Assessment of the genotoxicity of dietary acrylamide
thesisposted on 15.05.2014, 10:45 by Sandra Schumacher
The identification of the chemical acrylamide (AA) in food was followed by intense research which led to the discovery that AA forms in starch-rich foodstuff when roasted, fried and baked, due to the reaction of the amino acid asparagine and reducing sugars. Animal studies have shown the carcinogenic effect of AA leading to tumours in multiple sites and in 1994 the IARC classified AA as a probable human carcinogen. After ingestion AA is metabolised to the reactive epoxide glycidamide (GA) that is able to react with bio-macromolecules such as DNA and haemoglobin (Hb). Three major DNA adducts of GA have been reported of which the N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) is the most abundant. The aim of this project is to develop a sensitive method for the detection of the N7-GA-Gua adduct in human leukocyte DNA and urine with two different analytical techniques, namely mass spectrometry, applying LC-MS/MS, and the immunoassay ELISA. A mass spectrometric method was developed utilising online column-switching achieving a LOD of 7 adducts/10[superscript 8] nucleotides and a LOQ of 9 adducts/10[superscript 8] nucleotides for the detection of N7-GA-Gua in human leukocyte DNA. The developed method was applied to analyse the leukocyte DNA of 32 healthy volunteers. In a few samples peaks were detectable, indicating the presence of the N7-GA-Gua adduct but below the LOD and with a high variability. For 10 samples the AA- and GA-Hb adducts were analysed; AA-Hb adducts correlated with AA intake 24 hours prior to donation but there was a non-significant correlation between Hb adducts and N7-GA-Gua adduct levels. It was not feasible to develop a method for the detection of N7-GA-Gua in urine due to the high salt concentration of this matrix and difficult clean-up procedures prior to LC-MS/MS. Two polyclonal antibodies were raised against N7-GA-Gua to develop a competitive ELISA but due to very strong binding with low sensitivity towards the N7-GA-Gua adduct the antibodies were not suitable for use in an ELISA assay. Aiming for a better sensitivity might allow detecting the N7-GA-Gua in human samples.