New approaches to cancer chemoprevention with difluoromethylornithine and selenite.

New approaches to enhancing D,L-alpha-difluoromethylornithine (DFMO) inhibition of DMBA-induced mammary tumorigenesis were investigated. It is reasoned that perturbation of a second regulatory element in polyamine biosynthesis, i.e., the generation of propylamine groups from S-adenosylmethionine (AdoMet), would potentiate the effectiveness of DFMO. Precursor availability for AdoMet was limited when rats were fed a low methionine diet. The diet of control rats was supplemented with 0.3% methionine. DFMO, when added to the diet at 0.1%, suppressed tumorigenesis, regardless of methionine levels, although its efficacy was significantly enhanced by the low methionine diet. Selenite, another effective chemopreventive agent in this tumor model, also requires AdoMet for its metabolism. However, the anticarcinogenic action of selenite is not compromised by low dietary methionine. The differential sensitivity of DFMO and selenium chemoprevention to low dietary methionine, therefore, provides an opportunity to test for an additive effect. Results of the combination regimen study showed that the incidence and yield of DMBA-induced mammary tumors were significantly lower in the two-agent protocol compared to either DFMO or selenite alone. The mechanism(s) that accounts for the heightened efficacy of combined DFMO and selenite chemoprevention will be discussed.

Selenium inhibition of N-methyl-N-nitrosourea-induced mammary carcinogenesis in the rat.

The effect of supplemental dietary selenium on the postinitiation state of N-methyl-N-nitrosourea (MNU)-induced mammary carcinogenesis was investigated in noninbred female Sprague-Dawley rats. Mammary cancer was induced by a single iv injection of MNU. Supplemental selenium feeding was begun 7 days after carcinogen treatment. Feeding of selenium prolonged the latency of mammary cancer appearance and resulted in a reduction in the average number of cancers per rat. The results suggested an inhibitory effect of pharmacologic levels of dietary selenium on the postinitiation stage of mammary carcinogenesis.

Histogenesis and dose dependence of N-methyl-N-nitrosourea-induced carcinoma in a localized area of the hamster trachea.

The effect of the number of weekly intratracheal Instillations of N-methyl-N-nitrosourea (MNU) on induction of tracheal tumors was studied in male noninbred Syrian golden hamsters. The histogenesis of metaplastic and neoplastic lesions was also characterized. Treatment of hamsters once weekly for either 6, 8, 10, 12, or 14 weeks with a 0.5% solution of MNU resulted in the induction of a 0, 6, 11, 26, and 42% incidence of carcinoma, respectively, at 6 months after the first MNU treatment. Of the carcinomas induced, 87% were combined epidermoid and adenocarcinomas, whereas 13% were epidermoid carcinomas. In animals killed 1 week following either 1, 3, 6, 8, 10, 12, or 14 treatments, a continuum of metaplastic and neoplastic changes was observed that correlated well with the cancer incidence exhibited at the termination of the study. Mucous cells were found to be of prime importance in the development of the metaplastic and neoplastic tracheal lesions observed.

Carcinogenicity of N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea when applied to a localized area of the hamster trachea.

The carcinogenicity of N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU) was studied with the use of a hamster tracheal tumor model system. Hamsters received 15 or 10 once-weekly treatments of either a 0.5 or 0.25% solution of MNU and were killed 9 months after the first intratracheal instillation. Other hamsters received 15 once-weekly treatments of a 0.5, 0.25, or 0.125% solution of ENU and were killed at 6 months. Treatment with MNU resulted in a dose-dependent induction of tracheal carcinomas; 94% of the tumors induced were combined epidermoid and adenocarcinomas. Treatment of hamsters with a 0.5, 0.25, 0.125% solution of ENU induced an 83, 64, and 71% incidence of benign tracheal tumors, respectively (papillomas and polyps). No tracheal carcinomas were induced by ENU. The carcinogenicity of MNU and the reproducibility of tumor induction with the use of the localized tracheal washing, tumor model system were confirmed. Furthermore, the sensitivity of the localized tracheal washing technique for the detection of the tumorigenicity of compounds toward respiratory epithelium was demonstrated.

Diet, nutrition, and cancer: development of hypotheses and their evaluation in animal studies.

Research in diet, nutrition, and cancer is multidisciplinary. As a result there are many factors to consider in the conceptualization and design of experiments that may not be readily apparent to someone entering this field. In this article issues are identified that should be considered in the development of hypotheses about the effects of diet and nutrition on the carcinogenic process and the evaluation of these hypotheses using animal models. The questions considered are: what comprises an acceptable rationale for diet, nutrition, and cancer experiments in which animal models are used; what factors should be considered in the design of these experiments; and what constitutes a mechanistic component in such animal studies.

Effect of exercise intensity and duration on the induction of mammary carcinogenesis.

Physical activity is defined as bodily movement due to skeletal muscle contraction that results in quantifiable energy expenditure. Both epidemiological and laboratory data indicate that the level of physical activity in which an individual engages may affect cancer risk. Exercise is distinguished from other types of physical activity by virtue of the fact that the intensity, duration, and frequency of the activity(s) is specifically designed to improve physical fitness. Based on available data, a role for exercise in specifically reducing cancer risk has been conjectured and is referred to as the exercise-cancer hypothesis. However, the amount of scientific evidence in support of this hypothesis is still quite limited, and there are conflicting reports about the nature of the association. The exercise-cancer hypothesis was evaluated in two experimental animal models for breast cancer using treadmill running as the exercise paradigm. The data presented indicate that both the intensity and duration of exercise affect the development of experimentally induced breast cancer. In general, as exercise intensity increased, the likelihood that such physical activity inhibited carcinogenesis increased. Exercise at lower intensities resulted in either inhibition, no effect, or enhancement of the tumorigenic response depending on the duration of exercise. Inhibitory conditions of exercise affected both the initiation and promotion/progression stages of the disease process.

Dose-responsive induction of mammary gland carcinomas by the intraperitoneal injection of 1-methyl-1-nitrosourea.

Dose-response relationships for the induction of mammary tumors by a single i.p. injection of 1-methyl-1-nitrosourea (MNU) were studied. Groups of 30 female Sprague-Dawley rats were given i.p. injections of 50, 37.5, 25, 12.5, or 0 mg MNU/kg body weight at 50 days of age. Animals were palpated for tumor detection twice weekly throughout a 28-week observation period. Administration of MNU i.p. caused no acute toxicity. Both benign and malignant mammary tumors were induced by MNU, but malignant tumors appeared earlier and at a faster rate than benign tumors. The incidence and numbers of mammary carcinomas increased whereas median cancer-free time decreased with increasing dose of MNU. Approximately twice as many mammary cancers were observed in the cervical-thoracic as in the abdominal-inguinal mammary gland chains irrespective of carcinogen dose, while the frequency of tumor occurrence in the left versus right chains was similar. Tumor latency decreased with increasing dose of MNU, but the quartiles for time to detection of all tumors within each carcinogen dose group were similar irrespective of anatomical region in which the tumors occurred. The mammary tumor response attained via i.p. injection was similar but the coefficient of variation for tumor multiplicity within a carcinogen dose group was lower in comparison to that observed when MNU was administered i.v. or s.c. Among these techniques for carcinogen injection, the i.p. route is the most rapid method and offers an added advantage of ease of administration with quantitative, reproducible delivery of the desired amount of carcinogen and a decrease in variability of tumor response among animals within a treatment group. This method is well suited for the technically less experienced investigator and for those in need for a rapid method of injecting MNU to large numbers of animals.

Induction of mammary gland carcinomas by the subcutaneous injection of 1-methyl-1-nitrosourea.

The efficacy of s.c. administration of 1-methyl-1-nitrosourea (MNU) for the induction of mammary carcinomas was compared with the i.v. method of carcinogen injection in female Sprague-Dawley rats. Group-housed animals fed a laboratory chow diet and distilled water ad libitum throughout the study were injected at 50 days of age with 50 mg MNU per kg body weight. The carcinogen was given either s.c. or i.v., via the jugular vein, to one of the two groups of 20 rats each. Animals were palpated for tumor detection weekly and necropsied 180 days after injection with the carcinogen. At the termination of the study, 180 days postcarcinogen, cancer incidences were similar, 95 versus 90% in animals given MNU either s.c. or i.v. with an average of 3.9 and 3.9 cancers per rat, respectively. Time of tumor appearances were essentially identical under both treatment conditions. Using either method of carcinogen administration resulted in the induction of approximately 2.4 times more carcinomas in the cervical-thoracic mammary glands than in the abdominal-inguinal glands with no differences observed in cancer occurrence in the left versus the right mammary gland chains. The data indicate that s.c. administration of MNU is as effective and specific in the induction of mammary carcinomas as is i.v. administration. The s.c. method has the advantage of being easier and faster to perform and permits reproducible treatment of large numbers of rats by a small technical staff.

Effect of an inorganic and organic form of dietary selenium on the promotional stage of mammary carcinogenesis in the rat.

The relative effectiveness of either sodium selenite or selenomethionine in the inhibition of mammary carcinogenesis was studied in virgin female Sprague-Dawley rats. In one experiment, rats were given 50 mg of 1-methyl-1-nitrosourea per kg of body weight s.c. at 50 days of age. Beginning 7 days post-1-methyl-1-nitrosourea, they were assigned to a basal diet containing 0.1 ppm of selenium or basal diet supplemented to contain either 4, 5, or 6 ppm of selenium as sodium selenite or 5 or 6 ppm of selenium as selenomethionine. Selenium treatment was continued until termination of the study 135 days after 1-methyl-1-nitrosourea treatment. Sodium selenite, at the 5-ppm level, was the most effective chemopreventive agent. The highest level of selenomethionine (6 ppm) caused grossly apparent liver damage. No liver damage was noted in sodium selenite-treated rats. In a second experiment, rats were given 5 mg of 7,12-dimethylbenz(a)anthracene at 50 days of age. Beginning 7 days after 7,12-dimethylbenz(a)anthracene treatment, rats were assigned randomly to the control group or to one of two selenium treatment groups receiving either 3.4 ppm of selenium as sodium selenite or 3.4 ppm as selenomethionine in their drinking water. Selenium supplementation was continued throughout the study until its termination at 111 days postcarcinogen . Sodium selenite significantly reduced cancer incidence and the average number of cancers per rat. Treatment with selenomethionine was less effective and caused severe liver damage. Although both sodium selenite and selenomethionine can inhibit some aspect of the postinitiation stage(s) of mammary carcinogenesis, selenium provided as sodium selenite was the more effective and less toxic of the two chemicals. Increasing the dose of sodium selenite above 5 ppm did not enhance the inhibitory activity of selenium.

Effect of combined selenium and retinyl acetate treatment on mammary carcinogenesis.

The influence of feeding a pharmacological level of either selenium (SE), retinyl acetate (RA), or selenium and retinyl acetate (SE + RA) on N-methyl-N-nitrosourea-induced mammary carcinogenesis was studied in female Sprague Dawley rats. Rats received 50 mg N-methyl-N-nitrosourea per kg body weight at 50 days of age. Seven days after carcinogen treatment, groups of 25 rats each were placed on laboratory chow diet supplemented with either a placebo or 300 mg RA, 4 mg SE, or 300 mg RA plus 4 mg SE per kg diet. Animals were palpated for detection of mammary tumors twice each week, and the study was terminated 130 days after N-methyl-N-nitrosourea was given. In comparison to the placebo group, treatment with RA or RA + SE reduced tumor incidence, lessened the average number of tumors per rat, and prolonged tumor latency. RA + SE had the greatest inhibitory effect on the carcinogenic process. The effect of combined treatment with RA and SE was additive in nature. The length of the estrous cycle was increased by treatment with RA + SE, and some pathological changes of the ovary and uterus were noted. This investigation provides the first evidence of an additive inhibitory effect resulting from combined treatment with RA and SE.

Conjugated linoleic acid suppresses mammary carcinogenesis and proliferative activity of the mammary gland in the rat.

Conjugated linoleic acid (CLA) is a collective term which refers to a mixture of positional and geometric isomers of linoleic acid. It is naturally occurring in meat and dairy products. We have previously reported (Ip, C., Chin, S. F., Scimeca, J. A., and Pariza, M. W. Cancer Res., 51: 6118-6124, 1991) that 1% CLA in the diet suppressed mammary carcinogenesis in rats given a high dose (10 mg) of 7,12-dimethylbenz(a)anthracene. In the present study, dietary CLA between 0.05 and 0.5% was found to produce a dose-dependent inhibition in mammary tumor yield when fed chronically to rats treated with a lower dose (5 mg) of 7,12-dimethylbenz(a)anthracene. Short-term CLA feeding for 5 weeks, from weaning to the time of carcinogen administration at 50 days of age, also offered significant protection against subsequent tumor occurrence. This period corresponds to maturation of the mammary gland to the adult stage in the rat. The inhibitory response to short-term CLA exposure was observed with the use of 2 different carcinogens: 7,12-dimethylbenz(a)anthracene and methylnitrosourea. The fact that CLA was protective in the methylnitrosourea model suggests that it may have a direct modulating effect on susceptibility of the target organ to neoplastic transformation. The proliferative activity of the mammary epithelium was assessed by the incorporation of bromodeoxyuridine. Immunohistochemical staining results showed that CLA reduced the labeling index of the lobuloalveolar compartment, but not that of the ductal compartment of the mammary tree. Since the lobuloalveolar structures are derived from the terminal end buds which are the sites of carcinogenic transformation, the above finding is consistent with the bioassay data of tumor inhibition. Thus, changes in gland development and morphogenesis may be a locus of action of CLA in modulating mammary carcinogenesis. CLA is a unique anticarcinogen because it is present in foods from animal sources. Furthermore, its efficacy in cancer protection is manifest at dietary concentrations which are close to the levels consumed by humans.

Effect of type and amount of dietary fat on the enhancement of rat mammary tumorigenesis by exercise.

The effect(s) of treadmill exercise and type and amount of dietary fat on the process of mammary tumorigenesis was investigated. Female Sprague-Dawley rats were fed a purified 5% fat diet (AIN-76A) from 21 to 64 days of age. At 50 days of age each rat was intubated p.o. with 5 mg 7,12-dimethylbenz(a)anthracene (DMBA). Fourteen days after DMBA, the rats were randomized into one of three diet groups: 5% fat as corn oil, 24.6% fat as corn oil, or 24.6% fat as a mixture of palm (21.8%) and corn oil (2.8%). The combination of palm and corn oil provided the same amount of linoleic acid per g as the 5% corn oil diet. Half the animals receiving each diet were exercised on a treadmill at a speed of 20 m/min, 1 degree incline, 15 min/day, 5 days/week, and were designated as the moderate intensity treadmill exercise group (MITE). The remaining animals were exercised at a speed of 2 m/min, 1 degree incline, 15 min/day, 5 days/week, and were designated as the low intensity treadmill exercise group (LITE). The experiment was terminated 154 days after DMBA was administered. The median tumor-free time was significantly shortened in MITE rats receiving the 24.6% fat, corn oil-formulated diet in comparison to LITE rats receiving the same diet (43 day vs. 62 day, P = 0.028). Similarly, tumor appearance was more rapid in MITE rats consuming the low fat corn oil diet in comparison to the low fat diet-fed LITE group (57 day vs. 67 day, P = 0.046). Exercise exerted no effect on the rate of tumor appearance in rats that received the 24.6% palm and corn oil mixture, (58 day, MITE, vs. 62 day, LITE, P = 0.502). Mean body weight gains were similar among groups, although MITE rats consistently weighed more than LITE rats consuming the same diet. Gross carcass composition was unaffected by either the level of exercise or the amount of dietary fat consumed. The data indicate that moderate intensity treadmill exercise for a short duration, that is without effect on carcass fat content, can stimulate mammary tumorigenesis in rats fed low or high fat diets. This effect can be influenced by the type of dietary fat ingested.

Effect of short-term feeding of sodium selenite on 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis in the rat.

The inhibitory activity of short-term feeding of one of four concentrations of dietary selenium against the induction of mammary gland carcinomas by 7,12-dimethylbenz(a)anthracene (DMBA) was studied in female Sprague-Dawley rats. When 28 days old, the animals were placed on a Torula yeast diet formulation which contained, by analysis, either 0.05, 0.15, 1.05, or 2.06 microgram of selenium, as sodium selenite, per g of diet. Mammary cancer was induced by a single p.o. administration of either 7.5 or 15.0 mg DMBA at 50 days of age. The animals were maintained on the above diets until 14 days after carcinogen treatment at which time all animals were transferred to a chow diet containing 0.21 microgram of selenium per g of diet. The study was terminated 120 days after DMBA administration. The concentrations of selenium in the liver and mammary tissue measured at the time of DMBA treatment increased with increasing levels of dietary selenium (p less than 0.05). At the low dose of DMBA, there was a trend towards reduction in the number of cancers with increased amounts of selenium, but the only significant difference occurred between groups fed the next to lowest and the highest level of selenium. At the high dose of DMBA, the number of observed cancers showed a strong dose effect (p less than 0.05). In addition, tumor load was significantly reduced in selenium-supplemented rats (p less than 0.05), and there was a significant delay (p less than 0.05) in the time to appearance of the cancers of animals receiving the highest level of selenium when compared with those receiving the lowest level. The dietary concentrations of selenium shown to inhibit the early stage(s) of cancer induction in this system were both significantly lower and fed for a shorter time interval than that which was previously reported.

In vitro and in vivo studies of methylseleninic acid: evidence that a monomethylated selenium metabolite is critical for cancer chemoprevention.

Previous research suggested that the beta-lyase-mediated production of a monomethylated selenium metabolite from Se-methylselenocysteine is a key step in cancer chemoprevention by this agent. In an attempt to affirm the concept, the present study was designed to evaluate the activity of methylseleninic acid, a compound that represents a simplified version of Se-methylselenocysteine without the amino acid moiety, thereby obviating the need for beta-lyase action. The in vitro experiments showed that methylseleninic acid was more potent than Se-methylselenocysteine in inhibiting cell accumulation and inducing apoptosis in TM12 (wild-type p53) and TM2H (nonfunctional p53) mouse mammary hyperplastic epithelial cells, and these effects were not attributable to DNA damage, as determined by the comet assay. In general, methylseleninic acid produced a more robust response at one-tenth the concentration of Se-methylselenocysteine. It is possible that these cell lines may have only a modest ability to generate a monomethylated selenium species from Se-methylselenocysteine via the beta-lyase enzyme. In contrast, methylseleninic acid already serves as a preformed active monomethylated metabolite, and this could be an underlying reason why methylseleninic acid acts more rapidly and exerts a more powerful effect than Se-methylselenocysteine in vitro. Interestingly, the distinction between these two compounds disappeared in vivo, where their cancer chemopreventive efficacies were found to be very similar to each other [in both methylnitrosourea and dimethylbenz(a)anthracene rat mammary tumor models]. The beta-lyase enzyme is present in many tissues; thus, animals have an ample capacity to metabolize Se-methylselenocysteine systemically. Therefore, Se-methylselenocysteine would be expected to behave like methylseleninic acid if beta-lyase is no longer a limiting factor. Taken together, the present in vitro and in vivo results provide strong evidence in support of our earlier hypothesis that a monomethylated selenium metabolite is important for cancer chemoprevention. Methylseleninic acid could be an excellent tool, especially for molecular mechanism studies in cell culture, and some of these attributes are discussed.

Effect of the duration of retinyl acetate feeding on inhibition of 1-methyl-1-nitrosourea-induced mammary carcinogenesis in the rat.

The effect of the duration of retinoid treatment on the inhibition of 1-methyl-1-nitrosourea-induced mammary carcinogenesis was studied. Female Sprague-Dawley rats were given i.v. injections of 50 mg 1-methyl-1-nitrosourea per kg body weight at both 50 and 57 days of age. Feeding of a placebo diet or diet supplemented with 323 mg retinyl acetate per kg diet (retinoid treatment) was initiated at 10 days after the first carcinogen injection. Retinoid treatment was either continued or discontinued after 60 days postcarcinogen, and the study was terminated at 182 days postcarcinogen. Retinoid treatment between 10 and 60 days postcarcinogen prolonged the cancer latency and reduced the average number of cancers per rat in comparison to that in placebo-treated rats. Continuation or cessation of retinoid treatment in 60-day tumor-bearing rats had no effect on the time of appearance of additional cancers. In 60-day tumor-free rats, continuation of retinoid treatment prolonged cancer latency in comparison to either 60-day tumor-free rats changed to placebo or rats continuously treated with placebo. The cessation of retinoid treatment in 60-day tumor-free rats resulted in a rapid increase in the appearance of cancers; at the termination of the study, the average number of cancers per rat was similar to that of animals fed only the placebo. The data indicated that some rats are more responsive to the retinoid than are others. Retinoid treatment apparently prevented the progression of early neoplastic lesions, and a continuous daily intake of the retinoid appears necessary to sustain the chemopreventive effect under the experimental conditions imposed.

Effect of retinyl acetate on the occurrence of ovarian hormone-responsive and -nonresponsive mammary cancers in the rat.

The inhibitory activity of retinyl acetate against the induction of ovarian hormone-responsive and -nonresponsive mammary gland adenocarcinomas was studied in intact and castrated female Sprague-Dawley rats. Three experiments were conducted. Mammary cancer was induced by a single p.o. administration of 7,12-dimethylbenz(a)anthracene (DMBA) at 50 days of age. Animals in Experiments 1 and 2 each received 20 mg DMBA, whereas those in Experiment 3 received 15 mg. In all experiments, animals were fed a chow diet supplemented per kg with either a placebo or 328 mg retinyl acetate starting 7 days after carcinogen treatment. In Experiment 1, rats were castrated at either 7, 60, or 90 days postcarcinogen and were killed 120 days after DMBA was given. In Experiment 2, rats were castrated 30 days after DMBA and were killed 240 days after carcinogen treatment. In Experiment 3, rats were castrated when a detected tumor attained a measurable diameter, and the hormone responsiveness of their tumors was subsequently determined. The experiment was terminated 279 days after DMBA treatment. In both intact and castrated rats, mammary tumor occurrence was inhibited by treatment with retinyl acetate. However, there were no differences in the latency to appearance time of hormone-responsive and -nonresponsive cancers in intact animals receiving either placebo or retinyl acetate. The data indicate that retinyl acetate inhibits DMBA-induced mammary tumorigenesis in either the presence or the absence of the ovaries. It appears that retinyl acetate is effective in inhibiting both ovarian hormone-responsive and -nonresponsive mammary tumors.

Effect of exercise on the induction of mammary carcinogenesis.

Although data reported in several epidemiological investigations indicate that reduced consumption of dietary fat and increased levels of physical activity are associated with reduced risk for breast cancer, the results of some studies do not support these observations. Underlying this situation is the unanswered question about whether degree of body fatness, which is affected by dietary composition, total caloric intake, and energy expenditure, is the critical determinant affecting breast cancer risk. The objective of this work was to establish whether increasing energy expenditure by exercise would reduce the occurrence of mammary carcinomas induced by 7,12-dimethylbenz[a]anthracene (DMBA) in animals consuming a high fat diet to the level of occurrence observed in sedentary animals consuming a low fat diet. Female Sprague-Dawley rats were obtained at 21 days of age and maintained on a 5% (w/w) corn oil diet (AIN-76A) until they were 64 days of age. At 50 days of age, rats received either 5 mg DMBA or the solvent in which the carcinogen was dissolved. Fourteen days after DMBA intubation they were randomized into one of three groups: 5% fat (w/w), sedentary; 24.6% fat (w/w), sedentary; or 24.6% fat (w/w), exercised. Animals were exercised on a motor-driven treadmill at a belt speed of 20 m/min and a 1-degree incline for 15 min/day, 5 days/week for 18 weeks. Feeding a high fat versus a low fat diet increased the number of breast cancers induced and the rate at which they appeared in agreement with previous investigations. However, rather than retarding the development of tumors as was hypothesized, moderate treadmill exercise increased the incidence and number of cancers induced and shortened cancer latency in comparison to animals that received either the high fat or low fat diet and were sedentary. Body composition was not altered by the exercise regime imposed, although these animals weighed more than either sedentary group. These data document a heretofore unreported effect of a moderate level of aerobic work on breast cancer induction.

Effect of delay in administration of 13-cis-retinoic acid on the inhibition of urinary bladder carcinogenesis in the rat.

The effect of a delay in starting 13-cis-retinoic acid treatment on the inhibition of urinary bladder carcinoma induced by N-butyl-N-(4-hydroxybutyl)nitrosamine was studied in male Fischer 344 rats. Animals received a total p.o. dose of either 1200, 1800 or 2400 mg N-butyl-N-(4-hydroxybutyl)nitrosamine over a period of six weeks. At either one, five, and nine weeks after the last N-butyl-N-(4-hydroxybutyl)nitrosamine intubation, animals were started on a diet supplemented with 13-cis-retinoic acid (240 mg/kg of laboratory chow) or continued on laboratory chow. Animals were killed at one year after the first carcinogen intubation for histological evaluation of the bladder. Feeding of 13-cis-retinoic acid reduced the incidence, average number, and severity of transitional cell carcinomas as well as hyperplasia and cellular atypia. Furthermore, even a nine-week delay in starting the retinoid feeding did not diminish the ability of 13-cis-retinoic acid to inhibit bladder carcinogenesis.