Complexities in the Rat Iron Loading Model for Pre-Clinical Testing of Iron Chelators.

Abstract

Transfusion-dependent iron overload, such as occurs in beta-thalassaemia (Cooley's Anaemia), leads to lethal cardiac toxicity in the second decade of life if not treated by iron chelation, but even with subcutaneous desferrioxamine (DFO) cardiac disease remains a problem, although delayed by 1–2 decades. As we design novel iron chelators, we are testing them in various animal models of iron overload. While assessing outcomes we have observed relatively sparse reports of systematic studies on organs, tissues, cellular, or subcellular iron distribution. Therefore we initiated a series of studies to characterize iron distribution using various approaches. Multiple intraperitoneal injections of iron dextran, 200 mg/kg/week X 4 − 16 weeks, followed by an equilibration period of minimum 1–2 weeks was studied as a means of increasing total body iron load in hundreds of rats under various conditions. Sacrifice varied from 6 weeks to 1 year post iron loading and the concentration of iron in liver, heart, and other tissues, organs, cells and subcellular organelles was examined. Quantitatively, in untreated rats (no chelators), the liver/heart iron ratio was about 10:1, consistent with the accumulation observed in post-mortem studies in humans prior to extensive use of iron chelation. Much less-well described has been the distribution of iron in lymphatic tissues. Our studies revealed that lymph nodes become visibly enlarged. In addition, randomly distributed brown spots appeared in the omentum. Such changes persisted up to one year after iron loading, regardless whether they were treated daily with chelators (DFO or deferiprone) in standard doses for four months. Even after a single intraperitoneal iron-dextran injection of 200 mg/kg, changes were visible. Histopathological analysis (hematoxilin-eosin for general histology and Perl's Prussian Blue for iron) showed extensive iron accumulation in the omentum, and in the cortical and subcortical regions of the enlarged lymph nodes. Electron microscopy revealed cellular (macrophages) and subcellular (mitochondria) iron localization in the lymph nodes. When iron was administered as iron sucrose (single ip dose), iron accumulation was more extensive in the omentum and in the peritoneal fat in comparison to iron dextran, but the enlargement of the lymph nodes was not observed. Quantitative iron measurement (via validated HPLC method) in the liver and heart after a single iron dextran (N=30, up to 29th day) and iron sucrose dose (N=6 up to 50th day) was in agreement with the histological observations. Iron accumulation in the omentum and lymph nodes after four months of chelation treatment and one year after iron loading indicated the resistance of these unusual iron “pools” to chelation therapy. These studies confirm that different iron formulations may result in different patterns of iron distribution and they also raise questions about the suitability of rats as an animal model for transfusional iron overload in humans.