La tetracaína (Ttc), cuyas moléculas en solución fisiológica se encuentran mayoritariamente (97 %) en forma protonada, bloquea la corriente (IACh) evocada por acetilcolina (ACh) en ovocitos a los que se ha microtrasplantado receptores nicotínicos de acetilcolina (nAChRs) de la electroplaca de Torpedo marmorata. El bloqueo del nAChRm por Ttc fue muy potente, en el rango submicromolar (IC50= 0.5 μM) y reversible, recuperándose las respuestas a valores control tras un periodo de varios minutos. A concentraciones tan bajas como 0.1 μM, la Ttc ejerció un bloqueo que fue dependiente de voltaje, indicando que ejerce un bloqueo a canal abierto. El sitio de unión se pudo determinar en el interior del canal mediante técnicas de acoplamiento molecular. A concentraciones mayores (0.7 μM) se pudo observar un mecanismo de bloqueo distinto, a canal cerrado, que es independiente de voltaje y que se puede explicar por la unión de la Ttc a lugares situados en el ECD del nAChRm, que fueron determinados en los experimentos de docking virtual. Además, a esta concentración la Ttc aceleró la cinética de desensibilización de la IACh, cuando las células se mantuvieron en presencia sostenida del agonista. Esta se evocó cuando se co-aplicó la Ttc junto a la ACh a potenciales negativos. Por el contrario, cuando solamente se pre-aplicó la Ttc (aplicación previa a la de ACh), o cuando se co-aplicó a potenciales positivos, no se modificó la cinética de desensibilización, a pesar de que sí hubo una cierta inhibición de la IACh. Estos experimentos permitieron determinar que el sitio de unión de la Ttc que acelera la desensibilización se encuentra en el interior del canal. El ensayo de docking permitió localizar los residuos a los que su une la Ttc dentro del canal a altas concentraciones (con menor afinidad), que es más superficial que el implicado en el bloqueo a canal abierto. El lugar de unión determinado por anclaje virtual incluye la interacción de Ttc con αE262, γN224, γK271, y γE274, residuos que han sido previamente involucrados en el proceso de activación y desensibilización (Bouzat y cols., 2008; Forman y cols., 2007). El otro anestésico local (LA) estudiado, la benzocaína (Bzc), no posee carga al pH al que se efectúan los registros electrofisiológicos. La Bzc, al igual que la Ttc, inhibió la IACh, pero con una potencia menor, en el rango submilimolar y, a diferencia de la Ttc, su bloqueo fue independiente de voltaje. A pesar de mediar un bloqueo independiente de voltaje, la Bzc, evoca una corriente de rebote (IRb), similar a la que median moléculas que ejercen un bloqueo de canal abierto, sugiriendo que la Bzc podría estar uniéndose en el interior del canal. Otro efecto destacado de la Bzc sobre el nAChRms fue la aceleración de la desensibilización, haciéndola marcadamente más rápida incluso a potenciales positivos (a diferencia del efecto mediado por la Ttc). Además, se observó que, tras su pre-aplicación, la cinética de activación de la IACh se enlenteció y hubo un bloqueo de nAChRs, a canal cerrado, cuya recuperación fue especialmente lenta. Los efectos de ambos LAs fueron muy diferentes sobre los GABAAR. Así, la Ttc apenas tuvo efectos sobre este receptor, incluso a una concentración 10 veces superior a la IC50 determinada para el nAChRm. Por el contrario, la Bzc, aplicada a concentraciones similares a las que inhiben la IACh, aumentó la desensibilización y evocó una IRb similar a la observada en los nAChRs. Adicionalmente, la Bzc tuvo efectos sobre otros canales, como el ClC-0 y el CaCC. En relación con la Bzc, es interesante destacar que debido a su estructura química tiene una muy baja solubilidad al agua y, por tanto, debe solubilizarse en solventes como el etanol (EtOH) o el DMSO. Debido a que estos solventes pueden no ser totalmente inertes se probaron, en las mismas condiciones experimentales. No observándose efectos sobre los nAChRms.

No description available.

Die weltweit mehr als 7500 Amphibienarten sind durch anthropogene Ursachen wie Habitatzerstörung, Krankheitsverbreitung, Klimawandel und Umweltverschmutzung in ihrem Bestand bedroht. Einige der Ursachen sind kaum erforscht, so die Verschmutzung aquatischer Ökosysteme durch endokrine Disruptoren (EDs), Substanzen, die mit dem Hormonsystem interagieren. Ausgehend von neuen molekularen Markern, welche die Ermittlung des genetischen Geschlechts erstmals bei einigen Hyliden und Bufoniden erlauben, wurde in der vorliegenden Arbeit auf die Wirkung des synthetischen Östrogens 17α- Ethinylestradiol (EE2)und des Weichmachers Bisphenol A (BPA) fokussiert. Für drei Bufonidenarten wurde zunächst die Geschlechtsgebundenheit von Mikrosatelliten getestet und ein XX/XY-System nachgewiesen. Diese und bereits etablierte Marker wurden anschließend in ein neu entwickeltes Versuchsdesign für ED-Studien integriert: Nach gleichzeitiger Aufzucht von Modell- (Xenopus laevis) und Nicht-Modell-Arten (Hyla arborea, Bufo viridis) unter EE2- bzw. BPA- Exposition wurde das genetische Geschlecht bestimmt und mit dem anatomisch und histologisch ermittelten phänotypischen Geschlecht erglichen. Die drei Anuren zeigten starke Empfindlichkeitsunterschiede gegenüber beiden EDs. Umweltrelevante Konzentrationen beeinflussten die somatische Entwicklung und führten zu artspezifischen Gonaden-Fehlbildungen. EE2 bewirkte zahlreiche partielle und komplette Geschlechtsumwandlungen, mit stärkeren Effekten bei X. laevis. Diese Arbeit zeigt somit, dass bereits niedrige EE2- und BPA-Konzentrationen zu starken Schädigungen führen können und die Substanzen aufgrund ihrer erheblichen aquatischen Präsenz als ernstzunehmende Faktoren der Amphibienkrise anzusehen sind. Die Ermittlung des genetischen Geschlechts wird als wichtig eingestuft, um verlässliche Aussagen über ED-Effekte zu treffen. Zudem sollten an der Modell-Art X. laevis gewonnene Erkenntnisse nicht vorbehaltlos auf andere Amphibienarten extrapoliert werden. / The more than 7500 known amphibian species are globally threatened, mainly due to anthropogenic causes like habitat destruction, dispersing diseases, climate change and environmental pollution. Some of the causes are barely investigated, e.g. the pollution of aquatic ecosystems with endocrine disrupting compounds (EDCs), substances that interfere with the hormone system. Based on new molecular markers, for the first time allowing genetic sexing in some hylids and bufonids, this thesis focused on the effects of the synthetic estrogen 7α-ethinylestradiol(EE2) and the plasticizer bisphenol A (BPA). Initially, several microsatellite markers were tested for sex-linkage in three bufonid species, and an XX/XY system could be revealed. Subsequently, these and other established markers were integrated into a newly developed experimental design for EDC-research: after simultaneous exposure of model (Xenopus laevis) and non-model species (Hyla arborea, Bufo viridis) to EE2 or BPA, metamorphs were genetically sexed. Anatomically and histologically determined phenotypic sexes were directly compared with the genetic sex of each individual. The three anurans showed striking differences in their susceptibilities in both EDCexperiments. Environmentally relevant concentrations affected the somatic development and led to species-specific gonadal anomalies. In addition, EE2 provoked high numbers of mixed sex and completely sex-reversed individuals, with more pronounced effects in X. laevis than in the two non-model species. This work shows that low concentrations of EE2 and BPA lead to severe damages. Due to their widespread presence in the aquatic environment, these substances might contribute to the worldwide amphibian crisis. To produce reliable results in EDC-studies, genetic sexing is considered important. Furthermore, findings gained with the model species X. laevis should not unreservedly be extrapolated to other amphibian species.

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No description available.

The anatomy of the cranial peripheral nervous system has been studied for over a century, yet surprisingly little is known about how the nerves are guided to their targets. The study of the development of these nerves has important implications for our understanding of craniofacial anomalies and possible treatments for both injury and genetic disorders of nerve development such as Goldenhar-Gorlin syndrome. We have discovered that retinoic acid (RA) may play a role in the development of the trigeminal nerve. Inhibition of retinoic acid receptors (RAR) results in trigeminal nerves that become unbundled or defasciculated in the eye region. To further understand how RA is affecting trigeminal development we searched for genes downregulated in response to RAR inhibition by the inhibitor BMS-453 and have identified neurotrophin-3 (NT-3), activated leukocyte cell adhesion molecule (ALCAM) and Semaphorin 4B (Sema4B). We have analyzed the expression patterns of Sema4B and NT-3 by in situ hybridization and have found NT-3 expression in the eye and Sema4B in the embryonic target of the trigeminal nerve, lens of the eye and in the pharyngeal arches. ALCAM has been analyzed via qRT-PCR and its transcription is downregulated just prior to the observed defasciculation phenotype. The pattern of expression of these genes combined with known expression of NT-3 receptors allows us to suggest a model whereby RA signaling regulates Sema4B, ALCAM and NT-3, which support the survival, guidance and fasciculation of the trigeminal nerve. This work has the potential to better understanding of the complex nature of cranial nervous system development.

The diploid amphibian Xenopus tropicalis represents a significant model organism for studies of early development, genes function and evolution. Such techniques as gynogenesis, injection of morpholino antisense oligonucleotide into fertilized eggs or transgenesis were established. In the recent ten years, many efforts have been made to complete the sequence information. X. tropicalis genome has been sequenced but the completion of its assembly only on the basis of sequence data has been impossible. Therefore, our first work was focused on one of approaches for a genome completing- genetic mapping. First of all, the genetic map of Xenopus tropicalis was established pursuant linkage and physical positions of markers. Since the map contained gaps, we developed a new method for genetic mapping based on the next generation sequencing of laser microdissected arm. Using Illumina next generation sequencing of fifteen copies of a short arm of chromosome 7, we obtained new insights into its genome by localizing previously unmapped genes and scaffolds as well as recognizing mislocalized portions of the genome assembly. This was the first time laser microdissection and sequencing of specific chromosomal regions has been used for the purpose of genome mapping. These data were also used in the evolution study of...

The teratogenic potential of commercial formulations of atrazine (40.8%) and 2,4-D was evaluated using FETAX (frog embryo teratogenic assay--Xenopus). Because these herbicides have been detected in ground and surface water, this study was designed to determine the adverse effects in buffer and natural water for both herbicides. All treatments showed a significant concentration-response effect on exposed embryos, except for the 2,4-D natural water sample. Atrazine (solubility of the commercial formula used 70 mg/L at 20 degrees C), compared to 2,4-D (solubility = 311 mg/L at pH = 1 and 25 degrees C), had a significantly greater teratogenic effect in both the buffer (atrazine EC50 = 33 mg/L, LC50 = 100 mg/L, TI = 3.03; 2,4-D EC50 = 245 mg/L, LC50 = 254 mg/L, TI = 1.04) and natural water samples (atrazine EC50 < 8 mg/L, LC50 = 126 mg/L; 2,4-D EC50 and LC50 > 270 mg/L). The 2,4-D EC50 and LC50 values for the buffer were similar at 245 mg/L and 254 mg/L. These similar values and the teratogenic index (TI) of 1.04 suggested that 2,4-D was more embryotoxic than teratogenic to frog embryos at high concentrations. Atrazine in natural water demonstrated a significantly greater EC50 (100% abnormality at 8 mg/L, the lowest test concentration) to frog embryos than the buffer experiment (EC50 = 33 mg/L). The extrapolated lowest observable adverse effect concentration (LOAEC) for the natural water experiment was 1.1 mg/L. These results suggest that atrazine toxicity is enhanced by the synergistic or additive effects of some component of the water or atrazine was already present in the sample. In contrast to atrazine, 2,4-D was less toxic in natural water than buffer. These results suggest that both atrazine and 2,4-D pose little threat, since their embryotoxicity and teratogenicity to frog embryos occur at high concentrations approaching their maximum solubility levels in water.

Surfactants are one of the more ubiquitous contaminants in aquatic systems. Their importance as toxic components of pesticide formulations has, however, been largely overlooked. Amphibians particularly, as inhabitants of shallow, temporary and often lentic aquatic environments may be at risk from exposure to these chemicals when they enter aquatic systems. This thesis presents data on the toxicity of surfactants to amphibians. Several experimental exposures were conducted with embryo-larval, tadpole and adult developmental stages of the Australian species- Crinia insignifera, Helcioporus eyrei, Limnodynastes dorsalis and Litoria moorei and the exotic species- Bufo marinus and Xenopus laevis. Animals were variously exposed to glyphosate formulations that contain a high proportion of nonionic surfactants, or commercial pesticide wetting agents (alcohol alkoxylate and nonylphenol ethoxylate (NPE) surfactants). Feeding stage tadpoles of C.insignifera, H. eyrei, L. dorsalis and L. moorei were exposed to three commercial glyphosate formulations, glyphosate isopropylamine and glyphosate acid in static-renewal acute toxicity tests. The 48-h LC50 values for Roundup Herbicide (MON 2139) tested against tadpoles of C. insignifera, H. eyrei, L. dorsalis and L. moorei ranged between 8.1 and 32.2 mg/L (2.9 and 11.6 mg/L glyphosate acid equivalent (ae)), while the 48-h LC50 values for Roundup Herbicide tested against adult and newly metamorphosed C. insignifera ranged from 137-144 mg/L (49.4-51.8 mg/L ae). / Touchdown Herbicide (4 LC-E) tested against tadpoles of C. insignifera, H. eyrei, L. dorsalis and L. moorei was slightly less toxic than Roundup with 48-h LC50 values ranging between 27.3 and 48.7 mg/L (9.0 and 16.1 mg/L ae). Roundup Biactive (MON 77920) was practically non-toxic to tadpoles of the same four species producing 48-h LC50 values of 911 mg/L (328 mg/L ae) for L. moorei and >1000 mg/L (>360 mg/L ae) for C. insignifera, H. eyrei and L. dorsalis. Glyphosate isopropylamine was practically non-toxic producing no mortality amongst tadpoles of any of the four species over 48 h, at concentrations between 503 and 684 mg/L (343 and 466 mg/L ae). The toxicity of technical grade glyphosate acid (48-h LC50, 81.2-121 mg/L) is likely to be due to acid intolerance. Feeding stage tadpoles of B. marinus, X laevis, C. insignifera, H.eyrei, L. dorsalis and L. moorei were exposed to NPE and alcohol alkoxylate in static renewal acute toxicity tests. All species exhibited non-specific narcosis following exposure to both these surfactants. The 48-h EC50 values for NPE ranged between 1.1 mg/L (mild narcosis) and 12.1 mg/L (full narcosis). The 48-h EC50 values for alcohol alkoxylate ranged between 5.3 mg/L (mild narcosis) and 25.4 mg/L (full narcosis). Xenopus laevis was the most sensitive species tested. The sensitivity of the other five species was size dependent with larger species displaying greater tolerance. Replicate acute toxicity tests with B. marinus exposed to NPE at 30 degrees celsius over 96 hours indicated that the narcotic effects were not particularly time dependant. / The mean 24, 48, 72 and 96-h EC50 (mild narcosis) were 3.6, 3.7, 3.5 and 3.5 mg/L respectively. The mean 24, 48, 72, and 96-h EC50 (full narcosis) values were 4.0, 4.1, 4.2 and 4.0 respectively. Acute toxicity tests with B. marinus exposed to NPE at 30 degrees celsius under conditions of low dissolved oxygen (0.8-2.3 mg/L) produced a two to threefold increase in toxicity. The 12-h EC50 values ranged from 1.4 to 2.2 mg/L. The embryotoxicity of NPE was determined in X. laevis, L. adelaidensis and C. insignifera using a Frog Embryo Teratogenesis assay-Xenopus (FETAX). The 96-h LC50, EC50 and MCIG (LOEC) values for X. laevis were 3.9 to 5.4 mg/L, 2.8 to 4.6 mg/L and 1.0 to 3.0 mg/L respectively. The 140-h LC50, ECSO and MCIG values for L. adelaidensis were 9.2 mg/L, 8.8 mg/L and 5.1 to 6.0 ing/L respectively. The 134-h LC50, EC50 and MCIG values for C. insignifera were 6.4 mg/L, 4.5 mg/L and 4.0 mg/L respectively. Teratogenicity indices for the three species ranged between 1.0 and 1.6 indicating either no or low teratogenicity. Xenopus laevis was the more sensitive of the three species and the only species that displayed indisputable terata. The acute toxicity data indicated that the amphibian species tested were of similar sensitivity to fish and some invertebrates. Developmental retardation and oestrogenic effects following exposure to nonylphenol ethoxylate were indicated by sublethal toxicity tests. Crinia insignifera embryos were exposed during early embryogenesis to sublethal concentrations of NPE. / Exposure to NPE did not affect either weight nor size (snout-vent length) at metamorphosis. Exposure to 5.0mg/L NPE resulted in a significant delay in the time required to reach metamorphosis. Also, exposure to 3.0 mg/L NPE for the first 6 days of embryonic development or exposure to 5.0 mg/L NPE from day 2 to day 6 resulted in a statistically significant predominance in the female phenotype amongst metamorphosing froglets. Exposure for the first five days to 1.5 ing/L or 3.0 mg/L NPE had no effect on sex ratio. The results indicated that exposure to NPEs has endocrine disruptive effects in this species and that a narrow window of susceptibility exists for the induction of predominantly female phenotype. This study has also followed the degradation of a mixture of NPE oligomers and the concomitant formation of individual oligomers in static die-away tests with and without illumination in freshwater. Over 33 days in darkness there was a progressive and complete loss of long chain oligomers (NPEO(subscript)8-17), transient increases and subsequent loss of short to medium chain oligomers (NPE0(subscript)4-7), and large persistent increases (approximately 1000%) in short chain oligomers (NPE0(subscript)1-3). In the presence of illumination, biodegradation was retarded and heterotrophic bacterial proliferation was inhibited. After 33 days there was complete loss of long chain oligomers (NPE0(subscript)9-17), incomplete loss of medium chain oligomers (NPE0(subscript)6.8) and increases in short chain oligomers (NPE0(subscript)1-5). / This thesis discusses the importance of persistent metabolites of NPE degradation as it pertains to the habitat, developmental time frame and ecology of amphibians. Degradation of NPE is likely to occur over a time frame that is longer than that required for complete embryogenesis and metamorphosis of many species of amphibians, and may easily encompass those critical stages of development during which oestrogenic metabolites can affect development.