P. Dostert

A novel enzyme enantio-selectively synthesizes (R)salsolinol, a precursor of a dopaminergic neurotoxin, N-methyl(R)salsolinol.

In the human brain, only (R)enantiomer of 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline ((R)salsolinol) and N-methyl-salsolinol, a dopaminergic neurotoxin, were detected, suggesting their enzymatic biosynthesis. This paper reports the isolation and characterization of a novel enzyme, which enantio-selectively synthesizes (R)salsolinol from dopamine and acetaldehyde. Dopamine, acetaldehyde, formaldehyde and pyruvic acid were the substrates of this synthase, whereas N-methyldopamine, adrenaline, noradrenaline and L-DOPA were not. The possible function of this enzyme under physiological and pathological conditions in the brain is discussed.

Differential Changes in Superoxide Dismutase Activity in Brain and Liver of Old Rats and Mice

Superoxide dismutase (SOD) activity was measured in the brain and liver of 24–26‐ and 3‐month‐old rats. No significant age‐related differences in Cu/Zn‐SOD activity were found in any of the tissues studied. A small but significant increase in total SOD activity was observed in the whole brain (10‐20%), cerebral cortex (11%), and hypothalamus (18%) of old rats, whereas a much more important increase in Mn‐SOD activity was found in the whole brain (48%), cerebral cortex (70%), striatum (60%), and hypothalamus (30%). The increase of Mn‐SOD activity in the brain of old rats suggests the enzyme may play an important role in the process of aging. Mn‐SOD is found only in the mitochondrion, which could be an important site of oxygen free radical production, and a significant increase in the enzyme activity was also found in the lung of hypoxic rats. A significant decrease in total SOD and Mn‐SOD activity was observed in the liver of old rats. Preliminary experiments in 23–24‐month‐old mice similarly showed an increase and a decrease in total SOD and Mn‐SOD activity, respectively, in the whole brain and liver. These results suggest that the regulatory mechanisms of Mn‐SOD in the brain and liver vary differentially with age.

An endogenous dopaminergic neurotoxin, N-methyl-(R)-salsolinol, induces DNA damage in human dopaminergic neuroblastoma SH-SY5Y cells.

Abstract: Recently, an endogenous neurotoxin, 1(R),2(N)‐dimethyl‐6,7‐dihydroxy‐1,2,3,4‐tetrahydroisoquinoline [N‐methyl‐(R)‐salsolinol], was found to elicit parkinsonism in rats with selective depletion of dopamine neurons in the substantia nigra without necrotic tissue reaction. The mechanism of the cell death was examined by detection of DNA damage using a single‐cell gel electrophoresis (comet) assay in human dopaminergic neuroblastoma SH‐SY5Y cells. Only N‐methylsalsolinol was found to induce DNA damage, whereas other catechol isoquinolines, such as (R)‐salsolinol, (S)‐salsolinol, and 1,2‐dimethyl‐6,7‐dihydroxyisoquinolinium ion, did not. The (R)‐enantiomer of N‐methylsalsolinol damaged DNA much more profoundly than the (S)‐enantiomer. Cycloheximide protected the cells from DNA damage, suggesting that an apoptotic process may account for the DNA damage. Morphological changes indicating apoptotic cell death were also confirmed. Antioxidants and deprenyl reduced DNA damage, indicating that the damage was initiated by oxidative stress and that neuroprotection by deprenyl may be partially ascribed to its prevention of DNA damage. Apoptosis induced by neurotoxins may be a mechanism underlying the cell death of dopamine neurons in the substantia nigra of Parkinsons disease.

Dopamine-derived endogenous 1(R),2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, N-methyl-(R)-salsolinol, induced parkinsonism in rat: biochemical, pathological and behavioral studies

Dopamine-derived 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol, Sal) and related compounds were examined for their selective neurotoxicity to dopamine neurons by injection into the rat striatum. Among salsolinol analogs examined, only N-methyl-(R)- salsolinol (NM(R)Sal) induced behavioral changes very similar to those in Parkinsons disease: hypokinesia, stiff tail, limb twitching at rest and postural abnormality. Biochemical analysis showed that after NM(R)Sal injection, NM(R)Sal itself and its oxidation product, 1-2-dimethyl-6,7-dihydroxyisoquinolinium ion (DMDHIQ+) accumulated in the striatum, and also in the substantia nigra definite amount of DMDHIQ+ was detected. Dopamine and noradrenaline were reduced in the striatum and more markedly in the substantia nigra, whereas serotonin and its metabolite were not affected. Morphological analysis revealed selective reduction of tyrosine hydroxylase (TH)-containing neurons in the substantia nigra after continuous NM(R)Sal administration in the striatum. These results demonstrate the selective cytotoxicity of NM(R)Sal to the dopamine neurons in the substantia nigra, and the possible involvement of this 6,7-dihydroxy-isoquinoline in the pathogenesis of Parkinsons disease is discussed.

Presence of 2-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline and 1,2-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, novel endogenous amines, in parkinsonian and normal human brains

2-Methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline and 1,2-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline were identified for the first time as novel endogenous amines in parkinsonian and normal human brains by gas chromatography-mass spectrometry. It is of interest that these tetrahydroisoquinolines are analogues of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) which produces Parkinsons disease.

N-methyl(r)salsolinol produces hydroxyl radicals: Involvement to neurotoxicity

Recently, (R)-1,2-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline [N-methyl-(R)salsolinol, NM(R)Sal] and 1,2-dimethyl-6,7-dihydroxyisoquinolinium ion [DiMeDHIQ+] were found to cause a syndrome similar to parkinsonism in rodents. NM(R)Sal is produced in the brain by N-methylation of a naturally occurring catechol isoquinoline, 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline [(R)salsolinol, (R)Sal], which is formed from dopamine. The mechanism of NM(R)Sal cytotoxicity to dopamine neurons was examined using in vitro experiments. NM(R)Sal was found to be nonenzymatically oxidized into DiMeDHIQ+, with concomitant formation of hydroxyl radicals. The oxidation and the radical production were completely inhibited by the antioxidants, ascorbic acid and reduced glutathione, and the radical formation was enhanced by Fe(II) and, to a less extent, by Fe(III). The oxidation of NM(R)Sal into DiMeDHIQ+ and the production of hydroxyl radicals may be essential for neurotoxicity to develop in dopamine neurons. The possible involvement of this catechol isoquinoline in the pathogenesis of Parkinsons disease is discussed.

Uptake of a neurotoxin-candidate, (R)-1,2-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline into human dopaminergic neuroblastoma SH-SY5Y cells by dopamine transport system

Uptake of catechol isoquinolines to dopamine cells was studied using human dopaminergic neuroblastoma SH-SY5Y cells. Only (R)-1,2-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline [(R)-1,2-DiMeDHTIQ] was transported by dopamine uptake system, while (S)-1,2-DiMeDHTIQ, (R)- and (S)-1-methyl-6,7-dihydroxy-tetrahydroisoquinoline, and 1,2-dimethyl-6,7-dihydroxyisoquinolinum ion were not. Kinetical study showed that the uptake of (R)-1,2-DiMeDHTIQ followed the Michaelis-Menten equation, and the values of the Michaelis constant and the maximal velocity were obtained to be 102.6 ± 36.9 μM and 66.0 ± 2.8 pmol/min/mg protein. Dopamine was found to inhibit (R-1-DiMeDHTIQ uptake competitively. These results suggest that the selective uptake by dopamine transporter may account for the specific neurotoxicity of (R)-1,2-DiMeDHTIQ to dopamine neurons.

Dopamine-derived alkaloids in alcoholism and in Parkinson's and Huntington's diseases

Tetrahydroisoquinoline (TIQ) alkaloids and 1-carboxy TIQ derivatives have been found in human fluids and/or tissues. The possible biosynthetic pathways of salsolinol (Sal), taken as an example of TIQs, are discussed, and the possibility that biosynthesis occurs through a stereospecific enzymatic reaction is considered. In this respect, it is reported that the R enantiomer of Sal predominates in urines of healthy volunteers, whereas the S enantiomer predominates in port wine and possibly in other beverages and foods, suggesting that Sal present in humans could have, at least partially, and endogenous enzymatic origin. TIQs and other dopamine-derived alkaloids are weak MAO inhibitors, the R enantiomer of Sal and salsolidine being more potent than the S form. The changes in monoamine oxidase activity and the nigrostriatal concentrations of dopamine and homovanillic acid in Parkinsons and Huntingtons diseases and in alcoholism are reviewed. In these pathological situations, changes in the levels of dopamine-derived alkaloid levels may occur. The possibility that the modifications found might cause or contribute to changes in mental and/or neurophysiological states in these pathological situations is considered.

A dopaminergic neurotoxin, 1(R), 2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, N-methyl(R)salsolinol, and its oxidation product, 1,2(N)-dimethyl-6,7-dihydroxyisoquinolinium ion, accumulate in the nigro-striatal system of the human brain

N-Methyl(R)salsolinol was found to be an endogenous dopaminergic neurotoxin inducing parkinsonism in rodents and to increase in the cerebrospinal fluid of parkinsonian patients. The amounts of N-methyl(R)salsolinol and related compounds in the human brain regions were quantitatively analyzed. Only the (R)-enantiomer of salsolinol derivatives were detected, which suggests their enzymatic synthesis in situ. In the nigro-striatal system, the concentration of N-methyl(+)salsolinol was higher than in the frontal cortex, and its oxidized catechol isoquinolinium ion was detected only in the substantia nigra significantly. The accumulation of these neurotoxins in the nigro-striatal region might account for selective cell death of dopamine neurons in the substantia nigra of Parkinsons disease.

Ratio of the R and S enantiomers of salsolinol in food and human urine

Salsolinol is present in human fluids and tissues as well as in some foods and beverages. It was found previously that the R enantiomer of salsolinol predominates in human urine whereas the S enantiomer predominates in Port wine. In this study a new methodology for measuring the proportion of the R and S salsolinol enantiomers in dried banana and human urine is described. In dried banana, a food particularly rich in salsolinol, the R/S ratio was found to be very near to 1. In urine from additional healthy volunteers, the presence of only the R enantiomer was detected. The origin of urinary salsolinol and its enantiomeric composition are discussed with respect to exogenous salsolinol.