The expression of major human apurinic/apyrimidinic DNA endonuclease (APEX) from its cDNA in E. coli (DH5 alpha) was attempted in order to obtain a biologically active recombinant APEX. E. coli cells were transformed by a prokaryotic translation vector (pGEX-4T-3) harboring APEX cDNA. GST-APEX fusion protein with a molecular weight of 6.3 KDa was induced by IPTG (1.0 mM) treatment. Western blot immunodetection identified the induced protein as the GST-APEX fusion protein. The survival rate of E. coli cells (DH5 alpha) transformed with pGEX-4T-3-APEX increased when the cells were treated with N-diethyl-N-nitrosamine (DENA) or 3'-methyl-4-monomethylaminoazobenzene (3'-MeMAB), indicating that APEX expression had a protective effect on the cytotoxicity of these carcinogens. The fusion protein extracted from E. coli cells and purified by GSH-agarose gel affinity chromatography exhibited APEX activity. Treatment of thrombin to the GST-APEX fusion protein and affinity purification followed by Sephacryl S-100 gel filtration resulted in APEX peptide with MW 36 KDa, which exhibited AP DNA repair activity (8,7000 EU/mg protein). N-ethylmaleimide (0.1 mM) or AMP (0.98 mM) inhibited APEX activity by 50% and kinetic analysis indicated that the recombinant APEX (rAPEX) had a Km value of 0.022 microM (AP sites for AP DNA) and the Ki value was 0.48 mM for AMP. These results indicated that E. coli cells expressing biologically active GST-APEX were resistant to the cell damage caused by chemical carcinogens and that rAPEX purified from E. coli cells transformed with APEX cDNA-inserted translation vector was similar to native APEX in some properties.
Carbon-Oxygen Lyases/biosynthesis* ; Diethylnitrosamine/pharmacology ; Escherichia coli/genetics ; Escherichia coli/drug effects ; Human ; Recombinant Fusion Proteins/biosynthesis*

A wide-spectrum initiation model was investigated in mice. Sequential treatments with diethylnitrosamine, urethane and N-methylnitrosourea, with or without a promoter, phenobarbital, resulted in tumor formation in the lungs in 85-90% of animals, but did not produce any tumorous lesions in other organs. The lung tumors were adenomas and the mean number of adenomas was 2.2-2.6 per mouse. Phenobarbital combination had no additive effect on lung tumor incidence and multiplicity. Splenic NK cell activity showed inconsistent increment in the carcinogen plus phenobarbital-treated group during the experiment (P less than 0.05).
*Adenoma/chemically induced/immunology ; Animals ; Diethylnitrosamine/pharmacology ; Female ; *Killer Cells, Natural/drug effects ; *Lung Neoplasms/chemically induced/immunology ; Methylnitrosourea/pharmacology ; Mice ; Phenobarbital/pharmacology ; Random Allocation ; Urethane/pharmacology

Iron is essential for the growth of all living cells. One of the most important intracellular roles of iron is the activation of ribonucleotide reductase, which is indispensible to the production of deoxyribonucleotide necessary for DNA synthesis. Deferoxamine (DFO) is an iron chelating agent and has been known to have an antiproliferative effect in various malignant cells including hepatocellular carcinoma and the effect seems to be related to depletion of iron. This study was undertaken to investigate the effect of DFO on preneoplastic lesions in chemically induced hepatocarcinogenesis. The resistant hepatocyte model was used and Sprague Dawley rats were divided into the following groups; I: normal control, II: carcinogen administered group, III: carcinogen and DFO administered group. Rats were sacrificed at 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks and 8 weeks after partial hepatectomy (PH). DFO (50 mg/kg/day, I.P.) was daily injected from 3 weeks before administration of carcinogen to the time when rats were sacrificed. Hepatic iron content was higher in group II than in group III, especially at 3 days and 1 week after PH. Hyperplastic lesions of resistant hepatocytes were less well developed in group III than in group II. Bromodeoxyuridine labelling indices of oval cells and hyperplastic lesions of resistant hepatocytes were higher in group II than in group III except for rats examined at 3 days after PH. The results suggest that DFO has an antiproliferative effect on preneoplastic lesions in hepatocarcinogenesis and it might be related to reduction of the hepatic iron.
Animal ; Deferoxamine/*pharmacology ; Diethylnitrosamine ; Liver Neoplasms, Experimental/chemically induced/*prevention & control ; Male ; Precancerous Conditions/chemically induced/*prevention & control ; Rats ; Rats, Sprague-Dawley ; Support, Non-U.S. Gov't

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.
Animal ; Body Weight ; Carcinogens/pharmacology* ; Diethylnitrosamine/pharmacology* ; Female ; Glutathione Transferase/metabolism* ; Killer Cells, Natural/immunology* ; Liver/enzymology* ; Liver/cytology ; Liver Neoplasms/physiopathology* ; Organ Weight ; Placenta ; Rats ; Rats, Sprague-Dawley

The effects of ethyl alcohol and pig serum administration on the development of preneoplastic hepatic enzyme-altered foci were examined in an in vivo mid-term assay system. Rats were initially given a single dose (200 mg/Kg) intraperitoneal injection of diethylnitrosamine (DEN). Two weeks later, treatment was started with 10% ethanol + 10% sucrose solution, 10% sucrose solution, or tap water as drinking water for 6 weeks with or without intraperitoneal injection of porcine serum twice a week. All rats were subjected to a two-thirds partial hepatectomy at week 3. The modification potentials were evaluated by comparing the number and area per cm2 of glutathione S-transferase placental form-positive (GST-P+) foci in the liver of each group. As a result, ethanol significantly enhanced the development of GST-P+ foci. Unfortunately, the porcine serum injection produced no hepatic fibrosis and no significant alteration in GST-P+ foci.
Animals ; Diethylnitrosamine/*toxicity ; Ethanol/*pharmacology ; Glutathione Transferase/*metabolism ; Immune Sera/pharmacology ; Liver Cirrhosis, Alcoholic/enzymology ; Male ; Placenta/drug effects/*enzymology ; Precancerous Conditions/*chemically induced/enzymology ; Rats ; Rats, Inbred F344 ; Survival Rate ; Swine

Anticarcinogenic effect of red ginseng (Panax ginseng C.A. Meyer cultivated in JiLin, China) on the development of liver cancer induced by diethylnitrosamine (DEN) in rats was studied, especially in preventive and curative groups. In the preventive group, the rats were given with DEN concomitantly with red ginseng fluid, and in the curative group, the rats were administered with red ginseng fluid after they developed liver cancer nodules induced by DEN. The result of the preventive group revealed that the developmental rate of liver cancer in the experimental group was 14.3%, while 100% in the control group, with the difference being statistically significant. DNA, RNA, glycogen, gamma-GT, SDH, and 5'-NT were maintained at relatively normal level in experimental group, and decreased or increased in the control group. The result of curative group showed that hepatoma nodules of the DEN-red ginseng group I were smaller than those of control group I, the structure of hepatic tissue was well preserved, the area with gamma-GT positive was smaller, and the ultrastructure of hepatocytes was normal. The average life span the DEN-red ginseng group II and the DEN control group II were 72.8 and 42.3 days, respectively. To sum up, all findings on preventive and curative groups had clearly proved that the red ginseng had the anticarcinogenic effect on the development of liver cancer induced by DEN in rats.
Animal ; Anticarcinogenic Agents/*pharmacology ; Carcinoma, Hepatocellular/chemically induced/*pathology ; Data Interpretation, Statistical ; Diethylnitrosamine/adverse effects ; Liver/pathology/ultrastructure ; Liver Neoplasms, Experimental/chemically induced/*pathology ; Male ; *Panax ; Plant Extracts/pharmacology ; Rats ; Rats, Wistar

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 modifying potential of capsaicin (CAP) on lesion development was examined in a rat multiorgan carcinogenesis model. Groups 1 and 2 were treated sequentially with diethylnitrosamine (DEN) (100 mg/kg, ip, single dose at commencement), N-methylnitrosourea (MNU) (20 mg/kg, ip, 4 doses at days 2, 5, 8, and 11), and N,N-dibutylnitrosamine (DBN) (0.05% in drinking water during weeks 3 and 4). Group 3 received vehicles without carcinogens during the initiation period. Group 4 served as the untreated control. After this initiating procedure, Groups 2 and 3 were administered a diet containing 0.01% CAP. All surviving animals were killed 20 weeks after the beginning of the experiment and the target organs examined histopathologically. The induction of GST-P+ hepatic foci in rats treated with carcinogens was significantly inhibited by treatment with CAP. CAP treatment significantly decreased the incidence of adenoma of the lung but increased the incidence of papillary or nodular (PN) hyperplasia of the urinary bladder. The tumor incidence of other organs, such as the kidney and thyroid, was not significantly different from the corresponding controls. These results demonstrated that concurrent treatment with CAP not only can inhibit carcinogenesis but can also enhance it depending on the organ. Thus, this wide-spectrum initiation model could be used to confirm organ-specific modification potential and, in addition, demonstrate different modifying effects of CAP on liver, lung, and bladder carcinogenesis.
Animals ; Capsaicin/pharmacology/*toxicity ; Cocarcinogenesis ; Diethylnitrosamine ; Liver Neoplasms, Experimental/chemically induced/prevention & control ; Lung Neoplasms/chemically induced/prevention & control ; Male ; Methylnitrosourea ; Neoplasms, Experimental/*chemically induced/prevention & control ; Nitrosamines ; Rats ; Rats, Inbred F344 ; Urinary Bladder Neoplasms/chemically induced

The effects of A-L tonic capsule on DNA content in rat experimental hepatocarcinogenesis induced by diethylnitrosamine (DENA) were observed. The experimental rats were divided into 4 groups. With exception of group D in which the rats were only administered with DENA, the rats in the groups A, B, C were previously, simultaneously and subsequently fed with A-L tonic capsule respectively while they were administered with DENA. The DNA content of all rat livers was measured using automatic image analysis technique 20 weeks after administration of DENA. The results showed that the highest and lowest DNA contents were respectively seen in the groups D and A. There was significant difference in DNA contents between the groups A or B or C and D, and also between the groups A and B or C (both P < 0.01). 4 components (4C) and > or = 5C cells were predominant in the group D, while 2C cells were the minority. The number of 2C cells in the groups A, B, C was significantly higher than that in the group D, and the number of > or = 5C cells in the groups A, B, C was markedly lower than that in the group D (P < 0.01). Also, there was very significant difference in the number of 2C and > or = 5C cells between group A and B or C (P < 0.01). It was concluded that A-L tonic capsule could effectively inhibit the increase of DNA content of hepatocytes and improve the distribution of DNA content in rat hepatocarcinogenesis, especially in group A.
Animals ; Astragalus membranaceus ; chemistry ; Capsules ; Carcinogenicity Tests ; DNA, Neoplasm ; analysis ; Diethylnitrosamine ; Drugs, Chinese Herbal ; pharmacology ; Hepatocytes ; chemistry ; Ligustrum ; chemistry ; Liver Neoplasms, Experimental ; chemically induced ; chemistry ; genetics ; Male ; Rats ; Rats, Sprague-Dawley

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.
Actins ; metabolism ; Alanine Transaminase ; blood ; Animals ; Antineoplastic Agents ; pharmacology ; Aspartate Aminotransferases ; blood ; Bile ; chemistry ; Bilirubin ; blood ; Body Weight ; drug effects ; Carcinoma, Hepatocellular ; blood ; chemically induced ; pathology ; Diethylnitrosamine ; Liver ; metabolism ; pathology ; Liver Cirrhosis ; chemically induced ; pathology ; Liver Neoplasms, Experimental ; blood ; chemically induced ; pathology ; Male ; Powders ; pharmacology ; Proliferating Cell Nuclear Antigen ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Ursidae