Transfer and Metabolism of the Xenoestrogen Zearalenone in Human Perfused Placenta.

BACKGROUND: Pregnancy is a sensitive condition during which adverse environmental exposures should be monitored thoroughly and minimized whenever possible. In particular, the hormone balance during gestation is delicate, and disturbance may cause acute or chronic long-term health effects. A potential endocrine disruption may be provoked by in utero exposure to xenoestrogens mimicking endogenous estrogens. The mycoestrogen zearalenone (ZEN), a toxic fungal secondary metabolite and mycotoxin found frequently in food and feed, constitutes a prominent example. OBJECTIVES: We performed a comprehensive assessment of the transfer as well as phase I and phase II metabolism of ZEN at the human placental barrier. METHODS: Human placentas were perfused with 1µM (318µg/L) ZEN for 6 h. Samples from the maternal and fetal compartment, placental tissue, and fetal plasma were analyzed by a highly sensitive UHPLC-MS/MS assay to detect ZEN as well as nine key metabolites (α-zearalenol, ß-zearalenol, zearalanone, α-zearalanol, ß-zearalanol, ZEN-14-glucuronide, α-zearalenol-14-glucuronide, ß-zearalenol-14-glucuronide, ZEN-14-sulfate). RESULTS: The model revealed a fast maternofetal transfer of ZEN across the human placental barrier. We also unraveled phase I and phase II metabolism of the parent toxin ZEN into the approximately 70-times more estrogenic α-zearalenol and the less active ZEN-14-sulfate conjugate, which are effectively released into the maternal and fetal circulation in considerable amounts. CONCLUSIONS: Our findings suggest that exposure to ZEN (such as through consumption of ZEN-contaminated cereal-based products) during pregnancy may result in in utero exposure of the fetus, not only to ZEN but also some of its highly estrogenically active metabolites. In the light of the known affinity of ZEN and potentially co-occurring xenoestrogens to the estrogen receptor, and our results demonstrating placental transfer of ZEN and its metabolites in an ex vivo model, we recommend further research and more comprehensive assessment of gestational exposures in women. https://doi.org/10.1289/EHP4860.

Naturally occurring mixtures of Alternaria toxins: anti-estrogenic and genotoxic effects in vitro.

Alternaria molds can produce a variety of different mycotoxins, often resulting in food contamination with chemical mixtures, posing a challenge for risk assessment. Some of these metabolites possess estrogenic properties, an effect whose toxicological relevance is questioned in the light of the strong genotoxic and cytotoxic properties of co-occurring toxins. Thus, we tested a complex extract from A. alternata for estrogenic properties in Ishikawa cells. By assessing alkaline phosphatase activity, we did not observe estrogen receptor (ER) activation at non-cytotoxic concentrations (≤ 10 µg/ml). Furthermore, an extract stripped of highly genotoxic perylene quinones also did not mediate estrogenic effects, despite diminished genotoxic properties in the comet assay (≥ 10 µg/ml). Interestingly, both extracts impaired the estrogenicity of 17ß-estradiol (E2) at non-cytotoxic concentrations (5-10 µg/ml), indicating anti-estrogenic effects which could not be explained by the presence of known mycoestrogens. A mechanism for this unexpected result might be the activation of the aryl hydrocarbon receptor (AhR) by Alternaria metabolites, as indicated by the induction of CYP1A1 transcription. While a direct influence on the metabolism of E2 could not be confirmed by LC-MS/MS, literature describing a direct interplay of the AhR with estrogenic pathways points to a corresponding mode of action. Taken together, the present study indicates AhR-mediated anti-estrogenic effects as a novel mechanism of naturally co-occurring Alternaria toxin mixtures. Furthermore, our results confirm their genotoxic activity and raise questions about the contribution of still undiscovered metabolites to toxicological properties.

A Generic Liquid Chromatography-Tandem Mass Spectrometry Exposome Method for the Determination of Xenoestrogens in Biological Matrices.

We are constantly exposed to a variety of environmental contaminants and hormones, including those mimicking endogenous estrogens. These highly heterogeneous molecules are collectively referred to as xenoestrogens and hold the potential to affect and alter the delicate hormonal balance of the human body. To monitor exposure and investigate potential health implications, comprehensive analytical methods covering all major xenoestrogen classes are needed but not available to date. Herein, we describe a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of multiple classes of endogenous as well as exogenous estrogens in human urine, serum, and breast milk to enable proper exposure and risk assessment. In total, 75 analytes were included, whereof a majority was successfully in-house validated in the three matrices. Extraction recoveries of validated analytes ranged from 71% to 110% and limits of quantification from 0.015 to 5 µg/L, 0.03 to 14 µg/L, and 0.03 to 4.6 µg/L in urine, serum, and breast milk, respectively. The applicability of the novel method was demonstrated in proof-of-principle experiments by analyzing urine from Austrian individuals and breast milk from Austrian and Nigerian individuals. Thereby, we proved the methods' feasibility to identify and quantify different classes of xenoestrogens simultaneously. The results illustrate the general importance of multiclass exposure assessment in the context of the exposome paradigm. Specifically, they highlight the need for estimating total estrogenic burden rather than single analyte or chemical class measurements and its potential impact in endocrine disruption and hormone related diseases including cancers.