Hepatic Ploidy, Nuclearity, and Distribution of DNA Synthesis: A Comparison of Nongenotoxic Hepatocarcinogens with Noncarcinogenic Liver Mitogens

Abstract

Most nongenotoxic hepatocarcinogens such as diethylhexyl phthalate (DEHP) and chlorendic acid (CEA) induce hepatocyte replicative DNA synthesis. However, not all mitogens are carcinogenic; in National Toxicology Program (NTP) studies 1,4-dichlorobenzene (DCB) was not hepatocarcinogenic in the rat despite the induction of substantial hepatic DNA synthesis. We have examined the hypothesis that the profile of hepatocyte labeling index (LI) may dictate hepatocarcinogenic potential. Using flow cytometry, we investigated the effects of DEHP, CEA, and DCB on hepatocyte ploidy, nuclearity, and LI distribution among the ploidy/nuclearity classes in male Fischer 344 rats. Dosing was for 7 days at the dose and route employed previously in the NTP cancer bioassays. Unlike DEHP and CEA, DCB reduced the proportion of tetraploid cells (4N plus 2 × 2N) and increased the proportion of mononucleated octoploid (8N) cells. DCB and DEHP increased the mean hepatic LI (17 ± 2 and 23 ± 3%, respectively, compared with 1.4 ± 0.4% in controls), whereas, as expected for a chronic inducer of S-phase, CEA did not. LI was increased in all hepatocyte ploidy/nuclearity classes except the binucleated tetraploid cells (2 × 2N) and was elevated most in the mononucleated octoploid population (8N) (LI = 44 ± 11, 49 ± 14, and 1.3 ± 0.4% of 8N hepatocytes for DCB, DEHP, and control, respectively). In general, the effects of DCB on LI could not be distinguished from those of DEHP. However, DEHP tended to induce DNA synthesis in a greater proportion of 2N and 2 × 4N cells; future studies will analyze whether the induction of DNA synthesis in these small populations may be more relevant to hepatocarcinogenesis.