OBJECTIVES: To investigate the inhibitory effect of Zheng GanDecoction (ZGF) on tumor progression in a rat model of diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) and explore the possible mechanism. METHODS: Seventy SD rats were subjected to regular intraperitoneal injections of DEN (50 mg/kg) for 12 weeks to induce HCC tumorigenesis, with another 10 rats receiving saline injections as the normal control. After successful modeling, the rats were randomized into 5 groups (n=10) for daily treatment with distilled water ( model group), Huaier Granules (4 g/kg; positive control group), or ZGF at low, medium, and high doses (2, 4, and 8 g/kg, respectively) via gavage for 17 weeks. Body weight changes of the rats were monitored, and after completion of the treatments, the rats were euthanized for measurement of liver, spleen and thymus indices and morphological and histopathological examinations of the liver tissues using HE staining. The expressions of YAP, p-YAP, MST1, LATS1 and p-LATS1 in the liver tissues were detected using immunohistochemistry and Western blotting. RESULTS: Compared with the normal control rats, the rat models with DEN-induced HCC exhibited much poorer general condition with a significantly reduced survival rate, increased body weight and liver and spleen indices, and a lowered thymus index. ZGF treatment obviously reduced liver and spleen indices, increased the thymus index, and improved pathologies of the liver tissues of the rat models. Immunohistochemistry and Western blotting showed a dose-dependent reduction of YAP expression and an increment of p-YAP expression in ZGF-treated rats, which also exhibited significantly upregulated hepatic expressions of MST1, LATS1 and p-LATS1. CONCLUSIONS ZGF inhibits DEN-induced HCC in rats by activating the Hippo/YAP pathway via upregulating MST1 and LATS1 expression, which promotes YAP phosphorylation and degradation to suppress proliferation and induce apoptosis of the tumor cells.
Animals ; Drugs, Chinese Herbal/pharmacology* ; Diethylnitrosamine ; Rats, Sprague-Dawley ; Rats ; Signal Transduction/drug effects* ; Protein Serine-Threonine Kinases/metabolism* ; Carcinoma, Hepatocellular/drug therapy* ; YAP-Signaling Proteins ; Liver Neoplasms/drug therapy* ; Hippo Signaling Pathway ; Male ; Liver Neoplasms, Experimental/metabolism* ; Transcription Factors/metabolism* ; Adaptor Proteins, Signal Transducing/metabolism*

Animals ; Diethylnitrosamine/toxicity* ; Liver ; Liver Neoplasms/chemically induced* ; Rats

OBJECTIVE: To study the inhibitory effect of pills (BJJ) agaisnt diethylnitrosamine (DEN)-induced hepatocarcinogenesis and explore the relation between this effect and the inflammasome signaling pathway. METHODS: Sixty-five male SD rats were randomly divided into control group, DEN model group, and 3 BJJ treatment groups at low, medium and high dose (with daily dose of 0.55, 1.1 and 2.2 g/kg, respectively, for 12 consecutive weeks starting from the 5th week after modeling). The pathological changes of the liver tissue were observed with HE and Masson staining, and serum levels of alanine transaminase (ALT), glutamic oxaloacetic transaminase (AST), alkaline phosphatase (ALP) and total bilirubin (TBIL) of the rats were detected using ELISA. Oxidation stress in the liver tissue was assessed with ELISA, and Western blotting and ELISA were used to detect the molecular expressions of inflammasome-related pathway. RESULTS: BJJ significantly inhibited tumor growth in the liver of the rats. HE and Masson staining showed that BJJ treatment obviously ameliorated liver fibrosis and reduced cancer cell and inflammatory cell infiltration in the liver. BJJ significantly reduced elevations of serum ALT, AST, ALP and TBIL levels, increased the contents of superoxide dismutase, catalase and glutathione peroxidase in the liver and suppressed malondialdehyde in Den-treated rats. BJJ also dose-dependently decreased the expressions of NLRP3, apoptosis-associated speck-like protein (ASC), caspase-1, pro-IL-1β, pro-IL-18, IL-1β and IL-18 in the liver of Den-treated rats. CONCLUSIONS BJJ treatment can dose-dependently inhibit DEN-induced hepatocarcinogenesis by enhancing antioxidant capacity and down-regulating inflammatory-related pathways in rats.
Animals ; Aspartate Aminotransferases ; Diethylnitrosamine ; Liver ; Liver Neoplasms ; Male ; Rats ; Rats, Sprague-Dawley

European Medicines Agency withdrew valsartan from European market in July 2018 because it was contaminated with carcinogen, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA). Medicines and Healthcare Products Regulatory Agency also found the same contamination and withdrew it from England market. US Food and Drug Administration followed the action after confirming its contamination. Ministry of Food and Drug Safety (MFDS) conducted testing all the valsartans at Korean market and withdrew some of them from market after confirming the contamination with NDMA. MFDS provided the pharmaceutical companies and laboratory institutions with the manual for testing both NDMA and NDEA and educated relevant personnels. MFDS also evaluated the health impact of the contaminated valsartan on the hypertensive patients who took the valsartan, which was shown to be very low risk of additional cancer incidence. MFDS pronounced strengthening of the safety management for the raw materials of the medicines. For guaranteeing the safety of medicines, more comprehensive drug safety management system from developing new drugs to consuming the medicines should be established. For achieving such a goal, active participation of all the stakeholders of the medicines including governmental agencies including MFDS and Ministry of Health and Welfare, the National Assembly, healthcare professionals, pharmaceutical companies, mass media, and general population including patients should be needed.
Antihypertensive Agents ; Delivery of Health Care ; Diethylnitrosamine ; Dimethylnitrosamine ; England ; Humans ; Incidence ; Mass Media ; Safety Management ; United States Food and Drug Administration ; Valsartan

To explore the renal metabolic markers relavant to the renal toxicity of diethylnitrosamine and the metabolic pathways involved in the renal metabolic markers.
 Methods: Nineteen Sprague Dawley rats were assigned into 2 groups: A normal control group (n=9) and a diethylnitrosamine (DEN) administration group (n=10). The rats in the normal control group were given sterilized water for free drinking. The rats in the DEN administration group were given 0.1 mg/mL DEN solution for free drinking. After 18 weeks, the kidney tissues were collected and tested for nuclear magnetic resonance detection and pathological examination.
 Results: The content of kidneys metabolites in the rats with the DEN administration was changed significantly. The levels of alanine, taurine, pyruvate, acetate, and choline were significantly reduced compared with rat in the normal control group, while the levels of creatine, glycine, TMAO, methionine, proline, lactate, valine, leucine and isoleucine were significantly increased.
 Conclusion: Metabolicomics studies have revealed significant differences in five metabolic pathways, including valine, leucine and isoleucine biosynthesis, glycine serine and threonine metabolism, pyruvate metabolism, glycolysis or gluconeogenesis, cysteine and methionine metabolism.
Alkylating Agents ; toxicity ; Animals ; Diethylnitrosamine ; toxicity ; Glycine ; Kidney ; drug effects ; physiology ; Metabolic Networks and Pathways ; drug effects ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study whether the ethanol extract of Phellinus merrillii (EPM) has chemopreventive potential against liver carcinogenesis.

METHODSThirty male Spraque-Dawley rats were randomly divided into control group, EPM control group, hepatocarcinoma control group, low-dose EPM group and high-dose EPM group, 6 in each group. Using the Solt and Farber protocol in a rat model of hepatocarcinogenesis, the chemopreventive effect of EPM on diethylnitrosamine (DEN)-initiated, 2-acetylaminofluorene (2-AAF) and partial hepatectomy (PH)-promoted liver carcinogenesis in rats was evaluated. Basic pathophysiological and histological examinations, together with the serum levels of glutamic oxaloacetic transaminase (sGOT), glutamic pyruvic transaminase (sGPT) and gamma-glutamyl transpeptidase (γ-GT) were measured.

RESULTSTreatment of EPM at the concentration of 2 g/kg body weight in the diet for 8 weeks clearly prevented the development of carcinogenesis and reduced the levels of sGOT, sGPT, and serum γ-GT of rats as compared with the hepatocarcinoma control group (P<0.05 or P<0.01). These phenotypes were accompanied by a significant increase in natural killer cell activity.

CONCLUSIONEPM showed a strong liver preventive effect against DEN+2-AAF+PH-induced hepatocarcinogenesis in a rat model.

2-Acetylaminofluorene ; Animals ; Basidiomycota ; chemistry ; Carcinogenesis ; chemically induced ; Cytoprotection ; drug effects ; Diethylnitrosamine ; Ethanol ; chemistry ; Liver Neoplasms, Experimental ; chemically induced ; prevention & control ; Male ; Plant Extracts ; chemistry ; pharmacology ; Protective Agents ; pharmacology ; Rats ; Rats, Sprague-Dawley

The objective of this study was to determine the effect of ionizing radiation (IR) exposure of parents on carcinogenesis of the next generation focusing on the epigenetic perspective to clarify the relationship between radiation dose and carcinogenesis in F1 generation SD rats. F1 generations from pregnant rats (F0) who were exposed to gamma rays were divided into three groups according to the dose of radiation: 10 rad, 30 rad, and untreated. They were intraperitoneally injected with 50 mg/kg of diethylnitrosamine (DEN). Carcinogenesis was analyzed by examining expression levels of tumor suppressor genes (TSG) and other related genes by methylation-specific polymerase chain reaction (MSP). DNA methylation in liver tissues was evaluated to discern epigenetic regulation of transgenerational carcinogenesis vulnerability following IR exposure. Numerous studies have proved that transcriptional inactivation due to hypermethylation of TSG preceded carcinogenesis. Results of this study revealed hypermethylation of tumor suppressor gene SOCS1 in group treated with 30 rad. In addition, genes related to DNA damage response pathway (GSTP1, ATM, DGKA, PARP1, and SIRT6) were epigenetically inactivated in all DEN treated groups. In the case of proto-oncogene c-Myc, DNA hypermethylation was identified in the group with low dose of IR (10 rad). Results of this study indicated that each TSG had different radiation threshold level (dose-independent way) and DEN treatment could affect DNA methylation profile irrelevant of ionizing radiation dose.
Animals ; Carcinogenesis* ; Diethylnitrosamine ; DNA ; DNA Damage ; DNA Methylation ; Epigenomics* ; Family Characteristics ; Gamma Rays ; Genes, Tumor Suppressor ; Humans ; Liver ; Parents ; Polymerase Chain Reaction ; Proto-Oncogenes ; Radiation, Ionizing* ; Rats

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: BMSP may have a protective effect against acute liver injury by inhibiting necrosis and inflammatory response in DEN-treated mice.
Alanine Transaminase ; Alkaline Phosphatase ; Animals ; Aspartate Aminotransferases ; Bombyx* ; Carcinoma, Hepatocellular ; Chemokines ; Cytokines ; Diet ; Diethylnitrosamine ; Liver ; Macrophages ; Mice* ; Necrosis

OBJECTIVETo establish a zebrafish model of liver fibrosis via diethylnitrosamine (DEN)-induced liver injury.

METHODSA total of 120 wild-type 3-month-old zebrafish were randomly divided into DEN-treated group and control group. The survival rate and behavioral changes of each group were observed. After treatment with DEN for 2, 4, and 6 weeks, liver index was measured, and liver fibrosis was evaluated with HE staining, Gomori staining and Sirius red staining.

RESULTSNo obvious behavioral change was observed in DEN-treated group during the experiment. Compared with that in control group, the liver index of zebrafish in DEN-treated group showed no significantly changes at the time points of observation. Proliferation of reticulate fibers was found in 30% of zebrafish treated with DEN for 4 weeks, and the rate increased to 80% at 6 weeks when reticulate fibers and collagen fibers actively proliferated to result in fiber collapse and formation of fibrotic nodules.

CONCLUSIONA stable zebrafish liver fibrosis model was successfully established by inducing liver damage to facilitate studies of the pathogenesis of liver fibrosis and screening therapeutic drugs.

Animals ; Diethylnitrosamine ; Disease Models, Animal ; Liver Cirrhosis ; chemically induced ; Zebrafish

OBJECTIVEThe present paper aims to investigate the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and N-nitrosodiethylamine (DEN) on tumorigenesis and its potential mechanism.

METHODSThe potentials of TCDD and DEN in separation or in combination to induce malignant transformation were tested in Balb/c 3T3 cells by using a cell transformation assay method. The possible mechanism of observed effects was studied further by adding α-naphthoflavone (α-NF), a competitive binding agent of TCDD, to the Aryl hydrocarbon receptor (AhR) pathway. The mRNA expressions of Cyp1a1 and Cyp2a5 gene in Balb/c 3T3 cells treated by DEN and TCDD in separation or in combination with or without presence of α-NF were measured with fluorescence quantification RT-PCR technique.

RESULTSThe cell transformation frequency (TF) was significantly higher in case of induction with TCDD in combination with DEN, as compared to that with either TCDD or DEN alone. These effects were not inhibited via α-NF. The mRNA expression levels of both Cyp1a1 and Cyp2a5 were enhanced by TCDD treatment alone, but this inducible effect was blocked in cells treated by TCDD and DEN in combination.

CONCLUSIONTCDD and DEN had a significant synergistic effect on tumorigenesis when they were used in combination. AhR pathway may not be the key mechanism of this synergistic effect. Thus, it is necessary to further test the potential mechanism involved in cancer development.

3T3 Cells ; Animals ; Base Sequence ; Carcinogens ; toxicity ; Cell Transformation, Neoplastic ; Cytochrome P-450 Enzyme System ; genetics ; DNA Primers ; Diethylnitrosamine ; toxicity ; Drug Synergism ; Mice ; Mice, Inbred BALB C ; Polychlorinated Dibenzodioxins ; toxicity ; RNA, Messenger ; genetics ; Real-Time Polymerase Chain Reaction ; Receptors, Aryl Hydrocarbon ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction