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The study was focused on assessment of potential health risks of paper-based food contact materials (FCMs) in a step-wise approach using three toxicological bioassays in vitro and chemical analyses of migrating contaminants. 3T3 NRU cytotoxicity test showed high sensitivity to detect basal toxicity of FCMs extracts and served as a first-line test for selection of samples for further testing. The reconstructed human intestine model EpiIntestinal showed more realistic tissue response than cell culture monolayer and higher resistance despite prolonged exposure to the selected 6 samples, i.e. negligible decrease of viability and intestinal penetration, nevertheless an increase of IL-8 after exposure to black printed sample extract. Yeast based assays identified weak agonistic/antagonostic activity to human androgen receptor of the black printed sample. In accordance with the biological effects, the targeted LC and GC analytical methods confirmed the presence of high amounts of phthalates, photoinitiators and PAHs that could justify the hazard of the black printed sample. Heavily printed uncoated FCMs are recognized not to be suitable for direct contact with food. The selected bioassays and chemical analyses might be useful tools to detect targeted biological effects of xenobiotics suspected to contribute to human exposure from food.

Researchers have found that decreased levels of circulating citrulline could be an indicator of intestinal failure. Typically, this amino acid, which is produced by the intestinal mucosa cells, circulates in the blood at a physiological level of ∼40 μM. The current methodology for measuring this level involves the use of bulky equipment, such as mass spectroscopy and analysis at a central laboratory, which can delay diagnosis. Therefore, the current detection method is unsuited for routine monitoring at a doctor’s office. Our research group proposes the development of a point-of-care (POC) device to overcome this issue. The proposed device utilizes surface-enhanced Raman spectroscopy (SERS) coupled with a specifically designed aptamer, capable of binding to citrulline, conjugated to colloidal gold nanoparticles. The assay is then embedded within a vertical flow paper-fluidic platform as a deliverable at the POC, and a handheld Raman spectrometer (638-nm excitation) was used to interrogate the sample. Results showed good dynamic range and specificity with an average 73% decrease in SERS signal intensity with increasing concentrations of citrulline (0 to 50 μM) in phosphate-buffered saline compared to its controls: glycine, glutamine, histidine, and valine, which showed less than 10% average decrease in the presence of 200 μM of each analyte. Further, the limit of detection (LOD) within a chip was determined to be 0.56 μM, whereas the LOD across chips was below 10 μM.

The duodenal mucosa is exposed to endogenous and exogenous chemicals, including acid, CO2, bile acids and nutrients. Mucosal chemical sensors are necessary to exert physiological responses such as secretion, digestion, absorption, and motility. We propose a mucosal chemosensing system by which luminal chemicals are sensed via mucosal acid sensors and G-protein-coupled receptors. Luminal acid/CO2 sensing consists of ecto- and cytosolic carbonic anhydrases, epithelial ion transporters, and acid sensors expressed on the afferent nerves in the duodenum. Furthermore, a luminal L-glutamate signal is mediated via mucosal L-glutamate receptors, including metabotropic glutamate receptors and taste receptor 1 family heterodimers, with activation of afferent nerves and cyclooxygenase, whereas luminal Ca2+ is differently sensed via the calcium-sensing receptor in the duodenum. Recent studies also show the involvement of enteroendocrine G-protein-coupled receptors in bile acid and fatty acid sensing in the duodenum. These luminal chemosensors help activate mucosal defense mechanisms in or- der to maintain the mucosal integrity and physiological responses. Stimulation of luminal chemosensing in the duodenal mucosa may prevent mucosal injury, affect nutrient metabolism, and modulate sensory nerve activity.

Most afferent signals from the viscera do not give rise to conscious experience and yet they participate in the complex neural control of visceral functions. Surprisingly little information is available on the origin, morphology, and receptor functional characteristics of the nerve endings of most primary afferent neurones to the digestive tract. This review deals with the morphological nature of the afferent neurones that supply the gastrointestinal tract specifically.

Possible mechanisms by which the intestinal contents may influence afferent nerve fibres in the lamina propria of the intestinal mucosa are discussed in this brief review. After addressing intestinal epithelial permeability, endocrine and brush cells are discussed, as well as enterocytes, as sensors for luminal microbes.

The management of abdominal pain remains a major challenge for clinicians despite an explosion of knowledge regarding the physiology and pathophysiology of sensory neural circuits. Initial progress was made in the field of somatic pain and this provided broad hypotheses that could be tested in the field of visceral pain. The major advances in our understanding of basic mechanisms underlying visceral pain originated in the urinary tract and have been applied to the gut. As yet, this increased understanding of enteric sensory physiology has yet to generate new effective treatments for abdominal pain. This review addresses novel insights into peripheral mechanisms underlying visceral hyperalgesia and their applicability to the development of novel therapeutic approaches to the treatment of chronic abdominal pain.

In normal conditions, human gut mucosa is infiltrated with a large number of mononuclear cells. This is a reflection of the fact that human intestine is continuously subjected to a massive stimulation by luminal antigens. This state of "physiological" inflammation is a tightly controlled phenomenon, as several mucosal cells interact to generate and maintain an appropriate local immune response. Changes in cell type number and/or function, including the release of soluble mediators, have been associated with the development of chronic inflammatory diseases, such as Crohn's disease (CD) and ulcerative colitis (UC), the two major forms of inflammatory bowel disease. Evidence also indicates that the type of inflammatory response occurring in the intestine of patients with CD differs from that in UC, and this probably reflects distinct pathways of immune activation. In CD mucosa, a Th1 response with high IL-12 and IFNgamma production prevails, while in UC a humoral immunity appears to be predominant. Despite this, CD and UC share downstream inflammatory events, characterised by high levels of inflammatory cytokines, free radicals, matrix-degrading enzymes and growth factors.

Bacterial pathogens have evolved two major strategies to colonise the intestinal epithelium. Adherent microorganisms bind to the apical pole of the intestinal epithelium, whereas invasive microorganisms disrupt and invade the epithelium. Recognition of the genetic bases of bacterial pathogenicity and analysis of the molecular cross talks established between pathogens and their mammalian target cells have illuminated this diversity of interactions. We have compared the strategies of enteroinvasive pathogens, with emphasis on bacterial species such as Shigella, Yersinia, and Salmonella, that represent paradigms of interaction. Cross talks leading to alteration of the epithelial cell actin cytoskeleton appear as a recurrent theme during entry and dissemination into epithelial cells. Other cross talks alter the trafficking of cellular vesicles and induce changes in the intracellular compartment in which they reside, thus creating niches favourable to bacterial survival and growth. Finally, a variety of strategies also exist to deal with other components of the epithelial barrier, such as macrophages. Pro-phagocytic, anti-phagocytic, and pro-apoptotic processes appear to be of particular importance.

Microscopic analysis of endoscopically obtained tissue samples is important for the diagnosis of several gastrointestinal disorders such as gastritis and chronic inflammatory bowel disease (IBD). Histologically disease activity is based on the presence of neutrophilic polymorphonuclear leucocytes in conjunction with epithelial damage. Effective eradication treatment for Helicobacter pylori related gastritis reduces active inflammation rapidly whereas chronic inflammation decreases only slowly. Similar findings have been obtained for IBD. A literature review of clinical drug trials in IBD and the effect of various drugs on the microscopic features of Crohn's disease and immunohistochemistry for different markers was performed. Diagnostic microscopic features and the features characteristic for disease activity vary with time and treatment. The more recently developed drugs used for Crohn's disease can induce mucosal healing.

A number of phenanthrolines and benzoquinolines were examined for their ability to activate epithelial chloride secretion by measuring short circuit current (SCC) using the mouse colon epithelium. 1,10 phenanthroline stimulated electrogenic chloride secretion with an EC50 of 612±10 μ and a Hill slope of 4.9±0.3. A similar pharmacology was demonstrated by both 1,7 and 4,7 phenanthrolines, 7,8 benzoquinoline and phenanthridine. Evidence that the increase in SCC caused by 1,10 phenanthroline was due to chloride secretion is based upon (a) inhibition of the current by furosemide, (b) failure of cystic fibrosis (CF) colons to respond and (c) an associated net flux of 36Cl−. 1,10 Phenanthroline affected neither the generation of cyclic AMP or the concentration of intracellular Ca2+ in colonic epithelial cells. 1,10 phenanthroline affected the chloride conductance of the apical membrane, as shown by an increase in chloride current in ‘apical membrane only' preparations in the presence of an apical to basolateral chloride gradient. The increase in chloride current was inhibited by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and was not present in CF colons. Additionally, 1,10 phenanthroline activated basolateral K+ channels, both Ca2+- and cyclic AMP-sensitive channels, as shown by inhibitor studies with charybdotoxin (ChTX) and XE991, and after the apical membrane was permeabilized with nystatin. The phenanthrolines and benzoquinolines described here, with dual actions affecting CFTR and basolateral K+ channels, may constitute useful lead compounds for adjunct therapy in CF. Keywords: CFTR, cystic fibrosis, chloride channel openers, phenanthrolines, 7,8 benzoquinoline, phenanthridine, potassium channels, cyclic AMP, intracellular Ca2+ Introduction Increased interest in agents affecting epithelial chloride channels arose after it was shown that the cystic fibrosis transmembrane conductance regulator (CFTR) is also a chloride channel in many epithelial membranes (Anderson et al., 1991). Not only have blockers of CFTR been described but considerable interest has also focused on CFTR chloride channel openers (Schultz et al., 1999). The most common mutation in cystic fibrosis (CF) is ΔF508, in which phenylalanine is absent from position 508. This mutant protein (ΔF508CFTR) can function as a chloride channel (Drumm et al., 1991), however only a minute fraction of this protein is transported to the apical domain of epithelial cells (Ward & Kopito, 1994). Instead the quality control mechanisms of the cell cause the mutant protein to be degraded in the proteasome. Therefore attempts are being made to increase the delivery of ΔF508 CFTR to the membrane (Brodsky, 2001; Fischer et al., 2001; Fuller & Cuthbert, 2000). Thus agents which open CFTR channels are of interest, particularly so if they also activate ΔF508 CFTR. The present classes of CFTR openers are as follows; phosphatase inhibitors (Becq et al., 1998), xanthines, particularly CPX (Guay-Broder et al., 1995), benzimidazolones such as EBIO (Devor et al., 1996; Cuthbert et al., 1999), flavones, for example genistein (Illek et al., 1995), benzoxazolones like chlorzoxazone (Singh et al., 2000) and finally the MPB compounds described by Becq et al. (1999). The purpose of this study was to further investigate the actions of 1,10 phenanthroline on chloride secretion in the mouse colon following the observations made by Duszyk et al. (1999) that 1,10 phenanthroline, a powerful inhibitor of matrix metalloproteases (MMPs) activated a chloride conductance, measured as whole cell current, in human airway epithelial cells, Calu-3. No effect was found in airway cells lacking CFTR, namely A549 cells. Furthermore, an antibody to MMP2 caused a similar effect in Calu-3 cells as did 1,10 phenanthroline, suggesting metalloproteinase inhibition was responsible. A further aim was to explore structure activity relations in the phenanthroline isomers and to ask if both heterocyclic nitrogens in the tricyclic aromatic molecules were necessary for the effect. From various experimental approaches we conclude that the phenanthrolines and related structures constitute a new class of agents that stimulate chloride secretion.

DNA adducts were analyzed by (32)P-postlabeling in liver and intestine of perch (Perca fluviatilis). Fish were collected in the receiving water of a bleached kraft pulp mill at Norrsundet, Sweden, on the coast of the Bothnian Sea, and in three different reference areas. Sampling was carried out the last week in September 1993, 1995, and 1997. Since 1984/1985 the pulping process at Norrsundet has been successively modified and an external waste-water treatment has been installed, resulting in lowered and altered effluent discharges. Nevertheless, perch captured in the water area closest to the mill (2 km) had significantly elevated levels of aromatic/hydrophobic DNA adducts in both liver and intestine when compared to perch captured 8 km away and in the reference areas. Autoradiograms indicated a diagonal zone consisting of multiple overlapping adducts, a pattern typical of exposure to a complex mixture of aromatic/hydrophobic genotoxic substances. No significant difference in adduct levels was detected between the years. Results from this study reveal that fish in the area closest to the mill are exposed to substances with a genotoxic potential.

Male rats were fed 100 nM dichlorodiphenyltrichloroethane-14C in oil by gastric tube. Recovery of dichlorodiphenyltrichloroethane-14C in thoracic duct lymph was 60% in 12 hr. Lymph dichlorodiphenyltrichloroethane-14C (97%) occurred in lipoproteins of d

131I-insulin was given intravenously to normal rats, and the passage of radioactivity into the duodenal loop containing a proteasis inhibitor was studied by two techniques. In the first, the 131I-insulin was separated from the intestinal content by electrophoresis; whereas in the second, the radioactivity that remained on an ion-exchange resin, directly introduced into the small intestine, was measured. It was found that within 15 minutes from the injection a small amount of 131I-insulin passes into the gut lumen. — The absorption of 131I-insulin from small intestine into the blood was studied after introduction of 131I-insulin along with a proteasis inhibitor into the intestinal loop. The autoradiograph of the electrophoresis of serum showed plasma-protein-bound radioactivity but no radioactivity due to unbound (free) insulin-131I. — These findings show that the passage of insulin through the intestinal wall of rats can be demonstrated by neutralizing the proteolytic activity in the intestinal tract. The possible role of this passage in protein-losing enteropathy is pointed out.

Few studies have been published on peptide hydrolase activities of human small intestine mucosa. We developed methods to screen tissue extracts for such enzymes and to quantitate hydrolase activities for dipeptides containing the aromatic amino acid L-phenylalanine. The screening procedure indicated glycyl-L-proline hydrolase activity was reduced in biopsy specimens from patients with flattened intestinal mucosa. To explore this further, we established optimal assay conditions for hydrolase activities (a) glycyl-L-proline, (b) L-phenylalanyl-L-proline, (c) L-alanyl-L-phenylalanine, and (d) L-phenylalanylglycine. Biopsy specimens from patients with various intestinal disorders, but without flattened mucosa, and from three patients with flattened mucosa, showed a disproportionate reduction in activities (a) and (b), with the reduction being significantly more marked in the latter patients. We suggest that intestinal imidopeptide hydrolase activities, such as (a) and (b), are sensitive to changes in intestinal disease generally, particularly to the altered physiology associated with flattening of the mucosa, and are secondary to, rather than a cause of, the intestinal pathology. Our finding that intestinal alkaline phosphatase activity tended to parallel imidopeptide hydrolase activity, and that activity (a) was partially localized to the particulate fraction of mucosal homogenate, suggested that imidopeptide hydrolase activities may be located in the microvilli of the intestinal epithelium and that, like alkaline phosphatase activity, they may be reduced in flattened mucosae, in part at least because of the pathologic changes in the microvilli. In our studies of control subjects we did not detect peptide hydrolase activity deficiency analogous to asymptomatic disaccharidase deficiency.

1. 1. Serine ethanolamine phosphate is synthesised in the microsomes of the chicken intestinal mucosa according to the reaction: CDP-ethanolamine + l -serine → CMP + l -serine ethanolamine phosphate 2. 2. In the intact microsomes CDP-ethanolamine participates in two other concomitant reactions: phosphatidylethanolamine biosynthesis and CDP-ethanolamine cleavage. Differential denaturation studies have now shown these three activities to be due to distinct enzymes. 3. 3. Serine ethanolamine phosphate synthetase is absolutely specific for serine, and both stereoisomers are active. 4. 4. In the microsomal system CDP-choline and CDP-serine could not replace CDP-ethanolamine as substrates for serine ethanolamine phosphate synthetase. On the other hand, its phosphonic analogue (CMP-aminoethylphosphonate) and CDP-2-amino-2-methylpropanol were used as substrates and formed the corresponding phosphodiesters with serine. The phosphonic analogue was also an effective substrate for phospholipid biosynthesis. 5. 5. Bivalent cations are essential for activity. Mg 2+ are most effective, Mn 2+ and Co 2+ are less so. Ca 2+ are inhibitory. 6. 6. Kinetic studies of product inhibition show that the reaction mechanism is ordered, CDP-ethanolamine being the first reactant. The apparent K m values for the substrates CDP-ethanolamine and l -serine have been calculated to be 0.085 and 1 mM, respectively. 7. 7. A mechanistic model for the reaction is suggested on the basis of these findings.