Enhanced induction of mucin-depleted foci in estrogen receptor {beta} knockout mice

Abstract

The role of the estrogen receptor beta (ERbeta) in the colon has received considerable interest, yet in vivo models are needed to better define its protective actions. In the present study, wild-type (WT), ERalpha, and ERbeta knockout (alphaERKO and betaERKO) mice were injected with azoxymethane, a colon chemical carcinogen. Fourteen weeks after azoxymethane exposure, the incidence of aberrant crypt foci (ACF) was assessed by methylene blue staining. betaERKO mice showed significantly higher incidence (P < 0.001) of ACF (15.0 +/- 2.5) compared with alphaERKO (3.4 +/- 1.0) and WT (4.6 +/- 1.0) mice. The colons in several betaERKO mice had increased thickness and loss of normal morphology. It has been reported that ERbeta plays a role in the maintenance of the colonic crypt architecture; this may explain the loss of crypt organization in the colonic epithelium of betaERKO mice. The presence of mucin-depleted foci (MDF) has been shown, both in humans and in rodents, as an early event in colon cancer. Therefore, to surpass the limitations with ACF scoring, we performed Alcian blue-neutral red staining to assess the presence of MDF. This assay allowed the assessment of precancerous lesions on all the betaERKO mice colons (38.3 +/- 4.0; P < 0.001), comparing to WT and alphaERKO mice (6.6 +/- 1.5 and 10.0 +/- 1.9, respectively), and served to confirm the ACF results. Together, these data support the use of MDF staining as a biomarker for precancerous lesions and the protective role of ERbeta in colon carcinogenesis.

Fig. 1

Influence of ERα and ERβ-deficiency on incidence of ACF and MDF in AOM-treated WT and ERKO mice. A, number of ACF/colon; B, number of MDF/colon for WT, αERKO and βERKO mice. The values are means (+SE). Statistical differences are shown between WT and βERKO and between αERKO and βERKO groups.

Fig. 2

Topographical features of AOM-induced ACF and MDF presenting different multiplicity. A– C, ACF composed of 1 and 3 crypts (original magnification, ×20) and more than 4 crypts (original magnification, ×40) respectively as indicated by black arrows, observed in colons of WT mice stained with MB. D–F, MDF composed of 4, 10 and more than 12 crypts, respectively, indicated by white arrows, observed in colons of βERKO mice after AB-NR staining (original magnification, ×20).

Fig. 3

Topographical features of the observed colonic epithelium of AOM-treated WT, αERKO and βERKO mice after AB-NR staining. The background epithelium stains in red (NR), while the colonic crypts appear as dark-blue spots due to the high production of mucin stained with AB (WT, original magnification ×20; αERKO, original magnification ×10). In the ERβ-deficient colons there was a loss of normal crypt organization observed (βERKO, original magnification ×10). Despite this, MDF (eg. indicated by white arrow) were easily scored with AB-NR staining and used as biomarkers for precancerous lesions in the colon. βERKO (a) shows the higher magnification of the white rectangular region in the left βERKO panel.

Fig. 4

A. Expression of Ki-67 in colon sections of WT and ERKO mice (original magnification, ×20). B. Apoptosis detection by TUNEL in WT and ERKO colonic sections (original magnification, ×40). Note that the images show the top of the colonic crypts where the number of apoptotic cells is higher. C. Ki-67 immunostaining quantification. No significant statistical differences were observed between the three ER genotypes (p > 0.05). D. Quantitative analyses for TUNEL. Fewer apoptotic cells were observed in βERKO. Comparison of apoptotic cells in βERKO colonic epithelium with that of WT and αERKO showed statistically significant differences (p < 0.01 for βERKO Vs WT and p < 0.05 for βERKO Vs αERKO). All values are presented as mean ± SE.