Division of Physiology, Department of Physiology and Medical Physics, Innsbruck Medical University, Innsbruck, Austria.
Toxicol In Vitro. 2011 Dec;25(8):1855-62. doi: 10.1016/j.tiv.2011.05.018. Epub 2011 May 24.
Technological developments are driving in vitro methods towards integrated "omic" strategies. However, there is still an over reliance on classical viability assays for dose range finding. Such assays are not readily suited to the investigation of subtle alterations in cell function and most require termination of the experiment, which makes it difficult to monitor temporal alterations in repeat-dose long term exposure experiments. To this end, we investigated the use of lactate production as a marker of cell stress in long term repeat dose experiments. We conducted daily exposures to eight compounds at five concentrations for 14 days on human renal proximal tubular cells (RPTEC/TERT1), human hepatoma cells (HepaRG) and mouse fibroblasts (BALB-3T3) cells. Compounds were chosen from a training set used in the 7th EU Framework project Predict-IV and consisted of amiodarone, diclofenac, troglitazone, cadmium chloride, cephaloridine, cidofovir, cyclosporine A and buflomedil. At days 1, 3, 7 and 14, lactate was measured in the supernatant medium. At day 14, cells were assayed for resazurin reduction capability and subsequently lysed in methanol for ATP determination. Compound-induced loss of viability was comparable across all cell lines. For all cell types, when cell viability was compromised at day 14, lactate production was induced during the treatment period. In some situations, lactate also fell below control values, indicating cell death. Thus, temporal alterations in supernatant lactate provides information on the time and concentration of stress induction and the time and concentration where cell death becomes the dominant factor. Supernatant lactate production is a simple, cheap and non-invasive parameter. Since many molecular pathways converge on the glycolytic pathway, enhanced lactate production may be considered as a global marker of sub-lethal injury and thus an ideal marker for investigating temporal alterations in long term repeat dose testing in vitro regimes.
技术发展正在推动体外方法向综合的“组学”策略发展。然而，对于剂量范围的确定，仍然过度依赖于经典的生存能力测定。这些测定方法不易用于研究细胞功能的细微变化，而且大多数都需要终止实验，这使得在重复剂量长期暴露实验中很难监测时间变化。为此，我们研究了在长期重复剂量实验中使用乳酸生成作为细胞应激的标志物。我们对人肾近端小管细胞（RPTEC/TERT1）、人肝癌细胞（HepaRG）和小鼠成纤维细胞（BALB-3T3）进行了为期 14 天的每日暴露实验，实验中使用了 8 种化合物，浓度设置了 5 个梯度。这些化合物是从 7 个欧盟框架项目 Predict-IV 中使用的训练集中选择的，包括胺碘酮、双氯芬酸、曲格列酮、氯化镉、头孢菌素、西多福韦、环孢菌素 A 和布美他尼。在第 1、3、7 和 14 天，测量上清液中的乳酸。在第 14 天，用resazurin 还原能力测定法测定细胞活力，然后用甲醇裂解细胞测定 ATP。在所有细胞系中，化合物诱导的细胞活力丧失具有可比性。对于所有细胞类型，当第 14 天细胞活力受损时，在治疗期间会诱导乳酸生成。在某些情况下，乳酸也低于对照值，表明细胞死亡。因此，上清液中乳酸的时间变化提供了关于应激诱导的时间和浓度以及细胞死亡成为主要因素的时间和浓度的信息。上清液中乳酸的产生是一种简单、廉价且非侵入性的参数。由于许多分子途径都集中在糖酵解途径上，因此增强的乳酸生成可被视为亚致死损伤的通用标志物，因此是研究体外重复剂量试验中时间变化的理想标志物。
