Developmental exposure to mancozeb induced neurochemical and morphological alterations in adult male mouse hypothalamus.

Mancozeb, a dithiocarbamate widely used in agriculture, is considered a developmental hazard in humans; however, more evidences are still needed concerning the consequences of chronic exposure to this pesticide. Mancozeb neurotoxicity in developing mouse hypothalamus was evaluated by subchronic exposure of male Mus musculus mice to low and high doses of mancozeb (30 and 90 mg/kg body weight, respectively) from late neonatal until adolescence. Variations in hypothalamic amino acid neurotransmitter levels and changes in histological as well as cytological characteristics were analyzed in young adult experimental mice and compared with control. A dose-dependent increase in excitation/ inhibition ratio was observed in mancozeb-exposed hypothalamus, indicating an overall state of excitoxicity. Histopathological and ultrastructural studies showed increased apoptosis, neuroinflammation and demyelination, demonstrating mancozeb-induced cytotoxicity in hypothalamic neurosecretory cells. In summary, both neurochemical and morphological data revealed mancozeb-induced alterations during development of hypothalamic circuitry that are critical for maturation of the neuroendocrine system.

Development of an UPLC-MS/MS micromethod for quantitation of cinitapride in plasma and its application in a pharmacokinetic interaction trial.

AIM: Cinitapride (CIN) is a benzamide-derived molecule used for the treatment of gastroesophageal reflux and dyspepsia. Its pharmacokinetics are controversial due to the use of supratherapeutic doses and the lack of sensitive methodology. Therefore, a sensitive and accurate micromethod was developed for its quantitation in human plasma. RESULTS: CIN was extracted from 300 µl of heparinized plasma by liquid-liquid extraction using cisapride as internal standard, and analyzed with an ultra performance liquid chromatograph employing positive multiple-reaction monitoring-MS. CONCLUSION: The method proved to be rapid, accurate and stable within a range between 50 and 2000 pg/ml and was successfully validated and applied in a pharmacokinetic interaction trial, where it was demonstrated that oral co-administration of simethicone does not modify the bioavailability of CIN.

[Effects of occupational exposure to pesticides on semen quality of workers in an agricultural community of Merida state, Venezuela]. / Efectos de la exposición ocupacional a plaguicidas sobre la calidad del semen en trabajadores de una comunidad agrícola del estado Merida, Venezuela.

Numerous studies report adverse effects of pesticides on male reproductive health. The objectives of this study were to investigate whether there is a relationship between occupational exposure to pesticides and semen quality, and to determine whether chronic exposure to pesticides differentially affects semen quality in men of different ages. A comparative study of 64 farmers and 64 control men was performed. The farmers were interviewed to determine their occupational history and particularly, activities that may involve exposure to pesticides. Semen parameters were evaluated and a comparative analysis of semen variables between exposed and control groups, as well as between age groups: 18-29, 30-37 and 38-60 years was done. Significant alterations of some semen parameters in the exposed group were found, such as: decreases in sperm concentration, slow progressive motility and sperm membrane integrity; at the same time, increases in eosin Y positive and sperm DNA fragmentation index. The results obtained by age groups showed significant differences between exposed and control groups for the parameters of membrane integrity, eosin Y positive and sperm DNA fragmentation index, being the exposed group between 18-29 years that showed the highest altered cases of these parameters. Our results prove that occupational pesticide exposure is associated with alterations in sperm quality, creating a risk to farm workers in their reproductive capacity.

Efectos de la exposición ocupacional a plaguicidas sobre la calidad del semen en trabajadores de una comunidad agrícola del estado Mérida, Venezuela. / Effects of occupational exposure to pesticides on semen quality of workers in an agricultural community of Merida state, Venezuela.

Numerosos estudios informan de los efectos adversos de plaguicidas sobre la salud reproductiva masculina. Los objetivos de este estudio fueron investigar si existe una relación entre exposición ocupacional a plaguicidas y la calidad del semen, y determinar si la exposición crónica a plaguicidas afecta diferencialmente la calidad del semen de trabajadores de diferentes edades. Se realizó un estudio comparativo entre 64 agricultores y 64 hombres control. Los trabajadores agrícolas fueron entrevistados para determinar su historia ocupacional, particularmente las actividades que pueden involucrar exposición a plaguicidas. Se evaluaron los parámetros seminales y se hizo un análisis comparativo entre el grupo expuesto y control, así como entre los grupos de edad 18-29, 30-37 y 38-60 años. Se encontraron alteraciones significativas de algunos parámetros del semen en el grupo expuesto, tales como: disminuciones en la concentración, motilidad lenta progresiva e integridad de membrana espermática; a su vez, incrementos en eosina Y positiva e índice de fragmentación del DNA espermático. Los resultados obtenidos por grupo de edad mostraron diferencias significativas entre los grupos expuesto y control, para los parámetros de integridad de membrana, eosina Y positiva e índice de fragmentación del DNA espermático, siendo el grupo expuesto entre 18-29 años el que mostró mayores casos alterados de estos parámetros. Los resultados de este estudio comprueban que la exposición ocupacional a plaguicidas está asociada con alteraciones en la calidad espermática, creando riesgo para la capacidad reproductiva de los trabajadores del campo.

Numerous studies report adverse effects of pesticides on male reproductive health. The objectives of this study were to investigate whether there is a relationship between occupational exposure to pesticides and semen quality, and to determine whether chronic exposure to pesticides differentially affects semen quality in men of different ages. A comparative study of 64 farmers and 64 control men was performed. The farmers were interviewed to determine their occupational history and particularly, activities that may involve exposure to pesticides. Semen parameters were evaluated and a comparative analysis of semen variables between exposed and control groups, as well as between age groups: 18-29, 30-37 and 38-60 years was done. Significant alterations of some semen parameters in the exposed group were found, such as: decreases in sperm concentration, slow progressive motility and sperm membrane integrity; at the same time, increases in eosin Y positive and sperm DNA fragmentation index. The results obtained by age groups showed significant differences between exposed and control groups for the parameters of membrane integrity, eosin Y positive and sperm DNA fragmentation index, being the exposed group between 18-29 years that showed the highest altered cases of these parameters. Our results prove that occupational pesticide exposure is associated with alterations in sperm quality, creating a risk to farm workers in their reproductive capacity.

Neurotoxicidad de los plaguicidas como agentes disruptores endocrinos: una revisión / Neurotoxicity of pesticides as endocrine disruptors agents: a review

Los plaguicidas se encuentran en el medio ambiente como contaminantes formando mezclas complejas, los estudios recientes se han centrados en la evaluación de los efectos y riesgos que pueden causar estas mezclas de plaguicidas en el ser humano y los ecosistemas.Esta revisión hace hincapié a tres plaguicidas específicos, representativos de los grupos químicos organofosforados, carbamato de tipo ditiocarbamato y triazinas, como son: Paratión® etílico, Mancozeb® y Atrazina®, respectivamente. Ha sido demostrado en animales de experimentación la neurotoxicidad de estos plaguicidas por alteración de la transmisión sináptica y de los mecanismos de la homeostasis del sistema neuroendocrino, lo cual explica la acción de estos plaguicidas como disruptores endocrinos. Varios trabajos demuestran que estos plaguicidas son potencialmente tóxicos a la salud humana, y aún en bajas concentraciones, pueden afectar al organismo causando alteraciones en el sistema neuroendocrino, especialmente en los ejes hipotálamo-hipófisis-gónada e hipotálamo-hipófisis-tiroides. Los investigadores destacan que los momentos de particular sensibilidad de la exposición a plaguicidas disruptores endocrinos son las etapas tempranas de la vida como: durante el desarrollo embrionario y la primera infancia, períodos en los cuales los procesos de crecimiento son controlados por las hormonas afectando el desarrollo del cerebro, el sistema inmune y otros órganos como el sistema reproductor y tiroideo. Los cambios neuroquímicos provocados por la exposición a estos plaguicidas durante el desarrollo cerebral constituyen un alto riesgo porque son capaces de interferir en los procesos de neurogénesis y sinaptogénesis, afectando de manera adversa las funciones cognitivas y motoras, cuyos efectos se observan durante las etapas posteriores de la vida.

Pesticides are found in the environment as complex mixtures forming pollutants, recent studies have focused on evaluating the effects and risks that may cause these mixtures of pesticides on humans and ecosystems. This review highlights three specific, representative of organophosphorus pesticide chemical groups carbamate, dithiocarbamate and triazine type, such as: ethyl Paratión®, Mancozeb® and Atrazina®, respectively.It has been demonstrated in experimental animal’s neurotoxicity of these pesticides by altering synaptic transmission and mechanisms of homeostasis of the neuroendocrine system, which explains the action of these pesticides as endocrine disruptors. Several studies show that these pesticides are potentially toxic to human health, and even in low concentrations, can affect the body causing alterations in the neuroendocrine system, especially in the hypothalamic-pituitary-gonadal and hypothalamic-pituitary-thyroid axis. The researchers point out that the moments of particular sensitivity of exposure Endocrine disruptor pesticide are the early stages of life as during embryonic development and early childhood periods in which growth processes are controlled by hormones affecting the development brain, immune system and other organs such as the thyroid and reproductive system. The neurochemical changes induced by exposure to these pesticides during brain development are at high risk because they are able to interfere with the processes of neurogenesis and synaptogenesis, adversely affecting cognitive and motor functions, whose effects are observed during the later stages of life.

Occupational exposure to organophosphate and carbamate pesticides affects sperm chromatin integrity and reproductive hormone levels among Venezuelan farm workers.

OBJECTIVES: Several reports suggest that chronic pesticide exposure may affect semen quality and male fertility in humans. The objective of this study was to evaluate the association between occupational exposure to organophosphate (OP) and carbamate (CB) pesticides and semen quality, as well as levels of reproductive and thyroid hormones of Venezuelan farm workers. METHODS: Thirty-five healthy men (unexposed group) and 64 male agricultural workers (exposed group) were recruited for clinical evaluation of fertility status. Fresh semen samples were evaluated for sperm quality and analyzed for DNA fragmentation index (DFI) by flow cytometry. Pesticide exposure was assessed by measuring erythrocyte acetylcholinesterase (AChE) and plasma butyrylcholinesterase (BuChE) with a Test-mate ChE field kit. Serum levels of total testosterone (Tt), follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), thyroid stimulating hormone (TSH) and free thyroxine (FT4) were analyzed using enzyme immunoassay kits. RESULTS: Evidence of pesticide exposure was found in 87.5% of farmers based on AChE and BuChE inhibition. Significant increments were observed in sperm DFI with significant decreases in some semen parameters. DFI was negatively correlated with BuChE, sperm concentration, morphology and vitality in these workers. The levels of Tt, PRL, FT4 and TSH appeared to be normal; however, there was a tendency for increased LH and FSH levels in exposed workers. CONCLUSIONS: Our results confirm the potential impact of chronic occupational exposure to OP/CB pesticides on male reproductive function, which may cause damage to sperm chromatin, decrease semen quality and produce alterations in reproductive hormones, leading to adverse reproductive health outcomes.

Residuos de plaguicidas en aguas para consumo humano en una comunidad agrícola del estado Mérida, Venezuela / Pesticide residues in drinking water of an agricultural community in the state of Mérida, Venezuela

El objetivo del presente trabajo fue determinar la presencia de plaguicidas en agua potable, provenientes de seis acueductos en una región de intensa actividad agrícola del Estado Mérida, Venezuela. El estudio fue realizado durante cuatro semanas continuas, entre mayo y junio de 2008. Los residuos de plaguicidas fueron analizados mediante extracción en fase sólida y HPLC con detector de arreglo de diodos. El método SPE-HPLC-DAD cumplió con los criterios de validación analítica: linealidad (R²: 0,9840-0,9999), precisión (coeficiente de variabilidad inter-día 1,47-6,25%), exactitud (desviación estándar relativa 0,9-9,20%) y sensibilidad (límite de detección ≤ 0,012 µg/L; límite de cuantificación ≤ 0,030 µg/L, excepto mancozeb con 0,400 µg/L). Siete de los trece plaguicidas seleccionados tienen un porcentaje de recuperación entre 100% y 70%, el resto, entre 61% y 37%. En 72 muestras analizadas, se detectaron diez plaguicidas de los grupos químicos: organofosforados, carbamatos, triazinas y derivados de urea. Los plaguicidas con mayor frecuencia de detección fueron: carbofuran y atrazina (39%), malation (25%), dimetoato y metribuzin (19%). Los plaguicidas que se encontraron en niveles más altos fueron: diazinon (26,31 µg/L), metamidofos (10,99 µg/L), malation (2,03 µg/L) y mancozeb (1,27 µg/L). Los niveles de plaguicidas no superaron los valores máximos permitidos por la Legislación Venezolana, sin embargo, fueron superiores al nivel máximo permitido por la Unión Europea y EPA-USA. Este estudio demuestra la urgente necesidad de hacer un monitoreo sistemático de la calidad del agua para consumo humano en las regiones de alta productividad agrícola.

The aim of this study was to determine the presence of pesticides in drinking water from six aqueducts in a region of intense agricultural activity in the state of Merida, Venezuela. The study was conducted for four continuous weeks, between May and June 2008. Pesticide residues were analyzed by solid phase extraction (SPE) and liquid chromatography (HPLC) with diode array detector (DAD). The method SPE-HPLC-DAD met the criteria of analytical validation, with good linearity (R²: 0.9840 to 0.9999), precision (coefficient of inter-day variability from 1.47 to 6.25%), accuracy (relative standard deviation 0.9 to 9.20%) and sensitivity (LOD ≤ 0.012 µg/L; LOQ ≤ 0.030 µg/L, except mancozeb with 0.400 µg/L). Seven of the thirteen selected pesticides have a recovery rate between 100% and 70%, the rest between 61% and 37%. Ten pesticides of the following chemical groups, were detected in 72 samples analyzed: organophosphates, carbamates, triazines and urea derivatives. The pesticides with the highest frequency of detection were: carbofuran and atrazine (39%), malathion (25%), dimethoate and metribuzin (19%). The pesticides found at high levels were diazinon (26.31 µg/L), methamidophos (10.99 µg/L), malathion (2.03 µg/L) and mancozeb (1.27 µg/L). Pesticide levels did not exceed the maximum allowed by Venezuelan law, however, according to international standards (EU and EPA-USA) values were above the maximum permissible levels. This study demonstrates the urgent need for systematic monitoring of the quality of water for human consumption in regions of high agricultural productivity.

Efectos de exposición ocupacional a plaguicidas sobre la integridad de la cromatina espermática

Objetivo: Evaluar los efectos de la exposición a plaguicidas organofosforados y carbamatos sobre la integridad de cromatina espermática en trabajadores agrícolas. Métodos: Se evaluaron 64 trabajadores del campo, con edades entre 18 y 55 años, de la comunidad rural de Bailadores, Municipio Rivas Dávila, Estado Mérida, Venezuela, expuestos directamente a plaguicidas. Para el diagnóstico de exposición a plaguicidas, fueron determinados los niveles de las colinesterasas eritrocitaria (AChE) y plasmática (PChE). Para evaluar la fertilidad masculina, a cada trabajador se le realizó seminograma y se les evaluó la integridad de la estructura de cromatina espermática mediante la técnica "Sperm Chromatin Structure Assay" (SCSA). Resultados: El 25% de los trabajadores agrícolas presentó niveles deprimidos de AChE y el 83% con niveles anormales de PChE, con una reducción <75% del nivel normal. El grupo con edades entre 18 y 28 años fue el más afectado. Diferencias significativas fueron obtenidas en los promedios de los niveles de colinesterasas entre los casos normales y alterados, tanto para AChE (3,95±0,42 vs. 2,72±0,16, ρ<0,0001) así como para PChE (2,08±0,25 vs. 1,43±0,32, ρ<0,0001). El 69,7% de los trabajadores con niveles anormales de PChE presentó alteración en el ADN espermático. Se encontró una correlación negativa significativa entre el Índice de Fragmentación de ADN (IDF) espermático y los niveles de PChE (ρ=0,02). Conclusiones: En trabajadores agrícolas expuestos directamente a plaguicidas organofosforados y carbamatos, un alto porcentaje presentó niveles anormalmente deprimidos de PChE, junto con alteración en el ADN espermático. Estos resultados demuestran que los trabajadores se encuentran con alto riesgo de exposición a los efectos tóxicos de plaguicidas, lo cual efectivamente se comprueba con los resultados de análisis de las enzimas colinesterasas y la aplicación de la técnica SCSA para determinar la integridad de la cromatina espermática.

Objective: To evaluate the effects of exposure to organophosphate and carbamate pesticides on the integrity of sperm chromatin in farm workers. Methods: In this study, we evaluated 64 farm workers, aged between 18 and 55 years, from the rural community of Bailadores, Municipality of Rivas Dávila, Mérida State, Venezuela, directly exposed to pesticides. For the diagnosis of pesticide exposure, levels of erythrocyte (AChE) and plasma (PchE) cholinesterases. were determined. To evaluate male fertility, each worker underwent semen analysis and the integrity of sperm chromatin structure was assessed using the "Sperm Chromatin Structure Assay (SCSA) technique. Results: The results of this study showed that 25% of agricultural workers had depressed levels of AChE and a majority, 83%, was found with abnormal levels of PChE. The group with corresponding ages between 18 and 28 years was the most affected. Significant differences were obtained in the average cholinesterase levels between normal and altered cases for both AChE (3.95 ± 0.42 vs. 2.72 ± 0.16, p < 0.0001) and PChE (2.08 ± 0.25 vs. 1.43 ± 0.32, ρ < 0.0001). Among workers with abnormal PChE levels, 69.7% presented alterations of sperm chromatin structure. There was a significant negative correlation between sperm DNA Fragmentation Index (DFI) and the levels of PChE (ρ = 0.02). Conclusions: In agricultural workers directly exposed to organophosphate and carbamate pesticides, a high percentage showed abnormally depressed PChE, along with altered sperm chromatin structure. These results show that farm workers are at high risk of exposure to the toxic effects of pesticides, which were effectively demonstrated with the results of analysis of cholinesterase enzymes and the implementation of the SCSA technique for determining the status of the sperm chromatin structure.

[Pesticide residues in drinking water of an agricultural community in the state of Mérida, Venezuela]. / Residuos de plaguicidas en aguas para consumo humano en una comunidad agrícola del estado Mérida, Venezuela.

The aim of this study was to determine the presence of pesticides in drinking water from six aqueducts in a region of intense agricultural activity in the state of Merida, Venezuela. The study was conducted for four continuous weeks, between May and June 2008. Pesticide residues were analyzed by solid phase extraction (SPE) and liquid chromatography (HPLC) with diode array detector (DAD). The method SPE-HPLC-DAD met the criteria of analytical validation, with good linearity (R2: 0.9840 to 0.9999), precision (coefficient of inter-day variability from 1.47 to 6.25%), accuracy (relative standard deviation 0.9 to 9.20%) and sensitivity (LOD < or = 0.012 microg/L; LOQ < or = 0.030 microg/L, except mancozeb with 0.400 microg/L). Seven of the thirteen selected pesticides have a recovery rate between 100% and 70%, the rest between 61% and 37%. Ten pesticides of the following chemical groups, were detected in 72 samples analyzed: organophosphates, carbamates, triazines and urea derivatives. The pesticides with the highest frequency of detection were: carbofuran and atrazine (39%), malathion (25%), dimethoate and metribuzin (19%). The pesticides found at high levels were diazinon (26.31 microg/L), methamidophos (10.99 microg/L), malathion (2.03 microg/L) and mancozeb (1.27 microg/L). Pesticide levels did not exceed the maximum allowed by Venezuelan law, however, according to international standards (EU and EPA-USA) values were above the maximum permissible levels. This study demonstrates the urgent need for systematic monitoring of the quality of water for human consumption in regions of high agricultural productivity.

Efectos de la exposición prenatal a paraquat sobre el desarrollo de la transmisión sináptica aminoacídica en la corteza cerebral parietal del ratón / Effects of prenatal expossure to paraquat on the development of amino acid synaptic transmission in mouse cerebral parietal cortex

Se estudiaron los efectos de la exposición prenatal al paraquat (PQ), sobre el desarrollo postnatal de la transmisión sináptica aminoacídica de la corteza cerebral parietal del ratón. Las ratonas NMRI preñadas del grupo experimental recibieron 5 dosis de 10mg/kg de peso corporal de PQ, entre el día de gestación (G)12 y G20, y el grupo control recibió solución salina. Mediante HPLC, se determinaron los niveles de aspartato, glutamato, glicina, GABA y taurina de las crías, entre la edad postnatal (P)1 y P30. Entre P3 y P15, se observó un incremento significativo de los neurotransmisores excitatorios, aspartato y glutamato, en los ratones expuestos a PQ. Con respecto a la neurotransmisión inhibidora, los cambios más importantes se observaron en glicina: sus niveles se mantuvieron significativamente por debajo del grupo control entre P1 y P7, y significativamente por encima en P11 y P15. Para taurina, entre P1 y P7 sus niveles se mantuvieron significativamente altos con respecto al grupo control. En P30, los niveles de todos los neurotransmisores se encontraron significativamente por debajo del grupo control. En conclusión, podríamos decir que la exposición prenatal a PQ tuvo efectos tóxicos que se reflejaron en una alteración de los niveles basales de los neurotransmisores aminoacídicos durante el desarrollo postnatal de la corteza parietal del ratón, predominando la excitación sobre la inhibición durante todo el período estudiado. Estas alteraciones podrían indicar la ocurrencia de importantes daños corticales, tales como la disminución de algunas poblaciones neuronales, la inadecuada formación de los circuitos corticales y alteraciones en el proceso de sinaptogénesis.

The effects of prenatal expossure to paraquat (PQ) were studied on postnatal development of mouse parietal cerebral cortex, in particular, the ontogenesis of amino acid synaptic transmission. Pregnant NMRI mice were separated into two groups: the experimental group received 5 doses of 10mg PQ/kg body weight, between days of gestation (G)12 and G20, whereas the control group received physiological saline solution. Levels of neurotransmitter amino acids: Asp, Glu, Gly, GABA and Tau were determined by HPLC between postnatal (P) days P1 and P30. Between P3 and P15, a significant increment in the levels of excitatory amino acids, Asp and Glu, were observed in mice exposed to PQ, as compared with the control group. With respect to the inhibitory neurotransmitter levels, in the group exposed to PQ, the more important changes were observed in Gly between P1 and P15. In relation to taurine, its levels remained significantly higher between P1 and P7 with respect to the control group. It is important to emphasize that at P30, the levels of all neurotransmitters in the experimental group were significantly lower than those of control. In conclusion, prenatal exposure to PQ caused neurotoxicity in the developing mouse parietal cortex, as shown by the alterations in the basal levels of amino acid neurotransmitters, with the excitatory predominating over inhibitory neurotransmission, throughout the studied developmental period. These alterations could indicate the occurrence of important cortical injuries, such as decrement in some neuronal populations, inadequate formation of intrinsic cortical circuits and alterations in synaptogenic processes.

[Effects of prenatal exposure to paraquat on the development of amino acid synaptic transmission in mouse cerebral parietal cortex]. / Efectos de la exposición prenatal a paraquat sobre el desarrollo de la transmisión sináptica aminoacídica en la corteza cerebral parietal del ratón.

The effects of prenatal expossure to paraquat (PQ) were studied on postnatal development of mouse parietal cerebral cortex, in particular, the ontogenesis of amino acid synaptic transmission. Pregnant NMRI mice were separated into two groups: the experimental group received 5 doses of 10 mg PQ/kg body weight, between days of gestation (G)12 and G20, whereas the control group received physiological saline solution. Levels of neurotransmitter amino acids: Asp, Glu, Gly, GABA and Tau were determined by HPLC between postnatal (P) days P1 and P30. Between P3 and P15, a significant increment in the levels of excitatory amino acids, Asp and Glu, were observed in mice exposed to PQ, as compared with the control group. With respect to the inhibitory neurotransmitter levels, in the group exposed to PQ, the more important changes were observed in Gly between P1 and P15. In relation to taurine, its levels remained significantly higher between P1 and P7 with respect to the control group. It is important to emphasize that at P30, the levels of all neurotransmitters in the experimental group were significantly lower than those of control. In conclusion, prenatal exposure to PQ caused neurotoxicity in the developing mouse parietal cortex, as shown by the alterations in the basal levels of amino acid neurotransmitters, with the excitatory predominating over inhibitory neurotransmission, throughout the studied developmental period. These alterations could indicate the occurrence of important cortical injuries, such as decrement in some neuronal populations, inadequate formation of intrinsic cortical circuits and alterations in synaptogenic processes.

Antibiotic resistance patterns and SDS-PAGE protein profiles of Burkholderia cepacia complex isolates from nosocomial and environmental sources in Venezuela.

BACKGROUND: Antibiotic resistance and SDS-PAGE protein patterns were determined in nosocomial and environmental isolates of the Burkholderia cepacia complex to assess similarities between them and to identify common protein bands that could be associated with resistance to certain antimicrobial agents. MATERIAL/METHODS: Antibiotic resistance patterns were determined by the disk diffusion method on Mueller-Hinton medium and minimum inhibitory concentrations were obtained by the dilution method on agar. Electrophoresis of whole-cell protein extracts and purified external membrane proteins were performed by SDS-PAGE. Based on resistance to nine antibiotics and the presence or absence of specific protein bands, dendrograms were constructed by the unweighted pair-group using the average linkage clustering method. RESULTS: Both environmental and nosocomial Bcc isolates showed resistance to multiple antibiotics; however, clinical isolates demonstrated two times higher resistance levels than environmental isolates. The Dice index similarity coefficients between environmental and nosocomial strains ranged from 72% to 91.4%. Comparative analysis between common protein bands and antibiotic resistance patterns revealed close association of Mr 135, 76, 72, 53, and 50 kDa with imipenem and aztreonam, Mr 53 and 31 kDa with trimethoprim/sulfamethoxazole, and Mr 50 kDa with ceftazidime resistance. CONCLUSIONS: 1. The environmental and nosocomial Bcc isolates showed a high degree of similarity in their protein profiles. 2. Three common protein bands, i.e. Mr of 31, 50, and 53 kDa, detected in the Bcc isolates from both clinical and natural sources could be associated with resistance to the antimicrobial agents trimethoprim/sulfamethoxazole, ceftazidime, and aztreonam, respectively.

Histotypic mouse parietal cortex cultures: excitation/inhibition ratio and ultrastructural analysis.

Primary cultures of mouse parietal cortex, prepared between postnatal day 3 (P3) and P9, were studied using transmission electron microscopy and HPLC of excitatory (aspartate and glutamate) and inhibitory neurotransmitters (glycine, GABA and taurine) to determine their morphological and functional development. Relations between excitation and inhibition (E/I) were contrasted with ultrastructural features over the time course of in vitro development. After 6 days in vitro, cultured parietal cortex neurons prepared from mice at P3 had immature morphological characteristics, whereas P5 cultures showed a more developed histological structure but still with scarce synapses. The acquirement of histotypic characteristics was seen in P7 cultures, which contained numerous symmetric and asymmetric synaptic contacts. On P9, the cultures showed signs of tissue damage. In terms of neurotransmitter levels and E/I ratios, P7 cultures had relatively low E/I ratio as compared with the rest of the cultures prepared before or after P7. These results demonstrated that the development of inhibitory synaptic transmission, as indicated in the fall of E/I ratio, marked the maturation of cerebral cortical tissue and that the critical period to obtain histotypic cultures of mouse parietal cerebral cortex coincides between P5 and P7. This work provides useful information regarding the balance between excitation and inhibition as an indicative parameter for in vitro nerve cell survival, differentiation and maturation and reinforces the great value of histotypic cultures in the study of central nervous system development.

Effects of prenatal paraquat and mancozeb exposure on amino acid synaptic transmission in developing mouse cerebellar cortex.

The goal of this study was to analyze the effects of prenatal exposure to the pesticides paraquat (PQ) and mancozeb (MZ) on the development of synaptic transmission in mouse cerebellar cortex. Pregnant NMRI mice were treated with either saline, 10 mg/kg PQ, 30 mg/kg MZ or the combination of PQ + MZ, between gestational days 12 (E12) and E20. Variation in the levels of amino acid neurotransmitters was determined by HPLC, between postnatal day 1 (P1) and P30. Motor coordination was assessed by locomotor activity evaluation of control and experimental pups at P14, P21 and P30. Significant reductions in the levels of excitatory neurotransmitters, aspartate and glutamate, were observed in PQ-, MZ- or combined PQ + MZ-exposed pups, with respect to control, during peak periods of excitatory innervation of Purkinje cells: between P2-P5 and P11-P15. However, at P30, lower aspartate contents, in contrast with increased glutamate levels, were detected in all experimental groups. During the first two postnatal weeks, delays in GABA and glycine ontogenesis were observed in PQ- and PQ + MZ-exposed pups, whereas notable decrements in GABA and glycine levels were seen in PQ + MZ-exposed animals. Decreased taurine contents were detected at P3 and P11 in PQ- and PQ + MZ-exposed mice. Pups in different experimental groups all showed hyperactivity at P14 and then exhibited reduced locomotor activity at P30. Taken together, our results indicate that prenatal exposure to either PQ or MZ or the combination of both could alter the chronology and magnitude of synaptic transmission in developing mouse cerebellar cortex.

Prenatal and postnatal contents of amino acid neurotransmitters in mouse parietal cortex.

This study documents the variation in the amino acid neurotransmitter contents during mouse parietal cortex development, from embryonic day 13 (E13) until young adulthood, between postnatal day 21 (P21) and P30. Taurine, an inhibitory neurotransmitter and neuromodulator, is the most abundant neurotransmitter in the developing neocortex, whereas, at the adult stage, glutamate is the more prominent neurotransmitter playing an excitatory role, and GABA is the major inhibitory neurotransmitter. During the proliferative stage of neurogenesis in the mouse cerebral cortex, between E13 and E17, relatively high levels of glutamate, aspartate, taurine and glycine were detected, consistent with a possible trophic influence of these neurotransmitters during cortical development prior to synaptogenesis. Between E17 and E19, a significant decline in the contents of these neurotransmitters was observed, consistent with earlier reports of cell death in the ventricular and subventricular zones during this stage of development. During the perinatal period, a progressive increment in glutamate level was seen between E21 and P5, and then the values remained constant until the second postnatal week. Glutamate also decreased by about 25% between P11 and P15, on the other hand, aspartate diminished by about 20% between P7 and P9. These results were consistent with previous reports of histogenetic cell death during the first 2 postnatal weeks in mouse neocortex. GABA increased from the embryonic period until young adulthood, in contrast, the glycine content decreased; thus, in the adult parietal cortex, the GABA content was about 2.6-fold higher than that of glycine. During the first postnatal week, the concentrations of glutamate and GABA showed significant increments between P0 and P5, while those of aspartate and glycine remained constant. During this period, amino acids are predominantly excitatory and the cerebral cortex is vulnerable to epileptiform activity; the significant increment in taurine content between P0 and P3 suggests a neuroprotective action of taurine against excitotoxicity. At P15, coinciding with the period of maximum cortical synaptogenesis, significant increments in GABA and glycine contents were observed which could be related to the maturation of inhibitory synaptic transmission. At the young adult stage, there was a rise in the levels of both excitatory neurotransmitters, glutamate and aspartate, and a significant reduction in the contents of all three inhibitory neurotransmitters, GABA, glycine and taurine.

Levels of amino acid neurotransmitters during neurogenesis and in histotypic cultures of mouse spinal cord.

The development of spinal cord interneurons and the formation of interneuronal synaptic connections has received little attention; the most comprehensively studied developing circuit has been the connection between motoneurons and the muscle they innervate. All motoneurons are cholinergic whereas spinal interneurons are mostly glutamatergic, glycinergic or GABAergic neurons. In this study, we show quantitative data, obtained by high-pressure liquid chromatography (HPLC), on the levels of amino acid neurotransmitters during mouse spinal cord neurogenesis, from embryonic day (E) 12 until postnatal day (P) 30. At E12, high levels of glutamate, glycine and taurine were already detected but between E16 and P3, significant increments in their contents were observed, indicating the occurrence of maximum synaptogenesis during this period. Important reductions in their contents were also observed in two stages: between E12-E16 and P3-P7. These results suggest that the apoptotic death of interneurons and motoneurons in the developing brain or the synapse refinement of neural circuitry during maturation reduced the number of synapses, thereby decreasing the levels of neurotransmitters. The contents of these neurotransmitters were also analyzed in primary cultures of mouse spinal cord prepared from embryos between E13 and E19. As deduced from light microscopy, ultrastructural studies, as well as results from HPLC analysis, the cultures derived from E15-E16 embryos showed the highest degree of histotypic features and neurotransmitter contents comparable with those obtained in situ. Although glycine, GABA and taurine levels reached about 80-90% of normal in situ values, the contents of aspartate and glutamate were lower by about 40%, which could be mainly due to deafferentation of both sensory and supraspinal afferent axon terminals. These results indicate that intrinsic synaptic circuits can be maintained in histotypic spinal cord cultures prepared from E15-E16 mouse embryos. Histotypic cultures of the spinal cord will serve as a good model for studies on the pathophysiology of amino-acid-based neurotransmission and repair strategies in many CNS disorders.