TACE/TGF-α/EGFR regulates CXCL8 in bronchial epithelial cells exposed to particulate matter components.

Airborne particulate matter (PM) may induce or exacerbate neutrophilic airway disease by triggering the release of inflammatory mediators, such as CXC chemokine ligand (CXCL)8, from the airway epithelium. It is still unclear which PM components are driving CXCL8 responses, as most candidates occur at low concentrations in the dusts. We therefore hypothesised that different PM constituents may contribute through common mechanisms to induce CXCL8. Human bronchial epithelial cells (BEAS-2B) were exposed to different PM components (Zn²âº/Fe²âº salts, 1-nitropyrene, lipopolysaccharide and diesel exhaust/mineral particles). Gene expression patterns were detected by real-time PCR array. CXCL8 responses were measured by real-time PCR and ELISA. CXCL8 regulation was assessed with a broad inhibitor panel and neutralising antibodies. Epidermal growth factor receptor (EGFR) phosphorylation was examined by immunoprecipitation and Western blotting. Component-induced gene expression was mainly linked to nuclear factor-κB, Ca²âº/protein kinase C, phospholipase C, low-density lipoprotein and mitogenic signalling. Many inhibitors attenuated CXCL8 release induced by all PM components, but to varying extents. However, EGFR inhibition strongly reduced CXCL8 release induced by all test compounds and selected compounds increased EGFR phosphorylation. Interference with transforming growth factor (TGF)-α or tumour necrosis factor-α-converting enzyme (TACE), which mediates TGF-α ectodomain shedding, also attenuated CXCL8 release. Different PM constituents induced CXCL8 partly through similar signalling pathways but the relative importance of the different pathways varied. However, TACE/TGF-α/EGFR signalling appears to be a convergent pathway regulating innate immune responses of airway epithelial cells upon exposure to multiple airborne pollutants.

Differential effects of nitro-PAHs and amino-PAHs on cytokine and chemokine responses in human bronchial epithelial BEAS-2B cells.

Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are found in diesel exhaust and air pollution particles. Along with other PAHs, many nitro-PAHs possess mutagenic and carcinogenic properties, but their effects on pro-inflammatory processes and cell death are less known. In the present study we examined the effects of 1-nitropyrene (1-NP), 3-nitrofluoranthene (3-NF) and 3-nitrobenzanthrone (3-NBA) and their corresponding amino forms, 1-AP, 3-AF and 3-ABA, in human bronchial epithelial BEAS-2B cells. The effects of the different nitro- and amino-PAHs were compared to the well-characterized PAH benzo[a]pyrene (B[a]P). Expression of 17 cytokine and chemokine genes, measured by real-time PCR, showed that 1-NP and 3-NF induced a completely different cytokine/chemokine gene expression pattern to that of their amino analogues. 1-NP/3-NF-induced responses were dominated by maximum effects on CXCL8 (IL-8) and TNF-alpha expression, while 1-AP-/3-AF-induced responses were dominated by CCL5 (RANTES) and CXCL10 (IP-10) expression. 3-NBA and 3-ABA induced only marginal cytokine/chemokine responses. However, 3-NBA exposure induced considerable DNA damage resulting in accumulation of cells in S-phase and a marked increase in apoptosis. B[a]P was the only compound to induce expression of aryl hydrocarbon receptor (AhR)-regulated genes, such as CYP1A1 and CYP1B1, but did not induce cytokine/chemokine responses in BEAS-2B cells. Importantly, nitro-PAHs and amino-PAHs induced both qualitatively and quantitatively different effects on cytokine/chemokine expression, DNA damage, cell cycle alterations and cytotoxicity. The cytokine/chemokine responses appeared to be triggered, at least partly, through mechanisms separate from the other examined endpoints. These results confirm and extend previous studies indicating that certain nitro-PAHs have a considerable pro-inflammatory potential.

Cytokine and chemokine expression patterns in lung epithelial cells exposed to components characteristic of particulate air pollution.

Airborne particulate matter (PM) has a complex composition, and the relative contribution of different compounds to PM-induced effects is only partly understood. The present study compared the capability of selected components commonly found in PM, to induce pro-inflammatory responses in lung epithelial cells. Ultrafine carbon black (ufCB), ZnCl(2), FeSO(4), 1-nitropyrene (1-NP), lipopolysaccharide (LPS), and crystalline silica (positive control) were screened for effects on the expression of 84 inflammation-related genes in the bronchial epithelial cell line, BEAS-2B. A total of 22 genes were up-regulated by one or more of the tested compounds, and 5 cytokine and 11 chemokine genes were selected for further studies. After 10h exposure, silica induced significantly increased expression of CCL20, CXCL1/-3/-8/-10/-11, lymphotoxin (LT)beta and interleukin (IL)-6; ufCB induced CXCL8/-10 and -11; ZnCl(2) induced CCL11/-20/-26, CXCL1/-5/-8/-14 and tumor necrosis factor (TNF)-alpha; FeSO(4) induced a weak up-regulation of CXCL8 and TNF-alpha; LPS induced CCL20, CXCL1/-5/-8/-10/-11, LTbeta and IL-6; and 1-NP induced expression of CCL20, CXCL1/-3/-8, TNF-alpha and IL-6. Despite obvious differences, all compounds induced response-patterns that correlated relatively well with that of silica, the positive control. The predominant response appeared to be increased gene expression of neutrophil-recruiting CXC-chemokines. CXCL8 was the only gene induced by all tested PM-components, the most up-regulated on average, and also dominating the gene-expression patterns induced by coarse PM. The data show quantitative, and to a certain extent qualitative differences in cytokine/chemokine gene-expression profiles of the compounds tested. However, there were also striking similarities in the response-patterns induced by these physically/chemically widely different compounds.

Hyphae fragments from A. fumigatus sensitize lung cells to silica particles (Min-U-Sil): Increased release of IL-1ß.

Exposure to particulate matter (PM), such as mineral particles and biological particles/components may be linked to aggravation of respiratory diseases, including asthma. Here we report that exposure to Aspergillus fumigatus hyphae fragments (AFH) and lipopolysaccharide (LPS) induced both mRNA synthesis and release of pro-inflammatory interleukin-1 beta (IL-1ß) in both human THP-1 monocytes (THP-1 Mo) and phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 monocytes (THP-1 macrophages; THP-1 Ma); while Min-U-Sil alone enhanced the release of IL-1ß only in THP-1 Ma. Co-exposure to LPS or AFH with Min-U-Sil caused a synergistic release of IL-1ß when compared to single exposures. In contrast, Min-U-Sil did not markedly change LPS- and AFH-induced release of tumor necrosis factor alpha (TNF-α). The combined exposures did not increase the LPS- and AFH-induced expression of IL-1ß mRNA. Notably, the AFH- and LPS-induced IL-1ß responses with and without co-exposure to Min-U-Sil in THP-1 Mo were found to be caspase-dependent as shown by inhibition with zYVAD-fmk. Furthermore, co-exposure with AFH and Min-U-Sil resulted in similar synergistic releases of IL-1ß in primary human airway macrophages (AM; sputum), peripheral blood monocyte-derived macrophages (MDM) and in the human bronchial epithelial cell line (BEAS-2B). In conclusion, AFH induce both the synthesis and release of IL-1ß. However, Min-U-Sil further enhanced the cleavage of the induced pro-IL-1ß.

The effects of 1st and 2nd generation biodiesel exhaust exposure on hematological and biochemical blood indices of Fisher344 male rats - The FuelHealth project.

Diesel exhaust emissions (DEE), being one of the main causes of ambient air pollution, exert a detrimental effect on human health and increase morbidity and mortality related to cardiovascular and pulmonary diseases. Therefore, the objective of the present study was to investigate potential adverse effects of exhausts emissions from B7 fuel, the first-generation biofuel containing 7% of fatty acid methyl esters (FAME), and SHB20 fuel, the second-generation biofuel containing 20% FAME/hydrotreated vegetable oil (HVO), after a whole-body exposure with and without diesel particle filter (DPF). The experiment was performed on 95 male Fischer 344 rats, divided into 10 groups (8 experimental, 2 control). Animals were exposed to DEE (diluted with charcoal-filtered room air to 2.1-2.2% (v/v)) for 7 or 28 days (6 h/day, 5 days/week) in an inhalation chamber. DEE originated from Euro 5 engine with or without DPF treatment, run on B7 or SHB20 fuel. Animals in the control groups were exposed to clean air. Our results showed that the majority of haematological and biochemical parameters examined in blood were at a similar level in the exposed and control animals. However, exposure to DEE from the SHB20 fuel caused an increase in the number of red blood cells (RBC) and haemoglobin concentration. Moreover, 7 days exposure to DEE from SHB20 fuel induced genotoxic effects manifested by increased levels of DNA single-strand breaks in peripheral blood lymphocytes. Furthermore, inhalation of both types of DEE induced oxidative stress and caused imbalance of anti-oxidant defence enzymes. In conclusion, exposure to DEE from B7, which was associated with higher exposure to polycyclic aromatic hydrocarbons, resulted in decreased number of T and NK lymphocytes, while DEE from SHB20 induced a higher level of DNA single-strand breaks, oxidative stress and increased red blood cells parameters. Additionally, DPF technology generated increased number of smaller PM and made the DEE more reactive and more harmful, manifested as deregulation of redox balance.

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.

Air pollution-related metals induce differential cytokine responses in bronchial epithelial cells.

Different transition metals have been shown to induce inflammatory responses in lung. We have compared eight different metal ions with regard to cytokine responses, cytotoxicity and signalling mechanisms in a human lung epithelial cell model (BEAS-2B). Among the metal ions tested, there were large differences with respect to pro-inflammatory potential. Exposure to Cd(2+), Zn(2+) and As(3+) induced CXCL8 and IL-6 release at concentrations below 100µM, and Mn(2+) and Ni(2+) at concentrations above 200µM. In contrast, VO4(3-), Cu(2+) and Fe(2+) did not induce any significant increase of these cytokines. An expression array of 20 inflammatory relevant genes also showed a marked up-regulation of CXCL10, IL-10, IL-13 and CSF2 by one or more of the metal ions. The most potent metals, Cd(2+), Zn(2+) and As(3+) induced highest levels of oxidative activity, and ROS appeared to be central in their CXCL8 and IL-6 responses. Activation of the MAPK p38 seemed to be a critical mediator. However, the NF-κB pathway appeared predominately to be involved only in Zn(2+)- and As(3+)-induced CXCL8 and IL-6 responses. Thus, the most potent metals Cd(2+), Zn(2+) and As(3+) seemed to induce a similar pattern for the cytokine responses, and with some exceptions, via similar signalling mechanisms.

Role of size and surface area for pro-inflammatory responses to silica nanoparticles in epithelial lung cells: importance of exposure conditions.

The present study compared non-crystalline silica particles of nano (50nm)- and submicro (500nm)-size (Si50 and Si500) for the potential to induce cytokine responses in bronchial epithelial lung cells (BEAS-2B). The cell cultures were exposed to equal mass and surface area concentrations of the two particles in different exposure media; LHC-9 and DMEM:F12. The state of agglomeration was different in the two media; with marked agglomeration in LHC-9 and nearly no agglomeration in DMEM:F12. On a mass basis, Si50 was more potent than Si500 in inducing cytokine responses in both exposure media. In contrast, upon exposure to similar surface area concentrations, Si500 was more potent than Si50 in DMEM:F12. This might be due to different agglomeration/sedimentation properties of Si50 versus Si500 in the two media. However, influence of differences in particle reactivity or particle uptake cannot be excluded. The data indicated no qualitative changes in the cytokine gene-expression patterns induced by the two particles, suggesting effects through similar mechanisms. These aspects might be of importance for interpretation of in vitro studies of nanomaterials.

Mechanisms linked to differences in the mutagenic potential of 1,3-dinitropyrene and 1,8-dinitropyrene.

This study explores and characterizes the toxicity of two closely related carcinogenic dinitro-pyrenes (DNPs), 1,3-DNP and 1,8-DNP, in human bronchial epithelial BEAS-2B cells and mouse hepatoma Hepa1c1c7 cells. Neither 1,3-DNP nor 1,8-DNP (3-30 µM) induced cell death in BEAS-2B cells. In Hepa1c1c7 cells only 1,3-DNP (10-30 µM) induced a mixture of apoptotic and necrotic cell death after 24 h. Both compounds increased the level of reactive oxygen species (ROS) in BEAS-2B as measured by CM-H2DCFDA-fluorescence. A corresponding increase in oxidative damage to DNA was revealed by the formamidopyrimidine-DNA glycosylase (fpg)-modified comet assay. Without fpg, DNP-induced DNA damage detected by the comet assay was only found in Hepa1c1c7 cells. Only 1,8-DNP formed DNA adduct measured by 32P-postlabelling. In Hepa1c1c cells, 1,8-DNP induced phosphorylation of H2AX (γH2AX) and p53 at a lower concentration than 1,3-DNP and there was no direct correlation between DNA damage/DNA damage response (DR) and induced cytotoxicity. On the other hand, 1,3-DNP-induced apoptosis was inhibited by pifithrin-α, an inhibitor of p53 transcriptional activity. Furthermore, 1,3-DNP triggered an unfolded protein response (UPR), as measured by an increased expression of CHOP, ATF4 and XBP1. Thus, other types of damage possibly linked to endoplasmic reticulum (ER)-stress and/or UPR could be involved in the induced apoptosis. Our results suggest that the stronger carcinogenic potency of 1,8-DNP compared to 1,3-DNP is linked to its higher genotoxic effects. This in combination with its lower potency to induce cell death may increase the probability of causing mutations.

Inflammation-related effects of diesel engine exhaust particles: studies on lung cells in vitro.

Diesel exhaust and its particles (DEP) have been under scrutiny for health effects in humans. In the development of these effects inflammation is regarded as a key process. Overall, in vitro studies report similar DEP-induced changes in markers of inflammation, including cytokines and chemokines, as studies in vivo. In vitro studies suggest that soluble extracts of DEP have the greatest impact on the expression and release of proinflammatory markers. Main DEP mediators of effects have still not been identified and are difficult to find, as fuel and engine technology developments lead to continuously altered characteristics of emissions. Involved mechanisms remain somewhat unclear. DEP extracts appear to comprise components that are able to activate various membrane and cytosolic receptors. Through interactions with receptors, ion channels, and phosphorylation enzymes, molecules in the particle extract will trigger various cell signaling pathways that may lead to the release of inflammatory markers directly or indirectly by causing cell death. In vitro studies represent a fast and convenient system which may have implications for technology development. Furthermore, knowledge regarding how particles elicit their effects may contribute to understanding of DEP-induced health effects in vivo, with possible implications for identifying susceptible groups of people and effect biomarkers.

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.

Partial characterization of a sex steroid-binding protein in plasma from arctic charr (Salvelinus alpinus L.).

A sex steroid-binding protein (SBP) that binds 17beta-estradiol with high affinity and moderate capacity was identified in the plasma from Arctic charr (Salvelinus alpinus L.) sampled during the early stage of gonadal maturation in June and prior to spawning in October. Maximum specific binding (B(max)) and equilibrium dissociation constant (K(d)) of males (B(max) = 2122 fmol E(2)/mg protein, K(d) = 1.9 nM), females (B(max) = 4115 fmol E(2)/mg protein, K(d) = 3.0 nM), and juveniles (B(max) = 4355 fmol E(2)/mg protein, K(d) = 1.8 nM) resembled binding characteristics of SBP from related species. The early-maturing females displayed both B(max) and K(d) values significantly higher than those of males (June samples). No significant differences in binding characteristics between fully matured males or females and immature juveniles were observed in the October samples. Interestingly, despite large individual variations there was a strong correlation between SBP levels and affinity. The association rate for 17beta-estradiol was rapid (t(1/2) approximately 1-2 min) compared with the dissociation rate (t(1/2) approximately 3 h). Several native hormones (estrogens, androgens, and progesterone) were able to compete with tritiated 17beta-estradiol for the binding site. Gel filtration chromatography demonstrated a peak of estradiol binding at approximately 60 kDa, when eluted on a Sephadex S-200 HR column.

Binding of xenoestrogens to the sex steroid-binding protein in plasma from Arctic charr (Salvelinus alpinus L.).

A specific sex steroid-binding protein (SBP) is believed to be involved in regulation of circulating sex steroids, steroid delivery to target cells and intracellular signalling in sex steroid-sensitive tissues. In the present work, interactions between xenoestrogens and the plasma SBP in Arctic charr (Salvelinus alpinus L.) were determined using ligand-protein binding studies. The test compounds were all able to displace tritiated 17 beta-estradiol (E2) from the Arctic charr SBP (acSBP) in a competitive and dose-dependent manner. The acSBP affinities for the xenoestrogens ranged over several orders of magnitude (17 beta-estradiol>>ethynylestradiol (EE2)>zearalenone (ZEA)>diethylstilbestrol (DES)>genistein (GEN)>bisphenol A (BPA), 4-t-octylphenol (OP)>>o,p'-DDT, and dieldrin (DIN)), but were consistently lower than that of 17 beta-estradiol (about 4 x 10(2) -10(6)-fold less potent). The relative binding affinity (RBA) for selected chemicals were independent of both gender, age and maturation status, as well as variations of acSBP binding affinity. The affinity of endogenous steroids and estrogen mimics for the acSBP shows a high correlation to the affinity for the rainbow trout SBP, thus suggesting a phylogenetically conserved ligand-binding site between closely related species. Furthermore, it is argued that interaction with the acSBP- and SBP-mediated processes may introduce novel pathways for endocrine disruption, which may work in concert with the classical receptor-mediated effects.