* Division of Neurotoxicology and
Division of Biochemical Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas 72079;
Laboratory of Toxicology, Environmental Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Received October 19, 1999; accepted February 4, 2000
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ABSTRACT |
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Key Words: estrogen; phytoestrogen; isoflavone; endocrine disruptor; nutrition; chronic exposure.
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INTRODUCTION |
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Although the effects of estrogens on behavior are well documented, reports of phytoestrogen effects are limited. Recent reports from our laboratory suggest genistein effects on adult male mating behavior, but limited effects on other behaviors (Flynn et al., 1999a,b). A similar hormonally active phytoestrogen, coumestrol, has also been shown to affect mating behavior in male rats (Whitten et al., 1995). Sexually dimorphic behaviors, (i.e., those that differ according to sex), can vary across species but many are known to be estrogen sensitive in both rodents and humans (Collaer and Hines, 1995 for review). In rodents, such sexually dimorphic behaviors include not only mating and maternal behaviors, but also open field exploration, running wheel activity, and preference for flavored solutions, all of which increase following estrogen exposure (Beatty, 1979; Schulkin, 1999 for review). Because behavior represents the sum outcome of events at the molecular, cellular, and organ levels, several recent workshops have recommended including behavioral assessments in studies of the adverse effects of endocrine disruptors (Kavlock et al., 1996
; Tilson, 1998
). The sensitivity of sexually dimorphic behaviors to estradiol suggests that alterations may follow exposure to a weak environmental estrogen such as genistein.
Human genistein exposure comes primarily from the consumption of soy products. Although other factors associated with soy-containing diets may be involved, the multiple biochemical activities of genistein have focused attention on this compound. In the typical Asian diet, 1.5 mg of genistein or other isoflavones/kg/day may be ingested, whereas the typical Western diet contains less than 0.2 mg/kg/day (Coward et al., 1993). Soy-containing infant formulas and breast milk of mothers consuming soy foods contain isoflavones such as genistein (Franke et al., 1998
; Setchell et al., 1998
), and isoflavones have been quantified in amniotic fluid, making placental transfer likely (Adlercreutz et al., 1999
). Genistein-fed adult rats are reported to have blood genistein levels ranging up to 8.9 µM, similar to blood levels in adult and infant humans (up to 1.2 and 7 µM, respectively) (Holder et al., 1999
). Thus, genistein is a relatively common component of the human diet for certain adults and infants, and is present in rodent and human blood after dietary exposure. Its estrogenic activity raises the possibility of genistein-induced effects on the estrogen-sensitive sexual differentiation of the central nervous system, including the neural basis of sexually dimorphic behaviors. The current study examined four such behaviors in rats exposed to genistein from gestation through adulthood: open field activity, running wheel activity, and taste preference, which are estrogen sensitive, and play, which is androgen sensitive.
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MATERIALS AND METHODS |
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Food and water were provided ad libitum. Two weeks prior to mating, dams were shifted from the standard autoclaved NIH-31 pellet diet to an irradiated soy- and alfalfa-free diet (5K96, purchased from Purina Mills, St. Louis, MO). This diet is based on the NIH-31 formula, except that casein replaces the protein contributed by soy and alfalfa, soy oil is replaced by corn oil, and the vitamin mix is adjusted for irradiation. The control diet was assayed for genistein and daidzein after hydrolysis of conjugates. The concentrations of both genistein and daidzein were found to be below the limit of detection (0.5 ppm). Beginning on GD 7 and continuing through offspring weaning on postnatal day (PND) 22, dams consumed 5K96 chow containing 0 (n = 12), 25 (n = 11), 250 (n = 12), or 1250 (n = 12) ppm genistein (Toronto Research Chemicals, North York, Ontario, Canada). For a 250-g rat consuming 20 g chow per day, these doses are approximately equivalent to 0, 2, 20, and 100 mg genistein/kg/day, respectively. Genistein purity was greater than 99% as assessed by the Division of Chemistry at NCTR using HPLC analytical methods, mass spectrometry, and 1H-NMR. Genistein was mixed into the standard 5K96 feed by the Diet Preparation Staff, Bionetics at NCTR, and batches of feed were analyzed by the Division of Chemistry.
The day of birth was designated PND 1. On PND 2, litters were culled to eight pups, four males and four females, and the pups were tattooed on the dorsal surface of the paw for identification purposes. Litters remained with their biologic dam whenever possible; cross-fostering of a pup with another dam to maintain litter size and sex distribution was rare and was only done within treatment groups. Offspring were weaned on PND 22 and housed two per cage with a same-sex sibling. Weaned pups continued on the dosed 5K96 diet until sacrifice on PND 77.
All animal procedures were conducted under an animal protocol approved by the NCTR Institutional Animal Care and Use Committee (IACUC).
Physical Measures
Body weight and food intake were measured weekly for each dam on GD 1, 7, 14, and 21, and on postparturitional days (PPD) 8, 15, and 21. Weight gain during pregnancy was calculated by subtracting the day 7 body weight measurement from the day 21 measurement (if parturition had not occurred).
Gestational duration and reproductive outcomes (total and live pups/litter, total and live sex ratios, and average live pup birth weight) were assessed on the day of birth. Offspring weights were measured on PND 2, 8, 15, 21, 28, 42, 56, 70, and 77.
Behavioral Assessments of Offspring
All nonautomated assessments were conducted by testers blind to the experimental treatment.
Open field activity.
One male and one female from each of the 47 litters were tested before puberty (PNDs 2224), a different pair were tested at approximately the time of puberty (PNDs 4345), and a third pair were tested as young adults (PNDs 6567). Activity was measured for individual animals in a Plexiglas cube (46.5 x 46.5 cm) bisected by photobeams, as previously described (Ferguson et al., 1993). Three consecutive daily sessions of 60 min were run in lighted conditions. Activity was recorded as the number of photobeam breaks per 3-min period (total of 20 3-min periods/test session).
Play behavior.
Play behavior was assessed in 94 dyads of prepubertal animals (PND 35) using methodology previously described (Pellis et al., 1994). On PND 34, two males and two females from each of the 47 litters were individually housed in clean cages. Twenty-four hours later, the animals were reunited with their same-sex sibling in a clean cage and the total number of pins exhibited during the subsequent 5-min test period was recorded. A pin was defined as one animal having its dorsal surface to the ground while the other animal was on top (Panksepp, 1981
).
Residential running wheel activity.
From PNDs 6377, one male and one female from each litter were individually housed in a residential running wheel as previously described (Ferguson et al., 1993). Each apparatus was a standard polycarbonate housing cage, with all conditions as described above, but equipped with a running wheel (34.3-cm diameter). Number of wheel revolutions per 12-h dark period and 12-hr light period was recorded for each of 14 consecutive days.
Intake of flavored solutions.
For 3 consecutive days over the course of 1 week (PNDs 6975), intake of two flavored solutions was determined in one male and one female from each litter. The animals were individually housed in their home vivarium. Intake of a sweet solution containing 0.03% saccharin (ICN Biochemicals Inc., Aurora, OH) in water was measured on PNDs 6971 by placing two bottles on each animal's cageone containing regular water and the other containing the saccharin solution. Intake of a salt solution containing 3.0% sodium chloride (ICN Biochemicals Inc., Aurora, OH) in water was measured on PNDs 7375 by placing two bottles on each animal's cageone containing regular water and the other containing the salt solution. If spillage was observed, that day's bottle weight was not used and a fresh bottle weight was taken. All bottles were weighed once daily and amount consumed in milliliters per day was divided by PND 70 body weight to yield milliliters consumed/day/kg.
Statistical Analyses
All analyses were conducted using the litter as the unit of analysis. Analyses of variance (ANOVA) were used to determine treatment effects using genistein dose as a between-groups variable. Several analyses involved repeated measures over days and were done with multivariate techniques, which were implemented using a mixed model. Post-hoc tests (two-sided Dunnett's or Student-Newman-Keuls) were applied only if analysis of variance attained significance at or below the 0.05 level. Homogeneity of variance was tested using a likelihood ratio test based on the negative log likelihood from two different models, a mixed auto regressive, and a mixed heterogeneous auto regressive model. If chi square yielded p < 0.05, the heterogeneous variance model was rejected.
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RESULTS |
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Gestational duration, total and live pups/litter, and total and live sex ratios did not differ significantly among treatment groups (Table 1). There was a significant effect of genistein treatment on the litter mean birth weight/live pup (F(3,60) = 4.24, p < 0.01), and post-hoc tests revealed a significantly lower mean birth weight in the 1250 ppm group compared to the control group (p < 0.05) (Table 1
).
There was a significant interaction of genistein dose with age on offspring body weight (p < 0.0001; F(9,690) = 119.77; Fig. 2). Post-hoc tests showed that for both sexes the 1250 ppm group had significantly lower body weights than same-sex control groups on PNDs 4277 (p < 0.05). On PNDs 70 and 77, males of the 25 ppm group also had significantly decreased body weights when compared to the control group, and on PND 70 males of the 250 ppm group also differed from controls (p < 0.05).
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DISCUSSION |
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Food intake in the 1250 ppm dams was decreased between GD 714, and animals were decreased in body weight at GD 21,s although no dam had delivered by that date. Thus, the decreased body weight of the high-dose dams was unrelated to early parturition, and may have been the result of the earlier decrease in food consumption or of a genistein-induced decrease in fetal growth. The latter is supported by the low birth weight of the male and female 1250 ppm pups. This was followed by a decreased weight during nursing and a leveling off at about 85% of control weight by PND 77. A parallel study using the same experimental conditions also found decreased body weight in offspring exposed to 1250 ppm genistein (Delclos et al., 1999), although food intake was not recorded in either study. Despite the 15% decrease in body weight in high-dose pups, genistein-induced effects on behavior were not extensive. Undernutrition during development can cause alterations in aggression (Lucion et al., 1996
), but appears to have little effect on open field exploration, social, or mating behaviors (Tonkiss et al., 1987
), and had no effect on running wheel activity in this study.
The genistein effect on pinning frequency during PND 35 play assessment was not treatment related, in that no treated group differed from the control group, nor was it sexually dimorphic, in that the treatment effect did not interact with sex. Play behavior or play fighting is more common in juvenile males than females in several diverse species (Beatty, 1984 for review), and our control data show the expected sexual dimorphism, with males pinning an average of 21% more than females. The absence of a genistein effect on play is not surprising, given that the organizational effects of early androgen exposure are thought to mediate play behavior in rats. Neonatal castration decreased play in males, but treatment with an aromatase inhibitor to prevent estrogen formation did not (Meaney et al., 1983).
Animals exposed to high-dose genistein consumed more of a salt-flavored solution, which was not attributable to an overall increase in fluid consumption. Rodents are known to ingest salt even when not sodium deprived, with females showing a greater intake of salty solutions than males (Krecek et al., 1972). As gonadectomy of adults does not change their pattern of salt intake (Chow et al., 1992
), and estrogen treatment of ovariectomized adults likewise has no effect (Mascarenhas et al., 1992
), perinatal hormone effects appear paramount in the regulation of this behavior.
Neonatal females treated with testosterone developed a male pattern of salt intake (i.e., decreased) as adults (Krecek, 1973), but those treated with the nonaromatizable androgen dihydrotestosterone did not (Chow et al., 1992
). Thus it is estrogen, aromatized from testosterone, that appears to organize the male brain for a pattern of lower salt consumption in adulthood. Females ovariectomized as neonates, however, also developed a male pattern of sodium consumption as adults (i.e., decreased), and neonatal males exposed to estrogen became feminized in their salt intake, suggesting a need for early exposure to estrogens for the normal female pattern of high salt intake to develop (Krecek, 1978
). Following this reasoning, an estrogenic compound such as genistein would be expected to feminize males and hyperfeminize females, thus increasing adult salt consumption. Such a hypothesis would predict the current results.
Male offspring of the 1250 ppm group consumed an average of 68% more of the salt solution than controls, and the effect was more robust in females, which consumed 90% more than same-sex controls. These results suggest that sodium chloride intake in the adult rat may be an indicator of neonatal exposure to weak estrogenic agents. The apparent disruption of salt intake patterns by genistein suggests an interference with the normal patterns of sexually dimorphic central nervous system development by this weak estrogen. This is consistent with recent observations from our laboratory of increased salt solution consumption in rats exposed to high doses of three other estrogen mimics, ethinyl estradiol, nonylphenol, and methoxychlor (Ferguson et al., 1999), and with reports of genistein effects on other sexually dimorphic systems (Levy et al., 1995).
The absence of genistein-related effects on open field exploration, running wheel activity, and saccharin intake suggest that these behaviors, though sensitive to endogenous estrogens, are not as sensitive to weak environmental estrogens. The current data confirm that these three behaviors were sexually dimorphic in untreated adult animals. Although not statistically significant, control females were somewhat more active in the open field (563 beam breaks per test session at PND 6567 versus 432 for control males), supporting previous reports of increased open field activity in female rats appearing around PND 5060, shortly after puberty (Blizard et al., 1975). Control females exhibited increased activity in running wheel tests as well, running an average of 1679 revolutions per 12-h dark period versus 877 for males, and control females ingested more sweet solution than males, 231 ml/day/kg versus 175. The absence of any genistein-related effects on these behaviors may be due to the relatively weak biologic activity of genistein compared to estradiol. Genistein has a much lower affinity for the estrogen receptor, 95% less than estradiol for the
receptor and 60% less for ß (Kuiper et al., 1997
), making it possible that some receptor-mediated estrogenic effects could require very high doses of genistein. In fact, other reports have noted that 500 mg/kg is the minimum neonatal dose required to produce adult abnormalities in hypothalamic structure and reproductive physiology (Faber and Hughes, 1993
; Lamartiniere et al., 1998
).
These results suggest that there are some alterations in nonreproductive sexually dimorphic behaviors in rats exposed to genistein at doses approximating relatively high human exposure estimates. Genistein is one of several potential endocrine disruptors now being studied under an interagency agreement among the National Institute of Environmental Health Sciences (NIEHS), the National Toxicology Program (NTP), and the National Center for Toxicological Research (NCTR) (Newbold and Delclos, 1999). These studies will evaluate the potential adverse effects of continuous exposure to endocrine-disrupting chemicals. Range-finding studies will evaluate several physiologic, histologic, and behavioral endpoints, as well as establish doses for later multigenerational studies. It has long been known that sexually dimorphic behaviors may reflect an animal's current hormonal status as well as reflecting underlying organizational differences in neuroanatomy that occur following exposure to hormones during critical developmental periods. These sensitive periods occur around the time of birth in rodents and can be disrupted by inappropriate types or amounts of sex-specific hormones. The current study does not distinguish between organizational and activational hormone effects, but future studies will address this confound. As there is evidence of adverse effects from developmental exposure to other estrogen mimics (e.g., DES) (Robboy et al., 1977
), and there is increased attention to the possible beneficial effects of genistein, it is particularly prudent to fully investigate possible deleterious effects resulting from prolonged genistein exposure.
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ACKNOWLEDGMENTS |
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NOTES |
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