Antimalarial action of artesunate involves DNA damage mediated by reactive oxygen species

Abstract

Artemisinin-based combination therapy (ACT) is the recommended first-line treatment for Plasmodium falciparum malaria. It has been suggested that the cytotoxic effect of artemisinin is mediated by free radicals followed by the alkylation of P. falciparum proteins. The endoperoxide bridge, the active moiety of artemisinin derivatives, is cleaved in the presence of ferrous iron, generating reactive oxygen species (ROS) and other free radicals. However, the emergence of resistance to artemisinin in P. falciparum underscores the need for new insights into the molecular mechanisms of antimalarial activity of artemisinin. Here we show that artesunate (ART) induces DNA double-strand breaks in P. falciparum in a physiologically relevant dose- and time-dependent manner. DNA damage induced by ART was accompanied by an increase in the intracellular ROS level in the parasites. Mannitol, a ROS scavenger, reversed the cytotoxic effect of ART and reduced DNA damage, and modulation of glutathione (GSH) levels was found to impact ROS and DNA damage induced by ART. Accumulation of ROS, increased DNA damage, and the resulting antiparasite effect suggest a causal relationship between ROS, DNA damage, and parasite death. Finally, we also show that ART-induced ROS production involves a potential role for NADPH oxidase, an enzyme involved in the production of superoxide anions. Our results with P. falciparum provide novel insights into previously unknown molecular mechanisms underlying the antimalarial activity of artemisinin derivatives and may help in the design of next-generation antimalarial drugs against the most virulent Plasmodium species.

FIG 1

Artesunate (ART) causes DNA damage in P. falciparum. (A) Comet assay measurement of the olive tail moment (OTM) at 12 h and 24 h after ART treatment (1, 5, 100, and 1,000 IC₅₀s) compared to untreated (Control) parasites. The error bars represent standard errors of the means. (B) Comet assay visualization of DNA damage in P. falciparum parasites upon treatment with 2 nM (1 IC₅₀) and 10 nM (5 IC₅₀s) ART compared to untreated parasites. (C) Parasite growth and OTM values determined by a comet assay during the recovery period compared to those of untreated control parasites maintained in parallel. Parasites were treated with 2 nM ART for 30, 45, 60, 90, and 120 min (left to right), after which they were washed to remove ART, returned to culture, and sampled at 24, 48, and 72 h for determination of percent parasitemia (%P) (black bars, untreated cultures; gray bars, ART-treated cultures) and DNA damage, measured as the OTM, in comet assays (solid line, untreated cultures; dashed line, ART-treated cultures) (*, P < 0.015; **, P < 0.005). (D) Percent recovery of damaged parasite nuclei was analyzed by a comet assay at 24, 48, and 72 h during the reculture phase of parasites after ART treatment. Images from comet assays (minimum n = 10) were randomly scored as damaged (detectable comet) and undamaged (no detectable comet) nuclei for each sample. Shown are results indicating DNA repair as percent recovery of nuclei in control (black bars) and ART-treated (gray bars) cultures. The error bars represent standard errors of the means.

FIG 2

ART affects intracellular reactive oxygen species (ROS) levels, leading to DNA damage and parasite death. (A and B) Concentration-dependent oxidant activity of ART in elevating ROS levels in P. falciparum. Uninfected red blood cells (RBCs) and infected red blood cells (iRBC) (2% and 5%) were treated with the indicated concentrations of ART (2, 4, 10, and 20 nM) for 2 h (A) and 6 h (B), and ROS levels were measured by using DCF-DA, a fluorescent dye (*, P < 0.015; **, P < 0.005; ***, P < 0.0001). (C) Attenuation of ROS by mannitol. ROS induced by ART were effectively attenuated by coincubation with increasing concentrations of mannitol. Uninfected or infected red blood cells were coincubated with ART and mannitol for 2 h, followed by measurement of ROS levels (*, P < 0.05). (D) Attenuation of ART-induced ROS in the presence of mannitol was accompanied by significantly reduced DNA damage in P. falciparum (**, P <0.005; ***, <0.0001). OTM values were determined by a comet assay in the parasites coincubated with ART (0, 2, and 5 nM) and mannitol (10 μM) for 2 and 6 h. (E) Attenuation of ART-induced ROS by mannitol results in protection from the antimalarial effect of ART. Shown are percentages of infected red blood cells 48 h after treatment with ART (0, 2, and 5 nM) with or without mannitol (0, 1, and 10 μM). The error bars represent standard errors of the means (**, P < 0.005 for 10 μM mannitol).

FIG 3

Time kinetics of the antimalarial effect of ART and role of ROS. (A) Mannitol, a scavenger of ROS, was used to probe the early kinetics of the antimalarial effect of ART. Parasites were treated with ART (2 nM) and mannitol (1 and 10 μM), added at either time zero or 2 and 4 h after treatment with ART. Percent parasitemia was determined at the 48-h time point. The error bars represent standard errors of the means. (B) Mechanisms leading to the ART-mediated antiparasite effect manifest during the first 2 h. Parasites were treated with increasing concentrations of ART (0, 2, and 5 nM) for 2 h, washed to remove extracellular ART, and then returned to culture with increasing concentrations of mannitol (0, 1, and 10 μM) for 48 h, and parasitemia was measured. The error bars represent standard errors of the means. (C) A minimum of 45 min is needed before mannitol gradually becomes ineffective in scavenging ROS during the ART-mediated antiparasite effect. Parasites were treated with 2 nM ART for 15 to 120 min, washed to remove extracellular ART, and then returned to culture with 10 μM mannitol for 48 h to assess parasitemia. The error bars represent standard errors of the means. (D) ART-induced ROS levels steadily increase with time. Parasites were treated with 2 nM ART, and the relative fluorescence intensity was measured every 15 min. The error bars represent standard errors of the means.

FIG 4

ART affects total glutathione (GSH) in parasites. (A) ART treatment decreases total GSH levels and increases levels of the oxidized form of GSH (GSSG). Parasites were treated with increasing concentrations of ART (0, 2, and 5 nM) for the indicated times. Parasite lysates were prepared, and GSH levels were determined by using a DTNB assay. Total GSH concentrations (filled bars) and percentages of the oxidized form (solid lines) were determined. (B) GSH depletion causes an increase in intracellular ROS production in the presence of ART. Parasites were treated with GSH-depleting inhibitors (125 μM BSO, 10 μM DMF, and 1 mM NAC) in the presence or absence of ART for 2 h, and fluorescence intensity was measured. The error bars represent standard errors of the means. (C) GSH depletion causes an increase in DNA damage. Parasites were treated with various GSH-modulating agents in the presence or absence of ART for 2, 12, and 24 h, and OTM values were determined by a comet assay and compared to values for untreated parasites. The error bars represent standard errors of the means. (D) NADPH oxidase-catalyzed ROS are potentially involved in ART-mediated ROS production. Parasites were treated with ART (0, 2, and 5 nM) in the absence or presence of apocynin (0, 10, and 100 μM) for 2 h, and ROS levels were measured. The error bars represent standard errors of the means. DMSO, dimethyl sulfoxide.