Induction of altered mRNA expression profiles caused by fibrous and granular dust
- Authors:
- Published online on: October 29, 2013 https://doi.org/10.3892/mmr.2013.1765
- Pages: 217-228
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Copyright: © Helmig et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].
Abstract
Introduction
Human populations are exposed to environmental and occupational fibrous and granular dust. The number of synthetic or natural fibres and particles being introduced into the environment is continuously increasing. Due to the increasing number and compositional heterogeneity of potentially harmful fibres and particles, there is a crucial need to understand the mechanisms of their pathogenicity.
Lately, nano-sized particles with a diameter below 100 nm have become the focus of attention, as they are predicted to have a higher toxic potential as a result of their high surface/mass ratios. However, the crystalline structure, surface properties, solubility and particle size are also known to be relevant parameters (1). Therefore an accurate characterization of the particles is essential to allow an interpretation of the results of a study.
It is reasonable to categorise particles and fibres by their molecular effects. To identify the molecular characteristics of particles and fibres we used Union Internationale Contre le Cancer (UICC) crocidolite and chrysotil asbestos as fibered dust and titanium dioxide (TiO2) as well as zirconium dioxide (ZrO2) as representatives for bio-persistent granular dust. Hematite (Fe2O3) represents a nano-sized ultrafine dust with an iron (Fe) content of ~70%. Asbestos is known to be a carcinogen associated with the induction of lung cancer, mesothelioma and lung fibrosis (2). DNA damage and apoptosis are important downstream effects of asbestos, which occur in all the major lung target cells studied (3). Exposure to asbestos fibres causes alterations in cell signalling (4) and induction of various pro-inflammatory molecules, such as cytokines (5,6). The pathogenicity of various types of asbestos fibres is thought to be associated with fibre size, geometry and surface composition (7). The iron content in particular has to be considered when assessing the toxicity of asbestos fibres. Crocidolite (Na2[Fe3+]2[Fe2+]3Si8O22[OH]2) typically has a high iron content of ~26%, while the iron content in chrysotile (Mg6Si4O10[OH]8) ranges between 1 and 6%, and is primarily present as a surface contaminant (8).
In order to verify that the evoked effects are not only due to the iron content of the investigated particles, we used hematite with an iron content of ~70%. Hematite, the hexagonal modification of iron (III) oxide (α-Fe2O3) is the most important industrial iron oxide used.
Titanium dioxide, also known as titanium (IV) oxide, is the naturally occurring oxide of titanium, which is commercially used in a wide range of products, such as paint, varnishes, paper coating and cosmetics (9,10). Micro-sized titanium dioxide is suggested to be biologically inert (11,12), although an inflammatory response has been described (10). Particles can generate reactive oxygen species; particularly in the case of nano-sized particles, DNA adducts are observed in human lung cells (9,13). Additionally, increased micronucleus formation and DNA breakage, as well as activation of DNA damage checkpoint kinases in nano-TiO2-treated lymphocytes, have been demonstrated (14).
Zirconium dioxide, also known as zirconia, is used in various products, such as ceramic materials, scratch resistant varnishes and coatings, as well as in medical implants (15,16).
The aim of this study was to compare the effects of well-defined fibres (UICC, crocidolite and chrysotile ‘A’) and different size particles (titanium dioxide, zirconium dioxide and hematite) on human bronchial epithelial cells (BEAS-2B). We focused on the mRNA expression of 84 signalling molecules attributed to pathways such as ‘DNA damage and repair’, ‘oxidative/metabolic stress’, ‘growth arrest and senescence’, ‘inflammation’, ‘proliferation and carcinogenesis’, ‘heat shock’ and ‘apoptosis’.
Materials and methods
Materials
Crocidolite asbestos (UICC, South African NB #4173-111-3) and chrysotil asbestos (UICC, Rhodesian NB #4173-11-2) were used as standard references for bio-persistent fibrous dust. Titanium dioxide anatase (Sigma-Aldrich Chemie GmbH, Steinheim, Germany; AL232033) and zirconium dioxide (Sigma-Aldrich Chemie GmbH; AL230693) represented bio-persistent granular dust. Hematite, α-Fe2O3 (Nanopowder 544884, Sigma-Aldrich Chemie GmbH) was used to represent ultrafine particles.
Characterization of dust materials
For a detailed description of the characterization method, refer to a former paper (17). Scanning electron microscopy (SEM; Hitachi S-2700, Chiyoda, Japan) was used to identify particle geometry as well as the microstructure of the samples. The element analysis resulted from energy dispersive X-ray (EDX). To optimize the conductivity (electron beam), all samples were deposited with a very fine gold (Au) layer using a sputtering technique. Transmission electron microscopy (TEM) analysis combined with electron diffraction (detection of crystallinity) was performed using a transmission electron microscope H-600 (Hitachi, Japan). Thermogravimetry (TG) measurements (corundum crucibles, heating rate 5 K/min and synthetic air atmosphere) for controlling impurities such as water were conducted using a thermo balance TG 209 F1 Iris (NETZSCH-Gerätebau GmbH, Selb, Germany).
Culture conditions
SV-40 virus-transformed BEAS-2B cells were obtained from the European collection of cell cultures (ECCC, 95102433). Approximately 10 million cells (10×106) after trypsinization and counting using a haemocytometer were plated in 75 cm2 flasks (Falcon; Franklin Lakes, NJ, USA). The cells were grown in 15 ml Gibco® RPMI 1640 media containing 10–15% fetal calf serum (FCS), 0.5% gentamycin, 1% L-glutamine and 1% amphotericin. The cultures were maintained at 37°C and 5% CO2. After a 24-h pre-incubation, the cells were exposed to crocidolite (5 μg/cm2), chrysotil (1 μg/cm2), zirconium dioxide (10 μg/cm2), titanium dioxide (10 μg/cm2) or hematite (10 μg/cm2) for 48 h. Unexposed cells served as negative controls. All experiments were repeated 4 times. Cytotoxicity and genotoxicity analyses were investigated intensively in various cell systems for crocidolite and chrysotile (18–21), titanium dioxide (22,23) and hematite (22,24). Based on the results of the above study, the particle concentrations did not show any loss of viability in the BEAS-2B cell line. Additionally, the same concentrations and incubation times were used in numerous published studies and therefore the results are comparable.
mRNA extraction and reverse transcription
After washing twice with PBS (37°C), cells were trypsinized for ~30 sec with 10 ml of 0.05% trypsin and incubated for 10 min in 37°C. Detached cells then were resuspended in 5 ml ice-cold PBS and centrifuged at 400 × g (without brakes) for 10 min in 15-ml centrifuge tubes. This step was repeated with 1 ml of ice-cold PBS in 1.5 ml Eppendorf tubes. mRNA was extracted immediately with RNeasy Mini kit® (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. Reverse transcription was accomplished with the RT2 First Strand kit (Qiagen) as suggested by the manufacturer.
RT2 Profiler PCR Arrays®
The RT2 RNA QC PCR Array® (SaBiosciences, Qiagen) was used to test for RNA quality and inhibitors of RT-PCR analyses. For quantitative comparison of mRNA levels, real-time PCR was performed using RT2 Profiler PCR Arrays® Human Stress & Toxicity PathwayFinder PCR Array® (SaBiosciences). For each condition, four assays were carried out as independent samples. Gene expression was related to the mean expression of β2 microglobulin (B2M) and hypoxanthine phosphoribosyltransferase 1 (HPRT) as housekeeping genes, since these were the two most stable of the five housekeeping genes included in the array. Only Ct values <35 were included in the calculations.
Statistical analysis
Calculations of expression were performed with the 2−ΔΔCT method according to Pfaffl (25). For analysis the PCR Array Data Analysis Software (Excel & Web-based) provided by SaBiosciences was used. The cut-off was set to CT>35. The P-values are calculated based on a Student’s t-test of the replicate 2−ΔCt values for each gene in the control and treatment groups. Results are shown as the mean of four samples for each condition in relation to the mean of four control samples. All statistical analyses were performed using the statistical software package, SPSS, 17.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.
Results
Characterization of dust samples
UICC crocidolite South African (Na2(Fe32+Fe23+[(OH)2|Si8O22]) was shown to have 3,800 fibres/ml at a length of >5 μm and a diameter of <3 μm. The length to diameter ratio was at least 3:1 (WHO fibres). Crocidolite is a rigid and rod-like fibre with characteristic iron content (Fig. 1). Gold (Au) was detected in all EDX analyses due to the sputtering preparation technique.
UICC chrysotile ‘A’ Rhodesian (Mg6[(OH)8|Si4O10]) was shown to have 200 fibres/ml at a length of >5 μm and a diameter of <3 μm. The length to diameter ratio was at least 3:1 (WHO fibres). Chrysotile has a curly, pliable structure with nearly equal magnesium (Mg)/silicon (Si) distribution (Fig. 2).
Irregularly shaped crystalline titanium dioxide aggregates (diameter, 1–3 μm) were observed (Fig 3). The micro-sized aggregates were composed of ~20 primary particles with a diameter between 100 and 200 nm. The specific surface (BET) of titanium dioxide was 9.9 m2/g. Evaluation of the BET (for titanium dioxide and zirconium dioxide) was performed by K.-P- Company for surface- and solid state analysis mbH (o.f.u), Hamburg, Germany (Report B0104014 for the Federal Institute of Occupational Safety and Medicine, May 2001).
For zirconium dioxide, an aggregate diameter of 1–2 μm was determined. The crystalline aggregates were composed of ~50 primary particles with a diameter of ~100 nm (Fig. 4). The specific surface (BET) of zirconium dioxide was 5.9 m2/g.
Hematite was found to be a spherical formed, nano-sized material. Agglomerates of 0.2–2 μm were formed by 50–500 primary particles with a diameter of ~20 nm. Additionally, smaller aggregates (<100 nm) were detected by electron microscopy (Fig. 5). The usually observed integration of water within the crystal lattice, caused by the production (precipitation) process of hematite, was excluded by TG (26–28).
After a 48-h exposure to the described fibres and particles, relative mRNA expression of 84 genes were determined four times. The average and standard deviation of the Ct values of each gene are shown in Table I.
 | Table IRelative mRNA expression of 84 genes after incubation of fibrous and granular dust in bronchial epithelial cells (BEAS-2B). |
Direct genotoxicity
Molecules of the ‘DNA damage and repair’ pathway were assigned to direct genotoxicity. In BEAS-2B cells, UGT1A4 mRNA expression could not be detected. mRNA expression of the ‘DNA damage and repair’ pathway was induced mainly by crocidolite, followed by chrysotile. Zirconium dioxide significantly upregulated UNG (1.31; P=0.043) but downregulated RAD50 (−1.45; P=0.038), while neither titanium dioxide nor hematite altered mRNA expression of ‘DNA damage and repair’ pathway signalling molecules (Table II).
Indirect genotoxicity
Molecules of the ‘oxidative or metabolic stress’, ‘growth arrest and senescence’ as well as ‘inflammation’ pathway were assigned to indirect genotoxicity. In BEAS-2B cells, Cyp7A1, FMO1, CCL21, CCL4, CXCL10 and LTA mRNA expression could not be detected. The majority of changes in the signalling molecule mRNA expression of the ‘oxidative or metabolic stress’ pathway were due to crocidolite. Notably, changes in signalling molecule expression were comparable for chrysotile, zirconium dioxide, titanium dioxide and hematite (Table III).
Both fibres showed a moderate increase in signalling molecule expression of the ‘growth arrest and senescence’ pathway, while titanium dioxide only induced DDIT3 (1.4, P=0.048). There was no significant change in mRNA expression due to zirconium dioxide and hematite (Table IV).
Molecules belonging to the ‘inflammation’ pathway were induced mainly by crocidolite. Chrysotile and hematite provoked a comparable moderate increase in gene expression. Titanium dioxide distinctly induced CCL3 (2.79, P=0.007), while there were no expression changes due to zirconium dioxide (Table V).
Initiation and promotion of carcinogenesis
Molecules of the ‘proliferation and carcinogenesis’ pathway were assigned to initiation and promotion of carcinogenesis. The only gene of this pathway, which was induced was Cyclin D1 (CCND1). Cyclin D expression was induced by crocidolite (1.57, P=0.004), chrysotile (1.89, P=0.019) and titanium dioxide (2.36, P=0.007) (Table VI).
Acute toxicity and/or genotoxicity
Molecules of the ‘heat shock’ and ‘apoptosis’ pathways were assigned to acute toxicity and/or genotoxicity. Of all investigated pathways, the greatest changes were found within these two pathways. Crocidolite, titanium dioxide and hematite provoked the most changes in mRNA expression of signalling molecules of the ‘heat shock’ pathway, while crocidolite and zirconium dioxide provoked the most changes in mRNA expression of signalling molecules of the ‘apoptosis pathway’. Chrysotile showed a moderate increase of ‘heat shock’ genes and only a moderate increase of ‘apoptosis’ genes (Tables VII and VIII).
A comparison of the pathways induced by crocidolite, chrysotile, titanium dioxide, zirconium dioxide and hematite is provided in Table IX.
Discussion
In this study, we compared the ability of two different fibres (crocidolite and chrysotile) and three different sized particles (titanium dioxide, zirconium dioxide and hematite) to induce the mRNA expression of signalling molecules involved in diverse pathways. We characterized the toxicologically relevant chemical and physical properties of the fibres and particles to ensure the comparability of the present results. UICC crocidolite South African and UICC chrysotile ‘A’ are asbestos fibres, and their cytotoxic and genotoxic potential is well studied. The selected bio-persistent dust particles, titanium dioxide (100–200 nm) and zirconium dioxide (50–100 nm), were of the same origin as formerly used in vivo(29). After intratracheal installation, both particles induced lung tumours in female SPF Wistar rats (29). Hematite (20 nm), the smallest of all particles, was investigated, to observe whether the obtained reaction may be provoked by the iron content.
Asbestos fibres caused the most relevant changes in gene expression of all tested pathways. This finding is in accordance with the general knowledge that crocidolite as well as chrysotile are asbestos fibres with a high cytotoxic and genotoxic effect (2,20,21,30,31). A literature search, including in vitro analysis, animal experiments and epidemiological studies, confirmed that all fibre types show comparable harmful effects (32). Chrysotile is, due to its higher solubility, less bio-persistent than the crocidolite (33). Since our study determines the early effects (48 h) of fibres and particles, the 5-year clearance rate is of minor relevance to our results. In accordance with our study, it appears that chrysotile and crocidolite develop their genotoxicity due to direct and indirect (inflammatory driven) molecular mechanisms (18,19,34–36).
The iron content appears to not to be of major relevance for the observed induction of direct genotoxicity or the initiation and promotion of carcinogenesis, since these pathways are not induced by hematite (Fe 70%) but by zirconium dioxide (Fe 0%). In a study by Schürkes et al, the iron content appeared not to be relevant for the induction of 8-hydroxydeoxyguanosine (8-OHdG), since fibres with different iron amounts (0.025–20%) revealed comparable results (35). In the present study, nano-sized hematite and titanium dioxide showed an inflammatory and oxidative stress response and a high increase in gene expression attributed to the ‘heat shock’ pathway. These findings are in accordance with the results of Park et al, where single intratracheal instillation of iron nanoparticles (NP) in mice elevated the expression of many genes related to inflammation or tissue damage, such as heat shock proteins (37). Additionally, significant generation of ROS was described for titanium dioxide-NP and hematite (9,24,38). None of the investigated genes of the ‘DNA damage and repair’ pathway were induced by hematite or titanium dioxide in our study. Nanoparticles of hematite but not those of titanium dioxide induced significant DNA-breakage, measured by the Comet-assay in IMR-90 cells. DNA-damage and cytotoxic effects by hematite in BEAS-2B cells were not observed until a concentration of 50 μg/cm2 was used (9). On the contrary to ultrafine titanium dioxide, there were no significant alterations in micronuclei induction by fine titanium dioxide observed in Syrian hamster embryo cells (23). Incorporation into human lung cells was described for fine and ultrafine titanium dioxide as well as for hematite (24,39).
Notably, Cyclin D1 which, as a regulatory subunit of CDK4 or CDK6, promotes cell cycle progression through G1-phase is significantly upregulated by titanium dioxide (relative expression 2.36) correspondingly with chrysotile (relative expression 1.89) and crocidolite (relative expression 1.57). The deregulation of cyclin D1 plays an important role in tumorigenesis and has frequently been linked to various types of human cancer (40).
Zirconium dioxide with particle sizes between 50 and 100 nm induced molecules attributed to the ‘oxidative or metabolic stress’ pathway, which suggests an indirect genotoxicity. We also found a high increase of apoptotic molecules. Zirconium dioxide induced UNG, which eliminates uracil from DNA molecules by cleaving the N-glycosylic bond and initiates the base-excision repair (BER) pathway. Uracil appearing in DNA, for example as a result of cytosine deamination, is potentially mutagenic and deleterious for cell regulation (41).
In particular, properties such as size, geometry, chemical composition and surface behaviour of particles play important roles in interaction with cells and modify their pathogenicity. Many published studies are missing detailed information on properties and the concentration of the particles used, which makes it difficult to compare results.
Our study and recent reports in the literature demonstrate that gene expression profiling in human lung epithelial cells can be an important tool for analyzing the pathogenicity of potentially harmful fibres and particles (42–44). Gene expression profiling, for example in response to asbestos, is valuable to define early molecular effects as demonstrated in diverse human cells, such as normal human bronchial epithelial cells (NHEC) (45), human lung adenocarcinoma cells (A549) (46,47), SV40-transformed human bronchial epithelial cells (BEAS-2B) and SV40-immortalized pleural mesothelial cells (MET5A) (47). Changes in gene expression are also valuable to determine the pathogenicity pathway of asbestos fibres, as demonstrated in the human mesothelial (LP9/TERT-1) cell line (42).
In further studies, new particles can be screened to complete the toxicological knowledge on the molecular effects and to assess potentially hazardous risks. Altogether, analysis of gene expression profiles may play an important role in the early detection of fibres or potential hazards of particles to human health.
Acknowledgements
This study was supported by the E.W. Baader-Stiftung supervised by the German Stiftungszentrum, Barkhovenallee 1, 45239 Essen, Germany, Az. T007/20368/2010/sm.
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