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Cite this article: de Souza JM, da Silva WAM, de Oliveira Mendes B, Batista Guimarães AT, de Almeida SF, et al. (2016) Neurobehavioral Evaluation of C57BL/6J Mice Submitted to Tannery Effluents Intake. JSM Anxiety Depress 1(2): 1006.

*Corresponding authorGuilherme Malafaia, Programa de Pós-Graduação em Conservação de Recursos Naturais do Cerrado, Laboratório de Pesquisas Biológicas, Instituto Federal Goiano – Campus Urutaí. Rodovia Geraldo Silva Nascimento, s/n, Zona Rural, Urutaí, GO, Brazil, Tel: 55 64 3465 1996; Email:

Submitted: 06 May 2016

Accepted: 11 May 2016

Published: 13 May 2016

Copyright© 2016 Malafaia et al.

OPEN ACCESS

Keywords•Industrial waste•Animal model•Anxiety•Xenobiotics•Toxicity

Research Article

Neurobehavioral Evaluation of C57BL/6J Mice Submitted to Tannery Effluents IntakeJoyce Moreira de Souza1,2, Wellington Alves Mizael da Silva1,2, Bruna de Oliveira Mendes2, Abraão Tiago Batista Guimarães1,2, Sabrina Ferreira de Almeida2, Dieferson da Costa Estrela2,3, Anderson Rodrigo da Silva4, Aline Sueli de Lima Rodrigues2,3 and Guilherme Malafaia2,3,5,6*1Programa de Pós-Graduação em Conservação de Recursos Naturais do Cerrado, Instituto Federal Goiano, Brazil2Laboratório de Pesquisas Biológicas, Instituto Federal Goiano, Brazil3Departamento de Ciências Biológicas, Instituto Federal Goiano, Brazil4Departamento de Agronomia, Laboratório de Estatística Experimental, Brazil5Departamento de Ciências Biológicas and Programa de Pós-Graduação de Conservação de Recursos Naturais do Cerrado, Instituto Federal Goiano, Brazil6Programa de Pós-Graduação em Biodiversidade Animal, Universidade Federal de Goiás, Brazil

Abstract

The emission of tannery effluents into the environment may cause serious harm to human and environmental health. Nevertheless, few studies have investigated the possible effects of intake of these effluents in mammalian. Thus, this study aimed to evaluate the neurobehavioral effects of chronic intake of different tannery effluents concentrations (0.1%, 1% and 5%) (120 days) in male C57BL/6J mice. The animals were subjected to behavioral tests, predictive of anxiety, depression and memory deficits. By the elevated plus maze test was observed that the mice in the 5% of tannery effluents group showed higher anxiety scores. At neophobia test, all the animals exposed to chronic intake of tannery effluents showed higher latency time to start eating, which corresponds to an anxiogenic behavior. Regarding the forced swim test, it was observed that the animals of groups 1% and 5% of tannery effluents had longer in immobility behavior. Finally, the object recognition test showed that the treatments did not cause damage to the animals’ memory. The recognition rate of the new object did not differ among the experimental groups. Thus, it is concluded that male C57BL/6J mice exposed to tannery effluents have predictive neurobehavioral changes of anxiety and depression, with no memory deficit.

INTRODUCTIONIndustrial processes and human activities usually generate

specific waste, which are composed of different substances. Depending on the nature of these substances, such wastes may be harmful to the environment and human health [1,2]. Among the various types of waste generated to include those produced by profitable industrial activities, such as processing of bovine hide.

Although tannery activities generate significant profits, contributing to economic and social development of a country, such activities have been the subject of concern, mainly due to the generation of large amounts of waste/effluent during the processing of bovine hide. As discussed by Godecke et al. [3],

the leather tanning process requires several mechanical and chemical treatment processes that result in large amounts of waste with high concentrations of organic matter and various potentially toxic chemicals.

The amount of effluent generated is quite high, since, in the chemical process is spent a considerable amount of water, 20-35 m³ for each kilo of processed skin [4-6]. According to Buljan [7], it is estimated that worldwide per year, considering all existing tanneries, is generated around 300 million tons of waste water.

The problems linked to the generation of large amounts of this effluents are intensified by the fact that in many tannery industries there is disposal of waste directly into water bodies

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without any treatment, which implies a high risk of environmental contamination [3,8,9]. Even after the treatment in a particular installation, it was observed that tannery residues, including effluents, exhibit considerable organic and inorganic fillers, such as acids, phenols, sulfates and sulfides, as well as highly toxic elements such as chromium, which is used during the tanning process [10,11].

Studies in humans have shown that analysis of loss of health in workers exposed to organic solvents and heavy metals attached to effluents or tannery waste, is a widely discussed topic by the occupational toxicology field. Salazar [12], analyzing the impact on the environment and health of the Mexican workers from tannery that use chromium, shows that there is no ongoing training for workers, much less hygiene guidelines and security of these individuals. Cuberos et al. [13] emphasize that the aggravations caused by exposure to chromium cause otorhinolaryngological, dermatological and ophthalmological changes. Shahzad et al. [14] found a high prevalence of occupational asthma in workers of Pakistan’s leather industry and Chandrasekaran et al. [15] found a decrease in lung function of Indian workers. According to Das et al. [16] and Greene et al. [17], the exposure to different types of chemicals used in the tanning industry also causes reproductive health problems.

In the experimental field involving ecotoxicology related to tannery effluents, it has already been demonstrated teratogenicity in species of sea urchin, reducing the growth of microalgae and a variety of toxic effects on microcrustaceans [18]. Other studies involving exposure of fish, plants and bacteria to tannery effluents have also shown harmful effects [19-23].

However, it is important to consider that these organisms are suitable for determining, e. g., the lethality, but does not allow evaluation of signs and symptoms observed in mammals to be accomplished, opening a gap in the knowledge that deserves investigation. Regarding the effects of exposure to tannery effluents in mammalian experimental models, highlights only the studies of Kumar et al. [24], Siqueira et al. [25], Moysés et al. [26], Silva et al. [27], Lemos et al. [28], Ferreira et al. [29] and Rabelo et al. [30]. In the pioneering study by Kumar et al. [24], while chronically exposing male Wistar rats to tannery effluents, the authors observed increased mass of all androgen-dependent organs evaluated, such as the prostate and seminal vesicle, and have evidenced deformations in the seminiferous tubules with testicular hyperplasia signals. Associated with this, increases were observed in daily sperm production and serious levels of testosterone, these results are attributed to androgenic effects of one or more xenobiotic present in the used tannery effluents.

In the study by Siqueira et al. [25], the authors demonstrated that male Swiss mice (adults) exposed to 1% concentration of untreated tannery effluent, diluted in water for 15 days, showed predictive behavior for anxiety, suggesting that this type of effluent affects negatively the animals’ central nervous system. However, Moysés et al. [26] investigated possible hepatotoxic and neurological effects induced by chronic exposure to tannery effluents and predictive behaviors for anxiety, depression and memory deficits in adult male Wistar rats. These authors did not observe any change in the evaluated parameters and suggest that the experimental model used in the study may not be appropriate

for toxicological studies involving tannery effluents. Rabelo et al. [30] demonstrated that the exposure to tannery effluent caused memory deficit in Swiss mice in a similar way for both sexes, reinforcing previous findings that these pollutants affect the central nervous system.

Moreover, the studies by Silva et al. [27], Lemos et al. [28] and Ferreira et al. [29] evaluated lethal doses of tannery effluents diluted in water at different concentrations, using different mice strains and sex. Although they are considered pioneering studies related to the evaluation of acute toxicity and determination of the legal dose of tannery effluent in mice, these studies did not investigate any neurobehavioral effects in animals related to neuropsychiatric disorders such as anxiety and depression, neither to memory deficits.

In this context, this study aimed to assess the neurobehavioral effects of chronic exposure of different concentrations of tannery effluents diluted with water in C57BL/6J mice, seeking to expand the knowledge about the relationship between exposure to these xenobiotics and neuropsychiatric and cognitive disorders. It started from the hypothesis that chronic ingestion of tannery effluent diluted with water could induce predictive behavioral changes for anxiety, depression and memory deficits in male mice of the C57BL/6J strain.

MATERIALS AND METHODS

Animals and experimental groups

The herein presented study used male C57BL/6J mice to assess neurobehavioral effects of chronic exposure to tannery effluents. While it is known that both males and females can have access to water contaminated by tannery effluents, the choice to using mice males is due to the fact that several studies have pointed to the higher prevalence of men as workers in leather processing industries [16,17,31-33]. This finding puts males at the greater risk group for the effects of exposure to tannery effluents.

32 animals (21 and 30 days) were divided into four groups: control group, in which the animals received only fresh water containing 0% of untreated tannery effluent (see characterization in Table 1), and 0.1%, 1% and 5% groups, receiving raw tannery effluent diluted in fresh water. The concentrations of 0.1%, 1% and 5% correspond respectively to 1/600, 1/60 and 1/12 of the median lethal dose (LD50) given by the oral route to mice, as Ferreira et al. [29]. The fresh water used in this study came from the water treatment plant located on the dependences of Instituto Federal Goiano (IF Goiano) - Câmpus Urutaí (Urutaí, GO, Brazil).

The animals used in the herein study were kept in the Animal Facility of the Biological Research Laboratory at IF Goiano – Câmpus Urutaí (Urutaí, GO, Brazil) in sanitation conventional animal room with controlled temperature (22 to 24ºC) and luminosity (12h light cycle). The animals were kept in collective standard mice polypropylene crates (30 x 20 x 13 cm) with latticed galvanized wire caps with antioxidant treatment with a maximum of 4 animals each. The crates were cleaned three times a week, with change of sawdust and food. The standard rodent diet (Nuvilab CR 1) and water (with or without tannery effluent)

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Table 1: Characterization of raw tannery effluent and fresh water used in the herein study.Parameters1 Tannery Effluent Fresh Water

pH at 25ºC (UpH) 8.19 7.19

Turbidity (NTU) 382.00 <1.00

Turbidity ammonia-nitrogen (mg.L-1) 2.10 0.01

Total Nitrogen (mg.L-1) 110.00 1.20

Nitrate (mg.L-1) 23.00 0.30

Electrical conductivity 25º C (µS.cm-1) 72.10 52.00

Total Phosphorus (mg.L-1) 33.61 0.11

Orthophosphate (mg.L-1) 77.09 0.26Biochemical Oxygen Demand (BOD) (mg.L-1) 9.333,33 0.50

Total Solids (mg.L-1) 82,190.00 30.00

Copper dissolved (mg.L-1) <0.01 0.04

Manganese dissolved (mg.L-1) <0.10 ND

Dissolved Iron (mg.L-1) 1.91 0.09

Zinc (mg.L-1) <0.01 1.06

Sodium (mg.L-1) 5,680.00 5.01

Magnesium (mg.L-1) 243.20 1.21

Calcium (mg.L-1) 2,805.00 4.00

Sulfur (mg.L-1) 833.33 1.00

Potassium (mg.L-1) 122.00 1.60

Total organic carbon (TOC) (mg.L-1) 93.32 8.201Analysis of raw tannery effluent and fresh water was performed according to the methodology recommended by the American Public Health Association [34]. All analyzes were performed in a commercial laboratory in Goiania, GO, Brazil

were offered ad libitum. It is noteworthy that all the procedures adopted in this study were approved by the Ethics Committee on Animal Use from IF Goiano, GO, Brazil (protocol n. 17/2014). All efforts were made to minimize the number of animals employed in the present study and their suffering.

The effluent supplied by tannery industry did not contain chromium, because it was obtained in the removal step of bovine skin, in other words, the step prior to the leather tanning stage, where large amounts of chromium salts are commonly used [34]. Is was decided to use this type of effluent, whereas many tannery industries make the disposal of this waste directly into water bodies bordering their properties, mainly because this effluent does not contain the element chromium. According to the grantor effluent industry, used in this study, the following chemicals were used in the removal step of bovine skin: sodium hydrosulfide, sodium hydroxide, dimethyl sulfate, thioglycolic acid and sodium glycolate.

The animals in the 0.1%, 1% and 5% groups of tannery effluent diluted in water were chronically exposed for 120 days. The water consumption or effluent diluted with water was not measured due to the viscosity of the effluent diluted with water (related to its chemical composition). The minimal contact with the drinking fountain or the handling of the crates by the handlers was enough to make water easily exit the drinking fountain. Thus, the measurement of fluid intake would not be very reliable.

Whereas the consumption of liquids was not measured in this study, conducted clinical analysis of a possible dehydration of the mice, since the animals, especially the 0.1%, 1% and 5% tannery effluent groups could have an aversion to the taste of contaminated water and it would lead consequently to lower consumption, and possible dehydration. To this, was evaluated clinically the moisture of the animals’ eyes and oral cavity (one of the most widely used indicator of dehydration), the presence of sunken eyes and loss of natural luster, lethargy on the crates, the absence of food intake (although this parameter has not been quantified) and immobility of the animals in the crates. All these evaluations were performed during the trial period. In addition, the animals had their body masses were measured at the beginning and end of the experiment.

From the 111th experimental day, the animals were subjected to different predictive neurobehavioural tests for anxiety, depression and memory deficit (Figure 1), considering the possibility that the contaminated water with tannery effluents, as discussed by Siqueira et al. [25] and Moysés et al. [26], could lead to damage the animals’ nervous system.

Elevated plus-maze (EPM)

The EPM test has been widely used to measure anxiety in rodents [35-40]. The plus maze consists of two opposing open arms (30 x 5 x 25 cm) and two opposing closed arms (30 x 5 x 25 cm) extending from a common central platform (5 x 5 cm). The apparatus used was made of wood and elevated to a 45 cm height above the floor level. To facilitate the operation, an edge (0.25 cm) surrounded the open arms. Behavioral rehearsal room was soundproof and the illumination level was maintained at 100 lx. All experimental groups stayed for 30 min in the testing room environment for acclimatization before the test sessions. Then, each animal was placed individually in the center of the EPM, facing an open arm, the animal being allowed to freely explore the apparatus for 5 min. All mice were tested only once. Before each test session, the EPM was cleaned with 70% ethanol. The anxiety index was calculated according to Cohen et al. [41], Contreras et al. [42] and Estrela et al. [43] as follows: Anxiety index = 1 - [([time the animal stayed on the open arms, in seconds / test duration in seconds (300 s)] + [input frequency on the open arms / total number of entries]) / 2]. Furthermore, was registered the percentage of entries into the open arms and the percentage of time of the animals in the open arms, considered a primary predictive parameter for anxiety evaluated in the EPM, according to Rodgers &Dalvi [36] and Walf& Frye [38]. It is noted that the total number of entries was defined as the sum of the frequency with which the animal entered in the open and closed arms, and is considered an input when the four animals’ paws overtake the initial limit of the arm.

Open-field test

The apparatus of the open-field test consisted of a circular arena, diameter 28 cm, surrounded by a circular wall, high 45 cm, divided into 12 quadrants. The animals were placed individually in the center of the arena and videotaped for 5 min (with a camera above the apparatus). The ratio (in percentage) of locomotion in the central quadrants/total of locomotion was calculated. According to Prut & Belzung [44], a lower percentage of movement in the central quadrants, and consequently, a high

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Figure 1 Temporal distributions of the tests conducted on male C57BL/6J mice exposed to different concentrations of tannery effluents diluted with water. The behavioral tests were performed with an interval of two days between one test and another.

A) B)

Figure 2 (A) Body mass and (B) body mass gain of male C57Bl/6J mice that were exposed or not to chronic ingestion of different tannery effluent concentrations dilutes with water. C: control group; 0.1 and 5%: groups exposed to 0.1%, 1% and 5% of tannery effluent diluted with water intake; s: start of the experiment; e: end of the experiment. The bars indicate the mean + standard deviation. The data presented were obtained from an experiment with n = 8 per group. In “A”, the initial and final data of each experimental group were compared using Student’s t-test at 5% probability. The asterisks indicate differences between the final and initial weights of each experimental group. For analysis of body weights among the experimental groups, both initially as the end of the experiment, it was used analysis of variance (one-way ANOVA) with Tukey post-test at 5% probability. Different letters indicate significant differences among the experimental groups. Lowercase letters comparing the different groups at the start of the experiment. Uppercase compare the groups at the end of the experimental period. In “B”, the data on the body mass gain were subjected to analysis of variance (one-way ANOVA) with Tukey post-test at 5% probability.

percentage of movement in the lateral quadrants can be used as an anxiety index.

Neophobiatest

In the neophobia food test, mice were food deprived (16 h) and then placed in an unfamiliar box that contained standard rodents’ food. For 5 min the animals were videotaped. We evaluated the latency to start feeding, considered a predictive parameter for anxiety, as discussed by Chen et al. [45].

Object recognition test

The object recognition test was performed in the arena of open environment, according to the methodology used by Moysés et al. [26]. In the training session, the animals were

exposed to two identical objects (F1 and F2), called familiar objects (F), in which was evaluated the exploration time for each object. As it reaches the exploration time of 30 seconds (in both the objects), the animal was removed from the arena. 24 hours after the training session (long-term memory), one of the familiar objects (F) was replaced by another object [new object (N) – with different color, texture and size of the objects used in the training session], being evaluated by the 5 min time exploration of the objects. Exploration was taken as acts of smell and touch the object with the nose or front legs [46]. The recognition rate of each object was calculated for each animal, as described by Pietá-Dias et al. [47] and expressed by the ratio TOX / (TF + TN) [TOX = time spent exploring the familiar (F) or new (N) object; TF = time spent exploring the familiar object; TN = time spent exploring

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the new object]. Between one session and another, the arena was cleaned with 70% alcohol.

Forced swim test

The forced swim test consisted in place the mice individually in a cylindrical tank (height 18.5 cm, diameter 12.5 cm) containing water at 25 °C (13.5 cm depth). After the test (lasting 6 minutes), the mice were removed from the water and dried in heated light before being returned to their crates. All test sessions were videotaped with a video camera located 30 cm above the tank, aiming to allow further evaluation of the time spent in immobility behavior, commonly used as a predictor for depression in the forced swim test [48-51]. Immobility was defined as the absence of movement of the whole body of the animal, when the mouse stopped fighting and kept motionless, floating in water, or when the animal was only doing necessary movements to keep its head above the water. Immobility behavior were recorded during the first two minutes of the test since, according to Costa et al. [51], these first minutes are considered a good time window to evaluate the effects of antidepressants in mice.

Statistical analysis

Initially the residual normality of all data was verified using the Shapiro-Wilk test and Bartlett’s test was used to check the homoscedasticity of the data. Then, the data related to behavioral parameters were submitted to one-way analysis of variance (one-way ANOVA) with Tukey post-test at 5% probability, using the ASSISTAT software, version 7.7 beta (distributed for free). For all the behavioral tests, the videos were watched by two trained observers, and the same video was analyzed twice, yielding a higher intra-observer agreement at 85%. The analysis of behavioral parameters observed in the EPM test was performed using the software PlusMZ and in the open-field test used the OpenFLD software. The initial and final body masses of the animals were compared using the Student’s t-test at 5% probability.

RESULTS AND DISCUSSIONBoth during the experimental period and the end of the

experiment, the clinical evaluation of the animals did not show signs or symptoms related to dehydration, ruling out the possibility that the animals could have lower fluid intake due to the treatments. Moreover, the physical examination of the animals showed a significant body weight gain of the animals of all groups, when compared to initial and final masses (Figure 2A). This result already expected, considering the growth/development of animals during the experimental period, since, began the exposure of the animals in the juvenile period. However, the body mass gain of the different experimental groups did not differ statistically (F3,28 = 0.115, p = 0.950) (Figure 2B), suggesting that the treatments did not affect the growth/development of the animals.

In the end of the experiment, the animals that were exposed to 1% and 5% of tannery effluents had lower body mass compared to the others experimental groups (F3,28 = 3.236, p = 0.037) (Figure 2A). These results suggest that exposure to tannery effluent in concentrations of 1% and 5% can cause physiological changes related to weight loss, such as the decrease in animal

feeding behavior, although the daily intake ratio of the mice has not been quantified in the present study.

Despite the existing studies on intake tannery effluents in mammalian models [25,26,30] have not estimated physical parameters in the animals, it is known that the weight change is one of the most used parameters in toxicological evaluation to indicate the onset, often premature, toxic effects of a particular substance in the animal organism [52]. Likewise, studies have evaluated the body weight gain and relative weights of organs in researches that aimed at evaluating the effect of certain substances in the body in the medical, food or environmental toxicology field [16,53-56].

Regarding the behavioral tests, the first test was the EPM test. The results revealed that the animals that received fresh water containing 5% of tannery effluent showed a higher rate of anxiety (F3,28 = 3.136, p = 0.035), lower percentage of open arm entries (F3,28 = 4.254, p = 0.028) and lower percentage of time spent in the open arms (F3,28 = 4.357, p = 0.029) compared to the animals that received only fresh water (Figure 3), consistent results with a anxiogenic behavior of the animals.

These results are similar to those disclosed by Siqueira et al. [25], which when the male Swiss mice (3 months old) were submitted to tannery effluent intake in concentration of 1% showed anxiogenic behavior in the animals ingesting untreated effluent diluted with water. However, it is important to note that the effluent used in the study by Siqueira et al. [25] was collected during the bovine leather tanning stage, unlike the effluent used in this study, which was obtained from the removal step of bovine skin. Although the authors have not provided information on the chemical composition of the effluent used, according to Siqueira et al. [25] the effluent used contained high organic and inorganic loads and high concentration of chlorides and chromium salts. Another difference between this study and the work of Siqueira et al. [25], refers to the exposure period. While the authors offered tannery effluent diluted with water to mice for a period of only 15 days, the animals in this herein study were exposed for 120 days.

Moreover, this study differs from the study by Moysés et al. [26], who found that in male Wistar rats (3 months old) exposed to different concentrations of tannery effluent (0.1%, 1% and 5%) also diluted with water, no changes were observed in any of the behavioral parameters evaluated during the EPM test. Besides the differences between the types of effluents used in this herein study and the study by Moysés et al. [26], and the period of exposure of animals, we must consider that the physiological and biochemical differences between the investigated rodents species, are also important aspects that can explain the differences observed between the studies. As discussed by Moysés [57], metabolic differences observed in rats and mice, with possible formation of more toxic metabolites in mice, is an important hypothesis that could explain the differential effects observed in the species investigated. Studies such as those by Mitchell et al. [58], Nogueira et al. [59] and Meotti et al. [60], subsidize this hypothesis by showing that physiological differences between species can result in variations in the forms of metabolizing drugs or xenobiotics. Another hypothesis that could explain the discrepancy between studies relates to the genetics of the animals investigated. While in the present study

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A)

C)

B)

Figure 3 (A) Anxiety rates, (B) percentage of open arm entries and (C) percentage of time spent in the open arms in the elevated plus maze by male C57Bl/6J mice exposed or not to chronic ingestion of different tannery effluent concentrations dilutes with water. Different letters indicate significant differences among the experimental groups. Data were subjected to analysis of variance (one-way ANOVA) with Tukey post-test at 5% probability The bars indicate the mean + standard deviation. n= 8, for each of the experimental groups.

Figure 4 Ratio (in percentage) of the locomotion in central quadrants / total locomotion in the open-field test by male C57Bl/6J mice exposed or not to chronic ingestion of different tannery effluent concentrations diluted with water. The absence of letters over the bars indicates no statistical difference among the experimental groups for behavioral parameter measured. Data were subjected to analysis of variance (one-way ANOVA) with Tukey post-test at 5% probability The bars indicate the mean + standard deviation. n= 8, for each of the experimental groups.

we used an isogenic strain of mice (C57BL/6J), in the study by Moysés et al. [26] the authors used a strain of heterogenic rat (Wistar), showing that this last strain as being naturally more resistant, cannot be considered a good model for ecotoxicological investigations involving tannery effluents.

The second performed test was the open-field, which evaluates changes in the animals’ locomotor and exploratory activity, correlating them with emotional disturbances such as fear and anxiety in rodents [44,47,61,62]. The results of this herein study

revealed no differences among the experimental groups in the proportion (percentage) of locomotion in the central quarters / total locomotion calculated, considered an indirect measure for anxiety (F3,28 = 4.254, p = 0.280) (Figure 4). Therefore, this evaluation did not indicate anxiogenic or anxiolytic behavior in the animals subjected to the different treatments.

In this herein study, it is noted that the EPM and open-field test, show different results. While the EPM test demonstrated anxiogenic behavior in the animals subjected to 5% of tannery effluent diluted with water intake; in the open-field test, were not disclosed anxiogenic or anxiolytic effects in the animals due to the treatments, a fact that may be explained by the nature of the different tests. As discussed by Carola et al. [63], the open-field test is more appropriate to evaluate the levels of motor activities (mechanical components) and less sensitive to evaluate the psychomotor behavior in rodents as compared with the EPM test, although both tests may be used to evaluate predictive behaviors for anxiety.

Komada et al. [39] point out to the fact that instead of manage to the behaviors directly related to anxiety, the open-field test measures the animals’ anxiety in open spaces (open-space anxiety behavior), which is not the case of the EPM test. The elevated plus maze test is based on exploratory behavior of rodents and its natural aversion to open spaces, which usually causes fear and anxiety [35,38]. This well-established paradigm has a long history of successful evaluation of anxiety behavior in mice or rats [35,36,64,65]. The test exploits the natural tendency of rodents to explore new environments, being given to the rodent the option to spend time in open arms, the unprotected arms of the maze, or enclosed, the protected arms, all raised to a

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considerable height from the ground. Rodents tend to avoid open areas, especially when they are illuminated, preferring darker and enclosed spaces. This conflict results in prevention approach behaviors that were correlated with increases in the physiological stress indicators [66]. Opposed to that, the administration of benzodiazepines and other anxiolytics treatments result in increased exploration of the open arms, without affecting the general motivation or locomotion [64].

Furthermore, some authors as Tanaka et al. [67], suggest that the open-field test cannot evaluate comprehensively all xenobiotics related to anxiogenic and anxiolytic effects, since it is not sensitive to compounds as alprazolam, an agent effective in diseases such as panic attacks, obsessive-compulsive disorder, social anxiety disorder and post-traumatic stress disorder. However, the open-field test appears to be sensitive to the effects produced by classical anxiolytic benzodiazepines and 5-HT1A receptor agonists [44].

In neophobia test was evaluated the latency of onset of food intake by the mice, which were subjected to a fasting period and were exposed to a new environment where there was food available to then. This test creates a conflict between eating behavior and the natural aversion of rodents facing to a new environment. The treatment with a variety of drugs used to control anxiety in humans reduces the latency time to start feeding [68,69]. It was noticed in this herein study that the latency time to start feeding differ among the experimental groups (F3,28 = 7.039, p = 0.015). The mice of the 0.1%, 1% and 5% groups showed higher latency for the initiation of feeding (in seconds), a result consistent with an anxiogenic behavior in these animals (Figure 5). The results herein also differ from those which were obtained in the study by Moysés et al. [26], which also made use of this test. Probably these results are due to different factors related to the experimental design by Moysés et al. (2014), such as rodent species used (Moysés et al. [26]: Wistar rats; Herein: C57BL/6J), period of exposure to tannery effluent (Moysés et al [26]: 28 days when performing the neophobia test; Herein: 115 days of exposure), as well the type and chemical composition of the effluents used (Moysés et al. [26]: treated and untreated tannery effluent, arising from leather tanning processing stage; Herein: untreated tannery effluent, arising from the liming stage), as well as the aspects discussed above.

Regarding the forced swim test, which is a predictive depression test in laboratory animals [70], it was observed that the animals of 1% and 5% of effluent tanning diluted with water groups showed longer time in immobility behavior in comparison with the control group (F3,28 = 7.036, p = 0.014) (Figure 6). Thus, these data show a predictive depression behavior in animals exposed to higher concentrations of tannery effluents diluted with water (1% and 5%), diverging from Siqueira et al. [25] and Moysés et al. [26]. In these studies, the authors suggest that tannery effluent rich in chrome (in short exposure time) does not induce in the animals predictive depression behavior in Swiss mice and Wistar rats respectively.

Although it was not specifically investigated the mechanisms by which the treatments caused a predictive depression behavioral in the animals, it can be assumed that the constituents

of tannery effluent have caused physiological changes in the functioning of the hypothalamic-pituitary-adrenal axis (HPA axis) or in the serotonergic neurotransmission, these aspects are directly linked to the pathogenesis of depression 71. On the other hand, considering that depression is associated with low brain levels of serotonin, it is assumed that the treatments (1% and 5% of tannery effluent) have affected the production or distribution of this neurotransmitter. Anyway, it is recommended that further investigations be carried out to elucidate the endocrine or neural mechanisms related to these results.

The object recognition test showed that the treatments did not cause damage to the memory of the animals (Figure 7). Regardless of the experimental group, all animals showed higher recognition rate of the new object (N) relative to the familiar object (F) and the new object recognition rates did not differ among the different treatments (Figure 7).

Figure 5 Latency time to start feeding by male C57Bl/6J mice exposed or not to chronic ingestion of different tannery effluent concentrations dilutes with water. Different letters indicate significant differences among the experimental groups. Data were subjected to analysis of variance (one-way ANOVA) with Tukey post-test at 5% probability The bars indicate the mean + standard deviation. n= 8, for each of the experimental groups.

Figure 6 Duration (in seconds) of immobility behavior recorded during the first two minutes of forced swim test in male C57Bl/6J mice exposed or not to chronic ingestion of different tannery effluent concentrations dilutes with water. Different letters indicate significant differences among the experimental groups. Data were subjected to analysis of variance (one-way ANOVA) with Tukey post-test at 5% probability The bars indicate the mean + standard deviation. n= 8, for each of the experimental groups.

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Siqueira et al. [25] evaluated only the effects related to anxiety and depression in male Swiss mice. However, the data from this study are similar to those obtained by Moysés et al. [26], which showed that exposure of male adult Wistar rats to tannery effluent (treated or not) did not alter the parameters of learning and memory evaluated in the inhibitory avoidance, discriminative avoidance and object recognition tests. Therefore, it is suggested that in the experimental conditions adopted herein the treatments seem not affected neuronal functions in a specific way that can cause cognitive damage in animals. It is suggested, however, that further research using different predictive tests of memory deficit in situations of chronic exposure to tannery effluent to be conducted in order to better understand the effects of these xenobiotics in animal cognition.

CONCLUSION It is concluded that the intake of tannery effluent diluted

with water by males C57BL/6J mice can cause neurobehavioral changes associated with anxiety and depression, but without memory deficits and growth/development. It is suggested that mechanisms by which the tannery effluent interferes with the body homeostasis, leading to physiological and behavioral changes, should be more thoroughly investigated, especially in investigations using different mammals’ models, various effluent doses and different times of exposure. Moreover, the necessity of research studies involving more variables (both physiological and behavioral) is evident, because xenobiotics may have different mechanisms of action in the organisms evaluated.

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