Exposure to Spectracide® causes behavioral deficits in Drosophila melanogaster: Insights from locomotor analysis and molecular modeling

Chemosphere 248 (2020) 126037

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Chemosphere journal homepage: www.elsevier.com/locate/chemosphere

Exposure to Spectracide® causes behavioral deficits in Drosophila melanogaster: Insights from locomotor analysis and molecular modeling
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d, e, Ankur Chaudhuri a, 1, Roishinique Johnson b, 1, Kuntol Rakshit c, Andrea Bedna f a d, * , Kimberly Lackey , Sibani Sen Chakraborty , Natraj Krishnan Anathbandhu Chaudhuri b, f, ** a

Department of Microbiology, West Bengal State University, Barasat, Kolkata, 126, India Biology Department, Stillman College, Tuscaloosa, AL, 35404, USA c Department of Physiology and Biomedical Engineering, Mayo Clinic School of Medicine, Mayo Clinic, Rochester, MN, USA d Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, MS, 39762, USA e 
31, 370 05, Cesk
Institute of Entomology, Biology Centre, Czech Academy of Sciences, Branisovska e Budĕjovice, Czech Republic f Department of Biological Sciences, University of Alabama, Tuscaloosa, AL, 35487, USA b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


Spectracide® exposure disrupted locomotor activity rhythms and movement behavior in Drosophila.
Protein carbonyls were increased with decreased expression and activity of carbonyl reductase.
Molecular modeling revealed that atrazine strongly binds to the active site of carbonyl reductase.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 November 2019 Received in revised form 24 January 2020 Accepted 25 January 2020 Available online 27 January 2020

This study was focused on gaining insights into the mechanism by which the herbicide- Spectracide®, induces oxidative stress and alters behavior in Drosophila melanogaster. Exposure to Spectracide® (50%) significantly (p < 0.05) reduced the negative geotaxis response, jumping behavior and dampened locomotor activity rhythm in adult flies compared to non-exposed flies. Protein carbonyl levels indicative of oxidative damage increased significantly coupled with down-regulation of Sniffer gene expression encoding carbonyl reductase (CR) and its activity in Spectracide®-exposed flies. In silico modeling analysis revealed that the active ingredients of Spectracide® (atrazine, diquat dibromide, fluazifop-pbutyl, and dicamba) have significant binding affinity to the active site of CR enzyme, with atrazine having comparatively greater affinity. Our results suggest a mechanism by which ingredients in Spectracide® induce oxidative damage by competitive binding to the active site of a protective enzyme and impair its ability to prevent damage to proteins thereby leading to deficits in locomotor behavior in Drosophila. © 2020 Elsevier Ltd. All rights reserved.

Handling Editor: Willie Peijnenburg Keywords: Behavioral toxicology Carbonyl reductase Ecotoxicology Drosophila melanogaster Spectracide® In silico modeling

* Corresponding author. ** Corresponding author. Department of Biological Sciences, University of Alabama, Tuscaloosa, AL, 35487, USA. E-mail addresses: [email protected] (N. Krishnan), [email protected] (A. Chaudhuri). 1 Both authors contributed equally to this work. https://doi.org/10.1016/j.chemosphere.2020.126037 0045-6535/© 2020 Elsevier Ltd. All rights reserved.

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1. Introduction Pesticides including insecticides, herbicides, and fungicides that prevent pests, weeds, and fungi, are mostly used in agricultural fields to protect crops. The popular over-the-counter herbicide, Spectracide®, is used for sugarcane, corn, potato vines, aquatic weeds, and to maintain right of ways as a weed-killer, and its residual toxicity can persist for years in the environment (Mnif et al., 2011). There are four most common active ingredients present in Spectracide®, namely atrazine, diquat dibromide, fluazifop-p-butyl and dicamba. Commercial formulations of Spectracide® contain approximately 4% atrazine, 0.12% diquat dibromide, 0.06% fluazifop-p-butyl, 0.04% dicamba dimethylamine salt with the rest being other ingredients (mostly surfactants) which are undisclosed proprietary information. The relative proportion of the active ingredients however vary depending on the commercial formulation of Spectracide® and the purpose for which it is marketed. Atrazine (6-chloro-N-methyl-N 0-isopropyl-1,3,5-triazine-2,4-diamine) is the subject of ongoing controversy, with increasing documentation of its potentially harmful health and environmental impacts. Several reports suggest that atrazine toxicity could cause endocrine disruption in different organisms (Cocco, 2002; Cooper, 2000; Thibaut and Porte, 2004). Although the Environmental Protection Agency (EPA) in the United States approved its continued use in October 2003, the European Union (EU) announced a ban of atrazine that same month because of the ubiquitous and unpreventable water contamination (Sass and Colangelo, 2006). This raises concerns of possible toxic effects of continued use of atrazine-based herbicides on non-target organisms in the U.S. Several studies have focused on elucidating the effects of atrazine on the model organism Drosophila melanogaster. A proteomic analysis supported the role of atrazine exposure in D. melanogaster affecting the mitochondrial electron transport system and inducing oxidative stress (Thornton et al., 2010). Reduction in male reproductive performance (Vogel et al., 2015), decreased longevity (Marcus and Fiumera, 2016), and with alterations in behavior by disruption of the dopaminergic system (Figueira et al., 2017) have been reported in D. melanogaster following exposure to atrazine. Diquat dibromide is a bipyridal compound with documented genotoxic effects on Drosophila through its ability to generate reactive oxygen species (Gaiv~ ao et al., 1999; Kaya et al., 2000). Studies in beetles have shown that dicamba increased mortality and decreased body weight in females and also reduced the proportion of male’s in the population (Freydier and Lundgren, 2016). Fluazifop-p-butyl diminishes renal and hepatic functions and triggers testicular oxidative stress in orally exposed rats (Ore and Olayinka, 2017). Therefore, all four common ingredients of Spectracide® represent a risk factor for non-target organisms. However, most of such studies on toxicity of the active ingredients of herbicidal formulations to non-target organisms such as Drosophila focus on utilizing the pure chemical instead of the commercial formulations (Vogel et al., 2015; Marcus and Fiumera, 2016; Figueira et al., 2017; Vimal et al., 2019). While the four primary ingredients discussed have been demonstrated to be toxic to non-target organisms, their combined effects in a herbicidal formulation such as Spectracide® should be examined, because such a formulation with multiple toxic ingredients may likely cause more deleterious effects than a single compound (Larsen et al., 2003). The fruit fly D. melanogaster serves as an excellent model nontarget organism for assessing the toxicity of xenobiotics and it has been utilized previously as an effective tool in toxicological tests and facilitates scientific research in many areas (Rand, 2010;
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et al., 2020). Thus, in this study Peterson and Long, 2018; Bedna we focused on elucidating the effects of the herbicidal formulation

Spectracide® on D. melanogaster as a model non-target organism, focusing on its influence on general movement behavior, distinct diurnal locomotor activity rhythms, and oxidative damage to proteins. We also employed in silico modeling studies such as molecular-docking and molecular dynamics simulation to assess interactions of different ingredients of Spectracide® with carbonyl reductase, a key enzyme responsible for prevention of protein carbonyl formation - an indicator of oxidative damage to proteins. 2. Materials and methods 2.1. Drosophila rearing and Spectracide® exposure Wild type, yellow-body and white-eye strain (y w1118) of Drosophila melanogaster were reared on fly diet with a mixture of 1% agar, 6.25% cornmeal, 6.25% molasses, and 3.5% Red Star yeast at 25  C in 12 h light: dark (LD, 12:12) cycles (with an average light intensity of ~2000 lx). Healthy adult male and female flies, 3e5 days after eclosion, were collected and fed on filter paper saturated with 5% sucrose as control, or Spectracide® (Spectrum Group, Division of United Industries Corporation, St. Louis, MO) at concentrations of 5%, 25%, 50%, 75% in 5% sucrose and 100% (containing 5% sucrose) as treatments. The Spectracide® formulation used in the study contained 4% atrazine, 0.08% other related compound and 95.92% other ingredients (probably surfactants) which were not disclosed by the manufacturer. The other related compounds could be diquat dibromide, fluazifop-p-butyl and dicamba dimethylamine salt which are usually components of commercial formulations of Spectracide®. Three independent concentration-response experiments consisted of 25 flies (male or female) in three independent replicates. Based on the concentration-response studies in males and females separately (Supplemental Fig. S1), we chose 50% Spectracide® for further experiments, since it was representative of approximately the LC50 of both males and females combined. Since atrazine was the major component in the Spectracide® formulation we used in this study, we also conducted a concentration mortality response (Supplemental Fig. S2) with pure atrazine (PESTANAL® Millipore-Sigma, St.Louis, MO, USA). 2.2. Negative geotaxis and movement behavior assay Three different types of tests were performed to substantiate the detrimental effects of Spectracide® on fly movement behavior. The assays for movement behavior were performed at 0 and 24 h (the latter, only in those individuals which did not show signs of severe toxicity such as not moving or unresponsive). This time period was also taken as the optimal cut-off time to record sexually dimorphic movement responses of flies. 2.2.1. Vertical mobility assay The mobility of adult male and female flies was assessed using a negative geotaxis climbing assay (Inamdar et al., 2012) at 0 and 24 h during the course of herbicide exposure (50% Spectracide® in 5% sucrose solution). 10 flies (in 3 replications) were placed in an empty plastic vial, tapped to the bottom, and the time required to climb 5 cm was recorded three times sequentially with a 10 min rest period between each measurement. Each replication value recorded was an average of the three trials. The trials were repeated three times independently. 2.2.2. Movement deficit assay Movement deficits were also assessed by deviation from normal negative geotaxis behavior (Chaudhuri et al., 2007). 10 adult flies were placed in an empty plastic vial and tapped to the bottom. The number of flies able to climb 5 cm in 60 s were recorded. The score

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for each replication (three) was an average of three repeats. This trial was repeated three times independently. 2.2.3. Jumping behavior test The number of jumps in a given 10 s time period was counted to confirm the effects of Spectracide® on flying/skipping behavior. 10 flies in an empty vial (3 replications) were tapped 3 times and the number of jumps was counted. The experiment was repeated 3x with a 10 min rest in between. Three independent experiments were conducted and the data averaged across the experiments.

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activity was measured as described by Botella et al. (2004). Briefly, CR activity (total protein content of samples was at least 80 mg) was measured spectrophotometrically using a Synergy H1M plate reader (BioTek, Winooski, VT) in 100 mM sodium phosphate, 100 mM NaCl, pH 7.4, in the presence of 0.1 mM NADPH at 25  C. NADPH consumption (extinction coefficient at 340 nm 6.22 mM1 cm1) was monitored using 0.4 mM of p-nitrobenzaldehyde as substrate. One unit (U) of CR was defined as the amount of enzyme that catalyzed the consumption of 1 mmol NADPH per minute. Protein concentration was determined by bicinchoninic acid reagent using BSA as standard.

2.3. Locomotor activity analysis 2.5. Quantification of gene expression by qRT-PCR Young adult (3e5 day old) male and female flies were continuously fed with 50% Spectracide® in an agar-sucrose diet and subjected to locomotor activity analyses. Controls were fed only an agar-sucrose diet. An agar-sucrose diet was used in locomotor activity assays as a substitute for 5% sucrose since activity tubes required a solid food media instead of a liquid media. Locomotor activity was recorded for 3e5 days under LD 12:12 at 25  C using the DAM2 Drosophila Activity Monitor (TriKinetics Inc., Waltham, MA) as previously described (Rakshit et al., 2012; Rakshit and Giebultowicz, 2013). For quantitative measurement of circadian rhythmicity, Fast Fourier Transform (FFT) analysis was conducted using ClockLab software (Actimetrics Coulbourn Instruments, Whitehall, PA). Flies with FFT values < 0.04 were classified as arrhythmic, while FFT values > 0.04 were considered rhythmic. The experiments were repeated thrice with 15e20 individuals in each experiment. 2.4. Oxidative damage assessment 2.4.1. Protein carbonyl assay
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The amount of protein carbonyls was quantified (Bedna et al., 2017) in whole-body homogenates (25 flies in each replicate in three bio-replicates repeated thrice independently) after 24 h of Spectracide® treatment. Briefly, samples were homogenized in 50 mM K-PO4 buffer using a Kontes pestle homogenizer. 50 mL of 10% streptomycin sulfate was added to 500 mL of the homogenate to precipitate nucleic acids by incubating at room temperature for 15 min followed by centrifugation at 6000 g. Sample supernatants were separated into equal portions of samples derivatized after reaction with 7 mM 2,4 dinitrophenylhydrazine (DNPH) in 2 M HCl or controls treated only with 2 M HCl for 1 h in dark followed by sequential precipitation of protein with 28% and 5% TCA, washing with 1:1 ethanol: ethyl acetate mixture and resuspension of pellets in 6 M guanidine hydrochloride. Equal aliquots of samples (derivatized with DNPH) and parallel controls (treated with only 2 M HCl) were then pipetted onto multi-well plates and absorbance read at 370 nm and 280 nm. The results were expressed as nmol.mg1 protein using an extinction coefficient of 22,000 M1 cm1 at absorbance maxima of 370 nm in a H1M Synergy plate reader (BioTek, Winooski, VT). A bovine serum albumin (BSA) standard curve was used for protein concentrations in guanidine solutions (Abs 280 nm). Protein carbonyl values were corrected for interfering substances by subtracting the Abs 370 nm mg1 protein measured in control samples. Data is represented as mean of three independent experiments. 2.4.2. Carbonyl reductase enzyme assay Whole body homogenates of flies (25 flies per replicate in three replicates, with three independent repeats) untreated and treated with Spectracide® were collected 24 h after treatment and prepared in 100 mM sodium phosphate buffer. Carbonyl reductase (CR)

The expression of Sniffer (Sni) gene was quantified in whole body tissues of control and Spectracide®-treated flies (25 flies per replicate in three replicates with three independent repeats) collected after 24 h of treatment. Total RNA was extracted from whole fly using TriReagent (Sigma-Aldrich, Saint Louis, MO). Samples were treated with Takara Recombinant DNAse I (Clonetech Laboratories Inc., Mountain View, CA) followed by cDNA synthesis with iScript cDNA synthesis kit (BioRad, Hercules, CA). Quantitative real-time PCR (qRT-PCR) was performed on an Eppendorf realplex2 Mastercycler (Eppendorf, Hauppauge, NY) using the following thermal cycling conditions: a hot start at 95  C for 10 min, with denaturation at 95  C for 15 s with primer annealing at 59  C for 20 s and extension at 72  C for 30 s (repeated 40 times) with data acquisition at end of extension step. This was followed by a dissociation curve step and melt curve analysis. Every reaction contained Power SYBR Green (Applied Biosystems, Carlsbad, CA), 10 ng cDNA and 400 nM primers. The primer sequences used for Sni gene were Forward: 50 -AAC TCG ATC GCG GAT CTT CGG-30 Reverse: 50 -ATC CTC GAT TGC AGC CGG TTA TCA-30 . Data were analyzed using the 2-DDCT with mRNA levels normalized to the housekeeping gene rp49 (Forward: 50 -ACG TTG TGC ACC AGG AAC TT-30 Reverse: 50 -CCA GTC GGA TCG ATA TGC TAA-30 ) (Hanna et al., 2015). Relative expression was calculated with respect to pooled data from untreated flies set as 1. 2.6. In silico binding analysis of toxic compounds with carbonyl reductase by molecular docking and molecular dynamics simulation studies 2.6.1. Molecular docking The crystal structure of Drosophila Sniffer protein, a carbonyl reductase with a resolution of 1.75 Å was retrieved from the Protein Data Bank (PDB ID: 1SNY) (Sgraja et al., 2004). All water molecules were removed from the crystal structure and hydrogen atoms added to induce the proper ionization state to the protein. The structure of four toxic ingredients atrazine, diquat, fluazifop-pbutyl and dicamba were retrieved from PubChem database with CID 2256, 6794, 3033674, 3030 respectively. The ligands were prepared by adding hydrogen atoms to standardize charges and generating 3D coordinates. The protein and the ligands were typed with CHARMM forcefield (Feller and Mackerell, 2000; Mackerell et al., 1998) before docking studies. The possible binding sites were identified based on the shape of the active site cleft of carbonyl reductase. The binding mode of all four ligands to carbonyl reductase was investigated by the CDOCKER program (Wu et al., 2003) implemented in Discovery Studio 3.5 (Accelrys Discovery Studio). Ten molecular docking poses of each ligand were ranked according to CDOCKER energy. Finally, energy minimization of the docked receptor-ligand complexes was performed for interaction analysis. The interaction energy between each ligand and carbonyl

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reductase was analyzed using the Calculate Interaction Energy protocol. This protocol allows calculation of the non-bonded interactions i.e. van der Waals and electrostatic contribution between two sets of atoms in a specific structure. Before calculating the interaction energy, the receptor-ligand complexes were typed with CHARMM force field. 2.6.2. Molecular dynamics simulation Molecular dynamics simulation is used to predict the structural stability and dynamic behavior of the protein-ligand complexes. The four best-predicted carbonyl reductase-herbicide complexes were subjected to MD simulation with solvation systems. The four systems were solvated with the TIP3P water (Jorgensen et al., 1983) molecules in a spherical boundary condition. The four complexes were neutralized by ions such as Naþ and Cl-. The solvated carbonyl reductase-herbicide complexes were first minimized using 200 cycles of steepest descent (SD) followed by 500 cycles of the conjugate gradient (CG) method until the root mean deviation (RMS) gradient was <0.05 kcal/mol/Å2. This was prepared to remove any short-range bad contacts present in the docked complex structures. The minimized structures were then subjected to heating from 50 K to 300 K for 100 ps and equilibrated for 200 ps. A spherical cut-off method was used to calculate long-range electrostatic interactions (Steinbach and Brooks, 1994). Non-bond higher and lower cut-off distances were maintained at 12 Å and 10 Å respectively for electrostatics and van der Waals interactions. After equilibration, four carbonyl reductase-herbicide complexes were simulated for 5 ns at 300 K with a time step of 0.002 ps for the production run. This was done by the NPT ensemble (constant Number of particles, Pressure, and Temperature). During the simulation, all covalent bonds involving hydrogens were constrained using the SHAKE algorithm (Ryckaert et al., 1977). 2.6.3. Virtual alanine mutagenesis In order to identify the contribution of key amino acid residues at the binding site of the carbonyl reductase-herbicide complexes, in silico alanine scanning mutagenesis study was performed (Kortemme and Baker, 2002). Calculations were performed for four docked complexes to derive hotspot residues at the interface region. This process evaluates the effect of a single point mutation at the binding site region of the protein complexes by mutating a set of interacting amino acids to alanine. 2.7. Statistical analyses Each experiment was repeated thrice independently, and each repeat had at least three replicates of samples. Student’s unpaired ttest was conducted for all analyses. All statistical tests were conducted using GraphPad Prism Instat v3.0 (GraphPad Software Inc., San Diego, CA). Graphs were generated using GraphPad Prism 6 v 6.07 (GraphPad Software, La Jolla, CA www.graphpad.com). 3. Results 3.1. Spectracide® alters negative geotaxis, jumping behavior and daily locomotor activity We tested different locomotor behavioral activity using a selective concentration of 50% of Spectracide® in 5% sucrose solution. Control flies were fed 5% sucrose solution (vehicle only). Negative geotaxis (vertical climbing) and fly movement was significantly decreased in both male and female flies fed with Spectracide® solution. Herbicide-fed flies took significantly more time to cross 5 cm distance against gravity compared to controls irrespective of sex at 24 h after continuous exposure to herbicide (Fig. 1A).

Moreover, the number of flies crossing the 5 cm distance at 24 h was lesser in herbicide -fed groups (66.2% in males and 56% in females) compared to controls where almost all flies crossed the 5 cm mark (Fig. 1B). This diminished fly activity with time upon exposure to Spectracide® was reflected in the percentage of flies climbing 5 cm distance (Fig. 1B) and the number of jumps (Fig. 1C). In sucrose-fed controls, all the flies were able to climb 5 cm distance while this was significantly reduced in males and females after 24 h of Spectracide® exposure. Control female flies jumped 5e8 times, while males jumped ~2e6 times in a given 10 s. The number of jumps drastically reduced to 1e2 times at 24 h in both the male and female flies when fed with Spectracide®. 3.2. Spectracide®-induced disruption in diurnal activity and fly rhythmicity We recorded locomotor activity rhythms for 3e5 days at 15 min intervals under continuous exposure to 50% Spectracide® in 5% sucrose solution. The diurnal pattern of locomotor activity was completely disrupted in both males and females exposed to 50% Spectracide®, as indicated by double-plotted actograms (Fig. 2A) and corresponding periodograms (Fig. 2B). While control flies of both sexes demonstrated bimodal peaks of activity at lights on and off in LD 12:12, these peaks were obliterated in Spectracide® treated flies and there was no noticeable difference between activity in the light and dark phases (Fig. 2C). There was a significant decrease in the strength of activity rhythms (p < 0.05) as indicated by Fast Fourier Transform (FFT) values (Fig. 2D). Flies with FFT values < 0.04 were considered arrhythmic, while >0.04 were categorized as rhythmic. Corresponding to the FFT values, a majority of Spectracide® treated flies of both sexes lost their circadian rhythmicity and there were only 0e8% rhythmic flies, compared to 89e100% rhythmic controls (Fig. 2E). In addition to loss of rhythmicity, Spectracide® treated flies also showed a significant reduction in total activity counts by day 3 compared to controls (data not shown). 3.3. Exposure to Spectracide® induces oxidative damage to proteins The formation of protein carbonyls is a key biomarker for oxidative damage to proteins. We recorded a significant increase (p < 0.001) in protein carbonyl formation in whole body mass of adult flies within 24 h of Spectracide® exposure (130.2% in males and 139.6% in females) (Fig. 3). There was also a significant reduction (p < 0.001) of carbonyl reductase enzyme activity in Spectracide® treated male (53.88%) and female (50.52%) flies. Reduced carbonyl reductase enzyme activity corresponds to a significant decline (63% in male and 62% in female) in the expression of carbonyl reductase gene Sniffer (Sni) in Spectracide® treated flies, confirming the presence of herbicide-induced oxidative damage to proteins in the fly model. 3.4. Molecular interactions and stability of toxic compounds within the active site cleft of carbonyl reductase 3.4.1. Molecular docking To confirm the correct binding mode and ensure geometric fit, four active ingredients of Spectracide® were docked to the binding site of carbonyl reductase (CR). The lowest CDOCKER energy conformation was selected from the ten docking conformations of each receptor-ligand complex (Supplemental Table S1) to analyze intermolecular interactions. The molecular docking of atrazine into carbonyl reductase revealed the CDOCKER energy of 47.4191 kcal/ mol and formed two hydrogen bonds with Val203 and Thr205. The non-covalent van der Waals contribution present within the

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Fig. 1. Behavioral analyses of male and female Drosophila melanogaster (y w 1118) following continuous exposure of intermediate concentration of Spectracide® (50%) in 5% sucrose solution. Control flies were fed vehicle only (5% sucrose). Bar graphs representing (A) Negative geotaxis assay that determines time taken by flies to cross 5 cm distance, (B) Percentage of flies that were able to climb 5 cm distance within 60 s, and (C) Number of jumps in 10 s at 0 and 24 h. Each bar represents mean ± SEM of 3 independent experiments, each experiment consisting of three biological repeats (n ¼ 8e10 flies/replication) conducted at 10 min intervals. Statistical significance was calculated using unpaired t-test and is denoted by *p < 0.05.

binding pocket of carbonyl reductase and atrazine were contributed by Arg12, Gly13, Gly15, Asn90, Gly92, Mse152, Ser154, Pro200, Trp202 and Asp206 residues, whereas residues Leu14, Ser153, Tyr170, Lys174, Gly201, Val203, Thr205, Mse207, and Gly208 were responsible for electrostatic interactions (Fig. 4A and B). The second ligand, diquat molecule was encompassed into the binding site of sniffer protein with CDOCKER energy of 0.0816 kcal/mol. The

residues involved in van der Waals interactions were Leu14, Asn90, Ala91, Gly92, His99, Mse152, Ser153, Ser154, Ile155, Tyr170, Lys174, Pro200, Gly201, Trp202, Val203, Thr205 and Gly208 whereas residue Mse207 alone was involved in electrostatic interaction. The predominance of hydrophobic residues involved in non-covalent interactions suggests the contribution of van der Waals interaction in imparting stability to the diquate-carbonyl reductase

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Fig. 2. Circadian rhythm and daily activity pattern of male and female Drosophila melanogaster (y w 1118) following continuous exposure of intermediate concentration of Spectracide® (50%) in 5% sucrose solution. Control flies were fed with vehicle only (5% sucrose). Both male and female flies were monitored in LD 12:12 for 3e5 days at 25  C. (A) Doubleplotted actograms representing locomotor activity rhythms of male and female flies during continuous exposure to Spectracide®. Shaded areas represent periods of darkness and indicate time of lights off (ZT 12). (B) Corresponding chi-squared periodograms representing bimodal distribution of activity peaks around lights on and off in control flies as expected and diminished peaks in Spectracide®-treated flies. (C) Bar graphs representing average daily activity during a 24 h cycle. Shaded bars represent period of darkness. Each bar is mean ± SEM of three independent experiments (n ¼ 15e20 for each experiment; average of 3e5 days depending on the sex and fly survival). (D) Rhythm strength and (E) Percentage of rhythmic flies was measured using Fast Fourier Transform (FFT) values. Flies with FFT values < 0.04 were considered arrhythmic. Bars represent mean ± SEM of three independent experiments (n ¼ 15e20, for each experiment). Statistical significance was calculated using Student’s unpaired t-test and is denoted by *p < 0.05.

complex (Fig. 5A, B). The third ligand, Fluazifop-p-butyl compound was enclosed within the binding cavity of carbonyl reductase forming pi-cation interaction with Arg12. The non-covalent interactions present in the binding pocket of carbonyl reductase with Fluazifop-p-butyl were from Gly13, Leu14, Gly15, Asn90, Ala91, Gly92, Ile93, Mse152, Ser154, His199, Pro200, Gly201, Trp202, Val203, Thr205 and Gly208 residues for van der Waals and Gly9,

Asn11, Arg12, Arg37, Ser153, Tyr170, Lys174 and Mse207 residues for electrostatic interactions (Fig. 6A, B). Finally, the binding mode of dicamba at the active site of carbonyl reductase showed various interactions which include hydrogen bonding, van der Waals and electrostatic interactions with key residues in close proximity to the ligand molecule with CDOCKER energy of 18.2918 kcal/mol. It forms two hydrogen bonds with amino acids Asn90 and Lys174 of

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Fig. 3. Accumulation of protein carbonyls, relative Sni mRNA expression and carbonyl reductase activity in fly tissues following continuous exposure of an intermediate dose of Spectracide® (50%) in 5% sucrose solution. Control flies were fed with vehicle only (5% sucrose). Bars represent mean ± SEM three independent experiments with each experiment consisting of 3 replications (n ¼ 75 per experiment). Statistical significance was calculated using Student’s unpaired t-test and is denoted by *p < 0.05.

Fig. 4. Interaction between carbonyl reductase (PDB ID: 1SNY) and atrazine as predicted by molecular docking. A. Close view of docked complex. Carbonyl reductase and atrazine are represented as cartoon and stick model respectively. B. 2D representation of intermolecular interaction. Residues involved in electrostatic and van der Waals interactions are represented by pink and green circles, respectively. Green and blue dashed lines show main-chain and side-chain hydrogen bond interactions respectively. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5. Interaction between carbonyl reductase (PDB ID: 1SNY) and diquat as predicted by molecular docking. A. Close view of docked complex. Carbonyl reductase and diquat are represented as cartoon and stick model respectively. B. 2D representation of intermolecular interaction. Residues involved in electrostatic and van der Waals interactions are represented by pink and green circles, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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Fig. 6. Interaction between carbonyl reductase (PDB ID: 1SNY) and fluazifop-p-butyl as predicted by molecular docking. A. Close view of docked complex. Carbonyl reductase and fluazifop-p-butyl are represented as cartoon and stick model respectively. B. 2D representation of intermolecular interaction. Residues involved in electrostatic and van der Waals interactions are represented by pink and green circles, respectively. Orange line represents pi-cation interaction. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

carbonyl reductase. The non-covalent interactions present in the binding pocket of carbonyl reductase with dicamba were from Gly13, Ala91, Ile93, Mse152, Ser154, Pro200, Val203, Mse207 and Gly208 residues for van der Waals and Leu14, Asn90, Gly92, Ser153, Tyr170, Lys174 and Thr205 residues for electrostatic interactions (Fig. 7A, B). 3.4.2. Molecular dynamics simulation The lower energy conformations from docking analysis of the four active ingredients at the binding site cavity of the receptor carbonyl reductase were selected as starting points for MD simulation. Molecular dynamics simulation was used to investigate the dynamic behavior and stability of the four active ingredients of Spectracide® within the active site cleft of the carbonyl reductase enzyme. All the four complexes were subjected to 5ns MD simulations with explicit solvation system. The structural stability of all the receptor-ligand complexes was investigated by calculating the trajectory of the potential energy (kcal/mol) and the RMSD (Å)

values of the protein backbone atoms. The potential energy profile of the four complexes was ranged from 17500 kcal/mol to 18150 kcal/mol and remained stable throughout the 5 ns simulation run (Supplemental Fig. S3A). The average RMSD values ranged from 1.3 Å to 1.7 Å (Supplemental Fig. S3B). An overall trend of potential energy profiles, backbone RMSD (below 2 Å), for four complexes indicated that all systems were well equilibrated and stable during the simulation run. 3.4.3. Virtual alanine scanning Virtual alanine scanning mutagenesis study assists to identify hotspot residues at the interface region of the protein-ligand complex. It performs amino-acid scanning mutagenesis on a set of interacting residues by mutating them to alanine. The binding affinity was determined in terms of mutation energy which is calculated by the difference between the binding free energy of the mutated structure and the wild type protein. Some specific amino acids at the binding region of the carbonyl reductase-herbicide

Fig. 7. Interaction between carbonyl reductase (PDB ID: 1SNY) and dicamba as predicted by molecular docking. A. Close view of docked complex. Carbonyl reductase and dicamba are represented as cartoon and stick model respectively. B. 2D representation of intermolecular interaction. Residues involved in electrostatic and van der Waals interactions are represented by pink and green circles, respectively. Green and blue dashed lines show main-chain and side-chain hydrogen bond interactions respectively. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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complexes show destabilizing effect (mutation energy>0.5 kcal/ mol) upon alanine mutation. The residues Gly13, Leu14, Asn90, Gly92, Tyr170, Lys174, and Thr205 of carbonyl reductase play a significant role in the stability of carbonyl reductase during the complex formation with the four investigated ingredients of Spectracide® (Supplemental Table S2).

4. Discussion The use of pesticides and/or herbicides is an anthropogenic addition to the ecosystem. Benefits of herbicide use has fueled the manufacture and sale of various formulations purportedly targeting only weeds and has also given rise to genetically engineered crops which are herbicide resistant or tolerant (Kniss, 2018). Research in the past couple of decades has highlighted the possible serious side-effects of some pesticides/herbicides on non-target organisms including human health (Nicolopoulou-Stamati et al., 2016). Herbicidal formulations (specifically atrazine-based) are one of the most common environmental contaminants in the U.S. due to their ubiquitous use (Solomon et al., 1996; Scribner et al., 2000; Thurman and Cromwell, 2000; Jablonowski et al., 2011). Atrazine-based herbicidal formulations have been documented to negatively affect a variety of non-target organisms in both aquatic and terrestrial ecosystems, and a mechanistic understanding of detrimental effects of atrazine exposure has started to emerge from a large body of work (Cheremisinoff and Rosenfeld, 2011). In the present study we conducted an in-depth locomotor activity analysis and observed that exposure to the Spectracide® herbicidal formulation significantly dampens the circadian rhythm of locomotor activity in D. melanogaster. This corroborates the observation that exposure to pure atrazine can result in altered behavior in D. melanogaster (Figueira et al., 2017). We also conducted a concentration-response study with pure-atrazine and the LC50 was about 3.3 mg/ml for Drosophila (Supplemental Fig. S2). However, in the final herbicidal formulation Spectracide®, the proportion of atrazine is 4% whereas the other components (diquat dibromide, fluazifop-p-butyl and dicamba dimethylamine) account for 0.08% and exact concentration is proprietary information. The toxic effects of any surfactant (about 95.92%) in the final formulation also
et al., cannot be discounted as demonstrated recently (Bedn
arova 2020). Spectracide® formulations offer a ready to spray option such that the herbicidal formulation is currently recommended to be used at 100%, whereas in our study we observed detrimental effects at half the concentration. Given that exposure to pure atrazine causes circadian locomotor deficits, it raises the question if atrazine-based herbicidal formulations impact the molecular clock mechanism disrupting the rhythm in activity or if they disrupt the dopaminergic system as reported earlier (Figueira et al., 2017) and consequently affect general locomotor activity. This remains to be investigated. Oxidative stress is arguably the most common mechanism in the toxicology of environmental agents. We focused on protein carbonylation as a read-out/biomarker of oxidative stress. Carbonylation of proteins is an irreversible post-translational modification that often leads to the loss of protein function and is a hallmark of oxidative stress resulting in oxidative damage (Stadtman, 2004). Proteins have many reactive sites that can be modified or damaged during oxidative stress. Modification of proteins then leads to the formation of carbonyl derivatives by direct oxidation of certain amino acid side chains and oxidation-induced peptide cleavage (Stadtman, 2004). The accumulation of carbonyl groups generated by many different mechanisms is thus a good measure of reactive oxygen species-mediated protein oxidation. Atrazine based herbicidal use since 1950’s has also increased the

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potential for environmental contamination because of its ability to persist for almost 3 weeks post application (de Albuquerque et al., 2020). Also, it has been demonstrated earlier that atrazine exposure causes oxidative stress in D. melanogaster (Figueira et al., 2017). Similarly, diquat is used in agriculture as a plant growth regulator for the control of broadleaf and grassy weeds in terrestrial environments and aquatic areas where it is highly soluble in water and is detected as residues in the environment (Siemering et al., 2008). Moreover, the high propensity of diquat to propagate reactive oxygen species (ROS) is a predominant mechanism underlying diquat-induced cellular damage in different cell models (Slaughter et al., 2002; Zhang et al., 2012; Nisar et al., 2015). Similar effects of fluazifop-p-butyl and dicamba have been reported in non-target organisms (Lackmann et al., 2018; de Arcaute et al., 2019). In our study, we observed that exposure to Spectracide® significantly elevates protein carbonyl content. The association between oxidative damage to proteins and its impact on motor ability was shown in mice (Forster et al., 1996). Protein oxidative damage in cerebellum of mice was positively correlated with loss of motor ability. We speculate that exposure to Spectracide® induces oxidative stress which results in oxidative damage to proteins leading to a decline in locomotor activity rhythms. Further studies targeting motor neurons which would conclusively demonstrate the oxidative damage is necessary. On the other hand, oxidative stress induced by herbicide exposure can also target dopaminergic neurons which may impact locomotor behavior (Chaudhuri et al., 2007). It has been shown that the gene Sniffer, encoding a short-chain dehydrogenase/reductase (SDR) family member is essential for protecting neurons from oxidative stress-induced neurodegeneration (Botella et al., 2004). We found in our study that exposure to Spectracide® caused a significant decline in Sni gene expression as well as its activity. Thus, down-regulation of carbonyl reductase (CR) gene expression and activity would lead to a concomitant increase in protein carbonyl levels as seen in our study. In silico analysis was conducted to correlate the role of active ingredients of Spectracide® in inhibition of carbonyl reductase (CR) enzyme activity. Thus, in order to unravel molecular underpinnings of the interaction between the active ingredients of the herbicidal formulation Spectracide®, we conducted molecular docking simulation studies of the four common ingredients of Spectracide® to the binding site cleft of the target protein carbonyl reductase (CR). The conformations of the docked complexes were analyzed by several parameters such as interaction energy, hydrogen bonding and non-covalent interactions between four ingredients of Spectracide® and CR. The strong binding affinity of Spectracide® with the CR corroborate with the experimental results. The interaction energy of CR with atrazine, diquat, fluazifop-p-butyl and dicamba were 21.0261 kcal/mol, 82.25588 kcal/mol, 14.6198 kcal/mol and 5.2392 kcal/mol respectively. It is clear from the interaction analysis that the atrazine compound strongly binds with Sniffer protein, CR than the other ingredients in Spectracide®. The results of MD simulation suggested that all the ingredients of Spectracide® may bind to the active site cavity of the CR enzyme. The important residues that were identified from the virtual alanine study may act as target site for future pharmacological studies. 5. Conclusions Taken together, our study shows that exposure to Spectracide® significantly affected movement behavior and locomotor activity rhythms in Drosophila. This raises the possibility that such herbicidal formulations may likely impact the molecular clock network and may have implications for non-target organisms including humans. However, this remains to be demonstrated. Molecular

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A. Chaudhuri et al. / Chemosphere 248 (2020) 126037

modeling studies indicated a strong binding affinity of atrazine (one of the active ingredients of Spectracide®) to the active site of the protein carbonyl reductase whose activity was inhibited upon exposure to Spectracide®, leading to elevated levels of oxidatively damaged proteins. The potential for such herbicides and the active ingredients alone or in combination to specifically damage motor neurons and clock neurons in non-target organisms warrants further investigation. Author Credit Statement Ankur Chaudhuri: Molecular docking studies, in silico analysis, Roishinique Johnson: Concentration-response and Behavioral assays, Kuntol Rakshit: Data curation, graphs and computational analysis, locomotor activity analysis, writing-reviewing editing,
rov
Andrea Bedna a: Concentration-mortality response validation, Biochemical assays and gene expression analysis, Kimberly Lackey: Writing-reviewing editing, Sibani Sen Chakraborty: Molecular docking studies, in silico-analysis, Natraj Krishnan: Conceptualization, methodology, writing-reviewing editing Anathbandhu Chaudhuri: Supervision, conceptualization, original draft preparation, data curation. Declaration of competing interest The authors declare that they have no conflict of interest. Acknowledgements This study was supported in part by funds from a Title III grant, United States Department of Education, Stillman College, USA (AC), the Office of Research and Economic Development, Mississippi State University, USA (NK). AB acknowledges Grant no. CZ.02.2.69/ 0.0/0.0/18_070/0008772 from the European Structural and Investing Funds Operational Programme Research, Development and Education, EU-CZ. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2020.126037. References Accelrys Discovery Studio Visualiser V 3.5.0.12158, San Diego: Accelrys Software Inc.
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