Luca Dellafiora

In silico approach to evaluate molecular interaction between mycotoxins and the estrogen receptors ligand binding domain: A case study on zearalenone and its metabolites

An association of virtual screening, docking and a good rescoring procedure is a well known technique to discover and design new lead compounds in medicinal chemistry. We have demonstrated that the study on the interactions of unsuspected molecules with estrogen receptors, using the same technique applied in medicinal chemistry could be a valuable choice to discover new hypothetical xenoestrogens. The same approach can be applied to a wide set of chemicals found in food and seed. We propose this approach using as a case study on the zearalenone family to food safety. Zearalenone and its reductive metabolites are a well known set of mycotoxins able to bind estrogen receptors (ERs) thereby interfering with the endogenous estrogenic response. Their endocrine disrupting behavior is tightly related to their capability to competitively bind the ligand binding pocket (LBP) and to stabilize at least one of the functionally active conformational assets of the ligand binding domain (LBD). Altought proposing an interactive model for three-dimensional complexes not yet solved, this kind of computational aided analysis is a potentially intriguing tool to predict the binding event and to evaluate the xenoestrogenic role of any kind of chemicals and derivatives.

Hybrid in Silico/in Vitro Approach for the Identification of Angiotensin I Converting Enzyme Inhibitory Peptides from Parma Dry-Cured Ham

The bioactivity assessment of foodborne peptides is currently a research area of great relevance, and, in particular, several studies are devoted to the antihypertensive effects through the inhibition of angiotensin I converting enzyme (ACE). In the present work, a straightforward workflow to identify inhibitory peptides from food matrices is proposed, which involves a hybrid in vitro/in silico tandem approach. Parma dry-cured ham was chosen as case study. In particular, the advantage of using the hybrid approach to identify active sequences (in comparison to the experimental trials alone) has been pointed out. Specifically, fractions obtained by in vitro gastrointestinal digestion of ham samples of 18 and 24 months of aging have been assessed for ACE inhibition. At the same time, the released peptidomic profiles, which cannot be entirely evaluated by using in vitro assays, have been screened for the inhibition by using an in silico model. Then, to identify novel inhibitory sequences, a series of strong candidates have been synthesized and assessed for their inhibitory activity through in vitro assay. On the one hand, the use of computational simulations appeared to be an effective strategy to find active sequences, as confirmed by in vitro analysis. On the other hand, strong inhibitory sequences were identified for the first time in Parma dry-cured ham (e.g., LGL and SFVTT with IC50 values of 145 and 395 μM, respectively), which is a product of international dietary and economic relevance. Therefore, these findings demonstrate the usefulness of in silico methodologies coupled to in vitro tests for the identification of potentially bioactive peptides, and they give an important contribution to the study of the overall nutritional value of Parma ham.

Hazard assessment through hybrid in vitro/in silico approach: the case of zearalenone

Within the framework of reduction, refinement and replacement of animal experiments, new approaches for identification and characterization of chemical hazards have been developed. Grouping and read across has been promoted as a most promising alternative approach. It uses existing toxicological information on a group of chemicals to make predictions on the toxicity of uncharacterized ones. In the present work, the feasibility of applying in vitro and in silico techniques to group chemicals for read across was studied using the food mycotoxin zearalenone (ZEN) and metabolites as a case study. ZEN and its reduced metabolites are known to act through activation of the estrogen receptor α (ERα). The ranking of their estrogenic potencies appeared highly conserved across test systems including binding, in vitro and in vivo assays. This data suggests that activation of ERα may play a role in the molecular initiating event (MIE) and be predictive of adverse effects and provides the rationale to model receptor-binding for hazard identification. The investigation of receptor-ligand interactions through docking simulation proved to accurately rank estrogenic potencies of ZEN and reduced metabolites, showing the suitability of the model to address estrogenic potency for this group of compounds. Therefore, the model was further applied to biologically uncharacterized, commercially unavailable, oxidized ZEN metabolites (6α-, 6β-, 8α-, 8β-, 13- and 15-OH-ZEN). Except for 15-OH-ZEN, the data indicate that in general, the oxidized metabolites would be considered a lower estrogenic concern than ZEN and reduced metabolites.

Modeling the Effect of Phase II Conjugations on Topoisomerase I Poisoning: Pilot Study with Luteolin and Quercetin

Topoisomerases are targeted by several drugs in cancer chemotherapy acting as key enzymes in cell viability. Some flavonoids and their glycosides may exert health protective effects through the poisoning of topoisomerases. However, previous studies did not consider the substantial modifications taking place after ingestion neglecting that only metabolites can interact with the internal compartments of the human body. Since the high number of possible metabolites hinders their systematic analysis, an in silico approach can be a valuable tool to prioritize compounds by identifying candidates for further characterization. Specifically focusing on luteolin and quercetin, among the most ubiquitous flavonoids in the human diet, this work reports a computational procedure to model the effect of hepatic phase II conjugative metabolism on poisoning of human Topoisomerase I. As a general effect, glucuronidation and sulphation might enhance and quench poisoning activity, respectively. Among all, quercetin-3-O-glucuronide represents a promising candidate to be analyzed more thoroughly.

On the masked mycotoxin zearalenone-14-glucoside. Does the mask truly hide?

In the matter of foodborne mycotoxins, beside a number of regulated compounds, regulations are totally missing for phase-II plant metabolites--the toxicological knowledge of which is still in its infancy. Currently, zearalenone-14-glucoside is in the pipeline and its toxicological role is under a glowing scientific debate. In our work it clearly showed high toxicological concerns as it is prone to conversion to well-known toxic compounds (i.e. zearalenone and both zearalenol isomers) when exposed to breast cancer cells culture. The need of future risk assessment studies has been pointed out accordingly.

Ergot alkaloids: From witchcraft till in silico analysis. Multi-receptor analysis of ergotamine metabolites

Graphical abstract

Forthcoming Challenges in Mycotoxins Toxicology Research for Safer Food—A Need for Multi-Omics Approach

The presence of mycotoxins in food represents a severe threat for public health and welfare, and poses relevant research challenges in the food toxicology field. Nowadays, food toxicologists have to provide answers to food-related toxicological issues, but at the same time they should provide the appropriate knowledge in background to effectively support the evidence-based decision-making in food safety. Therefore, keeping in mind that regulatory actions should be based on sound scientific findings, the present opinion addresses the main challenges in providing reliable data for supporting the risk assessment of foodborne mycotoxins.

Hazard identification of cis/trans-zearalenone through the looking-glass

Among the food-related health issues, the presence of contaminants has a prominent role, due to the wide range of exogenous compounds that can occur in food commodities and to their large differences in structure and biological activity. A comprehensive assessment of the related risk is thus actually demanding in terms of time and facilities involved. In this context, the use of computational strategies can be an effective choice for supporting the hazard identification procedure at the early stage. In this work, we focused on the food contaminant zearalenone by comparing the trans and cis isomers, respectively the well-known mycoestrogen and its still largely understudied isomer. We estimated the possible effects exerted by human metabolism on the xenoestrogenicity of cis-ZEN by using a validated in silico strategy based on docking simulations and rescoring procedures. Similarly, the exploitation of the most promising enzymatic detoxifying routes designed for trans-ZEN - which relies on the enzyme lactono hydrolase from Clonostachys rosea - has been assessed for the cis-isomer as well. Our results showed that both isomers can act as functional analogues with respect to xenoestrogenic activity, and several cis-ZEN metabolites with high biological potential have been identified. On the contrary, in spite of the high degree of structural analogy, the cis isomer showed a pattern of interaction with the degrading enzyme in stark contrast with that observed for trans-ZEN. For these reasons, the outcomes presented herein strongly support the inclusion of cis-ZEN in further studies of occurrence, metabolism and bioactivity assessment, and suggest the need for a dedicated handling for the cis isomer in risk assessment studies.

Masked mycotoxins: An emerging issue that makes renegotiable what is ordinary.

The masked mycotoxins issue is of increasing relevance in the field of food safety. Although under discussion, regulations are still to be set due to the lack of proper toxicological data. In this communication, we discuss the unmet needs to support regulatory bodies in the decision making on this class of compounds.

Degradation of Aflatoxins by Means of Laccases from Trametes versicolor: An In Silico Insight

Mycotoxins are secondary metabolites of fungi that contaminate food and feed, and are involved in a series of foodborne illnesses and disorders in humans and animals. The mitigation of mycotoxin content via enzymatic degradation is a strategy to ensure safer food and feed, and to address the forthcoming issues in view of the global trade and sustainability. Nevertheless, the search for active enzymes is still challenging and time-consuming. The in silico analysis may strongly support the research by providing the evidence-based hierarchization of enzymes for a rational design of more effective experimental trials. The present work dealt with the degradation of aflatoxin B1 and M1 by laccase enzymes from Trametes versicolor. The enzymes–substrate interaction for various enzyme isoforms was investigated through 3D molecular modeling techniques. Structural differences among the isoforms have been pinpointed, which may cause different patterns of interaction between aflatoxin B1 and M1. The possible formation of different products of degradation can be argued accordingly. Moreover, the laccase gamma isoform was identified as the most suitable for protein engineering aimed at ameliorating the substrate specificity. Overall, 3D modeling proved to be an effective analytical tool to assess the enzyme–substrate interaction and provided a solid foothold for supporting the search of degrading enzyme at the early stage.