N-Acetylcysteine Attenuates Cisplatin Toxicity in the Cerebrum and Lung of Young Rats with Artificially Induced Protein Deficiency.

Neurotoxicity is a major obstacle in the effectiveness of Cisplatin in cancer chemotherapy. In this process, oxidative stress and inflammation are considered to be the main mechanisms involved in brain and lung toxicity. The aim of the present work was to study the influence of the amount of protein on some oxidative parameters in the brain and lungs of rats treated with Cisplatin (CP) and N-Acetylcysteine (NAC) as neuroprotectors. Four groups of Wistar rats, each containing six animals, were fed with a protein diet at 7% for 15 days. Thereafter, the groups were given either a unique dose of CP® 5 mg/kg or NAC® 5 mg/kg as follows: group 1 (control), NaCl 0.9% vehicle; group 2, CP; group 3, NAC; and group 4, NAC + CP. The animals were sacrificed immediately after the treatments. Blood samples were collected upon sacrifice and used to measure blood triglycerides and glucose. The brain and lungs of each animal were obtained and used to assay lipid peroxidation (TBARS), glutathione (GSH), serotonin metabolite (5-HIAA), catalase, and the activity of Ca+2, and Mg+2 ATPase using validated methods. TBARS, H2O2, and GSH were found to be significantly decreased in the cortex and cerebellum/medulla oblongata of the groups treated with CP and NAC. The total ATPase showed a significant increase in the lung and cerebellum/medulla oblongata, while 5-HIAA showed the same tendency in the cortex of the same group of animals. The increase in 5-HIAA and ATPase during NAC and CP administration resulted in brain protection. This effect could be even more powerful when membrane fluidity is increased, thus proving the efficacy of combined NAC and CP drug therapy, which appears to be a promising strategy for future chemotherapy in malnourished patients.

Oleic acid reduces oxidative stress in rat brain induced by some anticancer drugs.

Oleic acid (OA) is a monounsaturated compound with many health-benefitting properties such as obesity prevention, increased insulin sensitivity, antihypertensive and immune-boosting properties, etc. The aim of this study was to analyze the effect of oleic acid (OA) and some anticancer drugs against oxidative damage induced by nitropropionic acid (NPA) in rat brain. Six groups of Wistar rats were treated as follows: Group 1, (control); group 2, OA; group 3, NPA + OA; group 4, cyclophosphamide (CPP) + OA; group 5, daunorubicin (DRB) + OA; and group 6, dexrazoxane (DXZ) + OA. All compounds were administered intraperitoneally route, every 24 h for 5 days. Their brains were extracted to measure lipoperoxidation (TBARS), H2O2, Ca+2, Mg+2 ATPase activity, glutathione (GSH) and dopamine. Glucose, hemoglobin and triglycerides were measured in blood. In cortex GSH increased in all groups, except in group 2, the group 4 showed the highest increase of this biomarker. TBARS decrease, and dopamine increase in all regions of groups 4, 5 and 6. H2O2 increased only in cerebellum/medulla oblongata of group 5 and 6. ATPase expression decreased in striatum of group 4. Glucose increased in group 6, and hemoglobin increased in groups 4 and 5. These results suggest that the increase of dopamine and the antioxidant effect of oleic acid administration during treatment with oncologic agents could result in less brain injury.

Neuropsychological Alterations in Patients with Congenital Hypothyroidism Treated with Levothyroxine: Linked Factors and Thyroid Hormone Hyposensitivity.

Eighty-five percent of the studies of patients with congenital hypothyroidism (CH) treated with Levothyroxine (L-T4) report neuropsychological sequelae throughout life. In neonates and infants, there is a deficit in sensorimotor skills (impaired balance). In preschool and elementary school children and adolescents, there are alterations in intellectual quotient (low scores), language (delayed phonological acquisition), memory (visual, verbal, visuospatial, visuoconstructive, autobiographical, and semantic), sensorimotor skills (impaired fine and gross motor control), and visuoconstructive-visuospatial domain (low scores in spatial location, block design, and object assembly). These neuropsychological domains are also affected in young adults, except for language (adequate verbal fluency) and visuoconstructive-visuospatial domain (no data). The onset and severity of neuropsychological sequelae in patients with treated CH depend on several factors: extrinsic, related to L-T4 treatment and social aspects, and intrinsic, such as severity and etiology of CH, as well as structural and physiological changes in the brain. In this review, we hypothesized that thyroid hormone hyposensitivity (THH) could also contribute to neuropsychological alterations by reducing the effectiveness of L-T4 treatment in the brain. Thus, further research could approach the THH hypothesis at basic and clinical levels to implement new endocrinological and neuropsychological therapies for CH patients.

Oseltamivir induces favorable response on oxidative damage in the brain of rats treated with Bezafibrate.

AIM: The purpose was to measure the effect of Oseltamivir on oxidative biomarkers and dopaminergic and serotonergic systems in brain of rats with induced hypotriglyceridemia by Bezafibrate.Male young Wistar rats were treated as follows: group 1, NaCl 0.9%, (Controls); group 2, Oseltamivir (100 mg/kg); group 3, single dose of Bezafibrate (150 mg/kg); group 4, four dose of Bezafibrate; group 5, single dose of Bezafibrate + Oseltamivir and group 6, four doses of Bezafibrate + Oseltamivir. Drugs were given orally. Triglycerides, Dopamine, 5-hydroxyindoleacetic acid (5-HIAA), Glutathione (GSH), Hydrogen peroxide (H2O2), lipid peroxidation, as well as total ATPase activity were measured using validated methods. RESULTS: Oseltamivir treated animals showed lower GSH and lipid peroxidation levels and an increment in 5-HIAA in the three evaluated brain regions. Treatment with Oseltamivir also reduces H2O2 in the cortex and cerebellum/medulla oblongata. ATPase enzyme increased in these regions in the groups that were administered with Bezafibrate in repeated doses and in combination with Oseltamivir in single dose. Dopamine concentrations decreased in groups treated with Oseltamivir in the three evaluated regions. Also, there was a decrease in dopamine concentrations in the cerebellum/medulla oblongata of the animals treated with the combination of Oseltamivir and Bezafibrate.Innovation and conclusion: Animals with bezafibrate induced hypo-triglyceridemia that received Oseltamivir, either in single or repeated doses, have a higher improvement of their antioxidant activity and also experienced changes in the dopaminergic and serotonergic system in their brain, intending establish the beneficial of joint administration of both drugs in obese patients.

Folic acid increases levels of GHS in brain of rats with oxidative stress induced with 3-nitropropionic acid.

Aim: This study tested the hypothesis that folic acid (FA) modulates biogenic amines and protects the brain against oxidative stress induced by 3-nitropropionic acid (3NPA).Methods: Male Wistar rats received (groups of six) for 5 d: FA (50 mg/kg); 3NPA (10 mg/kg); or FA +3NPA. At last day, rats were sacrificed, and their brain was obtained to measure the levels of dopamine, 5-hydroxiindol acetic acid (5-HIAA). Reduced glutathione (GSH), total ATPase, H2O2 and lipid peroxidation were measured.Results: GSH increased significantly in cortex of rats treated with FA. ATPase increased significantly in cerebellum/medulla oblongata and decreased in cortex of animal treated with 3NPA. 5-HIAA increased in striatum of rats that received 3NPA alone or combined with FA.Conclusion: 3NPA generates free radicals such effect can be counteracted with FA administration since this folate increases antioxidant capacity and modulates biogenic amines.

Biochemical and histological changes produced by sweeteners and cytarabine in the brain of young rats.

OBJECTIVE: The aim of this study was to evaluate the effect of splenda and stevia on dopamine and 5-HIAA levels, and some biomarkers of oxidative stress in the presence of cytarabine. METHODS: Forty-eight young male Wistar rats each with a weight of 80 g (four weeks of age), distributed in six groups of eight animals each, were treated as follows: group 1, control (NaCl 0.9% vehicle); group 2, cytarabine (0.6 g/kg); group 3, stevia (0.6 g/kg); group 4, cytarabine + stevia; group 5, splenda; and group 6, cytarabine + splenda. Cytarabine was given intravenously (IV) while stevia and splenda were administered orally for five days, using orogastric tube. At the end of treatment, the animals were sacrificed and glucose levels in blood were measured. The brains were dissected for histological analysis and homogenated to measure levels of dopamine, lipid peroxidation (TBARS), serotonin metabolite (5-HIAA), Na+, K+ ATPase activity, and glutathione (GSH), using validated methods. RESULTS: Sweeteners increased the glucose in animals that received cytarabine. Dopamine increased in cortex and decreased in striatum of animals that received stevia alone and combined with cytarabine. 5-HIAA decreased in striatum and cerebellum/medulla oblongata of animals that received sweeteners and cytarabine alone or combined. GSH increased in animals that received sweeteners and decreased with cytarabine. Lipoperoxidation decreased in groups that received sweeteners and cytarabine. Histopathological changes revealed marked degeneration of neuronal cells in animals treated with cytarabine. CONCLUSION: These results show that sweeteners as stevia or splenda may lead to the onset of unfavorable changes in dopamine and 5-HIAA. Antioxidant effects may be involved. Besides, histological changes revealed marked lesions of neuronal cells in experimental animals treated with cytarabine.

Biochemical and histological changes produced by sweeteners and cytarabine in the brain of young rats / Cambios bioquímicos e histológicos producidos por edulcorantes y citarabina en el cerebro de ratas jóvenes

Objective: The aim of this study was to evaluate the effect of splenda and stevia on dopamine and 5-HIAA levels, and some biomarkers of oxidative stress in the presence of cytarabine. Methods: Forty-eight young male Wistar rats each with a weight of 80 g (four weeks of age), distributed in six groups of eight animals each, were treated as follows: group 1, control (NaCl 0.9% vehicle); group 2, cytarabine (0.6 g/kg); group 3, stevia (0.6 g/kg); group 4, cytarabine + stevia; group 5, splenda; and group 6, cytarabine + splenda. Cytarabine was given intravenously (IV) while stevia and splenda were administered orally for five days, using orogastric tube. At the end of treatment, the animals were sacrificed and glucose levels in blood were measured. The brains were dissected for histological analysis and homogenated to measure levels of dopamine, lipid peroxidation (TBARS), serotonin metabolite (5-HIAA), Na+, K+ ATPase activity, and glutathione (GSH), using validated methods. Results: Sweeteners increased the glucose in animals that received cytarabine. Dopamine increased in cortex and decreased in striatum of animals that received stevia alone and combined with cytarabine. 5-HIAA decreased in striatum and cerebellum/medulla oblongata of animals that received sweeteners and cytarabine alone or combined. GSH increased in animals that received sweeteners and decreased with cytarabine. Lipoperoxidation decreased in groups that received sweeteners and cytarabine. Histopathological changes revealed marked degeneration of neuronal cells in animals treated with cytarabine. Conclusion: These results show that sweeteners as stevia or splenda may lead to the onset of unfavorable changes in dopamine and 5-HIAA. Antioxidant effects may be involved. Besides, histological changes revealed marked lesions of neuronal cells in experimental animals treated with cytarabine (AU)

Objetivo: el objetivo fue evaluar el efecto de edulcorantes (splenda y stevia) sobre los niveles de dopamina, acido 5-hidroxiindolacetico (HIAA) y algunos biomarcadores de estrés oxidativo en presencia de citarabina. Métodos: cuarenta y ocho ratas Wistar machos con un peso aproximado de 80 g (cuatro semanas de edad), distribuidas en seis grupos de ocho animales cada uno, fueron tratados como sigue: grupo 1, control (NaCl 0,9% vehículo); grupo 2, citarabina (0,6 g/kg); grupo 3, stevia (0,6 g/kg); grupo 4, citarabina + stevia; grupo 5, splenda; y el grupo 6, citarabina + splenda. La citarabina fue administrada por vía intravenosa y la stevia y la splenda, por vía oral durante cinco días, utilizando una sonda orogastrica. Al final del tratamiento, los animales fueron sacrificados y se midieron los niveles de glucosa en sangre. Los cerebros fueron disecados para su análisis histológico y homogenizados para medir los niveles de dopamina, peroxidacion lipidica (TBARS), metabolito de la serotonina (5-HIAA), actividad de la Na+, K+ ATPasa y glutatión (GSH), usando métodos validados. Resultados: los edulcorantes aumentaron la glucosa en los animales que recibieron citarabina. La dopamina aumento en la corteza y disminuyo en el estriado de los animales que recibieron stevia sola y combinada con citarabina. La 5-HIAA disminuyo en el estriado y el cerebelo/ medula oblongata de animales que recibieron edulcorantes y citarabina sola o combinada. El GSH se incrementó en los animales que recibieron edulcorantes. La lipoperoxidacion disminuyo en los grupos que recibieron edulcorantes y citarabina. Estudios histopatológicos revelaron una degeneración neuronal importante en animales tratados con citarabina. Conclusión: los resultados muestran que los edulcorantes como stevia o splenda pueden conducir a la aparición de cambios desfavorables en los niveles de dopamina y 5-HIAA. Los cambios histológicos revelaron, además, lesiones marcadas de células neuronales en animales tratados con citarabina (AU)

Effect of cerebrolysin on dopaminergic neurodegeneration of rat with oxidative stress induced by 3-nitropropionic acid.

The study tested the hypothesis that cerebrolysin protects the brain from free radicals in rats treated with 3-nitropropionic acid (3-NPA). To address this hypothesis, the levels of dopamine (DA) and some oxidative stress biomarkers were measured after administration of 3-NPA. Young male Fischer rats were treated for three days with cerebrolysin, 3-NPA or both substances. Their brains were extracted, and DA, lipid peroxidation (LP), glutathione (GSH), calcium, and H2O2 were measured using validated methods. In the cortex, hemispheres and cerebellum/medulla oblongata of the group treated with cerebrolysin and 3-NPA, the levels of DA and LP decreased. In addition, calcium and H2O2 levels decreased in the hemispheres of the same group, while GSH increased in cortex. The increased dopamine metabolism due to the administration of cerebrolysin led to increased formation of radical species and oxidative stress, especially when free radicals were generated by 3-NPA.

Comparative effects of catechin, epicatechin and N-Ω-nitroarginine on quinolinic acid-induced oxidative stress in rat striatum slices.

UNLABELLED: The aim of this work was to compare the effects of catechin (CAT), epicatechin (EPI) and N-ω-l-nitroarginine (L-NARG) on different endpoints of oxidative stress induced by quinolinic acid (QUIN) in a simple tissue preparation, rat striatal slices - with particular emphasis in the glutathione system - in order to provide revealing information on the antioxidant efficacy of these agents in an excitotoxic model. METHODS: Rat striatal slices were incubated for 1h in the presence of 100 µM QUIN and/or 85 µM CAT or EPI, or 100 µM L-NARG. Lipid peroxidation (LP) and the levels of reduced and oxidized glutathione (GSH and GSSG) were determined. RESULTS: The three agents tested completely blocked the QUIN-induced lipid peroxidation and recovered the QUIN-induced altered GSH/GSSG balance. No statistical differences were detected among the protective effects exerted by these antioxidants, suggesting similar efficacy and common antioxidant mechanisms. The antioxidant properties exhibited by these molecules on the excitotoxic model tested herein support an active role of glutathione and prompt their use as therapeutic tools in models of neurodegenerative disorders.

El pirofosfato de tiamina reduce el daño celular inducido por hipoxia en el cerebro de ratas neonatas / Thiamine pyrophosphate reduces hypoxia-induced cellular damage in neonatal rat brain

La encefalopatía por hipoxia es causa de discapacidad y requiere de nuevas estrategias terapéuticas. El pirofosfato de tiamina (PPT) es un cofactor esencial de enzimas fundamentales en el metabolismo de la glucosa, cuya disminución puede conducir a la falla en la síntesis de ATP y a la muerte celular. El objetivo de este estudio fue determinar si la administración de PPT, puede reducir el daño celular en un modelo de hipoxia neonatal en ratas. Animales de 11 días de edad fueron tratados con PPT (130 mg/kg) en dosis única o solución salina, una hora antes del protocolo de hipoxia o al término de ésta. Los cerebros fueron colectados para la evaluación del daño celular. Además, se tomaron muestras sanguíneas para evaluar los indicadores gasométricos de presión de dióxido de carbono (PaCO2) y de oxígeno (PaO2) en sangre arterial y pH. Los resultados muestran que la administración de PPT previa a la inducción de hipoxia, reduce el daño celular y restablece los indicadores gasométricos. Estos datos indican que el uso de PPT reduce el daño inducido por la hipoxia en animales neonatos.

Hypoxic encephalopathy is a leading cause of disability and requires new therapeutic strategies. Thiamine pyrophosphate (TPP) is an essential cofactor of fundamental enzymes involved in glucose metabolism. TPP reduction may lead to ATP synthesis failure and cell death. The objective of this study was to determine if TPP administration can reduce cellular damage in a model of neonatal hypoxia in rats. Eleven day old animals were treated with TPP (130 mg/kg) as a single dose or with saline solution one hour before the hypoxia protocol or after ending the protocol. The brains were collected to evaluate cellular damage. Blood samples were also collected to evaluate arterial oxygen tension (PaO2), carbon dioxide tension (PaCO2) and acidity (pH). The results showed that TPP administration previous to hypoxia induction reduces cellular damage and reestablishes arterial blood gases. These data indicate that TPP use reduces the damage induced by hypoxia in neonatal animals.

Disminución del número de espinas dendríticas en las neuronas piramidales de la corteza motora de ratas bajo estrés prenatal / Diminution of dedritic spines in the piramidal neurons of the motor curtex in rats with prenatal stress

En el presente estudio se analizó el efecto a ratas gestantes sometidos a estrés por inmovilización. Sobre las neuronas piramidales de la capa V motora certica de la progenie, se usaron ratas hembras de la cepa Wistar que se distribuyeron al azar en dos grupos: control y experimental (n=5). Las ratas del grupo experimental fueron sometidos a estrés por inmovilización durante 2 a 6 horas diarias a lo largo de toda la gestación; las ratas grupo control se mantuvieron en condiciones normales de bioterio. Las crías de cada grupo fueron sacrificadas a los 14 y 21 días de edad. Para extraer su cerebro y obtener bloques de corteza motora que se procesaron con la técnica de Golgi Rápido. Se cuantifico el número de espinas dendríticas en segmentos de 50 micras, de una sección de 250 m de la dendrita axonal de neuronas piramidales. En el grupo experimental de 14 días, hubo reducción significativa en el número de espinas dendríticas en los segmentos de 50 a 100 y de 100 a 150 micras respecto al grupo control. Estos hallazgos sugieren que las deficiencias en la capacidad de aprendizaje, comportamiento adaptativo y de alteraciones de la actividad locomotora, reportadas en animales descendientes de madres sometidas a estrés durante la gestación pueden ser resultado de la reducción en la complejidad neuronal.

Efectos del estrés prenatal sobre la ramificación neuronal en la corteza visual de ratas neonatas / Prenatal stress of effects on neuronal branching in the visual cortex of the pup rats

Se evaluó el efecto del estrés por inmovilización aplicado a ratas gestantes, sobre la morfología de las neuronas piramidales en la corteza visual de machos descendientes de dichas hembras, a los 14 y 21 días posnatales. Ratas hembra de la cepa Wistar fueron sometidas a estrés por inmovilización forzada durante toda la gestación, en periodos que variaron entre 2 y 6 horas por día. En neuronas del área visual, impregnadas con el método de Golgi se cuatificó el grado de ramificación dendrítica. Los resultados muestran disminución de ramificaciones dendríticas sobre todo en ratas de 21 días. Las deficiencias en la capacidad de aprendizaje y comportamiento adaptativo observadas en animales descendientes de madres sometidas a estrés durante la gestación podrían explicarse con base a estos resultados

Apertura palpebral prematura experimental y desarrollo neuronal en la rata recién nacida / Experimental premature eyelid openingand neuron development in new-born rats

Los extractos de glándula submaxilar (EGS) de ratón induce apertura prematura de los párpados en ratas recién nacidas. En animales sometidos a ciclos alternos de luz-obscuridad y con EGS, las neuronas de la corteza visual exhiben, en la dendrita principal, más espinas y un mayor grado de ramificación que los controles, en tanto que en animales mantenidos en obscuridad total y con EGS, únicamente disminuyen las espinas dendríticas, mientras que el grado de ramificación es mayor comparado con los controles.

Efecto del tolueno en el desarrollo neuronal de crías de rata, cuyas madres fueron expuestas a inhalación crónica durante gestación o durante su adolescencia / Effects of the neural development of rat pups whose moters were exposed to toluene via chronic inhalation either during pregnancy or adolescence

Desarrollo neuronal en crios de rata adolescentes y gestantes expuestas a inhalación crónica de tolueno. Se sometieron a inhalación de tolueno a tres ratas jóvenes de 70 días de edad y nueve ratas gestantes de 90 días de edad durante 15 minutos por día desde la cruza hasta un día antes de la fecha de parto. Las ratas jóvenes se cruzaron después del período de inhalación con machos que no inhalaron el solvente. Las crias de las hembras tratadas, así como las de un grupo testigo fueron sometidas a iguales condiciones pero sin tolueno. Fueron sacrificadas por perfusión intracardíaca 24 crias de cada grupo, 12 de 14 días y 12 de 21 días de edad. Fue cuantificado el grado de ramificación de los procesos neuronales de las neuronas piramidales de la V capa de la corteza visual impregnadas con el método rápido de Golgi. Se encontró decremento significativo en las ramificaciones en la porción distal de la dendrita principal en los grupos expuestos al solvente comprarados con los grupos testigo y control

Aparición de espinas en la dendrita apical en neuronas piramidales del área 17 de ratas Wistar en el período de lactancia / The appearance of fendritic spines on the apical dentrites of area 17 pyramidal neurons in suckling Wistar rats

Se cuantificó el número de espinas dendríticas en ratas Wistar lactantes normales. En el presente trabajo se describen cambios del número de espinas dendríticas en las neuronas piramidales de rata durante la lactancia. Fueron usadas 22 camadas de ratas Wistar, después del nacimiento las camadas se ajustaron a 8 críos, se obtuvieron un total de 176 críos. Las camadas fueron sacrificadas diariamente desde el nacimiento (día 0) hasta los 21 días posnatales. Se cuantificó el número de espinas en segmento de 50 micrómetros de las dendritas apicales de las células piramidales de la corteza visual impregnadas con el método de Golgi-Kopsch. Las espinas fueron evidentes a partir del día 9, para los días 14 a 19 de densidad de espinas se incrementa y del día 20 al 21 hay disminución de espinas en las dendritas.

Efectos del estrés por inmovilización sobre la corteza motora en la rata / The effects of immobilization-Stress on rat motor cortex

El presente estudio analiza el efecto del estrés por inmovilización aplicado a ratas gestantes sobre las neuronas piramidales de la V capa de la corteza motora de sus descendientes. Se usaron ratas hembra de la cepa Wistar que se distribuyeron al azar en dos grupos: control y experimental (con cinco ratas cada uno). El grupo experimental fue sometido a estrés por inmovilización en períodos que variaron de 2 a 6 horas diarias durante la gestación, el grupo control se mantuvo en condiciones normales de bioterio. Las crías de cada grupo fueron sacrificadas a los 14 y 21 días de edad. Se extrajo el cerebro y se colectaron bloques de la corteza motora que se procesaron con la técnica de Golgi Rápido. Se analizaron las neuronas piramidales de esta área cuantificando el número de intersecciones de las ramificaciones dendríticas con ocho círculos concéntricos. Los resultados mostraron en ambas edades reducción significativa del número de ramificaciones dendríticas en todos los círculos concéntricos en el grupo experimental en comparación con el grupo control. Estos hallazgos indican reducción de la complejidad neuronal que puede ser responsable de deficiencias en la capacidad de aprendizaje, comportamiento adaptativo y de alteraciones de la actividad locomotora reportadas en animales descendientes de madres sometidas a estrés durante la gestación.

Efecto del tolueno en espinas dendríticas descendientes de ratas tratadas en la adolescencia o la gestación / Effects in dendritic spines in rats with a chronic exposure to tolueno

La inhalación crónica de tolueno causa lesiones microscópicas en el sistema nervioso central, tales como acanalamieto del tallo de las neuronas piramidales, hipertrofia neurofibrilar. Tres ratas Wistar jóvenes fueron expuestas a tolueno durante 21 días antes del apareamiento. Otras tres ratas hembras inhalaron vapores de tolueno desde el día 5 hasta el día 21 de la gestación. Después del tratamiento durante 15 minutos/día doce crías de cada grupo se sacrificaron a los 14 y 21 días de edad. Se determinó el número de espinas de la dendrita principal en segmento de 50 micras, de neuronas piramidales de la corteza visual en preparaciones de Golgi. Se encontró reducción significativa del número de espinas dendríticas en los grupos expuestos a tolueno en comparación con animales mantenidos bajo condiciones normales de laboratorio o del grupo sometido a las mismas condiciones pero sin tolueno.