Hinson J A, Michael S L, Ault S G, Pumford N R
Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
Toxicol Sci. 2000 Feb;53(2):467-73. doi: 10.1093/toxsci/53.2.467.
The hepatic centrilobular necrosis produced by the analgesic/antipyretic acetaminophen correlates with metabolic activation of the drug leading to its covalent binding to protein. However, the molecular mechanism of the toxicity is not known. Recent immunohistochemical analyses using an antinitrotyrosine antiserum indicated that nitrotyrosine protein adducts co-localized with the acetaminophen-protein adducts in the centrilobular cells of the liver. Nitration of proteins is believed to occur by peroxynitrite, a substance formed by the rapid reaction of superoxide with nitric oxide. Nitric oxide and superoxide may be formed by activated Kupffer cells or by other cells. Because we were unable to successfully utilize the commercial antiserum in Western blot analyses of liver fractions, we developed a new antiserum. With our antiserum, liver fractions from saline-treated control and acetaminophen-treated mice were successfully analyzed for nitrated proteins. The immunogen for this new antiserum was synthesized by coupling 3-nitro-4-hydroxybenzoic acid to keyhole limpet hemocyanin. A rabbit immunized with this adduct yielded a high titer of an antiserum that recognized BSA nitrated with peroxynitrite. Immunoblot analysis of nitrated BSA indicated that nitrotyrosine present in a protein sample could be easily detected at levels of 20 pmoles. Immunohistochemical analyses indicated that nitrotyrosine protein adducts were detectable in the centrilobular areas of the liver. Immunoblot analysis of liver homogenates from both saline-treated and acetaminophen-treated mice (300 mg/kg) indicate that the major nitrotyrosine protein adducts produced have molecular weights of 36 kDa, 44 kDa, and 85 kDa. The 85-kDa protein stained with the most intensity. The hepatic homogenates of the acetaminophen- treated mice showed significantly increased levels of all protein adducts.
解热镇痛药对乙酰氨基酚所致的肝小叶中心性坏死与该药物的代谢活化有关，后者导致其与蛋白质共价结合。然而，毒性的分子机制尚不清楚。最近使用抗硝基酪氨酸抗血清进行的免疫组织化学分析表明，硝基酪氨酸蛋白加合物与对乙酰氨基酚 - 蛋白加合物在肝小叶细胞中共定位。蛋白质硝化被认为是由过氧亚硝酸盐引起的，过氧亚硝酸盐是超氧化物与一氧化氮快速反应形成的一种物质。一氧化氮和超氧化物可能由活化的库普弗细胞或其他细胞形成。由于我们无法在肝脏组分的蛋白质印迹分析中成功使用市售抗血清，我们制备了一种新的抗血清。使用我们的抗血清，成功分析了生理盐水处理的对照小鼠和对乙酰氨基酚处理的小鼠肝脏组分中的硝化蛋白质。这种新抗血清的免疫原是通过将3 - 硝基 - 4 - 羟基苯甲酸与钥孔血蓝蛋白偶联合成的。用这种加合物免疫的兔子产生了高滴度的抗血清，该抗血清可识别用过氧亚硝酸盐硝化的牛血清白蛋白（BSA）。对硝化BSA的免疫印迹分析表明，蛋白质样品中存在的硝基酪氨酸在20皮摩尔水平时很容易被检测到。免疫组织化学分析表明，在肝脏小叶中心区域可检测到硝基酪氨酸蛋白加合物。对生理盐水处理和对乙酰氨基酚处理（300 mg/kg）的小鼠肝脏匀浆进行的免疫印迹分析表明，产生的主要硝基酪氨酸蛋白加合物的分子量为36 kDa、44 kDa和85 kDa。85 kDa的蛋白染色强度最大。对乙酰氨基酚处理的小鼠肝脏匀浆显示所有蛋白加合物水平均显著升高。
