Ann E. Aust

In vitro effects of coal fly ashes: hydroxyl radical generation, iron release, and DNA damage and toxicity in rat lung epithelial cells

Oxygen radical generation due to surface radicals, inflammation, and iron release has been suggested as the mechanism of adverse effects of quartz, such as emphysema, fibrosis, and carcinogenic effects. Therefore, we measured iron release, acellular generation of hydroxyl radicals, and oxidative DNA damage and cytotoxicity in rat lung epithelial (RLE) cells by different coal fly ashes (CFA) that contain both quartz and iron. Seven samples of CFA with different particle size and quartz content (up to 14.1%) were tested along with silica (alpha-quartz), ground coal, and coal mine dust (respirable) as positive control particles, and fine TiO(2) (anatase) as a negative control. Five test samples were pulverized fuel ashes (PFA), two samples were coal gasification (SCG) ashes (quartz content <0.1%), and one sample was a ground coal. No marked differences between SCG and PFA fly ashes were observed, and toxicity did not correlate with physicochemical characteristics or effect parameters. Stable surface radicals were only detected in the reference particles silica and coal mine dust, but not in CFA. On the other hand, hydroxyl radical generation by all fly ashes was observed in the presence of hydrogen peroxide, which was positively correlated with iron mobilization and inhibited by deferoxamine, but not correlated with iron or quartz content. Also a relationship between acellular hydroxyl radical generation and oxidative DNA damage in RLE cells by CFA was observed. Differences in hydroxyl radical generation and oxidative damage by the CFA were not related to iron and quartz content, but the respirable ashes (MAT023, 38, and 41) showed a very extensive level of hydroxyl radical generation in comparison to nonrespirable fly ashes and respirable references. This radical generation was clearly related to the iron mobilization from these particles. In conclusion, the mechanisms by which CFA and the positive references (silica, coal mine dust) affect rat lung epithelial cells seem to be different, and the data suggest that quartz in CFA does not act the same as quartz in silica or coal mine dust. On the other hand, the results indicate an important role for size and iron release in generation and subsequent effects of reactive oxygen species caused by CFA.

Iron mobilization from asbestos by chelators and ascorbic acid

The ability of chelators and ascorbic acid to mobilize iron from crocidolite, amosite, medium- and short-fiber chrysotile, and tremolite was investigated. Ferrozine, a strong Fe(II) chelator, mobilized Fe(II) from crocidolite (6.6 nmol/mg asbestos/h) and amosite (0.4 nmol/mg/h) in 50 mM NaCl, pH 7.5. Inclusion of ascorbate increased these rates to 11.4 and 4.9 nmol/mg/h, respectively. Ferrozine mobilized Fe(II) from medium-fiber chrysotile (0.6 nmol/mg/h) only in the presence of ascorbate. Citrate and ADP mobilized iron (ferrous and/or ferric) from crocidolite at rates of 4.2 and 0.3 nmol/mg/h, respectively, which increased to 4.8 and 1.0 nmol/mg/h in the presence of ascorbate. Since ascorbate alone mobilized iron from crocidolite (0.5 nmol/mg/h), the increase appeared to result from additional chelation by ascorbate. Citrate also mobilized iron from amosite (1.4 nmol/mg/h) and medium-fiber chrysotile (1.6 nmol/mg/h). Mobilization of iron from asbestos appeared to be a function not only of the chelator, but also of the surface area, crystalline structure, and iron content of the asbestos. These results suggest that iron can be mobilized from asbestos in the cell by low-molecular-weight chelators. If this occurs, it may have deleterious effects since this could result in deregulation of normal iron metabolism by proteins within the cell resulting in iron-catalyzed oxidation of biomolecules.

Morphological and chemical mechanisms of elongated mineral particle toxicities.

Much of our understanding regarding the mechanisms for induction of disease following inhalation of respirable elongated mineral particles (REMP) is based on studies involving the biological effects of asbestos fibers. The factors governing the disease potential of an exposure include duration and frequency of exposures; tissue-specific dose over time; impacts on dose persistence from in vivo REMP dissolution, comminution, and clearance; individual susceptibility; and the mineral type and surface characteristics. The mechanisms associated with asbestos particle toxicity involve two facets for each particles contribution: (1) the physical features of the inhaled REMP, which include width, length, aspect ratio, and effective surface area available for cell contact; and (2) the surface chemical composition and reactivity of the individual fiber/elongated particle. Studies in cell-free systems and with cultured cells suggest an important way in which REMP from asbestos damage cellular molecules or influence cellular processes. This may involve an unfortunate combination of the ability of REMP to chemically generate potentially damaging reactive oxygen species, through surface iron, and the interaction of the unique surfaces with cell membranes to trigger membrane receptor activation. Together these events appear to lead to a cascade of cellular events, including the production of damaging reactive nitrogen species, which may contribute to the disease process. Thus, there is a need to be more cognizant of the potential impact that the total surface area of REMP contributes to the generation of events resulting in pathological changes in biological systems. The information presented has applicability to inhaled dusts, in general, and specifically to respirable elongated mineral particles.

The Formation of Reactive Oxygen Species Catalyzed by Neutral, Aqueous Extracts of NIST Ambient Particulate Matter and Diesel Engine Particles

ABSTRACT It is important to characterize the chemical properties of particulate matter in order to understand how low doses, inhaled by a susceptible population, might cause human health effects. The formation of reactive oxygen species catalyzed by neutral, aqueous extracts of two ambient par-ticulate samples, National Institute of Standards & Technology (NIST) Standard Reference Materials (SRM) 1648 and 1649, and two diesel particulate samples, NIST SRM 1650 and SRM 2975, were measured. The formation of reactive oxygen species was estimated by measuring the formation of malondialdehyde from 2-deoxyribose in the presence of ascorbic acid; H2O2 was not added to this assay. SRM 1649, ambient particulate matter collected from Washington, DC, generated the most malondialdehyde, while SRM 2975, diesel particulate matter collected from a forklift, yielded the least amount. Desferrioxamine inhibited the formation of malondialdehyde from the par-ticulate samples providing additional data to support the observation that transition metals were involved in the generation of reactive oxygen species. Six transition metal sulfates (iron sulfate, copper sulfate, vanadyl sulfate, cobalt sulfate, nickel sulfate, and zinc sulfate) were assayed

Reduced Lung Cell Proliferation Following Short-Term Exposure to Ultrafine Soot and Iron Particles in Neonatal Rats: Key to Impaired Lung Growth?

Particulate matter (PM) has been associated with a variety of negative health outcomes in children involving the respiratory system and early development. However, the precise mechanisms to explain how exposure to airborne particles may cause adverse effects in children are unknown. To study their influence on early postnatal development, a simple, laminar diffusion flame was used to generate an aerosol of soot and iron particles in the size range of 10 to 50 nm. Exposure of 10-day-old rat pups to soot and iron particles was for 6 h/day for 3 days. The lungs were examined following a single injection of bromodeoxyuridine (BrdU) 2 h prior to necropsy. Neonatal rats exposed to these particles demonstrated no effect on the rate of cell proliferation within terminal bronchioles or the general lung parenchyma. In contrast, within those regions arising immediately beyond the terminal bronchioles (defined as the proximal alveolar region), the rate of cell proliferation was significantly reduced compared with filtered air controls. These findings strongly suggest exposure to airborne particles during early neonatal life has significant direct effects on lung growth by altering cell division within critical sites of the respiratory tract during periods of rapid postnatal development. Such effects may result in altered growth in the respiratory system that may be associated with lifelong consequences.

BIOAVAILABILITY OF IRON FROM COAL FLY ASH: Mechanisms of Mobilization and of Biological Effects

Particulate air pollution contains iron that may be involved in the pathological effects after inhalation. This article reviews work demonstrating that ambient particulate samples (Standard Reference Material [SRM] 1648 and SRM 1649, from the National Institute of Science and Technology) contain iron that can be mobilized from the particle in vitro and inside human lung epithelial (A549) cells. The mobilized iron can then catalyze the formation of reactive oxygen species (ROS). Work is also reviewed on the generation and size fractionation of coal fly ash (CFA) from three commercially important coal types, as well as size fractionation of three types of noncombustion particles. The availability of iron from these particles to A549 cells was measured by citrate mobilization in vitro and induction of the iron storage protein ferritin in particle-treated cells. The amount of bioavailable iron decreased with increasing particle size. The ability of particles to induce synthesis of the proinflammatory cytokine interleukin-8 (IL-8) was also determined. As with the bioavailability of iron, there was an inverse correlation with size. Further work showed that iron in CFA is responsible for IL-8 induction. Mössbauer spectroscopy of a CFA sample before and after desferrioxamine B treatment to remove bioavailable iron showed that the bioavailable iron was associated with the glassy aluminosilicate fraction of the particle. In conclusion, this work shows that bioavailable iron is responsible for ROS production by SRMs and IL-8 induction by CFA in A549 cells. The source of this bioavailable iron in CFA is glassy aluminosilicates, which are found at higher levels in smaller sizes of CFA.

MOBILIZATION OF IRON FROM CROCIDOLITE ASBESTOS BY CERTAIN CHELATORS RESULTS IN ENHANCED CROCIDOLITE-DEPENDENT OXYGEN CONSUMPTION

The reactivity of iron on crocidolite asbestos with dioxygen was determined and compared with iron mobilized from crocidolite. Ferrozine, a strong Fe(II) chelator, was used to demonstrate that iron on crocidolite was redox active. More Fe(II) was mobilized from crocidolite (1 mg/ml) by ferrozine anaerobically (11.2 nmol/mg crocidolite/h) than aerobically (6.6 nmol/mg/h) in 50 mM NaCl, pH 7.5, suggesting that Fe(II) on crocidolite reacts with O2 upon aqueous suspension. However, suspension of crocidolite in 50 mM NaCl, pH 7.5, did not result in a measurable amount of O2 consumption. The addition of reducing agents (1 mM) increased the amount of Fe(II) on crocidolite, and addition of ascorbate resulted in 0.4 nmol O2 consumed/mg crocidolite/min. Therefore, iron on crocidolite had limited redox activity in the presence of ascorbate. However, mobilization of iron from crocidolite increased its redox activity. Citrate, nitrilotriacetate (NTA), or EDTA (1 mM) mobilized 79, 32, or 58 microM iron, respectively, in preincubations up to 76 h, and increased O2 consumption upon addition of ascorbate to 2.8, 7.6, or 22.0 nmol O2 consumed/mg/min, respectively. This activity depended only upon the presence of a component(s) mobilized from crocidolite by the chelators. Pretreatment of crocidolite with the iron chelator desferrioxamine B (10 mM) inhibited O2 consumption. The results of the present study suggest that iron on or in crocidolite is responsible for the redox activity of crocidolite, but that mobilization of iron by chelators such as citrate, NTA, or EDTA greatly enhances its redox activity. Thus, iron mobilization from crocidolite in vivo by low-molecular-weight chelators may lead to the increased production of reactive oxygen species which may damage biomolecules, such as DNA.

Oxidative injury in the lungs of neonatal rats following short-term exposure to ultrafine iron and soot particles

Greater risk of adverse effects from particulate matter (PM) has been noted in susceptible subpopulations, such as children. However, the physicochemical components responsible for these biological effects are not understood. As critical constituents of PM, transition metals were postulated to be involved in a number of pathological processes of the respiratory system through free radical-medicated damage. The purpose of this study was to examine whether oxidative injury in the lungs of neonatal rats could be induced by repeated short-term exposure to iron (Fe) and soot particles. Sprague Dawley rats 10 d of age were exposed by inhalation to two different concentrations of ultrafine iron particles (30 or 100 µg/m3) in combination with soot particles adjusted to maintain a total particle concentration of 250 µg/m3. Exposure at 10 d and again at 23 d of age was for 6 h/d for 3 d. Oxidative stress was observed at both Fe concentrations in the form of significant elevations in glutathione disulfide (GSSG) and GSSG/glutathione (GSH) ratio and a reduction in ferric/reducing antioxidant power in bronchoalveolar lavage. A significant decrease in cell viability associated with significant increases in lactate dehydrogenase (LDH) activity, interleukin-1-beta (IL-1β), and ferritin expression was noted following exposure to particles containing the highest Fe concentration. Iron from these particles was shown to be bioavailable in an in vitro assay using the physiologically relevant chelator, citrate. Data indicate that combined Fe and soot particle exposure induces oxidative injury, cytotoxicity and pro-inflammatory responses in the lungs of neonatal rats.

Novel functional view of the crocidolite asbestos-treated A549 human lung epithelial transcriptome reveals an intricate network of pathways with opposing functions

BackgroundAlthough exposure to asbestos is now regulated, patients continue to be diagnosed with mesothelioma, asbestosis, fibrosis and lung carcinoma because of the long latent period between exposure and clinical disease. Asbestosis is observed in approximately 200,000 patients annually and asbestos-related deaths are estimated at 4,000 annually[1]. Although advances have been made using single gene/gene product or pathway studies, the complexity of the response to asbestos and the many unanswered questions suggested the need for a systems biology approach. The objective of this study was to generate a comprehensive view of the transcriptional changes induced by crocidolite asbestos in A549 human lung epithelial cells.ResultsA statistically robust, comprehensive data set documenting the crocidolite-induced changes in the A549 transcriptome was collected. A systems biology approach involving global observations from gene ontological analyses coupled with functional network analyses was used to explore the effects of crocidolite in the context of known molecular interactions. The analyses uniquely document a transcriptome with function-based networks in cell death, cancer, cell cycle, cellular growth, proliferation, and gene expression. These functional modules show signs of a complex interplay between signaling pathways consisting of both novel and previously described asbestos-related genes/gene products. These networks allowed for the identification of novel, putative crocidolite-related genes, leading to several new hypotheses regarding genes that are important for the asbestos response. The global analysis revealed a transcriptome that bears signatures of both apoptosis/cell death and cell survival/proliferation.ConclusionOur analyses demonstrate the power of combining a statistically robust, comprehensive dataset and a functional network genomics approach to 1) identify and explore relationships between genes of known importance 2) identify novel candidate genes, and 3) observe the complex interplay between genes/gene products that function in seemingly different processes. This study represents the first function-based global approach toward understanding the response of human lung epithelial cells to the carcinogen crocidolite. Importantly, our investigation paints a much broader landscape for the crocidolite response than was previously appreciated and reveals novel paths to study. Our graphical representations of the function-based global network will be a valuable resource to model new research findings.

Apoptosis induced by crocidolite asbestos in human lung epithelial cells involves inactivation of Akt and MAPK pathways

Exposure of human lung epithelial (A549) cells to asbestos fibers causes apoptosis, which is largely attributed to release of iron and generation of reactive oxygen species (ROS) within the cells. To mimic the highly oxidative environment generated by asbestos exposure in the absence of the actual fibers, we used two chemicals; buthione sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis and ferric ammonium citrate (FAC), a source of iron. Here, we report that exposure of A549 cells to crocidolite asbestos led to a significant time-dependent inactivation of signaling proteins, i.e. Akt and all mitogen-activated protein kinases (MAPKs) (p38, ERK1/2 and SAPK/JNK), and subsequently to apoptosis. Unlike crocidolite treatment, the use of BSO and FAC, independently or combined, did not change the phosphorylation status of proteins, nor did it induce apoptosis. Taken together, our results presented herein point to the possibility that crocidolite-induced apoptosis of human lung epithelial cells is not a mere consequence of generation of oxidants but also requires inactivation of major cell growth and differentiation pathways.