- Email: [email protected]
Piperonyl butoxide potentiates the synaptosome ATPase inhibiting effect of pyrethrin
Chemosphere 40 (2000) 301±305
Piperonyl butoxide potentiates the synaptosome ATPase inhibiting eect of pyrethrin Irma Kakko, Tarja Toimela, Hanna T ahti* Medical School, University of Tampere, P.O. Box 607, FIN-33101 Tampere, Finland Received 24 March 1999; accepted 8 June 1999
Abstract Pyrethrins are widely used insecticides in both agriculture and households. In many commercial formulations piperonyl butoxide (PBO) is used with pyrethrins. PBO is a well-known synergist of pyrethrins, used to intensify their eects. One of the cellular targets of pyrethrins is the sodium channel in the membrane. In the present study, the activity of the membrane-bound integral protein ATPase was studied as a biomarker for the membrane eects of pyrethrin and PBO. Cerebral synaptosomes of rat brain were used in the study. The isolation of synaptosomes was performed with the Percoll gradient method. Both total ATPase and Mg2 activated ATPase were studied by determining inorganic phosphate. Exposure to 0.1±1000 lM of pyrethrin and to 0.4±4000 lM of PBO decreased ATPase activity dose-dependently. The most ecient mixture was the one consisting of one part of pyrethrin and four parts of PBO. The activity of total ATPase decreased 15% in concentrations of 0.1±10 lM pyrethrin, and a 50% decrease was found at 100 lM pyrethrin. The mixture of pyrethrin and PBO caused a 15±60% decrease in the total ATPase activity at 0.1±10 lM pyrethrin and 0.4±40 lM PBO. A 85% decrease was found after exposure to the mixture of 100 lM pyrethrin and 400 lM PBO. PBO alone had no eect at 0.4±40 lM concentrations, but a marked eect was seen at over 40 lM concentrations. The results indicate that PBO is an eective synergist of pyrethrin and that it is very toxic in high concentrations. The results also con®rm that neuronal sodium homeostasis is one target of the neurotoxic eect of pyrethroid compounds. Ó 1999 Elsevier Science Ltd. All rights reserved. Keywords: Pyrethrin; Piperonyl butoxide; ATPase; Synaptosome
1. Introduction Pyrethrins are naturally occurring insecticides that were already used by the Chinese in about 1000 BC in the form of powdered pyrethrum plant. A number of synthetic analogues of the pyrethrins called pyrethroids, have been made since 1940 (Elliott, 1980). Synthetic pyrethroids are in general chemically and biochemically more stable than natural pyrethrins, and they also have excellent insecticidal properties. Pyrethroids are mainly formulated as emulsi®able concen-
*
Corresponding author. Tel: +358-3-215-6672; fax: +358-3215-6170. E-mail address: blhata@uta.® (H. TaÈhti)
trates for spraying. They are used to control a wide variety of agricultural insect pests and in horticulture. They are extensively used against the insect vectors of disease (e.g. tsetse ¯y in parts of Africa) and to eliminate veterinary pests by topical application to animals (Walker et al., 1996). Pyrethroids have been considered to be the safest of the highly potent insecticides (Shaw and Chadwick, 1998), but they may not be as safe as previously claimed (Peter et al., 1996). Toxicity in humans has been reported in farmers involved in spraying insecticides and in households (Altenkirch et al., 1996; Chen et al., 1991; O'Malley, 1997). The symptoms are abnormal impairment of facial sensations, dizziness, headache, fatigue, nausea or loss of appetite. The main environmental concern relates to pyrethroid toxicity to ®sh and
0045-6535/00/$ - see front matter Ó 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 9 9 ) 0 0 2 6 4 - 7
302
I. Kakko et al. / Chemosphere 40 (2000) 301±305
non-target invertebrates (Walker et al., 1996). The toxicity to ®sh may re¯ect species dierences in metabolism. Cases of intoxication of water ecosystems have been reported (Kontreczky et al., 1997; Burridge and Haya, 1997). Pyrethroids can also bind to particles in soils and sediments and show some persistence in these locations. In most commercial products, the synergist of pyrethrin is piperonyl butoxide (PBO), a well-known synthetic methylenedioxyphenyl inhibitor of cytochrome P450 . PBO also has other toxic eects, such as enhancing the percutaneous absorption of pyrethrins (Baynes and Riviere, 1998) and increasing the relative liver weight associated with hypertrophy. At high doses, PBO is hepatotoxic (Phillips and Hayes, 1989). In previous studies on the neurotoxicity of pyrethroids, the voltage-sensitive sodium channel has been studied as a principal molecular target site for pyrethroids both in insects and in mammals (Shankland, 1976; Narahashi, 1986; Soderlund and Bloomquist, 1989; Narahashi, 1996). In the present study, the eects of pyrethrin and PBO on the activity of total ATPase and Mg2 -ATPase in rat brain synaptosomal membranes were examined. The possible synergistic action of PBO on ATPase activity was also evaluated. 2. Materials and methods 2.1. Synaptosome preparation Male Sprague-Dawley rats (weight 200±300 g) were used for cerebral synaptosome isolations. The rats were maintained in well-controlled animal facilities, and they were given standard rat food and tap water ad libitum. After the rats were decapitated, the cerebrum was gently separated from the cerebellum, brain stem and meninges. The cells of the cerebrum were dissociated and the synaptosomal membranes were isolated in non-toxic isoosmotic Percoll gradients (Nagy and Delgado, 1984). 2.2. Test compounds
determination of inorganic phosphate was used (Phillips and Hayes, 1989; Korpela and T ahti, 1988; Vaalavirta and T ahti, 1995). The test substances pyrethrin and PBO were added to the following 2 ml reaction mixture: 50 mM Tris±HCl buer pH 7.6, 1.5 mM ATP (Sigma), 5 mM MgCl2 , 100 mM NaCl, 20 mM KCl and 0.1 ml synaptosome suspension. The tubes were tightly stoppered, mixed and incubated for 1 h at 37°C with continuous shaking. The total ATPase activity was measured while sodium (Na ), potassium (K ) and magnesium (Mg2 ) were present in the reaction mixture. The activity of Mg2 -ATPase was determined in the presence of Mg2 ions in the reaction mixture only (50 mM Tris±HCl buer pH 7.6, 1.5 mM ATP, 15 mM MgCl2 and 0.1 ml synaptosome suspension). The activity of Mg2 -ATPase was also determined by inhibiting the Na , K -ATPase with ouabain. Ouabain binding was carried out by adding 1 mM of unlabeled ouabain (Sigma-03125) to the reaction mixture with 0.1, 1, 10, 100 and 1000 lM of pyrethrin. The probes were then incubated for 1 h at 37°C under continuous shaking. After 1 h incubation, 2 ml ice-cold 10% TCA was added, followed by centrifugation at 800 g for 10 min. To 1 ml supernatant, 0.2 ml of the reagent (1% NH4 6 Mo7 O24 4H2 O=0:5 NH2 SO4 solution) and 2 ml of 1% ascorbic acid were added. After a 25-minute reaction time, the absorbances of the samples were read at 770 nm. The enzyme activities were expressed as micromoles of inorganic phosphate formed per hr per mg of protein (lmol Pi/h/mg prot). The results were normalized and the ATPase activities of treated samples were given as percentages of the activities of control samples. 2.4. Protein determination The total protein content of the samples was determined by a commercial method by Pierce (BCA* Protein Assay). It is a highly sensitive method for the spectrophotometric determination of protein concentration, based on bicinchoninic acid (BCA) reagent. 2.5. Statistics
Two commercial products of pyrethrins (Kemira, Finland) were used. Product 1, Biospray (Bioruiskute, Finnish commercial name) contained pyrethrin and PBO (1 part of pyrethrin + 4 parts of PBO) and product 2, Biospray S (Bioruiskute S, Finnish commercial name) contained pyrethrin only. PBO (Lancaster) was used in a mixture with an emulsi®er (2 parts of stearic acid + 1 part of sorbitan monooleate).
The determinations were made in triplicates at each dose level. Three independent measurements were made. The means and standard errors (Mean SEM) of independent tests were calculated at each concentration. The analysis of variance was used in the calculations of the data.
2.3. Determination of adenosine triphoshatase activities
3. Results
In the determination of the ATPase activities, a modi®cation of the discontinuous method based on the
In the present study, the activity of the membranebound integral protein ATPase was studied as a target
I. Kakko et al. / Chemosphere 40 (2000) 301±305
for the toxic eect of commercial pyrethrin and PBO product. The total ATPase and Mg2 -activated ATPase were studied by determining the release of the inorganic phosphate from the substrate ATP. Pyrethrin decreased the activity of total ATPase by 15% in 0.1±10 lM concentrations and by 40% in 100±1000 lM concentrations. Pyrethrin did not aect Mg2 -activated ATPase in concentrations of 0.1±100 lM, but in concentrations of 1000 lM there was a marked increase in the activity of Mg2 -ATPase (Fig. 1). This result was con®rmed by measuring the activity of Mg2 -ATPase when inhibiting the Na , K -ATPase with ouabain. The concentrations of 0.1±100 lM pyrethrin had no eect on the activity of Mg2 -ATPase, but the activity increased 100% in the 1000 lM concentration. We also studied the possible eects of pyrethrin, PBO and emulsi®er alone without synaptosome preparation activities. The test solvents or the emulsi®er with other reagents in the incubation mixture had no eect on the ATPase. The mixture of 0.1±10 lM pyrethrin and 0.4±40 lM PBO decreased the activity of total ATPase from 15% to 60% and 85% in a mixture of 100 lM pyrethrin and 400 lM PBO (p < 0:001). The activity did not decrease any more in the concentration of 1000 lM pyrethrin and 4000 lM PBO. This mixture had no eect on the activity of Mg2 -ATPase in the concentrations 0.1±1 lM, but the activity decreased 40±50% in the concentration of 10±100 lM (p < 0:1±0.01). It did not decrease anymore in the concentration of 1000 lM (Fig. 2). Exposure to PBO had no eect on the activity of total ATPase in the concentration of 0.4±4 lM. In the concentration of 40±4000 lM (p < 0:1), the activity of total ATPase decreased 10±60%. PBO decreased the activity of Mg2 -ATPase, but this decrease was not statistically signi®cant (Fig. 3).
303
Fig. 2. The eect of pyrethrin and PBO (in 4 concentration of pyrethrin) on the activities of total ATPase and Mg2 -ATPase in synaptosomal preparations. * p < 0:01, ** p < 0:1, *** p < 0:001.
Fig. 3. The eect of PBO on the activities of total ATPase and Mg2 -ATPase in synaptosomal preparations. * p < 0:1.
4. Discussion
Fig. 1. The eect of pyrethrin on the activities of total ATPase and Mg2 -ATPase in synaptosomal preparations. Each point represents the mean SEM of three independent experiments, all made in triplicates. * p < 0:1, ** p < 0:01.
We have studied the toxic eects of commercial pyrethroid products (Biospray and Biospray-S). The reason for using these products is that people in general are exposed to mixtures of pyrethrins in households and gardening. The commercial pyrethrin products are formulated as emulsi®able concentrates for spraying. Emulsi®ers do not usually come out from the can at spraying and do not aect the users of the insecticide. The water solubility of pyrethroids is very low. They act as neurotoxins in the same way as DDT. Compared to other organic insecticides, pyrethroids show a higher insecticidal activity and lower toxicity to mammals (Casida et al., 1975). The reason for this is that they are readily biodegradable, and do not have long biological
304
I. Kakko et al. / Chemosphere 40 (2000) 301±305
half-lives (Casida et al., 1975). The hazards that they present relate mainly to short-term toxicity. The low toxicity in mammals suggests that there is an extensive metabolism of pyrethroids and that the metabolites are not very toxic. The metabolism of the pyrethroids involves cytochrome P450 -catalyzed oxidation at several positions of the molecule. The metabolic pathway results in a signi®cant increase in the water solubility of the pyrethroids, facilitating their rapid elimination (Shaw and Chadwick, 1998). PBO is the most important pyrethroid synergist and a classical inhibitor of the microsomal mixed function oxidase (MFO) system, involved in the detoxi®cation of every pyrethroid in mammals. At high doses PBO increases the eectiveness of pyrethrin I to house¯ies by 300-fold (Testa and Jenner, 1981) PBO may also have neurotoxic potency, and it has been shown to have adverse eects on the motor activity of the exploratory behaviour in male mice (Tanaka, 1993). Pyrethroids exert potent actions on neuronal sodium channels, thereby causing neurotoxic eects in animals (Soderlund and Bloomquist, 1989; Narahashi et al., 1995; Vijverberg and van den Bercken, 1990). Pyrethroids slow down the kinetics of both the opening and the closing of individual sodium channels, resulting in delayed and prolonged openings (Narahashi, 1996). These changes can account for the various symptoms of poisoning including hyperexcitation and convulsions. Experiments measuring Na uptake provide additional support to the theory that the sodium channel is a target for the toxic eects of pyrethrins (Bloomquist and Soderlund, 1988; Brown et al., 1988; Lombet et al., 1988). Besides sodium channels, pyrethroids have been shown to interact with many other membrane integral proteins (Narahashi, 1986, 1996; Vijverberg and van den Bercken, 1990; Bloomquist, 1993). ATP-utilizing enzymes and ion pumps (ATPases) may be involved in the neurotoxic actions of pyrethroids (Soderlund and Bloomquist, 1989; Rao et al., 1984). It has been suggested that in neural cells and several other tissues, Na , K -ATPase activity may be subject to modulation by receptor/second messenger pathways (Bertorello and Aperia, 1989). In the present paper, pyrethrin had an inhibiting eect on the total ATPase activity at over 10 lM concentrations. PBO had a dose-dependent, slightly decreasing eect on the ATPase activities. In a mixture with pyrethrin, PBO potentiated the ATPase inhibiting eect of dierent doses of pyrethrin. Again the eect on total ATPase was greater than the eect on Mg2 -ATPase. Our result shows that PBO is a potent synergistic agent of pyrethrin, and that this synergistic action is not only based on the disturbance in biotransformation, but also at the level of the target site at the neural membrane. The inhibition of ATPase may cause disturbance in ion bal-
ance across the neural membrane and may aect the sodium channel kinetics. In conclusion, the results of the present study show that the activity of total ATPase may be a target for the neurotoxic action of pyrethrins and PBO, and that PBO and pyrethrin together have a synergistic inhibiting effect on synaptosomal membrane-bound ATPase.
Acknowledgements We are grateful to Mrs. Paula Helpi ol a for skillful technical assistance. This research was supported by Jenny and Antti Wihuri Fund and the Finnish Ministry of Agriculture and Forestry. References Altenkirch, H., Hopmann, D., Brockmeier, B., Walter, G., 1996. Neurological investigations in 23 cases of pyrethroid intoxication reported to the German Federal Health Oce. Neurotoxicol. 17, 645±651. Baynes, R.E., Riviere, J.E., 1998. In¯uence of inert ingredients in pesticide formulations on dermal absorption of carbaryl. Am. J. Vet. Res. 59, 168±175. Bertorello, A., Aperia, A., 1989. Na -K -ATPase is an eector protein for protein kinase C in renal proximal tubule cells. Am. J. Physiol. 256, F370±F373. Bloomquist, J.R., 1993. Neuroreceptor mechanisms in pyrethroid mode of action and resistance. In: Roe, R.M., Kuhf, R.J. (Eds.), Reviews in Pesticide Toxicology. Toxicology Communications, Raleigh, NC, pp. 185±230. Bloomquist, J.R., Soderlund, D.M., 1988. Pyrethroid insecticides and DDT modify alkaloid-dependent sodium channel activation and its enhancement by sea anemone toxin. Mol. Pharmacol. 33, 543±550. Brown, G.B., Gaupp, J.E., Olsen, R.W., 1988. Pyrethroid insecticides: stereospeci®c allosteric interaction with the batrachotoxinin ± a benzoate binding site of mammalian voltage-sensitive sodium channels. Mol. Pharmacol. 34, 54±59. Burridge, L.E., Haya, K., 1997. Lethality of pyrethrins to larvae and postlarvae of the American lobster (Homarus americanus). Ecotoxicol. Environ. Saf. 38, 150±154. Casida, J.E., Ueda, K., Gaughan, L.C., Jao, L.T., Soderlund, D.M., 1975. Structure±biodegradability relationships in pyrethroid insecticides. Arch. Environ. Contam. Toxicol. 3, 491±500. Chen, S.Y., Zhang, Z.W., He, F.S., Yao, P.P., Wu, Y.Q., Sun, J.X., Liu, L.H., Li, Q.G., 1991. An epidemiological study on occupational acute pyrethroid poisoning in cotton farmers. Br. J. Ind. Med. 48, 77±81. Elliott, T., 1980. Established pyrethroid insecticides. Pestic. Sci. 11, 119±128. Kontreczky, C., Farkas, A., Nemcsok, J., Salanki, J., 1997. Short- and long-term eects of deltamethrin on ®ltering activity of freshwater mussel (Aanodonta cygnea L.). Ecotoxicol. Environ. Saf. 38, 195±199.
I. Kakko et al. / Chemosphere 40 (2000) 301±305 Korpela, M., Tahti, H., 1988. The eect of in vitro and in vivo toluene exposure on rat erythrocyte and synaptosome membrane integral enzymes. Pharmacol. Toxicol. 63, 30± 32. Lombet, A., Mourre, C., Lazdunski, M., 1988. Interaction of insecticides of the pyrethroid family with speci®c binding sites on the voltage-dependent sodium channel from mammalian brain. Brain Res. 459, 44±53. Nagy, A., Delgado, E.A., 1984. Rapid preparation of synaptosomes from mammalian brain using nontoxic isoosmotic gradient material (Percoll). J. Neurochem. 43, 1114±1123. Narahashi, T., 1986. Mechanisms of action of pyrethroids on sodium and calcium channel gating. In: Ford, M.G., Lunt, G.G., Reay, R.C., Usherwood, P.N.R. (Eds.), Neuropharmacology of Pesticide Action. Ellis Horwood, Chichester, UK, pp. 36±60. Narahashi , T., 1996. Neuronal ion channels as the target sites of insecticides.. Pharmacol. Toxicol. 79, 1±14. Narahashi, T., Frey, J.M., Ginsburg, K.S., Nagata, K., Roi, M.L. Tatebayashi, H., 1995. Sodium channels and caminobutyric acid activated channels as target sites of insecticides. In: Clark, J.M. (Ed.), Molecular Action of Insecticides on Ion Channels. American Chemical Society, Washington, DC, pp. 26±43. O'Malley, M., 1997. Clinical evaluation of pesticide exposure and poisonings. Lancet 349, 1161±1166. Peter, J.V., John, G., Cherian, A.M., 1996. Pyrethroid poisoning. J. Assoc. Physicians India 44, 343±344.
305
Phillips, T.D., Hayes, A.W., 1989. Techniques in membrane toxicology. In: Hayes, A.E. (Ed.), Principles and Methods of Toxicology. Raven Press, New York, pp. 761±776. Rao, K.S., Chetty, C.S., Desaiah, D., 1984. In vitro eects of pyrethroids on rat brain and liver ATPase activities. J. Toxicol. Environ. Health 14, 257±265. Shankland, D.L., 1976. The nervous system: comparative physiology and pharmacology. In: Wilkinson, C.F. (Ed.), Insecticide Biochemistry and Physiology. Plenum Press, New York, pp. 229±270. Shaw, I.C., Chadwick, J., 1998. Principles of Environmental Toxicology, 1st edition. Taylor & Francis, London. Soderlund, D.M., Bloomquist, J.R., 1989. Neurotoxic actions of pyrethroid insecticides. Annu. Rev. Entomol. 34, 77±96. Tanaka, T., 1993. Behavioural eects of piperonyl butoxide in male mice. Toxicol. Lett. 69, 155±161. Testa, B., Jenner, P., 1981. Inhibitors of Cytochrome P-450s and their mechanism of action. Drug Metab. Rev. 12, 1± 117. Vaalavirta, L., T ahti, H., 1995. Astrocyte membrane Na+, K(+)-ATPase and Mg(2+)-ATPase as targets of organic solvent impact. Life Sci. 57, 2223±2230. Vijverberg, H.P., van den Bercken, J., 1990. Neurotoxicological eects and the mode of action of pyrethroid insecticides. Crit. Rev. Toxicol. 21, 105±126. Walker, C.H., Hopkin, S.P., Sibly, R.M., Peakall, D.B., 1996. Principles of Ecotoxicology, 1st edition. Taylor & Francis, London.
Piperonyl butoxide potentiates the synaptosome ATPase inhibiting eect of pyrethrin Irma Kakko, Tarja Toimela, Hanna T ahti* Medical School, University of Tampere, P.O. Box 607, FIN-33101 Tampere, Finland Received 24 March 1999; accepted 8 June 1999
Abstract Pyrethrins are widely used insecticides in both agriculture and households. In many commercial formulations piperonyl butoxide (PBO) is used with pyrethrins. PBO is a well-known synergist of pyrethrins, used to intensify their eects. One of the cellular targets of pyrethrins is the sodium channel in the membrane. In the present study, the activity of the membrane-bound integral protein ATPase was studied as a biomarker for the membrane eects of pyrethrin and PBO. Cerebral synaptosomes of rat brain were used in the study. The isolation of synaptosomes was performed with the Percoll gradient method. Both total ATPase and Mg2 activated ATPase were studied by determining inorganic phosphate. Exposure to 0.1±1000 lM of pyrethrin and to 0.4±4000 lM of PBO decreased ATPase activity dose-dependently. The most ecient mixture was the one consisting of one part of pyrethrin and four parts of PBO. The activity of total ATPase decreased 15% in concentrations of 0.1±10 lM pyrethrin, and a 50% decrease was found at 100 lM pyrethrin. The mixture of pyrethrin and PBO caused a 15±60% decrease in the total ATPase activity at 0.1±10 lM pyrethrin and 0.4±40 lM PBO. A 85% decrease was found after exposure to the mixture of 100 lM pyrethrin and 400 lM PBO. PBO alone had no eect at 0.4±40 lM concentrations, but a marked eect was seen at over 40 lM concentrations. The results indicate that PBO is an eective synergist of pyrethrin and that it is very toxic in high concentrations. The results also con®rm that neuronal sodium homeostasis is one target of the neurotoxic eect of pyrethroid compounds. Ó 1999 Elsevier Science Ltd. All rights reserved. Keywords: Pyrethrin; Piperonyl butoxide; ATPase; Synaptosome
1. Introduction Pyrethrins are naturally occurring insecticides that were already used by the Chinese in about 1000 BC in the form of powdered pyrethrum plant. A number of synthetic analogues of the pyrethrins called pyrethroids, have been made since 1940 (Elliott, 1980). Synthetic pyrethroids are in general chemically and biochemically more stable than natural pyrethrins, and they also have excellent insecticidal properties. Pyrethroids are mainly formulated as emulsi®able concen-
*
Corresponding author. Tel: +358-3-215-6672; fax: +358-3215-6170. E-mail address: blhata@uta.® (H. TaÈhti)
trates for spraying. They are used to control a wide variety of agricultural insect pests and in horticulture. They are extensively used against the insect vectors of disease (e.g. tsetse ¯y in parts of Africa) and to eliminate veterinary pests by topical application to animals (Walker et al., 1996). Pyrethroids have been considered to be the safest of the highly potent insecticides (Shaw and Chadwick, 1998), but they may not be as safe as previously claimed (Peter et al., 1996). Toxicity in humans has been reported in farmers involved in spraying insecticides and in households (Altenkirch et al., 1996; Chen et al., 1991; O'Malley, 1997). The symptoms are abnormal impairment of facial sensations, dizziness, headache, fatigue, nausea or loss of appetite. The main environmental concern relates to pyrethroid toxicity to ®sh and
0045-6535/00/$ - see front matter Ó 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 9 9 ) 0 0 2 6 4 - 7
302
I. Kakko et al. / Chemosphere 40 (2000) 301±305
non-target invertebrates (Walker et al., 1996). The toxicity to ®sh may re¯ect species dierences in metabolism. Cases of intoxication of water ecosystems have been reported (Kontreczky et al., 1997; Burridge and Haya, 1997). Pyrethroids can also bind to particles in soils and sediments and show some persistence in these locations. In most commercial products, the synergist of pyrethrin is piperonyl butoxide (PBO), a well-known synthetic methylenedioxyphenyl inhibitor of cytochrome P450 . PBO also has other toxic eects, such as enhancing the percutaneous absorption of pyrethrins (Baynes and Riviere, 1998) and increasing the relative liver weight associated with hypertrophy. At high doses, PBO is hepatotoxic (Phillips and Hayes, 1989). In previous studies on the neurotoxicity of pyrethroids, the voltage-sensitive sodium channel has been studied as a principal molecular target site for pyrethroids both in insects and in mammals (Shankland, 1976; Narahashi, 1986; Soderlund and Bloomquist, 1989; Narahashi, 1996). In the present study, the eects of pyrethrin and PBO on the activity of total ATPase and Mg2 -ATPase in rat brain synaptosomal membranes were examined. The possible synergistic action of PBO on ATPase activity was also evaluated. 2. Materials and methods 2.1. Synaptosome preparation Male Sprague-Dawley rats (weight 200±300 g) were used for cerebral synaptosome isolations. The rats were maintained in well-controlled animal facilities, and they were given standard rat food and tap water ad libitum. After the rats were decapitated, the cerebrum was gently separated from the cerebellum, brain stem and meninges. The cells of the cerebrum were dissociated and the synaptosomal membranes were isolated in non-toxic isoosmotic Percoll gradients (Nagy and Delgado, 1984). 2.2. Test compounds
determination of inorganic phosphate was used (Phillips and Hayes, 1989; Korpela and T ahti, 1988; Vaalavirta and T ahti, 1995). The test substances pyrethrin and PBO were added to the following 2 ml reaction mixture: 50 mM Tris±HCl buer pH 7.6, 1.5 mM ATP (Sigma), 5 mM MgCl2 , 100 mM NaCl, 20 mM KCl and 0.1 ml synaptosome suspension. The tubes were tightly stoppered, mixed and incubated for 1 h at 37°C with continuous shaking. The total ATPase activity was measured while sodium (Na ), potassium (K ) and magnesium (Mg2 ) were present in the reaction mixture. The activity of Mg2 -ATPase was determined in the presence of Mg2 ions in the reaction mixture only (50 mM Tris±HCl buer pH 7.6, 1.5 mM ATP, 15 mM MgCl2 and 0.1 ml synaptosome suspension). The activity of Mg2 -ATPase was also determined by inhibiting the Na , K -ATPase with ouabain. Ouabain binding was carried out by adding 1 mM of unlabeled ouabain (Sigma-03125) to the reaction mixture with 0.1, 1, 10, 100 and 1000 lM of pyrethrin. The probes were then incubated for 1 h at 37°C under continuous shaking. After 1 h incubation, 2 ml ice-cold 10% TCA was added, followed by centrifugation at 800 g for 10 min. To 1 ml supernatant, 0.2 ml of the reagent (1% NH4 6 Mo7 O24 4H2 O=0:5 NH2 SO4 solution) and 2 ml of 1% ascorbic acid were added. After a 25-minute reaction time, the absorbances of the samples were read at 770 nm. The enzyme activities were expressed as micromoles of inorganic phosphate formed per hr per mg of protein (lmol Pi/h/mg prot). The results were normalized and the ATPase activities of treated samples were given as percentages of the activities of control samples. 2.4. Protein determination The total protein content of the samples was determined by a commercial method by Pierce (BCA* Protein Assay). It is a highly sensitive method for the spectrophotometric determination of protein concentration, based on bicinchoninic acid (BCA) reagent. 2.5. Statistics
Two commercial products of pyrethrins (Kemira, Finland) were used. Product 1, Biospray (Bioruiskute, Finnish commercial name) contained pyrethrin and PBO (1 part of pyrethrin + 4 parts of PBO) and product 2, Biospray S (Bioruiskute S, Finnish commercial name) contained pyrethrin only. PBO (Lancaster) was used in a mixture with an emulsi®er (2 parts of stearic acid + 1 part of sorbitan monooleate).
The determinations were made in triplicates at each dose level. Three independent measurements were made. The means and standard errors (Mean SEM) of independent tests were calculated at each concentration. The analysis of variance was used in the calculations of the data.
2.3. Determination of adenosine triphoshatase activities
3. Results
In the determination of the ATPase activities, a modi®cation of the discontinuous method based on the
In the present study, the activity of the membranebound integral protein ATPase was studied as a target
I. Kakko et al. / Chemosphere 40 (2000) 301±305
for the toxic eect of commercial pyrethrin and PBO product. The total ATPase and Mg2 -activated ATPase were studied by determining the release of the inorganic phosphate from the substrate ATP. Pyrethrin decreased the activity of total ATPase by 15% in 0.1±10 lM concentrations and by 40% in 100±1000 lM concentrations. Pyrethrin did not aect Mg2 -activated ATPase in concentrations of 0.1±100 lM, but in concentrations of 1000 lM there was a marked increase in the activity of Mg2 -ATPase (Fig. 1). This result was con®rmed by measuring the activity of Mg2 -ATPase when inhibiting the Na , K -ATPase with ouabain. The concentrations of 0.1±100 lM pyrethrin had no eect on the activity of Mg2 -ATPase, but the activity increased 100% in the 1000 lM concentration. We also studied the possible eects of pyrethrin, PBO and emulsi®er alone without synaptosome preparation activities. The test solvents or the emulsi®er with other reagents in the incubation mixture had no eect on the ATPase. The mixture of 0.1±10 lM pyrethrin and 0.4±40 lM PBO decreased the activity of total ATPase from 15% to 60% and 85% in a mixture of 100 lM pyrethrin and 400 lM PBO (p < 0:001). The activity did not decrease any more in the concentration of 1000 lM pyrethrin and 4000 lM PBO. This mixture had no eect on the activity of Mg2 -ATPase in the concentrations 0.1±1 lM, but the activity decreased 40±50% in the concentration of 10±100 lM (p < 0:1±0.01). It did not decrease anymore in the concentration of 1000 lM (Fig. 2). Exposure to PBO had no eect on the activity of total ATPase in the concentration of 0.4±4 lM. In the concentration of 40±4000 lM (p < 0:1), the activity of total ATPase decreased 10±60%. PBO decreased the activity of Mg2 -ATPase, but this decrease was not statistically signi®cant (Fig. 3).
303
Fig. 2. The eect of pyrethrin and PBO (in 4 concentration of pyrethrin) on the activities of total ATPase and Mg2 -ATPase in synaptosomal preparations. * p < 0:01, ** p < 0:1, *** p < 0:001.
Fig. 3. The eect of PBO on the activities of total ATPase and Mg2 -ATPase in synaptosomal preparations. * p < 0:1.
4. Discussion
Fig. 1. The eect of pyrethrin on the activities of total ATPase and Mg2 -ATPase in synaptosomal preparations. Each point represents the mean SEM of three independent experiments, all made in triplicates. * p < 0:1, ** p < 0:01.
We have studied the toxic eects of commercial pyrethroid products (Biospray and Biospray-S). The reason for using these products is that people in general are exposed to mixtures of pyrethrins in households and gardening. The commercial pyrethrin products are formulated as emulsi®able concentrates for spraying. Emulsi®ers do not usually come out from the can at spraying and do not aect the users of the insecticide. The water solubility of pyrethroids is very low. They act as neurotoxins in the same way as DDT. Compared to other organic insecticides, pyrethroids show a higher insecticidal activity and lower toxicity to mammals (Casida et al., 1975). The reason for this is that they are readily biodegradable, and do not have long biological
304
I. Kakko et al. / Chemosphere 40 (2000) 301±305
half-lives (Casida et al., 1975). The hazards that they present relate mainly to short-term toxicity. The low toxicity in mammals suggests that there is an extensive metabolism of pyrethroids and that the metabolites are not very toxic. The metabolism of the pyrethroids involves cytochrome P450 -catalyzed oxidation at several positions of the molecule. The metabolic pathway results in a signi®cant increase in the water solubility of the pyrethroids, facilitating their rapid elimination (Shaw and Chadwick, 1998). PBO is the most important pyrethroid synergist and a classical inhibitor of the microsomal mixed function oxidase (MFO) system, involved in the detoxi®cation of every pyrethroid in mammals. At high doses PBO increases the eectiveness of pyrethrin I to house¯ies by 300-fold (Testa and Jenner, 1981) PBO may also have neurotoxic potency, and it has been shown to have adverse eects on the motor activity of the exploratory behaviour in male mice (Tanaka, 1993). Pyrethroids exert potent actions on neuronal sodium channels, thereby causing neurotoxic eects in animals (Soderlund and Bloomquist, 1989; Narahashi et al., 1995; Vijverberg and van den Bercken, 1990). Pyrethroids slow down the kinetics of both the opening and the closing of individual sodium channels, resulting in delayed and prolonged openings (Narahashi, 1996). These changes can account for the various symptoms of poisoning including hyperexcitation and convulsions. Experiments measuring Na uptake provide additional support to the theory that the sodium channel is a target for the toxic eects of pyrethrins (Bloomquist and Soderlund, 1988; Brown et al., 1988; Lombet et al., 1988). Besides sodium channels, pyrethroids have been shown to interact with many other membrane integral proteins (Narahashi, 1986, 1996; Vijverberg and van den Bercken, 1990; Bloomquist, 1993). ATP-utilizing enzymes and ion pumps (ATPases) may be involved in the neurotoxic actions of pyrethroids (Soderlund and Bloomquist, 1989; Rao et al., 1984). It has been suggested that in neural cells and several other tissues, Na , K -ATPase activity may be subject to modulation by receptor/second messenger pathways (Bertorello and Aperia, 1989). In the present paper, pyrethrin had an inhibiting eect on the total ATPase activity at over 10 lM concentrations. PBO had a dose-dependent, slightly decreasing eect on the ATPase activities. In a mixture with pyrethrin, PBO potentiated the ATPase inhibiting eect of dierent doses of pyrethrin. Again the eect on total ATPase was greater than the eect on Mg2 -ATPase. Our result shows that PBO is a potent synergistic agent of pyrethrin, and that this synergistic action is not only based on the disturbance in biotransformation, but also at the level of the target site at the neural membrane. The inhibition of ATPase may cause disturbance in ion bal-
ance across the neural membrane and may aect the sodium channel kinetics. In conclusion, the results of the present study show that the activity of total ATPase may be a target for the neurotoxic action of pyrethrins and PBO, and that PBO and pyrethrin together have a synergistic inhibiting effect on synaptosomal membrane-bound ATPase.
Acknowledgements We are grateful to Mrs. Paula Helpi ol a for skillful technical assistance. This research was supported by Jenny and Antti Wihuri Fund and the Finnish Ministry of Agriculture and Forestry. References Altenkirch, H., Hopmann, D., Brockmeier, B., Walter, G., 1996. Neurological investigations in 23 cases of pyrethroid intoxication reported to the German Federal Health Oce. Neurotoxicol. 17, 645±651. Baynes, R.E., Riviere, J.E., 1998. In¯uence of inert ingredients in pesticide formulations on dermal absorption of carbaryl. Am. J. Vet. Res. 59, 168±175. Bertorello, A., Aperia, A., 1989. Na -K -ATPase is an eector protein for protein kinase C in renal proximal tubule cells. Am. J. Physiol. 256, F370±F373. Bloomquist, J.R., 1993. Neuroreceptor mechanisms in pyrethroid mode of action and resistance. In: Roe, R.M., Kuhf, R.J. (Eds.), Reviews in Pesticide Toxicology. Toxicology Communications, Raleigh, NC, pp. 185±230. Bloomquist, J.R., Soderlund, D.M., 1988. Pyrethroid insecticides and DDT modify alkaloid-dependent sodium channel activation and its enhancement by sea anemone toxin. Mol. Pharmacol. 33, 543±550. Brown, G.B., Gaupp, J.E., Olsen, R.W., 1988. Pyrethroid insecticides: stereospeci®c allosteric interaction with the batrachotoxinin ± a benzoate binding site of mammalian voltage-sensitive sodium channels. Mol. Pharmacol. 34, 54±59. Burridge, L.E., Haya, K., 1997. Lethality of pyrethrins to larvae and postlarvae of the American lobster (Homarus americanus). Ecotoxicol. Environ. Saf. 38, 150±154. Casida, J.E., Ueda, K., Gaughan, L.C., Jao, L.T., Soderlund, D.M., 1975. Structure±biodegradability relationships in pyrethroid insecticides. Arch. Environ. Contam. Toxicol. 3, 491±500. Chen, S.Y., Zhang, Z.W., He, F.S., Yao, P.P., Wu, Y.Q., Sun, J.X., Liu, L.H., Li, Q.G., 1991. An epidemiological study on occupational acute pyrethroid poisoning in cotton farmers. Br. J. Ind. Med. 48, 77±81. Elliott, T., 1980. Established pyrethroid insecticides. Pestic. Sci. 11, 119±128. Kontreczky, C., Farkas, A., Nemcsok, J., Salanki, J., 1997. Short- and long-term eects of deltamethrin on ®ltering activity of freshwater mussel (Aanodonta cygnea L.). Ecotoxicol. Environ. Saf. 38, 195±199.
I. Kakko et al. / Chemosphere 40 (2000) 301±305 Korpela, M., Tahti, H., 1988. The eect of in vitro and in vivo toluene exposure on rat erythrocyte and synaptosome membrane integral enzymes. Pharmacol. Toxicol. 63, 30± 32. Lombet, A., Mourre, C., Lazdunski, M., 1988. Interaction of insecticides of the pyrethroid family with speci®c binding sites on the voltage-dependent sodium channel from mammalian brain. Brain Res. 459, 44±53. Nagy, A., Delgado, E.A., 1984. Rapid preparation of synaptosomes from mammalian brain using nontoxic isoosmotic gradient material (Percoll). J. Neurochem. 43, 1114±1123. Narahashi, T., 1986. Mechanisms of action of pyrethroids on sodium and calcium channel gating. In: Ford, M.G., Lunt, G.G., Reay, R.C., Usherwood, P.N.R. (Eds.), Neuropharmacology of Pesticide Action. Ellis Horwood, Chichester, UK, pp. 36±60. Narahashi , T., 1996. Neuronal ion channels as the target sites of insecticides.. Pharmacol. Toxicol. 79, 1±14. Narahashi, T., Frey, J.M., Ginsburg, K.S., Nagata, K., Roi, M.L. Tatebayashi, H., 1995. Sodium channels and caminobutyric acid activated channels as target sites of insecticides. In: Clark, J.M. (Ed.), Molecular Action of Insecticides on Ion Channels. American Chemical Society, Washington, DC, pp. 26±43. O'Malley, M., 1997. Clinical evaluation of pesticide exposure and poisonings. Lancet 349, 1161±1166. Peter, J.V., John, G., Cherian, A.M., 1996. Pyrethroid poisoning. J. Assoc. Physicians India 44, 343±344.
305
Phillips, T.D., Hayes, A.W., 1989. Techniques in membrane toxicology. In: Hayes, A.E. (Ed.), Principles and Methods of Toxicology. Raven Press, New York, pp. 761±776. Rao, K.S., Chetty, C.S., Desaiah, D., 1984. In vitro eects of pyrethroids on rat brain and liver ATPase activities. J. Toxicol. Environ. Health 14, 257±265. Shankland, D.L., 1976. The nervous system: comparative physiology and pharmacology. In: Wilkinson, C.F. (Ed.), Insecticide Biochemistry and Physiology. Plenum Press, New York, pp. 229±270. Shaw, I.C., Chadwick, J., 1998. Principles of Environmental Toxicology, 1st edition. Taylor & Francis, London. Soderlund, D.M., Bloomquist, J.R., 1989. Neurotoxic actions of pyrethroid insecticides. Annu. Rev. Entomol. 34, 77±96. Tanaka, T., 1993. Behavioural eects of piperonyl butoxide in male mice. Toxicol. Lett. 69, 155±161. Testa, B., Jenner, P., 1981. Inhibitors of Cytochrome P-450s and their mechanism of action. Drug Metab. Rev. 12, 1± 117. Vaalavirta, L., T ahti, H., 1995. Astrocyte membrane Na+, K(+)-ATPase and Mg(2+)-ATPase as targets of organic solvent impact. Life Sci. 57, 2223±2230. Vijverberg, H.P., van den Bercken, J., 1990. Neurotoxicological eects and the mode of action of pyrethroid insecticides. Crit. Rev. Toxicol. 21, 105±126. Walker, C.H., Hopkin, S.P., Sibly, R.M., Peakall, D.B., 1996. Principles of Ecotoxicology, 1st edition. Taylor & Francis, London.