Effects of glyphosate, 2,4-D, chlorantraniliprole, and imidacloprid formulations, separately and in mixtures in Caiman latirostris hatchlings.

The present study demonstrated the potential of glyphosate (GLY), 2,4-dichlorophenoxyacetic acid (2,4-D), imidacloprid (IMI) and chlorantraniliprole (CAP) separately and in mixtures to induce oxidative stress and DNA damage in Caiman latirostris hatchlings. Under controlled condition, an embryonic exposure to these pesticides was done at concentrations recommended for soybean crops. Treatments were: negative control, GLY, 2,4-D, IMI, CAP, mixture 1 (M1): GLY + 2,4-D, M2: IM I + CAP and M3: GLY + 2,4-D + IMI + CAP. At hatching, blood samples were taken for the evaluation of genotoxicity, oxidative damage to lipids and DNA, the enzymatic activity of catalase (CAT) and superoxide dismutase (SOD), and the expression level of their corresponding genes (catalase: cat and superoxide dismutase: sod). It has been shown that IMI, M2 and M3 induced a significant inhibition of CAT activity while no effect was observed on SOD. In turn, lipid peroxidation was significantly higher in individuals exposed to IMI, and to all the mixtures. Besides, genotoxicity and oxidative DNA damage were observed in all exposed groups. The results of mRNA expression showed no difference at transcription levels. In the same way, no alterations in growth parameters were recorded at hatching. Regarding to the mixtures, we observed a potentiating action of IMI on M3 in lipid peroxidation as well as independent action on oxidative DNA damage and genotoxicity parameters. Our results highlight the importance of investigating the effect of pesticides and their mixtures considering the potential consequences to caimans living in natural environments.

How the exposure to environmentally relevant pesticide formulations affects the expression of stress response genes and its relation to oxidative damage and genotoxicity in Caiman latirostris.

This study aimed to analyze the molecular stress responses thought the expression levels of catalase (cat), superoxide dismutase (sod) and heat shock protein 70 (hsp70) genes, and how these relate with cellular stress response considering oxidative damage to lipids, DNA and genotoxicity in blood of Caiman latirostris hatchlings exposed to pesticide formulations under ex situ conditions. Treatments were: negative control (NC-tap water), glyphosate 2% (GLY), cypermethrin 0.12% (CYP), chlorpyrifos 0.8% (CPF), and their ternary mixture (Mx3). The concentrations and schedule of application were those recommended in soybean crops. Soil and water showed pesticides residues in all exposed groups. Results showed a statistically significant increase in the micronucleus frequency and DNA damage, with an important oxidation in all exposed groups. The expression level of cat gene was significantly higher in CYP while the expression of hsp70 was significantly lower in GLY, CYP and Mx3, compared to NC. Pesticides tested showed alterations in expression levels, growth parameters, DNA damage and base oxidation under realistic exposure conditions, and can threaten, in the long term, the health status of wild populations.

Identification and evaluation of antioxidant and reference genes for quantitative real-time PCR in blood of Caiman latirostris.

The quantitative real-time polymerase chain reaction (qPCR) has been one of the most promising approaches to perform rapid and accurate quantification of DNA in various biological systems. The aim of this study was to standardized the qPCR technique for the analysis of important genes involved in the main routes of antioxidant defense against reactive oxygen species (catalase: cat and superoxide dismutase: sod) and evaluate the stability of different reference genes in blood of Caiman latirostris hatchlings. The stability of the reference genes, ß-actin, glyceraldehyde 3-phosphate dehydrogenase (gapdh) and ribosomal protein L8 (rpl8) was determined using the comparative ΔCt, NormFinder, geNorm, BestKeeper and RefFinder. Then, cat and sod genes were normalized with each reference gene and their mRNA abundances were determined through the qPCR. Stability of genes was ranked through the different methods in the following order: ß-actin, rpl8 and gapdh , under normal physiological conditions. The results reveal that cat and sod genes present a similar relative mRNA abundance with ß-actin and rpl8. This is the first report of the analysis of antioxidant mRNA as potential biomarkers of oxidative stress in blood for all crocodilians species. Besides, we determined the stability of different reference genes that can be used for normalization of mRNA abundance patterns in blood of C. latirostris, without the need to sacrifice the animals.