Department of Biochemistry, Sri Venkateswara University, Tirupati, Andhra Pradesh, India.
College of Pharmaceutical Sciences, Sri Venkateswara University, Tirupati, Andhra Pradesh, India.
Ecotoxicol Environ Saf. 2019 Oct 30;182:109416. doi: 10.1016/j.ecoenv.2019.109416. Epub 2019 Jul 10.
The main objectives of this study were to purify the glutathione S-transfereses (GSTs) and assess the effect of high doses of acrylamide (ACR) on male albino Wistar rat liver, kidney, testis and bran GST activities, and expression analysis of GST. ACR (50 mg/300 ml) was ingested for 40 days (20 doses) in drinking water on alternative days, on 40 day post ingestion the control and treated tissues were collected for GST purification by affinity column and biochemical characterization of GSTs by substrate specificities, and GST expression by immuno dot blots. In the analysis of the purified GSTs, we observed that liver GSTs were resolved in to three bands known as Yc, Yb and Ya; kidney GSTs were resolved in to two bands known as Yc and Ya; testis and brain GSTs were resolved as four bands known as Yc, Yb, Yβ and Yδ on 12.5% sodium dodecyl sulfate polyacrylamide gel (SDS PAGE). In the analysis of biochemical characterization, we observed a significant decrease (p < 0.05) in the specific activities of liver GST isoforms with the substrates 1-chloro 2,4-dinitrobenzene (CDNB), bromosulfophthalein (BSP), p-nitrophenyl acetate (pNPA), p-nitrobenzyl chloride (pNBC) and cumene hydroperoxide (CHP), but showed no activity with ethacrynic acid (ECA) and significant decrease (p < 0.05) in the specific activities of kidney GST isoforms with the substrates CDNB, pNPA, pNBC and CHP, but showed no activity with BSP and ECA, and a significant decrease (p < 0.05) in the specific activities of testis and brain GST isoforms with the substrates CDNB, BSP, pNPA, pNBC, ECA and CHP. In the analysis of immuno dot blots, we observed a decreased expression of liver, kidney, testis and brain GSTs. Through the affinity purification and biochemical characterization, we observed a tissue specific distribution of GSTs that is liver GSTs possess Yc, Yb and Ya sub units known as alpha (α) and mu (μ) class GSTs; kidney GSTs possess Yc and Ya sub units known as (α) alpha class GST; testis and brain GSTs possess Yc, Yb, Yβ and Yδ sub units known as alpha (α), mu (μ) and pi (π) class GSTs. Purification studies, biochemical characterization and immuno dot blot analysis were revealed the GSTs were sensitive to high doses of ACR and the high level exposure to ACR cause the damage of detoxification function of GST due to decreased expression and hence lead to cellular dysfunction of vital organs.
本研究的主要目的是纯化谷胱甘肽 S-转移酶（GSTs），并评估高剂量丙烯酰胺（ACR）对雄性白化 Wistar 大鼠肝、肾、睾丸和 bran GST 活性的影响，以及 GST 的表达分析。在 40 天的时间里（20 个剂量），ACR（50mg/300ml）通过交替饮水的方式摄入，在摄入后第 40 天收集对照和处理组织，通过亲和柱纯化 GST 并通过底物特异性进行 GSTs 的生化特性分析，通过免疫斑点印迹法进行 GST 表达分析。在纯化 GSTs 的分析中，我们观察到肝 GSTs 分为三个已知的亚基 Yc、Yb 和 Ya；肾 GSTs 分为两个已知的亚基 Yc 和 Ya；睾丸和脑 GSTs 在 12.5%十二烷基硫酸钠聚丙烯酰胺凝胶（SDS-PAGE）上分为四个已知的亚基 Yc、Yb、Yβ和 Yδ。在生化特性分析中，我们观察到肝 GST 同工酶对 1-氯-2,4-二硝基苯（CDNB）、溴磺酞（BSP）、对硝基苯乙酸酯（pNPA）、对硝基苄氯（pNBC）和 cumene 过氧化物（CHP）的比活性显著降低（p<0.05），但对 ethacrynic 酸（ECA）没有活性，肾 GST 同工酶对 CDNB、pNPA、pNBC 和 CHP 的比活性显著降低（p<0.05），但对 BSP 和 ECA 没有活性，睾丸和脑 GST 同工酶对 CDNB、BSP、pNPA、pNBC、ECA 和 CHP 的比活性显著降低（p<0.05）。在免疫斑点印迹分析中，我们观察到肝、肾、睾丸和脑 GSTs 的表达降低。通过亲和纯化和生化特性分析，我们观察到 GST 的组织特异性分布，即肝 GSTs 具有 Yc、Yb 和 Ya 亚基，称为 alpha（α）和 mu（μ）类 GSTs；肾 GSTs 具有 Yc 和 Ya 亚基，称为（α）alpha 类 GST；睾丸和脑 GSTs 具有 Yc、Yb、Yβ和 Yδ 亚基，称为 alpha（α）、mu（μ）和 pi（π）类 GSTs。纯化研究、生化特性分析和免疫斑点印迹分析表明，GSTs 对高剂量 ACR 敏感，高水平暴露于 ACR 会导致 GST 解毒功能受损，从而导致 GST 表达降低，进而导致重要器官的细胞功能障碍。
