Acrylamide (AA) has been classified as a probable human carcinogen by IARC. AA is generated in carbohydrate-rich foodstuff upon heating and its discovery in a variety of fried foods has raised public health concerns since oral consumption of AA could be an additional risk factor for cancer. Glycidamide (GA), formed via epoxidation, presumably by cytochrome P450 2E1, is considered the ultimate genotoxic agent playing a central role in AA carcinogenesis. Previous studies done by our group reported a strong correlation between GA-induction of Sister Chromatid Exchanges (SCE) and GA-DNA adducts, suggesting the possible involvement of Base Excision Repair (BER) and Homologous Recombination Repair (HRR) pathways in AA/GA induced DNA damage. Presently there is a lot of information concerning the mechanisms of genotoxicity of AA and GA in mammalian cells, however this is not the case for the role of AA and GA in gene expression.
In view of this, we studied gene expression in non-malignant mammary cell line (MCF10A) and in isolated human lymphocytes using a high-throughput technique, the RT2 Profiler PCR arrays: Human DNA Damage Signaling Pathway.
In our experimental protocol we exposed MCF10A cells to AA and GA (10 μM) for 1, 2 and 24 h periods. Moreover, to discern the possible differences of cultured cell lines and isolated lymphocytes, we exposed isolated lymphocytes from two healthy donors to GA (10 μM) for a period of 2 h. A value of p ≤0.05, and a fold change greater than 1.1 and lower than -1.1, were considered to be significant in gene expression dysregulation.
Concerning MCF10A cell line, the results obtained in 24h exposure showed that neither AA nor GA influenced the expression of the genes considered when compared to respective non-treated controls. However, for 1 h exposure it was observed that of the 84 studied genes, only GADD45A was up-regulated and GTSE1 was down-regulated for both compounds. On the other hand, MRE11A was only up-regulated in GA exposure. In what respect to 2 h exposure, of all the genes studied, 7 were significantly up-regulated (DMC1, GADD45G, MPG, RAD9A, XRCC3, XPA and XRCC6) and 2 down-regulated (GTF2H1 and GTSE1) for AA exposure and 16 were up-regulated (AIFM1, ATRX, DMC1, GADD45G, MNAT1, MPG, PMS2L3, RAD18, RAD51, RAD51L1, RAD9A, TREX1, XPA, XPC, XRCC3 and XRCC6) and 1 down-regulated (GTSE1) for GA exposure.
The results obtained regarding the study done with isolated lymphocytes exposed to GA (10 μM) for a 2 h period showed that GA up-regulated 1 gene for each donor, RAD51 for donor 1 and UNG for donor 2. However, when individuals were pooled, no statistical significance results were observed, what may be related to the importance of inter-individual variability in gene expression. In fact, several Single Nucleotide Polymorphisms (SNP's) can modulate the level of DNA damage induced by AA and GA, and for that reason, this variability should be taking into account.
The overall results obtained from the genes identified in this methodology are in general associated with DNA repair, specifically with BER and HRR, but also with apoptosis. In the future, Western blots should be performed in order to analyze the protein levels associated with representative genes identified and further studies should be conducted in human lymphocytes concerning other exposure periods, to account for variations in reaction between cell lines and isolated human lymphocytes. Moreover the number of donors should be increased to ascertain the possible effect of inter-individual variations in our study.
Citation Format: Marta Pingarilho, João P. Lima, Célia Martins, José Rueff, Jorge F. Gaspar. Gene expression induced by acrylamide and glycidamide in mammalian cells. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr 82.