Integrative Assessment of Chlorine-Induced Acute Lung Injury in Mice

Abstract

The genetic basis for the underlying individual susceptibility to chlorine-induced acute lung injury is unknown. To uncover the genetic basis and pathophysiological processes that could provide additional homeostatic capacities during lung injury, 40 inbred murine strains were exposed to chlorine, and haplotype association mapping was performed. The identified single-nucleotide polymorphism (SNP) associations were evaluated through transcriptomic and metabolomic profiling. Using > 10% allelic frequency and > 10% phenotype explained as threshold criteria, promoter SNPs that could eliminate putative transcriptional factor recognition sites in candidate genes were assessed by determining transcript levels through microarray and reverse real-time PCR during chlorine exposure. The mean survival time varied by approximately 5-fold among strains, and SNP associations were identified for 13 candidate genes on chromosomes 1, 4, 5, 9, and 15. Microarrays revealed several differentially enriched pathways, including protein transport (decreased more in the sensitive C57BLKS/J lung) and protein catabolic process (increased more in the resistant C57BL/10J lung). Lung metabolomic profiling revealed 95 of the 280 metabolites measured were altered by chlorine exposure, and included alanine, which decreased more in the C57BLKS/J than in the C57BL/10J strain, and glutamine, which increased more in the C57BL/10J than in the C57BLKS/J strain. Genetic associations from haplotype mapping were strengthened by an integrated assessment using transcriptomic and metabolomic profiling. The leading candidate genes associated with increased susceptibility to acute lung injury in mice included Klf4, Sema7a, Tns1, Aacs, and a gene that encodes an amino acid carrier, Slc38a4.

Functional Genomic Assessment of Phosgene-Induced Acute Lung Injury in Mice

“…In a process that consumes 40% of a cell's energy, Na,K-ATPase maintains the sodium-potassium electrochemical gradient across the plasma membrane by exporting three Na 1 and importing two K 1 for each ATP hydrolyzed (77). In previous metabolomics analyses of acrolein-and chlorine-induced lung injury, we observed that the lung undergoes energetic stress and that resistant mice appear better able to use substrates for energy production (33,34). The Na,K-ATPase-generated electrochemical gradient is essential for edema fluid absorption from the alveolus (1, 3).…”

“…The G allele is the minor allele (15.3% allelic frequency) and could result in the gain of a putative DNA binding site for transcription factor 12. Expressed in the lung (64), transcription factor 12 can repress E-cadherin (cadherin 1, type 1, E-cadherin [epithelial]) (65) and may modulate SMAD signaling (66), which is often active during ALI by transforming growth factor (TGF)b1 (32,34).…”

“…Recently, we identified semaphorin 7a (Sema7a) as a candidate gene for susceptibility to chlorine-induced ALI in mice (34). Expressed in pulmonary vasculature, PLXND1 can function as a semaphorin receptor (99,100), although its relationship to SEMA7A has not been evaluated.…”

“…Genome-wide association analysis was performed using efficient mixed-models association corrected for confounding from population structure and genetic relatedness (43,44). Previously, we determined that one statistical association by chance will occur in a genome-wide scan when the threshold is decreased to 1.6 3 10 25 or 2log (P) ¼ 4.8 (25,(32)(33)(34). Therefore, we used a significance threshold of -log (P) .…”

“…Recently, nextgeneration whole genome sequencing information has been obtained on 17 mouse strains (29), which has strengthened the existing single nucleotide polymorphism (SNP) database for over 40 mouse strains (29)(30)(31). Using a genetically diverse panel of >40 inbred mouse strains, we previously conducted functional genomic analyses of acrolein-induced (32,33) and chlorine-induced (34) ALI. In this study, we sought to compare these findings with an additional chemical, phosgene, also known to produce ALI after accidental or intentional terrorist exposures.…”