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Human plasma can modulate micronucleus frequency in TK6 and OE33 cells in vitro

Hamsa Naser, Kathryn Munn, Rachel Lawrence, Rhiannon Wright, ETHAN GREWAL, Lisa Williams, Shareen Doak Orcid Logo, Gareth Jenkins Orcid Logo

Mutation Research - Genetic Toxicology and Environmental Mutagenesis, Volume: 896, Start page: 503766

Swansea University Authors: Hamsa Naser, Kathryn Munn, Rhiannon Wright, ETHAN GREWAL, Shareen Doak Orcid Logo, Gareth Jenkins Orcid Logo

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DOI (Published version): 10.1016/j.mrgentox.2024.503766

Abstract

In this paper, we studied the potential genotoxic effects of human plasma from healthy volunteers, as well as patients with gastro-oesophageal reflux disease, Barrett’s oesophagus (BO) and oesophageal adenocarcinoma (OAC) using the oesophageal adenocarcinoma cell line (OE33) and the lymphoblastoid c...

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Published in: Mutation Research - Genetic Toxicology and Environmental Mutagenesis
ISSN: 1383-5718
Published: Elsevier BV 2024
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Both TK6 and OE33 cells were treated with plasma (10 % volume, replacing foetal bovine serum (FBS) or horse serum (HS)) at different time points of 4 hours (for the micronucleus (Mn) assay and the invasion assay) and 24 hours (for the cell cycle studies). Plasma-induced effects on DNA damage levels, cell viability and the cell cycle were studied by the micronucleus assay, cytokinesis block proliferation index (CBPI) and flow cytometry respectively. The expression of IL-8 in supernatants of TK6 cells and IFN-β in OE33 cells was also analysed by enzyme-linked immunosorbent assay (ELISA). Finally, we carried out an assessment of cellular invasion of OE33 cells following plasma treatment.The results of the micronucleus assay confirmed the genotoxicity of direct plasma treatment from some participants through the increase in DNA damage in TK6 cells. Conversely, some individual patient plasma samples reduced background levels of TK6 cell Mn frequency, in an anti-genotoxic fashion. In TK6 cells, (on average) plasma samples from patients with Barrett’s oesophagus induced higher micronucleus levels than healthy volunteers (p= 0.0019). There was little difference in Mn induction when using plasma versus serum to treat the cells in vitro. Cell cycle results showed that direct plasma treatment had a marked impact on OE33 cells at 24h (p=0.0182 for BO and p=0.0320 for OAC) by decreasing the proportion of cells in the S phase, while plasma exposure was less impactful on the cell cycle of TK6 cells. Invasion of OE33 cells was also seen to be non-significantly affected by plasma treatment of OE33 cells.The addition of N-acetyl cysteine NAC in a dose-dependent matter did not alter the formation of Mn in TK6 cells, suggesting that reactive oxygen species (ROS) are not the root cause of plasma’s genotoxicity. The concentration of IL-8 in TK6 cells and IFN-β in OE33 cells was significantly higher in cells treated with OAC-derived plasma than in the untreated negative control. Collectively, our results demonstrate that plasma-specific effects are detectable which helps us better understand some important aspects of the biology of blood-based biomarkers under development.</abstract><type>Journal Article</type><journal>Mutation Research - Genetic Toxicology and Environmental Mutagenesis</journal><volume>896</volume><journalNumber/><paginationStart>503766</paginationStart><paginationEnd/><publisher>Elsevier BV</publisher><placeOfPublication/><isbnPrint/><isbnElectronic/><issnPrint>1383-5718</issnPrint><issnElectronic/><keywords>Genotoxicity; Cytotoxicity; DNA damage; micronucleus assay; cell cycle; plasma; serum</keywords><publishedDay>1</publishedDay><publishedMonth>6</publishedMonth><publishedYear>2024</publishedYear><publishedDate>2024-06-01</publishedDate><doi>10.1016/j.mrgentox.2024.503766</doi><url/><notes/><college>COLLEGE NANME</college><department>Medical School</department><CollegeCode>COLLEGE CODE</CollegeCode><DepartmentCode>MEDS</DepartmentCode><institution>Swansea University</institution><apcterm>SU Library paid the OA fee (TA Institutional Deal)</apcterm><funders>We thank Cancer Research Wales for funding this project and note that a PhD studentship awarded to Kathryn Munn by Swansea University contributed to this work. Dr Hamsa Naser was funded by the Faculty of Medicine, Health and Life Science at Swansea University.</funders><projectreference/><lastEdited>2024-06-17T14:38:23.6697958</lastEdited><Created>2024-05-20T10:27:01.7827750</Created><path><level id="1">Faculty of Medicine, Health and Life Sciences</level><level id="2">Swansea University Medical School - Medicine</level></path><authors><author><firstname>Hamsa</firstname><surname>Naser</surname><order>1</order></author><author><firstname>Kathryn</firstname><surname>Munn</surname><order>2</order></author><author><firstname>Rachel</firstname><surname>Lawrence</surname><order>3</order></author><author><firstname>Rhiannon</firstname><surname>Wright</surname><order>4</order></author><author><firstname>ETHAN</firstname><surname>GREWAL</surname><order>5</order></author><author><firstname>Lisa</firstname><surname>Williams</surname><order>6</order></author><author><firstname>Shareen</firstname><surname>Doak</surname><orcid>0000-0002-6753-1987</orcid><order>7</order></author><author><firstname>Gareth</firstname><surname>Jenkins</surname><orcid>0000-0002-5437-8389</orcid><order>8</order></author></authors><documents><document><filename>66474__30594__08d2b8887a0b439f8deb11864f7c2cca.pdf</filename><originalFilename>66474.pdf</originalFilename><uploaded>2024-06-11T09:46:04.1472958</uploaded><type>Output</type><contentLength>1666644</contentLength><contentType>application/pdf</contentType><version>Version of Record</version><cronfaStatus>true</cronfaStatus><documentNotes>© 2024 The Author(s). 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spelling v2 66474 2024-05-20 Human plasma can modulate micronucleus frequency in TK6 and OE33 cells in vitro 29b4023fc506f38e5d235994a50d4471 Hamsa Naser Hamsa Naser true false 1c7f25073bd2fc065aa2d9bb54062879 Kathryn Munn Kathryn Munn true false 163f0122a8a0a2b9903d18ca317912c2 Rhiannon Wright Rhiannon Wright true false b82af3f13e4933f9938bb0f025ae33b0 ETHAN GREWAL ETHAN GREWAL true false 8f70286908f67238a527a98cbf66d387 0000-0002-6753-1987 Shareen Doak Shareen Doak true false a44095d26187304e903da7ca778697b6 0000-0002-5437-8389 Gareth Jenkins Gareth Jenkins true false 2024-05-20 MEDS In this paper, we studied the potential genotoxic effects of human plasma from healthy volunteers, as well as patients with gastro-oesophageal reflux disease, Barrett’s oesophagus (BO) and oesophageal adenocarcinoma (OAC) using the oesophageal adenocarcinoma cell line (OE33) and the lymphoblastoid cell line (TK6). Both TK6 and OE33 cells were treated with plasma (10 % volume, replacing foetal bovine serum (FBS) or horse serum (HS)) at different time points of 4 hours (for the micronucleus (Mn) assay and the invasion assay) and 24 hours (for the cell cycle studies). Plasma-induced effects on DNA damage levels, cell viability and the cell cycle were studied by the micronucleus assay, cytokinesis block proliferation index (CBPI) and flow cytometry respectively. The expression of IL-8 in supernatants of TK6 cells and IFN-β in OE33 cells was also analysed by enzyme-linked immunosorbent assay (ELISA). Finally, we carried out an assessment of cellular invasion of OE33 cells following plasma treatment.The results of the micronucleus assay confirmed the genotoxicity of direct plasma treatment from some participants through the increase in DNA damage in TK6 cells. Conversely, some individual patient plasma samples reduced background levels of TK6 cell Mn frequency, in an anti-genotoxic fashion. In TK6 cells, (on average) plasma samples from patients with Barrett’s oesophagus induced higher micronucleus levels than healthy volunteers (p= 0.0019). There was little difference in Mn induction when using plasma versus serum to treat the cells in vitro. Cell cycle results showed that direct plasma treatment had a marked impact on OE33 cells at 24h (p=0.0182 for BO and p=0.0320 for OAC) by decreasing the proportion of cells in the S phase, while plasma exposure was less impactful on the cell cycle of TK6 cells. Invasion of OE33 cells was also seen to be non-significantly affected by plasma treatment of OE33 cells.The addition of N-acetyl cysteine NAC in a dose-dependent matter did not alter the formation of Mn in TK6 cells, suggesting that reactive oxygen species (ROS) are not the root cause of plasma’s genotoxicity. The concentration of IL-8 in TK6 cells and IFN-β in OE33 cells was significantly higher in cells treated with OAC-derived plasma than in the untreated negative control. Collectively, our results demonstrate that plasma-specific effects are detectable which helps us better understand some important aspects of the biology of blood-based biomarkers under development. Journal Article Mutation Research - Genetic Toxicology and Environmental Mutagenesis 896 503766 Elsevier BV 1383-5718 Genotoxicity; Cytotoxicity; DNA damage; micronucleus assay; cell cycle; plasma; serum 1 6 2024 2024-06-01 10.1016/j.mrgentox.2024.503766 COLLEGE NANME Medical School COLLEGE CODE MEDS Swansea University SU Library paid the OA fee (TA Institutional Deal) We thank Cancer Research Wales for funding this project and note that a PhD studentship awarded to Kathryn Munn by Swansea University contributed to this work. Dr Hamsa Naser was funded by the Faculty of Medicine, Health and Life Science at Swansea University. 2024-06-17T14:38:23.6697958 2024-05-20T10:27:01.7827750 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Medicine Hamsa Naser 1 Kathryn Munn 2 Rachel Lawrence 3 Rhiannon Wright 4 ETHAN GREWAL 5 Lisa Williams 6 Shareen Doak 0000-0002-6753-1987 7 Gareth Jenkins 0000-0002-5437-8389 8 66474__30594__08d2b8887a0b439f8deb11864f7c2cca.pdf 66474.pdf 2024-06-11T09:46:04.1472958 Output 1666644 application/pdf Version of Record true © 2024 The Author(s). This is an open access article under the CC BY license. true eng http://creativecommons.org/licenses/by/4.0/
title Human plasma can modulate micronucleus frequency in TK6 and OE33 cells in vitro
spellingShingle Human plasma can modulate micronucleus frequency in TK6 and OE33 cells in vitro
Hamsa Naser
Kathryn Munn
Rhiannon Wright
ETHAN GREWAL
Shareen Doak
Gareth Jenkins
title_short Human plasma can modulate micronucleus frequency in TK6 and OE33 cells in vitro
title_full Human plasma can modulate micronucleus frequency in TK6 and OE33 cells in vitro
title_fullStr Human plasma can modulate micronucleus frequency in TK6 and OE33 cells in vitro
title_full_unstemmed Human plasma can modulate micronucleus frequency in TK6 and OE33 cells in vitro
title_sort Human plasma can modulate micronucleus frequency in TK6 and OE33 cells in vitro
author_id_str_mv 29b4023fc506f38e5d235994a50d4471
1c7f25073bd2fc065aa2d9bb54062879
163f0122a8a0a2b9903d18ca317912c2
b82af3f13e4933f9938bb0f025ae33b0
8f70286908f67238a527a98cbf66d387
a44095d26187304e903da7ca778697b6
author_id_fullname_str_mv 29b4023fc506f38e5d235994a50d4471_***_Hamsa Naser
1c7f25073bd2fc065aa2d9bb54062879_***_Kathryn Munn
163f0122a8a0a2b9903d18ca317912c2_***_Rhiannon Wright
b82af3f13e4933f9938bb0f025ae33b0_***_ETHAN GREWAL
8f70286908f67238a527a98cbf66d387_***_Shareen Doak
a44095d26187304e903da7ca778697b6_***_Gareth Jenkins
author Hamsa Naser
Kathryn Munn
Rhiannon Wright
ETHAN GREWAL
Shareen Doak
Gareth Jenkins
author2 Hamsa Naser
Kathryn Munn
Rachel Lawrence
Rhiannon Wright
ETHAN GREWAL
Lisa Williams
Shareen Doak
Gareth Jenkins
format Journal article
container_title Mutation Research - Genetic Toxicology and Environmental Mutagenesis
container_volume 896
container_start_page 503766
publishDate 2024
institution Swansea University
issn 1383-5718
doi_str_mv 10.1016/j.mrgentox.2024.503766
publisher Elsevier BV
college_str Faculty of Medicine, Health and Life Sciences
hierarchytype
hierarchy_top_id facultyofmedicinehealthandlifesciences
hierarchy_top_title Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id facultyofmedicinehealthandlifesciences
hierarchy_parent_title Faculty of Medicine, Health and Life Sciences
department_str Swansea University Medical School - Medicine{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Medicine
document_store_str 1
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description In this paper, we studied the potential genotoxic effects of human plasma from healthy volunteers, as well as patients with gastro-oesophageal reflux disease, Barrett’s oesophagus (BO) and oesophageal adenocarcinoma (OAC) using the oesophageal adenocarcinoma cell line (OE33) and the lymphoblastoid cell line (TK6). Both TK6 and OE33 cells were treated with plasma (10 % volume, replacing foetal bovine serum (FBS) or horse serum (HS)) at different time points of 4 hours (for the micronucleus (Mn) assay and the invasion assay) and 24 hours (for the cell cycle studies). Plasma-induced effects on DNA damage levels, cell viability and the cell cycle were studied by the micronucleus assay, cytokinesis block proliferation index (CBPI) and flow cytometry respectively. The expression of IL-8 in supernatants of TK6 cells and IFN-β in OE33 cells was also analysed by enzyme-linked immunosorbent assay (ELISA). Finally, we carried out an assessment of cellular invasion of OE33 cells following plasma treatment.The results of the micronucleus assay confirmed the genotoxicity of direct plasma treatment from some participants through the increase in DNA damage in TK6 cells. Conversely, some individual patient plasma samples reduced background levels of TK6 cell Mn frequency, in an anti-genotoxic fashion. In TK6 cells, (on average) plasma samples from patients with Barrett’s oesophagus induced higher micronucleus levels than healthy volunteers (p= 0.0019). There was little difference in Mn induction when using plasma versus serum to treat the cells in vitro. Cell cycle results showed that direct plasma treatment had a marked impact on OE33 cells at 24h (p=0.0182 for BO and p=0.0320 for OAC) by decreasing the proportion of cells in the S phase, while plasma exposure was less impactful on the cell cycle of TK6 cells. Invasion of OE33 cells was also seen to be non-significantly affected by plasma treatment of OE33 cells.The addition of N-acetyl cysteine NAC in a dose-dependent matter did not alter the formation of Mn in TK6 cells, suggesting that reactive oxygen species (ROS) are not the root cause of plasma’s genotoxicity. The concentration of IL-8 in TK6 cells and IFN-β in OE33 cells was significantly higher in cells treated with OAC-derived plasma than in the untreated negative control. Collectively, our results demonstrate that plasma-specific effects are detectable which helps us better understand some important aspects of the biology of blood-based biomarkers under development.
published_date 2024-06-01T14:38:22Z
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