Name: in vivo alkaline comet assay and enzyme-modified alkaline comet assay for measuring dna strand breaks and oxidative dna damage in rat liver
Uploaded: 2016-05-04T14:00:00
Description: 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

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)

Etik erklæring: Procedurer, der involverer dyr er blevet godkendt af Institutional Animal Care og brug Udvalg (IACUC) på den amerikanske FDA / National Center for toksikologiske Research. BEMÆRK: Undersøgelsen design beskrives her, er baseret på protokol udviklet af den japanske Center for Validering af Alternative Metoder (JaCVAM) for deres validering af in vivo gnaver alkalisk komet assay 18, og yderligere ændret på grundlag af anbefalingerne i OECD guideline TG4...

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Denne protokol beskriver den samtidige måling af både direkte og oxidativ DNA-skader i rottelever på enkelt celle niveau. Den generelle protokol for enhver væv, hvorfra enkelte celler eller kerner kan isoleres med minimal forarbejdning-induceret DNA-beskadigelse (dvs. DNA-beskadigelse induceret ikke af testmidlet, men ved håndtering og forarbejdning af animalske væv). I vores forskning har vi udført alkaliske comet assays af celler fra knoglemarv 7,9, mave 6, nyre 9, blæ...

Den alkaliske komet assay måler DNA streng brud på single-celle niveau. Suspensioner af enkelte celler er indlejret i agarose på et mikroskopobjektglas, og cellerne lyseres til dannelse nucleoids, som indeholder supercoilede sløjfer af DNA. Elektroforese ved pH&gt; 13 resulterer i tab af supercoiling i DNA loops indeholdende strengbrud, idet de frigjorte DNA-strenge migrerer mod anoden, hvilket skaber komet-lignende strukturer, som kan observeres ved fluorescensmikroskopi. Fragmenteret DNA migrerer fra &quot;hoved&quot; af komet i &quot;hale&quot; baseret på størrelsen af fragmentet, og den relative fluorescens af komet hale sammenlignet med den samlede intensitet (hoved og hale) kan anvendes til at kvantificere DNA brud 1 , to. Assayet er enkelt, følsomme, alsidig, hurtig, og relativt billig 1. Påvisningen af ​​fragmenteret DNA forårsaget af DNA-ødelæggende midler anvendes et assay til kvantificering DNA-skader i celler eller isolerede kerner fra enkelte kdobbelt væv fra dyr behandlet med potentielt genotoksisk materiale (r). På grund af sine fordele, er in vivo comet assay anbefales som en anden in vivo genotoksicitet assay (parret med in vivo mikronukleusanalysen) for at gennemføre produkt sikkerhedsvurderinger i nuværende International Konference om Harmonisering (ICH) 3 og Den Europæiske Fødevaresikkerhedsautoritet (EFSA ) 4 regulatoriske retningslinier. I vores laboratorium har vi ansat analysen for at vurdere in vivo DNA skader fremkaldt af fødevareingredienser, lægemidler og nanomaterialer 5-10. Rottelever vil blive anvendt som et eksempel i denne protokol, men comet assay kan udføres med andre væv / organer fra forsøgsdyr, når blot intakte enkeltceller kan isoleres fra vævet. Visse typer af DNA-skader er vanskelige at detektere DNA-strengbrud uden at ændre den grundlæggende alkalisk comet assay. I tilfælde af oxidativ DNA-skade, linje Bsideskift kan laves på oxidative læsioner i DNA ved fordøjelse med reparation enzymer, såsom humant 8-oxoguanine-DNA-N-glycosylase 1 (hOGG1, der skaber pauser i 8-oxoguanine (8-oxoGua) og methyl-fapy-guanin 11. også, endonuclease III (Endo III) skaber pauser hovedsagelig på oxiderede pyrimidiner 1. tilsætningen af et enzym-fordøjelse trin gør assayet en specifik og sensitiv metode til måling af oxidativ DNA-skade in vivo 12. ved hjælp af disse assays har vi vist giftstof-induceret oxidativ DNA-beskadigelse i leveren hos rotter og mus 6-8 og i hjertet af rotter 10. Den alkaliske komet assay har mange anvendelser i genetisk toksikologi og human bioovervågning: 1) som en opfølgning in vivo assay for genotoxins identificeret ved følsomme in vitro tests 3,13, 2) at evaluere mekanismer af miljøfremmede-induceret DNA-skader i flere væv 14, 3) at undersøge, om enkræftfremkaldende fungerer ved hjælp af en genotoksisk eller en ikke-genotoksisk virkemåde (MOA) 7, 4) for at evaluere DNA skader reparation 15, 5) at undersøge menneskelige sygdomme og erhvervsmæssig eksponering 16, og 6) som en potentiel high-throughput screening assay for organspecifik genotoksicitet 17.

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			<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>
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		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Microscope Slides</td>
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			<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>
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			<td height="21" style="height:21px;width:232px;">EDTA, Disodium</td>
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			<td style="width:156px;">BP120-1</td>
			<td></td>
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			<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>
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			<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>
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			<td height="21" style="height:21px;width:232px;">HEPES</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:156px;">BP310-1</td>
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			<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>
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			<td height="21" style="height:21px;width:232px;">Normal Melting Agarose (NMA)&#160;</td>
			<td style="width:140px;">Fisher</td>
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			<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>
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		<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>
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		<tr height="21">
			<td height="21" style="height:21px;width:232px;">slide labels, (0.94 x 0.5 in.)</td>
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			<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>
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			<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>
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			<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>
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			<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>
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		<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>
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			<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>
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			<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>
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		<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>
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		<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>
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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.

In vivo alkaliske komet assay blev udført i samarbejde med enzym-modificeret komet assay til at måle både direkte og oxidativ DNA-skader i leveren hos rotter behandlet med cyproteronacetat (CPA) 5. CPA er en syntetisk hormonal lægemiddel, som inducerer rotte levertumorer i en sex-specifik måde, med fem gange højere doser er nødvendige for at inducere levertumorer hos hanrotter i forhold til kvinder 24. Vi fandt, at den direkte DNA-skader produceret a...

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

In vivo Alkaline Comet assay og Enzyme-modificerede Alkaline Comet assay til måling DNA Strand Pauser og Oxidative DNA skader opstået i 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.

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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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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Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Forstærkning, Næste generations sekventering, og Genomisk DNA Kortlægning af retroviral integration steder

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

Filtrering Isolering af nukleinsyrer: en enkel og hurtig DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

Modificerede metoder til belastning af høj-throughput DNA ekstraktionsplader reducere risikoen for forurening

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, ...