In vitro biological activity and structural analysis of 2,4-diamino-5-(2'-arylpropargyl)pyrimidine inhibitors of Candida albicans.

In order to develop new antifungal agents effective against two species of Candida, we have designed a series of dihydrofolate reductase (DHFR) inhibitors. Here, we explore the structure-activity relationships of these inhibitors toward Candida albicans DHFR by evaluating enzyme inhibition, antifungal activity and toxicity to mammalian cells. Analysis of docked complexes of the enzyme and inhibitors yields the structural basis of relative potency. The meta-biphenyl series of this class exhibits the greatest enzyme inhibition, selectivity and antifungal activity.

Effects of co-administration of a selective serotonin reuptake inhibitor and monoamine oxidase inhibitors on 5-HT-related behavior in rats.

5-hydroxytryptamine (5-HT) syndrome is a dangerous condition of 5-HT excess that can occur in the case of co-administration of a monoamine oxidase (MAO) inhibitor and a serotonin reuptake inhibitor (SSRI). The goal of the present study was to investigate the effects of acute administration of MAO inhibitors and subchronic administration of fluvoxamine on 5-HT-related behaviors (head shaking and 5-HT syndrome) in rats treated with 5-hydroxytryptophan (5-HTP). Administration of the non-selective MAO inhibitor, pargyline, and the selective MAO-A inhibitor, clorgyline, resulted in 5-HT syndrome in 5-HTP-treated rats, and subchronic co-administration of fluvoxamine intensified the syndrome. However, administration of the selective MAO-B inhibitor, selegiline, did not induce 5-HT syndrome with or without subchronic fluvoxamine co-administration. These data suggest that non-selective MAO and selective MAO-A inhibitors can induce 5-HT syndrome in humans when co-administered with SSRI. Further, the risk of 5-HT syndrome may be lower with the selective MAO-B inhibitor, selegiline.

Fumigant combinations for Cyperus esculentum L control.

The phase-out of methyl bromide as a soil fumigant has stimulated research into the use of other soil fumigants for weed control. Methyl bromide, methyl iodide, propargyl bromide, 1,3-dichloropropene (1,3-D) and metam-sodium were tested alone and in combination with chloropicrin in laboratory experiments to determine their efficacy against Cyperus esculentus L (yellow nutsedge) tubers. Propargyl bromide and metam-sodium were the most efficacious fumigants tested, with EC50 values of 3.7 and 6.5 microM, respectively. The relative potencies of methyl iodide and chloropicrin were not significantly different but were 2.6 and 2.9 times more potent than methyl bromide, respectively. The EC50 values for all fumigants other than 1,3-D were significantly lower than that of methyl bromide. Combining each fumigant with 17% chloropicrin resulted in a synergistic interaction. The greatest increase in potency between the expected result and the actual result was a relative potency of 3.8 with the methyl bromide/chloropicrin combination. The smallest increase in efficacy was with propargyl bromide and chloropicrin, with a relative potency of 1.5. There was no significant difference between the EC50 values of methyl bromide/chloropicrin and methyl iodide/chloropicrin combinations. Combining 1,3-D with 17% chloropicrin resulted in an EC50 value for C. esculentus control similar to that of methyl iodide applied alone.

Cytotoxicity, cytoprotection and neurotoxicity of novel deprenyl-related propargylamines, stable nitroxide free radicals, in vitro and in vivo.

Since novel synthesized deprenyl-related derivatives of nitroxides, named "JSAKs", have been shown to possess antioxidative properties, their cytotoxicity on neuronal-like PC-12 cells line was examined. The antiproliferative effect of two selected JSAKs was examined and expressed as IC10, IC50 and IC90, and compared with those of the parent nitroxide (Nx-640), model nitroxide TEMPO and deprenyl. There were substantial differences in the dose-dependence of all the observed antiproliferative and cytotoxic effects. Compared to anticancer drugs and apoptosis inducers with topoisomerase inhibitor properties (etoposide and camptothecin), novel compounds displayed cytotoxicity at considerably higher concentrations. The dose-dependent anti-apoptotic potency of JSAKs and Nx-640 was also investigated and compared to TEMPO and deprenyl effects. The observed structure-dependent correlation was very encouraging and prompted us to screen and to compare the in vivo time-dependent effects of JSAKs, Nx-640 and deprenyl administration on the rat intact nigrostriatal neurocytes. TH-immunochemistry was applied as the test method and marker for the changes in the state of the rat catecholaminergic system, also giving evidence that low-toxic and cell-permeable JSAKs can cross the blood-brain barrier, which is the mandatory prerequisite for the therapeutic application of antioxidants and drugs to the brain. Taken together, it can be concluded with great certainty that novel deprenyl-related JSAKs might be especially good candidates for further anticancer investigations in vitro and in vivo and future pharmacological applications.

Ovicide-induced serosa degeneration and its impact on embryonic development in Manduca sexta (Insecta: Lepidoptera).

Eggs of Manduca sexta treated with the ovicide Ov. 165049 turn orange, and the embryos later die. The orange pigmentation is at first confined to the serosa, and is accompanied by pathological changes of serosal cells. Lipid vesicles aggregate and spindle-shaped electron-lucent vesicles-normally forming a single layer below the apical cell surface-greatly accumulate. The mitochondria swell considerably, and their matrices become electron-lucent. Subsequently, the serosal cells develop additional features of necrosis. They form many autophagic vacuoles which contain mostly degradating mitochondria, but also segregated rough endoplasmic reticulum (rER) and glycogen granules. The whole cytoplasm vesiculates, and the cells shrink considerably. The nuclei become less irregular in shape, the chromatin disperses rather evenly whereas the nucleoli persist. Neither chromatin condensation nor the production of apoptotic bodies was observed-further evidence, that the serosal cells die by necrosis rather than apoptosis. At some stage of development the damaged serosa ruptures, retracts from the embryo and forms a sphere beneath it. It is only after the rupture of the serosa, that the embryo also turns orange and disintegrates rapidly. This shows impressively the protective function which the serosa plays for the embryo. Our physiological tests indicate, that the orange pigmentation of the serosa induced by the ovicide results from a disturbance of the tryptophan/ommochrome pathway serving the excretion of potentially toxic metabolites of tryptophan-rich proteins. The results demonstrate first that the serosa represents an important target for ovicide pesticides and second that it plays a vital role as an excretory organ during embryogenesis.

Pargyline pretreatment prevents immunological changes induced by MPTP in mice.

The relationship between the central dopaminergic and the immune system is poorly understood. Experimental work suggest that damage of the nigrostriatal system may influence immunity. Immunological abnormalities have been described in Parkinson's disease and in a mouse model of this disorder induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In this report, we present evidence that reduced numbers of L3T4 T cells in blood, and diminished primary antibody response to sheep erythrocytes in MPTP treated mice can be restored by pargyline pretreatment. Since pargyline prevents dopamine depletion in the striatum in MPTP treated animals, our data extend previous experimental observations and support a possible role for dopamine in immune regulation.

Monoamine oxidase released into plasma treated with the hepatotoxin allyl formate.

Monoamine oxidase (MAO) released from the rat liver into plasma and the activity levels of lipid peroxide (LPO) and superoxide dismutase (SOD) in liver tissue were investigated after pretreatment of rats with the perilobular hepatotoxin allyl formate (AF). When 3H-pargyline was given to AF-pretreated rats, the levels of 3H-pargyline labelled MAO in rat plasma were increased to 38% (p < 0.01 vs control), but the radioactivity was decreased to 35% (p < 0.05 vs control). The molecular weight of MAO subunits in plasma was similar to that of MAO subunits in liver (about 60,000) as determined by SDS electrophoresis. Liver tissue LPO levels in these rats were increased, whereas SOD activity was decreased. These results suggest that MAO in liver mitochondria was released into plasma as a consequence of membrane disorder.

Effects of acute acetaldehyde, chronic ethanol, and pargyline treatment on agonist responses of the rat aorta.

The purpose of this investigation was to determine the mechanism(s) underlying the vasorelaxant effects of acute versus chronic acetaldehyde (ACA) exposure, in particular, the role of intact endothelium on contractile responses of rat aortic ring segments to potassium chloride (KCl) or norepinephrine (NE). The acute effects of ACA were assessed in preparations from normal animals maintained on a standard rat chow (non-ethanol-ingesting). The monoamine oxidase inhibitor, pargyline, elevates plasma ACA levels by decreasing acetaldehyde dehydrogenase activity. Accordingly, preparations from pargyline-treated ethanol-ingesting animals (PE) were used to assess the effects of chronic (12 weeks) ACA and results were compared to those of pargyline-treated non-ethanol-ingesting (P) rats fed a standard liquid diet. In normal and P preparations, maximal inotropic response to KCl and NE were greater in endothelium-denuded than in endothelium-intact preparations. However, in aortic rings obtained from PE rats, the maximal inotropic response to KCl was depressed only in endothelium-denuded rings and that to NE was nearly identical in endothelium-denuded compared to endothelium-intact preparations. Acute ACA (30 mM) exposure significantly reduced both NE- and KCl-induced contractile responses in muscles from all groups. The magnitude of the vasorelaxant effect of this [ACA] on NE-induced responses was endothelium-independent and was similar between groups. However, the vasorelaxant effect of ACA (30 mM) on KCl-induced contractile responses was significantly attenuated in muscles from PE animals with greater inhibition occurring in endothelium-denuded preparations. These results suggest that chronic acetaldehyde exposure leads to an impairment in the inotropic response to membrane depolarization in endothelium-denuded preparations resulting in depressed responsiveness. In addition, the acute vasorelaxant effect of ACA on KCl-induced contractures is significantly attenuated in preparations chronically exposed to ACA which suggests a possible development of tolerance.

Interaction of pargyline with tolbutamide in rabbits.

Acute treatment of rabbits with pargyline (50 mg/kg, ip, 30 min before tolbutamide) significantly increased the elimination half life and AUC0-->infinity of tolbutamide resulting in prolonged hypoglycaemia. Similar treatment also prolonged the half life of antipyrine which is used as model drug to indicate hepatic microsomal enzyme activity in vivo confirming that pargyline treatment delayed the elimination of tolbutamide in rabbits by inhibiting its hepatic metabolism.

Depletion of retinal dopamine increases brightness perception in goldfish.

The effect of unilateral depletion of retinal dopamine on goldfish visual behavior was studied using a behavioral reflex, the dorsal light reaction (DLR). Retinal dopamine was depleted by intraocular injections of 6-hydroxydopamine (6-OHDA) on two successive days. By 2 weeks postinjection, dopamine interplexiform cells (DA-IPC) were not detected using tyrosine-hydroxylase immunoreactivity (TH-IR). By 6 weeks postinjection, generation of DA-IPC was observed at the marginal zone and by 9 months postinjection, 2-3 rows of DA-IPC were present at the marginal zone. Neurites extended several hundred micrometers toward the central retina. By 2 weeks postinjection, all 6-OHDA lesioned fish tilted 7-15 deg toward the injected eye under uniform overhead illumination. The tilting did not occur under scotopic illumination and reappeared within 1 min of light adaptation. Quantitation of the DLR showed that 6-OHDA lesioned fish behaved as if light were 2.4 log units more intense to the injected eye. Partial recovery was observed by 9 months postinjection, paralleling the reappearance of DA-IPC at the marginal zone. Tilting also was induced by unilateral intraocular injection with D1 and D2 dopamine receptor antagonists, SCH 23390 and S(-)-sulpiride, respectively. Fish did not tilt if they were light adapted at the time of injection. Tilting was observed if the animals were dark-adapted for 3 h and left in the dark for 1 h postinjection. Fish tilted toward the drug-injected eye within 2 min of light adaptation and gradually returned to vertical within 2 h. The tilting response to S(-)-sulpiride was stronger (approximately 20 deg vs. approximately 5 deg) and occurred at lower concentration (1 microM vs. 10 microM) compared to SCH 23390. We conclude that dopamine depletion mimics the dorsal light reaction by increasing the luminosity output of the eye and that dopamine is directly involved in photopic luminosity function.

Depletion of retinal dopamine does not affect the ERG b-wave increment threshold function in goldfish in vivo.

Increment threshold functions of the electroretinogram (ERG) b-wave were obtained from goldfish using an in vivo preparation to study intraretinal mechanisms underlying the increase in perceived brightness induced by depletion of retinal dopamine by 6-hydroxydopamine (6-OHDA). Goldfish received unilateral intraocular injections of 6-OHDA plus pargyline on successive days. Depletion of retinal dopamine was confirmed by the absence of tyrosine-hydroxylase immunoreactivity at 2 to 3 weeks postinjection as compared to sham-injected eyes from the same fish. There was no difference among normal, sham-injected or 6-OHDA-injected eyes with regard to ERG waveform, intensity-response functions or increment threshold functions. Dopamine-depleted eyes showed a Purkinje shift, that is, a transition from rod-to-cone dominated vision with increasing levels of adaptation. We conclude (1) dopamine-depleted eyes are capable of photopic vision; and (2) the ERG b-wave is not diagnostic for luminosity coding at photopic backgrounds. We also predict that (1) dopamine is not required for the transition from scotopic to photopic vision in goldfish; (2) the ERG b-wave in goldfish is influenced by chromatic interactions; (3) horizontal cell spinules, though correlated with photopic mechanisms in the fish retina, are not necessary for the transition from scotopic to photopic vision; and (4) the OFF pathway, not the ON pathway, is involved in the action of dopamine on luminosity coding in the retina.

Monoamine oxidase B inhibitor enhances experimental carcinogenesis in rat colon induced by azoxymethane.

The effects of prolonged administration of the monoamine oxidase (MAO)-A inhibitor N-methyl-N-propargyl-3-(2,4-dichlorophenoxy)propylamine (clorgyline) and the MAO-B inhibitor N-methyl-N-2-propynyl-benzylamine (pargyline) on the incidence, number and histology of colon tumors induced by azoxymethane (AOM), and on the norepinephrine (NE) concentration in the colon wall and the labeling index of colon mucosa were investigated in Wistar rats. Rats were treated s.c. with 7.4 mg/kg body weight of AOM once a week for 10 weeks, and also s.c. with 5 mg/kg body weight of clorgyline or 50 mg/kg body weight of pargyline in 0.9% NaCl until the end of the experiment. Treatment with pargyline significantly increased the incidence of colon tumors in week 35. However, it did not influence the histological appearance of the colon tumors or the histological types and depth of involvement of colon adenocarcinomas. Furthermore, it significantly increased the NE concentration in the colon wall and the labeling index of the colon mucosa during and after AOM-treatment. In contrast, clorgyline had no influence on the development or histological appearance of colon tumors. These findings indicate that the MAO-B inhibitor, but not the MAO-A inhibitor, enhanced colon carcinogenesis, and that its effect may be related to its effect in increasing the NE concentration in the colon wall and subsequently increasing the proliferation of colon epithelial cells.

Effects of mushroom toxins on glycogenolysis; comparison of toxicity of phalloidin, alpha-amanitin and DL-propargylglycine in isolated rat hepatocytes.

The effects of phalloidin and alpha-amanitin as toxins of Amanita species and DL-propargylglycine identified from A. abrupta on the glycogenolysis in isolated rat hepatocytes were investigated. Phalloidin decreased glycogen content and activated phosphorylase a activity remarkably. alpha-Amanitin also decreased glycogen content significantly but activated phosphorylase a activity slightly. DL-Propargylglycine slightly affected glycogenolysis. Phalloidin, which most affected glycogenolysis among the three compounds mentioned above, elevated cytosolic Ca2+ concentration ([Ca2+]i) and 45Ca uptake into cells. Phalloidin depressed slightly 3H-inositol incorporation into phosphatidylinositol (PI) and remarkably phosphatidylinositol 4,5-bisphosphate (PIP2) but increased phosphoinositides breakdown. These results suggest that phalloidin alters phosphoinositides turnover and intracellular Ca2+ homeostasis, subsequently activates phosphorylase a, resulting in glycogenolysis in isolated rat hepatocytes.

In vitro toxicity test of poisonous mushroom extracts with isolated rat hepatocytes.

Effects of poisonous mushroom extracts on isolated rat hepatocytes were studied. Though no significant decrease in the cell viability was observed during the incubation of hepatocytes with the extracts at a concentration of 5% (v/v) of Amanita abrupta, A. gymnopus, and A. virosa caused marked decreases in the intracellular glutathione content in sharp contrast to the extracts of A. volvata and A. flavipes. Comparative toxicity tests were carried out for the effects of the extract of A. abrupta, dl-propargylglycine, and alpha-amanitin. The extract of A. abrupta at a concentration of 1% (v/v) caused a marked decrease in the glycogen content, a noticeable elevation in the phosphorylase alpha activity, and a slight acceleration of lipid peroxidation in the hepatocytes. Although dl-propargylglycine decreased the intracellular glutathione content progressively with the incubation time, a significant effect of the chemical on lipid peroxidation and the glycogen content was observed only after prolonged incubation at a concentration of 5 mM. On the other hand, alpha-amanitin exerted a little effect on the hepatocytes at 1 microM. These results have indicated that the intoxication by the extract of A. abrupta on the hepatocytes might not due to independently each component, dl-propargylglycine and alpha-amanitin, but combined effect of these components or unidentified substances.

Manganese neurotoxicity: effects of L-DOPA and pargyline treatments.

Single, monolateral injection into rat substantia nigra of manganese chloride produced within two weeks from its administration a loss of dopamine in the striatum ipsilateral to the injected side. The effect was dose-dependent and was not extended to serotoninergic terminals present in this brain area, whose content in serotonin and 5-hydroxyindoleacetic acid was not affected. When L-DOPA + carbidopa or pargyline were given to these animals the decrease of striatal dopamine was more marked. Moreover, rats treated two weeks before with a dose of manganese chloride that produced a 70-80% drop in striatal dopamine concentrations, rotated ipsilaterally to the dopamine-depleted striatum when injected with apomorphine, suggesting that in these animals the stimulatory effects of apomorphine were more relevant in striatum where presynaptic dopaminergic neurons were not affected by manganese chloride. These data indicate that the alterations of dopaminergic postsynaptic receptors may be different in parkinsonian and in manganese-intoxicated patients and that current therapy used for Parkinson's disease could be a hazard in treating manganese poisoning.

Chronic but not acute treatment with antidepressants enhances the electroconvulsive seizure response in rats.

The effects of the chronic administration of antidepressants on threshold electroconvulsive (ECS) seizures were evaluated in rats. Initially, tonic-clonic seizures were induced in 90% of the animals. Of those animals responding with tonic-clonic seizures, 42% had hindlimb extension (extensors); the remainder showed only hindlimb flexion (flexors). No alteration in the pattern of seizures was observed 24 hr after a single oral dose of any of the antidepressant drugs. The rats were then treated with a total of 20 consecutive daily doses of antidepressants and threshold electroconvulsive seizure responses were evaluated 24 hr after the last dose. A significantly greater percentage of rats responded with extensor seizures after chronic treatment with amoxapine, chlorimipramine, parglyine and mianserin. There was no change in the pattern of seizures of the rats treated chronically with desipramine, but the duration of the clonic phase was reduced. After a 7 day period free of drugs a significantly greater percentage of animals had extensor seizures in the groups treated with amoxapine, chlorimipramine, pargyline and desipramine but not mianserin. In the light of evidence that chronic treatment with antidepressants reduces the activity of norepinephrine- or isoproterenol-sensitive adenylate-cyclase, and that the norepinephrine system is an important endogenous anticonvulsant factor in electroconvulsive seizures, these results suggest that the same mechanism may mediate both the therapeutic and proconvulsant effects of the chronic administration of antidepressants.

Microsomal formation and chemical decomposition of pargyline N-oxide.

Pargyline undergoes metabolic N-oxidation in rat and rabbit liver microsomal preparations. The reaction requires oxygen and is NADPH dependent. N-oxidation and N-demethylation are equal in both control and induced rat liver microsomes, while N-oxidation is more dominant in rabbit tissue. Experiments investigating the CO-sensitivity and the effects of metyrapone suggest that cytochrome P-450 systems are involved in both reactions in the rat while an additional enzyme is responsible for the N-oxidation in the rabbit. Pargyline N-oxide is characterized by chemical instability and undergoes two consecutive rearrangements to yield propenal and Schiff bases, the latter undergoing hydrolysis to aldehydes and primary amines. Accordingly, due to the inherent instability of the N-oxide, metabolic N-oxidation of pargyline is, in addition to alpha-carbon oxidation, indicated as a metabolic route to benzaldehyde. Similarly the ease with which pargyline N-oxide generates propenal implicates N-oxidation as a metabolic route to be considered when evaluating the toxicity of pargyline.

Sex differences in behavioral and thermal responses to pargyline and tryptophan.

The effects of parenterally injected pargyline and tryptophan on rectal temperature and behavior have been studied in male and female rats. Pargyline alone (50 mg/kg) produced hypothermia in both sexes. Pargyline (50 mg/kg) followed by low doses (20--50 mg/kg) of tryptophan caused a behavioral syndrome consisting of tremor, hindlimb abduction, forepaw treading, and straub tail. In females, but not in males, hypothermia was potentiated. The same dose of pargyline followed by higher doses (60--150 mg/kg) of tryptophan produced a short hypothermia followed by a dose-dependent behavioral syndrome, hyperthermia, and mortality. On all of these measures, females responded following shorter latencies and lower doses of tryptophan. Both hypothermia and hyperthermia were observed in treated animals following pretreatment with a peripheral decarboxylase inhibitor. The results suggest a complex role for serotonin in thermoregulation. The sex differences observed suggest higher activity of serotonin in female rat brains following the drug treatment, which may be accounted for by a higher utilization rate of tryptophan.

Influence of Steroidal enzyme inducers on the toxicity of pyrogallol, pargyline and nialamide.

In rats, the toxic manifestations of overdosage with with parcyline (a monoamine oxidase inhibitor) or pyrogallol (a catechol-o-methyltransferase inhibitor) were diminished by treatment with the more potent steroidal (pregnenolone-16alpha-carbonitrile, spironolactone, etc.) or nonsteroidal (phenobarbital) catatoxic substances. Except for significant protection offered by glucocorticoids (triamcinolene, prednisolone acetate) against pargyline, all other pretreatments (progesterone, estradiol, desoxycorticosterone acetate, etc.) either had no influence on or increase the deleterious effects of the two amine inhibitors. Nialamide intoxication was exacerbated by most of these conditioners.