Long-term impact of hypothyroidism during gestation and lactation on the mammary gland.

The functional differentiation of the mammary gland (MG) is fundamental for the prevention of mammary pathologies. This process occurs throughout pregnancy and lactation, making these stages key events for the study of pathologies associated with development and differentiation. Many studies have investigated the link between mammary pathologies and thyroid diseases, but most have ignored the role of thyroid hormone (TH) in the functional differentiation of the MG. In this work, we show the long-term impact of hypothyroidism in an animal model whose lactogenic differentiation occurred at low TH levels. We evaluated the ability of the MG to respond to hormonal control and regulate cell cycle progression. We found that a deficit in TH throughout pregnancy and lactation induces a long-term decrease in Rb phosphorylation, increases p53, p21, Cyclin D1 and Ki67 expression, reduces progesterone receptor expression, and induces nonmalignant lesions in mammary tissue. This paper shows the importance of TH level control during mammary differentiation and its long-term impact on mammary function.

Effects of thyroxine on apoptosis and proliferation of mammary tumors.

Hypothyroidism is a protective factor against breast cancer but long-term exposure or overdoses of thyroid replacement therapy with thyroxine (T4) may increase breast cancer risk. OBJECTIVE: to study, in vivo and in vitro, the effects of T4 on the proliferation and apoptosis of mammary tumors of hypo- and euthyroid rats, and the possible mechanisms involved in these effects. MATERIAL AND METHODS: Female Sprague-Dawley rats were treated with a single dose of dimethylbenzathracene (15 mg/rat) at 55 days of age and were divided into three groups: hypothyroidism (HypoT; 0.01% 6-N-propyl-2-thiouracil -PTU- in drinking water, n = 20), hypothyroidism treated with T4 (HypoT + T4; 0.01% PTU in drinking water and 0.25 mg/kg/day T4 via sc; n = 20) and EUT (untreated control, n = 20). At sacrifice, tumor explants from HypoT and EUT rats were obtained and treated either with 10-10 M T4 in DMEM/F12 without phenol red with 1% Charcoalized Fetal Bovine Serum or DMEM/F12 only for 15 min to evaluate intracellular signaling pathways associated with T4, and 24 h to evaluate changes in the expression of hormone receptors and proteins related to apoptosis and proliferation by immunohistochemistry and Western Blot. RESULTS: In vivo, hypothyroidism retards mammary carcinogenesis but its treatment with T4 reverted the protective effects. In vitro, the proliferative and anti-apoptosis mechanisms of T4 were different regarding the thyroid status. In EUT tumors, the main signaling pathway involved was the cross-talk with other receptors, such as ERα, PgR, and HER2. In HypoT tumors, the non-genomic signaling pathway of T4 was the chief mechanism involved since αvß3 integrin, HER2, ß-catenin and, downstream, PI3K/AKT and ERK signaling pathways were activated. CONCLUSION: T4 can regulate mammary carcinogenesis by mainly activating its non-genomic signaling pathway and by interacting with other hormone or growth factor pathways endorsing that overdoses of thyroid replacement therapy with T4 can increase the risk of breast cancer.

The epigenetic role of breastfeeding in mammary differentiation.

Maternal milk consumption can cause changes in the mammary epithelium of the offspring that result in the expression of molecules involved in the induction of differentiation, reducing the risk of developing mammary cancer later in life. We previously showed that animals that maintained a higher intake of maternal milk had a lower incidence of mammary cancer. In the present study, we evaluated one of the possible mechanisms by which the consumption of maternal milk could modify the susceptibility to mammary carcinogenesis. We used Sprague Dawley rats reared in litters of 3 (L3), 8 (L8), or 12 (L12) pups per mother in order to generate a differential consumption of milk. Whole mounts of mammary glands were performed to analyze the changes in morphology. Using real-time polymerase chain reaction (PCR), we analyzed the expression of mammary Pinc, Tbx3, Stat6, and Gata3 genes. We use the real-time methylation-specific polymerase chain reaction method to assess the methylation status of Stat6 and Gata3 CpG sites. Our findings show an increase in the size of the epithelial tree and a smaller number of ducts called terminal end buds in L3 vs. L12. We observed an increased expression of mRNA of Stat6, Gata3, Tbx3, and a lower expression of Pinc in L3 with respect to L12. Stat6 and Gata3 are more methylated in the CpG islands of the promoter analyzed in L12 vs. L3. In conclusion, the increased consumption of maternal milk during the postnatal stage generates epigenetic and morphological changes associated with the differentiation of the mammary gland.

Human renal adipose tissue induces the invasion and progression of renal cell carcinoma.

We evaluated the effects of conditioned media (CMs) of human adipose tissue from renal cell carcinoma located near the tumor (hRATnT) or farther away from the tumor (hRATfT), on proliferation, adhesion and migration of tumor (786-O and ACHN) and non-tumor (HK-2) human renal epithelial cell lines. Human adipose tissues were obtained from patients with renal cell carcinoma (RCC) and CMs from hRATnT and hRATfT incubation. Proliferation, adhesion and migration were quantified in 786-O, ACHN and HK-2 cell lines incubated with hRATnT-, hRATfT- or control-CMs. We evaluated versican, adiponectin and leptin expression in CMs from hRATnT and hRATfT. We evaluated AdipoR1/2, ObR, pERK, pAkt y pPI3K expression on cell lines incubated with CMs. No differences in proliferation of cell lines was found after 24 h of treatment with CMs. All cell lines showed a significant decrease in cell adhesion and increase in cell migration after incubation with hRATnT-CMs vs. hRATfT- or control-CMs. hRATnT-CMs showed increased levels of versican and leptin, compared to hRATfT-CMs. AdipoR2 in 786-O and ACHN cells decreased significantly after incubation with hRATfT- and hRATnT-CMs vs. control-CMs. We observed a decrease in the expression of pAkt in HK-2, 786-O and ACHN incubated with hRATnT-CMs. This result could partially explain the observed changes in migration and cell adhesion. We conclude that hRATnT released factors, such as leptin and versican, could enhance the invasive potential of renal epithelial cell lines and could modulate the progression of the disease.

Effects of parity and serum prolactin levels on the incidence and regression of DMBA-induced tumors in OFA hr/hr rats.

Prolactin (PRL) is a key player in the development of mammary cancer. We studied the effects of parity or hyperprolactinemia on mammary carcinogenesis in OFA hr/hr treated with 7,12-dimethylbenzanthracene. They were divided into three groups: nulliparous (Null), primiparous (PL, after pregnancy and lactation), and hyperprolactinemic rats (I, implanted in the arcuate nucleus with 17ß-estradiol). The tumor incidence was similar in the three groups. However, a higher percentage of regressing tumors was evident in the PL group. Serum PRL, mammary development, and mammary ß-casein content were higher in I rats compared to Null. The expression of hormone receptors was similar in the different groups. However, mammary tissue from PL rats bearing tumors had increased expression of PRL and estrogen alpha receptors compared to rats free of tumors. Our results suggest that serum PRL levels do not have relevance on the incidence of tumors, probably because the low levels of PRL in OFA rats are not further decreased by PL like in other strains. However, supraphysiological levels of PRL affect carcinogenesis. PL induces regression of the tumors due to the differentiation produced on the mammary cells. Alterations in the expression of hormonal receptors may be involved in progression and regression of tumors.

The inhibitory effect of progesterone on lactogenesis during pregnancy is already evident by mid- to late gestation in rodents.

Lactogenesis is a very complex process highly dependent on hormonal regulation. In the present study the time-course of the inhibitory actions of progesterone on prolactin secretion, mammary gland morphology and lactogenesis from mid- to late gestation in rodents was investigated. Groups of pregnant rats were luteectomised or administered with mifepristone on Day 10, 13, 15 or 17 of gestation and decapitated 28 or 48h later. Whole-blood samples and the inguinal mammary glands were taken for determinations of hormone levels and for measurement of mammary content of casein and lactose and for tissue morphology analyses, respectively. Luteectomy or mifepristone evoked prolactin increases only after Day 17 of gestation. Mammary content of casein was increased by both treatments regardless of timing or duration. Mifepristone was less effective than luteectomy in inducing lactose production and the effect was only observed after Day 15 of gestation. Analysis of mammary gland morphology confirmed the observed effect of progesterone on lactogenesis. Both treatments triggered remarkable secretory activity in the mammary gland, even without a parallel epithelial proliferation, demonstrating that the mammary epithelium is able to synthesise milk compounds long before its full lobulo-alveolar development is achieved, provided that progesterone action is abolished. Thus, the present study demonstrates that progesterone is a potent hormonal switch for the prolactin and prolactin-like effects on mammary gland development and its milk-synthesising capacity during pregnancy, and that its inhibitory action is already evident by mid-pregnancy in rodents.

Expression of NHERF1 in colonic tumors induced by 1,2-dimethylhydrazine in rats is independent of plasma ovarian steroids.

In normal embryonic fibroblasts, the Na(+)/H(+) exchanger regulator factor 1 (NHERF1) stabilizes E-cadherin/ß-catenin binding and the lack of NHERF1 expression promotes cell transformation thus acting as a tumor suppressor gene. We here tested the hypothesis that NHERF1 could act as a tumor suppressor gene in colon cancer as a mediator of estrogens' protective actions in colon carcinogenesis. We studied the expression and localization of NHERF1 and ß-catenin by immunohistochemistry in colonic tumors induced by 1,2 dimethylhidrazine (DMH) in Sprague-Dawley rats. One group of the rats treated with the carcinogen was ovariectomized (OVX) in the middle of the tumor induction, simulating a human menopausal condition. We observed a protective role of estrogens in colon cancer, as non-ovariectomized rats (DMH) had a reduced tumor area compared with the ovariectomized group (DMH + OVX; mean ± SE) 28.98 ± 4.65 vs. 67.58 ± 8.69 (p < 0.00380). Despite the lack of plasma estrogen stimulation, we found abundant expression of NHERF1 in colon tumors from ovariectomized rats. NHERF1 was mainly localized in the cytoplasm of the adenocarcinoma cells and lost the apical localization previously reported in normal colon tissue. We also detected expression of NHERF1 by western blot in the SW48, CACO-2, and HT29 colon cancer cell lines. Non-estrogenic factors in plasma or the tumor microenvironment may regulate NHERF1 expression in transformed colon epithelial cells. Further studies are required to understand the regulation of NHERF1 expression in colon cancer tissue.

Regulation of prolactin secretion during the estrus in rats: possible role of glucocorticoids.

Mifepristone (MIF) administration to cycling rats at proestrus induces hypersecretion of prolactin (PRL) at the following estrus. We aimed to assess whether this effect is due to the antiprogesterone or antiglucocorticoid action of MIF and to help underscore the nature of the circulating hormone(s) regulating PRL secretion at estrus. Female cycling rats in proestrus were treated with vehicle; the progesterone (Pg) and glucocorticoid receptor antagonists, MIF (5 mg/kg) or ORG-33628 (5 mg/kg); the glucocorticoid agonist dexamethasone (DEX; 27 mg/kg)±MIF; or the inhibitor of steroid synthesis aminoglutethimide (AG; 150 mg/kg)±MIF. The animals' blood was sampled the same day at 1800 h and at 1800 h of the following day to assess for circulating PRL and Pg levels. To distinguish antiglucocorticoid from antiprogesterone effects of MIF, we administered a highly specific neutralizing antibody against Pg. None of the antagonists modified serum PRL values at proestrus but increased PRL levels at estrus. DEX decreased the secretion of PRL at proestrus, yet the effect was entirely blocked by MIF. Furthermore, DEX decreased PRL at estrus in a MIF-reversible manner, suggesting that adrenal corticoids during proestrous may regulate PRL secretion at estrus. AG increased PRL secretion at estrus, whereas its association with MIF produced an even higher response. PRL concentration at estrus was not modified by the antiprogesterone antibody, suggesting that the effect of MIF is a consequence of its antiglucocorticoid effect and not due to its antiprogesterone properties. In conclusion, PRL secretion in the afternoon of the estrus is most likely regulated by glucocorticoids through an inhibitory action.

Estrogen inhibits tuberoinfundibular dopaminergic neurons but does not cause irreversible damage.

Dopaminergic neurons of the hypothalamic tuberoinfundibular dopaminergic (TIDA) system exert a tonic inhibitory control on prolactin (PRL) secretion whereas estrogen, known to inhibit TIDA neuron function, has been postulated to be toxic to TIDA neurons when it is chronically high. In order to determine whether estrogen in high doses can cause permanent damage to TIDA function, we submitted young female rats to continue high doses of estrogen administered, either centrally (intrahypothalamic estrogen implants) or peripherally (subcutaneous estrogen implants or weekly intramuscular (i.m.) injections for 7 weeks), subsequently withdrawing the steroid and observing the evolution of lactotrophes, serum PRL and TIDA neurons. Serum PRL was measured by radioimmunoassay whereas tyrosine hydroxylase positive (TH+) neurons and PRL cells were morphometrically assessed in sections of fixed hypothalami and pituitaries, respectively. After 30 days, hypothalamic estrogen implants induced a significant increase in serum PRL, whereas TH+ neurons were not detectable in the arcuate-periventricular hypothalamic (ARC) region of estrogen-implanted rats. Removal of implants on day 30 restored TH expression in the ARC and brought serum PRL back to basal levels 30 days after estrogen withdrawal. Subcutaneous or i.m. administration of estrogen for 7 weeks induced a marked hyperprolactinemia. However, 30 weeks after estrogen withdrawal, TH neuron numbers in the ARC were back to normal and serum PRL returned to basal levels. After peripheral but not central estrogen withdrawal, pituitary weight and lactotrophic cell numbers remained slightly increased. Our data suggest that estrogen even at high doses, does not cause permanent damage to TIDA neurons.

Neurotransmitters involved in the opioid regulation of prolactin secretion at the end of pregnancy in rats.

Using a pharmacological approach, we explored potential mechanisms for the regulation of prolactin secretion by opioid peptides at the end of pregnancy in rats. On day 19 of pregnancy, intracereboventricular administration of the mu-opioid receptor agonist (D-Ala2, NMe-Phe4, Gly-ol5)-enkephalin (DAMGO) or beta-endorphin (beta-END) induced a dose-related increase in serum prolactin levels 30 min later. Pretreatment with the opioid antagonist naloxone abolished the increase induced by DAMGO injection. At lower doses, DAMGO and beta-END did not modify the 3,4-dihydroxyphenylacetic acid/dopamine ratio, but at higher doses, the mu-agonists evoked a significant increase of the dopaminergic activity as compared with saline control. The time course of the effects of beta-END (2.5 microg/rat) showed a higher increase in serum prolactin levels at 15 min than at 30 min after treatment. The 3,4-dihydroxyphenylacetic acid/dopamine ratio increased 15 min after beta-END administration and was even higher 30 min later. Neither the selective kappa-agonist U50,488H nor the selective delta-agonist (D-Pen2, D-Pen5)- enkephalin were able to modify the serum prolactin levels at the doses studied. To evaluate potential neurotransmitters involved in the regulation of prolactin secretion at the end of pregnancy, we combined the administration of serotoninergic or GABAergic antagonists with the opioid agonist DAMGO. The serotonin 5-HT2 receptor antagonist ketanserin increased the serum prolactin levels and potentiated the effect of DAMGO. The intracerebroventricular administration of SR-95531 did not modify the serum prolactin concentration under basal conditions, but partially prevented the increase induced by DAMGO injection. The intracerebroventricular administration of the GABA(B) receptor antagonist phaclofen had no effect on the serum prolactin levels either in naive or DAMGO-treated rats. The present results support the proposal that activation of mu-opioid receptors stimulates prolactin secretion at the end of pregnancy. Although the exact mechanisms by which the opioid system modulates prolactin secretion at the end of pregnancy are unclear, these results suggest an interaction of the opioidergic system with serotoninergic and GABAergic systems, without ruling out a direct or indirect action on dopaminergic neurons. In conclusion, the opioid system may regulate prolactin secretion at the end of pregnancy through either stimulatory (present results) or inhibitory actions previously described.