Figure 3.

Detection of DMPO adducts on DNA oxidized with Cu²⁺ and H₂O₂ with and without DMPO and with and without DMSO- and glyoxal-denaturation of DNA. DNA was oxidized with 50 µM Cu²⁺ and varying H₂O₂ concentrations in the presence (▼,■) and absence (▲,●) of DMPO. DNA (0.5 µg/well) was bound to an ELISA plate overnight in a Reacti-Bind DNA coating solution (Pierce). The DNA was denatured by incubating in 66% (vol/vol) DMSO, 1 M glyoxal and 1.5 mM sodium phosphate (▼,▲) at 37 °C for 1 h, and control samples were incubated in PBS (■,●). Subsequently, the denaturant was washed away and the DMPO adducts were detected with a mouse monoclonal antibody. Results are means ± SD of triplicate measurements and are representative of three independent experiments. Two-way ANOVA with Bonferroni post-tests showed there was no significant difference between 0 DMPO samples with or without glyoxal treatment. In the absence of glyoxal treatment, there was a significant difference between 100 mM DMPO samples and 0 mM DMPO samples at 100 and 200 uM H₂O₂ (p<0.05). There was a significant difference between 100 mM DMPO samples with glyoxal treatment and 0 DMPO samples at 50, 100 and 200 uM H₂O₂ (p<0.05).