Zimmerman Lisa J, Valentine Holly L, Valentine William M
Department of Pathology and Center in Molecular Toxicology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2561, USA.
Chem Res Toxicol. 2004 Feb;17(2):258-67. doi: 10.1021/tx034209c.
Thiocarbamates are a major class of herbicides used extensively in the agricultural industry. It has been shown that thiocarbamates can form reactive sulfoxide and sulfone intermediates, which may be involved in the toxicity of thiocarbamates through covalent modification of cysteine and serine active sites of enzymes. Molinate has been shown to generate an S-hexahydro-1H-azepine-1-carbonyl adduct on the Cys-125 residue of the beta2- and beta3-chains of rat globin analogous to that reported for disulfiram and to inhibit aldehyde dehydrogenase and nonspecific esterase activity. The present study examined whether other thiocarbamate herbicides produce similar covalent protein modifications and enzyme inhibition to that reported for molinate and whether S-(N,N-dialkylaminocarbonyl)cysteine adduct levels are correlated to enzyme inhibition or the structure of thiocarbamate herbicides. Additionally, the potential of molinate to act as a peripheral demyelinating agent similar to disulfiram was evaluated. To address these aims, rats were exposed ip to molinate, vernolate, ethiolate, EPTC, or butylate for 5 days after which hemogloblin was isolated and analyzed for protein adducts using HPLC and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. In addition, brain, liver, and testes mitochondrial and microsomal fractions were assayed for nonspecific esterase, low Km ALDH, or total ALDH activities, and S-(N,N-dialkylaminocarbonyl)cysteine adducts were measured by LC/MS/MS. For the neurotoxicity assessments, rats were administered molinate parenterally for subchronic periods and morphological evaluations performed on peripheral nerves. All of the thiocarbamates except butylate produced S-(N,N-dialkylaminocarbonyl)cysteine adducts on globin and the quantity of adducts detected decreased with increasing size of the nitrogen substituents. In contrast, a clear relationship between cysteine modification in mitochondrial and microsomal samples to nitrogen substituents was not evident, and although molinate produced relatively high levels of adducts and esterase inhibition and butylate low levels of adducts and esterase inhibition for most samples, in general, the level of S-(N,N-dialkylaminocarbonyl)cysteine adducts did not appear to be related to enzyme inhibition. Molinate did not produce segmental demyelination in peripheral nerve, suggesting that molinate and possibly other thiocarbamates do not share the neurotoxic potential of dithiocarbamates.
硫代氨基甲酸盐是农业中广泛使用的一类主要除草剂。研究表明，硫代氨基甲酸盐可形成活性亚砜和砜中间体，这些中间体可能通过共价修饰酶的半胱氨酸和丝氨酸活性位点而参与硫代氨基甲酸盐的毒性作用。已证明禾草敌能在大鼠球蛋白β2和β3链的Cys-125残基上生成S-六氢-1H-氮杂卓-1-羰基加合物，类似于双硫仑报道的加合物，并能抑制醛脱氢酶和非特异性酯酶活性。本研究调查了其他硫代氨基甲酸盐除草剂是否会产生与禾草敌报道的类似的共价蛋白质修饰和酶抑制作用，以及S-(N,N-二烷基氨基甲酰基)半胱氨酸加合物水平是否与酶抑制作用或硫代氨基甲酸盐除草剂的结构相关。此外，还评估了禾草敌作为类似于双硫仑的外周脱髓鞘剂的潜力。为实现这些目标，给大鼠腹腔注射禾草敌、灭草猛、茵草敌、扑草净或丁草特5天，之后分离血红蛋白，并用高效液相色谱法和基质辅助激光解吸电离飞行时间质谱法分析蛋白质加合物。此外，检测脑、肝和睾丸的线粒体和微粒体部分的非特异性酯酶、低Km醛脱氢酶或总醛脱氢酶活性，并用液相色谱/串联质谱法测量S-(N,N-二烷基氨基甲酰基)半胱氨酸加合物。对于神经毒性评估，给大鼠非肠道注射禾草敌进行亚慢性给药，并对外周神经进行形态学评估。除丁草特外，所有硫代氨基甲酸盐均在球蛋白上产生S-(N,N-二烷基氨基甲酰基)半胱氨酸加合物，检测到的加合物数量随氮取代基尺寸的增加而减少。相比之下，线粒体和微粒体样品中的半胱氨酸修饰与氮取代基之间没有明显的关系，尽管对于大多数样品，禾草敌产生相对高水平的加合物和酯酶抑制作用，丁草特产生低水平的加合物和酯酶抑制作用，但总体而言，S-(N,N-二烷基氨基甲酰基)半胱氨酸加合物水平似乎与酶抑制作用无关。禾草敌在外周神经中未产生节段性脱髓鞘，这表明禾草敌以及可能的其他硫代氨基甲酸盐不具有二硫代氨基甲酸盐的神经毒性潜力。
