Toxicological Sciences 68, 339-348 (2002)
Copyright © 2002 by the Society of Toxicology
ENDOCRINE TOXICOLOGY |
Normal Sexual Development of Two Strains of Rat Exposed in Utero to Low Doses of Bisphenol A
* Syngenta Central Toxicology Laboratory, Alderley Park, Cheshire, SK10 4TJ, United Kingdom; and
National Institute of Environmental Health Sciences, Alexander Drive, Research Triangle Park, North Carolina 27709
Received February 8, 2002; accepted April 2, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pregnant Sprague-Dawley (SD) and Alderley Park (Wistar derived) rats were exposed by gavage during gestation days 6–21 to 20 µg/kg, 100 µg/kg, or 50 mg/kg body weight of BPA with ethinylestradiol (EE; 200 µg/kg) acting as a positive control agent. The sexual development of the derived pups was monitored until termination at postnatal day 90–98. The endpoints evaluated were litter size and weight, anogenital distance at birth, days of vaginal opening, first estrus and prepuce separation, weights of the liver, seminal vesicles, epididimydes, testes, ventral prostate, uterus, vagina, cervix and ovaries, and daily sperm production. Males were terminated at postnatal day 90 and females at postnatal day 98. The only statistically significant effects observed for any dose of BPA were a decrease in daily sperm production and an increase in the age of vaginal opening for the Alderley Park animals at the highest dose evaluated (50 mg/kg). The dose of EE evaluated proved to be maternally toxic in our laboratory, but provided gross evidence of endocrine disruption in the treated dams. These results diverge from those of Chahoud and his colleagues who indicated disturbances to the sexual development of both male and female SD rat pups administered the same 3 doses of BPA. This failure to confirm low dose endocrine effects for BPA is discussed within the context of similar divergent conclusions derived from other assessments of the endocrine toxicity of this agent to rats.
Key Words: bisphenol A; Sprague-Dawley rats; Alderley Park rats; sexual development; in utero exposure.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chahoud and his colleagues have reported that exposure of pregnant Sprague-Dawley (SD) rats to 20 or 100 µg/kg, or 50,000 µg/kg (50 mg/kg) body weight of BPA, from gestation day (GD) 6 to GD 21, led to a range of disturbances to the sexual development of the pups at all doses evaluated (summarized in Table 1
). Those data have been published as several abstracts (Chahoud et al., 2001; Fialkowski et al., 2000; Schönefelder et al., 2001; Talsness et al., 2000b, 2001) and as a single earlier article (Talsness et al., 2000a). Although all of the data appear to have been derived from a single large experiment, there were several discrepancies between the article and the abstracts (addition of lower dose levels of BPA and EE in the later abstracts and the additional use of estradiol as a positive control in the Schönefelder et al. (2001) abstract). In addition, there were some discrepancies in the results between the article and the abstracts, as shown in Table 1. Given that the abstracts postdated to the article we have relied on the abstracts for the definitive changes listed in Table 1. These data therefore add to the preexisting and conflicting database available for low dose effects of BPA in the rat (reviewed in Table 2).
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The results of studies designed to confirm those observations using both the originally reported strain of rats (SD), together with our own strain of Wistar derived Alderley Park (AP) rats, are presented herein. The doses of BPA evaluated and the period of exposure in utero used were as described by Chahoud and colleagues. All of the test parameters studied by Chahoud and colleagues were evaluated in the present study, with the exception of the age at testes descent, testosterone levels, and the mean duration of the estrous cycle. Age at first estrus was added as an endpoint. The dose of the positive control agent used, EE, was the same as the higher of the two doses described by Chahoud, but it proved to be maternally toxic in our laboratory and was lowered during the course of the experiments.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
BPA was purchased from Aldrich (Gillingham, Dorset, UK) as a solid (99+% pure; MP158–159°C). EE was purchased as a solid (>98% pure) from Sigma Chemical Company (Poole, Dorset, UK). The test vehicle, arachis oil (AO), was also purchased from Sigma. Both dosing solutions and vehicle were stored at room temperature for a maximum of 3 days whereupon fresh solutions were prepared. Dosing solutions of BPA were shown to be stable for at least 7 days when stored at room temperature using reverse phase HPLC. Briefly, samples were diluted with solvent (methanol/tetrahydrofuran [1:2; v/v]) to give sample solution concentrations within the range of the calibration standards selected. Samples were analyzed using a 125 mm x 4.6 mm i.d. Hypersil H30DS-125 column fitted to a 2487 Series UV detector (Waters) with a flow rate of 1 ml/min. The limit of detection was determined to be 0.2 µg/ml bisphenol A (equivalent to a 2 µg/kg dosing solution). Determination of the dosing solutions’ concentrations was performed in a similar manner using a single pooled sample for each dose level. Specifically, a 3-ml sample was taken from each fresh dosing solution at the appropriate concentration and frozen at -70°C until analyses could be performed. Each sample was then thawed overnight and pooled according to dose level for analyses.
Animals and housing.
Young (
8 weeks old) pregnant female rats were obtained either from Harlan Olac UK (SD rats; 185 ± 20 g) or from the Barriered Animal Breeding Unit, AstraZeneca, Alderley Park, Macclesfield UK (AP rats; 230 ± 20 g) and allowed 5 days acclimatization prior to dosing. All animals were individually housed in plastic solid bottomed cages with sawdust (Wood Treatments Ltd.; Macclesfield, Cheshire, UK) and shredded paper as bedding (SI Supplies, Poynton, Cheshire, UK). All animals were subjected to a 12-h light/dark schedule and controlled humidity and temperature. The females were allowed Rat and Mouse No. 3 (RM3) breeding diet (Special Diet Services Ltd.; Witham, Essex, UK; 18.5% soya content) and water ad libitum. The females were maintained on RM3, which is specifically designed for breeding, lactation, and growth of young stock, until weaning was complete. Pups were maintained on Rat and Mouse No 1 (RM1) diet (Special Diet Services Ltd.; Witham, Essex, UK; 6.5% soya content) following weaning.
Group allocation and dosing.
Animals were weighed upon arrival in the laboratory (before acclimatization) and again on GD 5. Females (n = 7/group) were placed into 1 of 5 groups based primarily on the rank ordering of the body weight gains between GD 0 and GD 5, while also ensuring that there were no significant differences in group mean body weight at GD 5.
The pregnant females were dosed by gavage from GD 6 through GD 21 with daily doses of either AO (Group 1), 20 µg/kg BPA in AO (Group 2), 100 µg/kg BPA in AO (Group 3), 50 mg/kg BPA in AO (Group 4), or EE in AO (Group 5) using a dosing volume of 10 ml/kg body weight. The doses selected for evaluation were as described by Chahoud and colleagues (Table 1
). Analyses of the BPA dosing solutions as described above indicated that the actual concentrations were 24 µg/kg; 109 µg/kg, and 50.65 mg/kg for those used to dose the AP rats and 23 µg/kg, 108 µg/kg, and 49.15 mg/kg for those used to dose the SD rats. The dose of EE (200 µg/kg body weight) caused vaginal bleeding coupled with mild signs of toxicity (e.g., piloerection, hunched stance) and body weight loss (3% for AP females and 1.4% for SD rats) during the first 9 days of dosing, a time when the pregnant control animals were gaining weight. The daily dose level was therefore reduced to 100 µg/kg on GD 11 for AP rats and on GD 14 for SD females, dependent on when toxicity was first observed.
Fresh dosing solutions were prepared on GD 6, 7, 9, 13, 16, and 19 for the AP rats and on GD 6, 8, 11, 14, 17, and 19 for the SD rats. Samples of all dosing solutions were stored at -70°C for further analysis.
Littering and weaning.
Parturition occurred between GD 21 and GD 23, with the majority occurring on GD 22. Those females that started to litter on GD 21 did not receive a final dose of compound. The pups in each litter were counted, sexed, weighed, and the anogenital distance (AGD) measured 24 h after birth (defined as postnatal day [PND] 1). Five days after birth (defined as PND 5) all pups were weighed again and each litter was culled to give a combination, where possible, of 4 males and 4 females, and where not possible, a maximum of 8 pups/litter. Culling took no account of pup size, although one AP male pup was removed from the study, as it was significantly smaller than its littermates (body weight of 3.7 g compared to a mean bodyweight of 9.7 ± 1.6); this rat would probably not have survived for the duration of the study. All litters and dams were maintained on RM3 and were not handled again until weaning (PND 23). At weaning, littermates were identified by a unique number, weighed, and then group-housed according to sex. All pups were placed on RM1 diet and water ad libitum at this time.
Weighing of pups, monitoring of puberty, and termination.
Pups were weighed at weaning (PND 23) and then every third day until termination (PND 90–91 for males and PND 98 for females). The onset of vaginal opening (VO) was monitored daily from weaning and the body weight for each female was recorded on the day that this was observed. As soon as VO was observed vaginal smears were taken from each female and assessed for first estrus (defined as the first day on which only cornified epithelial cells were observed on a vaginal smear). Preputial separation (PPS) was monitored daily in all males from PND 35 and body weights were recorded on the day that this occurred.
All females were terminated on PND 98 by an overdose of fluothane followed by cervical dislocation; each animal being weighed just prior to termination. The liver and organs of the reproductive tract (vagina, cervix, uterus, and ovaries) were removed and weighed. The vagina and the uterus were fixed in Bouins for histopathological examination as described by Talsness et al. (2000a,b). All males were terminated at PND 90 as described above. Body weights were recorded just prior to termination. Liver, testes, epididymides, seminal vesicles, and ventral prostate were removed and weighed. The left testis and, in the case of the SD rats, both epididymides, were fixed in Bouins for histopathological examination as described by Fialkowski et al.(2000). The prostate, as well as the decapsulated right testis, was flash frozen in liquid nitrogen and stored at -80°C for further investigation.
Termination of dams.
All dams that had littered were terminated as described above following weaning. Any dams that had not littered were terminated on either GD 24 or GD 25 and pregnancy confirmed by staining the uterus with 10% ammonium polysulphide to determine the presence of implantation scars (Salewski, 1964). Two SD dams exposed to EE were terminated before the end of dosing because of severe vaginal bleeding. At parturition, one AP rat and one SD rat were terminated because of difficulties encountered during littering. In these cases the females were terminated as described above and the contents of the uterus examined for the presence of fetuses and the number of macroscopically identifiable implantation sites.
Calculation of daily sperm production.
Counts of homogenization resistant sperm were determined as described previously (Ashby et al., 1997) using the method ofBlazak et al.(1993). Each frozen right testis (decapsulated) was homogenized in 50 ml 0.9% (w/v) NaCl containing merthiolate (0.01% w/v) and Triton X-100 (0.05%, v/v) with a Waring blender. The number of released sperm heads was determined in duplicate with an improved Neubauer haemocytometer. The number of sperm present in the area of the counting chamber was equivalent, when multiplied by 104, to the number of sperm/ml homogenate. The variation between duplicate readings was less than 10%. Daily sperm production (DSP) values were derived with the transit time factor of 6.1 (Blazak et al., 1993).
Statistical analyses.
ANOVA procedures were used to assess the impact of BPA and litter (dam) effects on the variables of interest. In some cases, the variance-stabilizing logarithmic transformation was used in these analyses. Analysis of covariance (ANCOVA) procedures (Snedecor and Cochran, 1980) were used for organ weights to adjust for differences in body weight. Pairwise comparisons were made by Dunnett’s test (Miller, 1966). The statistical analyses shown for the data are using the litter as the statistical unit. However, analyses were performed using the grouped individuals as the unit, and that revealed no additional statistically significant changes from those found using the litter as the statistical unit.
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RESULTS |
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Litter Size and % Males
No significant differences in either litter size or % males/litter were observed between control and those groups exposed to the various doses of BPA in either strain of rat (Table 3
). In addition, these data were commensurate with our own historical database for these two strains of rat. However, only a small number of dams exposed to EE successfully delivered (3/7 AP dams and 2/7 SD dams) of which only the 3 litters from the AP dams survived to puberty. Given the problems encountered in the two groups exposed to EE, all data generated from EE exposed pups are considered separately below.
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Birth Weight and AGD Measurements
BPA did not induce any significant effects on the weight or AGD of either sex of both rat strains measured at 24 h after birth (Table 4
). There was, however, a significant litter effect in both strains (p < 0.01).
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Sexual Maturation and Termination in Female Rats
BPA did not affect the age at which VO occurred nor did it affect the body weight of the SD females at the time of VO (Table 5
). There was a delay (p < 0.05) in the age at VO for the highest dose of BPA in the AP females. There was also a significant (p < 0.01) correlation between body weight and the day of VO and a significant litter effect for this strain (data not shown). First estrus was monitored by means of vaginal smearing from the day of VO onwards (Table 5). BPA did not affect the age at which this occurred in either strain of rat.
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The termination data for all females (independent of stage of the estrous cycle) are presented in Table 6
. BPA had no adverse effects on any of the tissues isolated. In addition, an indication of the number of animals in each particular stage of the estrous cycle is given in Table 6 based on the histological analysis of both vaginal and uterine tissue samples. As with the total database, there were no apparent effects on any of the reproductive tissues at any of the stages of the estrous cycle.
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Sexual Maturation and Termination in Male Rats
There were no significant effects induced by BPA on the age of PPS or the body weight at PPS in either strain of rat (Table 7
). There was, however, a significant correlation (p < 0.01) between body weight and the age at PPS as well as a significant litter effect (p < 0.01; data not shown).
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The terminal tissue weight data for the male rats (terminated between PND 90 and 91) are presented in Table 8
. There were no significant effects on the weights of any of the organs isolated. Histological examination of the left testis indicated that there were no significant effects induced by BPA. BPA did not affect sperm production in SD rats, but a reduction in total sperm counts and DSP was observed for the highest dose of BPA evaluated in AP rats (Table 9).
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EE Data
All data pertaining to animals exposed to EE in utero are given in Table 10
. Only litters from the AP strain of rat survived to the end of the study. There was a significant advance in age of sexual maturation for females exposed in utero to EE, but there was no effect on male sexual maturation. At termination, a significant (p < 0.05) reduction in the weights of both the right epididymis and ventral prostate was observed. There were no significant effects observed during histological examination of the left testis. There were no significant differences in the weights of any organs isolated from females. It is likely that the lack of EE effects may be due, in part, to the low sensitivity associated with reduced sample sizes as only 12 males and 13 females from 3 litters were born, not all of which survived to PPS/VO. The high incidence of fetal resorptions in the EE-exposed animals indicated that the dose selected for study (based on the data of Chahoud and colleagues; Table 1) was too high, but these same resorptions provided evidence that EE had induced gross impairment of reproduction in the pregnant dams.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present study was designed to evaluate the range of rat endocrine toxicities for BPA reported by Chahoud and his colleagues (Fialkowski et al., 2000
; Chahoud et al., 2001; Schönefelder et al., 2001; Talsness et al., 2000a,b, 2001; summarized in Table 1). Those data were reported within the context of conflicting conclusions derived from other studies designed to reveal the possible effects of BPA on the sexual reproduction and development of rats (summarized in Table 2). The repeat study reported here deviated from that reported by Chahoud and colleagues in two ways. First, AO was employed as a common vehicle while Chahoud used corn oil for the EE group and culinary cornstarch for the control and BPA groups. Second, an intermediate termination time of PND 90–91 (males) and PND 98 (females) was chosen based on the repeat observations made by Chahoud at PND 70 and 170. These intermediate termination times were fully compatible with the endpoints being assessed and the reports being evaluated, and the use of a single sampling time enabled larger group sizes to be used while reducing animal usage. We also paralleled the study in SD rats with our own strain of Wistar-derived AP rats to enable us to relate any positive findings in SD rats to our own strain of animals (Wistar and SD rats are primarily used internationally in regulatory reproductive toxicity studies).
The dose of EE used by Chahoud and colleagues (200 µg/kg) was very high given that the EC10 value for EE in oral rat uterotrophic assays is 0.5 µg/kg (Kanno et al., 2001). In the present experiments, the 200 µg/kg dose of EE gave gross evidence of reproductive impairment in the form of a high incidence of fetal resorptions. There were also effects at the level of the litter on prostate and right epididymis weights and an advance in the time of VO. However, among all of the endpoints evaluated for the 3 doses of BPA, the only statistically significant effects observed were a reduction in sperm count and DSP and a delay in VO for the 50 mg/kg dose of BPA in AP rats. This dose of BPA is within the active uterotrophic assay dose range for BPA (Ashby, 2001) and represents an unexceptional observation beyond providing another example of a strain difference in the response of rats to BPA (Long et al., 2000).
The general absence of effects for BPA in the present study is in clear contrast to the results reported by Chahoud and his colleagues (Table 1). Those investigators observed positive results for most of the endpoints determined and for all of the doses of BPA and EE evaluated. While some of these changes were consistent across the 5 test groups, some were not. Instances of changes that were not dose-related across the 3 doses of BPA were the day of PPS, male organ weights at PND 70, and progesterone levels in diestrus (Table 1). Interpretation of the often subtle and sometimes nondose-related changes shown in Table 1 must be influenced by the values for individual parameters recorded for concurrent and historical control animals. However, no concurrent control animals were available in the studies by Chahoud and colleagues (NTP, 2001).
The present results, within the context of those shown in Table 2, add to earlier examples of endocrine toxicity data for some other chemicals not being capable of independent confirmation (e.g., Ashby, 2000; NTP, 2001; Sharpe et al., 1998). Such conflicts of findings may eventually influence the interpretation of results observed for chemicals after their routine assessment for endocrine toxicity. Two separate influences may contribute to such conflicts of findings, and these are worthy of study. First, some manifestations of endocrine toxicity may be intrinsically dependent on subtle and unspecified conditions of the bioassay (for example, diet, housing conditions, noise levels, degree of animal handling, strain of animal, and changes in body weight). In these situations genuine positive responses will exist that are difficult to reproduce in apparently similar, but critically different, experiments. Second, the multiplicity of biological endpoints typically evaluated and the intrinsic variability among animals for these endpoints may result in some instances of statistical false positives. Pending the resolution of this problem of data reproducibility, adequate intra- and interlaboratory confirmation of endocrine toxicities is indicated, especially in cases of new and subtle effects being observed. Further, attention should be given to the use of adequate concurrent control groups and the compilation of historical control data against which to assess the biological significance of new findings.
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NOTES |
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1 To whom correspondence should be addressed. Fax: (0) 44 1625590996. E-mail: john.ashby{at}ctl.syngenta.com.
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J. Ashby, H. Tinwell, P. A. Lefevre, R. Joiner, and J. Haseman The Effect on Sperm Production in Adult Sprague-Dawley Rats Exposed by Gavage to Bisphenol A between Postnatal Days 91-97 Toxicol. Sci., July 1, 2003; 74(1): 129 - 138. [Abstract] [Full Text] [PDF]
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