Exposure to dithiocarbamate fungicide Ziram results in hepatic and renal toxicity in Long Evan rats.

Ziram is a dimethyldithiocarbamate fungicide that is complexed to the metal zinc. The focus of this study is to examine the effects of dimethyldithiocarbamate exposure on metal homeostasis, glutathione levels, and the physiological parameters of the kidney and liver in Long-Evan rats. Our results indicate significant accumulation of copper or zinc, and changes in total GSH or GSH/GSSG ratio in the liver and kidneys of animals treated with Ziram only. Histopathological examination of liver and kidney sections indicate the presence of infiltrates in the liver of animals treated with Ziram only, whereas protein aggregates, sloughing of cells and increased KIM-1 positive cells, an indicator of tubule deterioration, are seen in the kidneys of animals treated with Ziram and sodium-dimethyldithiocarbamate, the salt form of the dimethyldithiocarbmate backbone. These findings suggest that the overall toxicological effect of Ziram is mediated by an intrinsic property rather than to dimethyldithiocarbamate backbone or metal moiety.

The ethylene bisdithiocarbamate fungicides mancozeb and nabam alter essential metal levels in liver and kidney and glutathione enzyme activity in liver of Sprague-Dawley rats.

Mancozeb is a fungicide of the ethylene bisdithiocarbamate (EBDC) class complexed to the metals manganese and zinc. Nabam is the sodium salt of the EBDC backbone. The purpose of this study was to determine if these EBDC compounds alter essential metal homeostasis and glutathione status in Sprague-Dawley rats. Our findings indicate EBDCs caused accumulation of copper in kidneys, but not liver. EBDC compounds also increased glutathione reductase activity in liver, but not kidneys, whereas only mancozeb increased glutathione peroxidase activity in the liver. Mancozeb and nabam increased total glutathione in liver, but only mancozeb increased total glutathione in the kidney. Neither mancozeb nor nabam altered glutathione ratio in either liver or kidney compared to control. Our data suggest that the EBDC backbone of mancozeb, and not the zinc or manganese moieties, is responsible for changes in glutathione status and alteration of essential metal homeostasis in rat liver and kidney.

The role of transcription factor Nrf2 in the toxicity of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) in C57BL/6 mouse astrocytes.

Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are members of perfluoroalkyl substances (PFAS). This study aimed to determine the protective role of Nrf2 against the toxicity of these agents. Nrf2-/- and wild-type astrocytes were exposed to PFOS (75-600 µM) and PFOA (400-1000 µM) for 24 h. Lactate dehydrogenase (LDH) release was significantly higher in nrf2-/- than in the wild-type astrocytes. Exposure to 600 µM PFOS and 800 µM PFOA showed significant increases in reactive oxygen species, lipid peroxidation, and apoptosis in nrf2-/- astrocytes as compared to wild-type astrocytes. The GSH/GSSG ratio was significantly decreased in nrf2-/- astrocytes when compared to wild-type astrocytes. Additionally, PFOS and PFOS caused dramatic ultrastructural alterations to the mitochondria. BHT pretreatment in wild-type cells decreased ROS production with exposure to both agents. Results of the present study conclude that PFOS and PFOA are cytotoxic to astrocytes and that nrf2 -/- cells are more sensitive to toxicity by these agents.

Disruption of mitochondrial complexes, cytotoxicity, and apoptosis results from Mancozeb exposure in transformed human colon cells.

Ethylene bisdithiocarbamate pesticides, including Mancozeb (MZ), are used as fungicides. Effects of MZ on apoptosis induction and mitochondrial activity of HT-29 colon cells were investigated. MZ exposed cells exhibited blebbing and cellular membrane disruption in scanning electron micrographs. Positive fluorescent staining with Annexin V at doses of 60-140 µM supports apoptosis as the mechanism of cell death. Activity of all electron transport chain complexes were evaluated. Mitochondrial Complex I activity was decreased in 100 µM treated cells. Mitochondrial Complex III activity was decreased in 60 and 100 µM MZ treated cells. Mitochondrial Complex II and Complex IV activities were decreased in cells treated with 60, 100, and 140 µM. Cells treated with 60 µM exhibited a decrease in Complex V enzyme activity. It is concluded that MZ exposure inhibits all mitochondrial complexes of HT-29 cells and that positive fluorescent microscopy and blebbing support previous studies of cell death via apoptosis.

Alterations in antioxidant/oxidant gene expression and proteins following treatment of transformed and normal colon cells with tellurium compounds.

The current study evaluated the potential of TeCl4 and DPDT to accumulate within cells and cause oxidative stress. HO-1, antioxidant gene expression and protein alterations were studied.

Ethylene bisdithiocarbamate pesticides Maneb and Mancozeb cause metal overload in human colon cells.

Previous studies in our laboratory have shown that ethylene bisdithiocarbamate (EBDC) fungicides Maneb and Mancozeb are equipotent gastrointestinal toxicants that produce cell loss and metal accumulation within HT-29 and Caco2 colon cells. Nabam, MnCl2, CuCl2 and ZnCl2 exposure produced no loss of viability up to 200 µM and increases in metal levels were noted but not to the same extent as Maneb and Mancozeb. EBDC exposure caused increases in copper levels (20-200 µM). Maneb and Mancozeb treatment also caused increases in manganese and zinc concentrations (20-200 µM). Nabam plus MnCl2 and Nabam and MnCl2 plus ZnCl2 caused decreases in viability and increases in metal levels comparable to Maneb and Mancozeb. Decreases in the ratio of reduced glutathione to glutathione disulfide were observed with Maneb and Mancozeb (20-200 µM). Maneb and Mancozeb treatment results in intracellular metal accumulation leading to the oxidative stress. The metal moiety and the organic portion of EBDCs contribute to toxicity.

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.

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.

Ethylene bisdithiocarbamate pesticides cause cytotoxicity in transformed and normal human colon cells.

The effects of the fungicides Maneb, Mancozeb, and Zineb were investigated in transformed colon cells, HT-29, Caco2 and non-transformed cells, CCD-18Co. Significant decreases in viability were observed with Maneb and Mancozeb in HT-29 and CCD-18Co (80-260µM), and Caco2 cells (40-180µM). No significant decreases in viability were observed in all cell types up to 800µM with Zineb. MnCl(2) and ZnCl(2) exposure produced no loss of viability in all cell types up to 400µM. Light microscopy confirmed viability analysis. Lipid peroxidation was observed with Maneb and Mancozeb in cell types tested (60-200µM). Caspase 3/7, 8, and 9 activities were observed with Maneb and Mancozeb in cell types tested (40-200µM). Maneb and Mancozeb treated HT-29 and Caco2 cells demonstrated increases in manganese and zinc concentrations (20-200µM). The lack of toxicity observed with Zineb, MnCl(2), and ZnCl(2) suggests that both the metal moiety and the organic portion of these fungicides together contribute to toxicity.

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.

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.

Aerosolized recombinant human lysozyme ameliorates Pseudomonas aeruginosa-induced pneumonia in hamsters.

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.

The role of surface receptor stimulation on the cytotoxicity of resveratrol to macrophages.

The present work has evaluated the role of lipopolysaccharide (LPS) stimulation and of TLR4 receptors on the cytotoxicity of resveratrol (RES), a polyphenolic stilbene component of red wine, in TLR4-bearing (i.e., RAW 264.7) and TLR4-deficient (i.e., 10ScNCr/23) macrophages. Based on the results of the MTT assay, DNA fragmentation analysis, and scanning electron microscopic examination, cell stimulation with LPS was found to maintain the viability of, attenuate DNA fragmentation in, and preserve normal morphology of TLR4-proficient macrophages exposed to RES. In contrast, LPS failed to spare TLR4-deficient macrophages from the deleterious effects of RES. Moreover, while LPS treatment conferred protection from RES toxicity in TLR4-bearing macrophages, this stilbene inhibited the production of nitric oxide by stimulated cells in a concentration-dependent fashion. It is therefore likely that the cytotoxic effects of RES towards non-stimulated macrophages and the propensity of RES to inhibit nitric oxide production by activated macrophages are both contributing, at least in part, to the anti-inflammatory activity of this natural product.

Evaluation of the antimicrobial activity of ebselen: role of the yeast plasma membrane H+-ATPase.

Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is a selenium-containing antioxidant demonstrating anti-inflammatory and cytoprotective properties in mammalian cells and cytotoxicity in lower organisms. The mechanism underlying the antimicrobial activity of ebselen remains unclear. It has recently been proposed that, in lower organisms like yeast, the plasma membrane H+-ATPase (Pma1p) could serve as a potential target for this synthetic organoselenium compound. Using yeast and bacteria, the present study found ebselen to inhibit microbial growth in a concentration- and time-dependent manner, and yeast and Gram-positive bacteria to be more sensitive to this action (IC50 approximately 2-5 microM) than Gram-negative bacteria (IC50 < 80 microM). Washout experiments and scanning electron microscopic analysis revealed ebselen to possess fungicidal activity. In addition, ebselen was found to inhibit medium acidification by PMA1-proficient haploid yeast in a concentration-dependent manner. Additional studies comparing PMA1 (+/-) and PMA1 (+/+) diploid yeast cells revealed the mutant to be more sensitive to treatment with ebselen than the wild type. Ebselen also inhibited the ATPase activity of Pma1p from S. cerevisiae in a concentration-dependent manner. The interaction of ebselen with the sulfhydryl-containing compounds L-cysteine and reduced glutathione resulted in the complete and partial prevention, respectively, of the inhibition of Pma1p ATPase activity by ebselen. Taken together, these results suggest that the fungicidal action of ebselen is due, at least in part, to interference with both the proton-translocating function and the ATPase activity of the plasma membrane H+-ATPase.

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 approximately 27 micromol L(-1), 24 h; approximately 9 micromol L(-1); 48 h)>macrophages (LC50 approximately 29 micromol L(-1), 24 h; 39 micromol L(-1), 48 h)>skin cells (LC50 approximately 91 micromol L(-1), 24 h; approximately 66 micromol L(-1), 48 h). Paradoxically, a high concentration of resveratrol (50 micromol L(-1)) inhibited the proliferation of all three cell types, and a low concentration (5 micromol 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 micromol 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>or=25 micromol 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.

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 30 microM Eb was used as a cotreatment with HN2 in all cell models tested at the following doses of HN2: A431 skin cells,10-40 microM; rat astrocytes, 20 and 40 microM; rat mesothelia, 10-40 microM; and human T cells 4-16 microM. 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.

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

4-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.

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.