Toxicokinetics of lambda-cyhalothrin in rats

Transcription

1 Toxicology Letters 165 (2006) Toxicokinetics of lambda-cyhalothrin in rats A. Anadón, M. Martínez, M.A. Martínez, M.J. Díaz, M.R. Martínez-Larrañaga Department of Toxicology and Pharmacology, Faculty of Veterinary Medicine, Universidad Complutense de Madrid, Madrid, Spain Received 22 September 2005; received in revised form 23 January 2006; accepted 24 January 2006 Available online 28 February 2006 Abstract The toxicokinetics of lambda-cyhalothrin after single 20 mg kg 1 oral and 3 mg kg 1 intravenous doses were studied in rats. Serial blood samples were obtained after oral and intravenous administration. Liver, brain, spinal cord, sciatic nerve, vas deferens, anococcygeus and myenteric plexus tissue samples were also collected. Plasma, liver, hypothalamus, cerebellum, medulla oblongata, frontal cortex, striatum, hippocampus, midbrain, spinal cord, vas deferens, anococcygeus, myenteric plexus and sciatic nerve concentrations of lambda-cyhalothrin were determined by HPLC. The plasma and tissue concentration time data for lambdacyhalothrin were found to fit a two-compartment open model. For lambda-cyhalothrin, the elimination half-life (T 1/2 ) and the mean residence time from plasma were 7.55 and 8.55 h after i.v. and and h after oral administration. The total plasma clearance was not influenced by dose concentration or route and reached a value of l h 1 kg 1. After i.v. administration, the apparent volume of distribution and at steady state were 0.68 and 0.53 l kg 1, suggesting a diffusion of the pyrethroid into tissue. After oral administration, lambda-cyhalothrin was extensively but slowly absorbed (T max, 2.69 h). The oral bioavailability was found to be 67.37%. Significant differences in the kinetic parameters between nervous tissues and plasma was observed. The maximum concentrations in hypothalamus (C max, gg 1 ) and myenteric plexus (C max, gg 1 ) were about 1.5 times higher than in plasma (C max, gml 1 ) and 1.3 times higher than in liver (C max, gml 1 ). Nervous tissue accumulation of lambda-cyhalothrin was also reflected by the area under the concentration curve ratios of tissue/plasma (liver). The T 1/2 for lambda-cyhalothrin was significantly greater for the nerve tissues, including neuromuscular fibres, (range and h, after i.v. and oral doses) than for plasma (7.55 and h, respectively) Elsevier Ireland Ltd. All rights reserved. Keywords: Pyrethroids; Lambda-cyhalothrin; Central and peripheral nervous system disposition 1. Introduction Pyrethroid insecticides have been used in agricultural and home formulations for more than 30 years and account for approximately one-fourth of the world- This paper was presented in part at the 43rd Annual Meeting of the Society of Toxicology, Baltimore, MD, USA, March 21 25, Corresponding author. Tel.: ; fax: address: mrml@vet.ucm.es (M.R. Martínez-Larrañaga). wide insecticide market (Casida and Quistad, 1998). Pyrethroids may be classified into two large groups (Gassner et al., 1997; Miyamoto et al., 1995). Type I pyrethroids (e.g. allethrin, permethrin) lack a cyano moiety. Type II pyrethroids (e.g. deltamethrin, fenvalerate and cyhalothrin) have a cyano group in the -position. They are neurotoxic both for mammals and insects. In all species tested, pyrethroids show a pattern of toxic action typical of a strongly excitant effect on the nervous system. In mammals two distinct toxic syndromes have been /$ see front matter 2006 Elsevier Ireland Ltd. All rights reserved. doi: /j.toxlet

2 48 A. Anadón et al. / Toxicology Letters 165 (2006) described, the T-syndrome induced by type I pyrethroids and the CS syndrome induced by type II compounds (Gammon et al., 1981; Verschoyle and Aldridge, 1980; Aldridge, 1990). Type I pyrethroids cause hyperexcitation, ataxia, convulsion, paralysis (Gray, 1985; Verschoyle and Aldridge, 1980; Vijverberg and Van den Bercken, 1990), and repetitive nerve firing (Narahashi, 1985; Vijverberg and de Weille, 1985). In contrast, type II pyrethroids poisoning is characterized by hypersensitivity, profuse salivation, choreoathetosis, tremor and paralysis (Gray, 1985; Vijverberg and Van den Bercken, 1990) but no repetitive nerve firing in sensory nerves (Lawrence and Casida, 1982). Some pyrethroids produced tremors and salivation, classified as the intermediate TS-syndrome. Although the molecular aspects of pyrethroid action are not yet fully understood, detailed electrophysiological investigations strongly suggest that the voltage-dependent sodium channel in the nerve membrane is the common target in both insects and mammals, including man (Vijverberg and Van den Bercken, 1982; Casida et al., 1983; Narahashi, 1985; Chinn and Narahashi, 1986; Vijverberg and Van den Bercken, 1990). Type II pyrethroids also depress resting chloride conductance, thereby amplifying any effects of sodium or calcium (Ray et al., 1997; Forshaw et al., 2000; Burr and Ray, 2005). Although extensive physiological and neuropharmacological investigations have been carried out in mammalian an nonmammalian species, only few studies have been conducted to analyze the toxicokinetic properties of type II pyretroids in laboratory animals (Gray and Rickard, 1981, 1982; Anadón et al., 1996). In the publications and reports, two characteristics have been mentioned which are allegedly tipical of pyrethroids and responsible for the health risks involved: (a) due to their lipophilic properties, pyrethroids are supposed to be capable of accumulating in nerve tissue, and (b) there is no evidence for indicate that the nervous system is reversibly or irreversibly damaged (Appel et al., 1994; Shafer et al., 2005). Lambda-cyhalothrin [ -cyano-3-phenoxybenzyl 3-(2-chloro-3,3,3-trifluoroprop-1-enyl)-2,2-dimethylcyclopropanecarboxylate] appears to be an insecticide that represents a good compromise between efficacy and toxicity. It is a type II pyrethroid with a high level of activity against a wide range of Lepidoptera, Hemiptera, Diptera, and Coleoptera species. Lambdacyhalothrin has found extensive uses in public and animal health applications where it effectively controls a broad spectrum of insects and ectoparasites, including cockroaches, flies, lices, mosquitos, and ticks (Davies et al., 2000; Kroeger et al., 2003). To date, toxicokinetics date have not been reported for lambda-cyhalothrin in mammals. Because the information regarding kinetic profile improves the scientific basis for risk decisions, the objective of the study reported here was to determine tissue and plasma concentrations of lambda-cyhalothrin and information about elimination of lambda-cyhalothrin in nervous tissues of rats after intravenous and oral administration. 2. Materials and methods 2.1. Chemicals Lambda-cyhalothrin (mixture 1:1 of S and R isomers); molecular formula C 23 H 19 ClF 3 NO 3 CAS RN , purity 98.8% w/w. Lambda-cyhalothrin and the S and R isomers of lambda-cyhalothrin were provided by Zeneca Agrochemicals (Syngenta), Bracknell, Berks, England. All other chemicals were of the highest quality grade and obtained from commercial sources Animals and experimental design Adult male Wistar rats (Charles River Inc., Margate, Kent, UK) each weighing g were used. The animals were individually housed in polycarbonate cages with sawdust bedding and were maintained in environmentally controlled rooms (22 ± 2 C and 50 ± 10% relative humidity) with a 12 h light/dark cycle (light from 08:00 to 20:00 h). Food (A04 rodent diet, Panlab SL) and water were available ad libitum. The rats were divided into two groups of 80 animals each, one group (Group 1) received lambda-cyhalothrin orally and the other group (Group 2) intravenously. Group 1 rats were deprived of food for 12 h before the single oral administration of 20 mg kg 1 body weight but were allowed water ad libitum. Lambda-cyhalothrin was administrated by gavage in a volume of 0.5 ml corn oil/rat. Group 2 rats were given a single i.v. injection of 3 mg kg 1 body weight into the lateral tail vein (in 0.1 ml glycerol formal/rat; Sanderson, 1959). Toxic doses of lambdacyhalothrin were selected (WHO, 2000) based on preliminary investigations that indicated these doses make sure that there was sufficient compound in the tissue samples to be above the level of limit of quantification of the analytical method. The animals of Groups 1 and 2 were divided into eight subgroups (A, B, C, D, E, F, G and H) of 10 animals each, respectively. All animals of the subgroups (A, B, C, D, E, F, G and H) were killed by cervical dislocation (one animal at each time) and then exanguination and decapitation at 0.16, 0.33, 0.5, 1, 2, 4, 6, 8, 12 and 24 h after oral and i.v. administration of lambda-cyhalothrin. Blood samples were withdrawn and collected in heparinized tubes and the rats were then decapited. Plasma was separated by centrifugation and stored frozen until analyzed. The liver, brain, sciatic nerve, ileum longitudinal muscle strips with the myenteric plexus attached, and vas deferens and anococcygeus muscles were removed (Ambache, 1954; Graham et al., 1968; Gillespie, 1972). Brains were dissected into hypothalamus, cerebellum, medulla oblongata,

3 A. Anadón et al. / Toxicology Letters 165 (2006) frontal cortex, hyppocampus, striatum, midbrain and spinal cord (Glowinski and Iversen, 1966). Each of these tissue samples was carefully weighed and kept frozen until analyzed Assay of lambda-cyhalothrin Plasma and tissue concentrations of lambda-cyhalothrin were measured by the use of an extraction method reported by Liu and Pleil (2002) and high-performance liquid chromatography as described (Bissacot and Vassilieff, 1997). The tissue samples were separately homogenized in distilled water (1 ml) ultrasonically (2 min at 40 W using a titanium needle probe on a Labsonic U/Braun, B. Braun Melsungen AG). The plasma (1 ml) and homogenized tissue samples were extracted with three portions of n-hexane (6 ml) by vortexing for 1 min. After extraction, the mixture was then centrifuged at 1600 g for 30 min. The organic phase (top) was then separated from the aqueous phase. The combined hexane extracts were dried over anhydrous sodium sulfate. After separation from the drying agent, the remainig solutions were evaporated under N 2 flowat30 C, and the residue was finally taken up in 1 ml of acetonitrile water (8:2 v/v) for high-pressure liquid chromatographic analysis. Lambda-cyhalothrin in plasma and tissues was measured in our laboratory using a Shimadzu liquid chromatograph equipped with a system controller SCL-10A VP, two solvent delivery modules LC-10AAD VP, an auto injector SIL-10AD VP, an UV/VIS photodiode array detector SPD-M10A VP, a CLASS-VP Ver.6.1 data system, and a Teknokroma Nucleosil 120 column (C-18, 5 m, 12.5 cm 0.40 cm). The mobile phase was a mixture of acetonitrile and water (80:20 v/v) pumped at 1 ml min 1. Detection was effected using ultraviolet absorption at 266 nm. Peak areas in the sample chromatograms were quantitated by external standard technique using solutions of lambda-cyhalothrin, and S and R isomer reference standards. For these conditions, lambda-cyhalothrin, and S and R isomers were eluted at the same retention time. Calibration curves from standard solutions and from plasma and nerve tissue samples fortified with lambda-cyhalothrin, as well as with the isomers, were obtained at 266 nm. Results for the method are linear over the calibration range of gml 1 as determined by use of the linear least squares regression procedure. Overall mean recovery of lambda-cyhalothrin and isomers from plasma and tissues was greater than 94% and 85%, respectively. Within-day and day-to-day precision were <6%. Interference of endogenous compounds was verified on blank plasma and tissues from untreated rats which provided the specificity of the method. For plasma and tissues, the validated limit of quantification was gml Data analysis The plasma and tissue concentrations versus time data for lambda-cyhalothrin were sequentially fitted to one-, two-, and multiple-compartment models, using an extended least-squares nonlinear regression program (Sheiner, 1981). The model of best fit was selected on the basis of the Akaike and Schwartz information criteria (Schwartz, 1978; Yamaoka et al., 1978). The two-compartment model was the best fit for all animal groups. This model was used to establish toxicokinetic characteristics. The plasma and tissue concentrations versus time curves of lambda-cyhalothrin (one animal at each time) and the mean plasma and tissue curves of lambda-cyhalothrin (eight animals at each time) after a single oral and i.v. administration were fitted to the following exponential equations: C = A 1 e αt + A 2 e βt A 3 e Kat (oral) C = A 1 e αt + A 2 e βt (i.v.) where C is the concentration of lambda-cyhalothrin; A 1, A 2 and A 3 are mathematical coefficients; α is the hybrid rate constant for the distribution phase; β the hybrid rate constant for the elimination terminal phase; K a is the first-order absorption rate constant. Absorption half-life (T 1/2a ), half-life of phase (T 1/2 ), half-life of phase (T 1/2 ), distribution rate constants for transfer of the drug from the central to the peripheral compartment (K 12 ) and from the peripheral to the central compartment (K 21 ), and the elimination rate constant (K 10 ) were performed by standard equations as previously described (Wagner, 1975, 1976). After oral and i.v. administration, the area under the concentration time curves (AUC) was calculated as follows: AUC = (A 1 /α) + (A 2 /β) (A 3 /K a ); or AUC = (A 1 /α) + (A 2 /β) Total plasma clearance (CL) was calculated, using the following formula: CL = (dose kg 1 ) (F/AUC); or CL = (dose kg 1 )/AUC where bioavailability (F) is (dose i.v. AUC oral )/(dose oral AUC i.v. ). Mean residence time (MRT) was calculated as follows: MRT = ((A 1 /α 2 ) + (A 2 /β 2 ) (A 3 /K 2 a )) (1/AUC); or MRT = ((A 1 /α 2 ) + (A 2 /β 2 )) (1/AUC) Apparent volume of distribution (V d(area) ) was determined as follows: V d(area) = (dose kg 1 ) ([F/AUC] β); or V d(area) = (dose kg 1 )/(AUC β) Volume of distribution at steady state (V d(ss) ) (only for i.v. administration) was determined as follows: V d(ss) = MRT CL Maximal drug concentration after oral administration of lambda-cyalothrin (C max ) and the interval (time) from oral administration until C max was detected (T max ) also were estimated.

4 50 A. Anadón et al. / Toxicology Letters 165 (2006) Mean absorption time (MAT) was calculated by use of the following equation (Riegelman and Collier, 1980): MAT = MRT oral MRT i.v. Statistical analysis was carried out using the Student s t-test. 3. Results 3.1. Plasma lambda-cyhalothrin disposition The mean plasma concentrations of lambdacyhalothrin after single oral and i.v. administration of lambda-cyhalothrin were determined (Fig. 1). Following oral and i.v. administration, analysis of plasma concentration time curves indicated a biphasic decrease. A good fit of the observed data for a two-compartment open model was obtained. Values of the kinetic variables that described absorption and disposition kinetics of lambda-cyhalothrin in rats were determined (Table 1). After i.v. administration of lambda-cyhalothrin, a rapid distribution phase and a slower elimination phase were observed, with a half-life of distribution phase (T 1/2 ) of 0.11 ± 0.01 h and a half-life of elimination phase (T 1/2 ) of 7.55 ± 0.99 h (Table 1). When administered orally, the pyrethroid was extensively but some Fig. 1. Mean plasma concentrations of lambda-cyhalothrin after single oral: ( ) administration of 20 mg kg 1 of body weight and after single i.v.; ( ) administration of 3 mg kg 1 of body weight. Data represent mean ± S.D. for eight rats. Symbols without bars indicate that S.D. is within the symbols. slowly absorbed. The half-life of oral absorption (T 1/2a ) was calculated to be 0.87 ± 0.12 h. The bioavailability (F) of lambda-cyhalothrin after oral administration was ± 9.21% and the maximal plasma concentration of lambda-cyhalothrin was ± 1.78 gml 1, estimated 2.69 ± 0.40 h after oral administration. The MAT after oral administration was 5.87 ± 1.39 h. Lambda-cyhalothrin was distributed more slowly after oral than i.v. dosing (distribution half-lives, T 1/2, 1.88 ± 0.28 and 0.11 ± 0.01 h, respectively). The Table 1 Toxicokinetic parameters for lambda-cyhalothrin in plasma after single oral and intravenous administration Parameter Dose Oral (20 mg/kg) i.v. (3 mg/kg) (n = 8) (n = 8) A 1 ( gml 1 ) ± ± A 2 ( gml 1 ) ± ± A 3 ( gml 1 ) ± α (h 1 ) 0.38 ± ± β (h 1 ) ± ± K a (h 1 ) 0.80 ± T 1/2 (h) 1.88 ± ± T 1/2 (h) ± ± T 1/2a (h) 0.87 ± V d(area) (l kg 1 ) 0.90 ± ± V d(ss) (l kg 1 ) 0.53 ± K 12 (h 1 ) 0.10 ± ± K 21 (h 1 ) 0.22 ± ± K 10 (h 1 ) 0.11 ± ± AUC(mghl 1 ) ± ± F (%) ± MRT (h) ± ± CL (l h kg 1 ) ± ± C max ( gml 1 ) ± T max (h) 2.69 ± a Toxicokinetic parameters were calculated from the mean concentration time curve.

5 A. Anadón et al. / Toxicology Letters 165 (2006) elimination half-life (T 1/2 ) of lambda-cyhalothrin after oral dose was somewhat greater than that calculated after i.v. dose. The elimination half-lives were ± 0.96 and 7.55 ± 0.99 h, respectively, indicating a slow final dissapearance of the pyrethroid from blood. This was supported by the values calculated for the MRT which was ± 0.91 h for oral dose and 8.55 ± 1.30 h for i.v. dose. The apparent volume of distribution [V d(area) ] and the volume of distribution at steady state [V d(ss) ] were 0.68 ± 0.07 and 0.53 ± 0.05 l kg 1, respectively, after i.v. administration, suggesting a distribution into the tissues. Total body clearance (CL) for lambda-cyhalothrin was 0.06 l h 1 kg 1 (Table 1) irrespective of the dosing route Tissue lambda-cyhalothrin disposition Lambda-cyhalothrin [after single doses of 20 mg kg 1 (oral) or 3 mg kg 1 (i.v.)] was efficiently distributed to tissues. High lambda-cyhalothrin concentrations were reached in all regions of the brain studied as well as in the ileum longitudinal muscle strips (myenteric plexus), vas deferens and anococcygeus muscles and the liver. The mean tissue concentration time curves for lambda-cyhalothrin after oral and i.v. administration are shown in Figs. 2 and 3. The tissue disposition of lambda-cyhalothrin after oral and i.v. administration in rats could be described best with a two-compartment open model. Table 2 shows the toxicokinetic parameters obtained for lambda-cyhalothrin for tissues other than plasma. Fig. 2. Mean tissue concentration time curves of lambda-cyhalothrin in rats after single oral dose of 20 mg kg 1 of body weight. (A) Central nervous system: ( ) hypothalamus; ( ) cerebellum; ( ) medulla oblongata; ( ) frontal cortex; ( ) hyppocampus; ( ) striatum; (+) midbrain; ( ) spinal cord. (B) Peripheral nervous system: ( ) sciatic nerve; ( ) ileum longitudinal muscle strips with the myenteric plexus attached; ( ) vas deferens; ( ) anococcygeus muscles; ( ) liver. Each value is the mean of eight rats. The standard error of the mean is indicated by the bars. Symbols without bars indicate that S.D. is within the symbols. Fig. 3. Mean tissue concentration time curves of lambda-cyhalothrin in rats after single i.v. dose of 3 mg kg 1 of body weight. (A) Central nervous system: ( ) hypothalamus; ( ) cerebellum; ( ) medulla oblongata; ( ) frontal cortex; ( ) hyppocampus; ( ) striatum; (+) midbrain; ( ) spinal cord. (B) Peripheral nervous system: ( ) sciatic nerve; ( ) ileum longitudinal muscle strips with the myenteric plexus attached; ( ) vas deferens; ( ) anococcygeus muscles; ( ) liver. Each value is the mean of eight rats. The standard error of the mean is indicated by the bars. Symbols without bars indicate that S.D. is within the symbols.

6 Table 2 Toxicokinetic parameters for lambda-cyhalothrin in tissues after a single 20 mg/kg (oral) and 3 mg/kg (i.v.) doses Tissue Oral (20 mg kg 1 ) i.v.(3mgkg 1 ) C max ( gg 1 ) T max (h) T 1/2 (h) AUC (mg h l 1 ) T 1/2 (h) AUC (mg h l 1 ) Hypothalamus ± 2.54 *** ± ± 1.97 *** ± *** ± 2.04 *** ± *** Cerebellum ± 2.24 ** ± ± 1.46 *** ± 3.40 * ± 1.86 *** ± *** Medulla oblongata ± ± ± 2.95 *** ± ± 2.66 *** ± *** Frontal cortex ± 0.81 * ± ± 1.92 *** ± 2.30 *** ± 2.50 *** ± *** Hyppocampus ± ± ± 1.72 *** ± ± 2.33 *** ± *** Striatum ± 1.87 * ± ± 2.12 *** ± 6.37 ** ± 1.94 *** ± *** Midbrain ± 2.17 * ± ± 2.09 *** ± ** ± 2.70 *** ± *** Spinal cord ± ± ± 2.19 *** ± ± 1.39 *** ± *** Myenteric plexus ± 1.83 *** ± ± 1.79 *** ± *** ± 1.69 *** ± *** Vas deferens ± 3.32 *** ± ± 1.96 *** ± *** ± 1.39 *** ± *** Anococcygeus ± 2.09 *** ± ± 2.85 *** ± *** ± 1.38 *** ± *** Sciatic nerve ± 1.87 *** ± ± 2.95 *** ± *** ± 1.76 *** ± *** Liver ± ± ± ± ± ± Plasma ± ± ± ± ± ± C max for hypothalamus is significantly higher than C max for other nerve tissues at P < 0.01 or P < (excepting neuromuscular fibres). C max for neuromuscular fibres are significantly higher than C max for nerve tissues at P < (excepting hypothalamus and cerebellum). C max for hypothalamus (P < 0.001), myenteric plexus (P < 0.001), vas deferens (P < 0.05) and annococygeus (P < 0.01) muscles are significantly higher than C max for liver. AUC for hypothalamus is significantly higher than AUC for other nerve tissues at P < (excepting sciatic nerve and the neuromuscular fibres). After oral dose, AUC for nerve tissues (excepting hippocampus, medulla oblongata and spinal cord) and neuromuscular fibres are significantly higher than AUC for liver at P < 0.05, P < 0.01 or P < After i.v. dose, AUC for nerve tissues and neuromuscular fibres are significantly higher than AUC for liver at P < a Toxicokinetic parameters were calculated from the mean concentration time curve. * Parameters for nerve tissues and neuromuscular fibres (myenteric plexus, vas deferens and anococcygeus muscles) are significantly higher than plasma parameters at P < ** Parameters for nerve tissues and neuromuscular fibres (myenteric plexus, vas deferens and anococcygeus muscles) are significantly higher than plasma parameters at P < *** Parameters for nerve tissues and neuromuscular fibres (myenteric plexus, vas deferens and anococcygeus muscles) are significantly higher than plasma parameters at P < A. Anadón et al. / Toxicology Letters 165 (2006) 47 56

7 A. Anadón et al. / Toxicology Letters 165 (2006) After oral administration, in relation to the tissues from central nervous system studied, the highest concentration for lambda-cyhalothrin was found in hypothalamus tissue. After oral administration, the maximal lambda-cyhalothrin concentration (C max ) was reached in all regions of the brain studied at the same time. The C max (range, gg 1 ) was detected h (T max ) after pyrethroid administration (Table 2). The peak concentrations of lambda-cyhalothrin obtained in all nervous tissues studied (excepting hippocampus, spinal cord and medulla oblongata) were significantly higher than the maximum plasma level. The largest discrepancy was found for the hypothalamus. The peak concentration of lambda-cyhalothrin obtained in hypothalamus tissue (C max, gg 1 ) was about 1.5 times higher than in plasma (C max, gml 1 ) P < and 1.3 times higher than in liver (C max, gg 1 ) P < and was also significantly higher than other tissues excepting the neuromuscular fibres (myenteric plexus, vas deferens and anococcygeus muscles). The elimination half-lives (T 1/2 ) of the pyrethroid were in the range of h after oral administration and h after i.v. administration in the brain regions studied (Table 2). With regard to autonomic nervous system tissues studied (myenteric plexus, vas deferens and anococcygeus muscles), after oral dose, the maximum amount of lambda-cyhalothrin detected in tissues was significantly higher than the maximum plasma level as well as higher than the maximum brain region levels (excepting hypothalamus and cerebellum). The C max (range, gg 1 ) was detected h (T max ) after pyrethroid administration (Table 2). The elimination half-lives (T 1/2 ) of the pyrethroid were in the range of h after oral administration and h after i.v. administration in the myenteric plexus, vas deferens and anococcygeus muscles (Table 2). The peak concentration of lambda-cyhalothrin obtained in sciatic nerve (C max, gg 1 ) was also significantly higher than the maximum plasma level (Table 2). The results obtained in the central and peripheral nervous system tissues indicated a tendency of lambda-cyhalothrin to accumulate in nervous tissues. After oral administration, the AUC for lambdacyhalothrin in tissues (excepting medulla oblongata, hyppocampus and spinal cord) was significantly higher than the AUC in plasma (Table 2), as well as (excepting hyppocampus and spinal cord) was significantly higher than the AUC in liver. The largest discrepancy was found for the hypothalamus, myenteric plexus and vas deferens muscles, and sciatic nerve. After oral administration, the ratios of AUC tissue /AUC plasma for lambda-cyhalothrin were 1.99 for hypothalamus, 2.11 for myenteric plexus, 1.88 for vas deferens and 2.26 for sciatic nerve. The ratios of AUC tissue /AUC liver for lambda-cyhalothrin were 2.15 for hypothalamus, 2.28 for myenteric plexus, 2.03 for vas deferens and 2.45 for sciatic nerve. 4. Discussion Lambda-cyhalothrin, a type II pirethroid, is known to have high insecticidal activity and relatively low inherent toxicity in mammals. It is widely used for numerous applications, varying from plant protection to general pest control. Improper use of this agent can potentially lead to adverse effects. One notable form of toxicity associated with overexposure to lambdacyhalothrin and other type II pirethroids, has been a facial cutaneous paraesthesia and irritation-related respiratory symptoms mainly observed in workers spraying pyrethroids or in occupational settings (Flannigan and Tucker, 1985; Flannigan et al., 1985; Vijverberg and Van den Bercken, 1990; Moretto, 1991; Chester et al., 1992; Appel et al., 1994; Arunodaya et al., 1997). Behavioral and biochemical data (Spinosa et al., 1999; Righi and Palermo-Neto, 2003) provide experimental evidence that lambda-cyhalothrin, and other type II pyrethroids, induce anxiety-like symptoms, with this effect being dose-related, thus anxiety should be also included among the several signs and symptoms of pyrethroid intoxication. The limited information available on the kinetics of lambda-cyhalothrin (Harrison, 1983, 1984; WHO, 2000) makes it difficult to interpret toxicological findings and to make risk assessment for lambda-cyhalothrin, topics that are still under debate. Toxicokinetic characteristics combined with toxycodynamic patterns must be considered in the use safety evaluation of lambda-cyhalothrin. To our knowledge this is the first study to have used a rapid specific chemical assay for the analysis of lambdacyhalothrin in biological fluids and nerve tissues in order to evaluate its pharmacokinetics. In the study reported here the kinetics of lambda-cyhalothrin after a single i.v. (3 mg kg 1 ) and oral (20 mg kg 1 ) administration were determined in rats. Disposition of lambdacyhalothrin after i.v. and oral administration in rats was best described by use of a two-compartment model. Disappearance of lambda-cyhalothrin from plasma and nerve tissues of rats was characterized by an initial rapid distribution phase followed by a slower elimination phase. The T 1/2 of lambda-cyhalothrin (7.55 h after i.v. administration) was similar to that observed for permethrin (8.67 h), a type I pirethroid, (Anadón et al., 1991) but shorter to that reported for deltamethrin (33 h), a type II pirethroid, (Anadón et al., 1996).

8 54 A. Anadón et al. / Toxicology Letters 165 (2006) Lambda-cyhalothrin metabolism could explain, in part, this finding. In mammal s species, the metabolism of lambda-cyhalothrin is still not fully elucitated although the hydrolysis by esterases seems to be the centre of metabolism (Harrison, 1983). Deltamethrin is biodegraded by both oxidative and hydrolytic processes (Ruzo et al., 1978, 1979; Cole et al., 1982; Anadón et al., 1996). The T 1/2 of lambda-cyhalothrin increased by 36% (to h) after oral administration. The values of V d(area) (0.68 and 0.90 l kg 1 after i.v. and oral administration) indicate that lambda-cyhalothrin easily penetrated all tissues, in agreement with data reported also for the pyrethroids permethrin and deltamethrin (Anadón et al., 1991, 1996). When given orally, lambda-cyhalothrin was extensively but slowly absorbed (T max, 2.69 h). The absorption process continued after T max as indicated by the mean absorption time of 5.87 h. The maximal plasma concentration (15.65 gml 1 ) was greater to those of study in which deltamethrin administered at a comparable dose of 26 mg kg 1 yield a C max of 0.46 gml 1 after oral administration (Anadón et al., 1996). The oral bioavailability of lambda-cyhalothrin was 67.37% in rats, which was higher than 14.43% reported for deltamethrin (Anadón et al., 1996). The low bioavailability of deltamethrin may be caused by biliary excretion or deltamethrin degradation at the site of absorption. Oral bioavailability of lambda-cyhalothrin (67.37%) in this study was greater than 55% reported in rats after oral administration of 25 mg kg 1 of 14 C-cyhalothrin (Harrison, 1984). These differences may have been due in part to differences in study design and analytical procedures. Although the symptoms of toxicity of lambdacyhalothrin were not quantitated in the present study, the administration of lambda-cyhalothrin to rats at oral (20 mg kg 1 )ori.v.(3mgkg 1 ) dose produced slight whole body tremor, sign of toxicity typical of type II pyrethroids (Barnes and Verschoyle, 1974; Ray and Cremer, 1979; Verschoyle and Aldridge, 1980). The symptoms were observed 1 3 h and min after oral and i.v. administration, respectively. Complete recovery was not observed until 12 h after oral dosing and until 2 3 h after i.v. dosing. No fatalities were recorded at these dose levels. Because, tremors were observed, it can be assumed that autonomic function was affected and that cardiovascular function was likely altered during the course of the experiment. Hemodynamic responses to lambda-cyhalothrin were not evaluated in this study; therefore potential hemodynamic changes cannot be easily used to explain the kinetic behaviour of this pyrethroid. However, other studies with type II pyrethroids, like the deltamethrin, have reported that the concentrations of deltamethrin in plasma and brain tissue appeared to correlate with the severity of toxicity (Rickard and Brodie, 1985; Anadón et al., 1996). Lambda-cyhalothrin rapidly enters the central and peripheral nervous system after oral and i.v. administration. In attempts to identify those brain regions and peripheral nervous tissues including neuromuscular fibres most involved, lambda-cyhalothrin levels were monitored. The analysis of the tissue concentration time course then revealed a long elimination half-life of lambda-cyhalothrin in all tissues investigated (T 1/2 ranges, and h after i.v. and oral administration, respectively). On the other hand, a discrete selectivity for an anatomical brain region could be shown. The primary brain region for lambda-cyhalothrin appears to be the hypothalamus. After oral administration of lambda-cyhalothrin, the highest peak concentration and area under the tissue concentration time curve and the longest elimination half-life of lambdacyhalothrin were found in the hypothalamus tissue, finding similar to that observed for deltamethrin (Anadón et al., 1996). In contrast, the lowest area under the tissue concentration time curve of lambda-cyhalothrin was found in the liver tissue. The peak concentrations in all brain regions are achieved the first 3 h when blood concentrations are also the maximum levels. Peak concentrations of lambda-cyhalothrin in all nervous tissues investigated, with the exception of medulla oblongata, hyppocampus and spinal cord, exceeded that the blood. These differences in concentrations could be due to the nerve tissue to blood partition coefficients or to the binding of lambda-cyhalothrin to nerve tissue components. The study of the possible correlation between lambdacyhalothrin levels and changes in cerebral blood flow could be of interest to clarify the multiple effects of the pyrethroids (Uchida et al., 1997). Further work is necessary to clarify the possible mechanisms. Although the brain regions contain high levels of lambda-cyhalothrin, certainly high deposits of this pyretroid have been also observed in the peripheral nervous tissue, sciatic nerve as well as in the neuromuscular fibres, myenteric plexus (longitudinal smooth muscle), vas deferens and anococcygeus muscles. A similar kinetic profile was previously observed for permethrin and deltamethrin (Anadón et al., 1991, 1996). The presence of lambda-cyhalothrin in brain with the higher deposits in the hypothalamus as well as in the neuromuscular fibres, myenteric plexus, vas deferens and anococcygeus muscles could provide the basis for future efforts to study the mode of action of lambda-cyhalothrin within the nervous system. It is well known that different neurotransmitters are differentially distributed among brain regions (Glowinski and Iversen,

9 A. Anadón et al. / Toxicology Letters 165 (2006) ) and that the post-ganglionic innervations of the myenteric plexus and vas deferens muscles have been classified physiologically as predominantly cholinergic and adrenergic, respectively (Paton et al., 1968, 1971; Sjöstrand, 1965). The properties of the anococcygeus muscle have been described (Gillespie, 1972; Gillespie et al., 1989); the rat anococcygeus muscle receives an inhibitory non-adrenergic, non-cholinergic innervation, and the NO has been proposed as the inhibitory transmitter of the rat anococcygeus. In summary, this is the first report of the toxicokinetics characteristics of lambda-cyhalothrin in an animal model after single oral and i.v. dose. Assuming the toxicokinetic parameters identified, this study serve to better understand the mammalian toxicity of lambda-cyhalothrin and to design further studies to characterize neurotoxicity of this pyrethoid. 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