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</sup...

<ol>
	<li>Collins, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comet+assay+for+DNA+damage+and+repair:+principles,+applications,+and+limitations.">The comet assay for DNA damage and repair: principles, applications, and limitations.</a> Mol Biotechnol. 26 (3), 249-261 (2004).</li><li>Olive, P. L., Banath, J. P., Durand, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+radiation-induced+DNA+damage+and+repair+in+tumor+and+normal+cells+measured+using+the+'comet'+assay.">Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the 'comet' assay.</a> Radiat Res. 122 (1), 86-94 (1990).</li><li>Committee, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scientific+opinion+on+genotoxicity+testing+strategies+applicable+to+food+and+feed+safety+assessment.">Scientific opinion on genotoxicity testing strategies applicable to food and feed safety assessment.</a> EFSA Journal. 9 (9), 2379 (2011).</li><li>Ding, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-specific+dose-response+analysis+of+genotoxicity+in+cyproterone+acetate-treated+F344+rats.">Sex-specific dose-response analysis of genotoxicity in cyproterone acetate-treated F344 rats.</a> Mutat Res Genet Toxicol Environ Mutagen. 774, 1-7 (2014).</li><li>Ding, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+genotoxicity+of+estragole+in+male+F344+rats.">In vivo genotoxicity of estragole in male F344 rats.</a> Environ Mol Mutagen. 56 (4), 356-365 (2015).</li><li>Ding, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+genotoxicity+of+furan+in+F344+rats+at+cancer+bioassay+doses.">In vivo genotoxicity of furan in F344 rats at cancer bioassay doses.</a> Toxicol Appl Pharmacol. 261 (2), 164-171 (2012).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxicity+and+genotoxicity+assessment+of+silver+nanoparticles+in+mouse.">Cytotoxicity and genotoxicity assessment of silver nanoparticles in mouse.</a> Nanotoxicology. 8 (suppl 1), 36-45 (2014).</li><li>Ding, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methyleugenol+genotoxicity+in+the+Fischer+344+rat+using+the+comet+assay+and+pathway-focused+gene+expression+profiling.">Methyleugenol genotoxicity in the Fischer 344 rat using the comet assay and pathway-focused gene expression profiling.</a> Toxicol Sci. 123 (1), 103-112 (2011).</li><li>Manjanatha, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotoxicity+of+doxorubicin+in+F344+rats+by+combining+the+comet+assay,+flow-cytometric+peripheral+blood+micronucleus+test,+and+pathway-focused+gene+expression+profiling.">Genotoxicity of doxorubicin in F344 rats by combining the comet assay, flow-cytometric peripheral blood micronucleus test, and pathway-focused gene expression profiling.</a> Environ Mol Mutagen. 55 (1), 24-34 (2014).</li><li>Smith, C. C., O'Donovan, M. R., Martin, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hOGG1+recognizes+oxidative+damage+using+the+comet+assay+with+greater+specificity+than+FPG+or+ENDOIII.">hOGG1 recognizes oxidative damage using the comet assay with greater specificity than FPG or ENDOIII.</a> Mutagenesis. 21 (3), 185-190 (2006).</li><li>Collins, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+oxidative+damage+to+DNA+and+its+repair+with+the+comet+assay.">Measuring oxidative damage to DNA and its repair with the comet assay.</a> Biochim Biophys Acta. 1840 (2), 794-800 (2014).</li><li>Brendler-Schwaab, S., Hartmann, A., Pfuhler, S., Speit, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vivo+comet+assay:+use+and+status+in+genotoxicity+testing.">The in vivo comet assay: use and status in genotoxicity testing.</a> Mutagenesis. 20 (4), 245-254 (2005).</li><li>Hartmann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+conducting+the+in+vivo+alkaline+Comet+assay.+4th+International+Comet+Assay+Workshop.">Recommendations for conducting the in vivo alkaline Comet assay. 4th International Comet Assay Workshop.</a> Mutagenesis. 18, 45-51 (2003).</li><li>Collins, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+oxidative+DNA+damage+and+its+repair+using+the+comet+assay.">Investigating oxidative DNA damage and its repair using the comet assay.</a> Mutat Res. 681, 24-32 (2009).</li><li>Collins, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comet+assay+in+human+biomonitoring+studies:+reliability,+validation,+and+applications.">Comet assay in human biomonitoring studies: reliability, validation, and applications.</a> Environ Mol Mutagen. 30 (2), 139-146 (1997).</li><li>Gutzkow, K. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+comet+assay+using+96+minigels.">High-throughput comet assay using 96 minigels.</a> Mutagenesis. 28 (3), 333-340 (2013).</li><li>AVMA Guidelines for the Euthanasia of Animals: 2013 Edition. AVMA Panel on Euthanasia Available from: <a target="_blank" href="https://www.avma.org/KB/Policies/Documents/euthanasia.pdf">https://www.avma.org/KB/Policies/Documents/euthanasia.pdf</a> (2013)</li><li>Ruehl-Fehlert, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revised+guides+for+organ+sampling+and+trimming+in+rats+and+mice--part+1.">Revised guides for organ sampling and trimming in rats and mice--part 1.</a> Exp Toxicol Pathol. 55 (2-3), 91-106 (2003).</li><li>Carson, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Self-Instrumental+Text.">A Self-Instrumental Text.</a> Histotechnology. , 25-37 (1996).</li><li>Dusinska, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=THE+COMET+ASSAY,+modified+for+detection+of+oxidised+bases+with+the+use+of+bacterial+repair+endonucleases.">THE COMET ASSAY, modified for detection of oxidised bases with the use of bacterial repair endonucleases.</a> Dusinska Protocol [Internet]. , (2000).</li><li>Schuppler, J., Gunzel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+tumors+and+steroid+hormones+in+rats+and+mice.">Liver tumors and steroid hormones in rats and mice.</a> Arch Toxicol Suppl. 2, 181-195 (1979).</li><li>Hobkirk, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steroid+sulfotransferases+and+steroid+sulfate+sulfatases:+characteristics+and+biological+roles.">Steroid sulfotransferases and steroid sulfate sulfatases: characteristics and biological roles.</a> Can J Biochem Cell Biol. 63 (11), 1127-1144 (1985).</li></ol>

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.

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		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Coverslips (No. 1, 24 x 50 mm)&#160;</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">12-544-14</td>
			<td style="width:215px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Microscope Slides</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">12-550-123</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Dimethylsulfoxide (DMSO)&#160;</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">67-68-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">EDTA, Disodium</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">BP120-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Phosphate buffered saline</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">ICN1860454</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">1X Hanks Balanced Salt Solution (HBSS) (Ca++, Mg++ free)&#160;</td>
			<td style="width:140px;">HyClone</td>
			<td style="width:156px;">SH30588.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">HEPES</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">BP310-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Low Melting Point Agarose (LMP)</td>
			<td style="width:140px;">Lonza</td>
			<td align="right" style="width:156px;">50081</td>
			<td>NuSieve GTG Agarose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Normal Melting Agarose (NMA)&#160;</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">BP1356-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">pH testing paper strips (pH 7.5-14)</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">M95873</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Potassium Cloride</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">7447-40-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Potassium Hydroxide</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">1310-58-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">slide labels, (0.94 x 0.5 in.)</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">NC9822036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Sodium Chloride (NaCl)&#160;</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Sodium Hydroxide (NaOH)&#160;</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">1310-73-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">SYBR&#8482; Gold&#160;</td>
			<td style="width:140px;">Invitrogen</td>
			<td style="width:156px;">S11494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Triton X-100&#160;</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">9002-93-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Trizma Base&#160;</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">77-86-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">2.0 mL microcentrifuge tubes</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">05-402-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Cell strainer (40 &#181;m)</td>
			<td style="width:140px;">Fisher</td>
			<td align="right" style="width:156px;">22363547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Endonuclease III (Nth)&#160;</td>
			<td style="width:140px;">New England Biolabs</td>
			<td style="width:156px;">M0268S</td>
			<td>Dilution 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">hOGG1&#160;</td>
			<td style="width:140px;">New England Biolabs</td>
			<td style="width:156px;">M0241S</td>
			<td>Dilution 1:1000</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 ...

The overall goal of the in vivo alkaline comet assay and the enzyme-modified alkaline comet assay, is to concurrently measure direct and oxidative DNA damage at the single-cell level. This method was first proposed as a regulatory tool for safety analysis for FDA regulated products. It can help answer key questions in fields ranging from regulatory safety evaluation, to molecular epidemiology, to basic cancer research.This technique is sensitive and versatile, and also it is relatively inexpensive. It is capable of detecting DNA damage in virtually any animal tissue. Generally, individuals knew this method will struggle to quickly prepare single cell suspensions from various tissues, and it can be a challenge.Visual demonstration of this method is critical because it requires a certain amount of art, as well as science. After euthanizing rats with carbon dioxide gas, according to the text protocol, palpate to confirm that the rat has no heartbeat, and pinch the toes to confirm that the rat does not withdraw its limb. Use ethanol to disinfect the skin, then use surgical scissors to open the abdominal cavity, before removing the intact liver.With scissors, separate the left lateral lobe from the rest of the organ, and place it on a cutting board. Next, using a disposable single-edged razor blade, cut two adjacent 3 to 4 millimeter thick transverse sections. Place one transverse section on filter paper, then, into 10%neutral buffered formalin, or NBF.Place the second liver section into five milliliters of ice-cold mincing buffer in a six centimeter petri dish, and rinse three times to remove the residual blood. Then, transfer the liver section to a new two milliliter centrifuge tube, containing one milliliter of ice-cold mincing buffer. With fine scissors, mince the section to release the cells, then with a 40 micrometer cell strainer, strain the cell suspension to remove any tissue lumps.Place the single cell suspension on ice, and with a hemocytometer, verify that the concentration is approximately two times 10 to the sixth cells per milliliter. To prepare comet slides, mix one volume of the single cell suspension with 10 volumes of molten 37 degree Celsius 0.5%low-melting agarose solution. Immediately pipette 100 microliters onto a previously prepared agarose-coated slide, and use a cover slip to cover the agarose cell mixture.Lay the slides flat, and cool at four degrees Celsius for 30 minutes. After the gel has solidified, gently remove the cover slips and immerse the slides in pre-chilled lysis buffer. Incubate in the dark at four degrees Celsius for one hour to overnight.Following the incubation, remove six of the eight slides prepared for each tissue sample from the lysis buffer, and use room temperature enzyme buffer to rinse them three times for five minutes each. Then, use tissues to blot excess solution. Next, use enzyme buffer to dilute hOGG1 and Endo III enzyme stocks, to one to one thousand, and place on ice.Place the slides into petri plates lined with moisten paper towels, then apply 200 microliters of the following solutions to the slides in each treatment group. Enzyme buffer alone to two slides, hOGG1 solution to two slides, and Endo III solution to two slides, and use Parafilm to cover the slides. Then place the slides at 37 degrees Celsius, incubating the Endo III treated and reference slides for 45 minutes, and the hOGG1 treated slides for 30 minutes.Following the incubation, drain the slides and for the reference slides, use cold neutralization buffer to remove residual detergent and salts. Then use distilled water to rinse the enzyme treated slides. Randomly place the slides in a horizontal gel electrophoresis tank, avoiding any spaces.Then add freshly made electrophoresis buffer to the tank, while avoiding bubbles, until it just covers the slides. Set the power supply to 0.7 volts per centimeter, and incubate the slides in the alkaline buffer for 20 minutes, to allow unwinding of the DNA and expression of the Alkali-liable damage. Record the slide placement, the temperature of the buffer, and the current.After the incubation for unwinding, place the electrophoresis tank at four degrees Celsius. Then press the Run button on the power supply, and if needed, adjust the current to 300 milliamps by raising or lowering the buffer level, keeping it just above the slides. Run the samples for 20 minutes, before again recording the slide placement, the temperature of the buffer, and the current.Following electrophoresis, gently lift the slides from the tank, and immerse a neutralization buffer to neutralize the excess Alkali. Then drain the slides and repeat the neutralization two more times. With cold 100%ethanol, fix the slides for five minutes.Allow the slides to air dry and store in a dry environment. To stain the DNA, place the slides on a cardboard or metal tray, and add 200 microliters of nucleic acid fluorescent stain working solution to each slide, before covering with a cover slip. Protect the slides from light and incubate for at least 30 minutes.Then to read the slides, gently blot away the excess staining solution, taking care not to disturb the cover glass. Next, place the comet slide on the stage of a fluorescence microscope equipped with a video camera, and randomly choose a field containing cells. After opening the comet assay software, using the mouse, select a cell, then left-click the center of the comet head of the cell.The software will calculate parameters for the cell, and add to a data file. Once all the cells have been scored in the current field, move to another field and repeat the scoring of any suitable comet cell that appears without bias. Score 10 random fields and at least 75 comets per slide.Finally, save the data into a spreadsheet for later analysis. As previously reported, 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 females. As shown here, the in vivo alkaline comet assay, and the enzyme-modified comet assay, demonstrated that a five-fold higher dose of CPA was needed, to induce a significant increase in DNA damage in male livers, compared to females.Hydroxysteroid sulfotransferase, or HST, is expressed at 15-fold higher levels in adult female rats, compared to adult males. HST metabolism is a rate-limiting step in the activation of CPA to DNA-binding metabolites. As seen here, CPA-induced oxidative DNA damage was generally greater in male compared to female rat livers, and therefore, less likely to be rate-limiting in tumor formation in male rats.As reported in these tables, histopathology evaluation of livers from CPA-treated rats, showed no evidence of agent-induced apoptosis or necrosis, confirming previous conclusions that the positive comet assay results were not a secondary effect of cytotoxicity. While doing this procedure, it is important to collect single cell suspension, and prepare comet assay slides as quickly as possible. After its development, this technique paved the way for researchers in the field of genetic toxicology to explore DNA damage, and repair quantitatively in virtually any animal tissue and in human samples, as well.After watching this video, we hope you can have a better understanding of how to use the in vivo alkaline comet assay together with the enzyme-modified comet assay, to concurrently measure posts of the rat and oxidative DNA damage, at the single cell level in animal tissues. Please remember that working with DNA damage reagent can be extremely hazardous. Please always wear the personal protective equipment when you perform these procedures.

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JoVE Journal

Biology

15.2K vistas.  US FDA/National Center for Toxicological Research. The overall goal of the in vivo alkaline comet assay and the enzyme-modified alkaline comet assay, is to concurrently measure direct and oxidative DNA damage at the single-cell level. This method was first proposed as a regulatory tool for safety analysis for FDA regulated products. It can help answer key questions in fields ranging from regulatory safety evaluation, to molecular epidemiology, to basic cancer research.This technique is sensitive and versatile, and also it is relatively...