Molecular and genetic toxicology of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), formed when meat containing food is cooked, induces cancer of the colon, prostate and mammary gland of rats, tumours that are strongly associated with a Western diet. After consumption of a meat meal, PhIP is rapidly absorbed, metabolised and bioactivated to DNA damaging species. Thus, PhIP should be considered as a candidate etiological agent for human cancer. Studies in vitro in model mammalian cell culture systems, and in vivo in transgenic animals, have shown that mutation induced by PhIP is dose dependent and describes a mutational "fingerprint" that is characteristic of the chemical. This genetic toxicity is dependent upon CYP1 family metabolic activation and is detectable in these model systems at micro M concentrations. At early time points, PhIP treated cells show subtle signs of toxicity that lead to altered growth and cycling. Using co-culture systems where one cell line bioactivates PhIP with a second cell line as target, we showed in human lymphoblastoid target cells that PhIP induced a dose- and time-dependent S-phase delay of the cell cycle. With time, the cell population became increasingly apoptotic with remaining survivors carrying a mutated gene set. Transcript profiling of treated cells indicated differential expression of genes involved in cell cycle regulation, stress response, receptors and tumour related genes. Prominent was elevation of p21(cip1/waf1) transcript and Western blot analysis confirmed induction of p21(cip1/waf1) and p53 proteins. The dose dependency and temporal aspects of these changes indicate that manipulation of the cell cycle and growth in response to PhIP is a precursor to mutant selection. Reduction of the PhIP dose allows dissection of a different battery of cellular responses that favour cell growth rather than inhibition. This pro-growth stimulus is oestrogen-like and encompasses altered gene expression, proliferation and cell behaviour. In human breast cell lines, these PhIP-mediated pro-oestrogenic responses are inhibited by the anti-oestrogen ICI 182780. This range of molecular and genetic responses induced in cells by PhIP is quite remarkable. Its ability to activate S-phase cell cycle checkpoint, alter gene expression leading to apoptosis and an increased frequency of mutation are probably direct consequences of its genetic toxicity. In contrast, its pro-oestrogenic activity is likely to be a driver of clonal expansion. We suggest that these PhIP-induced genomic and cellular events contrive to manipulate cell cycle and survival. Understanding these molecular processes as well as the genetic toxicology of the chemical will help to define the involvement of PhIP in carcinogenesis and shed light upon its tissue specificity.