Chronic alcohol intake promotes tumor growth in a diethylnitrosamine-induced hepatocarcinogenesis mouse model through increased Wnt/Beta-catenin signaling

USDA-ARS?s Scientific Manuscript database

Ethanol (EtOH) metabolism is involved in both initiating and promoting mechanisms in hepatocellular carcinoma progression in chronic alcoholics. In this study, we developed a mouse model to test the hypothesis that chronic EtOH consumption promotes tumor growth irrespective of EtOH-related initiati...

Soy protein isolate protects against ethanol mediated tumor progression in diethylnitrosamine treated male mice

USDA-ARS?s Scientific Manuscript database

In this study, DEN-treated male mice were assigned to 3 groups: a 35% high fat ethanol liquid diet (EtOH) with casein as the protein source, the same EtOH liquid diet with soy protein isolate as the sole protein source (EtOH/soy) and a chow group. EtOH feeding continued for 16 wks. As expected, E...

Protective Effect of Boiled and Freeze-dried Mature Silkworm Larval Powder Against Diethylnitrosamine-induced Hepatotoxicity in Mice.

PubMed

Cho, Jae-Min; Kim, Kee-Young; Ji, Sang-Deok; Kim, Eun-Hee

2016-09-01

Hepatocellular carcinoma (HCC) is a representative inflammation-associated cancer and known to be the most frequent tumors. HCC may also induce important pro- and anti-tumor immune reactions. However, the underlying mechanisms are unsatisfactorily identified. We investigated the protective effect of boiled and freeze-dried mature silkworm larval powder (BMSP) on diethylnitrosamine (DEN)-induced hepatotoxicity in mice. Mice were fed with diet containing BMSP (0.1, 1, and 10 g/kg) for two weeks and DEN (100 mg/kg, intraperitoneally) was injected 18 hours before the end of this experiment. Liver toxicity was determined in serum and histopathological examination was assessed in the liver tissues. Infiltration of immune cells and expressions of inflammatory cytokines and chemokines were also examined. Pretreatment with BMSP reduced necrotic and histopathological changes induced by DEN in the liver. Measurement of serum biochemical indicators, the levels of alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase, showed that pretreatment with BMSP also decreased DEN-induced hepatotoxicity. In addition, BMSP inhibited the macrophage and CD31 infiltration in a dose-dependent manner. The expressions of interleukin-1β, IFN-γ and chemokines for T cell activation were decreased in BMSP pretreatment groups. BMSP may have a protective effect against acute liver injury by inhibiting necrosis and inflammatory response in DEN-treated mice.

Validation of a preclinical model of diethylnitrosamine-induced hepatic neoplasia in Yucatan miniature pigs

PubMed Central

Mitchell, Jennifer; Tinkey, Peggy T.; Avritscher, Rony; Van Pelt, Carolyn; Eskandari, Ghazaleh; George, Suraj Konnath; Xiao, Lianchun; Cressman, Erik; Morris, Jeffrey S.; Rashid, Asif; Kaseb, Ahmed O.; Amin, Hesham M.; Uthamanthil, Rajesh

2016-01-01

Objective The purpose of this study was to reduce time to tumor onset in a diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) swine model via partial liver embolization (PLE) and to characterize the model for use in translational research. Methods Eight Yucatan miniature pigs were injected intraperitoneally with either saline (n=2) or DEN (n=6) solution weekly for 12 weeks. Three of the DEN-treated pigs underwent PLE. Animals underwent periodic radiological evaluation, liver biopsy, and blood sampling, and full necropsy was performed at study termination (~29 months). Results All DEN-treated pigs developed hepatic adenoma and HCC. PLE accelerated the time to adenoma development but not to HCC development. Biomarker analysis results showed that IGF1 levels decreased in all DEN-treated pigs, as functional liver capacity decreased with progression of HCC. VEGF and IL-6 levels were positively correlated with disease progression. Immunohistochemical probing of HCC tissues demonstrated the expression of several important survival-promoting proteins. Conclusion To our knowledge, we are the first to demonstrate accelerated development of hepatic neoplasia in Yucatan miniature pigs. Our HCC swine model closely mimics the human condition (i.e., progressive disease stages and expression of relevant molecular markers) and is a viable translational model. PMID:27305144

Metformin inhibits early stage diethylnitrosamine-induced hepatocarcinogenesis in rats

PubMed Central

JO, WOORI; YU, EUN-SIL; CHANG, MINSUN; PARK, HYUN-KYU; CHOI, HYUN-JI; RYU, JAE-EUN; JANG, SUNGWOONG; LEE, HYO-JU; JANG, JA-JUNE; SON, WOO-CHAN

2016-01-01

Antitumor effects of metformin have recently emerged despite its original use for type II diabetes. In the present study, the effects of metformin on the development and recurrence of hepatocellular carcinoma (HCC) were investigated using the diethylnitrosamine (DEN)-induced rat model of HCC. Tumor foci were characterized by gross examination and by histopathological characteristics, including proliferation, hepatic progenitor cell content and the expression of hepatocarcinoma-specific molecular markers. Potential target molecules of metformin were investigated to determine the molecular mechanism underlying the inhibitory effects of metformin on chemically induced liver tumorigenesis. The antitumor effects of metformin were increased by the reduction of surface nodules and decreased the incidence of altered hepatocellular foci, hepatocellular adenoma and carcinoma. Also, decreased expression levels of glutathione S-transferase placental form, proliferating cell nuclear antigen and cytokeratin 8 described the inhibitory effects of metformin on HCC. In the present study, Wistar rats receiving treatment with DEN were administered metformin for 16 weeks. In addition, metformin suppressed liver tumorigenesis via an AMPK-dependent pathway. These results suggested that metformin has promising effects on the early stage of HCC in rats. Therefore, metformin may be used for the prevention of HCC recurrence following primary chemotherapy for HCC and/or for high-risk patients, including chronic hepatitis and cirrhosis. PMID:26548419

Orally administered lycopene attenuates diethylnitrosamine-induced hepatocarcinogenesis in rats by modulating Nrf-2/HO-1 and Akt/mTOR pathways.

PubMed

Sahin, Kazim; Orhan, Cemal; Tuzcu, Mehmet; Sahin, Nurhan; Ali, Shakir; Bahcecioglu, Ibrahim H; Guler, Osman; Ozercan, Ibrahim; Ilhan, Necip; Kucuk, Omer

2014-01-01

Hepatocarcinogenesis is one of the most prevalent and lethal cancers. We studied the mechanisms underlying the inhibition of diethylnitrosamine (DEN)-induced hepatocarcinogenesis by lycopene in rats. Hepatocarcinogenesis was induced by an intraperitoneal injection of DEN followed by promotion with phenobarbital for 24 successive wk. The rats were given lycopene (20 mg/kg body weight) 3 times a week orally for 4 wk prior to initiation, and the treatment was continued for 24 consecutive wk. Lycopene reduced incidence, number, size, and volume of hepatic nodules. Serum alanine transaminase, aspartate aminotransferase, total bilirubin, and malondialdehyde (MDA) considerably increased and hepatic antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase) and glutathione decreased in DEN-treated rats when compared with the control group. Lycopene significantly reversed these biochemical changes and increased the expression of NF-E-2-related factor-2)/heme oxygenase-1, and it decreased NF-κB/cyclooxygenase-2, inhibiting the inflammatory cascade and activating antioxidant signaling (P < 0.05). Lycopene also decreased DEN-induced increases in phosphorylated mammalian target of rapamycin (p-mTOR), phosphorylated p70 ribosomal protein S6 kinase 1, phosphorylated 4E-binding protein 1, and protein kinase B (P < 0.05). Lycopene is an active chemopreventive agent that offers protection against DEN-induced hepatocarcinogenesis by inhibiting NF-κB and mTOR pathways.

Ursolic acid attenuates oxidative stress-mediated hepatocellular carcinoma induction by diethylnitrosamine in male Wistar rats.

PubMed

Gayathri, Renganathan; Priya, D Kalpana Deepa; Gunassekaran, G R; Sakthisekaran, Dhanapal

2009-01-01

Hepatocellular carcinoma is the most common primary cancer of the liver in Asian countries. For more than a decade natural dietary agents including fruits, vegetables and spices have drawn a great deal of attention in the prevention of diseases, preferably cancer. Ursolic acid is a natural triterpenoid widely found in food, medicinal herbs, apple peel and other products it has been extensively studied for its anticancer and antioxidant properties. The purpose of this study was to evaluate the effect of ursolic acid in diethylnitrosamine (DEN) induced and phenobarbital promoted hepatocarcinogenesis in male Wistar rats. Antioxidant status was assessed by alterations in level of lipid peroxides and protein carbonyls. Damage to plasma membranes was assessed by levels of membrane and tissue ATPases. Liver tissue was homogenized and utilized for estimation of lipid peroxides, protein carbonyls and glycoproteins. Anticoagulated blood was utilized for erythrocyte membrane isolation. Oral administration of UA 20 mg/kg bodyweight for 6 weeks decreased the levels of lipid peroxides and protein carbonyls at a significance of p< 0.05. Activities of membrane and tissue ATPases returned to normal after UA administration. Levels of glycoproteins were also restored after treatment. Histopathological observations were recorded. The findings from the above study suggest the effectiveness of UA in reducing the oxidative stress mediated changes in liver of rats. Since UA has been found to be a potent antioxidant, it can be suggested as an excellent chemopreventive agent in overcoming diseases like cancer which are mediated by free radicals.

Perindopril, fosinopril and losartan inhibited the progression of diethylnitrosamine-induced hepatocellular carcinoma in mice via the inactivation of nuclear transcription factor kappa-B.

PubMed

Saber, Sameh; Mahmoud, Amr A A; Goda, Reham; Helal, Noha S; El-Ahwany, Eman; Abdelghany, Rasha H

2018-05-31

Hepatocellular carcinoma (HCC) is a major global health problem. Therapeutic interventions of HCC are still limited because of its complicated molecular pathogenesis. Many reports showed that renin-angiotensin system (RAS) contributes to the development of different types of malignancies. Therefore, the present study aimed to examine the effect of RAS inhibition using perindopril (1 mg/kg), fosinopril (2 mg/kg), or losartan (10 mg/kg) on diethylnitrosamine-induced HCC compared to sorafenib (30 mg/kg). The administration of RAS inhibitors resulted in improved liver function and histologic picture with a reduction in AFP levels. These effects found to be mediated through inactivation of NFкB pathway by the inhibition of NFĸB p65 phosphorylation at the Ser536 residue and inhibition of the phosphorylation-induced degradation of NFĸBia. Consequently, expression levels of cyclin D1 mRNA were significantly lowered. In addition, NFкB-induced TNF-α and TGF-β1 levels were reduced leading to lower levels of MMP-2 and VEGF. We concluded that RAS inhibition either through inhibiting the ACE or the blockade of AT1R has the same therapeutic benefit and that the tissue affinity of the ACEIs has no impact on its anti-tumor activity. These results suggest that ACEIs and ARBs can serve as promising candidates for further clinical trials in the management of HCC. Copyright © 2018 Elsevier B.V. All rights reserved.

Diethylnitrosamine initiation does not alter clofibric acid-induced hepatocarcinogenesis in the rat.

PubMed

Michel, Cecile; Desdouets, Chantal; Slaoui, Mohamed; Isaacs, Kevin Robert; Roberts, Ruth Angela; Boitier, Eric

2007-09-01

Clofibric acid (CLO) is a nongenotoxic hepatocarcinogen in rodents that causes altered hepatocellular foci and/or neoplasms. Initiation by DNA-damaging agents such as diethylnitrosamine (DEN) accelerates focus and tumor appearance and could therefore significantly contribute to shortening of the regulatory 2-year rodent carcinogenicity bioassays. However, it is crucial to evaluate the histological and molecular impact of initiation with DEN on hepatocarcinogenesis promoted by CLO. Male F344 rats were given a single nonnecrogenic injection of DEN (0 or 30 mg/kg) followed by Control diet or CLO (5000 ppm) in diet for up to 20 months. Histopathology and gene expression profiling were performed in liver tumors and surrounding nontumoral liver tissues. The molecular signature of DEN was characterized and its histopathological and immunohistopathological effects on focus and tumor types were also determined. Although foci and tumors appeared earlier in the DEN+CLO-treated group compared to the group treated with CLO alone, DEN had little impact on gene expression in nontumoral tissues since the gene expression profiles were highly similar between Control and DEN-treated rats, and DEN+CLO- and CLO-treated rats. Finally, tumors obtained from DEN+CLO and CLO-treated groups displayed highly correlated gene expression profiles (r>0.83, independently of the time-point). The pathways involved in tumor development revealed by Gene Ontology functional analysis are similar when driven either by spontaneous initiation or by a chemically induced initiation step. Our work described here may contribute to the design optimization of shorter preclinical tests for the evaluation of the nongenotoxic hepatocarcinogenic potential of drugs under development.

Preventive Effect of Geraniol on Diethylnitrosamine-Induced Hepatocarcinogenesis in Rats.

PubMed

Sawada, Shintaro; Okano, Jun-Ichi; Imamoto, Ryu; Yasunaka, Yuki; Abe, Ryo; Koda, Masahiko; Murawaki, Yoshikazu; Isomoto, Hajime

2016-03-01

Geraniol is a plant-derived phytochemical possessing anti-cancer action. The anti-carcinogenic effect of geraniol was investigated in the diethylnitrosamine (DEN)-induced hepatocarcinogenic rat model. Male Wistar rats were intraperitoneally injected with 300 μL of phosphate-buffered saline (PBS) (G1; n = 4) or DEN (100 mg/kg body weight) dissolved in PBS (G2; n = 8) every 2 weeks on experimental weeks 2, 4 and 6. The rats were treated with a low concentration (0.07%) of geraniol (G3; n = 9) and high concentration (0.35%) of geraniol (G4; n = 7) for 12 weeks. To evaluate the effects of geraniol on the DEN-induced hepatocarcinogenesis, we compared the relative liver weight, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels and expression levels of proliferating cell nuclear antigen (PCNA) and glutathione S transferase-P (GST-P) by immunohistochemical analyses among each group. Relative liver weight was significantly higher in G2 than in G1 (P < 0.01). Both serum AST and ALT levels were significantly higher in G2 than in G3 and in G4 (P < 0.05). Serum ALP levels did not show a significant difference among each group. Percentages of both PCNA- and GST-P- positive area were significantly decreased in G3 and in G4 compared to in G2 (P < 0.001, respectively), suggesting anti-hepatocarcinogenic effects of geraniol. Geraniol is a promising compound useful for suppression of hepatocellular carcinoma. The mechanisms of action are required to be clarified in the future intensive study.

Diethylnitrosamine-induced hepatocarcinogenesis is suppressed in lecithin:retinol acyltransferase-deficient mice primarily through retinoid actions immediately after carcinogen administration.

PubMed

Shirakami, Yohei; Gottesman, Max E; Blaner, William S

2012-02-01

Loss of retinoid-containing lipid droplets upon hepatic stellate cell (HSC) activation is one of the first events in the development of liver disease leading to hepatocellular carcinoma. Although retinoid stores are progressively lost from HSCs during the development of hepatic disease, how this affects hepatocarcinogenesis is unclear. To investigate this, we used diethylnitrosamine (DEN) to induce hepatic tumorigenesis in matched wild-type (WT) and lecithin:retinol acyltransferase (LRAT) knockout (KO) mice, which lack stored retinoid and HSC lipid droplets. Male 15-day-old WT or Lrat KO mice were given intraperitoneal injections of DEN (25 mg/kg body wt). Eight months later, Lrat KO mice showed significantly less liver tumor development compared with WT mice, characterized by less liver tumor incidence and smaller tumor size. 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 O(6)-methylguanine-DNA methyltransferase (MGMT), both before and after DEN treatment. Our results indicate that Lrat KO mice are less susceptible to DEN-induced hepatocarcinogenesis due to increased retinoid signaling and higher expression of p21, which is accompanied by altered hepatic levels of DEN-activating enzymes and MGMT in Lrat KO mice also contribute to decreased cancer initiation and suppressed liver tumor development.

DOE Office of Scientific and Technical Information (OSTI.GOV)

MadanKumar, Perumal; NaveenKumar, Perumal; Manikandan, Samidurai

The anti-fibrotic effect of morin was examined in LX-2 cells (culture-activated human hepatic stellate cells) and in diethylnitrosamine induced rat model of liver fibrosis. The in vitro study was designed to determine whether morin affects the survival of cultured LX-2 cells, while the in vivo study was designed to evaluate the antioxidant and anti-fibrotic efficacy of morin on diethylnitrosamine induced liver fibrosis in male albino Wistar rat. The activities of liver function enzymes in serum, liver lipid peroxide levels, activities of serum antioxidant enzymes and liver architecture were monitored to cast light on the antioxidant and hepatoprotective nature of morin.more » To establish the anti-fibrotic effects of morin, the levels of key Wnt signaling molecules which are strongly associated with the signal transduction pathway of HSC activation were measured. Overall, from the in vitro results, it was observed that morin at 50 μM concentration inhibited the proliferation of cultured LX-2 cells, inhibited Wnt signaling and induced G1 cell cycle arrest. The in vivo results further confirmed that morin by downregulating the expressions of GSK-3β, β-catenin and cyclin D1 ameliorated DEN-induced liver fibrosis. Hence morin could be employed as a promising chemopreventive natural supplement for liver fibrosis. - Highlights: • In vivo and in vitro results revealed the active participation of Wnt signaling. • Morin at 50 μM inhibited LX-2 cell proliferation by suppressing Wnt signaling. • Morin exhibited hepatoprotective effects against DEN induced liver fibrosis. • Morin inhibited HSC activation in vivo by downregulating Wnt/β-catenin signaling.« less

Structural and numerical chromosome aberration inducers in liver micronucleus test in rats with partial hepatectomy.

PubMed

Itoh, Satoru; Hattori, Chiharu; Nagata, Mayumi; Sanbuissho, Atsushi

2012-08-30

The liver micronucleus test is an important method to detect pro-mutagens such as active metabolites not reaching bone marrow due to their short lifespan. We have already reported that dosing of the test compound after partial hepatectomy (PH) is essential to detect genotoxicity of numerical chromosome aberration inducers in mice [Mutat. Res. 632 (2007) 89-98]. In naive animals, the proportion of binucleated cells in rats is less than half of that in mice, which suggests a species difference in the response to chromosome aberration inducers. In the present study, we investigated the responses to structural and numerical chromosome aberration inducers in the rat liver micronucleus test. Two structural chromosome aberretion inducers (diethylnitrosamine and 1,2-dimethylhydrazine) and two numerical chromosome aberration inducers (colchicine and carbendazim) were used in the present study. PH was performed a day before or after the dosing of the test compound in 8-week old male F344 rats and hepatocytes were isolated 4 days after the PH. As a result, diethylnitrosamine and 1,2-dimethylhydrazine, structural chromosome aberration inducers, exhibited significant increase in the incidence of micronucleated hepatocyte (MNH) when given either before and after PH. Colchicine and carbendazim, numerical chromosome aberration inducers, did not result in any toxicologically significant increase in MNH frequency when given before PH, while they exhibited MNH induction when given after PH. It is confirmed that dosing after PH is essential in order to detect genotoxicity of numerical chromosome aberration inducers in rats as well as in mice. Regarding the species difference, a different temporal response to colchicine was identified. Colchicine increased the incidence of MNH 4 days after PH in rats, although such induction in mice was observed 8-10 days after PH. Copyright © 2012 Elsevier B.V. All rights reserved.

Panax ginseng exerts antiproliferative effects on rat hepatocarcinogenesis.

PubMed

Kim, Hyemee; Lee, Hae-Jeung; Kim, Dae Joong; Kim, Tae Myoung; Moon, Hyun-Seuk; Choi, Haymie

2013-09-01

It has been proposed that ginseng has chemopreventive effects against several types of cancer in animals and humans. However, the mechanisms underlying the chemopreventive activities of fresh ginseng against hepatocarcinogenesis have not yet been elucidated. Therefore, we hypothesized that these ginseng species may prevent hepatocarcinogenesis but that the chemopreventive mechanisms may differ by species. To determine the chemopreventive and therapeutic potential of 3 different types of fresh ginseng on hepatocarcinogenesis, Sprague-Dawley rats were injected with diethylnitrosamine and fed diets containing 2% Panax japonicus CA Meyer (JN), P. quinquefolius L (QQ), or P. ginseng CA Meyer (GS) for 10 weeks. Glutathione S-transferase P form (GST-P)-positive foci, a stable marker for rat hepatocarcinogenesis, were shown in all carcinogen-injected rats; but only the GS diet significantly reduced the area and number (62% and 68%, respectively; P < .05) of GST-P-positive foci compared with the diethylnitrosamine control group. In addition, the number of proliferating cell nuclear antigen-positive hepatocytes in the GST-P-positive area was significantly decreased in the GS group but not in the JN or QQ groups. Using cDNA microarray analyses to investigate the underlying molecular mechanisms, we observed that the p53 signaling pathway was altered by the GS diet and that the expression of Cyclin D1, Cyclin G1, Cdc2a, and Igf-1, which are involved in the p53 signaling pathway, was downregulated by the GS diet. Our data demonstrate, for the first time, that GS, but not JN or QQ, induces cell cycle arrest in hepatocarcinogenesis. This study suggests that fresh GS has potential chemopreventive effects and may prove to be a therapeutic agent against hepatocarcinogenesis. © 2013.

Dietary supplementation with branched-chain amino acids suppresses diethylnitrosamine-induced liver tumorigenesis in obese and diabetic C57BL/KsJ-db/db mice.

PubMed

Iwasa, Junpei; Shimizu, Masahito; Shiraki, Makoto; Shirakami, Yohei; Sakai, Hiroyasu; Terakura, Yoichi; Takai, Koji; Tsurumi, Hisashi; Tanaka, Takuji; Moriwaki, Hisataka

2010-02-01

Obesity and related metabolic abnormalities, including insulin resistance, are risk factors for hepatocellular carcinoma in non-alcoholic steatohepatitis as well as in chronic viral hepatitis. Branched-chain amino acids (BCAA), which improve insulin resistance, inhibited obesity-related colon carcinogenesis in a rodent model, and also reduced the incidence of hepatocellular carcinoma in obese patients with liver cirrhosis. In the present study, we determined the effects of BCAA on the development of diethylnitrosamine (DEN)-induced liver tumorigenesis in obese C57BL/KsJ-db/db (db/db) mice with diabetes mellitus. Male db/db mice were given tap water containing 40 ppm DEN for an initial 2 weeks and thereafter they received a basal diet containing 3.0% of BCAA or casein, which served as a nitrogen content-matched control of BCAA, throughout the experiment. Supplementation with BCAA significantly reduced the total number of foci of cellular alteration, a premalignant lesion of the liver, and the expression of insulin-like growth factor (IGF)-1, IGF-2, and IGF-1 receptor in the liver when compared to the casein supplementation. BCAA supplementation for 34 weeks also significantly inhibited both the development of hepatocellular neoplasms and the proliferation of hepatocytes in comparison to the basal diet or casein-fed groups. Supplementation with BCAA improved liver steatosis and fibrosis and inhibited the expression of alpha-smooth muscle actin in the DEN-treated db/db mice. The serum levels of glucose and leptin decreased by dietary BCAA, whereas the value of the quantitative insulin sensitivity check index increased by this agent, indicating the improvement of insulin resistance and hyperleptinemia. In conclusion, oral BCAA supplementation improves insulin resistance and prevents the development of liver tumorigenesis in obese and diabetic mice.

Potential chemoprevention of diethylnitrosamine-induced hepatocarcinogenesis in rats: myrrh (Commiphora molmol) vs. turmeric (Curcuma longa).

PubMed

El-Shahat, Mohamed; El-Abd, Sabah; Alkafafy, Mohamed; El-Khatib, Gamal

2012-09-01

The aim of the present study was to assess the potential chemopreventive effects of myrrh (Commiphora molmol) vs. turmeric (Curcuma longa) in hepatocarcinogenic rats induced by a single intraperitoneal injection of diethylnitrosamine (DENA) (200 mg/kg body weight). Ninety male Wistar rats used in this study were randomly divided into six equal groups (n=15). Group 1 rats served as negative controls; group 2 received a single i.p. injection of DENA and served as positive controls. Rats in both groups were fed on basal diet. Group 3 rats were fed a diet containing 5% turmeric, whereas group 4 rats were fed a diet containing 2% myrrh. Rats in groups 5 and 6 received a single i.p. injection of DENA and were fed diets containing 5% turmeric and 2% myrrh, respectively. The study demonstrated that DENA caused a significant increase in serum indices of liver enzymes and also severe histological and immunohistochemical changes in hepatic tissues. These included disorganized hepatic parenchyma, appearance of pseudoacinar and trabecular arrays of hepatocytes and alterations in CD10-immunoreactivity. Dietary supplementation of turmeric relatively improved the biochemical parameters to values approximating those of the negative controls and delayed the initiation of carcinogenesis. In contrast, myrrh did not improve the biochemical parameters or delay the hepatocarcinogenesis. Both turmeric and myrrh induced significant biochemical and histological changes in non-treated rats. In conclusion, DENA significantly changes the biological enzymatic activities in serum and the integrity of hepatic tissues. Phytochemicals with potential hepatoprotective effects must be applied cautiously owing to their potential hepatotoxicity. Copyright © 2011 Elsevier GmbH. All rights reserved.

Dietary fructose enhances the incidence of precancerous hepatocytes induced by administration of diethylnitrosamine in rat

PubMed Central

2013-01-01

Background Nonalcoholic fatty liver disease (NAFLD) is a risk for hepatocellular carcinoma (HCC), but the association between a high-fructose diet and HCC is not fully understood. In this study, we investigated whether a high-fructose diet affects hepatocarcinogenesis induced by administration of diethylnitrosamine (DEN). Methods Seven-week-old male Sprague–Dawley rats were fed standard chow (controls), a high-fat diet (54% fat), or a high-fructose diet (66% fructose) for 8 weeks. All rats were given DEN at 50 μg/L in drinking water during the same period. Precancerous hepatocytes were detected by immunostaining of the placental form of glutathione-S-transferase (GST-P). The number of GST-P-positive hepatocytes was assessed in liver specimens. Results Serum levels of total cholesterol were similar among the three groups, but serum triglyceride, fasting blood glucose, and insulin levels were higher in the high-fructose group compared to the high-fat group. In contrast, hepatic steatosis was more severe in the high-fat group compared with the high-fructose and control groups, but the incidence of GST-P-positive specimens was significantly higher in the high-fructose group compared to the other two groups. The average number of GST-P-positive hepatocytes in GST-P positive specimens in the high-fructose group was also higher than those in the other two groups. This high prevalence of GST-P-positive hepatocytes was accompanied by higher levels of 8-hydroxydeoxyguanosine in serum and liver tissue. Conclusions These results indicate that dietary fructose, rather than dietary fat, increases the incidence of precancerous hepatocytes induced by administration of DEN via insulin resistance and oxidative stress in rat. Thus, excessive fructose intake may be a potential risk factor for hepatocarcinogenesis. PMID:24321741

Cell expression patterns of CD147 in N-diethylnitrosamine/phenobarbital-induced mouse hepatocellular carcinoma.

PubMed

Lu, Meng; Wu, Jiao; He, Feng; Wang, Xi-Long; Li, Can; Chen, Zhi-Nan; Bian, Huijie

2015-02-01

Overexpression of CD147/basigin in hepatic cells promotes the progression of hepatocellular carcinoma (HCC). Whether CD147 also expressed in liver non-parenchymal cells and associated with HCC development was unknown. The aim of the study was to explore time-dependent cell expression patterns of CD147 in a widely accepted N-diethylnitrosamine/phenobarbital (DEN/PB)-induced HCC mouse model. Liver samples collected at month 1-12 of post-DEN/PB administration were assessed the localization of CD147 in hepatocytes, endothelial cells, hepatic stellate cells, and macrophages. Immunohistochemistry analysis showed that CD147 was upregulated in liver tumors during month 1-8 of DEN/PB induction. Expression of CD147 was positively correlated with cytokeratin 18, a hepatocyte marker (r = 0.7857, P = 0.0279), CD31 (r = 0.9048, P = 0.0046), an endothelial cell marker, and CD68, a macrophage marker (r = 0.7619, P = 0.0368). A significant correlation was also observed between CD147 and alpha-smooth muscle actin (r = 0.8857, P = 0.0333) at DEN/PB initiation and early stage of tumor formation. Immunofluorescence and fluorescence in situ hybridization showed that CD147 co-expressed with cytokeratin 18, CD31, alpha-smooth muscle actin, and CD68. Moreover, there existed positive correlations between CD147 and microvessel density (r = 0.7857, P = 0.0279), CD147 and Ki-67 (r = 0.9341, P = 0.0022) in the development of DEN/PB-induced HCC. In conclusion, our results demonstrated that CD147 was upregulated in the liver parenchymal and mesenchymal cells and involved in angiogenesis and tumor cell proliferation in the development of DEN/PB-induced HCC.

Aberrant methylation accounts for cell adhesion-related gene silencing during 3-methylcholanthrene and diethylnitrosamine induced multistep rat lung carcinogenesis associated with overexpression of DNA methyltransferases 1 and 3a

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Wenbin; Cui Zhihong; Ao Lin

To evaluate the significance of alterations in cell adhesion-related genes methylation during lung multistep carcinogenesis induced by the genotoxic carcinogens 3-methylcholanthrene (MCA) and diethylnitrosamine (DEN), tissue samples microdissected from MCA/DEN-induced rat lung carcinogenesis model were subjected to methylation-specific PCR to evaluate the DNA methylation status of CADM1, TIMP3, E-cadherin and N-cadherin. Immunohistochemistry was used to determine protein expression of CADM1, TIMP3, N-cadherin and the DNA methyltransferases (DNMTs) 1, 3a and 3b. E-cadherin hypermethylation was not detected in any tissue. CADM1, TIMP3 and N-cadherin hypermethylation was correlated with the loss of their protein expression during the progression of pathologic lesions. Themore » prevalence of DNA methylation of at least one gene and the average number of methylated genes increased with the histological progression. DNMT1 and DNMT3a protein expression increased progressively during the stages of lung carcinogenesis, whereas DNMT3b overexpression was only found in several samples. Furthermore, DNMT1 protein expression levels were correlated with CADM1 methylation, and DNMT3a protein expression levels were correlated with CADM1, TIMP3 and N-cadherin methylation. The average number of methylated genes during carcinogenesis was significantly correlated with DNMT1 and DNMT3a protein expression levels. Moreover, mRNA expression of CADM1 significantly increased after treatment with DNMT inhibitor 5-aza-2'-deoxycytidine in CADM1-methylated primary tumor cell lines. Our findings suggest that an accumulation of hypermethylation accounts for cell adhesion-related gene silencing is associated with dynamic changes in the progression of MCA/DEN-induced rat lung carcinogenesis. We suggest that DNMT1 and DNMT3a protein overexpression may be responsible for this aberrant DNA methylation.« less

[Influence of bear bile on rat hepatocarcinoma induced by diethylnitrosamine].

PubMed

Zhou, Jian-Yin; Yin, Zhen-Yu; Wang, Sheng-Yu; Yan, Jiang-Hua; Zhao, Yi-Lin; Wu, Duan; Liu, Zheng-Jin; Zhang, Sheng; Wang, Xiao-Min

2012-11-01

To investigate the influence of bear bile on rat hepatocarcinoma induced by diethylnitrosamine (DEN), a total of 40 rats were randomly divided into 4 groups: normal control group, model group, and two bear bile treatment groups. The rat liver cancer model was induced by breeding with water containing 100 mg x L(-1) DEN for 14 weeks. The rats of the bear bile groups received bear bile powder (200 or 400 mg x kg(-1)) orally 5 times per week for 18 weeks. The general condition and the body weight of rats were examined every day. After 18 weeks the activities of serum alanine transaminase (ALT), aspartate transaminase (AST) and total bilirubin (TBIL) were detected. Meanwhile, the pathological changes of liver tissues were observed after H&E staining. The expression of proliferative cell nuclear antigen (PCNA) and a-smooth muscle actin (alpha-SMA) in liver tissue were detected by immunohistochemical method. After 4 weeks the body weights of rats in normal group were significantly more than that in other groups (P < 0.05); and that in the two bile groups was significantly more than that in the model group. Compared with normal group, the level of serum glutamic-pyruvic transaminase and total bilirubin increased significantly in other groups; compared with model group, these two indexes decreased significantly in two bile groups. Hepatocellular carcinoma occurred in all rats except for normal group; there were classic cirrhosis and cancer in model group while there were mild cirrhosis and high differentiation in two bile groups. There were almost no expressions of PCNA and alpha-SMA in normal group while there were high expressions in model group; the two bile groups had some expressions but were inferior to the model group, and alpha-SMA reduced markedly. It indicated that bear bile restrained the development of liver cancer during DEN inducing rat hepatocarcinoma, which may be related to its depressing hepatic stellate cell activation and relieving hepatic lesion and cirrhosis.

Epithelial Transforming Growth Factor-β Signaling Does Not Contribute to Liver Fibrosis but Protects Mice From Cholangiocarcinoma.

PubMed

Mu, Xueru; Pradere, Jean-Philippe; Affò, Silvia; Dapito, Dianne H; Friedman, Richard; Lefkovitch, Jay H; Schwabe, Robert F

2016-03-01

Transforming growth factor-β (TGFβ) exerts key functions in fibrogenic cells, promoting fibrosis development in the liver and other organs. In contrast, the functions of TGFβ in liver epithelial cells are not well understood, despite their high level of responsiveness to TGFβ. We sought to determine the contribution of epithelial TGFβ signaling to hepatic fibrogenesis and carcinogenesis. TGFβ signaling in liver epithelial cells was inhibited by albumin-Cre-, K19-CreERT-, Prom1-CreERT2-, or AAV8-TBG-Cre-mediated deletion of the floxed TGFβ receptor II gene (Tgfbr2). Liver fibrosis was induced by carbon tetrachloride, bile duct ligation, or disruption of the multidrug-resistance transporter 2 gene (Mdr2). Hepatocarcinogenesis was induced by diethylnitrosamine or hepatic deletion of PTEN. Deletion of Tgfbr2 from liver epithelial cells did not alter liver injury, toxin-induced or biliary fibrosis, or diethylnitrosamine-induced hepatocarcinogenesis. In contrast, epithelial deletion of Tgfbr2 promoted tumorigenesis and reduced survival of mice with concomitant hepatic deletion of Pten, accompanied by an increase in tumor number and a shift from hepatocellular carcinoma to cholangiocarcinoma. Surprisingly, both hepatocyte- and cholangiocyte-specific deletion of Pten and Tgfbr2 promoted the development of cholangiocarcinoma, but with different latencies. The prolonged latency and the presence of hepatocyte-derived cholangiocytes after AAV8-TBG-Cre-mediated deletion of Tgfbr2 and Pten indicated that cholangiocarcinoma might arise from hepatocyte-derived cholangiocytes in this model. Pten deletion resulted in up-regulation of Tgfbr2, and deletion of Tgfbr2 increased cholangiocyte but not hepatocyte proliferation, indicating that the main function of epithelial TGFBR2 is to restrict cholangiocyte proliferation. Epithelial TGFβ signaling does not contribute to the development of liver fibrosis or formation of hepatocellular carcinomas in mice, but restricts cholangiocyte proliferation to prevent cholangiocarcinoma development, regardless of its cellular origin. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.

Development of a novel mouse model of hepatocellular carcinoma with nonalcoholic steatohepatitis using a high-fat, choline-deficient diet and intraperitoneal injection of diethylnitrosamine.

PubMed

Kishida, Norihiro; Matsuda, Sachiko; Itano, Osamu; Shinoda, Masahiro; Kitago, Minoru; Yagi, Hiroshi; Abe, Yuta; Hibi, Taizo; Masugi, Yohei; Aiura, Koichi; Sakamoto, Michiie; Kitagawa, Yuko

2016-06-13

The incidence of hepatocellular carcinoma with nonalcoholic steatohepatitis is increasing, and its clinicopathological features are well established. Several animal models of nonalcoholic steatohepatitis have been developed to facilitate its study; however, few fully recapitulate all its clinical features, which include insulin resistance, inflammation, fibrosis, and carcinogenesis. Moreover, these models require a relatively long time to produce hepatocellular carcinoma reliably. The aim of this study was to develop a mouse model of hepatocellular carcinoma with nonalcoholic steatohepatitis that develops quickly and reflects all clinically relevant features. Three-week-old C57BL/6J male mice were fed either a standard diet (MF) or a choline-deficient, high-fat diet (HFCD). The mice in the MF + diethylnitrosamine (DEN) and HFCD + DEN groups received a one-time intraperitoneal injection of DEN at the start of the respective feeding protocols. The mice in the HFCD and HFCD + DEN groups developed obesity early in the experiment and insulin resistance after 12 weeks. Triglyceride levels peaked at 8 weeks for all four groups and decreased thereafter. Alanine aminotransferase levels increased every 4 weeks, with the HFCD and HFCD + DEN groups showing remarkably high levels; the HFCD + DEN group presented the highest incidence of nonalcoholic steatohepatitis. The levels of fibrosis and steatosis varied, but they tended to increase every 4 weeks in the HFCD and HFCD + DEN groups. Computed tomography scans indicated that all the HFCD + DEN mice developed hepatic tumors from 20 weeks, some of which were glutamine synthetase-positive. The nonalcoholic steatohepatitis-hepatocellular carcinoma model we describe here is simple to establish, results in rapid tumor formation, and recapitulates most of the key features of nonalcoholic steatohepatitis. It could therefore facilitate further studies of the development, oncogenic potential, diagnosis, and treatment of this condition.

Dietary Broccoli Lessens Development of Fatty Liver and Liver Cancer in Mice Given Diethylnitrosamine and Fed a Western or Control Diet123

PubMed Central

Chen, Yung-Ju; Wallig, Matthew A; Jeffery, Elizabeth H

2016-01-01

Background: The high-fat and high-sugar Westernized diet that is popular worldwide is associated with increased body fat accumulation, which has been related to the development of nonalcoholic fatty liver disease (NAFLD). Without treatment, NAFLD may progress to hepatocellular carcinoma (HCC), a cancer with a high mortality rate. The consumption of broccoli in the United States has greatly increased in the last 2 decades. Epidemiologic studies show that incorporating brassica vegetables into the daily diet lowers the risk of several cancers, although, to our knowledge, this is the first study to evaluate HCC prevention through dietary broccoli. Objective: We aimed to determine the impact of dietary broccoli on hepatic lipid metabolism and the progression of NAFLD to HCC. Our hypothesis was that broccoli decreases both hepatic lipidosis and the development of HCC in a mouse model of Western diet–enhanced liver cancer. Methods: Adult 5-wk-old male B6C3F1 mice received a control diet (AIN-93M) or a Western diet (high in lard and sucrose, 19% and 31%, wt:wt, respectively), with or without freeze-dried broccoli (10%, wt:wt). Starting the following week, mice were treated once per week with diethylnitrosamine (DEN; 45 mg/kg body weight intraperitoneally at ages 6, 7, 8, 10, 11, and 12 wk). Hepatic gene expression, lipidosis, and tumor outcomes were analyzed 6 mo later, when mice were 9 mo old. Results: Mice receiving broccoli exhibited lower hepatic triglycerides (P < 0.001) and NAFLD scores (P < 0.0001), decreased plasma alanine aminotransferase (P < 0.0001), suppressed activation of hepatic CD68+ macrophages (P < 0.0001), and slowed initiation and progression of hepatic neoplasm. Hepatic Cd36 was downregulated by broccoli feeding (P = 0.006), whereas microsomal triglyceride transfer protein was upregulated (P = 0.045), supporting the finding that dietary broccoli decreased hepatic triglycerides. Conclusion: Long-term consumption of whole broccoli countered both NAFLD development enhanced by a Western diet and hepatic tumorigenesis induced by DEN in male B6C3F1 mice. PMID:26865652

Dietary Broccoli Lessens Development of Fatty Liver and Liver Cancer in Mice Given Diethylnitrosamine and Fed a Western or Control Diet.

PubMed

Chen, Yung-Ju; Wallig, Matthew A; Jeffery, Elizabeth H

2016-03-01

The high-fat and high-sugar Westernized diet that is popular worldwide is associated with increased body fat accumulation, which has been related to the development of nonalcoholic fatty liver disease (NAFLD). Without treatment, NAFLD may progress to hepatocellular carcinoma (HCC), a cancer with a high mortality rate. The consumption of broccoli in the United States has greatly increased in the last 2 decades. Epidemiologic studies show that incorporating brassica vegetables into the daily diet lowers the risk of several cancers, although, to our knowledge, this is the first study to evaluate HCC prevention through dietary broccoli. We aimed to determine the impact of dietary broccoli on hepatic lipid metabolism and the progression of NAFLD to HCC. Our hypothesis was that broccoli decreases both hepatic lipidosis and the development of HCC in a mouse model of Western diet-enhanced liver cancer. Adult 5-wk-old male B6C3F1 mice received a control diet (AIN-93M) or a Western diet (high in lard and sucrose, 19% and 31%, wt:wt, respectively), with or without freeze-dried broccoli (10%, wt:wt). Starting the following week, mice were treated once per week with diethylnitrosamine (DEN; 45 mg/kg body weight intraperitoneally at ages 6, 7, 8, 10, 11, and 12 wk). Hepatic gene expression, lipidosis, and tumor outcomes were analyzed 6 mo later, when mice were 9 mo old. Mice receiving broccoli exhibited lower hepatic triglycerides (P < 0.001) and NAFLD scores (P < 0.0001), decreased plasma alanine aminotransferase (P < 0.0001), suppressed activation of hepatic CD68(+) macrophages (P < 0.0001), and slowed initiation and progression of hepatic neoplasm. Hepatic Cd36 was downregulated by broccoli feeding (P = 0.006), whereas microsomal triglyceride transfer protein was upregulated (P = 0.045), supporting the finding that dietary broccoli decreased hepatic triglycerides. Long-term consumption of whole broccoli countered both NAFLD development enhanced by a Western diet and hepatic tumorigenesis induced by DEN in male B6C3F1 mice. © 2016 American Society for Nutrition.

6,7-dimethoxy-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid attenuates heptatocellular carcinoma in rats with NMR-based metabolic perturbations.

PubMed

Kumar, Pranesh; Singh, Ashok K; Raj, Vinit; Rai, Amit; Maity, Siddhartha; Rawat, Atul; Kumar, Umesh; Kumar, Dinesh; Prakash, Anand; Guleria, Anupam; Saha, Sudipta

2017-08-01

6,7-dimethoxy-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid (M1) was synthesized and evaluated for in-vivo antiproliferative action in diethylnitrosamine-induced hepatocarcinogenic rats. The antiproliferative effect of M1 was assessed by various biochemical parameters, histopathology of liver and HPLC analysis. Proton nuclear magnetic resonance-based serum metabolic study was implemented on rat sera to explore the effects of M1 on hepatocellular carcinoma-induced metabolic alterations. M1 showed protective action on liver and restored the arrangement of liver tissues in normal proportion. HPLC analysis displayed a good plasma drug concentration after its oral administration. Score plots of partial least squares discriminate analysis models exhibited that M1 therapy ameliorated hepatocellular carcinoma-induced metabolic alterations which signified its antiproliferative potential. M1 manifested notable antiproliferative profile, and warrants further investigation for future anticancer therapy.

[Sodium glutamate on some physiological features and chemically induced hepatocarcinogenesis in neontal period in male mice].

PubMed

Kaledin, V I; Il'nitskaia, S I; Kuznetsova, E G; Amstislavskaia, T G

2005-05-01

A single injection of diethylnitrosamine 50 mg/kg to 12-day old CBA mice led to development of 50.7 +/- 4.8 liver tumor nodules in males and 3.6 +/- 0.8 nodules in females. Only 19.0 +/- 3.6 tumor nodules developed in the liver of males who, prior to the carcinogen, received 5 intraperitoneal injections of monosodium glutamate (2-4 mg/g on alternate days from 1st to 9th days after birth). The glutamate-treated animals' body size diminished, as well as their weights of testes and seminal vesicles and blood testosterone concentration but, as a rule, quantity of body fat increased. The data obtained indicate that neonatal administration of monosodium glutamate to mice leads to disturbance of functional activity of sex steroids and presumably other hormones taking part in regulation of metabolism of body fat and energy.

Evidence for a Role of the Transcriptional Regulator Maid in Tumorigenesis and Aging

PubMed Central

Fujisawa, Koichi; Terai, Shuji; Matsumoto, Toshihiko; Takami, Taro; Yamamoto, Naoki; Nishina, Hiroshi; Furutani-Seiki, Makoto; Sakaida, Isao

2015-01-01

Maid is a helix-loop-helix protein that is involved in cell proliferation. In order to further elucidate its physiological functions, we studied Maid activity in two small fish model systems. We found that Maid expression was greatest in zebrafish liver and that it increased following partial hepatectomy. Maid levels were also high in hepatic preneoplastic foci induced by treatment of zebrafish with diethylnitrosamine (DEN), but low in hepatocellular carcinomas (HCC), mixed tumors, and cholangiocarcinomas developing in these animals. In DEN-treated transgenic medaka overexpressing Maid, hepatic BrdU uptake and proliferation were reduced. After successive breedings, Maid transgenic medaka exhibited decreased movement and a higher incidence of abnormal spine curvature, possibly due to the senescence of spinal cord cells. Taken together, our results suggest that Maid levels can influence the progression of liver cancer. In conclusion, we found that Maid is important regulator of hepatocarconogenesis and aging. PMID:26107180

Chemoprevention of Diethylnitrosamine-Initiated and Phenobarbital-Promoted Hepatocarcinogenesis in Rats by Sulfated Polysaccharides and Aqueous Extract of Ulva lactuca.

PubMed

Hussein, Usama K; Mahmoud, Hamada M; Farrag, Asmaa G; Bishayee, Anupam

2015-11-01

Hepatocellular carcinoma (HCC) is one of the common cancers and lethal diseases worldwide. Both oxidative stress and chronic inflammation contribute to the pathogenesis of HCC. Because of limited treatment options and a grave prognosis of HCC, preventive management has been emphasized. The marine macroalgae Ulva lactuca (Ulvaceae) is consumed by humans and livestock because of its nutritional value. Recent studies showed that various extracts of U. lactuca possess antiviral, antiplasmodial, antinephrotoxic, antioxidant, and anti-inflammatory properties. However, very limited information is available on anticancer potential of U. lactuca with no reports on liver cancer chemopreventive efficacy of this marine algae. Accordingly, the present study was initiated to evaluate the possible antihepatocarcinogenic effects and antioxidant mechanisms of action of various U. lactuca extracts against a clinically relevant rodent model of HCC. Initiation of hepatocarcinogenesis was performed in Sprague-Dawley rats by a single injection of dietary carcinogen diethylnitrosamine (DENA, 200 mg/kg, intraperitoneally), followed by promotion with phenobarbital (0.05%) in drinking water. The rats were fed with daily oral dose (50 mg/kg) of polysaccharide sulfate or aqueous extract of U. lactuca for 2, 12, and 24 weeks. At these timepoints, blood samples were taken to measure hepatic injury markers, including alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, γ-glutamyl transferase, and bilirubin. The liver tissue was harvested for measurement of hepatic oxidative indices, including lipid peroxidation, reduced glutathione, nitric oxide, catalase, superoxide dismutase, glutathione reductase, and glutathione S-transferase. Hepatic histopathology, immunohistochemical analysis of cell proliferation and apoptosis by DNA fragmentation assay were performed. Our results clearly indicate that sulfated polysaccharides of U. lactuca exert a marked chemoprevention of DENA-initiated hepatocarcinogenesis through inhibition of abnormal cell proliferation and induction of apoptosis. A modest inhibition rat liver carcinogenesis was observed with the aqueous extract. The sulfated polysaccharides altered serum parameters of hepatic damage and modulated various components of the hepatic enzymatic and nonenzymatic antioxidant defense systems. The sulfated polysaccharides from U. lactuca may have unique properties of providing protection against DENA-induced oxidative stress which could contribute to chemoprevention of experimental hepatocarcinogenesis. U. lactuca sulfated polysaccharides could be developed as chemopreventive and therapeutic drug against human HCC. © The Author(s) 2015.

Microbiota transplantation reveals beneficial impact of berberine on hepatotoxicity by improving gut homeostasis.

PubMed

Qin, Chenjie; Zhang, Huilu; Zhao, Linghao; Zeng, Min; Huang, Weijian; Fu, Gongbo; Zhou, Weiping; Wang, Hongyang; Yan, Hexin

2017-11-29

Berberine has been shown to reduce acute liver injury although the underlying mechanism is not fully understood. Because of the anatomic connection, the liver is constantly exposed to gut-derived bacterial products and metabolites. In this study, we showed that berberine has beneficial effects on both hepatotoxicity and intestinal damage in a rat model of chronic or acute liver injury. Microbiota transplantation from the rats with chronic hepatotoxicity could aggravate acute hepatotoxicity in mice treated with diethylnitrosamine (DEN). In rat models with gut homeostasis disruption induced by penicillin or dextran sulfate sodium (DSS), their fecal microbiota could also cause an enhanced hepatotoxicity of recipient mice. When treated with berberine, the DSS-induced enteric dysbacteriosis could be mitigated and their fecal bacteria were able to reduce acute hepatotoxicity in recipient mice. This study indicates that berberine could improve intestinal dysbacteriosis, which reduces the hepatotoxicity caused by pathological or pharmacological intervention. Fecal microbiota transplantation might be a useful method to directly explore homeostatic alteration in gut microbiota.

6,7-dimethoxy-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid attenuates heptatocellular carcinoma in rats with NMR-based metabolic perturbations

PubMed Central

Kumar, Pranesh; Singh, Ashok K; Raj, Vinit; Rai, Amit; Maity, Siddhartha; Rawat, Atul; Kumar, Umesh; Kumar, Dinesh; Prakash, Anand; Guleria, Anupam; Saha, Sudipta

2017-01-01

Aim: 6,7-dimethoxy-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid (M1) was synthesized and evaluated for in-vivo antiproliferative action in diethylnitrosamine-induced hepatocarcinogenic rats. Materials & methods: The antiproliferative effect of M1 was assessed by various biochemical parameters, histopathology of liver and HPLC analysis. Proton nuclear magnetic resonance-based serum metabolic study was implemented on rat sera to explore the effects of M1 on hepatocellular carcinoma-induced metabolic alterations. Results: M1 showed protective action on liver and restored the arrangement of liver tissues in normal proportion. HPLC analysis displayed a good plasma drug concentration after its oral administration. Score plots of partial least squares discriminate analysis models exhibited that M1 therapy ameliorated hepatocellular carcinoma-induced metabolic alterations which signified its antiproliferative potential. Conclusion: M1 manifested notable antiproliferative profile, and warrants further investigation for future anticancer therapy. PMID:28884001

Chemical carcinogenesis studies in nonhuman primates

PubMed Central

Takayama, Shozo; Thorgeirsson, Unnur P.; Adamson, Richard H.

2008-01-01

This review covers chemical carcinogenesis studies in nonhuman primates performed by the National Cancer Institute, USA, to provide hitherto unavailable information on their susceptibility to compounds producing carcinogenic effects in rodents. From autopsy records of 401 breeders and untreated controls, incidences of spontaneous malignant tumors were found to be relatively low in cynomolgus (1.9%) and rhesus monkeys (3.8%), but higher in African green monkeys (8%). Various chemical compounds, and in particular 6 antineoplastic agents, 13 food-related compounds including additives and contaminants, 1 pesticide, 5 N-nitroso compounds, 3 heterocyclic amines, and 7 “classical” rodent carcinogens, were tested during the 34 years period, generally at doses 10∼40 times the estimated human exposure. Results were inconclusive in many cases but unequivocal carcinogenicity was demonstrated for IQ, procarbazine, methylnitrosourea and diethylnitrosamine. Furthermore, negative findings for saccharine and cyclamate were in line with results in other species. Thus susceptibility to carcinogens is at least partly shared by nonhuman primates and rodents. PMID:18941297

Senescence marker protein 30 (SMP30)/regucalcin (RGN) expression decreases with aging, acute liver injuries and tumors in zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fujisawa, Koichi; Terai, Shuji, E-mail: terais@yamaguchi-u.ac.jp; Hirose, Yoshikazu

2011-10-22

Highlights: {yields} Zebrafish SMP30/RGN mRNA expression decreases with aging. {yields} Decreased expression was observed in liver tumors as compared to the surrounding area. {yields} SMP30/RGN is important for liver proliferation and tumorigenesis. -- Abstract: Senescence marker protein 30 (SMP30)/regucalcin (RGN) is known to be related to aging, hepatocyte proliferation and tumorigenesis. However, expression and function of non-mammalian SMP30/RGN is poorly understood. We found that zebrafish SMP30/RGN mRNA expression decreases with aging, partial hepatectomy and thioacetamide-induced acute liver injury. SMP30/RGN expression was also greatly decreased in a zebrafish liver cell line. In addition, we induced liver tumors in adult zebrafish bymore » administering diethylnitrosamine. Decreased expression was observed in foci, hepatocellular carcinomas, cholangiocellular carcinomas and mixed tumors as compared to the surrounding area. We thus showed the importance of SMP30/RGN in liver proliferation and tumorigenesis.« less

Hepatoprotective effect of engineered silver nanoparticles coated bioactive compounds against diethylnitrosamine induced hepatocarcinogenesis in experimental mice.

PubMed

Prasannaraj, Govindaraj; Venkatachalam, Perumal

2017-02-01

Nanoparticle based drug delivery can rapidly improves the therapeutic potential of anti-cancer agents. The present study focused to evaluate the hepatoprotective activity of silver nanoparticles (AgNPs) synthesized using aqueous extracts of Andrographis paniculata leaves (ApAgNPs) and Semecarpus anacardium nuts (SaAgNPs) against diethylnitrosamine (DEN) induced liver cancer in mice model. The physico-chemical properties of synthesized AgNPs were characterized by Fourier transform infrared (FTIR) spectroscopy, Transmission Electron Microscopy (TEM), Selected Area Electron Diffraction (SAED), X-ray Diffraction (XRD), Energy Dispersive X-ray (EDX) spectrum, Zeta potential and Dynamic Light Scattering (DLS) analysis. The surface plasmon resonance (SPR) absorption spectrum revealed a strong peak at 420nm for both SaAgNPs and ApAgNPs. FTIR results exhibited the presence of possible functional groups in the synthesized AgNPs. TEM analysis determined the hexagonal, and spherical shape of the synthesized silver nanoparticles. The XRD and SAED pattern confirmed the crystalline nature and crystalline size of the AgNPs. EDX result clearly showed strong silver signals in the range between 2 and 4keV. Zeta potential measurements indicated a sharp peak at -3.93 and -13.8mV for ApAgNPs and SaAgNPs, respectively. DLS measurement expressed the particle size distribution was 70 and 60nm for ApAgNPs and SaAgNPs, respectively. DEN (20mg/kg b.wt.) was subjected to induce liver cancer in mice for 8weeks and treated with biosynthesized silver nanoparticles. Interestingly, ApAgNPs and SaAgNPs treated DEN induced animal groups show a decreased level of aspartate amino transferase (AST), alanine amino transferase (ALT), serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) activity and elevated level of catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST) and superoxide dismutase (SOD) activity over untreated DEN control animals group. Histopathological investigation reveals decreased fat accumulation, appearance of binucleated cells in nanoparticle treated animals and showed mere normal cells induced by DEN. Argyrophilic nucleolar organiser region (AgNORs) had a significant decrease in number of acidic proteins and mast cells assay showed decrease of metachromatic cells in nanoparticles treated animal groups over control. Present results strongly suggest that biomolecule coated silver nanoparticles exposure showed potential hepatoprotective effect against DEN induced liver cancer and could be used as an effective anticancer nanodrug. Copyright © 2017. Published by Elsevier B.V.

Exploring the Potential Role of Chemopreventive Agent, Hesperetin Conjugated Pegylated Gold Nanoparticles in Diethylnitrosamine-Induced Hepatocellular Carcinoma in Male Wistar Albino Rats.

PubMed

Gokuladhas, Krishnan; Jayakumar, Subramaniyan; Rajan, Balan; Elamaran, Ramasamy; Pramila, Chengalvarayan Subramani; Gopikrishnan, Mani; Tamilarasi, Sasivarman; Devaki, Thiruvengadam

2016-04-01

Liver cancer is the fifth most common cancer and is still one of the leading causes of death world wide, due to food additives, alcohol, fungal toxins, air, toxic industrial chemicals, and water pollutants. Chemopreventive drugs play a potential role in liver cancer treatment. Obviously in the production of anticancer drugs, the factors like poor solubility, bioavailability, biocompatibility, limited chemical stability, large amount of dose etc., plays a major role. Against this backdrop, the idea of designing the chemopreventive nature of bio flavanoid hesperetin (HP) drug conjugated with pegylated gold nanoparticles to increasing the solubility, improve bioavailability and enhance the targeting capabilities of the drug during diethylnitrosamine (DEN) induced liver cancer in male wistar albino rats. The dose fixation studies and the toxicity of pure HP and HP conjugated gold nanoparticles (Au-mPEG(5000)-S-HP) were analysed. After concluded the dose fixation and toxicity studies the experimental design were segregated in six groups for the anticancer analysis of DEN induced HCC for 16 weeks. After the experimental period the body weight, relative liver weight, number of nodules and size of nodules, the levels of tumor markers like CEA, AFP and the level of lipid peroxidation, lipid hydroperoxides and the activities of antioxidant enzymes were assessed. The administration of DEN to rats resulted in increased relative liver weight and serum marker enzymes aspartate transaminase, alanine transaminase, alkaline phosphatase, lactate dehydrogenase, and gamma glutamyl transpeptidase. The levels of lipid peroxides elevated (in both serum and tissue) with subsequent decrease in the final body weight and tissue antioxidants like superoxide dismutase, catalase, reduced glutathione, glutathione peroxidise, and glutathione reductase. HP supplementation (20 mg/kg b.wt) significantly attenuated these alterations, thereby showing potent anticancer effect in liver cancer and the HP loaded gold nanoparticels (Au-mPEG(5000)-S-HP) treated animals shows the better treatment than the pure HP due to the solubility of drug, bioavailability and the target drug delivery of the biodegradable polymer. Histological observations were also carried out, which added supports to the chemopreventive action of the pure HP and HP loaded gold nanoparticles (Au-mPEG(5000)-S-HP) against DEN induction during liver cancer progression. These findings suggest that HP loaded gold nanoparticels (Au-mPEG(5000)-S-HP) shows better efficacy than the pure HP against lipid peroxidation, hepatic cell damage and protects the antioxidant system in DEN induced hepatocellular carcinogenesis.

Liver carcinogenesis by FOS-dependent inflammation and cholesterol dysregulation

PubMed Central

Bakiri, Latifa; Hamacher, Rainer; Graña, Osvaldo; Guío-Carrión, Ana; Martinez, Lola; Dienes, Hans P.; Thomsen, Martin K.; Hasenfuss, Sebastian C.

2017-01-01

Human hepatocellular carcinomas (HCCs), which arise on a background of chronic liver damage and inflammation, express c-Fos, a component of the AP-1 transcription factor. Using mouse models, we show that hepatocyte-specific deletion of c-Fos protects against diethylnitrosamine (DEN)-induced HCCs, whereas liver-specific c-Fos expression leads to reversible premalignant hepatocyte transformation and enhanced DEN-carcinogenesis. c-Fos–expressing livers display necrotic foci, immune cell infiltration, and altered hepatocyte morphology. Furthermore, increased proliferation, dedifferentiation, activation of the DNA damage response, and gene signatures of aggressive HCCs are observed. Mechanistically, c-Fos decreases expression and activity of the nuclear receptor LXRα, leading to increased hepatic cholesterol and accumulation of toxic oxysterols and bile acids. The phenotypic consequences of c-Fos expression are partially ameliorated by the anti-inflammatory drug sulindac and largely prevented by statin treatment. An inverse correlation between c-FOS and the LXRα pathway was also observed in human HCC cell lines and datasets. These findings provide a novel link between chronic inflammation and metabolic pathways important in liver cancer. PMID:28356389

A NEW MEDIUM‐TERM BIOASSAY SYSTEM FOR DETECTION OF ENVIRONMENTAL CARCINOGENS USING DIETHYLNITROSAMINE‐INITIATED RAT LIVER FOLLOWED BY D‐GALACTOSAMINE TREATMENT AND PARTIAL HEPATECTOMY

PubMed Central

Ito, Nobuyuki; Imaida, Katsumi; de Camargo, Joao Lauro V.; Takahashi, Satoru; Asamoto, Makoto; Tsuda, Hiroyuki

1988-01-01

The effects of D‐galactosamine on induction of preneoplastic glutathione S‐transferase placental form positive liver foci were investigated in F344 rats pretreated with diethylnitrosamine (DEN) in an attempt to improve the predictive value of the medium‐term bioassay system developed in our laboratory. Two weeks after the initial single ip dose (200 mg/kg) of DEN, administration of test compounds was commenced simultaneously with an ip injection of D‐galactosamine at a dose of 300 mg/kg body wt. All rats were then subjected to two‐thirds partial hepatectomy (PH) at week 5 and sacrificed for assessment of lesion yield at week 8. Measurement and comparison of the numbers and areas of glutathione S‐transferase placental form positive (GST‐P+) foci per cm2 revealed a positive response to more carcinogens, including non‐hepatocarcinogens, than did the same bioassay system without injection of D‐galactosamine. Thus the results suggest that inclusion of this extra proliferative stimulus may improve the medium‐term detection of carcinogens and modifiers. PMID:3136108

Sewage sludge does not induce genotoxicity and carcinogenesis.

PubMed

Silva, Paula Regina Pereira; Barbisan, Luis Fernando; Dagli, Maria Lúcia Zaidan; Saldiva, Paulo Hilário Nascimento

2012-07-01

Through a series of experiments, the genotoxic/mutagenic and carcinogenic potential of sewage sludge was assessed. Male Wistar rats were randomly assigned to four groups: Group 1 - negative control; Group 2 - liver carcinogenesis initiated by diethylnitrosamine (DEN; 200 mg/kg i.p.); Group 3 and G4-liver carcinogenesis initiated by DEN and fed 10,000 ppm or 50,000 ppm of sewage sludge. The animals were submitted to a 70% partial hepatectomy at the 3(rd) week. Livers were processed for routine histological analysis and immunohistochemistry, in order to detect glutathione S-transferase positive altered hepatocyte foci (GST-P(+) AHF). Peripheral blood samples for the comet assay were obtained from the periorbital plexus immediately prior to sacrificing. Polychromatic erythrocytes (PCEs) were analyzed in femoral bone-marrow smears, and the frequencies of those micronucleated (MNPCEs) registered. There was no sewage-sludge-induced increase in frequency of either DNA damage in peripheral blood leucocytes, or MNPCEs in the femoral bone marrow. Also, there was no increase in the levels of DNA damage, in the frequency of MNPCEs, and in the development of GST-P AHF when compared with the respective control group.

NFκB1 is a suppressor of neutrophil-driven hepatocellular carcinoma

NASA Astrophysics Data System (ADS)

Wilson, C. L.; Jurk, D.; Fullard, N.; Banks, P.; Page, A.; Luli, S.; Elsharkawy, A. M.; Gieling, R. G.; Chakraborty, J. Bagchi; Fox, C.; Richardson, C.; Callaghan, K.; Blair, G. E.; Fox, N.; Lagnado, A.; Passos, J. F.; Moore, A. J.; Smith, G. R.; Tiniakos, D. G.; Mann, J.; Oakley, F.; Mann, D. A.

2015-04-01

Hepatocellular carcinoma (HCC) develops on the background of chronic hepatitis. Leukocytes found within the HCC microenvironment are implicated as regulators of tumour growth. We show that diethylnitrosamine (DEN)-induced murine HCC is attenuated by antibody-mediated depletion of hepatic neutrophils, the latter stimulating hepatocellular ROS and telomere DNA damage. We additionally report a previously unappreciated tumour suppressor function for hepatocellular nfkb1 operating via p50:p50 dimers and the co-repressor HDAC1. These anti-inflammatory proteins combine to transcriptionally repress hepatic expression of a S100A8/9, CXCL1 and CXCL2 neutrophil chemokine network. Loss of nfkb1 promotes ageing-associated chronic liver disease (CLD), characterized by steatosis, neutrophillia, fibrosis, hepatocyte telomere damage and HCC. Nfkb1S340A/S340Amice carrying a mutation designed to selectively disrupt p50:p50:HDAC1 complexes are more susceptible to HCC; by contrast, mice lacking S100A9 express reduced neutrophil chemokines and are protected from HCC. Inhibiting neutrophil accumulation in CLD or targeting their tumour-promoting activities may offer therapeutic opportunities in HCC.

A crucial role of constitutive androstane receptor (CAR) in liver tumor development by imazalil in mice.

PubMed

Tamura, Kei; Inoue, Kaoru; Takahashi, Miwa; Matsuo, Saori; Kodama, Yukio; Yoshida, Midori

2016-01-01

To clarify the major pathway of liver tumor development induced by imazalil (IMA), an imidazole fungicide, male constitutive androstane receptor (CAR)-knockout (CARKO) and wild-type (WT) mice were treated with IMA at 500 ppm in the diet up to 27 weeks after initiation by diethylnitrosamine. After 27 weeks of treatment, neither altered foci nor adenomas were significantly increased in CARKO mice, whereas both eosinophilic altered foci and adenomas were increased in WT mice. After 4 or 13 weeks of IMA treatment, liver hypertrophy was observed at the tumor-inducible dose without differences among genotypes or durations. Analysis of hepatic drug metabolite enzymes, performed after administration of multiple doses during a 1-week period, indicated that pregnane X receptor might be involved in liver hypertrophy because IMA markedly elevated Cyp3a11 and Cyp2b10 expression levels in a dose-dependent manner in both genotypes. Our results demonstrated that the CAR pathway was the main mechanism of liver tumor development induced by IMA. The carcinogenic pathway was different from that of liver hypertrophy.

Sewage sludge does not induce genotoxicity and carcinogenesis

PubMed Central

Silva, Paula Regina Pereira; Barbisan, Luis Fernando; Dagli, Maria Lúcia Zaidan; Saldiva, Paulo Hilário Nascimento

2012-01-01

Through a series of experiments, the genotoxic/mutagenic and carcinogenic potential of sewage sludge was assessed. Male Wistar rats were randomly assigned to four groups: Group 1 - negative control; Group 2 - liver carcinogenesis initiated by diethylnitrosamine (DEN; 200 mg/kg i.p.); Group 3 and G4-liver carcinogenesis initiated by DEN and fed 10,000 ppm or 50,000 ppm of sewage sludge. The animals were submitted to a 70% partial hepatectomy at the 3rd week. Livers were processed for routine histological analysis and immunohistochemistry, in order to detect glutathione S-transferase positive altered hepatocyte foci (GST-P+ AHF). Peripheral blood samples for the comet assay were obtained from the periorbital plexus immediately prior to sacrificing. Polychromatic erythrocytes (PCEs) were analyzed in femoral bone-marrow smears, and the frequencies of those micronucleated (MNPCEs) registered. There was no sewage-sludge-induced increase in frequency of either DNA damage in peripheral blood leucocytes, or MNPCEs in the femoral bone marrow. Also, there was no increase in the levels of DNA damage, in the frequency of MNPCEs, and in the development of GST-P AHF when compared with the respective control group. PMID:23055806

Prevention of rat liver fibrosis and carcinogenesis by coffee and caffeine.

PubMed

Furtado, Kelly S; Polletini, Jossimara; Dias, Marcos C; Rodrigues, Maria A M; Barbisan, Luis F

2014-02-01

Coffee has been inversely related to the incidence of human liver disease; however, whether caffeine is the component responsible for the beneficial effects of coffee remains controversial. This study evaluated the beneficial effects of coffee or caffeine in a medium-term bioassay for rat liver fibrosis/carcinogenesis induced by diethylnitrosamine (DEN) and carbon tetrachloride (CCl4). One week after the DEN injection, the groups started to receive conventional coffee, instant coffee or 0.1% caffeine ad libitum for 24 weeks. The groups receiving conventional coffee or caffeine presented a significant reduction in collagen content and mRNA expression of collagen I. The groups receiving instant coffee or caffeine had a significant reduction in the size and area of pre-neoplastic lesions and in the mean number of neoplastic lesions. A significant increase in liver bax protein levels was observed in the groups receiving instant coffee or caffeine as compared to the control group. These data indicate that the most pronounced hepatoprotective effect against fibrosis was observed in the groups receiving conventional coffee and 0.1% caffeine, and the greatest effects against liver carcinogenesis were detected in the groups receiving instant coffee and 0.1% caffeine. Copyright © 2013 Elsevier Ltd. All rights reserved.

Babao Dan attenuates hepatic fibrosis by inhibiting hepatic stellate cells activation and proliferation via TLR4 signaling pathway.

PubMed

Liang, Lei; Yang, Xue; Yu, Yang; Li, Xiaoyong; Wu, Yechen; Shi, Rongyu; Jiang, Jinghua; Gao, Lu; Ye, Fei; Zhao, Qiudong; Li, Rong; Wei, Lixin; Han, Zhipeng

2016-12-13

Babao Dan (BBD), a traditional Chinese medicine, has been widely used as a complementary and alternative medicine to treat chronic liver diseases. In this study, we aimed to observe the protective effect of BBD on rat hepatic fibrosis induced by diethylnitrosamine (DEN) and explore it possible mechanism. BBD was administrated while DEN was given. After eight weeks, values of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) indicated that BBD significantly protected liver from damaging by DEN and had no obvious side effect on normal rat livers. Meanwhile, BBD attenuated hepatic inflammation and fibrosis in DEN-induced rat livers through histopathological examination and hepatic hydroxyproline content. Furthermore, we found that BBD inhibited hepatic stellate cells activation and proliferation without altering the concentration of lipopolysaccharide (LPS) in portal vein. In vitro study, serum from BBD treated rats (BBD-serum) could also significantly suppress LPS-induced HSCs activation through TLR4/NF-κB pathway. In addition, BBD-serum also inhibited the proliferation of HSCs by regulating TLR4/ERK pathway. Our study demonstrated that BBD may provide a new therapy strategy of hepatic injury and hepatic fibrosis.

Cytochrome b 5 reductase and the control of lipid metabolism and healthspan.

PubMed

Martin-Montalvo, Alejandro; Sun, Yaning; Diaz-Ruiz, Alberto; Ali, Ahmed; Gutierrez, Vincent; Palacios, Hector H; Curtis, Jessica; Siendones, Emilio; Ariza, Julia; Abulwerdi, Gelareh A; Sun, Xiaoping; Wang, Annie X; Pearson, Kevin J; Fishbein, Kenneth W; Spencer, Richard G; Wang, Miao; Han, Xianlin; Scheibye-Knudsen, Morten; Baur, Joe A; Shertzer, Howard G; Navas, Placido; Villalba, Jose Manuel; Zou, Sige; Bernier, Michel; de Cabo, Rafael

2016-01-01

Cytochrome b 5 reductases (CYB5R) are required for the elongation and desaturation of fatty acids, cholesterol synthesis and mono-oxygenation of cytochrome P450 enzymes, all of which are associated with protection against metabolic disorders. However, the physiological role of CYB5R in the context of metabolism, healthspan and aging remains ill-defined. We generated CYB5R-overexpressing flies (CYB5R-OE) and created a transgenic mouse line overexpressing CYB5R3 (CYB5R3-Tg) in the C57BL/6J background to investigate the function of this class of enzymes as regulators of metabolism and age-associated pathologies. Gender- and/or stage-specific induction of CYB5R, and pharmacological activation of CYB5R with tetrahydroindenoindole extended fly lifespan. Increased expression of CYB5R3 was associated with significant improvements in several metabolic parameters that resulted in modest lifespan extension in mice. Diethylnitrosamine-induced liver carcinogenesis was reduced in CYB5R3-Tg mice. Accumulation of high levels of long-chain polyunsaturated fatty acids, improvement in mitochondrial function, decrease in oxidative damage and inhibition of chronic pro-inflammatory pathways occurred in the transgenic animals. These results indicate that CYB5R represents a new target in the study of genes that regulate lipid metabolism and healthspan.

Assessment of hemodynamics in a rat model of liver cirrhosis with precancerous lesions using multislice spiral CT perfusion imaging.

PubMed

Ma, Guolin; Bai, Rongjie; Jiang, Huijie; Hao, Xuejia; Ling, Zaisheng; Li, Kefeng

2013-01-01

To develop an optimal scanning protocol for multislice spiral CT perfusion (CTP) imaging to evaluate hemodynamic changes in liver cirrhosis with diethylnitrosamine- (DEN-) induced precancerous lesions. Male Wistar rats were randomly divided into the control group (n = 80) and the precancerous liver cirrhosis group (n = 40). The control group received saline injection and the liver cirrhosis group received 50 mg/kg DEN i.p. twice a week for 12 weeks. All animals underwent plain CT scanning, CTP, and contrast-enhanced CT scanning. Scanning parameters were optimized by adjusting the diatrizoate concentration, the flow rate, and the delivery time. The hemodynamics of both groups was further compared using optimized multislice spiral CTP imaging. High-quality CTP images were obtained with following parameters: 150 kV; 150 mAs; 5 mm thickness, 5 mm interval; pitch, 1; matrix, 512 × 512; and FOV, 9.6 cm. Compared to the control group, the liver cirrhosis group had a significantly increased value of the hepatic arterial fraction and the hepatic artery perfusion (P < 0.05) but significantly decreased hepatic portal perfusion and mean transit time (P < 0.05). Multislice spiral CTP imaging can be used to evaluate the hemodynamic changes in the rat model of liver cirrhosis with precancerous lesions.

Loss of estrogen-related receptor α promotes hepatocarcinogenesis development via metabolic and inflammatory disturbances

PubMed Central

Hong, Eui-Ju; Levasseur, Marie-Pier; Dufour, Catherine R.; Perry, Marie-Claude; Giguère, Vincent

2013-01-01

Estrogen-related receptor α (ERRα) is a key regulator of mitochondrial function and metabolism essential for energy-driven cellular processes in both normal and cancer cells. ERRα has also been shown to mediate bone-derived macrophage activation by proinflammatory cytokines. However, the role of ERRα in cancer in which inflammation acts as a tumor promoter has yet to be investigated. Herein we show that global loss of ERRα accelerates the development of diethylnitrosamine (DEN)-induced hepatocellular carcinoma. Biochemical and metabolomics studies revealed that loss of ERRα promotes hepatocyte necrosis over apoptosis in response to DEN due to a deficiency in energy production. We further show that increased hepatocyte death and associated compensatory proliferation observed in DEN-injured ERRα-null livers is concomitant with increased nuclear factor κB (NF-κB)–dependent transcriptional control of cytokine expression in Kupffer cells. In particular, we demonstrate that loss of ERRα-dependent regulation of the NF-κB inhibitor IκBα leads to enhanced NF-κB activity and cytokine gene activation. Our work thus shows that global loss of ERRα activity promotes hepatocellular carcinoma by independent but synergistic mechanisms in hepatocytes and Kupffer cells, implying that pharmacological manipulation of ERRα activity may have a significant clinical impact on carcinogen-induced cancers. PMID:24127579

Selective Toxicity of Apigenin on Cancerous Hepatocytes by Directly Targeting their Mitochondria.

PubMed

Seydi, Enayatollah; Rasekh, Hamid R; Salimi, Ahmad; Mohsenifar, Zhaleh; Pourahmad, Jalal

2016-01-01

hepatocellular carcinoma (HCC) is the third cause of mortality due to cancer throughout the world. The main goal of the current research was to evaluate the selective toxicity of apigenin (APG) on hepatocytes and mitochondria obtained from the liver of HCC rats). In this research, HCC induced by a single dose of diethylnitrosamine (DEN); 200 mg/kg, i.p, and 2-acetylaminofluorene (2-AAF) (0.02%, through dietary) for 14 days. For confirmation of HCC, histopathological evaluations and determination of serum concentrations of liver toxicity enzymes and specific liver cancer marker; alpha-fetoprotein (AFP) were performed. Then, cancerous and non- cancerous hepatocytes were isolated by using the collagen perfusion method. Eventually, mitochondria isolated from HCC and normal hepatocytes were tested for every eventual toxic effects of APG. After confirmation of HCC, the results of this research showed that APG (10, 20 and 40 μM) increased mitochondrial parameters such as, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) level, mitochondrial swelling and cytochrome c expulsion only in cancerous hepatocytes. Apoptotic effect of APG on HCC cells was confirmed by caspase-3 activation and Annexin V-FITC and PI double staining analysis. These results propose the eligibility of the flavonoid APG as a complementary therapeutic agent for patients with hepatocellular carcinoma.

Metabolic and microbial signatures in rat hepatocellular carcinoma treated with caffeic acid and chlorogenic acid.

PubMed

Zhang, Zhan; Wang, Di; Qiao, Shanlei; Wu, Xinyue; Cao, Shuyuan; Wang, Li; Su, Xiaojian; Li, Lei

2017-07-03

Hepatocellular carcinoma (HCC) treatment remains lack of effective chemopreventive agents, therefore it is very attractive and urgent to discover novel anti-HCC drugs. In the present study, the effects of chlorogenic acid (ChA) and caffeic acid (CaA) on HCC induced by diethylnitrosamine (DEN) were evaluated. ChA or CaA could reduce the histopathological changes and liver injury markers, such as alanine transarninase, aspartate aminotransferase, alkaline phosphatase, total bile acid, total cholesterol, high density lipoprotein cholesterol and low density lipoprotein cholesterol. The underlying mechanisms were investigated by a data integration strategy based on correlation analyses of metabonomics data and 16 S rRNA gene sequencing data. ChA or CaA could inhibit the increase of Rumincoccaceae UCG-004 and reduction of Lachnospiraceae incertae sedis, and Prevotella 9 in HCC rats. The principal component analysis and partial least squares discriminant analysis were applied to reveal the metabolic differences among these groups. 28 different metabolites showed a trend to return to normal in both CaA and ChA treatment. Among them, Bilirubin, L-Tyrosine, L-Methionine and Ethanolamine were correlated increased Rumincoccaceae UCG-004 and decreased of Lachnospiraceae incertae sedis and Prevotella 9. These correlations could be identified as metabolic and microbial signatures of HCC onset and potential therapeutic targets.

An Evaluation of Transplacental Carcinogenesis for Human ...

EPA Pesticide Factsheets

Risk assessments take into account the sensitivity of the postnatal period to carcinogens through the application of age-dependent adjustment factors (ADAFs) (Barton et al. 2005). The prenatal period is also recognized to be sensitive but is typically not included into risk assessments (NRC, 2009). An analysis by California OEHHA (2008) contrasted prenatal, postnatal and adult sensitivity to 23 different carcinogens across 37 studies. That analysis found a wide range of transplacental sensitivity with some agents nearly 100 fold more potent in utero than in adults while others had an in utero/adult ratio adult only exposure). Five carcinogens had more modest ratios to adult potency in both pre- and postnatal testing (vinyl chloride, ethylnitroso biuret, 3-methylcholanthrene, urethane, diethylnitrosamine, 3-10 fold). Only one chemical showed a pre- vs postnatal divergence (butylnitrosourea, prenataladult). Based upon this limited set of genotoxic carcinogens, it appears that the prenatal period often has a sensitivity that approximates what has been found for postnatal, and the maternal system does not offer substantial protection against transplacental carcinogenesis in most cases. This suggests that the system of ADAFs developed for postnatal exposure may be considered for prenatal exposures as well. An alternative approach may be to calculate cancer risk for the period of pregnancy rather than blend this risk into the calculation of lifetime risk. This

Modifying influence of swine-serum-induced liver fibrosis on development of preneoplastic lesions in rat liver.

PubMed

Wada, S; Kato, T; Mutai, M; Ozaki, K; Yamaguchi, S; Kim, D J; Baba-Toriyama, H; Asamoto, M; Ito, N; Tsuda, H

1996-03-01

Modifying effects of fibrosis or a cirrhotic state, caused by treatment with swine serum (SS), on the induction of preneoplastic focal lesions were assessed in a rat medium-term liver bioassay model for the detection of environmental carcinogens, in which the test compound is administered during the promotion phase after initiation with diethylnitrosamine. In experiment I, repeated intraperitoneal administration of SS concomitantly with the hepatopromoting agent deoxycholic acid (DCA) or phenobarbital (PB) resulted in a cirrhotic state and a significant increase in the number or size of preneoplastic glutathione S-transferase placental form (GST-P)-positive liver cell foci as compared to the corresponding DCA or PB alone groups. In experiment II, SS was given prior to commencement of the same medium-term bioassay system, in which a known hepatopromoting agent, DCA, 17-alpha-ethynylestradiol, or 2-acetylaminofluorene, was applied. In this case, the liver did not show obvious cirrhotic change and, rather than any enhancement, slight inhibition of promotion occurred. The results indicate that a coexisting, but not a pre-existing, cirrhotic condition acts to increase growth pressure on GST-P+ preneoplastic foci, and suggest that concomitant administration of SS with the promoting agent could be applied to improve the sensitivity of the assay protocol.

Involvement of constitutive androstane receptor in liver hypertrophy and liver tumor development induced by triazole fungicides.

PubMed

Tamura, Kei; Inoue, Kaoru; Takahashi, Miwa; Matsuo, Saori; Irie, Kaoru; Kodama, Yukio; Gamo, Toshie; Ozawa, Shogo; Yoshida, Midori

2015-04-01

We clarified the involvement of constitutive androstane receptor (CAR) in triazole-induced liver hypertrophy and tumorigenesis using CAR-knockout (CARKO) mice. Seven-week-old male CARKO and wild-type (WT) mice were treated with 200 ppm cyproconazole (Cypro), 1500 ppm tebuconazole (Teb), or 200 ppm fluconazole (Flu) in the diet for 27 weeks after initiation by diethylnitrosamine (DEN). At weeks 4 (without DEN) and 13 (with DEN), WT mice in all treatment groups and CARKO mice in Teb group revealed liver hypertrophy with mainly Cyp2b10 and following Cyp3a11 inductions in the liver. Teb also induced Cyp4a10 in both genotypes. Cypro induced slight and duration-dependent liver hypertrophy in CARKO mice. At week 27, Cypro and Teb significantly increased eosinophilic altered foci and/or adenomas in WT mice. These proliferating lesions were clearly reduced in CARKO mice administered both compounds. The eosinophilic adenomas caused by Flu decreased in CARKO mice. The present study indicates that CAR is the main mediator of liver hypertrophy induced by Cypro and Flu, but not Teb. In contrast, CAR played a crucial role in liver tumor development induced by all three triazoles. Copyright © 2015 Elsevier Ltd. All rights reserved.

Tauroursodeoxycholic acid dampens oncogenic apoptosis induced by endoplasmic reticulum stress during hepatocarcinogen exposure

PubMed Central

Vandewynckel, Yves-Paul; Laukens, Debby; Devisscher, Lindsey; Paridaens, Annelies; Bogaerts, Eliene; Verhelst, Xavier; Van den Bussche, Anja; Raevens, Sarah; Van Steenkiste, Christophe; Van Troys, Marleen; Ampe, Christophe; Descamps, Benedicte; Vanhove, Chris; Govaere, Olivier; Geerts, Anja; Van Vlierberghe, Hans

2015-01-01

Hepatocellular carcinoma (HCC) is characterized by the accumulation of unfolded proteins in the endoplasmic reticulum (ER), which activates the unfolded protein response (UPR). However, the role of ER stress in tumor initiation and progression is controversial. To determine the impact of ER stress, we applied tauroursodeoxycholic acid (TUDCA), a bile acid with chaperone properties. The effects of TUDCA were assessed using a diethylnitrosamine-induced mouse HCC model in preventive and therapeutic settings. Cell metabolic activity, proliferation and invasion were investigated in vitro. Tumor progression was assessed in the HepG2 xenograft model. Administration of TUDCA in the preventive setting reduced carcinogen-induced elevation of alanine and aspartate aminotransferase levels, apoptosis of hepatocytes and tumor burden. TUDCA also reduced eukaryotic initiation factor 2α (eIf2α) phosphorylation, C/EBP homologous protein expression and caspase-12 processing. Thus, TUDCA suppresses carcinogen-induced pro-apoptotic UPR. TUDCA alleviated hepatic inflammation by increasing NF-κB inhibitor IκBα. Furthermore, TUDCA altered the invasive phenotype and enhanced metabolic activity but not proliferation in HCC cells. TUDCA administration after tumor development did not alter orthotopic tumor or xenograft growth. Taken together, TUDCA attenuates hepatocarcinogenesis by suppressing carcinogen-induced ER stress-mediated cell death and inflammation without stimulating tumor progression. Therefore, this chemical chaperone could represent a novel chemopreventive agent. PMID:26293671

Inter-laboratory comparison of turkey in ovo carcinogenicity assessment (IOCA) of hepatocarcinogens.

PubMed

Enzmann, H; Brunnemann, K; Iatropoulos, M; Shpyleva, S; Lukyanova, N; Todor, I; Moore, M; Spicher, K; Chekhun, V; Tsuda, H; Williams, G

2013-09-01

In three independent laboratories carcinogens (diethylnitrosamine, DEN, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone, NNK) and non-carcinogens (N-nitrosoproline, nicotine) were evaluated in turkey eggs for in ovo carcinogenicity assessment (IOCA). Compounds were injected into aseptic fertilized eggs. After incubation for 24 days, foci of altered hepatocytes (FAH), some with a pseudoglandular structure and/or signs of compression of the surrounding tissue were observed in the fetal liver. All laboratories were able to distinguish unequivocally the hepatocarcinogen-exposed groups from those exposed to non-carcinogens or the vehicle controls, based on the pre-specified evaluation parameters: tumor-like lesions, pseudoglandular areas and FAH. In addition to focal changes, only the carcinogens induced hepatocellular karyomegaly. Lower doses of the carcinogens, which did not induce FAH, were sufficient to induce hepatocellular karyomegaly. After exposure to 4 mg DEN, gall bladder agenesis was observed in all fetuses. The IOCA may be a valuable tool for early investigative studies on carcinogenicity and since it does not use rodents may complement chronic rat or mouse bioassays. Test substances that are positive in both rodents and fertilized turkey eggs are most probably trans-species carcinogens with particular significance for humans. The good concordance observed among the three laboratories demonstrates that the IOCA is a reliable and robust method. Copyright © 2012 Elsevier GmbH. All rights reserved.

Amelioration of carcinogenesis and tumor growth in the rat liver by combination of vitamin K2 and angiotensin-converting enzyme inhibitor via anti-angiogenic activities.

PubMed

Yoshiji, Hitoshi; Kuriyama, Shigeki; Noguchi, Ryuichi; Yoshii, Junichi; Ikenaka, Yasuhide; Yanase, Koji; Namisaki, Tadashi; Kitade, Mitsuteru; Yamazaki, Masaharu; Akahane, Takemi; Asada, Kiyoshi; Tsujimoto, Tatsuhito; Uemura, Masahito; Fukui, Hiroshi

2006-01-01

Recent studies have revealed that angiogenesis plays a pivotal role in carcinogenesis and tumor growth. We previously reported that the clinically used vitamin K(2) (VK) and angiotensin-converting enzyme inhibitor (ACE-I) exerted potent anti-angiogenic activities. The aim of our current study was to examine the combination effect of VK and ACE-I on hepatocarcinogenesis induced by diethyl-nitrosamine, and orthotopic hepatocellular carcinoma (HCC) growth in rats. When used individually, both VK and ACE-I at clinically comparable low doses exerted significant inhibitory effects on tumor development in the liver. A combination treatment of VK and ACE-I showed a more potent suppressive effect against hepatocarcinogenesis. Neovascularization increased during hepatocarcinogenesis, and VK and ACE-I significantly attenuated angiogenesis in the tumor. In orthotopic HCC transplantation, VK and ACE-I also showed marked suppressive effects against HCC development similar to those against hepatocarcinogenesis. In both experiments, the suppressive effects of VK and ACE-I against angiogenesis were similar in magnitude to their inhibitory effects against hepatocarcinogenesis and orthotopic HCC development. In the orthotopic model, VK and ACE-I treatment resulted in a marked increase of apoptosis in the tumor, whereas tumor cell proliferation itself was not altered. Since both VK and ACE-I are widely used in clinical practice without serious side effects, this combination therapy may be an effective new therapeutic strategy against hepatocarcinogenesis and HCC growth in the future.

Sorafenib prevents liver fibrosis in a non-alcoholic steatohepatitis (NASH) rodent model

PubMed Central

Stefano, J.T.; Pereira, I.V.A.; Torres, M.M.; Bida, P.M.; Coelho, A.M.M.; Xerfan, M.P.; Cogliati, B.; Barbeiro, D.F.; Mazo, D.F.C.; Kubrusly, M.S.; D'Albuquerque, L.A.C.; Souza, H.P.; Carrilho, F.J.; Oliveira, C.P.

2015-01-01

Liver fibrosis occurring as an outcome of non-alcoholic steatohepatitis (NASH) can precede the development of cirrhosis. We investigated the effects of sorafenib in preventing liver fibrosis in a rodent model of NASH. Adult Sprague-Dawley rats were fed a choline-deficient high-fat diet and exposed to diethylnitrosamine for 6 weeks. The NASH group (n=10) received vehicle and the sorafenib group (n=10) received 2.5 mg·kg-1·day-1 by gavage. A control group (n=4) received only standard diet and vehicle. Following treatment, animals were sacrificed and liver tissue was collected for histologic examination, mRNA isolation, and analysis of mitochondrial function. Genes related to fibrosis (MMP9, TIMP1, TIMP2), oxidative stress (HSP60, HSP90, GST), and mitochondrial biogenesis (PGC1α) were evaluated by real-time quantitative polymerase chain reaction (RT-qPCR). Liver mitochondrial oxidation activity was measured by a polarographic method, and cytokines by enzyme-linked immunosorbent assay (ELISA). Sorafenib treatment restored mitochondrial function and reduced collagen deposition by nearly 63% compared to the NASH group. Sorafenib upregulated PGC1α and MMP9 and reduced TIMP1 and TIMP2 mRNA and IL-6 and IL-10 protein expression. There were no differences in HSP60, HSP90 and GST expression. Sorafenib modulated PGC1α expression, improved mitochondrial respiration and prevented collagen deposition. It may, therefore, be useful in the treatment of liver fibrosis in NASH. PMID:25714891

RNA Binding Protein CUGBP1 Inhibits Liver Cancer in a Phosphorylation-Dependent Manner.

PubMed

Lewis, Kyle; Valanejad, Leila; Cast, Ashley; Wright, Mary; Wei, Christina; Iakova, Polina; Stock, Lauren; Karns, Rebekah; Timchenko, Lubov; Timchenko, Nikolai

2017-08-15

Despite intensive investigations, mechanisms of liver cancer are not known. Here, we identified an important step of liver cancer, which is the neutralization of tumor suppressor activities of an RNA binding protein, CUGBP1. The translational activity of CUGBP1 is activated by dephosphorylation at Ser302. We generated CUGBP1-S302A knock-in mice and found that the reduction of translational activity of CUGBP1 causes development of a fatty liver phenotype in young S302A mice. Examination of liver cancer in diethylnitrosamine (DEN)-treated CUGBP1-S302A mice showed these mice develop much more severe liver cancer that is associated with elimination of the mutant CUGBP1. Searching for mechanisms of this elimination, we found that the oncoprotein gankyrin (Gank) preferentially binds to and triggers degradation of dephosphorylated CUGBP1 (de-ph-S302-CUGBP1) or S302A mutant CUGBP1. To test the role of Gank in degradation of CUGBP1, we generated mice with liver-specific deletion of Gank. In these mice, the tumor suppressor isoform of CUGBP1 is protected from Gank-mediated degradation. Consistent with reduction of CUGBP1 in animal models, CUGBP1 is reduced in patients with pediatric liver cancer. Thus, this work presents evidence that de-ph-S302-CUGBP1 is a tumor suppressor protein and that the Gank-UPS-mediated reduction of CUGBP1 is a key event in the development of liver cancer. Copyright © 2017 American Society for Microbiology.

Chronic inflammation-elicited liver progenitor cell conversion to liver cancer stem cell with clinical significance.

PubMed

Li, Xiao-Feng; Chen, Cheng; Xiang, Dai-Min; Qu, Le; Sun, Wen; Lu, Xin-Yuan; Zhou, Teng-Fei; Chen, Shu-Zhen; Ning, Bei-Fang; Cheng, Zhuo; Xia, Ming-Yang; Shen, Wei-Feng; Yang, Wen; Wen, Wen; Lee, Terence Kin Wah; Cong, Wen-Ming; Wang, Hong-Yang; Ding, Jin

2017-12-01

The substantial heterogeneity and hierarchical organization in liver cancer support the theory of liver cancer stem cells (LCSCs). However, the relationship between chronic hepatic inflammation and LCSC generation remains obscure. Here, we observed a close correlation between aggravated inflammation and liver progenitor cell (LPC) propagation in the cirrhotic liver of rats exposed to diethylnitrosamine. LPCs isolated from the rat cirrhotic liver initiated subcutaneous liver cancers in nonobese diabetic/severe combined immunodeficient mice, suggesting the malignant transformation of LPCs toward LCSCs. Interestingly, depletion of Kupffer cells in vivo attenuated the LCSC properties of transformed LPCs and suppressed cytokeratin 19/Oval cell 6-positive tumor occurrence. Conversely, LPCs cocultured with macrophages exhibited enhanced LCSC properties. We further demonstrated that macrophage-secreted tumor necrosis factor-α triggered chromosomal instability in LPCs through the deregulation of ubiquitin D and checkpoint kinase 2 and enhanced the self-renewal of LPCs through the tumor necrosis factor receptor 1/Src/signal transducer and activator of transcription 3 pathway, which synergistically contributed to the conversion of LPCs to LCSCs. Clinical investigation revealed that cytokeratin 19/Oval cell 6-positive liver cancer patients displayed a worse prognosis and exhibited superior response to sorafenib treatment. Our results not only clarify the cellular and molecular mechanisms underlying the inflammation-mediated LCSC generation but also provide a molecular classification for the individualized treatment of liver cancer. (Hepatology 2017;66:1934-1951). © 2017 by the American Association for the Study of Liver Diseases.

Deletion of sphingosine kinase 1 inhibits liver tumorigenesis in diethylnitrosamine-treated mice

PubMed Central

Chen, Jinbiao; Qi, Yanfei; Zhao, Yang; Kaczorowski, Dominik; Couttas, Timothy A.; Coleman, Paul R.; Don, Anthony S.; Bertolino, Patrick; Gamble, Jennifer R.; Vadas, Mathew A.; Xia, Pu; McCaughan, Geoffrey W.

2018-01-01

Primary liver cancer is the 3rd leading cause of cancer deaths worldwide with very few effective treatments. Sphingosine kinase 1 (SphK1), a key regulator of sphingolipid metabolites, is over-expressed in human hepatocellular carcinoma (HCC) and our previous studies have shown that SphK1 is important in liver injury. We aimed to explore the role of SphK1 specifically in liver tumorigenesis using the SphK1 knockout (SphK1−/−) mouse. SphK1 deletion significantly reduced the number and the size of DEN-induced liver cancers in mice. Mechanistically, fewer proliferating but more apoptotic and senescent cells were detected in SphK1 deficient tumors compared to WT tumors. There was an increase in sphingosine rather than a decrease in sphingosine 1-phosphate (S1P) in SphK1 deficient tumors. Furthermore, the STAT3-S1PR pathway that has been reported previously to mediate the effect of SphK1 on colorectal cancers was not altered by SphK1 deletion in liver cancer. Instead, c-Myc protein expression was down-regulated by SphK1 deletion. In conclusion, this is the first in vivo evidence that SphK1 contributes to hepatocarcinogenesis. However, the downstream signaling pathways impacting on the development of HCC via SphK1 are organ specific providing further evidence that simply transferring known oncogenic molecular pathway targeting into HCC is not always valid. PMID:29643998

Dietary zinc deficiency predisposes mice to the development of preneoplastic lesions in chemically-induced hepatocarcinogenesis.

PubMed

Romualdo, Guilherme Ribeiro; Goto, Renata Leme; Henrique Fernandes, Ana Angélica; Cogliati, Bruno; Barbisan, Luis Fernando

2016-10-01

Although there is a concomitance of zinc deficiency and high incidence/mortality for hepatocellular carcinoma in certain human populations, there are no experimental studies investigating the modifying effects of zinc on hepatocarcinogenesis. Thus, we evaluated whether dietary zinc deficiency or supplementation alter the development of hepatocellular preneoplastic lesions (PNL). Therefore, neonatal male Balb/C mice were submitted to a diethylnitrosamine/2-acetylaminefluorene-induced hepatocarcinogenesis model. Moreover, mice were fed adequate (35 mg/kg diet), deficient (3 mg/kg) or supplemented (180 mg/kg) zinc diets. Mice were euthanized at 12 (early time-point) or 24 weeks (late time-point) after introducing the diets. At the early time-point, zinc deficiency decreased Nrf2 protein expression and GSH levels while increased p65 and p53 protein expression and the number of PNL/area. At the late time-point, zinc deficiency also decreased GSH levels while increased liver genotoxicity, cell proliferation into PNL and PNL size. In contrast, zinc supplementation increased antioxidant defense at both time-points but not altered PNL development. Our findings are the first to suggest that zinc deficiency predisposes mice to the PNL development in chemically-induced hepatocarcinogenesis. The decrease of Nrf2/GSH pathway and increase of liver genotoxicity, as well as the increase of p65/cell proliferation, are potential mechanisms to this zinc deficiency-mediated effect. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification of (Z)-2,3-Diphenylacrylonitrile as Anti-Cancer Molecule in Persian Gulf Sea Cucumber Holothuria parva

PubMed Central

Amidi, Salimeh; Hashemi, Zahra; Motallebi, Abbasali; Nazemi, Melika; Farrokhpayam, Hoda; Seydi, Enayatollah

2017-01-01

Hepatocellular carcinoma (HCC), also named cancerous hepatoma, is the most common type of malignant neoplasia of the liver. In this research, we screened the Persian Gulf sea cucumber Holothuria parva (H. parva) methanolic sub-fractions for the possible existence of selective toxicity on liver mitochondria isolated from an animal model of HCC. Next, we purified the most active fraction. Thus the structure of the active molecule was identified. HCC was induced by diethylnitrosamine (DEN) and 2-acetylaminofluorene (2-AAF) protocol. Rat liver mitochondria for evaluation of the selective cytotoxic effects of sub-fractions of H. parva were isolated and then mitochondrial parameters were determined. Our results showed that C1 sub-fraction of methanolic extract of H. parva considerably increased reactive oxygen species (ROS) generation, collapse of mitochondrial membrane potential (MMP), swelling in mitochondria and cytochrome c release only on HCC liver mitochondria. Furthermore, the methanolic extract of H. parva was investigated furthermore and the active fraction was extracted. In this fraction, (Z)-2,3-diphenylacrylonitrile molecule, which is also known as α-cyanostilbene, was identified by mass analysis. This molecule increased ROS generation, collapse of MMP, swelling in mitochondria and finally cytochrome c release only on HCC liver mitochondria. The derivatives of (Z)-2,3-diphenylacrylonitrile in other natural products were also reported as an anti-cancer agent. These results suggest the eligibility of the (Z)-2,3-diphenylacrylonitrile as a complementary therapeutic agent for patients with HCC. PMID:29035293

Therapeutic role of calcium and vitamin K3 in chemically induced hepatocarcinogenesis - new tools for cancer treatment.

PubMed

Anwar, Firoz; Khan, Ruqaiyah; Sachan, Richa; Kazmi, Imran; Rawat, Alisha; Sabih, Abdullah; Singh, Rajbala; Afzal, Muhammad; Ahmad, Aftab; Al-Orab, Abdulaziz S; Al-Abbasi, F A; Bhatt, Prakash Chandra; Kumar, Vikas

2018-04-17

HCC has been reported to be immensely occurring carcinoma worldwide. Recent days the mortality occurred due to liver cancer has also been found to be increased at an alarming speed affecting mostly the young patients. The aim of the current study was to decipher the role of calcium and vitamin K3 in the treatment of chemically induced hepatocarcinogenesis in the male Wistar rats. Liver cancer was induced via a subnecrogenic dose of 160 mg/kg body weight, diethylnitrosamine (DENA) when associated with fasting/refeeding in male Wistar rats. It elevated the serum glutamate oxaloacetate (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP), bilirubin, total cholesterol (CH), triglycerides (TG), alfa-fetoprotein (AFP) and reduced high-density lipoprotein (HDL). Histopathological examination of liver tissue showed marked carcinogenicity of the chemical carcinogen. Food, water intake and animal weights were also assessed, respectively. The animals exposed to DENA showed a significant decrease in the body weight. The elevated levels of serum SGOT, SGPT, ALP, AFP, TC and TG were restored by administration of calcium and Vit K (ad libitum) combination at higher dose than the normal dietary requirement (3 mg/kg) daily for 12 weeks p.o. Physiological and biochemical analysis showed the beneficial effects of calcium and vitamin K3 combination in the animals exposed to DENA. The results deciphered the beneficial effects of calcium and vitamin K3 in combination.

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

PubMed

Chi, Hsiang-Cheng; Chen, Shen-Liang; Tsai, Chung-Ying; Chuang, Wen-Yu; Huang, Ya-Hui; Tsai, Ming-Ming; Wu, Sheng-Ming; Sun, Cheng-Pu; Yeh, Chau-Ting; Lin, Kwang-Huei

2016-12-01

Recent studies have demonstrated a critical association between disruption of cellular thyroid hormone (TH) signaling and the incidence of hepatocellular carcinoma (HCC), but the underlying mechanisms remain largely elusive. Here, we showed that disruption of TH production results in a marked increase in progression of diethylnitrosamine (DEN)-induced HCC in a murine model, and conversely, TH administration suppresses the carcinogenic process via activation of autophagy. Inhibition of autophagy via treatment with chloroquine (CQ) or knockdown of ATG7 (autophagy-related 7) via adeno-associated virus (AAV) vectors, suppressed the protective effects of TH against DEN-induced hepatic damage and development of HCC. The involvement of autophagy in TH-mediated protection was further supported by data showing transcriptional activation of DAPK2 (death-associated protein kinase 2; a serine/threonine protein kinase), which enhanced the phosphorylation of SQSTM1/p62 (sequestosome 1) to promote selective autophagic clearance of protein aggregates. Ectopic expression of DAPK2 further attenuated DEN-induced hepatoxicity and DNA damage though enhanced autophagy, whereas, knockdown of DAPK2 displayed the opposite effect. The pathological significance of the TH-mediated hepatoprotective effect by DAPK2 was confirmed by the concomitant decrease in the expression of THRs and DAPK2 in matched HCC tumor tissues. Taken together, these findings indicate that TH promotes selective autophagy via induction of DAPK2-SQSTM1 cascade, which in turn protects hepatocytes from DEN-induced hepatotoxicity or carcinogenesis.

Pomegranate Bioactive Constituents Suppress Cell Proliferation and Induce Apoptosis in an Experimental Model of Hepatocellular Carcinoma: Role of Wnt/β-Catenin Signaling Pathway

PubMed Central

Bhatia, Deepak; Thoppil, Roslin J.; Mandal, Animesh; Samtani, Karishma A.; Darvesh, Altaf S.

2013-01-01

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide, and chemoprevention represents a viable approach in lowering the mortality of this disease. Pomegranate fruit, an abundant source of anti-inflammatory phytochemicals, is gaining tremendous attention for its wide-spectrum health benefits. We previously reported that a characterized pomegranate emulsion (PE) prevents diethylnitrosamine (DENA)-induced rat hepatocarcinogenesis though inhibition of nuclear factor-kappaB (NF-κB). Since NF-κB concurrently induces Wnt/β-catenin signaling implicated in cell proliferation, cell survival, and apoptosis evasion, we examined antiproliferative, apoptosis-inducing and Wnt/β-catenin signaling-modulatory mechanisms of PE during DENA rat hepatocarcinogenesis. PE (1 or 10 g/kg) was administered 4 weeks before and 18 weeks following DENA exposure. There was a significant increase in hepatic proliferation (proliferating cell nuclear antigen) and alteration in cell cycle progression (cyclin D1) due to DENA treatment, and PE dose dependently reversed these effects. PE substantially induced apoptosis by upregulating proapoptotic protein Bax and downregulating antiapoptotic protein Bcl-2. PE dose dependently reduced hepatic β-catenin and augmented glycogen synthase kinase-3β expression. Our study provides evidence that pomegranate phytochemicals exert chemoprevention of hepatic cancer through antiproliferative and proapoptotic mechanisms by modulating Wnt/β-catenin signaling. PE, thus, targets two interconnected molecular circuits (canonical NF-κB and Wnt/β-catenin pathways) to exert chemoprevention of HCC. PMID:23606879

Effect of purified diets and phenobarbital withdrawal on the phenotypic stability of altered hepatic foci (AHF)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Glauert, H.P.; Schwarz, M.; Pitot, H.C.

1986-03-05

The effect of the short-term withdrawal of phenobarbital (PB) and of the feeding of purified diets during the long-term withdrawal of PB on the stability of AHF were studied. In both experiments, female CD rats initially received an intragastric dose of diethylnitrosamine (10 mg/kg) 20 hours after being subjected to partial hepatectomy. In the short-term study, rats were fed 0.05% PB in a cereal-based diet for 6 months; at this time, half of the rats were killed whereas the other half were withdrawn from PB for 10 days before sacrifice. Withdrawing PB for 10 days resulted in a decrease inmore » the number and volume of AHF, particularly those which stained positively for gamma-glutamyltranspeptidase (GGT). In the long-term experiment, rats were fed 0.05% PB in a cereal-based diet containing PB and fed either a low-fat or a high-fat purified diet without PB for 8 months. At this time, the number and volume of AHF were much less than that seen at the time of PB withdrawal, and the distribution of phenotypes was altered: the percentage of foci containing GGT as a marker decreased dramatically. These results indicate that the observable number and total volume of AHF rapidly decrease after the withdrawal of PB from rats fed a cereal-based diet and that the feeding of purified diets after such PB withdrawal does not result in the reappearance of AHF.« less

Prophylactic Role of Averrhoa carambola (Star Fruit) Extract against Chemically Induced Hepatocellular Carcinoma in Swiss Albino Mice.

PubMed

Singh, Ritu; Sharma, Jyoti; Goyal, P K

2014-01-01

Liver cancer remains one of the severe lethal malignancies worldwide and hepatocellular carcinoma (HCC) is the most common form. The current study was designed to evaluate the prophylactic role of the fruit of Averrhoa carambola (star fruit or Kamrak) on diethylnitrosamine- (DENA-) induced (15 mg/kg b.wt.; single i.p. injection) and CCl4-promoted (1.6 g/kg b.wt. in corn oil thrice a week for 24 weeks) liver cancer in Swiss albino mice. Administration of ACE was made orally at a dose of 25 mg/kg b.wt/day for 5 consecutive days and it was withdrawn 48 hrs before the first administration of DENA (preinitiational stage). CCl4 was given after 2 weeks of DENA administration. A cent percent tumor incidence was noted in carcinogen treated animals while ACE administration resulted in a considerable reduction in tumor incidence, tumor yield, and tumor burden. Further, ACE treatment brings out a significant reduction in lipid peroxidation (P < 0.001) along with an elevation in the activities of enzymatic antioxidants (superoxide dismutase, P < 0.001, and catalase, P < 0.001), nonenzymatic antioxidant (reduced glutathione, P < 0.001), and total proteins (P < 0.001) when compared to the carcinogen treated control. These results demonstrate that ACE prevents the DENA/CCl4 induced adverse physical and biochemical alterations during hepatic carcinogenesis in mice. This study suggests the prophylactic role of Averrhoa carambola against hepatocellular carcinoma in mice; therefore, it could be employed for the further screening as a good chemopreventive natural supplement against cancer.

Prophylactic Role of Averrhoa carambola (Star Fruit) Extract against Chemically Induced Hepatocellular Carcinoma in Swiss Albino Mice

PubMed Central

Singh, Ritu; Sharma, Jyoti; Goyal, P. K.

2014-01-01

Liver cancer remains one of the severe lethal malignancies worldwide and hepatocellular carcinoma (HCC) is the most common form. The current study was designed to evaluate the prophylactic role of the fruit of Averrhoa carambola (star fruit or Kamrak) on diethylnitrosamine- (DENA-) induced (15 mg/kg b.wt.; single i.p. injection) and CCl4-promoted (1.6 g/kg b.wt. in corn oil thrice a week for 24 weeks) liver cancer in Swiss albino mice. Administration of ACE was made orally at a dose of 25 mg/kg b.wt/day for 5 consecutive days and it was withdrawn 48 hrs before the first administration of DENA (preinitiational stage). CCl4 was given after 2 weeks of DENA administration. A cent percent tumor incidence was noted in carcinogen treated animals while ACE administration resulted in a considerable reduction in tumor incidence, tumor yield, and tumor burden. Further, ACE treatment brings out a significant reduction in lipid peroxidation (P < 0.001) along with an elevation in the activities of enzymatic antioxidants (superoxide dismutase, P < 0.001, and catalase, P < 0.001), nonenzymatic antioxidant (reduced glutathione, P < 0.001), and total proteins (P < 0.001) when compared to the carcinogen treated control. These results demonstrate that ACE prevents the DENA/CCl4 induced adverse physical and biochemical alterations during hepatic carcinogenesis in mice. This study suggests the prophylactic role of Averrhoa carambola against hepatocellular carcinoma in mice; therefore, it could be employed for the further screening as a good chemopreventive natural supplement against cancer. PMID:24696677

BOK promotes chemical-induced hepatocarcinogenesis in mice.

PubMed

Rabachini, Tatiana; Fernandez-Marrero, Yuniel; Montani, Matteo; Loforese, Giulio; Sladky, Valentina; He, Zhaoyue; Bachmann, Daniel; Wicki, Simone; Villunger, Andreas; Stroka, Deborah; Kaufmann, Thomas

2018-03-01

BCL-2-related ovarian killer (BOK) is a conserved and widely expressed BCL-2 family member with sequence homology to pro-apoptotic BAX and BAK, but with poorly understood pathophysiological function. Since several members of the BCL-2 family are critically involved in the regulation of hepatocellular apoptosis and carcinogenesis we aimed to establish whether loss of BOK affects diethylnitrosamine (DEN)-induced hepatocarcinogenesis in mice. Short-term exposure to DEN lead to upregulation of BOK mRNA and protein in the liver. Of note, induction of CHOP and the pro-apoptotic BH3-only proteins PUMA and BIM by DEN was strongly reduced in the absence of BOK. Accordingly, Bok -/- mice were significantly protected from DEN-induced acute hepatocellular apoptosis and associated inflammation. As a consequence, Bok -/- animals were partially protected against chemical-induced hepatocarcinogenesis showing fewer and, surprisingly, also smaller tumors than WT controls. Gene expression profiling revealed that downregulation of BOK results in upregulation of genes involved in cell cycle arrest. Bok -/- hepatocellular carcinoma (HCC) displayed higher expression levels of the cyclin kinase inhibitors p19 INK4d and p21 cip1 . Accordingly, hepatocellular carcinoma in Bok -/- animals, BOK-deficient human HCC cell lines, as well as non-transformed cells, showed significantly less proliferation than BOK-proficient controls. We conclude that BOK is induced by DEN, contributes to DEN-induced hepatocellular apoptosis and resulting hepatocarcinogenesis. In line with its previously reported predominant localization at the endoplasmic reticulum, our findings support a role of BOK that links the cell cycle and cell death machineries upstream of mitochondrial damage.

Environmental complex mixture toxicity assessment.

PubMed

Gardner, H S; Brennan, L M; Toussaint, M W; Rosencrance, A B; Boncavage-Hennessey, E M; Wolfe, M J

1998-12-01

Trichloroethylene (TCE) was found as a contaminant in the well supplying water to an aquatic testing laboratory. The groundwater was routinely screened by a commercial laboratory for volatile and semivolatile compounds, metals, herbicides, pesticides, and polychlorinated biphenyls using U.S. Environmental Protection Agency methods. Although TCE was the only reportable peak on the gas chromatograph, with average concentrations of 0.200 mg/l, other small peaks were also present, indicating the possibility that the contamination was not limited to TCE alone. A chronic 6-month carcinogenicity assay was conducted on-site in a biomonitoring trailer, using the Japanese medaka fish (Oryzias latipes) in an initiation-promotion protocol, with diethylnitrosamine (DEN) as the initiator and the TCE-contaminated groundwater as a promoter. Study results indicated no evidence of carcinogenic potential of the groundwater without initiation. There was, however, a tumor-promotional effect of the groundwater after DEN initiation. A follow-up laboratory study was conducted using reagent grade TCE added to carbon-filtered groundwater to simulate TCE concentrations comparable to those found in the contaminated groundwater. Study results indicated no promotional effects of TCE. These studies emphasize the necessity for on-site bioassays to assess potential environmental hazards. In this instance, chemical analysis of the groundwater identified TCE as the only reportable contaminant, but other compounds present below reportable limits were noted and may have had a synergistic effect on tumor promotion observed with the groundwater exposure. Laboratory toxicity testing of single compounds can produce toxicity data specific to that compound for that species but cannot take into account the possible toxic effects of mixtures of compounds.

Differential biodistribution of oncolytic poxvirus administered systemically in an autochthonous model of hepatocellular carcinoma.

PubMed

Baril, Patrick; Touchefeu, Yann; Cany, Jeannette; Cherel, Yan; Thorne, Steve H; Tran, Lucile; Conchon, Sophie; Vassaux, Georges

2011-12-01

Preclinical studies have demonstrated that, unlike oncolytic adenoviruses, oncolytic vaccinia viruses can reach implanted tumors upon systemic injection. However, the biodistribution of this oncolytic agent in in situ autochthonous tumor models remains poorly characterized. In the present study, we assessed this biodistribution in a model of mouse hepatocellular carcinoma (HCC) obtained after injection of the carcinogen diethylnitrosamine (DEN). Twelve months after DEN administration, histology, quantitative reverse transcription-polymerase chain reaction, in situ hybridization and viral titration were used to characterize tumors, as well as to assess the viral load of the livers upon either intravenous or intraperitoineal injection. The results obtained showed that the architecture of the liver was lost, with a noticeable absence of sinusoids, as well as the presence of steatosis and α-fetoprotein-positive HCC tumor nodules. Bioluminescence imaging and measures of the infective virus load demonstrated that intravenous injection of 10(8)  plaque-forming units of the recombinant vaccinia virus led to a predominant transduction of the liver, whereas intraperitoneal injection resulted in a lower level of liver transduction accompanied by an increased infection of the lungs, spleen, kidneys and bowels. Immunohistochemical analysis of liver sections of animals injected intravenously with the virus revealed a preferential localization of vaccinia-specific immunoreactivity in the tumors. The findings of the present study emphasize the importance of the route of administration of the vector and highlight the relevance of systemic injection of oncolytic vaccinia virus in the context of hepatocellular carcinoma. Copyright © 2011 John Wiley & Sons, Ltd.

Carcinogenicity of by-products of disinfection in mouse and rat liver

DOE Office of Scientific and Technical Information (OSTI.GOV)

Herren-Freund, S.L.; Pereira, M.A.

1986-11-01

By-products of disinfection were tested for initiating and/or promoting activity in rat liver by using the rat liver foci bioassay. The assay uses an increased incidence of ..gamma..-glutamyltranspeptidase-positive foci (GGT foci) as an indicator of carcinogenicity. The by-products of disinfection, including chloramine, halogenated humic acids, halogenated ethanes, halogenated acetonitriles, halogenated methanes, halogenated ethylene, and N-Cl piperidine, did not initiate GGT foci, which would indicate that they are not capable of initiating carcinogenesis. Chloroform and halogenated benzenes were tested in this assay for their ability to promote the occurrence of GGT foci and tumors initiated by diethylnitrosamine (DENA). Chloroform either hadmore » no effect or inhibited the occurrence of GGT foci when administered subsequent to a single dose of DENA. However, when the chloroform was administered in drinking water concurrently with weekly doses of DENA, it enhanced the formation of liver tumors. Of 20 halogenated benzenes tested, only 1,2,4,5-tetrachlorobenzene and hexachlorobenzene promoted the occurrence of DENA-initiated GGT foci. Thus in rat liver, the tested by-products of drinking water disinfection did not demonstrate tumor-initiating activity, although a few appeared to possess tumor-promoting activity. Chloroform was also tested for tumor-promoting activity in 15-days-old Swiss mice initiated with ethylnitrosourea (ENU). ENU at 5 and 20 ..mu..g/g caused a dose-dependent increase in liver tumors. In male mice, chloroform inhibited both spontaneous and ENU-induced liver tumors. When administered in the drinking water, chloroform inhibited, whereas phenobarbital promoted, hepatocarcinogenesis in mice.« less

Inhibition of Hepatocarcinogenesis by ArtinM via Anti-proliferative and Pro-apoptotic Mechanisms.

PubMed

Braz, Mariana M; Roque-Barreira, Maria Cristina; Ramalho, Fernando S; Oliveira, Carlos A; Augusto, Marlei J; Ramalho, Leandra N Z

ArtinM is a d-mannose-binding lectin found in the seeds of Artocarpus heterophyllus (jackfruit) that interacts with N-glycans, that is associated with receptors on the surface of phagocytic cells and induces the production of inflammatory mediators. Some of them are especially important because they may be required for antitumor immune response. This study aimed to evaluate the effect of ArtinM on hepatocellular preneoplastic foci. Wistar rats received 50 mg/kg of diethyl-nitrosamine (DEN) intraperitoneal weekly for 12 weeks. From the 14th week, the treated animals received 50 μg/kg of ArtinM subcutaneous every 2 weeks until the 18th week, whereas control animals were injected with vehicle alone. Preneoplastic-related factors were estimated using histological, western blotting and RT-PCR analysis. In comparison to the groups exposed to DEN, the ArtinM-treated rats showed diminution of preneoplastic foci, decreased expression of proliferating cell nuclear antigen (PCNA), increased number of nuclear p21 and p27 stained cells, augmented number of apoptotic cells, increased expression of p53, p42/44 MAPK and p21 proteins, reduced cyclin D1 (CCND1) protein levels and increased expression of TNFα and IFNγ genes. No difference was observed in interleukin 12 (IL12) protein levels. These findings indicate that ArtinM may provide protection against hepatocarcinogenesis as a result of the induction of cell-cycle blockage and pro-apoptotic mechanisms. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Pomegranate-mediated chemoprevention of experimental hepatocarcinogenesis involves Nrf2-regulated antioxidant mechanisms

PubMed Central

Bishayee, Anupam; Bhatia, Deepak; Thoppil, Roslin J.; Darvesh, Altaf S.; Nevo, Eviatar; Lansky, Ephraim P.

2011-01-01

Hepatocellular carcinoma (HCC), one of the most prevalent and lethal cancers, has shown an alarming rise in the USA. Without effective therapy for HCC, novel chemopreventive strategies may effectively circumvent the current morbidity and mortality. Oxidative stress predisposes to hepatocarcinogenesis and is the major driving force of HCC. Pomegranate, an ancient fruit, is gaining tremendous attention due to its powerful antioxidant properties. Here, we examined mechanism-based chemopreventive potential of a pomegranate emulsion (PE) against dietary carcinogen diethylnitrosamine (DENA)-induced rat hepatocarcinogenesis that mimics human HCC. PE treatment (1 or 10 g/kg), started 4 weeks prior to the DENA challenge and continued for 18 weeks thereafter, showed striking chemopreventive activity demonstrated by reduced incidence, number, multiplicity, size and volume of hepatic nodules, precursors of HCC. Both doses of PE significantly attenuated the number and area of γ-glutamyl transpeptidase-positive hepatic foci compared with the DENA control. PE also attenuated DENA-induced hepatic lipid peroxidation and protein oxidation. Mechanistic studies revealed that PE elevated gene expression of an array of hepatic antioxidant and carcinogen detoxifying enzymes in DENA-exposed animals. PE elevated protein and messenger RNA expression of the hepatic nuclear factor E2-related factor 2 (Nrf2). Our results provide substantial evidence, for the first time, that pomegranate constituents afford chemoprevention of hepatocarcinogenesis possibly through potent antioxidant activity achieved by upregulation of several housekeeping genes under the control of Nrf2 without toxicity. The outcome of this study strongly supports the development of pomegranate-derived products in the prevention and treatment of human HCC, which remains a devastating disease. PMID:21389260

Chrysin abrogates early hepatocarcinogenesis and induces apoptosis in N-nitrosodiethylamine-induced preneoplastic nodules in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khan, Mahaboob S.; Department of Biochemistry, Govt. Home Science College, Panjab University, Chandigarh; Devaraj, Halagowder

Flavonoids possess strong anti-oxidant and cancer chemopreventive activities. Chrysin (5,7-dihydroxyflavone) occurs naturally in many plants, honey, and propolis. In vitro, chrysin acts as a general anti-oxidant, causes cell cycle arrest and promotes cell death. However, the mechanism by which chrysin inhibits cancer cell growth and the subcellular pathways activated remains poorly understood. Effect of dietary supplementation with chrysin on proliferation and apoptosis during diethylnitrosamine (DEN)-induced early hepatocarcinogenesis was investigated in male Wistar rats. To induce hepatocarcinogenesis, rats were given DEN injections (i.p., 200 mg/kg) three times at a 15 day interval. An oral dose of chrysin (250 mg/kg bodyweight) wasmore » given three times weekly for 3 weeks, commencing 1 week after the last dose of DEN. Changes in the mRNA expression of COX-2, NFkB p65, p53, Bcl-xL and {beta}-arrestin-2 were assessed by quantitative real-time PCR. Changes in the protein levels were measured by western blotting. Chrysin administration significantly (P < 0.001) reduced the number and size of nodules formed. Also, a significant (P < 0.01) reduction in serum activities of AST, ALT, ALP, LDH and {gamma}GT was noticed. Expression of COX-2 and NFkB p65 was significantly reduced whereas that of p53, Bax and caspase 3 increased at the mRNA and protein levels. Likewise, a decrease in levels of {beta}-arrestin and the anti-apoptotic marker Bcl-xL was also noted. These findings suggest that chrysin exerts global hepato-protective effect and its chemopreventive activity is associated with p53-mediated apoptosis during early hepatocarcinogenesis.« less

(Z)-3,5,4'-Trimethoxystilbene Limits Hepatitis C and Cancer Pathophysiology by Blocking Microtubule Dynamics and Cell Cycle Progression

PubMed Central

Nguyen, Charles B.; Kotturi, Hari; Waris, Gulam; Mohammed, Altaf; Chandrakesan, Parthasarathy; May, Randal; Sureban, Sripathi; Weygant, Nathaniel; Qu, Dongfeng; Rao, Chinthalapally V.; Dhanasekaran, Danny N.; Bronze, Michael S.; Houchen, Courtney W.; Ali, Naushad

2016-01-01

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide. Chronic hepatitis C virus (HCV) infection causes induction of several tumor/cancer stem cell (CSC) markers and is known to be a major risk factor for development of HCC. Therefore, drugs that simultaneously target viral replication and CSC properties are needed for a risk-free treatment of advanced stage liver diseases including HCC. Here, we demonstrated that (Z)-3,5,4’-trimethoxystilbene (Z-TMS) exhibits potent anti-tumor and anti-HCV activities without exhibiting cytotoxicity to human hepatocytes in vitro or in mice livers. Diethylnitrosamine (DEN)/carbon tetrachloride (CCl4) extensively induced expression of DCLK1 (a CSC marker) in the livers of C57BL/6 mice following hepatic injury. Z-TMS exhibited hepatoprotective effects against DEN/CCl4-induced injury by reducing DCLK1 expression and improving histological outcomes. The drug caused bundling of DCLK1 with microtubules and blocked cell cycle progression at G2/M phase in hepatoma cells via downregulation of CDK1, induction of p21cip1/waf1 expression, and inhibition of Akt (Ser473) phosphorylation. Z-TMS also inhibited proliferation of erlotinib-resistant lung adenocarcinoma cells (H1975) bearing the T790M EGFR mutation most likely by promoting autophagy and nuclear fragmentation. In conclusion, Z-TMS appears to be a unique therapeutic agent targeting HCV and concurrently eliminating cells with neoplastic potential during chronic liver diseases including HCC. It may also be a valuable drug for targeting drug-resistant carcinomas and cancers of the lungs, pancreas, colon, and intestine in which DCLK1 is involved in tumorigenesis. PMID:27287718

Mice with hepatocyte-specific FXR deficiency are resistant to spontaneous but susceptible to cholic acid-induced hepatocarcinogenesis

PubMed Central

Zhu, Yan; Li, Guodong; Williams, Jessica A.; Buckley, Kyle; Tawfik, Ossama; Luyendyk, James P.

2016-01-01

Farnesoid X receptor (FXR) belongs to the nuclear receptor superfamily with its endogenous ligands bile acids. Mice with whole body FXR deficiency develop liver tumors spontaneously, but the underlying mechanism is unclear. Moreover, it is unknown whether FXR deficiency in liver alone serves as a tumor initiator or promoter during liver carcinogenesis. This study aims to evaluate the effects of hepatocyte-specific FXR deficiency (FXRhep−/−) in liver tumor formation. The results showed that FXRhep−/− mice did not show spontaneous liver tumorigenesis with aging (up to 24 mo of age). Therefore FXRhep−/− mice were fed a bile acid (cholic acid)-containing diet alone or along with a liver tumor initiator, diethylnitrosamine (DEN). Thirty weeks later, no tumors were found in wild-type or FXRhep−/− mice without any treatment or with DEN only. However, with cholic acid, while only some wild-type mice developed tumors, all FXRhep−/− mice presented with severe liver injury and tumors. Interestingly, FXRhep−/− mouse livers increased basal expression of tumor suppressor p53 protein, apoptosis, and decreased basal cyclin D1 expression, which may prevent tumor development in FXRhep−/− mice. However, cholic acid feeding reversed these effects in FXRhep−/− mice, which is associated with an increased cyclin D1 and decreased cell cycle inhibitors. More in-depth analysis indicates that the increased in cell growth might result from disturbance of the MAPK and JAK/Stat3 signaling pathways. In conclusion, this study shows that hepatic FXR deficiency may only serve as a tumor initiator, and increased bile acids is required for tumor formation likely by promoting cell proliferation. PMID:26744468

Chemopreventive effects of korean red ginseng extract on rat hepatocarcinogenesis.

PubMed

Kim, Hyemee; Hong, Mi-Kyung; Choi, Haymie; Moon, Hyun-Seuk; Lee, Hae-Jeung

2015-01-01

The objective of this study was to determine a chemopreventive activity of Korean red ginseng extract (KRG) in diethylnitrosamine (DEN) induced hepatocarcinogenesis in rats. After acclimatization for a week, Sprague-Dawley rats were randomized into five groups (n = 15) and fed either KRG (0.5, 1 or 2%) or control diets for 10 weeks. After two weeks of starting of experimental diets, the rats were initiated hepatocarcinogenesis by injection of DEN and were then subjected to two-thirds partial hepatectomy at five-week for developing the medium-term bioassay system. Both 0.5 and 1% KRG diets suppressed the area (55 and 60%; p= 0.0251 and 0.0144) and number (39 and 59%; p= 0.0433 and 0.0012) of glutathione S-transferase placental form (GST-P) positive foci when compared to the DEN-control group. The production of thiobarbituric acid reactive substances (TBARS) was significantly reduced in 0.5 and 1% KRG-treated rats. The supplementation of 1% KRG diet significantly elevated the levels of total glutathione (tGSH) and glutathione-related enzymes including cytosolic glutathione S-transferase (GST) and glutathione peroxidase (GPx) activities. It was also observed in cDNA microarray that the gene expressions (Cyp2c6, Cyp2e1, Cyp3a9, and Mgst1) involved in the xenobiotics metabolism via cytochrome P450 signaling pathway were down-regulated in the 1% KRG diet-treated group when compared to the DEN-control. The chemopreventive effects of KRG could be affected by 1) the decrease of lipid peroxidation, 2) the increase of tGSH content and GSH-dependent enzyme activities, and 3) the decrease of the gene expression profile involved in cytochrome P450 signaling pathway. These results suggest that KRG may prove to be a therapeutic agent against hepatocarcinogenesis.

Chemopreventive Effects of Korean Red Ginseng Extract on Rat Hepatocarcinogenesis

PubMed Central

Kim, Hyemee; Hong, Mi-Kyung; Choi, Haymie; Moon, Hyun-Seuk; Lee, Hae-Jeung

2015-01-01

The objective of this study was to determine a chemopreventive activity of Korean red ginseng extract (KRG) in diethylnitrosamine (DEN) induced hepatocarcinogenesis in rats. After acclimatization for a week, Sprague-Dawley rats were randomized into five groups (n = 15) and fed either KRG (0.5, 1 or 2%) or control diets for 10 weeks. After two weeks of starting of experimental diets, the rats were initiated hepatocarcinogenesis by injection of DEN and were then subjected to two-thirds partial hepatectomy at five-week for developing the medium-term bioassay system. Both 0.5 and 1% KRG diets suppressed the area (55 and 60%; p= 0.0251 and 0.0144) and number (39 and 59%; p= 0.0433 and 0.0012) of glutathione S-transferase placental form (GST-P) positive foci when compared to the DEN-control group. The production of thiobarbituric acid reactive substances (TBARS) was significantly reduced in 0.5 and 1% KRG-treated rats. The supplementation of 1% KRG diet significantly elevated the levels of total glutathione (tGSH) and glutathione-related enzymes including cytosolic glutathione S-transferase (GST) and glutathione peroxidase (GPx) activities. It was also observed in cDNA microarray that the gene expressions (Cyp2c6, Cyp2e1, Cyp3a9, and Mgst1) involved in the xenobiotics metabolism via cytochrome P450 signaling pathway were down-regulated in the 1% KRG diet-treated group when compared to the DEN-control. The chemopreventive effects of KRG could be affected by 1) the decrease of lipid peroxidation, 2) the increase of tGSH content and GSH-dependent enzyme activities, and 3) the decrease of the gene expression profile involved in cytochrome P450 signaling pathway. These results suggest that KRG may prove to be a therapeutic agent against hepatocarcinogenesis. PMID:25553083

Efficacy of AKT Inhibitor ARQ 092 Compared with Sorafenib in a Cirrhotic Rat Model with Hepatocellular Carcinoma.

PubMed

Roth, Gaël S; Macek Jilkova, Zuzana; Zeybek Kuyucu, Ayca; Kurma, Keerthi; Ahmad Pour, Séyédéh Tayébéh; Abbadessa, Giovanni; Yu, Yi; Busser, Benoit; Marche, Patrice N; Leroy, Vincent; Decaens, Thomas

2017-10-01

Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related mortality worldwide. The AKT pathway has been found activated in 50% of HCC cases, making it a promising target. Therefore, we assess efficacy of the allosteric AKT inhibitor ARQ 092 compared with untreated control and standard treatment, sorafenib, in vitro and in vivo ARQ 092 blocked phosphorylation of AKT in vitro and strongly inhibited cell growth with significantly higher potency than sorafenib. Similarly, apoptosis and cell migration were strongly reduced by ARQ 092 in vitro To mimic human advanced HCC, we used a diethylnitrosamine-induced cirrhotic rat model with fully developed HCC. MRI analyses showed that ARQ 092 significantly reduced overall tumor size. Furthermore, number of tumors was decreased by ARQ 092, which was associated with increased apoptosis and decreased proliferation. Tumor contrast enhancement was significantly decreased in the ARQ 092 group. Moreover, on tumor tissue sections, we observed a vascular normalization and a significant decrease in fibrosis in the surrounding liver of animals treated with ARQ 092. Finally, pAKT/AKT levels in ARQ 092-treated tumors were reduced, followed by downregulation of actors of AKT downstream signaling pathway: pmTOR, pPRAS40, pPLCγ1, and pS6K1. In conclusion, we demonstrated that ARQ 092 blocks AKT phosphorylation in vitro and in vivo In the HCC-rat model, ARQ 092 was well tolerated, showed antifibrotic effect, and had stronger antitumor effect than sorafenib. Our results confirm the importance of targeting AKT in HCC. Mol Cancer Ther; 16(10); 2157-65. ©2017 AACR . ©2017 American Association for Cancer Research.

FXR-Gankyrin axis is involved in development of pediatric liver cancer.

PubMed

Valanejad, Leila; Lewis, Kyle; Wright, Mary; Jiang, Yanjun; D'Souza, Amber; Karns, Rebekah; Sheridan, Rachel; Gupta, Anita; Bove, Kevin; Witte, David; Geller, James; Tiao, Gregory; Nelson, David L; Timchenko, Lubov; Timchenko, Nikolai

2017-07-01

The development of hepatoblastoma (HBL) is associated with failure of hepatic stem cells (HSC) to differentiate into hepatocytes. Despite intensive investigations, mechanisms of the failure of HSC to differentiate are not known. We found that oncogene Gankyrin (Gank) is involved in the inhibition of differentiation of HSC via triggering degradation of tumor suppressor proteins (TSPs) Rb, p53, C/EBPα and HNF4α. Our data show that the activation of a repressor of Gank, farnesoid X receptor, FXR, after initiation of liver cancer by Diethylnitrosamine (DEN) prevents the development of liver cancer by inhibiting Gank and rescuing tumor suppressor proteins. We next analyzed FXR-Gank-Tumor suppressor pathways in a large cohort of HBL patients which include 6 controls and 53 HBL samples. Systemic analysis of these samples and RNA-Seq approach revealed that the FXR-Gank axis is activated; markers of hepatic stem cells are dramatically elevated and hepatocyte markers are reduced in HBL samples. In the course of these studies, we found that RNA binding protein CUGBP1 is a new tumor suppressor protein which is reduced in all HBL samples. Therefore, we generated CUGBP1 KO mice and examined HBL signatures in the liver of these mice. Micro-array studies revealed that the HBL-specific molecular signature is developed in livers of CUGBP1 KO mice at very early ages. Thus, we conclude that FXR-Gank-TSPs-Stem cells pathway is a key determinant of liver cancer in animal models and in pediatric liver cancer. Our data provide a strong basis for development of FXR-Gank-based therapy for treatment of patients with hepatoblastoma. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Fetal Onset of Aberrant Gene Expression Relevant to Pulmonary Carcinogenesis in Lung Adenocarcinoma Development Induced by In Utero Arsenic Exposure

PubMed Central

Shen, Jun; Liu, Jie; Xie, Yaxiong; Diwan, Bhalchandra A.; Waalkes, Michael P.

2009-01-01

Arsenic is a human pulmonary carcinogen. Our work indicates that in utero arsenic exposure in mice can induce or initiate lung cancer in female offspring. To define early molecular changes, pregnant C3H mice were given 85 ppm arsenic in drinking water from days 8 to 18 of gestation and expression of selected genes in the fetal lung or in lung tumors developing in adults was examined. Transplacental arsenic exposure increased estrogen receptor-α (ER-α) transcript and protein levels in the female fetal lung. An overexpression of various estrogen-regulated genes also occurred, including trefoil factor-3, anterior gradient-2, and the steroid metabolism genes 17-β-hydroxysteroid dehydrogenase type 5 and aromatase. The insulin growth factor system, which can be influenced by ER and has been implicated in the pulmonary oncogenic process, was activated in fetal lung after gestational arsenic exposure. in utero arsenic exposure also induced overexpression of α-fetoprotein, epidermal growth factor receptor, L-myc, and metallothionein-1 in fetal lung, all of which are associated with lung cancer. Lung adenoma and adenocarcinoma from adult female mice exposed to arsenic in utero showed widespread, intense nuclear ER-α expression. In contrast, normal adult lung and diethylnitrosamine-induced lung adenocarcinoma showed little evidence of ER-α expression. Thus, transplacental arsenic exposure at a carcinogenic dose produced aberrant estrogen-linked pulmonary gene expression. ER-α activation was specifically associated with arsenic-induced lung adenocarcinoma and adenoma but not with nitrosamine-induced lung tumors. These data provide evidence that arsenic-induced aberrant ER signaling could disrupt early life stage genetic programing in the lung leading eventually to lung tumor formation much later in adulthood. PMID:17077188

Fetal onset of aberrant gene expression relevant to pulmonary carcinogenesis in lung adenocarcinoma development induced by in utero arsenic exposure.

PubMed

Shen, Jun; Liu, Jie; Xie, Yaxiong; Diwan, Bhalchandra A; Waalkes, Michael P

2007-02-01

Arsenic is a human pulmonary carcinogen. Our work indicates that in utero arsenic exposure in mice can induce or initiate lung cancer in female offspring. To define early molecular changes, pregnant C3H mice were given 85 ppm arsenic in drinking water from days 8 to 18 of gestation and expression of selected genes in the fetal lung or in lung tumors developing in adults was examined. Transplacental arsenic exposure increased estrogen receptor-alpha (ER-alpha) transcript and protein levels in the female fetal lung. An overexpression of various estrogen-regulated genes also occurred, including trefoil factor-3, anterior gradient-2, and the steroid metabolism genes 17-beta-hydroxysteroid dehydrogenase type 5 and aromatase. The insulin growth factor system, which can be influenced by ER and has been implicated in the pulmonary oncogenic process, was activated in fetal lung after gestational arsenic exposure. In utero arsenic exposure also induced overexpression of alpha-fetoprotein, epidermal growth factor receptor, L-myc, and metallothionein-1 in fetal lung, all of which are associated with lung cancer. Lung adenoma and adenocarcinoma from adult female mice exposed to arsenic in utero showed widespread, intense nuclear ER-alpha expression. In contrast, normal adult lung and diethylnitrosamine-induced lung adenocarcinoma showed little evidence of ER-alpha expression. Thus, transplacental arsenic exposure at a carcinogenic dose produced aberrant estrogen-linked pulmonary gene expression. ER-alpha activation was specifically associated with arsenic-induced lung adenocarcinoma and adenoma but not with nitrosamine-induced lung tumors. These data provide evidence that arsenic-induced aberrant ER signaling could disrupt early life stage genetic programing in the lung leading eventually to lung tumor formation much later in adulthood.

Inhibition of DNA synthesis by chemical carcinogens in cultures of initiated and normal proliferating rat hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Novicki, D.L.; Rosenberg, M.R.; Michalopoulos, G.

1985-01-01

Rat hepatocytes in primary culture can be stimulated to replicate under the influence of rat serum and sparse plating conditions. Higher replication rates are induced by serum from two-thirds partially hepatectomized rats. The effects of carcinogens and noncarcinogens on the ability of hepatocytes to synthesize DNA were examined by measuring the incorporation of (3H)thymidine by liquid scintillation counting and autoradiography. Hepatocyte DNA synthesis was not decreased by ethanol or dimethyl sulfoxide at concentrations less than 0.5%. No effect was observed when 0.1 mM ketamine, Nembutal, hypoxanthine, sucrose, ascorbic acid, or benzo(e)pyrene was added to cultures of replicating hepatocytes. Estrogen, testosterone,more » tryptophan, and vitamin E inhibited DNA synthesis by approximately 50% at 0.1 mM, a concentration at which toxicity was noticeable. Several carcinogens requiring metabolic activation as well as the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine interfered with DNA synthesis. Aflatoxin B1 inhibited DNA synthesis by 50% (ID50) at concentrations between 1 X 10(-8) and 1 X 10(-7) M. The ID50 for 2-acetylaminofluorene was between 1 X 10(-7) and 1 X 10(-6) M. Benzo(a)pyrene and 3'-methyl-4-dimethylaminoazobenzene inhibited DNA synthesis 50% between 1 X 10(-5) and 1 X 10(-4) M. Diethylnitrosamine and dimethylnitrosamine (ID50 between 1 X 10(-4) and 5 X 10(-4) M) and 1- and 2-naphthylamine (ID50 between 1 X 10(-5) and 5 X 10(-4) M) caused inhibition of DNA synthesis at concentrations which overlapped with concentrations that caused measurable toxicity.« less

Gpr110 deficiency decelerates carcinogen-induced hepatocarcinogenesis via activation of the IL-6/STAT3 pathway

PubMed Central

Ma, Benting; Zhu, Junjie; Tan, Juan; Mao, Yulei; Tang, Lingyun; Shen, Chunling; Zhang, Hongxing; Kuang, Ying; Fei, Jian; Yang, Xiao; Wang, Zhugang

2017-01-01

Hepatocarcinogenesis is a complex process that includes pronounced necroinflammation, unregulated hepatocyte damage, subsequent extensive fibrosis, and carcinogenesis. GPR110 was an adhesion G protein-coupled receptor. Analysis of the expression pattern of Gpr110 in mice displayed that Gpr110 was expressed highly in liver, implicating the tissue compartments where Gpr110 could execute its functions, the role of Gpr110 in the physiological and pathological state of liver remains unclear. Based on a Gpr110 knockout mouse model, we evaluated the role of Gpr110 in hepatocarcinogenesis by using a carbon tetrachloride (CCl4)-induced liver injury and fibrosis model, as well as diethylnitrosamine (DEN) plus CCl4-induced liver cancer model. In this study, we found subdued chronic liver injury, reduced compensatory proliferation, lower liver fibrosis, but enhanced inflammation occurred in Gpr110-/- mice during CCl4 challenge. In addition, Gpr110-/- mice were resistant to liver tumorigenesis induced by DEN plus CCl4 injection. Molecular mechanisms underlying these differences correlated with augmented activation of the IL-6/STAT3 pathway, which exerted hepatoprotective effects during liver damage, fibrosis, and oncogenesis in Gpr110-/- mice. Furthermore, pharmacological inhibition of the activation of the IL-6/STAT3 pathway enhanced hepatic fibrosis and promoted DEN plus CCl4-induced carcinogenesis in Gpr110-/- mice. In summary, absence of Gpr110 decelerates liver fibrosis/cirrhosis progressing into tumorigenesis, due to strengthening activation of the IL-6/STAT3 pathway, leading to a weaker liver injury and fibrosis microenvironment. It is indicated that targeting Gpr110 and activating the IL-6/STAT3 pathway may be considered to be preventive methods for some cirrhosis transition. PMID:28401002

Chicken Fetal Liver DNA Damage and Adduct Formation by Activation-Dependent DNA-Reactive Carcinogens and Related Compounds of Several Structural Classes

PubMed Central

Williams, Gary M.; Duan, Jian-Dong; Brunnemann, Klaus D.; Iatropoulos, Michael J.; Vock, Esther; Deschl, Ulrich

2014-01-01

The chicken egg genotoxicity assay (CEGA), which utilizes the liver of an intact and aseptic embryo-fetal test organism, was evaluated using four activation-dependent DNA-reactive carcinogens and four structurally related less potent carcinogens or non-carcinogens. In the assay, three daily doses of test substances were administered to eggs containing 9–11-day-old fetuses and the fetal livers were assessed for two endpoints, DNA breaks using the alkaline single cell gel electrophoresis (comet) assay and DNA adducts using the 32P-nucleotide postlabeling (NPL) assay. The effects of four carcinogens of different structures requiring distinct pathways of bioactivation, i.e., 2-acetylaminofluorene (AAF), aflatoxin B1 (AFB1), benzo[a]pyrene (B[a]P), and diethylnitrosamine (DEN), were compared with structurally related non-carcinogens fluorene (FLU) and benzo[e]pyrene (B[e]P) or weak carcinogens, aflatoxin B2 (AFB2) and N-nitrosodiethanolamine (NDELA). The four carcinogens all produced DNA breaks at microgram or low milligram total doses, whereas less potent carcinogens and non-carcinogens yielded borderline or negative results, respectively, at higher doses. AAF and B[a]P produced DNA adducts, whereas none was found with the related comparators FLU or B[e]P, consistent with comet results. DEN and NDELA were also negative for adducts, as expected in the case of DEN for an alkylating agent in the standard NPL assay. Also, AFB1 and AFB2 were negative in NPL, as expected, due to the nature of ring opened aflatoxin adducts, which are resistant to enzymatic digestion. Thus, the CEGA, using comet and NPL, is capable of detection of the genotoxicity of diverse DNA-reactive carcinogens, while not yielding false positives for non-carcinogens. PMID:24973097

Diphenylarsinic acid, a chemical warfare-related neurotoxicant, promotes liver carcinogenesis via activation of aryl hydrocarbon receptor signaling and consequent induction of oxidative DNA damage in rats.

PubMed

Wei, Min; Yamada, Takanori; Yamano, Shotaro; Kato, Minoru; Kakehashi, Anna; Fujioka, Masaki; Tago, Yoshiyuki; Kitano, Mistuaki; Wanibuchi, Hideki

2013-11-15

Diphenylarsinic acid (DPAA), a chemical warfare-related neurotoxic organic arsenical, is present in the groundwater and soil in some regions of Japan due to illegal dumping after World War II. Inorganic arsenic is carcinogenic in humans and its organic arsenic metabolites are carcinogenic in animal studies, raising serious concerns about the carcinogenicity of DPAA. However, the carcinogenic potential of DPAA has not yet been evaluated. In the present study we found that DPAA significantly enhanced the development of diethylnitrosamine-induced preneoplastic lesions in the liver in a medium-term rat liver carcinogenesis assay. Evaluation of the expression of cytochrome P450 (CYP) enzymes in the liver revealed that DPAA induced the expression of CYP1B1, but not any other CYP1, CYP2, or CYP3 enzymes, suggesting that CYP1B1 might be the enzyme responsible for the metabolic activation of DPAA. We also found increased oxidative DNA damage, possibly due to elevated CYP1B1 expression. Induction of CYP1B1 has generally been linked with the activation of AhR, and we found that DPAA activates the aryl hydrocarbon receptor (AhR). Importantly, the promotion effect of DPAA was observed only at a dose that activated the AhR, suggesting that activation of AhR and consequent induction of AhR target genes and oxidative DNA damage plays a vital role in the promotion effects of DPAA. The present study provides, for the first time, evidence regarding the carcinogenicity of DPAA and indicates the necessity of comprehensive evaluation of its carcinogenic potential using long-term carcinogenicity studies. © 2013.

Clock gene Per2 as a controller of liver carcinogenesis

PubMed Central

Mteyrek, Ali; Filipski, Elisabeth; Guettier, Catherine; Okyar, Alper; Lévi, Francis

2016-01-01

Environmental disruption of molecular clocks promoted liver carcinogenesis and accelerated cancer progression in rodents. We investigated the specific role of clock gene Period 2 (Per2) for liver carcinogenesis and clock-controlled cellular proliferation, genomic instability and inflammation. We assessed liver histopathology, and determined molecular and physiology circadian patterns in mice on chronic diethylnitrosamine (DEN) exposure according to constitutive Per2 mutation. First, we found that Per2m/m liver displayed profound alterations in proliferation gene expression, including c-Myc derepression, phase-advanced Wee1, and arrhythmic Ccnb1 and K-ras mRNA expressions, as well as deregulated inflammation, through arrhythmic liver IL-6 protein concentration, in the absence of any DEN exposure. These changes could then make Per2m/m mice more prone to subsequently develop liver cancers on DEN. Indeed, primary liver cancers were nearly fourfold as frequent in Per2m/m mice as compared to wild-type (WT), 4 months after DEN exposure. The liver molecular clock was severely disrupted throughout the whole carcinogenesis process, including the initiation stage, i.e. within the initial 17 days on DEN. Per2m/m further exhibited increased c-Myc and Ccnb1 mean 24h expressions, lack of P53 response, and arrhythmic ATM, Wee1 and Ccnb1 expressions. DEN-induced tumor related inflammation was further promoted through increased protein concentrations of liver IL-6 and TNF-α as compared to WT during carcinogenesis initiation. Per2 mutation severely deregulated liver gene or protein expressions related to three cancer hallmarks, including uncontrolled proliferation, genomic instability, and tumor promoting inflammation, and accelerated liver carcinogenesis several-fold. Clock gene Per2 acted here as a liver tumor suppressor from initiation to progression. PMID:27494874

Next-generation proteasome inhibitor oprozomib synergizes with modulators of the unfolded protein response to suppress hepatocellular carcinoma.

PubMed

Vandewynckel, Yves-Paul; Coucke, Céline; Laukens, Debby; Devisscher, Lindsey; Paridaens, Annelies; Bogaerts, Eliene; Vandierendonck, Astrid; Raevens, Sarah; Verhelst, Xavier; Van Steenkiste, Christophe; Libbrecht, Louis; Geerts, Anja; Van Vlierberghe, Hans

2016-06-07

Hepatocellular carcinoma (HCC) responds poorly to conventional systemic therapies. The first-in-class proteasome inhibitor bortezomib has been approved in clinical use for hematologic malignancies and has shown modest activity in solid tumors, including HCC. However, a considerable proportion of patients fail to respond and experience important adverse events. Recently, the next-generation orally bioavailable irreversible proteasome inhibitor oprozomib was developed. Here, we assessed the efficacy of oprozomib and its effects on the unfolded protein response (UPR), a signaling cascade activated through the ATF6, PERK and IRE1 pathways by accumulation of unfolded proteins in the endoplasmic reticulum, in HCC. The effects of oprozomib and the role of the UPR were evaluated in HCC cell lines and in diethylnitrosamine-induced and xenograft mouse models for HCC. Oprozomib dose-dependently reduced the viability and proliferation of human HCC cells. Unexpectedly, oprozomib-treated cells displayed diminished cytoprotective ATF6-mediated signal transduction as well as unaltered PERK and IRE1 signaling. However, oprozomib increased pro-apoptotic UPR-mediated protein levels by prolonging their half-life, implying that the proteasome acts as a negative UPR regulator. Supplementary boosting of UPR activity synergistically improved the sensitivity to oprozomib via the PERK pathway. Oral oprozomib displayed significant antitumor effects in the orthotopic and xenograft models for HCC, and importantly, combining oprozomib with different UPR activators enhanced the antitumor efficacy by stimulating UPR-induced apoptosis without cumulative toxicity. In conclusion, next-generation proteasome inhibition by oprozomib results in dysregulated UPR activation in HCC. This finding can be exploited to enhance the antitumor efficacy by combining oprozomib with clinically applicable UPR activators.

Potential Molecular Targets of Statins in the Prevention of Hepatocarcinogenesis.

PubMed

Ridruejo, Ezequiel; Romero-Caími, Giselle; Obregón, María J; Kleiman de Pisarev, Diana; Alvarez, Laura

2018-04-09

Hepatocellular carcinoma (HCC) represents 90% of liver tumors. Statins, may reduce the incidence of various tumors, including HCC. Antitumoral activities may be mediated by changes in transforming growth factor-beta (TGF-β1) and thyroid hormones (TH) regulation. The aim of our study is to establish the statins mechanism of action and the potential key molecules involved in an in vivo and in vitro HCC model. We used two models: in vivo (in rats) using diethylnitrosamine (DEN) and hexachlorobenzene (HCB) to develop HCC, we analyzed cell proliferation parameters (proliferating cell nuclear antigen, PCNA) and cholesterol metabolism (hydroxy-methylglutaryl-CoA reductase, HMGCoAR). In vitro (Hep-G2 cells) we evaluated the effects of different doses of Atorvastatin (AT) and Simvastatin (SM) on HCB induced proliferation and analyzed proliferative parameters, colesterol metabolism, TGF-β1 mRNA, c-Src and TH levels. In vivo, we observed that cell proliferation significantly increased as well as cholesterol serum levels in rats treated with HCB. In vitro, we observed the same results on PCNA as in vivo. The statins prevented the increase in HMG-CoAR mRNA levels induced by HCB, reaching levels similar to controls at máximum doses: AT (30 μM), and SM (20 μM). Increases in PCNA, TGF-β1, and pc-Src, and decreases in deiodinase I mRNA levels induced by HCB were not observed when cells were pre-treated with AT and SM at maximum doses. Statins can prevent the proliferative HCB effects on Hep-G2 cells. TGF-β1, c-Src and TH may be the statins molecular targets in hepatocarcinogenesis.

Predominant modifier of extreme liver cancer susceptibility in C57BR/cdJ female mice localized to 6 Mb on chromosome 17

PubMed Central

Peychal, Stephanie E.-M.; Bilger, Andrea; Pitot, Henry C.; Drinkwater, Norman R.

2009-01-01

Sex hormones influence the susceptibility of inbred mice to liver cancer. C57BR/cdJ (BR) females are extremely susceptible to spontaneous and chemically induced liver tumors, in part due to a lack of protection against hepatocarcinogenesis normally offered by ovarian hormones. BR males are also moderately susceptible, and the susceptibility of both sexes of BR mice to liver tumors induced with N,N-diethylnitrosamine relative to the resistant C57BL/6J (B6) strain is caused by two loci designated Hcf1 and Hcf2 (hepatocarcinogenesis in females) located on chromosomes 17 and 1, respectively. The Hcf1 locus on chromosome 17 is the predominant modifier of liver cancer in BR mice. To validate the existence of this locus and investigate its potential interaction with Hcf2, congenic mice for each region were generated. Homozygosity for the B6.BR(D17Mit164-D17Mit2) region resulted in a 4-fold increase in liver tumor multiplicity in females and a 4.5-fold increase in males compared with B6 controls. A series of 16 recombinants covering the entire congenic region was developed to further narrow the area containing Hcf1. Susceptible heterozygous recombinants demonstrated a 3- to 7-fold effect in females and a 1.5- to 2-fold effect in males compared with B6 siblings. The effect in susceptible lines completely recapitulated the susceptibility of heterozygous full-length chromosome 17 congenics and furthermore narrowed the location of the Hcf1 locus to a single region of the chromosome from 30.05 to 35.83 Mb. PMID:19255062

Predominant modifier of extreme liver cancer susceptibility in C57BR/cdJ female mice localized to 6 Mb on chromosome 17.

PubMed

Peychal, Stephanie E-M; Bilger, Andrea; Pitot, Henry C; Drinkwater, Norman R

2009-05-01

Sex hormones influence the susceptibility of inbred mice to liver cancer. C57BR/cdJ (BR) females are extremely susceptible to spontaneous and chemically induced liver tumors, in part due to a lack of protection against hepatocarcinogenesis normally offered by ovarian hormones. BR males are also moderately susceptible, and the susceptibility of both sexes of BR mice to liver tumors induced with N,N-diethylnitrosamine relative to the resistant C57BL/6J (B6) strain is caused by two loci designated Hcf1 and Hcf2 (hepatocarcinogenesis in females) located on chromosomes 17 and 1, respectively. The Hcf1 locus on chromosome 17 is the predominant modifier of liver cancer in BR mice. To validate the existence of this locus and investigate its potential interaction with Hcf2, congenic mice for each region were generated. Homozygosity for the B6.BR(D17Mit164-D17Mit2) region resulted in a 4-fold increase in liver tumor multiplicity in females and a 4.5-fold increase in males compared with B6 controls. A series of 16 recombinants covering the entire congenic region was developed to further narrow the area containing Hcf1. Susceptible heterozygous recombinants demonstrated a 3- to 7-fold effect in females and a 1.5- to 2-fold effect in males compared with B6 siblings. The effect in susceptible lines completely recapitulated the susceptibility of heterozygous full-length chromosome 17 congenics and furthermore narrowed the location of the Hcf1 locus to a single region of the chromosome from 30.05 to 35.83 Mb.

Nardilysin promotes hepatocellular carcinoma through activation of signal transducer and activator of transcription 3.

PubMed

Kasai, Yosuke; Toriguchi, Kan; Hatano, Etsuro; Nishi, Kiyoto; Ohno, Mikiko; Yoh, Tomoaki; Fukuyama, Keita; Nishio, Takahiro; Okuno, Masayuki; Iwaisako, Keiko; Seo, Satoru; Taura, Kojiro; Kurokawa, Masato; Kunichika, Makoto; Uemoto, Shinji; Nishi, Eiichiro

2017-05-01

Nardilysin (NRDC) is a metalloendopeptidase of the M16 family. We previously showed that NRDC activates inflammatory cytokine signaling, including interleukin-6-signal transducer and activator of transcription 3 (STAT3) signaling. NRDC has been implicated in the promotion of breast, gastric and esophageal cancer, as well as the development of liver fibrosis. In this study, we investigated the role of NRDC in the promotion of hepatocellular carcinoma (HCC), both clinically and experimentally. We found that NRDC expression was upregulated threefold in HCC tissue compared to the adjacent non-tumor liver tissue, which was confirmed by immunohistochemistry and western blotting. We also found that high serum NRDC was associated with large tumor size (>3 cm, P = 0.016) and poor prognosis after hepatectomy (median survival time 32.0 vs 73.9 months, P = 0.003) in patients with hepatitis C (n = 120). Diethylnitrosamine-induced hepatocarcinogenesis was suppressed in heterozygous NRDC-deficient mice compared to their wild-type littermates. Gene silencing of NRDC with miRNA diminished the growth of Huh-7 and Hep3B spheroids in vitro. Notably, phosphorylation of STAT3 was decreased in NRDC-depleted Huh-7 spheroids compared to control spheroids. The effect of a STAT3 inhibitor (S3I-201) on the growth of Huh-7 spheroids was reduced in NRDC-depleted cells relative to controls. Our results show that NRDC is a promising prognostic marker for HCC in patients with hepatitis C, and that NRDC promotes tumor growth through activation of STAT3. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Valerian Inhibits Rat Hepatocarcinogenesis by Activating GABA(A) Receptor-Mediated Signaling

PubMed Central

Kakehashi, Anna; Kato, Ayumi; Ishii, Naomi; Wei, Min; Morimura, Keiichirou; Fukushima, Shoji; Wanibuchi, Hideki

2014-01-01

Valerian is widely used as a traditional medicine to improve the quality of sleep due to interaction of several active components with the γ-aminobutyric acid (GABA) A receptor (GABA(A)R) system. Recently, activation of GABA signaling in stem cells has been reported to suppress cell cycle progression in vivo. Furthermore, possible inhibitory effects of GABA(A)R agonists on hepatocarcinogenesis have been reported. The present study was performed to investigate modulating effects of Valerian on hepatocarcinogenesis using a medium-term rat liver bioassay. Male F344 rats were treated with one of the most powerful Valerian species (Valeriana sitchensis) at doses of 0, 50, 500 and 5000 ppm in their drinking water after initiation of hepatocarcinogenesis with diethylnitrosamine (DEN). Formation of glutathione S-transferase placental form positive (GST-P+) foci was significantly inhibited by Valerian at all applied doses compared with DEN initiation control rats. Generation of 8-hydroxy-2′-deoxyguanosine in the rat liver was significantly suppressed by all doses of Valerian, likely due to suppression of Nrf2, CYP7A1 and induction of catalase expression. Cell proliferation was significantly inhibited, while apoptosis was induced in areas of GST-P+ foci of Valerian groups associated with suppression of c-myc, Mafb, cyclin D1 and induction of p21Waf1/Cip1, p53 and Bax mRNA expression. Interestingly, expression of the GABA(A)R alpha 1 subunit was observed in GST-P+ foci of DEN control rats, with significant elevation associated with Valerian treatment. These results indicate that Valerian exhibits inhibitory effects on rat hepatocarcinogenesis by inhibiting oxidative DNA damage, suppressing cell proliferation and inducing apoptosis in GST-P+ foci by activating GABA(A)R-mediated signaling. PMID:25419570

Genotoxic and carcinogenic risks associated with the dietary consumption of repeatedly heated coconut oil.

PubMed

Srivastava, Smita; Singh, Madhulika; George, Jasmine; Bhui, Kulpreet; Murari Saxena, Anand; Shukla, Yogeshwer

2010-11-01

Repeated heating of vegetable oils at high temperatures during cooking is a very common cooking practice. Repeated heating of edible oils can generate a number of compounds, including polycyclic aromatic hydrocarbons (PAH), some of which have been reported to have carcinogenic potential. Consumption of these repeatedly heated oils can pose a serious health hazard. The objectives of the present study were to evaluate the genotoxic and carcinogenic risks associated with the consumption of repeatedly heated coconut oil (RCO), which is one of the commonly consumed cooking and frying medium. The PAH were analysed using HPLC in fresh CO, single-heated CO (SCO) and RCO. Results revealed the presence of certain PAH, known to possess carcinogenic potential, in RCO when compared with SCO. Oral intake of RCO in Wistar rats resulted in a significant induction of aberrant cells (P<0·05) and micronuclei (P<0·05) in a dose-dependent manner. Oxidative stress analysis showed a significant (P<0·05) decrease in the levels of antioxidant enzymes such as superoxide dismutase and catalase with a concurrent increase in reactive oxygen species and lipid peroxidation in the liver. In addition, RCO given alone and along with diethylnitrosamine for 12 weeks induced altered hepatic foci as noticed by alteration in positive (γ-glutamyl transpeptidase and glutathione-S-transferase) and negative (adenosine triphosphatase, alkaline phosphatase and glucose-6-phosphatase) hepatospecific biomarkers. A significant decrease in the relative and absolute hepatic weight of RCO-supplemented rats was recorded (P<0·05). In conclusion, dietary consumption of RCO can cause a genotoxic and preneoplastic change in the liver.

DNA Alkylating Agent Protects Against Spontaneous Hepatocellular Carcinoma Regardless of O6-Methylguanine-DNA Methyltransferase Status.

PubMed

Herzig, Maryanne C S; Zavadil, Jessica A; Street, Karah; Hildreth, Kim; Drinkwater, Norman R; Reddick, Traci; Herbert, Damon C; Hanes, Martha A; McMahan, C Alex; Reddick, Robert L; Walter, Christi A

2016-03-01

Hepatocellular carcinoma is increasingly important in the United States as the incidence rate rose over the last 30 years. C3HeB/FeJ mice serve as a unique model to study hepatocellular carcinoma tumorigenesis because they mimic human hepatocellular carcinoma with delayed onset, male gender bias, approximately 50% incidence, and susceptibility to tumorigenesis is mediated through multiple genetic loci. Because a human O(6)-methylguanine-DNA methyltransferase (hMGMT) transgene reduces spontaneous tumorigenesis in this model, we hypothesized that hMGMT would also protect from methylation-induced hepatocarcinogenesis. To test this hypothesis, wild-type and hMGMT transgenic C3HeB/FeJ male mice were treated with two monofunctional alkylating agents: diethylnitrosamine (DEN; 0.025 μmol/g body weight) on day 12 of life with evaluation for glucose-6-phosphatase-deficient (G6PD) foci at 16, 24, and 32 weeks or N-methyl-N-nitrosurea (MNU; 25 mg MNU/kg body weight) once monthly for 7 months starting at 3 months of age with evaluation for liver tumors at 12 to 15 months of age. No difference in abundance or size of G6PD foci was measured with DEN treatment. In contrast, it was unexpectedly found that MNU reduces liver tumor prevalence in wild-type and hMGMT transgenic mice despite increased tumor prevalence in other tissues. hMGMT and MNU protections were additive, suggesting that MNU protects through a different mechanism, perhaps through the cytotoxic N7-alkylguanine and N3-alkyladenine lesions which have low mutagenic potential compared with O(6)-alkylguanine lesions. Together, these results suggest that targeting the repair of cytotoxic lesions may be a good preventative for patients at high risk of developing hepatocellular carcinoma. ©2015 American Association for Cancer Research.

The Association of Peroxiredoxin 4 with the Initiation and Progression of Hepatocellular Carcinoma.

PubMed

Guo, Xin; Noguchi, Hirotsugu; Ishii, Naoki; Homma, Takujiro; Hamada, Taiji; Hiraki, Tsubasa; Zhang, Jing; Matsuo, Kei; Yokoyama, Seiya; Ishibashi, Hiroaki; Fukushige, Tomoko; Kanekura, Takuro; Fujii, Junichi; Uramoto, Hidetaka; Tanimoto, Akihide; Yamada, Sohsuke

2018-06-11

Peroxiredoxin 4 (PRDX4) is a member of the peroxiredoxin family of antioxidant enzymes. Previously, we reported that PRDX4 can restrain the initiation and progression of nonalcoholic steatohepatitis by reducing local and systemic reactive oxygen species (ROS) levels. Oxidative stress is recognized as a key factor in hepatocarcinogenesis, and a high ROS level has also been found in hepatocellular carcinoma (HCC). Here, our aim is to investigate roles of PRDX4 in the initiation and progression of HCC. In this study, for hepatocarcinogenesis, wild-type (WT), PRDX4 knockout (PRDX4 -/y ), and human PRDX4 transgenic (hPRDX4 +/+ ) mice were given a weekly intraperitoneal injection of diethylnitrosamine for 25 weeks. The HCC incidence was higher in PRDX4 -/y mice than in WT or hPRDX4 +/+ mice. Intrahepatic and circulating oxidative stress and inflammatory cell infiltration in the liver were obviously decreased in hPRDX4 +/+ mice, compared with WT mice. Furthermore, in our cohort study, human HCC specimens with low expression of PRDX4 had higher ROS levels and a highly malignant phenotype, which was associated with a reduced overall survival, compared with those with high PRDX4 expression. However, in human HCC cell lines, PRDX4 knockdown led to a rapidly increased intracellular ROS level and suppressed cell proliferation, inducing cell death. Innovation and Conclusion: Our results clearly indicate that PRDX4 has an inhibitory effect in the initiation of HCC, but a dual (inhibitory or promoting) role in the progression of HCC, suggesting the potential utility of PRDX4 activators or inhibitors as therapy for different stages and phenotypes of HCC. Antioxid. Redox Signal. 00, 000-000.

Diphenylarsinic acid, a chemical warfare-related neurotoxicant, promotes liver carcinogenesis via activation of aryl hydrocarbon receptor signaling and consequent induction of oxidative DAN damage in rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wei, Min; Yamada, Takanori; Yamano, Shotaro

2013-11-15

Diphenylarsinic acid (DPAA), a chemical warfare-related neurotoxic organic arsenical, is present in the groundwater and soil in some regions of Japan due to illegal dumping after World War II. Inorganic arsenic is carcinogenic in humans and its organic arsenic metabolites are carcinogenic in animal studies, raising serious concerns about the carcinogenicity of DPAA. However, the carcinogenic potential of DPAA has not yet been evaluated. In the present study we found that DPAA significantly enhanced the development of diethylnitrosamine-induced preneoplastic lesions in the liver in a medium-term rat liver carcinogenesis assay. Evaluation of the expression of cytochrome P450 (CYP) enzymes inmore » the liver revealed that DPAA induced the expression of CYP1B1, but not any other CYP1, CYP2, or CYP3 enzymes, suggesting that CYP1B1 might be the enzyme responsible for the metabolic activation of DPAA. We also found increased oxidative DNA damage, possibly due to elevated CYP1B1 expression. Induction of CYP1B1 has generally been linked with the activation of AhR, and we found that DPAA activates the aryl hydrocarbon receptor (AhR). Importantly, the promotion effect of DPAA was observed only at a dose that activated the AhR, suggesting that activation of AhR and consequent induction of AhR target genes and oxidative DNA damage plays a vital role in the promotion effects of DPAA. The present study provides, for the first time, evidence regarding the carcinogenicity of DPAA and indicates the necessity of comprehensive evaluation of its carcinogenic potential using long-term carcinogenicity studies. - Highlights: • DPAA, an environmental neurotoxicant, promotes liver carcinogenesis in rats. • DPAA is an activator of AhR signaling pathway. • DPAA promoted oxidative DNA damage in rat livers. • AhR target gene CYP 1B1 might be involved in the metabolism of DPAA.« less

Efficacy, Safety and Anticancer Activity of Protein Nanoparticle-Based Delivery of Doxorubicin through Intravenous Administration in Rats

PubMed Central

Golla, Kishore; Cherukuvada, Bhaskar; Ahmed, Farhan; Kondapi, Anand K.

2012-01-01

Background and Aims Doxorubicin is a potent anticancer drug and a major limiting factor that hinders therapeutic use as its high levels of systemic circulation often associated with various off-target effects, particularly cardiotoxicity. The present study focuses on evaluation of the efficacy of doxorubicin when it is loaded into the protein nanoparticles and delivered intravenously in rats bearing Hepatocellular carcinoma (HCC). The proteins selected as carrier were Apotransferrin and Lactoferrin, since the receptors for these two proteins are known to be over expressed on cancer cells due to their iron transport capacity. Methods Doxorubicin loaded apotransferrin (Apodoxonano) and lactoferrin nanoparticles (Lactodoxonano) were prepared by sol-oil chemistry. HCC in the rats was induced by 100 mg/l of diethylnitrosamine (DENA) in drinking water for 8 weeks. Rats received 5 doses of 2 mg/kg drug equivalent nanoparticles through intravenous administration. Pharmacokinetics and toxicity of nanoformulations was evaluated in healthy rats and anticancer activity was studied in DENA treated rats. The anticancer activity was evaluated through counting of the liver nodules, H & E analysis and by estimating the expression levels of angiogenic and antitumor markers. Results In rats treated with nanoformulations, the numbers of liver nodules were found to be significantly reduced. They showed highest drug accumulation in liver (22.4 and 19.5 µg/g). Both nanoformulations showed higher localization compared to doxorubicin (Doxo) when delivered in the absence of a carrier. Higher amounts of Doxo (195 µg/g) were removed through kidney, while Apodoxonano and Lactodoxonano showed only a minimal amount of removal (<40 µg/g), suggesting the extended bioavailability of Doxo when delivered through nanoformulation. Safety analysis shows minimal cardiotoxicity due to lower drug accumulation in heart in the case of nanoformulation. Conclusion Drug delivery through nanoformulations not only minimizes the cardiotoxicity of doxorubicin but also enhances the efficacy and bioavailability of the drug in a target-specific manner. PMID:23284832

A Target-Specific Oral Formulation of Doxorubicin-Protein Nanoparticles: Efficacy and Safety in Hepatocellular Cancer

PubMed Central

Golla, Kishore; Bhaskar, Cherukuvada; Ahmed, Farhan; Kondapi, Anand K.

2013-01-01

Background/Aims: Hepatocellular carcinoma (HCC) also known as malignant hepatoma is a most common liver cancer. Doxorubicin (Doxo) is an anti-cancer drug having activity against a wide spectrum of cancer types. Clinical Utility of doxo has been limited due to its poor bioavailability and toxicity to heart and spleen. Furthermore, cancer chemotherapeutics have limited oral absorption. Transferrin family proteins are highly abundant and plays important role in transport and storage of iron in cells and tissues. Since apotransferrin and lactoferrin receptors are highly expressed on the surface of metabolically active cancer cells, the principal objective of present study is to evaluate efficacy of doxorubicin loaded apotransferrin and lactoferrin nanoparticles (apodoxonano or lactodoxonano) in oral treatment of HCC in rats. Study Design: HCC was induced in rats by supplementing 100 mg/L of diethylnitrosamine (DENA) in drinking water for 8 weeks. A week after the last day of DENA administration, rats were divided into four groups, each group comprising of five animals. Each group was administered with one of the drug viz., saline, doxorubicin (doxo), apodoxonano and lactodoxonano (4 mg/ kg equivalent of drug). In each case, they received 8 doses of the drug orally with six day interval. One week after the last dose, anticancer activity was evaluated by counting the liver nodules, H & E analysis of tissue sections and expression levels of angiogenic and antitumor markers. Results: In rats treated with apodoxonano and lactodoxonano, the number of neoplastic nodules was significantly lower than that of rats administered with saline or with doxo. Apodoxonano and lactodoxonano did not exhibit decrease in mean body weight, which was markedly reduced by 22% in the case of doxo administered rats. In rats treated with nanoformulations, the number of liver nodules was found reduced by >93%. Both nanoformulations showed significantly high localization in liver compared to doxo. Conclusions: Apodoxonano and lactodoxonano showed improved efficacy, bioavailability and safety compared to doxo for treatment of HCC in rats when administered orally. PMID:24155776

Characterization of an acute molecular marker of nongenotoxic rodent hepatocarcinogenesis by gene expression profiling in a long term clofibric acid study.

PubMed

Michel, Cécile; Roberts, Ruth A; Desdouets, Chantal; Isaacs, Kevin R; Boitier, Eric

2005-04-01

Evaluation of the nongenotoxic potential early during the development of a drug presents a major challenge. Recently, two genes were identified as potential molecular markers of rodent hepatic carcinogenesis: transforming growth factor-beta stimulated clone 22 (TSC-22) and NAD(P)H cytochrome P450 oxidoreductase (CYP-R) (1). They were identified after comparing the gene expression profiles obtained from the livers of Sprague-Dawley rats treated with different genotoxic and nongenotoxic compounds in a 5 day repeat dose in vivo study. To assess the potential of these two genes as acute markers of carcinogenesis, we investigated their modulation during a long-term nongenotoxic study in the rat using a classic initiation-promotion regime. Clofibric acid (CLO), which belongs to the broad class of chemicals known as peroxisome proliferators, was used as a nongenotoxic hepatocarcinogen. Male F344 rats were given a single nonnecrogenic injection of diethylnitrosamine (0 or 30 mg/kg) and fed a diet containing none or 5000 ppm CLO for up to 20 months. Necropsies of five rats per groups were performed at 18, 46, 102, 264, 377, 447 (control, DEN, and DEN + CLO rats), 524, and 608 days (for the CLO and control rats). Gross macroscopic and microscopic evaluation and gene expression profiling (on Affymetrix microarrays) were performed in peritumoral and tumoral liver tissues. Bioanalysis of the liver gene expression data revealed that TSC-22 was strongly down-regulated early in the study. Its underexpression was maintained throughout the study but disappeared upon CLO withdrawal. These modulations were confirmed by real-time polymerase chain reaction. However, CYP-R gene expression was not significantly altered in our study. Taken together, our results showed that TSC-22, but not CYP-R, has the potential to be an acute early molecular marker for nongenotoxic hepatocarcinogenesis in rodents.

Enhancement of preneoplastic lesion yield by Chios Mastic Gum in a rat liver medium-term carcinogenesis bioassay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Doi, Kenichiro; Wei, Min; Kitano, Mitsuaki

2009-01-01

The mastic (Pistacia lentiscus var. chia) tree is native throughout the Mediterranean region and has long proved a source of food additives and medical treatments. To investigate the modifying effects of Chios Mastic Gum on rat liver carcinogenesis, 6-week-old male F344 rats were subjected to the established rat liver medium-term carcinogenesis bioassay (Ito-test). At the commencement, rats (groups 1-4) were intraperitoneally injected with 200 mg/kg body weight of diethylnitrosamine (DEN). After two weeks, mastic was added to CRF (Charles River Formula)-1 powdered basal diet at doses of 0, 0.01, 0.1 and 1% in groups 1-4, respectively. At week 3, allmore » rats were underwent two-thirds partial hepatectomy. The experiment was terminated at week 8. As results show, liver weights were significantly increased in a mastic dose-dependent manner among groups 1-4. The numbers (/cm{sup 2}) and the areas (mm{sup 2}/cm{sup 2}) of glutathione S-transferase placental form (GST-P)-positive cell foci ({>=} 0.2 mm in diameter) were significantly increased in the DEN-1% group compared to the DEN-alone group, along with the average areas per foci and larger-sized foci ({>=} 0.4 mm). 5-Bromo-2'-deoxyuridine (BrdU) + GST-P double-immunohistochemistry showed the highest BrdU-labeling indices within GST-P foci in the DEN-1% group. 8-hydroxydeoxyguanosine (8-OHdG) levels in liver DNA did not vary, while real-time quantitative polymerase chain reaction (PCR) analysis of livers revealed many up- or down-regulated genes in the DEN-1% group. In conclusion, this is the first report to display a promotion potential of Chios Mastic Gum on the formation of preneoplastic lesions in the established rat liver medium-term carcinogenesis bioassay.« less

Lack of gp130 expression in hepatocytes attenuates tumor progression in the DEN model.

PubMed

Hatting, M; Spannbauer, M; Peng, J; Al Masaoudi, M; Sellge, G; Nevzorova, Y A; Gassler, N; Liedtke, C; Cubero, F J; Trautwein, C

2015-03-05

Chronic liver inflammation is a crucial event in the development and growth of hepatocellular carcinoma (HCC). Compelling evidence has shown that interleukin-6 (IL-6)/gp130-dependent signaling has a fundamental role in liver carcinogenesis. Thus, in the present study we aimed to investigate the role of gp130 in hepatocytes for the initiation and progression of HCC. Hepatocyte-specific gp130 knockout mice (gp130(Δhepa)) and control animals (gp130(f/f)) were treated with diethylnitrosamine (DEN). The role of gp130 for acute injury (0-144 h post treatment), tumor initiation (24 weeks) and progression (40 weeks) was analyzed. After acute DEN-induced liver injury we observed a reduction in the inflammatory response in gp130(Δhepa) animals as reflected by decreased levels of IL-6 and oncostatin M. The loss of gp130 slightly attenuated the initiation of HCC 24 weeks after DEN treatment. In contrast, 40 weeks after DEN treatment, male and female gp130(Δhepa) mice showed smaller tumors and reduced tumor burden, indicating a role for hepatocyte-specific gp130 expression during HCC progression. Oxidative stress and DNA damage were substantially and similarly increased by DEN in both gp130(f/f) and gp130(Δhepa) animals. However, gp130(Δhepa) livers revealed aberrant STAT5 activation and decreased levels of transforming growth factor-β (TGFβ), pSMAD2/3 and SMAD2, whereas phosphorylation of STAT3 at Tyr705 and Ser727 was absent. Our results indicate that gp130 deletion in hepatocytes reduces progression, but not HCC initiation in the DEN model. Gp130 deletion resulted in STAT3 inhibition but increased STAT5 activation and diminished TGF-dependent signaling. Hence, blocking gp130 in hepatocytes might be an interesting therapeutic target to inhibit the growth of HCC.

Flow cytometric method for scoring rat liver micronuclei with simultaneous assessments of hepatocyte proliferation.

PubMed

Avlasevich, Svetlana L; Khanal, Sumee; Singh, Priyanka; Torous, Dorothea K; Bemis, Jeffrey C; Dertinger, Stephen D

2018-04-01

The current report describes a newly devised method for automatically scoring the incidence of rat hepatocyte micronuclei (MNHEP) via flow cytometry, with concurrent assessments of hepatocyte proliferation-frequency of Ki-67-positive nuclei, and the proportion of polyploid nuclei. Proof-of-concept data are provided from experiments performed with 6-week old male Crl:CD(SD) rats exposed to diethylnitrosamine (DEN) or quinoline (QUIN) for 3 or 14 consecutive days. Non-perfused liver tissue was collected 4 days after cessation of treatment in the case of 3-day studies, or 1 day after last administration in the case of 14-day studies for processing and flow cytometric analysis. In addition to livers, blood samples were collected one day after final treatment for micronucleated reticulocyte (MN-RET) measurements. Dose-dependent increases in MNHEP, Ki-67-positive nuclei, and polyploidy were observed in 3- and 14-day DEN studies. Both treatment schedules resulted in elevated %MNHEP for QUIN-exposed rats, and while cell proliferation effects were subtle, appreciable increases to normalized liver weights were observed. Whereas DEN caused markedly higher %MNHEP when exposure was extended to two weeks, QUIN-induced MNHEP were slightly increased with protracted dosing. Parallel microscopy-based MNHEP frequencies were highly correlated with flow cytometry-based measurements (four study/aggregate R 2  = 0.80). No increases in MN-RET were seen in any of the four studies. Collectively, these results suggest liver micronuclei are amenable to an automated scoring technique that provides objective analyses and higher information content relative to conventional microscopy. Additional work is needed to expand the number and types of chemicals tested, identify the most advantageous treatment schedules, and test the transferability of the method. Environ. Mol. Mutagen. 59:176-187, 2018. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

Effect of chronic administration of mestranol, tamoxifen, and toremifene on hepatic ploidy in rats.

PubMed

Dragan, Y P; Shimel, R J; Bahnub, N; Sattler, G; Vaughan, J R; Jordan, V C; Pitot, H C

1998-06-01

The nonsteroidal antiestrogen tamoxifen increases the incidence of rat liver cancer through a variety of mechanisms. To compare the effects of tamoxifen (TAM) and a structurally similar analog toremifene (TOR) on rat liver, we determined the ploidy distribution for hepatocytes isolated from rats treated for 18 months with these antiestrogens or the estrogenic compound mestranol (MS). Female Sprague-Dawley rats were subjected to a 70% partial hepatectomy and administered the solvent, trioctanoin, or diethylnitrosamine (10 mg DEN/kg). After a 2-week recovery from the surgery, the rats were administered a basal diet or one containing TAM (250 or 500 ppm), TOR (250, 500, or 750 ppm), or MS (0.2 ppm) for 18 months. Pathologic changes in the liver were examined in the 15-22 rats per treatment group at the 18-month time point. An increased incidence of hepatocellular carcinomas (HCC) was detected in the 500 ppm TAM group, but not with the other treatments that did not include DEN. Both TOR and TAM promoted formation of DEN-initiated HCCs. At sacrifice, four to five rats per group were perfused and the hepatocytes isolated and cultured. Karyotypic analysis was performed on colcemid-blocked cells after 2 days in culture. The hepatic ploidy distribution was characterized in Giemsa-stained metaphase spreads. These studies indicated that chronic treatment with TAM alone resulted in a shift from tetraploid to diploid, as was also observed for rats treated once with DEN. TOR and MS alone did not cause this change in hepatic ploidy at the doses examined. A shift toward an increased content of diploid hepatocytes occurred in all rats treated once with DEN followed by TAM, TOR, or MS. These results indicate that tamoxifen administration results in a shift toward growth of diploid hepatocytes, thus contributing to its carcinogenic action in the rat liver.

Metallothionein expression is suppressed in primary human hepatocellular carcinomas and is mediated through inactivation of CCAAT/enhancer binding protein alpha by phosphatidylinositol 3-kinase signaling cascade.

PubMed

Datta, Jharna; Majumder, Sarmila; Kutay, Huban; Motiwala, Tasneem; Frankel, Wendy; Costa, Robert; Cha, Hyuk C; MacDougald, Ormond A; Jacob, Samson T; Ghoshal, Kalpana

2007-03-15

Reactive oxygen species (ROS) resulting from chronic inflammation cause liver injury leading to transformation of regenerating hepatocytes. Metallothioneins (MT), induced at high levels by oxidative stress, are potent scavengers of ROS. Here, we report that the levels of MT-1 and MT-2A are drastically reduced in primary human hepatocellular carcinomas (HCCs) and in diethylnitrosamine-induced liver tumors in mice, which is primarily due to transcriptional repression. Expression of the transcription factor, MTF-1, essential for MT expression, and its target gene Zn-T1 that encodes the zinc transporter-1 was not significantly altered in HCCs. Inhibitors of both phosphatidylinositol 3-kinase (PI3K) and its downstream target AKT increased expression of MT genes in HCC cells but not in liver epithelial cells. Suppression of MT-1 and MT-2A by ectopic expression of the constitutively active PI3K or AKT and their up-regulation by dominant-negative PI3K or AKT mutant confirmed negative regulation of MT expression by PI3K/AKT signaling pathway. Further, treatment of cells with a specific inhibitor of glycogen synthase kinase-3 (GSK-3), a downstream effector of PI3K/AKT, inhibited MT expression specifically in HCC cells. Short interfering RNA-mediated depletion of CCAAT/enhancer binding protein alpha (C/EBPalpha), a target of GSK-3, impeded MT expression, which could not be reversed by PI3K inhibitors. DNA binding activity of C/EBPalpha and its phosphorylation at T222 and T226 by GSK-3 are required for MT expression. MTF-1 and C/EBPalpha act in concert to increase MT-2A expression, which probably explains the high level of MT expression in the liver. This study shows the role of PI3K/AKT signaling pathway and C/EBPalpha in regulation of MT expression in hepatocarcinogenesis.

Macrolide antibiotics differentially influence human HepG2 cytotoxicity and modulate intrinsic/extrinsic apoptotic pathways in rat hepatocellular carcinoma model.

PubMed

Abdel-Hamid, Nagwa I; El-Azab, Mona F; Moustafa, Yasser M

2017-04-01

This study was designed to examine the potential antitumor effect of some macrolides: clarithromycin, azithromycin, and erythromycin on chemically induced hepatocellular carcinoma (HCC) in rats and on human hepatoma cells (HepG2) as well. The possible underlying antiapoptotic mechanisms were investigated. Antiproliferative activity was assessed in HepG2 using Sulforhodamine-B staining method. In vivo, HCC was induced in rats by initiation-selection-promotion protocol using diethylnitrosamine (200 mg/kg, single i.p. injection)/2-acetylaminofluorene (0.03% w/w supplemented-diet for 2 weeks)/carbon tetrachloride (2 ml/kg diluted in corn oil 1:1, single intra-gastric dose)/phenobarbitone sodium (0.05% w/w supplemented-diet for 28 weeks). Macrolides were administered once daily starting from the 3rd week until the 17th week at a dose of 100 mg/kg in the current 33-week study period. Clarithromycin showed a higher efficacy in the suppression of HepG2 proliferation with lower IC50 value than doxorubicin. In vivo, chemically-induced HCC rat model proved that clarithromycin suppressed HCC via induction of apoptosis through up-regulation of both extrinsic/intrinsic apoptotic pathways' proteins (TNFR1, cleaved caspase-3, and Bax with an increased Bax/Bcl-2 ratio) along with MMP-9 normalization. Similarly, azithromycin demonstrated antitumorigenic effect through both apoptotic pathways, however, to a lesser extent compared to clarithromycin. Moreover, azithromycin suppressed the proliferation of HepG2, however, at a higher IC50 than doxorubicin. Surprisingly, erythromycin increased HepG2 proliferation in vitro, along with worsened tumorigenic effect of the carcinogenic agents in the in vivo study with ineffective apoptotic outcome. Some macrolides represent potential antitumor agents; however, this evident anticancer activity is an individual effect rather than a group effect and involves modulation of both intrinsic and extrinsic apoptotic pathways.

ESTABLISHMENT AND EVALUATION OF ORTHOTOPIC HEPATOCELLULAR CARCINOMA AND DRUG-INDUCED HEPATOCELLULAR CARCINOMA IN MICE WITH SPLEEN-DEFICIENCY SYNDROME IN TRADITIONAL CHINESE MEDICINE.

PubMed

Luo, Haoxuan; Chen, Yan; Sun, Baoguo; Xiang, Ting; Zhang, Shijun

2017-01-01

Spleen-deficiency syndrome (SDS) in Traditional Chinese Medicine (TCM) played pivotal roles on the development of hepatocellular carcinoma (HCC). This study was performed to establish and evaluate HCC model in mice with SDS in TCM. A total of 90 C57BL/6 mice were randomized in six groups (n=15 for each group): A, Control group; B, SDS group; C, orthotopic HCC (OHCC) group; D, OHCC based on SDS (SDS-OHCC) group; E, Drug-induced HCC (DHCC) group; F, DHCC based on SDS (SDS-DHCC) group. The SDS model were established by subcutaneous injection of reserpine, followed by the OHCC or DHCC model establishment. The SDS scores, tumor formation rate and survival time were recorded and calculated, as well as the histochemical stain was performed. The SDS scores of mice in Group B, D, F were 17.57±4.86 (P<0.05 vs. Group A), 18.13±4.53 (P<0.05 vs. Group A and C) and 23.32±4.94 (P<0.05 vs. Group A and E) respectively. The tumor formation rate of mice in Group C, D, E and F were 73.33%, 100%, 60% and 80% respectively. The survival time of mice in Group C, D, E and F were 26.42±5.27, 17.33±4.76 (P<0.05 vs. Group C), 35.77±6.12 and 22.61±5.05 (P<0.05 vs. Group E) respectively. The SDS-oriented HCC mice models were simple and easily-operated models for further studies on SDS oriented tumor. Meanwhile, SDS was a pivotal factor for low outcome of hepatic tumor. Abbreviations: HCC, Hepatocellular carcinoma; OHCC, Orthotopic hepatocellular carcinoma; DHCC, Drug-induced hepatocellular carcinoma; SDS, Spleen-deficiency syndrome; TCM, Traditional Chinese Medicine; SPF, Specific pathogen-free; DEN, Diethylnitrosamine; CCl4, Carbon tetrachloride; HE, Hematoxylin-eosin; IACUC, Institutional Animal Care and Use Committee.

Chicken fetal liver DNA damage and adduct formation by activation-dependent DNA-reactive carcinogens and related compounds of several structural classes.

PubMed

Williams, Gary M; Duan, Jian-Dong; Brunnemann, Klaus D; Iatropoulos, Michael J; Vock, Esther; Deschl, Ulrich

2014-09-01

The chicken egg genotoxicity assay (CEGA), which utilizes the liver of an intact and aseptic embryo-fetal test organism, was evaluated using four activation-dependent DNA-reactive carcinogens and four structurally related less potent carcinogens or non-carcinogens. In the assay, three daily doses of test substances were administered to eggs containing 9-11-day-old fetuses and the fetal livers were assessed for two endpoints, DNA breaks using the alkaline single cell gel electrophoresis (comet) assay and DNA adducts using the (32)P-nucleotide postlabeling (NPL) assay. The effects of four carcinogens of different structures requiring distinct pathways of bioactivation, i.e., 2-acetylaminofluorene (AAF), aflatoxin B1 (AFB1), benzo[a]pyrene (B[a]P), and diethylnitrosamine (DEN), were compared with structurally related non-carcinogens fluorene (FLU) and benzo[e]pyrene (B[e]P) or weak carcinogens, aflatoxin B2 (AFB2) and N-nitrosodiethanolamine (NDELA). The four carcinogens all produced DNA breaks at microgram or low milligram total doses, whereas less potent carcinogens and non-carcinogens yielded borderline or negative results, respectively, at higher doses. AAF and B[a]P produced DNA adducts, whereas none was found with the related comparators FLU or B[e]P, consistent with comet results. DEN and NDELA were also negative for adducts, as expected in the case of DEN for an alkylating agent in the standard NPL assay. Also, AFB1 and AFB2 were negative in NPL, as expected, due to the nature of ring opened aflatoxin adducts, which are resistant to enzymatic digestion. Thus, the CEGA, using comet and NPL, is capable of detection of the genotoxicity of diverse DNA-reactive carcinogens, while not yielding false positives for non-carcinogens. © The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Liver tumor promoting effect of etofenprox in rats and its possible mechanism of action.

PubMed

Hojo, Yuri; Shiraki, Ayako; Tsuchiya, Takuma; Shimamoto, Keisuke; Ishii, Yuji; Suzuki, Kazuhiko; Shibutani, Makoto; Mitsumori, Kunitoshi

2012-01-01

To investigate the liver tumor-promoting effects of etofenprox (ETF), a pyrethroid-like insecticide, 6 week-old male F344 rats were given an intraperitoneal injection of N-diethylnitrosamine (DEN). After 2 weeks from the DEN treatment, 12 rats per group received a powdered diet containing 0, 0.25, 0.50, or 1.0% ETF for 8 weeks. At the time of 2nd week of ETF administration, all animals were subjected to two-thirds partial hepatectomy (PH). One rat per group except for the 0.25% ETF group died due to surgical operation of PH. The number and area of glutathione S-transferase placental form (GST-P) positive foci significantly increased in the livers of DEN-initiated rats given 0.50% and 1.0% ETF compared with the DEN-alone group. Quantitative real-time RT-PCR analysis revealed that the mRNA expression of phase I enzymes Cyp2b1/2, phase II enzymes such as Akr7a3, Gsta5, Ugt1a6, Nqo1 significantly increased in the DEN+ETF groups. The immunohistochemistry showed the translocation of CAR from the cytoplasm to the nuclei of hepatocytes in the ETF-treated groups. Reactive oxygen species (ROS) production increased in microsomes isolated from the livers of ETF-treated rats, and thiobarbituric acid-reactive substances (TBARS) levels and 8- hydroxy-2-deoxyguanosine (8-OHdG) content significantly increased in all of the ETF-treated groups and DEN+1.0% ETF group, respectively. The results of the present study indicate that ETF has a liver tumor-promoting activity in rats, and suggest that ETF activates the constitutive active/androstane receptor (CAR) and enhances microsomal ROS production, resulting in the upregulation of Nrf2 gene batteries; such an oxidative stress subsequently induces liver tumor-promoting effects by increased cellular proliferation.

In vivo antitumour potential of camel's milk against hepatocellular carcinoma in rats and its improvement of cisplatin renal side effects.

PubMed

El Miniawy, Hala M F; Ahmed, Kawkab A; Mansour, Sameeh A; Khattab, Marwa M Salah

2017-12-01

Camel milk (CM) is recommended for liver disease patients in Egypt for a strong belief that it has a curative effect. The effect of consumption of CM with or without chemotherapeutic drug cisplatin was evaluated on induced hepatocarcinogenesis in rats. Wistar male rats (56) were divided into eight groups (7 rats each). Group I was control. Hepatocarcinogenesis was initiated by a single dose of intraperitoneal injection of diethylnitrosamine (DENA) (200 mg/kg BW) and promoted by phenobarbitone (500 ppm) in drinking water in groups V, VI, VII and VIII. Treatment started from 28th till 38th week using CM (5 mL/day) and/or cisplatin (5 mg/kg/3 weeks) in groups II, III IV, VI, VII and VIII. Biochemical analysis, lipid peroxidation and superoxide dismutase (SOD) activity in liver tissue were performed. Histopathology of liver and kidney and immunohistochemistry of placental glutathione-S-transferase (P-GST) in liver were performed and analyzed using image analysis. Albumin concentration and SOD activity were 3.13 ± 0.23 and 311.45 ± 41.71 in group VII (DENA & cisplatin), whereas they were 4.3 ± 0.15 and 540.5 ± 29.94 in group VII (DENA, CM and cisplatin). The mean area of altered hepatocellular foci and P-GST altered foci decreased in group VI (DENA and CM) (1049.6 ± 174.78 and 829.1 ± 261) and group VIII (cisplatin and CM) (1615.12 ± 436 and 543.9 ± 127) compared to group V (DENA only) (4173.74 ± 510.7 and 3169.49 ± 538.61). Cisplatin caused chronic interstitial nephritis, which was slightly alleviated in group VIII (CM and cisplatin). CM had an antioxidant effect and together with cisplatin managed to decrease hepatocarcinogenesis.

6-shogaol-rich extract from ginger up-regulates the antioxidant defense systems in cells and mice.

PubMed

Bak, Min-Ji; Ok, Seon; Jun, Mira; Jeong, Woo-Sik

2012-07-04

The rhizome of ginger (Zingiber officinale Roscoe) is known to have several bioactive compounds including gingerols and shogaols which possess beneficial health properties such as anti-inflammatory and chemopreventive effects. Based on recent observations that 6-shogaol may have more potent bioactivity than 6-gingerol, we obtained a 6-shogaol-rich extract from ginger and examined its effects on the nuclear factor E2-related factor2 (Nrf2)/antioxidant response element (ARE) pathway in vitro and in vivo. 6-Shogaol-rich extract was produced by extracting ginger powder with 95% ethanol at 80 °C after drying at 80 °C (GEE8080). GEE8080 contained over 6-fold more 6-shogaol compared to the room temperature extract (GEE80RT). In HepG2 cells, GEE8080 displayed much stronger inductions of ARE-reporter gene activity and Nrf2 expression than GEE80RT. GEE8080 stimulated phosphorylations of mitogen-activated protein kinases (MAPKs) such as ERK, JNK, and p38. Moreover, the GEE8080-induced expressions of Nrf2 and HO-1 were attenuated by treatments of SB202190 (a p38 specific inhibitor) and LY294002 (an Akt specific inhibitor). In a mouse model, the GEE8080 decreased the diethylnitrosamine (DEN)-mediated elevations of serum aspartate transaminase and alanine transaminase as well as the DEN-induced hepatic lipid peroxidation. Inductions of Nrf2 and HO-1 by GEE8080 were also confirmed in the mice. In addition, the administration of GEE8080 to the mice also restored the DEN-reduced activity and protein expression of hepatic antioxidant enzymes such as superoxide dismutase, glutathione peroxidase and catalase. In conclusion, GEE8080, a 6-shogaol-rich ginger extract, may enhance antioxidant defense mechanism through the induction of Nrf2 and HO-1 regulated by p38 MAPK and PI3k/Akt pathway in vitro and in vivo.

Branched-Chain Amino Acids Ameliorate Fibrosis and Suppress Tumor Growth in a Rat Model of Hepatocellular Carcinoma with Liver Cirrhosis

PubMed Central

Cha, Jung Hoon; Bae, Si Hyun; Kim, Hye Lim; Park, Na Ri; Choi, Eun Suk; Jung, Eun Sun; Choi, Jong Young; Yoon, Seung Kew

2013-01-01

Purpose Recent studies have revealed that branched-chain amino acids (BCAA) reduce the development of hepatocellular carcinoma (HCC) in patients with obesity and hepatitis C virus infection by improving insulin resistance (IR). The aim of this study was to examine the anti-cancer and anti-fibrotic effects of BCAA on the development of diethylnitrosamine (DEN)-induced HCC and liver cirrhosis in a rat model. Methods Male SD rats received weekly intraperitoneal injections of DEN (50 mg/kg of body weight) for 16 weeks to induce HCC. They were fed a diet containing 3% casein, 3% or 6% BCAA for 13 weeks beginning 6 weeks after DEN administration. DEN was used to induce HCC through stepwise development from cirrhosis to HCC. The effect of BCAA was evaluated in tumor tissues by histopathologic analyses, reverse transcription-polymerase chain reaction, and Western blotting. Results The mean area and number of dysplastic nodules (DNs) and tumors in the casein group tended to be larger than those in the BCAA group 16 weeks after DEN administration. The mean fibrotic area in the BCAA group was smaller than that in the casein group. The BCAA group showed decreased mRNA levels for markers of fibrosis, angiogenesis, and apoptosis inhibition. Compared with the casein group, the BCAA group had lower levels of α-smooth muscle actin, vascular endothelial growth factor, p-β-catenin, p-p38 mitogen-activated protein kinase, proliferating cell nuclear antigen, and caspase-3 protein expression, as well as a higher level of cleaved caspase-3 protein expression. Conclusions BCAA supplementation of the diet ameliorated liver fibrosis and HCC development in a DEN-induced rat model of HCC with liver cirrhosis, but not in the IR model. These results provide a rationale for anti-fibrosis and chemoprevention using BCAA treatment for HCC with liver cirrhosis, as well as decreasing the ammonia level. PMID:24223741

Methylation of nuclear proteins by dimethylnitrosamine and by methionine in the rat in vivo

PubMed Central

Turberville, C.; Craddock, V. M.

1971-01-01

1. The incorporation of methyl groups into histones from dimethylnitrosamine and from methionine was studied by injection of the labelled compounds, isolation of rat liver and kidney histones, and analysis of hydrolysates by column chromatography. 2. Labelled methionine gave rise to labelled ∈-N-methyl-lysine, di-∈-N-methyl-lysine and an amino acid presumed to be ω-N-methyl-arginine. 3. Administration of labelled dimethylnitrosamine gave rise to labelled S-methylcysteine, 1-methylhistidine, 3-methylhistidine and ∈-N-methyl-lysine derived from the alkylating metabolite of dimethylnitrosamine. In addition, labelled formaldehyde released by metabolism of dimethylnitrosamine leads to the formation of labelled S-adenosylmethionine, and hence to labelling of ∈-N-methyl-lysine, di-∈-N-methyl-lysine and ω-N-methylarginine by enzymic methylation. 4. The formation of ∈-N-methyl-lysine by alkylation of liver histones was confirmed by using doubly labelled dimethylnitrosamine to discriminate between direct chemical alkylation and enzymic methylation via S-adenosylmethionine. These experiments also suggested the possibility that methionine residues in the histones were alkylated to give methylmethionine sulphonium residues. 5. The extent of alkylation of liver histones was maximal at about 5h after dosing and declined between 5 and 24h. The methylated amino acids resulting from direct chemical alkylation were preferentially lost: this is ascribed to necrosis of the more highly alkylated cells. 6. Liver histones were about four times as alkylated as kidney histones; the extent of alkylation of liver histones was similar to that of liver total nuclear proteins. 7. Methyl methanesulphonate (120mg/kg) alkylated liver histones to a greater extent than did dimethylnitrosamine. Diethylnitrosamine also alkylated liver histones. 8. The results are discussed with regard to the possible effects of alkylation on histone function, and the possible role of histone alkylation in carcinogenesis by the three compounds. PMID:5131729

Altered Phenotypes in Saccharomyces cerevisiae by Heterologous Expression of Basidiomycete Moniliophthora perniciosa SOD2 Gene.

PubMed

Melo, Sônia C; Santos, Regineide X; Melgaço, Ana C; Pereira, Alanna C F; Pungartnik, Cristina; Brendel, Martin

2015-06-01

Heterologous expression of a putative manganese superoxide dismutase gene (SOD2) of the basidiomycete Moniliophthora perniciosa complemented the phenotypes of a Saccharomyces cerevisiae sod2Δ mutant. Sequence analysis of the cloned M. perniciosa cDNA revealed an open reading frame (ORF) coding for a 176 amino acid polypeptide with the typical metal-binding motifs of a SOD2 gene, named MpSOD2. Phylogenetic comparison with known manganese superoxide dismutases (MnSODs) located the protein of M. perniciosa (MpSod2p) in a clade with the basidiomycete fungi Coprinopsis cinerea and Laccaria bicolor. Haploid wild-type yeast transformants containing a single copy of MpSOD2 showed increased resistance phenotypes against oxidative stress-inducing hydrogen peroxide and paraquat, but had unaltered phenotype against ultraviolet-C (UVC) radiation. The same transformants exhibited high sensitivity against treatment with the pro-mutagen diethylnitrosamine (DEN) that requires oxidation to become an active mutagen/carcinogen. Absence of MpSOD2 in the yeast sod2Δ mutant led to DEN hyper-resistance while introduction of a single copy of this gene restored the yeast wild-type phenotype. The haploid yeast wild-type transformant containing two SOD2 gene copies, one from M. perniciosa and one from its own, exhibited DEN super-sensitivity. This transformant also showed enhanced growth at 37 °C on the non-fermentable carbon source lactate, indicating functional expression of MpSod2p. The pro-mutagen dihydroethidium (DHE)-based fluorescence assay monitored basal level of yeast cell oxidative stress. Compared to the wild type, the yeast sod2Δ mutant had a much higher level of intrinsic oxidative stress, which was reduced to wild type (WT) level by introduction of one copy of the MpSOD2 gene. Taken together our data indicates functional expression of MpSod2 protein in the yeast S. cerevisiae.

Altered Phenotypes in Saccharomyces cerevisiae by Heterologous Expression of Basidiomycete Moniliophthora perniciosa SOD2 Gene

PubMed Central

Melo, Sônia C.; Santos, Regineide X.; Melgaço, Ana C.; Pereira, Alanna C. F.; Pungartnik, Cristina; Brendel, Martin

2015-01-01

Heterologous expression of a putative manganese superoxide dismutase gene (SOD2) of the basidiomycete Moniliophthora perniciosa complemented the phenotypes of a Saccharomyces cerevisiae sod2Δ mutant. Sequence analysis of the cloned M. perniciosa cDNA revealed an open reading frame (ORF) coding for a 176 amino acid polypeptide with the typical metal-binding motifs of a SOD2 gene, named MpSOD2. Phylogenetic comparison with known manganese superoxide dismutases (MnSODs) located the protein of M. perniciosa (MpSod2p) in a clade with the basidiomycete fungi Coprinopsis cinerea and Laccaria bicolor. Haploid wild-type yeast transformants containing a single copy of MpSOD2 showed increased resistance phenotypes against oxidative stress-inducing hydrogen peroxide and paraquat, but had unaltered phenotype against ultraviolet–C (UVC) radiation. The same transformants exhibited high sensitivity against treatment with the pro-mutagen diethylnitrosamine (DEN) that requires oxidation to become an active mutagen/carcinogen. Absence of MpSOD2 in the yeast sod2Δ mutant led to DEN hyper-resistance while introduction of a single copy of this gene restored the yeast wild-type phenotype. The haploid yeast wild-type transformant containing two SOD2 gene copies, one from M. perniciosa and one from its own, exhibited DEN super-sensitivity. This transformant also showed enhanced growth at 37 °C on the non-fermentable carbon source lactate, indicating functional expression of MpSod2p. The pro-mutagen dihydroethidium (DHE)-based fluorescence assay monitored basal level of yeast cell oxidative stress. Compared to the wild type, the yeast sod2Δ mutant had a much higher level of intrinsic oxidative stress, which was reduced to wild type (WT) level by introduction of one copy of the MpSOD2 gene. Taken together our data indicates functional expression of MpSod2 protein in the yeast S. cerevisiae. PMID:26039235

The first study on therapeutic efficacies of a vascular disrupting agent CA4P among primary hepatocellular carcinomas with a full spectrum of differentiation and vascularity: Correlation of MRI-microangiography-histopathology in rats.

PubMed

Liu, Yewei; De Keyzer, Frederik; Wang, Yixing; Wang, Fengna; Feng, Yuanbo; Chen, Feng; Yu, Jie; Liu, Jianjun; Song, Shaoli; Swinnen, Johan; Bormans, Guy; Oyen, Raymond; Huang, Gang; Ni, Yicheng

2018-04-29

To better inform the next clinical trials of vascular disrupting agent Combretastatin-A4-phosphate (CA4P) in patients with hepatic malignancies, this preclinical study aimed at evaluating CA4P therapeutic efficacy in rats with primary hepatocellular carcinomas (HCCs) of a full spectrum of differentiation and vascularity by magnetic resonance imaging (MRI), microangiography and histopathology. Ninety-six HCCs were raised in 25 rats by diethylnitrosamine gavage. Tumor growth was monitored by T2-/T1-weighted-MRI (T2WI, T1WI) using a 3.0T scanner. Early vascular response and later intratumoral necrosis were detected by dynamic-contrast-enhanced (DCE) MRI and diffusion-weighted-imaging (DWI) before, 1h and 12h after CA4P iv-administration. In-vivo MRI-findings were validated by postmortem-techniques. Multi-parametric MRI revealed rapid CA4P-induced tumor vascular shutdown within 1h, followed by variable intratumoral necrosis at 12h. Tumor volumes decreased by 10% at 1h (P<0.05), but resumed at 12h. Correlations of semi-quantitative DCE parameter initial-area-under-the-gadolinium-curve (IAUGC30) with histopathology proved partial vascular closure and compensational reopening (P<0.05). The higher grades of vascularity prevented those residual tumor tissues from CA4P-caused ischemic necrosis. By histopathology using a 4-scale cellular-differentiation criteria and a 4-grade tumor-vascularity classification, percentage of CA4P-induced necrosis negatively correlated with HCC differentiation (r=-0.404, P<0.001) and tumor vascularity (r=-0.370, P<0.001). Ordinal-logistic-regression helped to predict early tumor responses to CA4P in terms of tumoral differentiation and vascularity. This study demonstrated that CA4P could induce vascular shutdown in primary HCCs within 1h, resulting in various degrees of tumor necrosis at 12h. MRI as a real-time imaging biomarker may help to define tumor vascularity and differentiation and further to predict CA4P therapeutic outcomes. This article is protected by copyright. All rights reserved. © 2018 UICC.

Inhibition of c-Jun NH2-terminal kinase switches Smad3 signaling from oncogenesis to tumor- suppression in rat hepatocellular carcinoma.

PubMed

Nagata, Hiromitsu; Hatano, Etsuro; Tada, Masaharu; Murata, Miki; Kitamura, Koji; Asechi, Hiroyuki; Narita, Masato; Yanagida, Atsuko; Tamaki, Nobuyuki; Yagi, Shintaro; Ikai, Iwao; Matsuzaki, Koichi; Uemoto, Shinji

2009-06-01

Transforming growth factor beta (TGF-beta) signaling involves both tumor-suppression and oncogenesis. TGF-beta activates the TGF-beta type I receptor (TbetaRI) and c-Jun N-terminal kinase (JNK), which differentially phosphorylate the mediator Smad3 to become COOH-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). TbetaRI-dependent pSmad3C transmits a tumor-suppressive TGF-beta signal, while JNK-dependent pSmad3L promotes carcinogenesis in human chronic liver disorders. The aim of this study is to elucidate how SP600125, a JNK inhibitor, affected rat hepatocellular carcinoma (HCC) development, while focusing on the domain-specific phosphorylation of Smad3. The rats received subcutaneous injections of either SP600125 or vehicle 11 times weekly together with 100 ppm N-diethylnitrosamine (DEN) administration for 56 days and were sacrificed in order to evaluate HCC development 28 days after the last DEN administration. The number of tumor nodules greater than 3 mm in diameter and the liver weight/body weight ratio were significantly lower in the SP600125-treated rats than those in the vehicle-treated rats (7.9 +/- 0.8 versus 17.7 +/- 0.9: P < 0.001; 6.3 +/- 1.2 versus 7.1 +/- 0.2%: P < 0.05). SP600125 significantly prolonged the median survival time in rats with DEN-induced HCC (113 versus 97 days: log-rank P = 0.0018). JNK/pSmad3L/c-Myc was enhanced in the rat hepatocytes exposed to DEN. However, TbetaRI/pSmad3C/p21(WAF1) was impaired as DEN-induced HCC developed and progressed. The specific inhibition of JNK activity by SP600125 suppressed pSmad3L/c-Myc in the damaged hepatocytes and enhanced pSmad3C/p21(WAF1), acting as a tumor suppressor in normal hepatocytes. Administration of SP600125 to DEN-treated rats shifted hepatocytic Smad3-mediated signal from oncogenesis to tumor suppression, thus suggesting that JNK could be a therapeutic target of human HCC development and progression.

Aldo-Keto Reductases as Early Biomarkers of Hepatocellular Carcinoma: A Comparison Between Animal Models and Human HCC.

PubMed

Torres-Mena, Julia Esperanza; Salazar-Villegas, Karla Noemí; Sánchez-Rodríguez, Ricardo; López-Gabiño, Belém; Del Pozo-Yauner, Luis; Arellanes-Robledo, Jaime; Villa-Treviño, Saúl; Gutiérrez-Nava, María Angélica; Pérez-Carreón, Julio Isael

2018-04-01

The intrinsic heterogeneity of hepatocellular carcinoma (HCC) represents a great challenge for its molecular classification and for detecting predictive biomarkers. Aldo-keto reductase (Akr) family members have shown differential expression in human HCC, while AKR1B10 overexpression is considered a biomarker; AKR7A3 expression is frequently reduced in HCC. To investigate the time-course expression of Akr members in the experimental hepatocarcinogenesis. Using DNA-microarray data, we analyzed the time-course gene expression profile from nodules to tumors (4-17 months) of 17 Akr members induced by the resistant hepatocyte carcinogenesis model in the rat. The expression of six members (Akr1c19, Akr1b10, Akr7a3, Akr1b1, Akr1cl1, and Akr1b8) was increased, comparable to that of Ggt and Gstp1, two well-known liver cancer markers. In particular, Akr7a3 and Akr1b10 expression also showed a time-dependent increment at mRNA and protein levels in a second hepatocarcinogenesis model induced with diethylnitrosamine. We confirmed that aldo-keto reductases 7A3 and 1B10 were co-expressed in nine biopsies of human HCC, independently from the presence of glypican-3 and cytokeratin-19, two well-known HCC biomarkers. Because it has been suggested that expression of Akr members is regulated through NRF2 activity at the antioxidant response element (ARE) sequences, we searched and identified at least two ARE sites in Akr1b1, Akr1b10, and Akr7a3 from rat and human gene sequences. Moreover, we observed higher NRF2 nuclear translocation in tumors as compared with non-tumor tissues. Our results demonstrate that Akr7a3 mRNA and protein levels are consistently co-expressed along with Akr1b10, in both experimental liver carcinogenesis and some human HCC samples. These results highlight the presence of AKR7A3 and AKR1B10 from early stages of the experimental HCC and introduce them as a potential application for early diagnosis, staging, and prognosis in human cancer.

Oxidation of Methyl and Ethyl Nitrosamines by Cytochromes P450 2E1 and 2B1

PubMed Central

Chowdhury, Goutam; Calcutt, M. Wade; Nagy, Leslie D.; Guengerich, F. Peter

2012-01-01

Cytochrome P450 (P450) 2E1 is the major enzyme that oxidizes N-nitrosodimethylamine (N,N-dimethylnitrosamine, DMN), a carcinogen and also a representative of some nitrosamines formed endogenously. Oxidation of DMN by rat or human P450 2E1 to HCHO showed a high apparent intrinsic kinetic deuterium isotope effect (KIE), ≥ 8. The KIE was not attenuated in non-competitive intermolecular experiments with rat liver microsomes (DV 12.5, D(V/K) 10.9, nomenclature of Northrop, D.B. (1982) Methods Enzymol. 87, 607–625) but was with purified human P450 2E1 (DV 3.3, D(V/K) 3.7), indicating that C-H bond breaking is partially rate-limiting with human P450 2E1. With N-nitrosodiethylamine (N,N-diethylnitrosamine, DEN), the intrinsic KIE was slightly lower and was not expressed (e.g., D(V/K) 1.2) in non-competitive intermolecular experiments. The same general pattern of KIEs was also seen in the D(V/K) results with DMN and DEN for the minor products resulting from the denitrosation reactions (CH3NH2, CH3CH2NH2, and NO2−). Experiments with deuterated N-nitroso-N-methyl,N-ethylamine demonstrated that the lower KIEs associated for ethyl compared to methyl oxidation could be distinguished within a single molecule. P450 2E1 oxidized DMN and DEN to aldehydes and then to the carboxylic acids. No kinetic lags were observed in acid formation; pulse-chase experiments with carrier aldehydes showed only limited equilibration with P450 2E1-bound aldehydes, indicative of processive reactions, as reported for P450 2A6 (Chowdhury, G. et al. (2010) J. Biol. Chem. 285, 8031–8044). These same features (no lag phase for HCO2H formation, lack of equilibration in pulse-chase assays) were also seen with (rat) P450 2B1, which has lower catalytic efficiency for DMN oxidation and a larger active site. Thus, the processivity of dialkylnitrosamine oxidation appears to be shared by a number of P450s. PMID:23186213

Tumor suppression effects of bilberry extracts and enzymatically modified isoquercitrin in early preneoplastic liver cell lesions induced by piperonyl butoxide promotion in a two-stage rat hepatocarcinogenesis model.

PubMed

Hara, Shintaro; Morita, Reiko; Ogawa, Takashi; Segawa, Risa; Takimoto, Norifumi; Suzuki, Kazuhiko; Hamadate, Naobumi; Hayashi, Shim-Mo; Odachi, Ayano; Ogiwara, Isao; Shibusawa, Sakae; Yoshida, Toshinori; Shibutani, Makoto

2014-08-01

To investigate the protective effect of bilberry extracts (BBE) and enzymatically modified isoquercitrin (EMIQ) on the hepatocarcinogenic process involving oxidative stress responses, we used a two-stage hepatocarcinogenesis model in N-diethylnitrosamine-initiated and piperonyl butoxide (PBO)-promoted rats. We examined the modifying effect of co-administration with BBE or EMIQ on the liver tissue environment including oxidative stress responses, cell proliferation and apoptosis, and phosphatase and tensin homolog (PTEN)/Akt and transforming growth factor (TGF)-β/Smad signalings on the induction mechanism of preneoplastic lesions during early stages of hepatocellular tumor promotion. PBO increased the numbers and area of glutathione S-transferase placental form (GST-P)(+) liver cell foci and the numbers of Ki-67(+) proliferating cells within GST-P(+) foci. Co-administration of BBE or EMIQ suppressed these effects with the reductions of GST-P(+) foci (area) to 48.9-49.4% and Ki-67(+) cells to 55.5-61.4% of the PBO-promoted cases. Neither BBE nor EMIQ decreased microsomal reactive oxygen species induced by PBO. However, only EMIQ suppressed the level of thiobarbituric acid-reactive substances to 78.4% of the PBO-promoted cases. PBO increased the incidences of phospho-PTEN(-) foci, phospho-Akt substrate(+) foci, phospho-Smad3(-) foci and Smad4(-) foci in GST-P(+) foci. Both BBE and EMIQ decreased the incidences of phospho-PTEN(-) foci in GST-P(+) foci to 59.8-72.2% and Smad4(-) foci to 62.4-71.5% of the PBO-promoted cases, and BBE also suppressed the incidence of phospho-Akt substrate(+) foci in GST-P(+) foci to 75.2-75.7% of the PBO-promoted cases. These results suggest that PBO-induced tumor promotion involves facilitation of PTEN/Akt and disruptive TGF-β/Smad signalings without relation to oxidative stress responses, but this promotion was suppressed by co-treatment with BBE or EMIQ through suppression of cell proliferation activity of preneoplastic liver cells. Copyright © 2014 Elsevier GmbH. All rights reserved.

Mutational landscape of a chemically-induced mouse model of liver cancer.

PubMed

Connor, Frances; Rayner, Tim F; Aitken, Sarah J; Feig, Christine; Lukk, Margus; Santoyo-Lopez, Javier; Odom, Duncan T

2018-06-26

Carcinogen-induced mouse models of liver cancer are used extensively to study pathogenesis of the disease and have a critical role in validating candidate therapeutics. These models can recapitulate molecular and histological features of human disease. However, it is not known if the genomic alterations driving these mouse tumour genomes are comparable to those found in human tumours. Here, we provide a detailed genomic characterisation of tumours from a commonly used mouse model of hepatocellular carcinoma (HCC). We analysed whole exome sequences of liver tumours arising in mice exposed to diethylnitrosamine (DEN). DEN-initiated tumours had a high, uniform number of somatic single nucleotide variants (SNVs), with few insertions, deletions or copy number alterations, consistent with the known genotoxic action of DEN. Exposure of hepatocytes to DEN left a reproducible mutational imprint in resulting tumour exomes which we could computationally reconstruct using six known COSMIC mutational signatures. The tumours carried a high diversity of low-incidence, non-synonymous point mutations in many oncogenes and tumour suppressors, reflecting the stochastic introduction of SNVs into the hepatocyte genome by the carcinogen. We identified four recurrently mutated genes that were putative oncogenic drivers of HCC in this model. Every neoplasm carried activating hotspot mutations either in codon 61 of Hras, in codon 584 of Braf or in codon 254 of Egfr. Truncating mutations of Apc occurred in 21% of neoplasms, which were exclusively carcinomas supporting a role for deregulation of Wnt/β-catenin signalling in cancer progression. Our study provides detailed insight into the mutational landscape of tumours arising in a commonly-used carcinogen model of HCC, facilitating the future use of this model to understand the human disease. Mouse models are widely used to study the biology of cancer and to test potential therapies. Here, we have described the mutational landscape of tumours arising in a carcinogen-induced mouse model of liver cancer. Since cancer is a disease caused by genomic alterations, information about the patterns and types of mutations in the tumours in this mouse model should facilitate its use to study human liver cancer. Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Effects on the tumor specific growth factor and tumor necrosis factor α in rats' precancerous lesion of primary hepatocellular carcinoma by direct moxibustion at Ganshu (BL 18) acupoint.

PubMed

Wang, Pei; Zhu, Jiang; Xie, Xi-Liang; Sui, Ming-He; Zhang, Qiu-Ju; Jia, Wen-Rui; Xin, Si-Yuan; Liu, Yang; Hou, Zhong-Wei

2016-07-01

To investigate the effect of direct moxibustion at Ganshu (BL18) on the serum concentrations of tumor specific growth factor (TSGF) and tumor necrosis factor α (TNF-α) in a rat model with precancerous lesion of primary hepatocellular carcinoma (HCC), so as to explore the mechanism of moxibustion underlying improvement of HCC. Sixty male Wistar rats were randomly divided into control group (n=10), model group (n=20), prevention group 1 (n=15) and prevention group 2 (n=15). The normal rats were injected with physiological saline as blank control. At the same time, the rats of other three groups were injected with diethylnitrosamine to establish the HCC model. Direct moxibustion with grain-sized moxa was applied to bilateral Ganshu acupoint of the rats in the prevention group 1 (1 treatment course, 20 days) and prevention group 2 (2 treatment courses, 40 days), 5 doses for each acupoint, 0.5 mg/dose, once every other day. At each time point (before model establishment, the end of 1st course prevention, the end of 2nd course prevention and the end of model establishment), serum levels of TSGF and TNF-α were detected using enzyme-linked immunosorbent assay. Compared with the control group, there was a remarkably increase of serum TSGF and TNF-α contents in the model group at the end of the experiment (P<0.05). At the end of the 1st course of direct moxibustion, the contents of serum TSGF and TNF-α of rats in the prevention group 1 were significantly increased compared with that of the model group (P<0.05). At the end of the 2nd course of direct moxibustion, serum TSGF and TNF-α levels of rats in the model group were higher than the normal group with significantly difference (P<0.05), and the levels of TSGF and TNF-α in the prevention group 2 were significantly reduced in comparison with the model group (P<0.05). It was possible that direct moxibustion could inhibit precancerous lesion and postpone hepatocarcinogenesis, and the therapeutic effect of two courses were better than one course.

Lack of promotional effects of groundwater contaminant mixtures on the induction of preneoplastic foci in rat liver.

PubMed

Benjamin, S A; Yang, R S; Tessari, J D; Chubb, L W; Brown, M D; Dean, C E; Keefe, T J

1999-10-01

F344 rats were exposed to drinking water mixtures of seven of the most common groundwater contaminants associated with hazardous waste sites [arsenic, benzene, chloroform, chromium, lead, phenol, and trichloroethylene (TCE)] as the full mixture or submixtures of the organic and/or inorganic chemicals. The lowest concentrations (1x) of the individual chemicals were environmentally realistic and below what would be expected to induce significant short-term toxicity. This study was intended to determine if previously reported increases in localized hepatocellular proliferation in response to these chemicals might be correlated with increased risk for hepatocarcinogenesis. Rats were exposed via a drinking water solution to the full seven- chemical mixture (at 1x and 10x concentrations), submixtures of the organic or inorganic chemicals (at 10x concentrations), a mixture of TCE, lead, and chloroform (TLC submixture at 10x and 100x concentrations), or deionized water as a control. The rats were evaluated for promotion of placental glutathione-S-transferase (GST-P) positive preneoplastic liver cell foci after diethylnitrosamine (DEN) initiation and partial hepatectomy. Focus formation, cell proliferation, and apoptosis were evaluated after exposure to DEN or saline controls, the chemical mixtures or deionized water controls, or combinations of these treatments. The total number and area of GST-P positive foci in DEN-treated rats exposed to the full seven-chemical mixture was increased as compared with the DEN-water controls, but this was statistically significant only for total focus area in the 1x dose group. In DEN-treated rats, the inorganic or TLC submixtures resulted in a significant reduction in number and area of GST-P positive foci. Focus area also was decreased in the organic submixture-treated group, but not significantly. Hepatocellular proliferation was not significantly changed in the chemical mixture saline groups as compared with the mixture water controls. After DEN treatment, however, cell proliferation was significantly decreased after the 10x seven-chemical and organic mixture treatments and the 100x TLC mixture treatment. Different groups showed either increased or decreased apoptotic rates which did not correlate well with proliferation rates or focus formation. Mixtures of these seven chemicals, therefore, did not appear to act as promoters of hepatic foci at environmentally relevant concentrations, and some mixture combinations appeared to decrease promotional activity.

[Effects of moxibustion with seed-sized moxa cone at "Ganshu" (BL 18) on liver function in rats with precancerous lesion of hepatic cellular cancer].

PubMed

Liu, Yang; Hou, Zhongwei; Lu, Jun; Dong, Feng; Wang, Pei; Jia, Wenrui; Wang, Chaoyang

2015-07-01

To explore the effects of moxibustion with seed-sized moxa cone at "Ganshu" (BL 18) on liver furiction and morphology in rat with precancerous lesion of hepatic cellular cancer MCC). A total of 60 male Wistar rats were randomly divided into a normal group (10 rats), a model group (20 rats), a 20-day treatment group (15 rats) and a 40-day treatment group (15 rats). HCC model was established by intraperitoneal injection of diethylnitrosamine (DEN). Rats in the normal group received no treatment. Rats in the model group were treated with fixation. Rats in the 20-day treatment group and 40-day treatment group were treated by moxibustion with seed-sized moxa cone at "Ganshu" (BL 18), once every other day, for 20 days and 40 days, respectively. Blood sample in each group was collected 1 d before model establishment, 20 d, 40 d and 84 d after model establishment. Chemical method was applied to test the activity of ALT (alamine aminotransferase), AST (aspartate transaminase) and GGT (glutamyl transpeptidase); at the end of model establishment, all the rats were sacrificed to observe the liver morphology changes. After the first therapeutic course, the. content of ALT and AST in the 20-day treatment group was significantly lower than that in the model group (all P<0. 05); after the second therapeutic course, the content of ALT, AST and GGT in the 40-day treatment group was insignificantly lower than that in the model group (all P>0. 05). Under light microscope, the slice of liver tissue indicated that primary tumor was induced in the model group, and the tumor cells were stained and irregular; the cytoplasm in the 20-day treatment group was even, and the tumor cells were few with several nodules alone. In the 40-day treatment group the liver morphology was normal and the staining was even; the tumor cells were few without nodules or a few. Conclusion Moxibustion with seed-sized moxa cone at "Ganshu" (BL 18) could reduce the serum content of ALT, AST and GGT in rats with HCC, which could protect the liver and: delay the DEN-induced precancerous lesion on some levels.

Carcinogenicity of antioxidants BHA, caffeic acid, sesamol, 4-methoxyphenol and catechol at low doses, either alone or in combination, and modulation of their effects in a rat medium-term multi-organ carcinogenesis model.

PubMed

Hirose, M; Takesada, Y; Tanaka, H; Tamano, S; Kato, T; Shirai, T

1998-01-01

The carcinogenicity of low dietary levels of the antioxidants butylated hydroxyanisole (BHA), caffeic acid, sesamol, 4-methoxyphenol (4-MP) and catechol, known to target the forestomach or glandular stomach, were examined alone or in combination in a 2-year long-term experiment and their modifying effects assessed in a medium-term multiorgan model. In the carcinogenicity study, groups of 30-31 male F344 rats were treated with 0.4% BHA, 0.4% caffeic acid, 0.4% sesamol, 0.4% 4-MP and 0.16% catechol either alone or in combination for up to 104 weeks and then killed. In the medium-term multi-organ model, groups of 10 to 15 male F344 rats were given diethylnitrosamine (DEN), N-methylnitrosourea (MNU), 1,2-dimethylhydrazine (DMH), N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) and 2,2'-dihydroxy-di-n-propylnitrosamine (DHPN) for a total multiple initiation period of 4 weeks (DMBDD treatment). BHA, caffeic acid, sesamol and 4-MP, each at doses of 0.4% or 0.08%, and catechol at doses of 0.16% or 0.032% were administered in the diet either alone or in combination after completion of the initiation regimen. All surviving animals were killed at the end of week 28, and major organs were examined histopathologically. In the carcinogenicity study, slightly increased incidences of forestomach papillomas were found in the sesamol- (15.8%), caffeic acid- (14.8%), catechol- (3%) and 4-MP- (11.5%) treated groups as compared with basal diet (0%), and a significant increase was observed with the five antioxidants in combination (42.9%, P < 0.001). In a medium-term multiorgan carcinogenesis model, incidences of forestomach papillomas and/or carcinomas were increased in each high dose group, but additive or synergistic effects were not found in the combination group. In the low dose case, the incidence of forestomach papillomas was significantly increased only in the combination group. With regard to other organs, the incidence of colon tumors was significantly decreased only in the high dose combination group. The results indicate that even at low dose levels phenolic compounds can exert additive/synergistic effect on carcinogenesis.

Hepatic transforming growth factor beta gives rise to tumor-initiating cells and promotes liver cancer development.

PubMed

Wu, Kun; Ding, Jin; Chen, Cheng; Sun, Wen; Ning, Bei-Fang; Wen, Wen; Huang, Lei; Han, Tao; Yang, Wen; Wang, Chao; Li, Zhong; Wu, Meng-Chao; Feng, Gen-Sheng; Xie, Wei-Fen; Wang, Hong-Yang

2012-12-01

Liver cirrhosis is a predominant risk factor for hepatocellular carcinoma (HCC). However, the mechanism underlying the progression from cirrhosis to HCC remains unclear. Herein we report the concurrent increase of liver progenitor cells (LPCs) and transforming growth factor-β (TGF-β) in diethylnitrosamine (DEN)-induced rat hepatocarcinogenesis and cirrhotic livers of HCC patients. Using several experimental approaches, including 2-acetylaminofluorene/partial hepatectomy (2-AAF/PHx) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-elicited murine liver regeneration, we found that activation of LPCs in the absence of TGF-β induction was insufficient to trigger hepatocarcinogenesis. Moreover, a small fraction of LPCs was detected to coexpress tumor initiating cell (T-IC) markers during rat hepatocarcinogenesis and in human HCCs, and TGF-β levels were positively correlated with T-IC marker expression, which indicates a role of TGF-β in T-IC generation. Rat pluripotent LPC-like WB-F344 cells were exposed to low doses of TGF-β for 18 weeks imitating the enhanced TGF-β expression in cirrhotic liver. Interestingly, long-term treatment of TGF-β on WB-F344 cells impaired their LPC potential but granted them T-IC properties including expression of T-IC markers, increased self-renewal capacity, stronger chemoresistance, and tumorigenicity in NOD-SCID mice. Hyperactivation of Akt but not Notch, signal transducer and activator of transcription 3 (STAT3), or mammalian target of rapamycin (mTOR) was detected in TGF-β-treated WB-F344 cells. Introduction of the dominant-negative mutant of Akt significantly attenuated T-IC properties of those transformed WB-F344 cells, indicating Akt was required in TGF-β-mediated-generation of hepatic T-ICs. We further demonstrate that TGF-β-induced Akt activation and LPC transformation was mediated by microRNA-216a-modulated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) suppression. Hepatoma-initiating cells may derive from hepatic progenitor cells exposed to chronic and constant TGF-β stimulation in cirrhotic liver, and pharmaceutical inhibition of microRNA-216a/PTEN/Akt signaling could be a novel strategy for HCC prevention and therapy targeting hepatic T-ICs. Copyright © 2012 American Association for the Study of Liver Diseases.

Genetic inactivation of Nrf2 prevents clonal expansion of initiated cells in a nutritional model of rat hepatocarcinogenesis.

PubMed

Orrù, Claudia; Szydlowska, Marta; Taguchi, Keiko; Zavattari, Patrizia; Perra, Andrea; Yamamoto, Masayuki; Columbano, Amedeo

2018-06-21

Dysregulation of the Keap1-Nrf2 pathway has been observed in experimental and human tumors, suggesting possible roles of the pathway in cancer development. Herein, we examined whether Nrf2 (Nfe2l2) activation occurs at early steps of rat hepatocarcinogenesis, to assess critical contributions of Nrf2 to the onset of hepatocellular carcinoma (HCC). We used wild-type (WT) and Nrf2 knockout (Nrf2KO) rats treated with a single injection of diethylnitrosamine (DENA) followed by choline-devoid methionine-deficient (CMD) diet. This experimental model causes massive fatty liver and steatohepatitis with fibrosis and enables identification of early stages of hepatocarcinogenesis. We found that Nrf2 activation takes place in early preneoplastic lesions identified by the marker glutathione S-transferase placental form (GSTP). Nrf2 missense mutations, known to disrupt the Keap1-Nrf2 binding, were present in 65.7% of GSTP-positive foci. Nrf2KO rats were used to directly investigate whether Nrf2 is critical for initiation and/or clonal expansion of DENA-damaged hepatocytes. While Nrf2 genetic inactivation did not alter DENA-induced initiation, it led to increased liver injury and chronic compensatory hepatocyte regeneration when rats were fed a CMD diet. However, in spite of such a permissive environment, the livers of Nrf2KO rats did not display any preneoplastic lesion unlike those of WT rats. These results demonstrate that, in a model of hepatocarcinogenesis resembling human non-alcoholic fatty liver disease: i) Nrf2 is activated at early steps of the tumorigenic process and ii) Nrf2 is mandatory for the clonal expansion of initiated cells, indicating that Nrf2 is critical in the onset of HCC. Dysregulation of the Keap1-Nrf2 molecular pathway has been observed in human tumors. In a nutritional model of hepatocarcinogenesis, the protein Nrf2 is frequently mutated/activated at early steps of the tumorigenic process. Herein, we show that Nrf2 is mandatory for the development of preneoplastic lesions. These results suggest that Nrf2 has a critical role in the onset of hepatocellular carcinoma. Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

O-GlcNAcylation of RACK1 promotes hepatocellular carcinogenesis.

PubMed

Duan, Fangfang; Wu, Hao; Jia, Dongwei; Wu, Weicheng; Ren, Shifang; Wang, Lan; Song, Shushu; Guo, Xinying; Liu, Fenglin; Ruan, Yuanyuan; Gu, Jianxin

2018-06-01

Aberrant oncogenic mRNA translation and protein O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) are general features during tumorigenesis. Nevertheless, whether and how these two pathways are interlinked remain unknown. Our previous study indicated that ribosomal receptor for activated C-kinase 1 (RACK1) promoted chemoresistance and growth in hepatocellular carcinoma (HCC). The aim of this study is to examine the role of RACK1 O-GlcNAcylation in oncogene translation and HCC carcinogenesis. The site(s) of RACK1 for O-GlcNAcylation was mapped by mass spectrometry analysis. HCC cell lines were employed to examine the effects of RACK1 O-GlcNAcylation on the translation of oncogenic factors and behaviors of tumor cells in vitro. Transgenic knock-in mice were used to detect the role of RACK1 O-GlcNAcylation in modulating HCC tumorigenesis in vivo. The correlation of RACK1 O-GlcNAcylation with tumor progression and relapse were analyzed in clinical HCC samples. We found that ribosomal RACK1 was highly modified by O-GlcNAc at Ser122. O-GlcNAcylation of RACK1 enhanced its protein stability, ribosome binding and interaction with PKCβII (PRKCB), leading to increased eukaryotic translation initiation factor 4E phosphorylation and translation of potent oncogenes in HCC cells. Genetic ablation of RACK1 O-GlcNAcylation at Ser122 dramatically suppressed tumorigenesis, angiogenesis, and metastasis in vitro and in diethylnitrosamine (DEN)-induced HCC mouse model. Increased RACK1 O-GlcNAcylation was also observed in HCC patient samples and correlated with tumor development and recurrence after chemotherapy. These findings demonstrate that RACK1 acts as key mediator linking O-GlcNAc metabolism to cap-dependent translation during HCC tumorigenesis. Targeting RACK1 O-GlcNAcylation provides promising options for HCC treatment. O-GlcNAcylation of ribosomal receptor for activated C-kinase 1 at the amino acid serine122 promotes its stability, ribosome localization and interaction with the protein kinase, PKCβII, thus driving the translation of oncogenes and tumorigenesis of hepatocellular carcinoma. Increased O-GlcNAcylation of ribosomal receptor for activated C-kinase 1 is positively correlated with tumor growth, metastasis and recurrence in patients with hepatocellular carcinoma. Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Inhibition of the promotion of hepatocarcinogenesis by 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153) by the deletion of the p50 subunit of NF-{kappa}B in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Glauert, Howard P.; Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40506; Tharappel, Job C.

Polychlorinated biphenyls (PCBs) are persistent and ubiquitous environmental chemicals that bioaccumulate and have hepatic tumor promoting activity in rodents. The present study examined the effect of deleting the p50 subunit of NF-{kappa}B on the hepatic tumor promoting activity of 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153) in mice. Both wild-type and p50-/- male mice were injected i.p. with diethylnitrosamine (DEN, 90 mg/kg) and then subsequently injected biweekly with 20 i.p. injections of PCB-153 (300 {mu}mol/kg/injection). p50 deletion decreased the tumor incidence in both PCB- and vehicle-treated mice, whereas PCB-153 slightly (P = 0.09) increased the tumor incidence in wild-type and p50-/- mice. PCB-153 increased themore » total tumor volume in both wild-type and p50-/- mice, but the total tumor volume was not affected by p50 deletion in either PCB- or vehicle-treated mice. The volume of tumors that were positive for glutamine synthetase (GS), which is indicative of mutations in the beta-catenin gene, was increased in both wild-type and p50-/- mice administered PCB-153 compared to vehicle controls, and inhibited in p50-/- mice compared to wild-type mice (in both PCB- and vehicle-treated mice). The volume of tumors that were negative for GS was increased in p50-/- mice compared to wild-type mice but was not affected by PCB-153. PCB-153 increased cell proliferation in normal hepatocytes in wild-type but not p50-/- mice; this increase was inhibited in p50-/- mice. In hepatic tumors, the rate of cell proliferation was much higher than in normal hepatocytes, but was not affected by PCB treatment or p50 deletion. The rate of apoptosis, as measured by the TUNEL assay, was not affected by PCB-153 or p50 deletion in normal hepatocytes. In hepatic tumors, the rate of apoptosis was lower than in normal hepatocytes; PCB-153 slightly (P = 0.10) increased apoptosis in p50-/- but not wild-type mice; p50 deletion had no effect. Taken together, these data indicate that the absence of the NF-{kappa}B p50 subunit inhibits the promoting activity of PCB-153 and alters the proliferative and apoptotic changes in mouse liver in the response to PCBs.« less

Hexachlorobenzene induces cell proliferation, and aryl hydrocarbon receptor expression (AhR) in rat liver preneoplastic foci, and in the human hepatoma cell line HepG2. AhR is a mediator of ERK1/2 signaling, and cell cycle regulation in HCB-treated HepG2 cells.

PubMed

de Tomaso Portaz, Ana Clara; Caimi, Giselle Romero; Sánchez, Marcela; Chiappini, Florencia; Randi, Andrea S; Kleiman de Pisarev, Diana L; Alvarez, Laura

2015-10-02

Hexachlorobenzene (HCB) is a widespread environmental pollutant, and a liver tumor promoter in rodents. Depending on the particular cell lines studied, exposure to these compounds may lead to cell proliferation, terminal differentiation, or apoptosis. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is involved in drug and xenobiotic metabolism. AhR can also modulate a variety of cellular and physiological processes that can affect cell proliferation and cell fate determination. The mechanisms by which AhR ligands, both exogenous and endogenous, affect these processes involve multiple interactions between AhR and other signaling pathways. In the present study, we examined the effect of HCB on cell proliferation and AhR expression, using an initiation-promotion hepatocarcinogenesis protocol in rat liver and in the human-derived hepatoma cell line, HepG2. Female Wistar rats were initiated with a single dose of 100 mg/kg of diethylnitrosamine (DEN) at the start of the experiment. Two weeks later, daily dosing of 100 mg/kg HCB was maintained for 10 weeks. Partial hepatectomy was performed 3 weeks after initiation. The number and area of glutathione S-transferase-P (GST-P)-positive foci, in the rat liver were used as biomarkers of liver precancerous lesions. Immunohistochemical staining showed an increase in proliferating cell nuclear antigen (PCNA)-positive cells, along with enhanced AhR protein expression in hepatocytes within GST-P-positive foci of (DEN HCB) group, when compared to DEN. In a similar manner, Western blot analysis demonstrated that HCB induced PCNA and AhR protein expression in HepG2 cells. Flow cytometry assay indicated that the cells were accumulated at S and G2/M phases of the cell cycle. HCB increased cyclin D1 protein levels and ERK1/2 phosphorylation in a dose-dependent manner. Treatment of cells with a selective MEK1 inhibitor, prevented HCB-stimulatory effect on PCNA and cyclinD1, indicating that these effects are mediated by ERK1/2. Pretreatment with an AhR antagonist, prevented HCB-induced PCNA protein levels, ERK1/2 phosphorylation and alterations in cell cycle distribution. These results demonstrate that HCB-induced HepG2 proliferation and cell cycle progression depend on ERK1/2 phosphorylation which is mediated by the AhR. Our results provide a clue to the molecular events involved in the mechanism of action of HCB-induced hepatocarcinogenesis. Copyright © 2015. Published by Elsevier Ireland Ltd.

Fumagillin treatment of hepatocellular carcinoma in rats: An in vivo study of antiangiogenesis

PubMed Central

Sheen, I-Shyan; Jeng, Kuo-Shyang; Jeng, Wen-Juei; Jeng, Chi-Juei; Wang, Yi-Ching; Gu, Shu-Ling; Tseng, Shin-Yun; Chu, Chien-Ming; Lin, Chia-Hui; Chang, Kuo-Ming

2005-01-01

AIM: To investigate the effect and possible mechanisms of antiangiogenesis therapy for HCC in rats. METHODS: Adult male LEW/SsN rats were divided into 3 groups, 25 animals each. Group A was the control group. Groups B and C were given diethylnitrosamine, 5 mg/kg/d. In addition, group C rats received an intraperitoneal injection of fumagillin, 30 mg/(kg·d). Five animals in each group were killed at 6th, 12th, 18th, 20th and 24th wk to evaluate the development of HCC and metastasis. Weight of the rats, liver tumors, and number of organs involved by HCC were measured at each stage. We compared methionine aminopeptidase-2 (MetAP-2) mRNA, Bcl-2 mRNA, telomerase mRNA, and telomerase activity at 24th wk in the liver tissue of group A rats and tumor tissue of HCC from group B and C rats. RESULTS: No HCC developed in group A, but tumors were present in group B and C rats by the 18th wk. At wk 20 and 24, the median liver weight in group B was 0.64 g (range: 0.58-0.70 g) and 0.79 g (range: 0.70-0.90 g) (P = 0.04), and that in group C was 0.37 g (range: 0.35-0.42 g) and 0.39 g (range: 0.35-0.47 g) (P = 0.67). The liver weight in group C rats was significantly lower than that in group B rats (P = 0.009). At the same time, the median metastasis score (number of organ systems involved) was 3 (range 2-3) in group B, and 1 (range 1-2) in group C, a significant difference between the groups (P = 0.007, 0.004). The levels of MetAP-2 mRNA were significantly higher in groups B and C than in group A (P = 0.025), and significantly higher in group C than in group B (P = 0.047). The level of Bcl-2 mRNA was significantly higher in group B than in group A (P = 0.024), but lower in group C than in group B, although not significantly (P = 0.072). Telomerase mRNA was significantly higher in group B than in group A (P = 0.025), but significantly lower in group C than in group B (P = 0.016). The same inter-group relationship was also true for telomerase activity (P = 0.025 and 0.046). CONCLUSION: Fumagillin effectively inhibits both liver tumor growth and metastasis in rats in vivo. A possible mechanism is fumagillin-induced inhibition of MetAP-2, which plays an essential role in endothelial cell proliferation. Inhibition of MetAP-2 also results in inhibition of Bcl-2 and telomerase activity. PMID:15682466

N-nitrosamines induced infertility and hepatotoxicity in male rabbits.

PubMed

Sheweita, S A; El Banna, Y Y; Balbaa, M; Abdullah, I A; Hassan, H E

2017-09-01

N-nitrosamines are widely spread environmental pollutants of well-known toxicity and carcinogenicity in various animal species. These compounds are metabolically activated by cytochrome P450 system predominantly in the liver and in other tissues into more active metabolites leading to generation of both alkylating agents that alkylate DNA and reactive oxygen species. In the current study, we investigated the influence of four types of N-nitrosamines that are commonly present in the environment [methyethylnitrosamine, (MEN), diethylnitrosamine (DEN), diphenylnitroasamine (DPN) and dimethylnitrosamine (DMN)] on both livers and testes of male rabbits through assessment of 17 β-hydroxysteroid dehydrogenase (17 β-HSD) activity. The protein expression of the three cytochrome P450s (CYP11A1, CYP19A1, and CYP21A2) is involved in the steroidogenesis. The levels of testosterone (T) and estradiol (E2) were also determined in the plasma of N-nitrosamines-treated rabbits after one, four-, eight- and twelve weeks of treatment of male New Zealand rabbits with an oral dose of 0.5 mg/kg B.W/day of each compound. In addition, activities of glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT) and levels of free radicals measured as thiobarbituric acid reactive substances (TBARS), and reduced glutathione (GSH) level were quantified in both livers and testes. The present study showed that levels of free radicals (TBARS) were markedly increased, whereas GSH levels were depleted in the tissues of both livers and testes after treatment of rabbits with any of N-nitrosamines. In addition, all tested N-nitrosamines inhibited the activities of antioxidant enzyme activities (GR, GST, SOD, and CAT) in hepatic and testicular tissues of rabbits after 12 weeks of treatment. Histopathological examination showed that N-nitrosamines caused lymphocytic infiltration with vascular degeneration and necrosis, congestion of central vein with RBCs hemolysis, dilated sinusoids, as well as fibrosis around portal areas were seen in hepatic tissues. In the testes, histopathological examination displayed disorganized seminiferous tubules with degeneration of germinal epithelium and Sertoli cells. Also, spermatogenic cells had pyknotic nuclei and others were detached from basement membranes of seminiferous tubules, edema was seen between seminiferous tubules. Moreover, the present data showed that MEN and DEN down-regulated the protein expression of both CYP19A1 and 21A2 in both livers and testes of male rabbits. In addition, both MEN and DEN decreased levels of testosterone and estradiol in plasma of treated rabbits. On the one hand, DMN and DPN markedly up-regulated the protein expression of CYP19A1 in both hepatic and testicular tissues of treated rabbits. These compounds potentially increased estradiol and decreased testosterone levels. On the other hand, no correlation was found between the expression of CYP11A1 and levels of both testosterone and estradiol. It is concluded that most of tested N-nitrosamines induce different changes, which could be a new mechanism of infertility due to exposure to N-nitrosamines from different environmental sources. © 2017 Wiley Periodicals, Inc.

T2 relaxation time is related to liver fibrosis severity

PubMed Central

Siqueira, Luiz; Uppal, Ritika; Alford, Jamu; Fuchs, Bryan C.; Yamada, Suguru; Tanabe, Kenneth; Chung, Raymond T.; Lauwers, Gregory; Chew, Michael L.; Boland, Giles W.; Sahani, Duhyant V.; Vangel, Mark; Hahn, Peter F.; Caravan, Peter

2016-01-01

Background The grading of liver fibrosis relies on liver biopsy. Imaging techniques, including elastography and relaxometric, techniques have had varying success in diagnosing moderate fibrosis. The goal of this study was to determine if there is a relationship between the T2-relaxation time of hepatic parenchyma and the histologic grade of liver fibrosis in patients with hepatitis C undergoing both routine, liver MRI and liver biopsy, and to validate our methodology with phantoms and in a rat model of liver fibrosis. Methods This study is composed of three parts: (I) 123 patients who underwent both routine, clinical liver MRI and biopsy within a 6-month period, between July 1999 and January 2010 were enrolled in a retrospective study. MR imaging was performed at 1.5 T using dual-echo turbo-spin echo equivalent pulse sequence. T2 relaxation time of liver parenchyma in patients was calculated by mono-exponential fit of a region of interest (ROI) within the right lobe correlating to histopathologic grading (Ishak 0–6) and routine serum liver inflammation [aspartate aminotransferase (AST) and alanine aminotransferase (ALT)]. Statistical comparison was performed using ordinary logistic and ordinal logistic regression and ANOVA comparing T2 to Ishak fibrosis without and using AST and ALT as covariates; (II) a phantom was prepared using serial dilutions of dextran coated magnetic iron oxide nanoparticles. T2 weighed imaging was performed by comparing a dual echo fast spin echo sequence to a Carr-Purcell-Meigboom-Gill (CPMG) multi-echo sequence at 1.5 T. Statistical comparison was performed using a paired t-test; (III) male Wistar rats receiving weekly intraperitoneal injections of phosphate buffer solution (PBS) control (n=4 rats); diethylnitrosamine (DEN) for either 5 (n=5 rats) or 8 weeks (n=4 rats) were MR imaged on a Bruker Pharmascan 4.7 T magnet with a home-built bird-cage coil. T2 was quantified by using a mono-exponential fitting algorithm on multi-slice multi echo T2 weighted data. Statistical comparison was performed using ANOVA. Results (I) Histopathologic evaluation of both rat and human livers demonstrated no evidence of steatosis or hemochromatosis There was a monotonic increase in mean T2 value with increasing degree of fibrosis (control 65.4±2.9 ms, n=6 patients); mild (Ishak 1–2) 66.7±1.9 ms (n=30); moderate (Ishak 3–4) 71.6±1.7 ms (n=26); severe (Ishak 5–6) 72.4±1.4 ms (n=61); with relatively low standard error (~2.9 ms). There was a statistically significant difference between degrees of mild (Ishak <4) vs. moderate to severe fibrosis (Ishak >4) (P=0.03) based on logistic regression of T2 and Ishak, which became insignificant (P=0.07) when using inflammatory markers as covariates. Expanding on this model using ordinal logistic regression, there was significance amongst all 4 groups comparing T2 to Ishak (P=0.01), with significance using inflammation as a covariate (P=0.03) and approaching statistical significance amongst all groups by ANOVA (P=0.07); (II) there was a monotonic increase in T2 and statistical significance (ANOVA P<0.0001) between each rat subgroup [phosphate buffer solution (PBS) 25.2±0.8, DEN 5-week (31.1±1.5), and DEN 9-week (49.4±0.4) ms]; (III) the phantoms that had T2 values within the relevant range for the human liver (e.g., 20–100 ms), demonstrated no statistical difference between two point fits on turbo spin echo (TSE) data and multi-echo CPMG data (P=0.9). Conclusions The finding of increased T2 with liver fibrosis may relate to inflammation that may be an alternative or adjunct to other noninvasive MR imaging based approaches for assessing liver fibrosis. PMID:27190762