OXIDATIVE STRESS IN NON-TRANSFUSION DEPENDENT THALASSEMIA: A JOURNEY FROM NADPH OXIDASES TO CYTOCHROME P450

Abstract

Background: Oxidative damage by reactive oxygen species (ROS) is considered to be one of the main contributors to cell injury and tissue damage in thalassemia patients. Recent studies suggest that ROS generation in non-transfusion-dependent (NTDT) patients occurs as a result of iron overload. This increased ROS production in organs has been associated with multiple pathological outcomes. It has been proposed that sources of ROS production during pathophysiology are disease-specific. Among the different sources of ROS, the NADPH oxidases and CYPs 450 have been proposed to be the driving force in certain diseases. ROS sources in patients with NTDT remain poorly understood.

Aim: We aim to identify the exact source of ROS in NTDT.

Methods: Hbbth3/+ mice were used as a model of NTDT. Eight mice were divided into two groups (a control group and a thalassemia group receiving no treatment). A peripheral blood smear was used to confirm the diagnosis of thalassemia. Liver tissue iron content was measured using high-performance liquid chromatography (HPLC). Assessment of superoxide production was done using HPLC for H2O2 production. Enzymatic activity of NADPH oxidases was assessed using the NADPH oxidase assay. Detection and quantification of NADPH oxidase and CYP450 mRNA levels and protein levels were performed by Real-time Polymerase Chain Reaction and western blotting, respectively. Epoxyeicosatrienoic Acids (EET) and 20-Hydroxyeicosatetraenoic Acid (20-HETE) activity were assessed by HPLC. Degree of expression of CYP450 was also evaluated by immunohistochemistry. Statistical analyses were done via an unpaired t-test. p-values < 0.05 were considered as statistically significant. All experiments were performed on liver tissues of Hbbth3/+ mice.

Results: Increased tissue iron levels were detected in the liver of Hbbth3/+ mice compared to control. There was an increased state of oxidative stress and elevated NADPH oxidase enzymatic activity in Hbbth3/+ mice. At the mRNA level, no significant changes were observed in the expression of NOX1, NOX2 and NOX4 in Hbbth3/+ mice compared to control. However, there was a decreased expression of in the protein levels of NOX1, NOX2 and NOX4 in Hbbth3/+ mice compared to control. Additionally, no significant changes were observed in the expression of CYP1A, CYP2B, CYP4A and CYP4F mRNA levels in Hbbth3/+ mice. A significant over-expression of CYP1A, CYP2B, CYP4A and CYP4F protein levels were observed in Hbbth3/+ mice compared to control. There was an increase in EET activity, which correlated with the increased expression of its respective CYPs (CYP1A and CYP2B). There was also an increase in 20-HETE activity, which correlated with the increased expression of its respective CYPs (CYP4A and CYP4F). Immunohistochemistry staining of liver tissue sections also showed over expression of CYP1A and CYP4A in Hbbth3/+ mice.

Conclusions: This is the first report indicating that CYP450 is the NADPH oxidase dependent ROS-producer responsible for superoxide and H2O2 production in the liver of thalassemic (Hbbth3/+) mice. Targeting CYP450 (by activating EET production and inhibiting 20-HETE production) merits evaluation as a novel therapeutic approach in thalassemia.