Foreman Brooke D, Tarloff Joan B
Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, University of the Sciences in Philadelphia, Philadelphia, PA 19104, USA.
Toxicol Appl Pharmacol. 2008 Jul 15;230(2):144-9. doi: 10.1016/j.taap.2008.02.014. Epub 2008 Mar 4.
para-aminophenol (PAP) causes nephrotoxicity by biochemical mechanisms that have not been fully elucidated. PAP can undergo enzymatic or non-enzymatic oxidation to form reactive intermediates. Using modulators of reactive oxygen species (ROS), the role of ROS in PAP toxicity in LLC-PK(1) cells was investigated. ROS formation was determined using a fluorescein derivative and viability using alamarBlue. Following treatment of cells with PAP, ROS formation occurred prior to loss of cell viability. Several modulators of ROS were used to identify the pathways involved in PAP toxicity. Viability was improved with catalase treatment, while viability was decreased when cells were treated with superoxide dismutase (SOD). Both catalase and SOD exert their effects outside of cells in the incubation medium, since there was no evidence of uptake of these enzymes in LLC-PK(1) cells. In cell-free incubations, hydrogen peroxide (H(2)O(2)) was produced when 0.5 mM PAP was included in the incubation medium. Further, SOD greatly increased and catalase greatly decreased H(2)O(2) production in these cell-free incubations. These data suggest that H(2)O(2) formed in the incubation medium contributes to loss of viability following PAP treatment. When cells were coincubated with 0.5 mM PAP and tiron, pyruvate, bathocuproine, 1, 10-phenanthroline, or dimethylthiourea (DMTU), ROS formation was decreased. However, there was minimal improvement in cell viability. Paradoxically, DMTU exacerbated PAP-induced loss of viability. These data suggest that ROS are generated in cells exposed to PAP but these species are not the predominant cause of cellular injury.
对氨基酚（PAP）通过尚未完全阐明的生化机制导致肾毒性。PAP可经历酶促或非酶促氧化以形成反应性中间体。使用活性氧（ROS）调节剂，研究了ROS在LLC-PK(1)细胞中PAP毒性中的作用。使用荧光素衍生物测定ROS形成，使用alamarBlue测定细胞活力。用PAP处理细胞后，ROS形成先于细胞活力丧失。使用了几种ROS调节剂来确定参与PAP毒性的途径。过氧化氢酶处理可提高细胞活力，而用超氧化物歧化酶（SOD）处理细胞时细胞活力降低。过氧化氢酶和SOD均在孵育培养基中的细胞外发挥作用，因为没有证据表明LLC-PK(1)细胞摄取了这些酶。在无细胞孵育中，当孵育培养基中包含0.5 mM PAP时会产生过氧化氢（H(2)O(2)）。此外，在这些无细胞孵育中，SOD大大增加而过氧化氢酶大大降低了H(2)O(2)的产生。这些数据表明，孵育培养基中形成的H(2)O(2)导致PAP处理后细胞活力丧失。当细胞与0.5 mM PAP和钛铁试剂、丙酮酸、bathocuproine、1,10-菲咯啉或二甲基硫脲（DMTU）共同孵育时，ROS形成减少。然而，细胞活力的改善很小。矛盾的是，DMTU加剧了PAP诱导的细胞活力丧失。这些数据表明，暴露于PAP的细胞中会产生活性氧，但这些物质不是细胞损伤的主要原因。
