Importance of size and composition of particles for effects on cells in vitro.

A primary goal of current research on particle-induced health effects is to reveal the critical characteristics that determine their biological effects. Experimental studies have shown that smaller particles induce stronger biological effects than larger particles of similar composition, due to their larger surface area to mass ratio. However, correlation for variations in surface area could not account for variation in biological reactivity among particles of differential composition. Hence, the importance of size and surface area does not override the importance of particle composition. Moreover, different particle characteristics appear to be involved in different biological effects in vitro. Our studies show that mineral particle-induced apoptosis mostly seems to depend on particle size, whereas composition and surface reactivity appeared to be most important for the proinflammatory potential of the particles. The ability of the particles to generate reactive oxygen species in vitro was not correlated with either inflammatory markers or apoptosis, suggesting that other mechanisms are at play. A single, specific component of the mineral particles, explaining the differences in response, has not been identified. In European-wide studies such as the Respiratory Allergy and Inflammation due to Air Pollution (RAIAP) study, particles have been sampled in different locations to study season- and site-dependent variations in responses particles, such as markers of inflammatory and allergic reactions in cells and animals. The data indicate that coarse particles can induce at least as strong inflammatory responses as fine particles. The allergic responses tended to be more associated with the organic fraction (PAH) of particles, whereas the inflammatory reactions seemed to be more associated with metals and endotoxin. Overall, coarse PM was found to have an inflammatory potential similar to fine PM on an equal mass basis. Even though one has to take into account different concentrations in ambient air as well as differences in respiratory system deposition of the size fractions, the potential of coarse particles to induce pulmonary effects should not be neglected.

Iron release and ROS generation from mineral particles are not related to cytokine release or apoptosis in exposed A549 cells.

The generation of reactive oxygen species (ROS) by mineral particles is believed to be central to their toxicity and their ability to induce inflammation. Surface bound or soluble iron may contribute to the particle-effects by enhancing the ROS generation through the Fenton reaction. Nevertheless, the importance of ROS and transition metals to mineral particle-induced effects is still unclear and further investigations are needed. In the present study we have investigated different mineral particles for their total iron content, amount of soluble iron at pH 7.0 and 4.0, their ability to generate ROS in a cell-free environment, and their ability to induce cytokine release and apoptosis in a human alveolar epithelial cell line (A549). All the investigated parameters varied considerably between the different particles, with the exception of ability to induce apoptosis. Total iron content did not reflect the amount of soluble iron, and neither total nor soluble iron was correlated with ROS generation. Moreover, iron content and ROS was not correlated with the ability of particles to induce cytokine release or apoptosis. The present results suggest that there is no clear relationship between the particles iron content and ability to generate ROS. Moreover, neither iron content nor the ability to induce ROS generation appears to be a prerequisite for the inflammatory potential or cytotoxicity of mineral particles.

Particulate matter properties and health effects: consistency of epidemiological and toxicological studies.

Identifying the ambient particulate matter (PM) fractions or constituents, critically involved in eliciting adverse health effects, is crucial to the implementation of more cost-efficient abatement strategies to improve air quality. This review focuses on the importance of different particle properties for PM-induced effects, and whether there is consistency in the results from epidemiological and experimental studies. An evident problem for such comparisons is that epidemiological and experimental data on the effects of specific components of ambient PM are limited. Despite this, some conclusions can be drawn. With respect to the importance of the PM size-fractions, experimental and epidemiological studies are somewhat conflicting, but there seems to be a certain consistency in that the coarse fraction (PM10-2.5) has an effect that should not be neglected. Better exposure characterization may improve the consistency between the results from experimental and epidemiological studies, in particular for ultrafine particles. Experimental data indicate that surface area is an important metric, but composition may play an even greater role in eliciting effects. The consistency between epidemiological and experimental findings for specific PM-components appears most convincing for metals, which seem to be important for the development of both pulmonary and cardiovascular disease. Metals may also be involved in PM-induced allergic sensitization, but the epidemiological evidence for this is scarce. Soluble organic compounds appear to be implicated in PM-induced allergy and cancer, but the data from epidemiological studies are insufficient for any conclusions. The present review suggests that there may be a need for improvements in research designs. In particular, there is a need for better exposure assessments in epidemiological investigations, whereas experimental data would benefit from an improved comparability of studies. Combined experimental and epidemiological investigations may also help answer some of the unresolved issues.

Importance of soluble metals and reactive oxygen species for cytokine release induced by mineral particles.

The mechanisms for particle-induced health effects are not well understood, but inflammation seems to be of importance. Previously, we have shown that stone quarry particles with various mineral and metal content differed widely in potency to induce inflammatory cytokines (IL-6, IL-8 and TNF-alpha) in different types of lung cells. In this study we investigated if the observed cytokine responses were associated with the soluble or insoluble components of the stone particles and if there was a relationship between the differential cytokine release and generation of reactive oxygen species (ROS). Exposure of the human alveolar cell line A549 to the different particle leachates (pH 7.4 and 4.0) did not induce corresponding differential increases in the IL-8 release as observed with whole particles. Increase in ROS production, measured as dichlorofluorescein-fluorescence, was only demonstrated after exposure of A549 cells to the pH 4.0 extract from basalt. Furthermore, generation of ROS was found in neutrophils but not in A549 cells and primary macrophages after exposure to suspensions of the solid particles. However, no obvious differences in potency among the different particles were demonstrated. In summary, other mechanisms than particle-induced ROS formation seem to be responsible for the differential induction of IL-8. Furthermore, our findings indicate that the differential ability to induce IL-8 release in lung cells is attributed to the solid components of the stone particles.

Role of signal transduction pathways in lung inflammatory responses.

A variety of cell types participate in lung inflammation. Macrophages and epithelial cells play an important role in the inflammatory process by releasing cytokines in a complex cell to cell network. Interleukins are important mediators of this cell signalling. The interleukins IL-6 and IL-8 are released from epithelial cells in response to noxious agents such as particles, bacterial and fungal toxins and various chemicals. Though the involvement of, e.g. NF-IL-6 (C/EBP-beta) in the regulation of interleukins has been reported, the role of different signal transduction pathways in the regulation of these mediators has not been thoroughly investigated in lung epithelial cells. The involvement of different signal transduction pathways in the release of inflammatory markers is discussed with special emphasis on the effect of lung toxic compounds in human and rat lung epithelial cells.

Release of inflammatory cytokines, cell toxicity and apoptosis in epithelial lung cells after exposure to ambient air particles of different size fractions.

Several studies have shown that particles of smaller size may be more potent than larger to induce inflammatory and toxic responses in cultured lung cells. However, the relative importance of different size fractions of ambient PM to induce such effects is still not known. In this study, we investigated the potency of different size fractions of urban ambient air particles to induce release of inflammatory cytokines in the human alveolar cell line A549 and primary rat type 2 cells. A mineral-rich ambient air PM10 sample collected in a road tunnel (road PM10) was also included. The coarse fraction of the urban ambient air particles demonstrated a similar or higher potency to induce release of the proinflammatory cytokines IL-8/MIP-2 and IL-6 compared to the fine and ultrafine fractions. The coarse fraction was also the most toxic in both cell systems. In contrast to the A549 cells, no induction of cytokine release was induced by the ultrafine particles in the primary type 2 cells. The mineral-rich road PM10 may be equally or more potent than the various size fractions of the ambient air particles to induce cytokines in both cell types. In conclusion, the coarse fraction of ambient particles may be at least as potent by mass as smaller fractions to induce inflammatory and toxic effects in lung cells.

Mineral and/or metal content as critical determinants of particle-induced release of IL-6 and IL-8 from A549 cells.

Mineral particles in occupational exposure and ambient air particles may cause adverse health effects in humans. In this study the ability of different stone quarry particles to induce release of the proinflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8) from human epithelial lung cells (A549) was investigated. Size distribution within the PM10 fractions was quite similar for all particle samples, whereas mineral content and metal composition differed. Particles, containing minerals such as quartz, amphibole, chlorite, and epidote, induced a marked increase in IL-6 and IL-8 release. Particles composed mainly of plagioclase were much less effective. The most potent particle samples exhibited a relatively high content of transition metals such as iron. Exposure to identical masses or surface areas resulted in the same order of potency among the different particle samples. Significant cytotoxicity was observed only at higher concentrations of particle exposure. Thus, mineral composition and/or metal contents of particles from different stone quarries were critical determinants for the marked differences in potency to induce cytokine responses in human epithelial lung cells.

Silica-induced cytokine release from A549 cells: importance of surface area versus size.

Physical and chemical properties such as structure, composition and surface reactivity determine the biological activity of mineral particles. Long-term exposure to crystalline silica is known to cause persistent pulmonary inflammation leading to adverse health effects. There is less information about the potential health effects of amorphous (noncrystalline) silica. In this study, the inflammatory and cytotoxic potency of crystalline and amorphous silica in relation to particle size and surface area was assessed. Human epithelial lung cells (A549) were exposed to different size fractions of quartz ( aerodynamic diameter 0.5, 2 and 10 microm) and amorphous silica (diameter 0.3 microm). All particles induced increased release of the proinflammatory cytokines interleukin (IL)-6 and IL-8. When cells were exposed to equal masses of quartz, the smallest size fraction was the most potent. These differences, however, disappeared when cytokine release was related to equal surface areas. When amorphous silica and quartz were compared, the amorphous silica was most potent to induce IL - 6 regardless of how exposure was expressed, whereas the smallest size fraction of quartz was the most potent inducer of IL-8. Thus, the surface area seems to be the critical determinant when potency of different sizes of quartz is compared.

Mineral particles of varying composition induce differential chemokine release from epithelial lung cells: importance of physico-chemical characteristics.

Presently, little is known about the potential health effects of mineral particles other than asbestos and quartz. In this study, a human epithelial lung cell line (A549), primary human small airway epithelial cells (SAECs) and primary rat type 2 (T2) cells were exposed to stone quarry particles of two size fractions (<10 and <2.5 microm) from nine different rock samples. The ability to induce the release of chemokines from lung cells was investigated and compared with the particles' mineral and element composition and the amount of soluble elements. The stone particles induced the release of only low levels of interleukin (IL)-8 from A549 cells. In contrast, some of the other particles induced the release of high levels of macrophage inflammatory protein (MIP)-2 from T2 cells, and high levels of IL-8 from SAECs. Differences in particle surface area could account for differences in activity between the <10 and <2.5 microm fractions of six out of the nine rock samples. For two samples the <2.5 microm fraction was most active and for one sample the <10 microm fraction was most active. Content of the mineral plagioclase displayed a strong, negative correlation with the potential to induce MIP-2, whereas the mineral pyroxene was positively correlated with MIP-2 induction. However, neither plagioclase nor pyroxene content was sufficient to explain differences in bioactivity between the particles. No statistically significant correlation was found between the amounts of total or soluble elements and MIP-2 release. In conclusion, the results suggest that mineral particles with a high content of plagioclase have a low potential to induce a pro-inflammatory response. However, a particular mineral or element responsible for eliciting strong increases in chemokine release could not be identified. Thus, at present it appears that analysing mineral and element content is insufficient to predict stone particle bioactivity, and that biological testing is a necessity.

Rat lung inflammatory responses after in vivo and in vitro exposure to various stone particles.

Rat lung alveolar macrophages and type 2 cells were exposed for 20 h in vitro to various stone particles with differing contents of metals and minerals (a type of mylonite, gabbro, feldspar, and quartz). The capability to induce the release of the inflammatory cytokines interleukin-6 (IL-6), tumour necrosis factor-alpha (TNF-alpha), and macrophage inflammatory protein-2 (MIP-2) was investigated. We found marked differences in potency between the various particles, with mylonite being most potent overall, followed by gabbro, and with feldspar and quartz having an approximately similar order of lower potency. The results also demonstrated differences in cytokine release pattern between the two cell types. For all particle types including quartz, type 2 cells showed the most marked increase in MIP-2 and IL-6 secretion, whereas the largest increase in TNF-alpha release was observed in macrophages. To investigate possible correlations between in vitro and in vivo inflammatory responses, rats were instilled with the same types of particles and bronchoalveolar lavage (BAL) fluid was collected after 20 h. The results demonstrated a correlation between the in vitro cytokine responses and the number of neutrophilic cells in the BAL fluid. The BAL fluid also showed a strong MIP-2 response to mylonite. However, this was the only particle type to give a significant cytokine response in the BAL fluid. We further examined whether a similar graded inflammatory response would be continued in type 2 cells and alveolar macrophages isolated from the exposed animals. Again a differential cytokine release pattern was observed between type 2 cells and macrophages, although the order of potency between particle types was altered. In conclusion, various stone particles caused differential inflammatory responses after both in vitro and in vivo exposure, with mylonite being the most potent stone particle. The results suggest the alveolar type 2 cell to be an important participant in the inflammatory response following exposure to particles.