Assessment of benznidazole and nifurtimox toxicity: effect on in vitro maturation of bovine oocytes.

MARURI, A.; GULÍN, E.; MENDIZABAL, I.; LOMBARDO, DM.

Encuentro; Virtual event Summer School on Innovative Approaches in Science; 2020

he Physicians Committee for Responsible Medicine; Johns Hopkins University Center for Alternatives to Animal Testing; The European Commission Joint Research Centre

Current therapeutic options for Chagas disease treatment is limited to nifurtimox (NFX) and benznidazole (BZ). Given the limited information on potential effects on gametes and embryonic development, etiological treatment is not recommended in women during pregnancy. The generation of preclinical information could contribute to the cost-benefit analysis for further clinical studies aimed to establish the safety of treatment during pregnancy, ensuring the treatment of pregnant women during regular medical consults, and even preventing congenital transmission. As a first step, we studied the in vitro effect of BZ and NFX on bovine oocytes maturation. Oocytes were obtained by puncture-aspiration of ovaries from a slaughterhouse. Oocytes with homogeneous cytoplasm surrounded by a compact cumulus of at least three cell layers were selected. Subsequently, they were incubated at 39 °C in an atmosphere with 5% CO2, in the presence of decreasing concentrations of NFX, BZ (500-2 µM) or with the vehicle (NT) at the maximum working concentration (DMSO; ≤ 0.081%). After 22 h, viability was evaluated with trypan blue and the percentage of oocyte nuclear maturation with Hoechst 33342 staining, considering mature oocytes those that had reached metaphase II. The experiments were run in three independent replicates. The NT group showed 100% viability and 81% of nuclear maturation, while in treatment groups maturation was affected in a concentration-dependent manner. The drug concentration that reduced the oocytes maturity in 50% (IC50) was 222 and 163 µM for BZ and NFX, respectively. The oocytes viability at BZ different concentrations ranged from 91 to 100%, while for NFX was from 69 to 79%. The calculated IC50 values are above the previously established limit for potential toxic concentration in oocytes (≤ 50 µM) and it would indicate that BZ and NFX do not significantly affect the in vitro maturation of bovine oocytes. Further studies should be continued to establish the toxicity of BZ and NFX in male gametes and early and late stages of embryo development.