Neuropeptides and enzymes are targets for the action of endocrine disrupting chemicals in the vertebrate brain.

Endocrine-disrupting chemicals (EDC) are molecules that interfere with endocrine signaling pathways and produce adverse consequences on animal and human physiology, such as infertility or behavioral alterations. Some EDC act through binding to androgen or/and estrogen receptors primarily operating through a genomic mechanism regulating gene expression. This mechanism of action may induce profound developmental adverse effects, and the major targets of the EDC action are the gene products, i.e., mRNAs inducing the synthesis of various peptidic molecules, which include neuropeptides and enzymes related to neurotransmitters syntheses. Available immunohistochemical data on some of the systems that are affected by EDC in lower and higher vertebrates are detailed in this review.

Merkel cells and permanent disesthesia in the oral mucosa after soft tissue grafts.

Connective tissue grafts are routinely procedures in the treatment of gingival defects. The clinical success of the gingival tissue graft procedures anyway should ensure not only the aesthetic integration between the tissues but also the physiological activity of the graft in terms of sensitivity and immunity because the skin and the mucosae constitute the first natural aspecific borders against pathogens. The aim of this paper was to investigate nervous net recovery after connective graft procedure, in relation with sensorial alteration in the injured area. Results showed that there is a close link among the number of Merkel cells and the alteration of sensations. Merkel cells can be found isolated standing in the basal layer, supposed to have neuroendocrine functions in the epithelia or in larger group not associated with nerves; when found in association with nerves they are named Merkel complexes, acting as slow adapter mechanical receptor. Our data can be explained in two ways: Merkel cells increase as a consequence of tissue injury, a sort of "SOS cells" that secrete neuroendocrine signals to guide tissue healing; as an alternative the presence of the Merkel cells could be read as a derailment of tissue regeneration with the stop of cellular differentiation in the direction of an abnormal proliferation, a sort of mad stem cell.

Neuroprotective effects of dihydroprogesterone and progesterone in an experimental model of nerve crush injury.

A satisfactory management to ensure a full restoration of peripheral nerve after trauma is not yet available. Using an experimental protocol, in which crush injury was applied 1 cm above the bifurcation of the rat sciatic nerve for 20 s, we here demonstrate that the levels of neuroactive steroids, such as pregnenolone and progesterone (P) metabolites (i.e. dihydroprogesterone, DHP, and tetrahydroprogesterone, THP) present in injured sciatic nerve were significantly decreased. On this basis, we have focused our attention on DHP and its direct precursor, P, analyzing whether these two neuroactive steroids may have neuroprotective effects on biochemical, functional and morphological alterations occurring during crush-induced degeneration-regeneration. We demonstrate that DHP and/or P counteract biochemical alterations (i.e. myelin proteins and Na(+),K(+)-ATPase pump) and stimulate reelin gene expression. These two neuroactive steroids also counteract nociception impairment, and DHP treatment significantly decreases the up-regulation of myelinated fibers' density occurring in crushed animals. Altogether, these observations suggest that DHP and P (i.e. two neuroactive steroids interacting with progesterone receptor) may be considered protective agents in case of nerve crush injury.

Early postnatal genistein administration permanently affects nitrergic and vasopressinergic systems in a sex-specific way.

Genistein (GEN) is a natural xenoestrogen (isoflavonoid) that may interfere with the development of estrogen-sensitive neural circuits. Due to the large and increasing use of soy-based formulas for babies (characterized by a high content of GEN), there are some concerns that this could result in an impairment of some estrogen-sensitive neural circuits and behaviors. In a previous study, we demonstrated that its oral administration to female mice during late pregnancy and early lactation induced a significant decrease of nitric oxide synthase-positive cells in the amygdala of their male offspring. In the present study, we have used a different experimental protocol mimicking, in mice, the direct precocious exposure to GEN. Mice pups of both sexes were fed either with oil, estradiol or GEN from birth to postnatal day 8. Nitric oxide synthase and vasopressin neural systems were analyzed in adult mice. Interestingly, we observed that GEN effect was time specific (when compared to our previous study), sex specific, and not always comparable to the effects of estradiol. This last observation suggests that GEN may act through different intracellular pathways. Present results indicate that the effect of natural xenoestrogens on the development of the brain may be highly variable: a plethora of neuronal circuits may be affected depending on sex, time of exposure, intracellular pathway involved, and target cells. This raises concern on the possible long-term effects of the use of soy-based formulas for babies, which may be currently underestimated.

Effects of gonadal hormones on central nitric oxide producing systems.

Nitric oxide-containing neurons are widely distributed within the CNS, including regions involved in the control of reproduction and sexual behavior. The expression of neuronal nitric oxide synthase is influenced by testosterone in male rat, and by estrogens in female. Moreover, nitric oxide synthase may co-localize with gonadal hormones' receptors. Gonadal hormones may influence nitric oxide synthase expression in adulthood as well as during the development. In fact, in mice knockout for estrogen receptor alpha, the nitric oxide synthase-expressing population is deeply reduced in specific regions. In physiological conditions, the female in mammalian species is exposed to short-term changes of gonadal hormones levels (estrous cycle). Our recent studies, performed in the rat vomeronasal system and in mouse hypothalamic and limbic systems reveal that, in rodents, the expression of nitric oxide synthase-producing elements within regions relevant for the control of sexual behavior is under the control of gonadal hormones. The expression of nitric oxide synthase may vary according to the rapid variations of hormonal levels that take place during the estrous cycle. This seems in accordance with the hypothesis that gonadal hormone activation of nitric oxide-cyclic guanosine-monophosphate pathway is important for lordosis behavior, as well as that this system is activated during mating behavior. Finally, comparative data available for other vertebrates suggest that class-specific and species-specific differences occur in the nitric oxide synthase system of hypothalamus and limbic structures. Therefore, particular caution is needed to generalize data obtained from studies in rodents.

Adult exposure to tributyltin affects hypothalamic neuropeptide Y, Y1 receptor distribution, and circulating leptin in mice.

Tributyltin (TBT), a pesticide used in antifouling paints, is toxic for aquatic invertebrates. In vertebrates, TBT may act in obesogen- inducing adipogenetic gene transcription for adipocyte differentiation. In a previous study, we demonstrated that acute administration of TBT induces c-fos expression in the arcuate nucleus. Therefore, in this study, we tested the hypothesis that adult exposure to TBT may alter a part of the nervous pathways controlling animal food intake. In particular, we investigated the expression of neuropeptide Y (NPY) immunoreactivity. This neuropeptide forms neural circuits dedicated to food assumption and its action is mediated by Y1 receptors that are widely expressed in the hypothalamic nuclei responsible for the regulation of food intake and energy homeostasis. To this purpose, TBT was orally administered at a dose of 0.025 mg/kg/day/body weight to adult animals [male and female C57BL/6 (Y1-LacZ transgenic mice] for 4 weeks. No differences were found in body weight and fat deposition, but we observed a significant increase in feed efficiency in TBT-treated male mice and a significant decrease in circulating leptin in both sexes. Computerized quantitative analysis of NPY immunoreactivity and Y1-related ß-galactosidase activity demonstrated a statistically significant reduction in NPY and Y1 transgene expression in the hypothalamic circuit controlling food intake of treated male mice in comparison with controls. In conclusion, the present results indicate that adult exposure to TBT is profoundly interfering with the nervous circuits involved in the stimulation of food intake.

Oral butyrate for mildly to moderately active Crohn's disease.

BACKGROUND: Butyrate exerts anti-inflammatory effects in experimental colitis and on Crohn's disease lamina propria mononuclear cells in vitro. AIM: To explore the efficacy and safety of oral butyrate in Crohn's disease. METHODS: Thirteen patients with mild-moderate ileocolonic Crohn's disease received 4 g/day butyrate as enteric-coated tablets for 8 weeks. Full colonoscopy and ileoscopy were performed before and after treatment. Endoscopical and histological score, laboratory data, Crohn's disease activity index and mucosal interleukin (IL)-1beta, IL-6, IL-12, interferon-gamma, tumour necrosis factor-alpha and nuclear factor-kappa B (NF-kappaB) were assessed before and after treatment. RESULTS: One patient withdrew from the study, and three patients did not experience clinical improvement. Among the nine patients (69%) who responded to treatment, seven (53%) achieved remission and two had a partial response. Endoscopical and histological score significantly improved after treatment at ileocaecal level (P < 0.05). Leucocyte blood count, erythrocyte sedimentation rate and mucosal levels of NF-kappaB and IL-1beta significantly decreased after treatment (P < 0.05). CONCLUSIONS: Oral butyrate is safe and well tolerated, and may be effective in inducing clinical improvement/remission in Crohn's disease. These data indicate the need for a large investigation to extend the present findings, and suggest that butyrate may exert its action through downregulation of NF-kappaB and IL-1beta.

Testosterone and estradiol differentially affect cell proliferation in the subventricular zone of young adult gonadectomized male and female rats.

Steroid hormones are important players to regulate adult neurogenesis in the dentate gyrus of the hippocampus, but their involvement in the regulation of the same phenomenon in the subventricular zone (SVZ) of the lateral ventricles is not completely understood. Here, in male rats, we tested the existence of activational effects of testosterone (T) on cell proliferation in the adult SVZ. To this aim, three groups of male rats: castrated, castrated and treated with T, and controls were treated with 5-bromo-2'-deoxyuridine (BrdU) and killed after 24h. The density of BrdU-labeled cells was significantly lower in castrated animals in comparison to the other two groups, thus supporting a direct correlation between SVZ proliferation and levels of circulating T. To clarify whether this effect is purely androgen-dependent, or mediated by the T metabolites, estradiol (E2) and dihydrotestosterone (DHT), we evaluated SVZ proliferation in castrated males treated with E2, DHT and E2+DHT, in comparison to T- and vehicle-treated animals, and sham-operated controls. The stereological analysis demonstrated that E2 and T, but not DHT, increase proliferation in the SVZ of adult male rats. Quantitative evaluation of cells expressing the endogenous marker of cell proliferation phosphorylated form of Histone H3 (PHH3), or the marker of highly dividing SVZ progenitors Mash1, indicated the effect of T/E2 is mostly restricted to SVZ proliferating progenitors. The same experimental protocol was repeated on ovariectomized female rats treated with E2 or T. In this case, no statistically significant difference was found among groups. Overall, our results clearly show that the gonadal hormones T and E2 represent important mediators of cell proliferation in the adult SVZ. Moreover, we show that such an effect is restricted to males, supporting adult neurogenesis in rats is a process differentially modulated in the two sexes.

Androgen receptor deficiency alters the arginine-vasopressin sexually dimorphic system in Tfm rats.

In rodents as well as in many other mammalian and non-mammalian species, the arginine-vasopressin (AVP) system includes a parvocellular sexually dimorphic portion located within the bed nucleus of the stria terminalis (BST), the medial amygdaloid nucleus (MeA) and the lateral septum. In this system, males have more cells and denser projections than females, neurons show androgen and estrogen receptors, and gonadal hormones are required for the activation. However, the role of these hormones for the differentiation of the system is not clear. Previous studies performed on aromatase knockout mice suggested that estradiol is not necessary for the differentiation of the system, but it is important for its activation in adulthood. To elucidate the role of androgens on differentiation and functioning of AVP parvocellular system, we compared male and female rats with a non-functional mutation of androgen receptor (Tfm, testicular feminization mutation) to their control littermates. Our data show that the lack of a functional androgen receptor significantly decreases the expression of AVP immunoreactivity within the BST and MeA of male Tfm. Thus supporting the hypothesis that androgens, through the action of their receptor, should have a relevant role in the organization and modulation of the AVP parvocellular sexually dimorphic system.

Effects of estrous cycle and sex on the expression of neuropeptide Y Y1 receptor in discrete hypothalamic and limbic nuclei of transgenic mice.

In the present study we used a transgenic mouse model, carrying the neuropeptide Y (NPY) Y1 receptor gene promoter linked to the LacZ reporter gene (Y1R/LacZ mice) to test the hypothesis of its up-regulation by gonadal hormones. Y1 receptor gene expression was detected by means of histochemical procedures and quantitative image analysis in the paraventricular nucleus, arcuate nucleus, medial preoptic nucleus, ventromedial nucleus and bed nucleus of stria terminalis of two-month-old female mice at different stages of estrous cycle. Qualitative and quantitative analyses showed that Y1R/LacZ transgene expression was higher in the paraventricular, arcuate, and ventromedial nuclei of proestrus mice as compared to mice in the other stages of the estrous cycle. In addition, we performed a comparison with a group of sexually active males. In this comparison a significant difference (less in males) was observed between males and proestrus females in the same nuclei. In conclusion, these data indicate that fluctuations in circulating levels of gonadal hormones, depending by estrous cycle, are paralleled by changes in the expression of NPY Y1 receptor in the hypothalamic nuclei involved in the control of both energy balance and reproduction.

Effects of perinatal administration of Bisphenol A on the neuronal nitric oxide synthase expressing system in the hypothalamus and limbic system of CD1 mice.

Bisphenol A (BPA) is a well-known plastic-derived pollutant that can bind to oestrogen receptors and is considered an endocrine-disrupting chemical. Its impact on different behaviours in rodents has been largely investigated, however, only a few data are available on its effects upon neural circuits. In the present study, we investigated the long-term effects of early exposure of mice of both sexes to BPA on the nitrinergic system, one of the neural systems involved in the control of sexual behaviour and under the control of gonadal hormones. Mice of both sexes were exposed for eight prenatal and eight postnatal days to BPA that was administered to the mothers. The maternally-exposed mice were sacrificed at the age of 2 months and their brains were sectioned and immunohistochemically treated for the detection of neuronal nitric oxide synthase (nNOS). Significant effects of BPA exposure were detected for the number of immunoreactive cells in the medial preoptic nucleus and in the ventromedial subdivision of the bed nucleus of the stria terminalis, in a sex-oriented and dose-dependent way. These results indicate that BPA has a powerful effect on specific portions of the nNOS-immunoreactive system belonging to the accessory olfactory system that are particularly important for the control of sexual behaviour. In addition, they confirm that perinatal exposure to endocrine-disrupting chemicals, in particular to BPA, may have a high impact on the organisation of specific neural pathways that can later affect complex behaviours and functions.

Effects of xenoestrogens on the differentiation of behaviorally relevant neural circuits in higher vertebrates.

Several environmental chemicals have the capability of impacting endocrine function (endocrine disrupting chemicals [EDCs]), and therefore they may have long-term consequences, especially if exposure occurs during embryonic development. In this study we present data relative to two widely used animal models: the Japanese quail and the mouse. These two species have been used to understand neural, neuroendocrine, and behavioral components of reproduction and are optimal models to understand how these components are altered by precocious exposure to EDCs. In particular, we discuss the effects of embryonic exposure to diethylstilbestrol, genistein, or ethylene,1,1-dichloro-2,2-bis(p-chlorophenyl) on the sexually dimorphic parvocellular vasotocin system and male copulatory behavior in quail and the effects of bisphenol A on the nitrinergic and kisspeptin systems and their behavioral impact in the mouse. In both models the exposure to EDCs during the critical period (early embryonic period in birds, perinatal period in rodents) alters the differentiation of relevant sexually dimorphic pathways, often inducing the appearance of a sex-reversed neurochemical phenotype that is the most probable cause of the final alteration of sexually differentiated behaviors in the adult animal. In conclusion, the data presented here should stimulate a critical reanalysis of the way to determine the "safe" exposure levels to EDCs for wild species and humans, considering behavior and related neural circuits among the factors to be analyzed.