Ethinyl estradiol (EE) is a strong promoter of hepatocarcinogenesis in female rats. A common effect shared by many non-genotoxic hepatic promoters is the stimulation of hyperplastic growth. However, studies by others with several hepatic promoters have shown that following an initial, transient increase in liver growth, continued exposure causes an inhibition in basal and/or induced growth. We have shown that EE also causes an initial, transient increase in hepatocyte proliferation (Carcinogenesis, 7, 2007-20014, 1986). The objective of the investigation reported here was to determine whether chronic EE treatment also becomes inhibitory to basal and/or induced liver growth. Female Lewis rats were treated with EE at 2.5 and 5.0 μg/rat/day using time-release tablets. Seven days prior to sacrifice, the rats were implanted with osmotic minipumps containing bromodeoxyuridine (BrdU) to allow the cumulative labeling of replicating hepatocytes. At sacrifice, liver tissue was fixed, sectioned and the percent labeled hepatocyte nuclei determined by immunohistochemistry. The results of these experiments revealed that, as expected, during the first 7 days of treatment, EE increased hepatocyte proliferation. However, after 28 and 42 days of EE treatment, the basal level of liver growth was dramatically inhibited. Thus, hepatocyte nuclear labeling indices, compared to controls, were reduced by 72 and 88% after 28 and 42 days of treatment respectively. An analysis of l25I-labeled EGF binding to isolated liver membranes revealed that EGF receptor levels decreased during the initial period of growth, but had returned to control levels by day 21 when replication had ceased. In another experiment, rats were treated with EE at 5.0 μg/rat/day for 21 days; control rats received placebo time-release tablets. At the end of that time, the rats were surgically partially hepatectomized and the level of subsequent regenerative DNA synthesis was determined using [3H]thymidine administered 2 h prior to sacrifice 24, 48, 72 and 96 h later. The results showed that EE caused an inhibition of regenerative growth. While at each time point the level of DNA synthesis in EE-treated rats was not significantly less than in corresponding controls, statistical analysis indicated that overall, EE caused a significant reduction in liver growth. The results of these studies demonstrate that chronic EE treatment leads to the appearance of a mitosuppressed state in the liver which is characterized by reduced cell turnover and decreased growth responsiveness. These results indicate that ethinyl estradiol should be added to the list of promoters of hepatocarcinogenesis which, upon continued treatment, subsequently cause mitosuppressive effects. Furthermore, together with the findings of others with other hepatic promoters, our results illustrate that initial proliferative effects may have little bearing on promoting potential. It now becomes important to determine whether promotion by EE actually involves differential mitoinhibitory effects in non-initiated versus initiated hepatocytes.
ASJC Scopus subject areas
- Cancer Research