Induction of hepatic Bach1 mRNA expression by carbon tetrachloride-induced acute liver injury in rats

Abstract

Hepatic oxidative stress is a major contributor to the pathogenesis of several acute liver diseases. Diagnostic markers of hepatic oxidative stress may facilitate early detection and intervention. Bach1 is an oxidative stress-responsive transcription factor that represses heme oxygenase 1 (HO-1), the rate-limiting enzyme in the catabolism of heme, a potent pro-oxidant. We previously demonstrated that carbon tetrachloride (CCl₄) causes oxidative hepatic injury in rats, exacerbated by free heme, suggesting that CCl₄ may affect Bach1 gene expression. In the present study, we used northern blot analysis to measure Bach1, HO-1 and δ-aminolevulinate synthase (ALAS1; a heme biosynthesis enzyme) mRNA expression levels during acute hepatic injury induced by CCl₄ (at doses of 0.1, 1.0 and 2.0 ml/kg body weight). Oxidative injury was assessed by measuring serum alanine aminotransferase (ALT), hepatic malondialdehyde (MDA) and glutathione (GSH) content. Treatment with CCl₄ induced a significant dose-dependent increase in Bach1 mRNA 1-3 h after administration. Bach1 mRNA peaked at 6 h after CCl₄ treatment (1 ml/kg), followed by a rapid decrease and gradual return to baseline by 12 h after treatment. The timecourse of transient Bach1 mRNA induction roughly mirrored that of HO-1 mRNA, while ALAS1 mRNA was inversely downregulated. Serum ALT levels and hepatic MDA concentration were significantly increased at 24 h after CCl₄ treatment, while the hepatic GSH content was significantly reduced within 3 h of treatment. Serum ALT levels were positively correlated with Bach1 mRNA levels. These findings indicate that Bach1 mRNA is transiently induced in rat liver by CCl₄, possibly as a regulatory mechanism to restore HO-1 to baseline following free heme catabolism. Our findings also suggest that Bach1 mRNA expression may be a novel indicator of the extent of oxidative hepatic injury caused by free heme.

Keywords: Bach1; carbon tetrachloride; free heme; heme oxygenase-1; oxidative stress.

Figure 1

Changes in hepatic HO-1 and ALAS1 gene expression levels following CCl₄ treatment. The rats were sacrificed at 0, 1, 3, 6, 9, 12, 18 and 24 h after injection of CCl₄ (1.0 ml/kg, intraperitoneally), their livers were excised and total RNA (20 μg) was subjected to northern blot analysis. Autoradiographic signals of RNA blots hybridized with (A) [α-³²P]dCTP-labeled HO-1 or (B) ALAS1 cDNA probes are shown. Ethidium bromide staining of the same RNA is shown as the loading control. Closed arrowhead, 28S ribosomal RNA; and open arrowhead, 18S ribosomal RNA. Three independent experiments showed similar results and a typical example is shown. (C) Concentrations of HO-1 mRNA are expressed as relative values to the concentration of an untreated control spleen, in which HO-1 is known to be constitutively expressed; and (D) concentrations of ALAS1 mRNA are expressed as relative values to the concentration of an untreated control liver. HO-1, heme oxygenase-1; ALAS1, δ-aminolevulinate synthase; CCl₄, carbon tetrachloride.

Figure 2

Effect of CCl₄ treatment on Bach1 gene expression. (A) Dose-response. The rats were injected with either CCl₄ [0.1, 1.0 or 2.0 ml/kg body weight, intraperitoneally (i.p.)] or 2 ml of vehicle (corn oil), were sacrificed 6 h after injection and their livers were excised for northern blot analysis. (B) Timecourse of hepatic Bach1 gene expression after CCl₄ treatment. The rats were sacrificed at 0, 1, 3, 6, 9, 12, 18 and 24 h after CCl₄ injection (1.0 ml/kg, i.p.) and their livers were excised. Total RNA (20 μg) was subjected to northern blot analysis. Autoradiographic signals of RNA blots hybridized with a [α-³²P]dCTP-labeled Bach1 cDNA probe are shown. Ethidium bromide staining of the same RNA is shown as the loading control. Closed arrowhead, 28S ribosomal RNA; and open arrowhead, 18S ribosomal RNA; control, vehicle (corn oil)-treated rats. Three independent experiments yielded similar results and a typical example is shown. (C and D) Bach1 gene expression levels expressed as densitometric arbitrary units. Data are expressed as the means ± standard deviation and were statistically evaluated using analysis of variance followed by Tukey-Kramer’s honestly significant difference test. ^*P<0.05 vs. control group; ^†P<0.05 vs. 0.1 ml/kg CCl₄; and ^#P<0.05 vs. 1.0 ml/kg CCl₄. CCl₄, carbon tetrachloride.

Figure 3

Correlation between hepatic Bach1 gene expression and serum ALT levels following CCl₄ treatment. The rats were injected with CCl₄ (0.1, 1.0 or 2.0 ml/kg body weight, intraperitoneally) or vehicle (corn oil) and were sacrificed after 6 h. The livers were excised for northern blot analysis and whole blood was collected for examination of serum ALT activity 6 h after CCl₄ injection. A significantly positive correlation between Bach1 gene expression and serum ALT levels was observed (r²=0.503342; P<0.0001). Linear regression (solid line) with 95% confidence intervals (dotted lines) is presented. ALT, alanine aminotransferase; CCl₄, carbon tetrachloride.