Figure 7.

Chromosomal aberrations induced by N-OH-PhIP in human colonic epithelial cells (HCEC) and the proposed model of PhIP-induced replication stress. (A) HCEC were incubated with 2.5 μM N-OH-PhIP for 24 h in the absence or presence of 2.5 μM ATR inhibitor prior to the addition of demecolcine. After 14 h, the cells were fixed and processed as described. Metaphase spreads (50 per treatment group, n = 3) were evaluated by phase-contrast microscopy and representative pictures thereof are shown. (B and C) Aberration frequencies expressed as aberrations per cell and aberrations per metaphase (normalized to the mean number of chromosomes in 50 metaphases of each treatment variant); *P < 0.05, **P < 0.01, ***P < 0.001 versus control cells. (D) Proposed model of PhIP-induced replication stress. Upon metabolic activation, PhIP induces the formation of C8-PhIP-dG adducts in DNA. These bulky lesions stall replicative polymerases, leading to ssDNA formation and subsequent accumulation of RPA. In turn, ATR is activated and phosphorylates its downstream targets CHK1 and H2AX, thereby conferring protection against PhIP-induced replication stress and promoting cell survival. Inhibition of ATR potentiates the formation of DSBs, which are recognized by the DNA damage sensor MRN, leading to the recruitment and activation of ATM. The enhanced DSB level results in both increased chromosomal aberrations and cell death.