Panagiota Galanopoulou

Stress through handling for vaginal screening, serotonin, and ACTH response to ether.

The effect of duration of handling for vaginal smear screening on the adrenal weight and acute ACTH response to ether were examined in 4-day-cycling female rats, sacrificed at 97-103 days of age on diestrus-2 after evaluation of resistance to handling, thymus weight, and hypothalamic serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA). Prolonged handling paralleled increased resistance (behavioral response) to handling and adrenal weight but was inversely related to thymus weight. The hypothalamic 5-HT, 5-HIAA, and 5-HIAA/5-HT ratio, compared to controls with similar conditions of handling, were not modified after 2.5 min of ether despite the ACTH rise. In ether-stressed rats, the ACTH response to ether was lower after prolonged handling compared to short handling paralleling decreased thymus weight. In contrast, 5-HT, 5-HIAA, and the 5-HIAA/5-HT ratio were higher, paralleling increased resistance and adrenal weight. The results suggest chronic activation of the hypothalamo-pituitary-adrenal axis with positive serotonergic involvement after prolonged handling and resistance during vaginal screening and a negative implication of this activation on the acute ACTH response to ether.

Effects of short-term exposure to manganese on the adult rat brain antioxidant status and the activities of acetylcholinesterase, (Na,K)-ATPase and Mg-ATPase: modulation by L-cysteine.

Manganese (Mn) is an essential metalloenzyme component that in high doses can exert serious oxidative and neurotoxic effects. The aim of this study was to investigate the potential effect of the antioxidant L-cysteine (Cys, 7 mg/kg) on the adult rat brain total antioxidant status (TAS) and the activities of acetylcholinesterase (AChE), Na+,K+-ATPase and Mg2+-ATPase induced by short-term Mn administration (as Mn chloride, 50 mg/kg). Twenty-eight male Wistar rats were divided into four groups: A (saline-treated control), B (Mn), C (Cys) and D (Mn and Cys). All rats were treated once daily, for 1 week with intraperitoneal injections of the tested compounds. Rats were killed by decapitation and mentioned parameters were measured spectrophotometrically. Rats treated with Mn exhibited a significant reduction in brain TAS (-39%, P < 0.001, B versus A) that was partially reversed by Cys co-administration (-13%, P < 0.01, D versus A), while Cys (group C) had no effect on TAS. The rat brain AChE activity was found significantly increased by both Mn (+21%, P < 0.001, B versus A) and Cys (+61%, P < 0.001, C versus A), while it was adjusted into the control levels by the co-administration of Mn and Cys. The activity of rat brain Na+,K+-ATPase was not affected by Mn administration, while Mg2+-ATPase exhibited a slight but statistically significant reduction in its activity (-9%, P < 0.01, B versus A) due to Mn, which was further reduced by Cys co-administration. The above findings suggest that short-term Mn in vivo administration causes a statistically significant decrease in the rat brain TAS and an increase in AChE activity. Both effects can be, partially or totally, reversed into the control levels by Cys co-administration (which could thus be considered for future applications as a neuroprotective agent against chronic exposure to Mn and the treatment of manganism). The activity of Na+,K+-ATPase is not affected by Mn, while Mg2+-ATPase activity is slightly (but significantly) inhibited by Mn, possibly due to Mg replacement.

Effects of adult-onset streptozotocin-induced diabetes on the rat brain antioxidant status and the activities of acetylcholinesterase, (Na(+),K (+))- and Mg(2+)-ATPase: modulation by L-cysteine.

Uncontrolled diabetes is known to affect the nervous system. The aim of this study was to investigate the effect of the antioxidant L-cysteine (Cys) on the changes caused by adult-onset streptozotocin (STZ)-induced diabetes on the rat brain total antioxidant status (TAS) and the activities of acetylcholinesterase (AChE), (Na+,K+)-ATPase and Mg2+-ATPase. Thirty-eight male Wistar rats were divided into six groups: CA (8-week-control), CB (8-week-control + 1-week-saline-treated), C + Cys (8-week-control + 1-week-Cys-treated), DA (8-week-diabetic), DB (8-week-diabetic + 1-week-saline-treated) and D + Cys (8-week-diabetic + 1-week-Cys-treated). All diabetic rats were once treated with an intraperitoneal (i.p.) STZ injection (50xa0mg/kg body weight) at the beginning of the experiment, while all Cys-treated groups received i.p. injections of Cys 7xa0mg/kg body weight (daily, for 1-week, during the 9th-week). Whole rat brain parameters were measured spectrophotometrically. In vitro incubation with 0.83xa0mM of Cys or 10xa0mM of STZ for 3xa0h was performed on brain homogenate samples from groups CB and DB, in order to study the enzymes’ activities. Diabetic rats exhibited a statistically significant reduction in brain TAS (−28%, DA vs CA;−30%, DB vs CB) that was reversed after 1-week-Cys-administration into basal levels. Diabetes caused a significant increase in AChE activity (+27%, DA vs CA; +15%, DB vs CB), that was further enhanced by Cys-administration (+57%, D + Cys vs CB). The C + Cys group exhibited no significant difference compared to the CB group in TAS (+2%), but showed a significantly increased AChE activity (+66%, C + Cys vs CB). Diabetic rats exhibited a significant reduction in the activity of Na+,K+-ATPase (−36%, DA vs CA;−48%, DB vs CB) that was not reversed after 1-week Cys administration. However, in vitro incubation with Cys partially reversed the diabetes-induced Na+,K+-ATPase inhibition. Mg2+-ATPase activity was not affected by STZ-induced diabetes, while Cys caused a significant inhibition of the enzyme, both in vivo (−14%, C + Cys vs CB;−17%, D + Cys vs CB) and in vitro (−16%, DB + in vitro Cys vs CB). In vitro incubation with STZ had no effect on the studied enzymes. The present data revealed a protective role for Cys towards the oxidative effect of diabetes on the adult rat brain. Moreover, an increase in whole brain AChE activity due to diabetes was recorded (not repeatedly established in the literature, since contradictory findings exist), that was further increased by Cys. The inhibition of Na+,K+-ATPase reflects a possible mechanism through which untreated diabetes could affect neuronal excitability, metabolic energy production and certain systems of neurotransmission. As concerns the use of Cys as a neuroprotective agent against diabetes, our in vitro findings could be indicative of a possible protective role of Cys under different in vivo experimental conditions.

Effects of gestational and lactational choline deprivation on brain antioxidant status, acetylcholinesterase, (Na(+),K(+))- and Mg(2+)-ATPase activities in offspring rats.

Abstract Background: Choline plays an important role in brain development. Choline-deficient diet (CDD) is known to produce (among other effects) a decrease in acetylcholine in rat brains. The aim of our study was to investigate how CDD administration during gestation and lactation could affect total antioxidant status (TAS) and activities of acetylcholinesterase (AChE), (Na+,K+)- and Mg2+-ATPase in the brains of both male and female newborn and suckling (21-day-old) rats. Methods: Three different experiments were performed. Whole brains were obtained from: (a) newborn rats following gestational CDD (experiment I); (b) 21-day-old rats following gestational but not lactational CDD (experiment II); and (c) 21-day-old rats following gestational and lactational CDD (experiment III). Enzyme activities and TAS were measured spectrophotometrically. Results: In choline-deprived (CD) newborn rats, TAS and AChE and Na+,K+-ATPase activities were signi-ficantly reduced by 23%, 24% and 50%, respectively, in the brains of both sexes. Gestational CDD caused only a decrease in TAS (−27%, p<0.001) in suckling rat brains in both sexes. No changes were observed for the other enzyme activities. Moreover, gestational and lactational CDD also led only to a decrease in TAS (−24%, p<0.001) in the suckling rat brains of both sexes. Mg2+-ATPase activities showed no changes after any of the experimental procedures. Conclusions: Our data suggest that the lower enzyme activities in newborn CD brains were restored to normal after 21 days of either normal or CDD lactation, possibly due to novel synaptogenesis, endogenous neuroregulation, and/or to other substances acquired by lactation. The increase in homocysteine concentration due to choline deficiency reported in the literature may be the cause of the low antioxidant capacity observed in offspring rat brains. Brain Na+,K+-ATPase inhibition (induced by CDD) could result in modul-ations of neural excitability, metabolic energy production and neurotransmission. Clin Chem Lab Med 2007;45:651–6.

Choline-deprivation alters crucial brain enzyme activities in a rat model of diabetic encephalopathy

Diabetic encephalopathy describes the moderate cognitive deficits, neurophysiological and structural central nervous system changes associated with untreated diabetes. It involves neurotoxic effects such as the generation of oxidative stress, the enhanced formation of advanced glycation end-products, as well as the disturbance of calcium homeostasis. Due to the direct connection of choline (Ch) with acetylcholine availability and signal transduction, a background of Ch-deficiency might be unfavorable for the pathology and subsequently for the treatment of several metabolic brain diseases, including that of diabetic encephalopathy. The aim of this study was to shed more light on the effects of adult-onset streptozotocin (STZ)-induced diabetes and/or Ch-deprivation on the activities of acetylcholinesterase (AChE) and two important adenosinetriphosphatases, namely Na+,K+-ATPase and Mg2+-ATPase. Male adult Wistar rats were divided into four main groups, as follows: control (C), diabetic (D), Ch-deprived (CD), and Ch-deprived diabetic (D+CD). Deprivation of Ch was provoked through the administration of Ch-deficient diet. Both the induction of diabetes and the beginning of dietary-mediated provoking of Ch-deprivation occurred at the same day, and rats were killed by decapitation after 30xa0days (1xa0month; groups C1, D1, CD1 and D1+CD1) and 60xa0days (2xa0months; groups C2, D2, CD2 and D2+CD2, respectively). The adult rat brain AChE activity was found to be significantly increased by both diabetes (+10%, pu2009<u20090.001 and +11%, pu2009<u20090.01) and Ch-deprivation (+19%, pu2009<u20090.001 and +14%, pu2009<u20090.001) when compared to the control group by the end of the first (C1) and the second month (C2), respectively. However, the Ch-deprived diabetic rats’ brain AChE activity was significantly altered only after a 60-day period of exposure, resulting in a +27% increase (D2+CD2 vs. C2, pu2009<u20090.001). Although the only significant change recorded in the brain Na+,K+-ATPase activity after the end of the first month is attributed to Ch-deprivation (+21%, pu2009<u20090.05, CD1 vs. C1), all groups of the second month exhibited a statistically significant decrease in brain Na+,K+-ATPase activity (−24%, pu2009<u20090.01, D2 vs. C2; −21%, pu2009<u20090.01, CD2 vs. C2; −22%, pu2009<u20090.01, D2+CD2 vs. C2). As concerns Mg2+-ATPase, the enzyme’s activity demonstrates no significant changes, with the sole exception of the D2+CD2 group (+21%, pu2009<u20090.05, D2+CD2 vs. C2). In addition, statistically significant time-dependent changes concerning the brain Mg2+-ATPase activity were recorded within the diabetic (pu2009<u20090.05, D2 vs. D1) and the Ch-deprived (pu2009<u20090.05, CD2 vs. CD1) rat groups. Our data indicate that Ch-deprivation seems to be an undesirable background for the above-mentioned enzymatic activities under untreated diabetes, in a time-evolving way. Further studies on the issue should focus on a region-specific reevaluation of these crucial enzymes’ activities as well as on the possible oxidative mechanisms involved.

Acute liver acetaminophen toxicity in rabbits and the use of antidotes: a metabonomic approach in serum.

The metabonomic approach has been widely used in toxicology to investigate mechanisms of toxicity. In the present study alterations in the metabolic profiles, monitored by 1H‐NMR spectroscopy, on serum samples in acetaminophen (APAP)‐induced liver injury in rabbits were examined. Furthermore, the effect of the established antidote N‐acetylcysteine (NAC) and the proposed antidotes silybinin (SIL), cimetidine (CIM) and SIL/CIM was also investigated. A single dose of APAP (2 g kg−1 b.w., i.g.) was administered to rabbits and APAP combined with the antidotes SIL, CIM and NAC. Animals were sacrificed at 24 h post‐APAP treatment. Healthy untreated animals served as controls. 1H‐NMR spectra of serum samples were acquired and underwent principal component analysis (PCA). Acute liver injury was verified by histopathological examination and the alterations of serum biochemical enzymes AST and ALT. 1H‐NMR spectroscopy revealed variations in the serum metabolic profile of APAP‐intoxicated rabbits compared with controls. Co‐administration of APAP with NAC, CIM and SIL + CIM seems to ameliorate the metabolic profile of animals compared with simply APAP‐treated ones. In this study, the model of APAPinduced liver injury was successfully described using the 1H‐NMR based metabonomic approach in serum. Furthermore, the use of antidotes that reduced the toxic insult was also recorded using this technique. The combination of NMR spectroscopy and PCA is a rapid methodology, capable of detecting alterations in the metabolic profile, and produces adequate models that could be used for the characterization of unknown samples, both experimental and clinical, reinforcing its future use in clinical settings.Copyright

Effects of mesulergine treatment on diet selection, brain serotonin (5-HT) and dopamine (DA) turnover in free feeding rats

1. The effects of mesulergine, a 5-hydroxytryptamine (5-HT) receptor antagonist with dopamine (DA) agonistic properties, on rats diet selection over a seven day period and on 5-HT and DA turnover was studied. 2. Three groups of male Wistar rats were individually caged and ad libitum fed with a standard (SD) and 50% sweet carbohydrate enriched diet (CED). Food intake was measured daily 4 hrs and 24 hrs after i.p. injections of mesulergine (1 and 3 mg/kg) or vehicle. 5-HT and 5-HIAA in hypothalamus (Hy), Striatum (St) and hippocampus (Hi) as well as DA and DOPAC in (Hy) and (St) were assayed at the 8th day of the experiment. 3. There was a dose dependent increase of SD consumption 4 hrs after mesulergine treatment while the CED remained unchanged with total food intake dose dependently increased as a consequence. At 24 hrs measurements SD consumption was increased only for the dose of 1 mg/kg of mesulergine, while a dose dependent decrease of CED intake was observed. Total food intake was unchanged for the dose of 1 mg/kg and decreased with the dose of 3 mg/kg consequently. A dose dependent decrease of rats body weight was observed too. 4. A significant increase of 5-HIAA/5-HT ratio in (Hy) and (St) for the dose of 1 mg/kg and in (Hi) for the dose of 3 mg/kg with no changes of DA turnover were found. 5. The above data suggest a dual mode of action of mesulergine presented as a short term hyperphagia due to simultaneous antiserotonergic and dopaminergic activity and long-term hypophagia due to long-term agonistic effects of dopaminergic neurons.

Effects of choline-deprivation on paracetamol- or phenobarbital-induced rat liver metabolic response

Choline is an essential nutrient that seems to be involved in a wide variety of metabolic reactions and functions in both humans and rodents. Various pathophysiological states have been linked to choline deprivation (CD). The aim of the present study was to determine the effect of CD upon biochemical, histological and metabolic alterations induced by drugs that affect hepatic functional integrity and various drug metabolizing systems via distinct mechanisms. For this purpose, paracetamol (ACET) or phenobarbital (PB) were administered to male Wistar rats that were fed with standard rodent chow (normally fed, NF) or underwent dietary CD. The administration of ACET increased the serum aspartate aminotransferase levels in NF rats, while CD restricted this increase. On the other hand, ACET suppressed alkaline phosphatase levels only in CD rats. Moreover, CD prevented the PB‐induced increase of the mitotic activity of hepatocytes. The administration of ACET down‐regulated CYP1A2 and CYP2B1 expression in CD rats, while up‐regulating them in NF rats. The administration of PB suppressed CYP1A2 apoprotein levels in CD rats, whereas the drug had no effect on NF rats. The PB‐induced up‐regulation of CYP2B, CYP2E1 and CYP1A1 isozymes was markedly higher in CD than in NF rats. In addition, PB increased glutathione‐S‐transferase activity only in CD rats. Hepatic glutathione content (GSH) was suppressed by ACET in NF rats, whereas the drug increased GSH in CD rats. Our data suggest that CD has a significant impact on the hepatic metabolic functions, and in particular on those related to drug metabolism. Thus, CD may modify drug effectiveness and toxicity, as well as drug–drug interactions, particularly those related to ACET and PB. Copyright

Equilibrated diet restores the effects of early age choline-deficient feeding on rat brain antioxidant status and enzyme activities: the role of homocysteine, l -phenylalanine and l -alanine

Choline is an essential nutrient that seems to be involved in a wide variety of metabolic reactions and functions, that affect the developing brain. The aim of this study was to: (a)examine the effects of early age choline deficient diet (CDD) administration on the total antioxidant status (TAS) and the activities of acetylcholinesterase (AChE), (Na+,K+)-ATPase and Mg2+-ATPase in the rat brain, (b)investigate the effect of feeding restoration into an equilibrated diet on the above parameters, and (c)study the role of homocysteine (Hcy), l-phenylalanine (Phe) and l-alanine (Ala) in certain of the above effects. Male and female Wistar rats were continuously kept off choline (Ch) during their gestational period of life, as well as during the first 6xa0weeks of their post-gestational life. The animals were sacrificed by decapitation and their whole brains were rapidly removed and homogenated. Their enzyme activities were measured spectrophotometrically. Moreover, in vitro experiments were conducted in order to estimate the effects of Hcy (0.3xa0mM), Phe (1.2xa0mM) and/or Ala (1.2xa0mM) on the above parameters. The administration of CDD led to a statistically significant decrease of the rat brain TAS (−29%, pu2009<u20090.001) and to a significant increase of both AChE (+20%, pu2009<u20090.001) and (Na+,K+)-ATPase (+35%, pu2009<u20090.001) activities. Mg2+-ATPase activity was found unaltered. Equilibrated diet, administered to early age CDD-treated rats of both sexes for an additional period of 18xa0weeks, restored the above parameters to control levels. Moreover, the in vitro experiments showed that Hcy could simulate these changes (at least under the examined in vitro conditions), while both Phe and Ala act protectively against the CDD-induced effects on the examined rat brain enzyme activities. The effects of early age CDD-feeding on the examined parameters are proved to be reversible through restoration to equilibrated diet, while our data suggest a role for Hcy (as a causative parameter for the CDD-induced effects) and a possible protective role for Phe and Ala (in reversing the observed CDD-induced effects).

Combined thirty-day exposure to thioacetamide and choline-deprivation alters serum antioxidant status and crucial brain enzyme activities in adult rats.

Choline (Ch) is an essential nutrient that seems to be involved in a wide variety of metabolic reactions and functions that affect the nervous system, while thioacetamide (TAA) is a well-known hepatotoxic agent. The induction of prolonged Ch-deprivation (CD) in rats receiving TAA (through the drinking water) provides an experimental model of mild progressive hepatotoxicity that could simulate commonly-presented cases in clinical practice. In this respect, the aim of this study was to investigate the effects of a 30-day dietary CD and/or TAA administration (300xa0mg/L of drinking water) on the serum total antioxidant status (TAS) and the activities of brain acetylcholinesterase (AChE), Na+,K+-ATPase and Mg2+-ATPase of adult rats. Twenty male Wistar rats were divided into four groups: A (control), B (CD), C (TAA), D (CD+TAA). Dietary CD was provoked through the administration of Ch-deficient diet. Rats were sacrificed by decapitation at the end of the 30-day experimental period and whole brain enzymes were determined spectrophotometrically. Serum TAS was found significantly lowered by CD (−11% vs Control, pu2009<u20090.01) and CD+TAA administration (−19% vs Control, pu2009<u20090.001), but was not significantly altered due to TAA administration. The rat brain AChE activity was found significantly increased by TAA administration (+11% vs Control, pu2009<u20090.01), as well as by CD+TAA administration (+14% vs Control, pu2009<u20090.01). However, AChE was not found to be significantly altered by the 30-day dietary CD. On the other hand, CD caused a significant increase in brain Na+,K+-ATPase activity (+16% vs Control, pu2009<u20090.05) and had no significant effect on Mg2+-ATPase. Exposure to TAA had no significant effect on Na+,K+-ATPase, but inhibited Mg2+-ATPase (−20% vs Control, pu2009<u20090.05). When administered to CD rats, TAA caused a significant decrease in Na+,K+-ATPase activity (−41% vs Control, pu2009<u20090.001), but Mg2+-ATPase activity was maintained into control levels. Our data revealed that an adult-onset 30-day dietary-induced CD had no effect on AChE activity. Treatment with TAA not only reversed the stimulatory effect of CD on adult rat brain Na+,K+-ATPase, but caused a dramatic decrease in its activity (−41%). Previous studies have linked this inhibition with metabolic phenomena related to TAA-induced fulminant hepatic failure and encephalopathy. Our data suggest that CD (at least under the examined 30-day period) is an unfavorable background for the effect of TAA-induced hepatic damage on Na+,K+-ATPase activity (an enzyme involved in neuronal excitability, metabolic energy production and neurotransmission).