Micronucleus test in reptiles: Current and future perspectives.

Micronucleus (MN) cell counting emerged in 1973-1975 as a valid alternative for characterizing chromosomal damage caused by different agents. It was first described in mammals, but its application was rapidly extended to other vertebrates, mainly fish. However, it was not until 28 years later that this test was implemented in studies on reptiles. Nowadays, reptiles are found to be excellent non-target species from environmental contamination exposure and MN test has become a fundamental tool for analyzing genotoxic effects induced by various xenobiotics. In this article we provide an updated review of the application of the MN test in reptile species, from an ecotoxicological perspective. Therefore, we present (I) a bibliometric analysis of the available research on genotoxic-induced MN formation in reptile species; (II) the use of reptiles as sentinel organisms in ecotoxicological studies; and (III) the strength and weakness of the application of the MN test in this group. With this review, we aim to provide a comprehensive view on the use of the MN test in ecotoxicology and to encourage further studies involving reptile species.

Pesticide-induced alterations in zebrafish (Danio rerio) behavior, histology, DNA damage and mRNA expression: An integrated approach.

To assess the impact of glyphosate and 2,4-D herbicides, as well as the insecticide imidacloprid, both individually and in combination, the gills of adult zebrafish were used due to their intimate interaction with chemicals diluted in water. Bioassays were performed exposing the animals to the different pesticides and their mixture for 96 h. The behavior of the fish was analyzed, a histological examination of the gills was carried out, and the genotoxic effects were also analyzed by means of the comet assay (CA) and the change in the expression profiles of genes involved in the pathways of the oxidative stress and cellular apoptosis. The length traveled and the average speed of the control fish, compared to those exposed to the pesticides and mainly those exposed to the mixture, were significantly greater. All the groups exposed individually exhibited a decrease in thigmotaxis time, indicating a reduction in the behavior of protecting themselves from predators. Histological analysis revealed significant differences in the structures of the gill tissues. The quantification of the histological lesions showed mild lesions in the fish exposed to imidacloprid, moderate to severe lesions for glyphosate, and severe lesions in the case of 2,4-D and the mixture of pesticides. The CA revealed the sensitivity of gill cells to DNA damage following exposure to glyphosate, 2,4-D, imidacloprid and the mixture. Finally, both genes involved in the oxidative stress pathway and those related to the cell apoptosis pathway were overexpressed, while the ogg1 gene, involved in DNA repair, was downregulated.

DNA damage, alterations in the expression of antioxidant enzyme genes and in the histoarchitecture of gill cells of zebrafish exposed to 17-α-ethinylestradiol.

Endocrine disruptors, such as estrogen, are chemical substances with the potential to alter the hormonal balance of organisms. Their origin can be natural or artificial, and they can act at very low doses. The estrogen 17α-ethinylestradiol (EE2) is used worldwide as an oral contraceptive and is a potential contaminant in aquatic ecosystems. It is well documented that these environmental pollutants can act directly or indirectly on the reproductive system, impairing development and fertility. However, little is known about the alteration of the cell oxidative status induced by EE2. The main objective of this study was to evaluate the effect on the gill cells of adult zebrafish exposed in vivo to EE2, analyzing cell histology, DNA damage and the expression levels of genes encoding the main enzymes involved in oxidative stress pathways. The histological study showed that EE2 produces moderate to high damage to the gill tissue, an increase in gill cell DNA damage and the mRNA levels of the genes corresponding to the manganese superoxide dismutase (Mn-sod) and catalase (cat) after exposure to 5 ng/L EE2. The results indicate that EE2 causes tissue alterations, DNA damage and oxidative stress. EE2 produced important alterations in the gills, a fundamental organ for the survival of fish. There is a clear need for further research on the ecological consequences of EDCs on non-target organisms.

Molecular analysis and bioinformatic characterization of cooper, zinc-superoxide dismutase (Cu/Zn-sod) gene of Caiman latirostris.

Superoxide dismutase (SOD) is an antioxidant enzyme that acts as a component of first-line defense system against reactive oxygen species (ROS). Copper/Zinc superoxide dismutase (Cu/Zn-SOD) is one of the isoforms of SOD enzyme and is sensitive to the exposure of different environmental factors, in different species and tissues. Caiman latirostris is one of the two crocodilian species living in Argentina and no information is available on the molecular and biochemical characteristics of the Cu/Zn-sod gene in this species. In the present work, we reported the presence of the Cu/Zn-sod gene in C. latirostris, the nucleotide and amino acid sequences, the modelled protein structure, evolutionary distance among species and tissue specific expression patterns. Cu/Zn-sod gene was 620 bp open reading frame in length and encoded 178 amino acids. The nucleotide sequences of C. latirostris shared high similarity with the Cu/Zn-sod genes of other crocodilian species, so it showed to be highly conserved. PCR analysis showed that Cu/Zn-sod gene was expressed in all the tissues examined (liver, gonads, spleen, heart, and whole blood), suggesting a constitutively expressed gene in these tissues. This study allows further investigation into the structure-activity relationship and the mechanism of action of Cu/Zn-SOD, besides exploring the functional breadth and possible alteration factors, including xenobiotics.

Cypermethrin induction of DNA damage and oxidative stress in zebrafish gill cells.

Cypermethrin (CYP) is a synthetic pyrethroid insecticide, used to control pests in domestic, industrial and agricultural environments. According to recent reports, it is one of the most common contaminants in freshwater aquatic systems. The aim of this study was to evaluate its potential genotoxic effect and the activation of the superoxide dismutase (SOD) and catalase (CAT) systems of adult zebrafish gill cells after in vivo exposure. The comet assay (CA) demonstrated that gill cells are sensitive to DNA damage after in vitro exposure to hydrogen peroxide (H2O2), showing a dose-dependent response. We also found an increase in DNA damage of gill cells following a dose- and time-dependent treatment with CYP. Moreover, it was verified that SOD and CAT activities significantly increased after exposure to 0.6 µg/L CYP, both during six and nine days. The same treatment caused a significant up-regulation of the mRNA levels of Mn-sod and cat genes. These data indicate that CYP causes gill cell's DNA damage and oxidative stress, modifying the activities of the enzymes responsible for maintaining ROS balance, as well as in their corresponding gene expression levels.

Cypermethrin: Oxidative stress and genotoxicity in retinal cells of the adult zebrafish.

Cypermethrin (CM), widely used for control of indoor and field pests, is one of the most common contaminants in freshwater aquatic systems. We evaluated CM genotoxicity and the activities of superoxide dismutase (SOD) and catalase (CAT) in retinal cells of adult zebrafish. Histological and immunofluorescence techniques show the presence of apoptotic cells in the zebrafish retina after 9 d of treatment with 0.6 µg/L CM. Histone γ-H2AX, a marker of DNA damage, was detected in both outer and inner nuclear layers; caspase-3, an apoptotic marker, was detected in the outer nuclear layer. In the comet assay, the cells were sensitive to hydrogen peroxide-induced DNA damage, showing a dose-dependent response. We observed a positive comet assay response to CM that was dose- and time-dependent. Following exposure to CM, SOD and CAT enzyme activities, and sod and cat mRNA levels, increased. These results indicate that CM causes DNA damage and oxidative stress and can induce apoptosis in retinal cells.