Probing Long-Term Impacts: Low-Dose Polystyrene Nanoplastics Exacerbate Mitochondrial Health and Evoke Secondary Glycolysis via Repeated and Single Dosing.

Nanoplastics (NPs) are omnipresent in the environment and contribute to human exposure. However, little is known regarding the long-term effects of NPs on human health. In this study, human intestinal Caco-2 cells were exposed to polystyrene nanoplastics (nanoPS) in an environmentally relevant concentration range (102-109 particles/mL) under two realistic exposure scenarios. In the first scenario, cells were repeatedly exposed to nanoPS every 2 days for 12 days to study the long-term effects. In the second scenario, only nanoPS was added once and Caco-2 cells were cultured for 12 days to study the duration of the initial effects of NPs. Under repeated dosing, initial subtle effects on mitochondria induced by low concentrations would accrue over consistent exposure to nanoPS and finally lead to significant impairment of mitochondrial respiration, mitochondrial mass, and cell differentiation process at the end of prolonged exposure, accompanied by significantly increased glycolysis over the whole exposure period. Single dosing of nanoPS elicited transient effects on mitochondrial and glycolytic functions, as well as increased reactive oxygen species (ROS) production in the early phase of exposure, but the self-recovery capacity of cells mitigated these effects at intermediate culture times. Notably, secondary effects on glycolysis and ROS production were observed during the late culture period, while the cell differentiation process and mitochondrial mass were not affected at the end. These long-term effects are of crucial importance for comprehensively evaluating the health hazards arising from lifetime exposure to NPs, complementing the extensively observed acute effects associated with prevalent short-term exposure to high concentrations. Our study underlines the need to study the toxicity of NPs in realistic long-term exposure scenarios such as repeated dosing.

Cellular and bioenergetic effects of polystyrene microplastic in function of cell type, differentiation status and post-exposure time.

The ubiquity of microplastics (MPs) in food sources and personal care products increasingly raises concerns on human health. However, little is known about the duration of the effects of MPs and whether effects depend on cellular differentiation status. Herein, cellular and bioenergetic effects of MPs in different exposure scenarios on four types of human cell lines derived from lung (A549 and BEAS-2B), colon (Caco-2) and liver (HepG2) were investigated. These cell lines are models for the major exposure routes in the body (inhalation, ingestion and physiological transport through the liver by the portal vein). To this aim, different scenarios were implemented by exposing undifferentiated and differentiated cells to single dosing of 2-µm polystyrene (PS) (102-105 particles/mL) for 48 h and 12 days. The undifferentiated Caco-2 cells with short exposure (48 h) showed the highest uptake rate of PS yet without significant cellular and mitochondrial responses. The biological effects, with the exception of ROS production, were not influenced by differentiation states of A549 and Caco-2 cells although differentiated cells showed much weaker ability to internalize PS. However, PS had significantly long-term impacts on cellular and mitochondrial functions even after the initial exposure period. In particular, Caco-2 cells that were post-exposed for 12 days after single PS dosing suffered higher oxidative stress and exhibited mitochondrial dysfunction than that for short exposure. Correspondingly, we observed that PS particles still remained in cell membrane and even in nuclei with high retention rate by 14-d post exposure during which metabolism and exchange of internalization and release occurred in cells. This indicates PS could induce chronic stress and even harmful effects on human cells after single intake that persists for a long time. This study paves the way for assessing the influence of PS on human health at low particle concentrations and with multiple exposure scenarios.

New insights into the combined toxicity of aflatoxin B1 and fumonisin B1 in HepG2 cells using Seahorse respirometry analysis and RNA transcriptome sequencing.

Aflatoxin B1 (AFB1) and fumonisin B1 (FB1) are widely (co-)detected in food and known for their hepatotoxicity in humans. Still, their combined toxicity needs to be investigated, especially the impact on mitochondria. In our previous work, we examined the effect of short-term exposure to different doses of AFB1, FB1, and their binary mixture (MIX) on the bioenergetic status of HepG2 cells, a well-recognized in vitro model system for studying liver cell function. In the current work, we further investigated the (combined) effect of AFB1 and FB1 on the mitochondrial and glycolytic activity of HepG2 cells using Seahorse respirometry analysis and RNA transcriptome sequencing. The results showed that the co-exposure, especially at high doses, is more toxic due to a more inhibition of all parameters of mitochondrial respiration. However, FB1 contributes more to the MIX effects than AFB1. RNA transcriptome sequencing showed that the p53 signaling pathway, a major orchestrator of mitochondrial apoptosis, was differentially expressed. Moreover, the co-exposure significantly downregulated the genes encoding for Complexes I, II, III, and IV, representing the onset of the suppressed mitochondrial respiration in HepG2 cells.

Rare Sugar Metabolism and Impact on Insulin Sensitivity along the Gut-Liver-Muscle Axis In Vitro.

Rare sugars have recently attracted attention as potential sugar replacers. Understanding the biochemical and biological behavior of these sugars is of importance in (novel) food formulations and prevention of type 2 diabetes. In this study, we investigated whether rare sugars may positively affect intestinal and liver metabolism, as well as muscle insulin sensitivity, compared to conventional sugars. Rare disaccharide digestibility, hepatic metabolism of monosaccharides (respirometry) and the effects of sugars on skeletal muscle insulin sensitivity (impaired glucose uptake) were investigated in, respectively, Caco-2, HepG2 and L6 cells or a triple coculture model with these cells. Glucose and fructose, but not l-arabinose, acutely increased extracellular acidification rate (ECAR) responses in HepG2 cells and impaired glucose uptake in L6 cells following a 24 h exposure at 28 mM. Cellular bioenergetics and digestion experiments with Caco-2 cells indicate that especially trehalose (α1-1α), D-Glc-α1,2-D-Gal, D-Glc-α1,2-D-Rib and D-Glc-α1,3-L-Ara experience delayed digestion and reduced cellular impact compared to maltose (α1-4), without differences on insulin-stimulated glucose uptake in a short-term setup with a Caco-2/HepG2/L6 triple coculture. These results suggest a potential for l-arabinose and specific rare disaccharides to improve metabolic health; however, additional in vivo research with longer sugar exposures should confirm their beneficial impact on insulin sensitivity in humans.

In vitro bioaccessibility and uptake of ß-carotene from encapsulated carotenoids from mango by-products in a coupled gastrointestinal digestion/Caco-2 cell model.

ß-carotene is a carotenoid with provitamin A activity and other health benefits, which needs to become bioavailable upon oral intake to exert its biological activity. A better understanding of its behaviour and stability in the gastrointestinal tract and means to increase its bioavailability are highly needed. Using an in vitro gastrointestinal digestion method coupled to an intestinal cell model, we explored the stability, gastrointestinal bioaccessibility and cellular uptake of ß-carotene from microparticles containing carotenoid extracts derived from mango by-products. Three types of microparticles were tested: one with the carotenoid extract as such, one with added inulin and one with added fructooligosaccharides. Overall, ß-carotene was relatively stable during the in vitro digestion, as total recoveries were above 68 %. Prebiotics in the encapsulating material, especially inulin, enhanced the bioaccessibility of ß-carotene almost 2-fold compared to microparticles without prebiotics. Likewise, ß-carotene bioaccessibility increased proportionally with bile salt concentrations during digestion. Yet, a bile salts level above 10 mM did not contribute markedly to ß-carotene bioaccessibility of prebiotic containing microparticles. Cellular uptake experiments with non-filtered gastrointestinal digests yielded higher absolute levels of ß-carotene taken up in the epithelial cells as compared to uptake assays with filtered digests. However, the proportional uptake of ß-carotene was higher for filtered digests (24 - 31 %) than for non-filtered digests (2 - 8 %). Matrix-dependent carotenoid uptake was only visible in the unfiltered medium, thereby pointing to possible other cellular transport mechanisms of non-micellarized carotenoids, besides the concentration effect. Regardless of a filtration step, inulin-amended microparticles consistently resulted in a higher ß-carotene uptake than regular microparticles or FOS-amended microparticles. In conclusion, encapsulation of carotenoid extracts from mango by-products displayed chemical stability and release of a bioaccessible ß-carotene fraction upon gastrointestinal digestion. This indicates the potential of the microparticles to be incorporated into functional foods with provitamin A activity.

Bioenergetic Status of the Intestinal and Hepatic Cells after Short Term Exposure to Fumonisin B1 and Aflatoxin B1.

Fumonisin B1 (FB1) and aflatoxin B1 (AFB1) are frequent contaminants of staple foods such as maize. Oral exposure to these toxins poses health hazards by disrupting cellular signaling. However, little is known regarding the multifaced mitochondrial dysfunction-linked toxicity of FB1 and AFB1. Here, we show that after exposure to FB1 and AFB1, mitochondrial respiration significantly decreased by measuring the oxygen consumption rate (OCR), mitochondrial membrane potential (MMP) and reactive oxygen species (ROS). The current work shows that the integrity of mitochondria (MMP and ROS), that is the central component of cell apoptosis, is disrupted by FB1 and AFB1 in undifferentiated Caco-2 and HepG2 cells as in vitro models for human intestine and liver, respectively. It hypothesizes that FB1 and AFB1 could disrupt the mitochondrial electron transport chain (ETC) to induce mitochondrial dysfunction and break the balance of transferring H+ between the mitochondrial inner membrane and mitochondrial matrix, however, the proton leak is not increasing and, as a result, ATP synthesis is blocked. At the sub-toxic exposure of 1.0 µg/mL for 24 h, i.e., a viability of 95% in Caco-2 and HepG2 cells, the mitochondrial respiration was, however, stimulated. This suggests that the treated cells could reserve energy for mitochondrial respiration with the exposure of FB1 and AFB1, which could be a survival advantage.

Metabolism and Health Effects of Rare Sugars in a CACO-2/HepG2 Coculture Model.

Non-alcoholic fatty liver disease (NAFLD) has become the most prevalent liver disease worldwide and is impacted by an unhealthy diet with excessive calories, although the role of sugars in NAFLD etiology remains largely unexplored. Rare sugars are natural sugars with alternative monomers and glycosidic bonds, which have attracted attention as sugar replacers due to developments in enzyme engineering and hence an increased availability. We studied the impact of (rare) sugars on energy production, liver cell physiology and gene expression in human intestinal colorectal adenocarcinoma (Caco-2) cells, hepatoma G2 (HepG2) liver cells and a coculture model with these cells. Fat accumulation was investigated in the presence of an oleic/palmitic acid mixture. Glucose, fructose and galactose, but not mannose, l-arabinose, xylose and ribose enhanced hepatic fat accumulation in a HepG2 monoculture. In the coculture model, there was a non-significant trend (p = 0.08) towards higher (20-55% increased) median fat accumulation with maltose, kojibiose and nigerose. In this coculture model, cellular energy production was increased by glucose, maltose, kojibiose and nigerose, but not by trehalose. Furthermore, glucose, fructose and l-arabinose affected gene expression in a sugar-specific way in coculture HepG2 cells. These findings indicate that sugars provide structure-specific effects on cellular energy production, hepatic fat accumulation and gene expression, suggesting a health potential for trehalose and l-arabinose, as well as a differential impact of sugars beyond the distinction of conventional and rare sugars.

Quercetin Mitigates Endothelial Activation in a Novel Intestinal-Endothelial-Monocyte/Macrophage Coculture Setup.

Atherosclerosis initiation is associated with a pro-inflammatory state of the endothelium. Quercetin is a flavonoid abundantly present in plant-based foods, with a possible impact on cardiovascular health. In this study, the effects of quercetin on lipopolysaccharide (LPS)-mediated endothelial inflammation and monocyte adhesion and migration, which are initial steps of the atherogenic process, are studied. Novel in vitro multicellular models simulating the intestinal-endothelial-monocytes/macrophages axis allowed to combine relevant intestinal flavonoid absorption, metabolism and efflux, and the consequent bioactivity towards peripheral endothelial cells. In this triple coculture, quercetin exposure decreased monocyte adhesion to and macrophage migration through an LPS-stressed endothelium, and this was associated with significantly lower levels of soluble vascular cell adhesion molecule-1 (sVCAM-1). Furthermore, quercetin decreased the pro-inflammatory cell environment upon LPS-induced endothelial activation, in terms of tumor necrosis factor- α (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), and sVCAM-1 expression. These findings highlight a mode-of-action by which quercetin may positively impact the initial states of atherosclerosis under more physiologically relevant conditions in terms of quercetin concentrations, metabolites, and intercellular crosstalk.

Sweet Biotechnology: Enzymatic Production and Digestibility Screening of Novel Kojibiose and Nigerose Analogues.

In view of the global pandemic of obesity and related metabolic diseases, there is an increased interest in alternative carbohydrates with promising physiochemical and health-related properties as a potential replacement for traditional sugars. However, our current knowledge is limited to only a small selection of carbohydrates, whereas the majority of alternative rare carbohydrates and especially their properties remain to be investigated. Unraveling their potential properties, like digestibility and glycemic content, could unlock their use in industrial applications. Here, we describe the enzymatic production and in vitro digestibility of three novel glycosides, namely, two kojibiose analogues (i.e., d-Glcp-α-1,2-d-Gal and d-Glcp-α-1,2-d-Rib) and one nigerose analogue (i.e., d-Glcp-α-1,3-l-Ara). These novel sugars were discovered after an intensive acceptor screening with a sucrose phosphorylase originating from Bifidobacterium adolescentis (BaSP). Optimization and upscaling of this process led to roughly 100 g of these disaccharides. Digestibility, absorption, and caloric potential were assessed using brush border enzymes of rat origin and human intestinal Caco-2 cells. The rare disaccharides showed a reduced digestibility and a limited impact on energy metabolism, which was structure-dependent and even more pronounced for the three novel disaccharides in comparison to their respective glucobioses, translating to a low-caloric potential for these novel rare disaccharides.

Bioavailability and Health Impact of Ingested Amyloid-like Protein Fibrils and their Link with Inflammatory Status: A Need for More Research?

The use of amyloid-like protein fibrils (ALFs) in food formulations looks very promising in terms of improving techno-functional properties, but raises some concerns in terms of food safety, because of their structural resemblance to disease-related endogenous amyloids. This review focuses on the biological fate and potential health implications of ingested ALF structures in both healthy and predisposed individuals. A comprehensive overview of ALF gastrointestinal digestion, intestinal absorption, and systemic dissemination is provided, in addition to a thorough assessment of potential ALF cross-seeding of endogenous precursor proteins linked to (non)neurodegenerative amyloidosis. In general, this study concludes that the health impact of ALF consumption remains widely understudied and merits additional research efforts to determine the exact extent to which ALF ingestion may influence the general health status.

Flavonoids and cellular stress: a complex interplay affecting human health.

Flavonoid consumption has beneficial effects on human health, however, clinical evidence remains often inconclusive due to high interindividual variability. Although this high interindividual variability has been consistently observed in flavonoid research, the potential underlying reasons are still poorly studied. Especially the knowledge on the impact of health status on flavonoid responsiveness is limited and merits more investigation. Here, we aim to highlight the bidirectional interplay between flavonoids and cellular stress. First, the state-of-the-art concerning inflammatory stress and mitochondrial dysfunction is reviewed and a comprehensive overview of recent in vitro studies investigating the impact of flavonoids on cellular stress, induced by tumor necrosis factor α, lipopolysaccharide and mitochondrial stressors, is given. Second, we critically discuss the influence of cellular stress on flavonoid uptake, accumulation, metabolism and cell responses, which has, to our knowledge, never been extensively reviewed before. Next, we advocate the innovative insight that stratification of the general population based on health status can reveal subpopulations that benefit more from flavonoid consumption. Finally, suggestions are given for the development of future cell models that simulate the physiological micro-environment, including interindividual variability, since more mechanistic research is needed to establish scientific-based personalized food recommendations for specific subpopulations.

A two-front nutritional environment fuels colorectal cancer: perspectives for dietary intervention.

Colorectal cancer (CRC) develops and progresses in a nutritional environment comprising a continuously changing luminal cocktail of external dietary and microbial factors on the apical side, and a dynamic host-related pool of systemic factors on the serosal side. In this review, we highlight how this two-front environment influences the bioenergetic status of colonocytes throughout CRC development from (cancer) stem cells to cancer cells in nutrient-rich and nutrient-poor conditions, and eventually to metastatic cells, which, upon entry to the circulation and during metastatic seeding, are forced to metabolically adapt. Furthermore, given the influence of diet on the two-front nutritional environment, we discuss dietary strategies that target the specific metabolic preferences of these cells, with a possible impact on colon cancer cell bioenergetics and CRC outcome.

Intracellular quercetin accumulation and its impact on mitochondrial dysfunction in intestinal Caco-2 cells.

PURPOSE: Flavonoid bioavailability and bioactivity is associated with interindividual variability, which is partially due to differences in health status. Previously, it was demonstrated that cellular stress, especially mitochondrial stress, increases intracellular quercetin uptake and this is associated with beneficial health effects. Here, the impact of quercetin on mitochondrial dysfunction, induced by stressors targeting different sites of the electron transport chain, is investigated. The influence of the mitochondrial stress on quercetin uptake and subcellular location is studied and the accumulated quercetin metabolites in intestinal Caco-2 cells and mitochondria are characterized. PRINCIPAL RESULTS: It was observed that quercetin counteracted (i) the carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)-induced decrease in maximum oxygen consumption, (ii) the valinomycin-, oligomycin- and FCCP-induced reactive oxygen species production and (iii) the valinomycin-induced disruption of mitochondrial membrane potential. Using confocal microscopy, it was found that upon mitochondrial stress, the intracellular quercetin accumulation increased and was partially located in the mitochondria. Finally, it was demonstrated that quercetin was present as O-methyl, O-methylglucuronide and O-methylsulfate conjugates in the cell lysate and mitochondria-enriched fraction. MAJOR CONCLUSIONS: This study shows that quercetin can partially restore, especially FCCP-induced, mitochondrial dysfunction and this protective effect was linked with an intracellular quercetin accumulation in the mitochondria of intestinal cells.

Method for beta-carotene extraction from processed baby foods as a model for plant-based fatty food products.

Reliable quantitative determination of carotenoids in complex food matrices such as processed baby food products is challenging because of their incorporation in rigid cellular structures, their sensitivity to oxidation and their lipophilic character. A one-pot liquid-liquid ß-carotene extraction procedure is described for solid baby foods, in the presence of enzymes (Clara-Diastase and Rapidase) facilitating matrix disintegration. The combined extraction and enzymatic dissolution not only protected ß-carotene from oxidation compared to the sequential approach, but also reduced the use of solvents and amount of filtrations steps, favouring a higher recovery. The addition of phenolic antioxidants (BHT, TBHQ and BHA) to calibration solutions and during the procedure at 25 mg/mL resulted in an up to 2.5-fold higher absorbance of ß-carotene solutions which was not observed for trans-ß-apo-8'-carotenal (used as internal standard) solutions. When applying the full procedure on ß-carotene spiked sunflower oil, an apparent recovery of 80% for ß-carotene was obtained. Finally, this protocol was applied to 50 vegetable-based and 22 fruit-based processed baby foods (range 0 to 1179 and 504 µg/100 g, respectively), and it was concluded that this extraction procedure may be used for similar processed foods products. The procedure proved to be sensitive (LOD = 0.12 µg/mL) and reproducible (CV for baby foods: 4-10%).

Microplastics in the human digestive environment: A focus on the potential and challenges facing in vitro gut model development.

Plastic pollution is a major issue worldwide, generating massive amounts of smaller plastic particles, including microplastics (MPs). Their ubiquitous nature in the environment but also in foodstuff and consumer packaged goods has revealed potential threats to humans who can be contaminated mainly through air, food and water consumption. In this review, the current literature on human exposure to MPs is summarized with a focus on the gastrointestinal tract as portal of entry. Then, we discuss the vector effect of MPs, in their pristine versus weathered forms, with well-known contaminants as heavy metals and chemicals, or more emerging ones as antibiotics or microbial pathogens, like Pseudomonas spp., Vibrio spp., Campylobacter spp. and Escherichia coli. Comprehensive knowledge on MP fate in the gastrointestinal tract and their potential impact on gut homeostasis disruption, including gut microbiota, mucus and epithelial barrier, is reported in vitro and in vivo in mammals. Special emphasis is given on the crucial need of developing robust in vitro gut models to adequately simulate human digestive physiology and absorption processes. Finally, this review points out future research directions on MPs in human intestinal health.

Rare mono- and disaccharides as healthy alternative for traditional sugars and sweeteners?

Obesity and type 2 diabetes are major health problems affecting hundreds of millions of people. Caloric overfeeding with calorie-dense food ingredients like sugars may contribute to these chronic diseases. Sugar research has also identified mechanisms via which conventional sugars like sucrose and fructose can adversely influence metabolic health. To replace these sugars, numerous sugar replacers including artificial sweeteners and sugar alcohols have been developed. Rare sugars became new candidates to replace conventional sugars and their health effects are already reported in individual studies, but overviews and critical appraisals of their health effects are missing. This is the first paper to provide a detailed review of the metabolic health effects of rare sugars as a group. Especially allulose has a wide range of health effects. Tagatose and isomaltulose have several health effects as well, while other rare sugars mainly provide health benefits in mechanistic studies. Hardly any health claims have been approved for rare sugars due to a lack of evidence from human trials. Human trials with direct measures for disease risk factors are needed to allow a final appraisal of promising rare sugars. Mechanistic cell culture studies and animal models are required to enlarge our knowledge on understudied rare sugars.

Co-Ingestion of Black Carrot and Strawberry. Effects on Anthocyanin Stability, Bioaccessibility and Uptake.

Although the fate of anthocyanins along digestion has been a matter of research over the last decade, their bioaccessibility so far has been mainly assessed for single administered fruits or vegetables, which is far from the real scenario where they are co-ingested in a meal. Accordingly, the aim of this study was to evaluate the effect of simultaneous intake of fruit and vegetable on in vitro stability, bioaccessibility and uptake of anthocyanins. Black carrot and strawberry were used as food sources of anthocyanins. Anthocyanin identification and quantification were performed using HPLC-Qtof/HPLC-UV. Single matrices and mixtures thereof, were submitted to a standardized in vitro digestion procedure. Anthocyanin uptake was evaluated through an intestinal Caco-2 cell model. Our results showed an increased intestinal stability for specific anthocyanins as a consequence of co-digestion. The presence of the strawberry food matrix positively affected the bioaccessibility of the carrot associated cyanidin-based anthocyanins, whereas no reciprocal effect was observed for pelargonidin-based derivatives in the presence of the black carrot food matrix. Anthocyanin transport was maintained after co-administration. Overall, co-ingestion of black carrot and strawberry did not negatively affect the stability, bioaccessibility or uptake of cyanidin-based anthocyanins, although the effect on pelargonidin-based anthocyanins depended on the type of pelargonidin derivative.

Chemical composition, antioxidant, antimicrobial and antiproliferative activities of the extracts isolated from the pomace of rowanberry (Sorbus aucuparia L.).

In the present work, acetone, ethanol and water extracts of rowanberry (Sorbus aucuparia L.) pomace were evaluated for their antiproliferative, antimicrobial and antioxidative effects. Chemical composition of the extracts was determined by UPLC-ESI-MS/MS and spectrophotometric methods. Neochlorogenic and chlorogenic acids were the major phenolic compounds. The water extract contained the highest total proanthocyanidins content (301 ± 18.9 mg/g) and demonstrated the highest antioxidant activity in the all assays (DPPH, FRAP and ORAC). Extracts isolated from rowanberry pomace effectively inhibited the growth of undesirable microorganisms, especially Gram-positive bacteria. Acetone extract was the strongest antimicrobial agent followed by water and ethanol extracts. Acetone and water extracts demonstrated also higher cytotoxic potential in cell viability assays (SRB and MTT) using Caco-2 cells. In general, the results suggest that rowanberry pomace is a promising source of natural compounds with antioxidant and biological activities.

Cell line-dependent increase in cellular quercetin accumulation upon stress induced by valinomycin and lipopolysaccharide, but not by TNF-α.

As the interface between the luminal and internal environment, the intestinal epithelium is strongly exposed to food-related, host-related and microbial stress. Furthermore, the endothelial stress response plays an important role in vascular disease development, which may be improved upon consumption of dietary bioactives such as polyphenols. The impact of the latter, however, is largely individual-dependent and effects are, in most cases, only observed under mild diseased conditions. Here, it is hypothesized that the individual's stressor levels may contribute to this variable response. To this end, the impact of the stressors (i) valinomycin (as model for cereulide, food-related microbial metabolite), (ii) TNF-α (host-related) and (iii) lipopolysaccharide (gram-negative bacterial cell related) on flavonoid accumulation was investigated in several intestinal and endothelial cell lines. Flow cytometry, confocal microscopy and an in-house developed, robust and high-throughput spectrofluorometric method, showed that quercetin accumulated in all tested cell lines in a dose-dependent manner. Upon stress induced by valinomycin and to a lesser extent by lipopolysaccharide, but not by TNF-α, an increased quercetin accumulation was observed in proliferating intestinal and endothelial cells and not in differentiated intestinal or quiescent endothelial cells. Therefore, flavonoid accumulation may be a potential cellular stress response mechanism which strongly depends on the applied stressor, flavonoid, cell line and even growth conditions. This opens perspectives for further understanding the mechanisms by which cellular stress may shape the individual's response to bioactive compounds.

Synthesis of 1,4-Thiazepane-Based Curcuminoids with Promising Anticancer Activity.

Curcumin, the main component of turmeric (Curcuma longa) is known to display an interesting bioactivity profile, including pronounced anticancer properties. However, its low bioavailability, metabolic instability and nonspecific activity are concerns that have to be addressed before curcuminoids can be considered for therapeutic applications. Within that framework, intensive research has been carried out in the last decades to develop new curcumin derivatives, generally centered on standard modifications of the sp2 curcumin framework, with the aim to augment its bioavailability while maintaining or improving its anticancer properties. To find potential hit molecules by moving away from the classical flat curcumin framework, we investigated an unexplored modification to produce novel, out-of-plane 1,4-thiazepane-based curcuminoids and assessed the impact of this modification on the biological activity. In this way, 21 new, structurally diverse thiazepane scaffolds (4-aryl-1-(7-aryl-1,4-thiazepan-5-ylidene)but-3-en-2-ones) were synthesized, as well as some biologically interesting unexpected reaction products (such as 5-aryl-6-arylmethylene-3-ethoxycyclohex-2-en-1-ones and 4-acetyl-5-aryl-2-(3-arylacryloyl)-3-methylcyclohex-2-en-1-ones). All these analogues were subsequently tested on their antioxidant capacity, their cytotoxicity properties and their ROS (reactive oxygen species) production. Many compounds demonstrated interesting activities, with ten curcuminoids, whereof eight 1,4-thiazepane-based, showing better antiproliferative properties compared to their mother compounds, as well as an increased ROS production. This unprecedented 3D curcumin modification has thus delivered promising new hit compounds with good activity profiles eligible for further exploration.