PROLIFERATING CELL NUCLEAR ANTIGEN - A MARKER FOR HEPATOCELLULAR PROLIFERATION IN RODENTS

Two different markers for quantitating cell proliferation were evaluated in livers of control and chemically treated mice and rats. Proliferating cell nuclear antigen (PCNA), an endogenous cell replication marker, and bromodeoxyuridine (BrdU), an exogenously administered DNA precursor label, were detected in formalin-fixed, paraffin-embedded tissues using immunohistochemical techniques. The percentage of cells in S phase (labeling indexes, LI) evaluated as PCNA- or BrdU-positive hepatocellular nuclei was compared in recut tissue sections from animals given BrdU by a single IP injection 2 hr before killing the animals. Ten-week-old male B6C3F(1) mice and F344 rats were exposed to known mitogenic hepatocarcinogens, Wy-14,643 (WY) in the diet at 0.1% for 2 days or 1,4-dichlorobenzene (DCB) in corn oil by gavage for 2 days (600 mg/kg/day in mice; 300 mg/kg/day in rats). In mice, PCNA and BrdU hepatocyte LI were similar in control, WY-treated, and DCB-treated animals. In rats, PCNA and BrdU gave similar LI in controls and Wy-treated animals. Although PCNA LI was statistically lower than BrdU LI in DCB-treated rats, both PCNA and BrdU LI for DCB-treated rats was increased over LI in control rats. Different patterns of PCNA immunohistochemical staining, interpreted to represent different subpopulations of cells at various phases of the cell cycle, were quantitated using PCNA immunohistochemistry. The proliferating index (PI), defined as the percentage of cells in the cell cycle (G(1) + S + G(2) + M), was more sensitive than the LI (S phase only) in detecting a chemically induced cell proliferative response. Due to reports of adverse effects of BrdU on cell proliferation, PCNA immunohistochemical methods were used to determine the effect of duration of exogenously administered DNA precursor label (BrdU or [H-3]-thymidine [H-3-TdR]) on rodent hepatocyte proliferation measurements. PCNA LI were determined in control animals pulse labeled with BrdU or H-3-Tdr, or labeled continuously for 3 or 6 days. PCNA LI did not increase with duration of exposure to BrdU or H-3-Tdr, suggesting that these labeling conditions are not causing a hepatocellular proliferative response. These results demonstrate comparable hepatocyte labeling of cells in S phase in control and chemically treated mice and rats with PCNA and pulse-BrdU labeling methods, supporting the use of PCNA as an alternative marker in either retrospective or prospective cell proliferation studies.