Gene Expression Profile and Toxic Effects in Human Bronchial Epithelial Cells Exposed to Zearalenone

Zearalenone (ZEA), a mycoestrogen produced by Fusarium fungal species, is mainly found in cereal crops such as maize, wheat and barley. Although ZEA has been reported to be present in air, little is known about the health risk or the molecular basis of action when lung cells are exposed to ZEA. As ZEA has a similar structure to estrogen, its potential risk as an endocrine disrupting chemical (EDC) has thus aroused both environmental and public health concerns. The purpose of this study is to identify the responses and underlying molecular changes that occur when human bronchial epithelial BEAS-2B cells are exposed to ZEA. Differential gene expression profiles were identified in cells that were treated with 40 µM ZEA for 6 h and 24 h by high-throughput microarray analysis using Affymetrix Human Gene 2.0 GeneChip. The array results showed that after ZEA treatment, 262 genes at 6 h and 1073 genes at 24 h were invovled in the differential regulation. Pathway analysis revealed that diverse cellular processes were affected when lung cells were exposed to ZEA resulting in impaired response to DNA damage, cell cycle arrest, down-regulation of inflammatory responses and alterations of epigenetic marks. Results of further experiments indicated that 40 µM ZEA decreased cell viability, induced apoptosis and promoted reactive oxygen species (ROS) generation in a time-dependent manner. Immuno-suppressive effects of ZEA were further revealed through the suppression of lipopolysaccharide (LPS)-induced expression of pro-inflammatory cytokines (IL-6, IL-8 and IL-1β). Interestingly, the level of global DNA methylation was markedly decreased after 24 h exposure to ZEA. Collectively, these observations suggested that a broad range of toxic effects are elicited by ZEA. Particularly, ROS may play a pivotal role in ZEA-induced cell death. These adverse effects observed in lung cells suggest that exposure to ZEA may increase susceptibility of lung cells to diseases and required further investigations.


Introduction
Mycotoxin Zearalenone (ZEA) is a secondary metabolite produced by various Fusarium fungal species [1,2] which are usually found in contaminated maize, wheat and barley [3]. Due to its structural similarity to estrogen, ZEA competes with estradiol for binding to estrogen receptors (ERs) and provokes estrogenic activities. Extensive studies have found that ZEA caused endocrine disruption and reproductive disorders in in vitro models and in laboratory and farm animals [4,5,6,7]. In addition, other effects of ZEA including developmental toxicity, immunotoxicity and genotoxicity have also been reported [3]. Increasing evidence suggested that these effects are not exclusively due to the estrogenic potency of ZEA but that oxidative stress may be an important mediator of these observed toxic effects [8,9,10].
Besides foods and feeds, inhalation is another route of exposure to ZEA. The detection of ZEA-producing fungi and toxigenic spores in nasal cavity has been reported [11,12]. In addition, the detection of air-borne ZEA was also documented. In a Belgium study, the maximum level of ZEA detected was 2.4 mg/kg ZEA which meant that exposure through dust inhalation for workers in those companies was estimated to be 0.1% of the tolerable daily intake of ZEA [13]. In a study carried out in Dalian, China, it was reported that the daily inhaled ZEA by a worker in a poultry house was estimated to be 17.432-20.512 ng respectively [14].
Estrogens have been shown to induce proliferation of non-small cell lung cancer (NSCLC) through ER-mediated signaling pathways [15]. Additionally, estrogen is also involved in the activation of carcinogens via the metabolism of polycyclic aromatic hydrocarbonds (PAHs) which promotes the formation of catechol estrogens and potentially mutagenic DNA adducts [16,17]. Interestingly, large cohort epidemiological studies indicate that females are more susceptible to developing chronic lung diseases including asthma and Chronic Obstructive Pulmonary Disease (COPD) [18].
To date, the molecular basis of the effects of ZEA in lung cells has not been fully investigated. Using a toxigenomic approach, we attempted to study the mechanism of actions of ZEA on lung cells. In addition, we show that ZEA induces a broad range of toxic effect not solely because of its estrogenic potency but also through induction of oxidative stress. A BEAS-2B cell line over-expressing a free radical scavenger, cytoglobin (CYGB) confirm that ZEA generates free radicals.

Cell culture and treatments
Human bronchial epithelial BEAS-2B cell line [19] (from the American Type Culture Collection, ATCC) was cultured in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 100 U/ml of penicillin and 10 mg/ml of streptomycin. All cells were maintained in a 37uC humidified incubator with 5% CO 2 . DMEM with Geneticin (G418, 200 mg/ml) were used to maintain and select Cygb overexpressing cells.
ZEA powder (Sigma) was dissolved in DMSO, aliquoted and stored at 220uC. The stock solution of ZEA was freshly diluted by culture medium before use. BEAS-2B cells were seeded overnight to achieve confluency. Cells were exposed to different concentration of ZEA or 0.05% DMSO solvent control for different durations (6,12,24 and 48 h) depend on experiments. For immune-responsive experiments, cells were stimulated by 2 mg/ml lipopolysaccharide (LPS, Sigma) for 6 h.
Cell viability assay 1610 4 -1610 5 cells were seeded in 96-well plates. After incubation overnight, cells were exposed to a serial concentration of ZEA (from 0 to 160 uM) for 6 h, 24 h or 48 h. 20 ml of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Invitrogen) solution was then added to each well. After 3 hours incubation, the medium was removed and DMSO was added to dissolve the purple formazan. The optical density of each well was quantified by measuring absorbance at 540 nm. The viabilities of treated groups were expressed as a percentage of control group, which was assumed as 100%.
Sample preparation and RNA extraction 5610 5 cells were seeded onto 6-well plates. After incubation overnight, cells were treated with either 0.05% DMSO (solvent control) or 40 mM ZEA (treatment) for 6 h and 24 h. RNA was extracted by Trizol reagent (Invitrogen) according to the manufacturer's protocol. The quality of RNA for microarray analysis was analyzed by the RNA 6000 Nano total RNA Assay using the Agilent 2100 Bioanalyzer. Only RNA samples with an A 260 /A 280 ratio ,1.8, 28S/18S ratio larger than 1.8 and RNA integrity number (RIN) larger than 8.0 were used for downstream GeneChip analysis.

Gene expression profiling and data analysis
The gene expression profiles were determined using GeneChipH Human Gene 2.0 ST Arrays (Affymetrix). Samples were hybridized onto array chips, stained, washed, and scanned according to Affymetrix protocol. The array image and cell intensity files (.CEL files) were generated by Affymetric GeneChip  Command Console. Both the RNA quality control tests and GeneChip analysis were conducted by Center for Genomic Services, HKU.
The data of GeneChip were analyzed using Agilent GeneSpring GX11 Software, Affrymetrix Transcriptome Analysis Console (TAC) Software and R software (http://www.r-project.org). When using the GeneSpring Software, filtering was performed to remove background noise. Probes that have signals weaker than 20 th percentile of overall signal were not included into analysis. Differentially expressed genes were identified as fold change $ 1.5 and p,0.05 by unpaired t-test. Over-represented Gene Ontology (GO) terms and enriched pathways associated with the list of differentially expressed genes were generated by the built-in GO and Single Experiment Analysis (SEA) of GeneSpring. Only GO terms and pathways that have more than 2 entities ( §3) involved and p-value ,0.05 were considered.
To further analyze the gene expression data, the Gene Set Enrichment Analysis (GSEA) were used to identify cohorts of genes which are linked to certain biological processes/cellular signaling pathways. Differentially expressed genes with annotations and known features were subjected to GSEA. Pathways are ranked according to the significance of enrichment [61]. Gene sets with a p-value ,0.05 by one-way ANOVA and False Discovery Rate (FDR) ,0.05 by multiple test correction of were considered to be significantly affected.
Validation of gene expression by Quantitative Real-time PCR (qPCR) cDNA was synthesized from 1 mg RNA by using PrimeScript TM RT Master Mix (Takara) according to manufacturer's instructions. Primers were designed to avoid amplification of genomic DNA using Primer Premier 5 software. The sequences of the primers used are shown in Table S1. qPCR was performed using FastStart Universal SYBR Green Master mix (Roche) and reaction mixes were set up as per manufacturer's instructions. The cycles were set as 95uC, 10 min, 40 cycles of 95uCfor 15 sec, 60uCfor 20 sec and 72uCfor 45 sec followed by melting curve analysis. The change in gene expression was calculated by comparative C T method with the housekeeping gene b-actin used for normalization.

Measurement of Reactive Oxygen Species (ROS) levels by DCFH-DA assay
The intracellular ROS levels were quantified by a fluorescent probe, 29,79-dichlorofluorescein-diacetate (DCFH-DA, Molecular Probes). After treatment with 40 mM ZEA, cells were washed and resuspended in PBS at a concentration of 10 6 cells/ml and then incubated with 10 mM DCFH-DA at 37uC for 40 minutes in the dark. The ROS production was quantified by DCF fluorescence intensity from 10 4 cells by flow cytometry. Results were expressed as the percentage of ROS generation as compared to control.

Establishment of Cygb stably overexpressing cells
The stable BEAS-2B over-expressing Cytoglobin (Cygb) by comprising the coding region of mouse Cygb which was cloned to pcDNA 3.1/V5-His A vector (Invitrogen) between Hind III and Xho I sites. To establish stably Cygb expressing cells, a 1576 bp core ubiquitously-acting chromatin opening element (UCOE, a gift from Dr. Michael Antoniou, School of Medicine, King's College London, UK) was inserted to the upstream of the CMV promoter of pcDNA 3.1/V5-His A/Cygb. Successfully transfected cells were selected using selective medium (DMEM with 10% FBS and 600 mg/ml G418). After 14 days of selection, single colonies were picked and expression levels were checked to identify the clones with Cygb over-expression. The incorporation of the UCOE onto the Cygb-transgene construct resulted in the sustained high expression of cytogobin in the stable BEAS-2B cell line. This stable over-expression cell line was employed as a model system to investigate the effects of free radicals generated by ZEA on cell viability.

Cell death pathway analysis by Annexin V/PI double staining
The apoptotic statuses of cells were determined using Annexin V-FITC Apoptosis Detection Kit I (BD Pharmingen) following manufacturer's instructions. Briefly, cells treated with 40 mM ZEA were washed with phosphate buffered saline (PBS) and then resuspended in binding buffer at a concentration of 10 6 cells/ml. Annexin V-FITC and Propidium Iodide (PI) were added to the resuspended cells. Cells were incubated for 15 min at room temperature in the dark. Apoptotic cells were analysed from 10 4 cells with a Beckman-Coulter FACScan flow cytometer. The percentage of living, early apoptotic and dead cells were quantified using the Modfit LT program.

Global DNA methylation analysis
To obtain an insight into the epigenetic effects of ZEA, global DNA methylation levels were investigated. 5-aza-cytidine, a known DNA methylation inhibitor acting as a substitute substrate for DNA methyltransferase, was used as positive control. BEAS-2B cells were treated with DMSO, 40 mM ZEA or 1 mM 5-azacytidine for 24 h. After treatment, genomic DNA was extracted using UltraClean Tissue & Cells DNA Isolation Kit (MO Bio Laboratories, Inc.) according to the manufacturer's protocols. The concentrations and qualities of DNA were quantified by Nano-Drop ND-1000 Spectrophotometer (Nano-Drop Technologies) and checked by 0.7% agarose gel electrophoresis, respectively. The global DNA methylation levels were determined using MethylFlash Methylated DNA Quantification Kit (Colorimetric) (Epigentek Group Inc.) following manufacturer's instructions. DNA is bound to specifically treated strip wells that have high DNA affinity. The 5-methylcytosine of DNA is detected using antibodies and quantified using an ELISA-like reaction by reading absorbance at 450 nm.

Statistical analysis
Unless otherwise specified, all data are results of 3 independent experiments, each with 3 samples per group and represent as mean6 SD. Student t-test and one-way analysis of variance (ANOVA) followed by Duncan's post hoc test were conducted using SPSS v11.5 software. Values of p,0.05 were considered as significant.

ZEA reduced viability of BEAS-2B cells
The cytotoxic effects of ZEA on cell viability were determined using MTT assay. The results revealed that ZEA induced cell death in a time-and dose-dependent manner (p,0.05). After 48 h treatments, the Lowest Observable Adverse Effect Level (LOAEL) was 40 mM, with a percentage of viable cells of 62.9667.30% ( Figure 1). As 40 mM has no significant inhibitory effect on cell viability after 6 h and 24 h treatments but was the LOAEL after 48 h treatment, it was chosen as the concentration for use in subsequent experiments.

Identification of differentially expressed genes
The number of differentially expressed genes are observed to increase in a time-dependent manner. According to the Transcriptome Analysis Console (TAC) software, out of 53,617 gene probes on the Genechip, 262 (131 genes up-regulated and 131 genes down-regulated) and 1073 (357 genes up-regulated and 716 genes down-regulated) genes were differentially expressed at 6 h and 24 h, respectively. The complete list of the differentially expressed genes and their fold change at 6 h and 24 h are shown in Table S2 and S3, respectively. The number of commonly upregulated and down-regulated genes at both time points is 67 and 68, respectively ( Figure 2).
For both time-point, the most significantly up-regulated genes are heat shock proteins of 70 kDA in size (HSPA1B, HSPA1A) which increased by 3.7-fold and 6.54-fold at 6 h and 24 h, respectively (Table 1 and Table 2). On the other hand, the most significantly down-regulated gene common for both time points is serpin peptidase inhibitor, clade B (ovalbumin), member 2 and 10 (SERPINB2, B10) which decreased by 2.87-fold and 5.52-fold at 6 h and 24 h, respectively.

Functional classification of differentially expressed genes
The identification of enriched ''Biological Processes'' under GO category was performed using GeneSpring. The enriched GO terms at 6 h and 24 h are shown in Table S4 and Table S5 respectively. The most significantly enriched GO term at 6 h is ''protein folding'' (p = 5.59E-07) while that at 24 h is ''DNA dependent DNA replication'' (p = 1.35E-14).

Pathway analysis of differentially expressed genes
The pathways altered after ZEA treatments were identified by single experiment analysis (SEA) using GeneSpring. The overrepresented pathways at 6 h and 24 h are shown in Table 3. At 6 h, only the ''transforming growth factor-beta (TGF-b) signaling'' pathway is altered. At 24 h, the top five most significantly altered pathways are ''DNA Replication'', ''G1 to S cell cycle control'', ''cell cycle'', ''synthesis of DNA'' and ''cholesterol biosynthesis''. These results suggest that ZEA alters DNA replication and cell cycle progression in BEAS-2B cells. The differentially expressed genes associated with the progression of cell cycle from G1 to S phase, the replication, damage and repair of DNA and the apoptotic pathway are shown in Table 4, Table 5 and Table 6 respectively.
In addition, the analysis also revealed significantly enriched Keap1-Nrf2 pathway (p-value = 0.032, Table 3) which suggested that the oxidative status of cells is altered. The apoptotic pathway is another significantly altered biological process that was observed (p-value = 0.027, Table 3). The list of differentially expressed genes related to apoptosis is summarized in Table 6.
To further identify the dysregulated biological processes, differential regulated genes were subjected to Gene Set Enrichment Analysis (GSEA). GSEA enabled us to determine whether a priori defined set of genes is statistically significantly (with nominal p-value ,0.05 and FDR,0.25) enriched after treatment with ZEA. The detailed results are shown in Table S6. Interestingly, in addition to the pathways as identified by SEA, gene sets related to the extracellular matrix molecule tenascin C [20], histone deacetylation [21] and estrogenic responses [22] were also recognized to be enriched (Figure 3).

ZEA induced oxidative stress in BEAS-2B cells
The generation of ROS after treatment with ZEA was detected by flow cytometry. The fluorescence intensity in wild type cells that were exposed to 40 mM ZEA for 6, 12, 24 and 48 h increased in a time-dependent manner by 18.4, 29.3, 28.0 and 25.1%, respectively ( Figure 4A).
Concurred with the induction of ROS levels, the expressions of selected oxidative stress-related genes were up-regulated ( Figure 4B-E). The up-regulation of heat shock protein 27 (Hsp27), superoxide dismutase (SOD2) and catalase were more pronounced at 6 h and their expression were increased by 2.86, 1.81 and 1.92 folds, respectively. Moreover, the expression of heat shock protein 70 (Hsp70) was dramatically increased by 41.98 folds and the up-regulation was maintained after 12 and 24 h treatment. Over-expression of CYGB reduced ZEA-induced ROS generation and apoptosis in BEAS-2B cells Over-expression of CYGB, a free radical scavenger, was used to study the role of oxidative stress upon ZEA-induced cytotoxicity. Unlike the wild type BEAS-2B cells, the detection of significantly increased level of ROS was delayed to after 24 h and 48 h ZEA exposure. In addition, the level of induction was lowered to 14.0 and 23.9%, respectively ( Figure 4A). These results suggested that over-expression of CYGB can attenuate and postpone the increase of ROS levels induced by ZEA.
The detection of apoptotic cells was done by flow cytometry. Apoptotic cells were found in both wild type and CYGBoverexpressing cells after 24 and 48 h ZEA treatment ( Figure 5). Over-expression of the endogenous free radcial scavenger, CYGB, conferred protective effects against ZEA-induced cell death. After ZEA exposure for 48 h, 13.6862.96% of wild type whilst 74.5560.78% of CYGB-overexpressing cells were still alive. On the other hand, 72.5762.61% of wild type whilst 13.9561.15% of CYGB-overexpressing cells underwent apoptosis and were at late apoptotic states.

Validation of differentially expressed genes by qPCR
Disturbingly, many of the pro-inflammatory responsive genes were down-regulated while those anti-inflammatory genes were up-regulated ( Table 7). The anti-inflammatory effects of ZEA were further revealed in its ability to reduce lipopolysaccharide (LPS)induced release of pro-inflammatory cytokines. After 6 h treatment, LPS alone induced the expressions of IL-6, IL-8 and IL-1b by 1.49, 1.37 and 1.29 folds, respectively. However, the inductions of these cytokines were significantly suppressed by ZEA ( Figure 6).
After analyzing the microarray data, the expression changes of 10 selected genes including SERPINB2 and PLAU (apoptosis), CYP1B1 (aryl hydrocarbon receptor signaling), SMAD7 (TGF-b signaling), IL-8 and IL-37 (inflammatory response), JUN and EGR1 (regulation of transcription), CCNE2 (progression of cell cycle) and DDIT4 (response to DNA damage) were verified by quantitative real-time PCR (qPCR). The expression pattern (direction of regulation) showed a good agreement between the data of microarray and qPCR, although the fold-changes detected by qPCR appeared to be more pronounced (Figure 7).  As shown in Figure 8, the level of global DNA methylation was significantly lowered to 40.3611.1% (p,0.01) and 53.09633.75% (p,0.01) relative to control in cells treated with 1 mM 5-azacytidine or 40 mM ZEA respectively. These results suggest that global DNA demethylation occurs when BEAS-2B cells are exposed to ZEA.

Discussion
The aim of this study is to decipher the toxic effects and molecular mechanisms induced by ZEA in human bronchial epithelial cells (BEAS-2B). Our experiments and biological interpretation of the genome-wide transcriptome analysis indicated that induction of oxidative stress, arrest of cell cycle progression, initiation of apoptosis, suppression of inflammatory responses and changes of epigenetic marks are the consequences of exposure to ZEA.

Induction of oxidative stress by ZEA
The broad range of toxic events provoked by ZEA is believed not solely due to the interaction with estrogen receptors but also through the induction of oxidative stress. Our results indicated that the Keap1-Nrf2 pathway was suppressed (Table 3), as the levels of intracellular ROS was increased ( Figure 4A) and the expression of free radical scavengers, SOD2 and catalase, were upregulated ( Figure 4D and E) after ZEA treatment. Nrf2 is a transcription factor that binds to the antioxidant responsive element at the regulatory region of target genes that confer protection against oxidative stress [23]. Nrf2 is normally sequestered and inhibited at the cytoplasm by Keap1. However, under stress conditions, Nrf2 dissociates from Keap1 and translocates to the nucleus to control gene expression [23]. The expression of  SOD2 and catalase are up-regulated under oxidative stress by the binding of Nrf2 to their promoters [24]. In addition, our results show dramatic up-regulation of heat shock proteins, which are early markers of oxidative stress [25,26].
On the other hand, the generation of ROS ( Figure 4A) and the induction of apoptosis ( Figure 5) were significantly attenuated by the over expression of CYGB, a potent free radical scavenger. Collectively, our results suggest that ZEA is a strong inducer of ROS and oxidative stress is the underlying mediator of ZEAinduced cytotoxicity. These observations substantiate early reports showing that exogenous antioxidants including vitamin E and Tunisian radish protect against ZEA-induced oxidative damage and subsequent apoptosis [27,28].
The mechanism by which ZEA induces production of ROS is ambiguous. It is known that upon metabolism of ZEA, 3a-/3bhydroxysteroid dehydrogenases catalyze the formation of two major reductive metabolites, aand b-Zearalenol [29]. Pfeiffer et al [30] identified two highly unstable oxidative metabolites of ZEA, 13-hydroxy-ZEA and 15-hydroxy ZEA, which are demonstrated to possess the same potency for causing oxidative DNA damage (as measured by the level of 8-oxo-29-deoxyguanosine of DNA) as catechols of estradiols [31]. Therefore, we believe that ROS could be generated during the formation of these metabolites. In addition, ROS may be generated due to the alteration of mitochondrial NADPH-oxidase which functions to generate superoxide anions (O 2 2 ) from normal oxygen [32]. Our array results support this supposition as the expression of NADPHoxidase (NOX5) is up-regulated by 1.32 and 1.6 fold upon 6 and 24 h exposure to ZEA (Table S2 and S3).

DNA damages and inhibition of DNA repair by ZEA
Excessive generation of ROS can oxidize cellular macromolecules including DNA, protein and lipids. The ability of ZEA to cause oxidative DNA damage including DNA fragmentation, single-and double stranded breakage as well as formation of 8oxoguanine were reported [9,27,33]. Our microarray results also signified the suppression of DNA repair and induction of DNA damage (Table 3 and 5). The expression of breast cancer 1, early onset (BRCA1), RAD51, bloom syndrome, RecQ helicase-like (BLM), flap-endonuclease 1 (FEN1), uracil-DNA glycosylase (UNG), damage-specific DNA binding protein 2 (DDB2) and exonuclease 1 (EXO1) were significantly suppressed after 24 h ZEA treatment (Table 5).
BRCA1 functions in response to the signal of DNA damage and transcriptionally control downstream effectors [34,35]. RAD51 and BLM are involved in the repair of DNA double-strand break through homologous recombination [36,37]. BRCA1 and RAD51 also interact to control recombination and maintain the integrity of the genome [38]. FEN1 [39], UNG [40], DDB2 [41] and EXO1 [42] are involved in DNA base-excision repair, DNA mismatch repair and homologous recombination. On the other hand, DNA-damage-inducible transcript 4 (DDIT4) and never in mitosis gene a-related kinase 2 (NEK2) which are involved in controlling DNA damage checkpoint and proper DNA repair, are significantly induced by 2.62 and 2.17 folds respectively after 24 h ZEA treatment (Table 5). Our transcriptome analysis suggested that ZEA inhibited DNA repair and induced DNA damages.
Our microarray data also revealed that the progression of cell cycle and replication of DNA in BEAS-2B cells was suppressed by ZEA (Table 4 and 5). The expressions of cyclin E (CCNE1, CCNE2), which are essential for S phase progression, are significantly down-regulated by 2.11 and 2.93 folds respectively while p21 activated kinase 3 (PAK3), a known inhibitor of Cdk4, is simultaneously induced by 1.91 folds after 24 h of ZEA treatment. These observations suggest that cyclinD/cdk4 and cyclinE/cdk2 complexes which are essential for G1/S cell cycle progression have been suppressed.

Arrest of cell cycle by ZEA
In addition, many genes related to DNA replication, which are also essential for S phase progression, are down-regulated after 24 h of ZEA treatment ( Table 5). The essential components of the pre-replicative complex (pre-RC) including origin recognition complex, subunit 1 (ORC1), cell division cycle 6 (CDC6) and most member of minichromosome maintenance protein (MCM 2-7) show decreased levels of expression (Table 5). In addition, the assembly and activation complex comprising MCM 10, CDC45, replication factor C (activator 1) 2 (RFC2) and interaction with subunits of replicative polymerase, including polymerase (DNA directed), alpha 1 (POLA1), polymerase (DNA directed), epsilon 2 (POLE2) and primase, DNA, polypeptide 1 (PRIM1), are also down-regulated (Table 5). DNA helicase B (HELB), which is involved in DNA synthesis, is down-regulated by 1.78 folds ( Table 5). From the above results, it can be concluded that ZEA directly affects the machinery for DNA replication and synthesis in BEAS-2B cells.
Taken together, it is tempting to speculate that ZEA induces DNA damage and halts cell cycle at G1/S phase as attempts to repair the damage are unsuccessful. These findings are in agreement with earlier studies which reported that ZEA induced DNA fragmentation and cell cycle arrest [27,33]. The presence of irreparable DNA lesions may lead to the occurrence of subsequent apoptosis in the affected cells (Figure 9). Down-regulation of inflammatory responses by ZEA Due to the large surface structure of the lung, its innate immune response is essential as the first line of defense to act against environmental particles, allergens and invasion of pathogens. The responses often include production of cytokines and chemokines to recruit inflammatory effector cells such as macrophages, neutrophils, eosinophils and lymphocytes [43]. Our results indicated that ZEA weakened the innate immune response to bacterial pathogens (LPS) in bronchial epithelial cells through downregulating the expression of various inflammatory genes ( Figure 6 and Table 7). The mechanism of this down-regulation is possibly through the inhibition of the toll-like receptor (TLR) signaling pathway (Table 3). TLR is a cell surface member of the pattern recognition receptors (PRRs) which are essential as first line defense of the host's responses to allergens and pathogens [44,45]. IL-1 receptor-associated kinase 4 (IRAK4) is an intrinsic kinase which mediates inhibition of IL-1/TLR induced NF-kB activation [44]. Upon activation and stimulation of TLRs, association of MyD88 recruits IRAK4 which in turn induces the phosphorylation of IRAK1 and triggers downstream activation of NF-kB [44]. In our microarray results, the expression of both MyD88 and IRAK4 are respectively down-regulated by 1.45 and 1.69 folds, pointing to the upstream suppression of TLR signaling and subsequent activation of NF-kB.
Both suppressive and inductive effects of ZEA on inflammatory responses have been reported [46,47,48] albeit with different models, dosage and duration of incubation with ZEA. Oxidative stress could trigger inflammation signals through activation of transcription regulators, NF-kB and AP-1 [49]. In our results, however, ZEA induced ROS generations but suppressed inflammatory responses. These observations could be explained by the suppression of NF-kB activation through inhibition of the TLR signaling Myd88-dependent pathway as aforementioned. The finding is consistent with earlier proteomic study in H295R cells showing possible suppression of NF-kB pathway after ZEA exposure [50]. Importantly, our results also indicate that the expressions of JUN, FOSL1 and ATF3 which form the AP-1 transcription complex are significantly decreased (Table 7) suggesting that the AP-1 activation is possibly inhibited by ZEA.
These observations suggested that exposure to air-borne ZEA may increase susceptibility of bronchial epithelial cells to infections due to down-regulation of the expression of inflammatory cytokines and chemokines.

Potential epigenetic changes by ZEA
On the other hand, GSEA of the array results indicated that the histone deacetylation pathway is altered (Figure 3). Histone deacetlyation is associated with pathogenesis of lung diseases. For example, in COPD, the progressive reduction of HDAC activity is linked to the severity of the disease [51,52,53]. Methylated promoters are often coupled with regional histone deacetylation and contribute to transcriptional inactivation. Global hypomethylation was also observed in BEAS-2B cells incubated with ZEA ( Figure 8). Possibly, the presence of 8-OHdG [54,55] and O 6 -methylguanine formed during ROS-induced DNA damage prevented the methylation of adjacent cytosine residues [56,57,58]. Disturbingly, global DNA hypomethylation is a feature of tumorigenesis [59,60]. The role of DNA hypomethylation in the development of cancer is still a paradox. Demethylation of DNA usually occurs at intragenic regions and at repetitive DNA sequences. Three mechanisms including causing instability of chromosome, reactivation of transposable elements and loss of imprinting are proposed [60]. More importantly, altered DNA methylation levels can be stably inherited during DNA replication and disturb subsequent generations. Our results raised the concern on long-lasting effect of ZEA to lung cells which required further investigation.

Conclusions
Our results clearly pointed out the diverse biological responses that ensued when BEAS-2B lung epithelial cells are exposure to ZEA. It also gives us an insight into the molecular mechanisms underlying the adverse consequences of air-borne ZEA. Disturbingly, our results suggested that exposure to ZEA may increase susceptibility of bronchial epithelial cells to diseases through i) the down-regulation of inflammatory cytokines and ii) demethylation of DNA which is a feature of lung carcinogenesis.

Supporting Information
Table S1 Primers used for real-time quantitative PCR. (DOCX)