短链氯化石蜡对人体正常肝细胞的代谢干扰

doi:10.3724/SP.J.1123.2023.10037

摘要/Abstract

摘要：

短链氯化石蜡(SCCPs)是一类新兴的持久性有机污染物,广泛存在于环境基质和人体样本中。SCCPs具有环境持久性以及远距离迁移能力,能够在生物体内积累并具有广泛的生物毒性效应,威胁着人类健康。本研究采用代谢组学方法评估了低剂量组(1 μg/L)、中剂量组(10 μg/L)以及高剂量组(100 μg/L)的SCCPs暴露对人体正常肝细胞L02的代谢干扰。主成分分析(PCA)与代谢扰乱水平指数(MELI)计算结果表明3个剂量组的SCCPs均能够引起L02细胞代谢活动的紊乱。在3个暴露组中,72个差异代谢物经二级质谱图信息定性或标准品验证。其中,1 μg/L SCCPs暴露组与10 μg/L SCCPs暴露组、100 μg/L SCCPs暴露组分别有33个、36个相同的差异代谢物。10 μg/L SCCPs暴露组与100 μg/L SCCPs暴露组有46个相同的差异代谢物。3个暴露组有33个相同的差异代谢物。在72个经二级质谱图信息定性或标准品验证的差异代谢物中,参与氨基酸代谢、核苷酸代谢和脂质代谢通路的差异代谢物分别有9、9以及45个。富集通路分析结果表明:SCCPs对L02细胞的代谢干扰主要表现在脂质代谢、脂肪酸β氧化以及核苷酸代谢通路上,且中、高剂量的SCCPs暴露引起更广泛的代谢通路的紊乱。SCCPs暴露干扰了甘油磷脂以及鞘脂类的代谢通路,其中,磷脂酰胆碱、磷脂酰乙醇胺以及鞘磷脂丰度的显著变化表明SCCPs暴露对细胞的生物膜有一定的损伤。同时,SCCPs暴露通过降低短链和中链酰基肉碱的含量抑制L02细胞中脂肪酸β氧化,提示细胞通过氧化脂肪酸供能减少。值得注意的是,与中剂量和低剂量SCCPs相比,高剂量的SCCPs暴露对脂肪酸β氧化的抑制作用更强。此外,SCCPs暴露诱导了核苷酸代谢通路的紊乱。次黄嘌呤水平显著升高提示SCCPs暴露可能诱导了L02细胞的缺氧、活性氧增多或者致癌等相关不良效应。

关键词: 液相色谱, 质谱, 短链氯化石蜡, 肝细胞, 代谢组学, 脂质代谢, 核苷酸代谢

Abstract:

Short-chain chlorinated paraffins (SCCPs) are an emerging class of persistent organic pollutants (POPs) that are widely detected in environmental matrices and human samples. Because of their environmental persistence, long-range transport potential, bioaccumulation potential, and biotoxicity, SCCPs pose a significant threat to human health. In this study, metabolomics technology was applied to reveal the metabolomic interference in human normal hepatic (L02) cells after exposure to low (1 μg/L), moderate (10 μg/L), and high (100 μg/L) doses of SCCPs. Principal component analysis (PCA) and metabolic effect level index (MELI) values showed that all three SCCP doses caused notable metabolic perturbations in L02 cells. A total of 72 metabolites that were annotated by MS/MS and matched with the experimental spectra in the Human Metabolome Database (HMDB) or validated by commercially available standards were selected as differential metabolites (DMs) across all groups. The low-dose exposure group shared 33 and 36 DMs with the moderate- and high-dose exposure groups, respectively. The moderate-dose exposure group shared 46 DMs with the high-dose exposure group. In addition, 33 DMs were shared among the three exposure groups. Among the 72 DMs, 9, 9, and 45 metabolites participated in the amino acid, nucleotide, and lipid metabolism pathways, respectively. The results of pathway enrichment analysis showed that the most relevant metabolic pathways affected by SCCPs were the lipid metabolism, fatty acid β-oxidation, and nucleotide metabolism pathways, and that compared with low-dose exposure, moderate- and high-dose SCCP exposures caused more notable perturbations of these metabolic pathways in L02 cells. Exposure to SCCPs perturbed glycerophospholipid and sphingolipid metabolism. Significant alterations in the levels of phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins indicated SCCP-induced biomembrane damage. SCCPs inhibited fatty acid β-oxidation by decreasing the levels of short- and medium-chain acylcarnitines in L02 cells, indicating that the energy supplied by fatty acid oxidation was reduced in these cells. Furthermore, compared with low- and moderate-dose SCCPs, high-dose SCCPs produced a significantly stronger inhibition of fatty acid β-oxidation. In addition, SCCPs perturbed nucleotide metabolism. The higher hypoxanthine levels observed in L02 cells after SCCP exposures indicate that SCCPs may induce several adverse effects, including hypoxia, reactive oxygen species production, and mutagenesis in L02 cells.

Key words: liquid chromatography (LC), mass spectrometry (MS), short-chain chlorinated paraffins (SCCPs), hepatic cells, metabolomics, lipid metabolism, nucleotide metabolism

中图分类号:

O658

罗云, 耿柠波, 陈双双, 程琳, 张海军, 陈吉平. 短链氯化石蜡对人体正常肝细胞的代谢干扰[J]. 色谱, 2024, 42(2): 176-184.

LUO Yun, GENG Ningbo, CHEN Shuangshuang, CHENG Lin, ZHANG Haijun, CHEN Jiping. Metabolomic interference induced by short-chain chlorinated paraffins in human normal hepatic cells[J]. Chinese Journal of Chromatography, 2024, 42(2): 176-184.

图/表 6

图1 SCCPs暴露L02细胞24 h后引起的代谢扰动

Fig. 1 Effects of 24 h of exposure to short-chain chlorinated paraffins (SCCPs) on the metabolomic profiles of L02 cells a. Principal component analysis (PCA) score plot of metabolite levels in L02 cells; b. metabolic effect level index (MELI) values of different treatment groups; c. Venn diagram showing the number of shared differential metabolites (DMs) among groups. Significant differences (in comparison with the control) were determined using t-test (n=6). * P<0.05; ** P<0.01.

图2 对照组和SCCPs暴露组中72个差异代谢物的热图

Fig. 2 Heatmaps of 72 DMs in control and SCCP exposure groups False discovery rate (FDR)<0.05; variable importance in the projection (VIP)>1; P<0.05. FFAs: free fatty acids; PC: phosphatidylcholine; PE: phosphatidylethanolamine; SM: sphingomyelin; Lyso PC: lysophosphatidylcholine; Lyso PE: lysophosphatidylethanolamine; ADP: adenosine 5'-diphosphate; CDP: cytidine diphosphate; dAMP: deoxyadenosine monophosphate; dGDP: 2'-deoxyguanosine 5'-diphosphate; IMP: inosine monophosphate; NAD: nicotinamide adenine dinucleotide; UDP: uridine 5'-diphosphate; CoA: coenzyme A; MG: monoacylglyceride.

图3 通路富集分析显示的SCCPs暴露后显著干扰的代谢通路

Fig. 3 Metabolic pathways significantly perturbed by SCCP exposures based on pathway enrichment analysis

图4 SCCPs诱导的脂质代谢通路的紊乱

Fig. 4 Perturbations of lipid metabolism induced by SCCPs Significant differences were determined using t-test (n=6). * P<0.05.

图5 SCCPs诱导的脂肪酸代谢通路的紊乱

Fig. 5 Perturbations of fatty acid metabolism induced by SCCPs Significant differences were determined using t-test (n=6). * P<0.05.

图6 SCCPs暴露后核苷酸代谢通路相关代谢物的相对峰面积

Fig. 6 Relative peak areas of metabolites participating in the nucleotide metabolism pathway perturbed by SCCPs Significant differences were determined using t-test (n=6). * 0.01<P<0.05; ** P<0.01.

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