Glutathione prevented dopamine-induced apoptosis of melanocytes and its signaling
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.
Section snippets
Reagents
Dopamine (DA), vitamin C, vitamin E, Trolox (a water-soluble vitamin E analogue), quercetin, NAC and l-glutathione (GSH) were purchased from Sigma (St. Louis, MO, USA). Antibodies that recognize phospho-specific JNK1/2 (Thr183/Tyr185, G-7, sc-6254), total JNK2 (D-2, sc-7345), phospho-specific p38 (Tyr182, D-8, sc-7973), total p38 (A-12, sc-7972) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell cultures
Mel-Ab cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with
Dopamine cytotoxicity
Mel-Ab cells were treated with various concentrations of DA [(A) 0–400 μM, (B) 0–1000 μM] for 24 h and cell viability was measured. The viability of DA-treated cells significantly decreased in a dose-dependent manner, starting to show significant difference from the concentration of 10 μM (Fig. 1A). About 50% reduction of cell viability was observed with 500 μM of DA, and this concentration was chosen for further experiments (Fig. 1B). Fig. 2A shows the time-dependent decrease in cell viability by
Discussion
Parkinson disease (PD) is a neurodegenerative disorder associated with a progressive loss of dopaminergic neurons of the substantia nigra. DA is reported to be deleterious to neural tissue and the proposed mechanisms of DA-induced neurotoxicity are explained in three pathways: ROS generated by intra- or extracelluar autooxidation, hydrogen peroxide induced by monoamine oxidase (MAO) and direct inhibition of the mitochondrial respiratory chain [8], [9], [10], [18]. In vitiligo, neural factors
Acknowledgement
This research was supported by a grant (SC3260) from the Stem Cell Research Center of the 21st Century Frontier Program funded by the Ministry of Science and Technology, Republic of Korea.
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