Ryohei Hasegawa

HISTOPATHOLOGICAL ANALYSIS OF PRENEOPLASTIC CHANGES DURING N-BUTYL-N-(4-HYDROXYBUTYL)- NITROSAMINE-INDUCED URINARY BLADDER CARCINOGENESIS IN RATS

Sequential microscopic alterations of the urinary bladder epithelium during carcinogenesis were examined in rats after oral administration of 0.01% or 0.05% N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN). Simple hyperplasia appeared after 4 weeks of BBN administration. This regressed by 12 weeks after BBN discontinuation but reappeared focally in some areas after 20 weeks and persisted to the termination of the experiment. Preneoplastic papillary or nodular hyperplasia appeared earlier and more frequently in rats treated with 0.05% BBN than in those treated with 0.01%, but these lesions regressed gradually during a prolonged observation period after BBN was discontinued. These results suggest that 2 types of papillary or nodular hyperplasias exist, one reversible and the other irreversible. Tumors appeared earlier in rats treated with 0.05% BBN than in those with 0.01% BBN.

Gastric and intestinal phenotypic expression of human stomach cancers as revealed by pepsinogen immunohistochemistry and mucin histochemistry

Gastric and intestinal phenotypic expression in 223 surgically obtained primary gastric cancers and their histogenetic relationship to intestinal metaplasia in the surrounding gastric mucosa were studied by mucin histochemistry and pepsinogen (Pg) immunohistochemistry. Histochemical differentiation of mucins (paradoxical concanavalin A, the galactose oxidase‐Schiff sequence and sialidase galactose oxidase Schiff) and immunohisto chemical staining of Pgs I and II, allowed differentiation of gastric cancer cells from different histological categories into gastric elements including mucous neck cells, pyloric gland cells and surface mucous cells or intestinal elements including goblet cell and intestinal absorptive cell types. Of 122 papillary and tubular adenocarcinomas, 33 (27.1%) consisted mainly of gastric type cells and 42 (34.4%) predominantly of intestinal type cells. The remainder (38.5%) consisted of mixtures of gastric‐ and intestinal‐type cells. Of 101 poorly differentiated adenocarcinomas, signet ring cell carcinomas and mucinous adenocarcinomas, 59 (58.4%) consisted mainly of gastric‐type cells and 20 (19.8%) mainly of intestinal‐type cells. Seven out of 35 papillary and tubular adenocarcinomas consisting mainly of gastric type cancer cells were surrounded by mucosa with intestinal metaplasia. Conversely, 10 out of 40 papillary and tubular adenocarcinomas consisting mainly of intestinal‐type cancer cells were observed in non metaplastic gastric mucosa. Thus no relationship as regards intestinal phenotypic expression was found between gastric cancers and surrounding gastric mucosa.

RAPID BIOASSAY METHODS FOR CARCINOGENS AND MODIFIERS OF HEPATOCARCINOGENESIS

It is very important to detect environmental carcinogens in a short period. For this purpose, a rapid bioassay system based on two-step hepatocarcinogenesis has been developed in our laboratory. Rats were initially given a single dose (200 mg/kg) of diethylnitrosamine (DEN) i.p. and, starting 2 weeks later, were treated with test compounds for 6 weeks and then sacrificed, all rats being subjected to a two thirds partial hepatectomy at week 3. Carcinogenic potential was scored by comparing the glutathione S-transferase placental form-positive foci in the liver with those of the corresponding control. More than 90% of hepatocarcinogens showed positivity, and none of the compounds reported as noncarcinogenic demonstrated positivity. Furthermore, this system also detected inhibitory effects. In order to detect nonhepatocarcinogens, other appropriate systems also have been developed, for example, using methylnitrosourea or other multispectrum carcinogens. These rapid bioassay systems are particularly useful for the screening of environmental carcinogens.

Medium-term bioassay system for detection of carcinogens and modifiers of hepatocarcinogenesis utilizing the GST-P positive liver cell focus as an endpoint marker.

We have developed a medium-term bioassay system of 8 weeks duration utilizing male Fischer 344 (F344) rats for detection of liver carcinogens and modifiers of hepatocarcinogenesis. The system consists of a single intraperitoneal injection of diethylnitrosamine (DEN, 200 mg/kg), 6-weeks-administration of test chemical beginning 2 weeks after the DEN injection, and 2/3 partial hepatectomy (PH) performed at week 3. Carcinogenic potency of test chemicals is predicted based on the results of quantitative analyses of immunohistochemically-demonstrated glutathione S-transferase placental form (GST-P) positive liver cell foci. At present, a total of 140 chemicals have been tested using this system, and the findings show a good correlation with reported carcinogenic activities in long-term tests. Furthermore, the reliability of the system has been extensively examined: the results from the medium-term bioassay were compared with those from long-term experiments using the same doses of selected chemicals; the data from presently-used 2-dimensional analysis were compared with calculated values utilizing mathematical formulae for three-dimensional analysis: conformity of phenotypic expression of enzymes in preneoplastic lesions was examined in relation to their growth activity. In conclusion, although the results with non-hepatocarcinogens were less than satisfactory, the present experimental protocol, which requires far fewer animals and shorter duration than a long-term carcinogenicity test, appears of advantage for rapid screening of the large number of environmental chemicals which may possess hazard potential for induction of liver cancer in man.

Liver medium-term bioassay in rats for screening of carcinogens and modifying factors in hepatocarcinogenesis

The present report describes a study of the hepatocarcinogenic potential of a second large assay series of 94 compounds carried out using the rapid bioassay system (DEN-PH model) developed in this laboratory and based on the two-step concept of hepatocarcinogenesis. Male F344 rats were initially given a single dose of diethylnitrosamine (DEN, 200 mg/kg body weight ip) and, starting 2 wk later, were treated with test compounds for 6 wk and then killed, all rats being subjected to a two-thirds partial hepatectomy at wk 3. Carcinogenic potential was scored by comparing the numbers (no./cm2) and areas (mm2/cm2) of induced glutathione S-transferase placental form (GST-P) positive foci in the livers of groups of about 15 rats with those of corresponding control groups given DEN alone. Positive was scored for a significant increase (P < 0.05) in quantitative values of GST-P positive foci, negative for no change or a decrease. Results for the 94 compounds were also compared with previously published data from Salmonella/microsome (Ames) tests and long-term carcinogenicity studies in rats and mice. Of the known liver carcinogens, 14 out of 14 (100%) mutagenic (Ames test) compounds and 10 out of 12 (83%) non-mutagenic compounds gave positive results in our DEN-PH system (mean 92%). Two hepatocarcinogenic peroxisome proliferators did not enhance the development of GST-P positive foci. Carcinogens other than hepatocarcinogens gave fewer positive results (five out of 17, 29%). One of the 13 compounds reported as non-carcinogenic, malathion, gave positive results in the DEN-PH assay, suggesting that this compound is a weak hepatocarcinogen or tumour promoter for hepatocarcinogenesis based on the two-stage hypothesis for carcinogenesis. The present study also provided information regarding the inhibitory potential of nine compounds. The practical usefulness and benefits of the DEN-PH protocol for the rapid screening of carcinogenic agents are discussed.

Medium-term liver and multi-organ carcinogenesis bioassays for carcinogens and chemopreventive agents

To bridge the gap between long-term carcinogenicity tests and short-term screening assays such as the Ames test, several types of medium-term bioassay for rapid detection of carcinogenic agents have been developed using male F344 rats. The liver model, in which diethylnitrosamine initiation and acceleration of carcinogenesis by partial hepatectomy are essential components, requires only 8 weeks of animal experimentation and a few weeks for quantitative analysis of hepatic preneoplastic lesions. Using the model, a total of 250 chemicals have been analyzed and the efficacy of the system for hapatocarcinogens has thereby been well established. Other models are so-called multi-organ bioassays for detection of carcinogenic agents in multiple organs within relatively short periods. Among these, the DMBDD bioassay with 5 known carcinogens as initiators has been found to be most applicable and has now been introduced for practical use. Data from these bioassays and several single organ carcinogenesis systems have demonstrated that carcinogenic and modifying effects of individual exogenous agents may markedly differ from organ to organ. Therefore, research into chemoprevention should be based on a whole body level analysis. The present medium-term systems are very useful for this purpose.

Carcinogenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the rat.

A total of 10 highly-mutagenic heterocyclic amines have been identified to be carcinogenic in rodents. Among these, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), generally the most abundant with normal cooking procedures, induces mammary and colon carcinomas in rats in a clear dose-dependent manner. In a two-generation exposure (transplacental and trans-breast milk) experiment using Sprague-Dawley rats, an increased risk of mammary adenocarcinoma development was found in the second generation. Excretion of PhIP into the milk and transfer of PhIP to fetuses and neonates with resultant hepatic PhIP-DNA adduct formation were also confirmed. On the other hand, PhIP mammary carcinogenesis was significantly inhibited by coadministration of chlorophyllin or a synthetic antioxidant, 1-O-hexyl-2,3,5-trimethylhydroquinone, in long-term experiments using female F344 rats. The available findings strongly suggest that this food-derived carcinogen might be of importance as an environmental factor in the production of human cancers and that its carcinogenicity could be largely avoided by reducing intake of such compounds or by adoption of appropriate chemopreventive measures.

Stomach carcinogenicity of caffeic acid, sesamol and catechol in rats and mice

The carcinogenic potential of caffeic acid, sesamol and catechol was examined in male and female F344 rats and B6C3F1 mice, groups of 30 animals being treated with diets containing 2% caffeic acid, 2% sesamol or 0.8% catechol for 104 weeks (rats) or 96 weeks (mice). Histological examination revealed that caffeic acid induced forestomach squamous cell carcinoma in 57% (P<0.001 vs. controls) and 50% (P<0.001) of male and female rats, respectively, whereas sesamol was associated with squamous cell carcinoma at incidences of 31% (P<0.001) in male rats, and 38% (P<0.001) and 17% (P<0.05) in male and female mice, respectively. Catechol induced glandular stomach adenocarcinomas in 54% (P<0.001) and 43% (P<0.001) of male and female rats, respectively. The results thus clearly demonstrated that all three antioxidants are carcinogenic in rodent stomach epithelia.

Dose-dependent Induction of 8-Hydroxyguanine and Preneoplastic Foci in Rat Liver by a Food-derived Carcinogen, 2-Amino-3, 8-dimethylimidazo [4,5-f] quinoxaline, at Low Dose Levels

Male F344 rats were administered 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx) in the diet at doses of 200, 50, 12.5, 3.2, 0.8, 0.2 and 0.05 ppm for 6 weeks, and partially hepatectomized 1 week after the beginning of MeIQx administration. Quantitative values for glutathione S‐transferase placental form (GST‐P)‐positiye foci in the liver were dose‐dependently increased by the MeIQx treatment. 8‐Hydroxyguanine (8‐OHG) levels assessed after 1 week of dietary MeIQx administration were also dose‐dependently increased, although the effect was no longer observed at the end of the treatment period. The correlation between numbers of GST‐P‐positive foci at week 6 and 8‐OHG levels at week 1 was linear, values for both parameters being higher than the control levels even in the 0.8 ppm dose group. These findings indicate that, in addition to the previously reported MeIQx‐DNA adduct formation, DNA modifications due to oxidative damage may play an important role in MeIQx liver carcinogenesis in rats.

Histogenesis of human stomach cancers based on assessment of differentiation.

The histogenesis of human stomach cancer was assessed based on the determination of the differentiation of component cancer cells. Specimens of 229 surgically obtained primary gastric cancers were used. Histochemical staining of mucins [paradoxical concanavalin A, galactose oxidase–Schiff (GOS), and sialidase–GOS sequence] and immunohistochemical demonstration of pepsinogens (Pg) I and II allowed the differentiation of gastric elements including mucous neck cells, pyloric gland cells, and surface mucous cells as well as intestinal goblet and absorptive cell types. Of 122 papillary and tubular adenocarcinomas, the proportion consisting mainly of intestinal type cells increased with progression from 22.9% (early) to 41.9% (advanced). Similarly, intestinal features increased with progression from 8.3% (early) to 25.4% (advanced) in the 107 poorly differentiated adenocarcinomas, signet ring cell carcinomas, and mucinous adenocarcinomas studied. A phenotypic shift from gastric- to intestinal-type expression was thus observed with progression of each histologic type of gastric cancer. Furthermore, tumors consisting mainly of gastric-type cells were commonly found within intestinal metaplastic mucosa, suggesting that this latter is not a preneoplastic lesion for gastric cancers in humans.