העבודה נתמכה, בין השאר, על ידי המכון הלאומי של המרכז לבריאות של אקסלנס למחקר ביולוגי [מספר מענק 1P20GM109005], הקונסורציום ארקנסו שטח גרנט דרך התעופה הלאומית שטח המינהל [NNX15AK32A מספר מענק], לבין המכון הלאומי לבטיחות תעסוקתית בריאות (NIOSH) [מספר מענק 2T420H008436]. המחברים מבקשים להודות כריסטופר Fettes עבור הגהה ועריכה כתב היד הזה.

<ol>
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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>

The authors have nothing to disclose.

מחקר ציטוגנטית או ניתוח מיקרוסקופי של מספרים או מבנים של כרומוזומים, בעיקר מתפשט metaphase, מספק מידע חיוני עבור הפרוגנוזה, הערכת סיכונים, וטיפול במחלות שונות. עכשיו זה מבוסס היטב, כי אבנורמליות ציטוגנטית מקושרת עם ההתקדמות והתפתחות של מחלות שונות, לרבות סרטן. נכון להיום,...

הערכה הוא כי חשיפה לחומר חלקיקים (PM) גורמת מעל 3 מיליון מקרי מוות מדי שנה עודפת, בעיקר ממחלות לב-ריאה וסרטן ריאות סרטן 1. ואכן, אחר הצהריים הוכר מסרטנים לבני אדם על ידי הסוכנות הבינלאומית לחקר הסרטן (קבוצה 1), כמו סיכון מוגבר לסרטן ריאות עם העלייה ברמות החשיפה PM הוכח 2. מעניין לציין שכמעט כל תאי סרטן נמל ליקויים מספריים ו / או מבניים כרומוזומליות. פחמן אורגן חלקיקים הנו נגזר, מורכב, תערובת הטרוגנית, אשר תלוי הפצת רכב וגדל על פליטה גם לשינויים פיסיים וכימיים. זה החשבונות של 35-55% ממסת עירוני PM 2.5 (PM 2.5 מיקרומטר בגודל קטן יותר) ויותר מ 60% של PM רקע כפריים קונטיננטלית 2.5 המונית 3, 4. השבר המסיס במים מהווה 30-90% של תרסיס אורגני. מספר רב של תרכובות אורגניותזוהה, כולל פחמימנים אליפטיות, פחמימנים ארומטיים פוליציקליים, ונגזר מחומצן ו הניטריות שלהם, אלדהידים ו כהלי aliphatic, חומצות שומן חופשיות ומלחיהם, חומצות קרבוקסיליות-di, תרכובות רבות תכליתיות, חלבונים, מקרומולקולות דמוי גומין (HULIS) באמצעות שיטות chromatographic בשילוב עם טכניקות ספקטרומטריה 5-8. תרכובות אלה מהוות פחות מ 10-20% של פחמן אורגן חלקיקים, ובכך ביותר של פחמן אורגני היא 9 ידוע. ראיה נסיונית עולה כי cytotoxicity, סטרס חמצוני, ודלקת מעורבים בהתפתחות מצבים פתולוגיים הקשורים PM. הוכח לאחרונה, עם זאת, כי חשיפת PM גם תוצאות מספר שינויים אפיגנטיים, כוללים שינויי מתילציה DNA של אלמנטים חוזרים, בשני ניסיוני במערכות במבחנה בבני אדם 10-12. מעניין במיוחד הן ההשפעות שלPM על DNA לווין - לווינים ראשיים ומשניות - אשר נמצאים באזור הטרוכרומטין ברחבי centromere של הכרומוזומים. זה היה הראה כי תופעות אלה עשויות להיות עקביות מטבעו, כפי שהם יכולים להיות מזוהים לפחות 72 שעות לאחר חשיפת 12. שינויים מתילציה DNA, במיוחד סביב צנטרומר, עלול לגרום להצטברות של תעתיקי mRNA DNA לווין, פשרה שלמות כרומוזומליות במהלך חלוקת התא, ולאחר מכן, לגרום להתפתחות של מגוון מצבים פתולוגיים 13. התאמת הגישה ציטוגנטית לניתוח CAs כמו שינויי נקודת סיום של אפיגנטיים הנגרמים על ידי ראש הממשלה, ובכך, היא בעלת חשיבות גבוהה. כאן אנו מדווחים הגישה לאיסוף PM הסביבה והכנת, בחשיפה במבחנה וניתוח של רשויות אישורים באמצעות מערכת מודל בעכברים מקרופאג RAW264.7. המקרופאגים מהווים את קו ההגנה הראשון נגד הגופים הזרים בשאיפה, ולכן לאקו התאו משמש הקים את המודל הנפוץ ביותר ב טוקסיקולוגיה חלקיקי 11, 12, 14, 15.

<table><tbody><tr><td>Total suspended particulate sampler</td><td>Tisch Environmental</td><td>TE-5170</td><td></td></tr><tr><td>Bruker Avance III NMR spectrometer&amp;nbsp;</td><td>Bruker</td><td>NA</td><td></td></tr><tr><td>TopSpin 3.5/pl2 software</td><td>Bruker</td><td>NA</td><td></td></tr><tr><td>ACD/NMR Processor Academic Edition</td><td>ACD/Labs</td><td>NA</td><td></td></tr><tr><td>RAW264.7 murine macrophages</td><td>ATCC</td><td>ATCC TIB-71</td><td></td></tr><tr><td>High glucose DMEM GlutaMAX media</td><td>ThermoFisher</td><td>10569010</td><td>Warm in a 37 &amp;deg;C waterbath before use</td></tr><tr><td>Fetal Bovine Serum</td><td>ThermoFisher</td><td>16000044</td><td>Warm in a 37 &amp;deg;C waterbath before use</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>ThermoFisher</td><td>15140163</td><td>Warm in a 37 &amp;deg;C waterbath before use</td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>ThermoFisher</td><td>25200056</td><td>Warm in a 37 &amp;deg;C waterbath before use</td></tr><tr><td>PBS, pH 7.4</td><td>ThermoFisher</td><td>10010049</td><td>Warm in a 37 &amp;deg;C waterbath before use</td></tr><tr><td>KaryoMAX Colcemid Solution in PBS</td><td>ThermoFisher</td><td>15212012</td><td>Warm in a 37 &amp;deg;C waterbath before use</td></tr><tr><td>KaryoMAX Potassium Chloride Solution</td><td>ThermoFisher</td><td>10575090</td><td>Warm in a 37 &amp;deg;C waterbath before use</td></tr><tr><td>Methanol (HPLC)</td><td>Fisher Scientific</td><td>A452N1-19</td><td></td></tr><tr><td>Acetic Acid, Glacial</td><td>Fisher Scientific</td><td>BP1185-500</td><td></td></tr><tr><td>Decon Contrad 70 Liquid Detergent</td><td>Fisher Scientific</td><td>04-355-1</td><td></td></tr><tr><td>Wright and Wright-Giemsa Stain Solutions</td><td>Fisher Scientific</td><td>23-200733</td><td></td></tr><tr><td>Permount Mounting Medium</td><td>Fisher Scientific</td><td>SP15-100</td><td></td></tr><tr><td>Axio Imager 2</td><td>Zeiss</td><td>NA</td><td></td></tr></tbody></table>

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

חשיפה לחומר חלקיקי (PM) הוא החשש העיקרי בעולם הבריאות, אשר עשוי לגרום נזק לרכיבים תאיים שונים, לרבות החומר הגנטי גרעיני. כדי להעריך את ההשפעה של PM על שלמות גנטית גרעינית, סטיות כרומוזומליות מבניות הבקיע מתפשט metaphase של תאי מקרופאג העכבר RAW264.7. PM נאסף מאוויר הסביבה עם סמפלר חלקיקים מרחף הכולל בנפח גבוה. החומר שנאסף solubilized ומסוננים לשמור על מסיסים במים, חלק בסדר. החלקיקים מאופיינים עבור הרכב כימי על ידי ספקטרוסקופיה בתהודה מגנטית גרעינית (NMR). ריכוזים שונים של השעית חלקיק מתווספים על תרבות במבחנה של מקרופאגים עכבר RAW264.7 במשך זמן חשיפה כולל של 72 שעות, יחד עם לתאי קבוצת ביקורת. בסוף החשיפה, התרבות מטופלת עם colcemid לעצור תאי metaphase. תאים אז שנקטפו, שטופלו פתרון hypotonic, קבוע acetomethanol, דropped גבי שקופיות זכוכית ולבסוף מוכתם פתרון Giemsa. שקופיות נבדקות כדי להעריך את סטיות כרומוזומליות מבניות (CA) ב metaphase פורשת בהגדלה 1,000X באמצעות מיקרוסקופ שדה בהיר. 50 עד 100 התפשטות metaphase הם הבקיע עבור כל קבוצת הטיפול. טכניקה זו מותאמת לצורך זיהוי של סטיות כרומוזומליות מבניות (CA), כגון הפסקות כרומטידה מסוג כרומטידה מהסוג חילופי, שברי acentric, כרומוזומים dicentric וטבעת, דקות כפולות, endoreduplication, ו טרנסלוקציות Robertsonian במבחנה לאחר חשיפת PM. זוהי שיטה רבת עוצמה כדי לשייך הסיום ציטוגנטית ומבוססת לשינויים אפיגנטיים.

בזהירות רבה יש לנקוט בבחירת המיקום של סמפלר TSP, כמו גם את הזמן של השנה כאשר מתבצע איסוף. ההרכב הכימי כמו גם את הגודל של חלקיקים עשויים להשפיע על התוצאות באופן משמעותי. החומר שנאסף צריך להיות גלוי נגד המסנן הלבן. התפשט metaphase עכבר רגיל יהיו 40 כרומוזומים a...

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

פרוטוקול זה מתאר שיטות כימות ואפיון של סטיות כרומוזומליות במבחנה מקרופאגים העכבר RAW264.7 לאחר הטיפול עם חומר חלקיקי באוויר הסביבה.

ניתוח של סטיות כרומוזומליות Matter הנגרמת חלקיקי אמביינט שימוש<em&gt; במבחנה</em&gt; מערכת

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

analysis-ambient-particulate-matter-induced-chromosomal-aberrations

Universidad San Sebastian

Research

JoVE Journal

Genetics

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

8.6K Views.  University of Arkansas for Medical Sciences.  פרוטוקול זה מתאר שיטות כימות ואפיון של סטיות כרומוזומליות במבחנה מקרופאגים העכבר RAW264.7 לאחר הטיפול עם חומר חלקיקי באוויר הסביבה. 

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

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

הערכה מקיפה של תאי מין כימי רעילות שימוש נמטודות<em&gt; Elegans Caenorhabditis</em

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

Developmental Biology

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

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

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

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

איתור של סטיות יציבות אינטר-כרומוזומליות ידי קרינה נפוצה<em&gt; באתרו</em&gt; הכלאה (mFISH) ו ספקטרלית Karyotyping (SKY) בעכברים מוקרנים

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

ניתוח מהיר של סטיות כרומוזום בעכבר לימפוציטים מסוג B על ידי הרשות הפלסטינית-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Immunology and Infection

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

שיטה אוטומטית לביצוע השימוש בשיטת ההדמיה באמצעות הדמיה רב ספקטקטרלית באמצעות זרימה

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Bioengineering

The Lambda Select cII Mutation Detection System

A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of these, transgenic mice and the Lambda (&#955;) Select cII Mutation Detection System have been employed for mutagenicity experiments by many research groups worldwide. Here, we describe a detailed protocol for the Lambda Select cII mutation assay, which can be applied to cultured cells of transgenic mice/rats or the corresponding animals treated with a chemical/physical agent of interest. The protocol consists of the following steps: (1) isolation of genomic DNA from the cells or organs/tissues of transgenic animals treated in vitro or in vivo, respectively, with a test compound; (2) recovery of the lambda shuttle vector carrying a mutational reporter gene (i.e., cII transgene) from the genomic DNA; (3) packaging of the rescued vectors into infectious bacteriophages; (4) infecting a host bacteria and culturing under selective conditions to allow propagation of the induced cII mutations; and (5) scoring the cII-mutants and DNA sequence analysis to determine the cII mutant frequency and mutation spectrum, respectively.

The Lambda Select cII Mutation Detection System

We describe a detailed protocol for the Lambda Select cII mutation assay in cultured cells of transgenic rodents or the corresponding animals treated with a chemical/physical agent of interest. This approach has been widely used for mutagenicity testing of carcinogens in mammalian cells.

למדא בחר cII מערכת איתור מוטציה

A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of ...

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

Chromosomics: איתור של שינוי המספרי מבני 24 כרומוזומים כל אדם בו זמנית באמצעות Assay רומן דגים OctoChrome

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

Biology

Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are electrophilic, generate reactive electrophiles upon biotransformation, or induce oxidative stress. Among the oxidized nucleobases, the most studied one is 8-oxo-7,8-dihydroguanine (8-oxoGua) or 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), a biomarker of oxidatively induced base damage in DNA. Aldehydes and epoxyaldehydes resulting from the lipid peroxidation process are electrophilic molecules able to form mutagenic exocyclic DNA adducts, such as the etheno adducts 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-&#949;dGuo) and 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-&#949;dAdo), which have been suggested as potential biomarkers in the pathophysiology of inflammation. Selective and sensitive methods for their quantification in DNA are necessary for the development of preventive strategies to slow down cell mutation rates and chronic disease development (e.g., cancer, neurodegenerative diseases). Among the sensitive methods available for their detection (high performance liquid chromatography coupled to electrochemical or tandem mass spectrometry detectors, comet assay, immunoassays, 32P-postlabeling), the most selective are those based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-ESI-MS/MS). Selectivity is an essential advantage when analyzing complex biological samples and HPLC-ESI-MS/MS evolved as the gold standard for quantification of modified nucleosides in biological matrices, such as DNA, urine, plasma and saliva. The use of isotopically labeled internal standards adds the advantage of corrections for molecule losses during the DNA hydrolysis and analyte enrichment steps, as well as for differences of the analyte ionization between samples. It also aids in the identification of the correct chromatographic peak when more than one peak is present.
We present here validated sensitive, accurate and precise HPLC-ESI-MS/MS methods that were successfully applied for the quantification of 8-oxodGuo, 1,N6-dAdo and 1,N2-dGuo in lung, liver and kidney DNA of A/J mice for the assessment of the effects of ambient PM2.5 exposure.

We describe here methods for sensitive and accurate quantification of the lesions 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-dAdo) and 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-dGuo) in DNA. The methods were applied to the assessment of the effects of ambient fine particulate matter (PM2.5) in tissues (lung, liver and kidney) of exposed A/J mice.

קוונפיקציה של שלוש נגעים DNA ידי ספקטרומטר המוני והערכה של רמות שלהם ברקמות של עכברים חשופים החומר מקיף חלקיקים עדינים

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are ...

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop.
Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle.
The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, &gamma;-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint 4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling 9-12.
Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Xenopus egg extract is a useful model system to investigate the DNA damage checkpoint. This protocol is for the preparation of Xenopus egg extracts and DNA damage checkpoint inducing reagents. These techniques are adaptable to a variety of DNA damaging approaches in the study of the DNA damage checkpoint signaling.

חקר מחסום הניזק לדנ&quot;א באמצעות<em&gt; Xenopus</em&gt; תמציות ביצה

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

הכנת כרומוזום מתאים בתרבית

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

מדידת נזק לדנ&quot;א ותיקון בSplenocytes העכבר לאחר כרוני<em&gt; In vivo</em&gt; חשיפה למינונים נמוכים מאוד של בטא וגמא-קרינה

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

ניתוח של סטיות כרומוזומליות Matter הנגרמת חלקיקי אמביינט שימוש

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הערכה מקיפה של תאי מין כימי רעילות שימוש נמטודות

איתור של סטיות יציבות אינטר-כרומוזומליות ידי קרינה נפוצה