Gene-Expression Profiling of Human Mononuclear ...

Journal of Toxicology and Environmental Health, Part A

ISSN: 1528-7394 (Print) 1087-2620 (Online) Journal homepage: http://www.tandfonline.com/loi/uteh20

Gene-Expression Profiling of Human Mononuclear Cells from Welders Using cDNA Microarray Kyung Taek Rim , Kun Koo Park , Yang Ho Kim , Yong Hwan Lee , Jeong Hee Han , Yong Hyun Chung & Il Je Yu To cite this article: Kyung Taek Rim , Kun Koo Park , Yang Ho Kim , Yong Hwan Lee , Jeong Hee Han , Yong Hyun Chung & Il Je Yu (2007) Gene-Expression Profiling of Human Mononuclear Cells from Welders Using cDNA Microarray , Journal of Toxicology and Environmental Health, Part A, 70:15-16, 1264-1277, DOI: 10.1080/15287390701428986 To link to this article: http://dx.doi.org/10.1080/15287390701428986

Published online: 24 Jul 2007.

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Date: 30 November 2015, At: 18:13

Journal of Toxicology and Environmental Health, Part A, 70: 1264–1277, 2007 Copyright © Taylor & Francis Group, LLC ISSN: 1528-7394 print / 1087-2620 online DOI: 10.1080/15287390701428986

Gene-Expression Profiling of Human Mononuclear Cells from Welders Using cDNA Microarray UTEH

Kyung Taek Rim Human Gene-expression Profiling With Cdna Microarray

Laboratory of Occupational Toxicology, Chemical Safety & Health Research Center, Occupational Safety & Health Research Institute, KOSHA, Daejeon, Republic of Korea

Kun Koo Park Pharmacogenechips, Inc., Chuncheon, Republic of Korea

Yang Ho Kim Downloaded by [Kyung-Taek Rim] at 18:13 30 November 2015

Department of Industrial Medicine, College of Medicine, Ulsan University, Ulsan, Republic of Korea

Yong Hwan Lee Department of Preventive Medicine, College of Medicine, Kosin University, Busan, Republic of Korea

Jeong Hee Han and Yong Hyun Chung Laboratory of Occupational Toxicology, Chemical Safety & Health Research Center, Occupational Safety & Health Research Institute, KOSHA, Daejeon, Republic of Korea

Il Je Yu Biosafety Evaluation Headquarters, Korea Environment & Merchandise Testing Institute, Incheon, Republic of Korea

A toxicogenomic chip developed to detect welding-related diseases was tested and validated for field trials. To verify the suitability of the microarray, white blood cells (WBC) or whole blood was purified and characterized from 20 subjects in the control group (average work experience of 7 yr) and 20 welders in the welding-fume exposed group (welders with an average work experience of 23 yr). Two hundred and fifty-three rat genes homologous to human genes were obtained and spotted on the chip slide. Meanwhile, a human cDNA chip spotted with 8600 human genes was also used to detect any increased or decreased levels of gene expression among the welders. After comparing the levels of gene expression between the control and welder groups using the toxicogenomic chips, 103 genes were identified as likely to be specifically changed by welding-fume exposure. Eighteen of the 253 rat genes were specifically changed in the welders, while 103 genes from the human cDNA chip were specifically changed. The genes specifically expressed by the welders were associated with inflammatory responses, toxic chemical metabolism, stress pro-

This study was supported by a 2004 research grant from the Occupational Safety & Health Research Institute, KOSHA (Korea Occupational Safety & Health Agency). Address correspondence to Il Je Yu, Biosafety Evaluation Headquarters, Korea Environment & Merchandise Testing Institute, 7-44 Songdo-dong, Yeonsu-gu, Incheon 406-130, Republic of Korea. E-mail: [email protected]

teins, transcription factors, and signal transduction. In contrast, there was no significant change in the genes related to short-term welding-fume exposure, such as tumor necrosis factor (TNF)alpha and interleukin. In conclusion, if further validation studies are conducted, the present toxicogenomic gene chips could be used for the effective monitoring of welding-fume-exposurerelated diseases among welders.

Chest x-rays of welders often reveal an increased profusion of small opacities due to chronic inhalation of welding fumes. In most cases, exposure to iron oxide fumes seems to produce iron oxide pneumoconiosis (called siderosis), a prominently abnormal chest film with no impairment of the pulmonary function and attributable to welding-fume exposure (Bohlig, 1964; Beckett, 1996). Welding fumes also contain Mn, Cr, and Ni that produce lung damage (Antonini et al., 2003). A casual relationship between interstitial pulmonary fibrosis in welders and long-term exposure to high concentrations of welding fumes has also been reported (Buerke et al., 2002). Further welding-fume-exposure-related pneumoconiosis is still prevalent in certain countries (Steurich & Feyerabend, 1997; Ruegger, 1995; Lubianova, 1990; Nemery, 1990). In particular, welders

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HUMAN GENE-EXPRESSION PROFILING WITH cDNA MICROARRAY

working in confined spaces, like small cellars, tunnels, tanks, boilers, containers, and ship hulls, have a higher risk of exposure to high concentrations of welding fumes (Kwag & Paik, 1997; Buerke et al., 2002). Although the chest x-rays of welders suffering from siderosis exhibit a gradual recovery after an interruption of weldingfume exposure, it was reported that lung fibrosis may also result from exposure to silica dust, NO2, and other weldingfume components (Guidotti et al., 1978; Antonini et al., 2003). Furthermore, lung tissue analyses showed that chronic interstitial pulmonary fibrosis resulted from welding-fume exposure without silica-dust exposure (Funahashi et al., 1988), while a strong relationship was recently reported between interstitial fibrosis and high-concentration long-term exposure to welding fumes (Buerke et al., 2002). In addition to pulmonary disease, neurotoxicological effects may also result from long-term and high-dose welding-fume exposure. Although manganeseexposed workers were not found to exhibit unique symptoms, high-level T1 signals were observed in magnetic resonance imaging (MRI) (Kim et al., 1999), plus manganese-poisoned welders were reported to exhibit high-level MRI signals in brain basal ganglia (Nelson et al., 1993). In a recent study of 15 welders suffering from parkinsonism, it was reported that welding-fume exposure may have facilitated this condition (Racette et al., 2001). Furthermore, high concentrations of welding fumes were found to induce lung fibrosis in animals (Yu et al., 2001, 2003a, 2004), where welding-fume chronic exposure increased the manganese concentration in lungs, liver, and cerebellum, plus other central nervous system (CNS) parts, including the substantia nigra, basal ganglia, temporal lobe, and frontal lobe, although the results were not statistically significant (Yu et al., 2003b). Toxicogenomics using microarray technology may be used to determine the effects of toxic substances and no-effect levels, establish the mechanisms of action, identify susceptible tissues and cell types, and extrapolate the effects from one species to another (Young, 2002). Toxicogenomics has also proven successful in the genetic identification of disease phenotypes (Alizadeh et al., 2000; Bittner et al., 2000), by evaluating differences in mRNA expression between exposed and unexposed groups, and determining the biological effects of exposure to toxic chemicals. In a recent study, suppression-subtractive hybridization (SSH) and a cDNA microarray were used to investigate the gene-expression profiles of peripheral blood mononuclear cells from welding-fume-exposed rats that showed early signs of welder’s pneumoconiosis after 30 d of welding-fume exposure. As a result, the expression of 261 genes was increased, while the expression of 772 genes was decreased (Rim et al., 2004). As such, the microarray gene expression for rats might be useful for comparing or selecting genes showing significant expression after prolonged welding-fume exposure in welders. Accordingly, toxicogenomics was used to investigate the gene-expression profiles of welders with over 20 yr of welding

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experience. Although peripheral blood is not directly exposed to welding fumes, it was used in the present study to include the influence of various factors after welding-fume exposure. As a result, the gene-expression profiles elucidated the activation or suppression of gene expression in relation to fibrosis induced by welding-fume exposure, while providing valuable information for future development of microarrays for early detection of welder’s pneumoconiosis. Finally, the proposed chip may be a valuable tool for monitoring or diagnosing occupational diseases produced by welding-fume exposure. MATERIALS AND METHODS Treatment Groups The blood of welders was used to test a cDNA microarray for the early detection of welder’s pneumoconiosis or other welding-fume-exposure-related disorders. The control group consisted of 20 healthy clerical staff members with no previous exposure to dust or heavy metals, while the test group consisted of 20 welders from the shipbuilding or car manufacturing industries. Priority was given to welders with pneumoconiosis showing on their chest x-rays and those with long-term exposure to welding fumes. Welders with experience of cardiovascular or pulmonary diseases, except for pneumoconiosis, cancer within the last 5 yr, and alcoholism were excluded. Before the subjects were selected, the schedule and test plan were both verified as regards their scientific and ethical soundness by a university internal review board, and the subjects all gave written consent. The subject questionnaire included items related to age, job career, case history, smoking record, etc. Where possible, ambient monitoring data were checked to determine the welding-fume exposure level. Total RNA prepared and purified from the control and test groups was isolated using a Ficol Hypaq Plus (Amersham Biosciences, Buckinghamshire, UK) gradient solution (blood 6 ml; Ficol 4 ml) for verification of the cDNA microarray from the rat model system. Meanwhile, the total RNA from the human Ramos cell line (RPMI1640, 10% FCS, 24 mM NaHCO3, penicillin/streptomycin solution [Cellgro, Mediatech., VA], 5% CO2, 37°C) was used to standardize the chip. DH 5α, an Escherichia coli strain was cultured for 16 h and inoculated into fresh media. After transforming the purchased gene, a plasmid was prepared and purified. Two hundred and twenty-eight human plasmid clones (KRIBB, Daejeon, Korea) were purchased, mini-prepped, and some of the mini-prepped plasmids were sequenced using a BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, CA, USA). The base sequence homology was analyzed using the NCBI BLAST program, where each clone was compared to genes with a known size and function. The genes with the highest estimated homology were selected and analyzed for their functional relationship. The cDNA plasmids used to manufacture the cDNA microarray were separated and purified using a

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K. T. RIM ET AL.


Montage preparation kit (Billerica, MA) and were polymerase chain reaction (PCR) amplified. After precipitating and performing electrophoresis on the PCR products, they were used to manufacture the microarray via a pin microarrayer. cDNA Microarray Test The probe DNA was dotted on aminosilane-coated slides (GAPS II) to create the microarray. The slide was then baked, heated, and the deposition was confirmed using a fluorescent scanner (Cyto60). The welders’ gene expression was tested based on this microarray. Reverse transcription (RT) and PCR amplification were performed with 100 ng total RNA extracted from blood (1 pM 3′ CDS primer IIA (5′-AAGCAGTGGTATGACGCAGAGTACT30VN-3′), 10 pM template switching primer (5′-AAGCAGGGTATCAAGGCAGAGTACGCGGG-3′), heated at 70°C for 2 min, iced for 2 min). The total RNA from the control and welder groups was labeled with Cy3 and Cy5, respectively, during the RT-PCR cDNA synthesis. Unlabeled fluorescence with Cy3-dCTP or Cy5-dCTP was eliminated with a GFX column (Amersham Biosciences, Buckinghamshire, UK). The microarray slides were incubated (42°C, 19 h) in a hybridization chamber. Microarray Data Analysis The fluorescence at each spot was analyzed using a Scanarray 4000, along with the differences in the gene expression between the control and welder groups. The actual fluorescent intensity was calculated by subtracting a background value from the measured value. The Cy5/Cy3 ratio was then calculated, and an over 2-fold increase or 50% decrease was regarded as a significant change. The base sequence homology was analyzed using the NCBI BLAST program, where each clone was compared to genes with a known size and function. The genes with the highest estimated homology were selected and analyzed for their functional relationship. All data are represented by the mean ± SD, plus an analysis of variance (ANOVA) and Duncan’s multiple range test were used to compare the control and welder group parameters. The criterion for significance was set at p < .05. RESULTS Test Groups Table 1 shows the general characteristics of the control and welder groups. The welder group had a mean career length of 23 yr, and a significantly longer history of smoking than the control group. One worker from each group had liver disease. After agarose gel electrophoresis of the total RNA from each group, the RNA was minimally degraded (Figure 1). Single-stranded cDNA was synthesized by reverse transcription using the total RNA as the template, followed by a PCR with the cDNA as the template for 19 cycles as the optimum. Two

TABLE 1 General Characteristics of Subjects Control (n=20)

Welders (n=20)

Total (n=40)

37.30 ± 1.92 6.80 ± 1.36e 12(60.00%) 14.33 ± 1.92 15(75.00%) 17.57 ± 3.26

48.50 ± 0.57f 23.20 ± 0.73f 9(45.00%) 21.67 ± 2.67f 19(95.00%) 15.92 ± 3.93

42.90 ± 1.34 16.17 ± 1.56 21(60.00%) 17.48 ± 1.74 34(85.00%) 16.64 ± 2.59

1 (5.00%)d

1 (5.00%)d

2 (5.00%)

Parameter Age Working durationa Smoker PKYb Drinker Daily drinkc (alcohol g/d) Disease a

Working duration at present company. Pack year. c Amount of daily alcohol consumption. d Liver disease. e n=15. f Significant at p < .05 based on t-test (welder vs. control). b

M

1

2

3

4

5

6

7

8

9

10

FIG. 1. Analysis of total RNA preparation. Electrophoresis of total RNA extracted from subjects’ whole blood. Control group: lane 1, c11; lane 2, c12; lane 3, c13; lane 4, c14; lane 5, c15. Test (welders) group: lane 6, t11; lane 7, t12; lane 8, t13; lane 9, t14; lane 10, t15. M: marker.

hundred and fifty-three rat homologous human cDNA clones related to welding-fume exposure were selected and purchased. Gene-Expression Analysis Using cDNA Microarray The 20 control subjects were paired with the 20 welder subjects, and 10 of the pairs were tested using a cDNA microarray composed of 253 rat homologous human genes, while the other 10 pairs were tested using a cDNA microarray of 8400 human genes. The differences in the gene expression between the control and welder groups were analyzed and compared using the cDNA microarray. As a result, 78 genes were found to increase, while the expression of 25 genes was either decreased or unchanged. The marked increasing ratio ranged from 1 to over 40, whereas the decreasing ratio ranged from 0.3 to 0.016. The rat homologous human genes were coded from R1 to R18, and their ratio of gene expression is shown in Table 2. The mean expression ratio for these genes ranged from 3.3 to 12.3, and the genes that increased the most were xenobiotic metabolism-related genes, like cytochrome P-450 family 1 subfamily B polypeptide (12.3 times) and glutathione S-transferase

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Paraoxonase 1

Cytochrome P-450, family 1, subfamily B, polypeptide 1

Ribosomal protein S13 Glutathione S-transferase A2

R3

R4

R5 R6

Cysteine-rich protein 2 Tumor necrosis factor (TNF superfamily, member 2)

MAX interacting protein 1

R16 R17

R18

R9 R10 R11 R12 R13 R14 R15

Pyridoxine-5’-phosphate oxidase ATPase, Na+/K+ transporting, alpha 1 polypeptide Phospholipase A2-activating protein Thioredoxin reductase 2 cathepsin S Heat-shock 90-kD protein 1, alpha RAB14, member RAS oncogene family Aspartyl aminopeptidase Eukaryotic translation initiation factor 1A

APEX nuclease (multifunctional DNA repair enzyme) 1

R2

R7 R8

7-Dehydrocholesterol reductase

Name of genetic marker

R1

Code

KU026729

KU026980 KU003087

KU001868 KU001705 KU013599 KU019209 KU001868 KU001705 KU013599

KU010189 KU000382

KU000424 KU021908

KU013585

KU012976

KU000355

KU000966

Clone ID

1.23

1.49 1.4

3.77 3.31 10.82 4.12 1.45 1.38 1.63

3.73 5.41

5.8 6.62

12.3

5.15

6.42

5.53

Ratio of gene expression

Cytokine that binds to TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. Mainly secreted by macrophages and can induce cell death of certain tumor cell lines. Potent pyrogen causing fever by direct action or stimulation of interleukin 1 secretion, and is implicated in induction of cachexia, Under certain conditions it can stimulate cell proliferation and induce cell differentiation. Transcriptional repressor. MXI1 binds with MAX to form sequence-specific DNA-binding protein complex that recognizes core sequence 5′-CAC[GA]TG-3′. MXI1 thus antagonizes MYC transcriptional activity by competing for MAX.

Seems to be required for maximal rate of protein biosynthesis. Enhances ribosome dissociation into subunits and stabilizes binding of initiator Met-tRNA(I) to 40 S ribosomal subunits

Conjugates reduced glutathione to wide number of exogenous and endogenous hydrophobic electrophiles. Oxidizes PNP and PMP into pyridoxal 5′-phosphate (PLP).

Produces cholesterol by reducing C7-C8 double bond of 7-dehydrocholesterol (7-DHC). Repairs oxidative DNA damage in vitro. May protect against cell lethality and suppression of mutations. Removes blocking groups from 3′ termini of DNA strand breaks generated by ionizing radiation and bleomycin. Hydrolyzes toxic metabolites of variety of organophosphorus insecticides. Capable of hydrolyzing broad spectrum of organophosphate substrates and number of aromatic carboxylic acid esters. May mediate enzymatic protection of lowdensity lipoproteins against oxidative modification and resulting series of events leading to atheroma formation. Catalyzes conversion of 25-hydroxyvitamin D3 (25(OH)D) to 1-alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D), plays important role in normal bone growth, calcium metabolism, and tissue differentiation.

Function

TABLE 2 Average Fold Increase in Gene Expression From cDNA Microarray Deduced from Rat Genes Specific to Welding-Fume Exposure



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K. T. RIM ET AL.

(6.6 times), and stress-related genes, such as heat-shock protein 90 (4.1 times), paraoxonase (5.2 times) (Deakin et al., 2005), and cathepsin S (10.8 times) as a cystein protease working in antigen presentation and autoimmunity (Liu & Spero, 2004). However, the tumor necrdosis factor (TNF)-alpha (1.4 times) gene, which is known to increase its expression with shortterm welding-fume exposure in vitro (Antonini et al., 1997), was essentially unchanged. The genes analyzed using the 8400 human cDNA microarray were coded from H1 to H85, and their ratio of gene expression is shown in Table 3. Eighty-five genes changed (increased or decreased) their expression, while 55 of 67 genes that increased their expression rose over threefold. The genes that increased the most were the apoptosis WT1 regulator, interleukin (IL) 3 receptor alpha, cysteine sulfinic acid decarboxylase, and coronin. Meanwhile, the genes that decreased the most were the synuclein beta, ribonuclease H1, angiotensin receptor, and glycosyltransferase. It would appear that most of these genes were disease- and immunityrelated genes functioning for elimination of toxic material and disease reduction. With the 253 rat homologous human cDNA microarray, almost no genes exhibited a decreased expression, while most of the increased genes were unrelated to the subjects’ job. In contrast, with the 8400 human cDNA microarray, a shorter period of employment as a welder resulted in more genes with a decreased expression, while a longer period of employment resulted in more genes with an increased expression. Thus, gene expression ratio needs to be reviewed in relation to length of employment as a welder.

DISCUSSION The current study used a cDNA microarray from an animal system to investigate the gene-expression profiles of welders with over 20 yr of experience. In recent studies, SSH and a cDNA microarray were used to investigate the geneexpression profiles of peripheral blood mononuclear cells from welding-fume-exposed rats that showed early signs of welder’s pneumoconiosis (Sung et al., 2004; Yu et al., 2001, 2003a, 2004) after 30 d of welding-fume exposure, the critical period for the development of pneumoconiosis. Therefore, the resulting cDNA microarray may be useful for the early detection of welder’s pneumoconiosis. Although the previous studies used peripheral blood that was not directly exposed to the welding fumes, it was assumed that the blood came into contact with materials induced by or originating from the welding fumes when crossing the capillary vessels from the lung alveolar cells. The blood was also expected to be further influenced, either directly or indirectly, by inflammatory factors or fibrosis-related substances after the welding-fume exposure. Thus, the genes expressed in the peripheral blood from the rats exposed to welding fumes for 30 d may be useful tools for future welder-monitoring studies using microarrays (Rim et al., 2004).

Welding fumes include many components, such as heavy metals (e.g., Fe, Mn, Ni, and Cr), gases (e.g., ozone, Cr(VI), and nitrous fumes), and particulate matter (e.g., SiO2 and asbestos) (Antonini et al., 2004; Yu et al., 2001). Although the gene-expression profiles of various cell systems and organs in vitro after exposure to these compounds are beginning to be elucidated, little is known about the mixed effect on cells in the systemic circulation, like the blood. It was found that chronic exposure to glass silicate increases the gene expression of antioxidant enzymes (e.g., manganese superoxide dismutase, glutathione peroxidase), signal transduction-related enzymes (e.g., nitric oxide synthase, MAPK/ ERK kinase), cytokines (e.g., TNF-alpha, IL-1), and transcription factors (e.g., NF-κB, AP-1) in the alveolar sacs (Fubini & Hubbard, 2003). In addition, when human lung epithelial cells were previously exposed to Cr(VI) in vitro, 150 genes were u-regulated and 70 genes downregulated in high-density oligonucleotide arrays, indicating that Cr(VI) is involved in redox stress, calcium mobilization, energy metabolism, protein synthesis, cell cycle regulation, and carcinogenesis in cells (Ye & Shi, 2001). Meanwhile, the effect of exposure to nontoxic concentrations of Ni(II) acetate on gene expression in cultured human peripheral lung epithelial HPL1D cells was found to influence the genes related to gene transcription, cytoskeleton, cell signaling, the cell membrane, and extracellular matrix proteins (Cheng et al., 2003). To study the effects of diesel exhaust particle (DEP) exposure on the lungs, a microarray of alveolar macrophages from the lungs of DEP-exposed rats was previously examined. As a result, the DEPs were found to increase many genes, including heme oxygenase (HO)-1 and -2, thioredoxin peroxidase 2 (TDPX-2), glutathione S-transferase P subunit (GST-P), NAD(P)H dehydrogenase, and proliferating cell nuclear antigen (PCNA), indicating the induction of genes to resist oxidative stress (Koike et al., 2002). Further, the exposure of Sprague-Dawley rats to residual oil fly ass (ROFA, 0.5 mg/ rat) by intratracheal instillation was found to increase the pERK:ERK and p-IκB:IκB in the lung cells, suggesting changes in cell growth, transformation, and inflammation within the airway (Roberts et al., 2004). However, these studies only covered short-term exposure, making them insufficient to evaluate chronic exposure to hazardous materials. Thus, a method is needed that can monitor the long-term exposure of welders exposed to hazardous materials, such as welding fumes. In this study, changes were found in gene expression related to inflammatory responses, xenobiotic metabolism, stressassociated proteins, transcription factors, and signal transduction, while none of the short-term exposure-specific genes (e.g., TNF-alpha and interleukin-1) were significantly increased. The microarray may also be used to detect manganese poisoning from welding fumes, as welders exposed to welding fumes over a long period develop manganism. In conclusion, if the microarray is used in more studies on miners or

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LOC55974, stromal cell protein IL3RA, interleukin 3 receptor, alpha (low affinity) CSAD, cysteine sulfinic acid decarboxylase CORO2A, coronin, actin binding protein, 2A MOCS3, molybdenum cofactor synthesis 3

IFIT4, interferon-induced protein with tetratricopeptide repeats 4 CCT6B, chaperonin containing TCP1, subunit 6B (zeta 2)

MRPS25, mitochondrial ribosomal protein S25

LY75, lymphocyte antigen 75

H2 H3 H4 H5 H6

H7

H9

H10

H8

PAWR, PRKC, apoptosis, WT1, regulator


H1

Code

O60449

Q96Q22

Q92526

O14879

Q9BRV3 P26951 Q9Y600 Q92828 O95396

Q96IZ0

3.37

3.39

3.4

3.44

4 3.99 3.94 3.67 3.45

6.87

3.78

3.92

3.58

4.17

6.02 6 6.28 4.94 4.27

13.69

Genebank Ratio of gene Standard accession number expression deviation Function

(Continued)

Implicated in mitochondrial protein import and macromolecular assembly. May facilitate the correct folding of imported proteins. May also prevent misfolding and promote the refolding and proper assembly of unfolded polypeptides generated under stress conditions in the mitochondrial matrix Component of the mitochondrial ribosome small subunit (28S), which comprises a 12S rRNA and about 30 distinct proteins. Acts as an endocytic receptor to direct captured antigens from the extracellular space to a specialized antigen-processing compartment. Causes reduced proliferation of B-lymphocytes.

Activates MPT synthase by the ATP dependent adenylation of its C-terminal residue

Pro-apoptopic protein capable of selectively inducing apoptosis in cancer cells, sensitizing the cells to diverse apoptotic stimuli, and causing regression of tumors in animal models. Induces apoptosis in certain cancer cells by activation of the Fas prodeath pathway and coparallel inhibition of NF-kappaB transcriptional activity. Inhibits the transcriptional activation and augments the transcriptional repression mediated by WT1. Downregulates the antiapoptotic protein BCL2 via its interaction with WT1. Seems also to be a transcriptional repressor by itself. May be directly involved in regulating the amyloid precursor protein (APP) cleavage activity of BACE1.

TABLE 3 Average Fold Increase in Gene Expression from Human cDNA Microarray


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HIPK3, homeodomain interacting protein kinase 3

GNG4, guanine nucleotide binding protein 4

ENTPD5, ectonucleoside triphosphate diphosphohydrolase 5

H11

H12

H13

SCARF1, scavenger receptor class F, member 1

NIP30, NEFA-interacting nuclear protein NIP30 HSHUR7SEQ, UV-B repressed sequence, HUR 7 EGFL4, EGF-like-domain, multiple 4 HMGCS1, 3-hydroxy-3-methylglutarylcoenzyme A synthase 1 (soluble) SLC24A1, solute carrier family 24 (sodium/ potassium/calcium exchanger), member 1

H15 H16

H17

H18 H19 H20 H21

H22

CREBBP, CREB binding protein (Rubinstein– Taybi syndrome) PTGIS, prostaglandin I2 (prostacyclin) synthase GM2A, GM2 ganglioside activator protein

H14


Code

O60721

Q9GZU8 Q92661 Q7Z7M0 Q01581

Q14162

Q16647 P17900

Q92793

O75356

P50150

Q9H422

3.25

3.27 3.27 3.26 3.26

3.29

3.32 3.29

3.33

3.34

3.36

3.37

3.27

3.68 3.65 3.74 3.7

3.6

3.81 3.4

3.66

3.42

3.51

3.83

Genebank Ratio of gene Standard accession number expression deviation

TABLE 3 (Continued) Function

Binds gangliosides and stimulates ganglioside GM2 degradation. It stimulates only the breakdown of ganglioside GM2 and glycolipid GA2 by beta-hexosaminidase A. It extracts single GM2 molecules from membranes and presents them in soluble form to beta-hexosaminidase A for cleavage of N-acetyl-D-galactosamine and conversion to GM3 Mediates the binding and degradation of acetylated low density lipoprotein (Ac-LDL). Mediates heterophilic interactions, suggesting a function as adhesion protein

Seems to negatively regulate apoptosis by promoting FADD phosphorylation. Enhances androgen receptor-mediated transcription. May act as a transcriptional corepressor for NK homeodomain transcription factors. Guanine nucleotide-binding proteins (G proteins) are involved as a modulator or transducer in various transmembrane signaling systems. The beta and gamma chains are required for the GTPase activity, for replacement of GDP by GTP, and for G protein-effector interaction. Likely to promote reglycosylation reactions involved in glycoproteins folding and quality control in the endoplasmic reticulum. Hydrolyzes UDP, GDP, and IDP but not any other nucleoside di-, mono- or triphosphates, nor thiamine pyrophosphate.


1271

JAK3, Janus kinase 3 (a protein tyrosine kinase, leukocyte) SKP2, S-phase kinase-associated protein 2 (p45) FKBP6, FK506 binding protein 6, 36 kD MRPS12, mitochondrial ribosomal protein S12

LCAT, lecithin-cholesterol acyltransferase

N4BP3, Nedd4 binding protein 3 ELA2, elastase 2, neutrophil

NT5C2, 5’-nucleotidase, cytosolic II

MMP25, matrix metalloproteinase 25 RAB21, RAB21, member RAS oncogene family

CD84, CD84 antigen (leukocyte antigen)

CASP10, caspase 10, apoptosis-related cysteine protease

H25

H29

H30 H31

H32

H33 H34

H35

H36

H26 H27 H28

H24

ATP6V1A1, ATPase, H+ transporting, lysosomal 70 kD, V1 subunit A, isoform 1 AK1, adenylate kinase 1

H23

Q92851

O15430

Q9NPA2 Q9UL25

P48902

O15049 Q6DF10

P04180

Q13309 O75344 O15235

P52333

Q13131

P38606

3.15

3.15

3.17 3.17

3.17

3.2 3.19

3.2

3.22 3.22 3.21

3.23

3.23

3.23

3.6

3.36

3.46 3.63

3.19

3.25 3.26

3.12

3.32 3.84 3.45

3.64

3.7

3.29

(Continued)

Modifies the functions of natural killer cells, monocytes and granulocytes. Inhibits C5a-dependent neutrophil enzyme release and chemotaxis. May have a critical role in the maintenance of a constant composition of intracellular purine/ pyrimidine nucleotides in cooperation with other nucleotidases. Preferentially hydrolyzes inosine 5-prime-monophosphate (IMP) and other purine nucleotides. May activate progelatinase A. Displays low GTPase activity and exist predominantly in the GTP-bound form Plays a role as adhesion receptor functioning by homophilic interactions and by clustering. Recruits SH2 domain-containing proteins SH2D1A/SAP. Increases proliferative responses of activated T cells and SH2D1A/SAP does not seen be required for this process. Homophilic interactions enhance interferon gamma/IFNG secretion in lymphocytes and induce platelet stimulation via a SH2D1A/SAP-dependent pathway. May serve as a marker for hematopoietic progenitor cells.

Component of the mitochondrial ribosome small subunit (28S), which comprises a 12S rRNA and about 30 distinct proteins. Central enzyme in the extracellular metabolism of plasma lipoproteins. Among other substrates it esterifies the free cholesterol transported in plasma lipoproteins.

This small ubiquitous enzyme is essential for maintenance and cell growth.


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PIGR, polymeric immunoglobulin receptor

H41

H46

H44 H45

IDS, iduronate 2-sulfatase (Hunter syndrome)

ISG15, interferon-stimulated protein, 15 kD KCNJ5, potassium inwardly rectifying channel, subfamily J, member 5 NFX1, nuclear transcription factor, X-box binding 1 TFAP2C, transcription factor AP-2 gamma (activating enhancer binding protein 2 gamma)

KCNQ3, potassium voltage-gated channel, KQT-like subfamily, member 3

H40

H42 H43

CNNM4, cyclin M4 GTSE1, G-2 and S-phase expressed 1 STAT3, signal transducer and activator of transcription 3 (acute-phase response factor)


H37 H38 H39

Code

O60597

Q12986 Q92754

P05161 P48544

P01833

O43525

Q6P4Q7 Q9NYZ3 P40763

3.07

3.09 3.07

3.11 3.1

3.11

3.12

3.13 3.12 3.12

3.35

3.06 3.34

3.41 3.38

3.42

3.45

3.2 3.52 3.53



Sequence-specific DNA-binding protein that interacts with inducible viral and cellular enhancer elements to regulate transcription of selected genes. AP-2 factors bind to the consensus sequence 5’-GCCNNNGGC-3’ and activate genes involved in a large spectrum of important biological functions including proper eye, face, body wall, limbs, and neural tube development. They also suppress a number of genes including MCAM/MUC18, C/EBP alpha and MYC.

Transcription factor that binds to the interleukin-6 (IL-6)-responsive elements identified in the promoters of various acute-phase protein genes. Probably important in the regulation of neuronal excitability. Associates with KCNQ2 or KCNQ5 to form a potassium channel with essentially identical properties to the channel underlying the native M-current, a slowly activating and deactivating potassium conductance that plays a critical role in determining the subthreshold electrical excitability of neurons as well as the responsiveness to synaptic inputs. This receptor binds polymeric IgA and IgM at the basolateral surface of epithelial cells. The complex is then transported across the cell to be secreted at the apical surface. During this process a cleavage occurs that separate the extracellular (known as the secretory component) from the transmembrane segment.


1273

ENTPD2, ectonucleoside triphosphate diphosphohydrolase 2

CD68, CD68 antigen

H56

H57

H55

H54

H53

H51 H52

SLC31A1, solute carrier family 31 (copper transporters), member 1 IL10, interleukin 10

ITPR3, inositol 1,4,5-triphosphate receptor, type 3 BCKDHB, branched chain keto acid dehydrogenase E1, beta polypeptide (maple syrup urine disease) ATP1A2, ATPase, Na+/K+ transporting, alpha 2 (+) polypeptide CRTAP, cartilage-associated protein FRAT2, frequently rearranged in advanced T-cell lymphomas 2 IRAK1, interleukin-1 receptor-associated kinase 1

H48 H49

H50

RTKN, rhotekin

H47

P34810

Q9Y5L3

P22301

Q5T1M3

P51617

O75718 O75474

P50993

Q14573 Q5T2J3

Q8WVN1

2.98

2.99

3

3.02

3.03

3.05 3.04

3.05

3.06 3.05

3.06

3.09

3.27

3.14

3.17

3.14

3.33 3.22

3.22

3.13 3.12

3.16

(Continued)

Inhibits the synthesis of a number of cytokines, including IFN-gamma, IL-2, IL-3, TNF and GM-CSF produced by activated macrophages and by helper T cells. In the nervous system, could hydrolyze ATP and other nucleotides to regulate purinergic neurotransmission. Hydrolyzes ADP only to a marginal extent. The order of activity with different substrates is ATP > GTP > CTP = ITP > UTP >> ADP = UDP. Could play a role in phagocytic activities of tissue macrophages, in both intracellular lysosomal metabolism and extracellular cell–cell and cell–pathogen interactions. Bind to tissue- and organ-specific lectins or selectins, allowing homing of macrophage subsets to particular sites. Rapid recirculation of CD68 from endosomes, lysosomes to the plasma membrane may allow macrophages to crawl over selectin-bearing substrates or other cells.

Binds to the IL-1 type I receptor following IL-1 engagement, triggering intracellular signaling cascades leading to transcriptional upregulation and mRNA stabilization. Isoform 1 binds rapidly but is then degraded, allowing isoform 2 to mediate a slower, more sustained response to the cytokine. Isoform 2 is inactive, suggesting that the kinase activity of this enzyme is not required for IL-1 signaling. Once phosphorylated, IRAK1 recruits the adapter protein PELI1.

Mediates Rho signaling to activate NF-kappa-B and may confer increased resistance to apoptosis to cells in gastric tumorigenesis. May play a novel role in the organization of septin structures.


1274

IREB2, iron-responsive element binding protein 2

H61

CFLAR, CASP8, and FADD-like apoptosis regulator

CTSS, cathepsin S

H62

H63

H60

DHFR, dihydrofolate reductase GCNT2, glucosaminyl (N-acetyl) transferase 2, I-branching enzyme IL13RA1, interleukin 13 receptor, alpha 1


H58 H59

Code

P25774

O15519

P48200

P78552

P00374 Q06430

2.91

2.92

2.93

2.95

2.97 2.95

3.12

2.96

3.21

3.17

3.25 3.06



Binds IL13 with a low affinity. Together with IL4R-alpha can form a functional receptor for IL13. Also serves as an alternate accessory protein to the common cytokine receptor gamma chain for IL4 signaling, but cannot replace the function of gamma C in allowing enhanced IL2 binding activity. Binds to iron-responsive elements (IRES), which are stem–loop structures found in the 5’UTR of ferritin, and delta aminolevulinic acid synthase mRNAs, and in the 3’UTR of transferrin receptor mRNA. Binding to the IRE element in ferritin results in the repression of its mRNA translation. Binding of the protein to the transferrin receptor mRNA inhibits the degradation of this otherwise rapidly degraded mRNA. Apoptosis regulator protein which may function as a crucial link between cell survival and cell death pathways in mammalian cells. Acts as an inhibitor of TNFRSF6-mediated apoptosis. A proteolytic fragment (p43) is likely retained in the death-inducing signaling complex (DISC), thereby blocking further recruitment and processing of caspase-8 at the complex. Full-length and shorter isoforms have been shown either to induce apoptosis or to reduce TNFRSF-triggered apoptosis. Lacks enzymatic (caspase) activity. Thiol protease. Key protease responsible for the removal of the invariant chain from MHC class II molecules. The bond specificity of this proteinase is in part similar to the specificities of cathepsin L and cathepsin N.


1275

PIK4CB, phosphatidylinositol 4-kinase, catalytic, beta polypeptide ZNF197, zinc finger protein 197 UQCRC1, ubiquinol-cytochrome c reductase core protein I

T1A-2, lung type-I cell membrane-associated glycoprotein SNCB, synuclein, beta

H65

H68

RFX2, regulatory factor X, 2 (influences HLA class II expression) PTPN9, protein tyrosine phosphatase, nonreceptor type 9

MEP50, MEP50 protein

KIAA0010, ubiquitin-protein isopeptide ligase (E3) ITGA2, integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor) IMPA1, inositol(myo)-1(or 4)-monophosphatase 1

H71

H73

H74

H76

H75

H72

RNASEH1, ribonuclease H1

H70

H69

H66 H67

NTRK3, neurotrophic tyrosine kinase, receptor, type 3

H64

P29218

P17301

P22681

Q9BQA1

P43378

P48378

O60930

Q16143

Q5T3U7

O14709 P31930

Q9UBF8

Q16288

0.3

0.42

0.43

0.43

0.44

0.46

0.55

0.67

0.69

2.69 1.05

2.82

2.89

0.3

0.33

0.37

0.38

0.34

0.32

0.35

0.31

0.38

2.82 1.17

2.87

3.12

(Continued)

Protein-tyrosine phosphatase that could participate in the transfer of hydrophobic ligands or in functions of the Golgi apparatus. The methylosome may regulate an early step in the assembly of U snRNPs, possibly the transfer of Sm proteins to the SMN complex.

Nonamyloid component of senile plaques found in Alzheimer’s disease. Could act as a regulator of SNCA aggregation process. Protects neurons from staurosporine and 6-hydroxydopamine (6OHDA)stimulated caspase activation in a p53-dependent manner. Contributes to restore the SNCA antiapoptotic function abolished by 6OHDA. Not found in the Lewy bodies associated with Parkinson disease. Endonuclease that specifically degrades the RNA of RNA–DNA hybrids.

May be involved in transcriptional regulation. This is a component of the ubiquinol-cytochrome c reductase complex (complex III or cytochrome b-c1 complex), which is part of the mitochondrial respiratory chain. This protein may mediate formation of the complex between cytochromes c and c1.

Receptor for neurotrophin-3 (NT-3). This is a tyrosine-protein kinase receptor. Known substrates for the trk receptors are SHC1, PI-3 kinase, and PLCG1. The different isoforms do not have identical signaling properties.


1276

HLA-DRB3, major histocompatibility complex, class II, DR beta 3 GRLF1, glucocorticoid receptor DNA binding factor 1

H77

H84 H85

H83

AD-017, glycosyltransferase AD-017 CUL4A, cullin 4A

FLT3, fms-related tyrosine kinase 3 FGD1, faciogenital dysplasia (Aarskog–Scott syndrome) EHHADH, enoyl-coenzyme A, hydratase/3hydroxyacyl coenzyme A dehydrogenase AGTR2, angiotensin II receptor, type 2

H80 H81

H82

FST, follistatin

H79

H78


Code

Q9P0I5 Q6UP08

P50052

Q08426

Q5VTU6 P98174

Q6FHE1

Q9NRY4

O19593

0.14 0.13

0.15

0.17

0.21 0.21

0.23

0.24

0.25

0.27 0.22

0.25

0.24

0.25 0.27

0.29

0.27

0.3



Involved in ubiquitination and subsequent proteasomal degradation of target proteins. Plays a role in the polyubiquitination of CDT1 in response to radiation-induced DNA damage. Required for histone H3 and histone H4 ubiquitination in response to ultraviolet and may be important for subsequent DNA repair.

Receptor for angiotensin II. May have a role in cell morphogenesis and related events in growth and development.

Represses transcription of the glucocorticoid receptor by binding to the cis-acting regulatory sequence 5’-GAGAAAAGAAACTGGAGAAACTC-3’. May participate in the regulation of retinal development and degeneration. May transduce signals from p21-ras to the nucleus, acting via the ras GTPase-activating protein (GAP). May also act as a tumor suppressor. May modulate the action of some growth factors on cell proliferation and differentiation. Binds heparin.


HUMAN GENE-EXPRESSION PROFILING WITH cDNA MICROARRAY

welders, the chip may become a valuable tool for monitoring or diagnosing occupational diseases produced by weldingfume exposure.


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