The Phytoestrogenic Effect of Hibiscus sabdariffa Involves Estrogen Receptor α in Ovariectomized Wistar Rats.

The calyxes of Hibiscus sabdariffa present multiple pharmacological effects primarily attributed to their high anthocyanin content; however, little is known about their phytoestrogenic effect. Ovarian hypofunction (OH) is a process characterized by the rapid detention of the production of ovarian hormones, which compromises reproductive and cognitive functions. Hormone replacement therapy (HRT) efficiently compensates for OH; nevertheless, questions have been raised on its secondary effects and safety. One of the alternatives to tackling OH involves using phytoestrogens such as anthocyanins for their structural similarity to natural estrogens. In a Wistar rat model of ovariectomy (OVX), we recently reported the beneficial properties of an anthocyanin-rich extract prepared from the calyces of H. sabdariffa (HSE) in hindering the adverse effects of OH on memory performance and highlighted a possible phytoestrogenic impact through the modulation of estrogen receptor (ER) expression. We now report that HSE and estradiol differentially affected the expression of ERα and ERß. ERα was more sensitive to HSE; meanwhile, estradiol preferentially modulated ERß. Thus, our study leads to further research on using H. sabdariffa as a nutrition-based alternative to HRT.

Effect of consumption of blue maize tortilla on anxiety-like behaviour, learning, memory and hippocampal BDNF expression in a chronic stress model in rats.

BACKGROUND: Pigmented maize consumption is of much interest because of its high anthocyanin content and multiple health benefits. OBJETIVES: This study was aimed to assess the effect of consuming blue maize tortillas on the anxiolytic capacity, preserve emotional memory, and the expression of brain-derived neurotrophic factor (BDNF) in rats subjected to chronic stress. METHODS: Sixty-four 3-month-old male Wistar rats were used, divided into eight groups (n = 8). Four groups were subjected to chronic stress by movement restriction (7 h/daily/7 consecutive days) and the remaining four groups were subjected to standard management. The treatments were commercial food, blue tortilla, anthocyanin extract, or white tortilla, administered for nine weeks to stressed or unstressed animals. In the eighth week, the animals were subjected to the restraint stress model. Subsequently, anxiety-like behaviour was assessed using the elevated plus-maze, and memory and emotional learning were evaluated by the step-down passive avoidance test. The animals were then sacrificed to quantify the relative expression of hippocampal BDNF by RT-qPCR. RESULTS: The consumption of anthocyanin extract or tortilla made with blue corn decreased anxiety-like behaviours, additionally, it improved the ability to retain emotionally relevant information, and it upregulated BDNF mRNA expression. PERSPECTIVE: Thus, the analyse of the impact of blue tortilla consumption on the nervous system is now necessary to guarantee the nutraceutical value of this food.

Proliferation and apoptosis regulation by G protein-coupled estrogen receptor in glioblastoma C6 cells.

Glioblastoma is the most frequent primary tumor in the human brain. Glioblastoma cells express aromatase and the classic estrogen receptors ERα and ERß and can produce estrogens that promote tumor growth. The membrane G protein-coupled estrogen receptor (GPER) also plays a significant role in numerous types of cancer; its participation in glioblastoma tumor development is not entirely known. The present study investigated the effect of the agonists [17ß-estradiol (E2) and G1] and antagonist (G15) of GPER on proliferation and apoptosis of C6 glioblastoma cells. GPER expression was evaluated by immunofluorescence, western blotting and reverse transcription-quantitative PCR. Cell proliferation was determined using Ki67 immunopositivity. Cell viability was examined using the MTT assay and apoptosis using caspase-3 immunostaining and ELISA. C6 cells express GPER, and the immunopositivity increased after exposure to E2, G1, or their combination. GPER protein expression increased after treatment with E2 combined with G1. However, GPER mRNA expression decreased in treated cells compared with control. The percentage of Ki67 immunopositive C6 cells increased under the effect of E2 in combination with G1 or G1 alone. G15 significantly reduced Ki67 immunopositivity. Pearson's correlation analysis revealed a positive relationship between GPER and Ki67 immunopositivity across the study conditions. Additionally, the MTT assay showed a significant reduction in C6 cell viability after G15 treatment, alone or in combination with G1. The exposure to G15 increased the percentage of caspase-3 immunopositivity cells and caspase-3 levels. Pearson's correlation analysis demonstrated a negative correlation between GPER and caspase-3 immunopositivity across the study conditions. Glioblastoma C6 cells express GPER, and this receptor modulates cell proliferation and apoptosis. The GPER agonists E2 and G1 favored cell proliferation; meanwhile, the antagonist G15 reduced cell proliferation, viability and favored apoptosis. Therefore, GPER may be used as a biomarker of glioblastoma and as a target to develop new therapeutic strategies for glioblastoma treatment.

Effects of Hibiscus sabdariffa calyces on spatial memory and hippocampal expression of BDNF in ovariectomized rats.

Ovarian hypofunction is characterized by decay in brain-derived neurotrophic factor (BDNF), a neurotrophin associated with cognitive and memory function. Hormone replacement therapy is the most common treatment to counteract the negative effects of ovarian insufficiency; however, this therapy may increase the odds of endometrial cancer, blood clots, stroke, and breast cancer. Therefore, a safer alternative to synthetic estrogens is needed. One possible candidate may be phytoestrogens. Hibiscus sabdariffa L. (Malvaceae) is a source of natural food colorants; the calyces and leaves of the plant are consumed in drinks and culinary preparations and are recognized for several health benefits related to their high content of anthocyanins. In the present study, we used an ovariectomized rat model to assess the phytoestrogenic effect of H. sabdariffa, and evaluated spatial memory and BDNF expression. Ninety-day-old female Wistar rats were randomly separated into six groups. Rats from four groups were ovariectomized and injected with a physiological dose of estradiol, or given, in drinking water, an extract prepared from calyces of H. sabdariffa at doses of 50 or 100 mg/kg body weight. Both Intact and Sham groups were included as controls. At day 42, short- and long-term memories were assessed by the Barnes maze test, and hippocampal BDNF expression was evaluated by RT-qPCR and Western blot. Ovariectomy significantly decreased memory performance and BDNF expression, compared with controls. However, administration of H. sabdariffa extract reversed the negative effect of ovariectomy on short- and long-term memory parameters and BDNF expression. A stronger effect was observed at a lower dose of the extract. In conclusion, the extract from H. sabdariffa acted as a phytoestrogen in ovariectomized rats, improving spatial memory performance and hippocampal BDNF expression. Based on these promising results, further clinical experimentation is recommended to study the benefits of H. sabdariffa as an alternative hormonal therapy in patients with ovarian hypofunction.

Chronic stress decreases ornithine decarboxylase expression and protects against 1,2-dimethylhydrazine-induced colon carcinogenesis.

Biological response to stress depends on the type, timing, and severity of the stressor. Acute stressful environments may positively activate molecular and cellular mechanisms to favor adaptation; however, chronic stress is often associated with detrimental health effects. Colon cancer (CC) is one of the leading causes of death associated with cancer and has been mentioned as a stress-related disease. In the present work, the effect of chronic stress on the initial phase of CC was evaluated, and special emphasis was placed on ornithine decarboxylase (ODC) expression and polyamines for their role in hyperproliferative diseases. BALB/c mice (n = 5/group) were administered the pro-carcinogen 1,2-dimethylhydrazine (DMH) for 8 weeks (20 mg/kg body weight/week) to induce colon carcinogenesis, and then exposed for 4 weeks to two physical stressors: restraint and forced-swimming. Distal colon inflammatory lesions and histomorphological changes were evaluated by hematoxylin-eosin staining; plasma corticosterone levels, colon ODC expression, and urinary polyamines were determined by competitive ELISA, RT-qPCR, Western Blot, and HPLC, respectively. The short-term exposure to DMH triggered colon inflammation, initiated colon carcinogenesis and increased ODC expression; meanwhile, the exposure to chronic stress activated the hypothalamic-pituitary-adrenal (HPA) axis, elicited the production of plasmatic corticosterone, and decreased ODC expression. The exposure of DMH-treated mice to chronic stress counteracted the inflammatory effect of DMH and maintained ODC homeostasis. In early phase of carcinogenesis, the exposure of DMH-treated mice to chronic stress had a positive effect against colon inflammation and maintained ODC homeostasis. The cross-talk between corticosterone, ODC expression, and inflammation in a tumor environment is discussed.

The anticonvulsant effect of sparteine on pentylenetetrazole-induced seizures in rats: a behavioral, electroencephalographic, morphological and molecular study.

Abnormal synchronous activity in neurons generates epileptic seizures. Antiepileptic drugs (AEDs) are effective in 70% of patients, but this percentage is drastically lower in developing countries. Sparteine is a quinolizidine alkaloid synthesized from most Lupine species and has a probable anticonvulsive effect. For this reason, the objective of the present work was to study the anticonvulsant effect of sparteine using a dose-effect curve and to determine its effectiveness against seizures using behavioral, electroencephalographic, morphological and molecular data. Wistar rats were grouped into control [saline solution (0.9%), pentylenetetrazole (90 mg/kg), and sparteine (13, 20 and 30 mg/kg), intraperitoneal (i.p.)] and experimental (sparteine + pentylenetetrazole) groups. The rats were implanted with surface electrodes to register electrical activity, and convulsive behavior was evaluated according to Velisek's scale. The rats were perfused to obtain brain slices for cresyl violet staining and cellular density quantification as well as for immunohistochemistry for NeuN and GFAP. Other animals were used to determine the hippocampal mRNA expression of the M2 and M4 acetylcholine receptors by qPCR. Sparteine exhibited a better anticonvulsant effect at a dose of 30 mg/kg (i.p.) than at the other doses used. This anticonvulsant effect was characterized by a decrease in the severity of convulsive behavior, 100% survival, an inhibitory effect on epileptiform activity 75 min after pentylenetetrazole administration, and the conservation of the cellular layers of CA1, CA3 and the dentate gyrus (DG); however, astrogliosis was observed after 30 mg/kg sparteine treatment. In addition, sparteine treatment increased the mRNA expression of the M4 receptor three hours after administration. According to our findings, the effective dose of sparteine as an anticonvulsant agent by i.p. injection is 30 mg/kg. The astrogliosis that was observed after sparteine administration may be a compensatory mechanism to diminish excitability and maintain neuronal homeostasis, possibly through redistributing potassium and glutamate. The increase in the mRNA expression of the M4 receptor may suggest the participation of the M4 receptor in the anticonvulsive effect of sparteine, as the activation of this receptor may inhibit acetylcholine release and facilitate the subsequent release of GABA. However, the precise mechanisms by which sparteine produces these effects are not known, and therefore, further experiments are necessary.

Immunomodulatory Effect of Lactobacillus casei in a Murine Model of Colon Carcinogenesis.

We previously reported beneficial effects of the probiotic strain Lactobacillus casei 393 in hindering colon carcinogenesis in a 1,2-dimethylhydrazine (DMH)-induced BALB/c mouse model of colon cancer. In the present study, we investigated the effect of preventive administration of L. casei 393 on the levels of selected pro- and anti-inflammatory circulating cytokines, as well as subpopulations of splenic T cells. The resulting experimental data on IFNγ, TNFα, IL-10, and colon histological features demonstrated that administration of L. casei 2 weeks before DMH treatment impaired the pro-inflammatory effect of DMH, while maintaining the levels of the three cytokines as well as colon histology; it also modulated splenic CD4+, CD8+, and NK T cell subpopulations. The preventive administration of L. casei to DMH-treated mice increased IL-17A synthesis and Treg percentages, further indicating a tumor-protecting role. Together, the results suggest that the colon-cancer-protective properties of L. casei 393 involve the dampening of inflammation through cytokine homeostasis and the maintenance of a healthy T cell subpopulation dynamic. For these reasons, probiotics such as L. casei may contribute to the health of the host as they promote optimal control of the immune response. Further, they may be used as prophylactic agents in combination with standard therapies against colon cancer.

Protective Effect of Lactobacillus casei on DMH-Induced Colon Carcinogenesis in Mice.

The administration of probiotics is a promising approach to reduce the prevalence of colon cancer, a multifactorial disease, with hereditary factors, as well as environmental lifestyle-related risk factors. Biogenic polyamines, putrescine, spermidine, and spermine are small cationic molecules with great roles in cell proliferation and differentiation as well as regulation of gene expression. Ornithine decarboxylase is the first rate-limiting enzyme for polyamine synthesis, and upregulation of ornithine decarboxylase activity and polyamine metabolism has been associated with abnormal cell proliferation. This paper is focused on studying the protective role of Lactobacillus casei ATCC 393 in a chemically induced mouse model of colon carcinogenesis, directing our attention on aberrant crypt foci as preneoplastic markers, and on polyamine metabolism as a possible key player in carcinogenesis. BALB/c mice were administered 1,2-dimethylhydrazine dihydrochloride (DMH) to induce colon cancer (20 mg/kg body weight, subcutaneous, twice a week for 24 weeks). L. casei ATCC 393 was given orally (106 CFU, twice a week), 2 weeks before DMH administration. Hematoxylin and eosin staining, high-performance liquid chromatography, and Western blotting were used to evaluate aberrant crypt foci, urinary polyamines, and ornithine decarboxylase expression in the colon. The experimental data showed that the preventive administration of L. casei ATCC 393 may delay the onset of cancer as it significantly reduced the number of DMH-induced aberrant crypt foci, the levels of putrescine, and the expression of ornithine decarboxylase. Hence, this probiotic strain has a prospective role in protection against colon carcinogenesis, and its antimutagenic activity may be associated with the maintenance of polyamine metabolism.

Endosulfan decreases cytotoxic activity of nonspecific cytotoxic cells and expression of granzyme gene in Oreochromis niloticus.

The effect of the organochlorinated insecticide endosulfan, on the cytotoxic activity of Nile tilapia nonspecific cytotoxic cells (NCC) was assessed. Juvenile Nile tilapia were exposed to endosulfan (7 ppb) for 96 h and splenic NCC were isolated. Flow cytometric phenotyping of NCC was based on the detection of the NCC specific membrane signaling protein NCCRP-1 by using the monoclonal antibody Mab 5C6; granzyme expression was evaluated by quantitative RT-PCR. The cytotoxic activity of sorted NCC on HL-60 tumoral cells was assessed using propidium iodide (PI) staining of DNA in HL-60 nuclei, indicating dead cells. Nile tilapia splenic NCC had the ability to kill HL-60 tumoral cells, however, the exposure to endosulfan significantly reduced, by a 65%, their cytotoxic activity when using the effector:target ratio of 40:1. Additionally, the exposure to endosulfan tended to increase the expression of NCCRP-1, which is involved in NCC antigen recognition and signaling. Moreover, it decreased the expression of the granzyme gene in exposed group as compared with non-exposed group; however significant differences between groups were not detected. In summary, the acute exposure of Nile tilapia to sublethal concentration of endosulfan induces alteration in function of NCC: significant decrease of cytotoxic activity and a tendency to lower granzyme expression, severe enough to compromise the immunity of this species.

A chronic combinatory stress model that activates the HPA axis and avoids habituation in BALB/C mice.

A detailed protocol is described to induce chronic stress in BALB/c mice, which affects the hypothalamic-pituitary-adrenal (HPA) axis. The protocol is based on a combination of two mild physical stressors: restraint stress and forced swimming. Physical stressors were applied on an alternative schedule: one day restraint, next day swimming, during a 28-day period. Mice were sacrificed at days 7, 14, 21 and 28 and plasma was obtained. Optimized chromatographic system with electrochemical detection and a commercially available enzyme immunoassay kit were used to measure catecholamines [representative of the activation of the autonomic nervous system (ANS)] and corticosterone (representative of the activation of the HPA axis). Corticosterone levels increased in mice under stress and remained significantly higher in stressed mice compared to control animals throughout the experimental procedure, indicating that mice did not show habituation to the combined stress. In our experimental conditions, catecholamine levels were not useful as an index of stress. The stress model applied here provoked a steady activation of the HPA axis resulting in the constant secretion of corticosterone from the adrenal gland, which may prevent the activation of the ANS axis. The simple and economic stress model presented here affected the HPA axis but not the ANS of BALB/c mice and is useful for the study of metabolic stress-related gastric pathology and stress hormone secretion in mice.

Effects of low concentration of endosulfan on proliferation, ERK1/2 pathway, apoptosis and senescence in Nile tilapia (Oreochromis niloticus) splenocytes.

Endosulfan is a potent organochlorinated pesticide that is known to induce side effects in aquatic organisms, including Oreochromis niloticus (Nile tilapia). It has been previously shown that endosulfan induces oxidative stress and non-specific activation of splenic macrophages and exacerbated serum interleukin-2 synthesis in Nile tilapia. Endosulfan may promote proliferation of T cells through MAP kinase (MAPK) activated signal transductions. The ERK family of MAPKs includes ERK1 and ERK2. Phosphorylated ERK1/2 (pERK1/2) molecules are involved in many aspects of cellular survival, and are important for apoptosis or oxidative stress-induced senescence. In order to study the mechanisms by which endosulfan affects fish health, the present study was aimed at evaluating the in vitro effects of this insecticide on proliferation, the ERK1/2 pathway, apoptosis and cell senescence in splenocytes from Nile tilapia. Lymphoproliferation was evaluated by colorimetric method using the WST-1 assay. Flow cytometry was used to assess pERK1/2, apoptosis and senescence, using Annexin V-FITC and ß-galactosidase respectively. Experimental data showed that exposure to 7 µg mL(-1) of endosulfan per se increased cellular proliferation, but decreased the lymphoproliferative response to mitogenic stimulus with PMA + ionomycin. Splenocytes exposed to endosulfan for 15-180 min showed significantly higher levels of pERK1/2 than the non-exposed control. Endosulfan mediated a decrease in etoposide-induced apoptosis and provoked cell senescence. In conclusion, exposure of immune cells to a low concentration of endosulfan deregulates their function and may facilitate the development of multiple diseases.

Endosulfan increases seric interleukin-2 like (IL-2L) factor and immunoglobulin M (IgM) of Nile tilapia (Oreochromis niloticus) challenged with Aeromona hydrophila.

Endosulfan is a persistent organochlorine insecticide which is extremely toxic to fish. It is known to induce immunological alterations in juvenile Nile tilapia (Oreochromis niloticus) such as increases in phagocytic activity and reactive oxygen species production of spleen macrophages. The purpose of the present study was to demonstrate the effects of acute exposure to a sublethal concentration of endosulfan (7 ppb, 96 h) on parameters of the adaptive humoral immune response of the aforementioned aquatic organism. The effect of endosulfan on the capacity of immune cells to produce interleukin-2 like (IL-2L) factor and immunoglobulin M (IgM) in response to a challenge with (1/2) LD50 of the infectious bacteria Aeromonas hydrophila was evaluated. Experimental results indicate that short, sublethal, endosulfan exposure triggers a succession of events beginning with non-specific activation of macrophages followed by an exacerbated synthesis of the IL-2L factor by activated B cells. This leads to significantly increased secretion of IgM and could in turn facilitate autoantibody production and the development of autoimmune pathologies.

Oxidative stress in macrophages from spleen of Nile tilapia (Oreochromis niloticus) exposed to sublethal concentration of endosulfan.

Endosulfan is a widely used insecticide with immunosuppressive or immunopotentiating effects which alters the immune response of fish. The effects of the acute exposure to endosulfan on a series of parameters of the innate immune response (IIR) of Nile tilapia (Oreochromis niloticus) were investigated-phagocytosis, production of oxygen reactive species, lipoperoxidation as well as spleen cell viability, relative spleen weight and splenocyte concentration-to fully document the effects of this pesticide on Nile tilapia. Juvenile Nile tilapia were exposed in vivo and for 96h to each one of nine concentrations of endosulfan in order to determine the pesticide's acute toxicity level and calculate the lethal concentration of endosulfan to these organisms (LC(50)=12,795ppb). Functional assays showed that endosulfan, at a level equivalent to (1)/(2)LC(50), altered some parameters of the spleen macrophages of Nile tilapia. Phagocytosis, production of oxygen reactive species, and lipoperoxidation increased significantly in exposed fish. Spleen cell viability and relative spleen weight were lower in exposed organisms compared to non-exposed ones, without reaching statistical significance. Splenocyte concentration was not altered in the present experimental conditions. Thus, in vivo exposure (7ppb) of juvenile organisms stimulated the phagocytic activity up to significant oxidative stress levels as indicated by the increased lipid peroxidation in plasma. It can be concluded that short exposure to low concentration of endosulfan stimulated macrophage activity but that there was no significant reduction in the structural parameters of the IIR.

Effects of diazinon and diazoxon on the lymphoproliferation rate of splenocytes from Nile tilapia (Oreochromis niloticus): the immunosuppresive effect could involve an increase in acetylcholine levels.

The lymphoproliferation rate of spleen cells from Nile tilapia (Oreochromis niloticus) exposed to the organophosphorus pesticide diazinon, to its metabolite diazoxon and to the neurotransmitter acetylcholine, was evaluated in order to explore the immunotoxic mechanism of action of this widely used insecticide. The lymphoproliferative response of spleen cells to mitogenic stimulus was not affected by either diazinon or diazoxon, indicating that these xenobiotic substances do not have direct immunotoxic properties. Conversely, ex vivo assays showed that spleen from fish exposed to diazinon presented a lower acetylcholinesterase activity and a higher acetylcholine concentration than non-exposed controls. Lymphoproliferation assays also indicated that pre-exposure to acetylcholine depleted the proliferative function of spleen cells. Thus the combined information from in vitro and ex vivo experiments suggest that the immunotoxic properties of diazinon in Nile tilapia are indirect and could involve the cholinergic system of lymphocytes.

Polyamines as biomarkers of the antitumoral activity of Bursera fagaroides

En células normales y tumorales las poliaminas (Pas) putresina (Pu), espermidina (Spd) y espermina (Spm) son requeridas en múltiples funciones fundamentales del ciclo celular. Altos niveles de Pas han sido reportados en varios tipos de cáncer, por lo que han sido propuestas como biomarcadores del desarrollo tumoral. En el presente trabajo se reporta su utilidad como biomarcadores de la evolución del linfoma murino L5178Y en diferentes fluidos, células y tejidos. Los hallazgos también fueron aplicados al seguimiento del efecto antitumoral de Bursera fagaroides, ya reportado previamente. Se utilizó cromatografía de intercambio iónico para determinar los niveles de Pas en orina, células peritoneales linfocitos circulantes, esplenocitos, mesotelio e hígado de ratones BALB/c a los 10, 17 y 24 días de evolución del tumor y de ratones tratados con el exacto hidroalcohólico de la corteza de B. fagaroides administrado por vía oral o intaperitonealmente (i.p) los niveles urinarios de Spd no fueron detectables, mientras que el incremento de Pu en orina es el mejor biomarcador del crecimiento del linfoma. Además los niveles urinarios de Pu disminuyeron significativamente en ratones tratados intraperitonealmente con B. fagoroides, lo cual refuerza resultados anteriores. En este modelo la variación de los niveles urinarios de Pu es un biomarcador efectivo del desarrollo neoplástico, dado que permite seguir la evolución del linfoma L5178Y. Además, proporciona una herramienta para estudiar in vivo, el efecto antitumoral de B. fagaroides y de otros fármacos.

Immunotoxicity and hepatic function evaluation in Nile tilapia (Oreochromis niloticus) exposed to diazinon.

The LC(50) of the organophosphorus pesticides (OPs) diazinon to Nile tilapia (Oreochromis niloticus) was determined, thereafter, hepatic activity, phagocytic index, percentages of active cells, relative spleen weight, total IgM concentration and lymphoproliferation rates were compared between diazinon exposed groups (LC(50) and (1/2)LC(50)) and non-exposed control group. Experimental data show that diazinon is highly toxic for juvenile Nile tilapia (LC(50)=7.830 ppm) and presents immunotoxic properties which affect both the innate and cellular adaptive immune responses of this fish, as revealed by the fact that splenocyte proliferation and phagocytic indices were significantly decreased after acute exposure to the pesticide. However, the hepatic biochemical parameters and the total circulating IgM concentrations were not affected in this experimental model.

Traditional infrageneric classification of Gymnopilus is not supported by ribosomal DNA sequence data.

The traditional classification of Gymnopilus (Agaricales) recognizes two primary groups, Annulati and Gymnopilus, based on the presence or absence of a membranous partial veil. While our analyses of DNA sequence data from the nuclear ribosomal ITS1-5.8S-ITS2 (ITS) gene supports the monophyly of the genus, these traditional subgroups were not recovered. Five well-supported clades within the genus were identified through these analyses: 1) the spectabilis-imperialis group; 2) nevadensis-penetrans group; 3) a clade formed by G. underwoodii, G. validipes and G. cf. flavidellus; 4) aeruginosus-luteofolius group; and 5) lepidotus-subearlei group. Relationships among these subgroups were not resolved.