Trevisan Rafael, Mello Danielle F, Delapedra Gabriel, Silva Danilo G H, Arl Miriam, Danielli Naissa M, Metian Marc, Almeida Eduardo A, Dafre Alcir L
Department of Biochemistry, Federal University of Santa Catarina, 88040-900 Florianópolis, Brazil; Department of Aquaculture, Federal University of Santa Catarina, 88034-001 Florianópolis, Brazil.
Department of Biochemistry, Federal University of Santa Catarina, 88040-900 Florianópolis, Brazil.
Aquat Toxicol. 2016 Apr;173:105-119. doi: 10.1016/j.aquatox.2016.01.008. Epub 2016 Jan 29.
The mercapturic acid pathway (MAP) is a major phase II detoxification route, comprising the conjugation of electrophilic substances to glutathione (GSH) in a reaction catalyzed by glutathione S-transferase (GST) enzymes. In mammals, GSH-conjugates are exported from cells, and the GSH-constituent amino acids (Glu/Gly) are subsequently removed by ectopeptidases. The resulting Cys-conjugates are reabsorbed and, finally, a mercapturic acid is generated through N-acetylation. This pathway, though very well characterized in mammals, is poorly studied in non-mammalian biological models, such as bivalve mollusks, which are key organisms in aquatic ecosystems, aquaculture activities and environmental studies. In the present work, the compound 1-chloro-2,4-dinitrobenzene (CDNB) was used as a model electrophile to study the MAP in Pacific oysters Crassostrea gigas. Animals were exposed to 10μM CDNB and MAP metabolites were followed over 24h in the seawater and in oyster tissues (gills, digestive gland and hemolymph). A rapid decay was detected for CDNB in the seawater (half-life 1.7h), and MAP metabolites peaked in oyster tissues as soon as 15min for the GSH-conjugate, 1h for the Cys-conjugate, and 4h for the final metabolite (mercapturic acid). Biokinetic modeling of the MAP supports the fast CDNB uptake and metabolism, and indicated that while gills are a key organ for absorption, initial biotransformation, and likely metabolite excretion, hemolymph is a possible milieu for metabolite transport along different tissues. CDNB-induced GSH depletion (4h) was followed by increased GST activity (24h) in the gills, but not in the digestive gland. Furthermore, the transcript levels of glutamate-cysteine ligase, coding for the rate limiting enzyme in GSH synthesis, and two phase II biotransformation genes (GSTpi and GSTo), presented a fast (4h) and robust (∼6-70 fold) increase in the gills. Waterborne exposure to electrophilic compounds affected gills, but not digestive gland, while intramuscular exposure was able to modulate biochemical parameters in both tissues. This study is the first evidence of a fully functional and interorgan MAP pathway in bivalves. Hemolymph was shown to be responsible for the metabolic interplay among tissues, and gills, acting as a powerful GSH-dependent metabolic barrier against waterborne electrophilic substances, possibly also participating in metabolite excretion into the sea water. Altogether, experimental and modeled data fully agree with the existence of a classical mechanism for phase II xenobiotic metabolism and excretion in bivalves.
巯基尿酸途径（MAP）是主要的II相解毒途径，包括在谷胱甘肽S-转移酶（GST）催化的反应中亲电物质与谷胱甘肽（GSH）结合。在哺乳动物中，GSH结合物从细胞中输出，随后GSH组成氨基酸（Glu/Gly）被外肽酶去除。生成的半胱氨酸结合物被重新吸收，最终通过N-乙酰化生成巯基尿酸。这条途径在哺乳动物中已得到充分表征，但在非哺乳动物生物模型中研究较少，如双壳贝类，它们是水生生态系统、水产养殖活动和环境研究中的关键生物。在本研究中，化合物1-氯-2,4-二硝基苯（CDNB）被用作模型亲电试剂，以研究太平洋牡蛎（Crassostrea gigas）中的MAP。将动物暴露于10μM CDNB中，并在海水中和牡蛎组织（鳃、消化腺和血淋巴）中跟踪MAP代谢物24小时。在海水中检测到CDNB迅速降解（半衰期1.7小时），GSH结合物在牡蛎组织中15分钟内达到峰值，半胱氨酸结合物1小时达到峰值，最终代谢物（巯基尿酸）4小时达到峰值。MAP的生物动力学模型支持CDNB的快速摄取和代谢，并表明虽然鳃是吸收、初始生物转化以及可能的代谢物排泄的关键器官，但血淋巴可能是代谢物沿不同组织运输的介质。CDNB诱导的鳃中GSH消耗（4小时）后，鳃中GST活性增加（24小时），但消化腺中没有。此外，编码GSH合成限速酶的谷氨酸-半胱氨酸连接酶以及两个II相生物转化基因（GSTpi和GSTo）的转录水平在鳃中呈现快速（4小时）且显著（约6 - 70倍）增加。通过水体暴露于亲电化合物会影响鳃，但不影响消化腺，而肌肉注射暴露能够调节两个组织中的生化参数。本研究首次证明双壳贝类中存在功能完整且涉及多个器官的MAP途径。血淋巴被证明负责组织间的代谢相互作用，鳃作为对水体亲电物质强大的依赖GSH的代谢屏障，可能也参与将代谢物排泄到海水中。总之，实验和模型数据完全支持双壳贝类中存在II相异源物质代谢和排泄的经典机制。
