Original Contribution Cu/Zn-superoxide dismutase and glutathione are involved in response to oxidative stress induced by protein denaturing effect of alachlor in Saccharomyces cerevisiae Kasidit Rattanawong a,b , Kittikhun Kerdsomboon a,b , Choowong Auesukaree a,b,n a Department of Biology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand b Center of Excellence on Environmental Health and Toxicology, CHE, Ministry of Education, Thailand article info Article history: Received 30 June 2015 Received in revised form 13 October 2015 Accepted 26 October 2015 Available online 28 October 2015 Keywords: Alachlor Oxidative stress Superoxide dismutase Heat shock protein Protein aggregation Glutathione Saccharomyces cerevisiae abstract Alachlor is a widely used pre-emergent chloroacetanilide herbicide which has been shown to have many harmful ecological and environmental effects. However, the mechanism of alachlor-induced oxidative stress is poorly understood. We found that, in Saccharomyces cerevisiae, the intracellular levels of reactive oxygen species (ROS) including superoxide anions were increased only after long-term exposure to alachlor, suggesting that alachlor is not a pro-oxidant. It is likely that alachlor-induced oxidative stress may result from protein denaturation because alachlor rapidly induced an increased protein aggregation, leading to upregulation of SSA4 and HSP82 genes encoding heat shock proteins (Hsp) of Hsp70 and Hsp90 family, respectively. Although only SOD1 encoding Cu/Zn-superoxide dismutase (SOD), but not SOD2 encoding Mn-SOD, is essential for alachlor tolerance, both SODs play a crucial role in reducing alachlor- induced ROS. We found that, after alachlor exposure, glutathione production was inhibited while its utilization was increased, suggesting the role of glutathione in protecting cells against alachlor, which becomes more important when lacking Cu/Zn-SOD. Based on our results, it seems that alachlor primarily causes damages to cellular macromolecules such as proteins, leading to an induction of endogenous oxidative stress, of which intracellular antioxidant defense systems are required for elimination. & 2015 Elsevier Inc. All rights reserved. 1. Introduction Alachlor (C 14 H 20 ClNO 2 ) or 2-chloro-2,6-diethyl-N-[methox- ymethyl] acetanilide is a herbicide prevalently used as pre- or early post-emergence herbicide to control annual grasses and broadleaved seedlings of maize, cotton, brassica, oilseed rape, peanut, radish, soy bean, and sugar-cane [1]. Action modes of alachlor are inhibiting biosynthesis of long-chain fatty acid, iso- prenoid, flavonoid, and gibberellin [2]. Because of its medium to high mobility in soil and slow rate of mineralization and photo- degradation in soil and water, alachlor contamination in water and soil becomes a cause of serious environmental pollutions [3]. Alachlor is proposed as a probable human carcinogen by USEPA [2]. It is metabolized by human cytochrome P450 into various harmful carcinogenic metabolites, which causes cellular damages through oxidation of several biomolecules such as DNA [4,5]. In rats, it caused tumor formation in nasal cavity [6], possibly through the perturbation of the antioxidant system [7]. In Crucian Carp, the levels of several antioxidant enzymes, e.g. superoxide dismutase (SOD), glutathione S-transferase (GST) and catalase (CAT), were increased after an alachlor treatment [8]. Alachlor therefore has been assumed to induce the generation of in- tracellular reactive oxygen species (ROS), causing oxidative da- mages to several biological macromolecules [5,9]. However, the molecular mechanisms of alachlor toxicity are still unknown. Previously, some herbicides such as 2,4-dichlorophenoxy acetic acid (2,4-D) and paraquat have been shown to induce intracellular oxidative stress in the eukaryotic model Saccharomyces cerevisiae [10,11]. Intracellular ROS levels were increased after 2,4-D ex- posure, leading to an elevated activity of Cu/Zn-SOD (Sod1p), one of major antioxidant enzymes [10]. In the case of a redox-cycling herbicide paraquat, it disturbs the respiratory electron transport chain in the mitochondria, resulting in an increased ROS Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/freeradbiomed Free Radical Biology and Medicine http://dx.doi.org/10.1016/j.freeradbiomed.2015.10.421 0891-5849/& 2015 Elsevier Inc. All rights reserved. Abbreviations: 2,4-D, 2,4-dichlorophenoxy acetic acid; DCFH-DA, 2′,7′-dichloro- fluorescein diacetate; DHE, dihydroethidium; DTNB, 5,5′-dithiobis[2-nitrobenzoic acid]; CAT, catalase; GFP, green fluorescent protein; GSH, reduced glutathione; GSSG, oxidized glutathione; GST, glutathione S-transferase; HSP, heat shock pro- tein; O 2 ∙− , superoxide anion; ROS, reactive oxygen species; SOD, superoxide dismutase n Corresponding author at: Department of Biology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand. Fax: þ66 2 3547161. E-mail address: choowong.aue@mahidol.ac.th (C. Auesukaree). Free Radical Biology and Medicine 89 (2015) 963–971