Effect of cadmium on cellular viability in two species of microalgae (Scenedesmus sp. and Dunaliella viridis).

We determined the effect of several concentrations of cadmium (0, 5, 10, and 20 microg/l) on cellular viability in the microalgae Scenedesmus sp. and Dunaliella viridis, by measuring growth at 0, 24, 48, 72, and 96 h and pigment production at 10 days. Algae were obtained from the Nonvascular Plant Laboratory collection, in the Facultad Experimental de Ciencias, Universidad del Zulia, Venezuela. Growth was measured by cellular counting, while pigment content was evaluated using conventional spectrophotometric techniques. Growth of both species decreased in the exposed cultures comparing with the control, but its behavior was similar, because in both control and exposed cultures, its was observed an adaptive phase in the first hours, as well as a growth phase after 72 h. Cadmium concentrations above 10 microg/l produced an adverse effect on pigment production, depending on the concentration and/or exhibition time. However, even though cadmium inhibited growth and pigment production, levels of both parameters indicated cellular viability, demonstrating the adaptability of the algae cultures when they were exposed to the metal.

Correlation of toxicity with lead content in root tip cells (Allium cepa L.).

The present study determines lead content in onion root tip cells (Allium cepa L.), correlating it with its toxicity. The treatment was carried at 25 +/- 0.5 degrees C using aqueous solutions of lead chloride at 0.1, 0.25, 0.50, 0.75, and 1 ppm for 12, 24, 48, and 72 h. For each treatment, a control where the lead solution was substituted by distilled water was included. After treatment, the meristems were fixed with a mixture of alcohol-acetic acid (3:1) and colored according to the technique of Feulgen. Lead content was quantified by graphite furnace absorption atomic spectrometry. The lead content in the roots ranged from 3.25 to 244.72 microg/g dry weight, with a direct relation with the concentration and time of exposure. A significant negative correlation was presented (r = -0.3629; p < 0.01) among lead content and root growth increment, and a positive correlation (r = 0.7750; p < 0.01) with the induction of chromosomic aberrations. In conclusion, lead is able to induce a toxic effect in the exposed roots, correlated with its content.

Determination of vanadium accumulation in onion root cells (Allium cepa L.) and its correlation with toxicity.

The vanadium is a metal that presents great interest from the toxicological point of view, because of the numerous alterations that can take place in different biological systems. This work evaluated the capacity of vanadium accumulation and its correlation with genotoxic effects in root cells of Allium cepa L. The bulbs were cultivated in renovated filtered water each 24 h, at a temperature of 25 +/- 0.5 degrees C, in darkness and constant aeration. Treatments were carried out under the same experimental conditions, using water solutions of vanadium of 25, 50, 75 and 100 microg/g for 0, 12, 24, 48 and 72 h. A control was carried out where metal solution was substituted by distilled water. After the treatment, the meristems were fixed with alcohol--acetic acid (3:1) and stained according to the technique of Feulgen. The capacity of accumulation was determined by GFAAS. The analysis of the results revealed an accumulation of the metal for all times and concentrations. No correlation was presented among vanadium accumulation, growth and mitotic index; however, positive correlation was given with the induction of chromosomic aberrations. In conclusion, vanadium is able to induce cytotoxic effect in the exposed roots, but only genotoxic effect was correlated with metal accumulation.

Citotoxicidad del cadmio en hepatocitos de ratón albino y sus posibles implicaciones en ambientes tropicales / Cadmium citotoxicity in mice hepatocytes and implications on tropical environments

We analyzed phenotypic, structural and ultrastructural alterations induced by Cd+2 in hepatocytes extracted from Swiss Albino mice. Cadmium was given orally in watery solution of CdCl2 during 100 days at concentrations of 50 ppm, 100 ppm and 150 ppm. In controls, distilled water alone was used. The samples were processed with the paraffin inclusion and hematoxilin-eosin coloration techniques for light microscopy. For transmission electron microscopy we used the conventional technique. We found phenotypic (size and weight differences) and physiologic changes (muscular weakness, unrest); at the structural level we noticed loss of trabecular disposition and of lobulillar architecture, lymphocyte agglomeration, vacuolization, dilatation of sinusoid and central vein, among others. The ultrastructural study evidenced alterations coincident with those seen with light microscopy, which were accentuated with the increase of metal concentration: nucleolus with a high number of fibrillar centers (50 ppm); voluminous lipidic drops in the cytoplasm, loose endoplasmic rough reticulum, citoplasmatic vacuolization, altered lisosomes and peroxisomes (100 ppm); contracted nuclei with condensed cromatine, dilatation of intracellular space and mitochondria, and loss of fibrillar areas (150 ppm). Cadmium produces a toxic effect in the hepatic cells; the effect is more severe at higher concentration, leading to cellular necrosis.

Se realizó un análisis de las alteraciones fenotípicas, estructurales y ultraestructurales inducidas por Cd+2 en hepatocitos de ratón albino suizo. El metal fue suministrado vía oral en solución acuosa de CdCl2 durante 100 días a concentraciones de 50 ppm, 100 ppm y 150 ppm, en los controles la solución de cadmio fue sustituida por agua destilada. Las muestras fueron procesadas utilizando la técnica de inclusión en parafina y teñidas con hematoxilina- eosina para microscopía óptica y por la técnica convencional para microscopía electrónica de transmisión. Identificamos cambios fenotípicos (diferencias entre talla y peso) y fisiológicos (debilidad muscular e intranquilidad); a nivel histológico, pérdida de la disposición trabecular y de la arquitectura lobulillar, focos de aglomerados linfocíticos, vacuolización, dilatación de sinosoides y de la vena central. El estudio ultraestructural señala diversas alteraciones tales como: nucléolo con un elevado número de centros fibrilares (50 ppm); voluminosas gotas de lípidos en el citoplasma, retículo endoplasmático rugoso distendido, vacuolización citoplasmática, lisosomas y peroxisomas alterados (100 ppm); núcleos contraídos con cromatina condensada, dilatación en el espacio intracelular y áreas de pérdida mitocondrial y fibrilar (150 ppm). Sugerimos que el cadmio ejerce un efecto tóxico en las células hepáticas el cual se hace más severo con el aumento de la concentración, llevando a la necrosis celular.

[Cadmium citotoxicity in mice hepatocytes and implications on tropical environments]. / Citotoxicidad del cadmio en hepatocitos de ratón albino y sus posibles implicaciones en ambientes tropicales.

We analyzed phenotypic, structural and ultrastructural alterations induced by Cd+2 in hepatocytes extracted from Swiss Albino mice. Cadmium was given orally in watery solution of CdCl2 during 100 days at concentrations of 50 ppm, 100 ppm and 150 ppm. In controls, distilled water alone was used. The samples were processed with the paraffin inclusion and hematoxilin-eosin coloration techniques for light microscopy. For transmission electron microscopy we used the conventional technique. We found phenotypic (size and weight differences) and physiologic changes (muscular weakness, unrest); at the structural level we noticed loss of trabecular disposition and of lobulillar architecture, lymphocyte agglomeration, vacuolization, dilatation of sinusoid and central vein, among others. The ultrastructural study evidenced alterations coincident with those seen with light microscopy, which were accentuated with the increase of metal concentration: nucleolus with a high number of fibrillar centers (50 ppm); voluminous lipidic drops in the cytoplasm, loose endoplasmic rough reticulum, citoplasmatic vacuolization, altered lisosomes and peroxisomes (100 ppm); contracted nuclei with condensed cromatine, dilatation of intracellular space and mitochondria, and loss of fibrillar areas (150 ppm). Cadmium produces a toxic effect in the hepatic cells; the effect is more severe at higher concentration, leading to cellular necrosis.

Efecto de la exposición al cloruro de cadmio sobre la maduración in vitro de ovocitos bovinos / Effects of cadmium chloride exposure on in vitro maduration of bovine oocytes

Se estudió el efecto del cadmio sobre la maduración invitro de ovovitos bovinos. Ovocitos recuperados a partir de ovarios bovinos recolectados en mataderos y liberados mediante la técnica de slicing fueron madurados en TCM199 durante 24 h a 38,5°C en un atmósfera con 5% de Dioxido de carbono en aire saturado de humedad, en presencia de 0, 1, 5 y 10 ppm de cloruro de cadmio. Al finalizar el cultivo, los ovocitos fueron fijados en etanol: ácido acético (3/1 v/v) a 4°C durante al menos 24 h para luego teñirlos con aceto-orceína al 1,1 por ciento, para evaluar el estadio nuclear bajo microscopio óptico. El ANOVA demostró que el cadmio causa un bloqueo meiótico significativo (f= 150,996; p<0,01) e induce degeneración ovocitaria (F= 89,92; P<0,01) de manera dependiente de la concentración. El porcentaje de ovocitos que alcanzaron el estadio de MII fue superior en el grupo control (P<0,01) en comparación con los expuestos al metal a las diferentes concentraciones estudiadas. Se establece una correlación inversa altamente significativa entre el porcentaje de ovocitos madurados y la concentración del metal en el medio (r= 0,818; p<0,01), mientras que el porcentaje de ovocitos degenerados muestra una correlación directa altamente significativa (r= 0,842; P<0,01). Además se apreciaron aberraciones cromosómicas tales como: picnosis, stekinesisy puentes anafásicos. Se demostró el efecto deletéreo del cadmio sobre los ovocitos bovinos, reflejado en un bloqueo de la maduración in vitro, la inducción de degeneración y aberraciones cromosómicas.

Optimización del proceso de extracción de la lactasa de kluyveromyces marxianus ATTC 8554 para su aplicabilidad en la industria láctea / Optimization of the process of extraction of lactase from kluyveromyces marxianus ATTC 8554 for their applicability in the dairy industry

La lactasa o ß-galactosidasa (EC 3.2.1.23) es una enzima exclusivamente intracelular en Kluyveromyces marxianus ATCC 8554, por lo que para su liberación existen diversos métodos de extracción. El objetivo del presente trabajo consistió en optimizar el proceso de extracción de esta enzima para su uso en la industria láctea. La producción de la enzima fue inducida mediante el crecimiento de la levadura en un medio con lactosa como única fuente de carbono por 9 horas, donde alcanzó la fase logarítmica final de crecimiento. Se evaluó la combinación de los parámetros fisicoquímicos: temperaturas 30; 37 y 42°C; pH 6,5; 8,5 y 10 y tiempo 5; 10 y 20 horas con el método de extracción con tolueno al 2% en buffer fosfato 0,1 M. Se determinó la actividad enzimática de la lactasa a través del uso de un sustrato cromógeno el o-nitrophenyl-ß-D-galactopyranosido (ONPG) sobre los extractos libres de células. Se determinó la cantidad de proteínas totales por el método de Lowry, que permitió la valorización de la actividad específica. Los resultados fueron analizados empleando el paquete estadístico Statgraf versión 7.0, aplicando análisis de varianza trifactorial, LSD y prueba de Tukey. El análisis por microscopía electrónica de transmisión (MET de las células de K. marxianus reveló que el tolueno ejerce un efecto permeabilizante, liberando la enzima sin ruptura de la integridad celular. Se encontró un acortamiento significativo (P<=0,01) en el tiempo necesario para la extracción de la lactasa, siendo la mejor combinación: 37°C, pH 6,5 y 5 horas, generando en este tiempo la mayor cantidad de ONP: 0,4937 µmoles/mL/10 min y la mayor actividad específica 34,2095 Uß-gal/mg proteínas.