Studies on the methyl isocyanate adducts with globin
Studies on the methyl isocyanate adducts with globin
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DOI：
10.1016/j.cbi.2003.06.003
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发表时间：
2004-06-30
影响因子：
5.1
通讯作者：
Smigolová, P
中科院分区：
文献类型：
--
作者：
Mráz, J;Simek, P;Smigolová, P
异氰酸酯，如合成氨基甲酸酯类农药的中间体甲基异氰酸酯（MIC），或塑料生产中使用的二异氰酸酯，都是高度活性的有毒化合物，可以自发地与生物大分子结合。体内形成稳定的加合物与血蛋白球蛋白提供了生物监测内部暴露于各种活性物质的可能性。因此，通过测定异氰酸酯与珠蛋白的特定加合物来进行生物监测是一项挑战。在这项研究中，我们用100倍摩尔过量的MIC处理了人类珠蛋白中形成的加合物。用pronase酶解珠蛋白，水解产物用高效液相色谱-正大气压化学电离质谱（HPLC/APCI-MS）分析。两种主要的MIC加合物为n端Val加合物和侧链Lys加合物。另外还发现了20种加合物，其中一些通过质谱和质谱进行了初步鉴定。虽然含有Tyr和His的加合物的检测是预期的，但含有Trp和Phe的加合物以及含有两个MIC片段的Lys加合物可能是珠蛋白水解过程中氨基甲酰基化的分析产物，而不是直接氨基甲酰基化的产物。其他检测到的产物是来自珠蛋白β链n端二肽的MIC-Val-His，以及由连接Gly、Val、Len、Thr和Glu的MIC-Lys组成的二肽。未能检测到相应的未修饰二肽，表明pronase的作用可能受到氨基酸修饰的阻碍。MIC是人类和大鼠体内工业溶剂N，N-二甲基甲酰胺（DMF）和N-甲基甲酰胺（MF）的代谢中间体。用HPLC/APCI-MS对大鼠注射DMF或MF （1000 mg/kg）后的珠蛋白进行分析，发现含有Val和Lys的MIC加合物。使用Edman降解和牙龈测定的DMF剂量大鼠珠蛋白中的Val加合物水平约为40 nmol/g，这是职业暴露于DMF的工人的常见水平。这也表明，这种人类珠蛋白样品中的Lys加合物可以进行分析，因此可以考虑作为DMF暴露的潜在生物标志物进行进一步研究。2004爱思唯尔爱尔兰有限公司版权所有。
Isocyanates such as methylisocyanate (MIC), an intermediate in the synthesis of carbamate pesticides, or diisocyanates, used in the production of plastics, are highly reactive toxic compounds that spontaneously bind to biological macromolecules. In vivo formation of stable adducts with blood protein globin offers possibilities for biomonitoring of internal exposure to various reactive species. Thus, biomonitoring of the isocyanates through determination of their specific adducts with globin is a challenge. In this study, we characterized the adducts formed in human globin upon treatment with 100-fold molar excess of MIC. The globin was subject to enzymatic hydrolysis with pronase, and the hydrolysate was analysed by high performance liquid chromatography with positive atmospheric pressure chemical ionization mass spectrometric detection (HPLC/APCI-MS). The two major MIC adducts were those with N-terminal Val and side-chain of Lys, as confirmed by comparison with the synthetic standards. About 20 other adducts were observed, and several of them were tentatively identified using their MS and MS/MS spectra. Whereas detection of the adducts with Tyr and His was expected, the adducts with Trp and Phe, and a Lys adduct containing two MIC moieties, were probably analytical artifacts resulting from the transcarbamoylation during globin hydrolysis rather than products of direct carbamoylation. The other detected products were MIC-Val-His, derived from the N-terminal dipeptide of globin beta-chain, and dipeptides consisting of MIC-Lys attached to Gly, Val, Len, Thr, and Glu. Failure to detect the corresponding non-modified dipeptides suggests that the pronase action may be hampered by the amino acid modification. MIC is known as a metabolic intermediate of the industrial solvents N,N-dimethylformamide (DMF) and N-methylformamide (MF) in humans and rats. The HPLC/APCI-MS analysis of globin from rats injected with DMF or MF, 1000 mg/kg, revealed the presence of the MIC adducts with both Val and Lys. The level of the Val adduct in globin from the DMF-dosed rats, determined using Edman degradation and GUMS, was ca. 40 nmol/g, which is a level common in workers occupationally exposed to DMF. This suggests that also the Lys adduct in such human globin samples can be feasible to analysis and is therefore considered for further studies as a potential biomarker of exposure to DMF. (C) 2004 Elsevier Ireland Ltd. All rights reserved.