Acute and chronic effects of oral genistein administration in neonatal mice

Abstract

Soy-based infant formulas are widely used in the United States and some other countries. These formulas contain high levels of the estrogenic isoflavone genistein, leading to concern that neonatal genistein exposure could cause acute and/or long-term adverse effects on reproductive and other organs. However, previous work to assess genistein effects in rodent models has not typically replicated the route of delivery and/or serum genistein concentrations reported for soy formula-fed human infants. Our objective was to develop a mouse model that more closely mimics the oral genistein exposure and total serum genistein concentrations observed in soy formula-fed infants. Mouse pups were dosed orally with genistein in a soy formula-corn oil emulsion from Postnatal Day (PND) 1 to PND 5, then effects on reproductive and non-reproductive organs were assessed after dosing and during subsequent development. Neonatal treatment resulted in changes both at the completion of dosing (PND 5) and in adult animals. At PND 5, neonatal genistein treatment caused increased relative uterine weight and down-regulation of progesterone receptor in uterine epithelia. Estrogenic effects of genistein were also seen in the neonatal ovary and thymus, which had an increase in the incidence of multioocyte follicles (MOFs) and a decrease in thymic weight relative to body weight, respectively. The increased incidence of MOFs persisted into adulthood for neonatally treated genistein females, and estrous cycle abnormalities were seen at 6 mo of age despite normal fertility in these mice. The immediate and long-term effects in this neonatal animal model raise concerns that high serum concentrations of genistein are estrogenic and could potentially impact the development of human infants fed soy formula.

FIG. 1.

Pharmacokinetics of orally administered genistein in PND 5 female mice. Serum was collected at the indicated times after the final dosing on PND 5 from pups. The yellow shaded area indicates total genistein (GEN) serum concentrations reported for 4-mo-old soy formula-fed infants [9]. After oral dosing of genistein, PND 5 pups have between 8 and 16 h of total genistein serum concentrations comparable to that of infants fed soy-based formula. n = 6–10 pups/time point. Data are shown as mean ± SEM.

FIG. 2.

Orally administered genistein affects estrogen-sensitive targets in the neonate. A) Body weights from PND 1 to PND 21. Body weights in the two groups were comparable. Vehicle: n = 9; genistein: n = 9. B) Thymic weight normalized to body weight. Thymic weight:body weight ratio decreased by 28% in genistein-treated pups. Vehicle (white bar): n = 10; genistein (black bar): n = 15. C) Uterine weight normalized to body weight. Uterine weight:body weight ratio increased by 41% in genistein-treated pups. Vehicle (white bar): n = 9; genistein (black bar): n = 14. Data are shown as mean ± SEM. #P < 0.01. D) Control animals showed robust epithelial progesterone receptor expression (arrow), whereas this was nearly absent in neonatally exposed genistein-treated animals (E). Bar = 50 μm. B–E were collected and measured from PND 5 pups. For all results shown, n represents one animal from a different litter. n = 4 for both treatment groups.

FIG. 3.

Neonatal genistein increases the incidence of MOFs in neonates and adults. Top: PND 5 ovaries; vehicle: n = 12; genistein: n = 10. Bottom: 4-mo-old adult ovaries; n = 6 for both vehicle and genistein. Each n represents one animal from a different litter. Data are shown as mean ± SEM. *P < 0.05.

FIG. 4.

Neonatal genistein exposure decreases the overall number of estrous cycles in 6-mo-old females because of prolonged diestrous phase. A) Mice treated neonatally with genistein had a significant reduction in the number of cycles (#P = 0.002) over a 25-day period. B) Stacked bar graph showing the percentage of animals with prolonged diestrous phase, which was defined as diestrous phase lasting more than 3 days. White bar indicates percentage of animals with normal diestrous phase, and black bar represents the percentage of animals with prolonged diestrous phase. Vehicle: n = 12; genistein-treated: n = 20. Data are presented as the mean.