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)

Ethik-Anweisung: Verfahren mit Tieren wurden von der Institutional Animal Care und Use Committee (IACUC) bei der US FDA / Nationales Zentrum für toxikologische Forschung genehmigt. HINWEIS: Die hier beschriebene Studiendesign basiert auf dem Protokoll , das von dem japanischen Zentrum für die Validierung alternativer Methoden (JaCVAM) für ihre Validierung des Nagetiers in - vivo - Assay alkalische Komet 18 entwickelt und weiter in OECD - Richtlinie auf Empfehl...

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Dieses Protokoll beschreibt die gleichzeitige Messung der direkten und oxidative DNA-Schäden in der Rattenleber auf Einzelzellebene. Das allgemeine Protokoll ist anwendbar auf jedes Gewebe , aus dem einzelne Zellen oder Zellkerne können mit minimalen Verarbeitungs induzierte DNA - Schädigung (dh DNA - Schädigung induziert nicht durch das Testmittel, sondern durch die Handhabung und Verarbeitung der tierischen Geweben) isoliert werden. In unserer Forschung haben wir alkalischen Comet-Assay auf Zellen aus dem...

Die alkalische Comet-Assay misst DNA-Strangbrüche auf Einzelzellebene. Suspensionen einzelner Zellen in Agarose auf einem Mikroskop-Objektträger eingebettet und die Zellen lysiert Nukleoide zu bilden, die supercoiled Schleifen von DNA enthalten. Elektrophorese bei pH&gt; 13 führt zum Verlust von Supercoiling in DNA Schlaufen enthaltenden Strangbrüche, mit den befreit DNA-Stränge in Richtung der Anode wandernde, wodurch Kometen artigen Strukturen, die durch Fluoreszenzmikroskopie beobachtet werden kann. Fragmentierte DNA wandert aus dem &quot;Kopf&quot; des Kometen in den &quot;Schwanz&quot; auf der Basis der Größe des Fragments, und die relative Fluoreszenz des Kometenschweif im Vergleich zu der Gesamtintensität (Kopf und Schwanz) verwendet werden kann , um DNA - Bruch 1 quantifizieren , 2. Der Test ist einfach, empfindlich, vielseitig, schnell und relativ kostengünstig 1. Der Nachweis von fragmentierter DNA durch DNA-schädigende Mittel ist ein Assay zur Quantifizierung von DNA-Schäden in Zellen oder isolierten Zellkernen von indivi verwendetDoppelgewebe von Tieren, die mit potentiell genotoxischen behandelnde Material (s). Aufgrund seiner Vorteile ist die in - vivo - Comet - Assay als zweite in vivo Genotoxizität Test empfohlen (gepaart mit der in vivo - Mikrokerntest) für die Durchführung von Produktsicherheit Bewertungen in bestehenden International Conference on Harmonisation (ICH) 3 und European Food Safety Authority (EFSA ) 4 regulatorischen Vorgaben. In unserem Labor haben wir den Test zur Bewertung der in - vivo - DNA - Schäden , die durch Lebensmittelzutaten, Pharmazeutika und Nanomaterialien 5-10 induzierte eingesetzt. Rattenleber wird als ein Beispiel in diesem Protokoll verwendet werden, aber der Comet-Assay kann mit anderen Geweben / Organen von Versuchstieren, solange intakten Einzelzellen aus dem Gewebe isoliert durchgeführt werden werden. Bestimmte Arten von DNA-Schäden sind schwierig, wie DNA-Strangbrüche zu erfassen, ohne die grundlegende alkalischen Comet-Assay modifiziert wird. Im Falle der oxidative DNA-Schäden, Strang bReaks kann durch Verdau mit der Reparatur - Enzyme wie menschliches 8-Oxoguanin-DNA-N-Glycosylase - 1 (hOGG1, die Pausen bei der 8-Oxoguanin erzeugt (8-oxoGua) und Methyl-Fapy-Guanin - 11 bei oxidative Läsionen in DNA erstellt werden. auch Endonuclease III (Endo III) erzeugt Pausen hauptsächlich an oxidiertem Pyrimidine 1. Somit ist die Zugabe eines Enzym-Verdauungsschritt macht den Assay eine spezifische und sensitive Methode zur Messung oxidative DNA - Schädigung in vivo . 12 diese Assays verwendet wird , haben wir gezeigt , toxicant induzierte oxidative DNA - Schädigung in der Leber von Ratten und Mäusen , 6-8 und im Herzen von Ratten 10. Die alkalische Comet - Assay hat viele Anwendungen in der genetischen Toxikologie und Human - Biomonitoring: 1) als ein Follow-up in vivo - Test für Genotoxinen durch empfindliche identifiziert in - vitro - Tests 3,13, 2) Mechanismen der xenobiotischen-induzierte DNA - Schäden in mehreren Geweben zu bewerten 3) 14, zu untersuchen , ob einKarzinogen betreibt eine gentoxische oder nichtgenotoxisches Wirkungsweise (MOA) 7 verwenden, 4) Reparatur DNA - Schäden zu bewerten 15, 5) 16 menschliche Krankheiten und Belastungen am Arbeitsplatz zu untersuchen und 6) als potenzielle Hochdurchsatz - Screening - Test für organspezifische Genotoxizität 17.

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			<td height="21" style="height:21px;width:232px;">Coverslips (No. 1, 24 x 50 mm)&#160;</td>
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			<td style="width:156px;">12-544-14</td>
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			<td style="width:156px;">67-68-5</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>
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			<td height="21" style="height:21px;width:232px;">HEPES</td>
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			<td height="21" style="height:21px;width:232px;">Low Melting Point Agarose (LMP)</td>
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			<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>
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			<td height="21" style="height:21px;width:232px;">pH testing paper strips (pH 7.5-14)</td>
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			<td style="width:156px;">M95873</td>
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			<td height="21" style="height:21px;width:232px;">Potassium Cloride</td>
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			<td style="width:156px;">7447-40-7</td>
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			<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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			<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>
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			<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>
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			<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>
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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>
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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>
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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.

Die in vivo alkalischen Komet - Test wurde in Verbindung mit dem enzymmodifizierten Comet - Assay durchgeführt , sowohl direkt als auch oxidative DNA - Schädigung in der Leber von Ratten , die mit Cyproteronacetat (CPA) 5 behandelt , zu messen. CPA ist ein synthetisches hormonelle Medikament , das Lebertumoren bei männlichen Ratten zu induzieren benötigt höhere Dosen induziert im Vergleich zu Frauen 24 Rattenlebertumoren in einer geschlechtsspezifische...

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 und enzymmodifizierten Alkaline Comet Assay zur Messung von DNA - Strangbrüchen und oxidative DNA - Schäden in der Rattenleber

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 ...

Research

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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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Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

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Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatinimmunpräzipitation Assay zur Identifizierung von Arabidopsis-Protein-DNA-Wechselwirkungen<em&gt; In-vivo-</em

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

Amplification, Next-Generation-Sequenzierung und genomischer DNA Mapping von Retrovirale Integration Seiten

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

Filtration Isolierung von Nukleinsäuren: Eine einfache und schnelle DNA-Extraktionsmethode

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

Modifizierte Methoden zum Laden von DNA-Extraktionsplatten mit hohem Durchsatz reduzieren das Kontaminationspotenzial

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