- Email: [email protected]
The inhibition of HEOM epoxide hydrase in mammalian liver microsomes and insect pupal homogenates
PESTICIDE
BIOCHEMISTRY
The
AND
PHYSIOLOGY
6, 132-141
(1976)
Inhibition of HEOM Epoxide Liver Microsomes and Insect A. C. C. CRAVEN,
Department
of Physiology
Hydrase in Mammalian Pupal Homogenates
G. T. BROOKS,’ AND C. H. WALKER and Biochemistry, Reading, United
Reading Kingdom
University,
Whiteknights,
Received March 24, 1975; accepted August 18, 1975 A range of compounds were tested as inhibitors of the enzyme epoxide hydrase, using a cyclodiene epoxide (HEOM) as substrate. Rat and rabbit liver microsomes and pupal homogenates of the blowfly (Calliphora erythroeephula) and the yellow mealworm (Tenebrio molitor) were compared as sources of the enzyme. Only minor differences were found between the four enzyme preparations, when considering ISOvalues and percentage inhibition at standard concentration. The simple epoxide l,l,l-trichloropropse-2,3-epoxide and two glycidyl ethers p-nitrophenyl glycidyl ether and p-ethylphenyl glycidyl ether tended to have lower ZSO values (1.8X1O-B to 8.OX1O-6 M) than triphenyl phosphate and SKF 5258 (4.5X10-6 to 1.4X1O-4 M). Triphenyl phosphate and SKF 5258 were competitive inhibitors for both the rat and Tenebrio enzymes. The only clear difference found between these two epoxide hydrase preparations was with respect to their inhibition by 1,&l-trichloropropane-2,3-epoxide, which was an uncompetitive inhibitor with the rat enzyme, but showed kinetics of mixed inhibition with the insect preparation. INTRODUCTION
Enzymes that cleave epoxide rings by hydration to give mainly the corresponding trans-diols (14) are of current interest in relation to the mechanism of carcinogenesis mediated by chemicals (5, S), the variable environmental stability of certain chlorinated epoxides used as insecticides and the deactivation of insect P-9, juvenile hormones that contain epoxide rings (10-13). Inhibitors of these epoxide hydrases (EC 4.2.1.63) are of value for demonstrating the intermediate formation of labile epoxides during the oxidative metabolism of various olefines (4, 6, 14, 15) and for exploring the background to species differences in insecticide and juvenile hormone 1 Agricultural Research Council, Unit of Invertebrate Chemistry and Physiology, University of Sussex, Brighton, Sussex, England.
metabolism (11). Information about such species variations is important in the design of selective insecticides. The cyclodiene epoxide HEOM2 (see Fig. 1) has proved to be a useful substrate for the investigation of epoxide hydrase activity as it is readily metabolized to a single product and because both substrate and product can be accurately estimated at low concentration (7-9, 11, 12, 16). We report here on the effects of several inhibitors on the hydrase activity towards this substrate in tissue preparations from two mammalian species and two insect species. 2 Abbreviations used : HEOM, 1,2 ,3,4 t9 t9-hexachloro-6,7-epoxy-1,4,4a t5 , 6 77,8 7Sa-octahydro-1,4methanonaphthalene (See Fig. 1); TLC, thin-layer chromatography; GLC, gas-liquid chromatography; TMS, trimethyl silyl ether. Roman numerals are used in the text to indicate inhibitors (for full structure of all inhibitors see Table 1). 132
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
INHIHITION
OF
XEOM Ttii-DIOL
XEOM
FIG.
1. Enzymatic
MATERIALS
MAMMALIAN
hydration
AND
of HEOM.
METHODS
Animals. Wistar rats (200-300 g) were obtained from Charles River, Manston, Kent, while Old English rabbits (2-2.5 kg) were obtained from Goodchild Farms, Crawley, Sussex. All mammals were mature males, in good condition. White prepupae of the blowfly (Calliphora erythrocephala) and O-l hr old pupae of the yellow mealworm (Tenebrio molitor) were used as the sources of insect epoxide hydrase. Substrate. The cyclodiene epoxide HEOM was synthesized as described previ,ously (17). ‘*C-labeled HEOM was prepared from [14C]hexachlorocyclopentadiene supplied by The Radiochemical Centre, Amersham, Bucks. Because of the small quantities of the labeled epoxide involved, final purification of the [14C]HEOM was by TLC on Kieselgel G. TLC separations were repeated using different solvent systems (0.5 and 1% acetone in hexane) until constant specific activity was achieved, and a single peak was found on examination by GLC using two different stationary phases, SE52 and OV225 (see later under Extraction and analytical techniques). The HEOM substrate was employed as a 2 mg ml-’ ethanolic solution, either unlabeled, or labeled with a specific activity of 0.1 &Ji mg--’ . Enzyme preparations. In the case of the rat and the rabbit, washed liver microsomes were used as the source of epoxide hydrase. Animals were killed by breaking the neck, and the livers were rapidly removed, washed, and placed on ice. After removal of connective tissue and the gall bladder (present in rabbit, but not rat) the livers were thinly sliced and homogenized
AND
INSECT
EPOXIDE
HTDRASE
1X3
in isotonic (1.15%) KC1 (3 ml/g of liver), for 2 min at 0-4°C in a stainless steel and glass homogenizer (MSE Scient,ific Instruments, Crawley, Sussex). Washed microsomes were prepared from the homogenates as described elsewhere (9) and then resuspended in isotonic KCl. The concentrations of the microsomal preparations, with respect to weight of liver represent,ed, were 4 mg ml-l and 40 mg ml-’ for t,he rabbit and rat, respectively. Homogenates (lOyo w/v) in KH,POI/ NaOH buffer (0.1 M, pH 8.5) were prrpared from Calliphora pupae 0-i hr old (1 g; 12-15 pupae) as described elsewhere (12) and used as such, since centrifugat,ion resulted in a substantial (ca. 50%) loss of epoxide hydrase activity. In the case of Tenebrio pupae, homogenates were made in the same way, but here, heavy particles were removed by centrifugation at 15001~ for 5 min without loss of activity, and the resulting supernatant was used as the source of epoxide hydrase. Total protein in the mammalian and insect preparations was estimated by the method of Lowry et al. (18). Protein yields were within the following ranges: Rat, 8--9 mg microsomal protein per gram of liver; Rabbit, 10-12 mg microsomal protein per gram of liver ; CaEZiphora, 12 mg protein per milliliter homogenate, Tenebrio, 8-Y mg protein per milliliter supernatant . A sample of unspecific carboxylesterase prepared from pig liver mierosomes (EC 3.1.1.1) was supplied by K. Krisch, Institute fiir Physiologische Chemie und Physikochemie, der UniversitBt,, Kiel, Federal Republic of Germany. Inhibitors. Compounds tested as inhibitors of epoxide hydrase were prepared or obtained as described previously (12) and were employed in ethanolic solution at st,andard concentrations, except for VIII, which was dissolved in acetone because of its low solubility in ethanol. Incubation procedures. In the case of the mammalian studies, reactions were carried out in Erlenmeyer flasks open to the air
134
CRAVEN,
BROOKS
and held in a metabolic shaker at 37°C for 30 min. The reaction mixture consisted of 4.5 ml buffered incubation medium pH 7.4 (9), 0.5 ml microsomal suspension ( = 20 mg liver for rat and 2 mg liver for rabbit) plus varying amounts of inhibitors and HEOM substrate. Components were added rapidly in the stated order, without any preincubation. The HEOM substrate was kept constant at 40 gg (20 ~1 of 2 mg ml-l solution) per incubat,ion flask, except with the doublereciprocal plots for the rat where a range of substrate concentrations was employed. Incubations with the insect preparations were carried out at 30°C in Erlenmeyer flasks, open to the air. The Calliphora homogenate (0.5 ml) or Tenebrio supernatant (0.5 ml) was added to the KH,POJ NaOH buffer (0.1 M, pH 5.5, 4.5 ml), followed by the inhibitor and HEOM substrate. The enzyme was preincubated (10 min) w&h each inhibitor before addition of HEOM, and incubation continued for 12 min after addition of the substrate, without agitation beyond that required at addition. The HEOM substrate was kept constant at 100 pg (50 ~1 of 2 mg ml-’ solution) per incubation flask, except with the double reciprocal plots for Tenebrio supernatant. All activity was lost from both the mammalian and insect preparations when enzyme plus buffered medium was heated for 5 min at 100°C before addition of HEOM. Ethanol and acetone, used for addition of substrate and inhibitors to the incubation medium, had no measurable effect on enzyme activity at the levels employed. Enzyme activity was linear during the respective incubation periods, permitting measurement of initial reaction velocity. Appropriate volumes of ethanol and/or acetone were included in control incubations and these produced consistent values for control enzyme activity. Values for activities of inhibited enzyme preparations are means of at least two concordant replicate incubations. The SEM figures determined for the hydrase assay procedures were within the range &S%.
AND
WALKER
Extraction and analytical techniques. After 30 min incubation with the mammalian preparations, the reaction was stopped by partitioning the mixture with ca. 3 ml diethyl ether. After withdrawing the ether layer, the aqueous mixture was reextracted with ether (2 X 2 ml). The three ether extracts were combined and the volume adjusted to 10 ml with redistilled n-hexane, prior to addition of anhydrous NazSOl for drying. Aliquots (1 ml) of the dried ether extracts were taken for TMS derivatization of the HEOM trans-diol by the method of Oates and Schrager (9, 19, 20). To arrest enzymic hydration in the insect preparations, acetone (7 ml) was added to each incubation mixture, which was then extracted with ether (3 X 5 ml) and the total extract adjusted to 25 ml with ether and dried (NazSOr). The T&IS derivatization was performed as described above. For both the mammalian and insect studies, the products of TMS derivatization were examined by GLC, using authentic samples of t’he TMS derivative of t’he trans-dihydrodiol of HEOM as standards. The GLC was carried out using PerkinElmer Fll and Pye Panchromatograph instruments, fit’ted with electron-capture detectors, both t,ritium and 63Ni sources were used (9, 12, 20). All-glass columns (0.6 cm i.d. and varying lengths) were fitted and these were packed with one of three different stationary phases, as follows : 1. 2.5% SE52 and 0.5% Epikote 1001 on SO-100 mesh acid-washed DMCS-treated Chromosorb W ; 2. 1.5% SE52 and 0.25% Epikote 1001 on S&100 mesh acid-washed DMCS-treated Chromosorb W ; 3. 3.0yo OV225 and 0.25% Epikote 1001 on 100-120 mesh acid-washed DMCStreated Chromosorb W. Where the [14C]HEOM substrate was employed, both the ether extracts and the residual aqueous reaction mixtures were counted to ascertain the recovery of added radioactivity. This was done by liquid scin-
INHIBITION
OF
MAMMALIAN
AND
TABLE Inhibition Number
of Epoxide
Inhibitor
Hydrase
INSECT
EPOXIDE
1
at Standard
Inhibitor
structureb
Concentration” Inhibition
Rabbit I II III IV V VI VII VIII IX x XI XII
HTDRASE
CCl,R p-CsHrCsHaOCHaR p-Cl-CkHaOCHzR p-NO&,HnOCHzR p-(CH-CCHZO)CBH~OCHZR m-(CH=CCHzO)CaHaOCH2R c-(CH=CCH20)CsHaOCHzR p-(C,H,CH=CH)C,H,OCH,w p-(C,H,CH,O)C,H,OCH,R (p-ltCH,OC6H4)2C(CH,)2 (CzHsO)t(GHsO)P=S (CaHjO)aP=O
92
Rat
(%) Calliphma
Tenebrio
93 79 94 97 60 x7 97 98
4-i 16 49 92 64 82 :< 86 93 57
100 77 70 73 84 73 1.5 91 94 97
93 4s :
.io
70
66
-
?I8
.ki
411 76 90 44 2%
20
40
2h
H+ClXIII
C;~H7C(CaH&COOCH&HzN(CzH&
a HEOM substrate level in mammalian incubations was 40 pg (2.2 X 1OW 100 pg (.i.5 X 1OF ,W). Inhibitor concentration was 5.0 X 10-S df except for the (rabbit); I, 2..5 X 1OP M (Z'endwio); XI, 6.4 X 1OW fi’f (mammals); I, 7.0 X 5.0 X 10m4 icf (Calliphora); XI, 6.0 X 1OW Jf (’msects). Each figure is the mean of
fif), in insect, incubations following: I, 1.5 X 1O-5 &f IO@ M (Calliphora); VIII, at least, t.wo determind ion*.
/Ok\. b IL = -CH-CH,
tillation counting, employing a dioxanbased scintillator, KL354 (Koch-Light Laborat,oriea). A Tracer-Lab scintillation counter was used and efficiency was determined by the use of an external standard. Radioactive recovery in the extracts was consistently above Y47,, while only negligible amounts remained in the aqueous reaction mixtures following extraction. Thus, the GLC analysis gave a good measurement of total diol production during incubation, from which the initial reaction velocity of the enzyme was calculatSed. Comparison of inhibitors. Comparison was made with respect to : 1. Percentage inhibition of epoxide hydrase in the four species, employing all the compounds at standard concentrations (usually 5.0 X 1O-5 IV). HEOM substrat’e level was maintained at 40 pg (2.2 X lop5 M) in the mammalian studies and at 100 pg (5.5 X 10-j JI) in the insect studies.
2. The inhibitor concentrations rtxquired to reduce enzyme activity by 5O[:b (i.e., Ijo values) were det.ermined for srlected compounds with the four enzyme preparations. Again, the HE031 substrate levels Tvere 40 pg and 100 pg for m:unmals and insects, respectively. Percentage artivity w-as measured for different inhibitor concentrations and plotted against a logarithmic scale of concentration, lines Iwing fitted by eye. 3. The kinetics of inhibition for wlect)ed compounds were examined b\, the method of Lineweaver and Hurk (21) using the double-reciprocal plot, and employing a range of HEOJI substrate concentrations. There was very little scatter of points \V~C~II straight lines were fitted by eye mtl no statistical analysis was attempted. (InI> preparat,ions from Tenebrin and rat, liwr were used, values for l’,,,., and h’,r, twing calculated for bot,h the enzyme preparatit )ns.
136
CRAVEN,
RESULTS
AND
BROOKS
AND
WALKER
TABLE
DISCUSSION
Examination of Table 1 shows that the compounds under investigation can be divided into four groups, on a structural basis :
Summary Four
Figures for percentage inhibition at standard concentration, for all the compounds investigated, are given in Table 1. Although compound and species differences are evident, it is apparent that the results for the four epoxide hydrase preparations are similar to each other in many respects. The simple epoxide I was the most effective inhibitor studied in this investigation. A compound I concentration of 5.0 X lop5 M reduced enzyme activity to zero in the case of the rabbit liver microsomes and the two insect preparations. Only with the rat was activity measurable at this inhibitor concentration (Table 1). Apart from I, the most effective inhibitors were certain of the phenyl glycidyl ethers, notably IX and the bis-epoxide X, which were almost equally effective for all four of the hydrase preparations. Together with compound IV, IX proved to be the best inhibitor of the rat enzyme, in contrast to the results obtained with the other three preparations. Two of the phenyl glycidyl ethers, VII and II, showed a lower inhibitory effect, especially with the hydrase preparation from the rat. The three positional isomers V, VI, and VII (Table 1) showed interesting differences in their inhibitory properties. Isomer VII had very little effect on the rat enzyme and only slightly greater effect on the preparation from Catliphora, though with the rabbit enzyme, inhibition was 60%. Both V and VI were better inhibitors,
of
Inhibitor
IZO Values Obtained Enzyme Preparationsa la0 value
Rat IV
1. Phenyl glycidyl ethers (phenyl 2,3epoxy propyl ethers). This includes the bis-epoxide X. 2. Organophosphate compounds, i.e., XI and XII. 3. Compound I, a simple chlorinated, aliphatic epoxide. 4. Compound XIII, i.e., SKF 525A.
2
I XII XIII II
5.2 x 10-6 7.1 x 10-s 7.1 x 10-e 9.0 x 10-s -
1.0 x 10-s 3.0 x 10-s -
0 Results shown am taken Figs. 2a-2e. HEOM substrate in the mammaLian incubations the insect incubations.
(M)
Tenebrio
Rabbit
from level and
for the
8.0 x 10-s 1.4 x 10-s 1.4 x lo-’ 1.3 x lo-’ 5.2 x 10-s
Calliphom
1.8 4.5 8.0 2.1
x x x X
10-s 10-s 10-s 10-s
the graphs presented in ~88 40 #g (2.2 X 10-S M) 100 cog (5.5 X 10-S M) in
as indicated, especially for the rat and CaEZiphora preparations (Table 1). It appears that in the case of the simple glycidyl ethers, the enzyme from Tenebrio is less susceptible to inhibition compared with the other three preparations. This is true for III, IV, V, VIII and IX. Overall, the organophosphorus compounds and the general microsomal enzyme inhibitor XIII (SKF 5258) were less powerful inhibitors than the glycidyl ethers and compound I. (Note that XI was employed at higher than standard concentration-Table 1.) Representative compounds were selected for further study from each of the structurally different groups. Figures 2a-2e show the 150 plots for the four enzyme preparations after treatment with compounds IV, I, XII, XIII, and II. In Table 2, the Iso values determined from the graphs have been collected together for purposes of comparison. Similarities between the four enzyme preparations are evident from these results, as are differences in the effectiveness of the compounds as inhibitors. The two phenyl glycidyl ethers, i.e., IV and II, were found to have intermediate potencies, with Iso values in the range 1 X 1O-5 to 8 X lo+ M (Figs. 2a and 2e). The single exception to this was found with the rat preparation, where IV showed a low 16,, value of 5 X lo-+ M, and was the most effective inhibitor with this species (Fig. 2a). With the other three preparations, the best inhibitor was I (Fig. 2b).
ISHIBITION
OF
MAMMALIAN
AND
INSECT
EPOXIDE
HTDRASE
13
FIG. 2. Determination of I60 values for selected compounds. Znhibition of HEOM-hydrae by (a) p-nitrophenyl glycidyl ether (IV); (b) l,l,l-trichloropropane-2,3-epoxide (I); (c) triphenyl phosphate (XII); (d) SKF &%A (XIIZ); and (e) p-ethylphenyl glycidyl ether (ZZ), using the preparation-s from rat (X), rabbit ( l ), Tenebrio ( n ) and Calliphora (A). Percentage hydrate activity plotted against molar inhibitor concentration (log s&e), enzyme activities being determined as in the text. HEOM substrate lwel was 40 fig (2.2 X lO-& M) in the mammalian incubations and 100 119 (5.6 X 1p6 M) in the insed incubations.
The hydrase preparations were least affected by the compounds XII and XIII (Figs. 2c and 2d). In general, the compound having the least inhibitory effect was XIII with all Iso values in the region of 10m4M, while XII had ISo values in the range
5 X 10e5 to 10e4 M. The preparation from Tenebrio had generally high Is0 values in the range low5 to 10F4 M, whereas all the other preparations had 1r,0values down t,o lo+ M and thus, the hydrase preparation from this insect seemsto be somewhat more
J35
CBAvEN,
BROOKS
AND
WALKER
FIG. 3. Lineweaver-Burk plots of HEOM-hydrase inhibition. Inhibition of (A) the rat iiver preparation by p-nitrophenyl glycidyl ether (IV); and (B) the Tenebrio preparation by p-propargyloxyphenyl glycidyl ether (V). S, HEOM concentration (mM); V, HEOM-dial formed (nmoleslmg protein/min). Znhibitor concentrations are shown; unlabeled lines, no inhibitor. Incubation conditions and assay as described in the text.
resistant to inhibition by the compounds under study (Table 2). Investigation of the inhibition by these representative compounds was extended to kinetic studies, employing the epoxide hydrase preparations from rat liver and Tenebrio. In Figs. 3-6 are shown double reciprocal plots (21) for these two enzyme preparations. Kinetics of pure competitive inhibition were found with the phenyl glycidyl ethers IV and V, in the rat and Tenebrio preparations, respectively, (Fig. 3). Oesch et al. (3) have shown that inhibition
of a purified epoxide hydrase preparation from guinea pig liver by III using styrene oxide substrate, is again purely competitive in nature. This suggests that, in spite of quantitative differences, the mechanism of enzyme inhibition is similar for these three phenyl glycidyl ethers. In the case of compound I, the kinetics of enzyme inhibition were different between the two preparat’ions (Fig. 4). Inhibition of the rat enzyme was of the uncompetitive type, while inhibition of the Tenelwio enzyme showed mixed kinetics.
J
65
OO
130 3
FIG. 4. Lineweaver-Burk Tenebrio,
plots for inhibition by l,l,l-trichloropropane-.%‘,,T-epoxide
of the HEOM-hydrase (I).
Details
as for
Fig.
from 3.
(A)
rat
liver
and
(B)
INHIBITION
FIG.
Tenebrio,
OF
MAMMALIAN
AND
5. Lineweaver-Rurk plots for inhibition by triphenyl phosphate (XII). Details
This was the only major qualitative difference found between the mammalian and insect, enzymes. Further, Oesch et al. (3) demonstrated that, compound I inhibited t,he guinea pig enzyme in an uncompetitive manner, whereas HEOM epoxide ring hydration by the hydrase preparation from Caltiphora was noncompetitively inhibited by I (22). In the present study compound I was the only inhibitor that did not show competitive kinetics, which suggests that
FIG.
Tenebrio,
6. Lineweaver-Burk by SKF 6,%A
plots (XIII).
GOT inhibition
Details
INSECT
EPOXIDE
of the HEOM-hydrase as for Fig. 2.
139
HYDRASE
from
(A)
rut
liver
and
(H,
this inhibitor may interact with epoxide hydrase at a site distinct’ from the substratcbinding site. The uncompetitive kinetics found with the mammalian preparations may arise because compound I combines only with the enzyme-subst,rate complex (23). The unusual inhibition by I should be explicable in t,erms of its structure. It is the smallest molecule studied hew, nonaromatic, and possesses a strongly electronwithdrawing substitucnt, the tric~hloro-
of the HEOM-hydrasc as for Fig. 3.
from
(A)
rat
liver
end
(11)
140
CRAVEN,
BROOKS
methyl group ; features that distinguish it from the other inhibitors in this study. The kinetics of inhibition for XII and XIII are shown in Figs. 5 and 6, respectively. Here, inhibition is of a competitive nature, indicating that these compounds may function as alternative substrates for the epoxide hydrase enzymes from rat and Tenebrio. This competitive inhibition by XII contrasts with the type found when unspecific microsomal carboxyl esterases are treated with organophosphates (24). Here, inhibition is noncompetitive and I~O values are relatively low. Also, the unspecific carboxyl esterase preparation from pig liver microsomes (24) showed no epoxide hydrase activity towards HEOM (25). These observations tend to confirm that epoxide hydrase and unspecific carboxyl esterase are different microsomal enzymes. Many microsomal enzymes exhibit competitive inhibition by the general inhibitor XIII, which may act as a multifunctional inhibitor by virtue of the combination of an ester function with several large oxidizable groups (26 and references therein). Thus, the appearance of competitive kinetics with the HEOM epoxide hydrases from rat and Tenebrio may imply alternative substrate competition, or steric exclusion of HEOM by this bulky inhibitor. Kinetic constants V,,, and K, were determined for the enzymes from rat and Tenebrio, in the absence of any inhibitors. From a comparison of the double reciprocal plots it can be seen that the rat enzyme is many times more active towards HEOM than is the Tenebrio enzyme and this is reflected in the values obtained for V,., (V,., values of 12.3 nmole diol per mg protein per min, and 0.62 nmole diol per mg protein per min, respectively). Despite this difference in V,,,, the values obtained for K, are similar: the value for the rat enzyme was 15.4 fig HEOM (8.34 pM in the incubation), while the corresponding figure for the Tenebrio enzyme was 22.3 pg HEOM (12.2 FM). Again, this suggests a
AND
WALKER
similarity
of the enzymes
from rat and
Tenebrio. ACKNOWLEDGMENTS
This work was Grant B/RG/19830 Council.
supported from
in part by the Science
Research Research
REFERENCES
1. G. T. Brooks, Progress in metabolic studies of the cyclodiene insecticides and its relevance to structure-activity correlations, World Rev. Pest Control 5, 62 (1966). 2. D. M. Jerina, J. W. Daly, B. Witkop, P. Zaltzman-Nirenberg, and S. Udenfriend, 1,2-Naphthalene oxide as an intermediate in the microsomal hydroxylation of naphthalene, Biochemistry 9, 147 (1970). 3. F. Oesch, N. Kaubisch, D. M. Jerina, and J. W. Daly, Hepatic epoxide hydrase. Structureactivity relationships for substrates and inhibitors, Biochemistry 10, 4858 (1971). 4. F. Oesch, Mammalian epoxide hydrases: Inducible enzymes catalysing the inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and olefinic compounds, Xenobioticu 3, 305 (1973). 5. J. K. Selkirk, E. Huberman, and C. Heidelberger, An epoxide is an intermediate in the microsomal metabolism of the chemical carcinogen, dibenz (a,h)anthracene, Biochern. Biophys. Res. Commun. 43, 1010 (1971). 6. P. L. Grover, A. Hewer, and P. Sims, Formation of K region epoxides as microsomal metabolites of pyrene and benzo(a)pyrene, Biochem. Pharmocol. 21, 2713 (1972). 7. G. A. El Zorgani, C. H. Walker, and K. A. Hassall, Species differences in the in vitro metabolism of HEOM, a chlorinated cyclodiene epoxide, Life Sci. 9 (Part II), 415 (1970). 8. C. H. Walker, G. A. El Zorgani, A. C. C. Craven, J. D. R. Kenny, and Maysoon Kurukgy, Studies of comparative metabolism using dieldrin analogues, in “Symposium on Comparative Studies of Food and Environmental Contamination (Finland),” p. 529. International Atomic Energy Agency, Vienna, 1974. 9. C. H. Walker and G. A. El Zorgani, The comparative metabolism and excretion of HCE, a biodegradable analogue of dieldrin, by vertebrate species, Arch. Environ. Contam. Toxiwl. 2, 97 (1974). 10. M. Slade and C. H. Zibitt, Metabolism of Cecropia juvenile hormone in insects and in mammals, in “Insect Juvenile Hormones: Chemistry and Action,” (J. J. Menn and M.
INHIBITION
OF
MAMMALIAN
AND
Beroza, Eds.), p. 155. Academic Press, New York, 1972. 11. G. T. Brooks, Insect epoxide hydrase inhibition by juvenile hormone analogues and metabolic inhibitors, Nature 245, 382 (1973). 12. G. T. Brooks, Inhibitors of cyclodiene epoxide ring hydrating enzymes of the Blowfly, Calliphora
Prythroccphala,
Pestic.
Sci.
5,
177
(1974). 13. M. Rlade and C. F. Wilkinson, Juvenile hormone analogues: a possible case of mistaken identity? Science 181, 672 (1973). 14. K. C. Leibman and E. Ortiz, Oxidation of indene in liver microsomes, Mol. Pharmacol. 4, 201 (196X). 15. F. Oesch, D. M. Jerina, J. W. Daly, A. Y. H. Lu, It. Kuntzman, and A. H. Conney, A reconst,ituted microsomal enzyme system that converts naphthalene to trans-1,2-dihydroxy1,2-dihydronaphthalene via naphthalene-1,2oxide: presence of epoxide hydrase in cytochrome P450 and Pdaa fractions, Arch. Biothem. Biophys. 153, 62 (1972). 16. G. T. Brooks, Investigations with some biodegradable dieldrin analogues, PTOC. 6th Brit. Inswtic. Fungic. Conj. 2, 472 (1969). 17. G. T. Brooks and A. Harrison, The effect of pyrethrin synergists, especially sesamex, on t,he insecticidal potency of hexachlorocyclopentadiene derivatives (“cyclodiene insecticides”) in the adult housefly, Musca domestica L, Biochem. Pharmuwl. 13, 827 (1964). 18. 0. H. Lowry, N. J. Rosebrough, A. L. Farr, and II. J. Randall, Protein measurements with
INSECT
19.
20.
21. 22.
23. 24.
25. 26.
EPOXIDE
HYDRASE
111
the Folin phenol reagent, J. Riol. Chem. 193. 265 (19.51). M. D. G. Oates and J. Schrager, The use of gas-liquid chromatography in the analysis of neutral monosaccharides in hydrolysates ttf gastric mucopolysaccharides, Biochvm. .I 97, 697 (1965). G. T. Brooks, A. Harrison, and S. E. I,(iwib, Cyclodiene epoxide ring hydration by m&-clsomes from mammalian liver and hollsrHir+, Biochem. Pharmaw/. 19, 2.55 (1970). H. Lineweaver and I>. Burk, The determinalion of enzyme dissociat,ion constants, 1. .g IXW. Chrm. Sot. 56, 658 (1934). M. Slade, G. T. Brooks, H. Kryst,yna I-iet,nar&i, and C. F. Wilkinson, Inhibit,ion of the enzymatic hydration of t,he epoxide HEOM in insects, P&rids BiochPm. Ph?ysio/. 5. :S (1975). M. Dixon and R. C. Webb, Enzyme inhibiturq, in “Enzymes,” 2nd Ed., p. 315. Longman-;, New York, 1964. W. Junge and K. Krisch, Current problems on t#he structure and classification of mammalian liver carboxylesterases (EC 3.1.1.1). .Ilol. Cd. Biochrm. 1, 41 (1973). C. H. Walker, unpublished results. M. W. Anders and G. J. Mannering, Inhibit.itm of drug metabolism. I. Kinetics of the inhihition of the iii-demethylation of ethylmorphine by 2-diet,hyl-aminoethyl-2,2-diphenylvalerate HCL (SKF 525A) and related compounds, -Mol. Phurmuwl. 2, 319 (1966). Also palts II and III in t,his volume.
BIOCHEMISTRY
The
AND
PHYSIOLOGY
6, 132-141
(1976)
Inhibition of HEOM Epoxide Liver Microsomes and Insect A. C. C. CRAVEN,
Department
of Physiology
Hydrase in Mammalian Pupal Homogenates
G. T. BROOKS,’ AND C. H. WALKER and Biochemistry, Reading, United
Reading Kingdom
University,
Whiteknights,
Received March 24, 1975; accepted August 18, 1975 A range of compounds were tested as inhibitors of the enzyme epoxide hydrase, using a cyclodiene epoxide (HEOM) as substrate. Rat and rabbit liver microsomes and pupal homogenates of the blowfly (Calliphora erythroeephula) and the yellow mealworm (Tenebrio molitor) were compared as sources of the enzyme. Only minor differences were found between the four enzyme preparations, when considering ISOvalues and percentage inhibition at standard concentration. The simple epoxide l,l,l-trichloropropse-2,3-epoxide and two glycidyl ethers p-nitrophenyl glycidyl ether and p-ethylphenyl glycidyl ether tended to have lower ZSO values (1.8X1O-B to 8.OX1O-6 M) than triphenyl phosphate and SKF 5258 (4.5X10-6 to 1.4X1O-4 M). Triphenyl phosphate and SKF 5258 were competitive inhibitors for both the rat and Tenebrio enzymes. The only clear difference found between these two epoxide hydrase preparations was with respect to their inhibition by 1,&l-trichloropropane-2,3-epoxide, which was an uncompetitive inhibitor with the rat enzyme, but showed kinetics of mixed inhibition with the insect preparation. INTRODUCTION
Enzymes that cleave epoxide rings by hydration to give mainly the corresponding trans-diols (14) are of current interest in relation to the mechanism of carcinogenesis mediated by chemicals (5, S), the variable environmental stability of certain chlorinated epoxides used as insecticides and the deactivation of insect P-9, juvenile hormones that contain epoxide rings (10-13). Inhibitors of these epoxide hydrases (EC 4.2.1.63) are of value for demonstrating the intermediate formation of labile epoxides during the oxidative metabolism of various olefines (4, 6, 14, 15) and for exploring the background to species differences in insecticide and juvenile hormone 1 Agricultural Research Council, Unit of Invertebrate Chemistry and Physiology, University of Sussex, Brighton, Sussex, England.
metabolism (11). Information about such species variations is important in the design of selective insecticides. The cyclodiene epoxide HEOM2 (see Fig. 1) has proved to be a useful substrate for the investigation of epoxide hydrase activity as it is readily metabolized to a single product and because both substrate and product can be accurately estimated at low concentration (7-9, 11, 12, 16). We report here on the effects of several inhibitors on the hydrase activity towards this substrate in tissue preparations from two mammalian species and two insect species. 2 Abbreviations used : HEOM, 1,2 ,3,4 t9 t9-hexachloro-6,7-epoxy-1,4,4a t5 , 6 77,8 7Sa-octahydro-1,4methanonaphthalene (See Fig. 1); TLC, thin-layer chromatography; GLC, gas-liquid chromatography; TMS, trimethyl silyl ether. Roman numerals are used in the text to indicate inhibitors (for full structure of all inhibitors see Table 1). 132
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
INHIHITION
OF
XEOM Ttii-DIOL
XEOM
FIG.
1. Enzymatic
MATERIALS
MAMMALIAN
hydration
AND
of HEOM.
METHODS
Animals. Wistar rats (200-300 g) were obtained from Charles River, Manston, Kent, while Old English rabbits (2-2.5 kg) were obtained from Goodchild Farms, Crawley, Sussex. All mammals were mature males, in good condition. White prepupae of the blowfly (Calliphora erythrocephala) and O-l hr old pupae of the yellow mealworm (Tenebrio molitor) were used as the sources of insect epoxide hydrase. Substrate. The cyclodiene epoxide HEOM was synthesized as described previ,ously (17). ‘*C-labeled HEOM was prepared from [14C]hexachlorocyclopentadiene supplied by The Radiochemical Centre, Amersham, Bucks. Because of the small quantities of the labeled epoxide involved, final purification of the [14C]HEOM was by TLC on Kieselgel G. TLC separations were repeated using different solvent systems (0.5 and 1% acetone in hexane) until constant specific activity was achieved, and a single peak was found on examination by GLC using two different stationary phases, SE52 and OV225 (see later under Extraction and analytical techniques). The HEOM substrate was employed as a 2 mg ml-’ ethanolic solution, either unlabeled, or labeled with a specific activity of 0.1 &Ji mg--’ . Enzyme preparations. In the case of the rat and the rabbit, washed liver microsomes were used as the source of epoxide hydrase. Animals were killed by breaking the neck, and the livers were rapidly removed, washed, and placed on ice. After removal of connective tissue and the gall bladder (present in rabbit, but not rat) the livers were thinly sliced and homogenized
AND
INSECT
EPOXIDE
HTDRASE
1X3
in isotonic (1.15%) KC1 (3 ml/g of liver), for 2 min at 0-4°C in a stainless steel and glass homogenizer (MSE Scient,ific Instruments, Crawley, Sussex). Washed microsomes were prepared from the homogenates as described elsewhere (9) and then resuspended in isotonic KCl. The concentrations of the microsomal preparations, with respect to weight of liver represent,ed, were 4 mg ml-l and 40 mg ml-’ for t,he rabbit and rat, respectively. Homogenates (lOyo w/v) in KH,POI/ NaOH buffer (0.1 M, pH 8.5) were prrpared from Calliphora pupae 0-i hr old (1 g; 12-15 pupae) as described elsewhere (12) and used as such, since centrifugat,ion resulted in a substantial (ca. 50%) loss of epoxide hydrase activity. In the case of Tenebrio pupae, homogenates were made in the same way, but here, heavy particles were removed by centrifugation at 15001~ for 5 min without loss of activity, and the resulting supernatant was used as the source of epoxide hydrase. Total protein in the mammalian and insect preparations was estimated by the method of Lowry et al. (18). Protein yields were within the following ranges: Rat, 8--9 mg microsomal protein per gram of liver; Rabbit, 10-12 mg microsomal protein per gram of liver ; CaEZiphora, 12 mg protein per milliliter homogenate, Tenebrio, 8-Y mg protein per milliliter supernatant . A sample of unspecific carboxylesterase prepared from pig liver mierosomes (EC 3.1.1.1) was supplied by K. Krisch, Institute fiir Physiologische Chemie und Physikochemie, der UniversitBt,, Kiel, Federal Republic of Germany. Inhibitors. Compounds tested as inhibitors of epoxide hydrase were prepared or obtained as described previously (12) and were employed in ethanolic solution at st,andard concentrations, except for VIII, which was dissolved in acetone because of its low solubility in ethanol. Incubation procedures. In the case of the mammalian studies, reactions were carried out in Erlenmeyer flasks open to the air
134
CRAVEN,
BROOKS
and held in a metabolic shaker at 37°C for 30 min. The reaction mixture consisted of 4.5 ml buffered incubation medium pH 7.4 (9), 0.5 ml microsomal suspension ( = 20 mg liver for rat and 2 mg liver for rabbit) plus varying amounts of inhibitors and HEOM substrate. Components were added rapidly in the stated order, without any preincubation. The HEOM substrate was kept constant at 40 gg (20 ~1 of 2 mg ml-l solution) per incubat,ion flask, except with the doublereciprocal plots for the rat where a range of substrate concentrations was employed. Incubations with the insect preparations were carried out at 30°C in Erlenmeyer flasks, open to the air. The Calliphora homogenate (0.5 ml) or Tenebrio supernatant (0.5 ml) was added to the KH,POJ NaOH buffer (0.1 M, pH 5.5, 4.5 ml), followed by the inhibitor and HEOM substrate. The enzyme was preincubated (10 min) w&h each inhibitor before addition of HEOM, and incubation continued for 12 min after addition of the substrate, without agitation beyond that required at addition. The HEOM substrate was kept constant at 100 pg (50 ~1 of 2 mg ml-’ solution) per incubation flask, except with the double reciprocal plots for Tenebrio supernatant. All activity was lost from both the mammalian and insect preparations when enzyme plus buffered medium was heated for 5 min at 100°C before addition of HEOM. Ethanol and acetone, used for addition of substrate and inhibitors to the incubation medium, had no measurable effect on enzyme activity at the levels employed. Enzyme activity was linear during the respective incubation periods, permitting measurement of initial reaction velocity. Appropriate volumes of ethanol and/or acetone were included in control incubations and these produced consistent values for control enzyme activity. Values for activities of inhibited enzyme preparations are means of at least two concordant replicate incubations. The SEM figures determined for the hydrase assay procedures were within the range &S%.
AND
WALKER
Extraction and analytical techniques. After 30 min incubation with the mammalian preparations, the reaction was stopped by partitioning the mixture with ca. 3 ml diethyl ether. After withdrawing the ether layer, the aqueous mixture was reextracted with ether (2 X 2 ml). The three ether extracts were combined and the volume adjusted to 10 ml with redistilled n-hexane, prior to addition of anhydrous NazSOl for drying. Aliquots (1 ml) of the dried ether extracts were taken for TMS derivatization of the HEOM trans-diol by the method of Oates and Schrager (9, 19, 20). To arrest enzymic hydration in the insect preparations, acetone (7 ml) was added to each incubation mixture, which was then extracted with ether (3 X 5 ml) and the total extract adjusted to 25 ml with ether and dried (NazSOr). The T&IS derivatization was performed as described above. For both the mammalian and insect studies, the products of TMS derivatization were examined by GLC, using authentic samples of t’he TMS derivative of t’he trans-dihydrodiol of HEOM as standards. The GLC was carried out using PerkinElmer Fll and Pye Panchromatograph instruments, fit’ted with electron-capture detectors, both t,ritium and 63Ni sources were used (9, 12, 20). All-glass columns (0.6 cm i.d. and varying lengths) were fitted and these were packed with one of three different stationary phases, as follows : 1. 2.5% SE52 and 0.5% Epikote 1001 on SO-100 mesh acid-washed DMCS-treated Chromosorb W ; 2. 1.5% SE52 and 0.25% Epikote 1001 on S&100 mesh acid-washed DMCS-treated Chromosorb W ; 3. 3.0yo OV225 and 0.25% Epikote 1001 on 100-120 mesh acid-washed DMCStreated Chromosorb W. Where the [14C]HEOM substrate was employed, both the ether extracts and the residual aqueous reaction mixtures were counted to ascertain the recovery of added radioactivity. This was done by liquid scin-
INHIBITION
OF
MAMMALIAN
AND
TABLE Inhibition Number
of Epoxide
Inhibitor
Hydrase
INSECT
EPOXIDE
1
at Standard
Inhibitor
structureb
Concentration” Inhibition
Rabbit I II III IV V VI VII VIII IX x XI XII
HTDRASE
CCl,R p-CsHrCsHaOCHaR p-Cl-CkHaOCHzR p-NO&,HnOCHzR p-(CH-CCHZO)CBH~OCHZR m-(CH=CCHzO)CaHaOCH2R c-(CH=CCH20)CsHaOCHzR p-(C,H,CH=CH)C,H,OCH,w p-(C,H,CH,O)C,H,OCH,R (p-ltCH,OC6H4)2C(CH,)2 (CzHsO)t(GHsO)P=S (CaHjO)aP=O
92
Rat
(%) Calliphma
Tenebrio
93 79 94 97 60 x7 97 98
4-i 16 49 92 64 82 :< 86 93 57
100 77 70 73 84 73 1.5 91 94 97
93 4s :
.io
70
66
-
?I8
.ki
411 76 90 44 2%
20
40
2h
H+ClXIII
C;~H7C(CaH&COOCH&HzN(CzH&
a HEOM substrate level in mammalian incubations was 40 pg (2.2 X 1OW 100 pg (.i.5 X 1OF ,W). Inhibitor concentration was 5.0 X 10-S df except for the (rabbit); I, 2..5 X 1OP M (Z'endwio); XI, 6.4 X 1OW fi’f (mammals); I, 7.0 X 5.0 X 10m4 icf (Calliphora); XI, 6.0 X 1OW Jf (’msects). Each figure is the mean of
fif), in insect, incubations following: I, 1.5 X 1O-5 &f IO@ M (Calliphora); VIII, at least, t.wo determind ion*.
/Ok\. b IL = -CH-CH,
tillation counting, employing a dioxanbased scintillator, KL354 (Koch-Light Laborat,oriea). A Tracer-Lab scintillation counter was used and efficiency was determined by the use of an external standard. Radioactive recovery in the extracts was consistently above Y47,, while only negligible amounts remained in the aqueous reaction mixtures following extraction. Thus, the GLC analysis gave a good measurement of total diol production during incubation, from which the initial reaction velocity of the enzyme was calculatSed. Comparison of inhibitors. Comparison was made with respect to : 1. Percentage inhibition of epoxide hydrase in the four species, employing all the compounds at standard concentrations (usually 5.0 X 1O-5 IV). HEOM substrat’e level was maintained at 40 pg (2.2 X lop5 M) in the mammalian studies and at 100 pg (5.5 X 10-j JI) in the insect studies.
2. The inhibitor concentrations rtxquired to reduce enzyme activity by 5O[:b (i.e., Ijo values) were det.ermined for srlected compounds with the four enzyme preparations. Again, the HE031 substrate levels Tvere 40 pg and 100 pg for m:unmals and insects, respectively. Percentage artivity w-as measured for different inhibitor concentrations and plotted against a logarithmic scale of concentration, lines Iwing fitted by eye. 3. The kinetics of inhibition for wlect)ed compounds were examined b\, the method of Lineweaver and Hurk (21) using the double-reciprocal plot, and employing a range of HEOJI substrate concentrations. There was very little scatter of points \V~C~II straight lines were fitted by eye mtl no statistical analysis was attempted. (InI> preparat,ions from Tenebrin and rat, liwr were used, values for l’,,,., and h’,r, twing calculated for bot,h the enzyme preparatit )ns.
136
CRAVEN,
RESULTS
AND
BROOKS
AND
WALKER
TABLE
DISCUSSION
Examination of Table 1 shows that the compounds under investigation can be divided into four groups, on a structural basis :
Summary Four
Figures for percentage inhibition at standard concentration, for all the compounds investigated, are given in Table 1. Although compound and species differences are evident, it is apparent that the results for the four epoxide hydrase preparations are similar to each other in many respects. The simple epoxide I was the most effective inhibitor studied in this investigation. A compound I concentration of 5.0 X lop5 M reduced enzyme activity to zero in the case of the rabbit liver microsomes and the two insect preparations. Only with the rat was activity measurable at this inhibitor concentration (Table 1). Apart from I, the most effective inhibitors were certain of the phenyl glycidyl ethers, notably IX and the bis-epoxide X, which were almost equally effective for all four of the hydrase preparations. Together with compound IV, IX proved to be the best inhibitor of the rat enzyme, in contrast to the results obtained with the other three preparations. Two of the phenyl glycidyl ethers, VII and II, showed a lower inhibitory effect, especially with the hydrase preparation from the rat. The three positional isomers V, VI, and VII (Table 1) showed interesting differences in their inhibitory properties. Isomer VII had very little effect on the rat enzyme and only slightly greater effect on the preparation from Catliphora, though with the rabbit enzyme, inhibition was 60%. Both V and VI were better inhibitors,
of
Inhibitor
IZO Values Obtained Enzyme Preparationsa la0 value
Rat IV
1. Phenyl glycidyl ethers (phenyl 2,3epoxy propyl ethers). This includes the bis-epoxide X. 2. Organophosphate compounds, i.e., XI and XII. 3. Compound I, a simple chlorinated, aliphatic epoxide. 4. Compound XIII, i.e., SKF 525A.
2
I XII XIII II
5.2 x 10-6 7.1 x 10-s 7.1 x 10-e 9.0 x 10-s -
1.0 x 10-s 3.0 x 10-s -
0 Results shown am taken Figs. 2a-2e. HEOM substrate in the mammaLian incubations the insect incubations.
(M)
Tenebrio
Rabbit
from level and
for the
8.0 x 10-s 1.4 x 10-s 1.4 x lo-’ 1.3 x lo-’ 5.2 x 10-s
Calliphom
1.8 4.5 8.0 2.1
x x x X
10-s 10-s 10-s 10-s
the graphs presented in ~88 40 #g (2.2 X 10-S M) 100 cog (5.5 X 10-S M) in
as indicated, especially for the rat and CaEZiphora preparations (Table 1). It appears that in the case of the simple glycidyl ethers, the enzyme from Tenebrio is less susceptible to inhibition compared with the other three preparations. This is true for III, IV, V, VIII and IX. Overall, the organophosphorus compounds and the general microsomal enzyme inhibitor XIII (SKF 5258) were less powerful inhibitors than the glycidyl ethers and compound I. (Note that XI was employed at higher than standard concentration-Table 1.) Representative compounds were selected for further study from each of the structurally different groups. Figures 2a-2e show the 150 plots for the four enzyme preparations after treatment with compounds IV, I, XII, XIII, and II. In Table 2, the Iso values determined from the graphs have been collected together for purposes of comparison. Similarities between the four enzyme preparations are evident from these results, as are differences in the effectiveness of the compounds as inhibitors. The two phenyl glycidyl ethers, i.e., IV and II, were found to have intermediate potencies, with Iso values in the range 1 X 1O-5 to 8 X lo+ M (Figs. 2a and 2e). The single exception to this was found with the rat preparation, where IV showed a low 16,, value of 5 X lo-+ M, and was the most effective inhibitor with this species (Fig. 2a). With the other three preparations, the best inhibitor was I (Fig. 2b).
ISHIBITION
OF
MAMMALIAN
AND
INSECT
EPOXIDE
HTDRASE
13
FIG. 2. Determination of I60 values for selected compounds. Znhibition of HEOM-hydrae by (a) p-nitrophenyl glycidyl ether (IV); (b) l,l,l-trichloropropane-2,3-epoxide (I); (c) triphenyl phosphate (XII); (d) SKF &%A (XIIZ); and (e) p-ethylphenyl glycidyl ether (ZZ), using the preparation-s from rat (X), rabbit ( l ), Tenebrio ( n ) and Calliphora (A). Percentage hydrate activity plotted against molar inhibitor concentration (log s&e), enzyme activities being determined as in the text. HEOM substrate lwel was 40 fig (2.2 X lO-& M) in the mammalian incubations and 100 119 (5.6 X 1p6 M) in the insed incubations.
The hydrase preparations were least affected by the compounds XII and XIII (Figs. 2c and 2d). In general, the compound having the least inhibitory effect was XIII with all Iso values in the region of 10m4M, while XII had ISo values in the range
5 X 10e5 to 10e4 M. The preparation from Tenebrio had generally high Is0 values in the range low5 to 10F4 M, whereas all the other preparations had 1r,0values down t,o lo+ M and thus, the hydrase preparation from this insect seemsto be somewhat more
J35
CBAvEN,
BROOKS
AND
WALKER
FIG. 3. Lineweaver-Burk plots of HEOM-hydrase inhibition. Inhibition of (A) the rat iiver preparation by p-nitrophenyl glycidyl ether (IV); and (B) the Tenebrio preparation by p-propargyloxyphenyl glycidyl ether (V). S, HEOM concentration (mM); V, HEOM-dial formed (nmoleslmg protein/min). Znhibitor concentrations are shown; unlabeled lines, no inhibitor. Incubation conditions and assay as described in the text.
resistant to inhibition by the compounds under study (Table 2). Investigation of the inhibition by these representative compounds was extended to kinetic studies, employing the epoxide hydrase preparations from rat liver and Tenebrio. In Figs. 3-6 are shown double reciprocal plots (21) for these two enzyme preparations. Kinetics of pure competitive inhibition were found with the phenyl glycidyl ethers IV and V, in the rat and Tenebrio preparations, respectively, (Fig. 3). Oesch et al. (3) have shown that inhibition
of a purified epoxide hydrase preparation from guinea pig liver by III using styrene oxide substrate, is again purely competitive in nature. This suggests that, in spite of quantitative differences, the mechanism of enzyme inhibition is similar for these three phenyl glycidyl ethers. In the case of compound I, the kinetics of enzyme inhibition were different between the two preparat’ions (Fig. 4). Inhibition of the rat enzyme was of the uncompetitive type, while inhibition of the Tenelwio enzyme showed mixed kinetics.
J
65
OO
130 3
FIG. 4. Lineweaver-Burk Tenebrio,
plots for inhibition by l,l,l-trichloropropane-.%‘,,T-epoxide
of the HEOM-hydrase (I).
Details
as for
Fig.
from 3.
(A)
rat
liver
and
(B)
INHIBITION
FIG.
Tenebrio,
OF
MAMMALIAN
AND
5. Lineweaver-Rurk plots for inhibition by triphenyl phosphate (XII). Details
This was the only major qualitative difference found between the mammalian and insect, enzymes. Further, Oesch et al. (3) demonstrated that, compound I inhibited t,he guinea pig enzyme in an uncompetitive manner, whereas HEOM epoxide ring hydration by the hydrase preparation from Caltiphora was noncompetitively inhibited by I (22). In the present study compound I was the only inhibitor that did not show competitive kinetics, which suggests that
FIG.
Tenebrio,
6. Lineweaver-Burk by SKF 6,%A
plots (XIII).
GOT inhibition
Details
INSECT
EPOXIDE
of the HEOM-hydrase as for Fig. 2.
139
HYDRASE
from
(A)
rut
liver
and
(H,
this inhibitor may interact with epoxide hydrase at a site distinct’ from the substratcbinding site. The uncompetitive kinetics found with the mammalian preparations may arise because compound I combines only with the enzyme-subst,rate complex (23). The unusual inhibition by I should be explicable in t,erms of its structure. It is the smallest molecule studied hew, nonaromatic, and possesses a strongly electronwithdrawing substitucnt, the tric~hloro-
of the HEOM-hydrasc as for Fig. 3.
from
(A)
rat
liver
end
(11)
140
CRAVEN,
BROOKS
methyl group ; features that distinguish it from the other inhibitors in this study. The kinetics of inhibition for XII and XIII are shown in Figs. 5 and 6, respectively. Here, inhibition is of a competitive nature, indicating that these compounds may function as alternative substrates for the epoxide hydrase enzymes from rat and Tenebrio. This competitive inhibition by XII contrasts with the type found when unspecific microsomal carboxyl esterases are treated with organophosphates (24). Here, inhibition is noncompetitive and I~O values are relatively low. Also, the unspecific carboxyl esterase preparation from pig liver microsomes (24) showed no epoxide hydrase activity towards HEOM (25). These observations tend to confirm that epoxide hydrase and unspecific carboxyl esterase are different microsomal enzymes. Many microsomal enzymes exhibit competitive inhibition by the general inhibitor XIII, which may act as a multifunctional inhibitor by virtue of the combination of an ester function with several large oxidizable groups (26 and references therein). Thus, the appearance of competitive kinetics with the HEOM epoxide hydrases from rat and Tenebrio may imply alternative substrate competition, or steric exclusion of HEOM by this bulky inhibitor. Kinetic constants V,,, and K, were determined for the enzymes from rat and Tenebrio, in the absence of any inhibitors. From a comparison of the double reciprocal plots it can be seen that the rat enzyme is many times more active towards HEOM than is the Tenebrio enzyme and this is reflected in the values obtained for V,., (V,., values of 12.3 nmole diol per mg protein per min, and 0.62 nmole diol per mg protein per min, respectively). Despite this difference in V,,,, the values obtained for K, are similar: the value for the rat enzyme was 15.4 fig HEOM (8.34 pM in the incubation), while the corresponding figure for the Tenebrio enzyme was 22.3 pg HEOM (12.2 FM). Again, this suggests a
AND
WALKER
similarity
of the enzymes
from rat and
Tenebrio. ACKNOWLEDGMENTS
This work was Grant B/RG/19830 Council.
supported from
in part by the Science
Research Research
REFERENCES
1. G. T. Brooks, Progress in metabolic studies of the cyclodiene insecticides and its relevance to structure-activity correlations, World Rev. Pest Control 5, 62 (1966). 2. D. M. Jerina, J. W. Daly, B. Witkop, P. Zaltzman-Nirenberg, and S. Udenfriend, 1,2-Naphthalene oxide as an intermediate in the microsomal hydroxylation of naphthalene, Biochemistry 9, 147 (1970). 3. F. Oesch, N. Kaubisch, D. M. Jerina, and J. W. Daly, Hepatic epoxide hydrase. Structureactivity relationships for substrates and inhibitors, Biochemistry 10, 4858 (1971). 4. F. Oesch, Mammalian epoxide hydrases: Inducible enzymes catalysing the inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and olefinic compounds, Xenobioticu 3, 305 (1973). 5. J. K. Selkirk, E. Huberman, and C. Heidelberger, An epoxide is an intermediate in the microsomal metabolism of the chemical carcinogen, dibenz (a,h)anthracene, Biochern. Biophys. Res. Commun. 43, 1010 (1971). 6. P. L. Grover, A. Hewer, and P. Sims, Formation of K region epoxides as microsomal metabolites of pyrene and benzo(a)pyrene, Biochem. Pharmocol. 21, 2713 (1972). 7. G. A. El Zorgani, C. H. Walker, and K. A. Hassall, Species differences in the in vitro metabolism of HEOM, a chlorinated cyclodiene epoxide, Life Sci. 9 (Part II), 415 (1970). 8. C. H. Walker, G. A. El Zorgani, A. C. C. Craven, J. D. R. Kenny, and Maysoon Kurukgy, Studies of comparative metabolism using dieldrin analogues, in “Symposium on Comparative Studies of Food and Environmental Contamination (Finland),” p. 529. International Atomic Energy Agency, Vienna, 1974. 9. C. H. Walker and G. A. El Zorgani, The comparative metabolism and excretion of HCE, a biodegradable analogue of dieldrin, by vertebrate species, Arch. Environ. Contam. Toxiwl. 2, 97 (1974). 10. M. Slade and C. H. Zibitt, Metabolism of Cecropia juvenile hormone in insects and in mammals, in “Insect Juvenile Hormones: Chemistry and Action,” (J. J. Menn and M.
INHIBITION
OF
MAMMALIAN
AND
Beroza, Eds.), p. 155. Academic Press, New York, 1972. 11. G. T. Brooks, Insect epoxide hydrase inhibition by juvenile hormone analogues and metabolic inhibitors, Nature 245, 382 (1973). 12. G. T. Brooks, Inhibitors of cyclodiene epoxide ring hydrating enzymes of the Blowfly, Calliphora
Prythroccphala,
Pestic.
Sci.
5,
177
(1974). 13. M. Rlade and C. F. Wilkinson, Juvenile hormone analogues: a possible case of mistaken identity? Science 181, 672 (1973). 14. K. C. Leibman and E. Ortiz, Oxidation of indene in liver microsomes, Mol. Pharmacol. 4, 201 (196X). 15. F. Oesch, D. M. Jerina, J. W. Daly, A. Y. H. Lu, It. Kuntzman, and A. H. Conney, A reconst,ituted microsomal enzyme system that converts naphthalene to trans-1,2-dihydroxy1,2-dihydronaphthalene via naphthalene-1,2oxide: presence of epoxide hydrase in cytochrome P450 and Pdaa fractions, Arch. Biothem. Biophys. 153, 62 (1972). 16. G. T. Brooks, Investigations with some biodegradable dieldrin analogues, PTOC. 6th Brit. Inswtic. Fungic. Conj. 2, 472 (1969). 17. G. T. Brooks and A. Harrison, The effect of pyrethrin synergists, especially sesamex, on t,he insecticidal potency of hexachlorocyclopentadiene derivatives (“cyclodiene insecticides”) in the adult housefly, Musca domestica L, Biochem. Pharmuwl. 13, 827 (1964). 18. 0. H. Lowry, N. J. Rosebrough, A. L. Farr, and II. J. Randall, Protein measurements with
INSECT
19.
20.
21. 22.
23. 24.
25. 26.
EPOXIDE
HYDRASE
111
the Folin phenol reagent, J. Riol. Chem. 193. 265 (19.51). M. D. G. Oates and J. Schrager, The use of gas-liquid chromatography in the analysis of neutral monosaccharides in hydrolysates ttf gastric mucopolysaccharides, Biochvm. .I 97, 697 (1965). G. T. Brooks, A. Harrison, and S. E. I,(iwib, Cyclodiene epoxide ring hydration by m&-clsomes from mammalian liver and hollsrHir+, Biochem. Pharmaw/. 19, 2.55 (1970). H. Lineweaver and I>. Burk, The determinalion of enzyme dissociat,ion constants, 1. .g IXW. Chrm. Sot. 56, 658 (1934). M. Slade, G. T. Brooks, H. Kryst,yna I-iet,nar&i, and C. F. Wilkinson, Inhibit,ion of the enzymatic hydration of t,he epoxide HEOM in insects, P&rids BiochPm. Ph?ysio/. 5. :S (1975). M. Dixon and R. C. Webb, Enzyme inhibiturq, in “Enzymes,” 2nd Ed., p. 315. Longman-;, New York, 1964. W. Junge and K. Krisch, Current problems on t#he structure and classification of mammalian liver carboxylesterases (EC 3.1.1.1). .Ilol. Cd. Biochrm. 1, 41 (1973). C. H. Walker, unpublished results. M. W. Anders and G. J. Mannering, Inhibit.itm of drug metabolism. I. Kinetics of the inhihition of the iii-demethylation of ethylmorphine by 2-diet,hyl-aminoethyl-2,2-diphenylvalerate HCL (SKF 525A) and related compounds, -Mol. Phurmuwl. 2, 319 (1966). Also palts II and III in t,his volume.