Investigation of the protective effects of polyphenols on polychlorinated biphenyl-induced inflammation and oxidative stress: impact of biotransformation

多酚对多氯联苯诱导的炎症和氧化应激的保护作用的研究：生物转化的影响

• 批准号: RGPIN-2014-06291
• 负责人: Kubow, Stan
• 金额: $ 3.79万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=656068
• 关键词: Investigation; protective; effects; polyphenols; polychlorinated

Background: PCB exposure is linked with neurologic damage in offspring of PCB-exposed women, cognitive disorders in children and poorer memory in adults. Despite food as the main source of PCBs, little is known about direct effects of PCBs on the gut cells. The role of gut microflora in PCB biotransformation is sparsely studied. PCB toxicity mediated via oxidative stress might be protected by dietary polyphenols via an antioxidant response induced via the nuclear factor erythroid 2-related factor 2 (Nrf2). Polyphenols, however, are poorly absorbed until they undergo gut microbial biotransformation that leads to more bioavailable secondary metabolites. Polyphenol metabolites may alter metabolism of PCBs and subsequent conjugation pathways that can modulate PCB toxicity. A limitation of existing mechanistic studies using human cell culture studies is the direct use of PCBs or polyphenols on human intestinal, hepatic and neuronal cells without exposure of such compounds to intermediary digestive or metabolic processes. *Research Progress: PCBs and polyphenols separately underwent human simulated in vitro digestion using a Computer Controlled Dynamic Human Gastrointestinal Model (SHGI model) that mimics the different stages of digestion throughout the human GI tract. This model includes the use of colonic vessels containing human microbiota obtained from human fecal samples. Using LC-MS and HPLC, we showed that the common dietary polyphenols led to differing polyphenol metabolite profiles and antioxidant capacities in the ascending, transverse and descending colon vessels of the GI model. We developed Caco-2/HepG2 cell co-cultures to mimic human intestinal and hepatic first pass metabolism. After the GI model colonic digests of the polyphenols were subjected to the biotransformation and absorptive processes of the co-cultures, we detected microbial polyphenol by-products previously noted in plasma of humans ingesting polyphenols. Two structurally different PCB congeners, dioxin-like (PCB-126) and non-dioxin-like PCB (PCB-153), were separately subjected to the GI model as they are dominant congeners in the human diet and in human plasma. The LC-MS profiles of the PCB metabolites generated from the GI model and co-cultures and the human microbiota profiles seen in the GI model after the polyphenol and PCB exposures are being studied.*Objectives: The overall objectives are to combine the GI model with the simulated first pass metabolism co-culture system to: (1) identify biotransformed metabolites of PCBs in the study of mechanisms of PCB-induced toxicity on human intestinal, hepatic and neural cells; and (2) assess the effects of polyphenol microbial metabolites on PCB metabolism and toxicity with a focus on PCB metabolites, oxidative stress, inflammation and the Nrf2 signalling pathway.*Experimental Design: Human intestinal, hepatic and neuronal cells will be exposed to parent PCBs and polyphenols and their metabolites generated by GI model and first pass metabolism system. PCB and polyphenol metabolites will be assessed via LC-MS and HPLC. Cells will be pre-treated with the identified polyphenol metabolites to test for their effects on PCB-mediated metabolism and toxicity. Neural effects will be tested on human neural progenitor cells co-cultured onto a Multiwell insert system with the human brain endothelial capillary cell line hCMEC/D3 as a model for the human blood brain barrier. Cell viability, intracellular reactive oxygen species accumulation and inflammatory cytokines will be measured. Nrf2 levels will be assessed via Western blot. *Significance: This study will provide new insights of PCB-induced inflammation and oxidative-stress as mediated by gut metabolism and interactions with with dietary polyphenols.

背景：PCB 接触与接触 PCB 的女性后代的神经损伤、儿童认知障碍和成人记忆力较差有关。尽管食物是多氯联苯的主要来源，但人们对多氯联苯对肠道细胞的直接影响知之甚少。肠道微生物群在 PCB 生物转化中的作用研究很少。通过氧化应激介导的 PCB 毒性可能会受到膳食多酚的保护，通过核因子红细胞 2 相关因子 2 (Nrf2) 诱导的抗氧化反应。然而，多酚的吸收率很低，直到它们经过肠道微生物生物转化，产生更多生物可利用的次级代谢产物。多酚代谢物可能会改变 PCB 的代谢以及随后调节 PCB 毒性的结合途径。使用人类细胞培养研究的现有机制研究的一个局限性是直接在人类肠道、肝脏和神经元细胞上使用多氯联苯或多酚，而不将此类化合物暴露于中间消化或代谢过程。 *研究进展：使用计算机控制的动态人体胃肠模型（SHGI 模型）模拟整个人体胃肠道消化的不同阶段，对 PCB 和多酚分别进行人体模拟体外消化。该模型包括使用含有从人类粪便样本中获得的人类微生物群的结肠血管。使用 LC-MS 和 HPLC，我们发现常见的膳食多酚导致胃肠道模型的升结肠、横结肠和降结肠血管中的多酚代谢谱和抗氧化能力不同。我们开发了 Caco-2/HepG2 细胞共培养物来模拟人类肠道和肝脏的首过代谢。在对多酚的胃肠道模型结肠消化物进行共培养物的生物转化和吸收过程后，我们检测到先前在摄入多酚的人类血浆中注意到的微生物多酚副产物。两种结构不同的 PCB 同系物，二恶英类 PCB (PCB-126) 和非二恶英类 PCB (PCB-153)，分别接受胃肠道模型的研究，因为它们是人类饮食和人类血浆中的主要同系物。正在研究从胃肠道模型和共培养中生成的 PCB 代谢物的 LC-MS 谱，以及在多酚和 PCB 暴露后在 GI 模型中看到的人类微生物群谱。*目标：总体目标是将 GI 模型与模拟首过代谢共培养系统相结合，以：（1）在研究多酚和 PCB 的机制中识别 PCB 的生物转化代谢物。 PCB对人体肠道、肝脏和神经细胞的毒性； (2) 评估多酚微生物代谢物对 PCB 代谢和毒性的影响，重点关注 PCB 代谢物、氧化应激、炎症和 Nrf2 信号通路。*实验设计：人类肠道、肝脏和神经元细胞将暴露于胃肠道模型和首过代谢系统产生的母体 PCB 和多酚及其代谢物。 PCB 和多酚代谢物将通过 LC-MS 和 HPLC 进行评估。将用已鉴定的多酚代谢物对细胞进行预处理，以测试它们对 PCB 介导的代谢和毒性的影响。神经效应将在与人脑内皮毛细细胞系 hCMEC/D3 共培养到多孔插入系统上的人神经祖细胞上进行测试，作为人血脑屏障的模型。将测量细胞活力、细胞内活性氧积累和炎症细胞因子。 Nrf2 水平将通过蛋白质印迹进行评估。 *意义：这项研究将为肠道代谢以及与膳食多酚的相互作用介导的 PCB 诱导的炎症和氧化应激提供新的见解。