Abstract

Butylated hydroxyanisole (BHA) and the chemically similar butylated hydroxytoluene (BHT) are widely used as antioxidants. Toxicity of BHA and BHT has been reported under in vitro and in vivo experimental conditions. However, the mechanism of BHA-induced toxic effects in cells is unclear. In this study, the cytotoxic effects of BHA and differences in cell death mechanism for BHA and BHT were investigated in rat thymocytes by flow cytometric analysis using a fluorescent probe. We observed a significant increase in propidium iodide fluorescence in the population of cells treated with 100 μM and 300 μM BHA (dead cells). Thymocytes treated with 100 µM BHA showed increased intracellular Ca2+ and Zn2+ levels and depolarized cell membranes. BHA (30–100 µM) decreased non-protein thiol content of cells, indicating decreased glutathione content. Co-stimulation with 100 µM BHA and 300 µM H2O2 acted synergistically to increase cell lethality. Moreover, BHA significantly increased caspase-3 activity and the number of annexin-V-positive cells in a concentration-dependent manner, indicating apoptosis. However, BHT reduced caspase-3 activity and increased the number of annexin-V-negative dead cells, indicating non-apoptotic cell death. Our results reveal the toxicity of BHA could be attributed to increased levels of intracellular Ca2+ and Zn2+, resulting in an increased vulnerability of rat thymocytes to oxidative stress. In addition, we demonstrate that whereas BHA induced apoptosis, BHT induced non-apoptotic cell death in rat thymocytes. Therefore, these results may support the safety of BHA, but also demonstrate the importance of performing toxicity evaluation at the cellular level besides the tissue level.

摘要

丁基羟基茴香醚 (BHA) 和化学性质相似的丁基羟基甲苯 (BHT) 被广泛用作抗氧化剂。已在体外和体内实验条件下报告了 BHA 和 BHT 的毒性。然而，BHA 诱导的细胞毒性作用的机制尚不清楚。在这项研究中，通过使用荧光探针的流式细胞术分析在大鼠胸腺细胞中研究了 BHA 的细胞毒性作用以及 BHA 和 BHT 的细胞死亡机制的差异。我们观察到用 100 μM 和 300 μM BHA（死细胞）处理的细胞群中碘化丙啶荧光显着增加。用 100 µM BHA 处理的胸腺细胞显示细胞内 Ca2+ 和 Zn2+ 水平升高，细胞膜去极化。BHA (30–100 µM) 降低细胞的非蛋白质硫醇含量，表明谷胱甘肽含量降低。与 100 µM BHA 和 300 µM H2O2 共同刺激可协同作用以增加细胞杀伤力。此外，BHA 以浓度依赖性方式显着增加 caspase-3 活性和膜联蛋白 V 阳性细胞的数量，表明细胞凋亡。然而，BHT 降低了 caspase-3 活性并增加了膜联蛋白 V 阴性死细胞的数量，表明非凋亡细胞死亡。我们的研究结果表明，BHA 的毒性可归因于细胞内 Ca2+ 和 Zn2+ 水平的增加，导致大鼠胸腺细胞对氧化应激的脆弱性增加。此外，我们证明 BHA 诱导细胞凋亡，而 BHT 在大鼠胸腺细胞中诱导非凋亡细胞死亡。因此，这些结果可能支持 BHA 的安全性，