Renal carcinogenicity of concurrently administered fish meal and sodium nitrite in F344 rats.

The effects of long-term concurrent administration of powdered fish meal and sodium nitrite were examined in F344 rats. A total of 600, 6-week-old rats were divided into 6 male and 6 female groups, each consisting of 50 animals. Rats in groups 1-3 and 7-9 were respectively fed diets supplemented with 64%, 32% and 8% (basal diet) fish meal, and simultaneously given 0.12% sodium nitrite in their drinking water. Groups 4-6 and 10-12 were respectively given 64%, 32% and 8% fish meal and tap water. At the 104th week, all surviving animals were killed and examined histopathologically. Treatment with fish meal dose-dependently increased the incidences and multiplicities of atypical tubules, adenomas and renal cell carcinomas in sodium nitrite-treated males. Females were less susceptible than males for renal tumor induction. In males given the 64% fish meal diet alone, the incidence and multiplicity of atypical tubules were also significantly increased as compared with the 8% fish meal alone case. Nephropathy was apparent in fish meal-treated groups in a clear dose-dependent manner, irrespective of the sodium nitrite treatment, and was more prominent in males than in females. Dimethylnitrosamine was found in the stomach contents after 4-week treatment with 64% fish meal plus 0.12% sodium nitrite, at a level twice that in the 8% fish meal plus 0.12% sodium nitrite group. The results clearly indicate that concurrent administration of fish meal and sodium nitrite induces renal epithelial tumors. Further studies are required to elucidate how nephropathy and nitrosamines produced in stomach contents may contribute to the observed renal tumor induction.     

Renal Carcinogenicity of Concurrently Administered Fish Meal and Sodium Nitrite in F344 Rats

The effects of long-term concurrent administration of powdered fish meal and sodium nitrite were examined in F344 rats. A total of 600, 6-week-old rats were divided into 6 male and 6 female groups, each consisting of 50 animals. Rats in groups 1–3 and 7–9 were respectively fed diets supplemented with 64%, 32% and 8% (basal diet) fish meal, and simultaneously given 0.12% sodium nitrite in their drinking water. Groups 4–6 and 10–12 were respectively given 64%, 32% and 8% fish meal and tap water. At the 104th week, all surviving animals were killed and examined histopathologically. Treatment with fish meal dose-dependently increased the incidences and multiplicities of atypical tubules, adenomas and renal cell carcinomas in sodium nitrite-treated males. Females were less susceptible than males for renal tumor induction. In males given the 64% fish meal diet alone, the incidence and multiplicity of atypical tubules were also significantly increased as compared with the 8% fish meal alone case. Nephropathy was apparent in fish meal-treated groups in a clear dose-dependent manner, irrespective of the sodium nitrite treatment, and was more prominent in males than in females. Dimethylnitrosamine was found in the stomach contents after 4-week treatment with 64% fish meal plus 0.12% sodium nitrite, at a level twice that in the 8% fish meal plus 0.12% sodium nitrite group. The results clearly indicate that concurrent administration of fish meal and sodium nitrite induces renal epithelial tumors. Further studies are required to elucidate how nephropathy and nitrosamines produced in stomach contents may contribute to the observed renal tumor induction.     

Antagonistic effect of diethylmaleate on the promotion of forestomach carcinogenesis by butylated hydroxyanisole (BHA) in rats pretreated withN-methyl-N'-nitro-N-nitrosoguanidine

The effects of diethyhaleate (DEM), previously demonstratedto inhibit butylated hydroxyanisole (BHA)-induced forestomachhyperplasia, on BHA promotion of forestomach carcinogenesisin rats pretreated with N-methyl-N'-nitro-N- nitrosoguanidine(MNNG) were examined. Groups of male 6-week-old F344 animalswere given a single i.g. administration of 150 mg/kg body weightMNNG and starting 1 week later administered powdered diet containing1% BHA plus 0.2% DEM, 1% BHA, 0.2% DEM or basal diet alone for51 weeks. Further groups of rats were treated with 1% BHA plus0.2% DEM, 1% BHA, 0.2% DEM or basal diet alone without MNNGpretreatment. Histopathological assessment of lesions at week52 revealed enhancement of MNNG-initiated papilloma (100 versus50%) and squamous cell carcinoma (100 versus 0%) developmentby BHA as compared to controls. Additional treatment with DEM,however, significantly reduced the relative incidences of carcinomain situ (0 versus 35.7%) and squamous cell carcinoma (35.7 versus100%), as well as BHA-induced forestomach hyperplasia with orwithout prior MNNG treatment. The results thus clearly demonstratethat DEM acts as a potent antagonist to BHA-promotion of ratforestomach carcinogenesis.     

Animal feeding study with nitrite-treated meat

In order to detect possible formation of carcinogenic N-nitroso compounds from nitrite and nitrosatable compounds in meat, studies were carried out with 70 male and 140 female F0 rats, divided into six groups, and 60, 100, 70, 60, 60 and 66 of their male and female offspring. One control group received casein and other groups chopped pork as the sole protein source (45%, mass/mass) on a fresh basis, either salted (sodium chloride) or not. For test groups, nitrite was also added to the meat before autoclaving and storing the diet and represented mass fractions of 200, 1 000 and 4 000 mg/kg, as sodium nitrite. The results do not demonstrate any effect on reproduction and no significant carcinogenic effect was revealed. However, an observed tendency toward an increased number of tumour-bearing rats in the highest dose group, plus the possible formation of carcinogenic N-nitroso compounds in nitrite-treated meat products, led to a recommendation to reduce the use of nitrite. Results from a concomitant study demonstrate that it is possible to produce many cured-meat products with the addition of only 50 mg/kg nitrite.     

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.     

Cytotoxic Effects of Betel Quid and Areca Nut Aqueous Extracts on Mouse Fibroblast,Human Mouth-Ordinary-Epithelium 1 and Human Oral Squamous Cell Carcinoma Cell Lines

Background: Betel quid chewing is more common among the older generation in rural areas of Malaysia. Oral cancer in Asia has been associated with the habit of chewing betel quid and areca nut. Objective:  This study aims to investigate the cytotoxic effects of betel quid and areca nut extracts on the fibroblast (L929), mouth-ordinary-epithelium 1 (MOE1) and oral squamous cell carcinoma (HSC-2) cell lines. Methods: L929, MOE1 and HSC-2 cells were treated with 0.1, 0.2 and 0.4 g/ml of betel quid and areca nut extracts for 24, 48 and 72 h. MTT assay was performed to assess the cell viability. Results: Both extracts, regardless of concentration, significantly reduced the cell viability of L929 compared with the control (P<0.05). Cell viability of MOE1 was significantly enhanced by all betel quid concentrations compared with the control (P<0.05). By contrast, 0.4 g/ml of areca nut extract significantly reduced the cell viability of MOE1 at 48 and 72 h of incubation. Cell viability of HSC-2 was significantly lowered by all areca nut extracts, but 0.4 g/ml of betel quid significantly increased the cell viability of HSC-2 (P<0.05). Conclusion: Areca nut extract is cytotoxic to L929 and HSC-2, whereas the lower concentrations of areca nut extract significantly increased the cell viability of MOE1 compared to the higher concentration and control group. Although betel quid extract is cytotoxic to L929, the same effect is not observed in MOE1 and HSC-2 cell lines. Further investigations are needed to clarify the mechanism of action.     

Long-term feeding study in C17 mice administered saccharin coated betel nut and 1,4-dinitrosopiperazine in combination

The observations on the effect of 3 agents - 1,4-dinitrosopiperazine,betel nut and saccharin fed to C17 mice in combination is presentedin this report. A total of 119 inbred mice of both sexes wereput on long-term feeding trials. Group I consisted of 34 micegiven a standard diet; group II of 32 mice fed an experimentaldiet containing saccharin coated betel nut powder at 10% concentration;group III of 29 mice given 0.2 ml aqueous solution of 0.1% 1,4-dinitrosopiperazineby intubation daily and group IV of 24 mice fed a combinationof the experimental diet together with intubation of 1,4-dinitrosopiperazine.Feeding was continued for 40 weeks at which time all mice weregiven a standard diet and water ad libitum, and then observedfor their full life-span. The commonest neoplasm found was squamouscell carcinoma of the forestomach in groups III and IV. Malemice were more susceptible to the treatment than female mice.In female mice reticular cell neoplasm - Type A of the uteruswas the commonest tumour and was more common in group III thanin group IV. The diet of saccharin coated betel nut failed topotentiate the carcinogenicity of 1,4-dinitrosopiperazine.     

Promoting and synergistic effects of chrysazin on 1,2-dimethylhydrazine-induced carcinogenesis in male ICR/CD-1 mice

The modifying effects of chrysazin on 1,2-dimethylhydrazine(DMH)-indueed colon and liver carcinogenesis were examined inmate ICR/CD-1 mice. Starting at 6 weeks of age, mice were dividedinto four groups, two of which were treated with s.c. injectionsof DMH (20 mg/kg body wt) once a week for 12 weeks. A week afterthe final injection of DMH, one group was kept on the basaldiet throughout the study (group I), and the other group wasfed the diet containing chrysazin (mixed in basal diet at 0.2%concentration) alone for 42 weeks (group II). The other twogroups were injected with normal saline and given the diet containing0.2% chrysazin for 42 weeks (group III), or the basal diet duringthe experiment (group TV). The incidence and multiplicity ofcolon tumors of group II were significantly greater than thoseof group I (P<0.05, P<0.01). The incidence and multiplicityof the hepatocellular neoplasms of group II were larger thanthose of group I (P< 0.002, P< 0.02 respectively). Ingroup III, colon tumors were not found, though a few liver neoplasmsand severe inflammatory lesions of the colon were observed.The activity of ornithine decarboxylase of the colonk mucosain mice exposed to chrysazin was stronger than that of animalswithout chrysazin. The results suggest that the promoting effectof chrysazin is probably related to an increase of cell proliferationin the target organ. A synergistic effect of DMH with chrysazinwas also observed in liver tumorigenesis.     

Liver and forestomach tumors and other forestomach lesions in rats treated with morpholine and sodium nitrite, with and without sodium ascorbate

Administration to rats of ascorbate with morpholine and nitrite was previously shown to inhibit the liver tumor production and to enhance the induction of forestomach tumors, as compared to treatment with morpholine and nitrite. In a repetition of this experiment, 10 g morpholine/kg in the diet and 2 g sodium nitrite/liter in the drinking water were administered for life to male MRC-Wistar rats without (group 1) or with (group 2) 22.7 g sodium ascorbate/kg in the diet. Group 3 was untreated. Group 2 showed a lower liver tumor incidence with a longer latency than group 1, indicating a 78% inhibition by ascorbate of in vivo N-nitrosomorpholine (NMOR) formation. The incidence of forestomach papillomas was 3% in group 1, 38% in group 2, and 8% in group 3. The difference between groups 1 and 2 was not significant due to the shorter life-span of group 1. Group 1 and especially group 2 had more forestomach hyperplasia and hyperkeratosis than group 3. Ascorbate might have enhanced induction of these lesions because of an action synergistic with that of NMOR. However, it is most likely that the lowered NMOR dose and concomitantly increased survival produced by the ascorbate were solely responsible for the increased incidence of forestomach papillomas and other lesions in group 2.     

Retinyl acetate inhibits estrogen-induced mammary carcinogenesis in female ACI rats

Two groups of female ACI rats were placed on powdered AIN-76diets containing retinyl acetate (412000 i.u. per kg diet) andtwo groups of rats were placed on placebo diets. Two weeks laterone group from each diet was subcutaneously implanted with a20 mg pellet containing 1 mg of 17-ethinylestradiol (EE2) mixedwith cholesterol, and the remaining groups received 20 mg cholesterolpellet implants. The four groups of animals were maintainedon their respective diet for 24 weeks after pellet implantation.The EE2-treated rats were hyperphagic and weighed less thanthe cholesterol-treated rats. Retinyl acetate had no effecton food consumption or body wt changes. None of the rats thatreceived pellets composed of cholesterol only exhibited mammarycarcinomas (MC) or pituitary tumors. All rats with an EE2 implanthad pituitary tumors: 88% of the rats on the placebo diet hadone or more MC; 70% of the rats on the retinyl acetate diethad one or more MC. The difference between the two EE2-treatedgroups for incidence of animals with at least one MC was notsignificant (x²). However, the EE2-treated rats on the placebodiet had approximately twice as many MC as the EE2-treated ratson the retinyl acetate diet. Thus, retinyl acetate inhibitedestrogen-induced mammary carcinogenesis in female ACI rats,without evidence of gross toxicity.     

Lack of inhibition by retinoids of bis(2-oxopropyl)nitrosamine-induced carcinogenesis in Syrian hamsters

Syrian hamsters were treated with either a low (10 mg/kg bodyweight) or high (40 mg/kg body weight) single dose of bis(2-oxopropyl)nitrosamine(BOP) and beginning 1 week later fed either low (0.2 mmol/kgdiet) or high (0.4–1.0 mmol/kg diet) levels of one offour retinoids [13 cis retinoic acid (13-cis-RA), N-ethylretinamide(ERA), N-(2-hydroxy- ethyl)retinamide (OHERA) or N-(phenyl)retinamide(PRA)] for periods of 40 or 50 weeks. The high retinoid levels(0.4–1.0 mmol/kg diet) fed following the highest BOP treatmentenhanced pancreatic carcinoma yields (average number/effectiveanimal) in males fed all four retinoids, and in females fedERA and 13-cis-RA. Enhanced adenoma yields were also seen inall groups when high retinoid levels were fed following 40 mgBOP/kg body weight. However, these retinoid levels caused anincreased adenoma yield in male hamsters only and did not modifycarcinoma yields when fed following 10 mg HOP/kg body weight.Similarly, tumor yields at extra-pancreatic sites were elevatedin refinoid-fed hamsters of both sexes after 40 mg BOP/kg bodyweight and in males fed ERA and 13-cis-RA after 10 mg BOP/kgbody weight when retinoids were given at the high levels (0.4–1.0mmol/kg diet). Increased incidences of bile duct and liver tumorsin particular were found in hamsters given 40 mg BOP/kg bodyweight. Consumption of retinoid levels of 0.4 mmol/kg diet andabove was also associated with a high incidence of liver cellnecrosis, ovarian cysts and ovarian hemorrhage. Retinoids (ERA,OHERA, and PRA) fed at the low level (0.2 mmol/kg diet) followingthe low BOP dose did not enhance carcinogenesis in the pan creasor at other sites and did not cause alterations in morphologicobservations.     

How Do Adolescents Assess and Rank the Risk of Areca Nut Use? Findings from a Study in Mumbai,India

Objective: Areca nut use, along with tobacco, is a contributor to India’s high rates of oral cancer. Areca nut use is culturally accepted, often initiated early in adolescence, and said to lead to later tobacco use. Unlike tobacco prevention, there are scarce prevention or harm-reduction programmes or campaigns specifically targeted at areca nut. Methods: A participative ranking method was used to understand adolescents’ assessment of risks of areca nut. Five focus group discussions were conducted with 31 adolescents, 19 fe-male and 12 male, non-users and users of chewing tobacco, water-pipe (hookah) and areca nut. Participants categorized and ranked the risk of 16 activities, including the use of areca nut and various tobacco-products, and discussed reasons for these risk-rankings. Results: Despite differences between groups on the assessment of risks associated with the 16 different activities, all the groups, user and non-user, rated cigarette smoking as having the highest risk, chewing fennel and using mouth fresheners as no risk, and areca nut as low risk. The other activities were ranked differently by each group. Adolescents’ perceptions of smoking or online games as risky was influenced by greater exposure to messaging on harmful consequences of the activity through multiple channels such as mass media, interpersonal networks including parents, and classroom health-education sessions. Inadequate knowledge about the harmful consequences of areca nut use, greater social and cultural acceptability, and the sweet taste of commercially packaged areca nut influenced low-risk perceptions. Conclusion: Perceptions of risk from an activity often determines preventive behaviors. Presently, adolescents do not perceive areca use as risky. In comparison to smoking they con-sider it less harmful. More research is required to better understand areca nut use and its cul-tural determinants. However, targeted health communication messages and prevention poli-cies and programmes have to be initiated to reduce areca nut use and associated burden of oral cancer.     

Hamsters chewing betel quid or areca nut directly show a decrease in body weight and survival rates with concomitant epithelial hyperplasia of cheek pouch

Betel quid (BQ) chewing is strongly associated with the occurrence of oral leukoplakia, oral submucous fibrosis, and oral cancer. There are about 200-600 million BQ chewers in the world. Previous animal studies support the potential carcinogenicity of BQ in different test systems. However, little animal experiment has let hamsters or rats to chew BQ directly, similar to that in humans. In the present study, we established a hamster model of chewing BQ or areca nut (AN). A total of 81 2-week-old hamsters were randomly divided into three groups: 25 for control group, 28 for BQ-chewing group, and 28 for AN-chewing group. These animals were fed with powdered diet with/without BQ or AN for 18 months. Although the consumption of BQ or AN showed some variations, hamsters fed with powdered diet could chew and grind AN or BQ into small pieces of coarse fibers during the entire experimental period. The survival rate of AN-chewing hamsters decreased significantly after 6 months of exposure. The mean survival time was 15.6 +/- 0.9 months for control animals, 13.6 +/- 0.98 months for AN-chewing animals, and 15.7 +/- 0.55 months for BQ-chewing animals. The body weight of BQ- or AN-chewing animals also decreased after 4-13 months. Hamsters fed with AN for 18 months showed hyperkeratosis in 80% and acanthosis in 50% of cheek pouches. Animals fed with BQ for 18 months also showed hyperkeratosis in 93% and acanthosis in 14% of cheek pouches. These results indicate that AN and BQ components may induce alterations in proliferation and differentiation of oral epithelial cells. Animal model of chewing BQ or AN can be useful for future tumor initiation, promotion and chemoprevention experiments simulating the condition of BQ chewing in humans.     

Evaluation of Successfulness of Capacity Building Programmes on Smokeless Tobacco and Areca Nut Cessation

Background: Prevalence of smoking in Sri Lanka has shown a gradual reduction whilst the use of smokeless tobacco and areca nut exhibits an increasing trend. At present, only a few well-structured smokeless tobacco (SLT)/areca nut (AN) cessation programs have been conducted in Sri Lanka, which is a gross underachievement as betel chewing-related oral squamous cell carcinoma is the most common cancer in Sri Lankan males. As General Dental Practitioners (GDP) do not contribute significantly to SLT/AN cessation activities at present, capacity building programs on SLT/AN control were carried out. The study evaluated the knowledge, attitude and practices  imparted on SLT/AN control among dental surgeons. Methods: Following a single day capacity building program on smokeless tobacco / areca nut control, two self-administered questionnaires were used to assess the improvement of knowledge and change of attitudes among 663 GDPs. Results: Majority had a good knowledge on harmful effects of SLT but not on areca nut. Knowledge of the current legislation on SLT control in Sri Lanka and carcinogenicity of areca nut was not satisfactory. Almost all agreed that proper counseling leads to patient quitting the habit, a formal training is necessary to conduct tobacco control activities and it should be a part of the regular treatment modalities. More than 80% of the participants support strict legislation. Most important factors leading to poor involvement in tobacco cessation activities were lack of expertise and inadequate educational material and not breach of patient privacy and lack of financial incentives. 20.1% dental surgeons had consumed smokeless tobacco / areca nut products in the past and only a few were current users of tobacco and/or areca nut. Conclusions: Well planned workshops are efficient in improving knowledge, practices and attitudes of dental surgeons towards SLT/AN cessation.     

Initiation of hepatocarcinogenesis by endogenously formed N-nitrosobis(2-hydroxypropyl)amine, N-nitrosodiethanolamine and N-nitroso-2,6-dimethylmorpholine in rats

Initiation activities of endogenously formed N-nitrosobis(2-hydroxypropyl)amine(NBHPA), N-nitrosodiethanolamine (NDELA) and N-nitroso-2,6-dimethylmorpholine(NDMM) were investigated in a modified short-term assay forrat hepatocarcinogenesis. Male Wistar rats were fed 1 % bis(2-hydroxypropyl)amine,0.5% diethanolamine or 0.25% 2,6-dimethylmorpholine in the dietplus 0.3% sodium nitrite in the drinking water. Two weeks afterstarting the experimental regimen they underwent 2/3 partialhepatectomy and were then maintained on the respective dietsfor a further week. Following a 2 week recovery period on basaldiet the rats were subjected to a resistant hepatocyte regimenconsisting of 0.02% 2-acetylaminofluorene in the diet for 2weeks and 1 mg carbon tetrachloride/kg body wt by gavage atthe midpoint. Initiation activity was assayed by measuring hepaticfoci positive for     

Lack of influence of low blood cholesterol levels on pancreatic carcinogenesis after initiation with N-nitrosobis(2-oxopropyl)amine in Syrian golden hamsters

The effects of a cholesterol-free diet, a cholesterol-free dietsupplemented with sesamin, and a diet supplemented with sesaminon pancreatic carcinogenesis of N-nitrosobis(2-oxopropyl) amine(BOP) were investigated in 140 female Syrian golden hamsters.BOP (70 and 20 mg/kg body wt) was injected s.c. twice at aninterval of 2 weeks at the beginning of the experiment. Starting3 weeks thereafter, the animals were maintained on basal diet,cholesterol-free diet, basal diet plus sesamin, or cholesterol-freediet plus sesamin for a further 15 weeks. All surviving hamsterswere killed at week 18, and the pancreatic tissues examinedhistologically. The incidences of pancreatic neoplastic andpreneoplastic lesions in each group did not show any statisticallysignificant variation. The cholesterol-free diet significantlydecreased the cholesterol contents of the serum, pancreas andliver, and sesamin supplement significantly decreased the cholesterolcontents of the serum and liver. Both the cholesterol-free dietand sesamin decreased the serum lipoperoxide levels. The resultsthus indicated that low cholesterol per se and sesamin exertno significant influence on BOP-initiated pancreatic carcinogenesisin hamsters, at least within the 4 month period after carcinogentreatment.     

p53 mutation is frequent and might not give a growth advantage in rat bladder carcinogenesis in vivo

Abnormalities of the p53 gene are frequently observed in humantumors, including urinary bladder carcinoma, suggesting thatp53 plays an important role in human carcinogenesis. However,its role in rat bladder carcinogenesis is unclear. In this study,we investigated the presence of p53 mutations in 122 urinarybladder tumors induced in F344 rats in the following carcinogenesismodels: (i) 0.2% N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide(FANFT; 6 weeks) in the diet followed by 3% or 5% sodium saccharinin the diet, 5% sodium ascorbate, 3.12% calcium saccharin (CaSac),1.34% sodium chloride (NaCl), 5.2% CaSac plus 1.34% NaCl, orbasal diet alone (72 weeks); and (ii) 0.2% FANFT, 0.05% N-(4-hydroxybutyl)nitrosaminein the drinking water, N-methyl-N-nitrosourea 20 mg/kg bodywt, i.p. twice per week, or basal diet alone (4 weeks), followedby 3% uracil in the diet (20 weeks). Polymerase chain reaction-single-strandconformation polymorphism analysis and direct sequencing wereperformed for exons 5–8 in the ratp53 gene. We found ninetumors (7.4%) with p53 mutations. Two tumors had two mutationsin the p53 gene. The tumors that had p53 mutations were relativelysmaller than those that did not have p53 mutations. There wereno mutation clusters among the treatments or hot-spots for p53mutations. These results indicate that p53 mutation is infrequentin bladder carcinogenesis in rats, and when it does occur, itdoes not appear to provide a growth advantage.     

Long-term carcinogenicity of pan masala in Swiss mice.

Carcinogenicity of pan masala, a dry powdered chewing mixture of areca nut, catechu, lime, spices and flavoring agents was evaluated by means of the long-term animal bio-assay 6- to 7-week old male and female S/RVCri mice were divided randomly into intermediate and lifetime exposure groups and fed normal diet without pan masala-(zero dose) or diet containing 2.5% and 5% pan masala. Animals in the intermediate-exposure group (n = 10/gender/dose group) were killed after 6, 12 or 18 months of treatment, while those in the lifetime-exposure group (n = 54/gender/dose group) were killed when moribund or at the termination of the experiment at 24 months. Several tissues were processed for histopathological examination. The body weight and survival rate of mice fed pan masala were lower than that of the controls. Histopathological observations of tissues from control animals did not reveal any neoplastic alterations. However, lifetime feeding of pan masala induced adenoma of the liver, stomach, prostate and sebaceous glands, also forestomach papilloma, liver hamartoma, hepatoma and hemangioma, carcinoma of the forestomach, adenocarcinoma of the lung and liver, and testicular lymphoma. Neoplastic lesions appeared mainly in the liver (n = 13), stomach (n = 3) and lung (n = 8). Lung adenocarcinoma, the most frequent malignant tumor type, was observed in 2/120 mice in the intermediate-exposure group and in 8/216 animals in the lifetime-exposure group. Statistical analysis of tumor-induction data revealed a significant dose-related increase in lung adenocarcinomas but not in liver and stomach neoplasms indicating that lung is the major target tissue for the carcinogenic action of pan masala.     

Para-methoxyphenol strongly stimulates cell proliferation in the rat forestomach but is not a promoter of rat forestomach carcinogenesis

The modifying effects of para-methoxyphenol (PMP) second stagetreatment on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-initiatedrat forestomach carcinogenesis were investigated. Groups of15 6 week old male F344 rats were given a single intragastricadministration of 150 mg/kg body wt MNNG and starting 1 weeklater were administered powdered diet containing 2.0, 1.0, 0.5,0.25 or 0% PMP until they were killed at week 52. PMP causedepithelial damage and hyperplasia in a dose-dependent mannerin the fore-stomach epithelium, but nevertheless was not associatedwith any increase in the incidences of either papillomas orsquamous cell carcinomas. The results thus clearly indicatedthat stimulation of cell proliferation does not necessarilycorrelate with promotion in the second stage of two-stage forestomachcarcinogenesis.     

Effects of dietary retinoic acid on skin papilloma and carcinoma formation in female SENCAR mice

Previously we have shown that dietary retinoids are essentialfor papilloma formation induced by either an initiation-promotionor a complete skin carcinogenesis protocol. The present studywas conducted to further determine the effect of dietary retinoicacid (RA) on papilloma formation and the conversion of papillomasto carcinomas. Skin tumors were induced in 3 week old femaleSENCAR mice by an initiation-promotion protocol with one applicationof 20 µg of 7,12-dimethylbenz[a]anthracene (DMBA), followedby 20 weekly applications of 2 µg of 12-O-tetradecanoylphorbol-13-acetate(TPA). Mice were fed RA at one of the three doses: 0.3 (nutritionallymarginal dose), 3 (near physiological) and 30 (pharmacological)µg/g of diet. Mice fed 30 µg of RA/g of diet hadthe same survival rate as the other two groups despite a lowerbody weight and all three groups had similar papilloma incidence,which reached 100% at age 18 weeks. Mice fed 3 µg of RA/gof diet had the highest papilloma yield (