necrotic pathways and negative ECG variables compared to the control group. Looking for molecular explanation of these pathophysiological processes, we

oxidative stress biomarkers and caspase 3 and 9.

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recorded decrement of mitochondrial complex's activities and ATP levels as well as an increment of

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The THs play critical role in the modulation of oxidative energy metabolism at the level of the mitochondrion [62]. THs also take part in modulation of several physiological processes in the CV system including maintenance of cardiac contractility, electrophysiological functions and cardiac

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structure. T4 is a principal product of thyroid glands. In order to exert its effect in the periphery, it

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should be converted to its biologically active form, T3. Previous studies demonstrated that THs have crucial impact on heart cell mitochondria. Indeed,

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there are evolving data suggesting that THs administration stimulates cardiac mitochondrial biogenesis, and increases myocardial mitochondrial mass. Furthermore, T3 can increase mitochondrial enzyme activities,mitochondrial protein synthesis, particularly mitochondrial

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respiratory complexes (I, II, IV and V), cytochrome, phospholipid and mitochondrial DNA content. These processes can further elevate mitochondrial respiration, and oxidative phosphorylation (OXPHOS) [27,40]. As a consequence of TH stimulation, increasing levels of mitochondrial activities of bio-energetic enzymes can subsequently enhance ATP generation [42]. According to our results, phosphine poisoning caused a 50% decline in cellular ATP reserve. Whereas, treatment with T3 successfully restored the phosphine-induced depletion of ATP pool. Previous evidences suggested antinecrotic and antiapoptotic properties of T3 administration in animal models of cell loss in central nervous system (CNS), liver and heart [15,24,45]. Several possible mechanisms have been proposed for antinecrotic and antiapoptotic effects of T3 administration, such as decreasing NF-ϰB, P53, BAX, BCL-2, activation of phosphatidylinositol 3' kinase/protein kinase B (PI3K/Akt) and extracellular regulated kinase 1 and 2 (ERK1/2) signaling [11,39]. On the other hand, in vivo studies demonstrated that T3 can ameliorate acute myocardial infarction through decreasing ROS and increasing total antioxidant capacity and GPx in myocardial tissue [16]. In the light of flow cytometric assay, it is ascertained that cellular apoptosis is the main Page 14 of 30

ACCEPTED MANUSCRIPT reason of cardiomyocytes death by phosphine poisoning. This finding is further confirmed by caspases 3 and 9 assays. Caspases assays also show that phosphine-induced apoptosis is conducted through both intrinsic and extrinsic pathways. As shown in the results, phosphine poisoning led to 25% mortality in cardiomyocytes, while treatment with T3 resulted in cell viability compared to the

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control group.

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Hence, in the light of these findings, we believe that T3 might be a proper candidate to overcome

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the cardiotoxic effects of AlP ingestion. In this study, the acute T3 administration has reversed cell death and negative ECG variables, down to the control level. Furthermore, treatment with T3 led to a significant increase in mitochondrial complex's activities (II and IV), ATP levels as well as

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decrease in oxidative stress biomarkers and caspase 3 and 9.

Conclusion

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Taking all data together, T3 exerts its cardioprotective effects in AlP poisoning through improvement of mitochondrial activities and reduction of necrosis and apoptosis levels and

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oxidative stress biomarkers.Our current understanding in the field of T3 cardioprotection should provide the impetus to bring these hormonal treatments to clinical trials for establishing an evidence

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based approach to the management of the AlP poisoned patients. However, a long way may still lie

Conflict of interest

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ahead before such hormonal therapies make their way into routine clinical use.

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The authors declare that there are no conflicts of interest.

Acknowledgment This study was financially supported by a PhD student grant from International Campus coded (TUMS 89-03-33-11232). Author’s contributions Amir Hossein contributed to the work as the PhD student by doing all experimental parts of the study and drafting the article. Amir Baghaei and Maziar Gooshe contributed in the ECG monitoring of the study. Reza Solgi, Maryam Baeeri, Mona Navaei-Nigjeh, Shokoufeh Hassani and Abbas Jafari assisted in doing the analytical parts of the study and interpretation of data. Seyed Mehdi Rezayat, Ahmad Reza Dehpour, Shahram Ejtemaei Mehr made enough consultation during the study. Mohammad Abdollahi was the main supervisor and conceived the study. All agreed to be accountable for the accuracy and scientific authenticity of the manuscript.

Page 15 of 30

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ACCEPTED MANUSCRIPT Variable

Control

AlP

AlP+ T3-1µg

AlP+ T3-2µg

AlP+ T3-3µg

QRS (ms)

0.017±0.0002

0.017±0.0009

0.015±0.0005

0.015±0.0009

0.015±0.0000

QT (ms)

0.12±0.02

0.09±0.02

0.10±0.01

0.10±0.01

0.13±0.01

0.06±0.009

0.07±0.014

0.07±0.007

QRS (ms)

0.016±0.0002b

0.020±0.0002a

0.014±0.0000ab

0.015±0.0005ab

0.016±0.0000b

QT (ms)

0.13±0.02

0.09±0.02

0.10±0.01

0.09±0.02

0.11±0.02

ST(mv)

0.03±0.003b

0.07±0.002a

0.13±0.003ab

0.07±0.007a

0.07±0.017a

QRS (ms)

0.016±0.0002b

0.020±0.0002a

0.015±0.0010b

0.016±0.0006b

0.015±0.0000b

QT (ms)

0.12±0.02

0.12±0.02

0.10±0.01

0.09±0.02

0.11±0.02

0.017±0.0002

0.021±0.0018

QT (ms)

0.12±0.02

0.17±0.02 0.16±0.028

a

0.016±0.0000b 0.11±0.02

a

ST(mv)

0.03±0.003

QRS (ms)

0.017±0.0002b

0.028±0.0019a

QT (ms)

0.12±0.02b

ST(mv)

IP

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QRS (ms)

0.12±0.023

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0.04±0.003

b

0.17±0.002

a

ST(mv)

T

0.04±0.002

b

0.13±0.019

ab

ST(mv)

0.11±0.006

a

0.05±0.020

b

0.05±0.010b

0.019±0.0017

0.014±0.0008b

0.10±0.02b

0.13±0.01

0.03±0.011

b

0.04±0.012b

0.019±0.0005b

0.014±0.0000b

0.19±0.01a

0.12±0.02b

0.10±0.02b

0.14±0.01

0.03±0.003b

0.15±0.004a

0.10±0.023ab

0.04±0.005b

0.05±0.005b

QRS (ms)

0.017±0.0000b

0.028±0.0014a

0.016±0.0000b

0.018±0.0008b

0.014±0.0008b

QT (ms)

0.11±0.02b

0.20±0.01a

0.12±0.02b

0.09±0.02b

0.14±0.01b

ST(mv)

0.02±0.003b

0.14±0.003a

0.07±0.009ab

0.07±0.005ab

0.04±0.001ab

D

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0.016±0.0004b

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150-180

120-150

90-120

60-90

30-60

0-30

Time

CE P

Table 1.Changes in ECG parameters in various groups.

AC

Data are mean±SEM of six animals in each group. The Control group received almond oil alone; AlP group (LD50) received only aluminium phosphide; AlP+T3-1µg/kg group received AlP (LD50) +T3 (1 µg/kg); AlP+T3-2µg/kg group received AlP (LD50) +T3 (2 µg/kg); AlP+T3-3 µg/kg group received AlP (LD50)+T3 (3 µg/kg). a Significantly different from the control group at p < 0.05. b Significantly different from the AlP group at p < 0.05. AlP:Aluminum phosphide, T3:Triiodothyronine

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ACCEPTED MANUSCRIPT Group

Time (min) 30-60

60-90

90-120

120-150

150-180

Control

351.44±1.83b

364.62±5.26

343.16±2.70b

348.66±2.26b

346.22±3.73b

366.34±2.20b

AlP

287.93±14.07a

331.90±4.79

247.19±0.75a

233.32±4.08a

AlP+ T3-1µg

258.75±10.91a

237.73±4.77ab

224.78±4.04a

220.78±6.65a

202.77±4.62a

236.55±13.20a

AlP+ T3-2µg

289.20±11.51a

257.78±15.68ab

234.20±9.49a

259.77±10.04

283.38±8.44a

285.20±29.50ab

AlP+ T3-3µg

260.58±7.38a

238.80±11.34ab

247.02±7.01a

273.00±47.76

339.90±12.13ab

336.08±15.44b

168.58±16.17a

IP

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Table 2. Changes in heart rate in various groups.

T

0-30

142.74±7.00a

AC

CE P

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D

MA

NU

Data are mean±SEM of six animals in each group. The Control group received almond oil alone; AlP group (LD50) received only aluminium phosphide; AlP+T3-1µg/kg group received AlP (LD50) +T3 (1 µg/kg); AlP+T3-2µg/kg group received AlP (LD50) +T3 (2 µg/kg); AlP+T3-3 µg/kg group received AlP (LD50)+T3 (3 µg/kg). a Significantly different from the control group at p < 0.05. b Significantly different from the AlP group at p < 0.05. AlP:Aluminum phosphide, T3:Triiodothyronine

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ACCEPTED MANUSCRIPT Group

Time (min) 30-60

60-90

Control

94.76±1.08

89.50±2.43

99.30±1.97b

107.08±1.22b 109.97±2.53 b 104.59±1.18 b

AlP

90.46±2.15

72.50±4.24

69.77±2.61a

64.18±0.97a

54.72±1.17 a

59.07±1.85 a

AlP+ T3-1µg

76.83±5.27

72.20±8.53

75.67±5.20a

72.20±4.21a

73.25±6.55 a

68.60±3.78 a

AlP+ T3-2µg

77.83±8.11

75.33±2.73

75.33±4.27a

76.33±2.58a

83.67±13.23

82.67±2.07 ab

AlP+ T3-3µg

68.00±4.76ab

75.47±1.37

79.46±6.32a

85.86±13.22

96.04±9.91 b

93.60±7.33 b

120-150

IP

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Table 3. Changes in blood pressure in various groups.

90-120

T

0-30

150-180

AC

CE P

TE

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MA

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Data are mean±SEM of six animals in each group. The Control group received almond oil alone; AlP group (LD50) received only aluminium phosphide; AlP+T3-1µg/kg group received AlP (LD50) +T3 (1 µg/kg); AlP+T3-2µg/kg group received AlP (LD50) +T3 (2 µg/kg); AlP+T3-3 µg/kg group received AlP (LD50)+T3 (3 µg/kg). a Significantly different from the control group at p < 0.05. b Significantly different from the AlP group at p < 0.05. AlP:Aluminium phosphide, T3:Triiodothyronine

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ACCEPTED MANUSCRIPT T3 (ng/dl) 103.0±7.8

T4 (µg/dl) 3.79±0.57

AlP

102.3±19.2

3.72±0.31

AlP+ T3-1

313.0±51.3ab

3.09±0.28

AlP+ T3-2

436.1±29.0ab

4.31±0.44

AlP+ T3-3

603.3±76.2ab

2.54±0.51

Table 4. The levels of T3 and T4 in rat serum in various groups.

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Group Control

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Data are mean±SEM of six animals in each group. The Control group received almond oil alone; AlP group (LD50) received only aluminium phosphide; AlP+T3-1 group received AlP (LD50) +T3 (1 µg/kg); AlP+T3-2 group received AlP (LD50) +T3 (2 µg/kg); AlP+T3-3 group received AlP (LD50)+T3 (3 µg/kg). a Significantly different from the control group at p<0.05. b Significantly different from the AlP group at p<0.05. AlP: Aluminium phosphide, T3:Triiodothyronine

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Figure 1. Changes in ECG parameters in various groups. The Control group received almond oil alone; AlP group (LD50) received only aluminium phosphide; AlP+T3-3 µg/kg group received AlP (LD50) +T3 (3 µg/kg). AlP: Aluminium phosphide, T3:Triiodothyronine A QRS Complex B ST height

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Figure 2. Changes in heart tissue ferric-reducing antioxidant power (FRAP) (A), lipid peroxidation (LPO), (B), superoxide dismutase (SOD) activity (C), reactive oxygen species (ROS) (D), and total thiol molecules (E) in various groups. Data are mean ± SEM of six animals in each group. ##P<0.01, ###P<0.001 compared to control group. *P<0.05, **P<0.01, ***P<0.001 compared to AlP group. The control group received only almond oil alone; AlP group (0.25 LD50) received only aluminium phosphide; AlP+T3-1 group received AlP (0.25 LD50) + T3 (1 µg/kg); AlP + T3-2 group received AlP (0.25 LD50) + T3 (2 µg/kg); AlP + T3-3 group received AlP (0.25 LD50) + T3 (3 µg/kg).

CTRL: Control, AlP: Aluminium phosphide, T3: Triiodothyronine

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Figure 3. Changes in activity of mitochondrial NADH-ubiquinone oxidoreductase (A), succinateubiquinone oxidoreductase (B), cytochrome C oxidase (C), and ADP/ATP ratio (D) in the rat cardiac muscle of control, AlP and treatment groups. Data are mean ± SEM of six animals in each group. #P<0.05, ##P<0.01, ###P<0.001compared to control group. **P<0.01, ***P<0.001compared to AlP group. The control group received only almond oil alone; AlP group (0.25 LD50) received only aluminium phosphide; AlP + T3-1 group received AlP (0.25 LD50) + T3 (1 µg/kg); AlP+T3-2 group received AlP (0.25 LD50) + T3 (2 µg/kg); AlP + T3-3 group received AlP (0.25 LD50) + T3 (3 µg/kg). CTRL: Control, AlP: Aluminium phosphide, T3: Triiodothyronine

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Figure 4. Flow cytometric analysis of the cardiac cells. The numbers at the bottom right quadrant of each dot plot represent the percentage of cells in early apoptosis (annexin V-positive, PI-negative). Numbers at the top right quadrant represent the percentage of cells in late apoptosis and/or secondary necrosis (annexin V-positive, PI-positive). The data are representative of three separate experiments (A). Changes in caspase 3 and 9 activities in control, AlP and treatment groups (B). Data are mean ± SEM of six animals in each group. ##P<0.01, ###P<0.001compared to control group. *P<0.05, **P<0.01, ***P<0.001compared to AlP group. The control group received only almond oil alone; AlP group (0.25 LD50) received only aluminium phosphide; AlP + T3-1 group received AlP (0.25 LD50) + T3 (1 µg/kg); AlP + T3-2 group received AlP (0.25 LD50) + T3 (2 µg/kg); AlP+T3-3 group received AlP (0.25 LD50) +T3 (3 µg/kg). CTRL: Control, AlP: Aluminium phosphide, T3: Triiodothyronine

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Graphical Abstract Proposed mechanism of the cardioprotective effects of T3 on AlP toxicity at the cellular level. AlP: Aluminium phosphide, PH3:Phosphine gas, T3: Triiodothyronine, ECG: Electrocardiogram, HR: Heart rate, BP:Blood pressure, ATP: Adenosine triphosphate, ROS: Reactive oxygen species, SOD:Superoxide dismutase, LPO: Lipid peroxidation.

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