Early alterations of apoptosis and cell proliferation in azoxymethane-initiated rat colonic epithelium

Alterations of apoptosis and cell proliferation in the colonic epithelium of rats after exposure to azoxymethane (AOM) were estimated by means of the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method, measurement of 5-bromo-2'-deoxyuridine (BrdU) incorporation, immunohistochemical staining for proliferating cell nuclear antigen (PCNA), and counting of mitotic cells. F344 male rats were given a single s.c. injection of AOM (15 mg/kg body weight) at 6 week of age, and killed 4 h, 8 h, 3 days, and 7 days after the AOM treatment. At 4 h after the treatment, many damaged cells were already observed in the colonic epithelium, and they were positive by TUNEL staining. At 8 h, the number of TUNEL-positive cells was largest. The reduction of DNA synthesis in the colonic epithelium, confirmed by BrdU incorporation, was not distinct in comparison with the mitotic inhibition. There was no remarkable change in PCNA labeling index, except that strong expression of PCNA was detected in many damaged cells. On the 3rd day, the appearance of cell death became infrequent and an increase of cell proliferation occurred. On the 7th day, the expression of TUNEL and the cell proliferation biomarkers were at almost normal levels. These findings suggest that AOM induces apoptosis, which is associated with synchronous inhibition of mitosis. The data also indicate that PCNA immunostaining does not reflect the true proliferation state in the early phase after AOM exposure, probably due to the occurrence of cell cycle arrest or DNA repair.