* Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, SK10 4TJ, United Kingdom;
Toxicology/Regulatory Services, Inc., Charlottesville, Virginia 22911;
General Electric Company, Pittsfield, Massachusetts 01201; and
Bristol-Myers Squibb, Drug Safety Evaluation, 1090 Elkton Rd., Newark, Delaware 19714
Received November 29, 2001; accepted February 5, 2002
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ABSTRACT |
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Key Words: diethylstilbestrol; reproductive toxicity; in utero; neonatal period; lactational exposure; rat.
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INTRODUCTION |
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DES is one of the most widely studied of endocrine toxicants. Endocrine toxicity has been observed for DES in all species studied and for all endpoints evaluated. Further, activity has been reported over a wide dose range, extending from unconfirmed reports of effects on the mouse ventral prostate gland following exposure in utero to 0.02 µg DES/kg body weight, to carcinogenic effects on the mouse uterus following neonatal exposure to 1000 µg DES/kg (representative data are shown in Table 1). Despite the extent of this database there has been no systematic comparative evaluation of the relative sensitivity of the many different windows of exposure. The objective of the present study was to determine which exposure period produced the most marked effects of DES on the reproductive capacity and sexual development of the rat, with particular emphasis on the relative sensitivity of in utero and postnatal exposures. Hitherto, such comparisons have been made between studies, rather than within a study.
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MATERIALS AND METHODS |
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Dosing solution preparation and analysis.
DES (> 99% pure) was obtained from SIGMA (Poole, Dorset, UK). Stock solutions of DES (1 mg/ml in ethanol) were prepared weekly and used for the preparation of drinking water solutions. Drinking water solutions were made up fresh each day for both uterotrophic assays and the pre- and postnatal studies. Solutions for the DES uterotrophic assay via the sc route were prepared by dilution of a stock solution of DES in arachis oil with additional arachis oil and used for the (3 day) duration of the experiment. The stock solutions of DES for the pre- and postnatal exposure study were analyzed to ensure that the concentration of DES was correct. The stability of the stock solution over a 2-week period was also established. Analysis of DES was by HPLC using an Ultracarb 7 mm ODS 30 column (ID 25 cm x 4.6 mm; Phenomenex, Macclesfield, Cheshire, UK), and a mobile phase of methanol (55%), acetonitrile (25%), and water (20%) (v/v) with a flow rate of 1.0 ml/min. A UV detector (210 nm) was used.
Uterotrophic assays.
Immature rat uterotrophic assays were conducted using weanling rats (either 19 to 20 days of age, or 20 to 21 days of age on arrival) for sc and drinking water studies, respectively) as described previously (Odum et al., 1997). DES was administered sc in arachis oil (5 ml/kg), daily for 3 days at 0.012.5 µg DES/kg body weight/day to 20 to 21-day-old rats. Control animals received vehicle only. DES was administered at 550 µg DES/l in the drinking water, ad libitum for 3 days to 21 to 22-day-old animals. Control animals received water only. Water consumption was measured daily and the intake of DES was calculated. In all cases, on the fourth day, animals were killed by an overdose of halothane (AstraZeneca, PLC) followed by cervical dislocation. Uteri were removed, trimmed free of fat, blotted and weighed, as described earlier (Odum et al., 1997
).
DES Pre- and Postnatal Exposure Study
Administration of DES to F0 and F1 generations.
The experimental design is shown in Figure 2. Pregnant female rats (F0; 1012 weeks old) were assigned to 10 groups on gestational day (GD) 0 (day of sperm positive smear). Two extra groups of untreated rats (with the same specification) were used to supply foster mothers for rearing of pups in Groups 2 and 4 (Fig. 2
). The animals were administered DES in the drinking water (ad libitum) at different periods throughout pregnancy, lactation, or postweaning.
Group 1 (control) received water. Group 2 (cross-fostered control) received water. Group 3 received DES (60 µg DES/l) from GD 0 to birth. Group 4 received DES (60 µg DES/l) from GD 0 to birth but were cross-fostered onto untreated mothers. Group 5 received DES (60 µg DES/l) from GD 0 to weaning (PND 21). Group 6 received DES (60 µg DES/l) from birth to weaning (PND 21). Group 7 received DES (60 µg DES/l) from birth to PND 10 (this group represented exposure during lactation only, since after this time the pups would start to consume the drinking water). Group 8 received DES (60 µg DES/l) from weaning to PND 100. Group 9 received DES (30 µg DES/l) from weaning to PND 100. Group 10 received DES (10 µg DES/l) from weaning to PND 100.
Freshly prepared drinking water solutions were supplied daily. The maximum concentration of DES (60 µg DES/l in drinking water) was selected based on the results of preliminary studies that indicated that this concentration gave body weight reductions of no greater than 20% for treated pregnant rats compared to the concurrent control animals.
Birth occurred naturally, and pups in Groups 2 and 4 were cross-fostered onto untreated mothers immediately after birth. The pups (F1) were culled to 8 per litter (4 of each sex if possible) on PND 4 (day of birth = D 0). At weaning (PND 21), the sexes were separated and housed with littermates. The dams were killed. All animals were weighed at 3- or 4-day intervals from GD 0 until termination of the study. Consumption of drinking water was determined daily throughout the study and the intake of DES calculated. Food consumption per cage was recorded weekly throughout the study.
The following developmental landmarks were monitored: anogenital distance (AGD; within 24 h of birth, using the method described in Ashby [1997]), testis descent (TD, from PND 21), onset of vaginal opening (VO, from PND 21), and prepuce separation (PPS, from PND 35). Animals were also weighed at the time of the observation of these specific landmarks. Vaginal smears were taken between PND 52 and 69 to determine the percentage of days spent in estrus and cyclicity.
At PND 100, postweaning administration of DES to Groups 810 ceased. Animals were selected for the F1 breeding phase, and the remaining animals were terminated between PND 107 and 111. Sex organ weights were determined for up to 3 pups per sex per litter. Ovaries, uterus, cervix, vagina, ventral prostate, and seminal vesicles were fixed in formal/saline. Testes and epididymides were fixed overnight in Bouins. The tissues were then dehydrated and processed by standard histopathological procedures to haemotoxylin/eosin stained paraffin-mounted sections (5 microns). The tissues from groups 1, 3, 5, and 8 were processed for standard histopathological analysis. The right cauda epididymis was taken for sperm analysis.
Breeding of the F1 generation.
Twenty-six animals of each sex were selected from Groups 1, 5, and 8 for breeding within the groups (Figure 2). These groups were representative of control, gestational, and postweaning exposure to 60 µg DES/l. Animals were randomly selected taking siblings into account. Exposure to DES had ceased in "Administration of DES to F0 and F1 generations" above and was not resumed. The animals (114128 days old) were mated in pairs for up to 2 weeks. Females were examined daily and the presence of sperm in the vaginal smear was taken as evidence of mating. When this occurred the animals were separated, males killed, and the females singly housed. Culling of pups was conducted as described above. TD, VO, and PPS were determined as described above. Females were terminated on PND
50 (after VO was complete for all animals) and males on PND
85 (after PPS was complete for all animals). Sex organ weights were determined for all animals, as in the F1 generation, and tissues were processed for histopathological analysis as above.
Sperm analysis.
The right cauda epididymis was scissor minced in 10 ml of 199 medium containing 25mM HEPES (Invitrogen, Groningen, The Netherlands). Sperm were isolated and counted as described in Ashby (1997). Analyses were carried out on all F1 groups using 2 pups per litter.
Statistical Methods
Uterotrophic assays.
Uterine weights were analyzed by covariance with the terminal body weights. Terminal body weights were adjusted for covariance with initial body weights. Differences from control values were assessed statistically using a two-sided Student's t-test based on the error mean square from the analysis of covariance. The individual was considered to be the statistical unit.
DES pre- and postnatal exposure.
Initial body weights were analyzed by variance and subsequent body weights by covariance with the initial body weight (taken on the first day of pregnancy, at birth, or at weaning, as appropriate). Water and food consumption were analyzed by variance. Percentage pup survival and pup sex were analyzed by variance following the double arcsine transformation of Freeman and Tukey (1950). Litter size and pup weights at birth were analyzed by variance. AGD was analyzed by variance and by covariance with birth weight. The proportions of animals recorded each day with developmental landmarks were analyzed by Fisher's Exact test and the observed days for the developmental landmarks were analyzed by variance. Body weights recorded at the time of observation of the landmark were also analyzed by variance. Estrus cycle data and sperm numbers were analyzed by variance. Organ weights were analyzed by variance and by covariance with the terminal body weights (Shirley, 1996) because this analysis provides a better means of allowing for differences in terminal body weights than organ to body weight ratios. Differences from control Group 1 values were assessed for Groups 3 and 510, and differences from control Group 2 values were assessed for Group 4; using a two-sided Student's t-test based on the error mean square from ANOVA or covariance. In all cases the litter was considered to be the statistical unit. Analyses were carried out as described in SAS (1996).
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RESULTS |
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Administration of DES to F0 and F1 generations.
The changes described below are those that are statistically significant. Changes that do not attain statistical significance are not described unless they are considered to be of importance in understanding a trend, or lack thereof, in which case the lack of statistical significance is clearly stated.
Consumption of drinking water containing DES by F0 and F1 animals during the study is shown in Figure 3. Groups receiving DES at 60 µg DES/l consumed less water than control groups (up to 60% of concurrent control values). At 10 µg DES/l there was no effect on water consumption although 30 µg DES/l reduced water consumption in females, but not males. The dosage of DES received (as µg DES/kg body weight/day) is shown in Table 3
. Gestational and postweaning exposure to a concentration of 60 µg DES/l resulted in
6 µg DES/kg/day while the dosage during lactation rose to
10 µg DES/kg/day. These doses of DES produced no adverse clinical signs in any animals. Body weights of treated F0 dams were reduced in the groups exposed to DES but were no less than 80% of concurrent control values throughout pregnancy (Fig. 4
). Dams exposed only during pregnancy (Group 3) regained this weight, once exposure had ceased. The body weights of dams administered DES postpartum only (Groups 6 and 7) were unaffected. Dams exposed through pregnancy and lactation (Group 5) were consistently lighter than those in all the other groups (Fig. 4
).
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AGD was unchanged in males and females in all groups (Tables 6 and 7). Since body weights at birth were similar in all groups, AGD adjusted for body weight was also unchanged (data not shown).
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VO is a sexual developmental landmark in female rats. The age and weight at which this occurred was also analyzed independently (i.e., it was unadjusted for body weight changes). There were no changes observed in VO in F1 pups exposed to DES during the gestational period only (Table 7, Fig. 6
). All of the other postnatal treatment regimes, with the exception of exposure to DES from birth to PND 10 (Group 7), reduced the age and weight at which VO occurred. Advances of up to 8 days were observed in animals exposed to DES for the longest periods postnatally and in the postweaning period when the pups themselves were drinking water containing DES, rather than only receiving it lactationally (Groups 810). The effect on VO in the latter 3 groups was dose-related with the lowest concentration of DES giving the least change in VO (Table 7
). In contrast to the effects on VO, DES produced little effect on estrus cyclicity (Table 7
). No changes were observed in the number of days spent in estrus or cycle length. A small reduction was observed in the number of cycles counted over a 21-day period in groups exposed to 60 and 30 µg DES/l post-weaning (Groups 8 and 9; Table 7
, Fig. 6
). These effects are consistent with the irregular cycles reported by Kwon et al. (2000) for rats exposed to DES from GD 11 to PND 21, although there are problems inherent in the evaluation of estrus cyclicity by vaginal smears (as opposed to histopathological assessment of the female reproductive tract).
Male sex organ weights were determined at PND 107111 and are shown in Table 8 as either absolute weights or adjusted for covariance with terminal body weight. Although statistical significance is shown in Table 8
for both sets of parameters, only the latter is discussed below since this provided a better comparison because terminal body weights were reduced in some of the treatment groups. The adjusted weight of epididymides was reduced, while that of seminal vesicles was increased, in animals exposed to DES from GD 0 to birth (Group 3). The adjusted weight of the ventral prostate of animals exposed to 60 to 10 µg DES/l from PND 21 to 100 (Groups 8 to 10) was increased, although only Group 9 was statistically significant. No other changes were observed (Table 8
, Fig. 5
). No changes were observed in the caudal sperm numbers in any of the groups (Table 9
). Histopathological examination of testes, epididymides, seminal vesicles, and ventral prostates from Groups 3, 5, 8, and controls showed that there were no treatment-related effects.
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DISCUSSION |
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A particular strength of the study is that the use of multiple parallel test groups enabled the validity of individual observations to be assessed within a broad context. Instances of where statistically significant effects are considered to reflect natural control variability are shown as open arrows in Figures 57. For example, changes in the day of PPS and body weight at PPS in Group 8 F1 animals are both clear-cut and internally consistent (closed arrows in Fig. 5
). In contrast, changes in the day of PPS for F1 animals in Groups 5 and 6 are difficult to interpret because of decreased body weights at weaning and the absence of consistency between the day of PPS and the mean body weight of the animals on their individual day of PPS (open arrows in Fig. 5
). The complexity of adjusting the day of PPS for changes in body weight have been discussed elsewhere (Ashby and Lefevre, 2000
). Likewise, the reduction in the weight of the cervix in Group 3 F1 animals is the reverse of what would be expected of an estrogen, and given that this change occurred only once in the whole database, we concluded that it probably arose by chance (open arrow in Fig. 6
). Similar concerns are associated with the decrease in seminal vesicle weights of F2 animals in Group 8 (Fig. 7
) and the changes in the weights of the epididymides and seminal vesicles among F1 male pups in Group 3 (Fig. 5
). Of particular interest is the variability in litter size and birth weight among the litters of the 5 groups of untreated dams in this study (Table 16
), a variability that complicates assessment of these parameters in DES-treated dams (discussed later).
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The overall consonance of body weight changes and experimental observations between male and female F1 pups in all of the test groups indicates a high level of internal consistency for the study (Figs. 5 and 6). There is no apparent explanation for the absence of changes in AGD within the study. Small reductions in AGD for male rats exposed to marginally higher doses of DES in utero have been reported earlier (Ashby et al., 1997
; Levy et al., 1995
); however, AGD is generally regarded as being most sensitive to antiandrogenic effects (Mably et al., 1992
; Neumann et al., 1970
) and was not observed in reproduction studies with estradiol (Biegel et al., 1998
).
The strongest responses were changes in the developmental landmarks in the F1 females (VO and less evidently, estrus cyclicity) and F1 males (PPS and less evidently, TD; shaded boxes in Figs. 5 and 6). These changes were uniquely dependent upon exposure occurring after PND 10. These effects could therefore have been produced for DES using a simple peripubertal assay. There was a complete absence of effects in F1 pups lactationally exposed to DES via the dam in the period PND 110, apart from a reduction in body weight before weaning (Group 7). There was a near absence of effects on F1 animals only exposed to DES in utero (Groups 3 and 4). There were decreased weights of the cervix, as mentioned above, and changes to the weight of the epididymides and seminal vesicles in Group 3 F1 males. However, there were no changes in the weight of the epididymides or the seminal vesicles in Group 4, where the pups were cross-fostered at birth. This would suggest that the weight changes seen in Group 3 animals could have been induced by brief lactational exposure to DES. A similar possibility could explain the decrease in cervix weight in Group 3 animals and the absence of such a change in the cross-fostered Group 4 animals (Fig. 6
). However, no changes in the weights of the epididymides, seminal vesicles or cervix were seen in animals from Groups 57, where similar lactational exposure to DES occurred.
Postweaning exposures to DES (Groups 810) produced a range of effects in the F1 pups while no effects were observed in pups exposed between PND 110 (Group 7). This is in contrast to the similar sensitivity to DES (and genistein, both injected sc) of the neonatal mouse uterotrophic assay (Newbold et al., 2001) and the weanling mouse uterotrophic assay (Ashby, 2001
, Ashby et al., 2001
). The apparent nonequivalence of exposure to DES in utero and postnatally in the present study may have been caused by the postnatal (lactational) exposure to DES in the present study being considerably lower than in the neonatal sc injection studies conducted by Newbold et al. (1998, 2000, 2001; see Table 1
). No changes in F1 testes weights or caudal sperm counts, and no significant histopathological changes in testes, epididymides, or the ventral prostate gland were seen for any group. Inspection of the shaded boxes in Figures 5 and 6
for Groups 810 indicates a dose-response relationship for the individual developmental markers, which suggests that changes in the age of TD and in estrus cyclicity are less sensitive landmarks than are changes in the age of PPS and VO, respectively.
F1 animals exposed to DES in utero (Group 3) produced heavier pups and reduced litter sizes, the F2 pups showed earlier VO, the body weight of F2 males on the day of PPS was elevated and the weight of the epididymides was increased. F1 animals exposed to DES from PND 21 (Group 8) produced heavier male pups and reduced litter sizes, the F2 females showed earlier VO and the F2 males had changes in the weights of the testes, epididymides, and seminal vesicles. No organ weight changes were seen in female F2 pups from either group, and no histopathological changes were seen in male or female F2 pups from either group. There was, therefore, an approximate equivalence in the effects of exposure to DES in utero and exposure from weaning. Akingbemi et al. (2001) recently drew the same conclusion regarding the gonadal endocrine effects of diethylhexylphthalate in the rat for the periods GD 1221 and PND 2135.
The 1 day advance in VO in Group 3 and Group 8 F2 pups was statistically significant, but the interpretation of these small changes is complicated by the generally heavier weight of the female pups in the times before puberty (Table 11
). However, we consider that these effects on VO are probably related to DES treatment, given that potentially artifactual advances in the day of VO are more difficult to rationalize than are delays in VO. It is also interesting to note that the reductions in the F2 litter sizes and the increases in the F2 pup weights remain within the control ranges when all of the control groups in this study are considered (Table 16
). Nonetheless, these F2 effects are left as closed arrows in Figure 7
because, in a normal multigenerational assay, access to such extra control groups would not be possible.
It is assumed here that the major effects and conclusions discussed above can be extrapolated to other estrogens and other strains and species of rodent. However, possible differences in the specificity of individual estrogens for the different forms of the estrogen receptor, and possible sensitivity differences between strains and species of rodent (see Spearow et al., 1999; Long et al., 2000
; Ashby, 2001
) may influence the outcome of individual experiments.
Conclusions
There are 4 major conclusions from this study that relate to many preconceptions. First, exposure to 6.5 mg DES/kg body weight/day in utero, or during postnatal days 110, produced negligible developmental effects compared to the effects induced following exposure from weaning. Second, developmental landmarks, such as the day of VO or PPS, acted as superior early indicators of exposure to DES than did changes in organ weights or histopathology. Third, reproductive effects were seen in both F1 animals exposed to DES in utero and in animals exposed postweaning. Fourth, each of the parameters studied here is shown to be subject to natural variability, and this indicates that some statistically significant changes observed in reproductive toxicity studies may arise by chance. This study, therefore, does not establish a unique role for exposures in utero, or during the early neonatal period. It also shows that in studies involving exposures during all life stages, such as in a multigenerational study, effects on developmental landmarks may not necessarily have been induced in utero or during the early neonatal period.
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ACKNOWLEDGMENTS |
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NOTES |
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