Determination of 8-hydroxydeoxyguanosine in human cells under oxygen-free conditions

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Abstract
To establish an accurate 8-hydroxydeoxyguanosine (8OHdG) determination system, we examined two potential factors causing experimental error in 8OHdG determination. First, we examined the efficiency of the enzymatic digestion of DNA, that could cause misestimation of 8OHdG. Second, since we considered that the oxygen molecules in atmosphere and in reagents were the main factor contributing to the experimental errors, we carried out the 8OHdG determination under oxygen-free conditions and compared the 8OHdG value with that determined by the methods under ambient atmosphere. The calf thymus DNA was sufficiently digested in the condition we used and the yields of dG were constant, even when the DNA was damaged with H2O2 (80mM) and UV irradiation. By carrying out the DNA extraction manually, instead of using the DNA extractor, we could reduce the additional 8OHdG formation during sample processing. No trend was found in the difference betweenthe 8OHdG values determined under oxygen-free conditions and under ambient atmosphere. However, when the 8OHdG values werecompared in samples with asbestos, the value determined under oxygen-free conditions was significantly lower than that determined under ambient atmosphere. These findings suggest that the removal of oxygen molecules was effective in reducing accidental ROS generation by impurities in the sample, which could cause the additional 8OHdG formation, and that the oxygen-free system made the determination of 8OHdG reproducible and more accurate than before. When the oxygen-free system was applied to human leukocytes, the system showed good reproducibility (r = 0.535, P < 0.001), even though the 8OHdG level was low. With the system, we could detect a significant difference between 8OHdG in polymorphonuclear leukocytes(0.241± 0.129) and mononuclear leukocytes (0.188 ± 0.126, P < 0.01).

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