Antibacterial therapeutics for the treatment of chytrid infection in amphibians: Columbus's egg?

Mariska Muijsers
An Martel
Pascale Van Rooij
Kris Baert
Griet Vercauteren
Richard Ducatelle
Patrick De Backer
Francis Vercammen
Freddy Haesebrouck
Frank Pasmans
Antibacterial therapeutics for the treatment of chytrid infection in amphibians: Columbus's egg?
R E S E A R C H A R T I C L E Open Access
Background: The establishment of safe and effective protocols to treat chytridiomycosis in amphibians is urgently required. In this study, the usefulness of antibacterial agents to clear chytridiomycosis from infected amphibians was evaluated. Results: Florfenicol, sulfamethoxazole, sulfadiazine and the combination of trimethoprim and sulfonamides were active in vitro against cultures of five Batrachochytrium dendrobatidis strains containing sporangia and zoospores, with minimum inhibitory concentrations (MIC) of 0.5-1.0 μg/ml for florfenicol and 8.0 μg/ml for the sulfonamides. Trimethoprim was not capable of inhibiting growth but, combined with sulfonamides, reduced the time to visible growth inhibition by the sulfonamides. Growth inhibition of B. dendrobatidis was not observed after exposure to clindamycin, doxycycline, enrofloxacin, paromomycin, polymyxin E and tylosin. Cultures of sporangia and zoospores of B. dendrobatidis strains JEL423 and IA042 were killed completely after 14 days of exposure to 100 μg/ml florfenicol or 16 μg/ml trimethoprim combined with 80 μg/ml sulfadiazine. These concentrations were, however, not capable of efficiently killing zoospores within 4 days after exposure as assessed using flow cytometry. Florfenicol concentrations remained stable in a bathing solution during a ten day period. Exposure of Discoglossus scovazzi tadpoles for ten days to 100 μg/ml but not to 10 μg florfenicol /ml water resulted in toxicity. In an in vivo trial, post metamorphic Alytes muletensis, experimentally inoculated with B. dendrobatidis, were treated topically with a solution containing 10 μg/ml of florfenicol during 14 days. Although a significant reduction of the B. dendrobatidis load was obtained, none of the treated animals cleared the infection.Conclusions:We thus conclude that, despite marked anti B. dendrobatidis activity in vitro, the florfenicol treatment used is not capable of eliminating B. dendrobatidis infections from amphibians.

Background

Amphibian medicine is a relatively young veterinary discipline and, so far, few studies have been carried out in order to develop evidence-based treatment protocols. Current treatment protocols against amphibian pathogens are most often based on empirical evidence and lack well supported scientific evidence. This major gap in veterinary science has garnered attention with the global emergence of the deadly fungal infection chytridiomycosis, a disease linked to mass mortalities and extinctions of amphibian species during the last decades [1,2]. The causative agent, Batrachochytrium dendrobatidis, is an aquatic pathogen with two life stages: a uniflagellated motile zoospore and an immotile reproductive zoosporangium [3]. In infected amphibians, B. dendrobatidis zoosporangia are found in the upper layers of the epidermis, causing hyperkeratosis and excessive shedding of the skin [1]. In more severe cases, B. dendrobatidis' capacity to disrupt normal regulatory skin functions (e.g. exchange of respiratory gases, water and electrolytes) causes electrolyte depletion and osmotic imbalance inducing clinical signs, like dehydration and anorexia, and death [4,5].

Controlling this disease and stabilizing populations of endangered amphibian species in captivity and in the wild has become a priority in amphibian conservation. The importance of clinical trials to develop an effective and safe treatment for chytridiomycosis has recently been put forward by Berger et al. [6]. At this moment minimum inhibitory concentrations in vitro against B. dendrobatidis have been determined for 10 antifungal agents: benzalkonium chloride (0.78 μg/ml), povidone iodine (312.5 μg/ml), amphotericin B (3.125 μg/ml), fluconazole (1.56 μg/ml), itraconazole (1.56 μg/ml), enilconazole (1.56 μg/ml), mercurochrome (6.25 μg/ml), sodium chloride (12.5 mg/ml), voriconazole (0.0125 μg/ml) and caspofungin (16 μg/ml) [7][8][9]. The use of these agents on a large scale in amphibians is hindered mainly due to toxic side effects, high prices or in vivo failure of activity [7][8][9][10]. Benzalkonium chloride, amphotericin B and fluconazole failed to clear B. dendrobatidis in vivo [7,8]. Moreover, amphotericin B was acutely toxic to Alytes muletensis tadpoles [8]. Formaldehyde and malachite green, even though found useful by Parker et al. [11], are extremely toxic, especially to tadpoles [12].

At present, especially itraconazole and voriconazole are antifungal agents of choice [8,10]. However, the treatment schedule of itraconazole is laborious and depigmentation of treated tadpoles has been observed [10]. Voriconazole, while apparently safe and effective, is quite expensive, the intravenous formulation hard to obtain and moreover, it is considered vital for the treatment of human patients with e.g. aspergillosis and therefore not the drug of choice for high scale use in veterinary medicine [8].

In 2009, Bishop et al. discovered the efficacy of chloramphenicol in the treatment of chytridiomycosis [13]. However, chloramphenicol is known to cause bone marrow toxicity in humans [14] and can also induce leukemia in amphibians [15]. The discovery that an antibacterial compound can be effective against B. dendrobatidis offers new opportunities for the development of a treatment protocol.

The aim of this study was to evaluate the usefulness of 10 antimicrobial agents for the treatment of chytridiomycosis. The minimal inhibitory concentrations were determined to make a first selection of in vitro efficacy. Florfenicol and trimethoprim sulfadiazine were further selected to determine the minimum fungicidal concentration and the time needed to kill the fungus in vitro. Finally, for florfenicol, stability, toxicity and treatment efficacy in midwife toads (Alytes muletensis), experimentally infected with B. dendrobatidis, were determined.

Methods

Strains and culture conditions

Five B. dendrobatidis strains, kindly provided by Dr. J. Longcore, Dr. T. Garner and Dr. M. Fisher, were used in this study ( Table 1). The strains were grown in TGhL broth (16 g tryptone + 2 g hydrolysed gelatin + 4 g lactose in 1 l distilled water) in 25 cm 2 cell culture flasks at 20°C for 5 days. For the collection of zoospores for the experimental inoculation, one ml of a 5 day old broth culture was inoculated on TGhL agar (16 g tryptone + 2 g hydrolysed gelatin + 4 g lactose + 10 g agar in 1 l distilled water) and incubated for 5 to 7 days at 20°C. Zoospores were collected by flooding the agar with 2 ml of distilled water and collecting the supernatant.

Determination of minimum inhibitory concentrations of antimicrobial agents

The minimum inhibitory concentration (MIC) of antimicrobial agents, each belonging to a different pharmacological group, for the B. dendrobatidis isolates were determined using a macrodilution method in 24 well plates following the method described by Martel et al.  and a visible increase in the number of fungal organisms. The MIC value was determined as the lowest drug concentration at which no growth of the B. dendrobatidis strain was visible after 14 days of incubation at 20°C using inverted microscopic examination. The experiment was carried out three times in triplicate for two strains (IA042 and JEL423). If growth inhibition was noticed for a given compound, the experiment was repeated for this compound for all five B. dendrobatidis strains (IA042, JEL197, JEL277, JEL310 and JEL423).

Determination of the time to 100% killing of B. dendrobatidis by florfenicol and trimethoprim-sulfadiazin

Based on the results of the MIC determination, florfenicol and the commercially available combination of trimethorpim and sulfadiazine were selected for further testing. The time to 100% killing of B. dendrobatidis by florfenicol and trimethoprim -sulfadiazine (TMP-S) was assessed in 24 well plates as described previously [8]. For florfenicol, the strains IA042 and JEL423 were exposed to concentrations equal to the minimal inhibitory concentration (1 μg/ml), 10 times the minimal inhibitory concentration (10 μg/ml) and 100 times the minimal inhibitory concentration (100 μg/ml) during 1, 2, 4, 6, 8, 10 and 14 days. For TMP-S, the 2 strains were exposed to concentrations of 1.6, 3.2, 8 and 16 μg/ml trimethoprim in combination with 8 μg/ml, 16 μg/ml, 40 μg/ml and 80 μg/ml of sulfadiazine corresponding to 1, 2, 5 and 10 times the minimal inhibitory concentration of sulfadiazine respectively, during 1, 2, 4, 6, 8, 10 and 14 days. Growth in the wells was compared to that in untreated control wells, allowing estimation of the percentage of growth reduction. After the exposure time, the medium was replaced by fresh TGhL broth without antimicrobials and the plates were further incubated for 14 days at 20°C. The time to 100% killing at a given antimicrobial concentration was defined as the earliest time point of medium replacement at which no growth of the strain was observed after 14 days of incubation at 20°C. All experiments were carried out twice in triplicate.

Killing capacity of florfenicol and trimethoprimsulfadiazine towards B. dendrobatidis zoospores

To determine the killing capacity of florfenicol and trimethoprim sulfadiazine towards B. dendrobatidis zoospores of strain IA042, the uptake of propidium iodide (PI) after exposure to florfenicol or trimethoprim sulfadiazine was assessed using flow cytometry as described previously [8]. A suspension containing approximately 10 6 zoospores/ml distilled water was exposed to either 1 μg/ml, 10 μg/ml or 100 μg/ml florfenicol or 1.6, 3.2, 8 or 16 μg/ml trimethoprim in combination with 8 μg/ml, 16 μg/ml, 40 μg/ml or 80 μg/ml sulfadiazine, and incubated for 1, 2 and 4 days at 20°C. The suspensions were transferred into Falcon tubes and PI was added to achieve a final concentration of 2 μg/ml PI. After incubation for 5 minutes at room temperature in the dark, the samples were analyzed using a FACSCanto flowcytometry system (Becton Dickinson Biosciences, Erembodegem, Belgium). Analyses were performed using FACSDiva software v5.0.1 (Becton Dickinson Biosciences). Vital zoospores were used to set light scatter gates for zoospore characteristics and zoospores killed with heat were used to set gates for PI positivity.

Florfenicol stability in water

Based on the results obtained, florfenicol was selected for further experiments. To determine whether florfenicol treatment should include daily replacement of the bathing solution, the concentration of florfenicol was measured for 10 days in 20°C bathing solution containing either 10 μg/ml or 100 μg/ml florfenicol. Water concentrations of florfenicol were determined using a HPLC method with ultraviolet (UV) detection. The samples were analysed after appropriate dilution on a Thermo Separations Product (TSP, Fremont, CA, USA) HPLCsystem using a Spectrasystem gradient pump, a Model AS 3000 autosampler and a Spectrafocus diode array detector set at 223 nm. A reversed-phase C 18 column (100 x 4.6 mm ID, 5 μm Nucleosil, Varian, Harbor City, USA) and a guard column of the same type were used. The injection volume was 50 μl, the flow rate was 0.35 ml/min and the run time was set at 13 min. The mobile phase consisted of 80% water and 20% acetonitrile (VWR, Leuven, Belgium) [16]. All samples were taken in triplicate.

Toxicity of florfenicol for tadpoles

Fifteen Discoglossus scovazzi tadpoles (Gosner stage 26-30) were individually housed in 600 ml of water at a temperature of 20°C in a room with a day-night cycle of 12 h:12 h and five animals were either exposed for 10 days to 10 μg/ml or 100 μg/ml florfenicol or not exposed to any antimicrobial drug. Animals were fed daily with commercial fish food pellets (Sera GmbH, Heinsberg, Germany) and weights were measured one hour before exposure and on day ten of this experiment. Daily observations for abnormal behavior, pathological signs or death were carried out. At the end of the experiment all animals were humanely euthanized and haematoxylin and eosin staining of paraffin embedded formalin fixed transversal whole body sections were examined for the presence of histological signs of toxicity. Statistical analyses of the results were performed using SPSS 17. This experiment was approved by the ethical committee of the Faculty of Veterinary Medicine, Ghent University.

Treatment of experimentally infected midwife toads (Alytes muletensis) with florfenicol B. dendrobatidis strain and growth condition

For the experimental infection, B. dendrobatidis strain IA042, isolated from a dead Alytes obstetricans, was used. The strain was grown on TGhL-agar for 5 days at 20°C. Subsequently, zoospores were collected by flooding the agar with 2 ml distilled water. Determination of zoospore density in the suspension was assessed using a Bürker counting chamber. The amount of zoospores in the suspension was then adjusted to approximately 10 7 zoospores/ml.

Experimental animals

All animal experiments were approved by the ethical committee of the Faculty of Veterinary Medicine (Ghent University). Experiments were performed following all necessary ethical and biosecurity standards. Twelve captive bred A. muletensis at approximately 6 months post metamorphosis were used in this experiment. The animals were kept in filter top cages (32 x 17x 21 cm) lined with moist tissue, containing terracotta flower-pots as shelter and a petri-dish filled with dechlorinated tap water for bathing and were fed fruit flies with calcium supplementation ad libitum. Ambient temperature varied between 16-20°C and was monitored using an automatic data logger device (Escort intelligent mini 3.0 V data recorder, Escort Data Loggers Inc., Buchanan, VA, USA).

Experimental inoculation and treatment

The animals were inoculated three times with a four day interval by topical inoculation of 0.1 ml of the zoospore suspension per animal. To determine whether the experimental inoculation resulted in infection, samples of the drinking patch and plantar sides of the feet were collected using rayon tipped swabs (160 C, Copan Italia S.p.A., Brescia, Italy) and examined after 1 week for the presence of B. dendrobatidis DNA using the qPCR test described by Boyle et al. [17]. As soon as all animals tested positive for the presence of B. dendrobatidis DNA twice with an interval of 1 week, the animals were divided into two groups. The first group of six animals served as untreated positive controls. The second group of six animals was treated daily with florfenicol at a concentration of 10 mg/l water. The treatment consisted of transferring the frogs to a disinfected container lined with tissue paper and daily spraying of the frogs and the container contents with the respective solution. Rayon tipped swabs were used to collect samples from the pelvic region and toes of all animals from the first day after treatment onwards. These swabs were examined for the presence of B. dendrobatidis DNA using the qPCR method mentioned above.

Results

B. dendrobatidis is susceptible to florfenicol and sulfonamides

Growth inhibition was observed after exposure to florfenicol or sulfonamides but not clindamycin, doxycyclin, paromomycin, polymyxin-E, trimethoprim solely and tylosin. Florfenicol had the lowest MIC for B. dendrobatidis with three strains (IA042, JEL197 and JEL310) being susceptible to a concentration of 1 μg/ml and two strains (JEL277 and JEL423) to a concentration of 0.5 μg/ml. Inhibition of the development of the spo rangia was visible at 4-5 days of exposure to these concentrations. For sulfamethoxazole, marked inhibition of sporangium development of B. dendrobatidis-strains IA042 and JEL423 was observed after 3-4 days of expo sure to a concentration of 8 μg/ml but already at 2 days after exposure to 8 μg/ml when combined with 1.6 μg/ml trimethoprim. Visible growth of all 5 strains tested was inhibited by 1.6 μg/ml trimethoprim in combination with 8 μg/ml sulfamethoxazole or sulfadiazine after 2 days of exposure.

Limited fungicidal activity of florfenicol and trimethoprim -sulfadiazine against B. dendrobatidis

Based on the results of the MIC determination, florfenicol and the combination trimethoprim sulfadiazine were selected to evaluate their fungicidal activity. At a concentration of 100 but not at 1 and 10 μg/ml of florfenicol, B. dendrobatidis cultures were completely killed within 14 days of exposure. At 10 μg/ml, sporadic viable zoospores were observed. For trimethoprim sulfadiazine, exposure to 16 μg/ml trimethoprim combined with 80 μg/ml sulfadiazine during 14 days was necessary to kill the B. dendrobatidis culture completely. Exposure to 3.2 and 8 μg/ml trimethoprim and 16 and 40 μg/ml sulfadiazine resulted in near absence of viable zoospores at 14 days. No significant killing was observed after exposure during 14 days to 1.6 μg/ml of trimethoprim combined with sulfadiazine at 8 μg/ml. No differences between the two tested strains (IA042 and JEL423) were observed. Compared to untreated zoospores, zoospores exposed to florfenicol at a concentration of 1, 10 or 100 μg/ml were not killed within 4 days, as assessed using flow cytometry. Exposure to trimethoprim sulfadiazine at a concentration of trimethoprim/sulfadiazine of 1.6/8 μg/ml, 3.2/16 μg/ml, 8/40 μg/ml or 16/80 μg/ml also did not kill zoospores within 4 days.

Florfenicol remains stable during at least 10 days in tank water

The quantification of the florfenicol concentration in water samples using HPLC showed no decrease of the concentration during the ten day period at both concentrations (10 μg/ml and 100 μg/ml), rendering florfenicol suitable for a single bath treatment.

Exposure of tadpoles to high doses of florfenicol results in decreased weight gain Clinical observation

No clinical abnormalities were observed in tadpoles exposed to either 10 μg/ml or 100 μg/ml florfenicol.

Weights Table 2 shows the average weights of the tadpoles at days 1 and 10 and the average weight gain during this ten day period. No significant differences were found between the weights of the different groups at the start of this experiment. Using one-way ANOVA and a

Bonferroni test significant differences between means were found for weights at day 10 and mean weight gain between the group treated with 100 μg/ml and the control group. Also, weights at day 10 and mean weight gain significantly differed between the group treated with 100 μg/ml and the group treated with 10 μg/ml. No significant differences were found between the weights at day 10 and the mean weight gain between the group treated with 10 μg/ml florfenicol and the control group that did not receive florfenicol treatment. Histopathological examination of the tadpoles revealed the absence of lesions in the negative control animals and mild multifocal vacuolization of epithelial cells in gut and kidneys of the animals exposed to 10 μg/ml. In the tadpoles exposed to 100 μg/ml, diffuse and marked vacuolization of epithelial cells in the gut with marked apoptosis of enterocytes and mild to moderate vacuolization of the renal tubular cells were present.

Fourteen day treatment with 10 μg/ml florfenicol reduces but does not eliminate B. dendrobatidis from experimentally infected postmetamorphic Alytes muletensis

Immediately before the experimental treatment, all animals tested positive on qPCR with an average load of 5,4 ± 4,4 B. dendrobatidis genomic equivalents (GE) in Table 2 Means of weights ± standard deviation at day 1 and day 10 and average weight gain ± standard deviation of Discoglossus scovazzi tadpoles (n = 5 per treatment group) during a ten day treatment experiment   (Figure 1). The average load of genomic equivalents was lower in the treated than in the untreated animals. The Mann-Whitney U test showed statistically significant differences between both the 10 and 14 day treatment group and the control group (P < 0.011 and P < 0.006 respectively). No significant difference was found between the 10 and 14 day treatment groups. After treatment termination, increase in the number of GE in the treated frogs was noted (Table 3).

Discussion

In this study, a range of antimicrobial agents were tested for their in vitro efficacy against B. dendrobatidis. Only florfenicol, a chloramphenicol analog, sulfonamides and a combination of trimethoprim and sulfonamides showed in vitro efficacy against B. dendrobatidis. The use of antibiotics and chemotherapeutics against fungal diseases is not a new phenomenon. In previous studies, researchers found minocycline, polymyxin as well as doxycycline to inhibit growth in Candida albicans and Candida tropicalis in vitro [18][19][20]. Fusarium showed in vitro susceptibility to tobramycin and moxifloxacin [21]. Although trimethoprim in se had no visible effect on in vitro growth of B. dendrobatidis, the combination with sulfadiazine resulted in earlier growth arrest, which was clearly visible as impairment of the development of mature sporangia. Both agents thus have a synergistic effect, probably exerted on folic acid synthesis as shown in many prokaryotic taxa [22][23][24]. Although, the in vitro results obtained with trimethoprim -sulfadiazine might seem promising, not only this combination is not stable in water according to the manufacturer (Kela laboratoria nv, Hoogstraten, Belgium), we also observed acute toxicity in 2 A. muletensis exposed to a concentration of 16 μg/ml of TMP-S (data not shown). Indeed, acute toxicity of TMP-S has been reported for other animal species as well [25,26]. We therefore excluded TMP-S from further experiments. Because of the relatively low MIC value, the near absence of toxic effects at a concentration of 10 x the MIC value and its remarkable stability in water, florfenicol was selected for treatment of experimentally infected A. muletensis. Although a significant reduction of the number of B. dendrobatidis organisms was achieved, the treatment protocol did not eliminate the fungus from its amphibian host, resulting in a rebound effect post treatment. This finding emphasizes the importance of treatments that completely eliminate the fungus from the amphibian host. The reduction in zoospore count in skin samples in florfenicol treated animals found in this study, is similar to that found in the study using chloramphenicol (Bishop et al., 2009). Probably, this is due to the fungistatic activity of florfenicol, resulting only in growth inhibition. Indeed, both florfenicol and the combination trimethoprim sulfadiazine completely killed cultures of B. dendrobatidis in vitro at very high concentrations only. Skin defenses in amphibians appear not capable of clearing growth impaired B. dendrobatidis cells, which results in recrudescence of the infection after termination of treatment. Another explanation might be poor penetration of florfenicol in the anuran skin. Since clear toxicity symptoms were present in the highest treatment group, sufficient absorption is probably present. However, pharmacokinetic data of florfenicol in amphibians are lacking.

Conclusions

In conclusion, the use of florfenicol applied in the bath water at 10 and 100 μg/l seems inappropriate to establish chytrid free populations in captivity. Also, the use of higher concentrations of florfenicol can be excluded because of the apparent toxicity at 100 μg/l. Further research with individual oral or parenteral treatments of florfenicol, longer treatment regimes and quantification of levels in skin tissue may be interesting, although impractical for treatment of a frog colony. However, since it is mandatory to eliminate B. dendrobatidis organisms completely from infected animals, application of voriconazole or itraconazole appears to be the only option at present to reliably control chytridiomycosis in infected amphibians.

(2010) [8]. To each well, 200 μl of TGhL broth containing various concentrations of clindamycin (Sigma-Alderich Chemi Gmbh, Steinheim, Germany), doxycyclin (Sigma-Alderich Chemi Gmbh, Steinheim, Germany), enrofloxacin (Bayer B.V., Diegem, Belgium), florfenicol (20%), paromomycin (Sigma-Alderich Chemi Gmbh, Steinheim, Germany), polymyxin E (V.M.D., Arendonk, Belgium), sulfamethoxazole (Sigma-Alderich Chemi Gmbh, Steinheim, Germany), trimethoprim (Sigma-Alderich Chemi Gmbh, Steinheim, Germany), a combination of trimethoprim and sulfamethoxazole in a ratio of 1:5, a commercially available combination of trimethoprim and sulfadiazine in a ratio of 1:5 (trimazin 30%, Kela laboratoria nv, Hoogstraten, Belgium) or tylosin (Sigma-Alderich Chemi Gmbh, Steinheim, Germany), were added to 200 μl of a 3 to 4 day old growing culture containing approximately 10 5 B. dendrobatidis organisms consisting of a mixture of zoospores and zoosporangia. Addition of 1600 μl TGhL broth to each well, resulted in final assay antimicrobial concentrations of 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.063, 0.031 and 0.016 μg/ml. In the two combinations of trimethoprim and sulfonamides this dilution resulted in final concentrations of trimethoprim/sulfonamide of 1.0.0031/0.016 μg/ml. The B. dendrobatidis cultures were examined for visible growth at 1, 2, 4, 6, 8, 10 and 14 days of incubation. Growth was compared to wells, containing B. dendrobatidis in TGhL broth without antimicrobial compound and was defined both as microscopically visible development of zoospores to sporangia,

Figure 1
1Boxplots of genomic equivalents (GE) of B. dendrobatidis in skin swabs from Alytes muletensis that were either not treated (control, determined at the end of the 14 days treatment period) or treated during 10 days or 14 days with florfenicol at a concentration of 10 μg/ml. skin swabs. After 14 days of treatment, all animals were still positive

Table 1
1Strains of B. dendrobatidis used in this studyStrain
Origin

JEL197
Dendrobatus azureus

JEL277
Ambystoma tigrinum

JEL310
Smilisca phaeota

JEL423
Lithobates catesbeianus

IA042
Alytes obstreticans

Table 3
3Mean load of genomic equivalents (GE) ± standard deviation of B. dendrobatidis in skin swabs collected from Alytes muletensis, experimentally inoculated with B. dendrobatidis, after a 14 days treatment period with 10 μg/ml florfenicolDays post
treatment

No. of
animals

No. of
positives

Genomic
equivalents

1
6
6
1.6 ± 1.5

10
6
6
5.1 ± 10.3

24
5
5
9.4 ± 7.1

http://www.biomedcentral.com/1746-6148/8/175
AcknowledgementsWe would like to thank Dr. J. Longcore, Dr. T. GarnerCompeting interestsThe authors declare that they have no competing interests.Authors' contributions MM carried out the in vitro as well as the in vivo studies, the statistical analyses and drafted the manuscript. AM participated in the design of the study and helped with the interpretation of results. PVR participated in both the in vitro and in vivo studies. KB and PDB carried out the HPLC tests. GV and RD participated in the pathological analyses of the toxicology studies. FV and FH participated in the design of the study and revised the manuscript. FP conceived of the study and participated in its design and coordination and helped to write the manuscript. All authors read and approved the final manuscript.
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Parameters of Oxidative Stress and Behavior in Animals Treated with Dexametasone and Submitted to Pentylenetetrazol Kindling

Edson Fernando
MDMuller Guzzo
Graduate Program in Biological Sciences: Physiology
Institute of Basic Health Sciences University

MDGabriel De Lima Rosa
Graduate Program in Biological Sciences: Physiology
Institute of Basic Health Sciences University

MDRafael Padilha Bremm
Faculty of Veterinary

MD, PhDCaroline Paula Meska
Graduate Program in Pharmaceutical Sciences
Federal University of Rio Grande do Sul

Medical Genetics Service
Hospital de Clínicas de Porto Alegre

MD, PhDCarmen Regla Vargas
Medical Genetics Service
Hospital de Clínicas de Porto Alegre

Graduate Program in Biological Sciences
Biochemistry

MD, PhDAdriana Simon Coitinho
Graduate Program in Biological Sciences: Physiology
Institute of Basic Health Sciences University

Department of Microbiology, Immunology and Parasitology
Institute of Basic Health Sciences
Department of Microbiology, Immunology and Parasitology
Institute of Basic Health Sciences
University Federal of Rio Grande do Sul
Porto AlegreBrazil

University Federal of Rio Grande do Sul
500 Rua Sarmento Leite, CEP 90050-170Porto AlegreBrazil

MDAdriana Simon Coitinho
Parameters of Oxidative Stress and Behavior in Animals Treated with Dexametasone and Submitted to Pentylenetetrazol Kindling

Original Article Journal of Epilepsy Research
Received April 27, 2021 Revised June 29, 2021 Accepted October 20, 2021This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Corresponding author:Oxidative stressPentylenetetrazoleDexamethasone
Background and Purpose:Oxidative stress (OS) is defined as an excessive production of reactive oxygen species that cannot be neutralized by the action of antioxidants, but also as an alteration of the cellular redox balance. The relationship between OS and epilepsy is not yet fully understood. The objective of this study was to evaluate the effect of dexamethasone on OS levels and memory in the kindling model induced by pentylenetetrazole.Methods:The animals were divided in six groups: control group that received no treatment, vehicle group treated with vehicle, diazepam group, and groups treated with dexamethasone (1, 2 and 4 mg/kg). Treated animals received pentylenetetrazole in alternated days for 15 days. Inhibitory avoidance test was conducted in 2 hours and OS was evaluated after animal sacrifice.Results:Regarding the treatment with dexamethasone, there was no significant difference when compared to the control groups in relation to the inhibitory avoidance test. On OS levels, there was a decrease in catalase activity levels in the hippocampus and an increase in thiobarbituric acid reactive substances and glutathione peroxidase levels in the hippocampus.Conclusions:The anticonvulsant effect of dexametasone remains uncertain. Immunological mechanisms, with the release of cytokines and inflammatory mediators, seem to be the key to this process. The mechanisms that generate OS are probably related to the anticonvulsant effects found. (2021;11:113-119)

Introduction

Oxidative stress (OS) is defined as an excessive production of reactive oxygen species that cannot be neutralized by the action of antioxidants, but also as an alteration of the cellular redox balance. The main reactive species (oxidants) associated with OS are: the superoxide anion radical (O2-), hydroxyl radical (OH), hydrogen peroxide (H2O2), nitric oxide (NO) and peroxynitrite (ONOO-). 1 Among the various neurological diseases, those of long duration, such as Alzheimer's disease, Parkinson's disease and Huntington's disease and multiple sclerosis, are associated with significant oxidative components. An emerging target in the regulation of OS and neuroinflammation in neurodegenerative diseases are given by the Nrf2/Keap1 pathway (the main metabolic pathway that regulates cytoprotective responses to oxidative and electrophilic stress). 2 The relationship between OS and epilepsy is not yet fully understood. Experimental models of epilepsy show an increase in OS biomarkers, demonstrating a correlation between OS and epileptogenesis. 3 OS and cerebral inflammation are two phenomena that are closely associated since they are interconnected and reinforce each other. Like brain inflammation, OS occurs quickly after epileptogenic brain damage and persists for some time. OS markers are increased in blood and brain tissues in human epilepsy. OS contributes to neuropathology and behavioral deficits associated with epilepsy and plays a determining role in the seizure threshold in animal models. 4 The hippocampus is a brain structure that has been widely studied for understanding the processes of epileptogenesis because it is a region involved in the onset of many epileptic seizures and it was the brain structure studied in this work. Despite the known influence of OS in epilepsy and epileptogenesis, the understanding of the mechanisms involved in this process is still not completely clear. Treatment with anti-inflammatory drugs, already used in some cases of refractory epilepsy, is still done empirically, so further studies are needed to understand the pathophysiological mechanisms involved.

Methods

Animals and treatment

Male Wistar rats were selected from the central vivarium at Universidade Federal do Rio Grande do Sul at 8-9 weeks of age (250-300 g). The animals were handled under standard laboratory conditions consisting of 12 hours light and 12 hours dark cycle and fixed temperature (22-24ºC), with free access to food and water. They were divided into six experimental groups (n=9-10 animals per group) that were treated for a period of 14 days on alternate days with group 1, 2 and 3 being the controls, where group 1 is the control group that received no treatment, group 2 that received saline (vehicle of the tested drugs) and group 3 that received diazepam (2 mg/kg), an anticonvulsant drug that is usually one of the first to be used in patients with epilepsy. Group 4, 5 and 6 were treated with the drug dexamethasone, steroidal anti-inflammatory drug, in different concentrations (1, 2 and 4 mg/kg, respectively). The animals in group 1 did not receive treatments and were considered the baseline group for the analysis of OS. Fig. 1 is a schematic figure explaining experimental protocol.

Evaluation of anticonvulsant activity

The kindling model, considered a chronic model of epilepsy, was used. 5 In this model, the animals received the same doses as described for the treatments for 14 days and, on alternate days, also received subconvulsant doses of pentylenetetrazole (PTZ) intraperitoneally (30 mg/kg). PTZ is a pro-convulsant agent, used to mimic experimental models of seizure in vivo, which acts through the inhibition of gabaergic receptors, thus promoting neural hyperexcitation by blocking the influx of Cl-and K+ 6 efflux. PTZ was injected 30 minutes after the treatments were administered. In these groups, the latency time for the onset of the epileptic seizure (of any kind or intensity) and animal mortality was observed. In a previous study, we have already published the scores presented by the animals according to the Racine scale.

Assessment of memory

In order to assess the work memory (short term memory, STM), the inhibitory avoidance test was used. The animals are placed on a platform (2.5 cm high by 8.0 cm wide) in an acrylic box with dimensions of 50×25×25 cm. The floor of this box consists of a box of parallel steel bars, with a distance of 0.2 cm between these bars. The time it takes for the animal to descend the platform and place the four legs on the floor of this box was counted. At this point, the animal received a shock in its 0.5 mA paws for 2 seconds. This first moment is called the training section. 8 The time it takes the animal to descend from the platform the first time was not used for any analysis or any kind of segregation. Immediately after the training session, the animals received the pharmacological treatments evaluated via i.p. Two hours after the training session, the animals were placed on the platform again and the time it takes the animal to make the new descent to the ground was counted. At this time the animal did not receive a new electrical discharge. With this test it was possible to check the STM. 8

Evaluation of antioxidant activity

After the end of the anticonvulsant evaluation, the animals were sacrificed by decapitation and the hippocampus was removed to assess the antioxidant activity in the control and treated groups. The samples were stored in a freezer -80°C until the tests were performed. The evaluation of antioxidant activity was carried out through the tests of lipid peroxidation, sulfhydryl groups, activity of the enzymes superoxide dismutase (SOD), catalase and glutathione peroxidase. In these evaluations, 5 to 8 animals were used per group.

Oxidative damage

Lipid damage was determined by the method based on thiobarbituric acid reactive substances (TBARS), which is widely used as a sensitive method for measuring lipid peroxidation, described by Wills. 9 The results are expressed in nmol TBARS/mg protein. The content of sulfhydryl groups, a measure of non-enzymatic cell defense, was determined through its reduction with 5,5′-dithiobis-2-nitrobenzoic acid (Ellman's reagent) producing a colored compound, read spectrophotometrically at 412 nm. 9

Total proteins

The determination of total proteins was carried out by the method of Lowry 10 and the values were expressed in mg/mL. This method has the advantage of its high sensitivity, being used for the determination of proteins in different tissues. 10

Superoxide dismutase activity

The activity of the SOD enzyme was measured spectrophotometrically in all samples as described by Bannister and Calabrese. 11 SOD can be measured indirectly, following the decrease in absorbance of oxidized adrenaline at 480 nm. The results were expressed in U SOD/mg protein. One unit is defined as the amount of enzyme that inhibits the speed of adenochrome formation by 50%.

11

Catalase activity

The measurement method for catalase activity (CAT) was that described by AEBI, 12 in which the enzyme catalyzes the decomposition of hydrogen peroxide into H2O and O2. The decomposition speed of H2O2 was measured spectrophotometrically at 230 nm for 180 seconds. The results were expressed in mmol H2O2/mg protein/minute. 12

Glutathione peroxidase activity

The activity of glutathione peroxidase (GPX) was described by Paglia, 12 in which the measurement occurred through changes in the absorbance of nicotinamide adenine dinucleotide phosphate (NADPH) at 340 nm. The results were expressed in nmol NADPH/mg pro-tein/minute.

Statistical analysis

The data were presented as mean and standard error. After defining the subgroups, statistical analysis was performed using analysis of variance followed by the Tukey post-hoc test. Results with p<0.05 were considered statistically significant (F-values are presented only if p<0.05). All statistical analyzes were performed using a database that was assembled in the SPSS version 17 statistical package (IBM Corp., Armonk, NY, USA).

Ethical considerations

In compliance with Law No. 11,794/2008, chapter IV, art.14, §4, the number of animals to be used for the execution of a project and the duration of each experiment were the minimum necessary to produce the conclusive result, saving to the maximum, the suffering animal. Ethical procedures for the care and use of animals were adopted according to the regulations published by the Brazilian Society for Neuroscience and Behavior. The present study is part of a larger project called "influence of inflammation on the epileptogenic process" that was approved by the Health Sciences Research Committee and the Ethics Committee on the use of animals at UFRGS on 12/18/2012.

Results

Behavioral data

Memory

In the animals treated with dexamethasone in all dosages, there  was no significant difference when compared to the vehicle group and the diazepam group in relation to STM. Thus, the proposed treatment did not alter the STM (Fig. 2).

Latency

As for the latency time, that is, the time the animal took for the first seizure, there was a reduction in the diazepam, DEXA 1, DEXA 2 and DEXA 4 groups when compared to the vehicle group. There was no significant difference between the DEXA groups and the diazepam group, indicating that DEXA, in all dosages, was able to increase the latency for the first crisis in a similar way to diazepam (Fig. 3). It is noteworthy that the control group did not receive PTZ, so there were no seizures in it.

Antioxidant activity

Regarding the evaluation of the antioxidant activity in the hippocampus of the animals (performed through the lipid peroxidation tests, sulfhydryl groups, SOD, CAT and GPX) and the lipid damage (determined by the TBARS method), the following results were observed.

TBARS

It was observed that the levels of lipid peroxidation, in the hippocampus, of the animals treated with dexamethasone 4 mg/kg were significantly elevated compared to the animals treated with dexamethasone 2 mg/kg (Fig. 4).

Sulfhydryl

As for sulfhydryl levels, an increase was observed in the groups treated with vehicle, diazepam and dexamethasone (in all dosages) compared to the baseline group. However, a reduction was also observed in the group treated with diazepam, DEXA 2 mg/kg and 4 mg/kg, compared to the vehicle group (Fig. 4).

SOD

An increase in superoxide dismutase activity was observed in all treated groups (vehicle, diazepam, DEXA 1 mg/kg, 2 mg/kg and 4 mg/kg) when compared to the baseline group. There was a decrease in the diazepam, DEXA 1 mg/kg and DEXA 2 mg/kg, compared to the DEXA 4 mg/kg group and a decrease in the DEXA 1 mg/kg group compared to the vehicle group. It was possible to observe a tendency to increase SOD activity in relation to the increase in the concentration of dexamethasone (Fig. 4).

CAT

A decrease in the CAT enzyme was observed in the groups treated with vehicle, diazepam and dexamethasone (in all dosages) compared to the baseline group (Fig. 4).

GPX

In the hippocampus, a significant decrease in the GPX enzyme was observed in animals treated with dexamethasone 1 mg/kg compared to the control group and the vehicle group. There was a slight, not statistically significant, increase in GPX activity in relation to increased dexamethasone concentration (Fig. 4).

Discussion

The excess of positive charges promotes depolarization of the neuronal membrane, which causes voltage-dependent calcium channels to open; calcium, in turn, is able to activate intracellular enzymes re-sponsible for triggering harmful processes, in addition to activating the enzyme phospholipase A2 (responsible for the metabolism of membrane lipids, which will trigger an inflammatory response) and mobilizing synaptic vesicles, thus triggering the synaptic processes that will generate neuronal hyperexcitability. This fine line between OS and neuroinflammation, predisposing to seizures, has already been well established. With the understanding of this mechanism, there is the possibility of using anti-inflammatory drugs as an alternative therapy for the treatment and in certain refractory epilepsies. It is strongly believed that OS has a great influence on the occurrence of seizures. [13][14][15] The animals' ability to memorize was tested in one moment. Steroid-specific receptors are known to be largely concentrated in the hippocampus, septal area and amygdala parts of the brain intimately involved in behavior, mood, learning and memory. Despite this, dexamethasone, a steroidal anti-inflammatory drug, acting on steroid receptors, has no deleterious effect on working memory in doses tested. 16 Continuing the behavioral analysis, the latency time for the first seizure was compared in the groups that received PTZ. It can be seen that the vehicle group, which did not receive any pharmacological treatment, had the shortest latency times as compared to the Diazepam control group and to all DEXA groups. Among the groups tested, there were no statistical differences between the mean latency times, allowing to state that DEXA, in all dosages, had an effect similar to diazepam in increasing latency for the first seizure. It has been seen in a model similar to this study that turmeric 16 was able to increase the latency time for the first seizure due to its anti-inflammatory and antioxidant effects. On the other hand, neuroprotective compounds such as yerba mate 17 and grape juice 18 were not able to produce this effect. In another animal model of epilepsy, the pilocarpine model, a similar result was found. The dose of DEXA used was 10 mg/kg and no one anticonvulsive drug was used as a control. There was a reduction in the latency time for the beginning of seizure demonstrating the possible anticonvulsant effect of DEXA on the animal model of pilocarpine. 19 As already mentioned, in a previous study we have already published the scores presented by the animals according to the Racine scale; however, the analysis of the latency for the onset of conclusive crises is of utmost importance for understanding the mechanism of action of this drug. It was possible to verify that DEXA acts in reducing not only the intensity of seizures, but also the onset time for seizures, reinforcing the possible anticonvulsant and neuroprotective role of this drug. 7 As for OS, no group differed from the control groups (vehicle and diazepam) in relation to TBARS levels; however, an increase in lipid peroxidation was found in the DEXA group 4 mg/kg compared to the DEXA 2 mg/kg group. The SOD enzyme catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide and because of this, it is an important antioxidant defense. The increase in this enzyme in all groups treated with PTZ compared to the baseline group, found in this study, is an unexpected result. The use of PTZ generated an increase in SOD. This increase was seen in all groups that received PTZ. Neither the use of diazepam nor DEXA at any concentration was able to reverse this increase and return levels to baseline values. A trend towards a reduction in SOD levels was seen with the use of DEXA at the lowest concentration, whereas at the highest dose, the levels remained as high as in the saline group. Unlike the result found in this study, Qi et al. 20 (2018) found decreased levels of SOD related to PTZ treatment in the brain of rats. This difference can be explained by the difference in the experimental design since in the mentioned study there was only one admin-istration of PTZ and in the present study the animals received PTZ for 2 weeks. This increase is probably generated by the chronic use of PTZ. It is noteworthy that the mentioned study evaluated the total brain tissue, and the present study the hippocampus. The increase found in the groups that received DEXA can be explained by the OS-inducing effect of this drug. 21 As for sulfhydryl levels, an increase was found in all treated groups when compared to the baseline group. This increase, caused by PTZ, was attenuated in the groups that received diazepam and DEXA at the two highest doses; however, there was no return to baseline values. In the DEXA 1 group, there was an increase compared to baseline, but it remained similar to the vehicle group, demonstrating that DEXA at this dose did not attenuate the OS caused by PTZ administration. There was a decrease in the CAT enzyme caused by the administration of PTZ. This decrease was not reversed by the use of DEXA or diazepam. Other studies demonstrate an increase in CAT related to PTZ administration both in the hippocampus 22 and in all brain tissue. 21,22 Reversal of this effect was found by other drugs such as metformin (first-line therapy for patients with type 2 diabetes since it has antioxidant, anti-inflammatory and neuroprotective properties) 20 and by compounds such as pycnogenol (Pinus pinaster bark extract, which contains flavonoids and has antioxidant, anti-inflammatory, neuroprotective and cardio protective properties), 21 the peony extract (extracted from the root barks, which has antioxidant and anti-inflammatory effects) 22 and the yerba mate (Ilex paraguariensis, anti-inflammatory effects). 17 Regarding the activity of the GPX enzyme, there was a tendency to decrease the activity with the use of DEXA, and the dose of 1 mg generated a significant reduction compared to the vehicle and control groups. In the other DEXA concentrations and diazepam-treated group, there were no significant differences compared to the baseline and control groups. Compensatory mechanisms can explain the results obtained, comparing the groups in which PTZ doses were administered in relation to the baseline group. As the enzyme activity of SOD increases, H2O2 can accumulate in the tissue. This accumulation could be metabolized by the enzymatic action of GPX and CAT, but there was little difference in the levels of GPX and decreased activity of CAT. In this situation, there would be a notable increase in H2O2 levels, which could (for example by the Fenton reaction) generate a high degree of hydroxyl ion concentration. However, the OS marker used (TBARS) showed no difference between the groups. Sulfhydryl groups were high compared to baseline. This may mean a non-enzymatic compensatory mechanism on redox signaling since the enzyme system was flawed, but there was no increase in TBARS levels among all groups. The anticonvulsant effect of DEXA remains uncertain. Immunological mechanisms, with the release of cytokines and inflammatory mediators, seem to be the key to this process. Mechanisms that generate OS may also be related to the observed effect. New studies are needed to investigate a new therapeutic approach in the treatment of epilepsy with this drug.

Conflict of Interest

The authors declare that they have no conflicts of interest.

Figure 1 .
1Schematic figure explaining experimental protocol. DEXA, dexamethasone; PTZ, pentylenetetrazole.

Figure 2 .
2Effect of treatment with dexamethasone in short term memory. The data are represented as mean±standard error (n=5-8 per group). There were no significant differences between groups (p=0.55, Kruskal-Wallis test). DEXA, dexamethasone.

Figure 3 .
3Effect of dexamethasone treatment on latency, for the onset of seizure. The data are represented as mean±standard error (n=5-8 per group) (*p<0.05 compared to the vehicle group; Friedman's test followed by Bonferroni's Post hoc ). PTZ, pentylenetetrazole.

Figure 4 .
4Anti-oxidative activity of dexamethasone in the hippocampus. The data are represented as mean±standard error (n=5-8 per group). (A) Lipid peroxidation (F4, 30=4.849, * p ≤0.05); (B) levels of sulfhydryl (F4, 30=4.541, p ≤0.001); (C) activity of the superoxide dismutase enzyme (F4, 30=31.630, p ≤ 0.05); (D) enzyme catalase (F4, 30=2.082, p ≤0.001); (E) enzyme glutathione peroxidase (F4, 30=31.723, p ≤0.05). Analysis of variance followed by post-hoc Tukey. TBARS, thiobarbituric acid reactive substances; DEXA, dexamethasone; DTNB, 5,5′-dithiobis-2-nitrobenzoic acid (Ellman's reagent); SOD, superoxide dismutase; CAT, catalase activity; NADPH, nicotinamide adenine dinucleotide phosphate; GPX, glutathione peroxidase.
AcknowledgementsThis work was supported in part by grants from Pró-Reitoria de Pesquisa UFRGS. The authors of the manuscript declare that they have no conflicts of interest.
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