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Zearalenone (ZEN) is a mycotoxin produced by Fusarium spp. present in food and feed. ZEN shows in vivo and in vitro endocrine disrupting potential via the activation of estrogen receptors (ERs). In mammals, liver and intestinal microbiota metabolize ZEN to α-ZEL and β-ZEL, with the estrogenic potency of α-ZEL being 60-times higher than that of ZEN and β-ZEL being 5-times less potent than ZEN. The species differences in sensitivity to ZEN exposure, with pigs being the most sensitive species, might be ascribed to interspecies differences in the formation of α-ZEL and β-ZEL in combination with possible interspecies differences in toxicodynamics. EFSA proposed in 2016 a group health-based guidance value (HBGV), i.e. a tolerable daily intake (TDI) for ZEN and its modified forms, of 0.25 μg/ kg bw based on the no observed effect level (NOEL) of 10.4 µg/kg bw for estrogenic effects of ZEN in young gilts. Considering the uncertainty around the effects of ZEN in humans, the aim of this PhD project was to gain further insight into the metabolism of ZEN, including its metabolism in liver and intestinal microbiota of not only experimental animals but also human, and to include this information in physiologically-based kinetic (PBK) models to enable evaluation of the role of metabolism of ZEN in its estrogenic activity. To this end, intestinal microbial metabolism of ZEN was studied in vitro using anaerobic incubations of fecal samples from rats, pigs and humans, and scaled to the in vivo situation to enable comparison to metabolism in the liver. The apparent in vivo catalytic efficiencies (kcat) for the formation of α-ZEL and β-ZEL in the three species showed that overall metabolism was higher in pigs, followed by rats and humans. The comparison to liver metabolism showed that the intestinal microbiota of pigs contributes up to 36% to the overall formation of α-ZEL from ZEN, and might thus contribute to the bioactivation of ZEN. For rats, this contribution amounted to 27%, while in humans this was less than 0.1%. These interspecies differences highlighted the importance for the development of human specific models to assess the kinetics of ZEN. In further studies of the present thesis the kinetic constants for the metabolism of ZEN by liver and intestinal microbiota were included in newly developed physiologically-based kinetic (PBK) models for ZEN and its bioactive metabolite α-ZEL, for rats and humans. These models enabled prediction of the maximum blood concentrations of ZEN and α-ZEL, as well as a comparison of these concentrations to concentrations known to induce estrogenicity in in vitro bioassays like the estrogen receptor α-mediated reporter gene (ERα-CALUX) assay and cell proliferation assays. The results obtained revealed that at a range of doses of 2.4-29 ng/kg bw of ZEN, representing the estimated daily intake in Europe for the average adult population, the maximum levels reaching the blood circulation in humans are 3 orders of magnitude below the concentrations of ZEN and α-ZEL known to be active in in vitro studies for estrogenicity of these compounds (EC10). Additionally, the human PBK model was used to study interindividual differences in toxicokinetics of ZEN. To this end in vitro derived kinetic constants for conversion of ZEN by individual liver and intestinal microbiota samples were combined to define 400 individual PBK models enabling the prediction of a distribution of the maximum blood concentrations (Cmax) for ZEN and α-ZEL. Subsequently the PBK model outcomes were combined with Monte Carlo simulation to obtain a distribution for a larger population (n=9,879). The distribution of the Cmax at a dose equivalent to the current TDI of 0.25 μg/ kg bw was used to estimate a chemical-specific assessment factor (CSAF) for human interindividual differences in toxicokinetics of 2.45 for the 95th percentile of the adult population. The estimated CSAF obtained for the simulated population indicated that the current default uncertainty factor for interindividual differences in kinetics of 3.16 is sufficiently protective. Age and reproductive status are reported to influence the sensitivity towards ZEN. In pigs, prepubertal gilts seem to be more sensitive to ZEN exposure. In humans, the presence of ZEN in urine of young girls with cases of precocious puberty was suggested to have a causal relation, while this could not be proven. It remains of interest for future studies to characterize if the CSAF also adequately covers the interindividual differences for sensitive groups and whether the relatively high sensitivity in younger populations is in part also related to differences in toxicokinetics or solely due to differences in toxicodynamics. Overall, the work of this thesis presents a proof-of-principle for the inclusion of in vitro kinetics for intestinal microbial and host metabolism in PBK models. Additionally, combining the PBK model with Monte Carlo simulations was shown to provide a strong in vitro-in silico tool for the study of interindividual differences in metabolism and to evaluate the default uncertainty factors used in the current risk assessment. Furthermore, the rapid hydrolysis by the intestinal microbiota in vitro of zearalenone-14-glucoside (ZEN-14-G) to release ZEN indicates the importance of the intestinal microbiota upon exposure to ZEN-14-G and supports the inclusion of ZEN-14-G into the group HBGV (EFSA, 2016) of 0.25 µg/ kg bw.In conclusion, the insights provided in this PhD thesis open a series of possibilities for further research including the identification of potential sensitive human subpopulations to ZEN exposure due to possible differences in toxicokinetics between individuals. The present thesis provides a way forward how to study this through the application of a combined approach of in vitro, and in silico studies. |
