Nutrient Interactions and Toxicity Research Communication A Natural Antioxidant Mixture from Spinach Does Not Have Estrogenic or Antiestrogenic Activity in Immature CD-1 Mice

The use of natural antioxidants and flavonoids in nutritional and pharmaceutical applications is increasing. Because some phytochemicals such as genistein, found in soy products, have estrogenic activity, we investigated the estrogenic potential of a natural antioxidant mixture (NAO) isolated from spinach leaves, using an in vivo uterotrophic bioassay and an in vitro transcriptional activation assay for the estrogen receptor (ER). Outbred female CD-1 mice (17 d old) were given subcutaneous injections of 17-estradiol or genistein (500 and 500,000 g /(kgd), respectively) as posi- tive controls or NAO (1000 to 1,000,000 g/(kgd)) for 3 d. Uterine wet weight/body weight ratios were determined. Both 17-estradiol and genistein significantly increased uterine wet weight ratios compared with untreated controls, but NAO did not. Histological examination of the uterus showed that 17-estradiol and genistein increased epithelial cell height, number and gland development, but NAO did not. Estrogenic activity of NAO was investigated in vitro using the ER tran- scriptional activation assay. BG1Luc4E2 cells expressing ER were stably transfected with a luciferase reporter gene re- sponsive to estrogens. 17-estradiol dose dependently in- creased luciferase activity; NAO had no effect. When NAO was tested for antiestrogenic activity, it did not lessen the effects of 17-estradiol. These data suggest that NAO does not have estrogenic or antiestrogenic activity. Thus, an anti- oxidant mixture has been identified that does not have poten- tially adverse estrogenic activity. J. Nutr. 133: 3584-3587, 2003.