烷基化试剂,以芥子气(SM)为代表,可以直接损伤DNA分子以及导致氧化应激,间接导致DNA损伤。相应地,两种类型的生物标志物,包括烷基化DNA加合物和氧化DNA加合物,通常参与这些药物引起的DNA损伤评估的研究。然而,发生的相关性和差异,持续时间,严重程度,以及这两种类型的生物标志物所反映的DNA分子水平上的烷基化和氧化损伤之间的可追溯性尚未得到系统研究。一种同时测定四种烷基化DNA加合物的方法,即,N7-(2-羟乙基硫基)2'-鸟嘌呤(N7-HETEG),O6-(2-羟乙基硫基)-2'-鸟嘌呤(O6-HETEG),N3-(2-羟乙基硫基)-2'-腺嘌呤(N3-HETEA),和双(2-乙基-N7-鸟嘌呤)硫醚(Bis-G),通过同位素稀释高效液相色谱-串联质谱(ID-HPLC-MS/MS)建立尿液样品中的氧化加合物8-羟基-2'-脱氧鸟苷(8-OH-dG),检测下限为0.02ng/mL(除Bis-G,0.05ng/mL),回收率为79-111%。在SD大鼠皮肤暴露于三个剂量水平(1、3和10mg/kg)的SM后,同时在尿液样本中监测这些加合物的分布。时间效应和剂量效应实验表明,当暴露于SM时,DNA烷基化损伤比氧化损伤更早发生。对于这两种类型的生物标志物,烷基化DNA加合物表现出明显的剂量-效应关系,可作为内暴露剂量和效应生物标志物,虽然8-OH-dG与暴露剂量没有相关性,证明它更适合作为DNA氧化性病变的生物标志物,但不是细胞毒性和内部暴露程度的指标。
Alkylation reagents, represented by sulfur mustard (SM), can damage DNA molecules directly as well as lead to oxidative stress, causing DNA lesions indirectly. Correspondingly, two types of biomarkers including alkylated DNA adducts and oxidative DNA adducts are commonly involved in the research of DNA damage evaluation caused by these agents. However, the correlations and differences of the occurrence, duration, severity, and traceability between alkylation and oxidation lesions on the DNA molecular level reflected by these two types of biomarkers have not been systematically studied. A simultaneous determination method for four alkylated DNA adducts, i.e., N7-(2-hydroxyethylthioethyl)2\'-guanine (N7-HETEG), O6-(2-hydroxyethylthioethyl)-2\'-guanine (O6-HETEG), N3-(2-hydroxyethylthioethyl)-2\'-adenine (N3-HETEA), and bis(2-ethyl-N7-guanine)thioether (Bis-G), and the oxidative adduct 8-hydroxy-2\'-deoxyguanosine (8-OH-dG) in urine samples by isotope-dilution high-performance liquid chromatography-tandem mass spectrometry (ID-HPLC-MS/MS) was built with a lower limit of detection of 0.02 ng/mL (except Bis-G, 0.05 ng/mL) and a recovery of 79-111%. The profile of these adducts was simultaneously monitored in urine samples after SD rats\' dermal exposure to SM in three dose levels (1, 3, and 10 mg/kg). The time-effect and dose-effect experiments revealed that when exposed to SM, DNA alkylation lesions would happen earlier than those of oxidation. For the two types of biomarkers, alkylated DNA adducts showed an obvious dose-effect relationship and could be used as internal exposure dose and effect biomarkers, while 8-OH-dG did not show a correlation with exposure dose, demonstrating that it was more suitable as a biomarker for DNA oxidative lesions but not an indicator for the extent of cytotoxicity and internal exposure.