RESUMO
Lewisite (LEW) is an arsenical vesicant that can be a potentially dangerous chemical warfare agent (CWA). Eyes are particularly susceptible to vesicant induced injuries and ocular LEW exposure can act swiftly, causing burning of eyes, edema, inflammation, cell death and even blindness. In our previous studies, we developed a LEW exposure-induced corneal injury model in rabbit and showed increased inflammation, neovascularization, cell death, and structural damage to rabbit corneas upon LEW exposure. In the present study, we further assessed the metabolomic changes to delineate the possible mechanisms underlying the LEW-induced corneal injuries. This information is vital and could help in the development of effective targeted therapies against ocular LEW injuries. Thus, the metabolomic changes associated with LEW exposures in rabbit corneas were assessed as a function of time, to delineate pathways from molecular perturbations at the genomic and proteomic levels. New Zealand white rabbit corneas (n = 3-6) were exposed to LEW vapor (0.2 mg/L; flow rate: 300 ml/min) for 2.5 min (short exposure; low dose) or 7.5 min (long-exposure; high dose) and then collected at 1, 3, 7, or 14 days post LEW exposure. Samples were prepared using the automated MicroLab STAR® system, and proteins precipitated to recover the chemically diverse metabolites. Metabolomic analysis was carried out by reverse phase UPLC-MS/MS and gas chromatography (GC)-MS. The data obtained were analyzed using Metabolon's software. The results showed that LEW exposures at high doses were more toxic, particularly at the day 7 post exposure time point. LEW exposure was shown to dysregulate metabolites associated with all the integral functions of the cornea and cause increased inflammation and immune response, as well as generate oxidative stress. Additionally, all important metabolic functions of the cells were also affected: lipid and nucleotide metabolism, and energetics. The high dose LEW exposures were more toxic, particularly at day 7 post LEW exposure (>10-fold increased levels of histamine, quinolinate, N-acetyl-ß-alanine, GMP, and UPM). LEW exposure dysregulated integral functions of the cornea, caused inflammation and heightened immune response, and generated oxidative stress. Lipid and nucleotide metabolism, and energetics were also affected. The novel information about altered metabolic profile of rabbit cornea following LEW exposure could assist in delineating complex molecular events; thus, aid in identifying therapeutic targets to effectively ameliorate ocular trauma.
Assuntos
Arsenicais , Lesões da Córnea , Animais , Coelhos , Irritantes/efeitos adversos , Irritantes/metabolismo , Cromatografia Líquida , Proteômica , Espectrometria de Massas em Tandem , Córnea/metabolismo , Lesões da Córnea/induzido quimicamente , Lesões da Córnea/metabolismo , Arsenicais/efeitos adversos , Arsenicais/metabolismo , Inflamação/metabolismo , Nucleotídeos/efeitos adversos , Nucleotídeos/metabolismo , LipídeosRESUMO
Pulmonary irritants, such as cigarette smoke (CS) and sodium hypochlorite (NaClO), are associated to pulmonary diseases in cleaning workers. We examined whether their association affects lung mechanics and inflammation in Wistar rats. Exposure to these irritants alone induced alterations in the lung mechanics, inflammation, and remodeling. The CS increased airway cell infiltration, acid mucus production, MMP-12 expression, and alveolar enlargement. NaClO increased the number of eosinophils and macrophages in the bronchoalveolar lavage fluid, with cells expressing IL-13, MMP-12, MMP-9, TIMP-1, and iNOS in addition to increased IL-1ß and TNF-α levels. Co-exposure to both irritants increased epithelial and smooth muscle cell area, acid mucus production, and IL-13 expression in the airways, while it reduced the lung inflammation. In conclusion, the co-exposure of CS with NaClO reduced the pulmonary inflammation, but increased the acidity of mucus, which may protect lungs from more injury. A cross-resistance in people exposed to multiple lung irritants should also be considered.
Assuntos
Fumar Cigarros , Lesão Pulmonar , Pneumonia , Animais , Líquido da Lavagem Broncoalveolar , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Interleucina-13/metabolismo , Irritantes/metabolismo , Irritantes/farmacologia , Pulmão/metabolismo , Lesão Pulmonar/induzido quimicamente , Lesão Pulmonar/metabolismo , Metaloproteinase 12 da Matriz/metabolismo , Pneumonia/metabolismo , Ratos , Ratos Wistar , Hipoclorito de Sódio/metabolismo , Hipoclorito de Sódio/farmacologia , NicotianaRESUMO
Transient receptor potential channel subfamily A member 1 (TRPA1) is a Ca2+-permeable cation channel that serves as one of the primary sensors of environmental irritants and noxious substances. Many TRPA1 agonists are electrophiles that are recognized by TRPA1 via covalent bond modifications of specific cysteine residues located in the cytoplasmic domains. However, a mechanistic understanding of electrophile sensing by TRPA1 has been limited due to a lack of high-resolution structural information. Here, we present the cryoelectron microscopy (cryo-EM) structures of nanodisc-reconstituted ligand-free TRPA1 and TRPA1 in complex with the covalent agonists JT010 and BITC at 2.8, 2.9, and 3.1 Å, respectively. Our structural and functional studies provide the molecular basis for electrophile recognition by the extraordinarily reactive C621 in TRPA1 and mechanistic insights into electrophile-dependent conformational changes in TRPA1. This work also provides a platform for future drug development targeting TRPA1.
Assuntos
Acetamidas/metabolismo , Irritantes/metabolismo , Isotiocianatos/metabolismo , Canal de Cátion TRPA1/ultraestrutura , Tiazóis/metabolismo , Acetamidas/farmacologia , Microscopia Crioeletrônica , Cisteína/metabolismo , Células HEK293 , Humanos , Irritantes/farmacologia , Isotiocianatos/farmacologia , Modelos Moleculares , Nociceptores , Dor/metabolismo , Técnicas de Patch-Clamp , Fosfolipídeos/metabolismo , Domínios Proteicos , Estrutura Terciária de Proteína , Prurido/metabolismo , Canal de Cátion TRPA1/efeitos dos fármacos , Canal de Cátion TRPA1/metabolismo , Tiazóis/farmacologiaRESUMO
The vesicant sulfur mustard (SM) is a banned chemical warfare agent that is controlled by the Organisation for the Prohibition of Chemical Weapons (OPCW). Bioanalytical procedures are mandatory for proving an alleged use and incorporation of SM into the body. We herein present the development and application of a novel optimized procedure suitable for qualitative verification analysis of plasma targeting the SM-adduct of human serum albumin (HSA) alkylated at the cysteine(34) residue. Diluted human plasma is directly mixed with pronase in an ultrafiltration device (10kDa cut-off) for proteolysis (4h, 37°C). Following ultrafiltration the filtrate is diluted and analyzed by microbore liquid chromatography-electrospray ionization high resolution tandem-mass spectrometry (µLC-ESI HR MS/MS) targeting the alkylated dipeptide hydroxyethylthioethyl-CysPro (HETE-CP). A hybrid quadrupole time-of-flight mass spectrometer provided high mass spectrometric resolution in the MS/MS mode enabling highest selectivity and sensitivity (lower limit of detection corresponding to 9.8nM SM in plasma). Kinetics of HETE-CP formation from heparin-, citrate-, and EDTA-plasma as well as serum are presented and the influence of different EDTA and pronase concentrations was characterized. The novel procedure was applied to plasma samples provided by the OPCW as well as to patients plasma derived from real cases of SM-poisoning.
Assuntos
Substâncias para a Guerra Química/metabolismo , Irritantes/metabolismo , Gás de Mostarda/metabolismo , Albumina Sérica/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Substâncias para a Guerra Química/intoxicação , Cromatografia Líquida , Cisteína , Humanos , Irritantes/toxicidade , Cinética , Gás de Mostarda/intoxicação , Intoxicação/sangue , Intoxicação/diagnóstico , Pronase/metabolismo , Ligação Proteica , Proteólise , Albumina Sérica Humana , UltrafiltraçãoRESUMO
The opilionid Acanthopachylus aculeatus was shown to produce a defensive secretion containing quinones (2,3-dimethyl-1,4-benzoquinone, 2,5-dimethyl-1,4-benzoquinone and 2,3,5-trimethyl-1,4-benzoquinone), confirming the findings reported nearly a half century ago in a classic study. The mechanism by which the opilionid puts the secretion to use is described. When disturbed, the animal regurgitates enteric fluid, which it conveys by intercoxal clefts to the anterolateral corners of the carapace, where the two gland openings are situated. It then injects some of its quinonoid secretion into the fluid, and conveys the mixed liquid along the length of its flanks by way of two special channels. Such a discharge mechanism may be widespread among opilionids of the family Gonyleptidae (suborder Laniatores), to which A. aculeatus belongs. In a bioassay based on a scratch reflex in decapitated cockroaches (Periplaneta americana) the liquid effluent of A. aculeatus was shown to be potently irritating. Use of the effluent was demonstrated to protect the opilionid against ants (Formica exsectoides). Wolf spiders (Lycosa ceratiola) were shown to be minimally affected by the effluent (they showed little response when the fluid was added to their mouthparts as they fed on mealworms, their normal laboratory prey), although they proved to be aversive to mere contact with the opiliond itself, and to reject the animal without inducing it to discharge. A. aculeatus may therefore contain distasteful factors besides its glandular products.
Assuntos
Aracnídeos/fisiologia , Benzoquinonas/metabolismo , Líquidos Corporais/metabolismo , Glândulas Exócrinas/metabolismo , Animais , Aracnídeos/metabolismo , Aracnídeos/ultraestrutura , Bioensaio , Líquidos Corporais/química , Baratas , Glândulas Exócrinas/anatomia & histologia , Cromatografia Gasosa-Espectrometria de Massas , Irritantes/metabolismo , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de VarreduraRESUMO
Glutathione-S-transferase class Omega (GSTO 1-1) belongs to a new subfamily of GSTs, which is identical with human monomethylarsonic acid (MMA(V)) reductase, the rate limiting enzyme for biotransformation of inorganic arsenic, environmental carcinogen. Recombinant GSTO 1-1 variants (Ala140Asp and Thr217Asn) were functionally characterized using representative substrates. No significant difference was observed in GST activity towards 1-chloro-2,4-dinitrobenzene, whereas thioltransferase activity was decreased to 75% (Ala140Asp) and 40% (Thr217Asn) of the wild-type GSTO 1-1. For MMA(V) reductase activity, the Ala140Asp variant exhibited similar kinetics to wild type, while the Thr217Asn variant had lower V(max) (56%) and K(m) (64%) values than the wild-type enzyme. The different activities of the enzyme variants may influence both the intracellular thiol status and arsenic biotransformation. This can help explain the variation between individuals in their susceptibility to oxidative stress and inorganic arsenic.
Assuntos
Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Arsenicais/metabolismo , Sítios de Ligação , Dinitroclorobenzeno/metabolismo , Dissulfetos/metabolismo , Glutationa Transferase/química , Humanos , Irritantes/metabolismo , Isoenzimas/química , Modelos Moleculares , Estrutura Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Teratogênicos/metabolismoRESUMO
Adaptive cytoprotection in the stomach was originally defined by applying the exogenous irritants only. The contribution of endogenous irritants as inductors of initial lesions was not specially evaluated. No attempt was made to either focus antiulcer agent activity on adaptive cytoprotection, or split their 'cytoprotection' into complex adaptive cytoprotective activity and simple cytoprotective effects. Agents had so far not been applied simultaneously with the second challenge with ethanol (or irritant), when differences between cytoprotection and adaptive cytoprotection appear. Gastrojejunal anastomosis for 24 h in rats was introduced as new model for analyzing cytoprotection/adaptive cytoprotection. The contribution of the up-normal level of endogenous irritants and the endogenous small irritant-induced minor lesions during the adaptive cytoprotection were studied. The effect of late challenge with 96% ethanol in the presence of an up-normal level of endogenous irritants and endogenous small irritant-induced minor lesions was compared with results of classic studies of ethanol-induced gastric lesions in normal rats (1 ml/rat i.g.). Antiulcer agents or a prostaglandins-synthesis inhibitor, indomethacin, given once only in classic studies, were given at several points during injury induction: (i) surgery, (ii) mild ethanol, (iii) strong ethanol, (iv) strong ethanol applied after a suitable period following either mild ethanol or surgery). Their effects were compared in rats treated as follows: exogenous irritant studies (96% or 20% ethanol), exogenous/exogenous irritant studies (20% ethanol 1 h before 96% ethanol), endogenous irritant studies (gastrojejunal anastomosis for 24 h), and endogenous/exogenous irritant studies (gastrojejunal anastomosis for 24 h before 96% ethanol). Characteristic of the various irritants differed: the (preceding) small irritants (exogenous (i.e., mild ethanol in healthy intact rats) (exogenous irritant studies) vs. endogenous (e.g., (increased) gastric acid secretion, duodenal reflux in gastric content in rats with termino-lateral gastrojejunal anastomosis) (endogenous irritant studies)). These factors caused modifications of agents' activities not, as initially thought, giving simple 'cytoprotection', but being only cytoprotective, or adaptive cytoprotective, or both cytoprotective and adaptive cytoprotective. Atropine (10 mg/kg i.p.) and ranitidine (10 mg) had only cytoprotective activity (exogenous irritant-studies), whereas pentadecapeptide BPC157 (10 microg or 10 ng), and omeprazole (10 mg) had mainly adaptive cytoprotective activity (endogenous/exogenous irritant studies) or both cytoprotective and adaptive cytoprotective activities (exogenous/exogenous irritant studies). Augmentation of the lesions by indomethacin (5 mg/kg s.c.), showed that only events preceding the late challenge with ethanol may be prostaglandin-dependent in both models. The second, adaptive cytoprotective part, seen after late ethanol challenge, may be either prostaglandin-dependent (exogenous/exogenous irritant studies) or non-dependent (endogenous/exogenous irritant studies). Both spontaneous lesion reduction, as an essential mechanism of adaptive cytoprotection, and the further lesion reduction by agents, such as pentadecapeptide BPC 157 and omeprazole, suggests that these agents function as an essential link between the various reactions in cytoprotection/adaptive cytoprotection.
Assuntos
Antiulcerosos/farmacologia , Inibidores de Ciclo-Oxigenase/toxicidade , Citoproteção/efeitos dos fármacos , Indometacina/toxicidade , Irritantes/metabolismo , Úlcera Péptica/patologia , Sequência de Aminoácidos , Anastomose Cirúrgica , Animais , Atropina/farmacologia , Relação Dose-Resposta a Droga , Etanol/toxicidade , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Irritantes/toxicidade , Jejuno , Masculino , Dados de Sequência Molecular , Omeprazol/farmacologia , Úlcera Péptica/induzido quimicamente , Úlcera Péptica/prevenção & controle , Fragmentos de Peptídeos/farmacologia , Proteínas/farmacologia , Ranitidina/farmacologia , Ratos , Ratos Wistar , EstômagoAssuntos
Dinitroclorobenzeno/metabolismo , Brânquias/enzimologia , Glutationa Transferase/metabolismo , Irritantes/metabolismo , Xenobióticos/metabolismo , Animais , Peixes , Glutationa/metabolismo , Glutationa Transferase/química , Cinética , Água do Mar , Especificidade por Substrato , Emirados Árabes Unidos , Poluentes Químicos da Água/metabolismoRESUMO
Nickel (Ni) dermatitis is thought to involve the formation of complexes between Ni ions and suitable proteins. 4 groups of 30 subjects who gave positive patch test responses to NiSO4 2.9% aq. were each retested to 1 of 4 different solutions containing equimolar (0.1 M) amounts of NiSO4 plus MgSO4, NiSO4 plus CuSO4, NiSO4 plus ZnSO4, and NiSO4 plus Li2SO4, respectively. The results, evaluated at 2 days by visual scoring only, demonstrated that the 4 metals exerted a different influence on the nickel reactions, perhaps interfering with one or more factors affecting the formation of Ni+ + complexes.
Assuntos
Dermatite Alérgica de Contato/diagnóstico , Dermatite Alérgica de Contato/imunologia , Irritantes/efeitos adversos , Níquel/efeitos adversos , Testes do Emplastro , Cobre/efeitos adversos , Cobre/metabolismo , Sulfato de Cobre , Dermatite Alérgica de Contato/metabolismo , Interações Medicamentosas , Humanos , Transporte de Íons , Irritantes/metabolismo , Compostos de Lítio/efeitos adversos , Compostos de Lítio/metabolismo , Sulfato de Magnésio/efeitos adversos , Sulfato de Magnésio/metabolismo , Níquel/metabolismo , Sulfatos/efeitos adversos , Sulfatos/metabolismo , Compostos de Zinco/efeitos adversos , Compostos de Zinco/metabolismo , Sulfato de ZincoRESUMO
The vesicant agents of the unitary chemical munitions stockpile include various formulations of sulfur mustard [bis-(2-chloroethyl) sulfide; agents H, HD, and HT] and small quantities of the organic arsenical Lewisite [dichloro(2-chlorovinyl) arsine; agent L]. These agents can be dispersed in liquid, aerosol, or vapor form and are capable of producing severe chemical burns upon direct contact with tissue. Moist tissues such as the eyes, respiratory tract, and axillary areas are particularly affected. Available data summarizing acute dose response in humans and laboratory animals are summarized. Vesicant agents are also capable of generating delayed effects such as chronic bronchitis, carcinogenesis, or keratitis/keratopathy of the eye under appropriate conditions of exposure and dose. These effects may not become manifest until years following exposure. Risk analysis derived from carcinogenesis data indicates that sulfur mustard possesses a carcinogenic potency similar to that of benzo[a]pyrene. Because mustard agents are alkylating compounds, they destroy individual cells by reaction with cellular proteins, enzymes, RNA, and DNA. Once begun, tissue reaction is irreversible. Mustard agents are mutagenic; data for cellular and laboratory animal assays are presented. Reproductive effects have not been demonstrated in the offspring of laboratory rats. Acute Lewisite exposure has been implicated in cases of Bowen's disease, an intraepidermal squamous cell carcinoma. Lewisite is not known to generate reproductive or teratogenic effects.
Assuntos
Intoxicação por Arsênico , Arsenicais , Dano ao DNA , Dermatite Irritante/etiologia , Irritantes/toxicidade , Ceratite/etiologia , Neoplasias Pulmonares/etiologia , Gás de Mostarda/toxicidade , Reprodução/efeitos dos fármacos , Doenças Respiratórias/etiologia , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Guerra Química , Feminino , Humanos , Irritantes/química , Irritantes/metabolismo , Dose Letal Mediana , Masculino , Gás de Mostarda/química , Gás de Mostarda/metabolismo , Mutação , GravidezRESUMO
Receptor compartments can be hydrophilic or hydrophobic. The hydrophobic character can be revealed from the logarithmic relationship between the octanol-water partition coefficient (Po/w) and the equipotent equilibrium concentration (Cw) measured in the water compartment (Franke: Theoretical Drug Design Methods. Elsevier, Amsterdam 1984). For activation of the sensory irritant receptor during exposure to airborne chemicals the Cw values at equilibrium can be obtained from the gas or vapour concentrations [( A]a) and the water-gas partition coefficients (Pw/g). However, if the octanol-gas partition coefficients (Po/g) are used, the analysis can be carried out directly from the gas or vapour concentrations. The thermodynamic activity can also be used to reveal whether the environment of the receptor is hydrophobic or not. We have adapted Franke's theory to a series of homologous airborne sensory irritants. Our results suggest that the environment of the sensory irritant receptor is likely to be a hydrophobic site within the polar part of the nerve membrane. The extended theory is general and it is therefore suggested that it applies to other airborne exposure concentrations which are in equilibrium with a hydrophobic receptor.
Assuntos
Irritantes/metabolismo , Receptores de Droga/efeitos dos fármacos , Fenômenos Químicos , Físico-Química , Irritantes/farmacologia , Octanóis , ÁguaRESUMO
Upper airway exposure to cigarette smoke elicits reflex changes in breathing pattern. To examine whether laryngeal afferents are affected by cigarette smoke, neural activity was recorded from the peripheral cut end of superior laryngeal nerve in anesthetized dogs. A box-balloon system, connected to the breathing circuit, allowed smoke to be inhaled spontaneously through the isolated upper airway while preserving its normal respiratory flow and pressure. Our results showed the following. Inhalation of cigarette smoke (25-50% concentration, 300-400 ml) caused a marked increase in activity of laryngeal irritant receptors which were either silent or randomly discharging during control breathing [their activity increased from a control value of 1.67 +/- 0.50 (mean +/- SE; n = 21) to a peak of 5.03 +/- 0.85 impulses/s in 11-15 s]. The activity of laryngeal cold receptors was reduced to 77.3 and 63.8% of control (n = 9) during the two breaths of smoke inhalation, respectively. After returning toward the base-line activity, a more pronounced inhibition (26.3% of control) occurred at three to nine breaths after the smoke inhalation. A small but significant decrease (88.5% of control) in the inspiratory discharge of laryngeal mechanoreceptors was observed during the first test breath. These effects were independent of the CO2 content of the smoke. Furthermore, there was no difference between the responses of these laryngeal afferents to high- and low-nicotine cigarette smoke.