Andreoli S P, Mallett C P
Indiana University Medical Center, Department of Pediatrics, Indianapolis 46223, USA.
Am J Physiol. 1997 Jun;272(6 Pt 2):F729-35. doi: 10.1152/ajprenal.1997.272.6.F729.
To determine the mechanism(s) of oxidant-mediated cell lysis in renal tubular epithelial cells, we determined ATP depletion, DNA damage, lipid peroxidation, and cytotoxicity in LLC-PK1 cells exposed to 500 microM hydrogen peroxide for 1 h with and without inhibitors of lipid peroxidation including a lazaroid compound, 2-methylaminochroman (2-MAC), and Trolox, a vitamin E analog. ATP levels were determined by luciferin-luciferase, DNA damage by the alkaline unwinding technique, lipid peroxidation by the generation of malondialdehyde, and early cytotoxicity (5 h) by the release of 51Cr, whereas late cytotoxicity (24 h) was determined by release of [3H]leucine from prelabeled cells. Cells exposed to 500 microM hydrogen peroxide demonstrated significant (P < 0.01) ATP depletion, DNA damage, and lipid peroxidation, followed by cell death at 5 h. Concentrations of 0.1-25 microM 2-MAC or 25-500 microM Trolox each markedly and significantly (P < 0.01) inhibited lipid peroxidation and early cytotoxicity but had little to no effect on ATP depletion or DNA damage. Thus oxidant-stressed cells remained intact for several hours despite significant ATP depletion and DNA damage when lipid peroxidation was inhibited with the antioxidant compounds. At 24 h, 2-MAG and Trolox had lost their protective effect, suggesting that mechanisms other than lipid peroxidation play a role in later cytotoxicity. We conclude that ATP depletion and DNA damage are not the primary mediators of early cytotoxicity following oxidant stress, whereas lipid peroxidation plays an central role in mediating early cytotoxicity following oxidant injury.
为了确定氧化剂介导的肾小管上皮细胞裂解的机制，我们测定了暴露于500微摩尔过氧化氢1小时的LLC-PK1细胞中的ATP消耗、DNA损伤、脂质过氧化和细胞毒性，实验中使用和不使用脂质过氧化抑制剂，包括一种拉扎罗类化合物2-甲基氨基色满（2-MAC）和维生素E类似物Trolox。通过荧光素-荧光素酶测定ATP水平，通过碱性解旋技术测定DNA损伤，通过丙二醛的生成测定脂质过氧化，通过51Cr的释放测定早期细胞毒性（5小时），而晚期细胞毒性（24小时）则通过预标记细胞中[3H]亮氨酸的释放来测定。暴露于500微摩尔过氧化氢的细胞表现出显著（P < 0.01）的ATP消耗、DNA损伤和脂质过氧化，随后在5小时时细胞死亡。0.1 - 25微摩尔的2-MAC或25 - 500微摩尔的Trolox浓度均显著（P < 0.01）抑制脂质过氧化和早期细胞毒性，但对ATP消耗或DNA损伤几乎没有影响。因此，当用抗氧化剂化合物抑制脂质过氧化时，尽管存在显著的ATP消耗和DNA损伤，氧化应激细胞在数小时内仍保持完整。在24小时时，2-MAG和Trolox失去了它们的保护作用，这表明除脂质过氧化外的其他机制在后期细胞毒性中起作用。我们得出结论，ATP消耗和DNA损伤不是氧化应激后早期细胞毒性的主要介质，而脂质过氧化在介导氧化损伤后的早期细胞毒性中起核心作用。
