Estrogenic isoflavones in rodent diets.

Many rodent diets contain components such as soy isoflavones (daidzein and genistein) known to have estrogenic properties. The dietary background of phytoestrogens may modulate some responses to environmental estrogens when these compounds are tested in rodent bioassays. Thus, and since only few data were available on the phytoestrogen content of rodent diets commonly used in European laboratories, it was of interest to analyze the daidzein and genistein contents of our standard animal feeds. Isoflavone contents were determined in seven batches of rodent chow (from two suppliers in Germany, Altromin and Ssniff) by high-performance liquid chromatography, and also analyzed in six rodent diets from the United States. The soy-based rodent diets from Germany contained isoflavone (daidzein plus genistein) concentrations in the range of 0.3-0.55 mg/g feed. These isoflavone contents are similar to those analyzed in the US rodent diets, and similar to values reported by others, including one particular lot of feed (with 0.35 mg isoflavones per g) which produced a large uterotrophic response in immature ovariectomized rats [Environ. Health Perspect., 106 (1998) 369]. Coumestrol was found in a sample of commercial rabbit food at rather high levels (0.27 mg/g), but, this phytoestrogen was not detected (<1 microg/g feed) in any of the other samples we analyzed. The soy components in our rodent diet produce a measurable background of daidzein and genistein in blood of female DA/Han rats, total isoflavones (aglycone plus conjugates) ranging between 90 and 290 ng/ml plasma. The ovariectomized animals kept on this chow, showed no signs of estrogenization of the reproductive tract (uterus, vagina), and responded normally to (xeno-)estrogen administration in a uterotrophic assay [J. Steroid Biochem. Mol. Biol., 73 (2000) 1]. Moreover, ovariectomized Wistar rats on our standard rodent diet (Ssniff R/M H) had lower uterine weights than animals kept on the isoflavone-free (solvent extracted) chow; both groups of rats responded to genistein administration with an increase in uterine weights. These results suggest that--albeit the sensitivity of the rodent uterotrophic assay is not reduced by the use of a diet containing soy isoflavones at commonly encountered levels--attention should be given to a variable dietary phytoestrogen background.

Inhibition of melanoma growth and metastasis by combination with (-)-epigallocatechin-3-gallate and dacarbazine in mice.

(-)-Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, was shown to have cancer chemopreventive activity. In this study, we examined the antimetastatic effects of EGCG or the combination of EGCG and dacarbazine on B16-F3m melanoma cells in vitro and in vivo. First, the antimetastatic potentials of five green tea catechins were examined by soft agar colony formation assay, and the results show that EGCG was more effective than the other catechins in inhibiting soft agar colony formation. Second, EGCG dose-dependently inhibited B16-F3m cell migration and invasion by in vitro Transwell assay. Third, EGCG significantly inhibited the spread of B16-F3m cells on fibronectin, laminin, collagen, and Matrigel in a dose-dependent manner. In addition, EGCG significantly inhibited the tyrosine phosphorylation of focal adhesion kinase (FAK) and the activity of matrix metalloproteinase-9 (MMP-9). In animal experiments, EGCG alone reduced lung metastases in mice bearing B16-F3m melanomas. However, a combination of EGCG and dacarbazine was more effective than EGCG alone in reducing the number of pulmonary metastases and primary tumor growths, and increased the survival rate of melanoma-bearing mice. These results demonstrate that combination treatment with EGCG and dacarbazine strongly inhibits melanoma growth and metastasis, and the action mechanisms of EGCG are associated with the inhibition of cell spreading, cell-extracellular matrix and cell-cell interactions, MMP-9 and FAK activities.

Response of hypothalamic NPY mRNAs to a negative energy balance is less sensitive in cachectic mice bearing human tumor cells.

We selected three human cancer cell lines [human melanoma (SEKI), human melanoma (G361), and human neuroepithelioma (NAGAI)] that have an ability to develop cancer cachexia syndrome with and without accompanying anorexia and examined the hypothalamic levels of mRNAs for neuropeptide Y (NPY), melanin-concentrating hormone, and orexin. The body weight of sham-operated mice continued to increase, while mice of all tumor-bearing groups lost weight. Competitive reverse transcription-polymerase chain reaction analysis showed that, regardless of feeding status, NPY mRNA levels were elevated in all tumor-bearing mice compared with sham-operated mice, although to a lesser degree than weight-matched pair-weight mice. Melanin-concentrating hormone and orexin mRNA in the hypothalamus followed the same pattern as NPY, although most of the differences did not reach statistical significance. These results support the notion that the response of NPY mRNA to a negative energy balance is less sensitive in these rodent models of cancer cachexia.

Serum inhibition of proliferation of serum-free mouse embryo cells.

Serum-free mouse embryo (SFME) cells, derived in medium supplemented with insulin, transferrin, high density lipoprotein, epidermal growth factor, and fibronectin, do not undergo crisis, maintain a predominantly diploid karyotype with no detectable chromosomal abnormalities for well over 100 population doublings in vitro, and are growth inhibited by concentrations of serum that are growth-stimulatory for most cell lines in culture. Serum inhibition of SFME cell proliferation was reversible and was not prevented by addition of the supplements of the serum-free medium, even when added repeatedly during the culture period. The serum effect on SFME cell proliferation could be detected after incubation in serum-containing medium for as little as 8 h. SFME cells in serum-containing medium were arrested in the G1 phase of the cell cycle with a greatly reduced rate of incorporation of precursors into DNA and thymidine kinase activity, while a reduction in rate of incorporation of amino acids into protein was not observed. SFME cultures maintained for extended periods in serum-containing medium underwent a crisis-like period followed by the appearance of variant cells capable of growing in serum-supplemented medium. These cells exhibited abnormal karyotype and were resistant to several inhibitors of proliferation active on the parent SFME cell type.

Inhibition of growth of human breast cancer cell line MCF-7 by serum derived from calcium chloride-clotted plasma.

The growth of MCF-7 cells, established from a metastatic mammary carcinoma, and of HBL-100 cells, derived from a primary culture of human milk, was examined in medium supplemented with whole blood serum (WBS), defibrinogenated plasma, and plasma-derived serum (PDS). PDS obtained from platelet-poor plasma collected with the anticoagulant citrate phosphate dextrose and clotted by the addition of calcium chloride did not promote the growth of MCF-7 cells as well as did WBS or platelet-poor defibrinogenated plasma alone. Growth-inhibitory activity was observed when final cell densities in the presence of PDS combined with fetal bovine serum (FBS) were compared with those in control cultures maintained in FBS alone. The level of this activity in PDS varied among donors. Inhibition was not observed with HBL-100 cells. Calcium chloride did not induce inhibitory activity when added to WBS or defibrinogenated plasma, and platelet components did not alter the level of inhibition in PDS. However, platelet extracts did affect the expression of inhibition when added to plasma before clotting. Activity was diminished following dialysis of PDS, which suggested that inhibition stemmed from a small-molecular-weight factor for which expression depended on the mechanism of plasma coagulation.