Valverde P, Manning P, Todd C, McNeil C J, Thody A J
Department of Dermatology, University of Newcastle-upon- Tyne, UK.
Exp Dermatol. 1996 Oct;5(5):247-53. doi: 10.1111/j.1600-0625.1996.tb00125.x.
Reactive oxygen species (ROS) are potentially cytotoxic and several mechanisms have evolved to protect against their damaging effects. In melanocytes, tyrosinase may have such a rôle by utilising the superoxide anion (O2-) in the production of melanin. In the present study, we have examined the cytotoxic effects of O2- and hydrogen peroxide (H2O2) in human melanocytes both before and following the activation of tyrosinase. Xanthine oxidase (XO, 5-150 mU.ml-1) and glucose oxidase (GO, 0.1-20 mU.ml-1) were used to generate the O2- and H2O2 respectively, and the cytotoxic effects assessed by measuring cell survival using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium (MTT) assay. 3 h later, dose-related decreases in melanocyte survival were seen. Similar experiments with keratinocytes and fibroblasts showed that these cells were more resistant to the cytotoxic effects of O2- than were the melanocytes. The effect of increasing tyrosinase activity was examined by growing the melanocytes in the presence of an analogue of melanocyte-stimulating hormone (MSH) Nle4DPhe7 alpha-MSH (10(-8) M), for 48 h. This increased tyrosinase activity, melanin content, the ability to trap O2- and the resistance of the melanocytes to the cytotoxic effects of this ROS, but failed to alter their susceptibility to the damaging effects of H2O2.Nle4DPhe7 alpha-MSH had no effect on the resistance of keratinocytes and fibroblasts to either O2- or H2O2. After 3 h, XO, as opposed to GO, also increased the melanin content of human melanocytes; this effect was not accompanied by an increase in tyrosinase activity. The present results suggest that tyrosinase may utilise O2- to produce melanin and that this process may protect melanocytes from the potentially damaging effects of this ROS.
活性氧（ROS）具有潜在的细胞毒性，机体已进化出多种机制来抵御其破坏作用。在黑素细胞中，酪氨酸酶可能通过在黑色素生成过程中利用超氧阴离子（O2-）发挥这样的作用。在本研究中，我们检测了酪氨酸酶激活前后O2-和过氧化氢（H2O2）对人黑素细胞的细胞毒性作用。分别使用黄嘌呤氧化酶（XO，5 - 150 mU/ml）和葡萄糖氧化酶（GO，0.1 - 20 mU/ml）生成O2-和H2O2，并通过使用3 - [4,5 - 二甲基噻唑 - 2 - 基] - 2,5 - 二苯基四氮唑（MTT）法测量细胞存活率来评估细胞毒性作用。3小时后，观察到黑素细胞存活率呈剂量相关下降。对角质形成细胞和成纤维细胞进行的类似实验表明，这些细胞比黑素细胞对O2-的细胞毒性作用更具抗性。通过在黑素细胞刺激激素（MSH）类似物Nle4DPhe7α - MSH（10(-8) M）存在下培养48小时，研究了酪氨酸酶活性增加的影响。这增加了酪氨酸酶活性、黑色素含量、捕获O2-的能力以及黑素细胞对这种ROS细胞毒性作用的抗性，但未能改变它们对H2O2破坏作用的敏感性。Nle4DPhe7α - MSH对角质形成细胞和成纤维细胞对O2-或H2O2的抗性均无影响。3小时后，与GO不同，XO也增加了人黑素细胞的黑色素含量；这种作用并未伴随酪氨酸酶活性的增加。目前的结果表明，酪氨酸酶可能利用O2-产生黑色素，并且这一过程可能保护黑素细胞免受这种ROS的潜在破坏作用。
