Innovative In Vitro Strategies for Food and Environmental Safety

dis-rupting

species-specific in vitro models. Models for oral and topical exposure to emerging mycotoxins, alone or combined also with traditional mycotoxins, were applied, such as human intestinal barrier with Caco-2 cells differentiated on inserts in serum-free medium (Ferruzza et al., 2012), species-specific intestinal barrier with IPEC-J2 cells cultured on inserts (Zakrzewski et al., 2013), and human reconstructed skin and cornea (MatTek Corporation). The toxicological effects of the mycotoxins on trans-epithelial electrical resistance (TEER) and cytokine release in the intestinal barriers and on cell viability (MTT) in the human reconstructed skin and cornea models were evaluated. A novel integrated in vitro toxicological evaluation is proposed that combines human and species-specific models (i.e., bovine granulosa cell, swine intestinal models, human barriers, etc.).
Isabella De Angelis, Valentina Prota and Olimpia Vincentini, Istituto Superiore di Sanità (ISS), described a pilot study to develop an OECD Test Guideline on an in vitro approach for determining the gastrointestinal fate of ingested nanomaterials. Several parameters can influence the physical-chemical properties and bioavailability of nanomaterials (NMs) during their passage through the gastrointestinal tract. Caco-2 monoculture on inserts is an ideal system for rapid assessment of intestinal permeability, Our diet and the environment are relevant sources of xenobiotic exposure for humans (Alloisio et al., 2019). In recent years, the scientific community has become increasingly interested in non-occupational exposure to contaminants and their mixtures and in their adverse effects. Attention has focused primarily on chemical agents but, more recently, also on biological and physical contaminants. Several bioassays have been proposed as tools to investigate the hazards of these unintentional exposures. In vitro models for food and environmental safety were discussed at the meeting "In vitro toxicology: Innovative strategies for food and environmental safety" organized by CELLTOX Italian Association of in vitro Toxicology in May 2020, which was presented as a webinar in compliance with the containment measures of SARS-CoV-2 infection. The meeting was chaired by Diego Baderna and Susanna Alloisio and attracted more than 100 participants.
Francesca Caloni and Alessia Bertero, Università degli Studi di Milano (ESP), presented an integrated approach for testing the emerging mycotoxins beauvericin (BEA) and enniatin B1 (EN-NB1), natural food and feed contaminants of emerging concern (Caloni et al., 2020;Albonico et al., 2017;Prosperini et al., 2017), using an in vitro strategy based on the combination of human and

Meeting Report
Innovative In Vitro Strategies for Food and Environmental Safety doi:10.14573/altex.2006261 tagenicity, tumor promotion, cell transformation, endocrine disrupting compounds (EDCs) activity. The overall approach combines physicochemical parameters, biological and ecotoxicological tests to assess the impact on human and wildlife health and to determine strategies for purification systems to break down pollutants.
Diego Baderna, Istituto di Ricerche Farmacologiche Mario Negri and Italian Institute for Planetary Health, focused on the use of in vitro models for the investigation of landfill leachate toxicity (Baderna et al., 2011(Baderna et al., , 2019. A recent review, covering 23 papers published in the period 2003-2018, compiled cell models that reflect potential targets of direct exposure to leachate (e.g., liver, skin and blood) and models for the study of specific mechanisms of action (e.g., breast cancer cells for the study of endocrine interference) covering the endpoints basal toxicity (viability, antiproliferative effects, cytotoxicity and apoptosis), genotoxicity, and endocrine interference. The studies showed that leachate is toxic, genotoxic, and can act as an estrogen-like compound and that the leachate from uncontrolled landfills tends to be more toxic than that from controlled landfills. It is clear that leachate can be a serious danger to human health and that a good management strategy must be adopted to avoid human exposure. The review highlighted how in vitro models were used not only for the study of leachate toxicity but also to evaluate the efficiency of purification and detoxification treatments. Studies showed that these treatments can reduce leachate toxicity, but not all of them are efficient in removing genotoxicity, probably due to the formation of highly reactive by-products.
The meeting highlighted the importance of multidisciplinary approaches to deal with common and emerging pollutants and their effects on humans and wildlife. In fact, the value of highly sensitive in vitro assays (both human-related and ecological) for assessing hazards and identifying modes of action is being increasingly recognized, not only for the study of toxicity, but also for environmental monitoring and the evaluation of detoxifying treatments. This has made biological assays indispensable tools alongside chemical analyses in this area.

References
Albonico, M., Schutz, L. F., Caloni, F. et al. (2017). In vitro effects of the Fusarium mycotoxins fumonisin B 1 and beauvericin on bovine granulosa cell proliferation and steroid production.  (Beloqui et al., 2017). An advanced tri-culture model based on Caco-2 cells/HT29-MTX mucus secreting cells/lymphoblastoid Raji-B cells has been developed for qualitative/quantitative assessment of absorption and translocation of NMs (García-Rodríguez et al., 2018;Kämpfer et al., 2020). Conversion of Caco-2 cells into M-like cells, achieved by co-culturing Caco-2 with Raji B cells, has been assessed by electrophysiological, histological, biochemical and functional measurements. Different endpoints of M cell phenotype induction and mucus production in the tri-culture model, such as TEER decrease, ZO-1 protein expression, barrier permeability to FITC-dextran and fluorescent silica nanoparticles have been discussed. Current results highlight the need for further standardization of experimental protocols to increase robustness and data comparability. Susanna Alloisio, Cristina Scarone and Francesco Misurale presented an integrated in vitro/in vivo approach applied to the evaluation of the effects of harmful algal blooms (HABs) on human and environmental health. HABs occur when algae, unicellular photosynthetic organisms that live in the sea and freshwater, grow out of control, producing toxins which are harmful to people, fish, shellfish, marine mammals and birds (Perovic et al., 2000;Ben-Gharbia et al., 2016). In the proposed approach, four dinoflagellates were selected: two toxic species (Ostreopsis cf. ovata and Prorocentrum lima); one species with uncertain toxicity (Coolia monotis); and a non-toxic species (Scripsiella cf. trochoidea). The main objectives were i) the optimization of the protocol for a fast and simple algal preparation that allows direct administration of the toxin mixture to mammalian cell culture, ii) the in vitro evaluation of skin and eye irritation potential of the algae using reconstructed human epidermis and corneal tissue, iii) the investigation of the neurotoxic potential of the algae by means of MTT-assay and MEA-based electrophysiological approach on cultured primary murine neuronal networks, and iv) the evaluation of algal ecotoxicity by means of a lethal toxicity test performed on Artemia franciscana nauplii.
Nathalie Steimberg, University of Brescia, focused on the development of a battery of in vitro tests to assess the biological impact of wastewater. The characteristics of wastewater are studied primarily from a chemical-physical point of view, but biological tests and ecotoxicological tools are needed to better understand the environmental impact of these complex mixtures (Pedrazzani et al., 2020). While chemical analysis of the aqueous mixtures allows identification and quantification of known substances and their transformation products, biological tests assess the impact of the mixtures. The longstanding scientific collaboration within the Mistral interuniversity research center have allowed the development of a complex and multifaceted approach for the evaluation of the environmental footprint of wastewater and the efficacy of wastewater treatment plants (Bertanza et al., 2011(Bertanza et al., , 2013Papa et al., 2016;Pedrazzani et al., 2020). The biological endpoints investigated include: i) baseline toxicity: algal growth inhibition, Microtox, Daphnia magna motility assay, MTT and neutral red assays in mammalian cells, ii) specific target: genotoxicity/mu-