A Multistage Cyclone Array for the Collection of Size-segregated Silica Aerosols to Test the Hypothesis that Ultrafine Crystalline Silica Particles are More Efficient in Their Activation of Macrophages

摘要

Occupational exposure to crystalline silica is a well-established occupational hazard. Once in the lung, crystalline silica particles can result in the activation of alveolar macrophages potentially leading to silicosis, a fibrotic lung disease. Because the activation of alveolar macrophages is the beginning step in a complicated inflammatory cascade, it is necessary to define the particle characteristics resulting in this activation. In this study a serial multi-cyclone sampling array (MCSA) capable of simultaneously sampling particles of multiple size fractions, from an occupational environment, was developed and tested and then used to collect size-segregated crystalline silica particles to determine the effect of the size of crystalline silica particles on the activation of macrophages. The MCSA method is an improvement over current methods used to size-segregate occupational aerosols for characterization, due to its simplicity and its ability to collect sufficient masses of nano- and ultrafine sized particles for analysis. This method was evaluated in a chamber providing a uniform atmosphere of dust concentrations using crystalline silica particles. The multi-cyclone sampling array was used to segregate crystalline silica particles into four size fractions, from a chamber concentration of 10 mg/m3. The size distributions of the particles collected at each stage were confirmed, in the air, before and after each cyclone stage. Once collected, the particle size distribution of each size fraction was measured using light scattering techniques, to further confirm the size distributions. As a final confirmation, scanning electron microscopy was used to collect images of each size fraction. The results presented here, using multiple measurement techniques, show that this multi-cyclone system was able to successfully collect distinct size-segregated particles at sufficient masses to perform toxicological evaluations and physical/chemical characterization.udOnce the particles were collected using the MCSA they were used to determine the effect of the size of crystalline silica particles on the activation of macrophages. RAW 264.7 macrophages were exposed to four different sizes of crystalline silica and their activation was measured using electron microscopy, reactive oxygen species (ROS) generation by mitochondria, and cytokine expression. These data identified differences in particle uptake and formation of subcellular organelles based on particle size. In addition, these data show that the smallest particles, with a geometric mean of diameter 0.3 µm, significantly increase the generation of mitochondrial ROS and the expression of cytokines when compared to larger crystalline silica particles, with a geometric mean diameter of 4.1 µm.udWith regard to occupational and public health significance, our data indicate the necessity of properly characterizing occupational aerosols using regulatory exposure metrics that are effectively protective against particle exposure causing adverse effects on workers’ health.ud