ARCHIVES
OF
BIOCHEMISTRY
Substrates
AND
BIOPHYSICS
and inhibitors
TAR0
170, 438-451 (19%)
of Hepatic Transferase
HAYAKAWA’
Roche Institute of Molecular
HARUHIKO Laboratory
YAGI
AND
SIDNEY
Glutathione-S-Epoxide
UDENFRIEND*
Biology, Nutley, New Jersey 07110 AND
DONALD
M. JERINA’
of Chemistry, National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014 Received February 19, 1975
The specific activities of glutathionea-epoxide transferase present in the 100,OOOg supernatant from sheep liver and a highly purified preparation of this enzyme have been compared with a variety of epoxides as substrates. Purification factors of 30- to 60-fold were observed for nearly 50 simple epoxides and arene oxides. The similarity in purification factors suggests either that a single enzyme with activity toward simple epoxides and arene oxides has been purified or that several related enzymes have been copuriiied. Comparison of specific activities for benzene oxides, naphthalene oxide, phenanthrene oxides, and arene oxides of benzoralpyrene and dimethylbenzaanthracene has established that (i) benzene oxides are poor substrates unless strong electron withdrawing substituenta are present, (ii) naphthalene oxide is a good substrate, and (iii) increasing the size of polycyclic arene oxides causes a steady decrease in activity. Reactivity toward simple epoxides was compared to that for epoxide hydrase. The transferase was not readily inhibited by any of the compounds which were used and showed a strict requirement for glutathione.
The principal route of oxidative detoxication for drugs, pesticides, and other nonpolar environmental agents in mammals is via the P-450 monoxygenases in liver (1). For aromatic substrates such as naphthalene (2) and higher polycyclic aromatic hydrocarbons (3), arene oxides have been demonstrated as obligatory intermediates on the pathways to phenols, dihydrodiols, and mercapturic acid precursors. In addition, olefins are converted to epoxides by the P-450 system. The principal reactions of arene oxides in the cell consist of nonenzymatic isomerization to phenols, hydration to dihydrodiols by the enzyme epoxide hydrase, conjugation with glutathione by the enzyme glutathioneS-epoxide trans’ Present address: Department of Biochemistry, School of Dentistry, Aichi-Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya 464, Japan. * To whom reprint requests should be addressed. 436 Copyright 0 1975 by Academic Press, Inc. All rights
of reproduction
in any
form
reserved.
ferase, and binding to macromolecular cell constituents. As this last reaction has been invoked as a mechanism by which the cytotoxic, mutagenic, and carcinogenic properties of many hydrocarbons can be explained (3), the potential protective roles of epoxide hydrase and glutathioneS-epoxide transferase assume special importance. A wide variety of substrates of glutathione transferase activity are known; in each case displacements of good leaving groups occur at a carbon atom. Simple epoxides and arene oxides are opened, addition occurs to double bonds, and coupling takes place at metabolically activated centers (4, 5). The actual number of different transferases involved in these reactions and the cross-specificity for these enzymes toward common substrates is unknown. A homogeneous epoxide transferase was iso-
SPECIFICITY
OF
GSHS-EPOXIDE
lated from rat liver based on its ability to use 1,2-epoxy-3-(4-nitrophenoxy) propane as substrate (6). With this highly purified rat enzyme a number of monosubstituted ethylene oxides were found to have 8-114% relative activity compared with the substrate used for purification. In a subsequent study ,(7), three additional glutathione transferases were isolated in homogeneous form from rat liver. 3,4-Dichloronitrobenzene, methyl iodide, and menaphthy1 sulfate were used as substrates in these purifications. Substantial cross-reactivity for various substrates was found for the four preparations despite the fact that the first was isolated for its ability to open epoxides while the latter three catalyzed displacement reactions. Development of a sensitive radiometric assay (8) for glutathione-S-epoxide transferase activity with naphthalene 1,2-oxide and [35S]glutathione as cosubstrates has made possible the isolation of a purified form of this enzyme from sheep liver. The present paper describes the substrate specificity and the effects of some inhibitors of this enzyme in the 100,OOOg supernatant from sheep liver and in a purified preparation from the same source. MATERIALS
AND
METHODS
Substrates and inhibitors. Simple epoxides and epoxide analogs used in Tables I-III were either obtained commercially or were prepared by standard methods from available precursors. Many of these had been employed previously in an examination of the substrate specificity of epoxide hydrase (9). Radioactive [7-3H]styrene oxide (10) and [23H1naphthalene 1,2-oxide (11) were prepared as described and were >99% radiochemically pure. The following arene oxides were synthesized directly by the methods indicated or by slight modifications of the procedures described: benzene oxide (121, naphthalene 1,2-oxide (131, l-methyl, 3-methyl, and 4methylbenzene 1,2-oxide (141, 1-methylnaphthalene 1,2-oxide (131, 2-methylnaphthalene 1,2-oxide (141, 4-carbo-t-butoxybenzene l,2-oxide (15), phenanthrene 1,2- and 3,4-oxides (13), phenenthrene 9,10oxide (161, 7,12-dimethylbenzo[a]anthracene 5,6-0xide (16, 171, benzolalpyrene 7,8- and 9,10-oxides (131, and benzo [alpyrene 4,5-oxide (16, 17). Studies leading to the synthesis of 3-chloroand 4-chlorobenzene 1,2-oxide and to 3-carbo-t-butoxybenzene 1,2-oxide have not yet been submitted. All of the above epoxides, arene oxides, and substrate analogs were judged to be >95% pure prior to use, by thin-layer
TRANSFERASE
439
chromatography, nuclear magnetic resonance, and mass spectrometry. Most substrates, inhibitors, and substrate analogs were added as 0.3 M solutions in acetone or as previously described (81. For K-region arene oxides, dimethyl sulfoxide was employed as solvent. In all cases, the quantity of solvent added to the incubations had no effect on the rates of reaction Enzyme preparations. Incubations were conducted either with the 100,OOOg supernatant fraction from sheep liver or with the Sephadex G-75 column fraction 40-fold purified over activity present in supernatant. Details were reported in a previous publication (8). Assay procedure. Most of the assays for transferase activity were based on the sensitive radioisotope procedure (8) developed to measure conjugate formation between naphthalene 1,2-oxide and 135Slglutathione by simply replacing naphthalene oxide with other compounds. Incubation mixtures for the standard assay consisted of 0.5 pmole of [?S]glutathione (4-5 x lo5 cpm), 100 pmoles of pyrophosphate buffer (pH 8.01, the indicated amounts of substrate and enzyme preparation, and sufficient water to make a total volume of 1 ml. Products were analyzed radiometrically after paper chromatography, as previously described (8). Results are corrected for nonenzymatic conversion, and the R, values for the various conjugates are given in the tables. Conversions to products have not been corrected for recovery which is about 50% for naphthalene oxide (8). Recovery for the conjugates produced here are probably quite comparable since the peptide residue is the principle functionality common to all the products. With all substrates the amount of product formed in the presence of enzyme ranged from two to ten times that formed nonenzymatically with most cases being five to eight times blank. Blanks consisting of incubations with boiled enzyme or no added enzyme were no significantly different. The substantial nonenzymatic rates for reaction between substrates and glutathione limits the sensitivity of the assay. When radioactive oxides were used as substrates with nonradioactive glutathione, incubation mixtures consisted of the indicated amounts of 2-3Hnaphthalene 1,2-oxide (3 x 10’ cpmO.5 pmol) or [73Hlstyrene oxide (4-5 x lo5 cpml.2 pmol), 5 pmol of glutathione, 100 pmol of pyrophosphate buffer (pH 8.01, enzyme as indicated, and sufficient water to make a total volume of 1 ml. Unless otherwise indicated, incubations were initiated by addition of oxide and run for 5 min at 22°C. Termination and product analysis were as described (8). RESULTS
The data presented in Tables I and II compare the ability of nearly 50 epoxides,
440
HAYAKAWA TABLE
COMPARISON
OF ACTIVITY
OF
Substrate
CRUDE
AL..
I
AND PURIFIED SHEEP LIVER GLUTATHIONE-S-EP~XIDE VARIOUS SUBSTRATES Initial concn bM)
2
Specific
Crude Purified extract” enzyme” (nmol/mg/min)
Purification factor
R, value of product
4.5
188
42
3.0
7.2
363
50
0.6
14.8
504
34
0.31
34
0.45
56
0.22
0
Not detectable
0.6 (pH
activity
TRANSFERASE
0.6
OHC--Cc-hi,
6.1
0.30
Not detectable
207
6.5)
HOW*-CclH2 0
3.0
Cl-W-CC&,
3.0
0
ET
Not detectable
4.6
Not detectable
259
ON
SPECIFICITY
OF
GSHS-EPOXIDE
TABLE Substrate
Initial concn h-M
02&-) 0-CH,-C~=?H, O
CH 3 -,6%-C,
I-Continued Specific
activity
Crude Purified extract” enzymea (nmol/mg/min)
3.0
4.0
0.6
6.4
3.0
2.4
3.0
2.9
164
296
Purification factor
RI value of product
41
0.34
46
0.20
32
0.52
36
0.26
2C 2 H 5
0
0
441
TRANSFERASE
CH/-‘CH
3
0
77
o-
0
0
3.0
/ 0 \
0
Not detectable
104
Not detectable
3.0
0.37
22.0
60
0.37
3.0
0.46
25.9
56
0.43
TABLE Substrate
0
CH3
/ 0
Specific
activity
Crude Purified extract” enzyme” (nmol/mg/min)
3.0
Not detectable
Not detectabled
3.0
Not detectable
Not detectabled
\
yc
I-Continued
Initial c0ncn bnM)
Purification factor
RI value
of product
Cl /
3.0
2.8
140
50
0.29
3.0
3.4
172
51
0.31
5.0
219
44
0.43
14.0
556
40
0.43
3.0
2.2 5.6
101 183
46 33
0.44 0.55
3.0
3.8
178
47
0.56
0
6\
Cl / 0
-0 \
0.43
0 (pH
6.5)
(CH,),CO& 0
0.6
(PI-I 6.5)
CH3
442
TABLE Substrate
/ / a‘I/‘,
I-Continued
Initial concn (mhd
Specific
Purificpn
activity
Crude Purified extracta enzyme” (nmol/mg/min)
RI
value of product
1.5
5.6
219
39
0.39
1.5
5.5
200*
36
0.47
1.5
1.4
38.96
28
0.41
0.15
3.5
111
32
0.58
0.6
0.25'
9.Bb
39
0.39
0.6
0.02"
Not detectableb,
0.42
d
-
0
0.6
0.09b
>Ob. d
-
0.51
Assays were carried out with 700 pg of 100,OOOg supernatant or 11 /qg of Sephadex G-75 column preparation. Assay conditions are described in Methods. n Crude extract and purified enzyme indicate 100,OOOg supernatant and Sephadex G-75 column preparation respectively (see ref. 8). *Reaction mixtures contained 3 pmoles of %-glutathione (2 x 106cpm); 1 mg of 100,OOOg supematant or 21 @g of Sephadex G-75 column preparation as enzyme, and incubated for 20 min at 30”. Others are the same as standard assay system. ‘Two products were detected on chromatography. d These are the least stable substrates employed in this study. 443
TABLE
COMPARISON OF GLUTATHIONES-EPOXIDE
ACTIVITIES
AND RELATED
COMPOUNDS
Specific activity (nmol/mg/min)
Substrate
0”’
II
TRANSFE~USE
TOWARD I
SUBSTITUTED
Activity
STYRENE R, value of product
0 100
0.30
929
60
0.27
479
31
0.25
850
55
0.36
631
41
0.36
65
0.35
1538
0
O2N
OeN
0
fl
0
Er
“%y Cl
103
Not
6.7
-
detectable
4.4
68
1749
114
CH, 444
0.37
0.27
0.27
OXIDES
SPECIFICITY
OF GSHS-EPOXIDE TABLE
Substrate
II-Continued
Specific activity (nmol/mg/min)
% Activity
Rr value of product
22
337
26.9 H3C
445
TRANSFERASE
0.27
1.8
0.30
CH3
@.+QHx
28.0
0.29
1.8
H3C
221
d
14
73
s
0.36
0.57
4.7
Not detectable”
-
0.39b
22
0.26
-
-
Cc
/‘r: 0”
S
0
342
NH
0
Not detectable
Assays were conducted with 11 pg of purified enzyme (Sephadex initial concentration of substrates. For assay conditions see Methods a Cyclohexene episulfide was sufficiently reactive with glutathione difference in radioactivity of the major spot with or without enzyme. ’ The R, value of the major spot of the conjugate. Additionally, having Rf values of 0.53, 0.62, and 0.83.
G-75 column preparation) section. nonenzymatically that
there
three
were
radioactive
spots
using
3 mM were
observed
no
HAYAKAWA
ET AL.
TABLE EPOXIDE
ANALCJGS AND OCTENE ~&OXIDE NAPHTHALENE ~,%OXIDE
III
AS INHIBITORB OR i’-%STYRENE
Inhibitor
OF PURIFIED TRANSFERASE OXIDE AS SUBSTRATES”
Substrate (inhibitor [2-3HlNaphthalene 1,2-oxide (0.09 mM) Remaining activity (%)
none
100 (-1
2-3H-
concn in mM) [7-3Hl,S$~ieloxide Remaining 100
activity (%) (-)
0
0”
47 (0.9)
0
0 03
WITH
-
0
-
68 (0.03)
/ 61 (0.9)
46 (0.6)
80 (0.9)
65 (0.6)
93 (0.9)
102 (0.6)
NH
‘Substrates and inhibitors were all dissolved in acetone to yield 0.3 M solutions, except naphthalene 1,2oxide which was 10x diluted. Reaction mixtures contained 5 pmoles of nonradioactive glutathione; 100 pmoles of pyrophosphate buffer, pH 8.0; the indicated amounts of substrates and inhibitors and 11 pg of purified enzyme (Sephadex G-75 column preparation) in 1 ml total volume. Details of the assay conditions are described in Methods.
arene oxides, and analogs, in which oxygen has been replaced by sulfur or nitrogen, to act as substrates for a crude and a purified preparation of glutathione-S-epoxide transferase from sheep liver. The assay system employed (a), although sensitive, is sufficiently time consuming as to preclude obtaining true kinetic comparisons for all these substrates. Both naphthalene and styrene oxide have K, values of about 0.1 mM (8), the reactions studied here are therefore assumed to be under conditions which are saturating with regard to oxide substrate since initial concentrations of 3 mM were used in most cases. Most of these
reactions are probably linear over the 5min incubation period (8), and product recoveries are anticipated to be comparable. The question of substrate stability over the time course of the incubation is a serious one for unstable arene oxides that readily rearrange to phenols and will be discussed later. Half-lives of a number of arene oxides used here have been determined (3, 19). Extrapolation from these studies leads to the conclusion that the enzyme probably remains saturated throughout the course of the reactions with all the substrates which were used. The purification factor between crude
SPECIFICITY
OF
GSHS-EPOXIDE
100,OOOg supernatant and the purified enzyme are presented for 28 substrates in Table I. In general, most substrates show a 40- to 50-fold purification for the enzyme with extremes of 30- and 60-fold being observed. The range of purification is within a factor of 2: and may be considered comparable for all the substrates studied. Styrene and naphthalene oxides show competitive inhibition (Fig. 1) with each other in the 100,OOOg fraction. Styrene oxide proved to be the most effective epoxide substrate for the enzyme (Table I), and a specific requirement for glutathione was found. With 13Hlnaphthalene oxide as substrate, cysteine, P-mercaptoethanol, dithiothreitol, and coenzyme A, at a final concentration of 2 mM, gave no detectable product. Reactivity of a number of monosubstituted ethylene oxides, the substrate class which contains styrene oxide, was examined (Table I). When the substituent was formyl, hydroxymethyl, or epoxycyclohexyl (the carcinogen, 1-ethyleneoxy-3,4epoxycyclohexane) (18), no activity was detected. In contrast, ethylene oxides which had phenyl (styrene oxide), chloromethyl, trichloromethyl, or a nitrophenoxymethylene as the substituent were quite active. Octene 1,2-oxide (Table II) was relatively inactive. No apparent steric or electronic factors explain these trends. Two trans1,2-disubstituted ethylene oxides, ethyl butyrate - 2,3 - oxide and stilbene oxide, showed high and moderate activity. Several substituted benzene oxides and 1.0,
TRANSFERASE
447
related compounds were examined (Table I). Benzene oxide and 1-methylbenzene oxide showed about 5% of the activity of styrene oxide while 3- and 4-methylbenzene oxide were inactive. Formation of glutathione conjugates of the toluene ring has not been reported in Go. While instability of the substrate toward isomerization to phenols may, in part, explain the inactivity of the two latter compounds (19), this certainly is not the case for benzene oxide as evidenced by its time course (Fig. 2). Substitution of chlorine or carbo-t-butoxy groups at the 3- or 4-positions produced moderate to excellent substrates. In fact, 4-carbo-t-butoxybenzene oxide proved to be the best substrate in the entire study. For the substituted benzene oxides, there is a fairly strong indication that electron withdrawing groups greatly facilitate reaction. Cyclohexene oxide was found to be a moderately good substrate, about eight times as active as benzene oxide. The inactivity of 1 - ethyleneoxy - 3,4 - epoxycyclohexane, which is related to cyclohexene oxide, is even more surprising in light of the activity of cyclohexene oxide. The insecticide, chlordane epoxide, which is structurally related to cyclohexene oxide, showed no detectable product at a final concentration of 2.6 mM (data not shown). A number of arene oxides of polycyclic aromatic hydrocarbons were examined (Table Il. While naphthalene oxide is a good substrate, methyl substitution at C-l or C2 significantly decreased its activity. Two 0.2r
-2 .E
A
,E, -10
I
0
‘/[3H.Nophtholene-I,
IO Z-oxide]
20 (mM-1)
‘+H-St yrene oxide] (mM-1) B Fig. 1. Lineweaver-Burk plots of glutathione-S-epoxide transferase from sheep liver 100,OOOg supernatant for both 12-3Hlnaphthalene 1,2-oxide (A) without styrene oxide (-0-l or with 0.9 mM styrene oxide as an inhibitor (-0 -1, and [7-3Hlstyrene oxide (B) without naphthalene 1,2-oxide (-0-l or with 0.03 mM naphthalene 1,2-oxide as an inhibitor (-0-l. Incubations were with 11 pg of purified enzyme in a final volume of 1.0 ml.
448
HAYAKAWA
5ooor--yv-
0
L ._LI
-I, 5
TIME
(min)
FIG. 2. Time course of the formation of glutathione conjugate with benzene oxide. Assays were carried out with 32 pg of protein from the Sephadex G 75 column preparation in the presence of 0.6 pmole of benzene oxide. Counts were net values after subtracting the counts for non-enzymatic conjugate formation. Details of the assay are described in Methods.
products were detected from l-methylnaphthalene l&oxide. Both compounds, on treatment with 1 N HCl for 15 min at 6o”C, underwent dehydration to conjugates which migrated more rapidly on chromatography. For the three possible arene oxides of phenanthrene, the 9,10-(K-region) and 1,Zoxides had about 60% of the activity of naphthalene oxide while the most sterically hindered 3,4-oxide was relatively inactive and comparable to benzene oxide. The K-region oxide of 7,12-dimethylbenz[ulanthracene had half the reactivity of the corresponding phenanthrene oxide while the K-region oxide of benzo[ulpyrene was practically inactive. Non-K-region oxides of benzo[a]pyrene at the 7,8- and 9,10positions showed practically no detectable activity. Stability of substrate toward isomerization to phenols cannot explains these results since high activity for the more stable K-region oxides (19) would be expected on this basis. The simplest conclusion to be drawn is that large molecules are poor substrates. The effect of varying the substituents on styrene oxide was also examined (Table II). Any monosubstitution on the phenyl ring was found to be detrimental to activity. Substitution of halogen or a nitro group at the meta orpara positions showed no clear trend for the decrease in activity. A bulky phenyl group at the para position
ET AL.
decreased activity E-fold. Substitution of methyl or phenyl at C-7 produced poor substrates. However, substitution of a methyl group at C-8, which is truns to the phenyl ring, produced a substrate more active than styrene oxide. In contrast, cis substitution of a methyl group at C-8 decreased activity five-fold. The three possible dimethylstyrene oxides, with both methyl groups on the oxirane ring, were all of low activity. Isoelectronic substitutions of sulfur and nitrogen in styrene oxide were also examined (Table II). The nitrogen analog gave no product while substitution by sulfur showed 20% of the activity of styrene oxide. Cyclohexene episulfide was found to be an excellent scavenger for glutathione, reacting considerably without enzyme. However, enzyme did not augment this activity. The effect of potential inhibitors on transferase activity toward radioactive styrene and naphthalene oxides was examined (Table III). Naphthalene oxide was the most effective inhibitor of the reaction with styrene oxide, producing 30% inhibition at l/20 of the substrate concentration. Among the sulfur and nitrogen analogs and octene oxide, cyclohexene episulfide produced 54% inhibition at equimolar concentration with styrene oxide. Reaction of naphthalene oxide was difficult to inhibit by any compound. Styrene oxide at ten times the substrate concentration was the best inhibitor, but caused only 53% inhibition. DISCUSSION
Substrate specificity of glutathione-Sepoxide transferase activity has been examined in the 100,OOOgsupernatant from sheep liver and in a purified (8) preparation of this enzyme. Like the epoxide transferase preparation from rat (61, a strict requirement for glutathione has been observed. Comparison of activity in the crude supernatant to that in the purified enzyme for a wide variety of simple epoxides and arene oxides shows that for all compounds, the degree of purification ranged for 28- to 60-fold (Table I). The similarity in purification factors suggests that individual epox-
SPECIFICITY
OF
GSHS-EPOXIDE
ide transferase activities toward specific structural types have not been significantly lost on purification. Comparable ranges of purification factors for simple epoxides and for arene oxides indicate that these activities copurify and suggest that either a single enzyme or a clearly related group of enzymes is involved. A similar finding has been observed for epoxide hydrase, purified from the liver of guinea pigs, with the exception that a specific activity for benzene oxide was preferentially lost in the hydrase preparation (9, 26). Competitive inhibition between styrene and naphthalene oxides (Fig. 1) further support the view that the epoxide transferase(s) from sheep liver does not distinguish between simple epoxides and arene oxides. The chemical nature of the adducts formed from the epoxide substrates used in this study and glutathione have not been examined. For the simple epoxides, direct tram opening of the oxirane ring is assumed to occur. With unsymmetrical substrates, two products are possible although not necessarily separable by the chromatographic procedure employed. Two major products were detected when l-methylnaphthalene 1,2-oxide was used as substrate. Arene oxides with isolated double bonds, such as benzene oxides and non-Kregion arene oxides of polycyclic hydrocarbons, undergo nucleophilic addition reactions at sites remote from the oxirane ring (21). However, sulfur nucleophiles always add to the oxirane ring of arene oxides in a trans-1,2 fashion (21). Thus, the two separable products for 1-methylnaphthalene 1,2-oxide probably arise by direct trans opening resulting from attack by sulfur at C-l and at C-2. A comparison of the extent of reaction with glutathione in the absence of enzyme (data not shown) is of interest from both a chemical and a pharmacological point of view. The very high nonenzymatic reactivity of 3- and especially 4-carbo-t-butoxybenzene oxides suggests that direct nucleophilic attack by thiols occurs on arene oxides with strong electron withdrawing groups. This is in contrast to a study of the reaction of benzene oxide with thiols which indicated that trapping of intermediate car-
TRANSFERASE
449
bonium ions from the oxide was the major pathway (22). Thus, for deactivated aromatic rings, which form stable arene oxides (23), nonenzymatic reaction with glutathione could play an important role in preventing the binding of arene oxides to tissue constituents. Thus, for deactivated aromatic rings, which form stable arene oxides (23), nonenzymatic reaction with glutathione could play an important role in preventing the binding of arene oxides to tissue constituents. Transferase activity toward chloro- and carbo-t-butoxy substituted benzene oxides was moderate to excellent, with 4-carbo-t-butoxybenzene oxide being the best substrate found in this study. Similarity in transferase activity toward 3- and 4-chlorobenzene oxide suggests that selective detoxication of these arene oxides by glutathioneS-epoxide transferase does not occur. Rates of both enzymatic and nonenzymatic conjugation of the arene oxides of the polycyclic hydrocarbons steadily decreased as the size of the ring systems increased. Thus, a protective role for glutathione against polycyclic hydrocarbon induced carcinogenesis via arene oxides is not evident from these studies, in marked contrast to the clear protective role for glutathione in the hepatic necrosis induced by halobenzenes (24). Although there are reports (25) indicating that K-region arene oxides of large polycyclic aromatic hydrocarbons are substrates for glutathione-S-epoxide transferase in rat liver, these studies have not been accompanied by adequate controls to substantiate that the reactions are enzymatic rather than spontaneous chemical interaction. Whether the high tissue levels of glutathione and glutathione-S-epoxide transferase activities in vivo offset the decreased reactivity remains to be established. Finally, the very high rate of nonenzymatic reaction between glutathione and cyclohexene episulfide suggests the use of the episulfide as a new pharmacological tool for the depletion of glutathione. Recent studies on the structures of the mercapturic acid precursors formed from glutathione and arene oxides have indicated that some of the previous structural assignments are in error (26).
450
HAYAKAWA
A number of substituted styrene oxides were examined (Tables I and II) in an attempt to establish steric and electronic factors capable of accelerating or retarding the reaction. Substitution of various groups on the phenyl ring produced poorer substrates but no clear trends were evident. Substitution on the oxirane ring was more informative. Methyl or phenyl groups at C-7, the point of attachment for the aromatic ring in styrene oxide, produced very poor substrates. When methyl was substituted at C-8, the truns isomer showed higher activity than styrene oxide while the cis isomer was rather poor. Substitution by two methyl groups produced poor substrates, especially when one of the methyl groups was at C-7. These tendencies provide an interesting contrast to the specificity of epoxide hydrase (9) where two substituents at one carbon of the oxirane ring produce a better substrate than cis-1,2-disubstitution. Thus, the two enzymes appear to have complementary activity. Octene 1,2-oxide is one of the best substrates for the hydrase and is a very poor substrate for the transferase. The nitrogen and sulfur analogs of styrene oxide which were examined were very poor substrates. It was considered possible that phenyl aziridine could function as a potent inhibitor for an acid catalyzed pathway by the enzyme (9, 27, 28) but this was not the case, as shown in Table III. Potent inhibitors for conjugation of styrene and naphthalene oxides were not found. The enzyme preparation from sheep liver (8) has a much broader spectrum of reactivity than the preparation from rat liver (6). 1,2-Disubstituted oxiranes, ethyl 2,3-epoxybutyrate, trans-stilbene oxide, cyclohexene oxide, and several arene oxides are quite active with the enzyme from sheep but were reported to be inactive in the preparation from the rat (6). In contrast, glycidaldehyde was found to be inactive with the present preparation while it was moderately active with the enzyme from the rat. The sheep epoxide transferase was not checked to determine whether it could displace leaving groups at carbon as was the case for the epoxide transferase from the rat (7).
ET
AL.
Very recently Jakoby and his colleagues purified to homogeneity and characterized a series of glutathione transferase from rat liver (29-31). ACKNOWLEDGMENTS The authors express their appreciation to Drs. P. Dansette (National Institutes of Health), R. G. Harvey (University of Chicago), and G. A. Berchtold (Massachusetts Institute of Technology) for kindly supplying several of the substrates used in these studies. REFERENCES 1. For examples see “Handbook of Experimental Pharmacology, Vol. 28/2, Concepts in Biochemical Parmacology, Part 2” (B. B. Brodie and J. R. Gillette, eds.). Springer-Verlag, New York, 1971. 2. JERINA, D. M., DALY, J. W., WITKOP, B., ZALT% MAN-NIRENBERG, P., AND UDENFRIEND, S. (1970) Biochemistry 9, 147, and references therein. 3. For a current review see JERINA, D. M., AND DALY, J. W. (1974) Science 185, 573. 4. BOYLAND, E., AND CHASSEAUD, L. F. (1969) Advances in Enzymol. 32, 173. 5. CHASSEAUD, L. F. (1974) Drug Metab. Rev. 2, 185. 6. FJELLSTEDT, T. A., ALLEN, R. H., DUNCAN, B. K., AND JAKOBY, W. B. (1973) J. Biol. Chem. 248, 3702. 7. PABST, M. J., HABIG, W. S., AND JAKOBY, W. B. (1973) Biochem. Biophys. Res. Commun. 52, 1123. 8. HAYAKAWA, T., LEMAHIEU, R. A., AND UDENFRIEND, S. (1974) Arch. Biochem. Biophys. 162,223. 9. OESCH, F., KAUBISCH, N., JERINA, D. M., AND DALY, J. W. (1971) Biochemistry 10, 4858. 10. OESCH, F., JERINA, D. M., AND DALY, J. W. (1971) Biochim. Biophys. Acta 227, 685. 11. DANSETTE, P. M., YAGI, H., JERINA, D. M., DALY, J. W., LEVIN, W., Lu, A. Y. H., CONNEY, A., AND KUNTZMAN, R. (1974) Arch. Biochem. Biophys. 164,511. 12. JERINA, D. M., DALY, J. W., WITKOP, B., ZALTZMAN-NIRENBERG, P., AND UDENFRIEND, S. (1968) Arch. Biochem. Biophys. 128, 176. 13. YAGI, H., AND JERINA, D. M. (1975) J. Amer. Chem. Sot. 97, 3185. 14. KAUBISCH, N., DALY, J. W., AND JERINA D. M. (1972) Biochemistry 11, 3080. 15. DEMARINIS, R. M., FILER, C. N., WARASZKIEWICZ, S. M., AND BERCHTOLD, G. A. (1974) J. Amer. Chem. Sot. 96, 1193. 16. DANSETTE, P. M., AND JERINA, D. M. (1974) J. Amer. Chem. Sot. 96, 1224. 17. GOH, S. H., AND HARVEY, R. G. (1973) J. Amer.
SPECIFICITY
OF
GSH-S-EPOXIDE
Chem. Sac. 95, 242. 18. MILLER, J. A.(1970) Cancer Res. 30, 559. 19. JERINA, D. M., YAGI, H., AND DALY, J. W. (1973) Heterocycles 1, 267. 20. OESCH, F., JERINA, D. M., AND DALY, J. W. (1971) Arch. Biochem. Biophys. 144, 253. 21. JEFFREY, A. M., YEH, H. J. C., JERINA, D. M., DEMARINIS, R. M., FOSTER, C. H., PICCOLO, D. E., AND BERCHTOLD, G. A. (1974) J. Amer. Chem. Sot. 96, 6929. 22. RUBEN, D. M., AND BRUICE, T. C. (1974) J. Chem. Sot. Chem. Commun., 113. 23. KASPEREK, G. J., BRUICE, T. C., YAGI, H., AND JERINA, D. M. (1972) J. Chem. Sot. Chem. Commun., 784. 24. JOLLOW, D. J., MITCHELL, J. R., ZAMPAGLIONE, N., AND GILLETTE, J. R. (1974) Pharmacology,
TRANSFERASE
451
11,151. 25. BOYLAND, E., AND SIMS, P. (1965) Biochem. J. 95, 788; 97, 7 (1965) ibid. 26. JEFFREY, A. M., AND JERINA, D. M. (1975) J. Amer. Chem. Sot. 97, 4427. 27. COREY, E. J., KANG, L. AND JAUTELAT, M. J. (1968) J. Amer. Chem. Sot., 90, 2724. 28. COREY, E. J., DEMONTELLANO, ORTIZ, LINK, P. R., AND DEAN, P. D. (1967) J. Amer. Chem. sot. 89, 2797. 29. PABST, M. J., HABIG, W. H., AND JAKOBY, W. B. (1974) J. BioZ. Chem, 249, 7140. 30. HABIG, W. H., PABST, M. J., FLEISCHNER, G., GATMAITAN, Z., ARIAS, I. M., AND JAKOBY, W. B., (1974) PNAS 71, 3879. 31. HABIG, W. H., PABST, M. J., AND JAKOBY, W. B. (1974) J. Biol. Chem. 249, 7130.
OF
BIOCHEMISTRY
Substrates
AND
BIOPHYSICS
and inhibitors
TAR0
170, 438-451 (19%)
of Hepatic Transferase
HAYAKAWA’
Roche Institute of Molecular
HARUHIKO Laboratory
YAGI
AND
SIDNEY
Glutathione-S-Epoxide
UDENFRIEND*
Biology, Nutley, New Jersey 07110 AND
DONALD
M. JERINA’
of Chemistry, National Institute of Arthritis, Metabolism and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014 Received February 19, 1975
The specific activities of glutathionea-epoxide transferase present in the 100,OOOg supernatant from sheep liver and a highly purified preparation of this enzyme have been compared with a variety of epoxides as substrates. Purification factors of 30- to 60-fold were observed for nearly 50 simple epoxides and arene oxides. The similarity in purification factors suggests either that a single enzyme with activity toward simple epoxides and arene oxides has been purified or that several related enzymes have been copuriiied. Comparison of specific activities for benzene oxides, naphthalene oxide, phenanthrene oxides, and arene oxides of benzoralpyrene and dimethylbenzaanthracene has established that (i) benzene oxides are poor substrates unless strong electron withdrawing substituenta are present, (ii) naphthalene oxide is a good substrate, and (iii) increasing the size of polycyclic arene oxides causes a steady decrease in activity. Reactivity toward simple epoxides was compared to that for epoxide hydrase. The transferase was not readily inhibited by any of the compounds which were used and showed a strict requirement for glutathione.
The principal route of oxidative detoxication for drugs, pesticides, and other nonpolar environmental agents in mammals is via the P-450 monoxygenases in liver (1). For aromatic substrates such as naphthalene (2) and higher polycyclic aromatic hydrocarbons (3), arene oxides have been demonstrated as obligatory intermediates on the pathways to phenols, dihydrodiols, and mercapturic acid precursors. In addition, olefins are converted to epoxides by the P-450 system. The principal reactions of arene oxides in the cell consist of nonenzymatic isomerization to phenols, hydration to dihydrodiols by the enzyme epoxide hydrase, conjugation with glutathione by the enzyme glutathioneS-epoxide trans’ Present address: Department of Biochemistry, School of Dentistry, Aichi-Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya 464, Japan. * To whom reprint requests should be addressed. 436 Copyright 0 1975 by Academic Press, Inc. All rights
of reproduction
in any
form
reserved.
ferase, and binding to macromolecular cell constituents. As this last reaction has been invoked as a mechanism by which the cytotoxic, mutagenic, and carcinogenic properties of many hydrocarbons can be explained (3), the potential protective roles of epoxide hydrase and glutathioneS-epoxide transferase assume special importance. A wide variety of substrates of glutathione transferase activity are known; in each case displacements of good leaving groups occur at a carbon atom. Simple epoxides and arene oxides are opened, addition occurs to double bonds, and coupling takes place at metabolically activated centers (4, 5). The actual number of different transferases involved in these reactions and the cross-specificity for these enzymes toward common substrates is unknown. A homogeneous epoxide transferase was iso-
SPECIFICITY
OF
GSHS-EPOXIDE
lated from rat liver based on its ability to use 1,2-epoxy-3-(4-nitrophenoxy) propane as substrate (6). With this highly purified rat enzyme a number of monosubstituted ethylene oxides were found to have 8-114% relative activity compared with the substrate used for purification. In a subsequent study ,(7), three additional glutathione transferases were isolated in homogeneous form from rat liver. 3,4-Dichloronitrobenzene, methyl iodide, and menaphthy1 sulfate were used as substrates in these purifications. Substantial cross-reactivity for various substrates was found for the four preparations despite the fact that the first was isolated for its ability to open epoxides while the latter three catalyzed displacement reactions. Development of a sensitive radiometric assay (8) for glutathione-S-epoxide transferase activity with naphthalene 1,2-oxide and [35S]glutathione as cosubstrates has made possible the isolation of a purified form of this enzyme from sheep liver. The present paper describes the substrate specificity and the effects of some inhibitors of this enzyme in the 100,OOOg supernatant from sheep liver and in a purified preparation from the same source. MATERIALS
AND
METHODS
Substrates and inhibitors. Simple epoxides and epoxide analogs used in Tables I-III were either obtained commercially or were prepared by standard methods from available precursors. Many of these had been employed previously in an examination of the substrate specificity of epoxide hydrase (9). Radioactive [7-3H]styrene oxide (10) and [23H1naphthalene 1,2-oxide (11) were prepared as described and were >99% radiochemically pure. The following arene oxides were synthesized directly by the methods indicated or by slight modifications of the procedures described: benzene oxide (121, naphthalene 1,2-oxide (131, l-methyl, 3-methyl, and 4methylbenzene 1,2-oxide (141, 1-methylnaphthalene 1,2-oxide (131, 2-methylnaphthalene 1,2-oxide (141, 4-carbo-t-butoxybenzene l,2-oxide (15), phenanthrene 1,2- and 3,4-oxides (13), phenenthrene 9,10oxide (161, 7,12-dimethylbenzo[a]anthracene 5,6-0xide (16, 171, benzolalpyrene 7,8- and 9,10-oxides (131, and benzo [alpyrene 4,5-oxide (16, 17). Studies leading to the synthesis of 3-chloroand 4-chlorobenzene 1,2-oxide and to 3-carbo-t-butoxybenzene 1,2-oxide have not yet been submitted. All of the above epoxides, arene oxides, and substrate analogs were judged to be >95% pure prior to use, by thin-layer
TRANSFERASE
439
chromatography, nuclear magnetic resonance, and mass spectrometry. Most substrates, inhibitors, and substrate analogs were added as 0.3 M solutions in acetone or as previously described (81. For K-region arene oxides, dimethyl sulfoxide was employed as solvent. In all cases, the quantity of solvent added to the incubations had no effect on the rates of reaction Enzyme preparations. Incubations were conducted either with the 100,OOOg supernatant fraction from sheep liver or with the Sephadex G-75 column fraction 40-fold purified over activity present in supernatant. Details were reported in a previous publication (8). Assay procedure. Most of the assays for transferase activity were based on the sensitive radioisotope procedure (8) developed to measure conjugate formation between naphthalene 1,2-oxide and 135Slglutathione by simply replacing naphthalene oxide with other compounds. Incubation mixtures for the standard assay consisted of 0.5 pmole of [?S]glutathione (4-5 x lo5 cpm), 100 pmoles of pyrophosphate buffer (pH 8.01, the indicated amounts of substrate and enzyme preparation, and sufficient water to make a total volume of 1 ml. Products were analyzed radiometrically after paper chromatography, as previously described (8). Results are corrected for nonenzymatic conversion, and the R, values for the various conjugates are given in the tables. Conversions to products have not been corrected for recovery which is about 50% for naphthalene oxide (8). Recovery for the conjugates produced here are probably quite comparable since the peptide residue is the principle functionality common to all the products. With all substrates the amount of product formed in the presence of enzyme ranged from two to ten times that formed nonenzymatically with most cases being five to eight times blank. Blanks consisting of incubations with boiled enzyme or no added enzyme were no significantly different. The substantial nonenzymatic rates for reaction between substrates and glutathione limits the sensitivity of the assay. When radioactive oxides were used as substrates with nonradioactive glutathione, incubation mixtures consisted of the indicated amounts of 2-3Hnaphthalene 1,2-oxide (3 x 10’ cpmO.5 pmol) or [73Hlstyrene oxide (4-5 x lo5 cpml.2 pmol), 5 pmol of glutathione, 100 pmol of pyrophosphate buffer (pH 8.01, enzyme as indicated, and sufficient water to make a total volume of 1 ml. Unless otherwise indicated, incubations were initiated by addition of oxide and run for 5 min at 22°C. Termination and product analysis were as described (8). RESULTS
The data presented in Tables I and II compare the ability of nearly 50 epoxides,
440
HAYAKAWA TABLE
COMPARISON
OF ACTIVITY
OF
Substrate
CRUDE
AL..
I
AND PURIFIED SHEEP LIVER GLUTATHIONE-S-EP~XIDE VARIOUS SUBSTRATES Initial concn bM)
2
Specific
Crude Purified extract” enzyme” (nmol/mg/min)
Purification factor
R, value of product
4.5
188
42
3.0
7.2
363
50
0.6
14.8
504
34
0.31
34
0.45
56
0.22
0
Not detectable
0.6 (pH
activity
TRANSFERASE
0.6
OHC--Cc-hi,
6.1
0.30
Not detectable
207
6.5)
HOW*-CclH2 0
3.0
Cl-W-CC&,
3.0
0
ET
Not detectable
4.6
Not detectable
259
ON
SPECIFICITY
OF
GSHS-EPOXIDE
TABLE Substrate
Initial concn h-M
02&-) 0-CH,-C~=?H, O
CH 3 -,6%-C,
I-Continued Specific
activity
Crude Purified extract” enzymea (nmol/mg/min)
3.0
4.0
0.6
6.4
3.0
2.4
3.0
2.9
164
296
Purification factor
RI value of product
41
0.34
46
0.20
32
0.52
36
0.26
2C 2 H 5
0
0
441
TRANSFERASE
CH/-‘CH
3
0
77
o-
0
0
3.0
/ 0 \
0
Not detectable
104
Not detectable
3.0
0.37
22.0
60
0.37
3.0
0.46
25.9
56
0.43
TABLE Substrate
0
CH3
/ 0
Specific
activity
Crude Purified extract” enzyme” (nmol/mg/min)
3.0
Not detectable
Not detectabled
3.0
Not detectable
Not detectabled
\
yc
I-Continued
Initial c0ncn bnM)
Purification factor
RI value
of product
Cl /
3.0
2.8
140
50
0.29
3.0
3.4
172
51
0.31
5.0
219
44
0.43
14.0
556
40
0.43
3.0
2.2 5.6
101 183
46 33
0.44 0.55
3.0
3.8
178
47
0.56
0
6\
Cl / 0
-0 \
0.43
0 (pH
6.5)
(CH,),CO& 0
0.6
(PI-I 6.5)
CH3
442
TABLE Substrate
/ / a‘I/‘,
I-Continued
Initial concn (mhd
Specific
Purificpn
activity
Crude Purified extracta enzyme” (nmol/mg/min)
RI
value of product
1.5
5.6
219
39
0.39
1.5
5.5
200*
36
0.47
1.5
1.4
38.96
28
0.41
0.15
3.5
111
32
0.58
0.6
0.25'
9.Bb
39
0.39
0.6
0.02"
Not detectableb,
0.42
d
-
0
0.6
0.09b
>Ob. d
-
0.51
Assays were carried out with 700 pg of 100,OOOg supernatant or 11 /qg of Sephadex G-75 column preparation. Assay conditions are described in Methods. n Crude extract and purified enzyme indicate 100,OOOg supernatant and Sephadex G-75 column preparation respectively (see ref. 8). *Reaction mixtures contained 3 pmoles of %-glutathione (2 x 106cpm); 1 mg of 100,OOOg supematant or 21 @g of Sephadex G-75 column preparation as enzyme, and incubated for 20 min at 30”. Others are the same as standard assay system. ‘Two products were detected on chromatography. d These are the least stable substrates employed in this study. 443
TABLE
COMPARISON OF GLUTATHIONES-EPOXIDE
ACTIVITIES
AND RELATED
COMPOUNDS
Specific activity (nmol/mg/min)
Substrate
0”’
II
TRANSFE~USE
TOWARD I
SUBSTITUTED
Activity
STYRENE R, value of product
0 100
0.30
929
60
0.27
479
31
0.25
850
55
0.36
631
41
0.36
65
0.35
1538
0
O2N
OeN
0
fl
0
Er
“%y Cl
103
Not
6.7
-
detectable
4.4
68
1749
114
CH, 444
0.37
0.27
0.27
OXIDES
SPECIFICITY
OF GSHS-EPOXIDE TABLE
Substrate
II-Continued
Specific activity (nmol/mg/min)
% Activity
Rr value of product
22
337
26.9 H3C
445
TRANSFERASE
0.27
1.8
0.30
CH3
@.+QHx
28.0
0.29
1.8
H3C
221
d
14
73
s
0.36
0.57
4.7
Not detectable”
-
0.39b
22
0.26
-
-
Cc
/‘r: 0”
S
0
342
NH
0
Not detectable
Assays were conducted with 11 pg of purified enzyme (Sephadex initial concentration of substrates. For assay conditions see Methods a Cyclohexene episulfide was sufficiently reactive with glutathione difference in radioactivity of the major spot with or without enzyme. ’ The R, value of the major spot of the conjugate. Additionally, having Rf values of 0.53, 0.62, and 0.83.
G-75 column preparation) section. nonenzymatically that
there
three
were
radioactive
spots
using
3 mM were
observed
no
HAYAKAWA
ET AL.
TABLE EPOXIDE
ANALCJGS AND OCTENE ~&OXIDE NAPHTHALENE ~,%OXIDE
III
AS INHIBITORB OR i’-%STYRENE
Inhibitor
OF PURIFIED TRANSFERASE OXIDE AS SUBSTRATES”
Substrate (inhibitor [2-3HlNaphthalene 1,2-oxide (0.09 mM) Remaining activity (%)
none
100 (-1
2-3H-
concn in mM) [7-3Hl,S$~ieloxide Remaining 100
activity (%) (-)
0
0”
47 (0.9)
0
0 03
WITH
-
0
-
68 (0.03)
/ 61 (0.9)
46 (0.6)
80 (0.9)
65 (0.6)
93 (0.9)
102 (0.6)
NH
‘Substrates and inhibitors were all dissolved in acetone to yield 0.3 M solutions, except naphthalene 1,2oxide which was 10x diluted. Reaction mixtures contained 5 pmoles of nonradioactive glutathione; 100 pmoles of pyrophosphate buffer, pH 8.0; the indicated amounts of substrates and inhibitors and 11 pg of purified enzyme (Sephadex G-75 column preparation) in 1 ml total volume. Details of the assay conditions are described in Methods.
arene oxides, and analogs, in which oxygen has been replaced by sulfur or nitrogen, to act as substrates for a crude and a purified preparation of glutathione-S-epoxide transferase from sheep liver. The assay system employed (a), although sensitive, is sufficiently time consuming as to preclude obtaining true kinetic comparisons for all these substrates. Both naphthalene and styrene oxide have K, values of about 0.1 mM (8), the reactions studied here are therefore assumed to be under conditions which are saturating with regard to oxide substrate since initial concentrations of 3 mM were used in most cases. Most of these
reactions are probably linear over the 5min incubation period (8), and product recoveries are anticipated to be comparable. The question of substrate stability over the time course of the incubation is a serious one for unstable arene oxides that readily rearrange to phenols and will be discussed later. Half-lives of a number of arene oxides used here have been determined (3, 19). Extrapolation from these studies leads to the conclusion that the enzyme probably remains saturated throughout the course of the reactions with all the substrates which were used. The purification factor between crude
SPECIFICITY
OF
GSHS-EPOXIDE
100,OOOg supernatant and the purified enzyme are presented for 28 substrates in Table I. In general, most substrates show a 40- to 50-fold purification for the enzyme with extremes of 30- and 60-fold being observed. The range of purification is within a factor of 2: and may be considered comparable for all the substrates studied. Styrene and naphthalene oxides show competitive inhibition (Fig. 1) with each other in the 100,OOOg fraction. Styrene oxide proved to be the most effective epoxide substrate for the enzyme (Table I), and a specific requirement for glutathione was found. With 13Hlnaphthalene oxide as substrate, cysteine, P-mercaptoethanol, dithiothreitol, and coenzyme A, at a final concentration of 2 mM, gave no detectable product. Reactivity of a number of monosubstituted ethylene oxides, the substrate class which contains styrene oxide, was examined (Table I). When the substituent was formyl, hydroxymethyl, or epoxycyclohexyl (the carcinogen, 1-ethyleneoxy-3,4epoxycyclohexane) (18), no activity was detected. In contrast, ethylene oxides which had phenyl (styrene oxide), chloromethyl, trichloromethyl, or a nitrophenoxymethylene as the substituent were quite active. Octene 1,2-oxide (Table II) was relatively inactive. No apparent steric or electronic factors explain these trends. Two trans1,2-disubstituted ethylene oxides, ethyl butyrate - 2,3 - oxide and stilbene oxide, showed high and moderate activity. Several substituted benzene oxides and 1.0,
TRANSFERASE
447
related compounds were examined (Table I). Benzene oxide and 1-methylbenzene oxide showed about 5% of the activity of styrene oxide while 3- and 4-methylbenzene oxide were inactive. Formation of glutathione conjugates of the toluene ring has not been reported in Go. While instability of the substrate toward isomerization to phenols may, in part, explain the inactivity of the two latter compounds (19), this certainly is not the case for benzene oxide as evidenced by its time course (Fig. 2). Substitution of chlorine or carbo-t-butoxy groups at the 3- or 4-positions produced moderate to excellent substrates. In fact, 4-carbo-t-butoxybenzene oxide proved to be the best substrate in the entire study. For the substituted benzene oxides, there is a fairly strong indication that electron withdrawing groups greatly facilitate reaction. Cyclohexene oxide was found to be a moderately good substrate, about eight times as active as benzene oxide. The inactivity of 1 - ethyleneoxy - 3,4 - epoxycyclohexane, which is related to cyclohexene oxide, is even more surprising in light of the activity of cyclohexene oxide. The insecticide, chlordane epoxide, which is structurally related to cyclohexene oxide, showed no detectable product at a final concentration of 2.6 mM (data not shown). A number of arene oxides of polycyclic aromatic hydrocarbons were examined (Table Il. While naphthalene oxide is a good substrate, methyl substitution at C-l or C2 significantly decreased its activity. Two 0.2r
-2 .E
A
,E, -10
I
0
‘/[3H.Nophtholene-I,
IO Z-oxide]
20 (mM-1)
‘+H-St yrene oxide] (mM-1) B Fig. 1. Lineweaver-Burk plots of glutathione-S-epoxide transferase from sheep liver 100,OOOg supernatant for both 12-3Hlnaphthalene 1,2-oxide (A) without styrene oxide (-0-l or with 0.9 mM styrene oxide as an inhibitor (-0 -1, and [7-3Hlstyrene oxide (B) without naphthalene 1,2-oxide (-0-l or with 0.03 mM naphthalene 1,2-oxide as an inhibitor (-0-l. Incubations were with 11 pg of purified enzyme in a final volume of 1.0 ml.
448
HAYAKAWA
5ooor--yv-
0
L ._LI
-I, 5
TIME
(min)
FIG. 2. Time course of the formation of glutathione conjugate with benzene oxide. Assays were carried out with 32 pg of protein from the Sephadex G 75 column preparation in the presence of 0.6 pmole of benzene oxide. Counts were net values after subtracting the counts for non-enzymatic conjugate formation. Details of the assay are described in Methods.
products were detected from l-methylnaphthalene l&oxide. Both compounds, on treatment with 1 N HCl for 15 min at 6o”C, underwent dehydration to conjugates which migrated more rapidly on chromatography. For the three possible arene oxides of phenanthrene, the 9,10-(K-region) and 1,Zoxides had about 60% of the activity of naphthalene oxide while the most sterically hindered 3,4-oxide was relatively inactive and comparable to benzene oxide. The K-region oxide of 7,12-dimethylbenz[ulanthracene had half the reactivity of the corresponding phenanthrene oxide while the K-region oxide of benzo[ulpyrene was practically inactive. Non-K-region oxides of benzo[a]pyrene at the 7,8- and 9,10positions showed practically no detectable activity. Stability of substrate toward isomerization to phenols cannot explains these results since high activity for the more stable K-region oxides (19) would be expected on this basis. The simplest conclusion to be drawn is that large molecules are poor substrates. The effect of varying the substituents on styrene oxide was also examined (Table II). Any monosubstitution on the phenyl ring was found to be detrimental to activity. Substitution of halogen or a nitro group at the meta orpara positions showed no clear trend for the decrease in activity. A bulky phenyl group at the para position
ET AL.
decreased activity E-fold. Substitution of methyl or phenyl at C-7 produced poor substrates. However, substitution of a methyl group at C-8, which is truns to the phenyl ring, produced a substrate more active than styrene oxide. In contrast, cis substitution of a methyl group at C-8 decreased activity five-fold. The three possible dimethylstyrene oxides, with both methyl groups on the oxirane ring, were all of low activity. Isoelectronic substitutions of sulfur and nitrogen in styrene oxide were also examined (Table II). The nitrogen analog gave no product while substitution by sulfur showed 20% of the activity of styrene oxide. Cyclohexene episulfide was found to be an excellent scavenger for glutathione, reacting considerably without enzyme. However, enzyme did not augment this activity. The effect of potential inhibitors on transferase activity toward radioactive styrene and naphthalene oxides was examined (Table III). Naphthalene oxide was the most effective inhibitor of the reaction with styrene oxide, producing 30% inhibition at l/20 of the substrate concentration. Among the sulfur and nitrogen analogs and octene oxide, cyclohexene episulfide produced 54% inhibition at equimolar concentration with styrene oxide. Reaction of naphthalene oxide was difficult to inhibit by any compound. Styrene oxide at ten times the substrate concentration was the best inhibitor, but caused only 53% inhibition. DISCUSSION
Substrate specificity of glutathione-Sepoxide transferase activity has been examined in the 100,OOOgsupernatant from sheep liver and in a purified (8) preparation of this enzyme. Like the epoxide transferase preparation from rat (61, a strict requirement for glutathione has been observed. Comparison of activity in the crude supernatant to that in the purified enzyme for a wide variety of simple epoxides and arene oxides shows that for all compounds, the degree of purification ranged for 28- to 60-fold (Table I). The similarity in purification factors suggests that individual epox-
SPECIFICITY
OF
GSHS-EPOXIDE
ide transferase activities toward specific structural types have not been significantly lost on purification. Comparable ranges of purification factors for simple epoxides and for arene oxides indicate that these activities copurify and suggest that either a single enzyme or a clearly related group of enzymes is involved. A similar finding has been observed for epoxide hydrase, purified from the liver of guinea pigs, with the exception that a specific activity for benzene oxide was preferentially lost in the hydrase preparation (9, 26). Competitive inhibition between styrene and naphthalene oxides (Fig. 1) further support the view that the epoxide transferase(s) from sheep liver does not distinguish between simple epoxides and arene oxides. The chemical nature of the adducts formed from the epoxide substrates used in this study and glutathione have not been examined. For the simple epoxides, direct tram opening of the oxirane ring is assumed to occur. With unsymmetrical substrates, two products are possible although not necessarily separable by the chromatographic procedure employed. Two major products were detected when l-methylnaphthalene 1,2-oxide was used as substrate. Arene oxides with isolated double bonds, such as benzene oxides and non-Kregion arene oxides of polycyclic hydrocarbons, undergo nucleophilic addition reactions at sites remote from the oxirane ring (21). However, sulfur nucleophiles always add to the oxirane ring of arene oxides in a trans-1,2 fashion (21). Thus, the two separable products for 1-methylnaphthalene 1,2-oxide probably arise by direct trans opening resulting from attack by sulfur at C-l and at C-2. A comparison of the extent of reaction with glutathione in the absence of enzyme (data not shown) is of interest from both a chemical and a pharmacological point of view. The very high nonenzymatic reactivity of 3- and especially 4-carbo-t-butoxybenzene oxides suggests that direct nucleophilic attack by thiols occurs on arene oxides with strong electron withdrawing groups. This is in contrast to a study of the reaction of benzene oxide with thiols which indicated that trapping of intermediate car-
TRANSFERASE
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bonium ions from the oxide was the major pathway (22). Thus, for deactivated aromatic rings, which form stable arene oxides (23), nonenzymatic reaction with glutathione could play an important role in preventing the binding of arene oxides to tissue constituents. Thus, for deactivated aromatic rings, which form stable arene oxides (23), nonenzymatic reaction with glutathione could play an important role in preventing the binding of arene oxides to tissue constituents. Transferase activity toward chloro- and carbo-t-butoxy substituted benzene oxides was moderate to excellent, with 4-carbo-t-butoxybenzene oxide being the best substrate found in this study. Similarity in transferase activity toward 3- and 4-chlorobenzene oxide suggests that selective detoxication of these arene oxides by glutathioneS-epoxide transferase does not occur. Rates of both enzymatic and nonenzymatic conjugation of the arene oxides of the polycyclic hydrocarbons steadily decreased as the size of the ring systems increased. Thus, a protective role for glutathione against polycyclic hydrocarbon induced carcinogenesis via arene oxides is not evident from these studies, in marked contrast to the clear protective role for glutathione in the hepatic necrosis induced by halobenzenes (24). Although there are reports (25) indicating that K-region arene oxides of large polycyclic aromatic hydrocarbons are substrates for glutathione-S-epoxide transferase in rat liver, these studies have not been accompanied by adequate controls to substantiate that the reactions are enzymatic rather than spontaneous chemical interaction. Whether the high tissue levels of glutathione and glutathione-S-epoxide transferase activities in vivo offset the decreased reactivity remains to be established. Finally, the very high rate of nonenzymatic reaction between glutathione and cyclohexene episulfide suggests the use of the episulfide as a new pharmacological tool for the depletion of glutathione. Recent studies on the structures of the mercapturic acid precursors formed from glutathione and arene oxides have indicated that some of the previous structural assignments are in error (26).
450
HAYAKAWA
A number of substituted styrene oxides were examined (Tables I and II) in an attempt to establish steric and electronic factors capable of accelerating or retarding the reaction. Substitution of various groups on the phenyl ring produced poorer substrates but no clear trends were evident. Substitution on the oxirane ring was more informative. Methyl or phenyl groups at C-7, the point of attachment for the aromatic ring in styrene oxide, produced very poor substrates. When methyl was substituted at C-8, the truns isomer showed higher activity than styrene oxide while the cis isomer was rather poor. Substitution by two methyl groups produced poor substrates, especially when one of the methyl groups was at C-7. These tendencies provide an interesting contrast to the specificity of epoxide hydrase (9) where two substituents at one carbon of the oxirane ring produce a better substrate than cis-1,2-disubstitution. Thus, the two enzymes appear to have complementary activity. Octene 1,2-oxide is one of the best substrates for the hydrase and is a very poor substrate for the transferase. The nitrogen and sulfur analogs of styrene oxide which were examined were very poor substrates. It was considered possible that phenyl aziridine could function as a potent inhibitor for an acid catalyzed pathway by the enzyme (9, 27, 28) but this was not the case, as shown in Table III. Potent inhibitors for conjugation of styrene and naphthalene oxides were not found. The enzyme preparation from sheep liver (8) has a much broader spectrum of reactivity than the preparation from rat liver (6). 1,2-Disubstituted oxiranes, ethyl 2,3-epoxybutyrate, trans-stilbene oxide, cyclohexene oxide, and several arene oxides are quite active with the enzyme from sheep but were reported to be inactive in the preparation from the rat (6). In contrast, glycidaldehyde was found to be inactive with the present preparation while it was moderately active with the enzyme from the rat. The sheep epoxide transferase was not checked to determine whether it could displace leaving groups at carbon as was the case for the epoxide transferase from the rat (7).
ET
AL.
Very recently Jakoby and his colleagues purified to homogeneity and characterized a series of glutathione transferase from rat liver (29-31). ACKNOWLEDGMENTS The authors express their appreciation to Drs. P. Dansette (National Institutes of Health), R. G. Harvey (University of Chicago), and G. A. Berchtold (Massachusetts Institute of Technology) for kindly supplying several of the substrates used in these studies. REFERENCES 1. For examples see “Handbook of Experimental Pharmacology, Vol. 28/2, Concepts in Biochemical Parmacology, Part 2” (B. B. Brodie and J. R. Gillette, eds.). Springer-Verlag, New York, 1971. 2. JERINA, D. M., DALY, J. W., WITKOP, B., ZALT% MAN-NIRENBERG, P., AND UDENFRIEND, S. (1970) Biochemistry 9, 147, and references therein. 3. For a current review see JERINA, D. M., AND DALY, J. W. (1974) Science 185, 573. 4. BOYLAND, E., AND CHASSEAUD, L. F. (1969) Advances in Enzymol. 32, 173. 5. CHASSEAUD, L. F. (1974) Drug Metab. Rev. 2, 185. 6. FJELLSTEDT, T. A., ALLEN, R. H., DUNCAN, B. K., AND JAKOBY, W. B. (1973) J. Biol. Chem. 248, 3702. 7. PABST, M. J., HABIG, W. S., AND JAKOBY, W. B. (1973) Biochem. Biophys. Res. Commun. 52, 1123. 8. HAYAKAWA, T., LEMAHIEU, R. A., AND UDENFRIEND, S. (1974) Arch. Biochem. Biophys. 162,223. 9. OESCH, F., KAUBISCH, N., JERINA, D. M., AND DALY, J. W. (1971) Biochemistry 10, 4858. 10. OESCH, F., JERINA, D. M., AND DALY, J. W. (1971) Biochim. Biophys. Acta 227, 685. 11. DANSETTE, P. M., YAGI, H., JERINA, D. M., DALY, J. W., LEVIN, W., Lu, A. Y. H., CONNEY, A., AND KUNTZMAN, R. (1974) Arch. Biochem. Biophys. 164,511. 12. JERINA, D. M., DALY, J. W., WITKOP, B., ZALTZMAN-NIRENBERG, P., AND UDENFRIEND, S. (1968) Arch. Biochem. Biophys. 128, 176. 13. YAGI, H., AND JERINA, D. M. (1975) J. Amer. Chem. Sot. 97, 3185. 14. KAUBISCH, N., DALY, J. W., AND JERINA D. M. (1972) Biochemistry 11, 3080. 15. DEMARINIS, R. M., FILER, C. N., WARASZKIEWICZ, S. M., AND BERCHTOLD, G. A. (1974) J. Amer. Chem. Sot. 96, 1193. 16. DANSETTE, P. M., AND JERINA, D. M. (1974) J. Amer. Chem. Sot. 96, 1224. 17. GOH, S. H., AND HARVEY, R. G. (1973) J. Amer.
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Chem. Sac. 95, 242. 18. MILLER, J. A.(1970) Cancer Res. 30, 559. 19. JERINA, D. M., YAGI, H., AND DALY, J. W. (1973) Heterocycles 1, 267. 20. OESCH, F., JERINA, D. M., AND DALY, J. W. (1971) Arch. Biochem. Biophys. 144, 253. 21. JEFFREY, A. M., YEH, H. J. C., JERINA, D. M., DEMARINIS, R. M., FOSTER, C. H., PICCOLO, D. E., AND BERCHTOLD, G. A. (1974) J. Amer. Chem. Sot. 96, 6929. 22. RUBEN, D. M., AND BRUICE, T. C. (1974) J. Chem. Sot. Chem. Commun., 113. 23. KASPEREK, G. J., BRUICE, T. C., YAGI, H., AND JERINA, D. M. (1972) J. Chem. Sot. Chem. Commun., 784. 24. JOLLOW, D. J., MITCHELL, J. R., ZAMPAGLIONE, N., AND GILLETTE, J. R. (1974) Pharmacology,
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11,151. 25. BOYLAND, E., AND SIMS, P. (1965) Biochem. J. 95, 788; 97, 7 (1965) ibid. 26. JEFFREY, A. M., AND JERINA, D. M. (1975) J. Amer. Chem. Sot. 97, 4427. 27. COREY, E. J., KANG, L. AND JAUTELAT, M. J. (1968) J. Amer. Chem. Sot., 90, 2724. 28. COREY, E. J., DEMONTELLANO, ORTIZ, LINK, P. R., AND DEAN, P. D. (1967) J. Amer. Chem. sot. 89, 2797. 29. PABST, M. J., HABIG, W. H., AND JAKOBY, W. B. (1974) J. BioZ. Chem, 249, 7140. 30. HABIG, W. H., PABST, M. J., FLEISCHNER, G., GATMAITAN, Z., ARIAS, I. M., AND JAKOBY, W. B., (1974) PNAS 71, 3879. 31. HABIG, W. H., PABST, M. J., AND JAKOBY, W. B. (1974) J. Biol. Chem. 249, 7130.
