Evaluation of the Genotoxic Potential of the Mineral Coal Tailings ...

Arch Environ Contam Toxicol (2010) 59:614–621 DOI 10.1007/s00244-010-9512-7

Evaluation of the Genotoxic Potential of the Mineral Coal Tailings Through the Helix aspersa (Mu¨ller, 1774) Daniela Dimer Leffa • Adriani Paganini Damiani • Juliana da Silva • Jairo Jose´ Zocche • Carla Eliete Iochims dos Santos • Liana Appel Boufleur Johnny Ferraz Dias • Vanessa Moraes de Andrade

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Received: 11 January 2010 / Accepted: 22 March 2010 / Published online: 7 April 2010 Springer Science+Business Media, LLC 2010

Abstract Coal mining is an activity with a high potential for environmental pollution. Coal has been described as the most significant pollutant of all the fossil fuels, containing a heterogeneous mixture. Many elements present in coal byproducts as well as coal tailings are rich in potentially toxic and genotoxic metals, which ultimately lead to profound changes in cells, tissues, populations, and ecosystems. The purpose of this study was to assess the genotoxic potential of the mineral coal tailings using the land snail Helix aspersa. Animals were divided in three groups, clustered in plexiglass cages: control (animals fed with organic lettuce), coal tailings (animals living in a layer of pyrite tailings and fed with organic lettuce), and mine lettuce (animals fed with lettuce grown in an area located in a deposit of coal tailings). The hemolymph was collected at different exposure times (24 h, 48 h, 72 h, 96 h, 1 week, 2 weeks, 3 weeks, and 1 month) for comet assay analyses. Results showed that the animals of the coal tailings and D. D. Leffa A. P. Damiani V. M. de Andrade (&) Laborato´rio de Imunologia e Mutageˆnese, Programa de Po´s-Graduaçaõ em Cieˆncias da Sau´de, Unidade Acadeˆmica de Cieˆncias da Sau´de, Universidade do Extremo Sul Catarinense, Av. Universita´ria, 1105 (UNESC), Bairro Universita´rio, Criciu´ma, SC 88806-000, Brazil e-mail: [email protected] J. da Silva Laborato´rio de Gene´tica Toxicolo´gica, ULBRA, Canoas, RS, Brazil J. J. Zocche Laborato´rio de Ecologia de Paisagem, UNESC, Criciu´ma, SC, Brazil C. E. I. dos Santos L. A. Boufleur J. F. Dias Instituto de Fı´sica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

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mine lettuce groups presented higher levels of DNA damage in relation to the control group at all exposure times, but with a peak of DNA damage in 48 h and 96 h. These results demonstrate that the coal pyrite tailings are potentially genotoxic and that H. aspersa has proven to be a sensitive instrument for a better risk assessment of environmental pollution.

The Santa Catarina Coal Basin is a traditional mining area in southern Brazil (lat. 28150 –29000 S, long 48350 – 49000 W). Over the past 120 years, coal production has been vital to the region’s economic development. It has also had a major impact on the environment, which increased dramatically after 1973, when surface mining was intensified to increase domestic coal production as a replacement for imported oil (Santos et al. 2008). Coal, as a sedimentary rock, is a complex heterogeneous mixture of organic and inorganic constituents containing intimately mixed solid, liquid, and gaseous phases of allothigenic or authigenic origin (Vassilev and Vassileva 2009). When coal is separated from its impurities by cleaning processes, coal tailings are formed and deposited usually in close proximity to the mining area. According to Finney et al. (2009), this material presents elements, such as carbon, hydrogen, nitrogen, and sulfur, in addition to various metals, such as aluminum (Al), arsenic (As), calcium (Ca), chromium (Cr), copper (Cu), iron (Fe), potassium (P), magnesium (Mg), molybdenum (Mo), sodium (Na), phosphorus (P), lead (Pb), silicon (Si), and zinc (Zn). Some of these compounds are dangerous because they have impact on the environment and human health (Silva et al. 2008). The tailings from coal improvements constitute one of the major environmental problems faced by the mining industry. Both nitrogen and

Arch Environ Contam Toxicol (2010) 59:614–621

sulfur emissions can react in the atmosphere to form acid rain, which can then acidify lakes and streams, corrode buildings and monuments, and adversely impact plants. Acid mine drainage, a widespread environmental concern, is produced by the oxidation of pyrite in the coal or in the strata overlying the coal. Reaction of sulfur in pyrite with water and air forms sulfuric acid (Finkelman and Gross 1999). Moreover, adverse effects such as genotoxicity and carcinogenicity have been attributed to metals present in coal, caused by oxidative stress (Miadokova et al. 1999; Silva et al. 2000). Brazilian coals have considerable concentrations of pyrite (FeS2) and because the predominant sulfated mineral in the tailings is pyrite, the majority of the components of the aqueous solution obtained by percolation in the residue module is iron and sulfate. The pyrite has recently been shown to spontaneously generate hydrogen peroxide (H2O2) and hydroxyl radicals (•OH) when placed in water (Cohn et al. 2004, 2006).The formation of these reactive oxygen species (ROS) also explains the recent observation that aqueous pyrite slurries degrade yeast RNA, ribosomal RNA, and DNA. Pyrite is thought to form H2O2 through the iron-catalyzed Haber–Weiss reactions (Cohn et al. 2006). According to Schins and Borm (1999), the ROS, through oxidative damage in antiprotease, might cause destruction of tissue through proteases participating in the process of normal enzymatic degradation of proteins and thus suggest that coal is responsible for the generation of oxidants involved in various diseases in miners. The exposure to a combination of compounds is considered to present a higher health risk due to potential synergistic effects of the resulting mixture. Once in the environment, pollutants resulting from coal mining have the potential to penetrate into water sources of the biota or into the atmosphere in significant amounts, thus presenting potential hazards for the environment and human health (Leoń et al. 2007). In recent years, a great deal of effort has focused on the development of techniques that could be used to monitor the health of organisms chronically exposed to pollutants. Monitoring the environment by means of biological test systems, so-called biomonitors and biomarkers, provides promising ways to identify hazards to human health and the environment (Butterworth 1995). One approach is the evaluation of the possible consequences of hazardous pollution that involves the assessment of genotoxic and cytotoxic damages. Therefore, genotoxicity assays and biomonitoring of coal mining pollution are both very important authorities for environment monitoring and for any animal or human being who has or will have contact with this type of pollutant. Because the extraction and processing of coal cause different impacts on the environment, the aim of the present study was to evaluate the genotoxic potential of

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coal tailings from the beneficiation of coal mining in southern Santa Catarina by using the comet assay and the land mollusk Helix aspersa (Mu¨ller, 1774). In addition, metals, which are some of the main contamination agents in the site, were also studied.

Materials and Methods Mollusks The land snail H. aspersa (Mu¨ller, 1974) (Mollusca: Gastropoda: Pulmonata: Helicidae) is native to western Europe and was introduced in other continents, first by New Zealand through France in 1860, being today one of the most abundant land snails (Barker and Watts 2002). Adult H. aspersa land mollusks (n = 144), weighting 7.50 ± 1.50 g, were obtained from a snail breeder at ULBRA, the Lutheran University of Brazil, in Canoas, RS, Brazil. The mollusks were acclimatized to laboratory conditions (22 ± 3C) for 7 days, during which time they received Lactuca sativa L. leaves from organic cultures and water ad libitum. After acclimatization, the snails were clustered in plexiglass cages (36 9 26 9 10 cm) covered by a lid with small holes to promote air ventilation and were identified as control and test groups. Moreover, these boxes were transparent for better distinction of photoperiodicity and visualization of the animals. The light cycle was 12 h light/12 h dark. Experimental Designs and Hemolymph Sampling Coal pyrite tailings were obtained in a deposit of beneficiation of coal from a mine in southern Santa Catarina. In this same place, a vegetable garden was cultivated by the coal mine workers for nutrition purposes. The collected samples were brought to the laboratory for animal exposure. The first step was the determination of the exposure period. The exposure periods to coal tailings were selected after a pilot study with 30 mollusks exposed for 1 week. The animals were accommodated on a 6-cm-deep layer of coal tailings in the boxes. During this time, the hemolymph was sampled from five animals per period of 0 h, 6 h, 12 h, 24 h, 48 h, and 1 week, and a Comet assay was performed. The results of a pilot study determined the periods of 24 h, 48 h, 96 h, 1 week, 2 weeks, 3 weeks, and 1 month for H. aspersa exposure. Individuals (n = 144) were divided in three experimental groups: (1) negative control (animals fed with organic lettuce, n = 48); (2) coal tailings (animals placed in a layer of pyrite tailings and fed with organic lettuce, n = 48); and (3) mine lettuce (animals fed with lettuce grown in an area located on deposit of coal tailings, n = 48). Hemolymph were collected with a 1-ml

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syringe at the end of the shell spiral of the snail at the end of each exposure time per group, consisting of six animals, which were killed at the end of the experiment. Comet Assay The alkaline Comet assay was performed as described by Tice et al. (2000) with several modifications adapted to hemolymph based on the work of Ianistcki et al. (2009). In brief, 25 ll of each heparinized hemolymph sample were added to 75 ll of 0.75% (w/v) low-melting-point agarose and the mixture was added to a microscope slide precoated with 1.5% (w/v) of normal-melting-point agarose and covered with a coverslip. The slide was briefly placed on ice for the agarose to solidify and the coverslip was carefully removed. Next, the slide was immersed in lysis solution (2.5 M NaCl, 100 mM EDTA, and 10 mM Tris–HCl, pH 10.0–10.5) containing freshly added 1% Triton X-100 and 10% dimethyl sulfoxide (DMSO) for at least 1 h at 4C. Subsequently, the slides were incubated in freshly made alkaline buffer (300 mM NaOH and 1 mM EDTA, pH [13) for 20 min for DNA unwinding and were electrophoresed in the same buffer. The electrophoresis conditions were 15 min at 300 mA and 25 V (0.7 V/cm). All of these steps were carried out under dim indirect light. The electrophoresis slides were neutralized in 400 mM Tris–HCl (pH 7.5) and fixed (15% w/v trichloroacetic acid, 5% w/v zinc sulfate, 5% glycerol), washed in distilled water, and dried overnight. The gels were rehydrated for 5 min in distilled water and then stained for 15 min (37C) with a solution containing the following sequence: 34 ml of Solution B (0.2% w/v ammonium nitrate, 0.2% w/v silver nitrate, 0.5% w/v tungstosilicic acid, 0.15% v/v formaldehyde, 5% w/v sodium carbonate) and 66 ml of Solution A (5% sodium carbonate). The staining was stopped with 1% acetic acid and the gels were air-dried (Villela et al. 2006). To calculate a damage index (DI), cells were visually allocated into five classes, using optical microscopy, according to tail size (0 = no tails and 4 = maximumlength tails), which resulted in a single DNA damage score for each individual and, consequently, for each group studied. Thus, the DI of the individual could range from 0 (completely undamaged = 100 cells 9 0) to 400 (maximum damage = 100 cells 9 4) (Collins et al. 2008; Silva et al. 2000). The damage frequency [DF (in %)] was calculated for each sample based on the number of cells with tail versus those without. Chemical Analysis Analysis by particle-induced X-ray emission (PIXE) technique has been successfully employed to detect trace elements

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in plants and organisms because of its multielemental character, high sensitivity, simplicity, and high sample throughput (Johansson et al. 1995; Mireles et al. 2004). The metal content of the organic and coal mine lettuce leaf samples was analyzed using this technique. In short, the leaves were dried, crushed, homogenized, and pressed into thick pellets. The pellets were accommodated in the target holder inside the reaction chamber, which was kept at a pressure of about 10-5 mbar. The experiments were carried out at the Ion Implantation Laboratory of the Physics Institute of the Federal University of Rio Grande do Sul (IF-UFRGS). The standardization protocol adopted in this work followed the procedure described by Johansson et al. (1995). A 3-MV Tandetron accelerator provided a 2.0-MeV proton beam with an average current of 1 nA at the target. The X-rays produced in the samples were detected by a Si(Li) detector with an energy resolution of about 160 eV at 5.9 keV. The spectra were analyzed with the GUPIXWIN software package (Campbell et al. 2000; Maxwell et al. 1995) and the final results are expressed in parts per million (ppm). Statistical Analysis The normality of variables was evaluated by the Kolmogorov–Smirnov test. Statistical analysis for the DI and the DF, measured by the Comet assay, was carried out using one-way analysis of variance (ANOVA). When ANOVA showed significant differences, post hoc analysis was performed with the Tukey test. When non-normal distribution was observed, comparisons were made using the Kruskal–Wallis test with Dunn’s test as post hoc. The Student t-test was used to make comparisons between two samples with normal distribution and the Mann–Whitney U-test was used for nonparametric samples. The level of significance considered was 0.05. All analyses were conducted using BioEstat software, version 5.0.

Results The Comet assay results from the pilot test are presented in Fig. 1. Hemolymph samples were collected at exposure times of 0 h, 6 h, 12 h, 24 h, 48 h, and 1 week in this test and the Comet assay was performed with the aim of evaluating a possible effect of coal tailings on land snails’ DNA. The analysis of Comet assay values (mean ± SD) indicated a significant increase in the DI (p \ 0.05, Kruskal–Wallis–Dunn test) at 24 h, 48 h, and 1 week of exposure in relation to hour 0 (control). Samples of H. aspersa hemolymph were collected after 24 h, 48 h, 96 h, 1 week, 2 weeks, 3 weeks, and 1 month for all groups (Table 1). The data represent the mean and standard deviation for the DI and DF of the Comet assay


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relation to the control group, in both parameters of the Comet assay (DI and DF) at all exposure times (Student t-test or the U-test, with p \ 0.05, 0.01, and 0.001), showing a genotoxic potential of coal tailings for H. aspersa by both dermal and digestive exposition. Only the 2-week exposure time in the parameter of DF of mine lettuce group did not show significant results. Moreover, the single analysis of the coal tailings group showed that the 48-h exposure period led to a higher level of damage in the genome in relation to almost all other exposure times. The DI measured at the 48-h exposure period showed statistically significant results in relation to 96 h, 2 weeks, 3 weeks, and 1 month of exposure (Kruskal–Wallis–Dunn or ANOVA–Tukey, p \ 0.05 and 0.01). Similar to this are the results of the DI and in DF parameters in the mine lettuce group, which has shown increased levels of DNA damage in the exposure times of 48 h and 96 h in relation to the exposure time of 2 weeks (Kruskal–Wallis–Dunn or ANOVA– Tukey, p \ 0.05 and 0.01). In addition, for DF these times were also significantly higher in relation to the results obtained with samples collected at the 24-h, 2-week, 3-week, and 1-month exposure periods (Kruskal–Wallis–Dunn or ANOVA–Tukey, p \ 0.05 and 0.01), except for the 1-week samples, which was significantly different only in relation to

Fig. 1 Detection of DNA damage (mean ± SD) in hemolymph of snails exposed to mineral coal tailings in the pilot test. a Data significantly higher in relation to control (0 h) at p \ 0.05 (Kruskal– Wallis–Dunn)

scores. Table 1 shows the comparison between animals that during these times were exposed to direct contact with coal tailings or to lettuce grown in the mine in relation to the control group. The animals exposed to coal tailings and to mine lettuce showed a higher level of DNA damage in

Table 1 Mean values (±SD) observed in Comet assay (DI and DF) in cells of hemolymph of snails exposed to mineral coal tailings and/or mine lettuce Exposure time

Groups Negative control DI

Coal tailings DF

Mine lettuce

DI

DF c

24 h

30.4 ± 17.9

13.6 ± 6.2

80.2 ± 36.0

48 h

35 ± 27.7

14.6 ± 8.5

188.3 ± 72.2c,d

96 h 1 week

30.2 ± 17.9 44 ± 25.4

12.6 ± 5.4 18.1 ± 8.6

a

62.4 ± 31.9

b

87.5 ± 44.2

c

DI c

36.7 ± 14.0

59.2 ± 18.4c,d,e b

27.8 ± 11.3

b

35.8 ± 13.6

c

68.0 ± 9.0

DF b

27.3 ± 7.5b

231.7 ± 33.6c,f

71.7 ± 7.9c,g,h

168.2 ± 55.4

c,f

62.7 ± 13.4c,h

123.6 ± 36.6

c

50.0 ± 9.5c,i,j a

2 week

20.3 ± 18.3

11.2 ± 9.6

63.3 ± 45.8

25.5 ± 13.8

30.0 ± 13.46

14.4 ± 6.0

3 week

16.8 ± 11.7

8.7 ± 6.4

70.8 ± 58.5c

28.9 ± 16.7c

53.9 ± 20.0c

24.7 ± 6.5c,k

1 month

27.8 ± 19.6

13.6 ± 7.3

59.7 ± 29.2c

28.5 ± 11.3c

48.9 ± 32.4a

18.5 ± 9.6b

a

Significant data in relation to control group at the same exposure time at p \ 0.05

b

p \ 0.01 p \ 0.001(Student t-test or U-test)

c d

Significant data for the same group and parameter in relation to exposure times of 96 h, 2 weeks, 3 weeks, and 1 month, p \ 0.05 (Kruskal– Wallis–Dunn)

e

Significant data for the same group and parameter in relation to exposure times of 72 hs and 1 week, p \ 0.05 (Kruskal–Wallis–Dunn)

f

Significant data for the same group and parameter in relation to exposure times of 2 weeks, p \ 0.05 (Kruskal–Wallis–Dunn)

g

Significant data for the same group and parameter in relation to exposure times of 1 week, p \ 0.05 (ANOVA, Tukey)

h

Significant data for the same group and parameter in relation to exposure times of 24 h, 2 weeks, 3 weeks, and 1 month, p \ 0.01 (ANOVA, Tukey)

i j k

Significant data for the same group and parameter in relation to exposure times of 24 h, p \ 0.05 (ANOVA, Tukey) Significant data for the same group and parameter in relation to exposure times of 2 weeks, 3 weeks, and 1 month, p \ 0.01 (ANOVA, Tukey) Significant data for the same group and parameter in relation to exposure times of 2 weeks, p \ 0.05 (ANOVA, Tukey)

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1436 Mine lettuce

Limits of detection: Na = 871.50; Mg = 4.65; Al = 0.34; Si = 0.07; P = 3.00E-03; S = 3.00E-03; Cl = 1.15E-03; K = 9.14E-04; Ca = 2.60E-03; Ti = 9.75E-05; Mn = 5.50E-05; Fe = 1.02E-04; Ni = 1.13E-04; Cu = 4.03E-05; Zn = 3.63E-05; Sr = 4.51E-04. N.D = samples with content below of the limit of detection

5.09E-04 2.84E-04

1.35E-04 7.25E-05

9.85E-05 9.39E-05

5.86E-05 1.32E-03

2.24E-02 3.17E-04

4.60E-05 2.47E-04

4.55E-03 0.13

0.07 0.11

0.15 0.03

0.01 0.01

0.02 0.03

0.03 1.95

26.35 9.95

0.90 995.70 Organic lettuce

27.57

Mn (*) Ti Ca K Cl S P Si Al Mg Na Groups

15.31

Zn (*) Cu (*) Ni (*)

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Chemical elements

Coal mining is an activity with a high potential for environmental pollution. Coal has been described as the most significant pollutant of all the fossil fuels, containing a heterogeneous mixture of more than 50 elements, including oxides and other elements, like silica, polycyclic aromatic hydrocarbons (in products of liquefaction and combustion), metals, and ash (Leoń et al. 2007). Many of the elements described above, which are present in coal byproducts as well as in coal tailings, are rich in potentially toxic and genotoxic substances, which ultimately lead to profound changes in cells, tissues, populations, and ecosystems (Agostini et al. 1996; Sańchez-Chardi et al. 2007, 2008). Our results of the genotoxicity, shown in Fig. 1 and Table 1, demonstrate that both the coal tailing and the lettuce grown in the mine area can cause genetic damage, corroborating previous findings that described this harmful

Concentration in lettuce leaves (ppm)

Discussion

Table 2 Inorganic element detection by PIXE analyses in the lettuce leaves

the results observed in the 48-h samples (ANOVA–Tukey, p \ 0.05) (Table 1). Figure 2 presents only results of the DI for the control group with the coal tailing and mine lettuce groups, highlighting the difference between them. The results of the chemical analyses conducted by the PIXE technique to characterize metal contents in organic and mine lettuce offered to H. aspersa in the present study are shown in Table 2. Most of the chemical elements related with coal mining were present in the samples of both lettuces; however, the concentrations of chemical elements showed higher amounts in samples of mine lettuce, except for P. Among these, the most representative were Na, Mg, Al, and Si.

Fe (*)

Fig. 2 Mean (±SD) values of DI observed in hemolymph of snails exposed to mineral coal tailings. a Data significant in relation to the same exposure time of the control group at p \ 0.05; b p \ 0.01; c p \ 0.001(Student t-test or U-test)

N.D


Sr (*)

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effect using bacterial (Kleinjans et al. 1989; Whong et al. 1983) and human cells (Kleinjans et al. 1989; Le0 onard et al. 1984; Sra´m et al. 1985; Stierum et al. 1993) with different techniques, such as sister-chromatid exchange, HPRT expression, chromosomal abnormalities, and micronucleus. In a study with the Comet assay and micronucleus test, Silva et al. (2000) showed that coal and its byproducts cause DNA damage in different tissues of the Ctenomys torquatus rodent. For Leoń et al. (2007), the mammals Mus musculus and Rattus rattus exposed to coal mining areas showed a significantly higher value of DI and DF when compared to animals in the control area, also demonstrating the genotoxic effects of this mineral. In the study by Agostini et al. (1996) conducted in the workers of a mine in Criciu´ma (Santa Catarina), the data have shown the existence of an increase risk of mutagenicity observed in the high levels of micronuclei in oral cells and chromosomal damage in peripheral blood cells. This and other works conducted on genotoxicity of coal and its byproducts could not point to one single causal agent of genotoxic damage due to biological and environmental interactions involved. However, the metals and pyrite are some of the agents identified as the cause of observed damage (Beyersmann and Hartwig 2008; Cohn et al. 2006). In our study, we verified the presence of some chemical elements, including heavy metals with higher concentration in mine lettuce group than in organic lettuce group, with special relevance to Al. Although the Al is not considered a heavy metal, the Al toxicity to all forms of life is recognized, as reported by several authors (Bishop et al. 1997; Exley et al. 1991; Vasishta and Gill 1996). Several in vitro studies have shown that Al can induce DNA crosslinking in rat ascites hepatoma cells (Wedrychowski et al. 1986) micronuclei and chromosome aberrations in human peripheral blood lymphocytes (Banasik et al. 2005; Migliore et al. 1999; Roy et al. 1990). Virtually all metals can produce toxicity when ingested in sufficient quantities, but several elements, like Pb, Fe, Cu, Mn, Cd, Ni, and Al, are especially important because they either are very pervasive, or produce toxicity at considerably low concentrations (Beyersmann and Hartwig 2008). Villatoro-Pulido et al. (2009) detected genotoxicity in a vegetable grown in soils contaminated with metals. This vegetable presented contamination in the roots and shoots, constituting a risk to the entire food chain due to adverse effects of metals. Metals, in general, are known to cause harmful effects in the environment and in human health when imbalanced (Walker et al. 2006). The environmental contamination with compounds containing metals is a problematic issue, because they have high toxicity, the ability for bioaccumulation, and the potential to induce damage to genetic material (Pra´ et al. 2006). The toxicity of metals and their

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compounds largely depends on their bioavailability; in addition, some metal compounds even undergo metabolic transformation. In general, metal genotoxicity is caused by indirect mechanisms. In spite of diverse physicochemical properties of metal compounds, three predominant mechanisms are observed: (1) interference with cellular redox regulation and induction of oxidative stress, which mighty cause oxidative DNA damage or trigger signaling cascades leading to stimulation of cell growth; (2) inhibition of major DNA repair systems, resulting in genomic instability and accumulation of critical mutations; (3) deregulation of cell proliferation by induction of signaling pathways or inactivation of growth controls such as tumor suppressor genes (Beyersmann and Hartwig 2008). The high levels of acidification resulting from the oxidation of pyrite might cause dissolution of aluminosilicate minerals, increase the concentration of metals such as Al, Fe, Mn, Cu, Ni, and Zn to toxic levels (Campos et al. 2003). Pyrite is the most abundant and dominant sulfide in coal and its byproducts. Earlier studies have shown that pyrite/aqueous suspensions generate H2O2 in the absence of oxygen and during pyrite oxidation. Although the formation of H2O2 has been established in pyrite suspensions, its fate is not clear. Minerals that induce the formation of • OH have been shown to cause strand DNA breakage, which is of interest in genetic toxicologic research. This radical preferentially attacks the DNA or RNA molecules, for the addition or deletion of bases, both resulting in depolymerization (Cohn et al. 2004, 2006). As we have a common pathway of both genotoxic agents, metals and pyrite interact in a harmful way with the DNA molecule is through the formation of ROS. In Fig. 2 we can observe a peak of DNA damage tailings coal (48 h) and lettuce mine (48 h and 96 h) groups in relation to almost all other exposure times, suggesting that the DNA damage induced by coal tailings is reversible or that the animals could adapt with time. Our results behaved similarly to the findings of Guo et al. (2008), who investigated the oxidative DNA damage by Comet assay in mouse peripheral leukocytes; the total antioxidant enzymes capacity and ROS in whole blood were also detected. An increase in DNA damage in peripheral leukocytes was observed, reaching a peak 1 h after the administration of the oxidizing agent, when compared to the control group. Within 24 h of acute administration, the DNA damage was repaired and decreased to the same levels as those of the control group, showing that antioxidant capacity depends on the oxidative damage. The increased antioxidative ability might result in a new balance between DNA damage and the activities of the antioxidative system, this last perhaps playing an important role in DNA repair. Second is the tolerance of the stressor agent, which in our study was the coal tailing.

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In the present study, the repair system could be observed along the exposure times. After 48 h (coal tailings group) and 96 h (mine lettuce group) of exposure, a decrease in the genotoxic damage was observed, but these values remained significantly high in relation to the control group, probably due to the repair system, which over time of exposure seems to be able to act more effectively, demonstrating that it is not be fully inactivated by metals. However, even though the DNA damage would be repaired, it should be kept in mind that the possibility of mutations and cancer development might increase as a result of error-prone repair processes (Kido et al. 2006). Our study did not attempt to define which specific contaminants in the coal mining were responsible for genetic damage, but it was to detect overall genotoxic effects from multiple contaminants, because this anthropic activity is subjected to multiple sources of environmental pollution.

Conclusion The present study contributes to the evaluation of genotoxic activity of coal tailings, a byproduct of the coal mining industry, using H. aspersa as a measure to indicate possible effects on human health. Our work provides biological data of this land snail exposed to coal tailings and fed on lettuce grown in the coal mine, confirming the sensitivity of the Comet assay in the assessment of complex mixtures. Also indicated is the association between the chemical elements, mainly Al, from the extraction of coal, as the main cause of DNA damage in cells of hemolymph of this terrestrial mollusk. However, these are passive to the damage repair, leading these organism to adapt. We also suggest oxidative stress as the main agent causing the damage observed. Thus, these results demonstrate that the coal tailings coming from extraction and beneficiation of coal is potentially genotoxic to H. aspersa and probably to other trophic levels and that this mollusk is a good biomonitor of environmental genotoxicity, both in the dermal and digestive pathways. The vegetable Lactuca sativa L. presented genotoxicity when grown near impacted environments by coal mining and caused health hazards when consumed. Acknowledgments The present work was supported by grants from the Postgraduate Program in Health Sciences, University of Southern Santa Catarina (UNESC) and National Counsel of Technological and Scientific Development (CNPq).

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