Chem Res Toxicol. 2022 Nov 21;35(11):2152-2159. doi: 10.1021/acs.chemrestox.2c00246. Epub 2022 Oct 19.
In a series of previous studies we reported that black raspberry (BRB) powder inhibits dibenzo[,]pyrene (DBP)-induced DNA damage, mutagenesis, and oral squamous cell carcinoma (OSCC) development in mice. In the present study, using human oral leukoplakia (MSK-Leuk1) and squamous cell carcinoma (SCC1483) cells, we tested the hypothesis that BRB extract (BRBE) will enhance the synthesis of glutathione (GSH) and in turn increase GSH conjugation of the fjord-region DBP diol epoxide (DBPDE) derived from DBP leading to inhibition of DBP-induced DNA damage. The syntheses of DBPDE-GSH conjugate, DBPDE-dA adduct, and the corresponding isotope-labeled internal standards were performed; LC-MS/MS methods were used for their quantification. BRBE significantly ( < 0.05) increased cellular GSH by 31% and 13% at 6 and 24 h, respectively, in OSCC cells; in MSK-LeuK1 cells, the levels of GSH significantly ( < 0.05) increased by 55% and 22%, at 1 and 6 h. Since BRBE significantly enhanced the synthesis of GSH in both cell types, subsequent experiments were performed in MSK-Leuk1 cells. Western blot analysis was performed to determine the types of proteins involved in the synthesis of GSH. BRBE significantly ( < 0.05) increased the protein expression (2.5-fold) of the glutamate-cysteine ligase catalytic subunit (GCLC) but had no effect on the glutamate-cysteine ligase modifier subunit (GCLM) and glutathione synthetase (GSS). LC-MS/MS analysis showed that pretreatment of cells with BRBE followed by DBPDE significantly ( < 0.05) increased the levels of DBPDE-GSH conjugate (2.5-fold) and decreased DNA damage by 74% measured by assessing levels of DBPDE-dA adduct formation. Collectively, the results of this study clearly support our hypothesis, and the LC-MS/MS methods developed in the present study will be highly useful in testing the same hypothesis initially in our mouse model and ultimately in smokers.
在之前的一系列研究中，我们报道黑覆盆子（BRB）粉末可抑制二苯并[，]芘（DBP）诱导的 DNA 损伤、致突变和口腔鳞状细胞癌（OSCC）在小鼠中的发展。在本研究中，我们使用人口腔白斑（MSK-Leuk1）和鳞状细胞癌（SCC1483）细胞，测试了 BRB 提取物（BRBE）将增强谷胱甘肽（GSH）的合成，进而增加 DBP 诱导的 fjord 区 DBP 二醇环氧化物（DBPDE）的 GSH 缀合，从而抑制 DBP 诱导的 DNA 损伤的假设。合成了 DBPDE-GSH 缀合物、DBPDE-dA 加合物及其相应的同位素标记内标物；使用 LC-MS/MS 方法对其进行定量。BRBE 分别在 6 和 24 h 时显著（<0.05）增加了 OSCC 细胞中的细胞 GSH 31%和 13%；在 MSK-LeuK1 细胞中，GSH 水平分别显著（<0.05）增加了 55%和 22%，在 1 和 6 h。由于 BRBE 显著增加了两种细胞类型中 GSH 的合成，因此随后在 MSK-Leuk1 细胞中进行了实验。进行了 Western blot 分析以确定参与 GSH 合成的蛋白质类型。BRBE 显著（<0.05）增加了谷氨酸半胱氨酸连接酶催化亚基（GCLC）的蛋白表达（2.5 倍），但对谷氨酸半胱氨酸连接酶修饰亚基（GCLM）和谷胱甘肽合成酶（GSS）没有影响。LC-MS/MS 分析表明，用 BRBE 预处理细胞，然后用 DBPDE 处理，可显著（<0.05）增加 DBPDE-GSH 缀合物的水平（2.5 倍），并降低 74%的 DNA 损伤，通过评估 DBPDE-dA 加合物形成的水平来测量。总之，这项研究的结果清楚地支持了我们的假设，并且本研究中开发的 LC-MS/MS 方法将非常有助于首先在我们的小鼠模型中并最终在吸烟者中测试相同的假设。
