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

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-2methylpyridin-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.

Antioxidant and Anti-inflammatory Protective Properties of Thai Shallot (Allium ascalonicum cv. Chiangmai) Juice on Human Vascular Endothelial Cell Lines (EA.hy926)

“…EA.hy926 cells were seeded at a density of 1×10 4 cells/well onto a 96-well culture plate and incubated with various concentrations of SHE (25, 50, 100, 200, 500 µg/ml) or 200 µg/ml vitamin E, for 24 h at 37 °C, 5 % CO 2 . Afterwards, levels of intracellular ROS were measured using fluorescence probe dichlorofluorescin diacetate (DCFH-DA) in presence of H 2 O 2 [11]. In brief, following removal of the culture medium, the treated cells were washed twice with PBS.…”

Antioxidant Properties and Cytotoxicity of White Mugwort (Artemisia lactiflora) Leaf Extract in Human Hepatocellular Carcinoma Cell Line

“…Intracellular production of ROS was estimated using the oxidation-sensitive fluorescent probe; 2',7'dichlorodihydrofluorescein diacetate (DCFH-DA) [17]. Briefly, HepG2 cells were treated with a different concentration of WMLE, ranging from 0.312 to 20 μg/mL, at 37 °C for 24 h. Then, the treated cells were washed 3 times with sterile PBS and labeled with 10 µM DCFH-DA solution in the dark at 37 °C for 30 min.…”