Maillard products and coffee roasting products activate NF-κB and Nrf2 in cell culture and intact human gut tissue ex vivo
Year: 2011
Type: dissertation
Abstract: Coffee is one of the most consumed beverages worldwide. The characteristic flavour and the stimulating effect of coffee are responsible for its popularity. Recent epidemiological studies also correlated a reduced risk of oxidative stress-associated diseases such as Alzheimer’s and Parkinson’s disease, brain, liver and colon cancer and diabetes mellitus to coffee consumption. To date, the bioactive coffee compounds causing a beneficial health effects in these oxidative stress-related diseases have not been identified. In the present study, Maillard products, which are formed during the roasting process in coffee by carbonyls and amines, were analysed. In detail, the role of Maillard products as potential triggers of cell signalling was investigated. Therefore, the ability of a Maillard model system (a ribose-lysine mixture which was heated at 120°C for 30 min) and of coffee (a Maillard-rich beverage) to activate NF-κB and Nrf2 was analysed. Both, NF-κB and Nrf2 are redox-sensitive transcription factors. NF-κB regulates the expression of genes coding amongst others for cytokines which control diverse cellular processes including immune response, inflammation and stress response. Nrf2, on the other hand, plays a role in the induction of detoxifying and antioxidant enzymes such as γ-glutamylcysteine synthetase, glutathione synthetase and phase II NADPH:quinone oxidoreductase 1 counteracting the unbalanced redox homeostasis during oxidative stress. Since both NF-κB and Nrf2 are involved in the development and balance of oxidative stress, the activation of NF-κB and Nrf2 by Maillard products was analysed. It was shown before that physiological concentrations of reactive oxygen species (ROS) especially H2O2 trigger various cell signalling pathways involving NF-κB, PKC and MAPK kinase. Indeed, Maillard products generate H2O2. Thus, the development of H2O2 by Maillard products and the role of H2O2 in the activation of NF-κB and Nrf2 were investigated in the present study. In the first part of the study, the generation of H2O2 by Maillard products was analysed by the FOXPCA assay. In a Maillard reaction mixture (Mrmh), consisting of 0.5 M D-ribose and L-lysine, which was heated at 120°C for 30 min, up to 180 µM H2O2 was detected. Even though a clear mechanism for the generation of H2O2 by Maillard products is not published yet, some substructures of Maillard products are suggested to be involved in the H2O2 formation. Amongst others, Maillard products with enediol and enaminol substructures, which tautomerize from Amadori products and dicarbonyls such as methylglyoxal, as well as reductones and aminoreductones are associated with the production of ROS in the presence of metals. Moreover, a mechanism is proposed highlighting the formation of H2O2 by autoxidation of α-hydroxyaldehydes. Thus, it is not surprising that H2O2 was also formed during caramelization of a heated ribose solution (ribh) but to a lower extent than in Mrmh. H2O2 was also detected in an unheated Maillard reaction mixture (Mrmu). Since neither an unheated ribose solution (ribu) nor an unheated lysine solution (lysu) generated H2O2, the formation of H2O2 in Mrmu was related to a reaction between ribose and lysine which can take place to a minor extent even at room temperature. Indeed, the H2O2 concentration was significantly lower in Mrmu than in Mrmh underlining the enhanced generation of H2O2 by Maillard products at increased temperatures. The amount of H2O2 in Mrmh increased with the sample concentration up to 25 mM Mrmh. However in Mrmh > 25 mM, the concentration of H2O2 decreased. To rule out any interference between the FOX reagent and the Maillard products of the Mrmh, the concentration of H2O2 in Mrmh was not only measured by the FOXPCA assay but also by oxygen electrodes. In the case of the FOXPCA assay, the oxidation of ferrous ions of the FOX reagent to ferric ions by H2O2 was measured photometrically. For the electrochemical detection of H2O2, the Clark and the Luminescent Dissolved Oxygen (LDO) electrodes were used. For this purpose, H2O2 was decomposed by catalase into water and oxygen which was detected by the oxygen electrodes. Similar Mrmh concentration response curves were observed by all detection methods used. Thus, the reduced amount of H2O2 in the presence of high Mrmh concentrations can be attributed to the radical scavenging activity of antioxidative active Maillard products. In this context, Maillard products with reductone structures were already identified as structures which -depending on their concentration- exhibit pro- as well as antioxidative activity. Next, the formation of H2O2 in Maillard reaction mixtures was monitored under various reaction conditions such as pH and temperature. Enhanced generation of H2O2 was measured directly after preparation of the Maillard mixtures at basic pH values up to pH 10 and also at high temperatures up to 130°C. Also during prolonged incubation of Maillard mixtures at 37°C for 96 h, the H2O2 content increased up to 370 µM. To test the hypothesis that H2O2 is not only generated at elevated temperatures during preparation of the Mrmh but also permanently at lower temperatures during prolonged storage, de novo generation of H2O2 was monitored at 4°C, 25°C and 37°C. Therefore, any H2O2 was removed in Mrmh by catalase and the generation of H2O2 was monitored for 96 h in each sample. After 1 h, H2O2 was generated under all tested conditions reaching levels between 50 and 100 µM. During prolonged incubation of a 25 mM Mrmh for 96 h, the amount of H2O2 stayed almost constant at 4°C, decreased at 25°C and increased at 37°C up to 200 µM. The same trend but at a lower H2O2 level was observed when Mrmh was adjusted to pH 5.5 instead of pH 8 after heating. In order to enrich H2O2 generating Maillard products, Mrmh was fractionated by size exclusion chromatography on a D-SaltTM polyacrylamide desalting column (cut off 1.8 kDa). Five H2O2-generating fractions (F5 - F9) out of 20 fractions were collected and unified into one active fraction. Next, the de novo generation of the active fraction was tested likewise. The active fraction also permanently produced H2O2 for up to 96 h reaching higher levels than the crude Mrmh (100 and 260 µM H2O2). Moreover, the amount of H2O2 in the active fraction incubated at 25°C did not decrease as shown for the crude Mrmh but rather stayed constant or increased indicating a successful enrichment of H2O2-generating Maillard products in the active fraction. Subsequently, it was investigated if Maillard products modulate cellular signalling by a H2O2 dependent mechanism. The intestine as part of the gastrointestinal tract represents a primary target for food-induced inter- and intracellular reactions of the human body. Thus, macrophages, Caco-2 cells (colorectal epithelial cells) and HIMEC (primary cells of the human intestinal microvascular endothelium), cells which are all present in the intestine, were incubated with Maillard mixtures. The activation of the redox-sensitive transcription factors NF-κB and Nrf2 was measured by their nuclear translocation. Firstly, macrophages and Caco-2 cells were stimulated with Mrmh for 2 h (short-term). Both, NF-κB and Nrf2 were significantly activated in a concentration dependent manner in Caco-2 cells and in macrophages up to 5-fold compared to PBS treated control cells. In primary HIMEC, NF-κB was activated 2-fold compared to PBS treated control cells after exposure to Mrmh for 2 h. Even though Mrmh activated both transcription factors in various cell lines and primary cells, the degree of activation varied strongly with the cell type showing the maximum effect in macrophages. Thus, long-term experiments for up to 24 h were conducted on macrophages. First, macrophages were incubated for 2 h with Mrmh and the nuclear amount of Nrf2 was analysed after a subsequent 22 h post-incubation without stimulant. During the stimulant-free post-incubation, the nuclear translocation of Nrf2 continuously raised up to 8-fold higher levels compared to experiments without post-incubation. When macrophages were continuously exposed for 24 h to Mrmh (25 mM), the nuclear Nrf2 level was 26-fold higher compared to PBS treated control cells which corresponds a 3-fold higher activation compared to the experiment with stimulant-free post-incubation. Thus, it was concluded that Mrmh might induce Nrf2 by chain reactions which are still active after removing Mrmh. During long-term experiments for up to 24 h, the activation of Nrf2 increased with the Mrmh concentration and the incubation time. Exposure of macrophages to ≥ 25 mM Mrmh significantly enhanced nuclear Nrf2 translocation reaching 50-fold higher Nrf2 levels after stimulation for 24 h compared to PBS treated control cells. To test if nuclear translocation of Nrf2 was indeed induced by Maillard products or by a high concentration of amino acids and sugars, an unheated mixture of ribose and lysine (Mrmu) was tested in the same way as Mrmh. Mrmu, which was shown not to induce NF-κB in macrophages under applied conditions, increased nuclear Nrf2 levels up to 20-fold compared to PBS treated control cells. However, the nuclear Nrf2 level was profoundly lower after stimulation with Mrmu than after exposure with the equivalent concentration of Mrmh indicating the Maillard products of the Mrmh as key players in NF-κB and Nrf2 activation. Moreover, the contribution of caramelization products can be excluded since a heated ribose solution (ribh) did neither induce NF-κB nor Nrf2 in macrophages. This in vitro screening raised the question of how the transcription factors respond in human intestinal tissue consisting of a wide range of cell types present in the intestine. Thus, a method was successfully established which allowed the stimulation of human gut tissue ex vivo. During gastrointestinal endoscopy, human gut tissue samples were taken from two localisations in the lower gastrointestinal tract; one biopsy from the terminal ileum of the small intestine and one biopsy from the ascending colon of the large intestine. Next, the human gut tissue samples were stimulated ex vivo with Mrmh for 2 h during mucosa oxygenation. It was demonstrated that (i) the tissue samples were uniform and comparable, and (ii) cell viability of the tissue samples was not harmfully affected by the mucosa oxygenation ex vivo. Mrmh induced a 2.8-fold and 2-fold activation of NF-κB and Nrf2 ex vivo in intact human gut tissue compared to PBS treated control cells. Thus, Mrmh did not only activate NF-κB and Nrf2 in various cell types but also in human gut tissue which is considered a valuable model to study nutritional effects on complex tissue systems for in vivo evaluations. To date, defined mechanisms for NF-κB and Nrf2 activation by Maillard products had not been established. However, it was shown before that Maillard-induced NF-κB activation in macrophages was inhibited by catalase. Thus, there is growing evidence that H2O2, which is permanently generated by Maillard products, is involved in the activation mechanism of NF-κB. As a matter of fact, the activation of both NF-κB and Nrf2 by Mrmh was fully abolished in macrophages, Caco-2 cells and HIMEC and reduced in human gut tissue by co-treatment with catalase. Heat-inactivated catalase did not show any effect. These results reveal the involvement of extracellular H2O2, which is most certainly generated by the Maillard products, in the activation of NF-κB and Nrf2 in all cell types tested. Interestingly, pure solutions of H2O2 with concentrations up to 500 µM activated NF-κB in macrophages up to 13-fold after stimulation for 2 h but did not induce any Nrf2 response within 24 h. Thus, it was hypothesized that the activation of Nrf2 by Mrmh might be induced not only by H2O2 but rather an interaction of H2O2 with Maillard products. Since the generation of H2O2 by Maillard products seems to play a key role in NF-κB and Nrf2 activation in various cells and tissue, the amount of H2O2 in Mrmh was analysed in the presence and absence of cells and tissue respectively. First, macrophages were incubated with Mrmh at 37°C for up to 24 h and extracellular H2O2 content was measured. In the presence of cells, the concentration of H2O2 was significantly lower compared to the H2O2 concentration in the absence of cells. The same trend was found during incubation of Mrmh in the presence and absence of intact human gut tissue ex vivo. Cells can detoxify extracellular and intracellular reactive oxygen species (ROS) such as H2O2. However, Maillard products continuously generate H2O2 which leads to an incomplete detoxification. It was hypothesized that the residual extracellular H2O2 might penetrate the cell and increases the intracellular ROS level which might induce signalling pathways including NF-κB and Nrf2. In order to verify this assumption, the intracellular ROS level of macrophages after stimulation with Mrmh was analysed using 2’,7’-dichlorofluorescein (DCF). The DCF assay determines intracellular ROS including H2O2, peroxyl radicals, nitric oxide and peroxynitrite by fluorescence signal. Thus, the intracellular DCF fluorescence can be used as an index to quantify the overall oxidative stress in cells. Indeed, the oxidative stress level was elevated after exposure to Mrmh in a concentration and time dependent manner. After 4 h, the oxidative stress level plateaued and stayed constant for up to 24 h at 4-fold higher levels compared to PBS treated control cells. It seems likely that intracellular antioxidants, which are highly expressed as a consequence of Nrf2 activation, attenuate any further increase of the oxidative stress level after 4 h. Since the NADPH oxidase, a membrane-bound enzyme, is known to produce ROS during the oxidative burst, the involvement of the NADPH oxidase in the increase of the oxidative stress level by Maillard products was investigated. Therefore, macrophages were incubated with Mrmh in the presence and absence of diphenyleneiodonium chloride (DPI), a NADPH oxidase inhibitor. Since, the intracellular oxidative stress level also increased in the presence of DPI, a major impact of the NADPH oxidase on the oxidative stress level after Mrmh incubation of macrophages was excluded. Thus, it was proposed that Mrmh generate permanently H2O2, which can not be fully detoxified by cells and thus penetrate the cells. As a consequence, intracellular ROS increases causing the induction of intracellular, signalling pathways such as NF-κB and Nrf2. Not only Mrmh but also Mrmu activated Nrf2 in macrophages. Since Mrmu also generated H2O2 but to a lower extent than Mrmh, the role of H2O2 in Mrmu induced Nrf2 activation was determined likewise. In contrast to Mrmh, co-treatment of macrophages with Mrmu and catalase did not reverse Nrf2 activation within 24 h. Thus, it was concluded that, contrary to Mrmh, Mrmu activate Nrf2 in macrophages H2O2-independently. Moreover, exposure of macrophages to Mrmu did not increase the intracellular oxidative stress level. Likewise, the oxidative stress level was not elevated after stimulation with ribh or a pure solution of H2O2 up to 500 µM. The content of H2O2 in Mrmu, ribh as well as a pure solution of 500 µM H2O2 was less than in Mrmh especially when incubated at 37°C. Thus, it was proposed that cells may fully detoxify the concentration of H2O2 present in Mrmu, ribh, a pure solution of 500 µM H2O2 but not the amount of H2O2 present in Mrmh. Consequently, the activation of NF-κB and Nrf2 by Mrmh but not Mrmu involved H2O2 which was permanently generated by Maillard products. Since Maillard products clearly induce NF-κB and Nrf2, coffee was investigated as an example for foods rich in Maillard products in the same test systems. Similar to the Maillard reaction mixtures, the generation of H2O2 was monitored in coffee extracts. With both methods, the FOXPCA assay and the oxygen electrodes, up to 440 µM H2O2 was detected in roasted coffee extract. However, also up to 200 µM H2O2 was detected in raw coffee extract. It was not surprising that H2O2 was also found in raw coffee extract as polyphenols such as chlorogenic acid, which are major components of raw coffee beans, are also considered as potent generators of H2O2. Since the polyphenols highly degrade during roasting, the generation of H2O2 in roasted coffee extract was attributed to degradation products and the Maillard products which are formed during roasting. In earlier studies, caffeic acid, pyrogallol and hydroxyhydroquinone (1,2,4-Benzenetriol), the degradation products of chlorogenic acid, were already identified as H2O2 generating structures in roasted coffee. However, the overall concentration of H2O2 formed by these degradation products is significantly lower compared to the amount of H2O2 found in coffee extract in the present study. Thus, the formation of H2O2 was not only attributed to degradation products but also to Maillard products. Next, coffee extract was tested for its ability to activate NF-κB and Nrf2 in various cells and tissue. First, Caco-2 cells and macrophages were exposed for 2 h to 1 - 4 mg/mL coffee extract (short-term). In Caco-2 cells neither NF-κB nor Nrf2 were activated. In macrophages coffee significantly activated NF-κB up to 4-fold. Moreover, a trend towards Nrf2 activation was found in macrophages. Next, intact human gut tissue samples were stimulated with 1 - 4 mg/mL coffee extract for 2 h. In human gut tissue samples, coffee extract did not activate Nrf2 but induced a trend towards NF-κB activation. Short-term incubation with coffee showed the highest NF-κB and Nrf2 activation in macrophages amongst the cells and tissue samples tested. Thus, macrophages were incubated next with coffee extract between 30 min and 24 h and the activation of NF-κB and Nrf2 was investigated time-dependently. Nrf2 was activated by coffee extract in a concentration and time dependent manner reaching nearly 20-fold higher Nrf2 levels after 24 h compared to water treated control cells. NF-κB was activated up to 3-fold higher levels after 2 h but declined slightly thereafter. After 6 h, the nuclear NF-κB concentration was still 1.7-fold higher compared to control cells. It was suggested that the pronounced activation of Nrf2 might attenuate the nuclear translocation of NF-κB. This crosstalk between Nrf2 and NF-κB has been recently discussed for stimuli such as sulforaphane which regulate both NF-κB and Nrf2. It was reported that for example sulforaphane activate Nrf2 and concomitantly repress the activation of NF-κB. These results may explain the pronounced activation of Nrf2 but not NF-κB after long-term incubation of macrophages with coffee extract. On the contrary to roasted coffee extract, raw coffee extract did activate neither NF-κB nor Nrf2 after long-term incubation of macrophages. Thus, it was concluded that the roasting products of coffee including the Maillard products are the key players triggering the nuclear translocation of NF-κB and Nrf2. Since it was shown before that H2O2 was involved in the activation mechanisms of NF-κB and Nrf2 by Mrmh, the role of H2O2 in coffee-induced NF-κB and Nrf2 activation was investigated likewise. Similar to the experiments with Mrmh, the activation of NF-κB after stimulation of macrophages with coffee extract was abolished by co-treatment with catalase. However, co-treatment with catalase showed only a slight trend to block the coffee-induced Nrf2 translocation in macrophages. Thus, it was concluded that the generation of H2O2 by coffee might play a major role in coffee-induced NF-κB but not in Nrf2 activation. Hence, the amount of extracellular H2O2 after coffee stimulation was measured in the presence and absence of macrophages. After incubation of coffee extract (1 - 4 mg/mL) for 24 h at 37°C, H2O2 levels were higher in the absence than in the presence of macrophages. In the absence of cells, up to 800 µM H2O2 was measured whereas in the presence of cells H2O2 levels did not exceed 230 µM. A similar trend was observed after incubation of coffee extract with and without human gut tissue for 2 h. These results indicate that coffee-generated H2O2 was not fully detoxified by cells and tissue respectively leading to the activation of NF-κB as discussed above for Mrmh. Thus, it was proposed that coffee and especially Maillard products may rather counteract oxidative stress by Nrf2 activation than induce an inflammatory response by NF-κB activation after long-term incubation. Activated levels of Nrf2 lead to an enhanced transcription of antioxidant and detoxifying enzymes. Hence, coffee and Maillard-rich foods may exert not only direct but also indirect antioxidative activity by inducing the expression of cytoprotective proteins especially in the gut which represents a primary target for food-induced inter- and intracellular reactions of the human body. Indeed, as shown in a mouse model, activators of Nrf2 can improve colitis. Enhanced intestinal Nrf2 signalling was further associated with a beneficial effect on gut barrier dysfunction, intestinal mucosal injury and intestinal inflammation other than colitis. Moreover, there is growing evidence that Nrf2 is also a promising target for the prevention of colorectal cancer. Indeed, epidemiological studies suggested for coffee drinker with regular coffee consumption a reduced risk of colorectal cancer as well as other of oxidative stress-associated diseases such as Alzheimer’s disease and type 2 diabetes mellitus.
Author: Tanja Sauer
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2979533746