Dooley G P, Reardon K F, Prenni J E, Tjalkens R B, Legare M E, Foradori C D, Tessari J E, Hanneman W H
Department of Environmental and Radiological Health Sciences, Proteomics and Metabolomics Facility, Anatomy and Neurobiology Section, Colorado State University, Fort Collins, Colorado 80523, USA.
Chem Res Toxicol. 2008 Apr;21(4):844-51. doi: 10.1021/tx700386f. Epub 2008 Mar 28.
Atrazine (ATRA) is the most commonly applied herbicide in the United States and is frequently detected in drinking water at significant levels. After oral exposure, ATRA metabolism yields diaminochlorotriazine (DACT), an electrophilic molecule that has been shown to form covalent protein adducts. This research was designed to identify ATRA-induced protein adducts formed in the pituitary gland of ATRA-exposed rats and in DACT-exposed LbetaT2 rat pituitary cells. Immunohistochemistry showed diffuse cytoplasmic and nuclear staining in both pituitary sections and LbetaT2 cells indicating the formation of DACT protein adducts. Protein targets from both rat pituitaries and LbetaT2 cell culture were identified following two-dimensional electrophoresis (2DE), immunodetection, and matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. Western blots from both exposed rats and LbetaT2 cells revealed over 30 DACT-modified spots that were not present in control animals. Protein spots were matched to concurrently run 2DE gels stained with Sypro Ruby, excised, and in-gel-digested with trypsin. Mass spectrometry analysis of digest peptides resulted in the identification of 19 spots and 8 unique proteins in the rats and 21 spots and 19 unique proteins in LbetaT2 cells. The identified proteins present in both sample types included proteasome activator complex subunit 1, ubiquitin carboxyl-terminal hydrolase isozyme L1, tropomyosin, ERp57, and RNA-binding proteins. Each of these proteins contains active-site or solvent-exposed cysteine residues, making them viable targets for covalent modification by DACT.
阿特拉津（ATRA）是美国使用最广泛的除草剂，在饮用水中经常被检测到且含量显著。经口暴露后，ATRA代谢产生二氨基氯三嗪（DACT），这是一种亲电分子，已被证明可形成共价蛋白加合物。本研究旨在鉴定在暴露于ATRA的大鼠垂体以及暴露于DACT的LbetaT2大鼠垂体细胞中形成的ATRA诱导的蛋白加合物。免疫组织化学显示垂体切片和LbetaT2细胞中均有弥漫性细胞质和细胞核染色，表明形成了DACT蛋白加合物。通过二维电泳（2DE）、免疫检测和基质辅助激光解吸电离飞行时间质谱分析，确定了大鼠垂体和LbetaT2细胞培养物中的蛋白靶点。来自暴露大鼠和LbetaT2细胞的蛋白质印迹显示，有超过30个DACT修饰的斑点在对照动物中不存在。将蛋白斑点与同时运行的用Sypro Ruby染色的2DE凝胶进行匹配，切除后用胰蛋白酶进行胶内消化。对消化后的肽段进行质谱分析，在大鼠中鉴定出19个斑点和8种独特蛋白质，在LbetaT2细胞中鉴定出21个斑点和19种独特蛋白质。两种样品类型中都存在的已鉴定蛋白质包括蛋白酶体激活复合物亚基1、泛素羧基末端水解酶同工酶L1、原肌球蛋白、内质网蛋白57和RNA结合蛋白。这些蛋白质中的每一种都含有活性位点或溶剂暴露的半胱氨酸残基，使其成为DACT共价修饰的可行靶点。
