Integrative Assessment of Chlorine-Induced Acute Lung
Injury in Mice
George D. Leikauf1*, Hannah Pope-Varsalona1, Vincent J. Concel1, Pengyuan Liu4, Kiflai Bein1,
Annerose Berndt2, Timothy M. Martin1, Koustav Ganguly1, An Soo Jang1,5, Kelly A. Brant1,
Richard A. Dopico, Jr.1, Swapna Upadhyay1, Y. P. Peter Di1, Qian Li6, Zhen Hu6, Louis J. Vuga3,
Mario Medvedovic6, Naftali Kaminski3, Ming You4, Danny C. Alexander7, Jonathan E. McDunn7,
Daniel R. Prows8, Daren L. Knoell9, and James P. Fabisiak1*
1Department of Environmental and Occupational Health, Graduate School of Public Health,2Department of Medicine, and3Simmons Center for
Interstitial Lung Disease, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania;4Wisconsin Cancer Center, Medical College of
Wisconsin, Milwaukee, Wisconsin;5Department of Internal Medicine, Soon Chun Hyang University, Bucheon, South Korea;6Department of
Environmental Health, University of Cincinnati, Cincinnati, Ohio;7Metabolon, Inc., Durham, North Carolina;8Department of Pediatrics, University of
Cincinnati, and Division of Human Genetics, Cincinnati Children’s Hospital, Cincinnati, Ohio; and9Dorothy M. Davis Heart and Lung Research Institute,
Department of Pharmacy, and Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, Department of Internal Medicine, Ohio State University,
The genetic basis for the underlying individual susceptibility to
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)
associationswere evaluated through transcriptomicand metabolomic
profiling. Using > 10% allelic frequency and > 10% phenotype ex-
plained as threshold criteria, promoter SNPs that could eliminate pu-
tative transcriptional factor recognition sites in candidate genes were
assessed by determining transcript levels through microarray and re-
verse 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,
lung) and protein catabolic process (increased more in the resistant
C57BL/10J lung). Lung metabolomic profiling revealed 95 of the 280
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
Keywords: ARDS; countermeasures; glutamine; genetics; metabolo-
Accidental (e.g., railroad derailments) (1–3) or intentional (e.g.,
terrorism in Iraq) (4–6) chlorine exposures have led to acute lung
injury. Over 10 million tons/year of chlorine are manufactured in
the United States, and accidental releases have occurred in many
industries (7, 8). Moreover, widespread use requires the transport
of approximately 20,000 tank cars/year (340,000 L/car) (9). Rail
accidents are rare but can be catastrophic, because a ruptured car
can generate a lethal plume for several hours (10). Accidental
chlorine releases are approximately five times more likely to pro-
duce casualties and evacuations compared with other chemical
A major consequence of chlorine exposure is acute lung injury
(9–17). Acute lung injury, which comprises a heterogeneous syn-
drome caused by direct (chlorine) and indirect (sepsis) insults,
involves decreased epithelial/endothelial integrity, fluid clearance,
and surfactant function (18–21). Outcomes vary greatly, and sur-
vival is difficult to predict (22–26), which has stimulated investi-
gation into individual susceptibility (27–48). However, genetic
analyses are mainly limited to case-control studies because acute
lung injury occurs sporadically. Nonetheless, investigations indi-
cate that this syndrome is complex and governed by multiple
(Received in original form January 23, 2012 and in final form March 7, 2012)
*These two authors contributed equally to this study.
This study was supported by National Institutes of Health grants ES015675,
HL077763, and HL085655 (G.D.L.), HL091938 (Y.P.P.D.), HG003749 and
LM009662 (M.M.), HL084932 and HL095397 (N.K.), AT003203, AT005522,
CA113793, and CA134433 (M.Y.), HL075562 (D.R.P.), and HL086981 (D.L.K.).
Author Contributions: G.D.L., K.B., A.B., M.M., N.K., M.Y., D.R.P., and D.L.K. were
responsible for this study’s conception and design. H.P.-V., V.J.C., P.L., K.B., A.B.,
T.M.M., K.G., A.S.J., K.A.B., R.A.D., S.U., Y.P.P.D., Q.L., Z.H., L.J.V., M.M., N.K.,
M.Y., D.C.A., J.E.M., D.R.P., G.D.L., and J.P.F. were responsible for the analyses and
interpretation in this study. G.D.L., K.B., D.R.P., and J.P.F. were responsible for
drafting the manuscript for important intellectual content.
Correspondence and requests for reprints should be addressed to James P. Fabisiak,
Ph.D., Department of Environmental and Occupational Health, Graduate School of
Public Health, University of Pittsburgh, 100 Technology Dr., Suite 350, Pittsburgh,
PA 15219-3130. E-mail: firstname.lastname@example.org or email@example.com
This article has an online supplement, which is accessible from this issue’s table of
contents at www.atsjournals.org
Am J Respir Cell Mol Biol
Copyright ª 2012 by the American Thoracic Society
Originally Published in Press as DOI: 10.1165/rcmb.2012-0026OC on March 23, 2012
Internet address: www.atsjournals.org
Vol 47, Iss. 2, pp 234–244, Aug 2012
A major challenge to critical care involves the reliable pre-
diction of survival in patients with acute lung injury. Because
acute lung injury is a sporadic disease produced by hetero-
geneous precipitating factors, previous genetic analyses were
gene associations. This study functionally assesses single-
nucleotide polymorphism associations linked with survival
during acute lung injury in mice. Genetic associations from
haplotype mapping were strengthened by an integrated
assessment using transcriptomic and metabolomic profiling.
The leading candidate genes associated with increased sus-
ceptibility toacutelunginjury inmiceincluded Klf4,Sema7a,
Tns1, Aacs, and a gene that encodes an amino acid carrier,
In a previous study of 16 murine strains, we reported that in-
bred mice varied approximately 3-fold in mean survival time,
supporting the likelihood of a genetic basis of susceptibility
(49). Haplotype mapping, using a large, genetically diverse panel
of inbred murine strains (50–52), has emerged as a valuable tool
to identify the genes responsible for complex traits (53–59). Re-
cently, we used this method to identify the genetic determinants
of acrolein-induced lung injury (59). In this study, we integrate
haplotype mapping with transcriptomic and metabolomic profil-
ing to identify candidate genes associated with delayed pulmo-
nary edema resulting from chlorine-induced lung injury.
MATERIALS AND METHODS
This study was performed with the approval of the Institutional Animal
CareandUseCommitteeatthe UniversityofPittsburgh,and mice(6–8-
week-old females) were housed under specific pathogen-free condi-
tions. At high concentrations, chlorine can produce rapid, often lethal,
lung injury, whereas low concentrations may cause delayed pulmonary
edema. To model the delayed form of lung injury, we previously ex-
posed 16 inbred murine strains to 45 parts per million (ppm) chlorine
for 24 hours, and monitored survival hourly (49). In this study, these
data were combined with data from an additional 24 inbred strains (n ¼
334) for haplotype association mapping. After their 24-hour exposure,
mice were returned to microisolator cages (filtered room air), and their
survival was monitored hourly. To examine chlorine-induced changes
in bronchoalveolar lavage, as well as lung histology and transcripts/
metabolites, groups (n ¼ 5–8 mice/group) of sensitive C57BLKS/J or
resistant C57BL/10J mice were exposed to filtered air (0 hours) or
chlorine (6 or 12 hours). Microarrays (n ¼ 5 mice/strain/time) and
quantitative RT-PCR (n ¼ 8 mice/strain/time) were used to contrast
transcript levels of candidate genes. Metabolomic profiling was per-
formed as described previously (60–63), using lung tissue (n ¼ 5
mice/strain/time) that was homogenized in deionized water containing
recovery standards, extracted (80:20 methanol/water), and analyzed by
positive or negative ultrahigh performance liquid chromatography–mass
spectrometry/mass spectrometry (LC:Surveyor; ThermoFisher, Pittsburgh,
PA) (61), or by gas chromatography–mass spectrometry (Thermo-
Finnegan Mat-95XP; ThermoFisher) (61). In contrast to the accurate-
mass and elution-time tags used in shotgun proteomics, our library-based
approach combines accurate retention times and tandem mass spectromet-
ric fragmentation patterns to unambiguously identify .2,400 biochemicals
(63). LRpath (64) and CLEAN (65) were used to assess pathway enrich-
ment in transcriptomic and metabolomic profiling. To contrast the strains,
a difference in the mean response was considered significant at a threshold
of 20.58 , X , 0.58 log 2 change (i.e., 61.5-fold change; P , 0.05).
Additional details are provided in the online supplement.
Haplotype Mapping of Survival Times in 40 Murine Strains
respiratory distress during exposure (45 ppm 3 24 hours), or after
return to filtered room air. During gross pathologic observations at
death, the lung surface appeared red from hemorrhaging and coag-
ulation consistent with severe lung injury (66). The mean survival
time was distributed continuously among murine strains (supportive
of a complex trait), with the polar strains varying by approximately
5-fold from 7.6 6 0.8 (mean 6 SEM) hours (PWD/PhJ) to 38.1 6
0.5 hours (NON/ShiLtJ) (Figure 1A). A haplotype association map
was obtained (Figure 1B), and significant single-nucleotide poly-
morphisms (SNPs) (n ¼ 11 SNPs; 2log(P) . 4.8) were identified
on chromosomes 1, 4, 5, 9, and 15, with suggestive SNP associations
(n ¼ 56 SNPs; 4.8 > 2log(P) . 4.0) on chromosomes 1, 5, 9, 12, 14,
16, and 18 (please refer to Table E1 in the online supplement).
Because haplotype association mapping identifies SNP associations
in linkage with functional SNPs (67), we evaluated the nonsynon-
ymous SNPs and promoter SNPs in genes 6 0.5 megabase pairs of
the identified SNPs in 28 candidate genes.
To prioritize SNPs associated with survival, candidate genes were
lead to nonsynonymous SNPs (i.e., an amino acid substitution, in-
sertion, or deletion in the encoded protein). We identified 51 non-
synonymousSNPsin21 genes (Table E2).Ofthese, 17 SNPs in11
genes had a greater than 10% allelic frequency and could explain
Three genes exhibited SNPs that could lead to amino acid sub-
stitutions in functional domains (Figure 2). Genes with predicted
substitutions that could alter the protein hydropathy index or side
chain polarity included Aacs Thr321Ile (domain, acyl-protein syn-
thetase), Ikbkap Gly662Val (domain, the IKI3 family), and Tns1
Asn1882Ser (domain, pleckstrin homology–like).
Histological and Lavage Protein Assessment
Based on the 40-strain analysis and our capability to obtain ade-
quate numbers of mice, C57BLKS/J and C57BL10/J were selected
At12hours,lavageprotein increased insensitive C57BLKS/J mice,
but not in resistant C57BL/10J mice (P , 0.001) (Figure 3). At 24
hours, lavage protein increased in the C57BL/10J mice, compared
with strain-matched control mice. The lung tissue from the sensi-
tive C57BLKS/J strain demonstrated perivascular enlargement
(Figure 4C) and alveolar wall thickening (Figure 4E) within 12
hours, compared with strain-matched control mice (Figure 4A).
Neither perivascular enlargement nor alveolar wall thickening
was evident in C57BL/10J mice after 12 hours (Figures 4D and
4F), compared with strain-matched control mice (Figure 4B).
SNP Association in Promoters
In addition to nonsynonymous SNPs, the 28 candidate genes iden-
tified by haplotype mapping were evaluated for strain differences
in lung transcript levels before (0 hours) or after (6 or 12 hours)
chlorine exposure (n ¼ 8 mice/strain/time) (Table E3). Baseline
lung transcripts encoding acetoacetyl coenzyme A synthetase
(log2 ¼ 0.7 6 0.1) and cytochrome P450family 11, subfamily A,
polypeptide 1 (log2 ¼ 1.2 6 0.2) increased, and Kruppel-like
factor (gut)-4 (KLF-4) (log2 ¼ 21.5 6 0.3) decreased, in
C57BLKS/J compared with C57BL/10J mice.
At 6 hours, KLF4 and solute carrier family 38, member 4
(SLC38A4) transcripts increased less in C57BLKS/J than in
C57BL/10J mice (Figure 5). At 12 hours, KLF4, semaphorin-7A
(SEMA7A), SLC38A4, and tensin 1 (TNS1) transcripts increased
less in C57BLKS/J than in C57BL/10J mice. Transcripts for other
candidate genes either decreased similarly in both murine strains
(e.g., SLC35A5 or SLCO4C1; Figure E1 in the online supple-
ment), or were not significantly different from control values after
exposure. The interrogation of SNPs within the 59 untranslated
region (promoter) that could change putative DNA-binding sites
was evident in six of the differentially expressed genes (Table 2).
These SNPs (except those in Klf4) could explain approximately
14–35% of the phenotypic difference between polar strains. Using
microarrays, we identified 41 increased and 10 decreased tran-
scripts within 104 transcription factors that were related to the
binding sites identified in the promoter SNPs (Table E4).
Transcriptomic Pathway Enrichment Analysis
sitive and resistant strains before or during exposure (n ¼ 6 female
mice/strain/time) was assessed by microarray (Table E5). In general,
the baseline lung transcriptome of C57BLKS/J mice was similar to
Leikauf, Pope-Varsalona, Concel, et al.: Genetics of Acute Lung Injury235
that of C57BL10/J mice, with 161 increased and 106 decreased tran-
scripts in the C57BLKS/J compared with the C57BL/10J strain. The
only pathway with significant enrichment was that of natural killer–
mediated cytotoxicity. Members in this pathway included nine tran-
scripts that encoded killer cell lectin-like receptors (also known as
inhibitory LY49-proteins), which decreased in C57BLKS/J mice
compared with C57BL/10J mice.
After exposure, significantly increased lung transcripts in sen-
sitive C57BLKS/J mice were enriched in pathways that included
the Rous sarcoma oncogene (Src) homology–3 domain, tran-
scription factor activity, and cell death (Figure E2A). After
exposure, decreased transcripts in the sensitive C57BLKS/J mice
were enriched in pathways that included protein transport, trans-
lation, and the development of vasculature (Figure E2B). After
exposure, increased transcripts in resistant C57BL/10J mice were
enriched in pathways that included cell adhesion, cytoskeletal or-
ganization, and the protein catabolic process (Figure E3A). De-
creased transcripts in resistant C57BL/10J mice were enriched in
pathways that included RNA binding, transcription, and mitochon-
dria (Figure E3B).
the transcription factor activity pathway, which contained transcripts
that increased more in sensitive C57BLKS/J lungs, and the transcrip-
tion pathway, which contained transcripts that decreased more in re-
sistant C57BL/10J lungs. Similarly, the protein transport pathway
was enriched with decreased transcripts in sensitive C57BLKS/J
lungs, whereas the protein catabolic process pathway was enriched
with increased transcripts in resistant C57BL/10J lungs. Pathways
that were altered nearly equally in both strains included the nuclear
factor erythroid-derived–2–like–2 (NFE2L2, also known as NRF2)–
mediated oxidative stress response (Figure 6).
Lung metabolomic profiling of these strains identified 280 com-
pounds (Table E6). In general, basal lung metabolites were
Figure 1. Haplotype association mapping of
murine strains varying in sensitivity to chlo-
rine-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 sur-
vival times were recorded hourly. Values repre-
sent means 6 SE (n ¼ 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
2log(P) association probability for a single-
nucleotide polymorphism (SNP) at the indicated
chromosomal location. Dashed line, threshold of
significant SNP associations of 2log(P) . 4.8.
Solid line, threshold of suggestive SNP associa-
tions of 4.8 > 2log(P) . 4.0.
236 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 472012
similar between the sensitive and resistant strains (i.e., only
seven increased and six decreased molecules differed signifi-
cantly between strains). Compared with resistant C57BL/10J
mice, the basal metabolite in sensitive C57BLKS/J murine lungs
that was reduced comprised cytosine, and metabolites that were
elevated included phenylacetylglycine, S-methylglutathione, and
Exposure markedly altered the lung metabolomic profile. At
6 hours and 12 hours of chlorine exposure, 95 metabolites were
altered in at least one strain, compared with strain-matched con-
trol mice. LRpath analysis identified the amino acid pathway
to be significantly different between strains and with treatment
Noteworthy metabolites in the amino acid pathway that
changed with exposure included alanine, which decreased more
in C57BLKS/J than in C57BL/10J mice, and glutamine, which in-
creased more in C57BL/10J than in C57BLKS/J mice (Figure 7).
Subpathways enriched with exposure included (1) glycerolipid
metabolism (e.g., decreased glycerophosphocholine and glycerol
3-phosphate), (2) medium chain fatty acids (e.g., increased capry-
late [8:0] and laurate [12:0]), and (3) phenylalanine and tyrosine
metabolism. The metabolites that changed in these subpathways
did not differ between strains (except for phenol sulfate, which
increased more in C57BL/10J mice). Other metabolites changed
in both strains included increased 3-hydroxybutyric acid (BHBA),
and decreased lactate, 1-palmitoleoylglycerophosphocholine, and 1-
palmitoleoylglycerophosphoinositol. Small molecules that increased
more in the sensitive C57BLKS/J than in the resistant C57BL/10J
strain included two carbohydrates (sorbitol and fructose).
lavage protein at 12 hours in the sensitive C57BLKS/J strain, or at
24 hours in the resistant C57BL/10J strain (Figure 3). These find-
ings are similar to those of Zarogiannis and colleagues (17). Lung
histology also indicated that the C57BLKS/J mice developed in-
jury sooner than the C57BL/10J mice (Figure 4). The finding of
perivascular enlargement should be evaluated with caution, be-
cause it can result from tissue processing. The histological samples
obtained at 6 and 12 hours were selected mainly to coincide with
transcriptomic and metabolomic analyses. These times may have
been too early to uncover a great deal of lung injury or inflam-
matory infiltrate. Gross pathology revealed that lungs were
marked by focal hemorrhages at the time of death. This similarity
of this histological feature among strains suggests that the extent
of injury was the same at the time of death, and that the pheno-
type being measured is survival time.
Ascorbate decreased slightly, but not to the extent of statis-
tical significance. Both strains demonstrated nearly equivalent
decreases in gulono-1,4-lactone (an ascorbate precursor) and
dehydroascorbate (an ascorbate metabolite) (Table E6). Simi-
larly, both strains exhibited a nearly equivalent enrichment of
the NFE2L2-mediated oxidative stress pathway (Figure 4).
Thus, although the response to oxidative stress was similar in
both strains, resistance can be defined as the ability to prolong
survival. The objective of this study was to uncover the genetic
basis that could provide additional metabolic or other homeo-
static capacities during chlorine-induced lung injury.
oritized these genes by several criteria, including the phenotypic
difference associated with nonsynonymous SNPs in functional
domains or with promoter SNPs matched with variable expression
by transcriptomic analyses. In addition, we paired these relation-
ships with altered metabolites identified by metabolomic profiling.
This integrative approach revealed 13 candidate genes (Tables 1
and 2), and of these, Klf4, Sema7a, Tns1, Slc38a4, and Aacs were
more noteworthy, and could be associated with survival in several
For example, Kru ¨ppel-like factor (gut)–4 (KLF4) can protect
against lung injury (68). KLF4, a transcription factor, regulates
cadherin-5 expression in adherens junctions, and KLF4 knock-
down augments LPS-induced lung injury in mice. KLF4 mRNA
also can be induced by other stresses (69, 70). Here, lung KLF4
transcripts increased more in the resistant C57BL/10J mice than
in the sensitive C57BLKS/J mice (Figure 5).
Another candidate, semaphorin-7A (SEMA7A), can be in-
duced by transforming growth factor–b (TGFB1) and mediates
TGFB1-induced alveolar apoptosis (71). SEMA7A polymor-
phisms are associated with abnormal bone mineral density in
Korean women (72). During acute lung injury, TGFB1 can
increase endothelial and epithelial permeability (73–75), and
the inhibition of TGFB1 can diminish lung injury (66, 76–78).
SEMA7A can mediate AKT phosphorylation (71), which is
TABLE 1. GENES WITH NONSYNONYMOUS SINGLE-NUCLEOTIDE POLYMORPHISMS ASSOCIATED WITH CHLORINE SURVIVAL TIME
Name of Gene Chromosome
(%) Allele Frequency
Tns11 72920639 rs30218834.1 rs40124058
Definition of abbreviations: Tns1, tensin 1; Slco4c1, solute carrier organic anion transporter family, member 4C1; IkbKap, inhibitor of kappa light polypeptide enhancer
in B cells; Ncor2, nuclear receptor co-repressor 2; Aacs, acetoacetyl coenzyme A synthetase; Fry, furry homolog; Cyp11a1, cytochrome P450family 11, subfamily A,
polypeptide 1; Ttk, Ttk protein kinase; Tnfrsf19, tumor necrosis factor receptor superfamily, member 19; Slc38a4, solute carrier family 38, member 4; Slc35a5, solute
carrier family 35, member 5; dbSNP, Single Nucleotide Polymorphism Database at NCBI; rs number, dbSNP reference sequence.
Leikauf, Pope-Varsalona, Concel, et al.: Genetics of Acute Lung Injury237
associated with increased cell survival (79) and is protective dur-
ing lung injury (80). In this study, promoter Sema7a SNPs asso-
ciated with 12–16% of the difference in survival phenotype
(Table 2) and SEMA7A mRNA increased longer in the resistant
C57BL/10J strain, compared with C57BLKS/J strain (Figure 5).
TNS1 polymorphisms have been associated with lung func-
tion and chronic obstructive lung disease (81, 82). TNS1, a scaf-
fold protein, recruits and organizes enzymes at focal adhesions
and mediates cell migration in wound healing (83). The
C-terminal domain of TNS1 has a Src homology–2 domain that
binds focal adhesion kinase, and a phosphotyrosine-binding
domain that binds integrin-b. Osmotic stress alters the binding
partners to the Src homology–2 domain (84). In this study, 12–
16% of the difference in survival between polar strains associ-
ated with two Tns1 promoter SNPs (Table 2) and lungs from the
resistant C57BL/10J strain demonstrated a prolonged increase
in lung TNS1 mRNA after exposure to chlorine (Figure 5). In
addition, we detected a nonsynonymous SNP (N1882S) in the
integrin-b binding domain with an approximately 30% allelic
frequency that associated with approximately 20% of the phe-
notypic difference (Figure 2 and Table 1).
During injury, lung epithelial cells are likely to be challenged
by energetic stress (60). In general, cell survival can depend, in
part, on limiting energy expenditure through many defense strat-
egies (85, 86). Alternately, the activation of energy-yielding path-
ways for ATP production may be required for energetic needs
incurred upon injury. Lactate and alanine decreased after chlo-
rine exposure, and these metabolites are the precursors for pyru-
vate and subsequently acetyl-coenzyme A (acetyl-CoA). Thus,
this response implicates an increased utilization of aerobic me-
tabolism via the Krebs cycle. Interestingly, both strains exhibited
a marked elevation in Krebs-cycle intermediates (citrate and cis-
aconitate in C57BLKS/J, and fumarate in C57BL/10J).
Restricting energy-dependent solute carriers can conserve en-
ergy, but this may be counterproductive because they are critical
for energy substrate uptake and fluid absorption. Thus, cellular
stress may modulate the array of solute carriers. Several pathways
and candidate genes identified in this study include solute carrier
(SLC) proteins. In particular, SLC35A5, SLCO4C1, and SLC38A4
were associated with increased susceptibility to chlorine-induced
lung injury. Little is known about SLC35A5, which is a putative
nucleotide–sugar transporter, based on a shared homology with
SLC35A1 (87). SLCO4C1, an organic anion transporter, can trans-
port eicosanoids, thyroid hormone, and steroids (88). Although the
SLCO4C1 transcript and protein are present in the lung (Figures
E4 and E5), the role of SLOC4C1 in lung injury remains un-
clear. SLCO4C1 is protective in kidney disease (89), and a hu-
man SLCO4C1 SNP was associated with preeclampsia (90). In
this study, nonsynonymous SNPs were identified (Table 1), but
these SNPs did not occur in known functional domains. In ad-
dition, lung SLCO4C1 and SLC35A1 transcripts decreased
nearly equivalently in the resistant and sensitive strains during
chlorine injury (Figure E1).
Figure 2. 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 ¼ 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 ¼ 8 mice) and resistant BUB/BnJ (n ¼ 22
mice) murine strains exposed to 45 ppm chlorine (total ¼ 364 female
mice). The SNP identification “rs” number is indicated in each histo-
gram. The predicted amino acid is presented for either allele, with the
consequences to side-chain polarity or hydropathy index. Value repre-
sent means 6 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, inhib-
itor of k light polypeptide enhancer in B cells, kinase complex–associated
protein; Tns1, tensin 1. C ¼ cytosine, T ¼ thymidine, A ¼ adenine, and
G ¼ guanine at the SNP position.
238AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 472012
In contrast, lung SLC38A4 transcripts increased more in the
resistant compared with the sensitive mice (Figure 5). SLC38A4
is a sodium-coupled neutral amino acid (including alanine and
glutamine) transporter. The tagSNP (rs32255071) on chromo-
some 15 was associated with SLC38A4 (2log(P) ¼ 6.25). Pro-
moter Slc38a4 SNPs were associated with 13–16% of the
difference in survival (Table 2).
The transcriptomic profiling of lung transcripts that decreased
more in sensitive C57BLKS/J compared with resistant C57BL/10J
mice identified enrichment in the protein transport pathway (Fig-
ure E2B). In contrast, the protein catabolic process pathway con-
tained transcripts that increased more in the resistant C57BL/10J
strain than in the sensitive C57BLKS/J strain (Figure E3A). Sim-
ilarly, metabolomic profiling indicated enrichment in the amino
acid pathway, and individual amino acids, including glutamine,
increased more in the resistant C57BL/10J than in the sensitive
C57BLKS/J strain. Alanine decreased more in the sensitive
C57BLKS/J than in the resistant C57BL/10J strain. Alanine
can be used during energetic stress to generate pyruvate and glu-
tamate (91). Moreover, glutamine can attenuate acute lung in-
jury by inducing heat-shock proteins (92–94). Thus, of the three
solute carrier proteins identified, SLC38A4 is worthy of addi-
The ability to increase lung glutamine also may be important
in the improved survival of the resistant C57BL/10J strain
through additional roles in metabolism. Although glucose is
generally thought to be the primary substrate for energy metab-
olism in most tissues, energetics in the lung are complex, as
manifested by multiple substrate usage. Here, glucose was un-
changed,whereaslactate decreased equally between strains.In-
terestingly, a fatty acid b-oxidation ketone body, BHBA,
increased in both strains initially, but was maintained longer
in resistant C57BL/10J mice, possibly reflecting greater fatty-
acid b-oxidation in the resistant strain. Previously, Fox and
colleagues (95) measured oxidation rates of glucose, gluta-
mine, lactate, and BHBA in alveolar Type II cells from fetal
rats. The CO2formation from lactate was greater than from
glutamine, which in turn was greater than from BHBA. The
rate of glucose oxidation was lower than in all these substrates
(z 5 times less than that of glutamine). In addition, glucose,
but not lactate, inhibited the oxidation of glutamine. Similarly,
alanine is also a substrate for energy production in alveolar
Type II cells (96). Thus, glutamine, alanine, and other substrates
can be oxidized for added energy in alveolar epithelial cells.
The alveolar Type II cell is a critical target during lung injury
because it generates pulmonary surfactant, which maintains
alveolar patency. Pulmonary surfactant consists of phospho-
lipids (mainly dipalmitoylglycerophosphocholine), surfactant
proteins, electrolytes, and other biomolecules. Surfactant-asso-
ciated protein B (SFTPB) mRNA decreased in the sensitive
C57BLKS/J mice more rapidly than in the C57BL/10J mice
(i.e., log2 ¼ 21.2 versus 20.3 at 6 hours, respectively). This
is relevant because maintaining SFTPB is critical to survival
during acute lung injury in mice (19, 27, 39).
Surfactant lipid production uses glucose-dependent fatty-acid
synthesis, but fatty acids can also be generated from lactate or ke-
tone bodies (97). In the lung, ketone metabolism also can serve as
an energy source. Alternately, acetoacetate can be used in the
synthesis of phospholipids, including palmitoylglycerophospho-
cholines, and thus have a potential role in supplying adequate
Figure 3. Chlorine-induced acute lung injury increased bronchoalveolar
air control), 6, 12, or 24 (C57BL/10J only) hours and anesthetized, and
bronchoalveolar lavage was performed with Ca21, Mg21-free PBS. Bron-
choalveolar 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 6 SE (n ¼
6 mice/strain/time). *Significantly different (P , 0.05) from strain-matched
control mice, as determined by ANOVA with an all-pairwise multiple com-
parison procedure (the Holm-Sidak method).ySignificantly 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).
Figure 4. 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 infil-
tration (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-mm sections were prepared
with hematoxylin and eosin staining. Bars indicate magnification.
Leikauf, Pope-Varsalona, Concel, et al.: Genetics of Acute Lung Injury239
surfactant lipids. Cytosolic lipid synthesis from acetoacetate can
conserve energy by bypassing the pathway involving the ATP-
dependent supply of acetyl units from the mitochondria to
cytosol (98, 99). Nonsynonymous SNPs were identified in Aacs
that could lead to Thr321Ile substitution in the acyl-protein synthe-
tase domain (Table 1). AACS, a cytosolic acetoacetate (ketone
bodies)–specific ligase, catalyzes the formation of short-chain acyl-
CoA from acetoacetate,thereby providing acetyl-CoA for fatty-acid
Figure 5. Transcript levels of candidate
genes that differed between the C57BL/
10J and C57BLKS/J murine strains after
chlorine exposure. Female mice were ex-
posed 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, immuno-
globulin domain, and glycophosphatidyl
inositol membrane anchor (semaphorin)–
7A; SLC38A4, solute carrier family 38,
member 4; TNS1, tensin 1. Values repre-
sent means 6 SE (n ¼ 8 mice/strain/time),
normalized to the sensitive C57BLKS/J
control (filtered air, 0 hours). *Signifi-
cantly different (P , 0.05) from strain-
matched control mice, as determined
by ANOVA with an all-pairwise multiple
comparison procedure (the Holm-Sidak
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
ySignificantly different (P ,
TABLE 2. GENES WITH PROMOTER SINGLE-NUCLEOTIDE POLYMORPHISMS ASSOCIATED WITH CHLORINE SURVIVAL TIME
dbSNPSubstitution DNA Binding Site
Tns11 72920639rs30218834.1 rs52269246A/G Loss: AP2
Gain: RAF and CTCF
Loss: AP2 and CACCC-binding factor
Gain: AP-1, CEB/Pa
Loss: GAL4 and TFII-I
Gain: E2F 1 p107
gain: HES-1 and C/EBP-a
Gain: AP-1,TF68, LCR-F1, GCR1
Gain: GR and NF-E
Loss: AP-1 and RAR-b
Gain: GAL4, T-Ag, and NFE2/CAC-bp
Gain: RAF, PEA3, E1A-F, and MAF
Loss: NF-E and NF-S
Loss: ZP5 and T-Ag
Gain: CACCC-binding factor
NDKlf44 55986910rs27805492 5.0
Aacs5 125996775 rs295417194.6 rs36310016
Sema7a9 58075842rs3674363 5.0rs52190134
49.4 Cyp11a19 58075842rs3674363 5.0
rs50603668 G/T 15.914.1
20.4Slc38a4 1596687641 rs32255071 6.3
rs48402040A/G 16.2 10.0
Definition of abbreviations: Aacs, acetoacetyl coenzyme A synthetase; Cyp11a1, cytochrome P450, family 11, subfamily A, polypeptide 1; Klf4, Kruppel-like factor–4
(gut); ND, not done; Sema7a: sema domain, immunoglobulin domain, and glycophosphatidyl inositol membrane anchor (semaphorin)–7A; Slc38a4, solute carrier
family 38, member 4; Tns1, tensin-1; dbSNP, Single Nucleotide Polymorphism Database; rs number, dbSNP reference sequence.
240 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 47 2012
synthesis (100). Two lysophospholipids, 1-palmitoleoylglycerophos-
phocholine and 1-palmitoleoylglycerophosphoinositol, were de-
creased in the lungs of the sensitive C57BLKS/J murine strain
(Table E6). Overall, the use of similar substrates for energy
production and surfactant synthesis could create competition
between these pathways, especially in times of stress and
injury. Therefore, to better define these relationships in future
mechanistic studies is imperative.
Another candidate gene is the inhibitor of kB kinase com-
plex–associated protein (Ikbkap, also known as IKAP). Human
IKBKAP polymorphisms produce a truncated protein that has
been associated with familial dysautonomia, a recessive disease
that affects the nervous system (101, 102). Neuronal dysfunction
leads to several defects, including abnormal respiratory hypoxic
responses and pneumonia (103). IKBKAP was named on the
basis of its reported ability to bind to and assemble IkB kinases
into an active complex (104). Later studies, however, failed to
confirm a role in NF-kB activation, and instead reported IKB-
KAP to be involved in transcription elongation (105), Jun
N-terminal kinase-mediated stress signaling (106), and cell mi-
gration (107). Here, we identified three nonsynonymous SNPs
in murine Ikbkap that could lead to amino acid substitutions in
exons 11, 18, and 36, associated with a phenotypic difference in
survival (Table 1). The Gly662Val substitution is in the IKI3 do-
main likely to be involved in IKBKAP’s transcriptional elongation
This study provides an integrative strategy that combines
haplotype mapping, transcriptomics, and metabolomics to assess
chlorine-induced acute lung injury in mice. However, as with any
investigation, each approach has limitations that cannot be fully
overcome by our combined approach. First, we acknowledge
that each approach is essentially descriptive, and that our exper-
imental design did not provide information on a mechanistic ba-
high-content information that screened potential candidate genes,
transcriptional responses, and metabolic pathways related to
strain-specific differences in lung injury that can be followed up
in mechanistic studies.
Second, analyses of transcripts and metabolites detected
in lung tissue involve limitations. Lung homeostasis is complex
and requires the consideration of contributions from other tis-
sues such as liver, kidney, adipose tissue, and blood. The steady-
could reflect either the activation of an upstream process or the
Figure 6. Transcripts in the enriched nuclear factor erythroid-derived–
2–like 2 (NFE2L2, also known as Nrf2)–mediated oxidative stress re-
sponse 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, ery-
throid-derived–2–like 2 (NFE2L2); activating transcription factor–3
(ATF3); v-maf musculoaponeurotic fibrosarcoma oncogene family, pro-
tein F (avian) (MAFF); FK506 binding protein–5 (FKBP5); glutamate–
cysteine ligase, catalytic subunit (GCLC); thioredoxin reductase–1
(TXNRD1); glutathione peroxidase–2 (GPX2); v-maf musculoaponeur-
otic fibrosarcoma oncogene family, protein K (avian) (MAFK); and glu-
tathione S-transferase, a 1 (Ya) (GSTA1). Values represent means 6 SE
(n ¼ 6 female mice/strain/time), normalized to strain-matched control
mice (0 hours).
Figure 7. 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 fil-
tered air (0 hours, control) or chlorine (45 ppm, 6 or 12 hours), and
metabolome profiling was performed with lung tissue. Values are nor-
malized 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 inter-
vals (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).ySignificantly 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).
Leikauf, Pope-Varsalona, Concel, et al.: Genetics of Acute Lung Injury241
inhibition of a downstream process. Full interpretation of these
results will require further assessments of precursor/product rela-
tionships, biochemical sites of regulation, and combinatorial rate-
limiting steps in multienzymatic pathways.
Third, although chlorine-induced acute lung injury has rele-
vance to accidental human exposures, numerous other agents
can produce acute lung injury (18). The genetic and metabolo-
mic findings here may have been limited by the use of a single
agent. We recently identified candidate genes associated with
acrolein-induced acute lung injury (58) and these genes differed
from those identified with chlorine. Until several types of chem-
ically induced acute lung injury have been evaluated, general-
izations to other forms may not be warranted. Nonetheless,
a major candidate identified previously with acrolein was Acvr1
(Activin A receptor, type 1), which implicated TGFB1 signaling.
In the present study, TGFB1 signaling was also implicated with
chlorine by the identification of Sema7a.
In conclusion, mean survival times varied by approximately
5-fold among strains, and haplotype mapping identified SNP asso-
ciations on chromosomes 1, 4, 5, 9, and 15. Microarrays revealed
several enriched pathways, including protein transport, which de-
creased more in sensitive C57BLKS/J lungs, and the protein cata-
bolic process, which increased more in resistant C57BL/10J lungs.
which decreased more in the C57BLKS/J strain, and glutamine,
which increased more in the C57BL/10J than in the C57BLKS/J
strain. The results from haplotype mapping were evaluated by
an integrated assessment using transcriptomic and metabolomic
profiling. The identified candidate genes associated with increased
susceptibility to acute lung injury in mice included Klf4, Sema7a,
Tns1, Aacs, and an amino acid carrier, Slc38a4. These genes or
genes in related pathways may help direct future human genetic
studies to evaluate such pathways.
Author disclosures are available with the text of this article at www.atsjournals.org.
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