Background level of 8-oxo-2'-deoxyguanosine in lymphocyte DNA does not correlate with the concentration of antioxidant vitamins in blood plasma.

Antioxidant vitamins, being effective free radical scavengers, can protect cellular DNA from oxidative damage. Therefore, in the present study we report on the relationship between basal level of 8-oxo-2'-deoxyguanosine in human lymphocyte DNA and the concentration of antioxidant vitamins (A, C and E). The average level of 8-oxo-2'-deoxyguanosine in lymphocytes of the studied group (15 males and 20 females) was 9.57 per 10(6) dG molecules. The endogenous level of ascorbic acid (vitamin C) in the plasma was, on average, 56.78 microM, while the mean concentrations of retinol (vitamin A) and alpha-tocopherol (vitamin E) were 1.24 uM and 25.74,uM, respectively. No correlations were found between individual 8-oxo-2 micro-deoxyguanosine levels in lymphocyte DNA and endogenous concentration of the vitamins.

Further evidence that oxidative stress may be a risk factor responsible for the development of atherosclerosis.

There are numerous data suggesting that oxidative stress may be involved in the development of atherosclerosis. Therefore, in the present study we measured the amount of 8-hydroxy-2'-deoxyguanosine (8-OH-dG), one of the typical biomarkers of oxidative stress, in DNA isolated from lymphocytes of the patients and in the control group. Levels of antioxidant vitamins (A, C, and E) and intracellular labile iron pool (LIP), which can influence oxidative stress, were also determined. Blood samples were obtained from a control group of 55 healthy persons and from 43 atherosclerotic patients. 8-OH-dG and the vitamin levels were measured by high-performance liquid chromatography. Labile iron pool in lymphocytes was analyzed by fluorescent assay. The levels of 8-OH-dG and LIP were significantly higher and vitamin C concentration was significantly lower in the patient group than in the control group. The rest of the analyzed parameters do not significantly differ between the groups. A lower concentration of vitamin C and higher levels of labile iron pool in a group of atherosclerotic patients when compared with the control group may lead to oxidative stress, which is manifested by a higher level of 8-OH-dG in blood lymphocytes. All these factors may create an environment that promotes the development of atherosclerosis.

8-Oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine levels in human urine do not depend on diet.

In the present study, we used the method involving HPLC pre-purification followed by gas chromatography with isotope dilution mass spectrometric detection for the determination of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 8-oxo-7,8-dihydroguanine (8-oxoGua) in human urine. The mean levels of 8-oxoGua and 8-oxodGuo in the urine samples of the subjects on unrestricted diet were respectively 1.87 nmol/kg 24 h (+/-0.90) and 0.83 nmol/kg 24 h (+/-0.49), and in the case of the groups studied, they did not depend on the applied diet. The sum of the amounts of both compounds in urine can give information about the formation rate of 8-oxoGua in cellular DNA. It is also likely that the levels of modified nucleo-base/side in urine sample are reflective of the involvement of different repair pathways responsible for the removal of 8-oxodGuo from DNA, namely base excision repair (BER) and nucleotide excision repair (NER).

Endogenous oxidative DNA base modifications analysed with repair enzymes and GC/MS technique.

GC/MS technique was used to identify endogenous levels of oxidatively modified DNA bases. To avoid possible artefact formation we used Fpg and Endo III endonucleases instead of acid hydrolysis to liberate the base products from unmodified DNA samples. Several different DNA preparations were used: (i) commercial calf thymus DNA, (ii) DNA isolated from rat liver, (iii) DNA isolated from human lymphocytes and (iv) nuclei isolated from rat liver. In all DNA samples used in our assays the most efficiently removed bases by Fpg protein are FapyG and FapyA although 8-oxoG was also detected in all preparations. The amount of 8-oxoG in human lymphocytes and in rat liver DNA was 3 and 2 per 10(7)bases, respectively. It is reasonable to postulate that the presented method is one of the techniques which should be used to reveal the enigma of endogenous, oxidative DNA damage.