Lipids versus proteins as major targets of pro-oxidant, direct-acting hemolytic agents

David C. McMillan, Christine L. Powell, Zachary S. Bowman, Jason D. Morrow, David J. Jollow

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)274-283
Number of pages10
JournalToxicological Sciences
Volume88
Issue number1
DOIs
StatePublished - Nov 1 2005

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Hemolytic Agents
Phosphatidylserines
Lipid Peroxidation
Reactive Oxygen Species
Macrophages
Lipids
Erythrocytes
Proteins
F2-Isoprostanes
Membranes
Cytophagocytosis
Lipid bilayers
Annexin A5
Hemolytic Anemia
Wounds and Injuries
Erythrocyte Membrane
Lipid Bilayers
Thromboplastin
Membrane Lipids
Cytoskeleton

Keywords

  • Erythrocyte
  • Hemoglobin
  • Hemolytic anemia
  • Lipid peroxidation
  • Phosphatidylserine translocation
  • Reactive oxygen species

ASJC Scopus subject areas

  • Toxicology

Cite this

Lipids versus proteins as major targets of pro-oxidant, direct-acting hemolytic agents. / McMillan, David C.; Powell, Christine L.; Bowman, Zachary S.; Morrow, Jason D.; Jollow, David J.

In: Toxicological Sciences, Vol. 88, No. 1, 01.11.2005, p. 274-283.

Research output: Contribution to journalArticle

McMillan, David C. ; Powell, Christine L. ; Bowman, Zachary S. ; Morrow, Jason D. ; Jollow, David J. / Lipids versus proteins as major targets of pro-oxidant, direct-acting hemolytic agents. In: Toxicological Sciences. 2005 ; Vol. 88, No. 1. pp. 274-283.
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