DNA repair and the origins of urinary oxidized 2'-deoxyribonucleosides.

Monitoring oxidative stress in vivo is made easier by the ability to use samples obtained non-invasively, such as urine. The analysis of DNA oxidation, by measurement of oxidized 2'-deoxyribonucleosides in urine, particularly 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), has been reported extensively in the literature in many situations relating to various pathologies, populations and environmental exposures. Understanding the origins of urinary 8-oxodG, other than it simply being a marker of DNA oxidation or its synthetic precursors, is important to being able to effectively interpret differences in baseline urinary 8-oxodG levels between subject groups and changes in excretion. Diet and cell turnover play negligible roles in contributing to urinary 8-oxodG levels, leaving DNA repair as a primary source of this lesion. However, which repair processes contribute, and to what extent, to urinary 8-oxodG is still open to question. The most rational source would be the activity of selected members of the Nudix hydrolase family of enzymes, sanitizing the deoxyribonucleotide pool via the degradation of 8-oxo-7,8-dihydro-2'-deoxyguanosine-5'-triphosphate and 8-oxo-7,8-dihydro-2'-deoxyguanosine-5'-diphosphate, yielding mononucleotide products that can then be dephosphorylated to 8-oxodG and excreted. However, nucleotide excision repair (NER), transcription-coupled repair, nucleotide incision repair (NIR), mismatch repair and various exonuclease activities, such as proofreading function associated with DNA polymerases, can all feasibly generate initial products that could yield 8-oxodG after further metabolism. A recent study implying that a significant proportion of genomic 8-oxodG exists in the context of tandem lesions, refractory to repair by glycosylases, suggests the roles of NER and/or NIR remain to be further examined and defined as a source of 8-oxodG. 8-OxodG has been the primary focus of investigation, but other oxidized 2'-deoxyribonucleosides have been detected in urine, 2'-deoxythymidine glycol and 5-hydroxymethyl-2'-deoxyuridine; the origins of these compounds in urine, however, are presently even more speculative than for 8-oxodG.

Radiation metabolomics. 2. Dose- and time-dependent urinary excretion of deaminated purines and pyrimidines after sublethal gamma-radiation exposure in mice.

Gamma-radiation exposure of humans is a major public health concern as the threat of terrorism and potential hostile use of radiological devices increases worldwide. We report here the effects of sublethal gamma-radiation exposure on the mouse urinary metabolome determined using ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry-based metabolomics. Five urinary biomarkers of sublethal radiation exposure that were statistically significantly elevated during the first 24 h after exposure to doses ranging from 1 to 3 Gy were unequivocally identified by tandem mass spectrometry. These are deaminated purine and pyrimidine derivatives, namely, thymidine, 2'-deoxyuridine, 2'-deoxyxanthosine, xanthine and xanthosine. Furthermore, the aminopyrimidine 2'-deoxycytidine appeared to display reduced urinary excretion at 2 and 3 Gy. The elevated biomarkers displayed a time-dependent excretion, peaking in urine at 8-12 h but returning to baseline by 36 h after exposure. It is proposed that 2'-deoxyuridine and 2'-deoxyxanthosine arise as a result of gamma irradiation by nitrosative deamination of 2'-deoxycytidine and 2'-deoxyguanosine, respectively, and that this further leads to increased synthesis of thymidine, xanthine and xanthosine. The urinary excretion of deaminated purines and pyrimidines, at the expense of aminopurines and aminopyrimidines, appears to form the core of the urinary radiation metabolomic signature of mice exposed to sublethal doses of ionizing radiation.

Increased urinary level of oxidized nucleosides in patients with mild-to-moderate Alzheimer's disease.

OBJECTIVE: Oxidative stress may play an important role in the pathogenesis of Alzheimer's disease (AD). DESIGN AND METHODS: To investigate the possible role of oxidative DNA damage in the pathogenesis of AD, we measured the metabolite concentrations of oxidized nucleosides (pseudouridine, 1-methyladenosine, 5-methylcytidine, 5-methyl-2'-deoxycytidine, 3-methyluridine, N(2), N(2)-dimethylguanosine, 8-hydroxy-2'-deoxyguanosine, 5-deoxyadenosine and 2-deoxyguanosine) in urine between AD (n=36) and control subjects (n=34) using liquid chromatography-mass spectrometry (LC-MS) without urine preparation. RESULTS: In AD, the 3-methyluridine, 1-methyladenosine, 8-hydroxy-2'-deoxyguanosine (p<0.05, respectively), 2-deoxyguanosine (p<0.01) and pseudouridine, N(2), N(2)-dimethylguanosine (p<0.001, respectively) were significantly increased when compared with the control subjects. CONCLUSION: The results indicate that oxidized urinary nucleosides may be useful as biomarkers for AD in early stages.

Comparison of urinary modified nucleosides and bases in rats with hepatomas and nephroblastomas.

Hepatomas were induced in rats with aflatoxin B1, and nephroblastomas with dimethylnitrosamine. Microscopic examination of livers of aflatoxin-treated rats revealed multinodular hepatocyte hyperplasia at 8 months, and by 13 months all rats had hepatomas. Nephroblastomas were observed by 4 months and by 8 months all rats had developed them. The urinary excretion of several modified nucleosides and bases by normal rats is dependent on body weight and reflects, to a certain extent, their concentrations in tissue tRNA. Increased levels of several modified nucleosides and bases were found in all rats that had cancer. Rats with hepatomas excreted essentially the same modified nucleosides and bases as did those with nephroblastomas; the quantitative patterns of excretion were different, however, suggesting that the urinary modified nucleosides and bases may be used to differentiate between neoplasms. Although the increase in urinary modified nucleosides and bases by tumor-bearing animals results primarily from more rapid turnover of neoplastic tRNAs, the data indicate that increased turnover of mRNA and possibly rRNA may occur in neoplastic tissue. Preliminary data suggest that increases in urinary modified nucleosides and bases may occur during a precancerous stage. The urinary pattern of modified nucleosides and bases by rats with hepatomas is altered if another primary tumor is present. The results obtained from these studies support the use of modified nucleosides and bases in urine as biochemical markers of cancer.

High-performance liquid chromatographic determination of purine and pyrimidine bases, ribonucleosides, deoxyribonucleosides and cyclic ribonucleotides in biological fluids.

A method is presented for the separation and quantitative determination of compounds normally related to purine and pyrimidine metabolism in biological material. The retention behaviour of nucleobases, ribonucleosides, deoxyribonucleosides and cyclic ribonucleotides has been systematically investigated by reversed-phase high-performance liquid chromatography using a non-linear gradient. Ultimately a separation of the purine and pyrimidine compounds was achieved in a 35-min run with an average detection limit of 5-10 pmol per injection. Recoveries of standards added to urine, plasma or serum were 96 +/- 5%.

[Occurrence of purine deoxyribosides in urine (author's transl)]. / Uber das Vorkommen von Purindesoxyribosiden im Harn.

It was shown that purine deoxyribosides are normal constituents of human urine. Healthy probands excrete about 25 microgram (41% of the total urinary deoxyribosides) per 24 h. Purine deoxyribosides were determined as the acid-labile fraction of the total deoxyribosides by the microbiological assay with Lactobacillus acidophilus R 26.

[Simple method of determining total deoxyribonucleosides in human urine]. / Prostoi metod opredeleniia summarnykh dezoksiribonukleozidov v moche cheloveka.

A method for quantitative estimation of total deoxyribonucleosides in human urine is based on the preliminary purification of urine usina ion exchange resin Dowex 1X 8 in OH- form, subsequent treatment of urine with acetone-ethynol mixture and the quantitative estimation of deoxyribonucleosides content by a microbiologic method. Urine of healthy persons contained 95.4+/-12.7 microng of deoxyribonucleosides.

Increased urinary excretion of labelled deoxyribonucleosides in rats with stable pre-labelled DNA treated with methyl methanesulphonate or nitrogen mustard.

After the DNA of newborn female rats had been labelled by repeated injections of [14C]orotate (totalling 36 mu Ci) during the first 3 weeks of life, approximately 1,000,000 dpm were found in the DNA of the liver, lungs, kidneys, gut, brain, heart and spleen of 8-week-old rats. Methyl methanesulphonate (MMS) (80 mg/kg) and di-(2-chloroethyl)methylamine (HN2) (5 mg/kg) injection increased the amount of 14C-labelled DNA pyrimidine nucleosides excreted in the urine to 5000 dpm from 350 dpm before injection. The effect on RNA products was much less marked.

Urine excretion of nucleosides in Hodgkin's disease treated by combined chemotherapy.

The authors have followed the excretion of pyrimidine doexyribonucleosides in the urine of patients with generalized stages of Hodgkin's disease. They proved an increased excretion of deoxyuridine and thymidine in these patients compared to healthy persons. In the course of 14-days of combined chemotherapy a statistically significant increase in the excretion of DNA catabolites followed was found.