Aryl hydrocarbon receptor ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin enhances liver damage in bile duct-ligated mice

Abstract

The environmental pollutant 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) is known to cause a wide variety of toxic effects, including hepatotoxicity, by way of the aryl hydrocarbon receptor (AHR). Although inducible expression of cytochrome P450 (CYP) 1A1 and CYP1A2 is associated with liver injury caused by high-dose TCDD, the specific role of the AHR-CYP1 cascade in hepatotoxicity remains unclear. We investigated the effects of AHR activation under conditions of cholestasis. We administered oral TCDD to mice at a dose that can effectively induce Cyp1 gene expression without overt liver toxicity and then ligated their bile ducts. TCDD pretreatment enhanced bile duct ligation (BDL)-induced increases in liver and plasma bile acids, bilirubin, and aminotransferases. Histology of TCDD-pretreated BDL mice revealed massive hepatic necrosis without any increase in number of apoptotic cells. Whereas induction of AHR-target genes by TCDD was observed similarly in sham-operated as well as in BDL mice, TCDD pretreatment of BDL mice altered the expression of hepatic genes involved in bile acid synthesis and transport. Increased plasma proinflammatory cytokines, tumor necrosis factor and interleukin-1β, in BDL mice were further elevated by TCDD pretreatment. Liver injury by TCDD plus BDL, such as increased plasma bile acids, bilirubin and aminotransferases, liver necrosis, and increased tumor necrosis factor production, was exaggerated in Cyp1a1/1a2(-/-) double knockout mice. These findings indicate that TCDD aggravates cholestatic liver damage and that the presence of CYP1A1 and CYP1A2 plays a protective role in liver damage caused by TCDD and BDL.

Figure 1

Effects of TCDD pretreatment and BDL on (A) body weight, (B) liver weight and bile acids, (C) plasma bile acids, bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (γ-GT), and alkaline phosphatase (ALP) in mice. Mice (n = 6 per group) were administered the vehicle corn oil or TCDD (15 μg/kg) via gavage on “Day −5” and subjected to sham operation or BDL on “Day 0”. Liver and blood were collected on “Day +3”. Values represent the means ± S.D. (A) **P < 0.01; ***P < 0.001 vs. vehicle-pretreated sham-operated mice. There was no significant difference between vehicle-pretreated sham-operated mice and TCDD-pretreated BDL mice. (B, C) *P < 0.05; **P < 0.01; ***P < 0.001; between indicated groups.

Figure 1

Effects of TCDD pretreatment and BDL on (A) body weight, (B) liver weight and bile acids, (C) plasma bile acids, bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (γ-GT), and alkaline phosphatase (ALP) in mice. Mice (n = 6 per group) were administered the vehicle corn oil or TCDD (15 μg/kg) via gavage on “Day −5” and subjected to sham operation or BDL on “Day 0”. Liver and blood were collected on “Day +3”. Values represent the means ± S.D. (A) **P < 0.01; ***P < 0.001 vs. vehicle-pretreated sham-operated mice. There was no significant difference between vehicle-pretreated sham-operated mice and TCDD-pretreated BDL mice. (B, C) *P < 0.05; **P < 0.01; ***P < 0.001; between indicated groups.

Figure 2

TCDD pretreatment induces massive hepatic necrosis in BDL mice. Hematoxylin and eosin staining (H&E; x 20 magnification), TUNEL assays (x 20 magnification and x 60 magnification), and cleaved caspase-3 staining (x 60 magnification) were performed on liver sections from mice treated as described in Figure 1.

Figure 3

Effects of TCDD and BDL on hepatic mRNA expression of AHR-target genes. (A) CYP1A1, CYP1A2 and CYP1B1 mRNA levels. (B) AHR and ARNT mRNA levels. Total RNA was prepared from the mice shown in Figure 1. In this and subsequent figures, expression of the indicated mRNA levels was measured by qRT-PCR using glyceraldehyde-3-phosphate dehydrogenase as the internal control. Values for normalized mRNA expression are relative to those of corn oil-pretreated sham-operated mice. Effects of BDL on CYP1A1, CYP1A2 and CYP1B1 mRNA expression in corn oil-pretreated mice are shown in insets of (A). Values represent the means ± S.D. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

Expression of hepatic CYP1A1, CYP1A2 and ABCB11 proteins. Each lane was loaded with 15 μg of microsomal proteins and 40 μg of membrane proteins for CYP1A1/CYP1A2 and ABCB11, respectively. Microsomes and membrane fractions were prepared from the mice shown in Figure 1. Experiments were repeated with similar results.

Figure 5

Effects of TCDD and BDL on hepatic mRNA levels of genes involved in bile acid metabolism. (A) CYP7A1 and CYP3A11 mRNA. (B) Expression of SLC01A1, ABCB11, ABCC2, ABCC3, ABCC4 and OSTB mRNA. Values represent the means ± S.D. *P < 0.05; **P < 0.01.

Figure 6

TCDD pretreatment enhances release of TNF and IL1B in BDL mice. (A) Plasma levels of TNF and IL1B proteins. (B) Hepatic TNF, ILIB, TNFRSF10B, and TNFSF10 mRNA levels. Values represent the means ± S.D. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7

Liver damage by TCDD and BDL is exaggerated in Cyp1a1/1a2(−/−) double-knockout mice. (A) Body weight changes and liver weight. (B) Plasma bile acids, bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (γ-GT), and alkaline phosphatase (ALP). (C) Hematoxylin and eosin staining (H&E) of liver sections (x10 magnification). (D) Plasma TNF protein concentrations. Cyp1a1/1a2(−/−) mice (n = 4 per group) were administered corn oil alone or TCDD (15 μg/kg) via gavage on “Day −5” and subjected to sham operation or BDL on “Day 0”. Liver and blood were collected on “Day +3” by the same method as shown in Figure 1. Values represent the means ± S.D. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7

Liver damage by TCDD and BDL is exaggerated in Cyp1a1/1a2(−/−) double-knockout mice. (A) Body weight changes and liver weight. (B) Plasma bile acids, bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (γ-GT), and alkaline phosphatase (ALP). (C) Hematoxylin and eosin staining (H&E) of liver sections (x10 magnification). (D) Plasma TNF protein concentrations. Cyp1a1/1a2(−/−) mice (n = 4 per group) were administered corn oil alone or TCDD (15 μg/kg) via gavage on “Day −5” and subjected to sham operation or BDL on “Day 0”. Liver and blood were collected on “Day +3” by the same method as shown in Figure 1. Values represent the means ± S.D. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7

Liver damage by TCDD and BDL is exaggerated in Cyp1a1/1a2(−/−) double-knockout mice. (A) Body weight changes and liver weight. (B) Plasma bile acids, bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (γ-GT), and alkaline phosphatase (ALP). (C) Hematoxylin and eosin staining (H&E) of liver sections (x10 magnification). (D) Plasma TNF protein concentrations. Cyp1a1/1a2(−/−) mice (n = 4 per group) were administered corn oil alone or TCDD (15 μg/kg) via gavage on “Day −5” and subjected to sham operation or BDL on “Day 0”. Liver and blood were collected on “Day +3” by the same method as shown in Figure 1. Values represent the means ± S.D. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7

Liver damage by TCDD and BDL is exaggerated in Cyp1a1/1a2(−/−) double-knockout mice. (A) Body weight changes and liver weight. (B) Plasma bile acids, bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (γ-GT), and alkaline phosphatase (ALP). (C) Hematoxylin and eosin staining (H&E) of liver sections (x10 magnification). (D) Plasma TNF protein concentrations. Cyp1a1/1a2(−/−) mice (n = 4 per group) were administered corn oil alone or TCDD (15 μg/kg) via gavage on “Day −5” and subjected to sham operation or BDL on “Day 0”. Liver and blood were collected on “Day +3” by the same method as shown in Figure 1. Values represent the means ± S.D. *P < 0.05; **P < 0.01; ***P < 0.001.