ARCHIVES OF BIOCHEMISTRY Vol. 197, No. 2, October 15,
Comparison WILLIAM
AND BIOPHYSICS
pp. 436-446, 1979
of Nuclear
and Microsomal
Epoxide
Hydrase
from Rat Liver
A. BORNSTEIN ,*,l WAYNE LEVIN,1- PAUL E. THOMAS,t DENE E. RYAN,S AND EDWARD BRESNICK”
* Department of Biochemistry, TDepartment of Biochemistry
University of Vermont College of Medicine, Burlington, Vemnont 05405, and and Drug Metabolism, Hoffman-LaRoche Inc., Nutley, New Jersey 07110 Received May 8, 1979
The specific activities of hydration of nine arene and alkene oxides by purified nuclei prepared from the livers of 3-methylcholanthrene-pretreated rats were found to fall within the range of 2.2 to 9.1% of the corresponding microsomal values. Pretreatment with phenobarbital enhanced both the nuclear and microsomal hydration of phenanthrene-9,10-oxide, benzo(a)pyrene-11,12-oxide, and octene-1,2-oxide. 3-Methylcholanthrene pretreatment enhanced the nuclear hydration of these three substrates by 30-60% but had no significant effect on microsomal hydration. An epoxide hydrase modifier, metyrapone, stimulated the hydration of octene-1,2-oxide by the two organelles to quantitatively similar extents, but affected the nuclear and microsomal hydration of benzo(a)pyrene-4,5-oxide differentially. Cyclohexene oxide also exerted differential effects on nuclear and microsomal epoxide hydrase which were dependent both on the substrate and on the organelle. The inhibition by this agent of nuclear and microsomal epoxide hydrase was quantitatively similar only for a single substrate, benzo(a)anthracene-5,6-oxide. When purified by immunoaffinity chromatography, nuclear and microsomal epoxide hydrases from 3-methylcholanthrene-pretreated rats were shown to have identical minimum molecular weights (-49,000) on polyacrylamide gels in the presence of sodium dodecyl sulfate. These findings support the assertion that microsomal metabolism can no longer be considered an exclusive index of the cellular activation of polycyclic aromatic hydrocarbons.
Polycyclic aromatic hydrocarbons are environmental carcinogens that are ubiquitous in the modern industrial biosphere (1). These compounds must be metabolically activated to electrophilic substances prior to inducing malignant transformation or tumors (2-5). The details of the sequence of events leading to metabolic activation are best understood for benzo(a)pyrene. The concerted action of the NADPH-requiring monooxygenase, aryl hydrocarbon hydroxylase, and of epoxide hydrase leads to the formation of (-)-trctns-7,8-dihydro-7,8dihydroxy benzo(a)pyrene (6-g), a proximate carcinogen (lo- 12). Further catalytic action of aryl hydrocarbon hydroxylase on this compound results in the formation of the diastereomeric benzo( a)pyrene-7,8-diol 9,10-epoxides in which the 9,10-epoxide is either cis (diol epoxide-1) or truns (diol 1 Present address; Department of Medicine, Duke University Medical Center, Durham, N. C. 27710. 0003-9861/79/120436-11$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
epoxide-2) to the benzylic 7-hydroxy group (6, 7, 9, 13-17). An abundance of evidence suggests that these diol epoxides are ultimate carcinogens mediating benzo( a)pyrene tumorigenesis (10, 11, 18-21). Data are also accumulating suggesting that “bay region” diol epoxides (18) of other polycyclic hydrocarbons may be ultimate carcinogens as well (22-36). The elucidation of the identities of the ultimate carcinogens makes a detailed knowledge of the enzyme systems leading to their formation of cardinal importance in the basic understanding of chemical carcinogenesis. Aryl hydrocarbon hydroxylase and epoxide hydrase have, until recently, been considered as localized exclusively to the endoplasmic reticulum. More recent evidence has established the presence of this activating system in nuclei (37-43). Furthermore, we have demonstrated the capacity of nuclei to form (-)-truns-7,8-dihydro7,8-dihydroxy benzo(u)pyrene (44) and the 436
COMPARISON
OF NUCLEAR
AND MICROSOMAL
diol epoxides from the latter (14), thus indirectly revealing the presence of nuclear epoxide hydrase. Direct demonstration has been made in our laboratory of the nuclear hydration of benzo( a)pyrene-4,5-oxide (45) and in Orrenius’ laboratory of the nuclear hydration of styrene oxide (41). Comparisons of nuclear and microsomal aryl hydrocarbon hydroxylase have revealed that both systems metabolize benzo( a)pyrene in a qualitatively similarly manner (40). Moreover, nuclear and microsomal sources of cytochrome P-448 and epoxide hydrase are immunologically identical (46). Subtle differences between the nuclear and microsomal enzymes have been suggested but remain controversial (47-48).
The studies reported here were undertaken to (i) compare nuclear and microsomal epoxide hydrase activities as assayed with a number of arene and alkene oxide substrates, (ii) compare the effects of animal pretreatment on the nuclear and microsomal enzyme activity, (iii) investigate the comparative effects of modifiers of epoxide hydrase activity on the two enzyme systems, and (iv) compare the minimum molecular weights of epoxide hydrase isolated from nuclear and microsomal sources. The data further substantiate the existence of nuclear epoxide hydrase and demonstrate some intriguing differences between the nuclear and microsomal enzymes. MATERIALS
AND METHODS
Animals and isolation of organelles. Male rats of the CD strain were maintained on a 12 h on-12 h off light-dark cycle and were allowed free access to Purina Rat Chow and to water. Animals were injected intraperitoneally with a solution of 3-methylcholanthrene (20 mgikg) in corn oil on each of two successive mornings and were killed by cervical dislocation 24 h after the second injection. When phenobarbital was the inducing agent, this compound was administered ip in saline at a dose of 75 mgikg at 96, 72, 48, and 24 h prior to sacrifice. The livers were removed immediately, washed in cold 0.9% NaCl and then either homogenized in 0.25 M sucrose (l:lO, w/v) for the preparation of microsomes or in 2.4 M sucrose containing 3 mM CaCl, (1:12, w/v) for the preparation of nuclei. The microsomes were sedimented from a 12,000g supernatant fraction by centrifuging at 106,OOOg as described previously (40). The nuclei
EPOXIDE
HYDRASE
437
were prepared as described (40) and the procedure in brief consisted of repurification of the crude nuclear pellet obtained from the dense sucrose by sedimentation in 1 M sucrose-l mM CaCl, until the preparation was homogeneous as judged by phase microscopy. In our previous publication (40), we have dealt with the matter of contamination of nuclear preparations with microsomes. Our nuclei were free of cytoplasmic contamination as judged by either light or electron microscopy. The contamination of our nuclear preparation with microsomes ascertained by deliberately contaminating the former with radiolabeled microsomes. The latter was obtained from the livers of rats that had been injected intraperitoneally 2 h previously with L[4,5-3H]leucine (51.6 Wmmol; 67 &i/g body wt). The specific activity of the microsomes was 4.5 x lo4 dpm/mg protein. The labeled microsomes corresponding to 2.5 x 105 dpm were added to a rat liver homogenate and the purification of nuclei conducted as described above. The final purified nuclei contained 1.9 x lo3 dpm which corresponded to a 0.76% microsomal contamination. This level is insufficient to account for epoxide hydrase activity within nuclei. ImmunoaffLnity
purz$cation
of nuclear
microsomal
Epoxide hydrase was purified by immunoaffinity column chromatography from nuclei (1290 mg protein) and microsomes (100 mg protein) isolated from livers of rats pretreated with 3-methylcholanthrene. Nuclei were first digested with deoxyribonuclease I for 20 min (49) to reduce the DNA concentration and prevent interference with the chromatographic separation. The microsomal preparation was treated in an identical manner as the nuclear preparation. The samples were then dialyzed for 1.5 h against 4 liters of DNA digestion buffer (49) minus P-mercaptoethanol. The nuclear and microsomal fractions were then solubilized with 0.5 mg sodium cholate and 0.2 mg Emulgen 911/mg protein followed by dialysis against the column equilibration buffer (10 mM potassium phosphate, pH 7.4, 0.2 M KCl, 0.2 mM EDTA, 20% glycerol, 0.2% Emulgen 911, and 0.5% sodium cholate). The white precipitate in the nuclear preparation was removed by centrifugation at 30,OOOg for 10 min. All sample preparation and subsequent chromatographic procedures were performed at room temperature. Antibody was produced in sheep to purified rat liver microsomal epoxide hydrase (50). Immunoglobulin was purified (51) from high titer sera taken 5-12 months after immunization and bound (9 mgiml) to CNBr-activated Sepharose (52). Two columns of 6 ml were poured and equilibrated with the buffer described above. The solubilized, deoxyribonucleasetreated nuclear and microsomal preparations were passed through their respective columns twice to insure saturation of all antibody binding sites. The immunoaftlnity columns were washed with 2 column epoxide
hydrase.
438
BORNSTEIN
volumes of the following solutions sequentially to minimize nonspecific protein binding: column equilibration buffer; 0.1 M sodium acetate, pH 4.2, containing 0.5 M KCl; 0.1 M sodium borate, pH 8.4, containing 1.0 M KCl, 0.2% Emulgen 911 and 0.2 mM EDTA; 0.2 M KSCN. Catalytically inactive epoxide hydrase was released with 2 column volumes of 4 M KSCN. The released enzyme was dialyzed against 5.0 mM potassium phosphate, pH 7.4 and concentrated by ultrafiltration. Protein was estimated by the method of Lowry et al. (53), and sodium dodecyl sulfatepolyacrylamide gel electrophoresis was performed as described by Laemmli (54). Substrates. The sources and characteristics of the arene and alkene oxides used in these experiments have been previously reported (55). Specific activities of the substrates were as follows: [7-3H]styrene-7,8oxide, 0.5 &i/pmol; [7,8-3H]octene-1,2-oxide, 7.2 pCi/pmol; [6-3H]benzo(a)pyrene-4,5-oxide, 5.3 rCi/ pmol; [6-3H]benzo(a)pyrene-7,8-oxide, 8.4 &i/pmol; [6-3H]benzo(a)pyrene-9,10-oxide, 39.5 #X~mol; [ll, 12-3H]benzo(a)pyrene-ll,12-oxide, 5.1 &i/pmol; [73H]benzo(a)anthracene-5,6-oxide, 8.5 &i/pmol; [5,63H]dibenzo(a,h)anthracene-5,6-oxide, 6.2 #.X~mol; 3-[11,12-3H]methylcholanthrene-ll,12-oxide, 2.4 &i/~mol; [3-3H]phenanthrene-9,10-oxide, 6.6 $i/ pmol. Epotide hydrase assay. In a total volume of 80 ~1, each assay (55) contained: 0.5 M Tris-HCl (pH 8.2 at 37”C), 25 ~1; nuclei or microsomes diluted in 3 mM MgCl,-5 mM Tris-HCl (8.5 at 2O”C), 50 ~1 (25 ~1 for metyrapone experiments); and substrate (12-150 nmol) in acetonitrile: NH,OH (lOOO:l, v/v), 5 ~1. All substrate concentrations used in this assay were saturating. When present, metyrapone, (a gift from Dr. J. Daly) was added in water. Cyclohexene oxide was added in 2 ~1 of acetonitrile but was withheld until immediately prior to the addition of substrate in order to minimize its enzymatic hydration. Otherwise, the reaction mixture, excluding substrate, was allowed to reach room temperature; the reaction was initiated by the addition of substrate, and subsequent incubation was performed at 37°C for 2-15 min. The reactions were conducted under conditions of linearity with respect to time. The reaction was then terminated by the addition of 25 ~1 of tetrahydrofuran followed by immediate transfer to an ice bath. A 35~1 aliquot of each sample (one-third of the total volume) in addition to a small amount of unlabeled carrier diol or glycol was subjected to thin-layer chromatography on Quantum Industries 5 x 20-cm LQDF thin-layer plates using the solvent systems previously described (55). After development of the plates, the products were visualized under ultraviolet light and fluorescent activation of the plates was allowed to extinguish for 2 h in room light. The bands of product were then scraped into scintillation vials, 0.1 ml of methanol and 15 ml of Scintisol
ET AL. counting medium (Isolab) were added sequentially. Radioactivity was quantitated in a Packard TriCarb spectrometer with a counting efficiency, determined using [3H]toluene as an internal standard of greater than 30%. All analyses were performed in duplicate. In all cases, values obtained in assays which lacked tissue were subtracted in the calculation of product formation. The recovery of radioactivity with the various substrates has been discussed previously (55). RESULTS
The effects of pH on the nuclear-catalyzed hydration of alkene and arene oxides were found to be essentially identical to those previously reported for microsomes (55). The pH of 3.2 (at 37°C) used in the comparative studies falls within the broad range of optima for both nuclear and microsomal systems. Conditions were established for linearity of product formation with respect to nuclear protein concentration. For most of the substrates studied, linearity was observed with as much as 800 lug of nuclear protein per 80 ~1 assay. In the comparative studies, nuclear and microsomal protein concentrations were selected which were within the range of constant product formation per milligram protein per minute. Table I shows the specific activities of nuclear and microsomal epoxide hydrase and their ratios using the nine alkene and arene oxide substrates. Nuclear and microsomal enzyme activities were always determined in parallel in the same experiments; relatively little day to day variation in the ratios of nuclear to microsomal activity was observed. For all of the substrates studied, the ratios of nuclear to microsomal specific activities fell within the range of 2.2 to 9.1%. The effects of phenobarbital and 3methylcholanthrene pretreatment on the nuclear and microsomal hydration of three substrates is shown in Table II. Phenobarbital pretreatment consistently enhanced the rate at which product was enzymatically formed from the three substrates by both organelles. On the other hand, 3-methylcholanthrene pretreatment depressed slightly the microsomal hydration of the three substrates while stimulating the nuclear hydration of these substrates by 30-
COMPARISON
OF NUCLEAR
AND MICROSOMAL TABLE
NUCLEAR
AND MICROSOMAL
EPOXIDE
HYDRASE
AND ALKENE
OXIDES’
I
HYDRATION
OF ARENE
Epoxide hydrase* (nmoI/mg/min) Amount (nmol)
Substrate Phenanthrene 9,10-oxide Benzo(a)anthacene-5,6-oxide Benzo(a)pyrene-4,5-oxide Benzo(a)pyrene-7,&oxide Benzo(a)pyrene-9,10-oxide Benzo(a)pyrene-1 1,12-oxide 3-Methylcholanthrene-11,12-oxide Octene-1,2-oxide Styrene-7,8-oxide
439
Nuclear
Microsomal
2.48 0.81 0.61 0.28 0.39 0.056 0.20 0.97 0.20
71.05 21.04 13.39 5.47 8.90 0.79 2.16 26.22 8.86
150 20 15 15 15 12 15 100 40
Pereentage nuclear/ microsomal’ 3.5 3.9 4.5 5.2 4.4 7.1 9.1 3.8 2.2
(3.3-3.7) (3.3-4.6) (4.3-4.7) (4.5-5.8) (5.6-8.0) (3.0-4.4) (1.8-2.8)
n Nuclei or microsomes which were isolated from the livers of 3-methylcholanthrene-pretreated 100-g male rats were incubated with the indicated amount of substrate for 2 min (except for styrene 7,8-oxide and benzo(a)pyrene-11,12-oxide which were incubated for 15 and 4 min, respectively). The amounts of nuclear and microsomal protein per 80 /*l were up to 0.8 and 0.2 mg, respectively. * Except for benzo(ufpyrene-9,l@oxide and 3-methylcholanthrene-11,12-oxide, each value represents the mean from at least two separate experiments. c Mean (range of values). Ratios were determined for individual experiments and then averaged.
60%. Although 3-methylcholanthrene has been reported to enhance microsomal epoxide hydrase activity for these substrates, the induction was reported to be TABLE EFFECTS
OF INDUCTION
ON THE NUCLEAR
variable from experiment to experiment (55, 59). Metyrapone and cyclohexene oxide are modifiers of microsomal epoxide hydrase II
AND MICROSOMAL
HYDRATION
OF ARENE
AND ALKENE
Epoxide hydrase” (nmoYmg/min)
OXIDES=
Animal pretreatment
Nuclear
Microsomal
Percentage nuclear/ microsomal
lo-oxide
Control Phenobarbital 3-Methylcholanthrene
1.46 3.68 (2.5) 2.37 (1.6)
78.6 166.9 (2.1) 63.8 (0.8)
1.9 2.9 3.7
Benzo(a)pyrene-11,12oxide
Control Phenobarbital 3-Methylcholanthrene
0.03 0.04 (1.3) 0.04 (1.3)
Octene-1,2-oxide
Control Phenobarbital 3-Methyleholanthrene
0.60 1.63 (2.7) 0.92 (1.5)
Substrate Phenanthrene-9,
0.79 1.54 (1.9) 0.64 (0.8) 27.4 56.3 (2.1) 21.0 (0.8)
3.6 2.9 6.0 2.2 2.9 4.4
a Nuclei and microsomes were prepared from the livers of 100-g male rats which had been pretreated by the intraperitoneal injection of phenobarbital (75 mg/kg) at 96, 72,48, and 24 h prior to sacrifice; or which had been pretreated by the injection of 3-methylcholanthrene (20 mg/kg) at 48 and 24 h prior to sacrifice. Control animals received no pretreatment. Incubations were carried out as described in the legend of Table I. * The numbers in parentheses are the ratios of induced to control enzyme activities for each substrate and organelle.
440
BORNSTEIN
activity (56), the effects of which differ depending on substrate (50). In the present studies, we compared the effects of these modifiers on the nuclear and microsomal systems. Figures 1 and 2 depict the effects of increasing concentration of metyrapone on the nuclear and microsomal hydration of four arene and alkene oxides. The microsomal hydration of benzo( a)pyrene-4,5oxide (Fig. 1A) was not greatly affected by the presence of metyrapone up to 6 mM but was inhibited slightly at the higher metyrapone concentrations (9 and 15 mM) to, respectively, 89 and 83% of control values. On the other hand, nuclear epoxide hydrase activity for this substrate was stimulated 44-78% by metyrapone concentrations of 6 lllM or greater. As shown in Fig. lB, when benzo( a)pyrene-11, 1Zoxide was the substrate, microsomal epoxide hy-
ET AL. BA-5,6-OXIDE
A
L > 20-
% I
c>
-06
.25
-7
I I
”
5
I
NUC
60\
A
BP-4,5-OXIDE
300-
3
H
200
100 iz
9 BP-I
15
I,IE-OXIDE
NU A-
A MIC I5
METYR~PONE
(5,lUl)
B
A--o
3
OXIDE
FIG. 2. Effects of metyrapone on the nuclear (NUC, 0) and microsomal (MIC, A) hydration of (A) benzo(a)anthracene-5,6-oxide and (B) octene-1,2-oxide. The amounts of substrate were: benzo(a)anthraeene-5,6oxide, 20nmol; octene-1,2-oxide, 100 nmol. The amount of microsomal and nuclear protein per assay was 100-200 and 400 pg, respectively. The details are presented in the text.
5 s:
A MIC
‘“L-+-J CY&?WEXENE
200-
0 NUC b MIC
(mM)
FIG. 1. Effects of metyrapone on the nuclear (NUC, 0) and microsomal (MIC, A) hydration of (A) benzo(a)pyrene-4-5-oxide and (B) benzo(a)pyrene-11,12oxide. The amounts of substrate per 80 ~1 assay were: benzo(a)pyrene-4,5-oxide, 15 nmoi; benzo(a)pyrene11,12-oxide, 12 nmol. Nuclear incubations contained 400 pg of protein; microsomal assays were performed with 25 PLgof protein. Incubations were carried out at 37°C as described in the text. Results-are expressed as percentage’*activity relative to control incubations without metyrapone. These are representative data from three sets of experiments.
drase was highly sensitive to the effects of metyrapone and was inhibited 70% at a modifier concentration of 6 mM. However; the nuclear enzyme was relatively insensitive to metyrapone-remaining within a range of approximately 15% of control activity. Epoxide hydrase activity with benzo(a)pyrene-5,6-oxide as substrate was mildly increased by the addition of metyrapone to both microsomal and nuclear systems (Fig. 2A). Nuclei and microsomes responded similarly when catalyzing the hydration of octene-1,2-oxide, i.e., with a stimulation of 3.5- to 4.0-fold at a final metyrapone concentration of 15 mM (Fig. 2B). To insure that linearity of product formation was maintained in the presence of
COMPARISON
OF NUCLEAR
BP-4,5-0X I DE
AND MICROSOMAL
Al
60
k-
BP- Il,l2-OXIDE
Y
# 100
-B
0 “1L
/O\o
N”C
EPOXIDE
441
HYDRASE
activity was somewhat more sensitive. As shown in Fig. 3B, the microsomal hydration of benzo( a)pyrene-11,12-oxide was markedly inhibited by cyclohexene oxide. The nuclear enzyme, on the other hand, was most sensitive to cyclohexene oxide inhibition when catalyzing the hydration of benzo(a)anthracene-5,6-oxide (Fig. 4A). Moreover, only with respect to the latter substrate were the nuclear and microsomal enzymes inhibited to the same extent at all cyclohexene oxide concentrations. While the nuclear conversion of octene-1,2-oxide to octene-1,2-glycol was stimulated slightly by 60 PM and 250 mM cyclohexene oxide, the microsomal enzyme was inhibited at all modifier concentrations (Fig. 4B). Although nuclear and microsomal epoxide hydrase differed in response to metyrapone and cyclohexene oxide as modifiers of catalytic activity, the minimum molecular
FIG. 3. Effects of cyclohexene oxide on the nuclear (NUC, 0) and microsomal (MIC, A) hydration of (A) benzo(a)pyrene-4,5-oxide and (B) benzo(a)pyrenell,l2-oxide. Experimental details were as described in the legend to Figs. 1 and 2, except that cyclohexene oxide was added in 2 ~1 acetonitrile. These are representative data from three sets of experiments.
metyrapone, the hydration of benzo( a)pyrene-4,5-oxide was investigated at different protein concentrations in the presence of 3 or 9 mM metyrapone. Linearity was maintamed under these conditions. The effects of cyclohexene oxide on the nuclear and microsomal hydration of four arene and alkene oxides are shown in Figs. 3 and 4. The microsomal system appeared generally more sensitive to the effects of this modifier than did the nuclear system. However, this greater sensitivity was not parallel for the four substrates. With benzo(a)pyrene-4,5-oxide as the substrate, the nuclear and microsomal enzyme activities were both inhibited approximately 25% by 60 PM cyclohexene oxide (Fig. 3A). However, at higher concentrations, microsomal
I 3
I METdPONE
hn$
FIG. 4. Effects of cyclohexene oxide on the nuclear (NUC, 0) and mierosomal (MIC, A) hydration of (A) benzo(a)anthracene-5,Goxide and (B) octene-1,2oxide. The legends to Figs. l-3. These are representative data from three sets of experiments.
442
BORNSTEIN
ET AL.
FIG. 5. Sodium dodecyl sulfate-gel electrophoresis of nuclear and microsomal epoxide hydrase isolated by immunoaffinity chromatography is described under Materials and Methods. The content of each well was: 1, 4 pg microsomal protein; 2, 0.3 pg microsomal protein purified by immunoaffinity chromatography; 3, effluent from immunoaffinity column eluted with 4 M KSCN contained 1.0 pg of immunoglobulin; 4, 0.5 pg purified microsomal epoxide hydrase; 5, 4 pg nuclear protein; 6, 0.3 Fg nuclear protein purified by immunoafllnity chromatography; 7, same as well 3; 8, same as well 4; 9, 0.3 fig of microsomal enzyme purified by immunoaffinity chromatography and 0.5 pg purified microsomal epoxide hydrase; 10, same as well 9 except contained 0.3 pg of nuclear enzyme purified by immunoaffinity chromatography.
weight of the enzyme from both sources was shown to be identical. We previously reported (46) that nuclear epoxide hydrase was antigenically identical to microsomal epoxide hydrase purified to apparent homogeneity (57). Purification of catalytically inactive epoxide hydrase from nuclei was accomplished with an antibody prepared against the purified microsomal enzyme (Fig. 5). Most of the contaminating protein evident on the sodium dodecyl sulfategels was due to immunoglobulin eluting from the immunoaffinity column in the presence of 4 M KSCN. Both nuclear and
microsomal epoxide hydrase which had been purified by the immunoaffinity technique corn&rated with the purified catalytically active enzyme on sodium dodecyl sulfate-gels (M, = 49,000). DISCUSSION
The role of aryl hydrocarbon hydroxylase and epoxide hydrase in the activation of polycyclic hydrocarbons to ultimate carcinogens is now dogma. The presence of these enzymes within the nucleus is also relatively certain. We have discussed, else-
COMPARISON
OF NUCLEAR
AND
where, the dangers inherent in strictly morphologic arguments for nuclear purity (40) and have presented data arguing the attribution of these nuclear activities to microsomal contamination (40, 58). It would not be consistent with the economy of nature to expect, a priori, these nuclear enzymes to differ greatly from their microsomal counterparts. Indeed, the preponderance of data thus far collected supports a very close similarity of the two systems. Thus, nuclear and microsomal aryl hydrocarbon hydroxylase have been found to be similar, if not identical, with respect to their immunochemical characteristics (46), their spectral characteristics (40), and their qualitative benzo( a)pyrene metabolite profiles (14, 40). On the other hand, one might reasonably expect at least quantitative differences between the behavior of the nuclear and microsomal activating systems in situ within purified organelles. In the present study, we have demonstrated that the nuclear microsomal hydration of arene and alkene oxides is not always identically affected by pretreatment of rats with certain xenobiotics. Thus (Table II), pretreatment with 3-methylcholanthrene had no effect or slightly depressed the microsomal hydration of phenanthrene-9,10-oxide, benzo(a)pyrene-11, E-oxide, and octene-1,2-oxide but increased the nuclear hydration of these substrates. Phenobarbital pretreatment, on the other hand, increased the nuclear and microsomal enzyme activities for all three substrates. However, both nuclear and microsomal epoxide hydrase are immunochemically identical (46) and have the same minimum molecular weight on sodium dodecyl sulfate-gels, whether isolated from 3-methylcholanthrene or phenobarbitalpretreated rats. Furthermore, we have shown (Fig. 1) effects of metyrapone on epoxide hydrase that are dependent both on organelle and on substrate. The data represented in Fig. 1B indicate that the microsomal hydration of benzo( a)pyrene-ll, le-oxide was highly sensitive to inhibition by metyrapone, whereas the nuclear hydration of this substrate was relatively unaffected by metyrapone. Were epoxide hydrase present in the
MICROSOMAL
EPOXIDE
HYDRASE
443
nuclear preparations merely as a function of microsomal contamination, one would expect that the “nuclear” enzyme would be at least equally sensitive to the effects of metyrapone as the microsomal enzyme. Alternatively, it would be necessary to postulate that nuclei interfere with the action of metyrapone. That the latter is not the case is demonstrated in Fig. 3B where metyrapone is shown to affect the microsomal and nuclear hydration of octene-1,2-oxide to an equal extent. Indeed, as shown in Fig. lA, benzo(a)pyrene-4,5-oxide was the substrate, nuclear epoxide hydrase was more sensitive to metyrapone than was its microsomal counterpart. Similarly, when cyclohexene oxide was the modifier of epoxide hydrase activity, the nuclear and microsomal enzyme activities were differentially affected depending on substrate. Thus, the nuclear and microsomal hydration or benzo(a)anthracene-5,6oxide was inhibited to very similar extents at all of the cyclohexene oxide concentrations studied (Fig. 4A). On the other hand, the microsomal hydration of benzo(a)pyrene-11,12-oxide (Fig. 3B) was exquisitely sensitive to cyclohexene oxide inhibition whereas the nuclear enzyme was much less sensitive. The nuclear hydration of the latter substrate appeared maximally inhibited by a relatively low concentration of cyclohexene oxide (250 PM). The latter observation provides additional evidence against the possibility of nucleus-induced alterations in dose-response characteristics as an explanation for these data. These data do not necessarily argue for different nuclear and microsomal epoxide hydrases. However, they do establish that the nuclear and microsomal enzymes behave differently in situ within the purified organelles. The data provide additional evidence against the supposition that “nuclear” epoxide hydrase merely represents microsomal contamination. Whether epoxide hydrase exists in single or multiple forms is, at present, a moot question, although in a separate study, evidence was presented for multiple regulatory sites in the rat liver microsomal enzyme, and for different forms of epoxide hydrase in several species (50). If multiple
444
BORNSTEIN
forms of the enzyme exist within a single tissue, then a possible, but not exclusive, interpretation of the present data is that these forms are not distributed identically with respect to nuclei and endoplasmic reticulum . Interpretation of these data is complicated by a lack of knowledge of relative uptake by the two organelles of the substrates and modifiers which were utilized in our study. However, linearity of product formation with respect to time and protein concentration for all of the substances suggests that both nuclear and microsomal enzyme to metyrapone was demonstrated by its potent enhancement of the hydration of oxtene-l,Zoxide-an effect quantitatively similar to that observed with microsomes. Similarly, it is the differential response to cyclohexene oxide of the nuclear and microsomal enzyme activities relative to multiple substrates that is of most interest. Finally, in this regard, the study of Levin et al. (50) addressed the possibility of membrane altering the relative uptake of the modifiers. In their study, the Same concentration of modifier effected microsomal and purified epoxide hydrase in an identical manner. Consequently, it is highly unlikely that the difference between microsomal and nuclear epoxide hydrases would be explainable by differential uptake of the modifiers. We have proposed that the nuclear polycyclic hydrocarbon metabolizing system may act as a second-line cellular defense against lipophilic substances, but that in doing, it may lead to the formation of ephemeral highly reactive electrophiles in proximity to the cell’s genetic apparatus (40). The effects of modifiers of polycyclic hydrocarbon metabolism such as the potent epoxide hydrase inhibitor, l,l,l-trichloro-2-propene oxide, on tumorigenesis have not been entirely consistent with predictions based on their effects on microsomal metabolism (60). The data herein reported suggest that differential effects on nuclear and microsomal metabolism may help explain such inconsistencies.
ET AL. ACKNOWLEDGMENTS The authors appreciate the assistance of Mr. Dennis Lawler and Mr. Bruce Hassuk in the largescale isolations of organelles. We are indebted to Dr. D. M. Jerina for providing the substrates used in this study. These studies were supported in part by Grant CA 20711 from the National Cancer Institute. The data have been submitted by Dr. William Bornstein as part of the Ph.D. thesis requirement of the Department of Cell and Molecular Biology of the Medical College of Georgia. REFERENCES 1. HOFFMAN, D., AND WYNDER, E. L. (1976) in Chemical Carcinogenesis, ACS Monograph 173. (Searle, C. E., ed.), pp. 324-365, American Chemical Society, Washington, D. C. 2. SIMS, P., AND GROVER, P. L. (1974) Adv. in Cancer Res. 20, 164-274. 3. HEIDELBERGER, C. (1973) Advan. Cancer Res. 18, 317-366. 4. JERINA, D. M., AND DALY, J. W. (1974) Science 184, 573-582. 5. MILLER, E. C., AND MILLER, J. A. (1974) in The Molecular Biology of Cancer (Busch, H., ed.), pp. 377-402, Academic Press, New York. 6. THAKKER, D. R., YAGI, H., Lu, A. Y. H., LEVIN, W., CONNEY, A. H., AND JERINA, D. M. (1976) Proc. Nat. Acad. Sci. USA 73, 3381-3385. 7. THAKKER, D. R., YAGI, H., AKAGI, H., KOREEDA, ., Lu, A. Y. U., LEVIN, W., WOOD, A. LW., CONNEY, A. H., AND JERINA, D. M. (1977) Chem. Biol. Interact. 16, 281-300. 8. YANG, S. K., MCCOURT, D. W., ROLLER, P. P., AND GELBOIN, A. V. (1976) Proc. Nat. Acad. Sci. USA 73, 2594-2598. 9. YANG, S. K., MCCOURT, D. W., LEUTZ, J. C., AND GELBOIN, H. V. (1977) Science 196, 1199-1201. 10. LEVIN, W., WOOD, A. W., YAGI, H., JERINA, D. M., AND CONNEY, A. H. (1976) Proc. Nat. Acad. Sci. USA 73, 3867-3871. 11. KAPITULNIK, J., LEVIN, W., CONNEY, A. H., YAGI, H., AND JERINA, D. M. (1977) Nature (London) 266, 378-380. 12. SLAGA, T. J., VIAGE, A., BERRY, D. L., BRACKEN, W. M., BUTTY, S. G., AND SCRIBNER, J. D. (1976) Cancer Lett. 2, 115-122. 13. SIMS, P., GROVER, P. L., SWAISLAND, A., PAL, A., AND HEWER, A. (1974) Nature (London) 252, 326-327. 14. BRESNICK, E., STOMING, T. A., VAUGHT, J. B., THAKKER, D. R., AND JERINA, D. M. (1977) Arch. Biochem. Biophys. 183, 31-37. 15. DAUDEL, P., DUQUESNE, M., VIGNEY, P.,
COMPARISON
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
OF
NUCLEAR
AND
GROVER, P. L., AND SIMS, P. (1975)FEBSLett. 57, 250-253. KING, H. W. S., OSBORNE, M. R., BELAND, F. A., HARVEY, R. G., AND BROOKES, P. (1976) Proc. Nat. Acad. Sci USA 73, 2678-2681. WEINSTEIN, J. B., JEFFREY, A. M., JENNETTE, K. W., BLOBSTEIN, S. H., HARVEY, R. G., HARRIS, C., AUTRUP, H., KASAI, H., AND NAKANISHI, K. (1976) Science 193, 592-595. JERINA, D. M., LEHR, R., SCHAEFER-RIDDER, M., YAGI, H., KARLE, J. M., THAKKER, D. R., WOOD, A. W., Lu, A. Y. H., RYAN, D., WEST, S., LEVIN, W., AND CONNEY, A. H. (1977) in Origins of Human Cancer. B. Mechanisms of Carcinogenesis. (Hiatt, H. H., Watson, J. D., and Winsten, J. A., eds.), pp. 639-658, Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. LEVIN, W., WOOD, A. W., CHANG, R. L., SLAGA, T. J., YAGI, H., JERINA, D. M., AND CONNEY, A. H. (1977) Cancer Res. 37, 27212725. SLAGA, T. J., BRACKEN, W. M., VIAGE, A., LEVIN, W., YAGI, H., JERINA, D. M., AND CONNEY, A. H. (1977) Cancer Res. 37, 41304133. KAPITULNIK, J., WISLOCKI, P. G., LEVIN, W., YAGI, H., JERINA, D. M., AND CONNEY, A. H. (1978) Cancer Res. 38, 354-358. WOOD, A. W., LEVIN, W., Lu, A. Y. H., RYAN, D., WEST, S. B., LEHR, R. E., SCHAEFERRIDDER, M., JERINA, D. M., AND CONNEY, A. H. (1976) Biochem. Biophys. Res. Commun. 72, 680-686. WOOD, A. W., CHANG, R. L., LEVIN, W., LEHR, R. E., SCHAEFER-RIDDER, M., KARLE, J. M., JERINA, D. M., AND CONNEY, A. H. (1977) Proc. Nat. Acad. Sci. USA 74, 27462750. WISLOCKI, P. R., KAPITULNIK, J., LEVIN, W., LEHR, R., SCHAEFER-RIDDER, M., KARLE, J. M., JERINA, D. M., AND CONNEY, A. H. (1978) Cancer Res. 38, 693-696. WOOD, A. W., LEVIN, W., CHANG, R. L., LEHR, R. E., SCHAEFER-RIDDER, M., KARLE, J. M., JERINA, D. M., AND CONNEY, A. H. (1977) Proc. Nat. Acad. Sci. USA 74,3176-3179. LEVIN, W., THAKKER, D. R., WOOD, A. W., CHANG, R. L., LEHR, R. E., JERINA, D. M., AND CONNEY, A. H. (1978) Cancer Res. 38, 1705-1710. MALAVEILLE, C., TIERNEY, B., GROVER, P. L., SIMS, P., AND BARTSCH, H. (1977) Biochem. Biophys. Res. Commun. 75, 427-433. CHOUROULINKOV, I., GENTIL, A., TIERNEY, B., GROVER, P. L., AND SIMS, P. (1977) Cancer Lett. 3, 247-253. MARQUARDT, H., BAKER, S., TIERNEY, B.,
MICROSOMAL
30.
31.
32. 33.
34.
35.
36.
37.
38. 39. 40.
41. 42.
43. 44.
45.
46.
47. 48.
EPOXIDE
HYDRASE
445
GROVER, P. L., AND SIMS, P. (1977) Int. J. Cancer 19, 833-838. MOSCHEL, R. C., BAIRD, W. M., AND DIPPLE, A. (1977) Biochem. Biophys. Res. Commun. 76, 1092-1098. VIGNY, P., DUQUESNE, M., COULOMB, H., TIERNEY, B., GROVER, P. L., AND SIMS, P. L. (1977) FEBS Lett. 82, 278-282. KING, H. W. S., OSBORNE, M. R. AND BROOKES, P. (1977) ht. J. Cancer 20, 564-571. WOOD, A. W., LEVIN, W., RYAN, D., THOMAS, P. E., YAGI, H., MAH, H. D., THAKKER, D. R., JERINA, D. M., AND CONNEY, A. H. (1977) Biochem. Biophys. Res. Commun. 76,847-854. LEVIN, W., WOOD, A. W., CHANG, R. L., YAGI, H., MAH, H. D., JERINA, D. M., AND CONNEY, A. H. (1978) Cancer Res. 38, 1831-1834. WOOD, A. W., LEVIN, W., THOMAS, P. E., RYAN, D., KARLE, J. M., YAGI, H., JERINA, D. M., AND CONNEY, A. H. (1978) Cancer Res. 38, 1967-1973. WOOD, A. W., CHANG, R. L., LEVIN, W., THOMAS, P. E., RYAN, D., STOMING, T. A., THAKKER, D. R., JERINA, D. M., ANDCONNEY, A. H. Cancer Res., in press. KHANDWALA, A. S., AND KASPER, C. B. (1973) Biochem. Biophys. Res. Commun. 54, 12411246. ROGAN, E. G., AND CAVALIERI, E. (1974) Biothem. Biophys. Res. Commun. 58, 1119-1126. VAUGHT, J., AND BRESNICK, E. (1976) Biochem. Biophys. Res. Commun. 69, 587-591. BRESNICK, E., VAUGHT, J. B., CHUANG, A. H. L., STOMING, T. A., BOCKMAN, D., AND MUKHTAR, H. (1977) Arch. Biochem. Biophys. 181, 257-269. JERNSTROM, B., VADI, H., AND ORRENIUS, S. (1976) Cancer Res. 36, 4107-4113. ALEXANDROV, K., BROOKES, P., KING, M. W. S., OSBORNE, M. R., ANDTHOMPSON, M. H. (1976) Chem. Biol. Interact. 12, 269-277. PEZZUTO, J. M., LEA, M. A., AND YANG, C. S. (1977) Cancer Res. 37, 3427-3433. JENNETTE, K. W., BORNSTEIN, W. A., CHUANG, A. H. L., AND BRESNICK, E. (1979) Biochem. Pharmacol., in press. BORNSTEIN, W. A., CHUANG, A. H. L., BRESNICK, E., MUKHTAR, H., AND BEND, J. R. (1979) Chem. Biol. Interact., in press. THOMAS, P. E., KORZENIOWSKI, D., BRESNICK, E., BORNSTEIN, W. A., KASPER, C. B., FAHL, W. E., JEFCOATE, C. R., AND LEVIN, W. (1979) Arch. Biochem. Biophys. 192, 22-26. ROGAN, E. G., AND CAVALIERI, E. (1978) Mol. Pharmacol. 14, 215-219. ROGAN, E. G., MAILANDER, P., AND CAVALIERI, E. (1976) Proc. Nat. Acad. Sci. USA 73, 457-461.
446
BORNSTEIN
49. KAY, R. R., FRASER, D., AND JOHNSTON, I. R. (1972)&r. J. Biochem. 30, 145-154. 50. LEVIN, W., THOMAS, P. E., KORZENIOWSKI, D., SEIFRIED, JERINA, D. M., AND Lu, A. Y. H. (1979) Mol. Pharwmcol., in press. 51. THOMAS, P. E., Lu, A. Y. H., RYAN, D., WEST, S. B., KAWALEK, J. AND LEVIN, W. (1976) J. Biol. Chem. 251, 1385-1391. 52. NISHIKAWA, A. H., AND BAILON, P. (1975) Anal. Biochem. 64, 268-275. 53. LOWRY, 0. H., ROSEBROUGH, N. T., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 54. LAEMMLI, U. K. (1970) Nature (London) 227, 680-685. 55. JERINA, D. M., DANSETTE, P. M., Lu, A. Y. H.,
ET AL.
56.
57.
58.
59.
60.
AND LEVIN, W. (197’7) Mol. Pharmacol. 13, 342-351. OESCH, F., KANDISCH, N., JERINA, D. M., AND DALY, J. W. (1971) Biochemistry 10, 48584866. Lu, A. Y. H., RYAN, D., JERINA, D. M., DALY, J. W., AND LEVIN, W. (1975) J. Biol. Chem. 250,8283-8288. NUNNICK, J. C., CHUANG, A. H. L., AND BRESNICK, E. (1979) Chem. Biol. Interact., in press. MUKHTAR, H., STOMING, T. A., DANSETTE, P. M., JERINA, D. M., AND BRESNICK, E. (1977) Biochem. Pharmucol. 26, 891-892. BORKI, K., STOMING, T. A., AND BRESNICK, E. (1974) J. Nat. Cancer Inst. 52, 785-788.
Comparison WILLIAM
AND BIOPHYSICS
pp. 436-446, 1979
of Nuclear
and Microsomal
Epoxide
Hydrase
from Rat Liver
A. BORNSTEIN ,*,l WAYNE LEVIN,1- PAUL E. THOMAS,t DENE E. RYAN,S AND EDWARD BRESNICK”
* Department of Biochemistry, TDepartment of Biochemistry
University of Vermont College of Medicine, Burlington, Vemnont 05405, and and Drug Metabolism, Hoffman-LaRoche Inc., Nutley, New Jersey 07110 Received May 8, 1979
The specific activities of hydration of nine arene and alkene oxides by purified nuclei prepared from the livers of 3-methylcholanthrene-pretreated rats were found to fall within the range of 2.2 to 9.1% of the corresponding microsomal values. Pretreatment with phenobarbital enhanced both the nuclear and microsomal hydration of phenanthrene-9,10-oxide, benzo(a)pyrene-11,12-oxide, and octene-1,2-oxide. 3-Methylcholanthrene pretreatment enhanced the nuclear hydration of these three substrates by 30-60% but had no significant effect on microsomal hydration. An epoxide hydrase modifier, metyrapone, stimulated the hydration of octene-1,2-oxide by the two organelles to quantitatively similar extents, but affected the nuclear and microsomal hydration of benzo(a)pyrene-4,5-oxide differentially. Cyclohexene oxide also exerted differential effects on nuclear and microsomal epoxide hydrase which were dependent both on the substrate and on the organelle. The inhibition by this agent of nuclear and microsomal epoxide hydrase was quantitatively similar only for a single substrate, benzo(a)anthracene-5,6-oxide. When purified by immunoaffinity chromatography, nuclear and microsomal epoxide hydrases from 3-methylcholanthrene-pretreated rats were shown to have identical minimum molecular weights (-49,000) on polyacrylamide gels in the presence of sodium dodecyl sulfate. These findings support the assertion that microsomal metabolism can no longer be considered an exclusive index of the cellular activation of polycyclic aromatic hydrocarbons.
Polycyclic aromatic hydrocarbons are environmental carcinogens that are ubiquitous in the modern industrial biosphere (1). These compounds must be metabolically activated to electrophilic substances prior to inducing malignant transformation or tumors (2-5). The details of the sequence of events leading to metabolic activation are best understood for benzo(a)pyrene. The concerted action of the NADPH-requiring monooxygenase, aryl hydrocarbon hydroxylase, and of epoxide hydrase leads to the formation of (-)-trctns-7,8-dihydro-7,8dihydroxy benzo(a)pyrene (6-g), a proximate carcinogen (lo- 12). Further catalytic action of aryl hydrocarbon hydroxylase on this compound results in the formation of the diastereomeric benzo( a)pyrene-7,8-diol 9,10-epoxides in which the 9,10-epoxide is either cis (diol epoxide-1) or truns (diol 1 Present address; Department of Medicine, Duke University Medical Center, Durham, N. C. 27710. 0003-9861/79/120436-11$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
epoxide-2) to the benzylic 7-hydroxy group (6, 7, 9, 13-17). An abundance of evidence suggests that these diol epoxides are ultimate carcinogens mediating benzo( a)pyrene tumorigenesis (10, 11, 18-21). Data are also accumulating suggesting that “bay region” diol epoxides (18) of other polycyclic hydrocarbons may be ultimate carcinogens as well (22-36). The elucidation of the identities of the ultimate carcinogens makes a detailed knowledge of the enzyme systems leading to their formation of cardinal importance in the basic understanding of chemical carcinogenesis. Aryl hydrocarbon hydroxylase and epoxide hydrase have, until recently, been considered as localized exclusively to the endoplasmic reticulum. More recent evidence has established the presence of this activating system in nuclei (37-43). Furthermore, we have demonstrated the capacity of nuclei to form (-)-truns-7,8-dihydro7,8-dihydroxy benzo(u)pyrene (44) and the 436
COMPARISON
OF NUCLEAR
AND MICROSOMAL
diol epoxides from the latter (14), thus indirectly revealing the presence of nuclear epoxide hydrase. Direct demonstration has been made in our laboratory of the nuclear hydration of benzo( a)pyrene-4,5-oxide (45) and in Orrenius’ laboratory of the nuclear hydration of styrene oxide (41). Comparisons of nuclear and microsomal aryl hydrocarbon hydroxylase have revealed that both systems metabolize benzo( a)pyrene in a qualitatively similarly manner (40). Moreover, nuclear and microsomal sources of cytochrome P-448 and epoxide hydrase are immunologically identical (46). Subtle differences between the nuclear and microsomal enzymes have been suggested but remain controversial (47-48).
The studies reported here were undertaken to (i) compare nuclear and microsomal epoxide hydrase activities as assayed with a number of arene and alkene oxide substrates, (ii) compare the effects of animal pretreatment on the nuclear and microsomal enzyme activity, (iii) investigate the comparative effects of modifiers of epoxide hydrase activity on the two enzyme systems, and (iv) compare the minimum molecular weights of epoxide hydrase isolated from nuclear and microsomal sources. The data further substantiate the existence of nuclear epoxide hydrase and demonstrate some intriguing differences between the nuclear and microsomal enzymes. MATERIALS
AND METHODS
Animals and isolation of organelles. Male rats of the CD strain were maintained on a 12 h on-12 h off light-dark cycle and were allowed free access to Purina Rat Chow and to water. Animals were injected intraperitoneally with a solution of 3-methylcholanthrene (20 mgikg) in corn oil on each of two successive mornings and were killed by cervical dislocation 24 h after the second injection. When phenobarbital was the inducing agent, this compound was administered ip in saline at a dose of 75 mgikg at 96, 72, 48, and 24 h prior to sacrifice. The livers were removed immediately, washed in cold 0.9% NaCl and then either homogenized in 0.25 M sucrose (l:lO, w/v) for the preparation of microsomes or in 2.4 M sucrose containing 3 mM CaCl, (1:12, w/v) for the preparation of nuclei. The microsomes were sedimented from a 12,000g supernatant fraction by centrifuging at 106,OOOg as described previously (40). The nuclei
EPOXIDE
HYDRASE
437
were prepared as described (40) and the procedure in brief consisted of repurification of the crude nuclear pellet obtained from the dense sucrose by sedimentation in 1 M sucrose-l mM CaCl, until the preparation was homogeneous as judged by phase microscopy. In our previous publication (40), we have dealt with the matter of contamination of nuclear preparations with microsomes. Our nuclei were free of cytoplasmic contamination as judged by either light or electron microscopy. The contamination of our nuclear preparation with microsomes ascertained by deliberately contaminating the former with radiolabeled microsomes. The latter was obtained from the livers of rats that had been injected intraperitoneally 2 h previously with L[4,5-3H]leucine (51.6 Wmmol; 67 &i/g body wt). The specific activity of the microsomes was 4.5 x lo4 dpm/mg protein. The labeled microsomes corresponding to 2.5 x 105 dpm were added to a rat liver homogenate and the purification of nuclei conducted as described above. The final purified nuclei contained 1.9 x lo3 dpm which corresponded to a 0.76% microsomal contamination. This level is insufficient to account for epoxide hydrase activity within nuclei. ImmunoaffLnity
purz$cation
of nuclear
microsomal
Epoxide hydrase was purified by immunoaffinity column chromatography from nuclei (1290 mg protein) and microsomes (100 mg protein) isolated from livers of rats pretreated with 3-methylcholanthrene. Nuclei were first digested with deoxyribonuclease I for 20 min (49) to reduce the DNA concentration and prevent interference with the chromatographic separation. The microsomal preparation was treated in an identical manner as the nuclear preparation. The samples were then dialyzed for 1.5 h against 4 liters of DNA digestion buffer (49) minus P-mercaptoethanol. The nuclear and microsomal fractions were then solubilized with 0.5 mg sodium cholate and 0.2 mg Emulgen 911/mg protein followed by dialysis against the column equilibration buffer (10 mM potassium phosphate, pH 7.4, 0.2 M KCl, 0.2 mM EDTA, 20% glycerol, 0.2% Emulgen 911, and 0.5% sodium cholate). The white precipitate in the nuclear preparation was removed by centrifugation at 30,OOOg for 10 min. All sample preparation and subsequent chromatographic procedures were performed at room temperature. Antibody was produced in sheep to purified rat liver microsomal epoxide hydrase (50). Immunoglobulin was purified (51) from high titer sera taken 5-12 months after immunization and bound (9 mgiml) to CNBr-activated Sepharose (52). Two columns of 6 ml were poured and equilibrated with the buffer described above. The solubilized, deoxyribonucleasetreated nuclear and microsomal preparations were passed through their respective columns twice to insure saturation of all antibody binding sites. The immunoaftlnity columns were washed with 2 column epoxide
hydrase.
438
BORNSTEIN
volumes of the following solutions sequentially to minimize nonspecific protein binding: column equilibration buffer; 0.1 M sodium acetate, pH 4.2, containing 0.5 M KCl; 0.1 M sodium borate, pH 8.4, containing 1.0 M KCl, 0.2% Emulgen 911 and 0.2 mM EDTA; 0.2 M KSCN. Catalytically inactive epoxide hydrase was released with 2 column volumes of 4 M KSCN. The released enzyme was dialyzed against 5.0 mM potassium phosphate, pH 7.4 and concentrated by ultrafiltration. Protein was estimated by the method of Lowry et al. (53), and sodium dodecyl sulfatepolyacrylamide gel electrophoresis was performed as described by Laemmli (54). Substrates. The sources and characteristics of the arene and alkene oxides used in these experiments have been previously reported (55). Specific activities of the substrates were as follows: [7-3H]styrene-7,8oxide, 0.5 &i/pmol; [7,8-3H]octene-1,2-oxide, 7.2 pCi/pmol; [6-3H]benzo(a)pyrene-4,5-oxide, 5.3 rCi/ pmol; [6-3H]benzo(a)pyrene-7,8-oxide, 8.4 &i/pmol; [6-3H]benzo(a)pyrene-9,10-oxide, 39.5 #X~mol; [ll, 12-3H]benzo(a)pyrene-ll,12-oxide, 5.1 &i/pmol; [73H]benzo(a)anthracene-5,6-oxide, 8.5 &i/pmol; [5,63H]dibenzo(a,h)anthracene-5,6-oxide, 6.2 #.X~mol; 3-[11,12-3H]methylcholanthrene-ll,12-oxide, 2.4 &i/~mol; [3-3H]phenanthrene-9,10-oxide, 6.6 $i/ pmol. Epotide hydrase assay. In a total volume of 80 ~1, each assay (55) contained: 0.5 M Tris-HCl (pH 8.2 at 37”C), 25 ~1; nuclei or microsomes diluted in 3 mM MgCl,-5 mM Tris-HCl (8.5 at 2O”C), 50 ~1 (25 ~1 for metyrapone experiments); and substrate (12-150 nmol) in acetonitrile: NH,OH (lOOO:l, v/v), 5 ~1. All substrate concentrations used in this assay were saturating. When present, metyrapone, (a gift from Dr. J. Daly) was added in water. Cyclohexene oxide was added in 2 ~1 of acetonitrile but was withheld until immediately prior to the addition of substrate in order to minimize its enzymatic hydration. Otherwise, the reaction mixture, excluding substrate, was allowed to reach room temperature; the reaction was initiated by the addition of substrate, and subsequent incubation was performed at 37°C for 2-15 min. The reactions were conducted under conditions of linearity with respect to time. The reaction was then terminated by the addition of 25 ~1 of tetrahydrofuran followed by immediate transfer to an ice bath. A 35~1 aliquot of each sample (one-third of the total volume) in addition to a small amount of unlabeled carrier diol or glycol was subjected to thin-layer chromatography on Quantum Industries 5 x 20-cm LQDF thin-layer plates using the solvent systems previously described (55). After development of the plates, the products were visualized under ultraviolet light and fluorescent activation of the plates was allowed to extinguish for 2 h in room light. The bands of product were then scraped into scintillation vials, 0.1 ml of methanol and 15 ml of Scintisol
ET AL. counting medium (Isolab) were added sequentially. Radioactivity was quantitated in a Packard TriCarb spectrometer with a counting efficiency, determined using [3H]toluene as an internal standard of greater than 30%. All analyses were performed in duplicate. In all cases, values obtained in assays which lacked tissue were subtracted in the calculation of product formation. The recovery of radioactivity with the various substrates has been discussed previously (55). RESULTS
The effects of pH on the nuclear-catalyzed hydration of alkene and arene oxides were found to be essentially identical to those previously reported for microsomes (55). The pH of 3.2 (at 37°C) used in the comparative studies falls within the broad range of optima for both nuclear and microsomal systems. Conditions were established for linearity of product formation with respect to nuclear protein concentration. For most of the substrates studied, linearity was observed with as much as 800 lug of nuclear protein per 80 ~1 assay. In the comparative studies, nuclear and microsomal protein concentrations were selected which were within the range of constant product formation per milligram protein per minute. Table I shows the specific activities of nuclear and microsomal epoxide hydrase and their ratios using the nine alkene and arene oxide substrates. Nuclear and microsomal enzyme activities were always determined in parallel in the same experiments; relatively little day to day variation in the ratios of nuclear to microsomal activity was observed. For all of the substrates studied, the ratios of nuclear to microsomal specific activities fell within the range of 2.2 to 9.1%. The effects of phenobarbital and 3methylcholanthrene pretreatment on the nuclear and microsomal hydration of three substrates is shown in Table II. Phenobarbital pretreatment consistently enhanced the rate at which product was enzymatically formed from the three substrates by both organelles. On the other hand, 3-methylcholanthrene pretreatment depressed slightly the microsomal hydration of the three substrates while stimulating the nuclear hydration of these substrates by 30-
COMPARISON
OF NUCLEAR
AND MICROSOMAL TABLE
NUCLEAR
AND MICROSOMAL
EPOXIDE
HYDRASE
AND ALKENE
OXIDES’
I
HYDRATION
OF ARENE
Epoxide hydrase* (nmoI/mg/min) Amount (nmol)
Substrate Phenanthrene 9,10-oxide Benzo(a)anthacene-5,6-oxide Benzo(a)pyrene-4,5-oxide Benzo(a)pyrene-7,&oxide Benzo(a)pyrene-9,10-oxide Benzo(a)pyrene-1 1,12-oxide 3-Methylcholanthrene-11,12-oxide Octene-1,2-oxide Styrene-7,8-oxide
439
Nuclear
Microsomal
2.48 0.81 0.61 0.28 0.39 0.056 0.20 0.97 0.20
71.05 21.04 13.39 5.47 8.90 0.79 2.16 26.22 8.86
150 20 15 15 15 12 15 100 40
Pereentage nuclear/ microsomal’ 3.5 3.9 4.5 5.2 4.4 7.1 9.1 3.8 2.2
(3.3-3.7) (3.3-4.6) (4.3-4.7) (4.5-5.8) (5.6-8.0) (3.0-4.4) (1.8-2.8)
n Nuclei or microsomes which were isolated from the livers of 3-methylcholanthrene-pretreated 100-g male rats were incubated with the indicated amount of substrate for 2 min (except for styrene 7,8-oxide and benzo(a)pyrene-11,12-oxide which were incubated for 15 and 4 min, respectively). The amounts of nuclear and microsomal protein per 80 /*l were up to 0.8 and 0.2 mg, respectively. * Except for benzo(ufpyrene-9,l@oxide and 3-methylcholanthrene-11,12-oxide, each value represents the mean from at least two separate experiments. c Mean (range of values). Ratios were determined for individual experiments and then averaged.
60%. Although 3-methylcholanthrene has been reported to enhance microsomal epoxide hydrase activity for these substrates, the induction was reported to be TABLE EFFECTS
OF INDUCTION
ON THE NUCLEAR
variable from experiment to experiment (55, 59). Metyrapone and cyclohexene oxide are modifiers of microsomal epoxide hydrase II
AND MICROSOMAL
HYDRATION
OF ARENE
AND ALKENE
Epoxide hydrase” (nmoYmg/min)
OXIDES=
Animal pretreatment
Nuclear
Microsomal
Percentage nuclear/ microsomal
lo-oxide
Control Phenobarbital 3-Methylcholanthrene
1.46 3.68 (2.5) 2.37 (1.6)
78.6 166.9 (2.1) 63.8 (0.8)
1.9 2.9 3.7
Benzo(a)pyrene-11,12oxide
Control Phenobarbital 3-Methylcholanthrene
0.03 0.04 (1.3) 0.04 (1.3)
Octene-1,2-oxide
Control Phenobarbital 3-Methyleholanthrene
0.60 1.63 (2.7) 0.92 (1.5)
Substrate Phenanthrene-9,
0.79 1.54 (1.9) 0.64 (0.8) 27.4 56.3 (2.1) 21.0 (0.8)
3.6 2.9 6.0 2.2 2.9 4.4
a Nuclei and microsomes were prepared from the livers of 100-g male rats which had been pretreated by the intraperitoneal injection of phenobarbital (75 mg/kg) at 96, 72,48, and 24 h prior to sacrifice; or which had been pretreated by the injection of 3-methylcholanthrene (20 mg/kg) at 48 and 24 h prior to sacrifice. Control animals received no pretreatment. Incubations were carried out as described in the legend of Table I. * The numbers in parentheses are the ratios of induced to control enzyme activities for each substrate and organelle.
440
BORNSTEIN
activity (56), the effects of which differ depending on substrate (50). In the present studies, we compared the effects of these modifiers on the nuclear and microsomal systems. Figures 1 and 2 depict the effects of increasing concentration of metyrapone on the nuclear and microsomal hydration of four arene and alkene oxides. The microsomal hydration of benzo( a)pyrene-4,5oxide (Fig. 1A) was not greatly affected by the presence of metyrapone up to 6 mM but was inhibited slightly at the higher metyrapone concentrations (9 and 15 mM) to, respectively, 89 and 83% of control values. On the other hand, nuclear epoxide hydrase activity for this substrate was stimulated 44-78% by metyrapone concentrations of 6 lllM or greater. As shown in Fig. lB, when benzo( a)pyrene-11, 1Zoxide was the substrate, microsomal epoxide hy-
ET AL. BA-5,6-OXIDE
A
L > 20-
% I
c>
-06
.25
-7
I I
”
5
I
NUC
60\
A
BP-4,5-OXIDE
300-
3
H
200
100 iz
9 BP-I
15
I,IE-OXIDE
NU A-
A MIC I5
METYR~PONE
(5,lUl)
B
A--o
3
OXIDE
FIG. 2. Effects of metyrapone on the nuclear (NUC, 0) and microsomal (MIC, A) hydration of (A) benzo(a)anthracene-5,6-oxide and (B) octene-1,2-oxide. The amounts of substrate were: benzo(a)anthraeene-5,6oxide, 20nmol; octene-1,2-oxide, 100 nmol. The amount of microsomal and nuclear protein per assay was 100-200 and 400 pg, respectively. The details are presented in the text.
5 s:
A MIC
‘“L-+-J CY&?WEXENE
200-
0 NUC b MIC
(mM)
FIG. 1. Effects of metyrapone on the nuclear (NUC, 0) and microsomal (MIC, A) hydration of (A) benzo(a)pyrene-4-5-oxide and (B) benzo(a)pyrene-11,12oxide. The amounts of substrate per 80 ~1 assay were: benzo(a)pyrene-4,5-oxide, 15 nmoi; benzo(a)pyrene11,12-oxide, 12 nmol. Nuclear incubations contained 400 pg of protein; microsomal assays were performed with 25 PLgof protein. Incubations were carried out at 37°C as described in the text. Results-are expressed as percentage’*activity relative to control incubations without metyrapone. These are representative data from three sets of experiments.
drase was highly sensitive to the effects of metyrapone and was inhibited 70% at a modifier concentration of 6 mM. However; the nuclear enzyme was relatively insensitive to metyrapone-remaining within a range of approximately 15% of control activity. Epoxide hydrase activity with benzo(a)pyrene-5,6-oxide as substrate was mildly increased by the addition of metyrapone to both microsomal and nuclear systems (Fig. 2A). Nuclei and microsomes responded similarly when catalyzing the hydration of octene-1,2-oxide, i.e., with a stimulation of 3.5- to 4.0-fold at a final metyrapone concentration of 15 mM (Fig. 2B). To insure that linearity of product formation was maintained in the presence of
COMPARISON
OF NUCLEAR
BP-4,5-0X I DE
AND MICROSOMAL
Al
60
k-
BP- Il,l2-OXIDE
Y
# 100
-B
0 “1L
/O\o
N”C
EPOXIDE
441
HYDRASE
activity was somewhat more sensitive. As shown in Fig. 3B, the microsomal hydration of benzo( a)pyrene-11,12-oxide was markedly inhibited by cyclohexene oxide. The nuclear enzyme, on the other hand, was most sensitive to cyclohexene oxide inhibition when catalyzing the hydration of benzo(a)anthracene-5,6-oxide (Fig. 4A). Moreover, only with respect to the latter substrate were the nuclear and microsomal enzymes inhibited to the same extent at all cyclohexene oxide concentrations. While the nuclear conversion of octene-1,2-oxide to octene-1,2-glycol was stimulated slightly by 60 PM and 250 mM cyclohexene oxide, the microsomal enzyme was inhibited at all modifier concentrations (Fig. 4B). Although nuclear and microsomal epoxide hydrase differed in response to metyrapone and cyclohexene oxide as modifiers of catalytic activity, the minimum molecular
FIG. 3. Effects of cyclohexene oxide on the nuclear (NUC, 0) and microsomal (MIC, A) hydration of (A) benzo(a)pyrene-4,5-oxide and (B) benzo(a)pyrenell,l2-oxide. Experimental details were as described in the legend to Figs. 1 and 2, except that cyclohexene oxide was added in 2 ~1 acetonitrile. These are representative data from three sets of experiments.
metyrapone, the hydration of benzo( a)pyrene-4,5-oxide was investigated at different protein concentrations in the presence of 3 or 9 mM metyrapone. Linearity was maintamed under these conditions. The effects of cyclohexene oxide on the nuclear and microsomal hydration of four arene and alkene oxides are shown in Figs. 3 and 4. The microsomal system appeared generally more sensitive to the effects of this modifier than did the nuclear system. However, this greater sensitivity was not parallel for the four substrates. With benzo(a)pyrene-4,5-oxide as the substrate, the nuclear and microsomal enzyme activities were both inhibited approximately 25% by 60 PM cyclohexene oxide (Fig. 3A). However, at higher concentrations, microsomal
I 3
I METdPONE
hn$
FIG. 4. Effects of cyclohexene oxide on the nuclear (NUC, 0) and mierosomal (MIC, A) hydration of (A) benzo(a)anthracene-5,Goxide and (B) octene-1,2oxide. The legends to Figs. l-3. These are representative data from three sets of experiments.
442
BORNSTEIN
ET AL.
FIG. 5. Sodium dodecyl sulfate-gel electrophoresis of nuclear and microsomal epoxide hydrase isolated by immunoaffinity chromatography is described under Materials and Methods. The content of each well was: 1, 4 pg microsomal protein; 2, 0.3 pg microsomal protein purified by immunoaffinity chromatography; 3, effluent from immunoaffinity column eluted with 4 M KSCN contained 1.0 pg of immunoglobulin; 4, 0.5 pg purified microsomal epoxide hydrase; 5, 4 pg nuclear protein; 6, 0.3 Fg nuclear protein purified by immunoafllnity chromatography; 7, same as well 3; 8, same as well 4; 9, 0.3 fig of microsomal enzyme purified by immunoaffinity chromatography and 0.5 pg purified microsomal epoxide hydrase; 10, same as well 9 except contained 0.3 pg of nuclear enzyme purified by immunoaffinity chromatography.
weight of the enzyme from both sources was shown to be identical. We previously reported (46) that nuclear epoxide hydrase was antigenically identical to microsomal epoxide hydrase purified to apparent homogeneity (57). Purification of catalytically inactive epoxide hydrase from nuclei was accomplished with an antibody prepared against the purified microsomal enzyme (Fig. 5). Most of the contaminating protein evident on the sodium dodecyl sulfategels was due to immunoglobulin eluting from the immunoaffinity column in the presence of 4 M KSCN. Both nuclear and
microsomal epoxide hydrase which had been purified by the immunoaffinity technique corn&rated with the purified catalytically active enzyme on sodium dodecyl sulfate-gels (M, = 49,000). DISCUSSION
The role of aryl hydrocarbon hydroxylase and epoxide hydrase in the activation of polycyclic hydrocarbons to ultimate carcinogens is now dogma. The presence of these enzymes within the nucleus is also relatively certain. We have discussed, else-
COMPARISON
OF NUCLEAR
AND
where, the dangers inherent in strictly morphologic arguments for nuclear purity (40) and have presented data arguing the attribution of these nuclear activities to microsomal contamination (40, 58). It would not be consistent with the economy of nature to expect, a priori, these nuclear enzymes to differ greatly from their microsomal counterparts. Indeed, the preponderance of data thus far collected supports a very close similarity of the two systems. Thus, nuclear and microsomal aryl hydrocarbon hydroxylase have been found to be similar, if not identical, with respect to their immunochemical characteristics (46), their spectral characteristics (40), and their qualitative benzo( a)pyrene metabolite profiles (14, 40). On the other hand, one might reasonably expect at least quantitative differences between the behavior of the nuclear and microsomal activating systems in situ within purified organelles. In the present study, we have demonstrated that the nuclear microsomal hydration of arene and alkene oxides is not always identically affected by pretreatment of rats with certain xenobiotics. Thus (Table II), pretreatment with 3-methylcholanthrene had no effect or slightly depressed the microsomal hydration of phenanthrene-9,10-oxide, benzo(a)pyrene-11, E-oxide, and octene-1,2-oxide but increased the nuclear hydration of these substrates. Phenobarbital pretreatment, on the other hand, increased the nuclear and microsomal enzyme activities for all three substrates. However, both nuclear and microsomal epoxide hydrase are immunochemically identical (46) and have the same minimum molecular weight on sodium dodecyl sulfate-gels, whether isolated from 3-methylcholanthrene or phenobarbitalpretreated rats. Furthermore, we have shown (Fig. 1) effects of metyrapone on epoxide hydrase that are dependent both on organelle and on substrate. The data represented in Fig. 1B indicate that the microsomal hydration of benzo( a)pyrene-ll, le-oxide was highly sensitive to inhibition by metyrapone, whereas the nuclear hydration of this substrate was relatively unaffected by metyrapone. Were epoxide hydrase present in the
MICROSOMAL
EPOXIDE
HYDRASE
443
nuclear preparations merely as a function of microsomal contamination, one would expect that the “nuclear” enzyme would be at least equally sensitive to the effects of metyrapone as the microsomal enzyme. Alternatively, it would be necessary to postulate that nuclei interfere with the action of metyrapone. That the latter is not the case is demonstrated in Fig. 3B where metyrapone is shown to affect the microsomal and nuclear hydration of octene-1,2-oxide to an equal extent. Indeed, as shown in Fig. lA, benzo(a)pyrene-4,5-oxide was the substrate, nuclear epoxide hydrase was more sensitive to metyrapone than was its microsomal counterpart. Similarly, when cyclohexene oxide was the modifier of epoxide hydrase activity, the nuclear and microsomal enzyme activities were differentially affected depending on substrate. Thus, the nuclear and microsomal hydration or benzo(a)anthracene-5,6oxide was inhibited to very similar extents at all of the cyclohexene oxide concentrations studied (Fig. 4A). On the other hand, the microsomal hydration of benzo(a)pyrene-11,12-oxide (Fig. 3B) was exquisitely sensitive to cyclohexene oxide inhibition whereas the nuclear enzyme was much less sensitive. The nuclear hydration of the latter substrate appeared maximally inhibited by a relatively low concentration of cyclohexene oxide (250 PM). The latter observation provides additional evidence against the possibility of nucleus-induced alterations in dose-response characteristics as an explanation for these data. These data do not necessarily argue for different nuclear and microsomal epoxide hydrases. However, they do establish that the nuclear and microsomal enzymes behave differently in situ within the purified organelles. The data provide additional evidence against the supposition that “nuclear” epoxide hydrase merely represents microsomal contamination. Whether epoxide hydrase exists in single or multiple forms is, at present, a moot question, although in a separate study, evidence was presented for multiple regulatory sites in the rat liver microsomal enzyme, and for different forms of epoxide hydrase in several species (50). If multiple
444
BORNSTEIN
forms of the enzyme exist within a single tissue, then a possible, but not exclusive, interpretation of the present data is that these forms are not distributed identically with respect to nuclei and endoplasmic reticulum . Interpretation of these data is complicated by a lack of knowledge of relative uptake by the two organelles of the substrates and modifiers which were utilized in our study. However, linearity of product formation with respect to time and protein concentration for all of the substances suggests that both nuclear and microsomal enzyme to metyrapone was demonstrated by its potent enhancement of the hydration of oxtene-l,Zoxide-an effect quantitatively similar to that observed with microsomes. Similarly, it is the differential response to cyclohexene oxide of the nuclear and microsomal enzyme activities relative to multiple substrates that is of most interest. Finally, in this regard, the study of Levin et al. (50) addressed the possibility of membrane altering the relative uptake of the modifiers. In their study, the Same concentration of modifier effected microsomal and purified epoxide hydrase in an identical manner. Consequently, it is highly unlikely that the difference between microsomal and nuclear epoxide hydrases would be explainable by differential uptake of the modifiers. We have proposed that the nuclear polycyclic hydrocarbon metabolizing system may act as a second-line cellular defense against lipophilic substances, but that in doing, it may lead to the formation of ephemeral highly reactive electrophiles in proximity to the cell’s genetic apparatus (40). The effects of modifiers of polycyclic hydrocarbon metabolism such as the potent epoxide hydrase inhibitor, l,l,l-trichloro-2-propene oxide, on tumorigenesis have not been entirely consistent with predictions based on their effects on microsomal metabolism (60). The data herein reported suggest that differential effects on nuclear and microsomal metabolism may help explain such inconsistencies.
ET AL. ACKNOWLEDGMENTS The authors appreciate the assistance of Mr. Dennis Lawler and Mr. Bruce Hassuk in the largescale isolations of organelles. We are indebted to Dr. D. M. Jerina for providing the substrates used in this study. These studies were supported in part by Grant CA 20711 from the National Cancer Institute. The data have been submitted by Dr. William Bornstein as part of the Ph.D. thesis requirement of the Department of Cell and Molecular Biology of the Medical College of Georgia. REFERENCES 1. HOFFMAN, D., AND WYNDER, E. L. (1976) in Chemical Carcinogenesis, ACS Monograph 173. (Searle, C. E., ed.), pp. 324-365, American Chemical Society, Washington, D. C. 2. SIMS, P., AND GROVER, P. L. (1974) Adv. in Cancer Res. 20, 164-274. 3. HEIDELBERGER, C. (1973) Advan. Cancer Res. 18, 317-366. 4. JERINA, D. M., AND DALY, J. W. (1974) Science 184, 573-582. 5. MILLER, E. C., AND MILLER, J. A. (1974) in The Molecular Biology of Cancer (Busch, H., ed.), pp. 377-402, Academic Press, New York. 6. THAKKER, D. R., YAGI, H., Lu, A. Y. H., LEVIN, W., CONNEY, A. H., AND JERINA, D. M. (1976) Proc. Nat. Acad. Sci. USA 73, 3381-3385. 7. THAKKER, D. R., YAGI, H., AKAGI, H., KOREEDA, ., Lu, A. Y. U., LEVIN, W., WOOD, A. LW., CONNEY, A. H., AND JERINA, D. M. (1977) Chem. Biol. Interact. 16, 281-300. 8. YANG, S. K., MCCOURT, D. W., ROLLER, P. P., AND GELBOIN, A. V. (1976) Proc. Nat. Acad. Sci. USA 73, 2594-2598. 9. YANG, S. K., MCCOURT, D. W., LEUTZ, J. C., AND GELBOIN, H. V. (1977) Science 196, 1199-1201. 10. LEVIN, W., WOOD, A. W., YAGI, H., JERINA, D. M., AND CONNEY, A. H. (1976) Proc. Nat. Acad. Sci. USA 73, 3867-3871. 11. KAPITULNIK, J., LEVIN, W., CONNEY, A. H., YAGI, H., AND JERINA, D. M. (1977) Nature (London) 266, 378-380. 12. SLAGA, T. J., VIAGE, A., BERRY, D. L., BRACKEN, W. M., BUTTY, S. G., AND SCRIBNER, J. D. (1976) Cancer Lett. 2, 115-122. 13. SIMS, P., GROVER, P. L., SWAISLAND, A., PAL, A., AND HEWER, A. (1974) Nature (London) 252, 326-327. 14. BRESNICK, E., STOMING, T. A., VAUGHT, J. B., THAKKER, D. R., AND JERINA, D. M. (1977) Arch. Biochem. Biophys. 183, 31-37. 15. DAUDEL, P., DUQUESNE, M., VIGNEY, P.,
COMPARISON
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
OF
NUCLEAR
AND
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MICROSOMAL
30.
31.
32. 33.
34.
35.
36.
37.
38. 39. 40.
41. 42.
43. 44.
45.
46.
47. 48.
EPOXIDE
HYDRASE
445
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446
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ET AL.
56.
57.
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59.
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