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Alkylation represents a main form of DNA damage. The N2 position of guanine is frequently alkylated in DNA. The SOS-induced polymerases have been shown to be capable of bypassing various DNA damage products in Escherichia coli. Herein, we explored the influences of four N2-alkyl-dG lesions (alkyl = ethyl, n-butyl, isobutyl, or sec-butyl) on DNA replication in AB1157 E. coli cells and the corresponding strains with polymerases (Pol) II, IV, and V being individually or simultaneously knocked out. We found that N2-Et-dG is slightly less blocking to DNA replication than the N2-Bu-dG lesions, which display very similar replication bypass efficiencies. Additionally, Pol II and, to a lesser degree, Pol IV and Pol V are required for the efficient bypass of the N2-alkyl-dG adducts, and none of these lesions was mutagenic. Together, our results support that the efficient replication across small N2-alkyl-dG DNA adducts in E. coli depends mainly on Pol II.

The anti-leukemia agent forodesine causes cytotoxic overload of intracellular deoxyguanosine triphosphate (dGTP) but is efficacious only in a subset of patients. We report that SAMHD1, a phosphohydrolase degrading deoxyribonucleoside triphosphate (dNTP), protects cells against the effects of dNTP imbalances. SAMHD1-deficient cells induce intrinsic apoptosis upon provision of deoxyribonucleosides, particularly deoxyguanosine (dG). Moreover, dG and forodesine act synergistically to kill cells lacking SAMHD1. Using mass cytometry, we find that these compounds kill SAMHD1-deficient malignant cells in patients with chronic lymphocytic leukemia (CLL). Normal cells and CLL cells from patients without SAMHD1 mutation are unaffected. We therefore propose to use forodesine as a precision medicine for leukemia, stratifying patients by SAMHD1 genotype or expression.

The anti-leukemia agent forodesine causes cytotoxic overload of intracellular deoxyguanosine triphosphate (dGTP) but is efficacious only in a subset of patients. We report that SAMHD1, a phosphohydrolase degrading deoxyribonucleoside triphosphate (dNTP), protects cells against the effects of dNTP imbalances. SAMHD1-deficient cells induce intrinsic apoptosis upon provision of deoxyribonucleosides, particularly deoxyguanosine (dG). Moreover, dG and forodesine act synergistically to kill cells lacking SAMHD1. Using mass cytometry, we find that these compounds kill SAMHD1-deficient malignant cells in patients with chronic lymphocytic leukemia (CLL). Normal cells and CLL cells from patients without SAMHD1 mutation are unaffected. We therefore propose to use forodesine as a precision medicine for leukemia, stratifying patients by SAMHD1 genotype or expression.

The anti-leukemia agent forodesine causes cytotoxic overload of intracellular deoxyguanosine triphosphate (dGTP) but is efficacious only in a subset of patients. We report that SAMHD1, a phosphohydrolase degrading deoxyribonucleoside triphosphate (dNTP), protects cells against the effects of dNTP imbalances. SAMHD1-deficient cells induce intrinsic apoptosis upon provision of deoxyribonucleosides, particularly deoxyguanosine (dG). Moreover, dG and forodesine act synergistically to kill cells lacking SAMHD1. Using mass cytometry, we find that these compounds kill SAMHD1-deficient malignant cells in patients with chronic lymphocytic leukemia (CLL). Normal cells and CLL cells from patients without SAMHD1 mutation are unaffected. We therefore propose to use forodesine as a precision medicine for leukemia, stratifying patients by SAMHD1 genotype or expression.

Mass spectrometry and tandem MS (MS/MS) have been widely employed for the identification and quantification of damaged nucleosides in DNA, including those induced by alkylating agents. Upon collisional activation, protonated ions of alkylated nucleosides frequently undergo facile neutral loss of a 2-deoxyribose in MS/MS, and further cleavage of the resulting protonated nucleobases in MS3 can sometimes be employed for differentiating regioisomeric alkylated DNA lesions. Herein, we investigated systematically the collision-induced dissociation (CID) of the protonated ions of O4-alkylthymidine (O4-alkyldT), O2-alkyldT, O6-alkyl-2-deoxyguanosine (O6-alkyldG), and N2-alkyldG through MS3 analysis. The MS3 of O2- and O4-MedT exhibit different fragmentation patterns from each other and from other O2- and O4-alkyldT adducts carrying larger alkyl groups. Meanwhile, elimination of alkene via a six-membered ring transition state is the dominant fragmentation pathway for O2-alkyldT, O4-alkyldT, and O6-alkyldG adducts carrying larger alkyl groups, whereas O6-MedG mainly undergoes elimination of ammonia. The breakdown of N2-alkyldG is substantially influenced by the structure of the alkyl group, where the relative ease in eliminating ammonia and alkene is modulated by the chain length and branching of the alkyl groups. We also rationalize our observations with density functional theory (DFT) calculations.

The anti-leukemia agent forodesine causes cytotoxic overload of intracellular deoxyguanosine triphosphate (dGTP) but is efficacious only in a subset of patients. We report that SAMHD1, a phosphohydrolase degrading deoxyribonucleoside triphosphate (dNTP), protects cells against the effects of dNTP imbalances. SAMHD1-deficient cells induce intrinsic apoptosis upon provision of deoxyribonucleosides, particularly deoxyguanosine (dG). Moreover, dG and forodesine act synergistically to kill cells lacking SAMHD1. Using mass cytometry, we find that these compounds kill SAMHD1-deficient malignant cells in patients with chronic lymphocytic leukemia (CLL). Normal cells and CLL cells from patients without SAMHD1 mutation are unaffected. We therefore propose to use forodesine as a precision medicine for leukemia, stratifying patients by SAMHD1 genotype or expression.

The anti-leukemia agent forodesine causes cytotoxic overload of intracellular deoxyguanosine triphosphate (dGTP) but is efficacious only in a subset of patients. We report that SAMHD1, a phosphohydrolase degrading deoxyribonucleoside triphosphate (dNTP), protects cells against the effects of dNTP imbalances. SAMHD1-deficient cells induce intrinsic apoptosis upon provision of deoxyribonucleosides, particularly deoxyguanosine (dG). Moreover, dG and forodesine act synergistically to kill cells lacking SAMHD1. Using mass cytometry, we find that these compounds kill SAMHD1-deficient malignant cells in patients with chronic lymphocytic leukemia (CLL). Normal cells and CLL cells from patients without SAMHD1 mutation are unaffected. We therefore propose to use forodesine as a precision medicine for leukemia, stratifying patients by SAMHD1 genotype or expression.

Standard and targeted cancer therapies for late-stage cancer patients almost universally fail due to tumor heterogeneity/plasticity and intrinsic or acquired drug resistance. We used the telomerase substrate nucleoside precursor, 6-thio-2'-deoxyguanosine (6-thio-dG), to target telomerase-expressing non-small cell lung cancer cells resistant to EGFR-inhibitors and commonly used chemotherapy combinations. Colony formation assays, human xenografts as well as syngeneic and genetically engineered immune competent mouse models of lung cancer were used to test the effect of 6-thio-dG on targeted therapy- and chemotherapy-resistant lung cancer human cells and mouse models. We observed that erlotinib-, paclitaxel/carboplatin-, and gemcitabine/cisplatin-resistant cells were highly sensitive to 6-thio-dG in cell culture and in mouse models. 6-thio-dG, with a known mechanism of action, is a potential novel therapeutic approach to prolong disease control of therapy-resistant lung cancer patients with minimal toxicities.

Standard and targeted cancer therapies for late-stage cancer patients almost universally fail due to tumor heterogeneity/plasticity and intrinsic or acquired drug resistance. We used the telomerase substrate nucleoside precursor, 6-thio-2'-deoxyguanosine (6-thio-dG), to target telomerase-expressing non-small cell lung cancer cells resistant to EGFR-inhibitors and commonly used chemotherapy combinations. Colony formation assays, human xenografts as well as syngeneic and genetically engineered immune competent mouse models of lung cancer were used to test the effect of 6-thio-dG on targeted therapy- and chemotherapy-resistant lung cancer human cells and mouse models. We observed that erlotinib-, paclitaxel/carboplatin-, and gemcitabine/cisplatin-resistant cells were highly sensitive to 6-thio-dG in cell culture and in mouse models. 6-thio-dG, with a known mechanism of action, is a potential novel therapeutic approach to prolong disease control of therapy-resistant lung cancer patients with minimal toxicities.

A wide spectrum of DNA lesions can be generated from byproducts of endogenous metabolism and/or from environmental exposure. A DNA adductomic approach for the robust quantification of DNA adducts in cellular and tissue DNA may facilitate the use of DNA adducts for biomonitoring studies and enable comprehensive assessment about DNA repair. Normalized retention time (iRT) has been widely used in scheduled selected-reaction monitoring (SRM) methods for highly sensitive and high-throughput analyses of protein samples in complicated matrices. By using a similar method, we established the iRT scores for 36 modified nucleosides from the retention times of the four canonical 2-deoxynucleosides on a nanoflow liquid chromatography-nanospray ionization-tandem mass spectrometry (nLC-NSI-MS/MS) system. The iRT scores facilitated reliable prediction of retention time and were employed for establishing a scheduled SRM method for quantitative assessment of a subset of the DNA adductome. The quantification results of the scheduled SRM method were more accurate and precise than those from an unscheduled method.

Many cancers appear to activate intrinsic antioxidant systems as a means to counteract oxidative stress. Some cancers, such as clear cell renal cell carcinoma (ccRCC), require exogenous glutamine for growth and exhibit reprogrammed glutamine metabolism, at least in part due to the glutathione pathway, an efficient cellular buffering system that counteracts reactive oxygen species and other oxidants. We show here that ccRCC xenograft tumors under the renal capsule exhibit enhanced oxidative stress compared with adjacent normal tissue and the contralateral kidney. Upon glutaminase inhibition with CB-839 or BPTES, the RCC cell lines SN12PM-6-1 (SN12) and 786-O exhibited decreased survival and pronounced apoptosis associated with a decreased GSH/GSSG ratio, augmented nuclear factor erythroid-related factor 2, and increased 8-oxo-7,8-dihydro-2'-deoxyguanosine, a marker of DNA damage. SN12 tumor xenografts showed decreased growth when treated with CB-839. Furthermore, PET imaging confirmed that ccRCC tumors exhibited increased tumoral uptake of 18F-(2S,4R)4-fluoroglutamine compared with the kidney in the orthotopic mouse model. This technique can be utilized to follow changes in ccRCC metabolism in vivo Further development of these paradigms will lead to new treatment options with glutaminase inhibitors and the utility of PET to identify and manage patients with ccRCC who are likely to respond to glutaminase inhibitors in the clinic. Cancer Res; 77(23); 6746-58. ©2017 AACR.

SCOPE: Xanthohumol (XN) is a hop flavonoid found in beers and refreshment drinks. Results of in vitro and animal studies indicate that it causes beneficial health effects due to DNA protective, anti-inflammatory, antioxidant, and phytoestrogenic properties. Aim of the present study was to find out if XN causes alterations of health-related parameters in humans.METHODS AND RESULTS: The effects of the flavonoid were investigated in a randomized crossover intervention trial (n = 22) in which the participants consumed a XN drink (12 mg XN/P/day). We monitored alterations of the DNA stability in single cell gel electrophoresis assays in lymphocytes and of several health-related biomarkers. A decrease of oxidatively damaged purines and protection toward reactive oxygen species induced DNA damage was found after the consumption of the beverage; also the excretion of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-guanosine in urine was reduced. The assumption that the flavonoid causes DNA protection was confirmed in a randomized follow-up study with pure XN (n = 10) with a parallel design. Other biochemical parameters reflecting the redox- and hormonal status and lipid- and glucose metabolism were not altered after the intervention.CONCLUSION: Taken together, our data indicate that low doses of XN protect humans against oxidative DNA damage.

INTRODUCTION: High levels of oxidative stress can have considerable impact on the outcomes of in vitro fertilisation (IVF). Recent studies have reported that melatonin, a neurohormone secreted from the pineal gland in response to darkness, has significant antioxidative capabilities which may protect against the oxidative stress of infertility treatment on gametes and embryos. Early studies of oral melatonin (3-4 mg/day) in IVF have suggested favourable outcomes. However, most trials were poorly designed and none have addressed the optimum dose of melatonin. We present a proposal for a pilot double-blind randomised placebo-controlled dose-response trial aimed to determine whether oral melatonin supplementation during ovarian stimulation can improve the outcomes of assisted reproductive technology. METHODS AND ANALYSES: We will recruit 160 infertile women into one of four groups: placebo (n=40); melatonin 2 mg twice per day (n=40); melatonin 4 mg twice per day (n=40) and melatonin 8 mg twice per day (n=40). The primary outcome will be clinical pregnancy rate. Secondary clinical outcomes include oocyte number/quality, embryo number/quality and fertilisation rate. We will also measure serum melatonin and the oxidative stress marker, 8-hydroxy-2'-deoxyguanosine at baseline and after treatment and levels of these in follicular fluid at egg pick-up. We will investigate follicular blood flow with Doppler ultrasound, patient sleepiness scores and pregnancy complications, comparing outcomes between groups. This protocol has been designed in accordance with the SPIRIT 2013 Guidelines. ETHICS AND DISSEMINATION: Ethical approval has been obtained from Monash Health HREC (Ref: 13402B), Monash University HREC (Ref: CF14/523-2014000181) and Monash Surgical Private Hospital HREC (Ref: 14107). Data analysis, interpretation and conclusions will be presented at national and international conferences and published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: ACTRN12613001317785

INTRODUCTION: High levels of oxidative stress can have considerable impact on the outcomes of in vitro fertilisation (IVF). Recent studies have reported that melatonin, a neurohormone secreted from the pineal gland in response to darkness, has significant antioxidative capabilities which may protect against the oxidative stress of infertility treatment on gametes and embryos. Early studies of oral melatonin (3-4 mg/day) in IVF have suggested favourable outcomes. However, most trials were poorly designed and none have addressed the optimum dose of melatonin. We present a proposal for a pilot double-blind randomised placebo-controlled dose-response trial aimed to determine whether oral melatonin supplementation during ovarian stimulation can improve the outcomes of assisted reproductive technology. METHODS AND ANALYSES: We will recruit 160 infertile women into one of four groups: placebo (n=40); melatonin 2 mg twice per day (n=40); melatonin 4 mg twice per day (n=40) and melatonin 8 mg twice per day (n=40). The primary outcome will be clinical pregnancy rate. Secondary clinical outcomes include oocyte number/quality, embryo number/quality and fertilisation rate. We will also measure serum melatonin and the oxidative stress marker, 8-hydroxy-2'-deoxyguanosine at baseline and after treatment and levels of these in follicular fluid at egg pick-up. We will investigate follicular blood flow with Doppler ultrasound, patient sleepiness scores and pregnancy complications, comparing outcomes between groups. This protocol has been designed in accordance with the SPIRIT 2013 Guidelines. ETHICS AND DISSEMINATION: Ethical approval has been obtained from Monash Health HREC (Ref: 13402B), Monash University HREC (Ref: CF14/523-2014000181) and Monash Surgical Private Hospital HREC (Ref: 14107). Data analysis, interpretation and conclusions will be presented at national and international conferences and published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: ACTRN12613001317785

Oksidativni stres nastao usled nesklada u produkciji slobodnih radikala i antioksidativnoj zaštiti sve češće se prepoznaje kao biohemijski mehanizam potencijalno uključen u patogenetska zbivanja u velikom broju sistemskih bolesti, pa tako i parodontopatije. Sa druge strane, parodontopatija kao inflamatorno destruktivno oboljenje i sama dovodi do pojačanog stvaranja slobodnih radikala. Ispitivanja pokazuju da parodontopatiju ne prati samo poremećaj u redoks ravnoteži lokalno u usnoj duplji, već da reaktivni oblici kiseonika proizvedeni tokom parodontopatije difunduju u krv dovodeći do oksidacije biomolekula krvi i nastanka cirkulatornog oksidativnog stresa. Cilj ovog istraživanja bio je da uporedimo stepen oksidativnog stresa u pljuvački i krvi u grupi pacijenata sa parodontopatijom i grupi pacijenata sa zdravim parodoncijumom kao i da ispitamo uticaj kauzalne terapije parodontopatije na stepen oksidativnog stresa u pljuvački i krvi kod pacijenata sa parodontopatijom. U istraživanje je bilo uključeno 30 pacijenata sa hroničnom parodontopatijom i 20 ispitanika sa zdravim parodoncijumom.Za procenu stanja parodoncijuma korišćeni su: Plak indeks (PI), Gingivalni indeks (GI), Indeks krvarenja gingive (IK), Dubina sondiranja (DS) i Nivo pripojnog epitela (NPE). Laboratorijsko ispitivanje se sastojalo od određivanja markera oksidativnog oštećenja DNK-8-hidroksideoksiguanozina (8-OHdG), markera peroksidacije lipida-malondialdehida (MDA) i antioksidativnog enzima-superoksid-dismutaze (SOD) u pljuvački i krvi. Kod pacijenata sa parodontopatijom bila je preduzeta kauzalna terapija. Klinička procena stanja parodoncijuma i laboratorijska ispitivanja su vršena na početku istraživanja i 3 meseca nakon završetka kauzalne terapije kod pacijenata sa parodontopatijom, odnosno samo na početku istraživanja kod pacijenata sa zdravim parodoncijumom. Vrednosti svih ispitivanih markera oksidativnog stresa u pljuvački pacijanata sa parodontopatijom bile su statistički značajno više u odnosu n, Oxidative stress arises due to the imbalance between the free radical production and the antioxidant defenses and is increasingly being recognized as a biochemical mechanism potentially present in pathogenic processes in a significant number of systemic illnesses, including periodontitis. On the other hand, periodontitis, as a destructive inflammatory disease, also contributes to the increased free radical production. Research indicates that periodontitis is accompanied not only by the disruption in bedox balance, localized in the oral cavity, but that reactive oxygen species produced during the periodontal disease are diffused into the blood, leading to the oxidation of blood biomolecules and the development of circulating oxidative stress. Aim: The aim of this study was to compare the level of oxidative stress in a group of patients with periodontitis to that of a group of patients with healthy periodontium and to evaluate the impact of non-surgical periodontal therapy on the level of oxidative stress in patients with periodontitis. Materials and methods: Study participians were 30 patients with chronic periodontitis and 20 periodontally healthy individuals. Their periodontal condition was assessed: Plaque index (PI), Gingival index (GI), Papilla bleeding index (PBI), Probing depth (PD) and Clinical attachment level (CAL). Laboratory assessment consisted of determination of DNA oxidative damage marker-8-hydroxydeoxyguanosine (8 OHdG), marker of lipid peroxidation- malondialdehyde (MDA) and antioxidant enzyme-superoxid dismutase (SOD) in saliva and blood. Patients with periodontitis were subjected to the non-surgical periodontal therapy. Clinical evaluation of periodontal conditions and laboratory tests were performed at baseline and 3 months after the non-surgical periodontal therapy. Results: The values of all studied oxidative stress markers in saliva were significantly higher in patients with periodontitis, compared to those with healthy periodontium (8-OHdG

A systematic study involving the use and optimization of gas-phase modifiers in quantitative differential mobility-mass spectrometry (DMS-MS) analysis is presented using nucleoside-adduct biomarkers of DNA damage as an important reference point for analysis in complex matrices. Commonly used polar protic and polar aprotic modifiers have been screened for use against two deoxyguanosine adducts of DNA: N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP) and N-(deoxyguanosin-8-y1)-2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine (dG-C8-PhIP). Particular attention was paid to compensation voltage (CoV) shifts, peak shapes, and product ion signal intensities while optimizing the DMS-MS conditions. The optimized parameters were then applied to rapid quantitation of the DNA adducts in calf thymus DNA. After a protein precipitation step, adduct levels corresponding to less than one modification in 10(6) normal DNA bases were detected using the DMS-MS platform. Based on DMS fundamentals and ab initio thermochemical results, we interpret the complexity of DMS modifier responses in terms of thermal activation and the development of solvent shells. At very high bulk gas temperature, modifier dipole moment may be the most important factor in cluster formation and cluster geometry, but at lower temperatures, multi-neutral clusters are important and less predictable. This work provides a useful protocol for targeted DNA adduct quantitation and a basis for future work on DMS modifier effects.

AIMS: Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo) are biomarkers of oxidative stress with clinical potential in a variety of diseases. As part of their clinical validation, this study aimed to investigate whether the urinary excretion of 8-oxodG and 8-oxoGuo undergoes diurnal variation and to evaluate the validity of 6-hour sampling as well as creatinine corrected spot urine sampling.METHODS: A total of 23 healthy study subjects collecting their 24-h urine in four fractions covering 6 hours each. Urinary 8-oxodG and 8-oxoGuo levels were quantified using a modified version of UPLC-MS/MS.RESULTS: No significant difference in excretion levels between the 12-h diurnal and 12-h nocturnal state or between the four 6-h periods during the day was found for either biomarker. A strong linear relationship between the excretion levels in each of the 6-h periods and the 24-h excretion level was shown for both biomarkers. Creatinine correction of the 6-h levels reduced the biological variation of the excretion levels and weakened the linear relationship with the uncorrected 24-h excretion level for both biomarkers. The correlations were strengthened when the 24-h excretion level was expressed per kg body weight.CONCLUSION: The results showed that 8-oxodG and 8-oxoGuo did not undergo diurnal variation in the study population overall and hence that the time of sampling is not crucial. Furthermore, 6-h sampling can be used as a substitute for 24-h sampling, and creatinine corrected sampling may be rational due to the reduction in biological variation of the biomarkers and the reasonable correlation with body weight-adjusted 24-h levels.

AIMS: Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo) are biomarkers of oxidative stress with clinical potential in a variety of diseases. As part of their clinical validation, this study aimed to investigate whether the urinary excretion of 8-oxodG and 8-oxoGuo undergoes diurnal variation and to evaluate the validity of 6-hour sampling as well as creatinine corrected spot urine sampling.METHODS: A total of 23 healthy study subjects collecting their 24-h urine in four fractions covering 6 hours each. Urinary 8-oxodG and 8-oxoGuo levels were quantified using a modified version of UPLC-MS/MS.RESULTS: No significant difference in excretion levels between the 12-h diurnal and 12-h nocturnal state or between the four 6-h periods during the day was found for either biomarker. A strong linear relationship between the excretion levels in each of the 6-h periods and the 24-h excretion level was shown for both biomarkers. Creatinine correction of the 6-h levels reduced the biological variation of the excretion levels and weakened the linear relationship with the uncorrected 24-h excretion level for both biomarkers. The correlations were strengthened when the 24-h excretion level was expressed per kg body weight.CONCLUSION: The results showed that 8-oxodG and 8-oxoGuo did not undergo diurnal variation in the study population overall and hence that the time of sampling is not crucial. Furthermore, 6-h sampling can be used as a substitute for 24-h sampling, and creatinine corrected sampling may be rational due to the reduction in biological variation of the biomarkers and the reasonable correlation with body weight-adjusted 24-h levels.

IntroductionDuctal carcinoma in situ (DCIS) is a noninvasive breast cancer wherein malignant cells are confined within a ductal lobular unit. Although less than half the cases of DCIS will progress to invasive disease, most women are treated aggressively with surgery, radiation, and/or hormone therapy due to the inability to clinically evaluate the extent and location of the disease. Intraductal therapy, in which a drug is administered directly into the mammary duct through the nipple, is a promising approach for treating DCIS, but the feasibility of instilling drug into a diseased duct has not been established.Patients and methodsFour to 6 weeks before their scheduled surgery, 13 women diagnosed with DCIS were subjected to cannulation of the affected duct. After both the absence of perforation and presence of dye in the duct were confirmed by ductogram, pegylated liposomal doxorubicin was instilled. Histopathologic assessment was performed after surgery to assess the treatment effects.ResultsOf the 13 women enrolled in the study, 6 had their DCIS duct successfully cannulated without perforation and instilled with the drug. The treatment was well tolerated, and no serious adverse events have been reported. Biomarker studies indicated a general decrease in Ki-67 levels but an increase in annexin-1 and 8-hydroxydeoxyguanosine in the lumen of DCIS-containing ducts, which suggests a local response to pegylated liposomal doxorubicin treatment.ConclusionsIntraductal therapy offers a nonsurgical strategy to treat DCIS at the site of disease, potentially minimizing the adverse effects of systemic treatment while preventing development of invasive cancer.

BACKGROUND: Depression has been associated with increased oxidative stress and hypothesized to accelerate aging. Nucleic acid damage from oxidation is a critical part of the aging process, and a suggested early event in age-related somatic morbidities that are also prevalent in depression, such as dementia and type 2 diabetes. We hypothesized that increased severity of depression is associated with increased systemic oxidatively generated DNA and RNA damage, and that this increase is attenuated by an effective antidepressant treatment.METHODS: The urinary excretion of markers of systemic oxidatively generated DNA and RNA damage, 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo), respectively, were determined in healthy controls (N=28), moderately depressed, non-medicated patients (N=26) and severely depressed patients eligible for electroconvulsive therapy (ECT) (N=29). In the severely depressed patient group, samples were also obtained 1 week after the completion of ECT.RESULTS: Systemic RNA damage from oxidation, as measured by 8-oxoGuo excretion, was higher with increasing severity of depression (controls

Balkan endemic nephropathy (BEN) is a human chronic tubulointerstitial renal disease that occurs in rural areas of some Balkan countries. The disease is insidious and fatal, and mostly affects persons in their sixties or seventies. BEN areas have unusually high rates of otherwise rare upper urinary tract tumors (UTT). Since extensive production of reactive oxygen species leading to oxidative stress has been implicated in tumor development, the aim of this study was to see whether oxidative stress is involved in the development of BEN and UTT. Urine samples were collected from a BEN village (N = 22) and a control village (N = 16) residents and analyzed for malondialdehyde (MDA) and 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxodG). The levels of both oxidative stress parameters were significantly higher in the BEN village residents than controls. However, there was no correlation between MDA and 8-oxodG results. Our results confirm that oxidative stress could be implicated in development of both, BEN and UTT.

Oxidative stress may be important in carcinogenesis and a possible risk factor for breast cancer. The urinary excretion of oxidatively generated biomolecules, such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), represents biomarkers of oxidative stress, reflecting the rate of global damage to DNA in steady state.

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.

BACKGROUND: Depression has been associated with increased oxidative stress and hypothesized to accelerate aging. Nucleic acid damage from oxidation is a critical part of the aging process, and a suggested early event in age-related somatic morbidities that are also prevalent in depression, such as dementia and type 2 diabetes. We hypothesized that increased severity of depression is associated with increased systemic oxidatively generated DNA and RNA damage, and that this increase is attenuated by an effective antidepressant treatment.METHODS: The urinary excretion of markers of systemic oxidatively generated DNA and RNA damage, 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo), respectively, were determined in healthy controls (N=28), moderately depressed, non-medicated patients (N=26) and severely depressed patients eligible for electroconvulsive therapy (ECT) (N=29). In the severely depressed patient group, samples were also obtained 1 week after the completion of ECT.RESULTS: Systemic RNA damage from oxidation, as measured by 8-oxoGuo excretion, was higher with increasing severity of depression (controls

Balkan endemic nephropathy (BEN) is a human chronic tubulointerstitial renal disease that occurs in rural areas of some Balkan countries. The disease is insidious and fatal, and mostly affects persons in their sixties or seventies. BEN areas have unusually high rates of otherwise rare upper urinary tract tumors (UTT). Since extensive production of reactive oxygen species leading to oxidative stress has been implicated in tumor development, the aim of this study was to see whether oxidative stress is involved in the development of BEN and UTT. Urine samples were collected from a BEN village (N = 22) and a control village (N = 16) residents and analyzed for malondialdehyde (MDA) and 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxodG). The levels of both oxidative stress parameters were significantly higher in the BEN village residents than controls. However, there was no correlation between MDA and 8-oxodG results. Our results confirm that oxidative stress could be implicated in development of both, BEN and UTT.

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.

Oxidative stress may be important in carcinogenesis and a possible risk factor for breast cancer. The urinary excretion of oxidatively generated biomolecules, such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), represents biomarkers of oxidative stress, reflecting the rate of global damage to DNA in steady state.

Previous studies have shown an association between oxidative stress and various diseases in humans including cancer, cardiovascular disease, diabetes, and chronic respiratory disease. To what extents this damage is determined by genetic and environmental factors is unknown. In a classical twin study with 198 elderly twins we examined the contributions of genetic versus environmental factors to nucleic acid oxidation and lipid peroxidation. Urinary excretion of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydroguanosine (8-oxoGuo), and dinor,dihydro F2-isoprostane metabolites (F2-IsoP-M) was measured using liquid chromatography–tandem mass spectrometry. The environmental influence on nucleic acid oxidation and lipid peroxidation was predominant, leaving only little influence from genetic factors, as evidenced by no differences in intraclass correlations between monozygotic (MZ) and dizygotic (DZ) twins, neither for 8-oxodG (rMZ = 0.55, rDZ = 0.47; P = 0.43), F2-IsoP-M (rMZ = 0.33, rDZ = 0.22; P = 0.42), nor 8-oxoGuo (rMZ = 0.45, rDZ = 0.58; P = 0.21). Accordingly, heritability estimates for the three markers of oxidative damage were low (h2 = 0.17–0.22). The three urinary markers of oxidative stress were closely correlated (r = 0.60–0.84). In conclusion, we demonstrated in a large population of elderly Danish twins that “whole-body” oxidative damage to nucleic acids and lipids is predominantly determined by potentially modifiable nongenetic factors., Previous studies have shown an association between oxidative stress and various diseases in humans including cancer, cardiovascular disease, diabetes, and chronic respiratory disease. To what extents this damage is determined by genetic and environmental factors is unknown. In a classical twin study with 198 elderly twins we examined the contributions of genetic versus environmental factors to nucleic acid oxidation and lipid peroxidation. Urinary excretion of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydroguanosine (8-oxoGuo), and dinor,dihydro F2-isoprostane metabolites (F2-IsoP-M) was measured using liquid chromatography-tandem mass spectrometry. The environmental influence on nucleic acid oxidation and lipid peroxidation was predominant, leaving only little influence from genetic factors, as evidenced by no differences in intraclass correlations between monozygotic (MZ) and dizygotic (DZ) twins, neither for 8-oxodG (r(MZ)=0.55, r(DZ)=0.47; P=0.43), F(2)-IsoP-M (r(MZ)=0.33, r(DZ)=0.22; P=0.42), nor 8-oxoGuo (r(MZ)=0.45, r(DZ)=0.58; P=0.21). Accordingly, heritability estimates for the three markers of oxidative damage were low (h²=0.17-0.22). The three urinary markers of oxidative stress were closely correlated (r=0.60-0.84). In conclusion, we demonstrated in a large population of elderly Danish twins that "whole-body" oxidative damage to nucleic acids and lipids is predominantly determined by potentially modifiable nongenetic factors.

Previous studies have shown an association between oxidative stress and various diseases in humans including cancer, cardiovascular disease, diabetes, and chronic respiratory disease. To what extents this damage is determined by genetic and environmental factors is unknown. In a classical twin study with 198 elderly twins we examined the contributions of genetic versus environmental factors to nucleic acid oxidation and lipid peroxidation. Urinary excretion of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydroguanosine (8-oxoGuo), and dinor,dihydro F2-isoprostane metabolites (F2-IsoP-M) was measured using liquid chromatography–tandem mass spectrometry. The environmental influence on nucleic acid oxidation and lipid peroxidation was predominant, leaving only little influence from genetic factors, as evidenced by no differences in intraclass correlations between monozygotic (MZ) and dizygotic (DZ) twins, neither for 8-oxodG (rMZ = 0.55, rDZ = 0.47; P = 0.43), F2-IsoP-M (rMZ = 0.33, rDZ = 0.22; P = 0.42), nor 8-oxoGuo (rMZ = 0.45, rDZ = 0.58; P = 0.21). Accordingly, heritability estimates for the three markers of oxidative damage were low (h2 = 0.17–0.22). The three urinary markers of oxidative stress were closely correlated (r = 0.60–0.84). In conclusion, we demonstrated in a large population of elderly Danish twins that “whole-body” oxidative damage to nucleic acids and lipids is predominantly determined by potentially modifiable nongenetic factors., Previous studies have shown an association between oxidative stress and various diseases in humans including cancer, cardiovascular disease, diabetes, and chronic respiratory disease. To what extents this damage is determined by genetic and environmental factors is unknown. In a classical twin study with 198 elderly twins we examined the contributions of genetic versus environmental factors to nucleic acid oxidation and lipid peroxidation. Urinary excretion of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydroguanosine (8-oxoGuo), and dinor,dihydro F2-isoprostane metabolites (F2-IsoP-M) was measured using liquid chromatography-tandem mass spectrometry. The environmental influence on nucleic acid oxidation and lipid peroxidation was predominant, leaving only little influence from genetic factors, as evidenced by no differences in intraclass correlations between monozygotic (MZ) and dizygotic (DZ) twins, neither for 8-oxodG (r(MZ)=0.55, r(DZ)=0.47; P=0.43), F(2)-IsoP-M (r(MZ)=0.33, r(DZ)=0.22; P=0.42), nor 8-oxoGuo (r(MZ)=0.45, r(DZ)=0.58; P=0.21). Accordingly, heritability estimates for the three markers of oxidative damage were low (h²=0.17-0.22). The three urinary markers of oxidative stress were closely correlated (r=0.60-0.84). In conclusion, we demonstrated in a large population of elderly Danish twins that "whole-body" oxidative damage to nucleic acids and lipids is predominantly determined by potentially modifiable nongenetic factors.

Previous studies have shown an association between oxidative stress and various diseases in humans including cancer, cardiovascular disease, diabetes, and chronic respiratory disease. To what extents this damage is determined by genetic and environmental factors is unknown. In a classical twin study with 198 elderly twins we examined the contributions of genetic versus environmental factors to nucleic acid oxidation and lipid peroxidation. Urinary excretion of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydroguanosine (8-oxoGuo), and dinor,dihydro F2-isoprostane metabolites (F2-IsoP-M) was measured using liquid chromatography–tandem mass spectrometry. The environmental influence on nucleic acid oxidation and lipid peroxidation was predominant, leaving only little influence from genetic factors, as evidenced by no differences in intraclass correlations between monozygotic (MZ) and dizygotic (DZ) twins, neither for 8-oxodG (rMZ = 0.55, rDZ = 0.47; P = 0.43), F2-IsoP-M (rMZ = 0.33, rDZ = 0.22; P = 0.42), nor 8-oxoGuo (rMZ = 0.45, rDZ = 0.58; P = 0.21). Accordingly, heritability estimates for the three markers of oxidative damage were low (h2 = 0.17–0.22). The three urinary markers of oxidative stress were closely correlated (r = 0.60–0.84). In conclusion, we demonstrated in a large population of elderly Danish twins that “whole-body” oxidative damage to nucleic acids and lipids is predominantly determined by potentially modifiable nongenetic factors., Previous studies have shown an association between oxidative stress and various diseases in humans including cancer, cardiovascular disease, diabetes, and chronic respiratory disease. To what extents this damage is determined by genetic and environmental factors is unknown. In a classical twin study with 198 elderly twins we examined the contributions of genetic versus environmental factors to nucleic acid oxidation and lipid peroxidation. Urinary excretion of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydroguanosine (8-oxoGuo), and dinor,dihydro F2-isoprostane metabolites (F2-IsoP-M) was measured using liquid chromatography-tandem mass spectrometry. The environmental influence on nucleic acid oxidation and lipid peroxidation was predominant, leaving only little influence from genetic factors, as evidenced by no differences in intraclass correlations between monozygotic (MZ) and dizygotic (DZ) twins, neither for 8-oxodG (r(MZ)=0.55, r(DZ)=0.47; P=0.43), F(2)-IsoP-M (r(MZ)=0.33, r(DZ)=0.22; P=0.42), nor 8-oxoGuo (r(MZ)=0.45, r(DZ)=0.58; P=0.21). Accordingly, heritability estimates for the three markers of oxidative damage were low (h²=0.17-0.22). The three urinary markers of oxidative stress were closely correlated (r=0.60-0.84). In conclusion, we demonstrated in a large population of elderly Danish twins that "whole-body" oxidative damage to nucleic acids and lipids is predominantly determined by potentially modifiable nongenetic factors.

Of the DNA-derived biomarkers of oxidative stress, urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) is the most frequently measured. However, there is significant discrepancy between chromatographic and immunoassay approaches, and intratechnique agreement among all available chromatography-based assays and ELISAs is yet to be established. This is a significant obstacle to their use in large molecular epidemiological studies. To evaluate the accuracy of intra/intertechnique and interlaboratory measurements, samples of phosphate buffered saline and urine, spiked with different concentrations of 8-oxoG, together with a series of urine samples from healthy individuals were distributed to ESCULA members. All laboratories received identical samples, including 2 negative controls that contained no added 8-oxodG. Data were returned from 17 laboratories, representing 20 methods, broadly classified as mass spectrometric (MS), electrochemical detection (EC), or enzyme-linked immunosorbant assay (ELISA). Overall, there was good within-technique agreement, with the majority of laboratories' results lying within 1 SD of their consensus mean. However, ELISA showed more within-technique variation than did the chromatographic techniques and, for the urine samples, reported higher values. Bland-Altman plots revealed good agreement between MS and EC methods but concentration-dependent deviation for ELISA. All methods ranked urine samples according to concentration similarly. Creatinine levels are routinely used as a correction factor for urine concentration, and therefore we also conducted an interlaboratory comparison of methods for urinary creatinine determination, in which the vast majority of values lay within 1 SD of the consensus value, irrespective of the analysis procedure. This study reveals greater consensus than previously expected, although concern remains over ELISA. © FASEB.

Of the DNA-derived biomarkers of oxidative stress, urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) is the most frequently measured. However, there is significant discrepancy between chromatographic and immunoassay approaches, and intratechnique agreement among all available chromatography-based assays and ELISAs is yet to be established. This is a significant obstacle to their use in large molecular epidemiological studies. To evaluate the accuracy of intra/intertechnique and interlaboratory measurements, samples of phosphate buffered saline and urine, spiked with different concentrations of 8-oxoG, together with a series of urine samples from healthy individuals were distributed to ESCULA members. All laboratories received identical samples, including 2 negative controls that contained no added 8-oxodG. Data were returned from 17 laboratories, representing 20 methods, broadly classified as mass spectrometric (MS), electrochemical detection (EC), or enzyme-linked immunosorbant assay (ELISA). Overall, there was good within-technique agreement, with the majority of laboratories' results lying within 1 SD of their consensus mean. However, ELISA showed more within-technique variation than did the chromatographic techniques and, for the urine samples, reported higher values. Bland-Altman plots revealed good agreement between MS and EC methods but concentration-dependent deviation for ELISA. All methods ranked urine samples according to concentration similarly. Creatinine levels are routinely used as a correction factor for urine concentration, and therefore we also conducted an interlaboratory comparison of methods for urinary creatinine determination, in which the vast majority of values lay within 1 SD of the consensus value, irrespective of the analysis procedure. This study reveals greater consensus than previously expected, although concern remains over ELISA. © FASEB.

The main source of mitochondrial DNA (mtDNA) damage is reactive oxygen species (ROS) generated during normal cellular metabolism. The main mtDNA lesions generated by ROS are base modifications, such as the ubiquitous 8-oxoguanine (8-oxoG) lesion; however, base loss and strand breaks may also occur. Many human diseases are associated with mtDNA mutations and thus maintaining mtDNA integrity is critical. All of these lesions are repaired primarily by the base excision repair (BER) pathway. It is now known that mammalian mitochondria have BER, which, similarly to nuclear BER, is catalyzed by DNA glycosylases, AP endonuclease, DNA polymerase (POLgamma in mitochondria) and DNA ligase. This article outlines procedures for measuring oxidative damage formation and BER in mitochondria, including isolation of mitochondria from tissues and cells, protocols for measuring BER enzyme activities, gene-specific repair assays, chromatographic techniques as well as current optimizations for detecting 8-oxoG lesions in cells by immunofluorescence. Throughout the assay descriptions we will include methodological considerations that may help optimize the assays in terms of resolution and repeatability.

Of the DNA-derived biomarkers of oxidative stress, urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) is the most frequently measured. However, there is significant discrepancy between chromatographic and immunoassay approaches, and intratechnique agreement among all available chromatography-based assays and ELISAs is yet to be established. This is a significant obstacle to their use in large molecular epidemiological studies. To evaluate the accuracy of intra/intertechnique and interlaboratory measurements, samples of phosphate buffered saline and urine, spiked with different concentrations of 8-oxoG, together with a series of urine samples from healthy individuals were distributed to ESCULA members. All laboratories received identical samples, including 2 negative controls that contained no added 8-oxodG. Data were returned from 17 laboratories, representing 20 methods, broadly classified as mass spectrometric (MS), electrochemical detection (EC), or enzyme-linked immunosorbant assay (ELISA). Overall, there was good within-technique agreement, with the majority of laboratories' results lying within 1 SD of their consensus mean. However, ELISA showed more within-technique variation than did the chromatographic techniques and, for the urine samples, reported higher values. Bland-Altman plots revealed good agreement between MS and EC methods but concentration-dependent deviation for ELISA. All methods ranked urine samples according to concentration similarly. Creatinine levels are routinely used as a correction factor for urine concentration, and therefore we also conducted an interlaboratory comparison of methods for urinary creatinine determination, in which the vast majority of values lay within 1 SD of the consensus value, irrespective of the analysis procedure. This study reveals greater consensus than previously expected, although concern remains over ELISA. © FASEB.

The main source of mitochondrial DNA (mtDNA) damage is reactive oxygen species (ROS) generated during normal cellular metabolism. The main mtDNA lesions generated by ROS are base modifications, such as the ubiquitous 8-oxoguanine (8-oxoG) lesion; however, base loss and strand breaks may also occur. Many human diseases are associated with mtDNA mutations and thus maintaining mtDNA integrity is critical. All of these lesions are repaired primarily by the base excision repair (BER) pathway. It is now known that mammalian mitochondria have BER, which, similarly to nuclear BER, is catalyzed by DNA glycosylases, AP endonuclease, DNA polymerase (POLgamma in mitochondria) and DNA ligase. This article outlines procedures for measuring oxidative damage formation and BER in mitochondria, including isolation of mitochondria from tissues and cells, protocols for measuring BER enzyme activities, gene-specific repair assays, chromatographic techniques as well as current optimizations for detecting 8-oxoG lesions in cells by immunofluorescence. Throughout the assay descriptions we will include methodological considerations that may help optimize the assays in terms of resolution and repeatability.

Of the DNA-derived biomarkers of oxidative stress, urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) is the most frequently measured. However, there is significant discrepancy between chromatographic and immunoassay approaches, and intratechnique agreement among all available chromatography-based assays and ELISAs is yet to be established. This is a significant obstacle to their use in large molecular epidemiological studies. To evaluate the accuracy of intra/intertechnique and interlaboratory measurements, samples of phosphate buffered saline and urine, spiked with different concentrations of 8-oxoG, together with a series of urine samples from healthy individuals were distributed to ESCULA members. All laboratories received identical samples, including 2 negative controls that contained no added 8-oxodG. Data were returned from 17 laboratories, representing 20 methods, broadly classified as mass spectrometric (MS), electrochemical detection (EC), or enzyme-linked immunosorbant assay (ELISA). Overall, there was good within-technique agreement, with the majority of laboratories' results lying within 1 SD of their consensus mean. However, ELISA showed more within-technique variation than did the chromatographic techniques and, for the urine samples, reported higher values. Bland-Altman plots revealed good agreement between MS and EC methods but concentration-dependent deviation for ELISA. All methods ranked urine samples according to concentration similarly. Creatinine levels are routinely used as a correction factor for urine concentration, and therefore we also conducted an interlaboratory comparison of methods for urinary creatinine determination, in which the vast majority of values lay within 1 SD of the consensus value, irrespective of the analysis procedure. This study reveals greater consensus than previously expected, although concern remains over ELISA. © FASEB.

Oxidized bases are common types of DNA modifications. Their accumulation in the genome is linked to aging and degenerative diseases. These modifications are commonly repaired by the base excision repair (BER) pathway. Oxoguanine DNA glycosylase (OGG1) initiates BER of oxidized purine bases. A small number of protein interactions have been identified for OGG1, while very few appear to have functional consequences. We report here that OGG1 interacts with the recombination protein RAD52 in vitro and in vivo. This interaction has reciprocal functional consequences as OGG1 inhibits RAD52 catalytic activities and RAD52 stimulates OGG1 incision activity, likely increasing its turnover rate. RAD52 colocalizes with OGG1 after oxidative stress to cultured cells, but not after the direct induction of double-strand breaks by ionizing radiation. Human cells depleted of RAD52 via small interfering RNA knockdown, and mouse cells lacking the protein via gene knockout showed increased sensitivity to oxidative stress. Moreover, cells depleted of RAD52 show higher accumulation of oxidized bases in their genome than cells with normal levels of RAD52. Our results indicate that RAD52 cooperates with OGG1 to repair oxidative DNA damage and enhances the cellular resistance to oxidative stress. Our observations suggest a coordinated action between these proteins that may be relevant when oxidative lesions positioned close to strand breaks impose a hindrance to RAD52 catalytic activities.

Oxidized bases are common types of DNA modifications. Their accumulation in the genome is linked to aging and degenerative diseases. These modifications are commonly repaired by the base excision repair (BER) pathway. Oxoguanine DNA glycosylase (OGG1) initiates BER of oxidized purine bases. A small number of protein interactions have been identified for OGG1, while very few appear to have functional consequences. We report here that OGG1 interacts with the recombination protein RAD52 in vitro and in vivo. This interaction has reciprocal functional consequences as OGG1 inhibits RAD52 catalytic activities and RAD52 stimulates OGG1 incision activity, likely increasing its turnover rate. RAD52 colocalizes with OGG1 after oxidative stress to cultured cells, but not after the direct induction of double-strand breaks by ionizing radiation. Human cells depleted of RAD52 via small interfering RNA knockdown, and mouse cells lacking the protein via gene knockout showed increased sensitivity to oxidative stress. Moreover, cells depleted of RAD52 show higher accumulation of oxidized bases in their genome than cells with normal levels of RAD52. Our results indicate that RAD52 cooperates with OGG1 to repair oxidative DNA damage and enhances the cellular resistance to oxidative stress. Our observations suggest a coordinated action between these proteins that may be relevant when oxidative lesions positioned close to strand breaks impose a hindrance to RAD52 catalytic activities.

The effects of a basil extract on the sulfation and concomitant DNA adduct formation of the proximate carcinogen 1′-hydroxyestragole were studied using rat and human liver S9 homogenates and the human hepatoma cell line HepG2. Basil was chosen since it contains the procarcinogen estragole that can be metabolized to 1′-hydroxyestragole by cytochrome P450 enzymes. Basil extract addition to incubations of rat and human liver S9 homogenates with 1′-hydroxyestragole, the sulfotransferase cofactor PAPS, and 2′-deoxyguanosine resulted in a dose-dependent inhibition of N2-(trans-isoestragol-3′-yl)-2′-deoxyguanosine formation. Because the inhibition resembled the inhibition by the sulfotransferase inhibitor pentachlorophenol and since the inhibition was not observed in incubations with the direct electrophile 1′-acetoxyestragole it is concluded that the inhibition occurs at the level of the sulfotransferase mediated bioactivation step. Additional experiments in HepG2 cells revealed the same protective effect of basil extract in intact cells, demonstrating that the inhibitors are able to enter the cells. The results of this study suggest that bioactivation and subsequent adverse effects of 1′-hydroxyestragole might be lower in a matrix of other basil ingredients than what would be expected on the basis of experiments using 1′-hydroxyestragole as a single compound. © 2008 Elsevier Ltd. All rights reserved. Chemicals / CAS: 1' hydroxyestragole, 51410-44-7; cytochrome P450, 9035-51-2; deoxyguanosine, 961-07-9; DNA, 9007-49-2; estragole, 140-67-0; sulfotransferase, 9023-09-0; 1'-hydroxyestragole, 51410-44-7; Anisoles; Deoxyguanosine, 961-07-9; DNA Adducts; Enzyme Inhibitors; Plant Extracts; Sulfotransferases, EC 2.8.2.-; Tetrazolium Salts; Thiazoles; thiazolyl blue, 298-93-1

Presents a case study of a massive oral exposure to hexavalent chromium in which there was demonstrable toxic effects seen on the proximal renal tubule that remained limited. Initial treatment of the medical condition; Main manifestations of hexavalent chromium toxicity; Results of the early administration of ascorbic acid and N-acetylcysteine.

Identification of genetic polymorphisms responsible for reduced DNA repair capacity may allow better cancer prevention. We examined whether variations in genes involved in base-excision (hOGGI, XRCCI) and double strand break (XRCC3) DNA repair contribute to inter-individual differences in genotoxic effects induced in the lymphocytes of 21 cobalt (Co) exposed, 26 hard metal (WC-Co) exposed and 26 matched control male workers. Genotyping was performed by PCR-RFLP. DNA single strand breaks and alkali-labile sites were measured by the alkaline Comet assay. Chromosomal rearrangements resulting from chromosome loss or acentric fragments were assessed as micronucleated mononucleates (MNMC) and binucleates (MNCB) with the cytokinesis-block micronucleus test. Urinary 8-hydroxydeoxyguanosine (8-OHdG) levels were used as an indicator of systemic oxidative DNA damage. A significantly higher frequency of MNMC was observed in WC-Co exposed workers with variant hOGGI(326) genotype. Multivariate analysis performed with genotypes, age, exposure status, type of plant, smoking and their interaction terms as independent variables indicated that MNMC and Comet tail DNA (TD) were influenced by genetic polymorphisms. In the exposed and total populations, workers variant for both XRCC3 and hOGG1 had elevated MNMC frequencies. Further studies will demonstrate whether genotyping for hOGG1 and XRCC3 polymorphisms is useful for a better individual monitoring of workers. (c) 2004 Elsevier Ireland Ltd. All rights reserved.

Although peroxisome proliferators are considered non-genotoxic agents, most of them, nevertheless, were found to promote and/or induce, hepatocellular carcinoma (HCC) in rodents. The aim of the present study is, first, to investigate whether the peroxisome proliferator perfluorooctanoic acid (PFOA) possesses inherent liver cancer promoting activity, and second, to study the possible mechanisms involved. To acheive these aims two protocols have been applied, a biphasic protocol (initiation by diethyl-nitrozamine (DEN) 200 mg/kg i.p. followed by treatment with 0.005% or 0.02% perflourooctanoic acid (PFOA) for 14 and 25 weeks) and a triphasic initiation, selection-promotion (IS) protocol (initiation by giving 200 mg/kg DEN i.p. followed by a selection procedure for 2 weeks consisting of giving 0.03% 2-acetylaminofluorene (2-AAF) in diet). In the middle of this treatment a single oral dose of carbon tetrachloride (2.0 ml/kg) was given, followed by giving diet containg 0.015% of PFOA for 25 weeks. After applying both protocols, our results showed slight increase in the catalase activity while acyl CoA oxidase activity was markedly increased. Both experiments indicated that PFOA has a liver cancer promoting activity. Other groups of rats were given either basal diet or diet containing 0.02% PFOA. Five or nine weeks later they were sacrificed and the levels of 8-hydroxydeoxyguanosine in the isolated DNA were estimated. The data showed a slight nonetheless insignificant increase in 8-hydroxydeoxyguanosine. From the present data, it is concluded that PFOA is a true liver cancer promoter that may not require extensive initial DNA damage for its promoting activity.

The role of the genetic polymorphism of NAD(P)H:quinone oxidoreductase (NQO1) and glutathione-S-transferase micro-1 (GSTM1) in the responsiveness to O(3)-induced acute effects was investigated in 24 healthy nonsmokers performing 2-h bike rides at ambient O(3) varying from 32 to 103 ppb. Before and after rides, each subject performed spirometric tests and provided a blood sample for the measurement of the Clara cell protein CC16. NQO1 and GSTM1 polymorphisms were characterized by polymerase chain reaction- based methods. The 8-hydroxy-2'-deoxyguanosine (8-OHdG) adduct was also measured in DNA of peripheral leukocytes. Rides at O(3) > 80 ppb resulted in significant decrements of pulmonary function tests and increased levels of serum CC16, consistent with mild impairment in respiratory function and increased lung epithelial permeability, respectively. Whereas NQO1wt and GSTM1null subjects showed both functional changes and increased serum CC16 after acute O(3) exposure, people with other haplotypes showed a rise in serum CC16 but no changes in lung function tests. In NQO1wt and GSTM1null subjects, partial correlation analysis showed that functional decrements and increased serum CC16 are closely associated with each other and with O(3) levels, whereas no such relationships were found among subjects bearing other haplotypes. An increased reaction rate between O(3) and hydroquinones would be consistent with the greater increase in 8-OHdG after O(3) exposure in this "susceptible" group.

The present study investigated the effect of a single bout of exhaustive exercise on the generation of DNA strand breaks and oxidative DNA damage under normal conditions and at high-altitude hypoxia (4559 meters for 3 days). Twelve healthy subjects performed a maximal bicycle exercise test; lymphocytes were isolated for analysis of DNA strand breaks and oxidatively altered nucleotides, detected by endonuclease III and formamidipyridine glycosylase (FPG) enzymes. Urine was collected for 24 h periods for analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a marker of oxidative DNA damage. Urinary excretion of 8-oxodG increased during the first day in altitude hypoxia, and there were more endonuclease III-sensitive sites on day 3 at high altitude. The subjects had more DNA strand breaks in altitude hypoxia than at sea level. The level of DNA strand breaks further increased immediately after exercise in altitude hypoxia. Exercise-induced generation of DNA strand breaks was not seen at sea level. In both environments, the level of FPG and endonuclease III-sensitive sites remained unchanged immediately after exercise. DNA strand breaks and oxidative DNA damage are probably produced by reactive oxygen species, generated by leakage of the mitochondrial respiration or during a hypoxia-induced inflammation. Furthermore, the presence of DNA strand breaks may play an important role in maintaining hypoxia-induced inflammation processes. Hypoxia seems to deplete the antioxidant system of its capacity to withstand oxidative stress produced by exhaustive exercise.

The preparation of oligonucleotides containing 8-bromo-2′-deoxyguanosine is described. Substitution of G by 8-bromoguanine on an alternating CG decamer stabilizes the Z-form in such a way that the B-form was not observed. Melting temperatures showed that duplexes in which 8-bromo-2′-deoxyguanosine paired with natural bases were much less stable.

The present study investigated the effect of a single bout of exhaustive exercise on the generation of DNA strand breaks and oxidative DNA damage under normal conditions and at high-altitude hypoxia (4559 meters for 3 days). Twelve healthy subjects performed a maximal bicycle exercise test; lymphocytes were isolated for analysis of DNA strand breaks and oxidatively altered nucleotides, detected by endonuclease III and formamidipyridine glycosylase (FPG) enzymes. Urine was collected for 24 h periods for analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a marker of oxidative DNA damage. Urinary excretion of 8-oxodG increased during the first day in altitude hypoxia, and there were more endonuclease III-sensitive sites on day 3 at high altitude. The subjects had more DNA strand breaks in altitude hypoxia than at sea level. The level of DNA strand breaks further increased immediately after exercise in altitude hypoxia. Exercise-induced generation of DNA strand breaks was not seen at sea level. In both environments, the level of FPG and endonuclease III-sensitive sites remained unchanged immediately after exercise. DNA strand breaks and oxidative DNA damage are probably produced by reactive oxygen species, generated by leakage of the mitochondrial respiration or during a hypoxia-induced inflammation. Furthermore, the presence of DNA strand breaks may play an important role in maintaining hypoxia-induced inflammation processes. Hypoxia seems to deplete the antioxidant system of its capacity to withstand oxidative stress produced by exhaustive exercise.

Exposure of individual histone proteins (H1, H2A, H2B, H3, or H4) and histone octamers (consisting of two molecules each of H2A, H2B, H3, and H4) to hydroxyl radicals, generated by gamma-irradiation, in the presence of O(2) generates protein-bound hydroperoxides in a dose-dependent fashion; this is in accord with previous studies with other proteins. These histone hydroperoxides are stable in the absence of exogenous catalysts (e.g., heat, light, and transition metal ions), but in the presence of these agents decompose rapidly to give a variety of radicals which have been identified by EPR spin trapping. Histone hydroperoxide-derived radicals generated on decomposition of the hydroperoxides with Cu(+) react with both pyrimidine and purine nucleobases. Thus, with uridine the histone hydroperoxide-derived radicals undergo addition across the C(5)-C(6) double bond of the pyrimidine ring to give cross-linked adduct species which have been identified by EPR spectroscopy. HPLC analysis of the products generated on reaction of histone hydroperoxide-derived radicals with 2'-deoxyguanosine, or intact calf thymus DNA, has shown that significant levels of the mutagenic oxidized DNA base 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) are formed, with the yield dependent on the individual histone protein, the presence of hydroperoxide functions, and the concentration of metal ion. These studies demonstrate that initial oxidative damage to individual histone proteins or histone octamers can result in the transfer of oxidative damage to associated DNA via the formation and subsequent decomposition of protein hydroperoxides to reactive radicals, and provide a novel route for the formation of mutagenic lesions in DNA.

Exposure of individual histone proteins (H1, H2A, H2B, H3, or H4) and histone octamers (consisting of two molecules each of H2A, H2B, H3, and H4) to hydroxyl radicals, generated by gamma-irradiation, in the presence of O(2) generates protein-bound hydroperoxides in a dose-dependent fashion; this is in accord with previous studies with other proteins. These histone hydroperoxides are stable in the absence of exogenous catalysts (e.g., heat, light, and transition metal ions), but in the presence of these agents decompose rapidly to give a variety of radicals which have been identified by EPR spin trapping. Histone hydroperoxide-derived radicals generated on decomposition of the hydroperoxides with Cu(+) react with both pyrimidine and purine nucleobases. Thus, with uridine the histone hydroperoxide-derived radicals undergo addition across the C(5)-C(6) double bond of the pyrimidine ring to give cross-linked adduct species which have been identified by EPR spectroscopy. HPLC analysis of the products generated on reaction of histone hydroperoxide-derived radicals with 2'-deoxyguanosine, or intact calf thymus DNA, has shown that significant levels of the mutagenic oxidized DNA base 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) are formed, with the yield dependent on the individual histone protein, the presence of hydroperoxide functions, and the concentration of metal ion. These studies demonstrate that initial oxidative damage to individual histone proteins or histone octamers can result in the transfer of oxidative damage to associated DNA via the formation and subsequent decomposition of protein hydroperoxides to reactive radicals, and provide a novel route for the formation of mutagenic lesions in DNA.

Exposure of amino acids, peptides and proteins to radicals, in the presence of oxygen, gives high yields of hydroperoxides. These materials are readily decomposed by transition metal ions to give further radicals. We hypothesized that hydroperoxide formation on nuclear proteins, and subsequent decomposition of these hydroperoxides to radicals, might result in oxidative damage to associated DNA. We demonstrate here that exposure of histone H1 and model compounds to gamma-radiation in the presence of oxygen gives hydroperoxides in a dose-dependent manner. These hydroperoxides decompose to oxygen- and carbon-centred radicals (detected by electron paramagnetic resonance spectroscopy) on exposure to Cu(+) and other transition metal ions. These hydroperoxide-derived radicals react readily with pyrimidine DNA bases and nucleosides to give adduct species (i.e. protein-DNA base cross-links). Product analysis has demonstrated that radicals from histone H1-hydroperoxides, and other protein and amino acid hydroperoxides, can also oxidize both free 2'-deoxyguanosine and intact calf thymus DNA to give the mutagenic oxidized base 7, 8-dihydro-8-oxo-2'-deoxyguanosine (8-hydroxy-2'-deoxyguanosine, 8-oxodG). The yield of 7,8-dihydro-8-oxo-2'-deoxyguanosine is proportional to the initial protein-hydroperoxide concentration, and corresponds (for histone H1-hydroperoxide, 280 microM) to approx. 1. 4% conversion for free 2'-deoxyguanosine (200 microM), and 0.14% for 2'-deoxyguanosine in DNA (70 microgram/ml). Evidence has also been obtained with DNA for reaction at cytosine and thymine, but not adenine; the lack of damage to the latter may result from damage transfer to 2'-deoxyguanosine residues. These studies demonstrate that initial radical-induced damage to nuclear proteins can give rise to subsequent DNA damage; the latter includes both DNA-protein cross-links and formation of oxidized DNA bases.

Oxidative damage to DNA could be involved in the increased risk of cancer associated with exposure to polluted urban air, which contains a number of oxidants. CYP1A2 is induced by and metabolizes polyaromatic hydrocarbons (PAH) and aromatic amines and could modify effects of exposure to ambient air pollution. Similarly, DNA repair may be influenced by occupational and other exposures as well as modify the effect of DNA damaging agents. As part of a large investigation of the genotoxic burden to diesel exposed workers in transport sectors we studied oxidative DNA damage in 57 non-smoking bus drivers from the greater Copenhagen area. The drivers were studied on a workday and on a day off work. Comparisons were made between drivers from the central (n=30) and rural/suburban (n=27) areas of Copenhagen. The rate of oxidative DNA damage was estimated from 24 h urinary excretion of 8-oxo-2'-deoxyguanosine (8-oxodG), a repair product of the highly mutagenic oxidation of guanine in DNA or the cellular pool of GTP. CYP1A2 activity was estimated from the urinary excretion of metabolites of dietary caffeine. The DNA repair was estimated by unscheduled DNA synthesis (UDS) in mononuclear cells isolated on the workday. Repeated measures ANOVA and multifactorial ANCOVA with CYP1A2 activity, age and UDS as covariates were used for statistical evaluation. On the workday, the 8-oxodG excretion was 190+/-108 and 146+/-89 pmol/kg 24 h in the bus drivers from central and the suburban/rural areas Copenhagen, respectively (p<0.05). The 8-oxodG excretion was not significantly different between the workday and the day off. CYP1A2 activity was not affected by driving area but was correlated with the 8-oxodG excretion on the workday (r=0.53; p<0.05). UDS was not significantly affected by driving area or correlated with the 8-oxodG excretion. The increased excretion of 8-oxodG in bus drivers from central Copenhagen as compared with drivers from rural/suburban greater Copenhagen suggest