During Diethylnitrosamine Research Articles

Characterization of the inflammatory microenvironment and hepatic macrophage subsets in experimental hepatocellular carcinoma models.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. HCC typically develops on a background of chronic inflammation and fibrosis with tumor associated macrophages (TAMs) playing an important role in chronic inflammation-induced HCC and progression. However, the liver harbors unique macrophages, resident liver Kupffer cells (KCs) and monocyte-derived macrophages (Mo-Mφ), and their contribution to HCC and to the population of TAMs is incompletely known. Here, we characterized the tumor microenvironment and the proportion and transcriptional profile of hepatic macrophages (Mφ) in two commonly used HCC mouse models. A gradually increased expression of inflammatory, immune regulatory, fibrotic and cell proliferation pathways and markers was observed during diethylnitrosamine (DEN)- and non-alcoholic steatohepatitis (NASH)-induced HCC development. The transcriptional phenotypes of isolated hepatic Mφ subsets were clearly distinct and shifted during HCC development, with mixed pro-inflammatory and tumor-promoting expression profiles. There were marked differences between the models as well, with Mφ in NASH-HCC exhibiting a more immunomodulatory phenotype, in conjunction with an upregulation of lipid metabolism genes. Our data show that at least some infiltrated macrophages display expression of pro-tumoral markers, and that Kupffer cells are part of the population of TAMs and enhance tumor progression. These insights are useful to further unravel sequential pathogenic events during hepatocarcinogenesis and direct future development of new treatment strategies for HCC.

COX-2/iNOS regulation during experimental hepatic injury and its mitigation by cloudy apple juice

A number of enzymes and transcription factors have been correlated with disease etiology. In this study, involvement of cyclooxygenase-2 and inducible-nitric oxide synthase is examined during diethylnitrosamine (DEN)-induced hepatic injury and cloudy apple juice (CAJ) supplementation. Liver injury was administered in rats by single dose of DEN (10 ml/kg bwt of 1% DEN), while 10 ml/kg bwt CAJ daily was given after 2 h of latency in DEN-treated animals for two weeks. CAJ was characterized by HPLC and subsequently examined for antioxidant power. During the course of treatment liver function, collagen (hydroxyproline), malondialdehyde, protein oxidation, antioxidant enzymes, ATPases, nitrite levels were investigated along with liver histopathology and electron microscopy. COX-2 and iNOS proteins were also localized in liver specimens. The results demonstrated rich polyphenols and antioxidant activity in CAJ. CAJ supplementation significantly restored liver biochemistry and anatomy as revealed by the refurbished investigated parameters. CAJ treatment also declined COX-2 and iNOS activities in injured animals. Electron microscopy demonstrated rejuvenated hepatocytes, Kupffer cells, RER, mitochondria and nucleus in CAJ supplemented animals. The novel outcomes of this study suggest that CAJ potentiates hepatoprotection by stimulating antioxidant power and regulating the COX-2 and iNOS proteins in the liver during experimental liver injury.

Antioxidant and Anticancer potential of Methanolic extract of Kigelia pinnata fruits against DENA induced Hepatocellular Carcinoma in rats

The aim of the present study is to study the preventive nature of Kigelia pinnata methanolic extract (MEKP)during DiEthyl Nitros Amine (DENA) induced hepatocellular carcinoma in rats. Phytochemical screening for the methanolic extract of Kigelia pinnata was carried out. The methanolic extract of the fruits was tested for antioxidant property by in vitro DPPH radical scavenging assay and reducing power assay and also anticancer activity of methanolic extract of Kigelia pinnata was evaluated in DENA induced Sprague Dawley rats at the doses of 250 and 500 mg/kg body weight orally and both doses were administered for 14 weeks. After 24 h of the last dose, the rats were sacrificed and antitumor effect of KMPE extract was assessed by evaluating biochemical parameters such as AFP, AST, ALT, ALP and LDH to evaluate anticancer activity of the extract. MDA and SOD levels in the liver tissue of each group were estimated for determining in vivo antioxidant activity of the plant. Histopathological studies were carried out. MEKP showed the presence of alkaloids, glycosides, flavonoids, tannins, saponins, reducing sugars and phenolic compounds. MEKP showed concentration dependent free radical scavenging activity and increase in reducing power activity. Comparative analysis on the KPME treatment groups revealed that high dose (500mg/kg body weight showed significant reduction in the serum level of enzymes. Also, there was dose dependent effect of MEKP in the MDA and SOD level in the liver tissue. Histopathological studies showed the decrease in the damages to the liver tissue in the MEKP treatment groups. Hence the methanolic extract of Kigelia pinnata possesses anti-hepatocellular activity which may be due to its hepatoprotective action. This may be due to the presence of flavonoids. Further studies can be carried out to elucidate the exact mechanism of its activity as an antioxidant and anticancer agent. The methanolic extract of Kigelia pinnata possesses anti-hepatocellular activity which may be due to its hepatoprotective action. This may be due to the presence of flavonoids. Further studies can be carried out to elucidate the exact mechanism of its activity as an antioxidant and anticancer agent.

Hesperetin conjugated PEGylated gold nanoparticles exploring the potential role in anti-inflammation and anti-proliferation during diethylnitrosamine-induced hepatocarcinogenesis in rats

Liver cancer is the fifth most common cancer and one of the leading causes of death in the world, and second most common cause of death in men. Natural products emerge as the most enduring approaches in the development of anticancer targeting drug. Hesperetin (HP), one of the abundant flavonoids found naturally in citrus fruits, has received considerable attention in anti-cancer promotion and progression. The present study was conducted to decipher the role of 0.5 ml hesperetin conjugated gold nanoparticles (Au-mPEG(5000)-S-HP NPs) during diethylnitrosamine (DEN)-induced hepatocarcinogenesis in male Wistar albino rats and shows the better antioxidant that possesses anti-inflammatory, anti-proliferation and anticarcinogenic properties and may modulate signaling pathways. The confirmation of polymer functionalized gold nanoparticles and drug loaded polymer gold nanoparticles were characterized by HR-TEM with EDAX, and DLS with Zeta potential techniques. The drug encapsulation efficiency and release properties were carried out in PBS at pH 7.4 for Au- mPEG(5000)-S-HP and compared with the control pure hesperetin (HP). Here, we review the role of mast cell counts, tumor necrosis factor alpha (TNF-α), transcription factor nuclear factor-κB (NF-κB), levels of glycoconjugates, proliferating cell nuclear antigen (PCNA) and argyrophilic nucleolar organizing regions, are the master regulator of inflammation and proliferation, in the development of hepatocellular injury, liver fibrosis and HCC. DEN-administered animals showed increased mast cell counts, tumor necrosis factor alpha, transcription factor nuclear factor-κB, glycoconjugates, proliferating cell nuclear antigen, and argyrophilic nucleolar organizing regions. Whereas Au-mPEG(5000)-S-HP NPs supplementation considerably suppressed all the above abnormalities. These results suggest that the Au-mPEG(5000)-S-HP NPs exhibited the better potential anticancer activity by inhibiting cell inflammation and proliferation in DEN-induced hepatocellular carcinogenesis.

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

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 16weeks. 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 (20mg/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.

Carvacrol modulates instability of xenobiotic metabolizing enzymes and downregulates the expressions of PCNA, MMP-2, and MMP-9 during diethylnitrosamine-induced hepatocarcinogenesis in rats.

Hepatocellular carcinoma is the fifth most common malignant tumor in the world, both in terms of incidence and mortality in Asian and Western countries. There are currently limited therapeutic regimens available for effective treatment of this cancer. Carvacrol is a predominant monoterpenoic phenol believed to impede cancer promotion and progression. The present study was conducted to decipher the role of carvacrol during diethylnitrosamine (DEN)-induced hepatocarcinogenesis in male wistar albino rats. Carvacrol (15mg/kg body weight) suppressed the elevation of serum tumor marker enzymes, carcinoembryonic antigen, and α-feto protein induced by DEN. The activities of phase I enzymes increased markedly during DEN induction, but was found to be significantly lowered upon carvacrol treatment. On the contrary, the phase II enzymes decreased in DEN-administered animals, which was improved normalcy upon carvacrol-treated animals. DEN-administered animals showed increased mast cell counts, argyrophilic nucleolar organizing regions, proliferating cell nuclear antigen, and matrix metalloproteinases (MMPs-2/9), whereas carvacrol supplementation considerably suppressed all the above abnormalities. The results suggest that the carvacrol exhibited the potential anticancer activity by inhibiting cell proliferation and preventing metastasis in DEN-induced hepatocellular carcinogenesis.

Protein expression under sustained activation of signal transducer and activator of transcription-3 in diethylnitrosamine-induced rat liver carcinogenesis

The aim of the present study was to investigate the expression of proteins associated with the sustained activation of the signal transducer and activator of transcription (STAT)-3 pathway during diethylnitrosamine (DEN)-induced rat liver carcinogenesis. DEN was intermittently administered to rats to induce liver cancer, and light and electron microscopy were used to observe the morphological changes in the liver during carcinogenesis. Western blotting and quantitative polymerase chain reaction (qPCR) were used to detect the expression of STAT-3, phosphorylated (p)-STAT-3, matrix metalloproteinase (MMP)-10, vascular endothelial growth factor (VEGF), kinase insert domain receptor (KDR), hypoxia inducible factor (HIF)-1α, basic fibroblast growth factor (bFGF) and interleukin (IL)-10, in order to investigate the association between STAT-3 and p-STAT-3 expression and MMP-10, VEGF, KDR, HIF-1α, bFGF and IL-10. The western blotting and qPCR results revealed that the expression of STAT-3, p-STAT-3, MMP-10, VEGF, KDR, HIF-1α, bFGF and IL-10 proteins gradually increased during carcinogenesis. Furthermore, the STAT-3 and p-STAT-3 levels were found to positively correlate with MMP-10, VEGF, KDR, HIF-1α, bFGF and IL-10 protein expression. During DEN-induced rat liver carcinogenesis, STAT-3 protein continually activated MMP-10, VEGF, KDR, HIF-1α, bFGF and IL-10, and its expression was found to positively correlate with the expression of these proteins.

Preventive Effect of Tephrosia Purpurea Against N,N-Diethylnitrosamine Induced Hepatocellular Carcinoma In Swiss Albino Mice

The aim of the present study is to divulge the preventive nature of Tephrosia purpurea Methanolic Extract (TpME) during DiEthylNitrosAmine (DENA) induced liver cancer in male Swiss albino mice. Anticancer activity of methanolic extract of Tephrosia purpurea was evaluated in DENA induced Swiss albino mice at the doses of 200 and 300 mg/kg body weight orally and both doses were administered for 14 days. After 24 h of the last dose, the mice were sacrificed and antitumor effect of TpME was assessed by evaluating liver serum marker enzyme and haematological parameters. Decrease in Aspartate transaminase, Alanine transaminase, Alkaline phosphatase, Bilirubin and Total protein count were observed in TpME treated animals when compared to DENA treated animals. Haematological profiles such as haemoglobin, red blood cell (RBC) and white blood cell (WBC) count reverted to normal level in TpME treated mice. A comparative histopathology study of liver from test group exhibited almost normal architecture, as compared to DENA treated group. The results demonstrated that Tephrosia purpurea methanolic extract at 300 mg/kg dose exhibits significant antitumor activity than 200 mg/kg.

Preventive Effect of Tephrosia Purpurea Against N,N-Diethylnitrosamine Induced Hepatocellular Carcinoma In Swiss Albino Mice

The aim of the present study is to divulge the preventive nature of Tephrosia purpurea Methanolic Extract (TpME) during DiEthylNitrosAmine (DENA) induced liver cancer in male Swiss albino mice. Anticancer activity of methanolic extract of Tephrosia purpurea was evaluated in DENA induced Swiss albino mice at the doses of 200 and 300 mg/kg body weight orally and both doses were administered for 14 days. After 24 h of the last dose, the mice were sacrificed and antitumor effect of TpME was assessed by evaluating liver serum marker enzyme and haematological parameters. Decrease in Aspartate transaminase, Alanine transaminase, Alkaline phosphatase, Bilirubin and Total protein count were observed in TpME treated animals when compared to DENA treated animals. Haematological profiles such as haemoglobin, red blood cell (RBC) and white blood cell (WBC) count reverted to normal level in TpME treated mice. A comparative histopathology study of liver from test group exhibited almost normal architecture, as compared to DENA treated group. The results demonstrated that Tephrosia purpurea methanolic extract at 300 mg/kg dose exhibits significant antitumor activity than 200 mg/kg.

Comparative proteomic analysis of thiol proteins in the liver after oxidative stress induced by diethylnitrosamine

Conversion of protein –SH groups to disulfides is an early event during protein oxidation, which has prompted great interest in the study of thiol proteins. Chemical carcinogenesis is strongly associated with the formation of reactive oxygen species (ROS). The goal of this study was to detect thiol proteins that are sensitive to ROS generated during diethylnitrosamine (DEN) metabolism in the rat liver. DEN has been widely used to induce experimental hepatocellular carcinoma. We used modified redox-differential gel electrophoresis (redox-DIGE method) and mass spectrometry MALDI-TOF/TOF to identify differential oxidation protein profiles associated with carcinogen exposure. Our analysis revealed a time-dependent increase in the number of oxidized thiol proteins after carcinogen treatment; some of these proteins have antioxidant activity, including thioredoxin, peroxirredoxin 2, peroxiredoxin 6 and glutathione S-transferase alpha-3. According to functional classifications, the identified proteins in our study included chaperones, oxidoreductases, activity isomerases, hydrolases and other protein-binding partners. This study demonstrates that oxidative stress generated by DEN tends to increase gradually through DEN metabolism, causes time-dependent necrosis in the liver and has an oxidative effect on thiol proteins, thereby increasing the number of oxidized thiol proteins. Furthermore, these events occurred during the hepatocarcinogenesis initiation period.

Potential preventive effect of carvacrol against diethylnitrosamine-induced hepatocellular carcinoma in rats

Antioxidants are one of the key players in tumorigenesis, several natural and synthetic antioxidants were shown to have anticancer effects. The aim of the present study is to divulge the chemopreventive nature of carvacrol during diethylnitrosamine (DEN)-induced liver cancer in male wistar albino rats. Administration of DEN to rats resulted in increased relative liver weight and serum marker enzymes aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and gamma glutamyl transpeptidase (γGT). 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 (SOD), catalase (CAT), reduced glutathione (GSH), glutathione peroxidase (GPx), and glutathione reductase (GR). Carvacrol supplementation (15 mg/kg body weight) significantly attenuated these alterations, thereby showing potent anticancer effect in liver cancer. Histological observations and transmission electron microscopy studies were also carried out, which added supports to the chemopreventive action of the carvacrol against DEN-induction during liver cancer progression. These findings suggest that carvacrol prevents lipid peroxidation, hepatic cell damage, and protects the antioxidant system in DEN-induced hepatocellular carcinogenesis.

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

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) was 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 β-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 γGT 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 β-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.

Morin regulates the expression of NF-κB-p65, COX-2 and matrix metalloproteinases in diethylnitrosamine induced rat hepatocellular carcinoma

Morin—a bioflavonoid is a naturally available dietary agent believed to impede cancer promotion and progression. The present study was conducted to decipher, in vivo, the role of morin on the expression of matrix metalloproteinases, cyclooxygenase (COX)-2 and nuclear factor kappa B (NF-κB)-p65 during diethylnitrosamine (DEN)-induced hepatocarcinogenesis in Wistar albino rats. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that administration of DEN (200 mg/kg bodyweight in drinking water) to experimental animals caused inflammation of the liver due to up-regulation of NF-κB-p65 and COX-2. RT-PCR and immunoblot analysis also revealed that the oral supplementation of morin (500 ppm in diet) to DEN-induced hepatocellular carcinoma rats down-regulated the expression of COX-2 and NF-κB-p65, thereby preventing inflammation and angiogenesis mediated hepatocellular carcinogenesis. Further, immunohistological analysis for NF-κB-p65 nuclear localization confirms the above observations. Gelatin zymography was performed for matrix metalloproteinase MMP-2 and MMP-9 expression to confirm their role in angiogenesis in DEN induced hepatocellular carcinoma and its modulation by morin. Both MMP-2 and MMP-9 levels were found to be increased in DEN-induced animals when compared to control. MMP-2 and MMP-9 levels were down-regulated in morin post-treated animals when compared to DEN-induced animals favouring prevention of angiogenesis. In conclusion, our findings indicate that morin possessed anti-inflammatory and anti-cancer properties favouring suppression of DEN-induced hepatocellular carcinoma.

Antiproliferative potential of gallic acid against diethylnitrosamine-induced rat hepatocellular carcinoma

One of the focuses in current cancer chemoprevention studies is the search for nontoxic chemopreventive agents that inhibit the initiation of malignant transformation. Cancer biomarkers are quantifiable molecules involved in the physiologic or pathologic events occurring between exposure to carcinogens and the development, progression of cancer. Biomarkers may be the consequence of a continuous process, such as increased cell mass, or a discrete event, such as genetic mutation. Analysis of tumor markers can be used as an indicator of tumor response to therapy. Gallic acid is a naturally available polyphenol, possess strong antioxidant activity with a capacity to inhibit the formation of tumors in several cancer models. In the present study, we investigated the antiproliferative effect of gallic acid during diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) in male wistar albino rats. DEN treatment resulted in increased levels of aspartate transaminase, alanine transaminase, alkaline phosphatase, acid phosphatase, lactate dehydrogenase, gamma-glutamyltransferase, 5'-nucleotidase, bilirubin, alpha-fetoprotein, carcinoembryonic antigen, argyophillic nucleolar organizing regions, and proliferating cell nuclear antigen. Gallic acid treatment significantly attenuated these alterations and decreased the levels of AgNORs and PCNA. These finding suggests that gallic acid is a potent antiproliferative agent against DEN-induced HCC.

Suppression of diethylnitrosamine and 2-acetylaminofluorene-induced hepatocarcinogenesis in rats by tocotrienol-rich fraction isolated from rice bran oil

The anticancer efficacy of tocotrienol-rich fraction (TRF) was evaluated during diethylnitrosamine (DEN)/2-acetylaminofluorene (AAF)-induced hepatocarcinogenesis in male Sprague-Dawley rats. TRF treatment was carried out for 6 months, and was started 2 weeks before initiation phase of hepatocarcinogenesis. Morphological examination of the livers from DEN/AAF rats showed numerous off-white patches and few small nodules, which were significantly reduced by TRF treatment. Cytotoxic damage by DEN/AAF was estimated by alkaline phosphatase (ALP) release into the plasma from the cell membranes. DEN/AAF caused a twofold increase in the activity of ALP in plasma as compared with normal control rats, and this increase was prevented significantly by TRF treatment. We observed an increase of 79% in liver ALP activity in DEN/AAF rats, which was further increased by another 48% after the administration of TRF. Hepatic activity of glutathione S-transferase (GST) was also increased (3.5-fold) during the induction of hepatic carcinogenesis. Lipid peroxidation and low-density lipoprotein (LDL) oxidation increased threefold following initiation by DEN/AAF as compared with normal control rats. However, TRF treatment to DEN/AAF-treated rats substantially decreased (62-66%) the above parameters and thus limited the action of DEN/AAF. We conclude that long-term intake of TRF could reduce cancer risk by preventing hepatic lipid peroxidation and protein oxidation damage due to its antioxidant actions.

Correlation of changes in natural killer cell activity and glutathione S-transferase placental form positive hepatocytes in diethylnitrosamine-induced rat hepatocarcinogenesis

To evaluate the induction of preneoplastic hepatic foci in relation to natural killer cell (NK) activity, we sequentially analyzed glutathione S-transferase placental form positive (GST-P+) hepatocytes and NK activity during diethylnitrosamine (DEN) and phenobarbital (PB)-induced hepatocarcinogenesis in Sprague-Dawley rats. Previous studies have shown that NK activity can modulate the carcinogenic process induced by chemical carcinogens. Newborn females were initially given a single intraperitoneal injection of 15 mg DEN/kg and three weeks later, they were treated with 500 ppm phenobarbital (PB). From week 3, PB was administered in drinking water for 9 weeks. Interim and terminal sacrifices were performed at weeks 12, 15 and 30. GST-P+ hepatocytes increased with age in DEN-treated rats, especially in the population of more than two GST-P+ hepatocytes. The NK activity of DEN-treated rats did not significantly differ from that of control rats until week 12, but it progressively decreased from week 15 to 30. These results indicate that changes of NK activity inversely correlated with the induction of preneoplastic hepatic foci. This strong correlation of decreased NK activity with enhanced induction of GST-P+ foci suggests that NK activity is important in the early progression of hepatocarcinogenesis in rats.

Glutathione metabolism in hepatomous liver of rats treated with diethylnitrosamine

Glutathione metabolism was studied in rat liver during diethylnitrosamine (DEN) carcinogenesis. Some studies were also made in foetal rat liver. Endogenous GSH and non-protein thiols concentrations are increased in DEN-treated rats when compared to non-treated rats but no differences were found in cysteine, total thiols and protein thiols concentration. In foetal liver GSH concentration is only 35% of that in DEN-treated rat liver. The activities of several enzymes involved in glutathione metabolism are changed in DEN-treated rats. γ-Glutamyl transferase activity and cysteine formation from GSH by liver homogenates is increased sevenfold. γ-Glutamylcysteine synthetase activity, initial rate of [ 35S]cysteine incorporation in γ-glutamylcysteine and initial rate of GSH formation from [ 35S]cysteine are increased two-fold. Cytosolic GSH S-transferase activity is increased twofold in DEN-treated rats and so GSH S-conjugates concentration is probably also increased. In foetal rat liver γ-glutamyl transferase activity is about the same but γ-glutamylcysteine synthetase activity is only 10% of that in DEN-treated rat liver. The increased GSH concentration in DEN-treated rat liver is probably due to the simultaneous increase in the activities of γ-glutamyl transferase and γ-glutamylcysteine synthetase. Blood plasma total glutathione is increased 1.4 times in DEN-treated rats, but no differences are found in GSH hepatic arteriovenous gradient. This associated with the increased γ-glutamyl transferase activity suggests that sinusoidal GSH efflux is increased in DEN-treated rats.

Study on the role of mast cells in the development of liver fibrosis during diethylnitrosamine (DEN) -induced hepatocarcinogenesis in rats.

The mast cells (MC) and their roles in the development of liver fibrosis during experimental hepatocarcinogenesis in rats have been studied immunohistochemically and electron microscopically. Pronounced MC hyperplasia occurred after beginning of the treatment of DEN. The vast majority of mast cells in fibrotic livers was present in thickened fibrous septa. Intracellular or pericytial collagen type IV and laminin staining were found in mast cells, collagen types I, III and fibronectin were negative. Mast cells mainly distributed in proximity to basement membrane and the electron microscopical observation revealed a close topographic relationship between mast cells and fibroblasts. The fibroblasts phagocytized the granules released by mast cells and thus were activated, showing enhanced formation of collagenous fibrils. The mast cells may be derived from blood circulation or replicated in situ. Those findings indicated that mast cells take part in the process of fibrosis at least by two ways: (1) It directly synthesizes the basement membrane components; (2) It stimulates the fibroblasts or other types of cells which have the potential functions in fibrogenesis by releasing granule mediators or cytokines acting upon the fibrosis process.

Ultrastructural and Metabolic Determinants of Resistance to Azo-dye and Susceptibility to Nitrosamine Carcinogenesis of the Guinea-pig

During diethylnitrosamine (DEN) administration, a distinctive difference was observed between rats and guinea-pigs in the sequence of ultrastructural changes in the hepatic endoplasmic reticulum (ER). In DEN-induced hepatic tumour cells in the guinea-pig there was extensive proliferation of the rough ER, while the smooth ER was quite sparse, in the premalignant liver the opposite was noted. This is in contrast to the rat, in which administration of either DEN or 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB) brings about, in both premalignant and malignant hepatic tissue, proliferation of the smooth ER and sparsity of the rough ER. Yet, as in the rat, the number of ribosomes on the outer surface of the guinea-pig liver rough ER is greatly reduced and this is paralleled by a 49% decrease of the RNA/protein ratio as early as 4 weeks of nitrosamine administration. The decrease of RNA/protein ratio and ultrastructurally observed loss of ribosomes from the ER, following nitrosamine administration, correlate with a decrease of photometric response of microsomal suspensions to the sulphydryl probe, p-chloromercuribenzoate. While azo-dye-reductase activity is higher in untreated rats than in untreated guinea-pigs, feeding 3′-Me-DAB for 6 weeks brings about a 76% decrease in the rat, but no significant decrease in the guinea-pig, which is refractory to azo-dye carcinogenesis. Thus, the ability of the liver to inactivate the dye is greatly decreased in the rat, but not in the guinea-pig, as administration progresses toward the threshold dose for tumorigenesis. On the other hand, constitutive levels of nitrosamine dealkylase are identical in the 2 species and remain essentially unchanged following administration of DEN for 10 weeks. Inasmuch as nitrosamine dealkylation represents activating metabolism, this provides a rationale for the comparable susceptibility of the rat and guinea-pig to DEN carcinogenesis. Of the 2 enzymes in the 2 species, it is only azo-dye reductase in the guinea-pig which appears to be unregulated by glucose repression, since starvation brings about no change in this activity. Starvation-induced increase of azo-dye reductase in the rat is not influenced by administration of 3′-Me-DAB and only slightly by DEN. The starvation-induced increase of nitrosamine dealkylation is abolished, however, in both species by administration of DEN but only slightly decreased by 3′-Me-DAB.