Meier Brent W, Gomez Jose D, Zhou Angela, Thompson John A
Department of Pharmaceutical Sciences, Box C238, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Chem Res Toxicol. 2005 Oct;18(10):1575-85. doi: 10.1021/tx050108y.
Two quinone methide (QM) metabolites of the phenolic antioxidant butylated hydroxytoluene (BHT), 2,6-di-tert-butyl-4-methylenecyclohexa-2,5-dienone (BHT-QM) and the tert-butyl-hydroxylated derivative (BHTOH-QM), are believed to be responsible for promoting lung tumor formation in mice treated with BHT. QMs are strongly electrophilic and undergo Michael type additions with nucleophiles at the exocyclic methylene to form benzylic thioether adducts. Our goal was to identify intracellular protein targets of these QMs in order to gain insight into their effects on tumorigenesis. Cell line E10 of mouse lung epithelial origin and its spontaneous transformant, the tumorigenic E9 cell line, were treated with BHT-QM or BHTOH-QM, and cellular proteins were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Adducted proteins were detected on western blots with polyclonal antibodies developed to a conjugate of BHTOH-QM that recognized adducts of both QMs bound to thiol groups of Cys and side chain amino groups of Lys and His residues. Tryptic digests of immunoreactive proteins were analyzed by HPLC mass spectrometry (LC/MS) and identified by searching protein databases using MS/MS data. In a few cases, adducted peptides in these digests were detected by matrix-assisted laser desorption/ionization time-of-flight MS. A total of 37 immunoreactive proteins were identified including proteins involved in carbohydrate metabolism, nucleic acid synthesis, and RNA and protein processing, in addition to several cytoskeletal and stress-related proteins. About half of the protein adducts were found in both cell lines. Adducts detected only in transformed E9 cells include glutathione S-transferase P1, peroxiredoxin 2, nucleoside diphosphate kinase, and vinculin, whereas several alkylated cytoskeletal proteins such as tubulins, vimentin, calvasculin, and calcyclin were detected exclusively in E10 cells. Several of the proteins modified by BHT-derived QMs have been implicated in various aspects of tumorigenesis and are excellent candidates for further study into the consequences of alkylation on cellular transformation.
酚类抗氧化剂丁基化羟基甲苯（BHT）的两种醌甲基化物（QM）代谢产物，即2,6 - 二叔丁基 - 4 - 亚甲基环己 - 2,5 - 二烯酮（BHT - QM）和叔丁基羟基化衍生物（BHTOH - QM），被认为是在用BHT处理的小鼠中促进肺肿瘤形成的原因。醌甲基化物具有很强的亲电性，会在环外亚甲基处与亲核试剂发生迈克尔型加成反应，形成苄基硫醚加合物。我们的目标是确定这些醌甲基化物在细胞内的蛋白质靶点，以便深入了解它们对肿瘤发生的影响。用BHT - QM或BHTOH - QM处理小鼠肺上皮来源的细胞系E10及其自发转化体、致瘤性E9细胞系，并用二维十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分析细胞蛋白。用针对BHTOH - QM共轭物制备的多克隆抗体在western印迹上检测加合物蛋白，该抗体可识别与半胱氨酸的巯基以及赖氨酸和组氨酸残基的侧链氨基结合的两种醌甲基化物的加合物。通过高效液相色谱 - 质谱联用（LC/MS）分析免疫反应性蛋白的胰蛋白酶消化产物，并使用MS/MS数据搜索蛋白质数据库进行鉴定。在少数情况下，通过基质辅助激光解吸/电离飞行时间质谱检测这些消化产物中的加合物肽段。总共鉴定出37种免疫反应性蛋白，包括参与碳水化合物代谢、核酸合成以及RNA和蛋白质加工的蛋白，此外还有几种细胞骨架和应激相关蛋白。约一半的蛋白质加合物在两种细胞系中都能找到。仅在转化的E9细胞中检测到的加合物包括谷胱甘肽S - 转移酶P1、过氧化物酶体增殖物激活受体2、核苷二磷酸激酶和纽蛋白，而几种烷基化的细胞骨架蛋白，如微管蛋白、波形蛋白、血管平滑肌肌动蛋白和钙周期蛋白仅在E10细胞中检测到。几种被BHT衍生的醌甲基化物修饰的蛋白质与肿瘤发生的各个方面有关，是进一步研究烷基化对细胞转化影响的优秀候选对象。
