G.L. Poletta

Genotoxicity of the herbicide formulation Roundup® (glyphosate) in broad-snouted caiman (Caiman latirostris) evidenced by the Comet assay and the Micronucleus test

The genotoxicity of pesticides is an issue of worldwide concern. The present study was undertaken to evaluate the genotoxic potential of a widely used herbicide formulation, Roundup (glyphosate), in erythrocytes of broad-snouted caiman (Caiman latirostris) after in ovo exposure. Caiman embryos were exposed at early embryonic stage to different sub-lethal concentrations of Roundup (50, 100, 200, 300, 400, 500, 750, 1000, 1250 and 1750microg/egg). At time of hatching, blood samples were obtained from each animal and two short-term tests, the Comet assay and the Micronucleus (MN) test, were performed on erythrocytes to assess DNA damage. A significant increase in DNA damage was observed at a concentration of 500microg/egg or higher, compared to untreated control animals (p<0.05). Results from both the Comet assay and the MN test revealed a concentration-dependent effect. This study demonstrated adverse effects of Roundup on DNA of C. latirostris and confirmed that the Comet assay and the MN test applied on caiman erythrocytes are useful tools in determining potential genotoxicity of pesticides. The identification of sentinel species as well as sensitive biomarkers among the natural biota is imperative to thoroughly evaluate genetic damage, which has significant consequences for short- and long-term survival of the natural species.

Caiman latirostris (broad-snouted caiman) as a sentinel organism for genotoxic monitoring: basal values determination of micronucleus and comet assay.

Caiman latirostris is one of the two crocodilian species that inhabit Argentina. In this country, as a consequence of agricultural frontiers expansion during the last years, many areas of the geographic distribution of the broad snouted caiman overlap with regions of intensive agricultural activity. Contaminants released to the environment may induce genetic alterations in wildlife, which could lead to mutations and/or carcinogenesis. Up to the moment, no studies had been made concerning the possibbility to apply biomarkers of genotoxic evaluation in C. latirostris. The aim of this study was to adapt two widely used genotoxic techniques, the comet assay and the micronucleus test, for their application in C. latirostris and to determine the baseline values in this species, in order to establish its suitability as a sentinel organism for future genotoxic monitoring of environmental pollutants. A total of 41 juvenile caimans of 4 months old (FMO) and 10 months old (TMO) were used. Genotoxic techniques were applied on peripheral blood erythrocytes introducing the necessary modifications required by the material, which are presented here. Our results show that baseline values of DNA damage are quite stable among juvenile caimans (MN: FMO animals 0.87+/-0.74 and TMO animals 1.04+/-0.92; DI: FMO animals 103.40+/-3.36 and TMO animals 120.08+/-11.33), being independent of the nest of origin, sex and size of the animals and confirm the potential value of both short term tests as accurate screening tools for the evaluation of genotoxic agents in C. latirostris. This is the first reference to the application of genotoxic techniques on C. latirostris and the second in crocodilians. Data provided here will be useful for future studies involving the biomonitoring of natural regions where C. latirostris occurs, employing this species as a sentinel organism for genotoxic assessment of environmental pollutants.

Chapter Two – Pesticides Used in South American GMO-Based Agriculture: A Review of Their Effects on Humans and Animal Models

In South America, the incorporation of genetically modified organisms (GMO) engineered to be resistant to pesticides changed the agricultural model into one dependent on the massive use of agrochemicals. Different pesticides are used in response to the demands of the global consuming market to control weeds, herbivorous arthropods, and crop diseases. Here, we review their effects on humans and animal models, in terms of genotoxicity, teratogenicity, and cell damage. We also stress the importance of biomarkers for medical surveillance of populations at risk and propose the use of biosensors as sensitive resources to detect undesirable effects of new molecules and environmental pollutants. The compatibility of glyphosate, the most intensively used herbicide associated to GMO crops, with an integrated pest management for soybean crops, is also discussed.

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.

Comet assay in gill cells of Prochilodus lineatus exposed in vivo to cypermethrin.

Agricultural chemicals can induce genetic alterations on aquatic organisms that have been associated with effects on growth, reproduction and population dynamics. The evaluation of DNA damage in fish using the comet assay (CA) frequently involves the utilization of erythrocytes. However, epithelial gill cells (EGC) can be more sensitive, as they are constantly dividing and in direct contact with potentially stressing compounds from the aquatic environment. The aim of the present study was to evaluate (1) the sensitivity and suitability of epithelial gill cells of Prochilodus lineatus in response to different genotoxic agents through the application of the CA, (2) the induction of DNA damage in this cell population after in vivo exposure to cypermethrin. Baseline value of the CA damage index (DI) for EGC of juvenile P. lineatus was 144.68±5.69. Damage increased in a dose-dependent manner after in vitro exposure of EGC to methyl methanesulfonate (MMS) and H2O2, two known genotoxic agents. In vivo exposure of fish to cypermethrin induced a significant increase in DNA DI of EGC at 0.150μg/l (DI: 239.62±6.21) and 0.300μg/l (270.63±2.09) compared to control (150.25±4.38) but no effect was observed at 0.075μg/l (168.50±10.77). This study shows that EGC of this species are sensitive for the application of the CA, demonstrating DNA damage in response to alkylation (MMS), oxidative damage (H2O2), and to the insecticide cypermethryn. These data, together with our previous study on DNA damage induction on erythrocytes of this species, provides useful information for future work involving biomonitoring in regions where P. lineatus is naturally exposed to pesticides and other genotoxic agents.

Effects of in vivo exposure to Roundup® on immune system of Caiman latirostris

Abstract The present study aimed to evaluate the effect of Roundup® (RU, glyphosate-based formulation) on some parameters of the immune system and growth of Caiman latirostris. Seventy-two caimans (20-day-old) from Proyecto Yacaré (Gob. Santa Fe/MUPCN) were used. Two groups were exposed for 2 months to different concentrations of RU (11 or 21 mg/L; taking into account the concentration recommended for its application in the field), while one group was maintained as control. The RU concentration was progressively decreased through the exposure period to simulate glyphosate degradation in water. Animals were measured and weighed at the beginning and end of the experiment, and blood samples taken after exposure to determine total and differential white blood cell (WBC) counts as well as total protein concentration (TPC), and for performing protein electrophoresis. The results showed that, compared against control hosts, there was a decrease in WBC counts, a higher percentage of heterophils, a higher TPC (with a low percentage of F2 protein fraction), and a negative effect on growth in the young caimans exposed to RU. These results demonstrate that in vivo exposure to RU induced alterations in the selected immune parameters, plasma proteins, and growth of caimans, thereby providing relevant information about the effects of this type of pesticide in this important species in the Argentinian wetlands.

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.

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.

Micronuclei and other nuclear abnormalities on Caiman latirostris (Broad-snouted caiman) hatchlings after embryonic exposure to different pesticide formulations

This study aimed to evaluate the embryotoxicity and genotoxicity of pesticide commercial formulations widely used in soybean crops through the Micronucleus (MN) test and other Nuclear Abnormalities (NAs) in erythrocytes of broad-snouted caiman (Caiman latirostris), exposed by topical application through the eggshell. Embryos were exposed (during all incubation: 70 days approximately) to sub-lethal concentrations of two glyphosate formulations PanzerGold® (PANZ) and Roundup® Full II (RU) (500, 750, 1000µg/egg); to the endosulfan (END) formulation Galgofan® and the cypermethrin (CYP) formulation Atanor® (1, 10, 100, and 1000µg/ egg). Blood samples were taken at the moment of hatching from the spinal vein for the application of the MN test and analysis of other NAs in erythrocytes, as markers of genotoxicity. Results indicated a significant increase in the frequency of MN for PANZ1000, END 10, CYP 1 and CYP 100 (p<0.05), and in the frequency of other NAs including Buds: END 100, 1000 and CYP 10 (p<0.05), eccentric nuclei: END 1, 10, 1000, CYP 10, 100, 1000 (p<0.01) and END 100 (p<0.05), notched nuclei: END 1, 10 (p<0.01) and END 1000, CYP 10, 100,1000 (p<0.05), and total nuclear abnormalities: END 1, 10, 100, 1000, CYP 10, 100 and 1000 (p<0.01), and the positive control (PC) (p<0.05), compared with the negative control. It was demonstrated a concentration dependent-effect in MN frequency only for PANZ (R2=0.98; p<0.01). Our study demonstrated that commercial formulations of pesticides induced genotoxic effects on C. latirostris, and NAs are a good indicator of genotoxicity in this specie.