AUTHOR=Kirk Natalie M. , Vieson Miranda D. , Selting Kim A. , Reinhart Jennifer M. 

TITLE=Cytotoxicity of Cultured Canine Primary Hepatocytes Exposed to Itraconazole Is Decreased by Pre-treatment With Glutathione

JOURNAL=Frontiers in Veterinary Science

VOLUME=8

YEAR=2021

URL=https://www.frontiersin.org/journals/veterinary-science/articles/10.3389/fvets.2021.621732

DOI=10.3389/fvets.2021.621732

ISSN=2297-1769

ABSTRACT=<p><bold>Objective:</bold> To identify the effect of glutathione (GSH) on cell survival in a novel <italic>in vitro</italic> model of itraconazole (ITZ)-associated hepatotoxicity using canine primary hepatocytes.</p><p><bold>Sample:</bold> Commercially sourced, cryopreserved male dog (Beagle) primary hepatocytes from a single donor.</p><p><bold>Procedures:</bold> 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 <italic>post-hoc</italic> test was used to detect individual differences.</p><p><bold>Results:</bold> 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 (<italic>P</italic> &lt; 0.001) and time (<italic>P</italic> = 0.004) and significantly decreased with GSH treatment (<italic>P</italic> &lt; 0.001).</p><p><bold>Conclusions and Clinical Relevance:</bold> This <italic>in vitro</italic> model demonstrates dose- and time-dependent ITZ-induced cytotoxicity, which is similar to clinical changes observed in canine patients and in <italic>in vivo</italic> rodent studies. Pre-treating with GSH is protective against <italic>in vitro</italic> 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.</p>