Pathology and Gene Expression in the Uteri of Mice Dosed with Oestradiol and Tamoxifen.

There is a marked difference in the development of uterine pathologies following administration of tamoxifen to neonatal mice by different routes. Oral dosing causes adenomyosis while subcutaneous administration results in cancer. In order to establish if this compound has similar estrogenic agonist effects by the two routes, a standard uterotrophic assay was set up in immature mice to compare the effects of tamoxifen and estradiol by these two routes. In this study mice were dosed with oral estradiol to establish the effects of this compound on the development of adenomyosis and changes in gene expression after 3 months in comparison to tamoxifen.

For uterotrophic assays, 14 day old CD-1 female mice were dosed by oral or subcutaneous routes daily for 3 days in groups of 4 with -estradiol 3-benzoate at either 0, 1, 5, 10, 50, or 100g/kg. b.w.; or with tamoxifen at either 0, 0.25, 0.5, 1, 2.5 or 5mg/kg. b.w.. Animals received oral doses at 5l/g bodyweight. Subcutaneous doses were administered at 5l/g bodyweight. Animals were killed on the fourth day and uteri were removed, weighed and fixed in Carnoy’s. For the 3-month pathology study, CD-1 female mice were dosed on days 2-5 after birth with oral -estradiol 3-benzoate at the same doses used for the uterotrophic assays with 12 mice per dosing level and 40 control mice. Mice were culled after 3 months and uteri were either fixed in Carnoy’s for pathology or snap frozen and stored in liquid nitrogen for RNA isolation. Changes in gene expression were analysed using cDNA arrays, essentially described elsewhere (Turton, 2001) from 25g total RNA.

Uterotrophic assays of estradiol showed that uterine weight, expressed as a percentage of total body weight, increased in response to subcutaneous administration in a dose dependent manner with a maximal response at 50g/kg. b.w. (0.56%, ± 0.12 S.D.). In contrast orally administered estradiol was less effective, with a positive effect only at 50g /kg. b.w. (0.35%, ± 0.06 S.D.) and 100g/Kg (0.39%, ± 0.04 S.D.). Tamoxifen gave an equally positive uterotrophic effect at both the lowest (0.25mg/kg) and highest (5mg/kg) doses with a slightly greater effect following oral administration. The uteri of all animals were normal as assessed by histopathology. Gene microarrays identified NGFa as a gene of interest in which expression was increased after tamoxifen treatment and decreased after estradiol treatment.

These results show that both compounds are reaching the uterus and that the development of adenomyosis is not due to metabolic effects. Results of the 3 month study show that adenomyosis does not develop in the uteri of estradiol treated mice in contrast to tamoxifen treated mice. Therefore it is reasonable to suggest that adenomyosis is not simply the result of an estrogen agonist action of tamoxifen on the developing uterine tissue.