Winter fine particulate matter from Milan induces morphological and functional alterations in human pulmonary epithelial cells (A549)

Samples of PM_2.5 were gravimetrically collected during the winter 2005/2006 in the urban area of Milan (North Italy). Samples were chemically characterized and the particles were detached from filters to determine their cytotoxic effects on the A549 cell line. Based on the potential toxicological relevance of its components, Milan winter PM_2.5 contained high concentrations of pro-oxidant transition metals and PAHs, while re-suspended particles showed a relatively high frequency of dimensional classes ranging from 40 nm to 300 nm.     

Mouse lung inflammation after instillation of particulate matter collected from a working dairy barn

Coarse and fine particulate matter (PM_2.5-10 and PM_2.5, respectively) are regulated ambient air pollutants thought to have major adverse health effects in exposed humans. The role of endotoxin and other bioaerosol components in the toxicity of PM from ambient air is controversial. This study evaluated the inflammatory lung response in mice instilled intratracheally with PM_2.5-10 and PM_2.5 emitted from a working dairy barn, a source presumed to have elevated concentrations of endotoxin. PM_2.5-10 was more pro-inflammatory on an equal weight basis than was PM_2.5; both fractions elicited a predominantly neutrophilic response. The inflammatory response was reversible, with a peak response to PM_2.5-10 observed at 24 h after instillation, and a return to control values by 72 h after instillation. The major active pro-inflammatory component in whole PM_2.5-10, but not in whole PM_2.5, is heat-labile, consistent with it being endotoxin. A heat treatment protocol for the gradual inactivation of biological materials in the PM fractions over a measurable time course was developed and optimized in this study using pure lipopolysaccharide (LPS) as a model system. The time course of heat inactivation of pure LPS and of endotoxin activity in PM_2.5-10 as measured by Limulus bioassay is identical. The active material in both PM_2.5-10 and PM_2.5 remained in the insoluble fraction when the whole PM samples were extracted with physiological saline solution. Histological analysis of lung sections from mice instilled with PM_2.5-10 or PM_2.5 showed evidence of inflammation consistent with the cellular responses observed in lung lavage fluid. The major pro-inflammatory components present in endotoxin-rich PM were found in the insoluble fraction of PM_2.5-10; however, in contrast with PM_2.5-10 isolated from ambient air in the Central Valley of California, the active components in the insoluble fraction were heat-labile.     

p53-Dependent apoptosis induced in human bronchial epithelial (16-HBE) cells by PM2.5 sampled from air in Guangzhou,China

Epidemiological studies have shown that air pollution particulate matter (PM) is associated with increased respiratory morbidity and mortality. However, the mechanisms are not fully understood. Oxidative stress-mediated apoptosis plays an important role in the occurrence of respiratory diseases. In this study, human bronchial epithelial (16-HBE) cells were exposed to different concentrations (16–128?µg/ml) of PM_2.5 for 24?h to investigate the apoptosis induced by PM_2.5. The results showed that PM_2.5 exposure significantly induced apoptosis, DNA strand breaks, and oxidative damage in a dose-dependent manner in 16-HBE cells. The expression of p53 and p73 increased significantly along with the dose of PM_2.5 in 16-HBE cells, whereas the expression of p21^Cip1/WAF1 decreased; the expression of mdm2 increased and then decreased, but not significantly. Taken together, these observations indicate that PM_2.5 may lead to oxidative damage and induce apoptosis through the p53-dependent pathway in 16-HBE cells. p53-dependent apoptosis mediated by DNA strand breaks may be an important mechanism of PM_2.5-induced apoptosis in 16-HBE cells.     

Inflammatory effects of coarse and fine particulate matter in relation to chemical and biological constituents

There is conflicting evidence in the literature as to the predominant mechanism and also the compositional element(s) that drives the pulmonary inflammatory response of ambient particulate matter (PM). We have investigated the inflammogenic potential of coarse (2.5-10 microm) and fine (<2.5 microm) PM from both a rural and an industrial location in Germany, using bronchoalveolar lavage (BAL) of rat lungs 18 h post intratracheal instillation with PM. Irrespective of the sampling location, the coarse fraction of PM(10) but not its fine counterpart caused neutrophilic inflammation in rat lungs, in the absence of any severe pulmonary toxicity as indicated by the lack of an increase in lavage protein and lactate dehydrogenase levels. The rural sample of coarse PM also caused a significant increase in the tumor necrosis factor alpha (TNFalpha) content as well as glutathione depletion in the BAL fluid. The contrasting inflammatory responses of the different samples could not be explained by differences in the concentrations of soluble Fe, Cu, V, Ni, Cr, or Al or by the.OH generating capacities of the PM suspensions. However, the effects of the different PM samples were clearly associated with their endotoxin content, as well as their ability to induce interleukin (IL)-8 and TNFalpha from whole blood in vitro. In conclusion, on an equal mass basis, coarse but not fine PM samples from our sampling campaign induced an inflammatory reaction in the lung in the absence of gross cellular lung damage, following intratracheal instillation. Our data also indicate, in accordance with previous independent in vitro observations, that endotoxin or related contaminants may play a role in these in vivo effects.     

Gene expression profiling of A549 cells exposed to Milan PM2.5

Background

Particulate matter (PM) has been associated to adverse health effects in exposed population and DNA damage has been extensively reported in in vitro systems exposed to fine PM (PM2.5). The ability to induce gene expression profile modulation, production of reactive oxygen species (ROS) and strand breaks to DNA molecules has been investigated in A549 cells exposed to winter and summer Milan PM2.5.

Results

A549 cells, exposed to 10 μg/cm² of both winter and summer PM2.5, showed increased cytotoxicity at 24 h and a significant increase of ROS at 3 h of treatment. Despite these similar effects winter PM induced a higher number of gene modulation in comparison with summer PM. Both PMs modulated genes related to the response to xenobiotic stimuli (CYP1A1, CYP1B1, TIPARP, ALDH1A3, AHRR) and to the cell-cell signalling (GREM1) pathways with winter PM2.5 inducing higher fold increases. Moreover the winter fraction modulated also JUN (cell-cell signalling), GDF15, SIPA1L2 (signal transduction), and HMOX1 (oxidative stress). Two genes, epiregulin (EREG) and FOS-like antigen1 (FOSL1), were significantly up-regulated by summer PM2.5. The results obtained with the microarray approach have been confirmed by qPCR and by the analysis of CYP1B1 expression. Comet assay evidenced that winter PM2.5 induced more DNA strand breaks than the summer one.

Conclusion

Winter PM2.5 is able to induce gene expression alteration, ROS production and DNA damage. These effects are likely to be related to the CYP enzyme activation in response to the polycyclic aromatic hydrocarbons (PAHs) adsorbed on particle surface.     

The toxic effects of indoor atmospheric fine particulate matter collected from allergic and non‐allergic families in Wuhan on mouse peritoneal macrophages

Recent studies have shown that fine particulate matter (PM_2.5) is associated with multiple adverse health outcomes and PM_2.5‐induced oxidative stress is now commonly known as a proposed mechanism of PM_2.5‐mediated toxicity. However, the association between allergic symptoms in children and exposure to PM_2.5 has not been fully elucidated, particularly the role of PM_2.5 on the indoor environment involved in allergy or non‐allergy is unknown. The aim of the present study was to explore whether indoor PM_2.5 from the homes of children with allergic symptoms had more increased risks of allergy than that of healthy ones and then compare the toxicity and inflammatory response of them. In this study, indoor PM_2.5 was collected from the homes of schoolchildren with allergic symptoms and those of healthy ones respectively, and components of PM_2.5 were analyzed. PM_2.5‐mediated oxidative damage and inflammatory response were further evaluated in mouse peritoneal macrophages based on its effects on the levels of reactive oxygen species accumulation, lipid peroxidation, DNA damage or cytokine production. It seems that oxidative stress may contribute to PM_2.5‐induced toxicity, and PM_2.5 from the allergic indoor environment produced more serious toxic effects and an inflammatory response on mouse peritoneal macrophages than that from a non‐allergic indoor environment. Copyright © 2015 John Wiley & Sons, Ltd.     

In vitro evaluation of the toxicity induced by nickel soluble and particulate forms in human airway epithelial cells

Epidemiological studies show that exposure to nickel (Ni) compounds is associated with a variety of pulmonary adverse health effects, such as lung inflammation, fibrosis, emphysema and tumours. However, the mechanisms leading to pulmonary toxicity are not yet fully elucidated. In the current study we used Calu-3, a well differentiated human bronchial cell line, to investigate in vitro the effect of Ni in soluble form (NiCl₂) and in the form of micro-sized Ni particles on the airway epithelium. For this purpose, we evaluated the effect of Ni compounds on the epithelial barrier integrity by monitoring the transepithelial electrical resistance (TEER) and on oxidative stress pathways by measuring reactive oxygen species (ROS) formation and induction of stress-inducible genes. Our results showed that exposure to NiCl₂ and Ni particles resulted in a disruption of the epithelial barrier function observed by alterations in TEER, which occurred prior to the decrease in cell viability. Moreover, Ni compounds induced oxidative stress associated with ROS formation and up-regulation of the stress-inducible genes, Metallothionein 1X (MT1X), Heat shock protein 70 (HSP70), Heme oxygenase-1 (HMOX-1), and gamma-glutamylcysteine synthetase (γGCS). Furthermore, we have demonstrated that the induced effects by Ni compounds can be partially attributed to the increase in Ni ions (Ni²⁺) intracellular levels.     

Effects of PM2.5 components in the release of amphiregulin by human airway epithelial cells

Exposure to ambient particulate matter (PM) is responsible for airway inflammation and tissue remodeling. Urban PM(2.5) (aerodynamic diameter <2.5microm) is a complex mixture rich in soots and containing hydrosoluble and organic components. We previously showed that the exposure of airway epithelial cells to PM(2.5) triggers the release of amphiregulin (AR), ligand of the epidermal growth factor receptor (EGFR) involved in proinflammatory and repair responses. The effect of Paris PM(2.5) organic and aqueous fractions in AR expression and secretion was investigated on the bronchial epithelial cell line 16HBE and normal human nasal epithelial (NHNE) cells. Both a macroarray specific for inflammation pathways and RT-PCR showed an AR upregulation in organic extract-treated 16HBE cells. AR release is induced in 16HBE and NHNE cells grown on plastic and exposed to native PM(2.5), organic extract and to a lesser extent washed PM(2.5) (deprived of its hydrosoluble content) and aqueous extract. Furthermore, as assessed by using NHNE cells grown on Transwell inserts, this secretion is polarized toward the basolateral side where the EGFR is expressed. To conclude, both PM(2.5) organic and hydrosoluble components are involved in the expression and secretion of AR; organic compounds exhibiting a strong effect when they are easily bioavailable.     

Cytoplasmic p21, ERK1/2 activation,and cytoskeletal remodeling are associated with the senescence-like phenotype after airborne particulate matter (PM10) exposure in lung cells

The exposure to particulate matter with a mean aerodynamic diameter ≤10 μm (PM₁₀) from urban zones is considered to be a risk factor in the development of cancer. The aim of this work was to determine if PM₁₀ exposure induces factors related to the acquisition of a neoplastic phenotype, such as cytoskeletal remodeling, changes in the subcellular localization of p21^CIP1/WAF1, an increase in β-galactosidase activity and changes in cell cycle. To test our hypothesis, PM₁₀ from an industrial zone (IZ) and a commercial zone (CZ) were collected, and human adenocarcinoma lung cell cultures (A549) were exposed to a sublethal PM₁₀ concentration (10 μg/cm²) for 24 h and 48 h. The results showed that PM₁₀ exposure induced an increase in F-actin stress fibers and caused the cytoplasmic stabilization of p21^CIP1/WAF1 via phosphorylation at Thr¹⁴⁵ and Ser¹⁴⁶ and the phosphorylation of ERK1/2 on Thr²⁰². Changes in the cell cycle or apoptosis were not observed, but an increase in β-galactosidase activity was detected. The PM₁₀ from CZ caused more dramatic effects in lung cells. We conclude that PM₁₀ exposure induced cytoplasmic p21^CIP1/WAF1 retention, ERK1/2 activation, cytoskeleton remodeling and the acquisition of a senescence-like phenotype in lung cells. These alterations could have mechanistic implications regarding the carcinogenic potential of PM₁₀.     

Urban particulate matter induces pro-remodeling factors by airway epithelial cells from healthy and asthmatic children

Abstract

Context: Chronic exposure to ambient particulate matter pollution during childhood is associated with decreased lung function growth and increased prevalence of reported respiratory symptoms. The role of airway epithelium-derived factors has not been well determined.

Objective: To determine if urban particulate matter (UPM) stimulates production of vascular endothelial growth factor (VEGF) and transforming growth factor-β₂ (TGF-β₂), and gene expression of mucin 5AC (MUC5AC) and interleukin-(IL)-8 by primary airway epithelial cells (AECs) obtained from carefully phenotyped healthy and atopic asthmatic school-aged children.

Methods: Primary AECs from 9 healthy and 14 asthmatic children were differentiated in air--liquid interface (ALI) culture. The apical surface was exposed to UPM suspension or phosphate buffered saline (PBS) vehicle control for 96?h. VEGF and TGF-β₂ concentrations in cell media at baseline, 48 and 96?h were measured via ELISA. MUC5AC and IL-8 expression by AECs at 96?h was measured via quantitative polymerase chain reaction.

Results: Baseline concentrations of VEGF, but not TGF-β₂, were significantly higher in asthmatic versus healthy cultures. UPM stimulated production of VEGF, but not TGF-β₂, at 48 and 96?h; the magnitude of change was comparable across groups. At 96?h there was greater MUC5AC and IL-8 expression by UPM exposed compared to PBS exposed AECs.

Conclusions: Induction of the pro-remodeling cytokine VEGF may be a potential mechanism by which UPM influences lung function growth in children irrespective of asthma status. Respiratory morbidity associated with UPM exposure in children may be related to increased expression of MUC5AC and IL-8.     

Chemical partitioning of fine particle-bound As,Cd, Cr,Ni, Co,Pb and assessment of associated cancer risk due to inhalation,ingestion and dermal exposure

The bioavailability and human health risks of As, Pb, Ni, Co, Cr and Cd in fine particulate matter (PM_2.5) at an urban site on a National highway in Agra, India were investigated. Inductively coupled plasma-optical emission spectrometer was used for metal analysis in sequentially extracted samples to ascertain the highly mobile, reducible, bioavailable and immobile fractions of the metals. Cancer risk resulting from inhalation, dermal and ingestion exposure to each metal in these fractions was calculated according to US EPA models. The average mass concentration of PM_2.5 was 87.16?±?62.51?μg/m³. Cr, Ni and Pb were the most abundant metals. The results showed that Pb and Cr were higher in the mobile fraction. Cd and Co had high bioavailability. Ingestion is the major exposure pathway for all heavy metals except Cr to infants, children and adults followed by inhalation and dermal contact. The cumulative risk for Cr(VI) due to dermal and inhalation routes exceed the maximum acceptable limit for children of age 1–7?years, 8–15?years and adults when total concentration is considered, but the estimated risks are within the acceptable limit when the bioavailable, water soluble and mobile fraction are taken into account. Hence the study shows that children and adults living in the vicinity of this site are more susceptible, hence more attention should be paid to protect them from pollution hazards. The study indicates the importance of metal speciation in assessing associated human health risks.     

Gene expression profiling analysis reveals arsenic-induced cell cycle arrest and apoptosis in p53-proficient and p53-deficient cells through differential gene pathways

Arsenic (As) is a well-known environmental toxicant and carcinogen as well as an effective chemotherapeutic agent. The underlying mechanism of this dual capability, however, is not fully understood. Tumor suppressor gene p53, a pivotal cell cycle checkpoint signaling protein, has been hypothesized to play a possible role in mediating As-induced toxicity and therapeutic efficiency. In this study, we found that arsenite (As³⁺) induced apoptosis and cell cycle arrest in a dose-dependent manner in both p53^+/+ and p53^−/− mouse embryonic fibroblasts (MEFs). There was, however, a distinction between genotypes in the apoptotic response, with a more prominent induction of caspase-3 in the p53^−/− cells than in the p53^+/+ cells. To examine this difference further, a systems-based genomic analysis was conducted comparing the critical molecular mechanisms between the p53 genotypes in response to As³⁺. A significant alteration in the Nrf2-mediated oxidative stress response pathway was found in both genotypes. In p53^+/+ MEFs, As³⁺ induced p53-dependent gene expression alterations in DNA damage and cell cycle regulation genes. However, in the p53^−/− MEFs, As³⁺ induced a significant up-regulation of pro-apoptotic genes (Noxa) and down-regulation of genes in immune modulation. Our findings demonstrate that As-induced cell death occurs through a p53-independent pathway in p53 deficient cells while apoptosis induction occurs through p53-dependent pathway in normal tissue. This difference in the mechanism of apoptotic responses between the genotypes provides important information regarding the apparent dichotomy of arsenic's dual mechanisms, and potentially leads to further advancement of its utility as a chemotherapeutic agent.     

Microarray analysis of gene expression in rat alveolar epithelial cells exposed to fractionated organic extracts of diesel exhaust particles

Diesel exhaust particles (DEP) affect health adversely. Our previous studies revealed that DEP extracts up-regulated expression of genes related to drug metabolism, antioxidant enzymes, cell cycle/apoptosis and coagulation, and in addition, n-hexane soluble fraction (n-HSF) of DEP extracts contains aliphatic and polycyclic aromatic hydrocarbons, and n-hexane insoluble fraction (n-HISF) contains oxygenated compounds and has strong oxidative property. However, the relationship between the properties of chemicals in DEP extracts and the gene expression has not been fully elucidated. Here, we used a microarray analysis to identify and characterize genes whose expression is regulated by exposure to fractions of DEP extracts. A dichloromethane-soluble fraction (DMSF) of DEP was further fractionated into n-HSF and n-HISF. We exposed rat alveolar epithelial (SV40T2) cells to these fractions (30 μg/ml) for 6 h and identified regulated genes. DMSF predominantly up-regulated genes associated with drug metabolism (Cyp1a1, Gsta3), oxidative stress response (HO-1, Srxn1) and cell cycle/apoptosis (Okl38). Genes up-regulated by n-HSF were mainly associated with drug metabolism (Cyp1a1, Gsta3). The genes up-regulated by n-HISF included antioxidant enzymes (HO-1, Srxn1); genes response to cell damage, such as those functioning in cell cycle regulation or apoptosis (Okl38); and genes in coagulation pathways. Our present results suggested that n-HSF and n-HISF regulated characteristic genes which respond to chemical properties of each fraction.     

Water-soluble and organic extracts of ambient PM2.5 in Tehran air: assessment of genotoxic effects on human lung epithelial cells (A549) by the Comet assay

The main aim of the present study is to investigate the physicochemical characterization of water-soluble extract (WS) and organic extract (OE) of PM_2.5 ambient in Tehran city air in order to evaluate the genotoxicity effects and the potential attribution to these effects. The lung epithelial cells (A549) were exposed to WS and OE, while Comet assays were conducted to analyze the genotoxicity. The results show that the amount of DNA damage in WS fraction and solvent-extractable organic samples is significantly higher than the control group and the increase in concentration significantly contributes to increase in the amount of DNA damage.     

UVB light regulates expression of antioxidants and inflammatory mediators in human corneal epithelial cells

The cornea is highly sensitive to ultraviolet B (UVB) light-induced oxidative stress, a process that results in the production of inflammatory mediators which have been implicated in tissue injury. In the present studies, we characterized the inflammatory response of human corneal epithelial cells to UVB (2.5–25 mJ/cm²). UVB caused a dose-dependent increase in the generation of reactive oxygen species in the cells. This was associated with increases in mRNA expression of the antioxidants Cu,Zn superoxide dismutase (SOD), Mn-SOD, catalase and heme oxygenase-1 (HO-1), as well as the glutathione S-transferases (GST), GSTA1-2, GSTA3, GSTA4, GSTM1, and mGST2. UVB also upregulated expression of the proinflammatory cytokines, IFNγ, IL-1β, TGFβ and TNFα, and enzymes important in prostaglandin (PG) biosynthesis including cyclooxygenase-2 (COX-2) and the PG synthases mPGES-2, PGDS, PGFS and thromboxane synthase, and in leukotriene biosynthesis including 5-lipoxygenase (5-LOX), 15-LOX-2, and the epidermal and platelet forms of 12-LOX. UVB was found to activate JNK and p38 MAP kinases in corneal epithelial cells; ERK1/2 MAP kinase was found to be constitutively active, and its activity increased following UVB treatment. Inhibition of p38 blocked UVB-induced expression of TNFα, COX-2, PGDS and 15-LOX-2, while JNK inhibition suppressed TNFα and HO-1. These data indicate that UVB modulates corneal epithelial cell expression of antioxidants and proinflammatory mediators by distinct mechanisms. Alterations in expression of these mediators are likely to be important in regulating inflammation and protecting the cornea from UVB-induced oxidative stress.     

Integrative analysis of mRNA and microRNA expression of a human alveolar epithelial cell(A549) exposed to water and organic‐soluble extract from particulate matter (PM)2.5

MicroRNA (miRNA) is now attracting attention as a powerful negative regulator of messenger RNA(mRNA) levels, and is implicated in the modulation of important mRNA networks involved in toxicity. In this study, we assessed the effects of particulate matter 2.5 (PM_2.5), one of the most significant air pollutants, on miRNA and target gene expression. We exposed human alveolar epithelial cell (A549) to two types of PM_2.5[water (W‐PM_2.5) and organic (O‐PM_2.5) soluble extracts] and performed miRNA microarray analysis. A total of 37 miRNAs and 62 miRNAs were altered 1.3‐fold in W‐PM_2.5 and O‐PM_2.5, respectively. Integrated analyses of miRNA and mRNA expression profiles identified negative correlations between miRNA and mRNA in both W‐PM_2.5 and O‐PM_2.5 exposure groups. Gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses showed that the 35 W‐PM_2.5 target genes are involved in responses to nutrients, positive regulation of biosynthetic processes, positive regulation of nucleobase, nucleoside, and nucleotide, and nucleic acid metabolic processes; while the 69 O‐PM_2.5 target genes are involved in DNA replication, cell cycle processes, the M phase, and the cell cycle check point. We suggest that these target genes may play important roles in PM_2.5‐induced respiratory toxicity by miRNA regulation. These results demonstrate an integrated miRNA‐mRNA approach for identifying molecular events induced by environmental pollutants in an in vitro human model. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 302–310, 2017.