Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences, University of Alberta, Edmonton, Alberta T6G 2R3, Canada.
Applied Pharmaceutical Innovation, University of Alberta, Edmonton, Alberta T6G 2R3, Canada.
Chem Res Toxicol. 2024 Oct 21;37(10):1738-1746. doi: 10.1021/acs.chemrestox.4c00265. Epub 2024 Oct 3.
6-PPD (-[1,3-dimethylbutyl]-'-phenyl--phenylenediamine) is an industrial antioxidant reported to be an environmental contaminant. It was found to be highly toxic to coho salmon and potentially other aquatic organisms. The toxicity of 6-PPD in humans, however, remains unknown. The neutrophil enzyme myeloperoxidase (MPO) is known to catalyze xenobiotic metabolism; therefore, its role in 6-PPD cytotoxicity was investigated using the MPO-rich HL-60 cell line. UV-visible spectroscopy and liquid chromatography-mass spectrometry (LC/MS) were performed to investigate the MPO-mediated oxidation of 6-PPD and identify possible metabolites in the absence and presence of glutathione (GSH). 6-PPD's cytotoxicity, effect on mitochondrial membrane potential (MMP), and GSH-depleting ability in HL-60 cells were assessed. Electron paramagnetic resonance (EPR) was used to determine GSH radical formation using DMPO, and mitochondrial-derived superoxide was assessed with the mito-TEMPO-H probe. Evaluation of the 6-PPD-induced cellular injury pathways was performed by preincubating an antioxidant and an MPO inhibitor with HL-60 cells. UV-vis analysis of MPO-catalyzed oxidation of 6-PPD demonstrated changes in the 6-PPD spectrum, whereas the addition of GSH altered the spectrum, indicating possible GSH conjugate formation. LC/MS showed the formation of multiple products, including GSH-6-PPD conjugates and a GSH conjugate to a 4-hydroxydiphenylamine (a known 6-PPD degradant), which could potentially induce cytotoxicity. 6-PPD demonstrated concentration-dependent cytotoxicity, and cellular GSH levels were decreased by 6-PPD. Similarly, the level of MMP decreased, suggesting mitochondrial depolarization. Furthermore, the EPR spin probe for mitochondrial superoxide showed a positive relationship with 6-PPD concentration, and EPR spin-trapping demonstrated 6-PPD concentration-dependent GSH radical signal intensity using MPO/HO. The GSH precursor, NAC, demonstrated partial cytoprotection against 6-PPD; however, the MPO inhibitor PF-1355 surprisingly showed no significant cytoprotective effect. Our results suggest that MPO could be a potential catalyst for 6-PPD toxicity in humans. However, MPO inhibition did not significantly affect cellular viability, suggesting an MPO-independent toxicity pathway. These findings warrant a deeper investigation to determine 6-PPD mammalian toxicity pathways.
6-PPD（1,3-二甲基丁基）-苯基-对苯二胺是一种工业抗氧化剂，据报道也是一种环境污染物。它被发现对银大麻哈鱼和其他潜在的水生生物具有高度毒性。然而，6-PPD 对人类的毒性尚不清楚。中性粒细胞酶髓过氧化物酶（MPO）已知能催化外来生物的新陈代谢；因此，使用富含 MPO 的 HL-60 细胞系研究了其在 6-PPD 细胞毒性中的作用。进行了紫外可见光谱和液相色谱-质谱（LC/MS）分析，以研究 MPO 介导的 6-PPD 氧化，并在没有和存在谷胱甘肽（GSH）的情况下鉴定可能的代谢物。评估了 6-PPD 在 HL-60 细胞中的细胞毒性、对线粒体膜电位（MMP）的影响以及 GSH 耗竭能力。使用 DMPO 通过电子顺磁共振（EPR）测定 GSH 自由基的形成，并用 mito-TEMPO-H 探针评估线粒体衍生的超氧化物。通过用 HL-60 细胞预孵育抗氧化剂和 MPO 抑制剂来评估 6-PPD 诱导的细胞损伤途径。MPO 催化的 6-PPD 氧化的 UV-vis 分析表明 6-PPD 光谱发生了变化，而添加 GSH 则改变了光谱，表明可能形成了 GSH 缀合物。LC/MS 显示形成了多种产物，包括 GSH-6-PPD 缀合物和 4-羟基二苯胺（已知的 6-PPD 降解物）的 GSH 缀合物，这可能潜在地诱导细胞毒性。6-PPD 表现出浓度依赖性的细胞毒性，细胞内 GSH 水平因 6-PPD 而降低。同样，MMP 水平降低，表明线粒体去极化。此外，线粒体超氧化物的 EPR 自旋探针与 6-PPD 浓度呈正相关，并且 EPR 自旋捕获使用 MPO/HO 显示出与 6-PPD 浓度依赖性的 GSH 自由基信号强度。GSH 前体 NAC 对 6-PPD 表现出部分细胞保护作用；然而，令人惊讶的是，MPO 抑制剂 PF-1355 并没有显示出显著的细胞保护作用。我们的结果表明，MPO 可能是人类 6-PPD 毒性的潜在催化剂。然而，MPO 抑制并没有显著影响细胞活力，表明存在 MPO 非依赖性毒性途径。这些发现需要更深入的研究来确定 6-PPD 哺乳动物毒性途径。
