Oxygen Effects on Free Radicals and Cytotoxicity of Freshly Crushed Coal

"The biologic events leading to coal workers' pneumoconiosis (C'WP) are not yet fully understood. There is no good correlation between toxicity studies or animal models with human epidemiologic and pathologic data. Several studies have shown that the severity and prevalence of the CWP in miners correlate strongly with the rank and type of the coal mined. However this has not been found to be the case with laboratory animal models or in vitro studies. In 1980 Russian scientists Artemov and Resnik suggested that the discrepancy may be related to the excessive amount of short-lived free radicals present in freshly generated coal mine dust. Because of their short life, such free radicals would be absent in the ""stale"" coal dust used in most animal experiments. To test this hypothesis we have undertaken a systematic study of the generation, reactivity with oxygen and biological significance of free radicals in coal dust.Electron spin resonance (ESR) was used as a technique for the direct detection of radical production and kinetics of decay. ESR signals from the newly generated radicals decreased with time on exposure to oxygen or when stored in biological buffers at a rapid rate. In vitro biological tests were carried out which suggest that the reaction between the newly generated radicals and oxygen plays a significant role in the sample's cytotoxicity.Materials and MethodsESR measurements were made at X-band (~9.7 GHz) using a computer-controlled Bruker ER200D ESR spectrometer. 11te two coals studied in detail were obtained from the Pennsylvania State University Coal Depository. These were, a bituminous sample, PSOC-1172 (72% carbon) and an anthracite sample, PSOC 867 (95% carbon). Coal particles of several millimeter (mm) dimensions were evacuated to a pressure of ~10-3 mm Hg typically overnight. Without exposure to air, these particles were ground in an agate mortar with an agate pestle for about 30 minutes and then sieved through a 20 \1111 mesh filter before ESR measurements or cytotoxicity studies. As an index of cytotoxicity, hemolysis was measured according to the method of Harrington, with minor modifications. Sheep erythrocytes (2%) were incubated with test samples at 37°C for one hour, and the hemoglobin released was quantitatively estimated photometrically. Additionally, release of alveolar macrophage enzyme lactate dehydrogenase (LOH) was estimated after incubation of the macrophages with the coal particles at different time intervals after the grinding. Enzyme activity was assayed according to the method of Wrobleski and LaDue and expressed as percent of enzyme released in 2 hours per million cells with 1 mg of coal."