Department of Cancer Studies and Molecular Medicine, University of Leicester, Leicester, LE2 7LX, UK.
Mutat Res. 2009 Aug;678(2):129-37. doi: 10.1016/j.mrgentox.2009.05.011. Epub 2009 May 27.
Ethylene oxide (EO) is a widely used chemical intermediate also formed endogenously from ethylene metabolism. Despite conflicting epidemiological evidence, EO is classified by the IARC as a human carcinogen. The mutagenicity and carcinogenicity of EO is attributed to direct reaction with DNA and formation of multiple 2-hydroxyethyl (HE) DNA adducts. However, the actual lesions responsible for the reported mutagenicity of EO have not been established. This study used the supF mutation assay to investigate the biological relevance of low levels of EO-induced DNA adducts in human Ad293 cells, with respect to the type and level of each HE adduct present. Initial experiments were conducted using pSP189 plasmid containing up to 290 N7-HEGuanine (N7-HEG) adducts/10(6) nucleotides, which far exceeds that typically detected in human DNA. No other HE-lesions were detectable using our validated LC-MS/MS assay. Replication in cells failed to produce a statistically significant increase in relative mutation frequency, above background rates in the solvent control. Furthermore, the mutation spectrum compiled for EO-treated plasmid (10-2000muM) did not differ significantly from the spontaneous distribution, suggesting EO is not strongly mutagenic in this system. Under refined reaction conditions using higher EO concentrations capable of inducing detectable levels of N1-HEdA, O(6)-HEdG and N3-HEdU along with N7-HEG, there was a significant dose-related increase in relative mutation frequency above background (3.76- and 5.30-fold at 10 and 30mM, respectively). EO treatment appeared associated with an elevated frequency of GC-->CG mutations and the occurrence of substitutions at AT base pairs. Additionally, there was a distinct GC-->TA mutational hotspot in the 10mM EO spectrum. Overall, the results suggest a certain level of promutagenic adducts must be attained before mutations become detectable above background, indicating that N7-HEG is not a promutagenic lesion, and support a role for the minor products of DNA hydroxyethylation in the generation of base substitutions by EO.
环氧乙烷（EO）是一种广泛使用的化学中间体，也可以从乙烯代谢中内源性形成。尽管流行病学证据相互矛盾，但 IARC 将 EO 归类为人类致癌物。EO 的致突变性和致癌性归因于与 DNA 的直接反应和形成多种 2-羟乙基（HE）DNA 加合物。然而，报告的 EO 致突变性的确切病变尚未确定。本研究使用 supF 突变测定法，研究了人类 Ad293 细胞中低水平 EO 诱导的 DNA 加合物与每种 HE 加合物的类型和水平相关的生物学相关性。最初的实验使用包含高达 290 个 N7-HEGuanine（N7-HEG）加合物/10（6）个核苷酸的 pSP189 质粒进行，这远远超过了通常在人类 DNA 中检测到的水平。使用我们经过验证的 LC-MS/MS 测定法，无法检测到其他 HE 损伤。在细胞中复制未能产生相对突变频率相对于溶剂对照的背景率的统计学显着增加。此外，为 EO 处理的质粒（10-2000μM）编制的突变谱与自发分布没有显着差异，表明 EO 在该系统中不是很强的诱变剂。在使用能够诱导可检测水平的 N1-HEdA、O（6）-HEdG 和 N3-HEdU 以及 N7-HEG 的更高 EO 浓度的精细反应条件下，相对突变频率与背景相比呈现出显著的剂量相关性增加（分别在 10 和 30mM 时增加 3.76 倍和 5.30 倍）。EO 处理似乎与 GC-->CG 突变的发生频率增加和 AT 碱基对的取代有关。此外，在 10mM EO 谱中存在明显的 GC-->TA 突变热点。总的来说，结果表明，在背景以上检测到突变之前，必须达到一定水平的促突变加合物，这表明 N7-HEG 不是促突变病变，并支持 DNA 羟乙基化的次要产物在 EO 产生碱基取代中的作用。
