Original ContributionGlycochenodeoxycholate plays a carcinogenic role in immortalized mouse cholangiocytes via oxidative DNA damage
Introduction
Biliary tract carcinoma has a common characterisitic with colorectal carcinoma since both types of cancer are epithelial carcinomas in the presence of bile acids. It has been suggested that bile acids are involved in colorectal carcinogenesis [1], [2], [3]. Furthermore, it is well known that inflammatory and cholestatic conditions (e.g., primary sclerosing cholangitis, biliary stone disease, and Caroli's disease) are predisposing factors for the development of biliary tract carcinoma [4], [5], [6], [7], [8]. Such reports suggest that cytotoxic bile acids may be closely related to the development of biliary tract carcinoma, although the underlying mechanisms are yet to be established. We previously reported that glycochenodeoxycholate (GCDC) induced cyclooxygenase 2 (COX-2) expression in immortalized mouse cholangiocytes, indicating that GCDC may be related to the development of biliary tract carcinoma [9].
In cholestatic liver diseases, the concentrations of various bile acids in the bile are significantly elevated, causing damage to hepatocytes and cholangiocytes. We previously reported that GCDC could induce the apoptosis of immortalized mouse cholangiocytes [10]. Hydrophobic bile acids have been reported to cause oxidative stress in hepatocytes [11]. Reactive oxygen species (ROS) are thought to be implicated in the processes of inflammation, aging, and carcinogenesis [12]. 8-Hydroxydeoxyguanosine (8-oxoguanine: 8-OHdG) is a promutagenic DNA lesion produced by ROS that represents one of the major forms of oxidative DNA damage [13]. Accumulation of 8-OHdG induces errors during DNA replication (G:C to T:A transversion), and is thought to play an important role in carcinogenesis. In chronic liver disease, the in situ detection of 8-OHdG in hepatocytes and cholangiocytes has been previously reported [14].
The oxidative DNA repair system, comprising the enzymes MutM, MutY, and MutT, prevents 8-OHdG-induced mutagenesis. Human homologs of MutM (human 8-oxoguanine DNA glycosylase [OGG1]), MutY (human adenine DNA glycosylase [MUTYH]), and MutT (human 8-oxo-dGTPase [MTH1]) have already been characterized [15], [16], [17]. OGG1 removes the oxidized base from 8-OHdG:C base pairs [15], while MUTYH excises misincorporated adenine from 8-OHdG:A base pairs formed during DNA replication [16]. In addition, MTH1 eliminates 8-oxo-dGTP from the nucleotide pool to prevent the incorporation of 8-oxo-dGTP into nascent DNA [17]. Aberrations in the expression of these enzymes and mutations of their genes have been reported in several types of cancer [18], [19], [20], [21], [22], [23].
Recent studies have shown that inflammatory cytokines induce oxidative DNA damage and inhibit DNA repair in cholangiocarcinoma cells, suggesting a link between inflammation and carcinogenesis [24], [25], [26]. However, very little information has been obtained with regard to bile acid-induced oxidative DNA damage in cholangiocytes. Accordingly, the present study was performed with the following aims: (1) to clarify the relationship between bile acids and oxidative stress in cholangiocytes; (2) to elucidate whether bile acids induce oxidative DNA damage via ROS; (3) to investigate the expression of oxidative DNA repair enzymes after bile acid-induced oxidative DNA damage; and (4) to determine whether the long-term effects of oxidative DNA damage caused by bile acids can promote carcinogenesis.
Section snippets
Chemicals
Glycochenodeoxycholate, taurocholate (TC), taurochenodeoxycholate (TCDC), taurodeoxycholate (TDC), and tauroursodeoxycholate (TUDC) were kindly provided by Mitsubishi Pharma Corp. (Tokyo, Japan). These bile acids had a purity >99% when examined by high-performance liquid chromatography. 2′,7′-Dichlorofluorescein diacetate (DCF-DA), 2′,7′-dichlorofluorescein (DCF), and N-acetylcysteine (NAC) were obtained from Sigma (St. Louis, MO).
Cell culture
Preparation of immortalized mouse cholangiocytes was reported
Bile acid-induced oxidative stress in immortalized mouse cholangiocytes
First, we investigated the effect of bile acids on the generation of ROS by immortalized mouse cholangiocytes using DCF fluorescence analysis (Fig. 1). ROS generation was significantly increased (2-fold) by exposure to 200 μM GCDC for 2 h, while no such effect was found for the other bile acids tested (Fig. 1A). GCDC-induced generation of ROS showed a concentration-dependent increase (Fig. 1B). These changes were inhibited by treatment with the antioxidant NAC at 10 mM. We also examined the
Discussion
In cholestatic liver diseases, primary bile acids are higher concentrated than secondary bile acids. Because chenodeoxycholate (CDC) is thought to be cytotoxic in the primary bile acids, we have studied different carcinogenic effects on cholangiocytes between GCDC and TCDC [9], [10]. We have suggested that GCDC but not TCDC plays a carcinogenic role in the biliary tract.
In the present study, we examined the effect of bile acids on biliary carcinogenesis using immortalized mouse cholangiocytes
Acknowledgment
The authors thank Yoshiyuki Ueno for providing immortalized mouse cholangiocytes.
References (41)
- et al.
Effect of dietary deoxycholic acid and cholesterol on fecal steroid concentration and its impact on the colonic crypt cell proliferation in azoxymethane-treated rats
Cancer Lett.
(1998) - et al.
Liver fluke infection and cholangiocarcinoma: model of endogenous nitric oxide and extragastric nitrosation in human carcinogenesis
Mutat. Res.
(1994) - et al.
Bile acid-induced rat hepatocytes apoptosis is inhibited by antioxidants and blockers of the mitochondrial permeability transition
Hepatology
(2001) - et al.
In situ detection of oxidative DNA damage, 8-hydroxydeoxyguanosine, in chronic human liver disease
J. Hepatol.
(2001) - et al.
Genomic structure and chromosome location of the human mutT homologue gene MTH1 encoding 8-oxo-dGTPase for prevention of A:T to C:G transversion
Genomics
(1994) - et al.
Expression of human MutT homologue (hMTH1) protein in primary non-small-cell lung carcinomas and histologically normal surrounding tissue
Free Radic. Biol. Med.
(2003) - et al.
Nitric oxide-mediated inhibition of DNA repair potentiates oxidative DNA damage in cholangiocytes
Gastroenterology
(2001) - et al.
Activation of constitutive nitric-oxide synthase activity is an early signaling event induced by ionizing radiation
J. Biol. Chem.
(2002) - et al.
Direct detection of 8-oxodeoxyguanosine and 8-oxoguanine by avidin and its analogues
Anal. Biochem.
(1998) - et al.
Acute arsenite-induced 8-hydroxyguanine is associated with inhibition of repair activity in cultured human cells
Biochem. Biophys. Res. Commun.
(2002)