INCREASED NUCLEAR PLOIDY, NOT CELL-PROLIFERATION, IS SUSTAINED IN THEPEROXISOME PROLIFERATOR-TREATED RAT-LIVER

Peroxisome proliferators are believed to induce liver tumors in rodent s due to sustained increase in cell proliferation and oxidative stress resulting from the induction of peroxisomal enzymes. The objective of this study was to conduct a sequential analysis of the early changes in cell-cycle kinetics and the dynamics of rat liver DNA synthesis aft er treatment with a peroxisome proliferator. Immunofluorescent detecti on of proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) incorporation into DNA during S phase were used to assess rat h epatocyte proliferation in vivo during dietary administration of Wy-14 ,643, a known peroxisome proliferator and hepatocarcinogen in rodents. Rats were placed on a diet containing 0.1% Wy-14,643 and implanted su bcutaneously with 5-bromo-2'-deoxyuridine containing osmotic pumps 4 d ays prior to being sacrificed on days 4, 11, and 25 of treatment. Isol ated liver nuclei labeled with fluorscein isothiocyanate (FITC)-anti-B rdU/PI and FITC-anti-PCNA/PI were analyzed for S-phase kinetics using flow cytometry. Morphometric analysis was performed to evaluate nuclea r and cell size and enumeration of BrdU labeled cells, binucleated hep atocytes, and mitotic index. The BrdU labeling index increased 2-fold in livers of Wy-14,643-treated rats at day 4, but distribution of cell s in G(1), S phase, and G(2)-M did nor differ significantly from contr ols. PCNA-positive cells decreased from 36% on day 4 to 17% on day 25, whereas the percentage of PCNA-positive cells in controls increased 2 -fold from day 4 to day 11 and remained unchanged up to day 25. The di fferences in the number of PCNA-positive nuclei between control and Wy -14,643-treated groups were statistically significant only on day 4. B inucleated hepatocytes, determined by morphometric analysis, increased slightly on day 25 in treated rats parallel to an increase in the per centage of cells in G(2)-M phase. Significant shifts were noted in nuc lear diameter and nuclear area after 11 and 25 days of treatment with Wy-14,643. Hepatic cell populations with nuclei >9 mu m diameter and n uclear area >64 mu m(2) increased in Wy-14,643-fed rats during the tre atment period compared with the control, indicating hepatic karyomegal y and hyperploidy, whereas percentage of distribution of nuclei based on diameter and area remained consistently unchanged in control animal s from 4 through 25 days of sham treatment. The flow cytometric and mo rphometric analysis indicated an initial wave of DNA synthesis in resp onse to Wy-14,643. The hepatomegaly was sustained over the treatment p eriod accompanied by increase in ploidy with a significant shift towar d hyperploidic hepatocytes. The increase in DNA content was almost ent irely accounted for by the overall polyploidy increase rather than by an absolute increase in cells.