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.

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.

Specific 8-oxo-dGTPase activity of MTH1 (NUDT1) protein as a quantitative marker and prognostic factor in human colorectal cancer.

The MTH1 (NUDT1) gene, because it is frequently upregulated in many types of human cancers, has been considered a general marker of carcinogenesis for over two decades. The MTH1 protein hydrolyzes the oxidized mutagenic DNA precursor, 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP), to the corresponding 5'-monophosphate and inorganic pyrophosphate. This prevents its incorporation into DNA by DNA polymerases and protects cells from the accumulation of 8-oxo-dGTP-induced point mutations. Elevated MTH1 mRNA and protein in many types of human cancer indicate a worse prognosis. However, the enzymatic activity of MTH1 has remained largely uninvestigated in this context. Therefore, we have set out to determine the specific 8-oxo-dGTPase activity of MTH1 in 57 pairs of human colorectal cancers (CRC) and adjacent cancer-free tissues (CFCF). The goal was to ascertain the potential for measuring this enzymatic activity as a way to differentiate cancerous from non-cancerous specimens of the intestine, as well as defining its capabilities as a prognostic value for disease-free survival. We found that 79% of CRC tumors exhibited a higher MTH1 activity than did CFCF, with a significant 1.6-fold increase in overall median value (p < 1E-6). The 8-oxo-dGTPase in both tissues was proportional to the corresponding levels of MTH1 protein, as assayed by Western blotting. Activity higher than the ROC-optimized threshold (AUC = 0.71) indicated cancerous tissue, with a 54% sensitivity and an 83% specificity. Postoperative fate followed for up to 100 months showed that higher 8-oxo-dGTPase, in either the CFCF or the CRC tumor, clearly lowered the probability of a relapse-free survival, although borderline statistical significance (p < 0.05) was crossed only for the CFCF.

In vivo evidence of ascorbate involvement in the generation of epigenetic DNA modifications in leukocytes from patients with colorectal carcinoma, benign adenoma and inflammatory bowel disease.

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.

Characteristic profiles of DNA epigenetic modifications in colon cancer and its predisposing conditions-benign adenomas and inflammatory bowel disease.

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.

Correction: Profiles of a broad spectrum of epigenetic DNA modifications in normal and malignant human cell lines: Proliferation rate is not the major factor responsible for the 5-hydroxymethyl-2'-deoxycytidine level in cultured cancerous cell lines.

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

Oxidation Products of 5-Methylcytosine are Decreased in Senescent Cells and Tissues of Progeroid Mice.

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.

Profiles of a broad spectrum of epigenetic DNA modifications in normal and malignant human cell lines: Proliferation rate is not the major factor responsible for the 5-hydroxymethyl-2'-deoxycytidine level in cultured cancerous cell lines.

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.

Accurate, Direct, and High-Throughput Analyses of a Broad Spectrum of Endogenously Generated DNA Base Modifications with Isotope-Dilution Two-Dimensional Ultraperformance Liquid Chromatography with Tandem Mass Spectrometry: Possible Clinical Implication.

Our hereby presented methodology is suitable for reliable assessment of the most common unavoidable DNA modifications which arise as a product of fundamental metabolic processes. 8-Oxoguanine, one of the oxidatively modified DNA bases, is a typical biomarker of oxidative stress. A noncanonical base, uracil, may be also present in small quantities in DNA. A set of ten-eleven translocation (TET) proteins are involved in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxycytosine. 5-Hydroxymethyluracil may be formed in deamination reaction of 5-hydroxymethylcytosine or can be also generated by TET enzymes. All of the aforementioned modifications seem to play some regulatory roles. We applied isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS) for direct measurement of the 5-methyl-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, 5-carboxy-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxyuridine, 2'-deoxyuridine, and 8-oxo-2'-deoxyguanosine. Analyses of DNA extracted from matched human samples showed that the 5-(hydroxymethyl)-2'-deoxycytidine level was 5-fold lower in colorectal carcinoma tumor in comparison with the normal one from the tumor's margin; also 5-formyl-2'-deoxycytidine and 5-carboxy-2'-deoxycytidine were lower in colorectal carcinoma tissue (ca. 2.5- and 3.5-fold, respectively). No such differences was found for 2'-deoxyuridine and 5-(hydroxymethyl)-2'-deoxyuridine. The presented methodology is suitable for fast, accurate, and complex evaluation of an array of endogenously generated DNA deoxynucleosides modifications. This novel technique could be used for monitoring of cancer and other diseases related to oxidative stress, aberrant metabolism, and environmental exposure. Furthermore, the fully automated two-dimensional separation is extremely useful for analysis of material containing a considerable amount of coeluting interferents with mass-spectrometry-based methods.

Epigenetic modifications and NF-κB pathway activity in Cu,Zn-SOD-deficient mice.

The aim of this study was to examine the possible impact of Cu,Zn-SOD deficiency on the level of epigenetic modifications in different mouse tissues, and the relationship between these modifications and the NF-κB transcription factor activity. Cu,Zn-SOD deficiency did not influence the level of 5mdC or 5hmdC in the analyzed tissues. Statistically significant organ-/tissue-specific differences between the levels of 5mdC and 5hmdC were demonstrated within each genotype. Also correlations between analyzed parameters pointed to wide tissue/genotype variety; we observed a positive correlation between 5mdC and NF-кB proteins, p50 and RelA, in the liver of wild mice, as well as an inverse correlation between 5mdC and p65 in the brain of Cu,Zn-SOD-deficient animals. Moreover, a positive correlation was revealed between 5mdC and 5hmdC in the liver and brain of knockout mice. As the highest levels of both 5mdC and 5hmdC were observed in the brains of analyzed animals regardless of their genotype, and lower, comparable to each other, levels of these modifications were shown in the kidney and liver, active demethylation process seems to be tissue-/organ-specific and does not necessarily rely solely on the redox/oxidation state of cells. According to the most likely scenario, various tissues may differ in terms of their metabolic rates, which has potential influence on cofactors, and consequently on the activity of TET enzymes or activation of TET-independent mechanisms.

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.

Does morphology of carotid plaque depend on patient's oxidative stress?

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.

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.

Up-regulation of 8-oxo-dGTPase activity of MTH1 protein in the brain, testes and kidneys of mice exposed to (137)Cs gamma radiation.

Abstract Mammalian MTH1 protein is an antimutagenic (2'-deoxy)ribonucleoside 5'-triphosphate pyrophosphohydrolase that prevents the incorporation of oxidatively modified nucleotides into nucleic acids. It decomposes most specifically the miscoding products of oxidative damage to purine nucleic acid precursors (e.g. 8-oxo-dGTP, 2-oxo-dATP, 2-oxo-ATP, 8-oxo-GTP) that may cause point mutations or transcription errors when incorporated into DNA and RNA, respectively. The increased expression of MTH1 mRNA and MTH1 protein was previously proposed as a molecular marker of oxidative stress. Therefore, we hypothesized that increased 8-oxo-dGTPase activity of MTH1 protein in mouse organs could serve as a dose-dependent marker of exposure to ionizing radiation, which is known to induce oxidative stress. To test our hypothesis, we measured 8-oxo-dGTPase activity in six organs of male BL6 mice after exposure to 0, 10, 25 and 50 cGy and 1 Gy of (137)Cs gamma radiation given as a single whole-body dose (1 Gy/min). The mice were killed 4, 8 and 24 h after irradiation. A statistically significant induction of 8-oxo-dGTPase was found in brains, testes and kidneys but not in lungs, hearts or livers. Brains, which demonstrated the highest (4.3-fold) increase of 8-oxo-dGTPase activity, were shown to express approximately 50% higher levels of MTH1 protein. However, due to the lack of a simple positive correlation between the dose and the observed 8-oxo-dGTPase activity in brain, testes and kidneys, we conclude that measurements of 8-oxo-dGTPase activity in these organs may serve as a rough indicator rather than a quantifiable marker of radiation-induced oxidative stress.

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.