Article available at http://www.parasite-journal.org or http://dx.doi.org/10.1051/parasite/2002093261
A ctivity
of natural a n d synthetic n a p h t h o q u in o n e s
a g a in s t To x o p l a s m a g o n d ii , i n vitro a n d in m u r in e m o d e l s of in fectio n FERREIRA R.A.*, OLIVEIRA A .B .**, GUALBERTO S.A.** & VITOR R.W A .*
R ésum é :
Summary: The effect of 14 natural and synthetic naphthoquinones in the replication of Toxoplasma gondii was evaluated. In vitro studies were accomplished in cultures of 2C 4 fibroblasts infected with RHstrain. Enzyme-linked immunosorbent assay was used to quantify parasite growth. For the studies in vivo, mice were infected with tachyzoites of the RH strain or cysts of the T. gondii EGS strain. In vitro, seven naphthoquinones demonstrated significant inhibition of intracellular T. gondii growth at concentrations of 1 and 5 pg/m l. Only three compounds were significantly protective when tested in animals: 2-hydroxy-3'-(3'-pentenyl)-l ,4-naphthoquinone |PHNQ4), 2-hydroxy-3-(l'-vinylphenyl)-l,4-naphthoquinone (PHNQ5) and 2hydroxy-3-(l'-propen-3'-phenyl)-1,4- naphthoquinone (PHNQ6). In animals infected with the EGS strain and treated with PHNQ4 (50 m g /k g /d a y orally), a 7-day prolongation of the time to death was observed. Treatment with 100 m g /k g /d a y orally or 5 0 m g /k g /d a y i.p. of PHNQ5 resulted in a 5-day and 16-day prolongation of the time to death, respectively. Treatment with 5 0 m g /k g /d a y orally or 5 0 m g /k g /d a y i.p. of PHN Q6 resulted in a 4-day prolongation of the time to death or up to 30 days after treatment, respectively. Our results suggest that the naphthoquinones may be important therapeutic agents for the treatment of toxoplasmosis. KEY WORDS : naphthoquinones, models.
Toxoplasma gondii,
strain,
in vitro,
murine
A c tiv ité d e n aph th oquin on es naturelles et SYNTHÉTIQUES CONTRE TOXOPLASMA GONDII IN VITRO ET DANS UN MODÈLE MURIN
L'effet de 14 naphthoquinones naturelles et synthétiques a été évalué sur la multiplication de Toxoplasma gondii. Les études in vitro ont été éffectuées avec des cultures de fibroblastes 2C 4 infectés avec la souche RH. Un ELISA a été utilisé pour mesurer la croissance du parasite. Pour les études in vivo, les souris ont été infectées avec des tachyzoïtes de la souche RH ou des kystes de la souche EGS de T. gondii. Les essais in vitro avec sept naphthoquinones ont démontré une inhibition importante du développement intracellulaire de T. gondii aux concentrations de 1 et 5 μg /m l. Seulement trois composés ont démontré une activité protectrice in vivo : 2-hydroxy-3'-(3'-pentenyl)- 1,4-naphthoquinone (PHNQ4I, 2-hydroxy-3-(I'-vinylphenyl)- 1,4-naphthoquinone (PHNQ5I et 2-hydroxy-3-(l'-propen-3'-phenyl)- 1,4-naphthoquinone (PHNQ6). Dans le groupe des animaux infectés avec la souche EGS et traités avec PHN Q4 (50 m g /k g /jo u r per os), une augmentation de sept jours de la survie des animaux a été observée. Le traitement avec 100 m g /k g /jo u r per os ou 5 0 m g /k g /jo u r i.p. de PH N Q 5 a provoqué une augmentation de la survie de cinq et 16 jours respectivement. Le traitement avec 5 0 m g /k g /jo u r per os ou 5 0 m g /k g /jo u r i.p. de PH N Q 6 a entraîné une augmentation de la survie de quatre et de plus de 3 0 jours après le traitement, respectivement. Ces résultats montrent donc que les naphthoquinones peuvent être des molécules d'avenir pour le traitement de la toxoplasmose.
MOTS CLÉS : naphthoquinones, Toxoplasma
gondii,
souche,
in vitro,
modèle
murin.
INTRODUCTION o x o p la s m a g o n d ii is an intracellular protozoan capable o f invading all types o f nucleic cells. The immune systems o f im m unocom petent patients control tachyzoite multiplication, w hereas infections in im m unocom prom ised patients can result in fatal en ce phalitis. Pyrimethamine alone or in com bination with T
* Departamento de Parasitologia, Instituto de Ciencias Biológicas, Universidade Federal de Minas Gerais, Av. Antonio Carlos 6627, Belo Horizonte, Minas Gerais, Brazil. ** Faculdade de Farmacia, Departamento de Produtos Farmacéuticos, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. Correspondence: Ricardo Wagner de Almeida Vitor. Tel.: (31) 3499-2853 - Fax: (3D 3499-2970. E-mail:
[email protected] Parasite, 2002, 9, 261-269
sulfadiazine has been the conventional treatment for acute toxoplasm osis and toxop lasm ic en ceph alitis although adverse reactions are com m on (Katlama, 1991; W ong & Remington, 1994; W eller & Williams, 2001) w hich may lead to the interruption o f chem o therapy (Leport et al., 1988; W ong e t al., 1994). It is recognized that there is an urgent need for new therapeutic approaches to treat toxoplasm osis, parti cularly for patients with AIDS (Luft & Remington, 1992; Khan et al., 1998; Gherardi et al., 2000). Several naph thoquinones have b een reported to exhibit antitumor, anti-T ry p an osom a cru zi, antimalarial and anti-Leisbm a n ia c h a g a s i activities (Hammond et al., 1985; Jernigan et al., 1996; Dolabella, 1997; Gourlart et al., 1997). Importantly, the naphthoquinone atovaquone has been proven to be effective against P n eu m ocy stis c a r in i and T ox o p lasm a g o n d ii (Araujo et al., 1991;
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Vilar et al., 1996). The present study reports the eva luation o f 14 naphthoquinones against T. g o n d ii in vitro and in a murine model o f infection.
MATERIALS A N D METHODS M ice
O
utbred fem ale Swiss mice from the Centro de Bioterism o o f the Universidade Federal de Minas Gerais (B elo Florizonte, Brazil) were used and w eighed roughly 20 g at the beginning of each experiment. Food and water were available to the animals throughout the experiment. T. GONDII ISOLATES
Strains RH and EGS w ere used. In vitro and in vivo studies used tachyzoites obtained from the peritoneal cavities o f m ice infected with the RH strain (Sabin, 1941) for two days. The virulent EGS strain (Ferreira et al., 2001) used in the in vivo analysis was isolated in our laboratory from the amniotic fluid o f an infected human patient (Castro et al., 2001). The EGS strain was maintained at a chronic stage by treating infected mice with sulfadiazine in drinking water. Mice were infected either i.p. with 103 tachyzoites o f the RH strain or orally with 10 tissue cysts o f the EGS strain. C o m po un d s The p a r a -hydroxynaphthoquinones (PHNQs), orth ofuranonaphthoquinones (O FN Q s) and p a r a - furanonaphthoquinones (PFNQs) used in the experim ents w ere synthetized via a route starting with 2-hydroxy1,4-naphthoquinone and aliphatic aldehydes, followed by oxidation o f the condensation products (Ferreira et al., 1989). These com pounds w ere obtained at the Departam ento de Produtos Farm acéuticos, Faculdade de Farmácia, Universidade Federal de Minas Gerais (B elo Horizonte, Brazil). In addition to the synthetic naphthoquinones, three natural naphthoquinones - lapachol, a-lap achon e and p-lapachone (Fig. 1) - were tested. The Lapachol, a-lapachone and P-lapachone w ere extracted from plants belonging to the family B ignoniaceae, particularly to the genus T a b eb u ia . Sul fadiazine was used for com parison with the com pounds tested in this study. In
v itro
a c tiv ity
The com pounds w ere dissolved in a small amount o f dimethyl sulfoxide and D u lbecco ’s modified Eagle’s medium (DMEM) (Sigma Chemical Co., St. Louis, Mo.) containing penicillin, streptom ycin and 10 % fetal bovine serum. The concentrations w ere prepared in 262
DMEM and varied betw een 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50 and 100 μg/ml. The effect o f each com pound on intracellular T. g o n d ii replication was evaluated using enzym e-linked im m unosorbent assay (ELISA), with m odifications (Romand et al., 1993). Sem icon fluent monolayers o f 2C4 fibroblasts w ere prepared in 96-well tissue culture plates and inoculated with tachy zoites o f T. g o n d ii RH strain at a ratio o f the three para sites/cell at 37° C in 5 % CO,. Eight wells w ere used for each drug concentration. After four hours, the various concentrations o f com pounds w ere added to the medium and the cultures w ere incubated for 48 h. The ELISA was done directly on the fixed cultures. Each culture well was w ashed three times with 100 μl o f phosphate-buffered saline (PBS) containing 0.05 % Tw een 20 (pH 7.6). The plates w ere then fixed with cold methanol for 15 min and filled with 5 % pow dered skim milk in PBS-Tw een for 60 min at 37° C. Then the plates w ere w ashed tw ice for five minutes each time with PBS-Tw een 20 pH 7.6. Positive sera (1 0 0 μl) from m ice ex p erim en tally in fected w ith T. g o n d ii were diluted 1:50 in powdered skim milk in 5 % PBS-Tween and incubated for 45 min at 37° C then added to each well. Plates w ere w ashed four times fol lowed by the addition o f 100 μl o f peroxidase-labelled anti-mouse IgG conjugate (Sigma) diluted 1/15000 and incubated for 45 min at 37° C. Plates were washed four times and then 100 μl o f the substrate ( ortho-phenylenediamine) was added to each well. After 20 min the reaction was stopped by the addition o f 100 μl o f 4 N H2SO 4 and the optical density o f the supernatant was determined for each culture well by ELISA readings C4490 nm). In each experiment, eight positive control wells w ere made with no com pound but with DMEM and T. g o n d ii tachyzoites while eight wells with no para sites and no compound were used as negative controls. D rug
t o x ic it y f o r f ib r o b l a s t s
The toxicitiy o f the com pounds for 2C4 fibroblasts was determined by colorimetric assay using methylene blue (G om es et al., 1995). Cells w ere seeded in four repli cate wells, using 104 cells per well. After incubation for 24 h at 37° C in 5 % CO 2, the wells containing the test com pounds w ere w ashed three times in a solu tion o f PBS 0.05 % -Tw een 20 and fixed with cold methanol for 15 min. Cells that rem ained in the wells w ere stained with 0.1 % m ethylene blue in 0.1 M borate buffer (pH 8.7) for 10 min. Excess stain was rem oved by washing three times with 0.01 M borate buffer (pH 8.7) and stain incorporated in the cells was extracted through the addition o f 100 μl o f 0.1 M HC1 to each well. Cells that w ere not treated with the com pound w ere used as a control. Color intensity was measured by optical density reading at 660 nm in an ELISA reader.
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N a p h th o q u in o n e s a g a i n s t
Compound No.
Chemical structure
1 (PFNQ1) 2 (PFNQ2) 3 (PFNQ3)
2-ethyl-naphtho [2,3b]-furan-4,9-quinone 2-n-propyl-naphtho[2,3b ]-furan-4,9-quinone 2-isopropyl-naphtho [2,3b ]-furan-4,9-quinone
1 (OFNQ1)
2-n-propyl-naphtho |1,2b ]-furan-4,5-quinone 2-ethyl-naphtho [l,2 b]-furan-4,5-quinone 2-isopropyl-naphtho [1,2b ]-furan-4,5-quinone
1 (PHNQ1) 2 3 4 5
(PHNQ2) (PHNQ3) (PHNQ4) (PHNQ5)
6 (PHNQ6)
CH=CH CH
ÇH 3
CH2CH=CMe2 CH=CH- CHMe 2 CH=CH CH, CH, CH, CH=CHØ CH=CHCH , Ø
g o n d ii
Quinone
R
2 (OFNQ2) 3 (OFNQ3)
T.
2-hydroxy-3-( 1’-butenyl)-1,4-naphthoquinone 2-hydroxy-3-(3’-methyl-2’-butenyl)-1,4-naphthoquinone (Lapachol) 2-hydroxy-3-(l'-isopentenyl)-l.4-naphthoquinone 2-hydroxy-3-(3’-pentenyl)-1,4-naphthoquinone 2-hydroxy-3-(1'-vinylphenyl)-1,4-naphthoquinone 2-hydro xy-3-(1’-propen-3-phenyl)-1,4-naphthoquinone
PPNQ
α-Lapachone
3,4-dihidro-2,2-dimetil-2H-nafto-[2.3b]-pyran-5,10quinone
OPNQ
β- Lapachone
3,4-dihidro-2,2-dimetil-2H-nafto-[1,2b]-pyran-5,6quinone
Fig. 1. - Chemical structures of the assayed compounds. Parasite, 2002,
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I n VIVO ACTIVITY The com pounds that show ed activity in vitro against T. g o n d ii w ere selected for in vivo analysis. Evaluation was done with com pounds that w ere in the neutral or sodium salt forms and administered orally or i.p., res pectively. All the com pounds in the salt form were dis solved in PBS, pH 7.2. Concentrations o f the com pounds that w ere insoluble in PBS (e.g. neutral forms) w ere prepared using a small am ount o f 20 % ethanol and the desired concentrations w ere obtained by the addition o f 0.25 % carboxymethylcellulose. Prior to the in vivo experiments, toxicity tests w ere done using five uninfected m ice per group that w ere treated orally or i.p. for 10 days with 10, 50 and 100 mg/kg/day o f the com pounds. Signs o f toxicity such as piloerection, lethargy, w eight loss or death w ere observed daily. D oses that w ere determ ined not to be toxic to the uninfected m ice w ere tested in m ice infected with the RH or EGS strains. Treatm ent o f m ice infected with RH-strain tachyzoites was initiated 24 h after parasite inoculation. In mice infected with EGS-strain cysts, treatment was started two days post infection. In both groups treatm ent lasted 10 consecutive days. All experim ents included 10 infected mice per group. The m ice w ere observed for 30 days o f infection for mortality and time to death. S ta tistic a l
a n a lysis
Data plotting was used to describe the effect o f com pounds in vitro. Optical density was plotted as a loga rithmic function o f concentration. For this, regression analysis was calculated using MicroCal Origin Version 2.24. T he 50 % inhibitory concentrations w ere calcu lated from the regression curves. The toxic effect of the com pounds was analyzed using the Student’s t-test com paring cells exp o sed and not exp o sed to the drugs. Survival curves w ere evaluated with the KaplanM eier log-rank test (Kleinbaum , 1999).
RESULTS
Compound
Inhibition o f I. g o n d ii growth IC50 (pg/m l.)
Cell toxicity (jig/mL)
PFNQ1 PFNQ2 PFNQ3 OFNQ1 OFNQ2 OFNQ3 PHNQ1 PHNQ2 (Lapachol) PHNQ3 PHNQ4 PHNQS PHNQ6 a-Lapachona p-Lapachona
♦ * * ND ND ND 25,45 4,88 8,31 37,20 6,31 3,01 5,75 ND
10 5 1 0,01 0,01 0,01 > 50 10 > 50 > 50 > 50 > 50 20 0,01
* The compound was not inhibitory for Toxoplasm a gondii. ND = not done. Table I. - In vitro effect of 14 naphthoquinones on Toxoplasm a g on dii growth in human fibroblast 2C4.
concentration o f 5 pg/ml (Fig. 2). These results w ere confirmed in a second experiment (data not shown). From the regression curve analysis, IC50 % was 25,45 pg/ml for PHNQ1, 4,88 pg/ml for PHNQ2, 8,31 pg/ml for PHNQ3, 3 7 ,2 0 pg/ml for PHNQ4, 6 ,3 1 μ g/ml for PHNQ5, 3,01 pg/ml for PHNQ6 and 5,75 pg/ml for PPNQ (a lapachone). All the p a r a -naphthoquinones w ere selec ted for in vivo assays. PFNQ1, PFNQ2 and PFNQ3 did not present anti- T. g o n d ii activity in vitro. All the com pounds classified as OFNQs and OPNQ ( β-lapachone) w ere high toxic to 2C4 fibroblasts (100 % o f cells w ere dead by 24hs after addition o f com pound at a co n cen tration o f 0.01 pg/ml.), as evidenced by the m ethylene blue assay and alterations in cell morphology. The p a r a - hydroxynaphtho quinones (PHNQs) and p a r a - furanonaphthoquinones (PFNQs) had low (no toxicity at a much higher concentration o f the drug ≥ 50 pg/ml) and medium (a toxic effect was noted with ≥ 1 μg/ml o f com pound) toxicity, respectively. Table I show s the IC50 for T. g o n d ii growth in 2C4 fibroblasts cultures and the minimum concentration that was found toxic (m icroscopic exam ination and m ethylene blue assay) for the monolayers. IN VIVO EXPERIMENTS
I n v itro a c t i v i t y AGAINST INTRACELLULAR T. GONDII REPLICATION
A
significant inhibitory effect on T o x o p la s m a growth was seen with the p a r a - hydroxynaphthoquinones. PHNQ1, PHNQ2 (lapachol), PHNQ3, PHNQ4, PHNQ5, PHNQ6 and PPNQ (α -lapachone) presented in vitro activity against T. g o n d ii beginning at a concentration o f 5 pg/ml for the first two com pounds and 1 mg/ml for the others (P < 0.05). Sulfa diazine presented activity in vitro against T. g o n d ii at 264
D oses o f 10, 50 and 100 mg/kg/day o f lap achol (PHNQ2), PHNQ1, PHNQ5 and PPNQ (a-lap ach o n e) were used for i.p. (sodium salt o f the com pound) treat ment. D ue to its toxicity, the PHNQ3 com pound was used in doses o f 10 and 25 mg/kg/day. D osages o f PHNQ4 w ere 10 and 50 mg/kg/day and for PHNQ6 they w ere 25, 50 and 100 mg/kg/day. For treatment via the oral route, all compounds were used in concen trations o f 10, 50 and 100 mg/kg/day. All experim ents included normal m ice treated only with the diluent and
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Fig. 2 - In vitro effect of p a ra-hydroxynaphthoquinones PHNQ1 (A), PHNQ2 (B), PHNQ3 (C), PHNQ4 (D), PHNQ5 (E), PHNQ6 (F), PPNQ (G), on Toxoplasma growth. Absorbance values in the enzyme-linked immunosorbent assay of infected cultures were plotted versus the In of the concentrations of drugs. (H) = Reference drug: Sulfadiazine. Control = cultures without drug. Parasite, 2002, 9, 261-269
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Fig. 3 - Activities of different doses of PHNQ4 (A), PHNQ5 (B and C), PHNQ6 (D and E) in dissemined acute murine toxoplasmosis oral infection with cysts of the EGS strain. Treatment was initiated 48 h after infection and was conti nued for 10 days. For panel A, C and E the drug was administred orally by gavage and for panel B and D the drug was administered by injection i.p. There were 10 mice in each experimental and control group.
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N a p h th o q u in o n e s a g a i n s t T. g o n d i i
untreated infected mice as controls. There w ere 10 m ice in each experim ental and control group. All the infected untreated control animals died within a period o f five to seven days w hen infected with the RH strain and seven to 10 days w hen infected with the EGS strain. None o f the com pounds administered either orally or i.p had an effect on mice infected with the RH strain (data not show n). Oral or i.p. treatment with PHNQ1, PHNQ2 (lapachol) and PPNQ (α -lapachone) at 10, 50 or 100 mg/kg/day did not provide significant protec tion against death in animals infected with EGS cysts (data not shown). The PHNQ3 compound administered i.p. in mice infected with the EGS strain prolonged sur vival in only one (10 %) o f the animals treated with a dose o f 10 mg/kg/day ( P = 0.0567) (data not shown). Retarded mortality was observed w hen PHNQ4 was given orally in a dose o f 50 mg/kg/day ( P = 0.0004) to mice infected with the EGS strain (Fig. 3A). Survival was also prolonged in animals that received 50 mg/kg/day ( P = 0.0014) i.p. or 100 mg/kg/day (P < 0.001) of PHNQ5 administered as a single oral dose daily for 10 days. (Fig. 3B and 3C). The results obtained using PHNQ6 administered i.p. resulted in prolongation o f the time to death. In animals infected with the EGS strain, treatment with 50 mg/kg/day o f PHNQ6 i.p., for 10 consecutive days, resulted in a prolongation up to 30 days ( P = 0.0004) after the conclusion o f treatment (Fig. 3D). An oral dose o f ≥ 50 mg/kg/day o f PHNQ6 resulted in additional four days ( P < 0.0015) in the time to death (Fig. 3E). A 100 mg/kg/day dose o f PHNQ6 administered i.p. appeared to be toxic, since 20 % of the treated mice died earlier than the untreated mice (controls). As with the other naphthoquinones, PHNQ6 did not have an effect on the animals infected with the RH strain even though the m ice treated with this com pound show ed signals o f murine toxicity (e.g. weight loss, lethargy and piloerection) later than the controls.
DISCU SSIO N
O
ur results indicate that the PHNQ4, PHNQ5 and PHNQ6 com pounds presented in vitro and in vivo activity against T. g o n d ii. These compounds, termed as p a r a -hydroxynaphthoquinones (PHNQs), w ere not significantly toxic to 2C4 fibroblasts or m ice in a concentration that inhibited parasite mul tiplication. O n the other hand, the p a r a - furanonaphthoquinones (PFNQs) did not show anti- T. g o n d ii acti vity in vitro even though they presented medium toxicity to 2C4 fibroblasts. The ortho-furanonaphthoquinones (OFN Q s) w ere significantly toxic to host cells. Parasite, 2002, 9, 261-269
The results o f toxicity tests in mice indicate that the administration route is an important factor in determi ning toxicity. All the com pounds that w ere tested via the oral route during 10 consecutive days did not demonstrate any toxic effect in normal mice. However, concentrations o f 50 and 100 mg/kg/day o f PHNQ3 and 100 mg/kg/day o f PHNQ4 administered i.p. were extremely toxic to uninfected mice, killing them imme diately after administration. The administration route not only affected the toxicity o f the com pounds, it also altered their activity in pro tecting m ice against death from T. g o n d ii infection. PHNQ5 in a dose o f 50 mg/kg/day i.p., inhibited replication o f EGS-strain tachyzoites (P = 0.0014) but the same amount administered orally did not have any effect on the same strain. As previously reported, the pH o f gastric juices and sites within the m ucosa may be an important factor that potentially affects naph thoquinone activity (Kumiko et al., 1998). In one study that tested the anti-T, g o n d ii activity o f two naphtho q u in o n es (N SC52 and N SC 55), b o th co m p ou n d s dem onstrated significant protective activity in m ice infected i.p. or orally with T. g o n d ii. However, pro tection was noted only w hen the com pounds w ere administered i.p., oral administration was not effective (Khan et al., 1998). O f further interest is the difference that exists betw een the pathogenesis o f the infection produced by inocu lation o f tachyzoites or cysts. The i.p. inoculation of 103 tachyzoites o f the RH strain resulted in a fulminant infection with a large num ber o f tachyzoites being pro duced in the peritoneal cavity o f all the positive control animals. In contrast, mice infected by using an oral ino culum o f 10 cysts o f the EGS strain produced an infec tion that progressed more slowly. However, oral infec tion with cysts or oocysts may b e more appropriate to simulate a natural infection. The results demonstrate significant differences in sus ceptibility to infection with T ox o p la sm a betw een the two T. g o n d ii strains analyzed. It appears that each strain has intrinsic characteristics that contribute to its virulence. The T. g o n d ii RH strain cause significantly higher levels o f parasitemia (Derouin & Garin, 1991). This high parasitemia may increase the severity of infection and may be crucial in influencing the efficacy o f our anti- T ox o p lasm a therapy. The m echanism o f action o f PHNQ4, PHNQ5 and PHNQ6 against T ox o p lasm a has not yet b een investi gated. However, the hydroxynaphthoquinones appear to act as potent inhibitors o f the redox processes in the respiratory chain. It has b een show n that several natural and synthetic naphthoquinones exhibit anti protozoal activity by the generation o f active oxygen species such as hydroxyl radical (*OH) and superoxide anion ( 0 2 ) that cause the lipid peroxidation and alte rations in the electron transport with inhibition o f the
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parasite respiration (Morello, 1988; D ocam po et al., 1978). In light o f the problem s caused by toxoplasm ic infec tion, particularly am ong AIDS patients, a continued search for new com p ou nd s and n ew therapeutic approaches for treatment o f opportunistic infections caused by T. g o n d ii is clear. The present study demons trates that p a r a -naphthoquinones are effective in inhi biting replication o f the RH strain o f T. g o n d ii in cell cultures and suggest that p a r a -hydroxynaphthoquinones may also be effective against the parasite in vivo. Further research should focus on evaluation o f these p a r a -hydroxynaphthoquinones com pounds. Additio nally, studies must be conducted to determ ine w he ther synergic effects are observed w hen these com p o u n d s a re a d m in is te re d w ith o th e r e f f e c tiv e compounds.
F e r r e ir a V.F., P in t o A.V., P in t o M.C.F.R., D a C r u z M.C. & C la -
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ACKNOWLEDGEMENTS
T
his w ork was supported by FAPEMIG (CBB2383/97) and CNPQ. R.W.A.V. is research fellow from the CNPq. W e thank Rosálida Estevam Nazar Lopes for technical assistance and Alan Lane de Melo for reviewing the manuscript.
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Reçu le 24 octobre 2001 Accepté le 23 avril 2002
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