Up to date, the involvement of dietary PE such as genistein and daidzein in human estrogen-sensitive organs’ development has not been conclusive. Within a coordinated research concept we plan to investigate the influence of dietary PE exposure during critical periods of development. Therefore the impact of PE on the development of the male and female reproductive and hormone sensitive tissues, metabolism and epigenetic modulations will be studied in animal models simulating different exposure scenarios and suitable biomarkers will be identified. In addition molecular mechanisms of PE activity during development will be studied in adapted cell models. To transfer the mechanistically findings on the human situation, serum and blood cells from children consuming soy food (both in China and Germany) will be analyzed with the aim to discover universal biomarkers for dietary PE risk assessment and elucidate the differences of reactions to dietary PE in European and Chinese population groups.
Within the coordinated research concept we investigated the influence of dietary Phytoestrogen exposure (PE) during critical periods of development. The impact of PE (isoflavones, ISO) on the development of the male and female reproductive and hormone sensitive tissues, metabolism and epigenetic modulations was studied in animal models simulating different exposure scenarios. Two breeding experiments with Wistar rats were conducted. Additionally ISO biotransformation was studied in a study with children on a soy diet. In male rats a dose response experiment was performed starting exposure to low and high doses (Asian exposure scenario) of ISO in utero, maintaining it through adolescence into adulthood. Body composition of intact male rats was not affected by ISO but serum triglycerides and hepatic fatty acid synthase expression were decreased by high dose ISO. Interestingly bone mineral density was reduced in intact rats on low dose ISO but increased by ISO high. ISO increased androgen sensitivity of seminal vesicle and levator ani, but not of prostate. ISO high dose exposure resulted in significantly reduced mean testosterone levels compared to IDD group. ISO plasma level between intact and castrated male rats differed considerably. In castrated animals the sulfate conjugates were diminished and ISO plasma concentrations were reduced compared to intact male rats. The observed effects could be almost restored by testosterone propionate treatment supporting the conclusion that testosterone has a huge impact on availability and phase II metabolism of ISO. To study the effect of neonatal ISO exposure, Rats were exposed to ISO free diets and diets containing ISO during embryogenesis and/or adolescence. An increased vaginal epithelial height at postnatal day (PND) 23 and a premature vaginal opening were observed in rats treated with ISO during neonatal period. More often irregular estrus cycles were observed later in life in these groups. Further, follicle stimulating hormone levels were increased at PND 80 in rats treated with ISO during neonatal period. ISO intake resulted in ISO plasma levels which are achievable by dietary intake of high amounts of soy or soy based infant nutrition in humans. DNA methylation was quantitatively assessed in mammary glands, uteri and prostate tissue. Lifelong dietary exposure affected more sites than the additional neonatal ISO exposure. mRNA expression of various epithelial and stromal cell type markers, estrogen receptors, the proliferation marker Ki67 and epigenetic enzymes in mammary gland tissue at PNDs 23, 50 and 80 was analysed. Changes in mRNA expression between the diet groups appeared to be stronger than the detected methylation changes. We investigated also the influence of age and endogenous sex hormones on the phase II metabolite profile of the most abundant soy ISO daidzein and genistein in biofluids and tissues. ISO metabolite profiles in plasma were similar in male and female pups (mainly monoglucuronides, followed by sulfoglucuronides) but changed substantially during ageing. A marked change was observed between PND 23 and PND 50, suggesting that puberty and the associated change in hormonal balance are the reason for this observation. This was confirmed by the observation that ISO metabolite pattern as well as overall ISO plasma level between intact and castrated male rats differed considerably. In the intervention study with children affected with heterozygous familial hypercholesterolemia a decrease in Low Density Lipoprotein Cholesterol (LDL-C) by a soy-protein enriched diet was observed. The reduction in LDL-C correlated with both urine and plasma ISO but not with self-reported soy intake. The ISO phase II metabolite profile measured in the urine of the children was comparable with those in female and male adults. In summary the results of our studies provide clear evidence that dietary phytoestrogen exposure during critical periods of development (especially neonatal), in doses relevant to human exposure scenarios resulted in reproductive, metabolic and transcriptional alterations and weak epigenetic effects. ISO metabolism and serum concentrations were strongly effected by sex steroids. Our epigenetic findings indicate that alternative molecular mechanisms (hormonal activity, signal transduction, effects on gonadotropins, steroid and thyroid hormones, changes in body and tissue composition, and other epigenetic mechanisms) might be stronger determinants of ISO activity in the neonatal and pubertal rat than DNA methylation.