Koizumi T, Shirakura H, Kumagai H, Tatsumoto H, Suzuki K T
Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Toxicology. 1996 Dec 2;114(2):125-34. doi: 10.1016/s0300-483x(96)03477-4.
The present study was performed to further elucidate the mechanism of cadmium (Cd)-induced cytotoxicity in rat hepatocytes focusing on the effects of Cd-induced acidification on cellular production of H2O2 and the integrity of the plasma membrane. Exposure of cells of Cd levels < 50 microM stimulated cellular production of H2O2 in a dose-dependent manner. In cells exposed to 50 microM Cd, generation of the toxic oxygen increased from 5 min after exposure, and reached a plateau at 15 min. The acidic medium at pH 6.5, a value which is corresponding to the cellular pH at maximal acidification induced by Cd, also enhanced production of the active oxygen at almost the same level as 25 microM Cd. These treatments affected permeability barrier of plasma membranes as assessed by nuclear staining with propidium iodide (PI, MW 668) and release of intracellular lactic dehydrogenase (LDH) into surrounding medium. Cd at 50 microM caused nuclear staining by the fluorescent probe, beginning from 15 min at exposure, reaching a peak at 60 min. LDH leakage likewise started from 60 min of Cd exposure onward. The acidic partially prevented by L-ascorbic acid pretreatment. H2O2-induced nuclear staining increased with the increasing pH values from 6.7 to 7.1 Cd at 50 microM lowered the cellular pH within 5 min, but the decreased cellular pH returned to a value near physiological levels 25 min later. Pretreatment with Amiloride, an inhibitor of the Na+/H+ exchange, partially blocked this pH recovery after acidification. The results indicate that Cd caused H2O2 accumulation and H+, Cd and H2O2-related permeability changes of the plasma membrane. This may link to subsequent extensive membrane damage occurring at near physiological cellular pH.
本研究旨在进一步阐明镉（Cd）诱导大鼠肝细胞毒性的机制，重点关注镉诱导的酸化对细胞过氧化氢生成及质膜完整性的影响。暴露于镉水平低于50微摩尔的细胞以剂量依赖方式刺激了过氧化氢的细胞生成。在暴露于50微摩尔镉的细胞中，有毒氧的生成在暴露后5分钟开始增加，并在15分钟达到平台期。pH 6.5的酸性培养基，该值对应于镉诱导最大酸化时的细胞pH值，也能在几乎与25微摩尔镉相同的水平增强活性氧的生成。这些处理影响了质膜的通透屏障，通过碘化丙啶（PI，分子量668）核染色及细胞内乳酸脱氢酶（LDH）释放到周围培养基中来评估。50微摩尔的镉在暴露15分钟开始导致荧光探针核染色，60分钟达到峰值。LDH泄漏同样从镉暴露60分钟后开始。酸性可通过L-抗坏血酸预处理部分预防。过氧化氢诱导的核染色随着pH值从6.7增加到7.1而增加。50微摩尔的镉在5分钟内降低细胞pH值，但降低的细胞pH值在25分钟后恢复到接近生理水平的值。用钠/氢交换抑制剂氨氯吡咪预处理可部分阻断酸化后的pH值恢复。结果表明，镉导致过氧化氢积累以及与氢离子、镉和过氧化氢相关的质膜通透性变化。这可能与随后在接近生理细胞pH值时发生的广泛膜损伤有关。
