The present report demonstrates that lung injury, inflammation, and oxidative damage related to inhalation of CEES were ameliorated by AEOL 10150 treatment. Specifically, increased BALF LDH following CEES inhalation demonstrated increased cellular injury. Protein and IgM levels in BALF were increased following CEES exposure. All of these indicators of lung injury were diminished by AEOL 10150 treatment. CEES inhalation increased PMN in the BALF and lung tissue. Treatment with AEOL 10150 decreased PMN infiltration. CEES inhalation also increased oxidative stress, which was also diminished by AEOL 10150 treatment. Finally, we demonstrated in histopathologic studies the presence of variable proteinaceous exudate and epithelial loss following CEES inhalation. These changes also appeared to be diminished by AEOL 10150 treatment, consistent with the findings of BALF protein and IgM.
Real world SM exposures can vary dramatically due to ambient temperatures, proximity, and air flow [2
]. Injury can vary from mild to lethal, although human mortality due to SM exposure in military settings is normally low at 2–3% [2
]. In battlefield conditions initial aerosolization is usually via explosives, and accurate measurements of concentrations delivered are not available. Following human exposures, a symptom-free interval of several hours to days occurs in inverse correlation with the absorbed dose [25
]. This delay is also mirrored in rat toxicokinetic studies showing SM is eliminated in a two-compartment model with a quick distribution (t1/2α
=5.56 min), and a long terminal half life (t1/2β
]. As mortality is low and injury is delayed, these initial studies investigated a concentration of CEES that resulted in delayed lung injury with minimal mortality.
SM produces dose-dependent damage to the respiratory tract, beginning with the upper airways and descending to the lower airways as the exposure level increases [27
]. Previous studies of lung injury have utilized intratracheal (IT) delivery of CEES or SM [6
]. While this does allow for direct delivery to the airways, it also results in focal injury that differs from an actual inhalation exposure. SM has little effect on lung parenchyma, although distal airways involvement is noted in severe exposure cases [31
Lung injury, and specifically central airways injury, is a hallmark of SM inhalation injury. In the absence of inflammatory cell infiltration in the BALF, an increase in LDH would be indicative of epithelial damage. Our data shows increases in BALF RBCs and PMNs, which when lysed also could also contribute to elevated BALF LDH activities. Thus, care must be taken in interpreting these data as exclusively representing epithelial damage. Previous studies have demonstrated LDH can be oxidatively inactivated [32
]. This could lead to underestimation of LDH activity. Paradoxically, when antioxidant treatment is present, LDH levels could be even more elevated as compared to animals receiving vehicle. This was not the case in this study, as the vehicle-treated, CEES-exposed group had increased BALF LDH activities that were decreased by treatment with AEOL 10150.
Increases in BALF protein and IgM were evident following CEES exposure. Increased protein could be indicative of cellular necrosis, vascular leak of plasma contents, or both. However, the presence of the high molecular weight immunoglobin IgM in the BALF was strongly indicative of increased permeability of vascular endothelium and movement of fluid into airways. Luminal entry of plasma is often associated with epithelial damage and sloughing [33
]. However, it has also been shown that plasma exudates can move between epithelial cells into the airway lumen even when the epithelial barrier is visibly intact [35
]. At the light microscopic level, airway epithelium appeared mostly intact in both diluent- and AEOL 101050-treated groups. Occasionally, we saw minor epithelial damage in some airways while others appears undamaged at the light microscopic level. It is therefore possible that both loss of epithelium and leak between epithelial cells play a role in allowing increased plasma components to enter the airway lining fluid. BALF changes indicative of this type of lung injury were decreased by AEOL 10150 treatment.
Acute inflammation is typified by increases in PMNs at the site of injury. While PMN are beneficial in phagocytosis of bacteria and possibly even damaged cells, their production of large quantities of ROS can damage local tissue. In fact, in previous studies, depletion of PMN prior to CEES exposure reduced lung injury indices [6
]. While we cannot assume that injury is due exclusively to ROS production by PMN, the current study shows that AEOL 10150 treatment reduced PMN infiltration and, very possibly, PMN activation. This reduction in PMNs entry into lung should therefore result in reduced ROS production. A similar metalloporphyrin antioxidant (MnTBAP) also attenuated PMN increases in a model of paraquat-induced lung injury [37
]. Another catalytic metalloporphyrin reduced inflammation in an asthma model as well as a hyperoxia-induced model of bronchopulmonary dysplasia [38
]. Inflammatory cells contribute to increased ROS production, but are not the only source in SM or CEES exposure. Studies of CEES in vitro
have demonstrated diminished mitochondrial membrane potential and subsequent ROS production [15
]. AEOL 10150 protected lung epithelial cells from mitochondrial damage in that model. Besides direct scavenging of ROS, potential mechanisms by which AEOL 10150 could improve indices of injury and disease are still not clear.
One outcome of oxidative stress is an increase in oxidation of macromolecules. In this study, CEES inhalation resulted in increased lung DNA oxidation, as measured by 8-OHdG. Whether DNA oxidation was due directly to CEES, its metabolites, or ROS formed as a result of CEES is not clear. CEES exposure is also known to cause lipid peroxidation [8
]. 4-HNE is a biologically active aldehyde and a stable end product of lipid peroxidation. While often viewed as simply a marker of injury, 4-HNE has biological functions as well. Increased 4-HNE levels have been linked to endothelial barrier dysfunction resulting in increased permeability [40
]. Thus, lipid peroxidation may be a mechanism by which CEES-induced permeability occurred. AEOL 10150 may have reduced lung injury, in part, by limiting this process.
Microscopic examination of fixed airways following CEES inhalation showed the presence of proteinaceous exudate in varying levels of the conducting airways. By contrast, this material was not detected in alveolar spaces, as detected by light microscopy, consistent with previous reports of SM exposure. Following CEES exposure, the exudates were not consistently noted in all airways. Sectioning in consistent regions of the lung did not always yield protein exudates and these exudates were present in varying concentrations, even within the same animal. A few sloughed airway epithelial cells were noted in BALF, but different animals were used for fixation and lavage, so we could not determine correlation. Material in the exudates may not have been freely soluble and accessible by lavage. In addition, it is possible that some of these exudates could have been lost during fixation, potentially underestimating damage. There are parallels between airways injury by CEES and following burn or smoke inhalation, the latter of which results in plasma leak from the bronchial circulation [42
]. This provides an interesting potential avenue for future mechanistic investigation.
Despite extensive studies of SM/CEES exposure over more than a century, the mechanisms underlying airway injury have not been fully elucidated. Injury from SM was previously believed to be due to direct alkylation of macromolecules. In fact, the major in vivo
metabolites are GSH conjugates [44
]. Depletion of this important endogenous antioxidant can allow increases in oxidants to go unchecked. Antioxidants have shown efficacy in a number of models of SM/CEES exposure, although the mechanism of protection is not clear. In addition, recent studies have shown that sulfur and nitrogen mustards can react with cellular reductases, altering electron transfer and increasing free radical production [45
]. The efficacy of AEOL 10150 is often attributed to its ability to directly scavenge oxidants. AEOL 10150 and similar metalloporphyrin compounds also have the capacity to redox cycle with cellular reductase domains in enzymes including cytochrome P450 reductase, NADH oxidase, and nitric oxide synthases [9
]. Although redox cycling is typically considered deleterious, AEOL 10150 counteracts such effects with its antioxidant capacity.
The two dose protocol for AEOL 10150 treatment was based on success with previous studies of radiation-induced lung injury [17
]. Having initially found two doses of the compound to be effective in this short term model, we later investigated whether one dose, either at 1 hour or 9 hours post exposure would be effective. As the data demonstrate, one dose was not effective at the tested concentration. Investigation of pharmacokinetics of this compound indicates that by 8 hours post injection, blood levels of AEOL 10150 are minimal but still detectable. Given the 8 hour difference between doses in our two dose protocol, it appears that, in order to be effective, there must be at least a minimal level of drug in the plasma. In vitro
studies have shown an increase in mitochondrial ROS peaking at 12 hours after CEES exposure, which was ameliorated by AEOL 10150 treatment beginning one hour after exposure [15
]. This demonstration of delayed injury seen with in vitro CEES exposure may provide some basis for the requirement of a second dose of the drug.
The present results build on current literature that has demonstrated that oxidative stress plays a role in the pathogenesis of CEES-induced lung injury. Herein, we demonstrated that substantial rescue of CEES-induced lung injury, inflammation, and oxidative stress was possible with AEOL 10150. Further studies are needed to determine whether AEOL 10150 also can afford protection against lung injury due to inhalation of sulfur mustard.