microsomal sulfoxidation of phorate in the fall armyworm, spodoptera frugiperda (j. e. smith)

PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY 23, 273-281 (1985)

Microsomal Sulfoxidation of Phorate in the Fall Armyworm, Spodoptera frugiperda (J. E. Smith)’

S. J. Yu

Department of Entomology & Nematology, University of Florida, Gainesville, Florida 32611

Received June 12, 1984; accepted July 23, 1984

Microsomal s&oxidation in fall armyworm (Spodoptera frugiperda) larvae was examined using phorate as substrate. The system required NADPH and was inhibited by CO and by piperonyl butoxide. Sulfoxidase activity was found in the alimentary canal, fat body, and Malpighian tubules, with the midgut being the most active. Microsomal substrates such as aldrin, heptachlor, biphenyl, and methyl parathion significantly inhibited the enzyme activity whereas p-nitroanisole and p- choloro-N-methylaniline had no effect. Enzyme activity increased during larval development, reaching a maximum shortly before pupation. Allelochemicals (monoterpenes, indoles, and fla- vones), drugs (phenobarbital and 3-methylcholanthrene), and host plants (corn cotton, parsnip, and parsley) significantly increased the enzyme activity. Increased phorate sulfoxidation through enzyme induction was found to decrease oral toxicity of phorate to the larvae. Analyses of internal insecticide revealed that, at various intervals, induced larvae retained less phorate and its oxidative metabolites than the controls. It was concluded that induction of phorate sulfoxidase activity results in an overall increase in the rate of phorate detoxication in this insect. o 1985 Academic

Press, Inc

INTRODUCTION

The microsomal mixed-function oxidase (MFO) system located in the endoplasmic reticulum of the cell plays an important role in the metabolism of xenobiotics and insect hormones in insects (1). This system has a broad substrate specificity capable of oxi- dizing various functional groups of lipo- philic molecules. The versatility of this system is attributed to the multiplicity of cytochrome P-450, the key component of the MFO system. Of the reactions performed by the MFO system, epoxidation, sulfoxidation, desulfuration, N-dealkylation, 0-dealkylation, and hydroxylation have been regarded as most important in the metabolism of pesticides.

As to sulfoxidation, in vivo studies revealed that many thioether-containing pesticides, such as organophosphorus compounds and carbamates, were oxidized by insects to their corresponding sulfoxides (2,

i Florida Agricultural Experiment Station Journal Series No. 5631.

3). In general, sulfoxide formation represents an oxidative acivation process leading to an increase in anticholinesterase activity. Despite its significance in the toxicity of insecticides, very little work has been done regarding the nature of the enzyme system in insects. Nigg er al. (4) briefly reported an assay method for measuring microsomal sulfoxidase activity in house flies using p- chlorothioanisole as enzyme substrate, and the thioether oxidation product, p-chloro- phenyl methylsulfinyl ether, was determined by gas chromatography. However, due to the lack of detailed characterization of the system, its biochemical nature in insects are virtually unknown at present.

This report concerns a study of microsomal sulfoxidation with phorate as substrate in fall armyworm larvae. The effect of allelochemicals as well as host plants on the sulfoxidase system was also examined.

MATERIALS AND METHODS

Insects. Larvae of the fall armyworm [Spodopreru frugiperdu (J. E. Smith)] were reared on a meridic diet (5) and maintained

273 0048-3575185 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved

274 S. J. YU

in environmental chambers at 25°C with a 16:8, 1ight:dark photoperiod.

Chemicals. Phorate and its sulfoxide and sulfone, and phoratoxon and its sulfoxide and sulfone were obtained from American Cyanamid Company, Princeton, New Jersey. Indole 3-acetonitrile, indole 3-carbinol, flavone, p-naphthoflavone, 3-methylcholanthrene, and n-octylamine were purchased from Sigma Chemical Company, St. Louis, Missouri. cw-Pinene and I-menthone were from Aldrich Chemical Com- pany, Milwaukee, Wisconsin. Peppermint oil was from I. P. Callison & Sons, Inc., Chehalis, Washington. All other chemicals were of analytical quality and were purchased from commercial suppliers.

Treatment of insects. Newly molted sixth-instar larvae (less than 3 hr after ec- dysis) that had been maintained on an artificial diet were placed in individual plastic cups and fed a meridic diet containing various allelochemicals for 2 days. Controls were fed the artificial diet only. After 48 hr, larvae were removed from each diet and used for enzyme assays. No mortality was observed due to the treatments.

In some experiments, newly molted sixth-instar larvae were placed in plastic containers in groups of 25 larvae each and fed for 2 days on fresh leaves of soybeans, Glycine max (L.) Merrill (Bragg); broccoli, Brassica oleracera L. (Waltham 29); radish, Raphanus sativus L. (Early Scarlet Globe); potato, Solanum tuberosum L. (Idaho Resset); peanuts, Arachis hypogaea L. (Florunner); cowpeas, Vigna unguicu- lata L. (Blackeye No. 5); corn, Zea mays L. (Pioneer 3160); cotton, Gossypium aro- reum L. (McNair 220); parsnip, Pastinaca sativa L. (Improved Hollow Crown); and parsley, Petroselinum crispum (Mill) Nym. (Extra Triple Curled). All plants were grown under greenhouse conditions. Lar- vae fed on the artificial diet were used as controls. At the end of the experiments, larvae were removed from their respective diets and used for enzyme assays.

Enzyme assays. Unless otherwise stated,

microsomes were prepared from midguts of sixth-instar larvae as described previously (6) and suspended in 0.1 M sodium phosphate buffer, pH 7.2, prior to enzyme assays. For cytochrome P-450 measurements microsomes were suspended in 0.07 M sodium phosphate buffer, pH 7.5, containing 30% glycerol.

Microsomal sulfoxidase activity was determined with phorate as substrate. The 5- ml incubation mixture contained 1 ml of microsomal suspension (1 mg protein); 0.1 M sodium phosphate buffer, pH 7.2; an NADPH-generating system consisting of 1.8 pmol NADP, 18 p,mol glucose 6-phosphate, and 1 unit glucose 6-phosphate de- hydrogenase; and 100 p.g of phorate in 0.1 ml of methyl Cellosolve. Duplicate incu- bations were conducted in a water bath with shaking at 30°C in an atmosphere of air for 15 min. The reaction product, phorate sulfoxide, was extracted with 10 ml of ethyl acetate, dried over anhydrous sodium sulfate, and analyzed by gas chromatography. Analyses were performed on a Varian Model 3740 gas chromatograph equipped with a thermionic specific detector and a Hewlett-Packard Model 3390A integrator. The column used was 6 ft x 2 mm glass column packed with 2% OV-101 on 80-100 mesh Ultra-Bond 20M (7). The operating conditions were column, 185°C; injection port, 200°C; detector, 250°C; nitrogen carrier gas, 30 ml/min; air, 175 ml/min; and hydrogen, 4.5 ml/min. En- zyme activity was expressed as nanomoles phorate sulfoxide produced per minute per milligram protein. In experiments with boiled microsomes, 98.7 + 2.9, 96.6 ? 1.6, 96.2 ? 2.4,95.6 + 2.1% of added phorate, phoratoxon, phorate sulfoxide or phorate sulfone (50 kg each), respectively, were re- covered by the standard extraction proce- dures.

Microsomal cytochrome P-450 content was determined by the method of Omura and Sato (8) using a Beckman Model 5260 uv/vis spectrophotometer equipped with a scattered transmission accessory. Types I

MICROSOMAL SULFOXIDATION IN FALL ARMYWORMS 275

and II spectra were determined with menthone (9) and n-octylamine, respectively, by the direct addition of saturating concen- trations of the substrate (or ligand) to the sample cuvette. Protein was determined by the method of Bradford (lo), using bovine serum albumin as standard.

NADPH-cytochrome c reductase activity as assayed according to Masters et al. (11) with the following modification. The 2.5-ml reaction mixture contained 0.06 M sodium phosphate buffer, pH 7.5, 50 FM cytochrome c, and 0.1 ml of microsomal suspension (0.1 mg protein). The reaction was initiated by the addition of 50 ~1 of the NADPH-generating system as described earlier.

For the in viva phorate sulfoxidation experiments, newly molted sixth-instar larvae were divided into two groups. One group was reared on an artificial diet containing 0.2% indole 3-carbinol and the other was reared on the artificial diet as control. After 48 hr, larvae were topically treated with phorate in 1 ~1 of acetone and kept individually in a scintillation vial at 25°C with a photoperiod of 16:8 1ight:dark. At various time intervals after treatment, duplicate groups of three larvae each were rinsed twice with 30 ml of acetone each time and homogenized in 8 ml of distilled water in a motor-driven grinder for 30 sec. The ho- mogenizer was rinsed with 2 ml of water. The combined homogenate was then extracted with 10 ml of ethyl acetate, dried over anhydrous sodium sulfate, and analyzed by gas chromatography.

Bioassays. To study the effect of sulfoxidase induction on the toxicity of phorate in viva, newly molted sixth-instar larvae were divided into two groups. One group was fed an artificial diet containing 0.2% indole 3-carbinol as mentioned above and the other group was fed the artificial diet as control. After 48 hr, 10 larvae from each group were starved for 3 hr and then fed individually a leaf disk of cowpea, 0.7 cm in diameter, that had been topically treated with phorate in acetone. Mortality counts

0 2 4 6 6 IO TIME (min)

FIG. 1. Typical gas chromntogram of metabolites (solid line peaks) produced when midgut microsomes of fall armyworm larvae are incubated with phorute fortified with NADPH. (A) Phoratoxon; (B) phorate; (C) phoratoxon &oxide; (0) phorate sulfoxide; (E) phoratoxon sulfone; (E, phorate su!fone.

were made after 24 hr and analyzed by probit analysis (12).

RESULTS

Characteristics of Phorate Sulfoxidase System

Figure 1 shows the gas chromatographic analysis of a typical extract from incubation of phorate with fall armyworm microsomes fortified with NADPH. The peaks A, B, and D correspond to authentic standards of phoratoxon, phorate, and phorate sulfoxide, respectively. No other oxidative products or other metabolites of phorate were evident in the extracts. Under the gas chromatographic conditions employed, phoratoxon sulfoxide (C), phoratoxon sulfone (E), and phorate sulfone (F) can also be separated from the other oxidative metabolites, as shown in Fig. 1 by dotted line peaks.

As shown in Fig. 2, the sulfoxidase activity was mainly located in the midgut. On a per tissue basis, less than 4% of midgut activity was found in the foregut, hindgut, fat body, or Malpighian tubules. When ac-

276 S. J. YU

tivity was based on microsomal protein, it was found that the midgut still possessed the highest sulfoxidase activity, with the fat body being the lowest among the five types of tissues assayed.

Midgut microsomes from fall armyworm larvae required NADPH for activity. The activity was inhibited (82%) by bubbling CO gently into the incubation mixture for 1 min prior to incubation, indicating the in- volvement of cytochrome P-450. Piperonyl butoxide, a well-known inhibitor of microsomal oxidases, also inhibited the sulfoxidase system, showing an I,, value of 1.41 x lop5 M. The microsomes contained an average of 0.26 nmol cytochrome P-450/mg protein. These observations indicated that the phorate sulfoxidase was a typical microsomal mixed-function oxidase (cytochrome P-450-dependent monooxygenase).

The effect of pH on the sulfoxidase activity was determined with sodium phosphate (0.1 A4) over the pH range 5.7 to 8.0. It can be seen from Fig. 3a that the maximum enzyme activity was at pH 7.2, although there was no marked difference between pH 7.2 and 7.5. Enzyme activity was linear up to 2.83 mg protein/sample (Fig. 3b) and increased with incubation time up to 20 min (Fig. 3~). Figure 3d shows that the sulfoxidase had an apparent K, value

of 15.4 ~J,M and V,,, value of 1.38 nmol min-’ mg protein-‘. From Table 1, it is seen that MFO substrates such as aldrin (epoxidation), heptachlor (epoxidation), biphenyl (hydroxylation), and methyl parathion (desulfuration) significantly inhibited the sulfoxidase activity, whereas p-nitroanisole (0-demethylation) and p-chloro-l\r- methylaniline (N-demethylation) had no effect.

Effect of Larval Znstar

Midgut sulfoxidase activity in different instars of fall armyworm larvae is presented in Table 2. Larvae younger than fourth instar were not included in this study because of the difficulty in dissecting the midgut. Sulfoxidase activity varied during larval development, being low in the fourth and fifth instars but reached an activity almost a three-fold higher in the final larval instar and in the prepupae.

Induction by Host Plants and Allelochemicals

It has been demonstrated that various host plants induced microsomal monooxy- genases such as activities of epoxidase, N- and 0-demethylase (6), and hydroxylase (13) in fall armyworm larvae. Table 3 shows that corn, cotton, parsnip, and parsley leaves significantly stimulated the sulfoxidase activity in armyworms. On the other hand, soybean and broccoli leaves ap- peared to significantly reduce the enzyme activity, whereas radish, potato, peanuts, and cowpeas had no effect. A live-fold difference in sulfoxidase activity was observed between the soybean- and parsley- fed larvae.

Apparently the induction was due to allelochemicals in the host plants. As seen in Table 4, all the allelochemicals and two classical MFO inducers significantly induced the sulfoxidase activity in fall armyworm larvae. Two indole compounds, indole 3-carbinol and indole 3-acetonitrile, increased enzyme activity by 5-fold as


60 64 66 7.2 76

pH (0.1 M sodium phos@mte buffer) INCUBATION TIME (min)

6 d

FIG. 3. Effect of pH (a), enzyme level (b), incubation time (c), and substrate concentration (d) on phorate sulfoxidation in fall armyworm larvae. V, product formed (nmol min-’ mg protein-‘); bhorate], substrate concentration (mm. Each point represents the mean of at least two determinations.

compared with the controls. Flavone and its analog, @naphthoflavone, were the best inducers among those tested, resulting in 6.1- and 5.1-fold increases in enzyme activity. Monoterpenes such as a-pinene and peppermint oil, and the barbiturate, phenobarbital, were moderate inducers. How- ever, the induction by 3-methylcholanthrene was only slight (23%).

Attempts were made to learn whether the induction was correlated with levels of cytochrome P-450 and NADPH-cytochrome P-450 reductase. Table 5 shows that although the compounds significantly induce

the P-450 contents as compared with the controls, there was no positive correlation between the P-450 content and phorate sulfoxidase activity (see Table 4) caused by the inducers. For example, flavone was the best inducer of sulfoxidase (Table 4) but was a weak P-450 inducer among the four compounds studied. Menthone binding spectrum (Type I) was significantly en- hanced by indole 3-carbinol, flavone, and l3-naphthoflavone, whereas indole 3-acetonitrile significantly reduced the binding spectrum. The n-octylamine binding spectrum (Type II) was not affected with the

278 S. J. YU

TABLE 1 In Vitro Effect of Various Microsomal Oxidase Substrates on Microsomal Sulfoxidase Activity

in Fail Armyworm Larvae

Phorate sulfoxidase activity

Competing substratea (% of control)

Control 100 Aldrin 75.6 k O.lb,** Heptachlor 55.2 f 0.3*** Biphenyl 65.5 2 1.5*** Methyl parathion 48.2 k 0.5*** p-Nitroanisole 95.7 + 1.2 p-Chloro-N-methylaniline 94.9 -t 0.3

a An equimolar concentration (38.5 t&f) was used for the compounds and enzyme substrate (phorate). All were added in 0.1 ml methyl Cellosolve.

b Mean f SE of two experiments, each with duplicate determinations. See Table 3 (footnotes) for the level of statistical difference from the control.

exception of indole 3-acetonitrile, which significantly decreased the type II spectrum. The results also show that the NADPH-cytochrome c reductase activity was significantly increased by indole 3-carbinol, flavone, and P-naphthoflavone, but not by indole 3-acetonitrile.

Toxicity and Metabolism of Phorate as Influenced by Induction

From Table 6, it can be seen that the ac- tivation of phorate via sulfoxidation and de-

TABLE 2 Microsomal Phorate Sulfoxidase Activity in Various

Instars of Fall Armyworm Larvae

Phorate Larval Age sulfoxidase activity instar (days) (nmol mini mg protein-‘)

4th 1 1.03 2 0.09” 5th 1 1.05 -c 0.11 6th 0 0.70 f 0.03

1 1.95 f 0.01 2 2.38 f 0.05 3 2.39 + 0.10 4 2.83 2 0.14 5 (prepwa) 2.07 * 0.07

a Mean * SE of two experiments, each with duplicate determinations.

TABLE 3 Phorate Sulfoxidase Activity of Fall Armyworm

Larvae Fed Various Host Plants

Phorate sulfoxidase activity Host plant0 (rim01 min-r mg protein-i)

Artificial diet 2.50 f 0.13b Soybeans 1.81 ” 0.10* Broccoli 1.87 k 0.10* Radish 2.08 * 0.10 Potato 2.43 2 0.13 Peanuts 2.50 2 0.04 Cowpeas 2.83 k 0.28 Corn 3.46 + 0.22** Cotton 5.73 f 0.28** Parsnip 6.65 r 0.96** Parsley 9.04 * 0.57***

a Newly molted sixth-instar larvae were fed leaves of host plants for 2 days prior to enzyme assays.

b Mean k SE of two experiments, each assayed in duplicate.

* Value significantly different (P < 0.05) from the control (artificial diet).

** Value significantly different (P < 0.01) from the control.

*** Value significantly different (P < 0.001) from the control.

sulfuration did not increase the toxicity of phorate in larvae. In fact, a two-fold decrease in the toxicity was observed even

TABLE 4 Phorate Sulfoxidase Activity of Fall Armyworm Larvae Fed Various Allelochemicals and Drugs

Allelochemical Sulfoxidase activity (0.2% in diet)” (nmol mini mg protein-i)

Artificial diet 2.41 f O.OSb Indole 3-carbinol 12.19 f 1.24c,** Indole 3-acetonitrile 11.83 k 0.96** Flavone 14.73 f 1.54** B-Naphthoflavone 12.23 2 0.65*** a-Pinene 3.83 * 0.11** Peppermint oil 7.70 f 0.33*** Phenobarbital 4.71 f 0.18** 3-Methylcholanthrene 2.97 k 0.13”

’ Newly molted sixth-instar larvae were fed meridic diets containing the compounds for 2 days prior to enzyme assays.

b Mean 2 SE of two experiments, each with duplicate determinatioins.

c See Table 3 (footnotes) for the level of statistical difference from the control (artificial diet).


TABLE 5 Properties of Cytochrome P-450 and Activity of NADPH-Cytochrome P-450 Reductase in Microsomes of Fall

Armyworm Larvae Fed Various Inducers

Cytochrome P-450

Spectral sizeb (AOD nmol P-450-l ml-‘)

NADPH- cytochrome (

reductase Compound”

(0.2%) Specific content

(nmol/mg protein) Type 1

Spectrum Type II

Spectrum (nmol min- ’ mg

protein- 0

Artificial diet 0.258 + O.Olc 0.029 2 0.003 0.051 -t 0.002 86.0 t 5.8 lndole 3-carbinol 0.833 e O.lO** 0.039 2 0.001** 0.048 + 0.002 153.0 -t 3.5* Indole 3-acetonitrile 0.900 k 0.23d,* 0.024 k O.OOl* 0.012 + 0.0002** 127.0 2 16.2 Flavone 0.518 2 0.067* 0.036 + O.OOl* 0.052 2 0.001 151.8 rt 5.9* 8-Naphthoflavone 0.513 2 0.06* 0.050 c 0.001*** 0.044 c 0.003 143.3 -t 9.4*

a Newly molted sixth-instar larvae were fed meridic diets containing the compounds for 2 days prior to enzyme assays.

b Peak to trough. c Mean 2 SE of two experiments, each assayed in duplicate. d See Table 3 (footnotes) for the level of statistical difference from the control (artificial diet).

though mortality always occurred earlier in the induced larvae than in the control larvae. The desulfuration of phorate ap- peared to be rather insignificant since its activity was 102-136 times lower than the sulfoxidase activity. Analysis of internal phorate and its metabolites revealed that phorate was rapidly oxidized to its sulfoxidation products, phorate sulfoxide and phorate sulfone, and, to a lesser extent, its desulfuration product, phoratoxon (Table 7). In agreement with the in vivo toxicity results, the induced larvae retained less phorate and its oxidative metabolites in all instances up to 24 hr after treatment. As found in the in vitro assays (Table 6) the

rate of in vivo phoratoxon formation was 114-325 times lower than that of phorate sulfoxide formation in the control larvae.

DISCUSSION

The results of the present study demon- strate that fall armyworm microsomes oxidized phorate to phorate sulfoxide in the presence of NADPH. It appears that phorate is a suitable substrate for measuring microsomal sulfoxidase activity since the sulfoxide produced was not fur- ther oxidized to its sulfone under the assay conditions employed and the desulfuration of phorate was very minimal.

It has been reported that thioether-con-

TABLE 6 Effect of Microsomal Enzyme Induction on the Toxicity of Phorate in Fall Armyworm Larvae

Enzyme Phorate desulfurase (pmol phoratoxon Phorate sulfoxidase L&o

induceP mint mg protein-‘) (nmol min-* mg protein-‘) (pg phorate/g larva)b

Control 23.79 f 1.60c 2.43 f 0.06 97.1 Indole 3-carbinol 83.93 * 1.57 11.40 k 1.17 191.4

a Newly molted sixth-instar larvae were fed a meridic diet containing the compound (0.2%) for 2 days prior to enzyme assays and bioassays.

b Groups of larvae (after induction) were fed a cowpea leaf disk that had been topically treated with phorate. Mortality counts were made 24 hr later.

c Mean f SE of two experiments, each assayed in duplicate.

280 S. J. YU

TABLE 7 Effect of Microsomal Enzyme Induction’on the in Vivo Metabolism of Phorate in Fall Armyworm Larvae

Internal phorate and metabolite@

Enzyme induceI

Time after treatment

(hr) Phorate Phoratoxon

(wid~a) (ngkva)

Phorate sulfoxide Wlarva)

Phorate sulfone

(ngktrva)

Control 1 18.29 k 2.W 8.55 * 0.41 974.00 + 19.06 230.60 f 13.98 3 25.38 t 0.43 7.12 YL 0.38 2039.50 f 66.70 818.22 k 46.98 6 20.68 + 0.91 5.49 + 0.24 1783.10 + 188.26 1570.16 k 126.09 9 15.24 + 2.40 3.80 k 0.86 1113.10 ‘- 77.63 1173.53 2 11.07

24 3.96 -c 0.33 0 0.16 f 0.15 207.36 f 61.17

Indole 3-carbinol 1 8.62 f 1.56 3.49 -+ 0.60 430.50 * 134.50 229.94 2 82.8 3 10.64 f 1.45 6.20 k 0.33 987.35 2 82.80 641.33 rt 33.71 6 1.32 f 0.73 0.60 2 0.60 0.098 SI 0.06 102.63 2 37.87 9 1.10 f 0.07 0 0.075 * 0.009 66.40 f 7.64

24 0.31 k 0.27 0 0 0

’ Newly molted sixth-instar larvae were fed a meridic diet containing the compound (0.2%) for 2 days prior to in vivo metabolism study.

b Larvae (after induction) were topically treated with phorate at 30 &larva in 3 ul acetone. c Mean i SE of two experiments consisting of two to four determinations.

taining pesticides such as phorate and disulfoton can be oxidized by a microsomal flavin adenine dinucleotide (FAD)-dependent monooxygenase system to their corresponding sulfoxides in mammals (14). As found in microsomal cytochrome P- 450 mooxygenases, the system requires NADPH and oxygen for activity but is in- sensitive to methylenedioxyphenyl compounds (15). Since the sulfoxidation of phorate by armyworm microsomes was strongly inhibited by piperonyl butoxide and CO, it must be concluded that the observed activity in the present study was not related to the FAD-dependent monooxygenase .

Table 1 shows that sulfoxidase activity was inhibited by aldrin, heptachlor, biphenyl, and methyl parathion, but was not affected by p-nitroanisole and p-chloro-N- methylaniline. These observations clearly indicate that cytochrome P-450 isozymes responsible for the sulfoxidase activity were quite different from those of 0- and N-demethylase. On the other hand, the poor inhibition observed may have been due to differences in Km between the three substrates. The sulfoxidase system is also

likely to be different from aldrin epoxidase system based on the findings in which host plants were used as inducers of the enzyme. Although parsnip and parsley were found to be the best inducers of the sulfoxidase among those tested (Table 3), these two host plants had no marked effect on microsomal aldrin epoxidase activity when fed to armyworm larvae (Yu, unpublished results). One the other hand, corn which was a potent inducer of aldrin epoxidase activity (four-fold over the control) (6), caused a 38% increase in the sulfoxidase in fall armyworms. In this respect, it would be erroneous to use one type of enzyme activity to express general MFO levels in the induction studies since each enzyme activity may respond differently to an inducer.

That phorate sulfoxidase system is different from biphenyl hydroxylase system was indicated in our activity profile studies of fall armyworms (13). It was found that the hydroxylase activity in the fourth- and fifth-instar larvae was about one-tenth that in Day 3 larvae (the highest activity) of the sixth instar, and there was a 23.4-fold difference in the enzyme activity between Day


0 and Day 3 larvae of the final instar. As seen in Table 2, such dramatic changes were not observed for the sulfoxidase activity, a maximum of four-fold difference in the sixth instar.

It is interesting to note that increased ac- tivation of phorate by sulfoxidase and desulfurase resulted in a decrease in toxicity (Table 6). In vitro studies showed that microsomal oxidase system of fall armyworm larvae oxidized phorate sulfoxide to its sulfone, phorate sulfone to phoratoxon sulfone, and phoratoxon to its sulfoxide, but oxidation of phoratoxon sulfoxide to its sulfone was not evident. It was also observed that oxidation of phorate sulfoxide to its sulfone can be stimulated in microsomes prepared from larvae induced with indole 3-carbinol. Thus, it seems likely that induction of sulfoxidase and desulfurase activity caused an overall increase in the rate of phorate metabolism resulting in detoxication. This explanation is consistent with the results of Sun et al. (16), who found that sesamex, an inhibitor of microsomal oxidases, increased the toxicity of phorate by 8.3-fold in house flies. Apparently, microsomal oxidation was very important in the detoxication of phorate.

ACKNOWLEDGMENTS

This work was supported. in part, by U.S. Depart- ment of Agriculrure Grant 82-CRCR-l-1091. I thank Dr. J. L. Nation and Dr. D. L. Silhacek for reviewing the manuscript. The technical assistance of R. T. Ing is also appreciated.

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oxime in plant and insect. .Z. Agrir. Food Chem. 14, 579 (1960).

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5. R. L. Burton, “Mass Rearing the Corn Earworm in the Laboratory,” USDA, Agric. Res. Serv. ARS 33-134, Washington, D.C.. 1969.

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frugiperdu (J. E. Smith), Pestic. Biochem. Plzy- Got. 17, 59 (1982).

7. S. Y Szeto and M. J. Brown, Gas-liquid chromatographic methods for the determination of disulfoton, phorate, oxydemeton-methyl, and their toxic metabolites in asparagus tissues and soil. J. Agric. Food Chem. 30, 1082 (1982).

8. T. Omura and R. Sato, The carbon monoxide- binding pigment of liver microsomes, J Biol. Chem. 239, 2370 (1964).

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10. M. M. Bradford. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248 (1976).

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12. D. J. Finney, “Probit Analysis”, 2nd ed., p. 236. Cambridge Univ. Press, London/New York. 1952.

13. S. f. Yu and R. T. lng, Microsomal biphenyl hydroxylase of fall armyworm larvae and its induction by allelochemicals and host plants. Comp. Biochem. Physiol. C 78, 145 (1984).

14. N. P. Hajar and E. Hodgson, Flavin adenine dinucleotide-dependent monooxygenase: Its role in the sulfoxidation of pesticides in mammals. Science (Washington. D.C.) 209, 1134 (1980).

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16. Y. P. Sun, E. R. Johnson, and L. F. Ward. Jr.. Evaluation of synergistic mixtures containing sesamex and organophosphorus or chlorinated insecticides tested against house flies, .Z. &on. Entomol. 60, 828 (1967).

microsomal sulfoxidation of phorate in the fall armyworm, spodoptera frugiperda (j. e. smith)

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