Introduction

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Pyrethroid insecticides are synthetic analogues of the natural pyrethrins contained in flowers of the genus Chrysanthemum. They form, together with chlorinated hydrocarbons (DDT, dieldrin, lindane), organo-phosphorus compounds (parathion, malathion, diazinon) and methylcarbamate esters (aldicarb, carbofuran, carbaryl) the four major classes of insecticides. Pyrethroids are neurotoxins. Based on the symptomology after acute intoxication of insects and mammals, they fall into two classes: type I, non--cyano-pyrethroids, such as permethrin, show generally peripheral activities and type II pyrethroids, such as cyhalothrin, incline to central action (Leahey, 1985). The latter are characterized chemically by a cyano substituent (fig. 1).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Structural formulae of permethrin and cyhalothrin.

Although synthetic pyrethroids are classified as well known and safe substances (Casida et al., 1983), their widespread use, their high, nonselective potency, and their considerable stability in the environment make them potentially harmful. In fact, intoxications of mammals, including humans, by pyrethroids has been observed (Chen et al., 1991). The underlying mechanisms of these toxic effects are not known.

A number of biochemical interactions of pyrethroids have been described in the literature. Alteration of the sodium channel kinetics is supposed to be the principal molecular mode of action of synthetic pyrethroids (Vijverberg et al., 1982; Vijverberg and van den Bercken, 1990; Tatebayashi and Narahashi, 1994). But inhibitory effects have also been described for Ca⁺⁺-channels (Kadous et al., 1994), ATPases (Reddy et al., 1991; Michelangeli et al., 1990), and the receptors for acetylcholine (Abbassy et al., 1983), GABA (Lawrence and Casida, 1983), serotonin (Oortgiesen et al., 1989) and benzodiazepine (Devaud and Murray, 1988). However, whether any of these interactions are responsible for the toxicological effects of pyrethroids in higher animals remains unclear. Recently, an effect of pyrethroids on mitochondria was observed in fish exposed to these drugs in vivo. It was found that pyrethroids affected O₂-consumption and led to a decrease in the activity of mitochondrial enzymes (Reddy and Philip, 1992; Ghosh, 1989). However, effects of pyrethroids on energy conservation by mammalian mitochondria have never been reported. In several toxicological models, movement disorders were linked to a dysfunction of mitochondria. Also, models exist for the development of movement disorders, based on toxins inhibiting complexes I and II of mitochondria (Schulz and Beal, 1994). In this light it appeared important to investigate the effects of pyrethroids on the activity of respiratory chain enzymes of mammalian mitochondria.

Here, we show that submicromolar concentrations of pyrethroids inhibit the respiratory complex I of rat liver mitochondria. This disturbance of the mitochondrial respiratory chain by pyrethroids could provide a new explanation for some of the symptoms of pyrethroid intoxication.

	Methods

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Materials. Cyhalothrin, (3-(2-chlor-3,3,3,-trifluor-1-propenyl)-2,2-demethylcyclopropanecarboxylic acid [1,3(Z)]-(±)-cyano(3-phenoxyphenyl)methyl ester) and permethrin, (m-phenoxybenzyl(RS)-cis, trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate) from Riedel-de Haën (Seelze, Germany), were dissolved in dimethylsulfoxide and were made fresh every day. Rotenone, horse heart cytochrome c and antimycin A were purchased from Sigma Chemical Co. (St. Louis, MO), threonyltrifluoroaceton, 2,6-dichlorophenolindophenol and DCCD were delivered from Fluka (Buchs, Switzerland). Duroquinol was synthesized from duroquinone (Fluka) according to Ragan et al. (1987). Crystalline suspensions in ammonium sulfate solution of pyruvate kinase (EC2.7.1.40, 10 mg/ml) and lactate dehydrogenase (EC1.1.1.27, 5 mg/ml) from rabbit muscle were form Boehringer Mannheim (Mannheim, Germany). Biuret reagent (Merkotest, Total Protein) was from Merck (Darmstadt, Germany).

Isolation of rat liver mitochondria. Mitochondria were isolated by differential centrifugation according to Hoppel et al. (1979). Briefly, male Sprague-Dawley rats, 500 g, were killed by decapitation and the liver was quickly removed. It was placed in ice-cold buffer A (220 mM mannitol, 70 mM sucrose, 5 mM MOPS, pH 7.4). The liver was minced and washed with buffer A. After the addition of 2 mM EDTA, a 10% suspension (w/v) of the minced liver was homogenized using a Potter-Elvehjem homogenizer with a loose fitting Teflon pestle. Nuclei and cell debris were removed by centrifugation at 700 × g for 10 min. Mitochondria were separated by centrifugation of the supernatant at 7000 × g for 10 min. The resulting mitochondrial pellet was washed twice with buffer A by resuspension and centrifugation as before and suspended in buffer A to a concentration of approximately 100 mg mitochondrial protein per ml.

Preparation of SMP. SMP were obtained form mitochondria according to Ragan et al. (1987). Freshly prepared, coupled rat liver mitochondria were suspended at a protein concentration of 15 mg/ml in buffer B (300 mM sucrose, 5 mM MOPS, 5 mM KP_i, 1 mM EGTA, pH 7.4) and sonicated with a Labsonic U sonicator (Braun, Melsung, Germany) fitted with a microtip. Five bursts of 10 sec were applied at 50 W and 50% duty cycle. Between bursts, the suspension was allowed to cool. The sonicated sample was diluted with an equal volume of buffer B and centrifuged at 15,000 × g for 10 min. SMP were collected from the supernatant by centrifugation at 100,000 × g for 30 min. The resulting pellet was washed twice by resuspension and centrifugation as before and suspended in buffer B to a final concentration of 5 mg mitochondrial protein per ml.

Oxygen electrode studies. Oxygen consumption of coupled mitochondria was measured in a chamber equipped with a Clark-type oxygen electrode (Yellow Springs Instrument Co., Yellow Springs, OH) at 30°C. The incubation medium [250 mM sucrose, 10 mM KCl, 1 mM P_i (Tris-salt), 25 mM Tris-Cl, pH 7.2] contained 0.87 ± 0.05 mg/ml mitochondrial protein. Permethrin and cyhalothrin were added to the medium 2 min before starting the reaction with 3 mM glutamate or succinate. After the recording of stable basal respiration, state 3 respiration was transiently initiated by the addition of 200 nmol ADP. In all the experiments described in this report, the concentration of DMSO never exceeded 1.5% (v/v) and controls with the same amount DMSO were always conducted. To uncouple mitochondria, 1.5 µM carbonyl cyanide p-(tri-fluoromethoxy)phenylhydrazone were added to the assay from a 1000-fold concentrated stock solution in ethanol.

Spectrophotometric assay of enzyme activities. Mitochondrial enzyme activities were determined using freeze-thawed SMP (complex I) or freeze-thawed mitochondria (complexes II-IV). The experiments were performed at 30°C. Mitochondrial preparations and pyrethroids or specific inhibitors were incubated in 3 ml medium. After an equilibration period of 5 min, the reaction was initiated by the addition of the substrate. Changes in absorbance were registered using a Perkin Elmer Corp. (Norwalk, CT) Lambda 5 UV/VIS spectrophotometer.

Determination of protein content. Protein concentrations were determined by the Biuret Method, using bovine serum albumin as a standard.

Curve fitting. Curves were drawn and half-maximal and maximal inhibitory concentrations were calculated as follows. The data on the inhibition (y) as a function of the pyrethroid concentration (p) were fitted in a nonlinear regression analysis by applying the Marquardt-Levenberg algorithm (Brent, 1995) according to the Hill equation:

(1)

The value of n increases with the degree of cooperativity and indicates the number of binding sites in the system. Half maximal inhibition (y_.5) is reached at

(2)

K comprises the dissociation constant of pyrethroids in the system (Segel, 1975).

	Results

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Effect of pyrethroids on respiratory control of mitochondria. In a first set of experiments, we looked at effects of permethrin and cyhalothrin on the respiratory control of mitochondria by measuring the O₂-consumption. In quadruplicate assays with isolated, coupled rat liver mitochondria from four animals (respiratory quotient with glutamate (RQ_glutamate) = 4.1 ± 0.5), we measured glutamate and succinate sustained oxygen consumption (state 4 respiration), ADP-stimulated oxygen consumption (state 3 respiration) and the P/O ratio. Although state 4 respiration and the P/O ratio were not affected by permethrin or cyhalothrin (data not shown), state 3 respiration was inhibited by micromolar concentrations of these drugs (fig. 2). Oxygen consumption of controls using glutamate as the substrate was 47.8 ± 9.9 nmol^·min^1·mg¹. Half maximal inhibition occurred at [permethrin]_.5 = 22 µM, (max. inhibition 107%); [cyhalothrin]_.5 = 7 µM (max. inhibition 72%). Uncoupled mitochondrial respiration was similarly inhibited by cyhalothrin (not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Dose-response curves for the effects of permethrin () and cyhalothrin () on glutamate sustained O2-consumption during state 3 respiration of freshly isolated rat liver mitochondria. O2-consumption was monitored with a Clark-type electrode as described in "Methods." The data are the means ± S.E.M. of assay quadruplicates.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Dose-response curves for the effects of permethrin () and cyhalothrin () on succinate sustained O2-consumption of rat liver mitochondria during state 3 respiration. Data points are the means of assay duplicates. Details of the procedure are as described for figure 2.

Assay of NADH:duroquinone oxidoreductase. Complex I activity was determined in extensively washed SMP from two animals. In these preparations, the activity was 151 ± 18 nmol^·min^1·mg¹ and was 72 ± 8% rotenone sensitive (19 experiments, SMP from two animals). The rotenone sensitive activity of complex I was inhibited in a concentration dependent manner by permethrin and cyhalothrin (fig. 4). According to equation 1, half maximal inhibition occurred at [permethrin]_.5 = 0.73 µM (max. inhibition 76%, n = 2.4) and [cyhalothrin]_.5 = 0.57 µM (max. inhibition 75%, n = 3.5). Thus, complex I was substantially inhibited by micromolar concentrations of pyrethroids.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Dose-response curves for the effects of permethrin () and cyhalothrin () on rotenone sensitive NADH:duroquinone oxidoreductase of SMP. The oxidation of NADH was monitored spectrophotometrically as described in "Methods." The data are the means ± S.E.M. of assay triplicates.

Assay of succinate:2,6-dichloroindophenol oxidoreductase. Complex II activity was assessed in triplicates from three animals. The activity of controls was 76 ± 11 nmol min¹ mg¹ and was 97% inhibited by 1 mM of the complex II inhibitor threonyltrifluoroaceton. The observed inhibition of complex II by pyrethroids leveled off at 26% indicating it may not be a specific effect (fig. 5). Using the Hill model, half maximal inhibitions were calculated to be at (permethrin)_0.5 = 0.5 µM (max. inhibition 26%) and (cyhalothrin)_0.5 = 3 µM (max. inhibition 36%).

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Dose-response curves for the effects of permethrin () and cyhalothrin () on succinate:2,6-dichloroindophenol oxidoreductase of freeze-thawed mitochondria. The reduction of 2,6-dichloroindophenol was followed spectrophotometrically as described under Methods. The data are the means ± S.E.M. of assay triplicates.

Assay of duroquinol:ferricytochrome c oxidoreductase. In the assay of complex III (bc₁-complex, triplicates, three animals), the background activity from the spontaneous oxidoreduction of duroquinol:cytochrome c was 38% of the total activity and was subtracted. The enzymatic activity of complex III was 66 ± 7 nmol^·min^1·mg¹ for controls (three experiments) and was not affected by up to 50 µM pyrethroids (fig. 6). The measured activity of complex III was 98% inhibited by 10 µg/ml of antimycin A, a specific inhibitor of complex III.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Dose-response curves for the effects of permethrin () and cyhalothrin () on duroquinole:ferricytochrome c oxidoreductase of freeze-thawed mitochondria. The data are the means ± S.E.M. of assay triplicates. Details of the procedure are as described "Methods."

Assay of ferrocytochrome c:oxygen oxidoreductase. Complex IV (cytochrome c oxidase) activity was not inhibited by permethrin, but appeared somewhat inhibited by cyhalothrin in the concentration range tested (fig. 7). However, the difference between the inhibitory action of the two drugs was not statistically significant. The measured control activity was 238 ± 14 nmol^·min^1·mg¹ (triplicates, three animals) and 1.5 mM KCN, the classical inhibitor of complex IV, blocked the activity completely.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Dose-response curves for the effects of permethrin () and cyhalothrin () on ferrocytochrome c:oxygen oxidoreductase of freeze-thawed mitochondria. The data are the means ± S.E.M. of assay triplicates.

Assay of ATPase. Complex V activity was not influenced by permethrin or cyhalothrin in the concentration range tested (fig. 8). The control activity was determined to be 205 ± 14 nmol^·min^1·mg¹ and was 80% inhibited by 1 mM DCCD (triplicates, three animals). The mitochondrial F-type ATPase consists of the polar, detachable F₁ head portion and the membrane-embedded F_o part. DCCD blocks the proton channel in the F_o-part of the ATPase. Only in the complete structure, ATP hydrolysis is coupled to proton flux and thus inhibited by DCCD. The high degree of inhibition we observed with this reagent indicates that our preparation consisted of 80% complete complex V molecules.

View larger version (13K): [in this window] [in a new window]   Fig. 8.   Dose-response curves for the effects of permethrin () and cyhalothrin () on ATPase of freeze-thawed mitochondria. Enzyme activities were assessed as described in "Methods." The data are the means ± S.E.M. of assay triplicates.

	Discussion

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Our study was undertaken to answer the question of whether permethrin, a type I pyrethroid, and/or cyhalothrin, a type II pyrethroid, interfered with the mitochondrial respiratory chain. We found that micromolar concentration of both compounds inhibited glutamate and succinate sustained state 3 respiration in a concentration-dependent manner. This inhibition could in principle be due to an effect of pyrethroids on an essential mitochondrial transport system and/or on a component of the respiratory chain. We identified complex I as major site of inhibition by permethrin and cyhalothrin. Complexes II to IV were not or only marginally affected by these drugs. We cannot rule out the possibility that pyrethroids have additional inhibitory effects on components accessory to the electron transport chain, such as transport systems. However, the inhibition of complex I is very pronounced and could thus be mainly responsible for the overall toxic effects of pyrethroids on mitochondria.

Mathematical analysis of the inhibition kinetics of complex I by permethrin and cyhalothrin indicated substantial cooperativity. Fitting the data with equation 1 yielded n-values of 2.4 for permethrin and 3.5 for cyhalothrin, indicating the interaction of at least three and four molecules, respectively, of inhibitor with complex I. This finding is not surprising in view of the hydrophobic nature of the pyrethroids on the one hand and the structural complexity of complex I on the other: all of the more than 40 subunits of complex I are potential targets for binding of pyrethroids.

Dysfunction of mitochondria due to exposure to toxins has been postulated to play a key role in the pathogenesis of movement disorders (Schulz and Beal, 1994). Inhibition of the mitochondrial complexes I and II (Pecci et al., 1994; Veitch and Hue, 1994) has drawn particular attention in this context, as some neurotoxins are known to interact with these sites. Model systems that use this mode of action for the induction of neurodegenerative diseases have recently been described. Even a slight inhibition of complex I induced concentration dependent, cell specific apoptosis in PC12 cells (Hartley et al., 1994). Also, suppression of complex I activity was found to be the ultimate step in the mode of action of MPTP. MPTP was discovered as a contaminant of an illicit pethidine analogue by the appearance of parkinsonian symptoms in young drug abusers. The neurotoxicity of MPTP was subsequently found to be based on the accumulation of the metabolite MPP⁺ in the mitochondrial matrix of neuronal cells, where it inhibited complex I of the respiratory chain. Eventually, this appears to lead to degeneration of neuronal tissue (Tipton and Singer, 1993). Another point in case is the induction of insulin-dependent diabetis mellitus by intoxication with the rodenticide Vacor. As shown recently, Vacor also inhibits complex I and could thus lead to selective destruction of the high energy requiring cells of pancreatic islets (Degli Esposti et al., 1996). Vacor and pyrethroids are in fact structurally similar chemical compounds.

Synthetic pyrethroids are widely used in agriculture. They are increasingly being used in veterinary applications on farm and pet animals, for the protection of stored foodstuffs, for the control of endemics and parasites in public health programs as well as for household applications in kitchens and bedrooms. Specially in indoor applications, a sustained contamination results from the adsorption of pyrethroids to small dust particles and various other surfaces (Schwabe et al., 1994). Chronic exposure of unaware individuals to low doses of synthetic pyrethroids consequently results. Synthetic pyrethroid insecticides may therefore represent an important indoor and environmental toxin. Based on a clinical analysis, a causative link between the exposure to synthetic pyrethroids and the development of motor and sensory disorders, including Parkinson-like syndromes, has been postulated (Müller-Mohnssen and Hahn, 1995). The result of our study suggests a possible molecular mechanism for the clinically observed neurotoxic symptoms after intoxication with synthetic pyrethroid insecticides. Clearly, further studies of the inhibitory effects of pyrethroids on mitochondria from different tissues, particularly brain, are required to prove this point.