Proopiomelanocortin (POMC) is a precursor gene of the neuropeptide β-endorphin in the hypothalamus and is known to regulate various physiological functions including stress response. Several recent reports showed that fetal alcohol exposure programs the hypothalamus to produce lower levels of POMC gene transcripts and to elevate the hypothalamic-pituitary-adrenal (HPA) axis response to stressful stimuli. We investigated the role of methyl CpG binding protein (MeCP2) in the effects of prenatal ethanol on POMC gene expression and hypothalamic-pituitary-adrenal (HPA) axis function. Pregnant Sprague Dawley rats were fed between GD 7 and 21 with a liquid diet containing 6.7% alcohol, pair-fed with isocaloric liquid diet, or fed ad libitum with rat chow, and their male offsprings were used at 60 days after birth in this study. Fetal alcohol exposure reduced the level of POMC mRNA, but increased the level of DNA methylation of this gene in the arcuate nucleus (ARC) of the hypothalamus where the POMC neuronal cell bodies are located. Fetal alcohol exposed rats showed a significant increase in MeCP2 protein levels in POMC cells, MeCP2 gene transcript levels as well as increased MeCP2 protein binding on the POMC promoter in the arcuate nucleus. Lentiviral delivery of MeCP2 shRNA into the third ventricle efficiently reduced MeCP2 expression and prevented the effect of prenatal ethanol on POMC gene expression in the arcuate nucleus. MeCP2-shRNA treatment also normalized the prenatal ethanol-induced increase in corticotropin releasing hormone (CRH) gene expression in the hypothalamus and elevated plasma adrenocorticotrophic hormone (ACTH) and corticosterone hormone responses to lipopolysaccharide (LPS) challenge. These results suggest that fetal alcohol programming of POMC gene may involve recruitment of MeCP2 on to the methylated promoter of the POMC gene to suppress POMC transcript levels and contribute to HPA axis dysregulation.

Background Alcohol exposure has adverse effects on stress physiology and behavioral reactivity. This is suggested to be due, in part, to the effect of alcohol on β-endorphin (β-EP) producing neurons in the hypothalamus. In response to stress, β-EP normally provides negative feedback to the HPA axis and interacts with other neurotransmitter systems in the amygdala to regulate behavior. We examined whether β-EP neuronal function in the hypothalamus reduces the corticosterone response to acute stress, attenuates anxiety-like behaviors, and modulates alcohol drinking in rats. Methods To determine if β-EP neuronal transplants modulate the stress response, anxiety behavior and alcohol drinking, we implanted differentiated β-EP neurons into the paraventricular nucleus of the hypothalamus (PVN) of normal, prenatal alcohol exposed, and alcohol-preferring (P) and non-preferring (NP) rats. We then assessed corticosterone levels in response to acute restraint stress and other markers of stress response in the brain, and anxiety-like behaviors in the elevated plus maze and open-field assays. Results We showed that β-EP neuronal transplants into the PVN reduced the peripheral corticosterone response to acute stress and attenuated anxiety-like behaviors. Similar transplants completely reduced the hyper-corticosterone response and elevated anxiety behaviors in prenatal alcohol exposed adult rats. Moreover, we showed that β-EP reduced anxiety behavior in P rats with minimal effects on alcohol drinking during and following restraint stress. Conclusions These data further establish a role of β-EP neurons in the hypothalamus for regulating physiological stress response and anxiety behavior, and resembles a potential novel therapy for treating stress-related psychiatric disorders in prenatal alcohol exposed children and those genetically predisposed to increased alcohol consumption.

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Prolactin secreting pituitary adenomas (prolactinomas) is the most common pituitary tumors in humans. Animal studies have identified aggressive prolactinoma development in fetal alcohol exposed rats. We have recently identified a combination treatment of a μ opioid receptor antagonist naltrexone and a δ opioid receptor agonist D-Ala2-,N-Me-Phe4,Gly-ol Enkephalin (DPDPE) increases innate immune function. In this study, we tested whether naltrexone and DPDPE combination therapy is useful to control pituitary tumor growth. Fetal alcohol exposed and control Fischer 344 female rats at 60 days of age were ovariectomized and received an estrogen implant to induce prolactinomas. Six weeks after the estrogen implant, these animals received treatments of naltrexone and DPDPE or saline. The growth of the pituitary tumor prior to and after opioidergic agent treatments was visualized using magnetic resonance imaging (MRI). At the end of the treatment, pituitary weights, plasma prolactin and splenic levels of cytotoxic factors were determined. Both imaging data and weight data indicated that the volume and the weight of the pituitary were increased more after estrogen treatment in animals exposed to fetal alcohol than control. Naltrexone and DPDPE treatment reduced the weight and volume of the pituitary gland and plasma levels of prolactin in both fetal alcohol exposed and control-fed animals. The treatment of opioidergic agents also increased the levels of cytotoxic factors in the spleen. These data provide a novel possibility in treating pituitary tumors using a combination therapy of naltrexone and DPDPE.

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The idea that exposure to adverse environmental conditions and lifestyle choices during pregnancy can result in fetal programming that underlies disease susceptibility in adulthood is now widely accepted. Fetal alcohol exposed offspring display many behavioral and physiological abnormalities including neuroendocrine-immune functions, which often carry over into their adult life. Since neuroendocrine-immune system is critically involved in the regulation of tumor surveillance, we determined whether fetal alcohol exposure increases the susceptibility to estrogen-induced pituitary prolactin-secreting tumors (prolactinomas), commonly occurring pituitary tumors in humans. Pregnant Fischer 344 rats were fed between gestational days 7 and 21 with a liquid diet containing alcohol (AF), pair-fed with isocaloric liquid diet (PF), or fed ad libitum with rat chow (AD). At 90 days of age, female offspring rats were ovariectomized and received a subcutaneous estradiol implant. These rats were sacrificed between 4 and 5 months after the estradiol implants. At the time of sacrifice, pituitaries of these animals were inspected for tumor and whole body were inspected for any tumor metastasis. Estradiol treatment increased pituitary weight about 5-folds in AD and PF treated groups and increased 20-30-folds in the AF treated group. Most tumors in the AF group were hemorrhagic. About 30% AF rats had some tumors in the non-pituitary sites. AD and PF rats did not show any non-pituitary site tumor. When AF rat pituitaries were grown in cultures, cells rapidly grew and formed colonies. Colony formation and rapid growth rate were not observed in cells of the pituitary from PF and AD rats. Histopathological evaluation revealed that tumors in AF group were more densely packed cells as compare to the PF and AD groups who showed uniform cells with abundant cytoplasm. Pituitary tumor from alcohol exposed animals showed strong nuclear p53 and monoclonal Ki67 immunoreactivity in almost all tumor cells. Significantly higher mRNA levels of hemorrhage-associated genes (VEGF and MMP-9) were also observed in pituitary tumor tissues from AF group as compare with PF and AD groups. Tumor cells showed positivity to prolactin staining but showed negativity to other pituitary hormones staining. Histological evaluation of the pituitary tissues revealed that tumors were prolactin producing in all groups, and tumors in AF pituitaries were highly proliferative. These data provide evidence for aggressive, possible neoplastic, prolactinoma development in the pituitary after estrogen treatment in fetal alcohol exposed female rats. (This work is supported by a National Institute of Health grant R01 AA11591). Citation Format: Shaima Jabbar, George Maglakelidze, Dipak K. Sarkar. Fetal alcohol exposure increases susceptibility to carcinogenesis in the pituitary. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3460. doi:10.1158/1538-7445.AM2015-3460

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