The influence of N-acetyl-L-cysteine on oxidative stress and nitric oxide synthesis in stimulated macrophages treated with a mustard gas analogue

Background: Sulphur mustard gas, 2, 2′-dichlorodiethyl sulphide (HD), is a chemical warfare agent. Both mustard gas and its monofunctional analogue, 2-chloroethyl ethyl sulphide (CEES), are alkylating agents that react with and diminish cellular thiols and are highly toxic. Previously, we reported that lipopolysaccharide (LPS) significantly enhances the cytotoxicity of CEES in murine RAW 264.7 macrophages and that CEES transiently inhibits nitric oxide (NO) production via suppression of inducible NO synthase (iNOS) protein expression. NO generation is an important factor in wound healing. In this paper, we explored the hypotheses that LPS increases CEES toxicity by increasing oxidative stress and that treatment with N-acetyl-L-cysteine (NAC) would block LPS induced oxidative stress and protect against loss of NO production. NAC stimulates glutathione (GSH) synthesis and also acts directly as a free radical scavenger. The potential therapeutic use of the antibiotic, polymyxin B, was also evaluated since it binds to LPS and could thereby block the enhancement of CEES toxicity by LPS and also inhibit the secondary infections characteristic of HD/CEES wounds. Results: We found that 10 mM NAC, when administered simultaneously or prior to treatment with 500 μM CEES, increased the viability of LPS stimulated macrophages. Surprisingly, NAC failed to protect LPS stimulated macrophages from CEES induced loss of NO production. Macrophages treated with both LPS and CEES show increased oxidative stress parameters (cellular thiol depletion and increased protein carbonyl levels). NAC effectively protected RAW 264.7 cells simultaneously treated with CEES and LPS from GSH loss and oxidative stress. Polymyxin B was found to partially block nitric oxide production and diminish CEES toxicity in LPS-treated macrophages. Conclusion: The present study shows that oxidative stress is an important mechanism contributing to CEES toxicity in LPS stimulated macrophages and supports the notion that antioxidants could play a therapeutic role in preventing mustard gas toxicity. Although NAC reduced oxidative stress in LPS stimulated macrophages treated with CEES, it did not reverse CEES-induced loss of NO production. NAC and polymyxin B were found to help prevent CEES toxicity in LPS-treated macrophages

Inhibition of Inducible Nitric Oxide Synthase by a Mustard Gas Analog in Murine Macrophages

Background: 2-Chloroethyl ethyl sulphide (CEES) is a sulphur vesicating agent and an analogue of the chemical warfare agent 2,2′-dichlorodiethyl sulphide, or sulphur mustard gas (HD). Both CEES and HD are alkylating agents that influence cellular thiols and are highly toxic. In a previous publication, we reported that lipopolysaccharide (LPS) enhances the cytotoxicity of CEES in murine RAW264.7 macrophages. In the present investigation, we studied the influence of CEES on nitric oxide (NO) production in LPS stimulated RAW264.7 cells since NO signalling affects inflammation, cell death, and wound healing. Murine macrophages stimulated with LPS produce NO almost exclusively via inducible nitric oxide synthase (iNOS) activity. We suggest that the influence of CEES or HD on the cellular production of NO could play an important role in the pathophysiological responses of tissues to these toxicants. In particular, it is known that macrophage generated NO synthesised by iNOS plays a critical role in wound healing. Results: We initially confirmed that in LPS stimulated RAW264.7 macrophages NO is exclusively generated by the iNOS form of nitric oxide synthase. CEES treatment inhibited the synthesis of NO (after 24 hours) in viable LPS-stimulated RAW264.7 macrophages as measured by either nitrite secretion into the culture medium or the intracellular conversion of 4,5-diaminofluorescein diacetate (DAF-2DA) or dichlorofluorescin diacetate (DCFH-DA). Western blots showed that CEES transiently decreased the expression of iNOS protein; however, treatment of active iNOS with CEES in vitro did not inhibit its enzymatic activity. Conclusion: CEES inhibits NO production in LPS stimulated macrophages by decreasing iNOS protein expression. Decreased iNOS expression is likely the result of CEES induced alteration in the nuclear factor kappa B (NF-κB) signalling pathway. Since NO can act as an antioxidant, the CEES induced down-regulation of iNOS in LPS-stimulated macrophages could elevate oxidative stress. Since macrophage generated NO is known to play a key role in cutaneous wound healing, it is possible that this work has physiological relevance with respect to the healing of HD induced skin blisters

Inhibition of inducible Nitric Oxide Synthase by a mustard gas analog in murine macrophages

BACKGROUND: 2-Chloroethyl ethyl sulphide (CEES) is a sulphur vesicating agent and an analogue of the chemical warfare agent 2,2'-dichlorodiethyl sulphide, or sulphur mustard gas (HD). Both CEES and HD are alkylating agents that influence cellular thiols and are highly toxic. In a previous publication, we reported that lipopolysaccharide (LPS) enhances the cytotoxicity of CEES in murine RAW264.7 macrophages. In the present investigation, we studied the influence of CEES on nitric oxide (NO) production in LPS stimulated RAW264.7 cells since NO signalling affects inflammation, cell death, and wound healing. Murine macrophages stimulated with LPS produce NO almost exclusively via inducible nitric oxide synthase (iNOS) activity. We suggest that the influence of CEES or HD on the cellular production of NO could play an important role in the pathophysiological responses of tissues to these toxicants. In particular, it is known that macrophage generated NO synthesised by iNOS plays a critical role in wound healing. RESULTS: We initially confirmed that in LPS stimulated RAW264.7 macrophages NO is exclusively generated by the iNOS form of nitric oxide synthase. CEES treatment inhibited the synthesis of NO (after 24 hours) in viable LPS-stimulated RAW264.7 macrophages as measured by either nitrite secretion into the culture medium or the intracellular conversion of 4,5-diaminofluorescein diacetate (DAF-2DA) or dichlorofluorescin diacetate (DCFH-DA). Western blots showed that CEES transiently decreased the expression of iNOS protein; however, treatment of active iNOS with CEES in vitro did not inhibit its enzymatic activity CONCLUSION: CEES inhibits NO production in LPS stimulated macrophages by decreasing iNOS protein expression. Decreased iNOS expression is likely the result of CEES induced alteration in the nuclear factor kappa B (NF-κB) signalling pathway. Since NO can act as an antioxidant, the CEES induced down-regulation of iNOS in LPS-stimulated macrophages could elevate oxidative stress. Since macrophage generated NO is known to play a key role in cutaneous wound healing, it is possible that this work has physiological relevance with respect to the healing of HD induced skin blisters

Protective Effect of Liposome-Encapsulated Glutathione in a Human Epidermal Model Exposed to a Mustard Gas Analog

Sulfur mustard or mustard gas (HD) and its monofunctional analog, 2-chloroethyl ethyl sulfide (CEES), or half-mustard gas , are alkylating agents that induce DNA damage, oxidative stress, and inflammation. HD/CEES are rapidly absorbed in the skin causing extensive injury. We hypothesize that antioxidant liposomes that deliver both water-soluble and lipid-soluble antioxidants protect skin cells from immediate CEES-induced damage via attenuating oxidative stress. Liposomes containing water-soluble antioxidants and/or lipid-soluble antioxidants were evaluated using in vitro model systems. Initially, we found that liposomes containing encapsulated glutathione (GSH-liposomes) increased cell viability and attenuated production of reactive oxygen species (ROS) in HaCaT cells exposed to CEES. Next, GSH-liposomes were tested in a human epidermal model, EpiDerm. In the EpiDerm, GSH-liposomes administered simultaneously or 1 hour after CEES exposure (2.5mM) increased cell viability, inhibited CEES-induced loss of ATP and attenuated changes in cellular morphology, but did not reduce caspase-3 activity. These findings paralleled the previously described in vivo protective effect of antioxidant liposomes in the rat lung and established the effectiveness of GSH-liposomes in a human epidermal model. This study provides a rationale for use of antioxidant liposomes against HD toxicity in the skin considering further verification in animal models exposed to HD

Comparison of Rooting Strategies to Explore Rock Fractures for Shallow Soil-Adapted Tree Species with Contrasting Aboveground Growth Rates: A Greenhouse Microcosm Experiment

For tree species adapted to shallow soil environments, rooting strategies that efficiently explore rock fractures are important because soil water depletion occurs frequently. However, two questions: (a) to what extent shallow soil-adapted species rely on exploring rock fractures and (b) what outcomes result from drought stress, have rarely been tested. Therefore, based on the expectation that early development of roots into deep soil layers is at the cost of aboveground growth, seedlings of three tree species (Cyclobalanopsis glauca, Delavaya toxocarpa, and Acer cinnamomifolium) with distinct aboveground growth rates were selected from a typical shallow soil region. In a greenhouse experiment that mimics the basic features of shallow soil environments, 1-year-old seedlings were transplanted into simulated microcosms of shallow soil overlaying fractured bedrock. Root biomass allocation and leaf physiological activities, as well as leaf δ13C values were investigated and compared for two treatments: regular irrigation and repeated cycles of drought stress. Our results show that the three species differed in their rooting strategies in the context of encountering rock fractures, however, these strategies were not closely related to the aboveground growth rate. For the slowest-growing seedling, C. glauca, percentages of root mass in the fractures, as well as in the soil layer between soil and bedrock increased significantly under both treatments, indicating a specialized rooting strategy that facilitated the exploration of rock fractures. Early investment in deep root growth was likely critical to the establishment of this drought-vulnerable species. For the intermediate-growing, A. cinnamomifolium, percentages of root mass in the bedrock and interface soil layers were relatively low and exhibited no obvious change under either treatment. This limited need to explore rock fractures was compensated by a conservative water use strategy. For the fast-growing, D. toxocarpa, percentages of root mass in the bedrock and interface layers increased simultaneously under drought conditions, but not under irrigated conditions. This drought-induced rooting plasticity was associated with drought avoidance by this species. Although, root development might have been affected by the simulated microcosm, contrasting results among the three species indicated that efficient use of rock fractures is not a necessary or specialized strategy of shallow-soil adapted species. The establishment and persistence of these species relied on the mutual complementation between their species-specific rooting strategies and drought adaptations

Protective Effect of Liposome-Encapsulated Glutathione in a Human Epidermal Model Exposed to a Mustard Gas Analog

Sulfur mustard or mustard gas (HD) and its monofunctional analog, 2-chloroethyl ethyl sulfide (CEES), or “half-mustard gas,” are alkylating agents that induce DNA damage, oxidative stress, and inflammation. HD/CEES are rapidly absorbed in the skin causing extensive injury. We hypothesize that antioxidant liposomes that deliver both water-soluble and lipid-soluble antioxidants protect skin cells from immediate CEES-induced damage via attenuating oxidative stress. Liposomes containing water-soluble antioxidants and/or lipid-soluble antioxidants were evaluated using in vitro model systems. Initially, we found that liposomes containing encapsulated glutathione (GSH-liposomes) increased cell viability and attenuated production of reactive oxygen species (ROS) in HaCaT cells exposed to CEES. Next, GSH-liposomes were tested in a human epidermal model, EpiDerm. In the EpiDerm, GSH-liposomes administered simultaneously or 1 hour after CEES exposure (2.5 mM) increased cell viability, inhibited CEES-induced loss of ATP and attenuated changes in cellular morphology, but did not reduce caspase-3 activity. These findings paralleled the previously described in vivo protective effect of antioxidant liposomes in the rat lung and established the effectiveness of GSH-liposomes in a human epidermal model. This study provides a rationale for use of antioxidant liposomes against HD toxicity in the skin considering further verification in animal models exposed to HD

East Meets West: An International Dialogue on Mediation and Med-Arb in the United States and China

This Second Beijing Arbitration Commission (BAC)/Straus Institute for Dispute Resolution International Videoconference, following up on last year\u27s successful inaugural program, will provide different perspectives on the current BAC initiative and evolving attitudes toward mediation and med-arb. Topics include: (1) the development and current state of business mediation in the U.S.; (2) the challenges and opportunities confronting China in developing stand-alone business mediation; (3) reflections on the skills necessary for mediators; (4) common pitfalls in mediation; (5) perspectives on med-arb (as opposed to stand-alone mediation); and (6) how to most effectively use mediation in conjunction with arbitration procedures

Characterization of the Transcriptome of Hair Cell Regeneration in the Neonatal Mouse Utricle

Background/Aims: Hearing and balance deficits are mainly caused by loss of sensory inner ear hair cells. The key signals that control hair cell regeneration are of great interest. However, the molecular events by which the cellular signals mediate hair cell regeneration in the mouse utricle are largely unknown. Methods: In the present study, we investigated gene expression changes and related molecular pathways using RNA-seq and qRT-PCR in the newborn mouse utricle in response to neomycin-induced damage. Results: There were 302 and 624 genes that were found to be up-regulated and down-regulated in neomycin-treated samples. GO and KEGG pathway analyses of these genes revealed many deregulated cellular components, molecular functions, biological processes and signaling pathways that may be related to hair cell development. More importantly, the differentially expressed genes included 9 transcription factors from the zf-C2H2 family, and eight of them were consistently down-regulated during hair cell damage and subsequent regeneration. Conclusion: Our results provide a valuable source for future studies and highlighted some promising genes, pathways or processes that may be useful for therapeutic applications

Dynamic Sealing Behavior of Sand Self-Juxtaposition Windows on a Trap-Bounding Fault in a Natural Gas Storage Site

AbstractAn understanding of across-fault seals is essential for planning an injection/production strategy for a fault-bounded gas storage site. In addition, it is more likely to permit lateral leakage for a fault with sand self-juxtaposition windows. This paper is aimed at identifying the dynamic sealing behaviors of a sand self-juxtaposition fault on the geological and gas injection timescales. Banzhongbei gas storage site, China, was taken as a target area, and fault seals and hydrocarbon distributions within the original reservoirs were studied. The results showed that across-fault pressure differences of 0.085~0.146 MPa (equivalent to 41.6~71.5 m oil-column and 27.0~46.4 m gas-column heights) were supported by sand self-juxtaposition windows on the B816 fault, and the resultant absolute permeability (5.97×10−2~5.69×10−1 mD) of the fault was nearly 3~4 orders of magnitude lower than the average absolute permeability of reservoirs (1.16×102 mD). Gas composition contrasts, between the original and injection gas coupled with dynamic pressure monitoring data, indicated that lateral leakage occurred across sand self-juxtaposition windows under the condition of high across-fault pressure difference. However, the low-permeability fault showed strong negative influence on the efficiency of fluid flow in the model calculations and prolongs the timescales of pressure-difference decayed as much as 5 orders of magnitude relative to those of nonfault model calculations. These modeled dynamic sealing behaviors of sand self-juxtaposition windows may lead to a better understanding of the relative retardation of across-fault gas flow by weak sealing faults on the gas injection/production timescale