Studies on cigarette smoke induced oxidative DNA damage and reduced spermatogenesis in rats.

In the present work, the effect of exposure to cigarette smoke on male fertility in rats, as characterized by changes in the relative weight of sex organs, epididymal sperm count, activity of marker enzymes and DNA damage was evaluated. Exposure of rats to cigarette smoke caused a gradual decrease in total body weight gain and relative weight of the epididymis and seminal vesicles by 30 and 40% respectively. Epididymal sperm count was reduced significantly by 25% (P 0.05) after 2 weeks and by 41% (P 0.001) after 4 weeks of exposure. Exposure to cigarette smoke had reduced the activity of sorbitol dehydogenase by 18% (P < or = 0.05) and increased the activity of lactate dehydrogenase by 28% (P < or = 0.05). The changes in both key enzymes were significant, which reflected the inhibitory effect of cigarette smoke on spermatogenesis and sperm maturation. The toxic effect of exposure could be explained partially due to induction of DNA damage and oxidative stress as shown by the significant increase in serum 8-hydroxy-2'-deoxyguanosine from 22.83 to 37.33 ng ml(-1) blood.

Specific group II metabotropic glutamate receptor activation inhibits the development of kindled epilepsy in rats.

The effects of intracerebral administration of the group II metabotropic glutamate receptor agonist, 2R,4R-APDC, were tested on both the development of amygdaloid kindling and on fully developed stage 5 amygdala kindled seizures. The development of amygdaloid kindling was significantly retarded in 2R,4R-APDC (10 nmol in 0.5 microl) treated animals compared to control animals over a period of 8 days. At a low dose, 2R,4R-APDC (0.1 nmol) caused a 42.5+/-26.6% increase of the generalised seizure threshold in fully kindled animals. As higher doses were administered, however, the changes in generalised seizure threshold were less marked, and even a small decrease in the threshold was seen (-19.6+/-5.36% at 10 nmol). The agonist 2R,4R-APDC inhibited depolarization-induced release of [3H]d-aspartate from cortical synaptosomes with an IC50 value of 0. 29 microM. This effect was maximal at 1 microM, and decreased with dose thereafter. These findings suggest that the selective activation of the group II metabotropic glutamate receptors by agonists such as 2R,4R-APDC may be of therapeutic potential in the treatment of seizure disorders.

The effect of 2-amino-3-arsonopropionate and 2-amino-4-arsonobutyrate on the development and maintenance of amygdala kindled seizures.

The effects of 2-a-3-arsonopropionate and 2-a-4-arsonobutyrate, the arsono analogues of aspartate and glutamate respectively, on the development of electrically-induced kindling in the amygdala, and on seizures induced in fully kindled rats, were compared to the effects of 3-amino-propylarsonate the arsono analogue of GABA. Intra-amygdaloid micro-injection of 2-a-3-arsonopropionate and 2-a-4-arsonobutyrate (10 nmol in 0.5 microl buffer phosphate) reduced the rate of epileptogenesis without preventing the development of generalized seizure responses, after 14 daily stimulations. In fully electrically kindled animals with stage 5 amygdala-kindled seizures, 3-aminopropy-larsonate (10 nmol/0.5 microl) increased after-discharge threshold (ADT) by 82% (P< or =0.005) without having any effect on mean seizure score or after-discharge duration. Chemical reduction of 3-aminopropylarsonate with glutathione diminished the anti-seizure activity of the drug. 2-a-3-arsonopropionate and 2-a-4-arsonobutyrate the arsono analogues of aspartate and glutamate were not effective when they were micro-injected into the amygdala of fully kindled animals at equivalent doses i.e. (10 nmol/0.5 microl). Higher doses (100 nmol/0.5 microl) of 2-a-3-arsonopropionate the analogue of aspartate increased the generalized seizure threshold by 40% (P < or = 0.025), while 2-a-4-arsonobutyrate was not effective even at high doses.

Effect of aqueous extract of Commiphora opobalsamum on blood pressure and heart rate in rats.

The cardiovascular effects of aqueous extracts from the branches of Commiphora opobalsamum tree were investigated. The intravenous administration of 4 mg/kg of the aqueous extract depressed systemic arterial blood pressure by 20% (P < 0.01) and reduced heart rate of anaesthetised rats by 14% (P < 0.05). The hypotensive and the bradycardiac effects were immediate and in a dose related manner. The hypotensive effect of C. opobalsamum was inhibited by the pretreatment with atropine sulfate (1-4 mg/kg). These results suggest that the hypotensive effect of C. opobalsamum is due to the activation of muscarinic cholinergic receptors.

Anti-epileptogenic and anticonvulsant activity of L-2-amino-4-phosphonobutyrate, a presynaptic glutamate receptor agonist.

The protective effect of amygdaloid (focally administered) doses of the presynaptic metabotropic glutamate receptor agonist, L-2-amino-4-phosphonobutyrate (L-AP4) was tested on the development of electrical kindling and in fully kindled animals. L-AP4 inhibited epileptogenesis at 10 nmol in 0.5 microl buffer, by preventing the increase in both seizure score and afterdischarge duration. The effects were reversible after withdrawal of the drug, with all treated animals subsequently progressing to the fully kindled state at the same rate as control animals. The same concentration of the drug was also effective when injected into fully kindled animals. It significantly decreased the mean seizure score by 88% (P < 0.005) and increased the mean generalized seizure threshold (GST) by 85% (P < 0.005). The increase in GST was accompanied by a significant delay before the onset of generalized seizure and by a 37% reduction in generalized seizure duration. MPPG ((RS)-alpha-methyl-4-phosphonophenyl glycine) a selective antagonist of L-AP4 at glutamate pre-synaptic receptors inhibited the depressant effect of L-AP4 in a dose-dependent manner. MPPG (10 nmol) inhibited the antiseizure activity of L-AP4, whilst MPPG (40 nmol) reduced both the anti-epileptogenic and antiseizure activities of L-AP4. MPPG (40 nmol) by itself had no effect on generalized seizure activity, and it had no detectable influence on the normal rate of kindled epileptogenesis. During in vitro studies using a microsuperfusion method, L-AP4 inhibited depolarization-induced release of [3H]D-aspartate from rat cortical synaptosomes (IC50 125.1 microM) and decreased the depolarization-evoked uptake of 45Ca2+ in a dose-dependent manner. Both actions of L-AP4 were reduced by the selective antagonist MPPG. When applied alone MPPG (200 microM) had no detectable action on veratridine-evoked 45Ca2+ uptake by the synaptosomes. These results suggest the mechanisms by which presynaptically active glutamate receptor agonists block the development of the chronically epileptic state induced by electrical kindling, and indicate that their anticonvulsive activity is due to inhibition of presynaptic glutamate and/or aspartate release following blockade of presynaptic Ca2+ entry.

The protective effect of 2-chloroadenosine against the development of amygdala kindling and on amygdala-kindled seizures.

The influence of 2-chloroadenosine, a non-metabolizable adenosine A1 receptor agonist, was tested on the development of electrically kindled amygdala and on the seizure responses of fully kindled rats. Focal intra-amygdaloid injection of 2-chloroadenosine (1-10 nmol/0.5 microl) 20 min before applying the daily kindling stimulus prevented the development of the kindling process. The behavioural seizure score and the afterdischarge duration were reduced below their initial values. The antiepileptogenic effects of 1 and 10 nmol of 2-chloroadenosine were reversible 8-10 days after withdrawal of the drug. When 2-chloroadenosine was tested on fully developed stage 5 amygdala-kindled seizures, it increased the generalised seizure threshold in a dose-dependent manner. A maximum efficiency of 125% (P < 0.001) was achieved with 5 nmol and the median effective dose was 0.55 nmol. Higher doses resulted in the reduced anticonvulsant effect (P < 0.05). With the same daily stimulation, 2-chloroadenosine 5 nmol in 0.5 microl vehicle, significantly reduced the maximum seizure score by 90%, the afterdischarge duration by 88% and completely blocked the generalised seizure duration. The antiseizure activity of the drug lasted for 3 days. In conclusion, 2-chloroadenosine not only acts as an anticonvulsant against electrically induced kindled seizures as described here, and against audiogenic seizures, electroshock and a variety of chemical convulsants as described by others, it prevents the development of the epileptic state by kindling-stimulation, i.e., it is antiepileptogenic. We theorise here that this is due to its blockade of presynaptic glutamate release.

The anti-epileptic effect of 3-aminopropylarsonate on electrically-kindled and N-methyl-D-aspartate-kindled amygdala.

The effects of 3-aminopropylarsonate, an arsono analogue of GABA, was tested on the development of electrically-kindled amygdala and on the expression of generalized seizure activity in electrically and NMDA fully amygdala-kindled rats. Intra-amygdaloid microinjection of 3-aminopropylarsonate (10 nmol in 0.5 microl injection vehicle) inhibited electrical epileptogenesis by keeping the seizure score at or below stage 1 on the Racine scale, and the afterdischarge duration (ADD) at or below 19.70 +/- 4.59 s. The effect was reversible after withdrawal of the drug, since the animals developed a generalized seizure activity when kindling stimuli continued in the absence of drug. In fully electrically kindled animals with stage 5 amygdala-kindled seizures, the drug increased afterdischarge threshold (ADT) by 30-70%, without any effect on mean seizure score or ADD. The changes were reversible after 7 days. In fully NMDA-kindled rats, intra-amygdala administration of 3-aminopropylarsonate (10 nmol/0.5 microl) 20 min before injection of NMDA (4 nmol/0.5 microl) reduced the seizure score from 3.80 +/- 0.37(5) on the Racine scale to 0.83 +/- 0.40(6) (P < 0.01). The effect was partially reversible after washing with phosphate buffer. 2-Amino-4-arsonobutyrate, the analogue of glutamate, had no effect on seizure score following treatment with the same concentration of the drug and the same route of injection. The inhibitory effect of 3-aminopropylarsonate on NMDA kindled activity was dose-dependent, since higher doses of NMDA reduced the effect of the drug. The effect of 3-aminopropylarsonate was also selective to NMDA receptors since it had no effect on kainate-induced seizures. With both models of kindling, no gross behavioural abnormalities were observed 3-6 months after treatment with the drug. These findings show the potent antiepileptogenic and anti-convulsant activity of the arsonoanalogue of GABA which appears to be non-toxic and therefore potentially useful as the basis for developing a new family of clinically useful anticonvulsants for treating epilepsy.

Hypoglycemic effect of copper(II) acetate imidazole complexes.

The effect of copper(II) complexes on glucose metabolism was studied in normal and streptozotocin-induced diabetic rats. The copper(II) complexes used were bis(acetato)tetrakis(imidazole) copper (II), [Cu(OAc)2(Im)4], bis(acetato)bis(2-methylimidazole) copper(II), [Cu(OAc)2(2mIm)2], bis(acetato)bis(1,2-dimethylimidazole) copper(II), [Cu(OAc)2(1,2dmIm)2], and bis(acetato)bis(mu-acetato)tetrakis(N-methylimidazole) copper(II) hexaaquo, [Cu2(OAc)4-(NmIm)4].6H2O. Intramuscular administration of various doses of Cu(OAc)2(Im)4 ranging from 10 to 100 mg/kg body mass to overnight fasted rats decreased blood glucose levels in a dose-dependent manner. Maximum hypoglycemic effect was observed 3 h after administration and lasted for at least 6 h. Treatment with 100 mg/kg body mass of Cu(OAc)2(Im)4 caused hypoglycemic shock, which was irreversible and even lethal. Blood insulin levels were reduced sharply during this hypoglycemic shock. Similar changes in blood glucose level were achieved using Cu(OAc)2(2mIm)2. The same pattern of hypoglycemia, although less pronounced, was observed for Cu2(OAc)4(NmIm)4.6H2O and Cu(OAc)2(1,2dmIm)2. Binary copper(II) acetate complex, the ligand imidazole, and the inorganic form of copper, such as copper(II) chloride, had no significant effect on blood glucose level. These results indicate that the hypoglycemic activity of these complexes varies with the imidazole ligand and structure of the complex.

Effect of afferent brachial stimulation and photic stimulation on glycogen concentration in cortical brain tissue.

Afferent electrical stimulation (AES) of the brachial plexus caused a 41% increase in glycogen concentration in the contralateral sensory-motor cortex of rat brain. This increase in glycogen concentration was confined to the stimulated sensory-motor cortex; no change in glycogen concentration in adjacent areas, such as visual and frontal cortex was observed. The increase in glycogen concentration was completely reversed within 10 min after cessation of stimuli. Assays of relevant metabolites in the sensory-motor cortex demonstrated an increase in the concentrations of pyruvate and glucose-6-phosphate by 135 and 93%, respectively, and a slight decrease (14%) in the concentration of glucose. No change in the concentration of lactate was found. Photic stimulation of the eyes of dark adapted guinea pigs caused a 13% increase in glycogen concentration in the visual cortex and had no effect on glycogen concentration in other cortical areas. The same photic stimulation had no effect on glycogen concentration in the visual cortex of Sprague-Dawley rats. The results suggest that the increased glycogen concentration in cortical areas results from increased uptake of glucose following stimulation.

Changes in the activity of glutamate related enzymes in cerebral cortex, during insulin-induced seizures.

The activity of glutamate related enzymes and the concentration of glutamine, glutamate and gamma-amino n-butyric acid (GABA) were investigated in the cerebral cortex of rats, in different stages of insulin-induced hypoglycemia. Hypoglycemia was produced by intraperitoneal injection of insulin 0.05-100 units per kg body weight. The minimum required dose to produce irreversible severe hypoglycemia was 0.5 units/kg. In 85% of the cases an insulin induced hypoglycemic convulsion, was achieved 130-150 minutes after injection. Blood glucose levels during insulin induced seizures ranged between 8-15 mg%. In the range of 0.5-100 u insulin/kg the degree of hypoglycemia and the onset of convulsions were identical. The concentration of glutamine was significantly reduced during convulsive and postconvulsive stages. Glutamate and GABA concentrations were reduced significantly in all stages of insulin-induced hypoglycemia. The decrease in glutamine concentration was concurrent with an increase in the activity of its degradative enzyme, glutaminase. This was apparent at the preconvulsive, convulsive and postconvulsive stages. The activity of other enzymes related to energy production such as glutamate dehydrogenase (GDH), glutamate transaminase (GPT) and aspartate aminotransferase (AAT) were also increased. The activity of glutamine synthase (GS) was unaffected by hypoglycemia. Insulin induced changes in glutamine, glutamate and their related enzymes could not be attributed to convulsion since a similar pattern of changes was observed in the preconvulsive and postconvulsive stages, and no changes were detected following picrotoxin-induced seizures.

Species-directed variation and non-uniform distribution of glycogen in mammalian brains during starvation, diabetes and anesthesia.

An ordered distribution of glycogen across various areas of rat, guinea pig and mouse brain was found. The cortex contained the lowest concentration of glycogen, intermediate concentrations were found in the cerebellum, medulla oblongata, thalamus, pons and olfactory bulb, and the highest concentration of glycogen was in the hypothalamus. In rat brain glycogen concentration ranged from 0.07 to 0.21 mg/g tissue in different cortical areas to 1.17 +/- 0.07 mg/g in the hypothalamus. The concentration of glycogen in guinea pig and mouse brain was 1.5-3.5 and 1.5-4.3-fold higher than in rat brain, respectively. Starvation and diabetes caused a decrease in glycogen concentration in the thalamus, hypothalamus and medulla oblongata. Glycogen concentration in cortical areas was unchanged in starvation and increased in diabetic animals. The reduced glycogen concentration in thalamus, hypothalamus and medulla of diabetic rats could be corrected by insulin treatment, indicating that glycogen metabolism in these areas is insulin dependent. Pentobarbitone anesthesia caused a marked increase in glycogen concentration in all brain areas in normal and in diabetic rats. The effect of pentobarbitone was related to the depth of anesthesia and was reversed following recovery.

The vascular action of aqueous extracts of Foeniculum vulgare leaves.

The vascular effects of aqueous extracts of Foeniculum vulgare leaves were tested using pentobarbital-anaesthetised rats. An intravenous administration of the lyophilized boiled water extract of leaves produced a significant dose-related reduction in arterial blood pressure, without affecting the heart rate or respiratory rate. In contrast the non-boiled aqueous extract showed very little hypotensive activity. The hypotensive effect of the boiling water extract appeared not to be mediated via adrenergic, muscarinic, ganglionic or serotonergic receptors; however, histamine antagonists inhibited the hypotensive effect in a dose-related manner.

Effect of alpha-amino-4-phosphonobutyrate on the release of endogenous glutamate and aspartate from cortical synaptosomes of epileptic rats.

The effect of the glutamate antagonist alpha-amino-4-phosphonobutyrate (APBA) on the release of endogenous amino acids from sensorimotor cortical synaptosomes of rats with a cortical cobalt focus and from non-epileptic rats was studied: (1) The release of endogenous glutamate, aspartate, and gamma-aminobutyric acid (GABA) from synaptosomal preparations of cobalt-induced epileptogenic tissues was increased compared with the release from the contralateral (sensorimotor) region or the sensorimotor cortex of normal animals. The intrasynaptosomal content of these amino acids was reduced in proportion to the amount released. The levels of other amino acids were unaffected or showed much smaller changes. (2) APBA (0.5-1 mM) decreased significantly the spontaneous release of aspartate and glutamate from the epileptic foci without affecting GABA or any other amino acid. (3) APBA produced no effect whatsoever on the release of any amino acid from synaptosomal preparations of nonepileptic focus.

In vivo inhibition of incorporation of [U-14C]glucose into proteins in experimental focal epilepsy.

The in vivo incorporation of [14C] from [U-14C]-glucose into rat brain proteins from different cortical areas was examined in three different experimental focal epilepsies: cobalt, freeze-lesions, and tityustoxin. When [U-14C]-glucose was injected intraperitoneally into awake and unrestrained animals with marked signs of epileptic hyperactivity, the inhibition of incorporation of [14C]-amino acids into trichloracetic acid (TCA)-insoluble proteins was highest in the focal (sensorimotor) area when compared with distant regions (approx. 60%), but less when compared with the contralateral (sensorimotor) region (approx. 23%). Greatly decreased incorporation caused by both cobalt and freeze-lesion-induced epilepsies was also observed in the contralateral area when a comparison was made with distant regions (approx. 50%), but there were no significant differences in protein-specific radioactivity between the different distant areas.

Morphine suppression of neurotransmitter release evoked by sensory stimulation in vivo.

The effects of morphine and naloxone on the release of acetylcholine and amino acid neurotransmitters from sensorimotor cortex were studied employing an in vivo superfusion cannula. Morphine (20 mg/kg) reduced the spontaneous release of acetylcholine but had no detectable effect on the spontaneous release of amino acids. It also suppressed the release of acetylcholine and amino acid neurotransmitters evoked by sensory stimulation of the contralateral sensorimotor cortex via the brachial plexus. Naloxone (5 mg/kg) prevented all of these inhibitory actions of morphine. Naloxone also caused a significant increase in spontaneous acetylcholine release.

Effect of depolarizing agents on the incorporation of amino acids into soluble cytoplasmic and membrane-bound proteins of synaptosome fractions.

The incorporation of [U-14C] protein hydrolysate and [U-14C]leucine into the trichloroacetic acid (TCA)-insoluble membrane and the soluble synaptoplasm proteins of synaptosomes was studied. Following treatment with the depolarizing agents veratrine, Tityus toxin, or potassium, the specific radioactivity of both precursor pool and proteins were measured to examine the link between protein labeling and the fall in the free amino acid pool due to depolarization-induced release of glutamate and aspartate. By reducing the size of the fall in precursor pool due to depolarization by using a nontransmitter amino acid such as leucine (as compared with the usual use of protein hydrolysate), it was shown that the amount of which the pool is reduced is proportional to the change in the protein labeling observed. These results confirm that membrane depolarization causes a large increase in the labeling of membrane-bound proteins as compared with the soluble synaptosomal proteins.

Effect of beta-bungarotoxin on the release of endogenous amino aids from the sensorimotor cortex.

beta-Bungarotoxin, a snake neurotoxin purified from the venom of Bungarus multicinctus, caused a significant increase in the in vivo release of glutamate from the superfused sensorimotor cortex of awake animals. A smaller effect on GABA release was observed, but no change was detected in the release of six other amino acids measured. The effects on glutamate and GABA release were entirely blocked by tetrodotoxin (1 micrometer) and were reversible when the cortical tissue was washed with saline.