Name: analysis of the ambient particulate matter-induced chromosomal aberrations using an in vitro system
Uploaded: 2016-12-21T15:00:00
Description: Watch this Scientific Journal Video about Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System at JoVE.com

Arbejdet blev støttet delvist af National Institute of Health Center of Biological Research Excellence [tilskud nummer 1P20GM109005], Arkansas Space Grant Consortium gennem National Aeronautics and Space Administration [tilskud nummer NNX15AK32A], og det nationale institut for Occupational Safety and Sundhed (NIOSH) [tilskud nummer 2T420H008436]. Forfatterne vil gerne takke Christopher Fettes for korrekturlæsning og redigering dette manuskript.

1. Particle Indsamling og klargøring <ol><li> Kalibrering af den samlede høj lydstyrke suspenderede partikler sampler <ol><li> Afbryd sampler motor fra massestrømsstyreenheden og forbinde motoren til en stabil vekselstrømskilde (figur 1). BEMÆRK: bruges ikke Et filter under denne procedure. </li><li> Monter kalibrator blænde og top loading adapterplade til sampler. Stram de øverste lastning adapter hold-down nødder sikkert til sikre, at ingen utætheder er til stede. Ti...

<ol>
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Phys. 6 (3), 729-753 (2006).</li><li>P&#246;schl, U., 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=Atmospheric+aerosols:+composition,+transformation,+climate+and+health+effects.">Atmospheric aerosols: composition, transformation, climate and health effects.</a> Angew. Chem. Int. 44 (46), 7520-7540 (2005).</li><li>Hou, L., 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=Altered+methylation+in+tandem+repeat+element+and+elemental+component+levels+in+inhalable+air+particles.">Altered methylation in tandem repeat element and elemental component levels in inhalable air particles.</a> Environ. Mol. Mutagen. 55 (3), 256-265 (2014).</li><li>Miousse, I. 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=Epigenetic+alterations+induced+by+ambient+particulate+matter+in+mouse+macrophages.">Epigenetic alterations induced by ambient particulate matter in mouse macrophages.</a> Environ. Mol. Mutagen. 55 (5), 428-435 (2014).</li><li>Miousse, I. 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=In+Vitro+Toxicity+and+Epigenotoxicity+of+Different+Types+of+Ambient+Particulate+Matter.">In Vitro Toxicity and Epigenotoxicity of Different Types of Ambient Particulate Matter.</a> Toxicol. Sci. 148 (2), 473-487 (2015).</li><li>Ehrlich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+instability+in+cancer+development.">Genomic instability in cancer development.</a> Genome Instability in Cancer Development. , 363-392 (2005).</li><li>Michael, S., Montag, M., Dott, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pro-inflammatory+effects+and+oxidative+stress+in+lung+macrophages+and+epithelial+cells+induced+by+ambient+particulate+matter.">Pro-inflammatory effects and oxidative stress in lung macrophages and epithelial cells induced by ambient particulate matter.</a> Environ Pollut. 183, 19-29 (2013).</li><li>Li, N., 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=Induction+of+heme+oxygenase-1+expression+in+macrophages+by+diesel+exhaust+particle+chemicals+and+quinones+via+the+antioxidant-responsive+element.">Induction of heme oxygenase-1 expression in macrophages by diesel exhaust particle chemicals and quinones via the antioxidant-responsive element.</a> J Immunol. 165 (6), 3393-3401 (2000).</li><li>Pathak, R., Ramakumar, A., Subramanian, U., Prasanna, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+radio-sensitivities+of+human+chromosomes+1+and+2+in+one+donor+in+interphase-+and+metaphase-spreads+after+60Co+gamma-irradiation.">Differential radio-sensitivities of human chromosomes 1 and 2 in one donor in interphase- and metaphase-spreads after 60Co gamma-irradiation.</a> BMC Med. Phys. 9, 6649 (2009).</li><li>Pathak, R., Dey, S. K., Sarma, A., Khuda-Bukhsh, 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=Genotoxic+effects+in+M5+cells+and+Chinese+hamster+V79+cells+after+exposure+to+7+Li-beam+(LET=+60keV/&#181;m)+and+correlation+of+their+survival+dynamics+to+nuclear+damages+and+cell+death.">Genotoxic effects in M5 cells and Chinese hamster V79 cells after exposure to 7 Li-beam (LET= 60keV/&#181;m) and correlation of their survival dynamics to nuclear damages and cell death.</a> Mutat. Res. Genet. Toxicol. Environ. Mutagen. 628 (1), 56-66 (2007).</li><li>Pathak, R., Dey, S. K., Sarma, A., Khuda-Bukhsh, 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=Cell+killing,+nuclear+damage+and+apoptosis+in+Chinese+hamster+V79+cells+after+irradiation+with+heavy-ion+beams+of+16+O,+12+C+and+7+Li.">Cell killing, nuclear damage and apoptosis in Chinese hamster V79 cells after irradiation with heavy-ion beams of 16 O, 12 C and 7 Li.</a> Mutat. Res. Genet. Toxicol. Environ. Mutagen. 632 (1), 58-68 (2007).</li><li>Chalbot, M. C., Kavouras, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+magnetic+resonance+spectroscopy+for+determining+the+functional+content+of+organic+aerosols:+a+review.">Nuclear magnetic resonance spectroscopy for determining the functional content of organic aerosols: a review.</a> Environ. Pollut. 191, 232-249 (2014).</li></ol>

Cytogenetisk undersøgelse eller mikroskopisk analyse af tal eller strukturer af kromosomer, primært i metafase spreads, giver oplysninger afgørende for prognose, risikovurdering, og behandling af forskellige sygdomme. Det er nu veletableret, at cytogenetiske abnormaliteter er forbundet med progression og udviklingen af ​​flere sygdomme, herunder cancer. Til dato har CA&#39;er blevet fundet i alle større tumortyper. CA&#39;er kan opstå spontant eller ved enten eksterne eller interne stimuli, såsom ioniserende s...

Det er blevet anslået, at eksponering for partikler (PM) forårsager over 3 millioner overskydende dødsfald årligt, primært fra hjerte-sygdomme og lungekræft 1. Faktisk PM blev anerkendt som kræftfremkaldende for mennesker af Det Internationale Agentur for Kræftforskning (gruppe 1), en øget risiko for lungekræft med stigende eksponering for PM er blevet vist 2. Interessant, næsten alle kræftceller havnen numeriske og / eller strukturelle kromosomafvigelser. Partikler organisk kulstof er en variant, kompleks og heterogen blanding, hvis sammensætning og størrelse fordeling afhænger af emissioner samt fysiske og kemiske omdannelser. Den tegner 35-55% af byerne PM 2,5 masse (PM 2,5 um i størrelse og mindre) og mere end 60% af baggrunden landdistrikter og kontinentale PM 2,5 masse 3, 4. Den vandopløselige fraktion udgør 30-90% af organisk aerosol. Et stort antal organiske forbindelserer blevet identificeret, herunder alifatiske carbonhydrider, polycykliske aromatiske kulbrinter og deres oxygenerede og nitroderivater, alifatiske aldehyder og alkoholer, frie fedtsyrer og deres salte, dicarboxylsyrer, multifunktionelle forbindelser, proteiner og humus-lignende makromolekyler (HULIS) under anvendelse kromatografiske metoder kombineret med massespektrometrisk teknikker 5-8. Disse forbindelser udgør mindre end 10-20% af partikulært organisk kulstof, og dermed de fleste af organisk kulstof er ukendt 9. Eksperimentelle beviser antyder, at cytotoksicitet, oxidativt stress og inflammation er involveret i udviklingen af ​​PM-associerede patologiske tilstande. Det har for nylig vist, dog, at eksponering for PM resulterer også i en række epigenetiske ændringer, herunder ændringer i DNA-methylering af repetitive elementer, i både eksperimentelle in vitro systemer og i humane individer 10-12. Af særlig interesse er virkningerne afPM på satellit-DNA - større og mindre satellitter - som findes i heterochromatic regionen omkring centromeren af ​​kromosomer. Det blev vist, at disse virkninger kan være persistente af natur, da de kan påvises i mindst 72 timer efter eksponering 12. Ændringer i DNA-methylering, især omkring centromerer, kan føre til akkumulering af satellit-DNA-mRNA-transkripter, kompromis kromosomale integritet under celledeling og efterfølgende resultere i udvikling af en række forskellige patologiske tilstande 13. Tilpasning af cytogenetisk fremgangsmåde til analyse af CA&#39;er som et endepunkt af epigenetiske ændringer forårsaget af PM, således er af stor betydning. Her rapporterer vi tilgangen til den omgivende PM indsamling og forberedelse, in vitro eksponering og analyse af CA&#39;er hjælp af murine makrofag RAW264.7 model system. Makrofager omfatter den første linje i forsvar mod de inhalerede fremmedlegemer og dermed thans cellelinie tjener som en etableret og de hyppigst anvendte model i partikel toksikologi 11, 12, 14, 15.

<table border="0" cellpadding="0" cellspacing="0" width="726">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Total suspended particulate sampler</td>
			<td style="width:179px;">Tisch Environmental</td>
			<td style="width:119px;">TE-5170</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:261px;">Bruker Avance III NMR spectrometer&#160;</td>
			<td style="width:179px;">Bruker</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">TopSpin 3.5/pl2 software</td>
			<td style="width:179px;">Bruker</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:261px;">ACD/NMR Processor Academic Edition</td>
			<td style="width:179px;">ACD/Labs</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RAW264.7 murine macrophages</td>
			<td style="width:179px;">ATCC</td>
			<td style="width:119px;">ATCC TIB-71</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:261px;">High glucose DMEM GlutaMAX media</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">10569010</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Fetal Bovine Serum</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">16000044</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:261px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">15140163</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">25200056</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">PBS, pH 7.4</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">10010049</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:261px;">KaryoMAX Colcemid Solution in PBS</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">15212012</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:261px;">KaryoMAX Potassium Chloride Solution</td>
			<td style="width:179px;">ThermoFisher</td>
			<td align="right" style="width:119px;">10575090</td>
			<td>Warm in a 37&#176;C waterbath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Methanol (HPLC)</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">A452N1-19</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Acetic Acid, Glacial</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">BP1185-500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:261px;">Decon Contrad 70 Liquid Detergent</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">04-355-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Wright and Wright-Giemsa Stain Solutions</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">23-200733</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Permount Mounting Medium</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">SP15-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Axio Imager 2</td>
			<td style="width:179px;">Zeiss</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of the ambient particulate matter-induced chromosomal aberrations using an in vitro system

Udsættelse for partikler (PM) er en af ​​verdens største sundhedsproblem, der kan ødelægge forskellige cellulære komponenter, herunder den nukleare genetiske materiale. For at vurdere virkningen af ​​PM om nuklear genetiske integritet, er strukturelle kromosomafvigelser scoret i de metafasespredninger af mus RAW264.7 makrofagceller. PM er indsamlet fra den omgivende luft med en samlet høj lydstyrke suspenderede partikler sampler. Det opsamlede materiale solubiliseres og filtreres til at tilbageholde den vandopløselige, fin portion. Partiklerne er kendetegnet for kemisk sammensætning ved kernemagnetisk resonans (NMR) spektroskopi. Forskellige koncentrationer af partikelsuspension bliver tilføjet på en in vitro-kultur af RAW264.7 musemakrofager til en samlet eksponeringstid på 72 timer, sammen med ubehandlede kontrolceller. Ved slutningen af ​​eksponeringen, er kulturen behandlet med colcemid at anholde celler i metafase. Celler høstes dernæst, behandlet med hypotonisk opløsning, fikseret i acetomethanol, dropped på objektglas og endelig farvet med Giemsa-opløsning. Slides undersøges for at vurdere de strukturelle kromosomafvigelser (CAS) i metafase spreder på 1.000X forstørrelse ved hjælp af en lys-felt mikroskop. 50 til 100 metafasespredning scores for hver behandlingsgruppe. Denne teknik er tilpasset til detektion af strukturelle kromosomforandringer (CAS), såsom kromatidtypen-typen pauser, udveksling kromatidtypen-type, acentriske fragmenter, dicentric og ring kromosomer, dobbelt minutter, endoreduplikation og Robertsonian translokationer in vitro efter udsættelse for PM. Det er en kraftfuld metode til at knytte en veletableret cytogenetisk endpoint til epigenetiske ændringer.

Stor omhu bør tages i at vælge placeringen af ​​TSP sampler, samt den tid af året, hvor kollektionen er udført. Den kemiske sammensætning samt størrelsen af ​​partikler kan i det væsentlige påvirke resultatet. Det indsamlede materiale skal være synlig mod den hvide filter. En normal mus metafase spredning vil have 40 acentriske kromosomer. Målet med den teknik er at påvise en ændring (eller fravær deraf) i andelen af ​​unormale kromosomer i behandlede celler i fo...

Watch this Scientific Journal Video about Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System at JoVE.com

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

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JoVE Journal

Genetics

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

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In Vitro Transcription Assays and Their Application in Drug Discovery

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Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

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Påvisning af Inter-kromosomale Stabil Afvigelser fra Multiple Fluorescens<em&gt; In Situ</em&gt; Hybridisering (MFISH) og Spectral Karyotyping (SKY) i bestrålede mus

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The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

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DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

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Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

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Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

Ved hjælp af in Vitro og i celler form at undersøge lille molekyle induceret Pre-mRNA strukturelle ændringer

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

Brugen af mus mammary tumor celler i en in vitro invasion analyse som et mål for Oncogenic celle adfærd

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

In vitro biokemiske assays ved hjælp af biotin etiketter til at studere Protein-nukleinsyre interaktioner

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...