Diane Hardej

Induction of cellular glutathione and glutathione S-transferase by 3H-1,2-dithiole-3-thione in rat aortic smooth muscle A10 cells: protection against acrolein-induced toxicity.

There is increasing evidence that aldehydes, including acrolein generated endogenously during the degradation process of biological molecules or the metabolism of foreign chemicals may be involved in the pathogenesis of cardiovascular diseases, such as atherosclerosis. Because glutathione (GSH) and GSH S-transferase (GST) are a major cellular defense against the toxic effects of reactive aldehydes, in this study we have characterized the inducibility of GSH and GST by the unique chemoprotective agent, 3H-1,2-dithiole-3-thione (D3T) and their protective effects against acrolein-induced toxicity in rat aortic smooth muscle A10 cells. Incubation of rat aortic A10 cells with micromolar concentrations of D3T resulted in a concentration- and time-dependent induction of both GSH and GST. Treatment of A10 cells with D3T also led to induction of gamma-glutamylcysteine synthetase, the key enzyme involved in GSH biosynthesis. Notably, the levels of GSH and GST remained higher than basal levels 72 h after removal of D3T from the culture media. To examine the protective effects of D3T-induced GSH and GST against reactive aldehyde-mediated toxicity, A10 cells were pretreated with D3T and then exposed to acrolein. Pretreatment of A10 cells with D3T resulted in a marked decrease of acrolein-induced toxicity as determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction assay and morphological changes. To further demonstrate the involvement of GSH and GST in protecting against acrolein-induced toxicity, buthionine sulfoximine (BSO) and sulfasalazine were used to inhibit cellular GSH biosynthesis and GST activity, respectively. Either depletion of cellular GSH by BSO or inhibition of cellular GST by sulfasalazine led to a marked potentiation of acrolein-induced toxicity in A10 cells. Furthermore, co-treatment of cells with BSO was found to greatly abolish the protective effects of D3T on acrolein-induced toxicity. Taken together, our results demonstrate for the first time that both GSH and GST in aortic smooth muscle cells can be induced by D3T, and that this increased cellular defense affords great protection against reactive aldehyde-induced cardiovascular cell injury.

The synthesis of phenylalanine-derived C5-substituted rhodanines and their activity against selected methicillin-resistant Staphylococcus aureus (MRSA) strains.

A series of rhodanine compounds containing various substituents at the N3- and C5-positions were synthesized and their in vitro activity against a panel of clinically relevant MRSA strains was determined. The anti-MRSA activity of compounds 21 (MIC=3.9 μg/mL, MBC=7.8 μg/mL) and 22 (MIC=1.95 μg/mL, MBC=7.8 μg/mL) was significantly greater than that of the lead compounds, 1-3 and reference antibiotics penicillin G (MIC=31.25 μg/mL) and ciprofloxacin (MIC=7.8 μg/mL) and comparable to that of vancomycin (MIC=0.97 μg/mL). Compounds 21 and 22 were found to be bactericidal at only 2-4-fold higher than their MIC concentrations. In addition, their MIC values remained unchanged in the presence or absence of 10% serum. Overall, the results suggest that compounds 21 and 22 may be of potential use in the treatment of MRSA infections.

The role of chemically induced glutathione and glutathione S-transferase in protecting against 4-hydroxy-2-nonenal-mediated cytotoxicity in vascular smooth muscle cells.

Abstract4-Hydroxy-2-nonenal (HNE) has been suggested to contribute to the pathogenesis of atherosclerosis. One of the major metabolic transformation pathways of HNE involves conjugation with glutathione (GSH) catalyzed by GSH S-transferase (GST). In this study, we have characterized the induction of GSH and GST by 3H-1,2-dithiole-3-thione (D3T) and the protective effects of the D3T-elevated cellular defenses on HNE-mediated toxicity in rat aortic smooth muscle A10 cells. Incubation of A10 cells with D3T resulted in a marked concentration-dependent induction of both GSH and GST. The induction of cellular GST by D3T also exhibited a time-dependent response. Pretreatment of A10 cells with D3T led to a dramatic decrease of HNE-induced cytotoxicity, as assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay and scanning electron microscopy. Incubation of A10 cells with HNE for 0.5 h and 1 h resulted in a significant depletion of cellular GSH, which preceded the decrease of cell viability. To further demonstrate the involvement of GSH and GST in protecting against HNE-induced cytotoxicity, buthionine sulfoximine (BSO) and sulfasalazine were used to inhibit cellular GSH biosynthesis and GST activity, respectively. Either depletion of GSH by BSO or inhibition of GST by sulfasalazine caused great potentiation of HNE-mediated cytotoxicity. Moreover, cotreatment of A10 cells with BSO was found to completely block the D3T-mediated GSH induction and to largely reverse the cytoprotective effects of D3T on HNE-induced toxicity. Taken together, this study demonstrates that D3T can induce both GSH and GST in aortic smooth muscle cells, and that the D3T-augmented cellular defenses afford a marked protection against HNE-induced vascular cell injury.

Tellurium tetrachloride and diphenyl ditelluride cause cytotoxicity in rat hippocampal astrocytes

Tellurium tetrachloride (TeCl(4)) and diphenyl ditelluride (DPDT) cytotoxicity, was investigated in rat astrocytes. Concentrations of 0.24-250μM (24h) were tested for viability using MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and trypan blue exclusion. MTT showed significant decreases at all concentrations tested for both compounds. Significant decreases in viability were seen in 1.95-250μM of DPDT and 0.97-250μM of TeCl(4) with trypan blue exclusion. The LC(50) for both compounds was 62.5μM. Light and scanning microscopy confirm toxicity observed at higher concentrations. Thiobarbituric acid reactive substances (TBARs) assay, TUNEL, cytochrome c and caspase release were carried out. No significant increase in TBARS with either agent was observed (15.625-62.5μM). TUNEL and cytochrome c assays demonstrated apoptosis in TeCl(4) treated cells (31.25-125μM). Non-apoptotic cells were observed in DPDT treated cells. Studies of caspase 3/7 and caspase 9 indicated increased activity in TeCl(4) but not in DPDT treated cells. Optical Emission Spectroscopy of DPDT and TeCl(4) treated cells demonstrated significant accumulation of elemental tellurium in all treatment groups (31.25-125μM). We conclude that DPDT and TeCl(4) are cytotoxic to astrocytes. TeCl(4) treated cells die via the intrinsic apoptotic pathway. Accumulation of tellurium occurs with both compounds, but results in different mechanisms of cell death.

Aerosolized recombinant human lysozyme ameliorates pseudomonas aeruginosa–induced pneumonia in hamsters

ABSTRACT As an alternative to conventional antibiotics, aerosolized recombinant human lysozyme (rhLZ) was used to treat experimentally induced pneumonia. Syrian hamsters were inoculated intratracheally with a nonmucoid strain of Pseudomonas aeruginosa (PA), then exposed to a 1.0%% solution of rhLZ in water for 2 hours per day for 3 consecutive days (controls were treated with aerosolized water alone). Compared to controls, the rhLZ-treated group showed statistically significant reductions in the following parameters: (1) lung histopathological changes, (2) bacterial colony-forming units in whole lung and bronchoalveolar lavage fluid (BALF), (3) total BALF leukocytes, (4) percent BALF neutrophils, and (5) alveolar septal apoptosis. Exposure to aerosolized rhLZ also resulted in a large increase in BALF lysozyme activity. These findings indicate that aerosolized rhLZ may be potentially useful in reducing the level of bacterial colonization and inflammation in the lungs of patients with PA pneumonia.

Ebselen protects brain, skin, lung and blood cells from mechlorethamine toxicity

Nitrogen mustards are vesicants capable of burning the skin, eyes and respiratory tract of exposed individuals. While generally less toxic than sulfur mustards, these compounds have the potential for use as chemical warfare agents. Presently, no antidote exists for treatment against nitrogen mustard toxicity. The purpose of this study was to investigate the in vitro toxicity of the nitrogen mustard mechlorethamine (HN2) in four cell models: CEM-SS human T cells, A431 human skin epithelial cells, rat hippocampal astrocytes and rat pleural mesothelial cells. Furthermore, the efficacy of the synthetic seleno-organic compound ebselen (Eb) (2-phenyl-1,2- benzisoselenazol-3(2H)-one) as a cytoprotective agent against such toxicity was evaluated. Significant increases in cell viability, as assessed using an MTT assay for viability, was demonstrated when 30μM Eb was used as a cotreatment with HN2 in all cell models tested at the following doses of HN2: A431 skin cells,10—40μM; rat astrocytes, 20 and 40μM; rat mesothelia, 10—40 μM; and human T cells 4—16 μM. Decreases in cell viability and toxicity to HN2 were confirmed using light and scanning electron microscopy. Membrane damage, observed with HN2 exposure, such as blebbing and loss of cell projections, was ameliorated with Eb cotreatment. Our results demonstrate a generalized protective effect observed with Eb cotreatment that suggests that this agent may have potential as an antidote for HN2 exposure and toxicity.

Evaluation of Resveratrol and Piceatannol Cytotoxicity in Macrophages, T Cells, and Skin Cells

Evaluation of Resveratrol and Piceatannol Cytotoxicity in Macrophages, T Cells, and Skin Cells The cytotoxicity of resveratrol and of piceatannol, a structural analog of resveratrol, was examined in cultured cells. Using a MTT-based assay, which measures the conversion of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) to a colored formazan product in living cells, resveratrol was found to inhibit the viability of transformed mouse macrophages, tumor-derived human T cells and human epidermoid carcinoma cells in a concentration-dependent manner, with the effect decreasing in the order: T cells (LC50 ~27 μmol L-1, 24 h; ~9 μmol L-1; 48h) > macrophages (LC50~29 μmol L-1, 24 h; 39 μmol L-1, 48 h) > skin cells (LC50 ~91 μmol L-1, 24 h; ~66 μmol L-1, 48 h). Paradoxically, a high concentration of resveratrol (50 μmol L-1) inhibited the proliferation of all three cell types, and a low concentration (5 μmol L-1) stimulated the proliferation of macrophages. The viability of macrophages was also decreased by piceatannol in a concentration-dependent manner. The stimulation of macrophages with zymosan lowered the cytotoxicity of both resveratrol and piceatannol. Scanning electron microscopy of cells treated with resveratrol revealed changes in cellular morphology that were consistent with toxicity. In macrophages and skin cells, resveratrol (50 μmol L-1) induced a time-dependent increase in reduced glutathione levels but did not alter the background levels of thiobarbituric acid-reactive substances. Taken together, the present data indicate that resveratrol is toxic to cultured macrophages, T cells and skin cells at concentrations ≥25 μmol L-1, and that the cytotoxicity occurs via a mechanism that does not involve oxidative stress. Furthermore, the degree of toxicity of both resveratrol and piceatannol towards macrophages depends on the activation status of these cells, with zymosan-activated cells appearing more resistant than nonstimulated cells. Evaluacija Citotoksičnosti Resveratrola I Piceatanola U Makrofazima, T-Stanicama I Stanicama Kože Citotoksičnost resveratrola i piceatanola, strukturnog analoga resveratrola, ispitivana je u uzgojenim stanicama. Primjenom MTT-testa koji mjeri pretvorbu 3-[4,5-dimetiltiazol-2-il]2,5-difenil-tetrazolijeva bromida (MTT) u obojeni formazan produkt u živim stanicama, nađeno je da resveratrol inhibira preživljavanje transformiranih makrofaga miša, ljudskih tumorskih T-stanica i humanih stanica epidermoidnog karcinoma u ovisnosti o koncentraciji, a učinak opada redom: T-stanice (LC50 ~27 μmol L-1, 24 h; ~9 μmol L-1; 48 h) > makrofazi (LC50 ~29 μmol L-1, 24 h; 39 μmol L-1, 48 h) > stanice kože (LC50 ~91 μmol L-1, 24 h; ~66 μmol L-1, 48 h). Paradoksalno, pri visokoj koncentraciji resveratrola (50 μmol L-1) inhibirana je proliferacija svih triju tipova stanica, a pri niskim koncentracijma (5 μmol L-1) stimulirana je proliferacija makrofaga. Preživljavanje makrofaga bilo je također smanjeno primjenom piceatanola ovisno o koncentraciji. Stimulacija makrofaga zimosanom smanjila je citotoksičnost i resveratrola i piceatanola. Skenirajuća elektronska mikroskopija stanica tretiranih resveratrolom pokazala je promjene u morfologiji stanica, što je bilo u skladu s toksičnosti. U makrofazima i stanicama kože resveratrol (50 μmol L-1) inducirao je porast smanjenja razina glutationa ovisan o vremenu, ali nije mijenjao osnovne razine reaktivnih spojeva tiobarbiturne kiseline. Gledajući skupno, prikazani rezultati indiciraju da je resveratrol toksičan za uzgojene makrofage, T-stanice i stanice kože pri koncentracijama ≥25 μmol L-1 i da se citotoksičnost zbiva mehanizmom koji ne uključuje oksidativni stres. Nadalje, stupanj toksičnosti resveratrola i piceatanola prema makrofagima ovisi o aktivacijskom statusu tih stanica, pri čemu su stanice aktivirane zimosanom rezistentnije od nestimuliranih stanica.

Evaluation of tellurium toxicity in transformed and non-transformed human colon cells

Diphenyl ditelluride (DPDT) and tellurium tetrachloride (TeCl(4)) were evaluated for toxicity in transformed (HT-29, Caco-2) and non-transformed colon cells (CCD-18Co). Significant decreases in viability were observed with DPDT exposure in HT-29 (62.5-1000 μM), Caco-2 (31.25-1000 μM) and CCD-18Co cells (500-1000 μM) and with TeCl(4) in HT-29 (31.25-1000 μM), Caco-2 (31.25-1000 μM) and CCD-18Co cells (500-1000 μM). Light microscopy confirmed viability analysis. Significant increases in caspase 3/7 and 9 activity were observed with DPDT in HT-29 (500-1000 μM) and CCD-18Co cells (1000 μM) indicating apoptosis. No significant increases in caspases were seen with TeCl(4) indicating necrosis. Apoptosis or necrosis was confirmed with fluorescent staining (FITC-Annexin, Hoechst 33342 and Ethidium Homodimer). Significant decreases in GSH/GSSG ratio were observed with DPDT in HT-29 (62.5-1000 μM), and CCD-18Co cells (1000 μM) and with TeCl(4) in HT-29 (62.5-1000 μM) and CCD-18Co cells (250-1000 μM). We concluded that cells treated with DPDT resulted in apoptosis and TeCl(4) treatment in necrosis. GSH/GSSG ratio shifts indicate oxidative mechanisms are involved.

The synthesis and SAR study of phenylalanine-derived (Z)-5-arylmethylidene rhodanines as anti-methicillin-resistant Staphylococcus aureus (MRSA) compounds

A focused library of rhodanine compounds containing novel substituents at the C5-position was synthesized and tested in vitro against a panel of clinically relevant MRSA strains. The present SAR study was based on our lead compound 1 (MIC=1.95 μg/mL), with a focus on identifying optimal C5-arylidene substituents. In order to obtain this objective, we condensed several unique aromatic aldehydes with phenylalanine-derived rhodanine intermediates to obtain C5-substituted target rhodanine compounds for evaluation as anti-MRSA compounds. These efforts produced three compounds with significant efficacy: 23, 32 and 44, with MIC values ranging from 0.98 to 1.95 μg/mL against all tested MRSA strains as compared to the reference antibiotics penicillin G (MIC=15.60-250.0 μg/mL) and ciprofloxacin (MIC=7.80-62.50 μg/mL) and comparable to that of vancomycin (MIC=0.48 μg/mL). In addition, compounds 24, 28, 37, 41, 46 and 48 (MIC=1.95-3.90 μg/mL) were efficacious against all MRSA strains. The majority of the synthesized compounds had bactericidal activity at concentrations only two to fourfold higher than their MIC. Overall, the results suggest that compounds 23, 32 and 44 may be of potential use in the treatment of MRSA infections.

The effects of ebselen on cisplatin and diethyldithiocarbamate (DDC) cytotoxicity in rat hippocampal astrocytes

Ebselen is a seleno-organic compound with documented cytoprotective properties. Little work has been done, however, demonstrating ebselens cytoprotective properties in neural cell lines. In order to examine the effects of this compound and its mechanism of action, astrocytes were exposed to two known neurotoxicants, cisplatin and diethyldithiocarbamate (DDC). Cells were pretreated with 30 microM ebselen and subsequently treated with either 150 microM DDC for 1 h or 250 and 500 microM cisplatin for 24 h. Results indicate significant increases in viability in cells pretreated with ebselen and exposed to cisplatin. Ebselen pretreatment did not significantly increase viability in cells exposed to DDC. Light and scanning electron microscopy studies confirm the viability studies. Gross morphological damage was seen in cells treated with cisplatin, however, cells pretreated with ebselen and then exposed to cisplatin, appeared similar to controls. No differences were noted in cells pretreated with ebselen and then exposed to DDC or cells treated with DDC alone. In order to examine the mechanism of protection of this compound, glutathione status was examined. Results show that ebselen does not significantly increase reduced or oxidized glutathione (GSH, GSSG). All cell groups treated with cisplatin showed an increase in GSH levels. Ebselen showed protection in glutathione depleted cells at the 250 microM cisplatin dose. DDC treatment showed no significant increase in either reduced or oxidized glutathione. We conclude that ebselen significantly protects against cisplatin, but not DDC toxicity. We further conclude that this protection is not related to changes in glutathione status in the rat hippocampal cell line as has been reported in other cell types.