Assessment of DNA Damage on Columbian Coal Miners

Da Silvia et al. (2010) evaluated genotoxic effects in a population exposed to coal residues from the open-cast mine in “El Cerrejón” Columbia using a cytokinesis-blocked micronucleus test and a comet assay. The 200 individuals tested had no known exposure to previous toxins like coal, radiation, chemicals or cigarettes. One hundred of the individuals were workers exposed to and working in four different mining activities and one hundred individuals were non-exposed control individuals were which acted as controls. Blood samples were taken to investigate biomarkers of genotoxicity; specifically, primary DNA damage, tail length, and percent of tail. Both biomarkers showed statistically significantly higher values in the exposed group compared to the non-exposed control group. No difference was observed between the exposed groups executing different mining activities. These results indicate that exposure to coal mining residues may result in an increased genotoxic exposure in coal mining workers. The study did not find a correlation between age, alcohol consumption and service time with the biomarkers of genotoxicity.—Rosemary Kulp
          Da Silva, J.  Espitia-Pérez , L. Hartmann, A. Henriques, J. Hoyos-Giraldo, L. León-Mejía, G. Quintana, M. 2010.  Assessment of DNA damage in coal open-cast mining workers using the cytokinesis-blocked micronucleus test and the comet assay. doi:10.1016/j.scitotenv.2010.10.049 2010

Coal is one of the most abundant minerals in nature and is one of the largest fossil fuel sources of energy. Colombia, a country in South America, has large natural coal reserves and “El Cerrejón” located in northern Guajira, is the world’s largest open-cast mine.  Coal residues consist of a mixture of substances, containing carbon, hydrogen, nitrogen, oxygen and sulfur. Some basic parameters of coal from “El Cerrejón” are: total moisture (~10%), volatiles (~30%), ash (~8%), sulfur (~1%), carbon (~70%), hydrogen (~6%), oxygen (~5%), and nitrogen (~1%). The main operations carried out in “ El Cerrejón “ are extraction of coal, and mincing coal for transportation.  During coal extraction large quantities of particles of coal dust are emitted and when it is exposed to oxygen and sunlight a spontaneous combustion can occur sending large clouds of polycyclic aromatic hydrocarbons (PAHs) into the environment.
Chronic inhalation of complex mixtures, containing substances such as heavy metals, ash, iron, PAHs and sulfur, can result in lung disorders including pneumoconiosis, progressive massive fibrosis, bronchitis, loss of lung function, emphysema. In addition to direct cellular damage, compounds like PAHs have mutagenic properties that have been associated with an increased risk for cancer development. However, coal dust still remains classified as a non carcinogen for humans according to the International Agency for Research on Cancer.
The primary target cells of inhaled coal dust particles are macrophages and epithelial cells. Activated macrophages (phagocytosis toxicity) produce excessive amounts of reactive oxygen species (ROS) and cytokines.  Epithelial cells and fibroblasts which are the main producers of components of the extracellular matrix including collagens, proteoglycans and elastic fibres, are also known to produce cytokines and ROS upon stimulation. Additional phagocytotic cell may be recruited by chemokines produced by the alveolar macrophages as well as epithelial cells, and may amplify local production of ROS and cytokines. Both ROS and cytokines may cause damage or proliferation of local epithelial and mesenchymal tissue and may as such have consequences to lung tissue morphology, and cell turnover. When there is excessive production of ROS, or when there are insufficient defense mechanisms, oxidative stress may result in DNA damage, lipid peroxidation, protein modification, membrane disruption, and mitochondrial damage, all of which capable of affecting cytogenetic damage levels.
These types of DNA damages are usually induced by most of the genotoxic agents which induce DNA breaks at the phosphodiester skeleton or between bases and sugars resulting in abasic sites identifiable under microscopic inspection.
Coal dust has been evaluated in real world field tests in wild rodents in coal mining areas in Brazil and in Colombia however this is the first field test on human exposure to coal ash in Columbia thus far. The exposed group was divided into four groups: Extracted coal transport (n=50) in which the workers are involved in coal transport up to the arrival in the storing centers; equipment field maintenance (n=18) the drivers that spread water on the roads where large quantities of coal dust are generated,  coal stripping (n=17) where workers accumulate the coal material after it’s been stripped for transportation (and put out the fires caused by coal spontaneous combustion), and coal embarking (n=15) where the workers ship coal in containers to be exported to other countries.
All the individuals included in the study were non-smokers. The exposed group was composed of volunteers between the ages of 24 and 60, medically evaluated and deemed healthy for at least five years at the time of the assessment, with no previous recorded of prescriptions that are known or suspected to have mutagenetic properties.  Peripheral blood samples from all 200 individuals were collected by venipuncture with 20 mL of blood being drawn.  Thirty micro-liters of isolated lymphocytes were mixed with 270 μL 0.5% of LMA-Invitrogen at 37 °C. This mixture was placed into a slide previously coated with 1.5% of agarose(NMA-Cambrex Bioscience Rockland) and processed at 60 °C. The slides were immersed overnight in lysis solution (2.5 M NaCl, 100 mM EDTA and 10 mM Tris, pH 10.0–10.5, 1% with freshly added 1% Triton X-100 and 10% DMSO) at 4 °C in the dark. Afterwards, the slides were placed for 30 minutes in an alkaline buffer at 4 °C (300 mM NaOH and 1 mM EDTA, pHN13) in order to unwind the DNA. The alkaline electrophoresis was carried out for 30 min at 25 V and 300 mA. This standard alkaline procedure allows single-strand DNA breaks to be detected and lesions are converted to strand breaks under these conditions as well. The gels were neutralized with 0.4 M Tris (pH 7.5) with 3 washes of 5 min each. Finally, the slides were stained with 50 μL ethidium bromide (2 μL/mL) and examined at 40× magnification under a fluorescence microscope.
For each individual 50 cells from each of two replicate slides were analyzed. The cells were classified according to tail size into five classes ranging from undamaged (0) to maximally damaged (4). The damage index (DI) calculation was carried out according to a pre-existing visual classification system. The values for the damage index could range from 0 (100 cells all at class 0) up to 400 (100 cells all at class 4).
The second test, the MN test using the cytokinesis-block technique was chosen because it allows reliable data scoring because only the MN of those cells that have completed one nuclear division are analyzed. Cultures were prepared with whole blood in duplicate. Cultures were incubated at 37 °C in the dark for 46 hours, under 5% CO2 in a humidified atmosphere. Two parallel cultures were then set in tubes for each sample. The cells were harvested after 72 hours before being treated with a hypotonic solution (0.075 M KCl) and were immediately centrifuged and fixed three times with methanol/acetic acid. The fixed cells were then dropped onto humidified slides and MN registration was performed on coded slides in a double blind test.
The mean values of both biomarkers parameters in the exposed group demonstrated significant differences when compared to the values of the non-exposed control group (p > 0.001) using the non-parametric Mann Whitney U-test. The Spearman correlation coefficients for MN frequency with age, and DNA damage (tail length, % of tail DNA, DI) for non-exposed control and exposed groups were not significant (p>0.05). The correlations between MN frequency with time of service, and DNA damage with respect to tail length, % of tail DNA and DI, for the subdivided exposed groups, were not significant (p>0.05). The effect of alcohol consumption on MN frequencies and DNA damage in all groups using Mann Whitney U-test and was not found to be statistically significant in any of the groups (p>0.05). There was also no statistically significant difference between the DNA damage between the four different coal mining activities (pN0.05) using a t-test.

The results obtained in Micronucleus frequency show that the values in the exposed to coal mining residuals group (8.6±4.8) are higher compared with the non-exposed control group (2.9±4.0). This clearly demonstrates higher levels of DNA damage in the exposed group (mean tail length=23.4±6.5; mean% of tail DNA=13.1±7.9; mean DI=60.0±39.5) compared to the nonexposed control group (mean tail length=14.3±2.5; mean% of tail DNA=2.9±1.5; mean DI=9.0±6.4). These differences were all statistically significant in the evaluation using a Mann Whitney U-test (p<0.001).

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