Name: quantification of three dna lesions by mass spectrometry and assessment of their levels in tissues of mice exposed to ambient fine particulate matter
Uploaded: 2019-05-29T14:00:00
Description: Watch this Scientific Journal Video about Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter at JoVE.

FAPESP (Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado de S&#227;o Paulo, Proc. 2012/22190-3 and 2012/08616-8), CNPq (Proc. 454214/2014-6 and 429184/2016-6), CAPES, PRPUSP (Pr&#243;-Reitoria de Pesquisa da Universidade de S&#227;o Paulo), INCT INAIRA (MCT/CNPq/FNDCT/CAPES/FAPEMIG/FAPERJ/FAPESP; Proc. 573813/2008-6), INCT Redoxoma (FAPESP/CNPq/CAPES; Proc. 573530/2008-4), NAP Redoxoma (PRPUSP; Proc. 2011.1.9352.1.8) and CEPID Redoxoma (FAPESP; Proc. 2013/07937-8). T. F. Oliveira and A. A. F. Oliveira received scholarships from FAPESP (Proc. 2012/21636-8, 2011/09891-0, 2012/08617-4) and CAPES (Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior). M. H. G. Medeiros, P. Di Mascio, P. H. N. Saldiva, and A. P. M. Loureiro received fellowships from CNPq.
Some figures and tables present in this work were originally published in Oliveira A.A.F. et al. Genotoxic and epigenotoxic effects in mice exposed to concentrated ambient fine particulate matter (PM2.5) from S&#227;o Paulo city, Brazil. Particle and Fibre Toxicology.&#160;15, 40 (2018).

Four week old male A/J mice, specific pathogen free, were obtained from the Breeding Center of Laboratory Animals of Funda&#231;&#227;o Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil, and were treated accordingly to the Ethics Committee of the Faculty of Medicine, University of S&#227;o Paulo (protocol no 1310/09).
1. Collection of mice tissues
<ol>
	<li>Anesthetize the animal with xylazine and ketamine. For a mouse with 30 g of body weight, inject a solution (no more than 2 mL) c...

A major problem found&#160;in the 8-oxodGuo analyses by HPLC methods is the possible induction of its formation during the workup procedures of DNA extraction, DNA hydrolysis, and concentration of DNA hydrolysates22,38. In order to minimize the problem of 8-oxodGuo artifactual formation, it is recommended the addition of deferoxamine to all DNA extraction, storage and hydrolysis solutions, the use of the sodium iodide chaotropic method and avoidance of phenol in ...

Some reactive oxygen species (ROS) are able to oxidize carbon double bonds of DNA bases and some carbons in the deoxyribose moiety, generating oxidized bases and DNA strand breaks1. As a negatively charged molecule rich in nitrogen and oxygen atoms, DNA is also a target for electrophilic groups that covalently react with the nucleophilic sites (nitrogen and oxygen), giving products that are called DNA adducts2. So,&#160;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 stress1,2. Although the modified DNA bases can be removed from DNA by base or nucleotide excision repair (BER or NER), the induction of an imbalance between the generation and removal of DNA lesions in favor of the former leads to a net increase of their levels in DNA overtime3. Outcomes are the increase of DNA mutation rates, reduced gene expression, and diminished protein activity2,4,5,6,7, effects that are closely related to the development of diseases. DNA mutations may affect diverse cellular functions, such as cell signaling, cell cycle, genome integrity, telomere stability, the epigenome, chromatin structure, RNA splicing, protein homeostasis, metabolism, apoptosis, and cell differentiation8,9. Strategies to slow down cell mutation rates and chronic disease development (e.g., cancer, neurodegenerative diseases) pass through the knowledge of the mutation sources, among them, DNA lesions and their causes.
ROS generated endogenously in excess, due to pollutant exposure, persistent inflammation, disease pathophysiology (e.g., diabetes), etc., are important causes of biomolecule damage, including DNA and lipid damage1. As an example, the highly reactive hydroxyl radical (OH) formed from H2O2 reduction by transition metal ions (Fe2+, Cu+) oxidizes the DNA bases, DNA sugar moiety and polyunsaturated fatty acids at diffusion-controlled rates10. Among the 80 already characterized oxidized nucleobases3, the most studied one is 8-oxo-7,8-dihydroguanine (8-oxoGua) or 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo, Figure 1), a lesion that is able to induce GT transversions in mammalian cells10,11. It is formed by the mono electronic oxidation of guanine, or by hydroxyl radical or singlet oxygen attack of guanine in DNA1. Polyunsaturated fatty acids are other important targets of highly reactive oxidants, such as &#8226;OH, which initiate the process of lipid peroxidation1,12. It gives rise to fatty acid hydroperoxides that may decompose to electrophilic aldehydes and epoxyaldehydes, such as malondialdehyde, 4-hydroxy-2-nonenal, 2,4-decadienal, 4,5-epoxy-(2E)-decenal, hexenal, acrolein, crotonaldehyde, which are able to form mutagenic exocyclic DNA adducts, such as malondialdehyde-, propano-, or etheno adducts1,12,13. The etheno adducts 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-&#949;dGuo,&#160;Figure 1) and 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-&#949;dAdo, Figure 1) have been suggested as potential biomarkers in the pathophysiology of inflammation14,15.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59734/59734fig1.jpg" /> 
Figure 1.&#160;Chemical structures of the DNA lesions quantified in the present study. dR = 2&#180;-deoxyribose. This figure has been modified from Oliveira et al.34. <a href="https://www.jove.com/files/ftp_upload/59734/59734fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Studies carried out in the early 1980s allowed the sensitive detection of 8-oxodGuo by high performance liquid chromatography coupled to electrochemical detection (HPLC-ECD). Quantification of 8-oxodGuo by HPLC-ECD in several biological systems subjected to oxidizing conditions led to the recognition of 8-oxodGuo as a biomarker of oxidatively induced base damage in DNA1,16. Although robust and allowing the quantification of 8-oxodGuo in the low fmol range17, HPLC-ECD measurements rely on the accuracy of the analyte retention time for analyte identification and on the chromatography resolution to avoid interferences of other sample constituents. As the electrochemical detection requires the use of salt (e.g., potassium phosphate, sodium acetate) in the mobile phase, the maintenance of adequate analytical conditions needs routine column and equipment cleaning time.
Alternatively, the use of the bacterial DNA repair enzyme formamidopyrimidine DNA glycosylase (FPG) and, afterwards, human 8-oxoguanine glycosylase 1 (hOGG1), for detection and removal of 8-oxoGua from DNA, emerged as a way for the induction of DNA alkali labile sites. The alkali labile sites are converted to DNA strand breaks and allow the very high sensitive indirect quantification of 8-oxoGua by alkaline single cell gel electrophoresis (&#34;comet assay&#34;). The high sensitivity and the accomplishment of the analyses without the need of cellular DNA extraction are the main advantages of this type of assay. It gives the lowest steady-state levels of 8-oxoGua in DNA, typically 7-10 times lower than the levels obtained by bioanalytical methods based on HPLC. However, it is an indirect measurement of 8-oxoGua and some drawbacks are the lack of specificity or the unknown efficiency of the repair enzymes used1,16,18.
Immunoassays are other set of methods used for the detection of 8-oxoGua1 and exocyclic DNA adducts, such as 1,N6-dAdo and 1,N2-dGuo12. Despite the sensitivity, a shortcoming of the use of antibodies for detection of DNA lesions is the lack of specificity due to cross-reactivity to other components of biological samples, including the normal DNA bases1,12. The exocyclic DNA adducts, including 1,N6-dAdo and 1,N2-dGuo, may also be detected and quantified by highly sensitive 32P-postlabeling assays12. The high sensitivity of 32P-postlabeling allows the use of very small amounts of DNA (e.g., 10 &#181;g) for detection of about 1 adduct per 1010 normal bases19. However, the use of radio-chemicals, lack of chemical specificity and low accuracy are some disadvantages19,20.
A shared limitation of the methods cited above is the low selectivity or specificity for the detection of the desired molecules. In this scenario, HPLC coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS and HPLC-MS3) evolved as the gold standard for quantification of modified nucleosides in biological matrices, such as DNA, urine, plasma and saliva1,19,20. Advantages of HPLC-ESI-MS/MS methods are the sensitivity (typically in the low fmol range) and the high specificity provided by i) the chromatographic separation, ii) the characteristic and known pattern of molecule fragmentation inside the mass spectrometer collision chamber, and iii) the accurate measurement of the selected mass to charge ratio (m/z) in multiple reaction monitoring mode1,19. 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 present1,12,19,20.
Several methods based on HPLC-ESI-MS/MS have been used for quantification of 8-oxodGuo, 1,N6-dAdo and 1,N2-dGuo in DNA extracted from different biological samples12,15,20,21,22,23,24,25,26,27,28,29. Fine particles (PM2.5) carry organic and inorganic chemicals, such as polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs, aldehydes, ketones, carboxylic acids, quinolines, metals, and water-soluble ions, which may induce inflammation and oxidative stress, conditions that favor the occurrence of biomolecule damage and disease30,31,32,33. We present here validated 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 exposure34.

<table border="0" cellpadding="0" cellspacing="0" style="width:1260px;" width="1262">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">[15N5]-2&#8217;-deoxyadenosine</td>
			<td style="width:369px;">Cambridge Isotope Laboratories</td>
			<td style="width:472px;">NLM-3895-25</td>
			<td style="width:65px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">[15N5]-2&#8217;-deoxyguanosine</td>
			<td style="width:369px;">Cambridge Isotope Laboratories</td>
			<td style="width:472px;">NLM-3899-CA-10</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">acetonitrile</td>
			<td style="width:369px;">Carlo Erba Reagents</td>
			<td style="width:472px;">412413000</td>
			<td></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:353px;">alkaline phosphatase from bovine intestinal mucosa</td>
			<td style="width:369px;">Sigma</td>
			<td style="width:472px;">P5521</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">ammonium acetate</td>
			<td style="width:369px;">Merck</td>
			<td style="width:472px;">101116</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">calf thymus DNA</td>
			<td style="width:369px;">Sigma</td>
			<td style="width:472px;">D1501</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">cell lysis solution</td>
			<td style="width:369px;">QIAGEN</td>
			<td style="width:472px;">158908</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">chloroform</td>
			<td style="width:369px;">Carlo Erba Reagents</td>
			<td style="width:472px;">412653</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">deferoxamine</td>
			<td style="width:369px;">Sigma</td>
			<td style="width:472px;">D9533</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">deoxyribonuclease I (DNase I)</td>
			<td style="width:369px;">Bio Basic Inc</td>
			<td style="width:472px;">DD0649</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">ethanol</td>
			<td style="width:369px;">Carlo Erba Reagents</td>
			<td style="width:472px;">414542</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">formic acid</td>
			<td style="width:369px;">Sigma-Aldrich</td>
			<td style="width:472px;">F0507</td>
			<td></td>
		</tr>
		<tr height="242">
			<td height="242" style="height:242px;width:353px;">HPLC-ESI-MS/MS system</td>
			<td style="width:369px;">HPLC: Agilent 1200 series&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; ESI-MS/MS: Applied Biosystems/MDS Sciex Instruments</td>
			<td style="width:472px;">HPLC: binary pump (G1312B), isocratic pump (G1310A), column oven with a column switching valve (G1316B), diode array detector (G1315C), auto sampler (G1367C).&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; ESI-MS/MS: Linear Quadrupole Ion Trap mass spectrometer, Model 4000 QTRAP.</td>
			<td></td>
		</tr>
		<tr height="138">
			<td height="138" style="height:138px;width:353px;">HPLC/DAD system</td>
			<td style="width:369px;">Shimadzu</td>
			<td style="width:472px;">Two pumps (LC-20AT), photo diode array detector (DAD-20AV), auto-injector (Proeminence SIL-20AC), column oven (CTO-10AS/VP)</td>
			<td></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:353px;">HPLC column (50 x 2.0 mm i.d., 2.5 &#181;m, C18)</td>
			<td style="width:369px;">Phenomenex</td>
			<td>00B-4446-B0</td>
			<td></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:353px;">HPLC column (150 x 2.0 mm i.d., 3.0 &#181;m, C18)</td>
			<td style="width:369px;">Phenomenex</td>
			<td style="width:472px;">00F-4251-B0</td>
			<td></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:353px;">HPLC column (250 x 4.6 mm i.d., 5.0 &#181;m, C18)</td>
			<td style="width:369px;">Phenomenex</td>
			<td style="width:472px;">00G-4252-E0</td>
			<td></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:353px;">HPLC C18 security guard cartridge (4.0 x 3.0 mm i.d.)</td>
			<td style="width:369px;">Phenomenex</td>
			<td style="width:472px;">AJO-4287</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">isoamyl alcohol</td>
			<td style="width:369px;">Sigma-Aldrich</td>
			<td style="width:472px;">M32658</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">isopropyl alcohol (isopropanol)</td>
			<td style="width:369px;">Carlo Erba Reagents</td>
			<td style="width:472px;">A412790010</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">ketamine</td>
			<td style="width:369px;">Ceva</td>
			<td style="width:472px;">Commercial name: Dopalen</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">magnesium chloride</td>
			<td style="width:369px;">Carlo Erba Reagents</td>
			<td style="width:472px;">349377</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">magnesium chloride</td>
			<td style="width:369px;">Sigma</td>
			<td style="width:472px;">M2393</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">methanol</td>
			<td style="width:369px;">Carlo Erba Reagents</td>
			<td style="width:472px;">L022909K7</td>
			<td></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:353px;">phosphodiesterase I from Crotalus atrox</td>
			<td style="width:369px;">Sigma</td>
			<td style="width:472px;">P4506</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">protein precipitation solution</td>
			<td style="width:369px;">QIAGEN</td>
			<td style="width:472px;">158912</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">proteinase K</td>
			<td style="width:369px;">Sigma-Aldrich</td>
			<td style="width:472px;">P2308</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">ribonuclease A</td>
			<td style="width:369px;">Sigma</td>
			<td style="width:472px;">R5000</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">sodium chloride</td>
			<td style="width:369px;">Sigma-Aldrich</td>
			<td style="width:472px;">S9625</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">SPE-C18 (Strata-X)</td>
			<td style="width:369px;">Phenomenex</td>
			<td style="width:472px;">8B-S100-TAK</td>
			<td></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:353px;">tris(hydroxymethyl)-aminomethane</td>
			<td style="width:369px;">Carlo Erba Reagents</td>
			<td style="width:472px;">489983</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:353px;">xylazine</td>
			<td style="width:369px;">Syntec do Brasil</td>
			<td style="width:472px;">Commercial name: Xilazin</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The average DNA concentrations (&#177; SD) obtained from mice liver (~ 1 g tissue), lung (~ 0.2 g tissue) and kidney (~ 0.4 g tissue) were, respectively, 5,068 &#177; 2,615, 4,369 &#177; 1,021, and 3,223 &#177; 723 &#181;g/mL in the final volume of 200 &#181;L. A representative chromatogram obtained by HPLC-DAD of the purified DNA is shown in Figure 3. The presence of the four 2&#39;-deoxynucleosides, free from the RNA ribonucleosides, which elute immediately...

Watch this Scientific Journal Video about Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter at JoVE.

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

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.

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

The methods presented here allow the quantification of DNA lesions induced by oxidative stress, contributing to the understanding of pathophysiological mechanisms that may be target for prevention and treatment of diseases. Selectivity and sensitivity are the main advantages. Selectivity is an essential advantage when working with complex biological samples.HPLC coupled to tandem mass spectrometry evolved as the gold standard for this type of quantification with biological matrices. Oxidative stress is a common event to different pathophysiological processes. Increased levels of etheno adducts have been detected in tissues or urine of patients with inflammatory diseases.The DNA lesions also provide information on endogenous events triggered by exposure to pollutants. If the DNA lesions are increased, the exposure may be at the level that increases the risk of cancer development, for example. The lesions can be quantified in DNA from any cellular system and alternatively in urine, plasma, saliva culture medium with adaptations in sample preparation.To begin, use a culture plate placed on ice as a base to cut a piece of tissue with a scalpel. Weigh one gram of the tissue in each 50-milliliter capped tube for immediate use. Keep the remaining tissue on dry ice before storing at minus 80 degrees Celsius.To each tube, add 10 milliliters of the commercial cell lysis solution containing 0.5-millimolar deferoxamine, and keep on ice. Set the tissue homogenizer to a low speed, around 60 to 90 rpm. Homogenize the tissues on ice for a few minutes until the solution is without tissue fragments.Then, add 150 microliters of proteinase K solution to each homogenized sample. Shake the tubes by inversion, and keep them at room temperature overnight. In the morning, add 40 microliters of ribonuclease A solution, shake by inversion, and keep the tubes at room temperature for two hours.Add five milliliters of commercial protein precipitation solution, vortex vigorously, and centrifuge at 2, 000 times g, four degrees Celsius, for 10 minutes. Transfer the supernatants to 50-milliliter capped tubes containing 10 milliliters of cold isopropanol. Invert the tubes gently several times until observation of precipitated DNA.Use a Pasteur pipette closed at the end to collect the precipitated DNA. Transfer the collected DNA to tubes containing four milliliters of 10-millimolar Tris buffer and one-millimolar deferoxamine at pH seven to dissolve. After the DNA is completely dissolved in the tubes, add four milliliters of a chloroform solution containing 4%isoamyl alcohol to each tube.Invert the tubes 10 times for homogenization, centrifuge at 2, 000 times g, four degrees Celsius, for 10 minutes, and transfer the upper phases to new tubes. Repeat the wash of the upper phase with the chloroform solution two more times. Then, add eight milliliters of absolute ethanol and 0.4 milliliters of a five-molar sodium chloride solution to precipitate the DNA.Again, collect the precipitated DNA, and transfer it to three milliliters of 70%ethanol. Repeat the wash with 70%ethanol one more time. Discard the ethanol solution with caution, and invert the tubes containing the precipitated DNA on absorbent paper to remove excess solution.After preparing DNA hydrolysis samples with 15N5 isotopic standard of 1, N6-etheno-deoxyadenosine and 15N5 isotopic standard of 1, N2-etheno-deoxyguanosine according to the manuscript, transfer 10 microliters of each sample into tubes for quantification of normal deoxynucleosides by HPLC-UV. Subject the residual volume to solid-phase extraction. To perform HPLC, first elute a C18 column attached to a C18 SecurityGuard cartridge with a gradient of 0.1%formic acid and methanol.Set up the gradient program to run from zero to 25 minutes with zero to 18%methanol, from 25 to 27 minutes with 18 to 0%methanol, then from 27 to 37 minutes with 0%methanol at a flow rate of one milliliter per minute and 30 degrees Celsius. After that, inject a volume between two and six microliters for each sample reserved for normal deoxynucleosides quantification. Set the DAD detector at 260 nanometers for integration of the deoxyguanosine and deoxyadenosine peaks.To perform solid-phase extraction for analyzes of 1, N6-etheno-deoxyadenosine and 1, N2-etheno-deoxyguanosine, first load the cartridges with a series of solutions at a volume of one milliliter. Add 100%methanol, deionized water, hydrolyzed DNA sample, deionized water, 10%methanol, 15%methanol, and lastly 100%methanol for collection. Then, proceed according to the manuscript.After preparing DNA hydrolysis samples 15N5 isotopic standard of 8-oxo-deoxyguanosine, transfer 80 microliters of each sample into vials for analysis in the HPLC-ESI-MS/MS system. Reserve the remaining 20 microliters for quantification of deoxyguanosine on HPLC-UV, as done previously. For analysis of 8-oxo-deoxyguanosine on HPLC-ESI-MS/MS, elute the C18 column A coupled to a C18 SecurityGuard cartridge with a gradient of solvent A and B at a flow rate of 150 microliters per minute and 25 degrees Celsius.Run the binary pump with zero to 15%of solvent B during the first 25 minutes, 15 to 80%of solvent B from 25 to 28 minutes, 80%of solvent B from 28 to 31 minutes, 80 to 0%of solvent B from 31 to 33 minutes, and 0%of solvent B from 33 to 46 minutes. Direct the first 16 minutes of column A eluent to waste. Condition column B by the isocratic pump with a solution of 15%methanol in water containing 0.1%formic acid at a flow rate of 150 microliters per minute.After six minutes, check the chromatogram to ensure the 8-oxo-deoxyguanosine standard elutes from column A.During the 16 to 32 minutes interval, switch the valve to the position allowing connection between column A and column B.Close the valve at 32 minutes to elute 8-oxo-deoxyguanosine from the second column and get a sharp chromatographic peak. For analysis of 1, N6-etheno-deoxyadenosine and 1, N2-etheno-deoxyguanosine, elute a C18 column coupled to a C18 SecurityGuard cartridge with a gradient of solvent A and B at a flow rate of 130 microliters per minute and 25 degrees Celsius. Run the binary pump with 0%of solvent B for the first 10 minutes, zero to 20%of solvent B for 10 to 39 minutes, 20 to 75%of solvent B from 39 to 41 minutes, 75%of solvent B from 41 to 46 minutes, 75 to 0%of solvent B from 46 to 47 minutes, and 0%of solvent B from 47 to 60 minutes.Use the switching valve to direct the first 35 minutes of eluent to waste and the 35 to 42 minutes fraction to the ESI source. Be sure that the adduct standards elute from the column in the set interval. Integrate the peaks of 8-oxo-deoxyguanosine 15N5 isotopic standard of 8-oxo-deoxyguanosine, 1, N6-etheno-deoxyadenosine, 15N5 isotopic standard of 1, N6-etheno-deoxyadenosine, 1, N2-etheno-deoxyguanosine, and 15N5 isotopic standard of 1, N2-etheno-deoxyguanosine from the HPLC-ESI-MS/MS analyses.Calculate the area ratios for the calibration curves and the samples. Establish the calibration curves, and calculate the amounts of lesions in each injected sample. Integrate the peaks of deoxyguanosine and deoxyadenosine from the HPLC-UV analyses.Use the areas to establish the calibration curves, and calculate the amounts of deoxyguanosine and deoxyadenosine in animals in each injected sample. Calculate the molar fractions for 8-oxo-deoxyguanosine over deoxyguanosine, 1, N6-etheno-deoxyadenosine over deoxyadenosine, and 1, N2-etheno-deoxyguanosine over deoxyguanosine, which give the number of lesions per one million normal deoxyguanosine or deoxyadenosine. A representative chromatogram of the purified DNA obtained by HPLC-UV shows the presence of the four normal deoxynucleosides, free from the RNA ribonucleosides, demonstrating the DNA purity.Representative chromatograms from HPLC-ESI-MS/MS of 8-oxo-deoxyguanosine, 1, N6-etheno-deoxyadenosine, and 1, N2-etheno-deoxyguanosine in mice tissue DNA samples are shown. The chromatogram obtained with UV detection shows the four normal deoxynucleosides eluting from the first column until about 10 minutes, with a good separation from 8-oxo-deoxyguanosine, eliminating undesired interferences. An important thing to remember for exact quantifications is to have always the same quantities of internal standards in the injection volume in the calibration curves and samples.The methods presented here may be adapted for the quantification of other modified deoxynucleosides. Expanding the band of modified deoxynucleosides allows a better understanding of the underlying pathophysiological mechanisms. We can investigate the role of oxidative damage in different situations such as exposure to xenobiotics, diabetes, and cell malignant transformation, therefore aiding the development of preventive and treatment strategies.

quantification-three-dna-lesions-mass-spectrometry-assessment-their

Research

JoVE Journal

Immunology and Infection

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct a tree to show the evolutionary relationship among different taxa (classification units) at the molecular level. Phylogenetic analysis can also be used to investigate domain relationships within an individual taxon, particularly for organisms that have undergone substantial change in morphology and physiology, but for which researchers lack fossil evidence due to the organisms&#39; long evolutionary history or scarcity of fossilization.
In this text, a detailed protocol is described for using the phylogenetic method, including amino acid sequence alignment using Clustal Omega, and subsequent phylogenetic tree construction using both Maximum Likelihood (ML) of Molecular Evolutionary Genetics Analysis (MEGA) and Bayesian Inference via MrBayes. To investigate the origin of eukaryotic Sugars Will Eventually be Exported Transporters (SWEET) genes, 228 SWEETs including 35 SWEET proteins from unicellular eukaryotes and 57 SemiSWEET proteins from prokaryotes were analyzed. Interestingly, SemiSWEETs were found in prokaryotes, but SWEETs were found in eukaryotes. Two phylogenetic trees constructed using theoretically distinct methods have consistently&#160;suggested that the first eukaryotic SWEET gene might stem from the fusion of a bacterial SemiSWEET gene and an archaeal SemiSWEET gene.&#160;It is worth noting that one should be cautious to draw a conclusion based only on phylogenetic analysis, although it is useful to explain the underlying relationship between different taxa, which is difficult or even impossible to discern through experimental means.

A method of constructing a phylogenetic tree based on sequence homology of SWEETs from eukaryotes and SemiSWEETs from prokaryotes is described. Phylogenetic analysis is a useful tool for explaining the evolutionary relatedness between homologous proteins or genes from different organism groups.

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their significance to both cell physiology and immunology, much remains to be discovered about novel lipoprotein forms, how they are synthesized, and the effect of the various forms on host immunity. To enable thorough studies on lipoproteins, this protocol describes a method for bacterial lipoprotein enrichment and preparation of N-terminal tryptic lipopeptides for structural determination by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Expanding on an established Triton X-114 phase partitioning method for lipoprotein extraction and enrichment from the bacterial cell membrane, the protocol includes additional steps to remove non-lipoprotein contaminants, increasing lipoprotein yield and purity. Since lipoproteins are commonly used in Toll-like receptor (TLR) assays, it is critical to first characterize the N-terminal structure by MALDI-TOF MS. Herein, a method is presented to isolate concentrated hydrophobic peptides enriched in N-terminal lipopeptides suitable for direct analysis by MALDI-TOF MS/MS. Lipoproteins that have been separated by Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) are transferred to a nitrocellulose membrane, digested in situ with trypsin, sequentially washed to remove polar tryptic peptides, and finally eluted with chloroform-methanol. When coupled with MS of the more polar trypsinized peptides from wash solutions, this method provides the ability to both identify the lipoprotein and characterize its N-terminus in a single experiment. Intentional sodium adduct formation can also be employed as a tool to promote more structurally informative fragmentation spectra. Ultimately, enrichment of lipoproteins and determination of their N-terminal structures will permit more extensive studies on this ubiquitous class of bacterial proteins.

The enrichment of bacterial lipoproteins using a non-ionic surfactant phase partitioning method is described for direct use in TLR assays or other applications. Further steps are detailed to prepare N-terminal tryptic lipopeptides for structural characterization by mass spectrometry.

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

Lung microRNA Profiling Across the Estrous Cycle in Ozone-exposed Mice

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a promising future of using miRNAs in the diagnosis and care of lung diseases. Here, we define a protocol for miRNA profiling to measure the relative abundance of a group of miRNAs predicted to regulate inflammatory genes in the lung tissue from of an ozone-induced airway inflammation mouse model. Because it has been shown that circulating sex hormone levels can affect the regulation of lung innate immunity in females, the purpose of this method is to describe an inflammatory miRNA profiling protocol in female mice, taking into consideration the estrous cycle stage of each animal at the time of ozone exposure. We also address applicable bioinformatics approaches to miRNA discovery and target identification methods using limma, an R/Bioconductor software, and functional analysis software to understand the biological context and pathways associated with differential miRNA expression.

Here we describe a method to assess lung expression of miRNAs that are predicted to regulate inflammatory genes using mice exposed to ozone or filtered air at different stages of the estrous cycle.

MicroRNA (miRNA) profiling has become of interest to researchers working in various research areas of biology and medicine. Current studies show a ...

Detection of Extravascular Trypanosoma Parasites by Fine Needle Aspiration

Fine Needle Aspiration (FNA) is a routine diagnostic procedure essential to both medical and veterinary practices. It consists of the percutaneous aspiration of cells and/or microorganisms from palpable masses, organs or effusions (fluid accumulation in a body cavity) using a thin needle similar to the regular needle used for the venous puncture. The material collected by FNA is in general highly cellular, and the retrieved aspirate is then smeared, air dried, wet-fixed, stained and observed under a microscope. In the clinical context, FNA is an important diagnostic tool that serves as a guide to the appropriate therapeutic management. Because it is simple, fast, minimally invasive and requires limited investment in the laboratory and human resources, it is extensively used by veterinary practitioners, mostly in domestic, but also in farm animals. In studies using animal models, FNA has the advantage that it can be performed repeatedly in the same animal, enabling longitudinal studies through the collection of cells from tumors and organs/tissues over the course of the disease. In addition to routine microscopy, retrieved material can also be used for immunocytochemistry, electron microscopy, biochemical analysis, flow cytometry, molecular biology or in vitro assays. FNA has been used to identify the protozoan parasite Trypanosoma brucei in the gonads of infected mice, opening the possibility for a future diagnosis in cattle.

Detection of Extravascular Trypanosoma Parasites by Fine Needle Aspiration

Fine Needle Aspiration is a technique, whereby cells are obtained from a lesion or organ using a thin needle. Aspirated material is smeared, stained and examined under a microscope for diagnosis or used for molecular biology, cytometry or in vitro analysis. It is cheap, simple, quick and causes minimal trauma.