1-Methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol) is toxic to dopaminergic neuroblastoma SH-SY5Y cells via impairment of cellular energy metabolism

The endogenous neurotoxin 1-methyl-6,7-dihydroxy-1,2,3, 4-tetrahydroisoquinoline (salsolinol), which is structurally similar to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been reported to inhibit mitochondrial complex I (NADH-Q reductase) activity as does the MPTP metabolite 1-methyl-4-phenylpyridinium ion (MPP(+)). However, the mechanism of salsolinol leading to neuronal cell death is still unknown. Thus, we correlated indices of cellular energy production and cell viability in human dopaminergic neuroblastoma SH-SY5Y cells after exposure to salsolinol and compared these results with data obtained with MPP(+). Both toxins induce time and dose-dependent decrease in cell survival with IC(50) values of 34 microM and 94 microM after 72 h for salsolinol and MPP(+), respectively. Furthermore, salsolinol and MPP(+) produce a decrease of intracellular net ATP content with IC(50) values of 62 microM and 66 microM after 48 h, respectively. In contrast to MPP(+), salsolinol does not induce an increase of intracellular net NADH content. In addition, enhancing glycolysis by adding D-glucose to the culture medium protects the cells against MPP(+) but not salsolinol induced cellular ATP depletion and cytotoxicity. These results suggest that cell death induced by salsolinol is due to impairment of cellular energy supply, caused in particular by inhibition of mitochondrial complex II (succinate-Q reductase), but not complex I.     

A systematic regional study of brain salsolinol levels during and immediately following chronic ethanol ingestion in rats

Regional contents of salsolinol and catecholamines in the brain of normal and ethanol-treated rats were studied. Male Sprague Dawley rats were given ethanol solution as sole drinking fluid for 3, 4, 5 or 6 months. Salsolinol determined by gas chromatography mass spectrometry was found to be present in the hypothalamus and the striatum of control rats. The levels of salsolinol in these regions increased significantly by long-term ethanol drinking and rapidly decreased to control levels following its removal. Salsolinol levels in other regions of rat brain were extremely low or negative and unaltered upon chronic ethanol treatment. In ethanol-treated rats the hypothalamic salsolinol, although generally higher than in the striatum, increased along with the ethanol exposure, whereas the striatal salsolinol was constant during those periods of study. Brain dopamine (DA) and norepinephrine contents remained unaltered during and immediately after chronic ethanol treatments. No correlation of salsolinol levels with DA contents or blood ethanol concentrations was observed. The occurrence of salsolinol in selected areas of rat brain with lack of changes in catecholamine level but as a result of an in vivo formation by long-term ethanol drinking was considered to be due to an alteration of acetaldehyde metabolism in the liver and brain.     

A Critical Evaluation of Influence of Ethanol and Diet on Salsolinol Enantiomers in Humans and Rats

Background: (R/S)‐Salsolinol (SAL), a condensation product of dopamine (DA) with acetaldehyde, has been speculated to have a role in the etiology of alcoholism. Earlier studies have shown the presence of SAL in biological fluids and postmortem brains from both alcoholics and nonalcoholics. However, the involvement of SAL in alcoholism has been controversial over several decades, since the reported SAL levels and their changes after ethanol exposure were not consistent, possibly due to inadequate analytical procedures and confounding factors such as diet and genetic predisposition. Using a newly developed mass spectrometric method to analyze SAL stereoisomers, we evaluated the contribution of ethanol, diet, and genetic background to SAL levels as well as its enantiomeric distribution. Methods: Simultaneous measurement of SAL enantiomers and DA were achieved by high performance liquid chromatography‐tandem mass spectrometry (HPLC/MS/MS). Plasma samples were collected from human subjects before and after banana (a food rich in SAL) intake, and during ethanol infusion. Rat plasma and brain samples were collected at various time points after the administration of SAL or banana by gavage. The brain parts including nucleus accumbens (NAC) and striatum (STR) were obtained from alcohol‐non‐preferring (NP) or alcohol‐preferring (P) rats as well as P‐rats which had a free access to ethanol (P‐EtOH). Results: Plasma SAL levels were increased significantly after banana intake in humans. Consistently, administration of banana to rats also resulted in a drastic increase of plasma SAL levels, whereas brain SAL levels remained unaltered. Acute ethanol infusion did not change SAL levels or R/S ratio in plasma from healthy humans. The levels of both SAL isomers and DA were significantly lower in the NAC of P rats in comparison to NP rats. The SAL levels in NAC of P rats remained unchanged after chronic free‐choice ethanol drinking. There were decreasing trends of SAL in STR and DA in both brain regions. No changes in enantiomeric ratio were observed after acute or chronic ethanol exposure. Conclusions: SAL from dietary sources is the major contributor to plasma SAL levels. No significant changes of SAL plasma levels or enantiomeric distribution after acute or chronic ethanol exposure suggest that SAL may not be a biomarker for ethanol drinking. Significantly lower SAL and DA levels observed in NAC of P rats may be associated with innate alcohol preference.     

Biosynthesis of salsolinol,a tetrahydroisoquinoline alkaloid,in healthy subjects

Summary The R enantiomer of salsolinol was detected in the urine of two out of six healthy subjects, whereas 1,2-dehydrosalsolinol was present in the urine of all the subjects. (S)-salsolinol was never detected. Administration of Madopar for 7 days resulted in the presence of large amounts of (R)- and (S)-salsolinol in the urine of five out of the six subjects, the urinary excretion of 1,2-dehydrosalsolinol being generally not markedly increased.The presence of 1,2-dehydrosalsolinol in urine suggests that the biosynthesis of salsolinol in healthy volunteers should occur by condensation of dopamine with pyruvic acid, in keeping with Hahn's hypothesis. The absence of salsolinol in the urine of one subject after Madopar administration seems to indicate that the biological system(s) involved in the reduction of the C = N bond in 1,2-dehydrosalsolinol can be missing or not, or poorly, functional in some individuals, and suggests that there is no alternative pathway for the formation of salsolinol in healthy volunteers.     

Salsolinol differentially affects mice selected for sensitivity to alcohol

Salsolinol, a compound putatively formed following alcohol ingestion, differentially decreased the activity of lines of mice after 18 generations of genetic selection for alcohol sensitivity. Low doses of salsolinol produced significantly lower activity levels in the alcohol-sensitive long-sleep (LS) line than in the alcohol-insensitive short-sleep (SS) line. A hypnotic dose of salsolinol induced significantly longer sleep-times in the LS line than in the SS line. Results are interpreted as supporting the hypothesis that salsolinol-like substances may mediate some of the effects of alcohol on the central nervous system.     

Effects of ethanol and salsolinol on catecholamine function in LS and SS mice

Adrenal and urinary levels of adrenaline and noradrenaline were determined in rats subjected to severe ethanol intoxication for periods of up to 96 hours, in rats undergoing withdrawal and in a post-withdrawal period, and in controls. Adrenaline and noradrenaline content of adrenal glands fell markedly to less than eight and twenty percent, respectively, after four days of intoxication. Noradrenaline content, but not adrenaline content, had recovered after a subsequent four day period of recovery. The depletion in adrenal catecholamine levels was coincident with increases in urinary adrenaline and noradrenaline levels over the first 48 hours of intoxication. Urinary catecholamine levels remained higher than control values for the next 48 hours of intoxication. Adrenal glands were larger after 12 hours of intoxication, although there was no increase in dry weight. At later times adrenal enlargement was associated with increased dry weight and protein content. This increase in mass was found to be of cortical origin. These results demonstrate that severe ethanol intoxication promotes an intense stimulation of the rat adrenal gland with enhanced synthesis and release of catecholamines, and cortical hypertrophy.     

The reinforcing properties of salsolinol in the ventral tegmental area: evidence for regional heterogeneity and the involvement of serotonin and dopamine

Background:  Salsolinol (SAL), the condensation product of acetaldehyde and dopamine, may be a factor contributing to alcohol abuse. Previous research indicated that both ethanol and acetaldehyde are self-administered into the posterior ventral tegmental area (VTA). The current study examined SAL self-infusions into the VTA, and determined the involvement of dopamine neurons and 5-HT₃ receptors in this process.
Methods:  The intracranial self-administration technique was used to determine the self-infusion of SAL into the VTA of adult, male Wistar rats. The rats were placed in 2-lever (active and inactive) experimental chambers, and allowed to respond for the self-infusion of 0, 0.03, 0.1, 0.3, 1.0 or 3.0 μM SAL into the posterior or anterior VTA. In a second experiment, rats self-administered 0.3 μM SAL for the initial 4 sessions, co-administered SAL with ICS-205,930 (a 5-HT₃ receptor antagonist) or quinpirole (a D_2,3 receptor agonist) for sessions 5 and 6, and then only 0.3 μM SAL for session 7.
Results:  Wistar rats, given 0.03 to 0.3 μM SAL, received more infusions per session than did the group given artificial cerebrospinal fluid (aCSF) alone (e.g., 41 infusions for 0.1 μM SAL versus 9 infusions for the aCSF group), and responded more on the active than inactive lever. These effects were observed in the posterior but not in anterior VTA. Co-infusion of 100 μM ICS-205,930, or quinpirole significantly reduced self-infusions and active lever responding.
Conclusions:  SAL produces reinforcing effects in the posterior VTA of Wistar rats, and these effects are mediated by activation of DA neurons and local 5-HT₃ receptors.     

Characterization of the in vivo actionof(R)-salsolino, an endogenous metabolite of alcohol, on serotonin and dopamine metabolism: A microdialysis study

Using a microdialysis-HPLC technique in conscious rats, we examined the action of (R)-1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, (R)-salsolinol (R-Sal), a possible endogenous metabolite of alcohol, on serotonin (5-HT) and dopamine (DA) metabolism in four regions of the brain: the striatum, the substantia nigra, the hippocampus and the hypothalamus. Following 1 mM R-Sal perfusion, the dialysate level of 5-HT in the striatum markedly increased from non-detectable levels to 4259.2 ± 617.5nM, while DA increased from 3.4 ± 0.9nM to206.0 ± 56.5nM. The increase was one order of magnitude larger in 5-HT than in DA. Conversely, the output of 5-hydroxyindoleacetic acid decreased markedly to non-detectable levels, while 3,4-dihydroxyphenylacetic acid and homovanillic acid outputs decreased below 40% of basal levels. These effects were dose-related to R-Sal (1 μM to 1 mM) and were confirmed also in 3 other brain regions. The R-Sal-induced responses in the striatum were observed even after pretreatment of 2 μM tetrodotoxin, a blocker of nerve-firing activity, via the dialysis membrane. The repetitive perfusion with 1 mM R-Sal into the striatum induced the reproducible response of 5-HT and DA. Furthermore, the potencies of 1 mM R-Sal to increase the output of 5-HT and DA were approximately 783.0-fold and 2.6-fold stronger, respectively, than those of the same dose of methamphetamine. The results suggest that R-Sal acts to stimulate a release of monoamines, 5-HT preferentially, with inhibition of monoamine oxidase and catechol-O-methyltransferase activities.     

Plasma and Urine Salsolinol in Humans: Effect of Acute Ethanol Intake on the Enantiomeric Composition of Salsolinol

The tetrahydroisoquinoline (TIQ) salsolinol (SAL), a condensation product of dopamine and pyruvate or acetaldehyde, is one of the neuropharmacologically active alkaloids in mammals. Previous HPLC studies have shown that the R -enantiomer of SAL is largely predominant, or is the only enantiomer in the urine of healthy subjects, whereas the S -enantiomer was found predominant in the urine of alcoholics. An enzymatic pathway for SAL formation that is influenced by chronic alcohol intake was proposed. However, our analyses showed that the SAL detectable in human urine and plasma is racemic, at least in healthy subjects. No change of the enantiomeric distribution was observed after an acute alcohol ingestion (1 g alcohol/kg body weight). Using a new method for the resolution of the SAL enantiomers and gas chromatography/mass spectrometry analysis, the SAL enantiomers were quantified in the urine and plasma of 24 subjects before and after the intake of alcohol. Special dietary conditions were observed to avoid interferences by the SAL of the foodstuff. Although the distribution of SAL enantiomers was not changed after alcohol intake, the total urinary SAL output and the plasma concentration of SAL were significantly influenced in different ways. Only five subjects showed a significant increase both in plasma SAL concentration and in the total urinary SAL output, whereas 19 subjects showed decreased or unchanged SAL levels after alcohol administration. Data also show that only the subjects with low baseline levels (mean of 0.148 ng SAL/ml plasma) tend to increase SAL levels after ethanol ingestion, which may imply some genetic basis for the response.