Objective: To identify the effect of glutathione (GSH) on cell survival in a novel in vitro model of itraconazole (ITZ)-associated hepatotoxicity using canine primary hepatocytes.

Sample: Commercially sourced, cryopreserved male dog (Beagle) primary hepatocytes from a single donor.

Procedures: Using a sandwich culture technique, canine primary hepatocytes were exposed to serial dilutions of ITZ. Calcein AM, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), and neutral red were investigated as potential cell viability assays. Hepatocytes were then pre-incubated with GSH, exposed to serial dilutions of ITZ, and cell viability determined at 4 and 24 h post-ITZ exposure. Each condition was performed in technical triplicate and the effect of time, GSH concentration, and ITZ concentration on % cytotoxicity assessed using a multivariate linear regression model. Tukey's post-hoc test was used to detect individual differences.

Results: The neutral red cell cytotoxicity assay was chosen based on its superior ability to detect dose-dependent changes in viability. Hepatocyte cytotoxicity significantly increased with ITZ concentration (P < 0.001) and time (P = 0.004) and significantly decreased with GSH treatment (P < 0.001).

Conclusions and Clinical Relevance: This in vitro model demonstrates dose- and time-dependent ITZ-induced cytotoxicity, which is similar to clinical changes observed in canine patients and in in vivo rodent studies. Pre-treating with GSH is protective against in vitro cell death. These results suggest that GSH precursors may have a role in the management or prevention of ITZ-associated hepatotoxicity in dogs. Clinical trials are needed to evaluate their utility for this adverse drug reaction.

客观的： 鉴定谷胱甘肽（GSH）对细胞存活的影响 体外 犬原代肝细胞的伊曲康唑（ITZ）相关肝毒性模型。

样本： 商业来源的冷冻保存的雄性狗（Beagle）原代肝细胞。

程序：使用三明治培养技术，将犬原代肝细胞暴露于ITZ系列稀释液中。研究了钙黄绿素AM，3-（4,5-二甲基噻唑-2-基）-2，溴化5-二苯基四唑鎓（MTT）和中性红作为潜在的细胞活力测定方法。然后将肝细胞与GSH预温育，暴露于ITZ系列稀释液中，并在ITZ暴露后4和24小时测定细胞活力。每种条件均一式三份进行，并使用多元线性回归模型评估时间，GSH浓度和ITZ浓度对％细胞毒性的影响。图基的事后 测试用于检测个体差异。

结果：选择中性红细胞细胞毒性试验是基于其检测生存能力的剂量依赖性变化的出色能力。肝细胞毒性随着ITZ浓度的增加而显着增加（P <0.001）和时间（P = 0.004），并且经GSH治疗后显着降低（=P <0.001）。

结论与临床意义： 这 体外 该模型证明了剂量依赖性和时间依赖性的ITZ诱导的细胞毒性，这与犬科动物和 体内啮齿动物研究。用谷胱甘肽预处理可预防体外细胞死亡。这些结果表明，GSH前体可能在犬的ITZ相关肝毒性的管理或预防中起作用。需要临床试验来评估其对这种药物不良反应的效用。