Accepted Manuscript Title: The comet assay in animal models: From bugs to whales – (Part 2 Vertebrates) Author: Goran Gajski Bojana Zeguraˇ Carina Ladeira Matjazˇ Novak Monika Sramkova Bertrand Pourrut Cristian Del Bo’ Mirta Milic´ Kristine Bjerve Gutzkow Solange Costa Maria Dusinska Gunnar Brunborg Andrew Collins PII: S1383-5742(18)30108-X DOI: https://doi.org/doi:10.1016/j.mrrev.2019.04.002 Reference: MUTREV 8273 To appear in: Mutation Research Received date: 19 November 2018 Revised date: 26 March 2019 Accepted date: 10 April 2019 Please cite this article as: G. Gajski, B. Zegura,ˇ C. Ladeira, M. Novak, M. Sramkova, B. Pourrut, C. Del Bo’, M. Milic,´ K.B. Gutzkow, S. Costa, M. Dusinska, G. Brunborg, A. Collins, The comet assay in animal models: From bugs to whales ndash (Part 2 Vertebrates), Mutation Research-Reviews in Mutation Research (2019), https://doi.org/10.1016/j.mrrev.2019.04.002 This is a PDF file of an unedited manuscript that has been accepted for publication. 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Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 The comet assay in animal models: From bugs to whales – 2 (Part 2 Vertebrates) 3 4 5 Goran Gajski 1, * , Bojana Žegura 2, Carina Ladeira 3,4 , Matjaž Novak 2, Monika Sramkova 5, 6 Bertrand Pourrut 6, Cristian Del Bo' 7, Mirta Mili ć1, Kristine Bjerve Gutzkow 8, Solange 7 Costa 9,10 , Maria Dusinska 11, Gunnar Brunborg 8, Andrew Collins 12 8 9 10 1Mutagenesis Unit, Institute for Medical Research and Occupational Health, Zagreb, Croatia 11 2Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, 12 Ljubljana, Slovenia 13 3H&TRC- Health & Technology Research Center, Escola Superior de Tecnologia da Saúde, 14 Instituto Politécnico de Lisboa, Lisbon, Portugal 15 4Centro de Investigação e Estudos em Saúde de Publica, Universidade Nova de Lisboa, 16 Lisbon, Portugal 17 5Biomedical Research Center, Cancer Research Institute, Slovak Academy of Sciences, 18 Bratislava, Slovakia 19 6EcoLab, Université de Toulouse, CNRS, INPT, UPS, Toulouse, France 20 7DeFENS-Division of Human Nutrition, University of Milan, Milan, Italy 21 8Department of Molecular Biology, Norwegian Institute of Public Health, Oslo, Norway 22 9National Health Institute Dr. Ricardo Jorge, Environmental Health Department, Porto, 23 Portugal 24 10 EPIUnit - Instituto de Saúde Pública, Universidade do Porto, Porto, Portugal 25 11Health Effects Laboratory, Department of Environmental Chemistry-MILK, NILU- 26 Norwegian Institute for Air Research, Kjeller, Norway 27 Accepted12Department of Nutrition, University Manuscript of Oslo, Oslo, Norway 28 29 30 31 *Address correspondence to: Goran Gajski, Institute for Medical Research and 32 Occupational Health, Mutagenesis Unit, Ksaverska cesta 2, 10000 Zagreb, Croatia, Tel. +385 33 1 4682 500, Fax. +385 1 4673 303, e-mail address: [email protected] 1 Page 1 of 128 34 Abstract 35 36 The comet assay has become one of the methods of choice for the evaluation and 37 measurement of DNA damage. It is sensitive, quick to perform and relatively affordable for 38 the evaluation of DNA damage and repair at the level of individual cells. The comet assay can 39 be applied to virtually any cell type derived from different organs and tissues. Even though 40 the comet assay is predominantly used on human cells, the application of the assay for the 41 evaluation of DNA damage in yeast, plant and animal cells is also quite high, especially in 42 terms of biomonitoring. The present extensive overview on the usage of the comet assay in 43 animal models will cover both terrestrial and water environments. The first part of the review 44 was focused on studies describing the comet assay applied in invertebrates. The second part of 45 the review, (Part 2) will discuss the application of the comet assay in vertebrates covering 46 cyclostomata, fishes, amphibians, reptiles, birds and mammals, in addition to chordates that 47 are regarded as a transitional form towards vertebrates. Besides numerous vertebrate species, 48 the assay is also performed on a range of cells, which includes blood, liver, kidney, brain, gill, 49 bone marrow and sperm cells. These cells are readily used for the evaluation of a wide 50 spectrum of genotoxic agents both in vitro and in vivo . Moreover, the use of vertebrate models 51 and their role in environmental biomonitoring will also be discussed as well as the 52 comparison of the use of the comet assay in vertebrate and human models in line with ethical 53 principles. Although the comet assay in vertebrates is most commonly used in laboratory 54 animals such as mice, rats and lately zebrafish, this paper will only briefly review its use 55 regarding laboratory animal models and rather give special emphasis to the increasing usage 56 of the assay in domestic and wildlife animals as well as in various ecotoxicological studies. 57 58 59 60 61 Accepted Manuscript 62 63 64 65 Keywords: Comet assay, DNA damage, Animal model, In vivo , In vitro , Vertebrates, 66 Biomonitoring 67 2 Page 2 of 128 68 1. Introduction 69 70 Over the past decades, the comet assay (single-cell gel electrophoresis) has become 71 one of the methods of choice for the evaluation and measurement of DNA damage [1–3]. In 72 epidemiological molecular and biomonitoring studies, primary DNA damage, evaluated by 73 the comet assay, is used as a biomarker of exposure that provides information on the 74 biologically effective dose of various physical and/or chemical mutagens/carcinogens [4–6]. 75 The comet assay is quite simple, quick, cost effective, accurate and reliable; in other words, it 76 fulfils all the criteria for use in routine laboratory, field as well as clinical testing. The comet 77 assay is a predictive test that allows for the detection of DNA alterations of diverse kinds, 78 such as single-strand DNA breaks, double-strand DNA breaks, alkali-labile sites, altered 79 bases, incomplete repair sites, and interstrand cross-links [5,7–10]. 80 Quantification of DNA damage by lysing cells embedded in agarose gel under alkaline 81 conditions was first introduced by Rydberg and Johanson [11]. To improve the sensitivity of 82 the single-cell DNA damage detection, Östling and Johanson developed the micro-gel 83 electrophoresis technique, also called the comet assay [12]. Cells are embedded in an agarose 84 matrix and lysed to produce nucleoids of supercoiled DNA attached to the nuclear matrix. 85 Breaks in the DNA relax the supercoiling and allow DNA loops to expand, and on 86 electrophoresis to move towards the anode. Along the way, the DNA path resembles the 87 shape of a comet, which gave the assay its final name. This is followed by either visual (after 88 classification into different categories based on tail length and shape), semi-automatic and/or 89 automatic (which automatically recognise the extent of damage) analyses of stained DNA and 90 calculation of fluorescence to determine the DNA damage extent. This is done by using 91 appropriate software that enables commercially available image analysing systems to be 92 connected through a camera to a fluorescence microscope, which facilitates the evaluation of 93 DNA damage. Each trace resembles a comet with a brightly fluorescent head and a tail whose 94 length and intensity are proportional to the frequency of DNA breaks present in the cell 95 [1,13–19]. Accepted Manuscript 96 At least 50 comets are analysed per sample, and several types of descriptors can be 97 employed, including tail length, relative tail intensity and tail moment. The migration length 98 is proportional to the extent of DNA damage but reaches a maximum at a relatively low level 99 of damage. Tail intensity is expressed as % of total DNA fluorescence in the tail of the comet. 100 Tail moment is calculated as the product of the tail length and the fraction of total DNA in the 101 comet tail. The use of software enables the measurement of a range of different descriptors, 3 Page 3 of 128 102 many of which might not be relevant in determining the extent of DNA damage. A large 103 number of factors could influence the shape, size, identification and determination of induced 104 damage, including the scoring criteria, staining techniques, selection of descriptors and/or 105 appearance of 'hedgehog' or 'clouds' [14,16]. Tail length, tail DNA and DNA distribution 106 profile in the tail are primary comet assay measurements (obtained by fluorescent 107 densitometric profiles of the comets). All other measurements are derived from the three 108 primary comet assay measurements [16,20]. Today, % tail DNA is recommended as the best 109 descriptor for DNA break frequencies. However, many researchers still prefer the use of tail 110 moment [21]. It has to be pointed out that these two descriptors are similarly influenced by 111 assay conditions [22–24]. 112 The most common comet assay application today is the one under alkaline conditions 113 allowing the detection of alkali labile sites in addition to DNA double- and single-strand 114 breaks. The less frequently used neutral comet assay allows both single- and double-strand 115 break detection but with less sensitivity than the alkaline version [1,2,25–27]. The sensitivity 116 and specificity of this assay may be further improved by using lesion-specific enzymes 117 capable of converting damaged bases to DNA strand breaks. For example, enzymes able to 118 recognize and remove oxidized bases induced by reactive oxygen radicals, which may arise as 119 a result of mutagenic exposure are used.