INCREASED RATE OF ACETYLCHOLINESTERASE SYNTHESIS IN DIFFERENTIATING NEUROBLASTOMA CELLS

When neuroblastoma cells (N18) in vitro are maintained in the absence of serum, the specific activity of AChE begins to rise rapidly after an initial lag period of about 2–3 days, reaching a maximum level (10–20-fold increase) by 7 days after induction. In order to clarify the mechanism of induction, it was necessary to measure the rate of AChE synthesis and its sensitivity to metabolic inhibitors. Return of enzymatic activity after irreversible inhibition of AChE in "differentiated" cells was blocked by cycloheximide, but not by cordycepin or actinomycin D, suggesting that protein but not mRNA synthesis was required for replacement. By using the initial rate of this replacement as a measure of the rate of synthesis of the enzyme, it was shown that cells which had differentiated in the absence of serum synthesized AChE 50-fold faster on a specific activity basis than their undifferentiated counterparts. In contrast, cordycepin effectively blocked the increase in the rate of AChE synthesis that occurs as a result of serum deprivation, indicating that the induction process itself requires the synthesis of new mRNA. Axonation, another index of differentiation, was not completely blocked by inhibition of RNA or protein synthesis and presumably utilizes only pools of pre-existing structural proteins

N -acetylcysteine and endothelial cell injury by sulfur mustard

Understanding the underlying mechanisms of cell injury and death induced by the chemical warfare vesicant sulfur mustard (HD) will be extremely helpful in the development of effective countermeasures to this weapon of terror. We have found recently that HD induces both apoptosis and necrosis in endothelial cells ( Toxicol. Appl. Pharmacol . 1996; 141: 568–583). Pretreatment of the endothelial cells for 20 h with the redox-active agent N -acetyl-L-cysteine (NAC) selectively prevented apoptotic death induced by HD. In this study, we tested the hypotheses that pretreatment with NAC acts through two different pathways to minimize endothelial injury by HD: NAC pretreatment acts via a glutathione (GSH)-dependent pathway; and NAC pretreatment acts to suppress HD-induced activation of the nuclear transcription factor NFΚB. We used a fluorescence microscopic assay of apoptotic nuclear features to assess viability and electrophoretic mobility shift assays (EMSAs) to assess the activity of NFΚB following exposure to HD. The cells were treated with 0–10 mM GSH for 1 h prior to and during exposure to 0 or 500 ΜM HD for 5–6 h. Cells were also treated with 50 mM NAC or 200 ΜM buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, alone or in combination overnight prior to exposure to 0 or 500 ΜM HD for 5–6 h. Externally applied GSH up to a concentration of 5 mM had no toxic effect on the cells. Mild toxicity was associated with 10 mM GSH alone. There was a dose-related enhancement of viability when 2.5 and 5 mM GSH were present during the HD exposure. Pretreatment with BSO alone had no discernible toxicity. However, pretreatment with this inhibitor of GSH synthesis potentiated the toxicity of HD. Pretreatment with 50 mM NAC, as previously reported, provided substantial protection. Combining pretreatment with both BSO and NAC eliminated the protective effect of NAC pretreatment alone on HD injury. These observations are highly suggestive that NAC enhances endothelial survival via GSH-dependent effects and confirms and extends the work of others with different models that externally supplied GSH alone may be a fairly effective countermeasure against HD injury of endothelium. We next examined the hypothesis that HD may activate the nuclear transcription factor NFΚB by performing EMSAs with nuclear extracts of endothelial cells following exposure to 0, 250 or 500 ΜM HD. This demonstrated an up to 2.5-fold increase (scanning densitometry) in activation of NFΚB binding to its consensus sequence induced by 500 ΜM HD after 5 h of HD exposure. Paradoxically, treatment of the endothelial cells alone with 50 mM NAC activated NFΚB, although HD-induced activation of NFkB was partially suppressed by NAC at 5 h. Factor NFΚB is an important transcription factor for a number of cytokine genes (e.g. tumor necrosis factor, TNF), which can be activated following stress in endothelial cells. Taken together, these observations suggest that the protective effects of NAC may be mediated by enhanced GSH synthesis. The increased GSH may act to scavenge HD and also prevent oxidative activation of NFΚB. Under some conditions, NAC may act as an oxidizing agent and thus increase NFΚB activity. The NFΚB-dependent gene expression may be important in inducing endothelial cell death as well as in generating a local inflammatory reaction associated with the release of endothelial-derived cytokines. Copyright © 2000 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34703/1/671_ftp.pd

Assessment of the role of DNA damage and repair in the survival of primary cultures of rat cutaneous keratinocytes exposed to bis(2-chloroethyl)sulfide

Toxicity manifests itself as vesication in human skin exposed topically to bis(2-chloroethyl)sulfide (BCES). The destruction of the proliferating population of epidermal cells is a major component of the pathogenic process. Available data strongly suggest that damage to cellular DNA is a critical factor in the loss of these cells. However, the influence of DNA repair on this toxic response has not been adequately studied. Therefore, a study was undertaken to ascertain the influence of DNA repair on the survival of primary monolayer cultures of rat cutaneous keratinocytes exposed to BCES. The sensitive nucleoid sedimentation assay was employed for the determination of DNA damage in cultures exposed to very low levels of BCES. Initial experiments demonstrated that within 1 hr of exposure to as little as 0.1 [mu] BCES the structural integrity of cellular DNA was compromised, presumably resulting from the appearance of single-strand breaks in the nucleic acid. This same effect was demonstrated in basal cells derived from a stratified, cornified culture grown at the air-liquid interface and exposed topically to the vesicant. Further studies with the monolayer culture demonstrated that the gross structural integrity of the DNA in cells exposed to as much as 5 [mu] BCES was completely restored within the first 22 hr following the exposure. However, this repair process appeared to be inefficient since a depression of thymidine incorporation into DNA and a significant loss of DNA were exhibited in exposed cultures as long as 72 hr after the initial exposure.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29057/1/0000090.pd

Loss of expression of TGF-βs and their receptors in chronic skin lesions induced by sulfur mustard as compared with chronic contact dermatitis patients

<p>Abstract</p> <p>Background</p> <p>Sulfur mustard (SM) is a blister-forming agent that has been used as a chemical weapon. Sulfur mustard can cause damage in various organs, especially the skin, respiratory system, and eyes. Generally, the multiple complications of mustard gas result from its alkalizing potency; it reacts with cellular components like DNA, RNA, proteins, and lipid membranes.</p> <p>TGF-β is a multi-functional cytokine with multiple biological effects ranging from cell differentiation and growth inhibition to extracellular matrix stimulation, immunosuppression, and immunomodulation. TGF-β has 3 isoforms (TGF-β 1, 2, 3) and its signaling is mediated by its receptors: R1, R2 and intracellular Smads molecules.</p> <p>TGF-β has been shown to have anti-inflammatory effects. TGF-βs and their receptors also have an important role in modulation of skin inflammation, proliferation of epidermal cells, and wound healing, and they have been implicated in different types of skin inflammatory disorders.</p> <p>Methods</p> <p>Seventeen exposed SM individuals (48.47 ± 9.3 years), 17 chronic dermatitis patients (46.52 ± 14.6 years), and 5 normal controls (44.00 ± 14.6 years) were enrolled in this study.</p> <p>Evaluation of TGF-βs and their receptors expressions was performed by semiquantitative RT-PCR. Only TGF1was analyzed immunohistochemically.</p> <p>Results</p> <p>Our results showed significant decreases in the expression percentages of TGF-β 1, 2 and R1, R2 in chemical victims in comparison with chronic dermatitis and normal subjects and significant decreases in the intensity of R1 and R2 expressions in chemical victims in comparison with chronic dermatitis and normal controls. (P value < 0.05)</p> <p>Conclusions</p> <p>TGF-βs and their receptors appear to have a noticeable role in chronic inflammatory skin lesions caused by sulfur mustard.</p

The percutaneous toxicokinetics of sulphur mustard in a damaged skin porcine model and the evaluation of WoundStat™ as a topical decontaminant

This is the peer reviewed version of the following article: Charlotte A. Hall, et al, 'The percutaneous toxicokinetics of Sulphur mustard in a damaged skin porcine model and the evaluation of WoundStat™ as a topical decontaminant', Journal of Applied Toxicology, July 2017, which has been published in final form at DOI: 10.1002/jat.3453. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. Copyright © 2017 John Wiley & Sons, Ltd.This study used a damaged skin, porcine model to evaluate the in vivo efficacy of WoundStat™ for decontamination of superficial (non-haemorrhaging), sulphur mustard-contaminated wounds. The dorsal skin of 12 female pigs was subjected to controlled physical damage and exposed to 10 μL 14C–radiolabelled sulphur mustard (14C–SM). Animals were randomly assigned to either a control or a treatment group. In the latter, WoundStat™ was applied 30 s post exposure and left in situ for 1 h. Skin lesion progression and decontaminant efficacy were quantified over 6 h using a range of biophysical measurements. Skin, blood and organ samples were taken post mortem for histopathological assessment, 14C–SM distribution and toxicokinetic analyses. Application of SM to damaged skin without decontamination was rapidly followed by advanced signs of toxicity, including ulceration and decreased blood flow at the exposure site in all animals. WoundStat™ prevented ulceration and improved blood flow at the exposure site in all decontaminated animals (n = 6). Furthermore, significantly smaller quantities of 14C–SM were detected in the blood (45% reduction), and recovered from skin (70% reduction) and skin surface swabs (99% reduction) at 6 h post-challenge. Overall, the distribution of 14C–SM in the internal organs was similar for both groups, with the greatest concentration in the kidneys, followed by the liver and small intestine. WoundStat™ significantly reduced the amount of 14C–SM recovered from the liver, a key organ for SM metabolism and detoxification. This study demonstrates that WoundStat™ is a suitable product for reducing the ingress and toxicity of a chemical warfare agent.Peer reviewedFinal Accepted Versio