Lambert G R, Padgett W T, George M H, Kitchin K T, Nesnow S
Biochemistry and Pathobiology Branch (MD-68), National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, 27711, USA.
Anal Biochem. 1999 Mar 15;268(2):289-96. doi: 10.1006/abio.1998.3060.
This study examined the potential use of hemoglobin (Hb)- and serum-protein adducts of alachlor as potential biomarkers of alachlor exposure, a genotoxic and carcinogenic herbicide. The method developed was based on the observation that cleavage of S-cysteinyl alachlor-protein adducts by methanesulfonic acid gave the rearrangement product 3-(2',6'-diethylphenyl)-1, 3-thiazolidine-4-one (TZO). The structure of TZO was confirmed by mass spectroscopy, NMR spectroscopy, and independent synthesis. In the assay, treatment of alachlor-cysteinyl protein adducts by methanesulfonic acid was followed by extraction and analysis. TZO was detected and quantitated by electron-impact GC/MS in the single ion-monitoring mode. [ring-13C6]Alachlor-N-acetylcysteine was added as an internal standard prior to treatment and was converted to [ring-13C6]TZO, allowing response factors to be used to quantitate TZO concentrations. Incubations of alachlor (0-1000 microM) with human albumin and bovine serum albumin (BSA) resulted in linear adduct formation with both proteins. Maximal adduction levels of 613-1130 pmol alachlor-albumin adducts/mg protein were observed, with BSA binding close to twice that of human albumin. A linear concentration response of alachlor-Hb adducts was observed when whole blood from female CD rats was incubated with alachlor in vitro at concentrations up to 300 microM. Maximal binding was 1860 pmol alachlor-Hb adducts/mg globin. Male CD rats treated with alachlor at 150 mg/kg body wt/day ip for 0, 1, 2, and 3 days were sacrificed 4 days after final dosing. A maximal binding of 2250 pmol alachlor-Hb adducts/mg globin was observed. This assay provides a new approach for biomonitoring alachlor levels in experimental animals and has the potential for use in humans.
本研究检测了甲草胺的血红蛋白（Hb）加合物和血清蛋白加合物作为甲草胺暴露潜在生物标志物的可能性，甲草胺是一种具有遗传毒性和致癌性的除草剂。所开发的方法基于以下观察结果：甲磺酸裂解S-半胱氨酰甲草胺-蛋白质加合物可得到重排产物3-(2',6'-二乙基苯基)-1,3-噻唑烷-4-酮（TZO）。通过质谱、核磁共振光谱和独立合成确定了TZO的结构。在该测定中，用甲磺酸处理甲草胺-半胱氨酰蛋白质加合物后进行萃取和分析。通过电子轰击气相色谱/质谱在单离子监测模式下检测和定量TZO。在处理前加入[环-13C6]甲草胺-N-乙酰半胱氨酸作为内标，并将其转化为[环-13C6]TZO，从而可使用响应因子来定量TZO浓度。甲草胺（0 - 1000 microM）与人血清白蛋白和牛血清白蛋白（BSA）孵育会导致两种蛋白质形成线性加合物。观察到甲草胺-白蛋白加合物的最大加合水平为613 - 1130 pmol甲草胺-白蛋白加合物/mg蛋白质，BSA的结合量接近人血清白蛋白的两倍。当雌性CD大鼠的全血在体外与浓度高达300 microM的甲草胺孵育时，观察到甲草胺-Hb加合物呈线性浓度响应。最大结合量为1860 pmol甲草胺-Hb加合物/mg球蛋白。以150 mg/kg体重/天的剂量腹腔注射甲草胺，分别处理雄性CD大鼠0、1、2和3天，在末次给药后4天处死大鼠。观察到最大结合量为2250 pmol甲草胺-Hb加合物/mg球蛋白。该测定为生物监测实验动物中甲草胺水平提供了一种新方法，并且有可能用于人类。
