An engineered cell line lacking OGG1 and MUTYH glycosylases implicates the accumulation of genomic 8-oxoguanine as the basis for paraquat mutagenicity
Paraquat (1,1′-dimethyl, 4,4′-bipyridinium dichloride; PQ), a widely used herbicide, is toxic to mammals through ingestion, inhalation and skin contact. Epidemiological data suggest that PQ is also mutagenic and carcinogenic, especially in high doses. The toxic and mutagenic properties of PQ are att...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier BV
2021
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| Online Access: | https://hdl.handle.net/1721.1/134921 |
| _version_ | 1811074884879515648 |
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| author | Tajai, Preechaya Fedeles, Bogdan I Suriyo, Tawit Navasumrit, Panida Kanitwithayanun, Jantamas Essigmann, John M Satayavivad, Jutamaad |
| author2 | Massachusetts Institute of Technology. Department of Biological Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Biological Engineering Tajai, Preechaya Fedeles, Bogdan I Suriyo, Tawit Navasumrit, Panida Kanitwithayanun, Jantamas Essigmann, John M Satayavivad, Jutamaad |
| author_sort | Tajai, Preechaya |
| collection | MIT |
| description | Paraquat (1,1′-dimethyl, 4,4′-bipyridinium dichloride; PQ), a widely used herbicide, is toxic to mammals through ingestion, inhalation and skin contact. Epidemiological data suggest that PQ is also mutagenic and carcinogenic, especially in high doses. The toxic and mutagenic properties of PQ are attributed to the ability of the molecule to redox-cycle, which generates reactive oxygen species (ROS) and subsequent oxidative stress. ROS also cause oxidative DNA damage such as 8-oxoguanine (8OG), a mutagenic base that, when replicated, causes G to T transversion mutations. The present study employed the CHO-derived cell line AS52 to quantify the mutagenic properties of low doses of PQ. By containing a functional, chromosomally-integrated copy of the bacterial gpt gene, AS52 cells a facile system for evaluating the mutagenic properties of genotoxicants. To bolster the sensitivity of this system for detecting mutagenesis of weak mutagens like PQ, and to provide a tool for mechanistic evaluation of the mutagenic process, we constructed a new AS52-derived cell line defective for 8OG DNA repair. Specifically, we employed CRISPR-Cas9 technology to knock out 8-oxoguanine DNA glycosylase (OGG1) and MUTYH glycosylase, two key enzymes involved in the base excision repair of 8OG. The double knock-out (DKO) AS52 cells were found to be more sensitive to PQ toxicity than the parental (WT) AS52 cell line. They experienced higher levels of ROS, which translated into more DNA double-strand breaks, which explained the PQ toxicity. The increased ROS levels also led to more 8OG genomic accumulation, and a higher level of mutations in the DKO cells, suggesting that PQ mutagenesis is mediated primarily by 8OG genomic accumulation. Consistent with this view, antioxidant co-treatment lowered induced cellular ROS and PQ-induced mutagenesis. Taken together, our data demonstrate the strong protective role of OGG1 and MUTYH against PQ-induced mutagenesis. Moreover, our experiments establish the engineered OGG1 MUTYH AS52 cell line and associated methods as a versatile cellular system for studying in quantitative terms the mutagenesis of other agents, environmental or endogenous, that induce oxidative stress. -/- -/- |
| first_indexed | 2024-09-23T09:56:51Z |
| format | Article |
| id | mit-1721.1/134921 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T09:56:51Z |
| publishDate | 2021 |
| publisher | Elsevier BV |
| record_format | dspace |
| spelling | mit-1721.1/1349212023-09-26T20:19:33Z An engineered cell line lacking OGG1 and MUTYH glycosylases implicates the accumulation of genomic 8-oxoguanine as the basis for paraquat mutagenicity Tajai, Preechaya Fedeles, Bogdan I Suriyo, Tawit Navasumrit, Panida Kanitwithayanun, Jantamas Essigmann, John M Satayavivad, Jutamaad Massachusetts Institute of Technology. Department of Biological Engineering Massachusetts Institute of Technology. Department of Chemistry Massachusetts Institute of Technology. Center for Environmental Health Sciences Paraquat (1,1′-dimethyl, 4,4′-bipyridinium dichloride; PQ), a widely used herbicide, is toxic to mammals through ingestion, inhalation and skin contact. Epidemiological data suggest that PQ is also mutagenic and carcinogenic, especially in high doses. The toxic and mutagenic properties of PQ are attributed to the ability of the molecule to redox-cycle, which generates reactive oxygen species (ROS) and subsequent oxidative stress. ROS also cause oxidative DNA damage such as 8-oxoguanine (8OG), a mutagenic base that, when replicated, causes G to T transversion mutations. The present study employed the CHO-derived cell line AS52 to quantify the mutagenic properties of low doses of PQ. By containing a functional, chromosomally-integrated copy of the bacterial gpt gene, AS52 cells a facile system for evaluating the mutagenic properties of genotoxicants. To bolster the sensitivity of this system for detecting mutagenesis of weak mutagens like PQ, and to provide a tool for mechanistic evaluation of the mutagenic process, we constructed a new AS52-derived cell line defective for 8OG DNA repair. Specifically, we employed CRISPR-Cas9 technology to knock out 8-oxoguanine DNA glycosylase (OGG1) and MUTYH glycosylase, two key enzymes involved in the base excision repair of 8OG. The double knock-out (DKO) AS52 cells were found to be more sensitive to PQ toxicity than the parental (WT) AS52 cell line. They experienced higher levels of ROS, which translated into more DNA double-strand breaks, which explained the PQ toxicity. The increased ROS levels also led to more 8OG genomic accumulation, and a higher level of mutations in the DKO cells, suggesting that PQ mutagenesis is mediated primarily by 8OG genomic accumulation. Consistent with this view, antioxidant co-treatment lowered induced cellular ROS and PQ-induced mutagenesis. Taken together, our data demonstrate the strong protective role of OGG1 and MUTYH against PQ-induced mutagenesis. Moreover, our experiments establish the engineered OGG1 MUTYH AS52 cell line and associated methods as a versatile cellular system for studying in quantitative terms the mutagenesis of other agents, environmental or endogenous, that induce oxidative stress. -/- -/- 2021-10-27T20:09:52Z 2021-10-27T20:09:52Z 2018 2019-09-18T12:51:00Z Article http://purl.org/eprint/type/JournalArticle https://hdl.handle.net/1721.1/134921 en 10.1016/J.FREERADBIOMED.2017.12.035 Free Radical Biology and Medicine Creative Commons Attribution-NonCommercial-NoDerivs License http://creativecommons.org/licenses/by-nc-nd/4.0/ application/pdf Elsevier BV PMC |
| spellingShingle | Tajai, Preechaya Fedeles, Bogdan I Suriyo, Tawit Navasumrit, Panida Kanitwithayanun, Jantamas Essigmann, John M Satayavivad, Jutamaad An engineered cell line lacking OGG1 and MUTYH glycosylases implicates the accumulation of genomic 8-oxoguanine as the basis for paraquat mutagenicity |
| title | An engineered cell line lacking OGG1 and MUTYH glycosylases implicates the accumulation of genomic 8-oxoguanine as the basis for paraquat mutagenicity |
| title_full | An engineered cell line lacking OGG1 and MUTYH glycosylases implicates the accumulation of genomic 8-oxoguanine as the basis for paraquat mutagenicity |
| title_fullStr | An engineered cell line lacking OGG1 and MUTYH glycosylases implicates the accumulation of genomic 8-oxoguanine as the basis for paraquat mutagenicity |
| title_full_unstemmed | An engineered cell line lacking OGG1 and MUTYH glycosylases implicates the accumulation of genomic 8-oxoguanine as the basis for paraquat mutagenicity |
| title_short | An engineered cell line lacking OGG1 and MUTYH glycosylases implicates the accumulation of genomic 8-oxoguanine as the basis for paraquat mutagenicity |
| title_sort | engineered cell line lacking ogg1 and mutyh glycosylases implicates the accumulation of genomic 8 oxoguanine as the basis for paraquat mutagenicity |
| url | https://hdl.handle.net/1721.1/134921 |
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