Impacts of nickel mining on the DNA and hematological parameters of two species of bat in central Brazil

doi:10.21203/rs.3.rs-2367486/v1

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Impacts of nickel mining on the DNA and hematological parameters of two species of bat in central Brazil

https://doi.org/10.21203/rs.3.rs-2367486/v1

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Exposure to heavy metals in mining zones is a significant threat, which can affect ecosystem services and contribute to the decline of wild bat populations. The present study investigated the impacts caused by mining on two bat species in central Brazil, the nectarivorous Glossophaga soricina and the frugivorous Carollia perspicillata. The bats were collected from a nickel miningzone (treatment) and protected area (control). The leukocyte profile of each species was compiled and genotoxicity (comet assay) and mutagenicity (micronucleus test) were determined using the appropriate procedures. Glossophaga soricina presented significantly higher frequencies of neutrophils and lymphocytes in the mining zone in comparison with the protected area, whereas C. perspicillata presented higher frequencies of lymphocytes in the mining zone, but significantly lower frequencies of monocytes. Concomitantly, G. soricina also presented a higher frequency of DNA damage, although no variation was found in this parameter in C. perspicillata when comparing environments. We also found no significant between populations in terms of the frequency of micronuclei and other nuclear abnormalities. Overall, the results of the study indicate that bats are susceptible to immunological disorders and DNA damage in mining zones, with the nectarivorous G. soricina appearing to be relatively more susceptible, and thus a potentially effective bioindicator of the impact of contamination in these environments.

Chiroptera

heavy metals

comet assay

micronucleus

leukocytes

Studies of the health of Neotropical bats are still infrequent, in particular, in populations that inhabit mining zones. Potentially toxic metals are of considerable environmental concern due to their capacity for bioaccumulation in many different types of organism, such as earthworms, Eisenia fetida (Sivakumar 2015), plants, Phragmites australis (Bonanno and Giudice 2010), fish, Salmo trutta fario (Jabee et al. 2012) and bees, Bombus impatiens (Sivakoff et al. 2020). This process also often leads to the biomagnification of the metals to higher trophic levels. In addition to bioaccumulation, these metals can cause morphological and genetic alterations in mammals, and pollute water sources and soils (Voutsas et al. 2002). Exposure to these environmental stressors can also damage other aspects of the health of a species, such as its blood cells and breeding patterns (Sotero et al. 2022), which may have irreversible consequences for the local wildlife.

Bats, for example, which are relatively long-lived in comparison with other small-bodied mammals (Zukal et al. 2015), tend to be at serious risk in mining zones. A number of studies have alerted to the potential impact of heavy metals on these animals, which can trigger a variety of adverse effects. In the case of insectivorous bats, Zukal et al. (2015) found that the principal form of contamination is bioaccumulation in the food chain, in which heavy metals are transferred from the sediments present in water, soils, and plants to insect larvae and thence to the bats. This means that these animals can be usedas indicators of environmental health, and can provide important insights into the impacts of human activities on natural environment. In this context, the analysis of the leukocyte profile of animals exposed to environments subject to anthropogenic impacts provides valuable data for the understanding of the effects of stress on the immune system. This analytical capacity makes the method useful for research on virtually any vertebrate species, and in particular, for systematic comparisons between taxa (Davis and Maney 2018). Overall, then, pollution can have a negative effect on the vertebrate immune system (Pilosof et al. 2014; de Gregorio et al. 2021; Menéndez-Blázquez et al. 2021).

Leukocytes, or white blood cells, consist of a number of different and functionally distinct types of cell, including neutrophils, eosinophils, basophils, monocytes, and lymphocytes (Overmann 2017). In addition to the leukocyte profile, the analysis of genetic biomarkers, such as the comet assay and the micronucleus test, can provide important parameters for the assessment of bat health (Sotero et al. 2022). While considered to be non-invasive, these tests are effective for the assessment of DNA damage, and have been applied successfully in a number of different taxonomic groups (Li et al. 2018; Russo et al. 2020; Motta et al. 2020; Zhisong et al. 2021; Lopes et al. 2021), including bats (Benvindo-Souza et al. 2022).The comet assay is a biomarker of genotoxicity that quantifies DNA damage, and is widely used due to its relatively simple procedure, sensitivity, agility, and its capacity to detect genetic damage, ranging from the rupture of DNA strands to cross-links and cellular apoptosis (Collins 2004). As a complement to the comet assay, the micronucleus and erythrocyte nuclear abnormality tests are used to investigate mutagenic and cytotoxic damage in cells exposed to stressors (Ribeiro 2003; Matsumoto et al. 2006). In bats, Benvindo et al. (2019a) found that the micronucleus test applied to the oral mucosa provides an effective biomarker for the detection of cell damage and other abnormalities.

Given these considerations, the present study evaluated, for the first time, the leukocyte profile of two bat species, Glossophaga soricina and Carollia perspicillata, in a nickel mining zone and compared this profile with that recorded in a well-preserved protected area. Nickel is a heavy metal with a density of 8.5 g/cm³, whichis extracted from lateritic ores to serve as a raw material for a range of applications, such as the production of batteries, coins, and auto parts, as well as galvanization and the application of metal finishes (Clancy et al. 2012). In Brazil, the state of Goiás, in which the present study was conducted, produces 42% of the country’s nickel ore (IBRAM 2010). Nickel is considered to be highly toxic, with immunotoxic, neurotoxic, genotoxic, nephrotoxic, hepatotoxic, and carcinogenic effects (see Khan et al. 2022). In addition to the white cell counts, the present study also evaluates genotoxicity using the comet assay and mutagenicity using the micronucleus test.

Study areas and bat sampling

We selected two common species of bat, the nectarivorous Glossophaga soricina and the frugivorous Carollia perspicillata, from the Brazilian Cerrado savanna for analysis in the present study. We sampled the nickel mining zone in two municipalities of the Brazilian state of Goiás(Fig. 1). We captured five G. soricina and seven C. perspicillata in the municipality of Barro Alto (15°3’4.01” S, 48°57’20.01” W), and three G. soricina and one C. perspicillata in Niquelândia (14°21’47.56” S, 48°25’42.13” W). As both collecting sites were located within the nickel mining zone, we summed these specimens to represent the mining (treatment) environment. The third site was located within a protected area (control environment), the Silvânia National Forest (FLONA Silvânia) in the municipality of Silvânia (16º 38’30.0” S, 48º 39’02.5” W), where we collected eight G. soricina and five C. perspicillata. The economies of Barro Alto and Niquelândia are based primarily on the extraction of ferro-nickel, and these municipalities correspond to the nucleus of the nickel mining industry in Goiás. The Silvânia National Forest is a protected area of approximately 486.37 hectares, whichis surrounded by soybean and tomato plantations, reflecting the importance of the agricultural sector for the economy of the municipality of Silvânia.

We captured bats at all three sites using mist nets, with a total of 10 nets (9 m x 2.5 m), which were deployed for four hours in the early night (18:00–22:00 h), and inspected every thirty minutes. At each location, the capture effort lasted 10 nights. The present study was authorized by the Animal Ethics Committee of the Federal University of Goiás (permit number 004/21), and the Brazilian Chico Mendes Institute (SISBIO), through license number 75819-1.

Leukocyte Profile

. We collected 10 µl of blood from each animal to analyze the immune cell defense. Blood was placed on a clean slide and spread with the help of another glass slide, thus making a blood smear. The slides were dried at room temperature and fixed with methanol (Miguel et al. 2019). Subsequently, they were stained with the panoptic kit and analyzed under a microscope using immersion oil in a 100-x objective. One hundred leukocytes per individual were quantified, scoring the frequency of lymphocytes, neutrophils, eosinophils, and monocytes (Paksuz et al. 2009).

Comet Assay

We applied the alkaline comet assay method of Singh (1988) in the present study with some modifications. The first step was to pre-coat microscope slides with 1.5% standard melting agarose. We obtained a blood sample of approximately 20µL from the radial artery of each bat, and diluted it in 120µL of 0.5% low melting point agarose, which we preheated to 40°C. We pipetted this solution onto the pre-coated slides (two per animal), covered it with a coverslip, and then transferred the slide to the refrigerator for five minutes to allow the material to solidify and adhere to the slide. Once the material had solidified, we removed the coverslips, and transferred the slides to a lysis solution consisting of 1 mL of Triton X-100, stock lysis (146.1g of sodium chloride [2.5M], 37.2g of disodium salt [100Mm], 1.2g of hydroxymethyl [10Mm], 8.0g of sodium hydroxide, 10g of sodium lauryl sarcosinate), and 89 ml of dimethyl sulfoxide. This solution exposes the genetic material and permits the identification of changes in the DNA. The samples were then incubated at 4°Cfor 12 hours. After lysis, we electrophoresed the slides inan alkaline running buffer in a horizontal tray, with the slides being left to settle for 30 minutes prior to the run. We ran the electrophoresis in the dark for 30 min at 25V and 300mA.After the electrophoresis, we immersed the slides in neutral buffer (0.4 M Tris-HCl, pH 7.5) three times for 5 minutes to neutralize the pH. We then washed the slides with distilled water and fixed them in absolute alcohol for 10 min.

We stained the DNA with SYBR Gold I, prepared from 1µL of the product diluted in 30mL of TBE Buffer. We stained each slide for 30 min with 100Μl of SYBR Green dye, and then washed the slides with distilled water and dried them in an oven at 37°C. We analyzed the slides under an Imager D2® epifluorescence microscope (Carl Zeiss, Germany), using a set of 515–560 nmexcitation filters for green fluorescence. We analyzed the slides from each bat in duplicate and counted 100 nucleoids using the Comet Imager program, version 2.2 (Meta Systems GmbH). A single researcher analyzed all the slides under a 20x lens. In the analysis of the nucleoids, we evaluated three parameters of genomic damage, following Motta et al. (2020), that is, tail length, the percentage of DNA in the tail (% DNA), and the olive tail moment (Olive).

Micronucleus Test

We obtained buccal cells from the bats using the protocol of Benvindo-Souza et al. (2019a), in which cotton swabs are rubbed lightly against the right and left jugal mucous membranes, the floor of the mouth, and the gums. We transferred the cells of the oral mucosa to clean glass slides (four per individual) prepared with a drop of saline solution (0.9% NaCl). We then fixed the slides in a 100% methanol solution (P.A. methyl alcohol) for approximately 10 min and stained them with a Rapid Dye kit (Panotic/Laborclin). Subsequently, we rinsed the slides with distilled water to remove the excess staining solution. We counted 1,000 cells per subject under a Leica DM 500 light microscope at a magnification of 100×. We registered the presence of micronucleated and binucleated cells, and karyolysis (Benvindo-Souza et al. 2019a,b).

Statistical analysis

The data obtained in the genotoxicity, mutagenicity, and hematological tests are presented as the mean ± standard error. The normality and homoscedasticity of the data were verified using the Shapiro-Wilk and Levene tests, respectively. Student’s t or the Mann-Whitney U test (for nonparametric data) was applied to compare the means between environments (mining zone vs. control – protected area), with a statistical significance of p < 0.05. All the analyses were run in Statistica v. 7.0.

Leukocyte profiles

Comparing areas (mining zone vs. control), G. soricina had significantly higher mean frequencies of neutrophils (3.63 ± 0.86) and lymphocytes (3.63 ± 0.65) in the mining zone in comparison with the protected area (0.63 ± 0.26 and 2.13 ± 0.30, respectively; Fig. 5A). In C. perspicillata, by contrast, while the mean frequency of neutrophils (4.25 ± 0.84) was also significantly higher in the mining zone in comparison with the protected area (0.60 ± 0.49), the mean frequency of monocytes (2.00 ± 1.26) was significantly higher in the protected area (Fig. 5B).

Comet Assay

We observed twice as much DNA damage in the G. soricina samples from the mining zone in comparison with those from the protected area (Fig. 4A). In the case of the frugivorous C. perspicillata, however, we found no significant variation in the comet assay parameters between the environments (Fig. 4B). Comparing the samples from the protected area, the C. perspicillata specimens presented significantly greater DNA damage – %DNA in the tail (t = -2.24; p = 0.04) and tail length (t = -2.39; p = 0.03) – than that recorded in G. soricina. No significant variation was found between the two species in the samples from the mining zone.

Micronucleus Test

We found no significant variation between environments in the results of the micronucleus test (Table 1). However, the sum of the most common abnormalities (binucleated cells and karyolysis) recorded in the C. perspicillata samples was significantly higher in the mining zone in comparison with the Silvânia NF (Table 1).

Table 1

Frequency (%) of micronuclei and the total number of nuclear abnormalities observed in the exfoliated cells of the buccal mucosa of the bats collected in the present study in the Cerrado savanna of the Brazilian state of Goiás. The asterisk (*) indicates a statistically significant difference between environments
	Mean ± standard error recorded in the:
Species	Silvânia NF	Mining zone	Statistic	p
Frequency (%) of micronuclei
Carollia perspicillata	0.00 ± 0.00	0.01 ± 0.01	U = 17.50	0.7144
Glossophaga soricina	0.00 ± 0.00	0.03 ± 0.02	U = 24.00	0.4008
Total nuclear abnormalities
Carollia perspicillata	0.48 ± 0.09	1.15 ± 0.16	t = -3.112	0.0098*
Glossophaga soricina	0.79 ± 0.12	0.86 ± 0.31	U = 30.00	0.8336

Biometric Parameters

Most of the C. perspicillata and G. soricina specimens collected in both environments were adult females. The G. soricina specimens from the mining zone weighed 8–18 g and those from the Silvânia NF weighed 10–15 g, while the C. perspicillata specimens from the mining zone weighed 12–18 g and the specimens from the national forest weighed 15–20 g. We were unable to evaluate possible gender-based variation in mutagenic and genotoxic damage in either species due to the small samples of male specimens collected in both cases. We also found no systematic variation in the DNA damage in relation to the body size (weight) of the bats.

In the present study, we evaluated the leukocyte profiles of C. perspicillata and G. soricina from a nickel mining zone for the first time, together with an analysis of biomarkers of genotoxicity and mutagenicity. The lymphocyte and neutrophil counts were significantly higher in the bats from the mining zone in comparison with the control, whereas the monocyte count in C. perspicillata was higher in the protected area. These findings may reflect chronic inflammation, although this level of inflammation is considered normal in frugivorous bats, due to their metabolism and environment such as Eidolon helvum (Olopade et al. 2021). Lymphocytes are responsible for immunity surveillance and the genetic continuity of the internal environment (Liudmila et al. 2017) and, as they are involved in adaptive immunity, lymphocytes act in a specific fashion to defend the organism from each pathogen (Becker et al. 2019). The neutrophils are sentinel cells that patrol, phagocytose, and kill pathogens (Burn et al. 2021), while the monocytes are circulating, mononuclear phagocytes that are part of the innate immune system (Guilliams et al. 2014; Vegting et al. 2021), producing cytokines and presenting antigens (Ziegler-Heitbrock 2015).

The cell counts recorded in the present study are consistent with the findings of previous studies of free-living bats (Santos et al. 2007). Continuous exposure to heavy metals can lead to immunosuppression in wild animals, which increases the vulnerability of the organism to contamination by pathogens (Grasman, 2002; Becker et al. 2021). In a studyof fish exposed to nickel contamination, Marenkov et al. (2021) found that the density of white blood cells tends to increase, which is consistent with the findings of the present study, although these authors did not distinguish the different types of cells. In Australia, Sanchez et al. (2022) demonstrated that higher concentrations of chromium and nickel in the fur of flying foxes were associated with increased infection by ectoparasites due to a decrease in the effectiveness of the immune system.

In the present study, we demonstrated a more significant proliferation of signaling and phagocytic cells, whereas Pilosof et al. (2014) found a decrease in the relative levels of neutrophils and an increase in lymphocytes in bats exposed to sewage water for 30 days. In humans, nickel, for example, may interact with the toll-like receptor-4 in immune and non-immune cells, triggering a pro-inflammatory cytokine cascade (Magrone et al. 2020), and a similar process is likely to occur in bats.

In the genotoxicity test, we observed that the nectarivous species (G. soricina) was more sensitive to the mining environment than the protected area. This species also presented higher levels of DNA damage than that observed in C. perspicillata in both study areas. This implies that G. soricina is the better bioindicator for the assessment of DNA damage caused by mining, as well as the importance of using well-preserved protected areas as reference sites. One possible explanation for the interspecific difference in DNA damage may be the faster metabolism of the nectarivorous bat, which has a high-energy diet. In any case, exposure to heavy metals affects the energy metabolism, as well as damaging cells, tissue, and organs, and enhances the bioaccumulative capacity of bats (Ferrante et al. 2018; Resongles et al. 2014), which means that these animals are highly sensitive to exposure to these environmental stressors.

In mining zones, bats may be contaminated by either direct or indirect exposure to pollutants (O'Shea, 2001). The foraging behavior of a species, including habitat use patterns, or its physiological traits may all influence the level of its exposure to metals (Walker et al. 2002).Previous studies have shown that nickel is one of the metals that may reach the highest concentrations in bats, resulting in the most significant DNA damage (Meehan et al. 2004). Nickel can be found naturally in the environment, although it may also be exposed by human activities such as mining or industrial processes (Genchi et al. 2020). Nickel is extracted by heating the ore in the air, typically using sulphide compounds, and converting it to nickel oxide (Lee 1999). This process obviously results in atmospheric pollution in nickel smelting areas, but may also contribute to the contamination of the soil and damage to the local vegetation (Koptsik et al. 2003).

The toxic effects of nickel on an organism will depend on a number of different factors, such as the chemical species, physical form, concentration, source of exposure, and the amount of nickel to which the animal was exposed (Schaumlöffel 2012). In a study of rodents with a similar body size and weight to microchiropteran bats, Tovar-Sánchez et al. (2012) showed that animals from a mining zone had higher concentrations of zinc, nickel, iron, and manganese than those from a non-mining area, as well as increased DNA damage. Exposure of rats to nickel tetracarbonyl (Ni(CO)₄) resulted in approximately is 50% of nickel in the viscera and blood,30% in muscle and adipose tissue, and 15% in the bone and connective tissue (Azevedo and Chasin 2003), which demonstrates that this metal can bioaccumulate in different organs and tissues.

Both short- and long-term studies have shown that significant amounts of nickel can bioaccumulate in the kidneys of rodents (Dhundasi 2008; Clancy and Costa 2012). In other mammals, nickel is known to be absorbed by the lungs, gastrointestinal tract (Khan et al. 2022), and skin (Begum et al. 2022).Pulmonary absorption is the principal form of induced toxicity, which may harm human health (Clancy et al. 2012). Clancy and Costa (2012) recorded DNA damage and ultrastructural ovarian damage in beetles (Blaps polycresta) caused by nickel oxide nanoparticles. Given these observations, while the present study did not assess the capacity of bats for the bioaccumulation of metals, further research should focus on the quantification of this process in animals, and its relationship with DNA damage. Many studies have shown that the response of different genera or even species to toxic stress may vary widely (Tovar-Sánchez et al. 2012).

Although G. soricina is a nectarivore, it supplements its diet with insects (Benvindo-Souza et al. 2022), which may amplify its potential for contact with metals in the environment. Biomagnification across trophic levels is a typical process of the bioaccumulation of substances in an ecosystem. Trace metals, for example, may accumulate in insects and then be transferred to insectivorous bats (Zukal et al. 2015). A number of studies have demonstrated the capacity of insects to accumulate metals when exposed to them in the environment (Skaldina and Sorvari 2019; Mebane et al. 2020; Parikh et al. 2021). This not only puts the insects themselves at risk, but also their predators, at higher trophic levels, including arachnids, birds, and mammals.

In the present study, we also showed that the samples of the frugivorous C. perspicillata from the protected area had significantly higher levels of DNA damage than G. soricina, even though they did not differ significantly in the mining zone, which indicates that these two species may be under the same level of pressure from the mining environment. While C. perspicillata is considered to be a generalist, it also complements its diet with insects. One other factor that may have contributed to the DNA damage found in this species in the protected area is that the Silvânia National Forest is surrounded by plantations of tomato, soybean, and other grains, which are sprayed with pesticides to increase productivity. This use of pesticides constitutes a potential source of contamination, given that the bats fly long distances in search of food, and are unlikely to remain within the area of the national forest. Particles of the pesticides and other xenobiotic substances may also drift into the protected area, although further research is needed to confirm the potential for this type of contamination.

No significant variation was found in the results of the micronucleus test between the two environments, although the C. perspicillata samples from the mining zone had a significantly higher frequency of total nuclear abnormalities. Gonçalves et al. (2010) found a high frequency of nuclear abnormalities in an analysis of anuran amphibians in the same region as the present study. In a study of the Nile tilapia, Oreochromis niloticus contaminated with nickel, Batista (2012) confirmed that nickel chloride has considerable genotoxic and mutagenic potential, being associated with the presence of micronuclei and other nuclear abnormalities, such as cells with nuclear buds.

The application of the micronucleus test to exfoliated cells of the buccal mucosa has become a promising benchmark for the biomonitoring of non-human organisms, including rodents (Benvindo-Souza et al. 2017), birds (Shepherd and Somers 2012), bats (Benvindo-Souza et al. 2019a,b; Benvindo-Souza et al. 2022), dogs and cats (Santovito et al. 2022). The presence of micronucleated cells indicates the occurrence of chromosome breaks or the existence of entire chromosomes that did not adhere to the cell nucleus (Russo et al. 2004). The micronucleus test can be applied to many different types of tissue, although most studies use erythrocytes (Fenech 2000). Zuniga-Gonzalez et al. (2000) showed that bats from the area of a monazite mine had an increase in micronuclei, which may have been caused by radiation, providing evidence of mutagenic damage caused by environmental stressors, and confirming the potential of bats as bioindicators.

Nickel is known to have toxic, genotoxic, and carcinogenic properties that impact the immune defenses of many different types of animal (Binkowski 2019). Given this, the evidence of the genotoxic and mutagenic effects and the impacts on immune defense mechanisms recorded in the present study indicate a response to the reduced environmental quality of the mining zone. The results of the present study also emphasize the importance of protected areas as a means of minimizing xenobiotic impacts on wildlife, such as nectar-feeding bats. More research is still needed on frugivorous species, however, as well as other trophic guilds, such as insectivores and omnivores, in order to provide a more comprehensive overview of the environmental scenario. It will also be necessary to quantify the presence of other metals in the mining zone and their relationship with the genetic damage observed in flying mammals.

Mining zones are major sources of environmental impact. The present study provides conclusive evidence of ecotoxicological damage caused by a heavy metal, nickel, in two bat species that represent different trophic guilds. The findings of the present study indicate that exposure to heavy metals in mining zones is reflected in the increased frequency of some leukocytes and may cause DNA damage in nectar-feeding bats, such as G. soricina. This study also reinforces the importance of Conservation Units in the preservation of bats. Further research is needed to better understand the patterns of ecotoxicological damage in different bat species and trophic guilds, given the enormous variety of foraging adaptations found in this mammalian order, which may influence the characteristics of their response to environmental stressors. Identifying and understanding the varying impacts of mining on different species of bats will be necessary to compile action plans for the effective conservation, given the fundamental contribution of these animals to the equilibrium of terrestrial ecosystems.

Acknowledgments

We thank the Coordination for Higher Education Personnel Training (CAPES). M.B.S. thanks the Brazilian Fund for Biodiversity (FUNBIO) and the Humaniza Institute for their support, and D.M.S. thanks the National Council for Scientific and Technological Development (CNPq) for a scholarship.

Author contribution

D.F.S., M.B.S and D.M.S. took the lead in writing the manuscript. All authors provided critical feedback and helped shape the research. D.F.S., M.B.S., A.T.C.L and R.M.F collected the samples and carried out the assays. D.M.S and M.B.S analyzed the data. D.M.S obtained the financial support.

Funding: Fundação de Apoio à Pesquisa (FUNAPE), PRONEX/CAPES/927-2022

Competing interests The authors have no conflicting interests in relation to this study.

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Impacts of nickel mining on the DNA and hematological parameters of two species of bat in central Brazil

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Abstract

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Introduction

Material And Methods

Study areas and bat sampling

Comet Assay

Micronucleus Test

Statistical analysis

Results

Leukocyte profiles

Comet Assay

Micronucleus Test

Biometric Parameters

Discussion

Conclusions

Declarations

References

Additional Declarations

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