Plasmodium falciparum: Isolate-Specific Radiosensitivity

Experimental Parasitology 99, 108–110 (2001) doi:10.1006/expr.2001.4649, available online at http://www.idealibrary.com on

RESEARCH BRIEF Plasmodium falciparum: Isolate-Specific Radiosensitivity

D. Sean Geoghegan,* Tina Skinner-Adams,† and Timothy M. E. Davis† *Department of Medical Physics, Royal Perth Hospital, GPO Box X2213, Perth, Western Australia 6847, Australia; and †Department of Medicine, University of Western Australia, Fremantle Hospital, PO Box 480, Fremantle, Western Australia 6959, Australia

Geoghegan, D. S., Skinner-Adams, T., and Davis, T. M. E. 2001. Plasmodium falciparum: Isolate-specific radiosensitivity. Experimental Parasitology 99, 108–110. 䉷 2001 Elsevier Science

parasites were simultaneously irradiated in separate petri dishes at room temperature and ambient oxygen concentration. All samples were stored and transported together when outside the candle jar. A leadshielded area within the irradiation room was set aside for those samples not being irradiated. The petri dishes were arranged as a stack of five, and doses were delivered to each respective layer with increasing dose in order of decreasing focus to sample distance. The dose rates at each layer ranged from 1.13 to 1.55 Gy/min, with the higher dose rates used for the samples receiving the higher doses. A sixth petri dish was set aside as a control. The doses were delivered in five successive stages, with the sample furthest from the focus being removed at the end of each stage and replaced with an empty dish. This procedure was calibrated with thermoluminescent dosimeters with an error in the dose rate at each level of 0.06 Gy/min. In vitro assays of parasite activity were performed in 96-well microtiter plates with each well containing 200 ␮l of the cell culture. Each well was labeled with 0.5 ␮Ci of [3H]hypoxanthine (Amersham International, England), harvested onto filter mats (Harvester 96; Tomtec Inc., U.S.A.) after 48 h of incubation, and counted in a 1450 MicroBeta Plus liquid scintillation counter. Experiments were performed at least twice. Media and vehicle control wells were included on each plate. The relationship between radiation dose and parasite growth inhibition for each isolate was determined from [3H]-hypoxanthine incorporation at 48 h relative to that of untreated cultures and the dose–response equation

The radiation sensitivity of malaria parasites has three potential clinical applications, namely (i) to prevent the transmission of malaria by blood transfusion (Ferreira-da-Cruz et al. 1997, Braz et al. 1998), (ii) to serve as an as adjunctive therapy when a radioactive isotope is complexed to a conventional antimalarial drug (Dadachova 1999), and (iii) to attenuate the pathogenicity of specific parasite stages as part of the development of a vaccine (Rieckmann 1990). In the first two applications, detailed information relating to parasite radiosensitivity is of vital importance since dosimetry must allow for the exposure of normal cells. The stage-specific and asynchronous radiosensitivity of Plasmodium falciparum has been described previously (Waki et al. 1983, 1985) but these reports apply to a single isolate (GGG). Because the sensitivity of P. falciparum to antimalarial agents can vary widely between isolates, parasite radiosensitivity may also be isolate dependent. Consistent with this hypothesis, a report of the strain-specific radiosensitivity of Plasmodium berghei has indicated that chloroquineresistant strains are also relatively radiation resistant compared to chloroquine-sensitive strains (Song et al. 1995). Since there are no published data on strain-specific radiosensitivity in the case of P. falciparum, we have assessed this in three commonly used laboratory-adapted isolates, 3D7, CS2, and K1, of which K1 is chloroquine resistant. The three isolates of P. falciparum were maintained in asynchronous continuous in vitro culture as previously described (Trager and Jensen 1976). For stage-specific experiments, cultures were synchronized by use of sorbitol lysis (Lambros and Vanderberg 1979). Parasite irradiation was carried out with a Siemens Stabilipan orthovoltage radiotherapy unit configured to deliver 2.1 mm Cu quality X-ray radiation. The

I ⫽ (100 ⭈ D)/(D ⫹ ID50),

(1)

where I is the inhibition of [3H]-hypoxanthine incorporation expressed as a percentage, D is the radiation dose in Gy, and ID50 is the dose required for 50% inhibition of [3H]-hypoxanthine incorporation. The measured inhibition of [3H]-hypoxanthine incorporation for each isolate is shown in Fig. 1 together with the hypothetical curve described by Eq. (1) with an ID50 equal to the weighted average of the isolatespecific ID50s. The isolate-specific ID50s are reported as mean and 95%

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P. falciparum: ISOLATE-SPECIFIC RADIOSENSITIVITY

FIG. 1. Inhibition of [3H]-hypoxanthine incorporation 48 h postirradiation as a function of ionizing radiation dose for 3D7 (open circles), CS2 (open triangles), and K1 (closed diamonds). Also shown is the model curve described by Eq. (1), with ID50 equal to the confidence-weighted mean of the isolate-specific ID50s listed in Table I.

confidence intervals in Table I. The ID50s for each of the isolates were all within the 95% confidence intervals of the ID50s for the other isolates. The confidence-weighted mean ID50 (⫾95% CI) for the three isolates studied is 24.7 ⫾ 3.0 Gy, which is in agreement with the equivalent LD50 (⫽ D0/ln 2) of 26 Gy calculated from the reported D0 of 18 Gy for asynchronous cultures of the GGG isolate (Waki et al. 1983). We found trophozoites to be the stage that was most radiosensitive, but all stages (rings, trophozoites, and schizonts) of each isolate had a mean ID50 between 14 and 26 Gy. Measurements of hypoxanthine incorporation in 5 ml of 1% parasitized red blood cells (RBCs) at 25% hematocrit at 48-h intervals after irradiation for up to 8 days showed that a dose of 100 Gy was required to inactivate parasite development. Taking into account the stochastic nature of the effects of ionizing radiation, i.e., that a linearly larger dose is required to inactivate a logarithmically greater number of parasites, we estimate that a dose of 400 ⫾ 40 Gy is required to inactivate 1% parasitized RBCs in a 430-ml blood bag at 100% hematocrit. This calculated dose is supported by the report that, for 6 ml of 3–5% parasitized RBCs at 8% hematocrit, a dose of 200 Gy is required to abolish replication of erythrocytic forms of the Palo Alto P. falciparum strain (Ferreira-da-Cruz et al. 1997).

TABLE I The Ionizing Radiation Dose Required to Inhibit [3H]-Hypoxanthine Incorporation by 50% (ID50) 48 h Post-treatment for Each of the Isolates 3D7, CS2, and K1 Isolate 3D7 CS2 K1

ID50 (⫾95% CI)(Gy) 24.5 ⫾ 4.3 23.1 ⫾ 6.5 25.9 ⫾ 4.3

Our data provide evidence that isolates of P. falciparum with different drug resistance profiles have uniform radiation sensitivity. This is in contrast to preliminary data from rodent malaria (Song et al. 1990). The clinical implication of this finding is that irradiation of blood prior to transfusion may not require consideration of the drug sensitivity patterns of local parasite strains. This may also apply to potential adjunctive radiotherapy such as that in the form of a chloroquine– 198Au complex (Dadachova 1999). Because the K1 isolate is chloroquine resistant and since ionizing radiation interacts with biological matter through the creation of free radicals, our results imply that the antimalarial action of chloroquine is not oxidative in nature. The relatively simple methodology that we used in the present study may facilitate further studies of the antimalarial effect of radiation in combination with antimalarial drugs.

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