316
J. Agric. Food Chem., Vol. 27, No. 2, 1979
Shono, Ohsawa, Casida
Metabolism of trans- and cis-Permethrin, trans- and cis-Cypermethrin, and Decamethrin by Microsomal Enzymes Toshio Shono,’ Kanju Ohsawa, and John E. Casida*
Microsomal systems are used to compare the metabolism of trans- and cis-permethrin by mouse liver, rat liver, and housefly and cabbage looper preparations and of trans- and cis-cypermethrin and decamethrin by mouse liver. Esteratic cleavage is more extensive for trans pyrethroids than for cis pyrethroids, while the relative extent of oxidative metabolism of the two isomers depends on the enzyme source. Species and isomer differences are noted in the preferred sites of permethrin hydroxylation, i.e., trans or cis methyl group and 2’, 4’, or 6 position of the alcohol moiety. With the cyanopyrethroids, mouse microsomes hydroxylate the 5 position in addition to the 4‘, trans methyl, and cis methyl sites. Several hydroxymethyl derivatives are further oxidized to the corresponding aldehydes and carboxylic acids. Four isomers of 2-carboxy-3-(2,2-dichlorovinyl)-2-methylcyclopropanecarboxylic acid are described. Thirteen to twenty-one metabolites of each pyrethroid are identified in the mouse microsomal systems.
,
Pyrethroid metabolites in microsomal esterase and oxidase systems are usually the same as those detected in vivo, except for conjugate formation (Casida et al., 1975/76; Miyamoto, 1976). These pyrethroid-hydrolyzing esterases are readily inhibited by tetraethyl pyrophosphate (TEPP) and the oxidases are only active when fortified with NADPH. This allows independent examination of products formed by esterase action (normal microsomes), oxidase action (TEPP-treated microsomes with NADPH), and esterase-plus-oxidase action (normal microsomes with NADPH) (Soderlund and Casida, 1977a,b,c; Ueda et al., 1975). Microsomal studies are therefore a useful adjunct to organismal investigations in understanding pyrethroid structure-biodegradability relationships and the sites most susceptible to metabolic attack. Several chrysanthemates (e.g., allethrin, dimethrin, pyrethrin I, resmethrin, and tetramethrin) are hydroxylated at a methyl group in the isobutenyl substituent by liver and housefly oxidases (Elliott et al., 1972; Suzuki and Miyamoto, 1974; Ueda et al., 1975; Yamamoto and Casida, 1966; Yamamoto et al., 1969). The oxidatively susceptible isobutenyl moiety is replaced by a dihalovinyl group in several highly potent pyrethroids (Elliott, 1977; Itaya et al., 1977, Ruzo andd Casida, 1977), including trans- and cis-permethrin without a cyano group and trans- and cis-cypermethrin and decamethrin with a cyano group (Figure 1). It is therefore of interest to define the sites of microsomal metabolism in this series of structurally related dihalovinyl pyrethroids. This report and a preliminary communication on this study (Shono and Casida, 1978) consider the metabolism of trans- and cis-permethrin in microsomal systems from mouse and rat liver, housefly (Musca domestica L.) thoraces plus abdomens, and cabbage looper (Trichoplusia ni Hubner) guts. It also uses the mouse liver microsomal system to compare the metabolism of trans- and cispermethrin, trans- and cis-cypermethrin, and decamethrin. MATERIALS AND METHODS
Spectroscopy. Infrared (IR) spectra reported in cm-l Pesticide Chemistry and Toxicology Laboratory, Department of Entomological Sciences, University of California, Berkeley, California 94720. l 0 n leave from the Laboratory of Applied Entomology, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan. 0021-856117911427-0316$0 1.OOlO
were determined on a Perkin-Elmer Model 457 grating spectrophotometer using chloroform solutions. Nuclear magnetic resonance (NMR) spectra were recorded for chloroform-d solutions, using tetramethylsilane as the internal standard (6 = 0 ppm), with a Perkin-Elmer Model R32 spectrometer at 90 MHz. Chemical ionization-mass spectra (CI-MS) were obtained on the Finnigan Corp. Model 1015D mass spectrometer with the System Industries Model 150 control system, using a direct introduction probe and isobutane as the reactant gas at a source pressure of 0.6-1.0 torr. The quasimolecular ion, [M + 1]+, is reported as m/e (relative intensity) based on the chlorine-35 isotope. Thin-Layer Chromatography (TLC). Silica gel 60 F-254 chromatoplates (EM Laboratories Inc., Elmsford, NY) with 0.25- and 0.50-mm gel thickness for analysis and preparative isolations, respectively, were used with the following solvent systems: BC, benzene-carbon tetrachloride (1:l);BE, benzene-ethyl acetate (6:l); BE’, two developments with benzene-ethyl acetate (12:l); BEM, benzene-ethyl acetate-methanol (15:5:1); BFE, benzene (saturated with formic acid)-ether (10:3); BFE’, two developments with benzene (saturated with formic acid)ether (10:3); CB, three developments with carbon tetrachloride-benzene (3:2); CE, carbon tetrachloride-ether (3:l); CE’, two developments with carbon tetrachlorideether (1O:l); CFE, chloroform (saturated with formic acid)-ether (10:3); EH, ether-hexane (1:l);HE, two developments with hexane-ether (4:l). Chemicals. Figure 1shows the labeling positions of the pyrethroids examined. Specific activities and radiochemical purities of the permethrin isomers are given by Shono et al. (1978) and of decamethrin by Ruzo et al. (1978). The labeled cypermethrin isomers were provided by D. H. Hutson (Shell Toxicology Laboratory, Sittingbourne, Kent, U.K.) with specific activities of 4.6 and 12-14 mCi/mmol for the I4C-acid- and 14C-alcohol-labeled preparations, respectively; their radiochemical purities were >99% after TLC cleanup (BC; R, 0.61 and 0.68 for the trans and cis isomers, respectively). Figures 2 and 3 give the structures and abbreviations for the compounds and metabolites considered. The suffixes Me and Et are used to designate methyl and ethyl esters, respectively. Most of the unlabeled standard compounds used for TLC cochromatography with I4C metabolites are previously described (Ruzo et al., 1977, 1978; Shono et al., 1978; Unai and Casida, 1977). Additional chemicals utilized were 4’-HO-t- and -c-cyper and 0 1979 American Chemical Society
Microsomal Metabolism of Pyrethroids
J. Agric. Food Chem., Vol. 27, No. 2, 1979
317
Table I. Analytical Data for Four Isomers of 2-Carboxy-3-(2,2-dichlorovinyl)-2-methylcyclopropanecarboxylic Acid and Their Dimethyl Esters
isomer of COOR-C1,CA-R' ( R = H or CH,; R' = CH, or none) condition or parameter "C
t-CI, CA-R' t-COOR
c-C1,CA-R' C-COOR
t-COOR
Mp for Dicarboxylic Acidsa 163-164 181-183
196-197
e-COOR 139-140
TLC R f Values for Dicarboxylic Acids solvent system BFE' CFE
0.37 0.33
CE EH
0.48' 0.46'
0.09 0.13
0.36 0.32
0.16 0.17
TLC R f Values for Dimethyl Esters 0. 34' 0.47 0.33b 0.46
0.39 0.39
GLC t~ Values, Min., for Dimethyl Esters column OV 25'
18.2
20.1
18.6
19.1
NMR Chemical Shifts ( 6 ) and Coupling Constants (Hz) for Dimethyl Esters substituentd 1-H 3-H 1'-H
2.66 (d, J = 6 . 8 ) 2.45 (dd, J = 8 . 5 and 6 . 8 ) 5.83 (d, J = 8.5)
1.87 ( d , J = 6 . 8 ) 2.93 (dd, J = 8 . 5 and 6.8) 5.61 (d, J = 8 . 5 )
CH, l-COOCH,e 2-COOCH3e
1.49 3.75 3.73
1.41 3.69 3.71
2.54-2.73 ( m )
2.12-2.34 ( m )
6.24 (dd, J = 8.5 and 1 . 5 ) 1.45 3.73 3.71
6.36 (d, J = 8 . 5 ) 1.47 3.69 3.72
Cochromatograph with Melting points are for compounds recovered from TLC without recrystallization. methylated metabolites of trans-permethrin in mouse microsomal oxidase systems. Glass column ( 2 m x 3 mm i.d.) of 5% OV 25 on Chromosorb W (A/W 80-100 mesh) with column temperature programming (80-180 "C, 4 "C/min), Substituent positions are designated as shown in N, as carrier gas (30 mL/min), and flame ionization detector. Figure 4. e Carbomethoxy protons of the 1-substituent resonate at lower field than those of the 2-substituent with t-COOMe-C1,CA-Me and vice versa with c-COOMe-C1,CA-Me o n analogy with the corresponding t- or c-CH,OH-C1,CA-Me isomers (Unai and Casida, 1977).
[ IRS,
c,s]Perrnethrin
"W&
[ I R S , ?runs, a R S ] C y p e r m e t h r i r ~
CI
CN
Decamethri n (the IR,cis,aS isomer)
Br
Figure 1. Structures of pyrethroids examined showing sites of radiocarbon labels.
t-CH20H-c-cyper provided by D. H. Hutson and the 3HO-benzyl ester of t-Cl,CA provided by T. Unai (formerly of this laboratory). Synthesis of Four Isomers of 2-Carboxy-3-(2,2-dichlorovinyl)-2-methylcyclopropanecarboxylicAcid (COOH-C12CA) and Their Dimethyl Esters (COOMe-C1,CA-Me). These dicarboxylic acids are possible metabolites of cis- and trans-permethrin and cisand trans-cypermethrin, so procedures were developed for preparing or isolating them and their dimethyl esters. Appropriate analytical data and chromatographic properties are recorded in Table I. On CI-MS analysis, each of the four dimethyl esters gives [M + 11' as the base peak
and only the least stable cis,cis isomer (c-COOMe-cC12CA-Me)yields a prominent 235 ion corresponding to M - OCH,. The (lRS)-transcompounds (t-or c-COOH-t-Cl,CA and t- or c-COOMe-t-Cl,CA-Me) were obtained via the corresponding diethyl ester (COOEt-Cl,CA-Et) (prepared by R. K. Huff, Plant Protection Division, Imperial Chemical Industries Limited, Jealott's Hill Research Station, Bracknell, Berkshire, U.K.) which consisted of tCOOEt-t-Cl2CA-Et (60%), c-COOEt-t-Cl2CA-Et (14%), and t-COOEt-c-Cl,CA-Et (25%)based on NMR and
