Soybeans are a common ingredient of animal feed. They contain isoflavones, which are known to act as phytoestrogens in animals. Isoflavones were described to have beneficial effects on farm animals. However, there are also reports of negative outcomes after the consumption of isoflavones. This review summarizes the current knowledge of metabolization of isoflavones (including the influence of the microbiome, phase I and phase II metabolism), as well as the distribution of isoflavones and their metabolites in tissues. Furthermore, published studies on effects of isoflavones in livestock species (pigs, poultry, ruminants, fish) are reviewed. Moreover, published studies on occurrence of isoflavones in feed materials and co-occurrence with zearalenone are presented and are supplemented with our own survey data.

Humans and animals are exposed to multiple substances in their food and feed that might have a negative health impact. Among these substances, the Fusarium mycoestrogen zearalenone (ZEN) and its metabolites α-zearalenol (α-ZEL) and α-zearalanol (α-ZAL) are known to possess endocrine disruptive properties. In a mixed diet or especially animal feed, these potential contaminants might be ingested together with naturally occurring phytoestrogens such as soy isoflavones. So far, risk assessment of potential endocrine disruptors is usually based on adverse effects of single compounds whereas studies investigating combinatorial effects are scarce. In the present study, we investigated the estrogenic potential of mycoestrogens and the isoflavones genistein (GEN), daidzein (DAI) and glycitein (GLY) as well as equol (EQ), the gut microbial metabolite of DAI, in vitro alone or in combination, using the alkaline phosphatase (ALP) assay in Ishikawa cells. In the case of mycoestrogens, the tested concentration range included 0.001 to 10 nM with multiplication steps of 10 in between, while for the isoflavones 1000 times higher concentrations were investigated. For the individual substances the following order of estrogenicity was obtained: α-ZEL > α-ZAL > ZEN > GEN > EQ > DAI > GLY. Most combinations of isoflavones with mycoestrogens enhanced the estrogenic response in the investigated concentrations. Especially lower concentrations of ZEN, α-ZEL and α-ZAL (0.001—0.01 nM) in combination with low concentrations of GEN, DAI and EQ (0.001—0.1 µM) strongly increased the estrogenic response compared to the single substances.

Risk assessment primarily relies on toxicological data of individual substances, with limited information on combined effects. Recent in vitro experiments using Ishikawa cells, an endometrial carcinoma cell line expressing both estrogen receptor isoforms, demonstrated interactive effects of phyto- and mycoestrogens. The mycoestrogens, zearalenone (ZEN), and α-zearalenol (α-ZEL) exhibited significantly enhanced estrogenic responses in the presence of isoflavones (ISF), depending on substance ratios and concentrations. This study investigated the impact of phyto- and mycoestrogen combinations on estrogenic response following OECD guideline 455, utilizing hERα-HeLa-9903 cells. Test substances included mycoestrogens (ZEN and α-ZEL) and isoflavones (genistein (GEN), daidzein (DAI), and S-equol (EQ), a gut microbial metabolite of DAI). Mycoestrogens were tested in the range of 0.001 to 100 nM, while isoflavones were used at concentrations 1000 times higher based on relevant occurrence ratios. Results showed that ZEN and α-ZEL induced ERα-dependent luciferase expression in concentrations above 1 nM and 0.01 nM, respectively. However, ISF caused a superinduction of the luciferase signal above 1 µM. A superinduction is characterized by an unusually strong or heightened increase in the activity of the luciferase enzyme. This signal is not affected by the estrogen receptor antagonist 4-hydroxytamoxifen (4-OH-TAM), which was additionally used to verify whether the increase of signal is a true reflection of receptor activation. This superinduction was observed in all combinations of ZEN and α-ZEL with ISFs. Contrary to the luciferase activity findings, RT-qPCR experiments and a stability approach revealed lower real ERα activation by ISFs than measured in the ONE-Glo™ luciferase test system. In conclusion, the OECD protocol 455 appears unsuitable for testing ISFs due to their superinduction of luciferase and interactions with the test system, resulting in experimental artifacts. Further studies are necessary to explore structure–activity relationships within polyphenols and clarify the test system’s applicability.

Humans and animals are frequently exposed to dietary compounds that can adversely affect intestinal health. The Fusarium mycotoxin deoxynivalenol (DON) is known to compromise epithelial barrier integrity. DON frequently co-occurs in food and feed with the mycoestrogen zearalenone (ZEN) and soy-derived isoflavones (ISFs) such as genistein (GEN) and daidzein. Therefore, this study investigates the effects of ZEN (10 nM), its metabolites hydrolyzed ZEN (HZEN) and decarboxylated hydrolyzed ZEN (DHZEN) (each 10 nM), and ISFs (GEN and equol (EQ), 10 µM), alone and in combination with DON (10 µM), on intestinal barrier function using the porcine epithelial cell line IPEC-J2. Barrier integrity was assessed via transepithelial electrical resistance (TEER) and paracellular permeability (lucifer yellow). DON (10 µM) significantly reduced barrier integrity, confirming its disruptive effect. Conversely, treatment with ZEN, HZEN, DHZEN, GEN, and EQ did not impair barrier function. Co-exposure of DON with ZEN or HZEN yielded TEER values similar to those observed with DON alone. In contrast, the DHZEN + DON combination resulted in higher TEER values compared to DON alone, suggesting a mitigating effect under the experimental conditions. Quantification of GEN by LC-MS/MS showed consistent levels across treatments, except for reduced basolateral GEN during co-incubation with DHZEN, suggesting altered transport or metabolism. These findings highlight the ability of DHZEN to mitigate the intestinal barrier disruption induced by DON and underscores the complex interaction of maintaining epithelial barrier integrities.

Multiple mycotoxins in feed threaten animal health and food safety, demanding sustainable mitigation strategies. This study evaluated acid-modified mangosteen peel (AMP), an agricultural by-product, as a potential multi-mycotoxin adsorbent. Physicochemical characterization using scanning electron microscopy (SEM), Brunauer–Emmett–Teller (BET) surface area analysis, and Fourier transform infrared spectroscopy (FTIR) analyses demonstrated that acid modification increased surface area (1.9 to 9.03 m²/g), pore volume (0.005 to 0.027 cm³/g), and surface negativity, indicating enhanced adsorption properties. In vitro binding experiments assessed adsorption of aflatoxin B₁ (AFB₁), zearalenone (ZEA), ochratoxin A (OTA), T-2 toxin, deoxynivalenol (DON) and fumonisin B₁ (FB₁) under different pH conditions. AMP exhibited high adsorption efficiencies for AFB₁, ZEA, OTA, and T-2 toxin, particularly at pH 3, whereas DON and FB₁ showed limited binding. Adsorption behavior was dose-dependent and best described by Langmuir and Freundlich isotherm models. Simulated gastrointestinal digestion indicated stable binding of AFB₁ and ZEA under gastric conditions, with partial release of some toxins at neutral pH. Cytotoxicity assessment in porcine intestinal epithelial cells (IPEC J2) showed no apparent cytotoxic effects at 0.25–1 mg/mL. Therefore, AMP demonstrated improved multi-mycotoxin adsorption compared to the untreated material and showed no apparent cytotoxic effects in vitro within the tested concentration range, indicating its potential as a promising feed additive candidate.