DEN-treated Mice Research Articles

Abstract 248: Vitamin D deficiency promotes hepatocellular carcinoma tumor growth in TGF-β impaired mice by Smad3 heterozygous deletion

Abstract Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide. Vitamin D (VD) has been implicated in the prevention of multiple cancers, some of which are characterized by inactivation of TGF-β signaling. Recently, inactivation of TGF-β signaling has been associated with development of HCC. We examined the potential role of VD in HCC chemoprevention in the context of TGF-β inactivation in a mouse model. Methods: Wild type, Sptbn1+/- and Smad3+/- male mice were treated with 50 mg/kg of the chemical carcinogen diethylnitrosamine (DEN) via i.p. injection at 14 days of age. At 8 months, the animals were changed from regular chow (2,200 IU/kg VD) to one of two diets: lower than normal VD (200 IU/kg) or high VD (10,000 IU/kg). At one year of age, the livers were harvested for tissue analysis. Several metrics were assessed including expression levels of 164 proteins from liver tumors by Reverse Phase Protein Array (RPPA). Results: Twenty three mice were treated with lower than normal dose VD while 26 received high dose VD. We did not find significant differences in blood calcium levels in animals on either diet. As expected, high VD intervention after 8 months of DEN injection did not affect the total number of tumors the mice developed; however, the lower than normal VD regimen promoted tumor growth in the context of Smad3 disruption (liver-to-body weight ratio 18.37% in Smad3+/- mice treated with low dose VD vs. 6.25% in Smad3+/- animals treated with high dose, p=0.028). Furthermore, RPPA data revealed that the lower than normal VD intake in DEN-treated Smad3 heterozygous null mice results in repression of tumor suppressing genes such as PDCD4 and concomitant up-regulation of tumor promoting genes like Stat5A, Bcl2-XL, PDGFRB and PEA15. Conclusions: Remarkably, lower than normal VD in the context of Smad3 disruption promotes tumor growth possibly through repression of tumor suppressing genes such as PDCD4 and up-regulation of oncogenes like Stat5A, Bcl2-XL, PDGFRB and PEA15. These results suggest that VD treatment strategies could potentially be significant for chemoprevention and treatment of HCC in the context of inactivation of TGF-β signaling in patient populations with underlying deficiency of VD. Citation Format: Nina M. Muñoz, Lior H. Katz, Keigo Machida, Hidekazu Tsukamoto, Kirty Shetty, Aiwu R. He, Lynt B. Johnson, Asif Rashid, Sang Bae Kim, Ju-Seog Lee, Lopa Mishra. Vitamin D deficiency promotes hepatocellular carcinoma tumor growth in TGF-β impaired mice by Smad3 heterozygous deletion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 248. doi:10.1158/1538-7445.AM2014-248

Abstract 1592: Frequent BrafV637E mutation in hepatocarcinogenesis induced by neonatal treatment with diethylnitrosamine (DEN) in B6C3F1 mice

Abstract The genetic event associated to the initiation process of hepatocarcinogenesis has not been well elucidated. By whole-exome analysis, we identified BrafV637E mutation, which is equivalent to human BrafV600E mutation, in all the four DEN-induced mouse hepatic tumors examined. By Sanger sequencing we validated that the BrafV637E mutation is highly frequent in the hepatic tumors [17/18 (94.4%)] in 1 year-old DEN-treated mice. Using DNA isolated by microscopic dissection we detected the BrafV637E mutation in 14/16 (87.5%) and 7/9 (77.8%) small preneoplastic hepatocyte foci respectively at 5 and 8 months after DEN treatment, indicating that the BrafV637E mutation occurs in the early stage. On the other hand, although the BrafV637E mutation was frequent in hepatic tumors induced by neonatal DEN treatment followed by weekly-administration of CCl4 [5/7 (71.4%)], none of 6 spontaneous and 24 CCl4-induced liver tumors showed the BrafV637E mutation, indicating that the BrafV637E mutation is specific for DEN-induced tumors. Histological analysis revealed that BrafV637E mutation was quite common in the lesions consisted of basophilic cells, while it was much less frequent in the lesions consisted of eosinophilic cells. We then investigated how the BrafV637E mutation influences the properties of preneoplastic/neoplastic hepatocytes. Firstly, the qPCR analysis revealed that p15Ink4b and p19ARF were upregulated in the DEN-induced hepatic tumors as compared to the surrounding non-tumor tissues, indicating that the Braf mutation leads to oncogene induced senescence (OIS), which may be related to the dormancy of preneoplastic lesions in the early stage. Secondly, when investigated the intracellular signaling pathways, Akt phosphorylation was much increased in the hepatic tumors as compared to the surrounding hepatic tissue, while ERK1/2 phosphorylation was not increased, suggesting the possible link between BrafV637E mutation and Akt activation. Thirdly, vemurafenib suppressed proliferation of the BrafV637E-mutated liver tumor cells both in vitro and in vivo, but not the proliferation of the BrafV637E mutation negative cells, indicating that BrafV637E mutation is related to the growth capacity. In summary, our results demonstrated that BrafV637E mutation is an early event in DEN-induced hepatocarcinogenesis, and may be related both to growth suppression via OIS and proliferation through activation of intracellular signaling. (Supported by the grant from the Japanese Ministry of Education, Culture, Sports, Science and Technology) Citation Format: Masahiro Yamamoto, Hiroki Tanaka, Yuji Nishikawa, Keiko Shimizu, Katsuhiro Ogawa. Frequent BrafV637E mutation in hepatocarcinogenesis induced by neonatal treatment with diethylnitrosamine (DEN) in B6C3F1 mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1592. doi:10.1158/1538-7445.AM2014-1592

Maotai Ameliorates Diethylnitrosamine-Initiated Hepatocellular Carcinoma Formation in Mice

Consumption of alcohol is closely related to liver disease, such as hepatic fibrosis or even hepatocellular carcinoma (HCC). However, epidemiological and experimental studies indicated that consumption of Maotai, one of the famous liquors in China, exhibits no significant correlation with hepatic fibrosis or cirrhosis as other beverage sources do. This study detected the relationship of Maotai consumption and HCC development in a diethylnitrosamine (DEN)-initiated HCC animal model. DEN was given to mice at a dose of 100 mg/kg, ip, and 50 mg/kg, ip in the following week. Mice were simultaneously given Maotai or an equal amount of ethanol (53%, 5 ml/kg/day, 5days/week for up to 35weeks). At 3-week and 35- week of the experiment, serum and livers were collected for biochemical and histopathological examination of liver injury and incidence of HCC. Real-time RT-PCR, immunohistochemistry and Western blotting were used to examine the expression of metallothionein-1/2 (MT-1/2), NF-E2-related factor 2 (Nrf2), glutamate-cysteine ligase catalytic subunit (GCLC) and modified subunit (GCLM). We identified tissue damage and dysfunction of liver in ethanol + DEN-treated mice, whereas the extent of injury was reduced in Maotai+ DEN –treated mice. Significant Glypican-3(GPC3) expression and precancerous injury or HCC were seen in approximately 50% of mice with ethanol+ DEN, but barely be seen in Maotai + DEN-treated mice. A higher expression of MT-1/2, Nrf2 and GCLC could be seen in Maotai + DEN-treated mice. Thus, Maotai liquor ameliorates the formation of DEN-induced HCC in mice, and the protection mechanism is possibly related with the activation of anti-oxidation factors, such as MTs, Nrf2 and GCLC.

Plasma Fibroblast Growth Factor 23 Concentration Is Increased and Predicts Mortality in Patients on the Liver-Transplant Waiting List

High plasma fibroblast growth factor-23 (FGF23) concentration predicts the risk of death and poor outcomes in patients with chronic kidney disease or chronic heart failure. We checked if FGF23 concentration could be modified in patients with end stage liver disease (ESLD) and predict mortality. We measured plasma FGF23 in 200 patients with ESLD registered on a liver transplant waiting list between January 2005 and October 2008. We found that median plasma FGF23 concentration was above normal values in 63% of the patients. Increased FGF23 concentration was not explained by its classical determinants: hyperphosphataemia, increased calcitriol concentration or decreased renal function. FGF23 concentration correlated with the MELD score, serum sodium concentration, and GFR. Forty-six patients died before being transplanted and 135 underwent liver transplantation. We analyzed the prognostic value of FGF23 levels. Mortality was significantly associated with FGF23 levels, the MELD score, serum sodium concentration and glomerular filtration rate. On multivariate analyses only FGF23 concentration was associated with mortality. FGF23 levels were independent of the cause of the liver disease. To determine if the damaged liver can produce FGF23 we measured plasma FGF23 concentration and liver FGF23 mRNA expression in control and diethyl-nitrosamine (DEN)-treated mice. FGF23 plasma levels increased with the apparition of liver lesions in DEN-treated mice and that FGF23 mRNA expression, which was undetectable in the liver of control mice, markedly increased with the development of liver lesions. The correlation between FGF23 plasma concentration and FGF23 mRNA expression in DEN-treated mice suggests that FGF23 production by the liver accounts for the increased plasma FGF23 concentration. In conclusion chronic liver lesions can induce expression of FGF23 mRNA leading to increased FGF23 concentration, which is associated with a higher mortality in patients on a liver-transplant waiting list. In these patients FGF23 concentration was the best predictor of mortality.

Hepatocyte nuclear factor 4 alpha deletion promotes diethylnitrosamine-induced hepatocellular carcinoma in rodents

Hepatocyte nuclear factor 4 alpha (HNF4α), the master regulator of hepatocyte differentiation, has been recently shown to inhibit hepatocyte proliferation by way of unknown mechanisms. We investigated the mechanisms of HNF4α-induced inhibition of hepatocyte proliferation using a novel tamoxifen (TAM)-inducible, hepatocyte-specific HNF4α knockdown mouse model. Hepatocyte-specific deletion of HNF4α in adult mice resulted in increased hepatocyte proliferation, with a significant increase in liver-to-body-weight ratio. We determined global gene expression changes using Illumina HiSeq-based RNA sequencing, which revealed that a significant number of up-regulated genes following deletion of HNF4α were associated with cancer pathogenesis, cell cycle control, and cell proliferation. The pathway analysis further revealed that c-Myc-regulated gene expression network was highly activated following HNF4α deletion. To determine whether deletion of HNF4α affects cancer pathogenesis, HNF4α knockdown was induced in mice treated with the known hepatic carcinogen diethylnitrosamine (DEN). Deletion of HNF4α significantly increased the number and size of DEN-induced hepatic tumors. Pathological analysis revealed that tumors in HNF4α-deleted mice were well-differentiated hepatocellular carcinoma (HCC) and mixed HCC-cholangiocarcinoma. Analysis of tumors and surrounding normal liver tissue in DEN-treated HNF4α knockout mice showed significant induction in c-Myc expression. Taken together, deletion of HNF4α in adult hepatocytes results in increased hepatocyte proliferation and promotion of DEN-induced hepatic tumors secondary to aberrant c-Myc activation.

Abstract 181: Hepatic retinoid signaling in mouse hepatocarcinogenesis models.

Abstract Retinoids are known to possess anti-cancer effects and a clinical trial of a synthetic retinoid for hepatocellular carcinoma (HCC) is ongoing recently in Japan. Hepatic retinoid metabolism is usually impaired during development of liver diseases. Loss of retinoid-containing lipid droplets upon hepatic stellate cell (HSC) activation is one of the first events in the development of liver diseases and retinoid stores are progressively lost from HSCs leading to hepatic fibrosis, cirrhosis, and HCC. In the present study, we employed two types of genetically-modified mice for investigating hepatic retinoid metabolism and signaling in diethylnitrosamine (DEN)-induced mouse hepatocarcinogenesis models. One is lecithin:retinol acyltransferas (LRAT) knockout (KO) mouse which lacks HSC lipid droplets and has much less hepatic retinoid stores, and the other is a transgenic mouse which expresses dominant negative form of retinoic acid receptor (dnRAR) to block retinoid signaling in the liver. Male 15 day-old mice were given intraperitoneal injections of DEN (25 mg/kg body weight). Eight-month-old DEN-treated Lrat KO mice showed lower liver tumor incidence and dnRAR transgenic mice showed higher incidence than that of respective control mice. In the acute phase after DEN injection, dnRAR transgenic mice exhibited higher hepatic levels of inflammatory cytokines. Two days after DEN injection, lower serum levels of alanine aminotransferase and decreased hepatic levels of cyclin D1 were observed in Lrat KO mice. Lrat KO mice also exhibited increased levels of retinoic acid-responsive genes, including p21, lower levels of cytochrome P450 enzymes required for DEN-bioactivation, and higher levels of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Our results indicate that Lrat KO mice, instead of almost no hepatic retinoid storage, are less susceptible to DEN-induced hepatocarcinogenesis due to increased hepatic retinoid signaling which is accompanied by higher expression of p21 in the liver. In conclusion, retinoid signaling plays a key role in these hepatocarcinogenesis models. Citation Format: Yohei Shirakami, William S. Blaner, Masahito Shimizu, Hisataka Moriwaki, Mitsuru Seishima. Hepatic retinoid signaling in mouse hepatocarcinogenesis models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 181. doi:10.1158/1538-7445.AM2013-181

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

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 salineor DEN-treated mice was processed for histopathological examination and immunohistochemical staining of EpCAM and apoptosis. And the remaining liver tissue was snapfrozen 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 liversof 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.

Osteopontin is involved in estrogen-mediated protection against diethylnitrosamine-induced liver injury in mice

Diethylnitrosamine (DEN) is a potent hepatotoxin and hepatocarcinogen in animals and possible in humans. Estrogen has been reported to play a protective role against DEN exposure. Osteopontin (OPN), a downstream molecular of estrogen, plays a role in many pathophysiological processes. In this study, we evaluate the role of OPN in estrogen-mediated hepatoprotection in DEN-treated mice. DEN was administrated intraperitoneally to C57BL/6 and OPN−/− mice. Compared to male mice, female mice exhibited significantly higher hepatic OPN expression with less liver damage 48h after DEN treatment. Interestingly, enhanced OPN expression was predominantly detected in hepatocytes after DEN treatment. OPN deficiency enhanced the susceptibility to DEN, which was more apparent in females than males. Estrogen-mediated protection against DEN in males was abrogated by OPN deficiency. The protective activities of estrogen could be mimicked by exogenous OPN. Consistent with liver injury, oxidative stress in liver was enhanced with OPN depletion. OPN reduced DEN-induced oxidative stress likely through inhibition of CYP2A5 expression. In conclusion, we demonstrate that OPN may be involved in estrogen-mediated hepatoprotection in DEN-induced liver injury through enhancement of hepatocyte survival and inhibition of DEN biotransformation. Our findings may provide new insight into gender differences in chemical-induced liver injury and related diseases.

Adaptive immunity suppresses formation and progression of diethylnitrosamine-induced liver cancer

BackgroundHepatocellular carcinoma (HCC) is a typical inflammation-associated cancer, but may also provoke antitumour immune responses whose significance and underlying mechanisms are incompletely understood.ObjectiveTo characterise immune responses in the diethylnitrosamine (DEN)-liver...

Identification of biomarkers of chemically induced hepatocarcinogenesis in rasH2 mice by toxicogenomic analysis

Toxicogenomic approaches have been applied to chemical-induced heptocarcinogenesis rodent models for the identification of biomarkers of early-stage hepatocarcinogenesis and to help clarify the underlying carcinogenic mechanisms in the liver. In this study, we used toxiciogenomic methods to identify candidate biomarker genes associated with hepatocarcinogenesis in rasH2 mice. Blood chemical, histopathologic, and gene expression analyses of the livers of rasH2 mice were performed 7 and 91days after the administration of the genotoxic hepatocarcinogens 2-acetylaminofluorene (AAF) and diethylnitrosoamine (DEN), the genotoxic carcinogen melphalan (Mel), and the nongenotoxic noncarcinogen 1-naphthylisothiocynate (ANIT). Histopathologic lesions and a rise in accompanying serum marker levels were found in the DEN-treated rasH2 mice, whereas no neoplastic lesions were observed in the rasH2 mice. However, biological functional analysis using Ingenuity Pathways Analysis (IPA) software revealed that genes with comparable molecular and cellular functions were similarly deregulated in the AAF- and DEN-treated rasH2 mice. We selected 68 significantly deregulated genes that represented a hepatocarcinogen-specific signature; these genes were commonly deregulated in both the AAF- and DEN-treated rasH2 mice on days 7 and 91. Hierarchical clustering analysis indicated that the expression patterns of the selected genes in the hepatocarcinogen (AAF and DEN) groups were distinctive from the patterns in the control, Mel, and ANIT groups. Biomarker filter analysis using IPA software suggested that 28 of the 68 signature genes represent promising candidate biomarkers of cancer. Quantitative real-time PCR analysis confirmed that the deregulated genes, which exhibited sustained up- and down-regulation up to day 91, are likely involved in early-stage hepatocarcinogenesis. In summary, the common and significant gene expression changes induced by AAF and DEN may reflect early molecular events associated with hepatocarcinogenesis, and these "signature" genes may be useful as biomarkers of hepatocarcinogenesis in mice.

Abstract 5555: Diethylnitrosamine-induced hepatic injury and hepatocarcinogenesis: A role for hepatic retinoids

Abstract The liver is the main tissue site of retinoid storage in the body and the non-parenchymal hepatic stellate cell (HSC) is the major cellular site of retinoid storage within the liver. Loss of retinoid-containing lipid droplets (LDs) from HSCs is one of the first events in the development of hepatic diseases, leading progressively to liver fibrosis, cirrhosis and hepatocellular carcinoma. Although the loss of LDs is a hallmark of HSC activation, the contribution of hepatic retinoid storage to hepatic injury and hepatocarcinogenesis remains unknown. Lecithin:retinol acyltransferase (LRAT) is the sole enzyme responsible for storing hepatic retinoid and LRAT knockout (KO) mice lack liver retinoid stores. Retinol-binding protein (RBP) is the principal transport protein for retinol in the blood. RBP KO mice are unable to mobilize hepatic retinoid stores and have significantly more retinoid storage in the liver compared to wild type (WT) mice. In this study, we investigated the effects of endogenous retinoids on hepatic injury and hepatocarcinogenesis. We induced hepatic injury and HCC using diethylnitrosamine (DEN) in WT, LRAT KO and RBP KO mice. Both acute (for 24 and 48 hours after DEN administration) and long-term (for 8 months after DEN administration) studies were undertaken. After DEN injection of WT, LRAT KO, and RBP KO mice, serum levels of alanine transaminase (ALT) were significantly lower and Cyclin D1 and Ki-67 mRNA expression levels were decreased in the livers of the KOs compared to WT mice. This suggests that LRAT KO and RBP KO mice are less susceptible to DEN-induced liver injury and following compensatory proliferation. Furthermore, 8-month-old DEN-treated LRAT KO (n = 18) and RBP KO (n = 13) mice showed lower HCC incidence, fewer tumors, and smaller tumors than DEN-treated WT mice (n = 24). Five-month-old male LRAT KO and RBP KO mice showed higher expression levels of retinoic acid (RA) responsive genes, such as Cyp26A1 and RARβ, mRNA in their liver compared to WT, indicating that RA signaling is upregulated in the livers of the KOs’ mice. The expression levels of p21 mRNA in LRAT KO and RBP KO mice liver were higher than WT at several time points in both acute and long-term studies. Together these results suggest that increased RA signaling and associated higher p21 expression play important roles in the lower susceptibility of these mouse models to liver injury and hepatocarcinogenesis. We are presently investigating other possibilities that the presence/absence through which endogenous hepatic retinoids might have effects on hepatic diseases, probably by modulating several cancer signaling pathways, regulating apoptosis of hepatocytes and cancer cells, or influencing the tumor-surrounding microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5555. doi:10.1158/1538-7445.AM2011-5555

Inhibitory effect of phytoglycoprotein (24 kDa) on hepatocarcinogenesis in N-nitrosodiethylamine-treated ICR mice

Hepatocellular carcinoma (HCC) is becoming one of the most prominent types of cancer in the world. For a long time in Korea Zanthoxylum piperitum DC (ZPDC) has been used in folk medicine to cure several cancers and inflammation. This study was designed to investigate whether ZPDC glycoprotein protected liver tissues against hepatocarcinogenic compounds such as N-nitrosodiethylamine (DEN). To study the chemopreventive effect of ZPDC glycoprotein on hepatocarcinogenesis, ICR mice were injected intraperitoneally with DEN (50 mg/kg) for four weeks. We evaluated the indicators of liver tissue damage (the activity of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT), and thiobarbituric acid-reactive substances (TBARS)), antioxidative enzymes (activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx)), hepatocarcinogenic indicator (heat shock protein (HSP) 70) and hepatocarcinogenic signals (activity of nuclear factor (NF)-κB, cyclooxygenase (COX)-2, and matrix metalloproteinase (MMP)-9) using biochemical methods and immunoblot analysis. The results obtained from this study revealed that ZPDC glycoprotein (20 mg/kg) decreased the levels of LDH, ALT, and TBARS, whereas the activity of SOD and GPx increased in the DEN-treated ICR mice. With respect to the hepatocarcinogenic indicator and hepatocarcinogenic signals, HSP70, NF-κB, COX-2, and MMP-9 activity decreased. The findings suggested that ZPDC glycoprotein prevented damage to liver tissue caused by DEN in the experimental mouse model.

Helicobacter hepaticus–Induced Liver Tumor Promotion Is Associated with Increased Serum Bile Acid and a Persistent Microbial-Induced Immune Response

Chronic microbial infection influences cancer progression, but the mechanisms that link them remain unclear. Constitutive androstane receptor (CAR) is a nuclear receptor that regulates enzymes involved in endobiotic and xenobiotic metabolism. CAR activation is a mechanism of xenobiotic tumor promotion; however, the effects of chronic microbial infection on tumor promotion have not been studied in the context of CAR function. Here, we report that CAR limits the effects of chronic infection-associated progression of liver cancer. CAR knockout (KO) and wild-type (WT) male mice were treated with or without the tumor initiator diethylnitrosamine (DEN) at 5 weeks of age and then orally inoculated with Helicobacter hepaticus (Hh) or sterile media at 8 weeks of age. At approximately 50 weeks postinoculation, mice were euthanized for histopathologic, microbiological, molecular, and metabolomic analyses. Hh infection induced comparable hepatitis in WT and KO mice with or without DEN that correlated with significant upregulation of Tnfα and toll receptor Tlr2. Notably, DEN-treated Hh-infected KO mice exhibited increased numbers of liver lobes with dysplasia and neoplasia and increased multiplicity of neoplasia, relative to similarly treated WT mice. Enhanced tumor promotion was associated with decreased hepatic expression of P450 enzymes Cyp2b10 and Cyp3a11, increased expression of Camp, and increased serum concentrations of chenodeoxycholic acid. Together, our findings suggest that liver tumor promotion is enhanced by an impaired metabolic detoxification of endobiotics and a persistent microbial-induced immune response.

Effect of the Magnetized Water Supplementation on Lymphocyte DNA Damage in Mice Treated with Diethylnitrosamine

Water gets magnetically charged when it is contacted with a magnet. Although magnetic water products have been pro- moted since the 1930's, they have received very little recognition due to questionable effectiveness. Diethylnitrosamine (DEN) is a widely occurring nitrosamine that is one of the most important environmental carcinogens primarily induc- ing tumors of liver. In this study, the effect of magnetized water supplementation on lymphocyte DNA damage in ICR mice treated with DEN was evaluated using the Comet assay. Mice were divided into 3 groups: control, DEN, and DEN + magnetized water group. Fifteen mice were maintained in each group for the entire experimental period of 6, 12 and 18 weeks. Five mice in each group were sacrificed at 6, 12, and 18th weeks, followed by the Comet assay using the blood obtained from heart puncture of the mice. The level of lymphocyte DNA damage reflected by tail moment and other DNA damage indices of tail DNA (%) or tail length of the magnetized water group were significantly decreased after the 6th, 12th and 18th weeks of supplementation compared with the positive control, the DEN group. The relative DNA damage of the magnetized water groups compared to the DEN control group after 6th, 12th, and 18th weeks of supple- mentation were 42.2%, 40.8%, and 32.9% for DNA in tail, 31.2%, 32.6%, and 21.3% for tail length, and 33.8%, 33.8%, and 24.6% for tail moment, respectively. This is the first report demonstrating that magnetized water may be involved in the lowering effect of the DNA damage in DEN-treated ICR mice. This result suggests that the magnetized water might have minimized the DNA damage by improving the antioxidant status of the mice. However, further studies are needed to characterize the condition of the magnetization and examine the long-term effect of the water product. (Korean J Nutr 2010; 43(6): 570 ~ 577)

Inflammation, HCC and sex: IL-6 in the centre of the triangle

Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production. Naugler WE, Sakurai T, Kim S, Maeda S, Kim K, Elsharkawy AM, Karin M. Hepatocellular carcinoma (HCC), the most common liver cancer, occurs mainly in men. Similar gender disparity is seen in mice given a chemical carcinogen, diethylnitrosamine (DEN). DEN administration caused greater increases in serum interleukin-6 (IL-6) concentration in males than it did in females. Furthermore, ablation of IL-6 abolished the gender differences in hepatocarcinogenesis in mice. DEN exposure promoted production of IL-6 in Kupffer cells (KCs) in a manner dependent on the Toll-like receptor adaptor protein MyD88, ablation of which also protected male mice from DEN-induced hepatocarcinogenesis. Estrogen inhibited secretion of IL-6 from KCs exposed to necrotic hepatocytes and reduced circulating concentrations of IL-6 in DEN-treated male mice. We propose that estrogen-mediated inhibition of IL-6 production by KCs reduces liver cancer risk in females, and these findings may be used to prevent HCC in males. [Abstract reproduced by permission of Science 2007;317:121-124].

Gender Disparity in Liver Cancer Due to Sex Differences in MyD88-Dependent IL-6 Production

Hepatocellular carcinoma (HCC), the most common liver cancer, occurs mainly in men. Similar gender disparity is seen in mice given a chemical carcinogen, diethylnitrosamine (DEN). DEN administration caused greater increases in serum interleukin-6 (IL-6) concentration in males than it did in females. Furthermore, ablation of IL-6 abolished the gender differences in hepatocarcinogenesis in mice. DEN exposure promoted production of IL-6 in Kupffer cells (KCs) in a manner dependent on the Toll-like receptor adaptor protein MyD88, ablation of which also protected male mice from DEN-induced hepatocarcinogenesis. Estrogen inhibited secretion of IL-6 from KCs exposed to necrotic hepatocytes and reduced circulating concentrations of IL-6 in DEN-treated male mice. We propose that estrogen-mediated inhibition of IL-6 production by KCs reduces liver cancer risk in females, and these findings may be used to prevent HCC in males.

Is interleukin-6 a gender-specific risk factor for liver cancer?

Hepatocellular carcinoma (HCC), the most common liver cancer, occurs mainly in men. Similar gender disparity is seen in mice given a chemical carcinogen, diethylnitrosamine (DEN). DEN administration caused greater increases in serum interleukin-6 (IL-6) concentration in males than it did in females. Furthermore, ablation of IL-6 abolished the gender differences in hepatocarcinogenesis in mice. DEN exposure promoted production of IL-6 in Kupffer cells (KCs) in a manner dependent on the Toll-like receptor adaptor protein MyD88, ablation of which also protected male mice from DEN-induced hepatocarcinogenesis. Estrogen inhibited secretion of IL-6 from KCs exposed to necrotic hepatocytes and reduced circulating concentrations of IL-6 in DEN-treated male mice. We propose that estrogen-mediated inhibition of IL-6 production by KCs reduces liver cancer risk in females, and these findings may be used to prevent HCC in males.

Deletion of Bid Impedes Cell Proliferation and Hepatic Carcinogenesis

Mechanisms that control the proliferation capability of the initiated cells during hepatocarcinogenesis are still largely unclear. We investigated the role of a pro-death Bcl-2 family protein, Bid, in liver tumor development using a neonatal diethylnitrosamine model. Diethylnitrosamine was administrated to 15-day-old wild-type and bid-null mice. The development of microfoci at the early stage and of gross tumors at the later stage was compared between the two groups of mice. Both microfoci and gross tumor development were significantly retarded in the bid-null mice, despite reduced cell death as measured by TUNEL staining. Further studies indicated that there were significantly less proliferating cells in diethylnitrosamine-treated bid-null livers. The regulation of cell proliferation by Bid was confirmed in two other systems not involving carcinogenesis. Hepatocyte proliferation following partial hepatectomy and T lymphocyte proliferation following anti-CD3 stimulation were both retarded in bid-null mice. Thus, these studies revealed a previously undisclosed function of Bid in regulating cell proliferation, which can be important to tumor development. Furthermore, the role of Bid in promoting hepatocarcinogenesis is in contrast to its reported role in suppressing myeloid leukemia and thus suggests an organ- and/or etiology-specific role of the Bcl-2 family proteins in regulating oncogenesis.

Transgenic mice expressing hepatitis B virus X protein are more susceptible to carcinogen induced hepatocarcinogenesis

The hepatitis B virus X (HBx) protein is thought to be implicated in the development of human hepatocellular carcinoma (HCC), but its exact function remains controversial. To investigate whether the expression of the HBx gene alone can induce HCC on an inbred C57BL/6 strain that displays a lower spontaneous rate of liver cancer, and to determine if HBx transgenic mice are more susceptible to the effects of hepatocarcinogens, C57-TgN (HBx) X transgenic mice were bred with normal C57BL/6 mice strain. The F1 mice (about 50% HBx positive and 50% HBx negative) were treated with a single dose of diethylnitrosamine (DEN) at 7 days of age, or were untreated. Mice were killed at appropriate time points and were analyzed for histological change in the liver. The expression of HBx protein were examined by using immunohistochemical staining. Glycogen storage foci were examined by using periodic acid-Schiff (PAS) staining. In HBx transgenic mice untreated with DEN, HBx expression and glycogen storage foci were always observed in the liver after 8 weeks, but not obvious histological pathologic changes. Histological examination of liver tissue confirmed that DEN-treated HBx mice developed approximately twice as many focal lesions of basophilic hepatocytes as treated wild-type littermates. Hepatocellular adenomas and carcinomas were also more frequent in DEN-treated HBx-positive than HBx-negative mice. Taken together, our results suggest that HBx gene expression alone is not sufficient for carcinogenesis, but may act as a promoter for malignant transformation.

Expression profiling and identification of novel genes in hepatocellular carcinomas.

Liver cancer is the fifth most common cancer worldwide and unlike certain other cancers, such as colon cancer, a mutational model has not yet been developed. We have performed gene expression profiling of normal and neoplastic livers in C3H/HeJ mice treated with diethylnitrosamine. Using oligonucleotide microarrays, we compared gene expression in liver tumors to three different states of the normal liver: quiescent adult, regenerating adult, and newborn. Although each comparison revealed hundreds of differentially expressed genes, only 22 genes were found to be deregulated in the tumors in all three comparisons. Three of these genes were examined in human hepatocellular carcinomas and were found to be upregulated. As a second method of analysis, we used Representational Difference Analysis (RDA) to clone mRNA fragments differentially expressed in liver tumors versus regenerating livers. We cloned several novel mRNAs that are differentially regulated in murine liver tumors. Here we report the sequence of a novel cDNA whose expression is upregulated in both murine and human hepatocellular carcinomas. Our results suggest that DEN-treated mice provide an excellent model for human hepatocellular carcinomas.