Evaluation of In Vitro Cytotoxic and Apoptotic Effects of Miltefosine on the Toxoplasma gondii RH Strain

Abstract

Background: We aimed to investigate the cytotoxic and apoptotic effects of miltefosine on Toxoplasma gondii RH strain by various techniques.

Methods: The study was conducted at the Department of Parasitology and Mycology, Urmia University of Medical Sciences, Iran in 2020. Four groups of five BALB/c mice were selected. The cytotoxicity test was conducted by adding miltefosine to T. gondii tachyzoites; control tachyzoites received PBS and MTT assay was done on each suspension. For evaluating the Th1-type immune responses, the serum levels of IFN-γ and nitric oxide (NO) were assessed in mice after injecting tachyzoites and miltefosine, respectively. The flow cytometry technique was performed on T. gondii tachyzoites challenged with IC₅₀ and IC₉₀ doses of miltefosine and unchallenged cells. DNA fragments in T. gondii tachyzoites were detected by Terminal dUTPnick-end labeling (TUNEL) method.

Results: Overall, 256, 64, 32, and 16 µg concentrations of miltefosine, respectively could kill more than 50% of viable T. gondii tachyzoites. The infected mice group, treated with miltefosine, significantly produced more IFN-γ relative to other groups (P< 0.001). Moreover, a significant difference was found in inducible NO synthase between the experimental and control groups (P<0.05). The flow cytometry results demonstrated a concentration-dependent apoptosis rate in tachyzoites incubated with miltefosine, though the necrosis rate was non-significant. DNA fragmentation analysis indicated oligonucleotides (18-200 bp) in tachyzoites treated with 11µg of miltefosine for 24, 48 and 72 h. However, this pattern was not observed in untreated control microorganisms.

Conclusion: Miltefosine could be a favorable candidate for use as a new treatment for toxoplasmosis.