Establishment of a cell culture from the frog Leptodactylus fuscus as a model for (eco)toxicological assays

Abstract

Leptodactylus fuscus is a frog species widely distributed in the Neotropical region, occurring in several biomes, which makes it a potential biomonitor of environmental conditions. To advance the establishment of this species in this field, we developed a fibroblast-like cell line derived from the digits of the forelimbs (LFUfd) and evaluated its response to possible environmental stressors. An adult male L. fuscus was collected, anesthetized, and euthanized with eugenol. Digits from the forelimbs were extracted, decontaminated, and dissociated. Cells were plated and cultivated in vented flasks with DMEM/F12+GlutaMAX medium and 20% FBS at 28 °C. On the 15th passage, we made the chromosome preparations, where we placed them on slides and stained them with Giemsa for karyotype analysis. The cellular response to multiple stressors (Temperature, Hydrogen Peroxide, and Aluminum) was evaluated through the MTT assay. The temperature test evaluated two groups (28 °C and 37 °C for 24 h). The effect of hydrogen peroxide (H2O2) was analyzed by treatment for 1 h at 28 °C in different concentrations (control, 2 μM, 20 μM, 50 μM, 100 μM and 200 μM). The effects of aluminum have been tested at three concentrations (0.5 mg/L, 1 mg/L, and 10 mg/L). The karyotype obtained showed 22 chromosomes, according to the description of the species, although a small proportion of aneuploid cells was observed (∼7%). At 37 °C there was a reduction in cell survival and no effects on cell viability at the H2O2 concentrations tested; however, there was a decrease in cell viability when exposed to aluminum in all the concentrations tested. The growth behavior and karyotype observed indicate the establishment of a continuous cell culture with a stable genome that responds to environmental changes.