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Effects of pyrethroids and endosulfan on fluidity of mitochondria membrane in Chilo suppressalis (Walker)
Pesticide Biochemistry and Physiology 95 (2009) 72–76
Contents lists available at ScienceDirect
Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Effects of pyrethroids and endosulfan on fluidity of mitochondria membrane in Chilo suppressalis (Walker) Haiping Li, Tao Feng 1, Xueyan Shi, Pei Liang, Qi Zhang, Xiwu Gao * Department of Entomology, China Agricultural University, Beijing 100193, PR China
a r t i c l e
i n f o
Article history: Received 20 December 2008 Accepted 4 June 2009 Available online 10 June 2009 Keywords: Chilo suppressalis Pyrethroids Endosulfan Fluidity of mitochondrial membrane Temperature coefficient
a b s t r a c t The effects of pyrethroids and endosulfan on fluidity of mitochondrial membranes from Chilo suppressalis were investigated at different temperatures by steady-state fluorescence polarization using 1,6diphenyl-1,3,5-hexatriene (DPH) molecule as probe. The results showed that changes in DPH polarization caused by the pyrethroids tested were more at lower temperature than at higher temperature, which showed a negative temperature coefficient of the pyrethroids. On the contrary, change in DPH polarization caused by endosulfan was more at higher temperature than at lower temperature. Endosulfan displayed a positive temperature coefficient. Moreover, these effects of pyrethroids and endosulfan on changes of DPH polarization were dose-dependent in mitochondrial membranes of C. suppressalis. DPH polarization value increased in the mitochondrial membranes treated by all concentrations of pyrethroids and decreased by endosulfan, and changes caused by pyrethroids were more pronounced than by endosulfan. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Pyrethroids are derived from the naturally occurring substance, pyrethrin. Synthetic pyrethroid insecticides have been widely applied in agriculture, home pest control, protection of foodstuffs and disease vector control in the world [1]. Pyrethroids accounted for approximately one-fourth of the worldwide insecticide market before 1998 [2]. At present pyrethroids are still one kind of the most commonly used insecticides in China. Pyrethroids may be divided into two groups according to their chemical structures. Type I pyrethroids do not contain acyano group, which is present in type II pyrethroids such as deltamethrin [3]. Pyrethroids are toxic to the nervous systems of both insects and mammals, and the main target of pyrethroids appears to be the ionic channels of biomembranes. Insecticides have been shown to partition into membranes and cause change in membrane fluidity [4]. Extensive studies on interaction of insecticides with membranes in mammal have been carried out for DDT [5,6], lindane [7], endosulfan [8], parathion [9], fenvalerate [10], deltamethrin [11], etc. But so far the researches on interaction of pyrethroids with biomembrane of insects have received little similar attention and details of their membrane interactions are not known.
* Corresponding author. Fax: +86 10 62732974. E-mail address: [email protected] (X. Gao). 1 His contribution for this paper is equal with the first author. 0048-3575/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2009.06.004
Fluidity is determined by lipid composition and temperature. Moreover, fluidity of biomembranes is influenced greatly by temperature. At cold temperatures, the membranes ‘‘gel” and are not fluid, and at high temperatures, the membranes are too fluid and become ‘‘leaky” allowing ions to cross (http://www.esf.edu/efb/ course/EFB325/). Moreover, the toxicity of insecticides to pests is affected by many factors, and temperature is one of the important factors. Many pyrethroids and DDT exhibit a negative temperature coefficient [12–15]. In the previous study, we elucidated effects of temperature on toxicity of pyrethroids and endosulfan, and activity of mitochondrial Na+–K+-ATPase and Ca2+–Mg2+-ATPase in Chilo suppressalis [16]. The result showed that toxicities of deltamethrin, bifenthrin and endosulfan all possessed a positive temperature coefficient between 17 and 37 °C. The temperature coefficients of deltamethrin, bifenthrin and endosulfan were 5.59, 1.68 and 2.85, respectively. The inhibition of deltamethrin to the specific activities of Na+– K+-ATPase and Ca2+–Mg2+-ATPase showed a negative temperature coefficient, but endosulfan exhibited a positive temperature coefficient. Inhibition of bifenthrin exhibited the contrary temperature coefficients between Na+–K+–ATPase and Ca2+–Mg2+-ATPase and a negative temperature coefficient for the former and a positive temperature coefficient for the later. In this paper, we reported studies on the effects of the three pyrethroid insecticides, deltamethrin, bifenthrin and cyfluthrin, and one organochlorine insecticide, endosulfan on the fluid of mitochondrial membrane from the rice stem borer in different temperatures.
H. Li et al. / Pesticide Biochemistry and Physiology 95 (2009) 72–76
73
2. Materials and methods
G ¼ IHV =IHH
2.1. Insect
IHH(HV) is the intensity with horizontal excitation with horizontal (vertical) emission polarizer.
Rice stem borers were originally obtained from a rice paddy of Jiangsu Province in 2004 and have been reared in our laboratory. The larvae were reared on rice seedlings as described by Shang et al. [17]. Insects were maintained in a growth chamber at 28 ± 1 °C under a 16:8 (L:D) photoperiod and approximately 80% RH. 2.2. Chemicals Cyfluthrin (92.0% a.i.), bifenthrin (95.0% a.i.), and deltamethrin (99% a.i.) were provided kindly by Jiangsu Yangnong Chemical Group Co., Ltd., and endosulfan (90% a.i.) by Jiangsu Rongdong Pesticide Factory in China. Coomassie brilliant blue G-250 was purchased from Sigma (USA). DPH (1,6-diphenyl-1,3,5-hexatriene, 99% purity) was purchased from Fluca (USA).
2.6. Protein assay Concentrations of protein in homogenates were determined by the method of Bradford [21] using bovine albumin as a standard. Briefly, 2 ml of protein reagent (final concentrations in the reagent were 0.01% (w/v) Coomassie Brilliant Blue G-250, 4.7% (w/v) ethanol, and 8.5% (w/v) phosphoric acid.) was added to the test tube with 1 ml homogenates and the contents mixed by vortexing. The absorbance at 595 nm was measured after 2 min and before 1 h in 3 ml cuvettes against a reagent blank prepared from 1 ml of the appropriate buffer and 5 ml of protein reagent. The weight of protein was plotted against the corresponding absorbance resulting in a standard curve used to determine the protein in homogenates.
2.3. Preparation of mitochondria membrane
2.7. Data analysis
Mitochondrial membrane of C. supperssalis was isolated by differential centrifugation according to Voss et al. [18], using an extraction medium consisting of 10 mM Tris–HCl, 0.1 mM EDTA, 250 mM sucrose, NaCl 0.8 g%, BSA 0.5 g%, pH 7.4. Rice stem borers were congealed in 80 °C ultra low temperature freezer and a group of 18 fourth-instar larvae (body weight 9–11 mg) were homogenized in 2 ml of ice-chilled sucrose extracting solution with a hand-operated tissue grinder (Teflon pestle). The homogenate was centrifuged at 3000g for 10 min and then the supernatant was re-centrifuged at 10,000g for 30 min at 4 °C (By using an Eppendorf centrifuge, 5417R, Eppendorf AG, German). The sediment was suspended in 2 ml ice-chilled sucrosuml extracting solution and then centrifuged 10,000g for 20 min and last suspended in 2 ml ice-chilled sucrosuml extracting solution. The fresh mitochondria were prepared in all experiments of this study.
The data analysis was performed by One-way ANOVA with Turkey posttest, using GraphPad InStat version 3.00.
2.4. Preparation of DPH A solution of DPH (2 mM) in tetrahydrofuran was diluted 1000fold by injection into a vigorously stirred solution of PBS (10 mM buffer, pH 7.4; 0.14 mol/L NaCl), and stirred for 1 h before use. It must be shacked fiercely when DPH was diluted. 2.5. Fluorescence polarization studies Fluorescence polarization was measured by the method of Cheng and Levy [19] using DPH [20]. Samples were diluted to a protein concentration of 250 mg per milliliter with Tris–HCl solution, mixed with an equal volume of the DPH solution, and incubated in a shaking bath for 30 min at 25 °C. Steady-state fluorescence polarization measurements were performed with a Model LC-55 polarization spectrophotometer (Perkin-Elmer), which is equipped with two photomultipliers to detect separately each polarized component of the fluorescent light. Temperature in the cuvettes was controlled with a thermostated circulating-water pump. Excitation wavelength was 362 nm and emission was measured at 432 nm. The degree of fluorescence polarization (P) was calculated as:
P ¼ Ik GIj =Ik þ GIj Where I| and I|| are the intensities of emitted light with vertical excitation, vertical (horizontal) emission polarizer. G is the correction factor for the optical system, given by:
3. Results 3.1. Effects of pyrethroids on fluidity of mitochondrial membrane dependent on temperature in C. suppressalis The fluorescence polarization of mitochondrial membrane incorporated by DPH as molecule probe was monitored from 15 to 45 °C. The effects of pyrethroids and endosulfan occurred over all of the range of the studied temperatures, from 15 to 45 °C. Deltamethrin, bifenthrin and cyfluthrin (100 lM) showed a considerable increase in DPH polarization in mitochondrial membrane examined (Fig. 1). Changes in DPH polarization were more at lower temperature than at higher temperature. The slope of the Arrhenius plot decreased more by pyrethroids than by control and a further decrease was observed with deltamethrin and cyfluthrin which suggested a change in the activation energy of the probe molecule. On the contrary, endosulfan showed a considerable decrease in DPH polarization in mitochondrial membrane in the examined temperatures (Fig. 1). The change in DPH polarization is more at higher temperature than at lower temperature, which showed endosulfan possessed a positive temperature coefficient. 3.2. Effects of pyrethroids on fluidity of mitochondrial membrane dependent on concentration in C. suppressalis The effect of increasing concentrations of pyrethroids and endosulfan on DPH polarization of mitochondrial membrane at a particular temperature (25 °C) was shown in Fig. 2. Effects of pyrethroids and endosulfan on fluorescence polarization were dose-despondent. The changing in DPH polarization caused by pyrethroids was different from endosulfan. The changes of DPH polarization caused by bifenthrin and cyfluthrin were more, and the effect of deltamethrin on DPH polarization was little in less than 80 lM, and the curves of concentration-effect were non-linear. However, the effect of endosulfan on fluorescence polarization is reversed comparing with pyrethroids. DPH polarization decreased with the increasing of endosulfan concentrations. The result showed that pyrethroids tested can decrease fluidity of membrane and endosulfan increases fluidity of membrane.
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H. Li et al. / Pesticide Biochemistry and Physiology 95 (2009) 72–76
0.31
0.29
Fluoresence Polarization
Fluoresence Polarization
0.31
0.27 0.25 0.23 0.21 Control
0.19
Deltamethria
0.17
0.29 0.27 0.25 0.23 0.21 0.19
Control
0.17
Bifenthrin
0.15
0.15 3.1
3.2
3.3
3.4
3.1
3.5
3.2
0.31
0.31
0.29
0.29
0.27 0.25 0.23 0.21 0.19
Control
0.17
Cyfluthrin
0.15
3.4
3.5
1000/TK
Fluoresence Polarization
Fluoresence Polarization
1000/TK
3.3
0.27 0.25 0.23 0.21 0.19
Control
0.17
Endosulfan
0.15 3.1
3.2
3.3
3.4
3.5
1000/TK
3.1
3.2
3.3
3.4
3.5
1000/TK
Fig. 1. Fluorescence polarization of DPH in mitochondrial membrane as a function of temperature in the absence (0 lM) and presence of pyrethroids and endosulfan (100 lM).
Cyfluthrin
0.26
Bifenthrin Endosulfan
Fluorescence Polarization
0.25
Deltamethrin 0.24 0.23 0.22 0.21 0.2 0
20
40
60
80
100
120
Concentration (µM) Fig. 2. Fluorescence polarization of DPH in mitochondrial membrane as a function of increasing pyrethroids and endosulfan concentration at 25 °C.
4. Discussion Actually, the effect of temperature on biomembrane had been studied in rat by Sarkar et al. Fenvalerate could decrease fluorescence polarization value of DPH of microsomal membrane, and
the change of DPH polarization was more at lower temperatures than at higher temperatures [10]. Welligton et al. found that deltamethrin increased the fluorescence polarization of DPH in native membrane from 10.5 to 40.5 °C [11]. In our study, the effect of three pyrethroids on fluorescence polarizations of DPH were all occurred in the range of temperatures from 10 to 45 °C. The changes of the three pyrethroids on DPH polarization in different temperatures were similar to that described in rat by Sarkar et al and Welligton et al. [10,11]. Clark and Matsumura showed that type II pyrethroids mainly inhibit Ca2+–Mg2+-ATPase activities [22]. In previous studies, we compared the toxicity and ATPase inhibition of pyrethroids in different temperatures [16]. The result showed a better relationship of the temperature coefficient in toxicity and inhibition of Ca2+–Mg2+-ATPase by deltamethrin and bifenthrin in C. suppressalis. Moreover, Na+– K+-ATPase could be also inhibited by pyrethroids and the inhibition was likewise effected by temperature [16]. Compared the inhibition of Na+–K+-ATPase with the effects on fluorescence polarization by the pyrethroids, the effect of temperature on both inhibition of Na+–K+-ATPase and fluorescence polarization were similar (Table 1). Three pyrethroids showed a negative temperature coefficient and endosulfan showed a positive temperature coefficient. It was because that Na+–K+-ATPase transports Na+ ions and K+ ions against its concentration gradient across the membrane, and fluidity is one of the most important characters of membrane. It’s that fluidity is determined by lipid composition and temperature. So the temperature could affect the changing of membrane fluidity and inhibition to
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H. Li et al. / Pesticide Biochemistry and Physiology 95 (2009) 72–76 Table 1 Comparison of polarization and percentage of inhibition of Na+–K+-ATPase in mitochondria by insecticides in C. suppressalis in different temperatures. Compound
Temp. (°C)
Fluorescence polarization (P)
Anisotropy (c)
Microviscosity (g)
Na+–K+-ATPase inhibition (%)*
Temp. coeff.
Mitochondrial membrane
17 27 37 17 27 37 17 27 37 17 27 37 17 27 37
0.228 ± 0.003 0.212 ± 0.001 0.195 ± 0.002 0.254 ± 0.005 0.226 ± 0.002 0.201 ± 0.003 0.275 ± 0.002 0.249 ± 0.001 0.224 ± 0.009 0.244 ± 0.005 0.218 ± 0.005 0.200 ± 0.003 0.228 ± 0.005 0.206 ± 0.002 0.189 ± 0.003
0.163 ± 0.002 0.151 ± 0.001 0.140 ± 0.004 0.187 ± 0.005 0.164 ± 0.003 0.147 ± 0.002 0.201 ± 0.03 0.176 ± 0.011 0.161 ± 0.006 0.175 ± 0.002 0.152 ± 0.007 0.143 ± 0.001 0.161 ± 0.003 0.148 ± 0.001 0.136 ± 0.002
1.96 1.705 1.472 2.459 1.926 1.594 2.982 2.360 1.893 2.266 1.794 1.534 1.898 1.627 1.429
62.81 ± 5.68 45.69 ± 0.76 29.27 ± 0.06 59.54 ± 3.55 26.36 ± 4.46 39.51 ± 3.51 43.93 ± 0.39 11.82 ± 5.41 27.27 ± 1.84 15.03 ± 1.97 1.82 ± 2.71 17.46 ± 5.61
Deltamethrin
Bifenthrin
Cyfluthrin
Endosulfan
*
+
Data was from Ref. [16].
Na+–K+-ATPase by pyrethroids, implicating that membrane fluidity is one of the most important factors affecting negative temperature coefficient of pyrethroids. Generally, it had been accepted that DDT and pyrethroids acted directly on the nervous system causing disruption of normal ion permeabilities in the nerve membrane [23]. Endosulfan is one of organochlorine insecticides, but different from DDT in molecular structure. Martins et al.’s study exhibited that addition of endosulfan to liposome induced a decrease in the polarization values in the gel phase [8]. Our previous study proved that the toxicity and ATPase inhibition of endosulfan was a positive temperature coefficient [16], which is consistent with the effect of endosulfan on membrane fluidity in the rice stem borer. An obvious conclusion emerging from this study is that the effects of temperature on biological action of pyrethroids are quite complex. The proposed three hypotheses might be all attributed to the negative temperature coefficient of pyrethroids. First one, differential target site interactions, had been proved by many researches [24,25]. The second, differential metabolism was indicated by Scott et al. [12] and Mcintosh et al. [26]. The third, different lipid solubility, which proposed by Munson was considered the least plausible because the nerve membrane is composed largely of lipids and would therefore accumulate more pesticide at higher temperature, thus manifesting a positive temperature coefficient [27]. The results obtained from this study should be useful in order to explain the effect of temperature on pyrethroids toxicity. Pyrethroid insecticides are widely applied in agriculture from south to north in China, and the air temperature is very different in the various areas of China. Temperature often has a significant effect on the efficacy of insecticides when used on the field. But the effect of temperature on insecticides to pests had not received great attention in China before. Pyrethroids might be one of alternative insecticides in rice paddy since methamidophos was forbidden by the end of 2006 in China. So knowledge of a pyrethroid’s temperature coefficient will enable pest managers to select a pyrethroid that is efficacious under the given environmental conditions. Acknowledgments This research was supported by National Basic Research Program of China (Contract No. 2006CB102003), National Natural Science Foundation of China (Contract No. 30530530, 30571232, 30471153, and 30170621) and National Key Research Program of China for the Eleventh Five-years Plan (Contract No. 2006BAD08A03).
References [1] J.J. Herve, Agricultural public health and animal usage, in: J.P. Leathey (Ed.), The Pyrethroid Insecticides, Taylor & Francis Press, London, 1985, pp. 343–426. [2] J.E. Casida, G.B. Quistad, Golden age of insecticide research: past, present, or future?, Annu Rev. Entomol. 43 (1998) 1–16. [3] D.W. Gammon, M.A. Brown, J.E. Casida, Two classes of pyrethroid action in the cockroach, Pestic. Biochem. Physiol. 15 (1981) 181–191. [4] M.C. Antunes-Maderia, V.M.C. Maderia, Interaction of insecticides with lipid membranes, Biochem. Biophys. Acta 550 (1979) 384–392. [5] M.C. Antunes-Maderia, V.M.C. Maderia, Membrane fluidity as affected by the organochlorine insecticide DDT, Biochem. Biophys. Acta 1023 (1990) 469–474. [6] M.C. Antunes-Maderia, L.M. Almeida, V.M.C. Maderia, DDT-membrane interactions studied with two fluorescence probes, Pestic. Sci. 33 (1991) 347–357. [7] M.C. Antunes-Maderia, V.M.C. Maderia, Membrane fluidity as affected by the insecticide lindane, Biochem. Biophys. Acta 982 (1989) 161–166. [8] J.D. Martins, J.P. Monteiro, M.C. Antunes-Madeira, A.S. Jurado, V.M.C. Madeira, Use of the microorganism Bacillus stearothermophilus as a model to evaluate toxicity of the lipophilic environmental pollutant endosulfan, Biochim. Biophys. Acta 1617 (2003) 595–601. [9] M.C. Antunes-Maderia, V.M.C. Maderia, Partition of parathion in synthetic and native membranes, Biochem. Biophys. Acta 778 (1984) 49–56. [10] S.N. Sarkar, S.V. Balasubramanian, S.K. Sikdar, Effect of fenvalerate, a pyrethroid insecticide on membrane fluidity, Biochem. Biophys. Acta 1147 (1993) 137–142. [11] L.B. Welligton, M.S.C.C. Silvia, G.S.C. Eva, E.M. Maria, M.B. Rocha, M. de Olivira, Effects of deltamethrin on function of rat mitochondria and on native and synthetic model membrane, Toxic. Lett. 152 (2004) 191–202. [12] J.G. Scott, G.P. Georghiou, Influence of temperature on knockdown, Toxicity and tesistance to pyrethroids in the house fly, Musca domestica, Pestic. Biochem. Physiol. 21 (1984) 53–62. [13] T.C. Sparks, M.H. Shour, E.G. Wellemeyer, Temperature–toxicity relationships of pyrethroids on three lepidopterans, J. Econ. Entomol. 75 (1982) 643–646. [14] S.J. Toth Jr., T.C. Sparks, Influence of treatment technique on the temperature– toxicity relationship of cis- and trans- permethrin in the cabbage looper (Lepidoptera: Noctuidae), J. Econ. Entomol. 81 (1988) 115–118. [15] T.C. Sparks, A.M. Pavloff, R.L. Rose, D.F. Glower, Temperature–toxicity relationships of pyrethroids on Heliothis virescens (F.) (Lepidoptera: Noctuidae) and Anthonomus grandis grandis Boheman (Coleoptera: Curculionidae), J. Econ. Entomol. 76 (1983) 243–246. [16] H.P. Li, F. Tao, P. Liang, X.Y. Shi, X.W. Gao, H. Jiang, Effect of temperature on toxicity of pyrethroids and endosulfan, activity of mitochondrial Na+–K+ATPase and Ca+–Mg+-ATPase in Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), Pestic. Biochem. Physiol. 86 (2006) 151–156. [17] Z.Z. Shang, Y.S. Wang, Y.H. Zhou, Study on rearing method of rice stem borer Chilo suppressalis Walker, Acta Entomol. Sin. 22 (2) (1979) 164–167. [18] D.O. Voss, A.P. Campelo, M. Bacial, The respiratory chain and oxidative phosphorylation of rat brain mitochondria, Biochem. Biophys. Res. Commun. 4 (1961) 48–51. [19] S. Cheng, D. Levy, The effects of cell proliferation on the lipid composition and fluidity of hepatocyte plasma membrane, Arch. Biochem. Biophys. 196 (1979) 424–429. [20] R.A. Harrls, F.R. Simon, What should hepatologists know about membrane fluidity?, Hepatology 7 (1987) 177–180. [21] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding, Anal. Biochem. 72 (1976) 248–254. [22] J.M. Clark, F. Matsumura, Two different types of inhibitory effects of pyrethroid on nerve Ca2+ and Ca2+–Mg2+-ATPase activity in the squid Loligo pealei, Pestic. Biochem. Physiol. 18 (1982) 180–190.
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[23] C.M. Wang, T. Narahashi, M.J. Suka, Mechanisms of negative temperature coefficient of nerve blocking action of allethrin, J. Pharmacol. Exp. Ther. 182 (1972) 442–453. [24] J.G. Starkus, T. Narahashi, Temperature dependence of allethrin-induced repetitive discharges in nerves, Pestic. Biochem. Physiol. 9 (1978) 225– 230.
[25] M.E. Adams, T.A. Miller, Site of action of pyrethroids: repetitive ‘‘backfiring” in flight motor units of house fly, Pestic. Biochem. Physiol. 11 (1979) 218–225. [26] A.H. Mcintosh, Temperature and toxicity of insecticides, Chem. Ind. (1957) 2– 6. [27] S.C. Munson, G.M. Padilla, M.L. Weissmann, Insect lipids and insecticidal action, J. Econ. Entomol. 47 (1954) 578–587.
Contents lists available at ScienceDirect
Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest
Effects of pyrethroids and endosulfan on fluidity of mitochondria membrane in Chilo suppressalis (Walker) Haiping Li, Tao Feng 1, Xueyan Shi, Pei Liang, Qi Zhang, Xiwu Gao * Department of Entomology, China Agricultural University, Beijing 100193, PR China
a r t i c l e
i n f o
Article history: Received 20 December 2008 Accepted 4 June 2009 Available online 10 June 2009 Keywords: Chilo suppressalis Pyrethroids Endosulfan Fluidity of mitochondrial membrane Temperature coefficient
a b s t r a c t The effects of pyrethroids and endosulfan on fluidity of mitochondrial membranes from Chilo suppressalis were investigated at different temperatures by steady-state fluorescence polarization using 1,6diphenyl-1,3,5-hexatriene (DPH) molecule as probe. The results showed that changes in DPH polarization caused by the pyrethroids tested were more at lower temperature than at higher temperature, which showed a negative temperature coefficient of the pyrethroids. On the contrary, change in DPH polarization caused by endosulfan was more at higher temperature than at lower temperature. Endosulfan displayed a positive temperature coefficient. Moreover, these effects of pyrethroids and endosulfan on changes of DPH polarization were dose-dependent in mitochondrial membranes of C. suppressalis. DPH polarization value increased in the mitochondrial membranes treated by all concentrations of pyrethroids and decreased by endosulfan, and changes caused by pyrethroids were more pronounced than by endosulfan. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction Pyrethroids are derived from the naturally occurring substance, pyrethrin. Synthetic pyrethroid insecticides have been widely applied in agriculture, home pest control, protection of foodstuffs and disease vector control in the world [1]. Pyrethroids accounted for approximately one-fourth of the worldwide insecticide market before 1998 [2]. At present pyrethroids are still one kind of the most commonly used insecticides in China. Pyrethroids may be divided into two groups according to their chemical structures. Type I pyrethroids do not contain acyano group, which is present in type II pyrethroids such as deltamethrin [3]. Pyrethroids are toxic to the nervous systems of both insects and mammals, and the main target of pyrethroids appears to be the ionic channels of biomembranes. Insecticides have been shown to partition into membranes and cause change in membrane fluidity [4]. Extensive studies on interaction of insecticides with membranes in mammal have been carried out for DDT [5,6], lindane [7], endosulfan [8], parathion [9], fenvalerate [10], deltamethrin [11], etc. But so far the researches on interaction of pyrethroids with biomembrane of insects have received little similar attention and details of their membrane interactions are not known.
* Corresponding author. Fax: +86 10 62732974. E-mail address: [email protected] (X. Gao). 1 His contribution for this paper is equal with the first author. 0048-3575/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2009.06.004
Fluidity is determined by lipid composition and temperature. Moreover, fluidity of biomembranes is influenced greatly by temperature. At cold temperatures, the membranes ‘‘gel” and are not fluid, and at high temperatures, the membranes are too fluid and become ‘‘leaky” allowing ions to cross (http://www.esf.edu/efb/ course/EFB325/). Moreover, the toxicity of insecticides to pests is affected by many factors, and temperature is one of the important factors. Many pyrethroids and DDT exhibit a negative temperature coefficient [12–15]. In the previous study, we elucidated effects of temperature on toxicity of pyrethroids and endosulfan, and activity of mitochondrial Na+–K+-ATPase and Ca2+–Mg2+-ATPase in Chilo suppressalis [16]. The result showed that toxicities of deltamethrin, bifenthrin and endosulfan all possessed a positive temperature coefficient between 17 and 37 °C. The temperature coefficients of deltamethrin, bifenthrin and endosulfan were 5.59, 1.68 and 2.85, respectively. The inhibition of deltamethrin to the specific activities of Na+– K+-ATPase and Ca2+–Mg2+-ATPase showed a negative temperature coefficient, but endosulfan exhibited a positive temperature coefficient. Inhibition of bifenthrin exhibited the contrary temperature coefficients between Na+–K+–ATPase and Ca2+–Mg2+-ATPase and a negative temperature coefficient for the former and a positive temperature coefficient for the later. In this paper, we reported studies on the effects of the three pyrethroid insecticides, deltamethrin, bifenthrin and cyfluthrin, and one organochlorine insecticide, endosulfan on the fluid of mitochondrial membrane from the rice stem borer in different temperatures.
H. Li et al. / Pesticide Biochemistry and Physiology 95 (2009) 72–76
73
2. Materials and methods
G ¼ IHV =IHH
2.1. Insect
IHH(HV) is the intensity with horizontal excitation with horizontal (vertical) emission polarizer.
Rice stem borers were originally obtained from a rice paddy of Jiangsu Province in 2004 and have been reared in our laboratory. The larvae were reared on rice seedlings as described by Shang et al. [17]. Insects were maintained in a growth chamber at 28 ± 1 °C under a 16:8 (L:D) photoperiod and approximately 80% RH. 2.2. Chemicals Cyfluthrin (92.0% a.i.), bifenthrin (95.0% a.i.), and deltamethrin (99% a.i.) were provided kindly by Jiangsu Yangnong Chemical Group Co., Ltd., and endosulfan (90% a.i.) by Jiangsu Rongdong Pesticide Factory in China. Coomassie brilliant blue G-250 was purchased from Sigma (USA). DPH (1,6-diphenyl-1,3,5-hexatriene, 99% purity) was purchased from Fluca (USA).
2.6. Protein assay Concentrations of protein in homogenates were determined by the method of Bradford [21] using bovine albumin as a standard. Briefly, 2 ml of protein reagent (final concentrations in the reagent were 0.01% (w/v) Coomassie Brilliant Blue G-250, 4.7% (w/v) ethanol, and 8.5% (w/v) phosphoric acid.) was added to the test tube with 1 ml homogenates and the contents mixed by vortexing. The absorbance at 595 nm was measured after 2 min and before 1 h in 3 ml cuvettes against a reagent blank prepared from 1 ml of the appropriate buffer and 5 ml of protein reagent. The weight of protein was plotted against the corresponding absorbance resulting in a standard curve used to determine the protein in homogenates.
2.3. Preparation of mitochondria membrane
2.7. Data analysis
Mitochondrial membrane of C. supperssalis was isolated by differential centrifugation according to Voss et al. [18], using an extraction medium consisting of 10 mM Tris–HCl, 0.1 mM EDTA, 250 mM sucrose, NaCl 0.8 g%, BSA 0.5 g%, pH 7.4. Rice stem borers were congealed in 80 °C ultra low temperature freezer and a group of 18 fourth-instar larvae (body weight 9–11 mg) were homogenized in 2 ml of ice-chilled sucrose extracting solution with a hand-operated tissue grinder (Teflon pestle). The homogenate was centrifuged at 3000g for 10 min and then the supernatant was re-centrifuged at 10,000g for 30 min at 4 °C (By using an Eppendorf centrifuge, 5417R, Eppendorf AG, German). The sediment was suspended in 2 ml ice-chilled sucrosuml extracting solution and then centrifuged 10,000g for 20 min and last suspended in 2 ml ice-chilled sucrosuml extracting solution. The fresh mitochondria were prepared in all experiments of this study.
The data analysis was performed by One-way ANOVA with Turkey posttest, using GraphPad InStat version 3.00.
2.4. Preparation of DPH A solution of DPH (2 mM) in tetrahydrofuran was diluted 1000fold by injection into a vigorously stirred solution of PBS (10 mM buffer, pH 7.4; 0.14 mol/L NaCl), and stirred for 1 h before use. It must be shacked fiercely when DPH was diluted. 2.5. Fluorescence polarization studies Fluorescence polarization was measured by the method of Cheng and Levy [19] using DPH [20]. Samples were diluted to a protein concentration of 250 mg per milliliter with Tris–HCl solution, mixed with an equal volume of the DPH solution, and incubated in a shaking bath for 30 min at 25 °C. Steady-state fluorescence polarization measurements were performed with a Model LC-55 polarization spectrophotometer (Perkin-Elmer), which is equipped with two photomultipliers to detect separately each polarized component of the fluorescent light. Temperature in the cuvettes was controlled with a thermostated circulating-water pump. Excitation wavelength was 362 nm and emission was measured at 432 nm. The degree of fluorescence polarization (P) was calculated as:
P ¼ Ik GIj =Ik þ GIj Where I| and I|| are the intensities of emitted light with vertical excitation, vertical (horizontal) emission polarizer. G is the correction factor for the optical system, given by:
3. Results 3.1. Effects of pyrethroids on fluidity of mitochondrial membrane dependent on temperature in C. suppressalis The fluorescence polarization of mitochondrial membrane incorporated by DPH as molecule probe was monitored from 15 to 45 °C. The effects of pyrethroids and endosulfan occurred over all of the range of the studied temperatures, from 15 to 45 °C. Deltamethrin, bifenthrin and cyfluthrin (100 lM) showed a considerable increase in DPH polarization in mitochondrial membrane examined (Fig. 1). Changes in DPH polarization were more at lower temperature than at higher temperature. The slope of the Arrhenius plot decreased more by pyrethroids than by control and a further decrease was observed with deltamethrin and cyfluthrin which suggested a change in the activation energy of the probe molecule. On the contrary, endosulfan showed a considerable decrease in DPH polarization in mitochondrial membrane in the examined temperatures (Fig. 1). The change in DPH polarization is more at higher temperature than at lower temperature, which showed endosulfan possessed a positive temperature coefficient. 3.2. Effects of pyrethroids on fluidity of mitochondrial membrane dependent on concentration in C. suppressalis The effect of increasing concentrations of pyrethroids and endosulfan on DPH polarization of mitochondrial membrane at a particular temperature (25 °C) was shown in Fig. 2. Effects of pyrethroids and endosulfan on fluorescence polarization were dose-despondent. The changing in DPH polarization caused by pyrethroids was different from endosulfan. The changes of DPH polarization caused by bifenthrin and cyfluthrin were more, and the effect of deltamethrin on DPH polarization was little in less than 80 lM, and the curves of concentration-effect were non-linear. However, the effect of endosulfan on fluorescence polarization is reversed comparing with pyrethroids. DPH polarization decreased with the increasing of endosulfan concentrations. The result showed that pyrethroids tested can decrease fluidity of membrane and endosulfan increases fluidity of membrane.
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0.31
0.29
Fluoresence Polarization
Fluoresence Polarization
0.31
0.27 0.25 0.23 0.21 Control
0.19
Deltamethria
0.17
0.29 0.27 0.25 0.23 0.21 0.19
Control
0.17
Bifenthrin
0.15
0.15 3.1
3.2
3.3
3.4
3.1
3.5
3.2
0.31
0.31
0.29
0.29
0.27 0.25 0.23 0.21 0.19
Control
0.17
Cyfluthrin
0.15
3.4
3.5
1000/TK
Fluoresence Polarization
Fluoresence Polarization
1000/TK
3.3
0.27 0.25 0.23 0.21 0.19
Control
0.17
Endosulfan
0.15 3.1
3.2
3.3
3.4
3.5
1000/TK
3.1
3.2
3.3
3.4
3.5
1000/TK
Fig. 1. Fluorescence polarization of DPH in mitochondrial membrane as a function of temperature in the absence (0 lM) and presence of pyrethroids and endosulfan (100 lM).
Cyfluthrin
0.26
Bifenthrin Endosulfan
Fluorescence Polarization
0.25
Deltamethrin 0.24 0.23 0.22 0.21 0.2 0
20
40
60
80
100
120
Concentration (µM) Fig. 2. Fluorescence polarization of DPH in mitochondrial membrane as a function of increasing pyrethroids and endosulfan concentration at 25 °C.
4. Discussion Actually, the effect of temperature on biomembrane had been studied in rat by Sarkar et al. Fenvalerate could decrease fluorescence polarization value of DPH of microsomal membrane, and
the change of DPH polarization was more at lower temperatures than at higher temperatures [10]. Welligton et al. found that deltamethrin increased the fluorescence polarization of DPH in native membrane from 10.5 to 40.5 °C [11]. In our study, the effect of three pyrethroids on fluorescence polarizations of DPH were all occurred in the range of temperatures from 10 to 45 °C. The changes of the three pyrethroids on DPH polarization in different temperatures were similar to that described in rat by Sarkar et al and Welligton et al. [10,11]. Clark and Matsumura showed that type II pyrethroids mainly inhibit Ca2+–Mg2+-ATPase activities [22]. In previous studies, we compared the toxicity and ATPase inhibition of pyrethroids in different temperatures [16]. The result showed a better relationship of the temperature coefficient in toxicity and inhibition of Ca2+–Mg2+-ATPase by deltamethrin and bifenthrin in C. suppressalis. Moreover, Na+– K+-ATPase could be also inhibited by pyrethroids and the inhibition was likewise effected by temperature [16]. Compared the inhibition of Na+–K+-ATPase with the effects on fluorescence polarization by the pyrethroids, the effect of temperature on both inhibition of Na+–K+-ATPase and fluorescence polarization were similar (Table 1). Three pyrethroids showed a negative temperature coefficient and endosulfan showed a positive temperature coefficient. It was because that Na+–K+-ATPase transports Na+ ions and K+ ions against its concentration gradient across the membrane, and fluidity is one of the most important characters of membrane. It’s that fluidity is determined by lipid composition and temperature. So the temperature could affect the changing of membrane fluidity and inhibition to
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H. Li et al. / Pesticide Biochemistry and Physiology 95 (2009) 72–76 Table 1 Comparison of polarization and percentage of inhibition of Na+–K+-ATPase in mitochondria by insecticides in C. suppressalis in different temperatures. Compound
Temp. (°C)
Fluorescence polarization (P)
Anisotropy (c)
Microviscosity (g)
Na+–K+-ATPase inhibition (%)*
Temp. coeff.
Mitochondrial membrane
17 27 37 17 27 37 17 27 37 17 27 37 17 27 37
0.228 ± 0.003 0.212 ± 0.001 0.195 ± 0.002 0.254 ± 0.005 0.226 ± 0.002 0.201 ± 0.003 0.275 ± 0.002 0.249 ± 0.001 0.224 ± 0.009 0.244 ± 0.005 0.218 ± 0.005 0.200 ± 0.003 0.228 ± 0.005 0.206 ± 0.002 0.189 ± 0.003
0.163 ± 0.002 0.151 ± 0.001 0.140 ± 0.004 0.187 ± 0.005 0.164 ± 0.003 0.147 ± 0.002 0.201 ± 0.03 0.176 ± 0.011 0.161 ± 0.006 0.175 ± 0.002 0.152 ± 0.007 0.143 ± 0.001 0.161 ± 0.003 0.148 ± 0.001 0.136 ± 0.002
1.96 1.705 1.472 2.459 1.926 1.594 2.982 2.360 1.893 2.266 1.794 1.534 1.898 1.627 1.429
62.81 ± 5.68 45.69 ± 0.76 29.27 ± 0.06 59.54 ± 3.55 26.36 ± 4.46 39.51 ± 3.51 43.93 ± 0.39 11.82 ± 5.41 27.27 ± 1.84 15.03 ± 1.97 1.82 ± 2.71 17.46 ± 5.61
Deltamethrin
Bifenthrin
Cyfluthrin
Endosulfan
*
+
Data was from Ref. [16].
Na+–K+-ATPase by pyrethroids, implicating that membrane fluidity is one of the most important factors affecting negative temperature coefficient of pyrethroids. Generally, it had been accepted that DDT and pyrethroids acted directly on the nervous system causing disruption of normal ion permeabilities in the nerve membrane [23]. Endosulfan is one of organochlorine insecticides, but different from DDT in molecular structure. Martins et al.’s study exhibited that addition of endosulfan to liposome induced a decrease in the polarization values in the gel phase [8]. Our previous study proved that the toxicity and ATPase inhibition of endosulfan was a positive temperature coefficient [16], which is consistent with the effect of endosulfan on membrane fluidity in the rice stem borer. An obvious conclusion emerging from this study is that the effects of temperature on biological action of pyrethroids are quite complex. The proposed three hypotheses might be all attributed to the negative temperature coefficient of pyrethroids. First one, differential target site interactions, had been proved by many researches [24,25]. The second, differential metabolism was indicated by Scott et al. [12] and Mcintosh et al. [26]. The third, different lipid solubility, which proposed by Munson was considered the least plausible because the nerve membrane is composed largely of lipids and would therefore accumulate more pesticide at higher temperature, thus manifesting a positive temperature coefficient [27]. The results obtained from this study should be useful in order to explain the effect of temperature on pyrethroids toxicity. Pyrethroid insecticides are widely applied in agriculture from south to north in China, and the air temperature is very different in the various areas of China. Temperature often has a significant effect on the efficacy of insecticides when used on the field. But the effect of temperature on insecticides to pests had not received great attention in China before. Pyrethroids might be one of alternative insecticides in rice paddy since methamidophos was forbidden by the end of 2006 in China. So knowledge of a pyrethroid’s temperature coefficient will enable pest managers to select a pyrethroid that is efficacious under the given environmental conditions. Acknowledgments This research was supported by National Basic Research Program of China (Contract No. 2006CB102003), National Natural Science Foundation of China (Contract No. 30530530, 30571232, 30471153, and 30170621) and National Key Research Program of China for the Eleventh Five-years Plan (Contract No. 2006BAD08A03).
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