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Hydrolysis of permethrin, a pyrethroid insecticide, by rainbow trout and mouse tissues in vitro: A comparative study
I86-192(1981)
TOXlCOLOGYANDAPPLlEDPHARMACOLOCY60,
Hydrolysis
of Permethrin, a Pyrethroid Insecticide, by Rainbow Trout and Mouse Tissues in Vitro: A Comparative Study ANDREW
H.
AND JOHN
GLICKMAN
J.
LECH’
Department of Pharmacology and Toxicology, Medical College of Wisconsin, P.O. Box 26509, Milwaukee. Wisconsin 53226
Received November 25. 1980; accepted April 2, 1981
Hydrolysis of Permethrin, a Pyrethroid Insecticide, by Rainbow Trout and Mouse Tissues in Vitro: A Comparative Study. GLICKMAN, A. H., AND LECH, J. J. (1981). Toxicol. Appl. Pharmacol. 60, l86- 192. Permethrin, a pyrethroid insecticide, is highly toxic to fish relative to its toxicity to mammals and the role of permethrin metabolism in this differential toxicity has not been investigated. A previous study, however, has shown that little hydrolysis of permethrin occurs in vivo in rainbow trout in contrast to mammals where ester hydrolysis is a major detoxification reaction. In the present study, the rates of permethrin hydrolysis in rainbow trout and mouse tissues in vitro were estimated. Mouse liver, kidney, and plasma incubated at 37”C, hydrolyzed trans-[‘%]permethrin approximately 166, 38, and 59 times faster, respectively, than the same rainbow trout tissues incubated at 12°C. At an incubation temperature of 37°C trout liver microsomes hydrolyzed trans-permethrin approximately 45 times slower than mouse liver microsomes. When the total capacity of trout and mouse tissues to hydrolyze trans-permethrin was compared on a whole body basis mice hydrolyzed transpermethrin 184 times faster than rainbow trout. The results suggest that rainbow trout tissues have a much lower capacity than mouse tissues to hydrolyze permethrin, and this may explain the relative absence of permethrin hydrolysis products in permethrin-exposed rainbow trout. It is impossible that the high toxicity of permethrin to rainbow trout is in part related to a low capacity to hydrolyze permethrin.
Permethrin, 3-phenoxybenzyl-[ lR,S]-cis, truns- 3( 2,2 - dichlorovinyl)- 2,2 -dimethylcyclopropane carboxylate, is a highly potent, synthetic pyrethroid insecticide that possesses low mammalian toxicity, but like many other pyrethroids, is highly toxic to fish (Miyamoto, 1976). A previous study showed that, whereas the [ lR,S]-cis isomer was 5 times more potent to rainbow trout (LD50 = 22 mg/kg) than to mice (LD50 = 108 mg/kg), the [ lR,S]-trans isomer was greater than 110 times more toxic to trout (LD50 = 7 mg/kg) than to mice (LD50 > 800 mg/kg) (Glickman et al., 198 1). It is possible that this differential toxicity be’ To whom all correspondence should be sent. 0041-008X/81/110186-07$02.00/0 Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
186
tween trout and mice may be due to differences in permethrin in metabolism between the two species. Previous work in our laboratory suggested that little ester hydrolysis of cis- or truns-permethrin occurs in viva in rainbow trout, the major metabolite being the glucuronide conjugate of 4’-HO-permethrin (Glickman et al., 198 1). This is significantly different from the metabolite profiles reported in rats, cows, and hens where the great majority of permethrin metabolites were shown to be products of ester hydrolysis (Gaughan et al., 1977, 1978a,b). Hydrolysis of truns-permethrin has been shown to occur approximately 60 times faster than hydrolysis of cis-permethrin in mouse liver microsomes (Soderlund and Casida, 1977); and
PERMETHRIN
HYDROLYSIS
it has been suggested that the trans isomer of some pyrethroids may be less toxic than the respective cis isomer in mice because of the tran~ isomer’s rapid hydrolysis (Abernathy et al., 1973). The role of metabolism in the high toxicity of permethrin to rainbow trout is not known. Since ester hydrolysis may be an important means of permethrin detoxification, the present study was directed toward examining the rates of hydrolysis of the individual cis- and truns-permethrin isomers by rainbow trout and mouse tissues. Major emphasis was placed on examining the rate of rrans-permethrin hydrolysis since trout are at least 120 times more sensitive to this isomer than mice. In addition, low rates of permethrin hydrolysis by trout tissues may explain the absence of permethrin hydrolytic metabolites in vivo in trout.
METHODS Materials
[ 1R.S]-cis-Permethrin (299% purity), [ lR.S]-transpermethrin (199% purity), and 14C-labeled preparations of these isomers were donated by the FMC Corporation (Agricultural Chemical Division, Middleport, N.Y .). The site of the label on the “C-preparations was at the methylene carbon on the benzyl ring in the alcohol moiety and the specific activity of both “C-isomers was 55.9 mmol/mCi. Tests for purity of the permethrin preparations have been described previously (Glickman el al., 1981). m-Phenoxybenzyl alcohol was purchased from Aldrich Chemical Company (Milwaukee, Wise.) and 4’-HO-rrans-permethrin was a gift from the laboratory of Professor John E. Casida, University of California, Berkeley. Precoated silica gel plates with uv fluorescence indicator were purchased from Brinkmann Instrument Company (Westbury, N.Y.). Budget-solve liquid scintillation cocktail was obtained from RPI Corporation (Elk Grove Village, Ill.). Rainbow trout (Salmo gairdneri) were obtained from Silver Springs Fish Hatchery (Plymouth, Wise.) and were maintained at 12°C in charcoal filtered water with a pH of 7.3 and total hardness of 139 mg/liter measured as CaCO,. Male, ICR mice were purchased from Sprague-Dawley (Madison, Wise.).
BY TROUT Preparation
AND MICE
187
of Tissue Fractions
Rainbow trout (50-200 g) and mice (25-35 g) were killed by a blow to the head and cervical dislocation, respectively. For each experiment, the livers and kidneys were excised and the organs from at least three animals were pooled and rinsed in cold 0.15 M KCI. The tissues were then homogenized in 4 vol Tris-HCI buffer (66 mM, pH 7.4) using a Potter-Elvjhem-type glass-Teflon homogenizer. Tissue homogenates were centrifuged at 8500g X 20 min, and the supernatants were decanted and centrifuged at 165,000g X 60 min to obtain the microsomal fraction. The microsome pellets were resuspended in Tris-HCI buffer (1 ml buffer/g wet tissue). Blood was obtained by decapitation (mice) or collection at the caudal vein (trout), and was spun at 13OOg X 10 min to obtain plasma. All liver and kidney fractions and plasma were used on the day of preparation. Protein concentrations were estimated by the method of Lowry et al. (1951). Incubation
Conditions
Incubation mixtures consisted of the tissue preparation (0.3-2 mg protein, except in the time course experiment) and Tris buffer in a total volume of 2.5 ml in 25-ml Erlenmeyer flasks. Incubations were held in a Dubnoff shaking water bath, and with the exception of the temperature-dependence experiment, mouse tissue incubations were run at 37°C and rainbow trout tissue incubations were held at 22” and 12’C. The reactions were begun by adding 100 nmol [14C]permethrin (0.25 pCi) in IO ~1 dimethylformamide to the incubation flasks. Controls consisted of adding 300 nmol tetraethylpyrosphosphate (TEPP), an esterase inhibitor, in acetone to the bottom of the flasks, evaporating the acetone, and then adding the incubation contents (Soderlund and Casida, 1977). Other control experiments consisted of terminating the reaction at zero time or simply running the incubations in the absence of tissue. Little, if any, differences were observed among the different controls. Incubations were terminated at designated times with the addition of 0.5 ml 10% trichloroacetic acid and the incubates were immediately extracted three times with equal volumes of diethyl ether. Extraction efficiencies ranged from 95 to 99%. Aliquots of the ether extracts (10,000-l 5,000 cpm), along with authentic permethrin, phenoxybenzyl alcohol, and 4’-HO-tram-permethrin standards, were applied to silica gel thin-layer chromatography plates. The plates were developed twice in a solvent system of benzene saturated with formic acid:ether (l&3), a mobile phase that clearly separates hydrolytic products (phenoxybenzyl alcohol) from permethrin (Gaughan et al., 1977). Areas of silica gel were scraped from the plates corresponding to the R/‘s of the authentic standards,
188
GLICKMAN
AND LECH
which were detected under ultraviolet light, and measured for radioactivity by liquid scintillation counting. The percentage of permethrin hydrolyzed was calculated by dividing the amount of [“Clphenoxybenzyl alcohol formed by the sum of [“CJphenoxybenzyl alcohol and [“Clpermethrin on the silica gel plate and then subtracting the percentage hydrolysis that occurred in the control. When appropriate, statistical significance was evaluated by Student’s r test.
RESULTS Figure 1 is a composite thin-layer chromatogram which illustrates the stoichiometry of the permethrin hydrolysis reaction and the basis of the permethrin hydrolysis assay.
4 _ (+TEFP)
3-
RAINBOW 6
lo '2 f
TROUT
(+TEPW
6 4 2
.
r
6 I-- (-TEPP)
‘;4=LJ-J ,
2 3 4
5 6
7 8
CM FROM
I
9 IO II 12 I3 I4 I5 I6
ORIGIN
1. Thin-layer radiochromatograms of ether extracts from incubations of mouse and trout liver microsomes and rrans[“C]permethrin. Incubations containing tetraethylpyrophosphate (TEPP), an esterase inhibitor, served as controls. Striped and solid spots represent the mobility of authentic standards of m-phenoxybenzyl alcohol and trans-permethrin, respectively. The asterisks indicate the site of the “‘C label on the parent compound (tram-permethrin) and hydrolytic metabolite (m-phenoxybenzyl alcohol). FIG.
FIG. 2. Time course of cis-[‘4C]and rruns[‘4C]permethtin hydrolysis by rainbow trout (22’C) and mouse (37“C) liver microsomes. Microsomal protein content was approximately 20 mg for both trout and mouse incubations. Each point is the mean of at least four incubations in two separate experiments.
In incubations containing mouse or trout tissue preparations and [ 14C]permethrin only m[ “C]phenoxybenzyl alcohol was produced in measurable amounts. Incubations containing tetraethylpyrophosphate, an inhibitor of the permethrin-hydrolyzing esterase, served as controls. An initial experiment on the time course of cis- and trans-permethrin hydrolysis by trout and mouse liver microsomes suggested that trout liver microsomes hydrolyzed both tram and cis-permethrin considerably slower than mouse liver microsomes (Fig. 2). Within 15 min over 70% of trans-permethrin was hydrolyzed by mouse liver microsomes at 37°C while less than 10% of trans-permethrin was hydrolyzed by trout liver microsomes at 22°C. Hydrolysis of cis-permethrin was relative slow in mouse liver preparations with 23 + 4% of the cis-permethrin hydrolyzed over 4 hr. No more than 4% of the cis isomer was hydrolyzed by the rainbow trout liver microsomes in 4 hr (p < 0.05, when compared to hydrolysis in mouse microsomes). Data not shown here indicated that the cis isomer was also slowly hydrolyzed by both mouse and trout plasma, with the hydrolytic activity being higher in the mouse plasma. Figure 3 illustrates that trans-permethrin
PERMETHRIN
HYDROLYSIS
189
BY TROUT AND MICE
4
10
FIG. 3. Effect of protein concentration on the rate of trans-[‘%]permethrin hydrolysis by rainbow trout (22°C) and mouse (37°C) liver microsomes and plasma. Each point represents the mean of two to six incubations.
hydrolysis by trout and mouse liver microsomes and plasma was protein dependent. The rate of trans-permethrin hydrolysis appeared linear out to 1 mg protein in the mouse incubations while hydrolysis was linear out to 2 mg protein with the trout preparations. An increase in incubation temperature, as shown in Fig. 4, caused an increase in the rate of trans-permethrin hydrolysis by trout and mouse liver microsomes; however,
37-C
FIG. 4. Effect of incubation temperature on the rate of rrans-[‘4C]permethrin hydrolysis by rainbow trout and mouse liver microsomes. Each bar is the mean k SE of four incubations in two separate experiments.
there was a 30- to 40-fold difference in the rate of hydrolysis between trout and mouse at all three temperatures. Although a complete subcellular fractionation of hydrolytic activity in trout and mouse liver was not conducted, the data in Table 1 suggest that a large proportion of permethrin hydrolytic activity in mouse liver resided in the microsomal fraction (approximately 70% of total activity in whole liver homogenate). It was difficult to determine which trout liver fraction contained the highest esterase activity since the hydrolysis rates were so slow. Hydrolytic activity in mouse kidney microsomes was approximately onehalf that in mouse liver microsomes and was near the limit of detectability in trout kidney microsomes. Rainbow trout tissue incubations were performed at 22°C in addition to 12°C in order to accurately determine hydrolytic activity, since activity was very low and difficult to measure at 12°C. A considerable amount of trans-permethrin hydrolytic activity was observed in mouse plasma and the activity in trout plasma seemed greater than that in trout liver microsomes (p < O.Ol), though it was still much lower than levels found in mouse plasma.
190
GLICKMAN
AND LECH
TABLE
1
rr~fl~-PERMETHRIN HYDROLYSIS BY RAINBOW TROUT’ AND MOUSER LIVER AND KIDNEY FRACTIONS AND PLASMA trans-permethrin
Trout 12°C 22°C Mouse
nmol hydrolyzed/min/mg
protein
8500g Liver supernatant
165,OOOg Liver supernatant
Liver microsomes
Kidney microsomes
Plasma
0.007 t 0.001’ 0.032 t- 0.005 1.80 +- 0.11
0.006 k 0.001 0.017 + 0.001 0.22 + 0.01
0.013 f 0.001 0.061 + 0.010 5.29 f 0.48
0.002 * 0.000 0.005 + 0.000 2.01 + 0.27
0.050 f 0.004 0.132 1. 0.003 1.88 f 0.13
’ Rainbow trout tissue incubations were at 12 and 22°C for 120 min. b Mouse tissue incubations were at 37’C for 15 min. ’ Values represent the mean f SEM of at least four incubations in two separate experiments.
In Table 2 trans-permethrin hydrolytic activity is expressed in nanomoles hydrolyzed per gram or milliliter of tissue. These data were derived by multiplying the activity determined per milligram of protein by the tissue protein concentration (mg protein/g or ml). From these data, the nanomoles of truns-permethrin hydrolyzed per minute per kilogram of body weight was calculated and the tissue distribution of trans-permethrin TABLE
hydrolytic activity was examined. The mean percentage body weight values were obtained by weighing the organs upon sacrifice of the animals with the exception of plasma values which were obtained from published sources (Sassan et al., 1968; Conte et al., 1963). Note that the mouse liver was 5 times larger than the liver in rainbow trout when the organ weights were expressed as percentage body weight. When the mouse and 2
COMPARISON OF IrUnS-PERMETHRIN HYDROLYTIC ACTIVITY IN RAINBOW TROUT AND MICE IN VITRO’
nmol hydrolized/min/g ml tissue
or Percentage body weight
nmol hydrolized/min/kg body wt
Species
Live?
Kidney’
Plasma
Liver
Kidney
Plasma
Liver
Kidney
Plasma
Mouse Trout Mouse/ trout
116 0.7
15 0.4
118 2.0
5.0 1.0
1.5 0.7
3.W 2.P
5800 I
225 2.8
3540 42
166
38
5
2.1
1.4
829
80.4
59
’ Tissue incubations were at 12 and 37°C for trout and mice, respectively. b Values based on activity in 85OOg liver supernatant. ’ Values based on activity in kidney microsomal fraction. d Total body activity based on liver, kidney, and plasma activity. ’ Value obtained from Sassen et al. (1968). ‘Value obtained from Conte et ol. (1963).
84.3
Total transpermethrin hydrolytic activityd (nmol/min/kg MY w 9565 51.8 184
PERMETHRIN
HYDROLYSIS
trout tissue hydrolytic rates were expressed as a function of body weight, mouse liver and plasma, incubated at 37”C, had approximately an 830-fold and 80-fold greater capacity to hydrolyze trans-permethrin, respectively, than rainbow trout liver and plasma incubated at 12°C. Activity in muscle homogenates was low in both species, 0.2 and co.05 nmol truns-permethrin hydrolyzed/min/g tissue in mice and trout, respectively. In the last column of Table 2 the sum of the trans-permethrin hydrolytic activity in liver, plasma, and kidney is presented. Since it is likely that the great majority of trans-permethrin hydrolysis occurs in these three tissues, the capacity of the mouse to hydrolyze permethrin is substantially greater than the trout when expressed on a body weight basis. DISCUSSION The present study clearly indicates that various rainbow trout tissues have a considerably lower capacity to hydrolyze cis- and truns-permethrin than comparable mouse tissues. Both liver microsomal and plasma esterases from trout and mice were capable of hydrolyzing trans-permethrin, though the reaction was quite slow with trout tissues. The rate of truns-permethrin hydrolysis by mouse microsomes was similar to that found in an earlier study (Soderlund and Casida, 1977). Permethrin hydrolytic activity in mouse plasma was high and plasma appeared to contain a large proportion of the total hydrolytic activity of the mouse. The protein dependency curves of trunspermethrin hydrolysis (Fig. 2) by mouse and rainbow trout tissues were close to linear at relatively low protein concentrations ((2.0 mg protein). For this reason, 0.3-2.0 mg tissue protein were used in trout incubations and tl mg protein in mouse incubations to estimate the rate of permethrin hydrolysis. Permethrin hydrolysis rates appeared to plateau as protein exceeded 2 mg and it is pos-
BY TROUT
AND
MICE
191
sible that the deviation from linearity was due to depletion of substrate, end product inhibition, or increased nonspecific binding of permethrin to the tissue suspensions, rendering the compound inaccessible to the enzyme. Mouse incubations were run for only 15 min since, as shown in Fig. 2, the reaction was no longer linear with time beyond 30 min. In contrast to the situation with mouse tissues, trout tissue incubations had to be run for up to 2 hr to observe measurable hydrolysis. Additional experiments indicated that trout tissue hydrolysis of [14C]permethrin was linear to 2 hr and that the [ 14C]permethrin substrate was soluble in the trout tissue incubations at both 12 and 22°C. An increase in incubation temperature from 12 to 37°C caused an increase in the rate of truns-permethrin hydrolysis by liver microsomes in both species. It should be recognized that the holding temperature of the rainbow trout used in this study was 12°C and therefore the assays run at this temperature may best reflect the physiological capacity of these fish to hydrolyze truns-permethrin. Although trout tissue incubations run at temperatures higher than 12’C exhibited higher rates of hydrolysis these rates cannot be used directly to reflect the in uivo capacity of trout acclimated to higher temperatures to hydrolyze permethrin. The low permethrin hydrolytic activity in rainbow trout tissues could very well explain the relative absence of permethrin hydrolytic metabolites found in vivo in rainbow trout exposed to either [lR,S]-cisor [l&S]truns-permethrin (Glickman et al., 198 1). Whether the large difference in the rate of hydrolysis of truns-permethrin between rainbow trout and mouse can explain the 1 IOfold greater toxicity of trans-permethrin in rainbow trout cannot be adequately answered with the present data. Studies concerning the quantitative aspects of oxidative and hydrolytic pathways of permethrin metabolism and the effect of esterase and monooxygenase inhibition on permethrin acute toxicity to rainbow trout and mice in
192
GLICKMAN
vivo ultimately may provide an understanding of the role of metabolism in the high toxicity of permethrin to rainbow trout and possibly other fishes.
ACKNOWLEDGMENTS This research was supported by NIH Grant ES01080, NIH Aquatic Biomedical Research Center ES01985, and NIH Toxicology Training Grant ES07043.
REFERENCES ABERNATHY, C. O., UEDA, K., ENGEL, J. L., GAUGHAN, L. C., AND CASIDA, J. E. (1973). Substrate specificity and toxicological significance of pyrethroid-hydrolyzing esterasesof mouse liver microsomes. Pest. B&hem. Physiol. 3, 300-3 11. CONTE, F. P., WAGNER, H. H., AND HARRIS, T. 0. (1963). Measurement of blood volume in the fish (Salmo
gairdneri
gairdneri).
Amer.
J. Physiol.
205,
533-540. GAUGHAN, L. C., UNAI, T., ANDCASIDA, J. E. (1977). Permethrin metabolism in rats. J. Agri. Food Chem. 25, 9-17.
AND LECH GAUGHAN, L. C., ACKERMAN, M. E., UNAI, T., AND CASIDA, J. E. (1978a). Distribution and metabolism of tram- and cis-permethrin in lactating Jersey cows. J. Agri.
Food
Chem.
26, 613-618.
GAUGHAN, L. C., ROBINSON, R. A., AND CASIDA, J. E. (1978b). Distribution and metabolic fate of transand cis-permethrin in laying hens. J. Agri. Food Chem. 26, 1374-1380. GLICKMAN,A. H., HAMID, A. R., RICKERT, D. E., AND LECH, J. J. (1981). Elimination and metabolism of permethrin isomers in rainbow trout. Toxicol. Appl. Pharmacol.
57, 88-98.
LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. MIYAMOTO, J. (1976). Degradation, metabolism and toxicity of synthetic pyrethroids. Environ. Health Perspect.
14, 15-28.
SASSEN, A., REUTER, A. M., AND KENNES, F. (1968). Determination of plasma volume in the mouse with screened iodine-labeled proteins. Experienria 24,12031204. SODERLUND, D. M., AND CASIDA, J. E. (1977). Effects of pyrethroid structure on rates of hydrolysis and oxidation by mouse liver microsomal enzymes. Pesf. Biochem.
Physiol.
7, 39 I-40
1.
TOXlCOLOGYANDAPPLlEDPHARMACOLOCY60,
Hydrolysis
of Permethrin, a Pyrethroid Insecticide, by Rainbow Trout and Mouse Tissues in Vitro: A Comparative Study ANDREW
H.
AND JOHN
GLICKMAN
J.
LECH’
Department of Pharmacology and Toxicology, Medical College of Wisconsin, P.O. Box 26509, Milwaukee. Wisconsin 53226
Received November 25. 1980; accepted April 2, 1981
Hydrolysis of Permethrin, a Pyrethroid Insecticide, by Rainbow Trout and Mouse Tissues in Vitro: A Comparative Study. GLICKMAN, A. H., AND LECH, J. J. (1981). Toxicol. Appl. Pharmacol. 60, l86- 192. Permethrin, a pyrethroid insecticide, is highly toxic to fish relative to its toxicity to mammals and the role of permethrin metabolism in this differential toxicity has not been investigated. A previous study, however, has shown that little hydrolysis of permethrin occurs in vivo in rainbow trout in contrast to mammals where ester hydrolysis is a major detoxification reaction. In the present study, the rates of permethrin hydrolysis in rainbow trout and mouse tissues in vitro were estimated. Mouse liver, kidney, and plasma incubated at 37”C, hydrolyzed trans-[‘%]permethrin approximately 166, 38, and 59 times faster, respectively, than the same rainbow trout tissues incubated at 12°C. At an incubation temperature of 37°C trout liver microsomes hydrolyzed trans-permethrin approximately 45 times slower than mouse liver microsomes. When the total capacity of trout and mouse tissues to hydrolyze trans-permethrin was compared on a whole body basis mice hydrolyzed transpermethrin 184 times faster than rainbow trout. The results suggest that rainbow trout tissues have a much lower capacity than mouse tissues to hydrolyze permethrin, and this may explain the relative absence of permethrin hydrolysis products in permethrin-exposed rainbow trout. It is impossible that the high toxicity of permethrin to rainbow trout is in part related to a low capacity to hydrolyze permethrin.
Permethrin, 3-phenoxybenzyl-[ lR,S]-cis, truns- 3( 2,2 - dichlorovinyl)- 2,2 -dimethylcyclopropane carboxylate, is a highly potent, synthetic pyrethroid insecticide that possesses low mammalian toxicity, but like many other pyrethroids, is highly toxic to fish (Miyamoto, 1976). A previous study showed that, whereas the [ lR,S]-cis isomer was 5 times more potent to rainbow trout (LD50 = 22 mg/kg) than to mice (LD50 = 108 mg/kg), the [ lR,S]-trans isomer was greater than 110 times more toxic to trout (LD50 = 7 mg/kg) than to mice (LD50 > 800 mg/kg) (Glickman et al., 198 1). It is possible that this differential toxicity be’ To whom all correspondence should be sent. 0041-008X/81/110186-07$02.00/0 Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
186
tween trout and mice may be due to differences in permethrin in metabolism between the two species. Previous work in our laboratory suggested that little ester hydrolysis of cis- or truns-permethrin occurs in viva in rainbow trout, the major metabolite being the glucuronide conjugate of 4’-HO-permethrin (Glickman et al., 198 1). This is significantly different from the metabolite profiles reported in rats, cows, and hens where the great majority of permethrin metabolites were shown to be products of ester hydrolysis (Gaughan et al., 1977, 1978a,b). Hydrolysis of truns-permethrin has been shown to occur approximately 60 times faster than hydrolysis of cis-permethrin in mouse liver microsomes (Soderlund and Casida, 1977); and
PERMETHRIN
HYDROLYSIS
it has been suggested that the trans isomer of some pyrethroids may be less toxic than the respective cis isomer in mice because of the tran~ isomer’s rapid hydrolysis (Abernathy et al., 1973). The role of metabolism in the high toxicity of permethrin to rainbow trout is not known. Since ester hydrolysis may be an important means of permethrin detoxification, the present study was directed toward examining the rates of hydrolysis of the individual cis- and truns-permethrin isomers by rainbow trout and mouse tissues. Major emphasis was placed on examining the rate of rrans-permethrin hydrolysis since trout are at least 120 times more sensitive to this isomer than mice. In addition, low rates of permethrin hydrolysis by trout tissues may explain the absence of permethrin hydrolytic metabolites in vivo in trout.
METHODS Materials
[ 1R.S]-cis-Permethrin (299% purity), [ lR.S]-transpermethrin (199% purity), and 14C-labeled preparations of these isomers were donated by the FMC Corporation (Agricultural Chemical Division, Middleport, N.Y .). The site of the label on the “C-preparations was at the methylene carbon on the benzyl ring in the alcohol moiety and the specific activity of both “C-isomers was 55.9 mmol/mCi. Tests for purity of the permethrin preparations have been described previously (Glickman el al., 1981). m-Phenoxybenzyl alcohol was purchased from Aldrich Chemical Company (Milwaukee, Wise.) and 4’-HO-rrans-permethrin was a gift from the laboratory of Professor John E. Casida, University of California, Berkeley. Precoated silica gel plates with uv fluorescence indicator were purchased from Brinkmann Instrument Company (Westbury, N.Y.). Budget-solve liquid scintillation cocktail was obtained from RPI Corporation (Elk Grove Village, Ill.). Rainbow trout (Salmo gairdneri) were obtained from Silver Springs Fish Hatchery (Plymouth, Wise.) and were maintained at 12°C in charcoal filtered water with a pH of 7.3 and total hardness of 139 mg/liter measured as CaCO,. Male, ICR mice were purchased from Sprague-Dawley (Madison, Wise.).
BY TROUT Preparation
AND MICE
187
of Tissue Fractions
Rainbow trout (50-200 g) and mice (25-35 g) were killed by a blow to the head and cervical dislocation, respectively. For each experiment, the livers and kidneys were excised and the organs from at least three animals were pooled and rinsed in cold 0.15 M KCI. The tissues were then homogenized in 4 vol Tris-HCI buffer (66 mM, pH 7.4) using a Potter-Elvjhem-type glass-Teflon homogenizer. Tissue homogenates were centrifuged at 8500g X 20 min, and the supernatants were decanted and centrifuged at 165,000g X 60 min to obtain the microsomal fraction. The microsome pellets were resuspended in Tris-HCI buffer (1 ml buffer/g wet tissue). Blood was obtained by decapitation (mice) or collection at the caudal vein (trout), and was spun at 13OOg X 10 min to obtain plasma. All liver and kidney fractions and plasma were used on the day of preparation. Protein concentrations were estimated by the method of Lowry et al. (1951). Incubation
Conditions
Incubation mixtures consisted of the tissue preparation (0.3-2 mg protein, except in the time course experiment) and Tris buffer in a total volume of 2.5 ml in 25-ml Erlenmeyer flasks. Incubations were held in a Dubnoff shaking water bath, and with the exception of the temperature-dependence experiment, mouse tissue incubations were run at 37°C and rainbow trout tissue incubations were held at 22” and 12’C. The reactions were begun by adding 100 nmol [14C]permethrin (0.25 pCi) in IO ~1 dimethylformamide to the incubation flasks. Controls consisted of adding 300 nmol tetraethylpyrosphosphate (TEPP), an esterase inhibitor, in acetone to the bottom of the flasks, evaporating the acetone, and then adding the incubation contents (Soderlund and Casida, 1977). Other control experiments consisted of terminating the reaction at zero time or simply running the incubations in the absence of tissue. Little, if any, differences were observed among the different controls. Incubations were terminated at designated times with the addition of 0.5 ml 10% trichloroacetic acid and the incubates were immediately extracted three times with equal volumes of diethyl ether. Extraction efficiencies ranged from 95 to 99%. Aliquots of the ether extracts (10,000-l 5,000 cpm), along with authentic permethrin, phenoxybenzyl alcohol, and 4’-HO-tram-permethrin standards, were applied to silica gel thin-layer chromatography plates. The plates were developed twice in a solvent system of benzene saturated with formic acid:ether (l&3), a mobile phase that clearly separates hydrolytic products (phenoxybenzyl alcohol) from permethrin (Gaughan et al., 1977). Areas of silica gel were scraped from the plates corresponding to the R/‘s of the authentic standards,
188
GLICKMAN
AND LECH
which were detected under ultraviolet light, and measured for radioactivity by liquid scintillation counting. The percentage of permethrin hydrolyzed was calculated by dividing the amount of [“Clphenoxybenzyl alcohol formed by the sum of [“CJphenoxybenzyl alcohol and [“Clpermethrin on the silica gel plate and then subtracting the percentage hydrolysis that occurred in the control. When appropriate, statistical significance was evaluated by Student’s r test.
RESULTS Figure 1 is a composite thin-layer chromatogram which illustrates the stoichiometry of the permethrin hydrolysis reaction and the basis of the permethrin hydrolysis assay.
4 _ (+TEFP)
3-
RAINBOW 6
lo '2 f
TROUT
(+TEPW
6 4 2
.
r
6 I-- (-TEPP)
‘;4=LJ-J ,
2 3 4
5 6
7 8
CM FROM
I
9 IO II 12 I3 I4 I5 I6
ORIGIN
1. Thin-layer radiochromatograms of ether extracts from incubations of mouse and trout liver microsomes and rrans[“C]permethrin. Incubations containing tetraethylpyrophosphate (TEPP), an esterase inhibitor, served as controls. Striped and solid spots represent the mobility of authentic standards of m-phenoxybenzyl alcohol and trans-permethrin, respectively. The asterisks indicate the site of the “‘C label on the parent compound (tram-permethrin) and hydrolytic metabolite (m-phenoxybenzyl alcohol). FIG.
FIG. 2. Time course of cis-[‘4C]and rruns[‘4C]permethtin hydrolysis by rainbow trout (22’C) and mouse (37“C) liver microsomes. Microsomal protein content was approximately 20 mg for both trout and mouse incubations. Each point is the mean of at least four incubations in two separate experiments.
In incubations containing mouse or trout tissue preparations and [ 14C]permethrin only m[ “C]phenoxybenzyl alcohol was produced in measurable amounts. Incubations containing tetraethylpyrophosphate, an inhibitor of the permethrin-hydrolyzing esterase, served as controls. An initial experiment on the time course of cis- and trans-permethrin hydrolysis by trout and mouse liver microsomes suggested that trout liver microsomes hydrolyzed both tram and cis-permethrin considerably slower than mouse liver microsomes (Fig. 2). Within 15 min over 70% of trans-permethrin was hydrolyzed by mouse liver microsomes at 37°C while less than 10% of trans-permethrin was hydrolyzed by trout liver microsomes at 22°C. Hydrolysis of cis-permethrin was relative slow in mouse liver preparations with 23 + 4% of the cis-permethrin hydrolyzed over 4 hr. No more than 4% of the cis isomer was hydrolyzed by the rainbow trout liver microsomes in 4 hr (p < 0.05, when compared to hydrolysis in mouse microsomes). Data not shown here indicated that the cis isomer was also slowly hydrolyzed by both mouse and trout plasma, with the hydrolytic activity being higher in the mouse plasma. Figure 3 illustrates that trans-permethrin
PERMETHRIN
HYDROLYSIS
189
BY TROUT AND MICE
4
10
FIG. 3. Effect of protein concentration on the rate of trans-[‘%]permethrin hydrolysis by rainbow trout (22°C) and mouse (37°C) liver microsomes and plasma. Each point represents the mean of two to six incubations.
hydrolysis by trout and mouse liver microsomes and plasma was protein dependent. The rate of trans-permethrin hydrolysis appeared linear out to 1 mg protein in the mouse incubations while hydrolysis was linear out to 2 mg protein with the trout preparations. An increase in incubation temperature, as shown in Fig. 4, caused an increase in the rate of trans-permethrin hydrolysis by trout and mouse liver microsomes; however,
37-C
FIG. 4. Effect of incubation temperature on the rate of rrans-[‘4C]permethrin hydrolysis by rainbow trout and mouse liver microsomes. Each bar is the mean k SE of four incubations in two separate experiments.
there was a 30- to 40-fold difference in the rate of hydrolysis between trout and mouse at all three temperatures. Although a complete subcellular fractionation of hydrolytic activity in trout and mouse liver was not conducted, the data in Table 1 suggest that a large proportion of permethrin hydrolytic activity in mouse liver resided in the microsomal fraction (approximately 70% of total activity in whole liver homogenate). It was difficult to determine which trout liver fraction contained the highest esterase activity since the hydrolysis rates were so slow. Hydrolytic activity in mouse kidney microsomes was approximately onehalf that in mouse liver microsomes and was near the limit of detectability in trout kidney microsomes. Rainbow trout tissue incubations were performed at 22°C in addition to 12°C in order to accurately determine hydrolytic activity, since activity was very low and difficult to measure at 12°C. A considerable amount of trans-permethrin hydrolytic activity was observed in mouse plasma and the activity in trout plasma seemed greater than that in trout liver microsomes (p < O.Ol), though it was still much lower than levels found in mouse plasma.
190
GLICKMAN
AND LECH
TABLE
1
rr~fl~-PERMETHRIN HYDROLYSIS BY RAINBOW TROUT’ AND MOUSER LIVER AND KIDNEY FRACTIONS AND PLASMA trans-permethrin
Trout 12°C 22°C Mouse
nmol hydrolyzed/min/mg
protein
8500g Liver supernatant
165,OOOg Liver supernatant
Liver microsomes
Kidney microsomes
Plasma
0.007 t 0.001’ 0.032 t- 0.005 1.80 +- 0.11
0.006 k 0.001 0.017 + 0.001 0.22 + 0.01
0.013 f 0.001 0.061 + 0.010 5.29 f 0.48
0.002 * 0.000 0.005 + 0.000 2.01 + 0.27
0.050 f 0.004 0.132 1. 0.003 1.88 f 0.13
’ Rainbow trout tissue incubations were at 12 and 22°C for 120 min. b Mouse tissue incubations were at 37’C for 15 min. ’ Values represent the mean f SEM of at least four incubations in two separate experiments.
In Table 2 trans-permethrin hydrolytic activity is expressed in nanomoles hydrolyzed per gram or milliliter of tissue. These data were derived by multiplying the activity determined per milligram of protein by the tissue protein concentration (mg protein/g or ml). From these data, the nanomoles of truns-permethrin hydrolyzed per minute per kilogram of body weight was calculated and the tissue distribution of trans-permethrin TABLE
hydrolytic activity was examined. The mean percentage body weight values were obtained by weighing the organs upon sacrifice of the animals with the exception of plasma values which were obtained from published sources (Sassan et al., 1968; Conte et al., 1963). Note that the mouse liver was 5 times larger than the liver in rainbow trout when the organ weights were expressed as percentage body weight. When the mouse and 2
COMPARISON OF IrUnS-PERMETHRIN HYDROLYTIC ACTIVITY IN RAINBOW TROUT AND MICE IN VITRO’
nmol hydrolized/min/g ml tissue
or Percentage body weight
nmol hydrolized/min/kg body wt
Species
Live?
Kidney’
Plasma
Liver
Kidney
Plasma
Liver
Kidney
Plasma
Mouse Trout Mouse/ trout
116 0.7
15 0.4
118 2.0
5.0 1.0
1.5 0.7
3.W 2.P
5800 I
225 2.8
3540 42
166
38
5
2.1
1.4
829
80.4
59
’ Tissue incubations were at 12 and 37°C for trout and mice, respectively. b Values based on activity in 85OOg liver supernatant. ’ Values based on activity in kidney microsomal fraction. d Total body activity based on liver, kidney, and plasma activity. ’ Value obtained from Sassen et al. (1968). ‘Value obtained from Conte et ol. (1963).
84.3
Total transpermethrin hydrolytic activityd (nmol/min/kg MY w 9565 51.8 184
PERMETHRIN
HYDROLYSIS
trout tissue hydrolytic rates were expressed as a function of body weight, mouse liver and plasma, incubated at 37”C, had approximately an 830-fold and 80-fold greater capacity to hydrolyze trans-permethrin, respectively, than rainbow trout liver and plasma incubated at 12°C. Activity in muscle homogenates was low in both species, 0.2 and co.05 nmol truns-permethrin hydrolyzed/min/g tissue in mice and trout, respectively. In the last column of Table 2 the sum of the trans-permethrin hydrolytic activity in liver, plasma, and kidney is presented. Since it is likely that the great majority of trans-permethrin hydrolysis occurs in these three tissues, the capacity of the mouse to hydrolyze permethrin is substantially greater than the trout when expressed on a body weight basis. DISCUSSION The present study clearly indicates that various rainbow trout tissues have a considerably lower capacity to hydrolyze cis- and truns-permethrin than comparable mouse tissues. Both liver microsomal and plasma esterases from trout and mice were capable of hydrolyzing trans-permethrin, though the reaction was quite slow with trout tissues. The rate of truns-permethrin hydrolysis by mouse microsomes was similar to that found in an earlier study (Soderlund and Casida, 1977). Permethrin hydrolytic activity in mouse plasma was high and plasma appeared to contain a large proportion of the total hydrolytic activity of the mouse. The protein dependency curves of trunspermethrin hydrolysis (Fig. 2) by mouse and rainbow trout tissues were close to linear at relatively low protein concentrations ((2.0 mg protein). For this reason, 0.3-2.0 mg tissue protein were used in trout incubations and tl mg protein in mouse incubations to estimate the rate of permethrin hydrolysis. Permethrin hydrolysis rates appeared to plateau as protein exceeded 2 mg and it is pos-
BY TROUT
AND
MICE
191
sible that the deviation from linearity was due to depletion of substrate, end product inhibition, or increased nonspecific binding of permethrin to the tissue suspensions, rendering the compound inaccessible to the enzyme. Mouse incubations were run for only 15 min since, as shown in Fig. 2, the reaction was no longer linear with time beyond 30 min. In contrast to the situation with mouse tissues, trout tissue incubations had to be run for up to 2 hr to observe measurable hydrolysis. Additional experiments indicated that trout tissue hydrolysis of [14C]permethrin was linear to 2 hr and that the [ 14C]permethrin substrate was soluble in the trout tissue incubations at both 12 and 22°C. An increase in incubation temperature from 12 to 37°C caused an increase in the rate of truns-permethrin hydrolysis by liver microsomes in both species. It should be recognized that the holding temperature of the rainbow trout used in this study was 12°C and therefore the assays run at this temperature may best reflect the physiological capacity of these fish to hydrolyze truns-permethrin. Although trout tissue incubations run at temperatures higher than 12’C exhibited higher rates of hydrolysis these rates cannot be used directly to reflect the in uivo capacity of trout acclimated to higher temperatures to hydrolyze permethrin. The low permethrin hydrolytic activity in rainbow trout tissues could very well explain the relative absence of permethrin hydrolytic metabolites found in vivo in rainbow trout exposed to either [lR,S]-cisor [l&S]truns-permethrin (Glickman et al., 198 1). Whether the large difference in the rate of hydrolysis of truns-permethrin between rainbow trout and mouse can explain the 1 IOfold greater toxicity of trans-permethrin in rainbow trout cannot be adequately answered with the present data. Studies concerning the quantitative aspects of oxidative and hydrolytic pathways of permethrin metabolism and the effect of esterase and monooxygenase inhibition on permethrin acute toxicity to rainbow trout and mice in
192
GLICKMAN
vivo ultimately may provide an understanding of the role of metabolism in the high toxicity of permethrin to rainbow trout and possibly other fishes.
ACKNOWLEDGMENTS This research was supported by NIH Grant ES01080, NIH Aquatic Biomedical Research Center ES01985, and NIH Toxicology Training Grant ES07043.
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gairdneri
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533-540. GAUGHAN, L. C., UNAI, T., ANDCASIDA, J. E. (1977). Permethrin metabolism in rats. J. Agri. Food Chem. 25, 9-17.
AND LECH GAUGHAN, L. C., ACKERMAN, M. E., UNAI, T., AND CASIDA, J. E. (1978a). Distribution and metabolism of tram- and cis-permethrin in lactating Jersey cows. J. Agri.
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GAUGHAN, L. C., ROBINSON, R. A., AND CASIDA, J. E. (1978b). Distribution and metabolic fate of transand cis-permethrin in laying hens. J. Agri. Food Chem. 26, 1374-1380. GLICKMAN,A. H., HAMID, A. R., RICKERT, D. E., AND LECH, J. J. (1981). Elimination and metabolism of permethrin isomers in rainbow trout. Toxicol. Appl. Pharmacol.
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LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. MIYAMOTO, J. (1976). Degradation, metabolism and toxicity of synthetic pyrethroids. Environ. Health Perspect.
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SASSEN, A., REUTER, A. M., AND KENNES, F. (1968). Determination of plasma volume in the mouse with screened iodine-labeled proteins. Experienria 24,12031204. SODERLUND, D. M., AND CASIDA, J. E. (1977). Effects of pyrethroid structure on rates of hydrolysis and oxidation by mouse liver microsomal enzymes. Pesf. Biochem.
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