24-Hour rhythm in gene expression of nitric oxide synthase and heme-peroxidase in anterior pituitary of ethanol-fed rats.

Chronic exposure of rats to ethanol results in significant changes in pituitary hormone secretion. However, identification of the site(s) and mechanism of action of ethanol to induce these effects remains elusive. Free radical damage at the adenohypophyseal level may play a role in the decline in serum gonadotropin levels in ethanol-fed rats. Since 24-h changes in redox state occurred, we analyzed the 24-h changes in pituitary gene expression of the prooxidant enzymes nitric oxide synthase (NOS) 1 and 2, and of heme oxygenase-1 (HO-1) enzyme, and in plasma NO(2)(-) and NO(3)(-) (NO(x)) levels, in ethanol and control rats. Male rats, 35-day-old, received a liquid diet for 4 weeks. The ethanol-fed group received a similar diet to controls except for that maltose was isocalorically replaced by ethanol. Animals were killed at six time intervals during a 24-h cycle. Anterior pituitary mRNA levels encoding NOS1, NOS2 and HO-1 were measured by real-time PCR analysis. Plasma NO(x) concentration was determined by the Griess reaction. Ethanol feeding of prepubertal rats changed significantly the 24-h pattern of expression of NOS1, NOS2 and HO-1 in the adenohypophysis and augmented NOS2 and HO-1 mRNA levels. Peak values for the three enzymes in ethanol-fed rats occurred at the beginning of the scotophase (i.e., at 21:00 h). Ethanol feeding augmented mean values plasma NO(x) levels with a maximum at 13:00 h while in controls a biphasic pattern was observed, with peaks at 09:00 h and 17:00-21:00 h. One of the mechanisms by which ethanol augments oxidative damage in the adenohypophysis may include overproduction of nitric oxide and carbon monoxide.

Cadmium-induced changes in Per 1 and Per 2 gene expression in rat hypothalamus and anterior pituitary: effect of melatonin.

Chronic cadmium (Cd) administration affects the circadian release of pituitary hormones in rats. To assess whether Cd modifies expression of two major clock genes, period (Per) 1 and Per 2, in the hypothalamic-pituitary unit and to what extent the changes could be prevented by melatonin, rats were exposed to CdCl(2) (5ppm in drinking water) with or without melatonin (3 microg/mL drinking water) for 1 month and were killed at two time intervals, i.e. a the beginning of the rest span (09:00h) and at the middle of the activity span (01:00h). Hypothalamic and pituitary mRNA levels encoding Per 1 and Per 2 were measured by real-time PCR analysis. Cd treatment decreased expression of hypothalamic Per 1 gene at both time intervals, of hypothalamic Per 2 gene at 01:00h, and of adenohypophysial Per 1 and Per 2 genes at 09:00h. Melatonin administration counteracted most of the effects of Cd and augmented hypothalamic Per 2, and adenohypophysial Per 1 and Per 2 gene expression. The results indicate that Cd administered chronically in the drinking water to rats affected expression of clock genes in the hypothalamic-pituitary unit, an effect prevented by melatonin.

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 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.

Modulation of clock gene expression by the transcriptional coregulator receptor interacting protein 140 (RIP140).

Circadian rhythms are generated in central and peripheral tissues by an intracellular oscillating timing mechanism known as the circadian clock. Several lines of evidence show a strong and bidirectional interplay between metabolism and circadian rhythms. Receptor interacting protein 140 (RIP140) is a coregulator for nuclear receptors and other transcription factors that represses catabolic pathways in metabolic tissues. Although RIP140 functions as a corepressor for most nuclear receptors, mounting evidence points to RIP140 as a dual coregulator that can repress or activate different sets of genes. Here, we demonstrate that RIP140 mRNA and protein levels are under circadian regulation and identify RIP140 as a modulator of clock gene expression, suggesting that RIP140 can participate in a feedback mechanism affecting the circadian clock. We show that the absence of RIP140 disturbs the basal levels of BMAL1 and other clock genes, reducing the amplitude of their oscillations. In addition, we demonstrate that RIP140 is recruited to retinoid-related orphan receptor (ROR) binding sites on the BMAL1 promoter, directly interacts with RORα, and increases transcription from the BMAL1 promoter in a RORα-dependent manner. These results indicate that RIP140 is not only involved in metabolic control but also acts as a coactivator for RORα, influencing clock gene expression.

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.

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

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 microg/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.

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.