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.

The respiratory epithelium. II. Hamster trachea, bronchus, and bronchioles.

The normal female hamster respiratory epithelium at five airway levels was characterized with the use of coordinated morphologic and histochemical techniques. Five morphologic cell types were recognized in the trachea, stem bronchi, and primary bronchl: basal cells and neurosecretory cells that were basally located and did not reach the lumen and mucous cells [mucous goblet cells and small mucous granule cells (SMGC)], indifferent cells showing mucous-ciliary differentiation, and ciliated cells that reached the lumen. Two epithelial cell types were observed in the bronchioles, ciliated cells and nonciliated Clara cells, both of which reached the lumen. Mucous cells presented as either SMGC with a few small periodic acid-Schiff-positive granules (diastase-resistant neutral mucosubstances) or as goblet cells, filled with the same material. Mucous cells were columnar, and the cytoplasm was electron-dense and contained a well-developed endoplasmic reticulum and Golgi complex. The microvilli of the mucous cells were coated more thickly with colloidal iron than either the cilia or microvilli of ciliated cells. Approximately one-half the cells in the trachea, bronchi, and bronchioles were ciliated. Ciliated cells containing intracellular ciliated cysts with normal cilia projecting into a closed space or ciliated cells bearing compound cilia were observed infrequently. Neurosecretory cells were rarely observed. These cells contained characteristic dense-core granules.

The respiratory epithelium. VI. Histogenesis of lung tumors induced by benzo[a]pyrene-ferric oxide in the hamster.

Lung tumors were induced in female Syrian golden hamsters by intratracheal instillation of benzo[a]pyrene-Fe2O3. The tumors were characterized with the use of coordinated morphologic and histochemical techniques including electron microscopy. The lung carcinomas were classified according to their presumed cell of origin. Most were derived from mucous cells and/or basal cells, and they were classified as either epidermoid carcinomas or as combined epidermoid and adenocarcinomas. The tumors in the second group (57% of the total number of carcinomas) presented a wide spectrum of epidermoid and adeno components. The epidermoid component was characterized in well-differentiated tumors by the presence of intercellular bridges and/or keratinization. Well-developed desmosomes and numerous bundles of tonofilaments were observed ultrastructurally. In diagnosing adenocarcinoma, one no longer needs to depend on the presence of tubules or gross glandular structures as criteria for diagnosis. The presence of intracellular and/or extracellular alveoli, well-developed Golgi complex, and endoplasmic reticulum and/or evidence of mucous secretion provide more definitive criteria. A tumor composed of neurosecretory cells that morphologically resembled a bronchial carcinoid of man was observed. Nests of uniform, small, polygonal cells with round-to-oval nuclei were seen at the light microscopic level. Dense-core secretory granules 1,100-2,200 A were present in the cytoplasm of the tumor cells. Several fibrosarcomas were observed. The tumors showed a very cellular structure, composed of either densely packed ovoid or spindle-shaped cells. Ultrastructurally, the cells resembled fibroblasts. The results obtained in this study give strong support for a histogenetic classification, i.e., a classification based on the cell of origin.

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.

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.

The respiratory epithelium. VII. Epidermoid metaplasia of hamster tracheal epithelium during regeneration following mechanical injury.

Regeneration was studied in female Syrian golden hamster tracheal epithelium. The epithelium was focally removed in vivo by scraping it with a blunt probe. At 2 hours, virtually all cells had sloughed from the injured area leaving a bare basal lamina. At 6 and 12 hours, flattened cells that migrated from adjacent uninjured epithelium partially covered the denuded basal lamina. Increased cell division did not occur at these times. Many of the simple squamous cells contained well-developed endoplasmic reticulum, Golgi apparatus, and mucous granules. Other cells resembled basal cells. At 24 hours the defect was covered by one or two layers of simple squamous cells. At that time, many of those cells were in division, and cell division was also greatly increased in mucous cells and basal cells in the uninjured epithelium distant from the defect. At 48 hours the epithelium was stratified, composed of four or five layers of polygonal to flattened cells, typical of nonkeratinizing epidermoid metaplasia. The cells contained many tonofilament bundles, a large Golgi apparatus, and many tiny mucous granules. Mitoses were seen in all cell layers. At 72 hours, the surface layer of cells was undifferentiated (indifferent cells) overlying an epithelium that otherwise retained its epidermoid character. Indifferent cells were characterized by an electron-lucent cytoplasm and a lack of tonofilament bundles, mucous granules, or cilla. Cells similar in other respects to indifferent cells were seen that possessed mucous granules or early signs of cilla formation. Some cells showed mucous granules and cilla developing in the same cell. By 96 hours, the regenerated epithelium was fully differentiated and was indistinguishable from the normal epithelium. These observations show that mucous cells have a significant role in the regenerative response. Mucous cells have a dual potential; they can undergo epidermoid metaplasia and still retain the ability to secrete mucus. The study explains the universal occurrence of mucosubstances in areas of epidermoid metaplasia and makes more understandable the previously reported fact that many bronchogenic carcinomas are combined epidermoid and adenocarcinomas. In the presence of a carcinogen, the hypothesis has been forwarded that initiation of mucous cells and basal cells occurs, which leads to malignant transformation and produces tumors that show active secretory activity and keratinization, often in the same cell.

The respiratory epithelium. III. Histogenesis of epidermoid metaplasia and carcinoma in situ in the human.

The histogenesis of epidermoid metaplasia and carcinoma in situ was analyzed in human bronchial epithelium. The conclusion is that epidermoid metaplasia and carcinoma in situ can result from conversion of mucous cells. This implies the direct transformation of one type of fully differentiated cell to another. The study therefore emphasizes the differentiation potentialities of the mucous cells that can divide and undergo goblet cell hyperplasia and epidermoid metaplasia. Epidermoid metaplasia is a common reaction to injury in the bronchus. In our series of cases it was especially frequent in patients without neoplastic disease who had undergone intratracheal intubation or tracheostomy and who had been maintained on a respirator in the Shock Trauma Unit, University of Maryland. Future studies will be required to distinguish the difference, if any, between epidermoid metaplasia destined to become malignant carcinoma and that which is not. One difference noted in this study was the absence of overt cornification in epidermoid metaplasia in patients without neoplastic disease.

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.

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 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.

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 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.

N-butyl-N-(4-hydroxybutyl)nitrosamine-induced urinary bladder cancer in C57BL/6 X DBA/2 F1 mice as a useful model for study of chemoprevention of cancer with retinoids.

Highly invasive carcinomas of the urinary bladder were induced in male C57BL/6 X DBA/2 F1 (hereafter called B6D2F1) mice by gastric intubation of N-butyl-(4-hydroxybutyl)nitrosamine (OH-BBN) using a quantitative dosing schedule. Animals received either 5 or 10 mg OH-BBN per intubation, two times each week, for 9 weeks for a total dose of either 90 or 180 mg, and they were killed 6 months after the first carcinogen intubation. Seven days after the final intubation of OH-BBN, animals were fed either a placebo diet or diet supplemented with either 150 or 200 mg 13-cis-retinoic acid per kg of diet. A 41 and 43% incidence of urinary bladder cancer was observed in mice given the low and high dose of carcinogen, respectively, and fed a placebo diet. Sixty-seven % of the carcinomas induced in these animals invaded either into or through the urinary bladder wall. Varying degrees of transitional and either squamous or glandular or both squamous and glandular differentiation were observed in the carcinomas. Feeding of diet supplemented with 13-cis-retinoic acid reduced cancer incidence; the degree of reduction was proportional to the dose of retinoid administered. The highly invasive nature of the carcinomas induced by quantitative administration of OH-BBN in B6D2F1, mice provides a useful animal model of the highly invasive variant of human transitional cell urinary bladder cancer in which to study chemoprevention by retinoids as well as other compounds.

Inhibition of urinary bladder cancer by N-(ethyl)-all-trans-retinamide and N-(2-hydroxyethyl)-all-trans-retinamide in rats and mice.

The chemopreventive activity of two synthetic retinamides of relatively low toxicity against N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN)-induced urinary bladder cancer was studied in F344 rats and C57BL/6 X DBA/2 F1 mice. Female and male rats were given a total dose of either 1800 or 3200 mg OH-BBN over a period of 6 or 8 weeks, respectively. Male mice were given a total dose of either 90 or 180 mg OH-BBN over a period of 9 weeks. Seven days after the final intubation of a period of 9 weeks. Seven days after the final intubation of OH-BBN, animals were fed either a placebo diet or a diet supplemented with the following retinoids: for rats, 0.8 mmol 13-cis-retinoic acid, 2 mmol N-(ethyl)-all-trans-retinamide, or 2 mmol N-(2-hydroxyethyl)-all-trans-retinamide per kg diet; and for mice, either 0.5 or 1.0 mmol of N-(ethyl)-all-trans-retinamide or N-(2-hydroxyethyl)-all-trans-retinamide per kg diet. Animals were killed 6 months after the initial gastric intubation. In comparison to male and female rats fed placebo diets, all three retinoids reduced the incidence, number, and severity of the low-grade papillary transitional cell carcinomas of the urinary bladder. Similarly, treatment of mice with either of the two retinamides reduced the incidence of highly invasive urinary bladder carcinomas. The chemopreventive effect of the less toxic retinamides was equal to or greater than that of 13-cis-retinoic acid.

Inhibitory effect of 13-cis-retinoic acid on urinary bladder carcinogenesis induced in C57BL/6 mice by N-butyl-N-(4-hydroxybutyl)-nitrosamine.

The effect of 13-cis-retinoic acid on the induction of urinary bladder carcinoma by N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) was studied in male C57BL/6 mice. Animals received a total dose of either 90 or 140 mg of OH-BBN via gastric intubations of 7.5 or 10.0 mg of OH-BBN 2 times each week for 6 or 7 weeks, respectively. Seven days after the last OH-BBN intubation, animals were fed laboratory chow diet supplemented with either 200 mg of 13-cis-retinoic acid per kg or its placebo. Animals were killed at 6 months after the first carcinogen intubation. Highly invasive squamous and transitional cell carcinomas of the urothelium were found at autopsy. In the majority of these carcinomas, invasion of the bladder muscle wall by tumor cells had occurred. At the two dose levels of OH-BBN, feeding of 13-cis-retinoic acid reduced the incidence of both carcinomas and noninvasive papillomas, as well as the extent of neoplastic development in the urinary bladder. In mice receiving the lower dose of OH-BBN, the feeding of 13-cis-retinoic acid prevented the appearance of both squamous and transitional cell carcinomas with a reduction in incidence from 33 to 0% (p less than 0.01). The results of this study indicate that 13-cis-retinoic acid reduced not only the severity of highly invasive urinary bladder carcinomas but also the incidence of such cancers.

N-(4-Hydroxyphenyl)retinamide, a new retinoid for prevention of breast cancer in the rat.

The synethesis of a new retinoid, N-(4-hydroxyphenyl)-all-trans-retinamide, which has useful biological properties, is described. This retinoid was more potent than retinyl acetate in reversing keratinization caused by retinoid deficiency in tracheal organ culture. It was markedly less toxic than retinyl acetate when fed p.o. to rats over 2-week or 6-month periods. It was an effective agent for inhibition of the development of breast cancer induced in rats by N-nitroso-N-methylurea, although it was not as potent as retinyl acetate in this regard. However, the lesser toxicity of 4-hydroxyphenylretinamide makes it a superior agent for prevention of breast cancer. High-pressure liquid chromatographic analyses of liver and breast extracts from rats treated for 6 months with retinoids show the pharmacokinetic basis for the superiority of 4-hydroxyphenylretinamide; this retinoid and its metabolites were found in high concentrations in breast tissue, without any measurable accumulation in the liver or evident liver toxicity. In contrast, chronic feeding of retinyl acetate caused marked deposition of retinyl esters in the liver and severe hepatotoxicity. Whole mounts of rat mammary glands, made after chronic feeding of 4-hydroxyphenylretinamide, showed that it had a marked antiproliferative effect on mammary epithelium.

Effect of graded dietary levels of selenium on tracheal carcinomas induced by 1-methyl-1-nitrosourea.

An investigation was conducted to determine the effect of the dietary level of selenium on the induction of tracheal cancer by 1-methyl-1-nitrosourea (MNU). Male Syrian golden hamsters received intratracheal instilations of a 0.5% MNU solution once weekly for 12 weeks. Two weeks prior to the initiation of carcinogen treatment, animals were placed on a semisynthetic, 30% torula yeast diet to which either no selenium or 1 or 5 mg selenium/kg of diet as sodium selenite was added. Animals were maintained on their respective diets for the duration of the study which was terminated 195 days after the first MNU treatment. No significant differences among groups in the incidence of either benign lesions or carcinomas was observed and the distribution of tumor type was similar irrespective of selenium treatment. The results of this study indicate that selenium exerts no chemopreventive effect against MNU-induced tracheal carcinogenesis.

Reproduction study in rats or ginseng extract G115.

The effect of ginseng extract G115 on reproductive performance was studied in two generations of Sprague-Dawley rats. Animals of both sexes were fed wither control diet or diet supplemented with ginseng extract G115 at dose levels of 1 . 5, 5 or 15 mg/kg body weight/day. Parameters of reproduction and lactation in the treated groups were comparable to those of the controls for two generations of dams and pups. For F1 males and females, no treatment-related effects were seen in weekly body weights and food consumption, haematological and clinical chemical data, and ophthalmic, gross and histopathological examinations. The gross autopsies of F1 and F2 animals also revealed no significant treatment-related findings.

Subchronic feeding study of grape colour extract in beagle dogs.

The effect of feeding Welch's Special Grape Color Powder Type BW-AT at dose levels of 7.5 and 15% w/w in the diet for 90 days was studied in beagle dogs. Body-weight gain of male and female dogs at the high dose level was significantly decreased compared with control dogs. No other treatment-related effects were seen in food consumption, haematology, clinical chemistry, ophthalmology or gross and histopathological findings.

Reproduction study of grape colour extract in rats.

The effect of Welch's Special Grape Color Powder Type BW-AT on reproductive performance was studied through two generations of Sprague-Dawley rats and a subchronic study was carried out on the F1 animals. The grape colour powder at dietary levels of 7.5 and 15.0% (w/w) had no adverse effects on reproductive performance. Body weights for F0 and F1 generation pups at both dose levels were significantly lower (P less than 0.05) than those of control pups at 21 days after birth. During the 13-wk subchronic feeding study of F1 rats, the body-weight gain of female rats in the high-dose group was reduced compared with the controls (P less than 0.05). Food conversion data was comparable among groups, thus the decrease in body-weight gain during this phase was most likely the result of the lower calorific value (w/w) of the feed supplemented with the grape colour powder compared with the control feed. No toxic effects or pathological changes were noted in rats fed grape colour powder.