Acute Cardiotoxicity Evaluation of the Marine Biotoxins OA, DTX-1 and YTX

Toxins 2015, 7, 1030-1047; doi:10.3390/toxins7041030 toxins ISSN 2072-6651 www.mdpi.com/journal/toxins Article Acute Cardiotoxicity Evaluation of the Marine Biotoxins OA, DTX-1 and YTX Sara F. Ferreiro 1, Cristina Carrera 1,2, Natalia Vilariño 1,*, M. Carmen Louzao 1, Germán Santamarina 2,3, Antonio G. Cantalapiedra 2,3 and Luis M. Botana 1,* 1 Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain; E-Mails: sara.ferna[email protected] (S.F.F.); cristina.car[email protected] (C.C.); mcarm[email protected] (M.C.L.) 2 Hospital Veterinario Universitario Rof Codina, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain; E-Mails: [email protected] (G.S.); [email protected] (A.G.C.) 3 Departamento de Ciencias Clínicas Veterinarias, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain * Authors to whom correspondence should be addressed; E-Mails: [email protected] (L.M.B.); [email protected] (N.V.); Tel./Fax: +34-9828-22233 (L.M.B. & N.V.). Academic Editor: Michio Murata Received: 9 February 2015 / Accepted: 18 March 2015 / Published: 27 March 2015 Abstract: Phycotoxins are marine toxins produced by phytoplankton that can get accumulated in filter feeding shellfish. Human intoxication episodes occur due to contaminated seafood consumption. Okadaic acid (OA) and dynophysistoxins (DTXs) are phycotoxins responsible for a severe gastrointestinal syndrome called diarrheic shellfish poisoning (DSP). Yessotoxins (YTXs) are marine toxins initially included in the DSP class but currently classified as a separated group. Food safety authorities from several countries have regulated the content of DSPs and YTXs in shellfish to protect human health. In mice, OA and YTX have been associated with ultrastructural heart damage in vivo. Therefore, this study explored the potential of OA, DTX-1 and YTX to cause acute heart toxicity. Cardiotoxicity was evaluated in vitro by measuring hERG (human èter-a-go-go gene) channel activity and in vivo using electrocardiogram (ECG) recordings and cardiac damage biomarkers. The results demonstrated that these toxins do not exert acute effects on hERG channel activity. Additionally, in vivo experiments showed that these compounds do not alter cardiac biomarkers and ECG in rats OPEN ACCESS Toxins 2015, 7 1031 acutely. Despite the ultrastructural damage to the heart reported for these toxins, no acute alterations of heart function have been detected in vivo, suggesting a functional compensation in the short term. Keywords: okadaic acid; dynophysistoxins; yessotoxin; cardiotoxicity; hERG; ECG; cardiac biomarkers 1. Introduction Marine algal blooms are natural phenomena caused by the overgrowth of marine phytoplankton. Presently, their appearance seems to have increased in frequency and severity, suggesting a public health problem of worldwide distribution. Some phytoplankton species present in these blooms produce phycotoxins that get accumulated in edible tissues of filter feeding shellfish through the marine food webs. Human intoxication episodes occur when contaminated seafood is consumed. Diarrhetic shellfish poisoning (DSP) is the toxic syndrome caused by the ingestion of shellfish contaminated with okadaic acid (OA) or its analogues, the dynophysistoxins (DTXs). OA and DTXs are marine lipophilic phycotoxins produced by dinoflagellates of the genera Prorocentrum and Dinophysis. DSP’s first episode was documented in Japan in the 1970s and since then, many episodes have been reported worldwide [1]. DSP is a severe gastrointestinal intoxication, of which the main symptoms are diarrhea, nausea, vomiting and abdominal cramps. Until now, no deaths have been related to acute or chronic toxicity of these toxins [2]. In order to protect human consumers, the presence of DSPs in seafood is regulated in many countries [3]. The mechanism of action of OA and DTXs is most likely related to the potent inhibition of serine/threonine protein phosphatases [4,5], which leads to hyperphosphorylation of cell proteins and dis-regulation of many cellular processes. Several toxicological studies with these compounds have described many effects at different cellular, molecular and genetic levels. In fact, they have been reported to cause cytotoxicity, neurotoxicity, immunotoxicity and embryotoxicity, as well as genotoxicity, tumor promotion and carcinogenicity [2]. Yessotoxins (YTXs) are polycyclic ether compounds produced by the phytoplanktonic dinoflagellates Protoceratium reticulatum, Lingulodinium polyedrum and Gonyaulux spinifera. YTXs were initially included in the DSP toxin class because they were detected simultaneously with OA and DTXs; however, nowadays they are classified and regulated separately owing to their different chemical structure, the lack of phosphatase inhibition activity and the absence of gastrointestinal toxicity [3]. Actually, and even though these toxins are also distributed worldwide, no human intoxication episodes have been related to the presence of yessotoxins in seafood [6]. In vivo toxicological studies in mice showed that yessotoxin causes alterations mainly in heart muscle [7–10]. On the other hand, in vitro data indicate that YTX induces apoptosis in many different cell lines, and it has been implicated in alterations of calcium movement [11], cyclic nucleotides and phosphodiesterases [12], or E-cadherin pathway and cytoskeleton [6,13]. Although the complete mechanism of action is not yet elucidated, the interaction of YTX with PDE4 is linked to the translocation to membrane and nucleus of the AKAP 149-PKA-PDE4A complex [14,15], being as well this effect linked to mTOR in apoptosis or autophagia [16]. Toxins 2015, 7 1032 In spite of the reports of ultrastructural alterations of cardiac muscle after oral or intraperitoneal administrations of yessotoxin and okadaic acid in mice [7–10,17,18], the functional implications of these effects have not been studied. The evident mitochondrial damage in one of the tissues with the highest demands of energy suggests potential cardiotoxicity [7]. In fact, the recommendations of the last EFSA report on YTXs, elaborated by a panel of experts on this field, include the study of the toxicological significance of these ultrastructural changes described in the heart [19]. Guidelines for the evaluation of a compound potential cardiotoxicity can be found in the recommendations of the EMA [20] and in several articles [21,22], entailing both in vivo and in vitro experiments. The evaluation of the effect on hERG (human èter-a-go-go gene) channel function by patch clamp is the acute in vitro method of choice to assess cardiac safety in drug development [23]. HERG encodes the channel responsible for a critical current in cardiac action potential (AP) repolarization, the rapid delayed rectifier K+ current (IKr) [24]. Alterations of HERG channel currents have been related to the appearance of arrhythmias, specifically a type of fatal arrhythmia known as “Torsades de Pointes” (TdP) [25]. Additionally, some drugs can alter hERG by disruption of channel trafficking, but this is considered a chronic effect that usually takes hours or even days to occur [26]. Even though all cardiac channels contribute to the coordinated electrical activity of the heart, the implications on heart conductivity of hERG dysfunction have made hERG blockage evaluation essential to estimate potential cardiotoxicity. To evaluate in vivo heart toxicity, heart function and structural damage are considered. For functional alterations, electrocardiography is the technique of choice [21,22,27]. An electrocardiogram (ECG) represents the changes of electrical charge of the heart chambers for every beat; therefore it gives information about the overall electrical activity of the heart. The ECG may be altered by changes in biochemical and metabolic processes, by modifications of cardiac channels and cardiomyocyte membrane properties and by any structural injury that affect impulse generation and propagation [27]. In relation to structural damage, plasma cardiotoxicity biomarkers have also been recently included among the in vivo experiments for assessing cardiac toxicity [28]. Cardiac troponins I (cTnI) and T (cTnT) and the brain natriuretic peptide (BNP) are among the more accepted cardiac biomarkers nowadays [29,30]. Therefore, the aim of this work was to evaluate OA, DTX-1 and YTX acute cardiotoxicity using in vivo and in vitro methods. 2. Results and Discussion 2.1. OA and YTX Effects on hERG Channel Activity The ability to block hERG channel currents is one of the required tests for preliminary evaluation of cardiotoxicity in drug development. Therefore, the effects of OA and YTX (Figure 1) on hERG activity were explored using automated patch clamp for the measurement of hERG currents in a CHO cell line stably expressing this channel. HERG channels were activated with the voltage protocol shown in Figure 2A. After stabilization of hERG currents, the cells were exposed to 10 µM OA or YTX, or an equivalent concentration of DMSO (carrier) for 5 min. The Ionflux system warrants an immediate change of extracellular solution and therefore no time is needed to allow for drug diffusion. For routine screening of hERG inhibiting drugs it is common to test concentrations in this range that will cause a Toxins 2015, 7 1033 clear and fast inhibition if the drug has high to medium blocking potency. A representative image of the current displayed by hERG CHO cells after YTX addition appears in Figure 2B. OA (Figure 2C, grey bar) and YTX (Figure 2D, grey bar) did not alter IKr amplitude after 5 minutes of exposure to a toxin concentration of 10 µM, when compared to the current in the same cells just before toxin addition. Similar results were obtained in cells exposed to carrier alone; no difference of IKr before and after addition of DMSO was observed (Figure 2C,D, white bars). DTX-1 (Figure 1) is an analog of OA and it has been described to have similar potency [1,31,32], thereby it was not tested in this assay to save the limited amount available for in vivo studies, which will provide information about potential cardiotoxicity generated by several possible mechanisms. Concentrations higher than 10 µM were not tested considering that compounds with an IC50 between 1 and 100 µM are classified as low potency hERG blockers and therefore would be pathologically irrelevant [33]. An underestimation of blocking potency due to technique limitations is not probable because the IC50 obtained for cisapride, a well-known high-potency hERG blocker, in our experimental conditions was 3 nM [34]. Figure 1. (A) Okadaic acid (OA); (B) dynophysistoxin-1 (DTX-1); (C) yessotoxin (YTX) chemical structures. Toxins 2015, 7 1034 Figure 2. Effects of OA, DTX-1 and YTX on human èter-a-go-go gene (hERG) channel activity. Automated patch clamp experiments were performed using a CHO cell line stably expressing hERG. (A) Voltage clamp protocol for the activation of hERG; (B) Representative current trace obtained after YTX addition. Currents were monitored for 5 min after toxin addition. COI: current of interest; (C) No effect of OA on hERG currents. Current magnitude is expressed as percentage of pre-treatment current that remained after 5 min of exposure to 10 µM OA or carrier alone (mean ± SEM; n = 4); (D) No effect of YTX on hERG currents. Current magnitude is expressed as percentage of pre-treatment current that remained after 5 min of exposure to 10 µM YTX or carrier (mean ± SEM; n = 4). 2.2. Effects of OA, DTX-1 and YTX on Rat ECG Cardiotoxicity may be caused by many mechanisms different than hERG blockage. Therefore, studies of potential heart toxicity were performed in vivo. The animal model selected for this study was the rat, due to practical feasibility reasons, because, owing to its size, rats provided an adequate balance between toxin expense, a correct manipulation during surgery and catheter placement for blood sample collections and reliable ECG results in our experimental conditions. Moreover, females were used due to the smaller size of adult individuals in relation to males, which allowed reducing the expense of toxin. The toxins were administered intravenously because this administration route is the fastest and requires lower toxin amounts. The effects of OA, DTX-1 and YTX on heart functionality were evaluated by electrocardiography. Several ECG parameters were analyzed before and at different times after intravenous administration of each toxin. Lead II and an ECG recording speed of 50 mm/s were used to measure HR (bpm), PR interval, QT interval and T wave durations (ms) (Figure 3A). ECG activity was recorded before, and immediately and every hour after toxin administration. Each recording had a minimal length of 10 min (continuous) and the total experiment duration was 4 h. For these experiments five rats were injected with 20 µg/kg OA, five rats with 16 µg/kg DTX-1 and seven rats with 10 µg/kg YTX. Nine control rats followed the same experimental procedure with the administration Toxins 2015, 7 1035 of carrier (DMSO in saline) in the absence of toxin. None of the ECG parameters evaluated were altered after the administration of OA, DTX-1, YTX or carrier (Figure 3B–E). Rodents have been widely used for cardiotoxicity evaluation and they share many similarities with humans in terms of pathophysiological changes or disease [27]; however, in humans hERG channel is an important player in heart action potential repolarization, and this is not the case in rodents. In vitro testing of hERG was included as a complement of cardiotoxicity evaluation due to this well-known difference. In addition, the macrolide antibiotic clarithromycin, which induces QTc prolongation, was previously used as a positive control for QTc interval prolongation in our experimental conditions; a dose of 2.2 mg/kg caused an increase of QTc duration of 19% ± 3% [35,36]. Figure 3. Effects of OA, DTX-1 and YTX on rat electrocardiogram (ECG). ECG parameters (HR, PR interval, QTc interval and T wave) were analyzed before and at different times after intravenous administration of 20 µg/kg OA to 5 rats, 16 µg/kg DTX-1 to 5 rats and 10 µg/kg YTX to 7 rats. (A) Representative ECG recording at 50 mm/s. The landmarks and the measurements of PR interval, R-R interval, QT interval and T wave duration are indicated; (B) HR; (C) PR interval; (D) QTc interval and (E) T wave were measured for all treated rats in ECG recordings at the following times: −10 (before toxin administration), 0 (toxin administration), 50, 110, 170 and 230 min. At every time point ECG was recorded for 10 min. Toxins 2015, 7 1036 Cardiac rhythm alterations were also analyzed. Type of arrhythmia, time of appearance from toxin administration, duration of arrhythmia sequences, total number of arrhythmia episodes, ECG recording duration and survival of the animal along the experiment are reported in Table 1. The rats administered with DTX-1 did not evidence any arrhythmia episode. However, 1/5 rat administered with OA, 2/7 rats administered with YTX and 2/9 rats administered with carrier showed the appearance of ventricular extrasystoles (VES). The total number of VES was 5 for OA-treated rats, 4 for YTX-treated rats and 3 for controls. Three YTX-treated rats died prematurely during the experiment as judged by apnea onset, and one of them had 3 VES before death occurred. One OA-treated rat died during the experiment but it did not show VES. None of DTX-1 and carrier-treated rats died before the end of the experiment. Therefore, it does not seem to be a correlation between death and arrhythmia occurrence in these experiments. Table 1. Heart rhythm alterations in OA, DTX-1 and YTX-treated rats and controls. Rat Type Time of Appearance Duration (s) Total N° ECG Total Time (min) Death before 240 min Control 1 0 265 no 2 0 265 no 3 0 265 no 4 0 265 no 5 0 265 no 6 VES t68:45 3 2 265 no 7 VES t52:31 1 265 no 8 0 265 no 9 0 265 no OA 10 0 265 no 11 0 265 no 12 VES t110: 30 66 3 265 no VES t174:45 1 265 no VES t196:49 1 265 no 13 0 130 yes 14 0 265 no DTX-1 15 0 265 no 16 0 265 no 17 0 265 no 18 0 265 no 19 0 265 no YTX 20 0 160 yes 21 0 265 no 22 0 120 yes 23 VES t236:08 1 1 265 no 24 VES t77: 35 5 3 140 yes 25 0 265 no 26 0 265 no VES: ventricular extrasystole. Toxins 2015, 7 1037 The i.v. doses used in this study were 1/10 of mouse intraperitoneal LD50 for OA and YTX and 1/10 of mouse intraperitoneal MLD for DTX-1 (no lethality data available in rats) [32,37]. Actually, the i.v. dose of YTX is probably close to the LD50 by this route, since 3/7 rats died during the experiment. A fairly high dose was selected to be sure that if no effects were observed, there would be no need to repeat the experiment with a higher dose, since that kind of experimental design would require higher amounts of animals and toxin, and also to account for the possibly lower sensitivity of females to cardiovascular diseases and toxicity [38,39]. Thereby, if cardiotoxicity was an important component of acute YTX toxicity, some functional signs should have been observed at this dosing level. In the case of OA-treated rats, although this dose is clearly lower than the LD50, one death was recorded during the experiment. Therefore, this dose is enough to cause death of some individuals, but no cardiotoxicity signs were observed. DTX-1 toxic potency is considered similar to OA [1], although it might be higher by the oral route [40]. Thus, DTX-1 dosing levels would probably be sufficient to induce clinical signs if cardiotoxicity were an important component of DTX-1 toxicity. The lack of effects of YTX on the ECG is somehow surprising due to the abundant evidence of ultrastructural damage observed in cardiomyocytes of YTX-treated mice [7–10,17,18]. In fact, cardiomyocyte ultrastructural alterations have been described after intraperitoneal and oral administration of the toxin, even at doses that are far below the LD50 and at times as short as 1 h [8]. Furthermore, oral administrations of YTX at asymptomatic doses have demonstrated myocardial alterations at 24 h that persisted for at least 30 days [10,17]. The absence of effects on ECG recordings may be due to heart compensatory mechanisms to maintain functionality. Overall, OA, DTX-1 and YTX did not have in vivo functional effects on ECG parameters. Although some arrhythmia episodes were observed in toxin-treated rats, the frequency and number of animals affected were similar to controls, and therefore they would not be related to a toxin effect. Maintenance of functionality with this cardiomyocyte ultrastructural damage may not be feasible in the long term, considering that loss of cardiomyocyte function after injury is described as the principal etiology of heart failure [41], and therefore further studies are necessary to explore chronic cardiotoxic effects of these toxins. 2.3. Effects of OA, DTX-1 and YTX on the Levels of Cardiac Biomarkers Quantification of plasma cardiotoxicity biomarkers has also been recently included among the experiments for assessing in vivo structural heart damage. The levels of the cardiac biomarkers cTnI, cTnT and BNP were measured in rats treated with OA, DTX-1 or carrier. The quantification of these biomarkers was done in plasma samples collected during ECG experiments using panel 1 of the rat cardiovascular disease (CVD) kit from Millipore®. Plasma samples from YTX-treated rats were analyzed only for cTnI with a specific ELISA kit. Control samples from rats that received just the carrier were also included in the assays. Several plasma samples were collected for each rat along the experiment; one before toxin administration and the others every hour after toxin administration (see Methods section). Rats treated with OA or DTX-1 did not evidence any increase of plasmatic cTnI, cTnT or BNP during the experiment (Figure 4A–C, grey bars). The rats that received only the carrier (Figure 4A–C, white bars) showed similar levels of these biomarkers. The results for YTX-treated rats demonstrated no increase of cTnI during the first four hours following intravenous administration of the toxin in 6 rats (Figure 4D, rats 21–26), with the exception of one rat (Figure 4D, rat 20). Actually, no statistically Toxins 2015, 7 1038 significant differences were observed with regards to cardiac biomarkers in this study. In the case of rat 20, the animal entered apnea 140 min after toxin administration and cardiac activity continued for some time with increasing appearance of arrhythmias. Finally, the experiment was interrupted at time 160 min and a blood sample was collected that showed an increased concentration of cTnI. This increase that occurred after apnea onset was probably due to hypoxia and it should not be considered a direct effect of YTX. Isoproterenol, which is known to cause an increase of plasmatic cTnI [42], was used as positive control in these experimental conditions [36]. Two rats were injected with 4 mg/kg isoproterenol and after 2 h cTnI plasma levels were increased 3 times [36]. The detection of elevated cTnI plasma levels post-apnea and in isoproterenol-treated rats served as positive controls for the performance of immuno-detection techniques and experiment duration. Because the levels of cardiac cTnI and cTnT in plasma of rats treated with OA, DTX-1 and YTX were not altered, important myocardial injury after the acute administration of these toxins can be excluded [30]. Additionally, BNP levels, which are indicative of hemodynamic changes, ventricular damage or stress [43] were also not increased by OA and DTX-1. Figure 4. Effects of OA, DTX-1 and YTX on the levels of cardiac biomarkers. Blood samples were collected before and at different times (60, 120, 180 and 240 min) after i.v. administration of toxin or carrier. For OAand DTX-1-treated rats, cTnI, cTnT and BNP were quantified in plasma samples. For YTX-treated rats cTnI levels were measured. (A) cTnI; (B) cTnT and (C) BNP plasma levels measured in OA- (light grey bars) and DTX-1-treated rats (dark grey bars) and control rats (white bars) using immunodetection with a xMap rat CVD panel from Millipore®; (D) cTnI plasma levels measured in seven YTXtreated rats using a specific ELISA kit. 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