Matrix metalloproteinase gelatinases in sulfur mustard-induced acute airway injury in guinea pigs.
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Respiratory tract lesions induced by sulfur mustard (SM), a chemical warfare agent, are characterized by epithelial damage associated with inflammatory cell infiltration. To test the potential role of matrix metalloproteinase gelatinases in these lesions, we evaluated gelatinase activity, albumin content, and total cell count in bronchoalveolar lavage fluid of guinea pigs 24 h after an intratracheal injection of 0.2 mg/kg of SM. The bronchial lavage and alveolar lavage fluids were analyzed separately. The increase in inflammatory cell content of the bronchial lavage fluid, mainly macrophages, observed in SM-intoxicated guinea pigs was accompanied by an increase in albumin and in 92-kDa gelatinase activity. There was a significant correlation between albumin content and 92-kDa gelatinase activity (r = 0.67) and between 92-kDa gelatinase and the number of macrophages. Immunohistochemistry performed on tracheal sections showed the presence of 92-kDa gelatinase at the site of intraepithelial cleavages. Zymography analysis of culture medium conditioned by guinea pig tracheal epithelial cells demonstrated that these cells produced in vitro 92-kDa gelatinase on stimulation. Culture of human bronchial epithelial cells obtained by the explant technique showed a marked increase in 92-kDa gelatinase after exposure to 5 x 10(-5) M SM that reinforced the relevance of our animal results to human exposure to SM. These results suggest that in SM respiratory intoxication, 92-kDa gelatinase of both inflammatory and epithelial cell origins could be involved in epithelial cell detachment. (+info)
Apoptosis and necrosis induced by sulfur mustard in Hela cells.
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AIM: To study the apoptotic effect of sulfur mustard (SM) on Hela cells. METHODS: Exponentially growing Hela cells were treated with SM at various concentrations for 3 h, then apoptosis was examined by electron-microscope, DNA gel electrophoresis, and flow cytometry. RESULTS: SM 1 mumol.L-1 arrested cell growth. After treatment with SM 10-100 mumol.L-1, cells were mainly blocked at G1-phase with apoptosis. Agarose gel electrophoresis of DNA from cells treated with SM revealed "DNA Ladder." About 33% of the Hela cells showed apoptosis 12 h after 3-h treatment with SM 100 mumol.L-1 as determined by flow cytometry and the S-phase cells were more susceptible. However, SM 1000 mumol.L-1 caused marked necrosis in Hela cells. CONCLUSION: SM caused 2 distinct forms of cell death, apoptosis or necrosis, in Hela cells in a concentration-dependent manner. (+info)
Efficient protection of human bronchial epithelial cells against sulfur and nitrogen mustard cytotoxicity using drug combinations.
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The aim of this study was to test the efficacy of several candidate molecules against sulfur mustard (SM) and nitrogen mustard (HN2) using a human bronchial-epithelial cell line (16HBE14o-). Candidate molecules were chosen on the basis of the known cytotoxicity mechanisms of mustards or their efficacy previously observed on other cellular models. It included the sulfhydryl-containing molecules N-acetyl-cysteine (NAC) and WR-1065, the nucleophile hexamethylenetetramine (HMT), the energy-level stabilizer niacinamide (NC), the antioxidant dimethylthiourea (DMTU), L-arginine analogues such as L-thiocitrulline (L-TC) and L-nitroarginine methyl ester (L-NAME), and the anti-gelatinase doxycycline (DOX). Their efficacy was determined using 2-(4-[3-iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2Htetrazolium (WST-1) reduction by viable cells 24 h after initial exposure to 100 microM HN2 or SM. On individual immediate cotreatment, some molecules exhibited selective protection against only one mustard, such as DMTU and WR-1065 against HN2 and DOX against SM, whereas NAC and L-TC were effective against both SM and HN2 cytotoxicity. However, as the level of protection against SM was always weak compared to HN2, several combinations were investigated against SM to improve the protection. The effective combinations (L-TC + DOX, NAC + DOX, NAC + DMTU, NAC + HMT, NC + DOX) combined agents, reducing the bioavailability of the mustard with compounds possibly acting on the consequences of alkylation. One of these combinations, NAC + DOX, appeared to be the most interesting, as these agents are already used in human therapy. It exhibited good efficacy in delayed cotreatment (up to 90 min) against SM. (+info)
Repair of sulfur mustard-induced DNA damage in mammalian cells measured by a host cell reactivation assay.
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DNA damage is thought to be the initial event that causes sulfur mustard (SM) toxicity, while the ability of cells to repair this damage is thought to provide a degree of natural protection. To investigate the repair process, we have damaged plasmids containing the firefly luciferase gene with either SM or its monofunctional analog, 2-chloroethyl ethyl sulfide (CEES). Damaged plasmids were transfected into wild-type and nucleotide excision repair (NER) deficient Chinese hamster ovary cells; these cells were also transfected with a second reporter plasmid containing RENILLA: luciferase as an internal control on the efficiency of transfection. Transfected cells were incubated at 37 degrees C for 27 h and then both firefly and RENILLA: luciferase intensities were measured on the same samples with the dual luciferase reporter assay. Bioluminescence in lysates from cells transfected with damaged plasmid, expressed as a percentage of the bioluminescence from cells transfected with undamaged plasmid, is increased by host cell repair activity. The results show that NER-competent cells have a higher reactivation capacity than NER-deficient cells for plasmids damaged by either SM or CEES. Significantly, NER-competent cells are also more resistant to the toxic effects of SM and CEES, indicating that NER is not only proficient in repairing DNA damage caused by either agent but also in decreasing their toxicity. This host cell repair assay can now be used to determine what other cellular mechanisms protect cells from mustard toxicity and under what conditions these mechanisms are most effective. (+info)
PARP determines the mode of cell death in skin fibroblasts, but not keratinocytes, exposed to sulfur mustard.
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Sulfur mustard is cytotoxic to dermal fibroblasts as well as epidermal keratinocytes. We demonstrated that poly(ADP-ribose) polymerase (PARP) modulates Fas-mediated apoptosis, and other groups and we have shown that PARP plays a role in the modulation of other types of apoptotic and necrotic cell death. We have now utilized primary dermal fibroblasts, immortalized fibroblasts, and keratinocytes derived from PARP(-/-) mice and their wildtype littermates (PARP(+/+)) to determine the contribution of PARP to sulfur mustard toxicity. Following sulfur mustard exposure, primary skin fibroblasts from PARP-deficient mice demonstrated increased internucleosomal DNA cleavage, caspase-3 processing and activity, and annexin V positivity, compared to those derived from PARP(+/+) animals. Conversely, propidium iodide staining, PARP cleavage patterns, and random DNA fragmentation revealed a dose-dependent increase in necrosis in PARP(+/+) but not PARP(-/-) cells. Using immortalized PARP(-/-) fibroblasts stably transfected with the human PARP cDNA or with empty vector alone, we show that PARP inhibits markers of apoptosis in these cells as well. Finally, primary keratinocytes were derived from newborn PARP(+/+) and PARP(-/-) mice and immortalized with the E6 and E7 genes of human papilloma virus. In contrast to fibroblasts, keratinocytes from both PARP(-/-) and PARP(+/+) mice express markers of apoptosis in response to sulfur mustard exposure. The effects of PARP on the mode of cell death in different skin cell types may determine the severity of vesication in vivo, and thus have implications for the design of PARP inhibitors to reduce sulfur mustard pathology. (+info)
Transepidermal water loss does not correlate with skin barrier function in vitro.
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The purpose of this study was to investigate the relationship between transepidermal water loss and skin permeability to tritiated water (3H2O) and the lipophilic penetrant sulfur mustard in vitro. No correlation was found between basal transepidermal water loss rates and the permeability of human epidermal membranes to 3H2O (p = 0.72) or sulfur mustard (p = 0.74). Similarly, there was no correlation between transepidermal water loss rates and the 3H2O permeability of full-thickness pig skin (p = 0.68). There was no correlation between transepidermal water loss rate and 3H2O permeability following up to 15 tape strips (p = 0.64) or up to four needle-stick punctures (p = 0.13). These data indicate that transepidermal water loss cannot be unconditionally ascribed to be a measure of skin barrier function. It is clear that further work should be conducted to interpret the significance of measuring transepidermal water loss by evaporimetry. (+info)
An epidemiologic study to screen for chronic myelocytic leukemia in war victims exposed to mustard gas.
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Chemical agents such as mustard gas (or sulfur mustard), which has alkylating characteristics, were used against Iranian combatants in the Iraq-Iran war. Previous studies have not shown a strong link between these chemical agents and the development of chronic myelocytic leukemia (CML). The purpose of this study was to evaluate the increased risk of CML development in Iranian soldiers exposed to mustard gas during the war. Based on a descriptive study of 2,500 cases with documented exposure to various chemical warfare agents, 665 patients had documented exposure to mustard gas. We screened the latter using the leukocyte alkaline phosphatase (LAP) test and performed further cytochemical studies on cases with positive results. From among the 665 cases with documented exposure to mustard gas, 9 cases had LAP scores < 20; 2 of these 9 cases had CML and a score of zero (0.3%). We detected cytogenetic abnormalities in 7 patients with low LAP scores and atypical lymphocytes of 5-11% in 40 patients. The risk ratio of CML developing in victims exposed to mustard gas (cutaneous or respiratory) may be higher in comparison with the normal population, although confounding factors (e.g., the possibility of exposure to combined chemical agents, excluding patients who did not manifest blisters) limited our results. Because the increased development of CML in young patients with a documented history of exposure to mustard gas cannot be disregarded, further studies are needed. (+info)
Ocular injuries following sulfur mustard exposure: clinical characteristics and treatment.
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The chemical warfare agent sulfur mustard affects primarily the eyes, skin and respiratory tract. Of these, ocular injury is the most immediate and distressing. Learning to recognize ocular injury enables the treating physician to provide early and suitable treatment, which will reduce complications and allow the victim a rapid recovery. (+info)