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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).)

Reliable quantitative analysis of DNA modification using liquid chromatography coupled with tandem mass spectrometry requires stable isotope-labeled internal standards. Only some of them are commercially available. Here we present a method allowing for the synthesis of [ 13 C 10 , 15 N 2 ]-5-methyl-2'-deoxycytidine from [ 13 C 10 , 15 N 2 ]-2'-deoxythymidine. We also describe an approach for the oxidation of [ 13 C 10 , 15 N 2 ]-5-methyl-2'-deoxycytidine and [ 13 C 10 , 15 N 2 ]-2'-deoxythymidine with Na 2 S 2 O 8 , leading to the generation of [ 13 C 10 , 15 N 2 ]-5-formyl-2'-deoxycytidine, [ 13 C 10 , 15 N 2 ]-5-carboxy-2'-deoxycytidine or [ 13 C 10 , 15 N 2 ]-5-(hydroxymethyl)-2'-deoxyuridine, correspondingly. Moreover, we provide optimized protocols for the oxidation of [ 13 C 5 , 15 N 2 ]-thymine to [ 13 C 10 , 15 N 2 ]-5-hydroxymethyluracil, [ 13 C 10 , 15 N 2 ]-5-formyluracil, and [ 13 C 10 , 15 N 2 ]-5-carboxyuracil using Na 2 S 2 O 8 .

Stable-isotope-dilution tandem mass spectrometry is the most advanced technique used for quantitative determination of a wide spectrum of endogenously generated DNA nucleobase modifications. It is regarded as a gold standard for such analyses. Here, we consider the requirements for reliable identification and quantification of DNA adducts/modifications, whether endogenously derived or not, and discuss how their quantification can provide information on the mechanism of action and the biological relevance of individual nucleobase modifications. A clinical application of such measurements will only be possible after a full validation of the assay and once we have gained a better understanding of the exact role that these DNA modifications play in disease pathogenesis. Once these prerequisites are satisfied, DNA modification measurements may be helpful as clinical parameters for treatment monitoring, for risk group identification and for the development of prevention strategies.

The epithelial-to-mesenchymal transition (EMT) is a complex transcriptional program induced by transforming growth factor β1 (TGF-β1). Histone lysine-specific demethylase 1 (LSD1) has been recognized as a key mediator of EMT in cancer cells, but the precise mechanism that underlies the activation and repression of EMT genes still remains elusive. Here, we characterized the early events induced by TGF-β1 during EMT initiation and establishment. TGF-β1 triggered, 30-90 min post-treatment, a nuclear oxidative wave throughout the genome, documented by confocal microscopy and mass spectrometry, mediated by LSD1. LSD1 was recruited with phosphorylated SMAD2/3 to the promoters of prototypic genes activated and repressed by TGF-β1. After 90 min, phospho-SMAD2/3 downregulation reduced the complex and LSD1 was then recruited with the newly synthesized SNAI1 and repressors, NCoR1 and HDAC3, to the promoters of TGF-β1-repressed genes such as the Wnt soluble inhibitor factor 1 gene (WIF1), a change that induced a late oxidative burst. However, TGF-β1 early (90 min) repression of transcription also required synchronous signaling by reactive oxygen species and the stress-activated kinase c-Jun N-terminal kinase. These data elucidate the early events elicited by TGF-β1 and the priming role of DNA oxidation that marks TGF-β1-induced and -repressed genes involved in the EMT., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

R-loops are nucleic acid structures formed by an RNA:DNA hybrid and unpaired single-stranded DNA that represent a source of genomic instability in mammalian cells 1-4 . Here we show that N 6 -methyladenosine (m 6 A) modification, contributing to different aspects of messenger RNA metabolism 5,6 , is detectable on the majority of RNA:DNA hybrids in human pluripotent stem cells. We demonstrate that m 6 A-containing R-loops accumulate during G 2 /M and are depleted at G 0 /G 1 phases of the cell cycle, and that the m 6 A reader promoting mRNA degradation, YTHDF2 (ref. 7 ), interacts with R-loop-enriched loci in dividing cells. Consequently, YTHDF2 knockout leads to increased R-loop levels, cell growth retardation and accumulation of γH2AX, a marker for DNA double-strand breaks, in mammalian cells. Our results suggest that m 6 A regulates accumulation of R-loops, implying a role for this modification in safeguarding genomic stability.

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.

The DNA of all living cells undergoes continuous structural and chemical alteration, which may be derived from exogenous sources, or endogenous, metabolic pathways, such as cellular respiration, replication and DNA demethylation. It has been estimated that approximately 70,000 DNA lesions may be generated per day in a single cell, and this has been linked to a wide variety of diseases, including cancer. However, it is puzzling why potentially mutagenic DNA modifications, occurring at a similar level in different organs/tissue, may lead to organ/tissue specific cancers, or indeed non-malignant disease - what is the basis for this differential response? We suggest that it is perhaps the precise location of damage, within the genome, that is a key factor. Finally, we draw attention to the requirement for reliable methods for identification and quantification of DNA adducts/modifications, and stress the need for these assays to be fully validated. Once these prerequisites are satisfied, measurement of DNA modifications may be helpful as a clinical parameter for treatment monitoring, risk group identification and development of prevention strategies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.

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.)

The aim of this review is to describe the reactions which lead to generation of 5-hydroxymethyluracil, as well as the repair processes involved in its removal from DNA, and its level in various cells and urine. 5-hydroxymethyluracil may be formed during the course of the two processes: oxidation/hydroxylation of thymine with resultant formation of 5-hydroxymethyluracil paired with adenine (produced by reactive oxygen species), and reacting of reactive oxygen species with 5-methylcytosine forming 5-hydroxymethylcytosine, followed by its deamination to 5-hydroxymethyluracil mispaired with guanine. However, other, perhaps enzymatic, mechanism(s) may be involved in formation of 5-hydroxymethyluracil mispaired with guanine. Indeed, this mispair may be also formed as a result of deamination of 5-hydroxymethylcytosine, recently described "sixth" DNA base. It was demonstrated that 5-hydroxymethyluracil paired with adenine can be also generated by TET enzymes from thymine during mouse embryonic cell differentiation. Therefore, it is possible that 5-hydroxymethyluracil is epigenetic mark. The level of 5-hydroxymethyluracil in various somatic tissues is relatively stable and resembles that observed in lymphocytes, about 0.5/10(6) dN in human colon, colorectal cancer as well as various rat and porcine tissues. Experimental evidence suggests that SMUG1 and TDG are main enzymes involved in removal of 5-hydroxymethyluracil from DNA. 5-hydroxymethyluracil, in form of 5-hydroxymethyluridine, was also detected in rRNA, and together with SMUG1 may play a role in rRNA quality control. To summarize, 5-hydroxymethyluracil is with no doubt a product of both enzymatic and reactive oxygen species-induced reaction. This modification may probably serve as an epigenetic mark, providing additional layer of information encoded within the genome. However, the pool of 5-hydroxymethyluracil generated as a result of oxidative stress is also likely to disturb physiological epigenetic processes, and as such may be defined as a lesion. Altogether this suggests that 5-hydroxymethyluracil may be either a regulatory or erroneous compound., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Replication-independent active/enzymatic demethylation may be an important process in the functioning of somatic cells. The most plausible mechanisms of active 5-methylcytosine demethylation, leading to activation of previously silenced genes, involve ten-eleven translocation (TET) proteins that participate in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxylcytosine. Recently, 5-hydroxymethylcytosine was demonstrated to be a relatively stable modification, and the previously observed substantial differences in the level of this modification in various murine tissues were shown to depend mostly on cell proliferation rate. Some experimental evidence supports the hypothesis that 5-hydroxymethyluracil may be also generated by TET enzymes and has epigenetic functions., Results: Using an isotope-dilution automated online two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry, we have analyzed, for the first time, all the products of active DNA demethylation pathway: 5-methyl-2'-deoxycytidine, 5-hydroxymethyl-2'-deoxycytidine, 5-formyl-2'-deoxycytidine and 5-carboxyl-2'-deoxycytidine, as well as 5-hydroxymethyl-2'-deoxyuridine, in DNA isolated from various rat and porcine tissues. A strong significant inverse linear correlation was found between the proliferation rate of cells and the global level of 5-hydroxymethyl-2'-deoxycytidine in both porcine (R2 = 0.88) and rat tissues (R2 = 0.83); no such relationship was observed for 5-formyl-2'-deoxycytidine and 5-carboxyl-2'-deoxycytidine. Moreover, a substrate-product correlation was demonstrated for the two consecutive steps of iterative oxidation pathway: between 5-hydroxymethyl-2'-deoxycytidine and its product 5-formyl-2'-deoxycytidine, as well as between 5-formyl-2'-deoxycytidine and 5-carboxyl-2'-deoxycytidine (R2 = 0.60 and R2 = 0.71, respectively)., Conclusions: Good correlations within the substrate-product sets of iterative oxidation pathway may suggest that a part of 5-formyl-2'-deoxycytidine and/or 5-carboxyl-2'-deoxycytidine can be directly linked to a small portion of 5-hydroxymethyl-2'-deoxycytidine which defines the active demethylation process.

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.)

Background: Because patients with celiac disease face increased risk of cancer and there is considerable circumstantial evidence that oxidatively damaged DNA may be used as a marker predictive of cancer development, we decided, for the first time, to characterize oxidative stress/oxidative DNA damage in celiac disease patients., Methods: Two kinds of oxidatively damaged DNA biomarkers, namely, urinary excretion of 8-oxodG and 8-oxoGua, and the level of oxidatively damaged DNA in the leukocytes, as well as the level of antioxidant vitamins were analyzed using high-performance liquid chromatography (HPLC) and HPLC/gas chromatography with isotope dilution mass detection methods. These parameters were determined in three groups: (a) children with untreated celiac disease, (b) patients with celiac disease on a strict gluten-free diet, and (c) healthy children., Results: The mean level of 8-oxodG in DNA isolated from the leukocytes and in the urine samples of the two groups of celiacs was significantly higher than in controls, irrespective of diet. There was no statistically significant difference in these parameters between treated and untreated celiacs. The mean plasma retinol and alpha-tocopherol concentration in the samples of untreated celiacs was significantly lower than in treated celiacs., Conclusion: Our results suggest that although diet can be partially responsible for oxidative stress/oxidatively damaged DNA in celiac patients, there is a factor independent of diet., Impact: It is possible that celiac disease patients may be helped by dietary supplementation rich in vitamin A (and E) to minimize the risk of cancer development., ((c)2010 AACR.)

The repair of oxidatively damaged DNA is integral to the maintenance of genomic stability, and hence prevention of a wide variety of pathological conditions, such as aging, cancer and cardiovascular disease. The ability to non-invasively assess DNA repair may provide information regarding repair pathways, variability in repair capacity, and susceptibility to disease. The development of assays to measure urinary DNA lesions offered this potential, although it rapidly became clear that possible contribution from diet and cell turnover may influence urinary lesion levels. Whilst early studies attempted to address these issues, up until now, much of the data appears conflicting. However, recent work from our laboratories, in which human volunteers were fed highly oxidatively modified 15N-labelled DNA demonstrates that diet does not appear to contribute to urinary levels of 8-hydroxyguanine and 7,8-dihydro-8-oxo-2'-deoxyguanosine. Furthermore, we propose that a number of literature reports form an argument against a contribution from cell death. Indeed we, and others, have presented evidence, which strongly suggests the involvement of cell death to be minimal. Taken together, these data would appear to rule out various confounding factors, leaving DNA repair pathways as the principal source of urinary purine, if not DNA, lesions enabling such measurements to be used as indicators of repair.

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.

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.

There is evidence suggesting that patients infected with human immunodeficiency virus (HIV) are under chronic oxidative stress. In the present study, the level of oxidatively modified bases in lymphocyte DNA and some other parameters of oxidative stress were measured in HIV-infected patients (n = 30), as well as in control groups (10 healthy volunteers and 15 HIV-seronegative injected drug users). Additional experiments were conducted using lymphocyte DNA samples from asymptomatic seropositive, HIV-infected patients who were supplemented with antioxidant vitamins A, C, and E or received placebo. Significant increases in the amount of the modified DNA bases were observed in HIV-infected patients when compared with the control group. The concentration of thiobarbituric acid reactive substances (TBARS) was higher and activities of antioxidant enzymes (superoxide dismutase and catalase) were lower in the group of HIV-infected patients in comparison to the control group. Vitamin supplementation resulted in the significant decrease in the levels of all modified DNA bases when compared to the patients who received placebo. The reduction of TBARS and the restoration of the activity of the enzymes were also observed. Our data suggest that people infected with HIV can benefit from treatment with antioxidant vitamins.

Hypermethylation of tumour suppressors and other aberrations of DNA methylation in tumours play a significant role in cancer progression. DNA methylation can be affected by various environmental conditions, including hypoxia. The response to hypoxia is mainly achieved through activation of the transcriptional program associated with HIF1A transcription factor. Inactivation of Von Hippel-Lindau Tumour Suppressor gene (VHL) by genetic or epigenetic events, which also induces aberrant activation of HIF1A, is the most common driver event for renal cancer. With whole-genome bisulphite sequencing and LC-MS, we demonstrated that VHL inactivation induced global genome hypermethylation in human kidney cancer cells under normoxic conditions. This effect was reverted by exogenous expression of wild-type VHL. We showed that global genome hypermethylation in VHL mutants can be explained by transcriptional changes in MDH and L2HGDH genes that cause the accumulation of 2-hydroxyglutarate – a metabolite that inhibits DNA demethylation by TET enzymes. Unlike the known cases of DNA hypermethylation in cancer, 2-hydroxyglutarate was accumulated in the cells with the wild-type isocitrate dehydrogenases. [ABSTRACT FROM AUTHOR]

Abstract: Active mechanism of DNA demethylation can be responsible for the activation of previously silenced genes. Products of 5‐methylcytosine oxidation are released into the bloodstream and eventually excreted with urine. Therefore, whole‐body epigenetic status can be assessed non‐invasively on the basis of the urinary excretion of a broad spectrum of epigenetic modifications: 5‐hydroxymethylcytosine (5‐hmCyt), 5‐formylcytosine (5‐fCyt), 5‐carboxycytosine (5‐caCyt), and 5‐hydroxymethyluracil (5‐hmUra). We have developed a specific and sensitive, isotope‐dilution, automated, online, two‐dimensional ultra‐performance liquid chromatography system with tandem mass spectrometry (2D UPLC–MS/MS) to measure 5‐hmCyt, 5‐fCyt, 5‐caCyt, and their deoxynucleosides in the same urine sample. Human urine contains all of the modifications except from 5‐formyl‐2′‐deoxycytidine (5‐fdC) and 5‐carboxy‐2′‐deoxycytidine (5‐cadC). A highly significant difference in the urinary excretion of 5‐(hydroxymethyl)‐2’‐deoxycytidine (5‐hmdC) was found between healthy subjects and colorectal cancer patients (3.5 vs. 7.8 nmol mmol−1 creatinine, respectively), as well as strong correlations between the majority of analyzed compounds. [ABSTRACT FROM AUTHOR]

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. 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). 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. 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 α-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. 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. [ABSTRACT FROM AUTHOR]

Measurement of the products of oxidatively damaged DNA in urine is a frequently used means by which oxidative stress may be assessed non-invasively. We believe that urinary DNA lesions, in addition to being biomarkers of oxidative stress, can potentially provide more specific information, for example, a reflection of repair activity. We used high-performance liquid chromatography prepurification, with gas chromatography-mass spectrometry (LC-GC-MS) and ELISA to the analysis of a number of oxidative [e.g., 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydro-guanine, 5-(hydroxymethyl)uracil], non-oxidative (cyclobutane thymine dimers) and oligomeric DNA products in urine. We analysed spot urine samples from 20 healthy subjects, and 20 age- and sex-matched cancer patients. Mononuclear cell DNA 8-oxodG levels were assessed by LC-EC. The data support our proposal that urinary DNA lesion products are predominantly derived from DNA repair. Furthermore, analysis of DNA and urinary 8-oxodG in cancer patients and controls suggested reduced repair activity towards this lesion marker in these patients. [ABSTRACT FROM AUTHOR]

Abstract: 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 &y& Elsevier]

Abstract: 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 106 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. [Copyright &y& Elsevier]