Original Paper Pharmacology 2014;94:157–162 DOI: 10.1159/000367897
Received: October 18, 2014 Accepted after revision: August 25, 2014 Published online: October 3, 2014
In vitro Effects of the Organophosphorus Pesticide Malathion on the Reactivity of Rat Aorta Marco Tulio Menezes de Carvalho Andrea Carla Celotto Agnes Afrodite Sumarelli Albuquerque Luciana Garros Ferreira Verena Kise Capellini Ana Paula Cassiano Silveira Tales Rubens de Nadai Paulo Roberto Barbosa Evora Department of Surgery and Anatomy, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil
Abstract Background and Purpose: There is a remarkable paucity of studies analyzing the role of the endothelium-derived relaxing factors on the vascular effects of organophosphates. This study was carried out to evaluate the vascular effects of malathion and the role of nitric oxide (NO) and prostacyclin (PGI2). Methods: Vascular reactivity measuring isometric forces in vitro (‘organ chambers’) and flow cytometry (cells loaded with DAF-FM DA) were used. Results: In rat thoracic aorta segments contracted with phenylephrine (Phe) (10–7 mol/l), malathion (10–10 to 10–5 mol/l) induced concentration-dependent relaxation in arteries with intact endothelium (n = 7; p < 0.05). Malathion-mediated relaxation was blocked by N-nitro-L-arginine methyl ester (L-NAME; 10–4 mol/l), a nonspecific NO synthase inhibitor, and/or indomethacin (10–5 mol/l), a nonspecific cyclooxygenase inhibitor (n = 10, p < 0.05). In thoracic aorta rings, with and without endothelium, Phe (10–10 to 10–5 mol/l) evoked concentration-dependent contraction, which was reduced in the presence of malathion. In rings with or without endothelium, in-
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cubated with malathion, L-NAME and indomethacin, the Phe-induced contraction was restored. The role of NO was confirmed using flow cytometry. Malathion evokes endothelium-dependent relaxation through the M1 muscarinic receptor, since this relaxation was clearly blocked by atropine (M1 and M2 blocker) and pirenzepine (M1 blocker), but was less blocked by gallamine (M2 blocker) or 4-DAMP (M3 blocker). Conclusions: These findings suggest that the organophosphate compound effects on vascular reactivity depend of NO and PGI2. © 2014 S. Karger AG, Basel
Background
Organophosphorus compounds are chemicals present in pesticides and are known acetylcholinesterase inhibitors. The main toxic action of organophosphates includes inhibition of the cholinesterase enzyme promoting muscarinic and nicotinic effects. In the cardiovascular system, the nicotinic manifestations are tachycardia and hypertension while the muscarinic effects include bradycardia and hypotension [1]. In the respiratory system, cholinesterase inhibition is characterized by increased bronchial secretions, bronchospasm and the occurrence of respiratory muscle P.R.B. Evora Department of Surgery and Anatomy, Ribeirão Preto Medical School University of São Paulo, Avenida Bandeirantes, 3900 Ribeirão Preto, SP 14048-900 (Brazil) E-Mail prbevora @ gmail.com
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Key Words Endothelium · Nitric oxide · Prostacyclin · Vasorelaxation · Organophosphorus · Pesticides · Malathion
Materials and Methods Materials Indomethacin, phenylephrine (Phe), atropine, 4-DAMP, gallamine, pirenzepine and malathion were purchased from Sigma Chemical Company (St. Louis, Mo., USA). N-nitro-L-arginine methyl ester (L-NAME) was obtained from Calbiochem (San Diego, Calif., USA). Thiopental sodium was purchased from Cristália (São Paulo, Brazil). The salts used for the preparation of Krebs solution were furnished by Vetec Química Fina Ltd. (Duque de Caxias, Brazil). All drugs were prepared with distilled water, except for indomethacin that was dissolved in ethanol, thiopental sodium that was dissolved in physiological saline (0.9% NaCl) and 4-DAMP that was dissolved in dimethyl sulfoxide. Animals The experimental procedures and animal handling were reviewed and approved by the Institutional Animal Care Review Board (CETEA – Ethics Committees of Animal Experiments of the Ribeirão Preto Medical School, University of São Paulo). This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Before the experiments, the rats were housed under standard laboratory conditions (12-hour dark cycle at 21 ° C), with free access to food and water.
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Vessel Preparation and Isometric Tension Recording Male Wistar rats (230–280 g) were anesthetized with thiopental sodium (40 mg/kg i.p.), followed by laparotomy for exsanguination via the abdominal aorta and thoracotomy for thoracic aorta harvesting. The thoracic aorta was carefully dissected free of connective tissue and immersed in cooled and oxygenated Krebs solution (NaCl: 118.0 mmol/l, KCl: 4.7 mmol/l, CaCl2: 2.5 mmol/l, KH2PO4: 1.2 mmol/l, MgSO4: 1.66 mmol/l, glucose: 11.1 mmol/l, NaHCO3: 25.0 mmol/l, pH 7.4). Ring segments of the thoracic aorta (4 mm in length) were prepared with proper care, so as not to touch the intimal surface. In some segments, the endothelium was removed by gently rubbing the intimal surface of the blood vessel with a pair of watchmaker’s forceps. This procedure removes endothelium but does not affect the ability of the vascular smooth muscle to contract or relax. Endothelial integrity was assessed qualitatively by the level of relaxation caused by ACh (10–5 mol/l) in the presence of contractile tone induced by Phe (10–7 mol/l). Thoracic aorta segments were mounted in organ chambers (10 ml) filled with Krebs solution maintained at 37 ° C and bubbled with 95% O2/5% CO2 (pH 7.4). Each arterial ring was suspended by two stainless steel clips placed through the lumen. One clip was anchored to the bottom of the organ chamber while the other was connected to a strain gauge for the measurement of the isometric force using Grass FT03 (Grass Instrument Company, Quincy, Mass., USA). The rings were placed at an optimal length-tension of 2 g and allowed to equilibrate for 60 min with the bath fluid being changed every 15–20 min. For studies of endothelium-intact vessels, the ring was discarded if relaxation with ACh was not 80% or greater. For studies of endothelium-denuded vessels, rings were discarded if there was any measurable level of relaxation. Sequentially, each ring was washed and re-equilibrated for 30 min. Aortic rings were then precontracted with Phe (10–7 mol/l), and cumulative concentration-response curves were obtained after a stable degree of contraction was attained. Rat aortic rings were incubated with malathion (10–4 mol/l) before cumulative concentration-response curves of Phe were studied.
Study Design The study was designed using two main protocols. In the first protocol, cumulative concentration-response curves were obtained for malathion (10–10 to 10–4 mol/l) in aortic rings, with and without endothelium, precontracted to a stable plateau with Phe (10–7 mol/l). In addition, cumulative concentration-response curves for Phe (10–10 to 10–5 mol/l) and ACh (10–10 to 10–5 mol/l) were obtained in endothelium-intact and endothelium-denuded aortic rings, in the presence or absence of malathion (10–4 mol/l). In the second protocol, concentration-response curves were also obtained after pre-incubating the vascular rings with L-NAME (10–4 mol/l), a nonspecific nitric oxide synthase (NOS) inhibitor; indomethacin (10–5 mol/l), an unspecific cyclooxygenase inhibitor; atropine (10–6 mol/l), a competitive nonselective antagonist at central and peripheral muscarinic acetylcholine receptors; 4-DAMP (10–6 mol/l), an M3 preferring cholinergic receptor antagonist; gallamine (10–5 mol/l), a muscle relaxant, allosteric muscarinic receptor antagonist, and pirenzepine (10–5 mol/l), a selective M1 muscarinic acetylcholine receptor antagonist. In the relaxation study, changes in the vascular wall tension were expressed as a percentage of relaxation of the maximal contraction following exposure to Phe, a convention that corrects for interanimal variability in the response of
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paralysis [2]. Although the effects of organophosphates are best known as resulting from cholinesterase inhibition, in vitro studies have demonstrated organophosphate-mediated vasodilator effects and airway constriction, suggesting that organophosphates have peripheral activity independent of acetylcholinesterase inhibition [2–6]. A better understanding of the toxic effects of organophosphates on the vascular and respiratory systems, independent of cholinesterase inhibition, could identify novel opportunities to find a more effective treatment for organophosphate poisoning victims directed to the major causes of death (respiratory failure and impaired cardiovascular function). In addition, there is a notable paucity of scientific data (Medline database) investigating the vascular effects mediated by organophosphates and the role of endothelium-derived relaxing factors in mediating the vascular effects. Malathion was selected because it is a pesticide which is widely used in public health pest control programs such as mosquito eradication, beyond its use in agriculture, residential landscaping and public recreation areas [7]. Thus, this study was performed to evaluate the effects of this organophosphate on the vascular reactivity of the isolated rat aorta. The main objective was to investigate the endothelium-dependent mechanisms involved in the vascular response, including the role of nitric oxide (NO) and prostacyclin (PGI2).
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Fig. 1. Concentration-response curve to malathion (10–10 to 10–5
Fig. 2. Concentration-response curve to malathion (10–9 to 10–5
mol/l) in rat thoracic aortas with and without endothelium. Values represent means ± SEM (n = 7). * p < 0.05; # p < 0.01. Two-way ANOVA, Bonferroni post hoc test.
mol/l) in rat thoracic aortas with intact endothelium in the presence and absence of indomethacin 10–5 mol/l and L-NAME 10–4 mol/l. Values represent means ± SEM (n = 7). * p < 0.05, # p < 0.01 and † p < 0.001 between the indomethacin, L-NAME and indomethacin + L-NAME groups compared to control. Two-way ANOVA, Bonferroni post hoc test. Indo = Indomethacin.
the tissue to the drug. In the contraction study, changes in the vascular wall tension were expressed as grams of tension that generates the respective curve.
Statistical Analysis Data are reported as means ± SEM. In all experiments, n refers to the number of animals from which three or four blood vessel rings were taken. For the relaxation, the responses were expressed as percent change from the precontraction levels. The in vitro vascular reactivity data were evaluated by two-way analysis of variance (ANOVA) with the Bonferroni post hoc test. The NO measurements were carried out using the Student t test. The GraphPad Prism 4.0 software (GraphPad Software Inc., San Diego, Calif., USA) for personal computers was applied for the statistical calculations. The level of significance was set at p < 0.05 in all data analyses.
In thoracic aortic rings with intact endothelium, malathion induced approximately 50% concentration-dependent relaxation, and in aortic rings without endothelium there was no measurable relaxation (fig. 1). Pre-incubation with indomethacin and/or L-NAME blocked malathion-induced relaxation that was partially inhibited by indomethacin and totally inhibited by LNAME. There was no difference in inhibition of malathion-induced relaxation between L-NAME and L-NAME plus indomethacin (fig. 2). In thoracic aorta rings, with intact endothelium, Phe (10–10 to 10–5 mol/l) evoked concentration-dependent contraction that was considerably reduced in the presence of malathion. The Phe contractile response was restored in the presence of indomethacin 10–5 mol/l and L-NAME 10–4 mol/l (fig. 3). The same response was observed in endothelium-denuded rings. In thoracic aorta rings, without endothelium, Phe (10–10 to 10–5 mol/l) promoted concentration-dependent contraction that was reduced in the presence of malathion (10–4 mol/l), which was restored in the presence of indomethacin 10–5 mol/l and L-NAME 10–4 mol/l (fig. 4). Malathion evokes endothelium-dependent relaxation through M1 muscarinic receptor, since this relaxation was
Effects of the Organophosphorus Pesticide Malathion on Vascular Reactivity
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Results Endothelial Intracellular NO Measurement by Flow Cytometry The thoracic aorta, immersed in Hank’s buffer (composition: NaCl 145.0 mmol/l, KCl 5.0 mmol/l, CaCl2 1.6 mmol/l, NaH2PO4 0.5 mmol/l, MgCl2 0.5 mmol/l, dextrose 10.0 mmol/l, HEPES 10.0 mmol/l, pH 7.4), was cut longitudinally, and the endothelial cells were isolated by gentle rake friction. Hank’s solution containing the isolated cells was centrifuged at 200 g for 2 min, and the cells were resuspended in 1 ml of Hank’s buffer. The cells were then loaded with DAF-FM DA (5 μmol/l, a selective NO fluorescent dye) and maintained in a humidified incubator at 37 ° C gassed with 5% CO2 for 30 min. The cytofluorographic analysis was performed on an FAC Scan (Becton-Dickinson, San Jose, Calif., USA): the fluorescence was excited with the 488-nm line of an argon ion laser for both dyes, and the emitted fluorescence was measured at 515 nm for DAF-FM DA. The fluorescence intensity was evaluated using Cell Quest 1.2 software (Becton-Dickinson, Franklin Lakes, N.J., USA).
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Fig. 3. Concentration-response curve to Phe (10–10 to 10–5 mol/l)
Fig. 4. Concentration-response curve to Phe (10–10 to 10–5 mol/l)
in rat thoracic aortas with endothelium in the presence of malathion (10–4 mol/l) and incubated with indomethacin (10–5 mol/l) and/or L-NAME (10–4 mol/l). Values represent means ± SEM (n = 7). * p < 0.05, # p < 0.001 and † p < 0.01 between the malathion and control groups. Two-way ANOVA, Bonferroni post hoc test. Indo = Indomethacin.
in rat thoracic aortas without endothelium in the presence of malathion (10–4 mol/l) and incubated with indomethacin (10–5 mol/l) and/or L-NAME (10–4 mol/l). Values represent means ± SEM (n = 7). * p < 0.05 between the malathion + L-NAME + indomethacin and control groups; # p < 0.01 between the malathion + L-NAME + indomethacin and control groups; † p < 0.001 between the malathion and control groups. Two-way ANOVA, Bonferroni post hoc test. Indo = Indomethacin.
clearly blocked by atropine (M1 and M2 blockers) and pirenzepine (M1 blocker), but less blocked by gallamine (M2 blocker) or 4-DAMP (M3 blocker) (fig. 5). In order to investigate malathion-induced NO production in aortic endothelial cells, flow cytometry was performed using DAF-FM DA. The results showed that NO production, represented as F, was higher in malathion-stimulated aortic endothelial cells in the first minute compared to control cells (p < 0.05). After 5 min, NO production was still higher in malathion-stimulated aortic endothelial cells compared to control cells, but the difference was not significant (p > 0.05) (fig. 6). Table 1 shows the log EC50 obtained in each experiment measuring vascular reactivity.
endothelium-intact and endothelium-denuded rings and re-established to control levels with indomethacin, LNAME and the association of both, and (4) NO production in endothelial cells was confirmed using flow cytometry, since NO increased 1 min after the addition of malathion. These data provide evidence that malathion-induced relaxation of the rat aorta is mediated through endothelium-derived NO. Flow cytometry results confirm the increased production of NO upon stimulation of aortic endothelial cells with malathion and show that malathion can induce a direct effect on the endothelium NO production. In the present study, indomethacin inhibited the malathion-induced endothelium-dependent relaxation, providing evidence that malathion-induced relaxation of the rat artery aorta is also mediated by endothelium-derived PGI2. The differences between indomethacin and L-NAME in inhibiting malathion-induced relaxation were not significant, leading to the hypothesis that both PGI2 and NO pathways were involved [8, 9]. It is well known that NO and PGI2 are coreleased from endothelial cells by stimuli acting via membrane-bound receptors [10]. The receptor-mediated release of NO and PGI2 is calcium dependent and synergizes in the inhibition of platelet aggregation, but there is still little evidence of
Discussion
The main conclusions of this study were: (1) malathion induced an endothelium-dependent relaxation, which was partially inhibited by indomethacin and totally by LNAME and the association of both; (2) malathion evokes NO endothelium-dependent relaxation through M1 muscarinic receptors; (3) in the presence of malathion, the Phe-induced contraction was partially inhibited in both 160
Pharmacology 2014;94:157–162 DOI: 10.1159/000367897
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Contraction (g)
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Fig. 5. Concentration-response curve to malathion (10–10 to 10–5
Fig. 6. NO flow cytometry. * p < 0.05 between the control and
mol/l) in rat thoracic aortas with endothelium in the presence of atropine (10–6 mol/l), pirenzepine (10–5 mol/l), gallamine (10–5 mol/l) and 4-DAMP (10–6 mol/l). Values represent means ± SEM (n = 6). * p < 0.05 between the malathion and pirenzepine, gallamine and 4-DAMP; # p < 0.01 between the malathion and atropine, gallamine and 4-DAMP; † p < 0.001 between the malathion and atropine and pirenzepine. Two-way ANOVA, Bonferroni post hoc test.
malathion groups.
Table 1. The log EC50 from vascular reactivity groups
Malathion Malathion + LN Malathion + Indo Phe Phe + Malathion Phe + Malathion + LN + Indo
E+
E–
6.77 ± 0.36a 6.51 ± 0.14b 6.22 ± 0.73b 7.31 ± 0.05c 5.91 ± 0.08c 7.47 ± 0.08c
– – – 7.73 ± 0.10d 6.46 ± 0.07d 7.41 ± 0.05d
smooth muscle relaxation [11]. There is a strong possibility that constitutively expressed endothelial NOS is the involved isoform, based on the Ca2+ dependency of Phe smooth muscle contraction and the well-known concept that the inducible NOS isoform is Ca2+ independent [12]. Even if there is a high possibility of endothelial NOS expression evoked by malathion, one cannot consider it since L-NAME is a nonspecific NOS inhibitor. The results obtained from Phe concentration-response curves in aortic rings with and without endothelium showed the potent ability of malathion to induce NO and PGI2 production. Concerning the vessels without endothelium, it is essential to remember that NO and PGI2 production in smooth muscle cells is not new information [7, 8]. Vascular smooth muscle cells have also been demonstrated to possess the biochemical pathways that lead to NO production [13–15]. Since the 90s, it has been known that the NO/cGMP pathway can be expressed in the vascular smooth muscle cells. The data obtained from vessels without endothelium suggests this possibility. The flow cytometric technique used for NO measurement in endothelial cells cannot be used for NO measurement in the smooth muscle cell; it was unable to ‘digest’ the arterial wall in order to obtain isolated muscle cells, limiting the use of this technique. Unfortunately, we do not have
any other technical resource to do it, as also reported in the specialized literature. During the investigation, it was hypothesized that malathion would be acting through a membrane receptor. However, the concentration-response curve (fig. 1) was very shallow and extended over five orders of magnitude, suggesting that it was not a single receptor or even a generalized toxicological effect. To address whether this was specific to malathion, new experiments were conducted to obtain concentration-response curves in the presence of muscarinic blockers. These experiments led to the conclusion that malathion evokes endothelium-dependent relaxation through M1 muscarinic receptor, since this relaxation was clearly blocked by atropine (M1 and M2 block-
Effects of the Organophosphorus Pesticide Malathion on Vascular Reactivity
Pharmacology 2014;94:157–162 DOI: 10.1159/000367897
Values are expressed as means ± SEM. For some groups, it was not possible to calculate the log EC50. E+ = With endothelium; E– = without endothelium; LN = L-NAME; Indo = indomethacin. a See figure 1; b see figure 2; c see figure 3; d see figure 4.
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*
20 Relaxation (%)
#†
© fluorescence DAF
*
0
ers) and pirenzepine (M1 blocker), but less blocked by gallamine (M2 blocker) or 4-DAMP (M3 blocker). An extensive literature review on the effects of organophosphorus pesticides on vascular reactivity by stimulating muscarinic receptors evokes the feeling of a ‘neverending story’. Unlike studies involving the central nervous system, digestive tract and heart, studies on the effects of pesticides on isolated blood vessels are scarce. In order to functionally characterize the muscarinic vasodilator responses evoked by organophosphorus pesticides, including malathion, Ryberg et al. [16] studied isolated preparations of the rat submandibular and carotid artery. The different muscarinic receptors were assessed by immunohistochemistry. Staining for muscarinic M1 receptors occurred in the endothelium, while M1 and possibly M3 receptors were detected in the arterial smooth muscle. The NOS inhibitor N-ω-nitro-L-arginine (10–4 mol/l) markedly reduced the cholinergic-evoked relaxation of precontracted carotid arterial preparations. In the presence of 4-DAMP 10–7 mol/l, the relaxation to cholinergic agonists was inhibited. Pirenzepine (10–5 mol/l) did not only inhibit the relaxatory effects, but even reversed them. The authors concluded that the arterial NO-dependent response to muscarinic receptor stimulation consisted of two parts – one sensitive to pirenzepine and 4-DAMP and the other to 4-DAMP only. Inhibition of the former part only resulted in cholinergic arterial contraction.
Study Limitation Although the study of vascular reactivity in ‘organ chambers’ is a widely used methodology for investigating endothelial function, the limitation of not being able to separate biological phenomena that occur inside and outside the vessel remains. It is noteworthy to emphasize that the rat aorta is not a blood vessel primarily associated with blood pressure; therefore, it would be questionable if the results can be applicable to circulatory shock, and a more suitable model may be needed to confirm these results. Meanwhile, it is more scientifically correct to say that malathion has its primary action through the stimulation of M1 receptors, leaving open the discussion on the other muscarinic receptors.
Acknowledgements This study was supported in part by CAPES, CNPq, FAPESP and FAEPA.
Disclosure Statement None of the authors had any competing financial interests in relation to this work.
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1 Worek F, Koller M, Thiermann H, Szinicz L: Diagnostic aspects of organophosphate poisoning. Toxicology 2005,214:182–189. 2 Kurek A: Effect of cholinesterase (ChE) reactivators on hemodynamics in animals. 2. Reactions of the circulatory and respiratory systems of dogs after administration of ChE reactivators. Pol Arch Weter 1987;24:485–499. 3 Lim SL, Sim MK, Loke WK: Acetylcholinesterase-independent action of diisopropyl-flurophosphate in the rat aorta. Eur J Pharmacol 2000;404:353–359. 4 Preston E, Heath C: Atropine-insensitive relaxation and hypotension in the organophosphate-poisoned rabbit. Arch Int Pharmacodyn Ther 1972;200:231–244. 5 Preston E, Heath C: Depression of the vasomotor system in rabbits poisoned with an organophosphate anticholinesterase. Arch Int Pharmacodyn Ther 1972;200:245–254. 6 Takahashi H, Kojima T, Ikeda T, Tsuda S, Shirasu Y: Differences in the mode of lethality produced through intravenous and oral administration of organophosphorus insecti-
Received: October 18, 2014 Accepted after revision: August 25, 2014 Published online: October 3, 2014
In vitro Effects of the Organophosphorus Pesticide Malathion on the Reactivity of Rat Aorta Marco Tulio Menezes de Carvalho Andrea Carla Celotto Agnes Afrodite Sumarelli Albuquerque Luciana Garros Ferreira Verena Kise Capellini Ana Paula Cassiano Silveira Tales Rubens de Nadai Paulo Roberto Barbosa Evora Department of Surgery and Anatomy, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil
Abstract Background and Purpose: There is a remarkable paucity of studies analyzing the role of the endothelium-derived relaxing factors on the vascular effects of organophosphates. This study was carried out to evaluate the vascular effects of malathion and the role of nitric oxide (NO) and prostacyclin (PGI2). Methods: Vascular reactivity measuring isometric forces in vitro (‘organ chambers’) and flow cytometry (cells loaded with DAF-FM DA) were used. Results: In rat thoracic aorta segments contracted with phenylephrine (Phe) (10–7 mol/l), malathion (10–10 to 10–5 mol/l) induced concentration-dependent relaxation in arteries with intact endothelium (n = 7; p < 0.05). Malathion-mediated relaxation was blocked by N-nitro-L-arginine methyl ester (L-NAME; 10–4 mol/l), a nonspecific NO synthase inhibitor, and/or indomethacin (10–5 mol/l), a nonspecific cyclooxygenase inhibitor (n = 10, p < 0.05). In thoracic aorta rings, with and without endothelium, Phe (10–10 to 10–5 mol/l) evoked concentration-dependent contraction, which was reduced in the presence of malathion. In rings with or without endothelium, in-
© 2014 S. Karger AG, Basel 0031–7012/14/0944–0157$39.50/0 E-Mail [email protected] www.karger.com/pha
cubated with malathion, L-NAME and indomethacin, the Phe-induced contraction was restored. The role of NO was confirmed using flow cytometry. Malathion evokes endothelium-dependent relaxation through the M1 muscarinic receptor, since this relaxation was clearly blocked by atropine (M1 and M2 blocker) and pirenzepine (M1 blocker), but was less blocked by gallamine (M2 blocker) or 4-DAMP (M3 blocker). Conclusions: These findings suggest that the organophosphate compound effects on vascular reactivity depend of NO and PGI2. © 2014 S. Karger AG, Basel
Background
Organophosphorus compounds are chemicals present in pesticides and are known acetylcholinesterase inhibitors. The main toxic action of organophosphates includes inhibition of the cholinesterase enzyme promoting muscarinic and nicotinic effects. In the cardiovascular system, the nicotinic manifestations are tachycardia and hypertension while the muscarinic effects include bradycardia and hypotension [1]. In the respiratory system, cholinesterase inhibition is characterized by increased bronchial secretions, bronchospasm and the occurrence of respiratory muscle P.R.B. Evora Department of Surgery and Anatomy, Ribeirão Preto Medical School University of São Paulo, Avenida Bandeirantes, 3900 Ribeirão Preto, SP 14048-900 (Brazil) E-Mail prbevora @ gmail.com
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Key Words Endothelium · Nitric oxide · Prostacyclin · Vasorelaxation · Organophosphorus · Pesticides · Malathion
Materials and Methods Materials Indomethacin, phenylephrine (Phe), atropine, 4-DAMP, gallamine, pirenzepine and malathion were purchased from Sigma Chemical Company (St. Louis, Mo., USA). N-nitro-L-arginine methyl ester (L-NAME) was obtained from Calbiochem (San Diego, Calif., USA). Thiopental sodium was purchased from Cristália (São Paulo, Brazil). The salts used for the preparation of Krebs solution were furnished by Vetec Química Fina Ltd. (Duque de Caxias, Brazil). All drugs were prepared with distilled water, except for indomethacin that was dissolved in ethanol, thiopental sodium that was dissolved in physiological saline (0.9% NaCl) and 4-DAMP that was dissolved in dimethyl sulfoxide. Animals The experimental procedures and animal handling were reviewed and approved by the Institutional Animal Care Review Board (CETEA – Ethics Committees of Animal Experiments of the Ribeirão Preto Medical School, University of São Paulo). This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Before the experiments, the rats were housed under standard laboratory conditions (12-hour dark cycle at 21 ° C), with free access to food and water.
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Vessel Preparation and Isometric Tension Recording Male Wistar rats (230–280 g) were anesthetized with thiopental sodium (40 mg/kg i.p.), followed by laparotomy for exsanguination via the abdominal aorta and thoracotomy for thoracic aorta harvesting. The thoracic aorta was carefully dissected free of connective tissue and immersed in cooled and oxygenated Krebs solution (NaCl: 118.0 mmol/l, KCl: 4.7 mmol/l, CaCl2: 2.5 mmol/l, KH2PO4: 1.2 mmol/l, MgSO4: 1.66 mmol/l, glucose: 11.1 mmol/l, NaHCO3: 25.0 mmol/l, pH 7.4). Ring segments of the thoracic aorta (4 mm in length) were prepared with proper care, so as not to touch the intimal surface. In some segments, the endothelium was removed by gently rubbing the intimal surface of the blood vessel with a pair of watchmaker’s forceps. This procedure removes endothelium but does not affect the ability of the vascular smooth muscle to contract or relax. Endothelial integrity was assessed qualitatively by the level of relaxation caused by ACh (10–5 mol/l) in the presence of contractile tone induced by Phe (10–7 mol/l). Thoracic aorta segments were mounted in organ chambers (10 ml) filled with Krebs solution maintained at 37 ° C and bubbled with 95% O2/5% CO2 (pH 7.4). Each arterial ring was suspended by two stainless steel clips placed through the lumen. One clip was anchored to the bottom of the organ chamber while the other was connected to a strain gauge for the measurement of the isometric force using Grass FT03 (Grass Instrument Company, Quincy, Mass., USA). The rings were placed at an optimal length-tension of 2 g and allowed to equilibrate for 60 min with the bath fluid being changed every 15–20 min. For studies of endothelium-intact vessels, the ring was discarded if relaxation with ACh was not 80% or greater. For studies of endothelium-denuded vessels, rings were discarded if there was any measurable level of relaxation. Sequentially, each ring was washed and re-equilibrated for 30 min. Aortic rings were then precontracted with Phe (10–7 mol/l), and cumulative concentration-response curves were obtained after a stable degree of contraction was attained. Rat aortic rings were incubated with malathion (10–4 mol/l) before cumulative concentration-response curves of Phe were studied.
Study Design The study was designed using two main protocols. In the first protocol, cumulative concentration-response curves were obtained for malathion (10–10 to 10–4 mol/l) in aortic rings, with and without endothelium, precontracted to a stable plateau with Phe (10–7 mol/l). In addition, cumulative concentration-response curves for Phe (10–10 to 10–5 mol/l) and ACh (10–10 to 10–5 mol/l) were obtained in endothelium-intact and endothelium-denuded aortic rings, in the presence or absence of malathion (10–4 mol/l). In the second protocol, concentration-response curves were also obtained after pre-incubating the vascular rings with L-NAME (10–4 mol/l), a nonspecific nitric oxide synthase (NOS) inhibitor; indomethacin (10–5 mol/l), an unspecific cyclooxygenase inhibitor; atropine (10–6 mol/l), a competitive nonselective antagonist at central and peripheral muscarinic acetylcholine receptors; 4-DAMP (10–6 mol/l), an M3 preferring cholinergic receptor antagonist; gallamine (10–5 mol/l), a muscle relaxant, allosteric muscarinic receptor antagonist, and pirenzepine (10–5 mol/l), a selective M1 muscarinic acetylcholine receptor antagonist. In the relaxation study, changes in the vascular wall tension were expressed as a percentage of relaxation of the maximal contraction following exposure to Phe, a convention that corrects for interanimal variability in the response of
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paralysis [2]. Although the effects of organophosphates are best known as resulting from cholinesterase inhibition, in vitro studies have demonstrated organophosphate-mediated vasodilator effects and airway constriction, suggesting that organophosphates have peripheral activity independent of acetylcholinesterase inhibition [2–6]. A better understanding of the toxic effects of organophosphates on the vascular and respiratory systems, independent of cholinesterase inhibition, could identify novel opportunities to find a more effective treatment for organophosphate poisoning victims directed to the major causes of death (respiratory failure and impaired cardiovascular function). In addition, there is a notable paucity of scientific data (Medline database) investigating the vascular effects mediated by organophosphates and the role of endothelium-derived relaxing factors in mediating the vascular effects. Malathion was selected because it is a pesticide which is widely used in public health pest control programs such as mosquito eradication, beyond its use in agriculture, residential landscaping and public recreation areas [7]. Thus, this study was performed to evaluate the effects of this organophosphate on the vascular reactivity of the isolated rat aorta. The main objective was to investigate the endothelium-dependent mechanisms involved in the vascular response, including the role of nitric oxide (NO) and prostacyclin (PGI2).
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Fig. 1. Concentration-response curve to malathion (10–10 to 10–5
Fig. 2. Concentration-response curve to malathion (10–9 to 10–5
mol/l) in rat thoracic aortas with and without endothelium. Values represent means ± SEM (n = 7). * p < 0.05; # p < 0.01. Two-way ANOVA, Bonferroni post hoc test.
mol/l) in rat thoracic aortas with intact endothelium in the presence and absence of indomethacin 10–5 mol/l and L-NAME 10–4 mol/l. Values represent means ± SEM (n = 7). * p < 0.05, # p < 0.01 and † p < 0.001 between the indomethacin, L-NAME and indomethacin + L-NAME groups compared to control. Two-way ANOVA, Bonferroni post hoc test. Indo = Indomethacin.
the tissue to the drug. In the contraction study, changes in the vascular wall tension were expressed as grams of tension that generates the respective curve.
Statistical Analysis Data are reported as means ± SEM. In all experiments, n refers to the number of animals from which three or four blood vessel rings were taken. For the relaxation, the responses were expressed as percent change from the precontraction levels. The in vitro vascular reactivity data were evaluated by two-way analysis of variance (ANOVA) with the Bonferroni post hoc test. The NO measurements were carried out using the Student t test. The GraphPad Prism 4.0 software (GraphPad Software Inc., San Diego, Calif., USA) for personal computers was applied for the statistical calculations. The level of significance was set at p < 0.05 in all data analyses.
In thoracic aortic rings with intact endothelium, malathion induced approximately 50% concentration-dependent relaxation, and in aortic rings without endothelium there was no measurable relaxation (fig. 1). Pre-incubation with indomethacin and/or L-NAME blocked malathion-induced relaxation that was partially inhibited by indomethacin and totally inhibited by LNAME. There was no difference in inhibition of malathion-induced relaxation between L-NAME and L-NAME plus indomethacin (fig. 2). In thoracic aorta rings, with intact endothelium, Phe (10–10 to 10–5 mol/l) evoked concentration-dependent contraction that was considerably reduced in the presence of malathion. The Phe contractile response was restored in the presence of indomethacin 10–5 mol/l and L-NAME 10–4 mol/l (fig. 3). The same response was observed in endothelium-denuded rings. In thoracic aorta rings, without endothelium, Phe (10–10 to 10–5 mol/l) promoted concentration-dependent contraction that was reduced in the presence of malathion (10–4 mol/l), which was restored in the presence of indomethacin 10–5 mol/l and L-NAME 10–4 mol/l (fig. 4). Malathion evokes endothelium-dependent relaxation through M1 muscarinic receptor, since this relaxation was
Effects of the Organophosphorus Pesticide Malathion on Vascular Reactivity
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Results Endothelial Intracellular NO Measurement by Flow Cytometry The thoracic aorta, immersed in Hank’s buffer (composition: NaCl 145.0 mmol/l, KCl 5.0 mmol/l, CaCl2 1.6 mmol/l, NaH2PO4 0.5 mmol/l, MgCl2 0.5 mmol/l, dextrose 10.0 mmol/l, HEPES 10.0 mmol/l, pH 7.4), was cut longitudinally, and the endothelial cells were isolated by gentle rake friction. Hank’s solution containing the isolated cells was centrifuged at 200 g for 2 min, and the cells were resuspended in 1 ml of Hank’s buffer. The cells were then loaded with DAF-FM DA (5 μmol/l, a selective NO fluorescent dye) and maintained in a humidified incubator at 37 ° C gassed with 5% CO2 for 30 min. The cytofluorographic analysis was performed on an FAC Scan (Becton-Dickinson, San Jose, Calif., USA): the fluorescence was excited with the 488-nm line of an argon ion laser for both dyes, and the emitted fluorescence was measured at 515 nm for DAF-FM DA. The fluorescence intensity was evaluated using Cell Quest 1.2 software (Becton-Dickinson, Franklin Lakes, N.J., USA).
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Fig. 3. Concentration-response curve to Phe (10–10 to 10–5 mol/l)
Fig. 4. Concentration-response curve to Phe (10–10 to 10–5 mol/l)
in rat thoracic aortas with endothelium in the presence of malathion (10–4 mol/l) and incubated with indomethacin (10–5 mol/l) and/or L-NAME (10–4 mol/l). Values represent means ± SEM (n = 7). * p < 0.05, # p < 0.001 and † p < 0.01 between the malathion and control groups. Two-way ANOVA, Bonferroni post hoc test. Indo = Indomethacin.
in rat thoracic aortas without endothelium in the presence of malathion (10–4 mol/l) and incubated with indomethacin (10–5 mol/l) and/or L-NAME (10–4 mol/l). Values represent means ± SEM (n = 7). * p < 0.05 between the malathion + L-NAME + indomethacin and control groups; # p < 0.01 between the malathion + L-NAME + indomethacin and control groups; † p < 0.001 between the malathion and control groups. Two-way ANOVA, Bonferroni post hoc test. Indo = Indomethacin.
clearly blocked by atropine (M1 and M2 blockers) and pirenzepine (M1 blocker), but less blocked by gallamine (M2 blocker) or 4-DAMP (M3 blocker) (fig. 5). In order to investigate malathion-induced NO production in aortic endothelial cells, flow cytometry was performed using DAF-FM DA. The results showed that NO production, represented as F, was higher in malathion-stimulated aortic endothelial cells in the first minute compared to control cells (p < 0.05). After 5 min, NO production was still higher in malathion-stimulated aortic endothelial cells compared to control cells, but the difference was not significant (p > 0.05) (fig. 6). Table 1 shows the log EC50 obtained in each experiment measuring vascular reactivity.
endothelium-intact and endothelium-denuded rings and re-established to control levels with indomethacin, LNAME and the association of both, and (4) NO production in endothelial cells was confirmed using flow cytometry, since NO increased 1 min after the addition of malathion. These data provide evidence that malathion-induced relaxation of the rat aorta is mediated through endothelium-derived NO. Flow cytometry results confirm the increased production of NO upon stimulation of aortic endothelial cells with malathion and show that malathion can induce a direct effect on the endothelium NO production. In the present study, indomethacin inhibited the malathion-induced endothelium-dependent relaxation, providing evidence that malathion-induced relaxation of the rat artery aorta is also mediated by endothelium-derived PGI2. The differences between indomethacin and L-NAME in inhibiting malathion-induced relaxation were not significant, leading to the hypothesis that both PGI2 and NO pathways were involved [8, 9]. It is well known that NO and PGI2 are coreleased from endothelial cells by stimuli acting via membrane-bound receptors [10]. The receptor-mediated release of NO and PGI2 is calcium dependent and synergizes in the inhibition of platelet aggregation, but there is still little evidence of
Discussion
The main conclusions of this study were: (1) malathion induced an endothelium-dependent relaxation, which was partially inhibited by indomethacin and totally by LNAME and the association of both; (2) malathion evokes NO endothelium-dependent relaxation through M1 muscarinic receptors; (3) in the presence of malathion, the Phe-induced contraction was partially inhibited in both 160
Pharmacology 2014;94:157–162 DOI: 10.1159/000367897
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Contraction (g)
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Fig. 5. Concentration-response curve to malathion (10–10 to 10–5
Fig. 6. NO flow cytometry. * p < 0.05 between the control and
mol/l) in rat thoracic aortas with endothelium in the presence of atropine (10–6 mol/l), pirenzepine (10–5 mol/l), gallamine (10–5 mol/l) and 4-DAMP (10–6 mol/l). Values represent means ± SEM (n = 6). * p < 0.05 between the malathion and pirenzepine, gallamine and 4-DAMP; # p < 0.01 between the malathion and atropine, gallamine and 4-DAMP; † p < 0.001 between the malathion and atropine and pirenzepine. Two-way ANOVA, Bonferroni post hoc test.
malathion groups.
Table 1. The log EC50 from vascular reactivity groups
Malathion Malathion + LN Malathion + Indo Phe Phe + Malathion Phe + Malathion + LN + Indo
E+
E–
6.77 ± 0.36a 6.51 ± 0.14b 6.22 ± 0.73b 7.31 ± 0.05c 5.91 ± 0.08c 7.47 ± 0.08c
– – – 7.73 ± 0.10d 6.46 ± 0.07d 7.41 ± 0.05d
smooth muscle relaxation [11]. There is a strong possibility that constitutively expressed endothelial NOS is the involved isoform, based on the Ca2+ dependency of Phe smooth muscle contraction and the well-known concept that the inducible NOS isoform is Ca2+ independent [12]. Even if there is a high possibility of endothelial NOS expression evoked by malathion, one cannot consider it since L-NAME is a nonspecific NOS inhibitor. The results obtained from Phe concentration-response curves in aortic rings with and without endothelium showed the potent ability of malathion to induce NO and PGI2 production. Concerning the vessels without endothelium, it is essential to remember that NO and PGI2 production in smooth muscle cells is not new information [7, 8]. Vascular smooth muscle cells have also been demonstrated to possess the biochemical pathways that lead to NO production [13–15]. Since the 90s, it has been known that the NO/cGMP pathway can be expressed in the vascular smooth muscle cells. The data obtained from vessels without endothelium suggests this possibility. The flow cytometric technique used for NO measurement in endothelial cells cannot be used for NO measurement in the smooth muscle cell; it was unable to ‘digest’ the arterial wall in order to obtain isolated muscle cells, limiting the use of this technique. Unfortunately, we do not have
any other technical resource to do it, as also reported in the specialized literature. During the investigation, it was hypothesized that malathion would be acting through a membrane receptor. However, the concentration-response curve (fig. 1) was very shallow and extended over five orders of magnitude, suggesting that it was not a single receptor or even a generalized toxicological effect. To address whether this was specific to malathion, new experiments were conducted to obtain concentration-response curves in the presence of muscarinic blockers. These experiments led to the conclusion that malathion evokes endothelium-dependent relaxation through M1 muscarinic receptor, since this relaxation was clearly blocked by atropine (M1 and M2 block-
Effects of the Organophosphorus Pesticide Malathion on Vascular Reactivity
Pharmacology 2014;94:157–162 DOI: 10.1159/000367897
Values are expressed as means ± SEM. For some groups, it was not possible to calculate the log EC50. E+ = With endothelium; E– = without endothelium; LN = L-NAME; Indo = indomethacin. a See figure 1; b see figure 2; c see figure 3; d see figure 4.
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*
20 Relaxation (%)
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© fluorescence DAF
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ers) and pirenzepine (M1 blocker), but less blocked by gallamine (M2 blocker) or 4-DAMP (M3 blocker). An extensive literature review on the effects of organophosphorus pesticides on vascular reactivity by stimulating muscarinic receptors evokes the feeling of a ‘neverending story’. Unlike studies involving the central nervous system, digestive tract and heart, studies on the effects of pesticides on isolated blood vessels are scarce. In order to functionally characterize the muscarinic vasodilator responses evoked by organophosphorus pesticides, including malathion, Ryberg et al. [16] studied isolated preparations of the rat submandibular and carotid artery. The different muscarinic receptors were assessed by immunohistochemistry. Staining for muscarinic M1 receptors occurred in the endothelium, while M1 and possibly M3 receptors were detected in the arterial smooth muscle. The NOS inhibitor N-ω-nitro-L-arginine (10–4 mol/l) markedly reduced the cholinergic-evoked relaxation of precontracted carotid arterial preparations. In the presence of 4-DAMP 10–7 mol/l, the relaxation to cholinergic agonists was inhibited. Pirenzepine (10–5 mol/l) did not only inhibit the relaxatory effects, but even reversed them. The authors concluded that the arterial NO-dependent response to muscarinic receptor stimulation consisted of two parts – one sensitive to pirenzepine and 4-DAMP and the other to 4-DAMP only. Inhibition of the former part only resulted in cholinergic arterial contraction.
Study Limitation Although the study of vascular reactivity in ‘organ chambers’ is a widely used methodology for investigating endothelial function, the limitation of not being able to separate biological phenomena that occur inside and outside the vessel remains. It is noteworthy to emphasize that the rat aorta is not a blood vessel primarily associated with blood pressure; therefore, it would be questionable if the results can be applicable to circulatory shock, and a more suitable model may be needed to confirm these results. Meanwhile, it is more scientifically correct to say that malathion has its primary action through the stimulation of M1 receptors, leaving open the discussion on the other muscarinic receptors.
Acknowledgements This study was supported in part by CAPES, CNPq, FAPESP and FAEPA.
Disclosure Statement None of the authors had any competing financial interests in relation to this work.
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