Decreased Diethylnitrosamine-induced Liver Preneoplastic Lesions by Estradiol-3-benzoate Treatment

Jin Seok Kang,Ki Dae Park,Byeongwoo Ahn,Beom Seok Han 한국독성학회 2011 Toxicological Research Vol.27 No.4

To clarify whether inhibitory effect of estrogen on liver tumor is associated with cell proliferation, we investigated its role in diethylnitrosamine (DEN)-induced rat preneoplastic lesions, with time sequenced manners. F344 male rats (n = 90) were divided into three groups at 5 weeks of age. The mini-osmotic pumps providing a continuous infusion of DEN was implanted into the abdominal cavity of each animal in group 1, 2 and 3 at 6 weeks of age. To see the effect of estrogen, pellet containing 1 or 10μg of estradiol-3-benzoate (EB) was implanted subcutaneously in the animals of groups 2 or 3, respectively, one week prior to DEN treatment. Ten animals of each group were euthanized at 10, 14 and 18 weeks after DEN treatment. Liver tissues at each time point were fixed in 10% phosphate-buffered formalin and were processed and embedded in paraffin and 5μm sections mounted on a silanized slide. Glutathione S-transferase placental form (GST-P) positive foci and 5-bromo-2-deoxyuridine (BrdU) labeling cells were detected at each time point. Area of GST-P positive foci in DEN+EB 1 or 10μg group was significantly decreased compared to DEN alone at 14 weeks (p < 0.01 or p < 0.05, respectively) an at 18 weeks (p< 0.05 or p< 0.01, respectively). BrdU index in DEN+EB 1 or 10μg groups was significantly decreased compared to DEN alone at 14 weeks and at 18 weeks (p < 0.01). Taken together, we conclude that EB treatment decrease the DEN-induced liver preneoplastic lesions and this may be associated with decrease of cellular proliferation.

Expression of CD44 in mouse diethylnitrosamine (DEN)-induced hepatic tumors as well as in DEN-treated embryo stem cells and derived hepatic lineage cells

Jin Seok Kang,Dae Joong Kim 충북대학교 동물의학연구소 2013 Journal of Biomedical and Translational Research Vol.14 No.4

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.

Decreased Diethylnitrosamine-induced Liver Preneoplastic Lesions by Estradiol-3-benzoate Treatment

Kang, Jin-Seok,Park, Ki-Dae,Ahn, Byeong-Woo,Han, Beom-Seok Korean Society of ToxicologyKorea Environmental Mu 2011 Toxicological Research Vol.27 No.4

To clarify whether inhibitory effect of estrogen on liver tumor is associated with cell proliferation, we investigated its role in diethylnitrosamine (DEN)-induced rat preneoplastic lesions, with time sequenced manners. F344 male rats (n = 90) were divided into three groups at 5 weeks of age. The mini-osmotic pumps providing a continuous infusion of DEN was implanted into the abdominal cavity of each animal in group 1, 2 and 3 at 6 weeks of age. To see the effect of estrogen, pellet containing 1 or 10 ${\mu}g$ of estradiol-3-benzoate (EB) was implanted subcutaneously in the animals of groups 2 or 3, respectively, one week prior to DEN treatment. Ten animals of each group were euthanized at 10, 14 and 18 weeks after DEN treatment. Liver tissues at each time point were fixed in 10% phosphate-buffered formalin and were processed and embedded in paraffin and 5 ${\mu}g$ sections mounted on a silanized slide. Glutathione S-transferase placental form (GST-P) positive foci and 5-bromo-2-deoxyuridine (BrdU) labeling cells were detected at each time point. Area of GST-P positive foci in DEN+EB 1 or 10 ${\mu}g$ group was significantly decreased compared to DEN alone at 14 weeks (p < 0.01 or p < 0.05, respectively) an at 18 weeks (p < 0.05 or p < 0.01, respectively). BrdU index in DEN+EB 1 or 10 ${\mu}g$ groups was significantly decreased compared to DEN alone at 14 weeks and at 18 weeks (p < 0.01). Taken together, we conclude that EB treatment decrease the DEN-induced liver preneoplastic lesions and this may be associated with decrease of cellular proliferation.

Decreased Diethylnitrosamine-induced Liver Preneoplastic Lesions by Estradiol-3-benzoate Treatment

강진석,박기대,안병우,한범석 한국독성학회 2011 Toxicological Research Vol.27 No.4

To clarify whether inhibitory effect of estrogen on liver tumor is associated with cell proliferation, we investigated its role in diethylnitrosamine (DEN)-induced rat preneoplastic lesions, with time sequenced manners. F344 male rats (n = 90) were divided into three groups at 5 weeks of age. The mini-osmotic pumps providing a continuous infusion of DEN was implanted into the abdominal cavity of each animal in group 1, 2 and 3 at 6 weeks of age. To see the effect of estrogen, pellet containing 1 or 10 μg of estradiol-3-benzoate (EB) was implanted subcutaneously in the animals of groups 2 or 3, respectively, one week prior to DEN treatment. Ten animals of each group were euthanized at 10, 14 and 18 weeks after DEN treatment. Liver tissues at each time point were fixed in 10% phosphate-buffered formalin and were processed and embedded in paraffin and 5 μm sections mounted on a silanized slide. Glutathione S-transferase placental form (GST-P) positive foci and 5-bromo-2-deoxyuridine (BrdU) labeling cells were detected at each time point. Area of GST-P positive foci in DEN+EB 1 or 10 μg group was significantly decreased compared to DEN alone at 14 weeks (p < 0.01 or p < 0.05, respectively) an at 18 weeks (p < 0.05 or p < 0.01, respectively). BrdU index in DEN+EB 1 or 10 μg groups was significantly decreased compared to DEN alone at 14 weeks and at 18 weeks (p < 0.01). Taken together, we conclude that EB treatment decrease the DEN-induced liver preneoplastic lesions and this may be associated with decrease of cellular proliferation.

Expression of CD44 in mouse diethylnitrosamine (DEN)-induced hepatic tumors as well as in DENtreated embryo stem cells and derived hepatic lineage cells

강진석,김대중 충북대학교 동물의학연구소 2013 Journal of Biomedical and Translational Research Vol.14 No.4

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.

Expression of epithelial cell adhesion molecule in early phase of hepatocarcinogenesis of mice treated with diethylnitrosamine

Jin Seok Kang 충북대학교 동물의학연구소 2012 Journal of Biomedical and Translational Research Vol.13 No.3

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.

Expression of epithelial cell adhesion molecule in early phase of hepatocarcinogenesis of mice treated with diethylnitrosamine

강진석 충북대학교 동물의학연구소 2012 Journal of Biomedical and Translational Research Vol.13 No.3

The expression of epithelial cell adhesion molecule (EpCAM) was investigated in early phase of hepatocarcinogenesis induced by diethylnitrosamine (DEN). Sixty 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) at 14 days of age, and were sacrificed at 6 h and 1, 2, 3, 7, and 28 days after treatment of saline or DEN. At necropsy, half of the liver from saline- or DEN-treated mice was processed for histopathological examination and immunohistochemical staining of EpCAM and apoptosis. And 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 area, as well as hepatocytes in the livers of DEN-treated mice. And 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 ones at 6 h (P<0.01). Taken together, EpCAM expression and apoptosis were increased in liver by DEN treatment.

N-DEN에 의한 백서의 간섬유화 병변의 동모양혈관의 미세 구조의 변화

허방,김경원,김영옥,윤명희,이충한 고신대학교(의대) 고신대학교 의과대학 학술지 2004 고신대학교 의과대학 학술지 Vol.19 No.1

Background : Hepatic sinusoid is lined by specialized endothelial cells showing numerous tiny fenestrae, and is not supported by an intact basement membrane, which permit direct transportation of fluid and micromolecules between Disse space and sinusoidal vascular lumen. Material & Methods : The purpose of this study is to investigate the ultrastructural changes of hepatic sinusoid during experimentally induced hepatic fibrosis with 12 week-N-diethylnitrosamine (DEN, 200ppm) oral intake. The rat liver tissues were examined under the light microscope, transmission and scanning electron microscope. Results : Comparing with number of sieve plate (10±3) and number of fenestrae (42±15) per a sieve plate in control group, their numbers were reduced in all DEN-treated groups, which were significantly correlated with duration of DEN intake. And also noted were decreased size of fenestrae. Although discontinuous basal lamina around the sinusoidal endothelium and deposition of amorphous material in Disse space were initially noted in 6 week DEN-treated group, intact basal lamina was observed in 12 week DEN-treated group. Ito cells suggest that ultrastructural changes of fenestrae in sinusoidal endothelial cells precede the deposition of extracellular matrix in Disse space, leading to development of basement membrane, and that Ito cells are participated in the capillarization process of hepatic sinusoid. It could be speculated that inhibition of Ito cell proliferation might delay the process of hepatic fibrosis in cirrhosis.

The Possibility of Liver and Lung Carcinogenesis Induced by Diethylnitrosamine in Newborn B6C3F1 Mice

Dae Joong Kim, Tae Myoung Kim, Tae Jin Shim, Kyoung Mo Yang, Jin Seok Kang 충북대학교 동물의학연구소 2011 Journal of Biomedical and Translational Research Vol.12 No.2

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 80th 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.

茵陳五苓散약침이 DEN으로 유발된 흰쥐의 간암에 미치는 영향

류미선,김성필,김재홍,류혜선,윤여충,신정철 대한침구의학회 2011 대한침구의학회지 Vol.28 No.5

Objectives : Inchinohryungsan has been used for treatment of hepatobiliary diseases. This study was designed to investigate the effect of Inchinohryungsan pharmacopuncture on hepatocellular carcinoma in rats. Sprague Dawley(SD) rats of the control and experimental groups received intraperitoneal injection of 50 ㎎/㎏ DEN, weekly for 12 weeks. Methods : Rats were divided into 5 groups. Normal group was not induced hepatocellular carcinoma and not treated. Control group was induced hepatocellular carcinoma and injected with Inchinohryungsan pharmacopuncture into the root of tail. Experimental groups were induced hepatocellular carcinoma. BL group was injected with Inchinohryungsan pharmacopuncture into the BL_(18) and LR_(14), BG group was injected into the BL_(19) and GB_(24) and CSC group was injected into the CV_(12), ST_(25) and CV_4. Thereafter, the changes of the body weight, the liver weight and the weight of liver/100g body weight, WBC, neutrophil, lymphocyte and the activities of AST, ALT, ALP, LDH, AFP and SOD were measured. And gross anatomy, light and electron microscopy were performed. Results : The significant results were as follows,1. The activities of LDH were significantly decreased in CSC group compared with control group. 2. The activities of AFP were significantly decreased in the BL, BG, CSC groups compared with control group. 3. The activities of SOD were increased in the BL, BG, CSC groups compared with control group and CSC group was significantly increased than normal group. 4. According to the gross anatomical observation, the control and BL, BG, CSC groups showed multi-nodular hepatocellular carcinoma. But the size and numbers of the hepatocellular carcinoma in experimental groups were smaller than control group. 5. The numbers of hepatic p53 positive cells were decreased in the BL, BG groups compared with control group. 6. According to the light and electron microscopical observation, the BL, BG and CSC groups were mildly improved than control group in morphological and histopathological changes. Conclusions : These results suggested that Inchinohryungsan pharmacopuncture may have some effects on hepatocellular carcinoma induced by DEN in rats.