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Discrimination of vanadium from zinc using gene profiling in human bronchial epithelial cells.

We hypothesized that gene expression profiling may discriminate vanadium from zinc in human bronchial epithelial cells (HBECs). RNA from HBECs exposed to vehicle, V (50 [micro]M), or Zn (50 [micro]M) for 4 hr (n = 4 paired experiments) was hybridized to Affymetrix Hu133A chips. Using one-class t-test with p < 0.01, we identified 140 and 76 genes with treatment:control ratios [greater than or equal to] 2.0 or [less than or equal to] 0.5 for V and Zn, respectively. We then categorized these genes into functional pathways and compared the number of genes in each pathway between V and Zn using Fisher's exact test. Three pathways regulating gene transcription, inflammatory response, and cell proliferation distinguished V from Zn. When genes in these three pathways were matched with the 163 genes flagged by the same statistical filtration for V:Zn ratios, 12 genes were identified. The hierarchical clustering analysis showed that these 12 genes discriminated V from Zn and consisted of two clusters. Cluster 1 genes (ZBTB1, PML, ZNF44, SIX1, BCL6, ZNF450) were down-regulated by V and involved in gene transcription, whereas cluster 2 genes (IL8, IL1A, PTGS2, DTR, TNFAIP3, CXCL3) were up-regulated and linked to inflammatory response and cell proliferation. Also, metallothionein 1 genes (MT1F, MT1G, MT1K) were up-regulated by Zn only. Thus, using microarray analysis, we identified a small set of genes that may be used as biomarkers for discriminating V from Zn. The novel genes and pathways identified by the microarray may help us understand the pathogenesis of health effects caused by environmental V and Zn exposure. Key words: cell proliferation, inflammation, interleukin-1, interleukin-8, metal, microarray, transcription. Environ Health Perspect 113:1747-1754 (2005). doi:10.1289/ehp.7947 available via http://dx.doi.org/[Online 21 June 2005]

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The advancement of microarray technology has allowed investigators to examine simultaneously changes in thousands of genes induced by environmental toxins. McDowell et al. (2000), using gene array with more than 8,000 cDNAs, found patterns of gene expression consistent with acute lung injury in nickel-treated mice. Sato et al. (1999) showed changes in genes related to cell growth and possibly carcinogenesis in rat lungs treated with diesel particles. More recently, Andrew et al. (2003) demonstrated distinct expression patterns in human lung cells exposed to low and high doses of arsenic. The capability of microarrays to provide a snapshot view of expression of a large number of genes may help us generate mechanistic hypotheses as well as identify biomarkers of exposure specific to environmental toxins. The availability of such specific genomic biomarkers may be important in determining the nature of environmental exposures.

Vanadium is present in several environmental settings, for example, during overhauling of oil-fired boilers and burning of heavy fuel in power plants. Exposures to high levels of V-rich particles produce upper and lower respiratory symptoms (Levy et al. 1984; Woodin et al. 1999, 2000). Intratracheal administration of vanadyl sulfate ([VOSO.sub.4]) and a V-rich pollutant dust, residual oil fly ash (ROFA), increased pulmonary artery pressure acutely in buffer-perfused rabbit lungs (Huang et al. 2002) and constricted isolated rat aortic rings (Cadene et al. 1997). Particulate air V concentration correlated with increases in heart rate variability index in boilermakers (Magari et al. 2002). V or ROFA altered the expression of many genes and their protein products related to acute stress (Carter et al. 1997; Gavett et al. 1997, 1999; Nadadur et al. 2000; Samet et al. 1998) and cell survival and tissue growth in cultured cells (Chen et al. 2001; Huang et al. 2000; Zhang et al. 2001).

Zinc is ubiquitous in the natural environment, including ambient air (Walsh et al. 1994). Exposure to excessive Zn (via metal fumes) is a potential hazard for industrial workers who perform welding and smelting operations. Inhalation of high concentrations of zinc oxide or zinc chloride produce respiratory epithelial cell damage, inflammation, and acute injury (Doig and Challen 1964; Evans 1945; Kuschner et al. 1995; Matarese and Matthews 1986; Nemery 1990; Pare and Sandler 1954). Treatment of lung epithelial cells in vitro with Zn compounds enhanced inflammatory signaling and produced cytotoxicity and cell death (Riley et al. 2003; Samet et al. 1998, 1999).

Although V and Zn belong to different elemental classes in the periodic table, they share many biologic properties. For example, both metals are potent enhancers for phosphorylation of signaling proteins, including mitogen-activated protein kinase (Samet et al. 1998) and epidermal growth factor receptors (Wu et al. 1999), and both increase Ras activity (Wu et al. 2002) and interleukin-8 (IL8) release (Samet et al. 1998). Many of these effects may be attributed to the capability of these metals to inhibit protein tyrosine phosphatase activity (Samet et al. 1999). Both V and Zn also inhibit metabolic activity of the cells (Riley et al. 2003). V and Zn may coexist in the ambient environment after being released from different emission sources (Nriagu and Pacyna 1988). The development of a biomarker that discriminates these metals thus may help define the sources and nature of exposures. In this study we hypothesized that gene profiling may be used to discriminate V from Zn in human bronchial epithelial cells (HBECs). We sought to identify a small group of genes that may serve as biomarkers of exposure.

Materials and Methods

Cell culture. Two bronchoscopists obtained bronchial epithelial cells from normal volunteers through bronchoscopic bronchial brushings following the same operational guidelines (Ghio et al. 2000; Huang et al. 2003). Subjects were informed of the procedures and potential risks, and each gave written informed consent. The protocol was approved by the University of North Carolina School of Medicine Committee on Protection of the Rights of Human Subjects and by the U.S. Environmental Protection Agency. A single experienced technician processed all brushings by following the established standard of procedures in our laboratory. The cells (passage 2 or 3) were maintained in bronchial epithelial growth medium (BEGM) (Clonetics, San Diego, CA), supplemented with bovine pituitary extract, insulin 5 [micro]g/mL, hydrocortisone 0.5 [micro]g/mL, gentamicin 50 [micro]g/mL, retinoic acid 0.1 ng/mL, transferrin 10 [micro]g/mL, triiodothyrodine 6.5 ng/mL, epinephrine 0.5 [micro]g/mL, and human epidermal growth factor 0.5 ng/mL. Cells were judged to be 95-100% confluent at the time of metal treatment.

Metal treatment. Stock solutions of metals were prepared in sterile water (Baxter Healthcare Corp., Deerfield, IL) and were diluted with BEGM before experiments. Cells were grown in 100-mm diameter petri dishes and exposed to 5.5 mL of BEGM with or without 50 [micro]M [VOSO.sub.4] or zinc sulfate (Zn[SO.sub.4]) (Johnson Matthey Corp., Ward Hill, MA) for 4 hr.

Purification and hybridization of RNA. Total cellular RNA was extracted from HBECs with Trizol reagent (GIBCO BRL Life Technologies, Gaithersburg, MD) and further purified with phenol/chloroform. The RNA integrity was assessed with an Agilent 2100 bioanalyzer (Agilent Technologies, Inc., Palo Alto, CA). The 260:280-nm ratios for all RNAs were > 1.9. The RNA hybridization to the U133A GeneChip oligonucleotide microarray (Affymetrix, Inc., Santa Clara CA) was performed by Expression Analysis Inc. (Durham, NC). Affymetrix Hu133A 2.0 gene chips were used for the study. The chip contained probes for 14,500 human genes. Target was prepared and hybridized according to the Affymetrix technical manual (Affymetrix, Inc. 2004a). Total RNA (10 [micro]g) was converted into cDNA using reverse transcriptase (Invitrogen Corp., Carlsbad, CA) and a modified oligo(dT)24 primer that contains T7 promoter sequences (GenSet Corp., San Diego, CA). After first-strand synthesis, residual RNA was degraded by the addition of RNaseH and a double-stranded cDNA molecule was generated using DNA polymerase I and DNA ligase. The cDNA was then purified and concentrated using a phenol:chloroform extraction followed by ethanol precipitation. The cDNA products were incubated with T7 RNA polymerase, and biotinylated ribonucleotides using an in vitro transcription kit (Enzo Diagnostics Inc., New York, NY). Half the cRNA products were purified using an RNeasy column (Qiagen Inc., Valencia, CA) and quantified with a spectrophotometer. The cRNA target (20 [micro]g) was incubated at 94[degrees]C for 35 min in fragmentation buffer (Tris, magnesium acetate, potassium acetate). The fragmented cRNA was diluted in hybridization buffer (2-morpholinoethanesulfonic acid, NaCl, EDTA, Tween 20, herring sperm DNA, acetylated bovine serum albumin) containing biotin-labeled oligoB2 and eukaryotic hybridization controls (Affymetrix). The hybridization cocktail was denatured at 99[degrees]C for 5 min, incubated at 45[degrees]C for 5 min, and then injected into a GeneChip cartridge. The GeneChip array was incubated at 42[degrees]C for at least 16 hr in a rotating oven at 60 rpm. GeneChips were washed with a series of nonstringent (25[degrees]C) and stringent (50[degrees]C) solutions containing variable amounts of 2-morpholinoethanesulfonic acid, Tween 20, and SSPE (3 M NaCl, 0.2 M, Na[H.sub.2][PO.sub.4], 0.02 M EDTA). The microarrays were then stained with streptavidin phycoerythrin, and the fluorescent signal was amplified using a biotinylated antibody solution. Fluorescent images were detected in a GeneChip Scanner 3000 (Affymetrix), and expression data were extracted using the default settings in the MicroArray Suite 5.0 software (Affymetrix). All GeneChips were scaled to a median intensity setting of 500. Four independent sets of experiments were performed on HBECs obtained from four different individuals. Each set consisted of control (vehicle), [VOSO.sub.4], and Zn[SO.sub.4].

Quantitative polymerase chain reaction.

Quantitative polymerase chain reaction (Q-PCR) was performed for selected genes to validate microarray results. HBECs were lysed in guanidine isothiocyanate (GITC) buffer [4 M GITC (Boehringer Mannheim, Indianapolis, IN), 25 mM sodium citrate (pH 7.0), 0.5% sarkosyl, and 0.1 M DTT], and RNA was pelleted at 80,000 rpm through a cesium chloride gradient for 2 hr at 15[degrees]C. cDNAs were synthesized from 0.4 [micro]g of total RNA in 100 [micro]L of a buffer containing 5 [micro]M random hexaoligonucleotide primers (Pharmacia, Piscataway, NJ), 10 U/[micro]L Moloney murine leukemia virus reverse transcriptase (GIBCO BRL Life Technologies), 1 U/[micro]L RNase inhibitor (RNasin; Promega, Madison, WI), 0.5 mM dNTP (Pharmacia), 50 mM KCl, 3 mM Mg[Cl.sub.2], and 10 mM Tris-HCl (pH 9.3). After 1 hr of incubation at 39[degrees]C, the reverse transcriptase was heat inactivated at 94[degrees]C for 4 min.

Q-PCR of specimen cDNA and standard cDNA was performed using TaqMan master mix (Perkin Elmer, Foster City, CA), 1.25 [micro]M probe, 3 [micro]M forward primer, and 3 [micro]M reverse primer in a 50-[micro]L volume. The probe, which contains both a fluorescence reporter dye at the 5'-end (6-carboxyfluorescein, 6-FAM: maximum emission wavelength = 518 nm) and a quencher dye at the 3'-end (6-carboxytetramethyl rhodamine, TAMRA: maximum emission wavelength = 582 nm), is degraded by the 5'-3' exonuclease activity of the Taq DNA polymerase, and the resulting fluorescence is detected by a laser in the sequence detector (TaqMan ABI Prism 7700 Sequence Detector System; PerkinElmer). The relative abundance of mRNA levels was determined from standard curves generated from a serially diluted standard pool of cDNA prepared from BEAS-2B cells. The relative abundance of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used to normalize levels of the mRNAs of interest. Six additional sets of Q-PCR experiments consisting of control (vehicle), [VOSO.sub.4], and Zn[SO.sub.4] were performed using HBECs from six different individuals.

Microarray data analysis. The microarray data were deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus database (http://www. ncbi.nlm.nih.gov/geo/; accession number GSE2111). Gene expression values were background corrected and normalized globally using the default setting of the Affymetrix Microarray Suite 5.0 software, and [log.sub.2]-transformed according to the Affymetrix Statistical Algorithm Reference Guide (Affymetrix, Inc. 2004b). The [log.sub.2] ratios of treatment (V or Zn) over control and V over Zn for all probe sets were analyzed using the one-class t-test against the null hypothesis of 0 (ratio = 1) using the Multiexperiment Viewer (version 3.0; The Institute of Genomic Research, Rockville, MD). A p-value of < 0.01 was considered statistically significant. If more than one probe set for the same gene were flagged, their ratios were averaged.

Functional classification of genes. Biologic processes represented by the differentially expressed genes were compiled using the GOCharts in the Database for Annotation, Visualization and Integrated Discovery (DAVID) (http://apps1.niaid.nih.gov/david/) with the coverage and specificity set at level 5 (high) and the hits threshold at 1; with the classification of the Gene Ontology Consortium (http://www.geneontology.org); and with the human gene resources from NCBI (http://www.ncbi.nlm.nih.gov). Comparison of the probe sets in the biologic processes between V and Zn was determined by the Fisher's exact test (p < 0.05) (StatView 4.0; SAS Inc., Cary, NC).

Results

Differentially expressed genes associated with V treatment. Incubation of HBECs with [VOSO.sub.4] at 50 [micro]M for 4 hr showed no cytotoxicity as supported by the lack of lactate dehydrogenase (LDH) release (data not shown). There were 140 differentially expressed genes with known protein products. Seventy-six genes were up-regulated with a treatment:control ratio [greater than or equal to] 2.0 (Table 1), and 64 genes were downregulated with a treatment:control ratio [greater than or equal to] 0.5 (Table 2). The expression of five up-regulated genes (IL8), prostaglandin-endoperoxide synthase 2 (PTGS2), intercellular adhesion molecule 2 (ICAM2), diphtheria toxin receptor (heparin-binding epidermal growth factor-like growth factor) (DTR), and dual specificity phosphatase 1 (DUSP1) was confirmed by Q-PCR in additional experiments (Figure 1). The 140 genes could be further classified functionally into 28 biologic processes containing at least three gene hits.

[FIGURE 1 OMITTED]

Differentially expressed genes associated with Zn treatment. Incubation of HBECs with Zn[SO.sub.4] at 50 [micro]M for 4 hr also showed no LDH release (data not shown). There were 76 differentially expressed genes with known protein products. Forty-three genes were up-regulated with a treatment:control ratio [greater than or equal to] 2.0 (Table 3), and 33 genes were down-regulated with a treatment:control ratio [less than or equal to] 0.5 (Table 4). The up-regulation of metallothionein 1F (MT1F) and heine oxygenase 1 (HMOX1) was confirmed by Q-PCR (Figure 1). The 76 genes could be further classified into 14 biologic processes containing at least three gene hits.

Identification of genes differentiating V from Zn. To identify genes that would discriminate V from Zn, we first analyzed V:Zn ratios using the same statistical filtration method. A total of 163 genes were identified. The results of the hierarchical clustering analysis using these genes are shown in Figure 2. We next compared biologic processes associated with V with those associated with Zn. We found that four biologic processes, regulation of transcription (24 genes), DNA-dependent transcription (22 genes), inflammatory responses (11 genes), and regulation of cell proliferation (10 genes), contained a disproportionately greater number of V-induced genes. Because all genes involved in the DNA-dependent transcription pathway were also flagged in the regulation of transcription pathway, these two processes were combined into one, designated "gene transcription." The number of probe sets in the three biologic pathways associated with V and Zn treatment was compared using the Fisher's exact test. The p-values for these three pathways, gene transcription, inflammatory response, and regulation of cell proliferation, are 0.004, 0.037, and 0.013, respectively.

[FIGURE 2 OMITTED]

We next matched genes in these three pathways with the 163 genes and identified 12 candidate genes: B-cell CLL/lymphoma 6 (BCL6), IL1[alpha] (ILIA), IL8, PTGS2, DTR, chemokine (C-X-C motif) ligand 3 (CXCL3), promyelocytic leukemia (PML), sine oculis homeobox homolog 1 (Drosophila) (SIX1), tumor necrosis factor (TNF), [alpha]-induced protein 3 (TNFAIP3), Zn finger and BTB domain containing 1 (ZBTB1), Zn finger protein 44 (KOX 7) (ZNF44), and Zn finger protein 450 (ZNF450). The hierarchical cluster analysis showed that these 12 genes clearly discriminated the V group from the Zn group (Figure 2) and could be separated into two clusters (Figure 2). Cluster 1 contained ZBTB1, PML, ZNF44, SIX1, BCL6, and ZNF450 that were down-regulated by V and involved in gene transcription. Cluster 2 contained IL8, IL1A, PTGS2, DTR, TNFAIP3, and CXCL3 that were up-regulated and linked primarily to inflammatory response and cell proliferation. We also noted metallothionein 1 genes were up-regulated by Zn but not by V. Zn treatment increased the expression of MT1F by 4.6-fold, MT1G by 29-fold, and MT1K by 81-fold. These metallothionein 1 genes constituted the third group of candidate biomarker genes.

Discussion

In the present study we first determined the differential gene expression patterns in HBECs exposed to 50 [micro]M of V and Zn and found 140 and 76 genes altered by V and Zn, respectively, compared with control. These genes could be classified into 28 and 14 biologic pathways, respectively, that each had at least three gene hits. Seven differentially expressed genes were validated prospectively in six additional experiments using HBECs from six different individuals. When the numbers of genes in the pathways were compared between V and Zn, three biologic processes (gene transcription, inflammatory response, and regulation of cell proliferation) contained a disproportionately greater number of V-induced genes. We then matched the genes in these three pathways with the 163 genes that differentiated V from Zn, and identified 12 candidate genes.

These 12 genes clearly discriminated the V group from the Zn group based on the hierarchical clustering analysis and could be separated into two clusters. The first cluster consisted of 6 genes (ZBTB1, PML, ZNF44, SIX1, BCL6, ZNF436) that were down-regulated by V but mildly up-regulated by Zn. All 6 genes were involved in gene transcription, and BCL6 was also linked to inflammatory response and regulation of cell proliferation. The inhibitory effects of V on the expression of these genes have not been reported. Five of these genes encode Zn finger proteins (ZBTB1, ZNF44, BCL6, ZNF436) or proteins containing Zn-binding domains (PML) that play a role in DNA binding (Bray et al. 1991; Zhong et al. 2000). SIX1 encodes a protein characterized by a divergent DNA-binding homeodomain and an upstream SIX domain, which may be involved in determining DNA-binding specificity and protein-protein interactions. Mice lacking the SIX1 gene have impaired organogenesis of skeletal muscle and kidney during embryo development (Laclef et al. 2003; Xu et al. 2003). Multiple adult tissues, including the lung, also express SIX1 (Boucher et al. 1996), but its function is unclear. The BCL6 gene encodes a Zn finger transcription repressor frequently associated with B-lymphocytes. Translocation and hypermutation of this gene have been detected in B-cell lymphoma (Ohno 2004). BCL6 is also expressed in the epithelial lining of upper airways (Bajalica-Lagercrantz et al. 1998). Based on our results, BCL6 might be involved in gene transcription, inflammatory response, and cell proliferation in airway epithelial cells. The PML gene encodes a Zn-binding protein in the tripartite motif (TRIM) family and is often involved in the translocation with the retinoic acid receptor-[alpha] gene associated with acute promyelocytic leukemia. High levels of PML protein are expressed in human vascular endothelial cells, epithelial cells, and macrophages (Flenghi et al. 1995).

Cluster 2 contained six genes that were up-regulated by V but down-regulated or unchanged by Zn. Four (IL8, ILIA, PTGS2, CXCL3) were related to inflammatory response, three (IL8, ILIA, DTR) related to regulation of cell proliferation, and two (DTR, TNFAIP3) related to gene transcription. Vanadium is known to induce IL8 in cultured bronchial epithelial cells (Carter et al. 1997; Mukherjee et al. 2004) and in the nasal fluid of workers exposed to V-rich pollutant particles (Woodin et al. 1998). Exposure to pollutant particles with high concentrations of V and Ni increased expression of PTSG2 (COX2) in nasal epithelial cells of dogs (Calderon-Garciduenas et al. 2003). Vanadium also increased the expression of DTR [heparin-binding epidermal growth factor-like growth factor (HB-EGF)] in HBECs and fibroblasts (Ingrain et al. 2003; Zhang et al. 2001). The stimulatory effects of V on IL1A and TNFAIP3 gene expression, however, have not been reported. IL1A is one of the nine genes in the ILl gene family and is involved in various immune responses, inflammatory processes, and hematopoiesis (Arend 2002). TNFAIP3 (A20) is a Zn finger protein that is rapidly induced by TNF. It inhibits NF-[kappa]B activation as well as TNF-mediated apoptosis (Gon et al. 2004; He and Ting 2002; Wertz et al. 2004). The CXCL3 (GRO-[gamma]) gene is a member of a gene superfamily encoding a set of related cytokines with inflammatory and growth regulatory properties (Haskill et al. 1990). Constitutive expression of CXCL3 has been identified in infiltrating leukocytes, bronchial epithelial cells, alveolar type II cells, and alveolar macrophages (Becker et al. 1994; Johnson et al. 1996). Several inflammatory stimuli, including ILl, TNF, lipopolysaccharide, and silica, induce the expression of CXCL3 (Becker et al. 1994; Haskill et al. 1990; Johnson et al. 1996; Rangnekar et al. 1991). Note that chemokine (C-X-C motif) ligand 1 (CXCL1) was also up-regulated by V (Table 1). Thus, it appears that the signaling pathways involving IL1, TNF, and chemokines activation may be novel targets for V and may play an important role in V-induced acute respiratory syndrome in boilermakers and power plant workers (Levy et al. 1984; Woodin et al. 2000). Up-regulation of ILIA and other growth-related genes (e.g., DTR, FOS, CXCL1, and EDN1) also indicates that the ILIA pathway may be also involved in clinical conditions associated with cell growth, such as fibrosis (Bonner et al. 1998, 2000).

Although not selected because they were not matched to any known pathways, several metallothionein 1 genes (MT1F, MT1G, MT1K) were significantly up-regulated by Zn. Metallothioneins (MT) are low-molecular-weight metal- and sulfur-rich proteins widely distributed in the organs, including the lung (Courtade et al. 1998). These intracellular proteins are thought to be involved in heavy metal detoxification and the homeostasis of essential trace metals, such as Zn and copper (Kagi 1993; Karin 1985). Exposure to zinc oxide fume increased mRNA of MTs in rat lungs (Cosma et al. 1992). Systemic administration of Zn enhanced MT levels in the liver (Conrad et al. 1997). Mice lacking MTs were more sensitive to Zn toxicity compared with wild-type mice (Park et al. 2001). In our study, in addition to increases in MTIF (4.6-fold), MT1G (29-fold), and MT1K(81-fold), other MTs, although not identified by our statistical filtration, also had elevated ratios: 1.36 for metallothionein 1X (MT1X), 1.17 for metallothionein 1H (MT1H) and 1.21 for metallothionein 2A (MT2A). These results confirm that up-regulation of the MTs may represent early cellular defense against Zn (Irato et al. 2001; Park et al. 2001) and may be used to distinguish Zn and other heavy metals from V.

In our study, we used the one-class t-test with a p-value of < 0.01 and a ratio cutoff of [greater than or equal to] 2.0 or [less than or equal to] 0.5 to identify differentially expressed genes. This statistical algorithm selected 140 genes (1.0%) from V-treated cells, 76 genes (0.5%) from Zn-treated cells, and 163 genes (1.1%) that differentiated V from Zn out of 14,500 genes in the Affymetrix Hu133A 2.0 gene chip. We are not aware of other large-scale genomic studies on V and Zn. One study reported 65 differentially expressed genes out of 1,200 genes (5.4%) associated with 4-hr 50 [micro]M arsenic treatment in BEAS-2B cells, using a ratio cutoff of 2.0 and signal difference of 13 (Andrew et al. 2003). It is difficult to compare across the different studies, but the smaller percentage of recovery of significant genes in our study may indicate in part a more stringent filtration method. Also, the cells in our study were exposed to 50 [micro]M VOS[O.sub.4] and ZnS[O.sub.4], or 14 and 18 [micro]g of elemental V and Zn, respectively. These doses would be equivalent to working 3 hr in the environments of boilermakers and welders with the ambient V and Zn concentrations of 8 and 10 [micro]g/[m.sup.3], respectively (Marquart et al. 1989; Woodin et al. 2000), assuming ventilation of 10 L/min.

Conclusion

It has been estimated that there are approximately 25,000 boilermakers and 300,000 welders nationwide. These workers can be exposed to high concentrations of V and Zn, respectively, at their workplaces. Our study compared gene expression profiles induced by V and Zn in HBECs and identified a group of 12 genes and several metallothionein 1 genes that may be used as a biomarker for V and Zn exposure, respectively. Determining the applicability of these candidate genes as biomarkers will require exposure studies enrolling a large number of subjects. The gene expression profiles provided by our study also identified potentially novel genes and pathways involved in the pathogenesis of health effects caused by environmental V and Zn exposure.

We thank A. Ghio and L. Dailey of the Human Studies Division of the U.S. Environmental Protection Agency (U.S. EPA) for performing bronchoscopic brushings and cell culture, respectively.

Received 19 January 2005; accepted 21 June 2005.

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Zhuowei Li, (1) Jackie Stonehuerner, (2) Robert B. Devlin, (2) and Yuh-Chin T. Huang (2)

(1) Center for Environmental Medicine and Lung Biology, University of North Carolina, Chapel Hill, North Carolina, USA; (2) National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, USA

Address correspondence to Y.-C.T. Huang, Human Study Facilities, U.S. EPA, CB 7315, 104 Mason Farm Rd., Chapel Hill, NC 27599 USA. Telephone: (919) 843-9504. Fax: (919) 966-6271. E-mail: huang.tony@epa.gov

The research described in this article has been reviewed by the U.S. EPA Health Effects and Environmental Research Laboratory and has been approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the U.S. EPA, nor does mention of the trade names or commercial products constitute endorsement or recommendation for use.

The authors declare they have no competing financial interests.
Table 1. Genes up-regulated by [VOSO.sub.4].

Gene Fold Gene
accession change symbol
no. (a) (b) (a) Gene name (a)

Hs.624 8.04 IL8 interleukin 8
Hs.290873 6.67 PPEF2 protein phosphatase, EF hand
 calcium-binding domain 2
Hs.518417 5.52 STX6 syntaxin 6
Hs.233389 5.36 CM carboxypeptidase, vitellogenic-like
Hs.196384 4.67 PTGS2 prostaglandin-endoperoxide synthase 2
 (prostaglandin G/H synthase and
 cyclooxygenase)
Hs.248189 4.46 KRTHA6 keratin, hair, acidic, 6
Hs.211600 4.33 TNFAIP3 tumor necrosis factor, alpha-induced
 protein 3
Hs.477070 4.30 CSNK1D casein kinase 1, delta
Hs.431460 4.24 ICAM2 intercellular adhesion molecule 2
Hs.44385 4.24 SBLF stoned B-like factor
Hs.799 4.21 DTR diphtheria toxin receptor (heparin-
 binding epidermal growth factor-like
 growth factor)
Hs.418167 4.16 ALB albumin
Hs.246310 4.11 JAM2 junctional adhesion molecule 2
Hs.406990 4.06 PDE4D1P phosphodiesterase 4D interacting
 protein (myomegalin)
Hs.992 4.04 PLA2G1B phospholipase A2, group IB (pancreas)
Hs.496222 3.97 ANGPTLI angiopoietin-like 1
Hs.65758 3.78 ITPR3 inositol 1,4,5-triphosphate receptor,
 type 3
Hs.66713 3.70 DIPA hepatitis delta antigen-interacting
 protein A
Hs.519884 3.65 GCNT2 glucosaminyl (N-acetyl) transferase 2,
 protein A
Hs.157259 3.64 SIPA1L3 signal-induced proliferation-
 associated 1-like 3
Hs.436023 3.56 PRDM1 PR domain containing 1, with ZNF
 domain
Hs.303980 3.51 CVP11A1 cytochrome P450, family 11, subfamily
 A, polypeptide 1
Hs.236646 3.49 HOXD9 homeo box D9
Hs.171695 3.46 DUSP1 dual specificity phosphatase 1
Hs.197693 3.44 CACNG2 calcium channel, voltage-dependent,
 gamma subunit 2
Hs.485910 3.34 RARSL arginyl-tRNA synthetase-like
Hs.211238 3.30 IL1F9 interleukin 1 family, member 9
Hs.520319 3.30 SLC22A16 solute carrier family 22 (organic
 cation transporter), member 16
Hs.445555 3.22 SERPIN12 serine (or cysteine) proteinase
 inhibitor, clade I (neuroserpin),
 member 2
Hs.256667 3.20 PDK2 pyruvate dehydrogenase kinase,
 isoenzyme 2
Hs.248122 3.10 GPR24 G-protein-coupled receptor 24
Hs.511899 3.02 EDN1 endothelin 1
Hs.523506 2.99 WARS2 tryptophanyl tRNA synthetase 2
 (mitochondrial)
Hs.333175 2.86 PLA2G12B phospholipase A2, group XIIB
Hs.410817 2.78 RPL13 ribosomal protein L13
Hs.520942 2.77 CLDN4 claudin 4
Hs.50823 2.74 PDCD6 programmed cell death 6
Hs.550498 2.72 RCEI RCE1 homolog, prenyl protein protease
 (S. cerevisiae)
Hs.436023 2.67 PRDM1 PR domain containing 1, with ZNF
 domain
Hs.421724 2.66 CTSG cathepsin G
Hs.2250 2.63 LIF leukemia inhibitory factor
 (cholinergic differentiation factor)
Hs.282387 2.58 RPC32 polymerase (RNA) III (DNA directed)
 (32 kDa)
Hs.525389 2.56 ARHJ ras homolog gene family, member J
Hs.106019 2.54 PPP1R10 protein phosphatase 1, regulatory
 subunit 10
Hs.250281 2.52 SLC13A3 solute carrier family 13 (sodium-
 dependent dicarboxylate
 transporter), member 3
Hs.2128 2.48 DUSP5 dual-specificity phosphatase 5
Hs.89690 2.45 CXCL3 chemokine (C-X-C motif) ligand 3
Hs.11169 2.45 MIG-6 mitogen-inducible gene 6
Hs.789 2.41 CXCL 1 chemokine (C-X-C motif) ligand 1
 (melanoma growth-stimulating
 activity, alpha)
Hs.485004 2.37 ZNF306 zinc finger protein 306
Hs.534478 2.36 DUSP21 dual-specificity phosphatase 21
Hs.441972 2.34 IFNT1 interferon tau-1
Hs.503598 2.33 JMJD2D jumonji domain containing 2D
Hs.546252 2.25 EDG3 endothelial differentiation, sphingo-
 lipid G-protein-coupled receptor, 3
Hs.85862 2.23 PDLIM3 PDZ and LIM domain 3
Hs.445489 2.22 PLEKHB1 pleckstrin homology domain containing,
 family B (evectins), member 1
Hs.1722 2.21 IL1A interleukin 1, alpha
Hs.466871 2.21 PLAUR plasminogen activator, urokinase
 receptor
Hs.159291 2.20 DRP2 dystrophin-related protein 2
Hs.303649 2.19 CCL2 chemokine (C-C motif) ligand 2
Hs.111944 2.19 CYP3A7 cytochrome P450, family 3, subfamily
 A, polypeptide 7
Hs.533683 2.19 FGFR2 fibroblast growth factor receptor 2
Hs.50550 2.19 KBTBD10 kelch repeat and BTB (POZ) domain
 containing 10
Hs.78944 2.19 RGS2 regulator of G-protein signaling 2, 24
 kDa
Hs.190783 2.17 HAL histidine ammonia-lyase
Hs.463059 2.17 STAT3 signal transducer and activator of
 transcription 3 (acute-phase
 response factor)
Hs.25647 2.16 FOS v-fos FBJ murine osteosarcoma viral
 oncogene homolog
Hs.127022 2.14 PTPRE protein tyrosine phosphatase, receptor
 type, E
Hs.447899 2.13 SIGLEC8 sialic acid-binding Ig-like lectin 8
Hs.344812 2.13 TREX1 three prime repair exonuclease 1
Hs.528670 2.12 MMP25 matrix metalloproteinase 25
Hs.514913 2.11 SERPINB2 serine (or cysteine) proteinase
 inhibitor, clade B (ovalbumin),
 member 2
Hs.506381 2.07 FGD6 FYVE, RhoGEF and PH domain
 containing 6
Hs.278658 2.06 KRTHB6 keratin, hair, basic, 6 (monilethrix)
Hs.439060 2.08 CLDN1 claudin 1
Hs.507348 2.05 HS3ST1 heparan sulfate (glucosamine)
 3-O-sulfotransferase 1

Only genes with known protein products are shown.

(a) Gene annotations are from NCBI (http://www.ncbi.nlm.nih.gov).

(b) Fold changes are the average of four individuals.

Table 2. Genes down-regulated by [VOSO.sub.4].

Gene Fold Gene
accession change symbols
no. (a) (b) (a) Gene name (a)

Hs.441975 -11.75 HSXIAPAF1 XIAP-associated factor-1
Hs.370503 -8.11 FYB FYN-binding protein (FYB-120/130)
Hs.76884 -7.48 ID3 inhibitor of DNA binding 3, dominant
 negative helix-loop-helix protein
Hs.520506 -7.37 FBX05 F-box only protein 5
Hs.22393 -6.90 DENR density-regulated protein
Hs.433060 -6.86 ACPP acid phosphatase, prostate
Hs.37045 -6.77 PTH parathyroid hormone
Hs.282410 -6.69 CALM1 calmodulin 1 (phosphorylase kinase,
 delta)
Hs.474251 -6.60 SCARF2 scavenger receptor class F, member 2
Hs.534101 -5.89 MY03B myosin IIIB
Hs.442578 -5.53 LHX9 LIM homeobox 9
Hs.292356 -5.29 CYBB cytochrome b-245, beta polypeptide
 (chronic granulomatous disease)
Hs.1973 -5.21 CCNF cyclin F
Hs.24684 -5.12 ARRDC3 arrestin domain containing 3
Hs.350756 -4.44 STAU2 staufen, RNA-binding protein, homolog
 2 (Drosophila)
Hs.133892 -4.41 TPM1 tropomyosin 1 (alpha)
Hs.24120 -4.23 ZNF44 zinc finger protein 44 (KOX 7)
Hs.275243 -3.89 S100A6 S100 calcium-binding protein A6
 (calcyclin)
Hs.526464 -3.71 PML promyelocytic leukemia
Hs.522090 -3.55 GALT galactose-1-phosphate
 (calcyclin)
Hs.432424 -3.51 TPP2 tripeptidyl peptidase II
Hs.485233 -3.47 MAPK14 mitogen-activated protein kinase 14
Hs.434924 -3.44 RIMS3 regulating synaptic membrane
 exocytosis 3
Hs.75294 -3.38 CRH corticotropin-releasing hormone
Hs.173984 -3.16 TBX1 T-box 1
Hs.444106 -3.11 TOR2A torsin family 2, member A
Hs.254042 -3.02 BAT1 HLA-B associated transcript 1
Hs.75862 -2.96 MADH4 MAD, mothers against decapentaplegic
 homolog 4 (Drosophila)
Hs.498292 -2.89 SDCCAG8 serologically defined colon cancer
 antigen 8
Hs.1650 -2.78 SLC26A3 solute carrier family 26, member 3
Hs.293798 -2.69 ZNF436 zinc finger protein 436
Hs.397073 -2.66 PMS2L5 postmeiotic segregation increased
 2-like 5
Hs.54416 -2.63 SIX1 sine oculis homeobox homolog 1
 (Drosophila)
Hs.118513 -2.59 MGC21621 G-protein-coupled receptor MrgF
Hs.129634 -2.57 CINP cyclin-dependent kinase 2-interacting
 protein
Hs.21388 -2.55 ZDHHC21 zinc finger, DHHC domain
 containing 21
Hs.131846 -2.51 TAF6L TAF6-like RNA polymerase II, p300/
 CBP-associated factor
 (PCAF)-associated factor, 65 kDa
Hs.116622 -2.46 ZFP30 likely ortholog of mouse zinc finger
 protein 30
Hs.478588 -2.41 BCL6 B-cell CLL/lymphoma 6 (zinc finger
 protein 51)
Hs.47712 -2.41 ZNF555 zinc finger protein 555
Hs.487774 -2.41 HNRPA2B1 heterogeneous nuclear
 ribonucleoprotein A2/B1
Hs.339 -2.37 P2RY2 purinergic receptor P2Y, G-protein
 coupled, 2
Hs.501309 -2.35 CIRBP cold-inducible RNA-binding protein
Hs.534040 -2.33 KBTBD6 kelch repeat and BTB (POZ) domain
 containing 6
Hs.6093 -2.27 ARRDC4 arrestin domain containing 4
Hs.400802 -2.27 ZBTB1 zinc finger and BTB domain
 containing 1
Hs.474799 -2.25 PDXP pyridoxal (pyridoxine, vitamin 86)
 phosphatase
Hs.13982 -2.23 KCTD6 potassium channel tetramerisation
 domain containing 6
Hs.487561 -2.22 ICA1 islet cell autoantigen 1, 69 kDa
Hs.48297 -2.17 ZNF363 zinc finger protein 363
Hs.424926 -2.14 TM7SF1 transmembrane 7 superfamily member 1
 (up-regulated in kidney)
Hs.101937 -2.14 SIX2 sine oculis homeobox homolog 2
 (Drosophila)
Hs.409876 -2.12 ZNF450 zinc finger protein 450
Hs.63335 -2.12 TERF2 telomeric repeat binding factor 2
Hs.105633 -2.12 WINS1 WINS1 protein with Drosophila Lines
 (Lin) homologous domain
Hs.142167 -2.11 HSZFP36 ZFP-36 for a zinc finger protein
Hs.186424 -2.09 BCOR BCL6 co-repressor
Hs.518438 -2.08 SOX2 SRY (sex determining region Y)-box 2
Hs.195710 -2.08 ZNF503 zinc finger protein 503
Hs.535499 -2.02 RARA retinoic acid receptor, alpha
Hs.310640 -2.02 T2BP TRAF2-binding protein
Hs.513645 -2.02 PAK6 p21(CDKNIA)-activated kinase 6
Hs.131494 -2.00 ARNT aryl hydrocarbon receptor nuclear
 translocator

Only genes with known protein products are shown.

(a) Genes annotations are from NCBI (http://www.ncbi.nlm.nih.gov).

(b) Fold changes are the average of four individuals.

Table 3. Genes up-regulated by Zn[SO.sub.4]

Gene Fold Gene
accession change symbols
no. (a) (b) (a) Gene name (a)

Hs.188518 81.01 MT1K metallothionein 1K
Hs.433391 28.87 MT1G metallothionein 1G
Hs.283678 8.40 PCDHB14 protocadherin beta 14
Hs.412196 8.09 ESRRBL1 estrogen-related receptor beta-like 1
Hs.502182 5.46 BDNF brain-derived neurotrophic factor
Hs.517581 4.78 HMOX1 heme oxygenase (decycling) 1
Hs.165736 4.67 SCAND2 SCAN domain containing 2
Hs.519469 4.65 SLC30A1 solute carrier family 30 (zinc
 transporter), member 1
Hs.513626 4.58 MT1F metallothionein 1F (functional)
Hs.154296 4.58 TLL2 tolloid-like 2
Hs.303090 3.94 PPP1R3C protein phosphatase 1, regulatory
 (inhibitor) subunit 3C
Hs.118354 3.66 PRR3 proline-rich polypeptide 3
Hs.466891 3.55 ZNF233 zinc finger protein 233
Hs.59889 3.47 HMGCS2 3-hydroxy-3-methylglutaryl-coenzyme A
 synthase 2 (mitochondrial)
Hs.278973 3.33 ANGPT4 angiopoietin 4
Hs.73962 3.31 EPHA7 EphA7
Hs.445835 3.22 SERTAD4 SERTA domain containing 4
Hs.352241 3.09 TAS2R40 taste receptor, type 2, member 40
Hs.78036 3.08 SLC6A2 solute carrier family 6
 (neurotransmitter transporter,
 noradrenalin), member 2
Hs.89714 3.05 CXCL5 chemokine (C-X-C motif) ligand 5
Hs.195471 3.02 PFKFB3 6-phosphofructo-2-kinase/fructose-2,
 6-biphosphatase 3
Hs.460260 3.02 AKR1C2 aldo-keto reductase family 1, member
 C2
Hs.16064 2.98 MAGI1 membrane-associated guanylate kinase
 interacting protein-like 1
Hs.143036 2.81 CABP4 calcium-binding protein 4
Hs.488671 2.67 BAZ1B bromodomain adjacent to zinc finger
 domain, 1 B
Hs.444450 2.62 EGLN1 egl nine homolog 1 (C. elegans)
Hs.465642 2.59 SEMA6B sema domain, transmembrane domain
 (TM), and cyto plasmic domain,
 (semaphorin) 6B
Hs.32374 2.57 DTX3 deltex 3 homolog (Drosophila)
Hs.405667 2.49 CD8B1 CD8 antigen, beta polypeptide 1 (p37)
Hs.516664 2.48 EFNA1 ephrin-A1
Hs.487188 2.46 MLLT4 myeloid/lymphoid or mixed-lineage
 leukemia (trithorax homolog,
 Drosophila); translocated to, 4
Hs.6638 2.33 MYEF2 myelin expression factor 2
Hs.150136 2.25 MAPK7 mitogen-activated protein kinase 7
Hs.372000 2.24 NSMAF neutral sphingomyelinase (N-SMase)
 activation associated factor
Hs.194721 2.21 NCR2 natural cytotoxicity triggering
 receptor 2
Hs.508720 2.19 RAB20 RAB20, member RAS oncogene family
Hs.522610 2.18 LOC119180 lysozyme 2
Hs.75535 2.16 FOXN4 forkhead box N4
Hs.485572 2.11 SOCS2 suppressor of cytokine signaling 2
Hs.521171 2.09 HIG2 hypoxia-inducible protein 2
Hs.80288 2.05 HSPAIL heat-shock 70 kDa protein 1-like
Hs.123450 2.03 JPH3 junctophilin 3
Hs.441047 2.01 ADM adrenomedullin

Only genes with know protein products are shown

(a) Gene annotations are from NCBI (http://www.ncbi.nim.nih.gov).

(b) Fold changes are the average of four individuals.

Table 4. Genes down-regulated by Zn[SO.sub.4].

Gene Fold Gene
accession change symbol
no. (a) (b) (a) Gene name (a)

Hs.376873 -6.25 ZNF390 zinc finger protein 390
Hs.106513 -6.09 TLL1 tolloid-like 1
Hs.200929 -5.87 IL23R interleukin-23 receptor
Hs.268581 -5.47 LPIN2 lipin 2
Hs.112218 -5.36 CAPN10 calpain 10
Hs.532082 -5.23 IL6ST interleukin 6 signal transducer
 (gp130, oncostatin M receptor)
Hs.483136 -4.53 COMMD10 COMM domain containing 10
Hs.141308 -4.39 MOG myelin oligodendrocyte glycoprotein
Hs.7138 -4.1 CHRM3 cholinergic receptor, muscarinic 3
Hs.120633 -4.08 SESN3 sestrin 3
Hs.512587 -3.58 MST1 macrophage stimulating 1 (hepatocyte
 growth factor-like)
Hs.370510 -3.23 1GSF4 immunoglobulin superfamily, member 4
Hs.533040 -3.21 PDLIM7 PDZ and LIM domain 7 (enigma)
Hs.552578 -3.03 TCF1 transcription factor 1, hepatic;
 LF-B1, hepatic nuclear factor
 (HNF1), albumin proximal factor
Hs.472558 -2.92 SDBCA684 serologically defined breast cancer
 antigen 84
Hs.506394 -2.77 ubiquitin specific protease 44
Hs.438994 -2.69 ZNF544 zinc finger protein 544
Hs.32721 -2.61 SAG S-antigen; retina and pineal gland
 (arrestin)
Hs.74082 -2.48 KLRC3 killer cell lectin-like receptor
 subfamily C, member 3
Hs.382683 -2.47 PRG-3 plasticity-related gene 3
Hs.522291 -2.42 PRKWNK2 protein kinase, lysine deficient 2
Hs.493275 -2.34 TRIM31 tripartite motif-containing 31
Hs.129895 -2.29 TBX3 T-box 3 (ulnar mammary syndrome)
Hs.546263 -2.29 KIR3DL2 killer cell immunoglobulin-like
 receptor, three domains, long
 cytoplasmic tail, 2
Hs.546354 -2.21 RRP4 homolog of yeast RRP4 (ribosomal RNA
 processing 4), 3'-5'-exoribonuclease
Hs. 19385 -2.17 ABHD5 abhydrolase domain containing 5
Hs.344400 -2.19 MPHOSPH6 M-phase phosphoprotein 6
Hs.411311 -2.17 IL24 interleukin 24
Hs.492236 -2.17 H326 H326
Hs.255432 -2.06 CIB3 calcium and integrin binding family
 member 3
Hs.476052 -2.02 SNRK SNF-1 related kinase
Hs.432898 -2.01 MAP3K13 mitogen-activated protein kinase
 kinase kinase 13

Only genes with known protein products are shown.

(a) Gene annotations are from NCBI (http://www.ncbi.nlm.nih.gov).

(b) Fold changes are the average of four individuals.
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Title Annotation:Research
Author:Huang, Yuh-Chin T.
Publication:Environmental Health Perspectives
Geographic Code:1USA
Date:Dec 1, 2005
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