Short-term atrazine exposure causes behavioral deficits and disrupts monoaminergic systems in male C57BL/6 mice Zhoumeng Lin a,b , Celia A. Dodd a,1 , Nikolay M. Filipov a,b, ⁎ a Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA b Interdisciplinary Toxicology Program, University of Georgia, Athens, GA 30602, USA abstract article info Article history: Received 3 April 2013 Received in revised form 6 June 2013 Accepted 6 June 2013 Available online 14 June 2013 Keywords: Atrazine Pesticides Novel object recognition test Forced swim test Dopamine Excessive exposure to the widely used herbicide atrazine (ATR) affects several organ systems, including the brain, where neurochemical alterations reflective of dopamine (DA) circuitry perturbation have been reported. The present study aimed to investigate effects of short-term oral exposure to a dose-range (0, 5, 25, 125, or 250 mg/kg) of ATR on behavioral, neurochemical, and molecular indices of toxicity in adult male C57BL/6 mice. The experimental paradigm included open field, pole and grip tests (day 4), novel object recognition (NOR) and forced swim test (FST; day 9), followed by tissue collection 4 h post dosing on day 10. After 4 days of exposure, ATR decreased locomotor activity (≥125 mg/kg). On day 9, ATR-exposed mice exhibited dose-dependent decreased performance in the NOR test (≥25 mg/kg) and spent more time swimming and less time immobile during the FST (≥125 mg/kg). Neurochemically, short-term ATR exposure increased striatal DA and DA turnover (its metabolite homovanillic acid [HVA] and the HVA/DA ratio; ≥125 mg/kg). In addition, ATR exposure increased the levels of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the stria- tum (≥125 mg/kg) and it also increased DA turnover (≥125 mg/kg), 5-HIAA (125 mg/kg), and norepinephrine (≥125 mg/kg) levels in the prefrontal cortex. In the hippocampus, the only effect of ATR was to increase the norepinephrine metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG; 250 mg/kg). At the molecular level, the expression of key striatal (protein) or nigral (mRNA) markers associated with nigrostriatal DA function, such as tyrosine hydroxylase, DA transporter, vesicular monoamine transporter 2, and DA receptors, was not affected by ATR. These results indicate that short-term ATR exposure targets multiple monoamine pathways at the neurochemical level, including in the striatum, and induces behavioral abnormalities suggestive of impaired motor and cognitive functions and increased anxiety. Impaired performance in the NOR behavioral test was the most sensitive endpoint affected by ATR; this should be taken into consideration for future low-dose ATR studies and for the assessment of risk associated with overexposure to this herbicide. © 2013 Elsevier Inc. All rights reserved. 1. Introduction Atrazine [ATR; 2-chloro-4-(ethylamino)-6-(isopropylamino)-s- triazine, CAS# 1912-24-9] is a widely used chlorotriazine herbicide (LeBaron et al., 2008). ATR and/or its metabolites are frequently detected in the soil, ground, surface, and drinking water (Battaglin et al., 2009; Krutz et al., 2010; Mosquin et al., 2012), in farm house- holds (Lozier et al., 2012), and in urine samples from pesticide appli- cators and the general population (Curwin et al., 2007). ATR's wide use and frequent detection raise concerns about potential adverse health effects due to overexposure. In laboratory studies, excessive exposure to ATR is detrimental to several organ systems, including the immune (Filipov et al., 2005), reproductive (Cooper et al., 2007), and nervous systems. In the brain, ATR disrupts hypothalamic control of the hypothalamic–pituitary– gonadal axis by affecting luteinizing hormone release characteristics (Foradori et al., 2009b), apparently, indirectly (Cooper et al., 2007; Foradori et al., 2013). ATR also targets dopamine (DA) circuitries, including the nigrostriatal system. For example, ATR disrupts DA ho- meostasis in catecholaminergic PC12 cells (Das et al., 2000), rat striatal slices (Filipov et al., 2007) and striatal synaptic vesicles (Hossain and Filipov, 2008). ATR also disrupts the morphological differentiation of N27 dopaminergic cells (Lin et al., 2013). In vivo, short-term ATR Neurotoxicology and Teratology 39 (2013) 26–35 Abbreviations: AChE, acetylcholine esterase; ANOVA, analysis of variance; ATR, atrazine, 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine; DA, dopamine; DACT, didealkylatrazine, 2-chloro-4,6-diamino-1,3,5-triazine; DAT, dopamine transporter; DOPAC, 3,4-dihydroxyphenylacetic acid; Drd1, dopamine receptor D1; Drd2, dopamine receptor D2; Drd4, dopamine receptor D4; FST, forced swim test; 5-HIAA, 5-hydroxyindoleacetic acid; HRP, horseradish peroxidase; 5-HT, serotonin; HVA, homovanillic acid; ip, intraperitone- al; LOAEL, lowest observed adverse effect level; MHPG, 3-methoxy-4-hydroxyphenylglycol; 3-MT, 3-methoxytyramine; N, Newton; NE, norepinephrine; NOR, novel object recognition test; Nurr1, nuclear receptor related 1; PD, Parkinson's disease; TH, tyrosine hydroxylase; VMAT-2, vesicular monoamine transporter 2; VTA, ventral tegmental area. ⁎ Corresponding author at: Department of Physiology and Pharmacology, University of Georgia, 501 D. W. Brooks Dr., Athens, GA 30602, USA. Tel.: +1 706 542 3014; fax: +1 706 542 3015. E-mail address: filipov@uga.edu (N.M. Filipov). 1 Current affiliation: Department of Biology, Fort Valley State University, Fort Valley, GA 31030, USA. 0892-0362/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ntt.2013.06.002 Contents lists available at ScienceDirect Neurotoxicology and Teratology journal homepage: www.elsevier.com/locate/neutera