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ToxSci Advance Access published online on January 5, 2005
Toxicological Sciences, doi:10.1093/toxsci/kfi075
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Toxicological Sciences © Society of Toxicology 2004; all rights reserved.
Received July 29, 2004
Accepted November 18, 2004

Genetic Toxicology

A mechanistic basis for the role of cycle arrest in the genetic toxicology of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)

S. Creton 1, H Zhu 1, and NJ Gooderham 1*

1 Molecular Toxicology, Division of Biomedical Sciences, Faculty of Medicine, Imperial College London, London SW7 2AZ, UK

* To whom correspondence should be addressed.
NJ Gooderham, E-mail: n.gooderham{at}imperial.ac.uk


  Abstract

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), formed during the cooking of meat, induces tumors of the prostate, colon and mammary gland when fed to rats. PhIP is readily absorbed and efficiently metabolised to a genotoxic derivative by CYP1 enzymes. Although metabolism and mutational potential of PhIP have previously been well characterised, the intervening cellular and genomic responses to the chemical are not fully understood. We have examined the cellular response to PhIP exposure in human mammary epithelial MCF10A cells, which retain characterisics of normal breast epithelial cells. As these cells fail to activate PhIP, they were cocultured with a human lymphoblastoid cell line MCL-5, which constitutively express CYP1A1, and have been transfected to express human CYPs1A2, 2A6, 3A4 and 2E6. The MCL-5 cells were irradiated (2,000 rads) prior to coculture, rendering them unable to replicate yet still retaining metabolic competency. MCF10A cells were treated (in the presence of MCL-5 cells) with PhIP (1-100µM) and harvested at various time-points. Compared to DMSO control, treatment (24 or 48 h) with PhIP resulted in a significant dose-dependent fall in cell number. Cells treated for 48h then cultured in the absence of PhIP (and MCL-5 cells) for a further 6 days showed a much greater dose-dependent reduction in cell number. Flow cytometric analysis indicated that PhIP treatment (48h) resulted in a dose-dependent accumulation of cells in the G1 population. Western blotting revealed elevated expression of p53 and the cyclin dependent kinase inhibitor p21WAF1/CIP1 after PhIP treatment. Levels of MDM2, a negative regulator of p53, and the hypophosphorylated form of RB were also elevated, consistent with the triggering of G1 cell cycle checkpoint. These cell cycle effects are critical as they enable cells to effect genome repair, accept mutation or eliminate excessively damaged cells.

Keywords: 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine; PhIP; cell cycle; p53; p21; MDM2; RB; cytotoxicity; breast epithelial cells.
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