effects of dihydrosafrole on cytochromes p-450 and drug oxidation in hepatic microsomes from control...

TOXICOLOGY AND APPLIED PHARMACOLOGY 68, 66-76 (1983)

Effects of Dihydrosafrole on Cytochromes P-450 and Drug Oxidation in Hepatic Microsomes from Control and Induced Rats

MICHAEL MURRAY,~HRISTOPHER F. WILKINSON, AND CHRISTOPHER E. DUBI?

Cornell University. Department of Entomology, Comstock Hall, Ithaca. New York 14853

Received June 12, 1982; accepted November 4, 1982

Effects of Dihydrosafrole on Cytochromes P-450 and Drug Oxidation in Hepatic Microsomes from Control and Induced Rats. MURRAY, M., WILKINSON, C. F., AND DUB& C. E., (1983). Toxicol. Appl. Pharmacol. 68, 66-76. Changes in cytochromes P-450, aminopyrine N-demethylase (APDM), aromatic hydrocarbon (benzo(a]pyrene) hydroxylase (AHH), and type III spectral complex formation were measured in hepatic microsomes of control, phenobarbital (PB)-, and P-naphthoflavone (@NF)-induced rats after a single dose of dihydrosafrole (4-n-propyl-1,2-meth- ylenedioxybenzene, DHS). Time profiles of changes in these microsomal parameters were complex and showed that APDM activities and cytochrome P-450 levels decreased immediately after treatment and were associated with concurrent increases in the intensity of the type III methylenedioxyphenyl (MDP) metabolite/cytochrome P-450 spectral complex. In noninduced rats, both APDM activity and cytochrome P-450 levels returned to control levels between 12 and 24 hr after treatment with DHS and subsequently increased above control levels. In PB- and /3NF- induced animals, the inhibitory phases were more prolonged and activity never returned to levels higher than the corresponding controls. AHH activity was increased substantially (two- to three- fold) in al1 cases after DHS administration. Although displacement of the MDP metaboIite/ cytochrome P-450 complex with 2-methylbenzimidazole generally led to a marked restoration of cytochrome P-450 levels and partially reversed the inhibition of APDM, it had little or no effect on AHH activities.

Methylenedioxyphenyl (MDP) derivatives undergo complex interactions with microsomal mixed-function oxidases (MFOs). In- cubation of MDP compounds with NADPH- fortified microsomal suspensions in vitro typically results in the inhibition of certain oxidase activities (Hodgson and Philpot, 1974). Under similar incubation conditions many MDP compounds elicit a characteristic optical difference spectrum, known as a type III spectrum, that exhibits dual Soret peaks near 427 and 455 nm (Philpot and Hodgson, 197 1; Franklin 197 1) which have been as- cribed to an inhibitory complex formed when an active MDP metabolite, possibly a car- bene, interacts with cytochromes P-450 (Nas- tainczyk et al., 1978).

In vivo the administration of many MDP derivatives such as isosafrole produces a biphasic effect on hepatic MFO activity, an initial inhibitory phase followed by induction of cytochromes P-450 and of several associated MFO reactions (Kamienski and Murphy, 197 1; Skrinjaric-Spoljar eE al., 197 1). Of par- ticular toxicological interest are observations that in hepatic microsomes isolated from isosafrole-pretreated rats, metabolic activity toward benzolalpyrene and biphenyl (Parke and Rahman, 1970; Lake et al., 1973) and 2-acet- amidofluorene (Lotlikar and Wasserman, 1972) is markedly increased.

Recent reports suggest that MDP compounds exhibit some degree of selectivity in their interactions with microsomal fractions

004 1-008X/83 $3.00 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

66

DIHYDROSAFROLE EFFECTS ON MICROSOMES 67

FIG. 1. Dihydrosafrole (4-n-propyl- 1,2-methylenedi- oxybenzene, DHS).

from differently induced animals (Bornheim and Franklin, 1982) and with different forms of purified cytochromes P-450 (Ryan et al., 1980). Consequently, the present study was undertaken to investigate the interactions of dihydrosafrole (Fig. l), an MDP compound with similar properties to isosafrole, with cytochromes P-450 and drug oxidation in microsomes from rats treated with phenobarbital (PB) and @-naphthoflavone (PNF).

METHODS

Chemicals

Dihydrosafrole (4-n-propylmethylenedioxybenzene, DHS) was prepared by the catalytic reduction of isosafrole (Pfahz and Bauer, Stamford, Conn.) with palladium on charcoal. After extraction, the purified material distilled in almost quantitative yield at 228 to 230°C at ambient pressure; the literature value for the bp is 228°C (Bam- berger and Lenglield, 1890). Biochemicals were pur- chased from Boehringer Mannheim, Indianapolis, Indi- ana; all solvents were analytical reagent grade.

Animals

Male Sprague-Dawleyderived rats (200 to 250 g) were obtained from Blue Spruce Farms, Altamont, New York. Phenobarbital sodium (PB) was administered ip in nor- mal saline at a dose of 100 mg/kg once daily for 3 days, and &naphthoflavone (6NF) was given ip in corn oil at a dose of 40 mgjkg once daily for 3 days. Dihydrosafrole (DHS) was given as a single ip dose of 400 mg/kg in corn oil, and animals were killed at appropriate times after treatment. In the case of animals receiving both PB or BNF and DHS, the latter compound was given approx- imately 24 hr after the final dose of the appropriate inducer.

Preparation of Microsomal Fractions

Hepatic microsomal fractions were prepared as previously described (Yu et al., 1980) except that 0.1 M po-

tassium phosphate-buffered sucrose (pH 7.4) was used in place of Tris-HCl-buffered sucrose. Protein was determined by the Biuret method (Robinson and Hogden, 1940) with bovine serum albumin as standard.

Enzyme Assays

Aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity was determined by the spectrofluorometric assay of Yang and Kicha (1978) in an Aminco SPF-125 spec- trofluorometer. I-Phenylimidazole (PI) and a-naphthoflavone (aNF) used in inhibition studies were added to the cuvettes in 50 gl of absolute ethanol; ethanol alone (50 ~1) was added to control incubations.

Aminopyrine Ndemethylase (APDM) activity was determined by the method of Murray et al. (1982) employing the Nash (1953) method for measurement of formaldehyde. Incubations contained 1.6 1 rmol anino- pyrine, 10 rmol magnesium chloride, 5 pmol EDTA, 15.6 pmol glucose 6-phosphate, 1 unit glucose-6-phosphate de- hydrogenase, 1.4 pmol NADP, 25 rmol semicarbazide hydrochloride, and 5.6 mg microsomal protein in potassium phosphate buffer (0.1 M, pH 7.4 to 4.00 ml). In- cubation time was 15 min.

Cytochrome P-450 was measured by the method of Omura and Sato (1964) in an Aminco-Chance DW-2 spectrophotometer, with an extinction coefficient of 91 mM-’ cm-i for the cytochrome P-450-ferrous carbonyl spectral complex. The DHS metabolite/cytochromes P- 450 complex (455-nm peak) was measured by optical difference spectroscopy in microsomal suspensions (1 to 2 mg protein/ml) after addition of a few grains of sodium dithionite to the sample cuvette.

MDP Metabolite Displacement

Displacement of the MDP metabolite from the cytochromes P-450 complex has effected by two successive 30-min incubations (37°C) of a concentrated microsomal suspension (-60 mg protein/ml) each with 10 pmol of 2-methylbenzimidazole. Following incubation, the microsomal suspension was eluted through a Sephadex G- 50 column (u 18 cm) with 0.1 M potassium phosphate buffer (pH 7.4) as described by Fisher et al. (1981). The extent of complex displacement was determined from the decreases in spectral ratios of the 427- and 455~nm peaks relative to the peak at 556 nm.

RESULTS

Several series of time profiles with respect to cytochrome P-450 levels, AHH and APDM activities, and optical difference spectra were

68 MURRAY, WILKINSON, AND DUBE

a

/@

o-o

0

b

.-.

__ _ ------_-_--__-__.

10 .

.

,

4 12 24 36

1

501 C

F z 8 i

T

/

o/O

.fl. --------

z 5 0

i * ‘/

.

it

(P6() .a /

./

.

0

/\ .

/\

.

/ . 0

!u

0

-----------------_____

4 12 24 36

d

HOURS AFTER DHS ADMINISTRATION

FIG. 2. Time profiles of (a) cytochrome P-450 content, (h) aromatic hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity, (c) aminopyrine N-demethylase (APDM) activity, and (d) 455/566 nm ab sorbance ratio, in microsomes from noninduced rats given a single dose of dihydrosafrole. Before displacement (0); after displacement (0) with 2-methylhenzimidazole. Actual control values (X k SE) were: cytochrome P-450 (0.70 + 0.04 nmol/mg protein), AHH activity (0.99 f 0.06 nmol benzo[o]pyrene metabolized/mg protein/mitt), APDM activity (2.4 + 0.1 nmol formaldehyde produced/mg protein/min). The absorbance ratio was determined as AA455-4s+ nm:AAS56-575 nm.

determined before and after MDP-complex a single dose of DHS 24 hr after termination displacement in microsomes from control and of inducer treatment (Figs. 2 to 4). PB- and PNF-induced rats that had received Cytochrome P-450 levels in microsomes


80.

40-

12 24 38 48 12 24 3’6 48

c 7. o-

12 24 38 48 12 24 36 48

HOURS AFTER DHS ADMINISTRATION

FIG. 3. As described in Fig. 2 except microsomes were prepared from phenobarbital (PB)-induced rats given a single dose of dihydrosafrole. Actual PB-induced values were: cytochrome P-450 ( 1.47 + 0.04 nmol/ mg protein), AHH activity (2.11 + 0.11 nmol benzo[a]pyrene metabolized/mg protein/min), APDM activity (6.3 + 0.3 nmol formaldehyde produced/mg protein/min). The absorbance ratio 427/556 nm was determined as A&-409 nm:AA~~6-5750m. In microsomes from PB-induced rats not given dihydrosafrole, the value of this ratio was 5.43. “I” refers to the 427/556 nm absorbance ratio profile before (0) and after (0) displacement with 2-methylbenzimidazole; “II” refers to the 455/556 nm absorbance ratio profile before and after displacement.

70 MURRAY. WILKINSON. AND DUBE

I

12 24 36

i2 2’4

HOURS AFTER DWS AOMINISTRATION

FIG. 4. As described in Fig. 2 except microsomes were prepared from &naphthoflavone (flNP)-induced rats given a single dose of dihydrosafrole. Actual @IF-induced values were: q&chrome P-450 (1.25 + 0.04 nmol/mg protein), AHH activity (6.16 k 0.50 nmol benzo[nJpyrene metabolii/mg protein/min), APDM activity (3.2 k 0.2 nmol formaldehyde produced/mg protein/min).

from noninduced, DHS-treated rats exhibited 12 to 36 hr after treatment. Concomitant a biphasic profile (Fig. 2a), a maximum re- changes in APDM activity (Fig. 2c) closely duction to about 75% of control levels oc- followed cytochrome P-450 levels, i.e., an ini- cur-ring 2 to 6 hr after treatment followed by tial inhibitory phase followed by induction a 40 to 50% increase at 24 and 36 hr. Dis- and decreased inhibition, and increased in- placement of the DHS metabolite from its duction following displacement of the com- complex with cytochrome P-450 caused a plex. substantial increase in cytochrome P-450 lev- In sharp contrast to the profile observed els, both decreasing the inhibitory phase and with APDM, that for AHH activity showed increasing the apparent induction phase from no initial inhibitory phase following DHS


TABLE 1

IN VITRO DISPLACEMENT BY 2-METHYLBENZIMIDAZOLE OF THE MICROSOMAL MDP METABOLITE/~T~CHROME P-450 SPECTRAL COMPLEXES RIWJLTING FROM IN VIVO TREATMENT OF CONTROL AND PB-, AND BNF-INDUCED RATS WITH DIHYDROSAFR~LE (ONS)

Spectral ratio”

Treatment Hours after

DHS exposure

Before After displacement displacement

A B A B

Percentage peak remaining

A B

Control 4 - 0.35 - 0.01 - 3 12 - 0.99 - 0 - 0 24 - 2.06 - 0.09 - 4 38 - 1.85 - 0.09 - 5

PB-induced 12 6.19’ 0.49 5.85 0 55 0 24 6.46 1.03 6.05 0 60 0 38 6.81 0.99 6.48 0.10 76 10 48 6.33 1.60 6.03 0.10 67 13

BNF-induced 6 - 0.83 - 0.14 - I7 12 - 2.61 - 0.43 - I6 24 - 2.34 - 0.35 - 15 36 - 3.68 - 0.29 - 9

‘Absorbance of the dual Soret peaks at 427 nm (A) and 455 nm (B) relative to the absorbance of the reduced cytochrome br peak at 556 nm. Measurement of this ratio for peak A was not possible in control or &naphthoflavone (BNF)-induced microsomes.

b The value of this ratio in phenobarbital (PB)-induced microsomes was 5.43.

treatment despite the concurrent decline in cytochrome P-450 levels. Instead, AHH activity was markedly stimulated by DHS and attained a maximum level of about 285% of controls 24 hr after treatment. Somewhat un- expectedly, complex displacement resulted in a significant decrease in AHH activity.

The magnitude of the DHS metabolite/cytochrome P-450 complex, as measured by the absorbance difference between 455 and 556 nm, increased to a maximum 24 hr after DHS treatment and thereafter remained constant through the following 36 hr. Displacement with 2-methylbenzimidazole largely removed the 455-nm peak, although a very small re- sidual absorbance (about 3%, Table 1) indi- cates that displacement was not complete under the conditions employed.

Changes in hepatic cytochrome P-450 levels, APDM and AHH activities, and redox spectra a&r treatment of PB-induced rats with

DHS show both similarities to and differences from those observed in noninduced animals (Fig. 3). Thus, in contrast to the short-lasting initial decrease in cytochrome P-450 levels occurring in the latter, the DHS-related decrease in cytochrome P-450 observed in microsomes from PB-induced rats is considerably more intense and prolonged, a maximum decline of 40% being reached by 24 hr and thereafter being maintained through 48 hr. No induction phase was observed through the 48-hr experimental period, and even following displacement with 2-methylbenzimidazole, cytochrome P-450 levels were only increased to levels equal to, or slightly higher than, those in controls. APDM activities again appeared to reflect cytochrome P-450 levels, inhibition being prolonged and only slightly reversed following displacement. The profile for AHH activity (Fig. 3b) in PB-induced rats was qualitatively similar to that observed in

72 MURRAY, WILKINSON, AND DUBE

FIG. 5. Dihydrosafrole metabolite-ferrocytochrome P-450 spectral complexes generated in vitro in microsomes from (A) control and (B) phenobarbital- and (C) /3-naphthoflavone-induced rats. Reaction mix- tures (1 ml) contained microsomal suspensions (I mg protein) in 0.05 M Tris-HCl buffer (pH 7.4) containing 0.1s M KC1, 0.2 mM EDTA LOO PM dihydrosafrole, and 1.3 mM NADPH. After spectral development was complete (lower trace), a few grains of sodium dithionite were added to cuvettes to fix the dual Soret spectrum (upper trace).

noninduced animals, activity increasing to a level of 207% of controls 24 hr after DHS exposure and declining steadily during the following 24 hr. Displacement had no effect on AHH activities.

The DHS/cytochrome P-450 spectral complex in PB-induced microsomes exhibited a much larger 427/455-nm peak ratio than those in control or /3NF-induced microsomes (Fig. 5) and provided an excellent opportunity to compare the relative ease of displacement of each peak by 2-methylbenzimidazole. The results (Fig. 3d, Table 1) clearly show that although the 455-nm spectral complex is substantially displaced, the complex at 427-nm is considerably more refractory to the action of 2-methylbenzimidazole with only 24 to 45% displacement occurring under the conditions employed.

Time profiles of microsomal cytochrome P-450 levels, APDM and AHH activities, and redox spectra following exposure of @NF-induced rats to DHS are shown in Fig. 4. Cy- tochrome P-450 levels declined to about 70% of the corresponding control value about 6 hr after DHS treatment and showed only a slight recovery (5 to 10%) through the remainder of

the 36-hr experimental period. The levels of cytochrome P-450 were significantly enhanced following displacement, so that from 24 to 36 hr after DHS treatment an increase of 45 to 50% over control values (i.e., ,f3NF- induced) was apparent. As was the case in noninduced and PB-induced microsomes, APDM profiles resemble those observed with cytochrome P-450, although even following displacement, activities never returned to control values.

Since preliminary dose-response data (not shown) had indicated that rats injected on each of 3 successive days with 40 mg/kg of PNF were maximally induced with respect to AHH activity, the further marked increase in AHH activity following exposure to a single dose of DHS (Fig. 4b) was surprising. As was observed in PB-induced microsomes, the complex had no effect on AHH activity.

The large 455-nm spectral peak of the DHS/ cytochrome P-450 complex in microsomes from BNF-induced rats (Fig. 4d) increased during the 36 hr following DHS treatment. The 455-nm complex was displaced to an extent of about 85% on treatment with 2-methylbenzimidazole (Fig. 4d, Table 1).


TABLE 2

EFFECT OF PHENYLIMIDAZOLE (PI) AND a-NAPHTHOFLAVONE (aNF) ON ARYL HYDROCARBON (BENZO[U]PYRENE) HYDROXYLASE (AHH) ACTIVITY IN MICRO~~MES FROM RATS TREATED EITHER WITH PHENOBARBITAL (PB) OR ,3- NAPHTHOFLAVONE (PNF’) ALONE OR WITH THE INDUCER AND DIHYDROSAFROLE (DHS)

AHH activity (nmol benzo[a]pyrene metabolized/mg protein/min)

Treatments/addition None PI (loo PM) (YNF (10 PM)

Control 0.99 2 0.06 (100)” 0.64 + 0.05 (65) 1.84 k 0.18 (186)

PB-induced 2.11 f 0.11 (100) 0.64 + 0.04 (30) 2.57 + 0.15 (122)

PB-induced/DHS Not displaced 4.36 +- 0.15 (100) 3.16 + 0.22 (72) 3.98 + 0.30 (91) Displaced 4.32 f 0.09 (100) 2.89 k 0.09 (67) 2.86 + 0.18 (66)

PNF-induced 6.16 + 0.50 (100) 7.84 -e 0.44 (127) 1.81 + 0.33 (29)

/3NF-induced/DHS Not displaced 11.15 + 0.53 (100) 9.27 k 0.35 (83) 2.63 + 0.17 (24) Displaced 11.62 2 0.29 (100) 10.19 + 0.34 (88) 2.69 + 0.26 (23)

4 Details of treatments and complex displacements procedure were as described under Methods. b Figures in parentheses indicate percentage of control activity.

In view of the marked enhancement of hepatic microsomal AHH activity caused by administration of a single dose of DHS to control and PB- and ,f3NF-induced rats, studies employing the selective MFO inhibitors, phenylimidazole (PI, 100 PM) and a-naphthoflavone (aNF, 100 PM), were conducted in an attempt to clarify the nature of the cytochromes P-450 involved. The results (Table 2) show that as previously reported (Wiebel et al., 1971) aNF strongly inhibits AHH activity in BNF-induced microsomes and en- hances AHH activity induced by PB; PI has precisely the opposite effect, inhibiting AHH activity in PB-induced microsomes and stim- ulating AHH activity in microsomes from aNF-induced animals. Although the susceptibility to aNF of AHH activity in microsomes from rats receiving both PNF and DHS is similar to that in microsomes from rats receiving @NF alone, this activity is slightly sus- ceptible to inhibition by PI (N 12 to 17%). In addition, the inhibitory effects of PI and aNF on AHH activity in microsomes from rats receiving both PB and DHS are different from the effects observed in PB-induced microsomes, PI being markedly less inhibitory to-

ward the former and ~YNF showing slight to moderate inhibition instead of stimulation. With the exception of the different effects of cvNF in displaced and nondisplaced microsomes from rats receiving PB and DHS, the inhibitory potencies of both PI and aNF toward AHH activity were apparently indepen- dent of the presence or absence of the MDP/ cytochrome P-450 complex.

DISCUSSION

Although MDP compounds are perhaps best known for their inhibitory effects on MFO activity in vitro (Hodgson and Philpot, 1974) it has long been recognized that their effects in vivo are extremely complex and dependent on the time following treatment. Thus, the effects of piperonyl butoxide and other MDP compounds administered in vivo to mammals (Kamienski and Murphy, 197 1; Skrinjaric- Spoljar et al., 197 1) and insects (Brattsten and Wilkinson, 1973) are typically biphasic; their acute inhibitory effects on microsomal metabolism are usually followed by a stimulation of MFO activity. The results of the present study clearly confirm the complexity of the time-dependent interactions occurring in he-

74 MURRAY, WILKINSON, AND DUBI?

patic microsomes following treatment of rats with a single dose of DHS.

In confirmation of previous studies estab- lishing the biphasic nature of MDP interactions in vivo, the results reported here show that DHS leads to an initial decline in hepatic cytochrome P-450 levels and that this decline is followed by induction. The decreased levels of cytochromes P-450 observed shortly after DHS exposure undoubtedly reflect formation of the MDP metabolite/cytochrome P-450 complex and, indeed, this finding is con- firmed by the observed time-dependent increases in type III spectral complex formation. It is quite clear, however, that the nature and stability of the DHS complexes with different forms of cytochrome P-450 are different (Ryan et al., 1980); these differences have profound implications with respect to the in vivo effects of DHS on reactions mediated by these cytochromes.

Of the reactions included in this study, APDM activity is consistently inhibited following exposure to DHS, whereas AHH activity is consistently enhanced. This pattern immediately suggests that reactions catalyzed by the “PB-type” cytochrome P-450 are inhibited by DHS, and those that are catalyzed by the “3MC (or /3NF)-type” cytochrome P- 448 are not. That this pattern is indeed the case is shown by the data (Table 3) indicating that while DHS and two other MDP compounds are effective inhibitors of AHH activity in PB-induced microsomes (cytochromes P-450) they exhibit no inhibitory activity toward AHH activity in those induced by @NF (cytochromes P-448). The apparent inability of MDP compounds to inhibit reactions catalyzed by cytochromes P-448 has been con- firmed in studies with reconstituted systems incorporating purified cytochromes from PB- and /3NF-induced ra& (unpublished data). It is of importance to this discussion to note that the DHS spectral complexes with purified cytochromes P-450 and P-448 are quite distinct, the former exhibiting dual Soret peaks at 427 and 455 nm and the latter a single peak at 455 nm (Ryan et al., 1980). These data un-

TABLE 3

THE EFFECTOFMETHYLENEDIOXYPHENYL (MDP) COMPOUNDS ON ARYL HYDRWARBON (BENZLW- PYRENE) HYDROXYLASE (AHH) Acnvrn IN HEPATIC MICROSOMES FROM PHENOBARBITAL (PB)- AND PNA- PHTHOFLAVONE(PNF')-INDUCEDRATS

MDP (100 PM)

Dihydrosafroie 4-Bromomethylene-

Percentage control AHH activity’

PB-induced @NF-induced

34 111

dioxybenzene 2,3-Methylenedioxy-

46 101

naphthalene 35

u Each value is the mean of three assays.

94

doubtedly account for the marked differences in the ratios of the 427- and 455-nm peaks of the DHS spectral complexes in control, PB-, and PNF-induced microsomes (Fig. 5), the 427-nm peak being particularly marked in the PB-induced microsomes.

Although it is well established that aminopyrine is metabolized by more than one form of cytochrome P-450 in control (and probably BNF-induced) microsomes, a single active enzyme, probably the major “PB-type” cytochrome P-450, dominates APDM activity in PB-induced microsomes (Pederson and Aust, 1970). Since this enzyme is the form of cytochrome P-450 that is selectively inhibited by DHS, it might be anticipated that in gen- eral the degree, duration, and intensity of the inhibitory effect of DHS toward APDM activity would be directly related to the amount of this cytochrome in the microsomes. That this hypothesis appears to be the case is shown by the fact that maximum inhibition of APDM activity is maintained through 24 hr in PB-induced microsomes, whereas in those from control and @JF-treated rats the maximum inhibitory effect occurs at about 2 and 6 hr, respectively, following DHS exposure. The profiles for APDM inhibition are com- plicated to some extent by the fact that although DHS and other MDP compounds induce primarily a novel form of cytochrome


P-450, cytochrome P-450d (Ryan et al., 1980), they also induce to a lesser extent the classical “PB-type” (P-45Ob) and “MC (or @NF)-type” (P-450~) cytochromes. The slow spontaneous recovery of inhibition of APDM activity is undoubtedly the net result of DHS complex formation and the induction of new enzyme, although in this study an induction of APDM activity above the appropriate control level was only observed in rats receiving DHS alone.

In the light of data from other studies (El- combe et al., 1975; Bridges and Fennell, 1980) (unpublished data), it would appear that the elevated levels of cytochromes P-450 and AHH activity observed in this study (Figs. 2 to 4) 12 to 36 hr after DHS treatment are almost certainly due to induction of the novel isozyme, cy-tochrome P-450d (Ryan et al., 1980). An alternative explanation is that increased AHH activity results from the concomitant induction of cytochrome P-450~ by DHS. However, the fact that the susceptibility of the DHS-induced AHH activity to the inhibitors PI and aNF [known to be selective in their action toward the major PB- and 3MC (or @NF)-cytochromes, respectively (unpublished data)], is intermediate between that of the activities catalyzed by P-450~ and P-450b, and the fact that AHH activity is substantially enhanced by DHS in rats already fully induced by PNF strongly suggests that it is catalyzed by the novel hemoprotein.

The displacement of MDP complexes from ferricytochrome P-450 by 2-alkylbenzimidaz- oles and a variety of other compounds is well established (Elcombe et al., 1976; Dickins et al., 1979) and would be expected to result in a restoration of cytochrome P-450 levels and a reversal of associated enzyme inhibition. However, the results of the displacement studies reported here are highly complex since it has been established that (a) the 455~nm spectral complex with cytochromes P-448 is not inhibitory at least with respect to AHH activity and that (b) the 455-nm spectral complex (the major form in control and PNF microsomes) is much more readily displaced by 2-methylbenzimidazole than the 427-nm

complex (the major form of the complex in PB-induced microsomes). In light of these findings it would be expected that restoration of considerably more cytochrome P-450 would result from complex displacement in microsomes exhibiting 455-nm complexes than in those with complexes at 427 nm. The results in Figs. 2 to 4 show that this is the case, the lowest levels of restoration of cytochromes P- 450 being observed in PB-induced microsomes where the stable 427-nm peak is dom- inant. The greater stability of the 427~nm complex (compared with the 455~nm complex) toward the displacer 2-methylbenzimidazole (Fig. 3d) almost certainly accounts for the low level of restoration of APDM activity in microsomes from PB-induced rats (Fig. 3a) and strongly suggests that the 427~nm complex is considerably more important than the 455-nm complex in the inhibitory interactions of MDP compounds. The fact that AHH activity appears to remain largely unchanged following displacement with 2-methylbenzimidazole (except in the case of noninduced animals) is consistent with the fact that formation of the 455-nm complex is not associated with inhibition of catalytic activity in BNF-induced microsomes or in reconstituted systems incorporating purified cytochrome P- 448 (unpublished data).

The ability of DHS, and probably other MDP compounds, to effect concurrently the inhibition of some microsomal reactions and the induction of others following their administration to animals in vivo is of consid- erable interest and has many important implications with regard to the potential of these compounds to cause unanticipated toxicological interactions. In attempting to shed further light on the mechanisms through which these in vivo effects occur, it is important to obtain a better understanding of the precise nature of the molecular events occurring at the cytochromes P-450.

ACKNOWLEDGMENT

This work was supported by Grant ES 0 1902 from the National Institutes of Health.

76 MURRAY, WILKINSON, AND DUB6

REFERENCES

BAMBERGER, E., AND LENGFIELD, F. (1890). Neue re- ductionsproducte des chinolins. Berichfe 23, 1138-l 158.

BORNHEIM, L., AND FRANKLIN, M. R. (1982). Metabolic- intermediate complex formation reveals major changes in rat hepatic cytochrome P-450 subpopulations in addition to those forms previously purified after phenobarbital, P-naphthoflavone, and isosafrole induction. Mol. Pharmacol. 21, 521-532.

BRAT’TSTEN, L. B., AND WILKINSON, C. F. (1973). In- duction of microsomal enzymes in the southern ar- myworm (Prodenia eridania). Pestic. Biochem. Phy- siol. 3, 393-407.

BRIDGES, J. W., AND FENNELL, T. R. (1980). Character- ization of the form of P-450 induced by methylenedioxy compounds in rats and mice. In Microsomes, Drug Oxidations, and Chemical Carcinogenesis (M. J. Coon, A. H. Conney, R. W. Estebrook, H. V. Gelboin, J. R. Gillette, and P. J. O’Brien. eds.), pp. 993-996. Academic Press, New York.

DICKINS, M., ELCOMBE, C. R., MOLONEY, S. J., NETTER, K. J., AND BRIDGES, J. W. (1979). Further studies on the dissociation of the isosafrole metabolite-cytochrome P-450 complex. B&hem. Pharmacol. Z&23 I- 238.

ELCOMBE, C. R., BRIDGES, J. W.. GRAY, T. J. B., NIMMO- SMITH, R. H., AND NETTER, K. J. (1975). Studies on the interaction of safrole with rat hepatic microsomes. Biochem. Pharmacol. 24, 1427-2433.

ELCOMBE, C. R., BRIDGES, J. W., AND NIMMC-SMITH, R. H. (1976). Substrate-elicited dissociation of a complex of cytochrome P-450 with a methylenedioxyphenyl metabolite. Biochem. Biophys. Res. Commun. 71,915-919.

FISHER, G. J., FUKUSHIMA, H., AND GAYLOR, J. L. (1981). Isolation, purification, and properties of a unique form of cytochrome P-450 in microsomes of isosafrole-treated rats. J. Biol. Chem. 256, 4388-4394.

FRANKLIN, M. R. (1971). The enzymic formation of a methylenedioxyphenyl derivative exhibiting an isocyanide-like spectrum with reduced cytochrome P-450 in hepatic microsomes. Xenobiotica 1, 58 1-59 1.

HODGSON, E., AND PHILPOT, R. M. (1974). Interaction of methylenedioxyphenyl (1,3-benzodioxole) compounds with enzymes and their effects on mammals. Drug Metab. Rev. 3, 231-301.

KAMIENSIU, F. X., AND MURPHY, S. D. (197 1). Biphasic effects of methyienedioxyphenyl synergists on the action of hexobarbital and organophosphate insecticides in mice. Toxicol. Appl. Pharmacol. 18, 883-894.

LAKE, B. G., HOPKINS, R., CHAKRABORTY, J., BRIDGES, J. W., AND PARKE, D. V. (1973). The influence of some hepatic enzyme inducers and inhibitors on extra he-

patic drug metabolism. Drug Metab. Dispus. 1, 342- 349.

LOTLIKAR, P. D., AND WASSERMAN, M. B. (1972). Effects of safrole and isosafrole pretreatment on N- and ring- hydroxylation of 2-acetamidotluorene by the rat and hamster. Biochem. J. 129, 937-943.

MURRAY, M., RYAN, A., AND LITTLE, P. (1982). Inhi- bition of rat microsomal aminopytine Ndemethylase activity by benzimidazole derivatives: quantitative structure activity relationships. J. Med. Chem. 25,887- 896.

NASH, T. (1953). The calorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem. J. 55,416-421.

NASTAINCZYK, W., ULLRICH, V., AND SIES, H. (1978). Effect of oxygen concentration on the reaction of hal- othane with cytochrome P-450 in liver microsomes and isolated perfused rat liver. Biochem. Pharmacol. 27, 387-392.

OMURA, T., AND SATO, R. (1964). The carbon monoxide- binding pigment of liver microsomes, I. Evidence for its hemoprotein nature. J. Biol. Chem. 239,2370-2378.

PARKE, D. V., AND RAHMAN, H. (1970). The induction of hepatic microsomal enzymes by safrole. Biochem. J. 119, 53p-54~.

PEDERSON, T. C., AND AUST, S. D. (1970). Aminopyrine demethylase, kinetic evidence for multiple microsomal activities. B&hem. Pharmacol. 19, 2221-2230.

PHILPOT, R. M., AND HODGSON, E. (1971). A cytochrome P-450-piperonylbutoxide spectrum similar to that produced by ethyl isocyanide. L@Sci. lO(II), 503- 512.

ROBINSON, H. W., AND HOGDEN, C. A. (1940). The biuret reaction in the determination of serum proteins. J. Biol. Chem. 135,707-725.

RYAN, D. E., THOMAS, P. E., AND LEVIN, W. (1980). Hepatic microsomal cytochrome P-450 from rats treated with isosafrole. J. Biol. Chem. 255, 7941-7955.

SKRINJARIC-SFQLJAR, M., MATTHEWS, H. B., ENGEL, J. L., AND CASIDA, J. E. (197 1). Response of hepatic microsomal mixed-function oxidases to various types of insecticide chemical synergists administered to mice. Biochem. Pharmacol. 20, 1607- I6 18.

WEIBEL, F. J., LEUTZ, J. C., DIAMOND, L., AND GELBOIN, H. V. (197 1). Aryl hydrocarbon (benzo[a]pyrene) hydroxylase in microsomes from rat tissues: Differential inhibition and stimulation by benzoflavones and or- ganic solvents. Arch. B&hem. Biophys. 144, 78-86.

YANG, C. S., AND KICHA, L. P. (1978). A direct fluoro- metric assay of benzo[a]pyrene hydroxylase. Anal. Biochem. 84, 154- 163.

Yu, L. S., WILKINSON, C. F., AND ANDERS, M. W. (1980). Generation of carbon monoxide during the microsomal metabolism of methylenedioxyphenyl compounds. Biochem. Pharmacol. 29, 1113- 1122.

effects of dihydrosafrole on cytochromes p-450 and drug oxidation in hepatic microsomes from control...

Documents