Environmental Toxicology and Chemistry, Vol. 20, No. 6, pp. 1228–1235, 2001 q 2001 SETAC Printed in the USA 0730-7268/01 $9.00 1 .00
EFFECTS OF ANABAENA SPIROIDES (CYANOBACTERIA) AQUEOUS EXTRACTS ON THE ACETYLCHOLINESTERASE ACTIVITY OF AQUATIC SPECIES JOSE´ MARI´A MONSERRAT,*† JOA˜O SARKIS YUNES,‡ and ADALTO BIANCHINI† †Departamento de Cieˆncias Fisiolo´gicas, Fundac¸a˜o Universidade Federal do Rio Grande (FURG), Rio Grande 96201-900, Brazil ‡Departamento de Quı´mica, Unidade de Pesquisa em Cianobacte´rias, FURG, Rio Grande 96201-900, Brazil ( Received 5 May 2000; Accepted 11 October 2000) Abstract—The effects of aqueous extracts from a cyanobacteria species, Anabaena spiroides, on fish (Odontesthes argentinensis), crab (Callinectes sapidus), and purified eel acetylcholinesterase (AChE) activity were studied. In vitro concentrations of A. spiroides aqueous extract that inhibited 50% of enzyme activity (IC50) were 23.0, 17.2, and 45.0 mg/L of lyophilized cyanobacteria for eel, fish, and crab AChE, respectively. Eel AChE inhibition follows pseudo-first-order kinetics, the same expected for organophosphorus pesticides. Inhibition of purified eel AChE using mixtures of bioxidized malathion and aqueous extract of A. spiroides showed a competitive feature (p , 0.05), suggesting that the toxin(s) could be structurally similar to an organophosphorus pesticide and that toxins present in the aqueous extract inhibit the active site of the enzyme. The inhibition recovery assays using 2-PAM (0.3 mM) showed that (1) bioxidized malathion inhibited 27.0 6 1.1% of crab and 36.5 6 0.1% of eel AChE activities; (2) with bioxidized malathion 1 2-PAM the registered inhibition was 13.2 6 2.1% and 3.7 6 0.5% in crab and eel AChE, respectively; (3) the aqueous extract from A. spiroides inhibited 17.4 6 2.2% and 59.9 6 0.5% of crab and eel AChE activity, respectively; and (4) aqueous extract 1 2-PAM inhibited 22.3 6 2.6 and 61.5 6 0.2% of crab and eel AChEs. The absence of enzyme activity recovery after 2-PAM exposure could imply that the enzyme aging process was extremely quick. Keywords—Neurotoxins
Acetylcholinesterase
Cyanobacteria
Anabaena spiroides
Aquatic species
In the Patos lagoon (Southern Brazil), several blooms of toxic cyanobacteria have been reported [14]. The estuarine region of this lagoon is inhabited by many crustacean and fish species that could be directly or indirectly exposed to such toxins. Among them, species such as the blue crab (Callinectes sapidus, Portunidae) and the silverside fish from Odontesthes genus (Atherinidae) have been used as experimental organisms in bioassays with organophosphorus pesticides [15–17]. Also, it must be stressed that oxime reagents can remove organophosphorus molecules bound to the active site of acetylcholinesterase, resulting in an increase of enzyme activity [18,19]. In fact, the augmented activity after addition of reactivators like pyridine 2-aldoxime (2-PAM) is generally interpreted as a previous exposure of the organism to organophosphorus pesticides, being also used as a biomarker of these kind of compounds [20–22]. The samples obtained in the present work were from heavy blooms of A. spiroides in lakes of South Brazil. Such heavy blooms developments are a common feature in drinking-water reservoirs and other inland body waters in the country [23]. Considering the facts described previously, two objectives were established in the present work: (1) to determine the toxicity and anticholinesterase effects of the cyanobacterium A. spiroides aqueous extracts in different animal species (mice, fish, and crab) in vivo and in vitro (additionally, some of these effects were compared with those promoted by an organophosphorus pesticide, malathion) and (2) to characterize the inhibition of purified eel AChE (V-S type) by the A. spiroides aqueous extract on exposure to the oxime reactivator pyridine 2-aldoxime (2-PAM).
INTRODUCTION
The contamination of aquatic ecosystems as consequence of human activities is a well-established fact. Indirect pollution processes like eutrophication favors cyanobacterial blooms, some of which are toxin producers [1]. These toxins are believed to be products of the secondary metabolism of cyanobacteria, and it has been hypothesized that they constitute a chemical defense against herbivores [2]. Neurotoxins are a special class of cyanobacterial toxins, including analogues of the neurotransmitter acetylcholine (anatoxin-a), sodium channel blockers (saxitoxin and neosaxitoxin), and the acetylcholinesterase inhibitor (anatoxin-a[s]) [1,2]. Acetylcholinesterase inhibitors produced by soil microorganisms (Streptomyces antibioticus) and anticholinesterase activity of marine zoanthids pigments were reported [3,4]. Also, cyanobacterium anatoxin-a(s) was considered as an organophosphorus pesticide analogue [1], being its chemical structure determined by Matsunaga et al. [5]. As expected, its physicochemical characteristics are similar to organophosphorus pesticides, that is, low stability at high pH and temperature [6]. Anatoxin-a(s) is a highly toxic compound, with a LD50 (intraperitoneal) in rats of about 20 mg/kg and 31 mg/kg in mice, respectively [6,7]. To date, only toxins and whole extracts from Anabaena flos-aquae and Anabaena spiroides were established to produce anticholinesterase or neurotoxic effects [8–11]. In nature, toxic effects, including death, were cited in animal populations after blooms dominated by Anabaena genus [8,12]. Also, in vitro experimental exposure to aqueous extract of A. flos-aquae to crustacean species like Daphnia pulicaria resulted in acetylcholinesterase inhibition [13].
MATERIALS AND METHODS
Sampling and preparation of A. spiroides aqueous extracts Anabaena spiroides samples were collected from an ornamental lake inside the University Campus (Fundac¸a˜o Univ-
* To whom correspondence may be addressed (
[email protected]). 1228
Anticholinesterase effects of A. spiroides aqueous extracts
ersidade Federal do Rio Grande, Rio Grande, RS, Southern Brazil) directly from a unialgal surface scum using 2-L clean plastic water bottles. Samples were frozen and lyophilized to dryness in a Micromodulyo lyophilizer (London, Edwards, UK). Dry cyanobacterial powders were conserved at 2308C for further tests. To prepare the aqueous extract, the protocol employed was based on Carmichael et al. [9]. Lyophilized scum sample was dissolved (0.5% w/v) in absolute ethanol and sonicated in cold during 1 min at 20 kHz and maximum amplitude. Then the ethanolic extract was passed four times through a Whatman acetate filter (0.45-mm pore; Clifton, NJ, USA) and dried on rotary evaporator at 408C. The sample was resuspended with chloroform (1% w/v) and washed 10 times (2 ml each) with double-distilled water (pH adjusted to 3.30). At every step, the aqueous fraction was stored. Then the aqueous fraction was passed through a SepPack C-18 (Millipore, Milford, MA, USA) chromatographic column in order to extract toxins other than neurotoxins and chloroformic residues. This procedure was adopted as we have detected the presence (5.4 mg/g dry powder) of hepatoxins (microcystins) in the bloom of A. spiroides employed. The hepatoxin was quantified using a polyclonal enzyme-linked immunosorbent assays commercial kit (EnviroGardt, Strategic Diagnostic, Newark, DE, USA). The aqueous eluate was kept and then concentrated at 408C to obtain a solution of 25.0 mg/ml of lyophilized algae.
In vivo acute toxicity tests with A. spiroides whole extracts Whole aqueous extracts of A. spiroides were tested in male Swiss albine mice (19.0–21.0 g), using the liophilized powder described previously and injected (intraperitoneal; 0.2 ml) to obtain a 62.5-, 125-, 250-, 500-, or 1,000-mg/kg dose. Control mice were injected (intraperitoneal) with saline water (0.9% NaCl in distilled water). Animals were observed for neurotoxic symptoms (e.g., tremors, muscle contractions, and salivation) during the first 2 h, and deaths were recorded until the first 24 h.
Environ. Toxicol. Chem. 20, 2001
method employed for determination of enzyme activity was based on Ellman et al. [25]. Acetylthiocholine iodide (Sigma) was employed as substrate in a final concentration of 8.0 3 1024 M (O. argentinensis and V-S) or 1.6 3 1023 M (crab). The Ellman’s reagent 5,59-dithio-bis(2-nitrobenzoic acid) (Sigma) was used to develop a yellow color in a final concentration of 4.0 3 1024 M (O. argentinensis and V-S) or 8.0 3 1024 M (crab). Absorbance increments were registered at 412 nm during 90 s. Homogenate protein content was determined using a commercial kit (Microprotet, Doles, Brazil), based on the method described by Bradford [26]. The IC50 values were calculated employing nonlinear regression, according to the following formula [27]: Vmax · IC50 IC50 1 AEC where AEC represents the aqueous extract concentration (mg/ L) and Vmax is the estimated enzyme activity in absence of the inhibitor (control). The estimates of IC50 from the different AChEs were compared considering the higher IC50/lower IC50 ratio (a 5 0.05) [28].
In vitro inhibition kinetics of AChE by A. spiroides aqueous extract Several authors showed that AChE inhibition by organophosphorus pesticides follows pseudo-first-order kinetics [29,30]. Following this model, the logarithm of enzyme activity (n) decreases linearly as the incubation time increases at fixed inhibitor concentrations. Also, the reciprocal of the slope for the estimated linear function at each concentration (Dt/ln n) will be linearly related to the reciprocal of the concentration of the assayed inhibitor. The last linear function allows estimation of two constants, the affinity equilibrium constant (Ka) and the bimolecular inhibition constant (Ki). These constants arise from a model proposed by Main [29], which considered that the inhibition of an enzyme (E) by an inhibitor (I) follows the reaction
Enzyme extraction Adult fish (Odontesthes argentinensis) and crab (Callinectes sapidus) were obtained from local fisherman. We employed 45 fish and 36 crab individuals (both sex), with a mean weight (6 1 standard deviation) of 157.0 6 7.1 g and 81.7 6 4.2 g, respectively. Fish brains and crab thoracic ganglia were extracted in cold (48C) and stored in liquid nitrogen until use. A 10% (w/v) homogenate for both tissues was prepared, using cold phosphate buffer (0.05 M for fish and 0.25 M for crab) in 20% glycerol and pH 7.40, according to previous works [15,24]. The homogenate was then centrifuged at 1,000 g (48C) during 15 min (fish) or 20 min (crab). The supernatant was again centrifuged at 12,800 g (48C) for the same lengths of time. The supernatant of the last centrifugation was used as enzyme source. Eel-purified AChE (V-S type) was purchased from Sigma (St. Louis, MO, USA) and dissolved in the same buffer used for O. argentinensis.
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K1
Kp
u (EI)R → (EI)I E1IU K21 R
where (EI) represents a reversible enzyme-inhibitor complex and (EI)I an irreversible one. An affinity equilibrium constant is defined as Ka 5 K21 /K1 , where a lower Ka implies a higher affinity. The term Kp represents a phosphorylation constant, defined as Kp 5 Ki·Ka, where Ki is the bimolecular inhibition constant. In order to estimate the previously cited inhibition constants, five concentrations ranging from 35.5 to 568.0 mg/L of A. spiroides aqueous extract were tested, at least in duplicate. The inhibition time ranged from 5 to 90 min, according to the concentration of the aqueous extract employed. Enzyme activity was determined as explained previously at 258C and pH 7.40. Only V-S type eel AChE was assayed.
In vitro AChE inhibition by A. spiroides aqueous extract
In vitro competitive assays with A. spiroides aqueous extract and AChE inhibitors
The effect of aqueous extract from A. spiroides on fish and crab AChE was determined in vitro by estimating the extract concentration that inhibited 50% of enzyme activity (IC50). Extract concentrations ranged from 0 (control) to 142.0 mg/ L. Enzymes were incubated with the extract for 1 h at 258C and pH 7.40 before determinations, at least in duplicate. The
Two different assays were conducted to establish whether the enzyme inhibition by the A. spiroides aqueous extract was modified in the presence of well-known AChE inhibitors (oxidized malathion and eserine). The first assay was performed to determine whether the inhibition of eel V-S type and crab AChE activities with mix-
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tures of A. spiroides aqueous extract and oxidized malathion (or eserine) were additive. The inhibition of enzyme activity in respect to control activity (considered as 100%) is determined after 1 h exposure to eserine, A. spiroides aqueous extract, mixture of eserine (Sigma; 99% purity) and the aqueous extract, oxidized malathion, and mixture of oxidized malathion and aqueous extract. Eserine inhibition assays was conducted only with V-S type AChE. Final concentration of inhibitors (alone or in mixture) for V-S type assays were A. spiroides aqueous extract 35.5 mg/L oxidized malathion 2.1 3 1025 M and eserine 5.6 3 1026 M. For crab AChE assays, final concentration (alone or in mixture) of A. spiroides and oxidized malathion was 17.7 mg/L and 8.2 3 1025 M, respectively. After 1 h of exposure, aliquots of inhibited enzyme were incubated with 3.0 3 1024 M of 2-PAM (Sigma; 99% purity) during 1 h, at 258C and pH 7.40. The remaining inhibition was calculated considering control activity exposed only to 3.0 3 1024 M of 2-PAM as being of 100%. Inhibition data from both AChE, with or without 2-PAM treatment, were compared by means of analysis of variance, followed by Scheffe´ a posteriori test (a 5 0.05). The normality and variance homogeneity were checked before the analysis. The second assay was performed to determine whether the inhibition of V-S type AChE activity by A. spiroides aqueous extract competed with oxidized malathion inhibition. Purified AChE was exposed during 1 h at 258C and pH 7.40 to different oxidized malathion concentrations ranging from 4.2 to 84.5 3 1026 M, with or without the addition of A. spiroides aqueous extract (35.0 mg/L). The inhibition curves were statistically compared in order to evaluate competition between the two inhibitors. At the concentration assayed, the aqueous extract from the cyanobacteria alone produced an inhibition of 61.3%. In both assays a commercial formulation (Nitrosint from Indol, Curitiba, Brazil; 500 g/L active product) of malathion (99% purity) was employed. Malathion (0,0-dimethyl S-[1,2dicarboethoxyethyl] phosphorodithioate) was bioxidized before the inhibition assays in order to acquire its anticholinesterase properties [18]. An enzymatic preparation as described by Mirer et al. [31] and Levine and Murphy [32] was employed, consisting of Wistar rat liver homogenates incubated at 378C with malathion, 1.4 3 1023 M of nicotinamide adenine dinucleotide phosphate (Sigma), 3.8 3 1023 M of glucose-6phosphate (Sigma), and 2.0 3 1024 M ethylenediaminetetraacetic acid in phosphate buffer 0.04 M, pH 7.60.
The AChE inhibition by increasing concentrations of cyanobacterial extracts follows a dose–response relationship (r2 $ 0.93; Fig. 1A). The aqueous extract IC50 for crab AChE was significantly higher (p , 0.05) than those from O. argentinensis and V-S AChE. The estimated IC50 values (6 1 standard error [SE]) were 23.0 6 4.4, 17.2 6 2.4, and 45.0 6 2.8 mg/L for V-S type, O. argentinensis, and C. sapidus AChEs, respectively (Fig. 1B).
Aging determination of purified eel AChE (V-S type) using A. spiroides aqueous extract or bioxidized malathion
In vitro inhibition kinetics of AChE by A. spiroides aqueous extract
Purified eel AChE (V-S type) was inhibited with bioxidized malathion or A. spiroides aqueous extract and then reactivated with 2-PAM. In the assays with the pesticide, eel AChE was incubated for 30 min (258C; pH 7.40) with bioxidized malathion (4.7 3 1024 M). With these experimental conditions, an inhibition of 93% was obtained. After the exposure period, the enzyme:oxidized malathion mixture was diluted 50 times. The inhibitor remaining after the dilution would give less than 5% inhibition after 1 h exposure. After the dilution and at several incubation times (30–360 min), aliquots of the mixture were mixed with 2-PAM (1.5 3 1024 M) and reactivated during 1 h (258C; pH 7.40). At the same incubation times, aliquots of the enzyme:oxidized malathion mixture were incubated without adding 2-PAM. After the incubation, the absorbance increments per minute at 412 nm were immediately determined in triplicate, using the same substrate and 5,59-dithio-bis(2-
As shown in Figure 2, the time course of AChE activity inhibition fits pseudo-first-order kinetics (r2 $ 0.98) at each concentration of A. spiroides aqueous extract assayed. The bimolecular inhibition (Ki) constant, the affinity (Ka), and phosphorylation (Kp) constants were estimated as 4.4 3 1024 mg/ L/min, 381.8 mg/L, and 1.7 3 1022/min, respectively. In this case, the term phosphorylation constant (Kp) is used assuming that the A. spiroides aqueous extract acts as an organophosphorus pesticide.
nitrobenzoic acid) concentrations previously mentioned for VS type AChE. The same procedure, exposure time, and 2-PAM concentration were used when A. spiroides aqueous extract (520.0 mg/L) was assayed. With the extract concentration employed, an inhibition of 88% was registered after 30 min of incubation. The following formula was employed in order to estimate the aging rate (kag) [20,33]: ln
(A rt /0.69 2 A it ) · 100 5 kag t (A r0 2 A i0 )
where Art and Ait represent the absorbance increments per minute at time t (t 5 30, . . . , 360 min) for enzyme with (Art) and without (Ait) 2-PAM treatment, respectively. The absorbance of the 2-PAM-reactivated enzyme was corrected since previous assays (n 5 3) showed that a final concentration of 1.5 3 1024 M of 2-PAM inhibited 31% of enzyme activity. Finally, Ar0 and Ai0 represent the absorbance increments after the 30-min inhibition period in control (Ar0) and oxidized malathion or A. spiroides aqueous extract (Ai0) treatments, respectively. RESULTS
In vivo acute toxicity tests with A. spiroides whole extracts Whole cell aqueous extracts of Anabaena spiroides produced a 24-h LD50 of 369.0 mg/kg. Deaths at concentrations near and above the LD50 occurred within minutes and produced symptoms as tremors and intense muscle contractions. At values below the LD50, deaths also occurred, but irregularly within the 24 h of test. No neurotoxic signs or deaths were observed in the control animals during the experimental period.
In vitro acetylcholinesterase inhibition with A. spiroides aqueous extract
Competitive assays with A. spiroides aqueous extract and AChE inhibitors A mean inhibition (61 SE) of 82.6 6 0.2, 59.9 6 0.5, and 36.5 6 0.1% of V-S type AChE activity was observed after 1 h exposure to eserine, aqueous extract of A. spiroides, and bioxidized malathion, respectively (Fig. 3A). With eserine plus
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Fig. 1. (A) Mean inhibition of enzyme activity for the different acetylcholinesterase (AChE) sources, at different concentrations of the extract. (B) Estimated in vitro concentrations (mg/L) of Anabaena spiroides aqueous extract that inhibited 50% of AChE activity (IC50) from eel (V-S), brain of Odontesthes argentinensis (OA), and thoracic ganglia of Callinectes sapidus (CS). Same letter indicates absence of significant differences between IC50 values (p . 0.05).
the A. spiroides aqueous extract and bioxidized malathion plus the A. spiroides aqueous extract mixtures, inhibitions of 86.6 6 0.2 and 71.6 6 0.3% were registered, respectively. After 2-PAM exposure, the mean (61 SE) inhibition registered was 3.7 6 0.5 and 61.5 6 0.2% in the bioxidized malathion and A. spiroides aqueous extract treatments, respectively. In a mixture of both, the inhibition was of 53.5 6 0.3%. Statistical comparisons failed to show significant differences in the A. spiroides aqueous extract treatment, before and after adding 2-PAM (p . 0.05). All other treatments showed inhibition values statistically lower (p , 0.05) after 2-PAM addition (Fig. 3A). When crab AChE was employed as enzyme source, a mean inhibition (61 SE) of 17.4 6 3.8 and 27.0 6 1.1% was registered after 1 h exposure to A. spiroides aqueous extract and bioxidized malathion, respectively (Fig. 3B). The mixture of both inhibited 36.6 6 2.2 % of enzyme activity. After adding 2-PAM, the mean percentage inhibition (61 SE) was 22.3 6 2.1, 13.2 6 2.0, and 24.6 6 2.8% in A. spiroides aqueous extract, bioxidized malathion, and mixture treatments. As in the assay with V-S type AChE, only the A. spiroides aqueous extract treatment showed absence of statistical differences (p . 0.05) before and after 2-PAM addition (Fig. 3B).
Fig. 2. (A) Inhibition of purified eel acetylcholinesterase (AChE, VS type) activity (n), expressed as mmoles substrate/min/mg protein, at different concentrations of Anabaena spiroides aqueous extract and incubation times. (B) Regression function employed to estimate the affinity equilibrium (Ka) and bimolecular inhibition (Ki) constants, according to the model proposed by Main [29]. Dt/(2.303.log10 n) represents the inverse of the slope of the natural logarithm of enzyme activity against time. ●, 35.5 mg/L; V, 71.0 mg/L; n, 142.0 mg/L; ▫, 284.0 mg/L; m, 568.0 mg/L.
Inhibition of the V-S type AChE activity increased steadily from 2.7 to 72.5% after 1 h exposure to bioxidized malathion concentrations varying between 4.2 and 84.5 3 1026 M (Fig. 4A). At the same pesticide concentrations, the enzyme inhibition also increased after addition of 35.0 mg/L of A. spiroides aqueous extract but at a lower rate. Covariance analysis showed that the slopes of the inhibition curves were statistically different (p , 0.05).
Aging of purified eel AChE (V-S type) using A. spiroides aqueous extract or bioxidized malathion The recovery of AChE activity after 2-PAM exposure was different when bioxidized malathion and A. spiroides aqueous extract were used as inhibitors. As seen in Figure 4B, 2-PAM treatment could not reactivate enzyme activity after exposure
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Fig. 3. (A) Mean inhibition of purified eel acetylcholinesterase (AChE, V-S type) after 1 h exposure to different inhibitors. E: eserine; T: Anabaena spiroides aqueous extract; E1T: simultaneous inhibition with eserine and the aqueous extract; M: bioxidized malathion; M1T: simultaneous inhibition with bioxidized malathion and the aqueous extract. (B) Mean inhibition of AChE activity from thoracic ganglia of Callinectes sapidus after 1 h exposure to different inhibitors. Symbols are those from Figure 3A. In (A) and (B), same letters indicate absence of significant differences between mean values (p . 0.05). Inhibition before and after 2-PAM addition (3.0 3 1024 M) is also indicated in both figures.
J.M. Monserrat et al.
Fig. 4. (A) Inhibition of purified eel acetylcholinesterase (AChE, VS type) activity after 1 h exposure to bioxidized malathion concentrations ranging from 4.2 to 84.5 3 1026 M. Also represented is the response of mixtures of 35.0 mg/L of Anabaena spiroides aqueous extract and bioxidized malathion. The two inhibition curves are significantly different (p , 0.05). ●, oxidized malathion; ,, oxidized malathion 1 aqueous extract. (B) Activity of purified eel AChE (VS type) previously inhibited (30 min) with bioxidized malathion (4.7 3 1024 M) or by the A. spiroides aqueous extract (520.0 mg/L) and after treatment with 2-PAM (1.5 3 1024 M). ●, oxidized malathion; ,, aqueous extract. DISCUSSION
to aqueous extract from the cyanobacterium. Activity inhibition remained within 1 to 6% at the assay times after the inhibition period. Different assay conditions were tested (inhibition time, final 2-PAM, and aqueous extract concentration), and in all conditions, similar results were obtained, confirming that enzyme was quickly inactivated and 2-PAM exposure was ineffective for reactivating (data not shown). On the other hand, AChE activity after bioxidized malathion inhibition was reactivated more than 70% with 2-PAM after 30 min. The reactivation decreased with time, being of 20% after 6 h. The kag was estimated to be 4.4 3 1023 6 2.6 3 1024/min (r2 5 0.92) (Fig. 4B).
Matsunaga et al. [5] first determined the molecular structure of the anatoxin-a(s). Interestingly, its structure fits with that of organophosphorus pesticides, including a P5O bond, which interacts with the esteratic site of AChE [2]. This constitutes a qualitative difference from some organophosphorus pesticides like malathion, methyl parathion, or parathion, where their anticholinesterase properties are acquired after bioxidation. This process is mediated by the cytochrome P450, resulting in the oxidation of the P5S bond of these pesticides [19]. Livingstone [34] has pointed out that different levels of cytochrome P450 in aquatic species could explain differences in resistance to pollutants. In this context, neurotoxins like
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Table 1. Estimated phosphorylation constant (Kp) for different organophosphorus pesticides (or their oxon analogue, ‡) and acetylcholinesterase (AChE) sources. Assay temperature was 258C (*) or 278C (†) Inhibitor DFPa Fenitrothion (‡) Malathion (‡) Methyl parathion (‡)
Kp /min
Enzyme source
Reference [11] [37] [37] [37]
22.2 (†)
Purified eel AChE Purified eel AChE Purified eel AChE Purified eel AChE SMb of brain from Ictalurus sp. SM of brain from Ictalurus sp.
2.0 (*)
Purified eel ACHE
[11]
1.7 3 1022 (*)
Purified eel AChE
Present work
10.0 6.9 52.2 27.4 14.7
(*) (*) (*) (*) (†)
Parathion (‡) Anatoxin-a(s), 70–80% purity Aqueous extract from A. spiroides a b
[30] [30]
DFP 5 diisopropyl fluorophosphate. SM 5 synaptosomal membranes.
anatoxin-a(s) would not depend of the oxidative metabolism capabilities of the target organism to exert a noxious effect. Thus, differential affinities of neurotoxins to AChEs should be the key factor determining their toxicity. In the in vitro assays performed in this work, the sensitivity of O. argentinensis AChE was higher than that presented by C. sapidus one. Previous results in our laboratory also showed a higher sensitivity to eserine of AChE from fish (Odontesthes bonaeriensis) than crustacean AChEs. In other crustacean species (D. pulicaria), an AChE inhibition of about 50% after exposure to 1.12 mg/L of A. flos-aquae aqueous extract was determined [13]. A lower inhibition was found for C. sapidus AChE (IC50 5 45.0 mg/L) in the present work. Although specific differences in AChE sensitivity to neurotoxin cannot be discarded, these differences probably reflect the toxin contents of both cyanobacteria species. Extraction methods for cyanobacterial blooms of bacteriafree cultures for toxin(s) analysis of lyophilized Anabaena genus are very diverse [9,12,13]. Also, few studies have analyzed anatoxin-a(s) using high-performance liquid chromatography or mass spectral analysis. However, consistent support is obtained when neurotoxic effects, including signs of salivation, lacrimation, loss of muscle coordination, trembling, and so on, and AChE inhibition are observed along with the assays [8,9,12,13,35]. Although the present work cannot conclude that the inhibitory effects on AChE activity are due solely to the presence of anatoxin-a(s), the results obtained indicate the existence in the A. spiroides aqueous extract of compound(s) that produce similar effects to those described for organophosphorus pesticides [18]. This conclusion is based on the following facts: (1) The inhibition of AChE activity follows a dose–response relationship; (2) when applied in mixture, eserine or bioxidized malathion and A. spiroides aqueous extracts inhibited AChE activity to a lesser extent than when applied separately; and (3) the inhibition curve of eel V-S AChE activity after bioxidized malathion exposure was displaced when A. spiroides aqueous extract was added, indicating a clear competition between the inhibitors. Neurotoxic symptoms were observed in mice injected with cyanobacteria aqueous extract. Cook et al. [7,35] suggested that the polar character of neurotoxins like anatoxin-a(s) should make difficult the crossing of the blood–brain barrier by this molecule. In fact, it seems that toxic effects are exerted by the AChE inhibition of peripheral sites. The symptoms that we observed could be due to the presence of different toxin(s)
being present in the aqueous extract. Preliminary tests using high-performance liquid chromatography failed to detect the presence of anatoxin-a(s) (Matthiensen, unpublished data). As the enzymatic assays indicate the presence of an inhibitory toxin(s) in the aqueous extract, it also suggests the use of this enzyme assay as a valuable and quick tool for the detection of anatoxin-a(s). The inhibition kinetics of AChE by A. spiroides aqueous extract resembles those of an organophosphorus pesticide. At this point, it is important to compare the phosphorylation constant (Kp) estimated when using the A. spiroides aqueous extract with that obtained with organophosphorus pesticides. Note that, in order to compare the bimolecular inhibition (Ki) and the affinity constant (Ka), the molar concentration of the inhibitor used must be known. Several factors influence inhibitory power of organophosphorus pesticides. Generally, it is accepted that Ka quantifies steric constraints of inhibitors to form the complex with AChE, whereas the phosphorylation constant (Kp) quantifies reactivity of inhibitors against the active site of the enzyme [36]. Taking into account the Kp estimates when using A. spiroides aqueous extract (Table 1), it can be concluded that the cyanobacteria toxin(s) present lower reactivity against AChE than organophosphorus pesticides. Also, some evidence showed that steric constraints of the neurotoxin are higher than that presented by several organophosphorus pesticides. According to Mahmood and Carmichael [11], the Ka for anatoxin-a(s) is 1.98 3 1023 M using purified eel AChE as enzyme source. For the same enzyme type, de Bruijn and Hermes [37] found a Ka of 1.5 3 1024 M and 2.2 3 1025 M for the oxon analogues of methyl parathion and malathion, respectively. In another fish AChE (Ictalurus sp.), Ka values of 2.4 3 1024 and 2.0 3 1024 M were estimated for methyl paraoxon and paraoxon, respectively [30]. These values and those showed in Table 1 seem to reinforce the idea that the cyanobacteria toxin(s) posses both lower affinity and reactivity to AChE than do synthetic organophosphorus pesticides. As can also be seen in Table 1, striking differences exist in a previous Kp estimate [11] and in our work, differences that cannot be explained at this time. Coincidentally, Cook et al. [35] reported a toxin extracted from A. flos-aquae that produced cholinergic effects, but with a different high-performance liquid chromatography retention time than a standard of anatoxin-a(s). More studies on different molecular forms of
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between Ka and Kp constants with the kag rates remains to be studied since, as mentioned previously, both the affinity (Ka) and reactivity (Kp) constants seem to be lower for anatoxina(s) than for organophosphorus pesticides. Acknowledgement—J.M. Monserrat is a guest researcher of CNPq (300492/98-7 [NV]). A. Bianchini and J.S. Yunes are research fellows of CNPq (300536/90-9 and 301143/92-7, respectively). The research was supported by a grant from Fundac¸a˜o de Amparo a` Pesquisa do Estado do Rio Grande do Sul (98/1063.2). The authors wish to thank to L.E.M. Nery, A. Matthiensen, and two anonymous reviewers. REFERENCES
Fig. 5. Substituents and leaving group of two organophosphorus pesticides (fonofos and crufomate) and anatoxin-a(s) [2,20]. Ethoxy or methoxy substituents are indicated in bold. According to Mason et al. [20], organophosphorus pesticides with methoxy substituents possess a higher aging rate.
this kind of neurotoxins and their inhibitory power on AChE are due. Considering the 2-PAM reactivation assays, it must be pointed out that the success of AChE reactivation depends on whether the inhibited enzyme has undergone the aging process. Enzyme aging results in the dealkylation of the phosphorylated serine group at the active site of AChE after organophosphorus inhibition [20,33]. After aging, the enzyme is no longer reactivable. Several factors affect aging rate, including temperature and kind of organophosphorus molecule [20]. The estimated aging rate for bioxidized malathion (4.4 3 1023/min) was very similar to that estimated by Mason et al. [20] when evaluating inhibition of human plasma cholinesterase by fonofos at almost the same temperature (228C). Inversely, the absence of AChE recovery after 2-PAM treatment previously inhibited with cyanobacteria extract suggests an extremely quick aging of the enzyme. Mason et al. [20] pointed out that depending on the chemical structure of the substituents (other than the leaving group) around the phosphorus atom, the aging rate can be quite different. The authors experimentally determined that pesticides with methoxy substituents showed higher aging rates than pesticides with ethoxy or methoxy-phenyl substituents. Interestingly, anatoxin-a(s) contains methoxy substituents [2], suggesting that this neurotoxin structurally possesses the potential for a high aging rate of AChE (Fig. 5). The relationships
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