An engineered cell line lacking OGG1 and MUTYH glycosylases implicates the accumulation of genomic 8-oxoguanine as the basis for paraquat mutagenicity

Abstract

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.

Andrographolide, an Antioxidant, Counteracts Paraquat- Induced Mutagenesis in Mammalian Cells

“…Its toxic effects are believed to stem from its ability to generate intracellular reactive oxygen species (ROS) through redox cycling and disrupt the mitochondrial genetically engineered AS52 cell culture system that lacks the DNA repair proteins required to repair 8OG. This result suggests that genomic accumulation of 8OG is the main driver of PQ-induced mutagenesis (Tajai et al, 2018).…”

“…Several studies recommend antioxidant therapy for the treatment of PQ poisoning (Kim et al, 2003;Blanco-Ayala et al, 2014;Ortiz et al, 2016). In support of this view, our previous work demonstrates that the co-treatment with antioxidants such as N-acetylcysteine (NAC) alleviates the mutagenicity of PQ (Tajai et al, 2018). Many other natural or artificial antioxidants, such as silymarin, quercetin, ellagic acid, and L-ascorbic acid have been proposed as potential treatments for PQ poisoning.…”

“…The MPA-containing medium was the complete medium (above) supplemented with 10 µg/ml MPA, 250 µg/ml xanthine (Sigma-Aldrich), 22 µg/ml adenine (Sigma-Aldrich), 11 µg/ml thymidine (Sigma-Aldrich), and 1.2 µg/ml aminopterin (Sigma-Aldrich). Thereafter, cells were maintained in complete medium plus 11.5 µg/ml xanthine, 3 µg/ml adenine and 1.2 µg/ ml thymidine for 3 additional days (Tindall et al, 1986;Tindall and Stankowski, 1989;Wattanawaraporn et al, 2012;Tajai et al, 2018). All experiments in this study were carried out within 10 passages after the MPA treatment.…”

“…Paraquat (1,1'-dimethyl, 4,4'-bipyridinium dichloride; PQ) is one of the most widely used herbicides especially in developing countries including Thailand (Cocheme and Murphy, 2008;Blanco-Ayala et al, 2014;Tajai et al, 2018). Its toxic effects are believed to stem from its ability to generate intracellular reactive oxygen species (ROS) through redox cycling and disrupt the mitochondrial genetically engineered AS52 cell culture system that lacks the DNA repair proteins required to repair 8OG.…”

“…This cell line is engineered to carry the bacterial xanthine-guanine phosphoribosyltransferase (gpt) gene, while lacking the native homologous hprt gene. Using a forward mutagenesis assay, gpt mutants in AS52 cells can be selected by their ability to grow in the presence of 6-thioguanine (6-TG) (Tajai et al, 2018).…”