7,8-hydroxy-2'-deoxyguanosine/2'-deoxiguanosine ratio determined in hydrolysates of brain DNA by ultrachromatrography coupled to tandem mass spectrometry.

7,8-hydroxy-2'-deoxyguanosine (8-OHdG) is an abundant DNA lesion formed by oxidation of the nucleoside 2'-deoxyguanosine (2-dG) and one of the most studied and accepted oxidative stress biomarkers. 8-OHdG has a strong carcinogenic potential, and prolonged oxidative stress heightens pathological conditions and especially cancer risk. Our aim was to develop, validate and apply a reliable method to assess DNA oxidation in genomic cellular DNA of sensible target organs such as brain. A procedure to isolate and digest the DNA of brain tissue properly for further detection of 8-OHdG and 2-dG by Ultra Performance Liquid Chromatography-tandem Mass Spectrometry (UPLC-MS/MS) was optimized. The UPLC-MS/MS was validated following the American Food and Drug Administration (FDA) Guidelines using mice pups' brain samples. To demonstrate the applicability of the UPLC-MS/MS method, 8-OHdG/2-dG ratio was determined in brain tissue of 1day old newborn mice pups (P1) in a model of hypoxia pre-conditioning during fetal-to-neonatal transition. We found that hypoxia at birth (FiO2 0.14) and for 8h thereafter induced lower levels of DNA oxidation in mice pups and rendered even protective against a postnatal asphyxia/reoxygenation insult compared with fetal to neonatal transition in room air. We conclude that the UPLC-MS/MS method developed has proven suitable for the analysis of DNA oxidation biomarker 8-OHdG/2-dG ratio in tissue samples from newborn mice pups. We aim to apply this method in future studies aiming to provide a deeper insight into the mechanisms of oxidation DNA caused during neonatal asphyxia and resuscitation.

Antenatal steroids and antioxidant enzyme activity in preterm infants: influence of gender and timing.

Antenatal steroids have improved the survival of preterm infants; however, the mechanism of action is not fully understood. We aimed to establish an association between antenatal steroids and antioxidant activity and postnatal oxidative stress. In a prospective cohort study, extremely preterm neonates receiving antenatal steroids (CORT) or not (NOCORT) were enrolled. An association between antenatal steroids and activities of antioxidant enzymes and glutathione cycle enzymes in cord blood was found. In addition, reduced oxidative stress (GSH/GSSG ratio, CORT vs. NOCORT, 35.68 + or - 12.20 vs. 28.38 + or - 9.92; p < 0.01) and, decreased oxidation of proteins (ortho-tyrosine/phenylalanine ratio, CORT vs. NOCORT, 8.66 + or - 2.45 vs. 12.55 + or - 4.41; p < 0.01) and DNA (8oxodG/2dG ratio, CORT vs. NOCORT, 6.73 + or - 2.18 vs. 9.53 + or - 3.83; p < 0.01) also was found. Antenatal steroids were associated with reduced oxygen supplementation, mechanical ventilation, and conditions such as bronchopulmonary dysplasia, intra-periventricular hemorrhage, or retinopathy of prematurity. The maximal effectiveness was when steroids were administered 2-4 days before delivery. Female preterm infants had less oxidative stress and increased antioxidant activity and better clinical outcomes than did male infants, independent of receiving or not antenatal steroids. Antenatal steroids are accompanied by a reduction in postnatal oxidative-stress-derived conditions and increased antioxidant enzyme activity. Both these effects seem to be influenced by specific timing and female gender.