Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Università degli Studi di Urbino Carlo Bo, 61029 Urbino, PU, Italy.
Mutat Res. 2010 Jul 7;689(1-2):65-73. doi: 10.1016/j.mrfmmm.2010.05.003. Epub 2010 May 25.
Sulforaphane (SFR), an isothiocyanate from cruciferous vegetables, possesses growth-inhibiting and apoptosis-inducing activities in cancer cell lines. Recently, SFR has been shown to promote the mitochondrial formation of reactive oxygen species (ROS) in human cancer cell lines. The present study was undertaken to see whether SFR-derived ROS might cause DNA damage in cultured human cells, namely T limphoblastoid Jurkat and human umbilical vein endothelial cells (HUVEC). 1-3 h treatments with 10-30 microM SFR elicited intracellular ROS formation (as assayed with dihydrorhodamine, DHR, oxidation) as well as DNA breakage (as assessed with fast halo assay, FHA). These effects lacked cell-type specificity, since could be observed in both Jurkat and HUVEC. Differential-pH FHA analysis of damaged DNA showed that SFR causes frank DNA single strand breaks (SSBs); no DNA double strand breaks (DSBs) were found within the considered treatment times (up to 3 h). SFR-derived ROS were formed at the mitochondrial respiratory chain (MRC) level: indeed rotenone or myxothiazol (MRC Complex I and III inhibitors, respectively) abrogated ROS formation. Furthermore ROS were not formed in Jurkat cells pharmacologically depleted of respiring mitochondria (MRC-/Jurkat). Formation of ROS was causally linked to the induction of SSBs: indeed all the experimental conditions capable of preventing ROS formation also prevented the damage of nuclear DNA from SFR-intoxicated cells. As to the toxicological relevance of SSBs, we found that their prevention slightly but significantly attenuated SFR cytotoxicity, suggesting that high-dose SFR toxicity is the result of a complex series of events among which GSH depletion seems to play a pivotal role. In conclusion, the present study identifies a novel mechanism contributing to SFR toxicity which - since DNA damage is a prominent mechanism underlying the cytotoxic activity of established antineoplastic agents - might help to exploit the therapeutic value of SFR in anticancer drug protocols.
萝卜硫素（SFR）是十字花科蔬菜中的一种异硫氰酸盐，具有抑制癌细胞生长和诱导细胞凋亡的作用。最近的研究表明，SFR 可以促进人类癌细胞中线粒体中活性氧（ROS）的形成。本研究旨在观察 SFR 衍生的 ROS 是否会引起培养的人细胞（即 T 淋巴细胞白血病 Jurkat 细胞和人脐静脉内皮细胞（HUVEC））中的 DNA 损伤。用 10-30 μM 的 SFR 处理 1-3 小时会引起细胞内 ROS 的形成（用二氢罗丹明，DHR，氧化法测定）以及 DNA 断裂（用快速 halo 测定法，FHA 测定）。这些作用缺乏细胞类型特异性，因为在 Jurkat 和 HUVEC 中都可以观察到。受损 DNA 的差 pH FHA 分析表明，SFR 导致 DNA 单链断裂（SSBs）；在考虑的治疗时间内（最长 3 小时）没有发现 DNA 双链断裂（DSBs）。SFR 衍生的 ROS 在线粒体呼吸链（MRC）水平形成：实际上，鱼藤酮或米托噻唑（分别是 MRC 复合物 I 和 III 的抑制剂）可阻断 ROS 的形成。此外，在药理学上耗尽呼吸线粒体的 Jurkat 细胞（MRC-/Jurkat）中未形成 ROS。ROS 的形成与 SSBs 的诱导密切相关：实际上，所有能够阻止 ROS 形成的实验条件也阻止了 SFR 中毒细胞的核 DNA 损伤。至于 SSBs 的毒理学相关性，我们发现其预防可轻微但显著减轻 SFR 的细胞毒性，表明高剂量 SFR 毒性是一系列复杂事件的结果，其中 GSH 耗竭似乎起着关键作用。总之，本研究确定了一种新的 SFR 毒性机制，由于 DNA 损伤是已建立的抗肿瘤药物细胞毒性的主要机制之一，因此该机制可能有助于在抗癌药物方案中利用 SFR 的治疗价值。
