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El uso de biomarcadores hematológicos es importante para poder evaluar riesgos ambientales y la exposición a sustancias químicas potencialmente tóxicas y así poder desarrollar medidas que sirvan como señales de alarma temprana en ambientes contaminados. El objetivo de este trabajo fue determinar biomarcadores hematológicos en el sapo común Bufo (Rhinella) arenarum que proporcionen información para el diagnóstico de la salud de distintos ecosistemas de la provincia de Córdoba. Un total de 93 individuos adultos de Bufo (Rhinella) arenarum fueron capturados en cuatro sitios de la provincia de Córdoba y distintas variables ambientales del agua fueron medidas en cada sitio. Las muestras de sangre se obtuvieron mediante la punción de la vena angularis. Luego fueron teñidas con la tinción diferencial May Grünwald-Giemsa y fueron observadas mediante microscopio Zeiss Primo Star iLED para realizar el conteo de glóbulos blancos. Las variables ambientales del agua mostraron diferencias estadísticamente significativas entre las poblaciones, se registraron valores menores en “Alpa Corral”, mientras que “Villa Dálcar” presentó los mayores valores para dichas variables. No se encontraron diferencias estadísticamente significativas entre localidades en el recuento de glóbulos blancos (cada 1000 eritrocitos); sin embargo, en “Villa Dálcar” se registraron los ejemplares con mayor cantidad de leucocitos en el conteo de glóbulos blancos (425,48 ± 481,34). Mientras que para la fórmula leucocitaria, los linfocitos fueron las células más abundantes para los ejemplares de todos los sitios; encontrándose diferencias estadísticamente significativas entre cada localidad y “Alpa Corral”. Los ejemplares de “Villa Dálcar” presentaron diferencias estadísticamente significativas en relación a los monocitos y basófilos, registrándose altos valores para ambas células en los ejemplares de este sitio; mientras que en los ejemplares de “Cultivo” las diferencias se encontraron en eosinófilos y la relación neutrófilos/linfocitos. Estos resultados revelaron que de las cuatro localidades analizadas, “Villa Dálcar” representa la más afectada por las actividades antrópicas. Estas variaciones registradas en los parámetros hematológicos podrían considerarse evidencia de la posible presencia de agentes contaminantes en el lago “Villa Dálcar”.

Hematologic biomarkers are important to assess the environmental and health risks of exposure to potentially toxic chemicals, and for developing measures that serve as early warning signals in polluted areas. The aim of this study was to determine hematologic biomarkers of common toad Bufo (Rhinella) arenarum that provide information for the diagnosis of the health of ecosystems of Córdoba province. A total of 93 adult individuals of Bufo (Rhinella) arenarum were collected from four sites of Córdoba Province and hydric environmental variables were measured on each site. Blood samples were obtained by angularis vein puncture, smears of fresh blood were stained with May Grünwald-Giemsa and observed by using a microscope Carl Zeiss Primo Star (Pack 5), for white blood cell count. Water environmental variables showed statistically significant differences among localities, with the lowest values in “Alpa Corral” and highest values in “Villa Dálcar”. There were no statistically significant differences among individuals of different sites for white blood cells count. However, individuals from "Villa Dálcar" showed the highest number of white cells (425.48 ± 481.34). In the leukocyte formula, lymphocytes were most abundant white blood cells in individuals from all study sites. The blood parameters of toads from all sites showed statistically significant differences with individuals from “Alpa Corral”. "Villa Dálcar" Individuals exhibited significant differences in relation to monocytes and basophils with the highest values for both cell types. “Cultivo” locality showed significant differences for eosinophils and for the relationship neutrophils/ lymphocytes. These results revealed that “Villa Dálcar” represent the site most affected by human activities. In particular, changes recorded in hematological parameters could be giving evidence of the possible presence of contaminants in Lake “Villa Dálcar”.

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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.

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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...

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Lead is a multiple-source pollutant, well known for its toxicity, of great risk both for the environment and human health. The main target organs of lead are the hematopoietic, nervous, and renal systems; there are also reports in support of its impairment effects on the reproductive and immune systems. It is well known that most of the metal is accumulated in the blood cells and that many of the deleterious effects are related to its circulating concentrations. These adverse effects have been described not only in humans but also in a number of other vertebrates such as fish and birds. The purpose of the present work was to evaluate the effects of weekly administration of sublethal Pb (as acetate, 50 mg x kg(-1)) during 6 weeks on the profile of the serum proteins and blood cell counts of the adult South American toad, Bufo arenarum (Anura: Bufonidae). The electrophoretic patterns of serum proteins pointed out the presence of four fractions; the metal provoked a significant decrease in both total proteins and albumin fraction; among the globulin fractions, the G3 resulted augmented. These findings may be related to the impact of lead on the toads' hepatic cells and immune system. The number of total red blood cells (RBC) showed a tendency to decrease after the injections of the metal, whereas the number of white blood cells (WBC) increased significantly; the differential leukocyte counts showed a statistically significant increase in the absolute number and in the relative percentage of blast-like cells. The decrease in RBC was attributed to the negative impact of the metals on the hemoglobin synthesis. The increasing of the WBC counts may be interpreted as a consequence of the induction of proliferation of pluripotential hematopoietic cells.

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Lead (Pb) is a ubiquitous environmental pollutant, widely distributed, representing a high toxicological and ecotoxicological risk. Several morphological, functional, and biochemical parameters have been proposed as biomarkers of effect and exposure to Pb. The information related to adverse effects of Pb is not abundant for adult amphibians. These animals are of interest, because during their development they move from aquatic to terrestrial habitats, which may be polluted by the metal since they are receptors of products generated by anthropogenic activities. Previous studies carried out on the adult South American toad Bufo arenarum (Amphibia, Anura) showed that it has a high tolerance to lead and studied the effect of sublethal doses of the metal on the erythrocyte osmotic fragility and delta-ALAD activity. It was also shown that after a single injection of Pb, a significant increase in the number of reticulocytes was produced, suggesting the suitability of those cell counts as a biomarker of exposure to the metal; its impact on the immune system of the toads was also studied. In this work we extend our early studies on the same species evaluating the chronic effect of sublethal Pb (equivalent to 5.6% of the 120-h LD-50) on free erythrocyte protoporphyrin (FEP) and blood Pb and delta-ALAD activity; blood lead was positively associated with a significant decrease in the enzyme activity and to an increase in the FEP level. Pb concentration in target organs (liver, spleen, femur, and kidney) and the total cumulated amount as well as its impact over the mass of those organs were also determined. In addition, the magnitude of the possible depuration through urine and intestine was evaluated. Our results showed that FEP, delta-ALAD, and blood Pb are reliable biosensors of chronic metal intoxication, the former being the marker with the highest sensitivity.

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The changes in the serum levels of the sexual steroids estradiol-17beta (E(2)), testosterone (T), dihydrotestosterone (DHT), and progesterone (P) in Bufo arenarum females were determined by radioimmunoassay (RIA) during 3 consecutive cycles (1999-2001). The serum concentrations of T and DHT, which showed a close parallelism during the annual reproductive cycle, exhibited the highest levels during the preovulatory period, when oogenesis is advanced, while lowest serum levels of these hormones were found during the ovulatory period. The data obtained for E(2) showed a pattern contrary to that determined for androgens. The maximum E(2) concentrations detected in the early postovulatory period might be associated with vitellogenesis and follicular growth. Lowest E(2) concentrations were reached during the period in which B. arenarum undergoes its final hibernation stage. Serum P showed a peak during the preovulatoy period, related to the induction of nuclear maturation in full grown oocytes. A strong decrease in the levels of the circulating hormones was observed after ovariectomy. Our results showed that, out of the four hormones examined, T and DHT were the best indicators of ovarian and oviductal stage, as shown by the strong positive correlation found between androgen levels and organ weight, while E(2) showed a weak negative correlation with ovarian and oviductal weight.

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Lead is an element of risk for the environment and human health and has harmful effects that may exceed those of other inorganic toxicants. The immune system is one of the targets of lead. Its immunomodulatory actions depend on the level of exposure, and it has been demonstrated that environmental amounts of the metal alter immune function. Very little information is available regarding the effect of the metal on different aspects of the immune system of lower vertebrates, in particular of amphibians. The aim of this study was to investigate the effect of sublethal lead (as acetate) on the function of polymorphonuclear cells of Bufo arenarum. The results revealed that phagocytic and lytic functions of the adherent blood cells collected from sublethal lead-injected toads and incubated with suspensions of Candida pseudotropicalis were affected negatively. The decrease of the phagocytic activity was correlated with increased blood lead levels (P < 0.0001). Additional information referred to the total and differential leukocyte counts was presented; the only difference found was in the number of blast-like cells that resulted augmented in the samples of lead-injected toads. It was concluded that the evaluation of these parameters might be a reliable tool for the biological monitoring of the immune status of amphibians.

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INTRODUCTION: Immune function assays to screen immunotoxic effects of xenobiotics has recently become of major interest. In the framework of our studies, we standardized methods to quantify the humoral response of an amphibian species (Bufo arenarum, Amphibia, Anura) exposed to sublethal lead (as acetate). METHODS: The levels of agglutinins to heterologous red blood cells (RBC) were measured in serum from adult B. arenarum. Since agglutinin titers were very low, a noncompetitive enzyme-linked immunosorbent assay (ELISA) method was carried out. As toad serum showed marked nonspecific binding, we developed a new ELISA on microtiter plates for the quantitative determination of the heterophile antibodies. The method was based on that described by Hirvonen et al. [Vox Sang. 69 (1995) 341], employing sheep red blood cells (SRBC) sensitized with amphibian antibodies that were transferred to microplates; later the measurement of bound immunoglobulins was performed. Different variables such as the amount of antigen, blocking agents, and other experimental conditions (fixing solution and commercial plates) were studied. Toads (n=22) received a weekly subcutaneous injection of 50 mg/kg lead (acetate) for 6 weeks, and the control ones (n=26) were injected with Na acetate at the same time. RESULTS: The anti-sheep RBC antibodies titers of adult toads were obtained with the improved ELISA method, being the absorbance range 0.12 to 1.58 AU (1/200 diluted serum). Titers from lead-exposed toads were also determined, being the final titers (expressed as (-)x +/-S.E.M.) higher (0.79+/-0.06 AU), than those of Day 0 (0.57+/-0.06) (P<.01). DISCUSSION: It was concluded that the ELISA technique we developed was useful for measuring the humoral immune response in this animal model and that in these preliminary studies, lead showed an immunostimulating action on the humoral immune system.

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The effects of sublethal doses of lead (as acetate) on blood parameters of adult male Bufo arenarum were studied. Toads received one single injection with 10, 25, 50 or 100 mg/kg of body weight, equivalent to approximately 1/90-1/10 of the 120 h-LD50; seven days after the injections, the hematocrit and the blood delta-aminolevulinic acid dehydratase (ALAD) activity were measured. Hematocrit of lead-injected animals did not exhibit significant changes respective to controls that received sodium acetate (range 29.8-38.8%). Blood lead concentrations were positively and significantly correlated with the injected metal doses. Blood ALAD activity declined proportionately to the doses of the metal as well as to its whole blood concentration. Because of its sensitivity and specificity, it was concluded that the activity of delta-ALAD may be adopted as a reliable biomarker of Bufo arenarum experimental lead intoxication.

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The aim of the present investigation was to standardize a method for measuring delta-aminolevulinic acid dehydratase (ALAD) activity in circulating red blood cells of adult Bufo arenarum kept in controlled environmental conditions, and to obtain reference basal values suitable for environmental monitoring of lead exposure. The normal ALAD activity for B. arenarum was 131.86 +/- 14.47 U per liter of red blood cells (n = 38, mean +/- SEM; interval 72.98-236.33). In animals exposed to lead, ALAD activity decreased as lead dose increased.

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The aim of the present investigation was to standardize a method for measuring delta-aminolevulinic acid dehydratase (ALAD) activity in circulating red blood cells of adult Bufo arenarum kept in controlled environmental conditions, and to obtain reference basal values suitable for environmental monitoring of lead exposure. The normal ALAD activity for B. arenarum was 131.86 + 14.47 U per liter of red blood cells (n = 38, mean + SEM; interval 72.98 - 263.33). In animals exposed to lead, ALAD activity decreased as lead dose increased.

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Serum steroid binding properties of mature Bufo arenarum females were studied. Binding data obtained using charcoal adsorption assay and equilibrium dialysis methods indicates a single protein, named Bufo arenarum sex binding protein (Ba SBP), which binds 5 alpha-dihydrotestosterone (DHT), testosterone (T), and estradiol-17 beta (E2) with high affinity (10(-7) M-1 - 10(8) M-1) and fair capacity (10(-6) M). Scatchard plot analysis demonstrated the coexistence of two binding sites. Ba SBP has a sedimentation coefficient of 5.2 S in sucrose gradient centrifugation in low salt and under steady-state conditions. The specificity of this protein, determined by competitive binding experiments, is comparable to human SBP. DHT and T bind with higher affinity than E2. Estriol and estrone competed poorly, while diethylstilbestrol and C21 steroids did not compete. The binding capacity of this protein is under estrogenic control.

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The absence of DNA from the blood serum of the toad Bufo arenarum was confirmed by classical colorimetric techniques and by a new method which allows differentiation between DNA and the glycosaminoglycans (GAG). A component of serum which had some of the properties of DNA was identified as a GAG. This component gave positive reactions in colorimetric assays for DNA, but the absorption spectra were completely different from those of authentic DNA. The compound was also not hydrolyzed at all by treatment with deoxyribonuclease.

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