Kohsuke Horiguchi

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.

Inhibition by Green Tea Extract of Diethylnitrosamine–initiated but Not Cholinedeficient, L–Amino Acid–defined Diet–associated Development of Putative Preneo–plastic, Glutathione S–Transferase Placental Form–positive Lesions in Rat Liver

The effects of green tea extract (GTE) on exogenous and endogenous models of rat liver carcinogenesis using diethylnitrosamine (DEN) and a choline–deficient, L–amino acid–defined (CDAA) diet were studied. For the exogenous carcinogenesis study, male Fischer 344 rats, 6 weeks old, were given a single intraperitoneal dose of 200 mg/Kg body weight of DEN, partially hepatectomized at week 3, and administered GTE at doses of 0, 0.01 and 0.1% in the drinking water from week 2 for 10 weeks. For the endogenous carcinogenesis study, rate were fed the CDAA diet and simultaneously given GTE for 12 weeks. All rats were killed at the end of week 12. After DEN–initiation, the apparent numbers of glutathione S–transferase placental form–positive foci, assayed as putative preneoplastic lesions, were decreased by the administration of GTE, though their sizes were not altered. In contrast, GTE did not significantly reduce the numbers of the lesions induced by the CDAA diet or affect their sizes. While the levels of 8–hydroxyguanine, a parameter of oxidative DNA damage, were reduced by the GTE administration in both experimental models, GTE did not protect against the CDAA–diet–associated liver tissue damage in terms of either histology or plasma marker enzyme levels. We conclude that, while GTE may be a possible chemopreventive agent for nitrosamine–initiated hepato–carcinogenesis in the absence of chronic hepatocyte damage, it does not significantly inhibit lesion development in hepatocarcinogenesis associated with the CDAA diet, a cirrhosis–associated model.

Characterization of three cloned cell lines from aN-nitrosobis(2-hydroxypropyl)amine-induced transplantable hamster pancreatic ductal adenocarcinoma

SummaryTo investigate characteristics of pancreatic carcinoma growth behavior, the cloned cell lines, HPD1NR, HPD2NR, and HPD3NR, were established from a transplantable hamster ductal adenocarcinoma induced byN-nitrosobis(2-hydroxypropyl)amine (BHP). All three clones showed similar epithelial cell morphology and grew as sheets in culture with no differences in doubling times, ranging from 23-28 h. Mutation in the c-Ki-ras exon 1 was detected in common. The modal chromosome numbers were also found to be similar at 60, 62, and 60-62 in the less than tetraploid cells in the three clones. In contrast, a clear difference in frequencies of tetraploid or polyploid cells at 24.7, 22.5, and 75.5% in HPD1NR, HPD2NR, and HPD3NR, respectively, was evident. Tumorigenic potency evaluated by transplanting individual clones revealed HPD3NR to display pronouncedly less growth in syngeneic hamsters. The results suggest that increase in frequency of tetraploid or polyploid cells might be associated with a decreased in vivo growth potential of hamster pancreatic ductal adenocarcinomas, and suggest that these clones might become a valuable tool for understanding in vivo growth mechanisms of cancer cells.

Selective 8-hydroxyguanine formation in pancreatic DNA due to a single intravenous administration of 4-hydroxyaminoquinoline 1-oxide in rats

8-Hydroxyguanine (8-OHG) formation, a possible initiating event, was determined in pancreatic and liver DNA and compared with the genesis of acinar cell and hepatocyte necrosis in male Wistar rats given a single intravenous administration of 4-hydroxyaminoquinoline 1-oxide (4-HAQO). At the non-necrotic but tumorigenic dose of 7.0 mg/kg body weight, 8-OHG was selectively generated in pancreatic DNA, in the absence of acinar cell necrosis, at the 6 and 24 h time points and repaired by the 48 h time point. When rats were exposed to 4-HAQO at a necrotic dose of 14.0 mg/kg body weight, 8-OHG was also selectively formed in pancreatic DNA with the same time-dependence of generation and repair, while acinar cell necrosis became evident at the 24 h time point and progressed thereafter. Whereas no hepatocyte necrosis was detected in any rats, 8-OHG values for liver DNA merely expressed slight increases only at the 24 and 48 h time points in rats given 14.0 mg/kg body weight of 4-HAQO. The present data suggest that formation of oxidative DNA damage, assayed by 8-OHG, in pancreatic DNA is independent from toxicity and may be involved, along with quinoline adducts, in mutational events underlying 4-HAQO-induced rat acinar cell carcinogenesis.

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.

Preventive Effects of Various Antioxidants on Endogenous Liver Carcinogenesis in Rats Fed a Choline-Deficient, L-Amino Acid-Defined Diet

A choline-deficient, L-amino acid-defined (CDAA) diet is a unique, semisynthetic diet containing no choline and a limited amount of methionine in the absence of any known carcinogens. Chronic feeding of the CDAA diet to male Fischer 344 rats for 1 year causes a high incidence of hepatocellular carcinomas through the development of putative preneoplastic, enzyme-altered, focal hepatocellular lesions. During this process fat accumulation, hepatocyte necrosis followed by regenerative hepatocyte proliferation, oval cell proliferation, and fibrosis reaching a stage of frank cirrhosis occur sequentially in the liver. Although details of the liver carcinogenic mechanisms have not been fully elucidated, oxidative damage to DNA and extra-DNA components in hepatocytes has been believed to play critical roles independently or together. We introduce chronic feeding of the CDAA diet as a unique and useful model for investigating the mechanisms of endogenous carcinogenesis, which is now appreciated as important. We also present results on the preventive effects of various antioxidants on this endogenous rat liver carcinogenesis.