In this study, chlorine produced an approximately 3-fold increase in lavage protein at 12 hours in the sensitive C57BLKS/J strain, or at 24 hours in the resistant C57BL/10J strain (). These findings are similar to those of Zarogiannis and colleagues (17
). Lung histology also indicated that the C57BLKS/J mice developed injury sooner than the C57BL/10J mice (). The finding of perivascular enlargement should be evaluated with caution, because 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 inflammatory 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 phenotype being measured is survival time.
Ascorbate decreased slightly, but not to the extent of statistical significance. Both strains demonstrated nearly equivalent decreases in gulono-1,4-lactone (an ascorbate precursor) and dehydroascorbate (an ascorbate metabolite) (Table E6). Similarly, both strains exhibited a nearly equivalent enrichment of the NFE2L2-mediated oxidative stress pathway (). 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 homeostatic capacities during chlorine-induced lung injury.
We identified 28 candidate genes with SNP associations. We prioritized 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 relationships with altered metabolites identified by metabolomic profiling. This integrative approach revealed 13 candidate genes ( and ), and of these, Klf4, Sema7a, Tns1, Slc38a4, and Aacs were more noteworthy, and could be associated with survival in several ways.
For example, Krüppel-like factor (gut)–4 (KLF4) can protect against lung injury (68
). KLF4, a transcription factor, regulates cadherin-5 expression in adherens junctions, and KLF4 knockdown augments LPS-induced lung injury in mice. KLF4 mRNA also can be induced by other stresses (69
). Here, lung KLF4 transcripts increased more in the resistant C57BL/10J mice than in the sensitive C57BLKS/J mice ().
Another candidate, semaphorin-7A (SEMA7A), can be induced by transforming growth factor–β (TGFB1) and mediates TGFB1-induced alveolar apoptosis (71
polymorphisms are associated with abnormal bone mineral density in Korean women (72
). During acute lung injury, TGFB1 can increase endothelial and epithelial permeability (73
), and the inhibition of TGFB1 can diminish lung injury (66
). SEMA7A can mediate AKT phosphorylation (71
), which is associated with increased cell survival (79
) and is protective during lung injury (80
). In this study, promoter Sema7a
SNPs associated with 12–16% of the difference in survival phenotype () and SEMA7A mRNA increased longer in the resistant C57BL/10J strain, compared with C57BLKS/J strain ().
polymorphisms have been associated with lung function and chronic obstructive lung disease (81
). TNS1, a scaffold 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-β. 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 associated with two Tns1
promoter SNPs () and lungs from the resistant C57BL/10J strain demonstrated a prolonged increase in lung TNS1 mRNA after exposure to chlorine (). In addition, we detected a nonsynonymous SNP (N1882S) in the integrin-β binding domain with an approximately 30% allelic frequency that associated with approximately 20% of the phenotypic difference ( and ).
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 strategies (85
). Alternately, the activation of energy-yielding pathways for ATP production may be required for energetic needs incurred upon injury. Lactate and alanine decreased after chlorine exposure, and these metabolites are the precursors for pyruvate and subsequently acetyl-coenzyme A (acetyl-CoA). Thus, this response implicates an increased utilization of aerobic metabolism 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 energy, 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 transport 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 unclear. SLCO4C1 is protective in kidney disease (89
), and a human SLCO4C1
SNP was associated with preeclampsia (90
). In this study, nonsynonymous SNPs were identified (), but these SNPs did not occur in known functional domains. In addition, lung SLCO4C1 and SLC35A1 transcripts decreased nearly equivalently in the resistant and sensitive strains during chlorine injury (Figure E1).
In contrast, lung SLC38A4 transcripts increased more in the resistant compared with the sensitive mice (). SLC38A4 is a sodium-coupled neutral amino acid (including alanine and glutamine) transporter. The tagSNP (rs32255071) on chromosome 15 was associated with SLC38A4 (−log(P) = 6.25). Promoter Slc38a4 SNPs were associated with 13–16% of the difference in survival ().
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 (Figure E2B). In contrast, the protein catabolic process pathway contained transcripts that increased more in the resistant C57BL/10J strain than in the sensitive C57BLKS/J strain (Figure E3A). Similarly, 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 glutamate (91
). Moreover, glutamine can attenuate acute lung injury by inducing heat-shock proteins (92
). Thus, of the three solute carrier proteins identified, SLC38A4 is worthy of additional investigation.
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 metabolism in most tissues, energetics in the lung are complex, as manifested by multiple substrate usage. Here, glucose was unchanged, whereas lactate decreased equally between strains. Interestingly, a fatty acid β-oxidation ketone body, BHBA, increased in both strains initially, but was maintained longer in resistant C57BL/10J mice, possibly reflecting greater fatty-acid β-oxidation in the resistant strain. Previously, Fox and colleagues (95
) measured oxidation rates of glucose, glutamine, lactate, and BHBA in alveolar Type II cells from fetal rats. The CO2
formation 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 (~ 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 phospholipids (mainly dipalmitoylglycerophosphocholine), surfactant proteins, electrolytes, and other biomolecules. Surfactant-associated protein B (SFTPB) mRNA decreased in the sensitive C57BLKS/J mice more rapidly than in the C57BL/10J mice (i.e., log2 = −1.2 versus −0.3 at 6 hours, respectively). This is relevant because maintaining SFTPB is critical to survival during acute lung injury in mice (19
Surfactant lipid production uses glucose-dependent fatty-acid synthesis, but fatty acids can also be generated from lactate or ketone 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 palmitoylglycerophosphocholines, and thus have a potential role in supplying adequate 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
). Nonsynonymous SNPs were identified in Aacs
that could lead to Thr321Ile substitution in the acyl-protein synthetase domain (). 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 synthesis (100
). Two lysophospholipids, 1-palmitoleoylglycerophosphocholine and 1-palmitoleoylglycerophosphoinositol, were decreased 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 κB kinase complex–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
). 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 IκB kinases into an active complex (104
). Later studies, however, failed to confirm a role in NF-κB activation, and instead reported IKBKAP to be involved in transcription elongation (105
), Jun N-terminal kinase-mediated stress signaling (106
), and cell migration (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 (). The Gly662Val substitution is in the IKI3 domain likely to be involved in IKBKAP's transcriptional elongation function.
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 experimental design did not provide information on a mechanistic basis for acute lung injury. Rather, this study was designed to provide 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 tissues such as liver, kidney, adipose tissue, and blood. The steady-state level of transcripts or metabolites in a pathway is limited, and could reflect either the activation of an upstream process or the inhibition of a downstream process. Full interpretation of these results will require further assessments of precursor/product relationships, biochemical sites of regulation, and combinatorial rate-limiting steps in multienzymatic pathways.
Third, although chlorine-induced acute lung injury has relevance to accidental human exposures, numerous other agents can produce acute lung injury (18
). The genetic and metabolomic 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 chemically induced acute lung injury have been evaluated, generalizations 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 associations on chromosomes 1, 4, 5, 9, and 15. Microarrays revealed several enriched pathways, including protein transport, which decreased more in sensitive C57BLKS/J lungs, and the protein catabolic process, which increased more in resistant C57BL/10J lungs. Lung metabolomic profiling revealed that 95 of the 280 metabolites measured were altered by chlorine exposures, and included alanine, 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.