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2. Measuring DNA modifications with the comet assay: a compendium of protocols

Author

Collins, Andrew, Møller, Peter, Gajski, Goran, Vodenková, Soňa, Abdulwahed, Abdulhadi, Anderson, Diana, Bankoglu, Ezgi Eyluel, Bonassi, Stefano, Boutet-Robinet, Elisa, Brunborg, Gunnar, Chao, Christy, Cooke, Marcus S., Costa, Carla, Costa, Solange, Dhawan, Alok, de Lapuente, Joaquin, Bo’, Cristian Del, Dubus, Julien, Dusinska, Maria, Duthie, Susan J., Yamani, Naouale El, Engelward, Bevin, Gaivão, Isabel, Giovannelli, Lisa, Godschalk, Roger, Guilherme, Sofia, Gutzkow, Kristine B., Habas, Khaled, Hernández, Alba, Herrero, Oscar, Isidori, Marina, Jha, Awadhesh N., Knasmüller, Siegfried, Kooter, Ingeborg M., Koppen, Gudrun, Kruszewski, Marcin, Ladeira, Carina, Laffon, Blanca, Larramendy, Marcelo, Hégarat, Ludovic Le, Lewies, Angélique, Lewinska, Anna, Liwszyc, Guillermo E., de Cerain, Adela López, Manjanatha, Mugimane, Marcos, Ricard, Milić, Mirta, de Andrade, Vanessa Moraes, Moretti, Massimo, Muruzabal, Damian, Novak, Matjaž, Oliveira, Rui, Olsen, Ann-Karin, Owiti, Norah, Pacheco, Mário, Pandey, Alok K., Pfuhler, Stefan, Pourrut, Bertrand, Reisinger, Kerstin, Rojas, Emilio, Rundén-Pran, Elise, Sanz-Serrano, Julen, Shaposhnikov, Sergey, Sipinen, Ville, Smeets, Karen, Stopper, Helga, Teixeira, João Paulo, Valdiglesias, Vanessa, Valverde, Mahara, van Acker, Frederique, van Schooten, Frederik-Jan, Vasquez, Marie, Wentzel, Johannes F., Wnuk, Maciej, Wouters, Annelies, Žegura, Bojana, Zikmund, Tomas, Langie, Sabine A. S., and Azqueta, Amaya

Published
2023
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4. In vivo toxicity and genotoxicity of beauvericin and enniatins. Combined approach to study in vivo toxicity and genotoxicity of mycotoxins beauvericin (BEA) and enniatin B (ENNB)

Author

Maranghi, Francesca, Tassinari, Roberta, Narciso, Laura, Tait, Sabrina, Rocca, Cinzia, Felice, Gabriella Di, Butteroni, Cinzia, Corinti, Silvia, Barletta, Bianca, Cordelli, Eugenia, Pacchierotti, Francesca, Eleuteri, Patrizia, Villani, Paola, Hégarat, Ludovic Le, Fessard, Valérie, Reale, Océane, Istituto Superiore di Sanita [Rome], Agenzia Nazionale per le nuove Tecnologie, l’energia e lo sviluppo economico sostenibile (ENEA), Unité de Toxicologie des Contaminants, Laboratoire de Fougères - ANSES, Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)-Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), and Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES)

Subjects
enniatin B, Mycotoxin, immunotoxicity, Toxicity, mycotoxine, beauvericin, genotoxicity, [SDV.TOX.TVM]Life Sciences [q-bio]/Toxicology/Vegetal toxicology and mycotoxicology, test in-vivo, [SDV.TOX.TCA]Life Sciences [q-bio]/Toxicology/Toxicology and food chain, Food safety, Feed, In-vivo test, génotoxicité, toxicité, sécurité des aliments
Abstract

Beauvericin (BEA) and Enniatins (ENN) are mycotoxins produced by Fusarium fungi detected in food and feed; there are insufficient data to establish their reference values. To evaluate BEA and ENN oral toxicity, an integrated approach was applied. Among ENN, Enniatin B (ENNB) was selected as test substance. The approach is composed by: i) in vitro and acute in vivo genotoxicity tests; ii) a repeated-dose oral toxicity study focused on genotoxic, immune, endocrine, nervous endpoints and the reproductive/developmental toxicity screening. For BEA, all the genotoxicity endpoints yielded negative results excluding Comet assay in duodenum and kidney after repeated doses. BEA immunotoxicity was observed in female mice, concentrated in number and functional activity of effector T cells in the spleen. Based on the repeated-dose BEA study, the No Observed Adverse Effect Level (NOAEL) for female mice is 1 mg/kg b.w. per day (increased thyroid pycnotic nuclei and endometrial hyperplasia). In males, the NOAEL is 0.1 mg/kg b.w. per day (reduced colloid and altered T4 serum levels). Maternal NOAEL is 0.1 mg/kg b.w. per day (increased thymus weight), developmental NOAEL is 10 mg/kg b.w. per day. For ENNB, the results support a genotoxic effect in bone marrow and liver cells after acute treatment, but not after repeated exposure. Immunotoxic ENNB effects were observed in both genders, suggestive of a suppressive/inhibiting activity particularly evident in males. Based on the repeated-dose ENNB study, the NOAEL for females is 0.18 mg/kg b.w. per day (histomorphometrical effects on thymus, uterus and spleen). In male mice, the NOAEL is 1.8 mg/kg b.w. per day (enterocyte vacuolization in duodenum and increased Reactive Oxygen Species and reduced Glutathione brain levels). The maternal NOAEL is 1.8 mg/kg b.w. per day (decreased white pulp area and increased red/white pulp area ratio in spleen), developmental NOAEL is 18 mg/kg b.w. per day.

Published
2018

5. Synergic toxic effects of food contaminant mixtures in human cells.

Author

Kopp, Benjamin, Sanders, Pascal, Alassane-Kpembi, Imourana, Fessard, Valérie, Zalko, Daniel, Hégarat, Ludovic Le, and Audebert, Marc

Subjects
POISONS, ENVIRONMENTAL toxicology, BINARY mixtures, MIXTURES, GENETIC toxicology, DNA repair
Abstract

Humans are exposed to multiple exogenous substances, notably through food consumption. Many of these compounds are suspected to impact human health, and their combination could exacerbate their harmful effects. We previously observed in human cells that, among the six most prevalent food contaminant complex mixtures identified in the French diet, synergistic interactions between component appeared in two mixtures compared with the response with the chemicals alone. In the present study, we demonstrated in human cells that these properties are driven only by two heavy metals in each mixture: tellurium (Te) with cadmium (Cd) and Cd with inorganic arsenic (As), respectively. It appeared that the predicted effects for these binary mixtures using the mathematical model of Chou and Talalay confirmed synergism between these heavy metals. Based on different cell biology experiments (cytotoxicity, genotoxicity, mutagenesis and DNA repair inhibition experiments), a detailed mechanistic analysis of these two mixtures suggests that concomitant induction of oxidative DNA damage and decrease of their repair capacity contribute to the synergistic toxic effect of these chemical mixtures. Overall, these results may have broad implications for the fields of environmental toxicology and chemical mixture risk assessment. [ABSTRACT FROM AUTHOR]

Published
2020
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7. Combined Effects of Lipophilic Phycotoxins (Okadaic Acid, Azapsiracid-1 and Yessotoxin) on Human Intestinal Cells Models.

Author

Ferron, Pierre-Jean, Dumazeau, Kevin, Beaulieu, Jean-François, Hégarat, Ludovic Le, and Fessard, Valérie

Subjects
ALGAL toxins, SEAFOOD poisoning, SEAFOOD inspection, SEAFOOD microbiology, FOOD poisoning prevention
Abstract

Phycotoxins are monitored in seafood because they can cause food poisonings in humans. Phycotoxins do not only occur singly but also as mixtures in shellfish. The aim of this study was to evaluate the in vitro toxic interactions of binary combinations of three lipophilic phycotoxins commonly found in Europe (okadaic acid (OA), yessotoxin (YTX) and azaspiracid-1 (AZA-1)) using the neutral red uptake assay on two human intestinal cell models, Caco-2 and the human intestinal epithelial crypt-like cells (HIEC). Based on the cytotoxicity of individual toxins, we studied the interactions between toxins in binary mixtures using the combination index-isobologram equation, amethodwidely used in pharmacology to study drug interactions. Thismethod quantitatively classifies interactions between toxins in mixtures as synergistic, additive or antagonistic. AZA-1/OA, and YTX/OA mixtures showed increasing antagonism with increasing toxin concentrations. In contrast, the AZA-1/YTX mixture showed increasing synergism with increasing concentrations, especially for mixtures with high YTX concentrations. These results highlight the hazard potency of AZA-1/YTX mixtures with regard to seafood intoxication. [ABSTRACT FROM AUTHOR]

Published
2016
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10. Cytotoxic and genotoxic effects of cylindrospermopsin in mice treated by gavage or intraperitoneal injection.

Author

Bazin, Emmanuelle, Huet, Sylvie, Jarry, Gérard, Hégarat, Ludovic Le, Munday, John S., Humpage, Andrew R., and Fessard, Valérie

Subjects
CYTOTOXIC T cells, LABORATORY mice, INTRAPERITONEAL injections, TUBE feeding, GENETIC toxicology, CYANOBACTERIAL toxins
Abstract

Cylindrospermopsin (CYN), a cyanobacterial hepatotoxin mainly produced by Cylindrospermopsis raciborskii, has been involved in human intoxications and livestock deaths. The widespread occurrence of CYN in the water supplies lead us to investigate its genotoxicity to assess potential chronic effects. This study reports evaluation of CYN-induced in vivo DNA damage in mice using alkaline comet assay (ACA) and micronucleus assay (MNA) concomittantly. ACA measures DNA breakage from single and double strand breaks as well as alkali labile sites. Conversely, MNA detects chromosome damage events such as chromosomal breakage and numeric alterations. Male Swiss mice were treated with CYN concentrations of 50, 100, and 200 μg/kg by a single intraperitoneal (ip) injection or with 1, 2, and 4 mg/kg by gavage. Methyl methane sulfonate (MMS) was used as positive control at 80 mg/kg. Twenty-four hours after treatment, samples of liver, blood, bone marrow, kidney, intestine, and colon were taken to perform ACA, the bone marrow and the colon were also used for MNA. Parameters used to quantify DNA damage were % Tail DNA for ACA and both micronucleated immature erythrocytes and epithelial colon cells for MNA. DNA breaks and chromosome damage were significantly increased by MMS in all the organs evaluated. Significant DNA damage was detected within the colon by ACA after ip injection of 100 and 200 μg/kg CYN ( P < 0.01). DNA damage was also detected in colon samples after 4 mg/kg oral administration of CYN and in bone marrow after 1 and 2 mg/kg of orally administered CYN. Histological examination showed foci of cell death within the liver and the kidney from mice that received the two highest doses of CYN by either route of administration. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012. [ABSTRACT FROM AUTHOR]

Published
2012
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11. A strategy to study genotoxicity: application to aquatic toxins, limits and solutions.

Author

Fessard, Valérie and Hégarat, Ludovic Le

Subjects
*TOXICOLOGY of water pollution, *GENETIC toxicology, *MARINE toxins, *ALGAL toxins, *INDUSTRIAL contamination
Abstract

Humans can be exposed to aquatic toxins mainly through contamination of food and water (drinking and recreational). Among these toxins, contamination by both phycotoxins occurring in shellfish and cyanotoxins mostly involved in freshwater bodies are of concern for public health. Whereas regulations exist to evaluate the genotoxicity of most compounds to which humans are exposed, including drugs and chemicals, no regulations have been established for these compounds. In this paper, we show that the same strategy including both in vitro and in vivo tests can be followed to evaluate the genotoxicity of aquatic toxins (phycotoxins and cyanotoxins). However, this strategy encountered different limits which arise when completing an overview of the genotoxic potential of toxins. The most restrictive one is undoubtedly the low amount (even the lack sometimes) of purified toxins available. Solutions and recommendations for testing the genotoxicity of aquatic toxins are suggested to overcome the specific problems encountered with these compounds. It must be kept in mind that recent developments in drug toxicology should be considered and that experiments must be conducted in respect of the 3Rs principle of refinement, reduction and replacement for animal experimentation. [ABSTRACT FROM AUTHOR]

Published
2010
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12. Measuring DNA modifications with the comet assay: a compendium of protocols

Author

Andrew Collins, Peter Møller, Goran Gajski, Soňa Vodenková, Abdulhadi Abdulwahed, Diana Anderson, Ezgi Eyluel Bankoglu, Stefano Bonassi, Elisa Boutet-Robinet, Gunnar Brunborg, Christy Chao, Marcus S. Cooke, Carla Costa, Solange Costa, Alok Dhawan, Joaquin de Lapuente, Cristian Del Bo’, Julien Dubus, Maria Dusinska, Susan J. Duthie, Naouale El Yamani, Bevin Engelward, Isabel Gaivão, Lisa Giovannelli, Roger Godschalk, Sofia Guilherme, Kristine B. Gutzkow, Khaled Habas, Alba Hernández, Oscar Herrero, Marina Isidori, Awadhesh N. Jha, Siegfried Knasmüller, Ingeborg M. Kooter, Gudrun Koppen, Marcin Kruszewski, Carina Ladeira, Blanca Laffon, Marcelo Larramendy, Ludovic Le Hégarat, Angélique Lewies, Anna Lewinska, Guillermo E. Liwszyc, Adela López de Cerain, Mugimane Manjanatha, Ricard Marcos, Mirta Milić, Vanessa Moraes de Andrade, Massimo Moretti, Damian Muruzabal, Matjaž Novak, Rui Oliveira, Ann-Karin Olsen, Norah Owiti, Mário Pacheco, Alok K. Pandey, Stefan Pfuhler, Bertrand Pourrut, Kerstin Reisinger, Emilio Rojas, Elise Rundén-Pran, Julen Sanz-Serrano, Sergey Shaposhnikov, Ville Sipinen, Karen Smeets, Helga Stopper, João Paulo Teixeira, Vanessa Valdiglesias, Mahara Valverde, Frederique van Acker, Frederik-Jan van Schooten, Marie Vasquez, Johannes F. Wentzel, Maciej Wnuk, Annelies Wouters, Bojana Žegura, Tomas Zikmund, Sabine A. S. Langie, Amaya Azqueta, University of Oslo (UiO), University of Copenhagen = Københavns Universitet (UCPH), Institute for Medical Research and Occupational Health, Institute of Experimental Medicine of the Academy of Sciences of the Czech Republic, Charles University [Prague] (CU), Florida International University [Miami] (FIU), University of Bradford, University of Würzburg = Universität Würzburg, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS San Raffaele Pisana), Universita Vita Salute San Raffaele = Vita-Salute San Raffaele University [Milan, Italie] (UniSR), ToxAlim (ToxAlim), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Ecole d'Ingénieurs de Purpan (INP - PURPAN), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre for Environmental Radioactivity (CERAD CoE), Norvegian Institut of Public Health, Massachusetts Institute of Technology (MIT), University of South Florida [Tampa] (USF), Instituto Nacional de Saùde Dr Ricardo Jorge [Portugal] (INSA), Universidade do Porto = University of Porto, Centre for Biomedical Research, University of Hull, University of Hull, Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Service d'Etudes du Comportement des Radionucléides (SECR), Département de Physico-Chimie (DPC), CEA-Direction des Energies (ex-Direction de l'Energie Nucléaire) (CEA-DES (ex-DEN)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-CEA-Direction des Energies (ex-Direction de l'Energie Nucléaire) (CEA-DES (ex-DEN)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Scientific Committee for Consumer Safety (SCCS) (SCCS), University of Aberdeen, Laboratoire de Fougères - ANSES, Agence nationale de sécurité sanitaire de l'alimentation, de l'environnement et du travail (ANSES), Farmacologie en Toxicologie, RS: NUTRIM - R3 - Respiratory & Age-related Health, Collins, Andrew, Møller, Peter, Gajski, Goran, Vodenková, Soňa, Abdulwahed, Abdulhadi, Anderson, Diana, Bankoglu, Ezgi Eyluel, Bonassi, Stefano, Boutet-Robinet, Elisa, Brunborg, Gunnar, Chao, Christy, Cooke, Marcus S, Costa, Carla, Costa, Solange, Dhawan, Alok, de Lapuente, Joaquin, Bo', Cristian Del, Dubus, Julien, Dusinska, Maria, Duthie, Susan J, Yamani, Naouale El, Engelward, Bevin, Gaivão, Isabel, Giovannelli, Lisa, Godschalk, Roger, Guilherme, Sofia, Gutzkow, Kristine B, Habas, Khaled, Hernández, Alba, Herrero, Oscar, Isidori, Marina, Jha, Awadhesh N, Knasmüller, Siegfried, Kooter, Ingeborg M, Koppen, Gudrun, Kruszewski, Marcin, Ladeira, Carina, Laffon, Blanca, Larramendy, Marcelo, Hégarat, Ludovic Le, Lewies, Angélique, Lewinska, Anna, Liwszyc, Guillermo E, de Cerain, Adela López, Manjanatha, Mugimane, Marcos, Ricard, Milić, Mirta, de Andrade, Vanessa Morae, Moretti, Massimo, Muruzabal, Damian, Novak, Matjaž, Oliveira, Rui, Olsen, Ann-Karin, Owiti, Norah, Pacheco, Mário, Pandey, Alok K, Pfuhler, Stefan, Pourrut, Bertrand, Reisinger, Kerstin, Rojas, Emilio, Rundén-Pran, Elise, Sanz-Serrano, Julen, Shaposhnikov, Sergey, Sipinen, Ville, Smeets, Karen, Stopper, Helga, Teixeira, João Paulo, Valdiglesias, Vanessa, Valverde, Mahara, van Acker, Frederique, van Schooten, Frederik-Jan, Vasquez, Marie, Wentzel, Johannes F, Wnuk, Maciej, Wouters, Annelie, Žegura, Bojana, Zikmund, Toma, Langie, Sabine A S, and Azqueta, Amaya

Subjects
Comet assay, DNA damage, animals, plants, ENABLES HIGH-THROUGHPUT, [SDV]Life Sciences [q-bio], INDUCED OXIDATIVE STRESS, alkaline comet assay, protocols, EPITHELIAL-CELLS, DOUBLE-STRAND BREAKS, Article, General Biochemistry, Genetics and Molecular Biology, INTER-LABORATORY VARIATION, DROSOPHILA-MELANOGASTER, CELL GEL-ELECTROPHORESIS, IN-VIVO MODEL, CORD BLOOD, INTERSTRAND CROSS-LINKS
Abstract

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.The comet assay is commonly used to assess DNA damage. This collection of consensus protocols includes adaptations for a wide range of species and sample types, assay formats and detection of different types of DNA lesions.

Published
2023

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