Table 4.

Examples of in vitro studies related to salsolinol. DA dopamine, DAT dopamine transporter, EC50 the half maximal effective concentration, IC50 the half maximal inhibitory concentration, N/A not available, NE norepinephrine, PRL prolactin, SAL salsolinol, TH tyrosine hydroxylase. Salsolinol was applied as a racemic mixture unless otherwise stated

Cell-based in vitro models
Salsolinol source	Salsolinol concentration(hydrochloride unless otherwise stated)	Model	Main outcomes	References
Santa Cruz Biotechnology, Dallas, TX, USA	0.01 μM – 1 mM (racemic and purified R- and S-SAL)	commercially cell-based assays, composed by recombinant CHO-K1 cells that overexpress only the human μ-opioid receptor	SAL activated the μ-opioid receptor by the classical G protein-adenylate cyclase pathway with EC50 of 2 × 10−5 M. The agonist action of SAL was fully blocked by the μ-opioid antagonist naltrexone. The EC50 for the purified stereoisomers (R)-SAL and (S)-SAL were 6 × 10−4 M and 9 × 10−6 M, respectively. Molecular docking simulations predicted a morphine-like interaction of (R)-SAL and (S)-SAL stereoisomers with the μ-opioid receptor and favoured the interaction for the (S)-SAL stereoisomer.	Berríos-Cárcamo et al. (2017)
N/A	0–1000 μM	human neuroblastoma (SH-SY5Y), human primary glioblastoma (U87) and human monocytic (THP-1) cells	SAL was toxic to SH-SY5Y cells in a dose-dependent manner with 47.50% cell death at 500 μM. Similarly, 500 μM SAL induced 13.50 and 50.50% death in U87 and THP-1 cells, respectively.	Wang et al. (2015)
Synthesized (Beijing Institute of Technology, Beijing, China)	275 – 2200 μM	human neuroblastoma (SH-SY5Y) cells	The lethal dose (LD50) values for SAL = 1500 μM.	Arshad et al. (2014)
Synthesized (Semmelweis University, Budapest, Hungary)	0–100 μM	bacterial Escherichia coli (BL21 DE-3) cells	SAL completely inhibited DA binding, to both the high and low affinity DA binding sites. The concentration at which half the DA bound was 58 ± 4.4 nM of SAL. It produced 3.7-fold greater inhibition of Ser40-phosphorylated TH compared to DA by competing more strongly with tetrahydrobiopterin.	Briggs et al. (2013)
Sigma Aldrich, St. Louis, MO, USA	100-800 μM	human neuroblastoma (SH-SY5Y) cells	SAL caused a dose-dependent toxicity mediated by apoptosis (increase in caspase-3 levels).	Brown et al. (2013)
	25, 50, 100, 200, 400 and 800 μM		Maximum toxicity (about 50%) was achieved with 400 μM of SAL.	Qualls et al. (2014)
	1–100 μM, especially 10 μM	neural stem cells (NSCs)	Morphological impairment, cleaved caspase-3 and decreased Bcl-2:Bax suggested apoptosis. SAL toxicity coincided with reduced pAkt level and its downstream effectors: pCREB, pGSK-3b, Bcl-2, suggesting repressed PI3K/Akt signaling pathway, confirmed on adding the PI3K inhibitor (LY294002), which abolished the protection	Shukla et al. (2013)
N/A	0 – 400 μM	rat pheochromocytoma (PC12) and parkin knockdown (PC 20) cells	The elevated parkin knock down elevated cellular oxidative stress and SAL levels.	Su et al. (2013)
Sigma Aldrich, St. Louis, MO, USA	0–500 μM	human neuroblastoma (SH-SY5Y) cells	SAL neurotoxicity towards SH-SY5Y cells was potentiated during treatment with concentrations of glutathione below 250 μM, whereas glutathione concentrations above 250 μM resulted in protection against SAL-induced neuronal cell death.	Wszelaki and Melzig (2012)
	10–500 μM	human neuroblastoma (SH-SY5Y, SK-NSH) cells	The cell viability decreased in a concentration-dependent manner. 500 μM of SAL caused 49.08 ± 1.8% and 22.5 ± 4.5% cell death in undifferentiated and differentiated SH-SY5Y cells, respectively.	Wszelaki and Melzig (2011)
	250 μM	human neuroblastoma (SH-SY5Y) cells	The anti-apoptotic action of N-methyl-D-aspartate (NMDA) on SAL (250 μM)-evoked cell death in human SH-SY5Y cells was observed, without the influence on caspase-3 activity.	Jantas and Lason (2009)
Synthesized (Szent-Györgyi Albert University, Szeged, Hungary)	0,001 - 10 μM (hydrobromide)	bovine anterior pituitary cells	SAL significantly stimulated the release of PRL from cultured bovine anterior pituitary cells at doses of 1 - 10 μM, compared to control cells.	Hashizume et al. (2008a)
			SAL (1 μM), thyrotropin-releasing hormone (TRH, 0,01 μM) ), and SAL plus TRH significantly increased the release of PRL, but the additive effect of SAL and TRH detected in vivo was not observed in vitro. DA (1 μM) inhibited the TRH-, as well as SAL-induced PRL release in vitro.	Hashizume et al. (2008b)
	1–1 mM	human embryonic kidney (HEK-293), human neuroblastoma (SH-SY5Y) and human glioblastoma (HTZ-146 cells	SAL was the endogenous key substrate of the sodium-independent organic cation transporter (OCT2). OCT2 was preferentially expressed in the dopaminergic regions of the substantia nigra where it co-localized with DAT and TH. SAL exhibited a selective toxicity toward OCT2-expressing cells that was prevented by cyclo(his-pro).	Taubert et al. (2007)
Sigma Aldrich, St. Louis, MO, USA	50–500 μM	human neuroblastoma (SH-SY5Y) cells	SAL treatment caused up-regulation in the levels of c-Jun and phosphorylated c-Jun. The binding activity of NF-κB to DNA was enhanced by SAL in the concentration dependent manner. SAL decreased the levels of the anti-apoptotic protein Bcl-2 and increased pro-apoptotic protein Bax, while enhancing the release of cytochrome-c from mitochondria.	Wanpen et al. (2007)
	0 – 0.8 mM		Exposure to 0.4 mM of SAL resulted in approximately 65% reduction in cell viability. Maximal toxic effect was observed with 0.8 mM of SAL where approximately 80% of cells did not survive.	Copeland et al. (2005)
	0–500 μM	human neuroblastoma (SH-SY5Y) andmice fetal mesencephalic cell	SAL increased the production of reactive oxygen species and significantly decreased glutathione levels and cell viability in SH-SY5Y cells. SAL decreased intracellular ATP levels and induced nuclear condensation in these cells. SAL-induced depletion in cell viability was completely prevented by N-acetylcysteine.	Wanpen et al. (2004)
	100 μM	human neuroblastoma (SH-SY5Y) cell	Both exogenous IGF-1 and IGF-1 gene transfer significantly prevented the SAL-induced cell death and increased cell viability.	Shavali et al. (2003)
	10–200 μM	human melanoma (FRM, MNT and M14) and murine melanoma (B16) cells	SAL enhanced TH activity and melanin production.	De Marco et al. (2002)
	0.01–1000 μM	human embryonic kidney (HEK-293) and mouse neuroblastoma (Neuro-2A) cells	Only 2(N)-methylated isoquinoline derivatives structurally related to MPTP/MPP+ are selectively toxic to dopaminergic cells via uptake by the DAT.	Storch et al. (2002)
	1 mM	dopaminergic neuronal (SN4741) cells	SAL induced the moderate ROS activity compared to paraquat, and subsequently activated much lower level of JNK1/2 activity compared to MPP+ and paraquat treatments.	Chun et al. (2001)
	0–500 μM	rat pheochromocytoma (PC12) cells, pBR322 and X174 supercoiled DNA, calf thymus DNA	SAL in combination with Cu(II) induced strand scission in pBR322 and X174 supercoiled DNA, which was inhibited by the copper chelator, reactive oxygen species (ROS) scavengers, reduced glutathione and catalase. Reaction of calf thymus DNA with SAL plus Cu(II) resulted in substantial oxidative DNA damage as determined by 8-hydroxydeoxyguanosine (8-OH-dG) formation. Blockade of the dihydroxyl functional group of SAL abolished its capability to yield 8-OH-dG in the presence of Cu(II).	Jung et al. (2001)
		rat pheochromocytoma (PC12) cells	SAL causes reduced viability, which was exacerbated by Cu2+. Although SAL alone could cause apoptotic death in PC12 cells, cells treated with SAL together with Cu2+ became necrotic.	Kim et al. (2001)
	0-200 μM	dopaminergic neuronal (RCSN-3) cells	SAL was found to decrease survival in RCSN-3 cells (derived from adult rat substantia nigra) in a concentration-dependent manner (208 μM of SAL induced a 50% survival decrease). In vitro oxidation of salsolinol to o-quinone catalyzed by lactoperoxidase gave the quinone methide and 1,2-dihydro-1-methyl-6,7-isoquinolinediol as final products of salsolinol oxidation as determined by nuclear magnetic resonance spectroscopy (NMR) analysis	Martinez-Alvarado et al. (2001)
Synthesized (according to Haber et al. 1993)	1 mM (R- and S-SAL)	mouse anterior pituitary tumor (AtT-20) cells (clone D16v)	SAL bound to the D(2) receptor family, especially to the D(3) receptor with a K(i) of 0.48+/-0.021 μM. S-SAL significantly inhibited the formation of cyclic AMP and the release of beta-endorphin and ACTH in a pituitary cell system.	Melzig et al. (2000)
Sigma Aldrich, St. Louis, MO, USA	0–1000 μM	human neuroblastoma (SH-SY5Y) cells	SAL was cytotoxic to human SH-SY5Y cells via impairment of cellular energy production. The IC50 = 34.2 μM (after 72 h) was established for SAL.	Storch et al. (2000)
Synthesized (according to Teitel et al. 1972)	0.1 μM–10 mM (R- and SSAL)		The IC50 values were 540.2 μM for (R)-SAL and 296.6 μM for (S)-SAL.	Takahashi et al. (1997)
Synthetized (according to Haber et al. 1993)	0-500 μM (R- and S-SAL)	mouse anterior pituitary tumor (ArT-20) cells	A significant decrease in the proopiomelanocortin (POMC) gene expression by the S-SAL was noted. The basal secretion of adrenocorticotropin (ACTH) as well as the corticotropin-releasing factor-stimulated ACTH release remained unchanged after R- and S-SAL treatment. It was shown that a reduction of intracellular cAMP level occurred after the treatment of the cells with S-SAL whereas R-SAL did not affect the cAMP production.	Putscher et al. (1995)
Sigma Aldrich, St. Louis, MO, USA	0.001–1 mM	human neuroblastoma (SH-SY5Y) cells	SAL stimulated catecholamine uptake with EC50 values of 17 μM and 11 μM, for NA and DA, respectively. At concentrations above 100 μM, SAL inhibited the uptake of NA and DA, with IC50 values of 411 μM and 379 μM, respectively.	Willets et al. (1995)
N/A	0.001–10 mM	calf aortic endothelial (BKEz-7) cells	SAL damaged the cultivated calf aortic endothelial cells (cytotoxic effects estimated by cell counting after 72 h treatment with SAL, IC50 = 38 μM), especially the mitochondria, and inhibited the respiration measured as inhibition of the oxygen consumption. The damage of endothelial cells was confirmed by the electron microscopy with various disintegrations of mitochondria.	Melzig and Zipper (1993)
Other in vitro models
Salsolinol source	Salsolinol concentration	Model	Main outcomes	References
Sigma Aldrich, St. Louis, MO, USA	0.1–2 mM	human ceruloplasmin (hCP)	Incubation of hCP with SAL increased the protein aggregation and enzyme inactivation in a dosedependent manner. Reactive oxygen species scavengers and copper chelators inhibited the SALmediated hCP modification and inactivation. The formation of dityrosine was detected in SALmediated hCP aggregates. Amino acid analysis post the exposure of hCP to SAL revealed that aspartate, histidine, lysine, threonine and tyrosine residues were particularly sensitive.	Kim et al. (2016)
	0.01–1 μM	230 μm horizontal slices of CD-1 mice midbrain	SAL was able to excite pVTA DA cells mice treated with α-methyl-p-tyrosine (a DA biosynthesis inhibitor). SAL was needed for ethanol-induced pVTA DA cells activation since neither acetaldehyde nor ethanol was able to excite these neurons in the absence of DA.	Melis et al. (2015)
	0.05–1 mM	horse cytochrome c	Protein aggregation increased in a dose-dependent manner after incubation of cytochrome c with SAL. The formation of carbonyl compounds and the release of iron were obtained in salsolinoltreated cytochrome c. Reactive oxygen species scavengers and iron specific chelator inhibited the SAL-mediated cytochrome c modification and carbonyl compound formation.	Kang (2013)
	0.5 mM	neurofilament-L (NF-L)	NF-L exposure to SAL produced losses of glutamate, lysine and proline residues. Carnosine and anserine were shown to significantly prevent SAL-mediated NF-L aggregation.	Kang (2012)
	0-1000 μM alone or in presence of Cu or Fe	plasmid DNA pBR322 or calf thymus DNA	SAL in the presence of divalent copper induced strand scission and damage in both plasmid and genomic DNA.	Tharakan et al. (2012)
	0.01–1 μM	250–300 μm coronal slices of rat midbrain	SAL excited VTA-dopamine neurons indirectly by activating μ-opioid receptors, which inhibited GABA neurons in the VTA.	Xie et al. (2012)
		200–250 μm coronal slices of rat midbrain	SAL via the activation of presynaptic D1receptors and facilitation of glutaminergic transmission contributed to SAL-induced excitation of pVTA DA neurons.	Xie and Ye (2012)
	5 mM	equine spleen ferritin	The exposure of ferritin to SAL resulted in the generation of protein carbonyl compounds and the formation of dityrosine.	Kang (2010)
	0-0.2 mM	pUC19 plasmid DNA purified from Escherchia coli	SAL/ferritin system-mediated DNA cleavage and mutation was attributed to hydroxyl radical generation via the Fenton-like reaction of free iron ions released from oxidatively damaged ferritin.	Kang (2009)
	0–5 mM	human Cu,Zn-superoxide dismutase	SAL led to inactivation of Cu,Zn-superoxide dismuthase (SOD) in a concentration-dependent manner. Free radical scavengers and catalase inhibited the SAL-mediated Cu,Zn-SOD modification. Exposure of Cu,Zn-SOD to SAL led also to the generation of protein carbonyl compounds.	Kang (2007)
	10, 20, 50 nM	an Fe3+–EDTA–H2O2 complex and a NO–H2O2 system	The in vitro production of the cytotoxic hydroxyl radicals (*OH) was recorded during the autoxidation of SAL.	Nappi et al. (1999)
Synthetized (according to Teitel et al. 1972)	0.05–1 mM (R- and S-SAL; hydrobromide)	pig brain soluble and membrane-bound catechol-O-methyltransferase (COMT)	Kinetic analysis of the O-methylation by S-COMT yielded almost equivalent Km values of 0.138 mM [(R)-SAL] and 0.156 mM [(S)-SAL]. Both enantiomers had similar Vmax values (0.201 and 0.189 nmol min-1 mg protein-1, respectively).	Hötzl and Thomas (1997)
Sigma Aldrich, St. Louis, MO, USA	0–500 μM	øX174 RFI supercoiled DNA, calf thymus DNA, PC12 cells	Incubation of SAL and CuCl2 with calf thymus DNA caused strand breaks. SAL in combination with Cu(II) mediated the strand scission in øX174 RFI supercoiled DNA in a time-related manner. SAL induced cell death in cultured PC12 cells, which was exacerbated by Cu(II).	Kim et al. (1997)
Synthesized (King’s College of London, London, UK)	100 μM	male Wistar rat striata synaptosomes	SAL (100 μM) produced the 39.9% inhibition of the [3H]dopamine uptake.	McNaught et al. (1996a)
	0.5–10 mM	intact Wistar rat liver mitochondria	Isoquinoline derivatives may exert mitochondrial toxicity in vitro similar to that of MPTP/MPP+, however SAL is a weak inhibitor of mitochondrial respiration. Qualitative structure-activity relationship studies revealed that isoquinolinium cations were more active than isoquinolines in inhibiting mitochondrial respiration.	McNaught et al. (1996b)
N/A	0–0.5 mM	microsomal fractions of male Wistar rats livers	Histamine and SAL competitively inhibited the activity of debrisoquine 4-monooxygenase (Ki = 0.31 and 0.43 mM, respectively).	Iwahashi et al. (1993)
Synthetized from salsolidine		human placental MAO A and human liver MAO B	Stereoselective competitive inhibition of MAO (monoamine oxidase) type A was found with the (R)-SAL (Ki = 31 μM), but not MAO type B.	Bembenek et al. (1990)
Synthetized	10-30 μM	liver homogenate (human liver dihydropteridine reductase)	SAL inhibited human liver dihydropteridine reductase non-competitively.	Shen et al. (1982)
Synthesized (according to Craig et al. 1952)	10–200 μg/ml, 333 μg/ml (hydrobromide)	chick biventer cervicis nerve muscle preparation, guinea pig ileum, chick biventer cervicis homogenates	SAL produced agonist effects at muscarinic receptors. In the chick biventer cervicis preparation, SAL (10-200 pg/mL) produced initial twitch augmentation, followed by blockade accompanied by a slowly developing contracture. Responses to exogenous carbachol were unaffected while those to acetylcholine were augmented. The neuromuscular blockade was unable to be reversed by choline, caffeine, physostigmine or tetanus.	Rodger et al. (1979)