Effects of arctiin on PhIP-induced mammary, colon and pancreatic carcinogenesis in female Sprague-Dawley rats and MeIQx-induced hepatocarcinogenesis in male F344 rats

Chemopreventive effects of arctiin, a lignan isolated from Arctium lappa (burdock) seeds, on the initiation or post initiation period of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced mammary carcinogenesis in female rats and on 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-associated hepatocarcinogenesis in male rats were examined. In experiment 1, female Sprague-Dawley (SD) rats were given intragastric doses of 100 mg/kg body wt of PhIP once a week for 8 weeks as initiation. Groups of 20 rats each were treated with 0.2 or 0.02% arctiin during or after PhIP initiation. Control rats were fed 0.2 or 0.02% arctiin, or basal diet alone during the experimental period. Animals were killed at the end of week 48. Although the incidence of mammary carcinomas did not significantly differ among the PhIP-treated groups, multiplicity was significantly decreased in rats given 0.2 (0.7+/-0.7, P<0.05) or 0.02% (1.0+/-1.1, P<0.05) arctiin after PhIP initiation as compared with the PhIP alone controls (2.1+/-2.5). The average number of colon aberrant crypt foci was also significantly decreased in these two groups. Pancreas acidophilic foci were induced in PhIP treated animals with slight decrease in the multiplicity with arctiin during the initiation phase. For liver carcinogenesis, groups of 15 male F344 rats were given a single intraperitoneal injection of diethylnitrosamine (DEN) and starting 2 weeks later, they were administered 0.03% MeIQx in the diet, MeIQx together with 0.5% arctiin, 0.1% arctiin or basal diet for 6 weeks. They were subjected to two-third partial hepatectomy 3 weeks after DEN initiation and killed at the end of week 8 for glutathione S-transferase placental form (GST-P) immunohistochemistry. The numbers and areas of preneoplastic GST-P positive foci were elevated by the treatment with MeIQx, and further increased by the simultaneous treatment with arctiin. These results indicate that arctiin has a protective effect on PhIP-induced carcinogenesis particularly in the mammary gland in the promotion period. On the other hand, it may have a weak co-carcinogenic influence on MeIQx-induced hepatocarcinogenesis. In addition, the results suggested that PhIP is a weak pancreatic carcinogen in female SD rats, targeting acinar cells.     

A bitter diterpenoid furanolactone columbin from Calumbae Radix inhibits azoxymethane-induced rat colon carcinogenesis

Chemopreventive effects of conjugated fatty acids derived from safflower oil (CFA-S), which contains large amounts of conjugated linoleic acid, and from perilla oil (CFA-P) with abundant conjugated alpha-linolenic acid were examined in a 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced rat mammary carcinogenesis model. Groups of 20-22 6-week-old female Sprague-Dawley (SD) rats were given eight intragastric injections of PhIP at a dose of 100 mg/kg b.w. during the initial 8 week period. Powdered basal diets containing 0.1% CFA-S or CFA-P were applied during or after PhIP treatment until week 40. In the rats receiving CFA-S or CFA-P together with PhIP treatment, retardation of mammary tumor emergence was observed until week 27. The groups given CFA-S or CFA-P after PhIP treatment, in contrast, demonstrated significant decrease in the final incidences of mammary adenocarcinomas. The indices of proliferating cell nuclear antigen positive cells in mammary adenocarcinomas were significantly reduced with both CFA-S and CFA-P in the post-initiation phase. Formation of aberrant crypt foci in the colon and basophilic foci of the pancreas due to the PhIP treatment group were not affected by CFA-S or CFA-P. In a second short-term experiment, female SD rats were maintained on powdered basal diet containing 0.03% PhIP alone or together with 0.1% CFA-S or CFA-P for 4 weeks. Immunohistochemically, CFA-S and CFA-P were revealed to suppress PhIP-DNA adduct formation in the epithelial cells of mammary gland (duct and alveolar cells), colon and pancreas. These results indicated that CFA-P and CFA-S may retard development of PhIP-induced mammary tumors with inhibition of PhIP-DNA adduct formation, and decreased mammary carcinogenesis in the post-initiation period with inhibition of cell proliferation.     

Inhibitory Effects of Diallyl Bisulfide or Aspirin on 2-Amino-l-methyl-6-phenylimidazo[4,5-b]pyridine-induced Mammary Carcinogenesis in Rats

Modifying effects of diallyl disulfide (DAD), aspirin or DL-α-difluoromethylornithine (DFMO) on 2-amino-l-methyl-6-phenyIimidazo[4,5-b]pyridine (PhlP)-induced mammary carcinogenesis in SD rats were investigated. A total of 166 female rats, 6 weeks old, were divided into 8 groups. They were fed a high fat diet throughout the experiment. Starting at 7 weeks of age, groups 1–4 were given PhIP (85 rag/kg body weight in corn oil) by gavage 8 times in 10 days, and groups 58 were given corn oil alone. For the beginning 4 weeks, groups 2 and 5 were given DAD at 200 ppm in diet. Similarly groups 3 and 6, and groups 4 and 7 were given aspirin (400 ppm) and DFMO (400 ppm), respectively. Mammary carcinomas were only recognized in groups 1–4 at the termination (25 weeks after the start of experiment). Multiplicity (mean number/rat) of neoplasms in group 2 (PhIP+DAD, 0.90/rat) and group 3 (PhIP+aspirin, 1.37/rat) was significantly smaller than that in group 1 (PhIP alone, 2.457 rat) (P < 0.005 and P < 0.05, respectively). These results indicate that dietary intake of DAD or aspirin during the time corresponding to initiation phase has chemopreventive potential on PhlP-induced mammary carcinogenesis in rats.     

Organ-dependent modifying effects of caffeine, and two naturally occurring antioxidants alpha-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, alpha-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% alpha-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%, (1.50). Incidences of mammary tumors in the PhIP plus alpha-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 alpha-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.     

Effect of alpha-naphthyl isothiocyanate on 2-amino-3-methylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis in rats

The modifying effects of alpha-naphthyl isothiocyanate (ANIT) on 2-amino-3-methylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis were investigated in female Sprague-Dawley (SD) rats, and the hepatic activities of the phase II detoxifying enzymes glutathione S-transferase (GST) and quinone reductase (QR) were also assayed. Ninety-eight rats were divided into 4 groups. Starting at 6 weeks of age, rats were fed the high-fat diet without ANIT (Groups 1 and 4) or the experimental diet (high-fat diet mixed with 400 ppm ANIT, Groups 2 and 3). At 7 weeks of age, Groups 1 and 2 were given PhIP in corn oil (85 mg/kg body weight, 8 times for 11 days) by intragastric intubation. One week after the last PhIP injection, 5 rats in each group were sacrificed to assay GST and QR activities, and the experimental diets for Groups 2 and 3 were switched to the high-fat diet without ANIT until termination of the experiment. Group 4 served as the vehicle control. All rats were sacrificed at 24 weeks after the start of the experiment. At termination of the experiment, mammary tumours were detected in Groups 1 (PhIP alone) and 2 (PhIP + ANIT) and were shown histologically to be adenocarcinomas; their incidences (multiplicities) were 56.3% (1.66 +/- 2.31/rat) in Group 1 and 6.7% (0.07 +/- 0.25/rat) in Group 2 (p < 0.001). Mean sizes of the tumours were 10.6 +/- 5.3 mm in Group 1 and 6.5 mm in Group 2. No mammary tumours were observed in rats of Groups 3 and 4. In addition, ANIT treatment significantly increased the activities of GST and QR in the livers of rats in Groups 2 and 3 as compared to Groups 1 and 4. These results imply that the isothiocyanate compound ANIT shows potent inhibitory effects on mammary carcinogenesis induced by PhIP in female SD rats when administered during the initiation stage.     

Chemoprevention by Nimesulide, a Selective Cyclooxygenase-2 Inhibitor, of 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced Mammary Gland Carcinogenesis in Rats

Breast cancer is common in women all over the world, and exploration of chemopreventive approaches to this cancer is very important. Nimesulide, a selective inhibitor of cyclooxygenase-2 (COX-2), is a good candidate as a chemopreventive agent with low toxicity. We examined its effects on mammary tumor development in female Sprague-Dawley rats induced with the environmental carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5- b ]pyridine (PhIP). Rats at 7 weeks of age received intragastric intubations of PhIP (85 mg/kg body weight) 4 times weekly for 2 weeks and were maintained on control diet (high fat diet) or experimental diet (high fat diet supplemented with 400 ppm nimesulide) throughout the experiment. COX-2 protein was over-expressed in epithelial cancer cells and stromal cells of the PhIP-induced mammary carcinomas, but was weak or not apparent in normal mammary gland cells. The development of mammary carcinomas was clearly suppressed by administration of nimesulide. The carcinoma incidence was 51% as compared to 71% for the control diet group. The average multiplicity of carcinomas in the experimental diet group was 1.2±0.2 ( P < 0.05), significantly smaller than the control diet group value (2.6±0.5). The size of carcinomas was also clearly decreased; 1.1±0.4 cm³/rat in experimental diet group ( P < 0.05), 4.1±1.3 cm³/rat in the control diet group. The results therefore provide evidence that the selective COX-2 inhibitor, nimesulide, possesses chemopreventive activity against PhIP-induced mammary carcinogenesis in rats.     

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.     

Chemoprevention by nimesulide, a selective cyclooxygenase-2 inhibitor, of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary gland carcinogenesis in rats.

Breast cancer is common in women all over the world, and exploration of chemopreventive approaches to this cancer is very important. Nimesulide, a selective inhibitor of cyclooxygenase-2 (COX-2), is a good candidate as a chemopreventive agent with low toxicity. We examined its effects on mammary tumor development in female Sprague-Dawley rats induced with the environmental carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Rats at 7 weeks of age received intragastric intubations of PhIP (85 mg / kg body weight) 4 times weekly for 2 weeks and were maintained on control diet (high fat diet) or experimental diet (high fat diet supplemented with 400 ppm nimesulide) throughout the experiment. COX-2 protein was over-expressed in epithelial cancer cells and stromal cells of the PhIP-induced mammary carcinomas, but was weak or not apparent in normal mammary gland cells. The development of mammary carcinomas was clearly suppressed by administration of nimesulide. The carcinoma incidence was 51% as compared to 71% for the control diet group. The average multiplicity of carcinomas in the experimental diet group was 1.2 +/- 0.2 (P < 0.05), significantly smaller than the control diet group value (2.6 +/- 0. 5). The size of carcinomas was also clearly decreased; 1.1 +/- 0.4 cm(3) / rat in experimental diet group (P < 0.05), 4.1 +/- 1.3 cm(3) / rat in the control diet group. The results therefore provide evidence that the selective COX-2 inhibitor, nimesulide, possesses chemopreventive activity against PhIP-induced mammary carcinogenesis in rats.     

Chemoprevention of 2-amino-1-methyl-6-phenylimidazo- [4,5-b]pyridine-induced colon carcinogenesis by 1-O-hexyl-2,3,5-trimethylhydroquinone after initiation with 1,2-dimethylhydrazine in F344 rats

The aim of this study is to investigate the chemopreventive effects of the synthetic phenolic antioxidant 1-O-hexyl-2,3, 5-trimethylhydroquinone (HTHQ) on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-associated colon carcinogenesis in rats after initiation with 1,2-dimethylhydrazine (DMH) in male F344 rats. Groups of 20-22, 6-week-old male F344 rats were given four subcutaneous injections of 40 mg/kg body wt of DMH during the initial 4 weeks. They were then maintained on powdered basal diet containing 0.03% PhIP alone, PhIP together with 0.5 or 0.125% HTHQ, 0.5 or 0.125% HTHQ alone or basal diet for 32 weeks. A small number (1.1 +/- 1.1/rat) of colon tumors were induced by DMH treatment alone. After initiation with DMH, the number of colon tumors was greatly increased to 8.3 +/- 5.6 by the administration of PhIP. Additional treatment with HTHQ dose-dependently decreased the multiplicity of colon adenocarcinomas to 4.9 +/- 2.8 and 2.6 +/- 1.4 with 0.125 and 0.5%, respectively. This treatment similarly reduced atypical hyperplasias of the ventral prostate. Furthermore, HTHQ significantly reduced the multiplicity of duodenal adenocarcinomas induced by DMH + PhIP or DMH alone. Immunohistochemically, HTHQ was revealed to suppress PhIP-DNA adduct formation in the epithelial cells of the colon and prostate in a separate 2 weeks experiment. The present results clearly showed that HTHQ has chemopreventive potential for PhIP-associated colon and prostate carcinogenesis. The observed inhibition may largely be due to interference with PhIP-DNA adduct formation. In addition, HTHQ has been demonstrated to inhibit duodenal carcinogenesis in the post-initiation stage.     

Chemoprevention of 2-amino-1-methyl-6-phenylimidazo[4, 5-b]-pyridine (PhIP)-induced mammary gland carcinogenesis by antioxidants in F344 female rats

Chemopreventive effects of the antioxidants 1-O-hexyl-2, 3,5-trimethylhydroquinone (HTHQ), 3-0-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 examinedin female F344 rats. Groups of 20–21 6-week-old rats weremaintained on a powdered diet containing 0.02% PhIP alone, PhIPtogether with 0.5% HTHQ, 1% EAsA, 1% DAsA, 1% GTC or 0.1% ellagicacid, these antioxidants alone or basal diet alone without supplementfor 52 weeks. The survival rates of PhIP plus antioxidant groupsat the end of the experiment were higher than that of the PhIPalone group. Sequential observation of palpable mammary tumorsdemonstrated only one tumor by week 52 in the PhIP plus HTHQgroup, whereas 40% of the rats receiving PhIP alone had tumorsby this time point. The final incidence of mammary adenocarcinomaswas significantly decreased in the PhIP plus HTHQ group (4.8%,P<0.01) as compared to the PhIP alone value (40%). Althoughstatistically not significant, incidences of adenocarcinomasin the other antioxidant-treated groups (23.8–28.6%) werealso lower than in the PhIP alone group. Furthermore, the incidenceof 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, particularlyHTHQ, exert a potent chemopreventive action against PhIP-inducedcarcinogenesis.     

Inhibitory effects of Bifidobacterium-fermented soy milk on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced rat mammary carcinogenesis, with a partial contribution of its component isoflavones

High consumption of soybean and soybean-related products is hypothesized to contribute to protection against breast cancer. Soybean is a rich source of genistein, a putative cancer chemopreventive agent. Fermented soy milk (FSM), which is made of soy milk fermented with the Bifidobacterium breve strain Yakult, contains larger amounts of the isoflavone aglycones genistein and daidzein than unfermented soy milk. In the present study, we examined the effects of FSM and its component isoflavone mixture (genistein:daidzein 4:1) on 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP)-induced mammary carcinogenesis in rats. Starting at 7 weeks of age, female Sprague-Dawley rats were given PhIP at a dose of 85 mg/kg body wt by intragastric administration four times a week for 2 weeks. They were fed control high fat basal diet or experimental high fat diet containing 10% FSM or 0.02 or 0.04% isoflavone mixture during and after carcinogen exposure. The incidences (percentage of rats with tumors) of mammary gland tumors were 71% in the control diet group, 51% in the FSM group and 68 and 61% in the groups treated with isoflavone mixture at 0.02 and 0.04%, respectively. Mammary tumor multiplicities (number of tumors per rat) were 1.2 +/- 0.2 for 10% FSM, 2.2 +/- 0.4 for 0.02% isoflavone mixture and 1.5 +/- 0.3 for 0.04% isoflavone mixture, being clearly smaller than the control diet value (2.6 +/- 0.5). Furthermore, feeding of FSM and the isoflavone mixture at both doses reduced the sizes of mammary tumors. Since the amounts of isoflavones in 10% FSM are approximately equivalent to those in the 0.02% isoflavone mixture, the chemopreventive activity of FSM could be partly attributable to the presence of isoflavones such as genistein and daidzein.     

Lack of enhancing effects of sodium nitrite on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis in female Sprague-Dawley rats

A number of heterocyclic amines (HCAs) have been shown to exert enhanced carcinogenic and mutagenic potential when given simultaneously with sodium nitrite (NaNO(2)). In the present experiment, effects of combined treatment with NaNO(2) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), one of the most prevalent carcinogenic HCAs in the human environment, were assessed with regard to mammary tumor induction in female Sprague-Dawley (SD) rats. Animals at 6 weeks of age were given intragastric doses of 100mg/kg body weight of PhIP twice a week for 4 weeks, during which period 0 or 0.2% NaNO(2) was administered in the drinking water. Control rats received 0.2% NaNO(2) alone for the 4 weeks or non-supplemented water during the entire 48 week experimental period, without carcinogen treatment. The first tumor in the PhIP+NaNO(2) group appeared significantly later than with PhIP alone, and during the experimental period, the incidence, multiplicity and volume of mammary tumors in this group tended towards decreased, although values did not significantly differ at the terminal sacrifice. These results indicate that NaNO(2) does not enhance PhIP-induced rat mammary carcinogenesis, rather possessing some potential for inhibition.     

Lack of significant modifying effect of arctiin on prostate carcinogenesis in probasin/SV40 T antigen transgenic rats

Arctiin, a plant lignan that can be extracted from the Arctium lappa (burdock) seeds, is a possible environmental endocrine disruptor compounds and have been shown to influence sex hormone metabolism as well as protein synthesis, steroid biosynthesis. Modifying effects of arctiin on prostate carcinogenesis in probasin/SV 40 T antigen (Tag) transgenic (TG) rats were examined. A total of 64 male TG rats, 6 weeks old, were randomly divided to three experimental groups (soybean free Oriental MF diet with 0.1, 0.02, or 0.004% arctiin) and a control group (soybean free Oriental MF diet). Animals were killed at the end of week 18. Histopathological evaluation of prostate revealed that all the rats in any group developed adenocarcinoma in dorsolateral lobe of prostate, except two rats in 0.1% arctiin treated and one rat in 0.002% arctiin treated groups without prostate adenocarcinoma development. However, there were no definite treatment-related changes with statistical significance in all parameters for prostate carcinomas measured in this experiment. These results indicated that arctiin might not exert significant modifying effect on prostate carcinogenesis in SV 40 Tag TG rats at least under the present experiment.     

Chemoprevention of experimental mammary carcinogenesis by the synthetic organoselenium compound, benzylselenocyanate, in rats

The effect of the dietary organoselenium compound, benzylselenocyanate (BSC) along with its sulphur analogue, benzylthiocynanate (BTC) and sodium selenite (Na2SeO3), on 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis was examined in female Sprague-Dawley rats during the initiation phase of carcinogenesis. Semipurified diets containing 25 p.p.m. of BSC and 25 p.p.m. BTC, and 4 p.p.m. Selenium as Na2SeO3 in drinking water were given to 5-week-old rats for 3 weeks starting 2 weeks before, during and until 1 week after carcinogen treatment. At 7 weeks of age animals were given a single dose of DMBA (10 mg) in 1 ml olive oil by oral intubation. One week after DMBA treatment, the groups receiving BSC- and BTC-supplemented diets were transferred to the unsupplemented standard diets and the group of rats receiving Na2SeO3 in drinking water was transferred to regular tap water for the duration of the experiment. The results indicate that the rats receiving BSC in their diet showed a highly significant inhibition of tumor incidence and tumor multiplicity as well as a prolonged latency period when compared to the group fed the control diet. Neither BTC nor Na2SeO3 had any effect on the subsequent development of mammary tumors. These results indicate that dietary BSC inhibits mammary tumor incidence during the initiation phase of carcinogenesis and is a considerably more potent inhibitor than its sulphur analogue BTC and inorganic selenium. This is the first report that demonstrates the inhibition of mammary carcinogenesis by a synthetic organoselenium compound.     

Protective effects of benzyl isothiocyanate and sulforaphane but not resveratrol against initiation of pancreatic carcinogenesis in hamsters

Potential chemopreventive effects of naturally occurring agents were investigated using a new 16-week medium-term pancreatic carcinogenesis models in hamsters. Male 6-week-old Syrian hamsters were subcutaneously injected with 10mg/kg body weight N-nitrosobis(2-oxopropyl)amine (BOP) four times within a week, and fed a diet supplemented with 80ppm benzyl isothiocyanate (BITC), 80ppm sulforaphane (SFN) or 10ppm resveratrol (RES) during the initiation or post-initiation stages. For the initiation stage, each chemical was given for 3 weeks including 1 week before and after the BOP injections. With post-initiation exposure, the groups were changed from basal diet 1 week after the last BOP injection, and then fed each chemical for 14 weeks. All the animals were sacrificed after 16 weeks. The multiplicities of combined pancreatic lesions including atypical hyperplasias and adenocarcinomas were significantly decreased by BITC and SFN given in the initiation but not the post-initiation stage. On the other hand, RES, a naturally occurring inhibitor of cyclooxygenase-2 (COX-2) reported chemopreventive effects, failed to show significant effects on pancreatic carcinogenesis in either the initiation or post-initiation stages. Our data suggest that the naturally occurring isothiocyanates BITC and SFN can block BOP-initiation of hamster pancreatic carcinogenesis.     

Inhibition of PhIP-induced mammary carcinogenesis in female rats by ingestion of freeze-dried beer

This study evaluated the modulating effect of non-alcoholic constituents of beer on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mammary carcinogenesis. Female Sprague-Dawley (SD) rats at 6 weeks of age were divided into four groups (n=26-30) and fed either a high fat diet or high fat diets containing 1, 2 or 4% freeze-dried beer (FD beer). One week after the start of feeding, rats received PhIP at a dose of 85 mg/kg by gavage four times weekly for 2 weeks. There were no differences in the body weights or diet intakes of rats between the control and the experimental groups. Weekly observation of palpable tumors indicated that tumor incidence and tumor multiplicity in the 2 and 4% FD beer groups were lower than in the control group throughout the experiment. Neoplastic lesions were pathologically examined at the end of the 22-weeks experiment. Tumor development was inhibited by FD beer intake in a dose-dependent manner. Tumor incidence (38.5%) and tumor multiplicity (0.8+/-0.4) for the group fed with a diet containing 4% FD were significantly reduced as compared with the control group (73.3% and 1.8+/-0.7). Supplementation with FD beer for 3 weeks together with the PhIP treatments resulted in increased liver GST activity, decreased liver CYP1A2 activity and a decrease in the number of DNA adducts in the mammary tissue, though these values were not significant. In conclusion, our results suggest that intake of FD beer may reduce the risk of carcinogenesis caused by heterocyclic amines.     

Inhibitory Effects of Nabumetone, a Cyclooxygenase-2 Inhibitor, and Esculetin, a Lipoxygenase Inhibitor, on N-Methyl-N-nitrosourea-induced Mammary Carcinogenesis in Rats

We investigated the modifying effects of nabumetone, a relatively selective cyclooxygenase-2 inhibitor, and esculetin, a lipoxygenase inhibitor, on N -methyl- N -nitrosourea(MNU)-induced mammary carcinogenesis in female Sprague-Dawley rats. A total of 124 rats, 6 weeks old, were divided into 6 groups. At 50 days of age, groups 1, 2, and 3 were treated with MNU (50 mg/kg body weight) by subcutaneous injection. From the age of 8 weeks, groups 2 and 4 were given 0.03% nabumetone in the diet and groups 3 and 5 were given 0.03% esculetin in the diet. All rats were necropsied at the termination (25 weeks after the start of experiment). The incidence and multiplicity of neoplasms in group 2 were significantly smaller than those in group 1 ( P <0.005 and P <0.001, respectively). The incidence of neoplasms in group 3 was also significantly smaller than that in group 1 ( P <0.05). These results indicate that the intake of nabumetone or esculetin during the time corresponding to the post initiation phase has a chemopreventive effect on MNU-induced mammary carcinogenesis in rats.     

Proliferation, development and DNA adduct levels in the mammary gland of rats given 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and a high fat diet

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic amine derived from cooked meat that is a mammary gland carcinogen in rats. A carcinogenic dose-regimen of PhIP (75 mg/kg, p.o., 10 doses, once per day) was administered to 43-day old female Sprague-Dawley rats, and the rats were then placed on a defined high fat (23.5% corn oil) or low fat (5% corn oil) diet for up to 6 weeks. At various times after carcinogen and diet, and prior to carcinogenesis, we examined the percentage of proliferating cells in terminal end bud (TEB) epithelial structures of the rat mammary gland by proliferating cell nuclear antigen staining, mammary gland architecture by whole mounting, and PhIP-DNA adduct levels in mammary epithelial cells by the 32P-post-labeling assay. Immediately after dosing, the percentage of proliferating epithelial cells in TEBs was significantly higher in PhIP-treated rats than in control rats receiving vehicle only [7.5 +/- 0.9% (n = 99) versus 4.2 +/- 0.6% (n = 127), respectively]. The mammary glands of PhIP-treated rats showed a significantly lower density of alveolar buds (ABs) and a higher density of TEBs than control rats, which suggests that PhIP exposure partially inhibited the normal glandular differentiation of TEBs to ABs. After 6 weeks on the diet, proliferation in TEBs was statistically higher in rats given PhIP plus a high fat diet than in rats given vehicle plus a low fat diet. The mammary glands from rats on a high fat diet also showed a statistically higher density of TEBs when compared with rats on a low fat diet [2.08 +/- 0.34% versus 1.04 +/- 0.20%, respectively (n = 6)]. PhIP-DNA adduct levels were relatively high in mammary epithelial cells of treated rats. At 3 h after the last dose of PhIP, DNA adduct levels [relative adduct labeling (RAL) x 10(7), mean +/- SE] were 10.5 +/- 1.7 (n = 8) and 0.9 +/- 0.2 (n = 7) in epithelial cells isolated from mammary gland and in the liver, respectively. DNA adduct removal rates from the mammary gland were not different between rats on the high fat and low fat diets. Adducts were still detected after 6 weeks on either diet. Thus, events that occurred prior to neoplasia in the mammary glands of PhIP-treated rats include formation of PhIP-DNA adducts at relatively high levels, and enhanced proliferation in TEBs (putative sites of origin of mammary gland carcinomas) and partial inhibition of TEB differentiation. The high fat diet, a promoter of PhIP-induced mammary gland carcinogenesis, appeared to sustain the proliferative effect of PhIP in mammary gland TEBs at a time when PhIP- DNA adducts are still detectable. These early events may contribute to the targeting and carcinogenicity of PhIP to the mammary gland of rats.      

Combined treatment with green tea catechins and sodium nitrite selectively promotes rat forestomach carcinogenesis after initiation with N-methyl-N'- nitro-N-nitrosoguanidine

Combined treatment with several phenolic antioxidants and sodium nitrite (NaNO(2)) has already shown to enhance rat forestomach carcinogenesis. In the present experiment, effects of green tea catechins (GTC) alone or in combination with NaNO(2) on gastric carcinogenesis were investigated in a rat two-stage carcinogenesis model. Groups of eight, 6-week-old F344 male rats were given 0.01%N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in their drinking water and 5% NaCl in the diet for 10 weeks for glandular stomach initiation and a single intragastric administration of 100 mg/kg/bodyweight of MNNG at week 9 for forestomach initiation. From week 11, they received either drinking water containing 0.2% NaNO(2) and a diet supplemented with 1% GTC in combination, each individual chemical alone or a basal diet until the end of week 42. In the forestomach, incidences and multiplicities of neoplastic lesions were clearly increased by the combined treatment, in spite of GTC alone suppressing the occurrence of papillomas. In a short-term experiment with similar protocol without MNNG pretreatment, a significant increase of 8-hydroxydeoxyguanosine (8-OHdG) levels in forestomach DNA occurred 24 h after the combined treatment, concomitant with erosion and inflammatory cell infiltration. In an in vitro study, electron spin resonance demonstrated hydroxyl radical formation after incubation of epigallocatechin gallate or epicatechin gallate with the NO generator, NOC-7. Thus, GTC alone showed a weak chemopreventive effect on forestomach carcinogenesis, but in the presence of NaNO(2) it exerted a promotive effect which might involve hydroxyl-radical-associated oxidative DNA damage. However, no influence was exerted in the glandular stomach.     

Anticarcinogenic and hepatotoxic interactions between retinyl acetate and butylated hydroxytoluene in rats

The natural retinoid, retinyl acetate (RA), and the phenolic antioxidant, butylated hydroxytoluene (BHT), are both effective inhibitors of mammary carcinogenesis in rats. The present study was designed to determine if an increased inhibition of mammary carcinogenesis is obtained when RA and BHT are administered in combination. At age 50 days (time 0), virgin, female Sprague-Dawley rats received a single intragastric instillation of 16 mg of 7,12-dimethylbenz(a)anthracene dissolved in 1 ml sesame oil. Groups of 30 carcinogen-treated rats received Wayne Lab Chow supplemented with (per kg diet) 250 mg RA, 5000 mg BHT, or 250 mg RA plus 5000 mg BHT by the following schedule: -2 to +1 week; +1 week until the end of the experiment; -2 weeks to end; or none. Combined administration of RA plus BHT by the -2 weeks to end schedule was more effective in mammary cancer chemoprevention than was RA alone or BHT alone; the interaction of RA and BHT was additive. Similarly, administration of RA plus BHT by the -2 weeks to end protocol was more active in chemoprevention than was RA plus BHT administered either from weeks -2 to +1 or +1 week to end. Chronic exposure to RA plus BHT induced a high incidence of hepatic fibrosis and bile duct hyperplasia; these changes were not observed in controls and were seen in low incidence in animals exposed to RA only or BHT only. These data indicate that enhanced anticarcinogenic activity can be obtained through the use of "combination chemoprevention" regimens; however, chemopreventive compounds may interact not only to inhibit carcinogenesis but also to induce toxicity.