Wakazono H, Gardner I, Eliasson E, Coughtrie M W, Kenna J G, Caldwell J
Molecular Toxicology, Imperial College School of Medicine, Norfolk Place, London W2 1PG, U.K.
Chem Res Toxicol. 1998 Aug;11(8):863-72. doi: 10.1021/tx9702188.
Hepatic protein adducts derived from the allylbenzene food flavor estragole, which is hepatocarcinogenic when given to rodents at high doses, have been identified using immunochemical approaches. Male Fischer 344 rats were given estragole orally and hepatic protein adducts were detected by immunoblotting, using antisera raised by immunizing rabbits with 4-methoxycinnamic acid-modified rabbit serum albumin. A major 155-kDa adduct was expressed in livers of animals that had been treated with estragole at 100, 300, or 500 mg/kg. Levels of expression of the adduct increased disproportionately with respect to dose, and other adducts (170, 100, 44, and 35 kDa) were detected also in the high-dose group. Rats given estragole for 5 days, at 300 mg/kg/day, expressed predominantly 155- and 44-kDa adducts. The 155-, 100-, 44-, and 35-kDa adducts were detected in greatest abundance in liver microsomal fractions, while the 170-kDa adduct was most abundant in the nuclear fraction. Interestingly, whereas the 170-, 155-, 100-, and 35-kDa adducts were detected in cytosolic fractions, relatively low levels of the 44-kDa adduct were detected in nuclear fractions but not in cytosolic fractions. The various adducts were solubilized when microsomal fractions were extracted with sodium carbonate and were digested by trypsin. This implies that the target proteins are peripheral membrane proteins bound to the outer surface of microsomal membranes. Experiments undertaken with isolated rat hepatocytes and with V79 cells transfected with human monoamine phenol sulfotransferase cDNA revealed that adduct formation required 1'-hydroxylation of estragole, followed by sulfation. The pattern of adducts expressed when the transfected V79 cells were incubated with 1'-hydroxyestragole was very similar to that expressed in livers of estragole-treated rats. These cells should constitute a valuable in vitro model system for investigation of toxicological consequences arising from estragole-induced protein adduct formation.
已使用免疫化学方法鉴定出来自烯丙基苯类食品香料草蒿脑的肝蛋白加合物，草蒿脑在高剂量给予啮齿动物时具有致癌性。给雄性Fischer 344大鼠口服草蒿脑，并使用用4-甲氧基肉桂酸修饰的兔血清白蛋白免疫兔子产生的抗血清，通过免疫印迹法检测肝蛋白加合物。在以100、300或500mg/kg的剂量接受草蒿脑处理的动物肝脏中，表达了一种主要的155kDa加合物。加合物的表达水平相对于剂量不成比例地增加，并且在高剂量组中也检测到了其他加合物（170、100、44和35kDa）。以300mg/kg/天的剂量给大鼠喂食草蒿脑5天，主要表达155kDa和44kDa的加合物。155kDa、100kDa、44kDa和35kDa的加合物在肝微粒体组分中检测到的丰度最高，而170kDa的加合物在核组分中最丰富。有趣的是，虽然在胞质组分中检测到了170kDa、155kDa、100kDa和35kDa的加合物，但在核组分中检测到的44kDa加合物水平相对较低，而在胞质组分中未检测到。当用碳酸钠提取微粒体组分并用胰蛋白酶消化时，各种加合物可溶解。这意味着靶蛋白是与微粒体膜外表面结合的外周膜蛋白。用分离的大鼠肝细胞和转染了人单胺酚磺基转移酶cDNA的V79细胞进行的实验表明，加合物的形成需要草蒿脑的1'-羟基化，然后是硫酸化。当转染的V79细胞与1'-羟基草蒿脑一起孵育时表达的加合物模式与在经草蒿脑处理的大鼠肝脏中表达的模式非常相似。这些细胞应该构成一个有价值的体外模型系统，用于研究草蒿脑诱导的蛋白加合物形成所产生的毒理学后果。
