Figure 1.
Cadmium stimulates anterior pituitary lactotroph proliferation.
Anterior pituitary cells were treated with vehicle (control), 10 nM Cd or 1 nM E2 for 96 h. Cell growth was determined by ICC measuring 24 h-BrdU incorporation. Lactotrophs were identified by prolactin-specific antibody and cell nuclei were stained by DAPI. Pictures are representative of three independent experiments performed in triplicate. Bars represent the mean ± SE of BrdU-labeling index expressed as positive BrdU lactrotroph / total lactotroph cell number x100. ANOVA followed by Tukey-Kramer’s test, **p<0.001 vs. control (N=3).
Figure 2.
Cadmium increases gene expression of proliferation markers in anterior pituitary cells.
Anterior pituitary cells were treated with vehicle (control), 10 nM Cd or 1 nM E2. Gene expression was evaluated by PCR. Bars represent the mean ± SE of densitometric values of cyclins D1 and D3 after 72 h (A) or c-fos after 8-24 h (B) normalized to GAPDH expression and are expressed as percent of control. ANOVA followed by Tukey-Kramer’s test, *p<0.05, **p<0.001 vs. control (N=3).
Figure 3.
Cadmium increases cyclin D1 protein expression in anterior pituitary cells.
Anterior pituitary cells were treated with vehicle (control), 10 nM Cd or 1 nM E2 for 72 h. A representative western blot is shown. Bars represent the mean ± SE of densitometric values normalized to β-actin expression and are expressed as percent of control. ANOVA followed by Tukey-Kramer’s test, *p<0.05 vs. control (N=3).
Figure 4.
Cadmium stimulates GH3 cell proliferation.
GH3 cells were treated with vehicle (control), 10 nM Cd or 1 nM E2 for 96 h. Cell growth was determined by ICC measuring 3 h-BrdU incorporation. Cell nuclei were stained by DAPI. Pictures are representative of three independent experiments performed in triplicate. Bars represent the mean ± SE of BrdU-labeling index expressed as positive BrdU cells / total cell number x 100. ANOVA followed by Tukey-Kramer’s test, *p<0.05 vs. control (N=3).
Figure 5.
Cadmium increased 23 kDa prolactin (PRL) protein expression in anterior pituitary cells in culture.
Anterior pituitary cells were incubated with 10 nM Cd or vehicle (control) for 8 h. Protein expression was measured by western blot. Bars represent mean ± SEM of PRL densitometric values normalized to β-actin and are expressed as percent of control. **P<0.01, Student’s ‘t’ test (N=3).
Figure 6.
E2 receptors (ERs) mediates cadmium effect on cyclins D1 and D3 and PRL mRNA expression.
Anterior pituitary cells cultures were first incubated with 100 nM ICI 182,780 (ICI) for 20 min and then incubated with vehicle (control) or 10 nM Cd for 72 h. Cyc D1 and D3 (A) and PRL (B) mRNA expression was evaluated by PCR. Bars represent the mean ± SE of densitometric values normalized to GAPDH and are expressed as percent of control. ANOVA followed by Tukey-Kramer’s test, **p<0.01 vs. control, ##p<0.01 vs. Cd (N=3).
Figure 7.
Cadmium exposure increases ERα
protein expression in anterior pituitary cells. Anterior pituitary cells were treated with vehicle (control), 10 nM Cd or 1 nM E2 for 24h. A representative western blot is shown. Bars represent the mean ± SE of densitometric values of full-length ERα (open bars) and ERα46 (black bars) normalized to β-actin expression and are expressed as percent of control. ANOVA followed by Tukey-Kramer’s test, *p<0.05, **p<0.01, ***p<0.001 vs. respective control; #p<0.05, ###p<0.001 vs. Cd (N=3).
Figure 8.
Additive effect of cadmium and E2 co-treatment on ERα mRNA expression.
Anterior pituitary cell cultures were treated with vehicle (control), 10 nM Cd or 1 nM E2 or 10 nM Cd plus 1 nM E2 for 8 h or 24 h. ERα mRNA expression was evaluated by PCR. Bars represent the mean ± SE of densitometric values normalized to GAPDH. ANOVA followed by Tukey-Kramer’s test, *p<0.05,