Genotoxicity of advanced glycation end products in vitro is influenced by their preparation temperature, purification and cell exposure time

Advanced glycation end products (AGEs) are formed via non-enzymatic reactions between amino groups of proteins and the carbonyl groups of reducing sugars. Previous studies have shown that highly glycated albumin prepared using a glucose-bovine serum albumin (Glu-BSA) model system incubated at 60 degrees C for 6 weeks induces genotoxicity in WIL2-NS cells at 9 days of exposure measured by the cytokinesis-block micronucleus cytome (CBMNcyt) assay. However, this AGE model system is not physiologically relevant as normal body temperature is 37 degrees C and the degree of glycation may exceed the extent of albumin modification in vivo. We hypothesised that the incubation temperature and purification method used in these studies may cause changes to the chemical profile of the glycated albumin and may influence the extent of genotoxicity observed at 3, 6 and 9 days of exposure. We prepared AGEs generated using Glu-BSA model systems incubated at 60 degrees C or 37 degrees C purified using trichloroacetic acid (TCA) precipitation or ultrafiltration (UF) and compared their chemical profile (glycation, oxidation, and aggregation) and genotoxicity in WIL2-NS cells using the CBMNcyt assay after 3, 6 and 9 days of exposure. The number of micronuclei (MNi) was significantly higher for cells treated with Glu-BSA incubated at 60 degrees C and purified via TCA (12 +/- 1 MNi/1000 binucleated cells) compared to Glu-BSA incubated at 37 degrees C and purified using UF (6 +/- 1 MNi/1000 binucleated cells) after 9 days (P< 0.0001). The increase in genotoxicity observed could be explained by a higher level of protein glycation, oxidation, and aggregation of the Glu-BSA model system incubated at 60 degrees C relative to 37 degrees C. This study highlighted that the incubation temperature, purification method and cell exposure time are important variables to consider when generating AGEs in vitro and will enable future studies to better reflect in vivo situations of albumin glycation.