Histone H1 studies: 1) In vivo evidence of nuclear protein damage; 2) In vitro studies of natural product glycation inhibitors

Glycation is a reversible, non-enzymatic reaction between reducing sugars and amino groups of proteins that undergo rearrangements to stable ketoamines, which lead to the formation of advanced glycation end products (AGEs) such as fluorescent (argpyrimidine) and non-fluorescent (N -carboxymethyllysine, CML) protein adducts and protein crosslinks. Protein glycation, induced by hyperglycemia, has been implicated in the appearance of diabetic complications that involved kidney, eye and neuronal tissue damage. Intracellular glycation has not yet been investigated as a contributor factor to the process of ageing or the development of diabetes complications and other chronic diseases. In the other hand, it is well documented that oxidative stress causes DNA damage and that high local concentrations of ADP-ribose, a very reactive reducing sugar, are produced in the chromatin microenvironment during the induced DNA repair process, which could potentially glycate nuclear proteins such as histones, which have a great proportion of lysine and arginine residues in their structure. In the first part of the project, our studies focused in searching for in vivo evidence for the formation of AGEs in likely targets of nuclear protein damage such as histones. Using a variety of AGE biomarkers histone H1, isolated from calf thymus, was found to be modified in vivo. In the second part of the project our aim was to search for effective glycation inhibitors from natural sources. When aminoguanidine, a proven AGEs inhibitor, was tested as an anti-diabetic drug in human clinical trials presented important side effects and this suggested a need for new and improved AGEs inhibitors. Histone H1 and ADP-ribose were used as a model for protein glycation since it allows to distinguish true AGE inhibitors from general antioxidants. Rutin derivatives were tested as AGEs inhibitors since rutin, a common dietary flavonoid that is consumed in fruits, vegetables and plant derived beverages, is not absorbed intact due to gut micro flora degradation and the resulting phenolic compounds have no significant antioxidant activity. Our data showed that 3,4-dihydroxyphenylacetic acid and 3,4-dihydoxy toluene were powerful inhibitors of fluorescence and the formation of CML protein adducts in contrast to aminoguanidine, which was only effective inhibitor of fluorescence. Furthermore when these two rutin metabolites and aminoguanidine were tested as inhibitors of AGEs using glyoxal or methylglyoxal and histone H1 as a glycation model we found that both type of compounds were effective. These results present alternative effective natural product AGE inhibitors to aminoguanidine that might potentially be recommended as dietary supplements in the prevention of diabetes complications.