Protective efficacy of ramelteon on methotrexate-induced DNA damage.

Ramelteon (RMLT) is a melatonin receptor agonist that it has antioxidative and anti-inflammatory effects associated with DNA damage through different mechanisms of action. In this regard, we investigated the potential usefulness of RMLT as a protective agent against methotrexate (MTX)-induced DNA damage. Four groups were constituted from 32 Wistar albino rats: Negative control, RMLT, MTX, and MTX + RMLT. Twenty mg/kg MTX (i.p., single dose) and RMLT 10 mg/kg (oral, 7 days) was administered. Comet assay was used and the parameter %TailDNA was used to detect DNA damage. %TailDNA was 4.90 ± 0.19 in the control group, 7.85 ± 0.33 in the MTX group, 5.49 ± 0.24 in the RMLT group, and 5.86 ± 0.23 in the MTX + RMLT group. While there was a significant increase in DNA damage in the MTX-treated group compared to the control group, there was a significant reduction in DNA damage in the MTX + RMLT group, compared to the MTX group (p < 0.001). In conclusion, it was observed that combined treatment with RMLT significantly reduced MTX-induced DNA damage.

Investigate the possible protective effect of RMLT against DNA damage caused by MTX using the comet method.The DNA damage of RMLT treated group was significantly reduced compared to group and MTX group. (p < 0.001).Combined treatment with MTX significantly reduces MTX-induced DNA damage.

The association between gestational diabetes mellitus and DNA damage in umbilical cord leukocytes and placental samples.

Objective(s): To evaluate the relation between gestational diabetes mellitus (GDM) and maternal and/or fetal DNA integrity. Method: 59 pregnant women were classified into two groups on the basis of 75 g oral glucose tolerance test (OGTT) and glycemic profile (GP): Control group (OGTT and GP normal, n = 29) and GDM group (abnormal 75 g OGTT, n = 30). The umbilical cord blood and placental samples obtained from the maternal side were collected at the time of delivery. Alkaline comet assay was performed for the determination of DNA damage. The trial was approved with the protocol number 72867572.050.01.04-299082. Result(s): Body mass index (BMI), weight gain during pregnancy, glycemic means and fetal weight were increased in GDM group compared control group (p = .01, .0001, .04, and .01, respectively). In the GDM group, the number of large-for-gestational-age (LGA) infants was significantly higher compared to the nondiabetic group (p = .04). Tail DNA percentages in placental samples were higher in the GDM group compared to controls (p = .01); however, DNA integrity in umbilical cord leukocytes was similar between the groups (p = 0.1). In contrast to umbilical cord DNA damage, placental DNA damage showed positive correlation with maternal glycemia in the whole group and within each group. The positive association of placental DNA damage and GDM remained after adjusting for age, BMI, smoking, glycemia, gestational age at delivery, fetal weight at delivery, and delivery type (p = .01). Conclusion(s): Placental DNA damage is associated with GDM and placental cells seem to be more vulnerable to DNA damage compared to fetal blood cells.

The association between age-related infertility and deoxyribonucleic acid (DNA) integrity parameters of granulosa cells and lymphocytes.

This study aimed to investigate the association between deoxyribonucleic acid (DNA) integrity parameters and advanced maternal age (AMA)-related infertility. The granulosa cells and the lymphocytes obtained from 119 infertile women were recruited. Patients were divided into two groups: the AMA group (≥35 years, n = 26) and the non-AMA group (<35 years, n = 93). The tail length, tail moment and tail DNA percentage were evaluated as the DNA integrity parameters using comet assay. Infertility duration (p=.001), luteinising hormone (p=.01) and progesterone levels (p<.0001) were higher and smoking was more prevalent in the AMA group (p=.001). AMA group was stimulated with higher gonadotropin doses (p=.04) and had decreased anti-mullerian hormone levels (p<.0001). All of DNA integrity parameters were distributed homogenously between the groups; however, the tail length of lymphocytes was higher (p=.02) in the AMA group. Fertilisation was lower (p=.02), oocyte quality was tended to be poor (p=.03) and blastocyst transfer was lower in the AMA group (p=.03). Embryo quality was distributed homogenously between the groups. Implantation, clinical pregnancy and live birth rates were similar between the groups. Impact StatementWhat is already known on this subject? Advanced maternal age (AMA)-related infertility is associated with diminished ovarian reserve and alteration in follicular environment resulting in poor oocyte quality; however, the exact pathophysiologic mechanism is not clear.What do the results of this study add? Tail length, tail deoxyribonucleic acid (DNA) percentage, tail moment of granulosa cells were nonsignificantly higher in the AMA group compared to younger patients. All of the DNA integrity parameters of lymphocytes were nonsignificantly higher; however, only tail length of lymphocytes was statistically higher in the AMA group than the non-AMA group. A positive correlation was observed between DNA integrity parameters of lymphocytes and body mass index. There were no correlations between DNA integrity parameters of granulosa cells and lymphocyte and infertility duration, gonadotropin dose, duration of ovarian stimulation, oocyte score, embryo score, basal hormone levels and anti-mullerian hormone levels.What are the implications of these findings for clinical practice and/or further research? Our findings offer new insight for further understanding the role of granulosa cells in mediating the poor reproductive outcome of ageing patients. Understanding the mechanisms of ovarian ageing and poor oocyte quality in women with AMA may help to identify specific targets for improving oocyte quality with ageing.

The efficiency of oxerutin on apoptosis and kidney function in rats with renal ischemia reperfusion injury.

BACKGROUND: Background: Renal ischemia-reperfusion injury (RIRI) is the most frequent cause of acute renal failure in clinical conditions such as trauma and shock as well as renal surgeries. Oxerutin is a member of the flavonoid family and possesses antioxidant properties. The aim of this study was to investigate whether oxerutin has protective effects on RIRI. METHODS: Twenty-eight male Wistar albino rats were randomly divided into three groups: sham control group (n=8), RIRI group (n=10), and RIRI + oxerutin group (n=10). RIRI was achieved by clamping the left renal artery for 30 min, followed 1-h reperfusion period. Thereafter, blood samples and left kidney tissue samples were taken for histopathological and biochemical examination. Blood urea nitrogen (BUN), urea, creatinine, and cystatin C levels, which are indicators of kidney function, as well as tumor necrosis factor-alpha, which is an indicator of inflammation were analyzed in blood samples. Total antioxidant status and total oxidant status (TOS), which are indicators of oxidative stress were analyzed on renal tissues. The apoptotic index, an indicator of kidney damage, as well as histopathological changes were evaluated on renal tissues. RESULTS: The apoptotic index, TOS, tumor necrosis factor-alpha, BUN, and urea levels were lower in the RIRI + oxerutin group than in the RIRI group (p<0.05). The results demonstrated that the histopathological and biochemical properties of oxerutin protected rats from RIRI. CONCLUSION: The findings obtained in this study show that prophylactic administration of oxerutin has protective effects on apoptosis and renal failure caused by RIRI. Therefore, oxerutin can be used as an effective prophylactic agent in the treatment of RIRI.

Effect of methylenetetrahydrofolate reductase gene polymorphisms and oxidative stress in silent brain infarction.

Ischemic infarctions occur under the influence of genetic and environmental factors. In our study, the role of ischemia-modified albumin and thiol balance, which are new markers in determining oxidative damage together with MTHFR gene polymorphisms and homocysteine levels, in the development of SBI was investigated. White matter lesions in the magnetic resonance imaging (MRI) results of the patients were evaluated according to the Fazekas scale and divided into groups (Grade 0, 1, 2, and 3). Homocysteine, folate, B12, IMA, total thiol, and native thiol were measured by biochemical methods. The polymorphisms in MTHFR genes were investigated by the RT-PCR method. According to our results, a significant difference was found between the groups in age, homocysteine, folate, IMA, total thiol, and native thiol parameters (p < 0.05). When we compared the groups in terms of genotypes of the C677T gene, we found a significant difference in TT genotype between grades 0/3 and 1/3 (p < 0.05). We determined that homocysteine and IMA levels increased and folate levels decreased in CC/TT and CT/TT genotypes in the C677T gene (p < 0.05). Considering our results, the observation of homocysteine and IMA changes at the genotype level of the MTHFR C677T gene and between the groups, and the deterioration of thiol balance between the groups suggested that these markers can be used in the diagnosis of silent brain infarction.

DNA methylation profiles of genes associated with angiogenesis in the samples of placenta in pregnancies complicated by intrauterine growth restriction.

BACKGROUND: Impairment in placental angiogenesis is blamed for the etiopathogenesis of intrauterine growth restriction (IUGR). AIM: To assess the genes related to angiogenesis in placental biopsies of pregnancies complicated by IUGR that could be aberrantly methylated and adversely affect placental angiogenesis. METHODS: The methylation profiles of soluble fms-like tyrosine kinase-1 (sFLT-1), vascular endothelial growth factor (VEGF), and the placental growth factor (PIGF) were evaluated using Illumina MiSeq™ System in placental biopsies from term IUGR pregnancies without preeclampsia (n = 18) and healthy controls (n = 17). DNA was isolated from samples of tissue collected from the fetal side of the placenta. In the targeted regions, we have identified 30, 24, and 29 CpG islands (CpGi) within sFLT-1, VEGF and PIGF genes, respectively. CpGi which are most methylated in the promoter regions of three genes were selected for the study from the database http://www.ensembl.org. RESULT(S): IUGR fetuses had significantly lower placental and fetal birth weight than controls. The promoter of sFLT-1 at three CpGi and VEGF at six CpGi were the regions with significant methylation differences between IUGR and control placentas. sFLT-1 was hypermethylated at 265 and 352 CpGi; however, hypermethylation was lower in IUGR group compared to control group at this position. sFLT-1 was hypomethylated at 456 CpGi in IUGR group and hypermethylated at the same region in control group. VEGF was hypomethylated at 668, 703, and 710 CpGi in control and IUGR groups; however, hypomethylation at these positions was significantly higher in control group compared to IUGR. 776, 845, and 863 CpGi of VEGF promoter were significantly hypermethylated in IUGR group whereas hypomethylated in control group. The methylation profile of PIGF did not differ between the groups. After adjustment for the factors known to affect fetal birth weight, DNA methylation of VEGF 668 CpGi had a significant negative association with fetal birth weight in the control and the IUGR group and a positive association with IUGR pregnancies. CONCLUSION(S): Our results do not support the hypothesis that altered DNA methylation in the placental angiogenic genes is a major mechanism generally involved in IUGR. Only a specific region (at 668 CpGi) corresponding to the promoter of VEGF may serve as an epigenetic marker of IUGR and may be involved in the mechanism of IUGR. Large sample-sized studies are needed to understand the effects of DNA methylation on placental gene function and how they might influence fetal growth.

Comparison of DNA damage and proliferative capacities in smear samples of HPV positive and negative patients by micronucleus counting and AgNOR staining.

Human papillomavirus (HPV) is believed to cause cervical cancer. Thousands of women develop cancer and other diseases caused by HPV each year. HPV 16 and 18 types are found in approximately 70% of cervical cancers. Micronuclei are small chromosomal fragments that are considered indicators of DNA damage. AgNOR positive dots are useful for assessing proliferation. We investigated the relation between HPV-DNA, micronuclei and AgNOR in smear samples. Three groups were defined: HPV negative, 16/18 positive and other high-risk groups (31, 33, 35, 39, 45, 51, 52, 56, 58, 66 and 68) (HR). After typing, micronuclei were identified by Papanicolaou staining and AgNOR regions were detected by silver staining. Serum reactive protein (CRP) also was measured. We found that the average age of HPV negative patients was significantly greater than for the HPV positive groups. We also found that CRP levels were significantly higher in the HPV 16/18 positive group than HPV negative and other HPV group. We found that the number of micronuclei in the HPV 16/18 group was significantly greater than for the HPV negative group. Also, we found that AgNOR staining for the HPV 16/18 group was significantly greater than for the HPV negative group. We found that CRP level, cell proliferation and genome instability were increased in HPV positive patients. The AgNOR and micronucleus tests were useful for evaluating cell proliferation and DNA damage.

Selenium potentiates the anticancer effect of cisplatin against oxidative stress and calcium ion signaling-induced intracellular toxicity in MCF-7 breast cancer cells: involvement of the TRPV1 channel.

BACKGROUND: In breast cancers, calcium signaling is a main cause of proliferation and apoptosis of breast cancer cells. Although previous studies have implicated the transient receptor potential vanilloid 1 (TRPV1) cation channel, the synergistic inhibition effects of selenium (Se) and cisplatin in cancer and the suppression of ongoing apoptosis have not yet been investigated in MCF-7 breast cancer cells. This study investigates the anticancer properties of Se through TRPV1 channel activity in MCF-7 breast cancer cell line cultures when given alone or in combination with cisplatin. MATERIALS: The MCF-7 cells were divided into four groups: the control group, the Se-treated group (200 nM), the cisplatin-treated group (40 µM) and the Se + cisplatin-treated group. RESULTS: The intracellular free calcium ion concentration and current densities increased with TRPV1 channel activator capsaicin (0.01 mM), but they decreased with the TRPV1 blocker capsazepine (0.1 mM), Se, cisplatin, and Se + cisplatin incubations. However, mitochondrial membrane depolarization, apoptosis, and the caspase 3, and caspase 9 values increased in the Se-treated group and the cisplatin-treated group, although Western blot (procaspase 3 and 9) results and the cell viability levels decreased with the Se and Se + cisplatin treatments. Apoptosis and caspase-3 were further increased with the Se + cisplatin treatment. Intracellular reactive oxygen species production increased with the cisplatin treatment, but not with the Se treatment. CONCLUSION: This study's results report, for the first time, that at a cellular level, Se and cisplatin interact on the same intracellular toxic cascade, and the combination of these two drugs can result in a remarkable anticancer effect through modulation of the TRPV1.

The efficiency of Poly(ADP-Ribose) Polymerase (PARP) cleavage on detection of apoptosis in an experimental model of testicular torsion.

The aim of this study was to evaluate the histopathological and apoptotic changes occurring in the rat ipsilateral and contralateral testes, after experimental spermatic cord torsion, and to explore and the role of poly(ADP-ribose) polymerase (PARP) cleavage in testicular torsion-detorsion injury. A total of 37 Wistar albino rats were subjected to 720° unilateral spermatic cord torsion for 1, 2 and 4 h, followed by 4-h reperfusion, or else to a sham operation (control group). Histology of the testicle was evaluated using haematoxylin-eosin (H&E) staining and Johnsen's scoring system. Germ cell apoptosis was evaluated via active caspase-3 immunostaining, and PARP expression levels were evaluated via Western blotting. The mean Johnsen's tubular biopsy scores (JTBS) of the ipsilateral testicles were lower for all torsion groups than for the controls (P < 0.05), but the JTBS of the contralateral testicles were only lower in the 4-h torsion group (P < 0.05). The mean apoptosis score (AS) of the ipsilateral and contralateral testicles was significantly higher in the torsion groups than in the sham group. AS increased correlatively with torsion time, in both testicles. The effect of testicular torsion on PARP cleavage was time dependent, with the highest effect observed after 4 h of testicular torsion (P < 0.05). Testicular torsion caused time-dependent histological changes, apoptosis and increases in PARP cleavage. Our results suggest that testicular torsion-detorsion injury caused cell damage and germ cell apoptosis that apparently involved cleavage of PARP. Increased PARP cleavage could, in turn, lead to enhanced apoptosis.