Cadmium mimics estrogen-driven cell proliferation and prolactin secretion from anterior pituitary cells.

Cadmium (Cd) is a heavy metal of considerable occupational and environmental concern affecting wildlife and human health. Recent studies indicate that Cd, like other heavy metals, can mimic effects of 17β-estradiol (E2) involving E2 receptor (ER) activation. Lactotrophs, the most abundant cell type in anterior pituitary gland, are the main target of E2, which stimulates cell proliferation and increases prolactin secretion through ERα. The aim of this work was to examine whether Cd at nanomolar concentrations can induce cell proliferation and prolactin release in anterior pituitary cells in culture and whether these effects are mediated through ERs. Here we show that 10 nM Cd was able to stimulate lactotroph proliferation in anterior pituitary cell cultures from female Wistar rats and also in GH3 lactosomatotroph cell line. Proliferation of somatotrophs and gonadotrophs were not affected by Cd exposure. Cd promoted cell cycle progression by increasing cyclins D1, D3 and c-fos expression. Cd enhanced prolactin synthesis and secretion. Cd E2-like effects were blocked by the pure ERs antagonist ICI 182,780 supporting that Cd acts through ERs. Further, both Cd and E2 augmented full-length ERαexpression and its 46 kDa-splicing variant. In addition, when co-incubated Cd was shown to interact with E2 by inducing ERα mRNA expression which indicates an additive effect between them. This study shows for the first time that Cd at nanomolar concentration displays xenoestrogenic activities by inducing cell growth and stimulating prolactin secretion from anterior pituitary cells in an ERs-dependent manner. Cd acting as a potent xenoestrogen can play a key role in the aetiology of different pathologies of the anterior pituitary and in estrogen-responsive tissues which represent considerable risk to human health

E2 receptors (ERs) mediates cadmium effect on cyclins D1 and D3 and PRL mRNA expression.

<p>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).</p

Cadmium increases gene expression of proliferation markers in anterior pituitary cells.

<p>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 <i>c-fos</i> 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). </p

Cadmium exposure increases ERα

<p><b>protein expression in anterior pituitary cells</b>. 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).</p

Cadmium increased 23 kDa prolactin (PRL) protein expression in anterior pituitary cells in culture.

<p>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).</p

Cadmium stimulates anterior pituitary lactotroph proliferation.

<p>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). </p

Additive effect of cadmium and E2 co-treatment on ERα mRNA expression.

<p>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, </p><p>** p<0.01, *** p<0.001 vs. Control; #p<0.05 vs. Cd; <b>^</b>p<0.05 vs. E2 (N=3).</p><p></p

Cadmium increases cyclin D1 protein expression in anterior pituitary cells.

<p>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).</p

Cadmium induced-oxidative stress in pituitary gland is reversed by removing the contamination source

Cadmium (Cd2+) is one of the most important environmental contaminants and acts as an endocrine disruptor. Previously, we have demonstrated that the simultaneous administration of Cd2+ and melatonin (Mel) in drinking water impaired metal-induced oxidative stress in rat anterior pituitary gland. The aim of this study was to investigate if a treatment started after the toxic manifestations of Cd 2+ became evident could reverse the effects of the metal. Animals exposed to Cd2+ (5 parts per million [ppm], 30 days) were treated with Mel or without the metal during the next 1 or 2 months. Cd2+ exposure increased the expression of heme oxygenase-1 (HO-1), a biomarker of oxidative stress, and an a posteriori Mel treatment reversed oxidative stress induced by Cd2+. This effect was also observed 1 month after metal removal. The Cd2+-induced increase in metallothionein-1 (MT-1) and nitric oxide synthase 1 (NOS1) expression were also reversed by metal removal. In addition, serum prolactin and luteinizing hormone levels affected by Cd 2+ exposure were normalized. Considering that the manifestations of Cd2+ intoxication become evident only after a certain period of metal accumulation, these results show that metal removal is enough to reverse Cd2+ effects in anterior pituitary gland and bring to light the relevance of moving away the individual from the contamination source.Fil: Miler, Eliana Andrea. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina; ArgentinaFil: Nudler, Silvana Iris. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina; ArgentinaFil: Quinteros, Fernanda A.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina; ArgentinaFil: Cabilla, Jimena Paula. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina; ArgentinaFil: Ronchetti, Sonia Alejandra. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina; ArgentinaFil: Duvilanski, Beatriz Haydee. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Fisicoquímica Biológicas; Argentina; Argentin