Determinación de la frecuencia de micronúcleos en eritrocitos de Bufo arenarum que habitan ambientes urbanizados / Determination of micronuclei frequency in erythrocytes of Bufo arenarum inhabiting urbanized environments

Los anfibios son constituyentes integrales de ecosistemas naturales y agrícolas, y debido a su sensibilidad pueden ser utilizados como bioindicadores. La frecuencia de micronúcleos se emplea como un biomarcador que evalúa la respuesta a agentes genotóxicos. El objetivo del trabajo fue determinar y comparar la frecuencia de micronúcleos en eritrocitos de Bufo arenarum que habitan ambientes antrópicos de la ciudad de Río Cuarto. Se escogieron tres sitios modificados: un lago urbano Lago Villa Dálcar (33º06´S-64º22´W) y dos sitios de cultivo denominados Cultivo1 (33º05'S-64º26'W) y Cultivo 2 (33º05'S-64º25'W). Como control se escogió un área ubicada en las sierras de Comechingones, en el Campo Experimental Las Guindas (32º35'S- 64º42'W), pertenecientes a la UNRC. En cada sitio se recolectaron ocho individuos utilizando trampas de caída y relevamientos por encuentros visuales. A cada individuo se le extrajo sangre de la vena angularis inmediatamente después de la captura. Se realizaron dos extendidos por cada individuo, se dejaron secar al aire por 24 hs, luego se fijaron con metanol por 20 minutos y se colorearon con Giemsa al 5% durante 10 minutos. Posteriormente los frotis fueron analizados con microscopio (1000X) y se tomaron fotografías con cámara digital. La frecuencia de micronúcleos fue contabilizada para 4000 células por individuo. Se realizaron comparaciones utilizando el test de Kruskal-Wallis. Las frecuencias de micronúcleos encontradas son: Las Guindas: 0,17 ± 0,23; Cultivo1: 0,78 ± 0,28; Cultivo 2: 0,61 ± 0,45 y Villa Dálcar: 0,76 ± 0,47. Existen diferencias significativas entre el control y los demás sitios (p< 0,01). Los resultados obtenidos demuestran la relevancia en la aplicación del test de micronúcleos in vivo en anfibios anuros para comparar distintos ambientes. Estos resultados indicarían la necesidad de nuevas investigaciones y de un monitoreo más prolongado en el tiempo para estos ambientes, incluyendo por ejemplo otros tipos de biomarcadores, así como otras posibles especies representativas de la región.

Amphibians are integral constituents of natural and agricultural ecosystems, and because of their sensitivity can be used as bioindicators. Micronuclei test is used as a biomarker for determining the response to genotoxic agents. The purpose of this study was to determine and compare the frequency of micronuclei in peripheral blood erythrocytes of Bufo arenarum toads that inhabiting anthropic environments for the city of Rio Cuarto. Organisms used in the MN test were collected from three sites modified: an urban lakeVilla Dalcar (33º06´S-64º22´W) and two cultivation sites called Crop 1 (33º05'S-64º26'W) and Crop 2 (33º05'S-64º25'W). As a control site, an area in the mountains of Comechingones, Las Guindas (32º35'S-64º42'W), belonging to the UNRC was sampled. At each site, eight individuals were collected using pitfall traps and by visual encounter surveys. To each individual is bled from the vein angularis immediately after capture. We performed two extended for each individual, air-dried for 24 hours, then fixed with methanol for 20 minutes and stained with 5% Giemsa for 10 minutes. Then the smears were analyzed with microscope (1000X) and took pictures with digital camera. For each individual the frequency of micronuclei for 4000 cells was recorded. Comparisons were made using the Kruskal-Wallis test. Micronuclei frequencies found are: Las Guindas: 0.17± 0.23; Crop 1: 0.78 ± 0.28, Crop 2: 0.61 ± 0.45 and Villa Dalcar: 0.76 ± 0.47. There are significant differences between the control and the other sites (p< 0.01). These findings show the relevance in the application of micronucleus test in vivo for anuran amphibians to compare different environments. These results suggest the need for further research and monitoring longer time for these environments, including such other biomarkers, as well as other possible species representative of the region.

Water-balance response of Rhinella arenarum (Hensel, 1867) tadpoles to graduated increase in environmental osmolarity / Resposta de Rhinella arenarum larvae a meio electrolítico e não electrolítico com aumento escalonado da osmolaridade

The water balance and the upper limit of osmotic tolerance of premetamorphic Rhinella arenarum larvae (Gosner's stage 26) was evaluated after semistatic incubation in electrolyte (NaCl) and non-electrolyte (mannitol) media following a protocol of progressively increased osmotic pressure. Wet and dry weights were measured to calculate the water content as a derived variable indicative of the hydric balance. Statistical analysis was performed using univariate and integrated multivariate analysis. Tadpoles survived in electrolyte and non-electrolyte solutions up to 200 mOsm. The discriminant function was the best tool to describe the responses of the animals to external environmental stress under experimental conditions. The results were compared with those obtained in previous studies using a protocol of acute exposure to the same media used in this study. It was concluded that a) multivariate analysis is an appropriate approach to describe the responses of tadpoles to changes in the environmental physicochemical parameters, and b) progressive and acute acclimation to the experimental solutions induced similar responses.

Avaliou-se o balanço hídrico e o limite superior de tolerância osmótica em larvas pré-metamórficas do Rhinella arenarum (etapa 26 de Gosner) sob condições de incubação semiestáticas, num meio eletrolítico (NaCl) e não eletrolítico (manitol), seguindo um protocolo de aumento progressivo da pressão osmótica do meio. A quantificação das respostas se efetuou por meio da medição dos valores de peso úmido e seco e do cálculo, a partir destes, do conteúdo de água, como variável derivada indicativa do equilíbrio hídrico. A análise estatística foi realizada usando análise univariada e multivariada. As larvas conseguiram sobreviver em soluções eletrolíticas e não eletrolíticas até 200 mOsm. A função discriminante foi a melhor ferramenta para descrever as respostas dos animais ao estresse osmótico ambiental. Os resultados foram comparados com os obtidos em estudos anteriores, usando um protocolo de exposição aguda aos mesmos meios de incubação usados neste estudo. Concluiu-se: a) a análise multivariada é a aproximação adequada para descrever as respostas das larvas às mudanças nos parâmetros físico-químicos do seu meio; e b) tanto a aclimatação progressiva, como as exposições agudas às soluções experimentais induziram as respostas semelhantes.

Humoral immune alterations caused by lead: studies on an adult toad model

There is evidence that environmentalmetal levels affect the immune function. In the particular case of the impact of heavy metals, information available suggests that the immune system is a target for low-dose Pb exposure. Among vertebrates it was shown that amphibians are capable of forming antibodies against a variety of antigens, causing several responses such as anaphylactic response and rejectinggrafts. In this study, the production of antibodies was assessed against sheep red blood cells (SRBC) in the anuran Bufo arenarum after six weekly injections of sublethal doses of lead (50 mg.kg-1, as lead acetate). Natural antibodies (natural heteroagglutinins)were also quantified against SRBC. Both assessments were carried out employing an ELISA method developedto this end, measuring absorbance (A). For natural anti-SRBC antibodies in both control (C) and Pb treated (T) toads, there was a non significant tendency to increase the initial absorbances (C initial: 0.69+0.39 A; T initial: 0.54+0.30 A), relative to those registered at the end of the experiments (C final: 0.89+0.49 A; T final: 0.76+0.31A); the T/C ratios also did not show changes. The only significant difference was found between initial and final samples from lead-treated toads (p<0.014). The immune anti-SRBC antibody levels of toads immunized with SRBC showed a significant lower increase (p<0.05) in lead-treated animals (T final: 0.66+0.36 A), as compared to control toads (C final: 0.91+0.50 A) at the end of the experiment. It was thus concluded thatthe changes due to the assayed doses of Pb in the levels of antibodies cannot be explained on the basis of only one singleaction mechanism of the metal, but as the result of a conjunction of effects over different immunocompetent cell subpopulations. These different responses suggest that factors affecting animals exposed to a foreign stimulus are different from those influencing the response of wild animals.

Existe evidencia de que los niveles de metal ambientales afectan la función inmune. En el caso particular del impacto de metales pesados, la información disponible sugiere que el sistema inmune es un blanco para la exposición a bajas dosis de Pb. Entre los vertebrados, se ha mostrado que los anfibios son capaces de formar anticuerpos contra una variedad de antígenos, que causan diversasrespuestas, tales como respuesta anafiláctica y rechazo de injertos. En este estudio, la producción de anticuerpos fue evaluada contra eritrocitos de oveja (EO) en el anuro Bufo arenarum, luego de seis inyecciones semanales de dosis subletales de plomo (50 mg.kg-1, como acetato de Pb). Los anticuerpos naturales (heteroaglutininas naturales) fueron también cuantificados contra EO. Ambas evaluaciones fueron llevadas a cabo empleando un método de ELISA desarrollado a este fin, midiendo laabsorbancia (A). Para los anticuerpos anti-EO naturales, tanto en sapos controles (C) como en sapos tratados con Pb (T), hubo una tendencia significativa a incrementar las absorbancias iniciales (C inicial: 0,69+0,39 A; T inicial: 0,54+0,30 A); la relación T/C tampoco mostró cambios. La única diferencia significativa se encontró entre las muestras inicial y final de los sapos tratados con plomo (p<0,014). Los niveles de anticuerpos anti-EO inmune de sapos inmunizados con EO mostraron un bajo incrementosignificativo (p<0,05) en los animales tratados con plomo (T final: 0,66+0,36 A), al compararse con sapos control (C final: 0,91+0,50 A) al final del experimento. De este modo, se concluye que los cambios debidos a las dosis analizadas de Pb en los niveles de anticuerpos no pueden explicarse sólo sobre la base de un único mecanismo de acción del metal, sino como resultadode una conjunción de efectos sobre diferentes subpoblaciones de células inmunocompetentes. Estas diferentes respuestas sugieren que los factores que afectan los animales expuestos a un estimulo externo son diferentes...