Richard A. Wall

Partial protection from the dopaminergic neurotoxin N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine by four different antioxidants in the mouse

C57 black mice given a single injection of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 40 mg/kg, developed marked reduction of striatal dopamine content and loss of dopaminergic neurons in the zona compacta of the substantia nigra. However, pretreatment with any one of four different antioxidants, alpha-tocopherol, beta-carotene, ascorbic acid or N-acetylcysteine, significantly decreased MPTP-induced striatal dopamine loss, and alpha-tocopherol prevented neuronal loss in the substantia nigra. Four chemical analogues of MPTP (cinnamaldehyde, N,N-dimethylcinnamylamine, arecoline and 2-methyl-1,2,3,4-tetrahydro-6,7-isoquinolinediol) were all found to lack dopaminergic nigrostriatal neurotoxicity in the mouse.

Nigrostriatal dopaminergic neurons remain undamaged in rats given high doses of L-DOPA and carbidopa chronically.

Abstract: Rats were fed maximally tolerated doses of l‐3,4‐Dihydroxyphenylalanine (l‐DOPA) and carbidopa daily for 120 days in order to achieve a sustained elevation in brain dopamine levels. Some animals were also given buthionine sulfoximine, a γ‐glutamylcysteine synthetase inhibitor, in an unsuccessful effort to reduce brain glutathione contents. l‐DOPA‐ and carbidopa‐treated animals displayed no behavioral changes suggestive of nigrostriatal dopaminergic neuronal loss. When sacrificed 60 days after L‐DOPA treatment ended, all rats had normal tyrosine hydroxylase activities and dopamine contents in their striata, and cell counts were normal in the substantia nigra. It therefore seems unlikely that a model of Parkinsons disease, suitable for exploring the etiological importance of glutathione deficiency, can be produced in rats merely by administering the largest tolerable doses of l‐DOPA.

Role of hydrazine in the mechanism of isoniazid hepatotoxicity in rabbits.

Abstract Isoniazid (INH) continues to be a highly effective drug in the chemoprophylaxis and treatment of tuberculosis; however, its use is associated with hepatotoxicity (predominantly hepatic necrosis) in 1–2% of individuals. The INH metabolites, acetylhydrazine and hydrazine, have each been implicated as the causative hepatotoxin in INH-induced hepatotoxicity. Using a model of INH-induced hepatotoxicity in rabbits, in which INH-induced hepatotoxicity manifests as hepatic necrosis, hepatic steatosis (hepatic fat accumulation) and hypertriglyceridaemia (elevated plasma triglycerides), we compared the severity of these measures of toxicity with plasma levels of INH, acetylhydrazine and hydrazine. Plasma INH and acetylhydrazine were not correlated with markers of INH-induced hepatic necrosis or fatty changes. Plasma hydrazine at 32 h was correlated significantly with plasma argininosuccinic acid lyase (ASAL, a sensitive marker of hepatic necrosis) activity as area under the curve (r2=0.54, P<0.002) and log plasma ASAL activity at 48 h after the first dose of INH (r2=0.53, p<0.005), but not with fatty changes. These results show in this model of INH-induced hepatotoxicity in rabbits that hydrazine, and not INH or acetylhydrazine, is most likely involved in the pathogenic mechanism of hepatic necrosis.

Paraquat and two endogenous analogues of the neurotoxic substance N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine do not damage dopaminergic nigrostriatal neurons in the mouse

C57 black mice were injected repeatedly with maximal tolerated doses of 4 different chemical analogues of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), or its metabolite N-methyl-4-phenylpyridinium ion (MPP+), in order to assess their possible neurotoxicity for dopaminergic nigrostriatal neurons and their potential for causing idiopathic Parkinsons disease. The 4 analogues were the herbicide paraquat, reduced paraquat (having two N-methyl-tetrahydropyridine moieties), N-methyl-1,2,3,4-tetrahydroisoquinoline, and 2-methyl-1,2,3,4-tetrahydro-beta-carboline, the latter two compounds being possible endogenous neurotoxins. Contents of striatal dopamine, measured by high-performance liquid chromatography with electrochemical detection one month after injections were completed, were not depleted by any of these 4 compounds in mice. They might conceivably prove more neurotoxic in primates.

A model of isoniazid-induced hepatotoxicity in rabbits.

Isoniazid (INH) continues to be an effective drug used for chemoprophylaxis and treatment of tuberculosis. Unfortunately, INH is associated with significant hepatotoxicity in up to 2% of individuals exposed, and if this adverse event is not recognized early it can be fatal. Research on INH-induced hepatotoxicity has been hampered by the lack of a suitable animal model that closely resembles the toxicity in humans. The mechanism of INH-induced hepatotoxicity is still unknown. The present study describes the development of a reliable model of INH-induced hepatotoxicity in rabbits. The protocol involves repeated injections of INH over a 2-day period, resulting in significant hepatic necrosis as indicated by elevations of plasma argininosuccinic acid lyase activity. Pretreatment with phenobarbital increased the occurrence of INH-induced hepatic necrosis from approximately 60% (9 out of 15 rabbits) with INH alone to more than 90% (13 out of 14 rabbits). Morphological indices were used to demonstrate the presence of INH-induced hepatotoxicity, and biochemical indices were used to demonstrate both the presence and severity of INH-induced hepatotoxicity in this model. This model may prove useful for further investigations into the mechanism of INH-induced hepatotoxicity.

Effects of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and its metabolite, N-methyl-4-phenylpyridinium ion, on dopaminergic nigrostriatal neurons in the mouse

A single subcutaneous injection in the C57 black mouse of 40 mg/kg of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes a 90% depletion of striatal dopamine, as well as loss of 33% of neuronal cell bodies in the substantia nigra, zona compacta. By 4.5 months, there appears to be partial recovery of striatal dopaminergic function. After injection into mice, the MPTP metabolite, N-methyl-4-phenylpyridinium ion (MPP+) enters the striatum, and through some unknown mechanism is even more toxic than MPTP. However, repeated injections of maximally tolerated amounts of MPP+ do not damage dopaminergic nigrostriatal neurons.

Human CSF GABA Concentrations: Revised Dowd for Controls, but Not Decreased in Huntington's Chorea

Abstract: γ‐Aminobutyric acid (GABA) concentrations were measured in CSF specimens from two large groups of control subjects, one without neurological or psychiatric disease, and one with a variety of neurological disorders not known to involve altered GABAergic function in brain. CSF GABA was also measured in patients with Huntingtons chorea and in patients with other choreiform disorders. GABA was measured in CSF by a modification of the ion exchange‐fluorometric method that featured use of a relatively large cation exchange column, and a markedly decreased quantity of sulfosalicylic acid for deproteinization of CSF. Mean GABA concentrations in CSF were 87 and 77 nmol/liter for neurologically normal and abnormal control subjects, 82 nmol/liter for the Huntingtons chorea patients, and 105 nmol/liter for patients with other forms of chorea. The mean concentration of homocarnosine was not reduced in CSF of Huntingtons chorea patients as compared with controls. Mean CSF GABA concentrations found in control subjects were less than half the lowest control means previously reported. These low values are attributable in part to a reduction in on‐column hydrolysis of conjugated forms of GABA in CSF, which can be produced by excessive sulfosalicylic acid, and in part to improved chromatographic resolution of GABA from other unknown o‐phthalaldehyde‐reactive compounds in CSF. Analysis of free GABA in CSF does not appear useful for diagnosis of suspected Huntingtons chorea, nor as a possible predictive test for persons genetically at risk for Huntingtons chorea.

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) does not destroy nigrostriatal neurons in the scorbutic guinea pig

Guinea pigs were injected subcutaneously with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in maximal tolerated doses (8 mg/kg, once daily) for 10 or 15 days. No neurological effects were noted, other than sedation and hypotonia lasting a few hours after each injection, either in animals maintained on normal diet or in animals fed an ascorbate-deficient diet and rendered severely scorbutic. Subsequent chemical analyses of the striatum showed no evidence of lasting damage to nigrostriatal dopaminergic neurons in MPTP treated guinea pigs on normal diet, and minimal evidence of permanent damage to these neurons in scorbutic animals. MPTP was undetectable in the urine of MPTP-treated animals, although a metabolite, presumably 1-methyl-4-phenylpyridinium ion (MPP+) was regularly present in urine. The relative lack of neurotoxicity of MPTP in the guinea pig remains unexplained. This species clearly is not a suitable small animal for MPTP-induced parkinsonism.

Elevation of Brain GABA Content by Chronic Low-Dosage Administration of Hydrazine, a Metabolite of Isoniazid

Abstract: When γ‐aminobutyric acid aminotransferase (GABA‐T) activity was measured in vitro in rat brain, neither isoniazid (INH) nor four of its known metabolites (isonicotinic acid, acetylisoniazid, acetylhydrazine, diacetylhydrazine) inhibited the enzyme in concentrations (5 mM) far higher than those likely to be achieved when INH is administered to man. In contrast, hydrazine (5 μM) caused a 50% inhibition of GABA‐T without inhibiting glutamic acid decarboxylase (GAD). Rats were injected daily for 109 days with hydrazine (0.08 or 0.16 mmol/kg/day), after which amino acid contents and enzyme activities were measured in their brains. Both hydrazine doses caused significant elevations of whole brain GABA content and reductions of GABA‐T activity, but did not affect GAD activity. Chronic administration of hydrazine at thee doses did not reduce weight gain or alter rat behavior, nor did it produce any irreversible pathologic changes in liver or alterations in hepatic aryl hydrocarbon hydroxylase activity. However, hydrazine treatment caused changes in the contents of many brain amino acids besides GABA, and markedly increased concentrations of ornithine, tyrosine, and α‐aminoadipic acid in rat plasma. Inhibition of GABA‐T activity and the other biochemical alterations observed in patients given high doses of INH probably result from hydrazine formed in the metabolic degradation of INH. Thus administration of hydrazine might be a more direct means of elevating brain GABA content in patients where this seems indicated, and might not entail a greater risk of adverse effects.

Physicochemical Determinants for Drug Induced Blockade of HERG Potassium Channels: Effect of Charge and Charge Shielding

The data on the activities of all previously described HERG blockers and of the most widely cited I(Kr) blockers were analyzed with respect to the effect of potential charged center(s) and its shielding by surrounding structural elements. The following model was considered: the less shielding of the charged form of the drug occurs, the easier its deprotonation will be and the less potency of the blockade of HERG/I(Kr) channels will be. Tertiary amines which form ammonium ions shielded by two structural fragments of the drug molecule were found to be potent HERG/I(Kr) blockers with IC50 < 1 microM (16 of 19 compounds, 84%). However, if the charged center was found at the molecular periphery as such groups as dimethylamino, N-methylpiperidino, N-methylpiperazino, N-methylpyrrolidino, pyrrolidino, imidazolo and partial periphery (diethylamino), then only moderate potency for HERG blockade with 1 microM < IC50 < 10 microM (8 of 11 compounds; 73%) was observed. Similarly, 27 of 32 weak HERG blockers ( IC50 > 10 microM) were found to be primary or secondary amines, or neutral or very weakly basic compounds. Ions of primary and secondary amines are susceptible to the fast deprotonation of the charged center and they, as well as non-charged compounds, have a low probability of induction of Torsades de Pointes (TdP). Conformational analysis and modeling of the interaction of the charged fragment of the drugs with acetone, a system that mimics a ketone fragment of HERG/I(Kr) channel, supports preference of the conformation with the shielded charged center for potent HERG/I(Kr) blockers. The absence of stereospecificity of HERG/I(Kr) blockade observed in most of the published studies reinforces the importance of charged center shielding as a key parameter. We suggest that the introduction of a hydroxy group at position 3 relative to a tertiary ammonium charged center, or the introduction of hydroxy, alkoxy or amino groups at position 2 relative to the nitrogen center of an aromatic system, should provide easy access of a water molecule to the proton, thereby facilitating deprotonation and thus leading to a moderate or weak HERG/I(Kr) blockade and a reduced risk of TdP.