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Nephrotoxicity induced by the venom of Hypnale hypnale from Sri Lanka:Studies on isolated perfused rat kidney and renal tubular cell lines

Mauren Villaltaa, Tiago Lima Sampaiob, Ramon Róseo Paula Pessoa Bezerra de Menezesb,Dânya Bandeira Limab, Antônio Rafael Coelho Jorgec, Renata Sousa Alvesb,Helena Serra Azul Monteiroc, Indika Gawarammanad, Kalana Maduwaged, Roy Malleappahe,Guillermo Leóna, Alice M.C. Martinsb, José María Gutiérreza,∗

a Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa RicabDepartment of Clinical and Toxicological Analysis, Federal University of Ceará, Fortaleza, Ceará, Brazilc Department of Physiology and Pharmacology, Federal University of Ceará, Fortaleza, Ceara, Brazild Faculty of Medicine, University of Peradeniya, Sri LankaeAnimal Venom Research International (AVRI), California, USA

A R T I C L E I N F O

Keywords:Hypnale hypnaleHump-nosed pit viperNephrotoxicityCytotoxicityRenal tubular cells

A B S T R A C T

The hump-nosed pit viper Hypnale hypnale is responsible for a high number of snakebite cases in southwesternIndia and Sri Lanka. Although most patients only develop local signs and symptoms of envenoming, there is agrowing body of evidence indicating that these envenomings may be associated with systemic alterations, in-cluding acute kidney injury. In this study we evaluated the renal toxicity of H. hypnale venom by using a perfusedisolated rat kidney system and by assessing cytotoxicity in two different renal tubular cell lines in culture. Thevenom caused alterations in several renal functional parameters, such as reduction on perfusion pressure, renalvascular resistance, and sodium and chloride tubular transport, whereas glomerular filtration rate and urinaryflow initially decreased and then increased after venom perfusion. In addition, this venom was cytotoxic toproximal and distal renal tubular cells in culture, with predominance of necrosis over apoptosis. Moreover, thevenom affected the mitochondrial membrane potential and induced an increment in reactive oxygen species inthese cells. Taken together, our results demonstrate a nephrotoxic activity of H. hypnale venom in these ex-perimental models, in agreement with clinical observations.

1. Introduction

Snakebite envenoming is a serious medical hazard in the Indianpeninsula and Sri Lanka (Warrell, 1995). In addition to the traditionalso-called ‘big four’ species (Daboia russelii, Echis carinatus, Naja naja andBungarus caeruleus), which are responsible for the majority and mostsevere snakebite envenomings, the hump-nosed viper, Hypnale hypnale,causes a high number of snakebites in this region (Warrell, 1995;Ariaratnam et al., 2008, 2009; Shivanthan et al., 2014). In the past, H.hypnale was considered as incapable of inflicting severe envenomings inhumans, but growing clinical evidence clearly shows that this snake isable to cause systemic manifestations.

The majority of envenomings by H. hypnale are characterized byonly local effects, i.e. edema of variable extension and pain (Warrell,1995; Ariaratnam et al., 2008; Kularatne et al., 2011). In addition, anumber of cases also develop systemic effects of envenoming, such as

mild coagulopathy, bleeding, thrombotic microangiopathy, acutekidney injury (AKI), and chronic kidney injury (CKI) (de Silva et al.,1994; Kularatne and Ratnatunga, 1999; Joseph et al., 2007; Ariaratnamet al., 2008, 2009; Kularatne et al., 2011; Herath et al., 2012;Maduwage et al., 2013; Karunarathne et al., 2013; Shivanthan et al.,2014; Rathnayaka et al., 2018, 2019a, 2019b). AKI is one of the maincauses of morbidity and mortality in envenomings inflicted by manysnake species (Albuquerque et al., 2014).

The pathogenesis of AKI induced by the venom of H. hypnale hasremained elusive, although the effects of this venom in the kidney atexperimental level include damage to proximal convoluted tubules(Gunatilake et al., 2003; Silva et al., 2012; Tan et al., 2012). As withother snake venoms that affect the kidney, it is likely that ne-phrotoxicity by H. hypnale is of multi-factorial nature, probably invol-ving mechanisms such as (a) renal ischemia secondary to hemodynamicdisturbances, (b) thrombotic microangiopathy and hemolytic

https://doi.org/10.1016/j.toxicon.2019.04.014Received 1 March 2019; Received in revised form 19 April 2019; Accepted 22 April 2019

∗ Corresponding author.E-mail address: jose.gutierrez@ucr.ac.cr (J.M. Gutiérrez).

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microangiopathic anemia, (c) tubular cell toxicity induced by he-moglobin accumulated in the tubular lumen, and (d) direct nephrotoxicaction of venom components (Sitprija and Sitprija, 2012). In order tostudy the nephrotoxic action of H. hypnale venom at the experimentallevel, the present study was carried out by assessing the effect of thisvenom on a perfused isolated rat kidney system and on two differentrenal tubular cell lines in culture.

2. Materials and methods

2.1. Venom

A pool of venom from 35 adult specimens of H. hypnale was used inthis study. Venom was obtained from specimens collected in the field infour provinces of Sri Lanka [Central Province (wet zone); NorthWestern province (dry and intermediate zones); Southern province (wetzone); Sabaragamuaga province (wet zone)] following the granting of acollecting permit from the Department of Wildlife Conservations, SriLanka (WL/3/2/7), and kept at the serpentarium of Animal VenomResearch International (AVRI) in Sri Lanka. The male: female ratio was20 : 15. Upon collection, venom was frozen and then freeze-dried, andstored at −20 °C until used. The identification of the specimens asbelonging to H. hypnale was based on the keys previously published byMaduwage et al. (2009).

2.2. Kidney perfusion experiments

Adult male Wistar rats (260–320 g) were fasted for 24 h with freewater access. They were anesthetized with sodium pento-barbitone(50mg/kg, i.p.) and, after dissection of the right kidney, the renal ar-tery was cannulated through the mesenteric artery. The kidney wasperfused with a modified Krebs-Henseleit solution (MKHS) with thefollowing composition (in mmol/L): 114 NaCl, 4.96 KCl, 1.24 KH2PO4,0.5 MgSO4.7 H2O, 2.10 CaCl2, 25 NaHCO3. Bovine serum albumin(BSA, 6% w/v), urea (0.75 mg/mL), and glucose (1.5 mg/mL) wereadded to the solution, and the pH was adjusted to 7.4. Thereafter, inulin(0.75 mg/mL) was added. In each experiment, 100mL of MKHS wererecirculated during 120min through the system. The perfusion pressurewas kept between 120 and 140mmHg. Initially, perfusion was per-formed without venom for 30min in order to have basal levels of eachdetermination. After this, H. hypnale venom (10 μg/mL) was added tothe system and changes were observed. Samples of urine and perfusatewere collected every 10min and perfusion pressure (mmHg) and flow(L/min) were recorded every 5min. A control group perfused withMKHS only was followed during 120min. Five rats were used for eachtreatment.

Perfusion pressure (PP) was determined at the tip of the stainlesssteel cannula in the renal artery. Osmolality was measured by using aPZL-1000 osmometer (Wescor 5100C, USA), and concentrations of so-dium, potassium and chloride were determined by using a 9180Electrolyte Analizer (Rapidchem 744, Bayer Diagnostic, UK). Inulinconcentration was quantified according to Walser et al. (1955) asmodified by Fonteles et al. (1983). The following renal functionalparameters were determined: perfusion pressure (PP), renal vascularresistance (RVR), urinary flow (UF), glomerular filtration rate (GFR),and the percentages of sodium (%TNa+), potassium (%TK+), andchloride (%TCl−) total tubular transport and proximal tubular trans-port (Martínez-Maldonado and Opava-Stitzer, 1978). Results werecompared to the internal control group corresponding to the values atthe 30min perfusion period prior to venom introduction into thesystem. The protocol was approved the Ethics Committee on AnimalResearch (CEPA) of the Federal University of Ceara (Permit Number79/08).

2.3. Cytotoxicity of venom on kidney-derived cells

Cytotoxicity of venom was tested in cell culture using the cell linesLLC-MK2 (Monkey Kidney epithelial cell line) and MDCK (Madin-DarbyCanine Kidney epithelial cells), as previously described (Morais et al.,2013; Mello et al., 2014). These cells correspond to proximal and distalrenal tubular cells, respectively. Cells were grown in DMEM supple-mented with 10% bovine fetal serum, under 5% CO2 at 37 °C. Uponconfluence, cells were grown in DMEM without fetal serum for 24 h.Then, the confluent monolayers were treated with 10% trypsin and cellswere grown in 96 well plates at a seeding density of 105 cells/well. Cellswere left to adhere to the plate overnight and were then treated withvenom. For this, solutions of various venom concentrations were addedto wells and incubated for 24 h. Control wells were treated with 0.12MNaCl, 0.04M phosphate, pH 7.2 (PBS) solution. Assays were run intriplicates. Cytotoxicity was assessed by reduction of 3-(4,5-di-methyltiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, Sigma, St.Louis, MO, USA). Ten microliters of MTT solution (2.5 mg/mL in PBS)were added to each well, at the time intervals indicated, and incubatedat 37 °C for 4 h. The medium was removed and the precipitated for-mazan crystals were dissolved in 10% sodium dodecyl sulphate (SDS) in0.01 N HCl. After 17 h of incubation, absorbances at 570 nm were re-corded in a microplate reader (Biochrom® Asys Expert Plus). Cell via-bility was estimated in comparison with the control group. The MedianCytotoxic Concentration (CC50), i.e. the venom concentration that in-duced 50% cytotoxicity, was estimated using the program GraphPadPrism.

2.4. Annexin V-FITC staining

Staining of cells with Annexin was used to assess apoptosis. LLC-MK2 or MDCK cells were seeded at 105 cells/well, as described, andincubated for 24 h at 37 °C. Then, solutions of two venom concentra-tions, corresponding to 1 and 2 CC50, were added. Controls includedcells incubated with PBS. After an incubation period of 24 h, cells weretrypsinized, centrifuged at 500 g for 5min, and the supernatant wasdiscarded. This step was repeated twice using PBS. The cellular pelletwas labeled with the Annexin/7AAD using the kit BD Pharmingen PEAnnexin Apoptosis Kit I (BD Pharmigen, CA, USA). Controls includedcells incubated with either Annexin/7AAD alone. Samples were in-cubated for 30min at room temperature in the dark. Then, sampleswere analyzed by flow cytometry in a FACS Calibur (Becton-Dickinson,Mountain View, CA, USA) and analyzed using a Cell Quest software(Becton-Dickinson).

2.5. Rhodamine staining

Staining of cells with Rhodamine 123 (Sigma) was used to assess themitochondrial membrane potential (Baracca et al., 2003). Cells wereincubated with venom solutions, as described above. Then, after cen-trifugation, the cellular pellet were resuspended in 100 μL PBS, and100 μL of a Rhodamine 123, dissolved in PBS, were added. After anincubation of 30min at room temperature in the dark, 1 mL of PBS wasadded and the preparation was centrifuged at 500 g for 5min. Thepellets were resuspended in 500 μL PBS and flow cytometry analysiswas then carried out.

2.6. DCFH-DA staining

Staining of cells with 2,7-dichlorodihydro-fluorescein diacetate(DCFH-DA) (Sigma) was used to measure the cellular levels of reactiveoxygen species, as described by Morais et al. (2015). Cells were in-cubated with venom, as described above. After incubation, cells weretrypsinized and centrifuged at 500 g for 5min. After several washingsteps, cells were resuspended in PBS containing 5 μM DCFH-DA, andincubated for 30min. Controls included cell suspensions in which

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DCFH-DA was not added. After incubation, cells were analyzed by flowcytometry. The data based on the FL1 channel were analyzed with theCell Quest program.

2.7. Statistical analyses

Results were expressed as mean ± S.E.M. of three or five replicates,in the case of cell culture and renal perfusion experiments, respectively.The significance of the differences between mean values was assessedby ANOVA, using a Dunnett post-hoc test to compare pairs of means,using the program GraphPad Prism® 5.0. Values of p < 0.05 wereconsidered statistically significant.

3. Results

3.1. Effects of venom on kidney functional parameters

Control experiments carried out on kidneys perfused with Krebs-Henseleit solution only did not show any significant changes in theparameters evaluated along the 120min observation time. The perfu-sion of a venom solution (10 μg/mL) in the isolated kidney preparationinduced changes, as compared to controls, in almost all functionalparameters evaluated. There was a significant reduction in perfusionpressure at 90 and 120min (PP; Fig. 1A), renal vascular resistance at 90and 120min (RVR; Fig. 1B), percentage of transported sodium at 90and 120min (%Na+; Fig. 1E), percentage of proximal tubule trans-ported sodium at 60, 90 and 120min (%pNa+; Fig. 1F), percentage oftransported chloride at 120min (%Cl−; Fig. 1I), and percentage ofproximal tubule transported chloride at 120min (%pCl−; Fig. 1J).Regarding urinary flow, there was a slight initial reduction at 60 and90min (UF; Fig. 1C), followed by an increment at 120min, whereas theglomerular filtration rate had a reduction at 60 and 90min (GFR;Fig. 1D), followed by recovery at 120min. Of the evaluated parameters,only the percentage of transported potassium was not altered uponinfusion of the venoms (Fig. 1G and H).

3.2. Cytotoxicity on kidney-derived cells

When cytotoxicity of venom on two kidney-derived cell lines wasassessed by the MTT assay, a dose- and time-dependent cytotoxic effectwas observed in both cell lines. The dose-dependency was more evidentwhen incubation was carried out for 24 h and, therefore, the estimationof the Median Cytotoxic Concentration (CC50) was made at this timeinterval. CC50s corresponded to 9.0 ± 2.9 μg/mL and 19.7 ± 12 μg/mL μg/mL for LLC-MK2 and MDCK cell lines, respectively. Thus, venomwas cytotoxic for cell lines derived from proximal and distal renaltubular cells.

3.3. Evaluation of cell death mechanisms

Cells of both lines were exposed to a concentration of venom cor-responding to one CC50 for 24 h, after which cells were stained withannexin (to assess apoptosis) and with 7AAD (to assess plasma mem-brane disruption, i.e., necrosis). As depicted in Fig. 2, necrosis was thepredominant effect at the venom concentration tested.

Cells of both lines exposed to a concentration of venom corre-sponding to one and two CC50 were stained with Rhodamine, in orderto assess the effects on the mitochondrial membrane potential. Venominduced a drastic reduction in Rhodamine fluorescence at both con-centrations (Fig. 3), hence evidencing a drop in the mitochondrialmembrane potential. Moreover, venom induced an increment in theconcentration of reactive oxygen species in the two kidney-derived cellslines, at concentrations corresponding to one and two CC50, as revealedby an increment in the relative fluorescence of DCFH-DA (Fig. 4).

4. Discussion

The majority of envenomings caused by H. hypnale are characterizedonly by local effects, i.e. pain and edema, but a growing body of pub-lished clinical evidence demonstrates that this viperid species is alsocapable of inducing severe envenomings characterized by systemicmanifestations, such as mild coagulopathy, hemorrhage, thromboticmicroangiopathy, and AKI (de Silva et al., 1994; Ariaratnam et al.,2008, 2009; Kularatne et al., 2011; Herath et al., 2012; Maduwageet al., 2013; Shivanthan et al., 2014; Rathnayaka et al., 2018, 2019a,2019b). The pathogenesis of AKI in envenomings by this and othersnake species is likely to be multifactorial (Sitprija and Sitprija, 2012).The clinical expression of AKI is an oliguric or anuric state associatedwith acute tubular necrosis after envenoming (Albuquerque et al.,2014).

Previous studies have assessed the effects of venoms of variousspecies of snakes of the genus Bothrops in the isolated kidney modelused in the present investigation (Evangelista et al., 2010; Morais et al.,2013; Marinho et al., 2015). Bothrops sp venoms induced a drop inperfusion pressure, renal vascular resistance, urinary flow, glomerularfiltration rate, and sodium tubular transport. Bothrops moojeni and B.jararacussu venoms showed similar effects, but caused an increase inurinary flow (Havt et al., 2001; Barbosa et al., 2002). In the presentwork, H. hypnale venom promoted a reduction of perfusion pressure,renal vascular resistance, sodium and chloride tubular transport, and aninitial drop of glomerular filtration rate and urinary flow, followed byan increment in these two parameters. Our findings underscore theability of H. hypnale venom to induce renal damage. Although themechanisms behind these alterations remain to be elucidated, our studyprovides experimental evidence that this venom alters functionalparameters, such as reduction on RVR and PP. These alterations couldlead to renal ischemia secondary to hemodynamic disturbances. Thepathogenesis of AKI in envenomings by H. hypnale may be also medi-ated through the formation of microthrombi, i.e. thrombotic micro-angiopahy, which contribute to renal ischemia (Rathnayaka et al.,2019b).

The infusion of H. hypnale venom in the isolated rat kidney causedalterations in practically all the parameters investigated, hence under-scoring a widespread effect of the venom on renal function. Moreover,our observations suggest that this venom affects the function of both thetubular cells and the glomeruli, as evidenced by electrolyte transportand glomerular filtration rate. Previously, H. hypnale venom had beenshown to induce AKI in experimental models, as revealed by incrementsin serum urea and creatinine concentrations, hematuria, proteinuria,and by histolopathogical changes in the kidney (Gunatilake et al., 2003;Silva et al., 2012; Tan et al., 2012). Our observations on the effect ofthis venom on sodium tubular transport is in agreement with a recentclinical observation of a patient who developed urinary sodium lossassociated with hyponatremia (de Silva et al., 2018).

H. hypnale venom was cytotoxic to both renal tubular cell typestested in culture (MDCK and LLC-MK2), as judged by the MTT assay.Cytotoxicity involving necrosis and apoptosis has been described in thesame cell lines with the venoms of Bothrops sp (Morais et al., 2013;Mello et al., 2014; Marinho et al., 2015). It was previously shown thatthe venoms of H. hypnale, H. zara and H. nepa inhibited cell prolifera-tion of rat aorta smooth muscle cells at a relatively low concentration(Maduwage et al., 2011). In our observations, when cells were in-cubated with a venom concentration corresponding to one CC50, for24 h, more cells were positively stained with 7AAD than with annexin,thus indicating that at this dose and time of incubation the majority ofcells was affected by necrosis instead of apoptosis. It has to be con-sidered, however, that the balance between necrosis and apoptosis inthis model may be shifted when using other venom concentrations, asshown for a lymphoblastoid cell line incubated with a myotoxic PLA2

homologue from the venom of Bothrops asper (Mora et al., 2005). Thedirect toxicity on tubular epithelial cell lines by necrosis can lead to a

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Fig. 1. Effects of H. hypnale venom (10 μg/mL), on perfusion pressure (A), renal vascular resistance (B), urinary flow (C), glomerular filtration rate (D), percent ofsodium, potassium and chloride tubular transport (E, G, I) and percent of proximal tubule sodium, potassium and chloride transport (F, H, J), in the isolated perfusedrat kidneys. The columns and bars represent the mean ± S.E.M. for five rats. The results in samples treated with venom were compared to the control correspondingto the first 30 min of perfusion with Krebs-Henseleit solution before venom was added (black bar) (*p < 0.05 as compared to control).

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Fig. 2. Effects on cell death of the H. hypnale venom measured by Annexin V-PE and 7-AAD staining and detected by flow cytometry. MDCK (B, C) and LLC-MK2 (E,F) cells were treated with 1 CC50 of H. hypnale venom for 24 h. The control group corresponds to cells without venom treatment (A: MDCK, D: LLC-MK2). On the flowcytometric scatter graphs, the left lower quadrant represents the live cells, the right lower quadrant represents the population of early apoptotic cells, the right upperquadrant represents the accumulation of cells in terminal stage of cell death, and the left upper quadrant corresponds to the population of necrotic cells. Results in Cand F correspond to means ± S.E.M. of three replicates from three independent experiments (*p < 0.05 as compared to controls).

Fig. 3. H. hypnale venom effects on mitochondrial transmembrane potential. MDCK (A, B) and LLC-MK2 (C, D) cells were treated with 1 CC50 and 2 CC50 of H. hypnalevenom for 24 h. Then, cells were stained with rhodamine 123 (Rho123) and analyzed in the flow cytometer to measure the decrease in Rho123 accumulation inmitochondria. A and C show a histogram of representative mitochondrial transmembrane potential analysis in cell populations treated with venom. B and D presentthe fluorescence ratio relative to control ± SEM, of three replicates from three independent experiments (*p < 0.05 as compared to control).

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rapid cell loss in vivo, with a consequent electrolyte dysfunction, as acontributory factor to the development of AKI.

Experiments were also performed with Rhodamine 123 in order toassess the effect on the mitochondrial membrane potential and DCF toassess the ROS production. There was a drastic reduction in this po-tential and an increase on ROS generation, underscoring a process ofcell damage. The loss of mitochondrial membrane potential has beenassociated with the opening of the permeability transition pore in mi-tochondrial membranes (Bernardi and Rasola, 2007), a mega-channelthat can assemble as a consequence of mitochondrial calcium overload,owing to increments in cytosolic calcium concentration followingplasma membrane damage, and also following an increment in reactiveoxygen species in the cell (Rottenberg and Hoek, 2017). It is thereforesuggested that cytotoxicity induced by H. hypnale venom in these celllines is likely to depend on a direct action of venom cytotoxic compo-nents on the plasma membrane, with a disruption in its integrity. This isfollowed by a calcium influx which directly affects mitochondria andcontributes to the generation of reactive oxygen species, ending in ir-reversible cell damage. Owing to the high proportion of phospholipasesA2 in the proteome of this venom (Ali et al., 2013; Tan et al., 2015;Vanuopadath et al., 2018), it is proposed that venom cytotoxicity is dueto the action of this hydrolytic enzymes on plasma membrane phos-pholipids, a hypothesis that awaits the isolation of these components.

The antivenoms used in Sri Lanka and the Indian peninsula do notinclude Hypnale sp venom in their immunizing mixture, they are in-effective in envenomings by H. hypnale (Sellahewa et al., 1995; Keyleret al., 2013). Recently, a new polyspecific antivenom was developedwhich included the venom of H. hypnale in the immunizing scheme.This antivenom proved effective in the neutralization of lethal, he-morrhagic, in vitro coagulant, proteinase and phospholipase A2 activ-ities of H. hypnale venom (Villalta et al., 2016). It would be relevant to

assess whether this antivenom is effective at abrogating the renal effectsof the venom.

In conclusion, our findings provide evidence of a nephrotoxic actionof the venom of H. hypnale, as demonstrated by perturbation of severalrenal functions in an isolated kidney preparation and by a direct cy-totoxic effect on proximal and distal tubular epithelial cell lines. Theseexperimental observations are in line with previous experimental stu-dies describing nephrotoxicity by this venom, and by clinical reportshighlighting the potential of H. hypnale venom to induce AKI. Themechanisms of this pathophysiological effect and its neutralization byantivenoms deserve further investigations.

Declaration of interests

The authors declare that they have no known competing financialinterests or personal relationships that could have appeared to influ-ence the work reported in this paper.

Ethical statement

The experimental protocol used with rats was approved the EthicsCommittee on Animal Research (CEPA) of the Federal University ofCeara (Permit Number 79/08).

Acknowledgements

This study was supported by Vicerrectoría de Investigación,Universidad de Costa Rica (project No. 741-B4-102), and by theGraduate Studies System of this university for supporting a researchstay of M. Villalta at the Federal University of Ceará. Thanks are due toDaniel Keyler for fruitful discussions. This work was carried out by

Fig. 4. Intracellular ROS generation induced by H. hypnale venom (1 CC50 and 2 CC50) in MDCK (A, B) and LLC-MK2 (C, D) cells. After treatment with venom for24 h, cells were incubated with 5 mM DCFH-DA for 30 min and then immediately submitted to flow cytometric analysis. ROS generation was expressed as a ratio ofrelative fluorescent intensity compared to the control group. A representative histogram of three independent experiments for each sample is presented (A, C).Generation of ROS in the cytoplasm is demonstrated by an increment in fluorescence. Summarized data show intracellular ROS content, expressed as the ratio ofmean fluorescence of treated groups relative to that of untreated control (B, D). Data are means ± S.E.M. of three replicates from three independent experiments andcompared with sample without any treatment (*p < 0.05 as compared to controls).

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Mauren Villalta in partial fulfillment of the requirements for the PhDdegree at the University of Costa Rica.

Transparency document

Transparency document related to this article can be found online athttps://doi.org/10.1016/j.toxicon.2019.04.014.

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