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D J Reed

Publications and source records attributed to D J Reed.

At least 37 records · Page 2Linked to original sources
PubMed1995

Influence of metabolic inhibitors on mitochondrial permeability transition and glutathione status.

Treatment of isolated mitochondria with Ca2+ and inorganic phosphate (Pi) induces an inner membrane permeability that appears to be mediated through a cyclosporin A (CsA)-inhibitable Ca(2+)-dependent pore. Isolated mitochondria during inner membrane permeability undergo rapid efflux of matrix solutes such as glutathione as GSH and Ca2+, loss of coupled functions, and large amplitude swelling. Permeability transition without large amplitude swelling, a parameter often used to assess inner membrane permeability, has been observed. The addition of either oligomycin, antimycin, or sulfide to incubation buffer containing Ca2+ and Pi abolished large amplitude swelling of mitochondria. The GSH status during a Ca(2+)- and Pi-dependent mechanism of mitochondrial GSH release in isolated mitochondria was influenced significantly by metabolic inhibitors of the respiratory chain but did not prevent inner membrane permeability as demonstrated by the release of mitochondrial GSH and Ca2+. The release of GSH was inhibited by the addition of CsA, a potent inhibitor of permeability transition. Under these conditions we did not find GSSG; however, rapid oxidation of pyridine nucleotides and depletion of ATP and ADP with conversion to AMP occurred. The addition of CsA, prevented the oxidation of pyridine nucleotides and depletion of ATP and ADP. Since NADH and NADPH were extensively oxidized, protection against oxidative stress is reflected in maintenance of GSH and not observable lipid peroxidation. Evidence from transmission electron microscopy analysis, combined with the GSH release data, indicate that permeability transition can be observed in the absence of large amplitude swelling.

Adenine Nucleotides↗
PubMed1995

Synthesis of glutathione in the preimplantation mouse embryo.

Depletion and repletion of glutathione in two-cell to blastocyst stage mouse embryos was examined. Reduced (GSH) and oxidized glutathione (GSSG) were measured by fluorimetric HPLC after derivatization of extracted embryo samples with dansyl chloride. Addition of buthionine sulfoximine (BSO) to culture medium for 16 h decreased GSH levels in both two-cell and blastocyst stage embryos; however, GSH decreased more drastically in blastocysts. Addition of diethyl maleate (DEM) to culture medium depleted GSH in both two-cell and blastocyst stage embryos. After removal of DEM, GSH levels returned to normal in blastocysts, and addition of BSO or removal of cystine from medium blocked GSH repletion. Two-cell stage embryos were unable to recover GSH levels after depletion and exhibited decreased in vitro development. Addition of methionine to culture medium was unable to substitute for cystine as a source of cysteine in glutathione synthesis, indicating that the embryos do not use the cystathionine pathway. Embryos collected early on Day 3 of development were unable to recover GSH levels within 5 h, whereas embryos collected late on Day 3 recovered GSH within 5 h. Addition of cycloheximide to culture medium at noon on Day 3 of development decreased the ability of blastocysts to recover their GSH levels late on Day 3. These data indicate that GSH turnover and synthesis increases between the two-cell and blastocyst stages. The increase in the ability of embryos to synthesize GSH on Day 3 is dependent on protein synthesis. Cleavage stage embryos have limited capacity to synthesize GSH and appear susceptible to adverse effects of toxicants or conditions that deplete glutathione.

Animals↗
PubMed1995

Impact of nutrients on cellular lipid peroxidation and antioxidant defense system.

A symposium entitled Impact of Nutrients on Cellular Lipid Peroxidation and Antioxidant Defense System was held at the 33rd Annual Meeting of the Society of Toxicology (SOT) at the Loews Anatole Hotel in Dallas, Texas. The symposium was sponsored by the Food Safety Specialty Section and focused on the role of particular nutrients in cellular lipid peroxidation and antioxidant defense system. Emphasis was placed on defining underlying mechanisms for damage and protection, as well as potential ramification in human health issues. The following are extractions of some of the highlights from each presentation.

Antioxidants↗
PubMed1995

Adenovirus-based p53 gene therapy in ovarian cancer.

Mutations of the p53 tumor suppressor gene are the most common molecular genetic abnormality to be described in ovarian cancer. To determine the feasibility of mutant p53 as a molecular target for gene therapy in ovarian cancer, we constructed an adenovirus vector containing the wild-type p53 gene. The ability of this adenovirus construct (Ad-CMV-p53) to express p53 protein was examined by Western blot analysis in the H358 lung cancer cell line, which has a homozygous deletion of the p53 gene. The ability of the adenovirus vector system to infect ovarian cancer cells was tested using an adenovirus containing the beta-galactosidase reporter gene under the control of the CMV promoter (Ad-CMV-beta gal). The ovarian cancer cell line 2774, which contains an Arg273His p53 mutation, was infected with Ad-CMV-beta gal, and the infected cells were assayed for beta-galactosidase activity after 24 hr. To test the ability of wild-type p53 to inhibit cell growth, the 2774 cell line was infected with Ad-CMV-p53 or Ad-CMV-beta gal, and the effect of these agents on the growth of 2774 cells was determined using an in vitro growth inhibition assay. Western blot analysis of lysates from H358 cells infected with Ad-CMV-p53 showed expression of wild-type p53 protein. When 2774 cells were infected with Ad-CMV-beta gal at a multiplicity of infection (m.o.i.) of 10 PFU/cell, > 90% of cells showed beta-galactosidase activity, demonstrating that these cells are capable of efficient infection by the adenovirus vector. Growth of 2774 cells infected with Ad-CMV-p53 was inhibited by > 90% compared to noninfected cells. The ability of the adenovirus vector to mediate high-level expression of infected genes and the inhibitory effect of Ad-CMV-p53 on the 2774 cell line suggests that the Ad-CMV-p53 could be further developed into a therapeutic agent for ovarian cancer.

Adenoviridae↗
PubMed1995

Cystathionine.

Explore the source record for details and available documents.

Alkynes↗
PubMed1994

Release of mitochondrial glutathione and calcium by a cyclosporin A-sensitive mechanism occurs without large amplitude swelling.

Treatment of isolated mitochondria with calcium and inorganic phosphate induces inner membrane permeability that is thought to be mediated through a non-selective, calcium-dependent pore. The inner membrane permeability results in the rapid efflux of small matrix solutes such as glutathione and calcium, loss of coupled functions, and large amplitude swelling. We have identified conditions of permeability transition without large amplitude swelling, a parameter often used to assess inner membrane permeability. The addition of either oligomycin, antimycin, or sulfide to incubation buffer containing calcium and inorganic phosphate abolished large-amplitude swelling of mitochondria but did not prevent inner membrane permeability as demonstrated by the release of mitochondrial glutathione and calcium. The release of both glutathione and calcium was inhibited by the addition of cyclosporin A, a potent inhibitor of permeability transition. Transmission electron microscopy analysis, combined with the glutathione and calcium release data, indicate that permeability transition can be observed in the absence of large-amplitude swelling. Permeability transition occurring both with and without large-amplitude swelling was accompanied by a collapse of the membrane potential. We conclude that cyclosporin A-sensitive permeability transition can occur without obvious morphological changes such as large-amplitude swelling. Monitoring the cyclosporin A-sensitive release of concentrated endogenous matrix solutes, such as GSH, may be a sensitive and useful indicator of permeability transition.

Animals↗
PubMed1994

Role of differential mRNA stability in the regulated expression of IgM and IgD.

The expression of IgM and IgD heavy chain mRNA in resting vs activated B cells offers a unique tool for the assessment of the effect of translation on mRNA stability because mu and delta mRNA have identical VDJ sequences but differ substantially in the rest of the molecule. We have shown that despite the 5' UTR identity that allows equal access to the translation machinery, mu mRNA has a significantly higher turnover rate than delta mRNA. However, the short t1/2 of mu mRNA increases significantly after B cell activation. Furthermore, the induction of microS mRNA after B cell activation provides yet another related molecule for comparison. Thus, despite the fact that microS and microM mRNA differ at their 3' ends, they have identical turnover rates in activated B cells. In addition, because the turnover rates of delta mRNA and beta 2 and GAPDH mRNA remain unchanged, these experiments suggest that B cell activation results in the induction of regulatory factor(s) that target specific sequences within mRNA-mu to confer greater stability. They also argue against a more passive regulation of mRNA stability that is a consequence of alterations in the secretory machinery.

Animals↗
PubMed1994

Oxidation of pyridine nucleotides and depletion of ATP and ADP during calcium- and inorganic phosphate-induced mitochondrial permeability transition.

We have examined the pyridine and adenine nucleotide status during calcium- and inorganic phosphate-induced permeability transition. Calcium- and inorganic phosphate-induced permeability transition is accompanied by the rapid oxidation of pyridine nucleotides and depletion of ATP and ADP with conversion to AMP. The addition of cyclosporin A, a potent inhibitor of the permeability transition prevented the oxidation of pyridine nucleotides and depletion of ATP and ADP.

Adenine Nucleotides↗
PubMed1994

Effects of dietary oils and methyl ethyl ketone peroxide on in vivo lipid peroxidation and antioxidants in rat heart and liver.

Weanling male Sprague-Dawley rats were fed diets for four weeks which differed in their content of n-6 (corn oil; CO) and n-3 fatty acids (fish oil; FO), but were similar in their content of saturated and monounsaturated fatty acids and vitamin E. At the end of the four-week feeding period, each dietary group was subdivided into two groups. One group received a single placebo injection of alpha-tocopherol-stripped corn oil (TSCO); the other group received a single injection of the free radical generator; methyl ethyl ketone peroxide (MEKP), in TSCO. Twenty-four hours after injection, the effect of dietary oil and MEKP treatment on endogenous lipid peroxide (LPO) production (measured as methylene blue formed by the "Determiner LPO" assay), glutathione (GSH) and vitamin E content, and fatty acid composition of phosphatidylcholine and phosphatidylethanolamine in heart and liver from unfasted animals were measured. FO-fed rats had significantly heavier hearts and livers, increased levels of n-3 fatty acids in membrane phospholipids, and higher liver LPO levels than CO-fed rats. MEKP treatment resulted in significantly lower body weights and liver GSH levels. The data indicate that dietary n-3 fatty acids increase lipid peroxidation in liver somewhat more than in heart. The study also demonstrates that the effect of induced oxidative stress due to a single dose of MEKP on lipid peroxide formation and antioxidant status in tissues from unfasted animals was independent of the dietary oils.

Animals↗
PubMed1994

Status of glutathione during oxidant-induced oxidative stress in the preimplantation mouse embryo.

Experiments were conducted to elucidate the status of glutathione present in the oxidized (GSSG), reduced (GSH), and protein-mixed disulfide (GSSprotein) forms in preimplantation mouse embryos during development and after treatment with tertiary-butyl hydroperoxide (tBH) to cause oxidative stress. Glutathione was measured at picomolar levels by fluorimetric HPLC after derivatization of extracted embryonic samples with dansyl chloride. GSH content decreased approximately 10-fold from that in the unfertilized oocyte to 0.12 pmol/blastocyst, representing an estimated change in concentration from 7 to 0.7 mM. GSH levels were lower in embryos cultured in vitro than in embryos that developed in vivo. Addition of GSH to the culture medium improved in vitro development of mouse embryos, but surprisingly the addition of glutathione monoethyl ester did not. Addition of low levels of the oxidant tBH (13.2 microM) to culture medium decreased the percentage of two-cell and blastocyst stage embryos that exhibited further development. After 15-min exposure to 13.2 microM tBH, GSH levels were markedly decreased in the two-cell stage embryo (75%), but only slightly decreased (25%) in the blastocyst. The loss of GSH was accounted for by increases in GSSG and GSSprotein, indicating that the embryo was undergoing oxidative stress. These data indicate that preimplantation embryos are very sensitive to conditions that can cause oxidative stress and show also that their glutathione status changes dramatically during development.

Animals↗
PubMed1992

Glutathione disulfide reduction in tumor mitochondria after t-butyl hydroperoxide treatment.

Treatment of isolated mitochondria from rat hepatoma tumor cells (AS-30D) with the oxidant, t-butyl hydroperoxide (tBuOOH, 1 or 5 mumol/ml) resulted in the oxidation of glutathione (GSH to GSSG) and the formation of protein-glutathione mixed disulfides (ProSSG). The GSSG was retained inside of the hepatoma mitochondria. In the presence of ADP+succinate (5 or 10 mM), or ketoglutarate (10 mM) or malate (5 mM), the GSSG was reduced to GSH, but the amount of ProSSG stayed constant. With saline or ADP+glutamate (10 mM)/malate (0.1 mm) no reduction of GSSG to GSH occurred. The presence of antimycin (5 micrograms/ml) with ADP+succinate inhibited reduction. At a concentration of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, 0.5 mM) which inhibited a major portion of the glutathione reductase activity, the reduction of GSSG to replenish GSH was also inhibited. NADPH may play a critical role as well, for the addition of 2.4 mM NADPH to permeabilized hepatoma mitochondria fostered the reduction of GSSG after tBuOOH treatment. Therefore, hepatoma mitochondria possess a glutathione reductase-dependent system to reduce GSSG to GSH. The reaction only occurs with actively respiring mitochondria.

Adenosine Diphosphate↗
PubMed1992

Cyclosporin A protects hepatocytes subjected to high Ca2+ and oxidative stress.

Hepatocytes incubated with 0.8 mM t-butylhydroperoxide are protected by cyclosporin A when the medium Ca2+ concentration is 10 mM, but not when it is 2.5 mM. The highest Ca2+ level is associated with an inhibition of t-butylhydroperoxide-dependent malondialdehyde accumulation and with mitochondrial Ca2+ loading within the cells. These findings are new evidence that t-butylhydroperoxide can kill cells by peroxidation-dependent and -independent mechanisms, and suggest that the mitochondrial permeability transition and the resultant de-energization are components of the peroxidation-independent mechanism. Cyclosporin A may have considerable utility for the protection of cells subjected to oxidative stress.

Animals↗
PubMed1991

Involvement of glutathione in 1-naphthylisothiocyanate (ANIT) metabolism and toxicity to isolated hepatocytes.

1-Naphthylisothiocyanate (ANIT) is a model compound which causes cholestasis in laboratory animals. Various biochemical and morphological changes including biliary epithelial and parenchymal cell necrosis occur in the liver of animals treated with ANIT. Although the mechanism(s) for these effects is not understood, a role for glutathione (GSH) in toxicity has been implicated. The possible role of GSH in hepatocellular toxicity caused by ANIT was investigated in this study. Treatment of freshly isolated rat hepatocytes with ANIT caused a concentration- and time-dependent depletion of cellular GSH that preceded lactate dehydrogenase (LDH) leakage. Analysis of the incubation medium indicated that the majority of the cellular GSH which was lost was present extracellularly as GSH or as a GSH-releasing compound. Mixing ANIT with GSH at pH 7.5 yielded a compound that was characterized by HPLC and fast atom bombardment-mass spectrometry (FAB-MS) S-(N-naphthyl-thiocarbamoyl)-L-glutathione (GS-ANIT). When dissolved in aqueous solutions at neutral pH, 95% of GS-ANIT dissociated to yield free ANIT and GSH. Under conditions designed to maximize formation and stability of GS-ANIT, GS-ANIT was found in the extracellular medium of hepatocytes treated with ANIT. Treatment of hepatocytes with the GS-ANIT caused GSH depletion and LDH leakage similar to that observed with equimolar amounts of ANIT. These data suggest that ANIT depletes hepatocytes of GSH through a reversible conjugation process. Such a process may play a role in the toxicity of ANIT.

1-Naphthylisothiocyanate↗
PubMed1991

Modification of hepatic vitamin E stores in vivo. II. Alterations in plasma and liver vitamin E content by 1,2-dibromoethane.

Previous studies with methyl ethyl ketone peroxide (MEKP), a radical generator, showed depletion of plasma vitamin E and liver glutathione (GSH) levels prior to a decrease of liver vitamin E levels. Since hepatic pools of this vitamin may serve to maintain circulating levels of vitamin E under conditions of oxidative challenge, we have evaluated the similarity of response after treatment with 1,2-dibromoethane (DBE), a compound that is not known to generate oxyradicals or to induce lipid peroxidation in vivo. Treatment of normal rats with DBE caused a depletion in hepatic vitamin E levels 1 day after treatment; however, in contrast to our prior findings with MEKP this depletion after DBE treatment was observed in tandem with elevations in the plasma content of vitamin E. Liver vitamin E depletion was neither dependent upon a sustained liver GSH depletion nor upon hepatocellular death. Mobilization and export of hepatic vitamin E did not result in an immediate whole body redistribution of this vitamin in that pulmonary and renal levels of vitamin E remained normal under conditions of liver vitamin E depletion. Moreover, the stimulus that resulted in exportation of liver vitamin E was maintained by daily treatments with DBE. DBE caused a substantial elevation above control values in liver GSH content and these elevations were also maintained by daily DBE treatments. In experiments to assess the influence of prandial replacement of vitamin E on the extent of depletion in response to DBE treatment, rats were fed a vitamin E-deficient diet for 2 days prior to treatment. This short pulse of a vitamin E-deficient diet delayed (to 2 days) both the elevation in liver GSH content and the depletion of liver vitamin E and hastened (to 1 day) the elevation in plasma vitamin E concentration. These observations suggest the presence of at least two pools of liver vitamin E and that one of these pools, which comprises at least 30% of the total hepatic vitamin E content, is able to be mobilized and exported in response to chemical challenge. The stimulus that resulted in liver vitamin E exportation in response to DBE treatment seems to result from wholly intrahepatic processes and may not be a direct response to lipid peroxidation. Moreover, the similarity between the time-course and the extent of hepatic vitamin E depletion observed after treatment with either MEKP or DBE suggests a similarity in physiochemical processes that function to mobilize hepatic vitamin E stores.

Alanine Transaminase↗
PubMed1991

Modification of hepatic vitamin E stores in vivo. III. Vitamin E depletion by 1,2-dibromoethane may be related to initial conjugation with glutathione.

In the companion paper we demonstrated that hepatic vitamin E in rats becomes depleted and extrahepatic pools of vitamin E are altered by treatment with 1,2-dibromoethane (DBE). Vitamin E depletion may be dependent upon initial steps of DBE metabolism that are either oxidative (cytochrome P450 dependent) or conjugative (glutathione transferase dependent). That the liver content of glutathione (GSH) and vitamin E, the plasma concentration of vitamin E, and the serum activities of AST and ALT may be influenced by cytosolic metabolism of DBE was assessed by comparison of findings from rats treated with either 1,2-dichloroethane (DCE) or 1-bromo-2-chloroethane (BCE). The extent of oxidative metabolism was diminished by the use of tetradeutero-DBE (d4-DBE), and the availability of GSH for conjugative metabolism was diminished by pretreatment of rats with L-buthionine-S,R-sulfoximine (BSO) prior to treatment with DBE. Our results indicate that neither DCE nor BCE provokes a liver vitamin E depletion in rats, that d4-DBE treatment hastens but does not enhance the observed hepatic vitamin E depletion by comparison to animals treated with an equimolar dose of DBE, and that BSO pretreatment prevented the hepatic vitamin E depletion observed from animals treated with DBE alone. These results indicate that hepatic vitamin E depletion is the unique sequelae to conjugation of GSH with DBE, and we suggest the reactive episulfonium ion intermediate or a macromolecular adduct of this ion derived from DBE may play a role in liver vitamin E depletion associated with exposure to DBE.

Alanine Transaminase↗
PubMed1991

Calcium chelation induced glutathione efflux from tumor cells and prevention by ruthenium red or neomycin.

Cultured human lung carcinoma cells (A549) were incubated in a calcium-free medium containing calcium chelators (EGTA, 1-10 mM or BAPTA, 5 mM) for 1 hour at 37 degrees C. With limited toxicity, the presence of calcium chelators resulted in a decrease of cellular GSH and detachment of the cells from the tissue culture flask. The permeable EGTA tetraacetoxymethyl ester (0.5mM-5 mM) caused a decrease in the cellular GSH content without cell detachment. GSH was not oxidized to GSSG nor formed mixed disulfides with protein thiols. AT-125, a gamma-glutamyl transpeptidase inhibitor, prevented detachment, but not the efflux of cellular GSH. Pretreatment with two impermeable compounds (ruthenium red, 100 microM and neomycin, 0.5-10 mM) protected the cells from detachment and prevented the decrease in intracellular GSH. The presence of calcium in the medium during the EGTA and BAPTA treatments also protected the cells. Calcium associated with the cytoplasmic membrane phospholipids or proteins appears important to limit membrane permeability for GSH efflux and to maintain cell attachment.

Calcium↗
PubMed1991

Modification of hepatic vitamin E stores in vivo. I. Alterations in plasma and liver vitamin E content by methyl ethyl ketone peroxide.

Since experiments with freshly isolated rat hepatocytes have shown that cellular vitamin E is consumed in response to insult by compounds that induce an oxidative stress only after cellular glutathione (GSH) concentrations have been substantially depleted, experiments were performed to determine whether this sequence of events occurred in response to oxidative insult in vivo. The role that plasma vitamin E plays in the response to chemically induced oxidative injury in vivo was also assessed. Treatments with 40 mg/kg of methyl ethyl ketone peroxide (MEKP) quickly induced lipid peroxidation in vivo and from one to 4 h after treatment caused a depression in the plasma content of vitamin E and the liver content of GSH, as well as signs of toxicity (elevations in serum activities of alanine and aspartate aminotransferases). At these time points however, the liver content of vitamin E was either indistinguishable from or slightly elevated from controls. By 12 to 24 h after treatment the liver content of vitamin E was reduced by 20-25% whereas values for all other indicators had returned toward control levels. Pretreatment of rats with L-buthionine-S,R-sulfoximine, an inhibitor of GSH by 4 or 24 h after treatment, did not alter the time course or extent of hepatic vitamin E depletion that was observed after treatment with MEKP. Other compounds that induce oxidative stress and lipid peroxidation to the liver, carbon tetrachloride and menadione, did not provoke an alteration in hepatic vitamin E levels as compared to controls 1 day after treatment. These findings indicate that depletion of hepatic vitamin E may not occur as an immediate consequence of oxidative insult to the liver and that the depletion of hepatic vitamin E levels may not be related to the extent of prior GSH depletion. Moreover, these findings suggest that alterations in the plasma concentration of vitamin E may not reflect concurrent alterations in hepatic vitamin E levels. A mechanism whereby liver vitamin E stores are mobilized for the maintenance of plasma vitamin E levels is proposed.

Alanine Transaminase↗