Immuno-endocrine alterations and oxidative stress induced by commercial formulations of pesticides in the tegu lizard (Salvator merianae).

Recent research has shown the risk of environmental contamination by pesticides in Argentina. Salvator merianae is considered as "sentinel species" for monitoring their effects. We intended to investigate growth, immunological and endocrine functions, and oxidative stress in S. merianae exposed to two commercial brands of the herbicide glyphosate: Roundup and Panzer Gold, as well as, to two insecticides: chlorpyrifos, and cypermethrin. Our results showed a higher increase in growth in animals exposed to Roundup and chlorpyrifos and a lower increase in those exposed to Panzer Gold. Higher total leukocyte and lymphocyte count values were observed in lizards exposed to Panzer Gold. The heterophils/lymphocytes ratio decreased in all treated animals. Regarding lobularity index, natural antibodies titers, and complement system activity values, lizards exposed to cypermethrin had higher values. Low corticosterone levels were observed in animals exposed to Roundup. Our results reveal different toxic effects of pesticides contributing to the knowledge of the pesticides' potential deleterious impact in the habitat of S. merianae.

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.

DNA damage and oxidative stress in gill cells of Cnesterodon decemmaculatus exposed to pesticides by runoff source in an agricultural basin.

In our country, great concern exists about diffuse pollution cause by the great use of pesticides in rural environments. A thorough analysis is needed to generate information, know the real situation and thus, be able to make decisions with the purpose of reducing environmental pollution. In situ bioassays have been carried out using Cnesterodon decemmaculatus within limnocorrals located in a surface natural water system that receives rainfall excess flowing from an agricultural basin with a typical crop rotation, including corn, wheat and soy. Specimens were taken from the limnocorrals 72 h after a probed natural runoff event toward the water body, and the gill cells were used to evaluate the DNA damage (comet assay, CA), catalase enzyme activity (CAT), and lipid peroxidation (LPO). In addition, the physicochemical analysis of the water (pH, temperature) and the presence and concentration of pesticides were carried out. The results showed significant differences on DNA damage and oxidative stress on the gill cells of the exposed fish compared to controls, being the combination of the rain regime and the mixtures of pesticides used in corn and soy more toxic than in wheat. These results highlight the necessity to understand detrimental processes caused by pesticides used in extensive systems of primary production, in order to prevent and minimize diffuse contamination, contributing to environmental recovery and sustainability.

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.

Biomarkers of geno- and cytotoxicity in the native broad-snouted caiman (Caiman latirostris): Chromosomal aberrations and mitotic index.

We evaluated the sensitivity of the chromosomal aberration (CA) and mitotic index (MI) assays on peripheral blood lymphocytes (PBLs) of Caiman latirostris, following ex vivo exposure to the alkylating agent, MMS. Two concentrations of MMS were tested in cultured peripheral blood. Relative to controls, MMS exposure reduced the number of metaphases observed, but both the numbers of cells with MN and the percentages of aberrant metaphases increased. The types of CA identified were chromosome and chromatid breaks, chromosomal rearrangements, monosomies, and nullisomies, with significantly higher values in the MMS-exposed groups. The incorporation of the MI and CA tests in C. latirostris can provide information on damage caused by xenobiotic exposures.

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.

The problem with implementing fish farms in agricultural regions: A trial in a pampean pond highlights potential risks to both human and fish health.

The safety of creating fish farms in agricultural settings was evaluated by growing Piaractus mesopotamicus in a pond, while crops where cultivated in a nearby field under a pesticide application regime typical of the Pampa region. Atrazine, glyphosate and its metabolite, aminomethylphosphonic acid (AMPA), were detected in the water of the pond at concentrations ranging between 92 and 118 µg/L for atrazine, 12 and 221 µg/L for glyphosate and 21 and 117 µg/L for AMPA. Atrazine and malathion were detected in fish muscles at concentrations ranging between 70 and 105 µg/kg for atrazine and 8.6 and 23.7 µg/kg for malathion. Compared to fish raised in a pisciculture, fish from the agricultural pond presented reduced values of pack cell volume, hemoglobin, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration, together with significantly greater cholinesterase activity in both plasma and liver and reduced glutathione-S-transferase activity in the liver. A comet assay also demonstrated that P. mesopotamicus from the agricultural pond presented a significantly greater level of DNA damage in both erythrocytes and gill cells. Overall, the present study demonstrates that pisciculture ponds established in an agricultural setting may receive pesticides applied to nearby cultures and that these pesticides may be taken up by the fish and affect their physiology and health. The accumulation of pesticides residues in fish flesh may also present a risk to human consumers and should be closely controlled.

Effects of glyphosate, cypermethrin, and chlorpyrifos on hematological parameters of the tegu lizard (Salvator merianae) in different embryo stages.

Different studies have demonstrated effects of pesticides during embryo development in vertebrates and stage-dependent effects, but there is no information concerning this for Salvator merianae. We evaluated the effects of the herbicides Glyphosate Roundup (GLY-RU) and Glyphosate Panzer Gold (GLY-PZ); and the insecticides Chlorpyrifos (CPF) and Cypermethrin (CYP), and their complex mixtures, at different concentrations in hematological parameters of S. merianae embryos at two different development stages. The analyzed parameters were Total and Differential White Blood Cells Count, Heterophils/Lymphocytes index (H/L), Lobularity index, and Natural Antibodies (Nabs titres), as well as growth, embryo mortality and birth delay. Heterophils decreased in the intermediate concentrations tested of CYP and GLY-RU, in animals exposed at 33-days development. Lymphocytes increased in the intermediate concentration tested of GLY-RU, and the H/L index decreased in the maximum concentration tested of GLY-RU. NAbs titres increased in those animals exposed to the maximum CYP concentration tested. However, animals exposed at 3/5-days development showed no differences among treatments in most of the analyzed parameters, suggesting a stage-dependent response. Nevertheless, those animals exposed to GLY-PZ showed lower Nabs titres in relation to negative control. These results suggest effects on different hematological parameters related to the immune system of S. merianae, according to the used pesticide (herbicide or insecticide), its concentration and commercial formulation (GLY-RU or GLY-PZ), and the stages of development of the exposed animals. Our results reveal the importance of carrying out studies that evaluate the effects of permanent exposure of living beings and their environments to these toxics.

Genotoxicity induced by widely used pesticide binary mixtures on Caiman latirostris (broad-snouted caiman).

In this study, we investigated the effects of three binary mixtures of pesticide formulations commonly used in soybean crop: Cypermethrin Atanor® (25% -CYP), Chlorpyrifos Lorsban 48E® (48% -CPF) and Glyphosate Roundup® Full II (66.2% -GLY) on broad-snouted caiman (Caiman latirostris) hatchlings exposed by voluntary immersion under controlled condition. Genotoxicity was evaluated in peripheral blood erythrocytes through the micronucleus (MN) test and other nuclear abnormalities (NAs) and besides, growth of caiman was analyzed in each experimental group. The results showed that pesticide formulations tested, at concentrations similar to those recommended for application in the field, induced an increase in the frequency of micronucleus (FMN; p = 0.001) and Notched nuclei (NN; p = 0.010) in the mixture CYP + CPF, while an increase in the frequency of buds and NN was observed in the mixture of GLY + CYP (pbuds = 0.016 and pNN = 0.021), compared to the vehicle control (VC). On the contrary, a possible antagonistic action was observed between the components in the mixture GLY + CPF. Growth was not affected in any exposed groups (p > 0.05). There was a clutch effect in the frequency of binucleated erythrocytes (BiN; p = 0.011), total length (TL; p = 0.001) and snout-vent length (SVL; p = 0.031). Biomarkers used in this study are considered important predictive tools for the evaluation of xenobiotics. In this study, we demonstrated genotoxicity of pesticide mixtures under conditions that simulate the real situation of exposure suffered by caiman and other wild species in Argentina.

Effects of cypermethrin (pyrethroid), glyphosate and chlorpyrifos (organophosphorus) on the endocrine and immune system of Salvator merianae (Argentine tegu).

Several geographical areas where Salvator merianae is distributed in Argentina are included in regions with agricultural activity and exposed to pesticide formulations. Some pesticides could affect defense mechanisms being able alter structures of some components of immune and endocrine systems. To assess the potential effects of pesticides in this reptile under seminatural conditions, on the immune system and endocrine responses in S. merianae we analyzed several blood parameters. Total (TWBCC), differential (DWBCC) white blood cells count, heterophils/lymphocytes index (H/L), lobularity index (LI), natural antibodies (NAbs) titres, complement system (CS), and corticosterone concentration were analyzed in animals exposed to a mixture of cypermethrin (25%), glyphosate (66.2%) and chlorpyrifos (48%) formulations. In addition, body size was considered in these analyzes. TWBCC and NAbs revealed lower values in organisms exposed to pesticides respect to a control indicating a possible immunosuppression effect. Besides, the LI showed a greater number of lobes in organism exposed demonstrating symptoms of chronic infection. In addition, we observed a reduced growth in these animals possibly related to a less energy investment in body mass to maintain an active defense against pesticides. Finally, we found high levels of plasma corticosterone in animals exposed to mix formulation that could demonstrate neuroendocrine axis activation. Other parameters like DWBCC, H/L index and activity of CS showed no differences in treated animals respect to control group, which could indicate low sensibility of these parameters to the concentration of pesticides used. Our results provide evidence of the toxic effects of pesticides on different immune system parameters, but also a trade-off among these parameters, corticosterone levels and growth. In this way, we can conclude that the formulated pesticides applied widely and constantly in the areas occupied by S. merianae, would be affecting its immune and endocrine systems and therefore its ability to defend against external agents. This kind of studies is of great interest to know the possible responses of wild species to anthropogenic disturbances such as pesticide contamination.

Evaluation of Stage-Dependent Genotoxic Effect of Roundup® (Glyphosate) on Caiman latirostris Embryos.

The agricultural expansion over the past decades, along with the associated increase in the use of pesticides, represents a high risk for many wild species. Caiman latirostris is a South American caiman with many features that make it highly vulnerable to pesticide exposure. Considering previous finding on the genotoxicity of the glyphosate-based formulation Roundup® in this species, the aim of this study was to evaluate the possible stage-dependent effect of this compound on C. latirostris embryos through the Comet assay (CA), micronuclei (MN), and nuclear abnormalities (NA) tests. Caiman eggs were exposed to three effective concentrations of Roundup® (750, 1250, 1750 µg/egg) in three different stages of the incubation period (total duration 70 ± 3 days at 31 ± 2 °C) of approximately 23 days each. A statistically significant difference in DNA damage determined by the CA was found between groups exposed to different concentrations of RU (p < 0.05) and the negative control, but no difference was observed among the three stages of exposure within any treatment (p > 0.05). There was no differences in the MN or NA frequencies between the different groups and the negative control (p > 0.05), nor among the different stages within each treatment. The results obtained in this study indicate that RU produce DNA damage on C. latirostris embryos independently of the developmental stage where the exposure occurs, implying an important risk for the species during all its period of development, when pesticide application is at maximum rate.

Total and differential white blood cell counts in Caiman latirostris after in ovo and in vivo exposure to insecticides.

Agricultural activities associated mainly with soybean crops affect the natural environment and wildlife by habitat destruction and the extensive use of agrochemicals. The aim of this study was to evaluate immunotoxic effects of the insecticides cypermethrin (CYP) and endosulfan (END) in Caiman latirostris analyzing total blood cell count (TWBC) and differential white blood cell count (DWBC) after in ovo and in vivo exposure. Eggs (in ovo) and hatchlings (in vivo) from nests harvested in natural habitats were artificially incubated and reared under controlled conditions in the Proyecto Yacaré (Gob.Santa Fe/MUPCN) facilities. Exposure of embryos was performed by topication on the eggshell during the first stage of development. The treatments were distilled water (negative control; NC), ethanol (vehicle control; VC), four groups treated with different concentrations of CYP and four groups with END. In vivo exposure was performed by immersion; treatments were NC, VC, two groups exposed to CYP and two to END. After embryonic exposure to the insecticides, no differences were found in TWBC or DWBC among the neonates exposed to pesticides versus controls. In the in vivo scenario, similar results were obtained for TWBC, but DWBC data showed differences between NC hatchlings and CYP-1 hosts for heterophil, lymphocyte and monocyte levels, and between NC and END-1 hosts for lymphocyte and monocyte levels. Research on the effects of pesticide exposure on this species is of special interest not only to assess the impact on caiman populations, but also to further characterize the species as a potential sentinel of ecosystem health.

Evaluación de estrés oxidativo en pacientes con Lupus Eritematoso Sistémico y su posible relación con la exposición ambiental a agroquímicos / Assessment of oxidative stress in patients with Systemic Lupus Erythematosus and its possible relationship with environmental exposure to agrochemicals

Los agroquímicos son un método efectivo para controlar especies perjudiciales a los intereses del hombre, pero aplicados indiscriminadamente provocan diversos impactos a nivel ambiental y en la salud. Uno de los mecanismos más importantes por el cual los agroquímicos se ponen en contacto directo con las poblaciones de zonas periurbanas es mediante la deriva. El Lupus Eritematoso Sistémico (LES) es una enfermedad autoinmune que posee un amplio rango de presentaciones clínicas y severidad. Si bien su etiología es desconocida, se la asocia con diversas causas ambientales dentro de las que podrían estar presentes los agroquímicos. El objetivo de este estudio fue evaluar, el posible daño oxidativo generado por exposición ambiental a mezclas de agroquímicos en pacientes con LES y personas sanas que residen en zonas rurales expuestas a mezclas de plaguicidas y contrastar los resultados con pacientes y personas sanas de zonas urbanas no expuestas. Se evaluaron 44 pacientes con diagnóstico de LES y 58 personas sanas (controles), se utilizaron como marcadores: catalasa (CAT), superóxido dismutasa (SOD), la relación glutation oxidado/reducido (GSH/GSSG) y lipoperoxidación (TBARS). Se encontraron diferencias estadísticamente signifcativas para TBARS y SOD al comparar los resultados del grupo LES urbano con su control y el grupo LES rural con el suyo (p<0,01 en ambos). Al realizar las comparaciones entre LES urbano y LES rural se halló un incremento en TBARS del 18,3% (p=0,014). Esta investigación demuestra la importancia de la utilización de biomarcadores de daño oxidativo en el seguimiento clínico de pacientes con LES, con el fn de establecer pautas de tratamiento adecuadas y considerar la relación entre la exposición ambiental a plaguicidas y el aumento de daño oxidativo en pacientes con LES.

The agrochemicals are an effective method to control harmful species of interests to humans, but when they are applied indiscriminately, they can cause environmental and health damage to people. Systemic Lupus Erythematosus (SLE) is an autoimmune disease that has a wide range of clinical manifestations and severity. While the etiology of SLE is unknown, it is associated with various environmental causes, being one possibility the exposure to agrochemicals. The aim of this study was to evaluate the possible oxidative damage generated by environmental exposure to agrochemical mixtures in SLE patients and healthy people living in rural areas and compare them with patients and healthy people living in urban, unexposed areas. Forty four patients diagnosed with SLE and 58 healthy individuals (controls) were evaluated, using the following markers: Catalase (CAT), superoxide dismutase (SOD), the oxidized/reduced glutathione ratio (GSH / GSSG) and lipid peroxidation (TBARS). Statistically signifcant differences were found in TBARS and SOD by comparing the results of the urban SLE group with its control and rural SLE group with its control (p <0.01 for both cases). When making comparisons between urban SLE group and the rural SLE group, an increase of 18.3% was found in TBARS (p = 0.014). This research demonstrates the importance of using biomarkers of oxidative stress in the clinical monitoring of patients with SLE, in order to establish guidelines for appropriate treatment and consider the relationship between environmental exposure to pesticides and increased oxidative damage in patients with SLE.

Genotoxicity induced by Roundup® (Glyphosate) in tegu lizard (Salvator merianae) embryos.

Environmental contaminants produce multiple adverse consequences at individual, population and ecosystem levels. High volumes of agrochemicals applied to great variety of crops, together with agricultural expansion, generate great concerns due to the impact for the environment and large risk implicated for wildlife. The lack of data on these threats is striking. The tegu lizard (Salvator merianae) is one of the species that live in environments under contaminant effects. Several characteristics allow proposing this species as a potential sentinel organism for the monitoring of pesticides in their habitat. The present study is the first report about genotoxicity in tegu lizard neonates after embryonic exposure to Roundup® (glyphosate 66.2%). The micronucleus test (MN), nuclear abnormalities (NAs) assay and comet assay (CA) were used as biomarkers of genotoxic effects induced in erythrocytes by topical exposure of the eggs to the glyphosate commercial formulation Roundup® (RU), in laboratory controlled conditions. A total of 96 eggs were distributed in six groups exposed to RU (50, 100, 200, 400, 800, 1600µg/egg), one positive control (PC; 200µg cyclophosphamide/egg) and one negative control (NC; distilled water). No teratogenic effects were observed in any of the exposed or control neonates. A significant increase in DNA damage was observed in all concentrations higher than 100µg/egg with respect to NC (p<0.05). However, no statistical differences were found in the frequencies of MN and NAs in any group exposed to RU compared to the NC. No statistically significant differences were found in the size of the lizards at birth or after six months post-exposure (p>0.05). Our results provide new information about the undesirable effects of the glyphosate-based herbicide formulations RU on this lizard species that inhabits areas permanently exposed to several pesticide formulations. We consider of utmost necessity a strict regulation of the agrochemical application conditions in those environments near to places where wild populations of terrestrial and aquatic species live, in order to minimize the adverse effects on ecosystems.

Biomarkers of oxidative damage and antioxidant defense capacity in Caiman latirostris blood.

Several xenobiotics, and among them pesticides, can produce oxidative stress, providing a mechanistic basis for their observed toxicity. Chronic oxidative stress induces deleterious modifications to DNA, lipids and proteins that are used as effective biomarkers to study pollutant-mediated oxidative stress. No previous report existed on the application of oxidative damage and antioxidant defense biomarkers in Caiman latirostris blood, while few studies reported in other crocodilians were done in organs or muscles of dead animals. The aim of this study was to characterize a new set of oxidative stress biomarkers in C. latirostris blood, through the modification of conventional techniques: 1) damage to lipids by thiobarbituric acid reactive substances (TBARS), 2) damage to DNA by comet assay modified with the enzymes FPG and Endo III, and 3) antioxidant defenses: catalase, superoxide dismutase and glutathione; in order to apply them in future biomonitoring studies. We successfully adapted standard procedures for CAT, SOD, GSH and TBARS determination in C. latirostris blood. Calibration curves for FPG and Endo III showed that the three dilutions tested were appropriate to conduct the modified comet assay for the detection of oxidized bases in C. latirostris erythrocytes. One hour of incubation allowed a complete repair of the damage generated. The incorporation of these biomarkers in biomonitoring studies of caiman populations exposed to xenobiotics is highly important considering that this species has recovered from a serious endangered state through the implementation of sustainable use programs in Argentina, and represents nowadays a relevant economic resource for many human communities.

Immunotoxicity of commercial-mixed glyphosate in broad snouted caiman (Caiman latirostris).

The expansion and intensification of agriculture during the past 50 years is unprecedented, and thus environmental problems have been triggered at different scales. These transformations have caused the loss of habitat and biodiversity, and disruption of the structure and functioning of ecosystems. As a result of the expansion of the agricultural frontier in the recent past, many areas of the natural geographic distribution of the local wildlife, among them crocodilians and particularly the broad snouted caiman (Caiman latirostris), are being exposed to contaminants. The present study was designed to evaluate the effect of commercially-mixed glyphosate (RU) on some parameters of the immune system of C. latirostris. Two groups of caimans were exposed for two months to different concentrations of RU recommended for its application in the field, while one group was maintained as an unexposed control. The RU concentration was progressively decreased through the exposure period to simulate glyphosate degradation in water. After exposure, total and differential white blood cell (WBC), and complement system activity (CS) were determined. In addition, the animals were injected with a solution of lipopolysaccharide (LPS) from Escherichia coli to trigger an immune response and evaluate the parameters associated with it. The results showed that an effect of the herbicide on CS was observed, as animals exposed to RU showed a lower CS activity than animals from the negative control (NC) but not in total WBC. In the case of leukocyte population counts, differences were only found for heterophils and lymphocytes.

Evaluación de genotoxicidad a través de la frecuencia de Micronúcleos en eritrocitos de Piaractus mesopotamicus (pacúes) expuestos in vivo a nanopartículas de plata / Assessment of genotoxicity by Micronucleus frequency in Piaractus mesopotamicus erythrocytes exposed to silver nanoparticles in vivo

El uso creciente de nanomateriales en productos industriales y de consumo ha incrementado la preocupación mundial respecto a sus posibles efectos adversos en los sistemas biológicos. Como consecuencia de la falta de un marco legislativo y la ausencia de un consenso sobre los protocolos experimentales, las investigaciones ecotoxicológicas se llevan a cabo a un ritmo mucho más lento que la producción de nuevas nanopartículas. Por esta razón, existe una necesidad creciente de realizar estudios que aporten conocimiento sobre el riesgo de estos contaminantes emergentes de propiedades únicas. El objetivo del presente trabajo fue evaluar la frecuencia de micronúcleos (FMN) en eritrocitos de ejemplares juveniles de pacú (Piaractus mesopotamicus) expuestos a nanopartículas de plata (Nano-Ag) a las concentraciones de 0 μg·L-1 (control); 2,5 μg·L-1; 10 μg·L-1; y 25 μg·L-1, durante 24 horas. Se observó que la FMN se incrementó significativamente (p<0,01) en la concentración de 25 μg·L-1, mientras que no hubo diferencias significativas entre los grupos expuestos a 2,5 y 10 μg·L-1 y el control. Estos resultados sugieren que los eventos aneugénicos o clastogénicos podrían representar un posible mecanismo de toxicidad de Nano-Ag en esta especie.

The growing use of nanomaterials in consumer and industrial products has aroused global concern about possible adverse effects on biological systems. Due to the lack of a regulation framework and the absence of a consensus on the experimental protocols, ecotoxicological investigations are carried out much slower than the production of new nanoparticles. For this reason, there is a growing need for studies that provide knowledge about the risk of these emerging contaminants of unique properties. The objective of the present study was to evaluate the frequency of micronuclei (FMN) in erythrocytes of juvenile Piaractus mesopotamicus exposed to silver nanoparticles (nano-Ag; Nanotek SA) at concentrations of 0 μg·L-1 (control); 2.5 μg·L-1; 10 μg·L-1; and 25 μg·L-1, for 24 hours (n = 10 per treatment). The FMN show a significant increase (p <0.01) in fish exposed to 25 μg·L-1 of Nano-Ag, while there were no significant differences among the groups exposed to 2.5 and 10 μg·L-1 with the control. These results suggest that the aneugenics or clastogenics events may represent a possible mechanism of toxicity of Nano-Ag in this specie.

Neurologic dysfunction and genotoxicity induced by low levels of chlorpyrifos.

Chlorpyrifos (CPF) is an organophosphorus cholinesterase inhibitor widely used as an insecticide. Neuro and genotoxicity of this agent were evaluated following daily subcutaneous injections at 0.1, 1 and 10mg/kg or its vehicle to laboratory rats during one week, at the end of which somatosensory evoked potentials (SEP) and power spectrum of the electroencephalogram (EEGp) were recorded under urethane anesthesia. In another group of conscious animals, auditory startle reflex (ASR) was evaluated followed, after euthanasia, with measurements of plasma B-esterases, and genotoxicity with the alkaline comet assay (ACA) at the same CPF doses. The results indicated a CPF dose related inhibition of B-esterases. Enhanced inhibition of the ASR by a subthreshold pre-pulse was observed at all doses and ACA showed a significant higher DNA damage than vehicle controls in animals exposed to 10mg/kg CPF. A trend to higher frequencies of EEGp and an increase in amplitude of the first negative wave of the SEP were found at all doses. The first positive wave of the SEP decreased at the CPF dose of 10mg/kg. In summary, a shift to higher EEG frequencies and alterations of somatosensory and auditory input to the central nervous system were sensitive manifestations of CPF toxicity, associated with depression of B-esterases. The changes in electrical activity of the cerebral cortex and DNA damage observed at doses that do not elicit overt toxicity may be useful in the detection of CPF exposure before clinical signs appear.

Spontaneous genetic damage in the tegu lizard (Tupinambis merianae): the effect of age.

Several studies indicate that certain factors such as age, sex or nutritional status among others, may affect the level of DNA damage, both induced and spontaneous, so it is very important to consider them for a more accurate interpretation of the findings. The aim of this study was to analyze the influence of age, sex, and nest of origin on spontaneous genetic damage of Tupinambis merianae determined by the comet assay (CA) and the micronucleus (MN) test, in order to improve reference data for future in vivo studies of xenobiotics exposure in this species. Sixty-five tegu lizards of three different ages: newborns (NB), juveniles (JUV) and adults (AD), both sexes and from different nests of origin were used. Blood samples were collected from the caudal vein of all animals and the MN test and CA were applied on peripheral blood erythrocytes to determine basal frequency of MN (BFMN) and basal damage index (BDI). The comparison between age groups showed statistically significant differences in the BFMN and BDI (p<0.05). NB animals showed significantly higher BDI values in relation to JUV and AD (p<0.016), but no statistically differences were found between the latter two. NB showed lower BFMN respect to other age groups, being statistically significant only when compared to AD (p<0.016). BFMN or BDI showed no statistically significant differences between sexes or nests of origin (p>0.05). A weak negative relationship was found only between BFMN and weight of NB tegu lizard (p=0.014; R(2)=0.245). Basal values of genetic damage obtained with both biomarkers in the tegu lizard evidenced that age is an intrinsic factor that should be taken into account to avoid misunderstanding of the results in future biomonitoring studies.

Detection and characterization of phospholipase A(2) (PLA(2)) in Caiman latirostris and Caiman yacare plasma.

Reptiles have proven to have a versatile and efficient nonspecific immune system adapted to the environments in which they commonly live. Phospholipase A(2) (PLA(2)) is important hydrolytic enzyme involved in the regulation of specific types of messengers, with significant roles in the innate immune response. A number of agents that exert effects on cellular receptors emit a series of signals leading to the increased activity of PLA(2). Phospholipase A(2) has been identified and characterized in temperature, plasma concentration, and kinetic dependence in two species of caiman. The results of these studies suggest that the high PLA(2) activities observed in caiman plasma may be an important component of a well-developed innate immunity. Based on the knowledge of their properties, this powerful immunologic component should be evaluated as a possible application in the veterinary or even human therapeutic industry. Additionally, this is another reason to consider these animals excellent models for the study of immune phylogenetic mechanisms.