Role of allopregnanolone on cerebellar granule cells neurogenesis.

The role of allopregnanolone on immature cerebellar granule cells (CGC) proliferation was studied. Allopregnanolone (0.1-1 microM) increased [(3)H]thymidine incorporation and cell number determined by neuronal counting and by an MTT colorimetric assay. The effect of the neurosteroid was completely prevented by preincubation with 10 mM MgCl(2), 10 microM nifedipine, 10 microM picrotoxin or by 50 microM bicuculine. We conclude that ALLO affects cerebellar neurogenesis by increasing calcium influx through voltage-gated calcium channels and GABA(A) receptors activation.

Potential dietary exposure to p-nonylphenol from food-contact use of tris(nonylphenyl)phosphite (TNPP).

The potential exposure to p-nonylphenol from the use of tris(nonylphenyl)phosphite (TNPP) in food-contact materials was calculated. The calculation was made on the basis of migration data from moulded plaques prepared from linear low-density polyethylene (LLDPE), films prepared from LLDPE and polyvinyl chloride (PVC) films. The data were obtained using the customary expermental procedures developed by the US Food and Drug Administration (FDA). The potential migration of p-nonylphenol to food from the use of TNPP as a component of food-contact articles was determined using food-simulating solvents and time and temperature conditions recommended by the FDA. Using the data obtained from these studies, along with procedures based on the FDA's conventional method for estimating potential dietary exposure using food simulating migration data, the potential dietary exposure to p-nonylphenol from the use of TNPP was determined to be approximately 25.5 parts per billion (ppb). Because the conditions of the migration tests exaggerate actual use conditions, this value overstates the potential dietary exposure.

A single-cell model to study changes in neuronal fractal dimension.

Many methods have been developed to quantify neuronal morphology: measurement of neurite length, neurite number, etc. However, none of these approaches provides a comprehensive view of the complexity of neuronal morphology. In this work we have analyzed the evaluation of fractal dimension (D) as a tool to represent and quantify changes in complexity of the dendritic arbor, in in vitro cultures grown under low-density conditions. Neurons grown in isolation developed a bipolar morphology corresponding to a fractal dimension close to the unit. The analysis showed that neuronal complexity increased when cells were incubated with a depolarizing potassium concentration and there was a correlation with an increase in fractal dimension (D5 mM KCl = 1.08 +/- 0.01, D25 mM KCl =1.25 +/- 0.01). We conclude that fractal dimension is a suitable parameter to quantify changes in neuronal morphological complexity.

GABA induces proliferation of immature cerebellar granule cells grown in vitro.

The presence of GABA and its receptors early in rodent nervous system development has lead to speculation on the role of this transmitter system in neuroblast proliferation, migration and differentiation. We studied the effect of GABA and GABA agonists on immature cerebellar granule cell proliferation and survival. Cerebellar granule cell suspensions were obtained from 6-8-day-old rats and grown in culture for up to 7 days in serum-containing or serum-free medium. The addition of GABA (0.1-100 microM) or muscimol (0.01-10 microM) 2 h after inoculation and harvested 22 h later, lead to an increase in 3H-thymidine incorporation over control samples with the correspondent increase in granule cells number assayed 48 h later. The effect on cell proliferation exerted by GABAA agonists was blocked by MgCl2 and nifedipine, as well as by the chloride channel blocker, picrotoxin (50 microM), and the GABAA receptor specific blocker, bicuculline (50 microM). The increase on cell proliferation induced by GABA also was blocked by PD98059 (75 microM), a specific inhibitor of the mitogen-activated protein kinase kinase (MAPKK). GABAA receptor-mediated proliferation was consistently seen in cells inoculated in serum-containing medium supplemented with 25 mM KCl but not seen in serum-free medium, with 5 mM or 25 mM KCl. The presence of serum did not enhance the survival of cerebellar granule cells grown for 7 days in either 5 mM or 25 mM KCl. Additionally, neither GABA nor muscimol applied from day 2 to day 7 in vitro affected cell survival in any culture condition. We conclude that GABA and GABAA receptor agonists influence granule cell proliferation but not survival and that this effect is mediated by a calcium influx via voltage-dependent calcium channel activation, with a subsequent activation of the MAPK cascade.

Cu/Zn superoxide dismutase activity at different ages in sporadic amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive disorder resulting from degeneration of motor neurons in the brain and spinal cord. Sporadic ALS (SALS) accounts for the majority of patients and the familial form (FALS) represents fewer than 10% of all cases. Since it was found that there are Cu/Zn superoxide dismutase (SODI) gene mutations in 20% of FALS patients and that FALS and SALS patients show similar clinical features, it has been postulated that both may share a common physiopathological mechanism. We studied Cu/Zn SOD1 activity in cytosolic extracts of erythrocytes from 125 normal individuals and 40 SALS patients. We found that enzyme activity does not change with age in control subjects and tends to decrease in most SALS patients older than 60 years. A subpopulation of five SALS patients had significantly increased SOD1 activity; four of these patients over 70 years old. There was no correlation between enzyme activity and time of onset of the disease, or clinical forms of the illness. The variation in SOD1 activity in ageing SALS patients compared with younger patients suggests that they may undergo an oxidative disbalance contributing to the development of the disease.

Potential exposure to bisphenol A from food-contact use of polycarbonate resins.

The potential exposure to bisphenol A (BPA) from the use of consumer products or packages made from bisphenol A-derived polycarbonate resins was calculated. The calculation was made on the basis of migration data from moulded discs prepared from a composite of polycarbonate resins. The data were obtained using the customary experimental procedures developed by the United States Food and Drug Administration (FDA). The potential migration of BPA to food from its contact with articles made from polycarbonate resins was determined using food-simulating solvents and time and temperature conditions recommended by FDA. The study demonstrates that no detectable BPA was found in the extracts obtained under FDA's most severe default testing condition using a method sensitive to 5 parts per billion (ppb) in the food simulants. Using these data, along with FDA's conventional procedure for estimating potential dietary exposure using food simulating migration data, the potential dietary exposure to bisphenol A from use of polycarbonate resins was determined to be less than 0.25 ppb.

Extracellular potassium concentration regulates proliferation of immature cerebellar granule cells.

The present study examines the effect of depolarizing potassium concentrations on the proliferation of immature rat cerebellar neurons. Cells inoculated in serum free medium and 5 mM KCl (5 K) showed a high degree of 3H-thymidine incorporation that decreased 24-48 h after plating as differentiation began. During the first 24 h after inoculation, cells grown in high potassium (25 K), showed a 34 +/- 3% increase (mean +/- S.E.M., n = 12) in 3H-thymidine incorporation as compared with the values observed in 5 K. After 24 h in vitro, cells grown in 25 K showed 23 +/- 3% (mean +/- S.E.M., n = 3) less DNA synthesis than those inoculated in 5 K. The increase in DNA synthesis due to 25 K was blocked by MgCl2 and nifedipine, but not by omega-conotoxin GVIA, suggesting that it is mediated by a Ca2+ influx via voltage-gated calcium channels (VGCC) of the L-subtype. High potassium-induced cell proliferation was blocked by the mitogen-activated protein kinase kinase (MEK1) inhibitor (PD98059, 75 microM). The number of neurons counted after 48 h in vitro in 25 K was 35-100% above of the number obtained with 5 K and this increase also was blocked by MgCl2 and nifedipine. These data support the hypothesis that depolarizing activity during neurogenesis plays a role in the modulation of cerebellar granule cells proliferation.

Assessment of polystyrene extract for estrogenic activity in the rat uterotrophic model and an in vitro recombinant receptor reporter gene assay.

The purpose of the study was to determine whether polystyrene used in food-contact applications would elicit an estrogenic response when extracts simulating exaggerated conditions of use were subjected to in vivo and in vitro tests. A sample of polystyrene was subjected to extraction conditions that simulate, or exaggerate, the actual food-contact uses of polystyrene to maximize the amount of low molecular weight polystyrene extractables. The food-simulating solvent and the time and temperature conditions recommended by the Food and Drug Administration (FDA) were selected to maximize the level of extractable components from polystyrene. The extract was examined for its estrogenic response in vivo using the immature rat uterotrophic assay and in vitro using an estrogen receptor (ER)-mediated recombinant receptor reporter gene assay. In vivo, the uterine weights of juvenile female Sprague Dawley rats (10 rats/group) were determined after oral gavage exposure to the extract (two dosage levels: one represents the maximum potential daily human exposure to polystyrene extractables and the other represents one-tenth of the maximum exposure level), vehicle control (sesame oil), or positive control [diethylstilbestrol (DES), at 200 micrograms/kg body weight]. In addition, five treatment groups were dosed by subcutaneous injection of either estradiol (1, 50, and 500 micrograms/kg body weight) or DES (2 and 200 micrograms/kg body weight). Dosing began on postnatal day (pnd) 21 and continued daily through pnd 23. Body weights were collected at study initiation (pnd 21) and at necropsy (pnd 24). Body weights were not different statistically between treatment groups at study initiation or at necropsy. Uterine wet weights and uterine weights relative to body weights were significantly increased (p < 0.05) for estradiol at 50 and 500 micrograms/kg, DES at 2 and 200 micrograms/kg, and DES at 200 micrograms/kg (oral) over vehicle control. The polystyrene extract had no effect on uterine wet weight or uterine weights relative to body weights at either level tested. An in vitro recombinant estrogen receptor/reporter gene assay that involved transiently transfecting MCF-7 human breast cancer cells with the chimeric human ER, Ga14-HEGO, consisting of the yeast Ga14 DNA binding domain linked to the ligand binding domain of the human ER and a Ga14 response element (17mer)-regulated reporter gene (17m5-G-Luc) was employed. Dose-dependent induction of the reporter gene, 17m5-G-Luc, was observed with the positive control, 17 beta-estradiol (E2). Induction of greater than 100-fold was obtained following incubation of transfected MCF-7 cells with 10 nM E2 for 24 hours. No induction of reporter gene activity was observed with the polystyrene extracts dissolved in dimethylsulfoxide (0.01, 0.1 or 0.01 mg/ml) using the same assay conditions. These results indicate that polystyrene extract does not elicit ER-mediated activity using the Ga14-HEGO/17m5-G-Luc recombinant receptor/reporter gene assay. In conclusion, extracts from polystyrene produced no estrogenic response in either the rat uterotrophic assay or the MCF-7 cell assay for estrogen receptor-mediated activity.

Neurosteroid modulation of GABAA receptors in the developing rat brain cortex.

The allosteric modulation of GABAA receptors in the rat brain cortex by neurosteroids was studied at different developmental stages. GABAA receptors were identified using [3H]muscimol binding to membrane preparations obtained from embryos and neonates (postnatal day 0-PN0; postnatal day 5-PN5). Data analysis disclosed a unique population of binding sites at all ages tested. An increase in the number of receptors was observed during development reaching almost adult levels at PN5. The neurosteroids pregnanolone and allopregnanolone failed to modulate [3H]muscimol specific binding in embryos and neonates, but a positive modulation was obtained in 5-day old animals. The addition of 1 microM pregnanolone induced a 3-6-fold increase [3H]muscimol affinity in PN5 (n = 3; P < 0.03), and a 2-fold increase in receptors number in adults (n = 3; P < 0.03). The differences observed in allosteric modulation during development suggest that a change occurred during the first week of life, and this change might affect GABAA receptor function.

Using a simple diffusion model to predict residual monomer migration--considerations and limitations.

A simple mathematical equation based on a diffusion model has been utilized recently to estimate migration of both acrylonitrile and styrene from polymers produced from these monomers which are used under a wide variety of food-contact applications. These calculated migration values have subsequently been used to estimate the US consumer's exposure to acrylonitrile and styrene from food stored in these materials. The basic assumptions integral to the model are discussed in relation to potential errors in migration estimates that could be experienced if the assumptions are not true. In addition to the discussion of the basic assumptions, factors affecting the migration predictions such as polymer 'ageing', temperature changes during the lifetime of the polymeric article, the effects of polymer-modifying materials (plasticizers, impact modifiers), and the physical form of the article or test sample are discussed.