由于其特征性的肝脏独特功能,永生化的人肝细胞HepG2细胞系通常用于毒理学研究,作为动物测试的替代方法。然而,对毒素暴露后这些细胞内的基线代谢变化知之甚少。我们已经应用高分辨率1H核磁共振(NMR)光谱来表征HepG2细胞在基线和暴露于过氧化氢(H2O2)后的生化组成。活细胞的代谢概况,细胞提取物,使用1H高分辨率幻角旋转(HR-MAS)NMR和1HNMR光谱技术获得了它们的废培养基上清液。使用正交偏最小二乘判别分析(O-PLS-DA)来表征基线组和H2O2处理组之间不同的代谢物。结果表明,H2O2引起10种代谢物的改变,包括醋酸盐,谷氨酸,脂质,磷酸胆碱,和活细胞中的肌酸;25种代谢物,包括醋酸盐,丙氨酸,二磷酸腺苷(ADP),天冬氨酸,柠檬酸盐肌酸,葡萄糖,谷氨酰胺,谷胱甘肽,和细胞提取物中的乳酸,和22种代谢物,包括醋酸盐,丙氨酸,甲酸盐,葡萄糖,丙酮酸,苯丙氨酸,苏氨酸,色氨酸,酪氨酸,和细胞上清液中的缬氨酸。毒素暴露后,至少有10条与这些代谢物相关的生化途径被破坏,包括那些参与能源的人,脂质,和氨基酸代谢。我们的发现说明了永生化人类细胞的基于NMR的代谢谱检测毒素暴露对中枢代谢的影响的能力。建立的数据集将使HepG2模型细胞系统中更微妙的生化变化能够在未来的毒性测试中得到鉴定。
The immortalised human hepatocellular
HepG2 cell line is commonly used for toxicology studies as an alternative to animal testing due to its characteristic liver-distinctive functions. However, little is known about the baseline metabolic changes within these cells upon toxin exposure. We have applied high-resolution 1H Nuclear Magnetic Resonance (NMR) spectroscopy to characterise the biochemical composition of
HepG2 cells at baseline and post-exposure to hydrogen peroxide (H2O2). Metabolic profiles of live cells, cell extracts, and their spent media supernatants were obtained using 1H high-resolution magic angle spinning (HR-MAS) NMR and 1H NMR spectroscopic techniques. Orthogonal partial least squares discriminant analysis (O-PLS-DA) was used to characterise the metabolites that differed between the baseline and H2O2 treated groups. The results showed that H2O2 caused alterations to 10 metabolites, including acetate, glutamate, lipids, phosphocholine, and creatine in the live cells; 25 metabolites, including acetate, alanine, adenosine diphosphate (ADP), aspartate, citrate, creatine, glucose, glutamine, glutathione, and lactate in the cell extracts, and 22 metabolites, including acetate, alanine, formate, glucose, pyruvate, phenylalanine, threonine, tryptophan, tyrosine, and valine in the cell supernatants. At least 10 biochemical pathways associated with these metabolites were disrupted upon toxin exposure, including those involved in energy, lipid, and amino acid metabolism. Our findings illustrate the ability of NMR-based metabolic profiling of immortalised human cells to detect metabolic effects on central metabolism due to toxin exposure. The established data sets will enable more subtle biochemical changes in the
HepG2 model cell system to be identified in future toxicity testing.