Yasumoto Mizoguchi

Effects of green tea catechins on the progression or late promotion stage of mammary gland carcinogenesis in female Sprague-Dawley rats pretreated with 7,12-dimethylbenz(a)anthracene

Effects of the green tea catechins (GTCs) on the late promotion or progression stage of mammary gland carcinogenesis were examined in female Sprague-Dawley (SD) rats pretreated with 7,12-dimethylbenz(a)anthracene (DMBA). A total of 84 7-week-old rats received a 50 mg/kg body weight intra-gastric dose of DMBA, and starting 13 weeks thereafter, when the tumor incidence had reached 50%, three groups of 28 animals each were placed on diet containing 0.5% Polyphenon E (58.4% content (-)-epigallocatechin gallate (EGCG)) (groups 1a and 1b), 0.5% EGCG-80 (81% content of EGCG) (groups 2a and 2b) or basal diet alone (groups 3a and 3b) for 23 weeks. The experiment was terminated at week 36. The growth (i.e. change in mean diameter) of mammary tumors present at week 13 (groups 1a, 2a and 3a) was not influenced by the treatment with EGCGs, with no significant intergroup differences in the lesion incidences, multiplicity or size being observed. Values for these parameters did show a tendency for decrease in group 2b (Polyphenon E) as compared to group 3b (control) during the study, but they were not significantly reduced at the sacrifice time point. These results indicate that GTCs are not effective at inhibiting progression of rat mammary carcinogenesis, but Polyphenon E may exert a weak inhibitory effect on the early promotion stage.

Stage and organ dependent effects of 1‐ O‐hexyl‐2,3,5‐trimethylhydroquinone, ascorbic acid derivatives, N‐heptadecane‐8,10‐dione and phenylethyl isothiocyanate in a rat multiorgan carcinogenesis model

The effects of 1‐O‐hexyl‐2,3,5‐trimethylhydroquinone (HTHQ), phenylethyl isothiocyanate (PEITC), 3‐O‐ethylascorbic acid, 3‐O‐dodecylcarbomethylascorbic acid and n‐heptadecane‐8,10‐dione were analyzed in a rat multiorgan carcinogenesis model. Groups of 15 animals were given a single intraperitoneal (i.p.) injection of diethylnitrosamine and 4 i.p. injections of N‐methylnitrosourea as well as N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine in the drinking water during the first 2 weeks. Then 4 subcutaneous (s.c.) injections of dimethylhydrazine and 2,2′‐dihydroxy‐di‐n‐propylnitrosamine were given in the drinking water over the next 2 weeks for initiation. Test compounds were administered during the initiation or post‐initiation periods. The dietary dose was 1% except for n‐heptadecane‐8,10‐dione and PEITC (0.1%). Complete autopsy was performed at the end of experimental week 28. All 5 compounds reduced the number of lung hyperplasia, particularly PEITC when given during the initiation period. In addition, HTHQ lowered the incidence of esophageal hyperplasia in the initiation period, and of small and large intestinal adenomas in the post‐initiation period. However, it also enhanced the development of hyperplasia and papilloma in the forestomach and tongue. PEITC lowered the induction of esophageal hyperplasia, kidney atypical tubules and liver glutathione S‐transferase placental form (GST‐P)‐positive foci when given during the initiation period but enhanced the development of liver GST‐P positive foci and urinary bladder tumors in the post‐initiation period. Moreover, it induced hyperplasia of the urinary bladder. Our results indicate minor adverse effects for HTHQ in the forestomach and tongue, and demonstrate that PEITC, which inhibits carcinogenesis at the initiation stage in several organs, also exhibits promotion potential in liver and urinary bladder in the post‐initiation stage under the present experimental conditions. Int. J. Cancer 76:851–856, 1998.© 1998 Wiley‐Liss, Inc.

Highly metastatic hepatocellular carcinomas induced in male F344 rats treated with N-nitrosomorpholine in combination with other hepatocarcinogens show a high incidence of p53 gene mutations along with altered mRNA expression of tumor-related genes

The carcinogenic and metastatic processes are thought to consist of a sequence of steps, and animal models featuring highly metastatic lesions are clearly necessary to allow analysis of the whole process of transformation from preneoplastic changes to high grade metastatic tumors, and to access effectiveness of therapeutic treatments of advanced cancers in vivo. The purpose of the present study was to establish a model and to screen for reported genetic alterations in induced lesions. In the present study, it was confirmed that lung metastasis of hepatocellular carcinomas (HCCs) induced in male F344 rats by N-nitrosomorpholine (NNM), given in the drinking water at a dose of 120 ppm for 24 weeks, was significantly enhanced by additional carcinogenic pretreatments and that a single i.p. injection of 100 mg/kg body weight N-diethylnitrosamine (DEN) alone was sufficient for that purpose. Molecular biological analyses of the induced lesions revealed point mutations in the p53 gene in 60.9% of HCCs, and elevated expression of mRNAs for p53, c-myc, c-fos, TGF-alpha, TGF-beta1, alpha-fetoprotein, GST-P, and GGT, and decreased mRNA expression of EGF and EGFR in HCCs when compared to controls. No obvious association of gene alterations with metastatic potential of primary tumors was found except for an increase in the incidence of p53 mutations. Since the process of metastasis is thought to be sequential and selective, further comparative analysis of metastatic and primary lesions should clarify the mechanisms involved in the multi-step process of metastasis.

Lack of inhibitory effects of green tea catechins in 1,2-dimetylhydrazine-induced rat intestinal carcinogenesis model: comparison of the different formulations, administration routes and doses

Differences in the modifying effects of green tea catechins (GTC) on intestinal carcinogenesis by different formulations, doses and administration routes were investigated in male rats pretreated with 1,2-dimethylhydrazine (DMH). One hundred and eighty nine F344 male rats received subcutaneous injections of DMH at 40 mg/kg body weight twice a week for 3 weeks. Three days after completion of the carcinogen treatment, they were divided into nine groups. Each was administered a different source of 0.1% or 0.01% of GTC (Mitsui Norin Co. (M) or Taiyo Kagaku Co. (T)) either in the diet (D) or the drinking water (W), or basal diet and tap water alone without GTC for 33 weeks and then killed for autopsy. The survival rate tended to be lower with 0.01% MGTC (W) group than in the other groups. In the large intestine, although the multiplicity and/or incidences of adenomas showed tendencies for dose-dependent decrease in all GTC groups, and the average volumes of tumors tended to be decrease dose-dependently in the MGTC (W) and TGTC (W) groups, the multiplicity of carcinomas did not show such a trend, rather being significantly increased in the 0.01% MGTC (D) and 0.1% TGTC (W) groups. In the small intestine, the incidence and the multiplicity of tumors in all GTC treated groups had a tendency to decrease. On the other hand, the volume of tumors was increased with statistical significance in the 0.01% MGTC (W) and 0.1% TGTC (W) groups. Thus it can be concluded that GTC does not exert chemopreventive effects on intestinal carcinogenesis irrespective of its formulation, dose or route of administration.

Hepatocyte Growth Factor Enhancement of Preneoplastic Hepatic Foci Development in Rats Treated with Diethylnitrosamine and N-Ethyl-N-hydroxyethylnitrosamine

Effects of hepatocyte growth factor were investigated in a two‐stage rat liver carcinogenesis protocol. Male F344 rats were first treated with diethylnitrosamine (200 mg/kg, i.p.) and then, starting two weeks later, with N‐ethyl‐N‐hydroxyethylnitrosamine (EHEN) for 6 weeks at a dose of 0.01% in drinking water. Hepatocyte growth factor, which was injected i.v. at a dose of 200 μg/kg body weight one (at week 3) or two times (at weeks 3 and 4) during EHEN administration, significantly increased the development of preneoplastic glutathione S‐transferase placental form‐positive foci. Although the observed effects of hepatocyte growth factor were weaker than that of the two‐thirds partial hepatectomy (PH) performed at week 3, the present results suggest that the enhancing effects of PH performed during the promotion stage may be largely mediated through induction of hepatocyte growth factor.

Changes in blood parameters in New Zealand White rabbits during pregnancy.

Changes in clinical pathology parameters, particularly those related to blood coagulation, were examined throughout the gestation period in New Zealand White rabbits. As compared with the non-pregnant group, the following major changes were observed in the pregnant group. For blood coagulation-related parameters, platelets increased progressively and fibrinogen increased slightly from organogenesis, prothrombin time was significantly prolonged during organogenesis and shortened in the late fetal growth stage, activated partial thromboplastin time was significantly prolonged during the fetal growth stage, and antithrombin III increased during and after late organogenesis. Such changes in blood coagulation-related parameters during the later stages of gestation seem to be physiological responses in preparation for protecting against excessive haemorrhage or haemostasis at parturition. For the other haematological and blood chemical parameters as well as progesterone, red blood cell counts, haemoglobin and haematocrit began to decrease during organogenesis and continued to decrease thereafter. Reticulocyte counts significantly increased during organogenesis and decreased thereafter. White blood cell parameters, except for neutrophils, showed significant decreases during the fetal growth stage. Serum progesterone concentration reached its highest level early in organogenesis and decreased thereafter. Total protein, albumin, glucose, cholesterol, calcium, blood urea nitrogen and creatinine decreased significantly during the middle and/or late periods of gestation. In conclusion, the data obtained from the present study can be used as background data for effective evaluation of reproductive toxicology in rabbits, and pregnant rabbits may serve as models of pregnant women in research pertaining to clinical pathology and gestation.

Studies of initiation and promotion of carcinogenesis by N-nitroso compounds

Complete carcinogens must possess both initiating and promoting properties. Most N-nitroso compounds are mutagens and are considered to be initiators, but some are not mutagenic and yet are complete carcinogens. To investigate the two activities, brief treatments of male F344 rats with each of three mutagens, nitrosodimethylurea, nitrosodiethylurea and nitrosobis-(2-oxopropyl)-amine, were followed by chronic treatment (40 weeks) with one of four non-mutagens, nitrosomethyl-3-carboxypropylamine, nitrosodiethanolamine, nitrosomethyl-2-hydroxypropylamine or phenobarbital, the last being a well-known promotor of liver tumors in rats. Each treatment group consisted of 18 animals and there were control groups of 15 animals without initiation and 15 animals without promotion. All surviving rats were sacrificed at week 78. There were almost no tumors in untreated controls or in groups treated with the promotors, other than bladder tumors in one group. Certain tumors were numerous in the initiated groups, but there were only a few instances of increased incidences after treatment with the promotors. The action of the initiators appeared to be the dominant factor and there was scant indication in this experiment of the induction of tumors by the promotors of promotion of initiated cells in most organs (e.g. the liver). This indicates that it is unlikely that non-genotoxic carcinogens induce tumors by promotion of already-initiated cells, but that some other mechanism prevails.

Phenotypic alteration of hepatocellular foci in rats treated with clofibrate and phenobarbital

In male F344 rats pretreated with diethylnitrosamine (DEN), subsequent administration of clofibrate increased the proportion of eosinophilic foci, to become the most abundant type, and reduced numbers of basophilic, clear and vacuolated foci, the total not being changed. A similar shift towards eosinophilia was also observed in phenobarbital-treated animals, but in this case clear increases in total number and area were apparent. Expression of the glutathione S-transferase placental form (GST-P) in foci was much lowered by clofibrate treatment, while the proportion of positive foci was very high in both phenobarbital and control groups. A marked contrast was found with eosinophilic foci, with 74% positive after phenobarbital as compared to only 15% for clofibrate. Thus, the decrease in GST-P positive foci by clofibrate was mainly due to increased negativity in the most abundant eosinophilic type foci. In a long-term feeding study without DEN initiation, similar negativity of foci was observed and, furthermore, only minimal effects of clofibrate on foci development was revealed in both young and old animals.

Historical control data on prenatal developmental toxicity studies in rabbits

Historical control data on rabbit prenatal developmental toxicity studies, performed between 1994–2010, were obtained from 20 laboratories, including 11 pharmaceutical and chemical companies and nine contract laboratories, in Japan. In this paper, data were incorporated from a laboratory if the information was based on 10 studies or more. Japanese White rabbits and New Zealand White rabbits were used for prenatal developmental toxicity studies. The data included maternal reproductive findings at terminal cesarean sections and fetal findings including spontaneous incidences of morphological alterations. No noticeable differences between strains or laboratories were observed in the maternal reproductive and fetal developmental data. The inter‐laboratory variations in the incidences of fetal external, visceral, and skeletal alterations seem to be due to differences in the selection of observation parameters, observation criteria, and classification of the findings, and terminology of fetal alterations.

Dose Dependence of 1‐O‐Hexyl‐2,3,5‐trimethylhydroquinone Promotion of Forestomach Carcinogenesis in Rats Pretreated with N‐Ethylnitrosourethane

Post‐initiation dose‐dependent effects of the chemopreventive antioxidant 1‐O‐hexyl‐2,3,5‐trimethylhydroquinone (HTHQ), a potent inhibitor of heterocyclic amine‐induced mutagenesis and carcinogenesis, on the development of forestomach and tongue tumors were investigated in male F344 rats. Groups of 22 rats were treated with 0.01% ethylnitrosourethane (ENUR) as an initiator in the drinking water for 4 weeks, then placed on diet containing 1.0%, 0.5%, 0.25% or 0.125% HTHQ, or basal diet alone for 36 weeks. Further groups of 12 rats each were similarly treated with the different doses of HTHQ or given basal diet alone for 36 weeks without prior ENUR treatment. All animals were killed at week 40. Tongue papillary hyperplasia and papillomas tended to be increased in the groups treated with ENUR followed by 0.5–0.125% HTHQ, though there was no effect at the highest dose, in line with increased bromodeoxyuridine labeling indices. In the forestomach, the incidences of papillomas and carcinomas were also significantly elevated only in the group treated with ENUR followed by 0.125% HTHQ. Without ENUR pretreatment, papillary hyperplasia was found in the 1–0.125% HTHQ groups and the labeling index was also increased, though without clear dose dependence. The results indicate that HTHQ may have very weak or weak promotion potential for tongue and forestomach carcinogenesis, but that both minimum and maximum thresholds for active dose levels may exist.