Laboratorio de Biologia Celular y Molecular

Resumen Los antihelmínticos benzimidazoles (BZD) desestabilizan el equilibrio tubulina-microtúbulo originando en el parásito una pérdida de la homeostasis celular, desprendimiento y muerte. Su baja toxicidad se explica por una mayor afinidad por la tubulina del parásito, ...

Two common and widely used liquid fuels with hepatotoxic activity, and trimethyltin, a compound with neurotoxic activity which is used in several industrial processes, were tested for their toxicity in two species of Pygoscelis penguins. Gentoo penguins Pygoscelis papua were dosed with trimethyltin (TMT)(15 mg/kg) and Adelie penguins P. adeliae were dosed with fuel for air (JP1)(0.20 ml/kg) and land transport (Polar Diesel, PD)(0.20 ml/kg). We use the serum proteins as markers of contamination on Gentoo and Adelie penguins. The following hematological parameters were measured: hemoglobin, hematocrit, glucose, total lipids, total proteins and enzymes (aspartate transaminase and alanine transaminase). Normal hematological values obtained on all penguins are in agreement with previously published data. All tested birds showed significant toxic changes being seen clinically and by blood tests. No mortality was observed during the experiment. ß globulin increased in Gentoo penguins treated with TMT, showing highly significant differences. α 1 and α 2 globulin was fusioned. There was a marked drop in total proteins in Adelie penguins dosed with JP1. ß and γ globulin decreased significantly in Adelie penguins exposed to Polar Diesel. This study show that serum proteins and enzymes levels can be used as biological markers of contamination on penguins.

Fasciola hepatica is a cosmopolitan trematode parasite, which causes considerable losses in livestock all over the world. Triclabendazole (TCBZ) is a halogenated benzimidazole compound highly effective against both juvenile and adult F. hepatica . The ability of different helminths to biotransform xenobiotic compounds has been demonstrated [2,. We have previously shown that both the microsomal and cytosolic fractions of F. hepatica oxidize the sulphur-containing flukicidal benzimidazole anthelmintics, albendazole (ABZ) [3] and TCBZ [4]. The intensive use of TCBZ in endemic areas of fascioliasis has resulted in the emergence of resistant liver flukes. Recent work addressed to identify the mechanisms of resistance to TCBZ indicated that the transtegumental influx/efflux balance for TCBZ and its main metabolites is altered in resistant (R) flukes, which accounted for a reduced amount of active drug being recovered within the R compared to the susceptible (S) parasite [5]. Furthermore, a higher capacity to oxidize TCBZ into its sulphoxide metabolite (TCBZO) was observed in R flukes compared with the S strain of the trematode parasite. The assays described here were performed to assess the potential involvement of an altered metabolism as a resistance mechanism towards TCBZ chemically related compounds. This complementary work was addressed to characterize the pattern of ABZ microsomal sulphoxidation in TCBZ-susceptible (TCBZ-S) and TCBZ-resistant (TCBZ-R) strains of F. hepatica.

It is unknown if the antiparasitic activity of the chiral compound ABZO is due to both enantiomeric forms or if one of them presents greater pharmacological activity. This determined the need for further knowledge of ABZSO enantiomeric behavior, not only with its receptor (parasite β-tubulin subunit), but also with mammal tubulin. Knowledge of ABZSO protein binding behavior is relevant to understanding drug-protein interactions in both the host and target parasites, which may (?) its pharmacological-toxicological effect.

Fasciolasis is a zoonotic parasitic disease caused by Fasciola hepatica and its control is mainly based on the use of triclabendazole (TCBZ). Parasite resistance to different anthelmintics is growing worldwide, including the resistance of F. hepatica to TCBZ. In the present work we evaluate "in vivo" the activity of xenobiotic metabolizing enzymes of phase I (carboxyl esterases) and phase II (glutathione S-transferases and carbonyl reductases) recovered of flukes from sheep treated with TCBZ. All three enzymes showed increased activity in TCBZ flukes returning 60 h post-treatment at similar to baseline unexposed flukes. TCBZ action may induce secondary oxidative stress, which may explain the observed increment in activities of the analyzed enzymes as a defensive mechanism. The enzymes analyzed are candidates to participate actively in the development of resistance at TCBZ in F. hepatica.

Triclabendazole (TCBZ) is a halogenated benzimidazole compound that possesses high activity against immature and adult stages of the liver fluke, Fasciola hepatica. The intensive use of TCBZ in endemic areas of fascioliasis has resulted in the development of liver flukes resistant to this compound. TCBZ sulphoxide (TCBZSO) and TCBZ sulphone (TCBZSO 2 ) are the main molecules recovered in the bloodstream of TCBZ-treated animals. In order to gain some insight into the possible mechanisms of resistance to TCBZ, the goals of the work described here were : to compare the ex vivo transtegumental diffusion of TCBZ parent drug and its sulpho-metabolites (TCBZSO and TCBZSO 2 ) into TCBZ-susceptible and -resistant liver flukes ; and to assess the comparative pattern of TCBZ biotransformation by TCBZ-susceptible and -resistant F. hepatica. For the tegumental diffusion studies, TCBZ-susceptible (Cullompton) and -resistant (Sligo) adult flukes collected from untreated infected sheep were incubated (15-180 min) in KRT buffer containing either TCBZ, TCBZSO or TCBZSO 2 (5 nmol.ml x1 ). For the metabolism studies, microsomal fractions obtained from TCBZ-susceptible and -resistant flukes were incubated for 60 min with TCBZ (40 mM), and the amount of the formed metabolic product (TCBZSO) was measured. Drug/metabolite concentrations were quantified by HPLC. All the assayed TCBZ-related molecules penetrated through the tegument of both TCBZ-susceptible and -resistant flukes. However, significantly lower (approximately 50 %) concentrations of TCBZ and TCBZSO were recovered within the TCBZ-resistant flukes compared to the TCBZ-susceptible ones over the 180 min incubation period. The rate of TCBZ sulphoxidative metabolism into TCBZSO was significantly higher (39 %) in TCBZ-resistant flukes. The flavin-monooxigenase (FMO) enzyme system appears to be the main metabolic pathway involved in the formation of TCBZSO in both TCBZ-susceptible and -resistant flukes. The altered drug influx/efflux and enhanced metabolic capacity identified in TCBZ-resistant liver flukes may account for the development of resistance to TCBZ.

The aim of the present study was to genetically characterize Fasciola hepatica strains from diverse ecogeographical regions (America and Europe), susceptible and resistant to Triclabendazole, using the random amplified polymorphic DNA fragments (RAPDs-PCR) technique to elucidate genetic variability between the different isolates. Ten different oligonucleotide primers of 10 bases with GC content varying from 50-70 % were used. A polymerase chain reaction (PCR) was carried out in 25 μl of total volume. Duplicate PCR reactions on each individual template DNA were performed to test the reproducibility of the individual DNA bands. The size of the RAPD-PCR fragments was determined by the reciprocal plot between the delay factors (Rf) versus the logarithm of molecular weight ladder. The phenogram obtained showed three main clusters, the major of which contained European Strains (Cullompton and Sligo) showing a genetic distance of 27.2 between them. The American strains (Cedive and Cajamarca) on the other hand formed each their distinctive group but clearly maintaining a closer genetic relationship among them than that to their European counterparts, with which showed a distance of 33.8 and 37.8, respectively. This polymorphism would give this species enhanced adaptability against the host, as well as the environment. The existence of genetically different populations of F. hepatica could allow, against any selection pressure, natural or artificial (for use fasciolicides products and/or control measures), one or more populations of F. hepatica to be able to survive and create resistance or adaptability to such selective pressure.

Body water (BW) and extracellular water (ECW), were determined with tritiated water (THO) and 82Br injected into the vein, to 8 mature animals of both sexes during the winter season. The biological half-time of THO (T1/2 THO) and the daily water turnover (WT) were measured and the intracellular water (ICW) calculated. The studies with THO were repeated in the same animals and in 2 lactating females in spring and summer. Two calves were also studied during spring. The values obtained in winter were: BW 659 +/- 12 ml/kg; T1/2 THO 9.2 +/- 1 day; WT per 24 h 50 +/- 3 ml/kg or 116 +/- 5 ml/kg 82 and 163 +/- 9 ml per 1 of BW82; ECW 215 +/- 8 ml/kg or 32.5 +/- 3% BW; ICW 447 +/- 12 ml/kg or 67.7 +/- 4% BW. The results of the spring's studies showed a significant increase in the values of WT. In summer a further increment of this parameters was observed when expressed as ml/kg body solids. This differences were remarkable in those in lactation. The proportion of water in the body was si...

The aim of the present work was to evaluate the effects of methimazole (MTZ) on the enantioselective sulphoxidation of albendazole (ABZ) by rat liver microsomes and tissue slices. Albendazole sulphoxide (ABZSO) was the metabolite recovered after the incubation with ABZ in both liver preparations. MTZ significantly reduced ABZSO production both in microsomes and slices. ABZSO production decreased as a function of MTZ concentration. The sulphoxidation reaction performed by rat liver explants in the presence of MTZ was 65% lower than that observed in controls. The reduction in the production of ABZSO in the presence of MTZ was mainly due to a lower production of (+) ABZSO. The results reported further contribute to the understanding of the enantioselective metabolism of ABZ. In addition, the work presented provides information on the comparison of two different liver tissue preparations for the evaluation of xenobiotic metabolism.

La fasciolosis, enfermedad zoonotica causada por el trematodo Fasciola hepática es la mayor causa de pérdidas económicas ganaderas en todo el mundo. El tratamiento de dicha enfermedad es exclusivamente farmacológico siendo el Triclabendazole (TCBZ) la droga más eficaz. El mal uso del mismo ha llevado a la aparición de cepas resistentes, las cuales ya fueron reportadas en Australia, Europa y Sudamérica.Los resultados obtenidos al momento indican que la resistencia en F. hepatica sería un proceso multifactorial que implica cambios en la absorción del fármaco, su metabolismo y excreción. Se sabe que el aumento del eflujo celular del TCBZ se encuentra relacionado con trasportadores ABC tales como la Glicoproteína P (Gp-P). Basándose en secuencias disponibles en bases de datos se dedujo la secuencia completa de una Gp-P de F. hepatica, y se postuló que una mutación (T687G) resultante en un cambio del aminoacido 1144 podría estar asociada a la resistencia. En el presente trabajo evaluamos...

INTEGRACION DE LA PLATAFORMA MOODLE COMO HERRAMIENTA TECNOLOGICA QUE FORTALECE LA COMUNICACIÓN Y EL APRENDIZAJE EN CURSOS DE POSGRADO INTERUNIVERSITARIO Chávez A., Germán1; Ortíz O., Pedro2; Solana G., Hugo3; Scarcella O., Silvana.3 1 Secretaría Académica. Rectorado. Universidad Nacional del Centro de la Provincia de Buenos Aires (UNCPBA), Tandil, Argentina. 3 Laboratorio de Biología Celular y Molecular. Centro de Investigación Veterinaria de Tandil (CIVETAN), CONICET, Facultad de Ciencias Veterinarias, UNCPBA, Tandil, Argentina. 2 Facultad de Ciencias Veterinarias. Universidad de Cajamarca, Cajamarca, Perú. e-mail: [email protected] La utilización de sistemas de gestión de cursos como Moodle como soporte virtual en un curso de posgrado internacional e interuniversitario permite fortalecer la comunicación entre los participantes y a la vez potenciar el aprendizaje a través de la utilización de herramientas y recursos que provee la plataforma. El propósito de este trabajo es ...

La zoonosis parasitaria producida por Fasciola hepatica se controla con Triclabendazole (TCBZ). El mal uso del TCBZ ha provocado el desarrollo de cepas TCBZ-resistente (TCBZ-R). Los parásitos evaden los xenobióticos por: i) mutación del receptor, ii) sobreexpresión de bombas de eflujo, y/o iii) sobreexpresión y/o modificación genética de las vías de detoxificación. En nematodos, la resistencia a los benzimidazoles es causada por mutaciones sobre β-tubulina (receptor). En F. hepatica TCBZ-R no se han detectado dichas mutaciones, por lo cual la resistencia debería explicarse por otros cambios genéticos o metabólicos. Trabajos previos desarrollados en nuestro laboratorio demuestran que una de las causas de la expresión del fenómeno de resistencia a TCBZ por parte de F. hepatica sería debido a una sobreexpresión de la actividad enzimática de Flavin Monooxigenasas no descartándose la participación de otras vías enzimáticas detoxificativas. Las Glutatión S-Transferasas (GST) son enzimas implicadas en los procesos de detoxificación de endo y xenobióticos utilizando la conjugación con Glutatión y ha sido descripta su participación frente al fenómeno de resistencia a otros xenobióticos Fasciolas adultas sensibles (S) a TCBZ (Cepa Cullompton) y resistentes (R) a TCBZ (Cepa Sligo) fueron obtenidas del conducto biliar de ovejas inoculadas con 200 metacercarias de cada cepa. Los trematodos adultos se recolectaron luego de 16 semanas post inoculación. Para la determinación de la actividad GST, se utilizo el método en continuo (Habig et al.,1974). Para la identificación de GST por PCR, los análisis de RT-PCR se llevaron a cabo a partir de ARN total. Se extrajo el ARN utilizando TRI REAGENT SIGMA ® (T 9414). La calidad y cantidad de ARN se evaluó mediante geles de agarosa al 1% visualizados con SYBR Safe® y espectrofotométricamente a través de la relación de A 260 nm/A 280 nm. Los oligonucléotidos específicos utilizados fueron diseñados para regiones conservadas del gen GST y amplificaron un fragmento de 657 pb. Como control interno para comparar las diferentes condiciones de RT-PCR fue utilizado el gen actina. El análisis termodinámico de los oligonucléotidos fue realizado con el programa OligoTech con apoyo del software DNAstar. Dichos amplicones fueron recuperados, purificados y ligados al vector comercial TOPO TA cloning kit ® (Invitrogen K457501) y secuenciados en un ABI PRISM utilizando oligonucleótidos que hibridan en el vector.

This open access article is distributed under a Creative Commons Attribution (CC-BY) 3.0 license. Abstract: Fasciolosis, a parasitic zoonosis of intrahepatic location, is caused by the trematode Fasciola hepatica. Its control is mainly based on the use of the anthelminthic Triclabendazole (TCBZ). The indiscriminate use of this drug has favored the development of anthelmintic resistance. The Glutation S-Transferases (GSTs) are multifunctional enzymes involved in the detoxification of xenobiotics and endogenous compounds using conjugation with endogenous glutathione. Recently, it has been shown an active participation of this family of enzymes in the detoxification of TCBZ related to the phenomenon of resistance. In F. hepatica, eight isoenzymes of the GST are present. Since it is well known that different isoenzymes do not necessarily have the same metabolic activity, this study evaluated the cytosolic activity of mu and pi GST isoenzymes in TCBZ resistant (Sligo and Oberon strains) ...