Diane F. Birt

Anti-mutagenesis and anti-promotion by apigenin, robinetin and indole-3-carbinol

We assessed the anti-mutagenic and anti-promotion properties of two flavones, apigenin and robinetin, and of indole-3-carbinol, because these compounds have been reported in vegetables, the consumption of which has been associated with reduced rates of cancer. However, the active components of these foods and their effects on carcinogenesis have not been established. Anti-mutagenicity was determined in the Salmonella typhimurium assay by measuring the effects of the test compounds on bacterial mutagenesis induced by methyl-nitrosourea (MNU), methyl-n-nitro-N-nitrosoguanidine (MNNG), benzo[a]pyrene (BaP) or 2-aminoanthracene (2-AA). Inclusion of apigenin resulted in a 62% and a 43% inhibition of mutagenicity with 13 nmol of 2-AA and 30 nmol BaP respectively. Robinetin caused an 87% inhibition of mutagenicity by 2-AA, but indole-3-carbinol had little or no effect on the mutagenicity of any of the compounds. None of the three compounds inhibited mutagenesis by MNU or MNNG and none were mutagenic or toxic when tested in the absence of mutagenic compounds at doses up to 20 micrograms/plate. Anti-promotion properties were assessed by measuring the effects of apigenin, robinetin and indole-3-carbinol on induction of ornithine decarboxylase activity (ODC) in mouse epidermis by 17 nmol 12-O-tetradecanoyl phorbol-13-acetate (TPA). Pretreatment of the skin half an hour before TPA with apigenin, robinetin, butylated hydroxyanisole, 13-cis-retinoic acid (all at 50 mumol) or di-fluoromethylornithine (1.6 mumol) inhibited ODC induction at 6 h after TPA by 67-80%. Pretreatment with 50 mumol indole-3-carbinol caused a 78% elevation in the TPA induction at this time. Dose response measurements were conducted with apigenin, indole-3-carbinol and robinetin. Inhibition by 30-90% of TPA-induced ODC was observed at 6 h after TPA in mice pretreated with 12.5-100 mumol apigenin. Pretreatment with 37.5 or 50 mumol indole-3-carbinol or 0.5, 12.5 or 25 mumol robinetin resulted in elevated induction of epidermal ODC by TPA at 6 h after TPA. However, treatment with 50 or 100 mumol robinetin diminished ODC induction at 6 h after TPA. Treatment with 100 mumol apigenin or 50 or 100 mumol indole-3-carbinol in non-TPA-treated mouse skin caused elevations in epidermal ODC. In comparing the time course of ODC induction, indole-3-carbinol (50 mumol) pretreatment shifted the induction of epidermal ODC to earlier times, in addition to elevating ODC induction by TPA.(ABSTRACT TRUNCATED AT 400 WORDS)

In Vivo and In Vitro Percutaneous Absorption of Cancer Preventive Flavonoid Apigenin in Different Vehicles in Mouse Skin

AbstractPurpose.In vivo and in vitro percutaneous absorption of apigenin was investigated in three vehicles previously used in cancer prevention studies to determine the drug delivery properties for optimal chemo-preventive activity. Methods. In vivo percutaneous absorption of apigenin on SENCAR mice was studied with DMSO and acetone/DMSO (A/D, 4:1) vehicle. In vitro percutaneous absorption studies used whole mouse skin, without subcutaneous fat, mounted on Franz diffusion cells with 37°C Dulbeccos phosphate-buffered saline as the receptor fluid. The skin was treated with [G-3H]-apigenin in DMSO, A/D (4:1), or propylene glycol/DMSO (PG/D, 4:1). Results. Apigenin uptake by epidermal cells and distribution in epidermis following in vivo topical treatment in two vehicles was in the order of A/D > DMSO, while apigenin distribution in dermis and subcutaneous fat was not different between DMSO and A/D. Total apigenin absorption in mouse skin in vitro was in the order of A/D > DMSO > PG/D. However, apigenin sub-tissue distribution within epidermis determined by tape-stripping and by determination of apigenin in dermal and epidermal tissue indicated that DMSO delivered more apigenin into viable epidermis than A/D while A/D deposited more apigenin in the stratum corneum. Apigenin absorption in mouse skin with DMSO or A/D showed saturation kinetics while apigenin in PG/D showed very low absorption initially and non-saturated absorption in a period of 6 hr. HPLC-scintillation profiles of in vitro samples showed no evidence of apigenin metabolism in mouse skin. Conclusions. Delivering apigenin into viable epidermis appears to be a necessary property for an apigenin formulation to be effective in skin cancer prevention.

Modulation of Estrogen Action in the Rat Pituitary and Mammary Glands by Dietary Energy Consumption

We are investigating the mechanisms through which estrogens induce development of prolactin (PRL)-producing pituitary tumors and mammary carcinomas in rats and how these mechanisms are affected by dietary energy consumption. The hypothesis under examination is that dietary energy restriction inhibits tumorigenesis in estrogen-responsive tissues by altering cellular responsiveness to estrogenic hormones. In the Fischer 344 (F344) rat strain, a 40% restriction of energy consumption virtually abolishes development of estrogen-induced pituitary tumors. Inhibition of pituitary tumorigenesis in the F344 strain by energy restriction results from modulation of estrogen regulation of cell survival, not cell proliferation. In contrast, energy restriction has no inhibitory effect on estrogen-induced pituitary tumor development in the ACI rat strain. However, energy restriction markedly inhibits induction of mammary carcinomas in female ACI rats treated with 17beta-estradiol. Data presented herein indicate that dietary energy restriction modulates the responsiveness of specific cell populations to estrogenic hormones and thereby inhibits estrogen-induced tumorigenesis in a manner specific to both rat strain and tissue.

Acceleration of Papilloma Growth in Mice Fed High-Fat Diets During Promotion of Two-Stage Skin Carcinogenesis

The effect of feeding a high-fat diet during the promotion phase of skin tumorigenesis was assessed in SENCAR mice. Tumors were initiated on the backs of mice by application of 10 nmol 7,12-dimethylbenz[a]anthracene (DMBA); the tumors were then promoted beginning one week later with twice weekly treatments of 2 micrograms 12-O-tetradecanoylphorbol-13-acetate (TPA) each in 0.2 ml acetone. Control diet containing 5% corn oil was fed from four weeks before until one week after DMBA treatment in all groups. A high-fat diet (24.6% corn oil) was fed from one week after DMBA treatment; the other groups continued on the control diet. Mice were fed ad libitum, and those given the high-fat diet consumed more calories early in the study than the controls did. Treatment with TPA increased calorie consumption throughout the study. Body weights were elevated in mice fed high-fat diets and reduced by TPA treatment. The average number of papillomas per mouse did not differ between the low- and high-fat groups, but papillomas grew more rapidly on the mice fed a high-fat diet than those fed a low-fat diet. The feeding of a high-fat diet following DMBA treatment, in the absence of TPA administration, did not result in promotion of skin tumors. Therefore, a high-fat diet acted as a copromoter of skin tumors in SENCAR mice.

Tolerance of Diets Deficient or Excessive in Selenium by Syrian Hamsters

Syrian hamsters were fed torula yeast (TY) diets with 8 selenium (Se) supplement levels (0.0-10.0 ppm Se as sodium selenite) or casein (C) diets with 5 supplement levels (0.0-5.0 ppm Se as sodium selenite) for 25 weeks. Whole blood Se, plasma glutathione peroxidase (GSH-Px) activity and erythrocyte GSH-Px activity were measured after 5, 10, 15 and 25 weeks. At 25 weeks hematology was examined and tissue samples analyzed for Se and evaluated for histopathological lesions. While survival was not influenced by dietary Se, food consumption and body weight gain were altered in animals given TY, as those fed 0.0, 0.05 or 10.0 ppm Se consumed less diet. Weight gains at 25 weeks were highest in animals at the 0.1 ppm Se level and reduced in those given unsupplemented TY or 10.0 ppm Se supplements. Hemoglobin, hematocrit and red blood cell counts were reduced in females fed the lowest and highest Se supplements with TY diets. With both C and TY, whole blood Se rose with increasing dietary Se and in the case of TY, Se was elevated with each feeding increment, except between the 0.05 and 0.1 ppm or the 0.25 and 0.5 ppm levels. Plasma GSH-Px increased with rising Se up to 10 ppm, and erythrocyte GSH-Px activity increased up to 5 ppm Se. Erythrocyte GSH-Px values were higher in animals fed C diets. Histopathological observations were normal at all Se levels. Syrian hamsters tolerated dietary Se from 0.05 to 5.0 ppm Se for 25 weeks of observation without detrimental effects.

Dietary lignin, an insoluble fiber, enhanced uterine cancer but did not influence mammary cancer induced by N-methyl-N-nitrosourea in rats

Previous investigations suggested potential breast cancer-preventive properties of dietary fiber from cabbage. The purpose of the present investigation was to determine whether lignin, a component of cabbage fiber, would protect against mammary carcinogenesis by N-methyl-N-nitrosourea (MNU) in Sprague-Dawley rats. A six-week study was conducted using diets containing 0.5-5% dietary wood lignin (a readily available, purified source). These diets were well tolerated by the rats, and a carcinogenesis study using 5 mg MNU/100 g body wt i.v. at 50 days of age was conducted, with the 2.5% lignin diet fed from 6 through 8 weeks of age followed by 5% lignin diet until 20 weeks after MNU. Dietary lignin and MNU treatment increased food consumption (p < 0.05), and body weight was slightly reduced at 10 and 20 weeks after MNU in the MNU-5% lignin diet group (p < 0.05). Serum estradiol was not altered by dietary lignin or MNU treatment, but uterine weights were highest in the MNU-control diet group 4 and 12 weeks after MNU. Expression of creatine kinase B, and estrogen-responsive gene, was lower in eh uteri of the MNU-lignin diet group than in other groups at 20 weeks. Mammary carcinogenesis was not altered by dietary lignin. However, uterine endometrial adenocarcinoma was observed only in the MNU-lignin diet group (4 carcinomas/40 effective rats) (p < 0.05).

Methodologic issues, theoretical considerations, and design criteria for experimental animal and cell culture experiments.

This article provides background information that is important when evaluating the relevance to humans of particular animal or in vitro experiments designed to assess the relations between fatty acids and cancer. Considerations in designing carcinogenesis studies to assess the relation between dietary fatty acids and human cancer include selection of the animal model and design of the experimental diets. Animal carcinogenesis models are generally best for evaluating the early phases of cancer development: the initiation and promotion of cancer. Transplantation protocols have been developed for evaluating the effect of diet on the growth and metastasis of partially or fully transformed cells. The variables that are important in such models are the origin and biology of the cell line, the animal host used for the implantation, the site of transplantation, whether the primary tumor is excised after a period of time to allow for metastasis, and when the diets are fed relative to the different phases of tumor growth and metastasis. Studies in cultured cells have been particularly useful for assessing the mechanisms by which fatty acids affect cancer. Considerations in designing studies with cultured cells include selection of the cell line, cell culture conditions, selection of biological endpoints that are relevant to human cancer, and in vivo confirmation of the mechanisms observed in vitro. Design considerations for each of these experimental approaches are discussed and the contributions of each approach are summarized.

Comparative studies on the effects of semipurified and commercial diet on longevity and spontaneous and induced lesions in the Syrian golden hamster.

Syrian golden hamsters were fed a semipurified or commercial diet from weaning throughout life. Bis(2-oxopropyl)nitrosamine (BOP) was administered at 8 weeks of age (10 mg/kg body wt, sc). Longevity was improved by 26% and 36% increases in the mean life-spans of male and female hamsters, respectively, fed the semipurified diets. Carcinogen treatment did not alter survival. The age-adjusted occurrence rates of pancreatic ductular proliferation, carcinomas, adenomas, and common duct polyps were higher in hamsters fed commercial diet; this indicates an earlier onset of these BOP-induced lesions in hamsters fed this diet. However, their overall incidences were generally similar when the two diet groups were compared. Acinar cell nodules were observed only in hamsters fed semipurified diets and were elevated in BOP-treated females. The onset of pancreatic ductular proliferation and adenomas, bile duct proliferation, parathyroid hyperplasia, and common duct papillary hyperplasia was earlier in females than in male hamsters, especially in groups fed commercial ration. Generalized vascular calcification was observed at an elevated rate and reached a higher overall incidence in hamsters fed commercial ration. The age-adjusted rate of amyloidosis was high in female hamsters and elevated in groups that consumed the commercial ration. In addition, colitis and islet cell hyperplasia occurred more often and earlier in hamsters fed commercial ration, but gallbladder stones occurred most in animals fed semipurified diet. This paper discusses the possible association between these and other observed lesions and survival.