Methylenedioxyphenyl compounds as inducers of cytochrome P-450 and monooxygenase activity in the southern armyworm (Spodoptera eridania) and the rat

26, 310-322 (1986)

PESTICIDEBIOCHEMISTRYANDPHYSIOLOGY

Methylenedioxyphenyl Compounds as inducers of Cytochrome and Monooxygenase Activity in the Southern Armyworm (Spodoptera eridania) and the Rat CRAIG

Institute

for

Comparative

and

B. MARCUS,~ MICHAEL AND CHRISTOPHER Environmental

Toxicology Ithaca, Neut

MURRAY,*

CHENG

P-450

WANG,

F. WILKINSON and York

Department 14853

of Entomology,

Cornell

University.

Received December 17, 1985; accepted February 11, 1986 Exposure of sixth-instar southern armyworm larvae (Spodoptera eridania) to each of five methylenedioxphenyl (MDP) compounds (0.05%. w/v in the diet) led to increased levels of cytochrome P-450 and aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity in preparations of midgut microsomes. Isosafrole, the most potent compound evaluated. resulted in AHH levels almost 30-fold higher than in controls; it also enhanced levels of ethoxycoumarin O-deethylase activity (-15fold) but did not induce ethoxyresorutin O-deethylase or aldrin epoxidase. MDP-induced AHH activity was highly sensitive to inhibition by a-naphthoflavone (I,, = 0.4 pkf) but was not inhibited by I-phenylimidazole at 150 pM. Analysis of the major metabolites of. benzo[a]pyrene in control and isosafrole-induced midgut microsomes established that MDP compounds promote the formation of the 3-hydroxy, 9-hydroxy. and 7,Gdihydrodihydroxy metabolites. In contrast, the metabolite pattern resulting from pentamethylbenzene induction indicated enhanced formation of the 9,10-dihydrodiol of benzo[a]pyrene. These metabolite patterns indicate the presence of armyworm midguts of distinct isozymes with different regiospecifities toward benzo[a]pyrene, and establish that the isozymes are different from those present in hepatic microsomes from control and phenobarbital-. S-naphthoflavone-, and dihydrosafrole-induced rats.

0 1986 Academic

Press. Inc.

INTRODUCTION

Insects, like mammals, possess a versatile polysubstrate monooxygenase (PSMO) system that plays an important role in the metabolic biotransformations of a wide array of naturally occurring (1, 2) and synthetic (3, 4) xenobiotics. The overall biochemical characteristics of insect and mammalian PSMO systems are quite similar to one another (5-7) and both are responsive to induction following in viva exposure to a variety of chemicals of both natural and synthetic origin (8- 12). i Present address: Department of Pharmacology, University of Wisconsin Medical School. Madison, Wise. 53706. * Present address: Department of Medicine. University of Sydney, Westmead Hospital, Westmead, NSW 2145, Australia.

Methylenedioxyphenyl (MDP) compounds are best known for their inhibitory interactions with cytochrome P-450 and associated PSMO activities (13, 14) and as a consequence, some, such as piperonyl butoxide, have been developed commercially as insecticide synergists (15, 16). It has been recognized for some time, however, that single in viva administrations of MDP compounds to both mammals and insects typically elicit a biphasic response with respect to PSMO activity, a transient inhibitory action being followed by a more prolonged inductive phase leading to enhanced levels of cytochrome P-450 and several oxidase activities (17, 18). The ability of MDP compounds to induce various hepatic PSMO activities, following repeated in viva administration to animals has also been known for more than a decade (19, 20). 310

0048-3575/86 $3.00 Copyright All rights

0 1986 by Academic Press. Inc. of reproduction in any form reserved.

INDUCTIVE

EFFECTS

OF METHYLENEDIOXYPHENYL

Interest in MDP compounds as inducers of hepatic PSMO activity was stimulated by the finding that isosafrole promotes the induction of a novel isozyme of rat hepatic cytochrome P-450, termed cytochrome P-450d, with structural and catalytic properties distinct from those of isozymes induced by most other xenobiotics (21-23). In addition to cytochrome P-450d, isosafrole also induces cytochrome P-450~ and P-450b (23). Subsequent studies have demonstrated the induction of cytochrome P-450 by dihydrosafrole and other MDP compounds (24-26) and have established that this is associated with an increase in aryl hydrocarbon (benzolalpyrene) hydroxylase (AHH) activity. The present study was undertaken to evaluate the capacity of MDP compounds to induce cytochrome P-450 and PSMO activity in midgut tissues of the southern armyworm (Spodopteru eridaniu) and to attempt to characterize the induced enzymes and compare them with those in rat liver. MATERIALS

AND

METHODS

Animals. A colony of the southern armyworm, S. eridania, was maintained under greenhouse conditions as described by Krieger and Wilkinson (27). Male Sprague-Dawley rats (200-250 g) were purchased from Blue Spruce Farms, Altamont, New York. Chemicals. p-Naphthoflavone, o-naphthoflavone, unlabeled benzo[a]pyrene (BP), and pentamethylbenzene were purchased from Aldrich Chemical Company, Milwaukee, Wisconsin. Isosafrole and lphenylimidazole (PI) were purchased from Frinton Laboratories, Vineland, New Jersey, and Tram World Chemicals, Washington, D.C., respectively, and both were purified by vacuum distillation prior to use. Dihydrosafrole was prepared as described previously (24). [G3H]BP (sp act 27 Ci/ mmol) was purchased from the Amersham Corporation, Arlington Heights, Illinois. BP metabolite standards were obtained

COMPOUNDS

311

from the National Institutes of Health, Bethesda, Maryland. Biochemicals were purchased from Sigma Chemical Company, St. Louis, Missouri, and all other chemicals and solvents were either analytical or reagent grade. Animal treatments tion. Newly molted

and enzyme prepara-

sixth instar southern armyworm larvae were removed from bean plants in the greenhouse and transferred to containers held in an environmental chamber maintained at 26°C under conditions of 50-60% relative humidity and employing a 16-hr light&hr dark photoperiod. Larvae were fed ad libitum for 72 hr on semidefined diets into which appropriate concentrations of the selected inducers were incorporated (10). Microsomal fractions were prepared from larval midgut tissues as described previously (28) and were stored as frozen pellets for a maximum of 4 days prior to use. Hepatic microsomes were prepared as previously described (24) from rats treated for 3 days with either phenobarbital (100 mg/kg ip in 0.9% NaCl), p-naphthoflavone (40 mg/kg ip in corn oil), or dihydrosafrole or isosafrole (400 mg/kg ip in corn oil). Control rats did not receive any treatment. Microsomes were stored as frozen pellets and used within 2 weeks of preparation. Cytochrome P-450 purification. The major phenobarbital-inducible (P-450b) and p-naphthoflavone-inducible (P-450~) isozymes of rat hepatic microsomal cytochrome P-450 were purified as described by Marcus et al. (29). Polysubstrate

monooxygenase

(PSMO)

assays. Armyworm midgut or rat hepatic microsomal pellets were resuspended in 0.1 M potassium phosphate buffer, pH 7.4, at a concentration of 2 mg microsomal protein/ ml. Appropriate aliquots were added to individual reaction mixtures for the various assays of PSMO activity as described below. Assays of PSMO activity utilizing purified rat hepatic cytochrome P-450 were

312

MARCUS

conducted in reconstituted systems as described previously (29). Total aryl hydrocarbon (BP) hydroxylase (AHH) activity was determined by the modification of the spectrofluorometric method of Yang and Kicha (30) described by Denison et al. (31). 7-Ethoxycoumarin 0-deethylase and 7-ethoxyresorufin Odeethylase activities were determined by the spectrofluorometric methods of Ullrich and Weber (32) and Prough et al. (33), respectively. Aldrin epoxidase activity was determined by the gas chromatographic method of Lewis et al. (34). Inhibitors were added to the reaction mixtures in absolute ethanol (50 p,l) and molar I50 values were determined by leastsquares linear regression analysis of PSMO activity against log inhibitor concentration. Duplicate or triplicate incubations using a minimum of four different inhibitor concentrations were conducted for each experiment. Other assays. Cytochrome P-450 was measured by the method of Omura and Sato (35) using an Aminco DW-2 spectrophotometer and employing an extinction coefficient of 91 mM- ’ cm-* for the cytochrome P-450 ferrous carbonyl spectral complex. Protein concentrations were determined by the method of Lowry et al. (36). Benzo[a]pyrene metabolism and HPLC analysis. [G-3H]BP (sp act 27 Ci/mmol) was

incubated with microsomes at 37°C for 10 min under conditions similar to those described by Wiebel et al. (37). Incubations (5 ml) contained 5 mg microsomal protein, 400 nmol BP, 6.5 pmole NADP, 16.5 pmol glucose 6-phosphate, 0.5 units glucose-6phosphate dehydrogenase, and 1 pmol EDTA, in 50 pJ4 Tris-HCl buffer (pH 7.6) (38, 39). Reactions were terminated by addition of 0.2 ml of 6 N HCl, saturated with NaCl, and extracted with ethyl acetate (2 X 3 ml) to remove BP and its metabolites. The ethyl acetate extract was dried and evaporated under a stream of dry nitrogen,

ET AL.

and the residue was redissolved in 200 ~1 of acetonitrile for HPLC analysis. All incubations were performed in the dark due to the light sensitivity of some BP metabolites (40, 41). BP metabolites were analyzed on a programmable Varian 5000 liquid chromatograph, fitted with a Varian Micropak MCH 5 column (0.4 x 30 cm) operated at 22°C. Ten microliters of each sample was injected. Elution was with acetonitrile:water (45:55) for 5 min, followed by an increasing linear gradient of acetonitrile at a rate of 1% per minute up to 100% acetonitrile; a constant flow rate of I ml/min was maintained. The column was eluted with 100% acetonitrile for a further 20 min after which it was reequilibrated with acetonitrile:water (45:55) prior to the next injection. During sample elution, the uv absorbance was monitored at 254 nm and fractions were collected directly into scintillation vials at 30 set intervals. Ten milliliters of Liquiscint (National Diagnostics) was added to each vial and radioactivity was measured in a Packard Tri-Carb scintillation spectrometer Model 2425. BP metabolites were identified by their retention times, compared with authentic standards, and were quantitated by the radioactivity associated with each peak, assuming a uniform labeling of BP. Although the HPLC protocol employed was unable to resolve all possible BP metabolites, adequate separation and quantitation of the major metabolites was effected. The BP-I-, 3-, IO-, and I l-phenols cochromatographed in the HPLC system employed, as did the BP-4-, 5-, S-, 9-, and 12-phenols and the BP-1,6-, 3,6-, and 6,12quinones. Therefore, each group of phenols is referred to collectively as the major product in the group, i.e., BP-3-OH or BP-9-OH. Since only the l-, 3-, 7-, and 9-phenols are actually formed enzymatitally (at least in mammals), and the 3- and 9-phenols constitute the major phenolic

INDUCTIVE

EFFECTS OF METHYLENEDIOXYPHENYL

products, the limitation of the HPLC method are in reality minimal.

The results shown in Table 1 indicate that while in vivo treatment of southern armyworm larvae with a variety of MDP derivatives results in only a modest increase (no more than 2-fold) in levels of midgut cytochrome P-450, corresponding increases in AHH activity are frequently substantial. Isosafrole and the 4-bromo-Smethoxy derivative are particularly effective inducers of AHH leading to activities that are almost 30-fold greater than those in controls; these two compounds also cause the largest increases in cytochrome P-450 (1.8-fold). In contrast, the 4,Sdichloro MDP derivative appears to cause an almost 19-fold increase

TABLE P-450 and Aryf Hydrocarbon Midgut and Rut Liver following

Treatmentb Southern armyworm None (control) 4-CH,CH = CH-MDBd (isosafrole) 4,5-diCl-MDB 4-Br-5-CH,O-MDB I-C,H,-MDB (dihydrosafrole) 4-C,H,O-MDB Phenobarbital @-Naphthoflavone Pentamethylbenzene Rat None (control) Phenobarbital P-Naphthoflavone Isosafrole

313

in AHH activity without significantly enhancing cytochrome P-450 levels. Even the least effective of the MDP compounds evaluated in this study (dihydrosafrole and the 4-ethoxy derivative) were both potent inducers of AHH activity and elicited a response similar to that of pentamethylbenzene [hitherto the most active armyworm AHH inducer reported (12)] and considerably greater than that of either phenobarbital or P-naphthoflavone. Table 1 also shows data on the relative potencies of phenobarbital, P-naphthoflavone, and isosafrole as inducers of hepatic microsomal AHH activity in rats. In rats, isosafrole is a considerably less effective inducer of AHH activity than p-naphthoflavone. Under the conditions employed in this study, isosafrole is lo-fold more effective than l3-naphthoflavone as an inducer of

RESULTS

Cytochrome

COMPOUNDS

1

Hydroxylase (AHH) in Mirrosomal Exposure to Methylenedioxyphenyl

Cytochrome P-450 (nmolimg protein)

Suspensions from Armyworm and Other Compounds”

AHH activity (nmol BP metabolized mg protein/min)

0.18 2 0.01 (lOO)C 0.34 k 0.04 (190)

0.21 + 0.06 (100) 6.28 2 1.90 (2990)

0.21 * 0.03 (120) 0.33 2 0.03 (180) 0.23 k 0.04 (130)

3.95 2 1.30(1880) 6.17 t 0.28 (2940) 1.33 t 0.47 (630)

0.27 0.35 0.55 0.52

2 f 5 4

0.03 0.01 0.03 0.07

(150) (190) (300) (290)

2.49 0.29 0.69 2.25

‘+ + ?I

1.40(1190) 0.09 (140) 0.11 (330) 0.83 (1070)

0.70 1.81 1.41 1.22

* k * k

0.07 0.12 0.12 0.12

(100) (260) (200) (170)

0.99 2.11 6.16 3.98

e ” 2 ”

0.06 0.25 1.12 0.81

(100) (210) (620) (400)

0 Values are means t SD of 3-6 experiments. b Armyworm larvae were fed ad libitum for 72 hr on diets containing the compound indicated at the following concentrations w/v); all MDP compounds (O.OS%), phenobarbital (0.25%), P-naphthoflavone (O.l%), and pentamethylbenzene (0.2%). Rats were injected intraperitoneally as described under Materials and Methods. c Values in parentheses are percentage relative to controls. d MDB = 1,2-methylenedioxybenzene.

314

MARCUS ET AL.

AHH activity in armyworms. The actual level of AHH activity in isosafrole-induced armyworm midguts (6.28 nmol BP metabolized min/mg protein) is similar to that measured in p-naphthoflavone-induced rat liver microsomes despite the fact that the latter contain about four times more cytochrome P-450. In terms of activity per nanomole of cytochrome P-450, the values are 18.5 and 4.35 nmol BP metabolized/min/nmol of cytochrome P-450, respectively, for armyworm midgut and rat liver. The high specific activity of the cytochrome P-450 isozymes in isosafrole-induced armyworm midgut microsomes was further demonstrated by additional experiments in which the turnover numbers for selected PSMO reactions were compared with those measured in reconstituted systems containing purified rat liver cytochromes P-450b and P-450~. The results in Table 2 show that isosafrole-induced armyworm midgut microsomes were extremely active with respect to AHH and ethoxycoumarin O-deethylase activities and, indeed, the turnover numbers for these reactions were equal to or greater than those observed with purified cytochromes P-450~ or P-450b, respectively. In contrast, isosafrole failed to induce al-

drin epoxidase in southern armyworm larvae and there was no detectable ethoxyresorufin O-deethylase in midgut microsomes of control or induced larvae. In an attempt to further characterize the MDP-induced AHH activity in armyworm midgut microsomes its sensitivity toward 1-phenylimidazole and c-w-naphthoflavone was evaluated. In previous studies with hepatic microsomes from rats, l-phenylimidazole and a-naphthoflavone have been shown to exhibit a fairly high degree of inhibitory selectivity toward reactions catalyzed by cytochromes P-450b and P-450~ respectively (37, 42). The effects of l-phenylimidazole and a-naphthoflavone toward variously induced AHH activities in armyworm midgut and rat liver microsomes are shown in Table 3. In agreement with previous data (42), 1-phenylimidazole, with I,, values of 120 and 60 p&J, respectively, proved to be moderately effective inhibitor of AHH activity in microsomes from control and phenobarbital-induced rats. At a concentration of 150 p.M, however, I-phenylimidazole stimulated AHH activity in pnaphthoflavone-induced rat liver microsomes. a-Naphthoflavone exhibited the opposite effect, stimulating AHH activity in control and PB-induced rat liver micro-

TABLE 2 Turnover Numbers for Mixed-Function Oxidase Activities Cytochromes P-450 and Midgut Microsomal Cytochrome

Catalyzed P-450 from

by Highly Purified Isosafiole-Induced

Mixed-function

Rat Hepatic Armyworms

oxidase activity”

Cytochrome P-450 source

AE

AHH

ECOD

EROD

Cytochrome P-450b (rat) Cytochrome P-450~ (rat) Isosafrole-induced microsomes (armyworm)e Control microsomes (armyworm)

0.68 0.04 2.5 3.0

NDb 29 26 1.2

2.0 0.5 2s 1.7

ND 11 ND ND

0 AE = aldrin epoxidase (nanomole dieldrin formed per nanomole cytochrome P-450 per minute); AHH = aryl hydrocarbon hydroxylase (nanomole benzo[a]pyrene metabolized per nanomole cytochrome P-450 per minute); ECOD = 7-ethoxycoumarin 0-deethylase (nanomole umbelliferone formed per nanomole cytochrome P-450 per minute); EROD = 7-ethoxyresorutin 0-deethylase (nanomole resorufin formed per nanomole cytochrome P-450 per minute). Values indicated are means of 2-4 assays and in all cases the coefficient of variation was less than 5%. b Not detected. c Armyworms were fed ad libitum for 72 hr on a diet containing 0.05% (w/v) isosafrole.

INDUCTIVE

EFFECTS OF METHYLENEDIOXYPHENYL TABLE

Effects

of I-Phenylimidazole Microsomal

(PO and cu-Naphthojlavone Suspensions from Control

COMPOUNDS

315

3 (aNF) on Atyl Hydrocarbon and Induced Armyworms

Hydroxylase and Rats

Southern armyworm midgut

Activity

in

Rat liver

Treatment”

PI

rvNF

PI

aNF

None (control) Phenobarbital B-Naphthoflavone Isosafrole Dihydrosafrole

6.6 6.6 3.2 NI’ NI

60 60 76 0.4 0.4

120 60 Stim. >150 >150

Stim.* Stim. 5 10 10

a Inducers were administered as described in Table 1 and under Materials and Methods. b Stimulated AHH activity at 150 (*M. c No inhibition at 150 u&f.

somes and being a potent inhibitor (I,, = 5 FM) in microsomes from rats induced with p-naphthoflavone. MDP-induced AHH activity in rat hepatic microsomes was also highly sensitive to inhibition by cw-naphthoflavone (I,, = 10 FM) and exhibited a low degree of sensitivity to 1-phenylimidazole. The pattern of inhibitory potency toward AHH activity in midgut microsomes from armyworms treated with the MDP compounds isosafrole and dihydrosafrole was similar to that observed in P-naphthoflavone-induced rat liver microsomes. In both cases AHH activity was extremely sensitive to a-naphthoflavone (Is, = 0.4 PM) and was completely refractory to inhibition by 1-phenylimidazole even at a concentration of 150 l.N. This contrasts with the previous results (12), confirmed by the results in Table 3, that armyworm midgut AHH activities induced by phenobarbital, p-naphthoflavone, and pentamethylbenzene (results not shown) are all highly sensitive to inhibition by PI and only moderately inhibited by a-naphthoflavone. Thus, in contrast to all previous compounds evaluated as inducers in armyworm larvae, MDP compounds appear to cause the induction of an isozyme with at least some characteristics similar to those described for rat hepatic cytochrome P-450~.

Since, in rats, it is well established that different isozymes of cytochrome P-450 exhibit varying degrees of regiospecificity with respect to their preferred sites of attack on BP (43, 44), BP metabolite patterns are useful indicators of the major isozymes present in microsomal suspensions. Consequently, a comparative study was undertaken to evaluate the major BP metabolites in liver and midgut microsomes from rats and armyworms treated with selected inducers. Although, as described earlier (see Materials and Methods), the HPLC methodology employed was unable to resolve all possible metabolites and although a full spectrum of authentic standards was not available, the results obtained (Table 4) were quite informative. The BP metabolite patterns observed in hepatic microsomes from control, phenobarbital-, and p-naphthoflavone-induced rats (Table 4) were consistent with those reported by others (43, 44). While in all cases BP-3-OH and BP-quinones were the major metabolites produced, phenobarbital-treatment caused a preferential increase in BP-4,5-dihydrodiol and P-naphthoflavone-treatment led to preferential increases in the 7,8- and 9,10-dihydrodiols. Dihydrosafrole-induced microsomes also produced primarily BP-3-OH and BP-qui-

26 (4) 206 (17) 241 (9) 133 (13)

30 (2) 515 (18) ND

4 (1)

ND

45 (2)

1 (2) 14 (2)

BP-4.5Dihydrodiol

78 (6) 277 (8) 185 (18)

42 (6)

38 (6) 33 (3) 390 (14) 79 (8)

237 (15)

6 (9) 17 (3)

BP-9-OH

metabolite

313 (20)

3 (5) 37 (5)

BP-7,8 Dihydrodiol

Benzolalpyrene

519 (42) 1013 (36) 445 (43)

428 (64)

989 (64)

49 (82) 522 (81)

BP-3-OH

372 (30) 375 (13) I85 (18)

89 (13)

ND

ND ND

BP-Quinones

may not total 100%

TABLE 4 Produced by Micr-osornal Fractions from the Sorcthern Annyu~orm Midgut and Rat Liver”

ND’ 64 (9)

BP-9,10Dihydrodiol

Metabolites

y Data shown are pmollminimg protein. Numbers in parentheses indicate percentage of total BP metabolites formed. Metabolites due to rounding off and the presence of a small percentage of the total radioactivity as unidentified peaks. b Inducers were injected ip (rats) or fed in diet (armyworms) as described under Materials and Methods. c ND, Not detected.

Rat None (control) Phenobarbital P-Naphfhoflavone Dihydrosafrole

Southern armyworm None Pentamethylbenzene Isosafrole

1nducel-b

Mujor Benzo[a]pyrene

$

5

6

5

INDUCTIVE

EFFECTS

OF METHYLENEDIOXYPHENYL

COMPOUNDS

317

barbital and catalyzes a broad spectrum of PSMO reactions while cytochrome P-450~ is induced by 3-methylcholanthrene, P-naphthoflavone, and 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) and is associated primarily with AHH activity. This latter type of induction is associated with the so called Ah locus (48) and is mediated by a cytosolic receptor that binds the inducer and translocates it to the nucleus (49). The results of the present investigation have established that, following 72 hr dietary exposure of armyworm larvae to MDP compounds (0.05% w/v), midgut microsomes exhibit markedly enhanced levels of some PSMO activities including AHH and ethoxycoumarin O-deethylase. AHH in particular was dramatically induced with isosafrole and the 4-bromo-5-methoxyMDP derivative, the activities in induced larvae being 29- to 30-fold greater than those in controls and approximately the same as that in hepatic microsomes from l3-naphthoflavone-induced rats (Table 1). Most of the MDP compounds evaluated in this study were considerably more potent inducers of AHH activity than pentamethylbenzene, previously the most effective AHH inducer reported in armyworms (12). The relatively modest increases in cytochrome P-450 observed in MDP-induced midgut microsomes may be explained in part by the presence of a portion of the cytochrome in the form of a complex with the MDP metabolite. On the other hand, the DISCUSSION data are clearly indicative of qualitative differences in the composition of the cyIt is now well established that hepatic cytochrome P-450 in mammals exists in tochrome P-450 in control and induced multiple isozymic forms (45, 46) and that larvae. Isosafrole is known to induce at least catalytically, structurally, and immunothree different isozymes in mammalian hechemically distinct forms are preferentially patic microsomes-cytochrome P-450b, induced following in viva treatment with different chemicals. To date, several iso- P-4SOc, and P-450d (23). Cytochrome P-450d was first described as a novel form zymes have been isolated and partially induced specifically by isosafrole (21-23) characterized from control and variously induced rat hepatic microsomes (47). Of but subsequent studies have shown it to be these, cytochromes P-450b and P-450~ under coordinate control with cytochrome P-45& (50). As a result, it is probable that have received most attention. Cytochrome varying amounts of both cytochromes P-450b is the major form induced by pheno-

nones but compared with controls generated larger amounts of BP-9-OH and the BPd$dihydrodiol. BP metabolite patterns in midgut microsomes from variously treated armyworm larvae exhibited both similarities to and differences from those observed in control and induced rat liver microsomes (Table 4). Microsomes from control (untreated) larvae were relatively inactive toward BP, the only significant product, BP-3-OH accounting for about 80% of the total metabolites produced. BP-3-OH, was also the major BP metabolite generated by microsomes from pentamethylbenzeneand isosafrole-induced larvae although these preparations were IO- to 20-fold more active than controls in metabolizing BP. The BP metabolite pattern observed in pentamethylbenzene-induced microsomes was similar to that in controls with the exception that BP-9, lo-dihydrodiol, not detected in controls, was a significant (9%) product. In the case of isosafrole-induced microsomes, a different metabolite pattern was observed, BP-9-OH and BP-7JGdihydrodiol being major products constituting 15 and 20%, respectively, of the total BP metabolites produced . In contrast to rat liver microsomes, where quinones always constitute a major portion of the BP metabolites produced, no BP-quinones were ever detected in preparations of armyworm midgut microsomes.

318

MARCUS

P-450~ and P-450d are enhanced by most classical inducers of cytochrome P-450~ (“P-448") (51, 52). Since purified cytochrome P-450d exhibits little, if any, activity toward BP (22, 23) it has been suggested that the AHH activity found in MDP-induced rat hepatic microsomes is due mainly to the presence of cytochrome P-450~ (26). Although the probable existence of multiple forms of insect cytochrome P-450 has been suggested (53, 54) little or no work has been done to characterize these cytochromes or to compare them with those found in mammals. As a result of studies showing the general insensitivity of armyworm larvae to several well established inducers of cytochrome P-450~ and AHH activity in mammals (3-methylcholanthrene, P-naphthoflavone, and TCDD) it has been suggested that insects may not possess an isozyme that can be considered a precise counterpart of mammalian cytochrome P-450~ (12); this has been further supported by failure to demonstrate a TCDD receptor in cytosol from armyworm midgut tissues (55). Thus, while previous studies have established that several compounds such as pentamethylbenzene and naphthalene are effective inducers of AHH activity in armyworm midgut microsomes (12) the response of this activity to selective inhibitors (phenyiimidazole and a-naphthoflavone) have indicated that it is catalyzed by an isozyme(s) with properties similar to the “phenobarbital-type” cytochrome P-450b in mammals. In contrast, the isosafrole- and dihydrosafrole-induced AHH activity in armyworm midgut microsomes behave in a manner similar to that in P-naphthoflavoneand MDP-induced rat liver microsomes, being highly sensitive to inhibition by (Ynaphthoflavone and insensitive to the effects of I-phenylimidazole. MDP-induced AHH activity is the first insect MFO reported to be so highly sensitive to (Ynaphthoflavone and as such it bears a striking resemblance to the AHH activity

ET AL.

catalyzed by mammalian cytochrome P-450~. On the other hand, isosafrole-induced armyworm midgut microsomes exhibited no detectable levels of ethoxyresorufin O-deethylase activity (Table 2) and since in mammals this is a reaction characteristic of cytochrome P-450~ (Table 2) (56) the insect isozyme(s) clearly possess a spectrum of catalytic activity different from mammalian cytochrome P-450~. For some reason, ethoxyresorufin O-deethylase appears to be a reaction not readily catalyzed by insect microsomes since previous studies (57) have shown that, while microsomal preparations from adult house flies (Musca domestica) readily catalyze the demethylation of 7-methoxyresorufin, they are much less active toward the deethylation of the 7-ethoxy analog. The potent inducing action of isosafrole toward midgut ethoxycoumarin O-deethylase activity is difficult to interpret since, as previously reported (58), and confirmed by the data in Table 2, this reaction is catalyzed by both mammalian cytochromes P-450b and P-450~. While this might be considered evidence for the induction of a P-450b-type isozyme it must be weighed against the failure of isosafrole to induce aldrin epoxidase, a reaction associated primarily with mammalian cytochrome P-450b (59) (Table 2). It is apparent, therefore, that MDP compounds are effective inducers of armyworm cytochrome P-450 isozymes with activities characteristic of both mammalian cytochromes P-450b and P-450~. That armyworm midguts undoubtedly contain several different isozymes of cytochrome P-450 that differ from those in mammalian liver and are differentially induced in response to in viva exposure to xenobiotics, is further shown by the patterns of BP metabolites formed in induced microsomes (Table 4). Thus, while both pentamethylbenzene and isosafrole are capable of inducing AHH in armyworm midgut microsomes, the different regiospecificities of the induced cytochrome P-450s are quite apparent. In the case of

INDUCTIVE

EFFECTS

OF METHYLENEDIOXYPHENYL

isosafrole, the preferential formation of BP-7,8-dihydrodiol and BP-9-OH resembled the effect of p-naphthoflavone-induction in rats. Major differences were apparent, however, in the formation of BP-9, lo-dihydrodiol and BP-4,5-dihydrodiol which, in p-naphthoflavone-induced rat liver microsomes, constitute a considerably greater percentage of the total metabolites produced than in isosafrole-induced armyworm midgut microsomes. A difference in formation of the BP-4,5-dihydrodiol was also apparent in microsomes from isosafrole-induced armyworms and dihydro&role-induced rats. Pentamethylbenzeneinduced armyworm midgut microsomes exhibited catalytic activities indicating a regiospecificity intermediate between that of phenobarbital- and p-naphthoflavone-induced rat liver microsomes. Clearly the results of the BP metabolite studies are highly complex and indicative of the presence of several distinct blends of cytochrome P-450 isozymes exhibiting various degrees of regiospeciticity. The failure to detect BP-quinones in preparations of armyworm midgut microsomes may be indicative of fundamental differences in the enzymatic composition or character of the insect and mammalian MFO systems. It is also possible, however, that BP-quinones are formed and are either rapidly conjugated (sulfates, glucosides) or otherwise metabolized (60-62) or are rendered unextractable through complex formation with tissue macromolecules. In summary, the present study has shown that, in addition to their well established inhibitory action on insect cytochrome P-450 and PSMO activity, several MDP compounds are potent inducers of AHH and other activities. While in some ways this is similar to the overall effects of MDP compounds on PSMO activity in mammals, it is apparent that the catalytic and other properties of the MDP-induced insect cytochromes P-450 are quite distinct

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