The purpose of this study was to investigate diethylnitrosamine (DEN)-induced liver damage in zebrafish. Zebrafish larvae were divided into five groups after seventy-two hours fertilization: group 1 (G1) as control, group 2 (G2) as probe control, groups 3, 4, and 5 (G3, G4, and G5) as DEN treated at doses of 25, 50, and 100 μg/mL, respectively. At twenty-two hours after DEN treatment, groups 2, 3, 4, and 5 were treated with ApoFlamma H 675 at a dose of 100 μM/zebrafish. They were examined by fluorescence stereomicroscope at twenty-four hours after DEN treatment. After fixation, the zebrafish were processed, embedded, sectioned and stained with hematoxylin and eosin (HE) and terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) staining. Fluorescence intensity of the livers of G3, G4, and G5 was significantly increased compared with those of G1 (p<0.01). Furthermore, fluorescence intensity of the livers of G3 and G5 was significantly increased compared with those of G2 (p<0.05 and p<0.01). HE staining showed cell deaths in the livers of DEN-treated zebrafish and TUNEL staining confirmed cell death in the same location. Taken together, in vivo fluorescence bioimaging detected cell death in the liver of DEN-treated zebrafish. This outcome was confirmed with histopathological examination. The results of this study provide confidence for using zebrafish as a liver carcinogenesis model.

As diethylnitrosamine (DEN) effect on cell proliferation, DNA damage and stem cell marker(s) expression have been largely unknown in mouse normal hepatocytes (AML-12 cells) cultured over a short-term period, this study was conducted to examine the cell proliferation, Ataxia telangiectasia mutated (ATM) and epithelial cell adhesion molecule (EpCAM) and Neighbor of Punc E 11 (Nope) expression in AML-12 cells treated with DEN for 24 and 48 h. Cells were treated with DEN (25-800 μg/mL) and cell phenotype was determined, and the MTT assay was used to quantify the proliferation of cells treated with DEN. Expression and distribution of ATM in AML-12 cells were determined by indirect immunofluorescence microscopy. And Western blot analysis of EpCAM and Nope was performed. Cell viability was significantly increased in response to all doses of DEN treatment compared to control at 24 h (p<0.05 or p<0.01). However, there was no significant increase at 48 h, even though it showed increased trend. Immunofluorescence staining of ATM showed that there was an increase of ATM expression at doses of 50, 100 and 200 μg/mL of DEN treatment, showing strong nuclear staining. Furthermore, Western blot analysis showed that DEN treatment showed increased trend of EpCAM and Nope expression. Taken together, DEN treatment increased cell proliferation in AML- 12 cells, and it was associated with increased ATM expression.

Hepatocellular carcinoma (HCC) is a representative inflammation-associated cancer and known to be the most frequent tumor. However, the preventive agents for hepatocarcinogenesis are unsatisfactorily identified. We investigated the protective effect of steamed and freeze-dried mature silkworm larval powder (SMSP) on diethylnitrosamine (DEN)-induced hepatotoxicity in mice and compared the effect of three silkworm varieties: white-jade, golden-silk, and light-green strains. The mice were fed with diet containing 0.1, 1, and 10 g/kg of three types of SMSP for two weeks while DEN (100 mg/kg, i.p.) was injected 18 h before the end of this experiment. Liver toxicity was determined as serum indicator, histopathological examination, and expression of inflammatory enzyme. Pretreatment with SMSP reduced necrotic and histopathological changes induced by DEN in the liver. The measurement of serum biochemical indicators showed that pretreatment with SMSP also decreased DEN-induced hepatotoxicity, the levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). In addition, SMSP inhibited the expressions of inflammatory enzymes, cyclooxygenase-2 and inducible nitric oxide synthase. White-jade SMSP showed the most effective hepatoprotective results against hepatotoxicity among the three silkworm strains used in this study. SMSP may have a protective effect against acute liver injury by inhibiting necrosis and inflammatory response in DEN-treated mice.

To clarify the role of stem cells in hepatocarcinogenesis, CD44 expression was investigated in mouse livers as well as embryonic cell lineages treated with diethylnitrosamine (DEN). Liver tumors induced by DEN were analyzed by immunohistochemisty for CD44. Liver tissues were sampled at 6, 24, and 48 hr after treatment with saline or DEN. Mouse embryonic stem cells (ESCs), hepatic progenitor cells (HPCs), and hepatocyte like cells (HCs), representing 0, 22, and 40 days of differentiation, respectively, were treated with DEN at four doses (0, 1, 5, and 15 mM, respectively) for 24 hr, after which CD44 expression levels were examined by relative quantitative real-time PCR. CD44 expression was weakly detected in tumor cells as well as in some hepatocytes surrounding the tumor cells. However, CD44 expression was not detected in liver tissue treated with DEN at early time points. The CD44 mRNA expression level was significantly different among cells treated with 5 mM DEN at day 22 (P<0.01) as well as 1, 5, and 15 mM DEN at day 40 (P<0.01) compared with control. Taken together, CD44 expression slightly increased in mouse DEN-induced tumors. Furthermore, expression of CD44 in embryonic cell lineages treated with various doses of DEN significantly differed among embryo stem cells and derived hepatic lineage cells. This suggests that CD44 expression may be modulated in the progeny of stem cells during their differentiation toward hepatocytes, and its expression may increase in the tumor stage but not during early carcinogenesis.

Vitamin C (ascorbic acid) is an essential nutrient of most living tissues. We established a strain of Gulo-/- mice with known deficiency, in which vitamin C intake can be controlled by diet, like humans, and investigated the differen- tially expressed proteins following treatments with Helicobacter pylori and diethylnitrosamine (DENA) in the liver of Gulo-/- mice using a proteomic approach. Expression of p53, 14-3-3ε and 14-3-3δ in Gulo-/- mice liver tissue was analyzed by immunohistochemistry. 2-DE maps constructed from Gulo-/- mice liver and differentially expressed proteins in liver tissue were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF/MS). In Gulo- /- mice after H. Pylori infection, followed by treatment with DENA, no differences in p53, 14-3-3ε and 14-3-3δ were observed by immunohistochemistry. Proteome analyses us- ing MALDI-TOF/MS resulted in successful identification of 12 proteins (nine proteins were up-regulated and three were down-regulated). Specifically, peroxiredoxin-6 and Alpha-1-antitrypsin 1-4 were up-regulated in liver after H. Pylori infection followed by treatment with DENA. These results indicated that oral supplementation with vitamin C led to rescue of Gulo-/- mice from vitamin deficiency, and protected the liver from H.pylori infection and/or DENA ef- fect, and vitamin C also protected the liver against oxidative stress.

To clarify the role of stem cells in hepatocarcinogenesis, octamer-binding transcription factor 4 (Oct4) expression was investigated in mouse liver and embryonic cell lineages. In vivo, at 14 days of age, ten ICR mice were divided into two groups and treated with saline or diethylnitrosamine (DEN), and were sacrificed at 6 h after treatment. Livers were fixed in 10% neutral phosphate buffered formalin, embedded in paraffin, sectioned to a thickness of 5 μm, and immunohistochemical analysis of Oct4 was performed. In vitro, mouse embryonic stem cells, hepatic progenitor cells and hepatocytes, representing 0, 22, and 40 days of differentiation, respectively, were treated with DEN at four doses (0, 1, 5 and 15 mM; G1, G2, G3 and G4, respectively) for 24 h and RNA was isolated; Oct4 and Gadd45a mRNA were investigated. In vivo, Oct4 expression was not detected in saline-treated livers. However, its expression was observed in hepatocytes of mice treated with DEN, showing cytoplasmic staining. In vitro, Oct4 expression differed significantly for G4 on day 0 (P<0.05) and for G2 on day 22 (P<0.01) and G3 and G4 on day 40 (P<0.05 and P<0.01, respectively) compared with G1 at each time point. Gadd45a expression differed significantly in G4 (P<0.01) on day 0 and G4 on day 40 (P<0.01), compared with that of G1 at each time point. Taken together, Oct4 expression was increased by treatment with DEN in hepatocytes, however, not in embroyonic stem cells and hepatic progenitor cells. This finding suggests that Oct4 expression may be modulated in hepatocarcinogenesis induced by DEN.

Expression of epithelial cell adhesion molecule (EpCAM) in the early phase of hepatocarcinogenesis induced by diethylnitrosamine (DEN) was investigated. At 14 days of age, 60 ICR mice were divided into two groups and treated with saline (group 1) or DEN (group 2, 10 mg/kg of body weight, i.p. injection), and were sacrificed at 6 h and 1, 2, 3, 7, and 28 days after treatment with saline or DEN. During necropsy, half of the liver from saline- or DEN-treated mice was processed for histopathological examination and immunohistochemical staining of EpCAM and apoptosis. The remaining liver tissue was snap-frozen in liquid nitrogen for RNA extraction and analysis of EpCAM mRNA expression. Immunohistochemical examination showed that EpCAM expression was detected only in a small number of hepatocytes from saline-treated mice and its expression was detected in bile duct cells and round cells around portal areas, as well as hepatocytes in the livers of DEN-treated mice. In addition, multiple apoptotic cells were found in the livers of mice treated with DEN. EpCAM mRNA expression was significantly higher in DEN-treated mice at 1, 7, and 28 days compared to saline-treated mice at 6 h (P<0.01). Taken together, EpCAM expression and apoptosis were increased in liver by DEN treatment.

Liver cancer represents a major health problem with steadily increasing incidence rates. Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third most common cause of cancer-related death. This study was conducted in order to investigate the gross findings following treatment with diethylnitrosamine (DEN) in mice. Sixteen male and female mice (B6C3F₁), initially 20 days of age, received intraparietal injection (20 mg/kg three times for a period of two weeks, IP) or were given drinking water (DW) containing 50 ppm DEN; all mice were sacrificed at the 80^th week of experiments. Hepatocellular adenoma (HCA) and HCC were induced in B6C3F₁), mice by administration of DEN. The numbers of HCA and HCC were 7.4±1.72 (IP) and 7.2±0.86 (DW) in male mice. However, no significant difference was observed between the DW and IP groups. The numbers of HCA and HCC were 0.67±0.33 (IP) and 2.0±0.63 (DW) in female mice. This study showed a tendency for high incidences of liver tumor with long-term exposure of newborn animals by drinking water.

Diethylnitrosamine (DEN) is known as a potential hepatic carcinogen by single administration. This study was designed to measure the effects of DEN-induced cell damage on the triglyceride and cholesterol concentration in the liver, excluding dietary effects. Fertilized chicken eggs, 10 days before hatching, were randomly divided into three groups (n=20) and each egg was injected 10 ul of corn oil (vehicle control), 5 ug of DEN/10 ul and 10 ug of DEN/10 ul into yolk via air sac. After 48 hr and 96 hr incubation, the damage of the chick-embryo liver cell was investigated by electron microscopy and by measuring the concentration of lipid components (total cholesterol, free cholesterol, phospholipid and triglyceride). For eggs administered 10 ug of DEN and incuvated 96 hr, in hepatocyte, the nucleus membrane was roughed, the size of nucleolus was apparently increased and euchromatin was accumulated. Mitochondria were condensed and cristae, located mitochondrial inner membrane, were obscured. Additionally, the levels of triglyceride and cholesterol classes were significantly increased depend on the amount of DEN and incubation time. Especially, triglycerides were notably increased in the group treated with 10 ug DEN at 96 hr, but phospholipids, component of cell membrane, were decreased with significance. As a conclusion, carcinogen induced hepatic lesion was correlated with the changes in lipid component of liver.