In vivo xenoestrogenic actions of cadmium and arsenic in anterior pituitary and uterus.

Cadmium (Cd) and arsenic (iAs) are toxic metals ubiquitously present in the environment. Both pollutants exert nonmonotonic dose responses, being mostly cytotoxic at high concentrations but mimicking estrogen (E2) effects at low doses. Xenoestrogenic activity of Cd and iAs has been demonstrated in different hormone-dependent tumor cell lines; however, their actions in vivo remain largely unknown. Here, we investigated whether in vivo administration of low doses of Cd and iAs through drinking water would display xenoestrogenic effects in the anterior pituitary gland and uterus of ovariectomized rats. Cd (1ppm) and iAs (0.1ppm) exposure increased the wet weight of anterior pituitary gland and uterus and induced proestrus- and estrus-like vaginal smears. Both metals stimulate cell proliferation of these tissues as they increased the expression of proliferation markers. More importantly, they augmented soluble guanylyl cyclase α1 subunit expression, which has been linked to hormone-dependent tumor progression. Also, Cd and iAs modified protein levels of full-length estrogen receptor α and its truncated variants in an E2-like manner. Anterior pituitary hormone secretion was differentially affected by both metals. Luteinizing hormone synthesis and release were strongly diminished after Cd exposure and only mildly reduced by iAs. Both metals were able to increase prolactin synthesis, although only iAs augmented serum prolactin levels. This study shows for the first time that Cd and iAs exert strong xenoestrogenic effects on anterior pituitary gland at low doses. The differences between Cd and iAs E2-like behavior indicate that other Cd- and iAs-specific mechanisms could be involved. Altogether, these results contribute to the knowledge of reproductive disorders associated with Cd and iAs environmental contamination.

In Vivo and In Vitro Arsenic Exposition Induces Oxidative Stress in Anterior Pituitary Gland.

Inorganic arsenic (iAs) is at the top of toxic metalloids. Inorganic arsenic-contaminated water consumption is one of the greatest environmental health threats worldwide. Human iAs exposure has been associated with cancers of several organs, neurological disorders, and reproductive problems. Nevertheless, there are no reports describing how iAs affects the anterior pituitary gland. The aim of this study was to investigate the mechanisms involved in iAs-mediated anterior pituitary toxicity both in vivo and in vitro. We showed that iAs administration (from 5 to 100 ppm) to male rats through drinking water increased messenger RNA expression of several oxidative stress-responsive genes in the anterior pituitary gland. Serum prolactin levels diminished, whereas luteinizing hormone (LH) levels were only affected at the higher dose tested. In anterior pituitary cells in culture, 25 µmol/L iAs significantly decreased prolactin release in a time-dependent fashion, whereas LH levels remained unaltered. Cell viability was significantly reduced mainly by apoptosis evidenced by morphological and phosphatidylserine externalization studies. This process is characterized by early depolarization of mitochondrial membrane potential and increased levels of reactive oxygen species. Expression of some key oxidative stress-responsive genes, such as heme oxygenase-1 and metallothionein-1, was also stimulated by iAs exposure. The antioxidant N-acetyl cysteine prevented iAs-induced effects on the expression of oxidative stress markers, prolactin release, and apoptosis. In summary, the present work demonstrates for the first time that iAs reduces prolactin release both in vivo and in vitro and induces apoptosis in anterior pituitary cells, possibly resulting from imbalanced cellular redox status.

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.

Soluble Guanylyl Cyclase Alpha1 Subunit: A New Marker for Estrogenicity of Endocrine Disruptor Compounds.

Endocrine-disrupting chemicals (EDCs) include widespread naturally occurring and synthetic substances in the environment that adversely affect humans and wildlife. Because of the increasing numbers of EDCs, screening methods and ideal biomarkers to determine EDC potencies at relevant environmental concentrations need to be drastically improved. Soluble guanylyl cyclase α1 subunit (sGCα1) is an abundant cytosolic protein ubiquitously expressed in most tissues. We previously showed that sGCα1 is specifically and highly up-regulated by estrogen (E2) in vivo and in vitro, even though it lacks estrogen-responsive elements. The aim of the present study was to evaluate sGCα1 protein expression as a potential marker for xenoestrogenic EDC exposure in the E2-responsive lactosomatotroph-derived pituitary cell line GH3. Cells were incubated with a wide variety of EDCs such as heavy metals and a metalloid, synthetic E2 derivatives, plastic byproducts, and pesticides at a range of doses including those with proven xenoestrogenic activity. We demonstrated that E2 increased sGCα1 expression in GH3 cells as well as in other E2-responsive tumor cell lines. Moreover, this effect was fully dependent on estrogen receptor (ER) activation. Importantly, sGCα1 protein levels were strongly up-regulated by all the EDCs tested, even by those exhibiting low or null ER binding capacity. We provide evidence that the in vitro sGCα1 protein assay may be a very sensitive and powerful tool to identify compounds with estrogenic activity, which could improve current mammalian-based screening methods. Environ Toxicol Chem 2019;38:2719-2728. © 2019 SETAC.

Soluble guanylyl cyclase α1 subunit is a key mediator of proliferation, survival, and migration in ECC-1 and HeLa cell lines.

Soluble guanylyl cyclase (sGC) is a heterodimeric enzyme constituted by two subunits, α1 and ß1. Previously we have shown that 17ß-estradiol (E2) exerts opposite effects on these subunits by increasing α1 and decreasing both ß1 expression and enzymatic activity. To date, the physiological relevance of E2-induced sGC subunits' imbalance has not been addressed. Also, increased levels strongly correlate with E2-induced proliferation in E2-dependent tissues. The aim of the present study was to investigate the role of sGCα1 in proliferation, survival, and migration in two E2-responsive and non-responsive tumour cell lines. Here we showed that E2 stimulated sGCα1 expression in ECC-1 endometrial cancer cells. sGCα1 knock-down significantly reduced E2-dependent cell proliferation. Moreover, sGCα1 silencing caused G1 arrest together with an increase in cell death and dramatically inhibited cell migration. Surprisingly, disruption of sGCα1 expression caused a similar effect even in absence of E2. Confirming this effect, sGCα1 knock-down also augmented cell death and decreased proliferation and migration in E2-unresponsive HeLa cervical cancer cells. Our results show that sGCα1 mediated cell proliferation, survival, and migration in ECC-1 and HeLa cells and suggest that sGCα1 can not only mediate E2-tumour promoting effects but can also be involved in hormone-independent tumour progression.

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.

17 beta-estradiol modifies nitric oxide-sensitive guanylyl cyclase expression and down-regulates its activity in rat anterior pituitary gland.

Previous studies showed that 17 beta-estradiol (17 beta-E2) regulates the nitric oxide (NO)/soluble guanylyl cyclase (sGC)/cGMP pathway in many tissues. Evidence from our laboratory indicates that 17 beta-E2 disrupts the inhibitory effect of NO on prolactin release, decreasing sGC activity and affecting the cGMP pathway in anterior pituitary gland of adult ovariectomized and estrogenized rats. To ascertain the mechanisms by which 17 beta-E2 affects sGC activity, we investigated the in vivo and in vitro effects of 17 beta-E2 on sGC protein and mRNA expression in anterior pituitary gland from immature female rats. In the present work, we showed that 17 beta-E2 acute treatment exerted opposite effects on the two sGC subunits, increasing alpha1 and decreasing beta1 subunit protein and mRNA expression. This action on sGC protein expression was maximal 6-9 h after 17 beta-E2 administration. 17beta-E2 also caused the same effect on mRNA expression at earlier times. Concomitantly, 17 beta-E2 dramatically decreased sGC activity 6 and 9 h after injection. These effects were specific of 17 beta-E2, because they were not observed with the administration of other steroids such as progesterone and 17 alpha-estradiol. This inhibitory action of 17beta-E2 on sGC also required the activation of estrogen receptor (ER), because treatment with the pure ER antagonist ICI 182,780 completely blocked 17 beta-E2 action. 17 beta-E2 acute treatment caused the same effects on pituitary cells in culture. These results suggest that 17 beta-E2 exerts an acute inhibitory effect on sGC in anterior pituitary gland by down-regulating sGC beta 1 subunit and sGC activity in a specific, ER-dependent manner.

Nitric oxide protects the mitochondria of anterior pituitary cells and prevents cadmium-induced cell death by reducing oxidative stress.

Cadmium (Cd2+) is a highly toxic metal that affects the endocrine system. We have previously shown that Cd2+ induces caspase-3 activation and apoptosis of anterior pituitary cells and that endogenous nitric oxide (NO) protects these cells from Cd2+. Here we investigate the mechanisms by which NO exerts this protective role. Cd2+ (25 microM) reduced the mitochondrial membrane potential (MMP) as measured by flow cytometry. Cd2+-induced apoptosis was mitochondrial dependent since cyclosporin A protected the cells from this metal. Inhibition of NO synthesis with 0.5 mM L-NAME increased the effect of Cd2+ on MMP, whereas the NO donor DETANONOate (0.1 mM) reduced it. Cd2+ increased the production of reactive oxygen species (ROS) as measured by flow cytometry. This effect was electron-transfer-chain-dependent since it was inhibited by rotenone. In fact, rotenone reduced the cytotoxic effect of the metal. The action of Cd2+ on mitochondrial integrity was ROS dependent. Trolox, an antioxidant, inhibited the effect of the metal on the MMP. Cd2+-induced increase in ROS generation was reduced by DETANONOate. There are discrepancies concerning the role of NO in Cd2+ toxicity. Here we show that NO reduces Cd2+ toxicity by protecting the mitochondria from oxidative stress in a system where NO plays a regulatory role.

Nitric Oxide Plays a Key Role in Ovariectomy-Induced Apoptosis in Anterior Pituitary: Interplay between Nitric Oxide Pathway and Estrogen.

Changes in the estrogenic status produce deep changes in pituitary physiology, mainly because estrogens (E2) are one of the main regulators of pituitary cell population. Also, E2 negatively regulate pituitary neuronal nitric oxide synthase (nNOS) activity and expression and may thereby modulate the production of nitric oxide (NO), an important regulator of cell death and survival. Little is known about how ovary ablation affects anterior pituitary cell remodelling and molecular mechanisms that regulate this process have not yet been elucidated. In this work we used freshly dispersed anterior pituitaries as well as cell cultures from ovariectomized female rats in order to study whether E2 deficiency induces apoptosis in the anterior pituitary cells, the role of NO in this process and effects of E2 on the NO pathway. Our results showed that cell activity gradually decreases after ovariectomy (OVX) as a consequence of cell death, which is completely prevented by a pan-caspase inhibitor. Furthermore, there is an increase of fragmented nuclei and DNA cleavage thereby presenting the first direct evidence of the existence of apoptosis in the anterior pituitary gland after OVX. NO production and soluble guanylyl cyclase (sGC) expression in anterior pituitary cells increased concomitantly to the apoptosis. Inhibition of both, NO synthase (NOS) and sGC activities prevented the drop of cell viability after OVX, showing for the first time that increased NO levels and sGC activity observed post-OVX play a key role in the induction of apoptosis. Conversely, E2 and prolactin treatments decreased nNOS expression and activity in pituitary cells from OVX rats in a time- and E2 receptor-dependent manner, thus suggesting interplay between NO and E2 pathways in anterior pituitary.

Nitric oxide protects anterior pituitary cells from cadmium-induced apoptosis.

Cadmium (Cd2+) is a potent toxic metal for both plants and animals. Chronic exposure to low doses of Cd2+ results in damage to several organs. We have previously reported that Cd2+ induces apoptosis in anterior pituitary cells by a caspase- and oxidative stress-dependent mechanism. Nitric oxide (NO) synthesis is affected by Cd2+ in several systems. NO has been shown to be either cytoprotective or cytotoxic in many systems. The aim of this study was to evaluate the possible participation of NO in the cytotoxic effect of Cd2+ on rat anterior pituitary cells. Cell viability was evaluated by mitochondrial dehydrogenase activity assay and confirmed by microscopy, studying nuclear morphology. Here we show that DETA NONOate ((Z)-1-[2 (2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate), a long-term NO donor, at concentrations below 0.5 mM, reduces nuclear condensation and fragmentation and reverses the decrease in cellular activity induced by Cd2+. Cd2+, by itself, induced NO synthesis, and inhibition of this synthesis enhanced Cd2+ cytotoxicity. NO also prevented caspase-3 activation and lipidic peroxidation induced by Cd2+. The NO/cGMP pathway does not seem to be involved in the cytoprotective effect of NO. These results indicate that NO has a cytoprotective role in Cd2+ -induced apoptosis, suggesting that endogenous NO could have a physiological role in protecting anterior pituitary cells.

Nitric oxide decreases the production of inositol phosphates stimulated by angiotensin II and thyrotropin-releasing hormone in anterior pituitary cells.

OBJECTIVE: Nitric oxide (NO) affects the synthesis of several second messengers, such as cyclic nucleotides, arachidonic acid metabolites and the intracellular calcium concentration, involved in the anterior pituitary hormone release. The present study was performed to investigate the effect of NO on phosphoinositide metabolism. METHODS: The synthesis of inositol phosphates (IPs) was studied in primary cultures of anterior pituitary cells from Wistar male rats. IPs (mono, bis and tris phosphates) were determined by ionic exchange chromatography. RESULTS: Sodium nitroprusside and DETA NONOate (DETA/NO) significantly decreased IP synthesis and prolactin release stimulated by angiotensin II (AngII) and thyrotropin-releasing hormone (TRH). These effects were not observed with decayed DETA NONOate (unable to release NO). LY-83583, a guanylyl cyclase inhibitor, completely reversed the inhibitory effect of DETA/NO on AngII-induced IP production. However, BAY 41-2272, a novel stimulator of the soluble guanylyl cyclase, did not mimic the effect of NO donors. Likewise, neither 8-Bromine-cyclic GMP (8-Br-cGMP), an analog of cGMP, nor Sp-8-pCPT-cGMPS triethylamine, a cGMP-dependent protein kinase (PKG) stimulator, decreased IP synthesis stimulated by AngII. In addition, Rp-8-pCPT-cGMPS triethylamine, a PKG inhibitor, did not block the effect of NO. The decrease of IPs induced by DETA/NO was fully reversed by guanosine 5'-O-(3-thiotriphosphate) tetralithium salt, a non-hydrolyzable analog of GTP. CONCLUSIONS: The present work indicated that NO decreases IP synthesis stimulated by Ang II and TRH in anterior pituitary cells by a soluble guanylyl cyclase/cGMP/PKG-independent pathway, and suggested that NO affects some regulatory factor located between the plasma membrane receptor and G-protein.

The effect of D-aspartate on luteinizing hormone-releasing hormone, alpha-melanocyte-stimulating hormone, GABA and dopamine release.

Since D-aspartate stimulates prolactin and LH release, our objective was to determine whether D-aspartate modifies the release of hypothalamic and posterior pituitary factors involved in the control of their secretion and whether its effects on these tissues are exerted through NMDA receptors and mediated by nitric oxide. In the hypothalamus, D-aspartate stimulated luteinizing hormone-releasing hormone (LHRH), alpha-melanocyte-stimulating hormone (alpha-MSH) and GABA release and inhibited dopamine release through interaction with NMDA receptors. It increased nitric oxide synthase (NOS) activity, and its effects on LHRH and hypothalamic GABA release were blunted when NOS was inhibited. In the posterior pituitary gland, D-aspartate inhibited GABA release but had no effect on dopamine or alpha-MSH release. We report that D-aspartate differentially affects the release of hypothalamic and posterior pituitary factors involved in the regulation of pituitary hormone secretion.

The activation of metabotropic glutamate receptors differentially affects GABA and alpha-melanocyte stimulating hormone release from the hypothalamus and the posterior pituitary of male rats.

The aim of the present study was to investigate the effect of metabotropic glutamate receptor (mGluR) activation on gamma-aminobutyric acid (GABA) and alpha-melanocyte stimulating hormone (alpha-MSH) release from hypothalamic fragments and posterior pituitaries. The actions of a number of subtype-selective mGluR agonists were monitored. A group I mGluR agonist, (S)-3-hydroxyphenylglycine (3-HPG; 0.5 mM), decreased K+-induced hypothalamic GABA release. (RS)-1-Aminoindan-1,5-dicarboxylic acid (AIDA), a specific group I mGluR antagonist (0.2 mM), blocked the effect of 3-HPG. (2S, 1'S, 2'S)-2-(Carboxycyclopropyl) glycine (L-CCG-I) and L-serine-O-phosphate (L-SOP; 0.01-1 mM), agonists of group II and III mGluRs, respectively, did not modify hypothalamic evoked GABA release. Group I mGluR activation decreased, whereas group III increased and group II induced no changes in GABA release from the posterior pituitary. 3-HPG (1 mM) and L-CCG-I (0.1 mM) decreased, whereas L-SOP (0.01-0.1 mM) did not change alpha-MSH release from hypothalamic fragments. No agonists of the three mGluR groups modified alpha-MSH release from the posterior pituitary. These results indicate that activation of mGluRs differentially affects GABA and alpha-MSH release from the hypothalamus and the posterior pituitary.

Daily changes of GABA and taurine concentrations in various hypothalamic areas are affected by chronic hyperprolactinemia.

This study was designed to characterize, in anterior, mediobasal, and posterior hypothalamic and median eminence, the 24h changes of gamma aminobutyric acid (GABA) and taurine (TAU) contents in adult male rats and to analyze whether chronic hyperprolactinemia may affect these patterns. Rats were turned hyperprolactinemic by a pituitary graft. Plasma prolactin (PRL) levels increased after pituitary grafting at all time points examined. A disruption of the circadian rhythm was observed in pituitary-grafted rats, whereas GABA and TAU content followed daily rhythms in all areas studied in controls. In the mediobasal hypothalamus, two peaks for each amino acid were found at midnight and midday. In the anterior hypothalamus, GABA and TAU showed only one peak of concentration at midnight. In the posterior hypothalamus, the values of both GABA and TAU were higher during the light as compared to the dark phase of the photoperiod. In the median eminence GABA content peaked at 20:00h, the time when TAU exhibited the lowest values. Hyperprolactinemia abolished the 24h changes of GABA in the mediobasal hypothalamus and reduced its content as compared to controls. Hyperprolactinemia advanced the diurnal peak of TAU to 12:00h in the mediobasal hypothalamus and did not modify the 24:00h peak. In the anterior hypothalamus, hyperprolactinemia increased GABA and TAU contents during the light phase while it decreased them during the dark phase of the photoperiod. In the posterior hypothalamus hyperprolactinemia did not modify GABA or TAU patterns as compared to controls. In the median eminence hyperprolactinemia increased the 20:00h peak of GABA and shift advanced the decrease in TAU content at 20:00h and its maximum at 24:00h as compared to controls. These data show that GABA and TAU content exhibit specific daily patterns in each hypothalamic region studied. PRL differentially affects the daily pattern of these amino acids in each hypothalamic region analyzed.

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 17ß-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.

Nitric oxide-sensitive guanylyl cyclase is differentially regulated by nuclear and non-nuclear estrogen pathways in anterior pituitary gland.

17ß-estradiol (E2) regulates hormonal release as well as proliferation and cell death in the pituitary. The main nitric oxide receptor, nitric oxide sensitive- or soluble guanylyl cyclase (sGC), is a heterodimer composed of two subunits, α and ß, that catalyses cGMP formation. α1ß1 is the most abundant and widely expressed heterodimer, showing the greater activity. Previously we have shown that E2 decreased sGC activity but exerts opposite effects on sGC subunits increasing α1 and decreasing ß1 mRNA and protein levels. In the present work we investigate the mechanisms by which E2 differentially regulates sGC subunits' expression on rat anterior pituitary gland. Experiments were performed on primary cultures of anterior pituitary cells from adult female Wistar rats at random stages of estrous cycle. After 6 h of E2 treatment, α1 mRNA and protein expression is increased while ß1 levels are down-regulated. E2 effects on sGC expression are partially dependent on de novo transcription while de novo translation is fully required. E2 treatment decreased HuR mRNA stabilization factor and increased AUF1 p37 mRNA destabilization factor. E2-elicited ß1 mRNA decrease correlates with a mRNA destabilization environment in the anterior pituitary gland. On the other hand, after 6 h of treatment, E2-BSA (1 nM) and E2-dendrimer conjugate (EDC, 1 nM) were unable to modify α1 or ß1 mRNA levels, showing that nuclear receptor is involved in E2 actions. However, at earlier times (3 h), 1 nM EDC causes a transient decrease of α1 in a PI3k-dependent fashion. Our results show for the first time that E2 is able to exert opposite actions in the anterior pituitary gland, depending on the activation of classical or non-classical pathways. Thus, E2 can also modify sGC expression through membrane-initiated signals bringing to light a new point of regulation in NO/sGC pathway.

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

Cadmium (Cd(2+)) is one of the most important environmental contaminants and acts as an endocrine disruptor. Previously, we have demonstrated that the simultaneous administration of Cd(2+) 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 Cd(2+) (5 parts per million [ppm], 30 days) were treated with Mel or without the metal during the next 1 or 2 months. Cd(2+) 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 Cd(2+). This effect was also observed 1 month after metal removal. The Cd(2+)-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 Cd(2+) intoxication become evident only after a certain period of metal accumulation, these results show that metal removal is enough to reverse Cd(2+) effects in anterior pituitary gland and bring to light the relevance of moving away the individual from the contamination source.

Nitric oxide sensitive-guanylyl cyclase subunit expression changes during estrous cycle in anterior pituitary glands.

17beta-estradiol (E2) exerts inhibitory actions on the nitric oxide pathway in rat adult pituitary glands. Previously, we reported that in vivo E2 acute treatment had opposite effects on soluble guanylyl cyclase (sGC) subunits, increasing alpha1- and decreasing beta1-subunit protein and mRNA expression and decreasing sGC activity in immature rats. Here we studied the E2 effect on sGC protein and mRNA expression in anterior pituitary gland from adult female rats to address whether the maturation of the hypothalamus-pituitary axis influences its effects and to corroborate whether these effects occur in physiological conditions such as during estrous cycle. E2 administration causes the same effect on sGC as seen in immature rats, and these effects are estrogen receptor dependent. These results suggest that E2 is the main effector of these changes. Since the sGC alpha-subunit increases while the sGC activity decreases, we studied if other less active isoforms of the sGC alpha-subunit are expressed. Here we show for the first time that sGCalpha2 and sGCalpha2 inhibitory (alpha2i) isoforms are expressed in this gland, but only sGCalpha2i mRNA increased after E2 acute treatment. Finally, to test whether E2 effects take place under a physiological condition, sGC subunit expression was monitored over estrous cycle. sGCalpha1, -beta1, and -alpha2i fluctuate along estrous cycle, and these changes are directly related with E2 level fluctuations rather than to NO level variations. These findings show that E2 physiologically regulates sGC expression and highlight a novel mechanism by which E2 downregulates sGC activity in rat anterior pituitary gland.

Reactive oxygen species are key mediators of the nitric oxide apoptotic pathway in anterior pituitary cells.

We previously showed that long-term exposure of anterior pituitary cells to nitric oxide (NO) induces apoptosis. The intracellular signals underlying this effect remained unclear. In this study, we searched for possible mechanisms involved in the early stages of the NO apoptotic cascade. Caspase 3 was activated by NO with no apparent disruption of mitochondrial membrane potential. NO caused a rapid increase of reactive oxygen species (ROS), and this increase seems to be dependent of mitochondrial electron transport chain. The antioxidant N-acetyl-cysteine avoided ROS increase, prevented the NO-induced caspase 3 activation, and reduced the NO apoptotic effect. Catalase was inactivated by NO, while glutathione peroxidase (GPx) activity and reduced glutathione (GSH) were not modified at first, but increased at later times of NO exposure. The increase of GSH level is important for the scavenging of the NO-induced ROS overproduction. Our results indicate that ROS have an essential role as a trigger of the NO apoptotic cascade in anterior pituitary cells. The permanent inhibition of catalase may strengthen the oxidative damage induced by NO. GPx activity and GSH level augment in response to the oxidative damage, though this increase seems not to be enough to rescue the cells from the NO effect.

In vivo and in vitro effects of chromium VI on anterior pituitary hormone release and cell viability.

Hexavalent chromium (Cr VI) is a highly toxic metal and an environmental pollutant. Different studies indicate that Cr VI exposure adversely affects reproductive functions. This metal has been shown to affect several tissues and organs but Cr VI effects on pituitary gland have not been reported. Anterior pituitary hormones are central for the body homeostasis and have a fundamental role in reproductive physiology. The aim of this study was to evaluate the effect of Cr VI at the pituitary level both in vivo and in vitro. We showed that Cr VI accumulates in the pituitary and hypothalamus, and decreases serum prolactin levels in vivo but observed no effects on LH levels. In anterior pituitary cells in culture, the effect of Cr VI on hormone secretion followed the same differential pattern. Besides, lactotrophs were more sensitive to the toxicity of the metal. As a result of oxidative stress generation, Cr VI induced apoptosis evidenced by nuclear fragmentation and caspase 3 activation. Our results indicate that the anterior pituitary gland can be a target of Cr VI toxicity in vivo and in vitro, thus producing a negative impact on the hypothalamic-pituitary-gonadal axis and affecting the normal endocrine function.