Impact of the Phytoestrogen Content of Laboratory Animal Feed on the Gene Expression Profile of the Reproductive System in the Immature Female Rat

Abstract

The effect of the dietary background of phytoestrogens on the outcome of rodent bioassays used to identify and assess the reproductive hazard of endocrine-disrupting chemicals is controversial. Phytoestrogens, including genistein, daidzein, and coumestrol, are fairly abundant in soybeans and alfalfa, common ingredients of laboratory animal diets. These compounds are weak agonists for the estrogen receptor (ER) and, when administered at sufficient doses, elicit an estrogenic response in vivo. In this study, we assessed the potential estrogenic effects of dietary phytoestrogens at the gene expression level, together with traditional biologic end points, using estrogen-responsive tissues of the immature female rat. We compared the gene expression profile of the uterus and ovaries, as a pool, obtained using a uterotrophic assay protocol, from intact prepubertal rats fed a casein-based diet (free from soy and alfalfa) or a regular rodent diet (Purina 5001) containing soy and alfalfa. Estrogenic potency of the phytoestrogen-containing diet was determined by analyzing uterine wet weight gain, luminal epithelial cell height, and gene expression profile in the uterus and ovaries. These were compared with the same parameters evaluated in animals exposed to a low dose of a potent ER agonist [0.1 μg/kg/day 17α-ethynyl estradiol (EE) for 4 days]. Exposure to dietary phytoestrogens or to a low dose of EE did not advance vaginal opening, increase uterine wet weight, or increase luminal epithelial cell height in animals fed either diet. Although there are genes whose expression differs in animals fed the soy/alfalfa-based diet versus the casein diet, those genes are not associated with estrogenic stimulation. The expression of genes well known to be estrogen regulated, such as progesterone receptor, intestinal calcium-binding protein, and complement component 3, is not affected by consumption of the soy/alfalfa-based diet when assessed by microarray or quantitative reverse transcriptase–polymerase chain reaction analysis. Our results indicate that although diet composition has an impact on gene expression in uterus and ovaries, it does not contribute to the effects of an ER agonist.

Nutritional flavonoids impact on nuclear and extranuclear estrogen receptor activities

“…Meanwhile, the genes related to specific differentiation of the cell, such as those for neuronal proteins, were down regulated (Teresaka et al, 2004). Cluster analysis performed in the same cell line indicated that very similar profiles were obtained with 10 μM genistein (Ramanathan and Gray, 2003;Naciff et al, 2004;Teresaka et al, 2004). Naciff and coworkers (2002) sustaining that the gene expression changes are more sensitive than the classical uterotrophic assay for evaluating estrogenicity applied cluster analysis to the effects of genistein on the uterus of immature rats.…”

“…In 1997 the Organization for Economic Cooperation and Development (OECD) initiated a high priority activity to develop new and revised guidelines for screening and testing the estrogenicity of potential endocrine disrupters (OECD, 1998). One of the validated screening was the uterotrophic bioassay, which demonstrated the uterine growth of pre-pubertal or ovariectomized rats by following E2 or genistein treatment (i.e., gavage or intraperitoneal injection) (Owens et al, 2003;Naciff et al, 2004;Ashby and Odum, 2004).…”

“…Naciff and coworkers (2002) sustaining that the gene expression changes are more sensitive than the classical uterotrophic assay for evaluating estrogenicity applied cluster analysis to the effects of genistein on the uterus of immature rats. 227 genes were affected by genistein, the majority of which were down-regulated (Naciff et al 2004). Although genistein increased the expression of the progesterone receptor (an estrogenic effect), most of the remainder of its apparent effects in the developing uterus were not estrogenic.…”

Xenopus laevis is a potential alternative model animal species to study reproductive toxicity of phytoestrogens

“…Simultaneously, some authors reported that there are no significant effects of phytoestrogens in rodents (Roberts et al, 2000;Mitchell et al, 2001;Lamartiniere et al, 2002). One of the explanations for these inconsistent results is that commercial rodent diets all contain high and variable levels of abundant phytoestrogens, which could affect results of studies on reproductive toxicity of test phytoestrogens (Odum et al, 2001;Stroheker et al, 2003;Naciff et al, 2004;Thigpen et al, 2004). In this study, X. laevis tadpoles were fed Daphnia and Artemia, and frogs were fed pork liver.…”

“…However, many studies involving endocrine disruptors (including phytoestrogens) with rats lack reproducibility and also show inter-laboratory inconsistencies. In addition to the complexity of the endocrine system, some researchers think that different sensitivities of model animals and variable levels of phytoestrogen in diets should be responsible for the variable results (Everitt and Foster, 2004;Naciff et al, 2004;Thigpen et al, 2004). Stokes suggests that it is critical to select appropriate animal models and diets for endocrine disruptor studies (Stokes, 2004).…”