Combination Treatments of 1-(N-Acetyl-6-Aminohexyl)-3-Hydroxy-2- Methylpyridin-4-One (CM1) With Deferiprone and Desferrioxamine Reduced Labile Iron Pool and Protected Oxidative Stress in Iron-Loaded Cultured Hepatocytes

Kanokwan Kulprachakarn,Kanjana Pangjit

doi:10.4172/vms.1000115

Abstract

Iron overload associated with oxidative stress is a serious problem in transfusion-dependent patients with β-thalassemia major. The increased iron overload in several organs may be caused by higher intestinal absorption along with less intensive chelation therapy. Liver iron overload could in turn facilitate the development or persistence of chronic progressive liver disease. Previous studies have shown that chelation with desferrioxamine (DFO) and deferiprone (DFP) substantially reduced body-iron scores in β-thalassemia patients with transfusional iron overload. We have synthesized and characterized a new bidentate iron chelator, 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2- methylpyridin-4-one (CM1). The compound can efficiently scavenge iron from both ferrous and ferric salts and plasma non-transferrin bound iron (NTBI). In this study we have studied the efficacy of the CM1 treatment on the decrease of levels of the labile iron pool (LIP) and reactive oxygen species (ROS) in iron-loaded mouse hepatocyte and HepG2 cell cultures. The isolated hepatocytes were treated with DFP, DFO and CM1 at different concentrations. The treated cells were analyzed for intracellular LIP using the calcein fluorescent technique and ROS levels using the dichlorofluorescein (DCF) fluorescent method. It was found that CM1 reduced the levels of intracellular LIP and hydrogen peroxideinduced ROS in both treated cells in a concentration-dependent manner. The combination treatment of CM1 with 25 μM DFP and DFO was demonstrated to decrease the levels of the LIP in both cells and tended to reduce the levels of ROS in HepG2 cells. Our findings support the evidence of iron-chelating and free radical-scavenging activities of CM1 in the livers with iron overload, which potentially can protect against oxidative liver inflammation and fibrosis. The efficacy of the CM1 treatment needs to be further investigated intensively under in vivo conditions.

Highlights

Iron is vital for almost all living organisms by participating in a various metabolic processes, including oxygen transport, DNA synthesis, and electron transport
We have investigated the effects of Combination Treatments of 1-(N-Acetyl-6-Aminohexyl)-3-Hydroxy-2Methylpyridin-4-One (CM1) on labile iron pool (LIP) and reactive oxygen species (ROS) levels in primary hepatocyte and HepG2 cell cultures
The LIP levels of primary hepatocytes pretreated with ferric ammonium citrate (FAC) for 24 hrs was decreased markedly in the presence of DFP and CM1 chelators in a concentration-dependent manner at both 12 and 24 hrs (Figure 1a)

Summary

Introduction

Iron is vital for almost all living organisms by participating in a various metabolic processes, including oxygen transport, DNA synthesis, and electron transport. Since there is no active mechanism to excrete iron from primates, long-term repeated blood transfusions in anemic patients with genetic disorders such as thalassemia, sickle cell disease (SCD), Diamond Blackfan syndrome, and bone-marrow failures such as aplastic anemia (AA) and myelodysplastic syndromes (MDS) can result in iron overload [1]. Elevated LIP causes damage to a variety of cells and tissues which accumulate NTBI including heart, liver, pancreas, erythrocytes and endocrine glands resulting in organ dysfunction. Without treatment, such iron overload becomes fatal. Combined therapy with DFP and DFO can decrease severe iron overload in patients with β-thalassemia major [10] and resulted in greater iron excretion and decreased adverse effects [11].

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