Pegg A E, Scicchitano D, Dolan M E
Cancer Res. 1984 Sep;44(9):3806-11.
The rates of loss of O6-methylguanine and O6-ethylguanine from rat liver DNA were determined over a time period of 15 min to 4 hr after various doses (5 micrograms/kg to 2 mg/kg) of dimethylnitrosamine and diethylnitrosamine which produced total amounts of these adducts in the range of 300 to 16,000 molecules/cell. This amount is considerably less than the content of O6-alkylguanine-DNA alkyltransferase protein (approximately 60,000 molecules/hepatocyte), and during the time period studied, the adducts were found to be lost with pseudo-first order kinetics. The half-life for O6-methylguanine was 47 min. O6-Ethylguanine was removed 3.6 times more slowly with a half-life of 172 min. The ability of partially purified rat liver O6-alkylguanine-DNA alkyltransferase to remove O6-methylguanine and O6-ethylguanine from [3H]alkyl-labeled DNA substrates in vitro was measured, and it was found that O6-methylguanine was removed 3.4 times more rapidly than was O6-ethylguanine. These results are consistent with the hypothesis that most, if not all, of the repair of these adducts which occurs within the first 4 hr after treatment is due to the alkyltransferase protein. Diethylnitrosamine, which is slightly more potent as a carcinogen to rat liver, produced a total amount of O6-ethylguanine of 3.7 mumol/mol guanine/mg compared to O6-methylguanine (28 mumol/mol guanine/mg) given by dimethylnitrosamine. The slower rate of loss of the ethyl adduct is not sufficient to account for this difference, and the results, therefore, support the concept that other DNA adducts (possibly O-alkylpyrimidines) contribute to the initiation of tumors by diethylnitrosamine. Preliminary evidence that the rat liver alkyltransferase can also remove hydroxyethyl groups from DNA at a rate slower than removal of ethyl groups was also obtained. Bacterial O6-alkylguanine-DNA alkyltransferase was shown to remove methyl, ethyl, and hydroxyethyl groups from the O6 position of guanine in DNA using fluorescence detection to quantitate these adducts. The bacterial protein removed methyl groups very rapidly but was much slower than the rat liver protein on the larger adducts. These results suggest that the relative rates of repair of different alkyl groups may be species specific and must be determined experimentally in the cell of interest before conclusions concerning biological effects can be drawn.
在给予不同剂量（5微克/千克至2毫克/千克）的二甲基亚硝胺和二乙基亚硝胺后15分钟至4小时的时间段内，测定了大鼠肝脏DNA中O6 - 甲基鸟嘌呤和O6 - 乙基鸟嘌呤的损失率。这些剂量产生的这些加合物总量在300至16,000个分子/细胞范围内。该量远低于O6 - 烷基鸟嘌呤 - DNA烷基转移酶蛋白的含量（约60,000个分子/肝细胞），并且在所研究的时间段内，发现加合物以准一级动力学损失。O6 - 甲基鸟嘌呤的半衰期为47分钟。O6 - 乙基鸟嘌呤的去除速度慢3.6倍，半衰期为172分钟。测定了部分纯化的大鼠肝脏O6 - 烷基鸟嘌呤 - DNA烷基转移酶在体外从[3H]烷基标记的DNA底物中去除O6 - 甲基鸟嘌呤和O6 - 乙基鸟嘌呤的能力，发现O6 - 甲基鸟嘌呤的去除速度比O6 - 乙基鸟嘌呤快3.4倍。这些结果与以下假设一致：即在处理后的前4小时内发生的这些加合物的大部分（如果不是全部）修复是由于烷基转移酶蛋白。作为大鼠肝脏致癌物稍强的二乙基亚硝胺，产生的O6 - 乙基鸟嘌呤总量为3.7微摩尔/摩尔鸟嘌呤/毫克，而二甲基亚硝胺产生的O6 - 甲基鸟嘌呤为（28微摩尔/摩尔鸟嘌呤/毫克）。乙基加合物较慢的损失速度不足以解释这种差异，因此，结果支持这样的概念，即其他DNA加合物（可能是O - 烷基嘧啶）促成了二乙基亚硝胺引发的肿瘤。还获得了初步证据，表明大鼠肝脏烷基转移酶也能以比去除乙基慢的速度从DNA中去除羟乙基。使用荧光检测定量这些加合物，结果表明细菌O6 - 烷基鸟嘌呤 - DNA烷基转移酶能从DNA中鸟嘌呤的O6位置去除甲基、乙基和羟乙基。细菌蛋白非常快速地去除甲基，但在处理较大的加合物时比大鼠肝脏蛋白慢得多。这些结果表明，不同烷基的相对修复速率可能具有物种特异性，在得出有关生物学效应的结论之前，必须在感兴趣的细胞中通过实验确定。
