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).

The authors have nothing to disclose.

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 DNA extraction, as well as the use of DNA amounts close to 100 &#181;g in the hydrolysis procedure to minimize the contribution of spurious oxidation to the final result39. We took into account the recommendations cited above, except the use of the sodium iodide chaotropic method for DNA extraction. Instead, for simplicity, we used commercial solutions for DNA extraction, adding deferoxamine to them before use. In addition, the obtained DNA hydrolysates were directly injected into a first column of the HPLC-ESI-MS/MS system for a previous separation of 8-oxodGuo from the normal nucleosides. Immediately before the elution of 8-oxodGuo, a switching valve was used to divert the first column eluent to a second column where further separation and peak narrowing were achieved. This approach allowed adequate sensitivity for 8-oxodGuo quantification free from interferences. The most similar approach for quantification of 8-oxodGuo in DNA was described by Chao and coworkers22, who used a trap column for sample cleanup and 8-oxodGuo retention prior to sample elution into the analytical column, using a switching valve between the columns. Alternatively, a concentration step of 8-oxodGuo collected from fractions eluted from HPLC separations of DNA hydrolysates prior to HPLC-ESI-MS/MS analyses was performed15, which is much more laborious.
Reported basal levels of 8-oxodGuo in rodent lung tissue, based on HPLC analyses, range from 180 - 450/108 dGuo23,39,41,42,43, 1,340 - 2,120/108 dGuo44, or approximately 3,000/108 dGuo45,46, with the lowest values obtained from DNA extraction methods by using sodium iodide. The mean 8-oxodGuo level found here in the lung of mice exposed to ambient air was 2,124/108 dGuo. The level increased to 2,466/108 dGuo in the animals exposed to ambient air enriched in PM2.5 (Table 3)34. It is possible that the sensitivity for detection of differences between groups could be improved by extracting the DNA with the sodium iodide chaotropic method. In the present study, the mean 8-oxodGuo levels found in control mice lung, kidney, and liver DNA were, respectively, 2.0, 1.8, and 2.7 times higher than the median basal level (1,047/108 dGuo) obtained by the European Standards Committee on Oxidative DNA Damage (ESCODD) in an inter-laboratory assessment of 8-oxodGuo in DNA extracted from standard samples of pig liver38.
The main limitation for detection of 1,N6-&#949;dAdo and 1,N2-&#949;dGuo in DNA is the method sensitivity, as these lesions occur at very low levels. The lowest levels of 1,N2-dGuo in DNA, quantified by HPLC-ESI-MS/MS, were in the range of 0.87 - 4 lesions per 108 dGuo in a human cell line and rat tissues25,47. One way to improve the sensitivity and selectivity for their quantification is to concentrate them from large samples of DNA hydrolysates, using solid phase extraction. This cleanup step solves chromatographic troubles that could arise from injections of more than 100 &#181;g DNA hydrolysates into HPLC analytical columns. We used this approach in the validated method presented here.The levels of 1,N6-&#949;dAdo detected in this study34 fall within the range obtained in studies employing ultrasensitive immunoaffinity/32P-postlabeling48,49,50,51&#160;and are lower than those described by other groups employing HPLC-ESI-MS/MS21,23,24. Similarly, the 1,N2-&#949;dGuo levels quantified here34 are consistent with the lowest levels reported by Garcia25 and Angeli26 by using HPLC-ESI-MS/MS.
HPLC-ESI-MS/MS systems with higher sensitivity than the equipment used in this study are available. The use of such systems allows the analyses of smaller amounts of DNA, which broadens the applications of the methods presented here for situations in which tissue availability is a limitation. The methods presented here may be adapted for the quantification of other modified deoxynucleosides, depending on the availability of their standards and isotopic standards. Adjustment of the chromatographic conditions would be necessary in order to obtain sharp peaks of all molecules included in the analyses.

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.remotexs.ntu.edu.sg/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><tbody><tr><td>[&lt;sup&gt;15&lt;/sup&gt;N&lt;sub&gt;5&lt;/sub&gt;]-2&amp;rsquo;-deoxyadenosine</td><td>Cambridge Isotope Laboratories</td><td>NLM-3895-25</td><td></td></tr><tr><td>[&lt;sup&gt;15&lt;/sup&gt;N&lt;sub&gt;5&lt;/sub&gt;]-2&amp;rsquo;-deoxyguanosine</td><td>Cambridge Isotope Laboratories</td><td>NLM-3899-CA-10</td><td></td></tr><tr><td>acetonitrile</td><td>Carlo Erba Reagents</td><td>412413000</td><td></td></tr><tr><td>alkaline phosphatase from bovine intestinal mucosa</td><td>Sigma</td><td>P5521</td><td></td></tr><tr><td>ammonium acetate</td><td>Merck</td><td>101116</td><td></td></tr><tr><td>calf thymus DNA</td><td>Sigma</td><td>D1501</td><td></td></tr><tr><td>cell lysis solution</td><td>QIAGEN</td><td>158908</td><td></td></tr><tr><td>chloroform</td><td>Carlo Erba Reagents</td><td>412653</td><td></td></tr><tr><td>deferoxamine</td><td>Sigma</td><td>D9533</td><td></td></tr><tr><td>deoxyribonuclease I (DNase I)</td><td>Bio Basic Inc</td><td>DD0649</td><td></td></tr><tr><td>ethanol</td><td>Carlo Erba Reagents</td><td>414542</td><td></td></tr><tr><td>formic acid</td><td>Sigma-Aldrich</td><td>F0507</td><td></td></tr><tr><td>HPLC-ESI-MS/MS system</td><td>HPLC: Agilent 1200 series&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; ESI-MS/MS: Applied Biosystems/MDS Sciex Instruments</td><td>HPLC: binary pump (G1312B), isocratic pump (G1310A), column oven with a column switching valve (G1316B), diode array detector (G1315C), auto sampler (G1367C).&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; ESI-MS/MS: Linear Quadrupole Ion Trap mass spectrometer, Model 4000 QTRAP.</td><td></td></tr><tr><td>HPLC/DAD system</td><td>Shimadzu</td><td>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><td>HPLC column (50 x 2.0 mm i.d., 2.5 &amp;micro;m, C18)</td><td>Phenomenex</td><td>00B-4446-B0</td><td></td></tr><tr><td>HPLC column (150 x 2.0 mm i.d., 3.0 &amp;micro;m, C18)</td><td>Phenomenex</td><td>00F-4251-B0</td><td></td></tr><tr><td>HPLC column (250 x 4.6 mm i.d., 5.0 &amp;micro;m, C18)</td><td>Phenomenex</td><td>00G-4252-E0</td><td></td></tr><tr><td>HPLC C18 security guard cartridge (4.0 x 3.0 mm i.d.)</td><td>Phenomenex</td><td>AJO-4287</td><td></td></tr><tr><td>isoamyl alcohol</td><td>Sigma-Aldrich</td><td>M32658</td><td></td></tr><tr><td>isopropyl alcohol (isopropanol)</td><td>Carlo Erba Reagents</td><td>A412790010</td><td></td></tr><tr><td>ketamine</td><td>Ceva</td><td>Commercial name: Dopalen</td><td></td></tr><tr><td>magnesium chloride</td><td>Carlo Erba Reagents</td><td>349377</td><td></td></tr><tr><td>magnesium chloride</td><td>Sigma</td><td>M2393</td><td></td></tr><tr><td>methanol</td><td>Carlo Erba Reagents</td><td>L022909K7</td><td></td></tr><tr><td>phosphodiesterase I from &lt;em&gt;Crotalus atrox&lt;/em&gt;</td><td>Sigma</td><td>P4506</td><td></td></tr><tr><td>protein precipitation solution</td><td>QIAGEN</td><td>158912</td><td></td></tr><tr><td>proteinase K</td><td>Sigma-Aldrich</td><td>P2308</td><td></td></tr><tr><td>ribonuclease A</td><td>Sigma</td><td>R5000</td><td></td></tr><tr><td>sodium chloride</td><td>Sigma-Aldrich</td><td>S9625</td><td></td></tr><tr><td>SPE-C18 (Strata-X)</td><td>Phenomenex</td><td>8B-S100-TAK</td><td></td></tr><tr><td>tris(hydroxymethyl)-aminomethane</td><td>Carlo Erba Reagents</td><td>489983</td><td></td></tr><tr><td>xylazine</td><td>Syntec do Brasil</td><td>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 before the corresponding 2&#39;-deoxynucleosides, demonstrates the DNA purity.
Representative chromatograms from HPLC-ESI-MS/MS analyses for quantification of 8-oxodGuo, 1,N6-&#949;dAdo and 1,N2-&#949;dGuo in mice tissue DNA samples are shown in Figures&#160;4 to&#160;6. The chromatogram obtained with UV detection in Figure 4 shows the four 2&#39;-deoxynucleosides eluting from the first column until ~ 10 min, with a good separation from 8-oxodGuo, eliminating undesired interferences. The normal 2&#39;-deoxynucleosides were not present in the analyses of 1,N6-&#949;dAdo and 1,N2-&#949;dGuo, as they were eliminated in the solid phase extraction procedure. Mass spectra of the standards used in this work are shown in Figure 7.
Typical linear calibration curves for quantification of 8-oxodGuo, 1,N6-&#949;dAdo and 1,N2-&#949;dGuo are shown in Figure 834. The methods were accurate and precise, as presented in Table 234. The inter-day precision calculated for DNA aliquots supplemented with 367 fmol of 8-oxodGuo was 16.97%, supplemented with 10 fmol of 1,N2-&#949;dGuo was 14.01%, and supplemented with 1 fmol of 1,N6-&#949;dAdo was 16.66%. The limits of quantification (S/N = 10) for the standards injected on-column were 25 fmol for 8-oxodGuo, 0.3 fmol for 1,N6-&#949;dAdo, and 1 fmol for 1,N2-&#949;dGuo34.
The methods were applied to the quantification of 8-oxodGuo, 1,N2-&#949;dGuo and 1,N6-&#949;dAdo in lung, liver, and kidney DNA samples of A/J mice tissues exposed whole body to ambient air enriched in PM2.5, compared to those exposed to in situ ambient air as the study control34. The levels found are shown in Table 3, and indicate the induction of DNA lesions in lung, liver and kidney by PM2.5 exposure34.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59734/59734fig3.jpg" /> 
Figure 3. Chromatogram of the hydrolysate of a DNA sample extracted from mouse lung. The chromatogram was obtained at 260 nm from the HPLC-DAD system. The four 2&#39;-deoxynucleosides are indicated: dC, 2&#39;-deoxycytidine; dA, 2&#39;-deoxyadenosine; dG, 2&#39;-deoxyguanosine; dT, 2&#39;-deoxythymidine. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59734/59734fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59734/59734fig4.jpg" /> 
Figure 4.&#160;Representative chromatograms showing the detection of 8-oxo-7,8-dihydro-2&#180;-deoxyguanosine (8-oxodGuo) and the internal standard [15N5]8-oxodGuo by HPLC-ESI-MS/MS, as well as the normal 2&#39;-deoxynucleosides eluting from the first column and diverted to DAD detection (&#955; = 260 nm) and waste. The DNA sample was extracted from mouse lung. The analyses by HPLC-ESI-MS/MS were performed with multiple reaction monitoring (MRM) using the fragmentations specified in the images. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59734/59734fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59734/59734fig5.jpg" /> 
Figure 5.&#160;Representative chromatograms showing the detection of 1,N6-etheno-2&#180;-deoxyadenosine (1,N6-&#949;dAdo) and the internal standard [15N5]1,N6-&#949;dAdo by HPLC-ESI-MS/MS. The DNA sample was extracted from mouse kidney. The analyses were performed with multiple reaction monitoring (MRM) using the fragmentations specified in the images. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59734/59734fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59734/59734fig6.jpg" /> 
Figure 6.&#160;Representative chromatograms showing the detection of 1,N2-etheno-2&#180;-deoxyguanosine (1,N2-&#949;dGuo) and the internal standard [15N5]1,N2-&#949;dGuo by HPLC-ESI-MS/MS. The DNA sample was extracted from mouse liver. The analyses were performed with multiple reaction monitoring (MRM) using the fragmentations specified in the images. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59734/59734fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59734/59734fig7.jpg" /> 
Figure 7.&#160;Mass spectra of the standards used in this work. The spectra were obtained in MS2 using the collision energy of 20 eV to fragment the [M+H]+ ions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59734/59734fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/59734/59734fig8.jpg" /> 
Figure 8. Calibration curves obtained by HPLC-ESI-MS/MS for quantification of 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), 1,N2-etheno-2&#180;-deoxyguanosine (1,N2-&#949;dGuo) and 1,N6-etheno-2&#180;-deoxyadenosine (1,N6-&#949;dAdo). Relative Area means the area ratios between the lesion and its respective [15N5] internal standard. This figure has been modified from Oliveira et al.34. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59734/59734fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ESI-MS/MS parameters</td>
			<td>8-oxodGuo</td>
			<td>Etheno adducts</td>
		</tr>
		<tr>
			<td>Curtain Gas</td>
			<td>20 psi</td>
			<td>20 psi</td>
		</tr>
		<tr>
			<td>Nebulizing Gas</td>
			<td>55</td>
			<td>50</td>
		</tr>
		<tr>
			<td>Ion Source Gas</td>
			<td>50 psi</td>
			<td>40 psi</td>
		</tr>
		<tr>
			<td>Collision-induced Dissociation Gas</td>
			<td>Medium</td>
			<td>Medium</td>
		</tr>
		<tr>
			<td>Ion Spray Voltage</td>
			<td>5000</td>
			<td>4500</td>
		</tr>
		<tr>
			<td>ESI Probe Temperature</td>
			<td>450</td>
			<td>450</td>
		</tr>
		<tr>
			<td>Declustering Potential</td>
			<td>31 V, 8-oxodGuo</td>
			<td>41 V, 1,N6-&#949;dAdo</td>
		</tr>
		<tr>
			<td></td>
			<td>31 V, [15N5]8-oxodGuo</td>
			<td>41 V, [15N5]1,N6-&#949;dAdo</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>45 V, 1,N2-&#949;dGuo</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>45V, [15N5]1,N2-&#949;dGuo</td>
		</tr>
		<tr>
			<td>Collision Energy</td>
			<td>23 eV, 8-oxodGuo</td>
			<td>25 eV, 1,N6-&#949;dAdo</td>
		</tr>
		<tr>
			<td></td>
			<td>23 eV, [15N5]8-oxodGuo</td>
			<td>25 eV, [15N5]1,N6-&#949;dAdo</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>27 eV, 1,N2-&#949;dGuo</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>27 eV, [15N5]1,N2-&#949;dGuo</td>
		</tr>
		<tr>
			<td>Collision Cell Exit Potential</td>
			<td>16 V, 8-oxodGuo,</td>
			<td>8 V, 1,N6-&#949;dAdo</td>
		</tr>
		<tr>
			<td></td>
			<td>16 V, [15N5]8-oxodGuo</td>
			<td>8 V, [15N5]1,N6-&#949;dAdo</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>16 V, 1,N2-&#949;dGuo</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>16 V, [15N5]1,N2-&#949;dGuo</td>
		</tr>
		<tr>
			<td>Entrance Potential</td>
			<td>10 V</td>
			<td>10 V</td>
		</tr>
	</tbody>
</table>
Table 1. Parameters used in the ESI-MS/MS equipment for detection of the DNA lesions. This table has been modified from Oliveira et al.34.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Basal level</td>
			<td>Added</td>
			<td colspan="3">Detected</td>
			<td>Detected(-) Basal</td>
			<td>Accuracy</td>
			<td>CV</td>
		</tr>
		<tr>
			<td colspan="3">Average &#177; SD (fmol)</td>
			<td>fmol</td>
			<td colspan="3">Average &#177; SD (fmol)</td>
			<td>Average (fmol)</td>
			<td>%</td>
			<td>%</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="10">8-oxodGuo</td>
		</tr>
		<tr>
			<td>373.00</td>
			<td>&#177;</td>
			<td>2.71</td>
			<td>0</td>
			<td>372.79</td>
			<td>&#177;</td>
			<td>50.6</td>
			<td>-</td>
			<td>-</td>
			<td>13.57</td>
		</tr>
		<tr>
			<td>373.98</td>
			<td>&#177;</td>
			<td>4.86</td>
			<td>367</td>
			<td>755.41</td>
			<td>&#177;</td>
			<td>107.92</td>
			<td>381</td>
			<td>103.93</td>
			<td>14.29</td>
		</tr>
		<tr>
			<td>374.84</td>
			<td>&#177;</td>
			<td>5.19</td>
			<td>734</td>
			<td>1069.57</td>
			<td>&#177;</td>
			<td>108.51</td>
			<td>695</td>
			<td>94.65</td>
			<td>10.14</td>
		</tr>
		<tr>
			<td>357.94</td>
			<td>&#177;</td>
			<td>15.05</td>
			<td>1469</td>
			<td>1671.67</td>
			<td>&#177;</td>
			<td>44.27</td>
			<td>1314</td>
			<td>89.43</td>
			<td>2.65</td>
		</tr>
		<tr>
			<td>371.07</td>
			<td>&#177;</td>
			<td>2.43</td>
			<td>2204</td>
			<td>2272.01</td>
			<td>&#177;</td>
			<td>40.2</td>
			<td>1901</td>
			<td>86.25</td>
			<td>1.77</td>
		</tr>
		<tr>
			<td colspan="10">1,N2-&#949;dGuo</td>
		</tr>
		<tr>
			<td>0.54</td>
			<td>&#177;</td>
			<td>0.01</td>
			<td>0</td>
			<td>0.54</td>
			<td>&#177;</td>
			<td>0.09</td>
			<td>-</td>
			<td>-</td>
			<td>16.88</td>
		</tr>
		<tr>
			<td>0.54</td>
			<td>&#177;</td>
			<td>0.01</td>
			<td>1</td>
			<td>1.47</td>
			<td>&#177;</td>
			<td>0.16</td>
			<td>0.93</td>
			<td>93.39</td>
			<td>11.17</td>
		</tr>
		<tr>
			<td>0.55</td>
			<td>&#177;</td>
			<td>0.01</td>
			<td>5</td>
			<td>5.30</td>
			<td>&#177;</td>
			<td>0.72</td>
			<td>4.76</td>
			<td>95.11</td>
			<td>13.50</td>
		</tr>
		<tr>
			<td>0.53</td>
			<td>&#177;</td>
			<td>0.01</td>
			<td>10</td>
			<td>10.60</td>
			<td>&#177;</td>
			<td>0.39</td>
			<td>10.06</td>
			<td>100.63</td>
			<td>3.67</td>
		</tr>
		<tr>
			<td>0.54</td>
			<td>&#177;</td>
			<td>0.01</td>
			<td>20</td>
			<td>20.20</td>
			<td>&#177;</td>
			<td>0.93</td>
			<td>19.66</td>
			<td>98.29</td>
			<td>4.60</td>
		</tr>
		<tr>
			<td colspan="10">1,N6-&#949;dAdo</td>
		</tr>
		<tr>
			<td>2.08</td>
			<td>&#177;</td>
			<td>0.10</td>
			<td>0</td>
			<td>2.29</td>
			<td>&#177;</td>
			<td>0.39</td>
			<td>-</td>
			<td>-</td>
			<td>17.05</td>
		</tr>
		<tr>
			<td>2.05</td>
			<td>&#177;</td>
			<td>0.04</td>
			<td>1</td>
			<td>3.06</td>
			<td>&#177;</td>
			<td>0.47</td>
			<td>1.01</td>
			<td>100.89</td>
			<td>15.31</td>
		</tr>
		<tr>
			<td>1.99</td>
			<td>&#177;</td>
			<td>0.06</td>
			<td>5</td>
			<td>7.87</td>
			<td>&#177;</td>
			<td>1.66</td>
			<td>5.88</td>
			<td>117.60</td>
			<td>21.10</td>
		</tr>
		<tr>
			<td>2.03</td>
			<td>&#177;</td>
			<td>0.07</td>
			<td>10</td>
			<td>12.43</td>
			<td>&#177;</td>
			<td>1.25</td>
			<td>10.41</td>
			<td>104.06</td>
			<td>10.06</td>
		</tr>
		<tr>
			<td>1.97</td>
			<td>&#177;</td>
			<td>0.03</td>
			<td>20</td>
			<td>22.42</td>
			<td>&#177;</td>
			<td>3.89</td>
			<td>20.46</td>
			<td>102.29</td>
			<td>17.34</td>
		</tr>
	</tbody>
</table>
Table 2.&#160;Method accuracy and coefficient of variation (CV) for quantification of 8-oxodGuo, 1,N2-&#949;dGuo and 1,N6-&#949;dAdo in DNA. This table has been modified from Oliveira et al.34.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">Ambient Air</td>
			<td colspan="3">PM2.5&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td>N</td>
			<td>P value</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="3">Average &#177; SEM</td>
			<td colspan="3">Average &#177; SEM</td>
			<td>&#160;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="9">Lung</td>
		</tr>
		<tr>
			<td>8-oxodGuo/108 dGuo</td>
			<td>2124</td>
			<td>&#177;</td>
			<td>56.96</td>
			<td>2466</td>
			<td>&#177;</td>
			<td>93.10</td>
			<td>6</td>
			<td>0.01</td>
		</tr>
		<tr>
			<td>1,N2-&#949;dGuo/108 dGuo</td>
			<td colspan="3">ND</td>
			<td colspan="3">ND</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>1,N6-&#949;dAdo/108 dAdo</td>
			<td>1.41</td>
			<td>&#177;</td>
			<td>0.23</td>
			<td>1.44</td>
			<td>&#177;</td>
			<td>0.13</td>
			<td>7</td>
			<td>NS</td>
		</tr>
		<tr>
			<td colspan="9">Liver</td>
		</tr>
		<tr>
			<td>8-oxodGuo/108 dGuo</td>
			<td>2848</td>
			<td>&#177;</td>
			<td>183.5</td>
			<td>2949</td>
			<td>&#177;</td>
			<td>223.8</td>
			<td>6; 5</td>
			<td>NS</td>
		</tr>
		<tr>
			<td>1,N2-&#949;dGuo/108 dGuo</td>
			<td>7.79</td>
			<td>&#177;</td>
			<td>2.49</td>
			<td>24.94</td>
			<td>&#177;</td>
			<td>5.21</td>
			<td>4</td>
			<td>0.02</td>
		</tr>
		<tr>
			<td>1,N6-&#949;dAdo/108 dAdo</td>
			<td>2.82</td>
			<td>&#177;</td>
			<td>0.30</td>
			<td>2.18</td>
			<td>&#177;</td>
			<td>0.25</td>
			<td>6</td>
			<td>NS</td>
		</tr>
		<tr>
			<td colspan="9">Kidney</td>
		</tr>
		<tr>
			<td>8-oxodGuo/108 dGuo</td>
			<td>1854</td>
			<td>&#177;</td>
			<td>87.13</td>
			<td>2363</td>
			<td>&#177;</td>
			<td>157.0</td>
			<td>6</td>
			<td>0.02</td>
		</tr>
		<tr>
			<td>1,N2-&#949;dGuo/108 dGuo</td>
			<td colspan="3">ND</td>
			<td colspan="3">ND</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>1,N6-&#949;dAdo/108 dAdo</td>
			<td>1.09</td>
			<td>&#177;</td>
			<td>0.15</td>
			<td>1.52</td>
			<td>&#177;</td>
			<td>0.12</td>
			<td>7</td>
			<td>0.04</td>
		</tr>
		<tr>
			<td colspan="3">(NS, Not Significant; ND, Not Detected)</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 3.&#160;Levels of the DNA lesions in A/J mice tissue samples. The mice were exposed to ambient air and to ambient air enriched in PM2.5 (PM2.5 concentrated 30 times). Means between the two groups (ambient air and PM2.5) were compared using t test. Results were considered statistically significant when P value was less than 0.05. This table has been modified from Oliveira et al.34.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.7K Views.  Universidade de São Paulo. De hier gepresenteerde methoden maken het mogelijk om DNA-laesies te kwantificeren die worden veroorzaakt door oxidatieve stress, wat bijdraagt aan het begrip van pathofysiologische mechanismen die gericht kunnen zijn op preventie en behandeling van ziekten. Selectiviteit en gevoeligheid zijn de belangrijkste voordelen. Selectiviteit is een essentieel voordeel bij het werken met complexe biologische monsters.HPLC in combinatie met tandemmassaspectrometrie evolueerde als de gouden stand...

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

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

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. To assess the impact of PM on nuclear genetic integrity, structural chromosomal aberrations are scored in the metaphase spreads of mouse RAW264.7 macrophage cells. PM is collected from ambient air with a high volume total suspended particles sampler. The collected material is solubilized and filtered to retain the water-soluble, fine portion. The particles are characterized for chemical composition by nuclear magnetic resonance (NMR) spectroscopy. Different concentrations of particle suspension are added onto an in vitro culture of RAW264.7 mouse macrophages for a total exposure time of 72 hr, along with untreated control cells. At the end of exposure, the culture is treated with colcemid to arrest cells in metaphase. Cells are then harvested, treated with hypotonic solution, fixed in acetomethanol, dropped onto glass slides and finally stained with Giemsa solution. Slides are examined to assess the structural chromosomal aberrations (CAs) in metaphase spreads at 1,000X magnification using a bright-field microscope. 50 to 100 metaphase spread are scored for each treatment group. This technique is adapted for the detection of structural chromosomal aberrations (CAs), such as chromatid-type breaks, chromatid-type exchanges, acentric fragments, dicentric and ring chromosomes, double minutes, endoreduplication, and Robertsonian translocations in vitro after exposure to PM. It is a powerful method to associate a well-established cytogenetic endpoint to epigenetic alterations.

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

This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter.

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

Genetics

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device.

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...

Quantifying Carbon in Pulmonary Macrophages via Induced Sputum Analysis

Measuring Carbon Content in Airway Macrophages Exposed to Carbon-Containing Particulate Matters

Pulmonary macrophages exhibit a dose-dependent pattern in phagocytizing particles. Following engulfment, these macrophages are subsequently excreted with sputum, rendering macrophages and particles visible and quantifiable under light microscopy. Notably, elemental carbon within the mammalian body originates exclusively from external contaminants. Consequently, the carbon content in airway macrophages (CCAM) serves as a valid exposure biomarker, accurately estimating individual exposure to carbon-containing particulate matter (PM). This article delineates a protocol involving sputum collection, preservation, processing, slide preparation, and staining, as well as macrophage photo acquisition and analysis. After removing the macrophage nuclei, the proportion of cytoplasm area occupied by carbon particles (PCOC) was calculated to quantify carbon content in each macrophage. The results indicate an elevation in CCAM levels after exposure to carbon-containing PM. In summary, this non-invasive, precise, reliable, and standardized method enables the direct measurement of carbon particles within target cells and is utilized for large-scale quantification of individual CCAM through induced sputum.

This article describes a detailed experimental protocol for measuring the carbon content of airway macrophages with the aim of assessing the internal exposure dose at the level of individual particulate matter exposure.

Pulmonary macrophages exhibit a dose-dependent pattern in phagocytizing particles. Following engulfment, these macrophages are subsequently excreted with ...

Environment

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Medicine

Using Immunofluorescence to Detect PM2.5-induced DNA Damage in Zebrafish Embryo Hearts

Ambient fine particulate matter (PM2.5) exposure can lead to cardiac developmental toxicity but the underlying molecular mechanisms are still unclear. 8-hydroxy-2&#39;deoxygenase (8-OHdG) is a marker of oxidative DNA damage and &#947;H2AX is a sensitive marker for DNA double strand breaks. In this study, we aimed to detect PM2.5-induced 8-OHdG and &#947;H2AX changes in the heart of zebrafish embryos using an immunofluorescence assay. Zebrafish embryos were treated with extractable organic matters (EOM) from PM2.5 at 5 &#956;g/mL in the presence or absence of antioxidant N-acetyl-L-cysteine (NAC, 0.25 &#956;M) at 2 h post fertilization (hpf). DMSO was used as a vehicle control. At 72 hpf, hearts were dissected from embryos using a syringe needle and fixed and permeabilized. After being blocked, samples were probed with primary antibodies against 8-OHdG and &#947;H2AX. Samples were then washed and incubated with secondary antibodies. The resulting images were observed under fluorescence microscopy and quantified using ImageJ. The results show that EOM from PM2.5 significantly enhanced 8-OHdG and &#947;H2AX signals in the heart of zebrafish embryos. However, NAC, acting as a reactive oxygen species (ROS) scavenger, partially counteracted the EOM-induced DNA damage. Here, we present an immunofluorescence protocol for investigating the role of DNA damage in PM2.5-induced heart defects that can be applied to the detection of environmental chemical-induced protein expression changes in the hearts of zebrafish embryos.

Using Immunofluorescence to Detect PM2.5-induced DNA Damage in Zebrafish Embryo Hearts

This protocol uses an immunofluorescence assay to detect PM2.5-induced DNA damage in the dissected hearts of zebrafish embryos.

Ambient fine particulate matter (PM2.5) exposure can lead to cardiac developmental toxicity but the underlying molecular mechanisms are still unclear. ...

Developmental Biology

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Biology

Shotgun Lipidomics of Rodent Tissues

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have made lipidomics a powerful analytical tool for monitoring tissue lipidome compositions in homeostatic as well as disease states. This paper presents a step-by-step protocol for a shotgun lipid analysis method that supports the simultaneous detection and quantification of a few hundred molecular lipid species in different tissue and biofluid samples at high throughput. This method leverages automated nano-flow direct injection of a total lipid extract spiked with labeled internal standards without chromatographic separation into a high-resolution mass spectrometry instrument. Starting from sub-microgram amounts of rodent tissue, the MS analysis takes 10 min per sample and covers up to 400 lipids from 14 lipid classes in mouse lung tissue. The method presented here is well suited for studying disease mechanisms and identifying and quantifying biomarkers that indicate early toxicity or beneficial effects within rodent tissues.

Shotgun mass spectrometry-based lipidomics delivers a sensitive quantitative snapshot of a broad spectrum of lipid classes simultaneously in a single measurement from various rodent tissues.

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have ...

Biochemistry

Identifying DNA Damage in Prophase-Arrested Oocytes of Mice

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis. This protracted state of arrest makes them extremely susceptible to accumulating DNA-damaging insults, which affect the genetic integrity of the female gametes and, therefore, the genetic integrity of the future embryo. 
Consequently, the development of an accurate method to detect DNA damage, which is the first step for the establishment of DNA damage response mechanisms, is of vital importance. This paper describes a common protocol to test the presence and progress of DNA damage in prophase-arrested oocytes during a period of 20 h. Specifically, we dissect mouse ovaries, retrieve the cumulus-oocyte complexes (COCs), remove the cumulus cells from the COCs, and culture the oocytes in &#924;2 medium containing 3-isobutyl-1-methylxanthine to maintain the state of arrest. Thereafter, the oocytes are treated with the cytotoxic, antineoplasmic drug, etoposide, to engender double-strand breaks (DSBs). 
By using immunofluorescence and confocal microscopy, we detect and quantify the levels of the core protein &#947;H2AX, which is the phosphorylated form of the histone H2AX. H2AX becomes phosphorylated at the sites of DSBs after DNA damage. The inability to restore DNA integrity following DNA damage in oocytes can lead to infertility, birth defects, and increased rates of spontaneous abortions. Therefore, the understanding of DNA damage response mechanisms and, at the same time, the establishment of an intact method for studying these mechanisms are essential for reproductive biology research.

Author Spotlight: Understanding DNA Damage Response in Mammalian Oocytes and Preimplantation Embryos 

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain ...

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high incidence in aging populations. The current conventional therapies for COPD focus mainly on symptom-modifying drugs; thus, the development of new therapies is urgently needed. Qualified animal models of COPD could help to characterize the underlying mechanisms and can be used for new drug screening. Current COPD models, such as lipopolysaccharide (LPS) or the porcine pancreatic elastase (PPE)-induced emphysema model, generate COPD-like lesions in the lungs and airways but do not otherwise resemble the pathogenesis of human COPD. A cigarette smoke (CS)-induced model remains one of the most popular because it not only simulates COPD-like lesions in the respiratory system, but it is also based on one of the main hazardous materials that causes COPD in humans. However, the time-consuming and labor-intensive aspects of the CS-induced model dramatically limit its application in new drug screening. In this study, we successfully generated a new COPD model by exposing mice to high levels of ozone. This model demonstrated the following: 1) decreased forced expiratory volume 25, 50, and 75/forced vital capacity (FEV25/FVC, FEV50/FVC, and FEV75/FVC), indicating the deterioration of lung function; 2) enlarged lung alveoli, with lung parenchymal destruction; 3) reduced fatigue time and distance; and 4) increased inflammation. Taken together, these data demonstrate that the ozone exposure (OE) model is a reliable animal model that is similar to humans because ozone overexposure is one of the etiological factors of COPD. Additionally, it only took 6 - 8 weeks, based on our previous work, to create an OE model, whereas it requires 3 - 12 months to induce the cigarette smoke model, indicating that the OE model might be a good choice for COPD research.

This study describes the successful generation of a new chronic obstructive pulmonary disease (COPD) animal model by repeatedly exposing mice to high concentrations of ozone.

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

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.

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

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

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