Toxicological evaluation of (processed) red meat ingredients and importance of heme oxygenase 1 in the context of colorectal carcinogenesis

结直肠癌（CRC）的形成与加工肉和红肉的食用有关。摄入肉类后，血红素从胃肠道的蛋白质中释放出来，导致肠上皮细胞氧化应激、DNA链断裂和突变。如果再用亚硝酸盐腌制红肉，血红素就会被亚硝基化，形成粉红色的no -血红素。一般认为亚硝酸盐会增加肉类化合物的毒性，因此目前正在测试ZnPP等色素替代品。不幸的是，ZnPP在肉类中的毒性潜能尚未得到分析。与预期相反，我们能够证明亚硝基化血红素在体外试验中具有与血红素相同甚至更低的毒性。此外，食物中亚硝酸盐含量越高，血红素诱导的尿脂质过氧化产物含量越低。亚硝酸盐的有益作用可能与血红素加氧酶1 （HO-1）有关，这是一种负责血红素降解的解毒酶。no -血红素与HO-1的相互作用从未被测试过，由此我们建立了唯一可靠的no -血红素合成方法。即使假设血红素、亚硝酸盐和HO-1等因素参与了加工肉和红肉相关肿瘤的形成，但大多数方面仍然未知。因此，该项目的目的是分析这些潜在的分子机制，以及由于包括多种因素的复杂食物基质可能发生的协同效应。将回答以下主要问题：1)亚硝酸盐的添加是否会影响红肉中血红素的毒性作用？2) HO-1在（加工过的）红肉相关CRC形成中的作用是什么？3) HO-1抑制剂ZnPP是否适合作为加工红肉中亚硝酸盐的无毒替代品？通过体外消化模型，将揭示亚硝基化反应等内源性修饰，同时将在广泛的体外毒性试验中评估消化后肉类样品的毒性以及血红素化合物、亚硝酸盐和ZnPP等选定因素。其中一个主要方面是不同血红素化合物和ZnPP与HO-1的相互作用，使用已建立的结肠细胞系以及无细胞模型。HO-1活性似乎也与肿瘤进展有关。因此，在HO-1存在和不存在的情况下，我们将分析肉类化合物在细胞迁移、上皮-间质转化和不依赖锚定生长方面的毒性作用。

Colorectal cancer (CRC) formation is associated with processed and red meat consumption. Upon meat intake, heme is released from protein in the gastrointestinal tract, which results in oxidative stress, DNA strand breaks and mutations in intestinal epithelial cells. If red meat is additionally cured using nitrite, heme will be nitrosylated resulting to the formation of pink-colored NO-heme. It is generally assumed that nitrite increase the toxicity of meat compounds and therefore coloring alternatives like ZnPP are currently tested. Unfortunately, the toxic potential of ZnPP in meat was not yet analyzed. Contrary to the expectation, we were able to show that nitrosylated heme possess an equal or even lower toxicity in in vitro assays than heme. Moreover, as higher the nitrite content in food, as lower was the heme-induced urinary content of lipid peroxidation products. The beneficial effects of nitrite may relate to heme oxygenase 1 (HO-1), an detoxification enzyme responsible for the degradation of heme. The interaction of NO-heme with HO-1 was never tested before, whereby we established the only reliable synthesis method for NO-heme. Even if it was hypothesized that factors like heme, nitrite and HO-1 are involved in processed and red meat-related tumor formation, most aspects are still unknown. Therefore the aim of the project is to analyze such underlying molecular mechanisms as well as possibly occurring synergistic effects due to the complex food matrix including several factors. The following main questions will be answered: 1) Does the addition of nitrite influence the toxic effects of heme from red meat? 2) What is the role of HO-1 in (processed) red meat-associated CRC formation? 3) Is the HO-1 inhibitor ZnPP suitable as non-toxic nitrite alternative in processed red meat? By using an in vitro digestion model, endogenous modifications like nitrosylation reactions will be revealed, while the toxicity of digested meat samples and selected factors like heme compounds, nitrite and ZnPP will be evaluated in a broad range of in vitro toxicity assays. One of the main aspects is the interaction of the different heme compounds and ZnPP with HO-1 using established colon cell lines as well as a cell-free model. The HO-1 activity seems also be involved in tumor progression. Therefore, the toxic effects of meat compounds in presence and absence of HO-1 will be analyzed regarding cell migration, epithelial-mesenchymal transition and anchorage-independent growth.