Maternal high-sodium intake alters the responsiveness of the renin-angiotensin system in adult offspring

Aims: The goal of the current study was to evaluate the impact of maternal sodium intake during gestation on the systemic and renal renin-angiotensin-aldosterone-system (RAAS) of the adult offspring. Main methods: Female Wistar rats were fed high- (HSD-8.0% NaCl) or normal-sodium diets (NSD-1.3% NaCl) from 8 weeks of age until the delivery of their first litter. After birth, the offspring received NSD. Tail-cuff blood pressure (TcBP) was measured in the offspring between 6 and 12 weeks of age. At 12 weeks of age, the offspring were subjected to either one week of HSD or low sodium diet (LSD-0.1 6%NaCl) feeding to evaluate RAAS responsiveness or to acute saline overload to examine sodium excretory function. Plasma (PRA) and renal renin content (RRC), serum aldosterone (ALDO) levels, and renal cortical and medullary renin mRNA expression levels were evaluated at the end of the study. Key findings: TcBP was higher among dams fed HSD, but no TcBP differences were observed among the offspring. Male offspring, however, exhibited increased TcBP after one week of HSD feeding, and this effect was independent of maternal diet. Increased RAAS responsiveness to the HSD and LSD was also observed in male offspring. The baseline levels of PRA. ALDO, and cortical and medullary renin gene expression were lower but the RRC levels were higher among HSD-fed male offspring (HSDoff). Conversely, female HSDoff showed reduced sodium excretion 4 h after saline overload compared with female NSDoff. Significance: High maternal sodium intake is associated with gender-specific changes in RAAS responsiveness among adult offspring. (C) 2012 Elsevier Inc. All rights reserved.

The effects of conjugated estrogen, raloxifene and soy extract on collagen in rat bones

Objective To evaluate the action of conjugated equine estrogen, raloxifene and isolated or combined genistein-rich soy extracts on collagen fibers in the bones of oophorectomized rats. Materials and methods Seventy female rats received testosterone propionate (0.1 mu g/g) on the 9th day after birth. At 6 months of age, the rats were administered the vehicle (propylene glycol, 0.5 ml/day), and ten of the rats were randomly chosen to comprise the non-oophorectomized control group (GI). The other 60 rats were ovariectomized and randomized into six groups of ten as follows: GII, vehicle; GIII, conjugated equine estrogen (CEE), 50 mu g/kg/day; GIV, raloxifene (RAL), 0.75 mg/kg/day; GV, genistein-rich soy extract (GSE), 300 mg/kg/day; GVI, CEE + GSE, 50 mu g/kg/day + 300 mg/kg/day; and GVII, CEE + RAL, 50 mu g/kg/day + 0.75 mg/kg/day. Three months after surgery, the drugs were administered for 60 consecutive days. All rats were euthanized, and their left tibiae were removed for histological routine. The histological sections were stained with hematoxylin-eosin, and picrosirius for evaluating bone microarchitecture. Types I and II collagen fibers were analyzed by immunofluorescence. Data analysis was carried out with ANOVA and Tukey's test. Results Collagen reduction was significant in the GIII animals when compared to the other groups (p < 0.05). There was no significant difference in the thickness of collagen fibers among the groups. There was a greater quantity of type III collagen in GVI than in the other groups. Conclusion Our data indicate that conjugated equine estrogen improves bone quality because it increases the quantity of type I collagen while reducing the quantity of thin collagen fibers. In addition, the combination of CEE and raloxifene or genistein-rich soy extract is not as efficient as CEE itself to improve bone quality.

Assessment of micronucleus frequency in the peripheral blood of female rats in persistent estrus treated with selective estrogen receptor modulators

The aim of this study was to evaluate micronucleus (MN) frequency in polychromatic erythrocytes (PCE) of female rats in persistent estrus (a model developed to mimic polycystic ovary syndrome) treated with selective estrogen receptor modulators (SERMs, tamoxifen, and raloxifene). Forty female Wistar-Hannover rats were divided into four groups of 10 animals each: Group I (normally cycling rats) and Group II (persistent estrus) both received only vehicle, while Group III (persistent estrus) was treated with tamoxifen (250 mu g/animal/day) and Group IV (persistent estrus) was treated with raloxifene (750 mu g/animal/day). Tamoxifen and raloxifene were given by oral gavage beginning on postnatal day 90 and continuing for 30 consecutive days. Peripheral blood samples were collected from tails 1 day following the last exposure. Blood smears were made on glass slides and stained with 10% Giemsa solution. ANOVA and a Tukey post-hoc test were used for data analysis. Mean percentages of MN were 1.82 +/- 0.13, 5.20 +/- 0.24, 3.32 +/- 0.13, and 3.04 +/- 0.12 in Groups I, II, III, and IV, respectively. The results indicate that tamoxifen and raloxifene similarly reduced the formation of MNPCE of female rats in persistent estrus (P < 0.0001 for Groups III and IV vs. Group II), using the dosages and time periods applied in the present study. The data suggest possibly antimutagenic effects of SERMs under high levels of estrogens. The findings also suggest that this is an interesting animal model for studying the genotoxicity of estrogens. Environ. Mol. Mutagen. 2012. (C) 2011 Wiley Periodicals, Inc.

Antioxidant treatment protects against matrix metalloproteinase activation and cardiomyocyte injury during acute pulmonary thromboembolism

Increased reactive oxygen species (ROS) promote matrix metalloproteinase (MMP) activities and may underlie cardiomyocyte injury and the degradation of cardiac troponin I (cTI) during acute pulmonary thromboembolism (APT). We examined whether pretreatment or therapy with tempol (a ROS scavenger) prevents MMP activation and cardiomyocyte injury of APT. Anesthetized sheep received tempol infusion (1.0 mg kg(-1) min(-1), i.v.) or saline starting 30 min before or 30 min after APT (autologous blood clots). Control animals received saline. Hemodynamic measurements were performed. MMPs were studied in the right ventricle (RV) by gelatin zymography, fluorimetric activity assay, and in situ zymography. The ROS levels were determined in the RV and cTI were measured in serum samples. APT increased the pulmonary arterial pressure and pulmonary vascular resistance by 146 and 164 %, respectively. Pretreatment or therapy with tempol attenuated these increases. While APT increased RV + dP/dt (max), tempol infusions had no effects. APT increased RV MMP-9 (but not MMP-2) levels. In line with these findings, APT increased RV MMP activities, and this finding was confirmed by in situ zymography. APT increased the RV ROS levels and tempol infusion, before or after APT, and blunted APT-induced increases in MMP-9 levels, MMP activities, in situ MMP activities, and ROS levels in the RV. cTI concentrations increased after APT, and tempol attenuated these increases. RV oxidative stress after APT increases the RV MMP activities, leading to the degradation of sarcomeric proteins, including cTI. Antioxidant treatment may prevent MMP activation and protect against cardiomyocyte injury after APT.

Lovastatin protects mithochondrial and renal function in kidney ischemia-reperfusion in rats

PURPOSE: To investigate the effect of lovastatin on renal ischemia followed by reperfusion. METHODS: Thirty one Wistar rats submitted to left renal ischemia for 60 minutes followed by contralateral nephrectomy were divided into two groups: A (n = 17, control, no treatment), and B (n = 14, lovastatin 15 mg/kg/day p.o. ten days before ischemia). The animals were sacrificed at the end of ischemia, after 24 hours and at seven days after reperfusion. Survival, serum urea and creatinine levels and renal mitochondrial function were evaluated. RESULTS: Mortality was 29.4% in group A and 0.7% in group B. Urea and creatinine levels were increased in both groups, but the values were significantly lower in group B. Mitochondrial function showed decoupling in 83.4% of group A, as opposed to 38.4/% of group B. CONCLUSIONS: The result shows a protective action of renal function by lovastatin administered before ischemia/reperfusion. Since most of the mitochondrial fraction presented membranes with the ability to maintain ATP production in group B, stabilization of the mitochondrial membrane should be considered as part of the protective action of lovastatin on renal function in ischemia/reperfusion.

Glucocorticoid and Estrogen Receptors Are Reduced in Mitochondria of Lung Epithelial Cells in Asthma

Mitochondrial glucocorticoid (mtGR) and estrogen (mtER) receptors participate in the coordination of the cell's energy requirement and in the mitochondrial oxidative phosphorylation enzyme (OXPHOS) biosynthesis, affecting reactive oxygen species (ROS) generation and induction of apoptosis. Although activation of mtGR and mtER is known to trigger anti-inflammatory signals, little information exists on the presence of these receptors in lung tissue and their role in respiratory physiology and disease. Using a mouse model of allergic airway inflammation disease and applying confocal microscopy, subcellular fractionation, and Western blot analysis we showed mitochondrial localization of GR alpha and ER beta in lung tissue. Allergic airway inflammation caused reduction in mtGR alpha, mtER beta, and OXPHOS enzyme biosynthesis in lung cells mitochondria and particularly in bronchial epithelial cells mitochondria, which was accompanied by decrease in lung mitochondrial mass and induction of apoptosis. Confirmation and validation of the reduction of the mitochondrial receptors in lung epithelial cells in human asthma was achieved by analyzing autopsies from fatal asthma cases. The presence of the mitochondrial GR alpha and ER beta in lung tissue cells and especially their reduction in bronchial epithelial cells during allergic airway inflammation suggests a crucial role of these receptors in the regulation of mitochondrial function in asthma, implicating their involvement in the pathophysiology of the disease.

Dietary carotenoid lutein protects against DNA damage and alterations of the redox status induced by cisplatin in human derived HepG2 cells

Several epidemiological and experimental studies has been reported that lutein (LT) presents antioxidant properties. Aim of the present study was to investigate the protective effects of LT against oxidative stress and DNA damage induced by cisplatin (cDDP) in a human derived liver cell line (HepG2). Cell viability and DNA-damage was monitored by MU and comet assays. Moreover, different biochemical parameters related to redox status (glutathione, cytochrome-c and intracellular ROS) were also evaluated. A clear DNA-damage was seen with cDDP (1.0 mu M) treatment. In combination with the carotenoid, reduction of DNA damage was observed after pre- and simultaneous treatment of the cells, but not when the carotenoid was added to the cells after the exposure to cDDP. Exposure of the cells to cDDP also caused significant changes of all biochemical parameters and in co-treatment of the cells with LT, the carotenoid reverted these alterations. The results indicate that cDDP induces pronounced oxidative stress in HepG2 cells that is related to DNA damage and that the supplementation with the antioxidant LT may protect these adverse effects caused by the exposure of the cells to platinum compound, which can be a good predict for chemoprevention. (C) 2011 Elsevier Ltd. All rights reserved.

N-Acetylcysteine protects the peritoneum from the injury induced by hypertonic dialysis solution

Background: Oxidative stress has been implicated in the development of peritoneal damage. The aim of this study was to evaluate the effects of N-acetylcysteine (NAC) in a rat peritoneal infusion model. Methods: Eighteen male Wistar rats were divided in 3 groups: (i) control group; (ii) HDS group, receiving peritoneal dialysis solution (PDS); and (iii) HDS+NAC group, receiving PDS and oral NAC. Six weeks later they were evaluated for dialysate to plasma urea ratio (D/P), ratio of glucose concentration in peritoneal fluid (G1/G0), thiobarbituric acid reactive substances in plasma and urine and histology of peritoneal membrane. Results: The HDS+NAC group presented a lower increase in solute transport (D/P 0.51 +/- 0.1, and G1/GO 0.35 +/- 0.06) in comparison with the HDS group (D/P 0.67 +/- 0.1; p=0.03, and G1/G0 0.27 +/- 0.07; p=0.01). The HDS+NAC group showed lower thiobarbituric acid reactive substance concentrations compared with the HDS group. In the treated group, the peritoneal membrane presented lower thickness. Conclusions: Functional and histological peritoneal changes were significantly reduced by the treatment with NAC.

C-Phycocyanin protects SH-SY5Y cells from oxidative injury, rat retina from transient ischemia and rat brain mitochondria from Ca2+/phosphate-induced impairment

Oxidative stress and mitochondrial impairment are essential in the ischemic stroke cascade and eventually lead to tissue injury. C-Phycocyanin (C-PC) has previously been shown to have strong antioxidant and neuroprotective actions. In the present study, we assessed the effects of C-PC on oxidative injury induced by tert-butylhydroperoxide (t-BOOH) in SH-SY5Y neuronal cells, on transient ischemia in rat retinas, and in the calcium/phosphate-induced impairment of isolated rat brain mitochondria (RBM). In SH-SY5Y cells, t-BOOH induced a significant reduction of cell viability as assessed by an MTT assay, and the reduction was effectively prevented by treatment with C-PC in the low micromolar concentration range. Transient ischemia in rat retinas was induced by increasing the intraocular pressure to 120 mmHg for 45 min, which was followed by 15 min of reperfusion. This event resulted in a cell density reduction to lower than 50% in the inner nuclear layer (INL), which was significantly prevented by the intraocular pre-treatment with C-PC for 15 min. In the RBM exposed to 3 mM phosphate and/or 100 mu M Ca2+, C-PC prevented in the low micromolar concentration range, the mitochondrial permeability transition as assessed by mitochondrial swelling, the membrane potential dissipation, the increase of reactive oxygen species levels and the release of the pro-apoptotic cytochrome c. In addition, C-PC displayed a strong inhibitory effect against an electrochemically-generated Fenton reaction. Therefore, C-PC is a potential neuroprotective agent against ischemic stroke, resulting in reduced neuronal oxidative injury and the protection of mitochondria from impairment. (C) 2012 Elsevier Inc. All rights reserved.

High-Fat Diet Obesity Associated With Insulin Resistance Increases Cell Proliferation, Estrogen Receptor, and PI3K Proteins in Rat Ventral Prostate

In this study, we evaluated the effects of obesity and insulin resistance induced by a high-fat diet on prostate morphophysiology, focusing on cell proliferation, expression of androgen (AR) and estrogen receptors (ER) and proteins of the insulin signaling pathway. Adult male Wistar rats were fed a high-fat diet (20% fat) for 15 weeks, whereas control animals received a balanced diet (4% fat). Both groups were then divided and treated for 2 weeks with 1 mg/kg body weight/day of the aromatase inhibitor letrozole or vehicle only. The ventral prostate was analyzed with immunohistochemical, histopathological, stereological, and Western blotting methods. Obese rats showed insulin resistance, hyperinsulinemia, and reduced plasma testosterone levels. The incidence of prostatic intraepithelial neoplasia (PIN) was 2.7 times higher in obese rats and affected 0.4% of the gland compared with 0.1% PIN areas found in control rats. Obesity doubled cell proliferation in both prostate epithelium and stroma. AR content decreased in the prostate of obese rats and estrogen receptor beta (ER beta) increased in this group. Protein levels of insulin receptor substrate 1 and protein kinase B diminished in the obese group, whereas phosphatidylinositol 3-kinase (PI3K) increased significantly. Most structural changes observed in the prostate of obese rats normalized after letrozole treatment, except for increased stromal cell proliferation and ER beta expression, which might be associated with insulin resistance. This experimental model of obesity and insulin resistance induced by a high-fat diet increases cell proliferation in rat prostate. Such alterations are associated with decreased levels of AR and increased ER beta and PI3K proteins. This change can facilitate the establishment of proliferative lesions in rat prostate.

Cannabidiol protects oligodendrocyte progenitor cells from inflammation-induced apoptosis by attenuating endoplasmic reticulum stress

Cannabidiol (CBD) is the most abundant cannabinoid in Cannabis sativa that has no psychoactive properties. CBD has been approved to treat inflammation, pain and spasticity associated with multiple sclerosis (MS), of which demyelination and oligodendrocyte loss are hallmarks. Thus, we investigated the protective effects of CBD against the damage to oligodendrocyte progenitor cells (OPCs) mediated by the immune system. Doses of 1 mu M CBD protect OPCs from oxidative stress by decreasing the production of reactive oxygen species. CBD also protects OPCs from apoptosis induced by LPS/IFN gamma through the decrease of caspase 3 induction via mechanisms that do not involve CB1, CB2, TRPV1 or PPAR gamma receptors. Tunicamycin-induced OPC death was attenuated by CBD, suggesting a role of endoplasmic reticulum (ER) stress in the mode of action of CBD. This protection against ER stress-induced apoptosis was associated with reduced phosphorylation of eiF2 alpha, one of the initiators of the ER stress pathway. Indeed, CBD diminished the phosphorylation of PKR and eiF2 alpha induced by LPS/IFN gamma. The pro-survival effects of CBD in OPCs were accompanied by decreases in the expression of ER apoptotic effectors (CHOP, Bax and caspase 12), and increased expression of the anti-apoptotic Bcl-2. These findings suggest that attenuation of the ER stress pathway is involved in the 'oligoprotective' effects of CBD during inflammation. Cell Death and Disease (2012) 3, e331; doi:10.1038/cddis.2012.71; published online 28 June 2012

VIP expression in vessels of the vaginal wall: relationship with estrogen status

Objective Vasoactive intestinal peptide (VIP) is a neuropeptide with elevated expression in regions that control urogenital functions. Estrogen appears to modulate VIP expression in various organs, but this effect has not been demonstrated in the vaginal wall. The aim of this study was to evaluate the influence of estrogen status on VIP expression in vessels of the vaginal wall. Methods Surgical specimens were removed from the vaginal walls of 18 premenopausal women and 12 postmenopausal women who were given surgery for genital prolapse grade I or II. Vaginal specimens were stained with estrogen receptor-alpha (ER-alpha) and VIP antibodies. Levels of follicle stimulating hormone (FSH), estradiol, prolactin, fasting glucose and serum thyroxine stimulating hormone were also measured. Estrogen status was assessed on the basis of FSH and ER-alpha scores. Results The vaginal walls of premenopausal women had significantly higher ER-alpha scores than those of menopausal women (premenopausal group, 3.6 +/- 2.2; menopausal group, 1.4 +/- 1.8; p = 0.01). Premenopausal women also had significantly higher levels of VIP in the vaginal wall than menopausal women (p = 0.02). Increasing age was associated with lower level of VIP staining (odds ratio 0.88; 95% confidence interval 0.78-0.99). Conclusion Levels of ER-alpha and VIP expression in the posterior vaginal wall were higher in premenopausal than in menopausal women, but VIP expression was not associated with estrogen status. Age was an independent predictor of VIP staining in vaginal wall biopsies.

Exclusive breastfeeding protects against postpartum migraine recurrence attacks?

Objectives: To observe postpartum migraine recurrence among migraine sufferers before pregnancy, its classifications and associated factors and to compare women, who were exclusively breastfeeding, with those that used other forms of infant feeding. Methods: Out of 686 consecutively assisted women, at the first postnatal week, 266 were identified as migraine sufferers before pregnancy. Among those, one in five that were exclusively breastfeeding (53) and all the ones consecutively using others forms of infant feeding (40) were interviewed at the first and forth postpartum weeks. Results: After multivariable analysis, exclusive breastfeeding, no breastfeeding problems, and low income were associated with decrease in migraine recurrence at the first postpartum week. At the fourth week, exclusive breastfeeding continued to be a protective factor. Conclusions: A decrease in postpartum migraine recurrence seems to be another advantage of exclusive breastfeeding.

Effect of the interaction between food state and the action of estrogen on oxytocinergic system activity

Increased plasma osmolality by food intake evokes augmentation of plasma oxytocin (OT). Ovarian steroids may also influence the balance of body fluids by acting on OT neurones. Our aim was to determine if estrogen influences the activity of OT neurones in paraventricular nucleus (PVN) and supraoptic nucleus (SON) under different osmotic situations. Ovariectomized rats (OVX) were treated with either estradiol (E-2) or vehicle and were divided into three groups: group I was fed ad libitum, group II underwent 48 h of fasting, and group III was refed after 48 h of fasting. On the day of the experiment, blood samples were collected to determine the plasma osmolality and OT. The animals were subsequently perfused, and OT/FOS immunofluorescence analysis was conducted on neurones in the PVN and the SON. When compared to animals which were fasted or fed ad libitum, the plasma osmolality of refed animals was higher, regardless of whether they were treated with vehicle or E-2. We observed neural activation of OT cells in vehicle-or E-2-treated OVX rats refed after 48 h of fasting, but not in animals fed ad libitum or in animals that only underwent 48 h of fasting. Finally, the percentage of neurones that co-expressed OT and FOS was lower in both the PVN and the SON of animals treated with E-2 and refed, when compared to vehicle-treated animals. These results suggest that E-2 may have an inhibitory effect on OT neurones and may modulate the secretion of OT in response to the increase of osmolality induced by refeeding. Journal of Endocrinology (2012) 212, 129-138

Glycosaminoglycan profiles in the uterus of adult ovariectomized rats treated with estrogen and progestagen

Objectives: To evaluate the effects of conjugated equine estrogens (CE) alone or in combination with medroxyprogesterone acetate (MPA) on glycosaminoglycans (GAGs) in the cervix and horns of the rat uterus. Study design: Thirty days after ovariectomy, adult rats were randomly divided into four groups: Cl, control (treated with drug vehicle); GII CE (50 mu g/kg per day); GIII, MPA (0.2 mg/kg per day), and GIV, CE + MPA (doses as in GII and Gill). Drugs and vehicle were given by gavage during 28 days. Afterwards the animals were anesthetized, the cervix and uterine horns were dissected out and the middle portion fixed in 10% formaldehyde solution; other portions were fixed in acetone for histological examination and glycosaminoglycan quantification, respectively. Agarose gel electrophoresis was used for sulfated GAG analyses, and hyaluronic acid was assayed with an ELISA-like method. Statistical analysis was done by the Student's t test and the Tukey-Kramer test (P < 0.05). Results: The cervix and uterine horn structures presented signs of atrophy in the control group (GI). The other groups, mainly groups III and IV, had histological aspects of proliferation. In all groups the concentration of sulfated GAGs (especially dermatan sulfate) was higher than that of non-sulfated GAGs, both in cervix and in uterine horns. Estrogens increased sulfated GAG concentration at the cervix and the horn, whereas in uterine horns the amounts of sulfated GAGs were decreased after estrogens plus MPA treatment. The concentration of hyaluronic acid in uterine horns was higher than in cervices. Conclusions: The profiling and amounts of glycosaminoglycans in the two portions of the rat uterus are uneven. Dermatan sulfate occurs in higher concentrations in both cervix and uterine horns. Sulfated GAGs in rat cervix were increased by estrogens plus MPA, but were decreased by MPA alone in uterine horns. (C) 2012 Elsevier Ireland Ltd. All rights reserved.