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.

中文翻译：

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建立来自青蛙 Leptodactylus fuscus 的细胞培养物作为（生态）毒理学测定的模型


Leptodactylus fuscus 是一种广泛分布在新热带地区的青蛙物种，分布在多个生物群落中，这使其成为环境条件的潜在生物监测器为了推进该物种在该领域的建立，我们开发了一种源自前肢手指 （LFUfd） 的成纤维细胞样细胞系，并评估了它对可能的环境压力源的反应。收集一只成年雄性 L. fuscus 并用丁香酚麻醉和安乐死。提取、去污和解离前肢的手指。将细胞接种在通气瓶中，加入 DMEM/F12+GlutaMAX 培养基和 20% FBS，在 28 °C 下培养。 在第 15 代，我们进行了染色体准备，将它们放在载玻片上并用 Giemsa 染色以进行核型分析。通过 MTT 测定评估细胞对多种应激源 （温度、过氧化氢和铝） 的反应。温度测试评估了两组 （28 °C 和 37 °C 持续 24 小时）。通过在 28 °C 下以不同浓度 （对照、2 μM、20 μM、50 μM、100 μM 和 200 μM） 处理 1 小时来分析过氧化氢 （H 2 O 2 ） 的影响。铝的效果已在三种浓度（0.5 mg/L、1 mg/L 和 10 mg/L）下进行了测试。根据物种的描述，获得的核型显示 22 条染色体，尽管观察到一小部分非整倍体细胞 （∼7%）。在 37 °C 时，在测试的 H 2 O 2 浓度下，细胞存活率降低，对细胞活力没有影响;然而，当暴露于所有测试浓度的铝时，细胞活力都会降低。观察到的生长行为和核型表明建立了具有响应环境变化的稳定基因组的连续细胞培养物。