PESTICIDE Biochemistry & Physiology
Pesticide Biochemistry and Physiology 80 (2004) 54–64 www.elsevier.com/locate/ypest
Synergistic and antagonistic effects of atrazine on the toxicity of organophosphorodithioate and organophosphorothioate insecticides to Chironomus tentans (Diptera: Chironomidae) Troy D. Anderson and Kun Yan Zhu* Department of Entomology, 123 Waters Hall, Kansas State University, Manhattan, KS 66506, USA Received 22 October 2003; accepted 11 June 2004 Available online 20 July 2004
Abstract The acute toxicities of two organophosphorodithioate (dimethoate and disulfoton) and two organophosphorothioate (omethoate and demeton-S-methyl) insecticides were evaluated individually and in binary combination with the herbicide atrazine using fourth-instar larvae of the aquatic midge, Chironomus tentans. Atrazine alone up to 1000 lg/L did not show significant toxicity to the midges in a 48-h bioassay. However, atrazine concentrations as low as 1 lg/L in combination with dimethoate at EC25 (concentration to affect 25% of tested midges), 100 lg/L in combination with disulfoton (EC25 ), and 10 lg/L in combination with demeton-S-methyl (EC25 ) significantly enhanced the toxicity of each organophosphate insecticide. In contrast, atrazine concentrations of 10 lg/L and above in combination with omethoate (EC25 ) significantly decreased the toxicity of the insecticide. Biochemical analysis indicated that increased toxicity of dimethoate, disulfoton, and demeton-S-methyl in binary combination with atrazine correlated to the increased inhibition of acetylcholinesterase. Furthermore, cytochrome P450-dependent O-deethylation activity in the midges exposed to atrazine at 1000 lg/L was 1.5-fold higher than that in the control midges. Thus, atrazine appeared to induce cytochrome P450 monooxygenases in the midges. Elevated cytochrome P450 monooxygenase activity may increase the toxicities of dimethoate, disulfoton, and demeton-S-methyl by enhancing the oxidative activation of dimethoate into omethoate, and disulfoton and demeton-S-methyl into their sulfoxide analogs with increased anticholinesterase activity. In contrast, atrazine reduced the toxicity of omethoate possibly by enhancing the oxidative metabolic detoxification since omethoate does not require oxidative activation. Ó 2004 Published by Elsevier Inc. Keywords: Atrazine; Organophosphates; Synergism; Cytochrome P450 monooxygenase
1. Introduction * Corresponding author. Fax: 1-785-532-6232. E-mail address:
[email protected] (K.Y. Zhu).
0048-3575/$ - see front matter Ó 2004 Published by Elsevier Inc. doi:10.1016/j.pestbp.2004.06.003
Atrazine is a selective pre- and post-emergence triazine herbicide applied to many major food
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crops including corn, sorghum, and sugar cane as well as residential and commercial landscaping projects to control broadleaf weeds and grasses [1,2]. According to the US Environmental Protection Agency (EPA),1 more than 85 million pounds of atrazine is applied annually and accounts for approximately 60% of the total mass of pesticides used each year [3–6]. In spite of its extensive use, atrazine is considered to be a relatively safe herbicide with a short half-life and insignificant bioaccumulation and biomagnification [4,7]. Atrazine is designed to hinder photosystem II and repress electron transport mechanisms necessary for photosynthesis. Organophosphorodithioates (e.g., dimethoate and disulfoton) and organophosphorothioates (e.g., omethoate and demeton-S-methyl) are members of the highly diverse class of organophosphate (OP) insecticides. They are extensively used in the urban and agricultural areas of the Midwest. According to the US EPA, approximately 2.5 million pounds of dimethoate is applied annually to an average of 5 million acres of vegetable, orchard, and ornamental crops in the Midwest whereas nearly 1.2 million pounds of disulfoton is utilized in the same areas to treat cotton, wheat, potato, and tobacco crops along with demeton-S-methyl [8–11]. Although these two types of OPs are structurally different, each is acutely toxic and is intended to interfere with normal cholinergic nerve transmission by inhibiting the enzyme acetylcholinesterase (AChE). Pharmacologically, organophosphorodithioates, such as dimethoate and disulfoton, require an in vivo oxidative activation into more toxic, AChEinhibiting O-analogs by cytochrome P450 monooxygenases in living organisms whereas the organophosphorothioates omethoate and demeton-S-methyl do not. These two organophosphorothioates are O-analogs in their parent form.
1
Abbreviations used: AChE, acetylcholinesterase; ATC, acetylthiocholine iodide; DTNB, 5,50 -dithio-bis(2-nitrobenzoic acid); EC, effective concentration; EPA, Environmental Protection Agency; GST, glutathione S-transferase; LSD, least significant differences; b-NADPH, b-nicotinamide adenine dinucleotide phosphate; OP, organophosphate; PBO, piperonyl butoxide.
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Pesticides applied to crops, lawns, and animals are commonly found in soil or water, and water contamination is often caused by more than one pesticide at relatively low concentrations. Recent reports have revealed that high doses of atrazine induce abnormalities and deformities in non-target organisms [7]. In addition, atrazine has been shown to act synergistically with the organophosphorothioates chlorpyrifos, methyl parathion, and diazinon thereby increasing the toxicities of these OPs to the aquatic midge larvae Chironomus tentans and the aquatic amphipod Hyalella azteca [12,13]. These increases in toxicity were determined to be associated with decreased AChE activity in both C. tentans and H. azteca [12,13]. Thus, atrazine may induce cytochrome P450 monooxygenases in order to confer the synergistic effects on the toxicity of organophosphorothioates [12,13]. However, we do not know whether these synergistic effects of atrazine are related to the chemical structures of OP insecticides. This study examined the structural features of organophosphorodithioate and organophosphorothioate insecticides in relation to the toxicity changes in the presence and absence of atrazine. Herein, we report: (1) the toxicities of the individual pesticides exposed to fourth-instar C. tentans larvae, (2) the effects of atrazine on the toxicity of dimethoate, disulfoton, omethoate, and demeton-S-methyl to C. tentans, (3) the AChE activity of C. tentans exposed to binary combinations of atrazine and each OP, and (4) the time- and dose-dependent increase in cytochrome P450 monooxygenase activity of atrazine-treated C. tentans.
2. Materials and methods 2.1. Organisms The fourth-instar larvae of the aquatic midge C. tentans were taken from the colonies cultured in the Department of Entomology at Kansas State University according to the standard operating procedures of the US EPA for static cultures [14], with slight modifications. Instead of separating each generation from the egg masses, the midges
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were reared in mixed-age brood cultures. The fourth-instar larvae were collected directly from the mixed-age cultures and used for the pesticide bioassays as well as the enzyme activity assays. 2.2. Chemicals Acetone (American Chemical Society certified) was purchased from Fisher Scientific (Pittsburgh, PA, USA). Acetylthiocholine iodide (ATC), bicinchoninic acid solution, bovine serum albumin, 5,50 -dithio-bis (2-nitrobenzoic acid) (DTNB), Triton X-100, umbelliferone (7-hydroxycoumarin), 7ethoxycoumarin (7-EC), oxidized glutathione, glutathione reductase, and b-nicotinamide adenine dinucleotide phosphate (reduced b-NADPH) were purchased from Sigma (St. Louis, MO, USA). Dimethoate [O,O-dimethyl S-methylcarbamoylmethyl phosphorodithioate] (99.5% purity), omethoate [O,O-dimethyl S-methylcarbamoylmethyl phosphorothioate] (98%), disulfoton [O,O-diethyl S-[2-(ethylthio) ethyl]phosphorodithioate] (98%), demeton-S-methyl [S-[2-(ethylthio) ethyl] O,O-dimethyl phosphorothioate] (98%), and atrazine [6chloro-N -ethyl-N 0 -(1-methylethyl)-1,3,5-triazine-2, 4-diamine] (99%) were obtained from Chem Service (West Chester, PA, USA). Piperonyl butoxide (PBO) was purchased from Fairfield Chemical (Baltimore, MD, USA). 2.3. Toxicity bioassays with individual pesticides and pesticide mixtures The acute toxicity bioassays were performed for 48 h using fourth-instar C. tentans larvae exposed to five concentrations of each pesticide. The appropriate dilutions of each pesticide were prepared in acetone. The pesticide was delivered by adding 100 ll of pesticide solution to 1-L reconstituted water containing 10 midges and 10 ml clean sand in a glass beaker. The same procedure was used to treat midges with corresponding concentrations of acetone in water as controls. The bioassay was repeated four times for each pesticide concentration and control. The treated midges were maintained in a growth chamber (Percival Scientific, Boone, IA, USA) at 25 1 °C with a 16:8-h light:dark photoperiod (maximum light intensity:
80 lmol/m2 /s) for 48 h. The endpoint for each bioassay was measured as an effective concentration (EC). The midges that were unable to perform an active movement upon gentle probing were considered as affected. Log-probit analysis was used to estimate the toxic endpoint concentrations for each OP [15]. In order to assess the combined effect of atrazine and each OP insecticide, fourth-instar C. tentans larvae were exposed to each OP at the EC25 level individually and in combination with atrazine treatments of 1, 10, 100, and 1000 lg/L. The pesticide exposure methods were the same as described above in the acute toxicity bioassays. The percentages of midges affected by the different pesticide treatments were statistically compared using Fisher’s least significant difference (LSD) multiple comparisons after arcsine square root transformations. In order to determine the magnitude of altered toxicity existing among treatments, synergism ratios were estimated by treating the midges with the five different concentrations of each OP individually and in combination with a fixed concentration of atrazine (200 lg/L) using the same techniques as described above. 2.4. In vivo inhibition of acetylcholinesterase activity The residual AChE activity was determined according to the method of Ellman et al. [16] as modified by Zhu et al. [17], using ATC as a substrate. The larvae of fourth-instar C. tentans were exposed to each OP at the EC25 level individually and in combination with atrazine at 1, 10, 100, or 1000 lg/L. The pesticide exposure methods were the same as described above in the acute toxicity bioassays. All surviving midges were collected from each beaker as a sample. Each sample was homogenized in ice-cold 0.1 M phosphate buffer (pH 7.5) containing 0.5% (v/v) Triton X-100. One hundred microliters of homogenizing buffer was used per midge. The homogenates were centrifuged at 15,000g for 15 min at 4 °C, and the supernatants were transferred to new tubes. The residual AChE activity in the supernatants then was measured using an enzyme kinetic microplate reader (Molecular Devices, Menlo Park, CA, USA) at 405 nm
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immediately after 100 ll of the mixture of ATC and DTNB was added to 50 ll of the supernatant. The final concentrations of ATC and DTNB in the reaction mixture were 0.25 and 0.40 mM, respectively. The concentration of total protein in each AChE preparation was determined using the method of Smith et al. [18] using bovine serum albumin as a standard. The measurement was carried out using the microplate reader (Molecular Devices) at 560 nm. 2.5. Fluorometric determination of cytochrome P450 monooxygenase activity The chemical exposure methods were generally the same as described above in the acute toxicity bioassays. Specifically, groups of 20 fourth-instar C. tentans larvae were exposed to PBO at 1000 lg/ L, dimethoate at 105.1 lg/L (EC25 ), or atrazine at 1000 and 10,000 lg/L for 24 and 48 h, respectively. Acetone (100 ll/L) and water-only controls were performed in parallel to chemical treatments. The bioassay was repeated five times for each chemical and control. All surviving midges were collected from each beaker as a sample. The guts were dissected from the midges in each sample and homogenized in 50 ll ice-cold 150 mM phosphate buffer (pH 7.4) containing 50 mM sucrose. Cytochrome P450 monooxygenase activity was determined using the method of Ulrich and Weber [19] as modified by de Sousa et al. [20] and later Stumpf and Nauen [21] using 7-ethoxycoumarin (7-EC) as a substrate. The gut homogenates were centrifuged at 10,000g for 20 min at 4 °C. The resulting supernatants were transferred to new tubes and used as the enzyme source. Eighty microliters of a mixture containing 50 mM 7-EC and 62.5 mM reduced b-NADPH were added to each microplate well containing 40 ll of enzyme preparation. The microplate was incubated for 30 min at 30 °C while shaking at 400 rpm using a Vortex-Genie 2 (Scientific Industries, Bohemia, NY, USA). The O-deethylation of 7-EC results in fluorescent umbelliferone (7-hydroxycoumarin). The absorption of light and emission of fluorescence by b-NADPH is similar to umbelliferone, therefore, b-NADPH was removed by oxidizing it to non-fluorescent
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NADP+ using the method of Chauret et al. [22]. Following incubation, 10 ll of 100 mM oxidized glutathione and 1.0 U glutathione reductase were added to each microplate well containing the reaction mixture and incubated for 15 min at 37 °C. The reaction was stopped using 120 ll of 50% (v/v) acetonitrile in 50 mM TRIZMA-base buffer (pH 10). The fluorescence of umbelliferone was measured with a FLX800TBIDE microplate fluorescence reader (Bio-Tek Instruments, Winooski, VT, USA) at 465 nm while exciting at 390 nm. The enzyme activity of 7-EC-O-deethylation was determined based on an umbelliferone standard curve and protein concentration of the sample determined using the method of Smith et al. [18] as described above. The differences in enzyme activity for each treatment and exposure period were statistically compared using a two-way analysis of variance (ANOVA) in combination with a Fisher’s least significant difference (LSD) multiple comparison test [15]. Enzyme activity ratios also were calculated by dividing the enzyme activity of each treatment by that of the control (water-only). These ratios were used to determine the magnitude of increase in enzyme activity existing among treatments.
3. Results 3.1. Toxicity bioassays with individual pesticides and pesticide mixtures Atrazine up to 1000 lg/L was not acutely toxic to the midges under our bioassay conditions (Fig. 1). However, atrazine concentrations as low as 1 and 10 lg/L in combination with dimethoate at EC25 (105.1 lg/L) and demeton-S-methyl at EC25 (57.3 lg/L), respectively, significantly increased the percentage of midges affected as compared with these OPs alone at EC25 (P < 0:05). The percentage of midges affected by disulfoton at EC25 (59.3 lg/L) also was significantly increased in combination with atrazine at 100 lg/L and above (P < 0:05). In contrast, atrazine concentrations as low as 10 lg/L in combination with omethoate at EC25 (218.9 lg/L) significantly reduced the
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Fig. 1. Binary combinations of atrazine (ATR; 1, 10, 100, and 1000 lg/L) with each OP (EC25 ) exposed to fourth-instar C. tentans for 48 h. One-hundred microliters of acetone was used for the control (CONT) treatment. Vertical bars indicate standard errors of the mean (n ¼ 4). Different letters on the bars indicate that the means are significantly different among the treatments (P < 0:05) in least significant difference multiple comparison tests.
percentage of midges affected (P < 0:05). Furthermore, the increased toxicities of dimethoate, disulfoton, and demeton-S-methyl correlated with the increased concentrations of atrazine, whereas the decreased toxicity of omethoate correlated with the increased concentrations of atrazine (P < 0:05). The EC50 concentrations estimated for each OP and each OP in combination with atrazine (200 lg/L) were used to calculate synergism ratios (Table 1). At the fixed concentration of atrazine (200 lg/L), the toxicity of demeton-S-methyl significantly increased by 1.42-fold (Table 1). Although atrazine synergized the toxicities of dimethoate and disulfoton by 1.14- and 1.31-fold, respectively, and antagonized the toxicity of omethoate by 1.34-fold, the alteration of the tox-
icity for each insecticide was not statistically significant due to the overlapping 95% confidence intervals of their EC50 (Table 1). 3.2. In vivo inhibition of acetylcholinesterase The residual AChE activity of fourth-instar C. tentans exposed to the EC25 treatment of each OP individually and in combination with atrazine treatments of 1, 10, 100, and 1000 lg/L is shown in Fig. 2. A significant reduction in AChE activity did not occur in response to atrazine-only exposures (up to 1000 lg/L) relative to the solvent controls. The treatments at EC25 of dimethoate (105.1 lg/L), disulfoton (59.3 lg/L), omethoate (218.9 lg/L), or demeton-S-methyl (57.3 lg/L) reduced AChE activity by 29.2, 39.2, 40.5, and
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Table 1 Effects of atrazine (ATR, 200 lg/L) on the toxicity of dimethoate (DMTH), omethoate (OMTH), disulfoton (DSFT), and demetonS-methyl (DMSM) to fourth-instar C. tentans a Pesticide
EC25 (95% CI)
EC50 (95% CI)
EC95 (95% CI)
Slope SE
v2 (probability)b
DMTH DMTH + ATR OMTH OMTH + ATR DSFT DSFT + ATR DMSM DMSM + ATR
105.1 (74.5–139.6) 94.4 (67.0–124.8) 218.9 (158.1–285.4) 245.2 (168.9–332.7) 59.3 (37.1–78.9) 48.1 (31.7–62.2) 57.3 (47.0–67.1) 40.8 (33.0–48.0)
248.9 (188.7–338.8) 218.6 (166.9–293.4) 483.8 (374.3–641.7) 650.0 (478.8–946.0) 109.9 (83.6–138.2) 83.6 (65.2–101.7) 92.4 (79.4–109.1) 65.3 (56.2–76.0)
1283.0 (830.4–2429.0) 1079.0 (713.7–1967.0) 2184.0 (1466.0–3927.0) 4144.0 (2390.0–9991.0) 495.3 (341.4–961.3) 322.6 (238.6–537.5) 229.2 (180.3–328.5) 159.6 (128.6–219.4)
0.54 0.02 0.55 0.02 0.58 0.03 0.48 0.03 2.51 0.88 2.80 0.42 0.95 0.06 0.98 0.05
0.13 1.32 0.09 0.46 4.06 3.75 2.24 2.14
(0.94) (0.52) (0.96) (0.80) (0.13) (0.15) (0.33) (0.34)
SRc —
1.14 —
0.74 —
1.31 —
1.42
a The OP toxicity data are presented as EC25 , EC50 , and EC95 and their 95% confidence intervals (95% CI) in micrograms per liter (lg/L), the effective concentrations at which 25, 50, and 95% of tested midges were affected, respectively, in a 48-h bioassay. Log-probit analysis was used to estimate the endpoint concentrations for each pesticide. b Pearson’s chi-square and the probability of v2 . The probability of >0.05 indicates that the observed regression model is not significantly different from the expected model (i.e., a significant fit between the observed and expected regression models). c Synergism ratio (SR) was calculated by SR ¼ EC50 OP-only /EC50 mixture: The asterisk next to the ratio indicates a significant difference between the EC50 s of the OP and its binary combination with atrazine (200 lg/L) based on the non-overlapping 95% confidence intervals of the EC50 values.
Fig. 2. Comparison of in vivo acetylcholinesterase (AChE) activity in fourth-instar C. tentans exposed to atrazine (ATR; 1, 10, 100, and 1000 lg/L) in binary combination with each OP (EC25 ). One-hundred microliters of acetone was used for the control (CONT) treatment. Vertical bars indicate standard errors of the mean (n ¼ 4). Different letters on the bars indicate that the means are significantly different among the treatments (P < 0:05) in least significant differences multiple comparison tests.
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Table 2 Comparison of cytochrome P450-dependent O-deethylation activity detected in gut homogenates dissected from untreated and piperonyl butoxide- (PBO; 1000 lg/L), dimethoate- (EC25 ; 105.1 lg/L), and atrazine-treated (1000 and 10,000 lg/L) fourth-instar C. tentans larvae Treatment
24 h exposure
48 h exposure a
Control (water-only) Control (100 ll acetone) PBO (1000 lg/L) Dimethoate (105.1 lg/L) Atrazine (1000 lg/L) Atrazine (10,000 lg/L)
Enzyme activity (fmol/min/mg protein)
Activity ratiob
Enzyme activity (fmol/min/mg protein)
Activity ratio
0.61 0.03bA 0.64 0.01bA 0.52 0.02aA 0.70 0.03bA 0.61 0.02bA 0.69 0.05bA
—
1.05 0.85 1.15 1.00 1.13
0.61 0.02bA 0.66 0.04bA 0.39 0.01aB 0.76 0.0cA 0.91 0.04 dB 0.91 0.01 dB
1.10 0.64 1.25 1.50 1.50
—
a
Cytochrome P450 enzyme activities are presented as the means standard error (n ¼ 5) by using 7-ethoxycoumarin (7-EC) as a substrate. Enzyme activity values, within a column (treatment) and within a row (exposure period), followed by the same lowercase and upper case letter, respectively, are not significantly different (P P 0:05, Fisher’s LSD multiple comparison test). b Activity ratios were calculated by dividing the enzyme activity of each treatment by that of the water-only control.
26.6%, respectively, compared with solvent controls. An atrazine concentration of 100 lg/L in combination with dimethoate (105.1 lg/L) reduced AChE activity by 29.6% compared with dimethoate-only treatments, whereas atrazine as low as 10 lg/L in combination with disulfoton (59.3 lg/L) and demeton-S-methyl (57.3 lg/L) reduced AChE activity by 8.3 and 31.5%, respectively, compared with disulfoton- and demeton-S-methyl-only treatments (Fig. 2). In contrast, increasing atrazine concentrations did not affect AChE inhibition by omethoate (P > 0:05) (Fig. 2). 3.3. Fluorometric determination of cytochrome P450 monooxygenase activity The cytochrome P450-dependent O-deethylation activity was determined in the homogenates of the guts dissected from fourth-instar C. tentans larvae exposed to PBO at 1000 lg/L, dimethoate at 105.1 lg/L (EC25 ), or atrazine at 1000 and 10,000 lg/L (Table 2). As expected, the enzyme activity in the midges exposed to PBO for 24 and 48 h was reduced by 14.3 and 35.7%, respectively, compared to the water-only control (P < 0:05). The enzyme activity was decreased by 25.0% in the midges exposed to PBO for 48 h compared to those exposed to PBO for 24 h. In contrast, the midges exposed to atrazine at 1000 or 10,000 lg/L did not show significant changes in enzyme activity at 24 h (P > 0:05). However, when the mid-
ges were exposed to the same concentrations atrazine for 48 h, cytochrome P450 monooxygenase activity was enhanced by 1.5-fold for both atrazine concentrations compared to the wateronly control (Table 2).
4. Discussion Due to the extent of pesticide application in the agricultural and residential areas of the US, the cooccurrence of atrazine and OP insecticides in surface and ground waters is inevitable. Atrazine residues have been reported as high 700 and 2300 lg/L in the ground water of 13 states and in the surface water of 31 states, respectively, during the peak application periods of spring and early summer [18,23,24]. However, surface and ground water concentrations of atrazine are typically below the US EPA’s drinking water standard maximum contaminant level of 3 lg/L [4,23–27]. On the other hand, disulfoton concentrations between 0.04 and 100 lg/L have been routinely detected in several municipal, community, and home wells with peak daily concentrations of 7.1–26.8 lg/L reported in the surface waters in the Midwest [10]. The water quality analysis of drinking water in several Midwestern states also reports dimethoate concentrations to range from 6.4 to 58.4 lg/L [8]. According to these reports, the concentrations of atrazine used in this study simulate a broad range
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of exposures in which non-target organisms may encounter in an aquatic environment. In addition, each of the OP concentrations that C. tentans were exposed to, individually and in combination with atrazine, correlate to environmentally realistic exposure levels. We report that atrazine up to 1000 lg/L itself did not show significant toxicity to C. tentans. However, we have determined that the toxicity of dimethoate, disulfoton, and demeton-S-methyl was increased by 15, 10, and 33% when in combination with atrazine (1 lg/L for dimethoate, 100 lg/L for disulfoton, and 10 lg/L for demetonS-methyl), respectively, as compared to OP-only treatments (Fig. 1). In contrast, atrazine (10 lg/L) decreased omethoate toxicity by 20% in the combination (Fig. 1). Our study indicates that the altered toxicity observed between atrazine and these OPs is associated with modified in vivo AChE inhibition in C. tentans. The AChE activity in C. tentans exposed to atrazine-only treatments (1000 lg/L) was not significantly lower than control C. tentans (Fig. 2). This indicates that atrazine is not an effective AChE inhibitor nor is the altered toxicity observed between atrazine and OP mixtures a result of direct inhibition of AChE by atrazine. However, the in vivo AChE activity of C. tentans exposed to disulfoton, demeton-S-methyl, and dimethoate, when in combination with atrazine (10 lg/L for disulfoton and demeton-S-methyl and 100 lg/L for dimethoate), was reduced by 8.3, 31.5, and 29.6%, respectively, when compared to OP-only treatments (Fig. 2). Therefore, the enhanced inhibition of AChE by the OPs may be the result of atrazine’s effect on an indirect mechanism. This current study reveals that cytochrome P450 monooxygenase activity is elevated by 50.0% in C. tentans exposed to atrazine (1000 lg/L) for 48 h compared to the water-only control whereas PBO (1000 lg/L) suppressed cytochrome P450 monooxygenase activity by 35.7% (Table 2). Piperonyl butoxide, a selective cytochrome P450 monooxygenase inhibitor, was chosen to validate that the O-deethylation activity of cytochrome P450 monooxygenases was occurring in C. tentans as well as determine the degree of increase in enzyme activity following atrazine exposure. A pre-
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vious study has demonstrated that atrazine is capable of facilitating enhanced in vivo biotransformation of the OP chlorpyrifos to the more toxic metabolite chlorpyrifos O-analog and increases the amount of chlorpyrifos metabolites in C. tentans [28]. In addition, Miota et al. [29] reported the induction of a 45-kDa protein in atrazine-treated C. tentans that is closely associated with the 45- to 60-kDa heme-thiolate membrane proteins that metabolize a variety of endogenous and exogenous compounds in insects while the induction of probable cytochrome P450 monooxygenases has been attributed to atrazine exposure in a variety of invertebrate and vertebrate species [29–31]. Furthermore, glutathione S-transferase (GST) induction has been confirmed in rats following chronic exposure to atrazine-treated diets as well as in various species of fish, insects, and mammals [31,32]. Atrazine also has been recognized to increase non-specific esterase activity in the southern armyworm Spodoptera eridania Cram [31]. Our findings validate our hypothesis that the alteration of OP toxicity is the result of atrazine enhancing the oxidative activation process of OP insecticides by inducing certain cytochrome P450 monooxygenases. However, diminished toxicity also may occur due to induced cytochrome P450 monooxygenases, GSTs, or increased esterase activity depending on the structure of the OP insecticides. We speculated that these modifications should increase the toxicity of certain organophosphorodithioate OPs and decrease the toxicity of certain organophosphorothioate OPs which is apparent in our results where atrazine increases dimethoate and disulfoton toxicity and decreases omethoate toxicity when exposed in combination to C. tentans as a result of modified AChE activity (Figs. 1 and 2). Alternatively, demeton-S-methyl exhibited increased toxicity and AChE inhibition when in combination with atrazine (Table 1 and Figs. 1 and 2). Figure 3 illustrates a hypothetical mechanism in which atrazine is influencing the toxicity of dimethoate and omethoate. According to this proposed mechanism, atrazine induces certain cytochrome P450 monooxygenases which enhance the oxidative activation of dimethoate into more toxic O-analog metabolites resulting in elevated AChE inhibition and increased toxicity.
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Fig. 3. Hypothetical mechanism of atrazine’s influence on dimethoate and omethoate toxicity in fourth-instar C. tentans.
However, the parent form of omethoate is a potent AChE inhibitor due to its O-analog structure. Atrazine could decrease omethoate toxicity by inducing cytochrome P450 monooxygenases resulting in the enhanced biotransformation of omethoate to less toxic metabolites (Fig. 3). Atrazine’s influence on OP toxicity becomes more complex when detailing a hypothetical mechanism of altered disulfoton and demeton-Smethyl toxicity. Disulfoton and demeton-S-methyl are OPs that can be readily oxidized by cytochrome P450 monooxygenases to produce sulfoxide metabolites that differentiate the toxicity and metabolic processes of these two OPs. For example, atrazine may induce certain cytochrome P450 monooxygenases that initially oxidize disulfoton into its organophosphorodithioate sulfoxide analog [33]. Such a sulfoxide metabolite generally has minimal or no effect on its inhibitory potency to AChE since it is not an O-analog (Fig. 4). Atrazine may induce cytochrome P450 monooxygenases that initially oxidize demeton-S-methyl into its
organophosphorothioate sulfoxide analog. Because demeton-S-methyl is already an O-analog, its sulfoxide metabolite generally is more toxic and has greater inhibitory potency to AChE compared to demeton-S-methyl. (Fig. 4). Therefore, increased oxidation of demeton-S-methyl will result in increased inhibition of AChE and inceased toxicity to the midges. In summary, our study reveals that atrazine significantly modifies the toxicity of structurally different OPs and that this altered toxicity may be the result of atrazine affecting one or more indirect mechanisms. However, cytochrome P450 monooxygenase induction is the most likely contributor to atrazine’s influence on OP toxicity, the biological activity of OP insecticides also may be modified by other mechanisms such as GSTs and esterases that are reportedly mediated by atrazine. Further analysis should be conducted to determine the relationship between other triazine herbicides and frequently used insecticides and their potential for synergistic or antagonistic interactions on non-
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Fig. 4. Hypothetical mechanism of atrazine’s influence on disulfoton and demeton-S-methyl toxicity in fourth-instar C. tentans.
target organisms. Furthermore, a comprehensive genomic profile of atrazine should be established in order to comparatively determine gene expression in non-target organisms and systematically identify the proteins that confer atrazine-mediated synergistic toxicity.
Acknowledgments We thank Sharon R. Starkey for her technical assistance and Dr. Yoonseong Park for reviewing an earlier version of the manuscript. This research was supported by the US Environmental Protection Agency Experimental Program to Stimulate Competitive Research (EPSCoR) (R827589-01-0) and the Kansas Agricultural Experiment Station, Kansas State University (KS559). The manuscript is contribution 04-098-J from the Kansas Agricultural Experiment Station. The C. tentans voucher specimens (110) are located in the Museum of Entomological and Prairie Arthropod Research,
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