Beatriz H. Duvilanski

Cadmium induces apoptosis in anterior pituitary cells that can be reversed by treatment with antioxidants.

Cadmium (Cd(2+)) is an ubiquitous toxic metal that is involved in a variety of pathological conditions. Several reports indicate that Cd(2+) alters normal pituitary hormone secretion; however, little is known about the mechanisms that induce this misregulation. This paper reports the effect of Cd(2+) on anterior pituitary cell viability and its relation to prolactin secretion. Cd(2+) concentrations above 10 microM were found to be cytotoxic for pituitary cells. Morphological studies as well as DNA ladder fragmentation and caspase activation showed that Cd(2+)-treated cells undergo apoptosis. Even though several hours were needed to detect Cd(2+)-induced cytotoxicity, the effect of the metal became irreversible very quickly, requiring only 3 h of treatment. Prolactin release (measured at 48 h) was inhibited when the cells were exposed to Cd(2+) for 1 h, before any change in cell viability was observed. The antioxidants N-acetyl-cysteine and Trolox (a hydrosoluble derivative of vitamin E), but not ascorbic acid, reversed both Cd(2+)-mediated cytotoxicity and the inhibition of prolactin release, supporting the involvement of oxidative stress in the mechanism of Cd(2+) action. In summary, the present work demonstrates that Cd(2+) is cytotoxic for anterior pituitary cells, that this effect is due to an induction of apoptosis, and that it can be reversed by antioxidants.

In vivo protective effect of melatonin on cadmium‐induced changes in redox balance and gene expression in rat hypothalamus and anterior pituitary

Abstract:  Cadmium (Cd) is widely used in industrial applications and is an important side contaminant of agricultural products. As an endocrine disruptor, Cd modifies pituitary hormone release. It has been shown that this metal causes oxidative stress in primary cultures of anterior pituitary cells. To examine whether Cd induces redox damage in the hypothalamic–pituitary axis in vivo and to evaluate the efficacy of the antioxidant molecule melatonin to prevent Cd activity, rats were exposed to Cd (5 p.p.m. in drinking water) with or without melatonin (3 μg/mL drinking water) for 1 month. In the anterior pituitary, Cd increased lipid peroxidation and mRNA levels for heme oxygenase‐1 (HO‐1) at both time intervals tested (09:00 and 01:00 hr, beginning of rest span and middle of activity span, respectively). Melatonin administration prevented the Cd‐induced increase in both parameters. In the hypothalamus, Cd affected the levels of mRNA for HO‐1 by decreasing it in the evening. Melatonin reduced hypothalamic HO‐1 gene expression. Cd treatment augmented gene expression of nitric oxide synthase (NOS)1 and NOS2 in the pituitary whereas melatonin decreased it, impairing the activity of Cd. Exposure to Cd increased the levels of hypothalamic NOS1 mRNA at 09:00 hr and decreased the levels of NOS2 mRNA at 01:00 hr, with melatonin treatment preventing Cd effects. Cd treatment decreased plasma thyroid‐stimulating hormone levels at both examined times, while melatonin reversed the effect of Cd at 09:00 hr and partially counteracted the effect at 01:00 hr. There were important variations between day and night in the expression of all the genes tested in both tissues. Melatonin treatment was effective reducing all examined effects of Cd, documenting its effectiveness to protect the rat hypothalamic–pituitary axis from the toxic metal effects.

Differential effects of glutamate agonists and D-aspartate on oxytocin release from hypothalamus and posterior pituitary of male rats.

In order to determine whether ionotropic (iGluRs) and metabotropic (mGluRs) glutamate receptor activation modulates oxytocin release in male rats, we investigated the effect of agonists of both types of glutamate receptors on oxytocin release from hypothalamus and posterior pituitary. Kainate and quisqualate (1 mM) increased hypothalamic oxytocin release. Their effects were prevented by selective AMPA/kainate receptor antagonists. NMDA (0.01–1 mM) did not modify hypothalamic oxytocin release. Group I mGluR agonists, such as quisqualate and 3-HPG, significantly increased hypothalamic oxytocin release. These effects were blocked by AIDA (a selective antagonist of group I mGluRs). In the posterior pituitary, oxytocin release was not modified by kainate, quisqualate, trans-ACPD (a broad-spectrum mGluR agonist) and l-SOP (a group III mGluR agonist). However, NMDA (0.1 mM) significantly decreased oxytocin release from posterior pituitary. d-Aspartate significantly increased oxytocin release from the hypothalamus, while it decreased oxytocin release from posterior pituitary. AP-5 (a specific NMDA receptor antagonist) reduced the d-Aspartate effect in the hypothalamus, but not in the posterior pituitary. Our data indicate that the activation of non-NMDA receptors and group I mGluRs stimulates oxytocin release from hypothalamic nuclei, whereas NMDA inhibits oxytocinergic terminals in the posterior pituitary. d-Aspartate also has a dual effect on oxytocin release: stimulatory at the hypothalamus and inhibitory at the posterior pituitary. These results suggest that excitatory amino acids differentially modulate the secretion of oxytocin at the hypothalamic and posterior pituitary levels.

GABA-related enzymes in the hypothalamus of rats treated with estradiol

The possibility that the activity of GABA-related enzymes in the hypothalamus, glutamic acid decarboxylase (GAD) and GABA-transaminase (GABA-T), may be modified by circulating prolactin levels was investigated in castrated female and male rats. Estradiol benzoate was chosen to induce an increase, while bromergocriptine was used to obtain a decrease of serum prolactin levels. Acute treatment with estradiol benzoate brought about 3 h later, a significant increase of serum prolactin, and a slight, non-significant increase of GAD activity in the hypothalamus. Chronic treatment with estradiol benzoate for 12 days induced significant increases in serum prolactin and GAD activity in the hypothalamus. Bromergocriptine significantly blocked this increase of GAD activity. Bromergocriptine-treated rats had significant increases of GABA-T activity in the hypothalamus. The results of this investigation support the possibility of the existence of a relationship between GABA-related enzymes in the hypothalamus and circulating prolactin levels.

Mechanisms of chromium (VI)-induced apoptosis in anterior pituitary cells.

Hexavalent chromium (Cr (VI)) is a highly toxic metal. Exposure to Cr (VI) compounds may affect reproductive functions. Due to the importance of anterior pituitary hormones on reproductive physiology we have studied the effects of Cr (VI) on anterior pituitary. We previously demonstrated that, after in vivo Cr (VI) administration, Cr accumulates in the pituitary gland and affects prolactin secretion. In vitro, Cr (VI) causes apoptosis in anterior pituitary cells due to oxidative stress generation. To better understand the mechanisms involved in Cr (VI)-induced apoptosis we studied: (a) whether Cr (VI) affects the intracellular antioxidant response and (b) which of the apoptotic factors participates in Cr (VI) effect. Our results show that Cr (VI) treatment induces a decrease in catalase and glutathione peroxidase (GPx) activity but does not modify glutathione reductase (GR) activity. Cr (VI) exposure causes an increase of GSH levels. p53 and Bax mRNA are also upregulated by the metal. Pifithrin alpha, a p53 transcriptional inhibitor, increases Cr (VI) cytotoxicity, suggesting a role of p53 as a survival molecule. The antioxidant N-acetyl-cysteine (NAC) could prevent Bax mRNA increase and caspase 3 activation, confirming that Cr (VI)-induced apoptosis involves oxidative stress generation.

Effect of sex steroids on GABA receptors in the rat hypothalamus and anterior pituitary gland

Our data indicate that sex steroids modify the number of GABA receptors, as detected by a [3H]muscimol binding assay, in the tuberoinfundibular GABAergic system. GABA binding was affected by chronic hormonal treatments in different ways depending on the sex of the rats and the steroids administered. Estradiol increased GABA binding in ovariectomized female rats while testosterone decreased the number of GABA binding sites in gonadectomized male rats. These results suggest a sex difference in the regulation of hypothalamic GABA receptors.

Chromium VI administration induces oxidative stress in hypothalamus and anterior pituitary gland from male rats

Hexavalent chromium (Cr VI)-containing compounds are known carcinogens which are present in industrial settings and in the environment. The major route of chromium exposure for the general population is oral intake. Previously we have observed that Cr VI affects anterior pituitary secretion and causes oxidative stress in vitro. The aim of the present work was to investigate if in vivo Cr VI treatment (100 ppm of Cr VI in drinking water for up 30 days) causes oxidative stress in hypothalamus and anterior pituitary gland from male rats. This treatment produced a 4-fold increase of chromium content in hypothalamus and 10-fold increase in anterior pituitary gland. Lipid peroxidation showed a significant increase in hypothalamus and anterior pituitary. Cr VI augmented superoxide dismutase activity in anterior pituitary gland and glutathione reductase activity in hypothalamus, but glutathione peroxidase and catalase activities remained unchanged in both tissues. Heme oxygenase-1 mRNA expression significantly rose in both tissues. Metallothionein 1 mRNA content increased in anterior pituitary and metallothionein 3 mRNA increased in hypothalamus. These results show, for the first time, that oral chronic administration of Cr VI produces oxidative stress on the hypothalamus and anterior pituitary gland which may affect normal endocrine function.

Effect of Interleukin-6 and Tumor Necrosis Factor-α on GABA Release from Mediobasal Hypothalamus and Posterior Pituitary

The release of cytokines during infection, inflammation and stress induces brain-mediated responses, including alterations of neuroendocrine functions. We examined the effect of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) on release of γ-aminobutyric acid (GABA) from mediobasal hypothalamic (MBH) explants and posterior pituitaries (PP) of male rats. IL-6 (10 ng/ml) did not modify basal GABA release from MBH and PP, but significantly increased GABA release under depolarizing conditions (40 mM K+). This effect was abolished by incubation of the tissue with indomethacin, an inhibitor of cyclooxygenase activity, indicating that prostaglandins could mediate the stimulation of GABA release induced by IL-6. On the contrary, TNF-α (50 ng/ml) significantly decreased K+-evoked GABA release from both MBH and PP. This inhibitory effect was not modified by indomethacin. Neither IL-6 nor TNF-α affected nitric oxide synthesis, as measured by [14C]citrulline production. The current results indicate that IL-6 stimulates GABA release from both hypothalamus and posterior pituitary by a mechanism mediated by prostaglandins. On the contrary, TNF-α inhibits GABA release from both tissues. These results suggest the possibility that GABAergic activity in the hypothalamic-pituitary axis could be involved in neuroendocrine responses to cytokines.

Effects of Passive Immunization against Vasoactive Intestinal Peptide on Serum Prolactin and LH Levels

Recent findings suggest that vasoactive intestinal peptide (VIP) may be a physiological regulator of prolactin secretion and may also be involved in the control of LH secretion. In the present work we have studied the effect of the blockade of endogenous VIP by means of the injection of a specific rabbit anti-VIP serum, in male and female rats with hyperprolactinemia. The administration of 0.5 ml of the VIP antiserum in ovariectomized rats given an acute or chronic treatment with 17 beta-estradiol induced a significant decrease in serum prolactin and LH levels as compared with estrogenized-control rats injected with normal rabbit serum. Anti-VIP serum also reduced serum LH levels in ovariectomized rats not treated with estrogens. The administration of the same antiserum decreased serum prolactin levels in male rats implanted with 2 anterior pituitary glands under the kidney capsule. On the other hand, the injection of the anti-VIP serum in the morning in proestrus rats brought about an increase in serum prolactin and LH levels in the afternoon of the same day. These results confirm previous data showing that VIP is a stimulator of prolactin release, and may also participate in the control of LH secretion in ovariectomized rats acting as a facilitatory factor. During proestrus however, VIP may act in an opposite way, inhibiting, rather than stimulating, prolactin and LH release.

Effect of lipopolysaccharide on tumor necrosis factor and prolactin release from rat anterior pituitary cells.

TNF-α plays a critical role in the cascade of neuroendocrine events during inflammation and septic shock. It also affects the release of pituitary hormones and acts as a growth factor in immune and nonimmune cells. The aim of the present study was to investigate the release of TNF-α from rat anterior pituitary cells and the effect of the steroid medium on its release. Cultured anterior pituitary cells from lactating rats spontaneously released TNF-α. The presence of lipopolysaccharide (LPS, 0.1 µg/mL) in the culture medium significantly increased TNF-α release and inhibited prolactin release. Chronic estrogenization of ovariectomized rats or the presence of 17 β-estradiol in the culture medium also increased TNF-α release. LPS significantly stimulated TNF-α release in all groups and abrogated the estrogen-induced prolactin release. We also investigated the effect of TNF-α on prolactin release. The presence of TNF-α (50 ng/mL) in the culture medium inhibited prolactin release from anterior pituitary cells. These data show that anterior pituitary cells in culture release TNF-α and that this release is stimulated by estrogens. Our results also indicate that LPS inhibits prolactin release in an estrogenic environment, suggesting that TNF-α could affect pituitary hormone release during endotoxemia.