This work was supported by the US Food and Drug Administration. We acknowledge the original publication of the CPA study by Elsevier B.V.: Ding W, Bishop ME, Peace MG, Davis KJ, White GA, Lyn-Cook LE, Manjanatha MG. Sex-specific dose-response analysis of genotoxicity in cyproterone acetate-treated F344 rats. Mutation Research 774: 1-7, 2014 (PMID: 25440904)

Ethics statement: Procedures involving animals have been approved by the Institutional Animal Care and Use Committee (IACUC) at the US FDA/National Center for Toxicological Research.
NOTE: The study design described here is based on the protocol developed by the Japanese Center for the Validation of Alternative Methods (JaCVAM) for their validation of the in vivo rodent alkaline comet assay18, and further modified based on recommendations in OECD guideline TG48919.
1. Preparation
<ol>
	<li>Slide preparation
	<ol>
		<li>Dissolve regular melting agarose at 1% (w/v) in phosphate buffer (Ca2+, Mg2+ free and phenol red free) by heating in a microwave oven.</li>
		<li>Place the molten 1% standard agarose solution in a 55 &#176;C water bath and equilibrate the temperature with the bath. Dip glass microscope slides into the agarose solution, drain off any excess agarose and wipe the back of the slides clean. Place the slides upright on a flat surface and allow the slides to dry at RT; store the slides in dry place.</li>
	</ol>
	</li>
	<li>Preparation of comet assay solutions
	<ol>
		<li>Prepare 0.5% low-melting agarose solution. Dissolve low-melting agarose in phosphate buffer (Ca2+, Mg2+, and phenol red free) by heating in a microwave oven. Keep the solution at 37-45 &#176;C during comet slide preparation; discard any remaining agarose solution afterwards.</li>
		<li>Prepare Lysis buffer. Make a 100 mM solution of disodium ethylenediaminetetraacetic acid (disodium EDTA, Mr 372.24 g/mol), 2.5 M sodium chloride (NaCl, Mr 58.44 g/mol), and 10 mM tris hydroxymethyl aminomethane (Tris Base, Mr 121.14 g/mol) in purified water, and adjust to pH 10 with 10 N sodium hydroxide solution (NaOH, Mr 40 g/mol). On the day of use, add Triton X100 and dimethyl sulfoxide (DMSO, Mr 78.13 g/mol) to the solution to achieve final concentrations of 1% and 10%, respectively, and stir at 4-10 &#176;C for at least 30 min prior to use.</li>
		<li>Make fresh enzyme buffer on the day of use. Prepare a solution containing 40 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES, Mr 238.3 g/mol), 100 mM potassium chloride (KCl, Mr 74.55 g/mol), 0.5 mM disodium EDTA, and 0.2% bovine serum albumin (BSA, Mr 66,463 g/mol) in purified water. Adjust the pH to 8.0 with 10 N potassium hydroxide solution (KOH, 56.1 g/mol). Stir at RT and verify complete dissolution prior to use.</li>
		<li>Prepare an alkaline buffer solution containing 300 mM NaOH and 1 mM disodium EDTA in purified water. Stir at 4-10 &#176;C for at least 30 min prior to use. Measure the pH of the solution prior to use and make sure the pH is &#62;13.</li>
		<li>Prepare a solution of 0.4 M Tris Base in purified water, and adjust to pH 7.5 with hydrochloric acid (HCl, Mr 36.46). Store the solution at 4-10 &#176;C. This is neutralizing buffer.</li>
		<li>Make fresh mincing buffer on the day of use. Prepare a solution containing 20 mM disodium EDTA and 10% DMSO in Hank&#39;s Balanced Salt Solution (HBSS, Ca2+, Mg2+, and phenol red free). Store on ice until use.</li>
		<li>To prepare staining solution, dilute nucleic acid fluorescent stain stock solution with Tris-borate-EDTA (TBE buffer) at 1:10,000 (v/v). Protect the solution from light using aluminum foil. Store at 2-8 &#176;C and use with 24 hr.</li>
	</ol>
	</li>
</ol>
2. Preparation of Single Cell Suspensions from Rat Liver
<ol>
	<li>Euthanize rats with CO2 gas following methods recommended in the American Veterinary Medical Guidelines for the Euthanasia of Animals: 2013 Edition20.
	<ol>
		<li>Set the CO2 flow rate at 2.75. Put the rat in the euthanasia chamber (e.g., desiccator jar, rat cage with vented lid) and turn on the CO2 by opening the tank valve. Wait for 1.5 to 2.5 min until the rat becomes unconscious. Increase the CO2 flow to its maximum setting. After cessation of respiration, leave the rat in the chamber for approximately 1 min to ensure euthanasia. Verify euthanasia by palpating to confirm that the rat has no heartbeat, and pinching the toes to determine that the rat does not withdraw its limb.</li>
	</ol>
	</li>
	<li>Disinfect the haired-skin with 70% ethanol. Open the abdominal cavity and remove the intact liver with surgical scissors. After removing the liver, remove the left lateral lobe from the remaining liver with surgical scissors. Place the left lateral lobe on a cutting board, and cut two adjacent 3-4 mm thick transverse sections using a disposable single-edged razor blade.</li>
	<li>Place one transverse section on filter paper (to prevent curling of the section), and place the section into 10% neutral buffered formalin (NBF). Ensure that the ratio of 10% NBF to tissue is 10:1 or greater. After a fixation time of at least 72 hr, process the liver for histopathology (see section 7, below).</li>
	<li>Place the second transverse liver section into 5 ml ice-cold mincing buffer (section 1.2.6) in a 6 cm Petri dish and rinse 3 times to remove residual blood.</li>
	<li>Transfer the liver section to a new 2 ml centrifuge tube containing 1 ml ice-cold mincing buffer. Mince the well-rinsed liver section with a pair of fine scissors to release the cells. Strain the cell suspension through a 40 &#181;m cell strainer to remove any tissue lumps.</li>
	<li>Place the single cell suspension on ice and use for making slides within 1 hr. Verify the concentration of cells by counting (e.g., with a hemocytometer or electronic cell counter) to ensure that its concentration is approximately 2 x106 cells/ml.</li>
</ol>
3. Preparation of Comet Slides
<ol>
	<li>Mix one volume of the single cell suspension with 10 volumes of molten, 37 &#176;C, 0.5% low-melting agarose solution (section 1.2.1). Immediately pipette 100 &#181;l onto a slide coated with regular melting point agarose (section 1.1). Immediately place a cover slip over the cell-agarose mixture. Make at least 8 slides for each sample. 
	NOTE: All slides should be identified by a number or similar code and their identity recorded, so that the slides can be scored in a blinded fashion (see 6.2.2, below).</li>
	<li>Lay the slides flat and cool to 4 &#176;C for 30 min.</li>
	<li>After the gel has solidified, gently remove the cover slips and immerse the slides in pre-chilled lysis buffer (section 1.2.2). Protect the slides from light and keep the slides in the lysis buffer at 4 &#176;C from 1 hr (minimum) to O/N (maximum).</li>
</ol>
4. Enzyme Treatment of Comet Slides
<ol>
	<li>Remove 6 of the 8 slides prepared from each tissue sample from the lysis buffer and rinse them 3x for 5 min each with enzyme buffer (section 1.2.3) at RT. Use the remaining two slides for the alkaline comet assay without enzyme-treatment (see 5.1, below). After rinsing, remove excess liquid by draining and blotting the slides with tissues.</li>
	<li>Dilute hOGG1 and Endo III enzyme stocks with enzyme buffer at 1:1,000 (v/v). Place on ice until use.</li>
	<li>Apply 200 &#181;l of the following solutions to the slides prepared from each treatment group: 200 &#181;l of enzyme buffer alone (2 slides used as reference slides), 200 &#181;l hOGG1 solution (2 slides) or 200 &#181;l Endo III solution (2 slides).</li>
	<li>Put the slides into Petri plates lined with moistened paper towels, cover the slides with Parafilm, and incubate at 37 &#176;C for 45 min for Endo III-treated and reference slides and 30 min for hOGG1-treated slides.</li>
	<li>After incubation, rinse the slides with distilled water and continue processing as described in 5.2, below.</li>
</ol>
5. Unwinding and Electrophoresis
<ol>
	<li>For slides not treated with enzymes, remove the slides from the lysis buffer, drain the slides and rinse once with cold neutralization buffer (section 1.2.5) for 5 min to remove residual detergent and salts.</li>
	<li>Place slides from Steps 4.5 and 5.1 randomly in a horizontal gel electrophoresis tank, avoiding any spaces. Fill the tank with freshly made pH &#62; 13 electrophoresis buffer (section 1.2.4) until the liquid level just covers the slides (avoid bubbles over the agarose and make sure the tank is level). Record the buffer pH and temperature at the start of DNA unwinding.</li>
	<li>Let the slides remain in the alkaline buffer for 20 min to allow for unwinding of the DNA and expression of alkali-liable damage.</li>
	<li>During unwinding, program the power supply to produce 0.7 V/cm in the electrophoresis tank (V/cm=Volts divided by the distance in cm between the positive and negative electrodes).</li>
	<li>After unwinding, press the run button on the power supply. If needed, adjust the current to 300 mA by raising or lowering the buffer level. Keep the buffer level just above the surface of the slides. Perform electrophoresis at 4 &#176;C for 20 min. Record the slide placement, the temperature of the buffer and the current (amps) at the start and end of electrophoresis. 
	NOTE: Too much buffer will result in lower voltages which can interfere with DNA migration.</li>
	<li>After electrophoresis, neutralize the excess alkali by gently lifting the slides from the tank and immersing in neutralization buffer (section 1.2.5) for 5 min. Drain the slides and repeat two more times.</li>
	<li>Fix the slides by immersing in cold 100% ethanol for 5 min. Allow the slides to air dry and place in a dry area for storage.</li>
</ol>
6. DNA Staining, Comet Visualization and Analysis
<ol>
	<li>DNA staining
	<ol>
		<li>Place the slides on a cardboard or metal tray and add 200 &#181;l of nucleic acid fluorescent stain working solution (section 1.2.7) to each slide and cover with a glass cover slip. Protect the slides from light allow the slides to remain in contact with the staining solution for at least 30 min before scoring. Stain the slides in batches of 20 at a time.</li>
		<li>Before reading each slide, gently blot away excess staining solution, being careful not to disturb the cover glass.</li>
	</ol>
	</li>
	<li>Comet Visualization and Scoring
	<ol>
		<li>Use a video camera attached to a fluorescence microscope to view live images of the comet slide on the computer monitor.</li>
		<li>Randomly choose a microscope field containing cells. Using a mouse, select a cell. Click the center of the &#34;comet head&#34; with left click of the mouse. 
		NOTE: The computer software calculates all measurement parameters for the cell and adds the data to a data file.</li>
		<li>Score all the cells in the current field (see step 6.2.4), and then use the microscope&#39;s stage controls and the onscreen video to find another set of cells. Score these cells as described in 6.2.2.</li>
		<li>Score the slide from right to left and down to up. Choose around 10 fields randomly. During this process, any suitable comet cell that appears in the view field should be scored without bias. 
		NOTE: Do not score a comet if any of the following is true: 1) it is located on any of the edges of the slide; 2) two or more comets overlap; 3) there is debris in the tail; 4) the software cannot differentiate between the head and the tail; or 5) the staining of the nucleus and/or tail differs greatly from other comets on the slide. 
		NOTE: Comets with almost all their DNA fluorescence in the tail are often referred to as &#39;hedgehog&#39; comets. Hedgehog comets should not be scored, but the number of hedgehogs detected during the slide scoring should be recorded and reported as a percentage of total comets.</li>
		<li>Analyze at least 75 comets per slide, reading two slides per organ/tissue to obtain at least 150 comet cells/sample in total. After the required number of cells from a slide have been scored, the result is saved as an spreadsheet file for later data analysis.</li>
	</ol>
	</li>
</ol>
7. Histopathology Analysis
<ol>
	<li>After a minimum of 72 hr fixation in 10% NBF, followed by tissue trimming21, routinely process and embed the left lateral liver lobe of the samples in a paraffin-wax based medium22. Section the samples at approximately 5 microns and collect on glass slides.</li>
	<li>Stain the sections with hematoxylin &#38; eosin (H&#38;E):
	<ol>
		<li>Heat the slides with the paraffin tissue sections in a 60 &#176;C oven for 2 hr prior to the staining procedure.</li>
		<li>Use xylene to deparaffinize the sides. Rehydrate the slides with graded ethanol (EtOH) to a running tap water rinse.</li>
		<li>Place the slides in 450 ml of hematoxylin for 2 min and 40 sec. Rinse slides with acid (2 ml glacial acetic acid to 450 ml de-ionized water) for 4 sec following by 5 min running tap water rinse.</li>
		<li>Place the slides in 450 ml 70% EtOH containing 1 ml ammonium hydroxide for 2 min, followed by running tap water rinse for 2 min and 30 sec, and 70% EtOH rinse for 1 min.</li>
		<li>Incubate the slides with 450 ml Eosin Y solution containing 4 ml glacial acetic acid for 3 min.</li>
		<li>Dehydrate the slides with graded EtOH. Clear the slides with xylene (three times 1 min each), and hold slides in xylene until cover slips are placed on the slides.</li>
	</ol>
	</li>
	<li>Examine the slides by light microscopy, record findings, and grade non-neoplastic lesions for severity as 1 (minimal), 2 (mild), 3 (moderate), or 4 (marked). 
	NOTE: The severity grade applied for a specific lesion is based upon the relative proportion of the liver with the lesion (minimal = &#60;1-15%; mild = 16-35%; moderate = 36-60%; and marked = 61-100%).</li>
</ol>
8. Data analysis
<ol>
	<li>The animal is considered the experimental unit for statistical evaluations. DNA damage is expressed as % DNA in tail.</li>
	<li>Oxidative DNA damage is calculated as follows: the reference slides (no enzyme treatment) provide an estimate of the background DNA strand breaks (SB). 
	NOTE: The enzyme-treated slides provide a measure of strand breaks and oxidized bases (SB + OX). Assuming a linear dose response for % DNA in tail as a function of DNA damage, subtraction of SB from SB + OX gives an estimate of DNA strand breaks from oxidized pyrimidines/altered purines23.</li>
	<li>Analyze % DNA in tail as a function of dose by one-way ANOVA, followed by Dunnett&#39;s test for pairwise comparisons of the responses in treated animals to the vehicle control. 
	NOTE: All tests are one-tailed and a p-value of less than 0.05 is set as the criterion for statistical significance.</li>
</ol>

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None of the authors have competing interests or conflicting interests. The information in these materials is not a formal dissemination of information by FDA and does not represent agency position or policy.

This protocol describes the concurrent measurement of both direct and oxidative DNA damage in rat liver at the single cell level. The general protocol is applicable to any tissue from which single cells or nuclei can be isolated with minimal processing-induced DNA damage (i.e., DNA damage induced not by the test agent, but by the handling and processing of the animal tissues). In our research, we have conducted alkaline comet assays on cells from bone marrow7,9, stomach6, kidney9...

The alkaline comet assay measures DNA strand breaks at the single-cell level. Suspensions of single cells are embedded in agarose on a microscope slide and the cells lysed to form nucleoids, which contain supercoiled loops of DNA. Electrophoresis at pH&#62;13 results in the loss of supercoiling in DNA loops containing strand breaks, with the freed strands of DNA migrating toward the anode, creating comet-like structures that can be observed by fluorescence microscopy. Fragmented DNA migrates from the &#34;head&#34; of the comet into the &#34;tail&#34; based on the size of the fragment, and the relative fluorescence of the comet tail compared to the total intensity (head and tail) can be used to quantify DNA breakage1,2. The assay is simple, sensitive, versatile, rapid, and relatively inexpensive1. The detection of fragmented DNA caused by DNA-damaging agents is used an assay for quantifying DNA damage in cells or isolated nuclei from individual tissues of animals treated with potentially genotoxic material(s). Due to its advantages, the in vivo comet assay is recommended as a second in vivo genotoxicity assay (paired with the in vivo micronucleus assay) for conducting product safety evaluations in current International Conference on Harmonisation (ICH)3 and European Food Safety Authority (EFSA)4 regulatory guidelines. In our lab, we have employed the assay for evaluating in vivo DNA damage induced by food ingredients, pharmaceuticals, and nanomaterials5-10. Rat liver will be used as an example in this protocol, but the comet assay can be performed with other tissues/organs of experimental animals, as long as intact single cells can be isolated from the tissue.
Certain types of DNA damage are difficult to detect as DNA strand breaks without modifying the basic alkaline comet assay. In the case of oxidative DNA damage, strand breaks can be created at oxidative lesions in DNA by digesting with repair enzymes such as human 8-oxoguanine-DNA-N-glycosylase 1 (hOGG1, which creates breaks at 8-oxoguanine (8-oxoGua) and methyl-fapy-guanine11. Also, Endonuclease III (Endo III) creates breaks mainly at oxidized pyrimidines1. Thus, the addition of an enzyme-digestion step makes the assay a specific and sensitive method for measuring oxidative DNA damage in vivo12. Utilizing these assays, we have demonstrated toxicant-induced oxidative DNA damage in the liver of rats and mice6-8 and in the heart of rats10.
The alkaline comet assay has many applications in genetic toxicology and human biomonitoring: 1) as a follow-up in vivo assay for genotoxins identified by sensitive in vitro tests3,13, 2) to evaluate mechanisms of xenobiotic-induced DNA damage in multiple tissues14, 3) to investigate if a carcinogen operates using a genotoxic or a non-genotoxic mode of action (MOA)7, 4) to evaluate DNA damage repair15, 5) to investigate human diseases and occupational exposures 16, and 6) as a potential high-throughput screening assay for organ-specific genotoxicity17.

<table><tbody><tr><td>Coverslips (No. 1, 24 x 50 mm)&amp;nbsp;</td><td>Fisher</td><td>12-544-14</td><td></td></tr><tr><td>Microscope Slides</td><td>Fisher</td><td>12-550-123</td><td></td></tr><tr><td>Dimethylsulfoxide (DMSO)&amp;nbsp;</td><td>Fisher</td><td>67-68-5</td><td></td></tr><tr><td>EDTA, Disodium</td><td>Fisher</td><td>BP120-1</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Fisher</td><td>ICN1860454</td><td></td></tr><tr><td>1x Hanks Balanced Salt Solution (HBSS) (Ca&lt;sup&gt;2+&lt;/sup&gt;, Mg&lt;sup&gt;2+&lt;/sup&gt; free)</td><td>HyClone</td><td>SH30588.02</td><td></td></tr><tr><td>HEPES</td><td>Fisher</td><td>BP310-1</td><td></td></tr><tr><td>Low Melting Point Agarose (LMP)</td><td>Lonza</td><td>50081</td><td>NuSieve GTG Agarose</td></tr><tr><td>Normal Melting Agarose (NMA)&amp;nbsp;</td><td>Fisher</td><td>BP1356-100</td><td></td></tr><tr><td>pH testing paper strips (pH 7.5-14)</td><td>Fisher</td><td>M95873</td><td></td></tr><tr><td>Potassium Cloride</td><td>Fisher</td><td>7447-40-7</td><td></td></tr><tr><td>Potassium Hydroxide</td><td>Fisher</td><td>1310-58-3</td><td></td></tr><tr><td>slide labels (0.94 x 0.5 in.)</td><td>Fisher</td><td>NC9822036</td><td></td></tr><tr><td>Sodium Chloride (NaCl)&amp;nbsp;</td><td>Fisher</td><td>7647-14-5</td><td></td></tr><tr><td>Sodium Hydroxide (NaOH)&amp;nbsp;</td><td>Fisher</td><td>1310-73-2</td><td></td></tr><tr><td>SYBR&amp;trade; Gold&amp;nbsp;</td><td>Invitrogen</td><td>S11494</td><td></td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>Fisher</td><td>9002-93-1</td><td></td></tr><tr><td>Trizma Base&amp;nbsp;</td><td>Fisher</td><td>77-86-1</td><td></td></tr><tr><td>2.0 ml microcentrifuge tubes</td><td>Fisher</td><td>05-402-6</td><td></td></tr><tr><td>Cell strainer (40 &amp;micro;m)</td><td>Fisher</td><td>22363547</td><td></td></tr><tr><td>Endonuclease III (Nth)&amp;nbsp;</td><td>New England Biolabs</td><td>M0268S</td><td>Dilution 1:1,000</td></tr><tr><td>hOGG1&amp;nbsp;</td><td>New England Biolabs</td><td>M0241S</td><td>Dilution 1:1,000</td></tr></tbody></table>

in vivo alkaline comet assay and enzyme-modified alkaline comet assay for measuring dna strand breaks and oxidative dna damage in rat liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

The in vivo alkaline comet assay was performed in conjunction with the enzyme-modified comet assay to measure both direct and oxidative DNA damage in the liver of rats treated with cyproterone acetate (CPA)5. CPA is a synthetic hormonal drug that induces rat liver tumors in a sex-specific manner, with five-fold higher doses needed to induce liver tumors in male rats compared to females24. We found that the direct DNA damage produced by CPA in the liver of ma...

Watch this Scientific Journal Video about In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver at JoVE.com

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

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Biology

15.4K Views.  US FDA/National Center for Toxicological Research. The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

Video: In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Evaluating In Vitro DNA Damage Using Comet Assay

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

Evaluating In Vitro DNA Damage Using Comet Assay

The comet assay is an efficient method to detect DNA damage including single and double-stranded DNA breaks. We describe alkaline and neutral comet assays to measure DNA damage in cancer cells to evaluate the therapeutic effect of chemotherapy.

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer ...

Cancer Research

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of carcinogenic processes. This covalent binding, which is considered the cause of tumorigenesis, is now evaluated as a central dogma of chemical carcinogenesis. Here, methods are described employing the reactions catalyzed by cytochrome P450 and additional biotransformation enzymes to investigate the potency of chemicals or drugs for their activation to metabolites forming these DNA adducts. Procedures are presented describing the isolation of cellular fractions possessing biotransformation enzymes (microsomal and cytosolic samples with cytochromes P450 or other biotransformation enzymes, i.e., peroxidases, NADPH:cytochrome P450 oxidoreductase, NAD(P)H:quinone oxidoreductase, or xanthine oxidase). Furthermore, methods are described that can be used for the metabolic activation of analyzed chemicals by these enzymes as well as those for isolation of DNA. Further, the appropriate methods capable of detecting and quantifying chemical/drug-derived DNA adducts, i.e., different modifications of the 32P-postlabeling technique and employment of radioactive-labeled analyzed chemicals, are shown in detail.

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Evaluating the potency of environmental chemicals and drugs, to be enzymatically bioactivated to intermediates generating covalent DNA adducts, is an important field in the development of cancer and its treatment. Methods are described for compound activation to form DNA adducts, as well as techniques for their detection and quantification.

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of ...

Biochemistry

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

Bioengineering

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device.

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...

Genetics

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

Neuroscience

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay

The DNA fiber assay is a simple and robust method for the analysis of replication fork dynamics, based on the immunodetection of nucleotide analogs that are incorporated during DNA synthesis in human cells. However, this technique has a limited resolution of a few thousand kilobases. Consequently, post-replicative single-stranded DNA (ssDNA) gaps as small as a few hundred bases are not detectable by the standard assay. Here, we describe a modified version of the DNA fiber assay that utilizes the S1 nuclease, an enzyme that specifically cleaves ssDNA. In the presence of post-replicative ssDNA gaps, the S1 nuclease will target and cleave the gaps, generating shorter tracts that can be used as a read-out for ssDNA gaps on ongoing forks. These post-replicative ssDNA gaps are formed when damaged DNA is replicated discontinuously. They can be repaired via mechanisms uncoupled from genome replication, in a process known as gap-filling or post-replicative repair. Because gap-filling mechanisms involve DNA synthesis independent of the S phase, alterations in the DNA fiber labeling scheme can also be employed to monitor gap-filling events. Altogether, these modifications of the DNA fiber assay are powerful strategies to understand how post-replicative gaps are formed and filled in the genome of human cells.

Here we describe two modifications of the DNA fiber assay to investigate single-stranded DNA gaps in replicating DNA after lesion induction. The S1 fiber assay enables the detection of post-replicative gaps using the ssDNA-specific S1 endonuclease, while the gap-filling assay allows visualization and quantification of gap repair.

The DNA fiber assay is a simple and robust method for the analysis of replication fork dynamics, based on the immunodetection of nucleotide analogs that ...

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell function, and therefore DNA damage may play a critical role in the development of, conceivably, all major human diseases, e.g., cancer, neurodegenerative and cardiovascular disease, and aging. Single-cell gel electrophoresis (i.e., the comet assay) is one of the most common and sensitive methods to study the formation and repair of a wide range of types of DNA damage (e.g., single- and double-strand breaks, alkali-labile sites, DNA-DNA crosslinks, and, in combination with certain repair enzymes, oxidized purines, and pyrimidines), in both in vitro and in vivo systems. However, the low sample throughput of the conventional assay and laborious sample workup are limiting factors to its widest possible application. With the "scoring" of comets increasingly automated, the limitation is now the ability to process significant numbers of comet slides. Here, a high-throughput (HTP) variant of the comet assay (HTP comet assay) has been developed, which significantly increases the number of samples analyzed, decreases assay run time, the number of individual slide manipulations, reagent requirements, and risk of physical damage to the gels. Furthermore, the footprint of the electrophoresis tank is significantly decreased due to the vertical orientation of the slides and integral cooling. Also reported here is a novel approach to chilling comet assay slides, which conveniently and efficiently facilitates the solidification of the comet gels. Here, the application of these devices to representative comet assay methods has been described. These simple innovations greatly support the use of the comet assay and its application to areas of study such as exposure biology, ecotoxicology, biomonitoring, toxicity screening/testing, together with understanding pathogenesis.

The comet assay is a popular means of detecting DNA damage. This study describes an approach to running slides in representative variants of the comet assay. This approach significantly increased the number of samples while decreasing assay run-time, the number of slide manipulations, and the risk of damage to gels.

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell ...

Alkaline Single Cell Gel Electrophoresis: A Sensitive Technique for Quantitative Estimation of DNA Damage in Individual Cancer Cells Following Chemotherapy

Source: Lu, Y., et al. <a href="https://www.jove.com/v/56450">Evaluating In Vitro DNA Damage Using Comet Assay</a> J. Vis. Exp. (2017)
In this video, we demonstrate an alkaline single-cell electrophoresis assay to quantify both single- and double-strand DNA breaks in doxorubicin-treated cells. Alkaline conditions disrupt the hydrogen bonds between DNA base pairs, resulting in the complete unwinding and separation of DNA strands. This allows differential migration of undamaged and damaged DNA fragments in agarose gel upon electrophoresis, revealing a comet-shaped structure of slowly migrating undamaged DNA forming the circular comet head, and rapidly migrating damaged DNA fragments forming the elongated comet tail.

Source: Lu, Y., et al. Evaluating In Vitro DNA Damage Using Comet Assay J. Vis. Exp. (2017)
In this video, we demonstrate an alkaline single-cell ...

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Electrophoresis Techniques

Comet Assay for DNA Damage Detection in Cells

Source: Ji, Y., et al. <a href="https://app.jove.com/v/63559">A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage.</a> J. Vis. Exp. (2022).
The video demonstrates the comet assay for detecting DNA damage following exposure to harmful agents. Under alkaline conditions, the DNA unwinds and denatures, and upon electrophoresis, the undamaged and damaged DNA migrate differently in the agarose gel, forming a comet-shaped structure. The slowly migrating undamaged DNA forms the circular comet head, while the rapidly migrating damaged DNA fragments form the elongated comet tail.

Source: Ji, Y., et al. A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage. J. Vis. Exp. (2022).
The video demonstrates the comet ...

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

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