Hepatotoxicity of Antimycotics Used for Invasive Fungal Infections: In Vitro Results

Abstract

Purpose. Drug-induced liver injury (DILI) is the most common cause of liver injury and a serious clinical problem; antimycotics are involved in approximately 3% of all DILI cases. The hepatotoxicity of many drugs, including the antimycotics, is poorly screened in human models. Methods. In a standardized assay the cytotoxicity on hepatocytes of different concentrations (Cmax, 5x Cmax, and 10x Cmax) of the antimycotics used for systemic infections was tested. Anidulafungin (ANI), liposomal amphotericerin B (L-AmB), caspofungin (CASPO), fluconazole (FLUCO), and voriconazole (VORI) were incubated with HepG2/C3A cells. After incubation, the viability of cells (XTT test, LDH release, trypan blue staining), the synthesis of albumin, the cytochrome 1A2 activity, and the cell death (DNA fragmentation) were determined. Kruskal-Wallis and Mann-Whitney tests were used for statistical analyses. Results. L-AmB, ANI, and CASPO showed a mild hepatotoxicity in the Cmax concentrations. Higher concentrations of anidulafungin led to a severe impairment of hepatocyte viability and function. The azoles FLUCO and VORI had a higher hepatotoxic potential in all concentrations. Conclusion. Antimycotics, especially azoles, used for systemic infections should be given with caution in patient with liver insufficiency or liver failure or high risk for this; therefore, therapeutic drug monitoring should be used. Further studies with this approach are encouraged.

Figure 1

Effects of antimycotics agents on cell vitality and cell count in medium. Cells in culture were treated with 5.7 mM liposomal amphotericerin B, 2.25 mM anidulafungin, 0.2 mM caspofungin, 4.5 mM fluconazole, and 0.25 mM voriconazole and untreated (negative control) and 15.24 mM APAP (positive control) for 144 hours. Cells were counted in each treatment ((a), Cmax) and percent cell vitality was determined with trypan blue staining ((b), Cmax). Cell vitality at different doses of anidulafungin was further measured by trypan blue staining (c). Results are expressed as median and 25th/75th percentile, n = 20 (biological and technical repeats). ∗ indicates significance of p < 0.05 against the negative control.

Figure 2

Effects of antimycotics agents on lactate dehydrogenase (LDH) release [U/l] in medium. Lactate dehydrogenase concentration in cultured C3A cells was measured after 72 hours of treatment with 5.7 mM liposomal amphotericerin B, 2.25 mM anidulafungin, 0.2 mM, caspofungin, 4.5 mM fluconazole, 0.25 mM voriconazole and untreated (negative control) and 15.25 mM APAP (positive control) (a). LDH was further measured in different doses of anidulafungin (b). Results are expressed as median and 25th/75th percentile (n = 20 biological and n = 40 technical repeats). ∗ indicates significance of p < 0.05 against the negative control.

Figure 3

Effects of antimycotics agents on the activity of mitochondrial dehydrogenases: XTT test [absorbance/well] in medium. XTT assay of treated hepatocytes with 5.7 mM liposomal amphotericerin B, 2.25 mM anidulafungin, 0.2 mM caspofungin, 4.5 mM fluconazole, and 0.25 mM voriconazole and untreated (negative control) and 15.25 mM APAP (positive control). LDH concentrations in medium of antifungal treated hepatocytes after 144 hours (a) and dose-dependent effects of anidulafungin incubated hepatocytes (b). Results are expressed as median and 25th/75th percentile (n = 20 biological and n = 40 technical repeats). ∗ indicates significance of p < 0.05 against the negative control.

Figure 4

Effects of antimycotics agents on the cytochrome 1A2 activity (7-ethoxyresorufin O-deethylation and conversion of ethoxyresorufin to resorufin) [pmol/l] in medium and plasma. Cells in culture were treated with 5.7 mM liposomal amphotericerin B, 2.25 mM anidulafungin, 0.2 mM caspofungin, 4.5 mM fluconazole, and 0.25 mM voriconazole, and untreated (negative control) and 15.25 mM APAP (positive control). Results are expressed as median and 25th/75th percentile (n = 20 biological and n = 40 technical repeats). ∗ indicates significance of p < 0.05 against the negative control.

Figure 5

Release of albumin [mg/l] in medium. Treatments of cells with Cmax concentrations of 5.7 mM liposomal amphotericerin B, 2.25 mM anidulafungin, 0.2 mM caspofungin, 4.5 mM fluconazole, 0.25 mM voriconazole, and untreated (negative control) and 15.25 mM APAP (positive control). Results are expressed as median and 25th/75th percentile, n = 20 (biological and technical repeats). ∗ indicates p < 0.05 against the negative control.