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

背景：多氯联苯暴露与多氯联苯暴露妇女后代的神经损伤、儿童认知障碍和成人记忆力下降有关。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。肠道菌群在PCB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2相关因子2 （Nrf2）诱导的抗氧化反应而受到膳食多酚的保护。然而，多酚很难被吸收，直到它们经历肠道微生物的生物转化，从而产生更多的生物可利用的次生代谢物。多酚代谢物可能改变多氯联苯的代谢和随后的偶联途径，从而调节多氯联苯的毒性。利用人类细胞培养研究的现有机制研究的一个局限性是，多氯联苯或多酚直接使用于人类肠道、肝脏和神经细胞，而不使这些化合物暴露于中间消化或代谢过程。研究进展：利用计算机控制的动态人体胃肠道模型（SHGI模型）模拟人体胃肠道消化的不同阶段，分别对多氯联苯和多酚进行了人体模拟体外消化。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。通过LC-MS和HPLC，我们发现常见的膳食多酚导致GI模型升、横、降结肠血管中多酚代谢物谱和抗氧化能力不同。我们开发了Caco-2/HepG2细胞共培养来模拟人类肠道和肝脏的第一次代谢。在多酚的GI模型结肠消化经历了共同培养的生物转化和吸收过程后，我们检测到了之前在摄入多酚的人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，二恶英样多氯联苯（PCB-126）和非二恶英样多氯联苯（PCB-153），分别受到GI模型的影响，因为它们是人类饮食和血浆中的主要同系物。目前正在研究多酚和多氯联苯暴露后，胃肠道模型和共培养物产生的多氯联苯代谢物的LC-MS谱，以及胃肠道模型中出现的人类微生物群谱。目的：总体目标是将胃肠道模型与模拟第一次代谢共培养系统相结合，：(1)鉴定多氯联苯的生物转化代谢物，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；(2)评估多酚微生物代谢物对多氯联苯代谢和毒性的影响，重点关注多氯联苯代谢物、氧化应激、炎症和Nrf2信号通路。实验设计：人体肠道、肝脏和神经细胞将暴露于母体多氯联苯和多酚及其代谢物，这些代谢物是由GI模型和一过代谢系统产生的。PCB和多酚代谢物将通过LC-MS和HPLC进行评估。细胞将用鉴定的多酚代谢物进行预处理，以测试其对多氯联苯介导的代谢和毒性的影响。神经效应将在与人脑内皮毛细血管细胞系hCMEC/D3共同培养在Multiwell插入系统上的人类神经祖细胞上进行测试，作为人类血脑屏障的模型。细胞活力，细胞内活性氧积累和炎症因子将被测量。Western blot检测Nrf2水平。意义：本研究将为多氯联苯诱导的炎症和氧化应激通过肠道代谢及其与饮食多酚的相互作用提供新的见解。

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.