Micronucleus as biomarker of genotoxicity in birds from Brazilian Cerrado

Ecotoxicology and Environmental Safety 115 (2015) 223–228

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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Micronucleus as biomarker of genotoxicity in birds from Brazilian Cerrado Camilla Queiroz Baesse a,b,n, Vitor Carneiro de Magalhães Tolentino a,b, Adriano Marcos da Silva a,b, Arthur de Andrade Silva a, Giancarlo Ângelo Ferreira a, Luís Pedro Mendes Paniago a,b, Júlio César Nepomuceno a,c, Celine de Melo a,nn a Universidade Federal de Uberlândia, Department of Biology, Laboratory of Ornithology and Bioacoustic, Umuarama Campus, Rua Ceará, s/n, Bairro Umuarama, CEP 38400-902 Uberlândia, Minas Gerais, Brazil b Graduate Program in Ecology and Conservation of Natural Resources, Brazil c Universidade Federal de Uberlândia, Department of Genetics and Biochemistry, Brazil

art ic l e i nf o

a b s t r a c t

Article history: Received 4 November 2014 Received in revised form 13 February 2015 Accepted 13 February 2015 Available online 23 February 2015

Birds are considered efficient bioindicators, by their behavioral characteristics, diversified diet, and use of several vegetation layers, including in contaminated environments. The accumulation of contaminants can harm the reproductive process and survival of species, in addition to causing severe metabolic disorders. Air pollution can also affect the birds' health. Micronucleus analysis, a technique able to evaluate the organisms' sensitivity to contaminant agents, has been regarded as a practical tool for evaluating and monitoring the clastogenic and aneugenic effects caused by pollutants. The purpose of this study was to evaluate the presence of micronuclei in bird species that use forest environments and their surroundings; and to verify if the frequency and amount of micronuclei varies between species, areas and populations. Birds transiting between the Brazilian Cerrado forest and open formations were analyzed, coming from four forest fragments of Triângulo Mineiro, two close to urban areas and two more distant. Birds were captured with mist-nets for collecting blood extensions, which were used for counting micronuclei. In total, 103 individuals of 21 species were captured, and the micronucleus rate for every 5000 erythrocytes analyzed was 1.30. Only six populations had sampling sufficiency. There was no difference between the number of individuals with and without micronuclei (χ² ¼3.18, df ¼1, p¼ 0.08). In areas closer to the urban perimeter, the micronuclei averages in birds were greater compared to the most isolated areas (H ¼27.534, df ¼ 3, po0,001). In São José, the individuals of Myiothlypis flaveola presented a number of micronuclei significantly greater than the Galheiro and Água Fria (H ¼9.601, df¼ 2, p ¼0.008). M. flaveola clearly reflected the area quality. The micronuclei analysis in birds was effective for evaluating the area quality as well as the intensity with which the birds respond to impacts caused by the surrounding matrix. & 2015 Elsevier Inc. All rights reserved.

Keywords: Anthropogenic actions Biomonitoring Farming activities Surrounding matrix Erythrocytes

1. Introduction Due to the hydrological and agricultural potential of the Brazilian Cerrado, anthropogenic actions are evident throughout all its geographical expanse (Klink et al., 1993; Stotz et al., 1996), transforming the landscape into fragmented environments. Most

n Corresponding author at: Universidade Federal de Uberlândia, Department of Biology, Laboratory of Ornithology and Bioacoustic, Umuarama Campus, Rua Ceará, s/n, Bairro Umuarama, CEP 38400-902, Uberlândia, Minas Gerais, Brazil nn Corresponding author. E-mail addresses: [email protected] (C.Q. Baesse), [email protected] (C.d. Melo).

http://dx.doi.org/10.1016/j.ecoenv.2015.02.024 0147-6513/& 2015 Elsevier Inc. All rights reserved.

forested areas are converted into agricultural fields or used for other activities (Primack, 1993), these being the most modified physiognomies (Pereira et al., 2011). Semi-deciduous forests, equivalent to dry forests, are formations that made up a large part of the Cerrado area (Pereira et al., 2011; Ribeiro and Walter, 1998) and currently are reduced to small remnants, of varying sizes and subject to several types of disturbance. Forest fragmentation unleashes various effects, such as habitat degradation, reduction in colonization, and increased mortality, leading to higher rates of extinction and loss of biological diversity (Marini, 2001). This process has generated several consequences for birds (Oliveira et al., 2004), leading to the decrease of more specialized bird species, leaving, mostly, those with generalist habits (Primack and

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Rodrigues, 2001; Stotz et al., 1996). As bioindicators of environmental quality, birds are among the most efficient animals due to their high potential for rapid detection of environmental damage (Dauwe et al., 2000; GarcíaFernández et al., 1995; Spahn and Sherry, 1999; Wayland et al., 2001), being sensitive (Grue and Shipley, 1984; Parker and Goldstein, 2000) and susceptible to contamination due to several features, such as their great displacement capacity and their place in the final link of the food chain (Valdes, 2010). Birds can concentrate heavy metals, acquired in the food (Oliveira et al., 2004), resulting in decreased reproductive success and survival (Eeva and Lehikoinen, 1996; Esselink et al., 1995). In addition to this, organochlorine compounds, resulting from leaching of pesticide residues, can be absorbed by birds (Valdes, 2010) and can cause serious metabolic disorders (Smith, 1991). Air pollution also affects birds’ health, because during flight they accumulate large volumes of air, and with this they absorb any gases or particles present in it, being therefore susceptible to environmental contaminants from the air (Brown et al., 1997). There are several techniques to evaluate the degree of contamination of organisms; some are more invasive and sacrifice of the animal may be necessary (Sutherland et al., 2004), while others can provide reliable estimates of exposure level to a pollutant without having to sacrifice the animal (Valdes, 2010). A technique able to evaluate organisms' sensitivity to contaminants is micronucleus analysis. Quantification of micronuclei has been considered a practical tool for the evaluation and monitoring of clastogenic and aneugenic effects caused by pollutants (Grisolia, 2002; Nepomuceno et al., 1997), generating the formation of acentric fragments and/or whole chromosomes that are delayed in anaphase and are separated from the nucleus (Fenech et al., 1999; Gattás, 2001; Schmid, 1975). The micronuclei have chromatinic characteristics similar to those of the main nucleus when evaluated by optical microscope (Gattás et al., 1992). Micronuclei formation can be due to spontaneous chromosomal changes or can arise from environmental changes (Rieger and Green, 1968). Evaluation of the presence and amount of micronuclei allows identification of the frequency of cell mutations, which are exposed to a wide variety of genotoxic agents (Benner et al., 1994; Muñoz et al., 1987). In addition, it can be used to evaluate environmental quality, because it has the potential to detect damage in organisms exposed to pollutants (Sutherland et al., 2004). Micronuclei evaluation has been developed in fish (Grisolia and Cordeiro, 2000; Nepomuceno et al., 1997), plants (Mcduffie et al., 2001; Pereira, 2012) and mammals (Ribeiro et al., 2004); however, few studies have been performed with wild birds (e.g., Quirós, et al., 2008; Skarphedinsdottira et al., 2010) and most are with captive animals (Pinhati et al., 2006; Zúñiga-González et al., 2000, 2001). It is important to evaluate the responses of birds in the Triângulo Mineiro region, because the use of pasture areas and agricultural crops predominate in the region of Uberlândia, corresponding to 71.22% of soil occupation (Brito and Prudente, 2005), which implies extensive use of agricultural inputs (Mcduffie et al., 2001). Whereas there are bird species that move between forest fragments and open areas, including pastures and agricultural crops, it is possible that they present micronuclei when exposed to places that have environmental contaminants.

2. Objectives Evaluate the micronuclei presence in bird species that use forest environments and its surroundings; and verify if the micronuclei frequency and amount vary between species, areas and populations.

3. Material and methods 3.1. Study area The study was carried out in four forest areas in the Triângulo Mineiro region, Minas Gerais State, Brazil. The region is located in the Cerrado area, with Aw type climate according to the Köppen climate classification, characterized by a marked seasonality, with well-marked rainy and dry seasons, annual rainfall of approximately 1500 mm, and average temperature of 22 °C (Rosa et al., 1991). 3.1.1. Gloria Gloria is located in the southeastern part of the city of Uberlândia, next to the urban perimeter and a highway. The forest fragment has about 30 ha, and it comprises semi-deciduous forest and gallery forest and the surrounding matrix for crops and pastures (Silva and Araujo, 2009). 3.1.2. São José São José forest is adjacent to the urban perimeter of Uberlândia, and the surrounding matrix comprises pasture, Eucaliptus sp. plantation, and urban area. The forest area has 20 ha, with a gradient between semi-deciduous seasonal forest and gallery forest (Lopes, 2010). 3.1.3. Água Fria Água Fria forest is located in the rural area of the city of Araguari, about 25 km from the urban area. The area has approximately 200 ha and is considered an area in an excellent state of conservation. Its vegetation is composed of gallery forests bordering water bodies, deciduous seasonal forests on the slopes, and semi-deciduous seasonal forests in the flood plains. The surrounding matrix is mainly composed of soy crops and pasture (Lopes, 2010). 3.1.4. Galheiro (Estação de Pesquisa e Desenvolvimento Ambiental Galheiro) Galheiro is located in the city of Perdizes, and is owned by the Companhia Energética de Minas Gerais (CEMIG), registered by the IBAMA as a “Reserva Particular do Patrimônio Natural” with a total area of 2800 ha (Castro, 1995). The forest fragment where the study was carried out has 260 ha and is 22 km from the urban area. It has forest vegetation formations characterized by semideciduous forests, gallery forests, and savannah forests (Castro, 1995). The surrounding matrix is basically composed of crops, especially soy crops. 3.2. Data collection The data collection occurred from June 2013 to February 2014. Birds were captured with mist-nets (12  3 m) exposed from 6 am to 4 pm, using from 16 to 25 nets per campaign, totaling four campaigns in each area. The species that displace among the forest and open formations were captured (Develey, 2003; Gwynne et al., 2010; Sick, 2001; Sigrist, 2007, 2009) and the individuals were identified in the species level and banded with metal rings (CEMAVE/ICMBio pattern). For to produce the blood extensions, the blood was collected through tarsal-metatarsal vein perforation with insulin syringes and the blood were directly dripped on the plate. To carry out the blood smear another plate was slipped at an inclination of 45° onto the plate with blood, so that the blood would be homogeneous (Braga et al., 2010). For every individual captured, two blood extension plates were made. When dry, the slides were fixed in methanol and colored in a

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5% solution of GIEMSA and phosphate lid with pH 5.8, rinsed in distilled water, dried at room temperature, and marked with the individual ring number. The use of GIEMSA dye evidences the nuclear and cytoplasmic components of erythrocytes, having a predominance of red (when acid) and blue (when basic) touches. The erythrocytes presented the cytoplasm with rose pigmentation and the nucleus and cell nuclei with a bluish pigmentation (Capitelli and Crosta, 2013; Mitchell and Johns, 2008). The micronucleus analysis was made with an optical microscope at  100 magnification using immersion oil, following the zig-zag model to avoid crossing the same field more than once. On each slide 2500 erythrocytes were counted, totaling 5000 erythrocytes per individual, being analyzed the presence or absence and amount of micronucleus. Each erythrocyte can contain one or more micronuclei and its identification followed the criteria proposed by Schmid (1975) and Wolf and Luepke (1997), which are: a) structure and intensity of colors similar to those of the main nucleus; b) evident edge, suggesting the existence of a nuclear membrane; c) rounded shape; d) intracytoplasmatic location, and e) diameter smaller than one-fifth of the main nucleus diameter.

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by chance. However, comparing all populations together, there was no difference between the number of individuals with and without micronuclei (χ² ¼3.18, df ¼1, p ¼0.08). There was a difference in the micronuclei average between birds of different areas (H¼ 27.534, df¼ 3, p o0.001). In areas closer to the urban perimeter, the averages were greater compared to the most isolated areas (Glória ¼2.3572.04, São José¼ 2.25 72.02, Água Fria¼ 0.427 0.69, and Galheiro¼0.60 70.90; Fig. 2). The amount of micronuclei differed between the populations with more than five individuals analyzed (H ¼26.142, df ¼7, po 0.001). The population of M. flaveola in São José had the highest number of micronuclei (3.40 71.82) and N. pallescens had the lowest (0 micronuclei). It was possible to compare the micronuclei amount only for populations of M. flaveola (Fig. 3A) of São José, Água Fria, and Galheiro. In São José, the individuals presented a significantly greater amount of micronuclei (H ¼9.601, df¼2, p ¼0.008). The micronuclei average for M. flaveola was 3.40 71.82 in São José, 0.6 70.89 in Água Fria, and 0.57 70.94 in Galheiro (Fig. 3B).

3.3. Statistical analysis 5. Discussion In order to verify if there was homogeneity in the presence of micronuclei within each population the Chi-square heterogeneity test was carried out only for those species that showed at least five captured individuals in the same area. To compare the micronuclei averages of each of the species that showed a minimum number of five individuals, the Chi-square test of adhesion, which is used to test the suitability of a probabilistic model to a set of observed data, was conducted. To test whether the amount of micronuclei varied between the four areas, regardless of the species, the Kruskal–Wallis test was carried out. The Kruskal–Wallis test was used to verify whether the micronuclei amount in Myiothlypis flaveola differed between Galheiro, São José, and Água Fria (areas in which this species was captured in sufficient numbers). In order to verify whether the populations with sampling sufficiency (nZ 5 individuals in the same area) differed from each other in relation to the amount of micronuclei, the Kruskal–Wallis test was carried out. Tests were carried out using Systat 10.2 (Wilkinson, 2002) and conducted at a significance level of 5%.

4. Results A total of 103 individuals belonging to 21 species of birds (12 families) which had characteristics of transiting between forest and open environments of Cerrado were captured. In the four evaluated areas, six populations presented five or more individuals: Arremon flavirostris, M. flaveola (in three areas: São José, Água Fria, and Galheiro), Neopelma pallescens, Saltator maximus, Synallaxis scutata, and Turdus leucomelas (Table 1). One hundred and thirty-four micronucleus were accounted (Fig. 1). Considering the 103 individuals, the general micronucleus rate was 1.30 (through 5000 erythrocytes analyzed per individual). Considering only the populations with five or more individuals captured, A. flavirostris, M. flaveola (in São José), and T. leucomelas showed the highest rate of the individuals with micronuclei, with 100% presenting at least one micronucleus. N. pallescens showed the lowest rate, with none of the five individuals presenting micronuclei in the analyzed cells. From the analyzed populations, four (A. flavirostris, T. leucomelas, M. flaveola in São José, and N. pallescens) were heterogeneous regarding the micronuclei frequency, that is, the proportion of individuals with micronuclei differed more than expected

The micronuclei average per individual in this study (0.93) is similar to in other works, when considering the same number of erythrocytes evaluated. For example, Pinhati et al. (2006) in a study carried out with Ara ararauna in captivity, found an average of 0.95 micronuclei per individual. In another study carried out with 30 species of birds in zoos and private breeding sites the average rate was 0.59 micronuclei (Zúñiga-González et al., 2000). Whereas in another study with zoo birds (15 species), the rate was 1.14 micronuclei (Zúñiga-González et al., 2001). The surrounding matrix, comprising crops and livestock, can influence the fitness of some species of birds that also use these areas to perform their biological activities (Gimenes and Anjos, 2003). In addition, the proximity to cities can be damaging to fauna and flora due to contamination caused by atmospheric gases (Assunção, 1998). In spite of the differences in sensitivity to genotoxic damage between organisms, a study carried out in the laboratory with sunflower cells culture tested the effect of adding herbicides, which caused increases of 15–19% in micronuclei rates depending on the concentration of herbicides (Binsfeld et al., 2000). The captured birds that were subject to greater pressure from surrounding matrix, such as farming activities and proximity to urban areas (São José and Glória), presented a greater amount of micronuclei than those from areas with less influence from the surrounding matrix (Água Fria and Galheiro). Gloria and São José are small forest areas near an urban area and with a surrounding matrix with a history of very intense use, both of agriculture and livestock (Lopes, 2010; Prieto and Colesanti, 2012). Due to the proximity to the urban area, they are heavily influenced by air, soil, and water pollution. Also, Água Fria and Galheiro are extensive forest areas and far from urban areas (420 km), despite the surrounding matrix of livestock and agriculture. Água Fria is a preserved forest fragment with primary formations and Galheiro is an area reserved for conservation, with 72% of its area covered by natural vegetation (Cardoso et al., 2002). Both are considered as good environmental quality areas, and due to their distance they are not affected by the pollution generated by urban areas. Only one species was compared among the four areas (M. flaveola); however it is possible that other species present similar patterns regarding micronuclei rates, that is, in more changed environments subject to greater pollution the micronuclei amount

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Table 1 Frequency of individuals with micronucleus (F), number of individuals (nΔ) and frequency of micronucleus (MN/1000) found in 1000 erythrocytes. Species

F (%)

Areas Glória nΔ

Arremon flavirostris Swainson, 1838 Cantorchilus leucotis (Lafresnaye, 1845) Casiornis rufus (Vieillot, 1816) Cnemotriccus fuscatus (Wied, 1831) Coereba flaveola (Linnaeus, 1758) Cyclarhis gujanensis (Gmelin, 1789) Elaenia parvirostris Pelzeln, 1868 Myiothlypis flaveola Baird, 1865 Neopelma pallescens (Lafresnaye, 1853) Nonnula rubecula (Spix, 1824) Saltator maximus (Statius Muller, 1776) Saltator similis d’Orbigny and Lafresnaye, 1837 Sittasomus griseicapillus (Vieillot, 1818) Synallaxis scutata Sclater, 1859 Tachyphonus coronatus (Vieillot, 1822) Tangara cayana (Linnaeus, 1766) Thamnophilus pelzelni Hellmayr, 1924 Tolmomyias sulphurescens (Spix, 1825) Turdus albicollis Vieillot, 1818 Turdus leucomelas Vieillot, 1818 Turdus rufiventris Vieillot, 1818 a

77.78 0 50 33.33 100 0 0 48 0 75 70 100 0 62.5 50 100 25 66.67 0 63.64 50

a

6 – – – 1 – – 1 – – 8a 4 1 – – 1 – 1 – 5a –

São José

Água Fria

Galheiro

MN/1000

nΔ

MN/1000

nΔ

MN/1000

nΔ

MN/1000

0.37 – – – 0.2 – – 0 – – 0.45 0.85 0 – – 1.2 – 0 – 0.52 –

– – – 1 – 1 – 5a – – 1 1 – – – 2 – 2 – 3 –

– – – 0 – 0 – 0.68 – – 0.8 0.8 – – – 0 – 0.6 – 0.33 –

2 1 – – – – – 5a 1 1 1 – – 3 – – 1 – – 3 1

0.1 0 – – – – 0.12 0 0 0 – – 0.07 – – 0.4 – – 0 0.2

1 – 2 2 – – 1 14a 5a 3 – – 1 5a 2 – 1 – 2 – 1

0 – 0.3 0.1 – – 0 0.11 0 0.27 – – 0 0.24 0.2 – 0 – 0 – 0

Population with five or more individuals analyzed in the same area.

Fig. 2. Mean (circle) and standard deviation (vertical bars) of number of micronucleus in birds in each area.

6. Conclusions

Fig. 1. Standard pigmentation cells (erythrocytes) of birds colored with GIEMSA and the presence of a micronuclei indicated by the arrow. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

is proportionally greater. Although this species occurs predominantly in forest environments and forest edges, the fact that there is foraging on the ground and in highly impacted edges (Sick, 2001) makes it susceptible to pollutants emitted in the vicinity or accumulated by invertebrates that are part of its diet (Oliveira et al., 2004).

In most of the bird species evaluated in this study, micronuclei presence was observed. The fact that these species have higher micronuclei rates in areas with lower environmental quality and with greater influence of the anthropized surrounding matrix highlights the potential use of birds in environmental biomonitoring, especially regarding pollution, through micronucleus analysis. The micronucleus analysis in birds was effective to evaluate the area quality as well as the intensity with which the birds respond to impacts caused by the surrounding matrix (e.g., M. flaveola). Therefore, the use of species occupying the stratum that has a higher probability of exposure is recommended, as well as those which are more abundant, in order to make sampling and environmental monitoring easier.

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Fig. 3. (A) Myiothlypis flaveola and (B) mean (circle) and standard deviation (vertical bars) of micronucleus of M. flaveola in São José, Galheiro and Água Fria.

Acknowledgments We thank Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG-APQ- 01654-12) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq – PELD – 403733/ 2012-0) for their financial support, Companhia Energética de Minas Gerais (CEMIG) for infrastructure and Programa de Pósgraduação em Ecologia and Conservação de Recursos Naturais da Universidade Federal de Uberlândia.

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