EEMGS Annual Meeting 2021
More info: https://www.eemgs.eu
In vitro and In vivo safety assessment of AgNPs-silica to be applied in animal feed as an alternative to antibiotics
Adriana Rodriguez-Garraus, Maria Alonso-Jáuregui, Amaya Azqueta, Adela López de Cerain
Machine Learning and Mechanistic information from a Multi Endpoint Genotoxicity Screen
Wilson, A, Elloway, J, Grabowski, P , Ebbles, T and Doherty, A.
Acoustic dosing (≤2mg) of compound is followed by a 24-hour treatment and a 24-hour recovery period. Confocal images are captured (Cell Voyager™ C7000 (Yokogawa, Japan)) and analysed using Columbus™ software (PerkinElmer). As standard the assay detects micronuclei (MN), cytotoxicity, nuclear fragmentation and condensation as well as cell-cycle profiles from Hoechst phenotypes. Mechanism of action information is primarily determined by kinetochore labelling in MN (aneugencity) and H2AX foci and pan analysis (DNA damage marker). Complex multiparametric data-sets containing 11,310 data points are generated per compound. PCA and t-SNE plots cluster compounds by mechanism of action (MOA). Applying computational approaches in R and implementing machine learning models alongside Bayesian classifiers allows the identification of, with 95% accuracy, the aneugenic, clastogenic and negative compounds within the data set (Matthews correlation coefficient: 0.9), reducing analysis time by 80% whilst concurrently minimising human bias. The data sets generated provide novel biomarkers for further compound classification and complimentary immunofluorescence analysis of multiple DNA damage response proteins (p53, 53BP1 and Rad50), as well as markers targeted to phosphorylated Histone H3, β-Tubulin and Aurora B, amongst others, provides further mechanistic understanding of genotoxic response and opportunities for further compound profiling. Combining high throughput screening, multiparametric image analysis and machine learning approaches has provided the opportunity to revolutionise early Genetic Toxicology assessment within AstraZeneca. By multiplexing assay endpoints, minimising data generation and analysis time and providing novel insights to the mechanisms and phenotypes that are associated with genotoxicity this assay enables complex genotoxicity safety assessments to be made sooner aiding the development of safer drug candidates.
COMPARISON OF SUBSTRATE SPECIFICITY OF HOMOLOGOUS AP-ENDONUCLEASES FROM INSECT, AMPHIBIAN, FISH AND HUMAN
Anastasiia T. Davletgildeeva1,2, Alexander A. Ishchenko3, Murat Saparbaev3, Olga S. Fedorova1, Nikita A. Kuznetsov1
Funding: this work was supported by Ph.D. grant 20-34-90008 from Russian Foundation for Basic Research.
UTILISING DUPLEX SEQUENCING TO ADVANCE THE ASSESSMENT OF MUTATIONS IN A GENETIC TOXICOLOGY ASSAY
Anne Ashford1, Toni Hering1, Emily Lythgoe1, Jan Thackray1, Raj Gandhi1, Rhiannon David1, Jo Elloway1, Ann Doherty1
In vitro carcinogenicity testing of complex Non-genotoxic carcinogens
Demi Pritchard1, Gareth Jenkins1
Equid milk: a well of safe probiotic bacteria and their metabolites with antioxidant properties
Deni Kostelac1, Marko Gerić2, Goran Gajski2, Jadranka Frece1
Funding: Faculty of Food Technology and Biotechnology, University of Zagreb funded this study.
Possible use of blood-derived biobank samples for analysis with the comet assay
Ezgi Eyluel Bankoglu, Johanna Gerber, Carolin Schuele, Franziska Stipp and Helga Stopper
REDUCED LEVELS OF HARMFUL AND POTENTIALLY HARMFUL CONSTITUENTS IN HEATED TOBACCO AEROSOL TRANSLATE TO REDUCED IN VITRO (GENO)TOXICOLOGICAL OUTCOMES
Fiona Chapman1 ; Roman Wieczorek2 ; Edgar Trelles Sticken2 ; Sarah Jean Pour2 ; Ole Dethloff2 ; Matthew Stevenson1
THE CYTOTOXIC POTENTIAL OF MESOPOROUS SILICA NANOPARTICLES LOADED ANTI-CANCER DRUG IN 3D COLON CANCER CELLS
Ibemusu Otele1, Elshaimaa Sayed2, Ketan Ruparelia2, Zeeshan Ahmad2, Neenu Singh1
Identification of Senolytic Agents for Sequential Treatment of GBM with TMZ
Lea C. Beltzig, Björn Stratenwerth, Markus Christmann, Bernd Kaina
Objectives. Here we tested natural substances for their senolytic capacity. Also, several proteins involved in the induction of senescence were tested for their importance in maintaining the senescent state.
Materials & Methods. The GMB cell line LN229 was used for analysis of cell death and senescence following TMZ with and without senolytic treatment. Cell death and senescence were measured via flow cytometry, using Annexin V-PI or C12FDG staining, respectively. Natural substances tested were fisetin and curcumin. The specific signaling pathway inhibitors were ABT-737, AZD1390, VE-821, UCN-01, Chk2i II, NF-κBi III, UC2288, 3-Methyladenine (3-MA), BV6 , pifithin-α, pifithrin-µ and chloroquine (Clq).
Results. Eight to ten days following TMZ treatment up to 80 % of senescent cells and 20-30 % of apoptotic cells were measured in the population. Fisetin, ABT 737, Clq and 3-MA significantly reduced senescence while cell death was increased significantly. The ATM and ATR inhibitors and pifithrin-α were also able to reduce senescence but only slightly increased cell death by apoptosis. No such effects were seen after treatment of senescent cells with IR, curcumin, the inhibitors of Chk1, Chk2, NF-κB or p21 and pifithrin-µ (data not shown).
Conclusion. Fisetin, Clq, ABT-737 and 3-MA were identified as senolytics by a decrease in senescence and increase in cell death. ATM, ATR and p53 play critical roles in the induction of senescence. They also seem to be important in maintaining senescence, since inhibition lead to a decrease of the senescent population. However, since no significant increase in the cell death rate was observed, the corresponding inhibitiors do not have senolytic properties. Inhibition of Chk1, Chk2, p21, NF-κB and p53 had no effect on senescenct cells, therefore they neither seem to play a critical role in maintaining senescence nor can they be used as senolytics. Since IR and curcumin did not reduce the senescence level, they cannot be considered to be senolytics.
Automated High Content Screening for Carcinogenicity Testing In Vitro
Linda Reilly*1, Amy Wilson2, Jo Elloway2, Ann Doherty2, Gareth J Jenkins1
This project aims to integrate traditional genotoxicity data with more advanced holistic data on cell phenotype using image analysis to provide an advanced multi-disciplinary in vitro testing platform. This novel testing platform is currently being validated at Swansea University and compared to a similar robotic Multi-Endpoint Genotoxicity Assessment (MEGA) system under development at AstraZeneca laboratories, Cambridge, UK.
The MEGA-Screen based on confocal microscopy and image analysis allows for a multiplexed assessment of both DNA damage and phenotypic markers in a single assay. At Swansea, another multi-endpoint system for carcinogenic mode action looks at morphological analysis, cell cycle and cell signaling perturbations, Micronuclei induction, ROS generation and mitochondrial toxicity separately measured with parallel platforms. By evaluating the two systems in parallel, we can refine the optimal testing platform for safety assessment in drug development and give a more reliable prediction of carcinogenicity.
Cisplatin and Temozolomide were two test compounds initially assessed for genotoxicity using the micronucleus assay and cell cycle perturbations using flow cytometry in both TK6 and A549 cell lines; and results were compared across both test systems as an initial phase of validation.
Funding: MRC Integrative Toxicology Training Partnership
PRODIAMESA OLIVACEA: NEW IMMUNE AND STRESS BIOMARKER GENES IN A POTENTIAL SENTINEL ORGANISM FOR ECOTOXICITY STUDIES IN NATURAL SCENARIOS
Lola Llorente1; Óscar Herrero1; Mónica Aquilino1; Rosario Planelló1
Assessing occuptional safety: a story of interventional radiology unit teams
Marko Gerić1; Jelena Popić2; Goran Gajski1, and Vera Garaj-Vrhovac1
Funding: Croatian Ministry of Science, Education and Sports (grant No. 022-0222148-2125) and the Institute for Medical Research and Occupational Health funded this study.
GENOTOXICITY AND C. ELEGANS – USING THE WORM FOR ASSESSMENT OF MN-INDUCED DNA DAMAGE AND DNA DAMAGE RESPONSE
Merle M. Nicolai1; Nicola Winkelbeiner2,3; Ann-Kathrin Weishaupt1,3, Anna Gremme1, Tanja Schwerdtle2,3,4, Julia Bornhorst1,3*
EVALUATION OF THE PHYSIOLOGICAL RESPONSE OF CHORTHIPPUS PARALLELUS (ORTHOPTERA) TO WOLBACHIA INFECTION: CHARACTERIZATION OF NEW MOLECULAR BIOMARKERS
Mónica Aquilino1; Lola Llorente1, Patricia Jiménez-Doradoz2, Óscar Herrero1, David Buckley2, José L. Bella2, Rosario Planelló1
This research is funded by the Spanish government (Ministerio de Ciencia, Innovación y Universidades), under the grant PID2019-104952GB-I00/AEI/10.13039/501100011033. Mónica Aquilino was awarded a postdoctoral contract (PEJD-2019-POST/AMB-16425) co-financed by UNED and the Government of the Community of Madrid.
ANALYSIS OF COHESIN COMPLEX PROTEINS IN RESPONSE TO GENOTOXICITY USING AUTOMATED IMAGE ANALYSIS
Natasha Irani1,2, Raj Gandhi1, Amy Wilson1, Rhiannon David1, Ann Doherty1 and Nigel J Gooderham2
ENVIRONMENTAL AND LIFESTYLE FACTORS IN THE DEVELOPMENT OF BIRTH DEFECTS
Olga Tselousova, Liudmila Ovsyannikova
GENOTOXIC AND IMMUNOMODULATORY EFFECTS IN HUMAN WHITE BLOOD CELLS AFTER EX VIVO EXPOSURE TO POLYSTYRENE NANOPLASTICS
Sandra Ballesteros1, Josefa Domenech1, Irene Barguilla1, Constanza Cortés1, Ricard Marcos1,2*, Alba Hernández1,2*
Funding: this work was partially supported by the Spanish Ministry of Education and Science [BFU2016-76831-R]. Sandra Ballesteros, Josefa Domenech, and Irene Barguilla hold fellowships from the Universitat Autònoma de Barcelona [PIF-UAB]
UV-PROTECTIVE EFFECT of in vitro GROWN Gentiana lutea METHANOLIC EXTRACTS
Stefana Đukanović 1*, Stefana Cvetković1, Branislav Nastasijević2, Dragana Mitić-Ćulafić1, Anita Klaus3, Jovana Vunduk3, Slađana Todorović4 and Biljana Nikolić1
Cu/Se combination study in human astrocytes cultured under Se-deficient conditions
Stefanie Raschke, Barbara Witt, Viktoria K. Wandt, Franziska Ebert, Anna P. Kipp, Tanja Schwerdtle