Toxicity of a Treatment Associating Dopamine and Disulfiram for Catecholaminergic Neuroblastoma SH-SY5Y Cells: Relationships with 3,4-Dihydroxyphenylacetaldehyde Formation

doi:10.1016/S0161-813X(03)00148-7

NeuroToxicology

Volume 25, Issue 3, March 2004, Pages 365-375

https://doi.org/10.1016/S0161-813X(03)00148-7 Get rights and content

Abstract

3,4-Dihydroxyphenylacetaldehyde (DOPAL) is formed by the oxidative deamination of dopamine (DA) catalyzed by monoamine oxidases (MAO); then, the aldehyde is oxidized to 3,4-dihydroxyphenylacetic acid (DOPAC) by aldehyde dehydrogenases (ALDH) or reduced to 3,4-dihydroxyphenylethanol (DOPET) by aldose/aldehyde reductases. The present work aimed at evaluating the in vitro toxicity of DOPAL on catecholaminergic neuroblastoma SH-SY5Y cells which accumulate DA. DOPAL synthesis was stimulated by incubating cells with DA and blocking DOPAL oxidation by disulfiram, an irreversible inhibitor of ALDH. As evidenced by MTT reduction assays, DA and disulfiram treatments produced cell losses which increased with time. 10^–2 M DA reduced by 40% cell viability after a 1 h treatment, when its TC₅₀ (concentration reducing viability by 50%) value was 7.3×10⁻⁵ M after a 24 h treatment. For the same treatment periods, TC₅₀ values for disulfiram were 8×10⁻⁵ and 8.7×10⁻⁷ M, respectively. MTT reduction assay performed after a 24 h treatment followed by a 24 h incubation in a drug-free medium evidenced that the toxicity of 10⁻⁴ M DA or 10⁻⁶ M disulfiram was potentiated by the second drug. HPLC measurements showed that DOPAL was produced at the early stages of the treatment by DA and disulfiram. This was evidenced by the significant increase in the (DOPAL+DOPET)/DOPAC ratio observed after a combined 3 h treatment by 10⁻⁴ M DA and 10⁻⁶ M disulfiram. Total contents in DA and DOPAL were greatly reduced at the end of a 15 h treatment, and disulfiram did not significantly enhanced the (DOPAL+DOPET)/DOPAC ratio. For both treatment durations, DOPAL and DOPET were detectable only in the extracellular medium. So, these results suggest that an early production of DOPAL could produce delayed toxic effects on SH-SY5Y cells. Production of DOPET and release of DOPAL could be important means for reducing DOPAL concentrations in dopaminergic neurons.

Section snippets

INTRODUCTION

Several reports suggest that an oxidative stress could be responsible for the rather specific loss of dopaminergic neurons which characterizes Parkinson’s disease. Mechanisms underlying this rather selective vulnerability remain unclear, but various experimental evidences suggest that a mitochondrial dysfunction (Orth and Shapira, 2002, Cassarino et al., 1997) could be associated with an altered dopamine (DA) metabolism and a decreased ability to scavenge toxic metabolites (Hornykiewicz and

Chemicals

[³H] DA (6.6 Ci/mmol) was purchased from Amersham. Culture medium, fetal calf serum and antibiotics (streptomycin and penicillin) were purchased from Biowhittaker (Belgium). Disulfiram, DA, DOPAC, NAD, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) and rotenone were obtained from Sigma (St. Louis, MO). Proprionaldehyde and pyrazole were purchased from Fluka. Rotenone was dissolved in methanol (0.2% of the final volume). Disulfiram solutions (10 mM) were prepared in a 45%

[³H] Dopamine Uptake by SH-SY5Y Cells

The specific uptake of [³H] DA by SH-SY5Y cells increased linearly with time during the first 20 min of incubation, at a mean rate of 22 fmol/min per 10⁵ cells. Thereafter, this rate slowed down (Fig. 2). Nomifensine blocked the specific uptake in a monophasic manner (not shown); its inhibitory concentration 50% was 4 × 10⁻⁸ M for a 5 min incubation period.

Effects of DA or Disulfiram on SH-SY5Y Viability

DA and disulfiram produced time- and concentration-dependent losses in SH-SY5Y viability (Table 1). A 1 h treatment in the presence of various DA

Discussion

The present study shows that a blockade of the ALDH activity can potentiate the DA toxicity on SH-SY5Y cells which display catecholaminergic features. This potentiation was observed in a situation which could occur in parkinsonians submitted to l-DOPA therapy. This treatment, aiming at increasing cerebral concentrations of DA, a substrate of MAO, can be associated with a blockade of the oxidation of DOPAL to DOPAC. This might result from a genetic deficit in ALDH (Goedde and Agarwal, 1987,

Acknowledgements

The authors wish to thank Prof. J.C. Quirion for providing the DOPET chemical synthesis. We would like to acknowledge Prof. C. Lange, Dr. C. Loutelier-Bourhis and Dr. P. Vérité for performing spectral analyses. This work was supported in part by grants from la Direction de la Recherche et de la Technologie (contract 97-187).

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Toxicity of a Treatment Associating Dopamine and Disulfiram for Catecholaminergic Neuroblastoma SH-SY5Y Cells: Relationships with 3,4-Dihydroxyphenylacetaldehyde Formation

Abstract

Section snippets

INTRODUCTION

Chemicals

[3H] Dopamine Uptake by SH-SY5Y Cells

Effects of DA or Disulfiram on SH-SY5Y Viability

Discussion

Acknowledgements

Chem. Biol. Interact.

Prog. Neurobiol.

Neuropharmacology

Brain Res.

Brain Res.

Free Rad. Biol. Med.

Biochim. Biophys. Acta

Eur. J. Pharmacol.

Biochem. Pharmacol.

Biochem. Pharmacol.

J. Biol. Chem.

Free Rad. Biol. Med.

Exp. Neurol.

Free Radic. Biol. Med.

Biochem. Pharmacol.

Eur. J. Pharmacol.

Mol. Brain Res.

Eur. J. Cancer

J. Biol. Chem.

Eur. J. Pharmacol.

FEBS Lett.

Blood

Biochem. Pharmacol.

J. Chromatogr.

Neurodegeneration

Biochem. Pharmacol.

J. Immunol. Meth.

Biochem. Pharmacol.

[³H] Dopamine Uptake by SH-SY5Y Cells