The effect of estrogen and tamoxifen on hepatocyte proliferation in vivo and in vitro

Abstract

We have previously shown that changes in estrogen-hepatocyte interaction occur during liver regeneration. Following 70% hepatectomy, estrogen levels in the blood were elevated, the number of estrogen receptors in the liver was increased and there was an active translocation of estrogen receptors from the cytosol to the nucleus. The injection of tamoxifen, an estrogen antagonist, inhibits hepatocyte proliferation following partial hepatectomy. The administration of 1 microgram tamoxifen per gm body weight at zero time or 6 hr after the operation resulted in a significant inhibition both of DNA synthesis and of the number of cells in mitosis. Injections of tamoxifen 12 hr or later after the operation had no effect. Concomitant injections of equimolar amounts of estrogen abolished the inhibition by tamoxifen. The effects of estrogen and tamoxifen were also tested on hepatocytes in primary culture. Estrogens in the presence of 5% normal rat serum stimulated hepatocyte DNA synthesis as determined by [3H]thymidine incorporation and the labeling index, whereas epidermal growth factor-induced DNA synthesis in the absence of normal rat serum was strongly inhibited. Tamoxifen, in contrast, inhibited DNA synthesis of hepatocytes in the presence of 5% normal rat serum and reversed the stimulatory effect of estrogen in the same system. Attempts to elucidate the mechanism of tamoxifen inhibition in vitro indicated that one effect of tamoxifen is to prevent the amiloride-sensitive Na+ influx necessary to initiate hepatocyte proliferation.

Fig. 1

Administration of tamoxifen at different times after 70% partial hepatectomy. All operations were performed at time 0, between 8 and 10 a.m. Injections of 1 μg per gm body weight tamoxifen were administered at indicated times after operation. All animals were given injections of 50 μCi [³H]thymidine at 23 hr after the operation. One hour later, animals were killed, and hepatic DNA synthesis and labeled nuclei were determined. The values represented by the bar are means of six rats ± S.D. * = significantly different from control value, p < 0.05.

Fig. 2

Reversal of tamoxifen inhibition of DNA synthesis by estrogen. All animals had 70% of the liver removed between 8 and 10 a.m. Where indicated, tamoxifen (1 μg per gm body weight) and/or estradiol (0.8 μg per gm body weight) were injected at zero time after the surgical procedure. DNA synthesis at 24 hr was determined as indicated for Fig. 1. The values represented by the bars are the means from at least five rats ± S.D. * = significantly different from control value, p < 0.01.

Fig. 3

Duration of tamoxifen inhibition of hepatocyte proliferation activity in vivo expressed as labeled nuclei following partial hepatectomy in male and female rats. All operations were performed between 8 and 10 a.m. Tamoxifen (1 μg per gm body weight) was injected at zero time after the operation. All animals were given injections of 50 μCi [³H]thymidine 1 hr before they were killed. At specified times after partial hepatectomy, indicated on the abscissa, the animals were killed and labeled nuclei were determined as described (6). The values represented by each bar were derived from four controls and four tamoxifen-injected rats. Open bars = controls; hatched bars = tamoxifen-injected animals.

Fig. 4

Effect of tamoxifen added to hepatocytes at various times after plating. Hepatocytes were isolated and plated as described in “Materials and Methods.” After 3 hr, the attachment medium was replaced with NRS-containing basal medium. At indicated times, three wells for each point received tamoxifen (4 mM). All wells received 3 μCi [³H]thymidine at 24 hr. The cells were harvested at 48 hr. The values shown are the averages of at least three determinations with individual values within 5% of the mean shown. Significantly different from control value: * = p < 0.01; ** = < 0.05.