Elsevier

Free Radical Biology and Medicine

Volume 116, 20 February 2018, Pages 64-72
Free Radical Biology and Medicine

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

https://doi.org/10.1016/j.freeradbiomed.2017.12.035Get rights and content

Highlights

  • Oxidative stress plays a role in paraquat (PQ) mutagenesis.

  • DNA repair enzymes OGG1 and MUTYH significantly protect against PQ-induced DNA damage and mutation.

  • Antioxidant treatment alleviates the mutagenicity of PQ by reducing available ROS.

  • The OGG1-/-MUTYH-/- model created by CRISPR/Cas9 genetic manipulation of CHO cells provides a sensitive tool to study the mutagenic effects of weak oxidative mutagens.

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.

Introduction

Oxidative stress is an important consequence of aerobic cellular metabolism; it can also be induced by environmental agents. A hallmark of oxidative stress is the excessive formation of reactive oxygen species (ROS), which can react with DNA to generate mutagenic lesions. An important oxidative DNA lesion is 8-oxoguanine (8OG), which results from oxidation at the C-8 position of guanine. In the absence of DNA repair, 8OG is highly mutagenic because, when traversed by replicative polymerases, it can base pair with adenine (in addition to cytosine), thus inducing G to T mutations [1], [2], [3], [4], [5], [6], [7].

Several DNA repair pathways have evolved to protect against oxidative DNA damage. Among these, base excision repair (BER) is the primary pathway that counteracts the mutagenic effects of oxidative DNA lesions by specifically recognizing and removing the modified bases by cleavage of the N-glycosyl bond. In the case of 8OG, BER involves the activity of 8-oxoguanine DNA glycosylase (OGG1) and MutY homolog (MUTYH), enzymes responsible for removing 8OG (when paired with C) and the mismatched adenine across from 8OG (which leads to mutations), respectively [3], [7], [8]. Defects in these glycosylases lead to excessive accumulation of oxidative stress-induced mutations, and an elevated cancer incidence, as shown in several mouse knockout models (Mutyh-/-Ogg1-/-) [3], [6], [9], [10], [11].

The BER pathway protects both the nuclear and the mitochondrial DNA. The mitochondrial BER employs isoforms of the nuclear enzymes and in general, involves simpler protein complexes [12]. Given that the inactivation of BER enzymes in most genetic models disrupts the repair of both nuclear and mitochondrial DNA, the specific role of the mitochondrial OG repair in toxicity of xenobiotics, or in cancer development is not completely established.

In addition to BER, oxidative-stress induced DNA lesions can be repaired by nucleotide excision repair (NER) [7], [13], [14]. Both pathways generate single-strand breaks (SSBs) in DNA as repair intermediates. If the level of lesions is high, or if the repair is happening concurrent with replication, much more lethal double-strand breaks (DSBs) can occur [13], [14], [15]. Therefore, when the capacity to repair SSBs is exceeded, or is compromised, the survival of the cell depends on its ability to repair DSBs using either homologous recombination (HR) or non-homologous end-joining (NHEJ) [15], [16].

Paraquat (1,1′-dimethyl, 4,4′-bipyridinium dichloride; PQ), one of the most widely used herbicides especially in developing countries, has been on the market for the past 60 years. PQ is known as an intracellular generator of ROS through redox cycling and/or mitochondrial electron transport chain disruption [17], which results in extensive mitochondrial damage and cell toxicity [17], [18], [19]. The cytotoxicity and genotoxicity of PQ, likely related to its ability to produce ROS, are well established [2], [19], [20], [21], [22], [23], [24]. However, the mutagenic consequences of PQ and the genetic and biochemical factors that protect against PQ are less understood. By generating oxidative DNA damage that can lead to mutations, PQ may be both mutagenic and carcinogenic [1], [2]. Epidemiological studies have correlated PQ exposure with an increased incidence of certain skin cancers such as lip cancer, penile cancer, non-melanomous skin cancer, skin melanoma, and skin squamous cell carcinoma [18], [25], [26]. A weak potential mutagenic and genotoxic activity of PQ has been documented in vitro and in cell culture. PQ significantly increased the frequency of chromosome aberrations (CA), micronuclei (MN), sister-chromatid exchanges (SCE), and DNA strand breaks in peripheral blood human lymphocytes [20], [21], [23] and human transformed cell lines (HeLa and Hep G2) [23]. PQ-induced CA, DNA damage, and mutation have been observed in human lung cancer cell lines [2] and V79 Chinese hamster cells [19], [24]. In some animal model studies, PQ was found to increase the level of 8OG in various rat organs [27], CA in mouse bone marrow [28], sperm-shape abnormalities in rodent spermatozoa [28], [29], and DNA damage in the erythrocytes of tadpoles [30]. Other studies, however, failed to detect an increase in hypoxanthine phosphoribosyl transferase (HPRT) gene mutation in V79 Chinese hamster cells [19] and the level of 8OG in rat organs even at toxic doses of PQ [31]. Therefore, additional research is warranted to clarify the mutagenic and genotoxic properties of PQ, as well as the mechanisms by which PQ induces mutations.

A versatile method for measuring mutagenesis in mammalian cells employs the AS52 cells, which are a Chinese hamster ovary (CHO) cell line engineered to carry the bacterial xanthine-guanine phosphoribosyltransferase (gpt) gene, instead of the mammalian homolog HPRT (which is knocked-out). 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) [32], [33], [34]. However, for a weak mutagen such as PQ, the AS52 cell model is sensitivity-challenged, owing to the presence of only a single chromosomally integrated copy of gpt gene. To increase the sensitivity of this model, the present study introduces an AS52-derived cell line in which the Ogg1 and Mutyh glycosylases have been knocked out using CRISPR-Cas9 technology. By lacking repair enzymes that counter oxidative stress, the new cell line (AS52DKO) is more suitable for quantifying the mutagenic effects of PQ, and may constitute a useful screening tool for studying oxidative stress-induced mutagenesis.

The key finding of the present study is that the toxicity and mutagenicity of PQ are significantly increased in our genetically engineered hamster cell culture system that lacks the OGG1 and MUTYH DNA glycosylases. This result implicates that the “GO” repair pathway (of which OGG1 and MUTYH are part of) is a critical modulator of PQ toxicity and mutagenicity, and suggests that genomic accumulation of 8OG is the main driver of PQ-induced mutagenesis. Moreover, the cellular system described here (the DKO cell line and associated methods) constitutes a versatile toolbox that enables to study in quantitative terms the role of the OGG1 and MUTYH glycosylases in modulating the toxicity and mutagenicity of other agents that induce oxidative stress (Fig. 1).

Section snippets

Cell culture and chemicals

AS52 cells were cultured in Ham's F-12 medium (Gibco, USA) supplemented with 10% heat-inactivated (56 °C for 30 min) fetal bovine serum (FBS) (Merck Millipore, Germany), 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine (Gibco) at 37 °C in a humidified 5% CO2 incubator. To remove the spurious or pre-existing 6-thioguanine resistant (6-TGr) mutants, cells were cultured in the presence of mycophenolic acid (MPA) (Sigma-Aldrich, USA) for 7 days. The MPA-containing medium was the

Generation of AS52 Ogg1-/-Mutyh-/- double knockout cell line

To render AS52 (WT) cells more sensitive to weak oxidative mutagens, we used CRISPR-Cas9 technology to generate AS52 double knockout cells that lack functional OGG1 and MUTYH glycosylases. Three sgRNAs were constructed for targeting Ogg1 in exons 1, 2, and 6 (Fig. 2(A)). Two sgRNAs were constructed for targeting Mutyh in exons 2 and 6 (Fig. S1(A)). All guides were selected using the “CRISPY” online tool [35]. The genotypes of DKO cells were confirmed by the SURVEYOR nuclease assay on the PCR

Discussion

The present study investigated the toxicity and mutagenicity of PQ in a mammalian cell culture system in which the cells were specifically modified in their ability to repair oxidatively damaged DNA. We showed that OGG1 and MUTYH DNA repair enzymes play an important role in the prevention of PQ-induced mutagenesis by generating (using CRISPR-Cas9 methods) and testing a double knock-out (Ogg1-/-Mutyh-/-) cell line.

The central goal of this study was to evaluate in quantitative terms the mutagenic

Conclusion

Our results suggest that the new cell model (AS52DKO) is a sensitive tool to study mutagenic effects of weak oxidative mutagens or low dose exposure to environmental agents that induce oxidative stress. Using this model, we have established that PQ is mutagenic and cytotoxic, its properties stemming from its ability to generate ROS and induce genomic accumulation of oxidative DNA lesions such as 8OG. The system described is well poised to enable deeper analysis of PQ mutagenesis in terms of the

Acknowledgements

Support for this work was provided by the National Institutes of Health grants P01 CA26731 (to J.M.E), R01 CA080024 (to J.M.E.) and P30 ES002109. P.T. was supported by a grant from the Graduate Program in Environmental Toxicology, Chulabhorn Graduate Institute (23/2555) and Center of Excellence on Environmental Health and Toxicology, Ministry of Education, Bangkok, Thailand EHT-R-5/2560(3). The authors thank Dr. Piyajit Watcharasit and Dr. Benjaporn Homkajorn for their special guidance and

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