* Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, SK10 4TJ, United Kingdom;
Union Carbide Corporation, Danbury, Connecticut 06817;
General Electric Company, Pittsfield, Massachusetts 01201;
§ Harlan Teklad U.K., Bicester, Oxfordshire, United Kingdom;
¶ Japanese Chemical Industries Association, Sumitomo Chemical Co., Ltd., 271, Shinkawa 2-chome, Chuo-ku, Tokyo 104-8260, Japan; and
|| Europeptides, 9 Avenue du Marais, 95108 Argenteuil, France
Received November 30, 2000; accepted January 23, 2001
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ABSTRACT |
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Marked differences in body weight, sexual development, and reproductive tissue weights were observed for rats maintained on AIN-76A or Purina 5001, with only minimal effects among rats maintained on the Global diet. These comparisons were against RM3/RM1 as the reference diet. However, using Purina 5001 as the reference diet reversed the direction of the differences seen when using RM3/RM1 as the reference diet. The differences observed when using RM3/RM1 as reference diet occurred mainly postnatally. In addition, the fact that similar differences were seen for the phytoestrogen-free diet, AIN-76A, and the phytoestrogen-rich diet, Purina 5001, indicate that these effects are more likely to be caused by nutritional differences between the diets that then have centrally mediated effects on rodent sexual development, rather than individual dietary components affecting peripheral estrogen receptors (ER). This proposal is supported by abolition of the uterotrophic activity of AIN-76A and Purina 5001 (relative to RM3/RM1) in the immature rat by coadministration of the gonadotrophin-releasing hormone (GnRH) antagonist Antarelix.
The present data indicate that choice of diet may influence the timing of sexual development in the rat, and consequently, that when evaluating the potential endocrine toxicity of chemicals, the components of rodent diets used should be known, and as far as is possible, controlled.
Key Words: phytoestrogen; sexual development; rat; endocrine toxicity; rodent diet..
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INTRODUCTION |
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The ability of phytoestrogens to influence the outcome of endocrine toxicity evaluations is illustrated by Boettger-Tong et al. (1998), who reported their inability to demonstrate a uterotrophic response to estradiol in rats receiving a diet high in phytoestrogens. Thigpen et al. (1999) corroborated these findings and stressed the importance of dietary phytoestrogens not only in studies of uterine growth but also in evaluations of the carcinogenicity of chemicals to the rodent mammary and prostate glands. We have also reported that the rodent diet selected for use can affect control uterine weights in the immature rat uterotrophic assay (Odum et al., 1997).
Thigpen et al. (1999) have suggested that the use of a semisynthetic rodent diet such as AIN-76A could eliminate the influence of phytoestrogens in laboratory-animal diets. However, when AIN-76A was fed for 3 days to immature rats, they had heavier uteri than animals maintained on our standard RM1 rat diet (Ashby et al., 1999). This uterotrophic activity of AIN-76A was abolished by coadministration of the antiestrogen Faslodex, thereby confirming a direct involvement of the estrogen receptor (ER) (Ashby et al., 1999
). Similar uterotrophic activity was observed for Purina 5001 (Ashby et al., 2001
), a diet reported to contain a relatively high phytoestrogen content (Thigpen et al., 1999). In addition, it was shown that the uterotrophic activity of AIN-76A could be abolished by coadministration of the gonadotrophin-releasing hormone (GnRH) antagonist Antarelix, indicating that, in addition to the involvement of ER, the uterotrophic activity is mediated centrally via effects on the hypothalamus (Ashby et al., 2000
). This finding for AIN-76A indicated that unknown dietary factors, in addition to phytoestrogens, can act as modulators of endocrine toxicity endpoints.
The above observations indicate that the diet selected for rodent endocrine toxicity studies may influence the outcome of those studies. Given this, and in the absence of agreement of a standard diet for use in endocrine toxicity studies, it became of interest to investigate a range of diets for their possible effects on the sexual development of male and female rats. The diets selected are commercially available and generally employed throughout the world. These were Rat and Mouse no. 3 (RM3) (used for "breeding, lactation and growth of young animals") and RM1 (a "general maintenance diet"), each being standard U.K. rodent diets, AIN-76A (a semisynthetic diet with no soy or alfalfa added), Teklad Global 2016 (a diet made from natural ingredients and containing no soy or alfalfa, intended primarily for growth and maintenance but shown in our study as supporting breeding), and Purina 5001 (a standard rodent diet used particularly in the U.S., suitable for "life-cycle nutrition" and reported to contain a high level of phytoestrogens; Thigpen et al., 1999). Combinations of these diets were fed to rats throughout pregnancy and to the offspring until they reached adulthood (Fig. 1). An additional group of animals were maintained on RM3 throughout pregnancy and on AIN-76A postnatally (RM3/AIN-76A). Sentinel developmental landmarks and reproductive organ weights in the offspring were then evaluated. The diets were also tested in the immature rat uterotrophic assay in the presence and absence of the GnRH antagonist Antarelix.
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MATERIALS AND METHODS |
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Diets and chemical.
The following diets were used. Rat and Mouse No.3 (RM3), and Rat and Mouse No.1 (RM1) were obtained from Special Diet Services, Ltd., Witham, Essex, U.K. AIN-76A and Teklad Global 2016 (Global), were obtained from Harlan Teklad U.K., Bicester, Oxfordshire, U.K. Purina Chow 5001 (5001) was from Purina Mills, Inc., Richmond, IN, U.S. All diets and drinking water were available ad libitum. The GnRH antagonist Antarelix was a gift from Europeptides, a Division of Asta Medica, Argenteuil Cedex, France.
Dietary analysis.
The diets were analyzed for genistein and daidzein content by GC-MS. Aliquots of the diets (10 pellets) were ground to a homogenous powder; 100mg of each was then extracted with 80% methanol (80 ml) by ultrasonication (3 min) followed by incubation at 60°C for 2 h and further ultrasonication (3 min). The mixtures were cooled, made up to 100 ml with methanol, and 0.1 ml samples taken and mixed with 0.05 ml methanol containing internal standards (deuterated d4-daidzein, d4-genistein, and dihydroxyflavone). Sodium acetate buffer (1 ml; 0.1 M pH 5.0) was added to the samples, which were then treated with ß-glucuronidase (Helix pomatia, 1000 units) to a final volume of 2.5 ml and incubated overnight at 37°C. The products were then extracted with ethyl acetate (2 x 4ml) and the combined extracts evaporated to dryness. The residues were reconstituted in chloroform:heptane:methanol 10:10:1. They were then applied to short columns of Sephadex LH20, washed with chloroform:heptane:methanol 10:10:1 (4ml), and eluted with methanol. After evaporation of the methanol, the samples were derivatized for GC-MS with n-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide containing 1% t-butyldimethylsilyl chloride (0.04 ml) in acetonitrile (0.04 ml) at 65°C for 2 h. After evaporation of the solvents the residues were reconstituted in ethyl acetate (0.02 ml) for GC-MS.
GC-MS was carried out on a DB5 MS-bonded silica capillary column (10 x 0.25 mm, phase thickness 0.25 µm) using helium as carrier gas and a temperature of 70300°C at 40°C per min. Isotope dilution MS was performed using selective ion monitoring at mass 425 for daidzein, 429 for d4-daidzein, 555 for genistein, and 559 for d4-genistein. Peak area ratios were determined for analytes and internal standards. Calibration curves were constructed and the concentrations of daidzein and genistein in the samples determined.
Sexual maturation study.
The experimental design for this study is shown in Figure 1. Sixty pregnant female rats (1012 weeks old) were assigned to 5 groups on day 0 of pregnancy (day of sperm-positive smear). Each group contained 12 pregnant females in order to achieve 10 litters per group, although this number was less than is recommended under ICH guidance criteria (where n = 16) and interim terminations meant that for some endpoints the numbers of litters were halved. Each group received a different diet combination through pregnancy, weaning, and up to postnatal day (PND) 68 (test phase: see Fig. 1
). Birth occurred naturally and no pup culling took place before weaning on PND 21 (day of birth = day 0). At weaning, the sexes were separated and housed with littermates. All females were retained at weaning, as the female offspring from 6 of the litters in each group were killed at PND 26 (the usual endpoint of the uterotrophic assay) and sex-organ weights determined. Males were culled to 4 per litter at weaning in order to standardize to 4 animals per cage. Animals were weighed at 4-day intervals from birth until weaning, and thereafter every 7 days. Food consumption per cage was monitored throughout the study and recorded as total food consumed per cage, weekly, from which average food consumption per group was calculated.
The following developmental landmarks were monitored: eye opening (from PND 8), testis descent (TD, from PND 21), vaginal opening (VO, from PND 21) and prepuce separation (PPS, from PND 35). The age at first estrus was determined by taking vaginal smears after vaginal opening, smearing ceasing when first estrus was defined. Smearing commenced again between PND 5269 in order to determine the percentage of days spent in estrus. When the male offspring were sexually mature (PND 68) males from 6 litters per group were killed and liver, kidney and sex organ weights determined. The remaining females were culled to 4 per litter at the same time (PND 68). On PND 70 all male and female animals were placed on RM1 diet to ascertain if any of the differences that might be seen would be reversible (Recovery Phase: Fig. 1). The males were killed at PND 128 and the females at PND 140144, as the growth curves indicated that plateaus had been reached. Females were killed when they were in estrus (established by vaginal smears). Liver, kidney, and sex organ weights were determined for both male and female animals at termination.
Uterotrophic assay.
An immature rat uterotrophic assay was carried out using weanling rats (2021 days old on arrival) as described previously (Odum et al., 1997). Animals were weaned on RM3 diet in the breeding unit and then fed RM1, AIN-76A, Global, or 5001 upon acceptance into the laboratory, and for the 4-day duration of the assay. Food consumption was monitored daily. Rats (2122 days old at the start of dosing) received Antarelix daily for 3 days by subcutaneous injection (300 µg/kg/day) in arachis oil (AO) (dosing volume 5 ml/kg). Control animals received AO only. Animals were killed by an overdose of halothane (AstraZeneca plc) 24 h after the final dose. Uteri were removed, blotted, and weighed, as described earlier (Odum et al., 1997
).
Statistical methods.
For the sexual maturation study, initial body weights were analyzed by variance and subsequent body weights by covariance with the initial body weight (taken at weaning). Food consumption was analyzed by variance. Organ weights were analyzed by variance and by covariance with the terminal body weights (Shirley, 1996). 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. Differences from control values in all cases were assessed statistically using a 2-sided Student's t-test based on the error mean square from the analysis of variance or covariance. Analyses were carried out twice, first taking the RM3/RM1 group as control and secondly taking the 5001/5001 group as control. In all cases the litter was considered to be the statistical unit. Analyses were carried out as described in SAS (1996).
For the 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 (RM1 + AO) were assessed statistically using a 2-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.
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RESULTS |
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The age and weight at TD was not markedly altered in any of the diet groups. Inconsistent changes in the day of TD of 1 day were seen in the AIN-76A groups and mean body weight at TD was slightly lower in the Global group (Tables 2 and 3
). Significant changes in the day of PPS were seen in both AIN-76A groups where PPS was advanced by
3 days whilst body weights at PPS were similar to the RM3/RM1 controls. PPS was advanced by
1.5 days in the 5001 group. PPS in the Global group was similar to the RM3/RM1 control (Tables 2 and 3
).
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Organ weights.
Female pups from 6 litters from all groups were terminated at PND 26 (the usual time of termination of the immature rat uterotrophic assay), and liver, kidney and sex organ weights were determined. Adjusted body weights, uterine and vaginal weights differed significantly for all the diets compared to the reference RM3/RM1 group. Adjusted cervix weight was also increased for the RM3/AIN-76A group, as was the adjusted ovarian weight for the RM3/AIN-76A group (Tables 2 and 4).
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DISCUSSION |
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Soy proteins (as used in some rodent diets) contain various levels of estrogenic phytoestrogens, of which the major are generally found to be genistein and daidzein (Thigpen et al., 1999). Of the diets studied here, Purina 5001 was found to have the highest genistein and daidzein content: approximately 180 µg and 150 µg/g diet, respectively. These values are somewhat lower than those reported by Thigpen et al. (1999; 214 µg and 277 µg/g diet, respectively), but are similar to those reported by Casanova et al. (2000) for NIH-07 diet (160 µg and 144 µg/g diet, respectively). Variation in reported levels may result from differences in analytical method, of which there are several for genistein and daidzein, and the variation in genistein and daidzein that is found in different batches of soybean meal. Boettger-Tong et al. (1998) reported levels of 210 µg and 140 µg/g genistein and daidzein, respectively, for a particular and nonrepresentative batch (rogue batch) of their standard diet, which they described as "a non-purified diet from a major U.S. manufacturer." According to Boettger-Tong et al. (1998), the phytoestrogens present in their rogue batch of diet led to significant increases in control immature rat uterine weights leading to a dramatic loss of assay sensitivity. Purina 5001, NIH-07, and Boettger-Tong's rogue batch of diet also contained alfalfa, which is a potential source of the phytoestrogen coumestrol (Bickoff et al., 1962, Tinwell et al., 2000), but this was not analytically determined in any of the above evaluations, or in the present study.
All of the diets studied here produced changes in one or another of the developmental landmarks or reproductive tissue weights, relative to the RM3/RM1 control animals, these changes being generally most marked for Purina 5001 (phytoestrogen-rich) and AIN-76A (phytoestrogen-free), and least marked for the Global diet. These changes are shown in Table 2 and are not discussed in detail here, the main point of relevance to emerge being that the choice of rodent diet can affect the sexual development in rats in a way that is not related directly to the phytoestrogen contents of the diets. As an example, the uterotrophic effects elicited by Purina 5001 and AIN-76A (Table 2
) are of a similar magnitude to the uterotrophic effects of weak synthetic estrogens such as nonylphenol in animals maintained on our standard RM/RM1 diet (Odum et al., 1997
). It is of interest that the majority of the effects induced by the AIN-76A/AIN-76A combination were also observed for the RM3/AIN-76A combination, suggesting that the postnatal period was the most sensitive to dietary influences on sexual development. Pup survival in the immediate postnatal period was also reduced with AIN-76A, indicating that this diet is not very suitable for breeding and lactation. The uterotrophic activity of AIN-76A (Table 8
) was reported earlier (Ashby et al., 1999
, 2001
) and is of particular interest given that this formulation contains nondetectable levels of phytoestrogens. Although the effects reported here for AIN-76A in female rats are typical of estrogenic compounds (Ashby et al., 1997
; Goldman et al., 2000
), the advance in PPS seen for both it and Purina 5001 in male rats was unexpected, given that estrogens generally delay PPS (Ashby and Lefevre, 2000
; Stoker et al., 2000
). This difference argues for a different (and perhaps nonestrogenic) mechanism of action for this effect.
In related studies, we have recently found that soy-based infant formulae also produced uterine growth and advanced VO and first estrus in immature female rats (Ashby et al., 2000). The levels of phytoestrogens present in the soy formulae were insufficient to account for these activities, and uterine growth did not occur in ovariectomized rats given soy formulae (Ashby et al., 2000
). However, the estrogen antagonist Faslodex inhibited these effects of the soy formulae, indicating that they were associated with increased exposure to estrogen. Use of a GnRH antagonist abolished these effects of the infant formula, indicating a centrally mediated mechanism of action associated with increased hypothalamic excretion of GnRH leading to premature synthesis of endogenous estrogen in immature rats, itself leading to premature entry of the rats into puberty (Ashby et al., 2000
). A similar mechanism may explain the effects reported here for these several diets. Thus, the uterotrophic effects of AIN-76A and Purina 5001 were abolished by the GnRH antagonist Antarelix, and body weights of the pups receiving AIN-76A or Purina 5001 were consistently heavier than those receiving RM3/RM1 or the Global diets. Further, both male and female sexual development occurred earlier in the AIN-76A and Purina 5001 groups. The reduced testes and epididymides weights in the AIN-76A/AIN-76A group cannot, however, be explained in these terms. Transfer of all of the test groups to RM1 diet (recovery phase) led to general maintenance of the body weight changes recorded for the two AIN-76A groups, the final body weights of the Global and Purina 5001 groups being similar to those of the RM3/RM1 control group.
The dietary component(s) responsible for effects reported here have yet to be established. There may be no single causative factor, because the sexual development of rats fed AIN-76A or Purina 5001 was similar, despite the composition of the diets being markedly different (Table 1). In particular, although centrally mediated increases in pup growth rates are suggested here to be a potentially critical stimulus to advanced sexual development, the metabolizable energies of the different diets, and the amount of diet consumed, do not provide an obvious explanation for the effects reported. Glucose and sucrose have been eliminated as the uterotrophic species in infant formulae and the AIN-76A diet (Ashby et al., 2000
).
Purina 5001 is widely used in the U.S. for both regulatory and research studies, and we therefore reanalyzed the present database using Purina 5001 as the reference diet (Table 9). This reversed the direction of the effects for the other diets, including RM3/RM1. This illustrates that the choice of reference diet is as important as the choice of a special diet for individual studies.
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In conclusion, administration of different diets to rats can affect the timing of both male and female sexual development. Phytoestrogens are not necessarily or wholly responsible for these effects. Although the present data indicate that choice of diet may influence some of the markers of endocrine toxicity, it cannot at this stage be concluded that any of the diets studied are inappropriate for use in endocrine toxicity studies. However, the components of rodent diets should be known, and as far as is possible, controlled. For investigators who do not have an historical database to prejudice by a change in diet, it would seem prudent to select a diet with low phytoestrogen levels. Among those suggested here to be suitable are cereal-based soy-free or low-soy diets, such as Teklad Global 2016, or the cereal-based soy-free version of NIH-07 used by Casanova et al. (2000), or low-soy diets, such as RM1. These diets give low control uterine weights in weanling rats, and this reduces the chance of these diets influencing the outcome of endocrine toxicity studies. However, when selecting a diet containing even small amounts of soybean meal, consideration should be given to the possibility that the use by a manufacturer of soybean meals of varying phytoestrogen content could produce resulting experimental variation.
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ACKNOWLEDGMENTS |
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NOTES |
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