Ilhan Onaran

High Levels of Cadmium and Lead in Seminal Fluid and Blood of Smoking Men are Associated with High Oxidative Stress and Damage in Infertile Subjects

We measured the levels of malondialdehyde (MDA), protein carbonyls, glutathione S-transferase (GST) and reducte glutathione (GSH) in seminal plasma and spermatozoa from 95 subjects including 50 infertile patients to evaluate the association between oxidative stress and damage and the components of the anti-oxidant defenses in seminal plasma and spermatozoa of infertile subjects and concentrations of cadmium (Cd) and lead (Pb) in the blood and seminal plasma because of tobacco smoke exposure. The reactive oxygen species (ROS) in spermatozoa were also evaluated by luminol (5-amino-2,3-dihydro-1,4-phthalazinedione)-enhanced chemiluminescence assay. The sperm count, motility, and morphology in the smokers infertile group were found to be lower than those in the fertile male group and nonsmokers infertile group (p < 0.001). Concentrations of Cd, Pb, MDA, protein carbonyls, and ROS levels in the smokers infertile group were significantly higher than those in the fertile male and nonsmokers infertile male groups (p < 0.001). However, GSH levels and GST activities were decreased in the smokers infertile male group than those in the fertile male and nonsmokers infertile male groups (p < 0.001). The results indicate that smoking could affect semen quality and oxidative lipid and protein damage in human spermatozoa. From Pearson correlation analysis, positive correlations were demonstrated between the seminal plasma Cd and seminal plasma protein carbonyls and between seminal plasma Pb and spermatozoa ROS levels in smokers of the subfertile group, while there was a significant positive correlation between blood Cd and ROS levels in smokers of the fertile group. There was also a significant negative correlation of the Cd level of the blood and GSH levels of the sperm and seminal plasma. These findings suggest that cigarette smoking enhances the levels of Cd and Pb in seminal plasma and blood and the extent of oxidative damage associated with a decrease in components of the anti-oxidant defenses in the sperm of infertile males.

Impact of Cu and Fe concentrations on oxidative damage in male infertility

Oxidative stress in the reproductive system is thought to have an effect on the fertilizing ability of sperm. The purpose of this study was to assess the interaction of iron (Fe) and copper (Cu) ions in suspected subfertile and fertile male groups and to find out the relationships of the semen parameters (sperm count, motility, and abnormal morphology), glutathione, malondialdehyde, and reactive oxygen species with these variables. Semen and blood obtained from 60 subfertile men and from 40 fertile volunteers were examined. The sperm count and motility in the subfertile male group were found lower than those in fertile male group (p<0.001). Cu levels in serum and seminal plasma in the subfertile male group were significantly higher than those in the fertile male group (p<0.001 and p<0.05, respectively). There was also a significant increase in the Fe level of seminal plasma in the subfertile male group (p<0.001). However, there was no significant difference in the Fe level of serum in the subfertile male group. In conclusion, these findings suggest that Cu and Fe might be mediators of the effects of oxidative damage and play an essential role in spermatogenesis and male infertility; the determination of Fe and Cu levels in serum and seminal plasma during infertility investigation is recommended.

Glioma risk associates with polymorphisms of DNA repair genes, XRCC1 and PARP1

Cancer develops through interactions between polygenic and environmental factors, and changes in DNA repair pathway can increase susceptibility to tumours. XRCC1 and PARP1 are two proteins that act cooperatively in base excision repair (BER) of DNA. The polymorphisms of genes coding these proteins may effect their action in BER pathway. In this study, we aimed to investigate the associations between glioma risk and XRCC1 Arg399Gln and PARP1 Val762Ala polymorphisms per se and in combination. XRCC1 Arg399Gln and PARP1 Val726Ala polymorphisms were investigated by PCR–RFLP method in 119 glioma patients and 180 cancer-free control subjects. The results were statistically analysed by calculating the odds ratios (OR) and their 95% confidence intervals (95% CI) using the χ2-tests. Glioma patients in this study had significantly higher frequencies of XRCC1 Arg399Gln polymorphism both in homozygote (GG) and heterozygote (AG) status (31% and 56%, respectively) (p < 0.001), and also increased frequency of 399Gln (G) allele (59%) (p < 0.001). Val/Ala (VA) genotype of PARP1 Val762Ala polymorphism was significantly more in the control group (p = 0.02). The combined genotypes of XRCC1 AG or GG with PARP1 VA or AA, and XRCC1 AG or GG with PARP1 VV were more represented in the glioma patients (p = 0.001 and 0.003, respectively). We conclude that XRCC1 Arg399Gln polymorphism is a significant risk factor, and 399Gln (G) allele carries a 3.5 times greater risk for glioma, while PARP1 Val/Ala genotype may be protective against it. We also suspect that in the presence of a polymorphic (G) allele of XRCC1, the plausible protective effect of PARP1 VA genotype may be greatly suppressed.

The Effect of Metformin on Insulin Receptors and Lipid Peroxidation in Alloxan and Streptozotocin Induced Diabetes

Biguanides are used for the treatment of non-insulin dependent diabetes mellitus but there is no evidence for an improving action of biguanide on the enhancement of peripheral glucose disposal in type 1 diabetes. It is known that biguanide agents reduce the oxidation of free fatty acids. Using alloxan and streptozotocin (STZ) induced diabetic rats as a model for type 1 diabetes mellitus, we measured insulin binding capacity and plasma lipid peroxidation levels before and after metformin induction. There was a significant increase in insulin binding capacity and lipid peroxidation levels in alloxan and STZ diabetes compared to controls. We examined the effect of metformin on alloxan and STZ-induced diabetic rats. In alloxan-induced diabetes metformin (Met) treatment led to an increase in insulin receptor number in liver plasma membranes (before Met: 46.50 +/- 2.69, after Met: 76.00 +/- 3.39 fmol/mg, p < 0.001) and a decrease in plasma lipid peroxidation levels compared to the non-treated group (before Met: 1.85 +/- 0.53, after Met: 1.10 +/- 0.09 nmol MDA/ml, p < 0.05). In STZ-induced diabetic rats metformin treatment did not change the lipid peroxidation levels (before Met: 1.26 +/- 0.31, after Met: 1.38 +/- 0.44 nmol MDA/ml, p > 0.05) whereas it did increase the receptor numbers (before Met: 41.60 +/- 4.33, after Met: 63.40 +/- 8.64 fmol/mg, p < 0.002).

The Ala allele at Val762Ala polymorphism in poly(ADP-ribose) polymerase-1 (PARP-1) gene is associated with a decreased risk of asthma in a Turkish population.

Background and objective. It has been suggested that inhibition of poly (ADP-ribose) polymerase-1 (PARP-1), either pharmacologically or by a gene knockout provides significant protection against systemic or tissue inflammation in animal models. The aim of this study was to analyze the association of the PARP-1 Val762Ala polymorphism, which has beenreported to be associated with decreased enzymatic activity, in Turkish patients with adult asthma. Methods. A total of 112 subjects with stable asthma and 180 normal controls from the same geographic region were studied and polymerase chain reaction-based restriction analysis was used to identify Val762Ala polymorphism of the PARP-1. Results. In univariate analysis, PARP-1 762 AA genotype conferred a 3.4 fold reduction in risk (OR = 0.297, 95% CI = 0.105-0.813; P = 0.014), while heterozygous VA genotype conferred an even greater level of protection (OR = 0.06; 95%CI, 0.026-0.14; P < 10−6). In addition, wild type PARP-1 762 V allele had 5 times the risk of developing asthma than those without the allele (OR 0.199, CI 0.118-0.334, P = 10−6). Conclusions. These findings suggest that PARP-1 V762A variants may be one of the factors participating in protection or susceptibility to asthma in our population.

Association of PARP-1, NF-κB, NF-κBIA and IL-6, IL-1β and TNF-α with Graves Disease and Graves Ophthalmopathy

BACKGROUND Graves Disease (GD) is an autoimmune disorder affected by an interaction of multiple genes such as Nuclear Factor-κB (NF-κB), Nuclear Factor-κB Inhibitor (NF-κBIA), Poly (ADP-ribose) polymerase-1 (PARP-1) and cytokines like Interleukin-1β (IL-1β), Interleukin-6 (IL-6) and Tumor Necrosis Factor-α (TNF-α) and mostly accompanied by an ocular disorder, Graves Ophthalmopathy (GO). We hypothesize that there is a relationship between GD, GO, polymorphisms of inflammatory related genes and their association with cytokines, which may play important roles in autoimmune and inflammatory processes. SUBJECTS AND METHODS To confirm our hypothesis, we studied the polymorphisms and cytokine levels of 120 patients with GD and GO using PCR-RFLP and ELISA methods, respectively. RESULTS We found that patients with GG genotype and carriers of G allele of PARP-1 G1672A polymorphism are at risk in the group having GD (p=0.0007) while having GA genotype may be protective against the disease. PARP-1 C410T polymorphism was found to be associated with GO by increasing the risk by 1.7 times (p=0.004). Another risk factor for development of GO was the polymorphism of del/ins of NFkB1 gene (p=0.032) that increases the risk by 39%. Levels of cytokines were also elevated in patients with GD, but no association was found between levels of cytokines and the development of GO as there was no change in levels of cytokines. CONCLUSIONS We suggest that, PARP-1 and NFkB1 gene polymorphisms may be risk factors for developing Graves Disease and Ophthalmopathy.

Does metformin prevent short-term oxidant-induced dna damage? In vitro study on lymphocytes from aged subjects.

Metformin(1-(diaminomethylidene)-3,3-dimethyl-guanidine), an anti-hyperglycemic agent, also has antioxidant effects. Although the origin is not clearly understood, the antioxidant activity of metformin might result from a direct effect on reactive oxygen species (ROS) or could have an indirect action on the superoxide anions produced by hyperglycemia. The ability of metformin to modulate DNA damage produced by oxidative stress is not known. For this reason, we examined the short term effect of metformin (50 microM, 2 h) on the DNA damage of cumene hydroperoxide (CumOOH)-induced lymphocytes from aged and young control groups (n = 10 each). In this study, DNA damage elicited by CumOOH (1 mM) was detected with the Comet Assay and the ELISA technique. Our results showed a significant increase in apoptotic DNA fragmentation and DNA strand breaks (Comet assay tail factor %) that was detected before and after CumOOH induction in lymphocytes of healthy elderly subjects when compared with healthy young control. Metformin significantly decreased CumOOH-induced apoptotic DNA fragmentation and DNA strand breaks in lymphocytes from aged subjects, although it did not produce a long-term effect. The in vitro results indicate that the short-term effect of metformin can protect against prooxidant stimulus-induced DNA damage in lymphocytes from elderly subjects.

The effect of genetic polymorphisms of TLR2 and TLR4 in Turkish patients with coronary artery disease.

Coronary artery disease (CAD), being a multifactorial disease process, has been suggested to be associated by the interaction of both environmental and genetic risk factors. Toll-like receptors (TLRs) are related to the receptors of the innate immune system which serves as the recognition of the conserved pathogen motifs and the activation of the signals that stimulate inflammatory genes. In this study, we investigated the relationship between the polymorphisms in the TLR2-Arg753Gly, TLR4-Asp299Gly and Thr399Ile gene and CAD. The study population consisted of 300 patients (149 men, 151 women) with angiographically documented CAD. The polymorphisms were genotyped by real time PCR. No association between TLR2-Arg677Trp or TLR4-Asp299Gly and -Thr399Ile gene polymorphisms and the presence or the severity of CAD was observed. On the other hand, the TLR2-Arg753Arg genotype seemed to have a protective effect against development of CAD (OR=0.17; 95% CI=0.04-0.83). Our findings suggest that TLR2-Arg753Gly polymorphism is associated with CAD susceptibility in Turkish patients.

Effect of donor age on the susceptibility of erythrocytes and erthrocyte membranes to cumene hydroperoxide-induced oxidative stress

A comparative study on erythrocytes and erythrocyte membranes of healthy elderly and young adults was carried out to understand how the antioxidant defense capacity is effected by aging. The levels of endogenous malondialdehyde and Ca(2+)-ATPase activity were taken as indices of oxidative damage. In addition, chemiluminescence measurements were performed on intact erythrocytes. The susceptibility of these parameters to in vitro cumene hydroperoxide, under low oxidant level that does not induce hemolysis, was also taken as an age-related indicator of the endogenous peroxidative potential of the erythrocytes. Our data showed that the content of malondialdehyde and Ca(2+)-ATPase activity did not change with age. Furthermore, the susceptibility of intact erythrocytes to oxidative stress did not change in the elderly group. However, under the same conditions erythrocyte membranes were more susceptible to oxidative damage in the elderly than young adults. Our results also showed that antioxidant defenses were overwhelmed in intact erythrocytes of the elderly at high concentrations of cumene hydroperoxide.

The association of MDR1 C3435T and G2677T/A polymorphisms with plasma platelet-activating factor levels and coronary artery disease risk in Turkish population

Increased levels of peripheral proinflammatory mediators can contribute to the development of coronary artery disease (CAD). Platelet activating factor (PAF) is an important proinflammatory mediator and plasma levels of PAF correlate with transmembrane transporter multidrug resistant 1 P-glycoprotein (MDR1 Pgp) expression and activity. MDR1 polymorphisms can affect the expression and activity of Pgp and plasma PAF levels. Therefore, we investigated the possible relationship between MDR1 C3435T and G2677T/A polymorphisms and plasma PAF levels and the risk of CAD. The study population consisted of 198 patients angiographically documented CAD, including 113 cases with at least 1 coronary artery with ≥50% luminal diameter stenosis and 85 control subjects with strictly normal coronary angiograms. Genotypes of the MDR1 C3435T and G2677T/A polymorphisms were determined by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP). Plasma PAF levels were detected by enzyme-linked immunosorbent assay (ELISA). There were no significant differences among plasma PAF levels in regard to MDR1 C3435T and G2677T polymorphisms in CAD patients and controls. No statistically significant difference was found for the genotypic and allelic distributions of the polymorphisms in the MDR1 gene between the patients and the control subjects. Furthermore, analysis of MDR1 haplotypes did not show any associations with increased plasma PAF levels and risk of CAD. Our results suggest that plasma PAF levels are not associated with MDR1 gene polymorphisms. There is no association between MDR1 C3435T and G2677T/A polymorphisms and the risk of CAD in Turkish patients.