Responses of representative midgut detoxifying enzymes from Heliothis zea and Spodoptera frugiperda to trichothecenes

Insect Biochem. Vol. 20, No. 4, pp. 349-356, 1990 Printed in Great Britain

0020-1790/90 $3.00+ 0.00 Pergamon Press pie

RESPONSES OF REPRESENTATIVE M I D G U T DETOXIFYING ENZYMES FROM H E L I O T H I S ZEA A N D S P O D O P T E R A FRUGIPERDA TO TRICHOTHECENES PATRICK F. DOWD Mycotoxin Research Unit, Northern Regional Research Center, U.S.D.A., Agricultural Research Service, Peoria, IL 61604, U.S.A. (Received 14 August 1989; revised and accepted 4 January 1990)

A~tract--The relative levels of representative midgut detoxifying enzymes were measured in last instar larvae of Heliothis zea and Spodopterafrugiperda after the insects had been fed for 48 h on diets containing representative trichothecenes, which are protein synthesis inhibitors. 4-Nitroanisole O-demethylation was induced (as indicated by increased enzyme activity) by 1.6-fold in H. zea and by 6. l-fold in S. frugiperda when fed 250 ppm of deoxynivalenol, a dose that severely retarded growth of the insects. Induction of this activity was also noted with lower levels of deoxynivalenol in both species, and with diacetoxyscirpenol and T-2 toxin in H. zea. 1-Chloro-2,4-dinitrobenzeneglutathione transferase conjugation was induced by c. 1.3-fold with deoxynivalenol at 250 ppm and 25 ppm, and with T-2 toxin at 25 ppm in H. zea; but was slightly inhibited (c. 10-20%) in S. frugiperda in some cases. l-Naphthyl acetate hydrolysis was generally unaffected by all trichothecenes tested at all doses in H. zea, but was inhibited by c. 30% in S. frugiperda by lower doses of all three trichothecenes. However, a new 1-naphthyl acetate esterase band was noted after exposure to 250 ppm of deoxynivalenol, and to some extent lower doses of deoxynivalenol and the other trichothecenes in both insect species. The hydrolysis of a radiolabeled model trichothecene, 4-monoacetoxyscripenol, was induced by 3.l-fold in H. zea and 2.7-fold in S.frugiperda with 250 ppm of deoxynivalenol (the only compound and dose tested). This activity could be inhibited by nearly 100% with 10-4 M paraoxon, suggesting a serine-hydroxyl esterase was involved. Although this activity could not be recovered from gels, indirect competitive inhibition assays with t-naphthyl acetate suggested the induced band could be responsible for some of the hydrolysis of the 4-monoacetoxyscirpenol. Key Word Index: enzyme induction, deoxynivalenol, diacetoxyscirpenol, T-2 toxin

INTRODUCTION

Enzymatic detoxification is a major means that insects use to avoid the toxicity of xenobiotics (Ahmad et al., 1986). In several instances, it appears that these enzymes may be induced (based on increases in activity on a per mg protein basis) by appropriate substrates in order to economize the costs of producing them (Yu, 1986). However, in some cases these enzymes may be inhibited by certain xenobiotics, such as quercetin inhibition of monooxygenases (e.g. Testa and Jenner, 1981), or organophosphate inhibition of esterases (e.g. F o n n u m et al., 1985). Thus, the overall response of detoxifying enzymes to a particular xenobiotic is likely to lie along a gradient of relative inducibility/inhibition of the different enzymes responsible for detoxification. The trichothecenes are mycotoxins produced by many species of Fusarium that are often plant pathogens (Committee on Protection Against Mycotoxins, 1983), and are toxic to a variety of insects (Wright et al., 1982). Trichothecenes are cytotoxic, and appear to act by inhibiting protein synthesis (Committee on

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Protection Against Mycotoxins, 1983). Thus, in cases where insects encounter trichothecenes by feeding on Fusarium-contaminated materials, the type of interaction that may occur is uncertain. Like other xenobiotics, they may have the appropriate functional groups to induce these enzymes. On the other hand, since they interfere with protein synthesis, levels of detoxifying enzymes may be reduced. In order to investigate this potentially antagonistic interaction, H. zea and S. frugiperda, which may potentially encounter trichothecenes when feeding on infected corn, were tested for relative activity of representative detoxifying enzymes after being fed diets containing naturally relevant concentrations of the representative trichothecenes deoxynivalenol, diacetoxyscirpenol and T-2 toxin (Fig. 1). MATERIALS AND METHODS Insects

Both insect species were maintained on pinto bean-based diet (Dowd, 1987) as described previously (Dowd, 1988a). Newly molted last instar larvae were placed on diets containing the xenobiotics for 48 h, and then assayed for enzyme activity. Chemicals

Diacetoxyscirpenol (DAS), T-2 toxin, deoxynivalenol (DON), I-naphthyl acetate, fast blue BB salt, NADP,

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PATRICK F. DOWD

M°U-

I

Fig. 1. Trichothecenes. DAS: R l = H 2, R 2 = H 2, R 3=CH2OAc, R 4=OAc, R 5 = O H ; T-2: R~ = H,OCOCH2CHMe2, R: = CH2OAc, R 3 = CH:OAc, R4 = OAc, R 5 = OH; DON: R~ = O, R: = OH, R 3 = CH2OH, R 4 = OH, R 5 = OH; MAS: R I = H 2 , R 2 = H2, R 3 = CH2OH, R 4 = OAc, R 5 = OH; scirpentriol: R 1 = H2, R 2 =H2, R 3 =CH2OH , R 4 = O H , R 5 = O H . glucose-6-phosphate, glucose-6-phosphate dehydrogenase, glutathione, bis-acrylamide, indole-3-carbinol, ammonium persulfate, TEMED and 4-nitroanisole were obtained from Sigma Chemical Company. The acrylamide and protein reagent were obtained from Bio-Rad, the Tris and glycine from Research Organics and the paraoxon from Aldrich Chemical Company. Radiolabeled [15-3H]-4-monoacetoxyscirpenol (MAS) (3 mCi/mmol), unlabeled MAS and scirpentriol (Fig. 1) were kindly provided by F. L. Van Middlesworth (present address, Merck, Sharp & Dohme Research Laboratories, Rahway, N.J.). All other chemicals were reagent grade.

Chemical incorporation All chemicals were incorporated by adding them in 125/~1 of acetone to 5 ml of unsolidified pinto bean-based diet (Dowd, 1988a). Diets were blended with a vortex mixer and dispensed into Petri plates for cooling (Dowd, 1988a). One quarter of the diet section (approx. 1.25 gm) was given individually to each insect. Concentrations used in this study were based on naturally reported concentrations, and included DON at 250, 25 and 2.5 ppm (Vesonder et al., 1976; Miller et al., 1983; Shotwell et al., 1985); and T-2 and DAS at 25 and 2.5 ppm (Siegfried, 1977; Puls and Greenway, 1976; respectively). Indole3-carbinol, a generally effective inducer of detoxifying enzymes in insects (Yu, 1986) was used at 250 ppm for comparison. Enzyme assays Midguts were dissected in chilled, 0.1 M, pH 7.8, phosphate buffer, 4 midguts were homogenized in 2 ml of the same buffer, centrifuged at 1200g for 5 min and the supernatant filtered through a 0.45 micron filter (Dowd 1988b). The filtrate was appropriately diluted to yield linear rates of enzyme activity, which depended on the assay (Dowd 1988b). Assays for the hydrolysis of l-naphthyl acetate, glutathione conjugation of CDNB and O-demethylation of 4-nitroanisole were performed as previously described (Dowd and Sparks, 1984; Dowd et al., 1986; Rose and Brindley, 1985; respectively--as modified in Dowd 1988b). The hydrolysis of l-naphthyl acetate was assayed for 20 min at 35°C. The product was detected by complexing with fast blue BB salt, and kept in solution with sodium dodecyl sulfate. The diazo complex was quantitated at 600 nm. The conjugation of CDNB with glutathione was continuously monitored at 35°C for 10 min at 345 nm. The O-demethylation of 4-nitroanisole was assayed for 30 min at 35°C. The reaction was stopped by adding acetone and pH 9.5 glycine-NaOH buffer, which also served to shift the pH of the mixture above 8.2, to maximize the intensity of the the yellow 4-nitrophenol product. The quantity of 4-nitrophenol present was determined at 410 nm. The hydrolysis of MAS was determined as reported previously (Dowd and Van Middlesworth, 1989). One tal of MAS stock in ethanol (3.09 x 10-1° mol) was added to 50/~1 of undiluted midgut filtrate, and incubated at 35°C for up to 1 h, depending on the preparation. The incubates were spotted directly on LK5DF thin layer plates (Whatman), air dried and chromatographed for c. 15cm with toluene:acetone

(1 : I). Unlabeled MAS and scirpentriol were used as standards. The appropriate areas were scraped and quantitated by liquid scintillation counting. Due to the limited amount of radiolabeled MAS available, MAS assays were only performed with insects fed solvent controls and DON at 250 ppm. T-2 and diacetoxyscirpenol were tested as inhibitors of l-naphthyl acetate hydrolysis by coincubating 1 x 10 -3 M concentrations with 1 x 10-3, 10 -4 and 10-5 M concentrations of l-naphthyl acetate and comparing activity ! to solvent controls. Paraoxon and l-naphthyl acetate were tested as inhibitors of MAS hydrolysis by incubating 1 x 10-4 concentrations with the previously indicated concentration of MAS and comparing activity to solvent controls. All enzyme assays were performed at least in duplicate on at least two separate occasions. Protein levels were determined with the Bio-Rad packaged assay (Bio-Rad, 1985). Values were tested for significant differences by analysis of variance. Electrophoretic separation and identification of enzymes capable of hydrolyzing 1-naphthyl acetate was performed according to previously published methods (Dowd and Sparks, 1986). Briefly, 10#1 of undiluted midgut homogenates was added to wells of 2 mm thick, 7.5% polyacrylamide gels. The gels were electrophoresed using pH 8.6, 0.1 M Tris-glycine buffer at 35 mA until the bromophenol tracking dye reached the opposite side of the gel. The gels were stained for protein using Coomassie brilliant blue R, and assayed for 1-naphthyl acetate esterase activity by incubating the gels in pH 7.4, 0.1 M buffer containing 0.04% 1-naphthyl acetate and 0.4% fast blue BB salt. The location of MAS hydrolase activity was assayed by cutting the gel in 1 cm sections, eluting the sections in 1 ml of phosphate buffer overnight, and assaying the buffer for activity as described previously. This methodology had proved successful previously in recovering permethrin-hydrolyzing activity from gels (Dowd and Sparks, 1986). All gel assays were performed at least in duplicate on at least two separate occasions. RESULTS

Toxicity A t 250 ppm, D O N was highly toxic to b o t h H. zea a n d S. frugiperda (Table 1). A l t h o u g h no mortality resulted, sublethal toxicity was a p p a r e n t since the larvae grew very little (initial weights were c. 200 mg for b o t h insect species). Some toxicity for all of the trichothecenes was noted at 25 p p m as well, but n o n e at 2.5 ppm. Indole-3-carbinol was essentially nontoxic to b o t h insect species at 250 ppm. Enzyme activity The P N A O-demethylase activity was c. 6-fold induced in S. frugiperda by D O N at 250 ppm, a n d induced by c. 3-fold when at 2 5 p p m (Table 2). Indole-3-carbinol at 2 5 0 p p m induced P N A Od e m e t h y l a t i o n activity in H. zea by c. 3-fold, while D O N at all c o n c e n t r a t i o n s tested a n d T-2 at 25 p p m

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Fig. 2. Protein banding patterns for midgut homogenates of H. zea exposed to dietary trichothecenes. 1, Control; 2, indole-3-carbinol, 250 ppm; 3, DON, 250 ppm; 4, DON, 25 ppm; 5, DON, 2.5 ppm; 6, T-2, 25 ppm; 7, T-2, 2.5 ppm; 8, DAS, 25 ppm; 9, DAS, 2.5 ppm.

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Fig. 3. Protein banding patterns for midgut homogenates of S. frugiperda exposed to dietary trichothecenes. 1, Control; 2, indole-3-carbinol, 250 ppm; 3, DON, 250 ppm; 4, DON, 25 ppm; 5, DON, 2.5 ppm; 6, T-2, 25 ppm; 7, T-2, 2.5 ppm; 8, DAS, 25 ppm; 9, DAS, 2.5 ppm. 351

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Fig. 4. 1-Naphthyl acetate esterase banding patterns for midgut homogenates of H. zea exposed to dietary trichothecenes. 1, Control; 2, indole-3-carbinol, 250 ppm; 3, DON, 250 ppm; 4, DON, 25 ppm; 5, DON, 2.5 ppm; 6, T-2, 25 ppm; 7, T-2, 2.5 ppm; 8, DAS, 25 ppm; 9, DAS, 2.5 ppm. Arrow indicates induced bands.

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Fig. 5. l-Naphthyl acetate esterase banding patterns for midgut homogenates of S. frugiperda exposed to dietary trichothecenes. 1, Control; 2, indole-3-carbinol. 250 ppm; 3, DON, 250 ppm; 4, DON, 25 ppm: 5, DON, 2.5 ppm; 6, T-2, 25 ppm; 7, T-2, 2.5 ppm; 8, DAS, 25 ppm; 9, DAS, 2.5 ppm. Arrow indicates induced bands. 352

Responses of representative midgut detoxifying enzymes Table I. Effect of trichothecenes on the development of H. zea and S. frugiperda larvae Weight of 2-day old last instars (mg) Compound Control I-3-ol DON DON DON T-2 T-2 DAS DAS

250 ppm 250 ppm 25 ppm 2.5 ppm 25 ppm 2.5 ppm 25 ppm 2.5 ppm

Heliothis zea

Spodoptera frugiperda

604 + 40 486 + 44 259 + 24* 470 _ 26* 558 + 15 476 + 47 545 + 14 434 -t- 31" 555 + 19

509 + 16 494 + 20 231 + 13" 458 __.21 515 + 21 374 + 29* 463 + 22 386 + 22* 520 +_ 18

Values are means + SE for at least 8 larvae. I-3-ol, indole-3-carbinol; DON, deoxynivalenol; DAS, diacetoxyscirpenol. Values followed by an asterisk are significantly different at P < 0.05 by analysis of variance.

also increased activity of this enzyme (by 1.2 to 1.6-fold). The CDNB glutathione conjugation activity was significantly induced in H. zea by indole-3carbinol and DON at 250ppm (both by 1.3-fold), and by both DON (1.3-fold) and T-2 (l.2-fold) at 25 ppm. Conversely, although slight (but nonsignificant) induction was noted for the two compounds when tested at 250 ppm, CDNB glutathione conjugation activity was significantly inhibited by DON (c. 25%) and T-2 (c. 16%) at 2.5ppm in

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not shown). The hydrolysis of l-naphthyl acetate by H. zea midgut homogenates was not inhibited by greater than 10% with either DAS or T-2 (data not shown) except for DAS when coincubated with 10 -5 M l-naphthyi acetate (remaining activity was 77.2 + 3.1% of the control). The hydrolysis of MAS by H. zea midguts was induced by 3. I-fold with DON at 250 ppm; that of S. frugiperda was induced by 2.7-fold (Table 3). Paraoxon inhibited the hydrolysis of MAS by greater than 95% in all cases (Table 3). Although l-naphthyl acetate inhibited the hydrolysis of MAS in all cases, the degree of inhibition was only significant in the case of the induced activity in S. frugiperda (Table 3). Gel electrophoresis

No obvious consistent differences were noted in protein banding patterns among the different treatments although high Rf bands were sometimes present (Figs 2 and 3). However, induction of an l-naphthyl acetate-hydrolyzing esterase at c. Rf 0.22 was noted for H. zea fed on trichothecene-containing diets, that appeared to increase in intensity relative to the rest of the bands (especially noted for DON) (Fig. 4). A similar response (c. Rr 0.40) was noted for S. frugiperda under the same conditions (Fig. 5). No MAS hydrolytic activity could be recovered from the gels.

S. frugiperda.

The rate of 1-naphthyl acetate hydrolysis was significantly increased in H. zea by DON at 25 ppm (1.2-fold), and similar increases were noted for DAS at 25 and 2.5 ppm. Conversely, the rate of l-naphthyl acetate hydrolysis in S. frugiperda was significantly inhibited by T-2 (c. 17%) and DAS (c. 12%) at 25 ppm; and by all three trichothecenes at 2.5 ppm (all by c. 35%). The hydrolysis of 1-naphthyl acetate by S. frugiperda midgut homogenates was not inhibited by more than 5% with either DAS or T-2 (data

DISCUSSION

Induction of detoxifying enzymes has been reported in a variety of insects (see Yu, 1986, for review). However, with the exception of xanthotoxin (Yu, 1984), levels of chemicals used in induction experiments are typically >0.1% (1000ppm); such concentrations are unrealistic in evaluating the potential inducing ability of naturally occurring levels of trichothecenes due to their much lower concentra-

Table 2. Effect of trichothecenes on representative midgut detoxifying enzymes in H. zea and S. frugiperda larvae Compound

PNA

CDNB

ANA

351 + 38 464 + 30* 453+16" 456 + 12" 324 __. 12 422 + 22* 347 +_ 38 347 + 6 416 _+ 25

1.62 + 0.08 1.49 + 0.01 1.78+0.04 1.99 + 0.19" 1.60 + 0.03 1.54 +_ 0.03 1.80 + 0.04 1.82 + 0.02* 1.82 __.0.12"

Heliothis zea Solvent I-3-ol DON DON DON T-2 T-2 DAS DAS

250 ppm 250 ppm 25 ppm 2.5 ppm 25 ppm 2.5 ppm 25 ppm 2.5 ppm

2.5 _+ 0.5 7.4 + 0.1" 4.0+0.1" 3.7 __.0.4* 3.1 + 0.2* 2.9 + 0.3 3.9 + 0.5* 3.2 +_ 0.8* 2.8 + 0.3

Spodoptera .[~'ugiperda Solvent I-3-ol DON DON DON T-2 T-2 DAS DAS

250 ppm 250 ppm 25 ppm 2.5 ppm 25 ppm 2.5 ppm 25 ppm 2.5 ppm

0.9 + 0.3 I.I + 0.8 5.5 + 0.5* 2.8 + 0.4* 0.7+0.6 1.2 _+ 0.3 1.0 + 0.5 1.0 _+0.2 1.2 + 0.1

209 + 3 283 + 44 283 + 19 192 _+ 18 156+__8" 196 + 8 176 + 9* 210 + 10 232 + 23

3.31 +_0.11 3.13 + 0.29 3.56 + 0.11 3.13 +__0.05 2.12+0.11" 2.76 + 0.04* 2.06 + 0.15" 2.92 __.0.06* 2.21 + 0.19"

Values are mean nmol/min/mg protein (,u tool for ANA) + SE for at least two assays performed in duplicate (triplicate for ANA). I-3-ol, indole-3-carbinol; DON, deoxynivalenol; DAS, diacetoxyscirpenol; PNA, 4-nitroanisole O-demethylation; CDNB, I-chloro-2,4-dinitrobenzene glutathione conjugation; A N A , I-naphthyl acetate hydrolysis. Values followed by an asterisk are significantly different from controls at P < 0.05 by analysis of variance.

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PATRICKF. DOWD Table 3. Hydrolysis of 4-monoacetoxyscirpenol by midguts of H. zea and S. frugiperda Treatment Solvent + 250 ppm DON Solvent +250 ppm DON

Solvent

+ Paraoxon

+ ANA

1.0 + 0.3* 1.9 + 0.6*

21.5 _+0.3 64.0 -+ 2.0

Spodoptera .frugiperda 31.7+1.0 0.5+0.2* 86.8_+2.5 3.1 _+ 1.2"

29.4_+0.4 74.8_+2.8*

Heliothis zea 22.3 + 0.3 69.0 + 2.2

Values are mean pmol/min/mg protein + SE for at least two assays performed in duplicate. ANA, I-naphthyl acetate; DON, deoxynivalenol. Values in rows followed by an asterisk are significantly different from controls at P < 0.05 by analysis of variance.

tion. Oxidative enzymes are frequently induced to the greatest extent relative to conjugating or hydrolytic enzymes by allelochemicals or other xenobiotics (Yu, 1986). This trend was also noted in the present study (e.g. DON induction of PNA O-demethylase activity in both insect species). Significant induction of glutathione transferases by plant allelochemicals has been reported as well (Yu, 1986). In the present study, significant induction of CDNB-glutathione conjugation was also noted for H. zea. The degree of induction of hydrolytic enzymes in insects is typically relatively low (less than 2 x ) compared to unspecific monooxygenase or glutathione transferase activity (Yu, 1986), although some mammalian esterases may be induced to a much greater degree (e.g. Hosokawa et al., 1988). Although low (but nonsignificant) levels of total 1-naphthyl acetate esterase induction occurred in the present study, many examples of apparent inhibitory responses occurred in S. frugiperda. It is possible that the trichothecenes did inhibit the synthesis of these enzymes. It is also possible the trichothecenes may bind to the hydrolytic enzymes but are not readily hydrolyzed, resulting in apparent overall inhibition, as appeared to have happened when DAS was coincubated with 10-SM 1-naphthyl acetate in H. zea gut preparations. Although spectrophotometric assays indicated either minor induction or inhibition in l-naphthyl acetate activity by the trichothecenes, gel electrophoresis indicated obvious induction of an esterase band in both insect species (especially for DON). The hydrolysis of the model trichothecene MAS was also significantly increased in both insects in the presence of DON at 250 ppm. This is relevant in that hydrolysis is reported to be a major route for trichothecene detoxification in a variety of organisms (Fonnum et al., 1985). For example, hydrolysis of T-2 (e.g. Ellison and Kotsonis, 1974; Ueno et al., 1983; Munger et al., 1987) and DAS (e.g. Ohta et aL, 1978; Kiessling et al., 1984; Bauer et al., 1985) has been reported in a variety of organisms. Although DON is not an ester, hydrolysis of acetylated DON could be an appropriate detoxification reaction because DON is less toxic than the acetylated form. Hydrolysis of trichothecene esters frequently results in significant reduction in cytotoxicity of the resulting metabolite (Grove and Hoskins, 1985; Joffe, 1986). Since MAS activity could not be recovered from the gel, it is not possible to definitely state whether the induced l-naphthyl acetate band is the source of the increased rate of MAS hydrolysis. However, the

trends show that the 1-naphthyl acetate was relatively more effective in inhibiting the MAS hydrolysis by the midgut homogenates of insects fed the DON, indicating that a relatively greater proportion of enzymes responsible for hydrolyzing the MAS could be tied up by the l-naphthyl acetate. Since the induced band is the only obvious qualitative change in l-naphthyl acetate hydrolysis compared to the total amount of l-naphthyl acetate hydrolysis that occurs, and since the total l-naphthyl acetate hydrolysis was not significantly different in control vs insects fed DON at 250 ppm, this suggests the l-naphthyl acetate band could be responsible for some of the increases in MAS hydrolysis. Further study is necessary to clarify this potential relationship. However, the enzyme(s) responsible for the MAS hydrolysis do appear to be carboxylesterases, since activity is effectively inhibited by paraoxon. Induction of the esterases may result in significant decreases in toxicity provided these esterases can hydrolyze naturally occurring trichothecenes. Other portions of these compounds are relatively resistant to metabolism. For example, the epoxide moiety is highly resistant to modification by glutathione transferases and epoxide hydrolases in mammals (Nakamura et al., 1977). Reduction of the epoxide to a diene (possibly performed by unspecific monooxygenases) has been reported in some cases (Yoshizawa et al., 1985; Sakamoto et al., 1986). Thus, the induction of PNA O-demethylase activity noted with DON and S. frugiperda may be important in determining relative toxicity of different trichothecenes if this activity is representative of other unspecific monooxygenases (i.e. those that are capable of epoxide reduction), even though this is not the major route of metabolism. For example, although hydrolysis was the major route of metabolism in both carbaryl-susceptible and resistant strains of S. frugiperda, resistance was apparently conferred by significant increases in oxidative enzyme activity in the resistant strain (McCord and Yu, 1987). Multiphasic responses to xenobiotics (i.e. the differential ability of the same compound to induce and inhibit different isozymes that act on the same substrate) is not uncommon (Testa and Jenner, 1981). This situation appears to have occurred in the present study as well in the case of CDNB-glutathione transferase and l-naphthyl acetate-esterase activity, since both inhibition and induction were noted, depending on compound and concentration. This response (as in the case of the esterase bands) may

Responses of representative midgut detoxifying enzymes reflect overall response of individual enzymes with varying substrate specificity, inhibitor susceptibility and inducibility as well. The results of the present study indicate that natural levels of trichothecenes are capable of inducing enzymes that could be appropriate in their detoxification. The known ability of trichothecenes to inhibit protein synthesis is not an obvious factor in affecting this response. A similar effect was noted for some rat tissues when rats were fed T-2 and DAS. Benzphetamine N-demethylase and ethoxycoumarin O-deethylase were induced in rat lung by T-2, and C D N B glutathione transferase was induced in rat liver and kidney by both D A S and T-2 (hydrolytic enzyme activity was not examined) (Galtier et al., 1989). The fact that toxicity was still noted suggests that these insects are not well adapted to trichothecenes, something that would be expected considering the unpredictable presence of these compounds in potential foods for these insects. However, when the trichothecenes cooccur with plant allelochemicals they may influence the toxicity of these compounds to insects through enzyme induction or inhibition. Acknowledgements--I thank C. L. Weber for technical assistance and T. C. Nelsen for suggestions on appropriate statistical analyses. REFERENCES

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