Asthmalike symptoms following intratracheal exposure of Guinea pigs to sulfur mustard aerosol: therapeutic efficacy of exogenous lung surfactant curosurf and salbutamol

The purpose of the present study was to investigate: (1) the acute effects of sulfur mustard on airway, lung, and surface tension of bronchoalveolar lavage fluid (BALfluid) in guinea pigs following intratracheal (i.t.) exposure to 1LD50 of an aerosolized solution of sulfur mustard in saline, and (2) the therapeutic efficacy of i.t. administration of the natural surfactant Curosurf and the broncholytic Salbutamol. Intratracheally aerosolized sulfur mustard solution induced two clinically relevant symptoms, that is, asthmalike symptoms reflected by an early bronchoconstriction and "late asthmatic responses" (LAR), and ARDS-like symptoms, that is, pulmonary edema and damage to the lung surfactant. The respiratory minute volume (RMV) was enhanced. Histologically, inflammation and severe epithelial injury in the upper airways were observed, whereas the lungs were homogeneously affected. The surface tension of BAL fluid derived at 24 h after sulfur mustard exposure was much higher (20 +/- 1 mN/m) than that of unexposed control animals (about 1.0 +/- 0.5 mN/m), indicating that the lung surfactant had been altered, and justifying treatment with exogenous surfactant. Intratracheal nebulization of a Salbutamol solution (10 microg/kg), or i.t. bolus administration of Curosurf (62.5 or 125 mg/kg), tended to reduce mortality, although Salbutamol appeared to be more effective than Curosurf in this respect. Although the present study does not give a definite answer to the question of whether the animal model used would be the most relevant for humans, a number of considerations in favor of i.t. aerosolization of sulfur mustard are discussed. Since it was noticed that sulfur mustard exposure induced damage to the lung surfactant, severe bronchoconstriction, and inflammation of the respiratory tract, the effectiveness of a combined treatment consisting of exogenous surfactant, anti-inflammatory drugs, and broncholytics is recommended to be further investigated.     

Standard operating procedure for immunuslotblot assay for analysis of DNA/sulfur mustard adducts in human blood and skin

A standard operating procedure has been developed for an immunoslotblot assay of sulfur mustard adducts to DNA in human blood and skin for use in a field laboratory. A minimum detectable level of exposure of human blood in vitro (> or = 50 nM) sulfur mustard is feasible with the assay. In the case of human skin, a 1 s exposure to saturated sulfur mustard vapor (830 mg/m(-3)) could still be detected.     

Airway epithelial damage and release of inflammatory mediators in human lung parenchyma after sulfur mustard exposure

This study was performed to evaluate the morphological effects of sulfur mustard on human lung parenchyma in vitro and to measure the metabolites of arachidonic acid which are released during acute exposure to the alkylating agent. Histological analysis of the tissue following exposure to sulfur mustard for a period of 45 min at 10 mM revealed the presence of paranuclear vacuoles in the epithelium, specifically, in the ciliated cells. The release of metabolites of arachidonic acid were determined in the bath fluids by an enzymo-immunoassay. The basal release of prostaglandin E2 (PGE2: 1.36 +/- 0.33 ng/g tissue) and 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha: 8.83 +/- 1.17 ng/g tissue) were not modified during tissue exposure to sulfur mustard (45 min, 0.1 mM). In addition, the basal release of cysteinyl-leukotriene E4 (LTE4: 1.55 +/- 0.44 ng/g tissue) was also not altered by challenge of the tissues with sulfur mustard. In contrast, when the human lung parenchyma was stimulated with anti human IgE (anti-IgE) only the basal release of the metabolite of the 5-lipoxygenase pathway was significantly increased (LTE4: 6.84 +/- 1.57 ng/g tissue). These data suggest that sulfur mustard may produce morphological alterations in epithelial cells and at the time point studied (45 min exposure), this effect is not associated with a release of arachidonic acid metabolites. However, the increased release of LTE4 by anti-IgE suggests that the target cells for sulfur mustard and anti-IgE in the human lung may be different.     

Evaluating the combinative effects on human lymphocyte DNA damage induced by ultraviolet ray C plus 1.8 GHz microwaves using comet assay in vitro

The objective of this study was to observe whether 1.8 GHz microwaves (MW) (SAR, 3 W/kg) exposure can influence human lymphocyte DNA damage induced by ultraviolet ray C (UVC). The lymphocytes, which were from three young healthy donors, were exposed to 254 nm UVC at the doses of 0.25, 0.5, 0.75, 1.0, 1.5 and 2.0 J m(-2), respectively. The lymphocytes were irradiated by 1.8 GHz MW (SAR, 3 W/kg) for 0, 1.5 and 4 h. The combinative exposure of UVC plus MW was conducted. The treated cells were incubated for 0, 1.5 and 4 h. Finally, comet assay was used to measure DNA damage of above treated lymphocytes. The results indicated that the difference of DNA damage induced between MW group and control group was not significant (P>0.05). The MTLs induced by UVC were 1.71+/-0.09, 2.02+/-0.08, 2.27+/-0.17, 2.27+/-0.06, 2.25+/-0.12, 2.24+/-0.11 microm, respectively, which were significantly higher than that (0.96+/-0.05 microm) of control (P<0.01). MTLs of some sub-groups in combinative exposure groups at 1.5-h incubation were significantly lower that those of corresponding UVC sub-groups (P<0.01 or P<0.05). However, MTLs of some sub-groups in combinative exposure groups at 4-h incubation were significantly higher that those of corresponding UVC sub-groups (P<0.01 or P<0.05). In this experiment it was found that 1.8 GHz (SAR, 3 W/kg) MW exposure for 1.5 and 4 h did not enhance significantly human lymphocyte DNA damage, but could reduce and increase DNA damage of human lymphocytes induced by UVC at 1.5-h and 4-h incubation, respectively.     

Assessment of DNA damage in peripheral blood lymphocytes of individuals susceptible to arsenic induced toxicity in West Bengal, India

Assessment of DNA damage was carried out using alkaline comet assay in lymphocytes of 30 individuals exposed to high levels of arsenic (247.12+/-18.93 microg/l) through contaminated groundwater in North 24 Parganas district, West Bengal, India. All of them exhibited high arsenic contents in nail (4.20+/-0.67 microg/g), hair (2.06+/-0.20 microg/g) and urine (259.75+/-33.89 microg/l) samples and manifested various arsenical skin lesions. Unexposed samples were collected from 30 residents of the unaffected East Midnapur district with very little or no exposure to arsenic (7.69+/-0.49 microg/l) in drinking water. The results were evaluated principally by manual analysis of comets and partly by computerized image analysis. Both the analytical methods exhibited a high degree of agreement in results. The exposed participants expressed significantly higher DNA damage (p < 0.01) in their lymphocytes than the unexposed participants. Alkaline comet assay was also combined with formamidopyrimidine-DNA glycosylase enzyme digestion to confirm that arsenic induced oxidative base damage in the lymphocytes. Significant positive trend effects of comet lengths in relation to arsenic levels in water prove that DNA damage can be used as a sensitive biomarker of arsenic exposure. This study demonstrates that arsenic induced significant DNA damage in the exposed participants, which could correspond to a higher susceptibility to arsenic induced toxicity and carcinogenicity.     

Asthmalike Symptoms Following Intratracheal Exposure of Guinea Pigs to Sulfur Mustard Aerosol: Therapeutic Efficacy of Exogenous Lung Surfactant Curosurf and Salbutamol

The purpose of the present study was to investigate: (1) the acute effects of sulfur mustard on airway, lung, and surface tension of bronchoalveolar lavage fluid (BALfluid) in guinea pigs following intratracheal (i.t.) exposure to 1LD50 of an aerosolized solution of sulfur mustard in saline, and (2) the therapeutic efficacy of i.t. administration of the natural surfactant Curosurf and the broncholytic Salbutamol. Intratracheally aerosolized sulfur mustard solution induced two clinically relevant symptoms, that is, asthmalike symptoms reflected by an early bronchoconstriction and “late asthmatic responses” (LAR), and ARDS-like symptoms, that is, pulmonary edema and damage to the lung surfactant. The respiratory minute volume (RMV) was enhanced. Histologically, inflammation and severe epithelial injury in the upper airways were observed, whereas the lungs were homogeneously affected. The surface tension of BAL fluid derived at 24 h after sulfur mustard exposure was much higher (20 ± 1 mN/m) than that of unexposed control animals (about 1.0 ± 0.5 mN/m), indicating that the lung surfactant had been altered, and justifying treatment with exogenous surfactant. Intratracheal nebulization of a Salbutamol solution (10 μ g/kg), or i.t. bolus administration of Curosurf (62.5 or 125 mg/kg), tended to reduce mortality, although Salbutamol appeared to be more effective than Curosurf in this respect. Although the present study does not give a definite answer to the question of whether the animal model used would be the most relevant for humans, a number of considerations in favor of i.t. aerosolization of sulfur mustard are discussed. Since it was noticed that sulfur mustard exposure induced damage to the lung surfactant, severe bronchoconstriction, and inflammation of the respiratory tract, the effectiveness of a combined treatment consisting of exogenous surfactant, anti-inflammatory drugs, and broncholytics is recommended to be further investigated.     

Evaluation of the potential genotoxicity of chromium picolinate in mammalian cells in vivo and in vitro.

Chromium picolinate (CrPic) is a synthetic nutritional supplement primarily used for weight loss and muscle building. Recent studies have indicated that CrPic might be genotoxic and these findings together with the wide-spread consumer use, have increased the concern about its safety. In the present study we investigated the potential genotoxicity of CrPic in mice given a single intraperitoneal injection (up to 3 mg/kgb.wt.) by evaluating the frequency of micronucleated polychromatic erythrocytes (fMNPCE) in peripheral blood, and DNA damage in lymphocytes and hepatocytes. The fMNPCE was evaluated after 42 h and DNA damage after 16 h. Using the Comet assay DNA damage was also monitored in extended-term cultures of human lymphocytes and in L5178Y mouse lymphoma cells that had been exposed for 3h to 500 microM CrPic under different exposure conditions. A slight, but significant CrPic-induced increase in DNA damage (P<0.001) was observed in the human lymphocytes, but only when these cells were exposed in the absence of serum. In all other experiments CrPic was found to be without genotoxic effects, both in vivo and in vitro. Taken together, our results suggest that a high concentration of CrPic might be DNA damaging, but only under non-physiological conditions.     

Demonstration of Benzo(a)pyrene-Induced DNA Damage in Mice by Alkaline Single Cell Gel Electrophoresis: Evidence for Strand Breaks in Liver but Not in Lymphocytes and Bone Marrow

Abstract: Alkaline single cell gel electrophoresis (also known as the‘comet assay') was used to measure DNA strand breaks and alkali-labile sites in peripheral lymphocytes, bone marrow and liver cells of C57BL/6 mice orally exposed to benzo(a)pyrene. Although this polycyclic aromatic hydrocarbon is a well-known genotoxic agent, little is known about to what extent it actually induces DNA strand breaks in peripheral lymphocytes and other tissues after in vivo exposure. Significant and dose-related damage was observed in liver cells after three days of exposure (lowest observed effect level being 3×100 mg benzo(a)pyrene/kg b.wt.). No such damage could be observed in the lymphocytes and bone marrow cells even after administration of 3×150 mg benzo(a)pyrene/kg b.wt. The reference substance cyclophosphamide produced pronounced DNA damage in lymphocytes and bone marrow cells already in a single dose of 100 mg/kg b.wt. The present mouse study questions the usability of DNA strand breaks in peripheral lymphocytes as an indicator of benzo(a)pyrene-induced genotoxicity.     

In vitro genotoxicity evaluation of 4-carboxyl-2,6-dinitrophenylazohydroxynaphthalenes using human lymphocytes

The genotoxicity of a new monoazo dye series, 4-carboxyl-2,6-dinitrophenylazohydroxynaphthalenes has been evaluated using human lymphocytes by alkaline comet assay. Freshly isolated human lymphocytes were exposed to the dyes (AZ-01, -02, -03 and -04) at concentrations ranging from 0 to 500 μM for 3h at 37 °C. Appropriate negative (culture medium) and positive (100 μM methyl methane sulfonate) controls were set up alongside with the dye-treated cells. Comet assay was performed to assess the extent of DNA damage. The four dyes gave varying results with respect to the parameters of DNA damage studied. AZ-01 showed concentration-dependent DNA damage (% Tail DNA) while lower concentrations (31.25-62.5 μM) did not produce any significant difference in the tail extent moment. AZ-02, the positional isomer of AZ-01, gave non-genotoxic effects at lower concentrations for the two DNA parameters. AZ-03 and AZ-04 (possessing additional C-7 substituents) did not produce significant genotoxic effect at all concentrations relative to the negative control. Two of these monoazo dyes show the potential of being used as edible colorants. The results revealed that genotoxicity of congeneric dyes bear a direct relationship to their chemical structure.     

Nitrogen mustard-mediated mutagenesis in human T-lymphocytes in vitro

The cytotoxic and mutagenic effect of the bifunctional alkylating agent nitrogen mustard (HN₂) was examined. Primary human lymphocytes were exposed to graded doses of HN₂ in vitro and relative survival was determined. Mutation induction at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus was measured by cloning the exposed T-cells in microtitre plates in the presence and absence of 6-thioguanine (TG). The IC₅₀-value determined for 30 min exposure to HN₂ was 1.34 μM. The mutant frequencies (MF) in exposed T-cell cultures were 10-fold (2 μM HN₂) to 32-fold (4 μM HN₂) higher than those of unexposed cultures (median values). Nitrogen mustard-mediated mutagenesis is discussed in terms of the current ideas about DNA damage and repair. Received: 14 May 1996 / Accepted: 28 August 1996     

Expression of proliferative and inflammatory markers in a full-thickness human skin equivalent following exposure to the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide

Sulfur mustard is a potent vesicant that induces inflammation, edema and blistering following dermal exposure. To assess molecular mechanisms mediating these responses, we analyzed the effects of the model sulfur mustard vesicant, 2-chloroethyl ethyl sulfide, on EpiDerm-FT™, a commercially available full-thickness human skin equivalent. CEES (100-1000 μM) caused a concentration-dependent increase in pyknotic nuclei and vacuolization in basal keratinocytes; at high concentrations (300-1000 μM), CEES also disrupted keratin filament architecture in the stratum corneum. This was associated with time-dependent increases in expression of proliferating cell nuclear antigen, a marker of cell proliferation, and poly(ADP-ribose) polymerase (PARP) and phosphorylated histone H2AX, markers of DNA damage. Concentration- and time-dependent increases in mRNA and protein expression of eicosanoid biosynthetic enzymes including COX-2, 5-lipoxygenase, microsomal PGE₂ synthases, leukotriene (LT) A₄ hydrolase and LTC₄ synthase were observed in CEES-treated skin equivalents, as well as in antioxidant enzymes, glutathione S-transferases A1-2 (GSTA1-2), GSTA3 and GSTA4. These data demonstrate that CEES induces rapid cellular damage, cytotoxicity and inflammation in full-thickness skin equivalents. These effects are similar to human responses to vesicants in vivo and suggest that the full thickness skin equivalent is a useful in vitro model to characterize the biological effects of mustards and to develop potential therapeutics.     

Assessment of alterations in barrier functionality and induction of proinflammatory and cytotoxic effects after sulfur mustard exposure of an in vitro coculture model of the human alveolo-capillary barrier

Acute lung injury after sulfur mustard (SM) inhalation is characterized by massive, localized hemorrhage and alveolar edema, which implies severe disruption of the vascular and distal airway barrier. In this study, we tested a recently established in vitro coculture model of the alveolo-capillary barrier for its applicability to investigate acute toxic effects of SM at the human respiratory unit. The epithelial compartment of cocultures was exposed to varying concentrations of SM (0-1000 microM; t = 30 min). Following exposure, functional and structural barrier integrity of cocultures was monitored over a period of 24 h. A 50% reduction of transbilayer electrical resistance (TER) within 12-24 h after exposure to 300 microM SM and within 8 h after 1000 microM SM revealed a time- and concentration-dependent impairment of barrier functionality, which was associated with structural loss of both cell layers. Subsequent quantification of interleukin (IL)-6 and IL-8 in cell culture supernatants of exposed cocultures showed enhanced liberation of proinflammatory markers. Highest mediator levels were detected after 300 microM SM, with pronounced stimulation in the endothelial compartment. SM-related cytotoxicity was determined by assessing adenylate kinase (AK) release and by quantifying the fraction of DNA-fragmented nuclei using terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling (TUNEL) and nuclear Hoechst staining. Both methods exposed a concentration-dependent increase of SM-mediated cytotoxic effects with high effects on endothelial cells. We conclude that the described in vitro model reflects important characteristics of SM-mediated acute lung injury in vivo and thus can be used to explore involved pathophysiological pathways.     

THE MOUSE EAR MODEL OF CUTANEOUS SULFUR MUSTARD INJURY

The mouse ear inflammation model was used to establish simple endpoints of skin injury following cutaneous exposure to sulfur mustard (bis(2- chloroethyl)sulfide, HD). Mouse ear edema and histopathologic response to a single topical application of 5 muL of 8, 16, 32, 64, or 128 mg/mL HD in dichloromethane was evaluated at 12, 18, and 24 h postexposure. Edema response was determined from weighed 8 mm diameter skin punch biopsies taken from the center of exposed and control ears. Key light microscopic histopathologic changes assessed on the inner (exposed side) and outer surfaces (contralateral side) of the exposed ear included epidermal necrosis (EN) and epidermal-dermal separation (subepidermal blister, SEB). HD produced statistically significant (p <. 05) dose- and time-dependent changes in edema and histopathologic features. A comparative evaluation of the endpoint responses from the various challenge doses indicated that a dose of 0.16 mg/ear HD evaluated at 24 h postexposure would be optimal for future studies using this mouse ear vesicant model (MEVM) to screen pharmacological compounds that may protect against HD-induced skin damage.     

DNA damage in T and B lymphocytes, bone marrow, spleens, and livers of rats exposed to benzene

Single-cell gel electrophoresis assays were performed in order to evaluate DNA damage occurring in the T and B lymphocytes, spleens, bone marrow, and livers of rats exposed to benzene at a concentration of 100, 200, or 400 ppm for 2 or 4 wk. The level of t,t-muconic acid (t,t-MA), which is a urinary benzene metabolite, was determined. In the control rats, mean Olive tail moments in the T and B lymphocytes were 1.507 +/- 0.398 and 1.579 +/- 0.206, respectively. DNA damage in the T and B lymphocytes exposed to 400 ppm benzene for 4 wk caused those rats to exhibit the highest Olive tail moments, with their values measured as 4.351 +/- 0.510 and 3.140 +/- 0.631, respectively. Also, the t,t-MA levels increased directly with increasing benzene exposure time and dose during the 4 wk. After 4 wk, the levels of t,t-MA in urine from rats exposed to 100, 200, and 400 ppm were 19.30 +/- 5.62, 30.36 +/- 4.46, and 46.93 +/- 9.10 mg/g creatinine. In conclusion, the present study demonstrates that benzene exposure results in significant DNA damage in the T and B lymphocytes, bone marrow, spleens, and livers of rats. DNA damage in the blood cells and organs was also discovered to vary directly with benzene exposure, in both a dose-dependent and time-dependent manner. In addition, a similar trend regarding DNA damage was found in the blood cells and organs, and evidenced a good association with the level of t,t-MA in the urine.     

Fluctuating estuarine conditions are not confounding factors for the Comet assay assessment of DNA damage in the mussel Mytilus edulis

The Comet assay is finding increasing application as a biomarker assay for the genotoxic potential of contaminants in field transplantation experiments involving mussels. Especially in estuaries, habitats that are of particular concern, environmental variables, such as salinity, can vary significantly. Although hinted at in the literature, there is a lack of clarification as to whether changes in salinity or emersion-induced hypoxia have the potential to alter background DNA damage in mussels, thus masking the extent of potential genotoxic effects following exposure to environmental contaminants. The present study exposed Mytilus edulis in the laboratory to static salinities (25, 50, 75, and 100?%) for 72?h. Mussels were also subjected to simulated tidal cycles, including periods of emersion, for 72?h. None of these treatments resulted in a significant change in the level of DNA damage expressed as % tail DNA. These experiments demonstrate that salinity, within the limits of the concentrations tested, and temporary emersion are not confounding factors for Comet assay data derived from M. edulis.     

Regulation of 1-alpha, 25-dihydroxyvitamin D3 on interleukin-6 and interleukin-8 induced by sulfur mustard (HD) on human skin cells

The regulatory effects of the active form of vitamin D, 1-alpha, 25-dihydroxyvitamin D3 (1-alpha, 25 (OH)2D3) were assessed on the cytokine and chemokine secretion induced by sulfur mustard on human skin fibroblasts and human epidermal keratinocytes. Stimulation of human skin fibroblasts with sulfur mustard (10(-4) M for 24 hr at 37 degrees ) resulted in approximately a 5 times increase in the secretion of interleukin-6 and over a 10 times increase for interleukin-8, which was inhibited by 1-alpha, 25 (OH)2D3, at 相似文献   

Regulation of 1‐α, 25‐Dihydroxyvitamin D3 on Interleukin‐6 and Interleukin‐8 Induced by Sulfur Mustard (HD) on Human Skin Cells*

Abstract: The regulatory effects of the active form of vitamin D, 1‐α, 25‐dihydroxyvitamin D₃ (1‐α, 25 (OH)₂D₃) were assessed on the cytokine and chemokine secretion induced by sulfur mustard on human skin fibroblasts and human epidermal keratinocytes. Stimulation of human skin fibroblasts with sulfur mustard (10^?4 M for 24 hr at 37°) resulted in approximately a 5 times increase in the secretion of interleukin‐6 and over a 10 times increase for interleukin‐8, which was inhibited by 1‐α, 25 (OH)₂D₃, at ≤10^?9 M. 1‐α, 25 (OH)₂D₃ also suppressed interleukin‐8 secretion by 5 times and interleukin‐6 by 4 times on sulfur mustard‐stimulated human epidermal keratinocytes at concentrations ≤ 10^?9 M. The effect of 1‐α, 25 (OH)₂D₃ was dose‐dependent for the suppression of interleukin‐6 and interleukin‐8 induced by sulfur mustard on human skin fibroblasts/human epidermal keratinocytes, apparent at nanomolar concentrations. Our results indicate that the suppression of these inflammatory mediators by 1‐α, 25 (OH)₂D₃ is dependent on the source of the primary cultures, cell densities, and kinetics of pretreatments. In contrast to the inhibition of cytokine/chemokine production, cell proliferation was enhanced by almost 1.7 times on treated human epidermal keratinocytes with 1‐α, 25 (OH)₂D₃ (1×10^?9 M) after sulfur mustard‐stimulation (10^?4 M for 24 hr at 37°C). The observed enhancement diversified based on cell density, and kinetics of pretreatment with a maximal synergism (s) observed at 1×10^?9 M. Photomicrographs show typical signs of cellular degeneration caused by sulfur mustard such as chromatin condensation. The observed cellular degeneration was lessened when human epidermal keratinocytes were treated with 1‐α, 25 (OH)₂D₃ (2×10^?9 M). 1‐α, 25(OH)₂D₃ could be an alternative treatment for cutaneous inflammation disorders caused by sulfur mustard because we have demonstrated its ability to suppress inflammatory mediators and enhanced cell proliferation in human skin cells stimulated with sulfur mustard.