Cell-based hepatotoxicity-testing of systemic antimycotics

Objective: Invasive fungal infections are associated with a high mortality rate. Systemic antimycotics for the treatment are poorly investigated for drug-induced hepatotoxicity. Therefore, we used an established in-vitro test with a standardised permanent cell line and tested the hepatotoxicity of acetaminophen and of systemic antimycotics. Methods: In a standardised mikrotiterplate assay the toxicity of different concentrations of acetaminophen (AM 7.62 mM | 15.24 mM) and of the antimycotics Anidulafungin (Ani), Liposomal Amphotericerin B (L-AmB), Fluconazole (Fluco)) were tested with human liver cells (HepG2/C3A, 500,000 cells/well; medium). The lowest test concentration of antimycotics was the mean plasma levels after induction of an i.v. therapy (Cmax), as well as the 5-times and 10-times concentrations of Cmax were analyzed. As controls served plasma with the different agents and a plasma plus medium control without agents (Control Group = CG). After incubation time of 2 × 3 days the standard parameters of vitality (e.g. LDH release, trypan blue staining) and DNA fragmentation evaluation by flow cytometry-based terminal transferase dUTP nick end-labeling (TUNEL) assay were tested. Results: (Median, 25/75 percentile): All antimycotics and AM showed a decreased vitality [%] (L-AmB 73, 66/77 | Ani 80, 76/82 | Fluco 75, 72/79) in the Cmax-concentration compared with the CG (94, 92/95) with corresponding increased LDH release [U/l] (L-AmB 70, 63.5/90.25 | Ani 66, 58.5/82.5 | Fluco 144, 107.75/182.25 | CG 70.5, 54/97 | AM 192, 163.75/214.5). With increasing concentrations of Ani a markedly decrease of cell vitality was seen; in contrast, Fluco und L-AmB showed a slightly increase. Comparing to the CG, AM, Fluco and L-AmB led to a significant lower number of DNA positive cells [%] (L-AmB 0.6, 0.2/1.1 | Fluco 4.9, 4.5/5.3 | CG 3.7, 1.7/8.4 | AM 2.3, 1.4/17.4). The number of DNA positive cells increased significantly in the Ani-group (10 × Cmax 58, 48.1/70). Conclusion: All antimycotics showed a slightly hepatotoxic potential in the Cmax concentration; Ani a stronger hepatotoxicity in higher concentrations. Therapeutic drug monitoring would be helpful for the use in critically ill patients.