pyrimidinones and phenylhydrazine

As discussed above, the process by which normal senescent red cells are selected for removal from the circulation is the subject of much ongoing research and is not yet well understood. This in turn creates a problem for studies on the enhanced removal that occurs in xenobiotic-induced hemolytic states; specifically, whether the enhanced removal should be considered as an increase in rate of the normal sequestration mechanism or as an unrelated process, in part or in whole. This difficulty bears directly on the interpretation of much of the mechanistic hemolytic literature. Because of its dual in vivo and in vitro hemolytic capability, and because of its capacity to induce frank lysis in the incubation mixture, phenylhydrazine has been used extensively as a model compound for mechanistic studies. These data have contributed heavily to our current concepts of how chemicals induce damage in the red cell. The comparison studies presented above cast doubt on the relevance of many of these phenylhydrazine studies for the in vivo hemolytic response. Phenylhydrazine, like divicine and DDS-NOH, shows an overwhelming predominance of uptake into the spleen, as distinct from removal by the RES system in general, as evidenced by relatively low liver uptake. This suggests strongly that damaged cells are removed intact by the spleen and do not lyse or fragment in the general circulation, at least to any significant extent. The studies with DDS-NOH indicate that neither Heinz body formation nor lipid peroxidation per se are essential steps in the process by which damaged red cells are removed from the circulation in the rat. It is not yet clear whether this lack of obligatory involvement of Heinz bodies and lipid peroxidation is peculiar to the arylhydroxylamine-induced hemolytic state or whether it will prove to be of general applicability. On the other hand, cysteamine failed to reverse the hemolytic damage caused by phenylhydrazine. Since cysteamine "rescued" DDS-NOH treated cells under the same experimental conditions, this observation raises the possibility that protein-thiol oxidation per se is also not an obligatory step in the sequence of events leading to premature sequestration. Clearly, the ratio of lipid to protein oxidation is markedly different in these three examples of hemotoxic compounds. DDS-NOH showed high protein oxidation with no discernible lipid oxidation, divicine showed both high protein and high lipid oxidation, and phenylhydrazine showed high

Topics: Dapsone; Erythrocytes; Free Radicals; Glucosephosphate Dehydrogenase; Humans; Oxidants; Oxidative Stress; Phenylhydrazines; Pyrimidinones

Lipid peroxidation and the accompanying translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the lipid bilayer have recently been identified as key components of a signaling pathway for phagocytosis of apoptotic cells by macrophages. Drug-induced hemolytic anemia has long been known to be caused by an accelerated uptake of damaged (but intact) erythrocytes by macrophages in the spleen, and this process has been associated with enhanced formation of reactive oxygen species (ROS). However, the role of lipid peroxidation in hemolytic injury has remained unclear, and the effect of hemolytic agents on the distribution of PS in the erythrocyte membrane is unknown. The present studies were undertaken to determine whether lipid peroxidation and PS translocation could be detected in rat and human erythrocytes by three types of direct-acting hemolytic agents--dapsone hydroxylamine, divicine hydroquinone, and phenylhydrazine. 2',7'-Dichlorodihydrofluorescein diacetate was employed as a probe for intracellular ROS formation; lipid peroxidation was assessed by GC/MS analysis of F2-isoprostanes; and PS externalization was measured by annexin V labeling and the prothrombinase assay. The data confirmed that all three hemolytic agents generate ROS within erythrocytes under hemolytic conditions; however, no evidence for lipid peroxidation or PS translocation was detected. Instead, ROS production by these hemolytic agents was associated with extensive binding of oxidized and denatured hemoglobin to the membrane cytoskeleton. The data suggest that the transmembrane signal for macrophage recognition of hemolytic injury may be derived from oxidative alterations to erythrocyte proteins rather than to membrane lipids.

Topics: Animals; Dapsone; Dose-Response Relationship, Drug; Erythrocytes; Hemoglobins; Hemolysis; Humans; In Vitro Techniques; Lipid Peroxidation; Lipids; Phenylhydrazines; Phosphatidylserines; Proteins; Pyrimidinones; Rats; Reactive Oxygen Species

Studies were carried out to characterize further the cytoplasmic ATP- and ubiquitin-independent proteolytic system in red blood cells that degrades hemoglobin damaged by exposure to oxidants (Fagan, J. M., Waxman, L., and Goldberg, A. L. (1986) J. Biol. Chem. 261, 5705-5713). Several proteases were ruled out as having a major role in the degradation of oxidant-treated hemoglobin (Ox-Hb). Acid hydrolases are not active in this process since the degradation of Ox-Hb has a pH optimum between 6 and 8. The calpains are also not involved since inhibitors of cysteine proteases (leupeptin and trans-epoxysuccinyl-L-leucylamido-(3-methyl)butane) did not diminish the increased proteolysis in intact erythrocytes treated with oxidants or in lysates to which Ox-Hb was added. The degradation of Ox-Hb was unaffected by inhibitors of serine and aspartic proteases. Removal of the high M(r) multicatalytic proteinase by immunoprecipitation also did not significantly affect the degradation of Ox-Hb in erythrocyte lysates. The degradation of Ox-Hb was sensitive to metal chelators and sulfhydryl-modifying reagents but not to specific inhibitors of known metalloproteases. Insulin, which is rapidly degraded in lysates, completely blocked the degradation of Ox-Hb. Insulin- and Ox-Hb-hydrolyzing activity was also inhibited following immunoprecipitation of the 100-kDa metalloinsulinase. The metalloinsulinase, which is inhibited by sulfhydryl-modifying reagents and which requires divalent metals, may therefore participate in the degradation of hemoglobin damaged by oxidants in erythrocytes.

Topics: Adenosine Triphosphate; Animals; Azides; Calcimycin; Cations, Divalent; Cysteine Proteinase Inhibitors; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Hemoglobins; Hydrogen Peroxide; Ionomycin; Kinetics; Leucine; Male; Oxidants; Phenylhydrazines; Pyrimidinones; Rabbits; Rats

Mouse erythrocytes were incubated with oxidizing agents, phenylhydrazine, divicine and isouramil. With all the oxidants a rapid release of iron in a desferrioxamine (DFO)-chelatable form was seen and it was accompanied by methaemoglobin formation. If the erythrocytes were depleted of GSH by a short preincubation with diethyl maleate, the release of iron was accompanied by lipid peroxidation and, subsequently, haemolysis. GSH depletion by itself did not induce iron release, methaemoglobin formation, lipid peroxidation or haemolysis. Rather, the fate of the cell in which iron is released depended on the intracellular availability of GSH. In addition, iron release was higher in depleted cells than in native ones, suggesting a role for GSH in preventing iron release when oxidative stress is imposed by the oxidants. Iron release preceded lipid peroxidation. The latter was prevented when the erythrocytes were preloaded with DFO in such a way (preincubation with 10 mM-DFO) that the intracellular concentration was equivalent to that of the released iron, but not when the intracellular DFO was lower (preincubation with 0.1 mM-DFO). Extracellular DFO did not affect lipid peroxidation and haemolysis, suggesting again that the observed events occur intracellularly (intracellular chelation of released iron). The relevance of iron release from iron complexes in the mechanisms of cellular damage induced by oxidative stress is discussed.

Topics: Animals; Barbiturates; Deferoxamine; Erythrocyte Membrane; Glutathione; Hemolysis; Iron; Lipid Peroxidation; Male; Methemoglobin; Mice; Oxidants; Phenylhydrazines; Pyrimidinones

Intact native red blood cells (RBC) and treated RBC preparations were labelled with MC 540 and irradiated in the presence of diaminobenzidine (DAB). The polymerized diaminobenzidine reaction product is permanently stable in comparison with the labile fluorescence labelling. The brownish stained DAB polymerization product (DAB brown) and osmium black (after conversion of DAB brown with OsO4) allow the densitometrical determination with the light microscope. The latter product can be directly observed in the electron microscope. A direct correlation exists between the fluorescence intensity and the polymerized diaminobenzidine staining. It can be deduced that the enhancement of the DAB mediated contrast is reflecting an increased fluidity of the red cell membrane. The reaction was successful with all red cell preparations tested. This method is also suitable for the determination of fluidity changes in other cell membranes.

Topics: Diamide; Erythrocyte Membrane; Fluorescence; Humans; In Vitro Techniques; Membrane Fluidity; Microscopy, Electron; Oxidation-Reduction; p-Dimethylaminoazobenzene; Phenylhydrazines; Photochemistry; Pyrimidinones; Regression Analysis

Phenylhydrazine-induced oxidative damage in red cells results in increased binding of merocyanine 540, a fluorescence probe sensitive to changes in lipid packing. Fluorescence polarization studies with diphenylhexatriene did not reveal major changes in order parameters both in intact red cells and lysates treated with phenylhydrazine. These fluorescence studies indicate that major changes are observed in membrane lipids. Analytical studies of membrane phospholipids revealed a significant decrease in phosphatidylethanolamine. The results of the fluorescence and lipid studies, taken in association with our previously reported findings on spectrin and other cytoskeletal protein degradation in red cells exposed to phenylhydrazine, suggests that degradation of cytoskeleton membrane proteins is also responsible for changes in the lipid bilayer surface of the red cell membrane.

Topics: Diphenylhexatriene; Erythrocyte Membrane; Fluorescence; Humans; In Vitro Techniques; Lipid Bilayers; Membrane Lipids; Phenylhydrazines; Phospholipids; Pyrimidinones; Spectrin