The effect of ozonised amino acids and their isolated active components on inflammation-associated signal transduction and in a model of periodontitis, together with analysis of mutagenesis
Final Report Abstract
The identification of the active oxidation products resulting from ozonation of tryptophan or cysteine proved more difficult than initially envisaged. Whilst several products were found after this process, none of the substances proved as effective in inhibition of the transcription factor NF-κB as the ozonized medium used in the initial experiments. It had been hoped that an effective substance could be isolated and eventually used in treatment of periodontitis. However, the difficulties in isolation of one single effective component of the mixture created following ozonation made this goal impossible to reach. The Identification of the numerous oxidation products resulting from ozone treatment of tryptophan in particular is still ongoing. Since a single effective component proved elusive, the experiments were continued using ozonized cell culture medium or the ozonized amino acids, as in the preliminary experiments. A mouse model of periodontitis was used to show the effectiveness of ozonized substances on this disease. Although a disease state was induced in the mice, the data were inconclusive regarding the ability of the ozonized substances to inhibit inflammation. The data showed a tendency towards a chronic inflammatory state in the mice following bacterial infection, which was, to some extent, reduced by pre-treatment with ozonized medium or tryptophan. However, the bone resorption seen in this disease was not particularly affected by the treatments. The model design may not have been ideal, but time restraints on the completion of the animal experiments prevented further investigation. To summarise, it was found that ozonized medium and ozonized tryptophan were able to inhibit the stimulus dependent secretion of IL-8 by fibroblast cells and that this inhibition was caused by an interruption in the signaling cascade at least at the level of IKK complex inhibition. Kinase assays showed that the ability of the IKK complex to phosphorylate IκBα was inhibited following ozonized medium/tryptophan treatment. This led to an inhibition of IκBα degradation, subsequent p65 nuclear translocation and activation, culminating in the reduction in κB dependent protein secretion seen in the ELISA experiments. Although a new treatment for periodontitis was not identified in this project, as had been hoped, the results deepened our knowledge of the mechanisms behind transcription factor NFαB inhibition caused by incubation with ozonized tryptophan and cysteine as well as cell culture medium. These results may lead to further research in this area, in addition to the ongoing identification of the active components of the ozonation mixture.
