Variations in phytoestrogen content between different mill dates of the same diet produces significant differences in the time of vaginal opening in CD-1 mice and F344 rats but not in CD Sprague-Dawley rats

Abstract

Background: The optimum test diet and rodent species/strain for evaluating endocrine-disrupting compounds (EDCs) are critical.

Objectives: We conducted studies to evaluate rodent species sensitivity and the effects of diets varying in phytoestrogen content on the time of vaginal opening (VO) in CD-1 mice, Fischer 344 (F344) rats, and CD Sprague-Dawley (S-D) rats.

Methods: Mice were weaned on postnatal day (PND) 15 and rats on PND19 and randomly assigned to control or test diets. Body weights, food consumption, and time of VO were recorded.

Results: The time of VO was significantly advanced in F344 rats fed diets containing daidzein and genistein, whereas these same diets did not advance VO in S-D rats. When animals were fed the AIN-76A diet spiked with genistein, time of VO was significantly advanced at all doses in CD-1 mice, at the two highest doses in F344 rats, and at the highest dose in S-D rats. The time of VO in F344 rats was more highly correlated with the phytoestrogen content than with the total metabolizable energy (ME) of 12 diets.

Conclusions: The S-D rat is less sensitive to dietary phytoestrogens compared with the F344 rat or the CD-1 mouse, suggesting that the S-D rat is not the ideal model for evaluating estrogenic activity of EDCs. The profound effects of dietary phytoestrogens on the time of VO, an estrogen-sensitive marker, indicate that a standardized open-formula phytoestrogen-free diet containing a low ME level should be used to optimize the sensitivity of estrogenic bioassays.

Keywords: dietary phytoestrogens; endocrine disruptors; rodent species/strain sensitivities in VO end points.

Figure 1

The effect of batch-to batch variation in the total D&G content in different mill dates (MD) of the same PMI 5002 diet on the timing of VO in F344 rats.

Figure 2

The effect of batch-to batch variation in the total D&G content in different mill dates (MD) of the same PMI 5002 diet on the timing of VO in S-D rats.

Figure 3

Effect of batch-to batch variation in total D&G content in different mill dates of the same PMI 5002 diet on the concentration of total isoflavones (mean ± SE) in the plasma of S-D rats.

Figure 4

Effect of AIN-76A diet spiked with 0, 150, 300, or 450 μg genistein/g diet on the time of VO in CD-1 mice.

Figure 5

Mean concentration of genistein (± SE) in the plasma of CD-1 mice, F344 rats, and S-D rats fed the AIN-76A diet spiked with 0, 150, 300, and 450 μg genistein/g diet.

Figure 6

Effect of AIN-76A diet spiked with 0, 150, 300, or 450 μg genistein/g diet on the time of VO in F344 rats.

Figure 7

Effect of AIN-76A diet spiked with 0, 150, 300 or 450 μg genistein/g diet on the time of VO in S-D rats.

Figure 8

Effect of AIN-76A diet spiked with 0, 150, 300, or 450 μg genistein/g diet on the body weights of F344 rats versus S-D rats at weaning and at the time of VO.

Figure 9

Comparison of VO times between S-D rats and F344 rats fed diets containing different concentrations of D&G or genistein. (A) Time of VO (mean ± SE) in S-D rats and F344 rats fed the control diet (5K96) or three different batches of the PMI 5002 natural ingredient diet containing different concentrations of D&G (< 7, 98, 223, and 431 μg/g diet) with similar ME levels. (B) Time of VO in S-D rats and F344 rats fed purified diets with high ME levels spiked with genistein at 0 (control), 150, 300, or 450 μg/g diet. *Significantly different at p < 0.05.

Figure 10

Future dietary considerations. Currently most experimental animals are fed at least two different diets containing variable levels of phytoestrogens and ME. Diet(s) should contain higher levels of ME for gestation and growth and lower levels of ME for VO and uterotrophic assays. Animal vendors should give serious consideration to providing animals that have been maintained on diets free of D&G for studies that can be affected by dietary estrogens.