Genomic and proteomic profiling of responses to toxic metals in human lung cells. (Article).

Eight of the top 50 substances on the 1997 Agency for Toxic Substances and Disease Registry (ATSDR) priority list (ATSDR 2001) are toxic metals, including arsenic, chromium, cadmium, and nickel. Exposure to these metals is associated with a variety of adverse health effects; however, the mechanisms that lead to the development of these diseases and the subcellular pathways modified in response to metal exposures are not well understood. Metal-specific biomarkers of exposure, effect, or susceptibility are needed for risk assessment and epidemiologic studies exploring the important health effects of exposure to these metals.

Arsenic exposure can occur through ingestion of contaminated drinking water, particularly in regions with geologic sources of arsenic, including Bangladesh, Taiwan, and Chile and parts of the United States such as New Hampshire, Michigan, Nevada, and California (Gebel 2000, 2001). Arsenic can also enter the body via inhalation, which is particularly important for certain occupational exposures (Abernathy et al,. 1999; ATSDR 1999a; IARC 1980). Dermal exposure does not appear to lead to significant systemic uptake, although local dermal exposure such as with Fowler's solution or arsenical pesticides has been associated with skin effects at the site of application (Baudouin et al. 2002). Chronic arsenic exposure has been associated with increased incidence of vascular and cardiovascular disease, diabetes, hyperkeratosis, and cancers of the skin, lung, liver, bladder, kidney, and colon (ATSDR 1999a; Byrd et al. 1996; Leonard and Lauwerys 1980).

The primary route of toxicologic concern for exposure to both nickel and chromium is inhalation, principally in occupational settings, although environmental exposures can also occur as a result of anthropogenic sources (IARC 1991; Leikauf 2002; Williams and Sandler 2001). It has been estimated that 1.5 million workers are exposed to nickel occupationally in the United States (IARC 1991). Particulate nickel is emitted into the atmosphere during oil and coal combustion, metal refining, nickel-alloy manufacturing and grinding, battery manufacturing, municipal incineration, electroplating, and stainless steel manufacturing, as well as from cigarette smoke and motor vehicle emissions, resulting in environmental inhalation exposure (Barceloux 1999; Laden et al. 2000; NiPERA 1999). Dermal exposure can occur through skin contact with soil, water, or metals, including stainless steel or coins containing nickel (ATSDR 1999b) and can result in allergic reactions. Occupational exposure to nickel via inhalation is associated with respiratory distress and lung and nasal cancer (ATSDR 1999b; Denkhaus and Salnikow 2002; Leikauf 2002).

Chromium(VI) enters the air principally as a result of coal and oil combustion, steel production, stainless steel welding, and chemical manufacturing (ATSDR 1998; Barnhart 1997; IARC 1991). Chromium exposure can also occur from cigarette smoke. Discharge from electroplating, leather tanning, textiles, and dye and pigment manufacturing can contaminate water sources. Occupational exposure to chromium(VI) through inhalation causes respiratory tract problems and lung cancer, whereas dermal contact can lead to allergic contact dermatitis and skin ulceration (Alcedo and Wetterhahn 1990; ATSDR 1998; Dayan and Paine 2001).

Cadmium inhalation can occur occupationally during battery manufacturing, metal soldering, or welding, as well as environmentally from burning fossil fuels, municipal waste, or cigarettes, and is associated with respiratory damage and cancer. Exposure can also occur through consumption of food or water containing cadmium, leading to gastrointestinal problems and kidney and bone disease, as well as increased body burdens of cadmium, which has a half-life of greater than 20 years in humans (ATSDR 2002; Beyersmann and Hechtenberg 1997; Jarup et al. 1998).

Identification of genes whose expression is specifically modified by toxic metal exposure would provide a better understanding of their mechanisms of action and allow development of sensitive and specific biomarkers of both exposure and susceptibility for use in both mechanistic laboratory and epidemiology studies. In the current work we used cDNA microarrays to compare the effects of the toxic metals arsenic, cadmium, chromium, and nickel on expression of 1,200 human genes in human bronchial BEAS-2B cells, as lung is a target for effects of all four of these metals. We also confirmed the expression of certain relevant genes at the protein level in both epithelial and vascular smooth muscle cell models.

Methods

Cell treatment and preparation. Human bronchial epithelial cells (BEAS-2B; ATCC, Rockville, MD) were grown to postconfluence in 75-[cm.sup.2] flasks (Corning Costar, Corning, NY) on a matrix of 0.01 mg/mL human fibronectin (Collaborative Biomedical Products, Bedford, MA), 0.03 mg/mL Vitrogen 100 (Collagen Biomaterials, Palo Alto, CA), and 0.01 mg/mL bovine serum albumin (Sigma Chemical Co., St. Louis, MO). The cultures were maintained in LHC-9 medium (Biofluids Inc., Rockville, MD) at 37[degrees]C under an atmosphere of 5% C[O.sub.2]/95% air, and medium was changed 24 hr before treatment.

Primary cultures of porcine smooth muscle cells (pSMC) were grown from medial explants of porcine aortas, using established methods (Ross 1971). Briefly, segments of thoracic aorta were cleaned of outer adventitia, opened longitudinally, and scraped to remove endothelial cells. Segments of the remaining intima and media were cut into 1-mm squares and allowed to adhere to scored plastic dishes. The squares were then cultured with complete Dulbecco's modified Eagle's medium (DMEM; Cellgro MediaTech Inc., Herndon, VA) containing 1 mmol/L glucose, 10% fetal bovine serum, and 1% penicillin/streptomycin under an atmosphere of 10% C[O.sub.2]/90% air. The explants were removed once cells began to grow out. The cells were harvested in trypsin/ EDTA and replated for continued subculturing or characterization by immunohistochemistry. All cultures stained greater than 99% positive for [alpha]-actin. The cells used in these experiments were from passage 3 or 4.

For both cell types, treatments were chosen that did not cause overt signs of toxicity or changes in cell survival or replication as measured by long-term colony-forming assays. The exception was the 50-[micro]M arsenic treatment used for dose-response comparisons. In all other cases, the doses of metal are relevant to those to which humans could be exposed. For example, levels of nickel found in the lungs of autopsied U.S. subjects with no known occupational exposure to nickel range between 1.8 and 2.1 mg Ni/[cm.sup.2] of lung surface area (Edelman and Roggli 1989). Nickel refinery workers had much higher levels of nickel in the lung (mean 15 mg Ni/[cm.sup.2]) (IARC 1991).

cDNA array analysis. One confluent flask of > [10.sup.7] cells BEAS-2B cells per treatment group of control cells or cells exposed to 5 or 50 [micro]M sodium arsenite, 3 [micro]M cadmium chloride, 10 [micro]M sodium dichromate (Aldrich, St. Louis, MO), 3 [micro]g/[cm.sup.2] of cell culture flask nickel subsulfide (Sigma), or 1 [micro]M mitomycin C (MMC) for 4 hr was washed and scraped in ice-cold phosphate-buffered saline. Cells were then centrifuged, and the cell pellet was snap frozen in liquid nitrogen. The expression of 1,200 genes was assessed by cDNA microarray analysis using Clontech nylon membrane-based Human Broad Coverage 1.2 I arrays (Clontech Laboratories Inc., Palo Alto, CA). Densitometry was performed on the hybridized membranes using a phospho-imager and the data were analyzed using AtlasImage software (Clontech Laboratories). The presence of nine housekeeping genes per array allowed us to discard housekeeping genes that were induced or repressed by a particular treatment (typically one to two of nine included on the array). The expression of each gene was normalized to the average of the remaining housekeeping genes. The housekeeping genes included on the array were ubiquitin, phospholipase A2, hypoxanthine-guanine, phosphoribosyl-transferase (HPRT), liver glyceraldehyde 3-phosphate dehydrogenase (GAPDH), brain-specific tubulin alpha 1 subunit (TUBA1), HLA class I histocompatibility antigen C-4 alpha subunit (HLAC), cytoplasmic beta-actin (ACTB), 23-kDa highly basic protein, 60S ribosomal protein L13A (RPL13A), and 40S ribosomal protein S9.

The normalized ratios (treated divided by control) and differences (treated minus control) in gene expression between treated and control samples were calculated for all genes. Microarray analyses were repeated using n = 7 independent cultures for the housekeeping genes as well as two untreated independent cultures for all 1,200 genes, and inter-array variability was estimated to be < 22%. Within a single array, the variability of housekeeping gene expression was estimated to be between 8.4 and 20.9%. The housekeeping genes were used to calculate thresholds for each treatment. Thresholds were determined using fold changes 2 standard deviations outside of the average housekeeping gene value. The following threshold values were assigned on the basis of the underlying distribution of the data: 5 [micro]M arsenic: ratio 1.69, difference 4; 50 [micro]M arsenic: ratio 2.0, difference 13; chromium, cadmium, nickel, and MMC: ratio 1.49, difference 4. In addition to setting a threshold for the ratios, the difference between the treated samples and the controls was used to examine genes with low expression levels in which a fold change would be less reliable (e.g., 400/200 units compared with 4/2 units).

Immunoblot. The effects of arsenic exposure on hypoxia inducible factor-1[alpha] (HIF-1[alpha]) or [beta]-actin protein levels (used as a loading control) were determined by Western blotting using a polyclonal antibody to HIF-1[alpha] (Transduction Laboratories, Lexington, KY) or a monoclonal antibody to [beta]-actin (Sigma). Immunoblotting was performed as described previously (Andrew et al. 2001; Barchowsky et al. 1997).

Kinase expression assay. For the kinase expression assay, the medium of 1-day postconfluent BEAS-2B cells was changed 12-18 hr prior to addition of 5 [micro]M potassium dichromate (Aldrich) and the cells were treated for a time course of 1, 4, and 24 hr. pSMC were grown to 80-90% confluence in 75-[cm.sup.2] flasks (Corning Costar), and the medium was changed to a serum-free DMEM containing 1 mg/mL bovine serum albumin 20 hr prior to the addition of sodium arsenite. Cells were treated with 2.5 [micro]M As for 1, 4, and 12 hr. A dose-response experiment using 1, 2.5, and 10 [micro]M As was performed at 24 hr. Following treatment, cells were rinsed with Tris-buffered saline containing protease inhibitors, as described previously. The cells were then prepared as described (Kinexus Bioinformatics Corp. 2001). Briefly, the cells were lysed in 20 mM 3-(N-morpholino) propanesulfonic acid, pH 7.0; 2 mM EGTA; 5 mM EDTA; 30 mM sodium fluoride; 40 mM [beta]-glycerophosphate, pH 7.2; 10 mM sodium pyrophosphate; 2 mM sodium orthovanadate; and 0.5% Nonidet P-40, supplemented with protease inhibitors. The cell lysates were sonicated twice for 15 sec and centrifuged for 2 hr at 19,000 rpm at 4[degrees]C. A protein assay was performed on the supernatant, and a cell lysate mixture was adjusted to a concentration of 1 [micro]g/[micro]L using a 4 x sample buffer (50% glycerol; 125 mM Tris-HCl, pH 6.8; 4% sodium dodecyl sulfate; 0.08% bromophenol blue; 5% [beta]-mercaptoethanol). Samples were heated at 100[degrees]C for 4 min. The samples were analyzed via the Kinetworks' Protein Kinase Screen 1.0, a multiplexed western blot service provided by Kinexus Bioinformatics Corp. (Vancouver, British Columbia, Canada). Kinexus loaded equal amounts of protein, quantified the blots by densitometry, and analyzed them using their proprietary software.

Results

BEAS-2B human lung bronchial epithelial cells were treated for 4 hr either with arsenic (as sodium arsenite, 5 or 50 [micro]M), cadmium (as cadmium chloride, 3 [micro]M), chromium (as sodium dichromate, 10 [micro]M), nickel (as nickel subsulfide, 3 [micro]g/[cm.sup.2]), or the genotoxic cancer chemotherapy drug MMC (1 [micro]M). cDNA array analysis with 1,200 human genes (Clontech 1.2; Clontech) was performed with each treatment. The selected threshold fold change for each treatment was outside the 1.49- to 2.0-fold range in expression seen in the housekeeping genes (average plus 2 standard deviations).

The genes listed in Figure 1 showed increased or decreased expression following each treatment (see "Materials and Methods" for details). This Boolean schematic representation lists the genes uniquely changed by each exposure within the appropriate shape, with overlapping regions (or underlining for arsenic) indicating genes that were modified by more than one treatment. To summarize the data, low-dose cadmium altered the expression of 25 genes; chromium, 44; nickel, 31; 5 [micro]M arsenic, 110 (Figure 2); 50 [micro]M arsenic, 65; and MMC, 16 genes.

[FIGURES 1-2 OMITTED]

As shown in Figure 1, although there was some overlap, overall each treatment modified expression of a largely unique set of genes. Only heat-shock protein 90A (HSP 90A) expression was modified by treatment with all four of the metals tested, and no gene's expression was modified by all five treatments. Several genes were differentially regulated in response to three metals: cadmium, chromium, and nickel. Specifically, these three metals induced expression of erythrocyte glucose transporter 1 (GLUT1) and decreased transcriptional activator (DB1), collagen type 4 (COL4A2), glutathione peroxidase (GSHPX1), hepatoma-derived growth factor (HDGF), and cytochrome P450 1B1 (CYP1B1) (Figure 1, overlapping region). Interestingly, when two or more exposures affected expression of the same gene, the expression was usually altered in the same direction, that is, increased or decreased, with each exposure. The only exception was that treatment with the organic DNA-damaging agent MMC induced expression of early growth response protein 1 (hEGR1), whereas chromium, arsenic, and nickel all suppressed expression of this gene (Figure 1, black font).

To explore the effects of dose on the gene expression profile, we exposed cells for 4 hr to two different doses of arsenic: 5 [micro]M, which caused little or no cytotoxicity, or 50 [micro]M, which was highly cytotoxic as determined by a colony-forming assay. Of the 1,200 genes examined at both doses, only 16 of 158 affected genes were altered at both doses (Figure 2, overlapping region). All genes altered by 50 [micro]M arsenic were increased in expression (red font) with the exception of monocyte chemotactic protein 1 precursor (MCP1) (blue font). In contrast, 5 [micro]M arsenic increased (red font) or decreased (blue font) expression of genes in similar numbers. Interestingly, more total genes were affected by the lower dose than by the higher cytotoxic dose. As might be expected, at the higher dose, stress response and apoptotic genes predominated. Interestingly, most of these genes were unaffected at the lower dose.

Western immunoblot and kinase expression assays were performed for certain genes to determine whether the altered gene expression seen in the microarray assays was paralleled by a change in protein expression. Immunoblots (Figure 3) demonstrated increases in protein levels of the transcription factor HIF-1[alpha], following exposure to arsenic for 4, 8, or 24 hr, which are consistent with the increased HIF-1[alpha] mRNA levels observed after 5-[micro]M arsenic exposure (Figure 2). This level of arsenic exposure did not affect [beta]-actin expression, which was used as a loading control.

[FIGURE 3 OMITTED]

Assays for kinase protein expression changes were performed on cells exposed to arsenic or chromium over a range of doses and time points in two separate cell types. The ratio of mRNA levels in exposed versus control cells (Figure 4) was compared with the ratio of protein levels observed in the kinase expression assay for each kinase (Figure 4). ERK3 gene and protein levels were increased at most arsenic doses and time points tested (Figure 4A), whereas RSK1, PKAC[alpha], and PKB[alpha]/aktl all showed consistent decreases in expression following arsenic exposure in both assays (Figure 4B-D). Chromium exposure also decreased mRNA and protein levels of PKB[alpha]/aktl (Figure 4E). Thus, for the genes and gene products examined, changes in mRNA expression were similar to changes in protein expression.

[FIGURE 4 OMITTED]

Discussion

Chronic exposure to the toxic metals arsenic, chromium, cadmium, and nickel has been associated with a wide variety of adverse health effects (ATSDR 1998, 1999a, 1999b, 2002). Previous studies of individual genes have demonstrated that these metals can each substantially alter gene expression in various cell and whole animal systems (Andrew and Barchowsky 2000; Hamilton and Wetterhahn 1989; Hamilton et al. 1998; Ihnat et al. 1997; McCaffrey et al. 1994). The development of gene array technology has provided a means for examination of alterations in gene expression on a more global level. The current study describes profiles of early changes in gene and protein expression that are observed in response to toxic metal exposure. These early changes may provide further insight into mechanisms underlying development of metal-induced diseases. These early gene and protein responses are also candidate biomarkers of metal exposure and/or effect that could potentially be used diagnostically in molecular and epidemiologic studies.

Results of the cDNA microarray experiments indicate that exposure to these toxic metals modifies expression of only a small subset of the 1,200 total genes examined (Figures 1 and 2), which is consistent with the concept that these were low, relatively nontoxic doses that did not activate large numbers of nonspecific pathways of toxicity response (with the exception of 50 [micro]M arsenic). Although there is some overlap in the genes modified between different metals, these data suggest that each metal modifies expression of a largely unique set of genes that may be characteristic of each treatment. Although this microarray does not contain all known metal-responsive genes, the results show metal-specific patterns of expression among the genes examined. No gene was modified by all five chemical treatments, and only HSP-90A was modified by all four metals. Only three to seven genes overlapped among any two treatments, and similarly, only a few genes were common to any three treatments. A similar unique pattern of gene expression has been observed in yeast exposed to equitoxic doses of several different alkylating agents (Jelinsky et al. 2000) as well as in rats treated with different classes of drugs (Hamadeh et al. 2002a, 2002b). Likewise, cadmium chloride, benzo[a]pyrene and trichloroethylene produced different patterns of gene expression in the livers of exposed mice (Bartosiewicz et al. 2001).

In this study the genes that were altered commonly by more than one treatment were all changed in the same direction, that is, either increased or decreased expression. This supports the idea that these represent biologically relevant responses to these treatments. Cadmium, chromium, and nickel exposures all increased expression of GL UTI and decreased levels of transcription activator DB1 (DB1), procollagen alpha 2(IV) subunit precursor (COL4A2), glutathione peroxidase (GSHPX1), hepatoma-derived growth factor (HDGF), and cytochrome P450 1B1 (CYP1B1). Despite the known ability of Cr(VI) and MMC to cause both monoadducts and cross-links in DNA, only 7 genes were modified in common by both of these agents. A previous 148-gene microarray experiment showed changes in expression of 12 genes in the liver following cadmium exposure (Bartosiewicz et al. 2001). Organ-specific effects as well as differences in the particular genes included in each microarray may explain the lack of overlap between these two studies. Previous studies indicate that 4-hr nickel exposure stabilizes HIF-l[alpha] protein resulting in transcriptional activation of hypoxia-inducible genes (Andrew et al. 2001; Salnikow et al. 2000). Consistent with these findings, HIF-l[alpha]-inducible genes, including the insulin-like growth factor binding protein (IGFBP3) and GLUT1, were up-regulated following nickel exposure (Figure 1) (Minet et al. 2001).

In addition to the metal-specific effects, we examined the effect of arsenic dose on gene expression. The lower-dose arsenic exposure (5 [micro]M) modified expression of a wide variety of genes representing a diverse range of protein classes such as transcription factors, inflammatory cytokines, kinases, and DNA repair proteins, as shown previously in human fibroblasts (Yih et al. 2002) and keratinocytes (Bae et al. 2002). The literature supports the observed induction of heme oxygenase (HO1) (Menzel et al. 1998; Taketani et al. 1989; Yih et al. 2002) and the transcription factor junD (junD) (Liu et al. 2001). In addition, the immunoblot in Figure 3 confirmed that the HIF-1[alpha] gene expression changes were correlated with higher protein levels. We have also demonstrated dose-dependent increases in HIF-1[alpha] protein and mRNA levels in vascular smooth muscle cells (data not shown), suggesting that these effects of low-level arsenic are not confined to a single cell type. Further investigation will be needed to determine the downstream consequences of increases in levels HIF-1[alpha] and the other 11 transcription factors induced in response to 5-[micro]M arsenic exposure. Arsenic exposure has also been associated with increased expression of the inflammatory cytokines, interleukin (IL)-6 and IL-8 via a mechanism that may also involve MAP kinase signaling pathways, as well as induction of other cytokines such as IL-12B, IL-7, and IL-2 (Wu et al. 1999).

The decreased expression of genes involved in DNA damage recognition and repair support the hypothesis that arsenic exposure may decrease the ability of exposed cells to recognize and repair DNA damage, potentially contributing to its carcinogenic and co-carcinogenic activity (Abernathy et al. 1999; Hartwig et al. 1997; Hartwig 1998; Rossman et al. 2001; Vogt and Rossman 2001). For example, the following DNA repair genes were altered after arsenic treatment: Xeroderma pigmentosum group D-complementing protein (XPD, DNA excision repair protein ERCC2), Xeroderma pigmentosum group C-complementing protein (XPC-), AP endonuclease 1 (APE1), DNA ligase-1 (DNL1), DNA polymerase delta catalytic subunit (DPD), DNA topoisomerase II alpha (TOP2A), DNA damage-inducible protein GADD45 (Chen et al. 2000; Liu et al. 2001), MCM DNA replication licensing factors 2 and 7 (MCM2, MCM7), proliferating cyclic nuclear antigen (PCNA), [O.sup.6]-methylguanine-DNA methyltransferase (MGMT), replication factor C large and 40-kDa subunits (RFC, RFC40), and uracil-DNA glycosylase precursor (UNG1). Other studies in our laboratory using human lymphocytes from an epidemiologic study have demonstrated a dose-dependent correlation between decreased expression of nucleotide excision repair genes and chronic exposure to arsenic in the drinking water (Andrew et al. 2003).

Surprisingly, increasing the dose of arsenic to 50 [micro]M did not simply increase the magnitude of the change in the same set of genes or add additional genes. Rather, we observed a striking shift in the gene response profile between the lower and the higher dose. Exposure to 50 [micro]M arsenic for 4 hr resulted in increased rather than decreased expression of nearly all genes that were modified, including many genes that prepare cells to deal with adverse conditions. Consistent with the concept of high-dose arsenic acting as a heat-shock mimetic, 50 [micro]M arsenic induced a variety of heat-shock proteins [HSP-40, HSP-71, HSP-70 (Liu et al. 2001), HSP-60 (Liu et al. 2001), HSP-27, HSP90A, HSP-70.1]. Many of the other genes induced in response to higher doses of arsenic are involved in stress response pathways. Higher doses of arsenic increase levels of jun kinases (JNKs), possibly via mitogen-activated protein kinase kinases, such as MAPKK3, and also activate MAP kinases such as extracellular signal regulated kinase (ERK3) (Cavigelli et al. 1996; Liu et al. 1996; Porter et al. 1999; Samet et al. 1998; Wu et al. 1999). Comparison between the microarray and kinase assay results shown in Figure 4 indicates a correlation between protein and gene-level changes in response to arsenic exposure for all genes that were examined in both studies: ERK3, ribosomal S6 kinase 1 (RSK1), cAMP-dependent protein kinase alpha-catalytic subunit (PKAC[alpha]), and protein kinase B (PKB/aktl). This correlation between gene- and protein-level changes was also seen for PKB/akt following chromium treatment. Further investigation is necessary to determine how the observed changes in kinase expression levels affect signal transduction pathways. These experiments were performed in the BEAS-2B human bronchial epithelial cell line as well as primary pSMC, indicating that the observed changes are universal rather than cell-type or cell-line specific.

This study demonstrates the feasibility of using gene expression profiling to understand toxin-induced biological responses.

Overall, the number of genes modified in response to metal exposures was relatively small. Although a few genes were modified in response to more than one metal, each metal largely altered expression of a unique set of genes. The profile of genes induced by high-dose arsenic exposure clearly indicated a stress response, whereas the other nonovertly toxic doses of metals led to more subtle modification of cell signaling pathways. Future work will focus on using these data to explore basic mechanisms of metal toxicity and to generate new hypotheses. We invite other researchers to consider our data (Table 1) from the perspective of their own specialized areas of expertise. These metal response patterns may shed new light on the mechanisms of toxic metal-induced human diseases and may also be useful for development of molecular biomarkers of exposure and/or effect in mechanistic, epidemiologic, and risk assessment studies.

Table 1. Relative expression of genes for cells treated with arsenic,
chromium, cadmium, nickel, or MMC. (a)

Gene
abbreviation (a)   Gene name (a)

3pK                MAPKAP kinase (3pK)

5-HT-3             5-Hydroxytryptamine 3 receptor
                   precursor (5-HT-3); serotonin-gated
                   ion channel receptor

A1ATR              Alpha-1 -antitrypsin precursor;
                   alpha-1 protease inhibitor;
                   alpha-1-antiproteinase

ABLL               Tyrosine-protein kinase ABL2;
                   tyrosine kinase ARG (ABLL)

ADA2               ADA2-like protein

AIM1               Aurora- & IPL1-like midbody-associated
                   protein kinase 1 (AIM1); ARK2

ALG-2              ALG-2 calcium-binding protein

AP-1               Proto-oncogene c-jun; transcription
                   factor AP-1

APE1               DNA-(apurinic or apyrimidinic site)
                   lyase; AP endonuclease 1; APEX
                   nuclease (APEN; APE1); REF-1 protein

AREB6              Transcription factor AREB6

ARHB               Transforming protein rhoB; ARHB; ARH6

ATF-3              Cyclic-AMP-dependent transcription
                   factor ATF-3 (activating factor 3)
                   sodium/potassium-transporting ATPase

ATPB3              Beta 3 subunit (ATPB3); sodium/
                   potassium-dependent ATPase

B94                B94 protein

BAG-1              BCL-2 binding athanogene-1 (BAG-1);
                   glucocorticoid receptor-associated
                   protein RAP46

BAX                Apoptosis regulator bax

bcl-6              B-cell lymphoma 6 protein (bcl-6); zinc
                   finger protein 51 (ZNF51); LAZ-3 protein

BCL-X              Apoptosis regulator bcl-x

BDNF               Brain-derived neurotrophic factor (BDNF)

BMP4               Bone morphogenetic protein 4 (BMP4) +
                   bone morphogenetic protein 2B (BMP2B)

BRCA1              BRCA1-associated ring domain protein

BRCA2              Breast cancer type 2 susceptibility
                   protein (BRCA2)

BSP1               Transforming growth factor-beta
                   signaling protein 1 (BSP1); mothers
                   against dpp homolog (MAD); MADR1;
                   MSMAD1

BTEB2              Basic transcription element-binding
                   protein 2 (BTEB2); GC-box binding
                   protein 2

BTF2p44            Basic transcription factor 2 44-kDa
                   subunit (BTF2p44)

CANP               Calpain 2 large (catalytic) subunit;
                   M-type calcium-activated neutral
                   proteinase (CANP)

CAP2               Cytoplasmic antiproteinase 2 (CAP2);
                   protease inhibitor 8

CASP2              Caspase-2 precursor (CASP2); ICH-1L
                   protease + ICH-1S protease

CASP4              Caspase-4 precursor (CASP4); ICH-2
                   protease; TX protease; ICE(REL)-II +
                   caspase-5 precursor (CASP5); ICH-3
                   protease; TY protease; ICE(REL)-III

CBF-B              CCAAT-binding transcription factor
                   subunit B (CBF-B); NF-Y protein subunit
                   A (NF-YA); Hap2; CAAT-box DNA-binding
                   protein subunit A

CCNB1              G2/mitotic-specific cyclin B1 (CCNB1)

CD40-L             CD40 ligand (CD40-L); tumor necrosis
                   factor (TNF,)-related activation protein
                   (TRAP); T-cell antigen GP39

C-ets-2            C-ets-2

C1-B18             NADH-ubiquinone oxidoreductase B18
                   subunit; complex I-B18 (CI-B18); cell
                   adhesion protein SQM1

CIP1               Cyclin-dependent kinase inhibitor 1
                   (CDKN1A); melanoma differentiation-
                   associated protein 6 (MDA6); CDK-
                   interacting protein 1 (CIP1); WAF1

CLK1               CDC-like kinase 1 (CLK1)

CLU                Clusterin precursor (CLU); complement-
                   associated protein SP-40,40; complement
                   cytotysis inhibitor (CLI); apolipoprotein
                   J (APO-J); TRPM-2; sulfated glycoprotein 2

c-myc              c-myc oncogene

COL[alpha]2        Procollagen alpha 2(IV) subunit precursor

Cortactin          Cortactin; amplaxin; ems-1 oncogene

CRAF1              CD40 receptor-associated factor 1 (CRAF1)

CREB2              cAMP-dependent transcription factor
                   ATF-4; DNA-binding protein TAXREB67;
                   cAMP-response element binding protein
                   (CREB2)

CTNNA1             Alpha1 catenin (CTNNA1); cadherin-
                   associated protein; alpha E-catenin

Cycin k            Cyclin K

CYP                Cytochrome P450 reductase

CYP1B1             Dioxin-inducible cytochrome P450
                   1B1 (CYP1B1)

DAD1               Defender against cell death 1 (DAD1)

DAXX               DAXX

DB1                Putative transcription activator DB1

DBP                DNA-binding protein TAXREB302;
                   albumin D box-binding protein (DBP)

DBP-A              DNA-binding protein A

DFF45              DNA fragmentation factor 45 (DFF45)

DIF-2              IEX-1L anti-death protein; PRG-1;
                   DIF-2

DPD                DNA polymerase delta catalytic subunit

DPP-1              Dipeptidyl-peptidase I precursor (DPP-I);
                   cathepsin C; cathepsin J; dipeptidyl
                   transferase

DRPLA              Atrophin-1; dentatorubral-paltidoluysian
                   atrophy protein (DRPLA)

E16                E16 amino acid transporter

E2F-3              E2F-3

EAR2               v-erbA-related protein (EAR2)

EB1                EB1 protein

ECK                Ephrin type-A receptor 2 precursor;
                   epithelial cell kinase (ECK); tyrosine-
                   protein kinase receptor ECK

EFNA4              Ephrin A4 precursor (EFNA4); EPH-
                   related receptor tyrosine kinase ligand
                   4 (EPLG4); LERK4

EPH                Ephrin type-A receptor 1 precursor;
                   tyrosine-protein kinase receptor eph

ERF1               TIS11B protein; EGF response factor 1
                   (ERF1)

ERK3               Extracellular signal-regulated kinase 3
                   (ERK3); MAP kinase 3 (MAPK3;
                   p97-MAPK); PRKM5

ETR101             Transcription factor ETRI01

ETS-1              Erythroblastosis virus oncogene homolog
                   1 (ETS-1); p54

ETV6               ets-related protein tel; ets translocation
                   variant 6 (ETV6)

FAST               fas-activated serine/threonine (FAST)
                   kinase

FGFR1              N-sam; fibroblast growth factor receptor1
                   precursor (FGFR1); basic fibroblast growth
                   factor receptor precursor (bFGFR); fms-like
                   tyrosine kinase-2 (FLT2) + heparin-binding
                   growth factor receptor (HBGF-R-alpha-A1) +
                   HBGF-R-alpha-A2 + HBGF-R-alpha-A3

FRA1               fos-related antigen (FRA1)

Fte-1              fte-1; yeast mitochondrial protein import
                   homolog; 40S ribosomal protein S3A
                   (RPS3A)

FX                 Thymosin beta 4; FX

GABP-[alpha]       GA-binding protein alpha subunit (GABP-
                   alpha); transcription factor E4TF1-47;
                   nuclear respiratory factor-2 alpha subunit

GADD153            Growth arrest and DNA-damage-inducible
                   protein 153 (GADD153); DNA-damage-
                   inducible transcript 3 (DDIT3); C/EBP
                   homologous protein (CHOP)

GADD45             Growth arrest and DNA-damage-inducible
                   protein (GADD45); DNA-damage-inducible
                   transcript 1 (DDIT1)

GADD45[beta]       Growth arrest and DNA-damage-inducible
                   protein 45 beta (GADD45 beta)

GALNR1             Galanin receptor type 1 (GALNR1; GALR1)

GAP                GAP-associated protein

GLUT1              Erythrocyte glucose transporter 1 (GLUT1)

GNBP               Guanine nucleotide-binding protein
                   G-i/G-s/G-t beta subunit 2; transducin
                   beta 2 subunit 2

GRRF1              Glucocorticoid receptor repression factor1

GSHPX1             Glutathione peroxidase (GSHPX1; GPX1)

GSR                Glutathione reductase (GRase; GSR; GR)

H2TF1              Nuclear factor NF-kappa-B p100 subunit;
                   nuclear factor NF-kappa-B p52 subunit;
                   H2TF1; oncogene lyt-10

HATB2              Histone acetyltransferase B subunit 2;
                   retinoblastoma-binding protein p46;
                   retinoblastoma-binding protein 7

HBEGF              Heparin-binding EGF-like growth factor
                   (HBEGF); diphtheria toxin receptor (DTR)

HDGF               Hepatoma-derived growth factor (HDGF)

hEGR1              Early growth response protein 1 (hEGR1);
                   transcription factor ETR103; KROX24;
                   zinc finger protein 225; AT225

HEIR-1             Helix-loop-helix protein HLH 1R21;
                   DNA-binding protein inhibitor Id-3; HEIR-1

HIF1-[alpha]       Hypoxia-inducible factor 1 alpha (HIF1
                   alpha); ARNT-interacting protein; member
                   of PAS protein 1 (MOP1)

HLAC               HLA class I histocompatibility antigen
                   C-4 alpha subunit (HLAC)

HO1                Heme oxygenase 1 (HO1); HSOXYGR

HOX-A5             Homeobox protein HOX-A5; HOX-1C

hSMN               Survival of motor neuron (hSMN)

HSP-27             Heat-shock 27-kDa protein (HSP27);
                   stress-responsive protein 27 (SRP27);
                   estrogen-regulated 24-kDa protein; HSPB1

HSP-40             Heat-shock protein 40 (HSP40)

HSP-60             Mitochondrial matrix protein P1 precursor;
                   p60 lymphocyte protein; chaperonin
                   homolog; HUCHA60; heat-shock protein
                   60 (HSP-60); HSPD1

HSP-70             Heat-shock 70-kDa protein 6 (heat-shock
                   70-kDa protein B)

HSP70.1            70-kDa heat-shock protein 1 (HSP70.1;
                   HSPA1)

HSP-71             Heat-shock cognate 71-kDa protein
HSP-90A            Heat-shock 90-kDa protein A (HSP9OA;
                   HSPCA); HSP86

HSR-70             Heat-shock-related 70-kDa protein 2

ICE-LAP3           Cysteine protease ICE-LAP3

Id-1H              DNA-binding protein inhibitor ID-1; Id-1H

IGFBP3             Insulin-like growth factor-binding protein
                   3 precursor (IGF-binding protein 3;
                   IGFBP3; IBP3)

IL-10              Interleukin-10 precursor (IL-10); cytokine
                   synthesis inhibitory factor (CSIF)

IL-11              Interleukin-11 (IL-11); adipogenesis
                   inhibitory factor (AGIF)

IL-12B             Interleukin-12 beta subunit precursor
                   (IL-12B); cytotoxic lymphocyte maturation
                   factor 40-kDa subunit (CLMF p40); NK
                   cell stimulatory factor subunit 2 (NKSF2)

IL-1R2             Interleukin-1 receptor type II precursor
                   (IL-1R2); IL-1 R-beta

IL-2               Interleukin-2 precursor (IL-2); T-cell
                   growth factor (TCGF)

IL2RA              Interleukin-2 receptor alpha subunit
                   precursor (IL-2 receptor alpha subunit;
                   IL2RA); p55; TAC antigen; CD25

IL-5RA             Interleukin-5 receptor alpha subunit
                   precursor (IL-5R-alpha; IL5RA); CD125
                   antigen

IL-6               Interleukin-6 precursor (IL-6); B-cell
                   stimulatory factor 2 (BSF2); interferon
                   beta-2 (IFNB2); hybridoma growth factor

IL-7               Interleukin-7 (IL-7)

IL-8               Interleukin-8 precursor (IL-8); monocyte-
                   derived neutrophil chemotactic factor
                   (MDNCF); T-cell chemotactic factor;
                   neutrophil-activating protein 1 (NAP1);
                   lymphocyte-derived neutrophil-activating
                   factor (LYNAP); protein 3-10C

ITGA4              Integrin alpha 4 precursor (ITGA4); VLA4;
                   CD49D antigen

ITGB4              Integrin beta 4 (ITGB4); CD104 antigen

JNKK               c-jun N-terminal kinase kinase 1 (JNKK);
                   JNK activating kinase 1 (JNKK1); MAP
                   kinase kinase 4 (MKK4)

JUN                jun activation domain binding protein

jun-D              jun-D

JUP,DP3            Junction plakoglobin (JUP); desmoplakin
                   III (DP3)

LIF                Leukemia inhibitory factor precursor (LIF);
                   differentiation-stimulating factor (D factor);
                   melanoma-derived LPL inhibitor (MLPLI);
                   HILDA

LIG1               DNA ligase I; polydeoxyribonucleotide
                   synthase (ATP)(DNL1) (LIG1)

LUCA2              LUCA2; lysosomal hyaluronidase 2
                   (HYAL2); PH-20 homolog

MAD                MAD protein; MAX dimerizer

MAPKAPK-2          MAP kinase-activated protein kinase
                   2 (MAPKAP kinase 2; MAPKAPK-2)

MAPKK3             Dual specificity mitogen-activated protein
                   kinase kinase 3 (MAP kinase kinase 3;
                   MAPKK 3; MKK3); ERK activator kinase 3;
                   MAPK/ERK kinase 3 (MEK3)

MCL-1              Induced myeloid leukemia cell
                   differentiation protein MCL-1

MCM2               MCM2 DNA replication licensing factor;
                   nuclear protein BM28; KIAA0030

MCM5               MCM5 DNA replication licensing factor;
                   CDC46 homolog

MCM7               MCM7 DNA replication licensing factor;
                   CDC47 homolog; p1.1-MCM3

MCP1               Monocyte chemotactic protein 1 precursor
                   (MCP1); monocyte chemotactic and
                   activating factor (MCAF); monocyte
                   secretory protein JE; monocyte
                   chemoattractant protein 1; HC11; small
                   inducible cytokine A2 (SCYA2)

MCT1               Monocarboxylate transporter 1 (MCT1)

MGMT               6-O-methylguanine-DNA methyltransferase
                   (MGMT); methylated-DNA-protein-cysteine
                   methyltransferase

MIP2[alpha]        Macrophage inflammatory protein 2 alpha
                   (MIP2-alpha); growth-regulated protein
                   beta (GRO-beta)

MMP-14             Matrix metalloproteinase 14 precursor
                   (MMP14); membrane-type matrix
                   metalloproteinase 1 (MT-MMPI); MMP-X1

MRP                macMARCKS; MARCKS-related protein
                   (MRP); MLP

NAK1               Early response protein NAK1; TR3 orphan receptor

NaKATPase          Sodium/potassium-transporting ATPase
                   alpha 1 subunit (Na+/K+ ATPase)

NFKB3              NF-kappaB transcription factor p65
                   subunit; RELA; NFKB3

NF-X1              Transcriptional repressor NF-X1

NHE1               Sodium/hydrogen exchanger 1 (Na+/H+
                   exchanger 1; NHE1); amiloride-sensitive
                   Na+/H+ antiporter

NIP3               NIP3 (NIP3)

NMBR               Neuromedin B receptor (NMBR);
                   neuromedin-B-preferring bombesin
                   receptor

NOL1               Proliferating cell nucleolar antigen P120;
                   NOL1

NRGN               Neurogranin (NRGN); RC3

p15, PC4           Activated RNA polymerase II transcriptional
                   coactivator p15; PC4

p78                p78 putative serine/threonine-protein
                   kinase

PAR-1              Thrombin receptor (TR); F2R; PAR1

PBX1               Pre-B-cell leukemia transcription factor-1;
                   homeobox protein pbx1; Homeobox
                   protein prl

PCNA               Proliferating cyclic nuclear antigen
                   (PCNA); cyclin

PDGFA              Platelet-derived growth factor A subunit
                   precursor (PDGFA; PDGF-1)

PI4K-[alpha]       Phosphatidylinositol 4-kinase alpha
                   (PI4-kinase; PTDINS-4-kinase; PI4K-alpha)

PI4PK              68-kDa type I phosphatidylinositol-4-
                   phosphate 5-kinase alpha (PTDINS(4)P-5-
                   kinase); 1-phosphatidylinositol-4-phosphate
                   kinase; diphosphoinositide kinase

PKAC[alpha]        cAMP-dependent protein kinase alpha-
                   catalytic subunit (PKA C-alpha)

PKB/akt            rac-alpha serine/threonine kinase (rac-
                   PK-alpha); protein kinase B (PKB); c-akt;
                   akt1

PLCG1              Phospholipase C gamma 1 (PLC-gamma 1;
                   PLCG1); 1-phosphatidylinositol 4,
                   5-bisphosphate phosphodiesterase
                   gamma 1; PLC-II; PLC-148

PN-II              Alzheimer's disease amyloid A4 protein
                   precursor; protease nexin-II (PN-II); APPI

POLG               DNA polymerase gamma (POLG);
                   mitochondrial DNA polymerase catalytic
                   subunit (MDP1)

PP-1A              Serine/threonine protein phosphatase
                   PP1-alpha 1 catalytic subunit (PP-1A)

PRL-1              PTPCAAX1 nuclear tyrosine phosphatase
                   (PRE-1)

Prot.c8            Proteasome component C8; macropain
                   subunit C8; multicatalytic endopeptidase
                   complex subunit C8

PRP                Major prion protein precursor (PRP);
                   PRP27-30; PRP33-35C; ASCR

PTMS               Parathymosin

RAP-1B             ras-Related protein RAP-1B; GTP-binding
                   protein SMG p21B

RFC                Activator 1 140-kDa subunit (A1 140-kDa
                   subunit); replication factor C large subunit;
                   DNA-binding protein PO-GA

RFC37              Activator 1 37-kDa subunit; replication
                   factor C 37-kDa subunit (RFC37); RFC4

RFC40              Activator 1 40-kDa subunit; replication
                   factor C 40-kDa subunit (RFC40); RFC2

RHO-GDI            rho GDP dissociation inihibitor 1
                   (RHO-GDI 1); RHO-GDI alpha (GDIA1);
                   ARHGDIA

R[kappa]B          R kappa B DNA-binding protein

ROB01              Roundabout 1 (ROB01)

RP-A               Replication protein A 14-kDa subunit
                   (RP-A) (RF-A); replication factor A protein 3

RPL6               60S ribosomal protein L6 (RPL6); TAX-
                   responsive enhancer element binding
                   protein 107 (TAXREB107); neoplasm-
                   related protein C140

RPS19              40S ribosomal protein S19 (RPS19)

RSK1               Ribosomal protein S6 kinase II alpha 1
                   (S6KII-alpha 1); ribosomal S6 kinase 1
                   (RSK1)

SAP2               ets Domain protein elk-3; NET; SRF
                   accessory protein 2 (SAP2)

SATT               Neutral amino acid transporter A
                   (SATT); alanine/serine/cysteine/threonine
                   transporter (ASCT1)

Shb                shb proto-oncogene

SMAD4              Mothers against dpp homolog 4 (SMAD4);
                   MADR4; pancreatic carcinoma gene
                   4 (DPC4)

SNK                Serum-inducible kinase (SNK)

Stratifin          14-3-3 Protein sigma; stratifin; epithelial
                   cell marker protein 1

Synapsin           Synapsin IIIa

TAFAP-2            Transcription factor AP-2 (TFAP2; AP2TF)

TIF1               Transcription intermediary factor 1 (TIF1)

tnkl               Tyrosine kinase tnk1

TOB                Transducer of ERBB2 (TOB)

TOP2A              DNA topoisomerase II alpha (TOP2A)

TR                 Thioredoxin reductase

TRRAP              TRRAP protein

TSC2               Tuberin; tuberous sclerosis 2 protein (TSC2)

TST                Thiosulfate sulfurtransferase; rhodanese

TTR                Transthyretin precursor (TTR); prealbumin;
                   TBPA

TYK2               tyk2 non-receptor protein tyrosine kinase

Ubiquitin          Ubiquitin

UNG1               Uracil-DNA glycosylase precursor (UNG1)

UPAR               Urokinase-type plasminogen activator
                   receptor GPI-anchored form precursor
                   (U-PAR); monocyte activation antigen MO3;
                   CD87 antigen

VEGF               Vascular endothelial growth factor
                   precursor (VEGF); vascular permeability
                   factor (VPF)

XPC                DNA-repair protein complementing XP-C
                   cells; xeroderma pigmentosum group C
                   complementing protein (p125)

XPD                Xeroderma pigmentosum group D
                   complementing protein (XPD); DNA
                   excision repair protein ERCC2

YWHA1              14-3-3n protein eta; protein AS1; YWHAH;
                   YWHA1

Zyxin              Zyxin + zyxin-2

                    Arsenic (5        Arsenic (50
                     [micro]M)         [micro]M)          Chromium
                       ratio             ratio              ratio

Gene
abbreviation (a)   Up       Down     Up       Down     Up       Down

3pK                         2.00

5-HT-3             2.00

A1ATR                                                            1.54

ABLL                                   2.44

ADA2               2.33

AIM1                        2.00

ALG-2                       2.40

AP-1                                  15.42

APE1                        2.07

AREB6              5.00

ARHB                        1.83

ATF-3                                  6.33

ATPB3              1.83

B94                         2.00

BAG-1                       1.70

BAX                         2.11

bcl-6              2.14

BCL-X                       1.89

BDNF                                   6.33

BMP4                                   2.00                      3.00

BRCA1              2.17

BRCA2              2.17

BSP1               3.50                2.44

BTEB2              2.26                2.59

BTF2p44            1.90

CANP                        1.82

CAP2               2.00

CASP2                       4.00

CASP4

CBF-B

CCNB1

CD40-L                      2.80

C-ets-2                                3.33

C1-B18                                                           1.67

CIP1                                   3.78

CLK1                                   2.78

CLU                                    2.79

c-myc                                  3.49                      3.42

COL[alpha]2                                                      2.00

Cortactin                   1.75

CRAF1                       5.00

CREB2              2.62                                1.72

CTNNA1                                                           1.76

Cycin k                                                          2.13

CYP                         3.00

CYP1B1                                                           3.50

DAD1                        2.07

DAXX                        2.50

DB1                                                              2.33

DBP                                    2.27

DBP-A              1.76

DFF45                       3.00

DIF-2                                  3.09                      2.60

DPD                         2.18

DPP-1              1.83

DRPLA

E16                                    2.63

E2F-3              2.17

EAR2                                   2.08

EB1                2.20                2.04

ECK                                    2.54                      2.00

EFNA4                       7.00

EPH                2.88

ERF1                                   3.37                      2.00

ERK3               1.70                3.92

ETR101                                 4.03                      3.00

ETS-1                                  3.21

ETV6                                   2.27

FAST                        2.75

FGFR1                                                            3.00

FRA1                                   8.59                      1.80

Fte-1                                                  1.50

FX                 2.21

GABP-[alpha]       2.33

GADD153                               10.35

GADD45             2.60               13.88

GADD45[beta]                           3.23

GALNR1                                 2.78

GAP                         2.00

GLUT1                                                  1.86

GNBP                        2.50

GRRF1              2.00

GSHPX1                                                 2.00

GSR

H2TF1              1.71

HATB2                                                  1.80

HBEGF                                 16.29

HDGF                                                             1.67

hEGR1                       2.36                                13.00

HEIR-1                                 2.41

HIF1-[alpha]       3.00

HLAC

HO1                50.93              54.95

HOX-A5             1.91

hSMN               2.33

HSP-27                                 4.28

HSP-40                                13.86            1.50

HSP-60                                 3.13            2.00

HSP-70                               108.50

HSP70.1            5.48               45.83

HSP-71                                 4.19

HSP-90A            2.33                3.96                      3.25

HSR-70                                 4.53

ICE-LAP3                    2.33

Id-1H                                                            5.00

IGFBP3

IL-10

IL-11                                 13.33

IL-12B             2.50                6.00

IL-1R2             2.40

IL-2               1.90

IL2RA              1.74

IL-5RA                                 2.66

IL-6               3.11                2.78                      2.45

IL-7               1.73

IL-8               2.95                4.46

ITGA4              2.54

ITGB4                                                            1.76

JNKK                                   3.00

JUN                2.43

jun-D                                  5.67

JUP,DP3                     2.33

LIF                2.15                7.63

LIG1                        2.94

LUCA2                                                            2.11

MAD                                   14.00

MAPKAPK-2                                                        1.50

MAPKK3                                 2.78

MCL-1                                  2.23

MCM2                        2.24

MCM5                                                             1.72

MCM7                        2.24

MCP1                                          2.21               7.00

MCT1               3.50

MGMT                        1.83

MIP2[alpha]        2.03                2.11

MMP-14

MRP                                                              1.80

NAK1                                   7.83

NaKATPase                                                        1.55

NFKB3                       2.00

NF-X1              3.00

NHE1               1.70

NIP3                        2.40

NMBR               1.76

NOL1                                                             1.83

NRGN                        1.75

p15, PC4

p78                                    2.36

PAR-1              2.40

PBX1               2.00

PCNA                        1.75

PDGFA                                  2.52

PI4K-[alpha]                3.00

PI4PK                                  2.86

PKAC[alpha]                 2.33

PKB/akt                     1.74                                 1.53

PLCG1                       2.11

PN-II              1.74

POLG                        3.00

PP-1A                                                            1.65

PRL-1              1.82                3.71

Prot.c8

PRP                2.05                2.54

PTMS

RAP-1B                                 2.25

RFC                         2.00

RFC37                                                            2.59

RFC40                       1.90

RHO-GDI                     1.81

R[kappa]B

ROB01              1.75

RP-A

RPL6               2.10

RPS19

RSK1                        1.72                                 1.75

SAP2               1.91

SATT                                   2.08

Shb                                    3.01

SMAD4              3.50

SNK                2.75

Stratifin                   1.88

Synapsin           1.90

TAFAP-2

TIF1               1.80

tnkl                                                             1.78

TOB                                    2.31

TOP2A                       3.50

TR                                     3.29

TRRAP                                                            2.00

TSC2                        2.22

TST                5.00

TTR                1.89

TYK2                        1.86

Ubiquitin          2.03                7.94            2.16

UNG1                        5.57

UPAR                                                             1.62

VEGF                                   5.86

XPC                         5.00

XPD                         2.33

YWHA1                       2.89                                 1.50

Zyxin                                                            2.25

                      Cadmium            Nickel             MMC
                       ratio             ratio             ratio

Gene
abbreviation (a)   Up       Down     Up       Down     Up       Down

3pK

5-HT-3

A1ATR                       1.65

ABLL

ADA2

AIM1

ALG-2

AP-1

APE1

AREB6

ARHB

ATF-3

ATPB3

B94                         3.50              2.33

BAG-1

BAX

bcl-6

BCL-X

BDNF

BMP4                                                            3.00

BRCA1

BRCA2

BSP1

BTEB2

BTF2p44

CANP

CAP2

CASP2

CASP4                                         1.53

CBF-B                                                  1.50

CCNB1                                                  1.51

CD40-L

C-ets-2

C1-B18

CIP1

CLK1

CLU

c-myc                                         1.95

COL[alpha]2                 1.75              1.75

Cortactin                                     1.76

CRAF1

CREB2                                                  1.58

CTNNA1

Cycin k                                       2.13

CYP

CYP1B1                      3.50              2.33

DAD1

DAXX

DB1                         1.75              1.75

DBP

DBP-A

DFF45

DIF-2                       1.80

DPD

DPP-1

DRPLA                                         3.00

E16

E2F-3

EAR2

EB1                                                    1.53

ECK                                           1.80              1.50

EFNA4

EPH

ERF1                                          2.00

ERK3

ETR101

ETS-1

ETV6

FAST

FGFR1                       3.00

FRA1

Fte-1                                1.52              1.55

FX                          1.74

GABP-[alpha]

GADD153

GADD45

GADD45[beta]

GALNR1

GAP

GLUT1              1.64              1.93

GNBP

GRRF1

GSHPX1                      1.68              1.62

GSR                         1.60

H2TF1

HATB2                                                  1.80

HBEGF

HDGF                        1.55              1.55

hEGR1                                2.17     1.62

HEIR-1

HIF1-[alpha]

HLAC                                                            1.52

HO1

HOX-A5

hSMN

HSP-27

HSP-40

HSP-60

HSP-70

HSP70.1

HSP-71

HSP-90A                     1.86              2.36

HSR-70

ICE-LAP3

Id-1H

IGFBP3                               2.00

IL-10                       1.54

IL-11

IL-12B

IL-1R2

IL-2

IL2RA

IL-5RA

IL-6

IL-7

IL-8

ITGA4

ITGB4                                         1.61

JNKK

JUN

jun-D

JUP,DP3                                       1.60

LIF

LIG1                                                            1.57

LUCA2

MAD

MAPKAPK-2                                     1.50

MAPKK3

MCL-1              2.00

MCM2

MCM5

MCM7

MCP1                                          2.33

MCT1

MGMT

MIP2[alpha]

MMP-14                                                          1.67

MRP

NAK1

NaKATPase                   1.55

NFKB3

NF-X1

NHE1                        1.80

NIP3               2.00

NMBR

NOL1

NRGN

p15, PC4                                               1.74

p78

PAR-1

PBX1

PCNA

PDGFA

PI4K-[alpha]

PI4PK

PKAC[alpha]

PKB/akt

PLCG1

PN-II                                                  1.86

POLG

PP-1A

PRL-1

Prot.c8                              1.80

PRP                1.62                                1.90

PTMS               2.10

RAP-1B

RFC

RFC37                                         1.73              1.54

RFC40

RHO-GDI                     1.67

R[kappa]B                   1.62

ROB01

RP-A               1.90              1.70

RPL6

RPS19              1.95

RSK1

SAP2

SATT

Shb                         1.62

SMAD4

SNK

Stratifin

Synapsin

TAFAP-2                                       1.80

TIF1

tnkl                                          1.78

TOB

TOP2A

TR

TRRAP                                         1.75

TSC2

TST

TTR

TYK2

Ubiquitin                            1.82

UNG1

UPAR

VEGF

XPC

XPD

YWHA1

Zyxin                                         3.00

Gene
abbreviation (a)   GenBank (a)

3pK                U09578

5-HT-3             D49394

A1ATR              X02920

ABLL               M35296

ADA2               AF069732

AIM1               AF008552

ALG-2              AF035606

AP-1               J04111

APE1               X59764;
                   X66133

AREB6              D15050

ARHB               X06820

ATF-3              L19871

ATPB3              U51478

B94                M92357

BAG-1              S83171;
                   Z35491

BAX                L22474

bcl-6              U00115

BCL-X              Z23115;
                   L20121;
                   L20122

BDNF               M61176

BMP4               D30751 +
                   M22490

BRCA1              X82200

BRCA2              U43746

BSP1               U57456

BTEB2              D14520

BTF2p44            Z30094

CANP               M23254

CAP2               L40377

CASP2              U13021 +
                   U13022

CASP4              U28014 +
                   U28015

CBF-B              M59079

CCNB1              M25753

CD40-L             L07414

C-ets-2            J04102

C1-B18             M33374

CIP1               U09579;
                   L25610
                   L29222

CLK1               M74816

CLU

c-myc              V00568

COL[alpha]2        X05562

Cortactin          M98343

CRAF1              U21092

CREB2              D90209

CTNNA1             D13866;
                   D14705;
                   L23805;
                   L22080

Cycin k            AF060515

CYP                S90469

CYP1B1             U03688

DAD1               D15057

DAXX               AF015956

DB1                D28118

DBP                D28468

DBP-A              M24069

DFF45              U91985

DIF-2              AF039067;
                   AF071596

DPD                M80397

DPP-1              X87212

DRPLA              D31840

E16                AF077866

E2F-3              Y10479

EAR2               X12794

EB1                U24166

ECK                M59371;
                   M36395

EFNA4              U14188

EPH                M18391

ERF1               X79067

ERK3               X80692

ETR101             M62831

ETS-1              J04101

ETV6               U11732

FAST               X86779

FGFR1              X66945;
                   M34641;
                   M34186;
                   M37722 +
                   M63887 +
                   M63888 +
                   M63889

FRA1               X16707

Fte-1              M77234

FX                 M17733

GABP-[alpha]       D13316

GADD153            S40706;
                   S62138

GADD45             M60974

GADD45[beta]       AF078077

GALNR1             L34339

GAP                U17032

GLUT1              K03195

GNBP               M36429

GRRF1              M73077

GSHPX1             Y00483;
                   M21304

GSR                X15722

H2TF1              X61498

HATB2              U35143

HBEGF              M60278

HDGF               D16431

hEGR1              X52541;
                   M62829

HEIR-1             X89111

HIF1-[alpha]       U22431

HLAC               M11886

HO1                X06985

HOX-A5             M26679

hSMN               U18423

HSP-27             X54079

HSP-40             D49547

HSP-60             M34664

HSP-70             X51757;
                   M11236

HSP70.1            M11717

HSP-71             Y00371

HSP-90A            X07270

HSR-70             L26336

ICE-LAP3           U39613

Id-1H              D13889

IGFBP3             M31159;
                   M35878

IL-10              M57627

IL-11              M57765

IL-12B             M65290

IL-1R2             X59770

IL-2               A14844

IL2RA              X01057;
                   X01058;
                   X01402

IL-5RA             M75914

IL-6               X04602;
                   M14584

IL-7               J04156

IL-8               Y00787

ITGA4              L12002;
                   X16983;
                   X15356

ITGB4              X53587;
                   X52186;
                   X51841

JNKK               L36870

JUN                U65928

jun-D              X56681

JUP,DP3            M23410;
                   Z68228

LIF                X13967;
                   M63420

LIG1               M36067

LUCA2              U09577

MAD                L06895

MAPKAPK-2          U1277
                   9

MAPKK3             L36719

MCL-1              L08246

MCM2               D21063

MCM5               X74795

MCM7               D55716

MCP1               M24545

MCT1               L31801

MGMT               M29971

MIP2[alpha]        X53799

MMP-14             D26512;
                   X83535

MRP                X70326

NAK1               L13740

NaKATPase          D00099

NFKB3              L19067

NF-X1              U15306

NHE1               M81768

NIP3               U15174

NMBR               M73482

NOL1               X55504

NRGN               Y09689

p15, PC4           U12979

p78                M80359

PAR-1              M62424

PBX1               M86546

PCNA               M15796;
                   J04718

PDGFA              X06374

PI4K-[alpha]       L36151

PI4PK              X80907

PKAC[alpha]        X07767

PKB/akt            M63167

PLCG1              M34667

PN-II              Y00264

POLG               X98093

PP-1A              M63960

PRL-1              U48296

Prot.c8            D00762

PRP                M13667

PTMS               M24398

RAP-1B             X08004

RFC                L14922

RFC37              M87339

RFC40              M87338

RHO-GDI            X69550

R[kappa]B          U08191

ROB01              AF040990

RP-A               L07493

RPL6               X69391

RPS19              M81757

RSK1               L07597

SAP2               Z36715

SATT               L14595

Shb                X75342

SMAD4              U44378

SNK                AF059617

Stratifin          AF029082

Synapsin           AF046873

TAFAP-2            M36711

TIF1               AF009353

tnkl               U43408

TOB                D38305

TOP2A              J04088

TR                 X91247

TRRAP              AF076974

TSC2               X75621

TST                D87292

TTR                K02091

TYK2               X54637

Ubiquitin          M26880

UNG1               X15653

UPAR               U08839;
                   M83246;
                   X51675

VEGF               M32977;
                   M27281

XPC                D21089

XPD                X52221

YWHA1              L20422

Zyxin              X94991;
                   X9573

(a) Information from GenBank (http://
www.ncbi.nlm.nih.gov/GenBank/index.html).

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Address correspondence to A.S. Andrew, Dept. of Pharmacology and Toxicology, Dartmouth Medical School, 7927 Rubin Bldg., 452M-3, One Medical Center Drive, Lebanon, NH 03756-0001. Telephone: [phone]. Fax: [phone]. E-mail: [e-mail]

We thank the technical staff at Clontech for assistance in analyzing the array data in these experiments. We also thank the Dartmouth Molecular Biology and Proteomics Core Facility and the Dartmouth Bioinformatics Group for their assistance and support.

This work was supported by the National Institute of Environmental Health Sciences Superfund Basic Research program grant P42 ES07373.

The authors state there is no conflict of interest.

Received 31 January 2003; accepted 6 May 2003.

Angeline S. Andrew, (1, 2, 3) Amy J. Warren, (1) Aaron Barchowsky, (1,2,3) Kaili A. Temple, (1, 2) Linda Klei, (1) Nicole V. Soucy, (1) Kimberley A. O'Hara, (1) and Joshua W. Hamilton (1, 2, 3)

(1) Department of Pharmacology and Toxicology, Dartmouth Medical School, and (2) Center for Environmental Health Sciences, Dartmouth College, Hanover, New Hampshire, USA; (3) Norris Cotton Cancer Center, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire, USA