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The Effect of Glutathione on Itraconazole‐Associated Hepatocyte Toxicity in an In Vitro Canine Model
Author(s) -
Kirk Natalie M.,
Vieson Miranda D.,
Selting Kimberly A.,
Reinhart Jennifer M.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.02119
Subject(s) - hepatocyte , glutathione , cytotoxicity , albumin , toxicity , pharmacology , serial dilution , itraconazole , in vitro , viability assay , chemistry , incubation , andrology , microbiology and biotechnology , biology , enzyme , biochemistry , medicine , pathology , organic chemistry , antifungal , alternative medicine
Background Itraconazole (ITZ) is used to treat many fungal infections in dogs. However, therapy is associated with dose‐dependent increases in liver enzymes in up to 42% of dogs. Glutathione (GSH) precursors are commonly used to treat ITZ‐associated hepatotoxicity, although no studies have investigated the efficacy of this practice. Objective To identify the effect of GSH on cell survival in an in vitro model of ITZ‐associated hepatotoxicity using canine primary hepatocytes. Methods Using a sandwich culture technique, pooled canine primary hepatocytes were incubated for 24 hours with 10‐fold dilutions of GSH (0 to 500 μM). Following GSH pre‐incubation, the cells were exposed to 5‐fold dilutions of ITZ (0 to 50 μM). Cell viability was determined using the neutral red assay after 4 and 24 hours of ITZ exposure (n=3). The effect of ITZ concentration, time, and GSH concentration on cytotoxicity was assessed using multivariate linear regression. Hepatocyte phenotype was assessed by measurement of albumin production using an enzyme‐linked immunosorbent assay (ELISA) kit for canine albumin and morphological assessment via phase contrast microscopy. Results Canine hepatocyte cytotoxicity significantly increased with ITZ concentration (p<0.001) and time (p=0.004). Whereas, hepatocyte cytotoxicity significantly decreased with GSH treatment (p<0.001). Maintenance of hepatocyte phenotype was confirmed by continued production of albumin and maintenance of cell morphology over time. Conclusions The in vitro model demonstrates dose‐ and time‐dependent ITZ cytotoxicity. Pre‐treating with GSH provides a protective effect against cell death in this model. These results suggest that GSH precursors may have a role in the management or prevention ITZ‐associated hepatotoxicity in dogs. Clinical trials are needed to evaluate their utility for this adverse drug reaction.