Oliveira Nuno G, Pingarilho Marta, Martins Célia, Fernandes Ana Sofia, Vaz Sofie, Martins Vanda, Rueff José, Gaspar Jorge Francisco
iMed.UL, Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal.
Mutat Res. 2009 May 31;676(1-2):87-92. doi: 10.1016/j.mrgentox.2009.04.009. Epub 2009 Apr 22.
Acrylamide (AA) is a suspected human carcinogen found to be generated during the heating of carbohydrate-rich foodstuffs. AA exhibits 'Michael-type' reactivity towards reduced glutathione (GSH), resulting in vivo in the urinary excretion of mercapturic acid conjugates. GSH is a key factor for mammalian cell homeostasis, with diverse functions that include, among others, the conjugation of electrophilic compounds and the detoxification of products generated by oxidative stress. Therefore, studies focusing on the modulation of GSH are of great importance for the understanding of the mechanisms of AA-induced toxicity. This report addresses this issue by analyzing cytotoxicity (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay) and clastogenicity (chromosomal aberrations) as endpoints in V79 cells after exposure to AA. The experiments described herein include the evaluation of the effect of buthionine sulfoximine (BSO), an effective inhibitor of GSH synthesis, GSH-monoethyl ester (GSH-EE), a compound that is taken up by cells and intracellularly hydrolysed to GSH, and also GSH exogenously added to culture medium. Pre-treatment with BSO increased the cytotoxicity and the frequency of aberrant cells excluding gaps (ACEG) induced by AA. While pre-treatment with GSH-EE did not modify the cytotoxicity or the frequency of ACEG induced by AA, co-treatment with AA and GSH decreased both parameters, rendering the cells less prone to the toxic effects of AA. In vitro studies in a cell-free system, using monochlorobimane (MCB), a fluorescent probe for GSH, were also performed in order to evaluate the role of AA in GSH depletion. The results show that spontaneous conjugation of AA with GSH in the extracellular medium is involved in the protection given by GSH. In summary, these results reinforce the role of GSH in the modulation of the cytotoxic and clastogenic effects induced by AA, which may be relevant in an in vivo exposure scenario.
丙烯酰胺（AA）是一种疑似人类致癌物，在富含碳水化合物的食品加热过程中会产生。AA对还原型谷胱甘肽（GSH）表现出“迈克尔型”反应性，导致体内硫醚氨酸共轭物经尿液排泄。GSH是哺乳动物细胞内稳态的关键因素，具有多种功能，包括亲电化合物的共轭作用以及氧化应激产生的产物的解毒作用。因此，专注于GSH调节的研究对于理解AA诱导毒性的机制非常重要。本报告通过分析暴露于AA后V79细胞中的细胞毒性（3-(4, ,5-二甲基噻唑-2-基)-2, ,5-二苯基-2H-四唑溴盐（MTT）还原试验）和致断裂性（染色体畸变）来解决这个问题。本文所述的实验包括评估丁硫氨酸亚砜胺（BSO）（一种有效的GSH合成抑制剂）、GSH-单乙酯（GSH-EE）（一种被细胞摄取并在细胞内水解为GSH的化合物）以及外源性添加到培养基中的GSH的作用。用BSO预处理会增加AA诱导的细胞毒性和异常细胞（不包括间隙）频率（ACEG）。虽然用GSH-EE预处理不会改变AA诱导的细胞毒性或ACEG频率，但AA与GSH共同处理会降低这两个参数，使细胞更不易受到AA的毒性作用。还使用无细胞系统进行了体外研究，使用单氯双氢杨梅素（MCB）（一种GSH荧光探针）来评估AA在GSH消耗中的作用。结果表明，细胞外培养基中AA与GSH的自发共轭作用参与了GSH提供的保护作用。总之，这些结果强化了GSH在调节AA诱导的细胞毒性和致断裂性效应中的作用，这在体内暴露情况下可能具有相关性。
