{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:24:47Z","timestamp":1760145887529,"version":"build-2065373602"},"reference-count":33,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2024,9,4]],"date-time":"2024-09-04T00:00:00Z","timestamp":1725408000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Malaysia Ministry of Higher Education","award":["A26611","603946 (HEALS)"],"award-info":[{"award-number":["A26611","603946 (HEALS)"]}]},{"name":"International Islamic University Malaysia","award":["A26611","603946 (HEALS)"],"award-info":[{"award-number":["A26611","603946 (HEALS)"]}]},{"name":"CR-UK","award":["A26611","603946 (HEALS)"],"award-info":[{"award-number":["A26611","603946 (HEALS)"]}]},{"name":"European Union\u2019s Seventh Programme for research, technological development and demonstration","award":["A26611","603946 (HEALS)"],"award-info":[{"award-number":["A26611","603946 (HEALS)"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxics"],"abstract":"<jats:p>Humans are unavoidably exposed to numerous different mutagenic DNA alkylating agents (AAs), but their role in the initiation of cancers is uncertain, in part due to difficulties in assessing human exposure. To address this, we have developed a screening method that measures promutagenic O6-alkylguanines (O6-AlkGs) in DNA and applied it to human DNA samples. The method exploits the ability of the Schizosaccharomyces pombe alkyltransferase-like protein (Atl1) to recognise and bind to a wide range of O6-AlkGs in DNA. We established an Atl1-based slot-blot (ASB) assay and validated it using calf thymus DNA alkylated in vitro with a range of alkylating agents and both calf thymus and human placental DNA methylated in vitro with temozolomide (TMZ). ASB signals were directly proportional to the levels of O6-meG in these controls. Pre-treatment of DNA with the DNA repair protein O6-methylguanine\u2013DNA methyltransferase (MGMT) reduced binding of Atl1, confirming its specificity. In addition, MCF 10A cells were treated with 500 \u03bcM TMZ and the extracted DNA, analysed using the ASB, was found to contain 1.34 fmoles O6 -meG\/\u03bcg DNA. Of six human breast tumour DNA samples assessed, five had detectable O6-AlkG levels (mean \u00b1 SD 1.24 \u00b1 0.25 O6-meG equivalents\/\u03bcg DNA. This study shows the potential usefulness of the ASB assay to detect and quantify total O6-AlkGs in human DNA samples.<\/jats:p>","DOI":"10.3390\/toxics12090649","type":"journal-article","created":{"date-parts":[[2024,9,4]],"date-time":"2024-09-04T08:46:22Z","timestamp":1725439582000},"page":"649","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development and Application of a Slot-Blot Assay Using the Damage Sensing Protein Atl1 to Detect and Quantify O6-Alkylated Guanine Bases in DNA"],"prefix":"10.3390","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2525-8425","authenticated-orcid":false,"given":"Hanum","family":"Yaakub","sequence":"first","affiliation":[{"name":"Epidemiology and Public Health Group, Division of Population Health, Health Services Research and Primary Care, School of Health Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PL, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anthony","family":"Howell","sequence":"additional","affiliation":[{"name":"Prevent Breast Cancer Centre, Wythenshawe Hospital Manchester Universities Foundation Trust, Wythenshawe, Manchester M23 9LT, UK"},{"name":"Manchester Breast Centre, The Christie NHS Foundation Trust, Wilmslow Road, Manchester M20 4GJ, UK"},{"name":"Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester M13 9PL, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-5471-918X","authenticated-orcid":false,"given":"Geoffrey P.","family":"Margison","sequence":"additional","affiliation":[{"name":"Epidemiology and Public Health Group, Division of Population Health, Health Services Research and Primary Care, School of Health Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PL, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9127-8328","authenticated-orcid":false,"given":"Andrew C.","family":"Povey","sequence":"additional","affiliation":[{"name":"Epidemiology and Public Health Group, Division of Population Health, Health Services Research and Primary Care, School of Health Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PL, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2024,9,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"114","DOI":"10.1038\/bjc.1956.15","article-title":"The production of malignant primary hepatic tumours in the rat by feeding dimethylnitrosamine","volume":"10","author":"Magee","year":"1956","journal-title":"Br. 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