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Titolo:
Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing
Autore:
Tice, RR; Agurell, E; Anderson, D; Burlinson, B; Hartmann, A; Kobayashi, H; Miyamae, Y; Rojas, E; Ryu, JC; Sasaki, YF;
Indirizzi:
Integrated Lab Syst Inc, Res Triangle Pk, NC 27709 USA Integrated Lab SystInc Res Triangle Pk NC USA 27709 gle Pk, NC 27709 USA AB Astra, Safety Assessment, Sodertalje, Sweden AB Astra Sodertalje Sweden Astra, Safety Assessment, Sodertalje, Sweden BIBRA Int, Surrey, England BIBRA Int Surrey EnglandBIBRA Int, Surrey, England Glaxo Wellcome, Ware, Herts, England Glaxo Wellcome Ware Herts EnglandGlaxo Wellcome, Ware, Herts, England Novartis Pharma AG, Basel, Switzerland Novartis Pharma AG Basel Switzerland rtis Pharma AG, Basel, Switzerland Shiseido Co Ltd, Safety & Analyt Res Ctr, Yokohama, Kanagawa, Japan Shiseido Co Ltd Yokohama Kanagawa Japan s Ctr, Yokohama, Kanagawa, Japan Fujisawa Pharmaceut Co Ltd, Toxicol Res Labs, Osaka 532, Japan Fujisawa Pharmaceut Co Ltd Osaka Japan 532 ol Res Labs, Osaka 532, Japan UNAM, Inst Invest Biomed, Mexico City, DF, Mexico UNAM Mexico City DF Mexico , Inst Invest Biomed, Mexico City, DF, Mexico Korea Inst Sci & Technol, Toxicol Lab, Seoul 130650, South Korea Korea Inst Sci & Technol Seoul South Korea 130650 ul 130650, South Korea
Titolo Testata:
ENVIRONMENTAL AND MOLECULAR MUTAGENESIS
fascicolo: 3, volume: 35, anno: 2000,
pagine: 206 - 221
SICI:
0893-6692(2000)35:3<206:SCGAGF>2.0.ZU;2-V
Fonte:
ISI
Lingua:
ENG
Soggetto:
ELECTROPHORESIS COMET ASSAY; SPRAGUE-DAWLEY RATS; INDUCED DNA-DAMAGE; GEL-ELECTROPHORESIS; MUTAGENICITY TEST; HUMAN-LYMPHOCYTES; INDIVIDUAL CELLS; STRAND BREAKS; GENOTOXICITY; APOPTOSIS;
Keywords:
single cell gel assay; Comet assay; DNA damage; genotoxicity; alkaline electrophoresis;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
63
Recensione:
Indirizzi per estratti:
Indirizzo: Tice, RR Integrated Lab Syst Inc, POB 13501, Res Triangle Pk, NC 27709 USAIntegrated Lab Syst Inc POB 13501 Res Triangle Pk NC USA 27709 SA
Citazione:
R.R. Tice et al., "Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing", ENV MOL MUT, 35(3), 2000, pp. 206-221

Abstract

At the International Workshop on Genotoxicity Test Procedures (IWGTP) heldin Washington, DC, March 25-26, 1999, an expert panel met to develop guidelines for the use of the single-cell gel (SCG)/Comet assay in genetic toxicology. The expert panel reached a consensus that the optimal version of theComet assay for identifying agents with genotoxic activity was the alkaline (pH, 13) version of the assay developed by Singh et al. [1988]. The pH > 13 version is capable of detecting DNA single-strand breaks (SSB), alkali-labile sites (ALS), DNA-DNA/DNAprotein cross-linking, and SSB associated with incomplete excision repair sites. Relative to at her genotoxicity tests, the advantages of the SCG assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, ifs ease of application, and the short time needed to complete a study. The expert panel decided that nosingle version of the alkaline (pH > 13) Comet assay was clearly superior. However, critical technical steps within the assay were discussed and guidelines developed for preparing slides with agarose gels, lysing cells to liberate DNA, exposing the liberated DNA to alkali to produce single-strandedDNA and to express ALS as SSB, electrophoresing the DNA using pH > 13 alkaline conditions, alkali neutralization, DNA staining, comet visualization, and data collection. Based on the current state of knowledge, the expert panel developed guidelines for conducting in vitro or in vivo Comet assays. The goal of the expert panel was to identify minimal standards for obtainingreproducible and reliable Comet data deemed suitable for regulatory submission, The expert panel used the current Organization for Economic Go-operation and Development (OECD) guidelines Far in vitro and in vivo genetic toxicological studies as guides during the development of the corresponding in vitro and in vivo SCG assay guidelines. Guideline topics considered included initial considerations, principles of the test method, description of thetest method, procedure, results, data analysis and reporting. Special consideration was given by the expert panel to the potential adverse effect of DNA degradation associated with cytotoxicity on the interpretation of Cometassay results. The expert panel also discussed related SCG methodologies that might be useful in the interpretation of positive Comet data, The related methodologies discussed included: (1) the use of different pH conditionsduring electrophoreses to discriminate between DNA strand breaks and ALS (2) the use of repair enzymes or antibodies to detect specific classes of DNA damage; (3) the use of a neutral diffusion assay to identify apoptotic/necrotic cells; and (4) the use of the acellular SCG assay to evaluate the ability of a test substance to interact directly with DNA. The alkaline (pH >13) Comet assay guidelines developed by the expert panel represent a work in progress. Additional information is needed before the assay can be critically evaluated for its utility in genetic toxicology. The information needed includes comprehensive data on the different sources of variability (e.g., cell to cell, gel to gel, run to run, culture to culture, animal to animal, experiment to experiment) intrinsic to the alkaline (pH > 3) SCG assay,the generation of a large database based on in vitro and in vivo testing using these guidelines, and the results of appropriately designed multilaboratory international validation studies. (C) 2000 Wiley-Liss, Inc.

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Documento generato il 28/09/20 alle ore 12:16:32