PMC

Histological assessment of lung tissue from (A) control C57BLKS/J mice, (B) control C57BL/10J mice, (C and E) chlorine-exposed C57BLKS/J mice, and (D and F) chlorine-exposed C57BL/10J mice. Consistent with acute lung injury, perivascular enlargement (C, arrows) and leukocyte infiltration (E) were more evident in the sensitive C57BLKS/J strain than in the resistant C57BL/10J strain (D and F). Female mice were exposed to filtered air (control) or chlorine (45 ppm, 12 hours) and anesthetized. Lung tissue was obtained and fixed in formaldehyde, and 5-μm sections were prepared with hematoxylin and eosin staining. Bars indicate magnification.

Chlorine-induced acute lung injury increased bronchoalveolar lavage protein. Female mice were exposed to 45 ppm chlorine for 0 (filtered air control), 6, 12, or 24 (C57BL/10J only) hours and anesthetized, and bronchoalveolar lavage was performed with Ca²⁺, Mg²⁺-free PBS. Bronchoalveolar lavage protein increased sooner in the sensitive C57BLKS/J murine strain than in the resistant C57BL/10J murine strain. Lavage fluid was centrifuged, and total protein in cell-free supernatants was measured according to a bicinchoninic acid assay. Values represent means ± SE (n = 6 mice/strain/time). *Significantly different (P < 0.05) from strain-matched control mice, as determined by ANOVA with an all-pairwise multiple comparison procedure (the Holm-Sidak method). ^†Significantly different (P < 0.05) between the sensitive C57BLKS/J and resistant C57BL/10J murine strain at indicated times, as determined by ANOVA with an all-pairwise multiple comparison procedure (the Holm-Sidak method).

Alanine decreased in sensitive C57BLKS/J murine lungs, but not in resistant C57BL/10J murine lungs, whereas glutamine increased more in the resistant C57BL10/J murine lungs, compared with the sensitive C57BLKS/J murine lungs. Female mice were exposed to filtered air (0 hours, control) or chlorine (45 ppm, 6 or 12 hours), and metabolome profiling was performed with lung tissue. Values are normalized to the sensitive C57BLKS/J control (filtered air, 0 hours) levels, and plots indicate the medians (lines within boxes) with 25% and 75% confidence intervals (borders of the boxes) and 95% confidence intervals (error bars). *Significantly different (P < 0.05) from strain-matched control mice, as determined by ANOVA with an all-pairwise multiple comparison procedure (the Holm-Sidak method). ^†Significantly different (P < 0.05) between the sensitive C57BLKS/J and resistant C57BL/10J murine strain at indicated times, as determined by ANOVA with an all-pairwise multiple comparison procedure (the Holm-Sidak method).

Transcripts in the enriched nuclear factor erythroid-derived–2–like 2 (NFE2L2, also known as Nrf2)–mediated oxidative stress response pathway in the lungs of C57BLKS/J and C57BL/10J mice after chlorine-induced acute lung injury. Microarrays increased transcripts (significantly different, at P < 0.05, from strain-matched control mice) that were enriched as determined by the logistic regression approach LRpath. Transcripts in this pathway were similar between strains, and included heme oxygenase (decycling)–1 (HMOX1); nuclear factor, erythroid-derived–2–like 2 (NFE2L2); activating transcription factor–3 (ATF3); v-maf musculoaponeurotic fibrosarcoma oncogene family, protein F (avian) (MAFF); FK506 binding protein–5 (FKBP5); glutamate–cysteine ligase, catalytic subunit (GCLC); thioredoxin reductase–1 (TXNRD1); glutathione peroxidase–2 (GPX2); v-maf musculoaponeurotic fibrosarcoma oncogene family, protein K (avian) (MAFK); and glutathione S-transferase, α 1 (Ya) (GSTA1). Values represent means ± SE (n = 6 female mice/strain/time), normalized to strain-matched control mice (0 hours).

Haplotype association mapping of murine strains varying in sensitivity to chlorine-induced acute lung injury. (A) Acute lung injury survival time of 40 murine strains. Female mice were exposed to 45 parts per million (ppm) of chlorine for up to 24 hours, and survival times were recorded hourly. Values represent means ± SE (n = 5–22 mice/strain, 6–8 weeks old). Numbers in parenthesis represent the number of mice/strain imputed at 48 hours. The NON/ShiLtJ strain was imputed at 42 hours. (B) Haplotype association map for chlorine-induced acute lung injury in mice. The scatter (Manhattan) plot displays the corresponding −log(P) association probability for a single-nucleotide polymorphism (SNP) at the indicated chromosomal location. Dashed line, threshold of significant SNP associations of −log(P) > 4.8. Solid line, threshold of suggestive SNP associations of 4.8 ≥ −log(P) > 4.0.

Transcript levels of candidate genes that differed between the C57BL/10J and C57BLKS/J murine strains after chlorine exposure. Female mice were exposed to filtered air (control, 0 hours), or to chlorine (45 ppm) for 6 or 12 hours, lung mRNA was isolated, and transcript expression levels were determined by quantitative real-time polymerase chain reactions. KLF4, Kruppel-like factor–4 (gut); SEMA7A, sema domain, immunoglobulin domain, and glycophosphatidyl inositol membrane anchor (semaphorin)–7A; SLC38A4, solute carrier family 38, member 4; TNS1, tensin 1. Values represent means ± SE (n = 8 mice/strain/time), normalized to the sensitive C57BLKS/J control (filtered air, 0 hours). *Significantly different (P < 0.05) from strain-matched control mice, as determined by ANOVA with an all-pairwise multiple comparison procedure (the Holm-Sidak method). ^†Significantly different (P < 0.05) between the sensitive C57BLKS/J and resistant C57BL/10J murine strain at indicated times, as determined by ANOVA with an all-pairwise multiple comparison procedure (the Holm-Sidak method).

Assessment of the phenotypic difference in survival times between polar sensitive PWD/PhJ and resistant BUB/BnJ murine strains produced by nonsynonymous single-nucleotide polymorphism (SNP) associations in exemplary candidate genes. The mean survival time was determined for mice carrying either allele (n = number of mice with either allele, as indicated below the abscissas). The difference between these groups was then compared with the difference of the means of polar sensitive PWD/PhJ (n = 8 mice) and resistant BUB/BnJ (n = 22 mice) murine strains exposed to 45 ppm chlorine (total = 364 female mice). The SNP identification “rs” number is indicated in each histogram. The predicted amino acid is presented for either allele, with the consequences to side-chain polarity or hydropathy index. Value represent means ± standard errors, and P values indicate the significance of the difference between the allele means as determined by ANOVA, according to an all-pairwise multiple comparison procedure (the Holm-Sidak method). Aacs, acetoacetyl-coenzyme A synthetase; Ikbkap, inhibitor of κ light polypeptide enhancer in B cells, kinase complex–associated protein; Tns1, tensin 1. C = cytosine, T = thymidine, A = adenine, and G = guanine at the SNP position.