Louis D. Trombetta

The protective effects of glutathione against methylmercury cytotoxicity

Mouse neuroblastoma cells exposed to 2.5 and 5.0 microM methylmercury for 24 h appeared rounded with the loss of processes. Immunohistochemical staining directed against beta-tubulin revealed severe alterations in microtubular architecture. Non-membrane-bound condensation product was visualized ultrastructurally in the treated cells and appeared similar to what was seen histochemically. Reduced and oxidized glutathione levels suggest that methylmercury may manifest its deleterious effects via oxidation of tubulin sulfhydryls, and by alterations due to peroxidative injury. Cells exposed to methylmercury showed a decrease in glutathione peroxidase activity. Simultaneous administration of 10 mM glutathione with 2.5 and 5.0 microM methylmercury dramatically prevented 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.

Protective effects of glutathione on diethyldithiocarbamate (DDC) cytotoxicity: A possible mechanism

Rat cerebral astrocytes grown in culture were exposed to 35 micrograms diethyldithiocarbamate (DDC)/ml of medium for 1 hr and treated with 0 or 10 mM reduced glutathione (GSH) 1 hr post-DDC. DDC treatment resulted in a 90% reduction in cell adherence within 24 hr and complete inhibition of growth. The most pronounced ultrastructural lesion in DDC-treated cells was on mitochondria. Numerous lipofuscin-like deposits were seen in these cells. In addition, DDC treatment resulted in a greater than 400% increase in cellular copper. The activity of the selenoenzyme glutathione peroxidase was reduced by about 40% with no concomitant effect on cytosolic superoxide dismutase activity. The data suggest that DDC cytoxicity is peroxidative in nature, presumably due to the massive influx of copper into the astrocyte. GSH treatment 1 hr after exposure of the cells to DDC completely prevented the DDC-induced reduction in cell adherence and growth inhibition. Ultrastructurally, cells post-treated with GSH prevented much of the damage caused by DDC. This protection was associated with marked reduction in cellular copper and a return to control glutathione peroxidase activity.

The effects of disulfiram on the hippocampus and cerebellum of the rat brain: a study on oxidative stress.

The purpose of this investigation was to study the relationship between disulfiram (DS) toxicity, lipid peroxidation, and copper in order to further elucidate the mechanisms of DS toxicity. Male Sprague-Dawley rats were dosed with either 375, 750 or 1500 mg/kg disulfiram via oral intubation for 1, 3 and 6 weeks. In the hippocampus and cerebellum, the increased accumulation of copper, increased production of malondialdehyde (MDA), increased activity of glutathione peroxidase along with alterations in glutathione and glutathione disulfide concentrations was indicative of oxidative stress. The production of MDA was directly related to the level of copper in both areas of the brain indicating that excess copper may be the primary cause of DS neurotoxicity.

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.

Biodegradation Behavior of Gellan Gum in Simulated Colonic Media

The objective of this investigation was to test the biodegradability of gellan gum in the presence of galactomannanase in order to explore its suitability for the development of colon‐specific controlled delivery systems. Gellan beads containing azathioprine (AZA) were prepared by ionotropic gelation in the presence of Ca2 + ions and were coated with an enteric polymer, Eudragit® S‐100. The effects of the simulated colonic fluid (SCF, pH 7.4 phosphate buffer) containing 15 mg/mL of galactomannanase on the in vitro release profiles of uncoated and enteric‐coated beads were investigated, and the morphological changes in the structure of uncoated beads were assessed by scanning electron microscopy (SEM). In addition, 1% solution of deacetylated gellan gum was prepared and several aliquots of the resulting solution were evaluated rheologically to determine the concentration‐ and time‐dependent effects of galactomannanase. Based on the percent drug released at 2 h, approximately 10% greater amount of drug was released in the SCF containing galactomannanase when compared with the enzyme‐free dissolution medium. Results of rheological studies demonstrated that effects of galactomannanase on the viscosity of gellan gum solution are concentration‐dependent rather than time‐dependent. A significant decrease in the viscosity was noted in the presence of galactomannanase at a concentration of 15 mg/mL, indicating that the polysaccharide degraded in an enzymatic reaction. SEM micrographs showed a distinct disruption of the polymeric network in the SCF. Overall, the results suggest that gellan gum undergoes significant degradation in the presence of galactomannanase which in turn facilitates the drug release from beads in the SCF in a controlled manner, thus approving the suitability of gellan gum as a carrier for controlled colonic delivery.

The relationship between stress protein induction and the oxidative defense system in the rat hippocampus following kainic acid administration

The time and dose-dependent effects of kainic acid (KA) induced excitotoxicity on the oxidative defense system and the relationship to the induction of stress proteins were investigated in the rat hippocampus. Male Long-Evans rats were injected subcutaneously with 5.0, 7.5, or 10 mg KA/kg. Rats were sacrificed and the hippocampus removed and processed for biochemical and electrophoretic analysis. The activity of glutathione peroxidase (GPx) increased significantly at the 5 mg KA/kg dose, while malondialdehyde (MDA) levels significantly increased at 7.5 mg KA/kg when measured at 24 h. A dose of 10 mg KA/kg depleted significantly hippocampal glutathione (GSH) levels at 8, 16 and 24 h post-treatment while GPx activity was increased significantly at 2, 4, 8 and 16 hr post-treatment. The 10 mg KA/kg increased significantly hippocampal MDA levels at 2 h post-treatment and decreased significantly thereafter. The induction of stress proteins increased in a dose and time dependent manner. The expression of Hp72 and Hsp32 increased significantly at 16 h with a maximum induction observed at 24 h post-treatment. The data suggests that KA toxicity is mediated through the formation of reactive oxygen species resulting in alterations in the oxidative defense system. The expression of stress proteins following KA administration may reflect a concomitant but alternate response to excitotoxic events.

The protective role of chelators and antioxidants on mancozeb-induced toxicity in rat hippocampal astrocytes:

Mancozeb, manganese ethylene-bis-dithiocarbamate with zinc salts, is one of the most commonly used fungicides in the United States. Epidemiological and experimental data showed that mancozeb causes detrimental effects on various organ systems including the reproductive, endocrine, immune, and central nervous system. Increasing evidence has shown a strong association between pesticides and neurodegenerative diseases. In this study, we examined the neurotoxic potential of mancozeb in rat hippocampal astrocytes. The cytotoxicity of mancozeb was found to be dose dependent and recovery studies showed that cells exposed to mancozeb for 1 h did not recover from mancozeb-induced insult. Atomic absorption data showed a significant accumulation of manganese in astrocytes after 1 h of treatment. This study further investigated whether various chelators and antioxidants could prevent mancozeb-induced cytotoxicity. Our data reported that butylated hydroxytoluene (BHT) was the most effective agent in protecting against mancozeb insult. BHT also increased total cellular antioxidants of astrocytes after 1-h mancozeb exposure. In summary, this study reported for the first time that the manganese portion of mancozeb might be, at least in part, responsible for the toxicity. Mancozeb-induced cytotoxicity in astroyctes can be protected by BHT and that this antioxidant increased the total cellular antioxidant capacity.

Effect of disulfiram (DS) on mitochondria from rat hippocampus: Metabolic compartmentation of DS neurotoxicity

This experiment was designed to study the acute effects of disulfiram on mitochondrial enzymes in nonsynaptic and synaptic mitochondria from rat hippocampus. Cytochromec oxidase, monoamine oxidase-B, glycerolphosphate acyltransferase and betahydroxybutyrate dehydrogenase were studied. Differences in enzyme activity were seen in controls. Cytochromec oxidase activity was higher in synaptic mitochondria whereas glycerolphosphate acyltransferase activity was higher in nonsynaptic mitochondria. Mitochondria from disulfiram treated rats, particularly synaptic mitochondria, exhibited lower specific activities of cytochromec oxidase and monoamine oxidase-B. These alterations were not limited to either the inner or outer mitochondrial membrane. Transmission electron microscopy revealed that mitochondria from disulfiram treated rats were severely altered in isolated preparations as well as in those from whole tissue. This study shows that disulfiram exerts a differential effect on mitochondrial subpopulations.

Acute toxicity and bioaccumulation of tributyltin in tissues of Urolophus jamaicensis (yellow stingray).

Tributyltin oxide (TBTO) is the main constituent of tin-based antifouling marine paint used on the hulls of ships to prevent the growth of fouling organisms. TBTO was shown to be hazardous to nontarget organisms. The stingray, Urolophus jamaicensis, may represent the ideal study organism for the adverse effects of TBTO to elasmobranches. This study investigated the toxicity and accumulation of tin in the gill tissue of the stingray U. jamaicensis after acute exposure to TBTO. This work demonstrates the alterations in the morphological architecture of the gill using electron and light microscopy, the induction of stress proteins, and peroxidative damage in response to tributyltin (TBT) exposure. A captured population of U. jamaicencis was housed in isolated, static tank systems. After a minimum 30-d acclimation period, the animals were exposed to one of 5 experimental doses of TBTO (4 μg/L, 2 μg/L, 1 μg/L, 0.5 μg/L, or 0.05 μg/L). A sixth group served as a control population. At 3h following treatment, animals were sacrificed and gill tissue was extracted, processed, and stored for analysis. Results indicate that U. jamaicensis is hypersensitive to TBT exposure. The elasmobranch gill showed a distorted, swollen epithelium with exfoliation following acute exposure to as little as 0.05 μg/L TBTO for 3 h. Graphite furnace atomic absorption spectroscopy (GFAAS) results indicate that tissues of treated animals contained a significantly increased tin concentration as compared to controls. Western blot analysis demonstrated the induction of the stress proteins Hsp 70 and HO1. 4‐Hydroxynonenol (4HNE) adduct formation determined by Western blot analysis provides further evidence that observed membrane degradation is a result of lipid peroxidation.