TOXICOLOGICAL SCIENCES 114(1), 133–148 (2010) doi:10.1093/toxsci/kfp266 Advance Access publication October 28, 2009

In Utero and Lactational Exposure to , In Contrast to Ethinyl , Does Not Alter Sexually Dimorphic Behavior, Puberty, Fertility, and Anatomy of Female LE Rats

Bryce C. Ryan,*,†,‡ Andrew K. Hotchkiss,*,† Kevin M. Crofton,§ and L. Earl Gray, Jr*,1

*Reproductive Toxicology Branch, TA Division, National Health and Environmental Effects Research Laboratory, ORD, U.S. Environmental Protection Agency Downloaded from https://academic.oup.com/toxsci/article/114/1/133/1629038 by guest on 29 September 2021 Office of Research and Development, Research Triangle Park, North Carolina 27711; †Department of Molecular Biosciences, Toxicity Assessment, North Carolina State University/U.S. Environmental Protection Agency Cooperative Training Program; ‡Department of Zoology, North Carolina State University, Raleigh, North Carolina 27695; and §Integrated Systems Division, National Health and Environmental Effects Research Laboratory, ORD, U.S. Environmental Protection Agency Office of Research and Development, Research Triangle Park, North Carolina 27711

1 To whom correspondence should be addressed at Office of Research and Development, MD 72, 2525 Highway 54, Research Triangle Park, NC 27711. Fax: (919) 541-4017. E-mail: [email protected].

Received September 14, 2009; accepted October 15, 2009

Key Words: ethinyl estradiol; bisphenol A; rat; reproductive Many chemicals released into the environment display estrogenic toxicology; sexually dimorphic behavior. 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 Many chemicals with estrogenic activity are present in the potential effects of BPA. The National Toxicology Program rated environment, including ethinyl estradiol (EE2; Jobling et al., the potential effects of low doses of BPA on behavior and central 1998, 2006) and bisphenol A (BPA; Kang et al., 2007). EE2 is nervous system (CNS) as an area of ‘‘some concern,’’ whereas most a used primarily in combination medications for a variety of effects were rated as of ‘‘negligible’’ or ‘‘minimal’’ concern. conditions in women. Unintentional exposure of the de- However, the number of robust studies in this area was limited. The current study was designed to determine if maternal exposure veloping human fetus to EE2 can occur if oral contraception to relatively low oral doses of EE2 or BPA in utero and during is continued through the early months of undetected pregnancy lactation would alter the expression of well-characterized sexually (reviewed in Smithells [1981]). Due to its widespread dimorphic behaviors or alter the age of puberty or reproductive pharmaceutical use and relatively long half-life, EE2 has been function in the female Long-Evans rat offspring. Pregnant rats were detected in river systems in the United States and Europe as gavaged with vehicle, EE2 (0.05–50 mg/kg/day), or BPA (2, 20, and a contaminant from sewage treatment works (Kolpin et al., 200 mg/kg/day) from day 7 of gestation to postnatal day (PND) 18, 2002; Zuo et al., 2006). In some aquatic systems, concen- and the female offspring were studied. EE2 (50 mg/kg/day) trations of are sufficient to induce adverse effects in increased anogenital distance and reduced pup body weight at fish, including ovotestes in males and reduced fecundity and PND2, accelerated the age at vaginal opening, reduced F1 fertility and F2 litter sizes, and induced malformations of the external population levels (Brion et al., 2004; Jobling et al., 1998, 2006; genitalia (5 mg/kg). F1 females exposed to EE2 also displayed Kidd et al., 2007; Lange et al., 2008). Effluents also may a reduced (male-like) saccharin preference (5 mg/kg) and absence of contain natural estrogens like estradiol and and (15 mg/kg), indications of defeminization of the occasionally industrial chemicals like alkylphenols. In fish, CNS. BPA had no effect on any of the aforementioned measures. for example, only 0.1 ng/l EE2 induces vitellogenin, 0.1–15 These results demonstrate that developmental exposure to phar- ng/l can affect reproductive development, and 2–10 ng/l can macologically relevant dosage levels of EE2 can permanently reduce fecundity (Nash et al., 2004). Thus, given that 5.7% of disrupt the reproductive morphology and function of the female rat. 139 U.S. streams had > 5 ng/l equivalents (EEQs; Kolpin et al., 2002), estrogens are a potential cause of reproductive dysfunction in fish. There are far less data on Disclaimer: The research described in this article has been reviewed by the levels of EEQs in drinking water. National Health and Environmental Effects Research Laboratory, ORD, U.S. BPA is a monomer used in the manufacturing of Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of polycarbonate products, and other uses, that displays estrogenic the Agency nor does the mention of trade names or commercial products activity in vitro and when administered by injection in vivo constitute endorsement or recommendation for use. (Chapin et al., 2008; Kanno et al., 2003; Gray and Ostby,

Published by Oxford University Press 2009. 134 RYAN ET AL.

1998). BPA is approximately a 105-fold less potent than However, the functional significance of these in vitro estradiol (Chapin et al., 2008) and 106-fold less potent than observations in vivo remains to be determined. EE2 in stimulating (ER)–dependent gene It also has been suggested that the fetus and neonate are far expression (EC50 for gene induction in T47D KBluc cells ¼ more sensitive to estrogens and other endocrine disrupters than 6.7 3 107 and 7.3 3 1013 for BPA and EE2, respectively are pubertal or adult female rodents (Guillette and Moore, 2006). (Vickie S Wilson and Dieldrich Bermudez, personal commu- In particular, the potential effects of exposure to BPA on the nication). In vivo, sc administration of BPA or EE2 stimulates development of sexually dimorphic behavior have been noted uterine weight (ED50 of 416 mg/kg/day for BPA versus 0.0008 as an area of special concern by the National Toxicology mg/kg/day for EE2; Kanno et al., 2001, 2003) and induces Program (NTP). The NTP expressed ‘‘some concern’’ for estrogen-dependent lordosis behavior in adult female rats (Gray adverse effects of BPA on the brain and behavior based upon

and Ostby, 1998; Spiteri et al., 1980). When given orally, EE2 ‘‘limited evidence of adverse effects’’ of low doses in rodent Downloaded from https://academic.oup.com/toxsci/article/114/1/133/1629038 by guest on 29 September 2021 stimulated uterine weight (ED50 of 2.5 lg/kg/day), whereas studies. When the Center for the Evaluation of Risk to Human oral BPA at doses as high as 1 g/kg/day were insufficient to Reproduction Expert Panel on BPA (Chapin et al., 2008) and achieve an ED50 (doses of 20–375 mg/kg/day had no effect NTP (2008) evaluated the studies on the neural and behavioral whereas 600 and 1000 mg/kg/day induced small but significant effects of BPA, the majority of the more than 40 studies were increases in weight; Kanno et al., 2003; Supplementary file 1a). found to be ‘‘inadequate’’ for further evaluation, and for this When administered to weanling female rat using the reason, the NTP monograph cites only seven rodent (four with weanling female rat using the U.S. Environmental Protection mice, three with rats) behavioral studies in its determination of Agency (USEPA) Screening Program ‘‘some concern’’ for low-dose neural and behavioral effects of protocol for the Pubertal Female Assay (http://www.epa.gov BPA. Relevant to the current study which examined the effects /endo/pubs/assayvalidation/status.htm), EE2 accelerated the of exposure to BPA on the behavior of the female rat, it is onset of vaginal opening (VO) by 10 days after only 2–3 days noteworthy that only one of these three studies exposed rats of treatment (Supplementary file 1b) and induced persistent during perinatal life (Negishi et al., 2004) and only male vaginal estrus, whereas dosage levels of BPA up to 600 mg/kg/ offspring were examined. day were without effect on these estrogen-regulated end points. One objective of the current study was to determine if In multigenerational studies, oral BPA administration also relatively low oral doses of EE2 or BPA would alter behavioral failed to accelerate the age at VO or induce persist vaginal sexual differentiation, the age at puberty, and the fertility of estrus in either Sprague-Dawley (SD) and Alderley Park female Long-Evans (LE) hooded rat offspring when adminis- (Wistar derived) rats (Ema et al., 2001; Tinwell et al., 2002; tered orally to the dam during gestation and lactation. Pregnant Tyl et al., 2002; Supplementary file 1c), whereas xenoestro- rats were gavaged once daily from gestation day (GD) 7 gens like (Gray and Ostby, 1998; Gray et al., through postnatal day (PND) 18 to expose their offspring 1989), 4 (Chapin et al., 1999), and during the period of sexual differentiation of the reproductive (Casanova et al., 1999) do accelerate the age at VO. organs as well as the neonatal period of sexual differentiation Although these results suggest that BPA may be too weak to of the brain. We selected the LE rat strain because the induce adverse effects during development via a nuclear ER- developing reproductive system is sensitive to both estrogenic mediated mechanism of action when administered orally, it has endocrine-disrupting chemicals (EDCs), such as methoxychlor been hypothesized that BPA may induce adverse effects via the (Gray et al., 1988, 1989), and anti-androgenic compounds seven-transmembrane orphan receptor GRP30 which has been (Gray et al., 1999). Furthermore, the LE rat strain is commonly purported to be responsive to estradiol and BPA (Thomas and used for the study of hormonal regulation of reproductive Dong 2006; Thomas et al., 2005). However, the functional role behaviors (Levine and Mullins, 1964). of GRP30 is itself controversial. In a recent review, Levin The doses of EE2 used herein spanned four log units, (2009) reported that at the cellular level, the early experimental including doses above, at, and below those pharmacologically support for this idea is simply not strong. In vitro, estradiol effective in humans, and the study follows the dosing protocol showed minimal binding to GRP30 and modest signal and dosing regime of a reproduction study which used the SD transduction, and in vivo, a putative GRP30 agonist, G1, did rat (Sawaki et al., 2003). not stimulate estrogen-like effects in the or mammary The dosage levels of BPA used in the current study are gland of mice and none of the four GRP30 knockout mouse commonly used in ‘‘low-dose’’ BPA studies with rats models shows cycling or fertility abnormalities (Levin 2009). (Akingbemi et al., 2004; Ema et al., 2001; Tinwell et al., In addition to GRP30, in vitro studies suggest that low doses 2002) with the lowest dose (2 lg/kg body weight/day) of BPA might produce adverse effects via activation of exceeding the median estimated intake by about 40-fold in membrane-bound ER alpha (Wozniak et al., 2005) or by the United States based upon National Health and Nutrition activating cyclic AMP dependent protein kinase (PKA) and Examination Survey data (maximum ¼ 3.47 lg/kg body cyclic GMP dependent protein kinase (PKG) via a membrane weight/day; median ¼ 0.0505; 25th to 95th percentile 0.0235 G-protein–coupled estrogen receptor (Bouskine et al., 2009). to 0.2722; from the NTP [2008] BPA Monograph derived from EE2 ALTERS SEX DIFFERENTIATION IN FEMALE RATS 135

Calafat et al. [2008] and Lakind and Naiman [2008]). It was that water samples stored for 1 week in old cages display estrogenicity in vitro; not the intention of this study to determine the sensitivity of the however, estrogenic effects were not seen in vivo as housing prepubertal mice female LE rat to developmental exposure to BPA over 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, a broader range of doses. and weanling and adult rats were fed Purina Rat Chow 5001 ad libitum. Since the organization of some rodent sexually dimorphic Animals were provided constant access to filtered (5-lm filter) municipal behaviors during perinatal life is imprinted by androgens and drinking water (Durham, NC) via an automatic watering system. Water was others by estrogens, exposure to EDCs that act via these tested monthly for Pseudomonas and every 4 months for a suite of chemicals, mechanisms can permanently imprint the nervous system and including and heavy metals. The current study was conducted under a protocol approved by the USEPA’s National Health and Environmental behavior. Although rodents display a wide range of sexually Effects Research Laboratory Institutional Animal Care and Use Committee dimorphic behaviors, the organizational role of neonatal in an animal facility approved by the Association for Assessment and Accreditation of Laboratory Animal Care. Control animals used in this study hormones has only been rigorously confirmed for a few Downloaded from https://academic.oup.com/toxsci/article/114/1/133/1629038 by guest on 29 September 2021 behaviors. We chose to study saccharin preference and lordosis purchased from the same supplier and maintained, as above. behavior in female rats because the defeminizing role of Dosage Levels and Administration of Chemicals neonatal hormones has been well established for these Pregnant rat dams were randomly assigned to treatment groups on GD7 behaviors and exposure to exogenous produces robust within each cohort prior to dosing. Laboratory-grade corn oil (CAS 8001-30-7; changes in the behavior of treated female rats. Female rats Lot 062K006), EE2 (CAS 57-63-6; Lot 103K1230; purity 98%), and BPA neonatally defeminized by steroidal hormones display reduced (CAS 80-05-7; Lot 03105ES; purity 99%) were purchased from Sigma- saccharin preference (Wade and Zucker, 1969), reduced Aldrich (St Louis, MO). Dams were dosed via oral gavage from GD7 through fertility (due to the loss of cyclicity of the hypothalamic- PND18 with 0 (corn oil, vehicle control), EE2 at 0.05, 0.5, 1.5, 5, 15, or 50 lg/ kg/day, or BPA at 2, 20, or 200 lg/kg/day. The doses were delivered in 0.5-ml pituitary-gonadal [HPG] axis), and lower levels of female sex corn oil/kg body weight; thus, all dams in the study received the same amount behavior (Blake and Ashiru, 1997; Herath et al., 2001; Kouki of vehicle per body weight. The dams were weighed daily during the dosing et al., 2003, 2005; Sierra and Uphouse, 1986). period to adjust the administered dose for body weight changes during The selection of well-characterized sexually dimorphic pregnancy and lactation and to monitor their health. behaviors that are imprinted by estrogens during development 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 lg/ enables us to clearly determine if EE2 and/or BPA have kg/day), or BPA (2, 20, or 200 lg/kg/day). The second block (block 2) was estrogen-like effects on organization of sexually dimorphic performed to expand the range of EE2 doses tested and involved 82 dams with behaviors. We also decided to determine if developmental 6–14 dams per treatment group: oil vehicle, EE2 (0.05, 0.15, 0.5, 1.5, 5, 15, or exposure to either EE2 or BPA would alter spontaneous 50 lg/kg/day), or BPA (20 or 200 lg/kg/day). locomotor activity levels in a Figure-8 maze. Preliminary data 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 indicated that female rats displayed significantly higher total during pregnancy was calculated from inception of dosing (GD7) to GD20, and 24-h activity levels in the Figure-8 maze than do male rats the analysis of these data included only those dams that were pregnant and (Gray Jr, unpublished observation). Although this behavior is survived through GD20. Maternal body weight gain during lactation was sexually dimorphic in adult rats under these test conditions, the calculated from PND2 through the end of dosing (PND18) and included only role of neonatal estrogens in the development of this sex those dams with pups surviving to weaning. difference is unknown. Maternal and F1 Neonatal and Infant Data The current study directly addresses some of the concerns A summary of the end points measured in F1 females is shown in Table 1. expressed by the NTP review of the literature on the potential On PND2, the sex, body weight, and anogenital distance (AGD) of each female ‘‘low-dose’’ effects of BPA and the effects of this chemical on pup were recorded; the observer measuring these end points was blind as to the the development of the brain behavior. The effects of EE2 and treatment group (samples sizes are given in Fig. 1). AGD was measured as lack of effect of BPA on the dams and their male offspring 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 have already been published (Howdeshell et al., 2008). 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. MATERIALS AND METHODS Vaginal Opening Animals After weaning at PND23, female rats were weighed and checked for VO every Adult female LE rats (Charles River Laboratories, Raleigh, NC) of other day until completion (sample sizes for weight and VO data are shown in approximately 90 days of age were mated by the supplier and shipped on Figs. 2 and 3, respectively). Animals in the 50-lg/kg/day EE2 treatment group GD2. Mating was confirmed by sperm presence in vaginal smears (day of were excluded from the statistical analysis of these data because their malformed sperm plug positive ¼ GD1). All animals were housed individually in genitalia made it difficult to determine when VO had occurred. transparent, 20 3 25 3 47-cm polycarbonate cages with laboratory-grade, heat- Fertility 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 At weaning, a subset of the developmentally exposed female and male rats in the current experiment was clear and without evidence of significant wear. from the second block of the study were paired with an age-matched, non- Only clear cages were used in the current study because some have expressed siblings for at least 4 months and monitored regularly for the presence of litters concern about BPA exposures from old polycarbonate cages. It has been shown (sample sizes for the females are shown in Fig. 4). Since the breeding pairs did 136 RYAN ET AL.

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 Downloaded from https://academic.oup.com/toxsci/article/114/1/133/1629038 by guest on 29 September 2021 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 No effect No effect (photocell counts): ovariectomized Locomotor activity in Figure-8 maze No effect No effect (photocell counts): ovariectomized plus oral EE2 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. 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. 2. Weights at 2 days of age (Fig. 2a) and weaning (Fig. 2b) were reduced in female F1 rats by EE2 treatment at 50 lg/kg/day. BPA had no Methods for the Study of Saccharin Preference effect. Weight at 2 days of age: F ¼ 2.38 (10,172 df), p < 0.015. Samples sizes Pilot study with control male and female LE rats. Prior to the study of for the groups were (number pups/numbers of litters) control (216,33), BPA2 animals treated with EE2 or BPA, a study of saccharin preference was (48,7), BPA20 (81,13), BPA200 (89,15), EE2 0.05 (114,19), EE2 0.15 (37,6), conducted with untreated LE rats to confirm that the behavioral methods we EE2 0.5 (186,29), EE2 1.5 (61,9), EE2 5 (176,30), EE2 15 (40,7), and EE2 50 were planning to use to assess the effects of in utero EE2 and BPA displayed (58,15). Weight at weaning: F ¼ 1.6 (9,59 df) from the ANOVA, p > 0.1. ‘‘#’’ the expected sexual dimorphism. This experiment also was designed to Indicates the mean differed from control by a Dunnett’s post hoc test p < 0.05. determine if female rats displayed a greater preference for either 0.50% wt/vol Samples sizes for the groups were (number pups/numbers of litters) control (5 g of saccharin was added to a liter of deionized, distilled water) or 0.25% wt/ (42,8), BPA20 (32,6), BPA200 (47,8), EE2 0.05 (33,6), EE2 0.15 (35,6), EE2 vol (2.5 g of saccharin was added to a liter of water) saccharin solutions. Adult 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.

male (n ¼ 3–5 per dose group) and adult female intact (n ¼ 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 FIG. 1. Effects of EE2 and BPA on female rat offspring AGD at 2 days of determine the consumption of each fluid. The position of the bottles within the age. AGD was increased by EE2 treatment at 50 lg/kg/day. BPA had no effect. cage was switched on a daily basis to control for any position biases. Saccharin F ¼ 1.6 (9,116 df) from the ANOVA, p < 0.12. Samples sizes for the groups preference was calculated by dividing the amount of saccharin solution were (number pups/numbers of litters) control (156,24), BPA20 (43,6), consumed by the total fluid (water and saccharin) for each day. 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 Saccharin preference in experimental females. Following the pilot study, figure are litter means and SEs. ‘‘#’’ indicates the mean differed from control saccharin preference was measured in female rats exposed during gestation and by a Dunnett’s post hoc test p < 0.05. lactation to varying doses of EE2 or BPA in the first block of the study (sample EE2 ALTERS SEX DIFFERENTIATION IN FEMALE RATS 137 Downloaded from https://academic.oup.com/toxsci/article/114/1/133/1629038 by guest on 29 September 2021

FIG. 3. Exposure to EE2 at 0.5 and 5 lg/kg/day accelerated the age at VO in F1 female rats (significant 5 lg/kg/day; Fig. 3a) and they attained this landmark at a lighter weight (3b). The external genitalia of the F1 females in the 50 lg/kg/day were too malformed to accurately determine the age or weight at VO. BPA had no effect. Age at VO: F ¼ 5.1 (5,32 df), p < 0.0015. Weight at VO: F ¼ 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. 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, FIG. 4. The fecundity (numbers of pups produced over four month and the average saccharin preference over the 5-day period was then analyzed continuous breeding period; Fig. 4a) and fertility (Fig. 4c) was reduced in F1 using litter means on PROC GLM. females exposed to EE2 treatment at 5, 15, and 50 lg/kg/day. BPA had no Sexual dimorphism of Figure-8 maze activity in untreated rats. Figure-8 effect. Fecundity over 4 months: F ¼ 5.25 (9,32 df), p < 0.0002. Samples sizes maze activity levels (Adams et al., 1985; Ruppert et al., 1985) were measured for the groups were (number pups/numbers of litters) control (11,5), BPA20 in untreated adult male and female LE rats to confirm our previous observations (6,5), BPA200 (9,6), EE2 0.05 (3,3), EE2 0.15 (6,5), EE2 0.5 (5,4), EE2 1.5 that females were more active in this apparatus than males (sample sizes are (6,4), EE2 5 (7,5), EE2 15 (6,4), and EE2 50 (2,1). Since some of the sample shown in Fig. 6). On the day of testing, the rats were transferred from the sizes were small, dose groups were pooled to display the consistency of effect animal room to the testing room and placed into small transfer cages. The rats on fecundity among the BPA, lower dose EE2, and higher dose EE2 groups were allowed to acclimate to the room for 5 min. After acclimation, the rats (Fig. 4b). Pooled fecundity over 4 months: F ¼ 14.0 (3,38 df), p < 0.0001. were quickly transferred into 16 Figure-8 mazes. The rats were allowed to Samples sizes for the groups were (number pups/numbers of litters) control freely explore the mazes for 10 h in the dark and the numbers of photocell beam (11,5), BPA: 20 and 200 (15,11), EE2 Low: 0.05, 0.15, 0.5, and 1.5 (18,16), interruptions electronically recorded on an hourly basis. The activity trial and EE2 High: 5.0, 15 and 50 (14,10). Values on the figure are litter means and started approximately an hour before the dark cycle began. SEs. ‘‘*’’ indicates the mean differs from control by p < 0.05. ‘‘**’’ indicates Subsequently, females were ovariectomized and the activity levels measured the mean differs from control by p < 0.01. 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 ll/g body weight), 175, or block of the study. After recovering from surgery, Figure-8 maze activity levels 275 lg/kg/day EE2 daily for 14 days. Based on the results of this experiment, were measured (trial 1), as above. Subsequently, females were treated orally by we administered 275 lg EE2/kg daily for 14 days to restore activity levels in gavage for 14 days with 275 lg/kg EE2 and retested in the Figure-8 mazes females developmentally exposed to EE2 and BPA. (trial 2; sample sizes are shown in Fig. 6). Assessment of Figure-8 maze activity in EE2- or BPA-treated females. Preliminary study of lordosis behavior and uterine weight in SD and LE Figure-8 maze activity was measured in ovariectomized females from the first rats after oral EE2 treatment: dose-response to EE2. Lordosis behavior was 138 RYAN ET AL.

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. Downloaded from https://academic.oup.com/toxsci/article/114/1/133/1629038 by guest on 29 September 2021 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. FIG. 5. Saccharin preference. Untreated female rats show a greater preference (about 80%) for 0.25% wt/vol and 0.50% wt/vol saccharin versus If 2 min elapsed without any mounts, the female was removed from the plain water than do untreated male rats (about 40%; Fig. 5a). Samples sizes: cage and placed into a test cage of another stimulus male. All lordosis testing took place in the same room, and the same 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 experimenter, blind to the treatment groups, scored every trial. 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) ¼ Surgical Methods for Ovariectomy of Experimental Females 11.8, p < 0.005. The F values for saccharin concentration and the interaction of Rats were anesthetized in a specially designed airtight Plexiglas container sex and concentration were not significant. In the F1 females from dams through which Halothane vapor (Bickford Halothane Vaporizer) and 10 exposed to either EE2 or BPA, saccharin preference was reduced by exposure standard cubic feet per hour of airflows. Initially, a low concentration (1% by to EE2 at 5 and 50 lg/kg/day. BPA had no effect (Fig. 5b). F ¼ 5.5 (6,25 df), volume) of halothane vapor was administered to avoid inducing stress in the p < 0.001. Samples sizes for the groups were (number pups/numbers of litters) rat. After the rat lost consciousness, the halothane concentration was increased control (10,6), BPA2 (6,3), BPA20 (15,7), BPA200 (8,4), EE2 0.5 (9,5), EE2 to 3% by volume to induce a surgical plane of anesthesia as determined by 5 (10,5), and EE2 50 (2,2). Values on the figure are litter means and SEs. reduced respiration rate and absence of any response to tactile stimuli. When ‘‘**’’ indicates the mean differs from control by p < 0.01. 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 studied using untreated adult ovariectomized females given EE2 followed by prior to surgery, the rats were injected with 0.05 mg/kg buprenorphine to progesterone to activate the behavior. Female rats that have been defeminized by provide pain control and placed on a heating pad. The dorsal lumbar region was neonatal estrogen exposures display low levels of lordosis behavior (Gorski, 1986). shaved, cleaned, and two bilateral incisions were made of about 0.5 cm. The Adult, ovariectomized SD and LE rats as well as adult, intact stimulus SD ovaries were identified, ligated with non-wicking, absorbable suture, and males were purchased from Charles River Laboratories. Female rats were removed. The incised muscle also was sutured together, and the incision was housed two per cage in a room on a reversed light cycle (lights on from 9:00 then closed with a wound clip. The rat was then removed from the nose cone, P.M. to 11:00 A.M.) and given at least 2 weeks to recover from surgery and to placed in a clean cage on a heating pad, and monitored until recovery. Prior to acclimate to the altered light cycle prior used for behavioral testing. In this surgery, all instruments were autoclaved for sterilization. The technician experiment, ovariectomized adult female LE rats were given varying doses of performing the surgery wore sterile gloves and performed the surgery through EE2 by oral gavage in oil (0, 2.5, 5, 10, 12.5, 19, 25, 37.5, 50, 65, 80, 95, 110, a sterile drape to minimize the risk of contaminating the surgical sites. Prior to 125, and 250 lg/kg) for 2 days, followed by 0.5 mg progesterone (sc) in oil on surgery, the incision sites were cleansed with betadine and alcohol swabs the third day in order to determine an optimal oral dose of EE2 that induces this utilizing a circular wiping motion three times with each solution. Instruments behavior reliably in control females. Females were retested at different dosage were sterilized using a hot bead sterilizer between animals. Following surgery, levels two to three times. This experiment (Fig. 7) included both LE and SD each rat was monitored until locomotion and other signs of activity appeared female rats to determine if there was a strain difference in the dose-response to normal and then checked twice daily to ensure recovery. The wound clips were EE2. removed after 7 days. 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 Lordosis Behavior of EE2- and BPA-Treated Females more sensitive to low doses of EE2, increased uterine weight, or induction of lordosis behavior. Ovariectomized female LE and SD rats were treated for After VO had been assessed in the second block of the study, rats were 2 days by gavage with EE2 at 0, 0.5, 1, 2.5, 5, 10, 25, 50, or 250 lg/kg and bilaterally ovariectomized and allowed to recover for at least 2 weeks prior to necropsied on the third day, and the wet weight of the uterus was measured. behavioral testing (sample sizes are shown in Fig. 8). Since administration of EE2 ALTERS SEX DIFFERENTIATION IN FEMALE RATS 139 Downloaded from https://academic.oup.com/toxsci/article/114/1/133/1629038 by guest on 29 September 2021

FIG. 6. 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 ¼ (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 ¼ 6) and 275 lg/kg (n ¼ 5) for 14 days increases the locomotor activity levels of untreated ovariectomized females (n ¼ 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 lg/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 ¼ 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 ¼ 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 ¼ 3.1 (5 df), p < 0.02, indicated by an ‘‘*’’.

50 lg/kg EE2 orally (approximately and ED80 dose; Fig. 7b) for 2 days data also were reanalyzed using a Dunnett’s post hoc test which, unlike followed by an injection of 0.5 mg progesterone induced LQ of nearly 1.0 in LSMEANS, does not require that the F value from the ANOVA be significant. control females, this dosing regime was selected to describe the effects of EE2 The statistical analyses were based upon litter mean values. The frequency of and BPA on the lordosis behavior of the experimental females. malformed external genitalia in individual F1 females was analyzed by Fisher exact test. Statistical significance was considered p 0.05. 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 RESULTS 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. Maternal and Pregnancy Data Urethral slit length: The length of the urethral slit. There was a significant main effect of EE2 treatment on Urethral slit depth: The depth of the urethral slit while viewing the maternal body weight gain during gestation (from Howdeshell phallus from the side. When viewed in this fashion, the urethral slit et al., 2008). Treatment with EE2 at 1.5 lg/kg/day and higher typically forms a ‘‘V’’ shape. Urethrovaginal distance: The distance from the urethral opening significantly decreased maternal body weight gain during (defined as the bottom of the urethral slit) to the posterior end of the gestation relative to controls. There were no treatment effects of vagina. BPA on maternal body weight gain during pregnancy or lactation. Anovaginal distance: The distance from the posterior vagina to the There was a significant main effect of treatment on the anterior anus. number of uterine implantations, with the 50-lg EE2/kg/day Statistical Analyses group having significantly fewer uterine implantation scars The F1 female data were analyzed using PROC GLM (a general linear (Howdeshell et al., 2008). There was a significant main effect models procedure) by one-way ANOVA using PC SAS (Statistical Analysis of treatment on the number and body weight of live pups on System), version 9.1 (SAS Institute, Cary, NC). If the overall F value of the PND2 (Figure 2; Howdeshell et al., 2008). Post hoc analysis 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 detected no significant effect of BPA treatment on the number end point. Significance was identified by an F value with p < 0.05, followed by of implantations and number of live pups at PND2 or fetal/ LSMEANS (t-test). If the F value of the ANOVA was ‘‘not significant,’’ the neonatal mortality. 140 RYAN ET AL.

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 lg 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 Downloaded from https://academic.oup.com/toxsci/article/114/1/133/1629038 by guest on 29 September 2021 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 file 2).

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 lg EE2/kg/day significantly reduced saccharin FIG. 7. Uterine (Fig. 7a) and lordosis (Fig. 7b) responses of control adult preference in intact F1 females to male-like levels (Fig. 5b). LE and SD ovariectomized female rats to two daily oral EE2 doses to identify The lowest-observed effect level for saccharin preference was a near-maximal dose to use to induce lordosis in the experimental females. The 5 lg EE2/kg/day. BPA did not significantly affect this behavior ED50 response to EE2 for uterine weight was about half the ED50 for lordosis (ANOVA and Dunnett’s tests were not significant for BPA at behavior. Values on the figure are means and SEs. There were no differences in any dose). the dose-response curves among these strains of rats. Figure-8 Maze Locomotor Activity Neonatal and Pup Data In untreated rats, males displayed significantly lower levels Table 1 displays a list of all the end points measured in of activity (photocell beam interruptions over 10 hrs) than did the F1 female rats exposed to EE2 and BPA from GD7 to female rats (Fig. 6a). The second pilot study demonstrated that PND18. EE2 at 50 lg/kg/day increased female rat AGD at oral administration of EE2 for 14 days increased maze activity PND2 (not significant by ANOVA, significant with a Dunnett’s in ovariectomized adult female rats to the levels seen in intact test, p < 0.05; Fig. 1) and decreased pup body weight on PND2 females in the first study (Fig. 6b). This demonstrates that and at weaning (Figs. 2a and 2b, respectively). BPA had no estrogen exposure during adult life is necessary to activate effect on body weight at any age or AGD at 2 days of age. female-like activity levels in our apparatus. Neither EE2 nor BPA induced areolae/nipple agenesis, a male In the EE2/BPA study, developmental exposure did not rat trait displayed among female rats—exposed in utero to affect maze activity in ovariectomized or ovariectomized EE2- androgens but not estrogens (data not shown). Since male treated females (Table 1 and Figs. 6c and d, respectively). weaning weight was not included in Howdeshell et al. (2008), it is shown in Table 2 for comparison to the body weight of the Lordosis Behavior weanling females. In the preliminary dose-response studies with EE2 on uterine weight and lordosis behavior, the ED50s for uterine weight and Age at Puberty (VO) induction of lordosis behavior were seen at doses of about 8 lg Prenatal treatment with 5 lg EE2/kg/day significantly EE2/kg/day (Fig. 7a) and 20 lg EE2/kg/day (Fig. 7b), accelerated the age at VO opening and the females attained respectively. The dose-response curves were similar for LE this landmark at a lighter body weight than control females and SD rats. Based upon these results, we used 50 lg EE2/kg/ (Figs. 3a and 3b, respectively). Since this end point was not day, which is approximately an ED80, to induce lordosis measured in F1 females in the second block, we do not know if behavior in the EE2/BPA study. the dose of 1.5 lg EE2/kg/day accelerated VO or not. In the EE2/BPA study, developmental exposure to EE2 at 15 Furthermore, the severity of the malformations of the external and 50 lg/kg/day (Fig. 8) reorganized (defeminized) the brain EE2 ALTERS SEX DIFFERENTIATION IN FEMALE RATS 141 0.49 0.37 0.81 ± ± 5.6 (3,2) ± 116 (7, 5) ± ± test without post hoc 4.1 (28,6) 44.4 ± 0.10 No data 6.31** 0.52 No data 20.5 0.13 No data 3.82** ± ± ± 2.8 (46,8) 59.0 Downloaded from https://academic.oup.com/toxsci/article/114/1/133/1629038 by guest on 29 September 2021 129 (24,12) No data 109 ± FIG. 8. The lordosis behavior of ovariectomized, EE2 primed F1 female ± rats was reduced by maternal treatment with EE2 at 15 and 50 lg/kg/day. BPA had no effect. F ¼ 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 0.01 or significant by Dunnett’s < 2.2 (39,9) 58.2 (6,3), BPA200 (15,6), EE2 0.05 (6,3), EE2 0.15 (6,3), EE2 0.5 (12,5), EE2 1.5 p (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. 0.05 or ** < 0.52 No data 18.2 0.07 No data 1.78* 0.08 No data 1.83 p ± ± ± 2.6 (39,7) 58.5 of the female LE rat such that administration of EE2 plus 174 (13,9) No data 205 ± progesterone was unable to activate lordosis behavior in adult ± F1 females. In contrast to EE2, BPA had no effect on lordosis behavior. 2.6 (36,6) 58.7 ± -test from control. * Malformations of the External Genitalia of F1 Females t F1 females exposed developmentally to EE2 at 5–50 lg

EE2/kg/day displayed (63–100%, respectively) malformations 0.7 No data 18.8 0.05 No data 0.93 0.06 No data 1.50 are presented. ± ± ± 1.6 (31,6) 60.7

of the external genitalia (Figs. 9 and 10a). Measurement of the F ± distance from the urethral opening to the VO (Fig. 10b) and the TABLE 2 length and depth of the urethral slit were altered (Table 2), confirming our visual assessment of the external genitalia. The 0.49 18.71 0.07 0.98 0.06 1.31 distance from the anal opening to the VO (Table 2) was not ± 1.5 (43,8) 60.1 ± ± 113 (26, 13) No data No data 325 affected by EE2 treatment. BPA did not affect any of these ± ± morphological end points in the F1 female rat. 0.36 19.7 0.07 0.85 0.05 1.34 ± ± ± DISCUSSION 3.4 (26,6) 57.8 159 (23,12) 247 Gestation and Lactation that Are Not Included in Any Figures ± ± Our results demonstrate that administration of EE2 in utero and during lactation permanently altered F1 female LE rat 0.47 19.9 0.06 1.03 reproductive morphology, function, and behavior (Table 1). 0.07 1.42 ± ± ±

Administration of EE2 at 5–15 lg/kg/day induced malforma- 110 (25,13) 316 tions of the female rat external genitalia (Figs. 9 and 10), ±

accelerated VO (Fig. 3b), defeminized lordosis (Fig. 8) and values with (treatment, error df) and probability values of saccharin preference (Fig. 5b) behaviors, and reduced fecundity F 0.5 19.5 0.10 1.05 0.05 1.37 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 ± 0 ± ± 237 (9,9) 239 and fertility (Figs. 4a–c). These results also suggest that the F1 2.0 (30,8) No data 58.5 ± ± ± g/kg/day BPA2 BPA20 BPA200 EE 0.05 EE 0.15 EE 0.5 EE 1.5 EE 5 EE 15 EE 50 l 19.6 1.07 females were more affected by perinatal EE2 than F1 males 1.48 686 (Howdeshell et al., 2008) in terms of the prevalence and 58.9 severity of the effects (Fig. 11). F1 LE male rats displayed 0.3 > 2.65, 98.5, 33.4, p value in ANOVA; 1.1, ¼ ¼ slight reductions in seminal vesicle and testis weights at 5 lg ¼ F ¼

EE2/kg/day and reduced sperm counts and histopathological 1.22, F (7,86) (7,86) (7,86) ¼ UVD, urethrovaginal distance in millimeter. Gray-shaded means differ significantly by ANOVA and a F F F

alterations in reproductive tissues, whereas doses as high as (9,56) 0.14 0.0001 0.0001 0.4 F < < < >

50 lg EE2/kg/day did not induce reproductive tract malforma- (6,66) Note. (mm); p urethral slit depth (mm); p urethral slit length (mm); p increase from 14 days of adult oral EE2 treatment; F (g); p Summary (litter means a significant (overall ANOVA) External genitalia—AVD End points; External genitalia— External genitalia— tions in F1 males (Howdeshell et al., 2008). It is noteworthy Figure-8 maze activity F1 male weaning weight 142 RYAN ET AL. Downloaded from https://academic.oup.com/toxsci/article/114/1/133/1629038 by guest on 29 September 2021

FIG. 9. 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 lg/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 lg/kg/day is shown in the top right panel. This effect was not displayed by any F1 females in the BPA groups. that exposure to estrogenic pesticides like methoxychlor (Gray group, whereas EE2 reduced fecundity in LE F1 females at et al., 1989) or toxic substances like nonylphenol (Tyl et al., doses of 5, 15, and 50 lg/kg/day. Subsequently, SD F1 female 2006; Chapin et al., 1999) do not cause similar malformations rats exposed to 50 lg/kg/day exhibited abnormal estrous of the reproductive tract although exposure to these xenoes- cyclicity, including persistent estrus, and histological examina- trogens during development does accelerate VO and reduce F1 tion revealed follicular cysts and absence of corpora lutea in the female fecundity (Supplementary file 1b). ovaries of the rats with persistent estrus (Sawaki et al., 2003). In contrast to EE2, perinatal BPA at 2, 20, and 200 lg/kg/ In addition to the studies of Sawaki et al. (2003 a, b), several day was without effect on the reproductive development of other studies with rats have reported on the adverse effects of either male or female F1 LE rat offspring. These results on the EE2 during development, the most robust being the study F1 females and those we reported earlier for the F1 male conducted at the National Center for Toxicological Research offspring from these litters (Howdeshell et al., 2008) for the NTP (Latendresse et al., 2009). They administered EE2 demonstrate that BPA did not disrupt reproductive develop- in a dietary multigenerational study at 2, 10, or 50 ppb in diet ment in an estrogenic manner in the F1 offspring when (estimated to be 0.2, 1.1, or 5.8 lg/kg/day) to SD rats. These administered orally to the rat dam in utero and during lactation. authors reported that EE2 reduced body weight of the females EE2 produced a wide variety of anatomical, histopathological, at 1.1 and 5.8 lg/kg/day, altered the age at VO, induced and functional alterations in F1 males and females and abnormal estrous cycles at 5.8 lg/kg/day, and induced non- behavioral alterations in the F1 females. Furthermore, none neoplastic uterine lesions at all doses. However, they did not of the effects of EE2 displayed a non-monotonic dose-response observe any reduction in fertility or fecundity as a consequence pattern over a dose range of four log units. of EE2 exposure. It is possible the dietary exposure that they used and gavage exposure used herein produce very different Effects of EE2 in Other Rat Transgenerational Studies maximum EE2 peak heights and areas under the curve in EE2-exposed F1 LE rat females in the current study were maternal, fetal, and offspring tissues. In another study with the more severely affected than similarly exposed SD rats (Sawaki SD rat strain, Fusani et al. (2007) dosed rats orally with EE2 at et al., 2003). Sawaki et al. (2003) observed cleft phallus in 0.004 or 0.4 lg/kg/day from GD5 to PND32. They reported almost all the female offspring at 50 lg/kg/day, but these that EE2 did not affect AGD, pup viability or body weight, females initially had fertility levels comparable to the control whereas fertility and estrous cyclicity were altered at 0.4 lg/kg/ EE2 ALTERS SEX DIFFERENTIATION IN FEMALE RATS 143 Downloaded from https://academic.oup.com/toxsci/article/114/1/133/1629038 by guest on 29 September 2021

FIG. 11. 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 ¼ ED50 for F1 female malformations of the genitalia < ED50 for the inhibition of F1 female lordosis behavior < ED50 F0 FIG. 10. Maternal treatment with EE2 at 5, 15, and 50 lg/kg/day increases maternal weight gain ¼ ED50 for F1 pup viability and ¼ F1 male seminal the percentage of F1 female rats displaying malformed external genitalia vesicle weights (one of the most affected end points in the F1 males. (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 lg/kg/day. The measurement was not taken in females treated with EE2 at 15 lg/kg/day. BPA had no effect on urethrovaginal distance (UVD) or the percentage of malformed females. Analysis of percent with cleft phallus: F ¼ 40.8 (9,59 df) from the ANOVA, that estrogens administered to neonatal female rats defeminizes p < 0.0001. Samples sizes for the groups were (number pups/numbers of litters) the hypothalamic-pituitary axis by abolishing the proestrus control (42,8), BPA20 (32,6), BPA200 (47,8), EE2 0.05 (33,6), EE2 0.15 luteinizing hormone (LH) surge (Gorski, 1986; Gorski and (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 ¼ 80.3 (7,86 df) from the ANOVA, p < 0.0001. Barraclough, 1962; Homma et al., 2009), resulting in infertility Samples sizes for the groups were (number pups/numbers of litters) control in treated female rats (McCarthy, 2008). (30,13), BPA2 (26,9), BPA20 (29,9), BPA200 (22,12), EE2 0.05 (24,10), EE2 Interestingly, the role of the E2-signaling pathway is quite 0.5 (46,15), EE2 5 (24,10), and EE2 50 (25,12). ‘‘**’’ indicates the mean diverse in mammals, and it even varies among rodents. For differs from control by p < 0.01. example, in the Syrian hamster, neonatal treatment with estrogens (estradiol [Whitsett et al., 1978], [Gray day in pairs of similarly treated males and females (Fusani et al., 1985], [kepone; Gray, 1982]) demasculi- et al., 2007). nizes male mating behavior such that the males do not mount sexually receptive females, whereas the ability of the female Effects of EE2 and BPA on Behavioral Sex Differentiation hamster to display lordosis behavior is not defeminized. In In mammals, some sexually dimorphic behaviors are contrast, administration of estrogens to the neonatal female rat permanently organized during the perinatal period of de- results in defeminization of female lordosis behavior. velopment by testicular or Although the organizational role of estradiol in the de- (DHT) or estradiol, metabolites of testosterone. The degree to velopment of rodent sexual behaviors is well established, what which the T, DHT, or E2 pathways each contribute to role, if any, estrogens play in primate brain development neurobehavioral sex differentiation varies greatly among remains to be determined (McCarthy, 2008). The review by mammalian species. In the rat, the E2 pathway is critical for McCarthy (2008) of the role of estradiol in the developing the normal differentiation of the central nervous system and brain concludes that ‘‘the lack of effect of DES on brain some behaviors. of T to E2 locally induces differentiation of human females is consistent with empirical masculinization and defeminization of specific sexually di- results generated in primates in which most results indicate no morphic behaviors. For example, it has been long established important role for estrogens in masculinization, this function 144 RYAN ET AL. instead being performed by prenatal androgens combined with saccharin solution, and only 20% was plain water. This is in social context and rearing conditions.’’ Baum (2006) also marked contrast to the male rats in which only 40% of the total reported that, in sexual differentiation of the human and fluid consumption was saccharin (Fig. 5a). These results are nonhuman primate brain, it is generally agreed that the consistent with numerous publications showing a sexual androgen-signaling pathway predominates in neurobehavioral dimorphism with intact female rats displaying a greater sex differentiation and a clear role for the E2 pathway, if any, preference for saccharin solutions versus plain water than has not yet been identified (Baum, 2006). control male rats. For example, Valenstein et al. (1967) first reported the sex differences in saccharin preference in 1967 Effects of EE2 and BPA on Lordosis Behavior and Wade and Zucker (1969) demonstrated that neonatal testosterone treatment could defeminize this behavior such that In addition to the LH surge, neonatal estrogens can

female rats no longer displayed a preference of saccharin Downloaded from https://academic.oup.com/toxsci/article/114/1/133/1629038 by guest on 29 September 2021 defeminize the brain such that when adult rats are ovariecto- solutions over plain water. However the current literature does mized, they fail to display lordosis behavior when activated not resolve whether defeminization of saccharin preference with the appropriate hormonal regime in adult life (McCarthy, results from androgenic or from estrogenic signaling during 2008). Lordosis behavior also has been studied in intact cycling neonatal life. female rats, with the behavior normally being displayed in the In the current study, maternal treatment with 5 and 50 lg dark phase of the light cycle on the evening of vaginal EE2/kg/day significantly reduced saccharin preference in intact proestrus. However, the advantage of using the ovariectomized, F1 females to male-like levels (Fig. 5b). It is not clear if the hormone-primed female, as done herein, is that this approach reduction in saccharin preference by in utero and lactational eliminates the possibility that the failure to display lordosis is EE2 treatment resulted from a direct effect on the brain regions due to a loss of estrous cyclicity in the intact rat. Hence, the fact that regulate the development of this behavior in the F1 female that EE2 exposure abolished lordosis behavior in the rat or via alterations of the HPG axis, estrous cyclicity, and ovariectomized, hormone-primed females herein is a clear serum estradiol levels. It would be interesting to examine the indication of a direct effect of this potent estrogen on the effects of perinatal EE2 on saccharin preference in ovariecto- developing nervous system. The lack of effect of BPA on this mized females with and without estrogen priming to determine behavior clearly indicates the estrogen pathway in the brain if this was a direct effect of EE2 on the organization of controlling this behavior was not defeminized by BPA. saccharin preference behavior or on the developing HPG axis. In future studies, we plan to use sc injection of estradiol BPA did not significantly affect this behavior at any dosage benzoate (E2B) rather than orally administered EE2 for level. 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 Effects of EE2 and BPA on Locomotor Activity in the E2B to determine the dose-response to determine if partially Figure-8 Maze defeminized females are less responsive to low doses as adults Results of the study revealed that control females had than untreated females. significantly higher spontaneous activity levels over the 10-h In addition to the current study, several other studies have assessment period than control male rats (Fig. 6a), ovariectomy examined the effects of BPA on the development of lordosis reduces activity to male levels (Fig. 6a), and administration of behavior in the female rat using a broad range of doses, an estrogen to an ovariectomized female rat restores the activity administered orally and by neonatal sc injection and failed to level to that seen in intact females (Fig. 6b). However, in utero find an effect. In total, none of six studies that have examined and lactational exposure to EE2 or BPA did not alter activity the effects of BPA on lordosis behavior have found that BPA levels in ovariectomized F1 females with or without EE2 defeminized this behavior. Kwon et al. (2000) administered priming (Figs. 6c and d, respectively). These results indicate BPA orally (Kwon et al., 2000) at oral doses of 3–300 mg/kg/ that neither of these chemicals defeminized the brain with day and found no effect of BPA, whereas the DES-positive respect to this sexually dimorphic behavior. Since we did not control was effective. Farabollini et al. (2002) and Kubo et al. examine the locomotor activity of the experimental females (2003) also reported that BPA did not decrease female lordosis prior to ovariectomy, we do not know if either chemical would behavior in an estrogenic manner. Furthermore, sc injection of have had an effect in the intact animal. It is possible that intact high doses of BPA ranging from 50 lg/kg to 50 mg/kg failed to EE2-treated females would have displayed altered estrous disrupt lordosis behavior in the female rats (Adewale et al., cycles, and locomotor levels would have been reduced as 2009; Monje et al., 2009). a consequence of reduced levels of endogenous estradiol. The fact that ovariectomized F1 females exposed to high doses of Effects of EE2 and BPA on Saccharin Preference EE2 in utero and during lactation were not defeminized (as Results of the study with control females reveal that about indicated by the observation that they responded normally to 80% of the total fluid consumed by intact female rats was the the activational effects of EE2 in adult life) suggests that EE2 ALTERS SEX DIFFERENTIATION IN FEMALE RATS 145 estrogens do not play a role during perinatal life in the (Calafat et al., 2008) did not alter any end point included in organization of this sexual dimorphic behavior. 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 Sensitivity of the LE Rat versus Humans to EE2 ranging from 2 to 200 lg/kg/day did not affect maternal When used as a human pharmaceutical, EE2 is often pregnancy or weight gain or F1 female birth weight, AGD, age combined with progestogens or other steroids to maximize the at VO, reproductive morphology, fertility, fecundity, or sexual effects and to reduce the adverse side effects. Over the years, dimorphic behaviors (lordosis, Figure-8 maze activity. or dosage levels have declined in order to eliminate adverse side saccharin preference). The lack of effect of BPA on female effects of EE2 in girls and women. For example, it was shown and male rat offspring after oral exposure to low doses in our that reducing EE2 levels from 100 to 50 lg/day in combination studies is consistent with the lack of adverse effects on growth, with a progestogen reduced the incidence of thromboembo- VO, fertility, and fecundity of low doses of BPA in several Downloaded from https://academic.oup.com/toxsci/article/114/1/133/1629038 by guest on 29 September 2021 lisms (Committee-on-Safety-of-Drugs, 1970). In some cases, other robust, well-designed, properly analyzed multigenera- relatively high dose levels of EE2 have been used alone or in tional studies (Cagen et al., 1999; Ema et al., 2001; Tinwell combination with doses ranging from 100 to 1000 lg/day to et al., 2002; Tyl et al., 2002). In future studies, we plan to limit final height in girls and boys (Rooman et al., 2005; assess the effects of these chemicals on estrous cyclicity, Schmitt et al., 1992; Svan et al., 1991). In addition, when EE2 ovarian function, and histology (not included in the current was administered as a postcoital contraceptive agent at 2–5 mg/ manuscript) and to more thoroughly interrogate the dose- day for 5 days, 2–3 mg was only partially effective while no response relationship of these environmental estrogens on the pregnancies occurred in the 5-mg group (Blye, 1973; Haspels, development of lordosis and saccharin preference behaviors. 1972). If one uses a body weight of 50 kg for reference, doses of EE2 of 100–5000 lg/day (equivalent to 2–100 lg/kg body weight/day) can result in adverse effects in humans. This dose SUPPLEMENTARY DATA range produced adverse effects in LE rat dams and F1 male and Supplementary data are available online at http://toxsci female offspring in our studies. Taken together, these results .oxfordjournals.org/. 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 FUNDING increased uterine weight and induced lordosis behavior in BR was funded by the NCSU/US EPA Cooperative Training untreated SD and LE rat strains at similar concentrations program in Environmental Sciences Research, Training Agree- (Figs. 7a and 7b, respectively), indicating that these rat strains ment CT826512010 with North Carolina State University. 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., ACKNOWLEDGMENTS 2001; ED50s for sc estradiol 17b are 22 and 7.8 lg/kg/day for SD rat vs. CD-1 mouse) and F344 rats (McKim et al., 2001; We would like to acknowledge Dr John Vandenbergh, ED50s for sc EE2 are 2.2 and 1.7 lg/kg/day for SD vs. F344 Professor Emeritus, North Carolina State University, Zoology rat). Taken together, these results indicate that there are no Department for his input on this research project and role as major differences among rat strains or among rats and CD-1 chairman of Dr Ryan’s PhD thesis committee. We also thank mice in their ability to respond to low doses of exogenously Dr Kembra Howdeshell for her assistance in preparation of the administered estrogens. manuscript and review of the data.

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