Hofer T, Möller L
Department of Biosciences, Unit for Analytical Toxicology, Karolinska Institute, SE-141 57 Huddinge, Stockholm, Sweden.
Chem Res Toxicol. 1998 Aug;11(8):882-7. doi: 10.1021/tx980041x.
The promutagenic base 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in DNA is known to be formed from oxygen radical attack on 2'-deoxyguanosine (dG) as a result of oxidative stress. Formation of 8-OH-dG from dG during workup is strongly dependent on temperature and transition metals and is mediated by oxygen radicals. The 8-OH-dG formation at temperatures between 0 and 140 degrees C for 1.5 h in an "ultrapure" solution followed a third-order equation. Fe2+ in the nM range mediated the formation of 8-OH-dG from dG without addition of H2O2. Fe3+, Cu+, and Cu2+ were shown to have weaker oxidative effects in comparison to Fe2+. The pH (5.0-9.0) had a very limited effect on 8-OH-dG formation. Acid phosphatase, which contains iron at its active site, caused the formation of 8-OH-dG, whereas alkaline phosphatase did not. Phenol was not found to be oxidative. Fe2+-catalyzed formation of 8-OH-dG was completely blocked by the nitroxide 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), whereas DMSO, mannitol, and DMPO had a significantly weaker protecting effect. Catalase cleaved the dG molecule and was not suitable for use. A simple, fast, and inexpensive method for 8-OH-dG workup and analysis was developed, and the background level seen in liver from 13-week-old male Sprague-Dawley rat was 0.23 +/- 0.020 8-OH-dG/10(5) dG, which is up to 200 times lower than reported values from some other methods and up to 26 times lower when compared to other reports using HPLC-EC methods. In summary, the TEMPO method reduces oxidation of dG to 8-OH-dG during workup by (1) using chemicals low in transition metals, (2) using a cold workup procedure, (3) limiting the incubation time, and (4) using the nitroxide TEMPO in all steps.
已知DNA中的前诱变碱基8-羟基-2'-脱氧鸟苷（8-OH-dG）是由于氧化应激导致氧自由基攻击2'-脱氧鸟苷（dG）而形成的。在处理过程中由dG形成8-OH-dG强烈依赖于温度和过渡金属，并由氧自由基介导。在“超纯”溶液中，于0至140摄氏度的温度下1.5小时内8-OH-dG的形成遵循三阶方程。在不添加过氧化氢的情况下，纳摩尔范围内的Fe2+介导了由dG形成8-OH-dG。与Fe2+相比，Fe3+、Cu+和Cu2+显示出较弱的氧化作用。pH值（5.0 - 9.0）对8-OH-dG的形成影响非常有限。在其活性位点含有铁的酸性磷酸酶会导致8-OH-dG的形成，而碱性磷酸酶则不会。未发现苯酚具有氧化性。Fe2+催化形成8-OH-dG被氮氧化物2,2,6,6-四甲基哌啶-N-氧基（TEMPO）完全阻断，而二甲基亚砜、甘露醇和DMPO的保护作用明显较弱。过氧化氢酶会裂解dG分子，不适合使用。开发了一种简单、快速且廉价的8-OH-dG处理和分析方法，13周龄雄性Sprague-Dawley大鼠肝脏中的背景水平为0.23±0.020 8-OH-dG/10(5) dG，这比其他一些方法报告的值低达200倍，与使用高效液相色谱 - 电化学方法的其他报告相比低达26倍。总之，TEMPO方法通过（1）使用过渡金属含量低的化学物质，（2）采用冷处理程序，（3）限制孵育时间，以及（4）在所有步骤中使用氮氧化物TEMPO，减少了处理过程中dG氧化为8-OH-dG的情况。
