Gergel D, Misík V, Ondrias K, Cederbaum A I
Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029, USA.
J Biol Chem. 1995 Sep 8;270(36):20922-9. doi: 10.1074/jbc.270.36.20922.
3-Morpholinosydnonimine (SIN-1) is widely used to generate nitric oxide (NO(x).) and superoxide radical (O2-.). The effect of SOD on the toxicity of SIN-1 is complex, depending on what is the ultimate species responsible for toxicity. SIN-1 (< 1 mM) was only slightly toxic to HepG2 cells. Copper, zinc superoxide dismutase (Cu,Zn-SOD) or manganese superoxide dismutase (Mn-SOD) increased the toxicity of SIN-1. Catalase abolished, while sodium azide potentiated, this toxicity, suggesting a key role for H2O2 in the overall mechanism. Depletion of GSH from the HepG2 cells also potentiated the toxicity of SIN-1 plus SOD. Although Me2SO, sodium formate, and mannitol had no protective effect, iron chelators, thiourea and urate protected the cells against the SIN-1 plus Cu,Zn-SOD-mediated cytotoxicity. The cytotoxic effect of Cu,Zn-SOD but not Mn-SOD, showed a biphasic dose response being most pronounced at lower concentrations (10-100 units/ml). In the presence of SIN-1, Mn-SOD increased accumulation of H2O2 in a concentration-dependent manner. In contrast, Cu,Zn-SOD increased H2O2 accumulation from SIN-1 at low but not high concentrations of the enzyme, suggesting that high concentrations of the Cu,Zn-SOD interacted with the H2O2. EPR spin trapping studies demonstrated the formation of hydroxyl radical from the decomposition of H2O2 by high concentrations of the Cu,Zn-SOD. The cytotoxic effect of the NO donors SNAP and DEA/NO was only slightly enhanced by SOD; catalase had no effect. Thus, the oxidants responsible for the toxicity of SIN-1 and SNAP or DEA/NO to HepG2 cells under these conditions are different, with H2O2 derived from O2-. dismutation playing a major role with SIN-1. These results suggest that the potentiation of SIN-1 toxicity by SOD is due to enhanced production of H2O2, followed by site-specific damage of critical cellular sites by a transition metal-catalyzed reaction. These results also emphasize that the role of SOD as a protectant against oxidant damage is complex and dependent, in part, on the subsequent fate and reactivity of the generated H2O2.
3-吗啉代非那明（SIN-1）被广泛用于生成一氧化氮（NOₓ）和超氧阴离子自由基（O₂⁻）。超氧化物歧化酶（SOD）对SIN-1毒性的影响较为复杂，这取决于最终导致毒性的物质是什么。SIN-1（<1 mM）对HepG2细胞仅有轻微毒性。铜锌超氧化物歧化酶（Cu,Zn-SOD）或锰超氧化物歧化酶（Mn-SOD）会增加SIN-1的毒性。过氧化氢酶可消除这种毒性，而叠氮化钠则会增强这种毒性，这表明H₂O₂在整个机制中起关键作用。从HepG2细胞中耗尽谷胱甘肽（GSH）也会增强SIN-1加SOD的毒性。尽管二甲基亚砜、甲酸钠和甘露醇没有保护作用，但铁螯合剂、硫脲和尿酸可保护细胞免受SIN-1加Cu,Zn-SOD介导的细胞毒性。Cu,Zn-SOD而非Mn-SOD的细胞毒性表现出双相剂量反应，在较低浓度（10 - 100单位/毫升）时最为明显。在存在SIN-1的情况下，Mn-SOD以浓度依赖的方式增加H₂O₂的积累。相比之下，Cu,Zn-SOD在低浓度而非高浓度的酶时会增加SIN-1产生的H₂O₂积累，这表明高浓度的Cu,Zn-SOD与H₂O₂相互作用。电子顺磁共振自旋捕获研究表明，高浓度的Cu,Zn-SOD可使H₂O₂分解形成羟基自由基。一氧化氮供体硝普钠（SNAP）和二乙胺/NO（DEA/NO）的细胞毒性仅被SOD轻微增强；过氧化氢酶则没有作用。因此，在这些条件下，导致SIN-1以及SNAP或DEA/NO对HepG2细胞毒性的氧化剂不同，由O₂⁻歧化产生的H₂O₂在SIN-1毒性中起主要作用。这些结果表明，SOD增强SIN-1毒性是由于H₂O₂生成增加，随后通过过渡金属催化反应对关键细胞位点进行位点特异性损伤。这些结果还强调，SOD作为抗氧化损伤保护剂的作用较为复杂，部分取决于所生成的H₂O₂的后续命运和反应性。
