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Objectives: Benign Prostatic Hyperplasia (BPH) and Prostate Cancer (PC) are very common pathologies among aging men. Both disorders involve aberrant cell division and differentiation within the prostate gland. However, the direct link between these two disorders still remains controversial. A plethora of works have demonstrated that inflammation is a major causative factor in both pathologies. Another key factor involved in PC development is DNA methylation and hydroxymethylation., Methods: A broad spectrum of parameters, including epigenetic DNA modifications and 8-oxo-7,8-dihydro-2'-deoxyguanosine, was analyzed by two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry in tissues of BPH, PC, and marginal one, as well as in leukocytes of the patients and the control group. In the same material, the expression of TETs and TDG genes was measured using RT-qPCR. Additionally, vitamin C was quantified in the blood plasma and within cells (leukocytes and prostate tissues)., Results: Unique patterns of DNA modifications and intracellular vitamin C (iVC) in BPH and PC tissues, as well as in leukocytes, were found in comparison with control samples. The majority of the alterations were more pronounced in leukocytes than in the prostate tissues., Conclusions: Characteristic DNA methylation/hydroxymethylation and iVC profiles have been observed in both PC and BPH, suggesting potential shared molecular pathways between the two conditions. As a fraction of leukocytes may be recruited to the pathological tissues and can migrate back into the circulation/blood, the observed alterations in leukocytes may reflect dynamic changes associated with the PC development, suggesting their potential utility as early markers of prostate cancer development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Prostate cancer (PC) represents one of the most common cancer types worldwide and many patients suffering from this kind of cancer are treated with radiotherapy (RTH). Ionizing irradiation is closely associated with reactive oxygen species (ROS) production and oxidative stress. Over the years the role of vitamin C (VC) in cancer prevention has been highlighted as it may be mediated by its ability to neutralize pro-carcinogenic ROS. However, the debate concerning the presence of VC in blood and its beneficial effect on the survival of cancer patients is inconsistent and controversial. To our best knowledge until recently there have been no studies concerning such a role of intracellular VC (iVC). In the present study, blood and intracellular concentrations of vitamin C were analyzed along with the level of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), as an established marker of the stress condition, in leukocytes of PC patients during the course of radiotherapy. The level of intracellular vitamin C significantly decreased in PC patients in comparison with the healthy group, while there were no differences in blood VC. It was observed that a sub-group of the PC patients reacted to RTH decreasing VC in leukocytes (group A), while the other sub-group acted the other way round, significantly increasing its level (group B). Under stressful conditions (RTH) leukocytes react in two different ways. Both ways are in good agreement with two well recognized functions, proposed for iVC; it may serve as a save factor, to protect the cellular DNA, increasing its concentration inside the cell (group B), and as a reservoir decreasing the VC level inside leukocytes and releasing VC into the plasma to rescue its physiological level (group A). It was also demonstrated that there was a relationship between the level of 8-oxodG in leukocytes' DNA and the markers of RTH toxicity., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background/aims: Seminal plasma composition is affected by the physiological state of the prostate, the major male reproductive gland. Semen components, like vitamin C, can modulate sperm function. Vitamin C is an effective scavenger of free radicals and is an essential component of enzymes such as TET proteins involved in the DNA demethylation process. In the present study, a broad range of parameters which may influence the metabolic state of the prostate gland were analysed including blood and prostate tissue vitamin C, epigenetic DNA modifications and 8-oxo-7,8-dihydro-2'-deoxyguanosine in DNA of leukocytes and prostate tissues., Methods: The experimental material were tissue samples from patients with benign prostatic hyperplasia (BPH), normal/marginal prostate tissues from prostate cancer patients, leukocytes from healthy donors, and blood plasma from BPH patients and healthy donors. We applied ultra-performance liquid chromatography methods with mass spectrometry and/or UV detection., Results: We found an unprecedentedly high level of intracellular vitamin C in all analysed prostatic tissues (benign prostatic hyperplasia and normal, marginal ones), a value much higher than in leukocytes and most human tissues. DNA epigenetic patterns in prostate cells are similar to other soft tissues like the colon, however, its uniqueness is the unprecedentedly high level of 5-(hydroxymethyl)-2'-deoxyuridine and a significant increase in 5-formyl-2'-deoxycytidine value compared to aforementioned tissues. Moreover, the level of 8-oxo-7,8-dihydro-2'-deoxyguanosine, an established marker of oxidative stress, is significantly higher in prostate tissues than in leukocytes and many previously studied soft tissues., Conclusion: Our results pointed out that prostatic vitamin C (regarded as the main supplier of the vitamin C to seminal plasma) and the DNA modifications (which may be linked to the regeneration of prostate epithelium) may play important role to maintain the prostate health., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

The active DNA demethylation process may be linked to aberrant methylation and may be involved in leukemogenesis. We investigated the role of epigenetic DNA modifications in childhood acute lymphoblastic leukemia (ALL) diagnostics and therapy monitoring. We analyzed the levels of 5-methyl-2'-deoxycytidine (5-mdC) oxidation products in the cellular DNA and urine of children with ALL (at diagnosis and during chemotherapy, n = 55) using two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry (2D UPLC-MS/MS). Moreover, the expression of Ten Eleven Translocation enzymes (TETs) at the mRNA and protein levels was determined. Additionally, the ascorbate level in the blood plasma was analyzed. Before treatment, the ALL patients had profoundly higher levels of the analyzed modified DNA in their urine than the controls. After chemotherapy, we observed a statistically significant decrease in active demethylation products in urine, with a final level similar to the level characteristic of healthy children. The level of 5-hmdC in the DNA of the leukocytes in blood of the patient group was significantly lower than that of the control group. Our data suggest that urinary excretion of epigenetic DNA modification may be a marker of pediatric ALL status and a reliable marker of chemotherapy response., (© 2021. The Author(s).)

DNA of all living cells undergoes continuous structural and chemical alterations resulting from fundamental cellular metabolic processes and reactivity of normal cellular metabolites and constituents. Examples include enzymatically oxidized bases, aberrantly methylated bases, and deaminated bases, the latter largely uracil from deaminated cytosine. In addition, the non-canonical DNA base uracil may result from misincorporated dUMP. Furthermore, uracil generated by deamination of cytosine in DNA is not always damage as it is also an intermediate in normal somatic hypermutation (SHM) and class shift recombination (CSR) at the Ig locus of B-cells in adaptive immunity. Many of the modifications alter base-pairing properties and may thus cause replicative and transcriptional mutagenesis. The best known and most studied epigenetic mark in DNA is 5-methylcytosine (5mC), generated by a methyltransferase that uses SAM as methyl donor, usually in CpG contexts. Oxidation products of 5mC are now thought to be intermediates in active demethylation as well as epigenetic marks in their own rights. The aim of this review is to describe the endogenous processes that surround the generation and removal of the most common types of DNA nucleobase modifications, namely, uracil and certain epigenetic modifications, together with their role in the development of hematological malignances. We also discuss what dictates whether the presence of an altered nucleobase is defined as damage or a natural modification.

Vitamin C is implicated in various bodily functions due to its unique properties in redox homeostasis. Moreover, vitamin C also plays a great role in restoring the activity of 2-oxoglutarate and Fe 2+ dependent dioxygenases (2-OGDD), which are involved in active DNA demethylation (TET proteins), the demethylation of histones, and hypoxia processes. Therefore, vitamin C may be engaged in the regulation of gene expression or in a hypoxic state. Hence, vitamin C has acquired great interest for its plausible effects on cancer treatment. Since its conceptualization, the role of vitamin C in cancer therapy has been a controversial and disputed issue. Vitamin C is transferred to the cells with sodium dependent transporters (SVCTs) and glucose transporters (GLUT). However, it is unknown whether the impaired function of these transporters may lead to carcinogenesis and tumor progression. Notably, previous studies have identified SVCTs' polymorphisms or their altered expression in some types of cancer. This review discusses the potential effects of vitamin C and the impaired SVCT function in cancers. The variations in vitamin C transporter genes may regulate the active transport of vitamin C, and therefore have an impact on cancer risk, but further studies are needed to thoroughly elucidate their involvement in cancer biology.

Background: A characteristic feature of malignant cells, such as colorectal cancer cells, is a profound decrease in the level of 5-hydroxymethylcytosine, a product of 5-methylcytosine oxidation by TET enzymes. Recent studies showed that ascorbate may upregulate the activity of TET enzymes in cultured cells and enhance formation of their products in genomic DNA., Methods: The study included four groups of subjects: healthy controls (n = 79), patients with inflammatory bowel disease (IBD, n = 51), adenomatous polyps (n = 67) and colorectal cancer (n = 136). The list of analyzed parameters included (i) leukocyte levels of epigenetic DNA modifications and 8-oxo-7,8-dihydro-2'-deoxyguanosine, a marker of oxidatively modified DNA, determined by means of isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry, (ii) expression of TET mRNA measured with RT-qPCR, and (iii) chromatographically-determined plasma concentrations of retinol, alpha-tocopherol and ascorbate., Results: Patients from all groups presented with significantly lower levels of 5-methylcytosine and 5-hydroxymethylcytosine in DNA than the controls. A similar tendency was also observed for 5-hydroxymethyluracil level. Patients with IBD showed the highest levels of 5-formylcytosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine of all study subjects, and individuals with colorectal cancer presented with the lowest concentrations of ascorbate and retinol. A positive correlation was observed between plasma concentration of ascorbate and levels of two epigenetic modifications, 5-hydroxymethylcytosine and 5-hydroxymethyluracil in leukocyte DNA. Moreover, a significant difference was found in the levels of these modifications in patients whose plasma concentrations of ascorbate were below the lower and above the upper quartile for the control group., Conclusions: These findings suggest that deficiency of ascorbate in the blood may be a marker of its shortage in other tissues, which in turn may correspond to deterioration of DNA methylation-demethylation. These observations may provide a rationale for further research on blood biomarkers of colorectal cancer development.

5-Hydroxymethylcytosine and 5-formylcytosine are stable DNA base modifications generated from 5-methylcytosine by the ten-eleven translocation protein family that function as epigenetic markers. 5-Hydroxymethyluracil may also be generated from thymine by ten-eleven translocation enzymes. Here, we asked if these epigenetic changes accumulate in senescent cells, since they are thought to be inversely correlated with proliferation. Testing this in ERCC1-XPF-deficient cells and mice also enabled discovery if these DNA base changes are repaired by nucleotide excision repair. Epigenetic marks were measured in proliferating, quiescent and senescent wild-type (WT) and Ercc1-/- primary mouse embryonic fibroblasts. The pattern of epigenetic marks depended more on the proliferation status of the cells than their DNA repair capacity. The cytosine modifications were all decreased in senescent cells compared to quiescent or proliferating cells, whereas 5-(hydroxymethyl)-2'-deoxyuridine was increased. In vivo, both 5-(hydroxymethyl)-2'-deoxyuridine and 5-(hydroxymethyl)-2'-deoxycytidine were significantly increased in liver tissues of aged WT mice compared to young adult WT mice. Livers of Ercc1-deficient mice with premature senescence and aging had reduced level of 5-(hydroxymethyl)-2'-deoxycytidine and 5-formyl-2'-deoxycytidine compared to aged-matched WT controls. Taken together, we demonstrate for the first time, that 5-(hydroxymethyl)-2'-deoxycytidine is significantly reduced in senescent cells and tissue, potentially yielding a novel marker of senescence.

Background: Active demethylation of 5-methyl-2'-deoxycytidine (5-mdC) in DNA occurs by oxidation to 5-(hydroxymethyl)-2'-deoxycytidine (5-hmdC) and further oxidation to 5-formyl-2'-deoxycytidine (5-fdC) and 5-carboxy-2'-deoxycytidine (5-cadC), and is carried out by enzymes of the ten-eleven translocation family (TETs 1, 2, 3). Decreased level of epigenetic DNA modifications in cancer tissue may be a consequence of reduced activity/expression of TET proteins. To determine the role of epigenetic DNA modifications in colon cancer development, we analyzed their levels in normal colon and various colonic pathologies. Moreover, we determined the expressions of TETs at mRNA and protein level.The study included material from patients with inflammatory bowel disease (IBD), benign polyps (AD), and colorectal cancer (CRC). The levels of epigenetic DNA modifications and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in examined tissues were determined by means of isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS). The expressions of TET mRNA were measured with RT-qPCR, and the expressions of TET proteins were determined immunohistochemically., Results: IBD was characterized by the highest level of 8-oxodG among all analyzed tissues, as well as by a decrease in 5-hmdC and 5-mdC levels (at a midrange between normal colon and CRC). AD had the lowest levels of 5-hmdC and 5-mdC of all examined tissues and showed an increase in 8-oxodG and 5-(hydroxymethyl)-2'-deoxyuridine (5-hmdU) levels. CRC was characterized by lower levels of 5-hmdC and 5-mdC, the lowest level of 5-fdC among all analyzed tissues, and relatively high content of 5-cadC. The expression of TET1 mRNA in CRC and AD was significantly weaker than in IBD and normal colon. Furthermore, CRC and AD showed significantly lower levels of TET2 and AID mRNA than normal colonic tissue., Conclusions: Our findings suggest that a complex relationship between aberrant pattern of DNA epigenetic modification and cancer development does not depend solely on the transcriptional status of TET proteins, but also on the characteristics of premalignant/malignant cells. This study showed for the first time that the examined colonic pathologies had their unique epigenetic marks, distinguishing them from each other, as well as from normal colonic tissue. A decrease in 5-fdC level may be a characteristic feature of largely undifferentiated cancer cells., Competing Interests: The study was conducted in accordance with the Declaration of Helsinki, its protocol was approved by the Local Bioethics Committee at Collegium Medicum, Nicolaus Copernicus University in Bydgoszcz (Poland), and written informed consent was sought from all the subjects.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

[This corrects the article DOI: 10.1371/journal.pone.0188856.].

Active demethylation of 5-methylcytosine moiety in DNA occurs by its sequential oxidation to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine, catalysed by enzymes of the Ten-Eleven Translocation family proteins (TETs 1, 2 and 3). Here we analyzed for the first time all the intermediate products of DNA demethylation pathway in the form of deoxynucleosides (5-methyl-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine and 5-carboxy-2'-deoxycytidine as well as 5-(hydroxymethyl)-2'-deoxyuridine) using automated isotope-dilution online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry. DNA was isolated from human malignant cell lines of colon adenocarcinoma (HCT 116), melanoma (Me45), myelogenous leukemia bone marrow blasts (K562), EBV-positive Burkitt's lymphoma lymphoblasts (Raji), EBV-negative Burkitt's lymphoma lymphoblasts (male-CA46 and female-ST486), as well as normal neonatal dermal fibroblasts (NHDF-Neo). The expression levels of TET1, TET2, TET3, SMUG1, and TDG genes were also assayed by RT-qPCR. Our results show a global erasure of 5-hydroxymethyl-2'-deoxycytidine and 5-carboxy-2'-deoxycytidine in DNA of cultured cells compared with DNA from primary malignant tissue. Moreover, malignant cells in culture have a quite different DNA epigenetic profile than cultured normal cells, and different types of malignant cells display different and characteristic profiles of DNA epigenetic marks. Similar analyses of a broader spectrum of epigenetic modifications, not restricted to 5-methyl-2'-deoxycytidine, could lead to better understanding of the mechanism(s) responsible for emergence of different types of cancer cells.

Background: The voltage-dependent anion channels (VDAC) play an essential role in the cross talk between mitochondria and the rest of the cell. Their implication in cell life and cell death has been studied extensively in recent years. In this work we studied the impact of mitochondrial membrane (VDACs) on cell survival and response to X-ionizing radiation (IR) of human lymphoblastoid K562 cells., Methods: The inhibition of VDACs was achieved by 4,4`-diisothiocyanostilbene-2,2`-disulfonic acid (DIDS) inhibitor and in vitro experiments including clonogenity assay, UV-visible spectrophotometry, comet assay and FACS analysis were implemented., Results: Inhibition of VDAC led to augmentation of IR-induced apoptosis and ROS production. Additionally, DIDS affected repair of IR-induced DNA strand breaks and was in line with both induction of apoptosis and caspase activity. The IR-induced NO production was potently reduced by inhibition of VDAC., Conclusion: Our results suggest that VDAC control cellular response to ionizing radiation through modulation of the ROS- and NO-dependent signaling pathways. Inhibition of VDAC with DIDS induced apoptosis in irradiated K562 lymphoblastoid cells points at DIDS, as a promising agent to enhance the effectiveness of radiotherapy., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

The most plausible mechanism behind active demethylation of 5-methylcytosine involves TET proteins which participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine; the latter is further oxidized to 5-formylcytosine and 5-carboxycytosine. 5-Hydroxymethyluracil can be also generated from thymine in a TET-catalyzed process. Ascorbate was previously demonstrated to enhance generation of 5-hydroxymethylcytosine in cultured cells. The aim of this study was to determine the levels of the abovementioned TET-mediated oxidation products of 5-methylcytosine and thymine after addition of ascorbate, using an isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with electrospray ionization tandem mass spectrometry. Intracellular concentration of ascorbate was determined by means of ultra-performance liquid chromatography with UV detection. Irrespective of its concentration in culture medium (10-100µM) and inside the cell, ascorbate stimulated a moderate (2- to 3-fold) albeit persistent (up to 96-h) increase in the level of 5-hydroxymethylcytosine. However, exposure of cells to higher concentrations of ascorbate (100µM or 1mM) stimulated a substantial increase in 5-formylcytosine and 5-carboxycytosine levels. Moreover, for the first time we demonstrated a spectacular (up to 18.5-fold) increase in 5-hydroxymethyluracil content what, in turn, suggests that TET enzymes contributed to the presence of the modification in cellular DNA. These findings suggest that physiological concentrations of ascorbate in human serum (10-100µM) are sufficient to maintain a stable level of 5-hydroxymethylcytosine in cellular DNA. However, markedly higher concentrations of ascorbate (ca. 100µM in the cell milieu or ca. 1mM inside the cell) were needed to obtain a sustained increase in 5-formylcytosine, 5-carboxycytosine and 5-hydroxymethyluracil levels. Such feedback to elevated concentrations of ascorbate may reflect adaptation of the cell to environmental conditions., (Copyright © 2016 Elsevier Inc. All rights reserved.)

5-Methylcytosine is one of the most important epigenetic modifications and has a profound impact on embryonic development. After gamete fusion, there is a widespread and rapid active demethylation process of sperm DNA, which suggests that the paternal epigenome has an important role during embryonic development. To better understand the epigenome of sperm DNA and its possible involvement in a developing embryo, we determined epigenetic marks in human sperm DNA and in surrogate somatic tissue leukocytes; the analyzed epigenetic modifications included 5-methyl-2'-deoxycytidine, 5-hydroxymethyl-2'-deoxycytidine, and 5-hydroxymethyl-2'-deoxyuridine. For absolute determination of the modification, we used liquid chromatography with UV detection and tandem mass spectrometry techniques with isotopically labeled internal standards. Our analyses demonstrated, for the first time to date, that absolute global values of 5-methyl-2'-deoxycytidine, 5-hydroxymethyl-2'-deoxycytidine, and 5-hydroxymethyl-2'-deoxyuridine in sperm are highly statistically different from those observed for leukocyte DNA, with respective mean values of 3.815% versus 4.307%, 0.797 versus 2.945 per 10⁴ deoxynucleosides, and 5.209 versus 0.492 per 10⁶ deoxynucleosides. We hypothesize that an exceptionally high value of 5-hydroxymethyluracil in sperm (>10-fold higher than in leukocytes) may play a not yet recognized regulatory role in the paternal genome., (© 2014 by the Society for the Study of Reproduction, Inc.)

The oxidatively modified DNA base 8-oxo-7,8-dihydroguanine (8-oxoGua) is nontoxic and weakly mutagenic. Here we report on new data suggesting a potential for 8-oxoGua to affect the expression of several genes via epigenetic changes resulting in chromatin relaxation. Using pig thymus extract, we analyzed the distribution of 8-oxoGua among different nuclei fractions representative of transcriptionally active and silenced regions. The levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) found in transcriptionally active euchromatin (4.37/10(6) nucleotides) and in the matrix fraction (4.16/10(6) nucleotides) were about 5 times higher than in transcriptionally silenced heterochromatin (0.91/10(6) nucleotides). Other experimental data are presented which suggest that 8-oxoGua present in specific DNA sequences may be widely used for transcription regulation. Like 8-oxoGua, 5-hydroxymethyluracil (5-hmUra) is another oxidatively modified DNA base (the derivative is formed by thymine oxidation). Recent experimental evidence supports the notion that 5-hmUra plays an important role in active DNA demethylation. This involves overexpression of activation-induced cytidine deaminase (AID) and ten-eleven translocation 1 (TET1) protein (the key proteins involved in active demethylation), which leads to global accumulation of 5-hmUra. Our preliminary data demonstrate a significant increase of the 5-hmUra levels in pig brain extract when compared with liver extract. The lack of 5-hmUra in Escherichia coli DNA also speaks for a role of this modification in the active demethylation process. It is concluded that 8-oxodG and 5-hmUra in DNA may be considered as epigenetic marks., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The aim of this work was to answer the question whether the broad range of parameters which describe oxidative stress and oxidatively damaged DNA and repair are appropriate prognosis factors of colon cancer (CRC) patients survival? The following parameters were analyzed for 89 CRC patients: concentration of uric acid and vitamins A, E, C in plasma; levels of 8-oxodGuo (8-oxo-7,8-dihydro-2'-deoxyguanosine) in DNA of leukocyte and colon tissues; urinary excretion rates of 8-oxodGuo and 8-oxoGua (8-oxo-7,8-dihydroguanine); the activity and mRNA or protein level of repair enzymes OGG1, APE1, ANPG, TDG and PARP1. All DNA modifications and plasma antioxidants were analyzed using high performance liquid chromatography (HPLC) or HPLC/gas chromatography-mass spectrometry techniques. Expression of repair proteins was analyzed by QPCR, Western or immunohistochemistry methods. Longer survival coincided with low levels of 8-oxodGuo/8oxoGua in urine and 8-oxodGuo in DNA as well as with high concentration of uric acid plasma level. In contrast to expectations, longer survival coincided with lower mRNA level in normal colon tissue of the main 8-oxoGua DNA glycosylase, OGG1, but no association was found for PARP-1 expression. When analyzing simultaneously two parameters the discriminating power increased significantly. Combination of low level of urinary 8-oxoGua together with low level of 8-oxodGuo in leukocyte (both below median value) or high concentration of plasma uric acid (above median value) have the best prediction power. Since prediction value of these parameters seems to be comparable to conventional staging procedure, they could possibly be used as markers to predict clinical success in CRC treatment., (Copyright © 2013 UICC.)

Objectives: This study explored the relationship between oxidative stress biomarkers and stability of carotid plaque. We decided to analyze the broad range of parameters describing oxidative stress in patients with carotid stenosis., Design and Methods: 124 consecutive patients undergoing carotid endarterectomy were enrolled in the study group. The control group consisted of 49 patients without symptoms of atherosclerosis. The stability of carotid plaques was assessed using GSM (gray-scale median) scoring system and the study group was divided into three subgroups according to echogenicity of the plaque. The following parameters of oxidative stress/DNA damage were analyzed: i) urinary excretion of the products of oxidative DNA damage repair; ii) the background level of 8-oxo-7,8-dihydro-2'-deoxyguanosine in leukocytes' DNA and in atherosclerotic plaques; and iii) the concentrations of antioxidant vitamins, uric acid and C-reactive protein in plasma., Results: Oxidative stress (described by redox status) was higher in the patient group than in the control group. There is a correlation between oxidative stress of the patients and stability of the plaque, echolucent plaques (GSM<25) being associated with the highest antioxidant level and lowest excretion of DNA repair markers., Conclusions: The plaque formation/morphology may depend on local environment and is independent of oxidative stress/inflammation observed on the level of the whole body., (Copyright © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Abnormal spermatozoa frequently display typical features of oxidative stress, i.e. excessive level of reactive oxygen species (ROS) and depleted antioxidant capacity. Moreover, it has been found that a high level of oxidatively damaged DNA is associated with abnormal spermatozoa and male infertility. Therefore, the aim of our study was the comparison of oxidative stress/DNA damage in semen and blood of fertile and infertile men. The broad range of parameters which describe oxidative stress and oxidatively damaged DNA and repair were analyzed in the blood plasma and seminal plasma of groups of fertile and infertile subjects. These parameters include: (i) 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanine (8-oxoGua) levels in urine; (ii) 8-oxodG level in DNA isolated from leukocytes and spermatozoa; (iii) antioxidant vitamins (A, C and E) and uric acid. Urinary excretion of 8-oxodG and 8-oxoGua and the level of oxidatively damaged DNA in leukocytes as well as the level of antioxidant vitamins were analyzed using HPLC and HPLC/GC/MS methods. The results of our study demonstrate that 8-oxodG level significantly correlated with every parameter which describe sperm quality: sperm count, motility and morphology. Moreover, the data indicate a higher level of 8-oxodG in sperm DNA compared with DNA of surrogate tissue (leukocytes) in infertile men as well as in healthy control group. For the whole study population the median values of 8-oxodG/10(6) dG were respectively 7.85 and 5.87 (p=0.000000002). Since 8-oxodG level in sperm DNA is inversely correlated with urinary excretion rate of 8-oxoGua, which is the product of OGG1 activity, we hypothesize that integrity of spermatozoa DNA may be highly dependent on OGG1 activity. No relationship between the whole body oxidative stress and that of sperm plasma was found, which suggests that the redox status of semen may be rather independent on this characteristic for other tissues.

DNA damage and DNA repair may mediate several cellular processes, like replication and transcription, mutagenesis and apoptosis and thus may be important factors in the development and pathology of an organism, including cancer. DNA is constantly damaged by reactive oxygen species (ROS) and reactive nitrogen species (RNS) directly and also by products of lipid peroxidation (LPO), which form exocyclic adducts to DNA bases. A wide variety of oxidatively-generated DNA lesions are present in living cells. 8-oxoguanine (8-oxoGua) is one of the best known DNA lesions due to its mutagenic properties. Among LPO-derived DNA base modifications the most intensively studied are ethenoadenine and ethenocytosine, highly miscoding DNA lesions considered as markers of oxidative stress and promutagenic DNA damage. Although at present it is impossible to directly answer the question concerning involvement of oxidatively damaged DNA in cancer etiology, it is likely that oxidatively modified DNA bases may serve as a source of mutations that initiate carcinogenesis and are involved in aging (i.e. they may be causal factors responsible for these processes). To counteract the deleterious effect of oxidatively damaged DNA, all organisms have developed several DNA repair mechanisms. The efficiency of oxidatively damaged DNA repair was frequently found to be decreased in cancer patients. The present work reviews the basis for the biological significance of DNA damage, particularly effects of 8-oxoGua and ethenoadduct occurrence in DNA in the aspect of cancer development, drawing attention to the multiplicity of proteins with repair activities.

Earlier experimental studies have demonstrated that: i) Cu,Zn-superoxide dismutase deficiency leads to oxidative stress and carcinogenesis; ii) dysregulation of NF-κB pathway can mediate a wide variety of diseases, including cancer. Therefore, we decided, for the first time, to examine the level of oxidative DNA damage and the DNA binding activity of NF-κB proteins in SOD1 knockout, heterozygous and wild-type mice. Two kinds of biomarkers of oxidatively damaged DNA: urinary excretion of 8-oxodG and 8-oxoGua, and the level of oxidatively damaged DNA were analysed using HPLC-GC-MS and HPLC-EC. The DNA binding activity of p50 and p65 proteins in a nuclear extracts was assessed using NF-κB p50/p65 EZ-TFA transcription factor assay. These parameters were determined in the brain, liver, kidney and urine of SOD1 knockout, heterozygous and wild-type mice. The level of 8-oxodG in DNA was higher in the liver and kidney of knockout mice than in wild type. No differences were found in urinary excretion of 8-oxoGua and 8-oxodG between wild type and the SOD1-deficient animals. The activity of the p50 protein was higher in the kidneys, but surprisingly not in the livers of SOD1-deficient mice, whereas p65 activity did not show any variability. Our results indicate that in Cu,Zn-SOD-deficient animals the level of oxidative DNA damage and NF-κB1 activity are elevated in certain organs only, which may provide some explanation for organ-specific ROS-induced carcinogenesis.

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is widely used as a marker of oxidative stress. Here we report the comparison of two, distinct chromatographic assays with an enzyme-linked immunosorbent assay (ELISA). The chromatographic assays displayed good agreement (r =:0.89, p < 0.0001), whereas there was markedly worse, albeit still significant, agreement with the ELISA (high-pressure liquid chromatography followed by gas chromatography (HPLC-GC/MS), r = 0.43; HPLC with electrochemical detection (HPLC-EC), r = 0.56; p < 0.0001). Mean values differed significantly between the chromatographic assays and the ELISA (HPLC-GC/MS 3.86, HPLC-EC 4.20, ELISA 18.70 ng mg(-1) creatinine; p < 0.0001). While it is reassuring to note good agreement between chromatographic assays, this study reveals significant short-comings in the ELISA, which brings into question its continued use in its present form.

It is possible that oxidatively damaged DNA which arises as a result of radiotherapy may be involved in the therapeutic effect of the ionizing radiation and in the side effects. Therefore, for the first time, the broad spectrum of oxidatively damaged DNA biomarkers: urinary excretion of 8-oxodG (8-oxo-7,8-dihydro-2'-deoxyguanosine), 8-oxoGua (8-oxo-7,8-dihydroguanine) as well as the level of oxidatively damaged DNA in leukocytes, was analyzed in head and neck cancer patients (n = 27) undergoing fractionated radiotherapy using methodologies which involve HPLC (high-performance liquid chromatography) prepurification followed by gas chromatography with isotope dilution mass spectrometry detection and HPLC/EC. Of all the analyzed parameters in the majority of patients, only urinary excretion of the modified nucleoside significantly increased over the initial level in the samples collected 24 hr after the last fraction. However, for the distinct subpopulation of 10 patients, a significant increase in the level of 8-oxodG in cellular DNA and a simultaneous drop in urinary 8-oxoGua (the repair product of oxidative DNA damage) were detected after completion of the therapy. Because 8-oxoGua is a repair product of the DNA damage, there is a possibility that, at least in the case of some patients with the lowest activity of OGG1 (8-oxo-7,8-dihydroguanine glycosylase), the combination of lower OGG1 repair efficacy and irradiation was associated with increased background level of 8-oxoGua in cellular DNA. Apparently reduced DNA repair is unable to cope with the radiation-induced, and the extra amount of 8-oxoGua leading to an increase of potentially mutagenic/carcinogenic lesions.

It has been known for a long time that DNA hypomethylation occurs in many human cancers and precancerous conditions. However, the mechanisms of hypomethylation are largely unknown. It is possible that endogenous 8-oxo-7,8-dihydroguanine (8-oxoGua) level may be linked to aberrant DNA methylation of adjacent cytosine and in this way influences carcinogenesis. Therefore, the aim of the present study was to assess a possible link between 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) background level and 5-methylcytosine content in DNA from human leukocytes of healthy subjects (n=105) as well as in patients with colon adenomas (n=39) and carcinomas (n=50). Our results demonstrated statistically significant negative correlation between background level of 8-oxodG and 5-methylcytosine content in DNA isolated from leukocytes of healthy donors (r=-0.3436, p=0.0003). The mean content of 5-methylcytosine was significantly lower, while 8-oxodG level was significantly higher in leukocytes DNA of patients with colon adenomas and carcinomas in comparison with healthy subjects. The mean values for 5-methylcytosine were: 3.59+/-0.173% (healthy subjects), 3.38+/-0.128% (patients with adenomas), 3.40+/-0.208% (colon cancer patients). The mean values of 8-oxodG in DNA were, respectively: 4.67+/-1.276, 5.72+/-1.787, 5.76+/-1.884 8-oxodG per 10(6) dG molecules. DNA from affected tissue (colon) suffered from significant, about 10% reduction in cytosine methylation in comparison with leukocytes of the paired subjects. Our work provides the first in vivo evidence suggesting that increased levels of 8-oxodG in DNA may lead to carcinogenesis not only via mispair/mutagenic potential of the modified base but also through its ability to influence gene expression by affecting DNA methylation.

Background: Vitamins A, E and C, and uric acid, which can scavenge free radicals should also protect DNA from the damage. It is reasonable to assume that agents that decrease oxidative DNA damage should also decrease subsequent cancer development., Aim of the Study: A relationship between basal level of antioxidants (vitamins A, C and E and uric acid) and oxidative DNA damage was assessed. For the first time, the broad spectrum of oxidative DNA damage biomarkers: urinary excretion of 8-oxodG, 8-oxoGua and 5HMUra as well as the level of oxidative DNA damage in leukocytes was analyzed in healthy subjects (n = 158)., Methods: Using HPLC prepurification/isotope dilution GC/MS methodology, we examined the amount of oxidative DNA damage products excreted into urine and the amount of 8-oxodG in leukocytes' DNA (with HPLC/EC technique). The level of antioxidant vitamins and uric acid was estimated by HPLC technique with fluorimetric and UV detection., Results: Analyses of relationship between the most common antioxidants (vitamins A, C, E and uric acid) and oxidative DNA damage products reveal weak, statistically significant negative correlation between retinol and all the measured parameters except 5HMUra. Vitamin C negatively correlates with urinary excretion of 8-oxodG and 8-oxoGua. Uric acid revealed statistically significant negative correlation with 8-oxodG in cellular DNA and urinary excretion of 5HMUra, while alpha-tocopherol correlates negatively only with 8-oxodG in cellular DNA. Good, significant (P < 0.0001), positive correlation (r = 0.61) was noted between urinary levels of the base, 8-oxoGua and the deoxynucleoside, 8-oxodG., Conclusion: Our results suggest that oxidative DNA damage shows limited but significant response to antioxidants analyzed in this study and is more affected by many other cellular functions like antioxidant enzymes or DNA repair enzymes as well as genetics.

Aging is a complex process involving morphologic and biochemical changes in single cells and in the whole organism. One of the most popular explanations of how aging occurs at the molecular level is the oxidative stress hypothesis. Oxidative stress leads in many cases to an age-dependent increase in the cellular level of oxidatively modified macromolecules including DNA, and it is this increase which has been linked to various pathological conditions, such as aging, carcinogenesis, neurodegenerative and cardiovascular diseases. It is, however, possible that a number of short-comings associated with gaps in our knowledge may be responsible for the failure to produce definite results when applied to understanding the role of DNA damage in aging and age-related diseases.

There is growing evidence suggesting that cytotoxic activity of cisplatin is closely associated with increased generation of reactive oxygen species (ROS). Therefore, this study was undertaken to examine oxidative DNA damage, which arises as a result of chemotherapy with cisplatin. Using HPLC prepurification/isotope dilution GC/MS methodology, we examined the amount of 8-oxoGua and 8-oxodG excreted into urine in cancer patients (n = 66) who received chemotherapy with cisplatin. One day after the infusion of the drug, significant increase in the amount of 8-oxoGua and 8-oxodG in urine of the patients was observed, when compared to the initial value (78%, p < 0.0001 and 22%, p = 0.0051). In the "nadir days" (when the most distinct cell death based on hematological cell counts can be observed), the level of modified base and nucleoside decreased in comparison with the aforementioned time point. These results, for the first time, indicate that oxidatively damaged DNA may be, at least in part, responsible for cisplatin induced cytotoxicity. Our results also demonstrate that cell death does not contribute to urinary 8-oxoGua and 8-oxodG in humans.

Numerous DNA repair pathways exist to prevent the persistence of damage, and are integral to the maintenance of genome stability, and hence prevention of disease. Excised lesions arising from repair may ultimately appear in the urine where their measurement has been acknowledged to be reflective of overall oxidative stress. The development of reliable assays to measure urinary DNA lesions, such as HPLC prepurification followed by gas chromatography/mass spectrometry, offers the potential to assess whole body oxidative DNA damage. However, some studies suggest a possibility that confounding factors may contribute to urinary levels of 7,8-dihydro-8-oxoguanine (8-oxoGua) and 7,8-dihydro-8-oxo-2 -deoxyguanosine (8-oxodG). This article considers several possible sources of urinary lesions: (a) the repair of oxidatively damaged DNA; (b) a possible dietary influence; and (c) cell death. The authors conclude that data from their laboratories, along with a number of literature reports, form an argument against a contribution from cell death and diet. In the absence of these confounding factors, urinary measurements may be attributed entirely to the repair of DNA damage and suggests their possible use in studying associations between DNA repair and disease.

Genome integrity is maintained via removal (repair) of DNA lesions and an increased load of such DNA damage has been linked to numerous pathological conditions, including carcinogenesis and ageing. 8-Oxo-7,8-dihydroguanine is one of the most critical lesions of this type. The free 8-oxo-7,8-dihydroguanine produced by the action of a specific DNA glycosylase is a potential source of this compound in urine. To date, there has been no direct, experimental evidence demonstrating that urinary 8-oxo-7,8-dihydroguanine is produced by the base excision repair pathway. For clarification of this issue, we applied a recently developed methodology which involved high performance liquid chromatography pre-purification followed by gas chromatography with isotope dilution mass spectrometric detection to compare the urinary excretion rate of 8-oxo-7,8-dihydroguanine in wild type and OGG1 glycosylase knock out mice. Our study revealed a 26% reduction in urinary level of 8-oxo-7,8-dihydroguanine in OGG1 deficient mice in comparison with the wild type strain. This clearly indicates that the mouse OGG1 glycosylase contributes significantly to the generation of urinary 8-oxo-7,8-dihydroguanine. Therefore, urinary measurements of 8-oxo-7,8-dihydroguanine may be attributed to DNA damage and repair, which in turn suggests that they may be useful in studying associations between DNA repair and disease.

Using recently developed methodology, which includes HPLC prepurification followed by GC/MS with isotope dilution, we analyzed urinary excretion of possible repair products of oxidative DNA damage-8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), and 5-(hydroxymethyl)uracil (5-HMUra)-in mammalian species that substantially differ in metabolic rate and longevity, namely, mice, rats, rabbits, dogs, pigs, and humans. We found highly significant, positive correlations between specific metabolic rates of the animals studied and their excretion rates for all the modifications analyzed with respective r values for the lesions of (8-oxoGua) r = .891, p < .01; (8-oxodG) r = .998, p < .001; and (5-HMUra) r = .949, p < .005. However, only 8-oxoGua significantly correlates negatively with maximum life span (MLSP) (r = -.928, p < .01). Despite substantial differences in MLSP between humans and pigs (120 and 27 years, respectively), the rates of excretion of all measured modifications were very similar. The urinary levels of all measured modifications found in our study for mouse and humans account respectively for about 34,000 and 2800 repaired events per average cell, per 24 h. It is therefore possible that the high metabolic rate in mice (or other short-lived animals) may be responsible for severe everyday oxidative DNA insults that may be accumulated faster than in long-lived species.

In order to eliminate the possibility that diet may influence urinary oxidative DNA lesion levels, in our experiments we used a recently developed technique involving HPLC pre-purification followed by gas chromatography with isotope dilution mass spectrometric detection. This methodology was applied for the determination of the lesions: 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 5-(hydroxymethyl)uracil (5HMUra) in the urine of mice fed with nucleic acid free diet and normal, unrestricted diet. The mean levels of 8-oxoGua, 8-oxodGuo and 5HMUra of the animals fed the normal diet reached the mean values of 15.6 +/- 3.5, 2.0 +/- 0.53 and 16.8 +/- 10.4 nmol/kg/24 h, After feeding the mice for 12 days with nucleic acid free diet the respective values were 18.8 +/- 4.6, 1.6 +/- 0.3 and 25.4 +/- 10.5 nmol/kg/24 h, respectively. The results clearly demonstrate that irrespective of the diet, the excretion rates were not statistically different during the course of feeding. The respective p values for the differences between lesions in the two types of diets were: 0.13 (8-oxoGua), 0.16 (8-oxodGuo), 0.18 (5-HMUra). Our results clearly indicate that diet does not contribute to urinary excretion of the lesions in mouse model.

A subject of current interest, especially in the development of androgen refractory prostate cancer, is the androgen receptor (AR) activation by growth factor receptors. Here, we report our work on the measurement of AR mRNA and protein expression in benign prostatic hyperplasia (BPH) and prostatic carcinoma (PCA) and evaluation of the relationship between AR, erbB-1 and erbB-2 gene expression determined in the same tissue. In order to define AR, erbB-1 and erbB-2 in human prostate neoplasms 36 benign prostatic hyperplasia, 46 prostatic carcinoma and 12 normal prostate gland samples were analysed. According to distant metastasis PCA tissues were divided into two categories: i) T1-4N0-3M0 (25 samples) and ii) T4N2-3M1 (21 samples). AR, erbB-1 and erbB-2 mRNA expression was estimated by RT-PCR. AR protein expression, both in nuclear and cytoplasmic fractions, was measured by Western blot technique. The association of AR mRNA and protein expression with erbB-1 and erbB-2 gene expression was evaluated. It was found that in clinically invasive (group II of PCA) prostate cancer cases AR mRNA expression was significantly correlated with erbB-2 mRNA expression (Spearman R coefficient 0.86, p<0.05). Interestingly, AR protein expression in this group of PCA was determined mainly in nuclear fraction. By Western blot AR protein was identified in 76.0% (16/21) and 23.8% (5/21) of PCA group II nuclear and cytoplasmic fractions, respectively. Furthermore, the mean AR protein level in nuclear fraction of clinically invasive (group II) PCA (0.82+/-0.04) was significantly higher (p<0.05) as compared to the normal group (0.56+/-0.11). In the case of T4N2-3M1 samples, significant correlation between AR protein level in nuclear fraction and erbB-2 mRNA expression (Spearman R coefficient 0.53, p<0.05) was stated.

A wide variety of oxidative DNA lesions are present in living cells. One of the best known lesions of this type is 8-oxoguanine (8-oxoGua) which has been shown to have mutagenic properties. An influence of antioxidative vitamins and labile iron pool on the background level of 8-oxoGua in cellular DNA is discussed and oxidative damage to free nucleotide pool as a possible source of 8-oxo-2'-deoxyguanosine in DNA and urine is described. An involvement of 8-oxoGua in the origin and/or progression of cancer is reviewed. It is concluded that a severe oxidative stress manifested as a high level of 8-oxoGua in cellular DNA as well as in urine of cancer patients is a consequence of development of many types of cancer. Although at present it is impossible to answer directly the question concerning involvement of oxidative DNA damage in cancer etiology it is likely that oxidative DNA base modifications may serve as a source of mutations that initiate carcinogenesis (i.e. they may be causal factors responsible for the process).

We assessed a relationship between the level of 8-oxodG in leukocyte DNA measured with the high performance liquid chromatography with electrochemical detection (HPLC/EC) technique and urinary excretion of the modified nucleoside/base analysed with a recently developed methodology involving HPLC prepurification followed by gas chromatography with isotope dilution mass spectrometric detection. No correlation was found between these markers of oxidative DNA damage commonly used in epidemiological studies. Several possible explanations of this finding are discussed.

Using high-performance liquid chromatography prepurification/isotope dilution gas chromatography/mass spectrometry technique, we examined whether the amount of 8-hydroxyguanine and 8-hydroxy-2'-deoxyguanosine excreted into urine is higher in cancer patients with advanced-stage disease than in the control group. The control group consisted of 38 healthy subjects, and the patient group comprised 42 cancer patients suffering from metastasis of their primary tumors into the bones. We have found that the amount of the modified base (but not the nucleoside) excreted into urine is about 50% higher in cancer patients than in the control group. Because the presence of the modified base in urine may represent the primary repair product of oxidative DNA damage in vivo, our results suggest an important role of DNA glycosylases (most likely OGG1) in removal of the damage induced as a result of cancer development.

Free radical attack upon DNA generates a multiplicity of DNA damage, including modified bases. Some of these modifications have considerable potential to damage the integrity of the genome. This article reviews recent data that suggest the involvement of oxidative DNA damage in carcinogenesis, atherosclerosis, and acquired immunodeficiency syndrome (AIDS). There is evidence that oxidative DNA damage may play a causative role in atherosclerosis. Oxidative DNA damage may lead to apoptotic cell death of patients infected with human immunodeficiency virus (HIV) and may influence the progression of AIDS. While many details regarding the role of reactive oxygen species-induced DNA damage in the etiology of complex multifactorial diseases like cancer are yet to be discovered, evidence suggests that oxidants act at several stages in the malignant transformation of cells. However, the quantitative relationship between the measured DNA damage and the development of cancer is still lacking.

In the present study we measured the amount of 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA isolated from lymphocytes of arteriosclerotic patients undergoing ozonetherapy. Treatment of the patients with therapeutic concentration of ozone caused a significant increase over the control value in the amount of 8-oxo-dG of DNA isolated from their lymphocytes. However, only three out of six patients examined responded positively to the treatment in terms of the base damage. The increases varied among patients, and were in the range of 100-450%. This interindividual difference may at least be partly explained by recently demonstrated heritable susceptibility to ozone., (Copyright 1999 Elsevier Science B.V.)

Anthracycline derivatives have been widely used in the treatment of several types of human malignancies. Cytotoxicity of these drugs has been attributed to inhibition of topoisomerase II as well as intracellular production of free radicals. In our work we used a gas chromatography/mass spectrometry technique to study free radical-induced DNA base modifications in chromatin isolated from lymphocytes of cancer patients who received chemotherapy with epirubicin (one of anthracycline's antitumor derivatives). The anticancer therapy caused significant increases in the amount of all four DNA base modifications over control levels in the lymphocytes of most of the patients. For the majority of the cases the base products returned to the control value 24 hr after the infusion of the drug, which suggests the removal of these lesions by cellular repair processes. However, some of the modified bases escaped repair. Because part of these modifications may possess premutagenic properties, they may be responsible for secondary cancers induced by chemotherapy.

The authors have studied DNA base damage and activities of antioxidant enzymes in human benign prostatic hyperplasia (BPH) tissues and surrounding disease-free tissues removed from prostate glands of 15 patients. In these tissues, endogenous levels of various typical hydroxyl radical-induced products of DNA bases and activities of catalase and superoxide dismutase were measured. The majority of patients had higher levels of DNA base lesions and lower activities of enzymes in BPH tissues than in normal prostate tissues. When activities of both enzymes were lower in BPH tissues than in normal tissues, the increases in the amounts of DNA base lesions over control levels were most prominent. In the case of similar enzyme activities in both BPH and normal tissues, no changes in levels of DNA base lesions were observed. These results suggest a possible association between antioxidant enzyme activities and levels of DNA base lesions in BPH tissues. Some of the identified DNA lesions are known to be premutagenic and may play a role in carcinogenesis. Although a possible link between BPH and prostate cancer is controversial, BPH patients with both decreased antioxidant enzyme activities and increased levels of DNA lesions may be at risk of developing prostate cancer.