1. Volume 119, Issue 6, Pages 1681-1691 (December 2000)
Estrogens stimulate proliferation of intrahepatic biliary epithelium in rats 
Domenico Alvaro, *, Gianfranco Alpini, ‡, Paolo Onori, §, Lucia Perego, ∥, Gianluca Svegliati Baroni, ¶, Antonio Franchitto, §, Leonardo Baiocchi, ‡, Shannon S. Glaser, ‡, Gene Le Sage, ‡, Franco Folli, ∥, Eugenio Gaudio, § 
Gastroenterology 
Volume 119, Issue 6, Pages (December 2000)
DOI: /gast
Copyright © 2000 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Immunohistochemistry for ER-α and ER-β in normal and 3-week BDL rat liver. Immunohistochemistry for ER-α shows a predominant nuclear positivity (arrows) that involves a higher (3-fold) percent of cholangiocytes in (B) 3-week BDL than in (A) normal liver. Scarce hepatocytes of normal and BDL livers showed nuclear immunoreactivity (arrowhead) for ER-α. Immunoreactivity for ER-β was virtually absent in cholangiocytes of (C) normal liver but involves approximately 80% of cholangiocytes (arrows) of (D) 3-week BDL liver with a predominant cytoplasmic localization. Hepatocytes are always negative for ER-β in both (C) normal and (D) 3-week BDL liver. Original magnification 50×.
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

3. Fig. 2
(A) RT-PCR and (B) Western blot analyses of ER-α and -β in hepatocytes and cholangiocytes. (A) RT-PCR showed a 129-bp message for ER-α in both cholangiocytes and hepatocytes (positive control, rat uterus; negative control, yeast transfer RNA). In contrast, a 371-bp transcript for ER-β was present in cholangiocytes but not hepatocytes (positive control, rat uterus; negative control, yeast transfer RNA). Sequence analysis showed a 100% homology with the reported sequence of rat ER-α or ER-β.5,6,20 (B) Western blot analysis showed the presence of the ER-α (~70 kilodaltons) in both hepatocyte and cholangiocyte lysates. In hepatocytes, after 1-week BDL, the expression of the ER-α decreased to 25% of normal (0). This reduction was even more evident after 2- and 3-week BDL, when it was approximately 5% of normal. In cholangiocytes from BDL rats, the intensity of the band decreased to 75% of control (0) at all time points studied. No reaction bands were observed in hepatocyte lysates challenged with the ER-β antibody. In cholangiocytes, a single band of approximately 70 kilodaltons reacting with the ER-β antibody was detected. The intensity of the band increased 4.5-fold at 1 week and 5-fold at 2 and 3 weeks of BDL compared with normal cholangiocytes (0). Quantitative data are means ± SEM (see text) from 3 cell preparations (each cell preparation is derived from 1 BDL rat or 2–5 normal rats).
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Immunohistochemistry for CK-19 in tamoxifen-treated, ICI 182,780–treated, and control 3-week BDL rat livers. Treatment with (B) tamoxifen or (C) ICI 182,780 in 3-week BDL rats induced a marked decrease in bile duct mass (CK-19) compared with (A) control BDL rats treated with tamoxifen carrier only. Original magnification 10×.
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Immunohistochemistry for PCNA and Fas antigen and TUNEL analysis in normal, tamoxifen-treated, ICI 182,780–treated, and control 3-week BDL rat livers. (A–D) PCNA. A strong nuclear positivity (arrows) is evident in cholangiocytes of (B) proliferating ducts (3-week BDL) but not in (A) normal liver. Treatment of 3-week BDL rats with (C) tamoxifen or (D) ICI 182,780 induced a marked decrease in the percentage of PCNA-positive cholangiocytes. (E–H) Fas. Immunoreaction for Fas antigen is negative in (E) normal and (F) 3-week BDL livers. In BDL rats treated with (G) tamoxifen or (H) ICI 182,780, 70% of cholangiocytes showed cytoplasmic positivity (arrows). (I and L–N) TUNEL. (I) Normal and (L) BDL livers do not show TUNEL positivity in cholangiocytes, whereas rat livers treated with (M) tamoxifen or (N) ICI 182,780 show a nuclear positivity (arrows) in more than 60% of cholangiocytes. (Counterstaining with methyl green; original magnification 50×.)
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions

6. Fig. 5
In vitro effect of 17β-estradiol on the proliferative capacity of isolated normal cholangiocytes. Cholangiocytes were isolated from normal rats, and proliferative capacities were assessed by evaluating [3H]thymidine incorporation into DNA and PCNA protein expression (Western blot). Incubation with 10 nmol/L 17β-estradiol (3 hours) increased PCNA protein expression and [3H]thymidine incorporation. This effect was completely blocked by 1 μmol/L tamoxifen and 1 μmol/L ICI 182,780, indicating a direct modulatory effect of estrogens on cholangiocyte proliferation. *P < 0.05 compared with basal, 17β-estradiol + tamoxifen or 17β-estradiol + ICI 182,780; *P < 0.01 compared with the other groups. Data are means ± SEM of 3 cell preparations (each cell preparation is derived from 1 BDL rat or 2–5 normal rats).
Gastroenterology  , DOI: ( /gast )
Copyright © 2000 American Gastroenterological Association Terms and Conditions