Brink Andreas, Schulz Berta, Stopper Helga, Lutz Werner K
Department of Toxicology, University of Würzburg, Versbacher Str. 9, 97078 Würzburg, Germany.
Mutat Res. 2007 Dec 1;625(1-2):94-101. doi: 10.1016/j.mrfmmm.2007.05.007. Epub 2007 May 21.
The biological significance of DNA adducts is under continuous discussion because analytical developments allow determination of adducts at ever lower levels. Central questions refer to the biological consequences of adducts and to the relationship between background DNA damage and exposure-related increments. These questions were addressed by measuring the two DNA adducts 7-methylguanine (7-mG) and O(6)-methyl-2'-deoxyguanosine (O(6)-mdGuo) by LC-MS/MS in parallel to two biological endpoints of genotoxicity (comet assay and in vitro micronucleus test), using large batches of L5178Y mouse lymphoma cells treated with methyl methanesulfonate (MMS). The background level of 7-mG was 1440 adducts per 10(9) nucleotides while O(6)-mdGuo was almost 50-fold lower (32 adducts per 10(9) nucleotides). In the comet assay and the micronucleus test, background was in the usual range seen with smaller batches of cells (2.1% Tail DNA and 12 micronuclei-containing cells per 1000 binucleated cells, respectively). For the comparison of the four endpoints for dose-related increments above background in the low-response region we assumed linearity at low dose and used the concept of the "doubling dose", i.e., we estimated the concentration of MMS necessary to double the background measures. Doubling doses of 4.3 and 8.7microM MMS were deduced for 7-mG and O(6)-mdGuo, respectively. For doubling the background measures in the comet assay and the micronucleus test, 5 to 15-fold higher concentrations of MMS were necessary (45 and 66microM, respectively). This means that the contribution of an increase in DNA methylation to biological endpoints of genotoxicity is overestimated. For xenobiotics that generate adducts without background, the difference is even more pronounced because the dose-response curve starts at zero and the limit of detection of an increase is not affected by background variation. Consequences for the question of thresholds in dose-response relationships and for the setting of tolerable exposure levels are discussed.
DNA加合物的生物学意义一直处于持续讨论之中，因为分析技术的发展使得能够测定出含量越来越低的加合物。核心问题涉及加合物的生物学后果以及背景DNA损伤与暴露相关增量之间的关系。通过液相色谱-串联质谱法（LC-MS/MS）同时测定两种DNA加合物7-甲基鸟嘌呤（7-mG）和O(6)-甲基-2'-脱氧鸟苷（O(6)-mdGuo），并结合两种遗传毒性生物学终点（彗星试验和体外微核试验），使用大量经甲磺酸甲酯（MMS）处理的L5178Y小鼠淋巴瘤细胞来解决这些问题。7-mG的背景水平为每10⁹个核苷酸1440个加合物，而O(6)-mdGuo几乎低50倍（每10⁹个核苷酸32个加合物）。在彗星试验和微核试验中，背景处于用较少量细胞观察到的通常范围内（分别为2.1%的拖尾DNA和每1000个双核细胞中有12个含微核的细胞）。为了比较低反应区域中高于背景的剂量相关增量的四个终点，我们假设低剂量时呈线性关系，并使用“加倍剂量”的概念，即我们估计使背景测量值加倍所需的MMS浓度。对于7-mG和O(6)-mdGuo，分别推导出4.3和8.7μM MMS的加倍剂量。为了使彗星试验和微核试验中的背景测量值加倍，需要高5至15倍的MMS浓度（分别为45和66μM）。这意味着DNA甲基化增加对遗传毒性生物学终点的贡献被高估了。对于产生无背景加合物的外源化学物，差异甚至更明显，因为剂量-反应曲线从零开始，增加的检测限不受背景变化的影响。讨论了剂量-反应关系中阈值问题以及可耐受暴露水平设定的后果。
