Pharma-Dev UMR 152, Université de Toulouse, IRD, UPS, 31000 Toulouse, France.
Department of Biomedical Sciences, University of Sassari, 07100 Sassari, Italy.
Int J Mol Sci. 2020 Jul 7;21(13):4799. doi: 10.3390/ijms21134799.
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.
在红细胞中，血红蛋白铁代表了最有可能的催化青蒿素激活的候选物，但与血红蛋白结合的铁的有限反应性不利于其直接参与。变性血红蛋白似乎是青蒿素氧化还原激活的更可能的候选物，因为它预计含有反应性铁，并且已经描述了它在红细胞中释放游离血红素和/或铁。我们的研究目的是使用三种不同的方法：荧光、电子顺磁共振和液相色谱-质谱联用，来研究增加红细胞中可及铁的水平如何增强青蒿素衍生的活性氧 (ROS) 产生。通过使用苯肼来实现铁的过度增加，苯肼是一种强氧化剂，会导致红细胞内氧化应激，导致氧合血红蛋白氧化，并导致高铁血红蛋白形成，随后转化为不可逆的血质（铁 (III) 化合物）。我们的研究结果使用铁 III 螯合剂去铁胺证实了铁 (III) 化合物在青蒿素激活过程中的间接参与。此外，在强还原条件下，青蒿素的激活和随后 ROS 的产生增强。总之，我们通过使用各种方法测量细胞内超氧化物和过氧化氢的产生，证明了红细胞的预氧化可以极大地增强青蒿素的激活。
