Controversy on health-based guidance values for bisphenol A-the need of criteria for studies that serve as a basis for risk assessment.

Since 2006, the responsible regulatory bodies have proposed five health-based guidance values (HBGV) for bisphenol A (BPA) that differ by a factor of 250,000. This range of HBGVs covers a considerable part of the range from highly toxic to relatively non-toxic substances. As such heterogeneity of regulatory opinions is a challenge not only for scientific risk assessment but also for all stakeholders, the Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG) analyzed the reasons for the current discrepancy and used this example to suggest improvements for the process of HBGV recommendations. A key aspect for deriving a HBGV is the selection of appropriate studies that allow the identification of a point of departure (PoD) for risk assessment. In the case of BPA, the HBGV derived in the 2023 EFSA assessment was based on a study that reported an increase of Th17 cells in mice with a benchmark dose lower bound (BMDL40) of 0.53 µg/kg bw/day. However, this study does not comply with several criteria that are important for scientific risk assessment: (1) the selected end-point, Th17 cell frequency in the spleen of mice, is insufficiently understood with respect to health outcomes. (2) It is unclear, by which mechanism BPA may cause an increase in Th17 cell frequency. (3) It is unknown, if an increase of Th17 cell frequency in rodents is comparably observed in humans. (4) Toxicokinetics were not addressed. (5) Neither the raw data nor the experimental protocols are available. A further particularly important criterion (6) is independent data confirmation which is not available in the present case. Previous studies using other readouts did not observe immune-related adverse effects such as inflammation, even at doses orders of magnitude higher than in the Th17 cell-based study. The SKLM not only provides here key criteria for the use of such studies, but also suggests that the use of such a "checklist" requires a careful and comprehensive scientific judgement of each item. It is concluded that the Th17 cell-based study data do not represent an adequate basis for risk assessment of BPA.

Bosea rubneri sp. nov. Isolated from Organically Grown Allium cepa.

Strain ZW T0_25T was isolated from an onion sample (Allium cepa var. Hytech F1) within a storage trial and proofed to be a novel, aerobic, Gram-stain negative, rod-shaped bacterial strain. Analyses of the 16S rRNA gene sequence and of the whole draft genome sequences, i.e., digital DNA-DNA hybridization (dDDH), Average Nucleotide Identity (ANI) and Average Amino Acid Identity (AAI) showed that this strain represents a new species of the genus Bosea. The genome size of strain ZW T0_25T is 6.19 Mbp, and the GC content is 66.9%. As whole cell sugars, rhamnose, ribose and glucose were identified. Ubiquinone Q-10 is the major respiratory quinone with 97.8%. Polar lipids in strain ZW T0_25T are composed of one phosphatidylethanolamine, one phosphatidylglycerol, one aminophospholipid, two aminolipids, one glycolipid and two phospholipids whereas the fatty acid profile predominantly consists of C18:1 w7c (63.3%), C16:1 w7c (19.5%) and C16:0 (7.1%). Phenotypic traits were tested in the wet lab as well as predicted in silico from genome data. Therefore, according to this polyphasic approach, the new name Bosea rubneri sp. nov. with the type strain ZW T0_25T (= DSM 116094 T = LMG 33093 T) is proposed.

Rathayibacter rubneri sp. nov. isolated from Allium cepa var. Rijnsburger, an onion landrace.

The novel, aerobic, Gram-stain-positive, rod-shaped bacterial strain, ZW T2_19T, was isolated from an onion sample (Allium cepa var. Rijnsburger). Analyses of the 16S rRNA gene sequence revealed that ZW T2_19T represented a member of the genus Rathayibacter but may represent a novel species of this genus. Analyses of the whole draft genome sequences, i.e. digital DNA-DNA hybridisation (dDDH) and average nucleotide identity (ANI) of ZW T2_19T and all type strains of species of the genus Rathayibacter confirmed that ZW T2_19T represents a novel species of the genus Rathayibacter. The genome size of ZW T2_19T is 4.01 Mbp and the DNA G+C content is 71.8 mol%. Glucose, mannose, rhamnose and ribose were detected as whole-cell sugars of ZW T2_19T. The major respiratory quinone of ZW T2_19T is menaquinone MK-10, at 78.9 %. The detected peptidoglycan type in ZW T2_19T is a variant of type B2γ with {Gly} [l-diaminobutyric acid (l-DAB)/l-homoserine (l-Hse)] d-Glu-l-DAB. Polar lipids in ZW T2_19T consisted of one diphosphatidylglycerol, one phosphatidylglycerol, seven glycolipids, one phospholipid and one lipid. The fatty acid profile of ZW T2_19T predominantly consisted of anteiso-C15 : 0 (53 %), iso-C16 : 0 (21 %) and anteiso-C17 : 0 (18 %). In addition, API 20NE, API 50CH, API Coryne, API ZYM, antibiotic susceptibility, haemolysis and growth at different temperatures and with different supplements was investigated. On the basis of the results obtained using this polyphasic approach, including molecular, phenotypic and biochemical analyses, we propose the novel species Rathayibacter rubneri with the type and only strain ZW T2_19T (= DSM 114294T = LMG 32700T).

Genome analysis reveals that the correct name of type strain Adlercreutzia caecicola DSM 22242T is Parvibacter caecicola Clavel et al. 2013.

The strain Adlercreutzia caecicola DSM 22242T (=CCUG 57646T=NR06T) was taxonomically described in 2013 and named as Parvibacter caecicola Clavel et al. 2013. In 2018, the name of the strain DSM 22242T was changed to Adlercreutzia caecicola (Clavel et al. 2013) Nouioui et al. 2018 due to taxonomic investigations of the closely related genera Adlercreutzia, Asaccharobacter and Enterorhabdus within the phylum Actinobacteria. However, the first whole draft genome of strain DSM 22242T was published by our group in 2019. Therefore, the genome was not available within the study of Nouioui et al. (2018). The results of the polyphasic approach within this study, including phenotypic and biochemical analyses and genome-based taxonomic investigations [genome-wide average nucleotide identity (gANI), alignment fraction (AF), average amino acid identity (AAI), percentage of orthologous conserved proteins (POCP) and genome blast distance phylogeny (GBDP) tree], indicated that the proposed change of the name Parvibacter caecicola to Adlercreutzia caecicola was not correct. Therefore, it is proposed that the correct name of Adlercreutzia caecicola (Clavel et al. 2013) Nouioui et al. 2018 strain DSM 22242T is Parvibacter caecicola Clavel et al. 2013.

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).

Contribution to the ongoing discussion on fluoride toxicity.

Since the addition of fluoride to drinking water in the 1940s, there have been frequent and sometimes heated discussions regarding its benefits and risks. In a recently published review, we addressed the question if current exposure levels in Europe represent a risk to human health. This review was discussed in an editorial asking why we did not calculate benchmark doses (BMD) of fluoride neurotoxicity for humans. Here, we address the question, why it is problematic to calculate BMDs based on the currently available data. Briefly, the conclusions of the available studies are not homogeneous, reporting negative as well as positive results; moreover, the positive studies lack control of confounding factors such as the influence of well-known neurotoxicants. We also discuss the limitations of several further epidemiological studies that did not meet the inclusion criteria of our review. Finally, it is important to not only focus on epidemiological studies. Rather, risk analysis should consider all available data, including epidemiological, animal, as well as in vitro studies. Despite remaining uncertainties, the totality of evidence does not support the notion that fluoride should be considered a human developmental neurotoxicant at current exposure levels in European countries.

Aspects of high hydrostatic pressure food processing: Perspectives on technology and food safety.

The last two decades saw a steady increase of high hydrostatic pressure (HHP) used for treatment of foods. Although the science of biomaterials exposed to high pressure started more than a century ago, there still seem to be a number of unanswered questions regarding safety of foods processed using HHP. This review gives an overview on historical development and fundamental aspects of HHP, as well as on potential risks associated with HHP food applications based on available literature. Beside the combination of pressure and temperature, as major factors impacting inactivation of vegetative bacterial cells, bacterial endospores, viruses, and parasites, factors, such as food matrix, water content, presence of dissolved substances, and pH value, also have significant influence on their inactivation by pressure. As a result, pressure treatment of foods should be considered for specific food groups and in accordance with their specific chemical and physical properties. The pressure necessary for inactivation of viruses is in many instances slightly lower than that for vegetative bacterial cells; however, data for food relevant human virus types are missing due to the lack of methods for determining their infectivity. Parasites can be inactivated by comparatively lower pressure than vegetative bacterial cells. The degrees to which chemical reactions progress under pressure treatments are different to those of conventional thermal processes, for example, HHP leads to lower amounts of acrylamide and furan. Additionally, the formation of new unknown or unexpected substances has not yet been observed. To date, no safety-relevant chemical changes have been described for foods treated by HHP. Based on existing sensitization to non-HHP-treated food, the allergenic potential of HHP-treated food is more likely to be equivalent to untreated food. Initial findings on changes in packaging materials under HHP have not yet been adequately supported by scientific data.

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.

Toxicity of fluoride: critical evaluation of evidence for human developmental neurotoxicity in epidemiological studies, animal experiments and in vitro analyses.

Recently, epidemiological studies have suggested that fluoride is a human developmental neurotoxicant that reduces measures of intelligence in children, placing it into the same category as toxic metals (lead, methylmercury, arsenic) and polychlorinated biphenyls. If true, this assessment would be highly relevant considering the widespread fluoridation of drinking water and the worldwide use of fluoride in oral hygiene products such as toothpaste. To gain a deeper understanding of these assertions, we reviewed the levels of human exposure, as well as results from animal experiments, particularly focusing on developmental toxicity, and the molecular mechanisms by which fluoride can cause adverse effects. Moreover, in vitro studies investigating fluoride in neuronal cells and precursor/stem cells were analyzed, and 23 epidemiological studies published since 2012 were considered. The results show that the margin of exposure (MoE) between no observed adverse effect levels (NOAELs) in animal studies and the current adequate intake (AI) of fluoride (50 µg/kg b.w./day) in humans ranges between 50 and 210, depending on the specific animal experiment used as reference. Even for unusually high fluoride exposure levels, an MoE of at least ten was obtained. Furthermore, concentrations of fluoride in human plasma are much lower than fluoride concentrations, causing effects in cell cultures. In contrast, 21 of 23 recent epidemiological studies report an association between high fluoride exposure and reduced intelligence. The discrepancy between experimental and epidemiological evidence may be reconciled with deficiencies inherent in most of these epidemiological studies on a putative association between fluoride and intelligence, especially with respect to adequate consideration of potential confounding factors, e.g., socioeconomic status, residence, breast feeding, low birth weight, maternal intelligence, and exposure to other neurotoxic chemicals. In conclusion, based on the totality of currently available scientific evidence, the present review does not support the presumption that fluoride should be assessed as a human developmental neurotoxicant at the current exposure levels in Europe.

Influence of salt concentration and iodized table salt on the microbiota of fermented cucumbers.

The fermentation of vegetables is a traditional preservation method, that experiences a renaissance even in domestic households. Table salt is added to the fermentation batches to favor the growth of lactic acid bacteria usually. On an industrial scale, the fermentation brine is typically prepared with non-iodized table salt. In our study, we investigated the microbiota of cucumber fermentations using culture-dependent and -independent methods. We could show that the fermentation process of cucumbers and the involved microbiota is influenced by the concentration of table salt and not by the use of iodized table salt. Therefore, we conclude that the use of iodized table salt does not negatively affect the fermentation process. We could verify that iodine permeates the cucumbers by diffusion, leading to satisfactory iodine concentrations in the final food product. The industrial use of iodized table salt in food fermentations could contribute to maintain a constant iodine supply to the general public.

Discovery and Validation of Banana Intake Biomarkers Using Untargeted Metabolomics in Human Intervention and Cross-sectional Studies.

BACKGROUND: Banana is one of the most widely consumed fruits in the world. However, information regarding its health effects is scarce. Biomarkers of banana intake would allow a more accurate assessment of its consumption in nutrition studies. OBJECTIVES: Using an untargeted metabolomics approach, we aimed to identify the banana-derived metabolites present in urine after consumption, including new candidate biomarkers of banana intake. METHODS: A randomized controlled study with a crossover design was performed on 12 healthy subjects (6 men, 6 women, mean ± SD age: 30.0 ± 4.9 y; mean ± SD BMI: 22.5 ± 2.3 kg/m2). Subjects underwent 2 dietary interventions: 1) 250 mL control drink (Fresubin 2 kcal fiber, neutral flavor; Fresenius Kabi), and 2) 240 g banana + 150 mL control drink. Twenty-four-hour urine samples were collected and analyzed with ultra-performance liquid chromatography coupled to a quadrupole time-of-flight MS and 2-dimensional GC-MS. The discovered biomarkers were confirmed in a cross-sectional study [KarMeN (Karlsruhe Metabolomics and Nutrition study)] in which 78 subjects (mean BMI: 22.8; mean age: 47 y) were selected reflecting high intake (126-378 g/d), low intake (47.3-94.5 g/d), and nonconsumption of banana. The confirmed biomarkers were examined singly or in combinations, for established criteria of validation for biomarkers of food intake. RESULTS: We identified 33 potentially bioactive banana metabolites, of which 5 metabolites, methoxyeugenol glucuronide (MEUG-GLUC), dopamine sulfate (DOP-S), salsolinol sulfate, xanthurenic acid, and 6-hydroxy-1-methyl-1,2,3,4-tetrahydro-ß-carboline sulfate, were confirmed as candidate intake biomarkers. We demonstrated that the combination of MEUG-GLUC and DOP-S performed best in predicting banana intake in high (AUCtest = 0.92) and low (AUCtest = 0.87) consumers. The new biomarkers met key criteria establishing their current applicability in nutrition and health research for assessing the occurrence of banana intake. CONCLUSIONS: Our metabolomics study in healthy men and women revealed new putative bioactive metabolites of banana and a combined biomarker of intake. These findings will help to better decipher the health effects of banana in future focused studies. This study was registered at clinicaltrials.gov as NCT03581955 and with the Ethical Committee for the Protection of Human Subjects Sud-Est 6 as CPP AU 1251, IDRCB 2016-A0013-48; the KarMeN study was registered with the German Clinical Trials Register (DRKS00004890). Details about the study can be obtained from https://www.drks.de.

Plasma metabolome analysis identifies distinct human metabotypes in the postprandial state with different susceptibility to weight loss-mediated metabolic improvements.

Health has been defined as the capability of the organism to adapt to challenges. In this study, we tested to what extent comprehensively phenotyped individuals reveal differences in metabolic responses to a standardized mixed meal tolerance test (MMTT) and how these responses change when individuals experience moderate weight loss. Metabolome analysis was used in 70 healthy individuals. with profiling of ∼300 plasma metabolites during an MMTT over 8 h. Multivariate analysis of plasma markers of fatty acid catabolism identified 2 distinct metabotype clusters (A and B). Individuals from metabotype B showed slower glucose clearance, had increased intra-abdominal adipose tissue mass and higher hepatic lipid levels when compared with individuals from metabotype A. An NMR-based urine analysis revealed that these individuals also to have a less healthy dietary pattern. After a weight loss of ∼5.6 kg over 12 wk, only the subjects from metabotype B showed positive changes in the glycemic response during the MMTT and in markers of metabolic diseases. Our study in healthy individuals demonstrates that more comprehensive phenotyping can reveal discrete metabotypes with different outcomes in a dietary intervention and that markers of lipid catabolism in plasma could allow early detection of the metabolic syndrome.-Fiamoncini, J., Rundle, M., Gibbons, H., Thomas, E. L., Geillinger-Kästle, K., Bunzel, D., Trezzi, J.-P., Kiselova-Kaneva, Y., Wopereis, S., Wahrheit, J., Kulling, S. E., Hiller, K., Sonntag, D., Ivanova, D., van Ommen, B., Frost, G., Brennan, L., Bell, J. Daniel, H. Plasma metabolome analysis identifies distinct human metabotypes in the postprandial state with different susceptibility to weight loss-mediated metabolic improvements.

Rubneribacter badeniensis gen. nov., sp. nov. and Enteroscipio rubneri gen. nov., sp. nov., new members of the Eggerthellaceae isolated from human faeces.

Two novel, anaerobic, Gram-positive, rod-shaped bacterial strains, ResAG-85T and ResAG-96T, were isolated from a faecal sample of a male human. 16S rRNA gene sequences analyses indicated that these strains represent a distinct lineage within the family Eggerthellaceae. Strain ResAG-85T showed 92.3 % similarity to the type strains of the genera Eggerthella and Gordonibacter. Strain ResAG-96T clustered together with Paraeggerthella hongkongensis and the newly (but not validly) published genus 'Arabia massiliensis' (94.8 % similarity). Analysis of quinones revealed that MK-5 (21 % in ResAG-85T and 95 % in ResAG-96T) and MK-7 (53 % in strain ResAG-85T) were present, which were described for the first time for members of the Eggerthellaceae. Furthermore, MK-6 was present in both strains (25 % ResAG-85T and 5 % in ResAG-96T). The polar lipids detected in ResAG-85T and ResAG-96T consisted of eight and six glycolipids, respectively. Both strains possessed three phospholipids, one phosphatidylglycerol and one diphosphatidylglycerol. Analysis of fatty acids revealed that the percentage of total branched fatty acids was relatively high in comparison to related strains with 42 and 50 % of strains ResAG-85T and ResAG-96T but comparable to the value obtained for Gordonibacter pamelaeae DSM 19378T. On the basis of this polyphasic approach including molecular (16S rRNA gene sequencing) and biochemical methods (analysis of fatty acids, quinones, polar lipids, Rapid ID 32A and API 20A), the new genera and species Rubneribacter badeniensis with ResAG-85T (=DSM 105129T=JCM 32272T) and Enteroscipio rubneri with ResAG-96T (=DSM 105130T=JCM 32273T) as the type and only strains are described.

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.

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.

[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.

Soy isoflavone exposure through all life stages accelerates 17ß-estradiol-induced mammary tumor onset and growth, yet reduces tumor burden, in ACI rats.

There is an ongoing debate whether the intake of soy-derived isoflavones (sISO) mediates beneficial or adverse effects with regard to breast cancer risk. Therefore, we investigated whether nutritional exposure to a sISO-enriched diet from conception until adulthood impacts on 17ß-estradiol (E2)-induced carcinogenesis in the rat mammary gland (MG). August-Copenhagen-Irish (ACI) rats were exposed to dietary sISO from conception until postnatal day 285. Silastic tubes containing E2 were used to induce MG tumorigenesis. Body weight, food intake, and tumor growth were recorded weekly. At necropsy, the number, position, size, and weight of each tumor were determined. Plasma samples underwent sISO analysis, and the morphology of MG was analyzed. Tumor incidence and multiplicity were reduced by 20 and 56 %, respectively, in the sISO-exposed rats compared to the control rats. Time-to-tumor onset was shortened from 25 to 20 weeks, and larger tumors developed in the sISO-exposed rats. The histological phenotype of the MG tumors was independent of the sISO diet received, and it included both comedo and cribriform phenotypes. Morphological analyses of the whole-mounted MGs also showed no diet-dependent differences. Lifelong exposure to sISO reduced the overall incidence of MG carcinomas in ACI rats, although the time-to-tumor was significantly shortened.

Phase II metabolism of the soy isoflavones genistein and daidzein in humans, rats and mice: a cross-species and sex comparison.

Soy isoflavones (IF) are in the focus of biomedical research since more than two decades. To assess their bioactivity, IF are investigated in rats and mice as a model. As the biological activity of IF is affected by their biotransformation, our aim was to comprehensively compare the conjugative and microbial metabolism of daidzein and genistein in adult humans, rats and mice of both sexes. One identical soy extract and a validated LC-MS method were used for all studies. We detected considerable differences between the three species. In rats and mice, sex-specific differences were observed in addition. The major plasma phase II metabolites in humans were the 7-sulfo-4'-glucuronides (39-49 %) and, in case of genistein, also the diglucuronide (34 %), whereas in mice monosulfates (33-41 %) and monoglucuronides (30-40 %) predominated. In male rats the disulfates (23-62 %) and 7-sulfo-4'-glucuronides (19-54 %) were predominant, while in female rats the 7-glucuronides (81-93 %) exhibited highest concentrations. The portion of aglycones was low in humans (0.5-1.3 %) and rats (0.5-3.1 %) but comparatively high in mice (3.1-26.0 %), especially in the case of daidzein. Furthermore, substantial differences were observed between daidzein and genistein metabolism. In contrast to humans, all rats and mice were equol producer, independent of their sex. In conclusion, there are marked differences between humans, rats and mice in the profile of major metabolites following IF phase II metabolism. These differences may contribute to resolve inconsistencies in results concerning the bioactivity of IF and should be considered when applying findings of animal studies to humans, e.g., for risk assessment.

Transport of the soy isoflavone daidzein and its conjugative metabolites by the carriers SOAT, NTCP, OAT4, and OATP2B1.

Soy isoflavones (IF) are phytoestrogens, which interact with estrogen receptors. They are extensively metabolized by glucuronosyltransferases and sulfotransferases, leading to the modulation of their estrogenic activity. It can be assumed that this biotransformation also has a crucial impact on the uptake of IF by active or passive cellular transport mechanisms, but little is known about the transport of IF phase II metabolites into the cell. Therefore, transport assays for phase II metabolites of daidzein (DAI) were carried out using HEK293 cell lines transfected with five human candidate carriers, i.e., organic anion transporter OAT4, sodium-dependent organic anion transporter (SOAT), Na(+)-taurocholate cotransporting polypeptide (NTCP), apical sodium-dependent bile acid transporter ASBT, and organic anion transporting polypeptide OATP2B1. Cellular uptake was monitored by UHPLC-DAD. DAI monosulfates were transported by the carriers NTCP and SOAT in a sodium-dependent manner, while OAT4-HEK293 cells revealed a partly sodium-dependent transport for these compounds. In contrast, DAI-7,4'-disulfate was only taken up by NTCP-HEK293 cells. DAI-7-glucuronide, but not DAI-4'-glucuronide, was transported exclusively by OATP2B1 in a sodium-independent manner. DAI-7-glucuronide-4'-sulfate, DAI-7-glucoside, and DAI were no substrate of any of the tested carriers. In addition, the inhibitory potency of the DAI metabolites toward estrone-sulfate (E1S) uptake of the above-mentioned carriers was determined. In conclusion, human SOAT, NTCP, OATP2B1, and OAT4 were identified as carriers for the DAI metabolites. Several metabolites were able to inhibit carrier-dependent E1S uptake. These findings might contribute to a better understanding of the bioactivity of IF especially in case of hormone-related cancers.

Quantification of soy isoflavones and their conjugative metabolites in plasma and urine: an automated and validated UHPLC-MS/MS method for use in large-scale studies.

The biotransformation of isoflavones by gut microbiota and by drug metabolizing enzymes plays a crucial role in the understanding of their potential health-promoting effects. The purpose of our work was to develop a simultaneous, sensitive, and robust automated ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method to quantify the soy isoflavones daidzein and genistein, their conjugative metabolites, as well as their major microbial degradation products in order to provide a method for use in large clinical trials or animal studies. An automated, 96-well solid-phase extraction method was used to extract the isoflavone analytes from plasma and urine. Separation of genistein, daidzein, and 19 of its metabolites, including five glucuronides, seven sulfates, and two sulfoglucuronides, as well as five microbial metabolites, was achieved in less than 25 min using a sub-2 µm particle column and a gradient elution with acetonitrile/methanol/water as mobile phases. Analysis was performed under negative ionization electrospray MS via the multiple reaction monitoring (MRM). Validation was performed according to the analytical method validation guidelines of Food and Drug Administration (FDA) and International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) consisting of selectivity, accuracy, precision, linearity, limit of detection, recovery, matrix effect, and robustness. All validated parameters essentially matched the FDA and ICH requirements. The application of this method to a pharmacokinetic study in postmenopausal women showed that isoflavones are extensively metabolized in vivo. A robust automated analytical approach was developed, which allows the handling of large sample sizes but nevertheless provides detailed information on the isoflavone metabolite profile leading to a better understanding and interpretation of clinical and animal studies.