The comet assay for DNA damage and repair. Applications in genotoxicity testing and human biomonitoring

Transcription

1 The comet assay for DNA damage and repair Applications in genotoxicity testing and human biomonitoring Andrew Collins Department of Nutrition, University of Oslo

2 Introduction measuring DNA damage Increasing throughput Genotoxicity testing DNA repair Applications in human biomonitoring

3 The comet assay (single cell gel electrophoresis) a simple and versatile method for measuring DNA breaks. Widely used in genotoxicity testing, human biomonitoring, ecogenotoxicology, and fundamental research into mechanisms of DNA damage and repair.

4 Introduction The comet assay is a quantitative method for measuring DNA damage - primarily strand breaks. Few genotoxic agents break DNA directly, but many lesions can be converted to breaks, and DNA repair pathways involve breaks as intermediates. DNA damage is a precursor of cancer, but the link is a weak one, as almost all damage is repaired

5 DNA damage can arise from exposure of cells to environmental mutagens/carcinogens. It can also occur as a consequence of endogenous processes notably oxidation. OH C N H 2 NC N HOH 2 C O N N C OH O DNA is readily oxidised most commonly by endogenous reactive oxygen (free radicals) released from mitochondria or as part of the inflammatory response. 8-oxoguanine potentially mutagenic, easily measured; a popular biomarker. 8-oxoguanine For an index of antioxidant status, cells are treated with H 2 O 2 and resulting strand breaks are measured.

6 % DNA in tail Visual score (arb. units) The comet assay (single cell gel electrophoresis) Cells embedded in agarose on microscope slide Lysis: Triton X-100, 2.5 M NaCl Nucleoids; supercoiled DNA ) s t i n u Alkaline incubation: 0.3 M NaOH, 10 mm EDTA l i a t n i A N D % y r a r t i b r a ( e r o c s l a u s i V % of DNA in tail depends on DNA break frequency Electrophoresis: 0.8 V/cm, 30 min Neutralisation, DAPI stain, fluorescence microscopy DNA breaks per 10 9 daltons

7 The comet assay (single cell gel electrophoresis) a simple and versatile method for measuring DNA breaks. Widely used in genotoxicity testing, human biomonitoring, ecogenotoxicology, and fundamental research into mechanisms of DNA damage and repair. The assay is made more sensitive and specific by incorporation of DNA repair enzymes that convert damaged bases to breaks (endonuclease III for oxidised pyrimidines, formamidopyrimidine DNA glycosylase [FPG] for oxidised purines, T4endoV for UV damage, AlkA for alkylated purines)

8 The comet assay (modified with lesion-specific enzymes) Cells embedded in agarose on microscope slide Lysis: Triton X-100, 2.5 M NaCl Nucleoids; supercoiled DNA Digestion with lesion-specific endonuclease Alkaline incubation: 0.3 M NaOH, 10 mm EDTA The frequency of damaged bases is given by the increase in DNA breaks in the presence of the specific endonuclease Electrophoresis: 0.8 V/cm, 30 min Neutralisation, DAPI stain, fluorescence microscopy This assay allows us to estimate the level of base damage in cells (e.g. lymphocytes)

9 D N A breaks (arbi trary uni ts) D N A breaks (arbi trary uni ts) The frequency of damaged bases is given by the increase in DNA breaks in the presence of the specific endonuclease Lymphocytes from ~30 healthy subjects: measurement of strand breaks ± FPG-sensitive sites pl us FPG no FPG net FPG - sensi t i ve si t es Subj ects Subj ects Net FPG-sensitive sites are calculated from the difference (± enzyme)

10 Increasing throughput A limitation of the standard comet assay is the number of samples that can be analysed in a single experiment; around 40 gels in a standard tank.

11 High throughput methods were developed in the recent COMICS project: Twelve mini-gels on a standard glass slide or a GelBond film with up to 96 mini-gels.

12 For the 12-gel system, we developed a chamber device (now commercially available) that allows individual treatment of gels with reagents or enzymes: The slide is clamped under a gasket with holes matching the gel positions.

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14 Genotoxicity testing

15 Enhancing sensitivity FPG is widely used in human biomonitoring to measure oxidised purines. However, the enzyme recognises other kinds of damage - either directly, or via secondary oxidative damage. It can greatly increase the sensitivity of detection of various lesions in genotoxicity testing. We carried out a trial with TK-6 cells treated with cytotoxic and non-cytotoxic chemicals, and with known genotoxins (+ S9 if necessary) at ~non-cytotoxic concentrations. [Azqueta et al. (2013) Mutagenesis 28, ]

16 Non-cytotoxic: Broken line: proliferation index Strand breaks (lysis only) FPG-sites (net) No strand breaks, and no FPG-sensitive sites

17 Cytotoxic: Broken line: proliferation index Strand breaks (lysis only) FPG-sites (net) DNA strand breaks, but at cytotoxic concentrations

18 Genotoxic: Broken line: proliferation index Strand breaks (lysis only) FPG-sites (net)

19 Genotoxic: Broken line: proliferation index Strand breaks (lysis only) FPG-sites (net) The use of FPG in the comet assay should significantly reduce the risk of false negatives when testing novel chemicals

20 DNA repair

21 DNA repair DNA damage as measured in cells represents a dynamic steady state a balance between input of damage and its repair. Input (free radicals) antioxidants apoptosis mutation Oxidised bases? in DNA DNA repair So DNA repair is an important factor in defining an individual s genetic stability status.

22 DNA repair DNA damage is the first step in carcinogenesis; but almost all damage that occurs is repaired. DNA repair capacity is regarded as an intrinsic biomarker of individual susceptibility; the higher the repair rate, the lower the cancer risk. It is likely that repair rates are influenced by genetic polymorphisms, but also by environmental factors exposure to DNA-damaging agents (inducing appropriate DNA repair pathways), and perhaps micronutrients.

23 DNA repair pathways that can be studied with the comet assay: Strand break (SB) rejoining insertion of missing nucleotide and ligation. Base excision repair (BER) (oxidised or alkylated DNA) a specific glycosylase removes the base, leaving an AP site, which is then cleaved. A DNA polymerase fills the gap; finally, ligation. Nucleotide excision repair (NER) (bulky adducts, UV-induced pyrimidine dimers) a repair protein complex removes an oligonucleotide containing the damage; gap-filling by DNA polymerase is followed by ligation.

24 The simplest approach to measuring DNA repair is to treat cells with a DNA-damaging agent, incubate the cells, and measure remaining damage at intervals. To measure SB rejoining, cells were treated with H 2 O 2. For BER of oxidised bases, cells were irradiated with visible light in the presence of photosensitiser Ro to induce 8- oxogua. Remaining damage was detected with FPG. SB rejoining Blue: HeLa cells Red: Caco2 cells BER of 8-oxoGua

25 For BER and NER, we developed an alternative in vitro assay, suitable for assaying repair capacity in samples of lymphocytes from human trials. A cell extract is incubated with nucleoids containing specific DNA damage - 8-oxoGua for BER, and UV-induced pyrimidine dimers for NER. Strand breaks (incision events incomplete repair sites) accumulate and are measured with the comet assay.

26 In vitro assay for BER DNA damage to substrate cells Lymphocytes Cell-free extract Incubation with extract: DNA substrate incised. Cells damaged with Ro (photosensitiser) + light, to induce 8-oxoGua, provide substrate nucleoids to measure activity of 8-oxoGua DNA glycosylase (OGG). Alkaline treatment Electrophoresis % DNA in comet tail indicates break frequency. Rate of accumulation of breaks is a measure of OGG repair activity. No extract Plus extract

27 DNA repair measured in humans Lymphocyte samples taken from ~30 subjects on two occasions. Assayed for BER and NER Correlation between 2 samples; i.e. subjects have characteristic repair rates. Wide range of values for different subjects; plenty of scope for investigating environmental and A promising biomarker assay genetic influences. [Gaivão et al. (2009) Cell Biol Toxicol 25, 45-52]

28 Applying the comet assay in human biomonitoring An example nutritional effects on DNA damage and repair

29 Kiwifruit intervention trial I II III Samples: * * * * * * Kiwifruit 3 weeks of supplementation 14 volunteers, in 3 groups. Crossover design, different doses, with washout periods between. [Collins et al. (2003) Carcinogenesis, 24, 511]

30 H 2 O 2 -induced breaks (arb. units) Ex vivo H2O2 challenge i n u y r a r t i b r a ( H 2 O 2 -induced DNA breaks decreased s k a e r b d e c u d n i - 2 O H P<0.001 P=0.05 P= Kiwifruits per day

31 FPG sites (arbitrary units) EndoIII sites (arbitrary units) ) s t i n u y r a r t i b r a ( s e t i s Endogenous damage Oxidised pyrimidines decreased P=0.04 t i b r a ( s e t i s I I I o d n E P<0.01 P<0.01 P= Kiwifruits per day G P F Kiwifruits per day Oxidised purines decreased

32 mrna ratio DNA repair rate (arb. units) DNA repair (BER) Base excision repair (OGG) enhanced y r a r t i b r a ( e t a r r i a p e r P=0.001 Before After P=0.03 P<0.001 A N D Kiwifruits per day but no effect at level of gene expression (APE1, OGG1)

33 A recent application in ophthalmology Lens epithelium from 11 cataract patients. FPG-sites were relatively high and endoiii-sites relatively low, compared with lymphocytes. [Osnes-Ringen et al. (2013) Acta Ophthalm. 91, ]

34 Leukocytes from frozen blood Till now, human biomonitoring has depended on isolation of peripheral blood mononuclear cells ( lymphocytes ). But it was recently shown that the comet assay could be performed on whole blood after freezing at -20 C (in small volumes). We improved this procedure by isolating leukocytes from the frozen/thawed blood. After thorough washing, the cells perform well in the comet assay, with or without FPG. This procedure has substantial advantages for biomonitoring: quick freezing, no need for -70 C freezer, small volumes of blood. How reliable is it?

35 Leukocytes from frozen blood Ten subjects, whole blood frozen for ~1 year, then leukocytes isolated. SBs, FPG-sites, and H 2 O 2 -induced breaks measured. SBs FPGsites H 2 O 2 - breaks [Akor-Dewu, MB et al. (2014) Cell Biochem. Funct. 32, ]

36 Leukocytes from frozen blood There was a good correlation between FPG-sites and H 2 O 2 -induced breaks both reflecting individual redox status. This concordance suggests that what we are measuring has real biological significance.

37 Summary: the comet assay in 2015 The assay of choice for specific DNA damage High throughput, miniaturisation Genotoxicity testing with extra sensitivity DNA repair - useful in vitro biomarker assays Ongoing applications, in nutrition, occupational and environmental exposure, ecogenotoxicology etc. Assays possible on leukocytes from frozen blood Novel clinical applications, e.g. in ophthalmology

38 Thanks to: Amaya Azqueta, Sergey Shaposhnikov, Isabel Gaivão, Torgrim Langleite, Yolanda Lorenzo, Maryam Dewu, Lena Bilyk, Naouale El-Yamani (Nutrition, University of Oslo) Bjørn Nicolaissen, Øyvind Ringen (Ophthalmology, University Hospital, Oslo) Maria Dusinska (NILU, Norway) Former colleagues in Aberdeen (Rowett Research Institute, Aberdeen, UK)