RESUMEN
Plants are widely existing in the environments and have been considered as potential sentinel species of toxic chemicals' exposure. In this study, the deadly toxic chemicals of three nitrogen mustards (NMs, including NH1, NH2 and NH3) were selected as the investigated targets. First, the reactivities of common endogenous plant components with NMs were examined in vitro. Then, the model plant Nicotiana benthamiana Domin was exposed to NMs. Three γ-aminobutyric acid-nitrogen mustard adducts (GABA-NMs) were identified in the living plant by high resolution mass spectrometry and comparison with the synthesized references. A sensitive detection method with the limits of quantification of 0.0500 ng mL-1 was developed using ultrahigh performance liquid chromatography-triple quadrupole mass spectrometry. The GABA-NMs could be detected after 120 days of the exposure and even in the dead leaves without obvious decrease. Furthermore, 20 different plant species grown in diverse climate zones were exposed to HN1, and the adduct of GABA-HN1 was identified in all the leaves. The results showed the good universality and specificity of GABA-NMs as plant biomarkers for NMs exposure. This work provides a new approach for the pollution investigation of toxic chemicals through analysing biomarkers in plant materials.
Asunto(s)
Biomarcadores , Espectrometría de Masas en Tándem , Ácido gamma-Aminobutírico , Ácido gamma-Aminobutírico/análisis , Ácido gamma-Aminobutírico/metabolismo , Biomarcadores/análisis , Cromatografía Líquida de Alta Presión , Hojas de la Planta/química , Hojas de la Planta/metabolismo , Mecloretamina/análisis , Mecloretamina/toxicidad , Mecloretamina/química , Nicotiana/química , Plantas/química , Plantas/metabolismo , Límite de Detección , Cromatografía Líquida con Espectrometría de MasasRESUMEN
Nitrogen mustard (NM) is a potent vesicating chemical warfare agent that is primarily absorbed through skin, inhalation, or ocular surface. Ocular exposure of NM can cause acute to chronic keratopathy which can eventually lead to blindness. There is a current lack of effective countermeasures against ocular exposure of NM despite their imperative need. Herein, we aim to explore the sustained effect of Dexamethasone sodium phosphate (DSP)-loaded polymeric nanoparticles (PLGA-DSP-NP) following a single subconjunctival injection in the management and prevention of corneal injury progression upon exposure to NM. DSP is an FDA approved corticosteroid with proven anti-inflammatory properties. We formulated PLGA-DSP-NP with zinc chelation ion bridging method using PLGA polymer, with particles of approximately 250 nm and a drug loading of 6.5 wt%. Under in vitro sink conditions, PLGA-DSP-NP exhibited a sustained drug release for two weeks. Notably, in NM injured cornea, a single subconjunctival (SCT) injection of PLGA-DSP-NP outperformed DSP eyedrops (0.1%), DSP solution, placebo NP, and saline, significantly mitigating corneal neovascularization, ulceration, and opacity for the two weeks study period. Through PLGA-DSP-NP injection, sustained DSP release hindered inflammatory cytokine recruitment, angiogenic factors, and endothelial cell proliferation in the cornea. This strategy presents a promising localized corticosteroid delivery system to effectively combat NM-induced corneal injury, offering insights into managing vesicant exposure.
Asunto(s)
Dexametasona , Mecloretamina , Nanopartículas , Dexametasona/análogos & derivados , Animales , Mecloretamina/toxicidad , Modelos Animales de Enfermedad , Lesiones de la Cornea/prevención & control , Lesiones de la Cornea/inducido químicamente , Lesiones de la Cornea/patología , Lesiones de la Cornea/tratamiento farmacológico , Glucocorticoides , Sustancias para la Guerra Química/toxicidad , Ratones , Quemaduras Químicas/prevención & control , Quemaduras Químicas/tratamiento farmacológico , Quemaduras Oculares/inducido químicamente , Quemaduras Oculares/prevención & control , Conejos , Córnea/efectos de los fármacos , Córnea/patología , Córnea/metabolismoRESUMEN
Nitrogen mustard (NM; mechlorethamine) is a cytotoxic vesicant known to cause acute lung injury which can progress to chronic disease. Due to the complex nature of NM injury, it has been difficult to analyze early responses of resident lung cells that initiate inflammation and disease progression. To investigate this, we developed a model of acute NM toxicity using murine precision cut lung slices (PCLS), which contain all resident lung cell populations. PCLS were exposed to NM (1-100 µM) for 0.5-3 h and analyzed 1 and 3 d later. NM caused a dose-dependent increase in cytotoxicity and a reduction in metabolic activity, as measured by LDH release and WST-1 activity, respectively. Optimal responses were observed with 50 µM NM after 1 h incubation and these conditions were used in further experiments. Analysis of PCLS bioenergetics using an Agilent Seahorse showed that NM impaired both glycolytic activity and mitochondrial respiration. This was associated with injury to the bronchial epithelium and a reduction in methacholine-induced airway contraction. NM was also found to cause DNA damage in bronchial epithelial cells in PCLS, as measured by expression of γ-H2AX, and to induce oxidative stress, which was evident by a reduction in glutathione levels and upregulation of the antioxidant enzyme catalase. Cleaved caspase-3 was also upregulated in airway smooth muscle cells indicating apoptotic cell death. Characterizing early events in NM toxicity is key in identifying therapeutic targets for the development of efficacious countermeasures.
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Pulmón , Mecloretamina , Animales , Mecloretamina/toxicidad , Pulmón/efectos de los fármacos , Pulmón/patología , Pulmón/metabolismo , Ratones , Daño del ADN , Ratones Endogámicos C57BL , Relación Dosis-Respuesta a Droga , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Sustancias para la Guerra Química/toxicidad , Glucólisis/efectos de los fármacos , Masculino , Apoptosis/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/patología , Lesión Pulmonar Aguda/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patologíaRESUMEN
Nitrogen mustard (NM) is a toxic vesicant that causes acute injury to the respiratory tract. This is accompanied by an accumulation of activated macrophages in the lung and oxidative stress which have been implicated in tissue injury. In these studies, we analyzed the effects of N-acetylcysteine (NAC), an inhibitor of oxidative stress and inflammation on NM-induced lung injury, macrophage activation and bioenergetics. Treatment of rats with NAC (150 mg/kg, i.p., daily) beginning 30 min after administration of NM (0.125 mg/kg, i.t.) reduced histopathologic alterations in the lung including alveolar interstitial thickening, blood vessel hemorrhage, fibrin deposition, alveolar inflammation, and bronchiolization of alveolar walls within 3 d of exposure; damage to the alveolar-epithelial barrier, measured by bronchoalveolar lavage fluid protein and cells, was also reduced by NAC, along with oxidative stress as measured by heme oxygenase (HO)-1 and Ym-1 expression in the lung. Treatment of rats with NAC attenuated the accumulation of macrophages in the lung expressing proinflammatory genes including Ptgs2, Nos2, Il-6 and Il-12; macrophages expressing inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and tumor necrosis factor (TNF)α protein were also reduced in histologic sections. Conversely, NAC had no effect on macrophages expressing the anti-inflammatory proteins arginase-1 or mannose receptor, or on NM-induced increases in matrix metalloproteinase (MMP)-9 or proliferating cell nuclear antigen (PCNA), markers of tissue repair. Following NM exposure, lung macrophage basal and maximal glycolytic activity increased, while basal respiration decreased indicating greater reliance on glycolysis to generate ATP. NAC increased both glycolysis and oxidative phosphorylation. Additionally, in macrophages from both control and NM treated animals, NAC treatment resulted in increased S-nitrosylation of ATP synthase, protecting the enzyme from oxidative damage. Taken together, these data suggest that alterations in NM-induced macrophage activation and bioenergetics contribute to the efficacy of NAC in mitigating lung injury.
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Acetilcisteína , Metabolismo Energético , Lesión Pulmonar , Mecloretamina , Estrés Oxidativo , Animales , Estrés Oxidativo/efectos de los fármacos , Acetilcisteína/farmacología , Mecloretamina/toxicidad , Masculino , Metabolismo Energético/efectos de los fármacos , Ratas , Lesión Pulmonar/inducido químicamente , Lesión Pulmonar/metabolismo , Lesión Pulmonar/patología , Ratas Sprague-Dawley , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/metabolismo , Sustancias para la Guerra Química/toxicidadRESUMEN
Vesicating chemicals like sulfur mustard (SM) or nitrogen mustard (NM) can cause devastating damage to the eyes, skin, and lungs. Eyes, being the most sensitive, have complicated pathologies that can manifest immediately after exposure (acute) and last for years (chronic). No FDA-approved drug is available to be used as medical counter measures (MCMs) against such injuries. Understanding the pathological mechanisms in acute and chronic response of the eye is essential for developing effective MCMs. Here, we report the clinical and histopathological characterization of a mouse model of NM-induced ocular surface injury (entire surface) developed by treating the eye with 2% (w/v) NM solution for 5 min. Unlike the existing models of specific injury, our model showed severe ocular inflammation, including the eyelids, structural deformity of the corneal epithelium and stroma, and diminished visual and retinal functions. We also observed alterations of the inflammatory markers and their expression at different phases of the injury, along with an activation of acidic sphingomyelinase (aSMase), causing an increase in bioactive sphingolipid ceramide and a reduction in sphingomyelin levels. This novel ocular surface mouse model recapitulated the injuries reported in human, rabbit, and murine SM or NM injury models. NM exposure of the entire ocular surface in mice, which is similar to accidental or deliberate exposure in humans, showed severe ocular inflammation and caused irreversible alterations to the corneal structure and significant vision loss. It also showed an intricate interplay between inflammatory markers over the injury period and alteration in sphingolipid homeostasis in the early acute phase.
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Lesiones Oculares , Gas Mostaza , Humanos , Animales , Ratones , Conejos , Mecloretamina/toxicidad , Lesiones Oculares/inducido químicamente , Párpados , Modelos Animales de Enfermedad , Gas Mostaza/toxicidad , Esfingolípidos , InflamaciónRESUMEN
Sulfur mustard (SM), a vesicating agent first used during World War I, remains a potent threat as a chemical weapon to cause intentional/accidental chemical emergencies. Eyes are extremely susceptible to SM toxicity. Nitrogen mustard (NM), a bifunctional alkylating agent and potent analog of SM, is used in laboratories to study mustard vesicant-induced ocular toxicity. Previously, we showed that SM-/NM-induced injuries (in vivo and ex vivo rabbit corneas) are reversed upon treatment with dexamethasone (DEX), a US Food and Drug Administration-approved, steroidal anti-inflammatory drug. Here, we optimized NM injuries in ex vivo human corneas and assessed DEX efficacy. For injury optimization, one cornea (randomly selected from paired eyes) was exposed to NM: 100 nmoles for 2 hours or 4 hours, and 200 nmoles for 2 hours, and the other cornea served as a control. Injuries were assessed 24 hours post NM-exposure. NM 100 nmoles exposure for 2 hours was found to cause optimal corneal injury (epithelial thinning [â¼69%]; epithelial-stromal separation [6-fold increase]). In protein arrays studies, 24 proteins displayed ≥40% change in their expression in NM exposed corneas compared with controls. DEX administration initiated 2 hours post NM exposure and every 8 hours thereafter until 24 hours post-exposure reversed NM-induced corneal epithelial-stromal separation [2-fold decrease]). Of the 24 proteins dysregulated upon NM exposure, six proteins (delta-like canonical Notch ligand 1, FGFbasic, CD54, CCL7, endostatin, receptor tyrosine-protein kinase erbB-4) associated with angiogenesis, immune/inflammatory responses, and cell differentiation/proliferation, showed significant reversal upon DEX treatment (Student's t test; P ≤ 0.05). Complementing our animal model studies, DEX was shown to mitigate vesicant-induced toxicities in ex vivo human corneas. SIGNIFICANCE STATEMENT: Nitrogen mustard (NM) exposure-induced injuries were optimized in an ex vivo human cornea culture model and studies were carried out at 24 h post 100 nmoles NM exposure. Dexamethasone (DEX) administration (started 2 h post NM exposure and every 8 h thereafter) reversed NM-induced corneal injuries. Molecular mediators of DEX action were associated with angiogenesis, immune/inflammatory responses, and cell differentiation/proliferation, indicating DEX aids wound healing via reversing vesicant-induced neovascularization (delta-like canonical Notch ligand 1 and FGF basic) and leukocyte infiltration (CD54 and CCL7).
Asunto(s)
Sustancias para la Guerra Química , Lesiones de la Cornea , Gas Mostaza , Animales , Humanos , Conejos , Mecloretamina/toxicidad , Irritantes/efectos adversos , Sustancias para la Guerra Química/toxicidad , Ligandos , Córnea , Lesiones de la Cornea/inducido químicamente , Lesiones de la Cornea/tratamiento farmacológico , Lesiones de la Cornea/metabolismo , Gas Mostaza/toxicidad , Dexametasona/farmacología , Dexametasona/uso terapéuticoRESUMEN
Nitrogen mustard (NM) is a known surrogate of sulfur mustard, a chemical-warfare agent that causes a wide range of ocular symptoms, from a permanent reduction in visual acuity to blindness upon exposure. Although it has been proposed that the two blistering agents have a similar mechanism of toxicity, the mode of NM-induced cell death in ocular tissue has not been fully explored. Therefore, we hypothesized that direct ocular exposure to NM in mice leads to retinal tissue injury through chronic activation of the unfolded protein response (UPR) PERK arm in corneal cells and VEGF secretion, eventually causing cell death. We topically applied NM directly to mice to analyze ocular and retinal tissues at 2 weeks postexposure. A dramatic decline in retinal function, measured by scotopic and photopic electroretinogram responses, was detected in the mice. This decline was associated with enhanced TUNEL staining in both corneal and retinal tissues. In addition, exposure of corneal cells to NM revealed 228 differentially and exclusively expressed proteins primarily associated with the UPR, ferroptosis, and necroptosis. Moreover, these cells exhibited activation of the UPR PERK arm and an increase in VEGF secretion. Enhancement of VEGF staining was later observed in the corneas of the exposed mice. Therefore, our data indicated that the mechanism of NM-induced ocular toxicity should be carefully examined and that future research should identify a signaling molecule transmitted via a prodeath pathway from the cornea to the retina. SIGNIFICANCE STATEMENT: This study demonstrated that NM topical exposure in mice results in dramatic decline in retinal function associated with enhanced TUNEL staining in both corneal and retinal tissues. We also found that the NM treatment of corneal cells resulted in 228 differentially and exclusively expressed proteins primarily associated with ferroptosis. Moreover, these cells manifest the UPR PERK activation and an increase in VEGF secretion. The latter was also found in the corneas of the cexposed mice.
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Sustancias para la Guerra Química , Gas Mostaza , Animales , Ratones , Mecloretamina/toxicidad , Mecloretamina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Neuropatía Óptica Tóxica , Córnea , Sustancias para la Guerra Química/toxicidad , Gas Mostaza/toxicidad , Gas Mostaza/metabolismo , Respuesta de Proteína DesplegadaRESUMEN
The chemical warfare agent sulfur mustard and its structural analog nitrogen mustard (NM) cause severe vesicating skin injuries. The pathologic mechanisms for the skin injury following mustard exposure are poorly understood; therefore, no effective countermeasure is available. Previous reports demonstrated the protective activity of carvedilol, a US Food and Drug Administration (FDA)-approved ß-blocker, against UV radiation-induced skin damage. Thus, the current study evaluated the effects of carvedilol on NM-induced skin injuries in vitro and in vivo. In the murine epidermal cell line JB6 Cl 41-5a, ß-blockers with different receptor subtype selectivity were examined. Carvedilol and both of its enantiomers, R- and S-carvedilol, were the only tested ligands statistically reducing NM-induced cytotoxicity. Carvedilol also reduced NM-induced apoptosis and p53 expression. In SKH-1 mice, NM increased epidermal thickness, damaged skin architecture, and induced nuclear factor κB (NF-κB)-related proinflammatory genes as assessed by RT2 Profiler PCR (polymerase chain reaction) Arrays. To model chemical warfare scenario, 30 minutes after exposure to NM, 10 µM carvedilol was applied topically. Twenty-four hours after NM exposure, carvedilol attenuated NM-induced epidermal thickening, Ki-67 expression, a marker of cellular proliferation, and multiple proinflammatory genes. Supporting the in vitro data, the non-ß-blocking R-enantiomer of carvedilol had similar effects as racemic carvedilol, and there was no difference between carvedilol and R-carvedilol in the PCR array data, suggesting that the skin protective effects are independent of the ß-adrenergic receptors. These data suggest that the ß-blocker carvedilol and its enantiomers can be repurposed as countermeasures against mustard-induced skin injuries. SIGNIFICANCE STATEMENT: The chemical warfare agent sulfur mustard and its structural analog nitrogen mustard cause severe vesicating skin injuries for which no effective countermeasure is available. This study evaluated the effects of US Food and Drug Administration (FDA)-approved ß-blocker carvedilol on nitrogen mustard-induced skin injuries to repurpose this cardiovascular drug as a medical countermeasure.
Asunto(s)
Sustancias para la Guerra Química , Gas Mostaza , Animales , Ratones , Mecloretamina/toxicidad , Mecloretamina/metabolismo , Carvedilol/farmacología , Carvedilol/uso terapéutico , Carvedilol/metabolismo , Sustancias para la Guerra Química/toxicidad , Gas Mostaza/farmacología , Gas Mostaza/toxicidad , Piel , Antagonistas Adrenérgicos beta/farmacologíaRESUMEN
The ocular surface comprises the wet mucosal epithelia of the cornea and conjunctiva, the associated glands, and the overlying tear film. Epitheliopathy is the common pathologic outcome when the ocular surface is subjected to oxidative stress. Whether different stresses act via the same or different mechanisms is not known. Dynasore and dyngo-4a, small molecules developed to inhibit the GTPase activity of classic dynamins DNM1, DNM2, and DNM3, but not mdivi-1, a specific inhibitor of DNM1L, protect corneal epithelial cells exposed to the oxidant tert-butyl hydroperoxide (tBHP). Here we report that, while dyngo-4a is the more potent inhibitor of endocytosis, dynasore is the better cytoprotectant. Dynasore also protects corneal epithelial cells against exposure to high salt in an in vitro model of dysfunctional tears in dry eye. We now validate this finding in vivo, demonstrating that dynasore protects against epitheliopathy in a mouse model of dry eye. Knockdown of classic dynamin DNM2 was also cytoprotective against tBHP exposure, suggesting that dynasore's effect is at least partially on target. Like tBHP and high salt, exposure of corneal epithelial cells to nitrogen mustard upregulated the unfolded protein response and inflammatory markers, but dynasore did not protect against nitrogen mustard exposure. In contrast, mdivi-1 was cytoprotective. Interestingly, mdivi-1 did not inhibit the nitrogen mustard-induced expression of inflammatory cytokines. We conclude that exposure to tBHP or nitrogen mustard, two different oxidative stress agents, cause corneal epitheliopathy via different pathologic pathways. SIGNIFICANCE STATEMENT: Results presented in this paper, for the first time, implicate the dynamin DNM2 in ocular surface epitheliopathy. The findings suggest that dynasore could serve as a new topical treatment for dry eye epitheliopathy and that mdivi-1 could serve as a medical countermeasure for epitheliopathy due to nitrogen mustard exposure, with potentially increased efficacy when combined with anti-inflammatory agents and/or UPR modulators.
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Síndromes de Ojo Seco , Hidrazonas , Mecloretamina , Naftoles , Quinazolinonas , Ratones , Animales , Mecloretamina/toxicidad , Mecloretamina/metabolismo , Síndromes de Ojo Seco/inducido químicamente , Síndromes de Ojo Seco/tratamiento farmacológico , Córnea , Lágrimas , DinaminasRESUMEN
Nitrogen mustard (NM) is a cytotoxic vesicant known to cause pulmonary injury that can progress to fibrosis. NM toxicity is associated with an influx of inflammatory macrophages in the lung. Farnesoid X receptor (FXR) is a nuclear receptor involved in bile acid and lipid homeostasis that has anti-inflammatory activity. In these studies, we analyzed the effects of FXR activation on lung injury, oxidative stress, and fibrosis induced by NM. Male Wistar rats were exposed to phosphate-buffered saline (vehicle control) or NM (0.125 mg/kg) by intratracheal Penncentury-MicroSprayer aerosolization; this was followed by treatment with the FXR synthetic agonist, obeticholic acid (OCA, 15 mg/kg), or vehicle control (0.13-0.18 g peanut butter) 2 hours later and then once per day, 5 days per week thereafter for 28 days. NM caused histopathological changes in the lung, including epithelial thickening, alveolar circularization, and pulmonary edema. Picrosirius red staining and lung hydroxyproline content were increased, indicative of fibrosis; foamy lipid-laden macrophages were also identified in the lung. This was associated with aberrations in pulmonary function, including increases in resistance and hysteresis. Following NM exposure, lung expression of HO-1 and iNOS, and the ratio of nitrates/nitrites in bronchoalveolar lavage fluid (BAL), markers of oxidative stress increased, along with BAL levels of inflammatory proteins, fibrinogen, and sRAGE. Administration of OCA attenuated NM-induced histopathology, oxidative stress, inflammation, and altered lung function. These findings demonstrate that FXR plays a role in limiting NM-induced lung injury and chronic disease, suggesting that activating FXR may represent an effective approach to limiting NM-induced toxicity. SIGNIFICANCE STATEMENT: In this study, the role of farnesoid-X-receptor (FXR) in mustard vesicant-induced pulmonary toxicity was analyzed using nitrogen mustard (NM) as a model. This study's findings that administration of obeticholic acid, an FXR agonist, to rats reduces NM-induced pulmonary injury, oxidative stress, and fibrosis provide novel mechanistic insights into vesicant toxicity, which may be useful in the development of efficacious therapeutics.
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Ácido Quenodesoxicólico/análogos & derivados , Lesión Pulmonar , Mecloretamina , Ratas , Masculino , Animales , Mecloretamina/toxicidad , Irritantes/efectos adversos , Ratas Wistar , Pulmón , Fibrosis , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/patología , Lesión Pulmonar/metabolismo , Estrés Oxidativo , LípidosRESUMEN
Exposure to mustard agents, such as sulfur mustard (SM) and nitrogen mustard (NM), often results in ocular surface damage. This can lead to the emergence of various corneal disorders that are collectively referred to as mustard gas keratopathy (MGK). In this study, we aimed to develop a mouse model of MGK by using ocular NM exposure, and describe the subsequent structural changes analyzed across the different layers of the cornea. A 3 µL solution of 0.25 mg/mL or 5 mg/mL NM was applied to the center of the cornea via a 2-mm filter paper for 5 min. Mice were evaluated prior to and after exposure on days 1, 3, 7, 14, and 28 for 4 weeks using slit lamp examination with fluorescein staining. Anterior segment optical coherence tomography (AS-OCT) and in vivo confocal microscopy (IVCM) tracked changes in the epithelium, stroma, and endothelium of the cornea. Histologic evaluation was used to examine corneal cross-sections collected at the completion of follow-up. Following exposure, mice experienced central corneal epithelial erosion and thinning, accompanied by a decreased number of nerve branches in the subbasal plexus and increased activated keratocytes in the stroma in both dosages. The epithelium was recovered by day 3 in the low dose group, followed by exacerbated punctuate erosions alongside persistent corneal edema that arose and continued onward to four weeks post-exposure. The high dose group showed persistent epitheliopathy throughout the study. The endothelial cell density was reduced, more prominent in the high dose group, early after NM exposure, which persisted until the end of follow-up, along with increased polymegethism and pleomorphism. Microstructural changes in the central cornea at 4 weeks post-exposure included dysmorphic basal epithelial cells and reduced epithelial thickness, and in the limbal cornea included decreased cellular layers. We present a mouse model of MGK using NM that successfully replicates ocular injury caused by SM in humans who have been exposed to mustard gas.
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Enfermedades de la Córnea , Edema Corneal , Úlcera de la Córnea , Gas Mostaza , Humanos , Animales , Ratones , Gas Mostaza/toxicidad , Mecloretamina/toxicidad , Córnea/patología , Enfermedades de la Córnea/inducido químicamente , Enfermedades de la Córnea/patología , Úlcera de la Córnea/patología , Trastornos de la Visión/patología , Microscopía ConfocalRESUMEN
Sulfur mustard (SM) and nitrogen mustard (NM) are vesicant agents that cause skin injury and blistering through complicated cellular events, involving DNA damage, free radical formation, and lipid peroxidation. The development of therapeutic approaches targeting the multi-cellular process of tissue injury repair can potentially provide effective countermeasures to combat vesicant-induced dermal lesions. MG53 is a vital component of cell membrane repair. Previous studies have demonstrated that topical application of recombinant human MG53 (rhMG53) protein has the potential to promote wound healing. In this study, we further investigate the role of MG53 in NM-induced skin injury. Compared with wild-type mice, mg53-/- mice are more susceptible to NM-induced dermal injuries, whereas mice with sustained elevation of MG53 in circulation are resistant to dermal exposure of NM. Exposure of keratinocytes and human follicle stem cells to NM causes elevation of oxidative stress and intracellular aggregation of MG53, thus compromising MG53's intrinsic cell membrane repair function. Topical rhMG53 application mitigates NM-induced dermal injury in mice. Histologic examination reveals the therapeutic benefits of rhMG53 are associated with the preservation of epidermal integrity and hair follicle structure in mice with dermal NM exposure. Overall, these findings identify MG53 as a potential therapeutic agent to mitigate vesicant-induced skin injuries.
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Irritantes , Mecloretamina , Ratones , Humanos , Animales , Mecloretamina/toxicidad , Mecloretamina/metabolismo , Irritantes/metabolismo , Queratinocitos/metabolismo , Cicatrización de Heridas/fisiología , Proteínas de la Membrana/metabolismoRESUMEN
Mustard agents are vesicants that were used in warfare multiple times. They are potent alkylating agents that activate cellular pathways of apoptosis, increase oxidative stress, and induce inflammation. Eyes are particularly susceptible to mustard exposure with a wide range of ocular surface damage. Three main categories of mustard-related eye injuries are acute, chronic, and delayed-onset manifestations. Mustard keratopathy (MK) is a known complication characterized by corneal opacification, ulceration, thinning, and neovascularization that can lead to severe vision loss and discomfort. Recently, a few reports demonstrated the role of senescence induction as a new pathological mechanism in mustard-related injuries that could affect wound healing. We ran the first murine model of delayed-onset MK and nitrogen mustard-induced senescence, evaluating the pathological signs of senescence in the cornea using beta-galactosidase staining. Our results suggest that nitrogen mustard exposure causes senescence in the corneal cells, which could be the underlying mechanism for chronic and late-onset ocular surface damage. We also found a significant correlation between the percentage of positive beta-galactosidase staining and the degree of fibrosis in the cornea. This provides valuable insight into the possible role of anti-senescence drugs in the near future for accelerating corneal healing and restricting fibrosis in patients with mustard keratopathy.
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Sustancias para la Guerra Química , Enfermedades de la Córnea , Gas Mostaza , Humanos , Animales , Ratones , Sustancias para la Guerra Química/toxicidad , Gas Mostaza/toxicidad , Mecloretamina/toxicidad , Enfermedades de la Córnea/patología , Córnea/metabolismo , Senescencia CelularRESUMEN
The vesicant sulfur mustard (SM) is a chemical warfare agent that causes acute and chronic injury to the cornea and proximal anterior segment structures. Despite clinical evidence of SM-exposure causing unexplained retinal deficits, there have been no animal studies conducted to examine the retinal toxicity of this vesciant. The cardinal hallmark of retinal response to stressors or injury is the activation of reactive gliosis, a cellular process largely governed by Müller glia. Previously we showed that corneal exposure to sodium hydroxide elicits rapid induction of reactive gliosis and results in retinal degeneration in a dose-related manner. Based on this evidence, we hypothesized that the vesicant nitrogen mustard (NM), an analog of SM, may also elicit reactive gliosis. To test this idea, we developed a mouse model of NM ocular injury and investigated corneal and retinal effects focusing on citrullination, a posttranslational modification (PTM) of proteins. This PTM was recently linked to alkali injury and has also been shown to occur in retinal degenerative conditions. Here, we demonstrate that corneal exposure to 1% NM causes a synchronous activation of citrullination in both the cornea and retina with hypercitrullination becoming apparent temporally and manifesting with altered cellular expression characteristics. A key finding is that ocular citrullination occurs acutely as early as 1-h post-injury in both the cornea and retina, which underscores a need for expeditious interception of this acute corneal and retinal response. Moreover, exploiting dose response and temporal studies, we uncoupled NM-induced retinal citrullination from its induction of retinal gliosis. Our findings demonstrate that hypercitrullination is a common corneo-retinal mechanism that sensitizes the eye to NM injury and suggests that counteracting hypercitrullination may provide a suitable countermeasure to vesicant injury.
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Lesiones Oculares , Gas Mostaza , Enfermedades de la Retina , Animales , Ratones , Mecloretamina/toxicidad , Irritantes/efectos adversos , Irritantes/metabolismo , Gliosis/inducido químicamente , Gliosis/metabolismo , Córnea/metabolismo , Lesiones Oculares/inducido químicamente , Lesiones Oculares/metabolismo , Retina , Gas Mostaza/toxicidad , Enfermedades de la Retina/inducido químicamente , Enfermedades de la Retina/metabolismoRESUMEN
Mechlorethamine (HN2) is a derivative of the chemical warfare agent sulfur mustard (SM) and cutaneous exposure to HN2 is associated with dermal-epidermal junction (DEJ) disruption (vesication). The primary purpose of the present study was to investigate the effect of HN2 on the mammalian target of rapamycin (mTOR) signaling pathway using an in vivo mouse ear vesicant model (MEVM). To this end, the ears of male C57BL/ 6 J mice were exposed to a single topical dose of HN2 (100 mM) or vehicle control (DMSO). Mice were then euthanized 30 min, 1 h or 24 h following exposure. Mouse ear skin exposed to HN2 and biopsied 24 h thereafter exhibited increased tissue expression of Raptor, an important member of the mTORC1 complex, relative to vehicle treated samples. HN2 reduced the downstream effectors phospho S6 (Ser 240/244) ribosomal protein and phospho 4E-BP1 (Thr 37/46) of the mTOR pathway in the epidermis at 30 min, 1 h and 24 h following HN2 exposure but not in the dermis. These results support the hypothesis that HN2-mediated cutaneous toxicity involves dysregulation of the mTOR signaling pathway in the epidermis.
Asunto(s)
Mecloretamina , Sirolimus , Masculino , Ratones , Animales , Mecloretamina/toxicidad , Sirolimus/farmacología , Ratones Endogámicos C57BL , Piel , Serina-Treonina Quinasas TOR , MamíferosRESUMEN
Ocular surface exposure to nitrogen mustard (NM) leads to severe ocular toxicity which includes the separation of epithelial and stromal layers, loss of endothelial cells, cell death, and severe loss of tissue function. No definitive treatment for mustard gas-induced ocular surface disorders is currently available. The research was conducted to investigate the therapeutic potential of mesenchymal stem cell-conditioned media (MSC-CM) in NM-induced corneal wounds. NM was added to different types of corneal cells, the ocular surface of porcine, and the ocular surface of mice, followed by MSC-CM treatment. NM significantly induced apoptotic cell death, cellular ROS (Reactive oxygen species), and reduced cell viability, metabolic gene expression, and mitochondrial function, and, in turn, delayed wound healing. The application of MSC-CM post NM exposure partially restored mitochondrial function and decreased intracellular ROS generation which promoted cell survival. MSC-CM therapy enhanced wound healing process. MSC-CM inhibited NM-induced apoptotic cell death in murine and porcine corneal tissue. The application of MSC-CM following a chemical insult led to significant improvements in the preservation of corneal structure and wound healing. In vitro, ex vivo, and in vivo results suggest that MSC-CM can potentially provide targeted therapy for the treatment of chemical eye injuries, including mustard gas keratopathy (MGK) which presents with significant loss of vision alongside numerous corneal pathologies.
Asunto(s)
Lesiones de la Cornea , Células Madre Mesenquimatosas , Gas Mostaza , Animales , Lesiones de la Cornea/inducido químicamente , Lesiones de la Cornea/metabolismo , Lesiones de la Cornea/terapia , Medios de Cultivo Condicionados/farmacología , Células Endoteliales/metabolismo , Mecloretamina/toxicidad , Células Madre Mesenquimatosas/metabolismo , Ratones , Gas Mostaza/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Factor de Células Madre/metabolismo , Porcinos , Cicatrización de HeridasRESUMEN
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, ß-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.
Asunto(s)
Lesión Pulmonar Aguda , Mecloretamina , Ratas , Masculino , Animales , Mecloretamina/toxicidad , Ratas Wistar , Lesión Pulmonar Aguda/inducido químicamente , Células Epiteliales Alveolares , Estrés Oxidativo , Metabolismo Energético , Tensoactivos/efectos adversosRESUMEN
Sulfur mustard (SM) is a notorious, bifunctional alkylating vesicant that was first used in warfare during World War I in 1917 and since then has been deployed in numerous skirmishes with its most recent documented use being during the Middle Eastern conflicts. Apart from its use in combat and terrorist activities, continual threat of accidental exposure from old stockpiles and improperly discarded munitions is ever present, especially to the innocent and unassuming civilian populations. SM can cause devastating injuries, depending on the dosage of SM exposure, route of exposure, as well as the physiological conditions of the individuals exposed. The most common routes of exposure are ocular, dermal, and exposure to the lungs and respiratory tissues through inhalation. Eyes are the most susceptible organ to SM-induced toxicities owing to their high moisture content and rapidly dividing cells. Additionally, ocular injury causes the most expeditious disablement of individuals even upon whole-body exposures. Therefore, it is imperative to understand the mechanisms underlying SM-induced ocular toxicity and design therapeutic interventions to prevent/mitigate ocular injuries. Ocular SM exposure may cause a wide range of symptoms such as inflammation, lacrimation, itching, dryness, photophobia, edema of the cornea/sclera/retina/iris, conjunctivitis, degradation of the corneal layer, fusion of two or more ocular layers, neovascularization, fibrosis, and temporary or permanent structural damage to one or more ocular layers. These symptoms may lead to vision impairments, resulting in partial or complete blindness that may be permanent. The highly toxic and exceedingly notorious nature of SM makes it a highly regulated chemical, requiring very expensive licensing, security, and safety requirements; thus, the more easily accessible analogue, nitrogen mustard (NM) that mimics SM-induced toxicity and injuries is employed in plethora of studies conducted in different animal models and culture systems. This review provides a comprehensive account of the injuries and symptoms that occur upon ocular SM exposures in human patients as well as studies in animal (in vivo, ex vivo) and cell (in vitro) models of SM and NM ocular exposures. Special emphasis has been laid on highlighting the strengths and lacunae in the research as well as the possible unexplored avenues of mechanisms underlying mustard-induced ocular injury that can be explored in future research endeavors. Furthermore, development of therapeutic interventions and targets of interest in the ocular system exposed to SM and NM, based on studies in human patients as well as in vivo, ex vivo, and in vitro models has been discussed in great depth, providing a valuable knowledge database to delineate pathways associated with vesicant-induced toxicity, and strategies/diagnostic tools against SM-induced toxicity.
Asunto(s)
Sustancias para la Guerra Química , Lesiones Oculares , Gas Mostaza , Animales , Sustancias para la Guerra Química/toxicidad , Córnea/metabolismo , Lesiones Oculares/inducido químicamente , Lesiones Oculares/metabolismo , Humanos , Irritantes/efectos adversos , Irritantes/metabolismo , Mecloretamina/toxicidad , Gas Mostaza/toxicidadRESUMEN
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-/- mice were treated intratracheally with PBS (control) or NM (0.08 mg/kg). Bronchoalveolar lavage fluid (BAL) and lung tissue were collected 3, 14 and 28 d later. NM caused progressive histopathologic alterations in the lung including inflammatory cell infiltration and alveolar wall thickening and increases in protein and cells in BAL; oxidative stress was also noted, as reflected by upregulation of heme oxygenase-1. These changes were more prominent in male FXR-/- mice. Flow cytometric analysis revealed that loss of FXR resulted in increases in proinflammatory macrophages at 3 d post NM; this correlated with upregulation of COX-2 and ARL11, markers of macrophage activation. Markers of anti-inflammatory macrophage activation, CD163 and STAT6, were also upregulated after NM; this response was exacerbated in FXR-/- mice at 14 d post-NM. These findings demonstrate that FXR plays a role in limiting macrophage inflammatory responses important in lung injury and oxidative stress. Maintaining or enhancing FXR function may represent a useful strategy in the development of countermeasures to treat mustard lung toxicity.
Asunto(s)
Lesión Pulmonar Aguda , Mecloretamina , Lesión Pulmonar Aguda/patología , Animales , Ciclooxigenasa 2/metabolismo , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Irritantes/toxicidad , Lípidos , Pulmón , Activación de Macrófagos , Masculino , Mecloretamina/toxicidad , RatonesRESUMEN
Nitrogen mustard (NM) is an alkylating vesicant that causes severe pulmonary injury. Currently, there are no effective means to counteract vesicant-induced lung injury. MG53 is a vital component of cell membrane repair and lung protection. Here, we show that mice with ablation of MG53 are more susceptible to NM-induced lung injury than the wild-type mice. Treatment of wild-type mice with exogenous recombinant human MG53 (rhMG53) protein ameliorates NM-induced lung injury by restoring arterial blood oxygen level, by improving dynamic lung compliance and by reducing airway resistance. Exposure of lung epithelial and endothelial cells to NM leads to intracellular oxidative stress that compromises the intrinsic cell membrane repair function of MG53. Exogenous rhMG53 protein applied to the culture medium protects lung epithelial and endothelial cells from NM-induced membrane injury and oxidative stress, and enhances survival of the cells. Additionally, we show that loss of MG53 leads to increased vulnerability of macrophages to vesicant-induced cell death. Overall, these findings support the therapeutic potential of rhMG53 to counteract vesicant-induced lung injury.