Microbial Metabolism of the Soy Isoflavones Daidzein and Genistein in Postmenopausal Women: Human Intervention Study Reveals New Metabotypes.

BACKGROUND: Soy isoflavones belong to the group of phytoestrogens and are associated with beneficial health effects but are also discussed to have adverse effects. Isoflavones are intensively metabolized by the gut microbiota leading to metabolites with altered estrogenic potency. The population is classified into different isoflavone metabotypes based on individual metabolite profiles. So far, this classification was based on the capacity to metabolize daidzein and did not reflect genistein metabolism. We investigated the microbial metabolite profile of isoflavones considering daidzein and genistein. METHODS: Isoflavones and metabolites were quantified in the urine of postmenopausal women receiving a soy isoflavone extract for 12 weeks. Based on these data, women were clustered in different isoflavone metabotypes. Further, the estrogenic potency of these metabotypes was estimated. RESULTS: Based on the excreted urinary amounts of isoflavones and metabolites, the metabolite profiles could be calculated, resulting in 5 metabotypes applying a hierarchical cluster analysis. The metabotypes differed in part strongly regarding their metabolite profile and their estimated estrogenic potency.

Pyrrolizidine alkaloids in borage (Borago officinalis): Comprehensive profiling and development of a validated LC-MS/MS method for quantification.

Pyrrolizidine alkaloids (PA) from borage (Borago officinalis) consumed as herb and tea, may pose a food safety risk. Therefore, the European Union (EU) set maximum levels of PA in borage, among other foodstuffs, which are applicable since July 1st, 2022. Here, a comprehensive LC-MS/MS based profiling of PA and their N-oxides (PANO) in B. officinalis leaves is presented. Based on these results a targeted, quantitative LC-MS/MS method for the determination of individual PA/PANO present in borage was developed. Chromatographic separation was achieved for all PA/PANO detected in B. officinalis. An easy and fast extraction procedure was developed using a design of experiments approach (DOE). The most efficient extraction was achieved using 0.2% formic acid in 10% methanol at a temperature of 47.5 °C for 60 min. The final method was validated and showed good overall accuracy (recoveries 85-121%) and precision (RDS ≤11%). The method was applied to B. officinalis leave material, demonstrating its suitability for the intended purpose. In these borage samples, the acetylated forms, which are not regulated by EU, were among the quantitatively most relevant PA.

Chemical Stability and Bioactivity of tanshinone I, tanshinone IIA, cryptotanshinone and dihydrotanshinone in in vitro test systems.

Danshen Si Wu is a Traditional Chinese Medicine used for menopausal complains. Beside tanshinone IIA (Tan IIA), Danshen also contains tanshinone I (Tan I), cryptotanshinone (CT) and dihydrotanshinone (DT). The aim of this study was to compare the biological activity of these tanshinones and to determine their cytotoxicity and genotoxicity. Purities and stabilities of the substances were analyzed by LC-DAD and LC-MS analyses. DT and CT concentrations decreased rapidly in dimethylsulfoxide and were converted to Tan I and Tan IIA, respectively. In aqueous solution concentration of all tanshinones decreased after 24 h. Tan I and Tan IIA showed dose-dependent bioactivity mediated by ERα and ERß. No cytotoxic and genotoxic effects for Tan I and Tan IIA were detected. In a yeast transactivation assay Tan I and Tan IIA showed antiandrogenic activity. A significant anabolic activity in C2C12 cells could be detected for Tan I and Tan IIA. In conclusion our data provide evidence that Tan I and Tan IIA are the most relevant bioactive tanshinones in Danshen. Our finding that all tanshinones display a certain instability in aqueous solutions is relevant when discussing their potential therapeutic benefits in humans.

Formation of B- and M-group aflatoxins and precursors by Aspergillus flavus on maize and its implication for food safety.

Aflatoxins count to the most toxic known mycotoxins and are a threat to food safety especially in regions with a warm and humid climate. Contaminated food reaches consumers globally due to international trade, leading to stringent regulatory limits of aflatoxins in food. While the formation of aflatoxin (AF) B1 by the filamentous fungus Aspergillus flavus is well investigated, less is known about the formation kinetics of its precursors and further aflatoxins. In this study, autoclaved maize kernels were inoculated with A. flavus and incubated at 25 °C for up to 10 days. Aflatoxins and precursors were analyzed by a validated UHPLC-MS method. Additional to AFB1 and AFB2, AFM1 and AFM2 were detected, confirming the ability of the formation of M-group aflatoxins on cereals by A. flavus. The measured relative levels of AFB2, AFM1, and AFM2 on maize compared to the level of AFB1 (mean of days 5, 7, and 10 of incubation) were 3.3%, 1.5%, and 0.2%, respectively. The occurrence and kinetics of the measured aflatoxins and their precursors sterigmatocystin, O-methylsterigmatocystin, 11-hydroxy-O-methylsterigmatocystin, aspertoxin, and 11-hydroxyaspertoxin (group 1) as well as of dihydrosterigmatocystin and dihydro-O-methylsterigmatocystin (group 2) supported the so far postulated biosynthetic pathway. Remarkable high levels of O-methylsterigmatocystin and aspertoxin (17.4% and 4.9% compared to AFB1) were found, raising the question about the toxicological relevance of these intermediates. In conclusion, based on the study results, the monitoring of O-methylsterigmatocystin and aspertoxin as well as M-group aflatoxins in food is recommended.

Metabolism of glyphosate by the human fecal microbiota.

Glyphosate is the most frequently used herbicide worldwide and its application is under discussion due to health concerns. As humans may be exposed to glyphosate, the present study investigated the metabolism of glyphosate by the human fecal microbiota in vitro. Human fecal samples were collected from 15 different volunteers and fecal suspensions were prepared. The human fecal suspension samples were incubated with glyphosate under strictly anaerobic conditions and glyphosate degradation was investigated. Neither a degradation of glyphosate, nor a formation of AMPA (aminomethylphosphonic acid), the known microbial metabolite in soil, was detected. In conclusion, the microbiota of human fecal suspensions did not metabolize glyphosate under the conditions used in our study which hints at the assumption that transformation of glyphosate by the gut microbiota seems to be negligible in humans.

Adlercreutzia rubneri sp. nov., a resveratrol-metabolizing bacterium isolated from human faeces and emended description of the genus Adlercreutzia.

The novel, anaerobic, Gram-positive, rod-shaped bacterial strain, ResAG-91T, was isolated from a faecal sample of a male human volunteer. Analysis of the 16S rRNA gene sequence revealed that strain ResAG-91T showed high similarity to the type strains of Adlercreutzia equolifaciens subsp. equolifaciens and Adlercreutzia equolifaciens subsp. celatus. Analysis of the whole draft genome sequences, i.e. digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI), of strain ResAG-91T and the type strains of Adlercreutzia species revealed that strain ResAG-91T represents a novel species of the genus Adlercreutzia. The genome size of strain ResAG-91T is 2.8 Mbp and the G+C content is 63.3 mol%. The major respiratory quinone of strain ResAG-91T was MMK-5 (methylmenaquinone). Major cellular fatty acids were C15 : 0 anteiso, C14 : 0 iso and C14 : 0 2-OH. Galactose and ribose were detected as major whole cell sugars. Furthermore, the peptidoglycan type of strain ResAG-91T was A1γ with meso-diaminopimelic acid. The polar lipids were phosphatidylglycerol, diphosphatidylglycerol, one unidentified lipid, three unidentified phospholipids and five unidentified glycolipids. Strain ResAG-91T was able to metabolize the stilbene resveratrol into dihydroresveratrol. On the basis of this polyphasic approach, including phenotypical, molecular (16S rRNA gene and whole genome sequencing) and biochemical (fatty acids, quinones, polar lipids, peptidoglycan, whole cell sugars, Rapid ID32A and API20A) analyses, we propose the novel species Adlercreutzia rubneri sp. nov. with the type and only strain ResAG-91T (=DSM 111416T=JCM 34176T=LMG 31897T).

DNA reactivity of altertoxin II: Identification of two covalent guanine adducts formed under cell-free conditions.

Fungi of the genus Alternaria infest many agricultural crops and produce numerous mycotoxins, of which altertoxin II (ATX II) is one of the most mutagenic metabolites. ATX II carries an epoxide group but the formation of DNA adducts has not been demonstrated to date. We report now that ATX II gives rise to two covalent adducts with guanine when incubated with DNA under cell-free conditions. These adducts were demonstrated by LC-high resolution MS after enzymatic degradation of the incubated DNA to deoxynucleosides. The major adduct results from the covalent binding of ATX II, presumably through the epoxide group, to guanine, whereas the minor guanine adduct is derived from the major one by the elimination of two equivalents of water. In addition, a third adduct was detected, formed through covalent binding of ATX II to cytosine followed by the loss of two equivalents of water. The direct DNA reactivity of ATX II may explain its high mutagenicity.

Glyphosate and AMPA levels in human urine samples and their correlation with food consumption: results of the cross-sectional KarMeN study in Germany.

Glyphosate (N-[phosphonomethyl]-glycine) is the most widely used herbicide worldwide. Due to health concerns about glyphosate exposure, its continued use is controversially discussed. Biomonitoring is an important tool in safety evaluation and this study aimed to determine exposure to glyphosate and its metabolite AMPA, in association with food consumption data, in participants of the cross-sectional KarMeN study (Germany). Glyphosate and AMPA levels were measured in 24-h urine samples from study participants (n = 301). For safety evaluation, the intake of glyphosate and AMPA was calculated based on urinary concentrations and checked against the EU acceptable daily intake (ADI) value for glyphosate. Urinary excretion of glyphosate and/or AMPA was correlated with food consumption data. 8.3% of the participants (n = 25) exhibited quantifiable concentrations (> 0.2 µg/L) of glyphosate and/or AMPA in their urine. In 66.5% of the samples, neither glyphosate (< 0.05 µg/L) nor AMPA (< 0.09 µg/L) was detected. The remaining subjects (n = 76) showed traces of glyphosate and/or AMPA. The calculated glyphosate and/or AMPA intake was far below the ADI of glyphosate. Significant, positive associations between urinary glyphosate excretion and consumption of pulses, or urinary AMPA excretion and mushroom intake were observed. Despite the widespread use of glyphosate, the exposure of the KarMeN population to glyphosate and AMPA was found to be very low. Based on the current risk assessment of glyphosate by EFSA, such exposure levels are not expected to pose any risk to human health. The detected associations with consuming certain foods are in line with reports on glyphosate and AMPA residues in food.

The Putative Caloric Restriction Mimetic Resveratrol has Moderate Impact on Insulin Sensitivity, Body Composition, and the Metabolome in Mice.

SCOPE: Data on resveratrol-(trans-3,5,4'-trihydroxystilbene)-induced caloric-restriction-(CR)-mimicking effects in mice receiving a high-fat diet (HFD) are contradictory. It is hypothesized that this can possibly stem from different bioactivities of resveratrol (RSV) microbial metabolites. METHODS AND RESULTS: C57BL/6Rj mice are fed an ad-libitum HFD supplemented with RSV or its metabolites, dihydroresveratrol (DHR) and lunularin (LUN) (≈28 mg (dihydro)stilbene kg-1 mouse per day). A 40% CR group was included in the study. While CR mice show robust changes in bodyweight and composition, hormone levels and mRNA expression, slight changes are found (more muscle, less adipose tissue) in body composition, leptin, and insulin levels in RSV-supplemented mice compared to ad libitum controls. LUN hardly and DHR does not change the hormone levels measured. Metabolome analysis of serum shows changes in CR mice but only slight, if any, changes in RSV-, DHR-, or LUN-supplemented mice compared to the controls. Evaluating the capability of RSV and its metabolites to inhibit carbohydrate-hydrolyzing enzymes in vitro, it is found that RSV reduced α-glucosidase activity to a stronger extent than DHR and LUN. CONCLUSION: Decelerated carbohydrate breakdown by RSV may have contributed to the moderate impact of dietary RSV on mouse insulin sensitivity (lowered fasting and post-glucose-bolus insulin levels).

Effects of Soy in Laboratory Rodent Diets on the Basal, Affective, and Cognitive Behavior of C57BL/6 Mice.

Soy is one of the most common sources of protein in many commercial formulas for laboratory rodent diets. Soy contains isoflavones, which are estrogenic. Therefore, soy-containing animal diets might influence estrogen-regulated systems, including basal behavioral domains, as well as affective behavior and cognition. Furthermore, the isoflavone content of soy varies, potentially unpredictably confounding behavioral results. Therefore researchers are increasingly considering completely avoiding dietary soy to circumvent this problem. Several animal studies have investigated the effects of soy free diets but produced inconsistent results. In addition, most of these previous studies were performed in outbred rat or mouse strains. In the current study, we assessed whether a soy-free diet altered locomotion, exploration, nesting, anxiety-related behaviors, learning, and memory in C57BL/6 mice, the most common inbred strain used in biomedical research. The parameters evaluated address measures of basic health, natural behavior, and affective state that also are landmarks for animal welfare. We found minor differences between feeding groups but no indications of altered welfare. We therefore suggest that a soy-free diet can be used as a standard diet to prevent undesirable side effects of isoflavones and to further optimize diet standardization, quality assurance, and ultimately increase the reproducibility of experiments.

Resveratrol, lunularin and dihydroresveratrol do not act as caloric restriction mimetics when administered intraperitoneally in mice.

Resveratrol as well as caloric restriction were shown to extend lifespan in some model organisms and may possibly delay onset of ageing-related diseases in humans. Yet, resveratrol supplementation does not always extend lifespan of animal models or improve health status of humans. Because of interindividual differences in human microbiota, resveratrol metabolite production in the gut differs. While some individuals produce lunularin and dihydroresveratrol in their gut, others produce dihydroresveratrol only. Therefore, we addressed the question whether these metabolites differ in their biological impact on ageing and intraperitoneally injected 13-month-old C57BL/6JRj mice on an ad-libitum (AL) HFD with resveratrol, dihydroresveratrol or lunularin (24 mg/kg bodyweight; 3 times/week). Compared to mice injected with vehicle (AL-control), resveratrol and dihydroresveratrol did not change bodyweight and had no impact on insulin or glucose levels while lunularin slightly reduced feed intake and bodyweight gain. CR-mice showed lowered cholesterol, insulin and leptin levels, elevated adiponectin and phosphorylated AMPK levels in liver as well as increased transcription of Pck1 and Pgc1α when compared to the AL-control. In contrast, injections with the test substances did not change these parameters. We therefore conclude that in our model, resveratrol, lunularin and dihydroresveratrol did not act as CR mimetics.

Anabolic Activity of a Soy Extract and Three Major Isoflavones in C2C12 Myotubes.

Isoflavones have been reported to stimulate muscle growth. The aim of this in vitro study was to examine anabolic activity and associated molecular mechanisms of a soy extract (SoyEx), isoflavone aglycones, and a mixture simulating the composition of SoyEx in C2C12 myotubes. C2C12 cells were differentiated into myotubes. The effects of SoyEx, genistein, daidzein, glycitein, and the mixture of genistein-daidzein-glycitein (Mix) on myotube diameter and number were determined. In addition, the expression of genes and proteins associated with anabolic activity was analyzed. Treatment with SoyEx, genistein, and Mix led to a significant increase of myotube diameter and an increase of the number of myotubes per area compared to the control cell. The increase of diameter by SoyEx was antagonized by an antiestrogen, not by an antiandrogen. Furthermore, gene expressions of insulin growth factor (IGF)-1 and its receptor (IGF-1R), as well as protein expression of myosin heavy chain (MHC), were significantly increased by SoyEx, genistein, and Mix. The effects induced by genistein and Mix were comparable to SoyEx. In conclusion, SoyEx displays an anabolic activity in C2C12 myotubes by binding to ER and modulating IGF-1 and MHC expression. Our studies with isoflavone aglycones and Mix indicate that the isoflavone aglycone with the highest anabolic bioactivity in SoyEx is genistein.

Dietary Resveratrol Does Not Affect Life Span, Body Composition, Stress Response, and Longevity-Related Gene Expression in Drosophila melanogaster.

In this study, we tested the effect of the stilbene resveratrol on life span, body composition, locomotor activity, stress response, and the expression of genes encoding proteins centrally involved in ageing pathways in the model organism Drosophila melanogaster. Male and female w1118 D. melanogaster were fed diets based on sucrose, corn meal, and yeast. Flies either received a control diet or a diet supplemented with 500 µmol/L resveratrol. Dietary resveratrol did not affect mean, median, and maximal life span of male and female flies. Furthermore, body composition remained largely unchanged following the resveratrol supplementation. Locomotor activity, as determined by the climbing index, was not significantly different between control and resveratrol-supplemented flies. Resveratrol-fed flies did not exhibit an improved stress response towards hydrogen peroxide as compared to controls. Resveratrol did not change mRNA steady levels of antioxidant (catalase, glutathione-S-transferase, NADH dehydrogenase, glutathione peroxidase, superoxide dismutase 2) and longevity-related genes, including sirtuin 2, spargel, and I'm Not Dead Yet. Collectively, present data suggest that resveratrol does not affect life span, body composition, locomotor activity, stress response, and longevity-associated gene expression in w1118 D. melanogaster.

Citrinin as an accessory establishment factor of P. expansum for the colonization of apples.

Penicillium expansum is the causal agent of blue mold decay of apples. This fungal species can produce the two important mycotoxins patulin and citrinin. It was previously shown that patulin represents a colonization factor for the infection of apples. No definitive information about the importance of citrinin for the colonization of apples is currently available. The pksCT gene of the citrinin cluster codes for the citrinin polyketide synthase. Mutants of P. expansum in which the pksCT was inactivated showed a drastic decrease in the citrinin production. In addition, the pksCT mutants were also reduced in the ability to colonize apples. Externally added citrinin restored the capacity of the mutants to colonize apples roughly to that of the wild type. A kinetic analysis of the expression of the two respective pks genes of patulin (patK) and citrinin (pksCT) revealed that both genes are highly expressed in the first phase during the colonization process. The production of patulin in the apple matrix coincides with the expression of the patK gene. Almost no citrinin could be identified analytically during the first phase but only at a later stage of the colonization. It could be demonstrated that citrinin is degraded in apples and can tightly be bound to pectin. Overall the results suggest that citrinin may have an accessory function for the establishment of the colonization guided by other factors.

An isoflavone enriched diet increases skeletal muscle adaptation in response to physical activity in ovariectomized rats.

SCOPE: This study was to investigate anabolic adaptation of skeletal muscle in response to an isoflavone (ISO) enriched diet, training and their combinations in ovariectomized (OVX) rats. METHODS AND RESULTS: Female Wistar rats were sedentary, performed treadmill uphill running, received ISOs, or a combination of ISOs and running after ovariectomy. Body weight was increased by OVX. Both ISO and training treatment antagonized this increase. The weights of soleus and gastrocnemius muscles were increased only when training and ISOs were combined. In soleus muscle insulin-like growth factor (IGF)-1R, MyoD and Myogenin expressions were only up-regulated by training in Sham groups. However, a stimulation of IGF-1R and MyoD expression could be observed when ISOs and training were combined. In gastrocnemius muscle MyoD and Myogenin expressions were stimulated by either training or ISOs. Additive effects were detected when combining the two interventions. CONCLUSION: Our results indicate that the combination of ISOs and exercise is more efficient in increasing relative skeletal muscle mass and the expression of molecular markers related to anabolic adaptation in the skeletal muscle of female rats.

[Isoflavone-containing dietary supplements]. / Isoflavonhaltige Nahrungsergänzungsmittel.

Isoflavones (IFs) from soy and other legumes have weak estrogenic properties. Isolated IFs are available as dietary supplements and advertised to alleviate symptoms of menopause. The present chapter provides an overview of the occurrence, the chemical structure of IFs and their metabolites, the market situation and reviews the current evidence on the efficacy and safety of IF-containing dietary supplements.The biological effectiveness of IFs is attributable to the activation of the estrogen receptor (ER). Studies on the influence of IFs on endogenous estrogen levels in women show inconsistent results. So far, the European Food Safety Authority (EFSA) has rejected all submitted health claims for IFs due to insufficient scientific evidence for any of the postulated health effects. Based on the results of their recent risk assessment, the EFSA concluded that the available human studies did not support the hypothesis of adverse effects of isolated IFs on the human mammary gland, uterus or thyroid in healthy postmenopausal women. However, the assessment does not allow a general statement on the safety of IF-containing dietary supplements. Studies in animal models are often not comparable with the complex interactions in humans due to differences in the metabolism of IFs, in the developmental stage at time of consumption and in the temporarily restricted uptake of IFs during certain stages of life. CONCLUSION: So far, for none of the advertised functions is unequivocal scientific evidence available. On the basis of available data, potential unwanted side effects cannot be fully excluded. This holds particularly true for women with undiagnosed diseases, especially for those with undetected precancerous lesions in the mammary gland.

Influence of testosterone on phase II metabolism and availability of soy isoflavones in male Wistar rats.

Genistein and daidzein are the main isoflavones in soy. Their potential beneficial or adverse effects in males like the prevention of prostate cancer or the impact on reproductive functions are controversially discussed. Major determinants of their bioactivity are the absorption and biotransformation of isoflavones. In this study, we focused on the influence of testosterone on plasma availability and phase II metabolism of isoflavones. Male Wistar rats, receiving an isoflavones rich diet, were randomized into three groups: Two groups were orchiectomized (ORX) at postnatal day (PND) 80 and treated for 11 days with testosterone propionate (TP) (ORX TP group) or a vehicle (ORX group) after a 7 days lasting hormonal decline. The third group served as control and remained intact. Rats were sacrificed at PND 98. ORX rats had reduced isoflavones plasma levels. Differently regulated mRNA expressions of transporters relevant for transport of phase II metabolites in liver and kidney may be responsible for this reduction, more precisely Slc10a1 and Slc21a1 in kidney as well as Slc22a8 in liver. While main phase II metabolites in intact rats were disulfates and sulfoglucuronides, the amount of sulfate conjugates was significantly diminished by ORX. In accordance with that, mRNA expression of different sulfotransferases was reduced in liver by ORX. The observed effects could be almost restored by TP treatment. In conclusion, testosterone, and likely further androgens, has a huge impact on phase II metabolism and availability of isoflavones by influencing the expression of different sulfotransferases and transporters.

Sulfoglucosides as Novel Modified Forms of the Mycotoxins Alternariol and Alternariol Monomethyl Ether.

The mycotoxins alternariol and alternariol-9-O-methyl ether have recently been reported to be extensively conjugated with glucose and malonyl glucose in tobacco suspension cells. However, only trace amounts of glucosylated conjugates were detected in tomatoes inoculated with Alternaria alternata in the present study. Instead, mostly sulfate conjugates were observed. In studies using cultures of A. alternata and incubations of alternariol and alternariol-9-O-methyl ether with tomato tissue in the absence of the fungus, it was clarified that sulfate conjugates were produced by the fungus, whereas tomato tissues converted alternariol and alternariol-9-O-methyl ether to glucosylated metabolites. Alternariol-3-sulfate, alternariol-9-sulfate, and alternariol-9-O-methyl ether-3-sulfate were unambiguously identified as fungal metabolites using MS and 1H and 13C NMR spectroscopy. When these sulfate conjugates were incubated with tobacco suspension cells or ex planta tomato tissues, three sulfoglucosides of alternariol and one sulfoglucoside of alternariol-9-O-methyl ether were formed. Using NMR spectroscopy, the chemical structures of alternariol-3-sulfate-9-glucoside, alternariol-9-sulfate-3-glucoside, and alternariol-9-O-methyl ether-3-sulfate-7-glucoside were established. These conjugates were also detected in the A. alternata-inoculated tomato. This is the first report on a mixed sulfate/glucoside diconjugate of a mycotoxin. Diconjugates of this novel type may be formed by all mycotoxins and their phase I metabolites with two or more hydroxyl groups and should be taken into account in the future analysis of modified mycotoxins.

Neonatal isoflavone exposure interferes with the reproductive system of female Wistar rats.

There is increasing concern about possible adverse effects of soy based infant formulas (SBIF) due to their high amount of isoflavones (ISO). The aim of the present study was to investigate effects of neonatal exposure to ISO on reproductive system of female Wistar rats. Animals were exposed to an ISO depleted diet or a diet enriched with an ISO extract (IRD; 508mg ISO/kg) during embryogenesis and adolescence. Pups of each group were fed daily by pipette with ISO-suspension (ISO+; 32mg ISO/kg bw) or placebo from postnatal day (PND) 1 until PND23 resulting in plasma concentrations similar to levels reported in infants fed SBIF. The visceral fat mass was reduced by long-term IRD. Vaginal epithelial height was increased at PND23 and vaginal opening was precocious in ISO+ groups. Later in life, more often irregular estrus cycles were observed in rats of ISO+ groups. In addition, FSH levels and uterine epithelial heights were increased at PND80 in ISO+ groups. In summary, the results indicate that neonatal ISO intake, resulting in plasma concentrations achievable through SBIF, has an estrogenic effect on prepubertal rats and influences female reproductive tract later in life.