Glutathione prevented dopamine-induced apoptosis of melanocytes and its signaling

Summary

Background

Dopamine (DA), a monoamine neurotransmitter, is a well-known neurotoxin and plays an etiologic role in neurodegenerative disorders such as Parkinson's disease. DA exerts its toxic effect by generation of reactive oxygen species and quinone product. Vitiligo, a depigmentary disorder of the skin and hair characterized by selective destruction of melanocytes, has been reported to show increased levels of DA with onset and progression of the disease.

Objective

The aim of this study is to investigate the cytotoxic effect of DA on melanocytes and to search for protective antioxidants against DA-induced toxicity. In addition, molecular mechanism of cell death was also investigated.

Methods

Cells were treated with DA and cell viabilities were measured by crystal violet staining method. To investigate the cytoprotective activity of various antioxidants, vitamin C, vitamin E, Trolox, quercetin, N-acetylcysteine (NAC) and l-glutathione (GSH) were used. To study cytoprotective effects of NAC and GSH, Mel-Ab cells and cultured normal human melanocytes were pretreated with NAC or GSH, then DA solution was added. DA-induced apoptosis and activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) were also observed by flow cytometric analysis and Western blotting.

Results

The viability of DA-treated Mel-Ab cells significantly decreased in a dose-dependent manner while keratinocytes were much more resistant to DA-toxicity, which was a consistent finding with the selective melanocyte loss observed in vitiligo. Among various antioxidants used in this study, only thiol-containing antioxidants such as NAC or GSH inhibited both JNK and p38 MAPK activation and apoptosis, indicating the unique protective capacity of thiol compounds. Cultured normal human melanocytes were also susceptible to DA and thiol compounds were very efficiently protective against DA-induced cytotoxicity.

Conclusion

DA-induced apoptosis and cytoprotective effect of thiol compounds shown in this study could be a clue to understand pathogenesis of viltigo and provide a new therapeutic strategy.

Introduction

Vitiligo is an acquired and progressive disorder manifested by circumscribed depigmented patches on the skin. The etiology is still unknown but several hypotheses (including neural, radical, autoimmune, self-destruction and inherent defect theory) have been proposed to explain the selective destruction of melanocytes. Although none of these hypotheses are sufficient to explain the pathogenesis of the disease, neural factors appear to play a major role, which is supported by clinical and biochemical findings [1], [2]. Moreover, the level of catecholamines and their metabolites have been reported to increase in plasma and urine of vitiligo patients as well as in lesional skin. The increase of these monoamines mainly occurs at the onset and progression phase, possibly contributing to the disappearance of melanocytes in vitiligo [3], [4], [5], [6].

Dopamine (DA) has a well-known neurotoxic property [7], [8], [9], [10]. Furthermore, DA is commonly used to investigate the neuropathological and biochemical characteristics of Parkinson's disease (PD). The pathogenesis of PD might have an implication for that of vitiligo because melanocytes originate from the neural crest and both diseases have a common pathology of melanin-containing cell loss. It was reported that DA initiated oxidative stress and induced neuronal cell death [9], [10]. In addition, there have been a number of attempts to reveal the mechanism of reactive oxygen species (ROS) production and their downstream pathways in PD [9], [10], [11], [12], [13]. However, no effort had been made to examine the effect of DA in melanocytes, until Chu et al. suggested DA-induced apoptosis in human melanocytes [14]. They revealed DA toxicity to melanocytes, involving generation of ROS, which was prevented by pretreatment with NAC, but did not elucidate its molecular mechanism.

The aim of this study was to investigate the toxic profile of DA and the molecular mechanism of DA-induced apoptosis in melanocytes. We used Mel-Ab cells, a mouse-derived spontaneously immortalized melanocyte cell line that produces large amounts of melanin [15]. In addition, cultured normal human melanocytes were used to examine sensitivity to DA toxicity. In our preliminary study, Mel-Ab cells were found to be sensitive to the toxic effects of DA. Previous reports concerning neuronal cells have pointed that various kinds of antioxidants have different efficacies in protecting against DA-induced neurotoxicity. Using this model, we tried to search for protective antioxidants against DA-induced toxicity in melanocytes. Especially, molecular mechanism of DA-induced apoptosis in melanocytes was investigated by Western blotting for stress signaling pathways.