Invited Symposium: Quinones and Other Reactive Oxygen Species in Neurobiologic, Apoptotic, and Neurotoxic Processes
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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.
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 Dulbecco’s modified Eagle medium with addition of 10% calf serum, 100 U/ml penicillin and 100 g/ml streptomycin at 37C 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.
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.Maher”personal communication”).
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 Parkinson’s 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 Parkinson’s 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 Parkinson’s disease (de Rijk et al.,1997) while ascorbic acid appeared to be significantly higher in Parkinson’s disease patients(King et al.,1992).
We may hypothesize that the pathogenesis of Parkinson’s 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) |