Güldal Kirkali

Formamidopyrimidines in DNA : Mechanisms of formation, repair, and biological effects

Oxidatively induced damage to DNA results in a plethora of lesions comprising modified bases and sugars, DNA-protein cross-links, tandem lesions, strand breaks, and clustered lesions. Formamidopyrimidines, 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), are among the major lesions generated in DNA by hydroxyl radical attack, UV radiation, or photosensitization under numerous in vitro and in vivo conditions. They are formed by one-electron reduction of C8-OH-adduct radicals of purines and thus have a common precursor with 8-hydroxypurines generated upon one-electron oxidation. Methodologies using mass spectrometry exist to accurately measure FapyAde and FapyGua in vitro and in vivo. Formamidopyrimidines are repaired by base excision repair. Numerous prokaryotic and eukaryotic DNA glycosylases are highly specific for removal of these lesions from DNA in the first step of this repair pathway, indicating their biological importance. FapyAde and FapyGua are bypassed by DNA polymerases with the insertion of the wrong intact base opposite them, leading to mutagenesis. In mammalian cells, the mutagenicity of FapyGua exceeds that of 8-hydroxyguanine, which is thought to be the most mutagenic of the oxidatively induced lesions in DNA. The background and formation levels of the former in vitro and in vivo equal or exceed those of the latter under various conditions. FapyAde and FapyGua exist in living cells at significant background levels and are abundantly generated upon exposure to oxidative stress. Mice lacking the genes that encode specific DNA glycosylases accumulate these lesions in different organs and, in some cases, exhibit a series of pathological conditions including metabolic syndrome and cancer. Animals exposed to environmental toxins accumulate formamidopyrimidines in their organs. Here, we extensively review the mechanisms of formation, measurement, repair, and biological effects of formamidopyrimidines that have been investigated in the past 50 years. Our goal is to emphasize the importance of these neglected lesions in many biological and disease processes.

Cockayne Syndrome Group B Protein Stimulates Repair of Formamidopyrimidines by NEIL1 DNA Glycosylase

Cockayne syndrome (CS) is a premature aging condition characterized by sensitivity to UV radiation. However, this phenotype does not explain the progressive neurodegeneration in CS patients. It could be due to the hypersensitivity of CSB-deficient cells to oxidative stress. So far most studies on the role of CSB in repair of oxidatively induced DNA lesions have focused on 7,8-dihydro-8-oxoguanine. This study examines the role of CSB in the repair of formamidopyrimidines 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4,6-diamino-5-formamidopyrimidine (FapyAde), which are substrates for endonuclease VIII-like (NEIL1) DNA glycosylase. Results presented here show that csb-/- mice have a higher level of endogenous FapyAde and FapyGua in DNA from brain and kidney than wild type mice as well as higher levels of endogenous FapyAde in genomic DNA and mtDNA from liver. In addition, CSB stimulates NEIL1 incision activity in vitro, and CSB and NEIL1 co-immunoprecipitate and co-localize in HeLa cells. When CSB and NEIL1 are depleted from HeLa cells by short hairpin RNA knockdown, repair of induced FapyGua is strongly inhibited. These results suggest that CSB plays a role in repair of formamidopyrimidines, possibly by interacting with and stimulating NEIL1, and that accumulation of such modifications may have a causal role in the pathogenesis of CS.

Nitric oxide as an activity marker in multiple sclerosis

Abstract. Nitric oxide (NO) molecules have one of the most important roles in the pathogenesis of multiple sclerosis (MS). It has been stated that a continuous and high concentration of NO metabolites in CSF and in the serum of MS patients in relapse may cause toxic damage to myelin and oligodendroglia. The aim of this study was to investigate whether NO is a marker of disease activity and is correlated with other disease activity markers such as active lesions on brain magnetic resonance imaging (MRI) and increased immunoglobulin G (IgG) index.Cerebrospinal fluid (CSF) and peripheral serum (PS) samples were taken from patients with definite MS (n = 24) during relapse and remission and from control subjects (n = 18). The Griess reaction was used to measure the NO metabolites, nitrite and nitrate in CSF and PS. Cranial MRI was carried out with triple dose (0,3 mmol/kg) gadolinium and the IgG index was determined.Nitrite and nitrate concentrations (NNCs) of CSF were 11.16 ± 8.60 μmol/ml in relapse and 6.72 ± 3.50 μmol/ml in remission, whereas in PS they were 12.89 ± 7.62 μmol/ml during relapse and 12.35 ± 6.62 μmol/ml during remission. In control subjects NNCs in CSF and PS were 7.42 ± 2.81 μmol/ml and 4.37 ± 1.63 μmol/ml respectively. NNCs in CSF during relapse period were significantly higher than those of both remission phase and control subjects (p = 0.000). Although serum NNCs did not differ in relapse and remission, they were still higher than normal controls. Validity analysis revealed that NNC measurement in CSF was 71 % specific and 66 % sensitive to disease activity. The most important result was the significant correlation of increased NNCs with the existence of active lesion in cranial MRI and an increase in IgG index (p < 0.05).In conclusion, these results add background data to assist in further outlining the possible role of NO in the pathogenesis of MS. Together with the other markers it may be used as an activity marker in relapses of MS.

Measurement of formamidopyrimidines in DNA

Formamidopyrimidines, 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), are among major lesions in DNA generated by hydroxyl radical attack, UV radiation, or photosensitization in vitro and in vivo. FapyAde and FapyGua exist in living cells at detectable background levels and are formed by exposure of cells to DNA-damaging agents. Numerous prokaryotic and eukaryotic DNA glycosylases exist for the repair of formamidopyrimidines by base excision repair pathways in cells, indicating their biological significance. Moreover, they are premutagenic lesions, albeit to different extents, revealing a possible role in disease processes. Methodologies using gas chromatography/mass spectrometry (GC/MS) with capillary columns have been developed to accurately measure FapyAde and FapyGua in DNA in vitro and in vivo. Stable isotope-labeled analogues of these compounds have been synthesized and are commercially available to be used as internal standards for accurate quantification. GC/MS with isotope dilution provides excellent sensitivity and selectivity for positive identification and accurate quantification, and has widely been applied in the past to the measurement of formamidopyrimidines under numerous experimental conditions. This paper reports on the details of this GC/MS methodology.

Reperfusion injury after detorsion of unilateral ovarian torsion in rabbits.

OBJECTIVE To determine if reperfusion injury takes place in ovarian tissue following the detorsion of the torsioned ovary. STUDY DESIGN Fifty-four New Zealand mature nonpregnant female rabbits were divided into six groups. One group served to determine the basal values of thiobarbituric acid reacting substance (TBARS) and another group was sham. In the third and fourth groups, ovarian torsions and subsequent oophorectomy were performed in 1 and 3h, respectively. In the fifth and sixth groups, detorsions were carried out after unilateral ovarian torsion lasting 1 and 3h, respectively, then oophorectomies of the detorsioned ovaries were performed 2h after detorsion. The level of TBARS in ovarian tissues was determined in all subjects. Statistical analysis was performed using analysis of variance and Duncans multiple range test. Differences were considered to be significant if P<0.05. RESULTS The levels of TBARS were not different in the basal and sham groups (P>0.05), while ovarian torsion caused significant increase in TBARS in the ovary (P<0.05), and detorsion caused a further significant increase in ovarian TBARS (P<0.05). CONCLUSION Reperfusion injury in ovarian tissue, following the detorsion after the torsion of the ovary lasting up to 3h was demonstrated biochemically in this study.

Serum ferritin as a clinical marker for renal cell carcinoma: influence of tumor size and volume.

Objectives: There is no established tumor marker for renal cell carcinoma (RCC). Ferritin is shown to be expressed by the tumor, and proposed as a tumor marker. The aim of this study is to assess the relation between ferritin levels and tumor volume, size and prognosis in RCC. Methods: We studied ferritin levels in serum from peripheral and renal veins of 52 patients with RCC who underwent surgery. Ferritin levels were measured by an enzyme immunoassay method. Tumor volume and the largest tumor diameter were calculated from the pathologic specimens. Results: The mean serum ferritin level from the renal vein (RVF) was statistically higher than the ferritin level from the peripheral vein (PVF) (p = 0.028). Although mean RVF level increased with increasing stage, it was not significant. While there was a correlation with tumor size, volume and RVF, PVF was in correlation with disease status. PVF in patients with metastatic and/or locally advanced disease was significantly higher than the patients with localized disease (p = 0.023). The initial RVF and PVF levels were predictive of survival (p = 0.028 and p = 0.034, respectively). Conclusions: Higher levels in the renal vein, its positive correlation with tumor size and volume suggest that ferritin is expressed by RCC. Initial peripheral serum values of ferritin can be indicative of disease status and also be a prognosticator of survival.

EFFECTS OF GRANULOCYTE-MACROPHAGE COLONY -STIMULATING FACTOR ON INCISIONAL WOUND HEALING IN AN EXPERIMENTAL DIABETIC RAT MODEL

The exact nature of poor wound healing in diabetes is uncertain. Neutrophils play a critical role in the host defense mechanism, and it is suggested that impaired neutrophil functions cause healing difficulties with or without infections in diabetic patients. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is used clinically when given systematically to increase the circulating neutrophils, but its wound-healing effects have not been systematically studied. This study was undertaken to examine the effects of GM-CSF on incisional wound healing in an experimental diabetic rat model. Forty rats were randomly divided into three groups, group I receiving saline as control, diabetes-induced group II receiving saline and diabetes-induced group III receiving GM-CSF. The anesthetized rats in all groups were wounded 21 days after diabetes induction by streptozotocin. Blood neutrophil counts and neutrophil fractions were also determined three days after wounding. Tensile strengths of wounded skin and the hydroxyproline (hyp) level of the wound were determined and wound healing processes were evaluated by light and electron microscopy, fourteen days after wounding. Neutrophil counts and phagocytosis were significantly increased in group III and neutrophil counts decreased in group II (p < 0.05). Although the hydroxyproline level of wound tissue significantly decreased in group II as compared with group III (p < 0.05), there was no differences of tensile strength between group II and III (p < 0.05). Wound score in group II was less than that in groups I and III (p < 0.05). It is concluded that PMN may have a role in modulating wound healing. GM-CSF may be useful for creating better wound healing healing. GM-CSF may be useful for creating better wound healing in risky patients such as diabetics.

Ferritin: a tumor marker expressed by renal cell carcinoma.

Renal cell carcinoma (RCC) has been shown to secrete several hormones and biologically active substances that influence the host metabolism or induce paraneoplastic syndromes. Observation of anemia in 20% of patients with RCC and the spontaneous recovery of anemia following nephrectomy drew attention to the body iron metabolism. Ferritin was previously proposed as a tumor marker for RCC. In order to determine whether RCC cells actually produce ferritin, we studied ferritin levels in serum from peripheral and renal veins as well as from the tumor tissue and the healthy parenchyma from radical nephrectomy specimens of 22 patients with RCC. Ferritin levels both in sera and cytosols were measured by an enzyme immunoassay method. The mean serum ferritin level from the renal vein was 419.9 +/- 72.4 ng/ml, and it was 157.3 +/- 18.3 ng/ml from the peripheral vein (p < 0.05). Renal vein ferritin correlated with stage and had a significant impact on prognosis (p < 0.05). The mean cytosolic ferritin level of the cancer tissue was 705.6 +/- 56.9 ng/mg cytosol protein, whereas in the normal parenchyma it was 95.9 +/- 10.1 ng/mg cytosol protein. This was also highly significant (p = 1.15 x 10(-13)), suggesting that RCC cells probably express ferritin. As currently there exists no reliable tumor marker for RCC, the value of ferritin as a marker should be investigated further before drawing any clinical conclusions.

The Relationship between Neutrophils and Incisional Wound Healing

The systemic administration of granulocyte-macrophage colony-stimulating factor (GM-CSF) is used clinically to increase circulating neutrophils, but its wound healing effects after intraperitoneal treatment have not been studied yet. We planned to investigate the effect of neutrophils on wound healing under cyclophosphamide and GM-CSF treatment. Forty rats were divided into three groups: control group (group I, n = 12) receiving saline, group II (n = 14) receiving cyclophosphamide and group III (n = 14) receiving GM- CSF. The rats in all groups underwent incisional wounding and were euthanized after 7 days. Blood neutrophil counts and functions, tensile strengths and the hydroxyproline level of skin were determined, and a histopathological evaluation of healing was made. Neutrophil counts and phagocytosis significantly increased in group III and decreased in group II. Although the skin hydroxyproline level did not differ, there was a difference in tensile strength of the wounded skin between group II and group III. The wound score in group II was lower than that in groups III and I. As a result we suggest that systemically given GM-CSF – by increasing the neutrophil count and neutrophil phagocytosis index – can enhance the tensile strength of surgical incisions.

Effect of ejaculation on prostate-specific antigen levels in normal men.

While several manipulations such as digital rectal examination and cystoscopy have been shown to increase prostate-specific antigen (PSA) levels, lack of ejaculation for a period of time has been implicated as a cause of decreased serum PSA concentrations. However, the effect of ejaculation on the serum PSA concentration is not clear. This work determined the effect of ejaculation on serum PSA levels in normal men. Blood samples of 19 healthy subjects were taken before and 5 consecutive days after ejaculation. One subject was withdrawn from the study because of ejaculation during sleep, since another ejaculation was prohibited during the study. The mean PSA levels of the men before and 5 consecutive days after ejaculation were 0.79, 0.72, 0.63, 1.01, 0.71, and 0.74 ng/ml--not statistically significantly different. We conclude that ejaculation does not seem to affect the serum PSA concentration.