Name: a general method for detecting nitrosamide formation in the in vitro metabolism of nitrosamines by cytochrome p450s
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Description: Watch this Scientific Journal Video about A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s at JoVE.com

Bu çalışmada grant tarafından desteklenen yok. CA-81301 Ulusal Kanser Enstitüsü'nden. Biz Bob Carlson Editör Yardım almak için Dr Peter Villalta ve Xun Ming için analitik Biyokimya paylaşılan kaynak Masonik Kanser Merkezi kütle spektrometresi yardım ve Dr Adam T. Zarth ve Dr Anna K. Michel onların değerli tartışmalar için teşekkür ederim ve giriş. Analitik Biyokimya paylaşılan kaynak kısmen Ulusal Kanser Enstitüsü Kanser Merkezi destek hibe CA-77598 tarafından desteklenmektedir

1. malzeme ve genel yordamları <ol><li>Synthesize NNN daha önce açıklandığı gibi 15. Norcotinine, P450 2A6 Baculosomes, NADPH rejenerasyon sistemi, 0.5 x reaksiyon tampon ve diğer kimyasal maddeler elde etmek veya reaktif sınıfta ticari kaynaklardan gelen solventler.</li>
	<li>Kayıt NMR spectra bir 500 MHz Spektrometre üzerinde. Rapor kimyasal vardiya başına parçaları olarak milyon (ppm). İç başvuru (7,26 ppm CDCl 3) için 1 H-NMR ve 13 C-NMR (77.2 ppm CDCl 3) kalan solvent doruklarına kullanın. 
	Not: Tepe bölme kullanılan aşağıdaki kısaltmalar: s singlet, d = eş, dd = eş birini, dt = üçüz, dq gelir = dörtlüsü, ddd gelir = kütleye kütleye birini ve m = multiplet =.</li>
	<li>Bir LTQ Orbitrap Velos ve rapor verilerini m/z olarak seçilen bileşikler için yüksek çözünürlüklü kütle spektrometresi (HRMS) gerçekleştirmek.</li>
	<li>Kullanım polygram silika jel TLC tabak (40 mm x 80 mm, 0.2 mm kalınlığında) ince katmanlı Kromatografi (TLC) 254 nm floresan göstergesi ön kaplamalı. TLC plakaları UV lamba ışınlama tarafından görselleştirmek.</li>
	<li>Flash Kromatografi bir 60 Å, 70-150 mesh silika jel 1,5 cm x 15 cm cam sütun kullanarak gerçekleştirmek.</li>
</ol> 2. Nitrosamide olumlu denetim hazırlanması (N &#8242;-nitrosonorcotinine, NNC) <ol><li>kuru, bir fırın gecede (16 h, ~ 140 ° C), 25 mL yuvarlak alt şişesi manyetik heyecan çubuğunu içeren. Sabah, serin altında N 2 akışı tutmak için bir petrol bubbler kullanırken bir akış N 2 şişeye oranı saniyede bir kabarcık. 
	Not: soğutma sonra şişeye N 2 altında tutmak için hiçbir önlem gereklidir.</li>
	<li>Bir duman başlık, soğutmalı şişeye norcotinine (31,8 mg, 0.196 mmol), asetik anhidrit (5 mL) ve Asetik asit (1 mL) ekleyin. 0 ° C su buz banyosu ile soğutma sırasında sürekli olarak bu karışımı ilave edin. 
	Uyarı: Asetik anhidrit ve Asetik asit aşındırıcı.</li>
	<li>NaNO eklemek 2 (33.3 mg, 0.483 mmol) bir bölümünü ve sürekli olarak 2.5 h. TLC tarafından tepki ilerlemeyi izleme için karışımı ilave edin (%100 EtOAc, R f = 0,19, bkz: adım 1.4). 
	Not: Bu zaman içinde karışım ara sıra köpüren ile giderek sarı olacak.</li>
	<li>Buz gibi H 2 O içine karışımı (18 mL) dökerek tepki gidermek. Hemen 18 mL CH 2 Cl 2 100 mL HCI'yi huni ile sulu çözüm ayıklayın. CH 2 Cl 2 (9 her mL) ile en az 2 ek kez sulu katman ayıklamak.</li>
	<li>Üzerinde ~ 100 mg MgSO 4 2 dk. filtre için havuza alınan Organik Kuru ve çözüm tarafından döner buharlaşma-ham, sarı yağ verimi için konsantre. Su banyosu ile 30 ° C arasında kalan Asetik asit kaldırmak için buharlaşma sırasında ısı. 
	Not: bileşik CH 2 Cl 2 ve 2-8 ° c karanlıkta saklanan çözüm içinde çözünmüş Eğer protokol burada duraklatılmış</li>
	<li>Arındırmak ham bileşik sütun Kromatografi tarafından (bkz. Adım 1.5) durağan faz ve % 100 silika jel kullanarak EtOAc olarak eluent 16. 
	Uyarı: NNC ve ilgili nitrosamides ele alınabilir özenle insan kanserojen olarak kabul edilir gibi.</li>
	<li>CDCl 3 ' te saf bileşik dağıtılması ve NMR kullanın (bkz. Adım 1.2) yapısı onaylamak ve bu çözüm 17 Derişim belirlemek için. 2-8 ° C'de gerekinceye kadar karanlıkta bu çözüm mağaza. 
	Not: Bu çözüm olumlu denetim vitro tahlil (Adım 3.5) olarak kullanılır. NMR spectra ve kimyasal vardiya destek bilgileri mevcuttur.</li>
	<li>Saf NNC çözümle, doğru onaylamak üst kitle ve ürün iyon kitleler tarafından yüksek çözünürlüklü bir Kütle Spektrometre (HRMS) üzerinde doğrudan infüzyon 1.3 ve 4.3 adımlarda açıklanan parametreleri altında belirlemek. 
	Not: Ürün kitleler vitro metabolizma tahlil (Adım 4.3) bu bileşik algılamak için kullanılan.</li>
</ol> 3. Bir örnek nitrosamine P450 vitro kuluçka <ol><li>-80 ° C dondurucudan P450 2A6 Baculosomes, canlı-NADPH-rejenerasyon sistemi ve 10 mM NADP + hisse senedi çözüm kaldırmak ve onları buza çözülme izin. Bir kez çözdürülen, Baculosomes ve NADPH rejenerasyon sistem 1 seyreltik: 10 ve 1:50, sırasıyla, bir tek 1 mL tüp ile 0.5 X reaksiyon arabellek içine. Benzer şekilde, reaksiyon arabellek X + hisse senedi çözüm 0,5 97 μL NADP 3 μL ekleyin. 
	Not: NADP + ışığa duyarlı olduğunu. Bu çözüm kapsayıcısının alüminyum folyo sarma tarafından korunan tutmak. Enzim çözümleri için donma-çözülme çevrimleri duyarlıdır; Bu fazla 2 geçer sınırlayın. Aliquots gelecekteki deneyler için gerektiği gibi hazırlayın.</li>
	<li>(5 pmol P450 içeren) enzim çözüm 1 ml 50 μL tüp tepki arabellek ile yapılan 4 mikron NNN çözüm içeren 40 μL her kuluçka için ekleyin. Bu yeni karışım bir su banyosu kullanarak 37 ° C'de 2 min için önceden kuluçkaya ve 10 μL seyreltilmiş NADP + çözüm ekleyin. 1-30 dk 37 için tam sistem kuluçkaya ° C. 
	Not: 5 dk aralıklarla kullanarak kuluçka kez için (örneğin 5, 10, 15 dk, vb) bir yeterli nitrosamide oluşumu saat ders verecek.</li>
	<li>İlk ekleyerek 10 μl 3.0 N ZnSO 4 ve 3.0 N Ba(OH) sonra 10 μL tarafından istenen kuluçka süre sonra susuzluğumu 2. Girdap bu çözüm ve beyaz çökelti oluşturacak. 8000 x g çökelti cips için 4 dakika için de örnek santrifüj kapasitesi. 
	Not: kurulan nitrosamides sulu çözümler istikrardır sınırlı olarak prosedür bu adımda duraklatmak gerekir değil. Uzun duraklar içinde yanlış negatif neden olabilir.</li>
	<li>Hemen süpernatant pipette ve 2 μL sıvı Kromatografi olumlu nanoelectrospray-iyonizasyon yüksek çözünürlüklü tandem kütle spektrometresi tarafından analiz (LC-NSI +-HRMS/MS, adımlar 4.1-4.3).</li>
	<li>Altında N akışı 2 1 mL tüp içine sentezlenmiş NNC çözeltinin 100 fmol içeren bir aliquot pozitif kontrol incubations için buharlaşır. Bu şişe için tam enzim sistemi 3.2 adımından ekleyin ve adımları 3.3-3.4 açıklandığı gibi devam edin.</li>
	<li>Negatif kontrol incubations için deneme açıklanan (adımları 3.2-3,4) Değiştir 50 μL enzim reaksiyon arabellek X 0.5 Çözümle dışında gerçekleştirin.</li>
</ol> 4. Örnek parametreleri LC-NSI + tarafından nitrosamide algılama için-HRMS/MS <ol><li>için LC bileşeni, bir ticari, el-Paketli 18 C18 kullanarak UPLC sisteme adım 4.2 aşağıdaki Multi-step degrade uygulayın (5 mikron), 100 mm x 75 mikron, 15 μm kalınlığında orifis kapiller sütun.</li>
	<li>Kullanarak 5 mM NH 4 OAc çözelti A ve çözücü B olarak MeCN olarak %5 B 1 μL/dk çalıştırarak örnek sütuna yükleme 0 - 5 min ve debisi 0,3 μL/dk sonra yavaşlama. Ardından, bir degrade üzerinde 4 dk ardından rampa %55 B 10 dk ve o zaman yeniden denge için % 5, % 20'si B 5 çalıştırın B.</li>
	<li>Gerçekleştirmek LC-NSI +-HRMS/MS LTQ Orbitrap üzerinde. Tam tarama ve MS 2 parçalanma monitör NNC için. 60.000 çözünürlükte tam tarama gerçekleştirmek ve bir kitle toleransı 5 ppm doğru üst kitle (192.07670) ayıklayın. Çarpışma kaynaklı ayrılma 25 ev, 15.000, çözünürlük tarafından (CID) bir çarpışma enerjisi ile üst iyonları (2.0 amu) ve parçası yalıtmak ve 30 Bayan zaman inceden inceye gözden geçirmek için kullanım MS 2 parçalanma NNC (m/z doğru kitlelerin ürün iyonları için ayıklamak 192 m/z 134.04739 ve 162.07874) adlı bir kitle hoşgörü 5 PPM.</li>
</ol>

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Nitrozaminler metabolizma elucidating onların carcinogenicity anlamak için önemli bir bileşenidir. Daha fazla yer sitokrom P450s ve diğer Metabolik enzimler Polimorfik olduğundan, bu bilginin uygulaması potansiyel olarak yüksek riskli bireyler1,4teşhis edebilir. Yeni veri α-hydroxynitrosamines, nitrozaminler DNA bağlamasında dahil tahmin ediliyor büyük metabolitleri daha fazla o oksidasyonunu gösterir, nitrosamides için mümkündür; Ancak, bu sa?...

N-nitrozamin çoğu diyet, tütün ürünleri ve genel çevre; bulunan kanserojen büyük bir sınıf Onlar da endogenously insan vücudu1' oluşturulması mümkündür. 300'den fazla N -nitrozo bileşikler test ettik ve &#62; % 90 gibi kanserojen olarak değerlendirilmiştir hayvan modelleri2,3. Onların carcinogenicity sergilemek, bu bileşiklerin tarafından sitokrom P450s1,2,3etkinleştirilmesi gerekir. Araştırma gösterir o sitokrom P450s kolayca okside nitrozaminler için α-hydroxynitrosamines (Resim 1), yarı-ömrü ~ 5 olan büyük ölçüde reaktif bileşikleri olan s kendiliğinden alkyldiazohydroxides için çürümeye başladı önce. İkinci DNA H2O ve N2kaybettikten sonra alkylate. Elde edilen DNA adducts, eğer unrepaired, kritik onko - veya Tümör baskılayıcı genler, Eğer kanser geliştirme1' e kurşun mutasyonlar neden olabilir. Bu nedenle, çok çaba metabolik yollar tam bir anlayış kazanmak için harcanan, DNA adducts ve sitokrom P450 oksidasyon kanserojen nitrozaminler ve aşağı akım metabolitleri. Bu bilgi bireysel kanser risk değerlendirme4' te potansiyel uygulama var.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56312/56312fig1.jpg"> 
Resim 1: Genel ve önerilen metabolizması nitrozaminler. Nitrozaminler (1) P450s tarafından α-hydroxynitrosamines (2) bu kendiliğinden alkyldiazohydroxides (3) ayrıştırmak için okside. Bu bileşikler DNA için DNA adducts forma bağlayabilirsiniz. Bu onaylanmadığına karar o 2 daha fazla nitrosamides 4' e P450s tarafından okside. Bunlar doğrudan DNA için roman DNA adducts veya 3 bilinen DNA adducts forma hidrolize formu bağlayabilirsiniz. R1 ve R2 herhangi bir alkil grubu temsil eder. <a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56312/56312fig1large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
α-hydroxynitrosamine hipotez sağlam geniş kapsamlı veriler tarafından desteklenir, ancak bazı tutarsızlıklar vardır; α-hydroxynitrosamines5,6kısa yarılanma büyük olanıdır. Bu bileşikler endoplazmik retikulum membran üretilir ve daha sonra nükleer DNA alkylate bilinir. Ömürleri birkaç saniyelik göz önüne alındığında, bu nasıl bu ara ürün gerekli seyahat gerçi sitozol hayatta şaşırtıcı. α-hydroxynitrosamines bir kısmını processively okside olduğunu nitrosamides7,8' e, hangi karşılaştırma9' oldukça istikrarlı bir hipotezdir. Bu muhtemelen α-hydroxynitrosamines sitokrom P450 etkin sitedeki tutma ile oluşacak. Bu tür bir oksidasyon için emsal nikotin10, alkoller11ve basit alkylnitrosamines12,13ile görülmüştür. Ayrıca, doğrudan etkili kanserojen2,3nitrosamides bulunmaktadır. Onların reaktivite9tarihinde dayanarak, bu bileşiklerin inanılıyor DNA üretmek için kullanılanlarla aynı α-hydroxynitrosamines ile birlikte yeni kaynaklanan adducts, keşfedilmemiş DNA (şekil 1) adducts. Böylece, bu hipotez sadece sitozol yoluyla ulaşım açıklar, ama Ayrıca DNA oluşumu zarar ürünleri.
Bu yazıda, vitro sitokrom P450-aracılı dönüşüm nitrozaminler nitrosamides değerlendirmek için genel bir protokol açıklanmıştır. N′- nitrosonorcotinine (NNC) sitokrom P450 2A6 tarafından N′- nitrosonornicotine (NNN) daha önce raporlanmış dönüşüm bir örnek14olarak sergiledi. Bu protokolün uygulamaya substrat enzim sistemleri geniş bir nitrosamides genel nitrosamine metabolizması önemini belirlemenize yardımcı olur.

<table><tbody><tr><td>Norcotinine</td><td>AKoS GmbH (Steinen, Germany)</td><td>CAS 17708-87-1, AKoS AK0S006278969</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>695092</td><td></td></tr><tr><td>Acetic Anhydride</td><td>Sigma-Aldrich</td><td>242845</td><td></td></tr><tr><td>Ammonium Acetate</td><td>Sigma-Aldrich</td><td>431311</td><td></td></tr><tr><td>Barium Hydroxide</td><td>Sigma-Aldrich</td><td>433373</td><td></td></tr><tr><td>D-Chloroform</td><td>Sigma-Aldrich</td><td>151823</td><td></td></tr><tr><td>HPLC Acetonitrile</td><td>Sigma-Aldrich</td><td>34998</td><td></td></tr><tr><td>Magnesium Sulfate</td><td>Sigma-Aldrich</td><td>M7506</td><td></td></tr><tr><td>Methylene Chloride</td><td>Sigma-Aldrich</td><td>34856</td><td></td></tr><tr><td>Sodium Nitrite</td><td>Sigma-Aldrich</td><td>237213</td><td></td></tr><tr><td>ViVid CYP2A6 Blue Screening Kit</td><td>Life Technologies</td><td>PV6140</td><td></td></tr><tr><td>Zinc Sulfate</td><td>Sigma-Aldrich</td><td>221376</td><td></td></tr><tr><td>0.5 mL tubes</td><td>Fisher</td><td>AB0533</td><td></td></tr><tr><td>100 mL round bottom flask</td><td>Sigma-Aldrich</td><td>Z510424</td><td></td></tr><tr><td>125 mL Erlenmeyer flask</td><td>Sigma-Aldrich</td><td>CLS4980125</td><td></td></tr><tr><td>125 mL Separatory Funnel</td><td>Sigma-Aldrich</td><td>Z261017</td><td></td></tr><tr><td>25 mL round bottom flask</td><td>Sigma-Aldrich</td><td>Z278262</td><td></td></tr><tr><td>500 MHz NMR Spectrometer</td><td>Bruker</td><td></td><td></td></tr><tr><td>Allegra X-22R Centrifuge</td><td>Beckman-Coulter</td><td></td><td></td></tr><tr><td>LC vials</td><td>ChromTech</td><td>CTC&amp;ndash;0957&amp;ndash;BOND</td><td></td></tr><tr><td>LTQ Orbitrap Velos</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Magnetic Stir bar</td><td>Sigma-Aldrich</td><td>Z127035</td><td></td></tr><tr><td>NMR tube</td><td>Sigma-Aldrich</td><td>Z274682</td><td></td></tr><tr><td>P1000, P200, and P10 pipettes</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Rotary evaporator</td><td>Sigma-Aldrich</td><td>Z691410</td><td></td></tr><tr><td>RSLCnano UPLC system</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Shaking Water Bath</td><td>Fisher</td><td>FSSWB15</td><td></td></tr><tr><td>Stir plate</td><td>Sigma-Aldrich</td><td>CLS6795420</td><td></td></tr><tr><td>PicoFrit Column</td><td>New Objective</td><td>PF3607515N5</td><td></td></tr><tr><td>Luna C18, 5 um</td><td>Phenomenex</td><td>535913-1</td><td></td></tr></tbody></table>

a general method for detecting nitrosamide formation in the in vitro metabolism of nitrosamines by cytochrome p450s

N-nitrozamin aktivite sergilemek sitokrom P450 oksidasyon gerektiren çevre kanserojen iyi kurulmuş bir grup vardır. Metabolik aktivasyon kabul edilen mekanizması oluşumu kendiliğinden DNA etmen acentelere ayrıştırmak α-hydroxynitrosamines içerir. DNA hasarı ve elde edilen mutasyonlar birikimi sonuçta kansere yol açabilir. Yeni kanıtlar α-hydroxynitrosamines daha fazla nitrosamides için processively sitokrom P450s tarafından okside olduğunu gösterir. Nitrosamides genellikle daha α-hydroxynitrosamines daha kararlı ve ayrıca DNA alkylate çünkü nitrosamides karsinojenezis içinde bir rol oynayabilir. Bu raporda, vitro sitokrom P450 katalize metabolizma üzerinden nitrosamide üretimini nitrozaminler değerlendirmek için genel bir iletişim kuralı açıklar. Bu iletişim kuralı ilgili nitrosamides ve algılama için sıvı Kromatografi-nanospray iyonlaşma-yüksek çözünürlüklü tandem kütle spektrometresi kullanılarak bir vitro sitokrom P450 metabolizma tahlil sentezi için genel bir yaklaşım kullanır. Bu yöntem N′- nitrosonorcotinine N′- nitrosonornicotine örnek çalışma küçük bir metaboliti olarak algılandı. Yüksek hassasiyet ve seçmeli olarak nedeniyle doğru kitle algılama yöntemi vardır. Bu yöntemin uygulama çok çeşitli nitrosamine sitokrom P450 sistemleri için bu dönüşüm genelliği belirlemenize yardımcı olur. Sitokrom P450s Polimorfik ve etkinliğini değişir çünkü nitrosamide oluşumu daha iyi bir anlayış içinde bireysel kanser risk değerlendirmesi yardımı olabilir.

Beyaz ve arkçalışmasına dayanmaktadır. 19, norcotinine nitrosated NNC için temiz bir şekilde ve yüksek verim (% 80-92) vitro deney için bir standart üretmek oldu. Başarılı bir tepki için yapısal kanıt spektroskopik 1H-NMR, 13C-NMR, rahat ve HSQC (destek bilgileri) ile birlikte üst kitle [M + H] teyit HRMS gibi analizler üzerinden elde edilen+ 5 ppm içinde Teorik değeri (Şek...

Watch this Scientific Journal Video about A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s at JoVE.com

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

Α-prokollajen sitokrom P450s tarafından kanserojen nitrozaminler ve mutasyonlar neden DNA-zarar intermediates üretir kabul edilen metabolik yoludur. Ancak, yeni verileri daha fazla nitrosamides oksidasyon oluşabilir gösterir. Biz genel bir tarif yöntemi nitrosamides tespit için üretilen vitro sitokrom P450 katalize metabolizması nitrozaminler.

Nitrosamide oluşumu nitrozaminler sitokrom P450s tarafından in Vitro metabolizma saptamak için bir genel yöntem

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted ...

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Universidad San Sebastian

Research

JoVE Journal

Cancer Research

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

10.0K Görüntüleme.  University of Minnesota. Α-prokollajen sitokrom P450s tarafından kanserojen nitrozaminler ve mutasyonlar neden DNA-zarar intermediates üretir kabul edilen metabolik yoludur. Ancak, yeni verileri daha fazla nitrosamides oksidasyon oluşabilir gösterir. Biz genel bir tarif yöntemi nitrosamides tespit için üretilen vitro sitokrom P450 katalize metabolizması nitrozaminler.

Video: A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

<em&gt; Drosophila</em&gt; Hayali Disk Tümör Modeli: Genetik Mozaikler kullanma Gen İfade ve Tümör invazivliğinin Görselleştirme ve

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Kanser Araştırmaları

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase solubility and facilitate excretion. On some occasions those structural modifications lead to the formation of new toxic products. In order to reduce animal testing, chemical risk can be assessed using metabolically competent cells. The expression of metabolic enzymes, however, is not stable over time in many in vitro primary culture systems and is often partial or absent in cell lines. Therefore, the study of medicines, additives, and environmental pollutants metabolism in vitro should ideally be conducted in cell systems where metabolic activity has been characterized. We explain here an approach to measure the activity of a class of metabolic enzymes (Human Phase I) in 2D cell lines and primary 3D cultures using chemical probes and their metabolic products quantifiable by UPLC mass spectrometry and luminometry. The method can be implemented to test the metabolic activity in cell lines and primary cells derived from a variety of tissues.

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Metabolic competency of in vitro systems is a key requirement for the biotransformation and disposition of drugs and toxicants. In this protocol, we describe the application of reference metabolic probes to assess phase I metabolism in cell cultures.

Kütle Spektrometre ve lüminojenik tabanlı Yaklaşımlar Faz I Metabolik Yetkinlik ait Characterize<em&gt; İn Vitro</em&gt; Hücre Kültürleri

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase ...

Biyokimya

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.

Kovalent DNA oluşumu enzimatik aktif kanserojen ve uyuşturucu Vitro ve kararlılıklarını 32P-postlabeling tarafından Adducts

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 ...

Analytical Techniques for Assaying Nitric Oxide Bioactivity

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be considered one of the most important signaling molecules produced in our body, regulating essential functions including but not limited to regulation of blood pressure, immune response and neural communication. Therefore its accurate detection and quantification in biological matrices is critical to understanding the role of NO in health and disease. With such a short physiological half life of NO, alternative strategies for the detection of reaction products of NO biochemistry have been developed. The quantification of relevant NO metabolites in multiple biological compartments provides valuable information with regards to in vivo NO production, bioavailability and metabolism. Simply sampling a single compartment such as blood or plasma may not always provide an accurate assessment of whole body NO status, particularly in tissues. The ability to compare blood with select tissues in experimental animals will help bridge the gap between basic science and clinical medicine as far as diagnostic and prognostic utility of NO biomarkers in health and disease. Therefore, extrapolation of plasma or blood NO status to specific tissues of interest is no longer a valid approach. As a result, methods continue to be developed and validated which allow the detection and quantification of NO and NO-related products/metabolites in multiple compartments of experimental animals in vivo. The established paradigm of NO biochemistry from production by NO synthases to activation of soluble guanylyl cyclase (sGC) to eventual oxidation to nitrite (NO2-) and nitrate (NO3-) may only represent part of NO's effects in vivo. The interaction of NO and NO-derived metabolites with protein thiols, secondary amines, and metals to form S-nitrosothiols (RSNOs), N-nitrosamines (RNNOs), and nitrosyl-heme respectively represent cGMP-independent effects of NO and are likely just as important physiologically as activation of sGC by NO. A true understanding of NO in physiology is derived from in vivo experiments sampling multiple compartments simultaneously. Nitric oxide (NO) methodology is a complex and often confusing science and the focus of many debates and discussion concerning NO biochemistry. The elucidation of new mechanisms and signaling pathways involving NO hinges on our ability to specifically, selectively and sensitively detect and quantify NO and all relevant NO products and metabolites in complex biological matrices. Here, we present a method for the rapid and sensitive analysis of nitrite and nitrate by HPLC as well as detection of free NO in biological samples using in vitro ozone based chemiluminescence with chemical derivitazation to determine molecular source of NO as well as ex vivo with organ bath myography.

The endogenous production of nitric oxide (NO) regulates a wide variety of biological functions. It is becoming increasingly clear that disruption or dysregulation of NO based signaling is involved in many human diseases. Methods to quantify relevant NO metabolites may provide novel diagnostic or prognostic biomarkers for human disease.

Nitrik Oksit Biyoaktivite içlin Analitik Teknikler

Nitric oxide (NO) is a diatomic free radical that is extremely short lived in biological systems (less than 1 second in circulating blood)1. NO may be ...

Tıp

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

3-nitrotyrosine (3-NT) is generated from the tyrosine residue in atmospheric bio-aerosol proteins via a reaction with ozone (O3) and nitrogen dioxide (NO2). Stable 3-NT is a specific marker for oxidative damage and is reported to have a promotive effect to elicit allergies. In the present study, we report the development of a highly sensitive assay to quantify 3-NT in air sampler filters to collect &#60; 2.5 &#181;m of particulate matter (PM2.5) from urban environmental air, including bio-aerosol. In this method, a 6 mm-diameter round hole was cut from the filters of air samplers and mixed with a nonspecific protease cocktail in order to hydrolyze proteins. Protein samples digested to the amino acid level were tested for 3-NT using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD). The maximum 3-NT content was detected in a prefilter for PM of sizes from 4.5 to 7.3 &#956;m, with a detection limit of 1.13 pg/m3.

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

We present a method to detect 3-nitrotyrosine chemical modifications of atmospheric proteins with 6 mm-diameter rounds cut from air sampler prefilters using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD).

3-Nitrotyrosine atmosferik ortamlar üzerinden yüksek performanslı sıvı kromatografi elektrokimyasal dedektör sistemi tespiti

3-nitrotyrosine (3-NT) is generated from the tyrosine residue in atmospheric bio-aerosol proteins via a reaction with ozone (O3) and nitrogen dioxide ...

Çevre

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide variety of pathological conditions. However, existing methods lack the sufficient sensitivity and/or specificity necessary to measure the low endogenous level of 3-NT reliably and are too cumbersome for clinical applications. Hence, analytical improvement is urgently needed to accurately quantify the levels of 3-NT and verify the role of 3-NT in pathological conditions. This protocol presents the development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) detection combined with a miniaturized solid phase extraction (SPE) for the rapid and accurate measurement of 3-NT in human urine as a non-invasive biomarker for oxidative stress. SPE using a 96-well plate markedly simplified the process by combining sample cleanup and analyte enrichment without tedious derivatization and evaporation steps, reducing solvent consumption, waste disposal, risk of contamination and overall processing time. The employment of 25 mM ammonium acetate (NH4OAc) at pH 9 as the SPE elution solution substantially enhanced the selectivity. Mass spectrometry signal response was improved through adjustment of the multiple reaction monitoring (MRM) transitions. Use of 0.01% HCOOH as additive on a pentafluorophenyl (PFP) column (150 mm x 2.1 mm, 3 &#181;m) improved signal response another 2.5-fold and shortened the overall run time to 7 min. A lower limit of quantitation (LLOQ) of 10 pg/mL (0.044 nM) was achieved, representing a significant sensitivity improvement over the reported assays. This simplified, rapid, selective and sensitive method allows two plates of urine samples (n = 192) to be processed in a 24 h time-period. Considering the markedly improved analytical performance, and non-invasive and inexpensive urine sampling, the proposed assay is beneficial for pre-clinical and clinical studies.

A selective and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method coupled with an efficient solid phase extraction on a mixed-mode cation-exchange (MCX) 96-well microplate was developed for the measurement of free 3-nitrotyrosine (3-NT) in human urine with high throughput, which is suitable for clinical applications.

Klinik Uygulamalar İçin İnsan İdrarında 3-Nitrotirosinin Minyatür Katı Faz Çıkarma ve LC-MS / MS Algılamasının Entegrasyonu

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide ...

Kimya

Measurement of the Potential Rates of Dissimilatory Nitrate Reduction to Ammonium Based on 14NH4+/15NH4+ Analyses via Sequential Conversion to N2O

The importance of understanding the fate of nitrate (NO3&#8722;), which is the dominant N species transferred from terrestrial to aquatic ecosystems, has been increasing because global nitrogen loads have dramatically increased following industrialization. Dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are both microbial processes that use NO3&#8722; for respiration. Compared to denitrification, quantitative determinations of the DNRA activity have been carried out only to a limited extent. This has led to an insufficient understanding of the importance of DNRA in NO3&#8722; transformations and the regulating factors of this process. The objective of this paper is to provide a detailed procedure for the measurement of the potential DNRA rate in environmental samples. In brief, the potential DNRA rate can be calculated from the 15N-labeled ammonium (15NH4+) accumulation rate in 15NO3&#8722; added incubation. The determination of the 14NH4+ and 15NH4+ concentrations described in this paper is comprised of the following steps. First, the NH4+ in the sample is extracted and trapped on an acidified glass filter as ammonium salt. Second, the trapped ammonium is eluted and oxidized to NO3&#8722; via persulfate oxidation. Third, the NO3&#8722; is converted to N2O via an N2O reductase deficient denitrifier. Finally, the converted N2O is analyzed using a previously developed quadrupole gas chromatography&#8211;mass spectrometry system. We applied this method to salt marsh sediments and calculated their potential DNRA rates, demonstrating that the proposed procedures allow a simple and more rapid determination compared to previously described methods.

Measurement of the Potential Rates of Dissimilatory Nitrate Reduction to Ammonium Based on 14NH4+/15NH4+ Analyses via Sequential Conversion to N2O

A series of methods to determine the potential DNRA rate based on 14NH4+/15NH4+ analyses is provided in detail. NH4+ is converted into N2O via several steps and analyzed using quadrupole gas chromatography&#8211;mass spectrometry.

14NH4+/15NH4+ Sıralı Dönüşüm Yoluyla N2O'ya Göre Amonyuma Nitrat Azaltma Nın Potansiyel Oranlarının Ölçülmesi

The importance of understanding the fate of nitrate (NO3&#8722;), which is the dominant N species transferred from terrestrial to aquatic ecosystems, has ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Anjiyotensin II tarafından gösterilen nitropeptid profil oluşturma ve tanımlama

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Chemiluminescence-based Assays for Detection of Nitric Oxide and its Derivatives from Autoxidation and Nitrosated Compounds

Nitric oxide (NO) activity in vivo is the combined results of its direct effects, the action of its derivatives generated from NO autoxidation, and the effects of nitrosated compounds. Measuring NO metabolites is essential to studying NO activity both at vascular levels and in other tissues, especially in the experimental settings where exogenous NO is administered. Ozone-based chemiluminescence assays allow precise measurements of NO and NO metabolites in both fluids (including plasma, tissue homogenates, cell cultures) and gas mixtures (e.g., exhaled breath). NO reacts with ozone to generate nitrogen dioxide in an excited state. The consequent light emission allows photodetection and the generation of an electric signal reflecting the NO content of the sample. Aliquots from the same sample can be used to measure specific NO metabolites, such as nitrate, nitrite, S-nitrosothiols, and iron-nitrosyl complexes. In addition, NO consumed by cell-free hemoglobin is also quantified with chemiluminescence analysis. An illustration of all these techniques is provided.

Here, we present protocols for detecting nitric oxide and its biologically relevant derivatives using chemiluminescence-based assays with high sensitivity.

Nitrik Oksit ve Türevlerinin Otoksidasyon ve Nitroslu Bileşiklerden Tespiti için Kemilüminesans Bazlı Testler

Nitric oxide (NO) activity in vivo is the combined results of its direct effects, the action of its derivatives generated from NO autoxidation, and the ...

Measuring Nitrite and Nitrate, Metabolites in the Nitric Oxide Pathway, in Biological Materials using the Chemiluminescence Method

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into nitrite and nitrate by interactions with various oxy-heme proteins and other still not well known pathways. The reverse process, reduction of nitrite and nitrate into NO had been discovered in mammals in the last decade and it is gaining attention as one of the possible pathways to either prevent or ease a whole range of cardiovascular, metabolic and muscular disorders that are thought to be associated with decreased levels of NO. It is therefore important to estimate the amount of NO and its metabolites in different body compartments &#8212; blood, body fluids and the various tissues. Blood, due to its easy accessibility, is the preferred compartment used for estimation of NO metabolites. Due to its short lifetime (few milliseconds) and low sub-nanomolar concentration, direct reliable measurements of blood NO in vivo present great technical difficulties. Thus NO availability is usually estimated based on the amount of its oxidation products, nitrite and nitrate. These two metabolites are always measured separately. There are several well established methods to determine their concentrations in biological fluids and tissues. Here we present a protocol for chemiluminescence method (CL), based on spectrophotometrical detection of NO after nitrite or nitrate reduction by tri-iodide or vanadium(III) chloride solutions, respectively. The sensitivity for nitrite and nitrate detection is in low nanomolar range, which sets CL as the most sensitive method currently available to determine changes in NO metabolic pathways. We explain in detail how to prepare samples from biological fluids and tissues in order to preserve original amounts of nitrite and nitrate present at the time of collection and how to determine their respective amounts in samples. Limitations of the CL technique are also explained.

Nitric oxide (NO) is an important signaling molecule in vascular homeostasis. NO production in vivo is too low for direct measurement. Chemiluminescence provides useful insight into NO cycle via measuring its precursors and oxidation products, nitrite and nitrate. Nitrite / nitrate determination in body tissues and fluids is explained.

Kemilüminesans Yöntemi kullanılarak biyolojik Malzemeleri, Nitrik Oksit Yolu&#39;ndaki nitrit ve nitrat, Metabolitler Ölçme

Nitric oxide (NO) is one of the main regulator molecules in vascular homeostasis and also a neurotransmitter. Enzymatically produced NO is oxidized into ...

Nitrosamide oluşumu nitrozaminler sitokrom P450s tarafından in Vitro metabolizma saptamak için bir genel yöntem

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