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The present study assessed the potential of a generic physiologically based kinetic (PBK) model to convert in vitro data for estrogenicity to predict the in vivo uterotrophic response in rats for diethylstibestrol (DES), ethinylestradiol (EE2), genistein (GEN), coumestrol (COU), and methoxychlor (MXC). PBK models were developed using a generic approach and in vitro concentration-response data from the MCF-7 proliferation assay and the yeast estrogen screening assay were translated into in vivo dose-response data. Benchmark dose analysis was performed on the predicted data and available in vivo uterotrophic data to evaluate the model predictions. The results reveal that the developed generic PBK model adequate defines the in vivo kinetics of the estrogens. The predicted dose-response data of DES, EE2, GEN, COU, and MXC matched the reported in vivo uterus weight response in a qualitative way, whereas the quantitative comparison was somewhat hampered by the variability in both in vitro and in vivo data. From a safety perspective, the predictions based on the MCF-7 proliferation assay would best guarantee a safe point of departure for further risk assessment although it may be conservative. The current study indicates the feasibility of using a combination of in vitro toxicity data and a generic PBK model to predict the relative in vivo uterotrophic response for estrogenic chemicals., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Despite various hypothesized benefits of dietary isoflavone genistein (GEN) from soy-based products, many questions surrounding GEN's immunotoxic effects, especially during perinatal exposure, have yet to be answered. The objective of the study was to determine if there existed a sex-specific effect of GEN on type 1 diabetes (T1D) following perinatal exposure. We exposed offspring of non-obese diabetic (NOD) mice to GEN per oral at a physiological dose (20 mg/kg body weight) from embryonic day 7 to postnatal day (PND) 21. In female offspring, perinatal GEN dosing significantly increased the incidence of T1D at early time points, and the exacerbation was associated with decreased serum levels of interleukin (IL)-10, IgG2a, and IgM. In male offspring dosed with GEN, a decrease in serum IgG1 was also observed. Flow cytometric analysis in females suggested an increased pro-inflammatory splenic CD5+CD24- and CD4-CD8+ cell counts, while both %T cells and %CD4+ T cells were significantly decreased in males, suggesting an anti-inflammatory effect. Gut microbiota (GMB) analysis indicated that fecal microbiota from PND 90 female offspring exhibited an increased level of Enterobacteriales (suggesting a pro-inflammatory response), while the similar changes were not found in PND 30 females. Moreover, RNA sequencing showed that intestinal α-defensin expression was down-regulated in GEN-treated females, supporting a pro-inflammatory response. However, perinatal GEN administration perturbed GMB toward an anti-inflammatory response in PND 90 males. Taken together, a strong sex-specific effect was found in the perinatal GEN exposure window, and the T1D exacerbation in NOD females was associated with GMB-related immunomodulatory mechanisms.

Genistein (Gen), the primary isoflavone in soy, has been shown to adversely affect various endocrine-mediated endpoints in rodents and humans. Soy formula intake by human infants has been associated with early age at menarche and decreased female-typical behavior in girls. Adipose deposition and expansion are also hormonally regulated and Gen has been shown to alter these processes. However, little is known about the impact of early-life soy intake on metabolic homeostasis in adulthood. The current study examined the impact of early-life Gen exposure on adulthood body composition (by magnetic resonance imaging) and the molecular signals mediating adipose expansion. From postnatal day (PND) 1 to 22, rat pups were daily orally dosed with 50mg/kg Gen to mimic blood Gen levels in human infants fed soy formula. Female but not male Gen-exposed rats had increased fat/lean mass ratio, fat mass, adipocyte size and number, and decreased muscle fiber perimeter. PND22 Gen-exposed females, but not males, had increased expression of adipogenic factors, including CCAAT/enhancer binding protein alpha (Cebpα), CCAAT/enhancer binding protein beta (Cebpβ), and peroxisome proliferator-activated receptor gamma (Pparγ). Furthermore, Wingless-related MMTV integration site 10b (Wnt10b), a critical regulator of adipogenic cell fate determination, was hypermethylated and had decreased expression in adipose of PND22 Gen-exposed females. These data suggest that developmental Gen exposure in rats has gender-specific effects on adiposity that closely parallel the effects of a postweaning high-fat diet and underscore the importance of considering timing of exposure and gender when establishing safety recommendations for early-life dietary Gen intake.

Endocrine-disrupting chemicals (EDC) are abundant in our environment. A number of EDCs, including bisphenol A (BPA) can bind to the estrogen receptors (ER), ERα and ERβ, and may contribute to estrogen-linked diseases such as breast cancer. Early exposure is of particular concern; many EDCs cross the placenta and infants have measurable levels of, eg, BPA. In addition, infants are frequently fed soy-based formula (SF) that contains phytoestrogens. Effects of combined exposure to xeno- and phytoestrogens are poorly studied. Here, we extensively compared to what extent BPA, genistein, and an extract of infant SF mimic estrogen-induced gene transcription and cell proliferation. We investigated ligand-specific effects on ER activation in HeLa-ERα and ERβ reporter cells; on proliferation, genome-wide gene regulation and non-ER-mediated effects in MCF7 breast cancer cells; and how coexposure influenced these effects. The biological relevance was explored using enrichment analyses of differentially regulated genes and clustering with clinical breast cancer profiles. We demonstrate that coexposure to BPA and genistein, or SF, results in increased functional and transcriptional estrogenic effects. Using statistical modeling, we determine that BPA and phytoestrogens act in an additive manner. The proliferative and transcriptional effects of the tested compounds mimic those of 17β-estradiol, and are abolished by cotreatment with an ER antagonist. Gene expression profiles induced by each compound clustered with poor prognosis breast cancer, indicating that exposure may adversely affect breast cancer prognosis. This study accentuates that coexposure to BPA and soy-based phytoestrogens results in additive estrogenic effects, and may contribute to estrogen-linked diseases, including breast cancer.

Flavonoids are potent antioxidants, freely available as high-dose dietary supplements. However, they can induce DNA double-strand breaks (DSB) and rearrangements in the mixed-lineage leukemia (MLL) gene, which are frequently observed in childhood leukemia. We hypothesize that a deficient DSB repair, as a result of an Atm mutation, may reinforce the clastogenic effect of dietary flavonoids and increase the frequency of Mll rearrangements. Therefore, we examined the effects of in vitro and transplacental exposure to high, but biological amounts of flavonoids in mice with different genetic capacities for DSB repair (homozygous/heterozygous knock-in for human Atm mutation [Atm-ΔSRI] vs. wild type [wt]). In vitro exposure to genistein/quercetin induced higher numbers of Mll rearrangements in bone marrow cells of Atm-ΔSRI mutant mice compared with wt mice. Subsequently, heterozygous Atm-ΔSRI mice were placed on either a flavonoid-poor or a genistein-enriched (270 mg/kg) or quercetin-enriched (302 mg/kg) feed throughout pregnancy. Prenatal exposure to flavonoids associated with higher frequencies of Mll rearrangements and a slight increase in the incidence of malignancies in DNA repair-deficient mice. These data suggest that prenatal exposure to both genistein and quercetin supplements could increase the risk on Mll rearrangements especially in the presence of compromised DNA repair.

In scientific literature, several estrogenic compounds are reported to induce responses in vitro that are significantly higher than that of estradiol (E2). These supramaximal (SPMX) estrogenic effects do not occur consistently and seem to differ depending on the cellular models applied. This study analyzes the possible underlying causes, mechanisms, and drivers for SPMX estrogenic effects in in vitro functional assays reported in the peer-reviewed literature. For the 21 natural and industrial chemicals identified as SPMX inducers, the culture and exposure conditions varied greatly among and between the assays. Detailed information on assay characteristics, however, sometimes lacked. Diethylstilbestrol, genistein, and bisphenol A were selected to build a database. The meta-analysis revealed that the occurrence of SPMX effects could be related to a number of specific assay characteristics: (1) the type of serum used to supplement the exposure medium, (2) the end point used to quantify the estrogenic potency (endogenous or transfected), (3) the number of estrogen response elements, and (4) and the promoter's nature. An SPMX response was not reported for expression of endogenous genes, assays that used African green monkey kidney (COS-1) cell line or with chloramphenicol transferase as the reporter gene. There were no indications that solvent concentration in culture, exposure period, or cell model influenced the occurrence of an SPMX effect. It is important to understand the mechanism behind this phenomenon because in vitro assays for estrogenicity are used extensively to characterize and quantify the estrogenic potency of compounds, mixtures and environmental extracts.

The potential teratogenic effects and fetal toxicity of environmental estrogenic endocrine disruptors have become a great concern in recent years, and they have yet to be fully characterized. In the present study, the teratogenic effects of bisphenol A (BPA) and genistein (GEN) on rat embryos during their critical period of organogenesis were investigated using a whole-embryo culture experiment. The combined exposure effects of BPA and GEN were explored using a 4 x 4 full factorial design. Both BPA and GEN produced concentration-dependent inhibition of embryonic development, beginning at 32.0 and 10.0 microg/ml, respectively. Full factorial and isobologram analyses revealed a significant synergistic interaction between BPA and GEN for most end points (12 out of 20 tested), as indicated by the enhanced developmental toxicity of BPA after coexposure with different dose levels of GEN. In particular, serious malformations and a higher abnormal frequency of the central nervous system were induced by the combination of BPA and GEN. Our findings suggest that GEN may be embryotoxic and teratogenic to humans. BPA alone may not be a potential teratogen, but these two estrogenic chemicals have a synergistic effect on embryonic development when present together during the critical period of major organ formation. The current findings suggest that pregnant women should not take soy supplements, but more studies are necessary to provide a conclusive recommendation.

The purpose of this study was to determine (1) the transcriptional program elicited by exposure to three estrogen receptor (ER) agonists: 17 alpha-ethynyl estradiol (EE), genistein (Ges), and bisphenol A (BPA) during fetal development of the rat testis and epididymis; and (2) whether very low dosages of estrogens (evaluated over five orders of magnitude of dosage) produce unexpected changes in gene expression (i.e., a non-monotonic dose-response curve). In three independently conducted experiments, Sprague-Dawley rats were dosed (sc) with 0.001-10 microg EE/kg/day, 0.001-100 mg Ges/kg/day, or 0.002-400 mg BPA/kg/day. While morphological changes in the developing reproductive system were not observed, the gene expression profile of target tissues were modified in a dose-responsive manner. Independent dose-response analyses of the three studies identified 59 genes that are significantly modified by EE, 23 genes by Ges, and 15 genes by BPA (out of 8740), by at least 1.5 fold (up- or down-regulated). Even more genes were observed to be significantly changed when only the high dose is compared with all lower doses: 141, 46, and 67 genes, respectively. Global analyses aimed at detecting genes consistently modified by all of the chemicals identified 50 genes whose expression changed in the same direction across the three chemicals. The dose-response curve for gene expression changes was monotonic for each chemical, with both the number of genes significantly changed and the magnitude of change, for each gene, decreasing with decreasing dose. Using the available annotation of the gene expression changes induced by ER-agonist, our data suggest that a variety of cellular pathways are affected by estrogen exposure. These results indicate that gene expression data are diagnostic of mode of action and, if they are evaluated in the context of traditional toxicological end-points, can be used to elucidate dose-response characteristics.

Distinction between dose addition and response addition for the analysis of the toxicity of mixtures may allow differentiation of the components regarding similar versus independent mode of action. For nonlinear dose responses for the components, curves of dose addition and response addition differ and embrace an "envelope of additivity." Synergistic or antagonistic interaction may then be postulated only if the mixture effect is outside this surface. This situation was analyzed for the induction of micronuclei in L5178Y mouse lymphoma cells by the two methylating agents methyl methanesulfonate (MMS) and N-methyl-N-nitrosourea (MNU) and the topoisomerase-II inhibitor genistein (GEN). All three chemicals reproducibly generated sublinear (upward convex) dose-response relationships. For the analysis of mixture effects, these genotoxic agents were investigated in the three binary combinations. Statistical testing for dose addition along parallel exponential dose responses was performed by linear regression with interaction based on the logarithm of the number of cells that contain micronuclei. For MMS+MNU, the mixture effect was compatible with dose addition (i.e., significantly larger than calculated for the addition of net responses). For MMS+GEN, the measured effect was larger than for response addition but smaller than for dose addition. For MNU+GEN, the measured effect was below response addition, indicative of true antagonism. In the absence of knowledge on the sublinear dose-response relationships for the individual components, a synergistic effect of MMS on both MNU and GEN would have been postulated erroneously. The observed difference between MMS and MNU when combined with GEN would not have been predicted on the basis of a simplistic interpretation of DNA methylation as the mode of action and may be due to differences in the profile of DNA methylations and/or epigenetic effects. We conclude that knowledge of nonlinearities of the dose-response curves of individual components of a mixture can be crucial to analyze for synergism or antagonism and that an in-depth mechanistic knowledge is useful for a prediction of similarity or independence of action.

Genistein is a plant-derived estrogenic isoflavone commonly found in soy-based products such as soymilk and soy-based dietary supplements for treating menopausal symptoms, for example. Vasopressin is a neurosecretory nonapeptide synthesized primarily in neurons of the hypothalamus and secreted into the bloodstream from the posterior lobe of the pituitary. The endogenous opiate peptide beta-endorphin is synthesized both in neurons of the hypothalamus and in pituitary cells, primarily of the neurointermediate lobe. It has been reported that exposure to 17beta-estradiol or diethylstilbesterol increased the vasopressin content of the hypothalamus, and that estradiol valerate selectively damages hypothalamic beta-endorphin-containing neurons. Since little was known of the potential effects of estrogenic endocrine-disruptor compounds on hypothalamic neuropeptides, we fed Sprague-Dawley fetuses from day 7 in utero until sacrifice at postnatal day 77, with either a control diet (<1 ppm) or an experimental diet containing 25, 250, or 1250 ppm of genistein. We then conducted ELISA assays for hypothalamic content of both beta-endorphin and vasopressin immunoreactivity. Whereas there were no statistically reliable effects of dietary genistein on hypothalamic beta-endorphin content, vasopressin levels were significantly elevated in the 1250-ppm genistein group (p < 0.05). Elevated vasopressin levels may be associated with fluid balance, altered blood pressure, and cardiovascular effects. These data are consistent with the known actions of estradiol and may serve to explain our finding in a previous study that estrogenic endocrine-disruptors such as genistein increased sodium preference in rats exposed through their diet.

The present studies report the effects on neonatal rats of oral exposure to genistein during the period from birth to postnatal day (PND) 21 to generate data for use in assessing human risk following oral ingestion of genistein. Failure to demonstrate significant exposure of the newborn pups via the mothers milk led us to subcutaneously inject genistein into the pups over the period PND 1-7, followed by daily gavage dosing to PND 21. The targeted doses throughout were 4 mg/kg/day genistein (equivalent to the average exposure of infants to total isoflavones in soy milk) and a dose 10 times higher than this (40 mg/kg genistein). The dose used during the injection phase of the experiment was based on plasma determinations of genistein and its major metabolites. Diethylstilbestrol (DES) at 10 micro g/kg was used as a positive control agent for assessment of changes in the sexually dimorphic nucleus of the preoptic area (SDN-POA). Administration of 40 mg/kg genistein increased uterus weights at day 22, advanced the mean day of vaginal opening, and induced permanent estrus in the developing female pups. Progesterone concentrations were also decreased in the mature females. There were no effects in females dosed with 4 mg/kg genistein, the predicted exposure level for infants drinking soy-based infant formulas. There were no consistent effects on male offspring at either dose level of genistein. Although genistein is estrogenic at 40 mg/kg/day, as illustrated by the effects described above, this dose does not have the same repercussions as DES in terms of the organizational effects on the SDN-POA.

The estrogenic isoflavone genistein is a common dietary component that has been shown to affect reproductive development in experimental animals at high doses. The objective of the present study was to examine interactions of genistein and the hormonally active pesticide methoxychlor on mammary gland development in juvenile rats. Timed-pregnant Sprague-Dawley rats were fed a soy- and alfalfa-free diet containing different combinations of genistein (300 and 800 ppm) and methoxychlor (800 ppm). Rats were fed these diets starting on gestation day (GD)1 and continuing through pregnancy and lactation until postnatal day (PND) 22, when the pups were killed. Inguinal mammary glands from both female and male pups were processed as whole-mount preparations for morphometric analysis. The total glandular area and the numbers of branch points, lateral buds, and terminal end buds in the male rats were found to be significantly greater in the groups exposed to methoxychlor than those exposed to genistein only. These effects were not observed in the female rats. In the male rats, methoxychlor had the most prominent effect on elongating the glandular ducts, while genistein enhanced the ductile branching. The 2 compounds in combination promoted the development of alveolar-lobular structure, an effect not observed with either compound alone. Immunostaining for proliferating cell nuclear antigen revealed a high percentage of immunopositive cells in the mammary epithelia of the males exposed to methoxychlor and genistein (800 ppm) compared to the controls. While no significant changes in serum levels of mammotrophic hormones were detected, increased immunostaining for insulin-like growth factor-1 receptor, estrogen receptor alpha, and progesterone receptor in the genistein + methoxychlor group suggested that local factors involved in regulating mammary growth may have played a role in propagating the endocrine effects of these two compounds. These results indicated that the mammary glands of juvenile male rather than juvenile female rats may be more sensitive to certain endocrine-active compounds and that high levels of phytoestrogens have the potential to alter the toxicological behaviors of other hormone mimics.

Humans and wildlife are frequently exposed to mixtures of endocrine active-compounds (EAC). The objective of the present study was to investigate the potential of the phytoestrogen genistein to influence the reproductive developmental toxicity of the endocrine-active pesticide methoxychlor. Three levels of genistein (0, 300, or 800 ppm) and two levels of methoxychlor (0 or 800 ppm) were used in this study. Sprague-Dawley rats were exposed to the two compounds, either alone or in combinations, through dietary administration to dams during pregnancy and lactation and to the offspring directly after weaning. Both compounds, methoxychlor in particular, were associated with reduced body growth at 800 ppm, but pregnancy outcome was not affected by either treatment. An acceleration of vaginal opening (VO) in the exposed female offspring was the only observed effect of genistein at 300 ppm. Exposure to 800 ppm genistein or 800 ppm methoxychlor caused accelerated VO and also altered estrous cyclicity toward persistent estrus in the female offspring. The estrogenic responses to genistein and methoxychlor administered together were apparently accumulative of the effects associated with each compound alone. Methoxychlor, but not genistein, delayed preputial separation (PPS) in the male rats. When administered with methoxychlor, genistein at 800 ppm enhanced the effect of methoxychlor on delaying PPS. Genistein and methoxychlor treatment did not change gender-specific motor activity patterns in either sex. To explore possible mechanisms for interaction between the two compounds on development, we performed estrogen receptor (ER)- and androgen receptor (AR)-based in vitro transcriptional activation assays using genistein and the primary methoxychlor metabolite 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE). While the in vitro assays supported the estrogenic effects of genistein and methoxychlor and the antiandrogenic effects of methoxychlor, the reactivity of these compounds with ERs alpha and beta could not predict the greater in vivo estrogenic potency of methoxychlor over genistein; nor could the potentiation of the methoxychlor effect on PPS by genistein be predicted based on in vitro HPTE and genistein reactions with the AR. Data from this study indicate that phytoestrogens are capable of altering the toxicological behaviors of other EACs, and the interactions of these compounds may involve complexities that are difficult to predict based on their in vitro steroid receptor reactivities.

Estrogenic isoflavones, such as genistein and daidzein, are present in virtually all natural-ingredient rodent diets that use soy as a source of protein. Since these compounds are endocrine-active, it is important to determine whether the amounts present in rodent diets are sufficient to affect sexual development. The present study consisted of in vitro and in vivo parts. In the in vitro portion, human hepatoma cells were transfected with either rat estrogen receptor (ER) alpha or beta plus an estrogen-responsive luciferase reporter gene. Genistein and daidzein were complete agonists at both ERs, genistein being more potent than daidzein, and both compounds were more potent at ER beta than ER alpha. In combined studies with estradiol, genistein exerted additive effects with estradiol in vitro. In the in vivo portion of the study, groups of six pregnant Sprague-Dawley females were fed one of the following four diets, and the pups were maintained on the same diets until puberty: (1) a natural-ingredient, open-formula rodent diet (NIH-07) containing 16 mg genistein and 14 mg daidzein per 100 g of feed; (2) a soy- and alfalfa-free diet (SAFD) in which casein and corn oil were substituted for soy and alfalfa meal and soy oil, respectively, that contained no detectable isoflavones; (3) SAFD containing 0.02% genistein (GE.02); or (4) SAFD containing 0.1% genistein (GE.1). In the GE.1 group, effects of dietary genistein included a decreased rate of body-weight gain, a markedly increased (2.3-fold) uterine/body weight (U/BW) ratio on postnatal day (pnd) 21, a significant acceleration of puberty among females, and a marginal decrease in the ventral prostate weight on postnatal day (pnd) 56. However, developmental differences among the groups fed SAFD, GE.02, or NIH-07 were small and suggested minimal effects of phytoestrogens at normal dietary levels. In particular, on pnd 21, the U/BW ratio of the GE.02 and NIH-07 groups did not differ significantly from that of the SAFD group. Only one statistically significant difference was detected between groups fed SAFD and NIH-07: the anogenital distance (AGD) of female neonates on pnd 1 whose dams were fed NIH-07 was 12% larger than that of neonates whose dams were fed SAFD. The results suggest that normal amounts of phytoestrogens in natural-ingredient rodent diets may affect one developmental parameter, the female AGD, and that higher doses can affect several other parameters in both males and females. Based on these findings, we do not suggest replacing soy- and alfalfa-based rodent diets with phytoestrogen-free diets in most developmental toxicology studies. However, phytoestrogen-free diets are recommended for endocrine toxicology studies at low doses, to determine whether interactive effects may occur between dietary phytoestrogens and man-made chemicals.