Toxicological Sciences 68, 147-163 (2002)
Copyright © 2002 by the Society of Toxicology
REPRODUCTIVE AND DEVELOPMENTAL TOXICOLOGY |
Comparison of the Developmental and Reproductive Toxicity of Diethylstilbestrol Administered to Rats in Utero, Lactationally, Preweaning, or Postweaning
* 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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The objective of the study was to determine which period of exposure produces the most marked effects on the reproductive capacity and sexual development of the rat, with particular emphasis on the relative sensitivity of in utero and postnatal exposures. The endocrine active chemical, diethylstilbestrol (DES) was used as an agent known to affect many of the endpoints examined. Hitherto, such comparisons have been made between studies, rather than within a study. Our data will be helpful in the interpretation of future multigenerational assay data. In preliminary studies, DES was shown to be active in the immature rat uterotrophic assay with a lowest detected dose of 0.05 mg DES/kg body weight by sc injection and 10 mg DES/l (1.6 mg DES/kg body weight) by administration in drinking water. A dose of 60 µg DES/l drinking water (
6.5mg DES/kg body weight/day) was selected for the main study since this represented the midpoint of the drinking water uterotrophic dose response and produced no overt maternal toxicity. The study used 10 groups of concomitantly pregnant animals, including 2 control groups. The first comparison was between the effects of exposure to DES in utero , and exposure from conception to weaning. Another group of animals was exposed to DES in utero and cross-fostered to untreated pregnant females to prevent lactational transfer of DES to pups. Two groups were exposed to DES neonatally, either from birth to postnatal day (PND) 10 (pups thus having only lactational exposure), or from birth until weaning (PND 21; pups thus having both lactational exposure and self-exposure via drinking water). In addition, a dose response study to DES was conducted on animals exposed from weaning to PND 100, when the first phase of the study was terminated. Pups exposed to DES in utero and pups exposed from weaning to PND 100 were bred to assess fertility of the F1 animals and the sexual development of F2 offspring. This last comparison was to determine the extent to which weanling rats could be used in endocrine toxicity studies to assess their potential to show activity in utero. The most sensitive period of exposure for inducing developmental effects in F1 animals was from weaning onwards. The neonatal to weaning period (PND 1–21) was the next most sensitive. Essentially no effects were induced in F1 animals exposed in utero . No effects of any kind were observed in animals only exposed over the early neonatal period of PND 1–10. The mean day of vaginal opening, testes descent, and prepuce separation was only altered in groups where postnatal exposure to DES continued beyond PND 10, or was started at weaning. No changes were observed in anogenital distance or caudal sperm counts. Some changes in organ weights were observed, but the interpretation of these was often confused by concomitant changes in body weight. In general, histopathological examination of tissues yielded no additional information. In breeding studies with animals exposed to DES in utero , or from weaning, reduced litter sizes and marginal advances in the day of vaginal opening were observed in the offspring, together with changes in organ weights. However, no unique sensitivity was noted for exposure in utero. Evaluation of the several exposure periods and the many markers monitored in this study may have individual strengths in individual cases, but when rigorously compared using the reference estrogen DES, many preconceptions regarding their absolute or relative value were not upheld. Further, each of these markers is subject to natural variability, as demonstrated by comparisons made among the 5 separate control groups available in parts of the present study. This variability increases the chance that small changes observed in endocrine toxicity studies employing small group sizes and a single control group, or no concomitant control group, may be artifactual. The most marked effects observed in this study were on the developmental landmarks in the F1 animals induced by exposures after PND 10. Some effects on developmental landmarks and organ weights were observed in F2 animals following exposure either in utero or postweaning. This study therefore does not establish a unique role for exposures in utero or during the early neonatal period. Key Words: diethylstilbestrol; reproductive toxicity; in utero; neonatal period; lactational exposure; rat.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One of the main uncertainties identified by the United States Endocrine Disrupter Screening and Testing Advisory Committee (EDSTAC), and others, was whether it is necessary to evaluate specifically the endocrine toxicity of chemicals in utero and in the early neonatal period, or whether exposures in other life stages will suffice to reveal endocrine toxicity (Goldman et al., 2000
; NTP, 2001; Pryor et al., 2000; Schmidt, 2001; Sharpe, 1994; Stoker et al., 2000; Williams et al., 2001). There are few, if any, comparative data available to answer this question. This question is particularly pertinent to interpretation of data derived from the rodent multigenerational assay (OECD, 1983), appropriately classed as an apical endocrine perturbation assay by EDSTAC. This assay requires constant administration of the test chemical through 2 generations, including exposures occurring in utero, during lactation, and during sexual maturation. As a consequence of the constant exposure, it is impossible to distinguish effects induced during gestation from those occurring postnatally, a distinction of relevance to human risk assessments. The multigenerational assay protocol was recently updated to include, among other things, observation of developmental landmarks such as the mean day of vaginal opening and prepuce separation (OECD, 2001a), landmarks not accessible in the prenatal developmental toxicity assay (OECD, 2001b) because of the culling of some newly born pups. These additions exacerbate the above problem because these newly added landmarks are modulated by exposure during the peripubertal period. In an attempt to address these uncertainties we have evaluated the comparative endocrine toxicity of diethylstilbestrol (DES) in different life stages of the rat.
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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Use of drinking water as the route of exposure to DES was chosen for the study to reduce stress to the treated animals and to be consistent with the general use of the po route of exposure in multigenerational studies. Likewise the rat was selected for study since this is the primary test species used in the multigenerational assay. Since most of the earlier studies on DES had used sc injection as the route of exposure, the relationship between these 2 routes for DES was first investigated using the immature rat uterotrophic assay. The uterotrophic activity of DES to the immature mouse is well established (Ashby, 2000
; Shelby et al., 1996), but its activity in the immature rat uterotrophic assay has not been reported. The dose of 60 µg DES/l drinking water ( 6.5 µg DES/kg body weight/day) was selected for the main part of the study as this was found to represent both the midpoint of the rat drinking water uterotrophic dose response curve and the midpoint of all previous endocrine toxicity studies on DES (see Fig. 1). This dose was also shown in preliminary experiments not to be overtly toxic to pregnant rats. The transplacental transfer of DES is known (Table 1
), and some degree of lactational exposure of pups of dams treated with DES may be assumed from the results of Vorherr et al. (1979).
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The study design (Fig. 2
) involved 10 test groups, including 2 control groups, comprising pregnant animals accepted into the study concomitantly. The first comparison was between the effects of exposure to DES in utero, and from conception to weaning (Groups 3 and 5). This comparison was devised to separate the 2 main exposure periods merged in a multigenerational study. Animals in Group 4 were exposed to DES in utero and cross-fostered to untreated pregnant females to prevent lactational exposure of the pups. Two groups were exposed to DES neonatally, either from birth until weaning at postnatal day (PND) 21(Group 6), thus including self-exposure of the pups to DES in the drinking water, or from birth to PND 10 (Group 7), to exclude self-exposure. In addition, a dose response study to DES was conducted on animals exposed from weaning to PND 100, at which time the first phase of the study was terminated (Groups 8–10). Pups derived from dams exposed to DES in utero (Group 3) and pups exposed from weaning to PND 100 (Group 8) were bred to assess fertility of the F1 animals and the sexual development of F2 offspring. All primary data are shown, and only statistically significant changes are discussed. The results are represented as a series of figures to facilitate absorption and understanding of this large database.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals.
Alpk:APfSD (Wistar derived) rats, obtained from the AstraZeneca breeding unit (Alderley Park) were used in both the uterotrophic asay and pre- and postnatal study. Animal studies were performed in accordance with the UK "Animals (Scientific Procedures) Act." Pregnant and lactating rats were housed (singly and with litters) in polypropylene cages and supplied with shredded paper bedding. Rat and Mouse No. 3 diet (Special Diet Services Ltd., Witham, Essex, UK) and water were available ad libitum. After weaning (see below), rats were housed (up to 5 per cage) in metal cages. Rat and Mouse No. 1 diet (Special Diet Services Ltd., Witham, Essex, UK) and water were available ad libitum. Animal care and procedures were conducted according to in-house standards as described previously (Odum et al., 1999
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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 0.01–2.5 µg DES/kg body weight/day to 20 to 21-day-old rats. Control animals received vehicle only. DES was administered at 5–50 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; 10–12 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 8–10 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 (114–128 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 5–10, 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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Uterotrophic Assays
DES was active in the immature rat uterotrophic assay when administered for 3 days by sc injection, giving a lowest statistically significant dose level of 0.05 µg DES/kg body weight/day and reaching a plateau at 1 µg DES/kg/day (Table 2
, Fig. 1). Administration of DES in drinking water gave a similar shaped dose response curve with a lowest statistically significant effect dose of 1.6 µg DES/kg/day (10 µg DES/l drinking water; Table 2, Fig. 1).
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DES Pre- and Postnatal Exposure Study
Dosing solution analysis.
The mean concentration for all batches of dosing preparations of DES analyzed were within 7% of the nominal concentration. The stability of the stock solutions used to prepare dosing preparations was 100% over 2 weeks.
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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The numbers of litters born and litter sizes at birth were similar for all groups. Pup survival to PND 4 (when numbers were culled to 8 per litter) among the groups which were not cross-fostered was similar among treated and untreated groups (Table 4
). Pup survival was significantly reduced in Group 4 (exposed through gestation, with cross-fostering) when compared with the cross-fostered controls (Group 2), but this was due to the higher survival rate of pups in Group 2 compared with the noncross-fostered control group (Group1) against which the other groups were compared. Body weights of male and female pups up to weaning were reduced in pups exposed to DES after birth (Groups 5–7), but not in pups exposed gestationally (Table 5, Figs. 5 and 6). In the postweaning period, body weights were reduced in pups exposed to 30 and 60 µg DES/l after weaning (Groups 8 and 9), no other groups were affected (Table 5, Figs. 5 and 6).
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Food consumption by the F0 dams was reduced to approximately 80% of control values during the DES exposure period, but returned to that of the control animals once exposure had ceased. Food consumption was similarly reduced in F1 animals exposed to 60 µg DES/l during the postweaning phase. F1 females exposed to 30 µg DES/l showed intermittent reductions (approximately 90% of control values) in food consumption (data not shown).
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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TD and PPS are sexual developmental landmarks in male rats. The age and weight at which these occur were analyzed independently (i.e., they were unadjusted for body weight changes). There were no changes observed in these parameters in F1 pups exposed to DES during the gestational period only (Groups 3 and 4). Animals exposed to DES postnatally were older, and sometimes heavier, at TD and PPS (Table 6
, Fig. 5). Exposure to 60 µg DES/l from GD 1 to PND 21 (Group 5) or exposure from birth to PND 21 (Group 6) gave a delay of 2–3 days for TD. These effects on these 2 groups were, however, considered to be of uncertain biological significance (Fig. 5) since they were not accompanied by increased body weight at TD. Group 5 animals were lighter but not statistically different from controls (Table 6). A delay in TD was also observed in animals exposed to 60 µg DES/l from PND 21 onwards (Group 8) and was accompanied by a body weight increase at the time of TD. An increase in body weight only at TD was observed in animals exposed to 30 µg DES/l from PND 21 onwards (Group 9), giving some indication of a dose response (Table 6, Fig. 5). PPS was also delayed in animals exposed to 60 µg DES/l from GD 1 to PND 21 (Group 5). However this group, and Group 6, also had reduced body weights at PPS. As with TD, the decreased body weights led us to consider the effects in Groups 5 and 6 to be of uncertain biological significance (Fig. 5). PPS was delayed by 6 days in animals exposed to 60 µg DES/l from PND 21 onwards (Group 8) and was also accompanied by a body weight increase. Exposure to lower concentrations of DES from PND 21 onwards (Groups 9 and 10) gave dose-related delays in PPS (Table 6). More of the groups showed delays in PPS than TD, which may be a reflection of the increased duration of exposure to DES in the period before PPS at PND 45. No changes in TD or PPS were observed in pups exposed to DES from birth to PND 10 (Group 7; Table 6, Fig. 5).
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 8–10). 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 107–111 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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Female sex organ weights were also determined at PND 107–111 (Table 10
). As with the males, terminal body weights were reduced in some of the treatment groups. The only change observed for the adjusted organ weights was a decrease in the weight of the cervix in animals exposed to DES from GD 0 to birth (Group 3), but this isolated result was not considered to be biologically significant (Table 10, Fig. 6). The ovaries, uterus, cervix, and vagina from Groups 3, 5, 8, and controls were examined histopathologically. There were no effects observed in animals exposed during gestation only (Group 3). Histopathological examination of ovaries revealed a reduction in corpora lutea in approximately 25% of Group 8 females indicating that administration of DES in the postweaning period produced an inhibitory effect on ovulation in some animals. Minor uterine changes of squamous metaplasia and dilatation with cellular debris were observed in Groups 5 and 8, consistent with the estrogenic effect of DES. There was a slightly higher (but not statistically significant) incidence of these changes in Group 8 compared with Group 5. These animals still exhibited normal cyclic changes in the reproductive tract. In Group 5, where exposure to DES ceased before pubertal growth of the uterus, these changes could reflect a permanent change in phenotype of the endometrial gland and luminal epithelium at the time when the uterus was developing from its embryonic tubular form to a more complex glandular structure.
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Breeding of the F1 generation.
The offspring from Groups 1 (control), 3 (60 µg DES/l F0 GD 0–birth), and 8 (60 µg DES/l F1 PND 21–100) were bred within their groups to determine whether gestational DES treatment or postweaning DES treatment would affect their breeding performance. All groups mated within 1 week. Maternal body weights at the start and end of pregnancy were similar for all groups (data not shown). The pregnancy indices, numbers of litters born, sex ratios, and pup survival were also similar for all groups (Table 11
). Overall litter size and weight (combined male and female pups) in the 2 treated groups were lower than the control group. Mean male pup weights in both treated groups and female pup weights in Group 3 were, however, higher at birth than the control group (Table 11, Figure 7). During the lactational and preweaning period the mean pup weights gradually equalized across the groups so that by PND 21 they were no longer different (Table 11).
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Testis descent and age at PPS were unaffected in the F2 generation, but males in Group 3 (60 µg DES/l F0 GD 0–birth) were heavier when PPS occurred (Table 12
, Figure 7). Vaginal opening in females from both DES treatment groups was advanced by approximately one day (Table 13, Fig. 7).
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The F2 males were terminated at PND 85. There were no differences in body weights between the groups. Increases in the absolute and adjusted weights of epididymides and the absolute weight of the ventral prostate were observed in Group 3 (60 µg DES/l F0 GD 0–birth), although the adjusted weights of the ventral prostate were not statistically significant. Significant increases in the absolute and adjusted weights of the testes and epididymides and a decrease in the adjusted weights of seminal vesicles were observed in Group 8 (60 µg DES/l F1 PND 21–100; Table 14
, Fig. 7). Sex organ weights of F2 females (terminated at PND 50) were unaffected (Table 15, Fig. 7).
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Histopathological examination of the F2 male and female reproductive organs (testis, epididymides, seminal vesicles, ventral prostate, ovary, uterus, cervix, and vagina) from Groups 1, 3, and 8 indicated that there were no treatment-related effects.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The following discussion is based on the data summarized in Figure 5
(F1 males), Figure 6 (F1 females), and Figure 7 (F1 reproduction and F2 male and female effects). Only statistically significant effects are shown in these figures and discussed in the text.
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 5–7. 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 main observations of this study, in relation to the questions posed in the Introduction, are itemized below with particular reference to Figures 5–7
. DES is active in the immature rat uterotrophic assay with a lowest detected dose for the sc route of administration of 0.05 µg DES/kg body weight. Administration of DES in the drinking water shifted the uterotrophic dose response curve to the right by about 1 log concentration, yielding a lowest detected dose of 1.6 µg DES/kg (Fig. 1). The dose of DES adopted for the main study (60 µg DES/l of water, 6.5 µg DES/kg body weight/day) was therefore about the EC50 value of the drinking water rat uterotrophic dose response to DES (Fig. 1), and the mean of previous positive endocrine toxicity studies of DES (Table 1).
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 1–10, 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 5–7, where similar lactational exposure to DES occurred.
Postweaning exposures to DES (Groups 8–10) produced a range of effects in the F1 pups while no effects were observed in pups exposed between PND 1–10 (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 8–10 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 12–21 and PND 21–35.
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 1–10, 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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We thank K. Jones and staff for carrying out the animal studies, J. Redwood for the formulation analysis, S. F. Moreland for histopathology, and S. Kirk for statistical analyses. We are grateful to the American Chemistry Council for financial contributions to the conduct of this study.
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NOTES |
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1 To whom correspondence should be addressed. Fax: (44) (0) 1625 590249. E-mail: john.ashby{at}syngenta.com.
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REFERENCES |
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