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Influence of organophosphorus pesticides on peroxidase and chlorination activity of human myeloperoxidase
Pesticide Biochemistry and Physiology 107 (2013) 55–60
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Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Influence of organophosphorus pesticides on peroxidase and chlorination activity of human myeloperoxidase Tamara Lazarevic´-Pašti a,⇑, Tatjana Momic´ a, Miloš M. Radojevic´ b, Vesna Vasic´ a a b
Department of Physical Chemistry, Vincˇa Institute of Nuclear Sciences, University of Belgrade, P.O. Box 522, 11001 Belgrade, Serbia University of Belgrade, School of Medicine, Dr. Subotica 8, 11000 Belgrade, Serbia
a r t i c l e
i n f o
Article history: Received 10 September 2012 Accepted 6 May 2013 Available online 14 May 2013 Keywords: Organophosphate Pesticide Inhibition Myeloperoxidase Malathion
a b s t r a c t Inhibitory effects of five organophosphorus pesticides (diazinon, malathion, chlorpyrifos, azinphosmethyl and phorate) and their oxo-analogs on human myeloperoxidase (MPO) activity were investigated. While inspecting separately peroxidase and chlorination activity, it was observed that investigated OPs affect peroxidase activity, but not chlorination activity. Among investigated pesticides, malathion and malaoxon have showed the highest power to inhibit MPO peroxidase activity with IC50 values of the order of 3 107 and 5 109 M, respectively. It was proposed that inhibition trend is rendered by molecular structure which invokes steric hindrance for OPs interaction with MPO active center responsible for peroxidase activity. In addition, it was concluded that physiological function of MPO is not affected by any of the investigated OPs. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Pesticides play an important role in high agricultural productivity, but due to inevitable toxicity, often towards non-target organisms, their uncritical release into the environment has serious consequences [1]. Organophosphorus pesticides (OP) cause neurotoxicity due to ability to irreversibly inhibit enzyme acetylcholinesterase (AChE) [2]. Presence of AChE in insects, birds, fishes and all mammals gives this class of pesticides enormous toxicity towards unintended targets. Beside AChE, OP-caused inhibition of other important enzymes enrolled in biochemical processes was also reported [3]. Apart from the toxic effects of many of these pesticides, long-term effects include capability to affect the endocrine system in mammals, birds and fishes [4]. The ability of pesticides to cause different types of endocrine malfunctions, to interact with estrogen and androgen receptors and thyroid function has been investigated to a limited extent, while many long-term effects are still unknown [5–8]. Myeloperoxidase (MPO) is a heme enzyme found in great amounts in neutrophils and also present in monocytes [9,10]. Depending on substrate availability, this enzyme can pass through the peroxidase and/or the halogenation cycle [11]. In the presence of halide and pseudo halide thiocyanate ions, MPO catalyzes the oxidation of these substrates with hydrogen peroxide, producing oxidizing and halogenation agents. Halogenation agents, especially hypochlorous acid (HOCl), promote oxidative killing of micro⇑ Corresponding author. Fax: +381 11 2447 207. E-mail address: [email protected] (T. Lazarevic´-Pašti). 0048-3575/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pestbp.2013.05.004
organisms by neutrophils, but also the destruction of host cells in inflamed tissue [12,13]. However, MPO is relatively nonspecific regarding its reducing substrates and some of them can cause inhibition of the enzyme activity. Suicide substrates undergo a structural change passing through the MPO catalytic cycle which results in irreversible binding to the enzyme. As a final result MPO is being inactivated [14]. In contrast to these substrates, numerous non-steroidal anti-inflammatory drugs, anilines and phenols inhibit MPO reversibly [15,16]. In our previous work we have showed that MPO, when present above certain concentration, can oxidize organophosphorus pesticides diazinon, malathion, chlorpyrifos, azinphos-methyl and phorate in vitro and transform them to corresponding oxo-analogues [17–20]. However, there are some indications that OPs can also inhibit MPO activity, but this effect was observed only through the ability of neutrophils of exposed humans to kill Candida albicans, which was related to MPO activity in neutrophils [21]. The subjects of former study have been exposed to carbamate and organophosphorus insecticides [21]. Due to high physiological importance of MPO it is of great significance to obtain detailed and reliable information regarding possible substrates and inhibitors of this enzyme, in this specific case, OPs, while accumulated knowledge can help to reduce undesired effects caused by OPs. The aim of this study was to investigate the influence of diazinon, malathion, chlorpyrifos, azinphos-methyl, phorate and their oxo-analogues on MPO isolated from human neutrophils in vitro. It was aimed to determine whether OPs are inhibitors of MPO since being its substrates, and whether investigated OPs can affect physiological function of MPO.
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2. Materials and methods 2.1. Chemicals and reagents MPO from human neutrophils purified to an Rz (A430/A280) of 0.84 was obtained from Planta Natural Products, Vienna, Austria. Its concentration was calculated using e430 = 91,000 M1cm1 per heme [22]. Catalase, from bovine liver, 3,30 ,5,50 -tetramethylbenzidine, taurine and o-dianisidine dihydrochloride were purchased from Sigma–Aldrich (St. Louis, MO, USA). Hydrogen peroxide solutions were prepared on a daily basis by diluting the 30% (m/v) stock solution and concentration was determined using e240 = 43.6 M1cm1 [23]. Investigated pesticides (of at least 93% purity) diazinon, malathion, chlorpyrifos, azinphos-methyl and phorate (Scheme 1), as well as their oxo-analogs diazoxon, malaoxon, chlorpyrifos oxon, azinphos-methyl oxon and phorate oxon were purchased from PestinalÒ, Sigma–Aldrich, Denmark. Pesticides’ working solutions were prepared diluting different amounts of stock solutions in water. In order to achieve higher OPs concentrations ethanol was added in some cases to improve OPs solubility. The final working solutions contained max. 1% of ethanol. Pesticide stock solutions were held in refrigerator until used. All chemicals were used without further purification. Deionized water was used throughout. 2.2. Measurement of myeloperoxidase activity O-dianisidine assay was used to investigate the influence of OPs on MPO peroxidase activity. The experiments were performed in vitro exposing the enzyme (7 nM) to OPs at different concentra-
tions (constant final volume of 3 mL). Incubation time of MPO with OPs was 1–60 min, before the reaction was started by adding 0.15 mM hydrogen peroxide. O-dianisidine (0.53 mM) was applied as enzyme substrate and chromogenic reagent, while the formation of colored product was monitored spectrophotometrically at 460 nm for 1 min. The control value was measured in the absence of OPs. The reaction was carried out in 50 mM phosphate buffer (pH 6.0) [24]. Concentration of HOCl produced by MPO in the presence of hydrogen peroxide and chloride was determined by measuring the conversion of taurine into taurine chloramines, which were assayed using 3,30 ,5,50 -tetramethylbenzidine in the presence of iodide (e645 = 30.23 M1 cm1) [25]. Reaction was carried out with 3 nM myeloproxidase in 20 mM phosphate buffer, pH 6.5, containing 100 mM chloride and 5 mM taurine. Reaction was started by adding hydrogen peroxide to a final concentration of 20 lM and stopped after 30 min by adding 100 lg/mL catalase. 2.3. Instrumentation and measurements The spectrophotometric measurements were performed on Perkin Elmer Lambda 35 UV–vis spectrophotometer, using 1 cm path length cuvette. pH measurements were performed using Metrohm pH meter, model 713, equipped with combined glass electrode. Waters ACQUITY Ultra Performance Liquid Chromatography (UPLC) system coupled with TUV detector was used, controlled by the Empower software. Chromatographic separations were run on the ACQUITY UPLC™ BEH C18 column, 1.7 lm, 100 mm 2.1 mm column (Waters). The analyses were performed under isocratic condition with mobile phase consisting of CH3COOH (0.1 vol% in
Scheme 1. Structure of (a) diazinon, (b) malathion, (c) chlorpyrifos, (d) azinphos-methyl and (e) phorate.
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water) and acetonitrile (J.T. Baker) (60:40, v/v). The elutions were monitored at 210 nm. The eluent flow rate was 0.3 mL min1 and the injection volume was 10 lL.
3. Results and discussion
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malaoxon IC50 value is 5 109 M. It is clear that malaoxon is much stronger inhibitor of MPO peroxidase activity as its IC50 value is two orders of magnitude lower. IC50 values were also determined using the Hill’s method of linear regression analysis (Fig. 2) and shown in Table 1. Comparing the results obtained using sigmoidal fit and Hill’s method of linear regression analysis, it is clear that the difference is on the level of experimental error.
3.1. Influence of OPs on MPO peroxidase activity The influence of OPs on MPO peroxidase activity was investigated for OPs concentrations ranging from 1 1011 to 1 104 M in phosphate buffer at pH 6. The effect was monitored through the oxidation of o-dianisidine in the presence of H2O2. Concentration of oxidized o-dianisidine is directly related to MPO activity and it was determined spectrophotometrically, as described earlier (Section 2.2.). Primarily, the optimal concentration of MPO for the inhibition test was determined. Our previous results [17] obtained using UPLC technique and AChE assay showed that the presence of MPO below 10 nM in reaction mixture did not induced measurable oxidation of investigated OPs in the concentration range from 1011 to 103 M. For the inhibition experiments it was necessary to choose the highest MPO concentration which would not lead to notable oxidation of OPs in specified concentration range. Therefore, the possible inhibition of MPO by parent OPs is the only observed phenomenon in this case. For this reason only the MPO concentrations below 10 nM were considered for inhibition experiments. The investigation of MPO activity vs. MPO concentration showed that it increased slowly up to 6 nM enzyme and then grew rapidly (Supplementary data, Fig. S1). As optimal MPO concentration for the inhibition test the concentration of 7 nM MPO was chosen. At that point, MPO activity increased dramatically compared to the lower concentrations. High MPO activity is very important for obtaining proper and reliable data. It was also necessary to determine the optimal incubation time of the enzyme and inhibitor in the inhibition test. In order to investigate the influence of incubation time between OPs and MPO on 7 nM MPO inhibition, the incubation time before the reaction was started varied from 1 to 60 min. The dependence of MPO activity after exposure to OPs as a function of incubation time in the presence of 1 106 M malathion or malaoxon is shown in Fig. S2 (Supplementary data). It can be seen that the MPO activity decreased with the increase of incubation time between the enzyme and OPs (Fig. S2). As an optimal incubation time for the inhibition test the period of 30 min of incubation was chosen, having in mind that it is important to keep the test as brief as possible and that the change in MPO activity between 30 and 60 min of incubation was negligible. The inhibition of 7 nM MPO peroxidase activity induced by investigated OPs in the concentration range from 1 1011 to 1 104 M was evaluated using above determined experimental conditions for the inhibition test. The incubation time of enzyme with OPs was 30 min. The obtained results are presented in Fig. 1. Activity of MPO is expressed as the mean value of the percentage of MPO activity relative to the corresponding control value measured in the absence of OPs. Using the UPLC analysis, OPs oxidation products were not detected in the reaction mixtures when OPs were in contact with 7 nM MPO (data not shown), unlike in the presence of higher enzyme concentration [17,18]. Among the investigated OPs, only malathion and malaoxon exerted the inhibitory power in the whole range of examined concentrations, hence only malathion and malaoxon were further examined. IC50 values for malathion and malaoxon (Table 1) were determined from the data given in Fig. 1B. For malathion, IC50 value calculated from the sigmoidal curve is 3 107 M, while for
3.2. Influence of OPs on MPO chlorination activity The influence of OPs on MPO chlorination activity was also examined. First, the optimal MPO and H2O2 concentration were determined. MPO in the concentration range from 1 to 10 nM in the presence of 10, 20 and 30 lM H2O2 were examined (Supplementary data, Fig. S3). From the results presented in Fig. S3 (Supplementary data), it is obvious that MPO chlorination activity increases with the increase of MPO and H2O2 concentration. In all cases, at certain MPO and H2O2 concentration ratios, a plateau of produced [HOCl] is reached, while the amount of produced HOCl was found to depend on initial [MPO]-to-[H2O2] ratio. In fact, at high H2O2 concentration and low [MPO]-to-[H2O2] the amount of HOCl starts to decay. This is due to the fact that H2O2, while it is MPO’s substrate, it is also MPO inhibitor if present at high concentration [26,27]. It was confirmed that H2O2 inhibits MPO following mechanism-based pattern [28]. Hence, 3 nM MPO in the presence of 20 lM H2O2 was chosen as optimal. MPO (3 nM) was incubated with OPs (diazinon, malathion, chlorpyrifos, azinphos-methyl, phorate and their oxo-analogs) in the concentration range from 1 1010 to 1 104 M during 30 min, in phosphate buffer pH 6.5 and in the presence of 100 mM NaCl and 5 mM taurine. After 30 min, the reaction was started by adding 20 lM H2O2. Neither of the investigated OPs inhibited MPO chlorination activity under specified experimental conditions. It was possible to conclude that, unlike peroxidase activity of MPO, chlorination activity is not affected by investigated OPs under given experimental conditions. This is of particular importance having in mind that the chlorination activity is the physiological function of MPO [12]. The results also confirmed that inhibition of the peroxidase activity of MPO by a certain compound do not necessarily indicate that the compound would also inhibit the chlorination activity of the enzyme [16]. On the other hand, majority of the known MPO inhibitors do affect both of the enzyme’s activities [16,29]. 4. Discussion In this section we analyze (i) observed trends in the inhibition of MPO peroxidase activity by investigated OPs, (ii) differences between the effects of investigated OPs on peroxidase and chlorination activity of MPO based on the MPO reaction mechanism and (iii) discuss previous reports on the effects of OPs on the action of human neutrophils. As can be seen from the inhibition curves (Fig. 1), peroxidase activity of MPO depends on OPs concentration in all examined cases, except in the case of azinphos-methyl and respective oxoform, which did not exert any inhibitory effect (data not shown). It is obvious that all oxo-forms of OPs are more powerful inhibitors of MPO peroxidase activity than respective thio-forms. Having in mind the structures of examined OPs (Scheme 1), it can be concluded that azinphos-methyl’s two rings, one aromatic and one heterocyclic, invoke large steric hindrance for the interaction with MPO. Furthermore, one can compare residual MPO activities when incubated with some particular OPs concentrations. For example, taking the OPs concentration of 107 M it can be seen that the
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Fig. 1. The concentration dependent inhibition of MPO peroxidase activity induced by (A) diazinon and diazoxon, (B) malathion and malaoxon, (C) chlorpyrifos and chlorpyrifos oxon and (D) phorate and phorate oxon. The MPO activities are expressed as the mean percentage of the activity relative to the corresponding control value in the absence of OPs.
Table 1 IC50 values obtained using sigmoidal fit and Hill’s method of linear regression analysis. OP
IC50 – sigmoidal fit (M)
IC50 – Hill’s analysis (M)
Malathion Malaoxon
(3 ± 1) 107 (5 ± 1) 109
(4 ± 1) 107 (7 ± 1) 109
Fig. 2. Hill’s analysis of MPO peroxidase activity inhibition induced by malathion and malaoxon.
lowest activities are observed in the cases of malathion and phorate, which have the simplest structure. MPO activity further increases, i.e. the degree of inhibition decreases, when going from diazinon to chlorpyrifos (having one aromatic ring within the structure) to azynphos-methyl (two rings). The same approximate trend is followed in the case of oxo-analogs too, leading to the conclusion that inhibition trend is rendered by molecular structure which invokes steric hindrance for OPs interaction with MPO’s active center responsible for peroxidase activity or allosteric site (see further discussion). For diazinon, chlorpyrifos and phorate it was not possible to achieve higher concentrations of OPs, due to their solubility (the final solutions must not contain more than 1% organic solvent in order to MPO have proper function). Additional insights regarding MPO peroxidase activity inhibition by investigated OPs could be provided by virtual docking analysis. As already mentioned, under specific conditions regarding the substrate availability, MPO displays its chlorination or peroxidase activity. Halide oxidation starts with reaction of native MPO (ferric-MPO) and H2O2 to form Compound I (Eq. (1)), having two oxidizing equivalents more than the native MPO. In halogenation cycle, Compound I oxidizes halides and pseudohalides via twoelectron process, while Compound I is reduced to native enzyme (Eq. (2)) [9]. In peroxidase cycle, Compound I is reduced in two one-electron steps, first to Compound II and then to native MPO (Eqs. (3) and (4)). It is believed that Compound II is inactive in halide and pseudohalide oxidation [30].
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Ferric-MPO þ H2 O2 ! Compound I þ H2 O
ð1Þ
Compound I þ X þ Hþ ! native MPO þ HOX
ð2Þ
Compound I þ AH2 ! Compound II þ AH
ð3Þ
Compound II þ AH2 ! native MPO þ AH
ð4Þ
o
Standard potentials (E ) of redox couples involved in halogenation and peroxidase cycles are considered to be: Eo (Compound I/ native MPO) = 1.16 V [31], Eo (Compound I/Compound II) = 1.35 V and Eo (Compound II/native MPO) = 0.97 V [32], indicating that Compounds I and II are potent oxidants which should be reduced easily in the presence of electron donors. Our recent work confirmed that investigated thio-OPs can be oxidized by electrochemically generated halogen X species (X = Cl, Br and I) [33,34]. In specific, these reactive species were generated from halide ions (Cl, Br or I) by anodic oxidation and, depending on the nature of the specific halide ion, different relative amounts of X2, X3, HOX and OX species were formed. Although Cl and Br species are potent antioxidants with standard redox potential close to the ones of MPO intermediates redox couples, iodine species were also able to oxidize thio-OPs although having Eo values well below that of MPO intermediates redox couples. This indicates that thioOPs should be good electron donors for both Compound I and Compound II. As chlorination activity of MPO was not affected by the presence of investigated OPs and peroxidase activity was influenced, it is suggested that reduction of Compound II by OPs cannot be the slow step in peroxidase cycle. Namely, if opposite holds, OPs could potentially trap MPO in the form of Compound II and consequently inhibit chlorination activity of MPO. Our previous work [17–20] suggests that OPs are essentially poor MPO peroxidase substrates as only a small fraction of OPs can be oxidized by MPO when peroxidase activity is forced by adequate experimental conditions. This points that, in spite the fact that oxidation of OPs is thermodynamically favorable, large kinetic barrier exists for the reaction of thio-OPs with Compound I. In the experiments where peroxidase activity of MPO was investigated, [MPO]-to-[OP] ratio was set in a way that no measurable amounts of OPs’ oxo-forms are formed in the course of the experiments. In addition, oxo-forms of investigated OPs are not MPO substrates, as cannot act as electron donors, but still affect peroxidase activity of MPO. Hence, it is suggested that investigated OPs and their corresponding oxoforms might actually affect MPO peroxidase activity following mixed inhibition pattern. Conformational changes of MPO upon binding of OPs might be subtle enough so that chlorination activity is not affected, but large enough to affect oxidation of o-dianisidine (used here to probe peroxidase activity of MPO) through the induction of steric hindrance for its bonding to active site. Referring to the work of Queiroz et al. [21] who examined neutrophil function of workers exposed to organophosphorus and carbamate pesticides, it is to be questioned whether the reduced ability of neutrophils to kill C. albicans and C. pseudotropicalis should be ascribed to the inhibitory action of organophosphorus pesticides towards MPO. It can be speculated that MPO deficiency upon the exposure to organophosphorus (and carbamate) pesticides [21] might be invoked by the mechanisms other than MPO inhibition by OPs, at least in the cases of five here investigated OPs and their oxo-analogs. 5. Conclusion Inhibitory action of five organophosphorus pesticides (diazinon, malathion, chlorpyrifos, azinphos-methyl and phorate) and their oxo-analogs, towards MPO was investigated. Obtained results suggest their separate effects when concerned with peroxidase and
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chlorination activity. Among investigated OPs only malathion and malaoxon delivered inhibitory power towards MPO peroxidase activity in the whole investigated concentration range, with IC50 values of the order of 3 107 and 5 109 M, respectively. Inhibition of peroxidase activity was correlated with the molecular structure of investigated OPs, concluding that inhibition trend is rendered by molecular structure which invokes steric hindrance for OPs interaction with MPO active center responsible for peroxidase activity. Complete absence of inhibitory effect on the MPO peroxidase activity was observed in the case of azinphos-methyl and azinphos-methyl oxon, OPs with two six-member rings within their structure. Interestingly, chlorination activity of MPO was completely unaffected by the presence of any of the investigated OPs, suggesting that physiological function of MPO is preserved in the presence of here selected OPs. Obtained results suggest that previously reported results regarding reduced ability of neutrophils to kill C. albicans and C. pseudotropicalis upon exposure to organophosphorus and carbamate pesticides might be invoked by the mechanisms other than inhibition of MPO. Hence, additional research is needed to resolve opened questions regarding modified action of human neutrophils upon exposure to OPs. Acknowledgment The authors would like to express their gratitude to the Ministry of Education, Science and Technological Development of the Republic of Serbia for their financial support, contract 172023. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.pestbp.2013. 05.004. References [1] J.S. Van Dyk, B. Pletschke, Review on the use of enzymes for the detection of organochlorine, organophosphate and carbamate pesticides in the environment, Chemosphere 82 (2011) 291–307. [2] T.R. Fukuto, Mechanism of action of organophosphorus and carbamate insecticides, Environ. Health Perspect. 87 (1990). [3] M. Colovic, D. Krstic, S. Petrovic, A. Leskovac, G. Joksic, J. Savic, M. Franko, P. Trebse, V. Vasic, Toxic effects of diazinon and its photodegradation products, Toxicol. Lett. 193 (2010) 9–18. [4] J. Lintellman, A. Katayama, N. Kurihara, L. Shore, A. Wenzel, Endocrine disruptors in the environment, Pure Appl. Chem. 75 (2003) 631–681. [5] H.R. Andersen, A.M. Vinggaard, T. Rasmussen, I.M. Gjermandsen, E.C. BonefeldJorgensen, Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro, Toxicol. Appl. Pharmacol. 179 (2002) 1–12. [6] G.H. Degen, H.M. Bolt, Endocrine disruptors: update on xenoestrogens, Int. Arch. Occup. Environ. Health 73 (2000) 433–441. [7] H. Kojima, E. Katsura, S. Takeuchi, K. Niiyama, K. Kobayashi, Screening for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells, Environ. Health Perspect. 112 (2004) 524–531. [8] J. Li, N. Li, M. Ma, J.P. Giesy, Z. Wang, In vitro profiling of the endocrine disrupting potency of organochlorine pesticides, Toxicol. Lett. 183 (2008) 65– 71. [9] S.J. Klebanoff, Myeloperoxidase, P. Assoc. Am. Phys. 111 (1999) 383–389. [10] A.J. Kettle, C.A. Gedye, C.C. Winterbourn, Mechanism of inactivation of myeloperoxidase by 4-aminobenzoic acid hydrazide, Biochem. J. 321 (1997) 503–508. [11] P.G. Furtmüller, M. Zederbauer, W. Jantschko, J. Helm, M. Bogner, C. Jakopitsch, C. Obinger, Active site structure and catalytic mechanisms of human peroxidases, Arch. Biochem. Biophys. 445 (2006) 199–213. [12] S.J. Klebanoff, Myeloperoxidase: occurrence and biological function, in: J. Everse, K.E. Everse, M.B. Grisham (Eds.), Peroxidase in Chemistry and Biology, CRC Press, Boca Raton, 1991, pp. 1–35. [13] M.D. Demling, H. Robert, The modern version of adult respiratory distress syndrome, Annu. Rev. Med. 46 (1995) 193–202. [14] S.G. Waley, Kinetics of suicide substrates, Biochem. J. 185 (1980) 771. [15] E. Shacter, R.L. Lopez, S. Pati, Inhibition of the myeloperoxidase-H2O2-Clsystem of neutrophils by indomethacin and other non-steroidal antiinflammatory drugs, Biochem. Pharmacol. 41 (1991) 975–984.
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Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Influence of organophosphorus pesticides on peroxidase and chlorination activity of human myeloperoxidase Tamara Lazarevic´-Pašti a,⇑, Tatjana Momic´ a, Miloš M. Radojevic´ b, Vesna Vasic´ a a b
Department of Physical Chemistry, Vincˇa Institute of Nuclear Sciences, University of Belgrade, P.O. Box 522, 11001 Belgrade, Serbia University of Belgrade, School of Medicine, Dr. Subotica 8, 11000 Belgrade, Serbia
a r t i c l e
i n f o
Article history: Received 10 September 2012 Accepted 6 May 2013 Available online 14 May 2013 Keywords: Organophosphate Pesticide Inhibition Myeloperoxidase Malathion
a b s t r a c t Inhibitory effects of five organophosphorus pesticides (diazinon, malathion, chlorpyrifos, azinphosmethyl and phorate) and their oxo-analogs on human myeloperoxidase (MPO) activity were investigated. While inspecting separately peroxidase and chlorination activity, it was observed that investigated OPs affect peroxidase activity, but not chlorination activity. Among investigated pesticides, malathion and malaoxon have showed the highest power to inhibit MPO peroxidase activity with IC50 values of the order of 3 107 and 5 109 M, respectively. It was proposed that inhibition trend is rendered by molecular structure which invokes steric hindrance for OPs interaction with MPO active center responsible for peroxidase activity. In addition, it was concluded that physiological function of MPO is not affected by any of the investigated OPs. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Pesticides play an important role in high agricultural productivity, but due to inevitable toxicity, often towards non-target organisms, their uncritical release into the environment has serious consequences [1]. Organophosphorus pesticides (OP) cause neurotoxicity due to ability to irreversibly inhibit enzyme acetylcholinesterase (AChE) [2]. Presence of AChE in insects, birds, fishes and all mammals gives this class of pesticides enormous toxicity towards unintended targets. Beside AChE, OP-caused inhibition of other important enzymes enrolled in biochemical processes was also reported [3]. Apart from the toxic effects of many of these pesticides, long-term effects include capability to affect the endocrine system in mammals, birds and fishes [4]. The ability of pesticides to cause different types of endocrine malfunctions, to interact with estrogen and androgen receptors and thyroid function has been investigated to a limited extent, while many long-term effects are still unknown [5–8]. Myeloperoxidase (MPO) is a heme enzyme found in great amounts in neutrophils and also present in monocytes [9,10]. Depending on substrate availability, this enzyme can pass through the peroxidase and/or the halogenation cycle [11]. In the presence of halide and pseudo halide thiocyanate ions, MPO catalyzes the oxidation of these substrates with hydrogen peroxide, producing oxidizing and halogenation agents. Halogenation agents, especially hypochlorous acid (HOCl), promote oxidative killing of micro⇑ Corresponding author. Fax: +381 11 2447 207. E-mail address: [email protected] (T. Lazarevic´-Pašti). 0048-3575/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pestbp.2013.05.004
organisms by neutrophils, but also the destruction of host cells in inflamed tissue [12,13]. However, MPO is relatively nonspecific regarding its reducing substrates and some of them can cause inhibition of the enzyme activity. Suicide substrates undergo a structural change passing through the MPO catalytic cycle which results in irreversible binding to the enzyme. As a final result MPO is being inactivated [14]. In contrast to these substrates, numerous non-steroidal anti-inflammatory drugs, anilines and phenols inhibit MPO reversibly [15,16]. In our previous work we have showed that MPO, when present above certain concentration, can oxidize organophosphorus pesticides diazinon, malathion, chlorpyrifos, azinphos-methyl and phorate in vitro and transform them to corresponding oxo-analogues [17–20]. However, there are some indications that OPs can also inhibit MPO activity, but this effect was observed only through the ability of neutrophils of exposed humans to kill Candida albicans, which was related to MPO activity in neutrophils [21]. The subjects of former study have been exposed to carbamate and organophosphorus insecticides [21]. Due to high physiological importance of MPO it is of great significance to obtain detailed and reliable information regarding possible substrates and inhibitors of this enzyme, in this specific case, OPs, while accumulated knowledge can help to reduce undesired effects caused by OPs. The aim of this study was to investigate the influence of diazinon, malathion, chlorpyrifos, azinphos-methyl, phorate and their oxo-analogues on MPO isolated from human neutrophils in vitro. It was aimed to determine whether OPs are inhibitors of MPO since being its substrates, and whether investigated OPs can affect physiological function of MPO.
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2. Materials and methods 2.1. Chemicals and reagents MPO from human neutrophils purified to an Rz (A430/A280) of 0.84 was obtained from Planta Natural Products, Vienna, Austria. Its concentration was calculated using e430 = 91,000 M1cm1 per heme [22]. Catalase, from bovine liver, 3,30 ,5,50 -tetramethylbenzidine, taurine and o-dianisidine dihydrochloride were purchased from Sigma–Aldrich (St. Louis, MO, USA). Hydrogen peroxide solutions were prepared on a daily basis by diluting the 30% (m/v) stock solution and concentration was determined using e240 = 43.6 M1cm1 [23]. Investigated pesticides (of at least 93% purity) diazinon, malathion, chlorpyrifos, azinphos-methyl and phorate (Scheme 1), as well as their oxo-analogs diazoxon, malaoxon, chlorpyrifos oxon, azinphos-methyl oxon and phorate oxon were purchased from PestinalÒ, Sigma–Aldrich, Denmark. Pesticides’ working solutions were prepared diluting different amounts of stock solutions in water. In order to achieve higher OPs concentrations ethanol was added in some cases to improve OPs solubility. The final working solutions contained max. 1% of ethanol. Pesticide stock solutions were held in refrigerator until used. All chemicals were used without further purification. Deionized water was used throughout. 2.2. Measurement of myeloperoxidase activity O-dianisidine assay was used to investigate the influence of OPs on MPO peroxidase activity. The experiments were performed in vitro exposing the enzyme (7 nM) to OPs at different concentra-
tions (constant final volume of 3 mL). Incubation time of MPO with OPs was 1–60 min, before the reaction was started by adding 0.15 mM hydrogen peroxide. O-dianisidine (0.53 mM) was applied as enzyme substrate and chromogenic reagent, while the formation of colored product was monitored spectrophotometrically at 460 nm for 1 min. The control value was measured in the absence of OPs. The reaction was carried out in 50 mM phosphate buffer (pH 6.0) [24]. Concentration of HOCl produced by MPO in the presence of hydrogen peroxide and chloride was determined by measuring the conversion of taurine into taurine chloramines, which were assayed using 3,30 ,5,50 -tetramethylbenzidine in the presence of iodide (e645 = 30.23 M1 cm1) [25]. Reaction was carried out with 3 nM myeloproxidase in 20 mM phosphate buffer, pH 6.5, containing 100 mM chloride and 5 mM taurine. Reaction was started by adding hydrogen peroxide to a final concentration of 20 lM and stopped after 30 min by adding 100 lg/mL catalase. 2.3. Instrumentation and measurements The spectrophotometric measurements were performed on Perkin Elmer Lambda 35 UV–vis spectrophotometer, using 1 cm path length cuvette. pH measurements were performed using Metrohm pH meter, model 713, equipped with combined glass electrode. Waters ACQUITY Ultra Performance Liquid Chromatography (UPLC) system coupled with TUV detector was used, controlled by the Empower software. Chromatographic separations were run on the ACQUITY UPLC™ BEH C18 column, 1.7 lm, 100 mm 2.1 mm column (Waters). The analyses were performed under isocratic condition with mobile phase consisting of CH3COOH (0.1 vol% in
Scheme 1. Structure of (a) diazinon, (b) malathion, (c) chlorpyrifos, (d) azinphos-methyl and (e) phorate.
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water) and acetonitrile (J.T. Baker) (60:40, v/v). The elutions were monitored at 210 nm. The eluent flow rate was 0.3 mL min1 and the injection volume was 10 lL.
3. Results and discussion
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malaoxon IC50 value is 5 109 M. It is clear that malaoxon is much stronger inhibitor of MPO peroxidase activity as its IC50 value is two orders of magnitude lower. IC50 values were also determined using the Hill’s method of linear regression analysis (Fig. 2) and shown in Table 1. Comparing the results obtained using sigmoidal fit and Hill’s method of linear regression analysis, it is clear that the difference is on the level of experimental error.
3.1. Influence of OPs on MPO peroxidase activity The influence of OPs on MPO peroxidase activity was investigated for OPs concentrations ranging from 1 1011 to 1 104 M in phosphate buffer at pH 6. The effect was monitored through the oxidation of o-dianisidine in the presence of H2O2. Concentration of oxidized o-dianisidine is directly related to MPO activity and it was determined spectrophotometrically, as described earlier (Section 2.2.). Primarily, the optimal concentration of MPO for the inhibition test was determined. Our previous results [17] obtained using UPLC technique and AChE assay showed that the presence of MPO below 10 nM in reaction mixture did not induced measurable oxidation of investigated OPs in the concentration range from 1011 to 103 M. For the inhibition experiments it was necessary to choose the highest MPO concentration which would not lead to notable oxidation of OPs in specified concentration range. Therefore, the possible inhibition of MPO by parent OPs is the only observed phenomenon in this case. For this reason only the MPO concentrations below 10 nM were considered for inhibition experiments. The investigation of MPO activity vs. MPO concentration showed that it increased slowly up to 6 nM enzyme and then grew rapidly (Supplementary data, Fig. S1). As optimal MPO concentration for the inhibition test the concentration of 7 nM MPO was chosen. At that point, MPO activity increased dramatically compared to the lower concentrations. High MPO activity is very important for obtaining proper and reliable data. It was also necessary to determine the optimal incubation time of the enzyme and inhibitor in the inhibition test. In order to investigate the influence of incubation time between OPs and MPO on 7 nM MPO inhibition, the incubation time before the reaction was started varied from 1 to 60 min. The dependence of MPO activity after exposure to OPs as a function of incubation time in the presence of 1 106 M malathion or malaoxon is shown in Fig. S2 (Supplementary data). It can be seen that the MPO activity decreased with the increase of incubation time between the enzyme and OPs (Fig. S2). As an optimal incubation time for the inhibition test the period of 30 min of incubation was chosen, having in mind that it is important to keep the test as brief as possible and that the change in MPO activity between 30 and 60 min of incubation was negligible. The inhibition of 7 nM MPO peroxidase activity induced by investigated OPs in the concentration range from 1 1011 to 1 104 M was evaluated using above determined experimental conditions for the inhibition test. The incubation time of enzyme with OPs was 30 min. The obtained results are presented in Fig. 1. Activity of MPO is expressed as the mean value of the percentage of MPO activity relative to the corresponding control value measured in the absence of OPs. Using the UPLC analysis, OPs oxidation products were not detected in the reaction mixtures when OPs were in contact with 7 nM MPO (data not shown), unlike in the presence of higher enzyme concentration [17,18]. Among the investigated OPs, only malathion and malaoxon exerted the inhibitory power in the whole range of examined concentrations, hence only malathion and malaoxon were further examined. IC50 values for malathion and malaoxon (Table 1) were determined from the data given in Fig. 1B. For malathion, IC50 value calculated from the sigmoidal curve is 3 107 M, while for
3.2. Influence of OPs on MPO chlorination activity The influence of OPs on MPO chlorination activity was also examined. First, the optimal MPO and H2O2 concentration were determined. MPO in the concentration range from 1 to 10 nM in the presence of 10, 20 and 30 lM H2O2 were examined (Supplementary data, Fig. S3). From the results presented in Fig. S3 (Supplementary data), it is obvious that MPO chlorination activity increases with the increase of MPO and H2O2 concentration. In all cases, at certain MPO and H2O2 concentration ratios, a plateau of produced [HOCl] is reached, while the amount of produced HOCl was found to depend on initial [MPO]-to-[H2O2] ratio. In fact, at high H2O2 concentration and low [MPO]-to-[H2O2] the amount of HOCl starts to decay. This is due to the fact that H2O2, while it is MPO’s substrate, it is also MPO inhibitor if present at high concentration [26,27]. It was confirmed that H2O2 inhibits MPO following mechanism-based pattern [28]. Hence, 3 nM MPO in the presence of 20 lM H2O2 was chosen as optimal. MPO (3 nM) was incubated with OPs (diazinon, malathion, chlorpyrifos, azinphos-methyl, phorate and their oxo-analogs) in the concentration range from 1 1010 to 1 104 M during 30 min, in phosphate buffer pH 6.5 and in the presence of 100 mM NaCl and 5 mM taurine. After 30 min, the reaction was started by adding 20 lM H2O2. Neither of the investigated OPs inhibited MPO chlorination activity under specified experimental conditions. It was possible to conclude that, unlike peroxidase activity of MPO, chlorination activity is not affected by investigated OPs under given experimental conditions. This is of particular importance having in mind that the chlorination activity is the physiological function of MPO [12]. The results also confirmed that inhibition of the peroxidase activity of MPO by a certain compound do not necessarily indicate that the compound would also inhibit the chlorination activity of the enzyme [16]. On the other hand, majority of the known MPO inhibitors do affect both of the enzyme’s activities [16,29]. 4. Discussion In this section we analyze (i) observed trends in the inhibition of MPO peroxidase activity by investigated OPs, (ii) differences between the effects of investigated OPs on peroxidase and chlorination activity of MPO based on the MPO reaction mechanism and (iii) discuss previous reports on the effects of OPs on the action of human neutrophils. As can be seen from the inhibition curves (Fig. 1), peroxidase activity of MPO depends on OPs concentration in all examined cases, except in the case of azinphos-methyl and respective oxoform, which did not exert any inhibitory effect (data not shown). It is obvious that all oxo-forms of OPs are more powerful inhibitors of MPO peroxidase activity than respective thio-forms. Having in mind the structures of examined OPs (Scheme 1), it can be concluded that azinphos-methyl’s two rings, one aromatic and one heterocyclic, invoke large steric hindrance for the interaction with MPO. Furthermore, one can compare residual MPO activities when incubated with some particular OPs concentrations. For example, taking the OPs concentration of 107 M it can be seen that the
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Fig. 1. The concentration dependent inhibition of MPO peroxidase activity induced by (A) diazinon and diazoxon, (B) malathion and malaoxon, (C) chlorpyrifos and chlorpyrifos oxon and (D) phorate and phorate oxon. The MPO activities are expressed as the mean percentage of the activity relative to the corresponding control value in the absence of OPs.
Table 1 IC50 values obtained using sigmoidal fit and Hill’s method of linear regression analysis. OP
IC50 – sigmoidal fit (M)
IC50 – Hill’s analysis (M)
Malathion Malaoxon
(3 ± 1) 107 (5 ± 1) 109
(4 ± 1) 107 (7 ± 1) 109
Fig. 2. Hill’s analysis of MPO peroxidase activity inhibition induced by malathion and malaoxon.
lowest activities are observed in the cases of malathion and phorate, which have the simplest structure. MPO activity further increases, i.e. the degree of inhibition decreases, when going from diazinon to chlorpyrifos (having one aromatic ring within the structure) to azynphos-methyl (two rings). The same approximate trend is followed in the case of oxo-analogs too, leading to the conclusion that inhibition trend is rendered by molecular structure which invokes steric hindrance for OPs interaction with MPO’s active center responsible for peroxidase activity or allosteric site (see further discussion). For diazinon, chlorpyrifos and phorate it was not possible to achieve higher concentrations of OPs, due to their solubility (the final solutions must not contain more than 1% organic solvent in order to MPO have proper function). Additional insights regarding MPO peroxidase activity inhibition by investigated OPs could be provided by virtual docking analysis. As already mentioned, under specific conditions regarding the substrate availability, MPO displays its chlorination or peroxidase activity. Halide oxidation starts with reaction of native MPO (ferric-MPO) and H2O2 to form Compound I (Eq. (1)), having two oxidizing equivalents more than the native MPO. In halogenation cycle, Compound I oxidizes halides and pseudohalides via twoelectron process, while Compound I is reduced to native enzyme (Eq. (2)) [9]. In peroxidase cycle, Compound I is reduced in two one-electron steps, first to Compound II and then to native MPO (Eqs. (3) and (4)). It is believed that Compound II is inactive in halide and pseudohalide oxidation [30].
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Ferric-MPO þ H2 O2 ! Compound I þ H2 O
ð1Þ
Compound I þ X þ Hþ ! native MPO þ HOX
ð2Þ
Compound I þ AH2 ! Compound II þ AH
ð3Þ
Compound II þ AH2 ! native MPO þ AH
ð4Þ
o
Standard potentials (E ) of redox couples involved in halogenation and peroxidase cycles are considered to be: Eo (Compound I/ native MPO) = 1.16 V [31], Eo (Compound I/Compound II) = 1.35 V and Eo (Compound II/native MPO) = 0.97 V [32], indicating that Compounds I and II are potent oxidants which should be reduced easily in the presence of electron donors. Our recent work confirmed that investigated thio-OPs can be oxidized by electrochemically generated halogen X species (X = Cl, Br and I) [33,34]. In specific, these reactive species were generated from halide ions (Cl, Br or I) by anodic oxidation and, depending on the nature of the specific halide ion, different relative amounts of X2, X3, HOX and OX species were formed. Although Cl and Br species are potent antioxidants with standard redox potential close to the ones of MPO intermediates redox couples, iodine species were also able to oxidize thio-OPs although having Eo values well below that of MPO intermediates redox couples. This indicates that thioOPs should be good electron donors for both Compound I and Compound II. As chlorination activity of MPO was not affected by the presence of investigated OPs and peroxidase activity was influenced, it is suggested that reduction of Compound II by OPs cannot be the slow step in peroxidase cycle. Namely, if opposite holds, OPs could potentially trap MPO in the form of Compound II and consequently inhibit chlorination activity of MPO. Our previous work [17–20] suggests that OPs are essentially poor MPO peroxidase substrates as only a small fraction of OPs can be oxidized by MPO when peroxidase activity is forced by adequate experimental conditions. This points that, in spite the fact that oxidation of OPs is thermodynamically favorable, large kinetic barrier exists for the reaction of thio-OPs with Compound I. In the experiments where peroxidase activity of MPO was investigated, [MPO]-to-[OP] ratio was set in a way that no measurable amounts of OPs’ oxo-forms are formed in the course of the experiments. In addition, oxo-forms of investigated OPs are not MPO substrates, as cannot act as electron donors, but still affect peroxidase activity of MPO. Hence, it is suggested that investigated OPs and their corresponding oxoforms might actually affect MPO peroxidase activity following mixed inhibition pattern. Conformational changes of MPO upon binding of OPs might be subtle enough so that chlorination activity is not affected, but large enough to affect oxidation of o-dianisidine (used here to probe peroxidase activity of MPO) through the induction of steric hindrance for its bonding to active site. Referring to the work of Queiroz et al. [21] who examined neutrophil function of workers exposed to organophosphorus and carbamate pesticides, it is to be questioned whether the reduced ability of neutrophils to kill C. albicans and C. pseudotropicalis should be ascribed to the inhibitory action of organophosphorus pesticides towards MPO. It can be speculated that MPO deficiency upon the exposure to organophosphorus (and carbamate) pesticides [21] might be invoked by the mechanisms other than MPO inhibition by OPs, at least in the cases of five here investigated OPs and their oxo-analogs. 5. Conclusion Inhibitory action of five organophosphorus pesticides (diazinon, malathion, chlorpyrifos, azinphos-methyl and phorate) and their oxo-analogs, towards MPO was investigated. Obtained results suggest their separate effects when concerned with peroxidase and
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chlorination activity. Among investigated OPs only malathion and malaoxon delivered inhibitory power towards MPO peroxidase activity in the whole investigated concentration range, with IC50 values of the order of 3 107 and 5 109 M, respectively. Inhibition of peroxidase activity was correlated with the molecular structure of investigated OPs, concluding that inhibition trend is rendered by molecular structure which invokes steric hindrance for OPs interaction with MPO active center responsible for peroxidase activity. Complete absence of inhibitory effect on the MPO peroxidase activity was observed in the case of azinphos-methyl and azinphos-methyl oxon, OPs with two six-member rings within their structure. Interestingly, chlorination activity of MPO was completely unaffected by the presence of any of the investigated OPs, suggesting that physiological function of MPO is preserved in the presence of here selected OPs. Obtained results suggest that previously reported results regarding reduced ability of neutrophils to kill C. albicans and C. pseudotropicalis upon exposure to organophosphorus and carbamate pesticides might be invoked by the mechanisms other than inhibition of MPO. Hence, additional research is needed to resolve opened questions regarding modified action of human neutrophils upon exposure to OPs. Acknowledgment The authors would like to express their gratitude to the Ministry of Education, Science and Technological Development of the Republic of Serbia for their financial support, contract 172023. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.pestbp.2013. 05.004. References [1] J.S. Van Dyk, B. Pletschke, Review on the use of enzymes for the detection of organochlorine, organophosphate and carbamate pesticides in the environment, Chemosphere 82 (2011) 291–307. [2] T.R. Fukuto, Mechanism of action of organophosphorus and carbamate insecticides, Environ. Health Perspect. 87 (1990). [3] M. Colovic, D. Krstic, S. Petrovic, A. Leskovac, G. Joksic, J. Savic, M. Franko, P. Trebse, V. Vasic, Toxic effects of diazinon and its photodegradation products, Toxicol. Lett. 193 (2010) 9–18. [4] J. Lintellman, A. Katayama, N. Kurihara, L. Shore, A. Wenzel, Endocrine disruptors in the environment, Pure Appl. Chem. 75 (2003) 631–681. [5] H.R. Andersen, A.M. Vinggaard, T. Rasmussen, I.M. Gjermandsen, E.C. BonefeldJorgensen, Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro, Toxicol. Appl. Pharmacol. 179 (2002) 1–12. [6] G.H. Degen, H.M. Bolt, Endocrine disruptors: update on xenoestrogens, Int. Arch. Occup. Environ. Health 73 (2000) 433–441. [7] H. Kojima, E. Katsura, S. Takeuchi, K. Niiyama, K. Kobayashi, Screening for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells, Environ. Health Perspect. 112 (2004) 524–531. [8] J. Li, N. Li, M. Ma, J.P. Giesy, Z. Wang, In vitro profiling of the endocrine disrupting potency of organochlorine pesticides, Toxicol. Lett. 183 (2008) 65– 71. [9] S.J. Klebanoff, Myeloperoxidase, P. Assoc. Am. Phys. 111 (1999) 383–389. [10] A.J. Kettle, C.A. Gedye, C.C. Winterbourn, Mechanism of inactivation of myeloperoxidase by 4-aminobenzoic acid hydrazide, Biochem. J. 321 (1997) 503–508. [11] P.G. Furtmüller, M. Zederbauer, W. Jantschko, J. Helm, M. Bogner, C. Jakopitsch, C. Obinger, Active site structure and catalytic mechanisms of human peroxidases, Arch. Biochem. Biophys. 445 (2006) 199–213. [12] S.J. Klebanoff, Myeloperoxidase: occurrence and biological function, in: J. Everse, K.E. Everse, M.B. Grisham (Eds.), Peroxidase in Chemistry and Biology, CRC Press, Boca Raton, 1991, pp. 1–35. [13] M.D. Demling, H. Robert, The modern version of adult respiratory distress syndrome, Annu. Rev. Med. 46 (1995) 193–202. [14] S.G. Waley, Kinetics of suicide substrates, Biochem. J. 185 (1980) 771. [15] E. Shacter, R.L. Lopez, S. Pati, Inhibition of the myeloperoxidase-H2O2-Clsystem of neutrophils by indomethacin and other non-steroidal antiinflammatory drugs, Biochem. Pharmacol. 41 (1991) 975–984.
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