Thursday 2 July 2009

T01009: Can cruciferous vegetables alter the genotoxicity of heterocyclic amines following human consumption of cooked meat?

Tuesday 18 January 2005

This research project investigates the effects of cruciferous vegetables upon the metabolism of potentially harmful genotoxins present in cooked meat.

Study Duration : June 1998 to October 2002

Contractor : Imperial College, London and BIBRA International Ltd

Background

Many forms of cancer may be partly attributable to diet. 2-Amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine (PhIP) is a rodent carcinogen that can be formed during high temperature cooking of meat and fish and may contribute to diet related cancers. PhIP is one of a group of compounds known as heterocyclic amines. Cruciferous vegetables (e.g. broccoli and brussels sprouts) have been reported to exhibit cancer protective properties possibly through the induction of specific enzymes involved in metabolism of compounds such as PhIP.

Research Approach

This project aimed to investigate effects of cruciferous vegetables on heterocyclic amine metabolism and excretion in humans. This involved carrying out a dietary intervention study that compared the metabolism of heterocyclic amines from a burger meal in individuals consuming cruciferous vegetables with those that did not eat cruciferous vegetables. This was carried out so that each volunteer participated in both regimes.

Several assays were developed to examine the effect of supplementing the diet with a known quantity of cruciferous vegetables. These included:

An examination of the effects of cruciferous vegetable supplementation on the enzymes involved in the metabolism of heterocyclic amines.
An examination of the effects of cruciferous vegetable supplementation on urinary excretion of PhIP.
The development of an assay to quantify the major PhIP metabolite in urine.
An analysis of the formation of PhIP DNA adducts and their excretion following excision and DNA repair.
The development of a robust antibody assay for the detection of DNA-PhIP adducts.

In addition the project included a limited study with resveratrol, a plant chemical found in grapes and various berries, which is reported to be chemoprotective. This included:

An evaluation of whether resveratrol can alter the frequency of PhIP induced DNA mutations in vivo .

Results and findings

Previous studies have shown that absorption of heterocyclic amines from a cooked beef meal is essentially complete and that urinary excretion of unchanged amine is indicative of the extent to which they are metabolised. This study confirmed increased excretion of metabolic products of heterocyclic amines in the urine of human volunteers who consumed cruciferous vegetables in addition to the cooked beef meal. Results suggested increased PhIP activation by the induction of enzymes (principally CYP1A2) involved in the activation of heterocyclic amines. Similarly, cruciferous vegetable consumption led to increased levels of N-hydroxy-PhIP glucuronide, reported to be the major detoxication metabolite of PhIP in human urine. This increase clearly shows that consumption of cruciferous vegetables enhanced overall clearance by glucuronidation (Phase II detoxication pathway).

Further work is needed to assess the net toxicological effect of these data.

An important aspect of this project involved significant improvements in methodology including:

Development of an assay for the analysis of the major urinary metabolite of PhIP. This involved the synthesis of unlabelled and deuterated N² -hydroxy-PhIP-N² -glucuronide and N-hydroxy-PhIP-N³ -glucuronide and analysis by LC-MS-MS.
Synthesis of guanine adducted-PhIP haptens and subsequent generation of polyclonal antibodies to N2-(deoxyguanosin-8-yl)-PhIP adducts. The antibodies were validated by detecting PhIP adducts in the urine of rats treated with 20mg/kg PhIP per day for 6 days. The antibody was not able to detect PhIP adducts in the urine of free-living humans presumably due to very low adduct levels. However, this may be possible with further refinement.

Limited studies were carried out (a) using resveratrol with heterocyclic amine treated mammalian cells; and (b) treating Muta™Mouse with PhIP and resveratrol.

This project illustrates:

the relevance of carefully controlled human volunteer studies;
the importance of high quality analytical methods; and
the need to extend work on biomarkers of toxicity for risk assessment of genotoxic compounds.

Dissemination information

The 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:

Walters DG, Young PJ, Agus C, Knize MG, Boobis AR, Gooderham NJ, Lake BG.
Cruciferous vegetable consumption alters the metabolism of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in humans.
Carcinogenesis. (2004) Sep;25( 9 ):1659-69; Epub 2004 Apr 8

Fang M, Edwards RJ, Bartlet-Jones M, Taylor GW, Murray S, Boobis AR.
Urinary N2-(2'-deoxyguanosin-8-yl)PhIP as a biomarker for PhIP exposure.
Carcinogenesis. (2004) Jun;25( 6 ):1053-62. Epub 2004 Jan 23.

Boyce A, Doehmer J, Gooderham NJ.
Phytoalexin resveratrol attenuates the mutagenicity of the heterocyclic amines 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline
Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences. 2004 Mar 25;802( 1 ):217-23.

Murray S, Lake BG, Gray S, Edwards AJ, Springall C, Bowey EA, Williamson G, Boobis AR, Gooderham NJ.
Effect of cruciferous vegetable consumption on heterocyclic aromatic amine metabolism in man.
Carcinogenesis. 2001 Sep;22( 9 ):1413-20.

Contact : Email : science@foodstandards.gsi.gov.uk

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T01009: Can cruciferous vegetables alter the genotoxicity of heterocyclic amines following human consumption of cooked meat?

Background

Research Approach

Results and findings

Dissemination information

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