Two‐dimensional differential gel electrophoresis/analysis of diethylnitrosamine induced rat hepatocellular carcinoma

Abstract

Diethylnitrosamine (DEN) is a known carcinogen that can alkylate DNA molecules. In rats, DEN-induced hepatocellular carcinoma (HCC) model is well established. In this study, we used a twodimensional differential gel electrophoresis (2D-DIGE) system and liquid chromatography/mass spectrometry/mass spectrometry to identify the differential expression protein profiles between the DEN-induced HCC and healthy liver cells. Western blotting and semiquantitative RT-PCR were used to further confirm the results. Seventeen differentially expressed spots were identified in DEN-induced HCC cells. Among all, the most prominent upregulated proteins include the members of the glutathione Stransferase super family, aldo-keto reductase superfamily and proteins involved in the response to oxidative stress. Downregulation was observed in 2 proteins that were known to contribute to hepatic dysfunction. This study provides the first comprehensive protein profiling of the DEN-induced HCC in rats. This model simulates the differential protein expression of human HCC and may be useful for further understanding the mechanism of HCC tumorigenesis. ' 2008 Wiley-Liss, Inc.Key words: hepatocellular carcinoma; diethylnitrosamine; twodimensional differential gel electrophoresis; glutathione S-transferase Hepatocellular carcinoma (HCC) is a major malignancy worldwide.1 It is the fourth most common malignant tumor in the world 2 and is the second leading fetal disease in Mainland China. 3 In last 2 decades, the incidence of HCC has increased substantially all over the North America and Europe. 4 Studies have indicated HCC is associated with a major risk factor of hepatitis B and C viral infections [i.e., hepatitis B virus (HBV) and hepatitis C virus (HCV)].5 However, other factors such as aflatoxin B1 exposure, alcohol drinking and genetic defects have also aroused research interests in recent years. 6,7 To further understand the disease, animal models have been established for the study of hepatocarcinogenesis. These include the two-stage initiation-promotion 8 and the three-stage initiation, promotion and progression 9 models. The initiative period is characterized by the mutation of cells, which then undergo tumor promotion via active cell division or altered genome expression induced by chemicals.10 Thereafter, mutations continue and lead to the development of HCC cells via a number of causes such as the presence of HBV, HCV or radiation. Changes such as oxidation, chemical adducts and errors in the DNA bases and repair are also implicated in the early cellular changes.11 They are the crucial changes in the initial stage of HCC, which correspond to the histological observations including fibrosis, cirrhosis, and hepatocellular adenoma. 11In this study, diethylnitrosamine (DEN) was employed to induce HCC in rats. It is an environmental carcinogen and a hepatotoxin. It is commonly used in inducing hepatocarcinogenesis as it causes degenerative, proliferative and neoplastic lesions in the liver. DEN is a chemical agent that can alkylate DNA mo...

Macrophages Regulate the Systemic Response to DNA Damage by a Cell Nonautonomous Mechanism

“…DEN is a DNA alkylating agent that leads to the formation of mutagenic DNA adducts. In addition, DEN metabolic activation by cytochrome P450 can generate ROS (19), which consequently ÃÃÃ , P < 0.001 by ANOVA. D-F, mice were either left untreated or injected with DEN and treated with either PBS-or clodronate-loaded liposomes.…”

Thyroid hormone suppresses hepatocarcinogenesis via DAPK2 and SQSTM1-dependent selective autophagy

“…16 As autophagy is regarded as an intracellular self-protection mechanism, we attempted to determine whether the hepatoprotective function of T 3 against DEN-induced toxicity is mediated through this process. After treatment with phosphate-buffered saline (PBS; euthyroid), T 3 (hyperthyroid), CQ or CQ plus T 3 for 14 d, mice were subjected to DEN to induce hepatic damage.…”

Preventive Effects of Vitamin C on Diethylnitrosamine-induced Hepatotoxicity in Smp30 Knockout Mice

“…DEN is a powerful carcinogenic agent in rodents that possibly causes its adverse effects by altering DNA structure and forming DNA adducts in the liver (8,27). The DEN-induced HCC experimental model is one of the most widely used models for screening anti-hepatocarcinogenic agents due to its similarity to human HCC (7,28). This animal model takes at least 8 months to develop typical HCC (1,29).…”