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1. Assessing non‐adjunctive CGM safety at home and in new markets (ANSHIN)

Author

Christy Chao, Sarah B. Andrade, Simon Bergford, Peter Calhoun, John B. Welsh, Tomas C. Walker, and ANSHIN Study Group

Subjects
blood glucose self‐monitoring, glycated haemoglobin, glycaemic control, Diseases of the endocrine glands. Clinical endocrinology, RC648-665
Abstract

Abstract Introduction Continuous glucose monitoring (CGM) can guide treatment for people with type 1 (T1D) and type 2 diabetes (T2D). The ANSHIN study assessed the impact of non‐adjunctive CGM use in adults with diabetes using intensive insulin therapy (IIT). Materials and Methods This single‐arm, prospective, interventional study enrolled adults with T1D or T2D who had not used CGM in the prior 6 months. Participants wore blinded CGMs (Dexcom G6) during a 20‐day run‐in phase, with treatment based on fingerstick glucose values, followed by a 16‐week intervention phase and then a randomized 12‐week extension phase with treatment based on CGM values. The primary outcome was change in HbA1c. Secondary outcomes were CGM metrics. Safety endpoints were the number of severe hypoglycaemic (SH) and diabetic ketoacidosis (DKA) events. Results Of the 77 adults enrolled, 63 completed the study. Those enrolled had mean (SD) baseline HbA1c of 9.8% (1.9%), 36% had T1D, and 44% were ≥65 years old. Mean HbA1c decreased by 1.3, 1.0 and 1.0 percentage points for participants with T1D, T2D or age ≥65, respectively (p

Published
2023
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2. Integrated Genotoxicity Testing of three anti-infective drugs using the TGx-DDI transcriptomic biomarker and high-throughput CometChip® assay in TK6 cells

Author

Julie K. Buick, Andrea Rowan-Carroll, Rémi Gagné, Andrew Williams, Renxiang Chen, Heng-Hong Li, Albert J. Fornace, Christy Chao, Bevin P. Engelward, Roland Frötschl, Heidrun Ellinger-Ziegelbauer, Syril D. Pettit, Jiri Aubrecht, and Carole L. Yauk

Subjects
genetic toxicology, TGx-28.65 genomic biomarker, toxicogenomics, nitrofurantoin, metronidazole, novobiocin, Toxicology. Poisons, RA1190-1270
Abstract

Genotoxicity testing relies on the detection of gene mutations and chromosome damage and has been used in the genetic safety assessment of drugs and chemicals for decades. However, the results of standard genotoxicity tests are often difficult to interpret due to lack of mode of action information. The TGx-DDI transcriptomic biomarker provides mechanistic information on the DNA damage-inducing (DDI) capability of chemicals to aid in the interpretation of positive in vitro genotoxicity data. The CometChip® assay was developed to assess DNA strand breaks in a higher-throughput format. We paired the TGx-DDI biomarker with the CometChip® assay in TK6 cells to evaluate three model agents: nitrofurantoin (NIT), metronidazole (MTZ), and novobiocin (NOV). TGx-DDI was analyzed by two independent labs and technologies (nCounter® and TempO-Seq®). Although these anti-infective drugs are, or have been, used in human and/or veterinary medicine, the standard genotoxicity testing battery showed significant genetic safety findings. Specifically, NIT is a mutagen and causes chromosome damage, and MTZ and NOV cause chromosome damage in conventional in vitro tests. Herein, the TGx-DDI biomarker classified NIT and MTZ as non-DDI at all concentrations tested, suggesting that NIT’s mutagenic activity is bacterial specific and that the observed chromosome damage by MTZ might be a consequence of in vitro test conditions. In contrast, NOV was classified as DDI at the second highest concentration tested, which is in line with the fact that NOV is a bacterial DNA-gyrase inhibitor that also affects topoisomerase II at high concentrations. The lack of DNA damage for NIT and MTZ was confirmed by the CometChip® results, which were negative for all three drugs except at overtly cytotoxic concentrations. This case study demonstrates the utility of combining the TGx-DDI biomarker and CometChip® to resolve conflicting genotoxicity data and provides further validation to support the reproducibility of the biomarker.

Published
2022
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3. 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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4. Applications of CometChip for Environmental Health Studies

Author

Bevin P. Engelward and Christy Chao

Subjects
medicine.medical_specialty, 010501 environmental sciences, Toxicology, 01 natural sciences, Article, 03 medical and health sciences, Environmental health, medicine, Animals, Humans, 030304 developmental biology, 0105 earth and related environmental sciences, 0303 health sciences, Extramural, business.industry, Public health, food and beverages, Heavy metals, General Medicine, Chemical industry, DNA, Particulates, Environmental science, Comet Assay, business, Environmental Health, DNA Damage
Abstract

Copyright © 2020 American Chemical Society. Environmental exposures have long been known to impact public health and safety. For example, exposures to airborne particulates, heavy metals in water, or certain industrial chemicals can contribute to aging and to risk of developing cancer and other diseases. Environmental factors can impact health in a variety of ways, but a key concern is DNA damage, which can lead to mutations that cause cancer. Cancer can take years to develop following chemical exposure; however, one way to predict carcinogenicity in a more practical time frame is by studying the chemical's ability to induce DNA damage. The comet assay (or single-cell gel electrophoresis assay) has been used successfully for genotoxicity testing. The comet assay allows for the detection of DNA strand breaks via analysis of DNA migration during electrophoresis. Previously, the Engelward laboratory, in collaboration with the Bhatia laboratory, developed the CometChip for measurements of DNA damage and repair. The CometChip is a high-throughput comet assay that improves user reproducibility and significantly shortens total assay time. Here, we describe how the high-throughput CometChip platform can be used to measure DNA damage in established cell lines, animal models, and human samples. We also discuss technical challenges associated with these studies and provide recommendations on how to achieve optimal results for researchers interested in adopting this assay.

Published
2020

5. SpheroidChip: Patterned Agarose Microwell Compartments Harboring HepG2 Spheroids are Compatible with Genotoxicity Testing

Author

Bevin P. Engelward, Christy Chao, P Ngo Le, Massachusetts Institute of Technology. Department of Chemical Engineering, and Massachusetts Institute of Technology. Department of Biological Engineering

Subjects
DNA damage, 0206 medical engineering, Cell Culture Techniques, Biomedical Engineering, 02 engineering and technology, Matrix (biology), Article, Biomaterials, Tissue culture, chemistry.chemical_compound, Spheroids, Cellular, Animals, Mutagenicity Tests, Chemistry, Sepharose, Spheroid, Hydrogen Peroxide, 021001 nanoscience & nanotechnology, 020601 biomedical engineering, Comet assay, Electrophoresis, embryonic structures, Biophysics, Agarose, 0210 nano-technology, DNA
Abstract

Three-dimensional tissue culture models are emerging as effective alternatives to animal testing. They are especially beneficial for liver toxicity studies, enabling hepatocytes to display improved levels of liver-specific functions. One common model is hepatocyte spheroids, which are spontaneously formed cell aggregates. Techniques for spheroid formation include the use of ultralow attachment plates and the hanging drop method, both of which are technically challenging and relatively low throughput. Here, we describe a simple-to-use platform that improves spheroid production and is compatible with genotoxicity testing by the comet assay. To achieve this, we created a chip containing a microwell array where dozens of spheroids are contained within a single well of a 96-well plate. The microwells are made from agarose, a nontoxic material suitable for cell growth and spheroid formation. HepG2 cells loaded into customizable microwells formed spheroids through agarose-assisted aggregation within one to two days. In addition, the agarose matrix allows the same platform to be used in DNA damage analysis. Specifically, the comet assay enables quantification of DNA breaks based on the increased migration of damaged DNA through agarose during electrophoresis. Here, we developed a modified comet assay and show that intact HepG2 spheroids cultured in microwells can be electrophoresed to reveal the extent of DNA damage following exposure to inflammatory chemicals (H2O2 and SIN-1). With this SpheroidChip analysis method, we detected a dose-dependent increase in DNA damage and observed rapid repair of H2O2-induced DNA damage. In summary, we utilized an agarose microarray to condense what had required an entire 96-well plate into a single well, enabling analysis techniques that were cumbersome or impossible under conditions of a single spheroid per well of a 96-well plate., National Institute of Environmental Health Sciences (Grants T32-ES007020, P42-ES027707, P30-ES002109)

Published
2019

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