Gill histopathology and micronucleus test of Astyanax jacuhiensis ( Cope , 1894 ) ( Teleostei , Characidae ) to evaluate effects caused by acute exposure to aluminum

The contamination of aquatic environments by aluminum (Al) is usually caused by several anthropogenic activities. Diverse morphological, physiological and biochemical alterations in aquatic organisms have been


Introduction
Pollution, loss of biodiversity and habitat destruction are probably the main environmental threats to aquatic ecosystems (LINDE-ARIAS et al., 2008).Increases in the number and amount of industrial, agricultural and commercial chemicals discharged into aquatic environments have led to various deleterious effects on aquatic organisms (MCGLASHAN;HUGHIES, 2001;OHE et al., 2004).Contamination of water resources by metals has caused various morphological, physiological and biochemical alterations in aquatic organisms (BORDAJANDI et al., 2003;SIRAJ BASHA;USHA RANI, 2003;DAUTREMEPUITS et al., 2004).
Aluminum (Al) is a harmful metal to aquatic ecosystems, and is responsible for events of toxicity with serious ecological consequences.No normal physiological functions in biological systems are attributed to this metal (NAYAK, 2002).High concentrations of Al in aquatic ecosystems might be attributed to rock and soil erosion and also to diverse anthropogenic activities, such as water treatment and lixiviation due to mining operations and industrial landills (KOVACEVIC et al., 2009;GARCÍA-MEDINA et al., 2011;FERNÁNDEZ-DÁVILA et al., 2012).Previous studies have shown that Al toxicity is mainly associated to low water temperature and low pH (VOURINEN et al., 1990;1991;PÓLEO et al., 1991;ALSTAD et al., 2005;CORREIA et al., 2010).Nevertheless, concentrations higher than permissive values established by the Brazilian legislation for Al (between 0.1 and 0.2 mg/L) have been observed in water resources where the pH is neutral (CAMARGO et al., 2009;BLUME et al., 2010).Thus, the evaluation of effects of Al at a neutral pH is important to better understand the toxic potential of this metal on living organisms.
Several biomarkers in fish have been used in ield and laboratory studies to assess the effects of contaminants.Fish gills are particularly sensitive to water quality, constituting the irst target of pollutants, due to their anatomic location, direct contact with the water and quick absorption (PANDEY et al., 2008).Alterations in gill epithelium are a consequence of exposure to contaminants, with the severity of alterations depending on the pollutant concentration and exposure period (SANTOS et al., 2012).Thus, the histopathological analysis of gills represents a reliable tool for evaluating the effects of pollutants, such as metals, in ish.Another tool often used to investigate the potential effects of environmental pollutants in these organisms is the micronucleus (MN) test.Micronuclei can originate both from acentric fragments resulting from chromosomal breaks and from whole chromosomes delayed, which are not incorporated into the main nucleus during cellular division.These characteristics allow the detection of damage provoked both by clastogenic and aneugenic chemicals (UDROIU, 2006).This analysis has been used in different environmental assessment studies to detect genotoxic effects of pure chemicals or mixtures of chemicals (ÇAVAS; ERGENE-GÖZÜKARA, 2005;LEMOS et al., 2007).The concomitant analysis of the occurrence of erythrocyte nuclear abnormalities (NA) allows for the evaluation of another toxicity biomarker, which has been successfully applied under ield and laboratory conditions (PACHECO; SANTOS, 1998;ÇAVAS;KÖNEN, 2008;OMAR et al., 2012).

Effects of aluminum exposure in ish
Therefore, the present work studied the effects of exposure to different concentrations of Al on the histology of gills and MN and NA in the erythrocytes of a Characidae ish species (Astyanax jacuhiensis, common name lambari) after acute exposure at neutral pH.

Specimen procurement and acclimation
Specimens of A. jacuhiensis (± 5 cm in length) were obtained from a local ish farm, transported to the laboratory, subjected to a 7-day acclimation period under a natural photoperiod at 22±1ºC, and fed with ish food every three days.The ish were maintained in oxygenated iltered water (iltration using sequential puriication with activated charcoal, resin of ion exchange, followed by reverse osmosis, reaching parameters for drinking water).Physicochemical parameters of the filtered water were pH 7.4, total dissolved solids 130.0 mg/L, conductivity 19.31 μS/cm, turbidity 0.1 NTU and hardness 0.7 mg CaCO 3 /L.

Toxicity assays
Aluminum toxicity was evaluated at two different concentrations, 0.3 mg/L and 30 mg/L, and prepared from a stock solution of AlCl 3 (Vetec ® , purity 99.99 percent) dissolved in iltered water.After the Al was added to the experimental media, the pH was measured and adjusted to 7.0 with NaOH (when necessary).The experimental media were left to stand for 24 h to allow decantation and to let the system equilibrate.The concentration 0.3 mg/L of Al was chosen due to the fact that it is the maximum level recommended for drinking water by decree No. 518/2004 of the Brazilian Ministry of Health (BRASIL, 2004).The concentration 30 mg/L of Al has been previously found in Sinos River, in southern Brazil (BLUME et al., 2010), and was chosen to evaluate the possible toxic effects induced by an environmentally relevant concentration of Al.
After the acclimation period, ish were randomly divided into three groups, control (n = 10, maintained in iltered well water), Al 0.3 mg/L (n = 10) and Al 30 mg/L (n = 10), and placed in 9-L aquaria under constant aeration.The pH was evaluated during the experiment, where no oscillation was observed.
Fish were exposed for 72 h, and then they were anesthetized by immersion in clove oil (1%) and killed by medullar transaction following the animal care protocols approved by the Ethics Committee for Animal Experimentation of Universidade Feevale (protocol No. 01.040.2013).The irst arch of gills was removed for histology analysis and blood samples from the caudal vein were collected for the MN test.

Gill histopathology
After 24 h of ixation in 4% formaldehyde, the gills were dehydrated in a graded ethanol series and embedded in parafin.The 5 μm thick sections, made with a rotatory microtome (Leica ® ), were stained with hematoxylin-eosin (H&E), examined with a light microscope (Olympus CX41) (400x magniication) and recorded using a CCD camera (Olympus DSC 3000).For the histopathological evaluation, an average of 10 ields per animal was analyzed, (each ield contained one primary lamella with eight secondary lamellae).The observed gill alterations were epithelial hyperplasia and hypertrophy, lamellar fusion, edema, epithelial lifting, aneurism and necrosis.For each ish, the following was recorded: the frequency of lamellae with each type of alteration (sum of lamellae with that alteration divided by the total number of lamellae analyzed) and the frequency of abnormal lamellae (sum of lamellae with one or more alterations divided by the total number of lamellae analyzed).

Micronucleus test
Peripheral blood smears were obtained from a caudal section.The samples were ixed in absolute ethanol for 10 min and stained with 5% Giemsa for 10 min.The relative frequency of micronucleated cells was evaluated with a light microscope (1000x magniication) by scoring an average of 2000 erythrocytes per ish.
Coded and randomized slides were scored using a blind review by a single observer.The criteria used for the identiication of micronuclei followed the protocol T. Dalzochio et al.
according to Grisolia (2002); briefly, (a) diameter smaller than approximately 1/3 of the main nucleus, (b) absence of contact with the nucleus and (c) color and texture similar to the nucleus.Other anomalies, such as invaginations, buds and binucleated cells, were recorded as nuclear abnormalities (NA).

Statistical analysis
Data are expressed as mean ± standard deviation.For the histological analysis, mean values for the different experimental conditions were compared using one-way analysis of variance (ANOVA) followed by Tukey's test.Considering the MN and NA test, mean values were compared using the Kruskal-Wallis test.The signiicance level adopted was 95% (p<0.05).

Gill histopathology
The microscopical examination of the gills of A. jacuhiensis exposed to different Al concentrations revealed several histopathological alterations, such as hyperplasia and hypertrophy of epithelial cells, edema, aneurysm, lamellar fusion, epithelial lifting and necrosis (Figure 1).Besides these lamellar alterations, proliferation of the ilament epithelium was found in groups exposed to Al, while it was absent in the control group.
The mean frequencies of secondary lamellae with each type of alteration observed in the gills of A. jacuhiensis are shown in Table 1.The frequency of lamellae with hyperplasia of epithelial cells was signiicantly higher in the ish exposed to 0.3 mg/L Al than in the control group (P<0.05).No statistical differences among groups were found in the frequency of lamellae with the other types of gill alterations.Considering the different types of gill alterations together, ish exposed to both Al concentrations showed signiicantly higher frequencies of abnormal lamellae compared to the control group (Figure 2).The frequency of abnormal lamellae for the control, Al 0.3 mg/L and Al A -General view of gills, presenting primary (PL) and secondary (SL) lamellae with no alterations.B -Lamella exhibiting epithelial lifting (thin arrow) and proliferation of the ilament epithelium (thick arrow).C -Aneurysms (*).Hematoxylin-eosin stain.Scale bar = 50 μm.

Micronucleus test
Table 2 shows the results for the micronucleus (MN) and nuclear abnormalities (NA) analysis in the erythrocytes of A. jacuhiensis after exposure to different Al concentrations and in the control group.Although ish exposed to both Al concentrations presented a higher mean frequency of MN and NA when compared to the control group, no statistical differences were observed (P = 0.19 for MN and P = 0.27 for NA).

Discussion
According to Brazilian law, the limits for dissolved aluminum in freshwater are between 0.1 and 0.2 mg/L (CONAMA, 2005).Nevertheless, some studies have reported much higher levels of aluminum in the environment, such as 5.7 mg/L (CAMARGO et al., 2009) and 30 mg/L (BLUME et al., 2010).Fish exposure to aluminum leads to osmoregulatory disturbances (CAMARGO et al., 2009), decrease in body weight (BRODEUR et al., 2001), alterations in swimming performance and blood chemistry (YTRESTØYL et al., 2001) and reduction of mucous cells (LEDY et al., 2003).However, most aluminum toxicity effects are known to be more intense under acidic pH conditions.In the present work, the occurrence of morphological alterations in gills and the frequency of micronuclei and nuclear abnormalities, in erythrocytes of A. jacuhiensis exposed to Al concentrations of 0.3 mg/L and 30 mg/L, were evaluated under neutral conditions, as found in most Brazilian rivers.

Gill histopathology
Histopathological alterations in the gills of A. jacuhiensis have been found, which demonstrates the potential of this metal to cause changes in this organ.Among other effects, Vourinen et al. (2003) showed that Al accumulated inside the gill tissue of vendance (Coregonus albula) exposed to an acidic pH and Al (200 µg/L -1 ), suggesting alterations in gill membrane properties.However, there is a lack of studies regarding the relationship between Al exposure at neutral pH and morphological alterations in ish gills.In the present study, a signiicantly higher frequency of alterations per secondary lamellae was found in both groups exposed to Al.The most prevalent alterations were hyperplasia and hypertrophy of epithelial cells.These alterations are known as a mechanism of defense because they increase the diffusion distance between the external medium and the bloodstream, acting as a barrier against the entry of the pollutant into the body (MALLATT, 1985;POLEKSIC;MITROCIC-TUTUNDZIC, 1994;FERNANDES;MAZON, 2003).A signiicantly higher frequency of hyperplasia of epithelial cells was observed in the Al 0.3 mg/L group in comparison with the control group; thus, a dose-response relationship was not observed.Monteiro et al. (2008) also did not observe this relationship in a study about the effects of copper exposure performed with Oreochromis niloticus.
The thickening of the ilament gill epithelium observed in groups exposed to Al may be due to the proliferation of cells rich in mitochondria and stem cells (DANG et al., 1999).Along with hyperplasia and hypertrophy of epithelial cells, this effect is also considered to be a protection mechanism against toxic agents (CENGIZ; UNLU, 2002).
Other responses observed include aneurisms, epithelial lifting, necrosis and lamellar fusion.Although no significant difference was observed among the groups, the frequency of such alterations was commonly higher in the groups exposed to the Al concentrations.The aneurisms result from extended vasodilatation, with the collapse of pillar cells and the breakdown of vascular integrity (MARTINEZ et al., 2004), and have been associated with the presence of irritants in the water (PEREIRA et al., 2013).Necrosis was also more frequent in the groups exposed to both Al concentrations.This lesion is characterized as irreversible and occurs following exposure to toxicants (TAKASHIMA; HIBIYA, 1995).Lamellar fusion and epithelial lifting were only observed in ish exposed to Al 30 mg/L.These lesions diminish the vulnerable gill surface area and are probably a defense strategy (MONTEIRO et al., 2008).
Possibly, even more severe lesions would be observed in ish after chronic exposure and/or higher aluminum concentrations.

Our histological indings are in accordance with
other laboratory studies about metal exposure (GALVEZ et al., 1998;CERQUEIRA;FERNANDES, 2002;SANTOS et al., 2012) because the alterations observed appear to be a general response to most irritant agents in ish gill tissue (MALLATT, 1985).The histopathology of ish gills is considered to be an important tool in evaluating the effects of toxicant exposure, because the injuries to this organ may reduce oxygen consumption and disrupt the osmoregulatory functions of the ish (MISHRA; MOHANTY, 2008).Nevertheless, a quantitative evaluation of gill alterations is important in order to perform a thorough analysis of morphological data, as performed in our study.

Micronucleus test
In a study by Galindo et al. (2010), micronuclei frequencies did not increase signiicantly in Prochilodus lineatus juveniles exposed to Al and an acidic pH for 6, 24 and 96 h, in comparison with the negative control group.However, in the same study, nuclear abnormalities frequencies increased signiicantly after all exposure periods.García-Medina et al. (2013), when evaluating the genotoxic effects of three Al concentrations (0.05 mg/L, 120 mg/L and 239 mg/L) on C. carpio, found a signiicant increase in the micronuclei frequencies after 12, 24 and 48 h.However, after 72 and 96 h, micronuclei frequencies decreased, suggesting that, for C. carpio, the peak induction of micronuclei caused by Al exposure occurs until 48 h and was very low.
There are no previous studies reporting the required time for the formation of micronuclei in A. jacuhiensis.The time required to reach a peak induction in micronuclei frequency varies among different species.Normally, micronucleated erythrocytes in ish occur 1 to 5 days after exposure, although in most ish species micronuclei are formed on days 2 and 3 (UDROIU, 2006).Considering that micronuclei can only be formed once cells undergo mitosis, detailed studies including different exposure periods are Effects of aluminum exposure in ish essential to understand the cell cycle and micronuclei induction of this species.
Several authors have suggested that variations in the shape of the nucleus could represent a manifestation of the effects of xenobiotics.Although the mechanisms underlying the formation of these abnormalities have not been fully explained, the induction of nuclear abnormalities has been reported in ish erythrocytes as a consequence of exposure to contaminants with cytotoxic, genotoxic, mutagenic or carcinogenic activity (AYLLON; GARCIA-VAZQUEZ, 2000;TALAPATRA;BANERJEE, 2007;MURANLI;GÜNER, 2011).Therefore, it has been suggested that in genotoxicity studies with ish models, the analysis of nuclear abnormalities should be considered and may complement micronuclei scoring (ÇAVAS; ERGENE GÖZÜKARA, 2005).In this study, nuclear abnormalities frequencies found in control animals and in animals exposed to both Al concentrations were considered low.However, our results are in accordance with several other authors, which also report a higher frequency of abnormalities in ish peripheral erythrocytes, in comparison with the micronuclei frequencies (FERRARO et al., 2004;CAVALCANTE et al., 2008;OLIVEIRA-MARTINS;GRISOLIA, 2009;MURANLI;GÜNER, 2011).Therefore, the concomitant analysis of nuclear abnormalities could represent an alternative to overcoming a possible lack of sensitivity related to the general low micronuclei frequency in ish erythrocytes (AYLLON, GARCIA-VAZQUEZ, 2000;GUILHERME et al., 2008;MONTEIRO et al., 2011).
In the present study, no evidence of genotoxicity was observed, as revealed by the micronucleus and nuclear abnormalities test.However, the acute exposure of A. jacuhiensis to aluminum at neutral pH was toxic, given the histological alterations found, even at the concentration allowed by Brazil for drinking water.However, more studies that include interactions such as water temperature, pH, and the physiology of ish species, in order to investigate the Al toxicity, are valuable and needed to improve what is known about the effects of this metal.

FIGURE 2 :
FIGURE 2: Frequency of normal and abnormal secondary lamellae in control group and groups exposed to different aluminum concentrations.

TABLE 1 :
Frequency of lamellae with histological alterations observed in the gills of Astyanax jacuhiensis after exposure for 72 h to different aluminum concentrations (mean ± standard deviation).
a Different letters indicate signiicant difference among groups (P<0.05).