Agnieszka Siomek

Severe oxidatively damaged DNA after cisplatin treatment of cancer patients.

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

Oxidative DNA damage and antioxidant vitamin level: Comparison among lung cancer patients, healthy smokers and nonsmokers

In the present study, we examined whether the level of 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine (8‐oxodGuo) in leukocyte DNA is higher in lung cancer patients compared to controls. Factors that may influence oxidative stress, such as antioxidant vitamins, were also determined. These parameters were analyzed in 4 groups of subjects: smokers with lung cancer, ex‐smokers with lung cancer, healthy smokers with comparable smoking status and healthy nonsmokers. The 8‐oxodGuo mean level in leukocytes of lung cancer patients reached values of 9.22/106 dGuo molecules (smokers) and 11.16/106 dGuo molecules (ex‐smokers). These values were significantly higher than in DNA of healthy smokers and nonsmokers, where mean levels reached 6.99/106 dGuo molecules and 5.98/106 dGuo molecules, respectively. Mean levels of vitamin C in the plasma of controls and lung cancer patients were 56.17 μM (nonsmokers), 26.34 μM (healthy smokers), 23.83 μM (cancer patients, smokers) and 29.19 μM (cancer patients, ex‐smokers). The difference between nonsmokers and the 3 other groups was statistically significant. Vitamin E level was significantly reduced in the plasma of cancer patients (smokers 19.94 μM, ex‐smokers 19.59 μM) compared to healthy smokers (28.93 μM). No changes in vitamin A concentration were found. Our results suggest that a high level of 8‐oxodGuo in leukocyte DNA and a low concentration of vitamin E in the blood may predict lung cancer risk. However, it is also possible that these phenomena may simply result from disease development.

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/106 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.

Oxidative damage DNA: 8-oxoGua and 8-oxodG as molecular markers of cancer

Summary Background The broad spectrum of oxidative damage 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 oxidative damage DNA in leukocytes, was analyzed in cancer patients and healthy subjects. Material/Methods 222 cancer patients and 134 healthy volunteers were included in the analysis, using methodologies which involve HPLC (high-performance liquid chromatography) prepurification followed by gas chromatography with isotope dilution mass spectrometry detection and HPLC/EC. Results For the whole patient population (n=222) the median values of 8-oxoGua and 8-oxodG in urine samples were 12.44 (interquartile range: 8.14–20.33) [nmol/24 hr] and 6.05 (3.12–15.38) [nmol/24 hr], respectively. The median values of 8-oxoGua and 8-oxodG in urine samples of the control group (n=85) were 7.7 (4.65–10.15) [nmol/24 hr] and 2.2 (1.7–2.8) [nmol/24 hr], respectively. The level of 8-oxodG in DNA isolated from leukocytes of the patient population (n=179) and of the control group (n=134) was 4.93 (3.46–9.27) per 10’6 dG and 4.46 (3.82–5.31) per 10’6 dG, respectively. Conclusions The results suggest that oxidative stress in cancer patients, demonstrated by augmented amounts of these modifications in urine, could be typical not only for affected tissue but also for other tissues and even the whole organism. An assay that enables the determination of levels of basic markers of oxidative stress might be applied in clinical practice as an additional, helpful marker to diagnose cancer.

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. (Cancer Epidemiol Biomarkers Prev 2009;18(11):2923–8)

Diet is Not Responsible for the Presence of Several Oxidatively Damaged DNA Lesions in Mouse Urine

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 [Formula: See Text], [Formula: See Text] and [Formula: See Text] After feeding the mice for 12 days with nucleic acid free diet the respective values were [Formula: See Text], [Formula: See Text] and [Formula: See Text] 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.

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.

Interlaboratory comparison of methodologies for the measurement of urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine

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

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.

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 α-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. Cancer Epidemiol Biomarkers Prev; 19(8); 1960–5. ©2010 AACR.