Enrofloxacin and Toltrazuril Are Able to Reduce Toxoplasma gondii Growth in Human BeWo Trophoblastic Cells and Villous Explants from Human Third Trimester Pregnancy

Abstract

Classical treatment for congenital toxoplasmosis is based on combination of sulfadiazine and pyrimethamine plus folinic acid. Due to teratogenic effects and bone marrow suppression caused by pyrimethamine, the establishment of new therapeutic strategies is indispensable to minimize the side effects and improve the control of infection. Previous studies demonstrated that enrofloxacin and toltrazuril reduced the incidence of Neospora caninum and Toxoplasma gondii infection. The aim of the present study was to evaluate the efficacy of enrofloxacin and toltrazuril in the control of T. gondii infection in human trophoblast cells (BeWo line) and in human villous explants from the third trimester. BeWo cells and villous were treated with several concentrations of enrofloxacin, toltrazuril, sulfadiazine, pyrimethamine, or combination of sulfadiazine+pyrimethamine, and the cellular or tissue viability was verified. Next, BeWo cells were infected by T. gondii (2F1 clone or the ME49 strain), whereas villous samples were only infected by the 2F1 clone. Then, infected cells and villous were treated with all antibiotics and the T. gondii intracellular proliferation as well as the cytokine production were analyzed. Finally, we evaluated the direct effect of enrofloxacin and toltrazuril in tachyzoites to verify possible changes in parasite structure. Enrofloxacin and toltrazuril did not decrease the viability of cells and villous in lower concentrations. Both drugs were able to significantly reduce the parasite intracellular proliferation in BeWo cells and villous explants when compared to untreated conditions. Regardless of the T. gondii strain, BeWo cells infected and treated with enrofloxacin or toltrazuril induced high levels of IL-6 and MIF. In villous explants, enrofloxacin induced high MIF production. Finally, the drugs increased the number of unviable parasites and triggered damage to tachyzoite structure. Taken together, it can be concluded that enrofloxacin and toltrazuril are able to control T. gondii infection in BeWo cells and villous explants, probably by a direct action on the host cells and parasites, which leads to modifications of cytokine release and tachyzoite structure.

Keywords: Toxoplasma gondii; enrofloxacin; placenta; toltrazuril; treatment; trophoblast.

Figure 1

Cellular viability in BeWo cells treated with different drugs. BeWo cells were cultured in 96-well plates (3 × 10⁴ cells/well/200 μL) for 24 h and treated or not with DMSO (0.26%), enrofloxacin (ENF) (A), toltrazuril (TOL) (B), sulfadiazine (SDZ) (C), pyrimethamine (PYR) (D), or combination of sulfadiazine plus pyrimethamine (SDZ+PYR) (E) in different concentrations (μg/mL). After 24 h of treatment, the cells were submitted to MTT assay and data were presented as percentage (%) of viable cell (cellular viability) in relation to untreated cells (100% of cellular viability). Data were shown as mean ± SEM from three independents experiments with nine replicates. Significant differences in relation to untreated cells (medium) (^#P < 0.05). Differences between groups were analyzed by One-Way ANOVA with the Bonferroni multiple comparison post-hoc test.

Figure 2

Percentage (%) of T. gondii proliferation (2F1 clone) in BeWo cells treated with different drugs. BeWo cells were cultured in 96-well plates (3 × 10⁴ cells/well/200 μL) for 24 h, infected with T. gondii (RH-2F1 clone) for 3 h, washed with medium to remove extracellular parasites and treated or not at the same time with different drugs (μg/mL): enrofloxacin (ENF), toltrazuril (TOL), sulfadiazine (SDZ), pyrimethamine (PYR), or combination of sulfadiazine and pyrimethamine (SDZ+PYR) for an additional 24 h. Next, the cells were submitted to T. gondii intracellular proliferation assay (β-galactosidase assay). Data were presented as mean ± SEM of the percentage (%) of T. gondii proliferation from three independent experiments in nine replicates. Significant differences in relation to infected and untreated cells (T. gondii) (^*P < 0.05), ENF (^&P < 0.05), and TOL (^$P < 0.05). Differences between groups were analyzed by One-Way ANOVA with the Bonferroni multiple comparison post-hoc test.

Figure 3

T. gondii infection (ME49 strain) in BeWo cells treated with different drugs. BeWo cells were cultured on round glass slides of 13 mm in 24-well plates (1 × 10⁵ cells/200 μL) for 24 h, infected with T. gondii (ME49 strain) for 3 h, washed with medium to remove extracellular parasites and treated or not with the different drugs (μg/mL): enrofloxacin (ENF), toltrazuril (TOL), sulfadiazine (SDZ), pyrimethamine (PYR), or combination of sulfadiazine plus pyrimethamine (SDZ+PYR) for an additional 24 h. Then, the cells were stained with 1% toluidine blue and counted using a light microscope. Data obtained were analyzed as infection index (% of infected cells) (A) and total number of tachyzoites (total number of tachyzoites per 200 cells) (B). Data were shown as mean ± SEM from three independents experiments in six replicates. Significant difference in relation to infected and untreated cells (T. gondii) (^*P < 0.05), ENF (^&P < 0.05), and TOL (^$P < 0.05). Differences between groups were analyzed by One-Way ANOVA with the Bonferroni multiple comparison post-hoc test. Representative photomicrographs of BeWo cells infected by T. gondii and untreated (C), or treated with ENF (D), TOL (E), SDZ (F), PYR (G), or combination of SDZ+PYR (H). Staining by blue toluidine. Arrows indicate parasites into parasitophorous vacuole. Bar scale: 20.0 μm.

Figure 4

Cytokines production in BeWo cells treated with different drugs and infected by T. gondii (RH-2F1 clone or ME49 strain). BeWo cells were maintained in culture plates for 24 h, infected or not with T. gondii (RH-2F1 clone or ME49 strain) for 3 h, washed with medium to remove extracellular parasites and treated or not with the different drugs (μg/mL): enrofloxacin (ENF), toltrazuril (TOL), sulfadiazine (SDZ), pyrimethamine (PYR), or combination of sulfadiazine and pyrimethamine (SDZ+PYR) for additional 24 h. The supernatants were collected for measurement of IL-6 (A–C) and MIF (D–F) by ELISA. Uninfected/treated cells and the respective control (medium) (A,D) or 2F1-infected/treated cells and the respective control (medium) (B,E) were cultured in 96-well plates (3 × 10⁴ cells/well/200 μL), while ME49-infected/treated cells and the respective control (medium) (C,F) were cultured in 24-well plates (1 × 10⁵ cells/well/200 μL). Data were expressed as pg/mL according to the standard curve. Data were shown as mean ± SEM from three independent experiments in nine (2F1 clone) or six replicates (ME49). Significant difference in relation to untreated and uninfected cells (medium) (^#P < 0.05), untreated and infected cells (T. gondii) (^*P < 0.05), ENF (^&P < 0.05), and TOL (^$P < 0.05). Differences between groups were analyzed by One-Way ANOVA with the Bonferroni multiple comparison post-hoc test.

Figure 5

Analysis of toxicity in human villous explants treated with different drugs.Villous explants were cultured during 24 h in 96-well plates and treated or not with different drugs (μg/mL): enrofloxacin (ENF), toltrazuril (TOL) or combination of sulfadiazine plus pyrimethamine (SDZ+PYR). After 24 h of treatment, supernatants were collected and lactate dehydrogenase (LDH) activity was measured using the LDH Liquiform Kit (A). Negative control was represented by villous treated with only RPMI medium. Data were expressed as mean ± SEM from three independent experiments in five replicates. Significant differences in relation to negative control (medium) (^#P < 0.05). Differences between groups were analyzed by One-Way ANOVA with the Kruskall Wallis Dunn's multiple comparison post-hoc test. Representative photomicrographs of untreated villous explants (B), or treated with ENF (700 μg/mL) (C), TOL (900 μg/mL) (D), or combination of SDZ+PYR (150 + 200 μg/mL) (E). Histological sections stained by hematoxylin and eosin. Arrows indicate the syncytiotrophoblast cells and asterisks(^*) the mesenchyme. Bar scale: 50.0 μm.

Figure 6

Percentage (%) of T. gondii proliferation in human villous explants treated with different drugs. Villi were collected and cultured for 24 h, infected with T. gondii (RH-2F1 clone), and after 24 h, treated or not with the different drugs (μg/mL): enrofloxacin (ENF), toltrazuril (TOL), or combination of sulfadiazine and pyrimethamine (SDZ+PYR) for additional 24 h. Next, the villous explants were macerated for β-galactosidase assay.Data were analyzed as (%) of T. gondii proliferation (A). Data were expressed as mean ± SEM of three independent experiments in nine replicates. Significant difference in relation to untreated and infected cells (T. gondii) (^*P < 0.05), ENF (^&P < 0.05), and TOL (^$P < 0.05). Differences between groups were analyzed by One-Way ANOVA with the Bonferroni multiple comparison post-hoc test. Representative photomicrographs of untreated and infected villous explants (B), or treated with ENF (C), TOL (D), or combination of SDZ+PYR (E). Immunohistochemical sections counterstained by Harris's hematoxylin. Arrows indicate immunolocalization of parasites by fast red naphtol. Bar scale: 20.0 μm.

Figure 7

Cytokines production in human villous explants treated with different drugs and infected by T. gondii. Villous explants were collected and cultured during 24 h, infected or not with T. gondii (RH-2F1 clone), and after 24 h were treated or not with the different drugs (μg/mL): enrofloxacin (ENF), toltrazuril (TOL) or combination of sulfadiazine and pyrimethamine (SDZ+PYR) for additional 24 h.The supernatants were collected for detection of IL-6 (A,B) and MIF (C,D). Data were expressed in pg/μg according to the protein concentration of each villous and the standard curve. Data were expressed as mean ± SEM of three independent experiments in nine replicates. Significant difference in relation to untreated and uninfected villous (medium) (^#P < 0.05), untreated and infected villous (T. gondii) (^*P < 0.05), and ENF (^&P < 0.05). Differences between groups were analyzed by One-Way ANOVA with the Bonferroni multiple comparison post-hoc test.

Figure 8

Viability of T. gondii tachyzoites. Tachyzoites were placed in microtubes (1 × 10⁶) and treated (μg/mL) or not with enrofloxacin (ENF), toltrazuril (TOL) or medium only for 3 h. After, the parasites were stained with trypan blue and counted in an optical microscope. Significant differences between viable and unviable parasites from the same group (^*P < 0.05), and between unviable parasites from the treated and untreated-group (^#P < 0.05).

Figure 9

Ultra-structural analyze of tachyzoites in BeWo cells treated with enrofloxacin or toltrazuril. BeWo cells were cultured in 24-well plates (5 × 10⁵ cells/well/200 μL) for 24 h, infected with T. gondii (RH-2F1 clone) for 3 h, washed with medium to remove extracellular parasites and treated (μg/mL) or not with enrofloxacin or toltrazuril for an additional 24 or 48 h.Then, the cells were submitted to transmission electron microscopy. Representative electromicrography of untreated BeWo cells infected by T. gondii by 24 (A) or 48 h (B), enrofloxacin-treated cells infected by T. gondii by 24 (C) or 48 h (D), and toltrazuril-treated cells infected by T. gondii by 24 (E) or 48 h (F). PV, Parasitophorous vacuole; DC, daughter cells; MC, mother cell; HCN, host cell nucleus; Rp, rhoptries; PN, nucleus; and M, mitochondria of the parasites. Arrows: basal ends (“tethered” parasites); arrowhead: duple membrane. Bar scale: 500 nm or 1.0 μm.