Objective: To explore the effects and related mechanisms of oral exposure titanium dioxide nanoparticles (TiO2 NPs) for 90 days on the intestinal and the gut microbiota of rats, through fecal metabolomics.
Methods: Twelve 4-week-old clean-grade Sprague Dawley (SD) rats were randomly de-vided into 2 groups by body weight, treated with TiO2 NPs at dose of 0 or 50 mg/kg body weight everyday respectively for 90 days. The solution of each infection was freshly prepared and shocked fully by ultrasonic. Characterization of the particle size, crystal form, purity, and specific surface area of TiO2 NPs was conducted. And the fresh feces of the rats were collected on the 90th day. After lyophilized and hydrophilic phase extraction, ultra performance liquid chromatography-Q-exactive orbitrap-high-resolution mass spectrometry system (UPLC-QEMS) was utilized for non-targeted determination of fecal meta-bolites. The metabolites were identified and labeled through Compound Discoverer 3.0 software, and used for subsequent metabolomics analysis. Bioinformatics analysis was carried out including unsupervised principal component analysis and supervised orthogonal projection to latent structure discriminant analysis for the differential metabolites between the two groups. The differential metabolites were followed-up for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis.
Results: Compared with the control group, the body weight of the rats was significantly reduced (P<0.05) in the treatment group. A total of 22 metabolites in fecal metabolomics showed significant changes. Among them, xanthine, 1-methyladenine, 3-hydroxypyridine, methionine sulfoxide, pyridoxine, 1,5-isoquinolinediol, N-acetylornithine, N-acetyl-D-galactosamine, L-citrulline, L-methionine, leucine, DL-tryptophan, L-ornithine, 4-methyl-5-thiazoleethanol, and L-glutamic acid totaled 15 metabolites increased significantly. N-acetylhistamine, D-pipecolinic acid, imidazolelactic acid, L-valine, 2,3,4,6-tetramethylpyrazine, caprolactam, and histamine totaled 7 metabolites decreased significantly. N-acetylhistamine, L-valine and methionine sulfoxide were changed more than 16 times. Analysis of KEGG pathway revealed that the two metabolic pathways arginine biosynthesis and aminoacyl-tRNA biosynthesis were significantly changed (false discover rate < 0.05, pathway impact > 0.1).
Conclusion: Oral exposure to TiO2 NPs for 90 days could disrupt the metabolism of the intestine and gut microbiota, causing significant changes in metabolites and metabolic pathways which were related to inflammatory response, oxidative stress, glucose homeostasis, blood system and amino acid homeostasis in rat feces. It is suggested that the toxic effect of TiO2 NPs on rats may be closely related to intestinal and gut microbiota metabolism.
目的: 通过粪便代谢组学,探索纳米二氧化钛(titanium dioxide nanoparticles,TiO2 NPs)经口染毒90 d对大鼠肠道及肠道菌群代谢的影响及其相关机制。
方法: 12只清洁级雄性Sprague Dawley(SD)大鼠按体质量随机分成两组,分别以0和50 mg/kg体质量的TiO2 NPs持续灌胃90 d,对TiO2 NPs的粒径、晶型、纯度、比表面积进行表征,并在第90天收集大鼠的新鲜粪便。经过冻干、亲水相萃取等前处理后,使用超高效液相色谱-轨道阱高分辨质谱仪联用系统(ultra performance liquid chromatography-Q-exactive orbitrap-high-resolution mass spectrometry system,UPLC-QEMS)对粪便代谢物进行非靶向测定,鉴定标注检测得到的代谢物,并进行代谢组学分析。
结果: 与对照组相比,TiO2 NPs染毒组大鼠体质量显著降低(P<0.05)。粪便代谢组学共发现22种代谢物浓度发生显著改变,其中黄嘌呤、甲基腺嘌呤、羟基吡啶、蛋氨酸亚砜等15种代谢物浓度显著上升,乙酰组胺、派可林酸、咪唑乳酸、缬氨酸等7种代谢物浓度显著下降。N-乙酰组胺、缬氨酸和蛋氨酸亚砜的改变倍数大于16倍。京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路分析发现,精氨酸生物合成通路和氨酰基-tRNA生物合成通路这两个代谢通路发生显著改变(错误发现率<0.05,通路受影响程度>0.10)。
结论: TiO2 NPs经口染毒90 d可扰乱肠道及肠道菌群代谢,并导致大鼠粪便中代谢物及代谢通路发生显著改变,提示TiO2 NPs经口暴露对大鼠的毒性作用可能与肠道及肠道菌群代谢改变密切相关。
Keywords: Feces; Metabolomics; Rats, Sprague-Dawley; Titanium dioxide nanoparticles.