Day B W, Jin R, Karol M H
Department of Environmental & Occupational Health, University of Pittsburgh, Pennsylvania 15238, USA.
Chem Res Toxicol. 1996 Apr-May;9(3):568-73. doi: 10.1021/tx9501703.
Guinea pig hemoglobin (Hb) adducts of 2,4- and 2,6-toluene diisocyanate (2,4- and 2,6-TDI) were individually prepared, each at a Hb tetramer to diisocyanate ratio of 1:1, and compared with adducts of Hb from animals exposed to 1 ppm 2,4-TDI vapor. Each Hb sample was subjected to C4 HPLC chain/heme separation with UV detection. Survey of the LC fractions using an antiserum prepared to a heterologous TDI-protein indicated a difference in chain specificity and product types formed in vitro by the two isomers. Ionspray mass spectrometry (MS) revealed the chemistry of the adduction products. Carbamoylated products, formed from adduction by one TDI molecule (with one isocyanato group hydrolyzed to an amino group), were detected by MS with 2,4-TDI on each chain and with 2,6-TDI on the beta chain. Additionally, a quasi-molecular ion of a bis carbamoylation adduct was noted by MS in the in vitro 2,4-TDI-Hb adduct in the form of TDI-cross-linked alpha and beta chains. Ionspray MS analysis of the Hb isolated from guinea pigs exposed in vivo to 2,4-TDI indicated carbamoylation products with both the alpha and beta chains in which one of the two original isocyanato groups had been hydrolyzed to the amine. We also found evidence of an amine-nitroso adduct on the alpha chain in the in vivo sample. These results indicate that at least one of the isocyanato moieties (or a masked derivative) of 2,4-TDI survived passage through the lung, into the serum, and through the erythrocyte membrane to form adducts with Hb that were stable to dialysis, gel filtration, and reversed phase HPLC separation under acidic conditions. The presence of an apparent amine-nitroso adduct indicated in vivo formation of 2,4-diaminotoluene, a recognized animal carcinogen.
分别制备了豚鼠血红蛋白（Hb）与2,4-和2,6-甲苯二异氰酸酯（2,4-TDI和2,6-TDI）的加合物，每种加合物的血红蛋白四聚体与二异氰酸酯的比例均为1:1，并将其与暴露于1 ppm 2,4-TDI蒸气的动物的血红蛋白加合物进行比较。每个血红蛋白样品都通过C4高效液相色谱进行链/血红素分离，并采用紫外检测。使用针对异源TDI-蛋白质制备的抗血清对液相色谱馏分进行检测，结果表明两种异构体在体外形成的加合物在链特异性和产物类型上存在差异。离子喷雾质谱（MS）揭示了加合产物的化学性质。通过MS检测到由一个TDI分子加合形成的氨基甲酰化产物（其中一个异氰酸酯基团水解为氨基），在每条链上都有2,4-TDI，在β链上有2,6-TDI。此外，在体外2,4-TDI-Hb加合物中，MS检测到一种双氨基甲酰化加合物的准分子离子，其形式为TDI交联的α链和β链。对体内暴露于2,4-TDI的豚鼠分离出的血红蛋白进行离子喷雾MS分析，结果表明α链和β链上均存在氨基甲酰化产物，其中两个原始异氰酸酯基团中的一个已水解为胺。我们还在体内样品的α链上发现了胺-亚硝基加合物的证据。这些结果表明，2,4-TDI的至少一个异氰酸酯部分（或一种掩蔽衍生物）在通过肺部、进入血清并穿过红细胞膜后仍能存活，从而与血红蛋白形成在透析、凝胶过滤和酸性条件下的反相高效液相色谱分离中均稳定的加合物。明显的胺-亚硝基加合物的存在表明体内形成了2,4-二氨基甲苯，这是一种公认的动物致癌物。
