pyrimidinones and 4-amino-4--hydroxylaminodiphenylsulfone

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