ToxSci Advance Access originally published online on August 6, 2007
Toxicological Sciences 2007 100(1):75-87; doi:10.1093/toxsci/kfm200
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Exposure to Arsenic at Levels Found in U.S. Drinking Water Modifies Expression in the Mouse Lung
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* Department of Community and Family Medicine, Dartmouth Medical School, Lebanon, New Hampshire 03756
Norris Cotton Cancer Center, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire 03756
Center for Environmental Health Sciences, Dartmouth Medical School, Hanover, New Hampshire 03755
Thayer School of Engineering/Computer Sciences Department, Dartmouth College, Hanover, New Hampshire 03755
¶ Health Informatics Department, Federal University of Sao Paulo/Escola Paulista de Medicina-UNIFESP/EPM, Sao Paulo, SP, Brazil
|| Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190
||| Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755
1 To whom correspondence should be addressed at Dartmouth Medical School, 7927 Rubin 860, One Medical Center Drive, Lebanon, NH 03756. Fax: (603) 653-9093. E-mail: angeline.andrew{at}dartmouth.edu.
Received April 6, 2007; accepted July 30, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGSUPPLEMENTARY DATAREFERENCES |
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The mechanisms of action of drinking water arsenic in the lung and the threshold for biologic effects remain controversial. Our study utilizes Affymetrix 22,690 transcript oligonucleotide microarrays to assess the long-term effects of increasing doses of drinking water arsenic on expression levels in the mouse lung. Mice were exposed at levels commonly found in contaminated drinking water wells in the United States (0, 0.1, 1 ppb), as well as the 50 ppb former maximum contaminant level, for 5 weeks. The expression profiles revealed modification of a number of important signaling pathways, many with corroborating evidence of arsenic responsiveness. We observed statistically significant expression changes for transcripts involved in angiogenesis, lipid metabolism, oxygen transport, apoptosis, cell cycle, and immune response. Validation by reverse transcription–PCR and immunoblot assays confirmed expression changes for a subset of transcripts. These data identify arsenic-modified signaling pathways that will help guide investigations into mechanisms of arsenic's health effects and clarify the threshold for biologic effects and potential disease risk.
Key Words: arsenic; apoptosis; cell cycle; drinking water; immune response; lung; microarray; oxygen transport.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGSUPPLEMENTARY DATAREFERENCES |
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Drinking water arsenic exposure is an established human health risk associated with cardiovascular disease, diabetes, and cancer at multiple organ sites including the lung, bladder, and skin (IARC, 2004
). Arsenic exposure has been associated with impaired lung function and bronchiectasis (De et al., 2004; Smith et al., 2006). In an Indian study, arsenic-associated skin lesions were independently associated with increased risk of respiratory illness (odds ratio 4.9; 95% confidence interval 3.2–7.5) compared to individuals with no skin lesions (Ghosh et al., 2007). Arsenic has also been found to synergize with cigarette smoking and other risk factors to induce mutations, DNA adducts, and cancer risk (Ahsan and Thomas, 2004; Chen et al., 2004; Evans et al., 2004; Rossman et al., 2002). The mechanisms of action and the threshold for biologic effects and disease risk, however, remain controversial (Kitchin, 2001).
Many of the epidemiologic studies and in vivo / in vitro experiments have been conducted at high doses with acute exposure. Thus far, microarray studies have explored arsenic-induced gene expression changes in cell culture models as well as several animal organs; however, the effects in the adult lung have not been reported (Durham and Snow, 2006; Shi et al., 2004). In contrast to a strengthening of similar effects with increasing dose that is observed with many compounds, arsenic may activate completely different pathways at low versus high doses (Andrew et al., 2003; Barchowsky et al., 1999; Lau et al., 2004; Soucy et al., 2003). Relatively little is known about the effects of arsenic at levels of exposure that are common to drinking water in the United States, particularly in the lung.
Our study utilizes oligonucleotide microarrays to assess the long-term effects of levels of drinking water arsenic commonly found in contaminated wells in the United States, as well as the 50 ppb former standard (0, 0.1, 1, and 50 ppb) on expression levels in the mouse lung. We present a functional approach to microarray analysis in which we focus on statistically significant expression modifications and investigate both the biologic function and how each protein regulates another using updated bioinformatics analysis tools.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGSUPPLEMENTARY DATAREFERENCES |
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Four groups of adult (7–8 weeks old) male C57/BL6 mice were exposed to increasing concentrations of inorganic arsenic (n = 4 at 0.1 µg/l [ppb], n = 3 at 1 µg/l [ppb], and n = 3 at 50 µg/l [ppb]) (sodium arsenite; LabTech, Pittsburgh, PA) in their drinking water for a period of 5 weeks. A control group of mice (n = 3) consumed uncontaminated drinking water. Lungs from additional groups of mice exposed to 0 or 10 µg/l (ppb) were used in the reverse transcription (RT)–PCR and immunoblot analyses. All mice were fed AIN-76A arsenic-free chow (Harlan-Teklad, Madison, WI) to control for other dietary sources of arsenic (total arsenic < 5 ppb). Levels of arsenic in food and water were confirmed by Inductively Coupled Plasma Mass Spectrometry analysis by the Dartmouth Trace Metal Facility. Lungs were removed and immediately placed in RNAlater to stabilize the RNA levels after sacrificing the animals. Exposed and unexposed animals were sacrificed at the same time of day. All protocols were approved by the University of Oklahoma Institutional Animal Care and Use Committee.
RNA was isolated using Qiagen RNeasy columns (Qiagen Inc., Valencia, CA), and DNAse treatment was performed using Ambion DNAfree reagents (Austin, TX) according to the manufacturer's instructions. The expression profiles were generated using the Affymetrix GeneChip Technology—chip GeneChip Murine Genome 430 oligonucleotide arrays (Affymetrix, Santa Clara, CA) which simultaneously tested 22,690 transcripts using one chip for each mouse on the integrated GeneChip Instrument System in the Dartmouth Microarray Core Facility. Our experiment was performed in compliance with the Minimum Information About a Microarray Experiment checklist for standardization guidelines for microarray experiments.
The raw microarray data were preprocessed and normalized using GeneTraffic version 3.2 which is a microarray data management and analysis client-server application (Stratagene, La Jolla, CA). The control (0 µg/l) group was set as the baseline, and data were normalized using Robust Multi-Chip Analysis (Irizarry et al., 2003).
The statistical significance of expression changes relative to the control group was assessed using methods based on modified t- or F-tests that adjust for multiple comparisons. This adjustment bounds a false discovery rate probability (FDR), i.e., chance that a transcript regarded as significant is a false positive, to select the transcripts (Benjamini and Hochberg, 1995). This method is implemented in the Significance Analysis of Microarrays (SAM) application version 1.13 (Tusher et al., 2001) implemented in The Institute of Genomic Research (TIGR) MultiExperiment Viewer (TIGR MeV) version 3.1 (TIGR, Rockville, MD). We performed SAM multiclass and two-class comparisons using 1000 permutations and selected significant transcripts at a FDR of 5%. We focused our attention on the group of differentially expressed transcripts that were called significant by the two-class SAM or multiclass SAM (dose response) analysis (provided as Supplementary Data). The transcripts that were significant by multiclass analysis or were modified at a minimum of two doses of arsenic are shown in Table 1. Transcripts are grouped by the lowest arsenic dose with statistically significant transcript expression modification compared with control. To identify subgroups of transcripts with similar patterns of expression among the statistically significant group, the multiclass selected transcripts were clustered by TIGR MeV with a hierarchical clustering with the complete linkage algorithm and Pearson correlation metric (Fig. 1).
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We selected transcripts for validation by real-time PCR using independent primer sets based on the microarray results and hypothesized involvement in arsenic pathogenesis in the lung (Fig. 2). Taqman primer–probe sets for each selected transcript were obtained from Applied Biosystems Inc. (ABI, Foster City, CA). Real-time RT–PCR was performed using the ABI PRISM sequence detection system and software. Briefly, total RNA (0.5 µg) was reverse transcribed using 100 U Moloney Murine Leukemia Virus reverse transcriptase in a mixture with oligo-dT and dNTPs according to the instructions provided with the Qiagen Omniscript kit (Qiagen Inc.). Samples were reverse transcribed in a MJ Research PTC-100 thermocycler (MJ Research Inc., Watertown, MA) for 60 min at 44°C and the reaction terminated by heating to 95°C for 10 min. Expression of specific genes was assessed by real-time PCR using 10 ng total RNA, 400nM primers, 200nM probe, and TaqMan Universal PCR Master Mix (ABI). Relative quantitation was performed using a standard curve consisting of serial dilutions of pooled sample cDNA from the same source as the test RNA with each plate. Relative expression levels of each gene were normalized to 18s rRNA. Statistical significance was assessed by one-way ANOVA with Newman-Keuls post-test using GraphPad Prism software (San Diego, CA).
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We further characterized the functional effects of all the statistically significant arsenic-modified transcripts (includes all of the SAM two-class selected transcripts) by implementing the Database for Annotation, Visualization, and Integrated Discovery (DAVID) gene ontology search engine (Table 2) (Dennis et al., 2003
). This bioinformatic tool identifies functional processes that are significantly overrepresented (p < 0.05) by the modified transcripts. Table 3 lists the biologic roles of all the significant arsenic-modified transcripts (all those selected by SAM multiclass or two-class analysis at any exposure) from the Pathway Studio ResNet 4.0 database (updated April 2007) (Ariadne Genomics, Rockville, MD). This Pathway Studio ResNet database catalogs relationships between biologic entities based on the published literature and was also used to identify the direct interactions between the arsenic-modified transcripts at each arsenic dose (Fig. 3 and Supplementary Data).
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Histologic slices of formalin-fixed, paraffin-embedded lung tissue from arsenic-exposed and unexposed animals were stained with hematoxylin and eosin and evaluated by a trained pathologist to assess the number of neutrophils (Supplementary Data).
The levels of Nr4a1, Hsp70, Ahr, and Cyclin D1 protein were assessed by immunoblotting using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) to resolve proteins from mouse lung tissue (Fig. 4). Frozen mouse lung was weighed and homogenized with EBC lysis buffer (50mM Tris, pH 8.0, 100mM NaCl) containing 10 µl/ml PMSF, 5 µl/ml aprotinin, and 1 µl/ml leupeptin. NP-40 Lysis buffer (10%) was added at a 5% vol/vol ratio. After centrifugation for 15 min at 14,000 rpm at 4°C the lysates were boiled for 5 min and clarified by centrifugation at 13,000 rpm for 10 min. Equal amounts of cell lysates were resolved by electrophoresis on 7.5% and 10–20% SDS–polyacrylamide gels. Electrophoresis was performed at constant voltage (200 V), then the resolved proteins were transferred from the polyacrylamide gel to polyvinylidene difluoride membrane (PVDF, Immobilon-P; Millipore, Bedford, MA) by semi-dry transfer (Hoeffer Semiphor, San Fransisco, CA) for 1 h at constant current (100 mA) using transfer buffer (25mM Tris, 192mM glycine, 20% (vol/vol) methanol, 0.01% SDS). To eliminate nonspecific interactions of antibodies with the membrane, the PVDF membrane was blocked with Tris-Tween Buffered Saline (TTBS) (10mM Tris–HCl, pH 8.0, 150mM NaCl, 0.05% Tween-20) containing 5% milk (7.5 g/150 ml) for 1 h at room temperature. Membranes were incubated with the primary antibody: anti-Nur77/Nr4a1 (Pharmingen, San Diego, CA), Hsp70/Hspa1b (Transduction Laboratories, Lexington, KY) diluted 1:1000, anti-Ahr (BIOMOL, Plymouth Meeting, PA) diluted 1:5000, or Cyclin D1 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) diluted 1:500 in TTBS overnight at 4°C. Actin was used as a loading control, and the antibody was diluted 1:50,000 in TTBS for 1 h (Calbiochem, San Diego, CA). The membranes were washed 3 times with TTBS. The Nur77/Nr4a1 and Hsp70 membranes were incubated with horseradish peroxidase (HRP)–linked goat anti-rabbit IgG (Santa Cruz Biotechnology Inc.) 1:3000 in TTBS with 5% milk (1.5 g/30 ml) for 1 h at room temperature. The Ahr, Cyclin D1, and Actin membranes were incubated with HRP-linked goat anti-mouse (Bio-Rad Laboratories, Inc., Hercules, CA) 1:2000, 1:3000, and 1:2000, respectively, in TTBS with 5% milk (1.5 g/30 ml). After 3 washes with TTBS, protein bands were visualized by enhanced chemiluminescence using the Amersham ECL Plus Western Blotting Detection system (GE Healthcare, Piscataway, NJ) and film (Lumi-Film; Roche Molecular Biochemicals, Indianapolis, IN).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGSUPPLEMENTARY DATAREFERENCES |
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To address the controversy over the biological effects of arsenic at levels commonly found in U.S. drinking water, we assessed expression patterns associated with arsenic exposure in the mouse lung. To evaluate the statistical significance of these changes in expression between the treated and control animals, we started with the hypothesis that arsenic would have a dose-responsive effect on expression. In Figure 1, we hierarchically clustered transcripts that were selected by the multiclass SAM analysis (59 transcripts) and observed that the data broke into four main branches, indicating subgroups of transcripts (labeled Clusters 1–4). Visual inspection of cluster 1 indicates decreased levels of transcripts involved in controlling apoptosis, fatty acid metabolism, and chemokines at the 0.1 and 1 ppb doses, but not at 50 ppb. Cluster 2 shows increased levels of transcripts involved in embryonic limb morphogenesis, glucocorticoid signaling, and antiapoptosis, particularly at the 1 and 50 ppb doses. The immune response transcripts in cluster 4 are decreased mainly at the 50 ppb dose (Igj, Igh-VJ558, Igk-V28). We observed a consistent pattern of decreased expression at all arsenic doses in the largest group, cluster 3. These genes are involved in a variety of processes including signal transduction, hemoglobin activity, glycolysis, transcription, apoptosis, and glycosylation. Arsenic exposure at 1 ppb strongly decreased Alas2, Siat8d, Agtrl, and Ear1 and increased expression of Zfp145, Fkbp5, and Hspa1b.
Two-class SAM analysis of each arsenic exposure dose compared with control (0 ppb) revealed 94 statistically significantly modified transcripts for 0.1 versus control. We chose this lowest dose (0.1 ppb) as a low level that has been associated with adverse health effects in a Finnish study and is found in U.S. drinking water sources (Kurttio et al., 1999). We detected 26 modified transcripts for 1 ppb arsenic versus control and 37 modified transcripts for the 50 ppb arsenic versus control analysis (FDR < 0.05). Table 1 lists the subset of these transcripts that were selected as statistically significant by the multiclass SAM or by two-class SAM analyses at a minimum of two arsenic doses, organized by the lowest dose with significant expression modification (all significantly modified transcripts are provided in the Supplementary Table). The direction of expression modification for each transcript that was significantly modified is marked with a "–" (indicating decreased expression) or a "+" (indicating increased expression). The ratio of each treated group versus control and the number of modified probe sets are shown in the adjoining columns. The modified transcripts were involved functional processes including transcription factors, immune response, oxygen transport, cell cycle, oxidative stress, or fatty acid metabolism.
While many transcripts showed consistent responses at all doses of arsenic, several were most dramatically decreased at the 0.1 ppb dose, including Angptl4 and Cxcl7 (Table 1, Down 0.1 ppb). Others such as Ear1 and Ear2 were decreased strongly at the 0.1 and 1 ppb doses, but not at 50 ppb (Table 1, Down 0.1, 1 ppb). A few of the transcripts had a differential response to the 0.1 ppb compared to 1 or 50 ppb arsenic (Table 1, Mixed multiclass only). Specifically, Egr1 levels were increased at 0.1 ppb, but decreased at the higher doses. In contrast, Hsp70 and Zfp145 levels were increased only above 1 ppb. In Table 1, the "Down 1, 50 ppb" section shows transcripts that were most strongly decreased at the higher doses, including Coro1a, Dbp, Plk2, and Ptprc. Likewise, the immune response transcripts (Igh-VJ558, Igj, Igk, Igk-V28, Ltb) were decreased almost exclusively with exposure to 50 ppb arsenic (Table 1, Down 50 ppb).
Since the number of modifications that we could validate by RT–PCR was limited by the quantity of sample remaining, we chose to validate changes in genes with known biologic functions that we hypothesized could be involved in arsenic pathogenesis in the lung. These functions included oxygen transport and control of cell proliferation/apoptosis. The validation graphs in Figure 2 show that for most of the transcripts, the expression profile derived from the array (blue line) closely mirrors the expression observed by real-time PCR (black line). For example, the decrease in aminolevulinic acid synthase 2 (Alas2) expression validated by real-time PCR was observed at all arsenic doses (Fig. 2). Likewise, decreases in expression of the oxygen transporter, hemoglobin alpha, adult chain 1 (Hba-a1) were also validated by real-time PCR.
We further characterized the functional processes represented by the transcripts that were significantly modified by arsenic treatment by implementing the DAVID search engine. We identified biologic functional processes that are overrepresented by the significantly modified genes from the two-class analysis for each arsenic dose. Expression of transcripts involved in response to abiotic stimulus was decreased at the lower doses (Table 2, 0.1 ppb row 6, 1 ppb row 3) and increased at 50 ppb (row 5). The 0.1 and 1 ppb doses both decreased expression of hydrolases (Table 2, 0.1 ppb row 3, 1 ppb row 1). Apoptosis and immune response were notable categories modified by the 50 ppb dose, with potential implications for disease risk (Table 2, 50 ppb rows 1, 3, 4). Analysis of the biologic roles of all statistically significantly modified transcripts (Table 3) indicated that arsenic modified a number of transcripts that encode receptors (row 1), ligands (row 3), metabolic enzymes (row 11), transcription factors (row 13), and transporters (row 16).
The pathway map in Figure 3 was generated by Pathway Studio and illustrates direct interactions between arsenic-modified transcripts by each dose compared with control and identifies common regulators. For example, Figure 3 shows that arsenic decreases expression of the Cyclin D1/CCND1 regulator HBP1 at all three doses. This pathway map also shows common regulators, for example, PPARA modifies the expression of three arsenic responsive genes—NR1D1, CPT1A, and Cd36. Other central nodes include MAPK1, SP1, and TP53.
Histologic slices of formalin-fixed, paraffin-embedded lung tissue from the mice were examined to look for structural differences between the arsenic unexposed versus exposed lungs (Supplementary Data). Pathologic examination did not reveal any visible abnormalities in the structure of the lungs. The pathologist did not detect differences between lung samples in the types of inflammatory cells present. Particularly, she did not observe any infiltration of neutrophils with arsenic exposure, indicating that the expression modification occurred within a static set of cells.
Lastly, we used immunoblot analysis to assess the protein levels associated with several arsenic-modified transcripts. As shown in Figure 4, mice exposed to arsenic had increased levels of Nr4a1/Nur77 and Hsp70 compared with unexposed animals, while Ahr and cyclin D1 levels were reduced in arsenic-exposed animals.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGSUPPLEMENTARY DATAREFERENCES |
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Drinking water arsenic exposure is associated with increased risk of lung diseases, particularly at levels exceeding 100 ppb found internationally (Smith and Smith, 2004
). The effects of lower doses and the biologic mechanisms remain unclear. In particular, few in vivo studies have examined the biologic effects of drinking water arsenic at levels 50 ppb and below that are highly relevant to U.S. drinking water levels. To address the need for data, our study evaluated differential expression patterns in the lungs of mice exposed to drinking water arsenic (0.1, 1, 50 ppb) for a period of 5 weeks using microarrays. The expression profiles revealed modification of a number of important signaling pathways, many with corroborating evidence of arsenic responsiveness. Many of the modified transcripts showed consistently decreased expression in arsenic exposed animals, while the few increases were strongest at the higher doses. The immune response transcripts showed clear evidence of a dose-responsive decrease at 1 and 50 ppb, but were unaffected, or even increased, at the 0.1 ppb dose. We also observed effects on several transcripts (e.g., S100a9, Ms4a8a, Angptl4, Cxcl7, Ear1, Ear2) that appear to be strongest at the lower doses of arsenic. In some cases, the responses even suggest a biphasic effect that could be interpreted as being beneficial at 0.1 ppb and adverse at higher levels (e.g., Egr1, Hsp70).
We observed significant modification of pathways that involve the central regulators MAPK1, CCND1, SP1, and TP53 (Fig. 3). Previous studies have reported that arsenic activates MAPK1/ERK signaling in lung cells (Samet et al., 1998). MAPK1/ERK regulates Egr1 expression in response to a number of stimuli (Guha et al., 2001). As observed previously in the bladder, arsenic had dose-dependent effects on Egr1 (increased at 0.1 ppb, decreased at 1 and 50 ppb), possibly via the epidermal growth factor receptor, MAPK1/ERK, and SP1 signaling pathways (Adnane et al., 1999; Luster and Simeonova, 2004; Simeonova et al., 2000). We observed the same effect of arsenic on Egr1 expression in vitro by RT–PCR following treatment of cultured epithelial cells (data not shown). Egr1 is a zinc finger transcription factor that regulates many processes, including differentiation, tumor suppression, cell cycle arrest, and apoptosis, and its expression level is related to lung cancer survival (Ferraro et al., 2005).
MAPK1/ERK signaling also plays a role in regulation of the cell cycle control protein cyclin D1 (CCND1) (Du et al., 2006). The decreased expression of the transcriptional repressor HBP1 that we observed with arsenic is consistent with previous reports that arsenic alters cyclin D1 levels (Sampson et al., 2001; (Vogt and Rossman, 2001). Arsenic exposure increased Hspa8/Hsc70, which binds to cyclin D1, as part of the catalytically active cyclin D1-cdk4 complex (Diehl et al., 2003). Although we did not observe statistically significant changes in CCND1 transcript levels by microarray, our immunoblot results show decreased cyclin D1 protein levels in the lungs of arsenic-exposed animals.
The decreased expression of transcripts involved in lipid metabolic processes was strongest in the animals exposed to the lowest dose, 0.1 ppb. In addition to other roles, Angptl4 inhibits lipoprotein lipase, an enzyme that regulates triglyceride clearance and lipid homeostasis (Koster et al., 2005). This low dose of arsenic was also associated with decreased Acyl-CoA thioesterases (Cte1), enzymes that regulate the levels of free fatty acid and coenzyme A by catalyzing the hydrolysis of acyl-CoAs (Hunt et al., 2006). Likewise, expression of carnitine palmitoyltransferase 1A (Cpt1a), the enzyme that catalyzes the primary rate controlling step of fatty acid oxidation, was decreased in arsenic-exposed animals (Cook and Park, 1999). Changes in the oxidative modification of lipoproteins may be related to the pathogenesis of peripheral artery disease (Steinberg et al., 1989).
Arsenic-exposed animals had decreased expression of a number of transcripts involved in oxygen transport (Alas2, Hbb-b1, Hba-a1, Bpgm). The effects on multiple hemoglobin complex and oxygen transporter transcripts were particularly notable at the lower doses. Alas2 catalyzes the first step in the heme biosynthetic pathway (Kramer et al., 2000). The hemoglobin complex members Hbb-b1 and Hba-a1 actually bind and transport oxygen. Bpgm is an enzyme that controls the levels of an allosteric effector of hemoglobin and the dissociation of oxygen (Garel et al., 1990).
We also observed differential expression of several apoptotic transcripts on the microarray, including decreases in Ahr, Stk17B, Nr4a1, Egr1, and Angptl4 and increases in Hspa1b/Hsp70 and ZFP145/ZBTB16 (above 1 ppb). Nr4a1 is a nuclear orphan steroid receptor that induces apoptosis (Rajpal et al., 2003). Interestingly, the protein levels of Nr4a1 were higher in the arsenic-exposed mice, indicating a possible role for posttranslational modification. We also observed increased levels of Hspa1b/Hsp70 with arsenic exposure levels 1 ppb and higher, as observed previously in the lymphocytes of arsenic-exposed individuals in Bangladesh (Argos et al., 2006). Immunoblot analysis confirmed increased protein levels of Hsp70 with arsenic exposure. Hsp70 is induced in response to stress and is important for DNA repair and maintaining genomic stability (Hunt et al., 2004).
The most dramatic effect of arsenic exposure in this experiment was a decrease in transcripts involved in immune response, including Igh-VJ558, Igj, Igk-V28, Igk-V8, Cd79b, Cxcl7, Ian6, s100a9, and Ahr (see Fig. 1, cluster 4). Igh-VJ558 is involved in B-cell antigen recognition and Igj, Igk-V28, and Igk-V8 in humoral immune response and antigen binding. Decreases in these transcripts were evident mainly at 50 ppb (Table 1, Down 50 ppb). Expression of the precursor plasma chemokine Cxcl7 that may affect tumor development by attracting immunocompetent cells was also decreased in the arsenic-exposed groups, particularly at 0.1 and 1 ppb (Van Damme et al., 2004). Similarly, the chemokines Cxcl2, Ccl3, Ccrl2, Cxcl3, Ccl4, and Ccl20 were decreased in the lymphocytes of arsenic-exposed individuals from Bangladesh (Argos et al., 2006). We also observed decreased expression of Calgranulin B/s100a9 with arsenic exposure, particularly at the lowest dose, 0.1 ppb. Calgranulin B is involved in the inflammatory response to stimuli, including the release of neutrophils and is regulated by Ahr signaling (Temchura et al., 2005). Ahr, which was also decreased at the transcript and protein level in arsenic-exposed mice, is involved in the generation of regulatory T cells (Funatake et al., 2005). SP1 cooperatively regulates expression of AHR, which modifies cell cycle progression. The direction of this effect is dependent on the presence or absence of exogenous ligands, such as Polycyclic Aromatic Hydrocarbons PAHs (Marlowe and Puga, 2005; Wang et al., 1999).
We also observed differential expression of several a priori hypothesized transcripts based on previous work. Induction of metallothionein by other toxic metals including cadmium, mercury, and copper is well documented; however, its response to arsenic is less clear. Consistent with previous reports in other organs and cells, the classic metal ion binding transcripts Mt1 and Mt2 were induced by low doses of drinking water arsenic exposure in the mouse lung. In utero exposure to arsenic also induces metallothionein levels in the lung (Shen et al., 2007). Mt knockout studies suggest that metallothionein protects mice against arsenic-induced toxicity (Liu et al., 2000; Zheng et al., 2003).
In summary, this study demonstrates that exposure to drinking water arsenic at levels commonly found in U.S. drinking water is associated with altered expression profiles in the mouse lung. Pathways involved in immune response, angiogenesis, lipid metabolism, and oxygen transport were particularly affected. With further validation, some of these genes may be useful as intermediate biomarkers in studies investigating arsenic-associated diseases. Future investigation of the effects of arsenic on the function of these pathways is warranted. In particular, little is known about the responses and health effects of arsenic exposure at doses below 0.1 ppb, and the possibility that arsenic may be an essential element at some very low trace level remains to be explored. These data identify pathways that will help guide investigations into mechanisms of arsenic's health effects and help clarify the threshold for biologic effects and potential disease risk.
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FUNDING |
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The National Cancer Institute, National Institutes of Health (NIH); the National Institute of Environmental Health Sciences, NIH; the National Center for Research Resources (NCRR), NIH (grant numbers CA099500, CA102327, P42 ES007373, P20RR018787).
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SUPPLEMENTARY DATA |
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Supplementary Table and other data are available online at http://toxsci.oxfordjournals.org/.
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ACKNOWLEDGMENTS |
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Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS or NIH. The authors do not have any competing financial interests.
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