Original Contribution
Glycochenodeoxycholate plays a carcinogenic role in immortalized mouse cholangiocytes via oxidative DNA damage

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Abstract

Bile acids have been suggested to be involved in biliary carcinogenesis, although the underlying mechanisms are yet to be established. The aim of this study was to investigate the carcinogenic effect of bile acids in the biliary tract in relation to oxidative stress. Immortalized mouse cholangiocytes were incubated with various bile acids, followed by measurement of reactive oxygen species (ROS) and the glutathione (GSH) level. As a marker of oxidative DNA damage, 8-hydroxydeoxyguanosine (8-OHdG) expression in cholangiocytes was analyzed by flow cytometry. Then the expression of oxidative DNA repair enzymes in cholangiocytes was examined by real-time PCR. In addition, the long-term effect of bile acid-induced oxidative DNA damage on cholangiocytes was investigated using a mouse oligo DNA microarray. It was found that glycochenodeoxycholate (GCDC) induced the generation of ROS and the depletion of GSH. In contrast, no marked changes were induced by the other bile acids. The percentage of 8-OHdG-positive cells was also increased by GCDC, but the expression of oxidative DNA repair enzymes was not up-regulated. DNA microarray analysis showed marked changes of various genes associated with carcinogenesis (genes related to cell proliferation, angiogenesis, invasion, and metastasis). In conclusion, the long-term effect of oxidative DNA damage due to GCDC may promote carcinogenesis in the biliary tract. Furthermore, accumulation of 8-OHdG due to GCDC might contribute to the dysfunction of oxidative DNA repair enzymes.

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.

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