NANOTOXICOLOGY AND NANOSAFETY GROUP
The research of the group is focused into the toxicological evaluation of nanomaterials and in the biological studies supporting the safe-by-design process of new nanomaterials for biomedical applications, using in vitro and in vivo biological models, alternative to the use of mammals
Staff: Paride Mantecca; Luisa Fiandra, Rossella Bengalli, Pamela Floris
1) BIO-INTERACTIONS AND TOXICITY OF NANOMATERIALS ON 2D AND 3D IN VITRO HUMAN RESPIRATORY BARRIER
- In vitro assays for testing nanoparticles pulmonary toxicity. In vitro testing of the hazardous properties of NPs on lung cells are performed to detect the adverse effects of nanodrugs for lung diseases (i.e. antimicrobials, anti-cancer nanodrugs), disclosing the relationships between the NPs physico-chemical properties and the toxicity outcomes. 2D cell models representative of the pulmonary apparatus including epithelial cells (human lung cells) and immune cells (monocytic THP-1) are used for screening cytotoxic, genotoxic and pro-inflammatory activity of the NPs. When possible, standard OECD or ISO tests are performed to match the regulatory frameworks. Interaction among NPs and cells and morphological alterations of are detected by withe-field and fluorescence optical microscopy and through transmission electron microscopy (TEM) and scanning electron microscopy (SEM),
together with correlative microscopy.
- Airborne NPs toxicity using 3D in vitro models of the pulmonary barriers. NPs lung toxicity can be also tested on models of human respiratory barriers. A 3D alveolar blood barrier has been developed to be exposed to NPs in submerged and/or air liquid interface (ALI). Epithelial barrier integrity (transepithelial electrical resistance– TEER), cell viability, pro-inflammatory cytokines release and microvascular endothelium activation (ICAM-1, VCAM-1, IL-8, MCP-1) are measured as toxicity endpoints. Predictive models for the evaluation of NPs distribution and dosimetry can be applied to evaluate real life exposure scenarios.
2) STUDIES ON THE BIOCOMPATIBILITY OF NANOPARTICLES AND NANOENABLED PRODUCTS (NEPs) USING SKIN MODELS
- OECD standard protocols for Epiderm 3D models. The toxicity of nanomaterials which come into contact with skin (i.e. antibacterials, cosmetics), such as of extracts from medical devices coated with nanoparticles, can be tested against human skin, using the standard in vitro protocols Corrosion Skin test (OECD 431) and Irritation Skin test for Medical Device extracts (ISO/TC 194/WG 8 for MD extracts). These tests are based on the exposure of commercially available in vitro 3D human reconstructed skin tissues, grown on transwell filters, to NP solutions and extracts from the coated materials, and the following measurement of cell viability by MTT. On the same 3D models is possible to apply the in vitro micronucleus (MN) test according to OECD TG 487 to evaluate genotoxicity.
- Assays on 2D in vitro skin models. Cytotoxicity and MN tests on 2D culture of human keratinocytes are also used to assess the adverse effects of nanomaterials and extracts on epidermis. Moreover, dermal cells can be employed to understand the cytotoxicity triggered by NPs and extracts in case of wounded skin. To this purpose, MTT and Colony Forming efficiency (CFE) assays are applied to Balb/c 3T3 fibroblasts. Interaction among NPs and cells and morphological alterations of are detected by withe-field and fluorescence optical microscopy and through TEM and SEM, together with correlative microscopy.
- Skin sensitization assays. Skin sensitization can be additionally evaluated by measuring the inflammatory cytokines released from epidermis and dermal cells, while the phototoxicity test Neutral Red Uptake assay on Balb/c 3T3 is useful to assess the photoactivation exerted by skin care products with and without UV irradiation (OECD TG. 432). Futhermore, the Human Cell Line Activation test (h-CLAT) can be performed according to the OECD TG 442E to evaluate skin sensitizers. The test is an in vitro assay based on the quantification of changes of cell surface marker expression (i.e. CD86 and CD54) by cytofluorimetric analysis on a human monocytic leukemia cell line (THP-1), following 24 hours exposure to the test chemical. These surface molecules are typical markers of monocytic THP-1 activation and mimic dendritic cells DC activation, which plays a critical role in skin sensitization.
3) ALTERNATIVE IN VIVO MODELS TO EVALUATE TOXICITY AND ADVERSE OUTCOME PATHWAYS (AOP) OF NMs AND NEPs
- The alternative model zebrafish. Zebrafish (Danio rerio) is a teleost fish from Southeast Asia, living in small rivers, paddy fields and streams. Zebrafish have a relatively short generation time, reaching the adulthood in nearly three months after fertilization, with a lifespan of two-three years. They are prolific breeders, generating approximately two hundred of embryos per week. In UNIMIB there is a recently founded facility, which holds some zebrafish lines, including wild type and transgenic ones.
- Fish Embryo Acute Toxicity (FET) test. Zebrafish is widely accepted as a model for toxicological studies. Organisation for Economic Co-operation and Development (OECD) recommends the FET test (OECD Guideline n. 236) to determine toxicity of a wide variety of chemicals on embryonic stages of zebrafish. Moreover, the NM toxicity can be investigated using a FET approach. During FET test, we expose zebrafish fertilized eggs to chemicals for 96 hours. Every 24 hours, we record different morphological observations as indicators of lethality. At the end of the exposure period, acute toxicity is determined based on a positive outcome in any of the observations recorded. After FET test, sublethal effects can be assessed in embryos exposed.
- Assessment of NM-induced AOPs and contribution to Safe-by-Design (SbD). In the last years adverse outcome pathways (AOPs) have gained more and more attention to provide mechanistic links and explanations of NM effects. In vitro models representative of the main expected human exposure routes (e.g. respiratory and skin barriers) will be used to characterize not only the dose-response curves according to the specific NM p-chem properties, but also the biomarkers able to specifically respond to NP-cell interactions at cell membrane level and/or after internalization. Among the main exploitable pathways, the cascade of the NMinduced ROS oxidative stress cascade leading to autophagic or apoptotic mechanisms are evaluated, to finally retrieve sensible cell markers for “specific” NP exposure. Such strategy is persecuted by using normal or transfected human cell lines and zebrafish (ZF) embryos (WT or mutant strains, i.e. GFP-Lc3 for autophagy). The study of the AOPs will be performed by coupling molecular biology analyses with advanced microscopy techniques, aimed at describing the bio-nano-interactions at subcellular level. This approach can improve the knowledge on the possible contribution of the NP chronic exposure to human diseases or environmental toxicity and in parallel adds relevant hazard performance attributes for the evaluation of the SbD solutions.
- Horizon 2020 Framework Program H2020 (H2020-720851 project PROTECT—Precommercial lines for production of surface nanostructured antimicrobial and anti-biofilm textiles, medical devices, and water treatment membranes) (www.protect-h2020.eu)
- Fondazione Cariplo grant to the project OverNanoTox (2013-0987) “Do new generations of nano-antibacterials OVERcome the epithelial barriers posing human health at risk? A predictive nanoTOXicology study (OVER NanoTOX)
RECENT PUBLICATIONS (from 2017 )
1. Colombo, A, Saibene, M, Moschini, E, Bonfanti, P, Collini, M, Kasemets, K, Mantecca, P. 2017. Teratogenic hazard of BPEI-coated silver nanoparticles to Xenopus laevis. NANOTOXICOLOGY 11 (3): 405-418, DOI: 10.1080/17435390.2017.1309703
2. Ahonen, M, Kahru, A, Ivask, A, Kasemets, K, Koljalg, S, Mantecca, P, Vrcek, IV, Keinanen-Toivola, MM, Crijns, F. 2017. Proactive Approach for Safe Use of Antimicrobial Coatings in Healthcare Settings: Opinion of the COST Action Network AMiCI. INTERNATIONAL JOURNAL OF ENVIRONMENTAL RESEARCH AND PUBLIC HEALTH 14 (4), 366. DOI: 10.3390/ijerph14040366
3. Mantecca, P, Kasemets, K, Deokar, A, Perelshtein, L, Gedanken, A, Bahk, YK, Kianfar, B, Wang, J. 2017. Airborne Nanoparticle Release and Toxicological Risk from Metal-Oxide-Coated Textiles: Toward a Multiscale Safe-by-Design Approach. ENVIRONMENTAL SCIENCE & TECHNOLOGY 51 (16): 9305-9317, DOI: 10.1021/acs.est.7b02390
4. Bengalli, R, Ferri, E, Labra, M, Mantecca, P. 2017. Lung Toxicity of Condensed Aerosol from E-CIG Liquids: Influence of the Flavor and the In Vitro Model Used. INTERNATIONAL JOURNAL OF ENVIRONMENTAL RESEARCH AND PUBLIC HEALTH 14 (10): 1254, DOI:10.3390/ijerph14101254
5. Bonfanti P., Saibene M., Bacchetta R., Mantecca P., Colombo A. 2018. A glyphosate micro-emulsion displays teratogenicity in Xenopus laevis. AQUATIC TOXICOLOGY 195: 103-113, DOI: 10.1016/j.aquatox.2017.12.007
6. Sheehan B., Murphy F., Mullins M., Furxhi I., Costa AL., Simeone FC., Mantecca P. 2018. Hazard Screening Methods for Nanomaterials: A Comparative Study. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 19, 649; doi:10.3390/ijms19030649
7. Furxhi, I, Murphy, F, Poland, CA, Sheehan, B, Mullins, M, Mantecca, P. 2019. Application of Bayesian networks in determining nanoparticle-induced cellular outcomes using transcriptomics. NANOTOXICOLOGY. DOI:10.1080/17435390.2019.1595206
8. Kasemets, K, Kaosaar, S, Vija, H, Fascio, U, Mantecca, P. 2019. Toxicity of differently sized and charged silver nanoparticles to yeast Saccharomyces cerevisiae BY4741: a nano-biointeraction perspective. NANOTOXICOLOGY 13, 8: 1041-1059
9. Bengalli, R, Ortelli, S, Blosi, M, Costa, A, Mantecca, P, Fiandra, L. 2019. In Vitro Toxicity of TiO2:SiO2 Nanocomposites with Different Photocatalytic Properties. NANOMATERIALS 9, 7:1041; DOI: 10.3390/nano9071041
10. Colombo, G, Cortinovis, C, Moschini, E, Bellitto, N, Perego, MC, Albonico, M, Astori, E, Dalle-Donne, I, Bertero, A, Gedanken, A, Perelsthein, I, Mantecca, P, Caloni, F. 2019. Cytotoxic and proinflammatory responses induced by ZnO nanoparticles in in vitro intestinal barrier. JOURNAL OF APPLIED TOXICOLOGY 39, 8: 1155-1163; DOI: 10.1002/jat.3800
11. Zerboni, A,, Bengalli, R, Baeri, G, Fiandra, L, Catelani, T, Mantecca, P. Mixture Effects of Diesel Exhaust and Metal Oxide Nanoparticles in Human Lung A549 Cells. NANOMATERIALS, In press