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As the most common and aggressive primary malignant brain tumor, glioblastoma is still lacking a satisfactory curative approach. The standard management consisting of gross total resection followed by radiotherapy and chemotherapy with temozolomide only prolongs patients' life moderately. In recent years, many therapeutics have failed to give a breakthrough in GBM treatment. In the search for new treatment solutions, we became interested in the repurposing of existing medicines, which have established safety profiles. We focused on the possible implementation of well-known drugs, metformin, and arginine. Metformin is widely used in diabetes treatment, but arginine is mainly a cardiovascular protective drug. We evaluated the effects of metformin and arginine on total DNA methylation, as well as the oxidative stress evoked by treatment with those agents. In glioblastoma cell lines, a decrease in 5-methylcytosine contents was observed with increasing drug concentration. When combined with temozolomide, both guanidines parallelly increased DNA methylation and decreased 8-oxo-deoxyguanosine contents. These effects can be explained by specific interactions of the guanidine group with m5CpG dinucleotide. We showed that metformin and arginine act on the epigenetic level, influencing the foreground and potent DNA regulatory mechanisms. Therefore, they can be used separately or in combination with temozolomide, in various stages of disease, depending on desired treatment effects.

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Currently, valproic acid (VPA) is known as an inhibitor of histone deacetylase (epigenetic drug) and is used for the clinical treatment of epileptic events in the course of glioblastoma multiforme (GBM). Which improves the clinical outcome of those patients. We analyzed the level of 5-methylcytosine, a DNA epigenetic modulator, and 8-oxodeoxyguanosine, an cellular oxidative damage marker, affected with VPA administration, alone and in combination with temozolomide (TMZ), of glioma (T98G, U118, U138), other cancer (HeLa), and normal (HaCaT) cell lines. We observed the VPA dose-dependent changes in the total DNA methylation in neoplastic cell lines and the lack of such an effect in a normal cell line. VPA at high concentrations (250-500 µM) induced hypermethylation of DNA in a short time frame. However, the exposition of GBM cells to the combination of VPA and TMZ resulted in DNA hypomethylation. At the same time, we observed an increase of genomic 8-oxo-dG, which as a hydroxyl radical reaction product with guanosine residue in DNA suggests a red-ox imbalance in the cancer cells and radical damage of DNA. Our data show that VPA as an HDAC inhibitor does not induce changes only in histone acetylation, but also changes in the state of DNA modification. It shows cross-reactivity between chromatin remodeling due to histone acetylation and DNA methylation. Finally, total DNA cytosine methylation and guanosine oxidation changes in glioma cell lines under VPA treatment suggest a new epigenetic mechanism of that drug action.

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Preterm infants face a significant risk of brain injury in the perinatal period, as well as potential long-term neurodevelopmental disabilities. However, preterm children with brain injury lack specific clinical manifestations in the early days. Therefore, timely and accurate diagnosis of brain injury is of vital importance. This study was to explore the diagnostic efficiency of myelin basic protein (MBP) and 8-oxo-deoxyguanosine (8-oxo-dG) serum levels in brain injury of premature infants. A total of 75 preterm infants with gestational age between 28 and 32 weeks and birth weight higher than 1,000 g were prospectively included. MBP serum levels were significantly higher in premature infants with white matter injury (WMI). 8-oxo-dG serum levels were significantly increased in both WMI and periventricular-intraventricular hemorrhages (PIVH). MBP and 8-oxo-dG were significantly correlated. The area under the curve was 0.811 [95% confidence interval (CI) 0.667-0.955; p = 0.002] in MBP and 0.729 (95% CI 0.562-0.897; p = 0.020) in 8-oxo-dG. Therefore, the results showed that high MBP levels indicated a possibility of WMI in the premature brain during the early postnatal period, while high 8-oxo-dG levels were closely related to both WMI and PIVH, thus suggesting that MBP and 8-oxo-dG could be used as potential neuro-markers of preterm brain injury.

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BACKGROUND/AIM: Nut consumption is associated with lower risk of colorectal cancer (CRC). Previously, single nut varieties have been investigated but there is limited research on the consumption of a nut mixture and the underlying mechanisms. This study examined mixed nut consumption's effect on colonic cell proliferation, apoptosis, and gene expression involved in CRC. MATERIALS AND METHODS: Thirty 21-day old Sprague Dawley rats were divided into three groups: control (no nuts), pistachio or mixed nut for 8 weeks. Ki-67 quantitative immunostaining was used to mark proliferative cells and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay for apoptotic cells. Real-time quantitative polymerase chain reaction analysis was used to determine colonic gene expression of prostaglandin endoperoxide synthase 2 (Ptgs2), nuclear factor kappa-B p65 subunit (Rela), cyclin D1 (Ccnd1), peroxisome proliferator-activated receptor gamma (Pparg), O6-methylguanine-DNA-methyltransferase (Mgmt), 8-oxoguanine glycosylase (Ogg1), superoxide dismutase (Sod), and catalase (Cat). RESULTS: DNA damage, determined using 8-oxo-deoxyguanosin, was found to be lower in the mixed nut group only (p<0.05). Differences in proliferation and apoptosis among all three groups were not significant. Lower levels of the inflammatory marker, Ptgs2, were observed between the pistachio group and the control (p=0.035). The pistachio and mixed nut groups had lower levels of Rela compared to the control (p=0.029). Differences among diets for Ccnd1, Pparg, Mgmt, Ogg1, Sod, and Cat were not significant. CONCLUSION: Mixed nut consumption reduced DNA damage possibly via down-regulation of Rela inflammation gene expression without changes to colonic cell proliferation and apoptosis.

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DNA modifications can be used to monitor pathological processes. We have previously shown that estimating the amount of the main DNA epigenetic mark, 5-methylcytosine (m5C), is an efficient and reliable way to diagnose brain tumors, hypertension, and other diseases. Abnormal increases of reactive oxygen species (ROS) are a driving factor for mutations that lead to changes in m5C levels and cancer evolution. 8-oxo-deoxyguanosine (8-oxo-dG) is a specific marker of ROS-driven DNA-damage, and its accumulation makes m5C a hotspot for mutations. It is unknown how m5C and 8-oxo-dG correlate with the malignancy of gliomas. We analyzed the total contents of m5C and 8-oxo-dG in DNA from tumor tissue and peripheral blood samples from brain glioma patients. We found an opposite relationship in the amounts of m5C and 8-oxo-dG, which correlated with glioma grade in the way that low level of m5C and high level of 8-oxo-dG indicated increased glioma malignancy grade. Our results could be directly applied to patient monitoring and treatment protocols for gliomas, as well as bolster previous findings, suggesting that spontaneously generated ROS react with m5C. Because of the similar mechanisms of m5C and guanosine oxidation, we concluded that 8-oxo-dG could also predict glioma malignancy grade and global DNA demethylation in cancer cells.

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The aim of our study was to determine the protective efficacy of the PrC-210 aminothiol radioprotector against X-ray-induced DNA damage in normal human cells and to establish dose- and time-effect models for future PrC-210 use in humans. The PrC-210 structure has a branched structure which enables scavenging of reactive oxygen species (ROS) away from DNA. Normal human blood lymphocytes, fibroblasts and naked genomic DNA were exposed to PrC-210 seconds to hours prior to irradiation. Biological (γ-H2AX foci), chemical (8-oxo-deoxyguanosine) and physical (genomic DNA electrophoretic migration) DNA damage endpoints were scored to determine the ability of PrC-210 to suppress radiation-induced DNA damage. X-ray-induced γ-H2AX foci in blood lymphocytes were reduced by 80% after irradiation with 10, 50 and 100 mGy, and DNA double-strand breaks in fibroblasts were reduced by 60% after irradiation with 20 Gy. Additionally, we observed a reduction of 8-oxo-deoxyguanosine (an ROS-mediated, DNA damage marker) in human genomic DNA to background in a PrC-210 dose-dependent manner. PrC-210 also eliminated radiation-induced cell death in colony formation assays after irradiation with 1 Gy. The protective efficacy of PrC-210 in each of these assay systems supports its development as a radioprotector for humans in multiple radiation exposure settings.

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The retina is sensitive for light damages, because of direct light exposure, especially intense blue and UV light, which increase level of ROS and other toxic phototransduction products in photoreceptor cells. In our previous work ( Damulewicz et al., 2017a and 2017b), we used 8-oxo-deoxyguanosine (8-OHdG) as a marker for oxidative stress to investigate the role of heme oxygenase in DNA protection against UV light. In this protocol, we showed how to determine the level of DNA damages in the retina using immunohistochemical staining.

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INTRODUCTION: Visfatin/nicotinamide phosphoribosyltransferase (Nampt), an enzyme involved in energy metabolism and sirtuins, SIRT1 and SIRT3, which are NAD-dependent deacetylases, are critical for cellular function. All three either regulate or are regulated by intracellular NAD+ levels and therefore available cellular energy, important for placental cell survival and successful pregnancy. This study investigates whether these protective proteins are involved in the placental pathophysiology of pre-eclampsia (PE) and if they are associated with 8-oxo-deoxyguanosine (8OHdG), a marker of oxidative damage or with placental telomere length. METHODS: Maternal blood and placental samples were collected from 31 patients with PE and 30 controls between 31 and 40 weeks gestation. Quantitative immunohistochemistry was performed on placental specimens for visfatin/Nampt, SIRT1, SIRT3, and nuclear 8OHdG. Plasma visfatin was measured by ELISA and telomere length by Southern blot analysis of telomere restriction fragments. RESULTS: Visfatin/Nampt and SIRT1 in syncytiotrophoblast decreased in PE compared to controls (p < 0.0001, p = 0.004 respectively). SIRT3 decreased in PE most significantly at preterm (p = 0.002). 8OHdG was only significantly lower in preterm controls compared to term controls (p = 0.01) and correlated with SIRT1 in all samples (r = 0.27). Telomere length was not different in PE and controls. DISCUSSION: Decreased visfatin/Nampt, SIRT1 and SIRT3 in syncytiotrophoblast in PE suggests a lack of placental reserve in metabolic energy efficiency, increased inflammation, and lower resistance to environmental stressors. However, there was little effect on nuclear function, or evidence of genomic DNA damage, which would lead to cellular senescence and death.

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8-oxo-7,8-dihydroxy-2'-deoxyguanosine (8-oxo-dG) has high mutagenic potential as it is prone to mispair with deoxyadenine (dA). In order to maintain genomic integrity, post-replicative 8-oxo-dG:dA mispairs are removed through DNA polymerase lambda (Pol λ)-dependent MUTYH-initiated base excision repair (BER). Here, we describe seven novel crystal structures and kinetic data that fully characterize 8-oxo-dG bypass by Pol λ. We demonstrate that Pol λ has a flexible active site that can tolerate 8-oxo-dG in either the anti- or syn-conformation. Importantly, we show that discrimination against the pro-mutagenic syn-conformation occurs at the extension step and identify the residue responsible for this selectivity. This residue acts as a kinetic switch, shunting repair toward long-patch BER upon correct dCMP incorporation, thus enhancing repair efficiency. Moreover, this switch also provides a potential mechanism to increase repair fidelity of MUTYH-initiated BER.

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Hyperthermophilic archaea offer certain advantages as models of genome replication, and Sulfolobus Y-family polymerases Dpo4 (S. solfataricus) and Dbh (S. acidocaldarius) have been studied intensively in vitro as biochemical and structural models of trans-lesion DNA synthesis (TLS). However, the genetic functions of these enzymes have not been determined in the native context of living cells. We developed the first quantitative genetic assays of replication past defined DNA lesions and error-prone motifs in Sulfolobus chromosomes and used them to measure the efficiency and accuracy of bypass in normal and dbh(-) strains of Sulfolobus acidocaldarius. Oligonucleotide-mediated transformation allowed low levels of abasic-site bypass to be observed in S. acidocaldarius and demonstrated that the local sequence context affected bypass specificity; in addition, most erroneous TLS did not require Dbh function. Applying the technique to another common lesion, 7,8-dihydro-8-oxo-deoxyguanosine (8-oxo-dG), revealed an antimutagenic role of Dbh. The efficiency and accuracy of replication past 8-oxo-dG was higher in the presence of Dbh, and up to 90% of the Dbh-dependent events inserted dC. A third set of assays, based on phenotypic reversion, showed no effect of Dbh function on spontaneous -1 frameshifts in mononucleotide tracts in vivo, despite the extremely frequent slippage at these motifs documented in vitro. Taken together, the results indicate that a primary genetic role of Dbh is to avoid mutations at 8-oxo-dG that occur when other Sulfolobus enzymes replicate past this lesion. The genetic evidence that Dbh is recruited to 8-oxo-dG raises questions regarding the mechanism of recruitment, since Sulfolobus spp. have eukaryotic-like replisomes but no ubiquitin.

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Metabolic syndrome describes a group of clinical features that together increase the incidence of coronary artery disease, stroke and type 2 diabetes. Insulin resistance is a major risk factor for developing metabolic syndrome. A chronic state of inflammation accompanies the accumulation of surplus lipids in adipose and liver tissue, frequently involved in insulin resistance. 8-Oxo-2'-deoxyguanosine (8-Oxo-dG) is a potent anti-inflammatory agent that inactivates both Rac1 and Rac2 which are critical to initiating the inflammatory responses in various cell types, including macrophages. In this study, we explored whether 8-Oxo-dG suppressed a series of systemic inflammatory cascades, resulting in the amelioration of typical features of metabolic syndrome in obese mice. The results demonstrate that 8-Oxo-dG effectively improved hyperglycemia, dyslipidemia and fatty liver changes in obese mice. The level of biochemical markers indicative of systemic inflammation were reduced in 8-Oxo-dG treated mice, whereas serum levels of adiponectin, a crucial factor associated with improved metabolic syndrome, were enhanced. Our results demonstrate that 8-Oxo-dG effectively disrupts the pathogenesis of insulin resistance and obesity-associated metabolic syndrome.

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Sporadic amyotrophic lateral sclerosis (ALS) is one of the most devastating neurological diseases; most patients die within 3 to 4 years after symptom onset. Oxidative stress is a disturbance in the pro-oxidative/antioxidative balance favoring the pro-oxidative state. Autopsy and laboratory studies in ALS indicate that oxidative stress plays a major role in motor neuron degeneration and astrocyte dysfunction. Oxidative stress biomarkers in cerebrospinal fluid, plasma, and urine are elevated, suggesting that abnormal oxidative stress is generated outside of the central nervous system. Our review indicates that agricultural chemicals, heavy metals, military service, professional sports, excessive physical exertion, chronic head trauma, and certain foods might be modestly associated with ALS risk, with a stronger association between risk and smoking. At the cellular level, these factors are all involved in generating oxidative stress. Experimental studies indicate that a combination of insults that induce modest oxidative stress can exert additive deleterious effects on motor neurons, suggesting that multiple exposures in real-world environments are important. As the disease progresses, nutritional deficiency, cachexia, psychological stress, and impending respiratory failure may further increase oxidative stress. Moreover, accumulating evidence suggests that ALS is possibly a systemic disease. Laboratory, pathologic, and epidemiologic evidence clearly supports the hypothesis that oxidative stress is central in the pathogenic process, particularly in genetically susceptive individuals. If we are to improve ALS treatment, well-designed biochemical and genetic epidemiological studies, combined with a multidisciplinary research approach, are needed and will provide knowledge crucial to our understanding of ALS etiology, pathophysiology, and prognosis.

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