Sulindac metabolism and synergy with tumor necrosis factor-alpha in a drug-inflammation interaction model of idiosyncratic liver injury

Abstract

Sulindac (SLD) is a nonsteroidal anti-inflammatory drug (NSAID) that has been associated with a greater incidence of idiosyncratic hepatotoxicity in human patients than other NSAIDs. In previous studies, cotreatment of rats with SLD and a modestly inflammatory dose of lipopolysaccharide (LPS) led to liver injury, whereas neither SLD nor LPS alone caused liver damage. In studies presented here, further investigation of this animal model revealed that the concentration of tumor necrosis factor-alpha (TNF-alpha) in plasma was significantly increased by LPS at 1 h, and SLD enhanced this response. Etanercept, a soluble TNF-alpha receptor, reduced SLD/LPS-induced liver injury, suggesting a role for TNF-alpha. SLD metabolites in plasma and liver were determined by LC/MS/MS. Cotreatment with LPS did not increase the concentrations of SLD or its metabolites, excluding the possibility that LPS contributed to liver injury through enhanced exposure to SLD or its metabolites. The cytotoxicities of SLD and its sulfide and sulfone metabolites were compared in primary rat hepatocytes and HepG2 cells; SLD sulfide was more toxic in both types of cells than SLD or SLD sulfone. TNF-alpha augmented the cytotoxicity of SLD sulfide in primary hepatocytes and HepG2 cells. These results suggest that TNF-alpha can enhance SLD sulfide-induced hepatotoxicity, thereby contributing to liver injury in SLD/LPS-cotreated rats.

Fig. 1.

Time course of TNF-α concentration in rat serum. Rats were treated with two administrations of SLD (50 mg/kg p.o.) or its vehicle (0.5% methyl cellulose) with a 16-h interval. One-half hour before the second administration of SLD, LPS (8.25 × 10⁵ EU/kg i.v.) or its saline vehicle was administered via a tail vein. TNF-α was evaluated by enzyme-linked immunosorbent assay in serum samples obtained from rats at 0, 1, 2, 4, or 8 h after the second administration of SLD. ∗, significantly different from Veh/Veh group at the same time. #, significantly different from Veh/LPS group at the same time. P < 0.05, n = 4–5 for all points except the 8-h group (n = 3), Veh/LPS and SLD/LPS at 1 h (n = 8 and 9, respectively), and SLD/LPS at 0 h (n = 8). Veh, vehicle.

Fig. 2.

Effect of TNF-α inhibition on liver injury induced by SLD/LPS. Rats were treated with etanercept (8 mg/kg s.c.) or its vehicle 1 h before LPS. SLD and LPS or their vehicles were administered to rats as described in Materials and Methods. ALT activity was determined at 12 h (A). ∗, significantly different from respective Veh/Veh group. #, significantly different from Veh/ SLD/LPS group. P < 0.05, n = 4 for all groups except SLD/LPS/Etan (n = 6). Liver sections from rats treated with Veh/Veh/Veh (B), Etan/Veh/Veh (C), Veh/SLD/LPS (D), and Etan/SLD/LPS (E) were examined. An arrow indicates a necrotic area. Veh, vehicle; Etan, etanercept.

Fig. 3.

Effect of LPS on plasma concentrations of SLD, SLD sulfone, and SLD sulfide. Rats were treated with SLD and with LPS or its saline vehicle as described in Fig. 1. They were euthanized, and plasma was collected at 0, 1, 2, 4, and 8 h after the second administration of SLD. The plasma concentrations of SLD, SLD sulfone, or SLD sulfide were determined as described in Materials and Methods. ∗, significantly different from SLD/Veh group at the same time. P < 0.05, n = 5 for all groups except SLD/LPS at 4 h (n = 7). Veh, vehicle.

Fig. 4.

Effect of LPS on liver concentrations of SLD, SLD sulfone, and SLD sulfide. Rats were treated with SLD and with either LPS or its saline vehicle as described in Fig. 1. Liver concentrations of SLD, SLD sulfone, and SLD sulfide were determined at 0, 1, 2, 4, and 8 h after the second administration of SLD. ∗, significantly different from SLD/Veh group at the same time. P < 0.05, n = 5 for all groups except SLD/LPS at 4 h (n = 8). Veh. vehicle.

Fig. 5.

The amounts of SLD, SLD sulfone, and SLD sulfide in GI tract and feces. Rats were treated with SLD and with either LPS or its saline vehicle as described in Fig. 1. Each rat was housed in a different cage after the LPS injection. Two hours after the second administration of SLD, feces in the cage and the whole GI tract and its contents were collected for each rat. The mixture was homogenized with acetonitrile, and the amounts of SLD, SLD sulfone, and SLD sulfide were determined by LC/MS/MS. ∗, significantly different from SLD/Veh group. P < 0.05, n = 4. Veh, vehicle.

Fig. 6.

Evaluation of cytotoxicity induced by SLD, SLD sulfone, or SLD sulfide. A, SLD, SLD sulfone, or SLD sulfide was administered at various concentrations to HepG2 cells. The percentage of LDH released into the medium after 24 h was determined as a marker of cytotoxicity. B, rat primary hepatocytes were treated with SLD, SLD sulfone, or SLD sulfide for 8 h, and the percentage of ALT activity released into medium was determined as described in Materials and Methods. ∗, significantly different from vehicle (0 concentration). #, significantly different from SLD or SLD sulfone at the same concentration. P < 0.05, n = 3.

Fig. 7.

Cytotoxicity induced by TNF-α and SLD or its metabolites. HepG2 cells were treated with SLD (A), SLD sulfone (B), or SLD sulfide (C) in the presence or absence of TNF-α (200 ng/ml). The percentage of LDH released was determined after 24 h as described in Materials and Methods. ∗, significantly different from vehicle (0 concentration). #, significantly different from value in the absence of TNF-α at the same concentration of SLD or metabolite. P < 0.05, n = 3. Veh, vehicle.

Fig. 8.

Effect of TNF-α on SLD sulfide-induced injury to rat primary hepatocytes. SLD sulfide (60 μM) and/or TNF-α (2 μg/ml) was administered to rat primary hepatocytes. The percentage of ALT released was determined as described in Materials and Methods. ∗, significantly different from corresponding vehicle group. #, significantly different from SLD sulfide alone group. P < 0.05, n = 3. Veh, vehicle.