Role of biotransformation in 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione-induced hepatotoxicity in Fischer 344 rats

Abstract

Cytochrome P450 (CYP)-mediated metabolism in the thiazolidinedione (TZD) ring may contribute to the hepatotoxicity of the insulin-sensitizing agents such as troglitazone. We were interested in determining if biotransformation could also be a factor in the liver damage associated with another TZD ring containing compound, 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT). Therefore, hepatotoxic doses of DCPT (0.6 or 1.0 mmol/kg, i.p.) were administered to male Fischer 344 rats after pretreatment with vehicle, 1-aminobenzotriazole (ABT, non-selective CYP inhibitor) and troleandomycin (TAO, CYP3A inhibitor). Alternatively, rats were pretreated with vehicle or the CYP3A inducer dexamethasone (DEX) prior to a non-toxic DCPT dose (0.2 mmol/kg, i.p.). Vehicle-, ABT-, TAO- and DEX-only control groups were also run. Toxicity was assessed 24 h after DCPT administration. Both hepatotoxic doses of DCPT induced elevations in serum alanine aminotransferase (ALT) levels that were attenuated by ABT or TAO pretreatment. Liver sections from rats that received vehicle+DCPT revealed areas of gross necrosis and neutrophil invasion, whereas sections from ABT+DCPT and TAO+DCPT rats showed minor changes compared to controls. DEX pretreatment potentiated ALT levels associated with the non-toxic DCPT dose. Furthermore, DEX+DCPT rat liver sections exhibited hepatic injury when compared against rats that received vehicle+DCPT. Blood urea nitrogen levels, urinalysis and kidney morphology were not markedly altered by any combination of pretreatments or treatments. Enzyme activity and Western blotting experiments with rat liver microsomes confirmed the effects of the various pretreatments. Our results suggest that hepatic CYP3A isozymes may be involved in DCPT-induced liver damage in male rats. We believe this is the first report demonstrating that modulation of the biotransformation of a TZD ring-containing compound can alter hepatotoxicity in a common animal model.

Fig. 1

Structures of 3-(3,5-Dichlorophenyl)-2,4-thiazolidinedione (DCPT) and Troglitazone (Rezulin^®).

Fig. 2

Effect of 1-Aminobenzotriazole (ABT) and Troleandomycin (TAO) Pretreatments on DCPT-induced Alterations in Rat Serum Alanine Aminotransferase (ALT) Levels. Pretreatment groups are indicated along the X-axis. Treatments are indicated by white (clear) bars (corn oil), grey bars (DCPT, 0.6 mmol/kg) and black bars (DCPT, 1.0 mmol/kg). Rats were pretreated with saline (vehicle, 4 ml/kg, i.p.), ABT (100 mg/kg, i.p. in saline) or TAO (500 mg/kg, i.p. in saline), and were then administered corn oil (vehicle, 4 ml/kg, i.p.) or DCPT (0.6 or 1.0 mmol/kg, i.p. in corn oil). Values indicate means ± standard errors (N = 4). An asterisk (*) indicates that the value is significantly different (p < 0.05) from the Saline + Corn oil control group. A dagger (†) indicates that the value is significantly different (p < 0.05) from the Saline + DCPT (0.6 mmol/kg) group. A double dagger (‡) indicates that the value is significantly different (p < 0.05) from the Saline + DCPT (1.0 mmol/kg) group.

Fig. 3

Effect of 1-Aminobenzotriazole (ABT) and Troleandomycin (TAO) Pretreatments on DCPT-induced Alterations in Rat Liver Histology. Groups are indicated by pretreatment + treatment as follows: (A) Saline + Corn oil; (B) ABT + Corn oil; (C) TAO + Corn oil; (D) Saline + DCPT (0.6 mmol/kg); (E) ABT + DCPT (0.6 mmol/kg); (F) TAO + DCPT (0.6 mmol/kg); (G) Saline + DCPT (1.0 mmol/kg); (H) ABT + DCPT (1.0 mmol/kg); (I) TAO + DCPT (1.0 mmol/kg). Rats were pretreated with saline (vehicle, 4 ml/kg, i.p.), ABT (100 mg/kg, i.p. in saline) or TAO (500 mg/kg, i.p. in saline), and were then administered corn oil (vehicle, 4 ml/kg, i.p.) or DCPT (0.6 or 1.0 mmol/kg, i.p. in corn oil). The black arrow heads point to swollen hepatocytes (panel D) or regions of necrosis (panel G); and the white arrow heads point to condensed nuclei. Sections were stained with hematoxylin-eosin. Magnification is 400×.

Fig. 4

Effect of Dexamethasone (DEX) Pretreatment on DCPT-induced Alterations in Rat Serum Alanine Aminotransferase (ALT) Levels. Pretreatment groups are indicated along the X-axis. Treatments are indicated by white (clear) bars (corn oil) or black bars (DCPT, 0.2 mmol/kg). Rats were pretreated with corn oil (vehicle, 4 ml/kg, i.p.) or DEX (50 mg/kg/day for 3 days, i.p. in corn oil), and were then administered corn oil (vehicle, 4 ml/kg, i.p.) or DCPT (0.2 mmol/kg, i.p. in corn oil). Values indicate means ± standard errors (N = 4). An asterisk (*) indicates that the value is significantly different (p < 0.05) from the Corn oil + Corn oil control group. A dagger (†) indicates that the value is significantly different (p < 0.05) from the Corn oil + DCPT (0.2 mmol/kg) group. A double dagger (‡) indicates that the value is significantly different (p < 0.05) from the DEX + corn oil group.

Fig. 5

Effect of Dexamethasone (DEX) Pretreatment on DCPT-induced Alterations in Rat Liver Histology. Groups are indicated by pretreatment + treatment as follows: (A) Corn oil + Corn oil; (B) DEX + Corn oil; (C) Corn oil + DCPT (0.2 mmol/kg); (D) DEX + DCPT (0.2 mmol/kg). Rats were pretreated with corn oil (vehicle, 4 ml/kg, i.p.) or DEX (50 mg/kg/day for 3 days, i.p. in corn oil), and were then administered corn oil (vehicle, 4 ml/kg, i.p.) or DCPT (0.2 mmol/kg, i.p. in corn oil). The black arrow heads point to condensed proteins in cells, and the black arrows point to vacuolization. Sections were stained with hematoxylin-eosin. Magnification is 400×.

Fig. 6

Effect of Dexamethasone (DEX) Pretreatment on DCPT-induced Alterations in Rat Liver Glycogen Content. Groups are indicated by pretreatment + treatment as follows: (A) Corn oil + Corn oil; (B) DEX + Corn oil; (C) Corn oil + DCPT (0.2 mmol/kg); (D) DEX + DCPT (0.2 mmol/kg). Rats were pretreated with corn oil (vehicle, 4 ml/kg, i.p.) or DEX (50 mg/kg/day for 3 days, i.p. in corn oil), and were then administered corn oil (vehicle, 4 ml/kg, i.p.) or DCPT (0.2 mmol/kg, i.p. in corn oil). The black arrows point to vacuolization due to loss of glycogen. Sections were stained with Periodic Acid-Schiff. Magnification is 400×.

Fig. 7

Effect of Dexamethasone (DEX) Pretreatment on CYP3A1/CYP3A2 Rat Liver Microsomal Protein Level Expression. Rats were pretreated with Corn oil (vehicle, 4 ml/kg, i.p.) or DEX (50 mg/kg/day for 3 days, i.p. in corn oil) before preparation of the microsomes. (A) Representative Western blots, (B) Quantitative results (means ± standard errors) following densitometric analysis. Protein levels were normalized to β-actin as a loading control. Asterisks (*) indicate values significantly different from Corn oil control. A dagger (†) indicates values different from corn oil pretreated CYP3A1. Lane 1 is a representative blot obtained using pooled corn oil pretreated microsomes, lanes 2–5 contain DEX pretreated microsomes from individual rats. The graphical representation summarizes the densitometry results obtained with each antibody from four separate blots using the DEX pretreated microsomes or pooled corn oil microsomes.