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Am. J. Trop. Med. Hyg., 70(2), 2004, pp. 119–124 Copyright 2004 by The American Society of Tropical Medicine and Hygiene A NOVEL DNA-BASED MICROFLUORIMETRIC METHOD TO EVALUATE YOLANDA CORBETT, LIURIS HERRERA, JOSE GONZALEZ, LUIS CUBILLA, TODD L. CAPSON, PHYLLIS D. COLEY, THOMAS A. KURSAR, LUZ I. ROMERO, AND EDUARDO ORTEGA-BARRIA Instituto de Investigaciones Científicas Avanzadas y Servicios de Alta Tecnología, Ciudad del Saber, Clayton, Panama; Laboratorio de Productos Naturales, Universidad de Panama, Panama City, Panama; Smithsonian Tropical Research Institute, Ancon, Panama; Department of Biology, University of Utah, Salt Lake City, Utah Abstract. This paper describes the development of a novel microfluorimetric assay to measure the inhibition of Plasmodium falciparum based on the detection of parasitic DNA by intercalation with PicoGreen®. The method was used to determine parasite inhibition profiles and 50% inhibitory concentration values of known or potential anti- malarial drugs. Values for parasite inhibition with known anti-malarial drugs using the PicoGreen® assay were compa- rable with those determined by the standard method based upon the uptake of 3H-hypoxanthine and the Giemsa stain microscopic technique. The PicoGreen® assay is rapid, sensitive, reproducible, easily interpreted, and ideally suited for screening of large numbers of samples for anti-malarial drug development.
chrome PicoGreen® into Plasmodium DNA. PicoGreen® is an ultrasensitive fluorescent nucleic acid stain for measuring Malaria is among the most life-threatening and widespread double-stranded DNA (dsDNA) in solution, and it enables diseases in the world, causing 250−300 million cases and ap- the detection of quantities as low as 25 pg/mL of dsDNA with proximately two million deaths annually.1 The disease is a moderately priced spectrofluorometer using fluorescein ex- caused by four Plasmodium species (i.e., P. falciparum, P. citation and emission wavelengths. Accordingly, the micro- vivax, P. ovale, and P. malariae) that are transmitted to hu- fluorimetric method described herein is ideally suited for anti- mans during the bite of the female anopheles mosquito. The malarial drug discovery programs based in developing na- growing resistance of the parasites to treatment with known anti-malarial agents such as chloroquine is of grave concern and is responsible for some of the worst cases of malaria in the tropical world.2 The spread of resistance of the mosquito vector to currently available insecticides and the limited suc- Cultivation of parasites. Two chloroquine-sensitive (Sierra
cess of potential anti-malarial vaccines contributes to the ur- Leone clone D6 and Tanzania F32) strains and a chloroquine- gent necessity of finding new chemotherapeutic agents for the resistant (Indochina clone W2) strain of P. falciparum were treatment of malaria, in particular, agents effective against P. used for this study. The D6 clone was provided by Philip J.
falciparum, the strain responsible of the most severe forms of Rosenthal (Division of Infectious Diseases, University of California, San Francisco, CA). The W2 clone was provided The standard test for screening potential drugs for anti- by Dennis Kyle (Division of Experimental Therapeutics, plasmodial activity is a radioactivity-based method that relies Walter Reed Army Institute of Research, Silver Spring, MD).
upon the incorporation of 3H-hypoxanthine into the DNA of The F32 strain was provided by Eric DeHaro (Institut de the parasite to measure parasitic replication in red blood Recherche pour le Développement Group, Instituto de In- cells.3 This method is very sensitive and it can be used to vestigaciones Fármaco Bioquímicas, Universidad Mayor de screen a large number of compounds, but requires hazardous radioactive materials that require special facilities and proce- The three strains were maintained in vitro by a modifica- dures. Alternatives to the 3H-hypoxanthine-based methodol- tion of the method of Trager and Jensen.10 The culture media ogy include a labor-intensive and time-consuming micro- consisted of standard RPMI 1640 (Gibco-BRL Laboratories, scopic method and several colorimetric assays.4–6 Colorimet- Gaithersburg, MD) supplemented with 10% heat-inactivated ric methods, however, are based on enzymatic activity rather human type O+ serum (Valley Biomedical, Inc., Winchester, than parasite replication, and in addition, may be subject to VA), 25 mM NaHCO3, 2 mM glutamine, and 25 HEPES artifacts caused by pigments present in crude plant extracts (Sigma, St. Louis, MO). Cultures were maintained in type O+ that are frequently used in drug screening programs.
human red blood cell suspensions obtained from healthy local Traditionally, natural products have been a rich source of donors and prepared in citrate-phosphate-dextrose antico- anti-plasmodial drugs, including quinine and artemisinin,7,8 agulant (Sigma) at a hematocrit of 2%. The parasite density many of which are derived from biodiversity-rich developing was maintained below 2% parasitemia under an atmosphere countries. Since the standard anti-plasmodial assay is based of a certified gas mixture containing 5% CO2, 5% O2, and on the use of radioactive isotopes, the same developing coun- 90% N2 at 37°C. For each experiment, samples of stock cul- tries are often not in a position to develop anti-malarial drug tures were further diluted in culture medium containing suf- discovery programs, limiting access to a large pool of scientific ficient noninfected type O+ human erythrocytes to yield a talent and emphasizing the need to develop cost-effective final hematocrit of 2% and a parasitemia of 1%. All assays techniques that do not require the use of radioactive iso- were carried out in microtiter plates. For those cases in which topes.9 The present study proposes a new, straightforward, assays were synchronized, sorbitol was used.11 efficient, and accurate method for the detection of anti- Radioactivity-based assay. Incorporation of 3H-hypo-
malarial agents based upon the intercalation of the fluoro- xanthine (specific activity ס 1.0 mCi/mL; American Radio- labeled Chemicals, Inc., St. Louis, MO) was used to measure higher or lower concentrations when necessary. The final di- growth of the parasites, as previously described by Desjar- lution contained less than 0.1 DMSO, which had no measur- dines and others.3 Different antimalarial compounds at final able effect on parasite survival in this system. DMSO at a final concentrations ranging from 1.95 nM to 2 ␮M were added in concentration of 0.1% in RPMI 1640 culture media was used duplicate to flat-bottom, 96-well microtiter plates (Corning as negative control, and represented 100% parasite viability.
Glass Works, Corning, NY) in a final volume of 25 ␮L. A The positive control consisted of chloroquine at concentra- 200-␮L volume of the culture parasite was added to each well tions of 1.0, 0.1, and 0.01 ␮g/mL, and provided a measure of and the plate was then placed in a humidified airtight cham- the susceptibility of the parasite to known antimalarial drugs.
ber (Bellco Glass Inc., Vineland, NJ) that was flushed with To measure the effect of each plant extract alone on the the gas mixture described earlier, sealed, and stored in an fluorescence signal, each extract concentration was incubated incubator at 37°C for 24 hours. Each compound was tested on in the absence of parasites and the signal was subtracted from at least two occasions against both chloroquine-sensitive and the value obtained in the presence of drug and parasite.
chloroquine-resistant strains. At the end of the incubation Data analysis. Data analyses were performed with a pre-
period, 25 ␮L of diluted 3H-hypoxanthine (final concentra- programmed calculus sheet on Microsoft (Redmond, WA) tion ס 1.5 ␮Ci) was added to each well. The plates were then Excel® 2000 that processes the relative fluorescence units ex- returned to the humidified airtight chamber, flushed again ported through the KC junior software from the microplate with the gas mixture described earlier, sealed, and incubated fluorimeter. The calculus sheet consists of 1) a formula that at 37°C for an additional 18 hours. The cultures were then calculated the mean of the two replicates per sample condi- harvested with a semi-automated PHD Cell harvester® tion, 2) subtraction of the respective color background of (American Instrument Exchange, Inc., Haverhill, MA) onto each dilution of the plant extract, 3) conversion of the mean fiberglass paper disks, washed with distilled water, and fixed RFU value to percentage of the response, taking as 100% the with ethanol. Each disk was placed in glass scintillation vials mean of the negative control, and 4) conversion of the per- containing 2 mL of Microscint scintillation cocktail (Micro- centage to the 50% inhibitory concentration (IC scint-High Efficiency LSC-Cocktail; Perkin Elmer Life and Analytical Science, Boston, MA) for one hour. The vials were regression. To adjust for the potential contribution of the then counted in a Packard microplate scintillation beta hemoglobin pigment from erythrocytes and the possible fluo- counter (American Laboratory Trading LLC, Niantic, CT).
rescence from the intrinsic pigments present in some plant The mean values for uptake of 3H-hypoxanthine in parasit- extracts, control wells were used that consisted of noninfected ized control and nonparasitized control erythrocytes were cal- erythrocytes alone, and samples of diluted drugs or extracts with noninfected erythrocytes. The inhibitory concentration Fluorimetric susceptibility test. Synchronized ring form cul-
(IC50) was defined as the drug concentration that results in tures (hematocrit ס 2% and parasitemia ס 1%) were used to 50% of the net fluorescence compared with nontreated con- test pure compounds or serial dilutions of plant extracts in 96-well culture plates. Cultures of P. falciparum were placed in a humidified, air-sealed container, flushed with the gas mixture described earlier, and incubated at 37°C. Parasites were allowed to grow for a 48-hour incubation period, after which a 150-␮L aliquot of culture was transferred to a new Relationship between parasite number and fluorescence. Pre-
96-well flat bottom plate. Fifty microliters of the fluoro- liminary experiments demonstrated that serial dilutions of chrome mixture, which consists of PicoGreen® (Molecular normal uninfected red blood cells did not emit significant Probes, Inc., Eugene, OR), 10 mM Tris-HCl, 1 mM EDTA, amount of fluorescence when incubated in the presence of pH 7.5 (TE buffer), and 2% Triton X-100 diluted with PicoGreen®, indicating that DNA from contaminating white double-distilled, DNAse-free water, was then added to liber- blood cells and the hemoglobin pigment from erythrocytes ate and label the parasitic DNA. The plates were then incu- does not interfere with the detection of Plasmodium DNA. In bated for 5−30 minutes in the dark. The fluorescence signal, addition, serial dilutions of crude plant extracts, either alone measured as relative fluorescence units (RFU) was quanti- or mixed with uninfected erythrocytes, also failed to produce tated with a fluorescence microplate reader (FL significant fluorescence, suggesting that any pigments associ- Tek Instruments, Inc., Winooski, VT) at 485/20 nm excitation ated with crude plant extracts do not interfere with the fluo- and 528/20 nm emission. Simultaneously, the RFU from posi- rescence signal associated with Plasmodium DNA.
tive and negative control samples were obtained, stored, and To test the sensitivity of the fluorimetric method as a means of detecting Plasmodium DNA in infected erythrocytes, we Preparation of crude plant extracts and microtitration
compared the percentage of infected erythrocytes as deter- plates. Plant samples were prepared according to standard
mined by microscopic counting with results obtained from the protocols.12 Lyophilized crude extracts were provided in in- fluorimetric technique. We used serial double dilutions of dividual vials of 3 mg (dry weight) and stored at −20°C until infected erythrocyte cultures to prepare Giemsa-stained thin ready for testing. Crude extracts and partially-purified frac- blood smears and the percentage of parasitemia was then tions were dissolved in dimethylsulfoxide (DMSO) (Research evaluated by light microscopy. Aliquots of the same or par- Organics, Cleveland, OH) at a stock concentration of 50 mg/ allel cultures were mixed in a 96-well plate with an equal mL. Known antimalarial compounds were dissolved in dis- volume of PicoGreen® cocktail and the amount of fluores- tilled water or ethanol according to published methods.13,14 cence was quantified as described in the Materials and Meth- Samples were tested in 96-well plates in duplicate at final ods. As shown in Figure 1, there is a direct relationship be- concentrations of 50, 10, and 2 ␮g/mL and re-evaluated at tween the percentage of infected red blood cells and the fluo- FLUORIMETRIC METHOD FOR DETECTION OF ANTI-MALARIAL DRUGS FIGURE 1. Comparison of the percentage of Plasmodium falciparum−infected erythrocytes determined by microscopic counting with fluo- rescence intensity obtained from the microfluorimetric technique. A serial two-fold dilution of a synchronized infected culture (15.0% ring stage) with noninfected erythrocytes was used. Bars indicate the standard deviation of the mean for four independently processed samples. The inset
shows the relationship below 1% of parasitemia.
rescence signal between 0.1% and 15% of ring stage infected presence of infected erythrocytes. No differences were ob- served when nonsynchronized or D-sorbitol-synchronized Time course for the assessment of parasitemia. Time
Plasmodium cultures were used, nor were differences ob- course experiments were then performed in which cultures of served when chloroquine-sensitive (F32 and D6) or chloro- P. falciparum- infected erythrocytes were initiated at a para- quine-resistant (W2) strains were tested. Based upon these sitemia of 0.5% and the number of parasites was determined experiments, a time point of 48 hours was chosen for the at different time intervals by both microscopic counting and evaluation of potential anti-plasmodial compounds.
the microfluorimetric technique. Figure 2 shows that both Determination of IC50 values of known antimalarial
methods of detection are equally effective in detecting the drugs. The microfluorimetric method was used to determine
FIGURE 2. Time course experiments with Plasmodium falciparum−infected erythrocytes by microscopic counting and the microfluorimetric techniques. Parallel cultures of synchronized parasites were initiated at a parasitemia of 0.5% and analyzed at 24 and 48 hours (h). Bars indicate the standard deviation of the mean for two independently processed samples. RFU ס relative fluorescence units.
the effect of known antimalarial drugs on the growth of P. its utility as a systematic and efficient means of screening falciparum by testing the effect of chloroquine and meflo- large numbers of crude extracts. We considered as active quine on the growth on the F32 strain, a chloroquine- those plant extracts with IC50 values < 50 ␮g/mL. Table 1 susceptible parasite. From dose-response experiments, an shows that there was a perfect correlation between the radio- IC50 of 31 ± 0.7 nM (mean ± SD) for chloroquine was deter- activity-based, microscopic, and microfluorimetric techniques mined using the microfluorimetric method, which is compa- with respect to their ability to detect plant extracts with anti- rable to the previously reported value of 29 ± 9 nM deter- plasmodial activity (seven of seven extracts tested with the mined by 3H-hypoxanthine incorporation.15 The IC50 for me- three assays and two of two extracts tested with the fluori- floquine was 15 ± 3.7 nM, which is comparable to the value of metric and radioactivity methods). While the IC50 levels of 9.2 ± 4.2 nM that was determined with the radioactivity-based crude extracts measured by the radioactivity-based and mi- method.14 The dose response curves obtained with the radio- croscopic methods tend to be lower than those values mea- activity-based and microfluorimetric methods for measuring sure by the microfluorimetric assay, no differences were ob- the effect of chloroquine on the growth of the chloroquine- served in IC50 values when pure compounds were evaluated resistant W2 clone are shown in Figure 3. We did not observe (Figure 3). We carried out the complementary experiment in any significant difference in the IC50 values determined by which plants shown to be inactive by the radioactivity-based either method, yielding IC50 values of 86.5 ± 9 and 88.7 ± 0.72 method were tested in the microfluorimetric assay. In every nM for the radioactivity-based and microfluorimetric meth- case (five of five), plants that were inactive in the radioactiv- ods, respectively. The IC50 values determined for chloroquine ity-based assay were also inactive in the microfluorimetric in these experiments are comparable to the published value of method, an observation relevant to the use of the latter 128 ± 73 nM for the chloroquine-resistant strains.5,15 method for drug discovery (Table 1).
Drug discovery. Natural products from plants have been a
The microfluorimetric assay was used to guide the purifi- rich source of anti-parasitic compounds.7,8 Therefore, we cation of a compound with anti-Plasmodium activity from the evaluated the ability of the microfluorimetric method to de- plant Coccoloba parimensis. Initial screening of a crude ex- tect plant extracts with anti-plasmodial activity and to assess tract of leaves of C. parimensis demonstrated significant anti- plasmodial activity (IC50 ס 6−12 ␮g/mL). The extract was subjected to liquid-liquid partition with hexane, ethyl acetate, methanol and water, a technique used to separate the chemi- cal constituents on the basis of their relative polarity12 and the fractions were tested for anti-plasmodial activity. Purifica- tion of the sample resultant from the ethyl acetate fraction (IC50 ס 10 ␮g/mL) led to the isolation of the methyl ester of gallic acid that showed IC50 values < 2 ␮g/mL.16 The microfluorimetric method for detecting anti-plas- modial compounds described herein has several advantages over the traditional assay that monitors the incorporation of 3H-hypoxanthine by the parasite.3 The radioactivity- based method requires the use of an expensive, hazardous radioactive compound, costly liquid ␤-scintillation counter Comparison of IC50 values for crude plant extracts by uptake of [3H]-hypoxanthine, microscopic counting of Giemsa-stained thin blood smears, and the microfluorimetric technique* FIGURE 3. Determination of the 50% inhibitory concentration (IC50) values for chloroquine by the incorporation of 3H- hypoxanthine (top) and the microfluorimetric technique (bottom).
Cultures of Plasmodium falciparum W2 strain-infected erythrocytes were initiated at a parasitemia of 0.5%, incubated with different con- centrations of chloroquine, and the number of parasites was deter- mined at 48 hours. IC50 values of 88.7 and 86.5 ␮g/mL were deter- mined for the microfluorimetric and radioactivity-based assays, re- spectively. Bars indicate the standard deviation from the mean for four independently processed samples. CPM ס counts per minute; * Values are in micrograms/milliliter.
IC50 ס 50% inhibitory concentration; ND ס not done.
FLUORIMETRIC METHOD FOR DETECTION OF ANTI-MALARIAL DRUGS equipment, and special local regulations for the introduction, Received April 4, 2003. Accepted for publication October 1, 2003.
management, and disposal of radioactive waste. An impedi- Acknowledgments: Special thanks are given to Phil Rosenthal, Den- ment for the development of drug discovery programs in de- nis Kyle, and Jeff Ryan for their continuous and generous support.
veloping countries is the lack of accessible and appropriate We also thank all members of the laboratory of Eduardo Ortega- technology that would permit the efficient testing of biologic Barria for helpful discussions and encouragement.
materials for anti-plasmodial activity. Although several non- Financial support: This work was supported by the International Co- radioactivity-based methods have been developed over the operative Biodiversity Groups Program, award #1U01 TW01021-01.
The laboratory of Eduardo Ortega-Barria is partially supported by years, they are cumbersome, multistep procedures.4,5 National Institutes of Health grant 1R03 TW01076.
The method described herein is based upon the detection of Plasmodium DNA in short-term cultures using a 96-well Authors’ addresses: Yolanda Corbett, Liuris Herrera, Jose Gonzalez, Luz I. Romero, and Eduardo Ortega-Barría, Instituto de Investiga- format, allowing the efficient and quantitative measurements ciones Científicas Avanzadas y Servicios de Alta Tecnología, Ciudad of anti-plasmodial activity in a large number of samples. The del Saber, PO Box 7250, Zona 5, Clayton, Panama City, Panama. Luis method uses PicoGreen®, an ultrasensitive fluorophore that Cubilla, Laboratorio de Productos Naturales, Universidad de intercalates into the double-stranded DNA of Plasmodium in Panama, Panama City, Panama. Todd L. Capson, Smithsonian Tropi- cal Research Institute, Apartado 2072, Balboa, Ancon, Panama.
solution, enabling the detection of as little as 25 pg/ml of Phyllis D. Coley and Thomas Kursar, Department of Biology, Uni- dsDNA, a 400-fold increase in sensitivity compared with the versity of Utah, Salt Lake City, UT 84112 and Smithsonian Tropical DNA intercalator Hoechst 33258 (Polysciences, Inc., War- Research Institute, Apartado 2072, Balboa, Ancon, Panama.
Reprint requests: Eduardo Ortega-Barría, Instituto de Investiga- The PicoGreen® method is straightforward and rapid. The ciones Científicas Avanzadas y Servicios de Alta Tecnología, Ciudad parasites are first incubated with the test drug for 48 hours, del Saber, PO Box 7250, Zona 5, Clayton, Panama City, Panama, Tele- phone: 507-317-0012, Fax: 507-317-0023, E-mail: eortega@senacyt.
followed by addition of PicoGreen®, followed by a 5−30- minute incubation period prior to the measurement of fluo-rescence. The PicoGreen® assay protocol presented herein issimpler than that for Hoechst 33258 since there is no require- ment to remove potentially interfering compounds such as 1. Greenwood B, Mutabingwa T, 2002. Malaria in 2002. Nature 415: hemoglobin and hemozoin, nor is there a chloroform extrac- tion step to prevent quenching of fluorescence.17 The repli- 2. Riddley RG, 1999. Planting the seeds of new antimalarial drugs.
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read at a more convenient time without a significant change in the fluorescence signal. Significantly, if a fluorescence micro- 5. Delhaes L, Lazaro JE, Gay F, Thellier M, Danis M, 1999. The microculture tetrazolium assay (MTA): another colorimetric plate reader is not available, determination of parasite growth method of testing Plasmodium falciparum chemosensitivity.
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We compared the microfluorimetric methodology with the 7. Klayman DL, 1993. Artemisia annua, from weed to respectable conventional radioactivity-based assay by using both methods antimalarial plant. Kinghorn AD, Balandron MA, eds. Human to test crude plant extracts for anti-plasmodial activity. We Medicinal Agents from Plants. Washington, DC: American found that for all of the extracts tested, both methods yielded identical results. We do not have an explanation for the small 8. Muñoz V, Sauvain M, Bourdy G, Callapa J, Bergeron S, Rojas I, Bravo JA, Balderrama L, Ortiz B, Gimenez A, DeHaro E, differences between the calculated IC50 values of crude plant 2000. A search for natural bioactive compounds in Bolivia extracts as determined by the two methods. One possible through a multidisciplinary approach Part I. Evaluation of the explanation is the presence of low levels of interfering sub- antimalarial activity of plants used by the Chacobo Indians. J stances in the extracts. Alternatively, the persistence of Plas- Ethnopharmacol 69: 127–137.
modium-derived DNA related to the initial parasite inoculum 9. Kursar TA, Capson TL, Coley PD, Corley DG, Gupta MB, Har- rison LA, Ortega-Barría E, Windsor DM, 1999. Ecologically may be responsible. However, no significant difference in guided bioprospecting in Panama. Pharmaceut Biol 37 (suppl): IC50 values were observed between the two methods when pure compounds (chloroquine and mefloquine) were tested, 10. Trager W, Jensen JB, 1976. Human malaria parasites in continu- supporting the utility the PicoGreen® assay for quantifying ous culture. Science 193: 673–675.
anti-plasmodial activity. The microfluorimetric method de- 11. Lambros C, Vanderberg JP, 1979. Synchronization of Plasmo- dium falciparum erythrocytic stages in culture. J Parasitol 65: scribed herein has been used successfully to guide the purifi- cation of compounds with anti-plasmodial activity from crude 12. Montenegro H, Gutiérrez M, Romero LI, Ortega-Barría E, Cap- plant extracts. It is hoped that the development of an effec- son TL, Cubilla-Rios L, 2003. Aporphine alkaloids from Guat- tive and straightforward method for measuring anti- teria spp. with leishmanicidal activity. Planta Med 69: 677–679.
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