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

背景：接触多氯联苯与暴露于多氯联苯的妇女的后代的神经损伤、儿童的认知障碍和成年人的记忆力减退有关。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。肠道微生物区系在多氯联苯生物转化中的作用研究较少。通过氧化应激介导的多氯联苯毒性可能通过核因子红系相关因子2(NRF2)诱导的抗氧化反应而被膳食多酚保护。然而，多酚很难被吸收，直到它们经过肠道微生物生物转化，产生更多可生物利用的次生代谢物。多酚代谢产物可能会改变多氯联苯的代谢和随后的结合途径，从而调节多氯联苯的毒性。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体肠道、肝脏和神经细胞上使用多氯联苯或多酚，而不将这些化合物暴露在中间的消化或代谢过程中。研究进展：使用计算机控制的动态人体胃肠模型(SHGI模型)分别对多氯联苯和多酚进行了体外模拟人体消化，该模型模拟了整个人体胃肠道的不同消化阶段。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区系的结肠血管。利用LC-MS和HPLC法，我们发现常见的膳食多酚导致了GI模型升、横、降结肠血管中不同的多酚代谢产物谱和抗氧化能力。我们开发了Caco-2/HepG2细胞共培养来模拟人类肠道和肝脏的首过代谢。在GI模型结肠消化多酚后，对共培养物进行生物转化和吸收过程，我们检测到先前在摄入多酚的人的血浆中发现的微生物多酚副产物。两种结构不同的多氯联苯同系物，类二恶英(PCB126)和非二恶英类多氯联苯(PCB153)分别符合GI模型，因为它们是人类饮食和血浆中的主要同系物。目的：将GI模型与模拟首过代谢共培养体系相结合，研究多氯联苯对人体肠道、肝脏和神经细胞的毒性作用机制；以及(2)评估多酚微生物代谢产物对多氯联苯代谢和毒性的影响，重点研究多氯联苯代谢产物、氧化应激、炎症和Nrf2信号通路。实验设计：将人的肠道、肝脏和神经细胞暴露于GI模型和First Pass代谢系统产生的亲本多氯联苯和多酚及其代谢物。多氯联苯和多酚代谢物将通过LC-MS和HPLC进行评估。细胞将用识别的多酚代谢物进行预处理，以测试它们对多氯联苯介导的新陈代谢和毒性的影响。神经效应将在人类神经前体细胞共培养到多孔插入系统上进行测试，该系统以人脑内皮毛细血管细胞系hCMEC/D3作为人血脑屏障的模型。将测量细胞存活率、细胞内活性氧积累和炎性细胞因子。NRF2水平将通过Western印迹进行评估。意义：这项研究将为多氯联苯引起的炎症和氧化应激提供新的见解，这些炎症和氧化应激是由肠道代谢和与饮食多酚的相互作用所介导的。

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.