Hikaru Tanaka

Induction of Renal Cell Tumors in Rats and Mice, and Enhancement of Hepatocellular Tumor Development in Mice after Long‐term Hydroquinone Treatment

Hydroquinone (HQ) was administered to F344 rats and B6C3F1 mice of both sexes at a level of 0.8% in the diet for two years. This treatment induced renal tubular hyperplasia as well as adenomas, predominantly in males of both species, and was associated with chronic nephropathy in rats. In addition, the occurrence of epithelial hyperplasia of the renal papilla was increased in male rats. Foci of cellular alteration of the liver were significantly reduced in number by HQ in rats, but in contrast, were increased in mice, where development of hepatocellular adenoma was also enhanced in males. The incidence of squamous cell hyperplasia of the forestomach epithelium was significantly higher in mice of both sexes given HQ than in the controls, but no corresponding increase in tumor development was observed. The present study strongly indicates potential renal carcinogenicity of HQ in male rats and hepatocarcinogenicity in male mice. Thus, it is possible that HQ, which is present in the human environment, may play a role in cancer development in man.

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.

Post-initiation inhibitory effects of green tea catechins on 7,12-dimethylbenz[a]anthracene-induced mammary gland carcinogenesis in female Sprague–Dawley rats

The dose-dependence of green tea catechin (GTC) effects on 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary gland carcinogenesis were investigated in female Sprague-Dawley rats. Groups of 20 6-week-old rats were treated with dietary 1, 0.1 or 0.01% GTC for 2 weeks and then basal diet alone for 35 weeks. At the end of week 1, they received a 25 mg/kg body weight intragastric dose of DMBA. Further groups of 20 7-week-old rats each were given an intragastric dose of 25 mg/kg body weight DMBA, and starting 1 week after DMBA treatment they were placed on diet containing 1, 0.1 or 0.01% GTC or basal diet alone for 35 weeks. Control rats were given 1% GTC or basal diet alone. The final incidences and multiplicities of mammary tumors were not significantly different between the groups treated with GTC at the same time as DMBA, compared to the DMBA alone control group. On the other hand, the final multiplicities of mammary tumors in groups treated with 1% GTC (P < 0.05) or 0.01% GTC (P < 0.01), but not 0.1% GTC, after DMBA treatment were significantly decreased as compared to the control value. These results indicate that whereas GTC may inhibit mammary carcinogenesis in the post-initiation stage, the effect is weak and not dose-dependent.

Changes in urine composition, bladder epithelial morphology, and DNA synthesis in male F344 rats in response to ingestion of bladder tumor promoters.

An investigation of changes in urine composition, morphology of bladder epithelium, and levels of DNA synthesis following 4 or 8 weeks oral administration of bladder tumor promoters or analogs without promotion potential was performed. The sodium salts of L-ascorbate, o-phenylphenate, and bicarbonate increased the pH value, sodium content, volume, and MgNH4PO4 crystalluria of the urine, while the parent compounds, L-ascorbic acid and o-phenylphenol, which in contrast are not tumor promoters, did not induce these changes. Sodium chloride ingestion caused natriuresis without increasing urinary pH. Diphenyl administration produced only microcalculi consisting of p-phenylphenol. Treatment with the antioxidants butylated hydroxytoluene, butylated hydroxyanisole, and ethoxyquin was also not associated with any changes in urinary pH or Na ions. However, tert-butylhydroquinone did cause an increase in pH. Administration of the strong bladder carcinogens N-butyl-N-(4-hydroxybutyl)nitrosamine and N-ethyl-N-(4-hydroxybutyl)nitrosamine did not result in alteration of urine composition, with the exception of a decrease in phosphorus concentration. However, all the bladder promoters and carcinogens, without exception, brought about an elevation of DNA synthesis in the urothelium and produced morphologic surface alterations such as formation of pleomorphic or short, uniform microvilli and ropy or leafy microridges. Thus, an ability to induce proliferation and cell surface alteration was characteristic of the complete range of bladder promoters investigated. The results suggest that considerable variation in the mechanisms underlying these changes may be involved for different individuals or groups of agents.

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.

Correlation between Medium-term Multi-organ Carcinogenesis Bioassay Data and Long-term Observation Results in Rats

The effects of four test chemicals [2‐acetylaminofluorene (2‐AAF), D, L‐ethionine (ethionine), butylated hydroxyanisole (BHA), and catechol] were compared in medium‐ and long‐term in vivo systems. In the medium‐term assay, animals were sequentially treated with N‐diethylnitrosamine (100 mg/kg body weight, i.p., single injection), N‐methylnitrosourea (20 mg/kg body weight, i.p., 4 times during weeks 1 and 2), N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (0.05% in the drinking water during weeks 1 and 2), 1,2‐dimethylhydrazine (40 mg/kg body weight, s.c., 4 times during weeks 3 and 4) and dihydroxy‐di‐N‐propylnitrosamine (0.1% in the drinking water during weeks 3 and 4) for multi‐organ initiation, and then treated with one of the four test chemicals for 24 weeks, and killed at week 28 (group 1). In the long‐term assay, animals were treated in the same manner and then given hasal diet and tap water (group 3) or test chemical continuously (group 4) for the remainder of the lifespan. Animals receiving multi‐organ initiation and then maintained on hasal diet for 24 weeks (group 2) or their lifespan (group 5) served as controls. Detailed histopathological examinations were performed on all rats. Hepatocellular carcinoma incidences in the long‐term assay were found to reflect closely the respective medium‐term results. Induction of proliferative forestomach or glandular stomach lesions by BHA and/or catechol, and bladder lesions by 2‐AAF and BHA in the mediumterm assay also correlated with tumor development in the long‐term. Furthermore, inhibition of thyroid proliferative lesions by all test chemicals corresponded with low thyroid tumor incidences in the long‐term assay. The observed strong correlation between medium‐ and long‐term results confirms the applicability of our medium‐term multi‐organ carcinogenesis bioassay system for detection of modifying effects of test chemicals in different organs.

Organ-dependent Modifying Effects of Caffeine, and Two Naturally Occurring Antioxidants α-Tocopherol and n-Tritriacontane-16,18-dione, on 2-Amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP)-induced Mammary and Colonic Carcinogenesis in Female F344 Rats

Modifying effects of caffeine, α‐tocopherol, and n‐tritriacontane‐16,18‐dione (TTAD) on 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP)‐induced mammary and colonic carcinogenesis were investigated in female F344 rats. Groups of 20 rats, 6 weeks old, were given 0.02% PhIP (in diet) alone, or together with 0.1% caffeine (in drinking water), 0.5%α‐tocopherol (in diet) or 0.1% TTAD (in diet) for up to 54 weeks. Groups of 10 females receiving basal diet or one of the test chemicals without PhIP supplementation were also maintained. The final combined incidences (adenomas plus adenocarcinomas) and multiplicity (No./rat) of mammary adenomas and adenocarcinomas were significantly lowered in the PhIP plus caffeine group (10%, 0.10) as compared to the PhIP alone value (40%, 0.50). Incidences of mammary tumors in the PhIP plus α‐tocopherol or TTAD groups tended to be decreased while their multiplicities were significantly lowered. With regard to colon tumor development, on the other hand, rats given PhIP plus caffeine exhibited an elevated incidence (75% versus 15% in the control), whereas α‐tocopherol and TTAD had no effect. Surprisingly, metabolic activation of PhIP was inhibited by addition of caffeine in an in vitro assay. The results indicate that caffeine exerts a potent chemopreventive action against PhIP‐induced mammary carcinogenesis, but acts as a co‐carcinogen for PhIP‐induced colonic carcinogenesis.

Rat strain differences in catechol carcinogenicity to the stomach.

The carcinogenic potential of catechol was compared in male Wistar, WKY, Lewis and SD strains of rats. Groups of 30 animals were treated with powdered diet containing 0.8% catechol for 104 wk and then examined histopathologically. Induction of glandular stomach adenocarcinomas occurred in 67, 73 and 77% of Wistar, Lewis and SD animals, respectively, but in only 10% of WKY rats. In addition, catechol induced forestomach papillomas in 20% (P < 0.05), and squamous cell carcinomas in 3% of SD rats. The results thus indicate that Wistar, Lewis and SD rats are much more susceptible than WKY rats to induction of glandular stomach adenocarcinomas by 0.8% catechol, and that this phenolic antioxidant also possesses weak carcinogenic activity for the SD rat forestomach.

Inhibitory effects of phenolic compounds on development of naturally occurring preneoplastic hepatocytic foci in long-term feeding studies using male F344 rats.

Five phenolic compounds, namely caffeic acid, sesamol, hydroquinone, catechol, and 4-methoxyphenol, were fed to groups of 30 male F344 rats at dietary levels of 2, 2, 0.3, 0.8, and 2%, respectively, for 2 years. Retardation of body weight and elevated relative liver weights were noted for all groups. Formalin-fixed and paraffin-embedded liver tissues from rats killed terminally were cut and stained for glutathione S-transferase placental form (GST-P) and tumor growth factor alpha (TGF alpha) immunohistochemically. Numbers and areas of GST-P-positive (GST-P+) foci per unit area of liver section were measured, and the respective treated/control proportional values were calculated to be 58 and 57% for caffeic acid. 58 and 54% for sesamol, 71 and 71% for hydroquinone. 58 and 133% for catechol, and 49 and 39% for 4-methoxyphenol. These data were comparable with results obtained with medium-term liver bioassays (Ito test). However, no intergroup differences were detected with regard to quantitative findings for TGF alpha foci, which were relatively rare. Long-term inhibitory effects of phenolic compounds on liver carcinogenesis, predicted from the Ito test, were thus confirmed in the present feeding studies using quantitative analysis of immunohistochemically demonstrable GST-P+ foci as end point marker lesions.

Forestomach Neoplasm Induction in F344/DuCrj Rats and B6C3F1 Mice Exposed to Sesamol

Sesamol was administered at a dietary level of 2% to groups of 30 male and female F344/DuCrj rats and B6C3F, mice for 104and 96 weeks, respectively. Squamous cell carcinomas in the forestomach were Induced in nine of 29 (31%) effective male rats, three of 30 (10%) female rats, eleven of 29 (38%) male mice and five of 30 (17%) female mice treated with sesamol. Papillomas developed in ten of 29 (34%) male rats and fourteen of 30 (47%) female rats, but not in any of the mice. Hyperplasias developed in almost all rats and mice of both sexes. Significant differences from control values were found for all three lesions in rats and for carcinoma and hyperplasia categories in mice. The incidences of other tumors in the 2% sesamol group were comparable with control values. In conclusion, sesamol induces squamous cell carcinomas in the forestomach of rats and mice, males being more susceptible than females.