MANNosylation of Mesoporous Silica Nanoparticles Modifies TLR4 Localization and NF-κB Translocation in T24 Bladder Cancer Cells.

D-mannose is widely used as non-antibiotic treatment for bacterial urinary tract infections. This application is based on a well-studied mechanism of binding to the type 1 bacterial pili and, therefore, blocking bacteria adhesion to the uroepithelial cells. To implement D-mannose into carrier systems, the mechanism of action of the sugar in the bladder environment is also relevant and requires investigation. Herein, two different MANNosylation strategies using mesoporous silica nanoparticles (MSNs) are described. The impact of different chemical linkers on bacterial adhesion and bladder cell response is studied via confocal microscopy imaging of the MSN interactions with the respective organisms. Cytotoxicity is assessed and the expression of Toll-like receptor 4 (TLR4) and caveolin-1 (CAV-1), in the presence or absence of simulated infection with bacterial lipopolysaccharide (LPS), is evaluated using the human urinary bladder cancer cell line T24. Further, localisation of the transcription factor NF-κB due to the MANNosylated materials is examined over time. The results show that MANNosylation modifies bacterial adhesion to the nanomaterials and significantly affects TLR4, caveolin-1, and NF-κB in bladder cells. These elements are essential components of the inflammatory cascade/pathogens response during urinary tract infections. These findings demonstrate that MANNosylation is a versatile tool to design hybrid nanocarriers for targeted biomedical applications.

Microplastics role in cell migration and distribution during cancer cell division.

Amidst the global plastic pollution crisis, the gastrointestinal tract serves as the primary entry point for daily exposure to micro- and nanoplastics. We investigated the complex dynamics between polystyrene micro- and nanoplastics (PS-MNPs) and four distinct human colorectal cancer cell lines (HT29, HCT116, SW480, and SW620). Our findings revealed a significant size- and concentration dependent uptake of 0.25, 1, and 10 µm PS-MNPs across all cell lines, with HCT116 cells exhibiting the highest uptake rates. During cell division, particles were distributed between mother and daughter cells. Interestingly, we observed no signs of elimination from the cells. Short-term exposure to 0.25 µm particles significantly amplified cell migration, potentially leading to pro-metastatic effects. Particles demonstrated high persistence in 2D and 3D cultures, and accumulation in non-proliferating parts of spheroids, without interfering with cell proliferation or division. Our study unveils the disturbing fact of the persistence and bioaccumulation of MNPs in colorectal cancer cell lines, key toxicological traits under REACH (Regulation concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals). Our observations underscore the potential of MNPs as hidden catalysts for tumor progression, particularly through enhancing cell migration and possibly fueling metastasis - a finding that sheds light on a significant and previously underexplored area of concern.

Cytotoxicity of Prymnesium parvum extracts and prymnesin analogs on epithelial fish gill cells RTgill-W1 and the human colon cell line HCEC-1CT.

Harmful algal blooms kill fish populations worldwide, as exemplified by the haptophyte microalga Prymnesium parvum. The suspected causative agents are prymnesins, categorized as A-, B-, and C-types based on backbone carbon atoms. Impacts of P. parvum extracts and purified prymnesins were tested on the epithelial rainbow trout fish gill cell line RTgill-W1 and on the human colon epithelial cells HCEC-1CT. Cytotoxic potencies ranked A > C > B-type with concentrations spanning from low (A- and C-type) to middle (B-type) nM ranges. Although RTgill-W1 cells were about twofold more sensitive than HCEC-1CT, the cytotoxicity of prymnesins is not limited to fish gills. Both cell lines responded rapidly to prymnesins; with EC50 values for B-types in RTgill-W1 cells of 110 ± 11 nM and 41.5 ± 0.6 nM after incubations times of 3 and 24 h. Results of fluorescence imaging and measured lytic effects suggest plasma membrane interactions. Postulating an osmotic imbalance as mechanisms of toxicity, incubations with prymnesins in media lacking either Cl-, Na+, or Ca2+ were performed. Cl- removal reduced morphometric rearrangements observed in RTgill-W1 and cytotoxicity in HCEC-1CT cells. Ca2+-free medium in RTgill-W1 cells exacerbated effects on the cell nuclei. Prymnesin composition of different P. parvum strains showed that analog composition within one type scarcely influenced the cytotoxic potential, while analog type potentially dictate potency. Overall, A-type prymnesins were the most potent ones in both cell lines followed by the C-types, and lastly B-types. Disturbance of Ca2+ and Cl- ionoregulation may be integral to prymnesin toxicity.

Autophagy modulation changes mechano-chemical sensitivity of T24 bladder cancer cells.

Bladder cancer cells possess unique adaptive capabilities: shaped by their environment, cells face a complex chemical mixture of metabolites and xenobiotics accompanied by physiological mechanical cues. These responses might translate into resistance to chemotherapeutical regimens and can largely rely on autophagy. Considering molecules capable of rewiring tumor plasticity, compounds of natural origin promise to offer valuable options. Fungal derived metabolites, such as bafilomycin and wortmannin are widely acknowledged as autophagy inhibitors. Here, their potential to tune bladder cancer cells´ adaptability to chemical and physical stimuli was assessed. Additionally, dietary occurring mycotoxins were also investigated, namely deoxynivalenol (DON, 0.1-10 µM) and fusaric acid (FA, 0.1-1 mM). Endowing a Janus' face behavior, DON and FA are on the one side described as toxins with detrimental health effects. Concomitantly, they are also explored experimentally for selective pharmacological applications including anticancer activities. In non-cytotoxic concentrations, bafilomycin (BAFI, 1-10 nM) and wortmannin (WORT, 1 µM) modified cell morphology and reduced cancer cell migration. Application of shear stress and inhibition of mechano-gated PIEZO channels reduced cellular sensitivity to BAFI treatment (1 nM). Similarly, for FA (0.5 mM) PIEZO1 expression and inhibition largely aligned with the modulatory potential on cancer cells motility. Additionally, this study highlighted that the activity profile of compounds with similar cytotoxic potential (e.g. co-incubation DON with BAFI or FA with WORT) can diverge substantially in the regulation of cell mechanotransduction. Considering the interdependence between tumor progression and response to mechanical cues, these data promise to provide a novel viewpoint for the study of chemoresistance and associated pathways.

Intracellular remodeling associated with endoplasmic reticulum stress modifies biomechanical compliance of bladder cells.

Bladder cells face a challenging biophysical environment: mechanical cues originating from urine flow and regular contraction to enable the filling voiding of the organ. To ensure functional adaption, bladder cells rely on high biomechanical compliance, nevertheless aging or chronic pathological conditions can modify this plasticity. Obviously the cytoskeletal network plays an essential role, however the contribution of other, closely entangled, intracellular organelles is currently underappreciated. The endoplasmic reticulum (ER) lies at a crucial crossroads, connected to both nucleus and cytoskeleton. Yet, its role in the maintenance of cell mechanical stability is less investigated. To start exploring these aspects, T24 bladder cancer cells were treated with the ER stress inducers brefeldin A (10-40nM BFA, 24 h) and thapsigargin (0.1-100nM TG, 24 h). Without impairment of cell motility and viability, BFA and TG triggered a significant subcellular redistribution of the ER; this was associated with a rearrangement of actin cytoskeleton. Additional inhibition of actin polymerization with cytochalasin D (100nM CytD) contributed to the spread of the ER toward cell periphery, and was accompanied by an increase of cellular stiffness (Young´s modulus) in the cytoplasmic compartment. Shrinking of the ER toward the nucleus (100nM TG, 2 h) was related to an increased stiffness in the nuclear and perinuclear areas. A similar short-term response profile was observed also in normal human primary bladder fibroblasts. In sum, the ER and its subcellular rearrangement seem to contribute to the mechanical properties of bladder cells opening new perspectives in the study of the related stress signaling cascades. Video Abstract.

Exposure to dietary fatty acids oleic and palmitic acid alters structure and mechanotransduction of intestinal cells in vitro.

Intestinal cells are continuously exposed to food constituents while adapting to peristaltic movement and fluid shear stress. Oleic acid (OA) and palmitic acid (PA) are among the most prevalent fatty acids with respect to dietary lipids. Despite the central importance of dietary lipids for a balanced diet, awareness about potential detrimental effects related to excessive consumption is increasing; this includes toxicity, metabolic deregulation, and, particularly for cancer cells, a benefit from the uptake of fatty acids related to promotion of metastasis. Expanding on this, we started elucidating the effects of OA and PA (25-500 µM) on non-transformed human intestinal epithelial cells (HCEC-1CT) in comparison to colon carcinoma cells (HCT116), with regard to the mechanosensory apparatus. Hence, intestinal cells' motility is on the one side essential to ensure adaption to peristaltic movement and barrier function, but also to enable metastatic progression. Incubation with both OA and PA (≥ 25 µM) significantly decreased membrane fluidity of HCT116 cells, whereas the effect on HCEC-1CT was more limited. Application of rhodamine-labelled PA demonstrated that the fatty acid is incorporated into the plasma membrane of HCT116, which could not be observed in the non-tumorigenic cell line. Down-streaming into the intracellular compartment, a pronounced rearrangement of actin cytoskeleton was evident in both cell lines (OA and PA; 25 and 100 µM). This was accompanied by a variation of translocation efficiency of the mechanosensitive co-transcription factor YAP1, albeit with a stronger effect seen for PA and the cancer cells. Untargeted proteomic analysis confirmed that exposure to OA and PA could alter the response capacity of HCT116 cells to fluid shear stress. Taken together, OA and PA were able to functionally modulate the mechanosensory apparatus of intestinal cells, implying a novel role for dietary fatty acids in the regulation of intestinal pathophysiology.

Activation of autophagy triggers mitochondrial loss and changes acetylation profile relevant for mechanotransduction in bladder cancer cells.

Bladder cells are constantly exposed to multiple xenobiotics and bioactive metabolites. In addition to this challenging chemical environment, they are also exposed to shear stress originating from urine and interstitial fluids. Hence, physiological function of bladder cells relies on a high biochemical and biomechanical adaptive competence, which, in turn, is largely supported via autophagy-related mechanisms. As a negative side of this plasticity, bladder cancer cells are known to adapt readily to chemotherapeutic programs. At the molecular level, autophagy was described to support resistance against pharmacological treatments and to contribute to the maintenance of cell structure and metabolic competence. In this study, we enhanced autophagy with rapamycin (1-100 nM) and assessed its effects on the motility of bladder cells, as well as the capability to respond to shear stress. We observed that rapamycin reduced cell migration and the mechanical-induced translocation potential of Krüppel-like transcription factor 2 (KLF2). These effects were accompanied by a rearrangement of cytoskeletal elements and mitochondrial loss. In parallel, intracellular acetylation levels were decreased. Mechanistically, inhibition of the NAD + -dependent deacetylase sirtuin-1 (SIRT1) with nicotinamide (NAM; 0.1-5 mM) restored acetylation levels hampered by rapamycin and cell motility. Taken together, we described the effects of rapamycin on cytoskeletal elements crucial for mechanotransduction and the dependency of these changes on the mitochondrial turnover caused by autophagy activation. Additionally, we could show that targeted metabolic intervention could revert the outcome of autophagy activation, reinforcing the idea that bladder cells can easily adapt to multiple xenobiotics and circumvent in this way the effects of single chemicals.

Markers for DNA damage are induced in the rat colon by the Alternaria toxin altertoxin-II, but not a complex extract of cultured Alternaria alternata.

Mycotoxins produced by Alternaria spp. act genotoxic in cell-based studies, but data on their toxicity in vivo is scarce and urgently required for risk assessment. Thus, male Sprague-Dawley rats received single doses of a complex Alternaria toxin extract (CE; 50 mg/kg bw), altertoxin II (ATX-II; 0.21 mg/kg bw) or vehicle by gavage, one of the most genotoxic metabolites in vitro and were sacrificed after 3 or 24 h, respectively. Using SDS-PAGE/Western Blot, a significant increase of histone 2a.X phosphorylation and depletion of the native protein was observed for rats that were exposed to ATX-II for 24 h. Applying RT-PCR array technology we identified genes of interest for qRT-PCR testing, which in turn confirmed an induction of Rnf8 transcription in the colon of rats treated with ATX-II for 3 h and CE for 24 h. A decrease of Cdkn1a transcription was observed in rats exposed to ATX-II for 24 h, possibly indicating tissue repair after chemical injury. In contrast to the observed response in the colon, no markers for genotoxicity were induced in the liver of treated animals. We hereby provide the first report of ATX-II as a genotoxicant in vivo. Deviating results for similar concentrations of ATX-II in a natural Alternaria toxin mixture argue for substantial mixture effects.

Combinatory Exposure to Urolithin A, Alternariol, and Deoxynivalenol Affects Colon Cancer Metabolism and Epithelial Barrier Integrity in vitro.

The human gastrointestinal tract is an important site of nutrient absorption and a crucial barrier against xenobiotics. It regularly faces "chemical cocktails" composed of food constituents, their human and microbial metabolites, and foodborne contaminants, such as mycotoxins. Hence, the colonic epithelium adapts to dietary molecules tuning its immune response, structural integrity, and metabolism to maintain intestinal homeostasis. While gut microbiota metabolites of berry ellagitannins, such as urolithin A (Uro A) might contribute to physiological epithelial barrier integrity, foodborne co-contaminating mycotoxins like alternariol (AOH) and deoxynivalenol (DON) could hamper epithelial function. Hence, we investigated the response of differentiated Caco-2 cells (clone C2BBe1) in vitro to the three compounds alone or in binary mixtures. In virtue of the possible interactions of Uro A, AOH, and DON with the aryl hydrocarbon receptor (AhR) pathway, potential effects on phase-I-metabolism enzymes and epithelial structural integrity were taken as endpoints for the evaluation. Finally, Liquid chromatography tandem mass spectrometry measurements elucidated the absorption, secretion, and metabolic capacity of the cells under single and combinatory exposure scenarios. Uro A and AOH as single compounds, and as a binary mixture, were capable to induce CYP1A1/1A2/1B1 enzymes triggered by the AhR pathway. In light of its ribosome inhibiting capacity, the trichothecene suppressed the effects of both dibenzo-α-pyrones. In turn, cellular responsiveness to Uro A and AOH could be sustained when co-exposed to DON-3-sulfate, instead of DON. Colonic epithelial structural integrity was rather maintained after incubation with Uro A and AOH: this was reinforced in the combinatory exposure scenario and disrupted by DON, an effect, opposed in combination. Passage through the cells as well as the metabolism of Uro A and AOH were rather influenced by co-exposure to DON, than by interaction with each other. Therefore, we conclude that although single foodborne bioactive substances individually could either support or disrupt the epithelial structure and metabolic capacity of colon cancer, exposure to chemical mixtures changes the experimental outcome and calls for the need of combinatory investigations for proper risk assessment.

Foodborne compounds that alter plasma membrane architecture can modify the response of intestinal cells to shear stress in vitro.

In order to ensure barrier function, intestinal cells need to respond promptly to biomechanical stimulation and to adapt constantly to physical cues. To this aim, cell membranes are essential and rely extensively on lipid metabolism and turnover. These can be tuned via nutrition, pharmacological treatment, or exposure to xenobiotics, however, knowledge on the impact of lifestyle and diet on intestinal cells' biomechanical compliance is relatively limited. Building on this, two intestinal cell models (non-transformed human colon epithelial cells HCEC-1CT and the colon adenocarcinoma cell line HT-29) were systematically compared in terms of cholesterol content, membrane fluidity, actin cytoskeletal organization, expression of mechano-gated PIEZO1 channels and caveolin-1. Biomechanical compliance was evaluated with the application of fluid shear stress (force response 0.75-1.5 dyn/cm2). As model substances the food contaminant mycotoxin alternariol (AOH, 0.01-10 µM) was chosen in virtue of its putative structural analogy with cholesterol. AOH was compared to the cholesterol lowering agent lovastatin (LOVA, 0.01-10 µM) and to water-soluble cholesterol (MßCD-CHOL, 0.01-10 µg/ml). Exposure to AOH, LOVA and MßCD-CHOL coherently modulated membrane cholesterol, expression of PIEZO1 and caveolin-1 as well as the formation of actin stress fibers. These effects were functionally relevant since they modified the force response profile to fluid shear stress (morphological adaption and [Ca2+]i). In sum, we could demonstrate a novel role for exogenous or endogenous molecules in shaping intestinal mechanotransduction via regulation of cholesterol homeostasis and plasma membrane architecture.

Alternaria alternata Mycotoxins Activate the Aryl Hydrocarbon Receptor and Nrf2-ARE Pathway to Alter the Structure and Immune Response of Colon Epithelial Cells.

After ingestion of food commodities, the gastrointestinal tract (GIT) poses the first barrier against xenobiotics and pathogens. Therefore, it is regularly confronted with external stressors potentially affecting the inflammatory response and the epithelial barrier. Alternaria mycotoxins such as alternariol (AOH) and altertoxin II (ATX-II) are frequently occurring food and feed contaminants that are described for their immunomodulatory capacities. Hence, this study aimed at exploring the effect of AOH and ATX-II as single compounds or binary mixtures on the immune response and epithelial homeostasis in noncancerous colon epithelial cells HCEC-1CT. Both toxins suppressed mRNA levels of proinflammatory mediators interleukin-8 (IL-8), tumor necrosis factor α (TNF-α), and secretion of IL-8, as well as mRNA levels of the matrix metallopeptidase 2 (MMP-2). Binary combinations of AOH and ATX-II reduced the response of the single toxins. Additionally, AOH and ATX-II modified immunolocalization of transmembrane proteins such as integrin ß1, zona occludens 1 (ZO-1), claudin 4 (Cldn 4), and occludin (Ocln), which support colonic tissue homeostasis and intestinal barrier function. Moreover, the cellular distribution of ZO-1 was affected by ATX-II. Mechanistically, these effects could be traced back to the involvement of several transcription factors. AOH activated the nuclear translocation of the aryl hydrocarbon receptor (AhR) and the nuclear factor erythroid 2-related factor 2 (Nrf2), governing cell metabolic competence and structural integrity. This was accompanied by altered distribution of the NF-κB p65 protein, an important regulator of inflammatory response. ATX-II also induced AhR and Nrf2 translocation, albeit failing to substantiate the effect of AOH on the colonic epithelium. Hence, both toxins coherently repress the intestinal immune response on the cytokine transcriptional and protein levels. Furthermore, both mycotoxins affected the colonic epithelial integrity by altering the cell architecture.

Persistence of the antagonistic effects of a natural mixture of Alternaria mycotoxins on the estrogen-like activity of human feces after anaerobic incubation.

Several Alternaria mycotoxins are believed to act as endocrine disruptive chemicals (EDCs), since they are reported to bind estrogen receptors in several experimental models. After ingestion of contaminated food commodities, the mycotoxins reach the intestine, where they come into direct contact with food constituents as well as the gut microbiota. Thus, the aim of the present work was to evaluate the modulatory potential of a complex extract of cultured Alternaria fungi (CE; containing eleven chemically characterized compounds) on the estrogenic signaling cascade of mammalian cells before and after anaerobic incubation with fecal slurries, in order to simulate an in vivo-like condition in the gut. Assessing alkaline phosphatase expression in Ishikawa cells as a measure for estrogenicity, we found the CE to partially quench the intrinsic estrogenic properties of fecal slurries and fecal waters, even after 3 h of fecal incubation. Investigation of the mechanisms underlying the effects observed carried out through an in vitro/in silico approach revealed the ability of the extract to decrease the ERα/ERß nuclear ratio, while a possible action of the mycotoxins as ER-antagonists was excluded. Our results suggest that Alternaria mycotoxins might act as EDCs in vivo, and warrant further investigation in animal models.

Cereulide and Deoxynivalenol Increase LC3 Protein Levels in HepG2 Liver Cells.

Food contaminants of bacterial or fungal origin frequently contaminate staple foods to various extents. Among others, the bacterial toxin cereulide (CER) and the mycotoxin deoxynivalenol (DON) co-occur in a mixed diet and are absorbed by the human body. Both toxins exert dis-tinctive mitotoxic potential. As damaged mitochondria are removed via autophagy, mitochondrial and lysosomal toxicity were assessed by applying low doses of single and combined toxins (CER 0.1-50 ng/mL; DON 0.01-5 µg/mL) to HepG2 liver cells. In addition to cytotoxicity assays, RT-qPCR was performed to investigate genes involved in lysosomal biogenesis and autophagy. CER and DON caused significant cytotoxicity on HepG2 cells after 5 and 24 h over a broad concentration range. CER, alone and in combination with DON, increased the transcription of the autophagy related genes coding for the microtubule associated protein 1A/1B light chain 3 (LC3) and sequestome 1 (SQSTM1) as well as LC3 protein expression which was determined using immunocytochemistry. DON increased LC3 protein expression without induction of gene transcription, hence it seems plausible that CER and DON act on different pathways. The results support the hypothesis that CER induces autophagy via the LC3 pathway and damaged mitochondria are therefore eliminated.

Elucidation of xenoestrogen metabolism by non-targeted, stable isotope-assisted mass spectrometry in breast cancer cells.

Environmental exposure to xenoestrogens, i.e., chemicals that imitate the hormone 17ß-estradiol, has the potential to influence hormone homeostasis and action. Detailed knowledge of xenobiotic biotransformation processes in cell models is key when transferring knowledge learned from in vitro models to in vivo relevance. This study elucidated the metabolism of two naturally-occurring phyto- and mycoestrogens; namely genistein and zearalenone, in an estrogen receptor positive breast cancer cell line (MCF-7) with the aid of stable isotope-assisted metabolomics and the bioinformatic tool MetExtract II. Metabolism was studied in a time course experiment after 2 h, 6 h and 24 h incubation. Twelve and six biotransformation products of zearalenone and genistein were detected, respectively, clearly demonstrating the abundant xenobiotic biotransformation capability of the cells. Zearalenone underwent extensive phase-I metabolism resulting in α-zearalenol (α-ZEL), a molecule known to possess a significantly higher estrogenicity, and several phase-II metabolites (sulfo- and glycoconjugates) of the native compound and the major phase I metabolite α-ZEL. Moreover, potential adducts of zearalenone with a vitamin and several hydroxylated metabolites were annotated. Genistein metabolism resulted in sulfation, combined sulfation and hydroxylation, acetylation, glucuronidation and unexpectedly adduct formation with pentose- and hexose sugars. Kinetics of metabolite formation and subsequent excretion into the extracellular medium revealed a time-dependent increase in most biotransformation products. The untargeted elucidation of biotransformation products formed during cell culture experiments enables an improved and more meaningful interpretation of toxicological assays and has the potential to identify unexpected or unknown metabolites.

A target fishing study to spot possible biological targets of fusaric acid: Inhibition of protein kinase-A and insights on the underpinning mechanisms.

Fusaric acid is a secondary metabolite produced by various Fusarium fungi, present with relatively high incidence in Fusarium-contaminated foods. It was already described as phytotoxic and cytotoxic. However, the understanding of its molecular mechanisms is still fragmentary and further data are needed to ensure an informed assessment of the risk related to its presence in food. This work applied an integrated in silico/in vitro approach to reveal novel potential biological activities of fusaric acid and to investigate the underpinning mechanisms. An in silico reverse screening was used to identify novel biological targets for fusaric acid. Computational results indicated as target protein kinase-A, which was confirmed with biochemical cell-free assays providing evidence of its actual inhibitory potential. Cell-based experiments on intestinal cells (HCEC-1CT cells) identified the mitochondrial network and cell membranes as potentially affected organelles, possibly resulting from PKA inhibition. The integration of 3D molecular modeling supported the plausibility of fusaric acid-dependent inhibition. From the hazard identification perspective, considering the Low Observed Adverse Effect Level described here (0.1 mM) and the possible level of contamination in food, fusaric acid might raise concern from a food safety standpoint and the gastrointestinal tract was described as a meaningful system to investigate with priority.

Morpho-metabotyping the oxidative stress response.

Oxidative stress and reactive oxygen species (ROS) are central to many physiological and pathophysiological processes. However, due to multiple technical challenges, it is hard to capture a comprehensive readout of the cell, involving both biochemical and functional status. We addressed this problem by developing a fully parallelized workflow for metabolomics (providing absolute quantities for > 100 metabolites including TCA cycle, pentose phosphate pathway, purine metabolism, glutathione metabolism, cysteine and methionine metabolism, glycolysis and gluconeogenesis) and live cell imaging microscopy. The correlative imaging strategy was applied to study morphological and metabolic adaptation of cancer cells upon short-term hydrogen peroxide (H2O2) exposure in vitro. The combination provided rich metabolic information at the endpoint of exposure together with imaging of mitochondrial effects. As a response, superoxide concentrations were elevated with a strong mitochondrial localization, and multi-parametric image analysis revealed a shift towards fragmentation. In line with this, metabolism reflected both the impaired mitochondrial function and shifts to support the first-line cellular defense and compensate for energy loss. The presented workflow combining high-end technologies demonstrates the applicability for the study of short-term oxidative stress, but it can be suitable for the in-depth study of various short-term oxidative and other cellular stress-related phenomena.

Endoplasmic Reticulum Adaptation and Autophagic Competence Shape Response to Fluid Shear Stress in T24 Bladder Cancer Cells.

Accumulation of xenobiotics and waste metabolites in the urinary bladder is constantly accompanied by shear stress originating from the movement of the luminal fluids. Hence, both chemical and physical cues constantly modulate the cellular response in health and disease. In line, bladder cells have to maintain elevated mechanosensory competence together with chemical stress response adaptation potential. However, much of the molecular mechanisms sustaining this plasticity is currently unknown. Taking this as a starting point, we investigated the response of T24 urinary bladder cancer cells to shear stress comparing morphology to functional performance. T24 cells responded to the shear stress protocol (flow speed of 0.03 ml/min, 3 h) by significantly increasing their surface area. When exposed to deoxynivalenol-3-sulfate (DON-3-Sulf), bladder cells increased this response in a concentration-dependent manner (0.1-1 µM). DON-3-Sulf is a urinary metabolite of a very common food contaminant mycotoxin (deoxynivalenol, DON) and was already described to enhance proliferation of cancer cells. Incubation with DON-3-Sulf also caused the enlargement of the endoplasmic reticulum (ER), decreased the lysosomal movement, and increased the formation of actin stress fibers. Similar remodeling of the endoplasmic reticulum and area spread after shear stress were observed upon incubation with the autophagy activator rapamycin (1-100 nM). Performance of experiments in the presence of chloroquine (chloroquine, 30 µM) further contributed to shed light on the mechanistic link between adaptation to the biomechanical stimulation and ER stress response. At the molecular level, we observed that ER reshaping was linked to actin organization, with the two components mutually regulating each other. Indeed, we identified in the ER stress-cytoskeletal rearrangement an important axis defining the physical/chemical response potential of bladder cells and created a workflow for further investigation of urinary metabolites, food constituents, and contaminants, as well as for pharmacological profiling.

Exploring the dermotoxicity of the mycotoxin deoxynivalenol: combined morphologic and proteomic profiling of human epidermal cells reveals alteration of lipid biosynthesis machinery and membrane structural integrity relevant for skin barrier function.

Deoxynivalenol (vomitoxin, DON) is a secondary metabolite produced by Fusarium spp. fungi and it is one of the most prevalent mycotoxins worldwide. Crop infestation results not only in food and feed contamination, but also in direct dermal exposure, especially during harvest and food processing. To investigate the potential dermotoxicity of DON, epidermoid squamous cell carcinoma cells A431 were compared to primary human neonatal keratinocytes (HEKn) cells via proteome/phosphoproteome profiling. In A431 cells, 10 µM DON significantly down-regulated ribosomal proteins, as well as mitochondrial respiratory chain elements (OXPHOS regulation) and transport proteins (TOMM22; TOMM40; TOMM70A). Mitochondrial impairment was reflected in altered metabolic competence, apparently combined with interference of the lipid biosynthesis machinery. Functional effects on the cell membrane were confirmed by live cell imaging and membrane fluidity assays (0.1-10 µM DON). Moreover, a common denominator for both A431 and HEKn cells was a significant downregulation of the squalene synthase (FDFT1). In sum, proteome alterations could be traced back to the transcription factor Klf4, a crucial regulator of skin barrier function. Overall, these results describe decisive molecular events sustaining the capability of DON to impair skin barrier function. Proteome data generated in the study are fully accessible via ProteomeXchange with the accession numbers PXD011474 and PXD013613.

Assessing Mixture Effects of Cereulide and Deoxynivalenol on Intestinal Barrier Integrity and Uptake in Differentiated Human Caco-2 Cells.

The human intestine is regularly exposed to ingested food contaminants, such as fungal and bacterial toxins, which have been described to co-occur in a mixed diet. Thus, it is of utmost importance to understand possible interactions between contaminants of different origin. Hence, we investigated the single and combined effects of one of the most abundant mycotoxins, deoxynivalenol (DON; 0.1 to 10 µg/mL), and the bacterial toxin cereulide (CER; 1 to 100 ng/mL) on differentiated human Caco-2 (C2BBe1) cells cultured in a transwell system. We tested the capacity of the two toxins to alter the intestinal integrity and further investigated the uptake of both compounds and the formation of selected DON metabolites. CER alone (10 and 100 ng/mL) and in combination with DON (10 ng/mL CER with 1 µg/mL DON) was found to alter the barrier function by increasing the transepithelial electrical resistance and the expression of the tight junction protein claudin-4. For the first time, DON-3-sulfate was identified as a metabolite of human intestinal cells in vitro. Moreover, co-incubation of CER and DON led to an altered ratio between DON and DON-3-sulfate. Hence, we conclude that co-exposure to CER and DON may alter the intestinal barrier function and biotransformation of intestinal cells.

Eicosanoid Content in Fetal Calf Serum Accounts for Reproducibility Challenges in Cell Culture.

Reproducibility issues regarding in vitro cell culture experiments are related to genetic fluctuations and batch-wise variations of biological materials such as fetal calf serum (FCS). Genome sequencing may control the former, while the latter may remain unrecognized. Using a U937 macrophage model for cell differentiation and inflammation, we investigated whether the formation of effector molecules was dependent on the FCS batch used for cultivation. High resolution mass spectrometry (HRMS) was used to identify FCS constituents and to explore their effects on cultured cells evaluating secreted cytokines, eicosanoids, and other inflammatory mediators. Remarkably, the FCS eicosanoid composition showed more batch-dependent variations than the protein composition. Efficient uptake of fatty acids from the medium by U937 macrophages and inflammation-induced release thereof was evidenced using C13-labelled arachidonic acid, highlighting rapid lipid metabolism. For functional testing, FCS batch-dependent nanomolar concentration differences of two selected eicosanoids, 5-HETE and 15-HETE, were balanced out by spiking. Culturing U937 cells at these defined conditions indeed resulted in significant proteome alterations indicating HETE-induced PPARγ activation, independently corroborated by HETE-induced formation of peroxisomes observed by high-resolution microscopy. In conclusion, the present data demonstrate that FCS-contained eicosanoids, subject to substantial batch-wise variation, may modulate cellular effector functions in cell culture experiments.