The level of active DNA demethylation compounds in leukocytes and urine samples as potential epigenetic biomarkers in breast cancer patients.

The active DNA demethylation process, which involves TET proteins, can affect DNA methylation pattern. TET dependent demethylation results in DNA hypomethylation by oxidation 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) and its derivatives. Moreover, TETs' activity may be upregulated by ascorbate. Given that aberrant DNA methylation of genes implicated in breast carcinogenesis may be involved in tumor progression, we wanted to determine whether breast cancer patients exert changes in the active DNA demethylation process. The study included blood samples from breast cancer patients (n = 74) and healthy subjects (n = 71). We analyzed the expression of genes involved in the active demethylation process (qRT-PCR), and 5-mC and its derivatives level (2D-UPLC MS/MS). The ascorbate level was determined using UPLC-MS. Breast cancer patients had significantly higher TET3 expression level, lower 5-mC and 5-hmC DNA levels. TET3 was significantly increased in luminal B breast cancer patients with expression of hormone receptors. Moreover, the ascorbate level in the plasma of breast cancer patients was decreased with the accompanying increase of sodium-dependent vitamin C transporters (SLC23A1 and SLC23A2). The presented study indicates the role of TET3 in DNA demethylation in breast carcinogenesis.

Urinary Measurement of Epigenetic DNA Modifications and 8-oxodG as Possible Noninvasive Markers of Colon Cancer Evolution.

The active DNA demethylation mechanism involves 5-methylcytosine (5-mCyt) enzymatic oxidation with the subsequent formation of 5-hydroxymethylcytosine (5-hmCyt), which can be further oxidized to 5-formylcytosine (5-fCyt) and 5-carboxylcytosine (5-caCyt). The products of active DNA demethylation are released into the bloodstream and eventually also appear in urine. We used online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS) to compare DNA methylation marks and 8-oxo-2'-deoxyguanosine (8-oxodG) in colorectal cancer and pre-cancerous condition in urine. The study included four groups of subjects: healthy controls, patients with inflammatory bowel disease (IBD), persons with adenomatous polyps (AD), and individuals with colorectal cancer (CRC). We have found that the level of 5-fCyt in urine was significantly lower for CRC and polyp groups than in the control group. The level of 5-hmCyt was significantly higher only in the CRC group compared to the control (2.3 vs. 2.1 nmol/mmol creatinine). Interestingly, we have found highly statistically significant correlation of 5-hydroxymethyluracil with 5-hydroxymethylcytosine, 5-(hydroxymethyl)-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxyuridine, and 5-methyl-2'-deoxycytidine in the CRC patients' group.

Diagnostic and Prognostic Power of Active DNA Demethylation Pathway Intermediates in Acute Myelogenous Leukemia and Myelodysplastic Syndromes.

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are characterized by genomic instability, which may arise from the global hypomethylation of the DNA. The active DNA demethylation process may be linked with aberrant methylation and can be involved in leukemogenesis. The levels of 5-methylcytosine oxidation products were analyzed in minimally invasive material: the cellular DNA from peripheral blood cells and urine of patients with AML and MDS along with the control group, using isotope-dilution two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry. The receiver operating characteristic curve analysis was used for the assessment of the ability to discriminate patients' groups from the control group, and AML from MDS. The most diagnostically useful for discriminating AML patients from the control group was the urinary excretion of 5-hydroxymethylcytosine (AUC = 0.918, sensitivity: 85%, and specificity: 97%), and 5-(hydroxymethyl)-2'-deoxyuridine (0.873, 74%, and 92%), while for MDS patients 5-(hydroxymethyl)-2'-deoxycytidine in DNA (0.905, 82%, and 98%) and urinary 5-hydroxymethylcytosine (0.746, 66%, and 92%). Multi-factor models of classification trees allowed the correct classification of patients with AML and MDS in 95.7% and 94.7% of cases. The highest prognostic value of the analyzed parameters in predicting the transformation of MDS into AML was observed for 5-carboxy-2'-deoxycytidine (0.823, 80%, and 97%) and 5-(hydroxymethyl)-2'-deoxyuridine (0.872, 100%, and 75%) in DNA. The presented research proves that the intermediates of the active DNA demethylation pathway determined in the completely non-invasive (urine) or minimally invasive (blood) material can be useful in supporting the diagnostic process of patients with MDS and AML. The possibility of an early identification of a group of MDS patients with an increased risk of transformation into AML is of particular importance.

The urinary excretion of epigenetically modified DNA as a marker of pediatric ALL status and chemotherapy response.

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.

Systemic oxidoreductive balance and vascular function in individuals without clinical manifestation of atherosclerosis.

INTRODUCTION: Endothelial dysfunction is recognized as the earliest disorder in the development of atherosclerosis, in the pathogenesis of which oxidative stress plays a crucial role. The aim of this study was to determine the relationships between non-invasive parameters of vascular dysfunction and oxidative stress. MATERIAL AND METHODS: Forty-eight individuals without clinical manifestation of atherosclerosis were studied. The plasma concentrations of the following were determined in all 48 subjects: retinol, ascorbic acid, α-tocopherol and uric acid, as well as the products of oxidative DNA damage repair: 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in blood leukocytes and urine, and 8-oxo-7,8-dihydroguanine (8-oxoGua) in urine. The following parameters of vascular dysfunction were also examined: flow- (FMD) and nitroglycerin- (NMD) mediated dilatation of the brachial artery, pulse pressure (PP), distensibility coefficient (DC), pulsation (PI) and resistance (RI) index, carotid intima-media thickness (cIMT), and ankle-brachial index (ABI). RESULTS: Individuals with an FMD value of ≥ 8.8% had significantly higher blood concentrations of antioxidative vitamins and lower concentrations of 8-oxodG in their urine and blood leukocytes than their counterparts. Blood concentration of alpha-tocopherol or ascorbic acid positively correlated with FMD, PI, RI, DC and ABI and negatively with PP and cIMT. The reverse was the case for 8-oxodG in urine and leukocytes. In multiple regression analysis, markers of oxidative DNA damage positively determined the variance in PP and ABI. CONCLUSIONS: In persons without clinical manifestation of atherosclerosis, oxidative stress was an independent factor associated with vascular wall dysfunction, and a better predictor than smoking and blood concentrations of glucose, lipids and creatinine.

Urinary Measurement of Epigenetic DNA Modifications: A Non-Invasive Assessment of the Whole-Body Epigenetic Status in Healthy Subjects and Colorectal Cancer Patients.

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.

Nucleotide excision repair of oxidised genomic DNA is not a source of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine.

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is a widely measured biomarker of oxidative stress. It has been commonly assumed to be a product of DNA repair, and therefore reflective of DNA oxidation. However, the source of urinary 8-oxodGuo is not understood, although potential confounding contributions from cell turnover and diet have been ruled out. Clearly it is critical to understand the precise biological origins of this important biomarker, so that the target molecule that is oxidised can be identified, and the significance of its excretion can be interpreted fully. In the present study we aimed to assess the contributions of nucleotide excision repair (NER), by both the global genome NER (GG-NER) and transcription-coupled NER (TC-NER) pathways, and sanitisation of the dGTP pool (e.g. via the activity of the MTH1 protein), on the production of 8-oxodGuo, using selected genetically-modified mice. In xeroderma pigmentosum A (XPA) mice, in which GG-NER and TC-NER are both defective, the urinary 8-oxodGuo data were unequivocal in ruling out a contribution from NER. In line with the XPA data, the production of urinary 8-oxodGuo was not affected in the xeroderma pigmentosum C mice, specifically excluding a role of the GG-NER pathway. The bulk of the literature supports the mechanism that the NER proteins are responsible for removing damage to the transcribed strand of DNA via TC-NER, and on this basis we also examined Cockayne Syndrome mice, which have a functional loss of TC-NER. These mice showed no difference in urinary 8-oxodGuo excretion, compared to wild type, demonstrating that TC-NER does not contribute to urinary 8-oxodGuo levels. These findings call into question whether genomic DNA is the primary source of urinary 8-oxodGuo, which would largely exclude it as a biomarker of DNA oxidation. The urinary 8-oxodGuo levels from the MTH1 mice (both knock-out and hMTH1-Tg) were not significantly different to the wild-type mice. We suggest that these findings are due to redundancy in the process, and that other enzymes substitute for the lack of MTH1, however the present study cannot determine whether or not the 2'-deoxyribonucleotide pool is the source of urinary 8-oxodGuo. On the basis of the above, urinary 8-oxodGuo is most accurately defined as a non-invasive biomarker of oxidative stress, derived from oxidatively generated damage to 2'-deoxyguanosine.

Urinary 5-hydroxymethyluracil and 8-oxo-7,8-dihydroguanine as potential biomarkers in patients with colorectal cancer.

CONTEXT: Oxidative stress linked with chronic inflammation is associated with etiology of the colorectal cancer. OBJECTIVES: To assess the diagnostic utility of urinary excretion of oxidatively modified DNA bases/nucleoside: 8-oxo-7,8-dihydroguanine (8-oxoGua), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 5-hydroxymethyluracil (5-hmUra). MATERIALS AND METHODS: Seventy-two healthy controls, 15 patients with adenomas and 56 colorectal cancer patients were recruited. RESULTS: The receiver operating characteristic (ROC) curve analysis showed that the area under the curve (AUC) for all markers tested separately was <0.7. The combination of these modifications showed better diagnostic power (AUC = 0.778 for 8-oxoGua + 8-oxodG)/5hmUra ratio). CONCLUSION: Urinary DNA modifications may reflect the oxidative stress/chronic inflammation in colorectal cancer but diagnostic performance for early-detection is moderate.

8-Oxo-7,8-dihydroguanine and uric acid as efficient predictors of survival in colon cancer patients.

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.

Comparison of oxidative stress/DNA damage in semen and blood of fertile and infertile men.

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.

Human and methodological sources of variability in the measurement of urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine.

AIMS: Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is a widely used biomarker of oxidative stress. However, variability between chromatographic and ELISA methods hampers interpretation of data, and this variability may increase should urine composition differ between individuals, leading to assay interference. Furthermore, optimal urine sampling conditions are not well defined. We performed inter-laboratory comparisons of 8-oxodG measurement between mass spectrometric-, electrochemical- and ELISA-based methods, using common within-technique calibrants to analyze 8-oxodG-spiked phosphate-buffered saline and urine samples. We also investigated human subject- and sample collection-related variables, as potential sources of variability. RESULTS: Chromatographic assays showed high agreement across urines from different subjects, whereas ELISAs showed far more inter-laboratory variation and generally overestimated levels, compared to the chromatographic assays. Excretion rates in timed 'spot' samples showed strong correlations with 24 h excretion (the 'gold' standard) of urinary 8-oxodG (rp 0.67-0.90), although the associations were weaker for 8-oxodG adjusted for creatinine or specific gravity (SG). The within-individual excretion of 8-oxodG varied only moderately between days (CV 17% for 24 h excretion and 20% for first void, creatinine-corrected samples). INNOVATION: This is the first comprehensive study of both human and methodological factors influencing 8-oxodG measurement, providing key information for future studies with this important biomarker. CONCLUSION: ELISA variability is greater than chromatographic assay variability, and cannot determine absolute levels of 8-oxodG. Use of standardized calibrants greatly improves intra-technique agreement and, for the chromatographic assays, importantly allows integration of results for pooled analyses. If 24 h samples are not feasible, creatinine- or SG-adjusted first morning samples are recommended.

Cu,Zn-superoxide dismutase deficiency in mice leads to organ-specific increase in oxidatively damaged DNA and NF-κB1 protein activity.

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.

Oxidatively damaged DNA/oxidative stress in children with celiac disease.

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.

Oxidative stress and 8-oxoguanine repair are enhanced in colon adenoma and carcinoma patients.

Oxidative stress is involved in the pathogenesis of colon cancer. We wanted to elucidate at which stage of the disease this phenomenon occurs. In the examined groups of patients with colorectal cancer (CRC, n = 89), benign adenoma (AD, n = 77) and healthy volunteers (controls, n = 99), we measured: vitamins A, C and E in blood plasma, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanine (8-oxoGua) in leukocytes and urine, leukocyte 8-oxoGua excision activity, mRNA levels of APE1, OGG1, 8-oxo-7,8-dihydrodeoxyguanosine 5'-triphosphate pyrophosphohydrolase (MTH1) and OGG1 polymorphism. The vitamin levels decreased gradually in AD and CRC patients. 8-OxodG increased in leukocytes and urine of CRC and AD patients. 8-OxoGua was higher only in the urine of CRC patients. 8-OxoGua excision was higher in CRC patients than in controls, in spite of higher frequency of the OGG1 Cys326Cys genotype, encoding a glycosylase with decreased activity. mRNA levels of OGG1 and APE1 increased in CRC and AD patients, which could explain increased 8-oxoGua excision rate in CRC patients. MTH1 mRNA was also higher in CRC patients. The results suggest that oxidative stress occurs in CRC and AD individuals. This is accompanied by increased transcription of DNA repair genes, and increased 8-oxoGua excision rate in CRC patients, which is, however, insufficient to counteract the increased DNA damage.

Selenium supplementation reduced oxidative DNA damage in adnexectomized BRCA1 mutations carriers.

Some experimental evidence suggests that BRCA1 plays a role in repair of oxidative DNA damage. Selenium has anticancer properties that are linked with protection against oxidative stress. To assess whether supplementation of BRCA1 mutation carriers with selenium have a beneficial effect concerning oxidative stress/DNA damage in the present double-blinded placebo control study, we determined 8-oxodG level in cellular DNA and urinary excretion of 8-oxodG and 8-oxoGua in the mutation carriers. We found that 8-oxodG level in leukocytes DNA is significantly higher in BRCA1 mutation carriers. In the distinct subpopulation of BRCA1 mutation carriers without symptoms of cancer who underwent adnexectomy and were supplemented with selenium, the level of 8-oxodG in DNA decreased significantly in comparison with the subgroup without supplementation. Simultaneously in the same group, an increase of urinary 8-oxoGua, the product of base excision repair (hOGG1 glycosylase), was observed. Therefore, it is likely that the selenium supplementation of the patients is responsible for the increase of BER enzymes activities, which in turn may result in reduction of oxidative DNA damage. Importantly, in a double-blinded placebo control prospective study, it was shown that in the same patient groups, reduction in cancer incidents was observed. Altogether, these results suggest that BRCA1 deficiency contributes to 8-oxodG accumulation in cellular DNA, which in turn may be a factor responsible for cancer development in women with mutations, and that the risk to developed breast cancer in BRCA1 mutation carriers may be reduced in selenium-supplemented patients who underwent adnexectomy.

Elevated level of 8-oxo-7,8-dihydro-2'-deoxyguanosine in leukocytes of BRCA1 mutation carriers compared to healthy controls.

Carriers of BRCA1 mutation face highly increased risk of breast and ovarian cancer and some studies with cell culture suggest that the encoded protein may be involved in oxidatively damaged DNA repair. However, no studies concerning a possible link between oxidatively damaged DNA and BRCA1 deficiency have been conducted with the mutations carriers. Therefore, to assess an involvement of BRCA in oxidative damage to DNA in the present study a broad spectrum of parameters reflecting oxidative stress/DNA damage were analyzed in 3 subject groups; (i) carriers of BRCA1 mutations without symptoms of the disease; (ii) patients with breast or ovarian cancer with the mutations and (iii) the group of healthy subjects recruited from among close relatives of the group of carriers without symptoms of the disease. We found that the endogenous levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in leukocytes DNA and excretion rates of urinary 8-oxodG were significantly higher in the cancer patients than in the healthy carriers. Similarly, to the cancer patient group, 8-oxodG level in leukocytes DNA is significantly higher in the carriers group in comparison with control group. That the control group comprised close relatives of the carriers gives further credit to our finding. Since we did not observe substantial differences in the analyzed markers of oxidative stress between the controls and the carriers, the observed increase in the level may be a result of a deficiency in the repair of 8-oxodG.

Small field radiotherapy of head and neck cancer patients is responsible for oxidatively damaged DNA/oxidative stress on the level of a whole organism.

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.

Antioxidant vitamins and cancer risk: is oxidative damage to DNA a relevant biomarker?

Oxidative damage to DNA is regarded as an important step in carcinogenesis. These lesions may arise as a consequence of exposure to xenobiotics, but are also generated as a consequence of endogenous generation of oxidizing compounds. Measurements of oxidative damage to guanines, such as 8-oxo-7, 8-dihydroguanine (8-oxodG) are increasingly being regarded as reliable biomarkers of oxidative stress and they may have a predictive value of cancer risk, although this needs to be established independently in several cohort studies. A survey of intervention studies of the ingestion of antioxidant-containing foods or tablets of antioxidants indicate that about one-third of the studies reported a protective effect in terms of lower levels of oxidative damage to DNA in white blood cells or decreased urinary excretion of 8-oxodG. Although firm conclusions cannot be reached, there appears to be links between ingestion of antioxidants, oxidative damage to DNA, and risk of cancer.

The relationship between 8-oxo-7,8-dihydro-2'-deoxyguanosine level and extent of cytosine methylation in leukocytes DNA of healthy subjects and in patients with colon adenomas and carcinomas.

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.

Effects of basal level of antioxidants on oxidative DNA damage in humans.

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.