Single and simultaneous exposure of acetylcholinesterase to diazinon, chlorpyrifos and their photodegradation products

Pesticide Biochemistry and Physiology 100 (2011) 16–22

Contents lists available at ScienceDirect

Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest

Single and simultaneous exposure of acetylcholinesterase to diazinon, chlorpyrifos and their photodegradation products Mirjana B. Cˇolovic´ a, Danijela Z. Krstic´ b, Gordana S. Ušc´umlic´ c, Vesna M. Vasic´ a,⇑ a

Department of Physical Chemistry, Vincˇa Institute of Nuclear Sciences, University of Belgrade, Belgrade, Serbia University School of Medicine, Institute of Chemistry, University of Belgrade, Belgrade, Serbia c Department of Organic Chemistry, Faculty of Technology and Metallurgy, University of Belgrade, Belgrade, Serbia b

a r t i c l e

i n f o

Article history: Received 11 June 2010 Accepted 30 January 2011 Available online 4 February 2011 Keywords: Diazinon Chlorpyrifos Photodegradation products Acetylcholinesterase Simultaneous exposure

a b s t r a c t In vitro inhibition of electric eel acetylcholinesterase (AChE) by single and simultaneous exposure to organophosphorus insecticides diazinon and chlorpyrifos, and their transformation products, formed due to photoinduced degradation, was investigated. Increasing concentrations of diazinon, chlorpyrifos and their oxidation products, diazoxon and chlorpyrifos-oxon, inhibited AChE in a concentration-dependent manner. IC50 (20 min) values, obtained from the inhibition curves, were (in mol/l): (5.1 ± 0.3)  108, (4.3 ± 0.2)  106 and (3.0 ± 0.1)  108 for diazoxon, chlorpyrifos and chlorpyrifos-oxon, respectively, while maximal diazinon concentration was lower than its IC50 (20 min). Calculated KI values, in mol/l, of 7.9  107, 9.6  106 and 4.3  107 were obtained for diazoxon, chlorpyrifos and chlorpyrifos-oxon, respectively. However, 2-isopropyl-4-methyl-6-pyrimidinol (IMP) and 3,5,6-trichloro-2-pyridinol, diazinon and chlorpyrifos hydrolysis products, did not noticeably affect the enzyme activity at all investigated concentrations. Additive inhibition effect was achieved for lower concentrations of the inhibitors (diazinon/diazoxon 61  104/1  108 mol/l i.e., chlorpyrifos/chlorpyrifos-oxon 62  106/3  108 mol/l), while an antagonistic effect was obtained for all higher concentrations of the organophosphates. Inhibitory power of 1  104 mol/l diazinon irradiated samples can be attributed mostly to the formation of diazoxon, while the presence of non-inhibiting photodegradation product IMP did not affect diazinon and diazoxon inhibitory efficiencies. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction Diazinon (O,O-diethyl-O-(2-isopropyl-4-methyl-6-pyrimidinyl) phosphorothionate) and chlorpyrifos (O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate) have been commonly used thionophosphorous organophosphate nonspecific insecticides for over 50 years, to control a variety of insects in agriculture and the household environment [1]. Although they have replaced organochlorine pesticides because of the persistence and accumulation of the latter in the environment, these compounds are essentially nerve poisons, highly toxic to animals and humans. Specifically, orgaonophosphates inhibit AChE (EC 3.1.1.7), by phosphorylation of the serine hydroxyl group in the substrate-binding domain of the enzyme, resulting in accumulation of acetylcholine at cholinergic synapses in the central and peripheral nervous systems (cholinergic syndrome) [2,3] and associated neurotoxicity [4,5].

⇑ Corresponding author. Address: Department of Physical Chemistry, Vincˇa Institute of Nuclear Sciences, P. O. Box 522, 11001 Belgrade, Serbia. Fax: +381 11 2447 207. E-mail address: [email protected] (V.M. Vasic´). 0048-3575/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2011.01.010

Furthermore, organophosphorus pesticides induce oxidative stress in animals and humans [5–7], generate free radicals and cause lipid peroxidation leading to genetic material damage and cell malformations [8–10]. Organophosphorus insecticides in the environment undergo the natural degradation pathway including mainly homogeneous and heterogeneous hydrolysis enhanced by the presence of dissolved metals, humic substances, microorganisms and other compounds present in soil [11–13]. Since organophosphates exhibit absorption maxima in UV region (240–310 nm), sunlight irradiation (direct photolysis) is expected to induce their environmental photodegradation pathways, which is catalyzed by oxygen or humic substances acting as natural sensitizers (indirect photolysis) [13]. Also, organophosphate degradation processes occur in chemical treatments for purification of polluted water, generally referred to as advanced oxidation processes: photolysis of hydrogen peroxide and ozone, Fenton reagent and radiolysis of water [14]. Degradation studies of direct [10,15–17] or indirect photolysis [18–20], and advanced oxidation processes [21–23], revealed different kinetics, mechanisms and transformation products, suggesting complete mineralization of the starting compound, but forming toxic products as well.

M.B. Cˇolovic´ et al. / Pesticide Biochemistry and Physiology 100 (2011) 16–22

Thio starting compounds can be transformed to their oxo analogues (several hundreds times more powerful AChE inhibitors compared to the parent compound) due to various degradation conditions [10,17,24], as well as the enzymatic reactions in birds, fish, insects and mammals [24]. The pathway of oxidation was assumed to be oxidative desulfuration by OH radical attack on thiono group or through oxidative mechanism acting directly on a thio organophosphate [19]. On the other hand, organophosphorus esters are very susceptible to hydrolysis, that is the most common degradation pathway [25] resulting in organophosphate covalent bond cleavage and producing non-inhibiting AChE compounds [11,26]. Hydrolysis occurs at several reactive centers in a given organophosphorus pesticide molecule. It can occur by a homogeneous mechanism, where H2O and OH (H+ catalysis is less common) act as nucleophiles. Therefore, the hydrolysis process is strongly dependent on medium pH (base- and acid-catalyzed hydrolysis) [24,27]. Alternatively, it can take place when dissolved metal ions enhance the rate of hydrolysis by catalysis [13]. This study deals with the in vitro investigation of the effects of single and simultaneous exposure of commercially purified electric eel AChE to diazinon, chlorpyrifos and their main photoinduced by-products: oxidation productsdiazoxon and chlorpyrifos-oxon, and hydrolysis productsIMP and 3,5,6-trichloro-2-pyridinol (Fig. 1). In addition, the enzyme activity alteration in the presence of synthetic mixtures of pure diazinon and its photolysis products,

17

at concentrations identified in the irradiated diazinon samples in the previously published photodegradation study [10], was investigated and evaluated. Diazinon and chlorpyrifos, commonly used insecticides with similar chemical structures, were chosen as model compounds because they have been frequently found in source water [28].

2. Materials and methods 2.1. Chemicals and reagents All chemicals were of analytical grade. Acetylcholinesterase (specific activity 288 IU/mg solid, 408 IU/mg protein) from electric eel, acetylthiocholine iodide (AChI) and 5,50 -dithio-bis-2-nitrobenzoic acid (DTNB) were purchased from Sigma Chemicals Co. (Germany). Chlorpyrifos (99.8%), chlorpyrifos-oxon solution (10 ppm in cyclohexane), diazinon (97.3%), O-analog diazinon (diazoxon) solution (100 ppm in acetonitrile) and 2-isopropyl-4-methyl-6pyrimidinol (IMP) (93%), 3,5,6-trichloro-2-pyridinol (99.4%) were from Pestanal (Germany). Other medium assay chemicals (sodium dodecyl sulfate (SDS), potassium hydrogen phosphate (K2HPO4), sodium dihydrogen phosphate (NaH2PO4  2H2O)) were from Merck (Germany). The chemical structures of the studied compounds are collected in Fig. 1.

Fig. 1. Chemical structures of studied compounds.

18

M.B. Cˇolovic´ et al. / Pesticide Biochemistry and Physiology 100 (2011) 16–22

2.2. AChE assay

20

-10

10

-9

10

-8

10

-7

10

-6

0.9 0.6 0.3 0.0

-7.5

-7.2

-6.9

logC (of inhibitor)

-0.3 -0.6 -0.9

10

-5

10

-4

10

-3

10

-2

10

-1

Inhibitor, mol/l Fig. 2. The concentration-dependent inhibition of electric eel AChE by diazinon (square), diazoxon (circle) and IMP (cross). The values are expressed as mean ± SEM. Inset: Hill analysis of inhibition of electric eel AChE activity induced by diazoxon (circle).

100 80 log[% activity/(100 - % activity)]

Analysis of variance (one-way ANOVA) was used to compare the mathematical sum of inhibitions caused by separate exposure to diazinon/diazoxon (or chlorpyriphos/chlorpyriphos oxon) with inhibitions induced by exposure to both organophosphates simultaneously. When a significant continuous probability distribution (F value) (p < 0.05) was obtained, a post hoc test (Bonferoni) was used to determine the differences. An antagonistic effect was defined as a statistically significant (p < 0.05) difference between inhibitions caused by simultaneous exposure and mathematically calculated means of enzyme inhibition for a pair of organophosphates, where the former is lower than the latter.

40

10

Activity (% of control)

2.4. Statistical analysis

60

0

2.3. Kinetic analysis Kinetic experiments were carried out according to the method introduced by Kitz et al. [30] using commercial electric eel AChE. The enzyme (0.02 IU) was exposed to various concentrations of the investigated inhibitors. The medium was the same composition as was used in the AChE assay. Remaining AChE activity was measured according to Ellman’s method described above.

80 log [% activity/(100 - % activity)]

Activity (% of control)

100 The inhibition of AChE activity was measured using slightly modified Ellman’s procedure [29] in the absence (control) and presence of chlorpyriphos, diazinon and their related compounds. The experiments were performed by in vitro (separate and simultaneous) exposure of 0.02 IU commercial enzyme to investigated compounds in 50 mmol/l phosphate buffer pH 8.0 (final volume 0.650 ml). The standard medium assays were preincubated for 20 min at 37 °C in the absence and presence of desired concentration of the inhibitors. The control tubes contained the corresponding concentration of organic solvents without the organophosphate. Ten microliter AChI (0.075 mol/l) was used as the enzyme substrate in combination with 20 ll DTNB (0.01 mol/l in 50 mmol/l phosphate buffer pH 7.0) as a chromogenic reagent. The reaction was started by the addition of AChI, and allowed to proceed for 5 min until stopped by 65 ll SDS (10%). The product 5-thio-2-nitrobenzoate, released in the reaction of thiocholineiodide (product of enzymatic reaction) and DTNB, was measured at 412 nm (in buffer solution) using Perkin Elmer Lambda 35 UV– VIS spectrophotometer (Shelton, USA). All experiments were performed in triplicate. Preliminary studies showed that diazinon, chlorpyrifos and their by-products did not interfere with quantization of the yellow product 5-thio-2-nitrobenzoate.

60 40 20 0 -10

10

-9

10

-8

10

-7

10

-6

10

-5

10

0.6

0.3

0.0

-8

-7

-6

-5

logC (of inhibitor) -0.3

-0.6

-0.9

-4

10

-3

10

-2

10

-1

10

0

10

Inhibitor, mol/l Fig. 3. The concentration-dependent inhibition of electric eel AChE by chlorpyrifos (triangle), chlorpyrifos-oxon (asterisk) and 3,5,6-trichloro-2-pyridinol (open circle). The values are expressed as mean ± S.E.M. Inset: Hill analysis of inhibition of electric eel AChE activity induced by chlorpyrifos (triangle) and chlorpyrifos-oxon (asterisk).

3. Results 3.1. In vitro effect on AChE activity induced by single exposure to diazinon, chlorpyrifos and their by-products The separate influence of diazinon, chlorpyrifos and their degradation products, usually formed due to the chemical conversion or photochemical treatment of the selected organophosphates, on AChE activity was investigated in the concentration range from 1  1010 to 1  102 mol/l. The obtained results show (Figs. 2 and 3) that diazinon, diazoxon, chlorpyrifos and chlorpyrifos-oxon inhibit AChE in a concentration-dependent manner but with different inhibitory potencies. Dependence of the AChE activity, expressed as the percent of control value (obtained without inhibitor), on the inhibitor concentration fitted a sigmoidal function in all cases (Figs. 2 and 3). The inhibition parameters, the concentrations of investigated com-

pounds with capability to inhibit 50% of the enzyme after given exposure time (IC50 values) and Hill coefficient nH determined using the Hill method (Figs. 2 and 3 (inset)) by linear regression analysis of log [% activity/(100  % activity)] vs. log (concentration of inhibitor) plots, are given in Table 1. The differences between values of IC50 obtained using the Hill method and by fitting the sigmoidal inhibition curves were in the range of experimental error. As can be seen (Figs. 2 and 3, Table 1) inhibitory potencies of diazinon and chlorpyrifos are quite different. Almost complete inhibition of AChE activity was achieved in the presence of 2  105 mol/l chlorpyrifos while the effect of the same concentration of diazinon on the enzyme activity was negligible. The maximal AChE inhibition induced by 2  104 mol/l diazinon was 34.6% compared to the control value. It is obvious that oxo forms of investigated organophosphates (diazoxon as well as chlorpyrifos-oxon) are several hundreds times more potent AChE activity

M.B. Cˇolovic´ et al. / Pesticide Biochemistry and Physiology 100 (2011) 16–22 Table 1 The inhibition parameters (IC50 values (20 min) and Hill coefficients nH) of diazinon, chlorpyrifos and their related compounds for AChE obtained by Hill analysis and fitting the experimental data by sigmoidal function. Compound

Diazinon Diazoxon IMP Chlorpyrifos Chlorpyrifos-oxon 3,5,6-trichloro-2-pyridinol

Sigmoidal fitting

Hill analysis

IC50, mol/l

IC50, mol/l

nH

>2.0  104 (5.1 ± 0.3)  108 – (4.3 ± 0.2)  106 (3.0 ± 0.1)  108 –

– 5.2  108 – 4.5  106 3.2  108 –

– 1.4 ± 0.1 – 1.2 ± 0.1 1.5 ± 0.1 –

According to the anticipated reaction scheme for the formation of phosphoryl enzyme [30], it is easy to derive the time-dependent equation for irreversible enzyme inhibition:

ln

inhibitors than their parent compounds. For example, at the concentration of 2  107 mol/l chlorpyrifos-oxon completely inhibited AChE activity, while the effect of the same concentration of chlorpyrifos on the enzyme activity was negligible. The IC50 value of the enzyme activity was achieved at (4.3 ± 0.2)  106 mol/l of chlorpyrifos, while the same effect was observed when the concentrations of diazoxon and chlorpyrifos-oxon were (5.1 ± 0.3)  108 mol/l and (3.0 ± 0.1)  108 mol/l, respectively. On the contrary, the presence of the highest investigated diazinon concentration (2  104 mol/l in 4% ethanol) was not able to reach half-maximum inhibition. The hydrolysis products of diazinon (IMP) and chlorpyrifos (3,5,6-trichloro-2-pyridinol) (Figs. 2 and 3) did not noticeably affect the enzyme activity at any of the investigated concentrations.

3.2. Kinetic analysis The rate of irreversible inhibition for diazoxon, chlorpyrifos and chlorpyrifos-oxon was measured according to the previously reported method [30]. The enzyme was exposed in vitro to diazoxon and chlorpyrifos-oxon in the concentration range from 2  108 to 2  107 mol/l (chlorpyrifos concentration range was 2  106 – 2  105 mol/l), at several preincubation times (enzyme-inhibitor contact time): 10, 15, 20, 25, 30 min. The results show that the inhibition of AChE activity by the investigated compounds increases as a function of exposure time within the investigated time interval (Fig. 4a).

E k3 t ¼ E0 1 þ K I =ðIÞ

1 1 KI 1 ¼ þ  kapp k3 k3 ðIÞ

ð2Þ

Fig. 4a represents the results obtained for diazoxon as an example. Irreversible inhibition, which progressed with time in accordance with Eq. (1), was obtained for diazoxon, chlorpyrifos and chlorpyrifos-oxon. The values of kapp obtained from the slope of the dependence of ln E/Eo vs. t where then plotted according to Eq. (2) and are presented in Fig. 4b. Reasonably good straight lines were obtained that intercept the positive y axis, confirming that reversible complexes are formed in the concentration range studied. The inhibition parameters KI and k3 were calculated from the slope and intercept of Eq. (2) and are given in Table 2. 3.3. Inhibition of AChE activity by simultaneous exposure to diazinon, chlorpyrifos and their photoinduced transformation products In our previously published photodegradation study [10], diazinon solution was photodegraded using 125 W Cermax xenon parabolic lamp, emitting light with wavelengths above 200 nm, during six different periods of time: 0, 5, 15, 30, 60 and 115 min. Quantification of diazinon and its break-down products, IMP and diazoxon, in aliquots of the irradiated samples was performed on Waters ACQUITY Ultra Performance Liquid Chromatography (UPLC) – UV/VIS detection system under described conditions [10]. According to the results of the analysis obtained in this study,

6

21 1 2

18

3

4

-1

kapp × 10 , s

0.36788

ð1Þ

where E/E0 represents the percent of the remaining enzyme activity in relation to the initial activity (E0), KI is the dissociation constant for the initial reversible enzyme inhibitor complex (EI), k3 is the first order rate constant for the conversion of the reversible complex to the irreversibly inhibited enzyme and t is the time interval after the mixing of the complex and the inhibitor. In our experiments, the condition (I) » (E0) was satisfied in all cases. Consequently, the slope of Eq. (1) (kapp) can be expressed in the form:

1

1

19

3

5

0.04979

0.01832 0

5

10

15

20

25

30

-1

0.13534

12

-1

ln (E/E 0)

4

kapp × 10 , s

-1

15

9

2

4 -1

-5

1

6 -1

6 3

35

0 0

Fig. 4a. Progressive development of inhibition of produced by reaction of AChE with different concentrations of diazoxon plotted as semi logarithmic curve in accordance with Eq. (1). Diazoxon concentrations: (1) 2  108 M, (2) 3  108 M, (3) 5  108 M, (4) 7.5  108 M, (5) 1  107 M and (6) 2  107 M.

2 0 0

Cinhibitor × 10 , mol/l

6

t, min

2

1

2

3 -1

4 -7

5

6

-1

Cinhibitor × 10 , mol/l

Fig. 4b. The dependence of kapp upon the concentration of diazoxon (1), chlorpyrifos-oxon (2) and chlorpyrifos ((3), inset) plotted as reciprocals in accordance with Eq. (2).

M.B. Cˇolovic´ et al. / Pesticide Biochemistry and Physiology 100 (2011) 16–22

20

45% of irradiated diazinon has been converted to IMP that does not affect enzyme activity (Fig. 2, Table 3). IMP does not alter diazinon and/or diazoxon inhibitory capacities in all investigated combinations (Table 3). (2) Synergism/antagonism between diazinon and diazoxon inhibition potencies were studied by 20-min preincubation of the enzyme with mixtures of diazinon and diazoxon at concentrations which, with individual exposure, produced around 50% inhibition or less. Table 4 presents the means of the experimental values and the mathematical data calculated as the sum of organophosphate-induced inhibitions determined separately. Analysis of variance (One-way ANOVA) was used to compare the mathematical sum of inhibitions caused by separate exposures to diazinon or diazoxon with inhibitions induced by exposure to both organophosphates in combination (simultaneously). When a significant F value (P < 0.05) was obtained, a post hoc Bonferoni test was used to determine the differences.

Table 2 Kinetic data (KI and k3) for irreversible inhibition of electric eel AChE by diazinon, chlorpyrifos and their transformation products. Compound

KI, mol/l

k3, s1

k3/KI (mol/l)1s1

Diazinon Diazoxon Chlorpyrifos Chlorpyrifos-oxon

– 7.9  107 9.6  106 4.3  107

– 0.48 ± 0.09 0.12 ± 0.06 0.23 ± 0.03

– 6.1  105 1.2  104 5.3  105

synthetic mixtures of pure degradation products (commercial diazinon, diazoxon and IMP) were composed in order to investigate the effects on AChE activity induced by simultaneous exposure toward diazinon and its photoinduced by-products. The concentrations of diazinon and its decomposition products in the mixtures were chosen using two criteria: (1) the concentrations of mixture components were similar to the concentrations identified by UPLC analysis in the irradiated samples after photodegradation of 1  104 mol/l diazinon at the time interval from 0 to 115 min and (2) concentrations of the diazinon and its inhibiting by-product diazoxon were in the concentration range around or lower than their IC50 (20 min) values. (1) The inhibitions of AChE activity obtained after 20 min of preincubation of the enzyme with the desired combinations of diazinon and/or its by-products are presented in Table 3 and compared to the inhibitions achieved in the presence of each single compound. As can be seen, the initial unirradiated 1  104 mol/l diazinon inhibits AChE activity by 25.1%. Five-minute irradiation causes almost complete AChE inhibition (98.6%). The rapid decrease of the enzyme activity is attributed to conversion of 0.2% of the initial parent compound to its oxo analogue, since the formed 2.0  107 mol/l diazoxon by itself induces 90.2% inhibition (Table 3). Diazinon (7.2  105 mol/l) and 4.0  108 mol/l diazoxon, inducing (by separate exposure) inhibitions by 20.2% and 41.1%, respectively, as well as non-inhibiting IMP have been detected after 15 min of photodegradation. The synthetic mixture of these compounds reduces AChE activity by 52.0% that is lower than the sum of their individual inhibition capacities indicating antagonistic effect of diazinon and diazoxon at these concentrations. Combinations of diazinon and IMP formed during 30–60 min induce the enzyme recovery (15.8% and 8.0% inhibition), while the complete regeneration is achieved after 115-min treatment when

As can bee seen from Table 4, with simultaneous exposure of enzyme to diazinon and diazoxon in various concentration ratios, additive inhibition effects are observed at lower concentrations of the inhibitors (in the presence of diazinon (mol/l)/diazoxon (mol/l) at concentrations: 2  105/5  109, 2  105/7.5  109, 2  105/1  108, 5  105/5  109, 5  105/7.5  109, 5  105/1  108, 1  104/5  109 and 1  104/7.5  109), while a statistically significant antagonistic inhibition (i.e., lower than Table 4 Inhibition of AChE activity induced by simultaneous exposure to diazinon and diazoxon in mixtures. Values in parenthesis represent diazinon/diazoxon induced inhibition measured separately (by single exposure). Inhibition (%) Diazinon (mol/l)

Diazoxon (mol/l) 5  109 (5.5%)

7.5  109 (7.7%)

1  108 (14.6%)

2  108 (25.5%)

3  108 (35.5%)

5  108 (50.7%)

2  105 (8.0%) 5  105 (15.5%) 1  104 (22.7%) 2  104 (34.2%)

13.0%

16.4%

17.9%

26.2%

33.3%

48.5%

22.4%

23.2%

25.3%

24.6%

37.8%

44.4%

28.6%

29.1%

22.4%

28.5%

36.7%

43.4%

31.8%

27.9%

30.1%

37.3%

38.7%

44.3%

Table 3 Inhibition of AChE by single and simultaneous exposure to different concentrations of diazinon and/or its photoinduced by-products. The values in parenthesis represent concentration of organophosphates identified by UPLC–UV/VIS detection system after several time periods of 1  104 mol/l diazinon photodegradation and used in inhibition experiments. Inhibition (%) [concentration of inhibitor, mol/l] Irradiation time, min [10] 0

5

15

30

60

115

9.2 [2.0  105] /

/

/

20.2 [7.2  105] 41.1 [4.0  108] 0 [6.4  106]

14.1 [4.1  105] /

IMP

23.4 [9.0  105] 90.2 [2.0  107] 0 [3.2  106]

/

Diazoxon

25.1 [1.0  104] /

0 [1.1  105]

0 [2.7  105]

0 [4.5  105]

Simultaneous exposureb Diazinon + diazoxon Diazinon + IMP Diazoxon + IMP Diazinon + dazoxon + IMP

/ / / /

97.5 24.1 88.7 98.6

49.7 21.7 43.2 52.0

/ 15.8 / /

/ 8.0 / /

/ / / /

Single exposurea [10] Diazinon

a

Data from Ref. [10]. Concentrations of mixture components used in the experiments of simultaneous exposure are the same as given in the data for single exposure after related time of irradiation. b

M.B. Cˇolovic´ et al. / Pesticide Biochemistry and Physiology 100 (2011) 16–22 Table 5 Inhibition of AChE activity induced by simultaneous exposure to chlorpyrifos and chlorpyrifos-oxon in mixtures. Values in parenthesis represent organophosphateinduced inhibition measured separately (by single exposure). Inhibition (%) Chlorpyrifos (mol/l)

Oxon (mol/l)

3  107 (5.5%) 5  107 (11.2%) 1  106 (16.2%) 2  106 (26.0%) 3  106 (44.3%) 5  106 (53.0%)

15.9%

23.5%

33.3%

44.8%

53.0%

55.0%

20.6%

27.4%

35.7%

45.5%

54.0%

58.1%

26.9%

37.9%

37.5%

47.5%

57.0%

60.4%

38.1%

42.9%

46.3%

50.7%

61.0%

68.2%

45.0%

49.9%

60.9%

64.7%

71.0%

78.0%

56.9%

64.1%

71.2%

73.6%

77.1%

84.8%

3  109 5  109 1  108 2  108 3  108 5  108 (12.5%) (20.7%) (28.4%) (41.0%) (50.6%) (60.0%)

the sum of organophosphate-induced inhibitions assayed separately) are obtained at higher concentrations of the inhibitors. Bavcon Kralj et al. investigated chlorpyrifos photodegradation during 120 min using the xenon light (125 W Cermax Xenon parabolic lamp) [17]. The analysis of chlorpyrifos irradiated solutions suggested the presence of the chlorpyrifos-oxon, as the main photodegradation product, and 3,5,6-trichloro-2-pyridinol, hydrolysis product of chlorpyrifos as a result of bond (PO) cleavage. The oxidation product was detected in the first 3.5 min of the irradiation, achieved maximal value at 15 min and was observed even in the 120-min irradiated sample at the end of experiment. In a toxicity evaluation of photodegraded chlorpyrifos, it is worthy to study the simultaneous influence of both chlorpyrifos and its inhibiting oxidation product, present in each of the investigated chlorpyrifos irradiated sample solutions, on the activity of AChE as their target enzyme. Therefore, synthetic mixtures of chlorpyrifos and chlorpyrifos-oxon (pure commercial compounds) were composed in various concentration ratios and applied in the AChE assay (20 min preincubation time). The final concentrations of the parent compound and its oxo analogue in the medium assays were around or lower than their IC50 (20 min) values (Table 5). The results of the influence of simultaneous and single (separate) exposure toward the selected chlorpyrifos/chlorpyrifos-oxon concentrations on AChE activity are presented in Table 5. According to the results of variance analysis (One-way ANOVA), as in the case of diazinon/diazoxon mixtures investigation described above, additive inhibition effects were obtained in the presence of lower chlorpyrifos (mol/l)/chlorpyrifos-oxon (mol/l) concentrations: 3  107/3  109, 3  107/5  109, 3  107/1  108, 3  107/2  108, 3  107/3  108, 5  107/3  109, 5  107/ 5  109, 5  107/1  108, 1  106/3  109, 1  106/5  109, 2  106/3  109, 2  106/5  109. Higher concentrations of the investigated organophosphates induced antagonistic enzyme inhibitions (Table 5). The presence of non-inhibiting (Fig. 3) chlorpyrifos hydrolysis product (identified in the irradiated solutions), 3,5,6-trichloro-2-pyridinol, in the chlorpyrifos/chlorpyrifos-oxon mixtures does not alter their inhibition potencies against AChE (data not shown). 4. Discussion The results of our study show that diazinon, chlorpyrifos and their oxidation products (diazoxon and chlorpyrifos-oxon), as previously reported [10,17,31], inhibited AChE activity in concentrationdependent manner but with varying potencies (Table 1). These findings are in agreement with previously published results about AChE

21

inhibition by malathion and its decomposition products [17,26,32,33], as well as findings that organophosphates toxicity increased by their break-down products [34–37]. Obtained Hill coefficients nH > 1 for diazoxon, chlorpyrifos and its inhibiting by-product (chlorpyrifos-oxon) are in accordance with previously reported nH values for chlorpyrifos and another organophosphates [36], as well as allosteric effects between the top and the bottom of the gorge [38]. It is generally considered that organophosphate pesticides are substrate analogues of acetylcholine. Like the natural substrate, organophosphates enter the active site, which is a 20 Å deep gorge with a catalytic triad (Ser 200, Glu 327 and His 440) at the bottom [39]. As in acetylation, the organophosphate is split and hydroxyl group of Ser 200 i.e., the enzyme is phosphorylated. The difference in substrate behavior lies in the next step; while the acyl enzyme quickly hydrolyzed to regenerate the enzyme, dephosphorylation is very slow. As phosphorylated enzyme cannot hydrolyze neurotransmitter, the post-synaptic membrane remains depolarized, synaptic transmission does not work and organophosphates are often considered as irreversible inhibitors [38]. Although diazinon and diazoxon (as well as chlorpyrifos and chlorpyrifos-oxon) have similar structures, it is clear that combination of the substituents at the central phosphorous atom is responsible for their different inhibition power. The replacement of sulfur with oxygen in the cases of both diazoxon and chlorpyrifos-oxon causes an increased positive charge density on the central phosphorous atom, facilitating the nucleophilic attack of the –OH group of serine. The consequence is the higher inhibitory potency of diazinon (i.e., chlorpyrifos) inhibiting by-products, diazoxon and chlorpyrifos-oxon (Tables 1 and 2). Obtained results are consistent with previously published inhibition parameters (IC50, KI and k3/KI) for malathion and its inhibiting transformation products [26]. The additive inhibition of AChE activity obtained by simultaneous exposure to low concentrations of diazinon/diazoxon (i.e., chlorpyrifos/chlorpyrifos-oxon) (Tables 4 and 5) confirms that both inhibitors bind to the same binding site and that there is an excess of inhibitor binding sites. The antagonistic inhibition of electric eel AChE in the presence of mixtures of inhibitors at higher concentrations (diazinon/diazoxon P1  104/1  108 mol/l i.e., chlorpyrifos/chlorpyrifos-oxon P2  106/3  108 mol/l) can be explained by competition between inhibitors for a limited number of inhibitor binding sites on the enzyme. The complete inhibition of the enzyme activity by simultaneous exposure to combinations of diazinon, diazoxon and IMP detected in 5-min 1  104 mol/l diazinon irradiation solution can be attributed to 2  107 diazoxon inhibiting by 90.2% AChE activity (by separate exposure) (Table 3). The obtained results after 5 and 15 min of diazinon phototreatment (Table 3) are consistent with previously reported findings [20,26,32] that reducing remaining AChE activity during organophopsphates photodegradation can be mostly assigned to their transformation to the oxo analogues, significantly more potent AChE inhibitors related to their parent compounds. It could be concluded, prolonged irradiation time is required for complete removal of more toxic oxidation products, formed due to photochemical treatment of the parent compounds, which induce noticeable effect on AChE activity at low concentrations. Moreover, the low concentrations of these compounds exhibit additive inhibition effect in combination with their parent compounds. On the contrary, non-inhibiting photoinduced hydrolysis products do not alter the inhibitory efficiencies of the parent compounds and their inhibiting transformation products. Acknowledgments Authors would like to thank to the Ministry of Science and Technological Development of the Republic of Serbia for their financial support (Project No. 172023).

22

M.B. Cˇolovic´ et al. / Pesticide Biochemistry and Physiology 100 (2011) 16–22

References [1] C. Cox, Diazinon, J. Pesticide Reform. 12 (1992) 30–35. [2] M. O’Malley, Clinical evaluation of pesticide exposure and poisonings, Lancet 349 (1997) 1161–1166. [3] Y. Solberg, M. Belkin, The role of excitotoxicity in organophosphorous nerve agents central poisoning, Trends Pharmacol. Sci. 18 (1997) 183–185. [4] M.H. Fulton, P.B. Key, Acetylcholinesterase inhibition in estuarine fish and invertebrates as an indicator of organophosphorus insecticide exposure and effects, Environ. Toxicol. Chem. 20 (2001) 37–45. [5] E.O. Oruc, D. Usta, Evaluation of oxidative stress responses and neurotoxicity potential of diazinon in different tissues of Cyprinus carpio, Environ. Toxicol. Pharmacol. 23 (2007) 48–55. [6] N.S. El-Shenawy, F. El-Salmy, R.A. Al-Eisa, B. El-Ahmary, Amelioratory effect of vitamin E on organophosphorus insecticide diazinon-induced oxidative stress in mice liver, Pestic. Biochem. Physiol. 96 (2010) 101–107. [7] E.O. Oruc, Oxidative stress, steroid hormone concentrations and acetylcholinesterase activity in Oreochromis niloticus exposed to chlorpyrifos, Pestic. Biochem. Physiol. 96 (2010) 160–166. [8] B.D. Banerjee, V. Seth, A. Bhattacharya, S.T. Pasha, A.K. Chakraborty, Biochemical effects of some pesticides on lipid peroxidation and free-radical scavengers, Toxicol. Lett. 107 (1999) 33–47. [9] A. Goel, V. Dani, D.K. Dhawan, Protective effects of zinc on lipid peroxidation, antioxidant enzymes and hepatic histoarchitecture in chlorpyrifos-induced toxicity, Chem. Biol. Interact. 156 (2005) 131–140. [10] M. Cˇolovic´, D. Krstic´, S. Petrovic´, A. Leskovac, G. Joksic´, J. Savic´, M. Franko, P. Trebše, V. Vasic´, Toxic effects of diazinon and its photodegradation products, Toxicol. Lett. 193 (2010) 9–18. [11] M. Bavcon, P. Trebse, L. Zupancic-Kralj, Investigations of the determination and transformations of diazinon and malathion under environmental conditions using gas chromatography coupled with a flame ionisation detector, Chemosphere 50 (2003) 595–601. [12] A. Dannenberg, S.O. Pehkonen, Investigation of the heterogeneously catalyzed hydrolysis of organophosphorus pesticides, J. Agric. Food Chem. 46 (1998) 325–334. [13] S.O. Pehkonen, Q. Zhang, The degradation of organophosphorus pesticides in natural waters: a critical review, Crit. Rev. Environ. Sci. Technol. 32 (2002) 17– 72. [14] H.D. Burrows, L.M. Canle, J.A. Santaballa, S. Steenken, Reaction pathways and mechanisms of photodegradation of pesticides (Invited review), J. Photochem. Photobiol. B: Biol. 67 (2002) 71–108. [15] H.B. Wan, M.K. Wong, C.Y. Mok, Comparative study on the quantum yields of direct photolysis of organophosphorus pesticides in aqueous solution, J. Agric. Food Chem. 42 (1994) 2625–2630. [16] C. Zamy, P. Mazellier, B. Legube, Phototransformation of selected organophosphorus pesticides in dilute aqueous solutions, Water Res. 38 (2004) 2305–2314. [17] M. Bavcon Kralj, M. Franko, P. Trebše, Photodegradation of organophosphorus insecticides–investigations of products and their toxicity using gas chromatography mass-spectrometry and AChE-thermal lens spectrometric bioassay, Chemosphere 67 (2007) 99–107. [18] H. Shemer, K.G. Linden, Degradation and by-product formation of diazinon in water during UV and UV/H2O2 treatment, J. Hazard. Mater. B 136 (2006) 553– 559. [19] V.N. Kouloumbos, D.F. Tsipi, A.E. Hiskia, D. Nikolic, R.B. van Breemen, Identification of photocatalytic degradation products of diazinon in TiO2

[20]

[21]

[22] [23]

[24]

[25]

[26]

[27]

[28] [29]

[30] [31]

[32]

[33]

[34] [35] [36]

[37] [38]

[39]

aqueous suspensions using GC/MS/MS and LC/MS with quadrupole time-offlight mass spectrometry, J. Am. Soc. Mass Spectr. 14 (2003) 803–817. M. Bavcon Kralj, U. Cˇernigoj, M. Franko, P. Trebše, Comparison of photocatalysis and photolysis of malathion, isomalathion, malaoxon and commercial malathion-products and their toxicity studies, Water Res. 41 (2007) 4504–4514. G.A. Penuela, D. Barcelo, Photosensitized degradation of organic pollutants in water: processes and analytical applications, Trends Anal. Chem. 17 (1998) 605–612. C. Dominguez, J. Garcia, M.A. Pedraz, A. Torres, M.A. Galan, Photocatalytic oxidation of organic pollutants in water, Catal. Today 40 (1998) 85–101. R.T. Meijers, E.J. Oderwald-Muller, P.A.N.M. Nuhn, J.C. Kruithof, Degradation of pesticides by ozonation and advanced oxidation, Ozone Sci. Eng. 17 (1995) 673–686. Q. Zhang, S.O. Pehkonen, Oxidation of diazinon by aqueous chlorine: kinetics, mechanisms, and product studies, J. Agric. Food Chem. 47 (1999) 1760– 1766. K.D. Racke, Degradation of organophosphorus insecticides in environmental matrices, in: J.E. Chambers, P.E. Levi (Eds.), Organophosphates, Chemistry, Fate, and Effects, Academic Press, San Diego, 1992, pp. 47–73. ˇ olovic´, M. Bavcon Kralj, M. Franko, K. Krinulovic´, P. Trebše, V. D. Krstic´, M. C Vasic´, Inhibition of AChE by Malathion and some structurally similar compounds, J. Enz. Inh. Med. Chem. 23 (2008) 562–573. M. Mansour, E.A. Feicht, A. Behechti, K.W. Schramm, A. Kettrup, Determination photostability of selected agrochemicals in water and soil, Chemosphere 39 (1999) 575–585. R.H. Coupe, J.D. Blomquist, Water-soluble pesticides in finished water of community water supplies, J. AWWA 96 (2004) 56–68. G.L. Ellman, K.D. Courtney, V. Andreas, R.M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7 (1961) 88–90. R. Kitz, I. Wilson, Esters of methanesulfonic acid as irreversible inhibitors of acetylcholinesterase, J. Biol. Chem. 237 (1962) 3245–3249. M. Bavcon Kralj, P. Trebše, M. Franko, Oxidation as a pre-step in determination of organophosphorus compounds by the AChE-TLS bioassay, Acta Chim. Slov. 53 (2006) 43–51. ˇ olovic´, K. Krinulovic´, D. Djuric´, V. Vasic´, Inhibition of AChE by D. Krstic´, M. C single and simultaneous exposure to malathion and its degradation products, Gen. Physiol. Biophys. 26 (2007) 247–253. ˇ olovic´, M. Bavcon Kralj, P. Trebše, K. Krinulovic´, V. Vasic´, The D. Krstic´, M. C influence of malathion and its decomposition products on free and immobilized acetylcholinesterase, Russ. J. Phys. Chem. A 82 (2008) 663– 668. L. Brenner, Malathion, J. Pesticide Reform. 12 (1992) 19–27. T.C. Marrs, Organophosphate poisoning, Pharmacol. Ther. 58 (1993) 51–66. C.J. Smulders, T.J. Bueters, S. Vailati, R.G. van Kleef, H.P. Vijverberg, Block of neuronal nicotinic acetylcholine receptors by organophosphate insecticides, Toxicol. Sci. 82 (2004) 545–554. J. Bajgar, Laboratory diagnosis of OPs/nerve agent poisoning, Klin. Biochem. Metab 13 (2005) 40–47. Y. Boublik, P. Saint-Aguet, A. Lougarre, M. Arnaud, F. Villatte, S. EstradaMondaca, D. Fournier, Acetylcholinesterase engineering for detection of insecticide residues, Protein Eng. Des. Sel. 15 (2002) 43–50. J.L. Sussman, M. Harel, F. Frolow, C. Oefner, A. Goldman, L. Toker, I. Silman, Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholinebinding protein, Science 253 (1991) 872–878.