| dc.description.abstract | Exposure to environmental toxicants is a contributing factor to health disorders such as cancer, asthma and allergy. A toxicant which has been studied more in depth the last years is the well known neurotoxicant acrylamide (AA) and its metabolite, glycidamide (GA). The reason for this is that AA have been traced in many different types of processed food and beverages. Although the genotoxic effect of AA has been widely studied, the molecular function remains mostly unclear. We therefore wanted to explore the molecular impact of the genotoxic effect of glycidamide, and to look more carefully into the mechanisms involved. In large biomonitoring studies, lymphocytes are an invaluable and often the only source of medium for such genotoxic analysis, and, therefore human lymphocytes and a lymphoblastoid cell line were used for the in vitro exposure studies.
In correspondence with results from ongoing studies in our laboratory, we found that very low exposure of the GA, but not AA alone, induced high levels of DNA damage recognised by the repair enzyme, Formamidopyrimidine glycosylase (Fpg). Further, when relatively high concentrations of GA (1 mM and 0.5 mM) was used, together with long exposure duration, a marked increase in DNA damage without the Fpg-enzyme was apparent. Thus, since GA is found to be highly genotoxic we wanted to study GAs effect on lymphoid cell cycle and survival when relatively low GA(0.1 mM) concentrations were used. No cell cycle arrest of the lymphoblastoid cell line, GM00130, was seen with low GA concentrations. Instead, by increasing the GA dose, starting from 0.5 mM, an accumulation of cells in early S-phase became apparent after 24 hours exposure time. Preliminary results also indicated a reduction in the incorporation of bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) following GA-exposure, indicating a halt in DNA-replication and an S-phase arrest. At the same GA-concentration used, the tumor suppressor protein p53 was highly phosphorylated at ser15, followed by a total p53 increase. An increase in the CDK inhibitor p21CIP1 was also notable at 0.5 mM GA concentration, while no significant changes were observed in the case of p27Kip1. Further, the expression of cyclin A that drives S-phase did not decrease as expected. When increasing the concentration of GA to 1 mM and also the time of exposure, an increased level of cells arresting in S-phase was observed together with an elevation of cell undergoing apoptosis. Additionally, since the transcription of the NER-enzymes, XPA and XPC, was previously found to be regulated by GA in other cell systems, we analysed the expression of these proteins after GA-exposure. However, in a preliminary experiment, no notable GA-induced increase in the protein level of these enzymes was noted.
Thus, unexpectedly, we found that the high number of Fpg-sensitive sites induced by low levels of GA did not lead to any cell cycle arrest or cell death or any changes in protein expression of important cell cycle parameters tested in the lymphoblastoma cell line. This may indicate that lymphoid cells are able to overcome or ignore these lesions at low levels of GA. However, continuous exposure may eventually lead to an accumulation of GA-induced lesions leading to mutations and cancerous development over time. When increasing the concentration and the exposure duration of GA, DNA-replication was markedly slowed down and cells were arrested in S-phase followed by a significant progression of apoptosis. This may be due to the observed GA-induced activation of p53 and the induction p21CIP1. All in all it shows that GA is genotoxic and affects the lymphoid cell cycle in a dose and time dependent matter. | eng |