Gene Expression Changes in the Olfactory Bulb of Mice Induced by Exposure to Diesel Exhaust Are Dependent on Animal Rearing Environment

<div><p>There is an emerging concern that particulate air pollution increases the risk of cranial nerve disease onset. Small nanoparticles, mainly derived from diesel exhaust particles reach the olfactory bulb by their nasal depositions. It has been reported that diesel exhaust inhalation causes inflammation of the olfactory bulb and other brain regions. However, these toxicological studies have not evaluated animal rearing environment. We hypothesized that rearing environment can change mice phenotypes and thus might alter toxicological study results. In this study, we exposed mice to diesel exhaust inhalation at 90 µg/m³, 8 hours/day, for 28 consecutive days after rearing in a standard cage or environmental enrichment conditions. Microarray analysis found that expression levels of 112 genes were changed by diesel exhaust inhalation. Functional analysis using Gene Ontology revealed that the dysregulated genes were involved in inflammation and immune response. This result was supported by pathway analysis. Quantitative RT-PCR analysis confirmed 10 genes. Interestingly, background gene expression of the olfactory bulb of mice reared in a standard cage environment was changed by diesel exhaust inhalation, whereas there was no significant effect of diesel exhaust exposure on gene expression levels of mice reared with environmental enrichment. The results indicate for the first time that the effect of diesel exhaust exposure on gene expression of the olfactory bulb was influenced by rearing environment. Rearing environment, such as environmental enrichment, may be an important contributive factor to causation in evaluating still undefined toxic environmental substances such as diesel exhaust.</p></div

Characterization of diesel exhaust.

<p>(A) Particle diameter distribution of diesel exhaust particles. (B) Concentrations of gaseous components.</p

Confirmation of microarray data in cluster C by RT-PCR.

<p>Data show mRNA expressions for (A) <i>Nr1i3</i>, and (B) <i>Sdad1</i> in the olfactory bulb of mice in each group. <i>Nr1i3</i> and <i>Sdad1</i> of the olfactory bulb of mice reared in a standard cage environment were significantly downregulated by exposure to diesel exhaust. There was no significant effect of exposure to diesel exhaust on gene expressions of mice reared in environmental enrichment. The data are expressed as relative target gene expression compared with <i>Gapdh</i> expression. Each column represents the mean ± standard deviation (C-C: n = 7, C-DE: n = 7, EE-C: n = 8, EE-DE: n = 9). Data were analyzed by two-way analysis of variance as described in the Methods section. An analysis of simple effects is as follows: <i>Nr1i3</i>: a indicates significant differences (Tukey–Kramer method, ^aaP<0.01, C-C vs. C-DE); b indicates significant differences (Tukey–Kramer method, ^bbP<0.01, C-DE vs. EE-C); and <i>Sdad1</i>: a indicates significant differences (Tukey–Kramer method, ^aP<0.05, C-C vs. C-DE and C-C vs. EE-DE).</p

Functional analysis of cluster C using Gene Ontology (GO).

<p>The results of the gene annotation of cluster C using GO identified gene sets correlated with inflammatory and immune systems.</p

Hierarchical clustering of gene expression data.

<p>Within each group, a fold change (C-DE/C-C, EE-DE/EE-C, and EE-DE/C-C) was calculated and log₂ transformed for all 116 spots on the microarray that did not have any missing values. These values were then hierarchically clustered using Euclidean distance metric and complete linkage. The colored images are presented as described: the color scale ranges from saturated green for log₂ ratios –3.0 and below to saturated red for log₂ ratios 3.0 and above. Gene expression profiles were divided into 3 clusters (clusters A, B, and C), and quantitative RT-PCR analysis was performed for genes surrounded by the red line in each cluster. Genes surrounded by black dotted line were the same gene derived from different spots.</p

Schematic diagram of design of the present study.

<p>Schematic diagram of design of the present study.</p

Confirmation of microarray data in cluster B by RT-PCR.

<p>Data show mRNA expressions for (A) <i>Cyp2f2</i>, (B) <i>Aqp3</i>, (C) <i>Mslnl</i>, (D) <i>Krt18</i>, and (E) <i>Umodl1</i> in the olfactory bulb of mice in each group. <i>Cyp2f2</i>, <i>Aqp3</i>, <i>Mslnl</i>, <i>Krt18</i>, and <i>Umodl1</i> of mice reared in a standard cage environment were significantly downregulated by exposure to diesel exhaust. There was no significant effect of exposure to diesel exhaust on gene expression of mice reared in environmental enrichment. The data are expressed as relative target gene expression compared with <i>Gapdh</i> expression. Each column represents the mean ± standard deviation (C-C: n = 7, C-DE: n = 7, EE-C: n = 8, EE-DE: n = 9). Data were analyzed by two-way analysis of variance as described in the Methods section. An analysis of simple effects is as follows: <i>Cyp2f2</i>: a indicates significant differences (Tukey-Kramer method, ^aP<0.05, C-C vs. C-DE); b indicates significant differences (Tukey–Kramer method, ^bbP<0.01, C-DE vs. EE-DE); <i>Aqp3</i>: a indicates significant differences (Tukey–Kramer method, ^aP<0.05, C-C vs. C-DE); b indicates significant differences (Tukey-Kramer method, ^bP<0.05, C-DE vs. EE-DE); <i>Mslnl</i>: a indicates significant differences (Tukey–Kramer method, ^aP<0.05, C-C vs. C-DE); b indicates significant differences (Tukey–Kramer method, ^bbP<0.01, C-DE vs. EE-DE); <i>Krt18</i>: a indicates significant differences (Tukey–Kramer method, ^aP<0.05, C-C vs. C-DE); b indicates significant differences (Tukey-Kramer method, ^bP<0.05, C-DE vs. EE-C); c indicates significant differences (Tukey–Kramer method, ^ccP<0.01, C-DE vs. EE-DE); <i>Umodl1</i>: a indicates significant differences (Tukey–Kramer method, ^aP<0.05, C-C vs. C-DE); and b indicates significant differences (Tukey–Kramer method, ^bbP<0.01, C-DE vs. EE-DE).</p