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Neuronal alterations resulting from exposure to Parkinsonian-inducing 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine (MPTP) in an in vitro model SH-SYSY human neuroblastoma cells were explored using cytotoxic effects, neurochemical changes and pathological injury as endpoints. The results suggested that: MPTP entered the SH-SYS5Y human neuroblastoma cells through a non-dopamine transport mechanism, and was metabolized to 1-methyl-4-phenyl-2,3-dihydropyridium (MPDP⁺) and 1-methyl-4-phenylpyridium (MPP⁺) by monoamine oxidase (MAO). MPP⁺, the neurotoxic analog of MPTP, was taken up into cells through a dopamine (DA) uptake mechanism. MPTP, via its metabolite MPP⁺, inhibited NADH dehydrogenase activity. The MPTP-induced alterations of morphology included formation of blebs, attenuated neutrites, abnormal mitochondria with electron-density of matrix and disorganization of cristae, and abnormal aggregation of filamentous material of the cytoskeleton. MPTP was neurotoxic to the dopaminergic system, inhibiting monoamine oxidase (MAO) activity, and decreasing levels of dopamine (DA) and other catecholamines. In addition, MPTP enhanced ³H-DA release from cells, and its metabolite MPP⁺ inhibited ³H-DA uptake. MPTP was found to directly act on the cholinergic system in SH-SY5Y cells, causing dose-related decreases in the binding at muscarinic and nicotinic receptors. MPTP also inhibited acetylcholinesterase (AChE) activity and increased choline levels. The MPTP-induced increase in DA release and the decreases in catecholamines in SH-SYSY cells were blocked by pretreatment with acetylcholine receptor antagonists atropine and d-tubocurarine. MPTP caused increases in tau proteins, and also caused an increased expression of the reverse transcriptase polymerase chain reaction (RT-PCR) product after treatment for 2 to 5 days at 10⁻³ to 10⁻⁴ M. The results, for the first time, demonstrated that MPTP affected cytoskeletal associated tau protein and altered its mRNA. These results demonstrated that the human neuroblastoma cell line, SH-SYSY, can be used as an in vitro model for the study of the neurotoxicity of MPTP, including the mechanisms associated with exposure to this neurotoxicitant. / Ph. D.

The principal psychoactive component of marihuana is Δ-9-tetrahy-drocannabinol. This compound at 10-5 molar concentration in the medium of human cell cultures appeared to inhibit DNA, RNA, and protein synthesis by 50, 40, and 30% respectively, as measured by incorporation of radioactive precursors into acid-insoluble cell fractions in human diploid fibroblasts, human neuroblastoma cells, and mouse neuroblastoma cells. While Δ-9-tetrahydrocannabinol inhibited semiconservative DNA synthesis, it had no effect on DNA repair synthesis in human cells as assayed by the photolysis of 5-bromodeoxyuridine incorporation into DNA during repair after ultraviolet radiation damage. Δ-9-tetrahydrocannabinol also had no effect on rejoining of DNA single-strand breaks induced by γ-rays. The nonspecificity of the inhibition of macromolecular synthesis by Δ-9-THC suggested a possible interference with uptake of radioactive precursors. However, experimentation has shown that this depression of macromolecular synthesis cannot be accounted for by reduced transport of radioactive precursors into the cell because the rate of transport of these precursors into the cell is essentially the same in the presence or absence of Δ-9-THC. Pool sizes of macromolecular precursors as measured radioisotopically (3Hthymidine, 3H-uridine, 14C-leucine) appear to be reduced about 50%, and this reduced pool size could fully account for the reduced macromolecular synthesis seen in the presence of Δ-9-THC. We do not know what causes this apparent reduction of pool sizes in the presence of Δ-9-THC.