Pourahmad Jalal, O'Brien Peter J, Jokar Farzaneh, Daraei Bahram
Faculty of Pharmacy, Shaheed Beheshti University of Medical Sciences, Tehran, PO Box 14155-6153, Iran.
Toxicol In Vitro. 2003 Oct-Dec;17(5-6):803-10. doi: 10.1016/s0887-2333(03)00123-1.
Severe chronic liver disease results from the hepatic accumulation of copper nickel, cobalt or iron in humans and on the other hand cadmium, dichromate and arsenic may induce lung or kidney cancer. Acute or chronic CdCl2, HgCl2 or dichromate administration induces hepatic and nephrotoxicity in rodents. Oxidative stress is often cited as a possible cause but has not yet been measured. For the first time we have measured the reactive oxygen species (ROS) formation induced when cells are incubated with metals and determined its source. Hepatocytes incubated with 2',7'-dichlorofluorescin diacetate resulted in its rapid uptake and deacetylation by intracellular esterases to form 2',7'-dichlorofluorescin. A marked increase in ROS formation occurred with LD50 concentrations of cadmium [Cd(II)], Hg(II) or arsenite [As(III)] which was released by proton ionophores that uncouple oxidative phosphorylation. Uncouplers or oxidative phosphorylation also inhibited ROS formation induced by these metals, which suggests that mitochondria are major contributors to endogenous ROS formation. Glycolytic substrates also inhibited Cd(II)/Hg(II)/As(III)-induced ROS formation and confirms that mitochondria are the site of ROS formation. By contrast ROS formation by LD50 concentrations of Cu(II), Ni(II), Co(II) or dichromate [Cr(VI)] were not affected by uncouplers or glycolytic substrates. However they were inhibited by lysosomotropic agents or endogenous inhibitors [in contrast to Hg(II), Cd(II) or As(III)]. Furthermore Cu(II), Ni(II), Co(II) or Cr(VI) accumulated in the lysosomes and the ROS formed caused a loss of lysosomal membrane integrity. The release of lysosomal proteases and phospholipases also contributed to hepatocyte cytotoxicity. ROS formation and cytotoxicity induced by added H2O2 or generated by the intracellular redox cycling of nitrofurantoin was also inhibited by lysosomotropic agents and ferric chelators suggesting that lysosomal Fe(II) contributes to H2O2-induced cytotoxicity. In conclusion, lysosomes are sites of cytotoxic ROS formation with redox transition metals (CuII, CrVI, NiII, CoII) whereas mitochondria are the ROS sites for non-redox or poor redox cycling transition metals (CdII, HgII, AsIII).
严重的慢性肝病是由人体肝脏中铜、镍、钴或铁的蓄积所致，另一方面，镉、重铬酸盐和砷可能诱发肺癌或肾癌。急性或慢性给予氯化镉、氯化汞或重铬酸盐会在啮齿动物中诱发肝毒性和肾毒性。氧化应激常被认为是一个可能的原因，但尚未得到测定。我们首次测定了细胞与金属孵育时诱导产生的活性氧（ROS）的形成，并确定了其来源。用二氯荧光素二乙酸酯孵育肝细胞会导致其被细胞内酯酶快速摄取并脱乙酰化，形成二氯荧光素。镉[Cd(II)]、汞(II)或亚砷酸盐[As(III)]的半数致死剂量（LD50）浓度会使ROS形成显著增加，而质子离子载体可释放这种增加，质子离子载体可使氧化磷酸化解偶联。解偶联剂或氧化磷酸化也抑制了这些金属诱导的ROS形成，这表明线粒体是内源性ROS形成的主要贡献者。糖酵解底物也抑制了Cd(II)/Hg(II)/As(III)诱导的ROS形成，并证实线粒体是ROS形成的场所。相比之下，Cu(II)、Ni(II)、Co(II)或重铬酸盐[Cr(VI)]的LD50浓度诱导的ROS形成不受解偶联剂或糖酵解底物的影响。然而，它们被溶酶体促渗剂或内源性抑制剂抑制（与Hg(II)、Cd(II)或As(III)相反）。此外，Cu(II)、Ni(II)、Co(II)或Cr(VI)在溶酶体中蓄积，形成的ROS导致溶酶体膜完整性丧失。溶酶体蛋白酶和磷脂酶的释放也导致了肝细胞毒性。溶酶体促渗剂和铁螯合剂也抑制了添加过氧化氢或由呋喃妥因细胞内氧化还原循环产生的ROS形成和细胞毒性，这表明溶酶体中的Fe(II)导致了过氧化氢诱导的细胞毒性。总之，溶酶体是具有氧化还原过渡金属（CuII、CrVI、NiII、CoII）的细胞毒性ROS形成的场所，而线粒体是非氧化还原或弱氧化还原循环过渡金属（CdII、HgII、AsIII）的ROS形成场所。
