Cancer Res. 2001 Jun 1;61(11):4325-8.
Uterine adenocarcinoma in mice treated neonatally with genistein.
Newbold RR, Banks EP, Bullock B, Jefferson WN.
Developmental Endocrinology Section, Laboratory of Toxicology, Environmental Toxicology Program, Division of Intramural Research, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA.
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The developing fetus is uniquely sensitive to perturbation with estrogenic chemicals. The carcinogenic effect of prenatal exposure to diethylstilbestrol (DES) is the classic example. Because phytoestrogen use in nutritional and pharmaceutical applications for infants and children is increasing, we investigated the carcinogenic potential of genistein, a naturally occurring plant estrogen in soy, in an experimental animal model previously reported to result in a high incidence of uterine adenocarcinoma after neonatal DES exposure. Outbred female CD-1 mice were treated on days 1-5 with equivalent estrogenic doses of DES (0.001 mg/kg/day) or genistein (50 mg/kg/day). At 18 months, the incidence of uterine adenocarcinoma was 35% for genistein and 31% for DES. These data suggest that genistein is carcinogenic if exposure occurs during critical periods of differentiation. Thus, the use of soy-based infant formulas in the absence of medical necessity and the marketing of soy products designed to appeal to children should be closely examined.

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Introduction Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

Studies in our laboratory have shown long-term consequences of exposure to estrogens during critical periods of development (1). In particular, DES,2 a potent synthetic estrogen, has been shown to cause adverse reproductive effects including structural abnormalities, infertility, and neoplasia after prenatal exposure (2). Furthermore, mice treated neonatally with 2 µg/pup/day (1000 µg/kg/day) DES on days 1–5 have a 90–95% incidence of uterine carcinoma at 18 months of age (3). The background incidence of this lesion is rare in this strain of mouse (3). The naturally occurring phytoestrogen, genistein, is found in many soy products. Humans may be exposed to high levels of genistein during development through soy-based infant formulas and soy products marketed specifically for children (4). The concentrations of genistein and other isoflavones found in some of these soy formulas can far exceed the amount found in an adult diet (4). Infants consuming a diet of soy-based formula may be exposed to 20–40 mg per day (4–6 mg/kg/day) of soy isoflavones, of which genistein makes up >65%, whereas adults consuming a moderate to large amount of soy in the diet are exposed to 1 mg/kg/day soy isoflavones (4). Earlier reports suggested beneficial effects of genistein exposure early in life, e.g., breast cancer prevention (5, 6) and improved cholesterol synthesis rates (7), but an increasing number of reports are now describing long-term deleterious effects of genistein (8) and of another phytoestrogen, coumestrol (9, 10). One recent report showed an increase in carcinogen-induced mammary carcinogenesis after prenatal exposure of rats to genistein (11), and another associated a diet high in phytoestrogens with the development of insulin-dependent diabetes (12). Still another report highlighted the increased incidence of hypospadias in male offspring of vegetarian mothers (13). To further study the effects of phytoestrogen exposure early in life, we treated mice neonatally with genistein or an equivalent estrogenic dose of DES and determined the long-term carcinogenic potential.

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Discussion

This report describes the induction of benign and malignant lesions of the reproductive tract, including uterine adenocarcinoma in the reproductive tract of mice treated neonatally with the phytoestrogen genistein. Similar malignant lesions have never been observed in control mice of this strain in our laboratory. The dose of genistein used in this study is within the range to which humans may be exposed in soy-based infant formulas (4). In addition, mice exposed to a dose of DES that is approximately equal in estrogenic activity gave similar incidences of uterine adenocarcinoma under the same treatment conditions. The association of estrogenicity and carcinogenicity in the neonate is further supported by the results of another study from our lab that compared metabolites of estradiol (17); based on the day-5 uterotropic bioassay, compounds with the highest estrogenic potency in the neonatal mouse uterus showed the highest percentage of uterine adenocarcinoma after neonatal exposure. The data shown in this report shows a close association between the estrogenic activity of the compound in the neonate and the incidence of uterine adenocarcinoma after neonatal exposure.

Of particular significance in this study is the incidence of uterine adenocarcinoma after neonatal exposure to genistein. This compound is readily available to many infants during the first year of life as a component of soy-based formulas. The amount of soy isoflavones found in some infant formulas, of which genistein and its conjugates account for >65%, approaches 40 mg/liter of formula (4). An infant consuming 1 liter of soy-based infant formula would ingest 27 mg of genistein per day. The amount of genistein used in our study (50 mg) is slightly higher than the amount consumed by infants, but it is certainly within one order of magnitude of the level of human exposure. We are currently evaluating the carcinogenic potential of lower doses of genistein as well as investigating the effects of genistein if exposure occurs through the diet instead of s.c. injection as described in this study.

In another report from our laboratory, lower doses of genistein caused alterations in the ovary such as multioocyte follicles (18); a dose-related increase in multioocyte follicles starting at a dose of 5 mg/kg/day was observed in 2-month-old mice. Previous work by Iguchi et al. (19) has shown that multioocyte follicles resulting from neonatal DES exposure are less fertile than single oocyte follicles. This data suggest that fertility could also be affected in mice exposed neonatally to genistein. Future studies in our laboratory will investigate fertility in genistein-treated mice as well as mechanisms involved in the formation of multioocyte follicles.

The findings of the present study raise concerns over the amount of phytoestrogens in soy-based infant formulas and other soy-based products that are fed to young children. Additional studies are needed to determine the potential effects in humans exposed to high quantities of phytoestrogens during critical stages of neonatal or early development. Ongoing National Toxicology Program (NTP) studies designed to address multigenerational effects of genistein,3 will help to determine the risks of developmental exposure to genistein and other endocrine-disrupting chemicals. The potential risks and benefits need to be thoroughly assessed to determine the appropriate balance of exposures of these chemicals during development and the permanent effects that may follow.

Categories: 2001, Soy, Cancer, Phytoestrogens, Hormone disruptors, Estrogen, Nutrition and diet