Toxicity of bisphenol A and its replacements in the mice Leydig cells in vitro.

The aim of the study was to examine the potential impacts of bisphenol A (BPA) and its analogues BPB, BPF, and BPS on mice TM3 Leydig cells, with respect to basal cell viability parameters such as metabolic activity, cell membrane integrity, and lysosomal activity after 48-h exposure. In addition, monitoring of potential bisphenol´s actions included evaluation of ROS production and gap junctional intercellular communication (GJIC) complemented by determination of testosterone secretion. Obtained results revealed significant inhibition in mitochondrial activity started at 10 microg/ml of bisphenols after 48-h exposure. Cell membrane integrity was significantly decreased at 5 microg/ml of BPA and BPF and 10, 25, and 50 microg/ml of BPA and BPS. The lysosomal activity was significantly affected at 10, 25, and 50 microg/ml of applied bisphenols. A significant overproduction of ROS was recorded mainly at 5 and 10 microg/ml of tested compounds. In addition, significant inhibition of GJIC was observed at 5 microg/ml of BPB followed by a progressive decline at higher applied doses. In the case of testosterone production, a significant decline was confirmed at 10, 25 and 50 microg/ml.

The effect of Apium graveolens L., Levisticum officinale and Calendula officinalis L. on cell viability, membrane integrity, steroidogenesis, and intercellular communication in mice Leydig cells in vitro.

Several plants have the potential to protect essential reproductive processes such as spermatogenesis or steroidogenesis, however, effective concentrations and main mechanisms of action are still unknown. This in vitro study was aimed to assess the effects of Apium graveolens L., Levisticum officinale, and Calendula officinalis L. extracts on the structural integrity, functional activity and gap junctional intercellular communication (GJIC) in mice Leydig cells. TM3 cells were grown in the presence of experimental extracts (37.5; 75; 150 and 300 µg/ml) for 24 h. For the present study, high-performance liquid chromatography analysis was used to quantify flavonoids or phenolic acids. Subsequently, Leydig cell viability was assessed by alamarBlue assay, while the cell membrane integrity was detected by 5-carboxyfluorescein diacetate-acetoxymethyl ester. The level of steroid hormones production was determined by enzyme-linked immunosorbent assay. Additionally, GJIC was assessed by scalpel loading/dye transfer assay. According to our results, Apium graveolens L. significantly increased the viability and cell membrane integrity at 75 µg/ml (109.0±4.3%) followed by a decline at 300 µg/ml (89.4±2.3%). In case of Levisticum officinale and Calendula officinalis L. was observed significant decrease at 150 µg/ml (88.8±11.66%; 87.4±6.0%) and 300 µg/ml (86.2±9.3%; 84.1±4.6%). Furthermore, Apium graveolens L. significantly increased the progesterone and testosterone production (75 and 150 µg/ml) however, Levisticum officinale and Calendula officinalis L. significantly reduced steroid hormones synthesis at 150 and 300 µg/ml. Finally, the disturbance of GJIC was significantly affected at 300 µg/ml of Levisticum officinale (82.5±7.7%) and Calendula officinalis L. (79.8±7.0%). The balanced concentration ratio may support the Leydig cell function, steroidogenesis as well as all essential parameters that may significantly improve reproductive functions.

Investigation of the Properties and Effects of Salvia Officinalis L. on the Viability, Steroidogenesis and Reactive Oxygen Species (ROS) Production in TM3 Leydig Cells in Vitro.

The aim of our study was to reveal the in vitro effects of Salvia officinalis L. (37.5, 75, 150, 200, 250, 300 and 600 µg/ml) extract on the TM3 Leydig cell viability, membrane integrity, steroidogenesis and reactive oxygen species production after 24 h and 48 h cultivation. For the present study, the extract prepared from Salvia officinalis L. leaves was analysed by high performance liquid chromatography (HPLC) for selected flavonoids and phenolic acids followed by a determination of its free radicals scavenging activity (DPPH). Furthermore, Leydig cell viability was assessed by the mitochondrial toxicity assay (MTT), while the membrane integrity was evaluated by 5- carboxyfluorescein diacetate-acetoxymethyl ester (5-CFDA-AM). The level of steroid hormones was performed by enzyme-linked immunosorbent assay (ELISA) from the culture media, while the superoxide radical generation was measured by the nitroblue tetrazolium chloride (NBT) assay. The results show that experimental concentrations did not damage the cell membrane integrity and viability when present at below 300 µg/ml, it was only at 600 µg/ml that a significant (P<0.05) cell viability decline was observed after a 48 h cultivation. A significant (P<0.05) stimulation of testosterone secretion was recorded at 250 µg/ml for 24 h, while the prolonged cultivation time significantly (P<0.05) increased the testosterone and progesterone production at 150, 200, 250 and 300 µg/ml. Furthermore, none of the selected doses exhibited significant ROS-promoting effects however, the highest dose of Salvia initiated the free radical scavenging activity in cultured mice Leydig cells.

Assessment of the effective impact of bisphenols on mitochondrial activity and steroidogenesis in a dose-dependency in mice TM3 Leydig cells.

The increasing worldwide production of bisphenols has been associated to several human diseases, such as chronic respiratory and kidney diseases, diabetes, breast cancer, prostate cancer, behavioral troubles and reproductive disorders in both sexes. The aim of the present in vitro study was to evaluate the potential impact bisphenols A, B, S and F on the cell viability and testosterone release in TM3 Leydig cell line. Mice Leydig cells were cultured in the presence of different concentrations of bisphenols (0.04-50 µg.ml-1) during 24 h exposure. Quantification of the cell viability was assessed using the metabolic activity assay, while the level of testosterone in cell culture media was determined by enzyme-linked immunosorbent assay. Within the panel of substances under investigations, the higher experimental concentrations (10; 25 and 50 µg.ml-1) significantly (P<0.001) decreased Leydig cells viability, while the same doses of BPA and BPB also reduced testosterone production significantly (P<0.001). Taken together, the results of our study reported herein is a consistent whit the conclusion that higher experimental doses of bisphenols have a cytotoxic effect and could have a dose-dependent impact on testosterone production.

Protective Effects of Quercetin on Selected Oxidative Biomarkers in Bovine Spermatozoa Subjected to Ferrous Ascorbate.

Quercetin (QUE) is a natural flavonol-type flavonoid with antibacterial, anti-inflammatory and anti-aggregatory properties. It is also a powerful reactive oxygen species (ROS) scavenger and chelating agent. The aim of this study was to assess the effectiveness of QUE to reverse ROS-mediated alterations to the motility, viability and intracellular antioxidant profile of bovine spermatozoa. Spermatozoa were washed out of fresh bovine semen, suspended in 2.9% sodium citrate and subjected to QUE treatment (7.5, 25, 50 and 100 µmol/l) in the presence or absence of a pro-oxidant, that is ferrous ascorbate (FeAA; 150 µmol/l FeSO4 and 750 µmol/l ascorbic acid) during a 6-h in vitro culture. Spermatozoa motion characteristics were assessed using the SpermVision computer-aided sperm analysis (CASA) system. Cell viability was examined with the metabolic activity (MTT) assay, ROS generation was quantified via luminometry, and the nitroblue tetrazolium (NBT) test was applied to quantify the intracellular superoxide formation. Cell lysates were prepared at the end of the in vitro culture to investigate the intracellular activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) as well as the concentrations of glutathione (GSH) and malondialdehyde (MDA). FeAA treatment led to a reduced sperm motility (p < 0.001), viability (p < 0.001) and decreased the antioxidant parameters of the sperm samples (p < 0.001) but increased the ROS generation (p < 0.001), superoxide production (p < 0.001) and lipid peroxidation (p < 0.001). QUE administration resulted in a preservation of the spermatozoa vitality and antioxidant characteristics (p < 0.01 with respect to the enzymatic antioxidants, p < 0.001 in relation to GSH) with a concentration range of 50-100 µmol/l QUE revealing to be the most effective. Our results suggest that QUE exhibits significant ROS-scavenging and metal-chelating properties which may prevent spermatozoa alterations caused by ROS, and preserve the functionality of male reproductive cells.

Aerosol radioactivity record in Bratislava/Slovakia following the Fukushima accident--a comparison with global fallout and the Chernobyl accident.

Results of radioactivity measurements in Bratislava aerosols following the Fukushima accident showed that at least three radioactive plumes arrived to Bratislava as indicated by (131)I/(137)Cs activity ratios. When compared with the Chernobyl results available for the Bratislava station, the Fukushima radionuclide levels were almost five orders of magnitude lower, with the maximum values for (131)I and (137)Cs of 0.5 and 0.07 mBq/m(3), respectively. The (131)I and (137)Cs vs. (7)Be aerosol activity records showed that the increases in (131)I and (137)Cs activity concentrations were accompanied by (7)Be increases, indicating that both the horizontal and vertical transports of radionuclides were responsible for observed radionuclide concentrations. The (134)Cs/(137)Cs activity ratio was close to 1, as has also been reported by other investigators.

[Doppler ultrasonographic velocimetry of the central retinal artery and central retinal vein]. / Doppler-ultrasonografická velocitometria arteria centralis retinae a vena centralis retinae.

A blood flow rate in the arteria and vena centralis retinae was measured by Doppler-ultrasonographic method in a group of 36 patients of an average age of 59.35 years. The patients did not suffer any cardiovascular disease, glaucoma, diabetes mellitus-factors which influence the blood viscosity. In the arteria centralis retinae, there was measured a maximum systolic rate of 10.25 +/- 2.66 cm/s and a end diastolic rate of 5.24 +/- 1.27 cm/s. In the vena centralis retinae, there was measured an average maximum flow rate of 5.89 +/- 1.72 cm/s and end diastolic rate of 4.1 +/- 1.28 cm/s (minimum 2.12; maximum 6.2 cm/s). A procedure of examination and terms of its optimalisation are described there.

Effect of supplemental sodium bicarbonate on nutrient digestibilities and ruminal pH measured continuously.

A technique was used to monitor continuously ruminal pH using a strip-chart recording pH meter. Ruminal pH measurements were made in four ruminal-cannulated crossbred wether lambs (ag initial weight, 42.5 kg). For l.5 h daily, lambs were given ad libitum access to 50% concentrate-50% chopped orchardgrass hay diets supplements with 0, l.5, 3.0 and 4.5% sodium bicarbonate (NaHCO3). A split-plot Latin-square design was used to evaluate NaHCO3 level and day of adaptation on the percentage of time (%T) that ruminal pH was less than 6.6, 6.2, 5.8, 5.4 and 5.0. No effect due to day of adaptation existed for ruminal pH measurements (P more than .10), while the effect of dietary NaHCO3 level was quadratic (P more than .01) for the %T that ruminal pH was less than 5.4. To evaluate the effects of NaHCO3 on nutrient digestion, the same diets were fed to eight wether lambs (avg initial weight, 38 kg) at 85% of their ad libitum intake in a replicated 4 x 4 Latin-square digestion trial. Digestibilities of dry matter (DM), organic matter, N and fiber fractions were not different due to level of NaHCO3 (P greater than .10). Ash digestibility increased with increasing levels of dietary NaHCO3 (P less than .01). Four ruminal-cannulated mature Hereford steers were also given ad libitum access to the diets in a split-plot Latin-square trial to evaluate effects of dietary NaHCO3 level on ruminal pH and in situ digestion of soybean meal N and orchardgrass DM. During incubation of the dacron bags for 3, 6, 9, 12, 24 and 36 h also increased linearly with increasing level of NaHCO3. Ruminal solid and liquid dilution rates were not affected by level of supplemental NaHCO3 (P greater than .10). The results of these trials suggest that increasing level of dietary NaHCO3 greatly increases the proportion of time ruminal pH is above critical levels for ruminal protein and dry matter digestion, but does not affect total tract nutrient digestion when 50% concentrate diets are fed.