diamide and Necrosis

Apoptosis was studied under conditions that mimic the steady state of H(2)O(2) in vivo. This is at variance with previous studies involving a bolus addition of H(2)O(2), a procedure that disrupts the cellular homeostasis. The results allowed us to define three phases for H(2)O(2)-induced apoptosis in Jurkat T-cells with reference to cytosolic steady state concentrations of H(2)O(2) [(H(2)O(2))(ss)]: (H(2)O(2))(ss) values below 0.7 microM elicited no effects; (H(2)O(2))(ss) approximately 0.7-3 microM induced apoptosis; and (H(2)O(2))(ss) > 3 microM yielded no additional apoptosis and a gradual shift towards necrosis as the mode of cell death were observed. H(2)O(2)-induced apoptosis was not affected by either BCNU, an inhibitor of glutathione reductase, or diamide, a compound that reacts both with low-molecular weight and protein thiols, or selenols. Glutathione depletion, accomplished by incubating cells either with buthionine sulfoximine or in cystine-free medium, rendered cells more sensitive to H(2)O(2)-induced apoptosis, but did not change the threshold and saturating concentrations of H(2)O(2) that induced apoptosis. Two unrelated metal chelators, desferrioxamine and dipyridyl, strongly protected against H(2)O(2)-induced apoptosis. It may be concluded that, under conditions of H(2)O(2) delivery that mimic in vivo situations, the oxidative event that triggers the induction of apoptosis by H(2)O(2) is a Fenton-type reaction and is independent of the thiol or selenium states of the cell.

Topics: 2,2'-Dipyridyl; Apoptosis; Carmustine; Chelating Agents; Deferoxamine; Diamide; Enzyme Inhibitors; Free Radicals; Glutathione; Glutathione Reductase; Humans; Hydrogen Peroxide; Jurkat Cells; Necrosis; Oxidation-Reduction; Selenium; Sulfhydryl Compounds

Oxidative stress induces a variety of cellular responses, including apoptosis, and caspase family proteases are known to be involved in apoptosis. Caspase-3(-like) protease activity was examined in Jurkat T cells to investigate the mechanism of apoptosis induced by a thioloxidant, diamide. Caspase-3 was activated when cells were cultured with 200 microM diamide that induced apoptosis, whereas no caspase-3 activation was detected with 500 microM diamide that induced necrosis. When apoptosis was induced in cells with exposure to 200 microM diamide, the intracellular thioredoxin (TRX) levels were maintained and the intracellular generation of reactive oxygen intermediates was marginal. The cytosolic fractions of cytochrome c were increased earlier than the activation of caspase-3. In contrast, when cells were exposed to 500 microM diamide, intracellular reactive oxygen intermediate generation was increased and processing of caspase-3 was not detected despite cytochrome c release, resulting in necrosis. Caspase-3 activity in cell lysate precultured with anti-Fas Ab was suppressed dose dependently by diamide and restored by thiol-reducing agents, DTT or TRX. When cells were precultured with 5 mM of buthionine sulfoximine, an inhibitor of glutathione synthesis, intracellular TRX levels were maintained, and as low as 20 microM diamide could induce apoptosis associated with the increase of cytosolic cytochrome c and the activation of caspase-3. These results indicate that the activation of caspase-3 in diamide-induced apoptosis is mediated, at least partly, by cytochrome c release from mitochondria, and the cellular reducing environment maintained by TRX, as well as glutathione, is required for caspase-3 activity to induce apoptosis.

Topics: Antibodies, Monoclonal; Apoptosis; Caspase 3; Caspases; Cytochrome c Group; Diamide; Dithiothreitol; Dose-Response Relationship, Drug; fas Receptor; Humans; Jurkat Cells; Mitochondria; Necrosis; Neoplasm Proteins; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species; Thioredoxins

In sepsis red blood cells (RBCs) have been shown to be less deformable (i.e., more rigid) and have been implicated in decreasing nutrient blood supply and possibly leading to organ dysfunction. However, no studies have demonstrated an association between organ dysfunction and rigid RBCs. This study examined cardiovascular physiologic and histologic changes in two different models to determine whether a relationship may exist between RBC deformability and organ function.. In the following two experiments, cardiac index (CI) was continuously measured, whereas both deformability index and histology were examined at the end of the experimental periods. The first experiment studied nonanesthetized, hydrated rats after a cecal ligation and puncture (CLP), a slow-developing means of inducing RBC rigidity. In a second experiment animals were anesthetized and received a 20% total blood volume transfusion of either diamide-treated (rigid) RBCs or normal RBCs.. CLP-treated animals' CI gradually decreased during 18 hours (232 +/- 60 ml/min/kg to 123 +/- 90 ml/min/kg; p = 0.05), with an increase in systemic vascular resistance (1459 +/- 517 dyne.sec/cm5.m2 to 2337 +/- 1213 dyne.sec/cm5.m2; p = 0.02). Diamide-treated animals had a rapid decrease in CI (86 +/- 7.0 ml/min/kg to 58 +/- 13 ml/min/kg; p = 0.05) and increase in SVR (2269 +/- 373 dyne.sec/cm5.m2 to 3897 +/- 988 dyne.sec/cm5.m2; p = 0.05) from baseline to 120 minutes after treatment respectively. The DI was significantly lower in both CLP and diamide groups (p < 0.03) when compared with control animals. Histologic evidence of subendocardial necrosis was shown in both the CLP- and Diamide-treated animals.. These data suggest an association with RBC deformability and organ function in both septic and nonseptic animal models.

Topics: Animals; Cecum; Diamide; Endocardium; Erythrocyte Deformability; Erythrocytes; Hemodynamics; Ligation; Lung; Male; Necrosis; Oxygen; Punctures; Rats; Rats, Sprague-Dawley; Sepsis; Vitamin E