Trovafloxacin enhances TNF-induced inflammatory stress and cell death signaling and reduces TNF clearance in a murine model of idiosyncratic hepatotoxicity

Abstract

Therapy employing the fluoroquinolone antibiotic, trovafloxacin (TVX) was curtailed due to idiosyncratic hepatotoxicity. Previous studies in mice showed that a nonhepatotoxic inflammatory stress induced by tumor necrosis factor alpha (TNF) synergized with a nonhepatotoxic dose of TVX to cause liver injury. The purpose of this study was to explore mechanisms by which TVX interacts with TNF to cause liver injury. TVX pretreatment prolonged the peak of plasma TNF after its administration. This prolongation of TNF by TVX was critical to the development of hepatotoxicity. The prolongation of TNF concentration in plasma was primarily due to reduced clearance when compared with secondary biosynthesis. TNF is cleared from plasma by binding to soluble TNF receptors (TNFRs) which are eliminated by the kidney; however, the plasma concentrations of soluble TNFRs were not reduced, and biomarkers of renal dysfunction were not elevated in TVX/TNF-treated mice. Two injections of TNF mimicked the prolongation of the TNF peak by TVX and caused liver injury, but injury was less severe than after TVX/TNF coexposure. TVX enhanced the induction of proinflammatory cytokines by TNF. Additionally, TVX sensitized Hepa1c1c7 cells to TNF-induced killing in a concentration-dependent manner and increased both potency and efficacy of TNF to activate effector caspases that were critically involved in cell death from TVX/TNF coexposure. In summary, TVX reduced the clearance of TNF independent of either receptor shedding or kidney dysfunction. Additionally, TVX interacted with TNF to enhance inflammation and sensitize hepatocytes to TNF-induced cell death.

FIG. 1.

TVX/TNF-induced hepatocellular oncosis and apoptosis. Mice were treated with TVX (150 mg/kg) or its saline vehicle 3 h before recombinant mTNF (50 μg/kg) or its saline vehicle as described in “Materials and Methods.” Plasma ALT activity was measured at various times. n = 4–7 animals per group. Mice were killed at 4 h for histopathology and immunohistochemistry. Photomicrographs were taken of representative liver sections stained with hematoxylin and eosin at ×200 (top row) and ×400 magnification (second row). Midzonal lesions of hepatocellular oncotic necrosis and apoptosis were observed in TVX/TNF-treated mice. Frozen liver sections were stained for DNA fragmentation (third row) using TUNEL staining. A representative photomicrograph at ×200 magnification is shown. Liver sections were stained for TUNEL (green) and nuclei (red). Staining colocalization (yellow), representing nuclei with DNA fragmentation, was observed only in livers from the TVX/TNF-treated mice. Paraffin-embedded liver sections were stained for cleaved caspase 3 (bottom row), which appears dark brown. A representative photomicrograph at ×200 magnification was taken of each treatment group. TVX/TNF-treated mice had greater cleaved caspase 3 staining compared with Veh/TNF-treated mice. *Significantly different from the respective treatment group at 0 h. ^#Significant difference between TVX/TNF and Veh/TNF groups at the same time.

FIG. 2.

Effect of TVX on plasma concentration of TNF after mTNF administration. Mice were treated with TVX (150 mg/kg) or its saline vehicle 3 h before recombinant mTNF (50 μg/kg) or its saline vehicle as described in “Materials and Methods.” (A) The plasma concentration of immunologically detectable TNF was measured at various times. n = 4–7 animals per group. *Significantly different from the respective treatment group at 0 h. ^#Significant difference between TVX/TNF and Veh/TNF groups at the same time. (B) Serum was collected at 3 h, and biological TNF activity was measured using the Hepa1c1c7 cell line as described in “Materials and Methods.” n = 5 animals per group. *Significantly different from Veh/TNF treatment group.

FIG. 3.

Effect of the timing of etanercept treatment on TVX/TNF-induced liver injury. Mice were treated with TVX and recombinant mTNF as described in “Materials and Methods.” Etanercept (12 mg/kg, i.p.) was administered at the indicated time relative to TNF dosing or not at all. Plasma ALT activity was measured at 15 h. n = 5–7 animals per group. Photomicrographs were taken of representative livers. *Significantly different from TVX/TNF treatment without etanercept.

FIG. 4.

TVX-induced liver injury and plasma TNF concentration after injection of rTNF or hTNF. Mice were treated with TVX (150 mg/kg) or its saline vehicle 3 h before recombinant rTNF (50 μg/kg), recombinant hTNF (50 μg/kg) or its saline vehicle as described in “Materials and Methods.” (A, C) Plasma ALT activity was measured 3 h later and compared with mice treated with TNF alone. n = 4–6 animals per group. *Significantly different from Veh group with same recombinant TNF treatment. (B, D) rTNF, hTNF, and mTNF concentrations were measured at 3 h. Cross-reactivity of antibodies used for measurement was below the detection limit. Total TNF was calculated by adding rTNF or hTNF and mTNF concentrations for each mouse. n = 4–6 animals per group. *Significantly different from Veh within TNF type.

FIG. 5.

Effect of TVX/TNF coexposure on plasma sTNFR concentrations and renal function. Mice were treated with TVX or Veh and with recombinant mTNF or Veh as described in “Materials and Methods.” (A, B) The plasma concentrations of sTNFR1 and sTNFR2 were measured at various times. n = 4–6 animals per group. *Significantly different from the respective treatment group at 0 h. (C, D) Plasma concentrations of creatinine and urea were measured at 3 h. n = 4–7 animals per group.

FIG. 6.

Effect of TVX pretreatment on TNF-induced increases in plasma cytokines. Mice were treated with TVX and/or TNF as described in “Materials and Methods.” The plasma concentrations of cytokines were measured at various times. n = 4–6 animals per group. *Significantly different from the respective treatment group at 0 h. ^#Significant difference between Veh/TNF- and TVX/TNF-treated groups at the same time.

FIG. 7.

Effect of TVX pretreatment on TNF-induced increases in plasma chemokines and hepatic neutrophil accumulation. Mice were treated with TVX and/or TNF as described in “Materials and Methods.” The plasma concentrations of chemokines were measured at various times. n = 4–6 animals per group. *Significantly different from the respective treatment group at 0 h. ^#Significant difference between Veh/TNF- and TVX/TNF-treated groups at the same time. Hepatic PMN accumulation was measured immunohistochemically at 4 h after TNF or Veh treatment. n = 4–6 animals per group. *Significantly different from the respective Veh group not treated with TNF. ^#Significantly different from Veh/TNF-treated group.

FIG. 8.

Effect of dual administration of TNF to mimic TVX prolongation of plasma TNF. Mice were treated with Veh/TNF, TVX/TNF, or TNF/TNF as described in “Materials and Methods.” (A) Plasma concentration of TNF was measured at various times. The timecourse of the plasma TNF concentration in Veh/TNF- and TVX/TNF-treated mice was also presented in Figure 2 and is represented here for comparison with TNF/TNF treatment. n = 4–6 animals per group. *Significantly different from the respective treatment group at 0 h. ^#Significantly different from Veh/TNF-treated mice at the same time. ^ϕSignificantly different from TVX/TNF-treated mice at the same time. (B) Plasma ALT activity was measured at 15 h. n = 6–8 animals per group. *Significantly different from Veh/Veh group. ^#Significantly different from TNF-treated mice. ^ϕSignificantly different from TVX/TNF-treated mice at the same time.

FIG. 9.

Comparison of TVX/TNF and TNF/TNF induction of cytokines and chemokines. Mice were treated with TVX/TNF or TNF/TNF as described in “Materials and Methods.” Plasma concentrations of cytokines and chemokines were measured at 3 h. n = 8–10 animals per group. *Significantly different from TNF/TNF group.

FIG. 10.

Effect of TVX on TNF-induced caspase activation and cell death. (A) Hepa1c1c7 cells were treated with various concentrations of TVX and with recombinant mTNF as described in “Materials and Methods.” Cell death was measured at 24 h. n = 4 separate experiments/group. Open symbols represent TNF-treated cells with 0.1% DMSO vehicle. Two-way ANOVA revealed a significant TVX interaction with TNF. (B, C) Hepa1c1c7 cells were treated with 200 μm TVX and with various concentrations of recombinant mTNF as described in “Materials and Methods.” (B) Caspase 3/7 activity was measured at 6 h. n = 4 separate experiments per group. Cells treated only with the vehicles were the control and set as a fold change of 1. *Significantly different from control group without TNF. #Significantly different from Veh-treated cells at the same TNF concentration. (C) Cells were treated with the caspase inhibitor, z-vad.fmk (40μM), TVX (200μM), and/or TNF (0.2 ng/ml) as TVX (200μM) as described in “Materials and Methods.” Cell death was measured at 24 h. n = 4 separate experiments per group. *Significantly different from all other treatment groups.