<?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Publishing DTD v1.2 20190208//EN" "http://jats.nlm.nih.gov/publishing/1.2/JATS-journalpublishing1.dtd"><article xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" article-type="research-article" dtd-version="1.2" xml:lang="en">
    <front>
        <journal-meta>
            <journal-id journal-id-type="pmc">F1000Research</journal-id>
            <journal-title-group>
                <journal-title>F1000Research</journal-title>
            </journal-title-group>
            <issn pub-type="epub">2046-1402</issn>
            <publisher>
                <publisher-name>F1000 Research Limited</publisher-name>
                <publisher-loc>London, UK</publisher-loc>
            </publisher>
        </journal-meta>
        <article-meta>
            <article-id pub-id-type="doi">10.12688/f1000research.157145.1</article-id>
            <article-categories>
                <subj-group subj-group-type="heading">
                    <subject>Research Article</subject>
                </subj-group>
                <subj-group>
                    <subject>Articles</subject>
                </subj-group>
            </article-categories>
            <title-group>
                <article-title>Eosinophilia and Leucocytic DNA damage</article-title>
                <fn-group content-type="pub-status">
                    <fn>
                        <p>[version 1; peer review: awaiting peer review]</p>
                    </fn>
                </fn-group>
            </title-group>
            <contrib-group>
                <contrib contrib-type="author" corresp="yes">
                    <name>
                        <surname>Szeto</surname>
                        <given-names>Yim Tong</given-names>
                    </name>
                    <role content-type="http://credit.niso.org/">Conceptualization</role>
                    <role content-type="http://credit.niso.org/">Data Curation</role>
                    <role content-type="http://credit.niso.org/">Formal Analysis</role>
                    <role content-type="http://credit.niso.org/">Funding Acquisition</role>
                    <role content-type="http://credit.niso.org/">Investigation</role>
                    <role content-type="http://credit.niso.org/">Methodology</role>
                    <role content-type="http://credit.niso.org/">Project Administration</role>
                    <role content-type="http://credit.niso.org/">Resources</role>
                    <role content-type="http://credit.niso.org/">Software</role>
                    <role content-type="http://credit.niso.org/">Validation</role>
                    <role content-type="http://credit.niso.org/">Visualization</role>
                    <role content-type="http://credit.niso.org/">Writing &#x2013; Original Draft Preparation</role>
                    <role content-type="http://credit.niso.org/">Writing &#x2013; Review &amp; Editing</role>
                    <uri content-type="orcid">https://orcid.org/0000-0002-1711-8510</uri>
                    <xref ref-type="corresp" rid="c1">a</xref>
                    <xref ref-type="aff" rid="a1">1</xref>
                </contrib>
                <aff id="a1">
                    <label>1</label>School of Medical &amp; Health Sciences, Tung Wah College, Kowloon, Hong Kong</aff>
            </contrib-group>
            <author-notes>
                <corresp id="c1">
                    <label>a</label>
                    <email xlink:href="mailto:ytszeto@alumni.vtc.edu.hk">ytszeto@alumni.vtc.edu.hk</email>
                </corresp>
                <fn fn-type="conflict">
                    <p>No competing interests were disclosed.</p>
                </fn>
            </author-notes>
            <pub-date pub-type="epub">
                <day>11</day>
                <month>10</month>
                <year>2024</year>
            </pub-date>
            <pub-date pub-type="collection">
                <year>2024</year>
            </pub-date>
            <volume>13</volume>
            <elocation-id>1216</elocation-id>
            <history>
                <date date-type="accepted">
                    <day>27</day>
                    <month>9</month>
                    <year>2024</year>
                </date>
            </history>
            <permissions>
                <copyright-statement>Copyright: &#x00a9; 2024 Szeto YT</copyright-statement>
                <copyright-year>2024</copyright-year>
                <license xlink:href="https://creativecommons.org/licenses/by/4.0/">
                    <license-p>This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p>
                </license>
            </permissions>
            <self-uri content-type="pdf" xlink:href="https://f1000research.com/articles/13-1216/pdf"/>
            <abstract>
                <p>Eosinophilia serves as an indicator of allergy and parasite infestation. Eosinophil granules are believed to have adverse effects on cells and contribute to oxidative stress. In our current study, we investigated the relationship between eosinophilia and healthy subjects in terms of nuclear DNA damage in peripheral leukocytes. The comet assay was employed to test whole blood samples from 52 subjects in each group. The results revealed that eosinophilia subjects exhibited significantly higher levels of nuclear DNA damage in leukocytes compared to healthy subjects. Additionally, a weak positive association was observed between eosinophil counts and DNA damage. Our findings suggest that eosinophilia is linked to systemic oxidative DNA damage.</p>
            </abstract>
            <kwd-group kwd-group-type="author">
                <kwd>Antioxidant</kwd>
                <kwd>Comet assay</kwd>
                <kwd>DNA Damage</kwd>
                <kwd>Eosinophils</kwd>
                <kwd>White blood cells</kwd>
            </kwd-group>
            <funding-group>
                <award-group id="fund-1">
                    <funding-source>Tung Wah College</funding-source>
                    <award-id>General</award-id>
                </award-group>
                <funding-statement>This work was financially supported by Tung Wah College. &#13;
&#13;
 </funding-statement>
                <funding-statement>
                    <italic>The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</italic>
                </funding-statement>
            </funding-group>
        </article-meta>
    </front>
    <body>
        <sec id="sec1" sec-type="intro">
            <title>1. Introduction</title>
            <p>Inflammation is a fundamental immune response. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced in response to pathogens, but they can also cause DNA damage (
                <xref ref-type="bibr" rid="ref12">Kay et al., 2019</xref>). The major immune cells involved in ROS production include neutrophils, macrophages, and eosinophils. Eosinophils have been found to exhibit a greater superoxide generation rate and longer latency period than neutrophils (
                <xref ref-type="bibr" rid="ref17">Petreccia et al., 1987</xref>), while eosinophilia may indicate a chronic health condition (
                <xref ref-type="bibr" rid="ref20">Valent et al., 2021</xref>).</p>
            <p>Eosinophilia refers to an abnormal increase in eosinophils in peripheral blood. These cells possess bactericidal, anti-parasitic, and cell-killing functions, primarily targeting microbial parasite infections and participating in inflammatory responses (
                <xref ref-type="bibr" rid="ref14">Lombardi et al., 2022</xref>). Activation of eosinophils by immunoglobulins, cytokines, and other lipid mediators triggers a cascade of inflammatory reactions in the body, leading to tissue damage (
                <xref ref-type="bibr" rid="ref18">Ramirez et al., 2018</xref>). The oxidizing intermediates resulting from eosinophil-mediated inflammation significantly contribute to increased oxidative stress and DNA damage (
                <xref ref-type="bibr" rid="ref11">Henderson et al., 2001</xref>). Basic proteins and cationic proteins released from eosinophil granules exert toxicity on mammalian cells (
                <xref ref-type="bibr" rid="ref15">Muniz et al., 2012</xref>). Eosinophil peroxidase can generate free radicals in the presence of hydrogen peroxide (
                <xref ref-type="bibr" rid="ref21">van Dalen et al., 2006</xref>). Animal studies demonstrate that allergens induce the formation of reactive oxygen species in human cell culture models (
                <xref ref-type="bibr" rid="ref3">Chan et al., 2017</xref>). In developed countries, helminthic infestations are not major health concerns, but an increase in eosinophil counts is likely due to allergic reactions. Allergy represents an undesirable inflammatory response, and oxidants are released during this process, contributing to oxidative stress in the body. Eosinophils have also been reported to contribute to oxidative stress in uncontrolled asthma (
                <xref ref-type="bibr" rid="ref5">de Groot et al., 2019</xref>), while children with allergic rhinitis are found to have higher plasma total oxidant status (
                <xref ref-type="bibr" rid="ref7">Emin et al., 2012</xref>).</p>
            <p>In the presence of chronic oxidative stress due to eosinophilia, the oxidative stress may contribute damage to peripheral cellular DNA. The aim of the current study was to investigate the association between eosinophils count and detectable leucocytic DNA damage if any.</p>
        </sec>
        <sec id="sec2" sec-type="methods">
            <title>2. Methods</title>
            <sec id="sec3">
                <title>2.1 Specimens and cell counting</title>
                <p>Healthy and diseased subjects were recruited from the clinics. Among both groups of subjects, no known diseases were observed except for a high eosinophil count and/or eosinophil percentage in the eosinophilia group (test group). Eosinophil counts exceeding 450/&#x03bc;L or greater than 6% in white blood cell differential counts were indicative of eosinophilia. Samples with eosinophil counts between 150 and 450/&#x03bc;L and differential count below 6% were categorized into the control group. A total of 104 EDTA peripheral blood samples were analyzed. Complete blood counts were conducted within 4 hours of blood collection using the XN-1000 Hematology Analyzer (Sysmex, Kobe, Japan). An aliquot of the whole blood was stored at room temperature and subsequently transferred to -20&#x00b0;C within 2 days of collection. Demographically, the control group age ranged from 6 to 77 years and test group from 1 to 83 years. The male and female numbers were 33 and 19 respectively for both groups.</p>
            </sec>
            <sec id="sec4">
                <title>2.2 Whole blood comet assay</title>
                <p>The microscopic slide was pre-coated with a layer of 1% regular agarose for adhesion enhancement. Eighty-five &#x03bc;L of 1% low gelling temperature agarose at about 40&#x00b0;C was mixed with 4 &#x03bc;L EDTA whole blood. The mixture was quickly transferred onto the slide in a circular motion to achieve about 1.5 cm diameter. Each slide had the capacity for accommodating 2 gels. The mixture of gel and blood was cooled down and solidified on the slide before transferring to the Coplin jar containing 40 mL cold lysis solution (2.5M NaCl, 0.1M EDTA, 10mM Tris, 1% Triton X-100, 10% DMSO, pH = 10). The slide remained in the lysis solution at 4&#x00b0;C for 1 hour and then transferred to another jar containing cold electrophoresis solution (0.3M NaOH, 1mM EDTA, pH &gt; 13) for the first 10 min of alkaline treatment at 4&#x00b0;C. Fresh electrophoresis solution replaced and further 10 min of alkaline treatment proceeded. The change of fresh electrophoresis solution was to remove excessive salt trapped in the gel left from the lysis step. The slide was then transferred to an electrophoresis tank containing cold electrophoresis solution. Electrophoresis took place at constant voltage 25 V and about 300 mA for 30 min. Slide was transferred to Coplin jar containing tap water for 15 min to remove alkaline solution. To fix the DNA, 75% alcohol was added onto the gel for 30 min. Excess alcohol was drained, and the slide was dried before staining. One part of stock Giemsa stain was diluted with 9 parts of pH 6.8 phosphate buffer (PB). About 200 &#x03bc;L of freshly prepared working Giemsa stain was added onto the gel for 30 min and followed by destaining with pH 6.8 PB. Finally, the slide was dried and kept at room temperature before scoring under light microscope at 100X (Primostar 3, Zeiss, Baden-W&#x00fc;rttemberg, German). Scoring of comet cells was described in the previous study (
                    <xref ref-type="bibr" rid="ref22">Chan et al., 2023</xref>).</p>
            </sec>
            <sec id="sec5">
                <title>2.3 Statistical analysis</title>
                <p>Unpaired t-test was used to compare the differences between 2 groups in age, white blood cell (WBC) count, eosinophil percentage, eosinophil count, and comet score. Pearson correlation was used to investigate the associations between comet score and eosinophil percentage, comet score and eosinophil count.</p>
            </sec>
        </sec>
        <sec id="sec6" sec-type="results">
            <title>3. Results</title>
            <p>Results showed that eosinophilia subjects demonstrated a higher leucocytic DNA damage than normal group. Comet scores were 92.8 (124.0) in normal subjects while 161.8 (151.1) in eosinophilia group [Mean (SD)] (
                <xref ref-type="fig" rid="f1">Figure 1</xref>). These findings support our initial hypothesis that DNA damage is elevated during inflammatory states. Additionally, weak positive associations between comet score and eosinophil percentage (r=0.3190, p=0.0010), comet score and eosinophil count (r=0.2608, p=0.0075) were observed. There were no statistically significant differences in age and WBC count between normal and test groups (p&gt;0.05) (
                <xref ref-type="table" rid="T1">Table 1</xref>) (
                <xref ref-type="bibr" rid="ref4">Szeto, 2024</xref>).</p>
            <fig fig-type="figure" id="f1" orientation="portrait" position="float">
                <label>Figure 1. </label>
                <caption>
                    <title>Comet score of normal subjects (n = 52); eosinophilia subjects (n = 52).</title>
                    <p>Statistically significant difference was seen between eosinophilia and normal subjects. Unpaired t-test (p&lt;0.0124).</p>
                </caption>
                <graphic id="gr1" orientation="portrait" position="float" xlink:href="https://f1000research-files.f1000.com/manuscripts/172556/7ee75984-dff7-4954-97f7-70d5072eb35e_figure1.gif"/>
            </fig>
            <table-wrap id="T1" orientation="portrait" position="float">
                <label>Table 1. </label>
                <caption>
                    <title>Demographics and comet scores of normal and eosinophilia subjects.</title>
                </caption>
                <table content-type="article-table" frame="hsides">
                    <thead>
                        <tr>
                            <th align="left" colspan="1" rowspan="1" valign="top">Variables</th>
                            <th align="left" colspan="1" rowspan="1" valign="top">Normal subject N = 52</th>
                            <th align="left" colspan="1" rowspan="1" valign="top">Eosinophilia subject N = 52</th>
                            <th align="left" colspan="1" rowspan="1" valign="top">p value Unpaired t-test</th>
                        </tr>
                    </thead>
                    <tbody>
                        <tr>
                            <td align="left" colspan="1" rowspan="1" valign="top">Male/Female</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">33/19</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">33/19</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">-</td>
                        </tr>
                        <tr>
                            <td align="left" colspan="1" rowspan="1" valign="top">Mean age &#x00b1; SD (yr)</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">38.1 &#x00b1; 23.4</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">38.4 &#x00b1; 23.3</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">0.9503</td>
                        </tr>
                        <tr>
                            <td align="left" colspan="1" rowspan="1" valign="top">Mean WBC count &#x00b1; SD (/&#x03bc;L)</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">6.5 &#x00b1; 1.4</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">6.9 &#x00b1; 1.6</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">0.2885</td>
                        </tr>
                        <tr>
                            <td align="left" colspan="1" rowspan="1" valign="top">Mean eosinophil count &#x00b1; SD (/&#x03bc;L)</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">0.18 &#x00b1; 0.10</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">0.63 &#x00b1; 0.30</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">&lt;0.0001</td>
                        </tr>
                        <tr>
                            <td align="left" colspan="1" rowspan="1" valign="top">Mean eosinophil percentage &#x00b1; SD (%)</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">2.8 &#x00b1; 1.4</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">9.3 &#x00b1; 3.5</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">&lt;0.0001</td>
                        </tr>
                        <tr>
                            <td align="left" colspan="1" rowspan="1" valign="top">Mean comet score &#x00b1; SD</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">92.8 &#x00b1; 124.0</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">161.8 &#x00b1; 151.1</td>
                            <td align="left" colspan="1" rowspan="1" valign="top">0.0124</td>
                        </tr>
                    </tbody>
                </table>
            </table-wrap>
        </sec>
        <sec id="sec7" sec-type="discussion">
            <title>4. Discussion</title>
            <p>Eosinophil can degranulate and release cytotoxic content at the site of injury which in turn worsens inflammation and tissue damage (
                <xref ref-type="bibr" rid="ref19">Rosenberg et al., 2013</xref>). Eosinophil peroxidase in eosinophils can generate potent oxidants to defend the invasion of parasite and likely cause eosinophil-mediated tissue damage (
                <xref ref-type="bibr" rid="ref11">Henderson et al., 2001</xref>). It has been reported that blood eosinophil count is associated with eosinophil infiltration in the nasal polyps (
                <xref ref-type="bibr" rid="ref6">El-Anwar et al., 2022</xref>) and in colorectal mucosal (
                <xref ref-type="bibr" rid="ref9">Haasnoot et al., 2023</xref>). Researchers have also demonstrated the positive correlation between peripheral blood eosinophil count and disease severity in bullous pemphigoid (BP) (
                <xref ref-type="bibr" rid="ref8">Gore Karaali et al., 2021</xref>).</p>
            <p>Allergens, such as house dust mites, are known to induce DNA damage in bronchial epithelium (
                <xref ref-type="bibr" rid="ref2">Chan et al., 2016</xref>). Although the mechanism remains unknown, plasma total antioxidant status is lower in children with asthma bronchiale (
                <xref ref-type="bibr" rid="ref23">Zeyrek et al., 2009</xref>). The current results imply that eosinophils might not only be associated with tissue damage but also contribute to DNA damage in peripheral white blood cells. However, there is no evidence to conclude a causal relationship between eosinophil count and leucocytic DNA damage. Pretreatment with antioxidants in mice with induced airway inflammation has been found to lower oxidative stress (
                <xref ref-type="bibr" rid="ref16">Park et al., 2009</xref>). Eosinophils in mild asthma have been suggested to contribute to oxidative stress, and treatment of oxidative stress could potentially relieve asthma in humans (
                <xref ref-type="bibr" rid="ref5">de Groot et al., 2019</xref>) or serve as beneficial adjunctive therapy (
                <xref ref-type="bibr" rid="ref1">Bowler &amp; Crapo, 2002</xref>). This highlights the potential application of peripheral blood cell DNA damage assessment to evaluate the effectiveness of oxidative stress treatment.</p>
        </sec>
        <sec id="sec8">
            <title>Ethics and consent</title>
            <p>This study was conducted in accordance with the principles of the Declaration of Helsinki, and all patients provided written informed consent prior to enrollment. Blood samples were from specimens for other laboratory tests and no additional blood sample was taken. The approval was obtained from the Research Ethics Committee. [Protocol No: 2022A]. Permission was obtained from the chairperson of Research Ethics Committee, Macao Society for the Study of Women&#x2019;s Health on 3rd Jan 2022. Reference number 202201.</p>
        </sec>
    </body>
    <back>
        <sec id="sec11" sec-type="data-availability">
            <title>Data availability</title>
            <p>The data and full results of this study are available in the Zenodo repository.</p>
            <p>Repository name: Eosinophilia and leucocytic DNA damage. DOI: 
                <ext-link ext-link-type="uri" xlink:href="https://doi.org/10.5281/zenodo.13841378">https://doi.org/10.5281/zenodo.13841378</ext-link> (
                <xref ref-type="bibr" rid="ref4">Szeto, 2024</xref>).</p>
            <p>The project contains the following underlying data:
                <list list-type="bullet">
                    <list-item>
                        <label>&#x2022;</label>
                        <p>Age (yr), WBC count (/&#x03bc;L), Eosinophil (%), Eosinophil count (/&#x03bc;L), and Comet score of normal and eosinophilia subjects.</p>
                    </list-item>
                </list>
            </p>
            <p>License: Creative Commons Attribution 4.0 International.</p>
        </sec>
        <ack>
            <title>Acknowledgement</title>
            <p>The authors would like to thank Tung Wah College for supporting this study.</p>
        </ack>
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