In vitro metabolism of etrimfos by house flies

PES’IICIDF

RIOC:HEMISTRY

AND

PHYSIOLOGY

10,

In Vitro Metabolism

31- 39 (1979)

of Etrimfos

by House Flies’

Y. M. IOANNOU AND W. C. DAUTERMAN T().~i~()k?

Progrutn.

Depurtmetu

Raleigh.

of Etltonmlogy.

North

Carolirw

North

Curolim

Stcrte

Ukwzsity,

27650

Received December 2. 1977: accepted April 4. 1978 The metabolism of etrimfos, O.O-dimethyl-O-(6-ethoxy-2-ethyl-4-pyrimidinyl) phosphorothioate was studied in vitro in a diazinon-resistant (Rutgers) and a susceptible (CSMA) strain of house flies. Practically no metabolism of etrimfos occurred without the addition of cofactors. However, the addition of the cofactor, reduced glutathione. resulted in a substantial amount of metabolism in both strains, the metabolism being higher in the resistant strain. The major route of metabolism was via the glutathione transferase system and the predominant metabolite was desmethyl etrimfos. Although the oxygen analog could not be isolated, microsomal oxidation of etrimfos resulted in the inhibition of acetylcholinesterase, suggesting the formation of the oxygen analog. Bovine serum albumin also degraded etrimfos yielding desmethyl etrimfos and 6-ethoxy-2-ethyl-4-hydroxypyrimidine. INTRODUCTION

pyrimidine), and EDHP (2-ethyl-4,6-dihydroxypyrimidine) were kindly supplied by Sandoz, Inc., East Hanover, New Jersey. Desmethyl etrimfos [tetramethyl ammonium, 0-methyl-0-(6-ethoxy-2-ethyl-4pyrimidinyl) phosphorothioatel was prepared by reacting trimethyl amine with etrimfos at room temperature (3). The S(6ethoxy-2-ethyl-4-pytimidinyl)-glutathione was prepared by coupling reduced glutathione (GSH) with 6-ethoxy-2-ethyl-4-pyrimidinyl) trimethyl ammonium chloride (4). The desmethyl oxygen analog of etrimfos was prepared by reacting sodium iodide with the oxygen analog of etrimfos MATERIALS AND METHODS (5). Chemicals. V4C1Etrimfos, labeled in the The cofactors, NADPH and GSH, were ring at C4 and Cg (specific activity of 11 obtained from ICN-pharmaceuticals, &i/mg), the oxygen analog of etrimfos, Cleveland, Ohio. The standard protein EEHP” (6-ethoxy-2-ethyl-4-hydroxybovine serum albumin, bovine erythrocyte acetylcholinesterase, and acetylthiocholine ‘Paper No. 5425 of the Journal Series of the North were purchased from Sigma Chemical Carolina Agricultural Experiment Station. Raleigh, Company, St. Louis, Missouri. 5.5’N. C. Work supported in part by PHS Research Grant Dithiobis-(2-nitrobenzoic acid) was obES-00044 from the Institute of Environmental Health Sciences. tained from Aldrich Chemical Company, ‘Abbreviations used: EEHP, 6-ethoxy-2-ethyl-4-hyMilwaukee, Wisconsin. droxypyrimidine; EDHP. 2-ethyl-4.6dihydroxyHouse flies and toxicity tests. TWO pyrimidine: GSH, reduced glutathione; BSA, bovine strains of Musca domestica (L.) were used serum albumin: AChE. acetylcholinesterase; mfo, throughout these experiments, a diazinonmixed-function oxidases. Etrimfos, O,O-dimethyl-0-(6-ethoxy-2ethyl-4-pyrimidinyl)phosphorothioate, is an experimental organophosphorus insecticide developed by Sandoz Ltd., Basel, Switzerland (1). Compared to other pyrimidinyl phosphate insecticides, etrimfos has a low mammalian toxicity. Its oral LDsO to male rats is 2040 mg/kg and to male mice 535 w&g (2). The present study was undertaken to investigate the metabolism of etrimfos in subcellular fractions from a pyrimidinyl phosphate-resistant and -susceptible strain of house flies.

31 0048-35751791010031-09802.0010 Copyright All rights

0 1979 by Academic Press. Inc. of reproduction m any form resewed.

32

IOANNOU

AND

resistant or Rutgers strain and the susceptible CSMA strain. Both strains were maintamed, as adults, on a diet of milk and sugar at 26°C and 55% relative humidity. The Rutgers strain has been reported to be multiresistant to various organophosphorus insecticides, chlorinated hydrocarbons, carbamates, etc. (6). Etrimfos toxicity to these strains of house flies was determined by topical application of 1 ~1 of an acetone solution with varying etrimfos concentrations to the thorax of 6-day-old adult female flies. Contact toxicity was based on 24-hr mortality. The LDsO values were computed by the method of Finney (7) for probit analysis. GSff determination. The GSH contents in the two strains of house flies were determined using a modification of the fluorometric method of McNeil and Beck (8). Enzyme preparation. Abdomens from 6-day-old female flies were homogenized in 0.1 M phosphate buffer, pH 7.4, 4°C at 12 abdomens/ml (approx. IO-12%, w/v). The homogenate was filtered through cheesecloth to remove the debris and the subcellular fractions 10,800g supernatant fraction, 300,OOOg supernatant (soluble) fraction and microsomes were obtained by differential centrifugation as previously described (9). The microsomes were washed, resuspended in buffer, and recentrifuged. An aliquot of the 100,OOOg (soluble) fraction was dialyzed against 100 vol of the buffer for 24 hr, at 4°C. Zncubation system. The reaction mixture consisted of 2 ml enzyme solution, 0.4 pmol of [14Cletrimfos, and either 2.6 pmol of the cofactor NADPH or 16 pmol of the cofactor GSH in a total volume of 4 ml. In a preliminary experiment the incubation system described above was used while the incubation time was varied from 0 to 150 min. In other experiments, higher concentrations of NADPH (13 pmol) or GSH (48 pmol) were also evaluated, and the reaction mixture was incubated for 2 hr at 37°C. In all cases, the final solution was

DAUTERMAN

extracted with 4 ml of benzene and the two layers were separated. The radioactivity in 0. I ml from each phase was quantitated in a Packard Model 3330 liquid scintillation counter using Triton X-100 cocktail (10). The efficiencies were corrected using 1r4Cln-hexadecane as the internal standard. All reactions were corrected for nonenzymatic hydrolysis. Characterization of metabolites. Materials and methods used for the characterization of the metabolites were the same as those employed in a previous study (2). The metabolites were separated by thin layer chromatography (tic) on 5 x 20 cm MNR Silica gel UVZs4 (0.25 mm precoated) plates. The tic plates were developed in solvent system I, ethyl acetate:ethanol:ammonium hydroxide (16:3: 1) and then redeveloped in solvent system II, cyclohexene:acetone (1: 1). The metabolites were detected by scanning the tic plates on a Packard 7201 radiochromatogram scanner. Known standards were visualized at 254 nm, with either iodine vapors, ninhydrin, or 2,6-dibromoquinone-4-chloroimide (11). The radioactive regions corresponding to the two major metabolites (desmethyl etrimfos and EEHP) were scraped from the tic plates, and purified and their structures were confirmed by mass spectra. Effect of abdomen concentration glutathione transferase activity.

on

The 100,000g (soluble) fraction from the Rutgers strain was studied at various concentrations, i.e., 1, 4, 9, and 12 female house fly abdomen equivalents per ml of 0.1 M phosphate buffer, pH 7.4. All concentrations were evaluated with or without GSH. The incubation system was the same as described previously. GSH was added either in the incubation mixture shortly before incubation or to the buffer prior to homogenization. In both cases, GSH was used at 48 pmol/4 ml of incubation mixture. Effect of bovine rimfos degradation.

serum albumin

serum

albumin

on et-

The effect of bovine was studied at different

IN

VITRO

METABOLISM

concentrations in 0.1 M phosphate buffer, pH 7.4. The reaction mixture consisted of 0.4 ~mol[14Cletrimfos (1 ml solution), 1 ml of phosphate buffer, and 2 ml of phosphate buffer containing various concentrations of bovine serum albumin [O-SO% (w/v)]. The mixture was incubated for 1 or 2 hr at 37”C, extracted with 4 ml benzene, and the radioactivity in each phase determined as described earlier. Inhibition of acetylcholinesterase. The inhibition of acetylcholinesterase (AChE) by products of microsomal oxidation of etrimfos was investigated. The method of Ellman et al. (12) was utilized to determine residual AChE activity. The incubation mixture was the same as described above, using microsomes from the Rutgers strain, NADPH, and different concentrations of etrimfos. AChE was either added to the incubation mixture prior to incubation of etrimfos or added to the cuvette just before the determination of residual AChE activity. The control AChE hydrolyzed 0.333 pmol acetylthiocholinelmin at pH 7.4 and 37°C. The microsomes with NADPH were incubated with etrimfos for 1 hr at 37” or 25°C or mixed a few minutes before the residual AChE activity was determined. A standard curve of AChE inhibition was ob-

tained using various concentrations oxygen analog of etrimfos. RESULTS

D 1

1

60

1

I

90

AND

of the

DISCUSSION

The in vitro degradation of etrimfos was examined in both Rutgers and CSMA strain 10,SOOg supernatant preparations. With or without the addition of cofactors the maximum amount of degradation in both strains was substantially lower than that observed in similar studies with rat and mouse 10,800g supernatant preparations (2). Etrimfos degradation in both strains of flies with or without cofactors increased gradually from 0 to 60 min with no significant changes between 60 to 150 minutes of incubation (Fig. 1). Hence 120 min of incubation was considered adequate for maximum degradation. Very little etrimfos was degraded into water-soluble metabolites when incubated with abdomen preparations from the two house fly strains. However, higher degradation was obtained with the resistant strain (Rutgers) when the cofactor GSH was added (Table 1). The enzyme(s) involved in etrimfos degradation are primarily glutathione transferases since the addition of GSH (a cofactor necessary for glutathione transferase activity) in 10,800g

A. RUTGERS

30

33

OF ETRIMFOS

6. CSMA

1

I

I20

I

I

ifI

I50

(III

30

0

60

90

1

fi

120

10

150

MINUTES

FIG. I. Rate oj‘degradation house jlies. (El) No cofactor

of etrimfos added, (

by /0,8OOg A) NADPH

supernatant fracfion of (A) Rutgers added, and IO) GSH added.

and (B) CSMA

strains

of

16.0 48.0 2.6 13.0 16.0 48.0 2.6 13.0 16.0 48.0

GSH GSH None NADPH NADPH GSH GSH None NADPH NADPH GSH GSH

a Average of three separate experiments.

Microsomes

100,008g (Soluble) Fraction

2.6 13.0

None NADPH NADPH

10,800g Supematant Fraction

Concentration (mol)

Cofactor added

1

28.7 34.2 4.6 4.2 6.3 55.5 61.4 0.0 1.6 5.5 6.0 7.8

3.4 6.1 5.7

100 100

81 95

195

EEHP m

15.6 18.3 3.9 4.0 6.3 33.9 39.2 0.7 1.5 3.8 4.7 5.5

5.3 7.0 5.1

Metabolites formed (%)

87 85

89 85

Desmethyl etrimfos (%)

Polar metabolites

Polar metabolites Desmethyl etrimfos (%)

CSMA

13 15

11 15

EEHP (%I

Fractions of CSMA and Rutgers Strains of House Flies”

Rutgers

of Etrimfos by Subcellular

Metabolites formed (%)

of Metabolites

Subcellular fraction

Effect of Cofactors on the Formation

TABLE

: 2 E Es

g

:

5 52

IN VITRO METABOLISM

35

OF ETRIMFOS

TABLE

2

Effect of Cofactors on the Degradation of Etrimfos by Dialyzed and Nondialyzed of Rutgers and CSMA Strains of House Flies”

Cofactor concentration Wnol)

Cofactor added None NADPH NADPH GSH GSH

2.6 13.0 16.0 48.0

lOO.OoOg (Soluble) Fraction

Rutgers

CSMA

Percentage polar metabolites formed

Percentage polar metabolites formed

Dialyzed

Nondialyzed

1.6 1.1

Dialyzed

4.6 4.2

4.0 3.8

3.3 55.3

6.3 55.5

66.1

61.4

2.6 37.6 39.7

_.--

Nondialyzed 3.9 4.0 6.3 33.9 39.2

n Average of three replicates.

supernatant and 100,OOOg supernatant resulted in a marked increase in etrimfos degradation. Glutathione transferases were less active in the susceptible strain (CSMA) since the addition of GSH resulted in less degradation when compared with the Rutgers strain. The mixed-function oxidases (mfo) contributed little to etrimfos degradation since the addition of NADPH in the 10,800g supernatant and microsomes resulted in only a minor increase in degradation (Table 1). A portion of the 100,OOOg supernatant from both fly strains was dialyzed against 100 vol of phosphate buffer for 24 hr in order to remove all endogenous cofactors including GSH. As can be seen in Table 2, the addition of the cofactor GSH resulted in higher degradation of etrimfos, comparable to that of the nondialyzed fraction. The above results show that glutathione trans-

ferases are active in both house fly strains and that the limiting factor in etrimfos degradation is the amount of GSH present. However, the resistant strain (Rutgers strain) has higher glutathione transferase activity than the susceptible strain (CSMA strain) as can be seen in Table 1. Motoyama er al. (13) reported that the glutathione transferase activity present in the 100,ooOg soluble fraction from 6-dayold female house fly abdomens from the Cornell-R strain decreased considerably as the tissue concentration increased, without the addition of GSH. With the addition of GSH (before homogenization) there was insignificant decrease as the homogenate concentration increased. Our results with the Rutgers strain (Table 3) show that glutathione transferase activity increased as the homogenate concentration increased from 1 to 9 abdomens/ml buffer with slight

TABLE Effect of Concentration Ply abdomen equivalents (ml)

Percentage polar metabolites formed NoGSH

added

1

0.7

4 9 12

2.1 5.9 4.5

n 100,OOOg supematant.

3

of Rutgers House Fly Abdomens on Etrimfos Metabolism”

pm01 GSH

48

27.3 57.0 68.9 61.4

48 pmol GSH added in the homogenizing buffer 30.2 51.6 70.2 -

36

IOANNOU

AND

decrease when 12 abdomens/ml were used. The increase in enzyme activity is much higher when GSH is added either before homogenization or just prior to incubation than when no GSH is added. These results suggest that the Rutgers strain, as opposed to Cornell-R strain, has a very low content of endogenous GSH which results in lower glutathione transferase activity. It is possible that the low glutathione transferase activity is due not only to low GSH content, but also to some inhibition by endogenous glutathione transferase inhibitors (possibly quinones) similar to those found in Cornell-R strain (13). However, these inhibitors must be found only in small quantities as compared to the Cornell-R strain, since the addition of GSH even minutes prior to incubation results in high glutathione transferase activity. In the aqueous layer, two metabolites were detected in both house fly strain abdomen preparations (Table 1). Desmethyl etrimfos (Rr = 0.22) was characterized in both the 10,800g supernatant fraction and the 100,OOOg (soluble) fraction from both fly strains. This was the major metabolite in vitro, and it is assumed to be the product of glutathione transferase activity since it occurs only when GSH is added. The second metabolite, EEHP, (Rf = 0.7) was found in very small quantities in association with 10,800g supernatant of both fly strains, and only when GSH was added. When NADPH was used (in 10,800g supernatant fraction and microsomes) the degradation was too low to be of value in determining the metabolites. After incubation, only the parent compound, etrimfos, was found in the organic extract of the subcellular fractions, even though the oxygen analog will partition into the organic phase. No oxygen analog of etrimfos could be detected in the organic extract by tic from either house fly strain. However, additional experiments have demonstrated that the oxygen analog of etrimfos is probably formed when etrimfos is incubated in the presence of microsomes

DAUTERMAN

(source of mfo), NADPH, and AChE. As can be seen in Table 4, an AChE inhibitor was formed rapidly (within minutes) at 25 and 37°C as long as the microsomes and NADPH were present. It was estimated (from a standard curve) that the incubation of 0.4 pmol (116.8 pg) of etrimfos with microsomes and NADPH for 1 hr at 37°C resulted in the formation of 0.04 pmol (11 pg) of the oxygen analog. Note that no inhibitor was present in the mixture after 1 hr of incubation, as demonstrated by the lack of AChE inhibition. Conversely, if AChE is added to the incubation mixture prior to incubation for 1 hr at either 25” or 37°C ACheE inhibition is obtained. This is also true when the mixture is incubated for only a few minutes at room temperature, i.e., AChE inhibition is obtained. These results suggest that the oxygen analog of etrimfos is formed very rapidly in the presence of microsomes and NADPH at 25 or 37°C but at the same time is unstable and breaks down rapidly (probably to EEHP and dimethyl phosphate) and therefore cannot be detected by tic even though sufficient amount of 14C-labeled oxygen analog was initially formed. Since the oxygen analog breaks down so rapidly (even before partitioning the reaction mixture with the organic solvent) the breakdown products partition into the aqueous layer and as a result only the parent compound (etrimfos) is found in the organic layer. Therefore, some EEHP found in the aqueous layer was probably formed via the oxygen analog (glutathione transferase or hydrolase activity) and not formed directly from etrimfos. Toxicity studies have shown that the CSMA strain of house flies was about 37fold more susceptible to etrimfos than the Rutgers strain as indicated by the LD5,, values, i.e., for CSMA strain the LD5,, value was 0.015 pg/fly while for the Rutgers strain it was 0.555 &fly. Part of this difference in susceptibility of the two strains to etrimfos might be due to differences in body weight between the two strains (the Rutgers strain

IN VITRO METABOLISM TABLE Acefylcholinesterase Etrimfos concentrationb (MM ml) 233.6 116.8 58.4 29.2 14.6 7.3 3.65 1.825 0.365 0.1825 0.0365

OF ETRIMFOS 4

Znhibition by Products of Microsomal

Oxidation of Etrimfos”

Percentage acetylcholinesterase AChE present during the incubation 51.7’ 36.9 28.9 22.1 11.4 12.7 12.7

96.0” 96.0 95.3 95.3 95.3 94.6 94.6 90.6 81.2 69.1 46.3

37

inhibition’

AChE added after completion of the incubation 0.7’ 3.1 2.0 1.3 0.7 0.1 0.0

0.7d 6.0 2.7 2.0 0.7 0.0 2.0

n Microsomes from the Rutgers Strain. b From each incubation mixture, 0.2 ml were used to determine residual AChE activity. (‘ Results in column at 25”C, S-10 min. d Results in column at 3X, 60 min.

is heavier than the CSMA strain by about 20%). However, this high resistance level of the Rutgers strain cannot be explained with information obtained from in vitro experiments and further studies are required for better understanding. Motoyama and Dauterman (9) have reported that the high activities of the mfo and the glutathione transferases in the Rutgers strain were responsible for the higher azinphosmethyl degradation and thus for the resistance of this strain to this insecticide as compared to the susceptible strain. However, in these experiments the activity of these enzymes was not sufficiently different between the two strains, at least without the addition of cofactors. Although the GSH content in the Rutgers strain was found to be higher than in CSMA (3 mg/g abdomen tissue for Rutgers as compared to 2.2 mg/g for CSMA) the difference in the glutathione transferase activity between the two strains was nonexistent without additional GSH. Thus, for all practical purposes, we cannot attribute the resistance of etrimfos in the Rutgers strain to differences in mfo and ghrtathione transferase activity between the two strains. The presence of endogenous inhibitors of

microsomal metabolism in insects has been reported by Wilkinson and Brattsten (14). To overcome this inhibition, BSA has been utilized in enzyme preparations to bind the inhibitor and thus increase enzyme activity (15). However, serum albumin from different animals has been known to catalyze carbamate hydrolysis. During the present studies, it was noted that a reaction mixture containing V4Cletrimfos, phosphate buffer, and 1% BSA formed water-soluble metabolites 30-fold higher in the presence of BSA than in the absence of BSA. Hence, a series of BSA concentrations were evaluated (see Materials and Methods). The results are given in Table 5. Note that as the BSA concentrations increased from 0.05 to 5%, the etrimfos degradation also increased, being much higher when the mixture was incubated for 2 hr rather than for 1 hr. Higher BSA concentrations (lO-50%) resulted in decreasing etrimfos degradation. The metabolites found in these studies vere the same as those observed in the in itro enzymatic studies (Table 5). The major metabolites were desmethyl etrimfos (- 90%) and EEHP (10%). As in the case

38

IOANNOU

AND DAUTERMAN TABLE

Effect of Various Concentrations

5

of Bovine Serum Albumin on the Formation of Polar Metabolites

of Etrimfos

Polar metabolites (%) BSA concentration (%I 0.0 0.05 0.1 0.5 1.0 5.0 10.0 15.0 20.0 25.0 40.0 50.0

I-hr Incubation Metabolites formed 1.3 7.0 12.5 31.2 43.7 45.1 32.4 29.3 24.7 18.3 -

Z-hr Incubation

Desmethyl etrimfos

EEHP

85 86 83 93 91 79 -

15 14 17 7 9 21 -

with carbamates (16), it is possible that BSA exhibits an “esterase-like” action when incubated with organophosphorus insecticides. The manner in which BSA exerts its action is not known, but it is possible that it forms an unstable complex with the insecticide resulting in the release of a methyl group. In summary, etrimfos is slowly degraded in vitro by both strains of house flies to mainly desmethyl etrimfos and EEHP. The cofactor GSH has been found to be rate limiting in etrimfos detoxication, and neither strain has shown appreciable degradation of etrimfos without the addition of GSH. On the contrary, mammalian systems, as exemplified by rats and mice (2), detoxify etrimfos very quickly with or without additional GSH. At the same time, rats and mice have a very active mfo system contributing to etrimfos degradation (oxidative dearylation) while in house flies these enzymes are not very active. These differences in glutathione transferase and mfo activity between the two systems (insect and mammalian) might explain the high toxicity of etrimfos to flies and the relatively low toxicity to rats and mice. These experiments have also shown that an AChE inhibitor (probably the oxygen analog of

Metabolites formed 2.8 6.3 15.0 40.0 55.4 73.4 69.4 61.8 56.1 49.6 34.3 27.7

Desmethyl etrimfos

EEHP

88 89 94 91 86 92 89 -

12 11 6’ 9 14 8 11 -

etrimfos) is formed via the mfo system. BSA also degrades etrimfos yielding primarily desmethyl etrimfos and EEHP. ACKNOWLEDGMENTS The authors are indebted to Drs. H. &helling and J. C. Karapally, Sandoz, Inc., East Hanover, New Jersey, for their valuable advice in this study and for supplying chemicals and samples used in this investigation. REFERENCES 1. H. J. Knutti and F. W. Reisser, Etrimfos -a new insecticide with low mammalian toxicity, Proc. 8th Brit. Insect. Fungic. Conf. 695 (1975). 2. Y. M. Ioannou and W. C. Dauterman, In vitro metabolism of etrimfos by rat and mouse liver, Pestic. Biochem. Physiol., 9, 190 (1978). 3. V. G. Hilgetag, G. Lehmann, A. Martini, Cl. Schramm, and H. Teichmann, Uber alkylienende ester spaltungen einiger dialkylaryl-und alkyldiaryl-thiophosphate,J. Prakt. Chem. 280, 207 (1959). 4. J. W. Crayford and D. H. Hutson, The metabolism of the herbicide, 2-chloro-4- (ethyl-amino)-6(I-cyano-I-methyl-ethylamino)-S-triazine in the rat, Pestic. Biochem. Physiol. 2, 295 (1972). 5. E. Y. Spencer. A. Todd, and R. F. Webb, Studies on phosphorylation, Pt. XVII: The hydrolysis of methyl 3-(O,O-dimethylphosphoryloxy) but-2-enoate, J. Chem. Sot. 2968 (1958). 6. A. J. Forgash, B. J. Cook, and R. C. Riley, Mechanisms of resistance in diazinon-selected multiresistant Musca domestica, J. Econ. Entomol. 55, 544 (1962).

IN

7.

D.

J.

Finney.

“Probit

Treatment

of

the

VITRO

Analysis, Sigmoid

METABOLISM

A

12.

Statistical

Response

OF

G. L. Ellman, R. M. orimetric

Curve,”

2nd ed.. Cambridge Univ. Press, London and New York, 1952. 8. T. L. McNeil and L. V. Beck, Fluorometric estimation of GSH-OPT. Anal. Biochem. 22, 431

13.

N.

(1%8). 9. N.

Motoyama metabolism and

resistant

Physiol. 10.

M.

and W. C. Dauterman, of azinphosmethyl in house

14.

S. Patterson

and

Measurement solution emulsions,

15.

by liq-

Anal.

J. J. Menn.

W.

reaction

R. Erwin, of

and

H. T. Gordon,

oneimine with thiophosphate paper chromatograms, J. Agr.

601 (1957).

inhibitors house flies,

Contam.

16. insecticides

Food

Chem.

Rev.

Tsukamoto hancement

on

5,

7, 88 (1961). and of &r/l.

in press. B. Brattsten. Microsomal

L.

enzymes

in

insects.

Irnra

1, 153 (1972).

and J. of oxidative

E.

Casida. metabolism

chemicals system.

Albumin enof methylby the house J. Ecorr. Err-

60, 617 (1967).

J. E. Casida plasma carbamate

Acta

and col-

Toxicol.,

and

metabolizing

tomol.

Color

2.6-dibromo-N-chloro-p-quin-

Jr..

W. C. Dauterman, Endogenous glutathione S-transferases in

carbamate insecticide fly microsome-NADPH*

Chem. 37,854(1965). 11.

M.

V. Andrts

A new and rapid of acetylcholinesterase E. Hodgson,

drug

Biochem.

D. Courtney,

activity. Biochem. Pharmacol. Motoyama. A. P. Kulkarni.

Metahol.

R. C. Greene,

K.

C. F. Wilkinson

2, 113 (1972).

of low beta emitters in aqueous uid scintillation counting of

39

Featherstone. determination

Environ.

In vitro susceptible

Pestic.

flies,

ETRIMFOS

and K. albumin and

B. Augustinsson, with I-naphthyl certain

36, 41 I (1959).

other

Reaction N-methylesters,

Biophy.s.

of