The food-derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine activates S-phase checkpoint and apoptosis, and induces gene mutation in human lymphoblastoid TK6 cells.

The mutagenic heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-blpyridine (PhIP) is formed at parts per billion levels when meat is cooked. It is efficiently absorbed from cooked food and extensively activated to its genotoxic N-hydroxy derivative by human cytochrome P4501A enzymes. It is also a rodent carcinogen. To better understand the genetic toxicity of PhIP, we have examined its effect on the cell cycle and gene mutation frequency using human lymphoblastoid cells (TK6) as a model. Because TK6 cells are unable to activate PhIP, we have cultured the cells in the presence of irradiated Chinese hamster XEMh1A2-MZ cells that have been genetically engineered to express human CYP1A2. Asynchronized TK6 cells were harvested at various times after treatment with PhIP (1.25-10 microg/ml), fixed and stained with propidium iodide for the examination of cell cycle by fluorescence-activated flow cytometry. After 20 h of PhIP treatment, a slight S-phase delay of the cell cycle was observed. Normal cell cycle recovered after the cells were washed and further cultured in the absence of PhIP for 5 days. However, PhIP treatment for 40 h induced a more pronounced S-phase arrest that was accompanied by a decrease in the level of cyclin A, an S-phase cyclin. This was followed by the appearance of a sub-G1 population (indicative of apoptotic cell death), range from 13 to 54% with PhIP concentrations from 1.25 to 10 microg/ml, compared with 5% in the vehicle control. A concomitant increase of mutation frequency at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus, assessed by colony formation assay in the presence of 6-thioguanine, was detected after 40 h-range, 16 to 45 x 10(-6) compared with 12 x 10(-6) in cultures without PhIP. In G1-enriched cell populations (synchronized culture), although PhIP induced S-phase delay, the induction of sub-G1 cells was substantially decreased. Our studies show that in TK6 cells, PhIP activates S-phase checkpoint, yet eludes G1 and G2-M checkpoints, and is accompanied by increased apoptosis and gene mutation. If treatment with PhIP induces similar cellular reactions in vivo, then activation of S-phase checkpoint with avoidance of G1 and G2-M checkpoints could be important factors in PhIP-induced genetic damage and neoplastic disease.