This thesis describes the development and use of a computer-automated microscopy routine for characterization of respirable dust particles from coal mines. Respirable dust in underground coal mining environments has long been known to pose an occupational health hazard for miners. Typically following years of exposure, coal workers' pneumoconiosis (CWP) and silicosis are the most common disease diagnoses. Although dramatic reductions in CWP and silicosis cases were achieved across the US between about 1970-1999 through a combination of regulatory dust exposure limits, improved ventilation and dust abatement practices, a resurgence in disease incidence has been noted more recently – particularly in parts of Appalachia. To shed light on this alarming trend and allow for better understanding of the role of respirable dust in development of disease, more must be learned about the specific characteristics of dust particles and occupational exposures. This work first sought to develop an automated routine for the characterization of respirable dust using scanning electron microscopy with energy dispersive x-ray (SEM-EDX). SEM-EDX is a powerful tool that allows determination of the size, shape, and chemistry of individual particles, but manual operation of the instrument is very time consuming and has the potential to introduce user bias. The automated method developed here provides for much more efficient analysis – with a data capture rate that is typically 25 times faster than that of the manual method on which it was based – and also eliminates bias between users. Moreover, due to its efficiency and broader coverage of a dust sample, it allows for characterization of a larger and more representative number of particles per sample. The routine was verified using respirable dust samples generated from known materials commonly observed in underground coal mines in the central Appalachian region, as well as field samples collected in this region. This effort demonstrated that particles between about 1-9μm were accurately classified with respect to defined chemical categories, and suggested that analysis of 500 particles across a large area of a sample filter generally provides representative results. The automated SEM-EDX routine was then used to characterize a total of 210 respirable dust samples collected in eight Appalachian coal mines. The mines were located in three distinct regions (i.e., northern, mid-central and south-central Appalachia), which differed in terms of primary mining method, coal seam thickness and mining height, and coal and/or rock mineralogy. Results were analyzed to determine whether number distributions of particle size, aspect ratio, and chemistry classification vary between and within distinct mine regions, and by general sampling location categories (i.e., intake, feeder, production, return). Key findings include: 1) Northern Appalachian mines have relatively higher fractions of coal, carbonate, and heavy mineral particles than the two central Appalachian regions, whereas central Appalachian mines have higher fractions of quartz and alumino-silicate particles. 2) Central Appalachian mines tended to have more mine-to-mine variations in size, shape, and chemistry distributions than northern Appalachian mines. 3) With respect to particle size, samples collected in locations in the production and return categories have the highest percentages of very small particles (i.e., 0.94-2.0μm), followed by the feeder and then the intake locations. 4) With respect to particle shape, samples collected in locations in the production and return categories have higher fractions of particles with moderate (i.e., length is 1.5 to 3x width) to relatively high aspect ratios (i.e., length is greater than 3x width) compared to feeder and intake samples. 5) Samples with relatively high fractions of alumino-silicates have higher fractions of particles with moderate aspect ratios than samples with low alumino-silicate fractions. 6) Samples with relatively high fractions of quartz particles have higher fractions of particles with moderate aspect ratios and higher percentages of very small particles than samples with no identified quartz particles. 7) Samples with high fractions of carbonates have higher percentages of particles with relatively low aspect ratios (i.e., length and width are similar) than samples with no identified carbonate particles. / Master of Science / This thesis describes the development and use of a computer-automated microscopy routine for characterization of respirable dust particles from coal mines. Overexposure to respirable dust has long been known to pose an occupational health hazard for miners, leading to the development of lung diseases such as coal workers’ pneumoconiosis (CWP, commonly called “black lung”) and silicosis. Incidence of such diseases amongst US coal miners declined for many years following regulation and development of mining best practices. However, a recent resurgence in disease incidence, particularly in parts of Appalachia, demonstrates a real need for greater understanding of the respirable dust in underground coal mines. This work first sought to develop an automated routine for characterizing coal mine dust using scanning electron microscopy with energy dispersive x-ray (SEM-EDX). SEM-EDX is a powerful tool that allows the size, shape and chemistry of individual particles to be determined. The developed routine is not only much faster than an analogous manual method, but it also reduces the possibility of user bias and provides for more representative results by examining more particles across a wider area of a sample. The method was verified using laboratorygenerated dust samples from known materials commonly observed in underground coal mines, as well as field samples collected in central Appalachia. This effort indicated that the method produces accurate and representative results. Next, the automated SEM-EDX method was used to scan 210 respirable dust samples. These were collected in eight mines in three different regions of Appalachia (i.e., northern, midcentral and south-central Appalachia), which differed by primary mining method, coal seam thickness and mining height, and coal and/or rock mineralogy. Results were analyzed to determine whether particle size, shape, and chemistry number distributions vary between and within distinct mine regions, and by general sampling location categories (i.e., intake, feeder, production, return). Key findings include: 1) Northern Appalachian mines have relatively higher fractions of coal, carbonate, and heavy mineral particles than the two central Appalachian regions, whereas central Appalachian mines have higher fractions of quartz and alumino-silicate particles. 2) Central Appalachian mines tended to have more mine-to-mine variations in size, shape, and chemistry distributions than northern Appalachian mines. 3) With respect to particle size, samples collected in locations in the production and return categories have the highest percentages of very small particles (i.e., 0.94-2.0μm), followed by the feeder and then the intake locations. 4) With respect to particle shape, samples collected in locations in the production and return categories have higher fractions of particles with moderate (i.e., length is 1.5 to 3x width) to relatively high aspect ratios (i.e., length is greater than 3x width) compared to feeder and intake samples. 5) Samples with relatively high fractions of alumino-silicates have higher fractions of particles with moderate aspect ratios than samples with low alumino-silicate fractions. 6) Samples with relatively high fractions of quartz particles have higher fractions of particles with moderate aspect ratios and higher percentages of very small particles than samples with no identified quartz particles. 7) Samples with high fractions of carbonates have higher percentages of particles with relatively low aspect ratios (i.e., length and width are similar) than samples with no identified carbonate particles.

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Made available in DSpace on 2011-05-04T12:36:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2010 / A sílica é um composto natural formado pelos dois elementos mais abundantes na Terra - oxigênio e silício. A exposição a partículas de sílica cristalina induz a uma inflamação pulmonar crônica, que pode evoluir para fibrose pulmonar, acarretando na doença conhecida como silicose. O estresse oxidativo desempenha um papel importante na patogênese desta fibrose pulmonar. Sendo assim, a expressão de genes antioxidantes, como glutationa S-transferases (GSTs), são importantes componentes de proteção das células contra o estresse oxidativo e são conhecidas como genes altamente polimórficos, podendo contribuir para a susceptibilidade a silicose. O polimorfismo da GSTP1 A/G resulta na substituição do aminoácido isoleucina por valina, diminuindo, substancialmente, a atividade da enzima GSTP1. O estudo teve como objetivo a determinação do polimorfismo da enzima GSTP1 em trabalhadores expostos à sílica e associação com silicose. A população foi composta por 82 trabalhadores expostos à sílica oriundos, principalmente, da indústria naval. O polimorfismo da GSTP1 foi analisado por PCR-RFLP. Como resultado verificou-se que 31,6 por cento dos trabalhadores tinham genótipo A/A, 57,9 por cento A/G e 10,5 por cento G/G. Observou-se que a média da atividade enzimática da GST foi menor (1,58 U/mL enzima) em indivíduos com o alelo G em relação ao alelo A (1,84 U/mL de enzima). Trabalhadores expostos à sílica portadores do alelo G mostraram um maior risco de desenvolver silicose, embora os resultados não tenham sido significativos, provavelmente, em função do universo amostral. Os indivíduos portadores do alelo G tiveram níveis menores na atividade da GST, independente do genótipo das enzimas GSTM1 e GSTT1. Em conclusão, estudos adicionais devem ser realizados para determinar o polimorfismo da GSTP1 em populações expostas à sílica em comparação com populações não-expostas, pois os resultados deste trabalho sugerem a utilização da determinação do polimorfismo da GSTP1, no processo de avaliação da exposição à sílica, como uma ferramenta complementar na identificação de subgrupos mais susceptíveis ao desenvolvimento da doença silicose. / Sílica is the natural compound of the two most abundant elements on Earth - oxygen and silicon. Exposure to crystalline silica particles induces chronic lung inflammation, which may progress to lung fibrosis, a disease known as silicosis. The oxidative stress plays an important role in the pathogenesis of pulmonary fibrosis. In this respect, the expression of antioxidant genes such as glutathione S-transferases (GSTs) are important to protect cells from oxidative stress and they are known as highly polymorphic genes, that may contribute to silicosis susceptibility. The GSTP1 A G polymorphism results in amino acid substitution isoleucine for valine which substantially diminishes GSTP1 enzyme activity. The objetive of study was the determination of enzyme GSTP1 polymorphism in workers exposed to silica and association with silicosis. The population was composed of 82 workers exposed to silica coming mainly from the shipbuilding industry.GSTP1 polymorphism was analyzed by PCR-RFLP. As a result it was found that 31,6% of workers had genotype A/A, 57,9% A/G and 10.5% G/G. It was observed that the mean enzyme activity of GST was lower (1.58 U / mL enzyme) in individuals with allele G compared with allele A (1.84 U / mL enzyme). Workers exposed to silica with allele G showed a higher risk of developing silicosis, although the results had not been significant, probably because of sample size. Individuals with allele G had lower levels of GST activity, independent of the genotype of GSTM1 and GSTT1 enzymes. In conclusion, further studies to determine the polymorphism of GSTP1 should be conducted in populations exposed to silica compared with non-exposed populations, because the results in this paper suggest the use of the determination of the polymorphism of GSTP1, in the process of evaluating the exposure to silica, as a complementary tool in the identification of subgroups more likely to develop the disease silicosis. / Sílica is the natural compound of the two most abundant elements on Earth - oxygen and silicon. Exposure to crystalline silica particles induces chronic lung inflammation, which may progress to lung fibrosis, a disease known as silicosis. The oxidative stress plays an important role in the pathogenesis of pulmonary fibrosis. In this respect, the expression of antioxidant genes such as glutathione S-transferases (GSTs) are important to protect cells from oxidative stress and they are known as highly polymorphic genes, that may contribute to silicosis susceptibility. The GSTP1 A G polymorphism results in amino acid substitution isoleucine for valine which substantially diminishes GSTP1 enzyme activity. The objetive of study was the determination of enzyme GSTP1 polymorphism in workers exposed to silica and association with silicosis. The population was composed of 82 workers exposed to silica coming mainly from the shipbuilding industry.GSTP1 polymorphism was analyzed by PCR-RFLP. As a result it was found that 31,6% of workers had genotype A/A, 57,9% A/G and 10.5% G/G. It was observed that the mean enzyme activity of GST was lower (1.58 U / mL enzyme) in individuals with allele G compared with allele A (1.84 U / mL enzyme). Workers exposed to silica with allele G showed a higher risk of developing silicosis, although the results had not been significant, probably because of sample size. Individuals with allele G had lower levels of GST activity, independent of the genotype of GSTM1 and GSTT1 enzymes. In conclusion, further studies to determine the polymorphism of GSTP1 should be conducted in populations exposed to silica compared with non-exposed populations, because the results in this paper suggest the use of the determination of the polymorphism of GSTP1, in the process of evaluating the exposure to silica, as a complementary tool in the identification of subgroups more likely to develop the disease silicosis.