Dietary biotin supplementation increases proliferation pathways in mice testes without affecting serum follicle-stimulating hormone levels and stem cell factor expression.

Supplements containing pharmacological concentrations of biotin are commercially available. The mechanisms by which biotin at pharmacological concentrations exerts its action have been the subject of multiple investigations, particularly for biotin's medicinal potential and wide use for cosmetic purposes. Several studies have reported that biotin supplementation increases cell proliferation; however, the mechanisms involved in this effect have not yet been characterized. In a previous study, we found that a biotin-supplemented diet increased spermatogonia proliferation. The present study was focused on investigating the molecular mechanisms involved in biotin-induced testis cell proliferation. Male BALB/cAnNHsd mice were fed a control or a biotin-supplemented diet (1.76 or 97.7 mg biotin/kg diet) for eight weeks. Compared with the control group, the biotin-supplemented mice presented augmented protein abundance of the c-kit-receptor and pERK1/2Tyr204 and pAKTSer473, the active forms of ERK/AKT proliferation signaling pathways. No changes were observed in the testis expression of the stem cell factor and in the serum levels of the follicle-stimulating hormone. Analysis of mRNA abundance found an increase in cyclins Ccnd3, Ccne1, Ccna2; Kinases Cdk4, Cdk2; and E2F; and Sp1 & Sp3 transcription factors. Decreased expression of cyclin-dependent kinase inhibitor 1a (p21) was observed but not of Cdkn2a inhibitor (p16). The results of the present study identifies, for the first time, the mechanisms associated with biotin supplementation-induced cell proliferation, which raises concerns about the effects of biotin on male reproductive health because of its capacity to cause hyperplasia, especially because this vitamin is available in large amounts without regulation.

Dietary Biotin Supplementation Impairs Testis Morphology and Sperm Quality.

Supplements containing pharmacological concentrations of biotin are commercially available over the counter. Classical toxicity studies have considered biotin administration as harmless; however, recent investigations have shown that biotin supplementation modifies tissue morphology without changes in toxicity markers, raising concerns about the consequences of morphological changes on tissues' functions and the safety of pharmacological concentrations of the vitamin. Testes are very sensitive to toxicants, and testicular histology is a reliable method to study its function. In this work, we investigated the effects of dietary biotin supplementation on testis morphology and spermatogenesis function using an experimental model, in which we have not observed unfavorable effects on other tissue functions or toxicity markers. Male BALB/cAnNHsd mice were fed a control or a biotin-supplemented diet (1.76 or 97.7 mg biotin/kg diet) for 8 weeks. Compared to the control group, the biotin-supplemented mice presented remarkable testis morphology changes, including increased spermatogonia layers; the cellular mechanism involved is related to increased proliferation. Sperm count and serum testosterone levels were not affected, but spermatozoa motility and morphology were significantly impaired in the biotin-supplemented mice. These results caution against the use of supplements with high concentrations of biotin and indicate that biotin's pharmacological effects on morphology need to be considered in toxicological studies.

Effects of Biotin Supplementation During the First Week Postweaning Increases Pancreatic Islet Area, Beta-Cell Proportion, Islets Number, and Beta-Cell Proliferation.

During maturation, pancreatic islets achieve their full capacity to secrete insulin in response to glucose, undergo morphological changes in which alpha-cells decrease and beta-cell mass increases, and they acquire the normal alpha- and beta-cell proportion changes that are important for islet functions later in life. In rodents, the first week of postweaning is critical for islet maturation. Multiple studies have documented the detrimental effects of several conditions on pancreatic maturation; however, few studies have addressed the use of pharmacological agents to enhance islet maturation. Biotin might have a potential action on islet maturation. Pharmacological concentrations of biotin have been found to modify islet morphology and function. In a previous study, we found that mice fed a biotin-supplemented diet for 8 weeks after weaning showed an increase in basal and glucose stimulated insulin secretion, enlarged islet size, and modified islet structure. In the present study, we investigated the effect of biotin on maturation features during the first week postweaning. Female BALB/cAnN Hsd mice were fed a control or a biotin-supplemented diet for 1 week after weaning. Compared with the control, biotin-supplemented mice showed an increase in pancreatic islet number and area in addition to an augmented proportion of beta-cells in the islet. These effects were related to an increase in beta-cell proliferation. No differences were found in insulin secretion, blood glucose concentrations, or serum insulin levels. These results indicate that biotin supplementation is capable of affecting beta-cell proliferation and might be a therapeutic agent for establishing strategies for regenerative medicine.

Dietary Biotin Supplementation Modifies Hepatic Morphology without Changes in Liver Toxicity Markers.

Pharmacological concentrations of biotin have pleiotropic effects. Several reports have documented that biotin supplementation decreases hyperglycemia. We have shown that a biotin-supplemented diet increased insulin secretion and the mRNA abundance of proteins regulating insulin transcription and secretion. We also found enlarged pancreatic islets and modified islet morphology. Other studies have shown that pharmacological concentrations of biotin modify tissue structure. Although biotin administration is considered safe, little attention has been given to its effect on tissue structure. In this study, we investigated the effect of biotin supplementation on hepatic morphology and liver toxicity markers. Male BALB/cAnN Hsd mice were fed a control or a biotin-supplemented diet for 8 weeks. Versus the control mice, biotin-supplemented mice had an altered portal triad with dilated sinusoids, increased vascularity, and bile conducts. Furthermore, we observed an increased proportion of nucleomegaly and binucleated hepatocytes. In spite of the liver morphological changes, no differences were observed in the serum liver damage indicators, oxidative stress markers, or antioxidant enzymes. Our data demonstrate for the first time that biotin supplementation affects liver morphology in normal mice, and that these modifications are not paralleled with damage markers.

Toxicological evaluation of the natural products and some semisynthetic derivatives of Heterotheca inuloides Cass (Asteraceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Heterotheca ineuloides Cass (Asteraceae), popularly known as árnica mexicana, is widely used in Mexican traditional medicine to treat bruises, dermatological problems, rheumatic pains, and other disorders as cancer. The major constituents in H. inuloides are cadinane type sesquiterpenes, flavonoids and phytosterols. Compounds with a cadinane skeleton have been proved to possess cytotoxic activity against human-tumor cell lines and brine shrimp, and display toxic effects in different animal species. Although this plant has been widely used, there is little available information on the safety and toxicity especially of pure compounds. AIM OF THIS STUDY: Evaluate the potential toxicity of the natural products isolated from H. inuloides and some semisynthetic derivatives. MATERIALS AND METHODS: The toxic aspects of the following natural products isolated from dried flowers of H. inuloides: 7-hydroxy-3,4-dihydrocadalene (1), 7-hydroxycadalene (2), 3,7-dihydroxy-3(4H)-isocadalen-4-one (3), (1R,4R)-1-hydroxy-4H-1,2,3,4- tetrahydrocadalen-15-oic acid (4), D-chiro-inositol (5), quercetin (6), quercetin-3,7,3'-trimethyl ether (7), quercetin-3,7,3',4'-tetramethyl ether (8), eriodictyol-7,4'-dimethyl ether (9), α-spinasterol (10), caryolan-1,9ß-diol (11) and 7-(3,3-dimethylallyloxy)-coumarin (12) as well as the toxic aspects of the semisynthetic compounds 7-acetoxy-3,4-dihydrocadalene (13), 7-benzoxy-3,4-dihydrocadalene (14), 7-acetoxycadalene (15), 7-benzoxycadalene (16), quercetin pentaacetate (17), 7-hydroxycalamenene (18), 3,8-dimethyl-5-(1-methylethyl)-1,2-naphthoquinone (19), and 4-isopropyl-1,6-dimethylbenzo[c]oxepine-7,9-dione (20). Toxic activities of compounds were determined by sulforhodamine B (SRB) assay, Artemia salina assay, RAW264.7 macrophage cells. Additionally, the acute toxicity in mouse of compound 1, the major natural sesquiterpene isolated from the acetone extract, was evaluated. RESULTS: The best cytotoxicity activity was observed for mansonone C (19) on K562 cell line with IC50 1.45 ± 0.14 µM, for 7-hydroxycadalene (2) on HCT-15 cell line with IC50 18.89 ± 1.2 µM, and for quercetin pentaacetate (17) on MCF-7 cell line with IC50 22.57 ± 2.4 µM. Sesquiterpenes mansonone C (19) and 7-hydroxy-3,4-dihydrocadalene (1) caused the strongest deleterious effects against A. salina with IC50 39.4 ± 1.07, and 45.47 ± 1.74 µM, respectively. The number of viable RAW 264.7 cells was reduced with sesquiterpenes 1 and 2 by more than 90%. In addition, the acute study of 1 revealed no lethal effects at 300 mg/kg body weight, however, a reduction in the body weight of mice, morphological changes in the tissues of the liver and kidney and toxic signs were observed at very high doses (2000 mg/kg). CONCLUSION: The results provided evidence for the cytotoxicity of Mexican arnica (H. inuloides) metabolites and may be correlated with one of the popular uses of this plant, in traditional Mexican medicine, as anticancer remedy. Among the active compounds contained in the acetone extract, the cytotoxic activity is mainly ascribable to cadinene type sesquiterpenes. In addition, evidence of acute toxicity suggests that 7-hydroxy-3,4-dihydrocadalene (1) may lead to toxicity at very high doses.

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

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

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.

Purkinje cell density in cerebella of alcoholized and non-alcoholized male rat offspring

The effect of alcohol intake by male rats was evaluated on Purkinje cell morphology and number in their offspring. Forty five male Wistar rats, 45 days old, were used and divided into three groups of 15 rats each: control group (CG), fed with conventional Purina rodent feed (CPRF) and water ad libitum; experimental group (EG), fed with CPRF ad libitum and a mixture of water/ethanol, which represented 36 per cent of kiloclories in food; and an equinergetic intake control group (ECG), which was given CPRF (in grams) and sugar in their drinking water, in order to substitute the energetic value provided by alcohol. Five subgroups (n=3) were created to be used for different treatment periods: 60, 90,120, 150 and 180 days; all groups started treatment period when they were 70 days old. At the end of each treatment period, male rats were mated with nulliparous females not having undergone treatment. Offspring were obtained and studied at 14 and 21 days of age. The Purkinje cells of the cerebella of 14-and 21-day-old offspring belonging to the CG and ECG showed no morphological changes. On the other hand, in 14-day-old offspring belonging to the experimental group of parents alcoholized during 90, 120, and 180 days, a large number of hyperchromatic Purkinje cells were seen, forming zones of cells undergoing a degenerative process. No significant differences in cellular density were determined between the CG and the ECG. When comparing the CG vs. EG and the ECG vs. Eg, significant differences were found in the 14-day-old offspring as well as in the 21-day-old ones with a p<0.05 of rats belonging to parents alcoholized for 90, 120, and 180 days. The results may indicate that there are changes in the germinal plasma of males fue to alcohol of males due to alcohol sunsumption; therefore, reflecting this effect on a drecrease of Purkinje cells and probably on ther cell populations