Oxidation of Erythrocytes Enhance the Production of Reactive Species in the Presence of Artemisinins.

Ioannis Tsamesidis,Pierre Pério,Antonella Pantaleo,Karine Reybier

doi:10.3390/ijms21134799

Abstract

In red blood cells, hemoglobin iron represents the most plausible candidate to catalyze artemisinin activation but the limited reactivity of iron bound to hemoglobin does not play in favor for its direct involvement. Denatured hemoglobin appears a more likely candidate for artemisinin redox activation because it is expected to contain reactive iron and it has been described to release free heme and/or iron in erythrocyte. The aim of our study is to investigate, using three different methods: fluorescence, electron paramagnetic resonance and liquid chromatography coupled to mass spectrometry, how increasing the level of accessible iron into the red blood cells can enhance the reactive oxygen species (ROS) production derived from artemisinin. The over-increase of iron was achieved using phenylhydrazine, a strong oxidant that causes oxidative stress within erythrocytes, resulting in oxidation of oxyhemoglobin and leading to the formation of methemoglobin, which is subsequently converted into irreversible hemichromes (iron (III) compounds). Our findings confirmed, using the iron III chelator, desferrioxamine, the indirect participation of iron (III) compounds in the activation process of artemisinins. Furthermore, in strong reducing conditions, the activation of artemisinin and the consequent production of ROS was enhanced. In conclusion, we demonstrate, through the measurement of intra-erythrocytic superoxide and hydrogen peroxide production using various methods, that artemisinin activation can be drastically enhanced by pre-oxidation of erythrocytes.

Highlights

Dioxygen and iron constitute two of the major components of human erythrocytes and for this reason red blood cells (RBCs) have the potential to catalyze the production of highly toxic reactive oxygen species (ROS)
In the erythrocyte, where OH free radical will lead to Hb denaturation and further release of heme iron, this process can be autocatalytic, leading an ever increasing oxidative stress once it is initiated by the release of threshold amounts of free iron [4,5]
Healthy RBCs are equipped with multiple mechanisms to inactivate potent oxidants (superoxide dismutase, catalase, glutathione peroxidase, peroxiredoxins, glutathione, methemoglobin reductase, etc.), thereby suppressing this auto-catalytic expansion of free iron, allowing them to circulate for 120 days before oxidative stress begins to promote their demise

Summary

Introduction

Dioxygen and iron constitute two of the major components of human erythrocytes and for this reason red blood cells (RBCs) have the potential to catalyze the production of highly toxic reactive oxygen species (ROS). One of the most potent drugs involving the production of ROS mediated by iron is artemisinin (ART) and its derivatives, which contain an endoperoxide moiety that can be activated by iron to form cytotoxic reactive species. This characteristic was applied for many pathologies including malaria [9], cancer [10] and osteoporosis [11]. Denatured hemoglobin appears a more likely candidate for artemisinin redox activation because it is expected to contain reactive iron and it has been described to release free heme and/or iron in erythrocytes [28]. We demonstrate, using three different methods: fluorescence, electron paramagnetic resonance (EPR) and LC-MS, how modifying the redox state of red blood cells can potentiate the production of ROS derived from artemisinin activation

Results and Discussion

Liquid Chromatography Coupled to Mass Spectrometry Analysis

EPR Assay

Hemichromes Analysis

Data Analysis