Participation of Phosphatidylinositol-3 Kinase Signalling in Human Chorionic Gonadotropin, Bovine Insulin (B-Insulin) and Human-Insulin-Like Growth Factor-I Induced Oocyte Maturation and Steroidogenesis in the Grey Mullet, Mugil Cephalus.

CONTEXT: The grey mullet, Mugil cephalus, is an edible fish of high economic importance. Breeding biology with reference to hormonal/growth factor regulation of oocyte maturation needs to be known for its commercial production. OBJECTIVE: The present study was conducted to examine the potency of maturation inducing hormones, chorionic gonadotropin (hCG), bovine-insulin, and insulin like growth factor1 (h-IGF-1) I on ovarian steroidogenesis and oocyte maturation. DESIGN: The role of hormones and growth factors on steroidogenesis and oocyte maturation was investigated using specific inhibitors, Wortmannin for phosphatidylinositol-3 (PI3) kinase, trilostane for 3ß-hydroxysteroid dehydrogenase, 1-octanol and 1-heptanol for gap junctions, actinomycin D for transcription and cycloheximide for translation of signal molecules. METHODS: Actions of hormonal and growth factors were examined for steroidogenesis, by radioimmunoassay and oocyte maturation by germinal vesicle breakdown (GVBD). Specific inhibitors were used to determine the cell signaling pathways, PI3 kinase. RESULTS: All the inhibitors attenuated the hCG-induced oocyte maturation (GVBD%), steroidogenesis including transcription, translation, gap junctions and PI3 kinase signaling. These inhibitors failed to inhibit h-IGF-I and b-insulin-induced oocyte maturation, steroidogenesis, translation and PI3 kinase signaling. CONCLUSION: hCG induces oocyte maturation via steroid dependent pathway involving gap junctions, transcription, translation and PI3 kinase signaling, unlike h-IGF-I and b-insulin in the mullet.

Arecoline inhibits pineal-testis function in experimentally induced hypothyroid rats.

Arecoline is known to cause endocrine dysfunction. In the current article role of arecoline on pineal-testis activity was investigated in hypothyroid rats induced by propylthiouracil (PTU). PTU treatment caused thyroid dysfunction ultrastructurally with a fall in T3 and T4 levels followed by a rise of thyroid stimulating hormone (TSH) level. Pineal activity was impaired by PTU treatment, as evident from degenerated synaptic ribbons and mitochondria of the pinealocytes with depletion of pineal and serum N-acetyl serotonin and melatonin levels. Leydig cell function was suppressed, evident from reduced smooth endoplasmic reticulum and depletion of testosterone level. Sex accessories function was impaired by showing scanty rough endoplasmic reticulum with depletion of fructose and sialic acid levels. Arecoline treatment that caused pineal dysfunction and testicular stimulation in control rats, suppressed both pineal and testis functions after PTU treatment. The findings suggest that arecoline inhibits pineal-testis function in experimentally induced hypothyroid rats.

Arecoline plays dual role on adrenal function and glucose-glycogen homeostasis under thermal stress in mice.

Arecoline has biomedical importance, but it has untoward side effects on endocrine functions. The aim is to investigate its role on adrenal activity under thermal stress by ultrastructural and hormonal parameters in mice. Cold (4 °C) or heat (37 °C) stress, or arecoline (10 mg/kg body wt), each for 7 days in cold or heat stress stimulated adrenocortical activity ultrastructurally with an elevation of corticosterone level. Adrenomedullary activity was suppressed in cold stress with depletion of catecholamine levels. In heat stress, adrenomedullary activity was stimulated ultrastructurally with an elevation of catecholamine levels. Arecoline treatment alone, or in cold or heat stress suppressed adrenomedullary activity, judged by ultrastructural and hormonal parameters. Arecoline treatment caused hypoglycemia with an elevation of glycogen level, but cold or heat stress, or arecoline treatment in thermal stress caused hyperglycemia, with a fall in glycogen profile. Thus, arecoline in thermal stress plays a dual role on adrenal function and glucose-glycogen homeostasis in mice.

Arecoline ameliorates hyperthyroid condition in mice under cold stress.

Betel nut of Areca catechu is chewed by millions of people for increased capacity to work and stress reduction, but it contains arecoline that causes hypothyroidism. The aim is to investigate the role of arecoline on thyroid activity in cold stress in mice. Arecoline treatment (10 mg/kg body wt/day, for 7 d) caused a reduction in thyroid weight and ultrastructural degeneration of thyro-follicular cells with depletion of T3 and T4 levels compared with the control mice. Cold stress (4 °C for 2 h, twice daily, for 7 d) stimulated thyroid activity ultrastructurally with an elevation of T3 and T4 levels. Arecoline treatment in cold stress suppressed thyroid activity by showing reversed changes to those of cold stress. In contrast, TSH concentrations were consistently increased under all experimental conditions. The findings suggest that cold stress causes hyperthyroidism which arecoline can ameliorate in mice.

Arecoline cannot alter testicular dysfunction and pineal activation caused by noise in wistar rat.

Millions of people consume betel nut for increased capacity to work and for stress reduction. The nut contains arecoline, which has multiple side effects on endocrine functions. Objective of the work is to investigate pineal-testicular responses to noise and after arecoline treatment in noise in rats. Noise exposure (100 dB, 6 h daily, 10 days) caused pineal stimulation ultrastructurally and at indoleamines level. Leydig cell dysfunction with fall of testosterone level and suppression of sex accessories were noticed. In contrast, pineal activity was inhibited and reproductive functions were stimulated after arecoline administration, confirmed from reversed changes to those of noise. Arecoline treatment in noise exposure showed same results as in noise both in pineal and in reproductive functions. It is concluded that noise causes testicular dysfunction probably by gonadotropin suppression induced by pineal melatonin in noise. Furthermore, arecoline cannot prevent it in noise in rats.

ARECOLINE CANNOT ALTER PINEAL-TESTICULAR RESPONSES TO METABOLIC STRESS IN WISTAR RATS.

CONTEXT: Betel nut is consumed by millions of people for stress reduction and increased capacity to work. One of its components is arecoline which is useful for Alzheimer and schizophrenia; it also influences endocrine and gonadal functions. OBJECTIVE: Objective is to examine whether arecoline can influence pineal-testicular function in metabolic stress. DESIGN: Rats were deprived of food or water or treated them with arecoline, each separately for 5 days. SUBJECTS: Pineal and testis with sex accessories were studied. METHODS: Ultrastructural (pineal, testis, Leydig cells and prostate), hormonal (melatonin and testosterone) and other parameters (fructose and sialic acid) were examined. Pineal indoleamines were quantitated by fluorometric method; testosterone by ELISA, and carbohydrate fractions by spectrophotometric methods. RESULTS: Inanition/ water deprivation caused pineal stimulation ultrastructurally (with enlarged synaptic ribbons) and elevation of melatonin level, but reproductive dysfunction by ultrastructural degeneration of Leydig cells and prostate with fall of testosterone, fructose and sialic acid concentrations. Arecoline treatment showed reversed changes to those of metabolic stress, but arecoline treatment in metabolic stress showed same results as in metabolic stress. CONCLUSION: The findings suggest that arecoline cannot alter the action of metabolic stress on pineal-testicular activity in rats.

DUAL ACTION OF ARECOLINE ON ADRENAL FUNCTION AND GLUCOSE-GLYCOGEN HOMEOSTASIS IN METABOLIC STRESS IN MICE.

BACKGROUND: People chew betel nut (Areca catechu) for physical work and stress reduction, but it contains arecoline, which has both therapeutic value and untoward effects on endocrine and gonadal functions. OBJECTIVE: Aim of the present study is to investigate its role on adrenal with its target in metabolic stress in mice. MATERIALS AND METHODS: Mice were deprived of water / food, each for 5 days / treated with arecoline (10 mg / kg body wt daily for 5 days) / arecoline after water or food deprivation, for 5 days each. RESULTS: Water or food-deprivation caused adrenocortical hyperactivity, evident from abundance of enlarged mitochondria and smooth endoplasmic reticulum (SER) with elevation of corticosterone level (C: 68.31 ± 2.30, WD: 159.31 ± 4.10 / FD: 194.12 ± 3.40 µg/ mL). Arecoline treatment alone or in water deprivation (C: 68.31 ± 2.30, AR: 144.50 ± 4.33, AR+WD: 194.42 ± 3.35 µg/ mL) / food deprivation (AR + FD: 180.89 ± 4.51 µg/ mL) stress also stimulated adrenocortical activity as recorded in metabolic stress. In contrast, adrenomedullary activity was not altered following water/ food deprivation. Arecoline treatment alone or in metabolic stress suppressed adrenomedullary activity by showing depletion of chromaffin granules (E/NE?), epinephrine (E) and norepinephrine (NE) concentrations. Both the stress decreased blood glucose and liver glycogen levels. Arecoline treatment decreased blood glucose level, with a rise in liver glycogen level, but elevated blood glucose level in water deprivation unlike in starvation. CONCLUSION: Arecoline alone or in metabolic stress involves adrenal and probably other endocrine glands (pancreas, posterior pituitary and rennin-angiotensin system) to maintain homeostasis in metabolic stress in mice.

Arecoline aggravates hypothyroidism in metabolic stress in mice.

Millions of people consume betel nut for increased capacity of work. It contains arecoline which is highly toxic and has several untoward side effects on endocrine functions. In this article, the role of arecoline on thyroid function under metabolic stress was investigated in mice. Water or food-deprivation, each for 5 days, caused ultrastructural degeneration of thyro-follicular cells, evident from pycnotic nuclei, scanty rough endoplasmic reticulum and mitochondria followed by depletion of blood serum T3 and T4 levels with alteration of TSH level as compared with control. Thyroid activity was also suppressed ultrastructurally as well as at hormonal level after arecoline administration. Further, arecoline treatment in water deprivation or food deprivation stress also caused thyroid dysfunction beyond that of metabolic stress, as evident from further ultrastructural degeneration of thyrocytes and depletion of thyroid hormones in mice. The findings suggest that arecoline aggravates hypothyroid condition in metabolic stress in mice.

In vitro induction of oocyte maturation and steroidogenesis by gonadotropins, insulin, calcitonin and growth factor in an estuarine flat head grey mullet, Mugil cephalus L.

In this article, an in vitro investigation was carried out to ascertain the roles of hormones and growth factor in the inductions of oocyte maturation and steroidogenesis of the postvitellogenic follicles in an Indian estuarine grey mullet, Mugil cephalus L. Oocyte maturation was evaluated by scoring the germinal vesicle breakdown (GVBD) percent of the postvitellogenic follicles. All the sex [17α,20ß-dihydroxy-4-pregnane-3-one (DHP), estradiol 17ß (E2), progesterone (P), 17α-OH progesterone (17-OH-P) and testosterone] and other [bovine-insulin and salmon-calcitonin, human chorionic gonadotropin (hCG), luteinizing hormone (LH) or hCG+DHP] hormones and insulin-like growth factor-I (IGF-I) significantly increased GVBD% in 9 h culture. DHP had a maximum effect (75 %) compared to other effectors. Some effectors (hCG: 82.14 %, LH: 78.94 %, hCG plus DHP: 81.81 %, E2: 80 % and IGF-I: 74.19 %) including DHP (79 %) further increased GVBD% in 15-h culture. All the hormones (except DHP) and IGF-I increased DHP, E2 and testosterone productions by the postvitellogenic ovarian follicles in vitro. DHP and testosterone productions were increased with the increase of incubation time from 9 h through 15 h. E2 production was not further increased beyond 12 h. DHP production was highest by hCG compared to other effectors. The hCG of all the test compounds was most effective in both the induction of GVBD% and steroid production. DHP is the most potent inducer of oocyte maturation in Indian estuarine flat head grey mullet. Involvement of estrogen in mullet oocyte maturation is indicated. hCG, like DHP, is equally potent and induces oocyte maturation via DHP production in vitro. hCG with DHP has synergistic action on oocyte maturation in mullet ovary. Interplay of several hormones (hCG, LH, and probably E2 and testosterone) and IGF-I on oocyte maturation is suggested in the mullet.

Ultrastructural and hormonal modulations of the thyroid gland following arecoline treatment in albino mice.

Arecoline is a plant alkaloid of betel nut Areca catechu. Arecoline has immunosuppressive, hepatotoxic, mutagenic and teratogenic effects, and disturbs some endocrine organs in rats. The objective is to investigate the untoward effects of arecoline on the thyroid gland in mice. Intraperitoneal injection of arecoline (10 mg/kg body weight only once) increased the serum T(3) and T(4) levels and decreased the serum TSH 20, 40 or 60 min after the treatment, with maximum effect at 40 min. Chronic arecoline treatment (10 mg/kg body weight daily for 15 days) caused light microscopic and ultrastructural degenerations of thyro-follicular cells with depletion of T(3) and T(4) levels followed by the elevation of the TSH level. Atropine (arecoline antagonist) injection prevented the changes (hyperactivity) induced by acute (40 min) arecline treatment. Arecoline initially stimulates thyroid activity, and eventually inhibits the activity; atropine prevents thyroid dysfunction induced by arecoline. Arecoline action is mediated probably via muscarinic cholinergic receptor-hypothalamic-pituitary-thyroid axis in mice.

The association of metabolic syndrome with triple-negative breast cancer.

Metabolic syndrome, a conglomerate of obesity, insulin resistance, dyslipidemia, and hypertension has been linked with an increased risk of breast cancer. We investigated the possible association of highly aggressive triple-negative breast cancer and the metabolic syndrome. Information on metabolic syndrome components and tumor characteristics were reviewed in a cohort of 176 patients (including 86 triple-negatives). Retrospective comparison was performed using Pearson Chi-square test or Student's t test for data analysis. A statistically significant association of triple-negative breast cancer with the metabolic syndrome was observed. In accordance with the NCEP (National Cholesterol Education Program) definition, 58.1% of triple-negative patients had metabolic syndrome compared to only 36.7% of non-triple-negative patients (P = 0.004). Consistently, by the AACE (American Association of Clinical Endocrinologists) criteria, 52.3% of triple-negative patients had metabolic syndrome as compared to 34.4% of non-triple-negative patients (P = 0.017). Blood glucose, triglyceride, and HDL levels but not hypertension or BMI (body mass index) showed significant independent association with triple-negative breast cancer. Additionally, triple-negative tumors displayed a significantly higher histological grade and relative paucity of ductal carcinoma in situ (DCIS) when compared to the non-triple-negative tumors (P < 0.001). Our study suggests that metabolic syndrome is significantly more prevalent in triple-negative breast cancer patients as opposed to non-triple-negative patients. Furthermore, triple-negative breast cancer showed a significantly higher histological grade and a relative absence of DCIS. Whether the presence of metabolic syndrome preferentially increases the risk of developing triple-negative-breast cancer remains to be elucidated with future prospective studies.

Cloning and expression of an outer membrane protein OmpW of Aeromonas hydrophila and study of its distribution in Aeromonas spp.

AIMS: The main aims of this study were to clone and express an outer membrane protein (OMP), OmpW, of Aeromonas hydrophila and to study its distribution in Aeromonas spp. METHODS AND RESULTS: The gene encoding OmpW in A. hydrophila has been cloned and expressed in Escherichia coli. Primers were designed for amplification of full-length ompW gene and used for identification of this gene in different Aeromonas spp. Of the 42 Aeromonas strains tested, all the isolates were positive by polymerase chain reaction (PCR) except one strain of Aeromonas veronii biovar veronii (VTE338). None of the other gram-negative bacteria were positive by PCR with primers specific to ompW gene of A. hydrophila. Polyclonal antibodies were raised in rabbit against the purified recombinant protein and the reaction of these antibodies was confirmed by western blotting using the purified recombinant protein and 42 Aeromonas cultures grown at various salt concentrations. CONCLUSIONS: The ompW-based PCR method developed in this study was found to be 100% specific and 97% sensitive. Expression of OmpW protein of Aeromonas was found to be salt-dependant. Recombinant OmpW protein was found to be highly immunogenic in fish. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report on cloning and expression of OmpW protein of A. hydrophila. Full-length ompW gene amplification by PCR can be used for the detection of Aeromonas. Recombinant OmpW protein can be useful for vaccination of fish against Aeromonas spp.

Adrenomedullary and glycemic alterations following diverse stress in soft-shelled turtles Lissemys punctata punctata Bonnoterre.

The objective of the current investigation was to study adrenomedullary and glycemic responses to stress in soft-shelled turtles, Lissemys p. punctata. Dehydration (7 days) and formalin (formaldehyde 1%, 0.1 mL/100 g body wt. daily for 7 days) stress-stimulated adrenomedullary activity at histological (by increasing the nuclear diameter and degranulation of chromaffin cells) and hormonal levels (by elevations of norepinephrine and epinephrine concentrations) with hyperglycemia in turtles. But salt loading (NaCl: 1%, 1 mL/100 g body wt. daily for 7 days) had no significant effect on adrenomedullary activity or glycemia presumably owing to the nonresponsiveness of adrenocortical activity to salt stress in turtles. It is suggested that dehydration and formalin stresses might have exerted their actions through the hypothalamo (CRF)-hypophysial (ACTH)-adrenocortical axis in turtles.

Thyroidal inhibition following diverse stress in soft-shelled turtle, Lissemys punctata punctata bonnoterre.

The current study was undertaken to ascertain the effects of diverse stress on thyroid activity in soft-shelled turtles, Lissemys punctata punctata. The findings revealed that starvation (10 days), dehydration (10 days) or exposure to electric shock (12 volts for 15 seconds at an interval of 30 min for 3 h) caused significant decrease in the body weight (except in electric shock), relative weight, peripheral and central epithelial heights of the follicles and peroxidase activity of the thyroid gland of turtles. The degree of change in the values of these parameters was nearly same in all the stress experiments, indicating that there is not much difference in the degree of thyroid responses to diverse stress in turtles. It is suggested that these stressors might have exerted their actions on thyroid activity presumably indirectly via adrenal medulla and/or substance in metabolic stress (starvation and dehydration) and via hypothalamo-hypophysial-adrenocortical axis in non-metabolic stress (electric shock) in Lissemys turtles.

Ultrastructural and hormonal changes in the pineal-testicular axis following arecoline administration in rats.

Arecoline is an alkaloid of betel nut of Areca catechu. Betel nut is chewed by millions of people in the world and it causes oral and hepatic cancers in human. It has therapeutic value for the treatment of Alzheimer and schizophrenia. Arecoline has immunosuppressive, mutagenic and genotoxic effects in laboratory animals. It also affects endocrine functions. The objective of this study was to investigate the effects of arecoline on pineal-testicular axis in rats. Since pineal activity is different between day and night, the current study is undertaken in both the photophase and scotophase. The findings were evaluated by ultrastructural and hormonal studies of pineal and testicular Leydig cells, with quantitations of fructose and sialic acid of sex accessories. Arecoline treatment (10 mg/kg body weight daily for 10 days) caused suppression of pineal activity at ultrastructural level by showing dilatation of the cisternae of the rough endoplasmic reticulum (RER), large autophagosome-like bodies with swollen mitochondrial cristae, numerous lysosomes, degenerated synaptic ribbons and reduced number of synaptic-like microvesicles. Moreover, pineal and serum N-acetylserotonin and melatonin levels were decreased with increased serotonin levels in both the gland and serum. In contrast, testicular Leydig cell activity was stimulated with abundance of smooth endoplasmic reticulum (SER), electron-dense core vesicles and vacuolated secretory vesicles, and increased testosterone level in the arecoline recipients. Consequently, the testosterone target, like prostate, was ultrastructurally stimulated with abundance of RER and accumulation of secretory vesicles. Fructose and sialic acid concentrations were also significantly increased respectively in the coagulating gland and seminal vesicle. These results were more significant in the scotophase than the photophase. The findings suggest that arecoline inhibits pineal activity, but stimulates testicular function (testosterone level) and its target organs presumably via muscarinic cholinergic receptor in rats.

Thyroid responses to altered photoperiod in the soft-shelled turtle Lissemys punctata punctata bonnoterre.

The aim of the current investigation was to investigate the effect of photoperiod on thyroid activity in soft-shelled turtles (Lissemys punctata punctata). Thirty days exposure of short photoperiod with 2L:22D increased relative weight, follicular epithelial height and peroxidase activity of the thyroid gland; whereas exposure of long photoperiod with 22L:2D for 30 days showed reversed changes to those of the short photoperiod in adult female turtles. These findings indicate that short photoperiod stimulates thyroid activity and long photoperiod inhibits its activity in soft-shelled turtles. It is suggested that photoperiod exerts its action on thyroid activity presumably via gonads and/or pineal-gonadal axis in turtles.

Immunosuppression, hepatotoxicity and depression of antioxidant status by arecoline in albino mice.

BACKGROUND: There are about 600 million betel quid chewers in the world. Betal quid chewing is one of the major risk factors of hepatocarcinoma, oropharyngeal and esophagus cancers. Arecoline, the main Areca alkaloid of the betel nut is reported to have cytotoxic, genotoxic and mutagenic effects in various cells. It shows strong correlation to the incidence of oral submucosal fibrosis, leukoplakia and oral cancer, and has also been found to impose toxic manifestations in immune, hepatic and other defense systems of the recipient. AIM: The precise molecular mechanisms underlying the toxic effects of arecoline deserve investigation. To clarify the action of arecoline on defense systems, immune, hepatic and detoxification system were studied in mice. METHOD: Cell count and cell cycle of the splenocytes were studied for evaluating cell immunity. Liver function test (LFT) was followed by assaying different enzyme systems from serum (SGPT, SGOT and ALP) and liver (GST for detoxication enzyme, SOD and catalase for antioxidant enzymes and GSH for non-enzymatic antioxidant) and by ultrastructural studies of hepatocytes. RESULTS: Here we report that arecoline arrested splenic lymphocyte cell cycle at lower concentration with induced apoptosis at higher concentration thereby causing immunosuppression in arecoline recipients. Besides, it resulted in hepatotoxicity in arecoline recipient mice by disrupting the hepatocyte ultrastructure, as judged by liver ultrastructural studies that showed decreased nuclear size, RER with profusely inflated cysternae and abundance of lipid droplets, and by up regulating hepatotoxic marker enzymes (SGOT and SGPT) in serum. Arecoline also caused depression of antioxidants, i.e., superoxide dismutase (SOD), catalase, reduced glutathione (GSH) and glutathione-S-transferase (GST) that are known to neutralize reactive oxygen species. CONCLUSION: All these above-mentioned results led us to conclude that arecoline attacks multiple targets to finally generate systemic toxicity in mice.

Roles of thyroid, adrenal and pancreatic hormones on thyroid activity of the soft-shelled turtles Lissemys punctata punctata Bonnoterre.

The effects of some exogenous peripheral hormones (thyroxine, corticosterone, epinephrine, norepinephrine and insulin) on thyroid activity were investigated in juvenile female soft-shelled turtles, Lissemys punctata punctata. Each hormone was injected in three different doses (25 microg, 50 microg or 100 microg each per 100 g body weight, once daily at 9 AM) for 10 consecutive days. Thyroid activity was evaluated by gravimetry, histology (epithelial height) and thyroperoxidase assay. The findings revealed that thyroxine in low dose (25 microg) stimulated thyroid activity by increasing the relative thyroid weight, epithelial height and thyroperoxidase activity, but inhibited gland activity at a high dose (100 microg) by decreasing the values of all these parameters. The medium dose (50 microg) had no significant effect. All other hormones, in all doses, significantly decreased thyroid activity by decreasing the values of all the parameters. Thyroid responses to exogenous hormones are generally dose-dependent in turtles. The mechanisms of actions of the hormones administered are suggested.

Separation and preconcentration of U(VI) on XAD-4 modified with 8-hydroxy quinoline.

Amberlite XAD-4 adsorber resin was modified with 8-hydroxy quinoline (Oxine) by equilibrating with methanol solution of the reagent and the modified resin was used as a support material for the solid phase extraction and preconcentration of UO(2)(2+) from aqueous solution at pH between 4 and 5.5. Ten micrograms of uranium from 300 ml of aqueous phase could be quantitatively extracted in to 1g of the modified resin giving an enrichment of 200. Uranium collected in the column could be eluted out with methanol-HCl mixture and determined spectrophotometrically using arsenazo(III) as the chromogenic reagent. The preconcentration could be made selective to uranium by using EDTA as a masking agent for transition metal ions and Th(IV).