Nano-sized TiO₂ (nTiO₂) exerts an oxidative effect on cells upon exposure to solar or UV irradiation and ecotoxicity of the nTiO₂ is an urgent concern. Little information is available regarding the effect of TiO₂ on cells under dark conditions. Metabolomics is a unique approach to the discovery of biomarkers of nTiO₂ cytotoxicity, and leads to the identification of perturbed metabolic pathways and the mechanism underlying nTiO₂ toxicity. In the present study, gas chromatography mass spectrometry (GC/MS)-based metabolomics was performed to investigate the effect of nTiO₂ on sensitive cells (P. polycephalum macroplasmodium) under dark conditions. According to the multivariate pattern recognition analysis, at least 60 potential metabolic biomarkers related to sugar metabolism, amino acid metabolism, nucleotide metabolism, polyamine biosynthesis, and secondary metabolites pathways were significantly perturbed by nTiO₂. Notably, many metabolic biomarkers and pathways were related to anti-oxidant mechanisms in the living organism, suggesting that nTiO₂ may induce oxidative stress, even under dark conditions. This speculation was further validated by the biochemical levels of reactive oxygen species (ROS), 8-hydroxy-2-deoxyguanosine (8-OHdG), and total soluble phenols (TSP). We inferred that the oxidative stress might be related to nTiO₂-induced imbalance of cellular ROS. To the best of our knowledge, the present study is the first to investigate the nTiO₂-induced metabolic perturbations in slime mold, provide a new perspective of the mechanism underlying nTiO₂ toxicity under dark conditions, and show that metabolomics can be employed as a rapid, reliable and powerful tool to investigate the interaction among organisms, the environment, and nanomaterials.

纳米级TiO ₂（nTiO ₂）在暴露于太阳或紫外线照射下会对细胞产生氧化作用，因此nTiO _2的生态毒性已成为迫切需要关注的问题。关于TiO ₂在黑暗条件下对细胞的作用的信息很少。代谢组学是发现nTiO ₂细胞毒性生物标志物的独特方法，并导致了扰动的代谢途径和nTiO ₂毒性的潜在机制的鉴定。在本研究中，进行了基于气相色谱质谱（GC / MS）的代谢组学研究，以研究nTiO ₂对敏感细胞（多头P）的影响。在黑暗条件下）。根据多变量模式识别分析，nTiO ₂显着干扰了至少60个与糖代谢，氨基酸代谢，核苷酸代谢，多胺生物合成和次生代谢途径相关的潜在代谢生物标志物。值得注意的是，许多代谢生物标志物和途径都与生物体内的抗氧化机制有关，这表明即使在黑暗条件下，nTiO ₂也会诱导氧化应激。通过活性氧（ROS），8-羟基-2-脱氧鸟苷（8-OHdG）和总可溶性酚（TSP）的生化水平进一步证实了这种推测。我们推断氧化应激可能与nTiO _2有关诱导的细胞ROS失衡。据我们所知，本研究是第一个研究粘液霉菌中nTiO ₂诱导的代谢扰动的方法，为黑暗条件下nTiO ₂毒性的潜在机理提供了新的视角，并表明代谢组学可以作为一种快速，可靠和强大的工具来研究生物，环境和纳米材料之间的相互作用。