EEMGS 2021

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.

Anastasiia T. Davletgildeeva <a.davletgildeeva@niboch.nsc.ru>

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

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.

Deni Kostelac <deni.kostelac@pbf.unizg.hr>

1Faculty of Food Technology and Biotechnology, Zagreb, Croatia 2Institute for Medical Research and Occupational Health, Zagreb, Croatia

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.

Ibemusu Otele <p2541097@my365.dmu.ac.uk>

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

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.

Lea Beltzig <lea.beltzig@uni-mainz.de>

University Medical Center Mainz, Department of Toxicology

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

Linda Reilly <946917@swansea.ac.uk>

1Swansea University, UK, 2 AstraZeneca laboratories, Cambridge, UK

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.

Monica Aquilino <maquilino@ccia.uned.es>

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

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.

Natasha Irani <natasha.irani17@imperial.ac.uk>

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

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.

Stefana Đukanović <stefana.d@bio.bg.ac.rs>

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

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.

Stefanie Raschke

University of Potsdam, Germany