Effect of Rodent Diets on the Sexual Development of the Rat

J. Odum*, H. Tinwell*, K. Jones*, J. P. Van Miller{dagger}, R. L. Joiner{ddagger}, G. Tobin§, H. Kawasaki, R. Deghenghi|| and J. Ashby*,1

* Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, SK10 4TJ, United Kingdom; {dagger} Union Carbide Corporation, Danbury, Connecticut 06817; {ddagger} General Electric Company, Pittsfield, Massachusetts 01201; § Harlan Teklad U.K., Bicester, Oxfordshire, United Kingdom; Japanese Chemical Industries Association, Sumitomo Chemical Co., Ltd., 27–1, 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Five rodent diets have been evaluated for their possible effect on the sexual development of the rat. Groups of 12 pregnant Alpk rats were fed one of the following combinations of diets during pregnancy and postnatally: RM3/RM1, AIN-76A/AIN-76A, RM3/AIN-76A, Teklad Global 2016 (Global)/Global and Purina 5001/Purina 5001. AIN-76A is phytoestrogen-free while the other diets contained varying amounts of phytoestrogens. The phytoestrogens genistein and daidzein were determined in the diets studied, and the concentrations found agreed with earlier estimates. RM3/RM1 was selected as the control group, as this has been used routinely in this laboratory for the past decade. Determinations were made in offspring of the times of vaginal opening and first estrus among the females, and of prepuce separation and testes descent among the males. At postnatal day (PND) 26 the females from 6 of the 12 litters were terminated and tissue weights measured. Males from 6 of the 12 litters were similarly studied at PND 68. Animals from the remaining litters were transferred to RM1 diet at PND 70. Termination of the study was at PND 128 (males) and PND 140 (females) when body weights and tissue weights were determined.

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..


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The development of standardized protocols to test for hormonally active compounds is a major goal of regulatory agencies worldwide. A strategy for testing for effects on male and female sexual maturation and thyroid activity, in accordance with EDSTAC (Endocrine Disrupter Screening and Testing Advisory Committee) recommendations has recently been published (Goldman et al., 2000Go; Stoker et al., 2000Go). Interpretation of results from such tests requires the acquisition of background/baseline data and an understanding of factors that may cause variation of these baselines. One of these factors is the phytoestrogen content and the energy of diet administered to the rodents. Most laboratory animal diets are formulated with constituents that contain phytoestrogens (plant derived estrogenic compounds); for example, soy extract containing the isoflavones genistein and daidzein, or alfalfa, which contains coumestrol (Patisaul and Whitten, 1999Go). These phytoestrogens are estrogenic to rodents, causing effects such as increased uterine weight and advanced vaginal opening in immature animals, similar to effects observed with xenobiotic estrogens (Tinwell et al., 2000; Bickoff et al., 1962Go; Casanova et al., 1999Go; Medlock et al., 1995Go; Whitten et al., 1992Go).

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., 1997Go).

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., 1999Go). 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., 1999Go). Similar uterotrophic activity was observed for Purina 5001 (Ashby et al., 2001Go), 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., 2000Go). 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. 1Go). 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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FIG. 1. Design of the sexual maturation study.

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Alpk:APfSD (Wistar derived) rats, obtained from the AstraZeneca breeding unit (Alderley Park), were used in both studies. Animal studies were performed in accordance with the U.K. "Animals (Scientific Procedures) Act." Animal care and procedures were carried out according to in-house standards as described previously (Odum et al., 1999Go).

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 70–300°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 1Go. Sixty pregnant female rats (10–12 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. 1Go). 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 52–69 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. 1Go). The males were killed at PND 128 and the females at PND 140–144, 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 (20–21 days old on arrival) as described previously (Odum et al., 1997Go). 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 (21–22 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., 1997Go).

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, 1996Go). 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Diet Analysis
RM1, RM3, Global, and 5001 are "closed formulae" (i.e., their precise components are known only to the manufacturer) natural ingredient diets. AIN-76A is an "open formula" (i.e., its exact specification is known) synthetic ingredient diet (Knapka, 1983Go). The manufacturers describe RM1, RM3, Global, and 5001 as suitable for maintenance of rodents. AIN-76A was devised by the American Institute of Nutrition (AIN) in 1973 and has been widely used for many years (Reeves et al., 1993Go). The ingredients of the five diets are shown in Table 1Go. With the exception of AIN-76A, all of the diets contain cereals as their primary ingredient. In contrast, AIN-76A contains, principally, sucrose and casein. The reported protein contents of RM3 and 5001 are greater than Global and RM1, as the former are described as being suitable for breeding where a higher protein content may be necessary. The calculated metabolizable energy values, which reflect the amount of energy available to the animals on consumption of the diets, are shown in Table 1Go. The values were similar in the natural ingredient diets (lowest, RM1; highest, Global) but that of AIN-76A was substantially higher, reflecting its high digestibility.


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TABLE 1 Ingredients, Protein Content, Metabolizable Energy, and Phytoestrogen Sources of the Diets Used in This Study
 
RM1, RM3 and 5001 contain soybean meal, which is a rich source of phytoestrogens, particularly isoflavones. Purina 5001 also contains alfalfa, which is a source of coumestrol, another phytoestrogen (Table 1Go). We have attempted to estimate the levels of soy and alfalfa where the manufacturer has not stated these levels. The isoflavone content of the diets (genistein and daidzein, determined by GC-MS) is shown in Figure 2Go. The quantities of genistein and daidzein correspond to the amounts of soybean meal in the diets: 5001 had the highest levels, followed by RM3 and RM1, while AIN-76A and Global had barely detectable levels.



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FIG. 2. Analysis of the diets for genistein and diadzein (by GC-MS, see Materials and Methods). Analyses were conducted on different batches, and on more than one occasion, with similar results. The data here represent one such set of results.

 
Sexual Maturation Study
Data analysis.
To compare effects observed with the different diets, the RM3/RM1 combination was taken as the control (reference) diet, because this combination has been used in most of our previous studies (Odum et al., 1997Go, 1999Go; Tinwell et al., 2000). RM3 is a diet suitable for breeding and lactation of rodents, and RM1 is more suitable for general maintenance. Statistically significant differences from the RM3/RM1 reference are described below and summarized in Table 2Go. Further analyses was carried out using 5001/5001 as the reference diet, because this is used in most regulatory and research studies in the U.S.


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TABLE 2 Statistically Significant Changes for Parameters When RM3/RM1 Is Taken as the Control Group
 
Body weights and food consumption.
All animals were pregnant and all gave birth normally. There were no convincing differences in the body weights of the pregnant dams given the different diets up to birth (some intermittent differences were seen but these were not sustained). After birth, during the lactational period, dams receiving AIN-76A or Global had significantly reduced body weights (Fig. 3Go). Food consumption in these groups over this time period was also marginally reduced (data not shown), perhaps reflecting their higher metabolizable energy density.



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FIG. 3. Body weights of pregnant and lactating dams up to pup weaning. Gestational day (GD) 0 = the day sperm were found in the vaginal smear; birth (GD 22) = PND 0. The first diet in each line of the key was used throughout gestation and up to weaning (PND 21). The second diet in each line of the key was used after weaning, and therefore was not used in the period shown in this figure. Data are ANCOVA-adjusted means; n = 12 for all groups up to birth, but by PND 21, had been reduced to 9, 11, and 11 in the AIN-76A/AIN-76A, RM3/AIN-76A, and Global/Global groups, due to litter losses. *Statistically significant changes when compared with RM3/RM1 as control group, p < 0.05.

 
Pup survival at birth was similar for all groups, but over the postnatal period from birth to PND 21, there were slightly more litter losses in the AIN-76A group: 9/12 litters were surviving at PND 21 compared with 11/12 or 12/12 litters in the other groups. Body weights for male and female pups receiving AIN-76A were reduced at birth and up to PND 13, but the body weights had recovered to control (RM3/RM1) values by PND 21 (Figs. 4 and 5GoGo). After weaning, both groups receiving AIN-76A (AIN-76A/AIN-76A and RM3/AIN-76A) maintained significantly increased body weights compared with the control group (RM3/RM1). Male and female pups receiving Global diet had similar body weights at birth compared to the control (RM3/RM1) group, but were significantly reduced by PND 21. After weaning, by PND 36, both sexes had similar weights to the RM3/RM1 control group, and this was maintained throughout the study (Figs. 4 and 5GoGo). Male and female pups receiving 5001 diet had similar body weights to the control (RM3/RM1) group throughout the pre-weaning period, but from PND 28 onwards, body weights increased and were always heavier than the control group; however, the increase was only statistically significant up to PND 56 (Figs. 4 and 5GoGo).



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FIG. 4. (A) Body weights of male pups from birth (PND 0) to weaning (PND 21); (B) body weights of male pups from weaning to PND 126. The first diet in each line of the key was used throughout gestation and up to weaning (PND 21). The second diet in each line of the key was used after weaning. All groups were given RM1 from PND 70. Data are ANCOVA-adjusted means; n = 12 litters for all groups at birth but by PND 21, there were 11, 9, 11, 11, and 12 litters for RM3/RM1, AIN/AIN, RM3/AIN, Global/Global, and 5001/5001 groups, respectively. Statistical significance is not shown (for clarity) but is described in Results. The RM3/RM1 group is regarded as the control group.

 


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FIG. 5. (A) Body weights of female pups from birth (PND 0) to weaning (PND 21); (B) body weights of female pups from weaning to PND 126. The first diet in each line of the key was used throughout gestation and up to weaning time (PND 21); the second diet in each line was used after weaning. All groups were given RM1 from PND 70. Data are ANCOVA-adjusted means. (A): n = 12 litters for all groups at birth but by PND 21 there were 9, 11, and 11 litters for AIN-76A/AIN-76A, RM3/AIN-76A, and Global/Global groups, respectively; (B): n = 6 litters for all groups except AIN-76A/AIN-76A where n = 5. Statistical significance is not shown (for clarity) but is described in Results. The RM3/RM1 group is regarded as the control group.

 
Food consumption for male and female pups in the post-weaning period is shown in Figure 6Go. Consumption of AIN-76A diet in the first few weeks of the study was significantly lower than control (RM3/RM1) diet animals; but by 8 weeks of age it was similar to control levels. Consumption of Global and 5001 diets was generally significantly greater than controls. At PND 70 (Recovery Phase, during which all animals were fed RM1) feeding patterns altered. Fluctuations occurred initially in both sexes, but afterwards, all female groups started to consume similar amounts within 2 weeks, while all male groups did not achieve this equality. Males from the AIN-76A groups, who originally consumed lower quantities of diet than the RM3/RM1 control group, consumed significantly more RM1 than the control group after the diet changeover.



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FIG. 6. Food consumption (post-weaning) for male and female pups. The first diet in each line of the key was used throughout gestation and up to weaning; the second diet in each line was used after weaning. All groups were given RM1 from PND 70. Data are means, n as shown in Figures 4 and 5GoGo. Statistical significance is not shown (for clarity) but is described in Results. The RM3/RM1 group is regarded as the control group.

 
Developmental landmarks.
The age at which all pups started and completed eye opening was determined as a general marker of development, but there were no differences between any of the diet groups. Eye opening (defined as full opening of the lid) started on PND 13 and was essentially complete by PND 17 in male and female pups from all groups (data not shown).

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 3GoGo). 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 3GoGo).


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TABLE 3 Developmental Landmark Results for Male and Female Pups
 
The ages at onset and completion of VO followed by day of first estrus were determined. Body weights at completion of VO were also determined, although in later studies we have used body weights at onset of VO as a preferred parameter (Ashby et al., 2000b). Onset of VO was accelerated by 2–3 days in both AIN-76A groups, compared to RM3/RM1, while body weight at VO was either unchanged or reduced compared to the RM3/RM1 control group. First estrus occurred earlier in the RM3/AIN-76A group (Tables 2 and 3GoGo). VO and first estrus were also advanced in the 5001 group, although the former was not statistically significant when using the litter as the experimental unit. The VO data for 5001 have previously been presented using the individual as the experimental unit, and in that case the advance in the mean day of VO was statistically significant (Ashby, 2000Go). Age at VO and first estrus in the Global group was unaffected (Tables 2 and 3GoGo). In all groups, a low proportion of the animals failed to complete VO, as evidenced by the retention of vaginal threads. Body weights at completion of VO, and the day of first estrus, were therefore not obtained for these animals that were excluded from all the female developmental landmark data in Table 2Go, but they were assessed with the other animals at termination. The total number of animals with vaginal threads was as follows: RM1/RM3, 17; AIN-76A/AIN-76A, 8; RM3/AIN-76A, 11; global, 11; and 5001, 12. The percentage of days spent in estrus during the estrus cycle was determined on all animals but was not different in any of the groups (data not shown).

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 4GoGo).


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TABLE 4 Organ Weights for Females Terminated at PND 26
 
Male pups from 6 litters from each group were terminated at PND 68. Liver, kidney, and male sex organ weights were determined. The most marked differences were seen for the AIN-76A/AIN-76A group and no changes were seen for the global group (Tables 2 and 5GoGo). The adjusted weights of the testes and epididymides were decreased in the AIN-76A/AIN-76A group.


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TABLE 5 Organ Weights for Males Terminated at PND 68
 
At PND 70 (recovery phase) all groups were placed on RM1 to determine whether the body and organ weight changes observed at PND 68 would be maintained or reversed. The remaining males were terminated at PND ~128, after ~8 weeks after the diet change to RM1. Adjusted body weights for AIN-76A/AIN-76A and RM3/AIN-76A were still significantly increased relative to the control RM3/RM1 group. Adjusted testes weights were still significantly decreased in the AIN-76A/AIN-76A group and adjusted kidney weights remained significantly elevated in the 5001/5001 group (Tables 2 and 6GoGo).


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TABLE 6 Organ Weights for Males Terminated at PND 128
 
The remaining females were terminated at PND ~140, 10 weeks after the diet change to RM1. Adjusted body weights for AIN-76A/AIN-76A and RM3/AIN-76A remained elevated relative to the control RM3/RM1 group. Adjusted kidney weights were increased in these two groups, but there were no other notable changes in any of the other groups compared to the RM3/RM1 control group (Tables 2 and 7GoGo).


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TABLE 7 Organ Weights for Females Terminated at PND 140
 
Uterotrophic Assay
Rats receiving AIN-76A or 5001 had significantly heavier terminal body weights than those receiving RM1 or Global. Food consumption for diets other than RM1 was increased (by 24–40%) relative to the RM1 + AO (vehicle only) control group. Absolute and adjusted uterine weights were significantly increased in animals receiving AIN-76A or 5001 and vehicle only compared to the RM1 + AO control group. After Antarelix treatment no uterotrophic effects were seen for any of the diets, uterine weights generally being lower than the RM1 + AO control group (Table 8Go).


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TABLE 8 Uterotrophic Assay for Weanling Rats Administered Different Diets in the Presence or Absence of Antarelix
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Uncertainties regarding the optimum diet for use in rodent endocrine disruption studies, and whether the phytoestrogen contents of diets should influence this decision (Ashby et al., 1999Go; Thigpen et al., 1999), led to the present study.

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., 1962Go, 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 2Go 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 2Go) 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., 1997Go). 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 8Go) was reported earlier (Ashby et al., 1999Go, 2001Go) 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., 1997Go; Goldman et al., 2000Go), 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, 2000Go; Stoker et al., 2000Go). 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., 2000Go). 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., 2000Go). 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., 2000Go). 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 1Go). 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., 2000Go).

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 9Go). 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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TABLE 9 Statistically Significant Changes for Parameters, When 5001/5001 Is Taken As the Control Group
 
In our studies with infant formulae, we observed a correlation between the quantity of formula consumed by immature rats and mice and the magnitude of the resultant uterotrophic effect (Ashby et al., 2000Go). That finding suggests that the amount of an endocrine-active diet (e.g., AIN-76A and Purina 5001) consumed by rodents may influence the timing of puberty. In some toxicity studies, palatability problems lead to the test animals consuming less diet than do the matched controls. Further, a general requirement for an adequate toxicity study is that the test animals should weigh less at the end of the study than do the matched control animals. Both of these situations may affect the timing of puberty of the test animals, with the chance of weak endocrine toxicity effects being inappropriately concluded for the test agent. In such situations it would be valuable to have access to food-intake data for the test and control animals, but this is often not available. In order to evaluate this speculative source of uncertainty, it would be necessary to conduct a rodent sexual maturation study that included a food-restricted group of animals and a group with enhanced food intake (via the use of a flavoring agent). Such a study, which will also monitor the potential involvement of insulin and leptin and changes in body fat, is currently being planned.

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.


    ACKNOWLEDGMENTS
 
We thank S. Kirk for the statistical analyses and Dr. M. Morton (Bioclinical Services, Ltd, Cardiff, U.K.) for the isoflavone analysis. We are grateful to the Alkyl Phenol Ethoxylate (APE) Research Council, the Endocrine Working Group of the Japanese Chemical Industries Association, and the Food Standards Agency (U.K.) for financial contributions to the conduct of this study.


    NOTES
 
1 To whom correspondence should be addressed. Fax: +44 0 1625 590249. E-mail: john.ashby{at}syngenta.com. Back


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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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