Toxicological evaluation of a fish oil concentrate containing Very Long Chain Fatty Acids.

Very long chain fatty acids (VLCFA) have a chain length ≥24 carbons. Fish contain low levels of these fatty acids. A commercial oil called EPAX® Evolve 05 with an up-concentration of VLCFAs of approximately 10 times, has been developed as a dietary supplement by Epax Norway AS. A series of toxicological studies were performed using mice and rats to determine the safety and toxicity of repeat dosing with a gavage administered VLCFA formulation. The results suggest transient lipid accumulation in kidneys and liver. Lipid accumulation was seen with the test item and with the soya control but was not dose related. Liver and kidney lipid accumulation, whilst present in 14- day repeat dose study, was absent in a 90-day rat study. No treatment-effect was seen in urine analysis in any of the studies. No treatment-related effects were seen with a functional observation battery, ophthalmological examination, haematology, urine analysis, oestrus cycle, thyroid hormones, organ weight, or histopathology. In the 90-day study the liver enzymes ALP, AST and ALT were statistically significantly increased with test item but within control values. There were no associated histological findings in the liver suggesting there was no toxic effect and the normalisation of values for all liver enzymes in the recovery groups suggests an adaptive response rather than a prevailing toxic response. The no-observed-adverse-effect level (NOAEL) was determined as 1200 mg VLCFA/kg b.w./day.

Genotoxicity evaluation of a fish oil concentrate containing Very Long Chain Fatty Acids.

Very long chain fatty acids (VLCFAs) are lipids found in fish with a chain length longer than C22. They represent a minor lipid fraction composing of less than 1% of the total lipid. EPAX® EVOLVE 05 is a concentrate of VLCFAs providing roughly 10 times the amount found in fish. Here we report genotoxocity studies performed in cell culture and using a rat model. No genotoxicity was noted in a bacterial reverse mutation test (AMES test). An in vitro micronucleus assay was negative with a 4-hr test item incubation but a 24-hr incubation resulted in a positive signal. This prompted further study using an in vivo Sprague Dawley rat model. Test item exposure was demonstrated by plasma measurements from Sprague Dawley rats with peak absorption at 2-4 h post administration, as expected for fatty acids. The micronucleus assay showed no genotoxicity for fish oil containing VLCFAs. Together, the data shows that VLCFAs up to the test dose of 1200 mg/kg b.w. do not show genotoxicity.

3D Cell Culture in Alginate Hydrogels.

This review compiles information regarding the use of alginate, and in particular alginate hydrogels, in culturing cells in 3D. Knowledge of alginate chemical structure and functionality are shown to be important parameters in design of alginate-based matrices for cell culture. Gel elasticity as well as hydrogel stability can be impacted by the type of alginate used, its concentration, the choice of gelation technique (ionic or covalent), and divalent cation chosen as the gel inducing ion. The use of peptide-coupled alginate can control cell-matrix interactions. Gelation of alginate with concomitant immobilization of cells can take various forms. Droplets or beads have been utilized since the 1980s for immobilizing cells. Newer matrices such as macroporous scaffolds are now entering the 3D cell culture product market. Finally, delayed gelling, injectable, alginate systems show utility in the translation of in vitro cell culture to in vivo tissue engineering applications. Alginate has a history and a future in 3D cell culture. Historically, cells were encapsulated in alginate droplets cross-linked with calcium for the development of artificial organs. Now, several commercial products based on alginate are being used as 3D cell culture systems that also demonstrate the possibility of replacing or regenerating tissue.

In situ gelation for cell immobilization and culture in alginate foam scaffolds.

Essential cellular functions are often lost under culture in traditional two-dimensional (2D) systems. Therefore, biologically more realistic three-dimensional (3D) cell culture systems are needed that provide mechanical and biochemical cues which may otherwise be unavailable in 2D. For the present study, an alginate-based hydrogel system was used in which cells in an alginate solution were seeded onto dried alginate foams. A uniform distribution of NIH:3T3 and NHIK 3025 cells entrapped within the foam was achieved by in situ gelation induced by calcium ions integrated in the foam. The seeding efficiency of the cells was about 100% for cells added in a seeding solution containing 0.1-1.0% alginate compared with 18% when seeded without alginate. The NHIK 3025 cells were allowed to proliferate and form multi-cellular structures inside the transparent gel that were later vital stained and evaluated by confocal microscopy. Gels were de-gelled at different time points to isolate the multi-cellular structures and to determine the spheroid growth rate. It was also demonstrated that the mechanical properties of the gel could largely be varied through selection of type and concentration of the applied alginate and by immersing the already gelled disks in solutions providing additional gel-forming ions. Cells can efficiently be incorporated into the gel, and single cells and multi-cellular structures that may be formed inside can be retrieved without influencing cell viability or contaminating the sample with enzymes. The data show that the current system may overcome some limitations of current 3D scaffolds such as cell retrieval and in situ cell staining and imaging.

Involvement of protein kinase C in chitosan glutamate-mediated tight junction disruption.

Chitosan has been successfully used as an excipient for trans-epithelial drug delivery systems. It is known to transiently open intercellular tight junctions thus increasing the permeability of an epithelium. In order to investigate the possible role of protein kinases in trans-epithelial delivery, changes in trans-epithelial electrical resistance ('TEER') of epithelial (Caco-2) cell monolayers were assessed in response to chitosan glutamate treatment, in the presence and absence of specific protein kinase inhibitors. Changes in subcellular localisation of the tight junction protein ZO-1 observed by immunofluorescence and western blotting of cellular fractions were also assessed. Inhibition of protein kinase C (PKC), but not mitogen activated protein kinase (MAPK) was found to prevent the chitosan-mediated decrease in TEER, and changes in localisation of ZO-1. In order to determine which PKC isozymes were responsible for the chitosan-mediated tight junction disruption, the activation of the PKC isozymes alpha, beta and delta was investigated. A chitosan-mediated translocation of PKC alpha but not PKC beta or delta from the cytosol to the membrane fraction, indicative of PKC alpha activation was observed. Thus, treatment of Caco-2 cells with chitosan may result in the activation of PKC-dependent signal transduction pathways which affect tight junction integrity.

Effect of chitosan on epithelial cell tight junctions.

PURPOSE: Chitosan has been proposed as a novel excipient for transepithelial drug-delivery systems. Chitosan is thought to disrupt intercellular tight junctions, thus increasing the permeability of an epithelium. The effect of chitosan on tight junction complex was investigated at the molecular level. METHODS: Changes in barrier properties of Caco-2 cell monolayers, including transepithelial electrical resistance and permeability to horseradish peroxidase (HRP), were assessed in response to chitosan treatment. Changes in subcellular localization of the tight junction proteins zona occludens 1 (ZO-1) and occludin by immunofluorescence and Western blotting of cellular fractions were also assessed. RESULTS: Chitosan was found to cause a dose-dependent reduction in transepithelial electrical resistance of Caco-2 monolayers of up to 83%. A corresponding increase in horseradish peroxidase permeability of up to 18 times greater than the control was also observed across the monolayer. Immunofluorescent localization of ZO-1 revealed loss of membrane-associated ZO-1 from discrete areas. Analysis of cellular fractions revealed a dose-dependent loss of ZO-1 and occludin from the cytosolic and membrane fractions into the cytoskeletal fraction. These changes did not occur because of chitosan-mediated ATP depletion. CONCLUSIONS: Chitosan-mediated tight junction disruption is caused by a translocation of tight junction proteins from the membrane to the cytoskeleton.

The effect of chitin and chitosan on fibroblast-populated collagen lattice contraction.

The effects of chitin [(1-->4)-2-acetamido-2-deoxy-beta-D-glucan] and its partially deacetylated derivatives, chitosans, on the human dermal fibroblast-mediated contraction of collagen lattices were examined in vitro as a model for the contraction of cutaneous wounds in vivo. Chitosan CL313A, a short-chain-length 89% deacetylated chitosan chloride, inhibited fibroblast-populated collagen lattice (FPCL) contraction at higher initial concentrations (500 and 1,000 microg/ml) in FPCLs fabricated with responsive dermal fibroblasts, while in FPCLs containing non-responsive fibroblasts inhibition of contraction was reduced. The responsive and non-responsive phenotype of human dermal fibroblasts to treatment with chitosan CL313A has been reported previously by us. The inhibition of fibroblast-mediated collagen lattice contraction by chitosan appeared to be strongly correlated with whether the cells were responsive or non-responsive. The effect of chitin-50A on fibroblast-mediated collagen lattice contraction was also examined to investigate whether the level of deacetylation was important for its inhibitory effect on contraction. However, this had no effect on contraction at the concentrations tested, supporting previous work that only chitosan samples with higher levels of deacetylation showed any biological activity. This work indicates that highly deacetylated chitosan inhibits fibroblast-mediated contraction of collagen lattices and may therefore be useful as a therapeutic agent to reduce contraction and therefore scarring in wound healing in vivo.