Corrigendum to: Betaine ameliorates high glucose-induced oxidative stress in granulosa cells.

CONTEXT: In diabetes, abnormalities of granulosa cells (GCs) and steroidogenesis are associated with hyperglycaemia-induced oxidative stress. Betaine has beneficial effect in experimental model of diabetes by reducing oxidative stress, inflammation, and apoptosis. AIMS: In this study we investigate the effects of betaine to prevent oxidative stress in GCs induced by high glucose and improve steroidogenesis. METHODS: Primary GCs, isolated from ovarian follicles of C57BL/6 mice were cultured in 5mM (control) and 30mM (hyperglycaemia) of glucose and in presence of 5mM of betaine for 24h. Then antioxidant enzymes, malondialdehyde, oestradiol and progesterone were measured. In addition, the expression of Nrf2 and NF-κB , antioxidant enzymes (Sod1 , Gpx and Cat ) were analysed by qRT-PCR assay. KEY RESULTS: We observed significant (P NF-κB and down-regulation of Nrf2 due to high concentration of glucose. Also significant (P Cat , Sod1 and GPx ) and activity reduction of these enzymes as well as significant (P NF-κB and up-regulating the expression of Nrf2 , Cat , Sod1 and GPx . It was also shown that betaine in the presence of FSH significantly (P Conclusion: Betaine compensated the antioxidant stress in mouse GCs under hyperglycaemic condition via regulation of Nrf2/NF-κB at transcription level. IMPLICATIONS: As betaine is a natural product and no side effect has been reported to today, we suggest more research needs to be carried out especially on patients whom suffer from diabetes to find the probability of using betaine as a therapeutic agent.

Betaine ameliorates high glucose-induced oxidative stress in granulosa cells.

CONTEXT: In diabetes, abnormalities of granulosa cells (GCs) and steroidogenesis are associated with hyperglycaemia-induced oxidative stress. Betaine has beneficial effect in experimental model of diabetes by reducing oxidative stress, inflammation, and apoptosis. AIMS: In this study we investigate the effects of betaine to prevent oxidative stress in GCs induced by high glucose and improve steroidogenesis. METHODS: Primary GCs, isolated from ovarian follicles of C57BL/6 mice were cultured in 5mM (control) and 30mM (hyperglycaemia) of glucose and in presence of 5mM of betaine for 24h. Then antioxidant enzymes, malondialdehyde, oestradiol and progesterone were measured. In addition, the expression of Nrf2 and NF-κB , antioxidant enzymes (Sod1 , Gpx and Cat ) were analysed by qRT-PCR assay. KEY RESULTS: We observed significant (P <0.001) up-regulation of NF-κB and down-regulation of Nrf2 due to high concentration of glucose. Also significant (P <0.001) down-regulation of related antioxidant genes (Cat , Sod1 and GPx ) and activity reduction of these enzymes as well as significant (P <0.001) elevation of malondialdehyde was observed. In addition, betaine treatment compensated the drastic effect of high glucose induced oxidative stress via down-regulating the expression of NF-κB and up-regulating the expression of Nrf2 , Cat , Sod1 and GPx . It was also shown that betaine in the presence of FSH significantly (P <0.001) restored the oestradiol and progesterone level. CONCLUSION: Betaine compensated the antioxidant stress in mouse GCs under hyperglycaemic condition via regulation of Nrf2/NF-κB at transcription level. IMPLICATIONS: As betaine is a natural product and no side effect has been reported to today, we suggest more research needs to be carried out especially on patients whom suffer from diabetes to find the probability of using betaine as a therapeutic agent.

Silymarin modulates cadmium-induced oxidative stress in human spermatozoa.

Environmental pollutants such as cadmium can negatively affect sperm parameters and decrease male fertility by inducing oxidative stress. Antioxidants are considered a useful strategy for oxidative stress conditions to neutralize free radicals and strengthen the antioxidant defence system. In this study, the effects of the common application of silymarin, as a natural antioxidant, with cadmium were assessed on human sperm. The washed human sperm samples were divided into five groups: (1) spermatozoa at 0- hour; (2) spermatozoa at 3 h; (3) spermatozoa treated with cadmium (20 µM) for 3 h; (4) spermatozoa treated with silymarin (2 µM) + cadmium (20 µM) for 3 h and (5) spermatozoa treated with silymarin (2 µM) for 3 h. Our results displayed that cadmium reduced sperm motility, viability, plasma membrane integrity and acrosome integrity by increasing malondialdehyde levels and decreasing the total antioxidant capacity and antioxidant enzymes activity. While silymarin attenuated oxidative stress biomarkers in human sperm treated with cadmium, and consequently improved the sperm quality. In summary, cadmium-induced oxidative stress impaired human sperm structures and silymarin with its antioxidant properties compensated for the adverse effects of oxidative stress on human spermatozoa.

Quantitative evaluation of human sperm viability using MTT assay: A laboratory study.

BACKGROUND: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay which evaluates cellular mitochondrial activity is widely used for the assessment of cell proliferation and viability. OBJECTIVE: This study was performed to assess human sperm viability using MTT assay. MATERIALS AND METHODS: In this laboratory study, human-ejaculated semen samples (n = 56 from different donors) were used. The sperm viability was determined using quantitative MTT assay and the sperm motility was assessed according to World Health Organization guidelines. Sperm viability and the correlation between sperm viability and motility were analyzed. RESULTS: Data revealed a marked positive correlation between MTT reduction rate and the percentage of viable spermatozoa. The Pearson's correlation coefficients also showed a significant correlation between sperm viability and motility. CONCLUSION: MTT assay which is based on mitochondrial functionality is a reliable method for evaluating human sperm viability and could be used as a diagnostic test for predicting sperm fertilization ability in clinical settings.

Exogenous Nitric Oxide Induced Early Mineralization in Rat Bone Marrow Mesenchymal Stem Cells via Activation of Alkaline Phosphatase

Background: Since the low concentration and short-time treatment with sodium nitroprusside (SNP), a nitric oxide (NO)­donor, cause no harm to rat bone marrow mesenchymal stem cells (MSCs), we studied the impact of SNP on MSCs differentiation. Methods: MSCs were treated with 100 and 1000 µM of SNP for 1 hour in every 48 hours and after 5, 10, 15, and 21 days in osteogenic media. The viability and the level of mineralization were determined using MTT assay and alizarin red staining, respectively. Morphology of the cells was studied using fluorescent dye. Concentration of calcium and the activity of alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) were evaluated by commercial kits. Results: SNP with the concentration of 1000 µM significantly reduced viability from day 5 to day 20, but 100 µM did not affect the viability until the day 15. The low concentration of SNP increased matrix deposition from day 10 and reached almost its maximum (4.40 ± 2.4) at the day 15. Also, increasing the activity of ALP (419 ± 2.2), due to low concentration of SNP, started at day 10 and continued till the day 20, while LDH (2026 ± 11) and AST (25.6 ± 0.4) elevations were observed from day 5 onwards. In case of ALT, we observed a significant decrease (36%) from day 5 till day 20. Conclusion: Based on our findings, low concentrations of SNP might be useful in the promotion of bone repair.

The toxic effect of gallic acid on biochemical factors, viability and proliferation of rat bone marrow mesenchymal stem cells was compensated by boric acid.

OBJECTIVE: Gallic acid (GA) and boron are found in many plants. Our previous studies showed 6 ng/ml boric acid (BA) had positive effect on biochemistry of rat bone marrow mesenchymal stem cells (MSCs) and their osteogenic differentiation. Therefore, we investigate the effect of different doses of GA alone and in the presence of BA on MSCs. MATERIALS AND METHODS: the viability of MSCs was assayed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue at 12, 24 and 36 h in presence of different concentration of GA. Then 30 and 120 µM of GA as well as 6 ng/ml of BA in 36 h were selected for further study. The proliferation, Morphology, sodium and potassium level, concentration of calcium, activity of alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) as well as malondialdehyde (MDA) concentration, total antioxidant capacity (FRAP) and activity of superoxide dismutase (SOD) and catalase (CAT) were estimated. RESULT: Results showed GA alone reduced viability, proliferation, nuclear diameter and cytoplasm area. In addition, GA showed anaerobic metabolic shift but no change in MDA and scavenging enzymes. Both concentration of GA caused elevation of FRAP, whereas only at 120 µM increased the sodium-potassium and reduced calcium. The co-treatment of GA and BA improves the viability, proliferation and morphology of the cells. In addition, co-treatment compensated the metabolic shift caused by GA and could balance the potassium level and FRAP as it was raised by GA. CONCLUSION: Although GA content of tea is harmful to the cells but simultaneous consumption of fruits and vegetables as a rich source of boron might compensate the damaging effect of GA.

Sodium Nitroprusside Changed The Metabolism of Mesenchymal Stem Cells to An Anaerobic State while Viability and Proliferation Remained Intact.

OBJECTIVE: We used sodium nitroprusside (SNP), a nitric oxide (NO) releasing molecule, to understand its effect on viability and proliferation of rat bone marrow mesenchymal stem cells (BM-MSCs). MATERIALS AND METHODS: This experimental study evaluated the viability and morphology of MSCs in the presence of SNP (100 to 2000 µM) at 1, 5, and 15 hours. We chose the 100, 1000, and 2000 µM concentrations of SNP for one hour exposure for further analyses. Cell proliferation was investigated by the colony forming assay and population doubling number (PDN). Na+, K+, and Ca2+ levels as well as activities of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), aspartate transaminase (AST), and alanine transaminase (ALT) were measured. RESULTS: The viability of MSCs dose-dependently reduced from 750 µM at one hour and 250 µM at 5 and 15 hours. The 100 µM caused no change in viability, however we observed a reduction in the cytoplasmic area at 5 and 15 hours. This change was not observed at one hour. The one hour treatment with 100 µM of SNP reduced the mean colony numbers but not the diameter when the cells were incubated for 7 and 14 days. In addition, one hour treatment with 100 µM of SNP significantly reduced ALT, AST, and ALP activities whereas the activity of LDH increased when incubated for 24 hours. The same treatment caused an increase in Ca2+ and reduction in Na+ content. The 1000 and 2000 µM concentrations reduced all the factors except Ca2+ and LDH which increased. CONCLUSION: The high dose of SNP, even for a short time, was toxic. The low dose was safe with respect to viability and proliferation, especially over a short time. However elevated LDH activity might increase anaerobic metabolism.

Boron Induces Early Matrix Mineralization via Calcium Deposition and Elevation of Alkaline Phosphatase Activity in Differentiated Rat Bone Marrow Mesenchymal Stem Cells.

OBJECTIVE: Boron (B) is essential for plant development and might be an essential micronutrient for animals and humans. This study was conducted to characterize the impact of boric acid (BA) on the cellular and molecular nature of differentiated rat bone marrow mesenchymal stem cells (BMSCs). MATERIALS AND METHODS: In this experimental study, BMSCs were extracted and expanded to the 3rdpassage, then cultured in Dulbecco's Modified Eagle's Medium (DMEM) complemented with osteogenic media as well as 6 ng/ml and 6 µg/ml of BA. After 5, 10, 15 and 21 days the viability and the level of mineralization was determined using MTT assay and alizarin red respectively. In addition, the morphology, nuclear diameter and cytoplasmic area of the cells were studied with the help of fluorescent dye. The concentration of calcium, activity of alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) as well as sodium and potassium levels were also evaluated using commercial kits and a flame photometer respectively. RESULTS: Although 6 µg/ml of BA was found to be toxic, a concentration of 6 ng/ml increased the osteogenic ability of the cell significantly throughout the treatment. In addition it was observed that B treatment caused the early induction of matrix mineralization compared to controls. CONCLUSION: Although more investigation is required, we suggest the prescription of a very low concentration of B in the form of BA or foods containing BA, in groups at high risk of osteoporosis or in the case of bone fracture.

Low dose and long term toxicity of sodium arsenite caused caspase dependent apoptosis based on morphology and biochemical character.

OBJECTIVE: Although arsenite is toxic it is currently recommended for the treatment of malignancies. In this study the effects of sub-micromolar concentrations of sodium arsenite on the viability, morphology and mechanism of cell death of rat bone marrow mesenchymal stem cells (BMCs) over 21 days was investigated. MATERIALS AND METHODS: In this experimental study, BMCs were extracted in Dulbecco's Modified Eagles Medium (DMEM) containing 15% of fetal bovine serum (FBS) and expanded till the 3(rd) passage. The cells were treated with 1, 10, 25, 50, 75 and 100 nM of sodium arsenite for 21 days and the viability of the cells estimated using 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium (MTT) and trypan blue staining. Cells were then treated with the selected dose (25 nM) of sodium arsenite to determine their colony forming ability (CFA) and population doubling number (PDN). Morphology of the cells was studied using florescent dyes, and the integrity of the DNA was investigated using the comet assay and agarose gel electrophoresis. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and the caspase 3 assay were then applied to understand the mechanism of cell death. Data was analyzed using one way ANOVA, Tukey test. RESULTS: A significant reduction of viability, PDN and CFA was found following treatment of BMCs with 25 nM sodium arsenite (p<0.05). Cytoplasm shrinkage and a significant decrease in the diameter of the nuclei were also seen. Comet assay and agarose gel electrophoresis revealed DNA breakage, while positive TUNEL and activated caspase 3 confirmed the apoptosis. CONCLUSION: A low concentration of sodium arsenite (25 nM) caused reduction of viability due to induction of apoptosis. Therefore, long term exposure to low dose of this chemical may have unwanted effects on BMCs.