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E-cigarette consumption is under scrutiny by regulatory authorities due to concerns about product toxicity, lack of manufacturing standards, and increasing reports of e-cigarette- or vaping-associated acute lung injury. In vitro studies have demonstrated cytotoxicity, mitochondrial dysfunction, and oxidative stress induced by unflavored e-cigarette aerosols and flavoring additives. However, e-cigarette effects on the complex lung parenchyma remain unclear. Herein, the impact of e-cigarette condensates with or without menthol flavoring on functional, structural, and cellular responses was investigated using mouse precision cut lung slices (PCLS). PCLS were exposed to e-cigarette condensates prepared from aerosolized vehicle, nicotine, nicotine + menthol, and menthol e-fluids at doses from 50 to 500 mM. Doses were normalized to the glycerin content of vehicle. Video-microscopy of PCLS revealed impaired contractile responsiveness of airways to methacholine and dampened ciliary beating following exposure to menthol-containing condensates at concentrations greater than 300 mM. Following 500 mM menthol-containing condensate exposure, epithelial exfoliation in intrabronchial airways was identified in histological sections of PCLS. Measurement of lactate dehydrogenase release, mitochondrial water-soluble-tetrazolium salt-1 conversion, and glutathione content supported earlier findings of nicotine or nicotine + menthol e-cigarette-induced dose-dependent cytotoxicity and oxidative stress responses. Evaluation of PCLS metabolic activity revealed dose-related impairment of mitochondrial oxidative phosphorylation and glycolysis after exposure to menthol-containing condensates. Taken together, these data demonstrate prominent menthol-induced pulmonary toxicity and impairment of essential physiological functions in the lung, which warrants concerns about e-cigarette consumer safety and emphasizes the need for further investigations of molecular mechanisms of toxicity and menthol effects in an experimental model of disease.

Exposure to ozone causes decrements in pulmonary function, a response associated with alterations in lung lipids. Pulmonary lipid homeostasis is dependent on the activity of peroxisome proliferator activated receptor gamma (PPARγ), a nuclear receptor that regulates lipid uptake and catabolism by alveolar macrophages (AMs). Herein, we assessed the role of PPARγ in ozone-induced dyslipidemia and aberrant lung function in mice. Exposure of mice to ozone (0.8 ppm, 3 hr) resulted in a significant reduction in lung hysteresivity at 72 hr post exposure; this correlated with increases in levels of total phospholipids, specifically cholesteryl esters, ceramides, phosphatidylcholines, phosphorylethanolamines, sphingomyelins, and di- and triacylglycerols in lung lining fluid. This was accompanied by a reduction in relative surfactant protein-B (SP-B) content, consistent with surfactant dysfunction. Administration of the PPARγ agonist, rosiglitazone (5 mg/kg/day, i.p.) reduced total lung lipids, increased relative amounts of SP-B, and normalized pulmonary function in ozone-exposed mice. This was associated with increases in lung macrophage expression of CD36, a scavenger receptor important in lipid uptake and a transcriptional target of PPARγ. These findings highlight the role of alveolar lipids as regulators of surfactant activity and pulmonary function following ozone exposure and suggest that targeting lipid uptake by lung macrophages may be an efficacious approach for treating altered respiratory mechanics.

A characteristic of cytochrome P450 (CYP) enzymes is their ability to generate H

Macrophage efferocytosis of apoptotic neutrophils (PMNs) plays a key role in the resolution of inflammation. In these studies, we describe a novel flow cytometric method to assess efferocytosis of apoptotic PMNs. Resident alveolar macrophages and PMNs were collected from lungs of mice exposed to inhaled ozone (0.8 ppm, 3 h) followed by lipopolysaccharide (3 mg/kg, i.v.) to induce acute lung injury. PMNs were labeled with PKH26 or DilC

Nitrogen mustard (NM) causes acute lung injury, which progresses to fibrosis. This is associated with a macrophage-dominant inflammatory response and the production of proinflammatory/profibrotic mediators, including tumor necrosis factor alpha (TNF-α). Herein, we refined magnetic resonance imaging (MRI) and computed tomography (CT) imaging methodologies to track the progression of NM-induced lung injury in rodents and assess the efficacy of anti-TNF-α antibody in mitigating toxicity. Anti-TNF-α antibody was administered to rats (15 mg/kg, every 8 days, intravenously) beginning 30 min after treatment with phosphate-buffered saline control or NM (0.125 mg/kg, intratracheally). Animals were imaged by MRI and CT prior to exposure and 1-28 days postexposure. Using MRI, we characterized acute lung injury and fibrosis by quantifying high-signal lung volume, which represents edema, inflammation, and tissue consolidation; these pathologies were found to persist for 28 days following NM exposure. CT scans were used to assess structural components of the lung and to register changes in tissue radiodensities. CT scans showed that in control animals, total lung volume increased with time. Treatment of rats with NM caused loss of lung volume; anti-TNF-α antibody mitigated this decrease. These studies demonstrate that MRI and CT can be used to monitor lung disease and the impact of therapeutic intervention.

Sulfur mustard (SM) inhalation causes debilitating pulmonary injury in humans which progresses to fibrosis. Herein, we developed a rat model of SM toxicity which parallels pathological changes in the respiratory tract observed in humans. SM vapor inhalation caused dose (0.2–0.6 mg/kg)-related damage to the respiratory tract within 3 days of exposure. At 0.4–0.6 mg/kg, ulceration of the proximal bronchioles, edema and inflammation were observed, along with a proteinaceous exudate containing inflammatory cells in alveolar regions. Time course studies revealed that the pathologic response was biphasic. Thus, changes observed at 3 days post-SM were reduced at 7–16 days; this was followed by more robust aberrations at 28 days, including epithelial necrosis and hyperplasia in the distal bronchioles, thickened alveolar walls, enlarged vacuolated macrophages, and interstitial fibrosis. Histopathologic changes were correlated with biphasic increases in bronchoalveolar lavage (BAL) cell and protein content and proliferating cell nuclear antigen expression. Proinflammatory proteins receptor for advanced glycation end product (RAGE), high-mobility group box protein (HMGB)-1, and matrix metalloproteinase (MMP)-9 also increased in a biphasic manner following SM inhalation, along with surfactant protein-D (SP-D). Tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS), inflammatory proteins implicated in mustard lung toxicity, and the proinflammatory/profibrotic protein, galectin (Gal)-3, were upregulated in alveolar macrophages and in bronchiolar regions at 3 and 28 days post-SM. Inflammatory changes in the lung were associated with oxidative stress, as reflected by increased expression of heme oxygenase (HO)-1. These data demonstrate a similar pathologic response to inhaled SM in rats and humans suggesting that this rodent model can be used for mechanistic studies and for the identification of efficacious therapeutics for mitigating toxicity.

A sensitive fluorescence assay based on Amplex Red (AR) oxidation by horseradish peroxidase (AR/HRP) is described which continuously monitor rates of H(2)O(2) production by microsomal enzymes in the presence of relatively high concentrations of NADPH. NADPH and NADH are known to interact with HRP and generate significant quantities of superoxide anion, a radical that spontaneously dismutates to form H(2)O(2) which interferes with the AR/HRP assay. Microsomal enzymes generate H(2)O(2) as a consequence of electron transfer from NADPH to cytochrome P450 hemoproteins with subsequent oxygen activation. We found that superoxide anion formation via the interaction of NADPH with HRP was inhibited by superoxide dismutase (SOD) without affecting H(2)O(2) generation by microsomal enzymes. Using SOD in enzyme assays, we consistently detected rates of H(2)O(2) production using microgram quantities of microsomal proteins (2.62 ± 0.20 picomol/min/μg protein for liver microsomes from naïve female rats, 12.27 ± 1.29 for liver microsomes from dexamethasone induced male rats, and 2.17 ± 0.25 picomol/min/μg protein for human liver microsomes). This method can also be applied to quantify rates of H(2)O(2) production by oxidases where superoxide anion generation by NADH or NADPH and HRP can interfere with enzyme assays.

The use of chemical warfare agents (CWAs) in military conflicts and against civilians is a recurrent problem. Despite ongoing CWA research using in vitro or in vivo models, progress to elucidate mechanisms of toxicity and to develop effective therapies, decontamination procedures, and general countermeasures is still limited. Novel scientific approaches to address these questions are needed to expand perspectives on existing knowledge and gain new insights. To achieve this, the use of ex vivo techniques like precision-cut tissue slices (PCTSs) can be a valuable approach. Existing studies employing this economical and relatively easy to implement method show model suitability and comparability with the use of in vitro and in vivo models. In this article, we review research on CWAs in PCTSs to illustrate the advantages of the approach and to promote future applications.

Nitrogen mustard (NM) and sulfur mustard are cytotoxic alkylating agents that cause severe and progressive damage to the respiratory tract. Evidence indicates that macrophages play a key role in the acute inflammatory phase and the later resolution/profibrotic phase of the pathogenic response. These diverse roles are mediated by inflammatory macrophages broadly classified as M1 proinflammatory and M2 anti-inflammatory that sequentially accumulate in the lung in response to injury. The goal of the present study was to identify signaling mechanisms contributing to macrophage activation in response to mustards. To accomplish this, we used RNA sequencing to analyze the gene expression profiles of lung macrophages isolated 1 and 28 days after intratracheal exposure of rats to NM (0.125 mg/kg) or phosphate-buffered saline control. We identified 641 and 792 differentially expressed genes 1 and 28 days post-NM exposure, respectively. These genes are primarily involved in processes related to cell movement and are regulated by cytokines, including tumor necrosis factor-α, interferon-γ, and interleukin-1β. Some of the most significantly enriched canonical pathways included STAT3 and NF-κB signaling. These cytokines and pathways may represent potential targets for therapeutic intervention to mitigate mustard-induced lung toxicity.

Laminin-332 is a basement membrane protein composed of three genetically distinct polypeptide chains that actively promote both skin epidermal cell adhesion and migration. Proteolytic fragments of the laminin γ2 chain stimulate migration and scattering of keratinocytes and cancer cells. Sulfur mustard (SM) is a bifunctional alkylating agent that induces separation of basal keratinocytes from the dermal-epidermal junction and invokes a strong inflammatory response leading to delayed wound repair. In the present studies, the role of laminin γ2 in SM-induced skin injury and wound repair was investigated using the mouse ear vesicant model. We found that laminin γ2 chain mRNA was preferentially upregulated in mouse ear skin exposed to SM. In situ hybridization confirmed overexpression of laminin γ2 transcript. Western blot analysis showed increased protein expression of the full-length proform of laminin γ2 and smaller processed fragments of laminin γ2 in skin exposed to SM. Dual immunofluorescence labeling indicated that laminin γ2 fragments are prevalent in suprabasal keratinocytes behind the leading edge in areas of hyperplasia in injured skin. In addition, co-expression of laminin γ2 and the senescent marker, p16-INK4a was found to overlap with the hyperplastic migratory epithelial sheet. This observation is similar to hypermotile keratinocytes reported in invasive carcinoma cells. Overall, our studies indicate that laminin γ2 is preferentially expressed in skin post SM exposure and that protein expression appears to become progressively more fragmented. The laminin γ2 fragments may play a role in regulating SM-induced skin wound repair. Anat Rec, 2020. © 2020 American Association for Anatomy.

Cytotoxic blistering agents such as sulfur mustard and nitrogen mustard (HN2) were synthesized for chemical warfare. Toxicity is due to reactive chloroethyl side chains that modify and damage cellular macromolecules including DNA and proteins. In response to DNA damage, cells initiate a DNA damage response directed at the recruitment and activation of repair-related proteins. A central mediator of the DNA damage response is p53, a key protein that plays a critical role in regulating DNA repair. We found that HN2 causes cytosolic and nuclear accumulation of p53 in HaCaT keratinocytes; HN2 also induced post-translational modifications on p53 including S15 phosphorylation and K382 acetylation, which enhance p53 stability, promote DNA repair, and mediate cellular metabolic responses to stress. HN2 also cross-linked p53, forming dimers and high-molecular-weight protein complexes in the cells. Cross-linked multimers were also modified by K48-linked ubiquitination indicating that they are targets for proteasome degradation. HN2-induced modifications transiently suppressed the transcriptional activity of p53. Using recombinant human p53, HN2 alkylation was found to be concentration- and redox status-dependent. Dithiothreitol-reduced protein was more efficiently cross-linked indicating that p53 cysteine residues play a key role in protein modification. LC-MS/MS analysis revealed that HN2 directly modified p53 at C124, C135, C141, C176, C182, C275, C277, H115, H178, K132, and K139, forming both monoadducts and cross-links. The formation of intermolecular complexes was a consequence of HN2 cross-linked cysteine residues between two molecules of p53. Together, these data demonstrate that p53 is a molecular target for mustard vesicants. Modification of p53 likely mediates cellular responses to HN2 including DNA repair and cell survival contributing to vesicant-induced cytotoxicity.

Ozone is a ubiquitous air pollutant that causes lung damage and altered functioning. Evidence suggests that proinflammatory macrophages contribute to ozone toxicity. Herein, we analyzed the role of extracellular vesicles (EVs) and microRNA (miRNA) cargo in ozone-induced macrophage activation. Exposure of mice to ozone (0.8 ppm, 3 h) resulted in increases in bronchoalveolar lavage fluid EVs, which were comprised predominantly of microvesicles (MVs). NanoFACS analysis revealed that MVs generated following both air and ozone exposure was largely from CD45+ myeloid cells; these MVs were readily taken up by macrophages. Functionally, MVs from ozone, but not air treated mice, upregulated mRNA expression of inflammatory proteins in macrophages including inducible nitric oxide synthase (iNOS), CXCL-1, CXCL-2, and interleukin (IL)-1β. The miRNA profile of MVs in bronchoalveolar lavage fluid (BALF) was altered after ozone exposure; thus, increases in miR-21, miR-145, miR320a, miR-155, let-7b, miR744, miR181, miR-17, miR-92a, and miR-199a-3p were observed, whereas miR-24-3p and miR-20 were reduced. Ingenuity pathway analysis revealed that these miRNAs regulate pathways that promote inflammatory macrophage activation, and predicted that let-7a-5p/let-7b, miR-24-3p, miR-21-5p, miR-17, and miR-181a-5p are key upstream regulators of inflammatory proteins. After ozone exposure, miR-199a-3p, but not precursor miR-199a-3p, was increased in lung macrophages, indicating that it is derived from MV-mediated delivery. Furthermore, lung macrophage mRNA expression of IL-1β was upregulated after administration of MVs containing miR-199a-3p mimic but downregulated by miR-199a-3p inhibitor. Collectively, these data suggest that MVs generated following ozone exposure contribute to proinflammatory macrophage activation via MV-derived miRNAs including miR-199a-3p. These findings identify a novel pathway regulating macrophage inflammatory responses to inhaled ozone.

Nitrogen mustard (NM) is a cytotoxic vesicant known to cause acute lung injury which progresses to fibrosis. Alveolar Type II cells are primarily responsible for surfactant production; they also play a key role in lung repair following injury. Herein, we assessed the effects of NM on Type II cell activity. Male Wistar rats were administered NM (0.125 mg/kg) or PBS control intratracheally. Type II cells, lung tissue and BAL were collected 3 d later. NM exposure resulted in double strand DNA breaks in Type II cells, as assessed by expression of γH2AX; this was associated with decreased expression of the DNA repair protein, PARP1. Expression of HO-1 was upregulated and nitrotyrosine residues were noted in Type II cells after NM exposure indicating oxidative stress. NM also caused alterations in Type II cell energy metabolism; thus, both glycolysis and oxidative phosphorylation were reduced; there was also a shift from a reliance on oxidative phosphorylation to glycolysis for ATP production. This was associated with increased expression of pro-apoptotic proteins activated caspase-3 and -9, and decreases in survival proteins, β-catenin, Nur77, HMGB1 and SOCS2. Intracellular signaling molecules important in Type II cell activity including PI3K, Akt2, phospho-p38 MAPK and phospho-ERK were reduced after NM exposure. This was correlated with dysregulation of surfactant protein production and impaired pulmonary functioning. These data demonstrate that Type II cells are targets of NM-induced DNA damage and oxidative stress. Impaired functioning of these cells may contribute to pulmonary toxicity caused by mustards.

Nitrogen mustard (NM) is a cytotoxic vesicant known to cause acute lung injury which progresses to fibrosis; this is associated with a sequential accumulation of pro- and anti-inflammatory macrophages in the lung which have been implicated in NM toxicity. Farnesoid X receptor (FXR) is a nuclear receptor involved in regulating lipid homeostasis and inflammation. In these studies, we analyzed the role of FXR in inflammatory macrophage activation, lung injury and oxidative stress following NM exposure. Wild-type (WT) and FXR

Sulfur mustard (SM) is a bifunctional alkylating agent that causes severe injury to the respiratory tract. This is accompanied by an accumulation of macrophages in the lung and the release of the proinflammatory cytokine, tumor necrosis factor (TNF)α. In these studies, we analyzed the effects of blocking TNFα on lung injury, inflammation and oxidative stress induced by inhaled SM. Rats were treated with SM vapor (0.4 mg/kg) or air control by intratracheal inhalation. This was followed 15–30 min later by anti-TNFα antibody (15 mg/kg, i.v.) or PBS control. Animals were euthanized 3 days later. Anti-TNFα antibody was found to blunt SM-induced peribronchial edema, perivascular inflammation and alveolar plasma protein and inflammatory cell accumulation in the lung; this was associated with reduced expression of PCNA in histologic sections and decreases in BAL levels of fibrinogen. SM-induced increases in inflammatory proteins including soluble receptor for glycation end products, its ligand, high mobility group box-1, and matrix metalloproteinase-9 were also reduced by anti-TNFα antibody administration, along with increases in numbers of lung macrophages expressing TNFα, cyclooxygenase-2 and inducible nitric oxide synthase. This was correlated with reduced oxidative stress as measured by expression of heme oxygenase-1 and Ym-1. Together, these data suggest that inhibiting TNFα may represent an efficacious approach to mitigating acute lung injury, inflammatory macrophage activation, and oxidative stress induced by inhaled sulfur mustard.

Nitrogen mustard (NM) is a vesicant known to target the lung, causing acute injury which progresses to fibrosis. Evidence suggests that activated macrophages contribute to the pathologic response to NM. In these studies, we analyzed the role of lung lipids generated following NM exposure on macrophage activation and phenotype. Treatment of rats with NM (0.125 mg/kg, i.t.) resulted in a time-related increase in enlarged vacuolated macrophages in the lung. At 28 days postexposure, macrophages stained positively for Oil Red O, a marker of neutral lipids. This was correlated with an accumulation of oxidized phospholipids in lung macrophages and epithelial cells and increases in bronchoalveolar lavage fluid (BAL) phospholipids and cholesterol. RNA-sequencing and immunohistochemical analysis revealed that lipid handling pathways under the control of the transcription factors liver-X receptor (LXR), farnesoid-X receptor (FXR), peroxisome proliferator-activated receptor (PPAR)-ɣ, and sterol regulatory element-binding protein (SREBP) were significantly altered following NM exposure. Whereas at 1–3 days post NM, FXR and the downstream oxidized low-density lipoprotein receptor, Cd36, were increased, Lxr and the lipid efflux transporters, Abca1 and Abcg1, were reduced. Treatment of naïve lung macrophages with phospholipid and cholesterol enriched large aggregate fractions of BAL prepared 3 days after NM exposure resulted in upregulation of Nos2 and Ptgs2, markers of proinflammatory activation, whereas large aggregate fractions prepared 28 days post NM upregulated expression of the anti-inflammatory markers, Il10, Cd163, and Cx3cr1, and induced the formation of lipid-laden foamy macrophages. These data suggest that NM-induced alterations in lipid handling and metabolism drive macrophage foam cell formation, potentially contributing to the development of pulmonary fibrosis.

Available studies, while limited in number, suggest that e-cigarette vaping induces oxidative stress, with one potential mechanism being the direct formation of reactive oxygen species (ROS) in e-vapor. In the present studies, we measured the formation of hydroxyl radical ((•)OH), the most destructive ROS, in e-vapor under a range of vaping patterns (i.e., power settings, solvent concentrations, flavorings). Study results show that increased power output and puff volume correspond with the formation of significantly higher amounts of (•)OH in e-vapor because of elevated coil temperature and oxygen supply. Vegetable glycerin (VG) e-liquids generated higher (•)OH levels than propylene glycol (PG) e-liquids, as did flavored e-liquids relative to nonflavored e-liquids. E-vapor in combination with ascorbic acid, which is an abundant biological molecule in human epithelial lining fluid, can also induce (•)OH formation. The dose of radical per puff associated with e-cigarette vaping was 10–1000 times lower than the reported dose generated by cigarette smoking. However, the daily average (•)OH dose can be comparable to that from cigarette smoking depending on vaping patterns. Overall, e-cigarette users who use VG-based flavored e-cigarettes at higher power output settings may be at increased risk for (•)OH exposures and related health consequences such as asthma and chronic obstructive pulmonary disease.

Nitrogen mustard, mechlorethamine (bis(2-chloroethyl)methylamine; HN2), and sulfur mustard are potent vesicants that modify and disrupt cellular macromolecules, including DNA leading to cytotoxicity and tissue injury. In many cell types, HN2 upregulates DNA damage signaling pathways including ataxia telangiectasia mutated (ATM), ataxia telangiectasia mutated- and Rad3-related (ATR), as well as DNA-dependent protein kinase catalytic subunit (DNA-PKcs). In the present studies, we investigated crosstalk between HN2-induced DNA damage response and cell cycle progression using human A549 lung epithelial cells. HN2 (1–20 µM; 24 h) caused a concentration-dependent arrest of cells in the S and G2/M phases of the cell cycle. This was associated with inhibition of DNA synthesis, as measured by incorporation of 5-ethynyl-2’-deoxyuridine (EdU) into S phase cells. Cell cycle arrest was correlated with activation of DNA damage and cell cycle checkpoint signaling. Thus, HN2 treatment resulted in time- and concentration-dependent increases in expression of phosphorylated ATM (Ser1981), Chk2 (Thr68), H2AX (Ser139), and p53 (Ser15). Activation of DNA damage signaling was most pronounced in S phase cells followed by G2/M phase cells. HN2-induced cell cycle arrest was suppressed by the ATM and DNA-PKcs inhibitors, KU55933 and NU7441, respectively, and to a lesser extent by VE821, an ATR inhibitor. This was correlated with abrogation of DNA damage checkpoints signaling. These data indicate that activation of ATM, ATR, and DNA-PKcs signaling pathways by HN2 are important in the mechanism of vesicant-induced cell cycle arrest and cytotoxicity. Drugs that inhibit activation of DNA damage signaling may be effective countermeasures for vesicant-induced tissue injury.

Sulfur mustard (SM; bis(2-chloroethyl) sulfide) is a highly reactive bifunctional alkylating agent synthesized for chemical warfare. The eyes are particularly sensitive to SM where it causes irritation, pain, photophobia, and blepharitis, depending on the dose and duration of exposure. In these studies, we examined the effects of SM vapor on the corneas of New Zealand white male rabbits. Edema and hazing of the cornea, signs of acute injury, were observed within one day of exposure to SM, followed by neovascularization, a sign of chronic or late phase pathology, which persisted for at least 28 days. Significant epithelial-stromal separation ranging from ~8-17% of the epithelial surface was observed. In the stroma, there was a marked increase in CD45

The study aims to develop high drug-loaded (about 15% lipid matrix) curcumin solid lipid nanoparticles (CSLNs) for wound healing. CSLNs prepared by hot, high-pressure homogenization, without using organic solvents, were optimized using the Taguchi design followed by the central composite design. The optimized CSLNs exhibited a high assay/drug content (0.6% w/w), solubility (6 × 105 times), and EE (75%) with a particle size &lt, 200 nm (PDI—0.143). The CSLNs were safe (in vitro and in vivo), photostable, autoclavable, stable up to one year at 30 °C and under refrigeration and exhibited a controlled release (zero-order, 5 days). XRD, FTIR, and DSC confirmed solubilization and entrapment of the curcumin within the SLNs. TEM and FESEM revealed a smooth and spherical shape. The CSLNs showed a significant antimicrobial effect (MIC of 64 µg/mL for planktonic cells, 512 µg/mL for biofilm formation, and 2 mg/mL for mature biofilm) against Staphylococcus aureus 9144, while free curcumin dispersion did not exhibit any effect. This is the first report on the disruption of mature biofilms by curcumin solid lipid nanoparticles (CSLNs). The cell proliferation potential of CSLNs was also evaluated in vitro while the wound healing potential of CSLNs (incorporated in a hydrogel) was assessed in vivo. In (i) nitrogen mustard gas and (ii) a full-thickness excision wound model, CSLNs exhibited (a) significantly faster wound closure, (b) histologically and immunohistochemically better healing, (c) lower oxidative stress (LPO) and (d) inflammation (TNFα), and (e) increased angiogenesis (VEGF) and antioxidant enzymes, i.e., catalase and GSH levels. CSLNs thus offer a promising modern wound therapy especially for infected wounds, considering their effects in mature biofilm disruption.

The natural product 8-methoxypsoralen (methoxsalen or 8-MOP) in combination with long wavelength ultraviolet light (UVA, 320–400 nm), also referred to as PUVA therapy, is used for the treatment of cutaneous proliferative disorders including psoriasis, vitiligo and mycosis fungoides. The use of 8-MOP (3) is limited by its poor water solubility and there remains a need to develop more water-soluble psoralens to enhance bioavailability following oral administration of the drug. In the present studies a water-soluble dimethylaminoethyl ether analog of 8-MOP was synthesized and analyzed for biological activity. This analog, (8-[2-(N,N-dimethylamino)ethoxy]-psoralen hydrochloride (1) [or CAS name: 9-[2-(dimethylamino)ethoxy]-7H-furo[3,2-g][1]benzopyran-7-one, hydrochloride], was found to be significantly more active than 3 in keratinocyte growth inhibition assays (IC(50) = 12 nM and 130 nM for 1 and 3, respectively). The partially reduced dihydro derivative of 1, 8-[2-(N,N-dimethylamino)ethoxy]-4’,5’-dihydropsoralen hydrochloride (2) [or CAS name: 9-[2-(dimethylamino)ethoxy]-2,3-dihydro-7H-furo[3,2-g][1]benzopyran-7-one, hydrochloride] and the partially reduced 4’,5’-dihydro-8-methoxypsoralen (4) lacking the water-solubilizing side-chain were significantly less active. As inhibitors of keratinocyte growth they ranked as IC(50) = 13,000 nM and 70,000 nM for 2 and 4, respectively, indicating that an unsaturated furan ring in the psoralen was required for maximal activity. Compound (1) was found to readily intercalate and damage DNA following UVA light treatment as determined by plasmid DNA nicking and unwinding experiments in neutral and alkaline agarose gels. Taken together, these data demonstrate that a water-soluble dimethylaminoethyl ether psoralen targets DNA, is highly active as a photosensitizer, and may be useful in the treatment of skin diseases involving abnormal keratinocyte proliferation.

Inflammatory macrophages are known to contribute to ozone toxicity. Farnesoid X receptor (FXR) is a nuclear receptor involved in regulating bile acid and lipid homeostasis; it also exerts anti-inflammatory activity by suppressing macrophage NF-κB. Herein, we analyzed the role of FXR in regulating macrophage activation in the lung following ozone exposure. Treatment of wild-type (WT) mice with ozone (0.8 ppm, 3 h) resulted in increases in proinflammatory (F4/80+CD11c+CD11b+Ly6CHi) and anti-inflammatory (F4/80+CD11c+CD11b+Ly6CLo) macrophages in the lung. The accumulation of proinflammatory macrophages was increased in FXR−/− mice compared with WT mice; however, anti-inflammatory macrophage activation was blunted as reflected by reduced arginase and mannose receptor expression, a response correlated with decreased Nur77. This was associated with prolonged oxidative stress, as measured by 4-hydroxynonenal-modified proteins in the lung. Loss of FXR was accompanied by protracted increases in lung NF-κB activity and its target, inducible nitric oxide synthase in response to ozone. Levels of Tnf-α, Il-1β, Ccr2, Ccl2, Cx3cr1, and Cx3cl1 were also increased in lungs of FXR−/− relative to WT mice; conversely, genes regulating lipid homeostasis including Lxrα, Apoe, Vldlr, Abcg1, and Abca1 were downregulated, irrespective of ozone exposure. In FXR−/− mice, ozone caused an increase in total lung phospholipids, with no effect on SP-B or SP-D. Dyslipidemia was correlated with blunting of ozone-induced increases in positive end-expiratory pressure-dependent quasi-static pressure volume curves indicating a stiffer lung in FXR−/− mice. These findings identify FXR as a regulator of macrophage activation following ozone exposure suggesting that FXR ligands may be useful in mitigating inflammation and oxidative stress induced by pulmonary irritants.

Acute respiratory distress syndrome (ARDS) is a highly morbid lung pathology induced by exposure to chemical warfare agents, including vesicants, phosgene, chlorine, and ricin. In this review, we describe the pathology associated with the development of ARDS in humans and experimental models of acute lung injury following animal exposure to these high-priority threat agents. Potential future approaches to disease-modifying treatment used in preclinical animal studies, including antioxidants, anti-inflammatories, biologics, and mesenchymal stem cells, are also described. As respiratory pathologies, including ARDS, are the major cause of morbidity and mortality following exposure to chemical threat agents, understanding mechanisms of disease pathogenesis is key to the development of efficacious therapeutics beyond the primary intervention principle, which remains mechanical ventilation.

Mustard vesicants, including sulfur mustard (2,2′-dichlorodiethyl sulfide, SM) and nitrogen mustard (bis(2-chloroethyl)methylamine, HN2) are cytotoxic blistering agents synthesized for chemical warfare. Because they contain highly reactive electrophilic chloroethyl side chains, they readily react with cellular macromolecules like DNA forming monofunctional and bifunctional adducts. By targeting DNA, mustards can compromise genomic integrity, disrupt the cell cycle, and cause mutations and cytotoxicity. To protect against genotoxicity following exposure to mustards, cells initiate a DNA damage response (DDR). This involves activation of signaling cascades including ATM (ataxia telangiectasia mutated), ATR (ataxia telangiectasia and Rad3-related) and DNA-PKcs (DNA-dependent protein kinase, catalytic subunit). Signaling induced by the DDR leads to the recruitment and activation of repair related proteins such as phospho H2AX and phospho p53 to sites of DNA lesions. Excessive DNA modifications by mustards can overwhelm DNA repair leading to single and double strand DNA breaks, cytotoxicity and tissue damage, sometimes leading to cancer. Herein we summarize DDR signaling pathways induced by SM, HN2 and the half mustard, 2-chloroethyl ethyl sulfide (CEES). At the present time, little is known about how mustard-induced DNA damage leads to the activation of DDR signaling. A better understanding of mechanisms by which mustard vesicants induce the DDR may lead to the development of countermeasures effective in mitigating tissue injury.

Sulfur mustard (SM) is a highly toxic blistering agent thought to mediate its action, in part, by activating matrix metalloproteinases (MMPs) in the skin and disrupting components of the basement membrane zone (BMZ). Type IV collagenases (MMP-9) degrade type IV collagen in the skin, a major component of the BMZ at the dermal-epidermal junction. In the present studies, a type IV collagenase inhibitor, N-hydroxy-3-phenyl-2-(4-phenylbenzenesulfonamido) propanamide (BiPS), was tested for its ability to protect the skin against injury induced by SM in the mouse ear vesicant model. SM induced inflammation, epidermal hyperplasia and microblistering at the dermal/epidermal junction of mouse ears 24–168 h post-exposure. This was associated with upregulation of MMP-9 mRNA and protein in the skin. Dual immunofluorescence labeling showed increases in MMP-9 in the epidermis and in the adjacent dermal matrix of the SM injured skin, as well as breakdown of type IV collagen in the basement membrane. Pretreatment of the skin with BiPS reduced signs of SM-induced cutaneous toxicity; expression of MMP-9 mRNA and protein was also downregulated in the skin by BiPS. Following BiPS pretreatment, type IV collagen appeared intact and was similar to control skin. These results demonstrate that inhibiting type IV collagenases in the skin improves basement membrane integrity after exposure to SM. BiPS may hold promise as a potential protective agent to mitigate SM induced skin injury.

A diverse group of toxicants has been identified that cause injury to the lung including gases (eg, ozone, chlorine), particulates/aerosols (eg, diesel exhaust, fly ash, other combustion products, mustards, nanomaterials, silica, asbestos), chemotherapeutics (eg, bleomycin), and radiation. The pathologic response to these toxicants depends on the dose and duration of exposure and their physical/chemical properties. A common response to pulmonary toxicant exposure is an accumulation of proinflammatory/cytotoxic M1 macrophages at sites of tissue injury, followed by the appearance of anti-inflammatory/wound repair M2 macrophages. It is thought that the outcome of the pathogenic responses to toxicants depends on the balance in the activity of these macrophage subpopulations. Overactivation of either M1 or M2 macrophages leads to injury and disease pathogenesis. Thus, the very same macrophage-derived mediators, released in controlled amounts to destroy injurious materials and pathogens (eg, reactive oxygen species, reactive nitrogen species, proteases, tumor necrosis factor α) and initiate wound repair (eg, transforming growth factor β, connective tissue growth factor, vascular endothelial growth factor), can exacerbate acute lung injury and/or induce chronic disease such as fibrosis, chronic obstructive pulmonary disease, and asthma, when released in excess. This review focuses on the role of macrophage subsets in acute lung injury and chronic fibrosis. Understanding how these pathologies develop following exposure to toxicants, and the contribution of resident and inflammatory macrophages to disease pathogenesis may lead to the development of novel approaches for treating lung diseases.

In the placenta, the breast cancer resistance protein (BCRP)/ABCG2 efflux transporter limits the maternal-to-fetal transfer of drugs and chemicals. Previous research has pointed to the estrogenic mycotoxin zearalenone as a potential substrate for BCRP. Here, we sought to assess the role of the BCRP transporter in the transplacental disposition of zearalenone during pregnancy. In vitro transwell transport assays employing BCRP/Bcrp-transfected Madine-Darby canine kidney cells and BeWo trophoblasts with reduced BCRP expression were used to characterize the impact of BCRP on the bidirectional transport of zearalenone. In both models, the presence of BCRP protein increased the basolateral-to-apical transport and reduced the apical-to-basolateral transport of zearalenone over a 2-h period. In vivo pharmacokinetic analyses were then performed using pregnant wild-type and Bcrp(−/−) mice after a single tail vein injection of zearalenone. Zearalenone and its metabolite α-zearalenol were detectable in serum, placentas, and fetuses from all animals, and β-zearalenol was detected in serum and fetuses, but not placentas. There were no significant differences in the maternal serum concentrations of any analytes between the two genotypes. In Bcrp(−/−) mice, the free fetal concentrations of zearalenone, α-zearalenol, and β-zearalenol were increased by 115%, 84%, and 150%, respectively, when compared with wild-type mice. Concentrations of free zearalenone and α-zearalenol were elevated 145% and 78% in Bcrp(−/−) placentas, respectively, when compared with wild-type placentas. Taken together, these data indicate that the placental BCRP transporter functions to reduce the fetal accumulation of zearalenone, which may impact susceptibility to developmental toxicities associated with in utero zearalenone exposure.

NADH cytochrome b(5) reductase mediates electron transfer from NADH to cytochrome b(5) utilizing flavin adenine dinucleotide as a redox cofactor. Reduced cytochrome b(5) is an important cofactor in many metabolic reactions including cytochrome P450-mediated xenobiotic metabolism, steroid biosynthesis and fatty acid metabolism, hemoglobin reduction, and methionine and plasmalogen synthesis. Using recombinant human enzyme, we discovered that cytochrome b5 reductase mediates redox cycling of a variety of quinones generating superoxide anion, hydrogen peroxide, and, in the presence of transition metals, hydroxyl radicals. Redox cycling activity was oxygen-dependent and preferentially utilized NADH as a co-substrate; NADH was 5–10 times more active than NADPH in supporting redox cycling. Redox cycling activity was greatest for 9,10-phenanthrenequinone and 1,2-naphthoquinone, followed by 1,4-naphthoquinone and 2-methyl-1,4-naphthoquinone (menadione), nitrofurantoin and 2-hydroxyestradiol. Using menadione as the substrate, quinone redox cycling was found to inhibit reduction of cytochrome b(5) by cytochrome b(5) reductase, as measured by heme spectral changes in cytochrome b(5). Under anaerobic conditions where redox cycling is inhibited, menadione had no effect on the reduction of cytochrome b(5). Chemical redox cycling by cytochrome b(5) reductase may be important in generating cytotoxic reactive oxygen species in target tissues. This activity, together with the inhibition of cytochrome b(5) reduction by redox-active chemicals and consequent deficiencies in available cellular cytochrome b(5), are likely to contribute to tissue injury following exposure to quinones and related redox active chemicals.

Sulfur mustard (SM, bis(2-chloroethyl sulfide) is a potent vesicating agent known to cause skin inflammation, necrosis and blistering. Evidence suggests that inflammatory cells and mediators that they generate are important in the pathogenic responses to SM. In the present studies we investigated the role of mast cells in SM-induced skin injury using a murine vapor cup exposure model. Mast cells, identified by toluidine blue staining, were localized in the dermis, adjacent to dermal appendages and at the dermal/epidermal junction. In control mice, 48–61% of mast cells were degranulated. SM exposure (1.4 g/m(3) in air for 6 min) resulted in increased numbers of degranulated mast cells 1–14 days post-exposure. Treatment of mice topically with an indomethacin choline bioisostere containing prodrug linked by an aromatic ester-carbonate that targets cyclooxygenases (COX) enzymes and acetylcholinesterase (1% in an ointment) 1–14 days after SM reduced skin inflammation and injury and enhanced tissue repair. This was associated with a decrease in mast cell degranulation from 90% to 49% 1–3 days post SM, and from 84% to 44% 7–14 days post SM. These data suggest that reduced inflammation and injury in response to the bifunctional indomethacin prodrug may be due, at least in part, to abrogating mast cell degranulation. The use of inhibitors of mast cell degranulation may be an effective strategy for mitigating skin injury induced by SM.

Photosensitizers are used in the treatment of epidermal proliferation and differentiation disorders such as psoriasis and vitiligo. In these studies, a ring-expanded carbon homolog of the linear psoralen (furo[3,2-g]benzopyran-7-one) class of photosensitizers, 4,10-dimethyl-2H,8H-benzo[1,2-b:5,4-b']dipyran-2-one (NDH2476), was synthesized and analyzed for biological activity. Following activation by ultraviolet light (UVA, 320-400 nm), NDH2476 was found to be a potent inhibitor of keratinocyte growth (IC50 = 9 nm). Similar derivatives methylated in the pyran ring, or containing a saturated pyran ring structure, were markedly less active or inactive as photosensitizers. NDH2476 was found to intercalate and damage DNA following UVA light treatment as determined by plasmid DNA unwinding and nicking experiments. Taken together, these data demonstrate that an intact furan ring in psoralen photosensitizers is not required for keratinocyte growth inhibition or DNA damage. Our findings that low nanomolar concentrations of a benzopyranone derivative were active as a photosensitizer indicates that this or a structurally related compound may be useful in the treatment of skin diseases involving aberrant epidermal cell growth and differentiation.

Sulfur mustard (SM; bis (2-chloroethyl) sulfide) is a potent vesicant which causes irritation of the conjunctiva and damage to the cornea. In the present studies, we characterized the ocular effects of SM in New Zealand white rabbits. Within one day of exposure to SM, edema and hazing of the cornea were observed, followed by neovascularization which persisted for at least 28 days. This was associated with upper and lower eyelid edema and conjunctival inflammation. The conjunctiva is composed of a proliferating epithelium largely consisting of stratified columnar epithelial cells overlying a well-defined dermis. Superficial layers of the conjunctival epithelium were found to express keratin 1, a marker of differentiating squamous epithelium, while in cells overlying the basement membrane expressed keratin 17, a marker of stratified squamous epithelium. SM exposure upregulated keratin 17 expression. Mucin 5 ac producing goblet cells were interspersed within the conjunctiva. These cells generated both acidic and neutral mucins. Increased numbers of goblet cells producing neutral mucins were evident after SM exposure; upregulation of expression of membrane-associated mucin 1 and mucin 4 in the superficial layers of the conjunctival epithelium were also noted. These data demonstrate that ocular exposure of rabbits to SM causes significant damage not only to the cornea, but to the eyelid and conjunctiva, suggesting multiple targets within the eye that should be assessed when evaluating the efficacy of potential countermeasures.

Increased levels of tumor necrosis factor (TNF) α have been linked to a number of pulmonary inflammatory diseases including asthma, chronic obstructive pulmonary disease (COPD), acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), sarcoidosis, and interstitial pulmonary fibrosis (IPF). TNFα plays multiple roles in disease pathology by inducing an accumulation of inflammatory cells, stimulating the generation of inflammatory mediators, and causing oxidative and nitrosative stress, airway hyperresponsiveness and tissue remodeling. TNFα-targeting biologics, therefore, present a potentially highly efficacious treatment option. This review summarizes current knowledge on the role of TNFα in pulmonary disease pathologies, with a focus on the therapeutic potential of TNFα-targeting agents in treating inflammatory lung diseases.

The breast cancer resistance protein (BCRP/ABCG2) is an efflux transporter in the placental barrier. By transporting chemicals from the fetal to the maternal circulation, BCRP limits fetal exposure to a range of drugs, toxicants, and endobiotics such as bile acids and hormones. The purpose of the present studies was to 1) determine whether BCRP localizes to highly-ordered, cholesterol-rich lipid raft microdomains in placenta microvillous membranes, and 2) determine the impact of cholesterol on BCRP-mediated placental transport in vitro.BCRP expression was analyzed in lipid rafts isolated from placentas from healthy, term pregnancies and BeWo trophoblasts by density gradient ultracentrifugation. BeWo cells were also tested for their ability to efflux BCRP substrates after treatment with the cholesterol sequestrant methyl-β-cyclodextrin (MβCD, 5 mM, 1 h) or the cholesterol synthesis inhibitor pravastatin (200 μM, 48 h).BCRP was found to co-localize with lipid raft proteins in detergent-resistant, lipid raft-containing fractions from placental microvillous membranes and BeWo cells. Treatment of BeWo cells with MβCD redistributed BCRP protein into higher density non-lipid raft fractions. Repletion of the cells with cholesterol restored BCRP localization to lipid raft-containing fractions. Treatment of BeWo cells with MβCD or pravastatin increased cellular retention of two BCRP substrates, the fluorescent dye Hoechst 33342 and the mycotoxin zearalenone. Repletion with cholesterol restored BCRP transporter activity. Taken together, these data demonstrate that cholesterol may play a critical role in the post-translational regulation of BCRP in placental lipid rafts.

Nitrogen mustard (NM)-induced lung injury is associated with an accumulation of proinflammatory/cytotoxic M1 and antiinflammatory/wound repair M2 macrophages, which have been implicated in tissue injury and repair. Herein, we analyzed the effects of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor with antiinflammatory and antioxidant activity, on lung macrophages responding to NM. Treatment of rats with NM (0.125 mg/kg, i.t.) resulted in structural alterations in the lung and a macrophage-rich inflammatory cell infiltrate, at 3 d and 7 d. This was accompanied by expression of PCNA, a marker of proliferation, and CYPb5, HO-1, and MnSOD, markers of oxidative stress. Administration of VPA (300 mg/kg/day; i.p.), beginning 30 min after NM, reduced increases in PCNA, CYPb5, HO-1, and MnSOD. This was associated with increases in immature CD11b+CD43+ M1 macrophages in the lung, and decreases in mature CD11b+CD43− M2 macrophages 3 d post NM, suggesting delayed maturation and phenotypic switching. VPA also attenuated NM-induced increases in lung iNOS+ and CCR2+ M1 macrophages, a response correlated with downregulation of NOS2, IL12B, PTGS2, MMP-9, and CCR2 expression. Conversely, numbers of CD68+, CD163+ , and ATR-1α+ M2 macrophages increased after VPA, along with the expression of IL10, ApoE, and ATR-1A. NM exposure resulted in increased HDAC activity and upregulation of HDAC2 and acetylated H3K9 in the lung. Whereas VPA blunted the effects of NM on HDAC2 expression, histone H3K9 acetylation increased. These data suggest that alterations in the balance between histone acetylases and deacetylases contribute to lung macrophage maturation and activation following NM exposure.

Extracellular vesicles (EVs) have emerged as key regulators of cell-cell communication during inflammatory responses to lung injury induced by diverse pulmonary toxicants including cigarette smoke, air pollutants, hyperoxia, acids, and endotoxin. Many lung cell types, including epithelial cells and endothelial cells, as well as infiltrating macrophages generate EVs. EVs appear to function by transporting cargo to recipient cells that, in most instances, promote their inflammatory activity. Biologically active cargo transported by EVs include miRNAs, cytokines/chemokines, damage-associated molecular patterns (DAMPs), tissue factor (TF)s, and caspases. Findings that EVs are taken up by target cells such as macrophages, and that this leads to increased proinflammatory functioning provide support for their role in the development of pathologies associated with toxicant exposure. Understanding the nature of EVs responding to toxic exposures and their cargo may lead to the development of novel therapeutic approaches to mitigating lung injury.

Sulfur mustard and related vesicants are cytotoxic alkylating agents that cause severe damage to the respiratory tract. Injury is progressive leading, over time, to asthma, bronchitis, bronchiectasis, airway stenosis, and pulmonary fibrosis. As there are no specific therapeutics available for victims of mustard gas poisoning, current clinical treatments mostly provide only symptomatic relief. In this article, the long-term effects of mustards on the respiratory tract are described in humans and experimental animal models in an effort to define cellular and molecular mechanisms contributing to lung injury and disease pathogenesis. A better understanding of mechanisms underlying pulmonary toxicity induced by mustards may help in identifying potential targets for the development of effective clinical therapeutics aimed at mitigating their adverse effects.