Endo Satoshi, Nishiyama Ayako, Suyama Miho, Takemura Mayuko, Soda Midori, Chen Huayue, Tajima Kazuo, El-Kabbani Ossama, Bunai Yasuo, Hara Akira, Matsunaga Toshiyuki, Ikari Akira
Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196, Japan.
Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196, Japan.
Chem Biol Interact. 2015 Jun 5;234:282-9. doi: 10.1016/j.cbi.2014.09.023. Epub 2014 Oct 5.
tert-Butylhydroquinone (BHQ), an antioxidant used as a food additive, exhibits an anticancer effect at low doses, but is carcinogenic in rodents at high doses. BHQ is metabolized into cytotoxic tert-butylquinone (TBQ), which is further converted to 6-tert-butyl-2,3-epoxy-4-hydroxy-5-cyclohexen-1-one (TBEH) through 6-tert-butyl-2,3-epoxy-4-benzoquinone (TBE). Both TBQ and TBE are cytotoxic, but their toxic mechanisms have not been fully characterized. In this study, we have investigated the toxic mechanisms of TBQ and TBE, and the defense system against the two p-quinones using lung cancer A549 cells. TBQ and TBE, but not BHQ and TBEH, showed cytotoxicity to A549 cells. Neither caspase-3 activation nor an increase in the expression of endoplasmic reticulum stress-associating target genes was observed. TBQ and TBE reacted with reduced glutathione, and significantly decreased the glutathione level in A549 cells, suggesting that the cytotoxicity of the p-quinones is caused by their high electrophilicity reacting with biomolecules. The A549 cells treated with the p-quinones also showed increased levels of autophagic vacuoles and LC3-II protein, which are specific autophagy markers. An autophagy inhibitor, 3-methyladenine (3MA), decreased the LC3-II production by the p-quinones, but enhanced the cytotoxicity induced by TBQ and TBE, suggesting that autophagy contributes to alleviating the p-quinone-triggered cytotoxicity. In addition, the TBE-induced cytotoxicity and autophagy activation in the cells were significantly suppressed by overexpression of aldo-keto reductase (AKR)1B10 that efficiently reduces TBE into TBEH, and were augmented by pretreatment with a potent AKR1B10 inhibitor, C1. The effects of 3MA and C1 on the TBE-induced cytotoxicity were additive. The data provides evidence for the first time that autophagy and AKR1B10 contribute to the defense system against the cytotoxicity caused by the electrophilic p-quinone metabolites of BHQ.
叔丁基对苯二酚（BHQ）是一种用作食品添加剂的抗氧化剂，低剂量时具有抗癌作用，但高剂量时对啮齿动物具有致癌性。BHQ代谢为具有细胞毒性的叔丁基醌（TBQ），后者通过6-叔丁基-2,3-环氧-4-苯醌（TBE）进一步转化为6-叔丁基-2,3-环氧-4-羟基-5-环己烯-1-酮（TBEH）。TBQ和TBE均具有细胞毒性，但其毒性机制尚未完全明确。在本研究中，我们利用肺癌A549细胞研究了TBQ和TBE的毒性机制以及针对这两种对苯醌的防御系统。TBQ和TBE对A549细胞具有细胞毒性，而BHQ和TBEH则无此作用。未观察到半胱天冬酶-3激活或内质网应激相关靶基因表达增加。TBQ和TBE与还原型谷胱甘肽反应，显著降低A549细胞中的谷胱甘肽水平，这表明对苯醌的细胞毒性是由其与生物分子的高亲电性反应所致。用对苯醌处理的A549细胞还显示出自噬空泡和LC3-II蛋白水平升高，这是自噬的特异性标志物。自噬抑制剂3-甲基腺嘌呤（3MA）可降低对苯醌诱导的LC3-II生成，但增强TBQ和TBE诱导的细胞毒性，这表明自噬有助于减轻对苯醌引发的细胞毒性。此外，通过高效将TBE还原为TBEH的醛酮还原酶（AKR）1B10的过表达，可显著抑制细胞中TBE诱导的细胞毒性和自噬激活，而用强效AKR1B10抑制剂C1预处理则可增强这种作用。3MA和C1对TBE诱导的细胞毒性的作用具有相加性。这些数据首次证明自噬和AKR1B10有助于抵御BHQ的亲电性对苯醌代谢产物引起的细胞毒性的防御系统。
