Studies on the potential neurotoxicity of tetrahydroisoquinoline and nicotinamide derivatives.
thesisposted on 19.11.2015, 08:51 by Jonathon. Willets
The naturally occurring compounds N'-methyl nicotinamide (NMN), 6,7-dihydro-1-methyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) and 1,2,3,4-tetrahydro-2-methyl-4,6,7-isoquinolinetriol (TMIQ) are reported to interact with catecholaminergic systems in vivo and have similar structure to 1-methyl-4-phenylpyridinimuM (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Three cell lines, rat B65, hmuMan IMR32 and SH-SY5Y, were examined to find the most appropriate with which to study such toxins. These data indicate that both B65 and IMR32 cells do not take up catecholamines (2% after one hour), in monolayer culture. In contrast, SH-SY5Y cells take up significant (P 0.05) amounts of both noradrenaline (NA) and dopamine (DA), which can be blocked by uptake1 inhibitors. Furthermore, SH-SY5Y cells store [3H]NA in a releasable pool, which can be stimulated through receptor-occupancy and depolarization. Therefore, the hmuMan SH-SY5Y neuroblastoma cell line was selected for further study. NMN induced cytotoxicity albeit at high concentrations (10mM), in SH-SY5Y cells. Lower concentrations had no significant (P 0.05) effect. Cytotoxicity was inhibited in a dose dependent manner by the addition of a-tocopherol. NMN had little effect upon catecholamine uptake and release. Both salsolinol and TMIQ significantly (P 0.05) inhibited MTT reduction and increased LDH release, at concentrations within the range of MPP+ cytotoxicity, in vitro. Cytotoxicity was not inhibited by the addition of monoamine oxidase inhibitors, antioxidants, or imipramine. TMIQ and salsolinol stimulated catecholamine uptake with EC50 values of 7muM (NA) and 54muM (DA), 17muM (NA) and 11muM (DA), respectively. However, above 100muM, salsolinol inhibited uptake with IC50 values of 411muM and 379muM for NA and DA, respectively. Inhibition of NA uptake by salsolinol, corresponded to the increased displacement of nisoxetine from the uptake1 recognition site, since the Ki (353muM) for displacement was similar to the IC50 values (411 and 379 muM) for uptake. TMIQ also displaced nisoxetine binding with Ki of 71muM. TMIQ and salsolinol stimulated catecholamine uptake does not involve the uptake recognition site, and is not inhibited by elevation of cAMP or cGMP, or the inhibition of PKC. Salsolinol inhibited both carbachol (CCH) and potassium, whilst TMIQ inhibited only potassium evoked [3H]NA release, from SH-SY5Y cells. Thus both TMIQ and salsolinol are cytotoxic to SH-SY5Y cells, although the exact mechanism of toxicity requires further investigation, it appears not to involve bioactivation by MAO, and is not mediated through membrane based free radical damage. NMN is also cytotoxic to neurones, but only at high concentrations, and appears to be membrane directed. The high concentration of NMN required to induce cytotoxicity casts doubt over the relevance of NMN as an in vivo toxin. In contrast, both TMIQ and salsolinol are cytotoxic in vitro and may have a role in neurodegeneration.