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

Two different markers for quantitating cell proliferation were evaluat ed in livers of control and chemically treated mice and rats. Prolifer ating cell nuclear antigen (PCNA), an endogenous cell replication mark er, and bromodeoxyuridine (BrdU), an exogenously administered DNA prec ursor label, were detected in formalin-fixed, paraffin-embedded tissue s using immunohistochemical techniques. The percentage of cells in S p hase (labeling indexes, LI) evaluated as PCNA- or BrdU-positive hepato cellular nuclei was compared in recut tissue sections from animals giv en 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 mitoge nic hepatocarcinogens, Wy-14,643 (WY) in the diet at 0.1% for 2 days o r 1,4-dichlorobenzene (DCB) in corn oil by gavage for 2 days (600 mg/k g/day in mice; 300 mg/kg/day in rats). In mice, PCNA and BrdU hepatocy te LI were similar in control, WY-treated, and DCB-treated animals. In rats, PCNA and BrdU gave similar LI in controls and Wy-treated animal s. Although PCNA LI was statistically lower than BrdU LI in DCB-treate d rats, both PCNA and BrdU LI for DCB-treated rats was increased over LI in control rats. Different patterns of PCNA immunohistochemical sta ining, interpreted to represent different subpopulations of cells at v arious phases of the cell cycle, were quantitated using PCNA immunohis tochemistry. The proliferating index (PI), defined as the percentage o f cells in the cell cycle (G(1) + S + G(2) + M), was more sensitive th an the LI (S phase only) in detecting a chemically induced cell prolif erative response. Due to reports of adverse effects of BrdU on cell pr oliferation, PCNA immunohistochemical methods were used to determine t he 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 labele d 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, sugg esting that these labeling conditions are not causing a hepatocellular proliferative response. These results demonstrate comparable hepatocy te labeling of cells in S phase in control and chemically treated mice and rats with PCNA and pulse-BrdU labeling methods, supporting the us e of PCNA as an alternative marker in either retrospective or prospect ive cell proliferation studies.