In vitro lung cell culture studies of particle-induced proinflammatory responses Paper # 541 Tyler A. Moss, John M. Veranth, William K. Nichols, Garold S. Yost Department of Pharmacology and Toxicology, University of Utah, 112 Skaggs, 30 South 2000 East, Salt Lake City, UT 84112 ABSTRACT Recent work with soil-derived mineral dusts and manufactured nanoparticles will be discussed to illustrate the types of toxicological information that can be obtained from treatment of cell cultures with low-solubility particles. Readily inhaled particulate matter (PM), which includes the enriched PM2.5 fraction of certain soil dusts or modified manufactured nanoparticles, was found to cause release of the proinflammatory cytokine IL-6 or induce cell death in human lung cell cultures. There is a need to include geological particulate matter when assessing adverse health effects of polluted air. Extensive prior cell culture research has been done by toxicologists to understand the affects and mechanisms of inhaled particles. Various surface-modifying treatments, including heat and leaching, were used to identify the particle components or characteristics that induced the cell signaling response. INTRODUCTION Numerous studies have reported adverse health effects correlated with increased inhaled particulate matter, but the toxicological mechanisms by which specific types of particles affect susceptible populations remain elusive. Pope et al.1 reported that differences in adverse health effects associated with the mass of particulate matter (PM10) in nearby Utah communities were due to the relative soil dust fraction. Schenker2 associated high exposure to agricultural dust with chronic bronchitis, but epidemiology studies in Spokane, WA found no correlation between ambient soil dust and mortality3 or hospital visits for asthma.4 The current ambient air quality standards for PM10 and PM2.5 are based largely on epidemiology studies that correlate particle mass in a specified size range, as measured by community air monitoring stations, with documented public health effects such as respiratory hospital admissions and cardiovascular deaths.5 Existing data are inadequate to identify the pollution sources that are the most relevant to decreased human health and there is no consensus on the biological mechanisms by which inhaled particles cause adverse effects in sensitive populations.6 There is increasing evidence suggesting that redox-active transition metals associated with particles can induce proinflammatory responses in lung cells.7-9, 10 , 11 Donaldson et al. 1 have concluded that ultrafine particles of low-solubility, low-toxicity materials are more inflammogenic in the rat lung than larger particles from the same material, and they hypothesize that the effects are related to surface area and involve oxidative stress.12 Titanium dioxide also shows increased response with ultrafine particles.13 Particleinduced tissue inflammation has been proposed as a central process connecting inhaled particles with adverse health effects.6 Numerous approaches may be used to identify induced inflammation from particles in sensitive populations. Epidemiology studies of humans or animals can be used, but will not identify the biological mechanisms involved in a particulate toxic response. Human and animal models are costly and it is uncertain which particles are potent or benign. In vitro cell culture models such as immortalized lung cell lines BEAS-2B14-16 and A549,7, 17 normal human bronchial epithelial cells,18 macrophages, and co-cultures of macrophages with epithelial cells19 are useful to screen substances for toxic effects, and provide an easily-manipulated system for studying biochemical signaling mechanisms. The approach in this paper is to assess the ability of ambient particulate matter (PM) and manufactured nanoparticles to induce proinflammatory signaling (cytokines) in BEAS-2B human bronchial epithelial cultured lung cells. BEAS-2B human immortalized lung cell lines are used as an alternative to normal human bronchial epithelial cells. BEAS-2B lung cell lines are virus transformed to produce immortalized growth. Immortalized cell lines are cost efficient and decrease the number of variables that may be introduced using various normal bronchial epithelial cells. Measuring particle-induced cytokines in cultured lung cells elucidates which particles cause inflammation and may provide information to determine individuals who may be susceptible to pulmonary problems upon inhalation of specific sources of soil dust. Surface modifying techniques help explain particle characteristics. Cytokines, such as interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor- (TNF-), are signaling molecules associated with many processes regulating cell growth, differentiation, and death, as well as physiological responses in tissues, and recruitment of neutrophils, macrophages and other mobile cells to specific sites.20-22 Cytokine assays are widely used in studies of lung cell responses to particles and other pollutants.23 Much of the recent in vitro work with environmental particles has used ELISA assays to measure IL-624-26 IL-8,7, 27, 28 and TNF-.29-31 Cytokine induction is associated with reactive oxygen species (ROS) formation, and there is evidence for both extracellular and intracellular reactions leading to ROS formation.31-33 Induction of the cytokine IL-6 has been observed in BEAS-2B cells treated with diesel exhaust particles,16 residual oil fly ash (ROFA),34 a range of ambient and combustion particles,35 negatively charged particles,36, 37 and capsaicin-related compounds.38 Titanium dioxide particles larger than 1 µm are inert and serve as a PM negative control,16, 39 but mineral dust from stone quarries induces cytotoxicity and IL-6 release in A549 cells24 and in rat lung type 2 alveolar cells.40 In vitro toxicology studies, motivated by specific biochemical signaling hypotheses, have attempted to link specific particle sources with cellular responses. Particle modifying 2 treatments are a means to understanding particle characteristics using human cell culture models. Physical treatments to modify particle toxicological properties include leaching particles with the metal chelator desferrioxamine7 and applying surface-modifying coatings.41 Various mechanisms have been proposed for particle-cell interactions.42 Mechanisms leading to pro-inflammatory signaling include: response to particle components dissolved in the extracellular fluid;14, 43 cell receptor recognition of materials adsorbed on the particle surface;26 particle uptake followed by induction of an intracellular response;27, 44 and particle surface charge activation of the TRPV1 receptor.25, 35 Particle mass in the ambient air correlates with a wide range of adverse effects, but the importance of geological dust inhalation to human health has not been resolved. Investigating the effects of inhaled PM helps to identify endangered populations and assist in developing programs that reduce risk. Physical modifying treatments, such as heat and leaching, of manufactured nanoparticles serve to simulate fire and wastewater conditions. The current studies contribute to our understanding of the biological effects of treated particles and help elucidate the environmental health impact of air born particles. METHODS PM2.5-enriched soil dusts were prepared from field samples by tumbling and aerodynamic separation to simulate the dust generated by mechanical attrition when tires contact the road surface, or when coarse particle bombardment generates fine dust during high wind events. Details are described by Veranth.46 Desert Research Institute, Reno, NV, laboratory procedures were used to determine chemical speciation measurements of samples. Elemental composition was determined by x-ray fluorescence. Soluble ions were measured by ion chromatography or by atomic adsorption spectroscopy. Eight carbon fractions based on desorption temperature were determined using thermal optical reflectance method. Carbonate carbon was determined by acid pretreatment. The accuracy of the chemical speciation measurements was determined through the use of blanks and duplicates and the existing DRI procedures were used to estimate measurement uncertainty using propagation of error analysis. Particle samples were weighed, sterilized, vacuum dried, and resuspended in cell culture media as described by Veranth.46 The thermally treated particles were prepared by heating in loosely covered borosilicate glass tubes in air in a muffle furnace at 150, 300, or 550°C for one hour. Leaching treatments involved suspending the particles in 5 mL of liquid and rotating the tubes overnight at 6 RPM. The samples were centrifuged at 750 x g for 10 minutes and decanted, and the cycle was repeated three times. The leaching liquid was LHC-9 cell culture media. The supernatant from the first 24 hr leaching was recovered and used to measure the effects of the soluble fraction of the original particles. Iron-silica nanoparticles were heat treated in a Thermolyne oven at 1100°C and slowly cooled. 3 BEAS-2B human bronchial epithelial cells were used as cell culture models. Cells were maintained and passaged as described by Veranth.46 Figure 1 describes the cell culture model. Cells were seeded in 24 or 48-well polystyrene plates (Costar, Fisher Scientific) at a concentration of about 30,000 cells/cm2. After two days new cell culture media was applied. The cell culture media containing particle samples was applied to the cells on the fourth day. On the fifth day the cell culture media was harvested for cytokine assays and viable cell count was measured. Experiments used triplicate wells for each treatment level, and allocated 6 or 9 wells per culture plate as controls. Positive controls were included to monitor changes in the cell response. Figure 1. Cell culture procedure. Cell viability and concentration of IL-6 was assessed by using the Dojindo assay and R&D kits as described by Veranth.46 RESULTS Cell culture can be used to screen a wide range of materials for the ability to induce proinflammatory signaling in lung cells. Figure 2 shows the cytotoxicity and proinflammatory signaling from twenty-six different soils and two positive controls (LPS and V80). The particle soils were taken from various places in New Mexico, Texas, and Utah. Each source of enriched soil dust for PM2.5 was applied at a concentration of 4 80µg/cm2. The cytotoxicity was measured as percent viability compared to control. IL-6 induction was measured as fold over control. The viability of cells was not directly correlated with induction of IL-6 for most soil samples. For example the soil dust JE seemed to be a potent inducer of IL-6 (30x), but did not appear to be toxic (80% viable compared to control). Whereas the soil dust 2SB appears to be both a potent inducer of IL-6 (25x) and toxic (40% viable compared to control). There seemed to be about ten soils that produced a proinflammatory response less than ten fold over control. Nine soils that produced a response between ten and twenty-fold over control and six soils that produced a response greater than twenty-fold over control. Ten soil samples produced significant cytotoxicity (below 60% viable compared to control). The diverse responses from the different non-soluble materials support the hypothesis that cell culture models may be used to identify the particle components or characteristics that induce the cell signaling response. 5 Figure 2. Proinflammatory and cytotoxic response of BEAS-2B cells exposed to different particles. Figure 2A is IL-6 production (fold over control). Figure 2B is cell viability (viable cells over control). Cell culture is a means to identify potent and benign particles for further study. x SD, N = 6 Figure 2A 90 IL-6 Fold over Control 80 70 60 50 40 30 20 10 JE V8 0 M N C on tro l LP S C FA AC TR SR R PH P PE SR M D V 2S A W F U N W TR M SR R 40 W R M T 14 1 PH R D D 8m i W W M F0 1 2S B 0 PM2.5 Soil Dusts (80µg/cm 2 ) Figure 2B 1.2 Viable Cells/Control 1 0.8 0.6 0.4 0.2 JE V8 0 M N C on tro l LP S C FA AC TR SR R PH P PE SR M D V 2S A W F U N W TR M SR R 40 W R M T 14 1 PH R D D 8m i W W M F0 1 2S B 0 PM2.5 Soil Dusts (80µg/cm 2 ) The particle characteristics or components that cause cell death and IL-6 release are robust, but can be altered by physical treatments. Figure 3 shows the effect of the thermal and leaching treatments of three diverse particle samples: DD, WM, and R4. Heating of the particles had no effect on cytotoxicity, but three-day aqueous washing did reduce the cytotoxicity of the particles. However, the effect of leaching was likely a combination of mechanical loss of particles during washing and partial dissolution. Separation of the 6 samples into an LHC-9-leached solid and a supernatant containing the components that dissolve in media showed that cytotoxicity was associated with the solid phase. Physical treatments also affected the particle factors controlling the IL-6 response. Figure 3 shows thermal treatment at 150, 300, and 550 °C to DD, WM and R4. Thermal treatment at 150 °C for 1 h had no significant effect on the IL-6 response. Heating to 300 and 550 °C attenuated the IL-6 release. Heating to 550 °C is used to prebake quartz filters for trace organic analysis, and is sufficient to oxidize most organic materials, but 550 °C treatment surprisingly did not reduce the IL-6 response to the control level for DD. The three-day leaching treatment attenuated the IL-6 release, but did not reduce it to control level. The IL-6 release response was predominantly associated with the solid phase for all three soil dusts. The LHC-9 supernatant caused a small, but statistically significant, IL-6 response for R4. 7 Figure 3. Cytotoxicity (A) and IL-6 (B) response of BEAS-2B cells exposed to untreated and physically modified particles of soil dust. Identifications: WT, wild-type unmodified particles, 150, 300, 550°C, oxidizing thermal treatment at indicated temperature for one hour; LHC-S, supernatant from initial leaching in cell culture media; LHC-P, particles recovered after three cycles of leaching in cell culture media. * designates statistically different from untreated particles, # designates not statistically different from media-only control, p<0.05. x SD, N = 6 Figure 3A Viable Cells / Control 1.2 * * * 1.0 * 0.8 0.6 0.4 0.2 0.0 WT 150 DD - Desert Dust 300 550 WM - West Mesa LHC-P LHC-S R4 - Range 40 Figure 3B IL-6 Fold over Control 10 8 6 * 4 * * * # 2 * * * # # * * * # # 0 WT 150 DD - Desert Dust 300 550 WM - West Mesa LHC-P LHC-S R4 - Range 40 8 Figure 4 presents the heating of iron-silica nanoparticle mixtures to flame temperatures. Concentrations of 10, 30, and 90µg/cm2 were used to treat BEAS-2B cells. Thermal treatment increased the toxicity of iron-silica particles suggesting that chemical reactions or mineral transformations are creating a more toxic species. When a carbon source was added toxicity of iron-silica particles appeared to decrease. The thermal treatment of iron-silica particles caused slight increases in IL-6 production by these lung cells at the 10 and 30µg/cm2 doses. 9 Figure 4. BEAS-2B cells exposed to untreated and physically modified iron-silica nanoparticles. Particles were modified by heat (HT) and heat with a carbon source (HT+C). Particles exposed to heat induce IL-6 (A) and are more toxic (B). x SD, N = 2 IL6 Fold over Control Figure 4A 10 8 6 4 2 0 SiFe 10 ug/cm^2 SiFe+HT 30 µg/cm^2 SiFe+HT+C 90 µg/cm^2 Viable Cells/ Control Figure 4B 1.5 1 0.5 0 SiFe 10 ug/cm^2 SiFe+HT 30 µg/cm^2 SiFe+HT+C 90 µg/cm^2 DISCUSSION Cell culture models can be used to derive information on particle components or characteristics that cause toxicological responses. Exposure to different sources of well characterized particles using human lung cell cultures provides a way to study biochemical mechanisms and to select particles for animal exposure studies. New insights have been gained from the physical treatment experiments, but the specific components or characteristics of the soil-derived particles that cause the IL-6 release remain elusive. Many papers have been published reporting in vitro cytokine induction in response to particles. The novel contributions of this paper are that a significant range of responses, from benign to highly inflammatory and cytotoxic, is caused by particles that are representative of fugitive dust from unpaved roads, and that physical treatments can be used to modify soil-derived PM2.5 particles and manufactured nanoparticles as a means of testing toxicology hypotheses related to potential active components or particle characteristics. 10 Geological dusts have often been assumed to be a benign or inert component of ambient air pollution. However this study strongly supports the hypothesis that the solid-phase PM2.5 component of certain soil dusts is responsible for both proinflammatory and cytotoxic responses in lung epithelial cells exposed to particles in vitro. Thus, studies of the health effects associated with the complex mixtures in ambient air pollution need to include assessment of the fine particles of geological origin. Similar methods will be used in a recently initiated study of the toxic effects of manufactured nanoparticles. Commercially manufactured nano-scale particles will be characterized using physical assays and cell culture assays that are motivated by the hypothesis that transition metals in particles induce lung inflammation through a cytokine signaling mechanism involving reactive oxygen species (ROS). Materials will be tested in the as-manufactured condition and after being subjected to surface-modifying treatments simulating fire and wastewater conditions. The future research focus of these studies will be on nanomaterials that: 1) contain transition metals, 2) produced and commercially distributed in powder or liquid suspension form, and 3) have identifiable uses in manufacturing, coatings, or consumer products. 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