Assessment of dna damage in coal open-cast mining workers using the cytokinesis-blocked micronucleus test and the comet assay

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s c i to t e n v Assessment of DNA damage in coal open-cast mining workers using thecytokinesis-blocked micronucleus test and the comet assay Grethel León-Mejía Lyda Espitia-Pérez , Luz Stella Hoyos-Giraldo , Juliana Da Silva ,Andreas Hartmann , João Antônio Pêgas Henriques ,, Milton Quintana a Laboratorio de Investigación Biomédica y Biología Molecular, Universidad del Sinú, Montería, Córdoba, Colombiab Department of Biology, Research Group Genetic Toxicology and Cytogenetics, Faculty of Natural Sciences and Education, Universidad del Cauca, Popayán, Cauca, Colombiac Laboratorio de Genética Toxicológica (PPGGTA), Universidade Luterana do Brasil (ULBRA), Canoas-RS, Brazild Novartis Pharma AG, Basel, Switzerlande Departamento de Biofísica, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil Coal mining is one of the most important causes of environmental pollution, as large quantities of coal dust particles are emitted. Colombia-South America has large natural coal reserves and “El Cerrejón” is the world's largest open-cast mine located in the northern department of Guajira. The aim of the present study was to evaluate genotoxic effects in a population exposed to coal residues from the open-cast mine “El Cerrejón”. 100exposed workers and 100 non-exposed control individuals were included in this study. The exposed group was divided according to different mining area activities: (i). Transport of extracted coal, (ii). Equipment field maintenance, (iii). Coal stripping and, (iv). Coal embarking. Blood samples were taken to investigate biomarkers of genotoxicity, specifically, primary DNA damage as damage index (DI), tail length and% of tail DNA using the Comet assay (alkaline version) and chromosome damage as micronucleus (MN) frequency in lymphocytes. Both biomarkers showed statistically significantly higher values in the exposed group comparedto the non-exposed control group. No difference was observed between the exposed groups executingdifferent mining activities. These results indicate that exposure to coal mining residues may result in anincreased genotoxic exposure in coal mining workers. We did not find a correlation between age, alcoholconsumption and service time with the biomarkers of genotoxicity. Our results are the first data of genotoxiceffects induced by coal mining exposure in Colombia, and thus, contribute to the exploration of test batteriesuse for monitoring of exposed populations and may stimulate designing control, hygiene and preventionstrategies for occupational health risk assessment in developing countries.
2010 Elsevier B.V. All rights reserved.
caloric power of this mineral, when it is exposed to ambient oxygenand sunlight a spontaneous combustion process may be initiated Coal is one of the most abundant minerals in nature and it which liberates large amounts of Polycyclic Aromatic Hydrocarbons constitutes the largest fossil fuel source used for the generation of (PAHs) into the environment. Coal residues consists of a mixture of energy. However, its extraction and use constitute an important substances, containing carbon, hydrogen, nitrogen, oxygen and sulfur pollution factor which represents a major threat for human health and (mineral particles of smaller size and inorganic extraction large quantities of particles of coal dust are emitted Colombia in South America has one of the world's largest natural contributing to environmental pollution. In addition, due to the high coal reserves in Latin America and “El Cerrejón” is the world's biggestopen-cast mine located in the northern department of Guajira (The main operations carried out in this mine are: stripping ⁎ Corresponding authors. Quintana is to be contacted at Laboratorio de Investigación (extraction of coal) and crushing (mincing of coal for transporting).
Biomédica y Biología Molecular, Universidad del Sinú, Campus Elías Bechara Zainúm,Calle 38 Cra.1W Barrio Juan XXIII, Montería, Córdoba, Colombia, South America. Tel./ During the extraction processes in coal open-cast mining, particulate fax: + 57 4 7841961. Henriques, Departamento de Biofísica, Prédio 43422, Laboratório matters and combustion products are released into the atmosphere, 210, Campus do Vale, Universidade Federal do Rio Grande do Sul, Avenida Bento where they constitute complex mixtures (These Gonçalves 9500, Bairro Agronomia-CEP 91501-970, Porto Alegre, RS, Brazil. Tel.: +55 mixtures are considered hazardous due to synergistic, additive and 0048-9697/$ – see front matter 2010 Elsevier B.V. All rights reserved.
doi: G. León-Mejía et al. / Science of the Total Environment 409 (2011) 686–691 The predominant route of coal mining residues exposure is Exposed workers were matched to non-exposed controls by age through inhalation. Today it is known that chronic inhalation of (±2 years) and similar social-economic status. Confounding and complex mixtures, containing substances such as heavy metals, ash, exclusion factors were collected from all participants who responded iron, PAHs and sulfur, can result in lung disorders including simple to an interviewer-administered, detailed, standard questionnaire pneumoconiosis, progressive massive fibrosis, bronchitis, loss of lung which included data of health status, cancer history, other chronic diseases, lifestyle, nutrition, smoking habits, medication intake, and Recent studies have postulated that some of frequency of alcohol consumption (total number of drinks and the these diseases may be a consequence of inhaling such material most widely alcoholic beverages consumed), occupational and time of resulting in activation of macrophages, interaction with epithelial service, protective measures, and previous exposure to medical X-rays cells and other cells, finally leading to generation of oxidative stress or treatment with known carcinogens. All individuals included into ). Besides direct cellular damage, compounds the study were non-smokers. The exposed group was selected released by coal mining activities such as PAHs present an important according to the following inclusion approaches: voluntary accep- mutagenic hazard that has been associated with an increased risk for tance, been healthy and time of service ≥5 years. Exclusion criteria for exposed and non-exposed groups were age over 60 years or less than findings, coal dust remains classified as non carcinogen for human 24 years, smoking (current and ex-smoking habits), medical treat- (Group 3) in International Agency for Research on Cancer ( ment up to 3 months or X-ray up to 1 year before sampling and therapeutic drugs intake, known to be mutagenic. All data was Biomonitoring studies in peripheral lymphocytes of coal workers organized and recorded in databases. There are no major differences demonstrated increased adduct formation and increased of non- regarding social-economic status or dietary habits were identified.
cellular and cellular sources of reactive oxygen species that can induce The whole study population was informed about the aim, benefits, risks and methodology details of the study though the informed There are only a few studies on the occupational hazard effects in consent, which was obtained from all individuals. We previously had coal miners. However, some studies have been conducted in animals the approval of the University of Sinú Ethic Committee for this of mining regions. For example, cytotoxic and genotoxic effects of coal research study. All the information identifying the study individuals is dust have been evaluated in vivo in wild rodents in coal mining areas kept at the “Laboratorio de Investigación Biomédica y Biología Molecular” of the University of Sinú in the city of Montería, Cordoba The aim of the present study was to evaluate potential genotoxic department, which is the only institution having full identifiable effects in peripheral blood lymphocytes in an exposed population to coal residues in the open-cast mine “El Cerrejón” in Guajira-Colombiausing Comet assay and Micronucleus (MN) test. The results obtained from this study present the first data for Colombia on a genotoxichazard generated by coal mining residuals exposure. In addition, our After informed consent was obtained from each individual, data represent an assessment of the feasibility of using tool peripheral blood samples from all 200 individuals were collected by biomarkers for evaluating potential occupational health risks. An venipuncture. 20 mL of blood were drawn into heparin tubes (Becton area of application of such methods is to investigate whether it can be Dickenson, vacutainer) for the Comet assay and MN test. All blood applied in coal mining activities in Colombia to support efforts for samples tubes were coded and kept in upright position at room applying cleaner production procedures which would have less temperature in dark during the transportation overnight to the negative impacts on the environment and the human population.
laboratory, where the samples were processed immediately uponarrival.
The Comet assay in the present study was carried out according to the original methodology (alkaline version) described by The exposed individuals included in this study were workers from “El Cerrejón” open-cast coal mine, in Guajira Department in the north coast of Colombia, South America. A group of 100 exposed individuals lymphocytes by Histopaque 1077, were mixed with 270 μL 0.5% of engaged in surface activities of the sub-bituminous open-cast coal low melting point (LMA-Invitrogen) at 37 °C. This mixture was placed mine with a minimum time of service of 5 years were selected. The into a slide previously coated with 1.5% of normal melting point non-exposed control group consisted of 100 individuals, from Guajira agarose (NMA-Cambrex Bioscience Rockland) processed at 60 °C. The department, with no known exposure to genotoxic agents including agarose layers were covered with a cover slip and after gel solidifying coal, radiation, chemicals or cigarettes. Both study populations the cover slips were removed. The slides were immersed overnight in (exposed and non-exposed group) lived in the same region lysis solution (2.5 M NaCl, 100 mM EDTA and 10 mM Tris, pH 10.0– considering that the two populations should have the same genetic 10.5, 1% with freshly added 1% Triton X-100 and 10% DMSO) at 4 °C in background and the same life habits.
dark. Afterwards, the slides were placed for 30 min in alkaline buffer The exposed group was divided according to the engaged activities at 4 °C (300 mM NaOH and 1 mM EDTA, pH N 13) to unwind the DNA.
in the mine: (i) Extracted coal transport (n = 50) in which the workers The alkaline electrophoresis was carried out for 30 min at 25 V and are involved in coal transport up to the arrival in the storing centers.
300 mA. This standard alkaline procedure allows single-strand DNA (ii) Equipment field maintenance (n = 18) these workers drive trucks breaks to be detected and alkali labile lesions (i.e., apurinic/ to spread water in the roads where large quantities of coal dust are apirimidinic sites) are converted to strand breaks under these generated the workers also fix the coal extraction equipment. (iii) conditions as well. The gels were neutralized with 0.4 M Tris (pH Coal stripping (n = 17) these workers are engaged in coal stripping 7.5) with 3 washes of 5 min each. Finally, the slides were stained with activities and the accumulation of the material for the transport in 50 μL ethidium bromide (2 μL/mL) and examined at 40× magnifica- trucks, they also extinguish fires generated by coal spontaneous tion under a fluorescence microscope equipped with a green filter of combustion. (iv) Coal embarking (n = 15) these workers are involved 540 nm. Direct light exposure of the samples was avoided during the in shipping of coal in containers to be exported to other countries.
whole process. For each individual we analyzed 100 randomly G. León-Mejía et al. / Science of the Total Environment 409 (2011) 686–691 selected Comets (50 cells from each of two replicate slides). For the analysis of the images we used a system image analysis software and Main demographic characteristics of the studied population: non-exposed control andexposed groups.
the considered endpoints as measurement to quantify DNA damage,were tail length and % of tail DNA. In addition, the cells were classified according to tail size into five classes ranging from undamaged (0) to maximally damaged (4), obtaining a measure of the individual damage for each animal and consequently for each analyzed group.
The damage index (DI) calculation was carried out according to the visual classification system (). The values for the damage index could range from 0 (100 cells class 0) up to 400 (100 cells class 4). For the statistical analysis we took into account the meanvalues for all the Comet assay parameters.
⁎ Drink more than three beers/day or drink in excess of once a week.
The MN test using the cytokinesis-block technique was performed deviation (SD) of the exposed group was 17.7 ± 6.9 years (range, 5– This approach allows reliable data scoring shows the percentage of alcohol consumption for non- because only the MN of those cells that have completed one nuclear exposed group (45%) and exposed group (55%), considered as alcohol division are analyzed. Cultures were prepared with whole blood in consumer that drink more than three beers per day or drink in excess duplicate and processed as described by .
Heparinized whole blood (0.5 mL) was added to 4.5 mL of RPMI summarizes the Comet assay data and Micronucleus 1640 medium (Sigma R8758, USA) supplemented with 2 mM frequency values for both study groups. The mean values of both glutamine (Sigma A5955,USA), 10% fetal bovine serum (Gibco/ biomarkers parameters in the exposed group demonstrated signifi- Invitrogen 15000-044, Brazil), 100 μL/mL antibiotic–antimycotic cant differences when compared to the values of the non-exposed (Sigma A5955, USA) and 2% phytohemaglutinin (Sigma L8754, USA) control group (p b0.001), which were analyzed using Kruskal–Wallis to stimulate the lymphocytes. Cultures were incubated at 37 °C in test (Dunn correction). There was no statistically significant differ- ence between the four different coal mining activities (p N 0.05).
2 in a humidified atmosphere. Two parallel cultures were set in tubes (Falcon 3033) for each sample. Cytocalasin The results obtained in Micronucleus frequency show that the B (Sigma, C6762) was added at 44 h of incubation at a final values in the exposed to coal mining residuals group (8.6 ± 4.8) are concentration of 6 μg/mL. The cells were harvested at 72 h, treated higher compared with the non-exposed control group (2.9 ± 4.0). The with hypotonic solution (0.075 M KCl) immediately centrifuged and differences were significant as is shown in evaluated through fixed three times with methanol/acetic acid (3:1). The fixed cells were Mann Whitney U-test (p b 0.001).shows the mean values of dropped onto humidified slides and air dried slides were stained with the Comet assay parameters of the two study groups. This compar- Giemsa for 10 min. MN registration was performed on coded slides ison, for each Comet assay parameter damage index, tail length and% with double blind. Two thousand binucleated cells (BN) per individual of tail DNA, clearly demonstrates higher levels of DNA damage in the (1000 BN per culture) were registered. All coded slides were analyzed exposed group (mean tail length = 23.4 ± 6.5; mean% of tail with an optical light microscope (40× magnification). All slides were DNA = 13.1 ± 7.9; mean DI = 60.0 ± 39.5) compared to the non- scored by one reader blinded to the exposure status of the individuals.
exposed control group (mean tail length = 14.3 ± 2.5; mean% of tail The scoring criteria followed those proposed by DNA = 2.9 ± 1.5; mean DI = 9.0 ± 6.4). These differences were allstatistically significant evaluated using Mann Whitney U-test(p b 0.001).
The Spearman correlation coefficients for MN frequency with age, and DNA damage (tail length, % of tail DNA, DI) for non-exposed The normality of the variables was evaluated using the Kolmo- control and exposed groups were not significant (p N 0.05). The gorov–Smirnov test; χ2 and t-tests were used to compare the correlations between MN frequency with time of service, and DNA demographic characteristics of study populations. The statistical damage (tail length, % of tail DNA and DI) for the subdivided exposed analysis of differences in MN test and DNA damage measured by groups, were not significant (p N 0.05). We analyzed the effect of Comet assay were carried out using the non-parametric Mann alcohol consumption on MN frequencies and DNA damage in all Whitney U-test and statistical differences between the five groups groups using Mann Whitney U-test and found no difference in any of (non-exposed control, extracted coal transport, equipment field maintenance, coal stripping, and coal embarking) were analyzedusing the non-parametric two-tailed Kruskal–Wallis test with the Dunn correction for multiple comparisons to perform a non-parametric analysis of variances. Correlations between different Occupational exposure to coal residues is a public health concern variables were determined by Spearman rank correlation test as in developing countries owing to the lack of regulation policies and appropriate. The critical level for rejection of the null hypothesis was epidemiologic surveillance programs and to the limited coal combus- considered to be p value of 5%. All analyses were performed with the tion residues and particulate matter emission management PRISMA 5.0 statistical software package.
During coal open-cast miningextraction processes significant amounts of these substances are released to the atmosphere where they constitute complex mixtures(). The main route of exposure to these particulate The main demographic characteristics of the study population are matters is through inhalation. Most of the published studies have shown in . The mean age of exposed group was 44.0 ± 7.5 years (range, 24–60 years), and non-exposed control group was 43.7 ± 7.8 years (range, 27–60 years).The mean time of service ±standard evaluated induction of chromosome aberrations by coal complex G. León-Mejía et al. / Science of the Total Environment 409 (2011) 686–691 Table 2Comet assay and Micronucleus test parameters in non-exposed control and Exposed group divided by work areas activities (mean ± standard deviation).
⁎ Significant difference in relation to the non-exposed control group; Kruskall Wallis–Dunn correction pb0.001.
mixtures in open-cast mining workers. Colombia possesses the largest matrix components. The formation and degradation of extracellular coal reserves in Latin America (however, to date, matrix may also be affected by ROS, as well as proteases and genotoxic endpoints related to coal open-cast extraction has only antiproteases produced by the various cell types present in the surrounding tissue ). When there is excessive In the present study we investigated potential genotoxic effects of production of ROS, or when there are insufficient in vivo defense coal exposure in mining workers. Biological monitoring of exposure to mechanisms, oxidative stress may occur. This stress may result in DNA chemical substances in the workplace is crucial to assess potential damage, lipid peroxidation, protein modification, membrane disrup- human health risks, as an integral strategy to improve occupational tion, and mitochondrial damage ), all modifications health, safety conditions and life quality in developing countries. We capable of affecting cytogenetic damage levels.
applied the Comet assay to compare the extent of primary DNA Other components associated to heavy metals toxicity such as damage and the Micronucleus test as cytogenetic effect biomarker in Copper, Lead, Cadmium, Nickel, Vanadium and Zinc appear in high peripheral blood lymphocytes from exposed and non-exposed concentrations in coal complex mixture (Large amounts of mutagenic compounds such as PAHs are produced during Our results demonstrate that the group exposed to coal mining the spontaneous coal combustions which is a common process in coal residuals exhibited a significantly higher extent of DNA damage in storing centers due to environmental factors as sunlight and climate peripheral lymphocytes in the Comet assay and MN test compared to conditions. Some PAHs related to coal mining such as chrysene and the control group. Previous studies with underground coal mine benzo(k)fluoranthene are indicators of coal combustion, and pyrene workers have demonstrated higher levels of chromosomal damage, and fluoranthene are associated with such processes ). PAHs evaluated through chromosomal aberration, MN and sister chromatid can induce DNA lesions as single-strand breaks via DNA repair ). There are no significant differences in ) and electrophilic metabolites that covalently interact with the DNA damage average (Comet assay and MN test) of control group were detected between this study and previous studies of our group with purines, especially with guanine after metabolic activation by It is known that coal mining activities release significant quantities Due to the complex mixture in occupational coal mining environ- of fugitive particles and toxic gases as sulfur dioxide into the ments it is difficult to relate genotoxic effects to a specific agent or environment ). Some quality parameters of coal from compound. The Comet assay is particularly sensitive towards the the open-cast mine “El Cerrejón” are total moisture (~10%), volatiles detection of direct and indirect DNA strand breakage and DNA (~30%), ash (~8%), sulfur (~1%), carbon (~ 70%), hydrogen (~6%), alkaline-labile sites. These types of DNA damages are usually induced oxygen (~ 5%), nitrogen (~1%), as well as different metals by most of the genotoxic agents which induce DNA breaks at the phosphodiester skeleton or between bases and sugars resulting in coal complex mixture release into the atmosphere () abasic sites (The MN assay in peripheral blood constitute one of the most important occupational health and safety lymphocytes is extensively used in biomonitoring and molecular risks of workers due to the potential synergistic toxic effects of this epidemiology studies to evaluate the presence and the extent of chromosomal damage in exposed human populations to genotoxic has suggested that reactive oxygen species (ROS) and their products agents, and also because an increased MN frequency has been are involved in the pathogenesis of lung disorders and cancer risk validated as a cancer risk biomarker in humans ( exposed workers. The primary target cells of inhaled coal dust In our study no significant difference in markers of genotoxicity particles are macrophages and epithelial cells. Activated macrophages between the four coal mine activities (extracted coal transport, (phagocytosis toxicity) produce excessive amounts of ROS and equipment field maintenance, coal stripping and coal embarking) was cytokines. ROS may additionally be generated via cell-independent observed. These observations indicate that exposed workers have a mechanisms due to intrinsic chemical properties of the coal dust (e.g.
comparable genotoxic response to complex mixture exposure surface radicals and iron). Epithelial cells and fibroblasts which are the independently to the area and activity in the mine. In addition to main producers of components of the extracellular matrix including these findings, there was no correlation between the time of service collagens, proteoglycans and elastic fibres, are also known to produce and Comet assay parameters or MN frequency. This observation is in cytokines and ROS upon stimulation. Additional phagocytotic cells agreement with the results obtained in other occupational settings (neutrophils, monocytes/macrophages) may be recruited by chemo- kines produced by the alveolar macrophages as well as epithelial cells, In our results, there was no influence of alcohol consumption, age and may amplify local production of ROS and cytokines. Both ROS and or time of service in the exposed individuals on Comet assay cytokines may cause damage or proliferation of local epithelial and parameters tail length, % of tail DNA and damage index or the MN mesenchymal tissue and may as such have consequences to lung tissue morphology, cell turnover and deposition of extracellular The results obtained with respect the alcohol consumption are in G. León-Mejía et al. / Science of the Total Environment 409 (2011) 686–691 agreement with other occupational studies in which there was no Fenech M. The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxicity studies in human populations. Mutat association between the alcohol consumption and the levels of DNA Fenech M. Micronucleus frequency in human lymphocytes is related to plasma vitamin B12 and homocysteine. Mutat Res 1999;428:299–304.
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