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Role of acetylcholinesterase and glutathione S-transferase following exposure to nicosulfuron and diazinon in Helicoverpa zea
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Ecotoxicology and Environmental Safety 71 (2008) 230–235 www.elsevier.com/locate/ecoenv
Role of acetylcholinesterase and glutathione S-transferase following exposure to nicosulfuron and diazinon in Helicoverpa zea Raffaella Massa, Stefania Blevins, Shirley L. Chao Department of Natural Sciences, Fayetteville State University, Lyons Science Bldg. Rm. 330, 1200 Murchison Road, Fayetteville, NC 28301, USA Received 22 March 2007; received in revised form 31 July 2007; accepted 22 August 2007 Available online 23 October 2007
Abstract The acute toxicity of diazinon combined with the herbicide nicosulfuron was determined using the corn earworm, Helicoverpa zea. Nicosulfuron significantly increased toxicity of diazinon to H. zea compared to diazinon alone based on dip bioassays to evaluate acute contact toxicity. Diazinon, an organophosphorous insecticide, controls insects by inhibiting acetylcholinesterase in the nervous system. Acetylcholinesterase activity was significantly altered in corn earworms exposed to binary mixtures compared to those exposed to diazinon alone; however, the activity did not correspond consistently with toxicity. Glutathione S-transferase (GST), also known to be altered by organophosphorous insecticides, did not exhibit significant changes following exposures to diazinon, nicosulfuron, or binary mixtures. Our results suggest that nicosulfuron increases toxicity of diazinon but the mechanism of toxicity does not appear to be correlated with acetylcholinesterase or GST inhibition. Published by Elsevier Inc. Keywords: Helicoverpa zea; Nicosulfuron; Diazinon; Acetylcholinesterase; Synergism; Glutathione S-transferase; Insecticide; Herbicide
1. Introduction Diazinon [O,O-diethyl-O-(2-isopropyl-6-methyl-4-pyrimidinyl) phosphorothioate] is a non-systemic organophosphate insecticide used in agricultural systems to control a wide variety of sucking and leaf-eating insects. It is applied on a variety of crops including corn, fruit trees, blueberries, lettuce, and tomatoes and on non-lactating cattle as an eartag. Residential product registrations have been canceled since 2004 due to unacceptable risks posed to agricultural workers, birds, and the environment. Despite cancellations, approximately 13 million lbs of active ingredient are used annually with rates of up to 4 lb active ingredient/acre (US EPA, 2004a). Diazinon is metabolized into an active oxon derivative, inhibiting acetylcholinesterase (AChE), an enzyme that catalyzes the hydrolysis of acetylcholine. The inhibition of AChE results in the accumulation of the neurotransmitter acetylcholine within cholinergic synapses, leading to Corresponding author. Fax: +1 910 672 1159.
E-mail address: [email protected] (S.L. Chao). 0147-6513/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.ecoenv.2007.08.014
synaptic blockage and disruption of signal transmission. Metabolism to its active oxon metabolite involves oxidative desulfuration whereas detoxification of diazinon involves dearylation. The balance between the rates of desulfuration and dearylation can result in very different levels of AChE inhibition (Larkin and Tjeerdema, 2000; Timchalk, 2001). Nicosulfuron [3-pyridinecarboxamide, 2-((((4,6-dimethoxypyrimidin-2-yl) amino carbonyl) aminosulfonyl))N,N-diethyl] is a member of the sulfonylurea (SU) family of herbicides and is used to control weeds on corn (US EPA, 2004b). Nicosulfuron was chosen for this study since it is registered for use on corn similar to diazinon and is applied alone or in formulation with other active ingredients to control annual and perennial grasses and broadleaf weeds with a maximum application rate of 0.06248 lb active ingredient/acre. The highest use of nicosulfuron is on corn and approximately 200,000 lb are used annually (US EPA, 2004b). Sulfonylurea along with other herbicides such as sulfonamide, and imidazolinone function by inhibiting the plant enzyme acetolactate synthase (ALS), stopping plant growth and eventually killing the plant (Battaglin
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et al., 2000). The ALS enzyme is found only in plants and not present in humans or other animals. Inhibition of the ALS enzyme system blocks the production of amino acids valine and isoleucine, essential building blocks of proteins and other plant components (US EPA, 1990). Corn earworms (Helicoverpa zea) were tested with these chemicals due to their economical importance for farmers. The earworm, also known as tomato fruitworm and cotton bollworm, is among the most destructive insect pests in the southern United States. The corn earworm infests over 100 plant species, but corn is the preferred host. First generation caterpillars attack the whorl stage, while the second generation is largely found in corn ears. They feed on a wide variety of crops including beans, peas, sweet corn, okra, tomatoes, cabbage, eggplant, and peppers. Damage caused by the corn earworm ranges from destruction of the host crop to cosmetic damage that may cause a crop to be unmarketable. These injuries can result in an unusable product for both the fresh and processing market (Fitt, 1989). Measurements of AChE activity have been used routinely as a biomarker of exposure to certain groups of contaminants, such as organophosphate and carbamate insecticides (Grue et al., 1997). Low concentrations of organophosphates are known to disrupt transmission in the central and peripheral cholinergic nervous system by inhibiting AChE (Hoy et al., 1991). In vertebrates, two kinds of cholinesterases are common. AChE is found only in erythrocytes and nervous and muscle tissue. If the AChE is inhibited significantly, the animal dies. Conversely, pseudocholinesterase is found in a number of tissues, including blood plasma, liver, and nervous tissue, and can be inhibited with no apparent effect on the animal (O’Brien, 1976). Interestingly, nicosulfuron has been found to inhibit AChE in the brain of goldfish up to 48 h in response to 50 mg/L. However, nicosulfuron exposure had no effect on AChE activity in the muscle of goldfish (Bretaud et al., 2000). In insects, AChE is abundant in nervous tissue. Unlike vertebrates, neuromuscular junctions in insects have shown clearly that there is no cholinesterase there and that transmission at the synapse does not include acetylcholine. AChE is confined to the ‘‘central’’ nervous system of insects (O’Brien, 1976). Glutathione S-transferase (GST) is a major detoxification factor in insects and dramatically increases the water solubility of metabolites of many pesticides. The most common metabolic resistance mechanisms for organophosphorous insecticides involve esterases, GSTs, or monooxygenases (Hemingway, 2000). Elevated GST levels in insects can produce resistance to a range of insecticides by conjugating reduced glutathione to the insecticide or its primary metabolic products. Because GSTs may also act together with an esterase-based mechanism (Hemingway, 2000), we explored the possibility of altered GST as a mechanism of toxicity for the binary mixtures of diazinon and nicosulfuron.
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Insecticides and herbicides are widely used in commercial and private use to control insect pests and weeds. The range of doses that results in toxic effects varies widely with formulation and with the individual species being exposed. Combined exposure of organophosphates and sulfonylurea herbicides are common in corn. No published results have yet been reported on toxicity of chemical mixtures such as nicosulfuron and diazinon. Based upon a possible shared mechanism of toxicity via the inhibition of AChE, one must consider the cumulative risk associated with organophosphate pesticide mixture exposures. In particular is a need to explore pesticide mixtures involving herbicides such as nicosulfuron that exhibit a possible shared mechanism of action with organophosphate insecticides. 2. Materials and methods 2.1. Chemicals Technical grade nicosulfuron, 3-pyridinecarboxamide, 2-((((4,6-dimethoxypyrimidin-2-yl) amino carbonyl) aminosulfonyl))-N,N-diethyl (75% purity) was generously donated by DuPont (Wilmington, DE). Technical grade diazinon, O,O-diethyl-O-(2-isopropyl-6-methyl-4-pyrimidinyl phosphorothioate (95% purity), was purchased from ChemService (West Chester, PA).
2.2. Animals Third instar larvae of H. zea and finished diet were purchased from the Department of Entomology at North Carolina State University. Corn earworms were maintained in the following conditions: photoperiod of 14 h light, 10 h darkness, temperature of 80 1F (27 1C) and at least 60% RH. The diet consisted of the following: 100 g wheat germ, 45 g (vitamin free) casein, 40 g sugar, 30 g Torula yeast, 15 g Wesson salt mix, 4 g L-ascorbic, 1.5 g sorbic acid, 1 g methyl paraben, and 0.5 g cholesterol. All the ingredients, once weighed out, were ground (in a rolling grinder or food processor) to break down the wheat germ and the ingredients were blended together to a fine consistency. All larvae, before and after treatments, were maintained on above diets until they were frozen for future assays.
2.3. Dip bioassay Third instar larvae H. zea were exposed to concentrations (1, 2, and 3 mg/L) of diazinon and nicosulfuron (22.5 mg/L) within 1–2 h upon their arrival. Groups of 10–12 H. zea were placed in cheesecloth and dipped for 10 s in the following test solutions: (1) nicosulfuron (22.5 mg/L) diluted in water, (2) three concentrations of diazinon: 1, 2, and 3 mg/L of diazinon in water, and (3) binary mixtures of diazinon concentrations (1, 2, and 3 mg/L) in combination with 22.5 mg/L nicosulfuron. Control larvae were dipped in water for 10 s at the same time as other treatment groups. The larvae were then removed from cheesecloth and placed in individual Petri dishes containing their diet. Mortality was determined 24 h after dipping worms in test solutions. All larvae were weighed and stored in 20 1C for further protein and enzyme analysis. Each treatment group was replicated three times and the bioassays were repeated twice.
2.4. Protein assay Total protein was determined by the Bradford method (Sigma Chemical Co., 1995) using Bovine Serum Albumin (Sigma) as the standard and the Bradford dye reagent (BioRad). Frozen whole bodies of third instar corn earworm larvae were weighed, thawed, and
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homogenized in buffer composed of: 0.2 M standard phosphate buffer at pH 7.4, 10% glycerol, and 1.0 mM EDTA. Tissues were homogenized using a polytron (Powergen, Fisher) for approximately 45 s. Tissues remained on ice during homogenization. The homogenate was then centrifuged for 5 min at 12,000g. Approximately 50 mL of the supernatant was then added to the Bradford dye, mixed, incubated for approximately 30 min to 1 h, and absorbance was read at 595 nm. The amount of absorbance was proportional to the amount of protein present.
benzene) were added at 4.33 mM and 1.12 mM, respectively. The negative control, a control sample without the substrate added, was also tested with treated samples. The total volume for this assay was 3 mL containing: 30 mL of supernatant from homogenized samples (approximately 40–100 mg of protein), 15 mL of CDNB, 130 mL of glutathione, and 0.1 M Tris–HCl at pH 8.0. Absorbance was read at 340 nm at the following time points: 2, 4, 6, and 8 min. Statistical analysis involved examining the main effects of the reaction time course and treatment in an overall ANOVA (SAS Institute, 2002–2003).
2.5. Acetylcholinesterase assays
2.6. Statistical analysis The results are presented as the mean of at least three separate experiments of at least three replicates for each experiment7standard error (S.E.). The main effects examined were reaction time course and treatment in an overall ANOVA (SAS Institute, 2002–2003) for mortality and AChE activity data. To ascertain which time points were different from one another by treatment, post hoc Fisher’s multiple comparisons were conducted with significant effects determined at po0.05.
2.7. Glutathione S-transferase determination The supernatants used for the protein assay were also used for determining GST activity. Glutathione and CDNB (1-chloro-2,4-dinitro-
3. Results Fig. 1 represents the percent of mortality of H. zea following exposure to three concentrations (1, 2, and 3 mg/L) of diazinon alone and in combination with nicosulfuron at 22.5 mg/L. Significant increases in mortality were observed after exposure with binary mixtures compared to diazinon alone. Following exposure to 1 mg/L diazinon, an average of 21% (73.1 S.E.) mortality resulted. When combined with nicosulfuron, the mortality of H. zea increased to 53% (72.0 S.E.). Following exposure to 2 mg/L diazinon an average of 51% (71.3 S.E.) mortality was observed compared to 80% (72.0 S.E.) mortality following exposure to the binary mixture of diazinon and nicosulfuron. Finally, at the highest concentration of diazinon (3 mg/L), there was a mortality of 87% (72.5 S.E.) compared to that of mortality at 100% (70 S.E.) following exposure to the binary mixture. No mortality resulted following the exposure to nicosulfuron alone at a concentration of 22.5 mg/L or to treatments with water. The time courses for the percent change of AChE activity relative to controls following exposures to diazinon at 1, 2, and 3 mg/L and nicosulfuron at 22.5 mg/L are presented in Fig. 2. Results are based on the Rappaport method and demonstrate significant decreases following exposures to the lower concentrations of diazinon (1 and 120 Diazinon Diazinon and Nicosulfuron
100
Mortality (%)
AChE activity was measured by two methods: a modified method developed by Rappaport et al. (1959) and a modified method based on the assay by Ellman et al. (1961). Cholinesterase activity was determined based on the procedure by Rappaport et al. (1959) in which m-nitrophenol was used as an indicator of the acetic acid produced by the hydrolysis of acetylcholine chloride (ACh). Since we used the substrate ACh, we assumed that the primary enzyme measured was AChE. Total cholinesterase activity was measured in H. zea following exposure to the pesticides indicated above. The increased AChE activity measured was proportional to decreasing absorbance, as the acidity was produced by the splitting of acetylcholine by the enzyme AChE or cholinesterase. Since the Rappaport method also measured cholinesterase activity, we confirmed the results by measuring AChE activity by the method of Ellman et al. (1961). Ellman’s assay measures the hydrolysis of acetylthiocholine by the release of sulfhydrylic groups able to react with bis-(3-carboxy-4nitrophenyl) disulfide (Ellman’s reagent). The reaction was followed by using a spectrophotometer at 412 nm in which 2-nitro-5-thiobenzoate anion formed from the reaction has maximal absorbance. The activities of endogenous sulfhydryls (the blank samples) and spontaneous hydrolysis of acetylthiocholine substrate (blank substrate) reacting with the dye were subtracted from the experimental samples to get true AChE measurements. Supernatants used to measure AChE activity were obtained from H. zea larvae that were prepared for protein measurements as described above. For the Rappaport method, AChE activity was measured from the supernatant, using ACh as the substrate in a 2 mL reaction medium consisting of 0.2 mM NaCl, 0.1 g/L nitrophenol, 0.3 mg/mL ACh, and 0.5 mL of supernatant at 25 1C. A Blank was prepared by adding 3 mM eserine for 10 min to compensate for background absorbance contributed by the sample. Absorbance for Blank and experimental samples was measured at 10, 15, 20, 25, and 30 min and the difference was used to estimate the AChE activity. For the Ellman method, AChE activity was measured from the supernatant, using acetylthiocholine iodide as the substrate in a 2 mL reaction medium consisting of 0.25 mM 5, 5-dithio-bis(2-nitrobenzoic acid) or DTNB, 0.1 M phosphate buffer, pH 7.5, and 0.001 M acetylthiocholine iodide. Absorbance was read at 412 nm for the following time points: 7, 12, 17, and 22 min. Both enzyme activities were standardized by total protein in the supernatant, as measured by the Bradford method. The AChE activity was replicated and repeated at least three times for each treatment.
80 60 40 20 0 0.5
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1.5 2.0 2.5 Pesticide Concentration (mg/L)
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Fig. 1. Mean percent of mortality of H. zea (7S.E.) following exposures to diazinon alone and in combination with nicosulfuron (22.5 mg/L). No mortality resulted in larvae following exposure to nicosulfuron alone and in control groups exposed to water. Significant differences between diazinon and binary mixture treatment groups were observed compared to controls based on Fisher’s LSD (po0.05).
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Nicosulfuron
1 mg/L Diazinon
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Fig. 2. Mean percent change of acetylcholinesterase activity (7S.E.) standardized by total protein in H. zea relative to control following treatments with diazinon alone at 1, 2, and 3 mg/L and nicosulfuron alone at 22.5 mg/L using the Rappaport method. Significant differences were observed between controls and lower concentrations of diazinon (1 and 2 mg/L) based on Fisher’s LSD (po0.05).
14 16 18 Time (minutes)
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Fig. 3. Mean acetylcholinesterase activity standardized by total protein in H. zea as expressed by percent of control following exposure to nicosulfuron at 22.5 mg/L determined by Ellman’s assay. The mean percent of control7S.E. is shown. Significant differences were observed between controls and nicosulfuron treatments based on Fisher’s LSD (po0.05).
2 mg/L) compared to controls. However, inhibition of AChE activity relative to controls was not observed for treatments with the highest dose of diazinon (3 mg/L) and nicosulfuron. Results based on Ellman’s assay (not shown), confirm results obtained using the Rappaport method in which a decrease of AChE activity was observed with treatments of diazinon at lower concentrations of diazinon. The mean AChE activity as expressed as the percent of control increased significantly with treatments of diazinon at the highest concentration (3 mg/L) compared to the decrease in activity observed with treatments at lower concentrations. Treatments with nicosulfuron significantly increased AChE activity compared to controls based on the Ellman’s method and agree with results obtained from assays using the Rappaport method (Fig. 3). Treatments with binary mixtures of nicosulfuron and diazinon in general produced intermediate responses regarding AChE activity when compared to treatments with pesticides separately. Based on the Rappaport method, when animals were treated with a binary mixture of nicosulfuron and diazinon at either 1 mg/L (Fig. 4) or 2 mg/L (not shown), an intermediate response of AChE activity was observed. In other words, the inhibition of AChE activity following treatments with diazinon alone (1 or 2 mg/L) was antagonized by nicosulfuron. Binary mixtures of diazinon (1 or 2 mg/L) and nicosulfuron produced higher AChE activity compared to the activity observed following treatments with diazinon alone. In contrast, binary mixtures of the nicosulfuron and the highest concentration of diazinon tested (3 mg/L) showed a significant increase in AChE activity compared to the control and either treatments of diazinon alone or nicosulfuron (Fig. 5). Results from the Ellman’s assay,
Percent Change Relative to Control
40 20 0 -20 Control Nicosulfuron 1 mg/L Diazinon 1 mg/L Diazinon + Nicosulfuron
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Fig. 4. Mean percent change of acetylcholinesterase activity (7S.E.) standardized by total protein in H. zea relative to control following treatments with diazinon alone at 1 mg/L, nicosulfuron alone at 22.5 mg/L, and binary mixtures of diazinon and nicosulfuron using the Rappaport method. Significant differences were observed between controls and treatment groups based on Fisher’s LSD (po0.05).
however, did not confirm the increase of AChE activity observed following treatments with binary mixtures of diazinon (3 mg/L) and nicosulfuron. GST activity in corn earworms was not significantly altered following exposure to pesticides alone (Fig. 6) or to binary mixtures of nicosulfuron and diazinon. 4. Discussion The widespread use of cholinesterase inhibiting pesticides in the environment presents increasing concerns about their effects on human, wildlife, and ecosystem health. As a group, these pesticides are generally highly
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Percent Change Relative to Control
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Fig. 5. Mean percent change of acetylcholinesterase activity (7S.E.) standardized by total protein in H. zea relative to control following treatments with diazinon alone at 3 mg/L, nicosulfuron alone at 22.5 mg/L, and binary mixtures of diazinon and nicosulfuron using the Rappaport method. Significant differences were observed between controls and treatment groups (po0.05).
0.12 Control 1 mgL Diazinon 2 mg/L Diazinon 3 mg/L Diazinon Nicosulfuron
0.10 0.08 0.06 0.04 0.02 0.00 -0.02 1
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4 6 5 Time (minutes)
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Fig. 6. Mean glutathione S-transferase activity standardized by total protein in H. zea following treatments with diazinon alone at 1, 2, and 3 mg/L and nicosulfuron alone at 22.5 mg/L. Significant differences were not observed based on an overall ANOVA.
toxic and have great potential for adversely affecting nontarget organisms. Nicosulfuron and sulfonylureas in general have low mammalian toxicity (US EPA, 2004b), but when combined with an organophosphate insecticide, toxicity of the insecticide may increase. Diazinon and nicosulfuron are registered for use on corn; however, studies examining the toxicity of mixtures of the two have not been published. Although there are no published studies on mixtures of these specific pesticides on corn earworms, there have been studies of other herbicides increasing the toxicity and AChE inhibition of organophosphates. Atrazine, an
herbicide, has been shown to increase effects on AChE activity in midges following exposure to organophosphorus insecticides. Although atrazine itself had no effects on AChE activity, when added to chlorpyrifos, AChE inhibition increased (Belden and Lydy, 2001). Our results demonstrate the unclear role of AChE and GST on the toxicity of diazinon and nicosulfuron and binary mixtures of the two. Mortality increased in a dose-dependent manner as expected with exposure to diazinon alone. If the inhibition of AChE were the only factor affecting the toxicity of diazinon, then the potency of diazinon to inhibit AChE should correlate with the acute toxicity level. Based on the mortality data alone, the concentration producing the highest mortality (3 mg/L diazinon) did not inhibit AChE activity, instead, AChE activity increased. In addition, no mortality was observed with exposures of nicosulfuron alone but like the diazinon exposure (3 mg/L) an induction of AChE activity was observed. Mortality increased when corn earworms were treated with binary mixtures of nicosulfuron and diazinon at several concentrations compared to treatments with diazinon alone. In particular, by increasing AChE activity of treatments with diazinon at the lower concentrations (1 and 2 mg/L) nicosulfuron appears to antagonize diazinon treatment effects. However, at higher concentrations of diazinon (3 mg/L), nicosulfuron appears to significantly increase AChE levels based on the Rappaport method; these results were not reproduced using the Ellman method. The Rappaport method measures general cholinesterase activity, including AChE activity while the Ellman method measures AChE specifically. Since cholinesterases serve a variety of functions including detoxification, the discrepancies could have resulted due to detoxification factors. Both methods (Rappaport and Ellman) demonstrated an increase in AChE activity following exposures to nicosulfuron alone. This contradicts previous reports of nicosulfuron inhibiting AChE activity (Bretaud et al., 2000). Since our study used a different animal species and a higher concentration of nicosulfuron, these factors could have contributed to the discrepancies observed. Organophosphate insecticides, like diazinon, that must be bioactivated do not necessarily exhibit the correlation between acute toxicity and AChE inhibition. Diazinon must be bioactivated by monooxygenases or cytochrome P450 enzymes in order to effectively inhibit AChE. In addition to activation, detoxification is also accomplished by cytochrome P450 enzymes. Thus, the ratio of activation to detoxification varies with different cytochrome P450s as Levi et al. (1988) suggested in their study of cytochrome p450 isozymes. In addition, Ca2+-dependent A-esterases which appear to display a high affinity for certain organophosphates, may also play a major role in detoxification in which the active metabolite can be hydrolyzed considerably before an accumulation in target sites as shown by Chambers et al. (1994). Finally, GST has also been shown to play a major role in the detoxification of
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organophosphate insecticides but GST activity did not demonstrate any relevance to toxicity in our current study. Our results are not the first indication of toxicity not always correlating with the degree of enzyme inhibition. Other studies have also questioned the usefulness of using AChE and GST as biomarkers of immobility or toxicity. Jemec et al. (2007) found no correlation between changes in activities of AChE and GST and the toxicity of diazinon to Daphnia magna. Printes and Callaghan (2004) observed no immobility of D. magna exposed to 100 mM of the organophosphate acephate despite a 70% inhibition of AChE activity compared to controls. Day and Scott (1990) demonstrated that some organophosphates did not change AChE activity in the stonefly Classenia sp. at concentrations that resulted in significant immobility. Keizer et al. (1995) showed that diazinon toxicity is species-dependent and dependent on the following: rate of bioactivation to the more potent oxon metabolite, detoxification in the organism, and the sensitivity of AChE for either the parent compound or its primary oxon metabolite. Further studies exploring the detoxification mechanisms associated with diazinon and nicosulfuron should be examined as well as the sensitivity of AChE to organophosphates in H. zea. 5. Conclusion This study demonstrates that nicosulfuron increases toxicity of diazinon in corn earworms, H. zea. Measurements of AChE activity do not appear to serve as a good predictor of acute toxicity in H. zea with diazinon exposure. In addition, GST does not appear to play a significant role in the toxicity of diazinon and binary mixtures of diazinon and nicosulfuron. Our results suggest the potential for herbicides such as nicosulfuron to increase toxicity of organophosphate insecticides; however, further studies understanding the metabolic enzymes and sensitivity of AChE in specific target species such as the agriculturally important pests like corn earworms need to be investigated. Acknowledgments This work was supported by the Fayetteville State University’s Sponsored Research and Programs Minigrant Initiative and by Grant P20 MD001089-01 from the National Institutes of Health, NCMHD, and the Department of Health and Human Services. Support for this research was also partially provided by the grant from FSU-REAP (Academy of Applied Science) program funded by DOD’s Army Research Office. References Battaglin, W.A., Furlong, E.T., Brukhardt, M.R., Peter, C.J., 2000. Occurrence of sulfonylurea, sulfonamide, imidazolinone, and other herbicides in rivers, reservoirs and ground water in the Midwestern United States, 1998. Sci. Total Environ. 248, 123–133.
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Belden, J.B., Lydy, M.J., 2001. Effects of atrazine on acetylcholinesterase activity in midges (Chironomus tentans) exposed to organophosphorus insecticides. Chemosphere 44, 1685–1689. Bretaud, S., Toutant, J.P., Saglio, P., 2000. Effects of carbofuran, diuron, and nicosulfuron on acetylcholinesterase activity in goldfish (Carassius auratus). Ecotoxicol. Environ. Saf. 47, 117–124. Chambers, J.E., Ma, T., Boone, J.S., Chambers, H.W., 1994. Role of detoxication pathways in acute toxicity levels of phosphorothionate insecticides in the rat. Life Sci. 54, 1357–1364. Day, K.E., Scott, I.M., 1990. Use of acetylcholinesterase activity to detect sublethal toxicity in stream invertebrates exposed to low concentrations of organophosphate insecticides. Aquat. Toxicol. 18, 101–113. Ellman, G.L., Courtney, K.D., Andres Jr., V., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95. Fitt, G.P., 1989. The ecology of Heliothis species in relation to agro ecosystems. Annu. Rev. Entomol. 34, 17–52. Grue, C.E., Gilbert, P.L., Seeley, M.E., 1997. Neuropsychological and behavioral changes in non-target wildlife exposed to organophosphate and carbamate pesticide: thermoregulation, food consumption and reproduction. Am. Zool. 37, 369–388. Hemingway, J., 2000. The molecular basis of two contrasting metabolic mechanisms of insecticide resistance. Insect Biochem. Mol. Biol. 30, 1009–1015. Hoy, T., Horseberg, T.E., Wichstrom, R., 1991. Inhibition of acetylcholinesterase in rainbow trout following dichlorvos treatment at different water oxygen levels. Aquaculture 95, 33–40. Jemec, A., Drobne, D., Tisler, T., Trebse, P., Ros, M., Sepcic, K., 2007. The applicability of acetylcholinesterase and glutathione S-transferase in Daphnia magna toxicity test. Comp. Biochem. Physiol. Part C 144, 303–309. Keizer, J., D’Agostino, G., Nagel, R., Volpe, T., Gnemi, P., Vittozzi, L., 1995. Enzymological differences of AChE and diazinon hepatic metabolism: correlation of in vitro data with the selective toxicity of diazinon to fish species. Sci. Total Environ. 171, 213–220. Larkin, D.J., Tjeerdema, R.S., 2000. Fate and effects of diazinon. Environ. Contam. Toxicol. 166, 49–82. Levi, P.E., Hollinworth, R.M., Hodgson, E., 1988. Differences in oxidative dearylation and desulfuration of fenitrothion by cytochrome P450 isozymes and in the subsequent inhibition of monooxygenase activity. Pestic. Biochem. Physiol. 32, 224–231. O’Brien, R.D., 1976. Acetylcholinesterase and its inhibition. In: Wilkinson, C.F. (Ed.), Insecticide Biochemistry and Physiology. Plenum Press, New York, pp. 271–296. Printes, L.B., Callaghan, A., 2004. A comparative study on the relationship between acetylcholinesterase activity and acute toxicity in Daphnia magna exposed to anticholinesterase insecticides. Environ. Toxicol. Chem. 23, 1241–1247. Rappaport, F., Fischl, J., Pinto, N., 1959. An improved method for the estimation of cholinesterase activity in serum. Clin. Chim. Acta 4, 227–230. SAS Institute, 2002–2003. PROC User’s Manual, SAS 9.1. SAS Institute, Cary, NC. Sigma Chemical Co., 1995. Technical Bulletin for Bradford Reagent, Product No. B 6916. Timchalk, C., 2001. Organophosphate pharmacokinetics. In: Krieger, R., Doull, J., Ecobichon, D., Gammon, D., Hodgson, E., Reiter, L., Ross, J. (Eds.), Handbook of Pesticide Toxicology, vol. 2. Academic Press, San Diego, pp. 929–951. US Environmental Protection Agency, 1990. Pesticide tolerances for nicosulfuron. Rules and regulations. Federal Register 55 (134). US Environmental Protection Agency, 2004a. Interim Reregistration Eligibility Decision: Diazinon. Prevention, Pesticides, and Toxic Substances (7508C). EPA 738-R-04-006. US Environmental Protection Agency, 2004b. Report of the Food Quality Protection Act (FQPA) Tolerance Reassessment Progress and Risk Management Decision (TRED) for Nicosulfuron. Prevention, Pesticides and Toxic Substances (7508C).
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Role of acetylcholinesterase and glutathione S-transferase following exposure to nicosulfuron and diazinon in Helicoverpa zea Raffaella Massa, Stefania Blevins, Shirley L. Chao Department of Natural Sciences, Fayetteville State University, Lyons Science Bldg. Rm. 330, 1200 Murchison Road, Fayetteville, NC 28301, USA Received 22 March 2007; received in revised form 31 July 2007; accepted 22 August 2007 Available online 23 October 2007
Abstract The acute toxicity of diazinon combined with the herbicide nicosulfuron was determined using the corn earworm, Helicoverpa zea. Nicosulfuron significantly increased toxicity of diazinon to H. zea compared to diazinon alone based on dip bioassays to evaluate acute contact toxicity. Diazinon, an organophosphorous insecticide, controls insects by inhibiting acetylcholinesterase in the nervous system. Acetylcholinesterase activity was significantly altered in corn earworms exposed to binary mixtures compared to those exposed to diazinon alone; however, the activity did not correspond consistently with toxicity. Glutathione S-transferase (GST), also known to be altered by organophosphorous insecticides, did not exhibit significant changes following exposures to diazinon, nicosulfuron, or binary mixtures. Our results suggest that nicosulfuron increases toxicity of diazinon but the mechanism of toxicity does not appear to be correlated with acetylcholinesterase or GST inhibition. Published by Elsevier Inc. Keywords: Helicoverpa zea; Nicosulfuron; Diazinon; Acetylcholinesterase; Synergism; Glutathione S-transferase; Insecticide; Herbicide
1. Introduction Diazinon [O,O-diethyl-O-(2-isopropyl-6-methyl-4-pyrimidinyl) phosphorothioate] is a non-systemic organophosphate insecticide used in agricultural systems to control a wide variety of sucking and leaf-eating insects. It is applied on a variety of crops including corn, fruit trees, blueberries, lettuce, and tomatoes and on non-lactating cattle as an eartag. Residential product registrations have been canceled since 2004 due to unacceptable risks posed to agricultural workers, birds, and the environment. Despite cancellations, approximately 13 million lbs of active ingredient are used annually with rates of up to 4 lb active ingredient/acre (US EPA, 2004a). Diazinon is metabolized into an active oxon derivative, inhibiting acetylcholinesterase (AChE), an enzyme that catalyzes the hydrolysis of acetylcholine. The inhibition of AChE results in the accumulation of the neurotransmitter acetylcholine within cholinergic synapses, leading to Corresponding author. Fax: +1 910 672 1159.
E-mail address: [email protected] (S.L. Chao). 0147-6513/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.ecoenv.2007.08.014
synaptic blockage and disruption of signal transmission. Metabolism to its active oxon metabolite involves oxidative desulfuration whereas detoxification of diazinon involves dearylation. The balance between the rates of desulfuration and dearylation can result in very different levels of AChE inhibition (Larkin and Tjeerdema, 2000; Timchalk, 2001). Nicosulfuron [3-pyridinecarboxamide, 2-((((4,6-dimethoxypyrimidin-2-yl) amino carbonyl) aminosulfonyl))N,N-diethyl] is a member of the sulfonylurea (SU) family of herbicides and is used to control weeds on corn (US EPA, 2004b). Nicosulfuron was chosen for this study since it is registered for use on corn similar to diazinon and is applied alone or in formulation with other active ingredients to control annual and perennial grasses and broadleaf weeds with a maximum application rate of 0.06248 lb active ingredient/acre. The highest use of nicosulfuron is on corn and approximately 200,000 lb are used annually (US EPA, 2004b). Sulfonylurea along with other herbicides such as sulfonamide, and imidazolinone function by inhibiting the plant enzyme acetolactate synthase (ALS), stopping plant growth and eventually killing the plant (Battaglin
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et al., 2000). The ALS enzyme is found only in plants and not present in humans or other animals. Inhibition of the ALS enzyme system blocks the production of amino acids valine and isoleucine, essential building blocks of proteins and other plant components (US EPA, 1990). Corn earworms (Helicoverpa zea) were tested with these chemicals due to their economical importance for farmers. The earworm, also known as tomato fruitworm and cotton bollworm, is among the most destructive insect pests in the southern United States. The corn earworm infests over 100 plant species, but corn is the preferred host. First generation caterpillars attack the whorl stage, while the second generation is largely found in corn ears. They feed on a wide variety of crops including beans, peas, sweet corn, okra, tomatoes, cabbage, eggplant, and peppers. Damage caused by the corn earworm ranges from destruction of the host crop to cosmetic damage that may cause a crop to be unmarketable. These injuries can result in an unusable product for both the fresh and processing market (Fitt, 1989). Measurements of AChE activity have been used routinely as a biomarker of exposure to certain groups of contaminants, such as organophosphate and carbamate insecticides (Grue et al., 1997). Low concentrations of organophosphates are known to disrupt transmission in the central and peripheral cholinergic nervous system by inhibiting AChE (Hoy et al., 1991). In vertebrates, two kinds of cholinesterases are common. AChE is found only in erythrocytes and nervous and muscle tissue. If the AChE is inhibited significantly, the animal dies. Conversely, pseudocholinesterase is found in a number of tissues, including blood plasma, liver, and nervous tissue, and can be inhibited with no apparent effect on the animal (O’Brien, 1976). Interestingly, nicosulfuron has been found to inhibit AChE in the brain of goldfish up to 48 h in response to 50 mg/L. However, nicosulfuron exposure had no effect on AChE activity in the muscle of goldfish (Bretaud et al., 2000). In insects, AChE is abundant in nervous tissue. Unlike vertebrates, neuromuscular junctions in insects have shown clearly that there is no cholinesterase there and that transmission at the synapse does not include acetylcholine. AChE is confined to the ‘‘central’’ nervous system of insects (O’Brien, 1976). Glutathione S-transferase (GST) is a major detoxification factor in insects and dramatically increases the water solubility of metabolites of many pesticides. The most common metabolic resistance mechanisms for organophosphorous insecticides involve esterases, GSTs, or monooxygenases (Hemingway, 2000). Elevated GST levels in insects can produce resistance to a range of insecticides by conjugating reduced glutathione to the insecticide or its primary metabolic products. Because GSTs may also act together with an esterase-based mechanism (Hemingway, 2000), we explored the possibility of altered GST as a mechanism of toxicity for the binary mixtures of diazinon and nicosulfuron.
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Insecticides and herbicides are widely used in commercial and private use to control insect pests and weeds. The range of doses that results in toxic effects varies widely with formulation and with the individual species being exposed. Combined exposure of organophosphates and sulfonylurea herbicides are common in corn. No published results have yet been reported on toxicity of chemical mixtures such as nicosulfuron and diazinon. Based upon a possible shared mechanism of toxicity via the inhibition of AChE, one must consider the cumulative risk associated with organophosphate pesticide mixture exposures. In particular is a need to explore pesticide mixtures involving herbicides such as nicosulfuron that exhibit a possible shared mechanism of action with organophosphate insecticides. 2. Materials and methods 2.1. Chemicals Technical grade nicosulfuron, 3-pyridinecarboxamide, 2-((((4,6-dimethoxypyrimidin-2-yl) amino carbonyl) aminosulfonyl))-N,N-diethyl (75% purity) was generously donated by DuPont (Wilmington, DE). Technical grade diazinon, O,O-diethyl-O-(2-isopropyl-6-methyl-4-pyrimidinyl phosphorothioate (95% purity), was purchased from ChemService (West Chester, PA).
2.2. Animals Third instar larvae of H. zea and finished diet were purchased from the Department of Entomology at North Carolina State University. Corn earworms were maintained in the following conditions: photoperiod of 14 h light, 10 h darkness, temperature of 80 1F (27 1C) and at least 60% RH. The diet consisted of the following: 100 g wheat germ, 45 g (vitamin free) casein, 40 g sugar, 30 g Torula yeast, 15 g Wesson salt mix, 4 g L-ascorbic, 1.5 g sorbic acid, 1 g methyl paraben, and 0.5 g cholesterol. All the ingredients, once weighed out, were ground (in a rolling grinder or food processor) to break down the wheat germ and the ingredients were blended together to a fine consistency. All larvae, before and after treatments, were maintained on above diets until they were frozen for future assays.
2.3. Dip bioassay Third instar larvae H. zea were exposed to concentrations (1, 2, and 3 mg/L) of diazinon and nicosulfuron (22.5 mg/L) within 1–2 h upon their arrival. Groups of 10–12 H. zea were placed in cheesecloth and dipped for 10 s in the following test solutions: (1) nicosulfuron (22.5 mg/L) diluted in water, (2) three concentrations of diazinon: 1, 2, and 3 mg/L of diazinon in water, and (3) binary mixtures of diazinon concentrations (1, 2, and 3 mg/L) in combination with 22.5 mg/L nicosulfuron. Control larvae were dipped in water for 10 s at the same time as other treatment groups. The larvae were then removed from cheesecloth and placed in individual Petri dishes containing their diet. Mortality was determined 24 h after dipping worms in test solutions. All larvae were weighed and stored in 20 1C for further protein and enzyme analysis. Each treatment group was replicated three times and the bioassays were repeated twice.
2.4. Protein assay Total protein was determined by the Bradford method (Sigma Chemical Co., 1995) using Bovine Serum Albumin (Sigma) as the standard and the Bradford dye reagent (BioRad). Frozen whole bodies of third instar corn earworm larvae were weighed, thawed, and
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homogenized in buffer composed of: 0.2 M standard phosphate buffer at pH 7.4, 10% glycerol, and 1.0 mM EDTA. Tissues were homogenized using a polytron (Powergen, Fisher) for approximately 45 s. Tissues remained on ice during homogenization. The homogenate was then centrifuged for 5 min at 12,000g. Approximately 50 mL of the supernatant was then added to the Bradford dye, mixed, incubated for approximately 30 min to 1 h, and absorbance was read at 595 nm. The amount of absorbance was proportional to the amount of protein present.
benzene) were added at 4.33 mM and 1.12 mM, respectively. The negative control, a control sample without the substrate added, was also tested with treated samples. The total volume for this assay was 3 mL containing: 30 mL of supernatant from homogenized samples (approximately 40–100 mg of protein), 15 mL of CDNB, 130 mL of glutathione, and 0.1 M Tris–HCl at pH 8.0. Absorbance was read at 340 nm at the following time points: 2, 4, 6, and 8 min. Statistical analysis involved examining the main effects of the reaction time course and treatment in an overall ANOVA (SAS Institute, 2002–2003).
2.5. Acetylcholinesterase assays
2.6. Statistical analysis The results are presented as the mean of at least three separate experiments of at least three replicates for each experiment7standard error (S.E.). The main effects examined were reaction time course and treatment in an overall ANOVA (SAS Institute, 2002–2003) for mortality and AChE activity data. To ascertain which time points were different from one another by treatment, post hoc Fisher’s multiple comparisons were conducted with significant effects determined at po0.05.
2.7. Glutathione S-transferase determination The supernatants used for the protein assay were also used for determining GST activity. Glutathione and CDNB (1-chloro-2,4-dinitro-
3. Results Fig. 1 represents the percent of mortality of H. zea following exposure to three concentrations (1, 2, and 3 mg/L) of diazinon alone and in combination with nicosulfuron at 22.5 mg/L. Significant increases in mortality were observed after exposure with binary mixtures compared to diazinon alone. Following exposure to 1 mg/L diazinon, an average of 21% (73.1 S.E.) mortality resulted. When combined with nicosulfuron, the mortality of H. zea increased to 53% (72.0 S.E.). Following exposure to 2 mg/L diazinon an average of 51% (71.3 S.E.) mortality was observed compared to 80% (72.0 S.E.) mortality following exposure to the binary mixture of diazinon and nicosulfuron. Finally, at the highest concentration of diazinon (3 mg/L), there was a mortality of 87% (72.5 S.E.) compared to that of mortality at 100% (70 S.E.) following exposure to the binary mixture. No mortality resulted following the exposure to nicosulfuron alone at a concentration of 22.5 mg/L or to treatments with water. The time courses for the percent change of AChE activity relative to controls following exposures to diazinon at 1, 2, and 3 mg/L and nicosulfuron at 22.5 mg/L are presented in Fig. 2. Results are based on the Rappaport method and demonstrate significant decreases following exposures to the lower concentrations of diazinon (1 and 120 Diazinon Diazinon and Nicosulfuron
100
Mortality (%)
AChE activity was measured by two methods: a modified method developed by Rappaport et al. (1959) and a modified method based on the assay by Ellman et al. (1961). Cholinesterase activity was determined based on the procedure by Rappaport et al. (1959) in which m-nitrophenol was used as an indicator of the acetic acid produced by the hydrolysis of acetylcholine chloride (ACh). Since we used the substrate ACh, we assumed that the primary enzyme measured was AChE. Total cholinesterase activity was measured in H. zea following exposure to the pesticides indicated above. The increased AChE activity measured was proportional to decreasing absorbance, as the acidity was produced by the splitting of acetylcholine by the enzyme AChE or cholinesterase. Since the Rappaport method also measured cholinesterase activity, we confirmed the results by measuring AChE activity by the method of Ellman et al. (1961). Ellman’s assay measures the hydrolysis of acetylthiocholine by the release of sulfhydrylic groups able to react with bis-(3-carboxy-4nitrophenyl) disulfide (Ellman’s reagent). The reaction was followed by using a spectrophotometer at 412 nm in which 2-nitro-5-thiobenzoate anion formed from the reaction has maximal absorbance. The activities of endogenous sulfhydryls (the blank samples) and spontaneous hydrolysis of acetylthiocholine substrate (blank substrate) reacting with the dye were subtracted from the experimental samples to get true AChE measurements. Supernatants used to measure AChE activity were obtained from H. zea larvae that were prepared for protein measurements as described above. For the Rappaport method, AChE activity was measured from the supernatant, using ACh as the substrate in a 2 mL reaction medium consisting of 0.2 mM NaCl, 0.1 g/L nitrophenol, 0.3 mg/mL ACh, and 0.5 mL of supernatant at 25 1C. A Blank was prepared by adding 3 mM eserine for 10 min to compensate for background absorbance contributed by the sample. Absorbance for Blank and experimental samples was measured at 10, 15, 20, 25, and 30 min and the difference was used to estimate the AChE activity. For the Ellman method, AChE activity was measured from the supernatant, using acetylthiocholine iodide as the substrate in a 2 mL reaction medium consisting of 0.25 mM 5, 5-dithio-bis(2-nitrobenzoic acid) or DTNB, 0.1 M phosphate buffer, pH 7.5, and 0.001 M acetylthiocholine iodide. Absorbance was read at 412 nm for the following time points: 7, 12, 17, and 22 min. Both enzyme activities were standardized by total protein in the supernatant, as measured by the Bradford method. The AChE activity was replicated and repeated at least three times for each treatment.
80 60 40 20 0 0.5
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Fig. 1. Mean percent of mortality of H. zea (7S.E.) following exposures to diazinon alone and in combination with nicosulfuron (22.5 mg/L). No mortality resulted in larvae following exposure to nicosulfuron alone and in control groups exposed to water. Significant differences between diazinon and binary mixture treatment groups were observed compared to controls based on Fisher’s LSD (po0.05).
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Fig. 2. Mean percent change of acetylcholinesterase activity (7S.E.) standardized by total protein in H. zea relative to control following treatments with diazinon alone at 1, 2, and 3 mg/L and nicosulfuron alone at 22.5 mg/L using the Rappaport method. Significant differences were observed between controls and lower concentrations of diazinon (1 and 2 mg/L) based on Fisher’s LSD (po0.05).
14 16 18 Time (minutes)
20
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Fig. 3. Mean acetylcholinesterase activity standardized by total protein in H. zea as expressed by percent of control following exposure to nicosulfuron at 22.5 mg/L determined by Ellman’s assay. The mean percent of control7S.E. is shown. Significant differences were observed between controls and nicosulfuron treatments based on Fisher’s LSD (po0.05).
2 mg/L) compared to controls. However, inhibition of AChE activity relative to controls was not observed for treatments with the highest dose of diazinon (3 mg/L) and nicosulfuron. Results based on Ellman’s assay (not shown), confirm results obtained using the Rappaport method in which a decrease of AChE activity was observed with treatments of diazinon at lower concentrations of diazinon. The mean AChE activity as expressed as the percent of control increased significantly with treatments of diazinon at the highest concentration (3 mg/L) compared to the decrease in activity observed with treatments at lower concentrations. Treatments with nicosulfuron significantly increased AChE activity compared to controls based on the Ellman’s method and agree with results obtained from assays using the Rappaport method (Fig. 3). Treatments with binary mixtures of nicosulfuron and diazinon in general produced intermediate responses regarding AChE activity when compared to treatments with pesticides separately. Based on the Rappaport method, when animals were treated with a binary mixture of nicosulfuron and diazinon at either 1 mg/L (Fig. 4) or 2 mg/L (not shown), an intermediate response of AChE activity was observed. In other words, the inhibition of AChE activity following treatments with diazinon alone (1 or 2 mg/L) was antagonized by nicosulfuron. Binary mixtures of diazinon (1 or 2 mg/L) and nicosulfuron produced higher AChE activity compared to the activity observed following treatments with diazinon alone. In contrast, binary mixtures of the nicosulfuron and the highest concentration of diazinon tested (3 mg/L) showed a significant increase in AChE activity compared to the control and either treatments of diazinon alone or nicosulfuron (Fig. 5). Results from the Ellman’s assay,
Percent Change Relative to Control
40 20 0 -20 Control Nicosulfuron 1 mg/L Diazinon 1 mg/L Diazinon + Nicosulfuron
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Fig. 4. Mean percent change of acetylcholinesterase activity (7S.E.) standardized by total protein in H. zea relative to control following treatments with diazinon alone at 1 mg/L, nicosulfuron alone at 22.5 mg/L, and binary mixtures of diazinon and nicosulfuron using the Rappaport method. Significant differences were observed between controls and treatment groups based on Fisher’s LSD (po0.05).
however, did not confirm the increase of AChE activity observed following treatments with binary mixtures of diazinon (3 mg/L) and nicosulfuron. GST activity in corn earworms was not significantly altered following exposure to pesticides alone (Fig. 6) or to binary mixtures of nicosulfuron and diazinon. 4. Discussion The widespread use of cholinesterase inhibiting pesticides in the environment presents increasing concerns about their effects on human, wildlife, and ecosystem health. As a group, these pesticides are generally highly
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Fig. 5. Mean percent change of acetylcholinesterase activity (7S.E.) standardized by total protein in H. zea relative to control following treatments with diazinon alone at 3 mg/L, nicosulfuron alone at 22.5 mg/L, and binary mixtures of diazinon and nicosulfuron using the Rappaport method. Significant differences were observed between controls and treatment groups (po0.05).
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Fig. 6. Mean glutathione S-transferase activity standardized by total protein in H. zea following treatments with diazinon alone at 1, 2, and 3 mg/L and nicosulfuron alone at 22.5 mg/L. Significant differences were not observed based on an overall ANOVA.
toxic and have great potential for adversely affecting nontarget organisms. Nicosulfuron and sulfonylureas in general have low mammalian toxicity (US EPA, 2004b), but when combined with an organophosphate insecticide, toxicity of the insecticide may increase. Diazinon and nicosulfuron are registered for use on corn; however, studies examining the toxicity of mixtures of the two have not been published. Although there are no published studies on mixtures of these specific pesticides on corn earworms, there have been studies of other herbicides increasing the toxicity and AChE inhibition of organophosphates. Atrazine, an
herbicide, has been shown to increase effects on AChE activity in midges following exposure to organophosphorus insecticides. Although atrazine itself had no effects on AChE activity, when added to chlorpyrifos, AChE inhibition increased (Belden and Lydy, 2001). Our results demonstrate the unclear role of AChE and GST on the toxicity of diazinon and nicosulfuron and binary mixtures of the two. Mortality increased in a dose-dependent manner as expected with exposure to diazinon alone. If the inhibition of AChE were the only factor affecting the toxicity of diazinon, then the potency of diazinon to inhibit AChE should correlate with the acute toxicity level. Based on the mortality data alone, the concentration producing the highest mortality (3 mg/L diazinon) did not inhibit AChE activity, instead, AChE activity increased. In addition, no mortality was observed with exposures of nicosulfuron alone but like the diazinon exposure (3 mg/L) an induction of AChE activity was observed. Mortality increased when corn earworms were treated with binary mixtures of nicosulfuron and diazinon at several concentrations compared to treatments with diazinon alone. In particular, by increasing AChE activity of treatments with diazinon at the lower concentrations (1 and 2 mg/L) nicosulfuron appears to antagonize diazinon treatment effects. However, at higher concentrations of diazinon (3 mg/L), nicosulfuron appears to significantly increase AChE levels based on the Rappaport method; these results were not reproduced using the Ellman method. The Rappaport method measures general cholinesterase activity, including AChE activity while the Ellman method measures AChE specifically. Since cholinesterases serve a variety of functions including detoxification, the discrepancies could have resulted due to detoxification factors. Both methods (Rappaport and Ellman) demonstrated an increase in AChE activity following exposures to nicosulfuron alone. This contradicts previous reports of nicosulfuron inhibiting AChE activity (Bretaud et al., 2000). Since our study used a different animal species and a higher concentration of nicosulfuron, these factors could have contributed to the discrepancies observed. Organophosphate insecticides, like diazinon, that must be bioactivated do not necessarily exhibit the correlation between acute toxicity and AChE inhibition. Diazinon must be bioactivated by monooxygenases or cytochrome P450 enzymes in order to effectively inhibit AChE. In addition to activation, detoxification is also accomplished by cytochrome P450 enzymes. Thus, the ratio of activation to detoxification varies with different cytochrome P450s as Levi et al. (1988) suggested in their study of cytochrome p450 isozymes. In addition, Ca2+-dependent A-esterases which appear to display a high affinity for certain organophosphates, may also play a major role in detoxification in which the active metabolite can be hydrolyzed considerably before an accumulation in target sites as shown by Chambers et al. (1994). Finally, GST has also been shown to play a major role in the detoxification of
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organophosphate insecticides but GST activity did not demonstrate any relevance to toxicity in our current study. Our results are not the first indication of toxicity not always correlating with the degree of enzyme inhibition. Other studies have also questioned the usefulness of using AChE and GST as biomarkers of immobility or toxicity. Jemec et al. (2007) found no correlation between changes in activities of AChE and GST and the toxicity of diazinon to Daphnia magna. Printes and Callaghan (2004) observed no immobility of D. magna exposed to 100 mM of the organophosphate acephate despite a 70% inhibition of AChE activity compared to controls. Day and Scott (1990) demonstrated that some organophosphates did not change AChE activity in the stonefly Classenia sp. at concentrations that resulted in significant immobility. Keizer et al. (1995) showed that diazinon toxicity is species-dependent and dependent on the following: rate of bioactivation to the more potent oxon metabolite, detoxification in the organism, and the sensitivity of AChE for either the parent compound or its primary oxon metabolite. Further studies exploring the detoxification mechanisms associated with diazinon and nicosulfuron should be examined as well as the sensitivity of AChE to organophosphates in H. zea. 5. Conclusion This study demonstrates that nicosulfuron increases toxicity of diazinon in corn earworms, H. zea. Measurements of AChE activity do not appear to serve as a good predictor of acute toxicity in H. zea with diazinon exposure. In addition, GST does not appear to play a significant role in the toxicity of diazinon and binary mixtures of diazinon and nicosulfuron. Our results suggest the potential for herbicides such as nicosulfuron to increase toxicity of organophosphate insecticides; however, further studies understanding the metabolic enzymes and sensitivity of AChE in specific target species such as the agriculturally important pests like corn earworms need to be investigated. Acknowledgments This work was supported by the Fayetteville State University’s Sponsored Research and Programs Minigrant Initiative and by Grant P20 MD001089-01 from the National Institutes of Health, NCMHD, and the Department of Health and Human Services. Support for this research was also partially provided by the grant from FSU-REAP (Academy of Applied Science) program funded by DOD’s Army Research Office. References Battaglin, W.A., Furlong, E.T., Brukhardt, M.R., Peter, C.J., 2000. Occurrence of sulfonylurea, sulfonamide, imidazolinone, and other herbicides in rivers, reservoirs and ground water in the Midwestern United States, 1998. Sci. Total Environ. 248, 123–133.
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