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Listen to this siteFriday 21 January 2005
This research project examines the effects of heterocyclic amines and oxidative agents on the level of DNA adduct formation and repair in liver cells
Study Duration : April 1999 to April 2002
Contractor : Institute of Food Research, Norwich
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).
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.
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.
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.
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 14 C-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.
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.
Treatment of liver cells (primary hepatocytes and HepG2) with 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) generated PhIP-DNA adducts in a dose dependent manner.
Co-treatments of HepG2 cells with 14C-PhIP, hydrogen peroxide and the phytochemicals, quercetin and sulforaphane and the antioxidant vitamin E were carried out. All four compounds resulted in fewer PhIP-DNA adducts than treatment with PhIP alone.
After a 6 hour treatment with hydrogen peroxide together with quercetin (20 µM) and sulforaphane (10 µM) the level of PhIP-DNA adducts was higher than with hydrogen peroxide alone but at 24 hours the results were reversed. This suggests that quercetin may have an antioxidant effect at six hours counteracting the effects of peroxide but that after 24 hours, when the peroxide has been metabolised, quercetin and sulforaphane continue to inhibit PhIP-DNA adduct levels.
Vitamin E was also able to modulate the effect of hydrogen peroxide after 6 hours co-treatment again suggesting an antioxidant effect.
Co-administration of sulforaphane (a dietary isothiocyante) and quercetin (a dietary flavonoid) resulted in a dose dependent reduction in adduct levels. It is concluded that the mechanism for this protective effect is that quercetin inhibits PhIP activation in the liver, thus reducing the levels of PhIP available for adduct formation. Sulforaphane upregulated production of PhIP detoxification enzymes, suggesting a role for these dietary compounds in detoxification of PhIP.
However, neither compound was involved in the repair of DNA adducts via regulation of the DNA repair enzymes.
These in vitro data suggest that though a diet high in flavonoids and isothiocyanates may be able reduce genotoxic damage in the liver it will not influence the rate of DNA repair.
Final report is available from the FSA Library and Information centre.
To obtain a copy, please contact the Enquiry Desk,
Dr Elsie Widdowson Library and Information Services, Food Standards Agency, tel: 020 7276 8181/8182 or email:
library&info@foodstandards.gsi.gov.uk
Results from this project have been published:
Bacon JR, Williamson G, Garner RC, Lappin G, Langouet S, Bao Y.
Sulforaphane and quercetin modulate PhIP-DNA adduct formation in human HepG2 cells and hepatocytes.
Carcinogenesis. 2003 Dec; 24(12): 1903-11. Epub 2003 Aug 29.
Bao Y, Bacon JR, Williamson G.
Effect of phytochemicals on PhIP-DNA adduct formation in human HepG2 and hepatocytes.
In: Biologically-active phytochemicals in food, 2001.
(Eds: Pfannhauser W, Fenwick GR and Khokar S)
Proceedings of the EUROFOODCHEM XI biologically active phytochemicals in food, Norwich, UK, 26-28 Sept 2001
Royal Society of Chemistry 2001.
pp 589-591
Contact
: For any enquiries concerning this research project, please contact the relevant Programme contact or email
science@foodstandards.gsi.gov.uk
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