Phytoestrogen Content of Purified, Open- and Closed-Formula Laboratory Animal Diets
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Laboratory Animal Science Vol 49, No 5 Copyright 1999 October 1999 by the American Association for Laboratory Animal Science Phytoestrogen Content of Purified, Open- and Closed-Formula Laboratory Animal Diets Julius E. Thigpen,1 Kenneth D. R. Setchell,2 Kathy B. Ahlmark,1 Jacqueline Locklear,1 Tamara Spahr,1 Gordon F. Caviness,1 Mary F. Goelz,1 Joseph K. Haseman,3 Retha R. Newbold,4 and Diane B. Forsythe1 Background and Purpose: Phytoestrogens exert estrogenic effects on the central nervous system, induce es- trus, and stimulate growth of the genital tract of female animals. Over 300 plants and plant products, includ- ing some used in laboratory animal diets, contain phytoestrogens. Therefore, the source and concentration of phytoestrogens in rodent diets were determined. Methods: Twelve rodent diets and six major dietary ingredients were assayed for phytoestrogens (daidzein, genistein, formononetin, biochanin A, and coumestrol), using high-performance liquid chromatography. Three rodent diets recently formulated to reduce phytoestrogen content also were assayed. Results: Formononetin, biochanin A, and coumestrol were not detected. Soybean meal was the major source of daidzein and genistein; their concentrations were directly correlated to the percentage of soybean meal in each diet. Conclusions: High, variable concentrations of daidzein and genistein are present in some rodent diets, and dietary phytoestrogens have the potential to alter results of studies of estrogenicity. Careful attention should be given to diet phytoestrogen content, and their concentration should be reported. A standardized, open- formula diet in which estrogenic substances have been reduced to levels that do not alter results of studies that are influenced by exogenous estrogens is recommended. Phytoestrogens are defined as plant compounds that exert phytoestrogens in plants or plant products varies and is in- estrogenic effects on the central nervous system, induce estrus, fluenced by species differences, portion of plant assayed, geo- and stimulate growth of the genital tract of female animals (1). graphic location, time of harvest, and method of processing Over 300 plants and plant products, including some used in (8, 9). In general, the relative potency of the phytoestrogens laboratory animal diets, contain phytoestrogens (2–4). The two is dependent on the type of assay (in vivo or in vitro) used to main classes of phytoestrogens are the lignans and the measure estrogenic potency, animal species and age, dosage, isoflavonoids. The isoflavonoids are further divided into the age at dosing, route, and duration of exposure (6, 12, 25–27). isoflavans, isoflavones, and coumestans (4–6). Natural dietary Previous studies in our laboratory have indicated that ingredients, such as soybeans (7–11), flax (12–14), wheat, bar- commonly used rodent diets differ significantly in estrogenic ley, corn, alfalfa, and oats, are major dietary ingredients that activity, as measured by their ability to increase immature have been reported to contain phytoestrogens (3). The mouse uterine-to-body weight ratio in a 7-day bioassay (27, isoflavones (daidzein, genistein, formononetin, and biochanin 28). The isoflavonoids—daidzein, genistein, coumestrol, A) and the coumestans (coumestrol) are the major biochanin A, and formononetin—have been documented to phytoestrogens found in dietary ingredients used in many labo- be estrogenic in the mouse bioassay, as determined by sig- ratory animal diets (10, 11). Structurally, phytoestrogens re- nificantly increasing mean uterine weight of treated mice semble endogenous steroidal estrogens of humans and animals over that of control mice (20, 29, 30). Also, the in vivo effects and share a common mechanism of action through binding to of phytoestrogens are of principal interest due to their po- the estrogen receptor (6, 12, 15). It is well established that tential health risks and/or beneficial effects on humans and phytoestrogens and the estrogenic mycotoxins can bind to the animals (12, 31–33). Some phytoestrogens (17, 33, 34), when estrogen receptor and induce estrogen-like effects in ani- consumed in large amounts, have been reported to cause re- mals (16–18), humans (19), and cells in culture (6, 18). The productive problems in sheep (35, 36) and liver disease and relative potency of phytoestrogens is significantly lower infertility in captive cheetahs (37). Phytoestrogens have also than that of steroidal estrogens, and animal species differ in been reported to be beneficial by acting as partial estrogen sensitivity to the various phytoestrogens (20–23). The antagonists (12, 31, 33, 38, 39); diets containing isoflavones phytoestrogens are weak estrogens, compared with diethyl- have been documented to reduce the rate of mammary tu- stilbestrol (DES), estradiol-17, or estrone, in mouse mor (40–42) and liver, colon, or prostate tumor (43) formation uterotropic assays (20, 24). The concentration of the in animal models of clinical carcinogenesis. Phytoestrogens have also been documented to reduce serum cholesterol con- Comparative Medicine Branch,1 Biostatistics Branch,3 and Laboratory of centration in monkeys (44), rats (45, 46), and humans (47– Toxicology,4 National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina; and Department of Pediatric Gastroenter- 50), and to increase bone density in aged rats (51–53). The ology, Children’s Hospital Medical Center, Cincinnati, Ohio2 phytoestrogens can act as antioxidants (38, 54–56) and sup- 530 Phytoestrogens in Rodent Diets press angiogenesis (57). Presence of 10% soybean meal in a Purified rodent diets: The following American Institute rodent diet, compared with a soy-free diet, resulted in a 40% of Nutrition (AIN) diets (63)—AIN-76A, a purified (casein) reduction in mammary tumors in two rat models of breast rodent diet; AIN-76A, a purified (soy-protein) rodent diet; cancer (41). The phytoestrogens genistein and daidzein were and AIN-93M, a purified (casein) maintenance diet—were reported to be responsible for this reduction in breast tu- purchased (Research Diets, Inc., New Brunswick, N.J.). For mors by acting as inhibitors of estrogen action or modulators future references, the diets will be identified by their vendor of hepatic metabolism (41). catalog numbers. Soy-based ingredients used in purified or natural-ingredi- Three new test diets: In the second experiment, three ent rodent diets have been reported to vary greatly in new test diets, recently formulated to contain reduced concen- isoflavone content (10, 11). The total isoflavone content in trations of phytoestrogens, were compared for phytoestrogen different varieties of soybeans varied from 116 to 309 mg/g content. Each of the three new test diets came from a differ- and varied from 46 to 195 mg/g within the same variety of ent rodent diet vendor and are identified as test diet A (ven- soybeans grown in different locations (9). The phytoestrogen dor A); test diet B (vendor B); and test diet C (vendor C). content of diets can vary greatly from batch to batch (58). We Dietary ingredients: Six commonly used dietary ingre- reported earlier that some laboratory animal diets contain dients, including whole wheat, whole oats, corn, soybean high concentrations of the phytoestrogens daidzein and meal, wheat middlings, and alfalfa meal (Zeigler Brothers, genistein (59). The biological role that phytoestrogens play Inc., Gardners, Pa.) were also assayed for phytoestrogens. in contributing to the estrogenic activity observed in rodent Phytoestrogen assays: The diets and dietary ingredients diets, and their effect on studies that are influenced by exog- were assayed for the phytoestrogens daidzein, genistein, enous estrogenic substances, is unclear. The objectives of the formononetin, biochanin A, and coumestrol. Soy flakes were as- study reported here were to determine the concentration sayed as a positive control. Dietary phytoestrogens were as- and source of the phytoestrogens daidzein, genistein, sayed, using a high-performance liquid chromatography formononetin, biochanin A, and coumestrol in rodent diets, (HPLC) procedure as described (64). Briefly, dietary estro- and to compare the phytoestrogen content in three new ro- gens were extracted from 5-g samples by prolonged reflux- dent diets recently formulated to reduce phytoestrogen con- ing in 80% ethanol. After extraction, the supernatants were tent. Results of these studies have led to recommendations cooled and filtered. A rotary evaporator was used to remove for the formulation and quality control of diets used when the bulk of ethanol from the extract. Lipids were removed by conducting studies investigating estrogenicity. liquid-liquid partitioning into hexane. The aqueous extract was taken to dryness, and a -glucosidase hydrolysis was Materials and Methods carried out in 0.1 M sodium acetate buffer (pH 5.0) over- In the initial experiment, some commonly used commer- night. The hydrolysate was then passed through Bond Elut- cially available, closed- and open-formula, natural-ingredi- C8 and Bond Elut-C18 cartridges (Varian, Inc., Harbor City, ent rodent diets and some purified rodent diets were Calif.) to extract the dietary estrogens, which were recovered examined for phytoestrogens. Also, lot-to-lot variability in by elution of the cartridges with 5 ml of methanol. The daidzein and genistein concentrations within four different methanolic extract was then subjected to anion exchange chro- milling dates for four selected diets was determined.