Oxidatively-induced DNA damage and base excision repair in euthymic patients with bipolar disorder.

Oxidatively-induced DNA damage has previously been associated with bipolar disorder. More recently, impairments in DNA repair mechanisms have also been reported. We aimed to investigate oxidatively-induced DNA lesions and expression of DNA glycosylases involved in base excision repair in euthymic patients with bipolar disorder compared to healthy individuals. DNA base lesions including both base and nucleoside modifications were measured using gas chromatography-tandem mass spectrometry and liquid chromatography-tandem mass spectrometry with isotope-dilution in DNA samples isolated from leukocytes of euthymic patients with bipolar disorder (n = 32) and healthy individuals (n = 51). The expression of DNA repair enzymes OGG1 and NEIL1 were measured using quantitative real-time polymerase chain reaction. The levels of malondialdehyde were measured using high performance liquid chromatography. Seven DNA base lesions in DNA of leukocytes of patients and healthy individuals were identified and quantified. Three of them had significantly elevated levels in bipolar patients when compared to healthy individuals. No elevation of lipid peroxidation marker malondialdehyde was observed. The level of OGG1 expression was significantly reduced in bipolar patients compared to healthy individuals, whereas the two groups exhibited similar levels of NEIL1 expression. Our results suggest that oxidatively-induced DNA damage occurs and base excision repair capacity may be decreased in bipolar patients when compared to healthy individuals. Measurement of oxidatively-induced DNA base lesions and the expression of DNA repair enzymes may be of great importance for large scale basic research and clinical studies of bipolar disorder.

Efficacy of Different Durations of Intravenous Methylprednisolone Treatment in Relapses of Multiple Sclerosis.

INTRODUCTION: Relapses of multiple sclerosis (MS) are usually treated with high-dose intravenous methylprednisolone (IVMP), given over 3-10 days. There is no consensus on the optimal duration of treatment. In this study, we aimed to investigate whether longer treatment provides additional short-term clinical benefits assessed by the change in plasma cytokine levels and EDSS scores in patients with relapsing-remitting MS (RRMS). METHODS: Forty RRMS patients during relapse were grouped into 3 and treated with 1 g/day of IVMP for either 5, 7, or 10 consecutive days. RESULTS: Levels of IL-10 and IL-12 were analyzed, and EDSS scores were noted before treatment, after treatment (on days 6, 8, or 11) and at the 4th week. IVMP treatment significantly induced anti-inflammatory IL-10 levels but had no effect on IL-12 levels. IVMP treatment for 7 or 10 consecutive days was not significantly different than that for 5 days in terms of the change in IL-12, IL-10 levels or clinical outcome. CONCLUSION: In conclusion, pulse high-dose IVMP treatment enhances functional recovery in patients with acute relapses of RRMS. In addition, IVMP treatment significantly increases the levels of IL-10 but has no effect on the levels of IL-12 in the short term.

Quantitative Tyrosine Phosphoproteomics of Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor-treated Lung Adenocarcinoma Cells Reveals Potential Novel Biomarkers of Therapeutic Response.

Mutations in the Epidermal growth factor receptor (EGFR) kinase domain, such as the L858R missense mutation and deletions spanning the conserved sequence 747LREA750, are sensitive to tyrosine kinase inhibitors (TKIs). The gatekeeper site residue mutation, T790M accounts for around 60% of acquired resistance to EGFR TKIs. The first generation EGFR TKIs, erlotinib and gefitinib, and the second generation inhibitor, afatinib are FDA approved for initial treatment of EGFR mutated lung adenocarcinoma. The predominant biomarker of EGFR TKI responsiveness is the presence of EGFR TKI-sensitizing mutations. However, 30-40% of patients with EGFR mutations exhibit primary resistance to these TKIs, underscoring the unmet need of identifying additional biomarkers of treatment response. Here, we sought to characterize the dynamics of tyrosine phosphorylation upon EGFR TKI treatment of mutant EGFR-driven human lung adenocarcinoma cell lines with varying sensitivity to EGFR TKIs, erlotinib and afatinib. We employed stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative mass spectrometry to identify and quantify tyrosine phosphorylated peptides. The proportion of tyrosine phosphorylated sites that had reduced phosphorylation upon erlotinib or afatinib treatment correlated with the degree of TKI-sensitivity. Afatinib, an irreversible EGFR TKI, more effectively inhibited tyrosine phosphorylation of a majority of the substrates. The phosphosites with phosphorylation SILAC ratios that correlated with the TKI-sensitivity of the cell lines include sites on kinases, such as EGFR-Y1197 and MAPK7-Y221, and adaptor proteins, such as SHC1-Y349/350, ERRFI1-Y394, GAB1-Y689, STAT5A-Y694, DLG3-Y705, and DAPP1-Y139, suggesting these are potential biomarkers of TKI sensitivity. DAPP1, is a novel target of mutant EGFR signaling and Y-139 is the major site of DAPP1 tyrosine phosphorylation. We also uncovered several off-target effects of these TKIs, such as MST1R-Y1238/Y1239 and MET-Y1252/1253. This study provides unique insight into the TKI-mediated modulation of mutant EGFR signaling, which can be applied to the development of biomarkers of EGFR TKI response.

Enhanced sensitivity of Neil1-/- mice to chronic UVB exposure.

Oxidative stress and reactive oxygen species (ROS)-induced DNA base damage are thought to be central mediators of UV-induced carcinogenesis and skin aging. However, increased steady-state levels of ROS-induced DNA base damage have not been reported after chronic UV exposure. Accumulation of ROS-induced DNA base damage is governed by rates of lesion formation and repair. Repair is generally performed by Base Excision Repair (BER), which is initiated by DNA glycosylases, such as 8-oxoguanine glycosylase and Nei-Endonuclease VIII-Like 1 (NEIL1). In the current study, UV light (UVB) was used to elicit protracted low-level ROS challenge in wild-type (WT) and Neil1-/- mouse skin. Relative to WT controls, Neil1-/- mice showed an increased sensitivity to tissue destruction from the chronic UVB exposure, and corresponding enhanced chronic inflammatory responses as measured by cytokine message levels and profiling, as well as neutrophil infiltration. Additionally, levels of several ROS-induced DNA lesions were measured including 4,6-diamino-5-formamidopyrimidine (FapyGua), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyAde), 8-hydroxyguanine (8-OH-Gua), 5,6-dihydroxyuracil (5,6-diOH-Ura) and thymine glycol (ThyGly). In WT mice, chronic UVB exposure led to increased steady-state levels of FapyGua, FapyAde, and ThyGly with no significant increases in 8-OH-Gua or 5,6-diOH-Ura. Interestingly, the lesions that accumulated were all substrates of NEIL1. Collectively, these data suggest that NEIL1-initiated repair of a subset of ROS-induced DNA base lesions may be insufficient to prevent the initiation of inflammatory pathways during chronic UV exposure in mouse skin.

IL-1ß, IL-6 and IL1Ra levels in temporal lobe epilepsy.

PURPOSE: There is now extensive evidence to support the involvement of inflammation in the course of epileptic seizures. Seizure-induced changes in serum IL-1ß, IL-6 and IL-1Ra levels are reported in several studies. Serum cytokine levels may also be disturbed in inter-ictal period due to seizure activity. METHODS: Twenty-one patients (12 women; mean age 35±12.3) with temporal lobe epilepsy (TLE), 17 patients (8 women; mean age 31.8±10.4) with extra-temporal lobe epilepsy (XLE) and 20 normal controls (10 women; mean age 35.6±8.8) were included in the study. Serum levels of IL-1ß, IL-6 and IL-1Ra of the TLE, XLE groups in inter-ictal period and of the normal control group were compared. RESULTS: All three cytokine levels are found to be significantly elevated in epilepsy patients when compared to controls (p<0.05). In TLE group, IL-1ß serum levels were significantly higher than in the XLE group (p<0001). CONCLUSION: The major findings in our study were increased levels of IL-1ß, IL-6 and IL-1Ra in epileptic patients and high levels of IL-1ß in TLE group. Our results support the existence of a chronic inflammatory state in epileptic patients.

Identification and quantification of DNA repair protein apurinic/apyrimidinic endonuclease 1 (APE1) in human cells by liquid chromatography/isotope-dilution tandem mass spectrometry.

Unless repaired, DNA damage can drive mutagenesis or cell death. DNA repair proteins may therefore be used as biomarkers in disease etiology or therapeutic response prediction. Thus, the accurate determination of DNA repair protein expression and genotype is of fundamental importance. Among DNA repair proteins involved in base excision repair, apurinic/apyrimidinic endonuclease 1 (APE1) is the major endonuclease in mammals and plays important roles in transcriptional regulation and modulating stress responses. Here, we present a novel approach involving LC-MS/MS with isotope-dilution to positively identify and accurately quantify APE1 in human cells and mouse tissue. A completely (15)N-labeled full-length human APE1 was produced and used as an internal standard. Fourteen tryptic peptides of both human APE1 (hAPE1) and (15)N-labeled hAPE1 were identified following trypsin digestion. These peptides matched the theoretical peptides expected from trypsin digestion and provided a statistically significant protein score that would unequivocally identify hAPE1. Using the developed methodology, APE1 was positively identified and quantified in nuclear and cytoplasmic extracts of multiple human cell lines and mouse liver using selected-reaction monitoring of typical mass transitions of the tryptic peptides. We also show that the methodology can be applied to the identification of hAPE1 variants found in the human population. The results describe a novel approach for the accurate measurement of wild-type and variant forms of hAPE1 in vivo, and ultimately for defining the role of this protein in disease development and treatment responses.

Interleukin-6, interleukin-1 beta and interleukin-1 receptor antagonist levels in epileptic seizures.

PURPOSE: Data are accumulating to support the involvement of inflammatory mechanisms in the pathogenesis and course of epilepsy. METHODS: The aim of this study was to examine seizure-induced changes in plasma concentrations of interleukin-6 (IL-6), interleukin-1 receptor antagonist (IL-1Ra), and interleukin-1 beta (IL-1ß) in 23 patients with epilepsy undergoing a video-electroencephalography (EEG) study. Patients were divided into groups based on epilepsy type as follows: temporal lobe epilepsy (TLE) (n=6), extra-temporal lobe epilepsy (XLE) (n=8) and idiopathic generalised epilepsy (IGE) (n=9). Serum levels of IL-1ß, IL-1Ra and IL-6 were measured at baseline, immediately after the epileptic seizure, and at 3h, 6h, 12h and 24h after the seizure. RESULTS: We demonstrated a significant increase in plasma levels of IL-6 and IL-1Ra that peaked at 12h into the post-ictal period (p<0.05). IL-1ß levels did not differ from the baseline levels. We did not observe any differences in post-ictal cytokine release patterns between the TLE, XLE and IGE groups. CONCLUSION: The present study confirms the findings that epileptic seizures induce the production of IL-6 and IL-1Ra.

Identification and quantification of human DNA repair protein NEIL1 by liquid chromatography/isotope-dilution tandem mass spectrometry.

Accumulated evidence points to DNA repair capacity as an important factor in cancer and other diseases. DNA repair proteins are promising drug targets and are emerging as prognostic and therapeutic biomarkers. Thus, the knowledge of the overexpression or underexpression levels of DNA repair proteins in tissues will be of fundamental importance. In this work, mass spectrometric assays were developed for the measurement in tissues of the human DNA repair protein NEIL1 (hNEIL1), which is involved in base excision and nucleotide excision repair pathways of oxidatively induced DNA damage. Liquid chromatography/isotope-dilution tandem mass spectrometry (LC-MS/MS), in combination with a purified and fully characterized recombinant (15)N-labeled analogue of hNEIL1 ((15)N-hNEIL1) as an internal standard, was utilized to develop an accurate method for the quantification of hNEIL1. Both hNEIL1 and (15)N-hNEIL1 were hydrolyzed with trypsin, and 18 tryptic peptides from each protein were identified by LC-MS/MS on the basis of their full-scan mass spectra. These peptides matched the theoretical peptides expected from trypsin hydrolysis of hNEIL1 and provided a statistically significant protein score that would unequivocally identify hNEIL1. The product ion spectra of the tryptic peptides from both proteins were recorded, and the characteristic product ions were defined. Selected-reaction monitoring was used to analyze mixtures of hNEIL1 and (15)N-hNEIL1 on the basis of product ions. Additional confirmation of positive identification was demonstrated via separation of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in-gel tryptic digestion followed by LC-MS/MS analysis. These results suggest that the developed assays would be highly suitable for the in vivo positive identification and accurate quantification of hNEIL1 in tissues.

Different responses of fluvastatin to cholesterol-induced oxidative modifications in rabbits: evidence for preventive effect against DNA damage.

Hypercholesterolemia is a major risk factor for atherosclerosis and related occlusive vascular diseases. We investigated the effect of low-dose fluvastatin (2 mg kg(-1) day(-1)) on antioxidant enzyme activities [superoxide dismutase (SOD), catalase], vascular reactivity changes and oxidatively induced DNA damage in early stage of atherosclerosis in hypercholesterolemic rabbits. The animals were divided into three groups each composed of 10 rabbits. The control group received a regular rabbit chow diet, and the cholesterol group had hypercholesterolemic diet (2%, 4 weeks). The fluvastatin group was given hypercholesterolemic diet plus fluvastatin. Dietary intake of cholesterol significantly increased total cholesterol levels in rabbits (control, 0.85 ± 0.29; cholesterol, 12.04 ± 4.61; fluvastatin, 8.07 ± 2.72 mmol l(-1)). Hypercholesterolemic diet revealed discernible fatty streaks in arcus aortae. Fluvastatin significantly reduced the areas of the lesions. The diet significantly increased SOD activities in both erythrocyte and tissue. Treatment with fluvastatin normalized the increased activity of SOD in both erythrocyte and aortic tissues from the cholesterol group. Cholesterol feeding decreased the sensitivity to acetylcholine, and treatment with fluvastatin significantly restored the diminished sensitivity to acetylcholine in thoracic aortae. Cholesterol feeding caused oxidatively induced DNA damage in liver tissues determined by the increased levels of 8-hydroxyguanine (8-OH-Gua) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). Fluvastatin decreased only FapyGua level in liver. In conclusion, our results may suggest that fluvastatin seems to play a protective role on high cholesterol-induced oxidative stress and DNA damage.

Evidence for upregulated repair of oxidatively induced DNA damage in human colorectal cancer.

Carcinogenesis may involve overproduction of oxygen-derived species including free radicals, which are capable of damaging DNA and other biomolecules in vivo. Increased DNA damage contributes to genetic instability and promote the development of malignancy. We hypothesized that the repair of oxidatively induced DNA base damage may be modulated in colorectal malignant tumors, resulting in lower levels of DNA base lesions than in surrounding pathologically normal tissues. To test this hypothesis, we investigated oxidatively induced DNA damage in cancerous tissues and their surrounding normal tissues of patients with colorectal cancer. The levels of oxidatively induced DNA lesions such as 4,6-diamino-5-formamidopyrimidine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 8-hydroxyguanine and (5'S)-8,5'-cyclo-2'-deoxyadenosine were measured by gas chromatography/isotope-dilution mass spectrometry and liquid chromatography/isotope-dilution tandem mass spectrometry. We found that the levels of these DNA lesions were significantly lower in cancerous colorectal tissues than those in surrounding non-cancerous tissues. In addition, the level of DNA lesions varied between colon and rectum tissues, being lower in the former than in the latter. The results strongly suggest upregulation of DNA repair in malignant colorectal tumors that may contribute to the resistance to therapeutic agents affecting the disease outcome and patient survival. The type of DNA base lesions identified in this work suggests the upregulation of both base excision and nucleotide excision pathways. Development of DNA repair inhibitors targeting both repair pathways may be considered for selective killing of malignant tumors in colorectal cancer.

Glutathione depletion by buthionine sulfoximine induces oxidative damage to DNA in organs of rabbits in vivo.

Glutathione (GSH) exists in mammalian tissues in vivo at high concentrations and plays an important protective role against oxidatively induced damage to biological molecules, including DNA. We investigated oxidatively induced damage to DNA by GSH depletion in different organs of rabbits in vivo. Rabbits were treated subcutaneously with buthionine sulfoximine (BSO), an effective GSH-depleting compound. GSH levels were measured in heart, brain, liver, and kidney of animals. BSO treatment significantly reduced GSH levels in heart, brain, and liver, but not in kidney. DNA was isolated from these tissues to test whether GSH depletion causes oxidatively induced DNA damage in vivo. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry with isotope dilution methods were applied to measure typical products of oxidatively induced damage in isolated DNA samples. Several such products were identified and quantified in all organs. BSO treatment caused significant formation of 8-hydroxyguanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 8-hydroxyadenine, and (5'S)-8,5'-cyclo-2'-deoxyadenosine in DNA of organs of rabbits. Animals were fed with the semiessential amino acid 2-aminoethanesulfonic acid (taurine) during BSO treatment. Taurine significantly inhibited GSH depletion and also formation of DNA products. Depletion of GSH correlated well with formation of DNA products, indicating the role of GSH in preventing oxidatively induced DNA damage. Our findings might contribute to the understanding of pathologies associated with DNA damage, oxidative stress, and/or defective antioxidant responses and improve our understanding of the effect of BSO in increasing the efficacy of anticancer therapeutics.

Targeted deletion of the genes encoding NTH1 and NEIL1 DNA N-glycosylases reveals the existence of novel carcinogenic oxidative damage to DNA.

We have generated a strain of mice lacking two DNA N-glycosylases of base excision repair (BER), NTH1 and NEIL1, homologs of bacterial Nth (endonuclease three) and Nei (endonuclease eight). Although these enzymes remove several oxidized bases from DNA, they do not remove the well-known carcinogenic oxidation product of guanine: 7,8-dihydro-8-oxoguanine (8-OH-Gua), which is removed by another DNA N-glycosylase, OGG1. The Nth1-/-Neil1-/- mice developed pulmonary and hepatocellular tumors in much higher incidence than either of the single knockouts, Nth1-/- and Neil1-/-. The pulmonary tumors contained, exclusively, activating GGT-->GAT transitions in codon 12 of K-ras of their DNA. Such transitions contrast sharply with the activating GGT-->GTT transversions in codon 12 of K-ras of the pathologically similar pulmonary tumors, which arose in mice lacking OGG1 and a second DNA N-glycosylase, MUTY. To characterize the biochemical phenotype of the knockout mice, the content of oxidative DNA base damage was analyzed from three tissues isolated from control, single and double knockout mice. The content of 8-OH-Gua was indistinguishable among all genotypes. In contrast, the content of 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) derived from adenine and guanine, respectively, were increased in some but not all tissues of Neil1-/- and Neil1-/-Nth1-/- mice. The high incidence of tumors in our Nth1-/-Neil1-/- mice together with the nature of the activating mutation in the K-ras gene of their pulmonary tumors, reveal for the first time, the existence of mutagenic and carcinogenic oxidative damage to DNA which is not 8-OH-Gua.

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.

Accumulation of (5'S)-8,5'-cyclo-2'-deoxyadenosine in organs of Cockayne syndrome complementation group B gene knockout mice.

Cockayne syndrome (CS) is a human genetic disorder characterized by sensitivity to UV radiation, neurodegeneration, premature aging among other phenotypes. CS complementation group B (CS-B) gene (csb) encodes the CSB protein (CSB) that is involved in base excision repair of a number of oxidatively induced lesions in genomic DNA in vivo. We hypothesized that CSB may also play a role in cellular repair of the DNA helix-distorting tandem lesion (5'S)-8,5'-cyclo-2'-deoxyadenosine (S-cdA). Among many DNA lesions, S-cdA is unique in that it represents a concomitant damage to both the sugar and base moieties of the same nucleoside. Because of the presence of the C8-C5' covalent bond, S-cdA is repaired by nucleotide excision repair unlike most of other oxidatively induced lesions in DNA, which are subject to base excision repair. To test our hypothesis, we isolated genomic DNA from brain, kidney and liver of wild type and csb knockout (csb(-/-)) mice. Animals were not exposed to any exogenous oxidative stress before the experiment. DNA samples were analysed by liquid chromatography/mass spectrometry with isotope-dilution. Statistically greater background levels of S-cdA were observed in all three organs of csb(-/-) mice than in those of wild type mice. These results suggest the in vivo accumulation of S-cdA in genomic DNA due to lack of its repair in csb(-/-) mice. Thus, this study provides, for the first time, the evidence that CSB plays a role in the repair of the DNA helix-distorting tandem lesion S-cdA. Accumulation of unrepaired S-cdA in vivo may contribute to the pathology associated with CS.

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.

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.

Oxidative DNA damage in polymorphonuclear leukocytes of patients with familial Mediterranean fever.

Familial Mediterranean fever (FMF) is an autosomal recessively inherited disorder characterized by recurrent, inflammatory self-limited episodes of fever and other symptoms. This disease is caused by more than 25 mutations in the gene MEFV. During fever attacks, there is a substantial influx of polymorphonuclear leukocytes into the affected tissues. Attack-free periods are accompanied by the up-regulation of neutrophil and monocyte phagocytic activity and oxidative burst. These facts led us to hypothesize that oxidative damage by free radicals to DNA may accumulate in FMF patients. To test this hypothesis, we investigated oxidative DNA damage in polymorphonuclear leukocytes of FMF patients during the attack-free period in comparison with FMF-free control individuals. DNA was isolated from polymorphonuclear leukocytes of 17 FMF patients and 10 control individuals. DNA samples were analyzed by liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry to measure the levels of various typical oxidatively induced products of DNA. We show, for the first time, that FMF patients accumulate statistically significant levels of these lesions in their DNA when compared to FMF-free control individuals. This work suggests that the persistent oxidative stress with excess production of free radicals in FMF patients may lead to accumulation of oxidative DNA damage. Defective DNA repair may also contribute to this phenomenon, perhaps due to mutations in the MEFV gene. The accumulation of mutagenic and cytotoxic DNA lesions may contribute to increased mutations and apoptosis in FMF patients, thus to worsening of the disease and well-being of the patients. Future research should deal with prevention of oxidative DNA damage and apoptosis in FMF patients, and also the elucidation of a possible role of DNA repair in this disease.

Effects of transdermal estrogen replacement therapy on plasma levels of nitric oxide and plasma lipids in postmenopausal women.

OBJECTIVE: Our purpose was to assess the effects of transdermal estrogen replacement therapy (TERT) on plasma levels of nitric oxide (NO) and plasma lipids in postmenopausal women. MATERIALS AND METHODS: The study designed as a randomized, double-blind, placebo-controlled trial, involved 43 postmenopausal healthy women who had previously undergone hysterectomy. Women received either transdermal 100 microg 17beta-estradiol (Climara forte TTS) or placebo once a week for 3 months. Plasma levels of NO metabolites, estradiol (E2), total cholesterol (TC), triglicerides (TG), low-density lipoproteins (LDL), high-density lipoproteins (HDL), HDL2 and HDL3 were measured in blood samples of all women which were collected before, after 24 h and after 3 months of therapy. RESULTS: We found significantly increased NO levels 24 h after therapy in TERT group. Moreover significantly higher NO levels were determined at 3rd month of therapy. Serum HDL and HDL2 levels of ERT group were significantly increased at 3rd month of therapy. Alteration of serum levels of HDL3, LDL and TC were not significantly different in groups. TG levels were significantly decreased in TERT group. DISCUSSION: NO-related mechanism may help to explain the cardio-protective effect of TERT in the postmenopausal period. TERT seems to have favorable effects on plasma lipids in surgical menopausal women.

Intrathecal sICAM-1 production in multiple sclerosis--correlation with triple dose Gd-DTPA MRI enhancement and IgG index.

In this study the aim was to evaluate the intrathecal sICAM-1 production in multiple sclerosis (MS) patients during relapse and remission. In addition to this, we assessed whether there is a correlation between intrathecal sICAM-1 production and other disease activity markers such as IgG index and gadolinium enhancement in magnetic resonance imaging (MRI). Twenty four relapsing-remitting MS patients were included in the study. Serum and cerebrospinal fluid (CSF) samples were obtained both during relapse and remission. The soluble form of ICAM (sICAM) was measured by the ELISA method in serum and CSF. Cranial MRI with triple dose gadolinium injection was performed for each patient both during relapse and remission. Serum levels of sICAM-1 (245.23 +/- 92.88 ng/ml) were higher during relapse than those in remission (219.90 +/- 110.94 ng/ml), but the difference was not statistically significant. In relapse periods CSF levels of sICAM-1 (1.304 +/- 0.92 ng/ml) were higher than those in remission (1.06 +/- 0.86 ng/ml), but this was not significant. However, during relapse periods patients had significantly higher sICAM-1 index values (1.76 +/- 0.60) than those found during remission periods (1.01 +/- 0.44) (p < 0.05). The IgG index values were higher in relapse periods than in remission (0.88 +/- 0.37 vs. 0.67 +/- 0.28) (p < 0.005). On T1 weighted images following triple dose Gd injection, at least two or more enhancing lesions were present in 22/24 of the patients (91%) in relapse and 4/24 of the patients (19%) in remission. There was strong correlation both between the sICAM-1 index and Gd enhancement (r =0 .72 p < 0.05) and sICAM-1 index and IgG index in relapse (r = 0.69 p < 0.05). In conclusion, there is association between high sICAM-1 and IgG indices, as well as between high sICAM-1 index and Gd enhancing MRI lesions in relapsing MS patients.

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 Duncan's 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.