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Specificity of mouse liver cytosolic epoxide hydrolase for K-region epoxides derived from polycyclic aromatic hydrocarbons
Cancer Letters, 9 (1980) 169-175 o Elsevier/North-Holland Scientific Publishers L,td.
169
SPECIFICITY OF MOUSE LIVER CYTOSOLIC EPOXIDE HYDROLASE FOR K-REGION EPOXIDES DERIVED FROM POLYCYCLIC AROMATIC HYDROCARBONS
FRANZ OESCH* and MARIO GOLAN Institute of Pharmacology, Mainz, (F.R.G.)
University of Mainz, Obere Zahlbacher
Strasse 67, D-6500
(Received 8 February 1980) (Accepted 14 February 1980)
SUMMARY
Mouse liver cytosol epoxide hydrolase, known to be very active for certain alkene oxides, had a specific activity which was 2.1-, ll- and 160-fold lower than that of the microsomal epoxide hydrolase for the arene oxides 7-methylbenz[a] anthracene 5,6-oxide, benz [a] anthracene 5,6-oxide and phenanthrene 9,10-oxide, respectively. For benzo[a] pyrene 4,5-oxide no activity (< 10 pmol product/mg protein/min) of cytoplasmic epoxide hydrolase was detectable. The specific activity of cytoplasmic epoxide hydrolase was much lower for all K-region epoxides investigated, compared to truns-stilbene oxide used as a positive control and for which a new assay is described. It is concluded from these rates combined with the fact that these lipophilic K-region epoxides are expected to stay preferentially at membranous sites where they are generated, that cytoplasmic epoxide hydrolase plays a minor role for their transformation compared to membrane-bound hydrolase. The data also show that for the substrates investigated the epoxide hydrolase activities in the cytoplasmic and microsomal fractions are complementary to some extent, but there is no quantitative inverse relationship.
INTRODUCTION
Aromatic and olefinic compounds can be metabolically transformed by mammalian monooxygenases to epoxides. These are electrophilically reactive to varying degrees and many of them exert mutagenic and carcinogenic effects. Some of them can be metabolically inactivated by epoxide hydrolases (EC’ 3.3.2.3). In the case of angular polycyclic aromatic hydrocarbons *To whom correspondence
and reprint requests should be addressed.
170
several classes of epoxides can be metabolically formed including K-region arene oxides and reactive dihydrodiol-bay-region epoxides. In the biosynthesis of the latter the formation of the precursor dihydrodiol by epoxide hydrolases is an essential prerequisite. Thus epoxide hydrolases play a pivotal role in both activation and inactivation [ l,-5,7-14,181. Microsomal epoxide hydrolase has been studied in considerable detail [5,11--141. The cytosol also shows epoxide hydrolase activity which is very efficient for certain alkene oxides [4,9,16]. However, it is unknown whether the cytoplasmic epoxide hydrolase catalyses the hydrolysis of arene oxides and, if so, whether its substrate preference tends to be similar or complementary to that of microsomal hydrolase. Information on this is of great importance to the improvement of rational risk assessment. MATERIALS
AND METHODS
Swiss-Webster mice were received from Professor Maltoni, Bologna, Italy. Male mice of 25-35 g were killed by cervical dislocation. Livers of 4 animals were pooled to prepare microsomes and 100,000 X g supernatant. The methods used for the preparation were: (A) as described [ 171; (B) 9000 X g supernatant was centrifuged at 300,000 X g for 2 h. The microsomal pellet was washed by rehomogenization in 10 mM phosphate buffer (pH 7.4) containing 1.15% KC1 followed by recentrifugation and the 300,000 X g supernatant was dialyzed 3 h against 3 X 3 1 of the same isotonic buffer. The final microsomal pellet was resuspended in isotonic KC1 buffer to give a protein concentration of 8-12 mg/ml while the protein concentration of 100,000 X g supernatant varied between 20-25 mg/ml, determined as described [6]. Assay for epoxide hydrolase activity using the four K-region epoxides derived from polycyclic aromatic hydrocarbon was performed as described [ 21. Truns-stilbene oxide hydrolysis was assayed as follows. To the incubation volume of total 250 ~1 were added 50 ~1 of appropriate buffer to measure cytoplasmic epoxide hydrolase, 0.1 M sodium phosphate buffer (pH 7.0) and for microsomal activity 0.5 M Tris-HCl (pH 9.0), furthermore, 10-100 ~1 cytoplasmic fraction (100-1200 pg protein) or 50-200 ~1 of microsomal suspension (400-2400 pg protein) were added. The incubation was started by addition of [3H] trans-stilbene oxide (3350 dpm/nmol, synthesized as described [ 151; chemical purity >95% and radiochemical purity >99%) in 10 ~1 acetonitrile to give a final concentration of 0.8 mM. After an incubation of 3-10 min at 37”C, the incubation was stopped by extraction into a mixture of 2.5 ml petroleum ether (b.p. 40-6O”C) and 250 ~1 dimethylsulfoxide by shaking the tube on a Vortex mixer for about 5 s followed by rotating the tubes for 3 min at 40 rev/min (Koto-Shaker, Kiihner, Basel, Switzerland). After centrifugation at 400 X g for 1 min, the petroleum ether was removed by aspiration and the extraction wit,h 2.5 ml petroleum ether was repeated. The product 1,2-dihydroxy-1,2_diphenylethane was then extracted into 1 ml of ethyl acetate by rotating the tubes for 5 min. The phases were
171
separated by centrifugation at 400 X g for 1 min and 0.5 ml of the ethyl acetate phase was used for quantitation of the diol by scintillation counting. To ascertain that the entire radioactivity above blank in the ethyl acetate phase was due to 1,2-dihydroxy-1,2_diphenylethane, this phase was concentrated under reduced pressure and chromatographed on silica gel thin layer plates (Merck, Darmstadt, F.R.G.) using benzene/methanol (93 : 7; v/v) as solvent system. RESULTS
AND DISCUSSION
Efficient radiometric extraction assays for the K-region epoxides to be investigated had earlier been established [2] and were used in this study. 4000
0.2
0.L
0.6
0.8
[STANDARD
RF
I DIOL
200’
1.d
TSO
B
I a u
Fig. 1. Radiochromatography of the ethyl acetate fraction obtained after incubation of [?H ] tiuns-stilbene oxide under the conditions adopted for the extraction assay as described in Materials and Methods. (A) Incubation with active cytoplaamic fraction. (B) Incubation with buffer only. The standards were truns-stilbene oxide (TSO) and erythro-1,2-dihydroxy-1,2_diphenylethane (DIOL). The solvent system used was benzene/methanol (93 : 7, v/v).
172
Truns-stilbene oxide is a very poor substrate of microsomal epoxide hydrolase [ 131, but was recently reported to be a good substrate of cytoplasmic epoxide hydrolase (with no indication of an assay since the report was in a meeting’s proceedings) [ 41. It was therefore decided to include truns-stilbene oxide as a positive control in the present study. The procedure for the assay of its hydrolysis is given in the Materials and Methods section. The separation of substrate and product proved to be very efficient in that the recovery of the substrate in the ethyl acetate phase was only 0.3 + 0.03%, while that of the product 1,2-dihydroxy-1,2_diphenylethane was 82 + 2% at either pH used for the incubations. Radiochromatography of the ethyl acetate phase (Fig. 1) showed that the entire radioactivity above blank was confined to the zone with the mobility of the product erythro-1,2-dihydroxy-1,2-diphenylethane. The product formation was linear with respect to time for at least 10 min and with respect to protein concentration at least for the range from 100 to 1200 and 400 to 2400 pg cytoplasmic and microsomal protein, respectively. Furthermore, substrate concentration used was shown to be saturating. All substrates were assayed both with the cytoplasmic and microsomal fractions at a minimum of 2 time points and 3 protein concentrations. For all substrates investigated it was possible to find conditions appropriate for true initial rate determinations where product formation proceeded linearly with respect to both time and amount of protein used. Thorough washing of the microsomal pellet by rehomogenization in fresh medium followed by recentrifugation (procedure ‘B’, see Materials and Methods) reduced the apparent specific epoxide hydrolase activity with truns-stilbene oxide as substrate from 240 + 32 pmol/mg protein/min (obtained after procedure ‘A’) to 45 ? 8 pmol/mg protein/min. The microsomal epoxide hydrolase activity was therefore tested using procedure ‘B’ with all substrates. Conversely the specific epoxide hydrolase activity of the cytoplasmic fraction with phenanthrene 9,10-oxide as substrate was not significantly different (even slightly higher after the more extensive centrifugation: 80 f 17 vs. 69 + 7 pmol/mg protein/min) whether the standard procedure ‘A’ or procedure ‘B’ was used. Nevertheless procedure ‘B’ was used for all substrates. Figure 2 shows that, in marked contrast to truns-stilbene oxide, the specific activity for the investigated K-region epoxides derived from polycyclic aromatic hydrocarbons was lower for the cytoplasmic than for the microsomal fraction. The ratios between the specific activities of the 2 subcellular fractions were 2 for 7-methylbenz[u] anthracene 5,6-oxide, 11 for benz[u] anthracene 5,6-oxide, 160 for phenanthrene 9,10-oxide and > 160 for benzo[u] pyrene 4,5-oxide. With this last epoxide no activity at all could be observed in the cytoplasmic fraction. The limit of sensitivity of the assay with this substrate was 10 pmol/mg protein/min. The activity of cytoplasmic epoxide hydrolase is also much lower for all investigated K-region epoxides compared to the positive control trans-stilbene oxide: 40-fold for 7-methyl-
173 12.5
12.0,
.5 E
2.0
cl a c i 1.5 !i F k % a
1.0
0 z E c 0.5
1I h
i’-MBA0
E
0
PO
d
BP0
Fig. 2. Specific activity of mouse liver cytoplasmic (%) and microsomal (0) epoxide hydrolase for 7-methylbenz[n]anthracene 5,6-oxide (7-MBAO), benz[a]anthracene 5,6oxide (BAO) phenanthrene 9,lOoxide (PO), benzo[a]pyrene 4,5-oxide (BPO) and for trans-stilbene oxide (TSO). Values are means + SD. (duplicate determinations each at 3 protein concentrations and 2 time points). The S.D. for the specific activity of microsomal epoxide hydrolase, which could not be indicated by a bar, was * 750 pmol/mg protein/min n.d. = not detectable (
benz[a] anthracene 5,6-oxide, 14-fold for benz[a] anthracene 5,6-oxide, 23-fold for phenanthrene 9,10-oxide and > 180-fold for benzo[a]pyrene 4,5-oxide. Since in comparison to other animal species, especially the rat, the mouse possesses an unusually high cytoplasmic epoxide hydrolase activity [4,16], it may be expected that for these substrates the contribution to epoxide hydrolysis of the cytoplasmic compared to microsomal hydrolase is much less in many other animal species. Moreover, due to the membranous site of formation and high degree of hydrophobicity of these K-region epoxides it is to be expected that their distribution between cytoplasm and membranes is much in favor of the membranes. Thus, it may well be that the importance of cytoplasmic epoxide hydrolase for the investigated K-region epoxides and possibly also for other epoxides derived from similar polycyclic aromatic hydrocarbons is minor compared to microsomal epoxide hydrolase. On the other hand, it becomes clear from the data presented in Fig. 2
174
that the substrate preference of cytoplasmic epoxide hydrolase is markedly different and, to some extent, complementary to that of microsomal hydrolase. Phenanthrene 9,10-oxide, benzo[a] pyrene 4,5-oxide and benz[a]anthracene 5,6-oxide very poor or no substrates of cytosolic but good substrates of microsomal epoxide hydrolase. Conversely, trans-stilbene oxide used as a positive control is a very poor substrate of microsomal [cf. 131 but a good substrate of cytosolic epoxide hydrolase [cf. 41. Finally, the data in Fig. 2 show that despite this complementarity the substrate specificities of epoxide hydrolase in the 2 subcell&r fractions are not strictly inversely related, but vary independently. Thus, the relative activity of cytosolic epoxide hydrolase must be included in a judgement of the overall situation. ACKNOWLEDGEMENTS
The authors thank A.J. Sparrow and K.L. Platt from their laboratory for radioactive substrates and B.D. Hammock, University of California, Riverside, for early disclosure of results. This work was supported by the Deutsche Forschungsgemeinschaft. REFERENCES Bentley, P. and Oesch, F. (1978) Enzymes involved in activation and inactivation of carcinogens and mutagens. In: Primary Liver Tumors, pp. 239-252. Editors: H. Remmer, H.M. Bolt, P. Bannasch and H. Popper. MTP Press, Falcon House, Lancaster, England. Bentley, P., Schmassmann, H.U., Sims, P. and Oesch, F. (1976) Epoxides derived from various polycyclic hydrocarbons as substrates of homogeneous and microsome bound epoxide hydratase. Eur. J. Biochem., 69,97-103. Gelboin, H.V., Kinoshita, N. and Wiebel, F. (1972) Microsomal hydroxylases: Induction and role in polycyclic hydrocarbon carcinogenesis and toxicity. Fed. Proc., 31,1298-1309. Hammock, B.D., El Tantawy, M., Gill, S.S., Hasagawa, L., Mullin, CA. and Ota, K. (1980) Extramicrosomal epoxide hydration. In: Microsomes, Drug Oxidations, and Chemical Carcinogenesis. Editors: M.J. Coon, A.H. Conney, R.W. Estabrook, H.V. Gelboin, J.R. Gillette and P.J. O’Brian. Academic Press, New York, in press. Jerina, D.M., Lehr, R., Schaefer-Ridder, M., Yagi, H., Karle, J.M., Thakker, D.R., Wood, A.W., Lu, A.Y.H., Ryan, D., West, S., Levin, W. and Conney, A.H. (1977) Bay-region epoxides of dihydrodiols: A concept explaining the mutagenic and carcinogenic activity of benzo(a)pyrene and benz(a)anthracene. In: Origins of Human Cancer, pp. 639-658. Editors: H.H. Hiatt, J.D. Watson and J.A. Winsten. Cold Spring Harbor Laboratory, Cold Spring Harbor. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randah, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275. Heidelberger, C. (1975) Chemical Carcinogenesis. Annu. Rev. Biochem., 44, 79-121. Miller, E.C. and Miller, J.A. (1974) Biochemical mechanisms of chemical carcinogenesis. In: Molecular Biology of Cancer, pp. 377-402. Editor: H. Busch. Academic Press, New York. Mumby, S.M. and Hammock, B.D. (1975) Substrate selectivity and stereochemistry of enzymatic epoxide hydration in the soluble fraction of mouse liver. Pestic. Biochem. Physiol., 11,275-284.
175 10
11
12
13
14
15
16 17
18
Nebert, D.W., Robinson, J.R., Niwa, A., Kumaki, K. and Poland, A.P. (1975) Genetic expression of aryl hydrocarbon hydroxylase activity in the mouse. J. Cell. Physiol., 85,393-414. Oesch, F. (1973) Mammalian epoxide hydrases: Inducible enzymes catalyzing the inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and olefinic compounds. Xenobiotica, 3,305-340. Oesch, F. (1979) Epoxide hydratase. In: Progress in Drug Metabolism, Vol. 3, pp. 253-301. Editors: J.W. Bridges and L.F. Chasseaud. John Wiley and Sons, London. Oesch, F., Kaubisch, N., Jerina, D.M. and Daly, J. (1971) Hepatic epoxide hydrase: Structure-activity relationship for substrates and inhibitors. Biochemistry, 10, 4858-4866. Oesch, F., Bentley, P. and Glatt, H.R. (1977) Epoxide hydratase: Purification to apparent homogeneity as a specific probe for the relative importance of epoxides among other reactive metabolites. In: Biological Reactive Intermediates, pp. 181206. Editors: D.J. Joiiow, J.J. Kocsis, R. Snyder and H. Vainio. Plenum Press, New York, 1977. Oesch, F., Sparrow, A.J. and Platt, K.L. (1980) Radioactively labelled epoxides part II. (1) Tritium-labelled cyclohexene oxide, trans-stilbene oxide and phenanthrene 9,10-oxide. J. Labelled Compd. Radiopharm., 17, 93-102. Ota, K. and Hammock, B. (1980) Epoxide hydration in the cytosolic fraction of mammalian liver. Science, in press. Schmassmann;H.U., Glatt, H.R. and Oesch, F. (1976) A rapid assay for epoxide hydratase activity with benzo(a)pyrene 4,5-(K-region)oxide as substrate. Anal. Biochem., 74,94-104. Sims, P. and Grover, P.L. (1974) Epoxides in polycyclic aromatic hydrocarbon metabolism and carcinogenesis. Adv. Cancer Res., 20,165-274.
169
SPECIFICITY OF MOUSE LIVER CYTOSOLIC EPOXIDE HYDROLASE FOR K-REGION EPOXIDES DERIVED FROM POLYCYCLIC AROMATIC HYDROCARBONS
FRANZ OESCH* and MARIO GOLAN Institute of Pharmacology, Mainz, (F.R.G.)
University of Mainz, Obere Zahlbacher
Strasse 67, D-6500
(Received 8 February 1980) (Accepted 14 February 1980)
SUMMARY
Mouse liver cytosol epoxide hydrolase, known to be very active for certain alkene oxides, had a specific activity which was 2.1-, ll- and 160-fold lower than that of the microsomal epoxide hydrolase for the arene oxides 7-methylbenz[a] anthracene 5,6-oxide, benz [a] anthracene 5,6-oxide and phenanthrene 9,10-oxide, respectively. For benzo[a] pyrene 4,5-oxide no activity (< 10 pmol product/mg protein/min) of cytoplasmic epoxide hydrolase was detectable. The specific activity of cytoplasmic epoxide hydrolase was much lower for all K-region epoxides investigated, compared to truns-stilbene oxide used as a positive control and for which a new assay is described. It is concluded from these rates combined with the fact that these lipophilic K-region epoxides are expected to stay preferentially at membranous sites where they are generated, that cytoplasmic epoxide hydrolase plays a minor role for their transformation compared to membrane-bound hydrolase. The data also show that for the substrates investigated the epoxide hydrolase activities in the cytoplasmic and microsomal fractions are complementary to some extent, but there is no quantitative inverse relationship.
INTRODUCTION
Aromatic and olefinic compounds can be metabolically transformed by mammalian monooxygenases to epoxides. These are electrophilically reactive to varying degrees and many of them exert mutagenic and carcinogenic effects. Some of them can be metabolically inactivated by epoxide hydrolases (EC’ 3.3.2.3). In the case of angular polycyclic aromatic hydrocarbons *To whom correspondence
and reprint requests should be addressed.
170
several classes of epoxides can be metabolically formed including K-region arene oxides and reactive dihydrodiol-bay-region epoxides. In the biosynthesis of the latter the formation of the precursor dihydrodiol by epoxide hydrolases is an essential prerequisite. Thus epoxide hydrolases play a pivotal role in both activation and inactivation [ l,-5,7-14,181. Microsomal epoxide hydrolase has been studied in considerable detail [5,11--141. The cytosol also shows epoxide hydrolase activity which is very efficient for certain alkene oxides [4,9,16]. However, it is unknown whether the cytoplasmic epoxide hydrolase catalyses the hydrolysis of arene oxides and, if so, whether its substrate preference tends to be similar or complementary to that of microsomal hydrolase. Information on this is of great importance to the improvement of rational risk assessment. MATERIALS
AND METHODS
Swiss-Webster mice were received from Professor Maltoni, Bologna, Italy. Male mice of 25-35 g were killed by cervical dislocation. Livers of 4 animals were pooled to prepare microsomes and 100,000 X g supernatant. The methods used for the preparation were: (A) as described [ 171; (B) 9000 X g supernatant was centrifuged at 300,000 X g for 2 h. The microsomal pellet was washed by rehomogenization in 10 mM phosphate buffer (pH 7.4) containing 1.15% KC1 followed by recentrifugation and the 300,000 X g supernatant was dialyzed 3 h against 3 X 3 1 of the same isotonic buffer. The final microsomal pellet was resuspended in isotonic KC1 buffer to give a protein concentration of 8-12 mg/ml while the protein concentration of 100,000 X g supernatant varied between 20-25 mg/ml, determined as described [6]. Assay for epoxide hydrolase activity using the four K-region epoxides derived from polycyclic aromatic hydrocarbon was performed as described [ 21. Truns-stilbene oxide hydrolysis was assayed as follows. To the incubation volume of total 250 ~1 were added 50 ~1 of appropriate buffer to measure cytoplasmic epoxide hydrolase, 0.1 M sodium phosphate buffer (pH 7.0) and for microsomal activity 0.5 M Tris-HCl (pH 9.0), furthermore, 10-100 ~1 cytoplasmic fraction (100-1200 pg protein) or 50-200 ~1 of microsomal suspension (400-2400 pg protein) were added. The incubation was started by addition of [3H] trans-stilbene oxide (3350 dpm/nmol, synthesized as described [ 151; chemical purity >95% and radiochemical purity >99%) in 10 ~1 acetonitrile to give a final concentration of 0.8 mM. After an incubation of 3-10 min at 37”C, the incubation was stopped by extraction into a mixture of 2.5 ml petroleum ether (b.p. 40-6O”C) and 250 ~1 dimethylsulfoxide by shaking the tube on a Vortex mixer for about 5 s followed by rotating the tubes for 3 min at 40 rev/min (Koto-Shaker, Kiihner, Basel, Switzerland). After centrifugation at 400 X g for 1 min, the petroleum ether was removed by aspiration and the extraction wit,h 2.5 ml petroleum ether was repeated. The product 1,2-dihydroxy-1,2_diphenylethane was then extracted into 1 ml of ethyl acetate by rotating the tubes for 5 min. The phases were
171
separated by centrifugation at 400 X g for 1 min and 0.5 ml of the ethyl acetate phase was used for quantitation of the diol by scintillation counting. To ascertain that the entire radioactivity above blank in the ethyl acetate phase was due to 1,2-dihydroxy-1,2_diphenylethane, this phase was concentrated under reduced pressure and chromatographed on silica gel thin layer plates (Merck, Darmstadt, F.R.G.) using benzene/methanol (93 : 7; v/v) as solvent system. RESULTS
AND DISCUSSION
Efficient radiometric extraction assays for the K-region epoxides to be investigated had earlier been established [2] and were used in this study. 4000
0.2
0.L
0.6
0.8
[STANDARD
RF
I DIOL
200’
1.d
TSO
B
I a u
Fig. 1. Radiochromatography of the ethyl acetate fraction obtained after incubation of [?H ] tiuns-stilbene oxide under the conditions adopted for the extraction assay as described in Materials and Methods. (A) Incubation with active cytoplaamic fraction. (B) Incubation with buffer only. The standards were truns-stilbene oxide (TSO) and erythro-1,2-dihydroxy-1,2_diphenylethane (DIOL). The solvent system used was benzene/methanol (93 : 7, v/v).
172
Truns-stilbene oxide is a very poor substrate of microsomal epoxide hydrolase [ 131, but was recently reported to be a good substrate of cytoplasmic epoxide hydrolase (with no indication of an assay since the report was in a meeting’s proceedings) [ 41. It was therefore decided to include truns-stilbene oxide as a positive control in the present study. The procedure for the assay of its hydrolysis is given in the Materials and Methods section. The separation of substrate and product proved to be very efficient in that the recovery of the substrate in the ethyl acetate phase was only 0.3 + 0.03%, while that of the product 1,2-dihydroxy-1,2_diphenylethane was 82 + 2% at either pH used for the incubations. Radiochromatography of the ethyl acetate phase (Fig. 1) showed that the entire radioactivity above blank was confined to the zone with the mobility of the product erythro-1,2-dihydroxy-1,2-diphenylethane. The product formation was linear with respect to time for at least 10 min and with respect to protein concentration at least for the range from 100 to 1200 and 400 to 2400 pg cytoplasmic and microsomal protein, respectively. Furthermore, substrate concentration used was shown to be saturating. All substrates were assayed both with the cytoplasmic and microsomal fractions at a minimum of 2 time points and 3 protein concentrations. For all substrates investigated it was possible to find conditions appropriate for true initial rate determinations where product formation proceeded linearly with respect to both time and amount of protein used. Thorough washing of the microsomal pellet by rehomogenization in fresh medium followed by recentrifugation (procedure ‘B’, see Materials and Methods) reduced the apparent specific epoxide hydrolase activity with truns-stilbene oxide as substrate from 240 + 32 pmol/mg protein/min (obtained after procedure ‘A’) to 45 ? 8 pmol/mg protein/min. The microsomal epoxide hydrolase activity was therefore tested using procedure ‘B’ with all substrates. Conversely the specific epoxide hydrolase activity of the cytoplasmic fraction with phenanthrene 9,10-oxide as substrate was not significantly different (even slightly higher after the more extensive centrifugation: 80 f 17 vs. 69 + 7 pmol/mg protein/min) whether the standard procedure ‘A’ or procedure ‘B’ was used. Nevertheless procedure ‘B’ was used for all substrates. Figure 2 shows that, in marked contrast to truns-stilbene oxide, the specific activity for the investigated K-region epoxides derived from polycyclic aromatic hydrocarbons was lower for the cytoplasmic than for the microsomal fraction. The ratios between the specific activities of the 2 subcellular fractions were 2 for 7-methylbenz[u] anthracene 5,6-oxide, 11 for benz[u] anthracene 5,6-oxide, 160 for phenanthrene 9,10-oxide and > 160 for benzo[u] pyrene 4,5-oxide. With this last epoxide no activity at all could be observed in the cytoplasmic fraction. The limit of sensitivity of the assay with this substrate was 10 pmol/mg protein/min. The activity of cytoplasmic epoxide hydrolase is also much lower for all investigated K-region epoxides compared to the positive control trans-stilbene oxide: 40-fold for 7-methyl-
173 12.5
12.0,
.5 E
2.0
cl a c i 1.5 !i F k % a
1.0
0 z E c 0.5
1I h
i’-MBA0
E
0
PO
d
BP0
Fig. 2. Specific activity of mouse liver cytoplasmic (%) and microsomal (0) epoxide hydrolase for 7-methylbenz[n]anthracene 5,6-oxide (7-MBAO), benz[a]anthracene 5,6oxide (BAO) phenanthrene 9,lOoxide (PO), benzo[a]pyrene 4,5-oxide (BPO) and for trans-stilbene oxide (TSO). Values are means + SD. (duplicate determinations each at 3 protein concentrations and 2 time points). The S.D. for the specific activity of microsomal epoxide hydrolase, which could not be indicated by a bar, was * 750 pmol/mg protein/min n.d. = not detectable (
benz[a] anthracene 5,6-oxide, 14-fold for benz[a] anthracene 5,6-oxide, 23-fold for phenanthrene 9,10-oxide and > 180-fold for benzo[a]pyrene 4,5-oxide. Since in comparison to other animal species, especially the rat, the mouse possesses an unusually high cytoplasmic epoxide hydrolase activity [4,16], it may be expected that for these substrates the contribution to epoxide hydrolysis of the cytoplasmic compared to microsomal hydrolase is much less in many other animal species. Moreover, due to the membranous site of formation and high degree of hydrophobicity of these K-region epoxides it is to be expected that their distribution between cytoplasm and membranes is much in favor of the membranes. Thus, it may well be that the importance of cytoplasmic epoxide hydrolase for the investigated K-region epoxides and possibly also for other epoxides derived from similar polycyclic aromatic hydrocarbons is minor compared to microsomal epoxide hydrolase. On the other hand, it becomes clear from the data presented in Fig. 2
174
that the substrate preference of cytoplasmic epoxide hydrolase is markedly different and, to some extent, complementary to that of microsomal hydrolase. Phenanthrene 9,10-oxide, benzo[a] pyrene 4,5-oxide and benz[a]anthracene 5,6-oxide very poor or no substrates of cytosolic but good substrates of microsomal epoxide hydrolase. Conversely, trans-stilbene oxide used as a positive control is a very poor substrate of microsomal [cf. 131 but a good substrate of cytosolic epoxide hydrolase [cf. 41. Finally, the data in Fig. 2 show that despite this complementarity the substrate specificities of epoxide hydrolase in the 2 subcell&r fractions are not strictly inversely related, but vary independently. Thus, the relative activity of cytosolic epoxide hydrolase must be included in a judgement of the overall situation. ACKNOWLEDGEMENTS
The authors thank A.J. Sparrow and K.L. Platt from their laboratory for radioactive substrates and B.D. Hammock, University of California, Riverside, for early disclosure of results. This work was supported by the Deutsche Forschungsgemeinschaft. REFERENCES Bentley, P. and Oesch, F. (1978) Enzymes involved in activation and inactivation of carcinogens and mutagens. In: Primary Liver Tumors, pp. 239-252. Editors: H. Remmer, H.M. Bolt, P. Bannasch and H. Popper. MTP Press, Falcon House, Lancaster, England. Bentley, P., Schmassmann, H.U., Sims, P. and Oesch, F. (1976) Epoxides derived from various polycyclic hydrocarbons as substrates of homogeneous and microsome bound epoxide hydratase. Eur. J. Biochem., 69,97-103. Gelboin, H.V., Kinoshita, N. and Wiebel, F. (1972) Microsomal hydroxylases: Induction and role in polycyclic hydrocarbon carcinogenesis and toxicity. Fed. Proc., 31,1298-1309. Hammock, B.D., El Tantawy, M., Gill, S.S., Hasagawa, L., Mullin, CA. and Ota, K. (1980) Extramicrosomal epoxide hydration. In: Microsomes, Drug Oxidations, and Chemical Carcinogenesis. Editors: M.J. Coon, A.H. Conney, R.W. Estabrook, H.V. Gelboin, J.R. Gillette and P.J. O’Brian. Academic Press, New York, in press. Jerina, D.M., Lehr, R., Schaefer-Ridder, M., Yagi, H., Karle, J.M., Thakker, D.R., Wood, A.W., Lu, A.Y.H., Ryan, D., West, S., Levin, W. and Conney, A.H. (1977) Bay-region epoxides of dihydrodiols: A concept explaining the mutagenic and carcinogenic activity of benzo(a)pyrene and benz(a)anthracene. In: Origins of Human Cancer, pp. 639-658. Editors: H.H. Hiatt, J.D. Watson and J.A. Winsten. Cold Spring Harbor Laboratory, Cold Spring Harbor. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randah, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275. Heidelberger, C. (1975) Chemical Carcinogenesis. Annu. Rev. Biochem., 44, 79-121. Miller, E.C. and Miller, J.A. (1974) Biochemical mechanisms of chemical carcinogenesis. In: Molecular Biology of Cancer, pp. 377-402. Editor: H. Busch. Academic Press, New York. Mumby, S.M. and Hammock, B.D. (1975) Substrate selectivity and stereochemistry of enzymatic epoxide hydration in the soluble fraction of mouse liver. Pestic. Biochem. Physiol., 11,275-284.
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