Comparative uterotrophic effects of endoxifen and tamoxifen in ovariectomized Sprague-Dawley rats

Abstract

Endoxifen (4-hydroxy-N-desmethyl-tamoxifen), one of the major active metabolites of tamoxifen, has substantially greater estrogen antagonist properties and antiproliferative effects in breast tumor cells than tamoxifen, a mixed estrogen agonist/antagonist. An associated risk of endometrial cancer and hyperplasia has been linked to the estrogen agonist properties of tamoxifen. We evaluated endoxifen using a classic uterotrophic effects method. Rats were given endoxifen or tamoxifen orally for 3 days. Estradiol was the positive control. Endoxifen and tamoxifen plasma levels exceeded those previously observed clinically. Uterine weight was 3-fold higher in the estradiol group than in the tamoxifen or endoxifen groups, which did not differ from vehicle controls. Tamoxifen and endoxifen caused a greater increase in luminal epithelial cell height than estradiol. Both tamoxifen and endoxifen produced an increase in the stromal BrdU labeling index (LI) that was ≤ estradiol and inversely related to dose, but did not affect luminal epithelial cell BrdU LI. As expected, estradiol increased luminal epithelial cell proliferation. These results indicate that endoxifen induces uterotrophic effects, but is less potent than estradiol in eliciting these effects. Given prior preclinical observations that endoxifen has superior antitumor activity than tamoxifen, the observations of similar uterine effects suggest that the endoxifen risk/benefit ratio may be superior to tamoxifen.

Keywords: endometrial cell proliferation.; endoxifen; tamoxifen.

Figure 1

Uterine weight (percentage of body weight) of ovariectomized rats after treatment with estradiol, tamoxifen, or endoxifen. Rats were administered tamoxifen or endoxifen once daily for three days by oral gavage, and euthanized on Day 4. The vehicle control groups for tamoxifen and endoxifen were given 10% Tween 80/15% PEG 400 or sterile water, respectively, via the same route and schedule. The positive control group was given 17-β-estradiol benzoate (estradiol) subcutaneously on the same schedule. No statistically significant changes were noted in uterine weight with tamoxifen or endoxifen compared to respective vehicle controls. Each data point represents the mean and standard error of the mean for five animals per group.

Figure 2

Hypertrophy as determined by luminal epithelial cell height in ovariectomized rats after treatment with estradiol, tamoxifen, or endoxifen. Rats were administered tamoxifen or endoxifen once daily for three days by oral gavage, and euthanized on Day 4. The vehicle control groups for tamoxifen and endoxifen were given 10% Tween 80/15% PEG 400 or sterile water, respectively, via the same route and schedule. The positive control group was given 17-β-estradiol benzoate (estradiol) subcutaneously on the same schedule. ‡Statistically significant at the 5% level compared to vehicle (Tween 80/PEG 400) control; *statistically significant at the 5% level compared to vehicle (water) control. Each data point represents the mean and standard error of the mean for five animals per group.

Figure 3

Hematoxylin and eosin stained sections of rat uterus demonstrating luminal epithelial cell hyperplasia as measured by cell height (arrow points to luminal epithelium). Compared to respective vehicle controls (A and B) and positive estradiol control (C), luminal cell height was significantly greater in all tamoxifen and endoxifen dose groups: 20 mg/kg/day tamoxifen (D), 200 mg/kg/day tamoxifen (E), 5 mg/kg/day endoxifen (F), 80 mg/kg/day endoxifen (G) and 200 mg/kg/day endoxifen (H). 20X objective; bar = 100 μm

Figure 3

Hematoxylin and eosin stained sections of rat uterus demonstrating luminal epithelial cell hyperplasia as measured by cell height (arrow points to luminal epithelium). Compared to respective vehicle controls (A and B) and positive estradiol control (C), luminal cell height was significantly greater in all tamoxifen and endoxifen dose groups: 20 mg/kg/day tamoxifen (D), 200 mg/kg/day tamoxifen (E), 5 mg/kg/day endoxifen (F), 80 mg/kg/day endoxifen (G) and 200 mg/kg/day endoxifen (H). 20X objective; bar = 100 μm

Figure 4

BrdU labeling index in the endometrial stroma of ovariectomized rats after treatment with estradiol, tamoxifen, or endoxifen. Rats were administered tamoxifen or endoxifen once daily for three days by oral gavage, and euthanized on Day 4. The vehicle control groups for tamoxifen and endoxifen were given 10% Tween 80/15% PEG 400 or sterile water, respectively, via the same route and schedule. The positive control group was given 17-β-estradiol benzoate (estradiol) subcutaneously on the same schedule. A continuous 3-day subcutaneous infusion of BrdU was given on Days 1–3 to all rats. ‡Statistically significant at the 5% level compared to vehicle (Tween 80/PEG 400) control; *statistically significant at the 5% level compared to vehicle (water) control. Each data point represents the mean and standard error of the mean for five animals per group.

Figure 5

BrdU labeling index in the luminal epithelium of ovariectomized rats after treatment with estradiol, tamoxifen, or endoxifen. Rats were administered tamoxifen or endoxifen once daily for three days by oral gavage, and euthanized on Day 4. The vehicle control groups for tamoxifen and endoxifen were given 10% Tween 80/15% PEG 400 or sterile water, respectively, via the same route and schedule. The positive control group was given 17-β-estradiol benzoate (estradiol) subcutaneously on the same schedule. A continuous 3-day subcutaneous infusion of BrdU was given on Days 1–3 to all rats. Each data point represents the mean and standard error of the mean for five animals per group.

Figure 6

BrdU immunostained sections demonstrating cell proliferation (brown staining) in luminal epithelial (L) and stromal (S) compartments of rat uterus. Compared to the positive estradiol control (A), cell proliferation was significantly less in both the luminal epithelium and stroma at 200 mg/kg/day tamoxifen (B) and 200 mg/kg/day endoxifen (C). Cell proliferation in the luminal epithelium was also significantly less than the positive control in the lower dose groups of both tamoxifen (20 mg/kg/day) and endoxifen (80 and 5 mg/kg/day) (see Figure 5 for data). Stromal cell proliferation was significantly less than the positive control at 200 mg/kg/day tamoxifen, but not at 20 mg/kg/day. Additionally, stromal cell proliferation was significantly less than the positive control at 80 mg/kg/day endoxifen, but not at 5 mg/kg/day (see Figure 4 for data). 20X objective; bar = 100 μm