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Effect of Hydroxyurea on the Intracellular Multiplication of Toxoplasma Gondii, Leishmania Amazonensis and Trypanosoma Cruzi

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Effect of Hydroxyurea on the Intracellular Multiplication of Toxoplasma Gondii, Leishmania Amazonensis and Trypanosoma Cruzi Brazilian Journal of Medical and Biological Research (2003) 36: 65-69 Hydroxyurea inhibits intracellular parasites 65 ISSN 0100-879X Short Communication Effect of hydroxyurea on the intracellular multiplication of Toxoplasma gondii, Leishmania amazonensis and Trypanosoma cruzi E.J.T. Melo and Laboratório de Biologia Celular, Centro de Biociências e Biotecnologia, H.J. Beiral Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ, Brasil Abstract Correspondence Toxoplasma gondii, Leishmania amazonensis and Trypanosoma Key words E.J.T. Melo cruzi are obligate intracellular parasites that multiply until lysis of host · Toxoplasma gondii Laboratório de Biologia Celular cells. The present study was undertaken to evaluate the effect of · Trypanosoma cruzi CBB, Universidade Estadual do hydroxyurea (an inhibitor of cell division at the G1/S phase) on the · Leishmania amazonensis Norte Fluminense multiplication of L. amazonensis, T. gondii, and T. cruzi in infected · Hydroxyurea Av. Alberto Lamego, 2000 host cells. Infected cells were treated with hydroxyurea (4 mM) for 48 28013-600 Campos dos Goytacazes, RJ h. Hydroxyurea arrested intracellular multiplication of all infective Brasil Fax: +55-022-2726-1514 forms of the parasites tested. In treated cultures, the percent of infected E-mail: [email protected] host cells decreased (50-97%) and most intracellular parasites were eliminated. Ultrastructural observations showed no morphologic change in host cells while intracellular parasites presented drastic morpho- logic alterations or disruption. The results strongly suggest that hy- Received May 29, 2001 droxyurea was able to interfere with the multiplication of intracellular Accepted October 14, 2002 parasites, leading to an irreversible morphological effect on L. amazo- nensis, T. gondii, and T. cruzi without affecting the host cells. The protozoan parasites Toxoplasma causes drastic effects on the host cells (5-7). gondii, Leishmania amazonensis and Trypa- Hydroxyurea is a drug that specifically in- nosoma cruzi are etiologic agents that cause hibits the cell cycle in the G1/S phase inacti- toxoplasmosis, leishmaniasis and Chagas vating the enzyme ribonucleotide reductase disease, respectively (1-3). These parasites and the synthesis of DNA (8). Different con- invade the host while being localized within centrations of hydroxyurea have been used a vacuole known as the parasitophorous vacu- for the treatment of several types of cancer ole (PV). L. amazonensis and T. gondii de- (8,9) and various concentrations have also velop within the PV whereas T. cruzi escapes been tested against trypomastigotes and to the cytoplasm of host cells. It is important epimastigotes of T. cruzi, promastigotes of to point out that the latter multiplies inside Crithidia luciliae (5-20 mM) (10), tachy- the cell cytoplasm and is released into the zoites of T. gondii (4 mM) (11), CD4 cell intercellular space after rupture of the host infected with HIV (100-200 mg/kg) and Tri- cell (4). In experimental animals, control of chomonas vaginalis (5 mM) (12). However, infection with therapeutic drugs generally hydroxyurea at a concentration up to 5 mM Braz J Med Biol Res 36(1) 2003 66 E.J.T. Melo and H.J. Beiral has been used to synchronize the cell and presence of medium 199 supplemented with parasite cultures without causing toxic ef- 5% FCS (11,13). All parasites were assumed fects (10,12). We have recently shown that to have interacted with the cells because the hydroxyurea, an inhibitor of DNA synthesis, parasite:host cell relationship was 10:1. inhibited the intracellular multiplication of Hydroxyurea (Merck Chemical Co., T. gondii followed by tachyzoite elimination Darmstadt, Germany) was dissolved in PBS (11). We have extended this study by also (4 M stock solution) and the solution was using hydroxyurea against the intracellular mixed with medium 199 in cultured cells at a parasites L. amazonensis and T. cruzi. final concentration of 4 mM. This drug con- Macrophages were collected from Swiss centration has been established for synchro- mice and cultivated for 24 h in medium 199 nization of the cell cycle, and has no toxic supplemented with fetal calf serum (FCS) at effects on the cells (10-13). The drug was 37ºC under a continuous flow of 5% CO2 added to the infected cells during intense (13). L. amazonensis (MHUM/Br/175/Josefa parasite proliferation. After incubation with strain) was cultivated for 7 days at 25ºC. The hydroxyurea, the cultures were washed with culture was infected for 1 h, washed with PBS, fixed with Bouins fixative, stained sodium phosphate-buffered saline (PBS) to with Giemsa and observed under a light remove extracellular parasites, and incubated microscope (63X objective Axioplan, Zeiss, for 72 h in medium 199 at 37ºC under a Jena, Germany). The percentage of infected continuous flow of 5% CO2. After this pro- cells and the mean number of intracellular cedure, most cells were infected. For infec- parasites were determined by examination tion with T. cruzi and T. gondii, Vero cells of at least 400 cells. (kidney fibroblasts from African green mon- Statistical analysis was carried out using keys) were obtained through periodic pas- the Student t-test, with the level of signifi- sages and used when semi-confluent on Fal- cance set at P<0.5. The data shown are rep- con plates or in flask cultures (11). The resentative of five experiments in triplicate. cultures were infected with T. cruzi trypo- The cultures in flasks were infected with mastigotes (Y strain obtained from a culture parasites and incubated with the drug as of Vero cells infected for 6-7 days at 37ºC) described above for transmission electron or T. gondii tachyzoites for 1 h. The culture microscopy. The cultures were fixed for 60 Figure 1. Effect of hydroxyurea on cultures of host cells infected was washed and incubated for 72 h (for T. min at room temperature in a solution con- with L. amazonensis, T. gondii, cruzi) or 48 h (for T. gondii) at 37ºC in the taining 1% glutaraldehyde, 4% paraformal- and T. cruzi. A, In the untreated dehyde, 5 mM CaCl2 and 5% sucrose in 0.1 culture, most host cells were in- fected (filled columns). After M cacodylate buffer, pH 7.2. Cells were treatment of infected cells with 100 post-fixed for 1 h in a solution containing 4 mM hydroxyurea for 48 h, the A 80 1% osmium tetroxide, 0.8% potassium fer- percent of infected cells was sig- 60 rocyanide and 5 mM CaCl in 0.1 M cacody- nificantly decreased (open col- 2 40 * umns). B, Mean number of in- * late buffer, pH 7.2. The cells were rinsed in tracellular parasites in infected 20 0.1 M cacodylate buffer, dehydrated in ac- 0 * host cells. The number of intra- L. amazonensis T. gondii T. cruzi % Infected host cells etone and embedded in Epon. Thin sections cellular parasites increased in untreated cells (filled columns) were stained with uranyl acetate and lead and was significantly decreased citrate and observed under a Zeiss 900 trans- 600 B in treated cells (open columns) mission electron microscope. after incubation with 4 mM hy- 400 droxyurea for 48 h. Data are re- When infected cells were incubated with 200 ported as means ± SD of four * * 4 mM hydroxyurea for 48 h, the number of independent experiments. Mean number of 0 * L. amazonensis T. gondii T. cruzi infected cells decreased significantly in all *P<0.05 compared to untreated intracellular parasites cells (Student t-test). treated cultures (Figure 1A, open columns) Braz J Med Biol Res 36(1) 2003 Hydroxyurea inhibits intracellular parasites 67 when compared with untreated control cells of infected cells and the number of intracel- (Figure 1A, filled columns). A similar sig- lular parasites. nificant decrease was observed in the mean Light and electron microscopy was used number of intracellular parasites in the three to observe the effects of the drug on parasite cultures (Figure 1B, open columns) when morphology and ultrastructure. Tachyzoites compared with control untreated cells (Fig- are elongated and curved, while amastigotes ure 1B, filled columns). A drastic reduction of T. cruzi and L. amazonensis have a spheri- in the number of infected cells was also cal shape. Light microscopy demonstrated observed in host cells infected with T. gondii. that treated Vero cells infected with T. gondii These results clearly show that the presence showed normal morphology, although intra- of hydroxyurea reduced both the percentage cellular parasites, when present, were dis- Figure 2. Effect of hydroxyurea on the intracellular multiplication of T. gondii, L. amazonensis and T. cruzi. a-d, Giemsa-stained host cells infected with para- sites. Macrophages infected with L. amazonensis (a) or Vero cells infected with T. cruzi (b) were treated (c,d) with 4 mM hydroxyurea for 48 h. A large number of parasites are ob- served in the cytoplasm of un- treated cultures (a,b, arrow- head). After incubation for 48 h in the presence of 4 mM hy- droxyurea, very few cells were infected with L. amazonensis (c, arrowhead) or T. cruzi (d, arrow- head). e-f, Ultrastructural obser- vation of macrophages or Vero cells infected with L. amazonen- sis (e) and T. cruzi (f), and treated with 4 mM hydroxyurea for 48 h. After incubation with 4 mM hydroxyurea, amastigotes of L. amazonensis with morpho- logical changes (arrow) or dis- rupted (arrowhead) were ob- served. Vero cells infected with T. cruzi showed intracellular parasites with morphological al- terations (arrow) or disrupted (arrowhead) in the cytoplasm of host cells. L = L. amazonensis, n = nucleus. Panels a-d = 900X; panels e,f = 15,000X. Braz J Med Biol Res 36(1) 2003 68 E.J.T. Melo and H.J. Beiral rupted (data not shown), in agreement with multiply within host cells, with parasite divi- earlier observations (11). Culture of macro- sion occurring at different times.
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