Cumulative Dietary Energy Intake Determines the Onset of Puberty in Female Rats
Odum, Jenny, Tinwell, Helen, Tobin, Graham, Ashby, John, Environmental Health Perspectives
Laboratory animal diets for studies to determine the endocrine-disrupting potential of chemicals are under scrutiny because they can affect both assay control values and assay sensitivity. Although phytoestrogen content is important, we have previously shown that a phytoestrogen-rich diet and a phytoestrogen-free diet were equally uterotrophic to rats and advanced vaginal opening (VO) when compared with the standard diet RM1. Abolition of the effects by the gonadotrophin-releasing hormone antagonist Antarelix indicated that these effects were mediated through the hypothalamus--pituitary-reproductive organ axis. In the present study, we investigated the relationship between cumulative energy intake and sexual maturation in female rats. Infant formula (IF) at different concentrations and synthetic diets, with a wide range of metabolizable energy (ME) values, were used to modulate energy intake. Increasing energy intake was associated with an increase in uterine weight (absolute and adjusted for body weight) for both IF and the synthetic diets. In both cases, the increased uterine weight was directly proportional to energy intake. Body weight was unaffected by IF consumption but, in the case of the diets, was increased proportionally with energy consumption. Antarelix abolished the uterine weight increases with both formula and the diets, whereas body weight was unaffected. The mean day of VO was also advanced by high-ME diets and IF, whereas body weight at VO was unaffected. VO occurred at an energy intake of approximately 2,300 kJ/rat determined by measuring total food intake from weaning to VO, indicating that this cumulative energy intake was the trigger for puberty. ME is therefore a critical factor in the choice of diets for endocrine disruption studies. Key words: energy intake, metabolizable energy, phytoestrogens, puberty, soy, uterotrophic assay. Environ Health Perspect 112:1472-1480 (2004). doi:10.1289/ehp.7039 available via http://dx.doi.org/[Online 21 July 20041
The choice of laboratory animal diet for rodent studies to determine the endocrine-disrupting potential of chemicals is currently under intense scrutiny (Lawton 2003; Odum et al. 2001; Owens et al. 2003; Owens and Koeter 2003; Thigpen et al. 2003). This is because the diet selected can affect both assay control values and assay sensitivity; for example, uterine weight in control animals needs to be low to maximize the dynamic range of the uterotrophic assay. One contributing factor is the phytoestrogen content of the diet. Most of the commonly used laboratory animal diets are formulated with soy extracts, which contain the isoflavones genistein (GEN) and daidzein, and/or alfalfa (lucerne), which contains coumestrol (Patisaul and Whitten 1999). These phytoestrogens are estrogenic to rodents, causing effects such as increased uterine weight and advanced vaginal opening (VO) in immature animals, similar to effects observed with xenobiotic estrogens (Bickoff et al. 1962; Boettger-Tong et al. 1998; Casanova et al. 1999; Medlock et al. 1995; Thigpen et al, 1999; Tinwell et al. 2000; Whitten et al. 1992).
An analysis conducted as part of the recent Organisation for Economic Co-operation and Development (OECD) evaluation of the immature rat uterotrophic assay indicated that isoflavone levels greater than 325-350 mg GEN equivalents/kg diet should be avoided to maintain optimal assay sensitivity and dynamic range (Owens et al. 2003). The phytoestrogen content of diets is not, however, the only factor of importance. This is shown by our earlier demonstration that the phytoestrogen-rich diet Purina 5001 (Purina Mills, Inc., Richmond, IN, USA) and the phytoestrogen-free diet AIN-76A are equally uterotrophic to rodents, compared with the standard diet RM1, and that each is able to advance the mean day of VO in rats, again compared with RM1 (Odum et al. 2001). Further, we showed that coadministration of the gonadotrophin-releasing hormone (GnRH) antagonist Antarelix (ANT; Europeptides, Argenteuil, France) abolished the uterotrophic activity of both diets, indicating that these effects were mediated at the level of the hypothalamus to influence GnRH secretion (Odum et al. …