Pubertal Development and Reproductive Functions of Crl:CD BR ...

55, 399 – 406 (2000) Copyright © 2000 by the Society of Toxicology

TOXICOLOGICAL SCIENCES

Pubertal Development and Reproductive Functions of Crl:CD BR Sprague-Dawley Rats Exposed to Bisphenol A during Prenatal and Postnatal Development S. Kwon, D. B. Stedman, B. A. Elswick, R. C. Cattley, and F. Welsch 1 Chemical Industry Institute of Toxicology, Six Davis Drive, Research Triangle Park, North Carolina 27709 –2137 Received October 15, 1999; accepted January 6, 2000

Bisphenol A (BPA) is used on a large scale in the manufacture of polycarbonate plastics. BPA has been shown to bind weakly to both estrogen receptor (ER)␣ and ER␤, and to transactivate reporter genes in vitro. The purpose of the present study was to determine whether exposure of rats to BPA during pre- and postnatal development affects estrogen-mediated end points related to pubertal development and reproductive functions. BPA was administered to pregnant Crl:CD BR Sprague-Dawley rats by gavage at 0, 3.2, 32, or 320 mg/kg/day from gestation day (GD) 11 through postnatal day (PND) 20. Diethylstilbestrol (DES) at 15 ␮g/kg/day was used as a reference chemical with known estrogenic effects. Female pubertal development was not affected by indirect BPA exposure of the offspring at any of the dose levels. Treatment with this chemical also did not produce detectable effects on the volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA), estrous cyclicity, sexual behavior, or male reproductive organ weights of F 1 offspring. However, DES at 15 ␮g/kg/day increased the volume of the SDN-POA of female offspring and affected their normal estrous cyclicity following puberty. In this study, pre- and postnatal exposure of rats to BPA at 3.2, 32, or 320 mg/kg/day from GD 11 through PND 20 did not have any apparent adverse effects on female rat pubertal development and reproductive functions. Key Words: bisphenol A; pubertal development; reproductive system; reproductive function.

Bisphenol A (BPA) is a high-production volume chemical used in the manufacture of polycarbonate plastics. BPA has been found in the liquor from canned food packed in lacquercoated cans (Brotons et al., 1995) and in saliva collected from subjects treated with composite dental sealants (Olea et al., 1996). BPA has been reported to be weakly estrogenic in both in vitro and in vivo systems. Krishnan et al. (1993) reported that BPA, leached from polycarbonate flasks, competed with [ 3H]-estradiol for binding to estrogen receptors (ER) from rat uterus, induced progesterone receptors, and promoted cell proliferation in cultured human mammary cancer cells (MCF-7). 1

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BPA binds to both ER␣ and ER␤ with low affinity and transactivates reporter genes in vitro (Gaido et al., 1997; Kuiper et al., 1998). Uterotrophic activity of BPA at high doses (3 daily oral doses at 400, 600, or 800 mg/kg/day) was also reported in immature female Alpk:AP rats (Ashby and Tinwell, 1998). Developmental and reproductive toxicity of high doses of BPA have been reported in rats and mice. Persistent estrus was observed in ovariectomized rats injected twice a day for 3 consecutive days with 100 mg of BPA (Dodds and Lawson, 1936). Intraperitoneal injection of BPA at 125 mg/kg on gestation day (GD) 1 through GD 15 also interfered with the establishment of pregnancy and reduced the number of live fetuses per litter in young adult female Sprague-Dawley rats (Hardin et al., 1981). However, no significant developmental toxicity of BPA was observed in CD rats and CD-1 mice exposed to BPA (rats, 640 mg/kg; mice, 1000 mg/kg) by gavage from GD 6 through GD 15 (Morrissey et al., 1987). A continuous breeding study conducted by the National Toxicology Program (NTP) of the USA showed that BPA at 0.5 or 1.0% in feed (approximate daily doses are 875 or 1750 mg/ kg/day) reduced the number of live pups per litter and litters per pair in Swiss CD-1 mice in the first generation mice (Morrissey et al., 1989). Oral administration of BPA at much lower doses has also been reported to affect male reproductive organ parameters such as prostate glands (increase in fresh tissue weights at 2 or 20 ␮g/kg/day), preputial glands (increase in tissue weights at 2 ␮g/kg/day), and epididymides (decrease in tissue weights at 2 ␮g/kg/day), and the efficiency of sperm production (decrease in daily sperm production per g testis at 20 ␮g/kg/day) in CF-1 mice exposed to BPA during prenatal development from GD 11 to GD 17 (Nagel et al., 1997; vom Saal et al., 1998). However, the low-dose effects of BPA have been controversial. Other researchers reported no treatmentrelated effects of BPA at the same and additional low-dose levels given at the same time of pregnancy to CF-1 mice (Ashby et al., 1999; Cagen et al., 1999). The purpose of the present study was to determine whether exposure of rats to BPA at high doses during pre- and postnatal development (from GD 11 to PND 20) affects estrogen-mediated end points related to pubertal development and reproduc-

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tive functions. The rationale for the chosen BPA-treatment window was based on the hypothesis that environmental estrogens have the potential to interact directly with target sites in the differentiating reproductive system and interfere with endocrine-regulated processes involved in the development and function of the reproductive system (Danzo, 1998; Gorski, 1986; Toran-Allerand, 1984). The dose levels of 0, 3.2, 32, or 320 mg BPA/kg/day were selected based upon the published weak estrogenic potency in both in vitro and in vivo systems. Kuiper et al. (1998) showed that the in vitro binding affinity of BPA was approximately 20,000-fold lower than that of diethylstilbestrol (DES) for both ER␣ and ER␤. Gaido et al. (1997) also reported that BPA has weak estrogenic activity in vitro, which is approximately 15,000-fold less potent than 17␤estradiol, using a yeast-based gene transcription assay. Oral administration of DES at 15 ␮g/kg/day was chosen as a reference chemical for estrogenic activity based on the relative in vitro estrogenic potency of DES compared to BPA, in which a DES dose of 15 ␮g/kg is equivalent to BPA at 320 mg/kg based on the in vitro binding affinity for both ER␣ and ER␤. In pilot studies, we had included the 320 mg BPA/kg dose given by oral bolus, because this was one of the doses in the preceding NTP study (Morrissey et al., 1989) and did not cause maternal toxicity under our experimental conditions. However, morphometric examination of a small sample size of female offspring whose mothers had been exposed to 320 mg/kg from GD 2 to PND 10 showed increases in the volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) (Liaw et al., 1997). In the present study, the estrogen-dependent parameters examined were the volume of the SDN-POA in a larger sample size, female pubertal development, estrous cyclicity, lordosis behavior, male reproductive organ weights, and the micromorphology of ovaries and ventral prostates. MATERIALS AND METHODS Chemicals. BPA (⬃99% purity) was purchased from Aldrich Chemical Company (Milwaukee, WI). DES (⬃99% purity), ␤-estradiol 3-benzoate (⬃98% purity), and progesterone (⬃99% purity) were obtained from Sigma Chemical Company (St. Louis, MO). Animals and treatments. Timed-pregnant Crl:CD BR Sprague-Dawley rats were obtained from Charles River Laboratories (Raleigh, NC) on GD 0 (GD 0 ⫽ sperm-positive), assigned to a treatment group (n ⫽ 8 dams per treatment group) by randomization of body weights, and then individually housed in polycarbonate cages with cellulose fiber-chip bedding (ALPHA-dri, Shepherd Specialty Papers, Kalamazoo, MI). This rat strain was chosen because it is the most commonly used rat in reproductive and developmental toxicity studies. Animals were kept in a facility accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC). The HEPA-filtered and mass air-displacement room was maintained within a temperature range of 22–25°C, at a relative humidity of 50 ⫾ 10%, and with a light-dark cycle of 12 h (light from 0700 to 1900 h). Rodent feed (NIH-07, Zeigler Brothers, Gardner, PA) and deionized water in glass bottles with Teflon-lined caps were provided ad libitum. The pregnant dams were administered BPA, DES, or corn oil as a vehicle at 5 ml/kg/day between 1030 and 1130 h from GD 11 through postnatal day (PND) 20 except on the day of parturition. Dose levels for BPA were 3.2, 32,

or 320 mg/kg/day. DES at 15 ␮g/kg/day was used as a reference chemical for estrogenic activity. Dams were examined for clinical signs of toxicity and weighed daily before dosing. After parturition, the pups were counted, weighed, and kept with their respective dams until weaning on PND 21. Body weights of pups were recorded on PND 1 and 7. At weaning, pups were identified individually by ear tag and housed in groups of 3– 4, according to treatment. Dams were killed by carbon dioxide asphyxiation when pups were weaned, and body weight and organ weights (liver, kidney, ovary, uterus, and adrenals) were determined. On PND 180, F 1 males were killed, and body and reproductive organ weights (testes, epididymides, ventral and dorsolateral lobes of the prostate, and seminal vesicles) were recorded. Ventral prostates collected from F 1 offspring at PND 180 were fixed in 10% neutral buffered formalin and embedded in paraffin. Histological sections (5 ␮m) were cut and stained with hematoxylin and eosin for micromorphological evaluation. The sexually dimorphic nucleus of the preoptic area. Brains were collected from F 1 females on PND 10 (1–3 pups/litter; n ⫽ 8, 7, and 6 litters at 0, 320 mg BPA/kg, and 15 ␮g DES/kg, respectively) and fixed in 10% neutral buffered formalin for a minimum of 2 weeks. Brains were then transferred to 10% sucrose-formalin solution for 48 h, frozen on dry ice, and stored at –70°C until sectioning. Frozen sections (50 ␮m) were stained with 0.5% cresyl violet acetate (Sigma). The volume of the SDN-POA was determined as described by Gorski et al. (1978) using Image-1威 software (Universal Imaging Corporation, West Chester, PA). Pubertal development. All female pups were examined daily for vaginal opening from PND 27 until each animal acquired this developmental landmark. Following the onset of vaginal opening, daily vaginal lavage was conducted for 10 days to determine first estrus. The ages and body weights at which vaginal opening or first ovulation in the F 1 offspring occurred were recorded. Estrous cyclicity. Estrous cyclicity all F 1 females was monitored for 22 days, from approximately 4 months of age. Vaginal smears were obtained daily from lavage fluid collected by flushing the female’s vagina with phosphatebuffered saline and were examined under a light microscope. The stage of the estrous cycle was determined based upon vaginal cytology as described by Everett (1989). Number of cycles, days in estrus, and cycle length were determined. Lordosis behavior. The lordosis response of the F 1 females exposed to BPA was examined at 6 months of age (1–2 rats/litter; n ⫽ 8, 7, 9, 7, and 8 rats at 0, 3.2, 32, and 320 mg BPA/kg and 15 ␮g DES/kg, respectively). Rats were first ovariectomized and then 2 weeks later were injected subcutaneously for 2 consecutive days with ␤-estradiol-3-benzoate (5 ␮g/0.05 ml/100 g body weight) and 24 h later with progesterone (200 mg/0.05 ml/100 g body weight). The lordosis behavior study was conducted within 4 –7 h following progesterone administration, under red light illumination. The intensity of the lordosis response was determined as described by Hardy and DeBold (1972) and recorded with a video camera (VM-5350A, Hitachi) for later detailed analysis and scoring. The lordosis intensity was scored using a 3-point scale (1 point: marginal lordosis; 2 points: normal lordosis; 3 points: maximal lordosis). The mean lordosis intensity was calculated as the total number of lordosis points divided by the total number of lordosis responses. Statistical analysis. Statistical analyses were conducted using the JMP statistical software package (SAS Institute, Cary, NC). The litter was used as an experimental unit except for maternal data such as body and organ weights of dams. Treatment effects on body and organ weights of dams and lordosis intensity were analyzed by one-way analysis of variance (ANOVA). When the F test was significant, it was followed by Dunnett’s test. Treatment effects on the volume of the SDN-POA, pubertal development, and estrous cyclicity were analyzed by nested ANOVA, where the individual pups were nested within the litter. Treatment effects on male reproductive organ weights were analyzed by nested analysis of covariance (ANCOVA), where body weight was used as a covariate. A pair-wise comparison of the least-square means was employed as a post hoc procedure. The significance level for all tests was set at p ⬍ 0.05.

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EFFECTS OF BPA ON REPRODUCTIVE FUNCTION

TABLE 1 Maternal and Reproductive Parameters in Rats Exposed to Bisphenol A (BPA) or Diethylstilbestrol (DES) during Pregnancy and Lactation (GD 11–PND 20) BPA (mg/kg/day) 0

3.2

32

320

DES (␮g/kg/day) 15

8 8 8

8 8 8

8 8 8

8 8 7b

8 7a 6c

200.1 ⫾ 6.38 240.3 ⫾ 5.05 276.3 ⫾ 6.0 337.7 ⫾ 7.57

199.3 ⫾ 8.32 248.6 ⫾ 7.81 288.4 ⫾ 8.89 361.8 ⫾ 15.18

203.7 ⫾ 7.89 243.5 ⫾ 9.27 274.8 ⫾ 9.90 339.9 ⫾ 12.61

201.9 ⫾ 7.01 243.6 ⫾ 6.34 261.2 ⫾ 4.02 311.3 ⫾ 6.14

200.1 ⫾ 8.16 250.6 ⫾ 11.39 275.0 ⫾ 10.35 328.9 ⫾ 15.01

283.9 ⫾ 5.52 302.5 ⫾ 5.61 304.4 ⫾ 3.61 298.6 ⫾ 5.20

302.3 ⫾ 9.93 322.6 ⫾ 10.98 329.4 ⫾ 9.93 312.5 ⫾ 9.52

277.8 ⫾ 11.21 302.6 ⫾ 13.21 306.7 ⫾ 10.60 297.4 ⫾ 9.17

258.2 ⫾ 5.46 280.5 ⫾ 4.87 280.9 ⫾ 8.19 284.3 ⫾ 8.63

269.5 ⫾ 9.0 292.0 ⫾ 10.44 301.6 ⫾ 8.08 301.8 ⫾ 8.78

15.10 ⫾ 0.53 2.15 ⫾ 0.08 68.63 ⫾ 10.97 89.5 ⫾ 9.16 400.0 ⫾ 28.45 12.6 ⫾ 0.75

16.10 ⫾ 0.89 2.14 ⫾ 0.09 70.88 ⫾ 8.32 115.0 ⫾ 6.84 429.3 ⫾ 32.50 12.6 ⫾ 1.28

14.93 ⫾ 0.56 2.04 ⫾ 0.08 72.0 ⫾ 5.46 108.3 ⫾ 6.71 290.8 ⫾ 26.60 13.0 ⫾ 0.65

15.18 ⫾ 0.66 2.08 ⫾ 0.06 71.86 ⫾ 4.72 98.0 ⫾ 8.07 379.3 ⫾ 59.62 11.25 ⫾ 0.90

17.70 ⫾ 0.61* 2.15 ⫾ 0.07 79.83 ⫾ 8.40 67.0 ⫾ 9.73 453.5 ⫾ 11.56 11.57 ⫾ 1.60

6.74 ⫾ 0.35

6.70 ⫾ 0.12

6.35 ⫾ 0.18

6.13 ⫾ 0.19

6.51 ⫾ 0.19

15.64 ⫾ 0.49 15.36 ⫾ 0.49

15.45 ⫾ 0.58 15.11 ⫾ 0.55

14.46 ⫾ 0.80 13.88 ⫾ 0.67

14.32 ⫾ 0.66 14.04 ⫾ 0.64

14.55 ⫾ 0.46 14.26 ⫾ 0.47

Parameter No. of dams Sperm-positive Pregnant At termination on PND 21 Body weight (g) a During pregnancy GD 0 GD 7 GD 14 GD 20 During lactation PND 5 PND 10 PND 15 PND 21 Organ weights at termination d Liver (g) Kidney (g) Adrenal (mg) Ovary (mg) Uterus (mg) No of live pups per litter d Live pup weight (g) d PND 1 PND 7 Males Females a

One dam (sperm-positive) was not pregnant and was euthanized on GD 23. One dam died on PND 16 due to gavage error; the pups were euthanized. c One dam died on PND 20 due to gavage error; the pups were weaned on PND 20. d Values are means ⫾ SE. e Values are litter means ⫾ SE. * Significantly different from control, p ⬍ 0.05. b

RESULTS

(Fig. 1). However, DES treatment significantly increased the volume of SDN-POA of F 1 female rats at 10 days of age.

Maternal Toxicity and Reproductive Performance There were no detectable effects of BPA at 3.2, 32, or 320 mg/kg/day on maternal body weights during either pregnancy or lactation or at termination on PND 21 (Table 1), even though there was a slight decrease in the mean body weights of dams at 320 mg BPA/kg/day on GD 20, PND 5, PND 10, and PND 15. No significant effects of BPA on maternal organ weights were observed at termination on PND 21. DES treatment significantly increased the liver weights of dams. BPA did not affect either the number of live pups per litter or body weights of live pups on PND 1 or 7. The Sexually Dimorphic Nucleus of the Preoptic Area The daily repeated gavage with 320 mg BPA/kg/day did not alter the volume of the SDN-POA of 10-day-old F 1 female rats

Pubertal Development None of the maternal BPA treatment regimens altered the age or body weight at which vaginal opening and first ovulation in the F 1 female offspring had occurred (Table 2). There was no significant difference in the average age at vaginal opening between control and BPA-treated groups. DES treatment also did not affect the pubertal development of F 1 offspring exposed in utero and during lactation. Estrous Cyclicity No significant effects of maternal BPA exposure on estrous cyclicity in 4-month-old female offspring were observed (Table 3). There was no significant difference in average cycle

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Male Reproductive Organ Weights

FIG. 1. Volume of the SDN-POA of 10-day-old F 1 female rats exposed to BPA or DES during pre- and postnatal development (GD 11–PND 10). Values are litter means ⫾ SE for 8, 7, and 6 litters at 0 and 320 mg BPA/kg/day, and 15 ␮g DES/kg/day, respectively. Asterisk indicates significant difference from control; p ⬍ 0.05.

length, days in estrus, the number of estrous cycles, and the number of F 1 females showing normal estrous cyclicity between BPA-treated and control groups. In contrast, 4-monthold F 1 offspring exposed to DES in utero and during lactation displayed irregular estrous cyclicity. Lordosis Behavior There were no significant differences in the mean lordosis intensity of either BPA- or DES-exposed F 1 female rats at approximately 6 months of age compared to age-matched control offspring (Fig. 2).

No significant effects on body and reproductive organ weights were observed in F 1 male offspring exposed to BPA in utero and during lactation when they were about 6 months old (Table 4). The high dose of 320 mg BPA/kg/day slightly increased the mean wet weight of the ventral prostate lobe by approximately 23% compared to that of the control group, but these mean values were not significantly different from one another. DES did not affect male body and reproductive organ weights. Furthermore, there was no correlation between body weights and reproductive organ weights of F 1 rats in any of the various groups. Microscopic examination of ventral prostates, ovaries, and uteri of approximately 6-month-old F 1 offspring exposed to BPA in utero and during lactation did not reveal any micromorphology that was significantly affected by BPA or DES treatment during development. There was no significant difference in the incidence and severity of atrophy characterized by extremely flattened acinar epithelium in ventral prostates of F 1 rats of control and BPA-treated groups at 6 months of age. The spontaneous occurrence of atrophy in the prostates of control Crl:CD Sprague-Dawley rats at 4.6 –7.5 months of age has been previously reported (Bosland, 1992). Absence of corpora lutea (CL) was found in some female offspring of the DEStreated group but there was no significant difference in the incidence of absence of CL among randomly selected animals from control, BPA, or DES-treated groups. No significant treatment-related effects were found in uteri from those animals (data not shown). DISCUSSION

The most notable finding of the present study is that daily gavage treatment of pregnant rats with BPA at 3.2, 32, or 320 mg/kg/day, and thus indirect exposure of their offspring during prenatal and postnatal sexual differentiation and development, did not affect estrogen-mediated end points related to female

TABLE 2 Pubertal Development of F 1 Female Rats Exposed to Bisphenol A (BPA) or Diethylstilbestrol (DES) during Prenatal and Postnatal Development (GD 11–PND 20) BPA (mg/kg/day) Parameter

0

3.2

32

320

DES (␮g/kg/day) 15

No. of F 1 females (litters) Age (PND) at vaginal opening a Body weight (g) at vaginal opening a Age (PND) at first estrus a

34 (8) 33.2 ⫾ 0.6 119.5 ⫾ 5.2 35.4 ⫾ 0.8

42 (8) 32.9 ⫾ 0.4 117.8 ⫾ 2.6 36.3 ⫾ 0.6

39 (8) 33.6 ⫾ 0.3 116.5 ⫾ 5.2 36.7 ⫾ 0.5

34 (7) 33.7 ⫾ 0.5 122.7 ⫾ 3.2 36.1 ⫾ 0.8

36 (7) 33.0 ⫾ 0.6 113.5 ⫾ 3.4 35.6 ⫾ 0.6

Note. The first estrus did not occur in some animals (2 in control, 1 in 32 mg BPA/kg, 3 in 3.2 mg BPA/kg, and 3 in DES-treated groups) within 10 days following vaginal opening. a Values are litter means ⫾ SE.

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EFFECTS OF BPA ON REPRODUCTIVE FUNCTION

TABLE 3 Estrous Cyclicity of F 1 Female Rats Exposed to Bisphenol A (BPA) or Diethylstilbestrol (DES) during Prenatal and Postnatal Development (GD 11–PND 20) BPA (mg/kg/day) Parameter

0

3.2

32

320

DES (␮g/kg/day) 15

No. of F 1 females (litters) Days in estrus a Number of cycles a Cycle length b Rats cycling normally c

33 (8) 5.1 ⫾ 0.14 4.1 ⫾ 0.14 4.2 ⫾ 0.07 94

41 (8) 4.9 ⫾ 0.22 3.9 ⫾ 0.22 4.2 ⫾ 0.05 90

38 (8) 4.7 ⫾ 0.22 3.7 ⫾ 0.21 4.4 ⫾ 0.40 84

33 (7) 5.0 ⫾ 0.16 4.0 ⫾ 0.16 4.1 ⫾ 0.12 91

35 (7) 3.2 ⫾ 0.62* 1.2 ⫾ 0.29* 1.9 ⫾ 0.30* 7

Note. Estrous cyclicity was monitored in F 1 females at approximately 4 months of age for 22 days. a Values are litter means ⫾ SE. b Normal cycle is defined as having a cycle length between 4 and 5 days with 1 to 2 days of estrus. c Values are % affected in F 1 females. * Significantly different from control, p ⬍ 0.05.

pubertal development and functions of the reproductive system in the F 1 generation. The reference chemical for estrogenic activity, DES, at 15 ␮g/kg/day, had significant effects on estrous cyclicity and the volume of SDN-POA but not on pubertal development or lordosis behavior. It appears that there are profound effects related to both route and mode of DES administration in rats. For example, treatment of pregnant Sprague-Dawley rats with DES at 50 ␮g/liter via drinking water (the DES exposure level based on estimated water consumption during gestation and lactation:

FIG. 2. The intensity of the lordosis response of F 1 female rats exposed to BPA or DES during pre- and postnatal development (GD 11–PND 20). Values are means ⫾ SE for 8, 7, 9, 7, and 8 rats at 0, 3.2, 32, and 320 mg BPA/kg/day and 15 ␮g DES/kg/day, respectively.

approximately 6 –10 ␮g/kg/day) from GD 2 to PND 21 caused premature vaginal opening in female offspring, with reduced body weights (Liaw et al., 1998). Ashby et al. (1997) also reported that premature vaginal opening occurred in pups whose mothers were exposed to DES from GD 1, at 100 ␮g/kg/day for the first 3 days of exposure, then reduced to 50 ␮g/kg/day from GD 4 to PND 20. However, in the present study, DES bolus administration given at 15 ␮g/kg/day to pregnant rats caused no such effects. Treatment of immature female Alpk:AP rats with 40 ␮g/kg/day by gavage for 3 consecutive days induced premature vaginal opening (Ashby and Tinwell, 1998). It should be noted that perinatal lethality was induced in pregnant Sprague-Dawley rats by prenatal exposure to DES at 45 ␮g/kg/day (Zimmerman et al., 1991). No treatment-related effects on pubertal development, estrous cyclicity, or reproductive organ weights have been reported in F 1 offspring following dietary exposure to 17␤-estradiol at 0.05 ppm in the 90-day/one-generation study (Biegel et al., 1998a,b; Cook et al., 1998). Therefore the sensitivity of target tissues for estrogens and estrogenic chemicals may depend on the dose levels of the specific chemicals as well as routes of administration. It has become apparent that there are end point-specific rat strain differences in response to estrogens and estrogenic agents. Steinmetz et al. (1997) reported that BPA induced hyperprolactinemia in Fischer 344 rats but not in SD rats. Recently, genetic variation in susceptibility to testicular development by 17␤-estradiol was reported in different strains of mice (Spearow et al., 1999). Therefore, it may be possible that the lack of BPA effects on the estrogen-mediated end points in the present study was due to insensitivity of Sprague-Dawley rats to endocrine-mediated toxicity by endocrine modulators. However, it is not clear whether Sprague-Dawley rats are insensitive in susceptibility to endocrine-mediated toxic effects caused by BPA or estrogenic compounds on pubertal develop-

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TABLE 4 Reproductive Organ Weights of 6-Month-Old F 1 Male Rats Exposed to Bisphenol A (BPA) or Diethylstibestrol (DES) during Prenatal and Postnatal Development (GD 11–PND 20) BPA (mg/kg/day) Parameter

0

3.2

32

320

DES (␮g/kg/day) 15

No. of F 1 females (litters) examined Body weight (g) a Organ weight (g) a Right testis Left testis Right epididymis Left epididymis Seminal vesicle Prostate lobes Ventral Dorso/lateral

44 (8) 691.1 ⫾ 11.97

51 (8) 735.8 ⫾ 17.69

47 (8) 682.8 ⫾ 20.83

28 (7) 668.3 ⫾ 16.18

38 (7) 697.7 ⫾ 26.17

1.88 ⫾ 0.04 1.89 ⫾ 0.05 0.68 ⫾ 0.02 0.67 ⫾ 0.02 1.84 ⫾ 0.10

1.96 ⫾ 0.06 1.95 ⫾ 0.06 0.70 ⫾ 0.02 0.69 ⫾ 0.02 1.84 ⫾ 0.06

1.89 ⫾ 0.08 1.90 ⫾ 0.08 0.69 ⫾ 0.02 0.68 ⫾ 0.02 1.83 ⫾ 0.05

1.85 ⫾ 0.04 1.83 ⫾ 0.03 0.70 ⫾ 0.02 0.67 ⫾ 0.01 1.86 ⫾ 0.07

1.82 ⫾ 0.06 1.81 ⫾ 0.05 0.67 ⫾ 0.02 0.65 ⫾ 0.02 1.90 ⫾ 0.08

0.60 ⫾ 0.03 0.75 ⫾ 0.03

0.64 ⫾ 0.05 0.79 ⫾ 0.02

0.61 ⫾ 0.03 0.78 ⫾ 0.04

0.74 ⫾ 0.03 0.78 ⫾ 0.01

0.53 ⫾ 0.03 0.78 ⫾ 0.03

a

Values are litter means ⫾ SE.

ment, estrous cyclicity, or lordosis behavior compared to other strains of rats. Molecular mechanisms controlling genetic differences in susceptibility to endocrine-mediated toxicity by estrogenic chemicals need to be defined. Gonadal steroids are involved in the sexual differentiation of the central nervous system (CNS) (MacLusky and Naftolin, 1981; Toran-Allerand, 1984). Estrogens aromatized from androgens in the brain are responsible for structural and functional masculinization of the CNS during the critical period of sexual differentiation of the rat brain. Exposure to environmental hormones during development and differentiation may modify structural and functional differentiation of the CNS with permanent and irreversible changes in reproductive functions or behavior in adults (Gorski, 1986). The SDN-POA is sexually dimorphic. The size of the SDN-POA is larger in males than in females and is regarded as a sex-specific morphological marker for the structural differentiation of the rat brain (Gorski et al., 1978). Treatment of pregnant rats with DES from GD 16 to PND 10 increased the volume of the SDN-POA in female offspring, indicating that sexual development and differentiation of the SDN-POA are susceptible to environmental estrogens during the critical period of sexual differentiation of the rat brain (Tarttelin and Gorski, 1988). No significant treatment effects on the volume of SDN-POA, vaginal opening, and first estrus in the BPA-exposed rat offspring were found in this study. These data indicate that the gavage dose levels of BPA at 3.2, 32, or 320 mg/kg/day were not potent enough to cause any treatment-related effects on the end points examined. Premature vaginal opening occurred in immature female Alpk:AP rats dosed with 800 mg BPA/kg/day for 3 consecutive days by sc injections but not by gavage (Ashby and Tinwell, 1998). However, BPA treatment at the same dose level by either gavage or sc injections significantly increased uterine weights. These results suggest that the in vivo

estrogenic potency of BPA may depend upon the sensitivity of target tissues as well as the route of administration. Early postnatal exposure of rats to exogenous hormones causes permanent adverse effects on estrous cyclicity upon reaching sexual puberty (Gorski, 1968). Female rats treated with 10 ␮g of testosterone propionate on PND 5 later became anovulatory and exhibited persistent estrus. Bjerke et al. (1994) also showed abnormal reproductive behavior in rats exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin during in utero and lactation. In our present study, we did not observe adverse effects of the 3 high BPA dose levels on estrous cyclicity and lordosis sexual behavior in F 1 females that were indirectly exposed to BPA in utero and during lactation. These findings suggest that the potency of BPA at any of those high-dose levels was insufficient to disturb the functional differentiation of the brain during the critical period of sexual differentiation. Furthermore, we did not find any changes in organ weights such as testis, epididymis, seminal vesicle, and ventral and dorsolateral prostates in male offspring exposed to BPA in utero and during lactation. It may be possible that there are species differences of rodent embryos/fetuses in response to BPA, because treatment of pregnant mice with BPA at 2 or 20 ␮g/kg/day from GD 11 to GD 17 affected certain male reproductive system parameters (Nagel et al., 1997; vom Saal et al., 1998). These investigators found that the in vivo potency of BPA for estrogenic activity was 1/100 that of DES (vom Saal et al., 1997) using total prostate gland fresh weights as the bioassay end point. Cagen et al. (1999) recently reported that in a much larger study of CF-1 mice treated orally with BPA at 0, 0.2, 2, 20, or 200 ␮g/kg/day from GD 11 to GD 17 showed no treatment-related effects of BPA on any of the parameters that were affected in the preceding mouse study. Similar conclusions were reached after another effort to replicate the original mouse data, using a study design that provided

EFFECTS OF BPA ON REPRODUCTIVE FUNCTION

more statistical power (Ashby et al., 1999). Our present observations that in utero and lactational BPA exposure had no detectable effects on male reproductive development indicate that the lack of in vivo potency of BPA is consistent with the low in vitro binding affinity to ER and the rapid in vivo metabolism of orally administered BPA to BPA glucuronide (Pottenger et al., 2000). The BPA glucuronide does not transactivate the estrogen-responsive luciferase reporter gene in HepG2 human hepatoma cells cotransfected with either ER␣ or ER␤ plasmids (Gaido, personal communication). The routedependent uterotropic activity of BPA, in which ip or sc injections are more effective than oral administration, may be due to rapid BPA metabolism as well as low estrogenic potency (Ashby and Tinwell, 1998). In conclusion, prenatal and postnatal exposure of rats to BPA at 3.2, 32, or 320 mg/kg/day did not have adverse effects on the estrogen-mediated end points examined here under our experimental conditions. Little is known about the bioavailability of BPA parent compound to embryos, fetuses, and nursing pups. Upon oral bolus administration, the plasma levels of the BPA glucuronide rose rapidly (Pottenger et al., 2000). Very low levels of BPA have also been found in milk from lactating rats administered BPA in drinking water (Gould et al., 1998). Thus, BPA transfer may be assumed to be very low, based on the few published data. BPA has been converted to DNA binding metabolites in vitro (Atkinson and Roy, 1995). However, it is not clear that these metabolites are responsible for potential adverse effects of BPA on reproductive functions. Further information regarding the bioavailability of BPA to fetuses, bioactivities of BPA metabolites, and strain/species differences of rodents in response to BPA will enhance our understanding of the reproductive toxicity of BPA and other environmental chemicals that interact with ER. ACKNOWLEDGMENTS The authors thank Dr. Derek Janszen for advice on statistical analyses, Drs. Rory Conolly, David Dorman, Paul Foster, and Li You for critical review of the manuscript, and Dr. Barbara Kuyper for editorial assistance. We also thank Elizabeth Gross Bermudez and the staff of the necropsy and histology unit as well as Paul Ross and the staff of the animal care facility.

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