Impact of nose-only exposure system on pulmonary gene expression

Abstract

Nose-only exposure is used to study the distribution and toxicity of airborne contaminants. Restraint of animals in nose-only tubes causes stress, but the impact on pulmonary mRNA levels is unknown. Since stress and xenobiotics activate common pathways, we assessed whether nose-only exposure would alter expression of toxicologically relevant genes in the lungs. To identify candidate genes for further analysis, we first interrogated microarray data to examine time-dependent changes in gene expression in air-control animals from a nose-only inhalation study involving male wild-type C57BL/6 mice and transgenic tumor necrosis factor (TNF)-α over-expressing littermates. Comparison of transcript levels immediately and 24 h after a single 4-h nose-only exposure to air revealed differential expression of 280 genes (false discovery rate-adjusted, p < .05). Functional analysis revealed enrichment of immune response, apoptosis, and signalling terms, consistent with effects of restraint stress. We then selected a subset of target genes for comparison of naive animals and air-exposed animals from the inhalation study by real-time polymerase chain reaction (PCR). Expression of genes involved in stress (BNIP, sestrin-1, CDKN1A [p21], GADD45γ), glucocorticoid-response (GILZ, Sgk), and signal transduction (MAP3K6, C/EBP-δ) was increased as a result of nose-only exposure (p < .05). In contrast, proinflammatory factors (lymphotoxin-β, chemokine receptor CXCR5) were decreased (p < .05). Immune gene responses observed in wild-type animals were reduced in animals with lung inflammation, indicating that pathological states can modify the response to nose-only exposure. Observed responses may warrant consideration in the evaluation of materials delivered by nose-only inhalation, and suggest that incorporation of naive animals into nose-only studies should be considered as a best practice.

Keywords::

• Gene expression
• lungs
• microarray
• mRNA
• nose-only inhalation
• restraint stress

Acknowledgments

The authors are grateful to Marcelle Phaneuf for animal genotyping and project planning, Josée Guénette and DJ MacIntyre for conducting the inhalation exposures, Pat Goegan and Erica Blais for technical assistance, and Lynn Berndt for microarray advice.

Declaration of interest: The author reports no conflicts of interest. The author alone is responsible for the content and writing of the paper.