Ollinger K, Brunk U T
Department of Pathology II, Faculty of Health Sciences, University of Linköping, Sweden.
Free Radic Biol Med. 1995 Nov;19(5):565-74. doi: 10.1016/0891-5849(95)00062-3.
Cultured primary hepatocytes pretreated (protected) with the iron chelator deferoxamine or the antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) were resistant to the toxicity of 5 microM naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) during a 180-min exposure. Hepatocytes exposed to naphthazarin without any protection were abruptly depleted of intracellular reduced glutathione, and the level of cytosolic Ca2+ was rapidly increased. This was followed by lipid peroxidation, measured as accumulation of malondialdehyde (MDA) and 4-hydroxyalkenals (4-HNA) intra- and extracellularly; decrease in ATP levels; destabilization of lysosomes; and finally cell death. The stability of the lysosomal membranes was evaluated by determining retention of the lysosomotropic weak base acridine orange (AO). Naphthazarin exposure caused leakage of protons from the acidic compartment, as indicated by relocalization of AO to the cytosol. Protection of the cell cultures with deferoxamine or DPPD prevented destabilization of lysosomes and cell killing. It also reduced the loss of ATP but did not prevent the depletion of glutathione or the increase in Ca2+. In cells subjected to naphthazarin exposure, DPPD protection also completely inhibited lipid peroxidation, whereas deferoxamine pretreatment only slightly reduced the intracellular accumulation of MDA and 4-HNA but completely prevented cell rupture and the leakage of these lipid peroxidation products to the medium that took place in large amounts from unprotected cells exposed to naphthazarin. Deferoxamine is taken up by endocytosis and is thus transported to the acidic vacuolar apparatus, whereas the lipophilic DPPD is rapidly distributed throughout the cells. Inhibiting endocytosis during deferoxamine pretreatment, by incubating at +4 degrees C or by preexposure to a mixture of the endocytosis-inhibitors cytochalasin B and monensin, abolished the protective effect of deferoxamine. The findings suggest that naphthazarin-induced cell killing is not caused directly by either thiol oxidation or an increase in cytosolic free Ca2+, but rather is preceded by lysosomal destabilization, which may be prevented either by inhibition of cellular peroxidation in general or by prevention of iron-catalyzed oxidative reactions, and involves peroxidation of cellular membranes, energy depletion, and leakage of lysosomal content. DPPD would protect against cell killing by preventing lipid peroxidation of cellular membranes in general, whereas deferoxamine seems to allow a limited general cellular peroxidation but specifically prevents peroxidation and fragmentation of lysosomal membranes by chelating intralysosomal iron and, consequently, leakage of destructive lysosomal contents with ensuing cell rupture and death. Thus, a certain degree of cellular peroxidation does not appear to be lethal as long as lysosomal membranes are protected, placing lysosomes into a category of cellular loci minora resistentia.
用铁螯合剂去铁胺或抗氧化剂N，N'-二苯基对苯二胺（DPPD）预处理（保护）的原代培养肝细胞，在180分钟的暴露期间对5微摩尔萘茜（5,8-二羟基-1,4-萘醌）的毒性具有抗性。未经任何保护而暴露于萘茜的肝细胞，其细胞内还原型谷胱甘肽突然耗尽，胞质Ca2+水平迅速升高。随后发生脂质过氧化，表现为细胞内和细胞外丙二醛（MDA）和4-羟基烯醛（4-HNA）的积累；ATP水平降低；溶酶体不稳定；最终细胞死亡。通过测定溶酶体促渗弱碱吖啶橙（AO）的保留情况来评估溶酶体膜的稳定性。萘茜暴露导致质子从酸性区室泄漏，这通过AO重新定位到胞质溶胶得以表明。用去铁胺或DPPD保护细胞培养物可防止溶酶体不稳定和细胞死亡。它还减少了ATP的损失，但不能防止谷胱甘肽的耗尽或Ca2+的增加。在暴露于萘茜的细胞中，DPPD保护还完全抑制了脂质过氧化，而去铁胺预处理仅略微降低了MDA和4-HNA的细胞内积累，但完全防止了细胞破裂以及这些脂质过氧化产物向培养基的泄漏，未受保护的暴露于萘茜的细胞会大量发生这种泄漏。去铁胺通过内吞作用被摄取，因此被转运到酸性液泡装置，而亲脂性的DPPD迅速分布于整个细胞。在去铁胺预处理期间，通过在+4℃孵育或预先暴露于内吞作用抑制剂细胞松弛素B和莫能菌素的混合物来抑制内吞作用，消除了去铁胺的保护作用。这些发现表明，萘茜诱导的细胞死亡不是直接由硫醇氧化或胞质游离Ca2+增加引起的，而是在溶酶体不稳定之前发生，这可以通过总体上抑制细胞过氧化或通过防止铁催化的氧化反应来预防，并且涉及细胞膜过氧化、能量消耗和溶酶体内容物泄漏。DPPD通常通过防止细胞膜脂质过氧化来防止细胞死亡，而去铁胺似乎允许有限的总体细胞过氧化，但通过螯合溶酶体内的铁来特异性地防止溶酶体膜的过氧化和破碎，从而防止具有破坏性的溶酶体内容物泄漏以及随之而来的细胞破裂和死亡。因此，只要溶酶体膜受到保护，一定程度的细胞过氧化似乎并不致命，这使溶酶体成为细胞抗性较小的位点类别。
