Radharaman Ray

Inhibition of sulfur mustard-induced cytotoxicity and inflammation by the macrolide antibiotic roxithromycin in human respiratory epithelial cells

BackgroundSulfur mustard (SM) is a potent chemical vesicant warfare agent that remains a significant military and civilian threat. Inhalation of SM gas causes airway inflammation and injury. In recent years, there has been increasing evidence of the effectiveness of macrolide antibiotics in treating chronic airway inflammatory diseases. In this study, the anti-cytotoxic and anti-inflammatory effects of a representative macrolide antibiotic, roxithromycin, were tested in vitro using SM-exposed normal human small airway epithelial (SAE) cells and bronchial/tracheal epithelial (BTE) cells. Cell viability, expression of proinflammatory cytokines including interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor (TNF), and expression of inducible nitric oxide synthase (iNOS) were examined, since these proinflammatory cytokines/mediators are import indicators of tissue inflammatory responses. We suggest that the influence of roxithromycin on SM-induced inflammatory reaction could play an important therapeutic role in the cytotoxicity exerted by this toxicant.ResultsMTS assay and Calcein AM/ethidium homodimer (EthD-1) fluorescence staining showed that roxithromycin decreased SM cytotoxicity in both SAE and BTE cells. Also, roxithromycin inhibited the SM-stimulated overproduction of the proinflammatory cytokines IL-1β, IL-6, IL-8 and TNF at both the protein level and the mRNA level, as measured by either enzyme-linked immunosorbent assay (ELISA) or real-time RT-PCR. In addition, roxithromycin inhibited the SM-induced overexpression of iNOS, as revealed by immunocytochemical analysis using quantum dots as the fluorophore.ConclusionThe present study demonstrates that roxithromycin has inhibitory effects on the cytotoxicity and inflammation provoked by SM in human respiratory epithelial cells. The decreased cytotoxicity in roxithromycin-treated cells likely depends on the ability of the macrolide to down-regulate the production of proinflammatory cytokines and/or mediators. The results obtained in this study suggest that macrolide antibiotics may serve as potential vesicant respiratory therapeutics through mechanisms independent of their antibacterial activity.

Macrolide antibiotics improve chemotactic and phagocytic capacity as well as reduce inflammation in sulfur mustard-exposed monocytes

BACKGROUND Sulfur mustard (SM) inhalation causes apoptosis and death of airway epithelial cells as well as inflammation in the airway. Efficient clearance of the cell debris by alveolar macrophages is necessitated to reduce the inflammation. Macrolide antibiotics have been reported to have anti-inflammatory properties by modulating the production of proinflammatory cytokines and mediators, and by improving macrophage functions. The present study investigated the effects of four commonly used macrolide antibiotics, namely azithromycin, clarithromycin, erythromycin, and roxithromycin, on chemotactic and phagocytotic function and on inflammatory cytokines/mediators production in vitro in SM-exposed monocyte THP-1 cells. RESULTS Chemotaxis and phagocytosis of the monocytes reduced upon exposure to 10microM SM (8.1% and 17.5%, respectively) were restored by treatment with 10microM of any of the four macrolides. Overexpression of inflammatory cytokines following SM exposure was decreased by 50-70% with macrolide treatment. Similarly, exaggerated iNOS expression and nitric oxide (NO) production induced by SM exposure was largely inhibited by treatment with macrolides. CONCLUSION The data demonstrate that macrolide antibiotics were effective in improving the degenerated chemotactic and phagocytotic functions of monocytes following SM exposure, and in reducing SM-induced overproduction of proinflammatory cytokines and mediators. Thus, treatment with macrolide antibiotics may lead to improved clearance of apoptotic material in the airway and ultimately result in reduced airway inflammation and injury caused by SM inhalation, suggesting that macrolide antibiotics may serve as potential vesicant respiratory therapeutics.

Sulfur Mustard Induces Apoptosis in Cultured Normal Human Airway Epithelial Cells: Evidence of a Dominant Caspase-8-Mediated Pathway and Differential Cellular Responses

We have shown that sulfur mustard (SM; bis-(2-chloroethyl) sulfide), an alkylating, vesicating chemical warfare agent, causes dermal toxicity, including skin microblisters, via the induction of both death receptor (DR) and mitochondrial pathways of apoptosis in human epidermal keratinocytes. While SM is known for its skin-vesicating properties, respiratory tract lesions are the main source of morbidity and mortality after inhalation exposure. We, therefore, investigated whether SM induces apoptotic cell death in normal human bronchial epithelial (NHBE) cells and small airway epithelial cells (SAEC) in vitro. Cells were exposed to various concentrations of SM (0, 50, 100, and 300 μM for 16 h) in the culture medium and then tested for the activation of apoptotic executioner caspase-3 and initiator caspases-8 and -9. Caspases-8 and -3 were activated by SM in both airway cell types, indicating the induction of a DR pathway of apoptosis in these cells; however, the levels of enzyme activation were different, depending on the cell type and the SM concentrations used. Consistent with enzyme activity results, immunoblot analyses revealed the proteolytic processing of the proenzymes to the active forms of caspases-8 and -3 in these cells after SM exposure. Interestingly, NHBE cells were found to be exquisitely sensitive to SM, compared to SAEC, with caspase-3 activities in SM-exposed NHBE cells ∼2-fold higher and caspase-8 activities ∼10-fold higher than in SAEC. Furthermore, SM activated caspase-9 in NHBE cells, but not in SAEC, indicating a possible role of the mitochondrial pathway only in the NHBE cells. The present study shows that both upper airway (NHBE cells) and deep lung (SAEC) epithelial cells undergo SM-induced apoptotic death in vitro, but distinct cell-type specific responses can be elicited, which may be attributed to intrinsic properties that characterize the response of these cells to SM. These findings need to be taken into consideration in the search for modulators of these pathways for the therapeutic intervention to reduce SM injury due to respiratory tract lesions.

Calmodulin, poly(ADP–ribose)polymerase and p53 are targets for modulating the effects of sulfur mustard

We describe two pathways by which the vesicating agent sulfur mustard (HD) may cause basal cell death and detachment: induction of terminal differentiation and apoptosis. Following treatment of normal human epidermal keratinocytes (NHEK) with 10 or 100 μmM HD, the differentiation‐specific keratin pair K1/K10 was induced and the cornified envelope precursor protein, involucrin, was cross‐linked by epidermal transglutaminase. Fibronectin levels were reduced in a time‐ and dose‐dependent manner. The rapid increase in p53 and decrease in Bcl‐2 levels was consistent not only with epidermal differentiation but with apoptosis as well. Further examination of biochemical markers of apoptosis following treatment of either NHEK or human papillomavirus (HPV)‐immortalized keratinocytes revealed a burst of poly(ADP–ribose) synthesis, specific cleavage of poly(ADP–ribose)polymerase (PARP) in vivo and in vitro into characteristic 89 and 24 kDa fragments, processing of caspase‐3 into its active form and the formation of DNA ladders. The intracellular calcium chelator BAPTA suppressed the differentiation markers, whereas antisense oligonucleotides and chemical inhibitors specific for calmodulin blocked both markers of differentiation and apoptosis. Modulation of p53 levels utilizing retroviral constructs expressing the E6, E7 or E6 + E7 genes of HPV‐16 revealed that HD‐induced apoptosis was partially p53‐dependent. Finally, immortalized fibroblasts derived from PARP −/− ‘knockout mice’ were exquisitely sensitive to HD‐induced apoptosis. These cells became HD resistant when wild‐type PARP was stably expressed in these cells. These results indicate that HD exerts its effects via calmodulin, p53 and PARP‐sensitive pathways. Copyright

An efficient drug delivery vehicle for botulism countermeasure

BackgroundBotulinum neurotoxin (BoNT) is the most potent poison known to mankind. Currently no antidote is available to rescue poisoned synapses. An effective medical countermeasure strategy would require developing a drug that could rescue poisoned neuromuscular synapses and include its efficient delivery specifically to poisoned presynaptic nerve terminals. Here we report a drug delivery strategy that could directly deliver toxin inhibitors into the intoxicated nerve terminal cytosol.ResultsA targeted delivery vehicle was developed for intracellular transport of emerging botulinum neurotoxin antagonists. The drug delivery vehicle consisted of the non-toxic recombinant heavy chain of botulinum neurotoxin-A coupled to a 10-kDa amino dextran via the heterobifunctional linker 3-(2-pyridylthio)-propionyl hydrazide. The heavy chain served to target botulinum neurotoxin-sensitive cells and promote internalization of the complex, while the dextran served as a platform to deliver model therapeutic molecules to the targeted neurons. Our results indicated that the drug delivery vehicle entry into neurons was via BoNT-A receptor mediated endocytosis. Once internalized into neurons, the drug carrier component separated from the drug delivery vehicle in a fashion similar to the separation of the BoNT-A light chain from the holotoxin. This drug delivery vehicle could be used to deliver BoNT-A antidotes into BoNT-A intoxicated cultured mouse spinal cord cells.ConclusionAn effective BoNT-based drug delivery vehicle can be used to directly deliver toxin inhibitors into intoxicated nerve terminal cytosol. This approach can potentially be utilized for targeted drug delivery to treat other neuronal and neuromuscular disorders. This report also provides new knowledge of endocytosis and exocytosis as well as of BoNT trafficking.

Role of poly(ADP–ribose) polymerase (PARP) in DNA repair in sulfur mustard-exposed normal human epidermal keratinocytes (NHEK)†‡

We previously reported that, in normal human epidermal keratinocytes (NHEK) cultures exposed to the alkylating compound sulfur mustard (bis‐(2‐chloroethyl) sulfide, HD, 0.3–1 mM), there is a rapid (≤1 h) activation (100% above unexposed control) of the DNA repair enzyme DNA ligase I (130 kD) followed by a first‐order decay (1–5 h). The DNA ligase activation is accompanied by a time‐dependent (0.5–4 h) and significant DNA repair. Inhibition of another putative DNA repair enzyme, poly(ADP–ribose) polymerase (PARP), by using 3‐amino benzamide does not affect DNA ligase activation following HD exposure, but increases the half‐life of the activated enzyme threefold. To examine the role of PARP in HD‐induced DNA ligase activation and subsequent DNA repair, we conducted studies using cultured keratinocytes in which the level of PARP had been selectively lowered (≥85%) by the use of induced expression of antisense RNA. In these cells, there was no stimulation of DNA ligase up to 3 h, and a small stimulation (ca. 30% above unexposed control at 5–6 h after HD exposure. A time‐course (0.5–6 h) study of DNA repair in HD‐exposed PARP‐deficient keratinocytes revealed a much slower rate of repair compared with HD‐exposed NHEK. The results suggest an active role of PARP in DNA ligase activation and DNA repair in mammalian cells, and also indicate that modulation of PARP‐mediated mechanisms may provide a useful approach in preventing HD toxicity. Published in 2000 by John Wiley & Sons, Ltd.

Inhibition of bioenergetics alters intracellular calcium, membrane composition, and fluidity in a neuronal cell line

The effect of inhibited bioenergetics and ATP depletion on membrane composition and fluidity was examined in cultured neuroblastoma-glioma hybrid NG108-15 cells. Sodium cyanide (CN) and 2-deoxyglucose (2-DG) were used to block oxidative phosphorylation and anaerobic glycolysis, respectively. Endoplasmic reticulum (ER) Ca2+-pump activity measured by45Ca2+ uptake was >92% inhibited in intact cells incubated with CN (1 mM) and 2-DG (20 mM) for 30 min. In addition, exposure of cells to CN and 2-DG caused a 134% increased release of isotopically labeled arachidonic acid (3H-AA) or arachidonate-derived metabolites from membranes. Removal of Ca2+ from the incubation medium ablated the CN/2-DG induced release of3H-AA or its metabolites. Membrane fluidity of intact cells was measured by electron spin resonance spectroscopy using the spin label 12-doxyl stearic acid. The mean rotational correlation time (τc) of the spin label increased 49% in CN/2-DG exposed cells compared to controls, indicating a decrease in membrane fluidity. These results show that depletion of cellular ATP results in inhibition of the ER Ca2+-pump, loss of AA from membranes, and decreased membrane fluidity. We propose that impaired bioenergetics can increase intracellular Ca2+ as a result of Ca2+-pump inhibition and thereby activate Ca2+-dependent phospholipases causing membrane effects. Since neurons derive energy predominantly from oxidative metabolism, ATP depletion during brain hypoxia may initiate a similar cytotoxic mechanism.

Suppression of Inducible Nitric Oxide Synthase Expression and Nitric Oxide Production by Macrolide Antibiotics in Sulfur Mustard-Exposed Airway Epithelial Cells

Sulfur mustard, a vesicant chemical warfare agent, causes airway injury due to massive release of destructive enzymes and mediators of inflammation. Nitric oxide plays an important yet controversial role in inflammation. An impressive number of reports suggest that excessive amount of nitric oxide may promote inflammation-induced cell injury and death. Overproduction of nitric oxide is catalysed by up-regulated expression of the inducible isoform of nitric oxide synthase (iNOS). In this study, we used quantum dot-mediated immunocytochemistry to analyse iNOS expression and flow cytometry to analyse the intracellular nitric oxide production in sulfur mustard-exposed normal human small airway epithelial cells and bronchial/tracheal epithelial cells and studied the effect of four US Food and Drug Administration-approved macrolide antibiotics, namely, azithromycin, clarithromycin, erythromycin and roxithromycin. Exposure to 100 microM sulfur mustard significantly up-regulated iNOS expression and resulted in overproduction of nitric oxide in these cells. Addition of macrolide antibiotics to 100 microM in the medium reduced both iNOS expression and nitric oxide production to near normal level. Thus, the current study provides in vitro evidence of the immunomodulatory effects of macrolide antibiotics in sulfur mustard-exposed airway epithelial cells. These results suggest that macrolide antibiotics may serve as potential vesicant respiratory therapeutics through mechanisms independent of their antibacterial activity.

Sulfur mustard induces apoptosis in lung epithelial cells via a caspase amplification loop

Sulfur mustard (SM [bis-(2-chloroethyl) sulfide]) is a chemical warfare agent that causes skin blisters presumably due to DNA alkylation and cross-links. We recently showed that SM also induces apoptotic death in cultured normal human bronchial/tracheal epithelial (NHBE) cells and small airway epithelial cells (SAEC) in vitro. In this process, caspases-8 and -3, but not caspase-9, were strongly activated; this suggests a death receptor pathway for apoptosis. We now show that rat lungs were induced to undergo apoptosis in vivo following exposure of rats to SM by inhalation. Further study of the mechanism of apoptosis due to SM was performed with cultured NHBE cells and SAEC using tetrapeptide inhibitors of caspases-3, and -8. Inhibition of caspase-8 drastically reduced the activation of caspase-3 and almost eliminated that of caspase-9. Moreover, caspase-3 inhibition markedly reduced the activation of caspase-8 and also almost completely inhibited activation of caspase-9. These results suggest a death receptor pathway of apoptosis that utilizes a feedback amplification mechanism involving an activated death receptor complex that leads to the activation of caspase-9 via a caspase-3 pathway. These results may be important for the design of inhibitors of these pathways for therapeutic intervention to attenuate SM injury in respiratory tract lesions.

Clostridial neurotoxins as a drug delivery vehicle targeting nervous system.

Several neuronal disorders require drug treatment using drug delivery systems for specific delivery of the drugs for the targeted tissues, both at the peripheral and central nervous system levels. We describe a review of information currently available on the potential use of appropriate domains of clostridial neurotoxins, tetanus and botulinum, for effective drug delivery to neuronal systems. While both tetanus and botulinum neurotoxins are capable of delivering drugs the neuronal cells, tetanus neurotoxin is limited in clinical use because of general immunization of population against tetanus. Botulinum neurotoxin which is also being used as a therapeutic reagent has strong potential for drug delivery to nervous tissues.