TY - JOUR
AU - Gray, L., Earl
AB - Abstract Many chemicals released into the environment display estrogenic activity including the oral contraceptive ethinyl estradiol (EE2) and the plastic monomer bisphenol A (BPA). EE2 is present in some aquatic systems at concentrations sufficient to alter reproductive function of fishes. Many concerns have been raised about the potential effects of BPA. The National Toxicology Program rated the potential effects of low doses of BPA on behavior and central nervous system (CNS) as an area of “some concern,” whereas most effects were rated as of “negligible” or “minimal” concern. However, the number of robust studies in this area was limited. The current study was designed to determine if maternal exposure to relatively low oral doses of EE2 or BPA in utero and during lactation would alter the expression of well-characterized sexually dimorphic behaviors or alter the age of puberty or reproductive function in the female Long-Evans rat offspring. Pregnant rats were gavaged with vehicle, EE2 (0.05–50 μg/kg/day), or BPA (2, 20, and 200 μg/kg/day) from day 7 of gestation to postnatal day (PND) 18, and the female offspring were studied. EE2 (50 μg/kg/day) increased anogenital distance and reduced pup body weight at PND2, accelerated the age at vaginal opening, reduced F1 fertility and F2 litter sizes, and induced malformations of the external genitalia (5 μg/kg). F1 females exposed to EE2 also displayed a reduced (male-like) saccharin preference (5 μg/kg) and absence of lordosis behavior (15 μg/kg), indications of defeminization of the CNS. BPA had no effect on any of the aforementioned measures. These results demonstrate that developmental exposure to pharmacologically relevant dosage levels of EE2 can permanently disrupt the reproductive morphology and function of the female rat. ethinyl estradiol, bisphenol A, rat, reproductive toxicology, sexually dimorphic behavior Many chemicals with estrogenic activity are present in the environment, including ethinyl estradiol (EE2; Jobling et al., 1998, 2006) and bisphenol A (BPA; Kang et al., 2007). EE2 is a used primarily in combination medications for a variety of conditions in women. Unintentional exposure of the developing human fetus to EE2 can occur if oral contraception is continued through the early months of undetected pregnancy (reviewed in Smithells [1981]). Due to its widespread pharmaceutical use and relatively long half-life, EE2 has been detected in river systems in the United States and Europe as a contaminant from sewage treatment works (Kolpin et al., 2002; Zuo et al., 2006). In some aquatic systems, concentrations of estrogens are sufficient to induce adverse effects in fish, including ovotestes in males and reduced fecundity and population levels (Brion et al., 2004; Jobling et al., 1998, 2006; Kidd et al., 2007; Lange et al., 2008). Effluents also may contain natural estrogens like estradiol and estrone and occasionally industrial chemicals like alkylphenols. In fish, for example, only 0.1 ng/l EE2 induces vitellogenin, 0.1–15 ng/l can affect reproductive development, and 2–10 ng/l can reduce fecundity (Nash et al., 2004). Thus, given that 5.7% of 139 U.S. streams had > 5 ng/l estrogen equivalents (EEQs; Kolpin et al., 2002), estrogens are a potential cause of reproductive dysfunction in fish. There are far less data on levels of EEQs in drinking water. BPA is a monomer used in the manufacturing of polycarbonate products, and other uses, that displays estrogenic activity in vitro and when administered by injection in vivo (Chapin et al., 2008; Kanno et al., 2003; Gray and Ostby, 1998). BPA is approximately a 105-fold less potent than estradiol (Chapin et al., 2008) and 106-fold less potent than EE2 in stimulating estrogen receptor (ER)–dependent gene expression (EC50 for gene induction in T47D KBluc cells = 6.7 × 10−7 and 7.3 × 10−13 for BPA and EE2, respectively (Vickie S Wilson and Dieldrich Bermudez, personal communication). In vivo, sc administration of BPA or EE2 stimulates uterine weight (ED50 of 416 mg/kg/day for BPA versus 0.0008 mg/kg/day for EE2; Kanno et al., 2001, 2003) and induces estrogen-dependent lordosis behavior in adult female rats (Gray and Ostby, 1998; Spiteri et al., 1980). When given orally, EE2 stimulated uterine weight (ED50 of 2.5 μg/kg/day), whereas oral BPA at doses as high as 1 g/kg/day were insufficient to achieve an ED50 (doses of 20–375 mg/kg/day had no effect whereas 600 and 1000 mg/kg/day induced small but significant increases in weight; Kanno et al., 2003; Supplementary Data). When administered to weanling female rat using the weanling female rat using the U.S. Environmental Protection Agency (USEPA) Endocrine Disruptor Screening Program protocol for the Pubertal Female Assay (http://www.epa.gov/endo/pubs/assayvalidation/status.htm), EE2 accelerated the onset of vaginal opening (VO) by 10 days after only 2–3 days of treatment (Supplementary Data) and induced persistent vaginal estrus, whereas dosage levels of BPA up to 600 mg/kg/day were without effect on these estrogen-regulated end points. In multigenerational studies, oral BPA administration also failed to accelerate the age at VO or induce persist vaginal estrus in either Sprague-Dawley (SD) and Alderley Park (Wistar derived) rats (Ema et al., 2001; Tinwell et al., 2002; Tyl et al., 2002; Supplementary Data), whereas xenoestrogens like methoxychlor (Gray and Ostby, 1998; Gray et al., 1989), 4 nonylphenol (Chapin et al., 1999), and genistein (Casanova et al., 1999) do accelerate the age at VO. Although these results suggest that BPA may be too weak to induce adverse effects during development via a nuclear ER-mediated mechanism of action when administered orally, it has been hypothesized that BPA may induce adverse effects via the seven-transmembrane orphan receptor GRP30 which has been purported to be responsive to estradiol and BPA (Thomas and Dong 2006; Thomas et al., 2005). However, the functional role of GRP30 is itself controversial. In a recent review, Levin (2009) reported that at the cellular level, the early experimental support for this idea is simply not strong. In vitro, estradiol showed minimal binding to GRP30 and modest signal transduction, and in vivo, a putative GRP30 agonist, G1, did not stimulate estrogen-like effects in the uterus or mammary gland of mice and none of the four GRP30 knockout mouse models shows cycling or fertility abnormalities (Levin 2009). In addition to GRP30, in vitro studies suggest that low doses of BPA might produce adverse effects via activation of membrane-bound ER alpha (Wozniak et al., 2005) or by activating cyclic AMP dependent protein kinase (PKA) and cyclic GMP dependent protein kinase (PKG) via a membrane G-protein–coupled estrogen receptor (Bouskine et al., 2009). However, the functional significance of these in vitro observations in vivo remains to be determined. It also has been suggested that the fetus and neonate are far more sensitive to estrogens and other endocrine disrupters than are pubertal or adult female rodents (Guillette and Moore, 2006). In particular, the potential effects of exposure to BPA on the development of sexually dimorphic behavior have been noted as an area of special concern by the National Toxicology Program (NTP). The NTP expressed “some concern” for adverse effects of BPA on the brain and behavior based upon “limited evidence of adverse effects” of low doses in rodent studies. When the Center for the Evaluation of Risk to Human Reproduction Expert Panel on BPA (Chapin et al., 2008) and NTP (2008) evaluated the studies on the neural and behavioral effects of BPA, the majority of the more than 40 studies were found to be “inadequate” for further evaluation, and for this reason, the NTP monograph cites only seven rodent (four with mice, three with rats) behavioral studies in its determination of “some concern” for low-dose neural and behavioral effects of BPA. Relevant to the current study which examined the effects of exposure to BPA on the behavior of the female rat, it is noteworthy that only one of these three studies exposed rats during perinatal life (Negishi et al., 2004) and only male offspring were examined. One objective of the current study was to determine if relatively low oral doses of EE2 or BPA would alter behavioral sexual differentiation, the age at puberty, and the fertility of female Long-Evans (LE) hooded rat offspring when administered orally to the dam during gestation and lactation. Pregnant rats were gavaged once daily from gestation day (GD) 7 through postnatal day (PND) 18 to expose their offspring during the period of sexual differentiation of the reproductive organs as well as the neonatal period of sexual differentiation of the brain. We selected the LE rat strain because the developing reproductive system is sensitive to both estrogenic endocrine-disrupting chemicals (EDCs), such as methoxychlor (Gray et al., 1988, 1989), and anti-androgenic compounds (Gray et al., 1999). Furthermore, the LE rat strain is commonly used for the study of hormonal regulation of reproductive behaviors (Levine and Mullins, 1964). The doses of EE2 used herein spanned four log units, including doses above, at, and below those pharmacologically effective in humans, and the study follows the dosing protocol and dosing regime of a reproduction study which used the SD rat (Sawaki et al., 2003). The dosage levels of BPA used in the current study are commonly used in “low-dose” BPA studies with rats (Akingbemi et al., 2004; Ema et al., 2001; Tinwell et al., 2002) with the lowest dose (2 μg/kg body weight/day) exceeding the median estimated intake by about 40-fold in the United States based upon National Health and Nutrition Examination Survey data (maximum = 3.47 μg/kg body weight/day; median = 0.0505; 25th to 95th percentile 0.0235 to 0.2722; from the NTP [2008] BPA Monograph derived from Calafat et al. [2008] and Lakind and Naiman [2008]). It was not the intention of this study to determine the sensitivity of the female LE rat to developmental exposure to BPA over a broader range of doses. Since the organization of some rodent sexually dimorphic behaviors during perinatal life is imprinted by androgens and others by estrogens, exposure to EDCs that act via these mechanisms can permanently imprint the nervous system and behavior. Although rodents display a wide range of sexually dimorphic behaviors, the organizational role of neonatal hormones has only been rigorously confirmed for a few behaviors. We chose to study saccharin preference and lordosis behavior in female rats because the defeminizing role of neonatal hormones has been well established for these behaviors and exposure to exogenous steroids produces robust changes in the behavior of treated female rats. Female rats neonatally defeminized by steroidal hormones display reduced saccharin preference (Wade and Zucker, 1969), reduced fertility (due to the loss of cyclicity of the hypothalamic-pituitary-gonadal [HPG] axis), and lower levels of female sex behavior (Blake and Ashiru, 1997; Herath et al., 2001; Kouki et al., 2003, 2005; Sierra and Uphouse, 1986). The selection of well-characterized sexually dimorphic behaviors that are imprinted by estrogens during development enables us to clearly determine if EE2 and/or BPA have estrogen-like effects on organization of sexually dimorphic behaviors. We also decided to determine if developmental exposure to either EE2 or BPA would alter spontaneous locomotor activity levels in a Figure-8 maze. Preliminary data indicated that female rats displayed significantly higher total 24-h activity levels in the Figure-8 maze than do male rats (Gray Jr, unpublished observation). Although this behavior is sexually dimorphic in adult rats under these test conditions, the role of neonatal estrogens in the development of this sex difference is unknown. The current study directly addresses some of the concerns expressed by the NTP review of the literature on the potential “low-dose” effects of BPA and the effects of this chemical on the development of the brain behavior. The effects of EE2 and lack of effect of BPA on the dams and their male offspring have already been published (Howdeshell et al., 2008). MATERIALS AND METHODS Animals Adult female LE rats (Charles River Laboratories, Raleigh, NC) of approximately 90 days of age were mated by the supplier and shipped on GD2. Mating was confirmed by sperm presence in vaginal smears (day of sperm plug positive = GD1). All animals were housed individually in transparent, 20 × 25 × 47-cm polycarbonate cages with laboratory-grade, heat-treated pine shavings (Northeastern Products, Warrensburg, NY) with a 14:10 light:dark cycle at 20°C–24°C and 40–50% relative humidity. All caging used in the current experiment was clear and without evidence of significant wear. Only clear cages were used in the current study because some have expressed concern about BPA exposures from old polycarbonate cages. It has been shown that water samples stored for 1 week in old cages display estrogenicity in vitro; however, estrogenic effects were not seen in vivo as housing prepubertal mice in old cages did not significantly increase uterine weight (Howdeshell et al., 2003). Pregnant and lactating dams were fed Purina Rat Chow 5008 ad libitum, and weanling and adult rats were fed Purina Rat Chow 5001 ad libitum. Animals were provided constant access to filtered (5-μm filter) municipal drinking water (Durham, NC) via an automatic watering system. Water was tested monthly for Pseudomonas and every 4 months for a suite of chemicals, including pesticides and heavy metals. The current study was conducted under a protocol approved by the USEPA's National Health and Environmental Effects Research Laboratory Institutional Animal Care and Use Committee in an animal facility approved by the Association for Assessment and Accreditation of Laboratory Animal Care. Control animals used in this study purchased from the same supplier and maintained, as above. Dosage Levels and Administration of Chemicals Pregnant rat dams were randomly assigned to treatment groups on GD7 within each cohort prior to dosing. Laboratory-grade corn oil (CAS 8001-30-7; Lot 062K006), EE2 (CAS 57-63-6; Lot 103K1230; purity 98%), and BPA (CAS 80-05-7; Lot 03105ES; purity ≥ 99%) were purchased from Sigma-Aldrich (St Louis, MO). Dams were dosed via oral gavage from GD7 through PND18 with 0 (corn oil, vehicle control), EE2 at 0.05, 0.5, 1.5, 5, 15, or 50 μg/kg/day, or BPA at 2, 20, or 200 μg/kg/day. The doses were delivered in 0.5-ml corn oil/kg body weight; thus, all dams in the study received the same amount of vehicle per body weight. The dams were weighed daily during the dosing period to adjust the administered dose for body weight changes during pregnancy and lactation and to monitor their health. This study was performed in two blocks. The first block involved 169 dams with 13–29 dams per treatment group: oil vehicle, EE2 (0.05, 0.5, 5, or 50 μg/kg/day), or BPA (2, 20, or 200 μg/kg/day). The second block (block 2) was performed to expand the range of EE2 doses tested and involved 82 dams with 6–14 dams per treatment group: oil vehicle, EE2 (0.05, 0.15, 0.5, 1.5, 5, 15, or 50 μg/kg/day), or BPA (20 or 200 μg/kg/day). The effects of EE2 and BPA on the dams and the F1 male offspring were previously published by Howdeshell et al. (2008). Maternal body weight gain during pregnancy was calculated from inception of dosing (GD7) to GD20, and the analysis of these data included only those dams that were pregnant and survived through GD20. Maternal body weight gain during lactation was calculated from PND2 through the end of dosing (PND18) and included only those dams with pups surviving to weaning. Maternal and F1 Neonatal and Infant Data A summary of the end points measured in F1 females is shown in Table 1. On PND2, the sex, body weight, and anogenital distance (AGD) of each female pup were recorded; the observer measuring these end points was blind as to the treatment group (samples sizes are given in Fig. 1). AGD was measured as previously described (Hotchkiss et al., 2004). The AGD was defined as the distance between the anterior end of the anus and the posterior end of the genital papilla. On PND14, the female rats were sexed and weighed and the number of areolae/nipples on the ventral surface of each female was determined. TABLE 1 End Points Evaluated in F1 Female LE Rats and F0 Dams Treated Orally with BPA or EE2 during Gestation and Lactation Chemical End points BPA EE2 AGD PND2 No effect Increased at 50 Female F1 pup body weight (g) PND2 No effect Reduced at 50 Age at vaginal opening No effect Accelerated at 5 Weight at vaginal opening No effect Reduced at 5 % Displaying cleft phallus at weaning No effect Increased at 5 External genitalia UVD (mm) at necropsy No effect Reduced at 5 Breeding data: fecundity No effect Reduced at 5 Saccharin preference No effect Male-like at 5 LQ: activated with 50 mg adult EE2 No effect Reduced at 15 Locomotor activity in Figure-8 maze (photocell counts): ovariectomized No effect No effect Locomotor activity in Figure-8 maze (photocell counts): ovariectomized plus oral EE2 No effect No effect F1 weaning weight (g) No effect Reduced at 50 Chemical End points BPA EE2 AGD PND2 No effect Increased at 50 Female F1 pup body weight (g) PND2 No effect Reduced at 50 Age at vaginal opening No effect Accelerated at 5 Weight at vaginal opening No effect Reduced at 5 % Displaying cleft phallus at weaning No effect Increased at 5 External genitalia UVD (mm) at necropsy No effect Reduced at 5 Breeding data: fecundity No effect Reduced at 5 Saccharin preference No effect Male-like at 5 LQ: activated with 50 mg adult EE2 No effect Reduced at 15 Locomotor activity in Figure-8 maze (photocell counts): ovariectomized No effect No effect Locomotor activity in Figure-8 maze (photocell counts): ovariectomized plus oral EE2 No effect No effect F1 weaning weight (g) No effect Reduced at 50 Note. If an effect was seen, the lowest-observed effect level is given for the chemical in micrograms/kg/day. Open in new tab TABLE 1 End Points Evaluated in F1 Female LE Rats and F0 Dams Treated Orally with BPA or EE2 during Gestation and Lactation Chemical End points BPA EE2 AGD PND2 No effect Increased at 50 Female F1 pup body weight (g) PND2 No effect Reduced at 50 Age at vaginal opening No effect Accelerated at 5 Weight at vaginal opening No effect Reduced at 5 % Displaying cleft phallus at weaning No effect Increased at 5 External genitalia UVD (mm) at necropsy No effect Reduced at 5 Breeding data: fecundity No effect Reduced at 5 Saccharin preference No effect Male-like at 5 LQ: activated with 50 mg adult EE2 No effect Reduced at 15 Locomotor activity in Figure-8 maze (photocell counts): ovariectomized No effect No effect Locomotor activity in Figure-8 maze (photocell counts): ovariectomized plus oral EE2 No effect No effect F1 weaning weight (g) No effect Reduced at 50 Chemical End points BPA EE2 AGD PND2 No effect Increased at 50 Female F1 pup body weight (g) PND2 No effect Reduced at 50 Age at vaginal opening No effect Accelerated at 5 Weight at vaginal opening No effect Reduced at 5 % Displaying cleft phallus at weaning No effect Increased at 5 External genitalia UVD (mm) at necropsy No effect Reduced at 5 Breeding data: fecundity No effect Reduced at 5 Saccharin preference No effect Male-like at 5 LQ: activated with 50 mg adult EE2 No effect Reduced at 15 Locomotor activity in Figure-8 maze (photocell counts): ovariectomized No effect No effect Locomotor activity in Figure-8 maze (photocell counts): ovariectomized plus oral EE2 No effect No effect F1 weaning weight (g) No effect Reduced at 50 Note. If an effect was seen, the lowest-observed effect level is given for the chemical in micrograms/kg/day. Open in new tab FIG. 1. Open in new tabDownload slide Effects of EE2 and BPA on female rat offspring AGD at 2 days of age. AGD was increased by EE2 treatment at 50 μg/kg/day. BPA had no effect. F = 1.6 (9,116 df) from the ANOVA, p < 0.12. Samples sizes for the groups were (number pups/numbers of litters) control (156,24), BPA20 (43,6), BPA200 (58,9), EE2 0.05 (109,17), EE2 0.15 (37,6), EE2 0.5 (122,19), EE2 1.5 (61,9), EE2 5 (113,21), EE2 15 (40,7), and EE2 50 (28,8). Values on the figure are litter means and SEs. “#” indicates the mean differed from control by a Dunnett's post hoc test p < 0.05. FIG. 1. Open in new tabDownload slide Effects of EE2 and BPA on female rat offspring AGD at 2 days of age. AGD was increased by EE2 treatment at 50 μg/kg/day. BPA had no effect. F = 1.6 (9,116 df) from the ANOVA, p < 0.12. Samples sizes for the groups were (number pups/numbers of litters) control (156,24), BPA20 (43,6), BPA200 (58,9), EE2 0.05 (109,17), EE2 0.15 (37,6), EE2 0.5 (122,19), EE2 1.5 (61,9), EE2 5 (113,21), EE2 15 (40,7), and EE2 50 (28,8). Values on the figure are litter means and SEs. “#” indicates the mean differed from control by a Dunnett's post hoc test p < 0.05. Vaginal Opening After weaning at PND23, female rats were weighed and checked for VO every other day until completion (sample sizes for weight and VO data are shown in Figs. 2 and 3, respectively). Animals in the 50-μg/kg/day EE2 treatment group were excluded from the statistical analysis of these data because their malformed genitalia made it difficult to determine when VO had occurred. FIG. 2. Open in new tabDownload slide Weights at 2 days of age (Fig. 2a) and weaning (Fig. 2b) were reduced in female F1 rats by EE2 treatment at 50 μg/kg/day. BPA had no effect. Weight at 2 days of age: F = 2.38 (10,172 df), p < 0.015. Samples sizes for the groups were (number pups/numbers of litters) control (216,33), BPA2 (48,7), BPA20 (81,13), BPA200 (89,15), EE2 0.05 (114,19), EE2 0.15 (37,6), EE2 0.5 (186,29), EE2 1.5 (61,9), EE2 5 (176,30), EE2 15 (40,7), and EE2 50 (58,15). Weight at weaning: F = 1.6 (9,59 df) from the ANOVA, p > 0.1. “#” Indicates the mean differed from control by a Dunnett's post hoc test p < 0.05. Samples sizes for the groups were (number pups/numbers of litters) control (42,8), BPA20 (32,6), BPA200 (47,8), EE2 0.05 (33,6), EE2 0.15 (35,6), EE2 0.5 (44,7), EE2 1.5 (47,9), EE2 5 (45,9), EE2 15 (34,7), and EE2 50 (11,3). Values on the figure are litter means and SEs. “**” indicates the mean differs from control by p < 0.01. FIG. 2. Open in new tabDownload slide Weights at 2 days of age (Fig. 2a) and weaning (Fig. 2b) were reduced in female F1 rats by EE2 treatment at 50 μg/kg/day. BPA had no effect. Weight at 2 days of age: F = 2.38 (10,172 df), p < 0.015. Samples sizes for the groups were (number pups/numbers of litters) control (216,33), BPA2 (48,7), BPA20 (81,13), BPA200 (89,15), EE2 0.05 (114,19), EE2 0.15 (37,6), EE2 0.5 (186,29), EE2 1.5 (61,9), EE2 5 (176,30), EE2 15 (40,7), and EE2 50 (58,15). Weight at weaning: F = 1.6 (9,59 df) from the ANOVA, p > 0.1. “#” Indicates the mean differed from control by a Dunnett's post hoc test p < 0.05. Samples sizes for the groups were (number pups/numbers of litters) control (42,8), BPA20 (32,6), BPA200 (47,8), EE2 0.05 (33,6), EE2 0.15 (35,6), EE2 0.5 (44,7), EE2 1.5 (47,9), EE2 5 (45,9), EE2 15 (34,7), and EE2 50 (11,3). Values on the figure are litter means and SEs. “**” indicates the mean differs from control by p < 0.01. FIG. 3. Open in new tabDownload slide Exposure to EE2 at 0.5 and 5 μg/kg/day accelerated the age at VO in F1 female rats (significant 5 μg/kg/day; Fig. 3a) and they attained this landmark at a lighter weight (3b). The external genitalia of the F1 females in the 50 μg/kg/day were too malformed to accurately determine the age or weight at VO. BPA had no effect. Age at VO: F = 5.1 (5,32 df), p < 0.0015. Weight at VO: F = 4.6 (5,32 df), p < 0.003. Samples sizes for the groups for VO and weight at VO were (number pups/numbers of litters) control (21,5), BPA2 (44,7), BPA20 (29,6), BPA200 (30,6), EE2 0.5 (30,7), and EE2 5 (30,7). Values on the figure are litter means and SEs. “**” indicates the mean differs from control by p < 0.01. FIG. 3. Open in new tabDownload slide Exposure to EE2 at 0.5 and 5 μg/kg/day accelerated the age at VO in F1 female rats (significant 5 μg/kg/day; Fig. 3a) and they attained this landmark at a lighter weight (3b). The external genitalia of the F1 females in the 50 μg/kg/day were too malformed to accurately determine the age or weight at VO. BPA had no effect. Age at VO: F = 5.1 (5,32 df), p < 0.0015. Weight at VO: F = 4.6 (5,32 df), p < 0.003. Samples sizes for the groups for VO and weight at VO were (number pups/numbers of litters) control (21,5), BPA2 (44,7), BPA20 (29,6), BPA200 (30,6), EE2 0.5 (30,7), and EE2 5 (30,7). Values on the figure are litter means and SEs. “**” indicates the mean differs from control by p < 0.01. Fertility At weaning, a subset of the developmentally exposed female and male rats from the second block of the study were paired with an age-matched, non-siblings for at least 4 months and monitored regularly for the presence of litters (sample sizes for the females are shown in Fig. 4). Since the breeding pairs did not necessarily contain females and males from the same treatment group, the data were analyzed for treatment effects on both sexes. F2 pups were removed at birth. If a breeding pair produced at least one litter during this period, they were considered fertile. Fecundity, the total number of pups produced over a 4-month period also was determined. FIG. 4. Open in new tabDownload slide The fecundity (numbers of pups produced over four month continuous breeding period; Fig. 4a) and fertility (Fig. 4c) was reduced in F1 females exposed to EE2 treatment at 5, 15, and 50 μg/kg/day. BPA had no effect. Fecundity over 4 months: F = 5.25 (9,32 df), p < 0.0002. Samples sizes for the groups were (number pups/numbers of litters) control (11,5), BPA20 (6,5), BPA200 (9,6), EE2 0.05 (3,3), EE2 0.15 (6,5), EE2 0.5 (5,4), EE2 1.5 (6,4), EE2 5 (7,5), EE2 15 (6,4), and EE2 50 (2,1). Since some of the sample sizes were small, dose groups were pooled to display the consistency of effect on fecundity among the BPA, lower dose EE2, and higher dose EE2 groups (Fig. 4b). Pooled fecundity over 4 months: F = 14.0 (3,38 df), p < 0.0001. Samples sizes for the groups were (number pups/numbers of litters) control (11,5), BPA: 20 and 200 (15,11), EE2 Low: 0.05, 0.15, 0.5, and 1.5 (18,16), and EE2 High: 5.0, 15 and 50 (14,10). Values on the figure are litter means and SEs. “*” indicates the mean differs from control by p < 0.05. “**” indicates the mean differs from control by p < 0.01. FIG. 4. Open in new tabDownload slide The fecundity (numbers of pups produced over four month continuous breeding period; Fig. 4a) and fertility (Fig. 4c) was reduced in F1 females exposed to EE2 treatment at 5, 15, and 50 μg/kg/day. BPA had no effect. Fecundity over 4 months: F = 5.25 (9,32 df), p < 0.0002. Samples sizes for the groups were (number pups/numbers of litters) control (11,5), BPA20 (6,5), BPA200 (9,6), EE2 0.05 (3,3), EE2 0.15 (6,5), EE2 0.5 (5,4), EE2 1.5 (6,4), EE2 5 (7,5), EE2 15 (6,4), and EE2 50 (2,1). Since some of the sample sizes were small, dose groups were pooled to display the consistency of effect on fecundity among the BPA, lower dose EE2, and higher dose EE2 groups (Fig. 4b). Pooled fecundity over 4 months: F = 14.0 (3,38 df), p < 0.0001. Samples sizes for the groups were (number pups/numbers of litters) control (11,5), BPA: 20 and 200 (15,11), EE2 Low: 0.05, 0.15, 0.5, and 1.5 (18,16), and EE2 High: 5.0, 15 and 50 (14,10). Values on the figure are litter means and SEs. “*” indicates the mean differs from control by p < 0.05. “**” indicates the mean differs from control by p < 0.01. Methods for the Study of Saccharin Preference Pilot study with control male and female LE rats. Prior to the study of animals treated with EE2 or BPA, a study of saccharin preference was conducted with untreated LE rats to confirm that the behavioral methods we were planning to use to assess the effects of in utero EE2 and BPA displayed the expected sexual dimorphism. This experiment also was designed to determine if female rats displayed a greater preference for either 0.50% wt/vol (5 g of saccharin was added to a liter of deionized, distilled water) or 0.25% wt/vol (2.5 g of saccharin was added to a liter of water) saccharin solutions. Adult male (n = 3–5 per dose group) and adult female intact (n = 4 per dose group) LE rats were purchased from Charles River Laboratories. Animals were housed individually upon receipt and allowed 2 weeks to acclimate before testing. All testing took place in the animal's home cage. To control for any novelty associated with the presence of two water bottles, each rat was given two water bottles (filled only with water) for at least three consecutive days prior to the start of the assay. The saccharin preference test consisted of providing each rat with two sources of water. One bottle of water contained deionized, distilled water, while the other bottle contained a 0.25% wt/vol or 0.5% wt/vol saccharin solution. Each bottle was weighed and refilled daily for five consecutive days to determine the consumption of each fluid. The position of the bottles within the cage was switched on a daily basis to control for any position biases. Saccharin preference was calculated by dividing the amount of saccharin solution consumed by the total fluid (water and saccharin) for each day. Saccharin preference in experimental females. Following the pilot study, saccharin preference was measured in female rats exposed during gestation and lactation to varying doses of EE2 or BPA in the first block of the study (sample sizes are shown in Fig. 5). Saccharin preference for 0.25% wt/vol saccharin solution versus deionized, distilled water was determined over a 5-day period, and the average saccharin preference over the 5-day period was then analyzed using litter means on PROC GLM. FIG. 5. Open in new tabDownload slide Saccharin preference. Untreated female rats show a greater preference (about 80%) for 0.25% wt/vol and 0.50% wt/vol saccharin versus plain water than do untreated male rats (about 40%; Fig. 5a). Samples sizes: five males were given 0.25% wt/vol saccharin and plain water, three males were given 0.5% wt/vol saccharin and plain water, and eight females (four per group) were given either 0.25% wt/vol or 0.5% wt/vol saccharin and plain water. Two-way ANOVA (sex and saccharin concentration) Fsex (1,12 df) = 11.8, p < 0.005. The F values for saccharin concentration and the interaction of sex and concentration were not significant. In the F1 females from dams exposed to either EE2 or BPA, saccharin preference was reduced by exposure to EE2 at 5 and 50 μg/kg/day. BPA had no effect (Fig. 5b). F = 5.5 (6,25 df), p < 0.001. Samples sizes for the groups were (number pups/numbers of litters) control (10,6), BPA2 (6,3), BPA20 (15,7), BPA200 (8,4), EE2 0.5 (9,5), EE2 5 (10,5), and EE2 50 (2,2). Values on the figure are litter means and SEs. “**” indicates the mean differs from control by p < 0.01. FIG. 5. Open in new tabDownload slide Saccharin preference. Untreated female rats show a greater preference (about 80%) for 0.25% wt/vol and 0.50% wt/vol saccharin versus plain water than do untreated male rats (about 40%; Fig. 5a). Samples sizes: five males were given 0.25% wt/vol saccharin and plain water, three males were given 0.5% wt/vol saccharin and plain water, and eight females (four per group) were given either 0.25% wt/vol or 0.5% wt/vol saccharin and plain water. Two-way ANOVA (sex and saccharin concentration) Fsex (1,12 df) = 11.8, p < 0.005. The F values for saccharin concentration and the interaction of sex and concentration were not significant. In the F1 females from dams exposed to either EE2 or BPA, saccharin preference was reduced by exposure to EE2 at 5 and 50 μg/kg/day. BPA had no effect (Fig. 5b). F = 5.5 (6,25 df), p < 0.001. Samples sizes for the groups were (number pups/numbers of litters) control (10,6), BPA2 (6,3), BPA20 (15,7), BPA200 (8,4), EE2 0.5 (9,5), EE2 5 (10,5), and EE2 50 (2,2). Values on the figure are litter means and SEs. “**” indicates the mean differs from control by p < 0.01. Sexual dimorphism of Figure-8 maze activity in untreated rats. Figure-8 maze activity levels (Adams et al., 1985; Ruppert et al., 1985) were measured in untreated adult male and female LE rats to confirm our previous observations that females were more active in this apparatus than males (sample sizes are shown in Fig. 6). On the day of testing, the rats were transferred from the animal room to the testing room and placed into small transfer cages. The rats were allowed to acclimate to the room for 5 min. After acclimation, the rats were quickly transferred into 16 Figure-8 mazes. The rats were allowed to freely explore the mazes for 10 h in the dark and the numbers of photocell beam interruptions electronically recorded on an hourly basis. The activity trial started approximately an hour before the dark cycle began. FIG. 6. Open in new tabDownload slide Figure-8 maze spontaneous locomotor activity levels (photocell beam interruptions over the 10-h observation period). Male and ovariectomized female rats display significantly lower activity levels than do intact female male rats (Fig. 6a). F = (2,43 df), p < 0.001. There we 16 intact and 16 ovariectomized females and 14 intact male LE rats. Oral administration of EE2 at 175 (n = 6) and 275 μg/kg (n = 5) for 14 days increases the locomotor activity levels of untreated ovariectomized females (n = 2; Fig. 6b). Maternal EE2 and BPA treatments did not alter the locomotor activity of ovariectomized F1 females before (Fig. 6c) or after 14 days of treatment with EE2 at 275 μg/kg (Fig. 6d) by oral gavage which should restore activity to that seen in intact female rats if the perinatal treatment did not defeminize the brain. Analysis of activity before adult EE2: F = 1.7 (6,71 df) from the ANOVA, p > 0.13. Samples sizes for the groups were (number pups/numbers of litters) control (19,10), BPA2 (26,13), BPA20 (25,13), BPA200 (26,13), EE2 0.5 (16,10), EE2 5 (26,13), and EE2 50 (10,6). Analysis of activity after adult EE2: F = 0.49 (7,66 df) from the ANOVA, p > 0.80. Samples sizes for the groups were (number pups/numbers of litters) control no adult EE2 (8,8), control plus adult EE2 (9,9), BPA2 (25,13), BPA20 (23,12), BPA200 (26,13), EE2 0.5 (13,9), EE2 5 (24,12), and EE2 50 (7,5). Values on the figures are litter means and SEs. “**” indicates the mean differs from control by p < 0.01. One tailed t-test of untreated versus 275 group = 3.1 (5 df), p < 0.02, indicated by an “*”. FIG. 6. Open in new tabDownload slide Figure-8 maze spontaneous locomotor activity levels (photocell beam interruptions over the 10-h observation period). Male and ovariectomized female rats display significantly lower activity levels than do intact female male rats (Fig. 6a). F = (2,43 df), p < 0.001. There we 16 intact and 16 ovariectomized females and 14 intact male LE rats. Oral administration of EE2 at 175 (n = 6) and 275 μg/kg (n = 5) for 14 days increases the locomotor activity levels of untreated ovariectomized females (n = 2; Fig. 6b). Maternal EE2 and BPA treatments did not alter the locomotor activity of ovariectomized F1 females before (Fig. 6c) or after 14 days of treatment with EE2 at 275 μg/kg (Fig. 6d) by oral gavage which should restore activity to that seen in intact female rats if the perinatal treatment did not defeminize the brain. Analysis of activity before adult EE2: F = 1.7 (6,71 df) from the ANOVA, p > 0.13. Samples sizes for the groups were (number pups/numbers of litters) control (19,10), BPA2 (26,13), BPA20 (25,13), BPA200 (26,13), EE2 0.5 (16,10), EE2 5 (26,13), and EE2 50 (10,6). Analysis of activity after adult EE2: F = 0.49 (7,66 df) from the ANOVA, p > 0.80. Samples sizes for the groups were (number pups/numbers of litters) control no adult EE2 (8,8), control plus adult EE2 (9,9), BPA2 (25,13), BPA20 (23,12), BPA200 (26,13), EE2 0.5 (13,9), EE2 5 (24,12), and EE2 50 (7,5). Values on the figures are litter means and SEs. “**” indicates the mean differs from control by p < 0.01. One tailed t-test of untreated versus 275 group = 3.1 (5 df), p < 0.02, indicated by an “*”. Subsequently, females were ovariectomized and the activity levels measured a second time to determine if the removal of the ovaries and endogenous estrogens reduced the behavior to male levels. Females were then tested a third time after oral exposure to EE2 at 0 (corn oil at 0.5 μl/g body weight), 175, or 275 μg/kg/day EE2 daily for 14 days. Based on the results of this experiment, we administered 275 μg EE2/kg daily for 14 days to restore activity levels in females developmentally exposed to EE2 and BPA. Assessment of Figure-8 maze activity in EE2- or BPA-treated females. Figure-8 maze activity was measured in ovariectomized females from the first block of the study. After recovering from surgery, Figure-8 maze activity levels were measured (trial 1), as above. Subsequently, females were treated orally by gavage for 14 days with 275 μg/kg EE2 and retested in the Figure-8 mazes (trial 2; sample sizes are shown in Fig. 6). Preliminary study of lordosis behavior and uterine weight in SD and LE rats after oral EE2 treatment: dose-response to EE2. Lordosis behavior was studied using untreated adult ovariectomized females given EE2 followed by progesterone to activate the behavior. Female rats that have been defeminized by neonatal estrogen exposures display low levels of lordosis behavior (Gorski, 1986). Adult, ovariectomized SD and LE rats as well as adult, intact stimulus SD males were purchased from Charles River Laboratories. Female rats were housed two per cage in a room on a reversed light cycle (lights on from 9:00 P.M. to 11:00 A.M.) and given at least 2 weeks to recover from surgery and to acclimate to the altered light cycle prior used for behavioral testing. In this experiment, ovariectomized adult female LE rats were given varying doses of EE2 by oral gavage in oil (0, 2.5, 5, 10, 12.5, 19, 25, 37.5, 50, 65, 80, 95, 110, 125, and 250 μg/kg) for 2 days, followed by 0.5 mg progesterone (sc) in oil on the third day in order to determine an optimal oral dose of EE2 that induces this behavior reliably in control females. Females were retested at different dosage levels two to three times. This experiment (Fig. 7) included both LE and SD female rats to determine if there was a strain difference in the dose-response to EE2. A similar experiment compared the dose-related effects of EE2 on uterine weight in LE and SD rats to determine which estrogen-dependent end point was more sensitive to low doses of EE2, increased uterine weight, or induction of lordosis behavior. Ovariectomized female LE and SD rats were treated for 2 days by gavage with EE2 at 0, 0.5, 1, 2.5, 5, 10, 25, 50, or 250 μg/kg and necropsied on the third day, and the wet weight of the uterus was measured. We used EE2 to activate estrogen-dependent behaviors and uterine weight (Fig. 7) because our initial studies demonstrated that it can be used to induce female-like maze activity as well as lordosis behavior, whereas based upon unpublished studies, we suspected that a single daily dose of estradiol would not induce activity as effectively. The lordosis assay followed a 3-day protocol: Days 1 and 2—Females were dosed orally with EE2 prior to 10 A.M. both days, Day 3 morning—All females were sc injected with a 0.5 mg of progesterone dissolved in 0.1 ml of corn oil at 7:30 A.M. All females, as well as the stud males, were moved into the behavioral observation room and allowed to acclimate. While in the holding room, rats had access to food and water. This room had an identical light cycle to that of the animal room. Day 3 afternoon—Animal testing began during the dark phase of the animal's activity cycle, rats being a nocturnal species, at 1:30 P.M. (3.5 h after lights out) under dim light. All observations were done blind to the treatment group. A stimulus male was placed into a clear cage, free of bedding, and allowed to acclimate for at least 5 min. Each female was placed in the test cage, and the male was allowed to mount the female five times. A rear mount was defined as a male placing his front paws onto the flank of the female followed by pelvic thrusting. Mounts directed at the side or head of the female were not counted. Lordosis quotients (LQs) were calculated as the number of lordosis/number of mounts. An LQ of 1.0 indicates that a female displayed a lordosis response to every mount. If 2 min elapsed without any mounts, the female was removed from the cage and placed into a test cage of another stimulus male. All lordosis testing took place in the same room, and the same experimenter, blind to the treatment groups, scored every trial. Surgical Methods for Ovariectomy of Experimental Females Rats were anesthetized in a specially designed airtight Plexiglas container through which Halothane vapor (Bickford Halothane Vaporizer) and 10 standard cubic feet per hour of airflows. Initially, a low concentration (1% by volume) of halothane vapor was administered to avoid inducing stress in the rat. After the rat lost consciousness, the halothane concentration was increased to 3% by volume to induce a surgical plane of anesthesia as determined by reduced respiration rate and absence of any response to tactile stimuli. When a surgical plane of anesthesia was reached, the rat was removed from the Plexiglas container and administered halothane vapor through a nose cone. The entire operation was conducted under a hood to avoid the possibility of halothane exposure to the technician performing this procedure. Immediately prior to surgery, the rats were injected with 0.05 mg/kg buprenorphine to provide pain control and placed on a heating pad. The dorsal lumbar region was shaved, cleaned, and two bilateral incisions were made of about 0.5 cm. The ovaries were identified, ligated with non-wicking, absorbable suture, and removed. The incised muscle also was sutured together, and the incision was then closed with a wound clip. The rat was then removed from the nose cone, placed in a clean cage on a heating pad, and monitored until recovery. Prior to surgery, all instruments were autoclaved for sterilization. The technician performing the surgery wore sterile gloves and performed the surgery through a sterile drape to minimize the risk of contaminating the surgical sites. Prior to surgery, the incision sites were cleansed with betadine and alcohol swabs utilizing a circular wiping motion three times with each solution. Instruments were sterilized using a hot bead sterilizer between animals. Following surgery, each rat was monitored until locomotion and other signs of activity appeared normal and then checked twice daily to ensure recovery. The wound clips were removed after 7 days. Lordosis Behavior of EE2- and BPA-Treated Females After VO had been assessed in the second block of the study, rats were bilaterally ovariectomized and allowed to recover for at least 2 weeks prior to behavioral testing (sample sizes are shown in Fig. 8). Since administration of 50 μg/kg EE2 orally (approximately and ED80 dose; Fig. 7b) for 2 days followed by an injection of 0.5 mg progesterone induced LQ of nearly 1.0 in control females, this dosing regime was selected to describe the effects of EE2 and BPA on the lordosis behavior of the experimental females. FIG. 7. Open in new tabDownload slide Uterine (Fig. 7a) and lordosis (Fig. 7b) responses of control adult LE and SD ovariectomized female rats to two daily oral EE2 doses to identify a near-maximal dose to use to induce lordosis in the experimental females. The ED50 response to EE2 for uterine weight was about half the ED50 for lordosis behavior. Values on the figure are means and SEs. There were no differences in the dose-response curves among these strains of rats. FIG. 7. Open in new tabDownload slide Uterine (Fig. 7a) and lordosis (Fig. 7b) responses of control adult LE and SD ovariectomized female rats to two daily oral EE2 doses to identify a near-maximal dose to use to induce lordosis in the experimental females. The ED50 response to EE2 for uterine weight was about half the ED50 for lordosis behavior. Values on the figure are means and SEs. There were no differences in the dose-response curves among these strains of rats. FIG. 8. Open in new tabDownload slide The lordosis behavior of ovariectomized, EE2 primed F1 female rats was reduced by maternal treatment with EE2 at 15 and 50 μg/kg/day. BPA had no effect. F = 18.6 (9,36 df) from the ANOVA, p < 0.0001. Samples sizes for the groups were (number pups/numbers of litters) control (16,6), BPA20 (6,3), BPA200 (15,6), EE2 0.05 (6,3), EE2 0.15 (6,3), EE2 0.5 (12,5), EE2 1.5 (15,7), EE2 5 (22,7), EE2 15 (9,5), and EE2 50 (2,1). “**” indicates the mean differs from control by p < 0.01. FIG. 8. Open in new tabDownload slide The lordosis behavior of ovariectomized, EE2 primed F1 female rats was reduced by maternal treatment with EE2 at 15 and 50 μg/kg/day. BPA had no effect. F = 18.6 (9,36 df) from the ANOVA, p < 0.0001. Samples sizes for the groups were (number pups/numbers of litters) control (16,6), BPA20 (6,3), BPA200 (15,6), EE2 0.05 (6,3), EE2 0.15 (6,3), EE2 0.5 (12,5), EE2 1.5 (15,7), EE2 5 (22,7), EE2 15 (9,5), and EE2 50 (2,1). “**” indicates the mean differs from control by p < 0.01. Reproductive Organ Morphology At approximately 10 months of age, female rats were anesthetized with carbon dioxide, euthanized by decapitation, and examined for abnormal external genitalia (sample sizes are shown in Fig. 9). In order to quantify the degree of abnormality in each animal, multiple measurements of the external genitalia were measured by the same prosector using calipers accurate to 0.1 mm. Urethral slit length: The length of the urethral slit. Urethral slit depth: The depth of the urethral slit while viewing the phallus from the side. When viewed in this fashion, the urethral slit typically forms a “V” shape. Urethrovaginal distance: The distance from the urethral opening (defined as the bottom of the urethral slit) to the posterior end of the vagina. Anovaginal distance: The distance from the posterior vagina to the anterior anus. FIG. 9. Open in new tabDownload slide The external genitalia of the female rat. Control photo on the upper left. The external genitalia of the F1 female rat exposed to EE2 at 5 μg/kg (middle panel, top) is mildly malformed and the distance from the urethral opening to the base of caudal end of the vaginal canal (UVD) is reduced (middle panel, bottom) as compared to the control UVD (bottom left panel). An example of a severely malformed genitalia from a female treated with EE2 at 50 μg/kg/day is shown in the top right panel. This effect was not displayed by any F1 females in the BPA groups. FIG. 9. Open in new tabDownload slide The external genitalia of the female rat. Control photo on the upper left. The external genitalia of the F1 female rat exposed to EE2 at 5 μg/kg (middle panel, top) is mildly malformed and the distance from the urethral opening to the base of caudal end of the vaginal canal (UVD) is reduced (middle panel, bottom) as compared to the control UVD (bottom left panel). An example of a severely malformed genitalia from a female treated with EE2 at 50 μg/kg/day is shown in the top right panel. This effect was not displayed by any F1 females in the BPA groups. Statistical Analyses The F1 female data were analyzed using PROC GLM (a general linear models procedure) by one-way ANOVA using PC SAS (Statistical Analysis System), version 9.1 (SAS Institute, Cary, NC). If the overall F value of the ANOVA was significant, then the EE2 and BPA data were analyzed separately to determine if either EE2 or BPA or both chemicals significantly affected an end point. Significance was identified by an F value with p < 0.05, followed by LSMEANS (t-test). If the F value of the ANOVA was “not significant,” the data also were reanalyzed using a Dunnett's post hoc test which, unlike LSMEANS, does not require that the F value from the ANOVA be significant. The statistical analyses were based upon litter mean values. The frequency of malformed external genitalia in individual F1 females was analyzed by Fisher exact test. Statistical significance was considered p ≤ 0.05. RESULTS Maternal and Pregnancy Data There was a significant main effect of EE2 treatment on maternal body weight gain during gestation (from Howdeshell et al., 2008). Treatment with EE2 at 1.5 μg/kg/day and higher significantly decreased maternal body weight gain during gestation relative to controls. There were no treatment effects of BPA on maternal body weight gain during pregnancy or lactation. There was a significant main effect of treatment on the number of uterine implantations, with the 50-μg EE2/kg/day group having significantly fewer uterine implantation scars (Howdeshell et al., 2008). There was a significant main effect of treatment on the number and body weight of live pups on PND2 (Figure 2; Howdeshell et al., 2008). Post hoc analysis detected no significant effect of BPA treatment on the number of implantations and number of live pups at PND2 or fetal/neonatal mortality. TABLE 2 Summary (litter means ± SEs) of End Points and Samples Sizes (numbers of pups/litters) for Female F1 LE Rat Data Exposed Orally via the Dam to BPA or EE during Gestation and Lactation that Are Not Included in Any Figures End points; F (overall ANOVA) 0 μg/kg/day BPA2 BPA20 BPA200 EE 0.05 EE 0.15 EE 0.5 EE 1.5 EE 5 EE 15 EE 50 External genitalia—AVD (mm); F(7,86) = 2.65, p < 0.14 19.6 ± 0.5 19.5 ± 0.47 19.9 ± 0.36 19.7 ± −0.49 18.71 ± 0.7 No data 18.8 ± 0.52 No data 18.2 ± 0.52 No data 20.5 ± 0.81 External genitalia—urethral slit depth (mm); F(7,86) = 98.5, p < 0.0001 1.07 ± 0.10 1.05 ± 0.06 1.03 ± 0.07 0.85 ± −0.07 0.98 ± 0.05 No data 0.93 ± 0.07 No data 1.78*± 0.10 No data 6.31** ± 0.49 External genitalia—urethral slit length (mm); F(7,86) = 33.4, p < 0.0001 1.48 ± 0.05 1.37 ± 0.07 1.42 ± 0.05 1.34 ± 0.06 1.31 ± 0.06 No data 1.50 ± 0.08 No data 1.83 ± 0.13 No data 3.82** ± 0.37 Figure-8 maze activity increase from 14 days of adult oral EE2 treatment; F(6,66) = 1.22, p > 0.3 686 ± 237 (9,9) 239 ± 110 (25,13) 316 ± 159 (23,12) 247 ± 113 (26, 13) No data No data 325 ± 174 (13,9) No data 205 ± 129 (24,12) No data 109 ± 116 (7, 5) F1 male weaning weight (g); F(9,56) = 1.1, p > 0.4 58.9 ± 2.0 (30,8) No data 58.5 ± 3.4 (26,6) 57.8 ± 1.5 (43,8) 60.1 ± 1.6 (31,6) 60.7 ± 2.6 (36,6) 58.7 ± 2.6 (39,7) 58.5 ± 2.2 (39,9) 58.2 ± 2.8 (46,8) 59.0 ± 4.1 (28,6) 44.4 ± 5.6 (3,2) End points; F (overall ANOVA) 0 μg/kg/day BPA2 BPA20 BPA200 EE 0.05 EE 0.15 EE 0.5 EE 1.5 EE 5 EE 15 EE 50 External genitalia—AVD (mm); F(7,86) = 2.65, p < 0.14 19.6 ± 0.5 19.5 ± 0.47 19.9 ± 0.36 19.7 ± −0.49 18.71 ± 0.7 No data 18.8 ± 0.52 No data 18.2 ± 0.52 No data 20.5 ± 0.81 External genitalia—urethral slit depth (mm); F(7,86) = 98.5, p < 0.0001 1.07 ± 0.10 1.05 ± 0.06 1.03 ± 0.07 0.85 ± −0.07 0.98 ± 0.05 No data 0.93 ± 0.07 No data 1.78*± 0.10 No data 6.31** ± 0.49 External genitalia—urethral slit length (mm); F(7,86) = 33.4, p < 0.0001 1.48 ± 0.05 1.37 ± 0.07 1.42 ± 0.05 1.34 ± 0.06 1.31 ± 0.06 No data 1.50 ± 0.08 No data 1.83 ± 0.13 No data 3.82** ± 0.37 Figure-8 maze activity increase from 14 days of adult oral EE2 treatment; F(6,66) = 1.22, p > 0.3 686 ± 237 (9,9) 239 ± 110 (25,13) 316 ± 159 (23,12) 247 ± 113 (26, 13) No data No data 325 ± 174 (13,9) No data 205 ± 129 (24,12) No data 109 ± 116 (7, 5) F1 male weaning weight (g); F(9,56) = 1.1, p > 0.4 58.9 ± 2.0 (30,8) No data 58.5 ± 3.4 (26,6) 57.8 ± 1.5 (43,8) 60.1 ± 1.6 (31,6) 60.7 ± 2.6 (36,6) 58.7 ± 2.6 (39,7) 58.5 ± 2.2 (39,9) 58.2 ± 2.8 (46,8) 59.0 ± 4.1 (28,6) 44.4 ± 5.6 (3,2) Note. UVD, urethrovaginal distance in millimeter. Gray-shaded means differ significantly by ANOVA and a t-test from control. *p < 0.05 or **p < 0.01 or significant by Dunnett's post hoc test without a significant F value in ANOVA; F values with (treatment, error df) and probability values of F are presented. Open in new tab TABLE 2 Summary (litter means ± SEs) of End Points and Samples Sizes (numbers of pups/litters) for Female F1 LE Rat Data Exposed Orally via the Dam to BPA or EE during Gestation and Lactation that Are Not Included in Any Figures End points; F (overall ANOVA) 0 μg/kg/day BPA2 BPA20 BPA200 EE 0.05 EE 0.15 EE 0.5 EE 1.5 EE 5 EE 15 EE 50 External genitalia—AVD (mm); F(7,86) = 2.65, p < 0.14 19.6 ± 0.5 19.5 ± 0.47 19.9 ± 0.36 19.7 ± −0.49 18.71 ± 0.7 No data 18.8 ± 0.52 No data 18.2 ± 0.52 No data 20.5 ± 0.81 External genitalia—urethral slit depth (mm); F(7,86) = 98.5, p < 0.0001 1.07 ± 0.10 1.05 ± 0.06 1.03 ± 0.07 0.85 ± −0.07 0.98 ± 0.05 No data 0.93 ± 0.07 No data 1.78*± 0.10 No data 6.31** ± 0.49 External genitalia—urethral slit length (mm); F(7,86) = 33.4, p < 0.0001 1.48 ± 0.05 1.37 ± 0.07 1.42 ± 0.05 1.34 ± 0.06 1.31 ± 0.06 No data 1.50 ± 0.08 No data 1.83 ± 0.13 No data 3.82** ± 0.37 Figure-8 maze activity increase from 14 days of adult oral EE2 treatment; F(6,66) = 1.22, p > 0.3 686 ± 237 (9,9) 239 ± 110 (25,13) 316 ± 159 (23,12) 247 ± 113 (26, 13) No data No data 325 ± 174 (13,9) No data 205 ± 129 (24,12) No data 109 ± 116 (7, 5) F1 male weaning weight (g); F(9,56) = 1.1, p > 0.4 58.9 ± 2.0 (30,8) No data 58.5 ± 3.4 (26,6) 57.8 ± 1.5 (43,8) 60.1 ± 1.6 (31,6) 60.7 ± 2.6 (36,6) 58.7 ± 2.6 (39,7) 58.5 ± 2.2 (39,9) 58.2 ± 2.8 (46,8) 59.0 ± 4.1 (28,6) 44.4 ± 5.6 (3,2) End points; F (overall ANOVA) 0 μg/kg/day BPA2 BPA20 BPA200 EE 0.05 EE 0.15 EE 0.5 EE 1.5 EE 5 EE 15 EE 50 External genitalia—AVD (mm); F(7,86) = 2.65, p < 0.14 19.6 ± 0.5 19.5 ± 0.47 19.9 ± 0.36 19.7 ± −0.49 18.71 ± 0.7 No data 18.8 ± 0.52 No data 18.2 ± 0.52 No data 20.5 ± 0.81 External genitalia—urethral slit depth (mm); F(7,86) = 98.5, p < 0.0001 1.07 ± 0.10 1.05 ± 0.06 1.03 ± 0.07 0.85 ± −0.07 0.98 ± 0.05 No data 0.93 ± 0.07 No data 1.78*± 0.10 No data 6.31** ± 0.49 External genitalia—urethral slit length (mm); F(7,86) = 33.4, p < 0.0001 1.48 ± 0.05 1.37 ± 0.07 1.42 ± 0.05 1.34 ± 0.06 1.31 ± 0.06 No data 1.50 ± 0.08 No data 1.83 ± 0.13 No data 3.82** ± 0.37 Figure-8 maze activity increase from 14 days of adult oral EE2 treatment; F(6,66) = 1.22, p > 0.3 686 ± 237 (9,9) 239 ± 110 (25,13) 316 ± 159 (23,12) 247 ± 113 (26, 13) No data No data 325 ± 174 (13,9) No data 205 ± 129 (24,12) No data 109 ± 116 (7, 5) F1 male weaning weight (g); F(9,56) = 1.1, p > 0.4 58.9 ± 2.0 (30,8) No data 58.5 ± 3.4 (26,6) 57.8 ± 1.5 (43,8) 60.1 ± 1.6 (31,6) 60.7 ± 2.6 (36,6) 58.7 ± 2.6 (39,7) 58.5 ± 2.2 (39,9) 58.2 ± 2.8 (46,8) 59.0 ± 4.1 (28,6) 44.4 ± 5.6 (3,2) Note. UVD, urethrovaginal distance in millimeter. Gray-shaded means differ significantly by ANOVA and a t-test from control. *p < 0.05 or **p < 0.01 or significant by Dunnett's post hoc test without a significant F value in ANOVA; F values with (treatment, error df) and probability values of F are presented. Open in new tab Neonatal and Pup Data Table 1 displays a list of all the end points measured in the F1 female rats exposed to EE2 and BPA from GD7 to PND18. EE2 at 50 μg/kg/day increased female rat AGD at PND2 (not significant by ANOVA, significant with a Dunnett's test, p < 0.05; Fig. 1) and decreased pup body weight on PND2 and at weaning (Figs. 2a and 2b, respectively). BPA had no effect on body weight at any age or AGD at 2 days of age. Neither EE2 nor BPA induced areolae/nipple agenesis, a male rat trait displayed among female rats—exposed in utero to androgens but not estrogens (data not shown). Since male weaning weight was not included in Howdeshell et al. (2008), it is shown in Table 2 for comparison to the body weight of the weanling females. Age at Puberty (VO) Prenatal treatment with 5 μg EE2/kg/day significantly accelerated the age at VO opening and the females attained this landmark at a lighter body weight than control females (Figs. 3a and 3b, respectively). Since this end point was not measured in F1 females in the second block, we do not know if the dose of 1.5 μg EE2/kg/day accelerated VO or not. Furthermore, the severity of the malformations of the external genitalia in high-dose females precluded assessment of VO. In contrast to EE2, BPA had no affect on the age at VO. Fecundity and Fertility Prenatal treatment with 5, 15, and 50 μg EE2/kg/day significantly reduced fecundity of F1 females (Fig. 4a). Lower dosage levels of EE2 and BPA did not affect the ability to produce viable F2 pups. Since some of the sample sizes were small, dose groups were pooled to display the consistency of effect on fecundity among the BPA, lower dose EE2, and higher dose EE2 groups (Fig. 4b). After correcting the data for the female effect on fertility (Fig. 4c) and fecundity, analysis of the data indicated that EE2 and BPA treatments did not affect male fertility; however, the sample sizes for some of the treatment groups are small (data not shown, Supplementary Data). Saccharin Preference In the first study with untreated females, females displayed a preference for 0.25 and 0.5% wt/vol solutions over deionized distilled water (about 80% of the total fluid consumed was the saccharin solution) whereas male rats preference for saccharin was much lower (about 40%; Fig. 5a). Prenatal treatment with 5 and 50 μg EE2/kg/day significantly reduced saccharin preference in intact F1 females to male-like levels (Fig. 5b). The lowest-observed effect level for saccharin preference was 5 μg EE2/kg/day. BPA did not significantly affect this behavior (ANOVA and Dunnett's tests were not significant for BPA at any dose). Figure-8 Maze Locomotor Activity In untreated rats, males displayed significantly lower levels of activity (photocell beam interruptions over 10 hrs) than did female rats (Fig. 6a). The second pilot study demonstrated that oral administration of EE2 for 14 days increased maze activity in ovariectomized adult female rats to the levels seen in intact females in the first study (Fig. 6b). This demonstrates that estrogen exposure during adult life is necessary to activate female-like activity levels in our apparatus. In the EE2/BPA study, developmental exposure did not affect maze activity in ovariectomized or ovariectomized EE2-treated females (Table 1 and Figs. 6c and d, respectively). Lordosis Behavior In the preliminary dose-response studies with EE2 on uterine weight and lordosis behavior, the ED50s for uterine weight and induction of lordosis behavior were seen at doses of about 8 μg EE2/kg/day (Fig. 7a) and 20 μg EE2/kg/day (Fig. 7b), respectively. The dose-response curves were similar for LE and SD rats. Based upon these results, we used 50 μg EE2/kg/day, which is approximately an ED80, to induce lordosis behavior in the EE2/BPA study. In the EE2/BPA study, developmental exposure to EE2 at 15 and 50 μg/kg/day (Fig. 8) reorganized (defeminized) the brain of the female LE rat such that administration of EE2 plus progesterone was unable to activate lordosis behavior in adult F1 females. In contrast to EE2, BPA had no effect on lordosis behavior. Malformations of the External Genitalia of F1 Females F1 females exposed developmentally to EE2 at 5–50 μg EE2/kg/day displayed (63–100%, respectively) malformations of the external genitalia (Figs. 9 and 10a). Measurement of the distance from the urethral opening to the VO (Fig. 10b) and the length and depth of the urethral slit were altered (Table 2), confirming our visual assessment of the external genitalia. The distance from the anal opening to the VO (Table 2) was not affected by EE2 treatment. BPA did not affect any of these morphological end points in the F1 female rat. FIG. 10. Open in new tabDownload slide Maternal treatment with EE2 at 5, 15, and 50 μg/kg/day increases the percentage of F1 female rats displaying malformed external genitalia (Fig. 10a). The distance from the urethral opening to the caudal end of the VO is reduced in females treated with EE2 at 5 and 50 μg/kg/day. The measurement was not taken in females treated with EE2 at 15 μg/kg/day. BPA had no effect on urethrovaginal distance (UVD) or the percentage of malformed females. Analysis of percent with cleft phallus: F = 40.8 (9,59 df) from the ANOVA, p < 0.0001. Samples sizes for the groups were (number pups/numbers of litters) control (42,8), BPA20 (32,6), BPA200 (47,8), EE2 0.05 (33,6), EE2 0.15 (35,6), EE2 0.5 (44,7), EE2 1.5 (47,9), EE2 5 (45,9), EE2 15 (34,7), EE2 50 (11,3). Analysis of UVD: F = 80.3 (7,86 df) from the ANOVA, p < 0.0001. Samples sizes for the groups were (number pups/numbers of litters) control (30,13), BPA2 (26,9), BPA20 (29,9), BPA200 (22,12), EE2 0.05 (24,10), EE2 0.5 (46,15), EE2 5 (24,10), and EE2 50 (25,12). “**” indicates the mean differs from control by p < 0.01. FIG. 10. Open in new tabDownload slide Maternal treatment with EE2 at 5, 15, and 50 μg/kg/day increases the percentage of F1 female rats displaying malformed external genitalia (Fig. 10a). The distance from the urethral opening to the caudal end of the VO is reduced in females treated with EE2 at 5 and 50 μg/kg/day. The measurement was not taken in females treated with EE2 at 15 μg/kg/day. BPA had no effect on urethrovaginal distance (UVD) or the percentage of malformed females. Analysis of percent with cleft phallus: F = 40.8 (9,59 df) from the ANOVA, p < 0.0001. Samples sizes for the groups were (number pups/numbers of litters) control (42,8), BPA20 (32,6), BPA200 (47,8), EE2 0.05 (33,6), EE2 0.15 (35,6), EE2 0.5 (44,7), EE2 1.5 (47,9), EE2 5 (45,9), EE2 15 (34,7), EE2 50 (11,3). Analysis of UVD: F = 80.3 (7,86 df) from the ANOVA, p < 0.0001. Samples sizes for the groups were (number pups/numbers of litters) control (30,13), BPA2 (26,9), BPA20 (29,9), BPA200 (22,12), EE2 0.05 (24,10), EE2 0.5 (46,15), EE2 5 (24,10), and EE2 50 (25,12). “**” indicates the mean differs from control by p < 0.01. DISCUSSION Our results demonstrate that administration of EE2 in utero and during lactation permanently altered F1 female LE rat reproductive morphology, function, and behavior (Table 1). Administration of EE2 at 5–15 μg/kg/day induced malformations of the female rat external genitalia (Figs. 9 and 10), accelerated VO (Fig. 3b), defeminized lordosis (Fig. 8) and saccharin preference (Fig. 5b) behaviors, and reduced fecundity and fertility (Figs. 4a–c). These results also suggest that the F1 females were more affected by perinatal EE2 than F1 males (Howdeshell et al., 2008) in terms of the prevalence and severity of the effects (Fig. 11). F1 LE male rats displayed slight reductions in seminal vesicle and testis weights at 5 μg EE2/kg/day and reduced sperm counts and histopathological alterations in reproductive tissues, whereas doses as high as 50 μg EE2/kg/day did not induce reproductive tract malformations in F1 males (Howdeshell et al., 2008). It is noteworthy that exposure to estrogenic pesticides like methoxychlor (Gray et al., 1989) or toxic substances like nonylphenol (Tyl et al., 2006; Chapin et al., 1999) do not cause similar malformations of the reproductive tract although exposure to these xenoestrogens during development does accelerate VO and reduce F1 female fecundity (Supplementary Data). FIG. 11. Open in new tabDownload slide Logistic regression curves demonstrate that EE2 more severely affected the F1 females than their dams or their male siblings (Howdeshell et al., 2008). ED50 for F1 female fecundity = ED50 for F1 female malformations of the genitalia < ED50 for the inhibition of F1 female lordosis behavior < ED50 F0 maternal weight gain = ED50 for F1 pup viability and = F1 male seminal vesicle weights (one of the most affected end points in the F1 males. FIG. 11. Open in new tabDownload slide Logistic regression curves demonstrate that EE2 more severely affected the F1 females than their dams or their male siblings (Howdeshell et al., 2008). ED50 for F1 female fecundity = ED50 for F1 female malformations of the genitalia < ED50 for the inhibition of F1 female lordosis behavior < ED50 F0 maternal weight gain = ED50 for F1 pup viability and = F1 male seminal vesicle weights (one of the most affected end points in the F1 males. In contrast to EE2, perinatal BPA at 2, 20, and 200 μg/kg/day was without effect on the reproductive development of either male or female F1 LE rat offspring. These results on the F1 females and those we reported earlier for the F1 male offspring from these litters (Howdeshell et al., 2008) demonstrate that BPA did not disrupt reproductive development in an estrogenic manner in the F1 offspring when administered orally to the rat dam in utero and during lactation. EE2 produced a wide variety of anatomical, histopathological, and functional alterations in F1 males and females and behavioral alterations in the F1 females. Furthermore, none of the effects of EE2 displayed a non-monotonic dose-response pattern over a dose range of four log units. Effects of EE2 in Other Rat Transgenerational Studies EE2-exposed F1 LE rat females in the current study were more severely affected than similarly exposed SD rats (Sawaki et al., 2003). Sawaki et al. (2003) observed cleft phallus in almost all the female offspring at 50 μg/kg/day, but these females initially had fertility levels comparable to the control group, whereas EE2 reduced fecundity in LE F1 females at doses of 5, 15, and 50 μg/kg/day. Subsequently, SD F1 female rats exposed to 50 μg/kg/day exhibited abnormal estrous cyclicity, including persistent estrus, and histological examination revealed follicular cysts and absence of corpora lutea in the ovaries of the rats with persistent estrus (Sawaki et al., 2003). In addition to the studies of Sawaki et al. (2003 a, b), several other studies with rats have reported on the adverse effects of EE2 during development, the most robust being the study conducted at the National Center for Toxicological Research for the NTP (Latendresse et al., 2009). They administered EE2 in a dietary multigenerational study at 2, 10, or 50 ppb in diet (estimated to be 0.2, 1.1, or 5.8 μg/kg/day) to SD rats. These authors reported that EE2 reduced body weight of the females at 1.1 and 5.8 μg/kg/day, altered the age at VO, induced abnormal estrous cycles at 5.8 μg/kg/day, and induced non-neoplastic uterine lesions at all doses. However, they did not observe any reduction in fertility or fecundity as a consequence of EE2 exposure. It is possible the dietary exposure that they used and gavage exposure used herein produce very different maximum EE2 peak heights and areas under the curve in maternal, fetal, and offspring tissues. In another study with the SD rat strain, Fusani et al. (2007) dosed rats orally with EE2 at 0.004 or 0.4 μg/kg/day from GD5 to PND32. They reported that EE2 did not affect AGD, pup viability or body weight, whereas fertility and estrous cyclicity were altered at 0.4 μg/kg/day in pairs of similarly treated males and females (Fusani et al., 2007). Effects of EE2 and BPA on Behavioral Sex Differentiation In mammals, some sexually dimorphic behaviors are permanently organized during the perinatal period of development by testicular testosterone or dihydrotestosterone (DHT) or estradiol, metabolites of testosterone. The degree to which the T, DHT, or E2 pathways each contribute to neurobehavioral sex differentiation varies greatly among mammalian species. In the rat, the E2 pathway is critical for the normal differentiation of the central nervous system and some behaviors. Metabolism of T to E2 locally induces masculinization and defeminization of specific sexually dimorphic behaviors. For example, it has been long established that estrogens administered to neonatal female rats defeminizes the hypothalamic-pituitary axis by abolishing the proestrus luteinizing hormone (LH) surge (Gorski, 1986; Gorski and Barraclough, 1962; Homma et al., 2009), resulting in infertility in treated female rats (McCarthy, 2008). Interestingly, the role of the E2-signaling pathway is quite diverse in mammals, and it even varies among rodents. For example, in the Syrian hamster, neonatal treatment with estrogens (estradiol [Whitsett et al., 1978], zearalenone [Gray et al., 1985], chlordecone [kepone; Gray, 1982]) demasculinizes male mating behavior such that the males do not mount sexually receptive females, whereas the ability of the female hamster to display lordosis behavior is not defeminized. In contrast, administration of estrogens to the neonatal female rat results in defeminization of female lordosis behavior. Although the organizational role of estradiol in the development of rodent sexual behaviors is well established, what role, if any, estrogens play in primate brain development remains to be determined (McCarthy, 2008). The review by McCarthy (2008) of the role of estradiol in the developing brain concludes that “the lack of effect of DES on brain differentiation of human females is consistent with empirical results generated in primates in which most results indicate no important role for estrogens in masculinization, this function instead being performed by prenatal androgens combined with social context and rearing conditions.” Baum (2006) also reported that, in sexual differentiation of the human and nonhuman primate brain, it is generally agreed that the androgen-signaling pathway predominates in neurobehavioral sex differentiation and a clear role for the E2 pathway, if any, has not yet been identified (Baum, 2006). Effects of EE2 and BPA on Lordosis Behavior In addition to the LH surge, neonatal estrogens can defeminize the brain such that when adult rats are ovariectomized, they fail to display lordosis behavior when activated with the appropriate hormonal regime in adult life (McCarthy, 2008). Lordosis behavior also has been studied in intact cycling female rats, with the behavior normally being displayed in the dark phase of the light cycle on the evening of vaginal proestrus. However, the advantage of using the ovariectomized, hormone-primed female, as done herein, is that this approach eliminates the possibility that the failure to display lordosis is due to a loss of estrous cyclicity in the intact rat. Hence, the fact that EE2 exposure abolished lordosis behavior in the ovariectomized, hormone-primed females herein is a clear indication of a direct effect of this potent estrogen on the developing nervous system. The lack of effect of BPA on this behavior clearly indicates the estrogen pathway in the brain controlling this behavior was not defeminized by BPA. In future studies, we plan to use sc injection of estradiol benzoate (E2B) rather than orally administered EE2 for hormone priming to activate lordosis behavior in adult female rats since this is more widely used for this purpose than is oral EE2. In addition, we would like to vary the priming dose of E2B to determine the dose-response to determine if partially defeminized females are less responsive to low doses as adults than untreated females. In addition to the current study, several other studies have examined the effects of BPA on the development of lordosis behavior in the female rat using a broad range of doses, administered orally and by neonatal sc injection and failed to find an effect. In total, none of six studies that have examined the effects of BPA on lordosis behavior have found that BPA defeminized this behavior. Kwon et al. (2000) administered BPA orally (Kwon et al., 2000) at oral doses of 3–300 mg/kg/day and found no effect of BPA, whereas the DES-positive control was effective. Farabollini et al. (2002) and Kubo et al. (2003) also reported that BPA did not decrease female lordosis behavior in an estrogenic manner. Furthermore, sc injection of high doses of BPA ranging from 50 μg/kg to 50 mg/kg failed to disrupt lordosis behavior in the female rats (Adewale et al., 2009; Monje et al., 2009). Effects of EE2 and BPA on Saccharin Preference Results of the study with control females reveal that about 80% of the total fluid consumed by intact female rats was the saccharin solution, and only 20% was plain water. This is in marked contrast to the male rats in which only 40% of the total fluid consumption was saccharin (Fig. 5a). These results are consistent with numerous publications showing a sexual dimorphism with intact female rats displaying a greater preference for saccharin solutions versus plain water than control male rats. For example, Valenstein et al. (1967) first reported the sex differences in saccharin preference in 1967 and Wade and Zucker (1969) demonstrated that neonatal testosterone treatment could defeminize this behavior such that female rats no longer displayed a preference of saccharin solutions over plain water. However the current literature does not resolve whether defeminization of saccharin preference results from androgenic or from estrogenic signaling during neonatal life. In the current study, maternal treatment with 5 and 50 μg EE2/kg/day significantly reduced saccharin preference in intact F1 females to male-like levels (Fig. 5b). It is not clear if the reduction in saccharin preference by in utero and lactational EE2 treatment resulted from a direct effect on the brain regions that regulate the development of this behavior in the F1 female rat or via alterations of the HPG axis, estrous cyclicity, and serum estradiol levels. It would be interesting to examine the effects of perinatal EE2 on saccharin preference in ovariectomized females with and without estrogen priming to determine if this was a direct effect of EE2 on the organization of saccharin preference behavior or on the developing HPG axis. BPA did not significantly affect this behavior at any dosage level. Effects of EE2 and BPA on Locomotor Activity in the Figure-8 Maze Results of the study revealed that control females had significantly higher spontaneous activity levels over the 10-h assessment period than control male rats (Fig. 6a), ovariectomy reduces activity to male levels (Fig. 6a), and administration of an estrogen to an ovariectomized female rat restores the activity level to that seen in intact females (Fig. 6b). However, in utero and lactational exposure to EE2 or BPA did not alter activity levels in ovariectomized F1 females with or without EE2 priming (Figs. 6c and d, respectively). These results indicate that neither of these chemicals defeminized the brain with respect to this sexually dimorphic behavior. Since we did not examine the locomotor activity of the experimental females prior to ovariectomy, we do not know if either chemical would have had an effect in the intact animal. It is possible that intact EE2-treated females would have displayed altered estrous cycles, and locomotor levels would have been reduced as a consequence of reduced levels of endogenous estradiol. The fact that ovariectomized F1 females exposed to high doses of EE2 in utero and during lactation were not defeminized (as indicated by the observation that they responded normally to the activational effects of EE2 in adult life) suggests that estrogens do not play a role during perinatal life in the organization of this sexual dimorphic behavior. Sensitivity of the LE Rat versus Humans to EE2 When used as a human pharmaceutical, EE2 is often combined with progestogens or other steroids to maximize the effects and to reduce the adverse side effects. Over the years, dosage levels have declined in order to eliminate adverse side effects of EE2 in girls and women. For example, it was shown that reducing EE2 levels from 100 to 50 μg/day in combination with a progestogen reduced the incidence of thromboembolisms (Committee-on-Safety-of-Drugs, 1970). In some cases, relatively high dose levels of EE2 have been used alone or in combination with doses ranging from 100 to 1000 μg/day to limit final height in girls and boys (Rooman et al., 2005; Schmitt et al., 1992; Svan et al., 1991). In addition, when EE2 was administered as a postcoital contraceptive agent at 2–5 mg/day for 5 days, 2–3 mg was only partially effective while no pregnancies occurred in the 5-mg group (Blye, 1973; Haspels, 1972). If one uses a body weight of 50 kg for reference, doses of EE2 of 100–5000 μg/day (equivalent to 2–100 μg/kg body weight/day) can result in adverse effects in humans. This dose range produced adverse effects in LE rat dams and F1 male and female offspring in our studies. Taken together, these results demonstrate that the LE rat provides a useful model for the study of EE2 since the sensitivity to this chemical, and likely other estrogens, is similar to the human sensitivity to EE2. In the current study, we also found that oral EE2 treatment increased uterine weight and induced lordosis behavior in untreated SD and LE rat strains at similar concentrations (Figs. 7a and 7b, respectively), indicating that these rat strains display similar sensitivities to the effects of EE2. In addition, the uterus of the SD rats displays similar sensitivities to sc injections of estrogens as do CD-1 mice (Padilla-Banks et al., 2001; ED50s for sc estradiol 17β are 22 and 7.8 μg/kg/day for SD rat vs. CD-1 mouse) and F344 rats (McKim et al., 2001; ED50s for sc EE2 are 2.2 and 1.7 μg/kg/day for SD vs. F344 rat). Taken together, these results indicate that there are no major differences among rat strains or among rats and CD-1 mice in their ability to respond to low doses of exogenously administered estrogens. CONCLUSIONS In conclusion, the current study demonstrates that maternal exposure to 5–50 μg EE2/kg/day during gestation and lactation produces permanent adverse effects on the developing female rat reproductive system. EE2 affected several reproductive measures at doses ranging from 5 to 50 μg/day, dosage levels within the dose range used by girls and women for therapeutic purposes. In contrast, exposure to BPA at dosage levels 40-, 400-, and 4000-fold above the estimated median human exposure (Calafat et al., 2008) did not alter any end point included in our studies in F1 male (Howdeshell et al., 2008) or in female LE rats. In the current study, we also found that doses of BPA ranging from 2 to 200 μg/kg/day did not affect maternal pregnancy or weight gain or F1 female birth weight, AGD, age at VO, reproductive morphology, fertility, fecundity, or sexual dimorphic behaviors (lordosis, Figure-8 maze activity. or saccharin preference). The lack of effect of BPA on female and male rat offspring after oral exposure to low doses in our studies is consistent with the lack of adverse effects on growth, VO, fertility, and fecundity of low doses of BPA in several other robust, well-designed, properly analyzed multigenerational studies (Cagen et al., 1999; Ema et al., 2001; Tinwell et al., 2002; Tyl et al., 2002). In future studies, we plan to assess the effects of these chemicals on estrous cyclicity, ovarian function, and histology (not included in the current manuscript) and to more thoroughly interrogate the dose-response relationship of these environmental estrogens on the development of lordosis and saccharin preference behaviors. FUNDING BR was funded by the NCSU/US EPA Cooperative Training program in Environmental Sciences Research, Training Agreement CT826512010 with North Carolina State University. We would like to acknowledge Dr John Vandenbergh, Professor Emeritus, North Carolina State University, Zoology Department for his input on this research project and role as chairman of Dr Ryan's PhD thesis committee. We also thank Dr Kembra Howdeshell for her assistance in preparation of the manuscript and review of the data. References Adams J , Buelke-Sam J , Kimmel CA , Nelson CJ , Reiter LW , Sobotka TJ , Tilson HA , Nelson BK . 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TI - In Utero and Lactational Exposure to Bisphenol A, In Contrast to Ethinyl Estradiol, Does Not Alter Sexually Dimorphic Behavior, Puberty, Fertility, and Anatomy of Female LE Rats
JF - Toxicological Sciences
DO - 10.1093/toxsci/kfp266
DA - 2010-03-01
UR - https://www.deepdyve.com/lp/oxford-university-press/in-utero-and-lactational-exposure-to-bisphenol-a-in-contrast-to-pqb9APJWMg
SP - 133
EP - 148
VL - 114
IS - 1
DP - DeepDyve
ER -