Angiotensin-converting enzyme inhibition reduces oxidative stress and protects dopaminergic neurons in a 6-hydroxydopamine rat model of Parkinsonism.

It is now established that the brain possesses a local renin-angiotensin system and that angiotensin II exerts multiple actions in the nervous system, including regulation of striatal dopamine release. Furthermore, angiotensin activates NADPH-dependent oxidases, which are a major source of superoxide, and angiotensin-converting enzyme inhibitors, commonly used in the treatment of hypertension and chronic heart failure, have shown antioxidant properties in several tissues. Oxidative stress is a key contributor to the pathogenesis and progression of Parkinson's disease. In the present study, we treated rats with intraventricular injections of the dopaminergic neurotoxin 6-hydroxydopamine and subcutaneous injections of the angiotensin-converting enzyme inhibitor Captopril to study the possible neuroprotective effect of the latter on the dopaminergic system and on 6-hydroxydopamine-induced oxidative stress. Rats treated with Captopril and 6-hydroxydopamine showed significantly less reduction in the number of dopaminergic neurons (i.e., immunoreactive to tyrosine hydroxylase) in the substantia nigra and in the density of striatal dopaminergic terminals than 6-hydroxydopamine-lesioned rats not treated with Captopril. In addition, Captopril reduced the levels of major oxidative stress indicators (i.e., lipid peroxidation and protein oxidation) in the ventral midbrain and the striatum of 6-hydroxydopamine-lesioned rats. Our results suggest that angiotensin-converting enzyme inhibitors may be useful for treatment of Parkinson's disease and that further investigation should focus on the neuroprotective capacity of these compounds.

Effect of iron and manganese on hydroxyl radical production by 6-hydroxydopamine: mediation of antioxidants.

6-Hydroxydopamine (6-OHDA) neurotoxicity has often been related to the generation of free radicals. Here we examined the effect of the presence of iron (Fe(2+) and Fe(3+)) and manganese and the mediation of ascorbate, L-cysteine (CySH), glutathione (GSH), and N-acetyl-CySH on hydroxyl radical (*OH) production during 6-OHDA autoxidation. In vitro, the presence of 800 nM iron increased (> 100%) the production of *OH by 5 microM 6-OHDA while Mn(2+) caused a significant reduction (72%). The presence of ascorbate (100 microM) induced a continuous generation of *OH while the presence of sulfhydryl reductants (100 microM) limited this production to the first minutes of the reaction. In general, the combined action of metal + antioxidant increased the *OH production, this effect being particularly significant (> 400%) with iron + ascorbate. In vivo, tyrosine hydroxylase immunohistochemistry revealed that intrastriatal injections of rats with 6-OHDA (30 nmol) + ascorbate (600 nmol), 6-OHDA + ascorbate + Fe(2+) (5 nmol), and 6-OHDA + ascorbate + Mn(2+) (5 nmol) caused large striatal lesions, which were markedly reduced (60%) by the substitution of ascorbate by CySH. Injections of Fe(2+) or Mn(2+) alone showed no significant difference to those of saline. These results clearly demonstrate the role of ascorbate as an essential element for the neurotoxicity of 6-OHDA, as well as the diminishing action of sulfhydryl reductants, and the negligible effect of iron and manganese on 6-OHDA neurotoxicity.

Inhibition of brain monoamine oxidase activity by the generation of hydroxyl radicals: potential implications in relation to oxidative stress.

Monoamine oxidase (MAO) is an enzyme involved in brain catabolism of monoamine neurotransmitters whose oxidative deamination results in the production of hydrogen peroxide. It has been documented that hydrogen peroxide derived from MAO activity represents a special source of oxidative stress in the brain. In this study we investigated the potential effects of the production of hydroxyl radicals (*OH) on MAO-A and MAO-B activities using mitochondrial preparations obtained from rat brain. Ascorbic acid (100 microM) and Fe2+ (0.2, 0.4, 0.8, and 1.6 microM) were used to induce the production of *OH. Results showed that the generation of *OH significantly reduced both MAO-A (85-53%) and MAO-B (77-39%) activities, exhibiting a linear correlation between both MAO-A and MAO-B activities and the amount of *OH produced. The reported inhibition was found to be irreversible for both MAO-A and MAO-B. Assuming the proven contribution of MAO activity to brain oxidative stress, this inhibition appears to reduce this contribution when an overproduction of *OH occurs.

Reduction of rat brain levels of the endogenous dopaminergic proneurotoxins 1,2,3,4-tetrahydroisoquinoline and 1,2,3,4-tetrahydro-beta-carboline by cigarette smoke.

1,2,3,4-Tetrahydroisoquinoline (TIQ) and 1,2,3,4-tetrahydro-beta-carboline (THbetaC) are two endogenous or exogenous dopaminergic proneurotoxicants supposedly involved in the etiology of Parkinson's disease. We investigated whether the chronic administration of a twice daily dose of a cigarette smoke solution might modify the endogenous concentrations of TIQ and THbetaC in rat brain. Using gas chromatography/mass spectrometry (GC-MS) we found a significant reduction in the brain levels of both proneurotoxins after 30 days of treatment. The reduction in the brain levels of both compounds was more significant using Burley rather than Bright tobacco. These results suggest that cigarette smoke may prevent the accumulation of these proneurotoxins in the brain, which corroborate the involvement of the reaction between both TIQ and THbetaC with some components of tobacco smoke as a neuroprotective mechanism for Parkinson's disease.

Autoxidation and neurotoxicity of 6-hydroxydopamine in the presence of some antioxidants: potential implication in relation to the pathogenesis of Parkinson's disease.

6-Hydroxydopamine (6-OHDA) is a dopaminergic neurotoxin putatively involved in the pathogenesis of Parkinson's disease (PD). Its neurotoxicity has been related to the production of reactive oxygen species. In this study we examine the effects of the antioxidants ascorbic acid (AA), glutathione (GSH), cysteine (CySH), and N-acetyl-CySH (NAC) on the autoxidation and neurotoxicity of 6-OHDA. In vitro, the autoxidation of 6-OHDA proceeds rapidly with the formation of H2O2 and with the participation of the H2O2 produced in the reaction. The presence of AA induced a reduction in the consumption of O2 during the autoxidation of 6-OHDA and a negligible presence of the p-quinone, which demonstrates the efficiency of AA to act as a redox cycling agent. The presence of GSH, CySH, and NAC produced a significant reduction in the autoxidation of 6-OHDA. In vivo, the presence of sulfhydryl antioxidants protected against neuronal degeneration in the striatum, which was particularly remarkable in the case of CySH and was attributed to its capacity to remove the H2O2 produced in the autoxidation of 6-OHDA. These results corroborate the involvement of oxidative stress as the major mechanism in the neurotoxicity of 6-OHDA and the putative role of CySH as a scavenger in relation to PD.

In vitro inhibition of catalase activity by cigarette smoke: relevance for oxidative stress.

The in vitro effects of cigarette smoke on catalase activity were investigated in biological preparations from rat liver and brain using a polarographic method. In both cases cigarette smoke solutions showed a potent ability to inhibit catalase activity with a slight time dependency. The reversibility of their inhibitory activity was demonstrated by in vitro dialysis tests. The catalase inhibitory compound(s) are formed in the smoking process, are not extracted with organic solvents and appear to have a relatively low molecular weight. We also examined the effects obtained by using two different commercial blends of tobacco, achieving a major inhibition with Burley tobacco in comparison to Bright tobacco. These data suggest that the cytotoxic and mutagenic effects of cigarette smoke may be mediated by its additional capacity to enhance the generation of free radicals by inhibiting catalase activity, thus contributing to cell damage particularly during oxidative stress.

Studies on the interaction between 1,2,3,4-tetrahydro-beta-carboline and cigarette smoke: a potential mechanism of neuroprotection for Parkinson's disease.

1,2,3,4-Tetrahydro-beta-carboline (TH beta C) is an endogenous or environmental neurotoxic factor putatively involved in the development of Parkinson's disease (PD). As part of our efforts to characterize the mechanism of the reported protection of smoking against PD, we have examined the interaction between TH beta C and cigarette smoke. We found that TH beta C reacts in vitro and under physiological conditions with some components of cigarette smoke to form N2-(cyanomethyl)-TH beta C (CM-TH beta C), N2-(gamma-cyanoethyl)-TH beta C (CE-TH beta C), N2-(1'-cyanopropyl)-TH beta C (CP-TH beta C), N2-(1'-cyanobutyl)-TH beta C (CB-TH beta C) and N2-formyl-TH beta C (F-TH beta C). Significant differences in the recovery of some of these TH beta C-derivatives were obtained for Burley and Bright tobacco. Several of the reported compounds showed reversible and competitive MAO-A inhibitory properties. The detection of some of these compounds in rat brain after chronic administration of TH beta C and a solution of cigarette smoke proved that the reported interactions also occur in vivo. These results are discussed as a potential mechanism of neuroprotection in the development of PD.

Extracellular amino acids in the rat hippocampus during picrotoxin threshold seizures in chronic microdialysis experiments.

The relation between changes in the concentrations of some of the neuroactive extracellular amino acids (glutamate, aspartate, gamma-aminobutyric acid, glycine and taurine) and epileptic seizures has been tested in a new experimental model of seizures induced by picrotoxin microdialysis in chronic freely moving rats. During ictal discharges (paroxysmal electroencephalographic discharges associated with behavioral seizures), a significant decrease in the levels of extracellular aspartate and glutamate was observed. However, no changes were found during the interictal discharges (paroxysmal electroencephalographic discharges, without concomitant seizures). Our results suggest that modifications in extracellular aspartate and glutamate may be related to neuronal synchronization rather than to paroxysmal activity, supporting the neurophysiological differences between non-ictal and ictal paroxysmal activity.

Inhibition of brain monoamine oxidase by adducts of 1,2,3,4-tetrahydroisoquinoline with components of cigarette smoke.

A series of adducts of 1,2,3,4-tetrahydroisoquinoline (TIQ) and some components of tobacco smoke were investigated for their ability to inhibit rat brain monoamine oxidase. 1-Cyano-TIQ (1CTIQ), N-(1'-cyanoethyl)-TIQ (CETIQ), N-(1'-cyanopropyl)-TIQ (CPTIQ), and N-(1'-cyanobutyl)-TIQ (CBTIQ) were found to act as competitive inhibitors for both MAO-A and MAO-B. Ki values ranged from 16.4 to 37.6 microM. N-(Cyanomethyl)-TIQ (CMTIQ) was not found to be an inhibitor (Ki > 100 microM). These findings may help to explain the in vivo inhibitory effects of tobacco smoke on MAO activity and the suggested protective effect of tobacco smoking against Parkinson's disease. They also appear to reinforce the usefulness of reversible MAO inhibitors in smoking cessation and abstinence. However, different results must be expected between Burley and Bright tobacco.

Interaction of 1,2,3,4-tetrahydroisoquinoline with some components of cigarette smoke: potential implications for Parkinson's Disease.

Tetrahydroisoquinoline (TIQ), a presumed proneutrotoxin linked with Parkinson's disease (PD), was found to interact with some components of cigarette smoke to give N-(cyanomethyl)-TIQ (CMTIQ), N-(1'-cyanoethyl)-TIQ (CETIQ), N-(1'-cyanopropyl)-TIQ (CPTIQ), N-(1'-cyanobutyl)-TIQ (CBTIQ), and 1-cyano-TIQ (1CTIQ). The in vitro formation of these compounds under physiological conditions occurs rapidly and with a high yield. Significant differences in the recovery of the different compounds were obtained when the data obtained from Burley tobacco were compared to those obtained from Bright tobacco. Following chronic administration of TIQ and a solution of cigarette smoke to rats, the presence of some of these compounds was also detected in the brain.

Quantitative analysis of neuroactive amino acids in brain tissue by liquid chromatography using fluorescent pre-column labelling with o-phthalaldehyde and N-acetyl-L-cysteine.

A high-performance liquid chromatographic method for the determination of aspartic acid, glutamic acid, glutamine, glycine, taurine, and gamma-aminobutyric acid in brain tissue is described. Amino acids were derivatized with o-phthalaldehyde/N-acetyl-L-cysteine, a fluorescent labelling mixture, in the presence of 0.1 M borate buffer pH 9.5. The derivatization reaction was sensitive to the pH and concentration of borate buffer. A drift in the fluorescent response less than 4% was obtained with the reported conditions after 4 h of reaction. The resolution of the amino acid derivatives was accomplished in a reversed-phase column with a methanol gradient in 50 mM acetate buffer pH 5.5. These conditions also allowed the separation of the major tissue free physiological amino acids. L-Norvaline was used as an internal standard for both peak identification and quantification. Within-day and between-day precision were less than 6.2%, and the accuracy ranged from 99.1 to 104%. The applicability of the method was demonstrated in a study in rats in which the levels of the assayed amino acids in discrete areas of brain were examined.

Correlation between ethosuximide brain levels measured by high performance liquid chromatography and its antiepileptic potential.

The concentration-time curve of ethosuximide (ESM) in several brain zones of Wistar rats (cortex, midbrain, cerebellum) showed a clear difference from the time course of the drug levels in plasma after administration of 50 mg/kg injected by intraperitoneal route. The brain/plasma ratio in the cortex changes drastically from 2.04 +/- 0.14 (5 m after injection) to 0.14 +/- 0.01 (2 h after injection). Similar behaviour was observed in the brain/plasma ratio of midbrain and cerebellum. The antiepileptic potential of the ESM was closely related to the evolution of the drug time-course in the brain. Reversible opening of the blood-brain barrier modifies the cortex/plasma ratio from 0.14 +/- 0.01 to 0.79 +/- 0.11 (2h after ESM injection). Similar changes were observed in midbrain and cerebellum. The results suggest that the blood-brain barrier plays some part in the time-course of ESM in the brain, and emphasizes the role of brain levels measured by high performance liquid chromatography (HPLC) in clarifying the clinical profile of the drug.

A reversed phase liquid chromatographic method for the simultaneous determination of several common penicillins in human serum.

A rapid and sensitive high performance liquid chromatographic method is described for the simultaneous determination of benzylpenicillin, ampicillin, phenoxymethylpenicillin, cloxacillin, dicloxacillin and nafcillin in small samples of human serum. The chromatographic system involves the use of a Spherisorb ODS reversed phase column and a gradient elution with 1 mM ammonium acetate buffer/acetonitrile (from 90:10 to 75:25 in 15 min). Detection and quantification are monitored by UV absorption at 208 nm. The compounds are extracted with dichloromethane, using tetrabutylammonium hydrogen sulfate neutralized with sodium hydroxide and buffered with borate as an ion pairing reagent; beta-hydroxyethyltheophylline is added as an internal standard. Our results show that the method is accurate and reproducible, allowing quantification of serum levels of assayed penicillins (0.5-50 micrograms/mL) without interference from other drugs commonly used in therapy. Recoveries were generally greater than 79.4%.

Reversed-phase high-performance liquid chromatography method for the determination of bemegride in serum and brain tissue: pharmacokinetics and brain distribution of an intraperitoneal subconvulsive dose in rats.

A simple and rapid HPLC method has been developed for the quantification of bemegride in serum and brain tissue, using p-methylphenobarbital as an internal standard. Serum and brain tissue homogenate samples were extracted with ethyl acetate and the evaporated and redissolved extracts injected into a reversed-phase column. The compounds were eluted with an acetonitrile-phosphate buffer mixture and monitored at 200 nm. A linear response was obtained in the range 1-40 micrograms ml-1 for serum and 1-40 micrograms g-1 for brain tissue. Within-day and between-day precisions were less than 5% and the analytical recovery greater than 76.4%. This method has been used to investigate the kinetic profiles of the drug in serum and discrete areas of rat brain after intraperitoneal administration of a subconvulsive dose of bemegride (10 mg kg-1). Peak concentrations occurred in the brain and serum at the same time (30 min), followed by a biphasic decay. The results also indicated the accumulation of the drug in the brain, with no significant differences (p greater than 0.05) in the impregnation of the different brain areas investigated.

Effect of phenobarbital on carbamazepine and its major metabolites in serum, different brain areas, and urine after acute and chronic administration to rats.

The influence of coadministration of phenobarbital (PB) on disposition of carbamazepine (CBZ) and carbamazepine-10,11-epoxide (CBZ-E) in serum and in discrete areas of rat brain, together with its effects on urinary excretion of CBZ, CBZ-E, and trans-10,11-dihydro-10,11-dihydroxycarbamazepine (CBZ-DIOL) were investigated after both acute and chronic administration. Acute coadministration of PB resulted in increased serum CBZ levels, whereas serum CBZ-E levels were initially lower and then higher. In daily urinary excretion, a reduction in both CBZ-E/CBZ ratio and CBZ-DIOL/CBZ ratio was observed. Chronic (30 day) coadministration of PB led to a decrease in serum CBZ levels after the first hour, whereas serum CBZ-E levels were initially higher and then lower. In daily urinary excretion, a decrease in CBZ-E/CBZ ratio and an increase in CBZ-DIOL/CBZ ratio were noted. These results are consistent with an inhibitory interaction and a metabolic induction on both CBZ epoxidation and CBZ-E metabolism in acute and chronic administration, respectively. However, effects on CBZ epoxidation were preferential. In the various brain areas, the effects observed were similar to those noted in serum. In addition, a relevant increase in brain/serum CBZ ratios was observed with chronic coadministration of PB.

Development of a microcomputer program for statistical evaluation of potential pharmacokinetic drug interactions.

A simple and self-explanatory program in BASIC for the statistical evaluation of potential pharmacokinetic drug interactions is described. This program, using the data (times and drug concentrations) obtained from two different populations, and with the help of graphic computing techniques, allows the determination and statistical comparison of pharmacokinetic parameters (disposition constants, transfer rate constants, area under the concentration-time curves, etc.) for one- and two-compartment open models after intravenous or extravasal administration. The program is organized in subroutines so that it can be easily modified or extended to other pharmacokinetic models by the user.

Experimental spike-and-wave discharges induced by pentylenetetrazol and tolerance to repeated injections: an electrophysiological and biochemical study.

This study was designed to obtain experimental data to correlate duration of spike-and-wave (SW) paroxysms with levels of pentylenetetrazol (PTZ) in several brain regions after intraperitoneal (i.p.) administration of subconvulsive doses of PTZ in Wistar rats. The influence of subconvulsive doses of PTZ on blood-brain barrier (BBB) permeability and tolerance of PTZ to repeated injections were also studied. Intraperitoneal administration of subconvulsive doses of PTZ (25 mg/kg) in single doses resulted in SW activity which accounted for 20% of the continuous electrical brain activity recorded during the first hour after i.p. administration. Brain PTZ levels (cortex, midbrain, cerebellum) were within the range 19.2-34.9 micrograms/g. Repeated doses of PTZ showed a significant decrease in SW activity with no change in PTZ brain levels. As PTZ bioavailability was the same after either a single dose or after repeated doses, the decrease in SW activity may be due to PTZ tolerance. No alterations in the BBB were induced by PTZ subconvulsive doses. The experimental data reported in this study may be useful to quantify modifications of biochemical parameters or to evaluate antiepileptic drugs.

Simultaneous determination of the two components of picrotoxin in serum by reversed-phase high-performance liquid chromatography with application to a pharmacokinetic study in rats.

A reversed-phase HPLC method is reported which allows the quantification of picrotin and picrotoxinin in serum. A linear response was obtained for both drugs in the range 0.2-20 micrograms ml-1. The within-day and between-day precisions were 0.8-3.7% and 1.3-4.9%, respectively. The mean recoveries were greater than 94.2%. The method was applied to a pharmacokinetic study following intraperitoneal (i.p.) administration of 3 mg kg-1 of picrotoxin in rats. The obtained data suggest a relatively slow absorption after i.p. administration followed by a rapid elimination from the central compartment according to a one-compartment open model. The elimination half-lives were 0.340 +/- 0.0308 h for picrotin and 0.312 +/- 0.0241 h for picrotoxinin.

High-performance liquid chromatographic procedure for quantitative determination of pentylenetetrazol in serum and discrete areas of rat brain.

A precise and reproducible high-performance liquid chromatographic method for the determination of pentylenetetrazol in serum and brain tissue is described. The procedure employs reversed-phase chromatography, monitoring the eluant at 202 nm. Quantification is based on peak-height ratio of the drug to the internal standard (p-methylphenobarbital). A linear response is obtained to 100 micrograms/ml (serum) or micrograms/g (brain tissue). Within-day and between-day precision are smaller than 5%, and analytical recovery is greater than 95%. Numerous drugs tested do not interfere with the assay. The method has been used to investigate the kinetics of pentylenetetrazol distribution in serum and in discrete areas of rat brain.

A rapid method for the determination of benzylpenicillin in serum by reversed-phase HPLC.

A simple procedure for the determination of benzylpenicillin in serum is described. The assay involves the extraction of the drug and the internal standard (phenoxymethylpenicillinic acid) from the sample into dichloromethane, using tetrabutylammonium hydrogen sulfate neutralized with NaOH and buffered with citrate as an ion-pairing reagent. RP-HPLC was performed on a Spherisorb 5 ODS column, eluting the drugs isocratically with 14% acetonitrile in 10 mM ammonium acetate buffer. Monitoring was by UV detection at 208 nm. Our results show that the method is accurate and reproducible, permitting quantification of serum levels of benzylpenicillin without interference from other drugs commonly used in therapy. Analytical recovery was greater than 79.2%.