The genotoxicity and cytotoxicity of tannery effluent in bullfrog (Lithobates catesbeianus).

Some of the most polluting activities occur in bovine skin processing. Tannery generates effluents containing high concentrations of heavy metals and organic compounds. The phases composing the leather production process generate a large volume of tannery effluents that are often discarded in aquatic environments without any previous treatment. However, the effect these xenobiotics have on adult representatives belonging to the class Amphibia remains unknown. Thus, the aim of the present study is to assess the geno- and cytotoxic effects of tannery effluent on adult male bullfrogs (Lithobates castesbeianus) exposed to it. Accordingly, the animals were divided into the following groups: negative control (tannery effluent-free water), positive control (cyclophosphamide), and effluent (water added with 5% tannery effluent). The animals were euthanized for blood collection, and erythrocyte analyses were conducted after 35 and 90 days of exposure. The micronuclei (MN) frequency and the frequency of other nuclear abnormalities in each of the animals in the experimental groups were assessed in 2000 erythrocytes. According to the present results, the exposure to tannery effluents increased MN frequency as well as other nuclear abnormalities (i.e., lobed nuclei, binucleated cell, kidney-shaped nuclei, notched nuclei, and apoptotic cell) in the erythrocytes of animals in the effluent group and in the positive control group after 35 and 90 exposure days. Thus, the current study corroborated the hypothesis that the tannery effluent has aneugenic and clastogenic potential in adult male bullfrogs (L. castesbeianus). The present study is the first to report such effect.

Immunocytochemical mapping of the hemoglobin biosynthesis site in amphibian erythroid cells.

During the past 25 years, several studies have attempted to determine the site of integration of the heme and the four globin chains in vertebrate erythroid cells that is important in the formation of the hemoglobin molecule. Mitochondrion-like organelles or hemosomes were pointed out as responsible for this task. We performed several experiments to investigate this hypothesis. The intracellular distribution of hemoglobin in amphibian erythroid cells was detected by post-embedding immuno-electron microscopy, using a polyclonal anti-human hemoglobin-proteinA-gold complex. Hemoglobin mapping showed an intense labeling in the cell cytoplasm, but none in cytoplasmic structures such as endoplasmic reticulum, mitochondria, mitochondrion-like organelles, Golgi complex, ribosomes or ferruginous inclusions. The mitochondrial fraction obtained according to the protocol described for some authors, showed by ultrastructural examination that this fraction has a heterogeneous content, also composed by microvesicles rich in cytoplasmic hemoglobin, an artifact generated by mechanical action during cell fractionation. Thus, when this fraction is lysed and its content submitted to electrophoresis, hemoglobin bands would be found inevitably, causing false-positive results, erroneously attributed to hemoglobin content of mitochondrion-like organelles. Our data do not confirm the hypothesis that the final hemoglobin biosynthesis occurs inside mitochondrion-like organelles. They suggest that the hemoglobin molecule be assembled in the erythrocyte cytoplasm outside of mitochondria or hemosomes.