(View plain language abstract)
The identification over 10 years ago that the chemical acrylamide (AA) was present in food was followed by intensive 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, and to many toxicological and epidemiological studies of AA. Animal studies have shown the carcinogenic effect of AA leading to tumours in multiple sites, and in 1994 the IARC classified it 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). The most abundant DNA adduct of GA is N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua). Measurement of this adduct in DNA would be a marker of the genotoxically active dose of acrylamide to which the DNA has been exposed.
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 and ELISA.
A mass spectrometric method was developed utilising online column-switching achieving a LOD of 7 adducts/108 nucleotides and a LOQ of 9 adducts/108 nucleotides for the detection of N7-GA-Gua in human leukocyte DNA. The method was applied to analyse leukocyte DNA of 32 healthy volunteers. In a few samples peaks were detectable, indicating the presence of the N7-GA-Gua adduct, but mostly below the LOQ of 9 adducts/108 nucleotides and with a high variability. 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.
This study demonstrated the possibility that the N7-GA-Gua adduct is present in human DNA but further improvements in instrumental sensitivity are needed to assess the extent of genotoxicity associated with human dietary consumption of AA.
The hypothesis is that there is a correlation between the dietary intake of acrylamide (assessed by a food frequency questionnaire) and the extent of acrylamide-induced DNA damage in human volunteers.
In 2002 it was discovered that cooking some foods rich in carbohydrate at high temperatures produces small amounts of a chemical called acrylamide, exposure to which is known to produce cancer in animals. Subsequent studies showed that many highly consumed food products such as potato chips and crisps, and coffee contain low levels of acrylamide. The significance to human health of this exposure is only partly understood so far. When acrylamide is consumed it is converted in part to a reactive metabolite glycidamde. One of the leading theories for the way in which acrylamide causes cancer is that it does so because glycidamide damages DNA. Determination of the extent of such DNA damage would give valuable information on the cancer risk associated with acrylamide intake, and of the association of this with particular dietary constituents. The plan of this project was to develop methods for measuring acrylamide-induced DNA damage, to determine the extent of this damage in human populations and see how it is associated with diet. This would be done both by using high sensitivity analytical equipment and by using antibodies to detect the DNA damage product (‘adduct’). DNA from both blood and urine will be examined for evidence of damage induced by acrylamide. The project was intended to lead to a clearer understanding of the extent of cancer risk associated with acrylamide ingestion and also to indicate ways by which to minimise this risk.
A compound containing this adduct bound to a carrier protein was synthesised, and antibodies were made to this. Extensive testing of these antibodies revealed that they were unfortunately not of sufficient sensitivity and specificity to develop an immunoassay for detection of the adduct in human populations. As an alternative approach an assay using liquid chromatography linked to mass spectrometry was successfully developed and was subjected to detailed validation.
Ethical permission was obtained for a pilot study on humans, in which volunteers provided a blood sample and completed a food frequency questionnaire to assess their recent consumption of acrylamide-containing foods.
The sensitivity achieved for our novel approach for the analysis of the acrylamide-induced DNA adduct was that it was able to detect 1 molecule of the acrylamide-induced adduct in DNA in about 14 million unchanged DNA molecules. The validation of the method was acceptable for its use on blood DNA. Analysis was undertaken on human volunteers (leukocyte DNA). There was an indication that the N7-GA-Gua adduct is present in these human samples. However the peaks that were detectable in many of the chromatograms are below the level at which they can be quantified. This study demonstrated the possibility that the acrylamide-induced adduct is present in human DNA but further improvements in instrumental sensitivity is needed to assess the extent of this damage associated with human dietary consumption of AA.