Subacute Cardiotoxicity of Yessotoxin: In Vitro and ... - ACS Publications

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Subacute cardiotoxicity of yessotoxin: in vitro and in vivo studies Sara F. Ferreiro, Natalia Vilariño, Cristina Carrera, M Carmen Louzao, Antonio G. Cantalapiedra, Germán Santamarina, J. Manuel Cifuentes, Andrés C. Vieira, and Luis M. Botana Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00012 • Publication Date (Web): 22 Apr 2016 Downloaded from http://pubs.acs.org on April 24, 2016

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Chemical Research in Toxicology

Subacute cardiotoxicity of yessotoxin: in vitro and in vivo studies Ŧ

Ŧ,*

Sara F. Ferreiro , Natalia Vilariño , Cristina Carrera Cantalapiedra

†, ‡,

Germán Santamarina

†, ‡

Ŧ, ‡

Ŧ

, M. Carmen Louzao , Antonio G. §

Ŧ

, J. Manuel Cifuentes , Andrés C. Vieira , Luis M.

Ŧ, *

Botana

†

Ŧ

Departamento de Farmacología , Departamento de Ciencias Clínicas Veterinarias , Hospital ‡

§

Veterinario Universitario Rof Codina and Departamento de Anatomía y Producción Animal , Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain

* To whom correspondence should be addressed: Luis M. Botana, Natalia Vilariño Departamento de Farmacología Facultad de Veterinaria Campus Universitario 27002 Lugo Spain e-mail: [email protected], [email protected] Telephone and Fax: +34 982822233

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ACS Paragon Plus Environment


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TOC

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ABSTRACT Yessotoxin (YTX) is a marine phycotoxin produced by dinoflagellates and accumulated in filter feeding shellfish. Although no human intoxication episodes have been reported, YTXs content in shellfish is regulated by many food safety authorities due to their worldwide distribution. YTXs have been related to ultrastructural heart damage in vivo, but the functional consequences in the long term have not been evaluated. In this study we explored the accumulative cardiotoxic potential of YTX in vitro and in vivo. Preliminary in vitro evaluation of cardiotoxicity was based on the effect on hERG (human ether-a-go-go related gene) channel trafficking. In vivo experiments were performed in rats that received repeated administrations of YTX followed by recording of electrocardiogram, arterial blood pressure and plasmatic cardiac biomarkers, and analysis of myocardium structure and ultrastructure. Our results showed that an exposure to 100 nM YTX for 12 or 24 hours caused an increase of extracellular surface hERG channels. Furthermore, remarkable bradycardia and hypotension, structural heart alterations and increased plasma levels of tissue inhibitor of metalloproteinases-1 were observed in rats after four intraperitoneal injections of YTX at doses of 50 or 70 µg/kg that were administered every 4 days along a period of 15 days. Therefore and for the first time, YTX-induced subacute cardiotoxicity is supported by evidence of cardiovascular function alterations related to its repeated administration. Considering international criteria for marine toxin risk estimation and that the regulatory limit for YTX has been recently raised in many countries, YTX cardiotoxicity might pose a health risk to humans and especially to people with previous cardiovascular risk.

Keywords: Yessotoxin, cardiotoxicity, subacute, heart, bradycardia, hypotension

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INTRODUCTION Yessotoxin (YTX) is a polycyclic ether first isolated from the scallop Patinopecten yessoensis.1 YTXs are produced by the phytoplanktonic dinoflagellates Protoceratium reticulatum, Lingulodinium polyedrum and Gonyaulux spinifera.2-4 Although no human intoxications have been reported, many food safety authorities have established maximum levels of YTXs in shellfish destined to human consumption5. Mice in vivo toxicological data point to the heart as one of the main target organs of YTXs due to ultrastructural alterations described in cardiomyocytes after oral and intraperitoneal administrations in acute and short-term studies.6-12 However, acute evaluation of YTX effects on heart function did not evidence cardiac dysfunction following intravenous administration of YTX to rats.13 In vitro data demonstrate that YTX induces cell death in many cell types, including rat cardiomyocytes, and alterations of calcium movement, cyclic nucleotides, E-cadherin and cytoskeleton.14-18 These toxicological data suggest that YTX repeated exposures might affect heart function in the long term, mainly considering the mitochondrial damage in cardiomyocytes. Actually, the study of the toxicological significance of these ultrastructural changes in the heart is recommended in the last EFSA report on YTXs.19 The European Medicines Agency (EMA) recommendations and several expert publications establish the guidelines for cardiotoxicity evaluation.20, 21 In vitro evaluation of human ether-ago-go related gene (hERG) channel function by patch clamp has been the in vitro method of choice for screening of potential acute cardiotoxicity; but hERG trafficking is also being evaluated nowadays for the detection of chronic effects.20 In vivo heart functionality is par excellence monitored by electrocardiography, which can evidence structural alterations that affect the normal cardiac impulse generation or propagation.22 Arterial blood pressure (ABP) measurement is also recommended in hemodynamic response evaluation. Structural alterations of the heart have been traditionally studied by histopathological analysis but quantification of cardiac biomarkers is presently required to assess cardiotoxic potential.23 Cardiac troponins (cTns) and brain natriuretic peptides (BNPs) are among the 4


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currently accepted biomarkers of heart damage. The cTns are considered the “gold standard” biomarkers of myocardial damage owed to their high specificity and stable release24 and the BNPs are highly sensitive to detect acute or chronic cardiac impairment.23 In addition, the tissue inhibitor of metalloproteinases-1 (TIMP-1) is considered a good plasma biomarker of heart tissue remodeling.25 Therefore, the aim of this study was to explore subacute cardiotoxicity of YTX evaluating hERG trafficking in vitro and heart function in vivo in the presence of YTX-induced structural alterations.

MATERIALS AND METHODS Reagents YTX certified reference material (CRM) was supplied by Laboratorio CIFGA S.A. (Lugo, Spain). Anti-Kv 11.1 (HERG) (extracellular) antibody was from Alomone Labs (Jerusalem, Israel). Anti-hERG antibody HERG (H-175) was from Santacruz Biotechnology, Inc. (Santa Cruz, CA). Annexin V-FITC apoptosis detection kit was purchased from Immunostep (Salamanca, Spain). Nut Mix F-12 W/GLUTAMAX-I medium, fetal bovine serum (FBS), geneticin, trypsin/EDTA, Dulbecco's phosphate-buffered saline (DPBS), phosphate-buffered saline (PBS), Cy3® goat anti-rabbit IgG (H+L), Tris–glycine SDS sample buffer 2x, Novex® sharp protein standard and Novex®tris glycine gels (8 %, 1.5 mm, 10 well) were purchased from Invitrogen®

(Madrid,

Spain).Bovine

serum

albumin

(BSA),

β-mercaptoethanol,

dimethylsulfoxide (DMSO), paraformaldehyde, trypan blue solution and monoclonal anti-βtubulin I clone SAP.4G5 antibody were from Sigma-Aldrich Química S.A. (Madrid, Spain). DetachinTM was purchased from Labclinics (Barcelona, Spain). Inmobilon®-FL transfer membrane and peroxidase conjugated goat anti-rabbit IgG was from Millipore® Iberica S.A. (Madrid, Spain). ECL mouse IgG HRP-linked whole Ab (from sheep) was purchased from GE Healthcare (Barcelona, Spain). SuperSignal® West Femto maximum sensitivity substrate and SuperSignal® West Pico chemiluminescent substrate were from Fisher Scientific (Madrid, 5



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Spain). Ionflux extracellular solution (145 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, 10 glucose (mM), EC solution), Ionflux intracellular solution (60 KCl, 70 KF, 15 NaCl, 5 HEPES, 5 EGTA (mM), IC solution) and Ionflux plates were obtained from Fluxion Bioscences Inc. (South San Francisco, CA). Sodium chloride solution 0.9 % was from Grifols Engineering, S.A. (Barcelona, Spain). General health profile (GHP) chemistry panel was obtained from IDEXX Laboratories (Barcelona, Spain). All chemicals were reagent grade quality. Cell line culture PrecisionTM hERG CHO (chinese hamster ovary) Recombinant cell line (Millipore® Iberica S.A., Madrid, Spain) was cultured and passaged as previously published.26 HERG channel trafficking External hERG and annexin-V signals were quantified in hERG-CHO cells by image flow cytometry and total hERG channel protein levels were evaluated by western blot analysis.

Imaging flow cytometry Cell cultures (0.2×106 cells per T25 cm2) were incubated with carrier (0.025% DMSO) alone or 100 nM YTX for 6, 12 or 24 h at 37 ºC. After washing the cells with warm DPBS, they were dissociated from the flask with warm DetachinTM for 10 min at 37 ºC. Then the cells were centrifuged at 900 rpm and 4 ºC for 5 min and the cell pellet was washed again with complete culture medium in the same conditions. Subsequently, two washes with cold DPBS and one with cold annexin-V binding buffer followed. All samples were first incubated with FITCannexin-V diluted (5:100) in annexin-V binding buffer for 15 min at 22 ºC in the dark. After two washes with cold DPBS, they were fixed with 2% paraformaldehyde in DPBS for 15 min on ice. Non-specific binding was blocked with 4% BSA in DPBS for 30 min on ice. The cells were then incubated with a 1:100 dilution of the anti-Kv 11.1 antibody (extracellular hERG) in 4% BSA/DPBS for 60 min at 37 ºC and, after two washes with DPBS; they were incubated with Cy3® goat anti-rabbit IgG (1:200) in the same conditions. Finally, the cell were washed twice with PBS and passed through a 50 µm nylon mesh filter. For flow cytometry analysis 2.5×106 6


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cells/ml in 50 µl of PBS was used. Flow cytometry data were acquired using an ImageStream multispectral imaging flow cytometer (Amnis Corporation, Seattle, WA). The intensity of the 488 nm laser was fixed at 20 mW. Cell classifiers were used for eliminating debris and clusters. The minimal number of events captured was 5000. Quantitative measurements were done using the data analysis software IDEAS (Amnis Corporation, Seattle, WA). After matrix compensation, only single and focused cells were used to generate final data. The results are reported as population median. DMSO (0.025%) did not affect the levels of surface hERG expression.

Western blot analysis HERG channel protein levels in hERG-CHO cells were evaluated by western blot analysis using a specific antibody. Cell cultures (0.2×106 cells per T25 cm2) were incubated with carrier (0.025% DMSO) alone or 100 nM YTX for 6 or 12 h at 37 ºC. Then, the cells were harvested by scraping and, after two washes with DPBS, they were centrifuged at 1000 rpm and 4 ºC for 5 min. The cell pellets were lysed immediately by adding 1× sample buffer with 2.5% βmercaptoethanol and boiling for 5 min. Cell proteins were separated by electrophoresis in 8% SDS polyacrylamide gels and transferred to polyvinyl difluoride (PVDF) membranes. The membranes were blocked overnight in 5% nonfat dry milk in TBST. HERG channel proteins were detected by incubation of the membranes with an anti-hERG (H-175) antibody (1:20000) for 60 min with constant shaking. After two washes, the membranes were incubated with an HRP-labeled goat anti-rabbit IgG antibody (1:15000) for 60 min with constant shaking. Detection was done with the HRP chemiluminescent West Femto maximum sensitivity substrate following the manufacturer instructions. The membranes were re-blotted for normalization of hERG expression levels to a reference protein, β-Tubulin. Chemiluminescence was detected using a Dyversity Imaging System (Syngene, Cambridge, UK) and quantified with the Genetools software from Syngene (Cambridge, UK).

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Automated patch clamp The effect of long exposures to YTX on hERG channel activity was tested using an IonFlux 16 automated patch clamp system (Fluxion Bioscences Inc, South San Francisco, CA). Current measurement experiments were performed in 16-well IonFlux plates that have 8 pairs of cell traps, each trap endowed with 20 trapping sites placed in parallel, so the measured current is the sum of an ensemble of 20 cells. Previously, the cells (1.5×106 cells per T175 cm2) were incubated with carrier (0.025% DMSO) alone or 100 nM YTX for 6 or 12 h at 37 ºC. Before every experiment, the cells were washed with toxin and/or carrier-free medium and placed for one h at 30 ºC in a humidified 5% CO2 incubator to remove the toxin and allow them to recover from a possible acute block. Then the cells were washed twice with 5 ml of warm DPBS and detached by incubation with 5 ml of warm DetachinTM solution for 10 min at 37 ºC. Immediately, 5 ml of warm CHO serum-free culture medium supplemented with 25 mM HEPES was added and the cell suspension was centrifuged at 700 rpm and 19 ºC for 5 min. Cell viability was determined using trypan blue. Viability was always higher than 99%. The cell pellet was suspended in CHO serum-free medium supplemented with 25 mM HEPES and centrifuged again in the same conditions. Finally the cells were washed twice with EC solution and suspended at a concentration of 20×106 cells/ml in EC before delivery in the appropriate IonFlux plate well. Experimental protocols previously optimized for hERG-CHO cells were used for cell trapping and whole cell configuration. The voltage protocol to record hERG currents was as follows: cells were clamped at −80 mV for 100 ms, pulsed to −100 mV for 90 ms and to −50 mV for 50 ms, then depolarized to +20 mV for 5 s and repolarized to −50 mV for 5 s, and finally returned to −80 mV. The hERG peak tail current (current of interest, COI) was measured in the second −50 mV step 550 s after whole cell configuration was achieved. Current data were accepted if cell resistances in each trap were at least 2 MΩ along all the duration of the experiment. All experiments were done at room temperature (22 ºC). DMSO did not affect hERG currents. 8


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Animals and in vivo experimental design Animals and housekeeping conditions were as in Ferreiro et al.26 The procedure lasted for 15 days. YTX and/or carrier (DMSO) were administered intraperitoneally (i.p.) on days 1, 5, 9, and 13. Doses of 50, 70 and 100 µg/kg YTX were injected to 8, 7 and 1 rats respectively. Each treated rat received the same dose in all injections. For i.p. injection the solvent of the YTX stock solution (methanol) was evaporated and the toxin was reconstituted with DMSO. Saline solution (Grifols Engineering, Barcelona, Spain) was added subsequently to provide a final concentration of 10 % DMSO (each rat received 50 µL of DMSO per 200 g of body weight) and 20, 40 or 61 µg/mL YTX (for the 50, 70 and 100 µg/kg doses respectively, final injection volume was 500 µL/ 200 g in all rats). Eight carrier control rats were injected with 10% DMSO in saline solution. The rats were evaluated for signs of toxicity twice a day. On day 15, they were anesthetized with isoflurane (FIISO 1.5-2%) and the cardiovascular function was monitored using electrocardiogram (ECG) and ABP measurements. ECG was monitored for one hour and then ABP was measured by placing a catheter in the right carotid artery. Afterwards, another catheter was placed in the jugular vein for blood sample collection. One blood sample of 400

µL was collected in EDTA tubes for the detection of cardiac biomarkers and another one of 500 µL was collected in a heparin tube for biochemical analysis. All animals were euthanized by exsanguination at the end of the experiment, except those that died during the treatment period or those euthanized prematurely to prevent suffering. All animal procedures were approved by the Institutional Animal Care Committee of the Universidad de Santiago de Compostela.

Electrocardiography

ECG was recorded with a MAC* 800 Resting ECG System (GE corporate, Madrid, Spain) using lead II as in Ferreiro et al.26 An ECG recording of every rat was obtained for 15 min, under anesthesia, 2-10 days before starting toxin administration (basal). Any abnormality at this point prompted the exclusion of the animal from the study. ECG was also registered on day 15 9



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of the experiment for 1h. The following ECG parameters were analyzed: heart rate (HR), QTc interval, PR interval, and T wave length. Arterial blood pressure ABP was measured in the right carotid artery using a direct method with a Diascope 2 S&W Medico Teknik instrument (Alberslund, Denmark). Three independent measurements of ABP were done for each rat. Data are reported as systolic arterial blood pressure (SAP), diastolic arterial blood pressure (DAP) and mean arterial blood pressure (MAP). Cardiac biomarkers Cardiac troponin I (cTnI), cardiac troponin T (cTnT), BNP and TIMP-1 were measured in one plasma sample collected before exsanguination using a Luminex XMap® assay. Blood samples were centrifuged immediately after collection to separate the plasma fraction and stored at -80 ºC until their analysis. A rat CVD panel 1, Milliplex® Map KIT was used for cTnI, cTnT, BNP and TIMP-1 quantifications in plasma following the manufacturer instructions. Samples were assayed in duplicate. Biochemistry analysis Biochemistry parameters were analyzed with the IDEXX VetTest® Chemistry Analyzer in the plasma fraction separated by centrifugation immediately after collection. A prepacked GHP panel was used to test albumin (ALB), alkaline phosphatase (ALKP), alanine aminotransferase (ALT), blood urea nitrogen (BUN), calcium (Ca), cholesterol (CHOL), creatine kinase (CK), creatinine (CREA), globulin (GLOB), glucose (GLU), phosphorus (PHOS) and total protein (TP). Histological damage evaluation Hearts were collected for histological evaluation immediately after euthanasia. Macroscopic examination was followed by preparation for light microscopy (LM) and transmission electron microscopy (TEM). 10


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For LM, samples were fixed by immersion in buffered 10% formalin and Bouin for 24 hours at 4º C. Then the tissues were processed for H&E staining and examined under the light microscope as in Ferreiro et al.26 Heart collagen content was evaluated with Sirius Red staining. Heart specimens were stained with 0.1% Sirius Red in saturated aqueous solution of picric acid for 1 hour. Subsequently, they were rinsed in acidified water (0.5% acetic acid in distilled water), dehydrated in dehydrated in graded ethanol solutions and cleared twice in xylene.27 The percentage of collagen in Sirius Red-stained heart sections was measured with ImageJ 1.43 software. Three non-consecutive analogous fields were randomly chosen in each heart section to obtain high-resolution images (40x). The surface corresponding to collagen staining was quantified in 3 images from 3 sections for each rat, obtaining 9 measurements per rat. Collagen content was reported as percentage of collagen area versus total tissue area.28 For TEM, samples (1 mm3) were fixed by immersion in 2.5% glutaraldehyde in 0.1 M cacodylate trihydrate buffer for 4 hours at 4 °C. Then, samples were rinsed with 0.1 M cacodylate trihydrate buffer. Post-fixation by immersion in 1% OsO4 in 0.1 M cacodylate trihydrate buffer was performed for 60 min. Finally, after a second rinse fixed tissues were dehydrated in graded ethanol solutions, including one bath with 70% ethanol and 0.5% uranyl acetate, rinsed in propylene oxide and embedded in Epon 812 (Momentive Specialty Chemicals Inc., Houston, TX). A Leica Ultracut UCT ultramicrotome from Leica Microsystems GmbH (Wetzlar, Germany) was used to obtain ultrathin sections of tissue samples and they were counterstained with uranyl acetate and lead citrate. Ultrastructural analysis of 1 mm2 samples was performed with a JEOL JEM-1011 Transmission Electron Microscope (Jeol Ltd, Tokyo, Japan). Data analysis Data were plotted as mean ± SEM. Statistical significance was determined using t test for unpaired data. ANOVA was used for multiple comparisons. P

Subacute Cardiotoxicity of Yessotoxin: In Vitro and ... - ACS Publications

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