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The Interactions of Genotoxins: A Mechanistic Approach Based on DNA Repair


<P>The project includes studies with primary human liver cell cultures (hepatocytes) and a cultured human liver cell line (HepG2). Using a range of treatments this project determines factors that modulate the level of PhIP-DNA adduct formation. PhIP- DNA adducts are formed by incubating the cells in 14C-labelled PhIP to create DNA adducts. These adducts are detected by Accelerator Mass Spectrometry (AMS) a very sensitive technique that can be used for accurate measurement of PhIP-DNA adduct formation in isolated cells.</P>

<P>Hydrogen peroxide is used to produce oxidative DNA damage. Protective effects are investigated using the plant chemicals quercetin (found in a wide variety of plants) and sulphorophane (which is particularly abundant in cruciferous vegetables) and the antioxidant vitamin E. Real-time polymerase chain reaction (PCR) is used to quantify activation of PhIP metabolising enzymes and the metabolic pathways involved in detoxification (the latter in conjunction with enzyme based assays). PhIP-DNA adducts can be repaired by known enzymes. Thus the project also examines the effects of protective dietary compounds on the degree of DNA repair enzyme expression. </P>

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<P>Diet is implicated in the formation of many forms of cancer. During the cooking or frying of meat and fish a group of carcinogenic heterocyclic amine compounds are produced. One of the most commonly occurring heterocyclic amines is 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).</P>

<P>PhIP is activated by the liver where it can bind to DNA forming potentially harmful PhIP-DNA adducts. Unless removed in a repair process genetic damage caused by such adducts can contribute to the development of cancer. In addition cells are continuously exposed to free radicals generated as a part of cellular metabolism. Though this oxidative DNA damage is routinely repaired as a function of cellular maintenance, it still represents an important factor in generating DNA lesions (i.e. as a source of mutations). There is some evidence to suggest that certain mutagenic compounds may stimulate enzymes involved in DNA repair. This raises the possibility that heterocyclic amine induced DNA damage could lead to enhanced overall DNA repair within the cell.</P>

<P>One of the initial aims of this project is to determine whether the heterocyclic amine, PhIP, influences the level of oxidative DNA damage caused by cellular free radicals and what the subsequent consequences for the cell will be.</P>

<P>Evidence suggests that a diet that is rich in certain vegetables and fruit, can contain substances that may reduce the risk of some cancers. Thus an additional aim of this project is to explore this hypothesis and to explore the mechanisms involved in possibly reducing the toxicity of PhIP compounds found in the diet. </P>

Institute of Food Research, UK
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