Asuman Sunguroglu

Effects of antioxidants on post-thawed bovine sperm and oxidative stress parameters: antioxidants protect DNA integrity against cryodamage.

This study was conducted to determine the effects of methionine, inositol and carnitine on sperm (motility, abnormality, DNA integrity and in vivo fertility) and oxidative stress parameters (lipid peroxidation, total glutathione and antioxidant potential levels) of bovine semen after the freeze-thawing process. Nine ejaculates, collected with the aid of an artificial vagina twice a week from each Simmental bovine, were included in the study. Each ejaculate, splitted into seven equal groups and diluted in Tris-based extender containing methionine (2.5 and 7.5 mM), carnitine (2.5 and 7.5 mM), inositol (2.5 and 7.5 mM) and no additive (control), was cooled to 5 °C and then frozen in 0.25 ml straws. Frozen straws were then thawed individually at 37 °C for 20s in a water bath for the evaluation. The extender supplemented with 7.5 mM doses of carnitine and inositol led to higher subjective motility percentages (61.9±1.3% and 51.3±1.6%) compared to the other groups. The addition of methionine and carnitine at doses of 2.5 and 7.5 mM and inositol at doses of 7.5mM provided a greater protective effect in the percentages of total abnormality in comparison to the control and inositol 2.5 mM (P < 0.001). As regards CASA motility, 7.5 mM carnitine (41.6±2.9% and 54.2±4.9%) and inositol (34.9±2.0% and 47.3±2.2%) caused insignificant increases in CASA and total motility in comparison to the other groups. All of the antioxidants at 2.5 and 7.5 mM resulted in lower sperm with damaged DNA than that of control, thus reducing the DNA damage (P < 0.05). No significant differences were observed in CASA progressive motility and sperm motion characteristics among the groups. In fertility results based on 59-day non-returns, no significant differences were observed in non-return rates among groups. As regards biochemical parameters, supplementation with antioxidants did not significantly affect LPO and total GSH levels in comparison to the control group (P > 0.05). The maintenance of AOP level in methionine 2.5 mM was demonstrated to be higher (5.06±0.38 mM) than that of control (0.96±0.29 mM) following the freeze-thawing (P < 0.001). Supplementation with these antioxidants prior to the cryopreservation process protected the DNA integrity against the cryodamage. Furthermore, future research should focus on the molecular mechanisms of the antioxidative effects of the antioxidants methionine, carnitine and inositol during cryopreservation.

Association of CYP1A1 and glutathione S-transferase polymorphisms with male factor infertility

OBJECTIVE To examine whether a relationship exists between genetic polymorphisms of glutathione S-transferase (GST) M1 and T1, CYP1A1(*)2C, and male factor infertility. DESIGN Genetic polymorphism analysis, case-control study. SETTING University research laboratory and andrology clinic. PATIENT(S) One hundred ten men with infertility and 105 healthy fertile men were recruited for the study. INTERVENTION(S) Physical examination of the genitalia of patients, scrotal colored Doppler ultrasound examination, and blood sampling were performed for DNA extraction and genotyping. MAIN OUTCOME MEASURE(S) CYP1A1(*)2C, GSTM1, and GSTT1 polymorphism genotypes were determined by polymerase chain reaction and restriction fragment length polymorphism methods. Seminal parameters were analyzed. RESULT(S) There were significant differences between infertility and GSTM1, CYP1A1(*)2C genotypes by univariate analyses. A subject carrying CYP1A1 Val/Val or CYP1A1 Ile/Val in association with GSTM null genotype has 6.90 times more risk to be infertile than a subject carrying CYP1A1 Ile/Ile in association with GSTM1 wild-type genotype (odds ratio: 6.90, 95% confidence interval: 2.29-19.3). No correlation was found between the seminal parameters and the genetic variability. CONCLUSION Our results suggest that genetic polymorphisms of xenobiotic-metabolizing enzymes could play an important role in infertility.

Impact of genetic variations of the CYP1A1, GSTT1, and GSTM1 genes on the risk of coronary artery disease.

Carcinogenic and toxic molecules produce DNA adducts that contribute to the development of atherosclerosis. Genetic polymorphisms of xenobiotic-detoxified enzymes, which control the level of DNA adducts, may affect both enzymatic activity and individual susceptibility to coronary artery disease (CAD). In this study we investigated the effects of genetic polymorphisms of the CYP1A1*2C, GSTT1, and GSTM1 enzymes on CAD risk in a Turkish population. Genotypes were determined for 132 CAD patients and 151 healthy controls by the polymerase chain reaction/restriction fragment length polymorphism method. There were no significant differences between patients and controls in terms of CYP1A1, GSTT1, and GSTM1 genotypes. Analysis of the possible interactions between the genotypes, after adjustment for the risk factors, demonstrated that individuals carrying CYP1A1 variant GSTT1 null genotypes had an 8.907-fold increased CAD risk compared to their wild status (p<0.05). We suggest that genetic polymorphisms of xenobiotic-metabolizing enzymes could play an important role in CAD. Therefore, CYP1A1 and GSTM1 polymorphisms should be considered as important parameters for the prediction of CAD.

Common fragile sites associated with the breakpoints of chromosomal aberrations in hematologic neoplasms.

Fragile sites are specific regions of chromosomes prone to breakage when cells are cultured under specific conditions. These sites are divided into two classes: common and rare. Common fragile sites are expressed in all individuals at different frequencies, whereas rare ones are found only in certain individuals. Common and rare fragile sites have been shown to display a number of characteristics of instability being preferential sites for chromosomal deletions, duplications, and rearrangements. Moreover, a majority of mapped oncogenes are located at or near these fragile sites. These observations have led to the suggestion that both classes of fragile sites may play a significant role in chromosomal rearrangements involved in oncogene activation or tumor supressor gene inactivation. For these reasons, involvement of common and rare fragile sites and their relevance to specific chromosome breakpoints in cancer have received much attention. In this study, which reports on the cytogenetic findings obtained from 256 patients with chronic myelocytic leukemia, 103 with acute myelocytic leukemia, 40 with acute lymphocytic leukemia, 33 with myelodysplastic syndrome, we documented the fragile sites involved in chromosomal aberrations involving oncogenes, tumor supressor genes, and other known genes important in cell cycle regulation localized at these sites.

Effect of L-carnitine on the synthesis of nitric oxide in RAW 264·7 murine macrophage cell line

L‐Carnitine (β‐hydroxy‐γ‐trimethyl aminobutyric acid) plays a critical role in inflammatory diseases by modulating inflammatory cell functions. Inducible nitric oxide synthase (iNOS), a proinflammatory enzyme responsible for the generation of nitric oxide (NO), has been implicated in the pathogenesis of inflammatory diseases. Mechanism of action of L‐carnitine on inflammation via iNOS and nuclear factor κB (NF‐κB) is unclear. In this study, we aimed to investigate the effect of L‐carnitine on nitric oxide synthesis in lipopolysaccharide (LPS)‐stimulated RAW 264·7 macrophage cells. For this purpose, cells were pretreated with various concentrations of L‐carnitine and subsequently incubated with LPS (1 µg·ml−1). NO levels, iNOS protein expression, and NF‐κB activity were determined using colorimetric detection, Western blotting and transfection assays. Our results showed that treatment with L‐carnitine suppressed nitric oxide production, iNOS protein expression and NF‐κB activity. We demonstrated that inhibitory effect of L‐carnitine on iNOS protein expression is at transcriptional level. This study may contribute to understanding the anti‐inflammatory effect of L‐carnitine. Copyright

Methylsulfonylmethane modulates apoptosis of LPS/IFN-γ-activated RAW 264.7 macrophage-like cells by targeting p53, Bax, Bcl-2, cytochrome c and PARP proteins

Abstract Methylsulfonylmethane (MSM) is a non-toxic, natural organosulfur compound, which is known to possess antioxidant and anti-inflammatory activities. In recent years, MSM has been widely used as a dietary supplement for its beneficial effects against various diseases, especially arthritis. Despite being a popular supplement product, the mechanism of action of MSM is not well known. This study was designed to investigate the effects of MSM on cytotoxic signals induced by lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) in RAW 264.7 macrophage-like cells. The results showed that MSM reversed apoptosis of RAW 264.7 macrophage-like cells at non-cytotoxic concentrations probably through the modulation of apoptotic proteins. After pre-treatment of cells with non-toxic doses of MSM; caspase-3 activation, p53 accumulation, cytochrome c release and Bax/Bcl-2 ratio were significantly decreased and full length poly ADP-ribose polymerase (PARP) was significantly increased. In addition, the loss of mitochondrial membrane potential was decreased with MSM pretreatment in activated macrophages. Since excess nitric oxide production causes apoptosis of macrophages, anti-apoptotic effects of MSM are thought to be mediated by its inhibitor effects on inducible nitric oxide synthase (iNOS) protein and nitric oxide levels. More interestingly, higher doses of MSM exhibited biphasic effects, inhibited cell viability, induced apoptosis of macrophages, increased caspase-3 activity and PARP cleavage. Thus, our results reveal the molecular mechanism of of MSM indicating that MSM supplementation may be beneficial for complications related to nitric oxide-dependent apoptosis in inflammatory conditions. However, the optimum concentration of MSM must be chosen carefully to elicit the desired effect.

The effect of CYP1A1, GSTT1 and GSTM1 polymorphisms on the risk of lung cancer A case–control study

Lung cancer, which is mainly affected by environmental factors, is a lethal malignancy. It is also important to investigate the effect of genetic factors on lung cancer aetiology. In this study, we aimed to investigate the distribution of CYP1A1*2C, GSTT1 and GSTM1 polymorphisms in Turkish lung cancer patients to determine whether any promoting effect of polymorphisms could cause development of lung cancer. For this purpose, genomic DNA samples obtained from peripheral blood of 128 patients with lung cancer and 122 healthy subjects were analyzed. Genotyping of polymorphic enzymes were carried out by polymerase chain reaction–restriction fragment length polymorphism methods. Although there were no significant differences between groups in terms of CYP1A1 polymorphism, the carriers of CYP1A1 Ile/Val genotype (odds ratio [OR] = 1.224, 95% confidence interval [CI]: 0.585–2.564) or CYP1A1 Val/Val genotype (OR = 3.058, 95% CI: 0.312–30.303) had an increased risk of lung cancer development. There was no statistical difference between groups in terms of both GSTT1 null genotype (OR = 1.114, 95% CI: 0.590–2.105) and GSTM1 null genotype (OR = 0.776, 95% CI: 0.466–1.290). This is the first case–control study investigating CYP1A1 Ile/Val, GSTT1 and GSTM1 polymorphisms in Turkish lung cancer patients. Although we suggest that other genes in addition to the proposed genes could play a role in lung cancer development, the results of our study will contribute to the possible associations between CYP1A1 Ile/Val, GSTT1 and GSTM1 gene polymorphism on the risk of lung cancer.

Variations in glutathione-S-transferase genes influence risk of chronic myeloid leukemia

Glutathione S‐transferases (GSTs) are phase II enzymes that detoxify hazardous xenobiotics including carcinogens. Inter‐individual variations in GSTM1 and GSTT1 loci have been associated with several types of cancer, including leukemias. In this study, we investigated the possible association between GSTM1 and GSTT1 polymorphisms and susceptibility to chronic myeloid leukemia (CML) in a Turkish population. In a case‐control study, 106 CML patients and 190 healthy controls were evaluated for GSTM1 and GSTT1 polymorphisms. GSTM1 null (GSTM1‐) genotype frequencies in CML cases and controls were 45.3% and 42.6%, respectively. GSTT1 null (GSTT1‐) genotype frequencies were 44.3% and 18.4%, respectively. The frequency of the GSTT1‐ genotype among CML patients was significantly higher than in controls [odds ratio (OR) 3.53, 95% confidence interval (CI) 2.08‐6.00; P < 0.0001]. Individuals with the GSTM1‐ genotype did not have increased risk of CML [OR: 1.11; 95% CI: 0.69‐1.80; P = 0.714]. The combined GSTM1‐/ GSTT1‐ genotype was significantly associated with risk of CML compared to the GSTM1+/GSTT1+ genotype which was most frequent in both cases and controls [OR: 9.47; 95% CI: 3.61‐24.87]. Similar findings have only been obtained in Turkish and Indian populations but not elsewhere. The GSTM1+/GSTT1‐ genotype was associated with a 2.5‐fold increased risk compared with the GSTM1‐/GSTT1+ genotype, the second most frequent genotype (OR; 2.46; 95% CI: 1.17, 5.20), suggesting a complex interaction between GSTM1 and GSTT1. Our results indicate an association between the GSTT1‐ genotype, either alone or in combination with GSTM1‐ genotype, and risk of CML, suggesting a possible interaction between GSTM1 and GSTT1. These findings, which are possibly restricted to Turkey and India, warrant further research. Copyright

Impact of follicle-stimulating hormone receptor variants in female infertility

PurposeFollicle-stimulating hormone (FSH) and its receptor play a major role in the development of follicles and regulation of steroidogenesis in the ovary and spermatogenesis in the testis. We aim to analyze the role of FSHR gene variants (single nucleotide polymorphisms (SNPs) in exon 10 (codon 307 and 680) and in the core promoter region (at position −29) and Ala189Val inactivating mutation) in Turkish infertile women. There were studies analyzing the effects of the SNPs in exon 10 (codon 307 and 680) and in the core promoter region (at position −29) of the FSHR gene on spermatogenesis, but to our knowledge, there were no studies analyzing the effects of these three SNP combinations on female fertility.MethodsIn this study, the allelic, genotype, and haplotype frequency distributions of these three SNPs in the FSHR gene were analyzed in 102 infertile women and 99 unrelated healthy control individuals. The distribution of the polymorphisms was conformed by Hardy–Weinberg equilibrium test.ResultsThere were no statistical differences (P > 0.05) in the allele, genotype, and haplotype frequencies of the polymorphisms and FSH, luteinizing hormone (LH), estradiol (E2), and prolactin (PRL) levels between the infertile patients and the controls. However, a significant relation was found between 307 SNP GA genotype and FSH level ≥12. We did not find any homozygous or heterozygote mutations in infertile patients and healthy fertile controls.ConclusionThe present study was the first study analyzing gma mutation and the polymorphism of the FSHR core promoter at position −29 alone and in combination with the two common SNPs in exon 10 in Turkish infertile women population. These findings indicate the significance of Ala307Thr GA genotype may be a predictive marker for poor ovarian reserve and infertility.

MDR1 gene polymorphisms may be associated with Behçet's disease and its colchicum treatment response

Behçets disease (BD) is a chronic multisystem disorder. Infectious agents, immune system mechanisms, and genetic factors are implicated in the etiopathogenesis of BD, which remains to be explained. The human MDR1 (ABCB1) gene encoder P-glycoprotein (P-gp) plays a key role in drug disposition, serves as a protective mechanism against xenobiotics, and provides additional protection for the brain, testis, and fetus. We investigated the genotype and haplotype distributions of three MDR1 gene polymorphisms (C1236T, G2677T/A, and C3435T) in 104 BD patients and 130 control subjects. The genotyping analysis was performed by using PCR-RFLP methods. No statistically significant differences were found for the genotypic and allelic distributions of three individual single nucleotide polymorphisms (SNPs) in the MDR1 gene between BD patients and control subjects in this study (p>0.05). However, combined genotype and haplotype frequencies have found statistically significant differences between BD and control subjects for some combinations (p<0.05). The CC-GG binary genotype for C1236T-G2677T/A loci couple in particular may have a high degree of predisposition to BD (p=0.009; OR, 3.03; 95% CI, 1.41-6.54). Furthermore, significant differences between colchicine-responsive and -nonresponsive groups were found. Genotypic and allelic distributions of C3435T and G2677T/A loci, as well as their genotype and haplotype combinations, were found to have statistically significant differences (p<0.05). The TT genotype for the C3435T locus (p=0.001; OR, 6.59; 95% CI, 1.86-23.30) and T allele (p=0.009; OR, 2.09; 95% CI, 1.18-3.70) plays a substantial role in the colchicine response. Our study showed that MDR1 genes and their polymorphisms may affect a patients BD susceptibility and colchicine response.