***************
Invited Symposium: Quinones and Other Reactive Oxygen Species in Neurobiologic, Apoptotic, and Neurotoxic Processes






Abstract

Introduction

Materials & Methods

Results

Discussion & Conclusion

References




Discussion
Board

INABIS '98 Home Page Your Session Symposia & Poster Sessions Plenary Sessions Exhibitors' Foyer Personal Itinerary New Search

Toxicity of Dopamine/dopaminochrome on Cultured Cells


Contact Person: Lauro Galzigna (gal@civ.bio.unipd.it)


Introduction

Recently we have found (Galzigna et al.,1998) that rat brain contains a dopamine peroxidase activity which converts dopamine to dopaminochrome using hydrogen peroxide as an oxidant. Other authors (Okun,1997) have found a brain peroxidase and claim that it is necessary for the synthesis of neuromelanin. Since the rat seems to be lacking neuromelanin (Bogerts,1981), in that case dopamine peroxidase might be a system necessary to eliminate hydrogen peroxide and in normal conditions dopaminochrome may be converted back to dopamine by bioreductive enzymes, e.g. DT-diaphorase. In the presence of hydrogen peroxide, ascorbic acid has been shown to act as a prooxidant (Sakagami and Satok,1997) and this should increase the formation of dopaminochrome; ascorbic acid might act similarly with other oxidant species as well.

This paper reports the effects of dopamine and dopaminochrome on different cells cultured in vitro in the presence and absence of ascorbic acid.

Back to the top.


Materials and Methods

Cell cultures: Primary fibroblasts were obtained from paws of newborn male Wistar rats by digestion with trypsin (2.25% in 15 mM phosphate buffer pH 7.2) and cultured in flasks with 10 ml of Dulbeccoís modified Eagle medium with addition of 10% calf serum, 100 U/ml penicillin and 100 g/ml streptomycin at 37C in the presence of 5% carbon dioxide. A line of monkey kidney tumor(Vero) and a rat neuroblastoma cell line were cultured on the same medium under the same conditions.

Before treatments, the cells were detached from the culture medium with trypsin, centrifuged at 400 x G and resuspended with the same medium. After counting in a Thoma-Zeiss chamber 30,000 cells were sowed in wells of plastic plates containing 24 wells/plate.

The treatment consisted of adding to the incubation medium 0.05 to 0.5 mmol/l dopamine or dopaminochrome for times ranging from 30 to 120 min in the presence or absence of 0.2 mmol/l ascorbic acid.

After the treatments the cells were dehydrated with methanol, stained with Toluidine bleu and counted by an inverted microscope. The total number of viable cells was estimated by counting 10 casual microscopic fields(20X).

Determination of glutathione: Total glutathione was determined spectrophotometrically at 412 nm with the recycling procedure described by Tietze(1969).

Enzyme activities:In cell extracts the DT-diaphorase activity was measured with substrate dopaminochrome 0.05 mmol/l and NADH 0.2 mmol/l pH 7.4 as described by Bindoli et al. (1990). Dopamine peroxidase was assayed as described by Galzigna et al. (1998) with 0.1 mmol/l dopamine and 0.03 mmol/l hydrogen peroxide. Protein was estimated by the biuret method according to Gornall et al. (1949) and 1-2 mg were used for the enzyme assays. Cell extracts were prepared by one cycle of freezing-thawing followed by addition of 2.2 U aprotinin (Antagosan,Behring) per 106 cells, mixing with Vortex and l min sonic irradiation with an Ultra-Turrax (Janke & Kunkle,GFR).

Dopaminochrome synthesis: Dopaminochrome was synthesized with a final yield of 80% from dopamine hydrochloride and sodium periodate (4:1) mixed in 20 ml of bd water for 30 min at room temperature. The mixture was lyophilized and an aqueous solution of 1 mg/ml of the product had a molar extinction of 1175 mol-1cm-1 at 475 nm.

Back to the top.


Results

The present results are compatible with parallel observations made on a clonal cell line derived from mouse hippocampus susceptible to the toxic action of glutamate. At concentration of 0.3 mmol/l dopaminochrome is twice more toxic than dopamine(P.MaherĒpersonal communicationĒ).

Table 1 summarizes the effect of increasing dopaminochrome concentrations at different incubation times on the viability of fibroblasts, Vero and neuroblastoma cells in the presence and absence of ascorbic acid. Table 2 show the effect of dopamine under the same conditions. Cells were always controlled for uniform growth before any treatment. Dopaminochrome treatment produced cell disruption and changes of shape suggesting dehydration.Control experiments with no addition showed no change in cell viability up to 2 h and with the addition of ascorbate alone no significant effect was observed. Table 3 shows the data of enzyme activities and glutathione content of fibroblasts, Vero and neuroblastoma cells.

It is known that in Parkinsonís disease dopaminergic neurons and the nigrostriatal pathway degenerate thus disrupting motor activity and inducing the typical symptoms of the disease. The nigrostriatal degeneration seems to be connected to dopamine oxidation which triggers apoptosis (Offen et al.,1995). Several authors have documented the toxicity of dopamine oxidation products (Hastings et al.,1996) (Linert et al.,1996) (Terland et al.,1997) showing that an imbalance between dopamine oxidation and antioxidant defence is related to the selective degeneration of dopamine neurons in Parkinsonís disease (Church and Ward,1994). The increase of dopamine-induced apoptosis by ascorbate and the protective role of glutathione have been observed on human neural cells (Gabby et al.,1996).

Free radicals are generated during melanin synthesis and their cytotoxic activity has been shown to depend on the level of glutathione (Fruehauf et al.,1998). In fact we see that lower levels of glutathione and DT-diaphorase are compatible with the higher cytotoxicity of dopaminochrome observed in our experiments.

Melanin, endowed with radical scavenging and antioxidant effects, strongly inhibits peroxidation (Avramidis et al.,1998) and the impairment of the cells antioxidant defences, together with the decrease of melanin formation may be a component of neuroendangerment(McIntosh et al.,1998).

A high intake of dietary vitamin E has been shown to exert a protective effect against the occurrence of Parkinsonís disease (de Rijk et al.,1997) while ascorbic acid appeared to be significantly higher in Parkinsonís disease patients(King et al.,1992).

We may hypothesize that the pathogenesis of Parkinsonís disease be related to a chain of events including a reduced bioreductive power affecting melanin biosynthesis and a decrease of melanin content which up-regulates dopamine peroxidation. The toxic dopamine derivatives(e.g. dopamine-o-semiquinone) thus produced may be responsible for the neurodegenerative effects leading to the clinical manifestation of the disease.

Table 1. Effect of 0.3 mmol/l dopaminochrome on cells viability in the presence and absence of 0.2 mmol ascorbic acid at different incubation times. Mean(SD) of 4 experiments
Percent of dead cells
Cells Time
0.5h 1h 2h
Fibroblasts 2(1) 5(2) 20(8)
" + ascorbate 20(5) 18(3) 80(10)
Vero 2(1) 2(1) 3(1)
" + ascorbate 8(3) 15(5) 90(12)
Neuroblastoma 10(2.5) 40(12) 70(15)
" + ascorbate 15(3) 60(15) 100(20)

Table 2: Effect of 0.3 mmol/l dopamine on cell viability in the presence and absence of 0.2 mmol/l ascorbic acid at different incubation times. Mean(SD) of 4 experiments.
Percent of dead cells
Cells Time
0.5h 1h 2h
Fibroblasts 0 0 5(2)
" + ascorbate 5(1) 10(3) 15(3)
Vero 0 2(0.5) 3(1)
" + ascorbate 1(0.5) 5(2) 8(4)
Neuroblastoma 3(1.5) 7(2) 10(3)
" + ascorbate 5(2) 15(4) 30(4)

Table 3: DT-diaphorase and dopamine peroxidase activities with total glutathione content of fibroblasts, Vero and neuroblastoma cells. Dopaminochrome = DC. Mean(SD) of 4 experiments.
Cells DT-diaphorase Dopamine peroxidase Glutathione
micro mol NADH/min/mg micro mol DC/min/mg micro mol/mg prot
Fibroblasts 0.012(0.005) 0.41(0.01) 236(60)
Vero 0.005(0.001) 0.83(0.01) 43(10)
Neuroblastoma 0.006(0.002)) 0.95(0.05) 40(15)

Back to the top.


References

  1. Avramidis N.,Kourounakis A.,Hadjipetrou L.,and Seuchuk V.(1998) Anti-inflammatory and immunomodulating properties of grape melanin.Inhibitory effects on paw edema and adjuvant induced disease.Arzneim.Forsch. 48,764-771

  2. Bindoli A.,Rigobello M.P.,and Galzigna L.(1990) Reduction of adrenochrome by rat liver and brain DT-diaphorase. Free Rad.Res.Comm. 8,295-298

  3. Bogerts B.(1981) A brain stem atlas of catecholaminergic neurons in man,using neuromelanin as a natural marker. J.Comp.Neurol. 197,63-80

  4. Church W.H. and Ward V.L.(1994) Uric acid is reduced in the substantia nigra in Parkinsonís disease:effect on dopamine oxidation.Brain Res.Bull. 33,419-425

  5. de Rijk M.C.,Breteler M.M.,den Breeijen J.H.,Launer L.J.,Grobbee D.E.,van der Meche F.G.,and Hofman A.(1997) Dietary antioxidants and Parkinsonís disease.The Rotterdam study.Arch.Neurol. 54,762-765

  6. Fruehauf J.P,Zonis S.,al-Bassau M.,Kyshtoobayeva A.,Dasgupta C.,Milovanovic T.,Parker R.J.,and Buzaid A.C.(1998) Melanin content and downregulation of glutathione-S-transferase contribute to the action of L-buthionine-S-sulfoximine on human melanin.Chem.-Biol.Interactions 111,277-305

  7. Gabby M.,Tauber M.,Porat S.,and Simantov R.(1996) Selective role of glutathione in protecting human neuronal cells from dopamine-induced apoptosis. Neuropharmacology 35,571-578

  8. Galzigna L.,Schiappelli M.P.,Rigo A.,and Scarpa M.(1998) Dopaminochrome formation catalyzed by purified peroxidases and by a rat brain preparation.Ēin publicationĒ

  9. Gornall A.G.,Bardawill C.J. and David M.M.(1949) Determination of serum protein by means of the biuret reaction. J.Biol.Chem. 177,751-766

  10. Hastings T.G.,Lewis D.A.,and Zigmond M.J.(1996) Role of oxidation in the neurotoxic effect of intrastriatal dopamine injections. Proc.Natl.Acad.Sci.USA 93,1956-1961

  11. King D.,Playfer J.R.,and Roberts N.B.(1992) Concentrations of vitamins A,C and E in elderly patients with Parkinsonís disease. Postgrad.Med.J 68,634-637

  12. Linert W.,Herlinger E.,Jameson R.F.,Kienzl E.,Jellinger K.,and Youdim M.B.(1996) Dopamine,6-hydroxydopamine,iron,and dioxygen:their mutual interactions and possible implication in the development of Parkinsonís disease. Bioch.Bioph.Acta 1316,160-168

  13. McIntosh L.J.,Cortopassi K.M.,and Sapolsky R.M.(1998) Glucocorticoids may alter antioxidant enzymes capacity in the brain:kainic acid studies.Brain Res. 791,215-222 Offen D.,Ziv I,Gorodin S.,Barzilai A.,Malik Z.,and Melamed E.(1995) Dopamine-induced programmed cell death in mouse thymocytes. Bioch.Bioph.Acta 1268,171-177

  14. Okun M.R. (1997) The role of peroxidase in neuromelanin synthesis:a review. Physiol.Chem.Phys.Med.NMR 29,15-22

  15. Sakagami H. and Satoh K. (1997) Prooxidant action of two antioxidants:ascorbic acid and gallic acid. Anticancer Res. 17,221-224

  16. Terland O.,Flatmark T.,Tangeras A.,and Gronberg M.(1997) Dopamine oxidation generates an oxidative stress mediated by dopamine semiquinone and unrelated to reactive oxygen species. J.Mol.Cell Cardiol. 29,1731-1738

Back to the top.


Back to the top.


| Discussion Board | Previous Page | Your Symposium |
Galzigna, L; (1998). Toxicity of Dopamine/dopaminochrome on Cultured Cells. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/kostrzewa/galzigna0211/index.html
© 1998 Author(s) Hold Copyright