Mechanisms Underlying the Effects of Chloroquine on Red Blood Cells Metabolism

Abstract

Chloroquine (CQ) is a 4-aminoquinoline derivative largely employed in the management of malaria. CQ treatment exploits the drug's ability to cross the erythrocyte membrane, inhibiting heme polymerase in malarial trophozoites. Accumulation of CQ prevents the conversion of heme to hemozoin, causing its toxic buildup, thus blocking the survival of Plasmodium parasites. Recently, it has been reported that CQ is able to exert antiviral properties, mainly against HIV and SARS-CoV-2. This renewed interest in CQ treatment has led to the development of new studies which aim to explore its side effects and long-term outcome. Our study focuses on the effects of CQ in non-parasitized red blood cells (RBCs), investigating hemoglobin (Hb) functionality, the anion exchanger 1 (AE1) or band 3 protein, caspase 3 and protein tyrosine phosphatase 1B (PTP-1B) activity, intra and extracellular ATP levels, and the oxidative state of RBCs. Interestingly, CQ influences the functionality of both Hb and AE1, the main RBC proteins, affecting the properties of Hb oxygen affinity by shifting the conformational structure of the molecule towards the R state. The influence of CQ on AE1 flux leads to a rate variation of anion exchange, which begins at a concentration of 2.5 μM and reaches its maximum effect at 20 µM. Moreover, a significant decrease in intra and extracellular ATP levels was observed in RBCs pre-treated with 10 µM CQ vs. erythrocytes under normal conditions. This effect is related to the PTP-1B activity which is reduced in RBCs incubated with CQ. Despite these metabolic alterations to RBCs caused by exposure to CQ, no signs of variations in oxidative state or caspase 3 activation were recorded. Our results highlight the antithetical effects of CQ on the functionality and metabolism of RBCs, and encourage the development of new research to better understand the multiple potentiality of the drug.

Keywords: antioxidant systems; band 3 protein; chloroquine; hemoglobin; oxygenation–deoxygenation cycle; red blood cells; sulfate transport.

Figure 1

Sulfate flux measured in oxygenated RBCs in the absence and presence of increasing CQ concentrations (2.5, 5, 10, and 20 μM). Values are presented as the mean ± SD (N = 5), * p < 0.05 vs. RBCs; ** p < 0.01 vs. RBCs; *** p < 0.0001 vs. RBCs.

Figure 2

Sulfate flow measured in deoxygenated RBCs in the absence and presence of CQ (10 and 20 μM). Values are presented as the mean ± SD (N = 5), * p < 0.05 vs. RBCs; ** p < 0.01 vs. RBCs.

Figure 3

Hill plot for the binding of oxygen to Hb in the absence (closed symbols) and in presence (open symbols) of 10 μM CQ. Conditions: 0.1 M HEPES buffer plus 0.1 M NaCl and 3 mM 2,3-biphosphoglycerate at pH 7.4 and 37 °C.

Figure 4

UV-visible absorption spectra of CQ (50 µM) at pH 7.4, in the absence or presence of increasing Hb concentrations (2.5 µM, 5 µM, 7.5 µM, 10 µM). The image shows an illustration of the variation of absorbance obtained with several different experiments. Section 1: shows the spectrum of Hb at different concentrations (2.5, 5, 7.5, 10 μM). Section 2: shows the CQ (50 μM) spectrum variation by adding increasing concentrations of Hb. Section 3: shows the CQ (50 μM) spectrum minus Hb spectra (5, 7.5, 10 μM).

Figure 5

Analysis of the effect of 0 and 20 µM CQ and 250 µM AAPH on ROS generation/oxidative stress calculated from fluorescence emission (section B). Representative morphological images of the changes induced by AAPH (3), or CQ (2) and no-oxidant treatments (1) (section A). Values are presented as the mean ± SD (N = 5). Scale bar: 25 µm.

Figure 6

Caspase 3 activity in RBCs in the absence and in the presence of CQ (10 and 20 µM). Results are from four independent experiments ± standard deviation.

Figure 7

Effect of CQ on the intracellular (A) and extracellular (B) ATP levels in RBCs. ATP concentrations were measured at the end of the incubation time of RBCs without and with 10 μM CQ. Results are from four independent experiments ± standard deviation. Asterisks indicate significant differences at p < 0.05 versus control. Values are presented as the mean ± SD (N = 5). ** p < 0.05 compared with control.

Figure 8

Phosphatase activity in normal human RBCs, incubated in the absence or in the presence of 10–20 μM of CQ. Values are the mean ± SD of at least three different experiments. *** p < 0.005, ## p < 0.05 and # p < 0.5 compared with control.

Figure 9

Effect of CQ (10 and 20 μM) on ghosts incubated at 37 °C and pH 7.4. Values are the mean ± SD of at least three different experiments. *** p < 0.005 compared to the control.

Figure 10

Schematical representation of RBC metabolism in the absence (A) and presence (B) of CQ.