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Spectral and inhibitory interactions of methylenedioxyphenyl compounds with southern armyworm (Spodoptera eridania) midgut microsomes
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
15, 32-42 (1981)
Spectral and Inhibitory Interactions of Methylenedioxyphenyl Compounds with Southern Armyworm (Spodoptera eridania) Midgut Microsomes KUO-MEI CHANG, Department
of Entomology,
C.F. WILKINSON, Cornell
AND KRYSTYNAHETNARSKI
University,
Cornsrock
Hall,
Ithaca,
New
York 14853
Received October 28, 1980; accepted January 7, 1981 Characteristics of the Type III optical difference spectra of 13 methylenedioxyphenyl compounds in NADPH-fortified armyworm midgut microsomes varied with the nature of the substituents in the aromatic ring. Compounds with electron-donating substituents yielded spectra with large 427/458 nm peak ratios, whereas those with electron-withdrawing groups exhibited low 427/ 458 nm peak ratios. Small amounts of carbon monoxide were generated during incubation of the 4,5-dihalo derivatives with midgut microsomes, and cis- and trans-methylenedioxycyclohexanes exhibited spectra with a major Soret peak at about 430 nm and a very weak absorbance maximum at about 480 nm. Formation of the Type III spectral complex occurred very rapidly and was associated with a marked decrease (up to 72%) in cytochrome P-450 levels as measured by carbon monoxide binding. Although a 24% reduction of cytochrome P-450 was observed in the absence of any measureable 458-nm spectral complex a linear relationship existed between further decreases in the cytochrome and the increase in Type III complex formation (458 nm). Inhibitory potencies of the compounds towards aldrin epoxidase and benzopyrene hydroxylase activities were not clearly correlated with either spectral complex formation or decrease in cytochrome P-450 and it is apparent that different factors are involved in the inhibition of different monooxygenase reactions. INTRODUCTION
optical difference spectrum. Subsequently, numerous studies have confirmed that, in the presence of NADPH and 0, (2, 12-15), or cumene hydroperoxide (16, 17), several MDP compounds undergo microsomal metabolism to reactive intermediates that bind to reduced cytochrome P-450 to give stable inhibitory complexes exhibiting Type III difference spectra. Considerable speculation has centered on the nature of the reactive intermediates involved and homolytic radicals (18), benzodioxolium ions (19), and carbanions (20) have all been implicated in the mechanism. The most recent suggestions favor the formation of a reactive carbene species (21). There seems little doubt that the Type III difference spectrum is indicative of the formation of a stable inhibitory complex with cytochrome P-450. However, a direct correlation between complex formation and inhibition of microsomal oxidation has yet to be demonstrated. In interpreting the Type III spectral complex and its relationIII’
Although it is now well established that methylenedioxyphenyl (MDP) compounds inhibit microsomal oxidations in vitro (l-6) and synergize the activity of many drugs and insecticides in vivo in both mammals and insects (2-6) their precise mode of action remains unclear. Following early suggestions that inhibition might occur simply through alternative substrate interactions (7) a more specific inhibitory mechanism was first indicated by the demonstration of decreased levels of cytochrome P-450 in microsomal preparations from mammals (8) and insects (9, 10) treated in vivo with piperonyl butoxide. Aerobic incubation of piperonyl butoxide with NADPH-fortified mouse liver microsomes was also found to cause a decrease in cytochrome P-450 and was associated with the formation of a spectrally observable complex with dual Soret peaks at 455 and 427 nm the relative peak heights being pHdependent (11); this was termed the ‘Type 32 0048-3575/81/010032-l 1$02.00/O Copyright All rights
@ 1981 by Academic Press, Inc. of reproduction in any form reserved.
INHIBITORY
INTERACTIONS
OF
METHYLENEDIOXYPHENYL
ship to inhibition of drug oxidation it is usually assumed that the 455nm peak represents the primary inhibitory complex between the active MDP intermediate and the ferrous heme iron of cytochrome P-450. This assumption is based mainly on the fact that several other types of compounds, including amphetamine derivatives (22-24), SKF 525A (22, 25), and some halogenated hydrocarbons (26) undergo metabolism to intermediates that give similar spectrally identifiable 455-nm complexes with concomitant inhibition of microsomal oxidation, and that several other r-acceptor ligands (CO, SnX,, GeX,, where X = halogen) give single Soret peaks in the 455-nm region (27). However, several methylenedioxy compounds which are inhibitors of oxidative metabolism and CO binding to cytochrome P-450 do not form typical Type III complexes in NADPH-reduced microsomes. Thus cis- and trans-methylenedioxycyclohexanes (28) and several aliphatic dioxolanes (29) exhibit stable optical difference spectra with a single peak at 427-430 nm and MDP compounds with electronwithdrawing groups in the aromatic ring yield mainly CO from the methylenic carbon during metabolism by rat liver microsomes (15). As a consequence it appears important to evaluate more carefully the relationship between spectral complex formation and inhibition of microsomal oxidation by MDP compounds. The studies reported in this manuscript were designed to investigate this in southern armyworm (Spodoprera eridunia) midgut microsomes. MATERIALS
AND
METHODS
Chemicals. 1,2-Methylenedioxybenzene (MDB) and its 4-bromo and 4-methyl derivatives were purchased from Frinton Laboratories, Vineland, New Jersey, and piperonyl nitrile (4-cyano-MDB), sesamol (Chydroxy-MDB) safrole, and isosafrole were from the Aldrich Chemical Company, Milwaukee, Wisconsin. Piperonyl butoxide was kindly provided by the FMC Corpora-
COMPOUNDS
33
tion (Niagara Chemical Division), Middleport, New York. All other MDB derivatives employed were synthesized by established procedures (30). cis-Hexahydro- 1,3-benzodioxolan, bp,, 77°C [reported bp,, 69-70°C (3 l)], and trans- hexahydro1,3-benzodioxolan, bp,, 68- 69°C [reported bp,, 66-67°C (32)] were prepared by reaction of the corresponding cis- and transcyclohexanediols with paraformaldehyde (31, 32). Biochemicals were purchased from Sigma Chemical Company, St. Louis, Missouri and all other chemicals and reagents employed were of analytical reagent grade. Enzyme preparation. Larvae of the southern armyworm (S. eridania) were reared on bean plants under greenhouse conditions through the first five instars as previously described (33). The sixth-instar larvae used in these experiments were kept in an environmental chamber and were fed on a semidefined artificial diet (34) for 48 h prior to use. Microsomal fractions of armyworm midguts were prepared as previously described (35) and the microsomal pellets were resuspended in 0.05 M TrisHCl buffer, pH 7.8, at 2 mg protein/ml. Protein concentrations were determined by the method of Lowry et al. (36) using bovine serum albumin as a standard. Spectral studies. Optical difference spectra were measured at 30°C with an Aminco DW-2 spectrophotometer using l-cm pathlength semimicro cuvettes and l-ml aliquots of microsomal suspensions (2 mg protein/ml). NADPH (1 pmol) was added to each cuvette and after baseline correction, the reaction was initiated by addition to the sample cuvette of the appropriate MDP compound (0.1 pmol) in 10 ~1 ethanol; the reference cuvette received 10 ~1 ethanol. Repetitive scans (5 nm/sec) between 400 and 500 nm were made at intervals of 1 min. After maximum spectral development a few grains of sodium dithionite were added to each cuvette and CO was bubbled through the sample for measurement of cytochrome P-450 (37). In other experiments, midgut micro-
34
CHANG,
WILKINSON,
somes (2 mg protein/ml) were incubated with the various MDP compounds (0.1 mM) at 30°C in Erlenmeyer flasks both with and without addition of NADPH (1 mM). After 5 min the reactions were terminated by addition of dithionite and cytochrome P-450 levels were determined in each incubation mixture. Spectral studies were also conducted using aliquots from incubations without NADPH (reference) vs those from incubations with NADPH (sample). Enzyme assays. Aldrin epoxidation assays were carried out aerobically at 30°C as described previously (34) using a j-ml reaction mixture at pH 7.8. Incubation time was 10 min and the reaction was initiated by addition of 100 pg aldrin in 25 ~1 ethanol. MDP compounds were added to the incubations in lo-50 ~1 of ethanol and Iso (M) values were usually determined from the means of duplicate incubations with at least four different inhibitor concentrations. Benzopyrene hydroxylase activity was assayed by the radiometric procedure described by DePierre et al. (38) using [G3H]benzo[a]pyrene diluted to 13.5 PCiI pmol. The l-ml incubation mixtures contained 0.5- 1.0 mg microsomal protein, 1 mM NADPH, 5 mM MgC&, 5 pM MnCl, in 50 mM Tris-HCl, pH 7-8; the reactions were initiated by addition of 50 nmol [3H]benzopyrene in 10 ~1 acetone. MDP compounds were added in 10 ~1 acetone to give a concentration of 0.1 mM. The aerobic incubations were at 30°C for 12 min and were terminated by addition of 1 ml of ice-cold 0.5 N NaOH in 80% ethanol. Extraction was carried out according to DePierre et al. (38) and counting was effected in a Packard Model 2425 scintillation counter using a scintillant consisting of a mixture of PPO (4.75 g), POPOP (0.32 g), and Bio-Solv (40 ml) dissolved in toluene (1000 ml). Counting efficiency was 48.5%.
AND
HETNARSKI
I.oos WAVELENGTH
(nm)
FIG. 1. Type III optical difference spectra obtained with 10m4 M, I.5dibromo-MDB (A), MDB (B), isosaf role (C), and trans-methyfenedioxycyclohexane (0) in NADPH-fortified armyworm midgut microsomes.
exception of sesamol (4-hydroxy-MDB), which showed no obvious spectral interaction, all of the MDP compounds exhibited Type III optical difference spectra in NADPH-reduced armyworm midgut microsomes. The spectra were characterized by absorbance peaks at 426-429 nm and 456-462 nm and a marked trough at 412-414 nm. However, the compounds can be divided arbitrarily into three major groups based on the relative intensities of the two major spectral peaks (Fig. 1). In the first group (Group I), consisting of the 4cyano, 4,5-dibromo, and 4,5-dichloro derivatives of MDB, the ratio of the 427/458 nm peaks’ is less than unity (Fig. lA, Table 1) and with the cyano compound, the 427-nm peak appears only as an indistinct shoulder on the predominant 458-nm peak. At the other extreme, Group II compounds exhibit spectra in which the 427-nm peak predominates and with 4271458 nm peak
RESULTS
Spectral studies were conducted with a total of 13 MDP derivatives and with cisand trans-methylenedioxycyclohexanes each at a concentration of 10e4 M. With the
1 The double Soret peaks in the Type III difference spectra are referred to throughout the text as the 427 and 458 nm peaks even though the wavelengths of maximum absorbance vary somewhat from compound to compound.
427.2 (0.009 426.8 (0.0035 427.8 (0.0019 427.7 (0.0185 427.6 (0.0152 427.5 (0.0175 427.5 (0.0072 427.3 (0.01 430.3 (0.0037 430.7 (0.0038
427.7 (0.008 428.5 (0.0129 427.4 (0.0119 Shoulder
456.8 -t 0.7 (0.0044 -c O.ooo6) 458.1 * 1.1 (0.0048 t 0.0006) 457.4 2 1.0 (0.0048 rt 0.0002) 458.6 If: 1.3 (0.0035 k O.ooo6) No detectable spectrum r 0.3 457.4 iz 0.8 r 0.0006) (0.0071 + 0.0004) k 0.8 458.1 k 0.3 -e 0.ooo6) (0.0063 2 0.0001) 5 0.1 457.0 2 0.9 t 0.0005) (0.0071 2 0.001) t 0.1 458.7 * 0.5 -c 0.0032) (0.0068 ” 0.0008) If: 0.2 459.2 k 0.6 r 0.0025) (0.0064 + 0.0007) e 0.3 459.5 k 0.3 rt 0.0015) (0.0065 2 0.005) -e 0.2 461.5 2 0.6 rf: 0.002) (0.0037 -c 0.0007) rt 0.1 461.2 z 1.2 t 0.004) (0.002 5 0.0005) 2 1.0 478.2 2 0.1 It 0.0009) (0.0016 2 0.0003) r 0.8 480 f 1.5 rt 0.0004) (0.0007 k 0.0002)
r 0.3 r 0.0017) 2 1.1 -+ 0.0012) k 0.0 & 0.0007) at 428
0 (6.3 x 10m3) 3 (1.9 x 10-S)
28.2 41.1
-
81 (2.7 x 1O-5)
32.2
5.0
74 (3.4 x 10-S)
95 (1.4 x 10-S)
66 (5.0 x 10-S)
100 (6.3 x 1O-6)
100 (2.1 x 10-S)
93 (7.5 x 10-G)
0 (>lO-2) 95 (7.5 x 10-G)
3 (1.3 x 10-Z)
55 (8.0 x lo-“)
31 (2.2 x IO-“)
0 (1.0 x 10-3)”
AE
50
30
ND’
56
57
89
49
56
55
5 58
15
51
51
23
BP
Percentage inhibition of microsomal oxidation at IO-* Mr
55.8
69.6
49.4
53.5
72.7
71.5
33.4 50.6
39.0
54.2
41.0
17.9
Percentage decrease in cytochrome P-450”
Midgut Microsomes
1.95
2.69
2.38
2.72
0.27
0.56
1.27
-
2.48
2.69
1.82
Absorbance ratio 4211458 nm
TABLE 1 of Methvlenedioxv Compounds uaith Armw,orm
Type III spectral character”
Interactions
I’ Upper figures denote peak position (nm), lower tigures in parentheses indicate maximum absorbance (vs 490 nm) in presence of 10m4M test compound. Values are means ? SEM of three or four replicates. h Measured as described in text. ’ AE, Aldrin epoxidase; BP, benzopyrene hydroxylase. I2Is, (M) values. ti Not determined.
2,3-Methylenedioxynaphthalene Piperonyl butoxide cis-hlethylenedioxycyclohexane trans-Methylenedioxycyclohexane
Dihydrosafrole
Isosafrole
Safrole
4,5-di-Cl-MDB
4,5-di-Br-MDB
COH-MDB 4-Br,S-CH,O-MDB
CCN-MDB
CCH,O-MDB
CCH,-MDB
MDB
Compound
Spectral and Inhibitory
36
CHANG,
WILKINSON,
ratios ranging from about 2.5 to 5.0 (Fig. 1C). Included in this group are safrole, isosafrole, dihydrosafrole, piperonyl butoxide, and the 4-methyl and 4-methoxy derivatives of MDB. Three compounds, MDB, 4-bromo-5-methoxy-MDB, and methylenedioxynaphthalene (Group III) are intermediate between these extremes and exhibit spectra in which the 427- and 458-nm absorbance peaks are of approximately equal intensity (427/458-nm ratio 1.3 to 1.95) (Fig. IB). cis- and fransMethylenedioxycyclohexanes behave differently from the aromatic compounds and as has been previously reported (28) exhibit difference spectra with a single major peak at about 431 nm and a very indistinct peak at 478-480 nm (Fig. 1D). In all cases spectral development was extremely rapid and was essentially complete within 1 to 2 min following addition of the methylenedioxy compound to the reaction mixture. Once formed the spectra were stable and continued incubation in the presence of NADPH for periods of at least 20 min resulted in no further qualitative or quantitative spectral changes. The characteristics of the Type III difference spectra with various methylenedioxy compounds are shown in Table 1. With compounds in Groups II and III the addition of dithionite to each cuvette after maximum Type III spectral development with NADPH had little or no effect on the observed spectrum with the exception of a slight (approximately 1 nm) bathochromic shift in the 427-nm peak with some compounds and a noticeable intensification of the trough in the region of 410 nm. Dithionite caused a more obvious shift to lower wavelength in Type III spectra formed from several compounds in Group I. Thus a 3- to 4-nm shift in both peaks occurred with the two dihalo compounds whilst in the case of 4-cyano-MDB, the 458-nm peak remained unchanged and the 427 shoulder disappeared entirely. The wavelength shift observed with the 4,5-dibromo and 4,5dichloro compounds on addition of dithio-
AND
HETNARSKI
nite was accompanied by a slight increase in absorbance at about 453 nm and suggested the possible formation of carbon monoxide as has been reported with rat liver microsomes (15). This possibility was strengthened by the finding that addition of heme (2 pmol) to each cuvette caused the 453nm peak to revert to about 457 nm and led to the concomitant development of a new peak at 418.5-420 nm in the region of carboxyhemoglobin. No attempt was made to quantitate the small amount of carbon monoxide produced, and of all the compounds tested it was apparent only with the two dihalo derivatives. Carbon monoxide production from MDP compounds in armyworm midgut microsomes is clearly much lower than in those from rat liver (15) and in contrast to the latter there was no evidence of any carbon monoxide from the 4-cyano derivative. Despite the lack of any obvious NADPH-mediated microsomal difference spectrum with 4-hydroxy-MDB (sesamol), addition of dithionite after incubation with NADPH caused the development of a small absorbance peak at about 453 nm suggesting the formation of low levels of carbon monoxide from this compound. As discussed elsewhere (15) it is probable that this occurs by a mechanism different from that followed by other MDP compounds. Addition of dithionite to microsomes incubated with cis- and transmethylenedioxycyclohexanes, and allowed to attain a full spectral development in the presence of NADPH, resulted in a marked decrease in the 430-nm peak and no obvious change in the small absorbance peak at about 480 nm. Attempts to measure residual cytochrome P-450 levels after incubation of the various test compounds with NADPH in the cuvettes were not successful due to the complex nature of the combined Type III/CO difference spectra produced. The absorbance maxima of the latter spectrum varied considerably from compound to compound, being as high as 458 nm in the case of isosafrole, and thus making any in-
INHIBITORY
INTERACTIONS
OF
METHYLENEDIOXYPHENYL
terpretation of corrected spectra extremely difficult. It was further complicated by the large absorbance peak which frequently appeared at 421-423 nm and which indicated significant degradation of the fairly labile armyworm cytochrome P-450 to cytochrome P-420. A more convenient method for estimation of residual levels of cytochrome P-450 was afforded by spectral measurements following incubation (5 min at 30°C) of the various test compounds (lo-* M) with armyworm midgut microsomes in individual flasks in either the presence or absence of NADPH. Levels of cytochrome P-450 measured in microsomes incubated with MDP compounds without NADPH were always the same as those in control nonincubated microsomes, indicating no inhibitory interaction under these conditions. In incubations of the test compounds with NADPHfortified microsomes levels of cytochrome P-450 were decreased significantly, the percentage reduction varying from about 18% in the case of MDB itself to approximately 70% with the 4,5-dibromo and 4,5dichloro derivatives and with dihydrosahole (Table 1). In all cases, the absorbance maximum of the CO difference spectra measured under these conditions was in the range of 450-452 nm, only slightly greater than that in control microsomes (449.8 nm). Furthermore, there appeared to be less conversion to cytochrome P450 under these conditions than in incubations carried out directly in the cuvettes. The inhibitory potencies of the MDP compounds towards aldrin epoxidation are indicated in Table 1 as ISo (M) values as well as by the percentage inhibition observed at lo-* M. It is apparent that considerable variation exists within the group of compounds tested, I,,, values ranging from 6.3 x lo-” M in the case of isosafrole to >10e2 M with 4-hydroxy-MDB (sesamol), and inhibition ranging from 100 to 0% at lo-* M. Inhibitory potency of the various compounds towards benzopyrene hydroxylase was somewhat less variable and at lo-* M
COMPOUNDS
37
only one, isosafrole, exhibited more than 60% inhibition (Table 1). Unfortunately, determination of inhibitory activity towards this reaction proved difficult due to the very low hydroxylase levels that exist in control armyworm midgut microsomes. In general, aldrin epoxidation was somewhat more sensitive to inhibition than benzopyrene hydroxylase although this was not the case with MDB, and its 4-methyl and 4-cyan0 derivatives or with isosafrole. Interestingly, in contrast to their lack of effect on aldrin epoxidase at 1Om1M both cis- and rrans-methylenedioxycyclohexanes were quite effective inhibitors of benzopyrene hydroxylase activity at this concentration. Attempts to demonstrate a correlation between inhibitory potency towards aldrin epoxidase and benzopyrene hydroxylase activity and either reduction in cytochrome P-450 or intensity of 458-nm absorbance at the same concentration (IO-” M) were not successful (Fig. 2). There appears to be a trend indicating greater inhibition with compounds exhibiting a more intense 458-nm absorbance and causing a greater reduction in cytochrome P-450 but the correlation is not clear-cut and there are several obvious exceptions such as piperonyl butoxide (Fig. 2A) which is an effective inhibitor of aidrin epoxidase but which causes only a slight reduction in cytochrome P-450 and only a relatively small 458-nm spectral interaction. With aldrin epoxidase (Fig. 2A) a reduction of approximately 25-30% of the cytochrome P-450 can apparently be sustained without significant loss of enzyme activity; this does not appear to be true for benzopyrene hydroxylase (Fig. 2B). A weak correlation (r = 0.696) exists between the 458-nm absorbance and percentage decrease in cytochrome P-450 with 12 MDP compounds (values for MDB not included) (Fig. 3) and the intercept of the y axis suggests that a 24.4% reduction of cytochrome P-450 occurs in the absence of any observable 458-nm absorbance. This value approximates the reduction in cyto-
38
CHANG,
CYTOCHFRME
WILKINSON,
P-454
DECREASE
AND
(K)
HETNARSKI
or
(AABSORBANCE
459 /49Onm)
2. Relationship between inhibition of aldrin epoxidase (A) and benzopyrene hydroxyluse (B) activities with reduction in cytochrome P450 (0) and Type 111458 nm absorbance ( 0) with MDP derivutives (10m4 M) in armyworm midgut microsomes. (I) MDB; (2) I-cyano-MDB; (3) I-hydroxy-MDB; (4) I-methyl-MDB; (5) I-methoxy-MDB; (6) isosafrole; (7) 2,3-methylenedioxynaphthalene (8) dihydrosufrole; (9) 4,5-dibromo-MDB; (10) 4,5-dichloro-MDB, (1 I) 4-bromo-5-methoxy-MDB; (12) sufrole; (13) piperonyl butoxide. FIG.
chrome P-450 observed with the cis- and trans-methylenedioxycyclohexanes (28.2 and 41.1%, respectively), which exhibit no 458 nm absorbance peak (Table 1) and the reduction with sesamol. DISCUSSION
The results of this investigation indicate some similarities and several important differences between the inhibitory interactions of MDP compounds with armyworm midgut microsomes and those previously reported for rat liver (15). The Type III optical difference spectra observed on incubation of most MDP compounds with NADPH-fortified armyworm midgut microsomes are qualitatively similar to those reported in microsomes from other species (2) with dual Soret peaks at about 427 and 458 nm. In contrast to rat liver microsomes, however, where in the presence of NADPH, MDP compounds containing electron-withdrawing groups in the aromatic ring generate significant amounts of carbon monoxide from the methylenic carbon atom (15), carbon monoxide formation in armyworm midgut microsomes occurs to a much
lower extent and appears to be restricted to the 4,5-dichloro and 4,5-dibromo derivatives; it also occurs to some extent with sesamol although this compound exhibits no obvious optical difference spectrum. The characteristics of the MDP Type III spectral complexes in armyworm midgut
pI,IIIIIIIL 0
,002
A ABSORBANCE
.004
.006
(459
.ooe
/a90
nm)
3. Relationship between reduction of cytochrome P-450 levels and Type ZZZ 458 nm absorbance with MDP derivatives (10V4 M) in armyworm midgut microsomes. Numerical identiftcation of compounds used is the same us in Table 2 and the line represents the best fit (linear regression) through all points exclusive of compound I (MDB). FIG.
INHIBITORY
INTERACTIONS
OF
METHYLENEDIOXYPHENYL
COMPOUNDS
39
microsomes, particularly the relative size of interaction of those with electron-withdrawthe 427- and 458~nm peaks, appears to be ing groups occurs mainly through the free related to the nature of the substituents in form of the ligand resulting in low 4271458 the aromatic ring. Compounds with elec- nm ratios. tron-withdrawing groups in the ring (Group A factor which complicates interpretaI) exhibit spectra with strong 458- and tions of 427/458 nm peak ratios in terms of weak 427-nm absorbance peaks, whereas chemical structure is that the ratios are also those with electron-donating substituents dependent on the pH of the incubation (Group II) give spectra in which the 427-nm mixture (11). This is observed in armyworm peak predominates. Group III compounds midgut microsomes as in those from other species, although detailed studies to detersuch as MDB and 2,3-methylenedioxynaphthalene with no substituents, or those con- mine and compare the effects of pH on taining both electron-withdrawing and -do- Type III spectra in microsomes of different nating groups (e.g., 4-bromo-5-methoxyspecies have not yet been conducted. It MDB) yield spectra intermediate between should be pointed out that since the data Groups I and II. The relative intensity of presented here were obtained at a single the 427- and 458-nm peaks, therefore, ap- pH, somewhat different results may have pear to be related in some way to the been observed had different pH values been electronic distribution within the molecule, employed. although a more complete interpretation The relationship between Type III specwill have to await further information on tral complex formation, reduction in cytothe nature of the ligand binding sites in- chrome P-450 and inhibition of microsomal volved. It has been suggested that the 427- oxidation remains unclear and it is apparent and 458-nm Type III spectral peaks ex- that factors other than those considered in this investigation are involved. The data hibited by most dioxoles (MDP compounds) shown in Fig. 3 suggest a linear relationship may result from ligand interaction with cytochrome P-450 of the bound and free between 458-nm absorbance and the obforms respectively of a relatively stable served decrease in cytochrome P-450. lipophilic r-acceptor ligand such as the However, extrapolation of these data to the proposed carbene intermediate (29, 39, 40). y axis indicates that an approximately 24% The existence of an equilibrium between the decrease in cytochrome P-450 is not dibound and free forms of the ligand, com- rectly attributable to the 458-nm absorbined with the instability of the free form of bance peak. Since both the 427- and 458-nm the ligand formed from dioxolanes (aliphatic peaks presumably represent ligand commethylenedioxy compounds and methyleneplexes with cytochrome P-450 it is possible dioxycyclohexanes) has been proposed as that the 24% reduction in cytochrome P-450 not associated with the 458-nm complex an explanation of the single 427-nm differrepresents the mean contribution of the ence spectra observed with these com427-nm ligand complex. The contribution of pounds (29). If this interpretation is correct, the 427/458 nm peak ratios may reflect the the latter to the observed reduction in cytoequilibrium between the bound and free chrome P-450 appears to have been given forms of the proposed carbene ligand, as- little attention to date, although it has been suming that the free ligands from different reported that the Ki value for the inhibition MDP compounds are of approximately of CO binding by piperonyl butoxide coinequal stability. Consequently, the data cides with the KS value for the formation of presented here may suggest that electronthe Type III spectrum calculated from the donating groups favor the association of the sum of the 427- and 455nm peaks (14). It ligand to the cytochromal protein (hence therefore seems clear that the 427-nm comthe large 427/458 nm ratios) whereas ligand plex plays some role in the observed reduc-
40
CHANG,
WILKINSON,
tion of cytochrome P-450 and should be taken into consideration in evaluating the overall inhibitory interactions of MDP compounds. Unfortunately, quantitation of the total spectral complex is not simple and summation of the absorbance differences between 427 and 490 nm and between 458 and 490 nm may not provide an adequate measure. Attempts to correlate the inhibition of microsomal oxidation with spectral complex formation and cytochrome P-450 reduction (Fig. 2) are also difficult since it is almost certain that both competitive (alternative substrate) and noncompetitive (complex formation) interactions contribute to the overall inhibitory activity. Thus Franklin (41) found that the inhibition of ethylmorphine N-demethylation by piperonyl butoxide was initially competitive in nature but subsequently became noncompetitive following formation of the complex. In view of the established existence of multiple forms of cytochrome P-450 (42) which catalyze the oxidative metabolism of different xenobiotic substrates, it is entirely possible that variations in inhibitory potency towards different substrates, or reaction types, might result from differences in the rate or extent of MDP complex formation with different forms of the cytochrome. The data presented here suggest that this might be the case, since with aldrin epoxidation, 25 - 30% reduction of cytochrome P-450 can apparently occur without causing significant inhibition of enzyme activity. This does not appear to be the case with benzopyrene hydroxylase, however, and the significant inhibition of this reaction by cis- and trans-methylenedioxycyclohexanes, which form only the 427-nm spectral complex, suggests that the latter may be of more significance in the inhibition of benzopyrene hydroxylase than of aldrin epoxidase. It is of considerable interest to speculate whether the previously discussed bound and free forms of the ligand interact differently with different types or pools of cytochrome P-450.
AND
HETNARSKI
Another factor which may complicate attempted correlations between spectral complex formation and inhibition of oxidase activity is the possible displacement of the Type III complex by some substrates and the consequent restoration of monooxygenase activity; this has been reported with several, but not all, microsomal enzyme substrates tested against the Type III isosafrole complex in rat liver microsomes (43) but has not yet been investigated in those from armyworm midgut. ACKNOWLEDGMENT
The work was supported in part by a grant (ES 01902) from the U.S. Public Health Service. REFERENCES
1. M. W. Anders, Inhibition of microsomal drug metabolism by methylenedioxybenzenes, Biochem. Pharmacol. 17, 2367 (1968). 2. E. Hodgson and R. M. Philpot, Interaction of methylenedioxyphenyl (1,3-benzodioxole) compounds with enzymes and their effects on mammals, Drug Metab. Rev. 3, 231 (1975). 3. J. E. Casida, Mixed-function oxidase involvement in the biochemistry of insecticide synergists, J. Agr. Food Chem. 18, 753 (1970). 4. M. R. Franklin, Methylenedioxyphenyl insecticide synergists as potential human health hazards, Environ. Health. Perspect. 14, 29 (1976). 5. C. F. Wilkinson, Insecticide synergists in “The Future for Insecticides: Needs and Prospects” (R. L. Metcalf and J. J. McKelvey, Jr., Eds.), p. 195, Wiley, New York, 1976. 6. C. F. Wilkinson, Effects of synergists on the metabolism and toxicity of anticholinesterases, Bull. WHO 44, 171 (1971). 7. J. E. Casida, J. L. Engel, E. G. Esaac, F. X. Kamienski, and S. Kuwatsuka, Methylene‘QZ-dioxyphenyl compounds: Metabolism in relation to their synergistic action, Science 153, 1130 (1966). 8. H. B. Matthews, M. Skrinjaric-Spoljar, and J. E. Casida, Insecticide synergist interactions with cytochrome P-450 in mouse liver microsomes, Life Sci. 9, 1039 (1970). 9. A. S. Perry and A. J. Buckner, Studies on microsomal cytochrome P-450 in resistant and susceptible houseflies, Life Sci. 9, 335 (1970). 10. H. B. Matthews and J. E. Casida, Properties of housefly microsomal cytochromes in relation to sex, strain, substrate specificity and apparent inhibition by synergist and insecticide chemicals, Life Sci. 9, 989 (1970).
INHIBITORY
INTERACTIONS
OF METHYLENEDIOXYPHENYL
11. R. M. Philpot and E. Hodgson, A cytochrome P-450- piperonyl butoxide spectrum similar to that produced by ethyl isocyanide, Life Sci. 10, 503 (1971). 12. M. R. Franklin, The enzymic formation of a methylenedioxyphenyl derivative exhibiting an isocyanide-like spectrum with reduced cytochrome P-450 in hepatic microsomes, Xenohiorica 1, 581 (1971). 13. M. R. Franklin, Piperonyl butoxide metabolism by cytochrome P-450: Factors affecting the formation and disappearance of the metabolite-cytochrome P-450 complex, Xenohiotira 2, 517 (1972). 14. R. M. Philpot and E. Hodgson, The effect of piperonyl butoxide concentration on the formation of cytochro ?e P-450 difference spectra in hepatic microso lies from mice, Mol. Pharmad. 8, 204 (1912). 15. Loh-Sheng Yu, C. F. Wilkinson, and M. W. Anders, Generation of carbon monoxide during the microsomal metabolism of methylenedioxyphenyl compounds, Biochem. Pharmacol. 29, 1113 (1980). 16. C. R. Elcombe, J. Bridges, R. H. Nimmo-Smith, and J. Werringloer, Cumene hydroperoxidemediated formation of inhibited complexes of methylenedioxyphenyl compounds with cytochrome P-450, Biochem. Sot. Trans. 3, 967 (1975). 17. A. P. Kulkarni and E. Hodgson. Cumene hydroperoxide-generated spectral interactions of piperonyl butoxide and other synergists with microsomes from mammals and insects, Pestic. Biochem. Physiol. 9, 75 (1978). 18. C. Hansch. The use of homolytic, steric and hydrophobic constants in a structure-activity study of 1,3-benzodioxole synergists, J. Med. Chem. 11, 920 (1968). 19. D. J. Hennessy. Hydride transferring ability of methylenedioxybenzenes as a basis of synergistic activity, J. Agr. Food Chem. 13, 218 (1965). 20. V. Ullrich and K. H. Schnabel, Formation and binding of carbanions by cytochrome P-450 of liver microsomes, Drug Metab. Dispos. 1, 176 (1973). 21. W. Nastainczyk, V. Ullrich, and H. Sies, Effect of oxygen concentration on the reaction of halothane with cytochrome P-450 in liver microsomes and isolated perfused liver, Biochem. Pharmacol. 27, 387 (1978). 22. M. R. Franklin, Inhibition of mixed-function oxidations by substrates forming reduced cytochrome P-450 metabolic-intermediate complexes. Pharmacol. Ther. 2, 227 (1977). 23. J. Werringloer and R. W. Estabrook, Evidence for an inhibitory product cytochrome P-450 complex generated during benzphetamine metab-
24.
25.
26.
27.
28. 29.
30.
31. 32. 33.
34.
35.
36.
COMPOUNDS
41
olism by liver microsomes, Life Sci. 13. 1319 (1973). M. R. Franklin, The formation of 455 nm complexes during the microsomai metabolism of substituted amphetamines, Pharmacologisf 16. 322 (1974). J. B. Schenkman, B. J. Wilson, and D. L. Cinn. Diethylaminoethyl 2,2-diphenylvalerate HCl (SKF 525-A)-& ~~i\~oand in vitro effects of metabolism by rat liver microsomes-formation of an oxygenated complex, Biochem. Pharmtrcol. 21, 2373 (1972). C. R. Wolf, D. Mansuy, W. Nastainczyk, and V. Ullrich, The interaction of polyhalogenated methanes with ferrous cytochrome P-450, in “Microsomes and Drug Oxidations” (V. Ullrich, A. Hildebrandt, I. Roots, R. W. Estabrook. and A. H. Conney. Eds.). p. 240. Pergamon, New York. 1977. A. R. Dahl and E. Hodgson, Complexes of stannous fluoride and other group IVA dihalides with mammalian enzymes, Scicnc,e 197. 1376 (1977). E. Hodgson, R. M. Philpot. R. C. Baker, and R. B. Mailman, Effect of synergists on drug metabolism, Drug Metab. Dispos. 1,301 (19731. A. R. Dahl and E. Hodgson, The interaction of aliphatic analogs of methylenedioxyphenyl compounds with cytochromes P-450 and P-420. Chem. Biol. Inferucr. 27. 163 (1979). C. F. Wilkinson. Penetration, metabolism and synergistic activity with carbaryl of some simple derivatives of 1,3-benzodioxole in the house fly. J. Agr. Food Chem. 15, 139 (1967). F. S. H. Head, Reactions of simple aldehydes with cis- and frans-cyclohexane-1.2-diol, ./. Chem. Sot. 1778 (1960). P. V. Bonsignore and M. D. Hurwitz. Cyclic formals of polyhydroxycyclohexane compounds. J. Org. Chem. 28, 3535 (1963). R. I. Krieger and C. F. Wilkinson, Microsomal mixed-function oxidases in insects 1: Localization and properties of an enzyme system effecting aldrin epoxidation in larvae of the southern armyworm (Prodenia rridanici ), Biochem. Pharmacol. 18, 1403 (1969). L. B. Brattsten and C. F. Wilkinson, Induction of microsomal enzymes in the southern armyworm (Spodoprera eridaniu). In.sc~~t Riothem. 9, 43 (1979). D. L. Crankshaw. H. K. Hetnarski, and C. F. Wilkinson, Microsomal NADPH-cytochrome ( reductase from the midgut of the southern armyworm (Spodoptera eridania ). In.trct Biochem. 9, 43 (1979). 0. H. Lowry, N. J. Rosebrough, A. L. Farr. and R. J. Randall, Protein measurement with the Folin phenol reagent, 1. Biof. Chem. 193, 265 (1951).
42
CHANG,
WILKINSON,
37. T. Omura and R. Sato, The carbon monoxidebinding pigment of liver microsomes, .I. Biol. Chem. 239, 2379 (1964). 38. J. W. DePierre, K. A. M. Johanneson, M. S. Moron, and J. Seidegrd, Radioactive assay of aryl hydrocarbon monooxygenase and epoxide hydrase in “Methods in Enzymology” (S. Fleischer and L. Packer, Eds.), Vol. LII, p. 412, Academic Press, New York, 1978. 39. A. R. Dahl and E. Hodgson, The affinity of cytochrome P-450 for ethyl isocyanide: An explanation for double Soret spectra, Chem. Biol. Interact. 20, 171 (1978). 40. A. R. Dahl and E. Hodgson, Complexes of trivalent oxygenated phosphorus compounds with cytochrome P-450 and cytochrome P-420: The
AND HETNARSKI origin of double Soret spectra, Chem. Biol. Interact. 21, 137 (1978). 41. M. R. Franklin, Inhibition of hepatic oxidative xenobiotic metabolism by piperonyl butoxide, Biochem. Phormacol. 21, 3287 (1972). 42. F. P. Guengerich, Isolation and purification of cytochrome P-450 and the existence of multiple forms, Pharmacol. Ther. 6, 99 (1979). 43. C. R. Elcombe, M. Dickins, B. C. Sweatman, and J. W. Bridges, Substrate-elicited dissociation of the isosafrole metabolite-cytochrome P-450 complex and the consequential reactivation of monooxygenation, in “Microsomes and Drug Oxidations” (V. Ullrich, A. Hildebrandt, I. Roots, R. W. Estabrook, and A. H. Conney, Eds.) p. 247, Pergamon, New York, 1977.
BIOCHEMISTRY
AND
PHYSIOLOGY
15, 32-42 (1981)
Spectral and Inhibitory Interactions of Methylenedioxyphenyl Compounds with Southern Armyworm (Spodoptera eridania) Midgut Microsomes KUO-MEI CHANG, Department
of Entomology,
C.F. WILKINSON, Cornell
AND KRYSTYNAHETNARSKI
University,
Cornsrock
Hall,
Ithaca,
New
York 14853
Received October 28, 1980; accepted January 7, 1981 Characteristics of the Type III optical difference spectra of 13 methylenedioxyphenyl compounds in NADPH-fortified armyworm midgut microsomes varied with the nature of the substituents in the aromatic ring. Compounds with electron-donating substituents yielded spectra with large 427/458 nm peak ratios, whereas those with electron-withdrawing groups exhibited low 427/ 458 nm peak ratios. Small amounts of carbon monoxide were generated during incubation of the 4,5-dihalo derivatives with midgut microsomes, and cis- and trans-methylenedioxycyclohexanes exhibited spectra with a major Soret peak at about 430 nm and a very weak absorbance maximum at about 480 nm. Formation of the Type III spectral complex occurred very rapidly and was associated with a marked decrease (up to 72%) in cytochrome P-450 levels as measured by carbon monoxide binding. Although a 24% reduction of cytochrome P-450 was observed in the absence of any measureable 458-nm spectral complex a linear relationship existed between further decreases in the cytochrome and the increase in Type III complex formation (458 nm). Inhibitory potencies of the compounds towards aldrin epoxidase and benzopyrene hydroxylase activities were not clearly correlated with either spectral complex formation or decrease in cytochrome P-450 and it is apparent that different factors are involved in the inhibition of different monooxygenase reactions. INTRODUCTION
optical difference spectrum. Subsequently, numerous studies have confirmed that, in the presence of NADPH and 0, (2, 12-15), or cumene hydroperoxide (16, 17), several MDP compounds undergo microsomal metabolism to reactive intermediates that bind to reduced cytochrome P-450 to give stable inhibitory complexes exhibiting Type III difference spectra. Considerable speculation has centered on the nature of the reactive intermediates involved and homolytic radicals (18), benzodioxolium ions (19), and carbanions (20) have all been implicated in the mechanism. The most recent suggestions favor the formation of a reactive carbene species (21). There seems little doubt that the Type III difference spectrum is indicative of the formation of a stable inhibitory complex with cytochrome P-450. However, a direct correlation between complex formation and inhibition of microsomal oxidation has yet to be demonstrated. In interpreting the Type III spectral complex and its relationIII’
Although it is now well established that methylenedioxyphenyl (MDP) compounds inhibit microsomal oxidations in vitro (l-6) and synergize the activity of many drugs and insecticides in vivo in both mammals and insects (2-6) their precise mode of action remains unclear. Following early suggestions that inhibition might occur simply through alternative substrate interactions (7) a more specific inhibitory mechanism was first indicated by the demonstration of decreased levels of cytochrome P-450 in microsomal preparations from mammals (8) and insects (9, 10) treated in vivo with piperonyl butoxide. Aerobic incubation of piperonyl butoxide with NADPH-fortified mouse liver microsomes was also found to cause a decrease in cytochrome P-450 and was associated with the formation of a spectrally observable complex with dual Soret peaks at 455 and 427 nm the relative peak heights being pHdependent (11); this was termed the ‘Type 32 0048-3575/81/010032-l 1$02.00/O Copyright All rights
@ 1981 by Academic Press, Inc. of reproduction in any form reserved.
INHIBITORY
INTERACTIONS
OF
METHYLENEDIOXYPHENYL
ship to inhibition of drug oxidation it is usually assumed that the 455nm peak represents the primary inhibitory complex between the active MDP intermediate and the ferrous heme iron of cytochrome P-450. This assumption is based mainly on the fact that several other types of compounds, including amphetamine derivatives (22-24), SKF 525A (22, 25), and some halogenated hydrocarbons (26) undergo metabolism to intermediates that give similar spectrally identifiable 455-nm complexes with concomitant inhibition of microsomal oxidation, and that several other r-acceptor ligands (CO, SnX,, GeX,, where X = halogen) give single Soret peaks in the 455-nm region (27). However, several methylenedioxy compounds which are inhibitors of oxidative metabolism and CO binding to cytochrome P-450 do not form typical Type III complexes in NADPH-reduced microsomes. Thus cis- and trans-methylenedioxycyclohexanes (28) and several aliphatic dioxolanes (29) exhibit stable optical difference spectra with a single peak at 427-430 nm and MDP compounds with electronwithdrawing groups in the aromatic ring yield mainly CO from the methylenic carbon during metabolism by rat liver microsomes (15). As a consequence it appears important to evaluate more carefully the relationship between spectral complex formation and inhibition of microsomal oxidation by MDP compounds. The studies reported in this manuscript were designed to investigate this in southern armyworm (Spodoprera eridunia) midgut microsomes. MATERIALS
AND
METHODS
Chemicals. 1,2-Methylenedioxybenzene (MDB) and its 4-bromo and 4-methyl derivatives were purchased from Frinton Laboratories, Vineland, New Jersey, and piperonyl nitrile (4-cyano-MDB), sesamol (Chydroxy-MDB) safrole, and isosafrole were from the Aldrich Chemical Company, Milwaukee, Wisconsin. Piperonyl butoxide was kindly provided by the FMC Corpora-
COMPOUNDS
33
tion (Niagara Chemical Division), Middleport, New York. All other MDB derivatives employed were synthesized by established procedures (30). cis-Hexahydro- 1,3-benzodioxolan, bp,, 77°C [reported bp,, 69-70°C (3 l)], and trans- hexahydro1,3-benzodioxolan, bp,, 68- 69°C [reported bp,, 66-67°C (32)] were prepared by reaction of the corresponding cis- and transcyclohexanediols with paraformaldehyde (31, 32). Biochemicals were purchased from Sigma Chemical Company, St. Louis, Missouri and all other chemicals and reagents employed were of analytical reagent grade. Enzyme preparation. Larvae of the southern armyworm (S. eridania) were reared on bean plants under greenhouse conditions through the first five instars as previously described (33). The sixth-instar larvae used in these experiments were kept in an environmental chamber and were fed on a semidefined artificial diet (34) for 48 h prior to use. Microsomal fractions of armyworm midguts were prepared as previously described (35) and the microsomal pellets were resuspended in 0.05 M TrisHCl buffer, pH 7.8, at 2 mg protein/ml. Protein concentrations were determined by the method of Lowry et al. (36) using bovine serum albumin as a standard. Spectral studies. Optical difference spectra were measured at 30°C with an Aminco DW-2 spectrophotometer using l-cm pathlength semimicro cuvettes and l-ml aliquots of microsomal suspensions (2 mg protein/ml). NADPH (1 pmol) was added to each cuvette and after baseline correction, the reaction was initiated by addition to the sample cuvette of the appropriate MDP compound (0.1 pmol) in 10 ~1 ethanol; the reference cuvette received 10 ~1 ethanol. Repetitive scans (5 nm/sec) between 400 and 500 nm were made at intervals of 1 min. After maximum spectral development a few grains of sodium dithionite were added to each cuvette and CO was bubbled through the sample for measurement of cytochrome P-450 (37). In other experiments, midgut micro-
34
CHANG,
WILKINSON,
somes (2 mg protein/ml) were incubated with the various MDP compounds (0.1 mM) at 30°C in Erlenmeyer flasks both with and without addition of NADPH (1 mM). After 5 min the reactions were terminated by addition of dithionite and cytochrome P-450 levels were determined in each incubation mixture. Spectral studies were also conducted using aliquots from incubations without NADPH (reference) vs those from incubations with NADPH (sample). Enzyme assays. Aldrin epoxidation assays were carried out aerobically at 30°C as described previously (34) using a j-ml reaction mixture at pH 7.8. Incubation time was 10 min and the reaction was initiated by addition of 100 pg aldrin in 25 ~1 ethanol. MDP compounds were added to the incubations in lo-50 ~1 of ethanol and Iso (M) values were usually determined from the means of duplicate incubations with at least four different inhibitor concentrations. Benzopyrene hydroxylase activity was assayed by the radiometric procedure described by DePierre et al. (38) using [G3H]benzo[a]pyrene diluted to 13.5 PCiI pmol. The l-ml incubation mixtures contained 0.5- 1.0 mg microsomal protein, 1 mM NADPH, 5 mM MgC&, 5 pM MnCl, in 50 mM Tris-HCl, pH 7-8; the reactions were initiated by addition of 50 nmol [3H]benzopyrene in 10 ~1 acetone. MDP compounds were added in 10 ~1 acetone to give a concentration of 0.1 mM. The aerobic incubations were at 30°C for 12 min and were terminated by addition of 1 ml of ice-cold 0.5 N NaOH in 80% ethanol. Extraction was carried out according to DePierre et al. (38) and counting was effected in a Packard Model 2425 scintillation counter using a scintillant consisting of a mixture of PPO (4.75 g), POPOP (0.32 g), and Bio-Solv (40 ml) dissolved in toluene (1000 ml). Counting efficiency was 48.5%.
AND
HETNARSKI
I.oos WAVELENGTH
(nm)
FIG. 1. Type III optical difference spectra obtained with 10m4 M, I.5dibromo-MDB (A), MDB (B), isosaf role (C), and trans-methyfenedioxycyclohexane (0) in NADPH-fortified armyworm midgut microsomes.
exception of sesamol (4-hydroxy-MDB), which showed no obvious spectral interaction, all of the MDP compounds exhibited Type III optical difference spectra in NADPH-reduced armyworm midgut microsomes. The spectra were characterized by absorbance peaks at 426-429 nm and 456-462 nm and a marked trough at 412-414 nm. However, the compounds can be divided arbitrarily into three major groups based on the relative intensities of the two major spectral peaks (Fig. 1). In the first group (Group I), consisting of the 4cyano, 4,5-dibromo, and 4,5-dichloro derivatives of MDB, the ratio of the 427/458 nm peaks’ is less than unity (Fig. lA, Table 1) and with the cyano compound, the 427-nm peak appears only as an indistinct shoulder on the predominant 458-nm peak. At the other extreme, Group II compounds exhibit spectra in which the 427-nm peak predominates and with 4271458 nm peak
RESULTS
Spectral studies were conducted with a total of 13 MDP derivatives and with cisand trans-methylenedioxycyclohexanes each at a concentration of 10e4 M. With the
1 The double Soret peaks in the Type III difference spectra are referred to throughout the text as the 427 and 458 nm peaks even though the wavelengths of maximum absorbance vary somewhat from compound to compound.
427.2 (0.009 426.8 (0.0035 427.8 (0.0019 427.7 (0.0185 427.6 (0.0152 427.5 (0.0175 427.5 (0.0072 427.3 (0.01 430.3 (0.0037 430.7 (0.0038
427.7 (0.008 428.5 (0.0129 427.4 (0.0119 Shoulder
456.8 -t 0.7 (0.0044 -c O.ooo6) 458.1 * 1.1 (0.0048 t 0.0006) 457.4 2 1.0 (0.0048 rt 0.0002) 458.6 If: 1.3 (0.0035 k O.ooo6) No detectable spectrum r 0.3 457.4 iz 0.8 r 0.0006) (0.0071 + 0.0004) k 0.8 458.1 k 0.3 -e 0.ooo6) (0.0063 2 0.0001) 5 0.1 457.0 2 0.9 t 0.0005) (0.0071 2 0.001) t 0.1 458.7 * 0.5 -c 0.0032) (0.0068 ” 0.0008) If: 0.2 459.2 k 0.6 r 0.0025) (0.0064 + 0.0007) e 0.3 459.5 k 0.3 rt 0.0015) (0.0065 2 0.005) -e 0.2 461.5 2 0.6 rf: 0.002) (0.0037 -c 0.0007) rt 0.1 461.2 z 1.2 t 0.004) (0.002 5 0.0005) 2 1.0 478.2 2 0.1 It 0.0009) (0.0016 2 0.0003) r 0.8 480 f 1.5 rt 0.0004) (0.0007 k 0.0002)
r 0.3 r 0.0017) 2 1.1 -+ 0.0012) k 0.0 & 0.0007) at 428
0 (6.3 x 10m3) 3 (1.9 x 10-S)
28.2 41.1
-
81 (2.7 x 1O-5)
32.2
5.0
74 (3.4 x 10-S)
95 (1.4 x 10-S)
66 (5.0 x 10-S)
100 (6.3 x 1O-6)
100 (2.1 x 10-S)
93 (7.5 x 10-G)
0 (>lO-2) 95 (7.5 x 10-G)
3 (1.3 x 10-Z)
55 (8.0 x lo-“)
31 (2.2 x IO-“)
0 (1.0 x 10-3)”
AE
50
30
ND’
56
57
89
49
56
55
5 58
15
51
51
23
BP
Percentage inhibition of microsomal oxidation at IO-* Mr
55.8
69.6
49.4
53.5
72.7
71.5
33.4 50.6
39.0
54.2
41.0
17.9
Percentage decrease in cytochrome P-450”
Midgut Microsomes
1.95
2.69
2.38
2.72
0.27
0.56
1.27
-
2.48
2.69
1.82
Absorbance ratio 4211458 nm
TABLE 1 of Methvlenedioxv Compounds uaith Armw,orm
Type III spectral character”
Interactions
I’ Upper figures denote peak position (nm), lower tigures in parentheses indicate maximum absorbance (vs 490 nm) in presence of 10m4M test compound. Values are means ? SEM of three or four replicates. h Measured as described in text. ’ AE, Aldrin epoxidase; BP, benzopyrene hydroxylase. I2Is, (M) values. ti Not determined.
2,3-Methylenedioxynaphthalene Piperonyl butoxide cis-hlethylenedioxycyclohexane trans-Methylenedioxycyclohexane
Dihydrosafrole
Isosafrole
Safrole
4,5-di-Cl-MDB
4,5-di-Br-MDB
COH-MDB 4-Br,S-CH,O-MDB
CCN-MDB
CCH,O-MDB
CCH,-MDB
MDB
Compound
Spectral and Inhibitory
36
CHANG,
WILKINSON,
ratios ranging from about 2.5 to 5.0 (Fig. 1C). Included in this group are safrole, isosafrole, dihydrosafrole, piperonyl butoxide, and the 4-methyl and 4-methoxy derivatives of MDB. Three compounds, MDB, 4-bromo-5-methoxy-MDB, and methylenedioxynaphthalene (Group III) are intermediate between these extremes and exhibit spectra in which the 427- and 458-nm absorbance peaks are of approximately equal intensity (427/458-nm ratio 1.3 to 1.95) (Fig. IB). cis- and fransMethylenedioxycyclohexanes behave differently from the aromatic compounds and as has been previously reported (28) exhibit difference spectra with a single major peak at about 431 nm and a very indistinct peak at 478-480 nm (Fig. 1D). In all cases spectral development was extremely rapid and was essentially complete within 1 to 2 min following addition of the methylenedioxy compound to the reaction mixture. Once formed the spectra were stable and continued incubation in the presence of NADPH for periods of at least 20 min resulted in no further qualitative or quantitative spectral changes. The characteristics of the Type III difference spectra with various methylenedioxy compounds are shown in Table 1. With compounds in Groups II and III the addition of dithionite to each cuvette after maximum Type III spectral development with NADPH had little or no effect on the observed spectrum with the exception of a slight (approximately 1 nm) bathochromic shift in the 427-nm peak with some compounds and a noticeable intensification of the trough in the region of 410 nm. Dithionite caused a more obvious shift to lower wavelength in Type III spectra formed from several compounds in Group I. Thus a 3- to 4-nm shift in both peaks occurred with the two dihalo compounds whilst in the case of 4-cyano-MDB, the 458-nm peak remained unchanged and the 427 shoulder disappeared entirely. The wavelength shift observed with the 4,5-dibromo and 4,5dichloro compounds on addition of dithio-
AND
HETNARSKI
nite was accompanied by a slight increase in absorbance at about 453 nm and suggested the possible formation of carbon monoxide as has been reported with rat liver microsomes (15). This possibility was strengthened by the finding that addition of heme (2 pmol) to each cuvette caused the 453nm peak to revert to about 457 nm and led to the concomitant development of a new peak at 418.5-420 nm in the region of carboxyhemoglobin. No attempt was made to quantitate the small amount of carbon monoxide produced, and of all the compounds tested it was apparent only with the two dihalo derivatives. Carbon monoxide production from MDP compounds in armyworm midgut microsomes is clearly much lower than in those from rat liver (15) and in contrast to the latter there was no evidence of any carbon monoxide from the 4-cyano derivative. Despite the lack of any obvious NADPH-mediated microsomal difference spectrum with 4-hydroxy-MDB (sesamol), addition of dithionite after incubation with NADPH caused the development of a small absorbance peak at about 453 nm suggesting the formation of low levels of carbon monoxide from this compound. As discussed elsewhere (15) it is probable that this occurs by a mechanism different from that followed by other MDP compounds. Addition of dithionite to microsomes incubated with cis- and transmethylenedioxycyclohexanes, and allowed to attain a full spectral development in the presence of NADPH, resulted in a marked decrease in the 430-nm peak and no obvious change in the small absorbance peak at about 480 nm. Attempts to measure residual cytochrome P-450 levels after incubation of the various test compounds with NADPH in the cuvettes were not successful due to the complex nature of the combined Type III/CO difference spectra produced. The absorbance maxima of the latter spectrum varied considerably from compound to compound, being as high as 458 nm in the case of isosafrole, and thus making any in-
INHIBITORY
INTERACTIONS
OF
METHYLENEDIOXYPHENYL
terpretation of corrected spectra extremely difficult. It was further complicated by the large absorbance peak which frequently appeared at 421-423 nm and which indicated significant degradation of the fairly labile armyworm cytochrome P-450 to cytochrome P-420. A more convenient method for estimation of residual levels of cytochrome P-450 was afforded by spectral measurements following incubation (5 min at 30°C) of the various test compounds (lo-* M) with armyworm midgut microsomes in individual flasks in either the presence or absence of NADPH. Levels of cytochrome P-450 measured in microsomes incubated with MDP compounds without NADPH were always the same as those in control nonincubated microsomes, indicating no inhibitory interaction under these conditions. In incubations of the test compounds with NADPHfortified microsomes levels of cytochrome P-450 were decreased significantly, the percentage reduction varying from about 18% in the case of MDB itself to approximately 70% with the 4,5-dibromo and 4,5dichloro derivatives and with dihydrosahole (Table 1). In all cases, the absorbance maximum of the CO difference spectra measured under these conditions was in the range of 450-452 nm, only slightly greater than that in control microsomes (449.8 nm). Furthermore, there appeared to be less conversion to cytochrome P450 under these conditions than in incubations carried out directly in the cuvettes. The inhibitory potencies of the MDP compounds towards aldrin epoxidation are indicated in Table 1 as ISo (M) values as well as by the percentage inhibition observed at lo-* M. It is apparent that considerable variation exists within the group of compounds tested, I,,, values ranging from 6.3 x lo-” M in the case of isosafrole to >10e2 M with 4-hydroxy-MDB (sesamol), and inhibition ranging from 100 to 0% at lo-* M. Inhibitory potency of the various compounds towards benzopyrene hydroxylase was somewhat less variable and at lo-* M
COMPOUNDS
37
only one, isosafrole, exhibited more than 60% inhibition (Table 1). Unfortunately, determination of inhibitory activity towards this reaction proved difficult due to the very low hydroxylase levels that exist in control armyworm midgut microsomes. In general, aldrin epoxidation was somewhat more sensitive to inhibition than benzopyrene hydroxylase although this was not the case with MDB, and its 4-methyl and 4-cyan0 derivatives or with isosafrole. Interestingly, in contrast to their lack of effect on aldrin epoxidase at 1Om1M both cis- and rrans-methylenedioxycyclohexanes were quite effective inhibitors of benzopyrene hydroxylase activity at this concentration. Attempts to demonstrate a correlation between inhibitory potency towards aldrin epoxidase and benzopyrene hydroxylase activity and either reduction in cytochrome P-450 or intensity of 458-nm absorbance at the same concentration (IO-” M) were not successful (Fig. 2). There appears to be a trend indicating greater inhibition with compounds exhibiting a more intense 458-nm absorbance and causing a greater reduction in cytochrome P-450 but the correlation is not clear-cut and there are several obvious exceptions such as piperonyl butoxide (Fig. 2A) which is an effective inhibitor of aidrin epoxidase but which causes only a slight reduction in cytochrome P-450 and only a relatively small 458-nm spectral interaction. With aldrin epoxidase (Fig. 2A) a reduction of approximately 25-30% of the cytochrome P-450 can apparently be sustained without significant loss of enzyme activity; this does not appear to be true for benzopyrene hydroxylase (Fig. 2B). A weak correlation (r = 0.696) exists between the 458-nm absorbance and percentage decrease in cytochrome P-450 with 12 MDP compounds (values for MDB not included) (Fig. 3) and the intercept of the y axis suggests that a 24.4% reduction of cytochrome P-450 occurs in the absence of any observable 458-nm absorbance. This value approximates the reduction in cyto-
38
CHANG,
CYTOCHFRME
WILKINSON,
P-454
DECREASE
AND
(K)
HETNARSKI
or
(AABSORBANCE
459 /49Onm)
2. Relationship between inhibition of aldrin epoxidase (A) and benzopyrene hydroxyluse (B) activities with reduction in cytochrome P450 (0) and Type 111458 nm absorbance ( 0) with MDP derivutives (10m4 M) in armyworm midgut microsomes. (I) MDB; (2) I-cyano-MDB; (3) I-hydroxy-MDB; (4) I-methyl-MDB; (5) I-methoxy-MDB; (6) isosafrole; (7) 2,3-methylenedioxynaphthalene (8) dihydrosufrole; (9) 4,5-dibromo-MDB; (10) 4,5-dichloro-MDB, (1 I) 4-bromo-5-methoxy-MDB; (12) sufrole; (13) piperonyl butoxide. FIG.
chrome P-450 observed with the cis- and trans-methylenedioxycyclohexanes (28.2 and 41.1%, respectively), which exhibit no 458 nm absorbance peak (Table 1) and the reduction with sesamol. DISCUSSION
The results of this investigation indicate some similarities and several important differences between the inhibitory interactions of MDP compounds with armyworm midgut microsomes and those previously reported for rat liver (15). The Type III optical difference spectra observed on incubation of most MDP compounds with NADPH-fortified armyworm midgut microsomes are qualitatively similar to those reported in microsomes from other species (2) with dual Soret peaks at about 427 and 458 nm. In contrast to rat liver microsomes, however, where in the presence of NADPH, MDP compounds containing electron-withdrawing groups in the aromatic ring generate significant amounts of carbon monoxide from the methylenic carbon atom (15), carbon monoxide formation in armyworm midgut microsomes occurs to a much
lower extent and appears to be restricted to the 4,5-dichloro and 4,5-dibromo derivatives; it also occurs to some extent with sesamol although this compound exhibits no obvious optical difference spectrum. The characteristics of the MDP Type III spectral complexes in armyworm midgut
pI,IIIIIIIL 0
,002
A ABSORBANCE
.004
.006
(459
.ooe
/a90
nm)
3. Relationship between reduction of cytochrome P-450 levels and Type ZZZ 458 nm absorbance with MDP derivatives (10V4 M) in armyworm midgut microsomes. Numerical identiftcation of compounds used is the same us in Table 2 and the line represents the best fit (linear regression) through all points exclusive of compound I (MDB). FIG.
INHIBITORY
INTERACTIONS
OF
METHYLENEDIOXYPHENYL
COMPOUNDS
39
microsomes, particularly the relative size of interaction of those with electron-withdrawthe 427- and 458~nm peaks, appears to be ing groups occurs mainly through the free related to the nature of the substituents in form of the ligand resulting in low 4271458 the aromatic ring. Compounds with elec- nm ratios. tron-withdrawing groups in the ring (Group A factor which complicates interpretaI) exhibit spectra with strong 458- and tions of 427/458 nm peak ratios in terms of weak 427-nm absorbance peaks, whereas chemical structure is that the ratios are also those with electron-donating substituents dependent on the pH of the incubation (Group II) give spectra in which the 427-nm mixture (11). This is observed in armyworm peak predominates. Group III compounds midgut microsomes as in those from other species, although detailed studies to detersuch as MDB and 2,3-methylenedioxynaphthalene with no substituents, or those con- mine and compare the effects of pH on taining both electron-withdrawing and -do- Type III spectra in microsomes of different nating groups (e.g., 4-bromo-5-methoxyspecies have not yet been conducted. It MDB) yield spectra intermediate between should be pointed out that since the data Groups I and II. The relative intensity of presented here were obtained at a single the 427- and 458-nm peaks, therefore, ap- pH, somewhat different results may have pear to be related in some way to the been observed had different pH values been electronic distribution within the molecule, employed. although a more complete interpretation The relationship between Type III specwill have to await further information on tral complex formation, reduction in cytothe nature of the ligand binding sites in- chrome P-450 and inhibition of microsomal volved. It has been suggested that the 427- oxidation remains unclear and it is apparent and 458-nm Type III spectral peaks ex- that factors other than those considered in this investigation are involved. The data hibited by most dioxoles (MDP compounds) shown in Fig. 3 suggest a linear relationship may result from ligand interaction with cytochrome P-450 of the bound and free between 458-nm absorbance and the obforms respectively of a relatively stable served decrease in cytochrome P-450. lipophilic r-acceptor ligand such as the However, extrapolation of these data to the proposed carbene intermediate (29, 39, 40). y axis indicates that an approximately 24% The existence of an equilibrium between the decrease in cytochrome P-450 is not dibound and free forms of the ligand, com- rectly attributable to the 458-nm absorbined with the instability of the free form of bance peak. Since both the 427- and 458-nm the ligand formed from dioxolanes (aliphatic peaks presumably represent ligand commethylenedioxy compounds and methyleneplexes with cytochrome P-450 it is possible dioxycyclohexanes) has been proposed as that the 24% reduction in cytochrome P-450 not associated with the 458-nm complex an explanation of the single 427-nm differrepresents the mean contribution of the ence spectra observed with these com427-nm ligand complex. The contribution of pounds (29). If this interpretation is correct, the 427/458 nm peak ratios may reflect the the latter to the observed reduction in cytoequilibrium between the bound and free chrome P-450 appears to have been given forms of the proposed carbene ligand, as- little attention to date, although it has been suming that the free ligands from different reported that the Ki value for the inhibition MDP compounds are of approximately of CO binding by piperonyl butoxide coinequal stability. Consequently, the data cides with the KS value for the formation of presented here may suggest that electronthe Type III spectrum calculated from the donating groups favor the association of the sum of the 427- and 455nm peaks (14). It ligand to the cytochromal protein (hence therefore seems clear that the 427-nm comthe large 427/458 nm ratios) whereas ligand plex plays some role in the observed reduc-
40
CHANG,
WILKINSON,
tion of cytochrome P-450 and should be taken into consideration in evaluating the overall inhibitory interactions of MDP compounds. Unfortunately, quantitation of the total spectral complex is not simple and summation of the absorbance differences between 427 and 490 nm and between 458 and 490 nm may not provide an adequate measure. Attempts to correlate the inhibition of microsomal oxidation with spectral complex formation and cytochrome P-450 reduction (Fig. 2) are also difficult since it is almost certain that both competitive (alternative substrate) and noncompetitive (complex formation) interactions contribute to the overall inhibitory activity. Thus Franklin (41) found that the inhibition of ethylmorphine N-demethylation by piperonyl butoxide was initially competitive in nature but subsequently became noncompetitive following formation of the complex. In view of the established existence of multiple forms of cytochrome P-450 (42) which catalyze the oxidative metabolism of different xenobiotic substrates, it is entirely possible that variations in inhibitory potency towards different substrates, or reaction types, might result from differences in the rate or extent of MDP complex formation with different forms of the cytochrome. The data presented here suggest that this might be the case, since with aldrin epoxidation, 25 - 30% reduction of cytochrome P-450 can apparently occur without causing significant inhibition of enzyme activity. This does not appear to be the case with benzopyrene hydroxylase, however, and the significant inhibition of this reaction by cis- and trans-methylenedioxycyclohexanes, which form only the 427-nm spectral complex, suggests that the latter may be of more significance in the inhibition of benzopyrene hydroxylase than of aldrin epoxidase. It is of considerable interest to speculate whether the previously discussed bound and free forms of the ligand interact differently with different types or pools of cytochrome P-450.
AND
HETNARSKI
Another factor which may complicate attempted correlations between spectral complex formation and inhibition of oxidase activity is the possible displacement of the Type III complex by some substrates and the consequent restoration of monooxygenase activity; this has been reported with several, but not all, microsomal enzyme substrates tested against the Type III isosafrole complex in rat liver microsomes (43) but has not yet been investigated in those from armyworm midgut. ACKNOWLEDGMENT
The work was supported in part by a grant (ES 01902) from the U.S. Public Health Service. REFERENCES
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11. R. M. Philpot and E. Hodgson, A cytochrome P-450- piperonyl butoxide spectrum similar to that produced by ethyl isocyanide, Life Sci. 10, 503 (1971). 12. M. R. Franklin, The enzymic formation of a methylenedioxyphenyl derivative exhibiting an isocyanide-like spectrum with reduced cytochrome P-450 in hepatic microsomes, Xenohiorica 1, 581 (1971). 13. M. R. Franklin, Piperonyl butoxide metabolism by cytochrome P-450: Factors affecting the formation and disappearance of the metabolite-cytochrome P-450 complex, Xenohiotira 2, 517 (1972). 14. R. M. Philpot and E. Hodgson, The effect of piperonyl butoxide concentration on the formation of cytochro ?e P-450 difference spectra in hepatic microso lies from mice, Mol. Pharmad. 8, 204 (1912). 15. Loh-Sheng Yu, C. F. Wilkinson, and M. W. Anders, Generation of carbon monoxide during the microsomal metabolism of methylenedioxyphenyl compounds, Biochem. Pharmacol. 29, 1113 (1980). 16. C. R. Elcombe, J. Bridges, R. H. Nimmo-Smith, and J. Werringloer, Cumene hydroperoxidemediated formation of inhibited complexes of methylenedioxyphenyl compounds with cytochrome P-450, Biochem. Sot. Trans. 3, 967 (1975). 17. A. P. Kulkarni and E. Hodgson. Cumene hydroperoxide-generated spectral interactions of piperonyl butoxide and other synergists with microsomes from mammals and insects, Pestic. Biochem. Physiol. 9, 75 (1978). 18. C. Hansch. The use of homolytic, steric and hydrophobic constants in a structure-activity study of 1,3-benzodioxole synergists, J. Med. Chem. 11, 920 (1968). 19. D. J. Hennessy. Hydride transferring ability of methylenedioxybenzenes as a basis of synergistic activity, J. Agr. Food Chem. 13, 218 (1965). 20. V. Ullrich and K. H. Schnabel, Formation and binding of carbanions by cytochrome P-450 of liver microsomes, Drug Metab. Dispos. 1, 176 (1973). 21. W. Nastainczyk, V. Ullrich, and H. Sies, Effect of oxygen concentration on the reaction of halothane with cytochrome P-450 in liver microsomes and isolated perfused liver, Biochem. Pharmacol. 27, 387 (1978). 22. M. R. Franklin, Inhibition of mixed-function oxidations by substrates forming reduced cytochrome P-450 metabolic-intermediate complexes. Pharmacol. Ther. 2, 227 (1977). 23. J. Werringloer and R. W. Estabrook, Evidence for an inhibitory product cytochrome P-450 complex generated during benzphetamine metab-
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COMPOUNDS
41
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37. T. Omura and R. Sato, The carbon monoxidebinding pigment of liver microsomes, .I. Biol. Chem. 239, 2379 (1964). 38. J. W. DePierre, K. A. M. Johanneson, M. S. Moron, and J. Seidegrd, Radioactive assay of aryl hydrocarbon monooxygenase and epoxide hydrase in “Methods in Enzymology” (S. Fleischer and L. Packer, Eds.), Vol. LII, p. 412, Academic Press, New York, 1978. 39. A. R. Dahl and E. Hodgson, The affinity of cytochrome P-450 for ethyl isocyanide: An explanation for double Soret spectra, Chem. Biol. Interact. 20, 171 (1978). 40. A. R. Dahl and E. Hodgson, Complexes of trivalent oxygenated phosphorus compounds with cytochrome P-450 and cytochrome P-420: The
AND HETNARSKI origin of double Soret spectra, Chem. Biol. Interact. 21, 137 (1978). 41. M. R. Franklin, Inhibition of hepatic oxidative xenobiotic metabolism by piperonyl butoxide, Biochem. Phormacol. 21, 3287 (1972). 42. F. P. Guengerich, Isolation and purification of cytochrome P-450 and the existence of multiple forms, Pharmacol. Ther. 6, 99 (1979). 43. C. R. Elcombe, M. Dickins, B. C. Sweatman, and J. W. Bridges, Substrate-elicited dissociation of the isosafrole metabolite-cytochrome P-450 complex and the consequential reactivation of monooxygenation, in “Microsomes and Drug Oxidations” (V. Ullrich, A. Hildebrandt, I. Roots, R. W. Estabrook, and A. H. Conney, Eds.) p. 247, Pergamon, New York, 1977.