The Ribonucleotide Reductase Inhibitor, Didox, Reduces the In Vivo Vascular Inflammation and Oxidative Stress Induced By Acute Hemolysis

Introduction: Several diseases and disorders, including hereditary and acquired hemolytic anemia, blood transfusion reactions, preeclampsia and some infections, incur intravascular hemolysis to varying degrees. The destruction of red blood cells and the release of hemoglobin (Hb) and heme into the circulation results in vascular inflammation, characterized by the recruitment of leukocytes to the vascular endothelium, reduced nitric oxide bioavailability and oxidative stress, all of which may contribute to complications seen in hemolytic diseases, such as pulmonary hypertension, cutaneous leg ulcers and priapism. Didox (3,4-dihydroxybenzohydroxamic acid), a ribonucleotide reductase inhibitor, has been shown to have anti-inflammatory and anti-oxidative effects. This molecule has potential as a cancer therapy and has demonstrated beneficial effects in numerous pathologies, diminishing vaso-occlusive processes in mice with sickle cell disease, and suppressing mast cell activation and degranulation, amongst other effects. Aims: The aim of this study was to evaluate the effects of didox on the vascular inflammatory effects of in vivo acute hemolysis. Methods: C57BL/6 mice received i.v. didox (10 mg/animal) or the same volume of saline vehicle immediately before receiving the hemolytic stimulus. In some mice, didox (10 mg) was given intraperitonally (i.p.) 30 min before hemolysis. Acute hemolysis (HEM) was induced by injecting mice (i.v.) with 100 µl sterile water; for non-hemolysis controls (CON), the same quantity of saline was administered i.v.. At 15 min post-hemolysis, blood was obtained by cardiac puncture for biochemical and flow cytometry analyses; other animals were submitted to cremaster muscle exposure followed by intravital microscopy. Results: Intravascular H2O administration successfully induced hemolysis within 15 min, as demonstrated by elevated plasma cell-free Hb and heme levels (plasma Hb: 0.20±0.05 and 0.66±0.16 mg/ml, P