Carvalho Márcia, Remião Fernando, Milhazes Nuno, Borges Fernanda, Fernandes Eduarda, Carvalho Félix, Bastos Maria Lourdes
REQUIMTE, Serviço de Toxicologia, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha 164, 4099/030, Portugal.
Toxicology. 2004 Aug 5;200(2-3):193-203. doi: 10.1016/j.tox.2004.03.016.
In the past decade, clinical evidence has increasingly shown that the liver is a target organ for 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy") toxicity. The aims of the present in vitro study were: (1) to evaluate and compare the hepatotoxic effects of MDMA and one of its main metabolites, N-methyl-alpha-methyldopamine (N-Me-alpha-MeDA) and (2) to investigate the ability of antioxidants, namely ascorbic acid and N-acetyl-L-cysteine (NAC), to prevent N-Me-alpha-MeDA-induced toxic injury, using freshly isolated rat hepatocytes. Cell suspensions were incubated with MDMA or N-Me-alpha-MeDA in the final concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 mM for 3 h. To evaluate the potential protective effects of antioxidants, cells were preincubated with ascorbic acid in the final concentrations of 0.1 and 0.5 mM, or NAC in the final concentrations of 0.1 and 1 mM for 15 min before treatment with 1.6 mM N-Me-alpha-MeDA for 3 h (throughout this incubation period the cells were exposed to both compounds). The toxic effects were evaluated by measuring the cell viability, glutathione (GSH) and glutathione disulfide (GSSG), ATP, and the cellular activities of GSH peroxidase (GPX), GSSG reductase (GR), and GSH S-transferase (GST). MDMA induced a concentration- and time-dependent GSH depletion, but had a negligible effect on cell viability, ATP levels, or on the activities of GR, GPX, and GST. In contrast, N-Me-alpha-MeDA was shown to induce not only a concentration- and time-dependent depletion of GSH, but also a depletion of ATP levels accompanied by a loss in cell viability, and decreases in the antioxidant enzyme activities. For both compounds, GSH depletion was not accompanied by increases in GSSG levels, which seems to indicate GSH depletion by adduct formation. Importantly, the presence of ascorbic acid (0.5 mM) or NAC (1 mM) prevented cell death and GSH depletion induced by N-Me-alpha-MeDA. The results provide evidence that MDMA and its metabolite N-Me-alpha-MeDA induce toxicity to freshly isolated rat hepatocytes. Oxidative stress may play a major role in N-Me-alpha-MeDA-induced hepatic toxicity since antioxidant defense systems are impaired and administration of antioxidants prevented N-Me-alpha-MeDA toxicity.
在过去十年中，临床证据越来越多地表明肝脏是3,4-亚甲基二氧甲基苯丙胺（摇头丸）毒性作用的靶器官。本体外研究的目的是：（1）评估和比较摇头丸及其主要代谢产物之一N-甲基-α-甲基多巴胺（N-Me-α-MeDA）的肝毒性作用；（2）使用新鲜分离的大鼠肝细胞，研究抗氧化剂（即抗坏血酸和N-乙酰-L-半胱氨酸（NAC））预防N-Me-α-MeDA诱导的毒性损伤的能力。将细胞悬液与终浓度为0.1、0.2、0.4、0.8和1.6 mM的摇头丸或N-Me-α-MeDA孵育3小时。为了评估抗氧化剂的潜在保护作用，在用1.6 mM N-Me-α-MeDA处理3小时之前（在整个孵育期间细胞暴露于这两种化合物），将细胞与终浓度为0.1和0.5 mM的抗坏血酸或终浓度为0.1和1 mM的NAC预孵育15分钟。通过测量细胞活力、谷胱甘肽（GSH）和谷胱甘肽二硫化物（GSSG）、ATP以及谷胱甘肽过氧化物酶（GPX）、谷胱甘肽二硫化物还原酶（GR）和谷胱甘肽S-转移酶（GST）的细胞活性来评估毒性作用。摇头丸诱导了浓度和时间依赖性的GSH消耗，但对细胞活力、ATP水平或GR、GPX和GST的活性影响可忽略不计。相比之下，N-Me-α-MeDA不仅诱导了浓度和时间依赖性的GSH消耗，还导致了ATP水平的消耗，同时伴有细胞活力丧失以及抗氧化酶活性降低。对于这两种化合物，GSH消耗并未伴随着GSSG水平的升高，这似乎表明是通过加合物形成导致GSH消耗。重要的是，抗坏血酸（0.5 mM）或NAC（1 mM）的存在可预防N-Me-α-MeDA诱导的细胞死亡和GSH消耗。结果提供了证据表明摇头丸及其代谢产物N-Me-α-MeDA对新鲜分离的大鼠肝细胞具有毒性作用。氧化应激可能在N-Me-α-MeDA诱导的肝毒性中起主要作用，因为抗氧化防御系统受损，而给予抗氧化剂可预防N-Me-α-MeDA的毒性。
