Molecular Characterization of the Allergenic Arginine Kinase from the Edible Insect Hermetia illucens (Black Soldier Fly).

SCOPE: Arginine kinase (AK) is an important enzyme for energy metabolism of invertebrate cells by participating in the maintenance of constant levels of ATP. However, AK is also recognized as a major allergen in insects and crustaceans capable of cross-reactivity with sera of patients sensitized to orthologous proteins. In the perspective of introducing insects or their derivatives in the human diet in Western world, it is of primary importance to evaluate possible risks for allergic consumers. METHODS AND RESULTS: This work reports the identification and characterization of AK from Hermetia illucens commonly known as the black soldier fly, a promising insect for human consumption. To evaluate allergenicity of AK from H. illucens, putative linear and conformational epitopes are identified by bioinformatics analyses, and Dot-Blot assays are carried out by using sera of patients allergic to shrimp or mites to validate the cross-reactivity. Gastrointestinal digestion reduces significantly the linear epitopes resulting in lower allergenicity, while the secondary structure is altered at increasing temperatures supporting the possible loss or reduction of conformational epitopes. CONCLUSION: The results indicate that the possible allergenicity of AK should be taken in consideration when dealing with novel foods containing H. illucens or its derivatives.

Effects of Chrysin and Chrysin-7-sulfate on Ochratoxin A-Albumin Interactions and on the Plasma and Kidney Levels of the Mycotoxin in Rats.

The nephrotoxic mycotoxin ochratoxin A (OTA) is a common food contaminant. OTA binds to the Sudlow's Site I region of serum albumin with very high affinity, resulting in its slow elimination. The displacement of OTA from albumin may be beneficial due to the faster excretion of the mycotoxin, while it may also lead to the increased tissue uptake of OTA. Furthermore, it is challenging to displace the mycotoxin from albumin even with high-affinity Site I ligands. In this study, we tested the impacts of Site I and Heme site ligands on OTA-albumin interactions by applying fluorescence spectroscopic, ultracentrifugation, and modeling studies. Chrysin-7-sulfate (C7S) strongly displaced OTA from both human and rat albumins; therefore, the impacts of C7S (single intravenous administration) and the parent flavonoid chrysin (repeated peroral treatment) were examined on the plasma and kidney levels of OTA in rats. Chrysin barely influenced the concentrations of mycotoxin in plasma and kidneys. In the first few hours, C7S significantly decreased the plasma levels of OTA compared to the control animals; while after 24 h, only minor differences were noticed. Our study highlights the superior displacing ability of C7S vs OTA regarding human and rat albumins.

Computational methods meet in vitro techniques: A case study on fusaric acid and its possible detoxification through cytochrome P450 enzymes.

Mycotoxins are known environmental pollutants that may contaminate food and feed chains. Some mycotoxins are regulated in many countries to limit the trading of contaminated and harmful commodities. However, the so-called emerging mycotoxins are poorly understood and need to be investigated further. Fusaric acid is an emerging mycotoxin, noxious to plants and animals, but is known to be less toxic to plants when hydroxylated. The detoxification routes effective in animals have not been elucidated yet. In this context, this study integrated in silico and in vitro techniques to discover potential bioremediation routes to turn fusaric acid to its less toxic metabolites. The toxicodynamics of these forms in humans have also been addressed. An in silico screening process, followed by molecular docking and dynamics studies, identified CYP199A4 from the bacterium Rhodopseudomonas palustris HaA2 as a potential fusaric acid biotransforming enzyme. Its activity was confirmed in vitro. However, the effect of hydroxylation seemed to have a limited impact on the modelled toxicodynamics against human targets. This study represents a starting point to develop a hybrid in silico/in vitro pipeline to find bioremediation agents for other food, feed and environmental contaminants.

Free fatty acid receptors beyond fatty acids: A computational journey to explore peptides as possible binders of GPR120.

Free fatty acids receptors, with members among G protein-coupled receptors (GPCRs), are crucial for biological signaling, including the perception of the so called "fatty taste". In recent years, GPR120, a protein belonging to the GPCR family, drew attention as an interesting pharmacological target to cope with obesity, satiety and diabetes. Apart from long chain fatty acids, which are GPR120 natural agonists, other synthetic molecules were identified as agonists expanding the chemical space of GPR120's ligands. In this scenario, we unveiled peptides as possible GPR120 binders toward a better understanding of this multifaceted and relevant target. This study analyzed a virtual library collecting 531 441 low-polar hexapeptides, providing mechanistic insights on the GPR120 activation and further extending the possible chemical space of GPR120 agonists. The computational pipeline started with a narrow filtering of hexapeptides based on their chemical similarity with known GPR120 agonists. The best hits were tested through docking studies, molecular dynamics and umbrella sampling simulations, which pointed to G[I,L]FGGG as a promising GPR120 agonist sequence. The presence of both peptides in food-related proteins was thoroughly assessed, revealing they may occur in mushrooms, food-grade bacteria and rice. Simulations on the counterparts with D-amino acids were also performed. Umbrella sampling simulations described that GdIFGGG may have a better interaction compared to its all-L counterpart (-13 kCal/mol ΔG and -6 kCal/mol ΔG, respectively). Overall, we obtained a predictive model to better understand the underpinning mechanism of GPR120-hexapeptides interaction, hierarchizing novel potential agonist peptides for further analysis and describing promising food sources worth of further dedicated investigations.

Virtual display of targets: A new level to rise the current understanding of ochratoxin A toxicity from a molecular standpoint.

Ochratoxin A (OTA) is a mycotoxin spread worldwide contaminating several food and feed commodities and rising concerns for humans and animals. OTA toxicity has been thoroughly assessed over the last 60 years revealing a variety of adverse effects, including nephrotoxicity, hepatotoxicity and possible carcinogenicity. However, the underpinning mechanisms of action have yet to be completely displayed and understood. In this framework, we applied a virtual pipeline based on molecular docking, dynamics and umbrella simulations to display new OTA potential targets. The results collected consistently identified OGFOD1, a key player in protein translation, as possibly inhibited by OTA and its 2'R diastereomer. This is consistent with the current knowledge of OTA's molecular toxicology and may fill some gaps from a mechanistic standpoint. This could pave the way for further dedicated analysis focusing their attention on the OTA-OGFOD1 interaction, expanding the current understanding of OTA toxicity at a molecular level.

Combined in vitro and in silico mechanistic approach to explore the potential of Alternaria mycotoxins alternariol and altertoxin II to hamper γH2AX formation in DNA damage signaling pathways.

Risk assessment of food and environmental contaminants is faced by substantial data gaps and novel strategies are needed to support science-based regulatory actions. The Alternaria mycotoxins alternariol (AOH) and altertoxin II (ATXII) have garnered attention for their possible genotoxic effects. Nevertheless, data currently available are rather scattered, hindering a comprehensive hazard characterization. This study combined in vitro/in silico approaches to elucidate the potential of AOH and ATXII to induce double-strand breaks (DSBs) in HepG2 cells. Furthermore, it examines the impact of co-exposure to AOH and the DSB-inducing drug doxorubicin (Doxo) on γH2AX expression. AOH slightly increased γH2AX expression, whereas ATXII did not elicit this response. Interestingly, AOH suppressed Doxo-induced γH2AX expression, despite evidence of increased DNA damage in the comet assay. Building on these observations, AOH was postulated to inhibit γH2AX-forming kinases. Along this line, in silico analysis supported AOH potential interaction with the ATP-binding sites of these kinases and immunofluorescence experiments showed decreased intracellular phosphorylation events. Similarly, in silico results suggested that ATXII might also interact with these kinases. This study emphasizes the importance of understanding the implications of AOH-induced γH2AX expression inhibition on DNA repair processes and underscores the need for caution when interpreting γH2AX assay results.

A mechanistic toxicology study to grasp the mechanics of zearalenone estrogenicity: Spotlighting aromatase and the effects of its genetic variability.

Zearalenone (ZEN) is a mycoestrogen produced by Fusarium fungi contaminating cereals and in grain-based products threatening human and animal health due to its endocrine disrupting effects. Germane to the mechanisms of action, ZEN may activate the estrogen receptors and inhibit the estrogens-producing enzyme aromatase (CYP19A1). Both show single nucleotide variants (SNVs) among humans associated with a diverse susceptibility of being activated or inhibited. These variations might modify the endocrine disrupting action of ZEN, requiring dedicated studies to improve its toxicological understanding. This work focused on human aromatase investigating via 3D molecular modelling whether some of the SNVs reported so far (n = 434) may affect the inhibitory potential of ZEN. It has been also calculated the inhibition capability of α-zearalenol, the most prominent and estrogenically potent phase I metabolite of ZEN, toward those aromatase variants with an expected diverse sensitivity of being inhibited by ZEN. The study: i) described SNVs likely associated with a different susceptibility to ZEN and α-zearalenol inhibition - like T310S that is likely more susceptible to inhibition, or D309G and S478F that are possibly inactive variants; ii) proofed the possible existence of inter-individual susceptibility to ZEN; iii) prioritized aromatase variants for future investigations toward a better comprehension of ZEN xenoestrogenicity at an individual level.

The bitter side of toxicity: A big data analysis spotted the interaction between trichothecenes and bitter receptors.

The bitter taste perception evolved in human and animals to rapidly perceive and avoid potential toxic compounds. This is mediated by taste receptors type 2 (TAS2R), expressed in various tissues, which recently proved to be involved in roles beyond the bitter perception itself. With this study, the interaction between food-related toxic compounds and TAS2R46 has been investigated via computational approaches, starting with a virtual screening and moving to molecular docking and dynamics simulations. The virtual screening analysis identified trichothecolone and the trichothecenes class it belongs to, which includes mycotoxins widespread in several commodities raising food safety concerns, as possible TAS2R46 binders. Molecular docking and dynamics simulations were performed to further explore the trichotecenes-TAS2R46 interaction. The results indicated that deoxynivalenol and its 15-acetylated derivative could activate TAS2R46. Eventually, this study provided initial evidence supporting the involvement of TAS2R46 in the underpinning mechanisms of deoxynivalenol action highlighting the need of digging into the involvement of TAS2R46 and TAS2Rs in the adverse effects of deoxynivalenol and congeners.

The Occurrence of Non-Regulated Mycotoxins in Foods: A Systematic Review.

The aim of this systematic review is to provide an update on the occurrence and co-occurrence of selected non-regulated mycotoxins and provide an overview of current regulations. Fifteen non-regulated mycotoxins were found in 19 food categories worldwide. On top of that, 38 different combinations of non-regulated mycotoxins were found, with mixtures varying from binary combinations up to 12 mycotoxins. Taking into consideration the amount of evidence regarding the prevalence and co-occurrence of non-regulated mycotoxins, future steps should be taken considering continuous monitoring, scientific exchange, and generation of high-quality data. To enhance data quality, guidelines outlining the minimum quality criteria for both occurrence data and metadata are needed. By doing so, we can effectively address concerns related to the toxicity of non-regulated mycotoxins. Furthermore, obtaining more data concerning the co-occurrence of both regulated and non-regulated mycotoxins could aid in supporting multiple chemical risk assessment methodologies. Implementing these steps could bolster food safety measures, promote evidence-based regulations, and ultimately safeguard public health from the potential adverse effects of non-regulated mycotoxins.

An in silico insight on the mechanistic aspects of gelsenicine toxicity: A reverse screening study pointing to the possible involvement of acetylcholine binding receptor.

Gelsedine-type alkaloids are highly toxic plant secondary metabolites produced by shrubs belonging to the Gelsemium genus. Gelsenicine is one of the most concerning gelsedine-type alkaloids with a lethal dose lower than 1 mg/Kg in mice. Several reported episodes of poisoning in livestock and fatality cases in humans due to the usage of Gelsemium plants extracts were reported. Also, gelsedine-type alkaloids were found in honey constituting a potential food safety issue. However, their toxicological understanding is scarce and the molecular mechanism underpinning their toxicity needs further investigations. In this context, an in silico approach based on reverse screening, docking and molecular dynamics successfully identified a possible gelsenicine biological target shedding light on its toxicodynamics. In line with the available crystallographic data, it emerged gelsenicine could target the acetylcholine binding protein possibly acting as a partial agonist against α7 nicotinic acetylcholine receptor (AChR). Overall, these results agreed with evidence previously reported and prioritized AChR for further dedicated analysis.

A computational study on the biotransformation of alkenylbenzenes by a selection of CYPs: Reflections on their possible bioactivation.

Alkenylbenzenes are aromatic compounds found in several vegetable foods that can cause genotoxicity upon bioactivation by members of the cytochrome P450 (CYP) family, forming 1'-hydroxy metabolites. These intermediates act as proximate carcinogens and can be further converted into reactive 1'-sulfooxy metabolites, which are the ultimate carcinogens responsible for genotoxicity. Safrole, a member of this class, has been banned as a food or feed additive in many countries based on its genotoxicity and carcinogenicity. However, it can still enter the food and feed chain. There is limited information about the toxicity of other alkenylbenzenes that may be present in safrole-containing foods, such as myristicin, apiole, and dillapiole. In vitro studies showed safrole as mainly bioactivated by CYP2A6 to form its proximate carcinogen, while for myristicin this is mainly done by CYP1A1. However, it is not known whether CYP1A1 and CYP2A6 can activate apiole and dillapiole. The present study uses an in silico pipeline to investigate this knowledge gap and determine whether CYP1A1 and CYP2A6 may play a role in the bioactivation of these alkenylbenzenes. The study found that the bioactivation of apiole and dillapiole by CYP1A1 and CYP2A6 is limited, possibly indicating that these compounds may have limited toxicity, while describing a possible role of CYP1A1 in the bioactivation of safrole. The study expands the current understanding of safrole toxicity and bioactivation and helps understand the mechanisms of CYPs involved in the bioactivation of alkenylbenzenes. This information is essential for a more informed analysis of alkenylbenzenes toxicity and risk assessment.

A Computational Inter-Species Study on Safrole Phase I Metabolism-Dependent Bioactivation: A Mechanistic Insight into the Study of Possible Differences among Species.

Safrole, a 162.2 Da natural compound belonging to the alkenylbenzenes class, is classified as a possible carcinogen to humans by IARC (group IIB) and has proven to be genotoxic and carcinogenic to rodents. Despite its use as a food or feed additive, it is forbidden in many countries due to its documented toxicity; yet, it is still broadly present within food and feed and is particularly abundant in spices, herbs and essential oils. Specifically, safrole may exert its toxicity upon bioactivation to its proximate carcinogen 1'-hydroxy-safrole via specific members of the cytochrome P450 protein family with a certain inter/intra-species variability. To investigate this variability, an in-silico workflow based on molecular modelling, docking and molecular dynamics has been successfully applied. This work highlighted the mechanistic basis underpinning differences among humans, cats, chickens, goats, sheep, dogs, mice, pigs, rats and rabbits. The chosen metric to estimate the likeliness of formation of 1'-hydroxy-safrole by the species-specific cytochrome P450 under investigation allowed for the provision of a knowledge-based ground to rationally design and prioritise further experiments and deepen the current understanding of alkenylbenzenes bioactivation and CYPs mechanics. Both are crucial for a more informed framework of analysis for safrole toxicity.

Experimental-theoretical study of laccase as a detoxifier of aflatoxins.

We investigate laccase-mediated detoxification of aflatoxins, fungal carcinogenic food contaminants. Our experimental comparison between two aflatoxins with similar structures (AFB1 and AFG2) shows significant differences in laccase-mediated detoxification. A multi-scale modeling approach (Docking, Molecular Dynamics, and Density Functional Theory) identifies the highly substrate-specific changes required to improve laccase detoxifying performance. We employ a large-scale density functional theory-based approach, involving more than 7000 atoms, to identify the amino acid residues that determine the affinity of laccase for aflatoxins. From this study we conclude: (1) AFB1 is more challenging to degrade, to the point of complete degradation stalling; (2) AFG2 is easier to degrade by laccase due to its lack of side products and favorable binding dynamics; and (3) ample opportunities to optimize laccase for aflatoxin degradation exist, especially via mutations leading to π-π stacking. This study identifies a way to optimize laccase for aflatoxin bioremediation and, more generally, contributes to the research efforts aimed at rational enzyme optimization.

Perfluoroalkyl substances (PFASs) are substrates of the renal human organic anion transporter 4 (OAT4).

Poly- and perfluoroalkyl substances (PFASs) are omnipresent in the environment and have been shown to accumulate in humans. Most PFASs are not biotransformed in animals and humans, so that elimination is largely dependent on non-metabolic clearance via bile and urine. Accumulation of certain PFASs in humans may relate to their reabsorption from the pre-urine by transporter proteins in the proximal tubules of the kidney, such as URAT1 and OAT4. The present study assessed the in vitro transport of 7 PFASs (PFHpA, PFOA, PFNA, PFDA, PFBS, PFHxS and PFOS) applying URAT1- or OAT4-transfected human embryonic kidney (HEK) cells. Virtually no transport of PFASs could be measured in URAT1-transfected HEK cells. All PFASs, except PFBS, showed clear uptake in OAT4-transfected HEK cells. In addition, these in vitro results were further supported by in silico docking and molecular dynamic simulation studies assessing transporter-ligand interactions. Information on OAT4-mediated transport may provide insight into the accumulation potential of PFASs in humans, but other kinetic aspects may play a role and should also be taken into account. Quantitative information on all relevant kinetic processes should be integrated in physiologically based kinetic (PBK) models, to predict congener-specific accumulation of PFASs in humans in a more accurate manner.

Sprouts of Moringa oleifera Lam.: Germination, Polyphenol Content and Antioxidant Activity.

(1) Background: In recent years, the consumption of sprouts, thanks to their high nutritional value, and the presence of bioactive compounds with antioxidant, antiviral and antibacterial properties, is becoming an increasingly widespread habit. Moringa oleifera Lam. (Moringa) seems to be an inexhaustible resource considering that many parts may be used as food or in traditional medicine; on the other hand, Moringa sprouts still lack a proper characterization needing further insights to envisage novel uses and applications. (2) Methods: In this study, a rapid and easy protocol to induce the in vivo and in vitro germination of Moringa seeds has been set up to obtain sprouts and cotyledons to be evaluated for their chemical composition. Moreover, the effects of sprouts developmental stage, type of sowing substrate, and gibberellic acid use on the chemical characteristics of extracts have been evaluated. (3) Results: Moringa seeds have a high germinability, both in in vivo and in vitro conditions. In addition, the extracts obtained have different total phenolic content and antioxidant activity. (4) Conclusions: This research provides a first-line evidence to evaluate Moringa sprouts as future novel functional food or as a valuable source of bioactive compounds.

Risk-benefit assessment of shifting from traditional meat-based diets to alternative dietary patterns.

The consumption of plant-based meat alternatives has recently transitioned from the niche market to the mainstream. However, changing the dietary patterns may lead to new health challenges referred to possible higher exposure to natural toxins found in plant-based food. The present project aimed to train the fellow in the field of chemical risk assessment and provide a comprehensive overview on how a change towards plant-based meat alternatives may represent a driver for emerging risks. Thus, within the EU-FORA programme the fellow engaged in the following activities: (i) perform a systematic review to analyse the distribution of natural toxins in the most common plant-based meat alternatives in Europe, (ii) risk-benefit assessment of shifting from meat-based diets to soy-based meat alternatives by modelling the substitution of meat with soy, and (iii) determine the occurrence of mycotoxins in plant-based meat alternatives from local markets in Parma, Italy. The fellow learnt the chemical risk assessment procedures applied by the researchers from the Department of Food and Drug of Parma University, thus gaining an in-depth expertise in all the steps. A risk-benefit assessment was performed modelling the intake of aflatoxin B1-contaminated soy-based meat analogues. The health impact due to intake of soy and exposure to aflatoxin B1 was estimated. Within the research group, the fellow also worked on developing a multi-mycotoxin determination method for plant-based meat alternatives matrices. The results of the project provide a picture reflecting the occurrence of natural toxins in plant-based meat alternatives and the need of upgraded regulation frameworks that take into account new products and dietary patterns. The EU-FORA fellowship was a great opportunity for the fellow to expand his professional network and increase his expertise in food safety by gaining new skills in chemical risk assessment, risk-benefit assessment and analytical chemistry.

A mechanistic investigation on kokumi-active γ-Glutamyl tripeptides - A computational study to understand molecular basis of their activity and to identify novel potential kokumi-tasting sequences.

Kokumi is an important taste perception whose chemical basis still needs clarifications and for which the development of high-throughput tools of analysis is desirable. The activation of Calcium-sensing receptor (CaSR) was described as the basis of kokumi perception allowing the use of molecular modelling to deepen its chemical rationale and related mechanisms. This study focused on γ-Glutamyl tripeptides, computationally providing mechanistic insights on their CaSR-activating properties and extended the comprehension of their structure-activity relationship. A library of 400 γ-Glutamyl tripeptides was also screened. γ-Glu-Pro-Ala and γ-Glu-Pro-Ser were identified for further dedicated investigations based on their promising CaSR-activating potential and their presence should be checked accordingly in food matrices to better profile the kokumi fingerprint. This work provided a meaningful tool for the top-down analysis of kokumi-active molecules that may support either the identification of kokumi molecules concealed in food or the rational design of kokumi-active molecules de novo.

An In Silico Framework to Mine Bioactive Peptides from Annotated Proteomes: A Case Study on Pancreatic Alpha Amylase Inhibitory Peptides from Algae and Cyanobacteria.

Bioactive peptides may exert beneficial activities in living organisms such as the regulation of glucose metabolism through the inhibition of alpha amylases. Algae and cyanobacteria are gaining a growing interest for their health-promoting properties, and possible effects on glucose metabolism have been described, although the underlying mechanisms need clarification. This study proposes a computer-driven workflow for a proteome-wide mining of alpha amylase inhibitory peptides from the proteome of Chlorella vulgaris, Auxenochlorella protothecoides and Aphanizomenon flos-aquae. Overall, this work presents an innovative and versatile approach to support the identification of bioactive peptides in annotated proteomes. The study: (i) highlighted the presence of alpha amylase inhibitory peptides within the proteomes under investigation (including ELS, which is among the most potent inhibitory tripeptides identified so far); (ii) mechanistically investigated the possible mechanisms of action; and (iii) prioritized further dedicated investigation on the proteome of C. vulgaris and A. flos-aquae, and on CSSL and PGG sequences.

Cleaning the Label of Cured Meat; Effect of the Replacement of Nitrates/Nitrites on Nutrients Bioaccessibility, Peptides Formation, and Cellular Toxicity of In Vitro Digested Salami.

Curing salts composed of mixtures of nitrates and nitrites are preservatives widely used in processed meats. Despite many desirable technological effects, their use in meat products has been linked to methemoglobinemia and the formation of nitrosamines. Therefore, an increasing "anti-nitrite feeling" has grown among meat consumers, who search for clean label products. In this view, the use of natural compounds as alternatives represents a challenge for the meat industry. Processing (including formulation and fermentation) induces chemical or physical changes of food matrix that can modify the bioaccessibility of nutrients and the formation of peptides, impacting on the real nutritional value of food. In this study we investigated the effect of nitrate/nitrite replacement with a combination of polyphenols, ascorbate, and nitrate-reducing microbial starter cultures on the bioaccessibility of fatty acids, the hydrolysis of proteins and the release of bioactive peptides after in vitro digestion. Moreover, digested salami formulations were investigated for their impacts on cell proliferation and genotoxicity in the human intestinal cellular model (HT-29 cell line). The results indicated that a replacement of synthetic nitrates/nitrites with natural additives can represent a promising strategy to develop innovative "clean label" salamis without negatively affecting their nutritional value.

Mechanistic Insights into Angiotensin I-Converting Enzyme Inhibitory Tripeptides to Decipher the Chemical Basis of Their Activity.

Food proteins are an important source of bioactive peptides, and the angiotensin I-converting enzyme (ACE) inhibitors are worthy of attention for their possible beneficial effects in subjects with mild hypertension. However, the chemical basis underpinning their activity is not well-understood, hampering the discovery of novel inhibitory sequences from the plethora of peptides encrypted in food proteins. This work combined computational and in vitro investigations to describe precisely the chemical basis of potent inhibitory tripeptides. A substantial set of previously uncharacterized tripeptides have been investigated in silico and in vitro, and LCP was described for the first time as a potent ACE inhibitory peptide with IC50 values of 8.25 and 6.95 µM in cell-free and cell-based assays, respectively. The outcomes presented could serve to better understand the chemical basis of already characterized potent inhibitory tripeptides or as a blueprint to design novel and potent inhibitory peptides and peptide-like molecules.