Teratogenic and anticonvulsant effects of zinc and copper valproate complexes in zebrafish

Teratogenic and anticonvulsant effects of zinc and copper valproate complexes in zebrafish

Epilepsy Research 139 (2018) 171–179 Contents lists available at ScienceDirect Epilepsy Research journal homepage: www.elsevier.com/locate/epilepsyr...

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Epilepsy Research 139 (2018) 171–179

Contents lists available at ScienceDirect

Epilepsy Research journal homepage: www.elsevier.com/locate/epilepsyres

Teratogenic and anticonvulsant effects of zinc and copper valproate complexes in zebrafish

T

Lauren D. Grünspana, Ben Hur M. Mussulinib, Suelen Baggiob, Paulo R. dos Santosa, ⁎ Françoise Dumasc, Eduardo P. Ricod, Diogo L. de Oliveirab, Sidnei Mouraa, LBIOP – Laboratory of Biotechnology of Natural and Synthetics Products, Technology Department, Biotechnology Institute, University of Caxias do Sul, Caxias do Sul, Brazil b Laboratory of Cellular Neurochemistry – Universidade Federal do Rio Grande do Sul, Programa de Pós-Graduação em Ciências Biológicas – Bioquímica, Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Porto Alegre, RS, Brazil c Laboratoire BioCIS, UMR CNRS 8076, Chimie des SubstancesNaturelles, IPSIT, Université Paris-Saclay, Université Paris-Sud, Faculté de Pharmacie, 5, rue Jean-Baptiste Clément, 92296, Châtenay-Malabry, France d Programa de Pós-Graduação em Ciências da Saúde, Universidade do Extremo Sul Catarinense UNESC, Av. Universitária, 1105, Bairro Universitário, 88806-000 Criciúma, SC, Brazil a

A R T I C L E I N F O

A B S T R A C T

Keywords: Valproate complex Zebrafish Teratogenic Anticonvulsant Pentylenetetrazole

Valproic acid (VPA) is an antiepileptic drug (AED) that has the broadest spectrum across all types of seizures and epileptic syndromes. Unfortunately, approximately 30% of epileptic patients are refractory to the classical AED. Metal ions have been frequently incorporated into pharmaceuticals for therapeutic or diagnostic purposes and research. In this preliminary study, we assess the embryo toxicity and the anticonvulsant activity of 4 novel metallodrugs, with Zn+2 and Cu+2, a derivative of valproic acid and the N-donor ligand in an adult zebrafish epileptic seizure model induced by pentylenetetrazole. The most toxic complex was [Cu(Valp)2Bipy], in which the LC50 was 0.22 μM at 48 h post fertilization (HPF) and 0.12 μM at 96 HPF, followed by [Zn(Valp)2Bipy] (LC50 = 10 μM). These same metallodrugs ([Cu(Valp)2Bipy] 10 mM/kg and [Zn(Valp)2Bipy] 30 mM and 100 mM/kg) displayed superior activity, thus reducing the seizure intensity by approximately 20 times compared to sodium valproate (175 mM/kg). Overall, [Cu(Valp)2Bipy] showed the best anticonvulsant effects. However, because of the toxicity of copper, [Zn(Valp)2Bipy] is considered the most promising anticonvulsant for future studies.

1. Introduction Valproic acid (VPA) is an antiepileptic drug (AED) that has the broadest spectrum across all types of seizures and epileptic syndromes (Belcastro and Striano, 2012). Unfortunately, approximately 30% of epileptic patients are refractory to classical pharmacological treatments (Langer et al., 2011). The development of new AEDs poses a great challenge, demanding screening for new compounds and/or chemical modifications of classical AEDs, such as VPA. Metal ions play a vital role in the life cycle by serving as essential cofactors and fulfilling cellular functions that cannot be achieved by organic molecules (Thompson and Orvig 2003); they are often incorporated into pharmaceuticals for therapeutic purposes (Barry and

Sadler, 2013; Lemire et al., 2013). The choice of metal ions with different oxidation states and coordination environments is a crucial factor, which may render new metallodrugs with different spectra of activity, such as in the treatment of cancer, epilepsy (Zhao et al., 2014), Alzheimer’s disease, Amyotrophic Lateral Sclerosis (ALS), diabetes, inflammatory states, and cardiovascular diseases (Chohan et al., 2005). No organometallic drug has been recently used in epilepsy treatment. However, new complex entities of valproic acid with divalent copper ions have demonstrated therapeutic effects that are much more effective than the original drug (Sylla-Iyarreta et al., 2009). The valproate compound bis(1,10-phenanthroline) copper (II) was effective in preventing minimal clonic convulsions (ED50 8 mM/kg) and the compound bis(1,10-phenanthroline) copper (II) displayed

Abbreviations: Zn+2, zinc; Cu+2, copper; HPF, hours post fertilization; FET, zebrafish toxicity test; Cu(Valp)2Phen, bis(2-propil-pentanoate)(1,10-phenanthroline)copper(II); Cu (Valp)2Bipy, bis(2-propil-pentanoate)(2,2-bipyridine)copper(II); Zn(Valp)2Phen, bis(2-propil-pentanoate)(1,10-phenanthroline)zinc(II); Zn(Valp)2Bipy, bis(2-propil-pentanoate)(2,2-bipyridine)zinc(II); VPA, sodium valproate (2-propil-pentanoate); Phen, phenanthroline; Bipy, bipyridine; PTZ, pentylenetetrazole; LD50, lethal dose50; AED, antiepileptic drug; GABA, gama-aminobutyric acid ⁎ Corresponding author at: Technology Department, Biotechnology Institute, University of Caxias do Sul, 1130 Francisco Getúlio Vargas st., CEP 95070-560, Caxias do Sul, Brazil. E-mail address: [email protected] (S. Moura). https://doi.org/10.1016/j.eplepsyres.2018.01.005 Received 21 February 2017; Received in revised form 5 December 2017; Accepted 3 January 2018 Available online 19 January 2018 0920-1211/ © 2018 Elsevier B.V. All rights reserved.

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2.3. Animals

anticonvulsant activity in the maximal electroshock model (MEM) (Sylla-Iyarreta et al., 2009). The synthesis of valproic acid derivatives with Zn, 2,2 – bipyridine and phenanthroline as potential ligands presented an LD50 of 409 μg/mL in a brine shrimp toxicity model. Addressing the need for novel experimental models and the search for a new AED, zebrafish offer a reasonable compromise between physiological complexity and throughput results. The zebrafish model can be utilized in screening for a wide range of anticonvulsants, offering potential advantages in comparison with mice (Berghmans et al., 2007). Embryos of zebrafish have been employed in toxicological studies due to their sensitivity to environmental changes and their physiological characteristics such as other vertebrates (Pamanji et al., 2015). The lack of a systematic approach to study the toxicity of metal compounds and general structural information on membrane transporters hampers the understanding of the complex mechanism of drug absorption and excretion, particularly in medicinal inorganic chemistry (Spreckelmeyer et al., 2014). The acute seizures induced in wild-type zebrafish by 4aminopyridine and pentylenetetrazole or heat closely resemble those induced in mammals, both physiologically and behaviorally (Baraban et al., 2005; Hunt et al., 2012). The same is observed in a more complex system regarding adult zebrafish (Mussulini et al., 2013) with potential i.p. injection compounds (Alfaro et al., 2011). The rules for the initiation and termination of neuronal electrical seizures in zebrafish, mice and humans are similar (Jirsa et al., 2014). In this study, we assess the embryo toxicity and the anticonvulsant activity of bis(2-propil-pentanoate) (1,10-phenanthroline) copper(II); bis(2-propil-pentanoate)(2,2-bipyridine)copper(II); bis(2-propil-pentanoate)(1,10-phenanthroline)zinc(II); and bis(2-propil-pentanoate) (2,2-bipyridine)zinc(II) compared to sodium valproate (2-propil-pentanoate) in an adult zebrafish epileptic seizure model induced by pentylenetetrazole.

Embryos and adult shortfin wild-type zebrafish (Danio rerio) were obtained from the university’s fish facility. Zebrafish embryos were obtained from pair-wise breeding and maintained in embryo medium (NaCl 5.03 mM, KCl 0.17 mM, CaCl2 0.33 mM, MgSO4 0.33 mM, methylene blue 0.1%, pH 7.5 ± 0.5, 28 ± 1° C) while awaiting the tests. Adult zebrafish were maintained in a recirculating housing system (ZebTech®, Tecniplast, Italy) at the following water quality parameters: 28 ± 1° C, pH 7.5 ± 0.5, conductivity 500 uS/cm, and a 14 h/10 h dark/light cycle. The animals were housed in 3- and 8-L tanks at 5 animals/L. The fish were fed three times a day with commercial fish food (once) and Artemia sp. (twice). All animals were experimentally naive, healthy and disease-free. All procedures were performed according to the Brazilian law for the care and use of laboratory animals (Law 11794/2008) and were previously approved by the Research Ethical Committee from the Federal University of Rio Grande do Sul (number #27725). 2.4. Embryo toxicity assay The fish embryo toxicity (FET) test was performed according to the OECD Guidelines for the Testing of Chemicals (2013). Toxicity was observed only when fertilization was ≥80%. Eggs fertilized within 1.5 h post-fertilization (HPF) (4–16-cell stage) were placed in a 24-well plate at one egg/well. Eggs were treated during 96 HPF with 1 mL of the following solutions: embryo medium (control group), ethanol 0.01% (vehicle group), sodium valproate (0.008–5000 μM) or organometallic complexes (0.00037–100 μM). Each solution was changed every 24 h. Lethality and morphological alterations (malformation of the head, tail, or heart; scoliosis, deformity of the yolk, and growth retardation) were assessed every 24 h using a Nikon SMZ 800 inverted stereomicroscope (Kimmel et al., 1995). According to the OECD Guidelines for the Testing of Chemicals (2013), the hatching rate of zebrafish embryos was evaluated at 72 HPF. The sample size was 20–24 embryos per group.

2. Material and methods 2.1. Chemicals

2.5. PTZ-induced seizure

All chemicals utilized in this work were purchased from SigmaAldrich (St. Louis, MO, USA). Ultra-pure water was obtained from a Milli-Q system (Millipore Corporate®, Billerica, MA, USA). Ethanol was purchased from MERCK (Darmstadt, Germany).

Adult animals were carefully weighed and measured to select the ones of a similar weight and size (350 ± 20 mg and 4 ± 0.5 cm, respectively) to avoid putative variations of pharmacodynamic and pharmacokinetic drugs. Animals were randomly handled from their home tanks and individually transferred to beakers filled with 160 μg/ mL tricaine. Then, the animals were anesthetized and injected intraperitoneally (i.p.) with ethanol 0.01%, sodium valproate (0.875 and 1.75 mM/kg), [Cu(Valp)2Phen]/[Cu(Valp)2Bipy] (0.1 and 0.3 mM/kg), or [Zn(Valp)2Phen]/[Zn(Valp)2Bipy] (0.1, 0.3 and 1.0 mM/kg). The volume of injection was 10 μL/g. Thirty min after i.p. injection, the animals were immersed in PTZ solution (10 mM) or vehicle for 20 min. Seizures were scored according to Mussulini et al. (2013): (0) short

2.2. Preparation of the zinc and copper valproate complexes The chemical synthesis was performed according to Santos et al. (2015) and Sylla-Iyarreta et al. (2009). The chemical structures (Fig. 1) were characterized by UV–vis FTIR, High Resolution Mass Spectrometry HRMS (ESI-QTOF), Nuclear Magnetic Resonance NMR for 1H and 13 C and X-ray. Metal concentrations were determined by Atomic Absorption Spectrophotometry Flame (FAAS).

Fig. 1. Chemical structures of the tested compounds: (1) [bis(2-propil-pentanoate)(1,10-phenanthroline)copper(II)] or [Cu(Valp)2Phen]; (2) [bis(2-propil-pentanoate)(2,2-bipyridine) copper(II)] or [Cu(Valp)2Bipy]; (3) [bis(2-propil-pentanoate)(1,10-phenanthroline)zinc(II)] or [Zn(Valp)2Phen], (4) [bis(2-propil-pentanoate)(2,2-bipyridine)zinc(II)] or [Zn (Valp)2Bipy]; and (5) sodium valproate (2-propil-pentanoate).

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Similarly, the LC50 values for [Zn(Valp)2Phen] at 48/96 HPF and [Zn (Valp)2Bipy] at 48 HPF could not be calculated (Fig. 2B). For [Zn (Valp)2Bipy] 96 HPF, LC50 was 10 μM (Fig. 2B). [Cu(Valp)2Bipy] showed a more prominent toxic effect, with an LC50 of 0.22 and 0.12 μM for 48 and 96 HPF, respectively (Fig. 2B). In the first 24 HPF, 40 and 80% of embryos treated with 30 μM of [Cu(Valp)2Phen] and [Cu(Valp)2Bipy] died, respectively (Mantel-Cox; Chi-square = 32.31, Chi-square = 67.94, respectively; p < 0.0001 compared to vehicle group) (Fig. 3). However, embryos treated with 100 μM of [Zn(Valp)2Phen] and [Zn(Valp)2Bipy] presented 40% and 74% mortality, respectively, at 96 HPF (Mantel-Cox; Chisquare = 20.50, Chi-square = 50.51, respectively; p < 0.001 compared to vehicle group). All control and vehicle embryos survived until the end of the experiment. Another important embryo toxicity endpoint evaluated was the hatching rate (Table 1). Vehicle-treated embryos started hatching at 48 HPF, and approximately 96% of embryos hatched at 72 HPF. Animals treated with copper organometallic complexes showed a reduced hatching rate in a concentration-dependent manner, such that the higher concentration of complexes, the lower the hatching rate. All embryos treated with 30 μM of [Cu(Valp)2Phen] and [Cu(Valp)2Bipy] did not hatch at 72 HPF. The hatching rate profile of zebrafish embryos treated with zinc organometallic complexes was similar to those observed for copper complexes, since a concentration-dependent reduction of the hatching rate was observed. However, some embryos hatched even at higher concentrations of [Zn(Valp)2Phen] and [Zn (Valp)2Bipy]. Regarding morphological alterations, sodium valproate induced embryo edema and a shortened bent tail in concentrations higher than 1000 μM (Fig. 4A). [Cu(Valp)2Phen] treatment induced a slight retardation in development, which was manifested as a delay in hatching (Fig. 4B). The compound [Cu(Valp)2Bipy] caused augmentation of the sac yolk area and heart malformation (Fig. 4C). Embryos exposed to the range between 0.37 and 100 μM of the organometallic complex [Zn (Valp)2Phen] showed heart malformations and yolk sac edema (Fig. 4D). Treatment with [Zn(Valp)2Bipy] caused heart malformation, yolk sac edema and extension. Fig. 4F depicts a vehicle-treated animal.

swim mainly at the bottom of the tank; (1) increased swimming activity and high frequency of opercular movement; (2) burst swimming, erratic, left and right movements; (3) circular movements; (4) clonic seizure-like behavior (abnormal whole-body rhythmic muscular contraction); (5) fall to the bottom of the tank, tonic seizure-like behavior (sinking to the bottom of the tank, loss of body posture, principally by rigid extension of the body); and (6) death. After PTZ exposure, the animals were transferred to another beaker containing ZebTech® system water and then moved to the recirculating housing system for 7 days for survival measurements (Mussulini et al., 2013; Alfaro et al., 2011). To compare doses of organometallic complexes with doses of sodium valproate, we converted all the treatments to mM/kg, since the molecular weight of the organometallic complexes was higher than that of sodium valproate. The N was 12 animals/group. 2.6. Statistical analysis 2.6.1. Embryo toxicity assay According to the OECD Guidelines for the Testing of Chemicals (2013), the LC50 of each compound was determined to be 48 HPF and 96 HPF. LC50 was defined as the concentration that led to death in 50% of all tested animals. The survival rate was analyzed by a Kaplan-Meier estimator followed by a log-rank (Mantel-Cox) post hoc test. The hatching rate was analyzed by Chi-square test. P < 0.05 was considered significant. 2.6.2. Adult PTZ-induced seizure Over time, the analysis of seizure scores was expressed as median ± interquartile range. The seizure intensity was expressed as the total area under the seizure score’s curves (AUC), which was expressed as the mean ± S.E.M. and analyzed by one-way ANOVA followed by Bonferroni’s post hoc test. The latency to score 4 was presented as the mean ± S.E.M. and analyzed by one-way ANOVA followed by Bonferroni’s post hoc test. Cumulative frequency was presented as the percentage of total animals that reached each score in each evaluated time point. The survival rate after PTZ-induced seizure was analyzed by the Chi-square test. The false positive alpha level for significance was 0.05.

3.2. Anti-seizure effect 3. Results Zebrafish injected with vehicle displayed a seizure profile similar to that observed by Mussulini et al. (2013). Animals started having a seizure (score 4) within approximately 150 s after PTZ exposure (Fig. 5), displaying seizure scores of approximately 270 s (Fig. 6). In the last fifteen minutes after PTZ, animals presented a continuous transition between score 4 and 5 (Fig. 7). Seizure intensity analysis indicated an AUC of 288 in the first interval, 532 in the second interval and 3456 in the third interval (Fig. 8). After 72 h of PTZ-induced

3.1. Embryo toxicity Neither morphological alterations nor death was observed in the embryos exposed to embryo medium or vehicle (ethanol 0.01%) (data not shown), and the LC50 values for VPA were 5000 μM and 2340 μM for 48 and 96 h, respectively (Fig. 2A). We were not able to determine LC50 for [Cu(Valp)2Phen] because of its low solubility (Fig. 2B).

Fig. 2. Lethal concentrations (LC50) for the organometallics and sodium valproate through accumulated mortality (48 HPF and 96 HPF). (A) Sodium valproate, accumulated mortality at 5000 μM and 2340 μM. (B) 48 HPF [Cu(Valp)2Phen] could not be determined due to solubility limits. [Cu(Valp)2Bipy] LC50 = 0.22 μM; [Zn(Valp)2Phen] and [Zn(Valp)2Bipy] could not be determined due to solubility limits. (C) LC50 values for 96 HPF: [Cu(Valp)2Phen] could not be determined due to solubility limits; [Cu(Valp)2Bipy] LC50 = 0.12 μM (96 HPF); [Zn (Valp)2Phen] could not be determined due to solubility limits and [Zn(Valp)2Bipy] LC50 value 10 μM.

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seizures, the mortality ratio was 33% in the vehicle group. Animals treated with sodium valproate (0.875 mM/kg) presented neither alterations in the seizure profile nor protection from mortality (Figs. 5–7). Conversely, treatment with 1.75 mM/kg of sodium valproate increased the time to transition between score 4–5 (Figs. 6 and 7) and increased the latency to seizure (score 4) compared to the vehicle group (one-way ANOVA; F [12,143] = 17.29, p < 0.0001; post hoc, p < 0.05) (Fig. 5). Seizure intensity was reduced in the first (oneway ANOVA; F [12,143] = 21.16, p < 0.0001; post hoc, p < 0.05) and second (one-way ANOVA; F [12,143] = 16.06, p < 0.0001; post hoc, p < 0.05) intervals compared to vehicle (Fig. 8). Animals treated with [Cu(Valp)2Phen] 0.1 mM/kg presented a slower seizure score progression compared to the vehicle (Figs. 6 and 7), taking 120 s in 100% of animals that presented a score of 2 and 450 s in 50% of the animals that presented a score of 5 (Fig. 7). However, there was no difference in latency to seizure compared to vehicle (Fig. 5). The seizure intensity was reduced in the first (one-way ANOVA; F [12,143] = 21.16, p < 0.0001; post hoc, p < 0.05) and second (oneway ANOVA; F [12,143] = 16.06, p < 0.0001; post hoc, p < 0.05) intervals compared to vehicle (Fig. 8). The mortality ratio was 25% (Fig. 5). On the other hand, animals treated with the same compound at a dose of 0.3 mM/kg presented a faster progression of seizure scores compared to vehicle (Fig. 7) and all animals presented a score of 5 within 180 s (Fig. 6). There was no significant difference regarding seizure intensity (Fig. 8) and latency to score 4 compared to vehicle, and the mortality ratio was 41% (Fig. 5). Animals treated with [Cu(Valp)2Bipy] 0.1 mM/kg presented a slower seizure score progression compared to control and a return to initial seizure scores in the last fifteen minutes of observation (Fig. 7). All animals presented a score of 1 at 120 s after PTZ exposure, and 33% of fish presented seizure within 300 s (Fig. 6). Seizure intensity was reduced in the first (one-way ANOVA; F [12,143] = 21.16, p < 0.0001; post hoc, p < 0.05), second (one-way ANOVA; F [12,143] = 16.06, p < 0.0001; post hoc, p < 0.05), and third (oneway ANOVA; F [12,143] = 6.736, p < 0.0001; post hoc, p < 0.05) intervals compared to vehicle (Fig. 8). Moreover, the animals also presented higher latencies to seizure (one-way ANOVA; F [12,143] = 17.29, p < 0.0001; post hoc, p < 0.05, mean time to seizure 515 s), and the mortality reached 8% only when compared to the vehicle group (Fig. 5). However, the animals treated with [Cu (Valp)2Bipy] 0.3 mM/kg presented no visible anti-seizure effect when compared to the 0.1-mM/kg [Cu(Valp)2Bipy] group. The seizure behavioral profile was similar to that of the vehicle (Fig. 7), and there was no difference in latency to score 4 (Fig. 5). Seizure intensity was slightly reduced in the first (one-way ANOVA; F [12,143] = 21.16, p < 0.05; post hoc, p < 0.05) and second (one-way ANOVA; F [12,143] = 16.06, p < 0.05; post hoc, p < 0.05) intervals compared to vehicle (Fig. 8). Mortality ratio was 25% (Fig. 5). Animals treated with [Zn(Valp)2Phen] 0.1 mM/kg presented no seizure profile alterations. At concentrations of 0.3 and 1.0 mM/kg, animals presented a slower seizure score progression compared to the vehicle group and 75% of animals displayed scores 1 and 2 in the last minute of observation (Fig. 7). For both doses, only 50% of animals presented with seizures (score of 4) at 300 s (Fig. 6). The latency to clonic seizures was higher than the vehicle for both groups (one-way ANOVA; F [12,143] = 17.29, p < 0.0001; post hoc, p < 0.05) (Fig. 5). Both doses presented a lower seizure intensity in the first (oneway ANOVA; F [12,143] = 21.16, p < 0.0001; post hoc, p < 0.05) and second (one-way ANOVA; F [12,143] = 16.06, p < 0.0001; post hoc, p < 0.05) intervals compared to the vehicle (Fig. 8). The mortality ratio was 25% and 0% for 0.3 and 1.0 mM/kg, respectively (Fig. 5). Animals treated with all doses of [Zn(Valp)2Bipy] presented seizure profile alterations (Fig. 7). All doses slowed down the progression of all scores in the first 300 s of observation (Fig. 6). Latencies to seizure were higher than the vehicle for animals treated with 0.30 and 1.0 mM/kg (one-way ANOVA; F [12,143] = 17.29, p < 0.0001; post hoc,

Fig. 3. Mortality across time. (A) Sodium valproate shows a constant mortality across time at higher concentrations (5000 μM). Organometallics containing copper displayed early embryo mortality at 24 HPF (hours post fertilization) (B) [Cu(Valp)2Phen] (1) and (C) [Cu(Valp)2Bipy] (2). The compounds containing zinc (D) [Zn(Valp)2Phen] (3) and (E) [Zn(Valp)2Bipy] (4) showed a higher mortality rate at the larvae stage (72 HPF). (KaplanMeier curves followed by log-rank analysis—compounds (A) p < 0.001. (B), (C) and (E) p < 0.0001. (D) p < 0.005).

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Table 1 Hatching rate of zebrafish embryos (at 72 hpf) exposed to organometallic complexes. Organometallic complexes Concentration (μM)

[Cu(Valp)2Phen]

[Cu(Valp)2Bipy]

[Zn(Valp)2Phen]

[Zn(Valp)2Bipy]

0 0.0037 0.037 0.37 1.11 3.33 10 30 100

20/20 n.t n.t 11/20a 09/18a 02/18a 0/18a 0/11a n.t

24/24 17/21 13/14 09/09 10/10 01/07b 0/06b 0/04b n.t

24/24 n.t n.t 24/24 22/22 15/20 10/19 03/19 14/17

24/24 n.t 15/22 06/21 05/19 11/18 10/16 03/11 08/12

Abbreviations: (hpf) hours post fertilization; (n.t.) not tested. a = p < 0.0001 as compared to the respective vehicle group (Chi-square; df = 25.01; 5). b = p < 0.05 as compared to the respective vehicle group (Chi-square; df = 12.54; 7). All data were presented according to the hatching rate of embryos (number of hatched embryos/total number of survived embryos in each treatment).

p < 0.05) (Fig. 5). All doses reduced the seizure intensity in the first interval (one-way ANOVA; F [12,143] = 21.16, p < 0.0001; post hoc, p < 0.05), and animals treated with 0.3 and 1.0 mM/kg presented lower seizure intensity in the second interval (one-way ANOVA; F [12,143] = 16.06, p < 0.0001; post hoc, p < 0.05) when compared to

the vehicle group (Fig. 8). The mortality ratio was 25%, 25% and 0%, respectively (Fig. 5). In comparison to sodium valproate 1.75 mM/kg, animals injected with [Cu(Valp)2Bipy] 0.1 mM/kg, [Zn(Valp)2Phen] 1.0 mM/kg and [Zn (Valp)2Bipy] (0.1, 0.3 and 1.0 mM/kg) showed a reduced seizure

Fig. 4. Photographs of embryos at 96 HPF exposed to maximum viable concentration. (A) Sodium valproate, 1000 μM caused malformations such as edema, a shortened bent tail, necrosis and unhatched embryos. (B) [Cu(Valp)2Phen], 30 μM, unhatched embryos. (C) [Cu(Valp)2Bipy], 30 μM, larger yolk sac area, heart malformation, pooling blood and unhatched embryos. (D) [Zn(Valp)2Phen] at 100 μM, heart malformation occurred with yolk sac edema. (E) [Zn(Valp)2Bipy] at 100 μM, embryos with heart malformations, pooling blood, necrosis, yolk sac edema and yolk sac extension. [F] Solvent control group, ethanol 0.001%. There was no malformation or teratogenic effect.

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Fig. 5. Seizure intensity evaluation. Seizure intensity was evaluated by the area under the curve at three different time intervals [0–150 s (A); 150–300 s (B); and 300–1200 s (C)]. Data were represented as the mean ± S.E.M and analyzed by one-way ANOVA followed by Bonferroni’s post hoc test, where the * and # indicate significant differences between the compounds.

p < 0.05) compared to sodium valproate 1.75 mM/kg.

intensity at 0–150 s (one-way ANOVA; F [12,143] = 21.16, p < 0.0001; post hoc, p < 0.05). For the second interval (150–300 s), treatment with [Cu(Valp)2Bipy] 0.1 mM/kg and [Zn(Valp)2Bipy] 0.3 and 1.0 mM/kg, reduced the seizure intensity compared to sodium valproate 1.75 mM/kg (one-way ANOVA; F [12,143] = 16.06, p < 0.0001; post hoc, p < 0.05). Only treatment with [Cu (Valp)2Bipy] 0.10 mM/kg could reduce the seizure intensity in the last interval (one-way ANOVA; F [12,143] = 6.736, p < 0.0001; post hoc,

4. Discussion The synthesis of metal complexes is a recent approach to obtain bioisostere drugs. The organocomplexes or coordination compounds are molecules with mixed chemical function, which consist of organic molecules bound to metal in a geometric arrangement known as

Fig. 6. Behavioral cumulative frequency score, seizure profile of PTZ and anti-seizure molecule pretreatment. Data are represented as the animal index (%) that reached the scores across time. The cumulative frequencies are depicted on the Y-axis. The X-axis shows two time intervals as follows: 0–300 s, when the beginning of the seizure is noticeable; and 300–1200 s, when the control showed stability of the seizure behavior profile between scores 4 and 5.

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Fig. 7. Behavioral score, seizure profile of PTZ and anti-seizure molecule pretreatment. Seizure scores are depicted on the Y-axis. The X-axis shows two time intervals as follows: 0–300 s, when the beginning of the seizure is evident, and 300–1200 s, when the vehicle reached stability of the seizure behavior profile, changing between scores 4 and 5. Data are represented as the median ± interquartile range.

such as eye contact, gill accumulation leading to breathing alterations, and, most importantly, it allows for dose comparison (Alfaro et al., 2011). The first aim of this study was to delineate a dose of sodium valproate that could elucidate an anti-seizure effect. We based our initial protocol on the study by Luszczki et al. (2006), in which the authors indicated the ED50 of Sodium Valproate as 0.875 mM/kg. As shown in Figs. 5–7, there was no significant anti-seizure effect at this concentration. Thus, the dose was doubled to find a clear anti-seizure effect by increasing the mean time to clonic seizure from 151 s to 297 s (Fig. 5). To compare the dose with the other reported works, all treatments were converted to mM/kg. As soon as we injected the

coordination spheres. The activity of the organic metal coordination compounds has a significant importance in life maintenance. However, investigating and establishing the activity of a new drug alone is not sufficient; the toxic action should also be evaluated for safety concerns. To predict toxicity in humans, different models have to be tested. The zebrafish emerged in the scenarios of pharmacologic screening therapy for epilepsy in 2005 (Baraban et al., 2005). In the last 10 years, the zebrafish epileptic seizure model has been deeply characterized (Grone and Baraban, 2015), and it has increased our knowledge of the model and the strength of the translation of this animal model to study epilepsy (Afrikanova et al., 2013). Adult zebrafish easily tolerate intraperitoneal injection, avoiding any extracorporeal effect of compounds,

Fig. 8. Latency to score 4 and mortality rate. Data for latency were represented as the mean ± S.E.M and analyzed by one-way ANOVA followed by Bonferroni’s post hoc test. Data for mortality rate were expressed as a percentage of live animals of the total animals in each group. * and # indicate significant differences compared to vehicle and sodium valproate 1.75 mM/kg, respectively.

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ion, and both compounds presented an anti-seizure effect inversely proportional to their dose; therefore, we aimed to change Cu+2 to Zn+2 in the molecules. Based on Figs. 5–7, the compounds [Zn(Valp)2Phen] and [Zn(Valp)2Bipy] did not affect latency to clonic-seizure in the 0.1mM/kg groups. On the other hand, both groups treated with 1.0 mM/kg showed full death prevention. An interesting result was observed in the treatment with [Zn(Valp)2Bipy]. All doses reduced the seizure intensity in the first interval of analysis (0–150 s) when compared to sodium valproate at 0.3 mM/kg, which still promoted a lower seizure intensity, even in the second interval of analysis compared to the classical treatment molecule. This may be due to differences between 1,10phenanthroline (Phen) and 2,2-bipyridine (Bipy) as the ligand. Popa and Lerche (2006) have shown that 1,10-phenanthroline is a sodium channel blocker. The strength and occurrence of hydrogen bonds formed between phenanthroline and a putative receptor site within the pore region are critically determined by the orientation of the molecules involved, so the local membrane electric field would directly act on the binding of Phen. Copper and zinc also play an important role in the CNS. Both ions can modulate synaptic transmission postsynaptically via direct actions on glutamate, GABA, or glycine receptors. Zinc and copper could have presynaptic effects on transmitter release via the inhibition of voltage-gated calcium channels. In addition to these effects on neurotransmission, zinc and copper can influence neuronal excitability by modulating voltage-gated ion channels, displaying distinct effects on voltage-gated K+ channels and input resistance, effects that may alter a neuron’s capacity to repetitively fire action potentials (Horning and Trombley, 2001).

compounds [Cu(Valp)2Phen] and [Cu(Valp)2Bipy] in the same dose as sodium valproate (1.75 mM/kg), the animals presented abnormal behaviors, such as burst, jumps and circular movements indicating possible toxicological effects (Ibrahim et al., 2014). This result led us to test the toxic doses of these compounds, which was performed by toxicological screening in larva zebrafish to detect the maximal concentration that works without such effects. The fish embryo toxicity test (FET) was designed to determine the acute toxicity of chemicals on embryonic stages of fish and it has been used for toxicity screening of new compounds, including AED (Johnson et al., 2007; Selderslaghs et al., 2009; Teixido et al., 2013). The first challenges of testing new compounds are the determination of solubility and maximum soluble concentration and finding an adequate solvent. As many compounds exhibit limited solubility in aqueous solution, the use of organic solvents is required to create stock solutions that can be diluted in the embryo medium (Maes et al., 2012). All the organometallic complexes were soluble in ethanol (99.9%), and the maximum solubility concentrations were as follows: [Cu(Valp)2Phen] 38 mM, [Cu(Valp)2Bipy] 190 mM, [Zn(Valp)2Phen] 180 mM and [Zn (Valp)2Bipy] 190 mM. It is well known that ethanol causes severe malformations in zebrafish embryos; therefore, the maximum final concentration in the test plate was 0.01%. The maximum tolerated concentration of [Cu(Valp)2Phen] was 10 μM and 100 μM for [Zn (Valp)2Phen]. Based on these results, we chose our concentration range. In this way, the lethal concentration (LC50) was calculated just for the complexes with bipyridine (Fig. 2). On the other hand, we could not determine the LC50 due to the solubility limit for the phenanthroline compounds. The mortality was evaluated across time, and the complexes with copper promoted an early death, at 24 HPF during the pharyngula period (Fig. 3). Differently, the zinc complexes induced death at the early larvae stage, 72 HPF when the embryo is formed (Fig. 3). In similar tests with embryos, Selderslaghs et al. (2009) tested sodium valproate with an LC50 of 2.09 mM. In the morphologic evaluation, the metals seemed to influence the hatching rate (Table 1), where the larvae exposed to copper complexes were unable to hatch, and even if the larvae did not hatch, the animal development continued normally or with some delay. Zinc did not alter the hatching rate as significantly as copper; however, it still induced some morphological alterations, such as heart malformations, enabling the organ to pump blood and therefore affecting the circulation, pooling blood in the yolk sac and promoting necrosis (Fig. 4D and E). Heart malformations were detected in the larvae exposed to copper organometallics, but the blood circulation was preserved; there was some necrosis and yolk sac edema, especially in the larvae treated with [Cu(Valp)2Bipy] (Fig. 4B and C). The highest viable sodium valproate (Fig. 4A) concentration tested provoked morphological defects as reported in the literature (Selderslaghs et al., 2009; Herrmann, 1993). The embryos treated with ethanol (0.001%) did not develop any morphological alterations (Fig. 4F). Anticonvulsant effects were tested in adult zebrafish, in which the [Cu(Valp)2Phen] had no effect on the seizure latency at a dose of 0.1 mM/kg. It presented the same effects on seizure intensity as sodium valproate at the dose of 1.75 mM/kg (Fig. 5). Conversely, the same compound at the dose of 0.3 mM/kg, presented a similar profile as the control and increased the seizure intensity in the first five minutes, with animals showing an increase to a score of 5 in the same period compared to controls (Figs. 7 and 8). A similar profile could be observed using [Cu(Valp)2Bipy] because animals treated with the lower doses presented a clear anti-seizure effect, and the higher dose started to lower this effect (Figs. 5–7). Furthermore, the [Cu(Valp)2Bipy] 0.1 mM/ kg group was the only one to present a higher seizure latency compared to the sodium valproate 1.75 mM/kg group, and it was the only treatment able to lower the seizure intensity in the last 15 min of observation when only 25% of the animals presented a score of 5. The only difference between the last two compounds was the metal

5. Conclusion In this work, we performed a screening of new organometallic drugs as anticonvulsants in the zebrafish epileptic seizure model induced by pentylenetetrazole, in which the results are summarized in Table 2. Regarding toxicity, copper compounds are more toxic compared to zinc compounds. In this preliminary work, all the organometallics tested were more effective as anticonvulsants than sodium valproate. [Cu (Valp)2Bipy] can be emphasized as having best anticonvulsant effects, but it is highly toxic; therefore, [Zn(Valp)2Bipy] should be considered the most promising anticonvulsant drug for future studies.

Table 2 Summary of activities and toxicity of organocomplexes tested as anticonvulsant in this work. Adult Zebrafisha Compound [Cu(Valp)2Phen] 10 30 [Cu(Valp)2Bipy] 10 30 [Zn(Valp)2Phen] 10 30 100 [Zn(Valp)2Bipy] 10 30 100 Sodium Valproate 175 Control

Embryosb

Antiseizure

Mortality

Hatching

Mortality

− +

+ ++

− −

− +

+++ −

+ ++

− −

+++ +++

− ++ ++

+ + −

++ + ++

− − −

+ ++ ++ ++ −

+ + − − +

++ + ++ − +++

++ ++ +++ − −

+++ = very high; ++ = high; + = moderate; − = not detected. a Adult zebrafish mM/Kg. b Compounds concentration range μM.

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Conflicts of interest

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