EEMGS 2021

EEMGS

EEMGS Annual Meeting 2021

More info: https://www.eemgs.eu

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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

Abstract: The increase of antimicrobial resistance is a concerning global health problem. The use of silver has re-emerged as an alternative to antibiotics in form of silver nanoparticles (AgNPs). AgNPs-silica, material composed of AgNPs and silica as carrier, has been developed to be used in animal feed instead of antibiotics. AgNPs-silica safety is being assessed following the EFSA guideline 20181. The in vitro genotoxicity evaluation was designed following the strategy suggested by the EFSA guideline for metallic NPs1, involving the mouse lymphoma assay (MLA) (OECD TG 490) for induction of point mutations and the micronucleus (MN) (OECD TG 487) test for induction of structural and numerical chromosome aberrations. The in vitro comet assay modified by the addition of Fpg was included to provide complementary information on pre-mutagenic DNA damage. Both AgNPs-silica and Ag/AgNPs released from the AgNPs-silica material after agitation in cell culture media, were evaluated at 3 and 24 h treatment. The three in vitro assays were performed without metabolic activation, under good laboratory practices (GLPs). Negative results were obtained from every in vitro assay. Due to the complexity of the AgNPs-silica, an in vivo evaluation is being carried out, following EFSA recommendations. An in vivo integrated strategy has been applied consisting on a repeated dose-28-day oral toxicity study (OECD TG 407) combined with the in vivo mammalian alkaline comet assay (OECD TG 489) and the in vivo mammalian erythrocyte MN test (OECD TG 474), under GLPs-like conditions. Male and female 8-week-old Wistar rats were orally administered with AgNPs-silica, daily, for 28 days at doses of 0, 50, 300 and 2000 mg/kg b.w. Only the females were used for the genotoxicity assays. Samples of liver, spleen, kidney and duodenum were obtained for the alkaline comet assay and bone marrow samples were obtained for the MN test. The preliminary results will be presented in the congress.
Presented by: Adriana Rodriguez-Garraus
Institution: University of Navarra, Department of Pharmacology and Toxicology

Chat with PresenterAvailable June 23 (9-11 h)

Machine Learning and Mechanistic information from a Multi Endpoint Genotoxicity Screen

Wilson, A, Elloway, J, Grabowski, P , Ebbles, T and Doherty, A.

Abstract: To provide a comprehensive analysis of small molecule genotoxic potential we have developed and validated an, image-based in vitro Micronucleus (IVM) assay. This automated, high-content, high-throughput assay simultaneously assesses hundreds of cellular properties associated with genotoxicity including micronuclei and multiple additional cellular markers.

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.
Presented by: Amy Wilson <amy.wilson3@astrazeneca.com>
Institution: Functional and Mechanistic Safety, Clinical Pharmacology and Safety Sciences, Biopharmaceuticals R&D, AstraZeneca, Cambridge, United Kingdom.

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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

Abstract: A significand amount of data was obtained on the nature of protein-DNA contacts that control the specificity of nucleotide incision repair implemented by apurinic/apyrimidinic (AP) endonucleases. Nevertheless, the question on how a given nucleotide is recognized by the active site of the enzyme remains unanswered. Therefore, the main purpose of our study was to compare kinetics of conformational changes of homologous APE1-like endonucleases belonging to representatives of various classes of animals (insect Drosophila melanogaster Rrp1, amphibian Xenopus laevis xAPE1, fish Danio rerio zAPE1, and human Homo sapiens hAPE1) during their interaction with number of damaged DNA substrates, i.e., DNA containing an F-site (an analog of natural AP-site), 1,N6-ethenoadenosine, 5,6-dihydrouridine, uridine, or the alpha-anomer of adenosine. Pre-steady-state analysis of fluorescence time courses obtained for the interaction of the APE1-like enzymes with DNA substrates containing various lesions and a FRET dye pair allowed us to outline a model of substrate recognition by this class of enzymes. It was found that binding constants for F-site-containing DNA proved to be similar among all the assayed AP endonucleases, suggesting that abasic site recognition by these enzymes most likely is based on a combination of DNA flexibility, the ability of the F-site to flip out from the duplex, and the absence of steric hindrances throughout the trajectory of eversion into the active site of the enzymes. However, Rrp1 manifested a significantly slower rate of the catalytic reaction with F-site-containing DNA compared with other enzymes. Unexpectedly, the differences in rates of DNA substrates’ binding do not lead to significant differences in the cleavage efficiency of DNA containing a damaged base. The results suggest that the formation of enzyme–substrate complexes is not the key factor that limits enzyme turnover; the mechanisms of damage recognition and cleavage efficacy are related to fine conformational tuning inside the active site.

Funding: this work was supported by Ph.D. grant 20-34-90008 from Russian Foundation for Basic Research.
Presented by: Anastasiia T. Davletgildeeva <a.davletgildeeva@niboch.nsc.ru>
Institution: 1 Institute of Chemical Biology and Fundamental Medicine, SB RAS, Novosibirsk, Russia 2 Department of Natural Sciences, Novosibirsk State University, Novosibirsk, Russia 3 Group Mechanisms of DNA Repair and Carcinogenesis, Equipe Labellisée LIGUE 2016, CNRS UMR9019, Université Paris-Saclay, Villejuif, France

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

Abstract: Traditional Genetic Toxicology mutation assays indirectly infer mutation frequency from cellular phenotypic changes, typically reflecting variations in a single gene. Next Generation Sequencing (NGS) complements these assays by providing direct information from the wider genome on not only mutation frequency but also mutation type and location. The abundant, but often unique, mutations that genotoxic chemicals induce are masked in standard NGS due to the technique’s inherent high error rate. Therefore, an error-corrected NGS method needs to be utilised, such as Duplex Sequencing (DS). Here we show ongoing validation of DS in comparison with a traditional mutation assay to define a mutational load which links to carcinogenicity. A successful initial DS experiment was performed on L5178Y cells treated with known mutagen EMS (Ethyl methanesulfonate) in the in vitro Pig-a mutation assay. Interestingly, in addition to providing direct mutational information, DS also demonstrated greater sensitivity when measuring mutation frequency, with a 14-fold change observed with DS compared with a 7-fold change with flow cytometry. 88% of the DS mutations observed with EMS were C>T, strongly aligning with the published mechanism of action. These preliminary findings indicate a great utility for DS in enabling enhanced mechanistic insights into the mutagenic potential of compounds.
Presented by: Anne Ashford <anne.ashford@astrazeneca.com>
Institution: 1Functional and Mechanistic Safety, Clinical Pharmacology & Safety Sciences, R&D, AstraZeneca, Cambridge, UK

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In vitro carcinogenicity testing of complex Non-genotoxic carcinogens

Demi Pritchard1, Gareth Jenkins1

Abstract: Around 70-90% of human cancers are induced due to environmental agent exposure. Carcinogens are split into two main groups, genotoxic carcinogens (GC) and non-genotoxic carcinogens (NGC). Currently there are no validated in vitro testing systems for NGCs and due to their complex nature they are largely ignored. Some carcinogens may have been mislabelled due to negative results in traditional in vitro genotoxicity testing. NGCs provide complex data and use a variety of different mechanisms to elicit an oncogenic response, making them difficult chemicals to study. The 2-year rodent bioassay is the only validated NGC test however there are a number of limitations. It uses >600 animals per chemical and is a timely and costly procedure, so not all chemicals will be subject to this test. This means a lot of chemicals are not properly regulated, posing a problem for human health. In order to try to understand the complex nature of NGCs, multiple mechanistic studies are required. Nickel Chloride, 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and two forms of Arsenic have been studied. Traditional acute dosing was compared with more human relevant chronic exposures, as well as exploring Micronuclei induction, cytotoxicity, cell cycle changes and Reactive oxygen species (ROS) levels. NiCl2 induced Mn and ROS in an acute manor with subtle cell cycle changes. Cacodylic acid induced Mn in a chronic setting as well as cell cycle changes. Sodium meta arsenite induced Mn chronically and changes to the cell cycle. TCDD was negative in all tests to date. As NGCs make up ∼12% of carcinogens and are poorly regulated, this highlights the need for the development of an in vitro testing approach. If NGC screening could be performed in vitro, it would reduce the number of animals needed to detect these chemicals and mechanistic understanding would allow for better classification of these carcinogens.

NC3Rs funded
Presented by: Demi Pritchard <822839@swansea.ac.uk>
Institution: 1 Swansea University, South Wales

Chat with PresenterAvailable June 24th 12:00-13:00 CET

Equid milk: a well of safe probiotic bacteria and their metabolites with antioxidant properties

Deni Kostelac1, Marko Gerić2, Goran Gajski2, Jadranka Frece1

Abstract: The human exposome represents an intriguing gene-environment interactions. Since the food is one of the most easily manageable human exposure, the search for food items that might have beneficial properties to human health is of high importance. This study aimed to assess the probiotic potential and the antioxidant properties of a new indigenous probiotic bacteria isolated from equid milk, known for its beneficial properties for ages. DPPH scavenging ability of live cells was determined and compared to antioxidant standard (L-ascorbic acid). Furthermore, the antioxidant activity of extracellular bacterial metabolites, with molecular mass smaller than 2000 Daltons (Da) that can cross the epithelial barrier was assessed accompanied by the investigation of cytotoxic and genotoxic effect of metabolites on peripheral blood lymphocytes. The battery of tests showed excellent probiotic potential. The DPPH free radical scavenging activity of L. plantarum M2 and L. plantarum KO9 cells was 7.25% and 9.51%. The tested bacterial media fractions removed 4.84% and 9.21% for investigated strains. L-ascorbic acid (20 mg/L) as a high positive standard removed 56.41% of free radicals. Examined metabolites exhibited no cytotoxic effects on peripheral blood mononuclear cells which was detected by acridine orange/ethidium bromide vital staining compared to controls. The results of comet assay also did not show induction of primary DNA damage of peripheral blood mononuclear cells exposed to bacterial extracellular metabolites. Taken together, we managed to present a new bacteria from donkey and mare milk with excellent probiotic potential and moderate to high antioxidative activity through combined activity of cells and produced metabolites. Furthermore, investigated strains do not posses any toxic properties so we confirmed Qualified Presumption Safety Status. The next steps in research will try to elucidate its possibility of minimizing effects of mutagens found in food.

Funding: Faculty of Food Technology and Biotechnology, University of Zagreb funded this study.
Presented by: Deni Kostelac <deni.kostelac@pbf.unizg.hr>
Institution: 1Faculty of Food Technology and Biotechnology, Zagreb, Croatia 2Institute for Medical Research and Occupational Health, Zagreb, Croatia

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

Abstract: The comet assay is a very efficient and sensitive method for detection of DNA damage. With the addition of FPG enzyme, it allows an assessment of the contribution of oxidative stress to the observed DNA damage. Modifications applying in vitro treatment of the cells can be used to measure the efficiency of DNA repair. The comet assay has been applied in the monitoring of human, usually analysing whole blood, or peripheral blood mononuclear cells (PBMCs). Ideally, all samples should be analysed at the same time. For larger studies or if samples become available at different times/locations, this can only be achieved by storing samples, but there is much less experience regarding analysis of frozen samples compared to fresh samples in the comet assay. We have now compared DNA damage in fresh and frozen samples in whole blood, buffy coat and PBMCs with and without the use of FPG enzyme. While all three sample types could be used well fresh and frozen, PBMCs yielded a slightly higher basal damage than the other two sample types. An in vitro hydrogen peroxide challenge worked well only in fresh PBMCs and to a lower extend in frozen PBMCs, but not in buffy coat or whole blood. The use of FPG enzyme detected increased damage in all three sample types, fresh and frozen. The attempt to apply a DNA repair assay for base and nucleotide excision repair was not successful in frozen samples due to elevated basal damage, which developed during the repair time, but a recovery time or a mitogen stimulation yielded similar results to fresh samples. In contrast to the successful utilization of cryopreserved samples in the alkaline and FPG comet assay, it was only possible to use cryopreserved PBMCs in repair comet assay with an introduction of a recovery time.
Presented by: Ezgi Eyluel Bankoglu
Institution: Institute of Pharmacology and Toxicology, University of Wuerzburg, Germany

Chat with PresenterAvailable June 24th, 14:15 - 14:45

Poster: REDUCED LEVELS OF HARMFUL AND POTENTIALLY HARMFUL CONSTITUENTS IN HEATED TOBACCO AEROSOL TRANSLATE TO REDUCED IN VITRO (GENO)TOXICOLOGICAL OUTCOMES

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

Abstract: Tobacco harm reduction (THR) involves offering adult smokers a potentially reduced harm mode of nicotine delivery compared to continued combustible cigarette use. Heated tobacco products (HTPs) form a category of products which heat tobacco to produce a nicotine-containing aerosol without the need for combustion. This lack of combustion has been evidenced to result in substantially reduced levels of harmful and potentially harmful constituents (HPHCs) in HTP aerosols compared to cigarette smoke. These reductions have also been correlated with reduced toxicological and disease-related outcomes associated with HTP use, compared to combustible cigarette smoking. In this study, selected HPHCs within the aerosol of a prototype HTP (p-HTP) and the smoke of the 1R6F Reference Cigarette were compared. The smoke/ aerosol was then applied in three regulatory toxicity assays, the Ames test (bacterial strains: TA98; TA100), the micronucleus assay (cell line: V79) and the neutral red uptake (NRU) cytotoxicity assay (cell line: Beas-2B). Upon comparison of the HPHCs measured, their levels were substantially reduced (by 90-98%) or below the limit of quantification within the p-HTP aerosol compared to in the 1R6F smoke. Furthermore, whilst significant mutagenicity was observed following exposure to 1R6F smoke, this was not present under the conditions of the test for the p-HTP aerosol. An 8/13 (+S9/-S9)-fold greater number of puffs was required to induce a three-fold increase in micronucleus frequency (ECMN3) above background levels with the p-HTP whole aerosol compared to 1R6F whole smoke exposure, and in the NRU assay, a 39-fold greater number of puffs were required to reach EC50 cytotoxicity. In conclusion, substantially reduced levels of selected HPHCs within the p-HTP aerosol, compared to 1R6F smoke, translate into greatly reduced in vitro toxicological effects, and support the HTP product category’s THR potential. Funding: This work was funded by Imperial Brands PLC.
Presented by: Fiona Chapman <Fiona.Chapman@uk.imptob.com>
Institution: 1 Imperial Brands PLC, 121 Winterstoke Road, Bristol, BS3 2LL, UK 2 Reemtsma Cigarettenfabriken GmbH, an Imperial Brands PLC Company, Albert-Einstein-Ring-7, D-22761, Hamburg, Germany

Chat with PresenterAvailable 24-06-21, 12.25-12.45pm

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

Abstract: The advent of nanomedicine has led to a significant breakthrough in overcoming the adverse effects observed in treating colorectal cancer (CRC), the third-largest cause of cancer-related mortality. Mesoporous Silica Nanoparticles (MSNs) is used as a carrier to deliver anti-cancer drugs into CRC cells. The poor prognosis and tumour invasion of CRC is associated with the aberrant expression of large glycoproteins, mucins (MUC). Cytochrome P450 (CYPs) also plays a significant role in anti-cancer drug metabolism and has shown differential expression in 2-dimensional (2D) and 3-dimensional (3D) cellular environment. This study investigates the expression of MUC1, CYPs and assesses the cytotoxicity potential of MSN-loaded anti-cancer drug on HCT116 colon cancer cells. The anti-cancer drug oxaliplatin was loaded into MSNs through the electrospraying technique. Cellular aggregates or spheroids were formed to represent 3D models of the tumour. Immunocytochemistry was used to detect the presence of MUC1 in colon cancer cells. Real-time PCR was used to determine the expression of CYPS in colon cancer cells. Cytotoxicity in 2D vs 3D cells was assessed using an MTT assay. 3D spheroids or 2D culture treated with MSN-loaded anti-cancer drug (compared to the drug only) showed a dose-dependent decrease in cell viability. 2D cultures showed a significant reduction in cell viability compared to 3D spheroids. Real-time PCR results showed increased expression of CYP1A1 and CYP1B1 in 3D spheroids compared to 2D cells. MUC-1 expression increased in 3D models compared to 2D cells. Moreover, MUC1 expression was time-dependent. Although the CYP enzymes showed an upregulated CYP expression, the increased expression of MUC-1 seems to serve as a barrier to MSN loaded anti-cancer drugs and their ability to induce cellular toxicity in 3D spheroids. Therefore, this study shows the importance of evaluating drug resistance and screening in 3D models and the potential application of MSN loaded cytotoxic drugs. Funding: Niger Delta Development Commission (NDDC) funded this study.
Presented by: Ibemusu Otele <p2541097@my365.dmu.ac.uk>
Institution: 1Leicester School of Allied Health Sciences, Faculty of Health and Life Sciences, De Montfort University, Leicester LE1 9BH, United Kingdom 2Leicester School of Pharmacy, Faculty of Health and Life Sciences, De Montfort University, Leicester LE1 9BH, United Kingdom

Identification of Senolytic Agents for Sequential Treatment of GBM with TMZ

Lea C. Beltzig, Björn Stratenwerth, Markus Christmann, Bernd Kaina

Abstract: Introduction. Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults. Despite a stringent treatment schedule, the median survival rate is only 15 months after diagnosis due to recurrances. Temozolomide (TMZ) induces survival pathways, one of which is senescence. If GBM cells escape the senescent state, this may give rise to recurrent tumors. Therefore, treatments that specifically target senescent cells, “senolytic drugs”, may lead to a better outome of GBM therapy.

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.
Presented by: Lea Beltzig <lea.beltzig@uni-mainz.de>
Institution: University Medical Center Mainz, Department of Toxicology

Automated High Content Screening for Carcinogenicity Testing In Vitro

Linda Reilly*1, Amy Wilson2, Jo Elloway2, Ann Doherty2, Gareth J Jenkins1

Abstract: Carcinogenicity testing is a regulatory requirement for pharmaceutical development and other regulated sectors. However, the current in vitro and in vivo testing paradigm fails to accurately predict carcinogenic potential in humans, with up to 95% of drugs failing at the clinical trial stage. Thus, there is an expressed need to change the current testing paradigm.

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
Presented by: Linda Reilly <946917@swansea.ac.uk>
Institution: 1Swansea University, UK, 2 AstraZeneca laboratories, Cambridge, UK

Poster: PRODIAMESA OLIVACEA: NEW IMMUNE AND STRESS BIOMARKER GENES IN A POTENTIAL SENTINEL ORGANISM FOR ECOTOXICITY STUDIES IN NATURAL SCENARIOS

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

Abstract: Toxicological studies on non-model organisms complement and provide powerful information in terms of natural ecosystems and confer a breakthrough in ecotoxicology. Chironomus (Diptera) has four OECD standardized tests for the evaluation of water and sediment toxicity, which assess classical parameters of toxicity. Prodiamesa olivacea (Diptera) is a non-model aquatic insect species not used in toxicity tests that frequently shares habitat with C. riparius but requires higher oxygen levels and lower extreme conditions. Therefore, it is of special interest to study possible differences in the response of both species to pollutant exposures. This work characterizes and tests the effectiveness of early effect biomarkers related to environmental pollution in water, in natural population of P. olivacea larvae under different stress conditions. It contributes to increase the knowledge about xenobiotics effects on benthic aquatic invertebrates. De novo RNAseq P. olivacea transcriptome was obtained and used for de novo identification and characterization of genes related to the stress and immune response: Hsp60, PGRP, Toll among others. Quantitative real-time PCR was used to evaluate the expression of selected genes. Benzyl butyl phthalate (BBP; CAS No. 85-68-7) toxicity was elucidated in natural populations of both species. Transcriptional effects of BBP acute exposures were evaluated and results revealed species-dependent gene responses. P. olivacea showed a greater BBP sensitivity since compared to C. riparius, more severe effects were observed in all the analysed biomarkers. This research provides new tools for assessing and monitoring water quality. It is essential to assess tolerance/sensitivity of natural populations of model organisms and non-model insect species chronically exposed to complex pollutant mixtures. This will avoid conferring incomplete conclusions considering high tolerant model organisms data. This kind of approach will allow us to have a broader view of the risk associated with the presence of pollutants in ecosystems in short, medium and long term.
Presented by: Lola Llorente <lolallorente@ccia.uned.es>
Institution: 1Biology and Environmental Toxicology Group, Faculty of Science, UNED, Madrid, Spain

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Assessing occuptional safety: a story of interventional radiology unit teams

Marko Gerić1; Jelena Popić2; Goran Gajski1, and Vera Garaj-Vrhovac1

Abstract: Occupational hazards range from Asbestos to Zidovudine and are usually grouped into three major spheres: biological, chemical, and physical. Interventional radiology unit teams are facing everyday occupational exposure to low-dose ionizing radiation. Despite personal protective equipment, physical dosimetry, and safety dose limits, incidence of some malignant diseases in this population is more frequent compared to public, highlighting the need for further studies. On the other hand, Multidisciplinary European Low Dose Initiative (MELODI) prioritises the research of low-dose ionizing radiation (≤100 mSv) effects to human populations. Therefore, the aim of our study was to assess cytogenetic status of interventional radiology unit teams from hospitals in Zagreb. To achieve the aim, 24 volunteers on average 41±10 years old participated in the study. We also recruited a control group that was matched in number of volunteers (N=24), sex, age, BMI, smoking status, season of sampling, and city of residence, in order to minimise the effects of possible confounders. The results of physical dosimetry showed that none of the volunteers reached set limits, while their average occupational annual effective dose was 1.82±3.60 mSv. As for the molecular changes on the level of DNA molecule and chromosomes, we observed higher frequency of nuclear buds (4.1±1.9 vs 3.0±1.7) in peripheral blood lymphocytes and longer comet assay’s tail length (15.5±1.5 µm vs 14.1±1.4 µm) in exposed group compared to controls (p<0.05). Differences in other cytokinesis-block micronucleus and comet assay descriptors, as well as their correlation with measured doses did not reach statistical significance. To sum up, we observed molecular changes in blood cells of occupationally exposed population with quite high inter-individual variability of the results. Further studies are needed in order to improve occupational safety of populations exposed to low-dose radiation while acknowledging ALARA principles.

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.
Presented by: Marko Gerić <mgeric@imi.hr>
Institution: 1Institute for Medical Research and Occupational Health, Mutagenesis Unit, Zagreb, Croatia; 2University of Zagreb, School of Medicine, Clinical Hospital Merkur, Zagreb, Croatia

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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*

Abstract: For this study, Caenorhabditis elegans (C. elegans) was used to establish an in vivo genotoxicity testing strategy with the aim of complementing the existing testing battery by low-cost follow-up in vivo tests. The nematode offers the possibility to overcome some of the limitations current assays show, including the absence of toxicokinetics in bacteria or cell lines, and the lack of repeated dosing or high-throughput analysis. By adapting various cell culture assays, we are able to provide a test strategy for manganese (Mn)-induced DNA damage/ repair in a multicellular, metabolizing organism without using animal experiments. Mn-induced neurodegeneration (manganism) is excessively discussed to be caused by oxidative stress, the subsequent oxidative DNA damage and restricted DNA damage response. For oxidative stress, various markers were determined including reduced and oxidized glutathione levels. The oxidative damage 8-oxo-dG can be quantified in the worms using an ELISA. We were able to establish an alkaline unwinding assay in order to quantify the amount of dsDNA in C. elegans. The assay was validated by using the positive control bleomycin. For investigations of the cellular DNA damage response, poly(ADP-ribosyl)ation (PAR) can be quantified analytically via LC-MS/MS. Furthermore, the incision activities of repair enzymes were determined in worm lysates. Additional to the detection of the direct DNA damage and DNA damage response, gene expression studies were used to point out the relevant DNA repair pathways and using transgenic DNA repair mutants helped to identify sensitivity towards Mn. In sum, Mn overexposure (250 mM for 1 h) caused oxidative stress, induced 8-oxo-dG, reduced the amount of dsDNA, upregulated the gene expression of BER genes and dysregulated the incision activity, but does not induce PARylation. Using this assembly of assays, it is possible to analyse the genotoxic potential of Mn – from cause to consequence.
Presented by: Merle M. Nicolai <merle.nicolai@uni-wuppertal.de>
Institution: 1Food Chemistry, Faculty of Mathematics and Natural Sciences, University of Wuppertal, Gaußstr. 20, 42119 Wuppertal, Germany 2Department of Food Chemistry, Institute of Nutritional Science, University of Potsdam, Arthur Scheunert-Allee 114-116, 14558 Nuthetal, Germany 3TraceAge – DFG Research Unit on Interactions of Essential Trace Elements in Healthy and Diseased Elderly (FOR 2558), Berlin-Potsdam-Jena-Wuppertal, Germany 4German Federal Institute for Risk Assessment (BfR), Berlin, Germany

Chat with PresenterAvailable June 23 + 24, 1 - 2 pm (CEST); Password: aKF2GKK6 ; Meeting ID: 931 2503 6009

Poster: EVALUATION OF THE PHYSIOLOGICAL RESPONSE OF CHORTHIPPUS PARALLELUS (ORTHOPTERA) TO WOLBACHIA INFECTION: CHARACTERIZATION OF NEW MOLECULAR BIOMARKERS

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

Abstract: The grasshopper Chorthippus parallelus (Orthoptera: Acrididae) forms well-known hybrid zones (HZs) both in the Pyrenees and in the Alps. These HZs provide a remarkable system to identify ‘key genes’ that actually maintain genetic boundaries between emerging species. The Iberian endemism C. p. erythropus and the subspecies C. p. parallelus, widely distributed throughout the rest of Europe, overlap and form the Pyrenean HZ. Both subspecies differ morphologically, as well as in behavioural, mitochondrial, nuclear and chromosomal traits, but also in the strains of the maternally transmitted bacterial endosymbiont Wolbachia infecting them. This research explores how Wolbachia may induce the expression of some major genes involved in development, reproduction, and in other important metabolic pathways, in addition to genes related to reproductive barrier in the C. parallelus in the Pyrenean HZ. More specifically, we have explored, through molecular biomarkers, the physiological responses in C. parallelus individuals infected by Wolbachia, with particular attention to hormonal pathway, immune, and stress cell responses. Quantitative real-time PCR was used to evaluate the expression of selected genes in the gonad of infected and uninfected adults of both sexes. The studies were performed in these tissues since they constitute the main target of Wolbachia infection. Our preliminary results are promising since we identified new sensitive biomarkers involved in the reproductive barrier that Wolbachia induces in the hybrid zone. Transcriptional effects show differences between sexes for all the analysed biomarkers in infected and non-infected adults.

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.
Presented by: Monica Aquilino <maquilino@ccia.uned.es>
Institution: 1 Grupo de Biología y Toxicología Ambiental. Facultad de Ciencias. Universidad Nacional de Educación a Distancia (UNED), Madrid, Spain. 2 Departamento de Biología (Genética), Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid, Spain

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

Abstract: The spatial organisation of the genome plays a crucial role in regulating genomic stability, supporting repair of DNA double strand breaks (DSBs) and epigenetic changes. Chromatin is organised into loops via loop extrusion, which involves cohesin, a multiple protein complex, and CTCF. Loop domains or topologically associated domains (TADs) have a functional role in transcription and repairing DSBs, cohesin is recruited to sites of DSBs. Loop formation allows DNA repair enzymes to target histones located thousands base pairs away. RAD21, a DSB repair protein and is recruited to cohesin binding sites after DSB induction. The aim of this study was to explore the hypothesis that expression levels of cohesin complex subunit proteins in the nuclei and micronuclei (MN) in A549 cells was altered over time following treatment with either etoposide or paclitaxel using a micronucleus assay coupled with imaging. Cells were treated with etoposide (0.05µM-1.5 µM) or paclitaxel (1nM-7nM) from 1h to 48h, fixed, incubated with cohesin complex antibodies and imaged using confocal microscopy. Analysis was performed using Columbus software. Treatment with etoposide and paclitaxel induced MN, maintaining viability >50% with a 2-fold (0.5µM) and 4-fold (3.5nM) increase in MN at 24 h, respectively. Interestingly, for RAD21, expression in the MN did not change over the time course following treatment with etoposide, but a dose-dependent increase in the proportion of RAD21-positive MN was observed with paclitaxel. Treatment with paclitaxel (4 nM) for 8h and 12h showed 50% of RAD21-positive MN which returned to baseline (10 %) by 24h. For etoposide the proportion remained 10% at all time points. The reason for this is unclear, but suggests a temporal relationship with MN formation and could reflect the aneugenic mode of action of paclitaxel. Further work to understand this is ongoing and will provide further insight into the mechanism of MN formation.
Presented by: Natasha Irani <natasha.irani17@imperial.ac.uk>
Institution: 1Functional and Mechanistic Safety, Clinical Pharmacology and Safety Sciences, R&D, AstraZeneca, Cambridge, United Kingdom; 2Department of Metabolism, Digestion & Reproduction, Imperial College London, London, United Kingdom

ENVIRONMENTAL AND LIFESTYLE FACTORS IN THE DEVELOPMENT OF BIRTH DEFECTS

Olga Tselousova, Liudmila Ovsyannikova

Abstract: Every year, approximately 6% of worldwide infants are born with birth defects. They are the leading cause of infant death and contribute to morbidity and long-term disability in the population. The most common causes of birth defects are chromosomal abnormalities, single gene defects, and multifactorial influences. However, the causal factors for the vast majority of nonsyndromic birth defect cases are still unknown. We tested the hypothesis about the influence of lifestyle and environmental factors on the incidence of nonsyndromic birth defect in a city with a high level of petrochemical pollution (Ufa, Russia). Within the eight -year study period, 1095 mothers of infants with birth defect participated in the study. The mean ages of the mothers and fathers were 26,59±6,20 and 29,63 ±6,51 years respectively. Majority (86,94%) of the mothers and fathers (77,96%) were younger than 35 years of age. We identified high risk for infants with birth defects in smoker mothers (OR = 4.76; 95%CI (3 - 7.54)). There were detected significant differences in the increased risk of congenital malformations when drinking alcohol during pregnancy (χ2 = 16.23, df 1, p = 7 * 10-5). In 1.64% of families with a infants birth defects, one of the parents used drugs (only the mother, or only the father) and in 0.27% of families, both parents suffered from drug addiction (p = 0.001). Analysis of environmental pollution demonstrated negative effects on the formation of birth defects. A high risk of infant mortality was revealed during long-term residence in areas with a developed petrochemical industry (OR = 16,25; 95%CI (2,17- 121,53); p = 0,0001). Our research finds evidence that nonsyndromic birth defects are the result of harmful environmental and lifestyle factors.
Presented by: Olga Tselousova <Olga.tselousova@gmail.com>
Institution: Bashkir State Medical University of the Ministry of Health of Russian Federation

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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*

Abstract: The increased presence of plastics in our environment, as waste after use, is of great ecological concern. Nevertheless, in addition to the visible part of this environmental contamination, plastic degrades to micro and nanosizes (MNPLs) forming a non-visible source of environmental pollutants. At these sizes, MNPLs can be easily intake by organisms, including humans, supposing a potential health concern. Independently of the exposure route, the uptaken environmental MNPLs end into the body general compartment (blood), potentially interacting with blood cells. Using whole blood samples from different donors, exposed ex vivo to polystyrene nanoplastics (PSNPLs), the effects in different subsets of white peripheral blood cells (WBCs) were analysed. Interestingly, the different cell lineages manifested sharp differences in PSNPLs uptake with very limited uptake in lymphocytes, and very high uptake in monocytes. Furthermore, significant increases in the levels of DNA damage were observed in monocytes and polymorphonuclear cells, but not in lymphocytes. Furthermore, our results showed that PSNPLs exposure-induced changes in the whole blood secretome. These findings were further confirmed when the expression of different cytokines was analysed, revealing a significant increase in the expression of different cytokines related to the inflammatory, immune, and stress response, as well as cell proliferation. Summarizing, our results support that the ex vivo model is a powerful strategy to study the nanoparticles effects on the human blood system.

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]
Presented by: Sandra Ballesteros <Sandra.Ballesteros@uab.cat>
Institution: 1Group of Mutagenesis, Department of Genetics and Microbiology, Faculty of Biosciences, Universitat Autònoma de Barcelona, Cerdanyola del Vallès (Barcelona), Spain. 2Consortium for Biomedical Research in Epidemiology and Public Health (CIBERESP), Carlos III Institute of Health, Madrid, Spain

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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

Abstract: Increased exposure to ultraviolet (UV) radiation has been recognized as a major factor that leads to skin cancer development. About 95% of solar UV radiation that reaches the surface of the earth is from UV-A spectrum. This kind of radiation induces damages by forming reactive oxygen species (ROS) consequently causing single-strand breaks, mutations and chromosomal aberrations. In order to improve and protect skin health, examination of natural products possessing strong antioxidant activity, which could be applied as dietary supplements or sunscreens, has become very intensive. The aim of the study was to investigate the antioxidant and antigenotoxic effect of chemically characterized in vitro grown Gentiana lutea methanolic root and shoot extracts against UV-A radiation, on normal lung fetal fibroblasts (MRC-5) and human melanoma cells (Hs294T). The UPLC – MS/MS analysis of extracts revealed that gentiopicroside was the most abundant component of both extracts. To investigate the antigenotoxic effect of G. lutea extracts, alkaline comet assay was applied. The obtained results demonstrated that root extract inhibited UV-A induced DNA damage (dose 3 J/cm2) for 49% in MRC-5 and 69% in Hs 294T cell lines, while shoot extract inhibited for approximately 60% in both cell lines. Next, the potential of extracts to scavenge free radicals was investigated in DPPH assay and EC50 values were 165 µg/mL and 73 µg/mL for root and shoot extract respectively. Furthermore, shoot extract demonstrated potential to inhibit the process of lipid peroxidation in TBA test up to 35%. The ability of extracts to perform the reduction of copper ions was investigated by CUPRAC assay and shoot extract had stronger effect, with EC50 value at 459 µg/mL. The results obtained in this study are encouraging for further investigation of protective role of in vitro grown G. lutea extracts since the UV radiation is serious problem that we are facing daily.
Presented by: Stefana Đukanović <stefana.d@bio.bg.ac.rs>
Institution: 1University of Belgrade – Faculty of Biology, Department of Microbiology, Studentski trg 16, 11000 Belgrade, Serbia 2University of Belgrade – VINČA Institute of Nuclear Science, Department of Physical Chemistry, Mike Petrovića Alasa 12-14, 11000 Belgrade, Serbia 3University of Belgrade – Faculty of Agriculture, Institute for Food Technology and Biochemistry, Nemanjina 6, 11080 Belgrade, Serbia 4University of Belgrade – Institute for Biological Research “Siniša Stanković”, Bulevar Despota Stefana 142, 11000, Belgrade, Serbia

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

Abstract: The trace elements selenium (Se) and copper (Cu) are essential for humans. While Cu directly acts as a co-factor for several essential enzymes, Se is incorporated into selenoproteins which are important e.g. in antioxidative defense and cellular redox homeostasis. Disturbances in the homeostasis of redox active Cu is associated with neurological diseases like Wilson´s or Alzheimer´s disease. The underlying mechanism of Cu toxicity is still not fully understood, but one proposed mode of action is supposed to be via induction of oxidative stress. Furthermore, an impaired selenoprotein expression under Se-deficient conditions can further contribute to an increase in oxidative stress. To elucidate potential protective effects of a Se-supplementation towards Cu-induced toxicity, this Cu/Se-combination study was conducted in a human astrocyte cell line. Therefore, suboptimally Se-supplied astrocytes were supplemented with selenite or selenomethionine for 48 h and subsequently treated with CuSO4 for additional 48 h. Endpoints regarding cytotoxicity, Se status (Glutathione peroxidase activity, total Se and Selenoprotein P content), marker of oxidative stress as well as DNA damage were investigated. Co-exposure studies indicated that Se could not mitigate Cu-induced toxicity and thus, provided no evidence for protective effects regarding cell viability. Cu treatment negatively influenced marker of Se status which could be ameliorated by Se supplementation. Investigation of mitochondrial membrane potential revealed a protective effect of Se against Cu-induced damage of mitochondria. Additionally, preliminary data indicated that Se might have a protective effect in the generation of Cu-induced oxidative DNA damage. Finally, this study provides small insights into the Se-mediated protective effects against Cu-induced toxicity. In further research, a qPCR-based gene expression screening will be used to elucidate the involved pathways in Cu toxicity as well as the potential influence of a Se-supplementation.
Presented by: Stefanie Raschke
Institution: University of Potsdam, Germany