Eisaku Kobayashi

Improved genomic/nuclear DNA extraction for 8-hydroxydeoxyguanosine analysis of small amounts of rat liver tissue

Using two different commercially available extraction kits, genomic/nuclear DNA could be recovered from rat liver samples as small as 10 mg. Background 8-hydroxydeoxyguanosine levels in such DNA were low and stable at 0.26-0.30 +/- 0.01-0.03/10(5) guanine residues. The minimum tissue wet weight required for the accurate 8-hydroxydeoxyguanosine analysis was 25 mg. The present results indicate that routine and reliable assessment of the involvement of oxidative DNA damage in the development of various diseases, including cancer, is feasible using a variety of tissue sources.

K‐ras Gene Mutation in Early Ductal Lesions Induced in a Rapid Production Model for Pancreatic Carcinomas in Syrian Hamsters

The presence of K‐ras gene mutation was examined in experimentally induced preneoplastic pancreatic ductal lesions. Syrian hamsters received 70 mg/kg of N‐nitrosobis(2‐oxopropyl)amine (BOP) followed by repeated exposure to an augmentation pressure regimen consisting of choline‐deficient diet combined with dl‐ethionine and l‐methionine and administration of 20 mg/kg BOP. After two augmentation pressure cycles, pancreatic ductal cell hyperplasias appeared and after three cycles, atypical hyperplasias of pancreatic ductal cells and intraductal carcinomas developed. K‐ras mutations were detected by single‐strand conformation polymorphism analysis of polymerase chain reaction products and nucleotide sequencing. The results showed that K‐ras mutation had occurred in one of 9 simple hyperplasias of pancreatic ductal epithelium, in 5 of 9 atypical hyperplasias, and in 4 of 8 intraductal carcinomas. The findings thus suggested that K‐ras is activated in association with very early stage malignant transformation of pancreatic ductal cells in hamsters.

Comparison of K-ras oncogene activation in pancreatic duct carcinomas and cholangiocarcinomas induced in hamsters by N-nitrosobis(2-hydroxypropyl)amine

The presence of K‐ras point mutations in pancreatic duct carcinomas and Cholangiocarcinomas induced by N‐nitrosobis(2‐hydroxypropyl)amine (BHP) in Syrian hamsters was investigated by single‐strand conformation polymorphism analysis of polymerase chain reaction products from frozen fresh materials in order to clarify the K‐ras mutation rates in those two carcinomas induced simultaneously by one carcinogen, BHP. In the examined pancreatic duct carcinomas, 10 out of 16 were positive for a mutation in codon 12 while 3 out of 12 Cholangiocarcinomas demonstrated mutation of K‐ras gene. G‐to‐A transition was detected in the second position of codon 12 in both pancreatic carcinomas and Cholangiocarcinomas. These results suggest that the role of genetic alteration in carcinogenesis may differ with the target organ, even when initiation is with the same carcinogen.

Different Roles of 8‐Hydroxyguanine Formation and 2‐Thiobarbituric Acid‐reacting Substance Generation in the Early Phase of Liver Carcinogenesis Induced by a Choline‐deficient, l‐Amino Acid‐defined Diet in Rats

The present study was performed to assess the roles of hepatocellular oxidative damage to DNA and constituents other than DNA in rat liver carcinogenesis caused by a choline‐deficient, l‐amino acid‐defined (CDAA) diet by examining the effects of the antioxidant N, N′‐diphenyl‐p‐phenylenediamine (DPPD). The parameters used for cellular oxidative damage were the level of 8‐hydroxyguanine (8‐OHGua) for DNA and that of 2‐thiobarbituric acid‐reacting substance (TBARS) for constituents other than DNA. A total of 40 male Fischer 344 rats, 6 weeks old, were fed the CDAA diet for 12 weeks with or without DPPD (0.05, 0.10 or 0.20%) or butylated hydroxytoluene (BHT, 0.25%). In the livers of the rats, the numbers and sizes of glutathione S‐transferasc (EC 2.5.1.18) placental form (GSTP)‐ and/or γ‐glutamyltransferase (GGT, EC 2.3.2.2)‐positive lesions and levels of 8‐OHGua and TBARS were determined. The GSTP‐positive lesions of 0.08 mm2 or larger were all stained positively for GGT as well in cross‐sectional area, whereas the smaller lesions were generally negative for GGT. DPPD and BHT reduced the size of the GSTP‐positive lesions without affecting their total numbers. At the same time, they reduced TBARS generation without affecting 8‐OHGua formation in DNA. The present results indicate that oxidative DNA damage (represented by 8‐OHGua formation) and damage to constituents other than DNA (represented by TBARS generation) may play different roles in rat liver carcinogenesis caused by the CDAA diet; the former appears to be involved in the induction of phenotypically altered hepatocyte populations while the latter may be related to the growth of such populations.

Prevention by Methionine of Enhancement of Hepatocarcinogenesis by Coadministration of a Choline-deficient L-Amino Acid-defined Diet and Ethionine in Rats

The effects of methionine on hepatocarcinogenesis induced by Coadministration of a choline‐deflcient L‐amino acid‐defined (CDAA) diet and ethionine were examined. F344 male rats were divided into 4 experimental groups. Groups 1 and 2 received the CDAA diet and a choline‐supplemented L‐amino acid‐defined (CSAA) diet, respectively. Group 3 received the CDAA diet containing 0.05% ethionine, and group 4 the CDAA diet containing 0.05% ethionine and 0.47% methionine. Animals were killed after 12 weeks of treatment. Histologically, the CDAA diet induced intracellular fat accumulation and foci. In contrast, ethionine caused not only foci, but also hyperplastic nodules, cholangiofibrosis and the proliferation of oval cells without such fat accumulation. Methionine abolished the development of all of the liver lesions induced by Coadministration of the CDAA diet and ethionine. To investigate the effects of methionine on induction of c‐myc and c‐Ha‐ras expression, as well as generation of 8‐hydroxyguanine (8‐OHGua) and 2‐thiobarbituric acid‐reacting substances (TBARS), by Coadministration of the CDAA diet and ethionine, subgroups of 3 to 5 animals were killed at 2, 4, 8 or 11 days after the beginning of the experiment. Coadministration of the CDAA diet and ethionine markedly enhanced the level of expression of c‐myc and c‐Ha‐ras, 8‐OHGua formation and TBARS generation as compared with the CDAA or CSAA diet within 11 days, and methionine blocked these actions. These results indicate that addition of methionine prevents the induction of c‐myc and c‐Ha‐ras expression, 8‐OHGua formation and TBARS generation, as well as hepatocellular lesions, by Coadministration of the CDAA diet and ethionine in rats, and suggest a possible involvement of oxidative stress and gene expression in hepatocarcinogenesis by these agents.

Comparative Changes in the Liver of Female Fischer-344 Rats after Short-Term Feeding of a Semipurified or a Semisynthetic L-Amino Acid-Defined Choline-Deficient Diet

Groups of female Fischer-344 rats were fed a semipurified choline-deficient (CD) diet, or a semisynthetic L-amino acid-defined choline-deficient (CDAA) diet, for up to 12 wk and effects of the 2 diets on the liver were compared. Steatosis was diffuse and more severe throughout in rats fed the CDAA diet than in rats fed the CD diet. Greater elevations in serum aspartate and alanine aminotransferase activities were also present in the former rats, along with higher 2-bromodeoxyuridine labeling indices in the liver. Discrete amounts of 8-hydroxyguanine were detected in liver DNA, but were not significantly different in rats fed the 2 diets, or from those present in a group of control rats killed at 0 time. Glutathione S- transferase placental form-positive focal lesions were not observed in any of the rats. The results show that the CDAA diet causes more severe degrees of steatosis and liver cell death and proliferation than the CD diet, raising the possibility that it may, in contrast to the CD diet, result in the eventual induction of hepatocellular carcinomas in female Fischer-344 rats.

Disturbance of the Cell Cycle with Colchicine Enhances the Growth Advantage of Diethylnitrosamine‐initiated Hepatocytes in Rats

The effect of cell cycle disturbance due to colchicine on the induction of enzyme‐altered foci during liver regeneration in rats was studied. For initiation, diethylnitrosamine (DEN) at a dose of 10 mg/ kg was injected intraperitoneally and partial hepatectomy (PH) was performed 4 h thereafter. Colchicine at doses of 0, 0.1, 0.25 and 0.5 mg/kg was injected intraperitoneally 1 and 3 days after the initiation, followed by application of selection pressure consisting of 2‐acetylaminofluorene (AAF) and carbon tetrachloride (CCl4) administration. As end point lesions, γ–glutamyltransferase (GGT)‐positive enzyme‐altered foci were assayed at week 5. There was no significant effect of colchicine on numbers of foci. However, a significant, dose‐dependent increase in the area of GGT‐positive lesions in the groups treated with colchicine was observed. Bromodeoxyuridine labeling indices were higher in foci induced in colchicine‐treated rats than in the untreated rats. In a separate experiment, serum glutamic pyruvic transaminase was not increased significantly after DEN and colchicine treatment, and the mitotic index at 6 days after PH was increased in the liver of colchicine‐treated rats. These results suggest that the cell cycle disturbance induced by colchicine causes more pronounced selective growth of cells initiated by DEN and colchicine, and this experimental model may be useful for analyzing the mechanisms underlying that growth advantage and the effects of cell cycle abnormalities in liver carcinogenesis.