Human Colonic Caco-2 Cells Research Articles

Valorization of Pichia spent medium via one-pot synthesis of biocompatible silver nanoparticles with potent antioxidant, antimicrobial, tyrosinase inhibitory and reusable catalytic activities.

The purpose of this study was to determine the feasibility of a novel biogenic synthesis of silver nanoparticles using Pichia pastoris spent medium - a major waste product from heterologous protein expression as the sole reducing and capping agent, with potential biological, antimicrobial and wastewater remediation applications. Using UV-vis spectroscopy, TEM, XRD, EDX, FTIR, SEM we demonstrated the successful bio-fabrication of Pichia spent medium encapsulated silver nanoparticles (PSM-AgNPs). PSM-AgNPs displayed substantial antiradical activity against DPPH and ABTS. The antiradical activity against ABTS was similar to that of the control, ascorbic acid. PSM-AgNPs also revealed potent anti-tyrosinase and antibacterial activity against some common foodborne pathogenic microbes. Human erythrocyte hemolytic and embryonic colon Caco-2 cell viability assays suggest that PSM-AgNPs was biocompatible. In addition, PSM-AgNPs, was also effective in the catalytic degradation of methyl orange and Congo red dyes with pseudo first order rate constants of 0.2301min-1 and 4.7×10-3s-1, respectively. These results present a clean, convenient, and inexpensive approach for the biosynthesis of silver nanoparticles with potential implications in the eco-friendly, safe and effective utilization of waste culture media, mitigation of pathogenic bacteria and management of industrial effluents.

Herbal components of Japanese Kampo medicines exert laxative actions in colonic epithelium cells via activation of BK and CFTR channels

Japanese Kampo medicines Junchoto and Mashiningan are mixtures of numerous herbal plant extracts and empirically known to exert laxative actions by stimulating fluid secretion in the colonic epithelium. However, it is unknown which and how the herbal components of these crude Kampo drugs are effective to stimulate ion effluxes causing fluid secretion. Here, we selected four herbal components of Junchoto and Mashiningan, Mashinin (MSN), Kyonin (KYN), Tonin (TON), and Daio (DIO), which are putatively laxatives, and examined their effects on the ion channel activity of human colonic epithelial Caco-2 cells. Patch clamp analyses revealed that MSN activated whole-cell current characteristics of the cystic fibrosis transmembrane conductance regulator (CFTR) channel, whereas KYN, TON, and DIO activated the large-conductance and voltage-activated K+ (BK) channel. Furthermore, electronic cell sizing showed that MSN induced secretory volume decrease (SVD) sensitivity to a CFTR blocker, whereas TON, KYN, and DIO induced SVD sensitivity to a K+ channel blocker. In conclusion, MSN and TON, KYN, and DIO promote fluid secretion from colonic epithelial cells by activating CFTR and BK channels. Thus, Japanese Kampo medicines, Junchoto and Mashiningan, exert anti-constipation actions by inducing KCl efflux through the combined actions of CFTR- and BK-stimulating herbal components.

Progesterone decreases gut permeability through upregulating occludin expression in primary human gut tissues and Caco-2 cells

Progesterone plays a protective role in preventing inflammation and preterm delivery during pregnancy. However, the mechanism involved is unknown. Microbial product translocation from a permeable mucosa is demonstrated as a driver of inflammation. To study the mechanism of the protective role of progesterone during pregnancy, we investigated the effect of physiologic concentrations of progesterone on tight junction protein occludin expression and human gut permeability in vitro and systemic microbial translocation in pregnant women in vivo. Plasma bacterial lipopolysaccharide (LPS), a representative marker of in vivo systemic microbial translocation was measured. We found that plasma LPS levels were significantly decreased during 24 to 28 weeks of gestation compared to 8 to 12 weeks of gestation. Moreover, plasma LPS levels were negatively correlated with plasma progesterone levels but positively correlated with plasma tumor necrosis factor-alpha (TNF-α) levels at 8 to 12 weeks of gestation but not at 24 to 28 weeks of gestation. Progesterone treatment increased intestinal trans-epithelial electrical resistance (TEER) in primary human colon tissues and Caco-2 cells in vitro through upregulating tight junction protein occludin expression. Furthermore, progesterone exhibited an inhibitory effect on nuclear factor kappa B (NF-κB) activation following LPS stimulation in Caco-2 cells. These results reveal a novel mechanism that progesterone may play an important role in decreasing mucosal permeability, systemic microbial translocation, and inflammation during pregnancy.

Functional characterization of SLC26A3 c.392C>G (p.P131R) mutation in intestinal barrier function using CRISPR/CAS9-created cell models

BackgroundCongenital chloride diarrhea (CCD) in a newborn is a rare autosomal recessive disorder with life-threatening complications, requiring early diagnostics and treatment to prevent severe dehydration and infant mortality. SLC26A3 rs386833481 (c.392C>G; p.P131R) gene polymorphism is an important genetic determinant of CCD. Here, we report the influence of the non-synonymous SLC26A3 variant rs386833481 gene polymorphism on the function of the epithelial barrier and the potential mechanisms of these effects.ResultsWe found that P131R-SLC26A3 increased dysfunction of the epithelial barrier compared with wild type SLC26A3 in human colonic Caco-2 and mouse colonic CMT-93 cells. When P131R-SLC26A3 was subsequently reverted to wild type, the epithelial barrier function was restored similar to wild type cells. Further study demonstrated that variant P131R-SLC26A3 disrupts function of epithelial barrier through two distinct molecular mechanisms: (a) decreasing SLC26A3 expression through a ubiquitination pathway and (b) disrupting a key interaction with its partner ZO-1/CFTR, thereby increasing the epithelial permeability.ConclusionOur study provides an important insight of SLC26A3 SNPs in the regulation of the epithelial permeability and indicates that SLC26A3 rs386833481 is likely a causative mutation in the dysfunction of epithelial barrier of CCD, and correction of this SNP or increasing SLC26A3 function could be therapeutically beneficial for chronic diarrhea diseases.

Determination of polycyclic aromatic hydrocarbon content in heat-treated meat retailed in Egypt: Health risk assessment, benzo[a]pyrene induced mutagenicity and oxidative stress in human colon (CaCo-2) cells and protection using rosmarinic and ascorbic acids

This study was undertaken to estimate the concentrations of the formed polycyclic aromatic hydrocarbons (PAHs) in heat-treated (boiled, pan-fried and grilled) meats collected from Egypt. Dietary intakes and cancer risks of PAHs among Egyptian adults were calculated. Benzo[a]pyrene (B[a]P)-induced mutagenicity and oxidative stress in human colon (CaCo-2) cell line and mechanisms behind such effects were also investigated. Finally, protection trials using rosmarinic (RMA) and ascorbic acids (ASA) were carried out. The results indicated formation of PAHs at high levels in the heat-treated meats. Calculated incremental life time cancer risk among Egyptian adults were 7.05179E−07, 7.00604 E−06 and 1.86069 E−05 due to ingestion of boiled, pan-fried and grilled meats, respectively. B[a]P-exposed CaCo-2 cells had high abilities for mutagenicity (490.05 ± 21.37 His + revertants) and production of reactive oxygen species. RMA and ASA protected CaCo-2 cells via reduction of B[a]P-induced mutagenicity and oxidative stress and upregulation of phase II detoxification enzymes and xenobiotic transporters.

Host-antibody inductivity of virulent Entamoeba histolytica and non-virulent Entamoeba moshkovskii in a mouse model

BackgroundDespite similarities in morphology, gene and protein profiles, Entamoeba histolytica and E. moshkovskii show profound differences in pathogenicity. Entamoeba histolytica infection might result in amoebic dysentery and liver abscess, while E. moshkovskii causes only mild diarrhea. Extensive studies focus on roles of host immune responses to the pathogenic E. histolytica; however, evidence for E. moshkovskii remains scarce.MethodsTo study differences in host-antibody response profiles between E. histolytica and E. moshkovskii, mice were immunized intraperitoneally with different sets of Entamoeba trophozoites as single species, mixed species and combinations.ResultsMice prime-immunized with E. histolytica and E. moshkovskii combination, followed by individual species, exhibited higher IgG level than the single species immunization. Mice immunized with E. moshkovskii induced significantly higher levels and long-lasting antibody responses than those challenged with E. histolytica alone. Interestingly, E. histolytica-specific anti-sera promoted the cytopathic ability of E. histolytica toward Chinese hamster ovarian (CHO) cells, but showed no effect on cell adhesion. There was no significant effect of immunized sera on cytopathic activity and adhesion of E. moshkovskii toward both CHO and human epithelial human colonic (Caco-2) cell lines. Monoclonal-antibody (mAb) characterization demonstrated that 89% of E. histolytica-specific mAbs produced from mice targeted cytoplasmic and cytoskeletal proteins, whereas 73% of E. moshkovskii-specific mAbs targeted plasma membrane proteins.ConclusionsThe present findings suggest that infection with mixed Entamoeba species or E. moshkovskii effectively induces an antibody response in mice. It also sheds light on roles of host antibody response in the pathogenic difference of E. histolytica and E. moshkovskii trophozoites, and cell surface protein modifications of the amoebic parasites to escape from host immune system.

Difference in Mono-O-Glucosylation of Ras Subtype GTPases Between Toxin A and Toxin B From Clostridioides difficile Strain 10463 and Lethal Toxin From Clostridium sordellii Strain 6018.

Clostridioides difficile toxin A (TcdA) and Toxin B (TcdB) trigger inflammasome activation with caspase-1 activation in cultured cells, which in turn induce the release of IL-6, IFN-γ, and IL-8. Release of these proinflammatory responses is positively regulated by Ras-GTPases, which leads to the hypothesis that Ras glucosylation by glucosylating toxins results in (at least) reduced proinflammatory responses. Against this background, data on toxin-catalyzed Ras glucosylation are required to estimate of pro-inflammatory effect of the glucosylating toxins. In this study, a quantitative evaluation of the GTPase substrate profiles glucosylated in human colonic (Caco-2) cells treated with either TcdA, TcdB, or the related Clostridium sordellii lethal toxin (TcsL) was performed using multiple reaction monitoring (MRM) mass spectrometry. (H/K/N)Ras are presented to be glucosylated by TcsL and TcdA but by neither TcdB isoform tested. Furthermore, the glucosylation of (H/K/N)Ras was detected in TcdA-(not TcdB)-treated cells, as analyzed exploiting immunoblot analysis using the Ras glucosylation-sensitive 27H5 antibody. Furthermore, [14C]glucosylation of substrate GTPase was found to be increased in a cell-free system complemented with Caco-2 lysates. Under these conditions, (H/K/N)Ras glucosylation by TcdA was detected. In contrast, TcdB-catalyzed (H/K/N)Ras glucosylation was detected by neither MRM analysis, immunoblot analysis nor [14C]glucosylation in a cell-free system. The observation that TcdA (not TcdB) glucosylates Ras subtype GTPases correlates with the fact that TcdB (not TcdA) is primarily responsible for inflammatory responses in CDI. Finally, TcsL more efficaciously glucosylated Ras subtype GTPase as compared with TcdA, reinforcing the paradigm that TcsL is the prototype of a Ras glucosylating toxin.

Butyrate interacts with benzo[a]pyrene to alter expression and activities of xenobiotic metabolizing enzymes involved in metabolism of carcinogens within colon epithelial cell models

Butyrate helps to maintain colon homeostasis and exhibits chemopreventive effects in colon epithelium. We examined the interactive effects of butyrate and benzo[a]pyrene (BaP), dietary carcinogen, in regulation of expression of a panel of phase I and II xenobiotic metabolizing enzymes (XMEs) in human colon cells. In human colon carcinoma HCT-116 and HT-29 cell lines, butyrate alone increased mRNA levels of some enzymes, such as N-acetyltransferases (in particular NAT2). In combination with BaP, butyrate potentiated induction of cytochrome P450 family 1 enzymes (CYP1A1), aldo-keto reductases (AKR1C1) or UDP-glucuronosyltransferases (UGT1A1). There were some notable differences between cell lines, as butyrate potentiated induction of NAD(P)H:quinone oxidoreductase 1 (NQO1) and UGT1A4 only in HCT-116 cells, and it even repressed AKR1C3 induction in HT-29 cells. Butyrate also promoted induction of CYP1, NQO1, NAT2, UGT1A1 or UGT1A4 in human colon Caco-2 cells, in a differentiation-dependent manner. Differentiated Caco-2 cells exhibited a higher inducibility of selected XME genes than undifferentiated cells. Butyrate increased induction of enzymatic activities of NATs, NQO1 and UGTs by BaP in HCT-116 and HT29 cells, whereas in differentiated Caco-2 cells it helped to increase only enzymatic activity of NQO1 and UGTs. Together, the present data suggest that butyrate may modulate expression/activities of several enzymes involved in metabolism of carcinogens in colon. In some cases (NAT2, UGT1 A1), this was linked to inhibition of histone deacetylases (HDAC), as confirmed by using HDAC inhibitor trichostatin A. These results may have implications for our understanding of the role of butyrate in regulation of XMEs and carcinogen metabolism in colon.

Strategies and limitations associated with in vitro characterization of vitamin D receptor activators

In vitro cell-based assays are common screening tools used for the identification of new VDR ligands. For 25-hydroxyvitamin D3 [25(OH)D3] and 1α-hydroxyvitamin D3 [1α(OH)D3], protein expressions of CYP2R1 and CYP27B1, respectively, that form the active 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] ligand were detected in human embryonic kidney (HEK293) cells expressing the GAL4-hVDR, the human brain microvessel endothelial (hCMEC/D3) and adenocarcinoma colonic (Caco-2) cells. The impact of bioactivation enzymes was shown upon the addition of ketoconazole (10 μM KTZ), a pan-CYP inhibitor, which reduced the apparent potency of 25(OH)D3 and increased the EC50 from 272 to 608 nM in HEK293 cells. EIA assays verified that 1,25(OH)2D3 was formed and contributed to VDR activity independently of its precursors. In hCMEC/D3 cells where enzyme protein levels were lowest, changes in MDR1/P-gp expression with KTZ were minimal. In Caco-2 cells, the induction of TRPV6 (calcium channel), CYP24A1, CYP3A4, OATP1A2 and MDR1 mRNA expression was 1,25(OH)2D3 > 1α(OH)D3 > 25(OH)D3, with the magnitude of change being blunted by KTZ. Upon inclusion of KTZ in the cell-based assays, high transcriptional activities were observed for synthetic VDR activators from Teijin Pharma. Cyclopentanone derivatives: TPD-003, TPD-005, TPD-006, TPD-008 and TPD-009 (EC50s 0.06 to 67 nM, unchanged with KTZ) were found more potent over straight chain and lactone derivatives (antagonists). Most TPD compounds activated OATP1A2, CYP24A1, CYP3A4, and MDR1 (28–67%) and TRPV6 transcriptionally in Caco-2 cells. The results identified that cell-based assays with added KTZ could accurately identify new VDR activators, although these may be hypercalcemic with strong TRPV6 inducing properties.

Transcriptional control of intestinal cholesterol absorption, adipose energy expenditure and lipid handling by Sortilin

The sorting receptor Sortilin functions in the regulation of glucose and lipid metabolism. Dysfunctional lipid uptake, storage, and metabolism contribute to several major human diseases including atherosclerosis and obesity. Sortilin associates with cardiovascular disease; however, the role of Sortilin in adipose tissue and lipid metabolism remains unclear. Here we show that in the low-density lipoprotein receptor-deficient (Ldlr−/−) atherosclerosis model, Sortilin deficiency (Sort1−/−) in female mice suppresses Niemann-Pick type C1-Like 1 (Npc1l1) mRNA levels, reduces body and white adipose tissue weight, and improves brown adipose tissue function partially via transcriptional downregulation of Krüppel-like factor 4 and Liver X receptor. Female Ldlr−/−Sort1−/− mice on a high-fat/cholesterol diet had elevated plasma Fibroblast growth factor 21 and Adiponectin, an adipokine that when reduced is associated with obesity and cardiovascular disease-related factors. Additionally, Sort1 deficiency suppressed cholesterol absorption in both female mice ex vivo intestinal tissue and human colon Caco-2 cells in a similar manner to treatment with the NPC1L1 inhibitor ezetimibe. Together our findings support a novel role of Sortilin in energy regulation and lipid homeostasis in female mice, which may be a potential therapeutic target for obesity and cardiovascular disease.

Abstract 595: Transcriptional Control of Intestinal Cholesterol Absorption, Adipose Energy Expenditure and Lipid Handling by Sortilin

Objective: The sorting receptor Sortilin functions in the regulation of glucose and lipid metabolism. Dysfunctional lipid uptake, storage, and metabolism contribute to several major human diseases including atherosclerosis and obesity. Sortilin associates with cardiovascular disease; however, the role of Sortilin in adipose tissue and lipid metabolism remains unclear. Approach and Results: Here we show that in the low-density lipoprotein receptor-deficient (Ldlr -/- ) atherosclerosis model, Sortilin deficiency ( Sort1 -/- ) in female mice inhibits intestinal Niemann-Pick type C1-Like 1 (Npc1l1) expression (-60.6 %, p<0.01), reduces body (-17.2 %, p<0.01) and white adipose tissue weight (-35.2 %, p<0.05), and improves brown adipose tissue function partially via transcriptional downregulation of Krüppel-like factor 4 and Liver X receptor (Figure). Female Ldlr -/- Sort1 -/- mice on a high fat/cholesterol diet had elevated plasma Fibroblast growth factor 21 (+89.1 %, p<0.05) and Adiponectin (+37.7 %, p<0.01), an adipokine that when reduced is associated with obesity and cardiovascular disease related factors. Additionally, Sortilin deficiency suppressed cholesterol absorption in both human colon Caco-2 cells (-16.5 %, p<0.05) and mouse ex vivo intestinal tissue (-16.9 %, p<0.05) in a similar manner to treatment with the Npc1l1 inhibitor - ezetimibe. Conclusions: Together our findings support a novel role of Sortilin in energy regulation and lipid homeostasis in female mice, which may be a potential therapeutic target for obesity and cardiovascular disease.

Hypoxia-induced intestinal barrier changes in balloon-assisted enteroscopy.

Balloon-assisted enteroscopy (BAE) is an emerging standard procedure by utilizing distensible balloons to facilitate deep endoscopy in the small and large intestine. Sporadic cases of bacteraemia were found after BAE. Balloon distension by BAE caused gut tissue hypoxia. The impact of balloon distension-induced hypoxia on intestinal barriers remains unclear. Murine models of BAE by colonic balloon distension showed that short- and long-term hypoxia evoked opposite effects on epithelial tight junctions (TJs). Short-term hypoxia fortified TJ integrity, whereas long-term hypoxia caused damage to barrier function. Our data showed for the first time the molecular mechanisms and signalling pathways of epithelial barrier fortification and TJ reorganization by short-term hypoxia for the maintenance of gut homeostasis. The findings suggest avoiding prolonged balloon distension during BAE to reduce the risk of hypoxia-induced gut barrier dysfunction. Balloon-assisted enteroscopy (BAE) is an emerging standard procedure that uses distensible balloons to facilitate deep endoscopy. Intestines are known to harbour an abundant microflora. Whether balloon distension causes perturbation of blood flow and gut barrier dysfunction, and elicits risk of bacterial translocation remains unknown. Our aims were to (1) conduct a prospective study to gather microbiological and molecular evidence of bacterial translocation by BAE in patients, (2) establish a murine model of colonic balloon distension to investigate tissue hypoxia and intestinal barrier, and (3) assess the effect of short- and long-term hypoxia on epithelial permeability using cell lines. Thirteen patients were enrolled for BAE procedures, and blood samples were obtained before and after BAE for paired comparison. Four of the 13 patients (30.8%) had positive bacterial DNA in blood after BAE. Post-BAE endotoxaemia was higher than the pre-BAE level. Nevertheless, no clinical symptom of sepsis or fever was reported. To mimic clinical BAE, mice were subjected to colonic balloon distension. Local tissue hypoxia was observed during balloon inflation, and reoxygenation after deflation. A trend of increased gut permeability was seen after long-term distension, whereas a significant reduction of permeability was observed by short-term distension in the proximal colon. Human colonic epithelial Caco-2 cells exposed to hypoxia for 5-20min exhibited increased tight junctional assembly, while those exposed to longer hypoxia displayed barrier disruption. In conclusion, sporadic cases of bacteraemia were found after BAE, without septic symptoms. Short-term hypoxia by balloon distension yielded a protective effect whereas long-term hypoxia caused damage to the gut barrier.

CYTOTOXICITY EVALUATION OF TITANIUM AND ZINC OXIDE NANOPARTICLES ON HUMAN CELL LINES

Objective: In vitro cytotoxicity evaluation of titanium dioxide, 20 nm (TNP 20) and zinc oxide, 20 nm (ZNP 20) nanoparticles (NP) were tested on different types of human skin (HaCat), lung (A549), liver (Hep G2) and colon (Caco-2) cell cultures in relevance to human risk assessmentMethods: The different concentrations of test TNP 20 and ZNP 20 1-300 µg/ml were exposed to determine the cell viability reduction on four human cell lines after 48 h post exposure using 3-(4, 5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). The mitochondrial membrane activities of the viable cells were determined with intensity of formazon formation by interpreting ELISA absorbance values at 470 nm.Results: The percent of cytotoxicity was determined by comparing percentage of cell viability reduction of test with that of control. The ZNP 20 produced higher cytotoxicity at the doses 100 (p<0.05) and 300 (p<0.001) µg/ml significantly on tested four human skin (HaCaT), lung (A549), liver (Hep G2) and colon (Caco-2) cells compared to TNP 20. The tested NP induced lesser cytotoxicity at lower concentrations with 1 and 3µg/ml in all the tested four cell lines. The induced cytotoxicity was an indicator for increased intracellular reactive oxygen species which further cause’s major cell damage and cell death.Conclusion: The tested NP were induced greater cytotoxicity in the colon, Liver, lung and skin cells at higher concentrations 100 and 300 µg/ml significantly. The cytotoxicity order of TNP 20 and ZNP 20 at the highest dose (300µg/ml) were concluded as Caco-2>Hep G2>A549>HaCaT for 48 h post exposed cells.

Probiotic Bacteria and their Supernatants Protect Enterocyte Cell Lines from Enteroinvasive Escherichia coli (EIEC) Invasion.

Probiotic microorganisms have attracted a growing interest for prevention and therapy of gastrointestinal disorders. Many probiotic strains have been shown to inhibit growth and metabolic activity of enteropathogenic bacteria as well as their adhesion and invasion to intestinal cells. In the present study, we evaluated the interference of bacteria-free supernatants (BFS) of cultures belonging to sixteen strains of lactobacilli and bifidobacteria, with invasion of enteroinvasive Escherichia coli (EIEC) strain, using human colonic adenocarcinoma cell lines, T84 and Caco2 cells. To assess invasion of Caco-2 and T84 cells by EIEC, and measure the number of pathogens inside the enterocytes, the gentamicin protection assay was conducted. In addition, three different invasion inhibition assays were designed; namely co-incubation, pre-incubation and treatment with the BFS of probiotics. Data obtained and theoretical calculation showed that the most effective assay in the prevention of pathogen invasion was treatment with BFS. Besides, co-incubation assay was more valid than pre-incubation assay in invasion prevention. The obtained results suggest that probiotics may produce some metabolites that strongly prevent invasion of enteroinvasive E.coli into the small and large intestine. Also, probiotics are able to compete with or exclude pathogen invasion.

Effects of pro-inflammatory cytokines, lipopolysaccharide and COX-2 mediators on human colonic neuromuscular function and epithelial permeability

Chronic colitis is associated with decreased colonic muscle contraction and loss of mucosal barrier function. Pro-inflammatory cytokines and bacterial lipopolysaccharide (LPS) are important in the generation and maintenance of inflammation. While colitis is associated with upregulated COX-2 -derived prostanoids and nitric oxide (NO), the direct activity of pro-inflammatory cytokines on human colonic neuromuscular function is less clear. This study investigated the effects of IBD-associated pro-inflammatory cytokines IL-17, TNF-α, IL-1β and LPS on human colonic muscle strip contractility, alone and following inhibition of COX-2 or nitric oxide production. In addition, human colonic epithelial Caco-2 cell monolayers were treated with LPS or COX-2 mediators including prostaglandins (PGE2, PGF2α) or their corresponding ethanolamides (PGE2-EA or PGF2α-EA) over 48h and trans-epithelial electrical resistance used to record permeability changes. Longitudinal muscle strips were obtained from healthy colonic resection margins and mounted in organ baths following IL-17, TNF-α, IL-1β and bacterial LPS incubations in an explant setting over 20h. Contraction in response to acetylcholine (ACh) was then measured, before and after either COX-2 inhibition (nimesulide; 10−5M) or nitric oxide synthase (NOS) inhibition (l-NNA; 10−4M). None of the cytokine or LPS explant incubations affected the potency or maximum cholinergic contraction in vitro, and subsequent COX-2 blockade with nimesulide revealed a significant but similar decrease in potency of ACh-evoked contraction in control, LPS and cytokine-incubated muscle strips. Pre-treatment with l-NNA provided no functional differences in the potency or maximum contractile responses to ACh in cytokine or LPS-incubated colonic longitudinal smooth muscle. Only PGE2 transiently increased Caco-2 monolayer permeability at 24h, while LPS (10μg/ml) increased permeability over 24–48h.These findings indicate that cholinergic contractility in the human colon can be decreased by the blockade of COX-2 generated excitatory prostanoids, but major pro-inflammatory cytokines or LPS do not alter the sensitivity or amplitude of this contraction ex vivo. While PGE2 transiently increase epithelial permeability, LPS generates a significant and sustained increase in permeability indicative of an important role on barrier function at the mucosal interface.

Hydrophobicity of Antifungal β-Peptides Is Associated with Their Cytotoxic Effect on In Vitro Human Colon Caco-2 and Liver HepG2 Cells.

The widespread distribution of fungal infections, with their high morbidity and mortality rate, is a global public health problem. The increase in the population of immunocompromised patients combined with the selectivity of currents treatments and the emergence of drug-resistant fungal strains are among the most imperative reasons to develop novel antifungal formulations. Antimicrobial β-peptides are peptidomimetics of natural antimicrobial peptides (AMPs), which have been proposed as developmental platforms to enhance the AMPs selectivity and biostability. Their tunability allows the design of sequences with remarkable activity against a wide spectrum of microorganisms such as the human pathogenic Candida spp., both in planktonic and biofilm morphology. However, the β-peptide’s effect on surrounding host cells remains greatly understudied. Assessments have mainly relied on the extent of hemolysis that a candidate peptide is able to cause. This work investigated the in vitro cytotoxicity of various β-peptides in the Caco-2 and HepG2 mammalian cell lines. Results indicated that the cytotoxic effect of the β-peptides was influenced by cell type and was also correlated to structural features of the peptide such as hydrophobicity. We found that the selectivity of the most hydrophobic β-peptide was 2–3 times higher than that of the least hydrophobic one, for both cell types according to the selectivity index parameter (IC50/MIC). The IC50 of Caco-2 and HepG2 increased with hydrophobicity, which indicates the importance of testing putative therapeutics on different cell types. We report evidence of peptide-cell membrane interactions in Caco-2 and HepG2 using a widely studied β-peptide against C. albicans.

Contribution of ionic silver to genotoxic potential of nanosilver in human liver HepG2 and colon Caco2 cells evaluated by the cytokinesis-block micronucleus assay.

Extensive human exposure to food- and cosmetics-related consumer products containing nanosilver is of public concern because of the lack of information about their safety. Genotoxicity is an important endpoint for the safety and health hazard assessment of regulated products including nanomaterials. The in vitro cytokinesis-block micronucleus (CBMN) assay is a very useful test for predictive genotoxicity testing. Recently, we have reported the genotoxicity of 20 nm nanosilver in human liver HepG2 and colon Caco2 cells evaluated using the CBMN assay. The objective of our present study was three-fold: (i) to evaluate if HepG2 and Caco2 cells are valuable in vitro models for rapid genotoxicity screening of nanosilver; (ii) to test the hypothesis that the nanoparticle size and cell types are critical determinants of its genotoxicity; and (iii) to determine if ionic silver contributes to the nanosilver genotoxicity. With these objectives in mind, we evaluated the genotoxic potential of 50 nm nanosilver of the same shape, composition, surface charge, obtained from the same commercial source, under the same experimental conditions and the same genotoxic CBMN endpoint used for the previously tested 20 nm silver. The ionic silver (silver acetate) was also evaluated under the same conditions. Results of our study show that up to the concentrations tested in these cell types, the smaller (20 nm) nanosilver induces micronucleus formation in both the cell types but the larger (50 nm) nanosilver and the ionic silver provide a much weaker response compared with controls under the same conditions.

Flow cytometric evaluation of the contribution of ionic silver to genotoxic potential of nanosilver in human liver HepG2 and colon Caco2 cells.

Exposure to nanosilver found in food- and cosmetics-related consumer products is of public concern because of the lack of information about its safety. In this study, two widely used in vitro cell culture models, human liver HepG2 and colon Caco2 cells, and the flow cytometric micronucleus (FCMN) assay were evaluated as tools for rapid predictive screening of the potential genotoxicity of nanosilver. Recently, we reported the genotoxicity of 20 nm nanosilver using these systems. In the current study presented here, we tested the hypothesis that the nanoparticle size and cell types were critical determinants of its genotoxicity. To test this hypothesis, we used the FCMN assay to evaluate the genotoxic potential of 50 nm nanosilver of the same shape, composition, surface charge and obtained from the same commercial source using the same experimental conditions and in vitro models (HepG2 and Caco2) as previously tested for the 20 nm silver. Results of our study show that up to the concentrations tested in these cultured cell test systems, the smaller (20 nm) nanoparticle is genotoxic to both the cell types by inducing micronucleus (MN). However, the larger (50 nm) nanosilver induces MN only in HepG2 cells, but not in Caco2 cells. Also in this study, we evaluated the contribution of ionic silver to the genotoxic potential of nanosilver using silver acetate as the representative ionic silver. The MN frequencies in HepG2 and Caco2 cells exposed to the ionic silver in the concentration range tested are not statistically significant from the control values except at the top concentrations for both the cell types. Therefore, our results indicate that the ionic silver may not contribute to the MN-forming ability of nanosilver in HepG2 and Caco2 cells. Also our results suggest that the HepG2 and Caco2 cell cultures and the FCMN assay are useful tools for rapid predictive screening of a genotoxic potential of food- and cosmetics-related chemicals including nanosilver.

Titanium dioxide nanoparticles induced activation of extracellular signal-regulated kinases signaling pathway in human colonic epithelial Caco-2 cells is mediated by epidermal growth factor receptor

Titanium dioxide nanoparticles induced activation of extracellular signal-regulated kinases signaling pathway in human colonic epithelial Caco-2 cells is mediated by epidermal growth factor receptor

Gut Lactobacillus protects against the progression of renal damage by modulating the gut environment in rats.

The role of gut microbiota in the progression of chronic kidney disease (CKD) has not been fully elucidated. Renal failure was induced in 6-week-old spontaneously hypertensive rats by 5/6 nephrectomy (Nx). We analyzed the gut microbiota population to identify the relevant species potentially involved in inducing renal damage. Human colon Caco-2 cells were used to delineate the mechanism involved in the molecular changes in the gut of Nx rats. Nx rats showed an increase in Bacteroides (Bact) and a decrease in Lactobacillus (Lact) species compared with sham-operated rats. Lact, but not Bact, populations were significantly associated with urinary protein excretion. Treatment of Nx rats with 1 × 10(10) CFU/kg/day Lact ameliorated increased urinary protein excretion and higher serum levels of the uremic toxins, indoxyl sulfate and p-cresyl sulfate, and serum urea nitrogen levels. Lact also attenuated systemic inflammation in Nx rats, as evaluated by serum lipopolysaccharide, interleukin-6 and C-reactive protein levels. Histologically, renal sclerosis in Nx rats was restored by Lact treatment. A reduction in the expression of tight junction proteins and the Toll-like receptor 2 (TLR2), a putative Lact receptor, in the colons of Nx rats were mitigated by Lact. Treatment of Caco-2 cells with indole downregulated tight junction protein expression, which was abolished by exposure to Lact. The effects of Lact were reversed by treatment with OxPAPC, a TLR inhibitor. Similarly, the increase in the permeability of the Caco-2 cell monolayer was reversed by the administration of Lact. Lact upregulated TLR2 expression in Caco-2 cells. Lact also attenuated the increase in serum indoxyl sulfate and urea levels and urinary protein excretion in Nx rats even in the pseudogerm-free environment. Lact supplementation mitigated the systemic inflammation and proteinuria associated with renal failure, suggesting that in the gut microbiota, Lact plays a protective role against the progression of CKD.