2,3,7,8-Tetrachlorodibenzo-p-dioxin enhances CCl4-induced hepatotoxicity in an aryl hydrocarbon receptor-dependent manner

Abstract

Cytochrome P4502E1 (CYP2E1) is involved in the biotransformation of several low molecular weight chemicals and plays an important role in the metabolic activation of carcinogens and hepatotoxins such as CCl(4). Induction of CYP2E1 is exerted mainly at posttranscriptional levels through mRNA and protein stabilization, and there is little evidence of xenobiotic induction at the transcriptional level. Previously, we reported microarray analysis data suggesting a decrease in Cyp2e1 gene expression on Ahr-null livers when compared to wild-type mouse livers. The goal of the present study was to determine whether 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increased mouse CYP2E1 levels in an AhR-dependent manner and the impact on CCl(4)-induced hepatotoxicity. TCDD treatment induced CYP2E1 mRNA and protein levels in mouse liver, and this effect was aryl hydrocarbon receptor (AhR)-dependent. Moreover, TCDD pre-treatment increased the CCl(4)-induced alanine aminotransferase (ALT) activity, the extent of CCl(4)-induced necrosis, and the number of sinusoidal cells in wild-type animals, while this potentiating effect was not observed in Ahr-null mice. In conclusion, this study revealed that TCDD, probably in an AhR-dependent manner, exacerbated CCl(4)-induced hepatotoxicity through induction of CYP2E1.

Figure 1.

TCDD increases CYP2E1 mRNA levels. Wild-type or Ahr-null mice were treated with TCDD (80 μg/kg) or vehicle. After 72 h, liver samples were removed and the relative expression of CYP1A1 (A) or CYP2E1 (B) mRNA levels were determined. mRNA levels were normalised with 18S ribosomal RNA. Data represent mean values ± SD. *p < 0.05.

Figure 2.

Effect of TCDD on CYP2E1 protein levels. Wild-type or Ahr-null mice were treated with TCDD (80 μg/kg) or vehicle. After 72 h, liver samples were removed and CYP2E1 protein levels were determined. (A) Relative values for CYP2E1 protein signals were normalised with actin. (B) Representative immuno-blot image. Data represent mean values ± SE. *p < 0.05.

Figure 3.

Body weights during TCDD treatment. Both wild-type and Ahr-null mice were treated with TCDD (80 μg/kg) as described in the Materials and Methods section, and the body weight was evaluated every day for 4 days. Data represent mean values ± SE from five animals of each genotype.

Figure 4.

Serum aminotransaminase activity after TCDD treatment. Wild-type or Ahr-null mice were treated with TCDD (80 μg/kg) or vehicle. After 72 h, serum levels of AST (A) or ALT (B) were determined. Data represent mean values ± SE. *p < 0.05.

Figure 5.

Histopathological analysis of livers of mice treated with TCDD. Representative H&E-stained liver sections are shown from wild-type and Ahr-null mice treated with corn oil or TCDD (80 μg/kg) for 72 h.

Figure 6.

Aminotransaminase activity after CCl₄ and TCDD treatments. Wild-type or Ahr-null mice were treated with CCl₄ (1.59 g/kg) for 24 h, or co-treated with TCDD (80 μg/kg) and CCl₄ (1.59 g/kg) as indicated in Materials and methods section. After treatments, serum levels of AST (A) or ALT (B) were determined. Data represent mean values ± SE. *p < 0.05. Wildtype-CCl₄ vs. AhR-null-CCl₄, §p < 0.05.

Figure 7.

Histopathological analysis of livers of mice after TCDD and CCl₄ treatments. Representative H&E-stained liver sections are shown from wild-type and Ahr-null mice treated with CCl₄ (1.59 g/kg) for 24 h, or co-treated with TCDD (80 μg/kg) and CCl₄ (1.59 g/kg) as indicated in Materials and methods section.