Patrick Goegan

Acute Effects of Inhaled Urban Particles and Ozone : Lung Morphology, Macrophage Activity, and Plasma Endothelin-1

We studied acute responses of rat lungs to inhalation of urban particulate matter and ozone. Exposure to particles (40 mg/m3 for 4 hours; mass median aerodynamic diameter, 4 to 5 microm; Ottawa urban dust, EHC-93), followed by 20 hours in clean air, did not result in acute lung injury. Nevertheless, inhalation of particles resulted in decreased production of nitric oxide (nitrite) and elevated secretion of macrophage inflammatory protein-2 from lung lavage cells. Inhalation of ozone (0.8 parts per million for 4 hours) resulted in increased neutrophils and protein in lung lavage fluid. Ozone alone also decreased phagocytosis and nitric oxide production and stimulated endothelin-1 secretion by lung lavage cells but did not modify secretion of macrophage inflammatory protein-2. Co-exposure to particles potentiated the ozone-induced septal cellularity in the central acinus but without measurable exacerbation of the ozone-related alveolar neutrophilia and permeability to protein detected by lung lavage. The enhanced septal thickening was associated with elevated production of both macrophage inflammatory protein-2 and endothelin-1 by lung lavage cells. Interestingly, inhalation of urban particulate matter increased the plasma levels of endothelin-1, but this response was not influenced by the synergistic effects of ozone and particles on centriacinar septal tissue changes. This suggests an impact of the distally distributed particulate dose on capillary endothelial production or filtration of the vasoconstrictor. Overall, equivalent patterns of effects were observed after a single exposure or three consecutive daily exposures to the pollutants. The experimental data are consistent with epidemiological evidence for acute pulmonary effects of ozone and respirable particulate matter and suggest a possible mechanism whereby cardiovascular effects may be induced by particle exposure. In a broad sense, acute biological effects of respirable particulate matter from ambient air appear related to paracrine/endocrine disruption mechanisms.

Regulation of Promoter-CAT Stress Genes in HepG2 Cells by Suspensions of Particles from Ambient Air

A panel of HepG2-derived cell lines (CAT-Tox [L] assay, Xenometrix), harboring stress genes consisting of a sequence for chloramphenicol acetyltransferase (CAT) under the transcriptional regulation from mammalian promoters and response elements, was exposed for 18-24 hr to aqueous suspensions of urban dusts (SRM-1648, SRM-1649, EHC-93) or PM2.5 particles (particulate matter < 2.5 micron). Expression of CAT protein was measured by enzyme-linked immunosorbent assay. Induction of the CAT genes was verified with benzo[a]pyrene (CYP1A1, cytochrome P450 1A1 promoter; GSTYa, glutathione transferase subunit Ya promoter; XRE, xenobiotic response element), cadmium sulfate, and copper sulfate (HMTIIa, metallothionein IIa promoter; HSP70, heat shock protein 70 promoter). The urban dust suspensions were active on CYP1A1, GSTYa, and XRE cell lines. SRM-1648 and SRM-1649 were twice as potent as EHC-93 per unit mass in inducing the xenobiotic-dependent responses, which correlated with contents in polycyclic aromatic hydrocarbons. These three reference particles, as well as six PM2.5 preparations collected on hi-vol filters in the Great Lakes basin, were also found to induce HMTIIa and HSP70, the magnitude of the responses correlating closely with the amount of soluble copper in the particulate preparations. The results indicate that bioavailable chemical species in the unfractionated particles can directly and quantitatively induce xenobiotic, metal, and stress-dependent responses in a target cell model, resulting in patterns of gene induction consistent with the chemical compositions of the environmental materials. We propose that cell culture models could be helpful for toxicodynamic inferences in adjunct to environmental monitoring and exposure assessments.

Effects of serum protein and colloid on the alamarBlue assay in cell cultures.

The reagent alamarBlue allows for real-time and repeated monitoring of cell proliferation and cell viability in cytotoxicity assays. Foetal bovine serum (FBS), bovine serum albumin (BSA) and, to a lesser extent, polyvinylpyrrolidone (40,000 mw) produce an apparent decrease in the rate of reduction of the reagent in cell cultures. The effect is attributable in part to a measurement artefact, possibly due to binding of the reduced and oxidized, extracellular forms of alamarBlue to these agents, resulting in absorbance and fluorescence spectral shifts. For dual wavelength spectrophotometric determination, this effect can be corrected using empirical absorbance ratios and applying a general equation of the form: AR (570) = ( A (570) - A (600)R (0)) ( R (RI570) - R (R)R (0)R (R)I570 ) , where AR(570)(0) is the standardized absorbance of the reduced product at zero extracellular protein, A(570) and A(600) are the absorbance at 570 and 600 nm of the culture supernatant, r(0) is the ratio of the absorbance at 570 nm to the absorbance at 600 nm for the oxidized substrate, R(R) is the ratio of the absorbance at 600 nm to the absorbance at 570 nm for the reduced product and R(RI570) is the ratio of the absorbance at 570 nm for the reduced form in the presence of interfering protein to the absorbance at 570 nm in the absence of protein. The factors R(0), R(R) and R(RI570) are determined empirically at defined protein concentrations. After correction of absorbance values, FBS and BSA added to culture medium were found to depress the reduction of alamarBlue in lung fibroblasts and mesothelial cells. The alamarBlue assay is thus sensitive to protein conditions in culture media and assay parameters should be standardized for reproducibility.

Effects of ambient air particles on nitric oxide production in macrophage cell lines

We assessed thein vitro toxicity of various particles on three murine macrophage cell lines, (J774A.1, WR19M.1, RAW264.7). The cells were exposed to aqueous suspensions (0–100 μg/30 mm2 well) of urban particulate matter (SRM-1648, SRM-1649, EHC-93), fine particulate matter (PM2.5), titanium dioxide (SRM-154b), and respirable cristobalite (SRM-1879) for 2 h and were then stimulated with lipopolysaccharide (LPS, 100 ng/ml) and recombinant interferon-gamma (IFN, 100 U/ml). After overnight incubation with the particles and LPS/IFN, nitric oxide production was estimated from culture supernatant nitrite. Cell viability was determined by monitoring the rate of AlamarBlue™ reduction. The dose-effect relationships for nitrite and viability were modeled as a power function (Fold change=[Dose+1]β), where β represents the slope of the dose-response curve. Potency was defined as the rate of change in nitrite production corrected for cell viability (βPOTENCY = βNITRITE − βVIABILITY). Overall, the urban particles decreased nitric oxide production (βPOTENCY < 0), while exposure of the cells to fine particulate matter or cristobalite increased the production of nitric oxide (βPOTENCY > 0). Titanium dioxide (TiO2) was essentially inactive (βPOTENCY ≈ 0). The decrease in nitric oxide production seen in cells exposed to the urban particles was directly correlated to a decrease in the expression of inducible nitric oxide (iNOS) as determined by Western blot analysis. The results indicate that particles are modulators of nitric oxide production in murine macrophages and may directly disrupt expression of iNOS during concomitant pathogen exposure. Pathways leading to enhanced NO production causing cell injury, and to decreased NO release resulting in lower bacterial clearance, may both be relevant to the health effects of ambient particles.

Proteomic changes in human lung epithelial cells (A549) in response to carbon black and titanium dioxide exposures.

This study combined cytotoxicity assays with proteomic analysis to characterize the unique biological responses of the A549 human lung epithelial cell line to two physicochemically distinct respirable particles titanium dioxide (TiO2) and carbon black (CB). Cellular LDH, ATP, BrdU incorporation and resazurin reduction indicated that CB was more potent than TiO2. Proteomic analysis was done using 2D-GE and MALDI-TOF-TOF-MS. Proteomic changes reflected common and particle-specific responses. Particle-specific proteomic responses were associated with cell death (necrosis and apoptosis), viability and proliferation pathways. Our results suggested that these pathways were consistent with the cytotoxicity data. For instance, increased expressions of anti-proliferative proteins LMNA and PA2G4 were in agreement with the decreased BrdU incorporation in A549 cells after exposure to CB. Similarly, increased expression of HSPA5 that is associated with ATPase activity was consistent with decreased cellular ATP levels in these cells. These findings reveal that proteomic changes can explain the cellular cytotoxicity characteristics of the particles. In essence, our results demonstrate that the in vitro toxicoproteomic approach is a promising tool to gain insight into molecular mechanisms underlying particle exposure-specific cytotoxicity. BIOLOGICAL SIGNIFICANCE In this study we have shown that toxicoproteomics is a sensitive and informative method to resolve the toxicity characteristics of particles with different physicochemical properties. This approach can be useful in the investigation of molecular mechanisms underpinning cellular cytotoxic responses elicited by particle exposures. Thus, the toxicoproteomic approach can be valuable in assessing the risk associated with particle exposures in vitro.

Alteration in aromatic hydroxylation and lipid oxidation status in the lungs of rats exposed to ozone.

Fischer 344 rats were exposed to ozone by inhalation to identify sensitive indices of acute exposure. 5-Aminosalicylic acid (5-ASA) hydroxylation in bronchoalveolar lavage (BAL), an indicator of hydroxyl radical (*OH) formation, and lipid oxidation in various regions of airways, representing oxidative stress, were measured to verify whether they can function as markers of exposure. BAL cells and supernatants taken from rats that received saline or 5-ASA (ip, 50 mg/kg) prior to ozone exposure (0, 0.4, or 0.8 ppm for 4 h) were analyzed for products of lipid oxidation. *OH formation was assessed by analysis of the BAL supernatant for 5-aminotetrahydroxybenzoic acid (5-ATHBA), a hydroxylation product of 5-ASA. The tetrahydroxy derivative of 5-ASA was higher in the BAL of ozone-treated rats than in air controls, reaching significance (p <. 05) at 0.8 ppm of ozone, The products of lipid oxidation propanal and hexanal were higher in BAL cells taken from rats exposed to ozone, reaching significance (p <. 05) at a 0.8 ppm ozone level, compared to air control animals, irrespective of whether they received saline or 5-ASA prior to ozone exposure. Increases in cholesterol levels were also seen in BAL cells after rats were exposed to ozone. However, there were no significant dose-related changes in the lipid oxidation products in BAL supernatants after exposure to ozone. Lipid oxidation products in BAL cells and 5-ATHBA in lavage exhibited the potential to serve as markers of ozone exposure. This work was supported by Health Canada (#4320105) and Toxic Substances Research Initiatives (TSRI #60).

Respiratory burst in alveolar macrophages exposed to urban particles is not a predictor of cytotoxicity

We examined the utility of respiratory burst measurements in alveolar macrophages to assess adverse cellular changes following exposure to urban particles. Cells were obtained by bronchioalveolar lavage of Fisher 344 rats and exposed (0-100 μg/well) to urban particles (EHC-93, SRM-1648, SRM-1649, PM2.5), the soluble (EHC-93sol) and insoluble (EHC-93insol) fractions of EHC-93 (EHC-93tot), mineral particles (TiO(2), SiO(2)) and metal oxides (iron III oxide, iron II/III oxide, copper II oxide, nickel II oxide). The particle-induced respiratory burst was measured by chemiluminescence for 2h after the addition of particles. The cells were then stimulated with phorbol 12-myristate 13-acetate (PMA), yeast Zymosan fragments (Zymosan), or lipopolysaccharide plus interferon-gamma (LPS/IFN-γ) and the stimulant-induced respiratory burst was measured. Independently of the potential of particles to induce directly a respiratory burst, exposure to most particles attenuated the subsequent stimulant-induced burst. The notable exception was SiO(2), which produced a strong respiratory burst upon contact with the macrophages and enhanced the subsequent response to PMA or LPS/IFN-γ. Based on the degree of inhibition of the stimulant-dependent respiratory burst, particles were clustered into groups of high (SRM-1649, iron III oxide), intermediate (EHC-93tot, EHC-93insol, SRM-1648, VERP, iron II/III oxide, copper II oxide), and low (EHC-93sol, SiO(2), TiO2 and nickel II oxide) potency. Across these clusters, the potency of the particles to inhibit the stimulant-dependent respiratory burst showed poor correlation with cytotoxicity determined by XTT reduction assay.

90-Day Repeated Inhalation Exposure of Surfactant Protein-C/Tumor Necrosis Factor-α (SP-C/TNF-α) Transgenic Mice to Air Pollutants

Tumor necrosis factor (TNF)-α, a cytokine present in inflammed lungs, is known to mediate some of the adverse effects of ozone and inhaled particles. The authors evaluated transgenic mice with constitutive pulmonary expression of TNF-αunder transcriptional regulation of the surfactant protein-C promoter as an animal model of biological susceptibility to air pollutants. To simulate a repeated, episodic exposure to air pollutants, wild-type and TNF mice inhaled air or a mixture of ozone (0.4 ppm) and urban particles (EHC-93, 4.8 mg/m3) for 4 h, once per week, for 12 consecutive weeks and were sacrificed 20 h after last exposure. TNF mice exhibited chronic lung inflammation with septal thickening, alveolar enlargement, and elevated protein and cellularity in bronchoalveolar lavage fluid (genotype main effect, p <.001). Repeated exposure to pollutants did not result in measurable inflammatory changes in wild-type mice and did not exacerbate the inflammation in TNF mice. The pollutants decreased recovery of alveolar macrophages in lavage fluid of both wild-type and TNF mice (exposure main effect, p < .001). Exacerbation of the rate of protein nitration reactions specifically in the lungs of TNF mice was revealed by the high ratio of 3-nitrotyrosine to L-DOPA after exposure to the air pollutants (Genotype × Exposure factor interaction, p = .014). Serum creatine kinase-MM isoform increased in TNF mice exposed to pollutants (Genotype × Exposure factor interaction, p = .043). The marked pollutant-related nitration in the lungs of the TNF mice reveals basic differences in free radical generation and scavenging in the inflamed lungs in response to pollutants. Furthermore, elevation of circulating creatine kinase-MM isoform specifically in TNF mice exposed to pollutants suggests systemic adverse impacts from lung inflamma-tory mediators, possibly on muscles and the cardiovascular system.

Responses of A549 human lung epithelial cells to cristobalite and α-quartz exposures assessed by toxicoproteomics and gene expression analysis.

In this study, we used cytotoxicity assays, proteomic and gene expression analyses to examine the difference in response of A549 cells to two silica particles that differ in physical properties, namely cristobalite (CR) and α‐quartz (Min‐U‐Sil 5, MI). Cytotoxicity assays such as lactate dehydrogenase release, 5‐bromo‐2′‐deoxyuridine incorporation and cellular ATP showed that both silica particles could cause cell death, decreased cell proliferation and metabolism in the A549 human lung epithelial cells. While cytotoxicity assays revealed little difference between CR and MI exposures, proteomic and gene expression analyses unveiled both similar and unique molecular changes in A549 cells. For instance, two‐dimensional gel electrophoresis data indicated that the expression of proteins in the cell death (e.g., ALDH1A1, HTRA2 and PRDX6) and cell proliferation (e.g., FSCN1, HNRNPAB and PGK1) pathways were significantly different between the two silica particles. Reverse transcription–polymerase chain reaction data provided additional evidence supporting the proteomic findings. Preliminary assessment of the physical differences between CR and MI suggested that the extent of surface interaction between particles and cells could explain some of the observed biological effects. However, the differential dose–response curves for some other genes and proteins suggest that other physical attributes of particulate matter can also contribute to particulate matter‐related cellular toxicity. Our results demonstrated that toxicoproteomic and gene expression analyses are sensitive in distinguishing subtle toxicity differences associated with silica particles of varying physical properties compared to traditional cytotoxicity endpoints. Copyright

Human lung epithelial cell A549 proteome data after treatment with titanium dioxide and carbon black

Here, we have described the dataset relevant to the A549 cellular proteome changes after exposure to either titanium dioxide or carbon black particles as compared to the non-exposed controls, “Proteomic changes in human lung epithelial cells (A549) in response to carbon black and titanium dioxide exposures” (Vuong et al., 2016) [1]. Detailed methodologies on the separation of cellular proteins by 2D-GE and the subsequent mass spectrometry analyses using MALDI-TOF-TOF-MS are documented. Particle exposure-specific protein expression changes were measured via 2D-GE spot volume analysis. Protein identification was done by querying mass spectrometry data against SwissProt and RefSeq protein databases using Mascot search engine. Two-way ANOVA analysis data provided information on statistically significant A549 protein expression changes associated with particle exposures.