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.

Spirogyra spp. (tao) is freshwater green algae which are consumed as an uncooked food especially in the north of Thailand. Aquatic plants especially algae are easily contaminated by pesticide residues from soil and water. Paraquat (1,1β-dimethyl, 4,4β-bipyridinium dichloride; PQ), a widely used herbicide among Thai farmers, is toxic, mutagenic, and carcinogenic to mammals through ingestion, inhalation and skin contact. This study aims to determine the concentrations of paraquat contaminated in Spirogyra spp. (tao) and health risk assessment on the consumption of Spirogyra spp. (tao) in Chiang Mai province, Thailand. The levels of paraquat were analyzed by using a simple, sensitive, and reliable method which was high performance liquid chromatography (HPLC). Seven samples were collected from the different districts. The levels of paraquat contamination were 5.07±0.00 to 12.69±0.10 mg/kg. The results showed that the concentrations of paraquat in all samples were lower than the standard level of the European Commission Regulation. Risk assessment of paraquat found that the hazard quotient was in the range of 0.18-0.46. It indicated that the consumption of Spirogyra spp. (tao) was expected to have no adverse health effects. However, the consumption of Spirogyra spp. (tao) should be a concern in terms of chronic exposure to toxic herbicide contaminated in the environment.

Paraquat (1,1'-dimethyl, 4,4'-bipyridinium dichloride; PQ), a commonly used herbicide worldwide, is both toxic and mutagenic. The mutagenic effect of PQ stems from its ability to redox-cycle, generating oxidative stress and subsequently oxidative DNA damage, which miscodes when replication is attempted. Andrographolide (AP 1), the major constituent in the leaves of the herbaceous plant Andrographis paniculata, is a diterpenoid with reported antioxidant activity. The present study employed the mammalian cell line AS52 to investigate the protective effect of AP 1 against PQ-induced mutagenesis. AP 1 induced cytotoxicity in AS52 cells in a dose-dependent manner (IC 50 = 15.7 µM), which allowed the selection of a non-lethal dose for the mutagenesis studies. While PQ was mutagenic in AS52 cells as evidenced by the increased levels of 6-TGr mutants, AP 1 by itself did not increase the mutation frequency. However, co-treatment with AP 1 (1-5 µM) or the antioxidant N-acetylcysteine (2 mM) almost completely counteracted the mutagenicity of PQ (10-100 µM) in AS52 cells. Taken together, these findings suggest that AP 1 , and likely by extension, A. paniculata extracts, are effective antioxidants that can protect against PQ-induced mutations, and thus could be a promising alternative treatment for PQ poisoning.