Alterations in kinetic and thermotropic properties of cerebral membrane-bound acetylcholineesterase during thioacetamide-induced hepatic encephalopathy: correlation with membrane lipid changes.

Acetylcholinesterase (AchE) is an important peripheral membrane-bound enzyme, crucial for cholinergic neurotransmission. Changes in AchE activity, kinetic and thermotropic properties were studied in hepatic encephalopathy (HE) associated with acute liver failure induced experimentally by the administration of the hepatotoxin thioacetamide (TAA). Activity of AchE decreased significantly following TAA administration. AchE from cerebral cortex membrane isolates of TAA-treated rats also showed a decrease in Vmax and an increase in Km. Arrhenius plots revealed considerable changes in the thermotropic behavior of AchE from membrane isolates obtained from TAA-treated rats as evident from the decreased transition temperature. A positive correlation was observed between changes in membrane cholesterol (r2=0.987), sphingomyelin (r2=0.99) levels and AchE activity, thus indicating that membrane lipid changes could underlie the observed changes in kinetic and thermotropic properties of membrane-bound AchE during TAA-induced HE.

Co-administration of C-Phycocyanin ameliorates thioacetamide-induced hepatic encephalopathy in Wistar rats.

Fulminant hepatic failure (FHF) is a condition with a sudden onset of necrosis followed by degeneration of hepatocytes, without any previously established liver disease, generally occurring within hours or days. FHF is associated with a wide spectrum of neuropsychiatric alterations ranging from stupor to coma, culminating in death. In the present study FHF was induced in rats by the administration of thioacetamide (TAA). Oxidative stress is thought to play a prominent role in the pathophysiology of cerebral changes during FHF leading to the assumption that antioxidants might offer protection. Hence, in the present study the protective effect of C-Phycocyanin (C-PC), a natural antioxidant, was evaluated on TAA-induced tissue damage. C-Phycocyanin was administered intraperitoneally twice at 24 h interval (50 mg/kg body weight) along with the hepatotoxin TAA (300 mg/kg body weight). The animals were sacrificed 18 h after the second injection of TAA treatment and various biochemical parameters were analysed in liver, serum and brain tissues. These studies revealed significant prevention of TAA-induced liver damage by C-PC, as evidenced by a) increase in survival rate; b) the prevention of leakage of liver enzymes (AAT and AST) and ammonia into serum; c) increase in prothrombin time and d) liver histopathology. Ultrastructural studies of astrocytes of different regions of brain clearly showed a decrease in edema after C-PC treatment. TAA-induced histopathological lesions in different regions of the brain namely cerebral cortex, cerebellum and pons medulla were significantly reduced by the co-administration of C-PC with TAA. Further C-PC treatment resulted in a) decrease in the levels of tryptophan and markers of lipid peroxidation and b) elevation in the activity levels of catalase, glutathione peroxidase in different regions of brain. These studies reveal the potential of C-PC in ameliorating TAA-induced hepatic encephalopathy by improving antioxidant defenses.

Phospholipid and cholesterol alterations accompany structural disarray in myelin membrane of rats with hepatic encephalopathy induced by thioacetamide.

Fulminant hepatic failure is often associated with a wide range of neurological symptoms which are collectively referred to as hepatic encephalopathy. Fulminant hepatic failure with associated hepatic encephalopathy has a poor prognosis with the currently available sure treatment being only liver transplantation. This is largely owing to the lack of understanding of critical factors involved in the etiology of the condition. Lipid changes have been implicated in cerebral derangements characteristic of hepatic encephalopathy. About 79% of the brain lipid is concentrated in the myelin fraction where they play an important role in ion balance and conduction of nerve impulses. Hence, in the present study we aimed to investigate changes in myelin lipid composition and structure. Myelin was isolated by sucrose density gradient centrifugation from cerebral cortex of male Wistar rats (250-300 g body weight) treated with 300 mg/kg body weight thioacetamide administered twice at 24h interval to induce hepatic encephalopathy. Significant decrease was observed in the cholesterol and phospholipids content of myelin from treated rats. Sphingomyelin, phosphatidylserine and phosphatidylethanolamine content also decreased significantly following 18 h of thioacetamide administration. However, phosphatidylcholine levels remained unaltered. Transmission electron microscopic observation of myelin membrane from cerebral cortex sections showed considerable disorganization in myelin structure. Increase in malondialdehyde levels precede lipid changes leading to the speculation that oxidative damage may be the critical factor leading to decrease in the anionic phospholipids. Changes in myelin were evident only in later stages of hepatic encephalopathy indicating that myelin alteration may not play a role in early stages of hepatic encephalopathy. Nevertheless, myelin alteration may have a crucial role to play in various psycho-motor alterations during later stages of hepatic encephalopathy.

Membrane alterations and fluidity changes in cerebral cortex during acute ammonia intoxication.

Acute ammonia intoxication is known to cause alterations in activities of several membrane bound enzymes like Na+ K+ ATPase, acetylcholine esterase and glutamate uptake in brain. The alteration in these membrane associated activities could be a consequence of altered membrane architecture. To probe this, the effect of pathophysiological concentrations of ammonia on lipid composition and fluidity of membranes isolated from cerebral cortex of rats, were investigated in the present study. Administration of acute doses of ammonium acetate caused depletion of membrane sphingomyelin and cholesterol levels thereby reducing cholesterol: phospholipid (C: PL) ratio. Levels of phosphatidylserine increased while those of phosphatidylcholine and phosphatidylethanolamine remain unaltered. Membrane fluidity estimations using 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) indicated no changes in core and surface membrane fluidity following ammonium acetate administration. Acute ammonia toxicity induced no alteration in bulk fluidity but a decrease in annular fluidity of membranes, as determined using pyrene fluorescence. Elevated levels of malondialdehyde and declined level of total thiols in cerebral cortex membranes of rats under acute ammonia intoxication indicated the existence of oxidative stress.

Effect of thyroid hormone depletion on monoamines and expression patterns of catfish GnRH in the air-breathing catfish, Clarias gariepinus.

Thyroid hormone is known to have profound effect on the efficient functioning of the reproductive system. The GnRH-gondaotropin (GTH) axis is the crucial axis involved in regulation of the various aspects of reproduction like spermatogenesis, synthesis of sex steroids and regulation of courting and spawning behavior. Earlier study from our laboratory has shown that thyroid hormone depletion causes a decrease in GTH and GnRH levels in pituitary and preoptic area, respectively. GnRH secretion in pre-optic area is largely regulated by the monoaminergic system mainly dopamine (DA), epinephrine (E), norepinephrine (NE) and serotonin (5-HT). The expression pattern of catfish GnRH transcripts tends to corroborate our earlier findings. Hence, in the current study we aimed to investigate the levels of monoamines in the preoptic area-hypothalamus (POA-H), so as to determine whether thyroid hormone depletion modulates the monoaminergic neurotransmission thereby affecting GnRH secretion. The levels of NE and L-dopa decreased significantly while that of DA was unaltered following thyroid hormone depletion. DA has an inhibitory effect on GnRH secretion. Findings from the present study suggest that the inhibitory tone is unaltered while the stimulatory tone influencing GnRH neurons in POA-H is decreased during thiourea induced thyroid hormone depletion.

Expression of 20beta-hydroxysteroid dehydrogenase and P450 17alpha-hydroxylase/c17-20 lyase during hCG-induced in vitro oocyte maturation in snake head murrel Channa striatus.

Partial cDNAs encoding carbonyl reductase like 20beta-hydroxysteroid dehydrogenase (20beta-HSD) and P450 17alpha-hydroxylase/c17-20 lyase (CYP17) were isolated from the ovary of snake head murrel and they exhibited high sequence identity to the Nile tilapia and rainbow trout, respectively. A low transcript level of both 20beta-HSD and CYP17 were detected in pre-vitellogenic follicles, while the transcript level was high in full-grown immature follicles. In hCG-induced in vitro oocyte maturation, we found a significant increase in 20beta-HSD transcript level after 2 h. The CYP17 transcripts also showed a considerable increase following hCG-induction compared to saline-treated controls. On the other hand, Western blot analysis demonstrated no significant change in the CYP17 protein level during hCG-induced in vitro oocyte maturation. Taken together, we suggest that in addition to 20beta-HSD, the CYP17 might have a role in the shift in steroidogenesis during meiotic maturation of snake head murrel.

Seabream GnRH: partial cDNA cloning, localization and stage-dependent expression in the ovary of snake head murrel, Channa striatus.

Vertebrate reproduction is under the neuroendocrine control of the hypothalamic decapeptide GnRH which synchronizes various reproductive events and influences other reproduction related aspects like spawning behavior and pheromonal action in fish. Multiple forms of GnRH peptides have been reported across diverse vertebrate and invertebrate classes. Here we report the partial seabream GnRH (sbGnRH) cDNA sequence cloned from the brain of Channa striatus (snake head murrel) a fresh water perciform with immense economic and medicinal value across Asiatic countries. sbGnRH mRNA was found in brain, gill and ovary of mature murrel with possible implications to the effect of GnRH on pheromonal phenomena and on reinitiation of oocyte meiosis. In keeping with the earlier reported role of GnRH in initiation of oocyte meiosis we here present evidence from RT-PCR, ICC demonstrating an increase in the level of sbGnRH mRNA in ovary from pre-vitellogenic to post-vitellogenic follicles.

Immunocytochemical localization of gonadotropins during the development of XX and XY Nile tilapia.

Gonadal development and steroidogenesis in teleosts is regulated by two gonadotropic hormones; luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Earlier studies in tilapia have shown that FSH-beta and LH-beta appear by 14 days after hatching (dah), results from the current study corroborate with these previous reports in tilapia. Here we demonstrate the appearance of LH in pituitary between 14 and 20 dah. In addition to this the present study primarily focuses on any possible differences in appearance of LH-beta and FSH-beta immunoreactivity between XX and XY population of Nile tilapia. LH immunoreactivity was found to be lower in pituitary of XX fish when compared to XY fish. The development of FSH-beta immunoreactivity in pituitary of the Nile tilapia is also presented. Overall, it remains to be established what significance these findings on the appearance of gonadotropins hold for sex differentiation in tilapia.

Thyroid hormone modulation of ovarian recrudescence of air-breathing catfish Clarias gariepinus.

In the present study, thiourea-induced thyroid hormone depletion and thyroxine (T(4)) 'overdose' were used as a strategy to understand the influence of thyroid hormones on ovarian recrudescence of juvenile (3-months-old), immature (8-months-old) and adult (1-year-old) air-breathing catfish, Clarias gariepinus. Thiourea-induced thyroid hormone depletion in juvenile catfish impaired ovarian development, but no significant effect was observed in immature catfish and during late stage of ovarian recrudescence of mature catfish. T(4) treatment in females undergoing late stages of ovarian recrudescence induced rapid oocyte growth by promoting its early entry into maturational phase as evident from the presence of more number of vitellogenic and post-vitellogenic follicles, decrease in aromatse immunoreactivity and reduced estradiol-17beta levels. Hence, thyroid hormones have an important role to play during early stages of ovarian development and vitellogenesis of catfish and also indicating that thyroid has a stage dependent effect on ovary.

Thiourea-induced alteration in the expression patterns of some steroidogenic enzymes in the air-breathing catfish Clarias gariepinus.

Previous study from our laboratory on thiourea-induced thyroid hormone depletion in mature male catfish demonstrated that thyroid hormones play a significant role in testicular function. In the present study, we aimed to analyze the changes in the expression pattern of several steroidogenic enzyme genes after thyroid hormone depletion using semi quantitative RT-PCR in both adult male and female catfish. There was a marked decrease in the 11beta-hydroxylase expression in the testis and liver while no change was observed in case of kidney. A significant decrease in 11beta-hydroxysteroid dehydrogenase transcript level in testis, liver and kidney were observed in the thiourea treated males. The results obtained corroborated with our earlier findings of testicular regression after thyroid hormone depletion. In females, expression of aromatase transcript increased in experimental group compared to control. There was no considerable change observed in the transcript level of 3beta-hydroxysteroid dehydrogenase, P450 17alpha-hydroxylase/C17-20-lyase, and 20beta-hydroxysteroid dehydrogenase in both males and females. Thus, thyroid hormones might exert modulating effect on steroidogenic enzyme genes at the transcription level.

Antifeedant activity of quinones from Ventilago madaraspatana.

The insect antifeedant activity of the quinones isolated from Ventilago madaraspatana was investigated by circular leaf disc dual choice bioassay. Amongst the quinones tested, Ventiloquinone A was the most effective antifeedant against Henosepilachna vigintioctopunctata and Spodoptera litura.

Influence of ethynylestradiol and methyltestosterone on the hypothalamo-hypophyseal-gonadal axis of adult air-breathing catfish, Clarias gariepinus.

Adult male and female air-breathing catfish Clarias gariepinus were treated with ethynylestradiol (EE(2)) and methyltestosterone (MT) at concentrations of 1microg/L, respectively for 21 days. EE(2) treatment caused disappearance of spermatids/sperm from several testicular lumen/lobules in males while MT treatment to females led to precocious ovarian development. EE(2) caused significant fluid retention in all tissues including peritoneal cavity and seminal vesicles. Immunocytochemical localization of catfish GnRH (cfGnRH) and luteinizing hormone (LH) in preoptic area-hypothalamus (POA-H) and pituitary, respectively, revealed decreased immunoreactivity (ir-) following EE(2) treatment in males. MT treatment however caused no observable change in cfGnRH ir- and a significant increase in LH ir- in females. Semi-quantitative RT-PCR analysis indicated that cfGnRH transcripts in POA-H decreased significantly following EE(2) and MT treatment in males and females, respectively. Levels of POA-H dopamine (inhibitory monoamine for gonadotropin [GTH] synthesis and release) increased following EE(2) and MT treatment in males and females while levels of serotonin and norepinephrine (GTH-stimulatory monoamines) decreased significantly. The results demonstrate a direct in vivo effect of sex steroid analogs on cfGnRH-LH axis and monoaminergic system vis-à-vis on gonads in addition to probable direct action on gonads.

Cloning and expression of StAR during gonadal cycle and hCG-induced oocyte maturation of air-breathing catfish, Clarias gariepinus.

Complementary DNAs encoding steroidogenic acute regulatory protein (StAR) have been isolated from different fish species, yet the relevance of StAR during gonadal cycle and more importantly in final oocyte maturation has not been assessed so far. A cDNA encoding StAR was isolated from the ovarian follicles of air-breathing catfish, Clarias gariepinus. Catfish StAR exhibited 55 to 72% identity at nucleotide level with other vertebrate orthologs. RT-PCR analysis of tissue distribution pattern demonstrated the presence of StAR mRNA in various tissues including gonads, kidney, liver, brain and intestine of catfish. Real-time RT-PCR analysis revealed high expression of StAR mRNA in the pre-spawning phase of ovary while it was low in preparatory, spawning and regressed phases. In testis, maximum expression was noticed during the preparatory phase. During human chorionic gonadotropin (hCG)-induced oocyte maturation, both in vitro and in vivo, StAR mRNA levels were augmented by 2 h and then declined gradually to reach basal levels by 12 h as that of saline-treated controls. Taken together, high level of expression during hCG-induced oocyte maturation vis-à-vis in spawning suggests a role for StAR, in addition to the steroidogenic enzyme genes in final oocyte maturation.

Seabream GnRH immunoreactivity in brain and pituitary of XX and XY Nile tilapia, Oreochromis niloticus during early development.

Seabream gonadotropin-releasing hormone (sbGnRH)-the chief preoptic area-hypothalamus (POA-H) form of GnRH in tilapia is involved in sexual maturation. In this study, we investigated the qualitative changes in ontogeny of sbGnRH immunoreactivity (ir-), between sexes to understand its impending role during sex differentiation. For this, the differences in immunocytochemical localization of sbGnRH in genetically male (XY) and female (XX) fish were studied from 1 day after hatching (dah), through the critical period of sex differentiation (7-21 dah) to 40 dah and mature Nile tilapia. Specific antisera against sbGnRH were used for immunolocalization. SbGnRH ir- neurons were observed in POA-H as early as 5 and 15 dah in XY fish and XX fish, respectively. Higher ir- was detected in the POA-H of XY tilapia compared with XX population till 10 dah. There was a qualitative drop in sbGnRH ir- neurons/cell bodies in POA-H around 20 dah till 30 dah in XY population compared with other durations. SbGnRH ir- cells were detected in pituitary of XX fish by 15 dah and in XY fish around 10 dah but seemed to drop down by 20 dah in XY whereas it continued to remain steady in XX fish. The sbGnRH ir- in XY fish showed a rise from 35 dah and thence till 40 dah. This study revealed subtle differences in POA-H and pituitary sbGnRH ir- during early development between genetic male and female fish with possible implications in sex differentiation.

Fulminant hepatic failure in rats induces oxidative stress differentially in cerebral cortex, cerebellum and pons medulla.

Hepatic Encephalopathy (HE) is one of the most common complications of acute liver diseases and is known to have profound influence on the brain. Most of the studies, available from the literature are pertaining to whole brain homogenates or mitochondria. Since brain is highly heterogeneous with functions localized in specific areas, the present study was aimed to assess the oxidative stress in different regions of brain-cerebral cortex, cerebellum and pons medulla during acute HE. Acute liver failure was induced in 3-month old adult male Wistar rats by intraperitoneal injection of thioacetamide (300 mg/kg body weight for two days), a well known hepatotoxin. Oxidative stress conditions were assessed by free radical production, lipid peroxidation, nitric oxide levels, GSH/GSSG ratio and antioxidant enzyme machinery in three distinct structures of rat braincerebral cortex, cerebellum and pons medulla. Results of the present study indicate a significant increase in malondialdehyde (MDA) levels, reactive oxygen species (ROS), total nitric oxide levels [(NO) estimated by measuring (nitrites + nitrates)] and a decrease in GSH/GSSG ratio in all the regions of brain. There was also a marked decrease in the activity of the antioxidant enzymes-glutathione peroxidase, glutathione reductase and catalase while the super oxide dismutase activity (SOD) increased. However, the present study also revealed that pons medulla and cerebral cortex were more susceptible to oxidative stress than cerebellum. The increased vulnerability to oxidative stress in pons medulla could be due to the increased NO levels and increased activity of SOD and decreased glutathione peroxidase and glutathione reductase activities. In summary, the present study revealed that oxidative stress prevails in different cerebral regions analyzed during thioacetamide-induced acute liver failure with more pronounced effects on pons medulla and cerebral cortex.

Changes in cerebral membrane lipid composition and fluidity during thioacetamide-induced hepatic encephalopathy.

Lipids are an essential structural and functional component of cellular membranes. Changes in membrane lipid composition are known to affect the activities of many membrane-associated enzymes, endocytosis, exocytosis, membrane fusion and neurotransmitter uptake, and have been implicated in the pathophysiology of many neurodegenerative disorders. In the present study, we investigated changes in the lipid composition of membranes isolated from the cerebral cortex of rats treated with thioacetamide (TAA), a hepatotoxin that induces fulminant hepatic failure (FHF) and thereon hepatic encephalopathy (HE). HE refers to acute neuropsychiatric changes accompanying FHF. The estimation of membrane phospholipids, cholesterol and fatty acid content in cerebral cortex membranes from TAA-treated rats revealed a decrease in cholesterol, phosphatidylserine, sphingomyelin, a monounsaturated fatty acid, namely oleic acid, and the polyunsaturated fatty acids gamma-linolenic acid, decosa hexanoic acid and arachidonic acid compared with controls. Assessment of membrane fluidity with pyrene, 1,6-diphenyl-1,3,5-hexatriene and 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene revealed a decrease in the annular membrane fluidity, whereas the global fluidity was unaffected. The level of the thiobarbituric acid reactive species marker for lipid peroxidation also increased in membranes from TAA-treated rats, thereby indicating the prevalence of oxidative stress. Results from the present study demonstrate gross alterations in cerebral cortical membrane lipid composition and fluidity during TAA-induced HE, and their possible implications in the pathogenesis of this condition are also discussed.

Thiourea-induced thyroid hormone depletion impairs testicular recrudescence in the air-breathing catfish, Clarias gariepinus.

We used thiourea-induced thyroid hormone depletion as a strategy to understand the influence of thyroid hormones on testicular recrudescence of the air-breathing catfish, Clarias gariepinus. Treatment with 0.03% thiourea via immersion for 21 days induced hypothyroidism (thyroid hormone depletion) as evidenced by significantly reduced serum T(3) levels. Thiourea-treated males had narrowed seminiferous lobules with fewer spermatozoa in testis, very little or no secretory fluid, reduced protein and sialic acid levels in seminal vesicles when compared to controls. The histological changes were accompanied by reduction in serum and tissue levels of testosterone (T) and 11-ketotestosterone (11-KT), a potent male specific androgen in fish. Qualitative changes in the localization of catfish gonadotropin-releasing hormone (cfGnRH) and luteinizing hormone (LH, heterologous system) revealed a reduction in the distribution of immunoreactive neuronal cells and fibers in thyroid depleted fish. Interestingly, thiourea-withdrawal group showed physiological and histological signs of recovery after 21 days such as reappearance of spermatozoa and partial restoration of 11-KT and T levels. These data demonstrate that thyroid hormones play a significant role in testicular function of catfish. The mechanism of action includes modulating sex steroids either directly or through the hypothalamo (GnRH)-hypophyseal (LH) axis.