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Distribution of radiolabel in rats after intravenous injection of a toxic dose of 14C-acid-, 14C-alcohol-, or 14C-cyano-labeled deltamethrin
PESTICIDE
BIOCHEMISTRY
Distribution
AND
PHYSIOLOGY
16, 79-85 (1981)
of Radiolabel in Rats after Intravenous Toxic Dose of 14C-Acid-, 14C-Alcohol-, 1%-Cyano-Labeled Deltamethrin A.J. GRAY*
*Division California
of Toxicology 92521; and
Injection or
of a
AND J.RICKARD?
and Physiology, Department of Entomolog.y, University of California, Riverside, tMRC Toxicology Unit, Medical Research Council Laboratories. Woodmansterne Road, Carshalton. Surrey, SMS 4EF, United Kingdom
Received February 16, 1981: accepted June 10. 1981 The tissue distribution of toxic doses of Y-acid-. “C-alcohol-. and ‘lC-cyano-labeled deltamethrin was followed after iv administration to rats. All three radiolabeled preparations were found in every tissue examined within 1 min of injection. Peak central nervous system (CNS) levels were achieved within l-5 min, but did not correspond to the onset of choreoathetosis. In the majority of the tissues examined the levels of deltamethrin label were similar, proportionately, to those found after administration of alcohol-labeled cismethrin. A major exception was the CNS which contained approximately 20% of the anticipated level of radiolabel suggesting that the CNS threshold for deltamethrin was approximately 0.55 1.O nmolig. Progressive accumulation of radioactivity in the erythrocyte fraction of the blood was observed following administration of cyano-labeled deltamethtin. This may contribute to the selective retention of r4C with this radiolabeled preparation. INTRODUCTION
Recent studies (1) have shown that. in general, pyrethroids containing an CXcyano-3-phenoxybenzyl alcohol and a halogen group in the acid moiety produce a writhing type of toxicity in rats (choreoathetosis) (2) that is usually associated with salivation (l)., These symptoms are quite distinct from the tremors produced by pyrethroids not containing such groups (1, 3, 4). The signs of toxicity observed in rats after deltamethrin’ [S-cY-cyano-3-phenoxybenzyl-3(2,2-dibromovinyl)-2,2-dimethylcyclopropanecarboxylate] administration include salivation and choreoathetosis (1,2, 5, 6) and have been designated as the CS syndrome of toxicity (1). Studies on the distribution of cismethrin (an early pyrethroid that produces tremors in rats) (3, 4, 7) after iv administration to rats have indicated that this pyrethroid was rapidly metabolized in vivo and that there was a threshold brain concentration at which tremors occurred (4, 7). However, ’ Previously known as decamethrin.
only limited studies on the tissue distribution of toxic doses of deltamethrin or any other pyrethroids that produce the CS type of toxicity in rats have been reported (8, 9). although the mammalian metabolism and excretion of this compound have been extensively investigated (8- 10). In the preliminary studies reported here the distributions of ‘“C-acid-, ‘“C-alcohol-, and ‘“C-cyano-labeled deltamethrin were followed in various tissues, including the central nervous system, after iv injection of a toxic dose, in order to determine whether the toxicity produced by this class of pyrethroid was due to a different tissue distribution pattern from that of cismethrin. MATERIALS
AND METHODS
Chemicals
Unlabeled deltamethrin and deltamethrin labeled with 14C in the acid, alcohol, and cyano moieties (Fig. 1) were supplied as gifts from Roussel Uclaf, Romainville, France. The radiochemical purity of the labeled deltamethrin was >99% and was
79 0048-3575/81/040079-07$02.00/O Copyright All rights
@ 1981 by Academic Press. Inc. of reproduction in any form reserved
80
GRAY
AND
14C alcohol
Specific
FIG. labeling
Acliwties
1. Strucruw positions.
Acid labelled 53.3 mCl/mmol Alcohol labelled 59.3 mCiimmol Cyano labelled 51.5 mCi/mmol
of dcltarnethrin
shmkg
the
‘“C-
checked and repurified as necessary by tlc2 in benzene. Glycerinformal (11) was obtained from Fluka A.G., and all other solvents of analytical grade from BDH Ltd and Fisons Ltd. Animals and Dosing Female Porton rats (140- 165 g), given free access to MRC 41B cubed diet and water, were used throughout these experiments. Deltamethrin was administered iv at 0.5 ml/kg as a 3.5 mg/ml solution in glycerinformal. Although the symptoms produced by deltamethrin were quite distinct from those produced by cismethrin, this dose (1.75 mg/kg) resulted in signs of toxicity that were of similar severity to those observed after iv injection of cismethrin at 2.5 mg/kg (7). The radiolabeled deltamethrin was diluted with unlabeled pyrethroid to prepare dosing solutions of specific activity 7.60-9.49 mCi/mmol (alcohol labeled), 9.46 mCi/mmol (acid labeled), and 6.34 mCi/mmol (cyan0 labeled). Tissue Distribution As previous work there was only slight animals in experiments only a limited amount tamethrin preparation cate values were not
(7) had shown that variation between of this type, and of each labeled delwas available, repliobtained. Animals
s Thin-layer chromatography. 3 Glycerinformal, 75% 5-hydroxy-1.3-dioxan 4-hydroxymethyl-l.3-dioxolan.
+ 25%
RICKARD
were killed, by decapitation, at intervals between 1 min and 8 hr after iv administration and samples of the following tissues taken: blood, plasma, liver, kidney, combined perirenal and ovarian fat, brown fat from the nape of the neck, leg muscle, submaxillary salivary glands. spinal cord. cerebrum, cerebellum, and sections of both sciatic nerves. A weighed portion of each tissue was burned in an Intertechnique Oxymat using the following scintillant mixture: 2-methoxyethylamine (330 ml, Hopkin and Williams), methanol (220 ml), toluene (450 ml), and 2,5-diphenyloxazole (10 g, Intertechnique). Brain Distribution In each animal half of the cerebrum was dissected by hand into nine anatomically defined parts consisting of the olfactory lobes, cortex, striatum, hypothalamus, amygdoloid region, hippocampus, thalamus, pons and medulla, and the midbrain. Each region of brain was weighed, solubilized in 1.5 ml of Soluene 350 (Packard) at 50°C overnight. and the radioactive content determined by lsc” after addition of 10 ml of Insta-gel (Packard) containing 1% acetic acid. Collection
of Saliva
Saliva was collected for the initial 12- 16 min following dosing with labeled deltamethrin. The saliva was either collected in 2-min fractions weighed and Insta-gel (10 ml) added for lsc, or pooled and extracted three times with 3 ml of ethyl acetate. The combined ethyl acetate extracts were evaporated under nitrogen gas at 35°C and the tube walls rinsed twice with a further 1 ml of ethyl acetate. The residue was finally suspended in 200 ~1 of ethyl acetate. A 50-~1 sample of this solution was added to Insta-gel (10 ml) and its radioactive content determined by lsc. A further 100 ~1 was spotted onto an aluminium-backed silicacoated tic plate (Merck, silica gel 60, 0.02 mm thick, uvZS4) and developed twice in ’ Liquid
scintillation
counting.
DISTRIBUTION
benzene, saturated (10:3, v/v) (BFE2).
with formic
OF
RADIOLABELED
81
DELTAMETHRIN
acid:ether
RESULTS 25
Toxicity
20'
The iv administration of deltamethrin to rats at 1.75 mgikg produced all the signs of toxicity typical of this type of pyrethroid (1, 2, 5, 6). Profuse salivation was observed from 2 min until 12- 16 min after dosing and was associated with chewing movements of the jaws and a “bulldozing” motion of the head and neck. After 5-10 min, animals displayed a coarse whole body tremor rapidly followed by auditory or sensoryinduced writhing, and finally spontaneous choreoathetosis (2, 6) occurred from 20 min. The writhing motions continued for approximately 1 hr. Although animals showed some signs of recovery by 2 hr. complete recovery was not observed until 4 hr. No deaths were recorded at this dose level.
-6
6
E
5
g 3-
4 3 2 1
L
Tissue Distribution
The three labeled deltamethrin preparations were found in all the tissues examined at 1 min (Figs. 2-4, Table 1). and in the majority of tissues the initial levels of radioactivity were similar. However, by 30 min some differences in the distribution of the three labeled deltamethrin preparations became apparent. The clearance of the alcohol-labeled compound from the blood (Fig. 2) plasma and kidney (Table 1) was slightly slower than deltamethrin labeled in either of the other two positions. Acidlabeled deltamethrin tissue radioactivity levels tended to be the lowest of the three preparations, except in the brown and ovarian fat deposits (Table 1). The distribution of cyanolabeled deltamethrin radiolabel was similar to the alcohol-labeled preparation except that higher levels of cyano label were found in the cerebrum, cerebellum, and spinal cord (Fig. 3) at all times, and in the blood and muscle at 4 and 8 hr. The sciatic nerve radioactivity content was maintained at a higher concentration
1
0
FIG.
2.
Lr~srls
of
rnc~taholitrs
it7
blood
ministration
of
acid
labeled
2
3
4
5
6
Time after dosing
(hrl
pyrcthroid ut (0).
ar7d
vurio7t.s
tirr7es
alcohol
(A),
its
7
radioactive after
or
a
cyar7o
iv
ad(m)-
deltrrmethrin.
after administration of cyano than after acid or alcohol-labeled compound (Table 1). The majority (>90%) of the radiolabel found in the blood (Fig. 2) with acid and alcohol-labeled preparations was in the plasma fraction, but with the cyano-labeled deltamethrin the radioactive content of the erythrocyte fraction accounted for 15% of the blood label at 5 min and increased to around 35% from 1 hr onward. The levels of radioactivity found in the brown fat were approximately three times those found in the combined ovarian and perirenal fat at 15 min, but had reduced to similar amounts by 8 hr. The distribution and clearance curves for the three regions of the CNS were similar except that the amounts of label found in the spinal cord
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DISTRIBUTION
of labeled found in maximum cleared to
OF
RADIOLABELED
deltamethrin and metabolites the liver (Fig. 4) achieved a within 5 min and then rapidly about 20% of this level by 2 hr.
Brain Distribution
The distribution of acid-, alcohol-, and cyano-labeled deltamethrin in the nine brain regions and the cerebellum are shown in Fig. 5. Similar to the findings of the gross tissue label content analysis, cyano label was found at higher concentrations at the early times, although all three labeled preparations showed similar uniform distribution and clearance of label in the brain. Saliva Analysis
Salivation was most profuse just after onset at around 2 min after deltamethrin
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FIG. 5. Levels of pyrethroid and its radioactive metabolites in 10 regions of the brain at various times after iv administration of acid (a), alcohol (O), or c.wno (a) -/abeled deltamerhrin. The numbers correspond to the following regions of the brain: olfactory lobes (I). cortex (2). striatum 0). hypothalamus (4). amygdoloid region (5). hippocampus (6), thalamus (7), pans and medulla (8). midbrain (9). and cerebellum (10).
DELTAMETHRIN
83
administration and the total amount of saliva collected in a 1Zmin period varied from 1 to 2 g. The amount of radiolabel collected was greatest between 6 and 8 min although these values were never more than three times that of the lowest level measured in a particular animal. The amount of deltamethrin and equivalent labeled metabolites excreted in the saliva per minute (picomole per minute per gram of saliva) increased steadily, reaching a maximum at around lo- 14 min when salivation ceased. The actual maximum rate achieved varied with the labeling position, being highest with cyano-labeled deltamethrin (2.50.-500 pmol/min/g saliva), with the acid (40-50 pmol/‘min/g saliva) and alcohol-labeled compounds (13 - 15 pmol/min/g saliva) being excreted at lower rates. Thin-layer chromatographic analysis of ethyl acetate extracts of saliva demonstrated the presence of parent deltamethrin with all three labeled preparations, varying between 2 and 6 pmol/g saliva. Some metabolites were also insolated, but in insufficient quantities for their structures to be determined. The majority of the radioactivity in saliva collected from animals administered acid-labeled deltamethrin was present as a single spot (I?, 0.72) on tic developed in the BFE2 system. This was a derivative of the acid moiety as no labeled compound of similar Rf was isolated with alcohol or cyano-labeled compound. Similarly, the labeled spot (R, 0.67) isolated after administration of alcohol-labeled deltamethrin was a derivative of the alcohol moiety not containing the a-cyano group. The majority of the radiolabel in the saliva after administration of cyano-labeled deltamethrin was not extracted by ethyl acetate although it was soluble in ethanol and remained at the origin in solvent system BFE2. DISCUSSION
All three labeled deltamethrin preparations rapidly entered all the tissues examined in this study. The distribution curves in the majority of tissues were similar to
84
GRAY
AND
those previously reported for alcohollabeled cismethrin but dissimilar to those of bioresmethrin (12). This would be expected as, like cismethrin, deltamethrin has the cis configuration across the cyclopropane ring and therefore undergoes less esterase cleavage than pyrethroids with the truns configuration (S- 10). As, on a molar basis, the dose of deltamethrin (520 nmoVl.50 g rat) administered was approximately 50% that of the equitoxic dose of cismethrin (1109 nmoYl50 g rat) used in a previous study (7), the radiolabel content of the majority of tissues examined in this study corresponded to approximately half those of the equitoxic cismethrin dose (7). The only major exceptions were the spinal cord, cerebellum. and cerebrum whose label content after iv injection of any of the three labeled deltamethrin preparations reached only 20% of the expected value. A possible explanation for this finding may be that deltamethrin is unable to cross the bloodbrain barrier as readily as cismethrin due to its greater polarity. It has been suggested from the signs of toxicity produced that deltamethrin has its primary action on the central nervous system (CNS) of mammals (1, 2, 6) and that direct injection of very small amounts of deltamethrin into the brains of mice (9) and spinal cord or brains of rats (13, 14) causes a similar toxicity to that observed after iv, ip, or oral administration of much larger doses. Hence, if it is assumed that, at least at the earlier times, the majority of the labeled compound is present as parent deltamethrin (i.e.. that amount common to all three labeled positions). the results of this study imply that the CNS threshold level of deltamethrin to produce writhing is about 0.5 - 1.O nmol/g. This value is in good agreement with the approximate figure of 1 nmol/g that was proposed for mice (10). Higher blood concentrations of cyano as opposed to acid- or alcohol-labeled deltamethrin have been previously reported in animals killed 8 days after oral dosing (8). Our experiments indicate that this may be due, at least partially, to the selective pres-
RICKARD
ence of cyano label in the erythrocytes, which was possibly a result of the formation of a complex between the cyanide released when the ester link was cleaved (8) and the low levels of methemoglobin present in erythrocytes (15). The different distribution of cyano label when compared to acid- and alcohol-labeled deltamethrin in the later stages of the experiment was probably due to the tissue distribution of the former mainly reflecting that of thiocyanate and 2iminothiazolidine-4-carboxylic acid formed from the released cyanide (8). The small amount of deltamethrin and metabolites found in the saliva would contribute very little to the overall clearance of the pyrethroid from the animal. All three labeled deltamethrin preparations rapidly entered the CNS, which is probably the major site of toxic action of this pyrethroid (1, 2, 5. 6, 9, 13, 14), and therefore the delay in onset of choreoathetosis was not due to the slow entry of compound. As the distribution of radiolabel throughout the brain was uniform at all the times examined it is also unlikely that the delay was due to gross redistribution of the pyrethroid within the CNS although it may be related to the micromolecular distribution of the compound. In conclusion, although the tissue distribution of labeled deltamethrin was very similar to an equitoxic dose of cismethrin (7), the CNS threshold for writhing was approximately 10% that of cismethrin (4, 7) and the onset of toxicity did not correspond to the peak brain radioactivity level. ACKNOWLEDGMENTS
We thank Roussel Uclaf for the gifts of radiolabeled and nonlabeled deltamethrin and Dr. T. A. Connors, Dr. V. J. Cunningham, and Mr. R. D. Verschoyle for their constructive critism of the draft manuscript. One of us (A.J.G.) also thanks the National Research Development Corporation for funding this project. REFERENCES
I. R. D. Verschoyle and W. N. Aldridge. Structure-activity relationships of some pyrethroids in rats, Arch. Tuxicol. 45, 325 (1980).
DISTRIBUTION
OF RADIOLABELED
2. D. E. Ray and J. E. Cremer, The action of decamethrin (a synthetic pyrethroid) on the rat. Pestic. Eiochem. Physiol. 10, 333 (1979). 3. R. D. Verschoyle and J. M. Barnes. Toxicity of natural and synthetic pyrethrins to rats. Pesfic. Biochem.
Physiol.
2, 308 (1972).
4. I. N. H. White, R. D. Verschoyle. M. H. Moradian. and J. M. Barnes, The relationship between brain levels of cismethrin and bioresmethrin in female rats and neurotoxic effects. Pcstic,.
Biocham.
10.
Physiol.
6, 491 (1976).
5. J. M. Barnes and R. D. Verschoyle. Toxicity of new pyrethroid insecticide, Nature (London) 248, 711 (1974). 6. D. E. Ray. An EEG investigation of decamethrin-induced choreoathetosis in the rat. E.rp. Brain Rcs. 38. 221 (1980). 7. A. J. Gray, T. A. Connors, H. Hoellinger, and Nguyen-Hoang-Nam. The relationship between the pharmacokinetics of intravenous cismethrin and bioresmethrin and their mammalian toxicity. Pestic. Eiochum. Physiol. 13, 281 (1980). 8. L. 0. Ruzo. T. Unai. and J. E. Casida. Decamethrin metabolism in rats, J. Agr. Food Chum. 26. 918 (1978). 9. L. 0. Ruzo. J. L. Engel, and J. E. Casida. Decamethrin metabolites from oxidative. hydrolyt-
11. 12. 13.
14. 15.
DELTAMETHRIN
85
ic and conjugative reactions in mice, J. Agr. Food Chrm. 27, 725 (1979). T. Shono, K. Ohsawa. and J. E. Casida, Metabolism of trans- and cis-permethrin, tram- and cis-cypermethrin. and decamethrin by microsomal enzymes, J. Agr. Food Chum. 27, 316 (1979). D. M. Sanderson, A note on glycerol formal as a solvent in toxicity testing. J. Pharm. Phurmud. 11, 150 (1959). A. J. Gray and T. A. Connors, Delayed toxicity after intravenous administration of bioresmethrin to rats. Pesfic. Sci. 11, 361 (1980). A. J. Gray, T. A. Connors. and J. Rickard, Mechanism of mammalian toxicity of the pyrethroids, irr “Neurotransmitters and Their Receptors: Proceedings of the EMBO workshop. February, 1980” (U. Z. Littauer, Y. Dudai, I. Silman. V. I. Teichberg, and Z. Vogel., Eds.), Wiley, London, 1980. A. J. Gray and J. Rickard. Toxicity of pyrethroids to rats after direct injection into the central nervous system. Submitted for publication. A. De Bruin, Metabolism of occupational agents, in “Biochemical Toxicology of Environmental Agents” (A. De Bruin, Ed.), p. 109. Elsevieri North-Holland. Amsterdam, 1976.
BIOCHEMISTRY
Distribution
AND
PHYSIOLOGY
16, 79-85 (1981)
of Radiolabel in Rats after Intravenous Toxic Dose of 14C-Acid-, 14C-Alcohol-, 1%-Cyano-Labeled Deltamethrin A.J. GRAY*
*Division California
of Toxicology 92521; and
Injection or
of a
AND J.RICKARD?
and Physiology, Department of Entomolog.y, University of California, Riverside, tMRC Toxicology Unit, Medical Research Council Laboratories. Woodmansterne Road, Carshalton. Surrey, SMS 4EF, United Kingdom
Received February 16, 1981: accepted June 10. 1981 The tissue distribution of toxic doses of Y-acid-. “C-alcohol-. and ‘lC-cyano-labeled deltamethrin was followed after iv administration to rats. All three radiolabeled preparations were found in every tissue examined within 1 min of injection. Peak central nervous system (CNS) levels were achieved within l-5 min, but did not correspond to the onset of choreoathetosis. In the majority of the tissues examined the levels of deltamethrin label were similar, proportionately, to those found after administration of alcohol-labeled cismethrin. A major exception was the CNS which contained approximately 20% of the anticipated level of radiolabel suggesting that the CNS threshold for deltamethrin was approximately 0.55 1.O nmolig. Progressive accumulation of radioactivity in the erythrocyte fraction of the blood was observed following administration of cyano-labeled deltamethtin. This may contribute to the selective retention of r4C with this radiolabeled preparation. INTRODUCTION
Recent studies (1) have shown that. in general, pyrethroids containing an CXcyano-3-phenoxybenzyl alcohol and a halogen group in the acid moiety produce a writhing type of toxicity in rats (choreoathetosis) (2) that is usually associated with salivation (l)., These symptoms are quite distinct from the tremors produced by pyrethroids not containing such groups (1, 3, 4). The signs of toxicity observed in rats after deltamethrin’ [S-cY-cyano-3-phenoxybenzyl-3(2,2-dibromovinyl)-2,2-dimethylcyclopropanecarboxylate] administration include salivation and choreoathetosis (1,2, 5, 6) and have been designated as the CS syndrome of toxicity (1). Studies on the distribution of cismethrin (an early pyrethroid that produces tremors in rats) (3, 4, 7) after iv administration to rats have indicated that this pyrethroid was rapidly metabolized in vivo and that there was a threshold brain concentration at which tremors occurred (4, 7). However, ’ Previously known as decamethrin.
only limited studies on the tissue distribution of toxic doses of deltamethrin or any other pyrethroids that produce the CS type of toxicity in rats have been reported (8, 9). although the mammalian metabolism and excretion of this compound have been extensively investigated (8- 10). In the preliminary studies reported here the distributions of ‘“C-acid-, ‘“C-alcohol-, and ‘“C-cyano-labeled deltamethrin were followed in various tissues, including the central nervous system, after iv injection of a toxic dose, in order to determine whether the toxicity produced by this class of pyrethroid was due to a different tissue distribution pattern from that of cismethrin. MATERIALS
AND METHODS
Chemicals
Unlabeled deltamethrin and deltamethrin labeled with 14C in the acid, alcohol, and cyano moieties (Fig. 1) were supplied as gifts from Roussel Uclaf, Romainville, France. The radiochemical purity of the labeled deltamethrin was >99% and was
79 0048-3575/81/040079-07$02.00/O Copyright All rights
@ 1981 by Academic Press. Inc. of reproduction in any form reserved
80
GRAY
AND
14C alcohol
Specific
FIG. labeling
Acliwties
1. Strucruw positions.
Acid labelled 53.3 mCl/mmol Alcohol labelled 59.3 mCiimmol Cyano labelled 51.5 mCi/mmol
of dcltarnethrin
shmkg
the
‘“C-
checked and repurified as necessary by tlc2 in benzene. Glycerinformal (11) was obtained from Fluka A.G., and all other solvents of analytical grade from BDH Ltd and Fisons Ltd. Animals and Dosing Female Porton rats (140- 165 g), given free access to MRC 41B cubed diet and water, were used throughout these experiments. Deltamethrin was administered iv at 0.5 ml/kg as a 3.5 mg/ml solution in glycerinformal. Although the symptoms produced by deltamethrin were quite distinct from those produced by cismethrin, this dose (1.75 mg/kg) resulted in signs of toxicity that were of similar severity to those observed after iv injection of cismethrin at 2.5 mg/kg (7). The radiolabeled deltamethrin was diluted with unlabeled pyrethroid to prepare dosing solutions of specific activity 7.60-9.49 mCi/mmol (alcohol labeled), 9.46 mCi/mmol (acid labeled), and 6.34 mCi/mmol (cyan0 labeled). Tissue Distribution As previous work there was only slight animals in experiments only a limited amount tamethrin preparation cate values were not
(7) had shown that variation between of this type, and of each labeled delwas available, repliobtained. Animals
s Thin-layer chromatography. 3 Glycerinformal, 75% 5-hydroxy-1.3-dioxan 4-hydroxymethyl-l.3-dioxolan.
+ 25%
RICKARD
were killed, by decapitation, at intervals between 1 min and 8 hr after iv administration and samples of the following tissues taken: blood, plasma, liver, kidney, combined perirenal and ovarian fat, brown fat from the nape of the neck, leg muscle, submaxillary salivary glands. spinal cord. cerebrum, cerebellum, and sections of both sciatic nerves. A weighed portion of each tissue was burned in an Intertechnique Oxymat using the following scintillant mixture: 2-methoxyethylamine (330 ml, Hopkin and Williams), methanol (220 ml), toluene (450 ml), and 2,5-diphenyloxazole (10 g, Intertechnique). Brain Distribution In each animal half of the cerebrum was dissected by hand into nine anatomically defined parts consisting of the olfactory lobes, cortex, striatum, hypothalamus, amygdoloid region, hippocampus, thalamus, pons and medulla, and the midbrain. Each region of brain was weighed, solubilized in 1.5 ml of Soluene 350 (Packard) at 50°C overnight. and the radioactive content determined by lsc” after addition of 10 ml of Insta-gel (Packard) containing 1% acetic acid. Collection
of Saliva
Saliva was collected for the initial 12- 16 min following dosing with labeled deltamethrin. The saliva was either collected in 2-min fractions weighed and Insta-gel (10 ml) added for lsc, or pooled and extracted three times with 3 ml of ethyl acetate. The combined ethyl acetate extracts were evaporated under nitrogen gas at 35°C and the tube walls rinsed twice with a further 1 ml of ethyl acetate. The residue was finally suspended in 200 ~1 of ethyl acetate. A 50-~1 sample of this solution was added to Insta-gel (10 ml) and its radioactive content determined by lsc. A further 100 ~1 was spotted onto an aluminium-backed silicacoated tic plate (Merck, silica gel 60, 0.02 mm thick, uvZS4) and developed twice in ’ Liquid
scintillation
counting.
DISTRIBUTION
benzene, saturated (10:3, v/v) (BFE2).
with formic
OF
RADIOLABELED
81
DELTAMETHRIN
acid:ether
RESULTS 25
Toxicity
20'
The iv administration of deltamethrin to rats at 1.75 mgikg produced all the signs of toxicity typical of this type of pyrethroid (1, 2, 5, 6). Profuse salivation was observed from 2 min until 12- 16 min after dosing and was associated with chewing movements of the jaws and a “bulldozing” motion of the head and neck. After 5-10 min, animals displayed a coarse whole body tremor rapidly followed by auditory or sensoryinduced writhing, and finally spontaneous choreoathetosis (2, 6) occurred from 20 min. The writhing motions continued for approximately 1 hr. Although animals showed some signs of recovery by 2 hr. complete recovery was not observed until 4 hr. No deaths were recorded at this dose level.
-6
6
E
5
g 3-
4 3 2 1
L
Tissue Distribution
The three labeled deltamethrin preparations were found in all the tissues examined at 1 min (Figs. 2-4, Table 1). and in the majority of tissues the initial levels of radioactivity were similar. However, by 30 min some differences in the distribution of the three labeled deltamethrin preparations became apparent. The clearance of the alcohol-labeled compound from the blood (Fig. 2) plasma and kidney (Table 1) was slightly slower than deltamethrin labeled in either of the other two positions. Acidlabeled deltamethrin tissue radioactivity levels tended to be the lowest of the three preparations, except in the brown and ovarian fat deposits (Table 1). The distribution of cyanolabeled deltamethrin radiolabel was similar to the alcohol-labeled preparation except that higher levels of cyano label were found in the cerebrum, cerebellum, and spinal cord (Fig. 3) at all times, and in the blood and muscle at 4 and 8 hr. The sciatic nerve radioactivity content was maintained at a higher concentration
1
0
FIG.
2.
Lr~srls
of
rnc~taholitrs
it7
blood
ministration
of
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deltamethrin and metabolites the liver (Fig. 4) achieved a within 5 min and then rapidly about 20% of this level by 2 hr.
Brain Distribution
The distribution of acid-, alcohol-, and cyano-labeled deltamethrin in the nine brain regions and the cerebellum are shown in Fig. 5. Similar to the findings of the gross tissue label content analysis, cyano label was found at higher concentrations at the early times, although all three labeled preparations showed similar uniform distribution and clearance of label in the brain. Saliva Analysis
Salivation was most profuse just after onset at around 2 min after deltamethrin
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FIG. 5. Levels of pyrethroid and its radioactive metabolites in 10 regions of the brain at various times after iv administration of acid (a), alcohol (O), or c.wno (a) -/abeled deltamerhrin. The numbers correspond to the following regions of the brain: olfactory lobes (I). cortex (2). striatum 0). hypothalamus (4). amygdoloid region (5). hippocampus (6), thalamus (7), pans and medulla (8). midbrain (9). and cerebellum (10).
DELTAMETHRIN
83
administration and the total amount of saliva collected in a 1Zmin period varied from 1 to 2 g. The amount of radiolabel collected was greatest between 6 and 8 min although these values were never more than three times that of the lowest level measured in a particular animal. The amount of deltamethrin and equivalent labeled metabolites excreted in the saliva per minute (picomole per minute per gram of saliva) increased steadily, reaching a maximum at around lo- 14 min when salivation ceased. The actual maximum rate achieved varied with the labeling position, being highest with cyano-labeled deltamethrin (2.50.-500 pmol/min/g saliva), with the acid (40-50 pmol/‘min/g saliva) and alcohol-labeled compounds (13 - 15 pmol/min/g saliva) being excreted at lower rates. Thin-layer chromatographic analysis of ethyl acetate extracts of saliva demonstrated the presence of parent deltamethrin with all three labeled preparations, varying between 2 and 6 pmol/g saliva. Some metabolites were also insolated, but in insufficient quantities for their structures to be determined. The majority of the radioactivity in saliva collected from animals administered acid-labeled deltamethrin was present as a single spot (I?, 0.72) on tic developed in the BFE2 system. This was a derivative of the acid moiety as no labeled compound of similar Rf was isolated with alcohol or cyano-labeled compound. Similarly, the labeled spot (R, 0.67) isolated after administration of alcohol-labeled deltamethrin was a derivative of the alcohol moiety not containing the a-cyano group. The majority of the radiolabel in the saliva after administration of cyano-labeled deltamethrin was not extracted by ethyl acetate although it was soluble in ethanol and remained at the origin in solvent system BFE2. DISCUSSION
All three labeled deltamethrin preparations rapidly entered all the tissues examined in this study. The distribution curves in the majority of tissues were similar to
84
GRAY
AND
those previously reported for alcohollabeled cismethrin but dissimilar to those of bioresmethrin (12). This would be expected as, like cismethrin, deltamethrin has the cis configuration across the cyclopropane ring and therefore undergoes less esterase cleavage than pyrethroids with the truns configuration (S- 10). As, on a molar basis, the dose of deltamethrin (520 nmoVl.50 g rat) administered was approximately 50% that of the equitoxic dose of cismethrin (1109 nmoYl50 g rat) used in a previous study (7), the radiolabel content of the majority of tissues examined in this study corresponded to approximately half those of the equitoxic cismethrin dose (7). The only major exceptions were the spinal cord, cerebellum. and cerebrum whose label content after iv injection of any of the three labeled deltamethrin preparations reached only 20% of the expected value. A possible explanation for this finding may be that deltamethrin is unable to cross the bloodbrain barrier as readily as cismethrin due to its greater polarity. It has been suggested from the signs of toxicity produced that deltamethrin has its primary action on the central nervous system (CNS) of mammals (1, 2, 6) and that direct injection of very small amounts of deltamethrin into the brains of mice (9) and spinal cord or brains of rats (13, 14) causes a similar toxicity to that observed after iv, ip, or oral administration of much larger doses. Hence, if it is assumed that, at least at the earlier times, the majority of the labeled compound is present as parent deltamethrin (i.e.. that amount common to all three labeled positions). the results of this study imply that the CNS threshold level of deltamethrin to produce writhing is about 0.5 - 1.O nmol/g. This value is in good agreement with the approximate figure of 1 nmol/g that was proposed for mice (10). Higher blood concentrations of cyano as opposed to acid- or alcohol-labeled deltamethrin have been previously reported in animals killed 8 days after oral dosing (8). Our experiments indicate that this may be due, at least partially, to the selective pres-
RICKARD
ence of cyano label in the erythrocytes, which was possibly a result of the formation of a complex between the cyanide released when the ester link was cleaved (8) and the low levels of methemoglobin present in erythrocytes (15). The different distribution of cyano label when compared to acid- and alcohol-labeled deltamethrin in the later stages of the experiment was probably due to the tissue distribution of the former mainly reflecting that of thiocyanate and 2iminothiazolidine-4-carboxylic acid formed from the released cyanide (8). The small amount of deltamethrin and metabolites found in the saliva would contribute very little to the overall clearance of the pyrethroid from the animal. All three labeled deltamethrin preparations rapidly entered the CNS, which is probably the major site of toxic action of this pyrethroid (1, 2, 5. 6, 9, 13, 14), and therefore the delay in onset of choreoathetosis was not due to the slow entry of compound. As the distribution of radiolabel throughout the brain was uniform at all the times examined it is also unlikely that the delay was due to gross redistribution of the pyrethroid within the CNS although it may be related to the micromolecular distribution of the compound. In conclusion, although the tissue distribution of labeled deltamethrin was very similar to an equitoxic dose of cismethrin (7), the CNS threshold for writhing was approximately 10% that of cismethrin (4, 7) and the onset of toxicity did not correspond to the peak brain radioactivity level. ACKNOWLEDGMENTS
We thank Roussel Uclaf for the gifts of radiolabeled and nonlabeled deltamethrin and Dr. T. A. Connors, Dr. V. J. Cunningham, and Mr. R. D. Verschoyle for their constructive critism of the draft manuscript. One of us (A.J.G.) also thanks the National Research Development Corporation for funding this project. REFERENCES
I. R. D. Verschoyle and W. N. Aldridge. Structure-activity relationships of some pyrethroids in rats, Arch. Tuxicol. 45, 325 (1980).
DISTRIBUTION
OF RADIOLABELED
2. D. E. Ray and J. E. Cremer, The action of decamethrin (a synthetic pyrethroid) on the rat. Pestic. Eiochem. Physiol. 10, 333 (1979). 3. R. D. Verschoyle and J. M. Barnes. Toxicity of natural and synthetic pyrethrins to rats. Pesfic. Biochem.
Physiol.
2, 308 (1972).
4. I. N. H. White, R. D. Verschoyle. M. H. Moradian. and J. M. Barnes, The relationship between brain levels of cismethrin and bioresmethrin in female rats and neurotoxic effects. Pcstic,.
Biocham.
10.
Physiol.
6, 491 (1976).
5. J. M. Barnes and R. D. Verschoyle. Toxicity of new pyrethroid insecticide, Nature (London) 248, 711 (1974). 6. D. E. Ray. An EEG investigation of decamethrin-induced choreoathetosis in the rat. E.rp. Brain Rcs. 38. 221 (1980). 7. A. J. Gray, T. A. Connors, H. Hoellinger, and Nguyen-Hoang-Nam. The relationship between the pharmacokinetics of intravenous cismethrin and bioresmethrin and their mammalian toxicity. Pestic. Eiochum. Physiol. 13, 281 (1980). 8. L. 0. Ruzo. T. Unai. and J. E. Casida. Decamethrin metabolism in rats, J. Agr. Food Chum. 26. 918 (1978). 9. L. 0. Ruzo. J. L. Engel, and J. E. Casida. Decamethrin metabolites from oxidative. hydrolyt-
11. 12. 13.
14. 15.
DELTAMETHRIN
85
ic and conjugative reactions in mice, J. Agr. Food Chrm. 27, 725 (1979). T. Shono, K. Ohsawa. and J. E. Casida, Metabolism of trans- and cis-permethrin, tram- and cis-cypermethrin. and decamethrin by microsomal enzymes, J. Agr. Food Chum. 27, 316 (1979). D. M. Sanderson, A note on glycerol formal as a solvent in toxicity testing. J. Pharm. Phurmud. 11, 150 (1959). A. J. Gray and T. A. Connors, Delayed toxicity after intravenous administration of bioresmethrin to rats. Pesfic. Sci. 11, 361 (1980). A. J. Gray, T. A. Connors. and J. Rickard, Mechanism of mammalian toxicity of the pyrethroids, irr “Neurotransmitters and Their Receptors: Proceedings of the EMBO workshop. February, 1980” (U. Z. Littauer, Y. Dudai, I. Silman. V. I. Teichberg, and Z. Vogel., Eds.), Wiley, London, 1980. A. J. Gray and J. Rickard. Toxicity of pyrethroids to rats after direct injection into the central nervous system. Submitted for publication. A. De Bruin, Metabolism of occupational agents, in “Biochemical Toxicology of Environmental Agents” (A. De Bruin, Ed.), p. 109. Elsevieri North-Holland. Amsterdam, 1976.