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Phenethyl isothiocyanate (PEITC)(1) and benzyl isothiocyanate (BITC), naturally occurring constituents of cruciferous vegetables, have been reported to exert inhibitory effects against development of tobacco-specific carcinogen-induced lung tumors and are regarded as promising chemopreventive agents for lung cancer. However, tumor promoting and carcinogenic activities in the rat urinary bladder have been detected in several animal models. The purpose of the present study was to investigate early changes in rat urinary bladder epithelium induced by PEITC and BITC and to explore promotion/carcinogenic mechanisms. In the first experiment, in order to assess acute toxic effects, PEITC or BITC at 0.1% each in the diet were administered to 6-week-old F344 rats for 1, 2, 3, and 7 days and sequential histopathological assessment and urinalysis were performed. In the second and third experiments, structure-activity relationships of PEITC, BITC and 8 other analogues, benzyl isocyanate and benzyl thiocyanate, and phenyl-, alpha-naphthyl-, tert-butyl-, butyl-, methyl-, and ethyl isothiocyanates (ITCs) were explored in a 14-day experiment. In the first experiment, the urinary pH was significantly lowered on day 1 by both PEITC and BITC. Striking features of toxicity, such as marked inflammatory changes characterized by cellular infiltration, apoptosis/single cell necrosis, cytoplasmic vacuolation, erosion, and hemorrhage in the urinary bladder were caused, with peaks apparent on days 2 or 3, respectively. Sequential change in 5-bromo-2'-deoxyuridine (BrdU) labeling indices was in line with the inflammatory response, but the thickness of the urinary bladder epithelium continued to gradually increase up to day 7. In the second and third experiments, simple and papillary or nodular (PN) hyperplasias were observed after 14-days treatment with PEITC, BITC, and phenyl- and butyl ITCs. These results suggest that continuous urinary epithelial cell proliferation due to cytotoxicity may play an important role in the early stage of rat urinary bladder carcinogenesis due to oral administration of ITCs. In addition, hydrophobic activity of ITCs, dependent on the alkyl carbon chain length, might strongly influence the induction of bladder lesions in rats.

Chemopreventive effects of the antioxidants 1-O-hexyl-2,3,5- trimethylhydroquinone (HTHQ), 3-O-ethylascorbic acid (EAsA), 3-O-dodecylcarbomethylascorbic acid (DAsA), green tea catechins (GTC) and ellagic acid on 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis were examined in female F344 rats. Groups of 20-21 6-week-old rats were maintained on a powdered diet containing 0.02% PhIP alone, PhIP together with 0.5% HTHQ, 1% EAsA, 1% DAsA, 1% GTC or 0.1% ellagic acid, these antioxidants alone or basal diet alone without supplement for 52 weeks. The survival rates of PhIP plus antioxidant groups at the end of the experiment were higher than that of the PhIP alone group. Sequential observation of palpable mammary tumors demonstrated only one tumor by week 52 in the PhIP plus HTHQ group, whereas 40% of the rats receiving PhIP alone had tumors by this time point. The final incidence of mammary adenocarcinomas was significantly decreased in the PhIP plus HTHQ group (4.8%, P < 0.01) as compared to the PhIP alone value (40%). Although statistically not significant, incidences of adenocarcinomas in the other antioxidant-treated groups (23.8-28.6%) were also lower than in the PhIP alone group. Furthermore, the incidence of large intestinal tumors in the PhIP plus HTHQ group (0%) showed a tendency to decrease relative to the PhIP alone group (16.7%). These results indicate that antioxidants, particularly HTHQ, exert a potent chemopreventive action against PhIP-induced carcinogenesis.

The effects of dietary administration of green tea catechins (GTC) were examined using a multi-organ carcinogenesis model. Groups of 15 F344 male rats were initially treated with a single i.p. administration of 100 mg/kg body wt N-diethyl-nitrosamine, 4 i.p. administrations of 20 mg/kg body wt N-methylnitrosourea, 4 s.c. doses of 40 mg/kg body wt 1,2-dimethylhydrazine, together with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine for 2 weeks and then 0.1% 2,2'-dihydroxy-di-n-propylnitrosamine for 2 weeks, both in the drinking water, for a total initiation period of 4 weeks. GTC in the diet, at doses of 1.0 or 0.1%, was administered from 1 day before and during carcinogen exposure, after carcinogen exposure or both during and after carcinogen exposure. Further groups of animals were treated with carcinogen, 1% GTC or basal diet alone as controls. All animals were killed at the end of week 36, and all major organs examined histopathologically. The numbers of small intestinal tumors (adenomas and carcinomas) per rat were significantly reduced in the groups treated with 1% GTC during (0.13 +/- 0.35) and after carcinogen exposure (0.31 +/- 0.48) and in those receiving 1% and 0.1% GTC both during and after carcinogen exposure (0.14 +/- 0.36, 0.46 +/- 0.97 respectively) as compared with the carcinogen alone group (1.07 +/- 1.21). On the other hand, numbers of glutathione S-transferase placental form positive liver foci per cm2 were slightly but significantly increased in the groups treated with 1 and 0.1% GTC during carcinogen exposure, 1% GTC after carcinogen exposure and 1% GTC both during and after carcinogen exposure. The results indicated that while GTC inhibits small intestinal carcinogenesis it slightly enhances hepatocarcinogenesis in a dose dependent manner when applied both during and after carcinogen exposure.

Phenethyl isothiocyanate (PEITC)(1) and benzyl isothiocyanate (BITC), naturally occurring constituents of cruciferous vegetables, have been reported to exert inhibitory effects against development of tobacco-specific carcinogen-induced lung tumors and are regarded as promising chemopreventive agents for lung cancer. However, tumor promoting and carcinogenic activities in the rat urinary bladder have been detected in several animal models. The purpose of the present study was to investigate early changes in rat urinary bladder epithelium induced by PEITC and BITC and to explore promotion/carcinogenic mechanisms. In the first experiment, in order to assess acute toxic effects, PEITC or BITC at 0.1% each in the diet were administered to 6-week-old F344 rats for 1, 2, 3, and 7 days and sequential histopathological assessment and urinalysis were performed. In the second and third experiments, structure-activity relationships of PEITC, BITC and 8 other analogues, benzyl isocyanate and benzyl thiocyanate, and phenyl-, alpha-naphthyl-, tert-butyl-, butyl-, methyl-, and ethyl isothiocyanates (ITCs) were explored in a 14-day experiment. In the first experiment, the urinary pH was significantly lowered on day 1 by both PEITC and BITC. Striking features of toxicity, such as marked inflammatory changes characterized by cellular infiltration, apoptosis/single cell necrosis, cytoplasmic vacuolation, erosion, and hemorrhage in the urinary bladder were caused, with peaks apparent on days 2 or 3, respectively. Sequential change in 5-bromo-2'-deoxyuridine (BrdU) labeling indices was in line with the inflammatory response, but the thickness of the urinary bladder epithelium continued to gradually increase up to day 7. In the second and third experiments, simple and papillary or nodular (PN) hyperplasias were observed after 14-days treatment with PEITC, BITC, and phenyl- and butyl ITCs. These results suggest that continuous urinary epithelial cell proliferation due to cytotoxicity may play an important role in the early stage of rat urinary bladder carcinogenesis due to oral administration of ITCs. In addition, hydrophobic activity of ITCs, dependent on the alkyl carbon chain length, might strongly influence the induction of bladder lesions in rats.

The effects of 1-O-hexyl-2,3,5-trimethylhydroquinone (HTHQ), green tea catechins (GTC), alpha-tocopherol, beta-carotene, chlorophyllin, phenylethylisothiocyanate (PEITC), 3-O-ethylascorbic acid (EAsA), 3-O-dodecylcarbomethyl ascorbic acid (DAsA), n-tritriacontane-16,18-dione (TTAD) and d-limonene on 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1)- or dimethylnitrosamine (DMN)-induced hepatocarcinogenesis, and the dose dependence of HTHQ inhibition of Glu-P-1- or 2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx)-influence on lesion development were examined in a rat medium-term liver bioassay system featuring diethylnitrosamine initiation and partial hepatectomy. At the end of week 8, the number and total area of glutathione S-transferase placental form (GST-P) positive liver foci in rats treated with 0.03% Glu-P-1 alone were increased significantly (46.8 +/- 11.0 and 12.0 +/- 5.6 respectively) as compared to the control values (3.8 +/- 1.6 and 0.4 +/- 0.2). Combined treatment with 1% HTHQ remarkably reduced both of these parameters (8.1 +/- 2.1 and 0.6 +/- 0.2). GTC (1%), PEITC (0.1%), beta-carotene (0.1%) and DAsA (1%) also demonstrated inhibition but less than HTHQ. On the other hand, these antioxidants did not influence development of foci initiated by 0.002% DMN. In the dose-response study, up to 0.125% HTHQ significantly reduced the effects of 0.02% Glu-P-1 or 0.03% MeIQx on the number and area of foci. These results indicate that several antioxidants exert chemopreventive effects against heterocyclic amine (HCA)-induced hepatocarcinogenesis, and particularly HTHQ which thus deserves further attention as a chemopreventor in the contest of the environmentally important HCA group of carcinogens.

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