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. 2009 Jul;117(7):1108-15.
doi: 10.1289/ehp.0800199. Epub 2009 Mar 4.

Chronic exposure to arsenic in the drinking water alters the expression of immune response genes in mouse lung

Courtney D Kozul  1 , Thomas H Hampton, Jennifer C Davey, Julie A Gosse, Athena P Nomikos, Phillip L Eisenhauer, Daniel J Weiss, Jessica E Thorpe, Michael A Ihnat, Joshua W Hamilton
Affiliations

Chronic exposure to arsenic in the drinking water alters the expression of immune response genes in mouse lung

Courtney D Kozul et al. Environ Health Perspect. 2009 Jul.

Abstract

Background: Chronic exposure to drinking water arsenic is a significant worldwide environmental health concern. Exposure to As is associated with an increased risk of lung disease, which may make it a unique toxicant, because lung toxicity is usually associated with inhalation rather than ingestion.

Objectives: The goal of this study was to examine mRNA and protein expression changes in the lungs of mice exposed chronically to environmentally relevant concentrations of As in the food or drinking water, specifically examining the hypothesis that As may preferentially affect gene and protein expression related to immune function as part of its mechanism of toxicant action.

Methods: C57BL/6J mice fed a casein-based AIN-76A defined diet were exposed to 10 or 100 ppb As in drinking water or food for 5-6 weeks.

Results: Whole genome transcriptome profiling of animal lungs revealed significant alterations in the expression of many genes with functions in cell adhesion and migration, channels, receptors, differentiation and proliferation, and, most strikingly, aspects of the innate immune response. Confirmation of mRNA and protein expression changes in key genes of this response revealed that genes for interleukin 1beta, interleukin 1 receptor, a number of toll-like receptors, and several cytokines and cytokine receptors were significantly altered in the lungs of As-exposed mice.

Conclusions: These findings indicate that chronic low-dose As exposure at the current U.S. drinking-water standard can elicit effects on the regulation of innate immunity, which may contribute to altered disease risk, particularly in lung.

Keywords: arsenic; inflammation; innate immune system; lung; migration.

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Figures

Figure 1
Figure 1
Chronic As exposure at the current drinking water standard alters gene expression in mouse lung: intensity-dependent data from the microarray analysis of mice exposed to 10 ppb As (A) or 100 ppb As (B) in drinking water compared with control. Genes identified by rank-product analysis to be significantly up-regulated by As exposure are highlighted in red, and genes identified to be significantly down-regulated are highlighted in green.
Figure 2
Figure 2
IPA of gene expression changes induced by As exposure indicates similar networks for both the 10-ppb exposure (A) and 100-ppb exposure (B). The top functions of these networks are cellular movement, hematologic system development and function, and immune response. Gene symbols are located in the center of each box; green shapes indicate down-regulated genes, and red shapes indicate up-regulated genes, compared with control. Nonshaded shapes indicate genes that IPA inserted to obtain the most connections within the network, but these genes were not regulated by the experimental treatment. See Supplemental Material, Table 4 (http://www.ehponline.org/members/2009/0800199/suppl.pdf) for legend to symbols.
Figure 3
Figure 3
Genomewide (A) and TLR/IL-1 pathway (B) transcriptome microarray analysis of immune response genes. DW, drinking water. Each column represents a separate animal, grouped manually by treatment; genes are clustered by relational analysis using standard Pearson correlation (see “Materials and Methods”). Individual Affymetrix probes that were differentially expressed are represented across each row. Green boxes indicate genes that were significantly down-regulated, and red boxes indicate genes that were up-regulated, relative to control. (A) Heat map of clustered, differentially regulated probes. The ANOVA function of R was used to generate a list of the differentially regulated genes. A comprehensive list of genes known to have a function in the immune response was intersected with a list of the top 5% of genes identified by ANOVA. (B) Heat map of raw RMA normalized probe data based in literature references used to create a list of genes known to play a role in the TLR/IL1R signaling pathway.
Figure 4
Figure 4
Genomewide transcriptome microarray analysis of cellular migration genes: heat map of clustered, differentially regulated probes. DW, drinking water. Genomewide transcriptome microarrays were run and treated as described in Figure 3. The ANOVA function of R was used to create a list of the differentially regulated genes. A comprehensive list of genes known to have a function in the cellular migration was created using Affymetrix NETAFFX. This list was intersected with a list of the top 20% of genes identified by ANOVA.
Figure 5
Figure 5
PCR confirmation of genes identified by microarray analysis to be altered by As exposure: transcript levels for Adam10 (A), Scn3b (B), Il1b (C), and Il1r2 (D) determined by quantitative real-time RT-PCR. Relative mRNA levels are expressed as arbitrary units, normalized to the data from control animals. Each bar represents mean + SEM of relative values from 4–6 animals per treatment. *p < 0.05 versus control (one-tailed Student’s t-test).
Figure 6
Figure 6
Low-dose As alters protein levels of IL1b and TNF-α in whole lung homogenate: densitometric quantification from all animals from two separate experimental repeats (n = 6 per experimental repeat; n = 12 total) in each group for levels of IL1b (A) and TNF-α (B) protein (representative Western blot results for three mice from each treatment group). (C) Data from two representative Ponceau bands averaged and used for normalization. Error bars represent mean + SEM of values for 12 mice per treatment. *p < 0.05, **p < 0.01, versus control, two-tailed Student’s t-test.
Figure 7
Figure 7
Low-dose As alters the protein levels of TLR and IL1R signaling pathway mediators: densitometric quantification from all animals from two separate experimental repeats (n = 6 per experimental repeat; n = 12 total) in each group for Myd88 (A), Traf6 (B), and IκBα (C) protein levels (representative Western blot results for three mice from each treatment group). (D) Data from two representative Ponceau bands were averaged and used for normalization. Error bars represent mean + SEM of values for 12 mice per treatment. *p < 0.05, **p < 0.01, *** p < 0.001, versus control, two-tailed Student’s t-test.
Figure 8
Figure 8
Low-dose As exposure increases lymphocytes in BALF. (A) Total cells recovered from the lungs of control and As-exposed animals by bronchoalveolar lavage. Cell differentials were determined by cellular morphology from 10 random fields after staining of cytospin slides. Macrophages (B), neutrophils (C), and lymphocytes (D) were identified (represented as a percentage of total cells). Error bars represent mean + SEM of values for five to six mice per treatment. *p < 0.05, **p < 0.01, versus control, two-tailed Student’s t-test.
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