Camp. Biochem. Physiol. Printed in Great Britain
Vol. 103C,
No. I, pp. 207-214,
0306~4492/92 $5.00+ 0.00 0 1992Pergamon Press Ltd
1992
BIOCHEMICAL CHARACTERIZATION OF A VARIANT FORM OF CYTOSOLIC EPOXIDE HYDROLASE INDUCED BY PARENTAL EXPOSURE TO N-ETHYL-N-NITROSOUREA JAFFAR NOUROOZ-ZADEH,*~ BRUCE S. WINDER,* ERIC C. DIETZE,*$ CAROL S. GIOMETTI,~ SANDRA L, TOLLAKSEN~and BRUCE D. HAMMOCK*11 *Departments of Entomology and Environmental Toxicology, University of California, Davis, CA 95616, U.S.A. (Received 24 December 199 1)
Abstract-l. ENU4 mice express a protein variant originally detected in a CBFl mouse sired by a C57BL/6 mouse exposed to N-ethyl-IV-nitrosourea. It appears to be an isolelectric point variant of cytosolic epoxide hydrolase. Affinity purified cytosolic epoxide hydrolase from ENU4 mice has a pI of approximately 5.1 compared to 5.6 in other mouse strains. 2. Clofibrate induced cytosolic epoxide hydrolase to similar levels in five strains of mice. However, CBFI and ENW mice were more sensitive to the induction of palmitoyl CoA oxidase activity. 3. Except for isoelectric point, the physico- and immunochemical properties of cytosolic epoxide hydrolase from ENU4 mice were similar to those of the other mouse strains. Substrate specificities for five of six substrates tested were also similar
INTRODUCTION Living organisms are continuously exposed to a variety of man-made and naturally occurring epoxides, some of which can spontaneously alkylate nucleophilic moieties of tissue macromolecules such as DNA, RNA and proteins. In mammals, epoxides are metabolized by two families of enzymes. GlutathioneS-transferases (GST) (EC 2.5.1.18) catalyze a nucleophilic attack of a sulfur anion on the epoxide ring leading to formation of glutathione conjugates. Epoxide hydrolases (EH) (EC 3.3.2.3) convert epox-
ides to 1,2-diols through addition of water to the oxirane ring. Different forms of EH have been characterized. For example, cholesterol epoxide hydrolase selectively hydrates cholesterol epoxides and is predominantly microsomal (Watabe et al., tPresent address: William Harvey Research Institute, St Bartholomew’s Hospital Medical College, Charterhouse Square, London EClM 6BQ. SPresent address: Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195, U.S.A. §Argonne National Laboratory, 9700 S. Cass Avenue, Argonne, Illinois 60439, U.S.A. /IAuthor to whom correspondence should be addressed. List of abbreviations: cEH, cytosolic epoxide hydrolase; CNEH, ci.s-4-nitrophenyl-3,4-epoxyhexanoate; DFP, diisopropyl fluorophosphate; EDTA, ethylenediaminetetraacetic acid; ENP-5, truns-1,2-epoxy-1-(4-nitrophenyl)-pentane; ENU, N-ethyl-N-nitrosourea; EQU-5, truns-1,2-epoxy-l-(2-quinolyl)-pentane; F-CHO, 4fluorochalcone oxide; mEH, microsomal epoxide hydrolase; PI, isoelectric point; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; S-NEPC, 4nitrophenyl-(2S,3S)-2,3-epoxy-3-phenylpropylcarbonate; TNEH, rruns-4-nitrophenyl-3,4_epoxyhexanoate; TSO, trans-stilbene oxide; 2DE, two-dimensional electrophoresis.
1981; Finley et nl., 1988) while epoxide hydrolases selective for leukotriene A4 are located in the cytosol (Haeggstrom et al., 1986; Miki et al., 1989; Pace-Asciak and Lee, 1989). Neither of these enzymes, however, appear to be involved in the metabolism of xenobiotics. Cytosolic epoxide hydrolase (cEH) rapidly metabolizes a variety of epoxides on aliphatic systems (Mumby and Hammock, 1979; Hammock and Hasagawa, 1983). Microsomal epoxide hydrolase (mEH) hydrates epoxides on arene systems and some epoxides on aliphatic systems (Oesch, 1973; Magdalou and Hammock, 1988). In addition, cEH and mEH are distinguished by pH optima, induction, isoelectric point, molecular weight and antigenic determinants. N-ethyl-N-nitrosoura (ENU) is known to be potent mutagen in vitro and in vivo (Russell et al., 1979). Using two-dimensional electrophoresis (2DE), ENU was shown to induce the heritable expression of liver protein variants in a small number of the offspring of exposed sires (Giometti et al., 1987). Each of these variants was characterized by a corresponding decrease of approximately 50% in the abundance of a neighboring protein. Of the five protein variants described, one has been identified as an isoelectric point variant of ornithine aminotransferase (OAT: Giometti et al., 1988). This identification was based on the comigration of OAT purified from a normal mouse with a normal liver protein observed as reduced in abundance by approximately 50% in mice expressing one of the ENU-induced liver protein variants, and on the comigration of a protein with OAT activity with the ENU2 variant protein. Western blot analysis with anti-OAT antisera confirmed the identification of both the normal and variant forms of OAT. 207
J. NOUROOZ-ZADEHet al.
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In this paper, we describe a similar characterization of a second ENU-induced liver protein variant referred to in the original paper as ENU4. A preliminary experiment (unpublished) indicated that cEH purified from CBFl mice (Fl-hybrid offspring of C57BL/6 males and Balb/c females) comigrated with the protein reduced by 50% in the original mouse expressing the ENU4 variant. We pursued this finding and present data indicating that the ENU4 variant is an altered form of cEH that has catalytic properties essentially identical to those of cEH purified from other mouse strains with the exception of an increased inducibility of palmitoyl CoA oxidase by clofibrate. MATERIALS AND METHODS
Reagents tram-Stilbene oxide (TSO) was purchased from Aldrich Chemical Co., Milwaukee, WI. 4-Fluorochalcone oxide (F-CHO) was synthesized according to Mullin and Hammock (1982). tram-1.2-Enoxv-1-(2-auinolvl)-oentane (EQUS) and ’ rrans-1,2-epoxy- i-(4-nitropheny&cntane (ENPS) were synthesized as described elsewhere (Wixtrom and Hammock, 1988). 4-Nitrophenyl-(2S,3S)-2,3-epoxy-3phenylpropylcarbonate (S-NEPC), cis-4-nitrophenyl-3,4epoxyhexanoate (CNEH) and tram-4-nitrophenyl-3,4epoxyhexanoate (TNEH) were prepared according to Dietze et al. (1991). [3H]-TS0 was prepared as described by Gill et al. (1983). Other chemicals were the highest quality commercially available. Animal treatment and subcellular fractionation Balb/c, C57BL/6 and Swiss-Webster mice (Mus musculus) were obtained from Charles River Labs, Cambridge, MA. CBFI and ENW (7-8 weeks old) were obtained from Argonne National Laboratory, Argonne, IL. The CBFl mice were the first generation offspring of crosses between C57BL/6 males and Balb/c females and represent the genetic background of the ENW stock. The ENU4 stock, homozygous for the liver protein variant (Giometti et a/., 1987) was generated first by backcrossing to C57BL/6 followed by sibling crosses of mice heterozygous for the ENU4 variant. The homozygous ENU4 stock is now maintained by sibling crosses of homozygous individuals. The mice were fed Purina Laboratories chow (ad lib&urn) for one week. Thereafter, control mice received ground chow containing 5% corn oil for 14 days while the treated mice received the chow containing 5% corn oil plus 0.5% clofibrate. The animals were killed by cervical dislocation. Livers were removed and were washed with chilled 75 mM Na/K phosphate buffer @H 7.4) containing 0.1 mM ethylenediaminetetraacetic acid (EDTA) and diisopropyl fluorophosphate (DFP). The livers were weighed and homogenized in the phosphate buffer to give a 20% (w/v) suspension. Subcellular fractionation was performed as described elsewhere (Hammock and Ota, 1983). The cytosolic fraction (100,000 g) was immediately stored at - 70°C until analysis. Preparation of ajinity gel and enzyme purification Benzylthiol-Sepharose CL-6B was prepared as described by Wixtrom et al. (1988). Enzyme purification was performed at 4°C. In a typical experiment, 1 ml of the benzylthiol gel was sequentially washed with ethanol, water and phosphate buffer (pH 7.4) containing 0.1 mM EDTA. The gel was poured into an Econo column (5 mm id., Bio-Rad Laboratories, Richmond, CA). Cytosol (10%; total volume 15 ml) was pumped through the-column at a flow rate of 0.2 ml/min and the gel was subsequently washed with 45 ml of phosphate buffer. cEH was specifically released by wash-
ing the gel with 4 ml 0.2 mM F-CHO. Aliquots (0.5 ml) were collected and were tested for protein content (Bradford reagents, Bio-Rad). Fractions with high protein content were pooled. After dialysis against phosphate buffer for 2 hr, the affinity purified cEH was stored at 4°C until used. Protein contents were determined using Bradford reagent with the modification suggested by Moody et al. (1985). Enzyme assays cEH activity was monitored using partitionand spectrophotometric assays. The partition assay with [3H]-TS0 was performed as described by Hammock et al. (1985). Photometric assays were carried out on a Varian/Cary 219 spectrophotometer. EQUS and ENPS assays were performed as described elsewhere (Wixtrom and Hammock, 1988). The EQUS assay is based on a shift in absorbance from 320 to 312nm. The ENPS assay was monitored at 302 nm and is based on a decrease in absorbance. Spectrophotometric assays with CNEH, TNEH and S-NEPC were performed according to Dietze et al. (1991). Release of p-nitrophenol was monitored at 405 nm. Palmitoyl-CoA assay was performed using the spectrophotometric method described by Small et al. (1985). The assay is based on oxidation of Leuco-DCF by H,O* produced by palmitoyl CoA-oxidase. Gel electrophoresis and Western blotting Discontinuous sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on a Bio-Rad minislab apparatus. Proteins (affinity purified cEH or cytosol) were separated on 10% SDS gels with 3% stacking gels. The gels were stained with 0.1% Coomassie Brilliant Blue R-250 in methanol/water/acetic acid (45/45/10; v/v/v). For Western blotting, affinity purified liver cEH or cytosol were separated by SDS-PAGE and subsequently electrophoretically transferred to a nitrocellulose membrane (Bio-Rad) at 110 mA for 1 hr in a Hoefer semi-dry blotter (Hoefer Sci., San Francisco, CA). Immunoblotting was carried out according to the procedure recommended by Bio-Rad using rabbit anti-rhesus monkey liver cEH and alkaline phosphatase-conjugated anti-rabbit IgG (ICN Immunobiological, Irvine, CA). A screen of antibodies to cEH from different sources revealed this one to be the most suitable for use on Western blots (data not shown). Flatbed isoelectric focusing (IEF) was nerformed on a LKB 2117 Multiphor apparatus using ire-packed gels (OH 3.5-10 or 556.5. LKB Bromma. Sweden). Lanes were ibaded (affinity purified cEH or cytosol) and the gel was focused according to the manufacturer’s recommendations. The lanes were cut into 2mm slices and incubated with 300~1 phosphate buffer containing 1OOpg bovine serum albumin/ml and 0.1 mM EDTA for 3 hr before measurement of activity. For determination of pH gradients, 5 mm slices were cut and incubated in 300~1 20mM potassium chloride for 2 hr. Amino acid analysis Analysis was performed at the UC Davis Protein Structure Laboratory. Purified mouse liver cEH (S&l00 pg) was hydrolyzed on 6 M HCl with or without prior performic acid oxidation. Amino acids were derivatized with ninhydrin and quantified on a Beckman 6300 amino acid analyzer using a-amino-guanidino propionic acid as the internal standard. RESULTS
Clofibrate treatment of CBFl and ENU4 mice for two weeks caused no significant change in body weight when compared to those receiving the unsupplemented diet for the same period. On the other hand, an increase in liver weights was observed in the
Altered epoxide hydrolase Table I. Effects of clofibrate
treatmenton body
209
weight, liver weight and hepatic cytosolic activities in five strains of mice
expoxide hydrolase
(cEH) and palmitoyl
CoA
Activities
Strain
Treatment
CBFI
Control Clolibrate Control Clofibrate Control Clolibrate Co”trol Clofibrate Control Clofibrate
ENW Balb/c C57/BL SW
Number of animals IO IO 10 IO IO
10 10 IO 10 10
Body weight In) 34.0 * 3.0 32.9 + 2.4 30.7 * 1.2 27.0 & 1.3 24.5 + 1.6 23.3 + 1.4 25.2 f I .8 25.2 _+ 1.8 27.6 f 15 28. I + 2.0
Liver weight In)
Liver weight as % of body weight
I .97 f 0.2 2.62 + 0.3’ 1.7kO.l 2.02 + 0.2c 1.2610.1 1.44+0.1C 1.62 + 0.2 2.13 + 0.3’ I .53 + 0.2 1.94+ 0.2”
5.76 f 0.1 7.96 + O.@ 5.5 + 0.3 7.48 + 0.4d 5.14 f 0.2 6.18f0.2’ 6.44 + 0.4 8.44 2 0.9d 5.56 f 0.3 6.89 + 0.4d
Palmitoyl CoA’ (mODimin/mn)
cEHb (fimol/min/mg) 0.006 0.018 0.007 0.013 0.008 0.026 0.012 0.026 0.015 0.023
11.1 + 1.6 173k29’ 22.2 + 5.4 440+51c 36.9 + 7.3 139 f 14e 46.6 _+3.5 652 + 82’ 57.7 + 2.0 537 + 104’
a: Palmitoyl CoA activity was monitored according to Small et al. (1985). The values are the mea” of quadruplicate b: cEH activity in cytosol was monitored with the TSO partition assay according to Hammock ef al. (1985). c,d,e,f: Means in control and treated groups within each strain were significantly different (P < 0.03).
clofibrate treated mice compared to the controls (Table 1). Balb/c, C57BL/6 and SW mice also responded to the dietary clofibrate in the same fashion, judged by comparing body and liver weights. In this study, none of the Balb/c, C57BL6, SW or CBFl mice showed any signs of toxicity during the exposure to dietary clofibrate. In contrast, the ENU4 strain was more susceptible to the exposure to clofibrate with only about 70% of the original stock surviving the drug treatment. The cause of death in these mice is not known. The induction of palmitoyl CoA oxidase by dietary clofibrate was monitored by measuring the rate of H,O, production. The analysis showed that the rates of H,O, production by palmitoyl-CoA oxidase in liver cytosol from untreated CBFl and ENU4 mice were 11 and 22 mOD/min/mg protein, respectively. The corresponding rates in the liver cytosols from the clofibrate-treated mice were 173 and 440mOD/min/mg protein. The rates of H202 production in liver cytosols from untreated Balb/c, C57BL/6 and SW mice varied from 36 to 57 mOD/min/mg protein. The corresponding rates of H,Oz production in the clofibrate-treated mice ranged from 139 to 652 mOD/min/mg protein (Table 1). Comparison of the ratios of the H,O, produced in the clofibrate-treated mice to that of the corresponding controls is presented in Table 1. To facilitate comparison of the hepatic cEH activities from the hybrid CBFl and ENU4 strains with the inbred strains of mice, pH profile studies were performed. Enzyme activity was monitored using the partition assay over a pH range of 5 to 10 using Na/K phosphate and Tris buffers. cEH activity in the cytosols of CBFl, ENU4 and SW strains (clofibratetreated and untreated) increased in catalytic activity towards TSO in the pH range between 6 and 7.8. Maximum catalytic activity was obtained at pH 7.0 (Fig. 1). In this study, the partition assay with TSO was performed at pH 7.4 to obtain specific activity data which allowed direct comparison with previous work. Liver cytosols from untreated CBFI and ENU4 mice hydrated TSO at a rate of 0.007 pmol/min/mg protein. Cytosol from clofibrate-treated CBFl mice hydrated TSO at a rate approximately three times higher when compared to the controls (Table 1). No significant induction of cEH activity was observed in
+ + + * + + * + * f
0.0003 0.002’ 0.002 0.002’ 0.002 0.005’ 0.001 0.006’ 0.003 0.005’
determinations.
the cytosols from clofibrate-induced ENU4 mice. Cytosols from untreated Balb/c, C57BL/6 and SW mice metabolized TSO at a rate between 0.008 and 0.015 pmol/min/mg protein. The corresponding values for the clofibrate-treated mice varied from 0.023 to O.O26pmol/min/mg (Table 1). Since the protein contents of the liver cytosols were not affected by the dietary clofibrate, the increase in specific activities reflects an increase in total cEH activities (data not shown). While these findings support earlier reports on the coupling between peroxisome proliferation and induction of cEH (Hammock and Ota,
1
E 4 2 _-
5
4
5
’
’
a
’
lo
g
10
ENU4 pH Profile
6
11
pH’Gra&mt
Fig. 1. pH profile of catalytic activity towards TSO in cytosol preparations. Epoxide hydrolase activity was measured at different pHs using 75 mM Na/K phosphate (PH 5-8) or 100 mM Tris-HCl (pH 8-10) buffers. Values are the means of triplicate analyses and expressed as nmol/min/mg protein. These experiments were repeated with different preparations with similar results. Keys: A, CBFl
liver cytosol; B, ENW liver cytosol; C, SwissWebster mouse (SW) liver cytosol.
J. NOUROOZ-ZADEH et al.
210
1983; Oesch et al., 1988), it is evident that the correlation between cEH and palmitoyl CoA oxidase is not the same among the five strains of mice and follows the order ENU4 < CBFl < C57BL/6 < SW < Balb/c. Figure 2 presents a Coomassie stained SDS gel and a Western blot of liver cytosols and purified cEH from CBFl, ENU4 and SW. SDS-PAGE analysis of the purified cEH from the five strains of mice indicated the presence of a single band at 62 kDa. Western blots of the affinity purified cEHs revealed that the protein at 62 kDa cross-reacted with rabbit antibodies raised to rhesus hepatic cEH. A similar pattern was also observed with the cytosol preparations from the different strains of mice (clofibrate-treated and untreated). To evaluate whether the affinity purified proteins with the same apparent molecular weight from the
different strains of mice were the same enzymes, specific activities were compared. Table 2 shows specific activities for purified liver cEH from the different strains of mice (untreated) using a battery of selected spectrophotometric substrates whose structures are shown (Fig. 3). The purified enzymes from the five different strains of mice possessed similar hydrolytic activities towards CNEH (approximately 1.5 pmol/min/mg protein), EQU5 (3.5-3.9 pmol/ min/mg) and S-NEPC (6.6-8.0 pmol/min/mg). With TNEH, the enzymes from CBFl, ENU4, C57BL/6 and SW metabolized the substrate at similar rates (5.9-6.5 flmol/min/mg) whereas a lower hydration rate was observed with the Balb/c enzyme (4.2 pmol/min/mg). Interestingly, the affinity purified enzymes from CBFl and ENU4 mice exhibited higher activity towards ENPS (12 and 13 pmol/ min/mg, respectively) than those obtained from
kDa 97 66 45 31 21 ABCDEFGHI
J
kDa
ABCDEFGHIJ Fig. 2. 10% SDS-PAGE and Western blot of mouse liver cytosol and purified cytosolic epoxide hydrolase (cEH). The gel (top) was loaded with 3 ng purified cEH and 25 pg of cytosol. An identical gel was blotted to nitrocellulose (bottom) and probed with rabbit-anti-rhesus monkey liver cEH (1: 250). The secondary antibody was goat-anti-rabbit-IgG (1: 2000). Separate gels indicated that purified cEH and cEH in cytosol from all strains of mice. with and without clofibrate treatment. showed the same mobility bv Western blot. Keys: A. Molecular weight standards, phosphorylase b, 9? kDa; BSA, 66 kDa; o&bumin, 45 kDa; carbonic anhvdrase. 31 kDa: sovbean trvnsin inhibitor. 21 kDa. B. ENU4 cEH. C. CBFl cEH. D. Swiss-Webster cEH. E. Swiss-Webster cytosol, clofibrate treated. F. ENU4 cytosol, clofibrate treated. G. ENU4 cytosol, no clofibrate. H. CBFl cytosol, clofibrate treated. I. CBFl cytosol, no clofibrate. J. Molecular weight standards as in A.
Altered epoxide hydrolase
211
Table 2. Soecific activitv of ourilied cvtosolic eooxide hvdrolase with selected substrates Substrate
Concentration (u M)
CNEHb EQU-5’ S-NEPCb TNEHb ENP-5’ TSO’
200 500 50 200 50 50
CBFI 1.6kO.l 3.8 f 0.1 8.0 + 0.3 6.5 f 0.1 I3 f I.5 2.6 k 0.3
Specific activity (~mol/min/mg) ENU4 Balbic C57B116 1.5 f 0.1 3.9 f 0.3 7.7 * 0.3 5.9kO.l 12kO.5 I.9 f 0.2
1.3+0.3 3.5* I.1 6.9 f 0.2 4.2 f 0.5 9.1 + 0.2 2.7 f 0.1
1.5kO.2 3.7 f 0.8 6.6 * 0.2 6.0 rt 0.3 9.4 f 0.5 2.0 f 0.3
Swiss-Webster 1.5 * 3.5 f 7.4 i 5.9 f II + 2.1 +
0.2 0.3 0.8 0.3 0.2 0.2
a: EQU-5, ENP-5 were monitored (25°C in 76 mM phosphate buffer, pH 7.4, 0. I mM EDTA, 0. I mg/ml BSA) according to Wixtrom and Hammock (1988). b: CNEH, TNEH and S-NEPC assayed (25°C in 76mM phosphate buffer, pH 6.4, 0.1 mM EDTA, 2.5 mg/ml BSA) according to Dietze et al. (1991). c: TSO was assayed (37°C in 76 mM phosphate buffer, pH 7.4.0.1 mM EDTA, 0.1 mg/ml BSA) according to Hammock et al. (1985). The values for a and b were the means for quadruplicate determinations. The values for c were the means for triplicate determinations. The assays were performed on one preparation of cEH from clofibrate-treated mice of each strain.
Balb/c, C57BL/6 and SW mice (9.0-l 1pmol/ min/mg). The affinity purified enzymes from CBFl and ENU4 mice hydrated TSO at a rate of 2.6 and 1.9 nmol/min/mg protein, respectively, which was similar to that for Balb/c, C57BL/6 and SW (2.0-2.7 ~mol/min/mg). Affinity purified cEHs from the five strains of mice were also analyzed to determine kinetic rate constants for TSO hydration using the partition assay. The concentration of TSO ranged between 0.1 and 12mM. Hydrolytic activities in the affinity purified cEHs from clofibrate-treated and untreated mice obeyed Michaelis-Menten kinetics. The data were plotted according to Lineweaver-Burke and apparent kinetic constants were determined. The K, and V,,,., for cEHs from the different strains of mice are presented in Table 3. Although there is variation in the values determined for K, and V,,,,,, these are within the range of experimental variation normally seen among enzyme preparations. Thus one can only conclude that there are no major differences in these kinetic parameters between the ENU4 or CBFl strains and the other strains tested. In addition, we observed no significant effect of clofibrate on these parameters. Isoelectric points have been reported for cEH from Balb/c, C57BL/6 and SW (Loury et al., 1985; Meijer and DePierre, 1985; Hammock et al., 1986; Dietze
et al., 1990). To check for similarity among the pIs of the hydrolytic enzymes from the five strains of mice (clofibrate-treated and untreated), liver cytosols were initially analyzed using both wide and narrow range IEF-gels. Analysis of hydrolytic activity towards TSO indicated that enzyme activities in liver cytosols from untreated Balb/c, C57BL/6, CBFl and SW mice focused as single peaks at a p1 of 5.6 (Fig. 4). Analysis of liver cytosols (clofibrate-treated and untreated) from ENU4 mice revealed that enzymatic activity focused at a p1 of 5.1 was consistent with the shift in p1 reported previously (Giometti et al., 1987). IEF analysis of the affinity purified cEH from the different strains of mice (clofibrate-treated and untreated) gave the same pI values as those obtained with cytosol preparations. A minor, more acidic peak was sporadically detected. Similar observations have been reported by other investigators. Affinity purified cEHs from clofibrate treated CBFl, ENU4 and SW mice were analyzed for amino acid composition (Table 4). Within the error of the analysis, the amino acid compositions of the cEHs from the five strains of mice were identical. DISCUSSION
A number of studies have shown that clofibrate belongs to a group of chemicals known as
TSO
EQUS
ENPS
S-NEPC
TNEH
CNEH
Fig. 3. Structures of substances for cEH. mm-Stilbene oxide (TSO). truns-1,2-Epoxy-I-(2-quinolyl)-pentane (EQUS). mm-1,2-Epoxy-1-(4-nitrophenyl)-pentane (ENPS). 4-Nitrophenyl-(2S,3S)-2,3-epoxy-3phenylpropylcarbonate (S-NEPC). mm-4-Nitrophenyl-3,4-epoxyhexanoate (TNEH). cis4-Nitrophenyl3,4-epoxyhexanoate (CNEH).
J. NOUROOZ-ZADEH
212
Table 3. Table of kinetic values from clolibrate-treated and untreated mutagenized and normal mice with TSO as the substrate*
strain ENU4 CBFI C57/Bl6 Balb/c Swiss-Webster
Clofibrate
Km (u M)
+ f _ + + + -
I1 * 1.3 23 + I.7 6.2 + 1.9 10 + 6.0 7.0 ?c 0.8 25i 12 8.5 i 3.6 6.6 + I .6 4.3 + 0.3 9.6 + 3.5
VInPI (umoliminim~) 5.3 3.0 6.5 3.7 3.6 4.2 7.5 3.7 3.2 2.6
* * f f f k + + f +
0.5 0.2 3.2 0.9 0.4 1.6 3.0 0.8 0.2 0.6
*Assays carried out in triplicate with one CEH preparation. All assays performed at 37” in 76 mM phosphate buffer (pH = 7.4, 0.1mM EDTA, 0.1mg/ml BSA). Kinetic values determined from Lineweaver-Burke plots.
peroxisome proliferating agents, some of which have een shown to induce cEH. Among the enzymes associated with peroxisomes are palmitoyl-CoA oxidase and catalase. In several studies, palmitoyl-CoA oxidase has been shown to be induced by clofibrate while catalase appears not to be and may actually decrease (Butler et al., 1988; Lundgren and DePierre, 1989). One of the consequences of the apparent imbalance between elevated H,O, production by palmitoylCoA oxidase and lower degradation of H,Oz by catalase is increased levels of hydroxyl radicals. These may in turn lead to lipid peroxidation and damage to DNA and cellular membranes (Reddy and Lalwai, 1983; Bentley et al., 1988; Moody et al., 1991). Since epoxidized fatty acids have been shown to be excellent substrates for cEH (Halarnkar et al., 1989), the induction of cEH by peroxisome proliferating agents may be critical to the protection of the cell from damage by oxidized lipids. 6,000 ,
, 6.5
Fig. 4. Plot of cytosolic epoxide hydrolase activities on a typical narrow range isoelectric focusing gel for five mouse strains. Gels were prefocused for 30 min. Cytosols from five strains of mice (100 pg) were loaded and focused for 3 hr. The gel was cut into 2 mm slices and the slices eluted in phosphate buffer (ph 7.4) for 3 hr. The pH gradient was measured from 5mm slices of a parallel channel eluted into 20mM KCl. Only a single acidic peak was detected on wide range gels. Multiple runs of cytosols with measurement of activity or runs with affinity purified material indicated that cEHs from four strains of mice were indistinguishable by this technique. However, purified cEH and cEH activity in cytosol from the ENU4 mice were clearly more acidic.
et
al. Table 4. Amino acid composition of affinity purified cytosolic epoxide hydrolase from different strains of mice* ENU4
SW
CBFI
Asx CYS GIU Glx His Ile Leu LYS Met Phe Pro Ser Thr Trp Tyr Val
44.3 25.6 54.7 11.4 70.8 41.8 9.8 32.1 51.9 36. I 20.9 33.2 39.4 34.9 28.6 ND 13.6 34.6
44.8 26.3 55.4 11.6 70.5 39.2 9.7 32.7 52.3 36. I 22.4 32.4 40.4 32.9 28.7 ND 13.7 34.7
44.0 26.4 55.2 I I.2 65.2 42.5 9.4 33. I 53.9 33.3 23.0 32.2 38.5 34.2 32.0 ND 15.6 36.0
Sum
583.8
583.8
585.7
Amino
Ala
Arg
acid
-
‘Values are the mean of duplicate determinations, each with HCl, with and without performic acid. ND: not determined.
The cEH from Balb/c, C57BL/6 and SW mice is well characterized and thus served as a control for our analysis of cEH from the Fl hybrid strain CBFl and the variant stock ENU4. Peroxisomal inducibiity (measured as an increase in oxidase production of H20,), catalytic activity, immunoreactivity and physicochemical properties of cEH from the five different strains of mice were compared. Affinity purified enzymes from all five strains of mice (clofibrate-treated and untreated) were identical on SDSPAGE with an apparent molecular weight of 62 kDa. The similarity among the enzymes from the different strains of mice was reflected in similar pH optima and immunological characteristics. No interstrain differences in the specific activities and kinetic constants (K,,, and V,,,,X)were observed in the purified enzymes from the five strains of mice. Although there were significant differences in hydrolytic activities towards certain substrates (e.g. ENPS) the differences do not allow us to distinguish among the inbred strains on the basis of activity. The purified cEH from Balb/c, C57BL/6, SW and CBFl mice had a p1 of 5.6 which is in the same range reported by other investigators for cytosolic epoxide hydrolases (Loury et al., 1985; Meijer and DePierre, 1985; Dietze et al., 1990). In contrast, the enzyme from ENU4 mice had a p1 of 5.1 when run on the same gel. Apparently, the cEH from ENU4 mice is distinguished from the cEH of the other strains of mice primarily by its lower PI. ENU treatment may have altered the amino acid composition of cEH in ENU4 mice in a fashion similar to that reported for ornithine aminotransferase in ENU2 mice (Giometti et al., 1988). However, the change of one amino acid reported for the ENU2 mice would not have been detected by our amino acid composition analysis. Of course other subtle changes in the protein, such as a change in the protein’s folding pattern, could also cause the observed change in PI. Studying responses of coupled enzyme systems to treatment is another approach in evaluating the regulation of given enzymes, and in the comparison
Altered epoxide hydrolase
of given enzymes in several systems. In this study, the mice were exposed to clofibrate, a pcroxisome proliferating agent, and the activity of palmitoyl-CoA oxidase was monitored. Exposure to dietary clofibrate caused a 4-20-fold elevation in the rate of HzOz production by palmitoyl-CoA oxidase in the different strains of mice when compared to the controls (Table 1). The CBFl and ENU4 strains showed the highest increase (19 and 15 times, respectively) followed by C57BL/6, SW and Balb/c in decreasing order. These results suggest that dietary clofibrate is affecting the endogenous mechanism for hepatic peroxisome proliferation in the CBFl and ENU4 strains in a manner similar to the other mouse strains. Cytosols from all five strains of mice metabolized TSO at approximately the same rate (Table 1). However, a quantitative increase in hydrolytic activity towards TSO was observed in all strains after exposure to clofibrate. The relative increase in cEH activity varied from approximately 2-4-fold when compared to the controls (Table 1). Balb/c had the highest increase in cEH activity followed by CBFl and C57BL/6 mice. ENU4 and SW mice were the least responsive to the clofibrate induction of cEH while, in contrast, ENU4 showed the greatest increase in clofibrate induced palmitoyl-CoA oxidase activity. On the one hand, this weaker “coupling” of peroxisome proliferation and induction of cEH suggests a slight alteration in the regulation of cEH relative to palmitoyl-CoA oxidase which will be helpful in dissecting the factors important in the regulation of these two enzymes. On the other hand, the imbalance between the producers of free radicals and free radical scavengers in ENU4 mice might cause these mice to have higher numbers of tumours caused by peroxisome proliferating agents such as clofibrate. In this context, ENU4 mice may be a good model in which to study the role of cEH in the prevention of tumors following treatment with agents that lead to peroxisome proliferation. In addition, if ENU4 represents allelic variation, standard linkage analysis can be used to establish the map location of the cEH locus on one of the mouse chromosomes (Peters, 1982). Acknowledgements-This
work was supported in part by NIEHS grant No. ES02710, NIEHS Superfund grant No. ES04699 and a Burroughs Wellcome Toxicology Scholar Award to B.D.H., in part by the United States Department of Energy, Office of Health and Environmental Research under Contract No. W-31-l09-ENG-38 to C.S.G., and in part by a NIEHS predoctoral training grant No. ES07059 to E.C.D. REFERENCES
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Chacos N., Capdevila J., Falck J. R., Manna S., MartinWixtrom C., Gill S. S., Hammock B. D. and Estabrook R. W. (1983) The reaction of arachidonic acid epoxides
213
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Gill S. S., Ota K. and Hammmock B. D. (1983) Radiometric assays for mammalian epoxide hydrolases and glutathione-S-transferases. Anal. Biochem. 131, 273-282. Giometti C. S., Gemmell M. N., Nance S. L., Tollaksen S. L. and Taylor J. (1987) Detection of heritable mutations as quantitative changes in protein expression. J. biol. Chem. 262, 12764-12767.
Giometti C. S., Tollaksen S. L., Gemmell M. A., Burcham J. and Peraino C. (1988) A heritable variant of mouse liver ornithine aminotransferase induced by ethylnitrosourea. J. biol. Chem. 263. 15781-15784. Haeggstrom J., Meijer J. and Radmark 0. (1986) Enzymatic conversion into 5,6-dihydroxy-7,9,11,14-eicosatetraenioc acid by mouse liver cytosohc epoxide hydrolase. J. biol. Chem. 261, 6332-6337.
Halarnker P. P., Wixtrom R. N., Silva M. H. and Hammock B. D. (1989) Catabolism of epoxy fatty esters by the purified epoxide hydrolase from mouse and human liver. Arch. Biochem. Biophys. 272, 226-236.
Hammock B. D. and Hasagawa L. S. (1983) Differential substrate selectivity of murine cytosolic and microsomal epoxide hydrolases. Biochem. Pharmacol. 32, 1155-l 164. Hammock B. D. and Ota K. (1983) Differential induction of cytosolic epoxide hydrolase, microsomal epoxide hydrolase and glutathione-S-transferase activities. Toxicol. Appl. Pharmacol. 71, 254-265.
Hammock B. D., Moody D. E. and Sevanian A. (1985) Epoxide hydrolases in the catabolism of sterols and isoprenoids. In Methods in Enzymology (edited by Law J. H. and Rilling H. C.), Vol III, pp. 487-494. Academic Press, Orlando, FL. Hammock B. D., Prestwich G. D., Loury D. N., Cheung P. Y. K., Eng W-S., Park S-K., Moody D. E., Silva M. H. and Wixtrom R. N. (1986) Comparison of crude and affinity purified cytosolic epoxide hydrolases from hepatic tissue of control and clofibrate-fed mice. Arch. Biochem. Biophys. 244, 292-309.
Loury D. N., Moody D. E., Kim B. W. and Hammock B. D. (1985) Effect of dietary clofibrate on epoxide hydrolase activity in tissues of mice. Biochem. Pharmacol. 34, 1827-1833.
Lundgren B. and Depierre J. W. (1989) Proliferation of peroxisomes and induction of cytosolic epoxide hydrolase and microsomal epoxide hydrolases in different strains of mice and rats after treatment with clofibrate. Xenobiotica 19, 867-88
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Magdalou J. and Hammock B. D. (1988) 1,2-Epoxycycloalkanes: substrates and inhibitors of microsomal and cytosolic epoxide hydrolases in mouse liver. Biochem. Pharmacol. 37, 2717-2722.
Meijer J. and DePierre J. W. (1985) Properties of cytosolic epoxide hydrolase purified from the liver of untreated and clofibrated-treated mice. Eur. J. Biochem. 148, 421-430. Miki I., Shimizu T., Seyama Y., Kitamura S., Yamaguchi K., Sano H., Ueno H.. Hiratsuka A. and Watabe T. (1989) Enzymatic conversion of ll,l2-leukotriene A4 to 11,12-dihydroxy-5,14-cis-7,9-rrans-eicosatetraenoic acid. J. biol. Chem. 264, 5799-5805.
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Moody D. E., Loury D. N. and Hammock B. D. (1985) Epoxide metabolism in the liver of mice treated with clofibrate (ethyl-a-@-chlorophenoxyisobutyrate)), a peroxisome proliferator. Toxicol. Appl. Pharmacol. 78, 351-362. Moody D. E., Reddy J. K., Lake B. G., Popp J. and Reese D. H. (1991) Peroxisome proliferation and nongenotoxic carcinogenesis: Commentary on a Symposium. Fundam. Appl. Toxicol. 16, 233-248. Mullin C. A. and Hammock B. D. (1982) Chalcone oxidespotent selective inhibitors of cytosolic epoxide hydrolase. Arch. Biochem. Biophys. 216, 423-439. Mumby S. M. and Hammock B. D. (1979) Substrate selectivity and stereochemistry of enzymatic epoxide hydration in the soluble fraction from mouse liver. Peslic. Biochem. Physiol. 11, 275-284. Oesch F. (1973) Mammalian epoxide hydrases: inducible enzymes catalysing the inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and olefinic compounds. Xenobiotica 3, 305-340. Oesch F., Schladt L., Steinberg P. and Thomas H. (1988) Concomitant induction of cytosolic epoxide hydrolase and peroxisomal /?-oxidation by hypolipidemic compounds in rat and guinea pig liver. Arch. Toxicol. Suppl. 12,248-255. Pace-Asciak C. R. and Lee W. S. (1989) Purification of hepoxilin epoxide hydrolase from rat liver. J. biol. Chem. 264,9310-9313. Peters J. (1982) Nonspecific esterases of Mus musculus. Biochem. Genetics 20, 585-606.
Reddy J. K. and Lalwai N. D. (1983) Carcinogenesis by hepatic peroxisome proliferators: Evaluation of the risk of hypolipidimic drugs and industrial plasticizers to humans. Critical Ret&w Toxicol. 12, I-is. Russell W. L., Kelly E. M., Hunsicker P. R., Bangham J. W., Maddux S. C. and Phipps E. L. (1979) Specificlocus test shows ethylnitrosourea to be the most-potent mutagen in the mouse. Proc. Nat1 Acad. Sci. (U.S.A.) 76, 5818-5819. Small G. M., Burdett K. and Connock M. J. (1985) A sensitive spectrophotometric assay for peroxisomal acylCoA oxidase. Biochem. J. 227, 205-210. Watabe T., Isobe M., Kanai M. and Ozawa N. (1981) The hepatic microsomal biotransformation of A5-steroids to 5u,6a-glycols via c(- and /?-epoxides. J. biol. Chem. 256, 2900-2907. Wixtrom R. N. and Hammock B. D. (1985) Membranebound and soluble fraction epoxide hydrolases: methodological aspects. In: Biochemical Pharmacology and Toxicology, Vol. 1: Methodological Aspects of Drug Metabolizing Enzymes (edited by Zakim D. and Vessey D. A.), pp. l-93. John Wiley, NY. Wixtrom R. N. and Hammock B. D. (1988) Continuous spectrophotometric assays for cytosolic epoxide hydrolase. Anal. Biochem. 174, 291-299. Wixtrom R. N., Silva M. H. and Hammock B. D. (1988) Affinity purification of cytosolic epoxide hydrolase using derivatized epoxy-activated Sepharose gels. Anal. Biothem. 169, 71-80.
Vol. 103C,
No. I, pp. 207-214,
0306~4492/92 $5.00+ 0.00 0 1992Pergamon Press Ltd
1992
BIOCHEMICAL CHARACTERIZATION OF A VARIANT FORM OF CYTOSOLIC EPOXIDE HYDROLASE INDUCED BY PARENTAL EXPOSURE TO N-ETHYL-N-NITROSOUREA JAFFAR NOUROOZ-ZADEH,*~ BRUCE S. WINDER,* ERIC C. DIETZE,*$ CAROL S. GIOMETTI,~ SANDRA L, TOLLAKSEN~and BRUCE D. HAMMOCK*11 *Departments of Entomology and Environmental Toxicology, University of California, Davis, CA 95616, U.S.A. (Received 24 December 199 1)
Abstract-l. ENU4 mice express a protein variant originally detected in a CBFl mouse sired by a C57BL/6 mouse exposed to N-ethyl-IV-nitrosourea. It appears to be an isolelectric point variant of cytosolic epoxide hydrolase. Affinity purified cytosolic epoxide hydrolase from ENU4 mice has a pI of approximately 5.1 compared to 5.6 in other mouse strains. 2. Clofibrate induced cytosolic epoxide hydrolase to similar levels in five strains of mice. However, CBFI and ENW mice were more sensitive to the induction of palmitoyl CoA oxidase activity. 3. Except for isoelectric point, the physico- and immunochemical properties of cytosolic epoxide hydrolase from ENU4 mice were similar to those of the other mouse strains. Substrate specificities for five of six substrates tested were also similar
INTRODUCTION Living organisms are continuously exposed to a variety of man-made and naturally occurring epoxides, some of which can spontaneously alkylate nucleophilic moieties of tissue macromolecules such as DNA, RNA and proteins. In mammals, epoxides are metabolized by two families of enzymes. GlutathioneS-transferases (GST) (EC 2.5.1.18) catalyze a nucleophilic attack of a sulfur anion on the epoxide ring leading to formation of glutathione conjugates. Epoxide hydrolases (EH) (EC 3.3.2.3) convert epox-
ides to 1,2-diols through addition of water to the oxirane ring. Different forms of EH have been characterized. For example, cholesterol epoxide hydrolase selectively hydrates cholesterol epoxides and is predominantly microsomal (Watabe et al., tPresent address: William Harvey Research Institute, St Bartholomew’s Hospital Medical College, Charterhouse Square, London EClM 6BQ. SPresent address: Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195, U.S.A. §Argonne National Laboratory, 9700 S. Cass Avenue, Argonne, Illinois 60439, U.S.A. /IAuthor to whom correspondence should be addressed. List of abbreviations: cEH, cytosolic epoxide hydrolase; CNEH, ci.s-4-nitrophenyl-3,4-epoxyhexanoate; DFP, diisopropyl fluorophosphate; EDTA, ethylenediaminetetraacetic acid; ENP-5, truns-1,2-epoxy-1-(4-nitrophenyl)-pentane; ENU, N-ethyl-N-nitrosourea; EQU-5, truns-1,2-epoxy-l-(2-quinolyl)-pentane; F-CHO, 4fluorochalcone oxide; mEH, microsomal epoxide hydrolase; PI, isoelectric point; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; S-NEPC, 4nitrophenyl-(2S,3S)-2,3-epoxy-3-phenylpropylcarbonate; TNEH, rruns-4-nitrophenyl-3,4_epoxyhexanoate; TSO, trans-stilbene oxide; 2DE, two-dimensional electrophoresis.
1981; Finley et nl., 1988) while epoxide hydrolases selective for leukotriene A4 are located in the cytosol (Haeggstrom et al., 1986; Miki et al., 1989; Pace-Asciak and Lee, 1989). Neither of these enzymes, however, appear to be involved in the metabolism of xenobiotics. Cytosolic epoxide hydrolase (cEH) rapidly metabolizes a variety of epoxides on aliphatic systems (Mumby and Hammock, 1979; Hammock and Hasagawa, 1983). Microsomal epoxide hydrolase (mEH) hydrates epoxides on arene systems and some epoxides on aliphatic systems (Oesch, 1973; Magdalou and Hammock, 1988). In addition, cEH and mEH are distinguished by pH optima, induction, isoelectric point, molecular weight and antigenic determinants. N-ethyl-N-nitrosoura (ENU) is known to be potent mutagen in vitro and in vivo (Russell et al., 1979). Using two-dimensional electrophoresis (2DE), ENU was shown to induce the heritable expression of liver protein variants in a small number of the offspring of exposed sires (Giometti et al., 1987). Each of these variants was characterized by a corresponding decrease of approximately 50% in the abundance of a neighboring protein. Of the five protein variants described, one has been identified as an isoelectric point variant of ornithine aminotransferase (OAT: Giometti et al., 1988). This identification was based on the comigration of OAT purified from a normal mouse with a normal liver protein observed as reduced in abundance by approximately 50% in mice expressing one of the ENU-induced liver protein variants, and on the comigration of a protein with OAT activity with the ENU2 variant protein. Western blot analysis with anti-OAT antisera confirmed the identification of both the normal and variant forms of OAT. 207
J. NOUROOZ-ZADEHet al.
208
In this paper, we describe a similar characterization of a second ENU-induced liver protein variant referred to in the original paper as ENU4. A preliminary experiment (unpublished) indicated that cEH purified from CBFl mice (Fl-hybrid offspring of C57BL/6 males and Balb/c females) comigrated with the protein reduced by 50% in the original mouse expressing the ENU4 variant. We pursued this finding and present data indicating that the ENU4 variant is an altered form of cEH that has catalytic properties essentially identical to those of cEH purified from other mouse strains with the exception of an increased inducibility of palmitoyl CoA oxidase by clofibrate. MATERIALS AND METHODS
Reagents tram-Stilbene oxide (TSO) was purchased from Aldrich Chemical Co., Milwaukee, WI. 4-Fluorochalcone oxide (F-CHO) was synthesized according to Mullin and Hammock (1982). tram-1.2-Enoxv-1-(2-auinolvl)-oentane (EQUS) and ’ rrans-1,2-epoxy- i-(4-nitropheny&cntane (ENPS) were synthesized as described elsewhere (Wixtrom and Hammock, 1988). 4-Nitrophenyl-(2S,3S)-2,3-epoxy-3phenylpropylcarbonate (S-NEPC), cis-4-nitrophenyl-3,4epoxyhexanoate (CNEH) and tram-4-nitrophenyl-3,4epoxyhexanoate (TNEH) were prepared according to Dietze et al. (1991). [3H]-TS0 was prepared as described by Gill et al. (1983). Other chemicals were the highest quality commercially available. Animal treatment and subcellular fractionation Balb/c, C57BL/6 and Swiss-Webster mice (Mus musculus) were obtained from Charles River Labs, Cambridge, MA. CBFI and ENW (7-8 weeks old) were obtained from Argonne National Laboratory, Argonne, IL. The CBFl mice were the first generation offspring of crosses between C57BL/6 males and Balb/c females and represent the genetic background of the ENW stock. The ENU4 stock, homozygous for the liver protein variant (Giometti et a/., 1987) was generated first by backcrossing to C57BL/6 followed by sibling crosses of mice heterozygous for the ENU4 variant. The homozygous ENU4 stock is now maintained by sibling crosses of homozygous individuals. The mice were fed Purina Laboratories chow (ad lib&urn) for one week. Thereafter, control mice received ground chow containing 5% corn oil for 14 days while the treated mice received the chow containing 5% corn oil plus 0.5% clofibrate. The animals were killed by cervical dislocation. Livers were removed and were washed with chilled 75 mM Na/K phosphate buffer @H 7.4) containing 0.1 mM ethylenediaminetetraacetic acid (EDTA) and diisopropyl fluorophosphate (DFP). The livers were weighed and homogenized in the phosphate buffer to give a 20% (w/v) suspension. Subcellular fractionation was performed as described elsewhere (Hammock and Ota, 1983). The cytosolic fraction (100,000 g) was immediately stored at - 70°C until analysis. Preparation of ajinity gel and enzyme purification Benzylthiol-Sepharose CL-6B was prepared as described by Wixtrom et al. (1988). Enzyme purification was performed at 4°C. In a typical experiment, 1 ml of the benzylthiol gel was sequentially washed with ethanol, water and phosphate buffer (pH 7.4) containing 0.1 mM EDTA. The gel was poured into an Econo column (5 mm id., Bio-Rad Laboratories, Richmond, CA). Cytosol (10%; total volume 15 ml) was pumped through the-column at a flow rate of 0.2 ml/min and the gel was subsequently washed with 45 ml of phosphate buffer. cEH was specifically released by wash-
ing the gel with 4 ml 0.2 mM F-CHO. Aliquots (0.5 ml) were collected and were tested for protein content (Bradford reagents, Bio-Rad). Fractions with high protein content were pooled. After dialysis against phosphate buffer for 2 hr, the affinity purified cEH was stored at 4°C until used. Protein contents were determined using Bradford reagent with the modification suggested by Moody et al. (1985). Enzyme assays cEH activity was monitored using partitionand spectrophotometric assays. The partition assay with [3H]-TS0 was performed as described by Hammock et al. (1985). Photometric assays were carried out on a Varian/Cary 219 spectrophotometer. EQUS and ENPS assays were performed as described elsewhere (Wixtrom and Hammock, 1988). The EQUS assay is based on a shift in absorbance from 320 to 312nm. The ENPS assay was monitored at 302 nm and is based on a decrease in absorbance. Spectrophotometric assays with CNEH, TNEH and S-NEPC were performed according to Dietze et al. (1991). Release of p-nitrophenol was monitored at 405 nm. Palmitoyl-CoA assay was performed using the spectrophotometric method described by Small et al. (1985). The assay is based on oxidation of Leuco-DCF by H,O* produced by palmitoyl CoA-oxidase. Gel electrophoresis and Western blotting Discontinuous sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on a Bio-Rad minislab apparatus. Proteins (affinity purified cEH or cytosol) were separated on 10% SDS gels with 3% stacking gels. The gels were stained with 0.1% Coomassie Brilliant Blue R-250 in methanol/water/acetic acid (45/45/10; v/v/v). For Western blotting, affinity purified liver cEH or cytosol were separated by SDS-PAGE and subsequently electrophoretically transferred to a nitrocellulose membrane (Bio-Rad) at 110 mA for 1 hr in a Hoefer semi-dry blotter (Hoefer Sci., San Francisco, CA). Immunoblotting was carried out according to the procedure recommended by Bio-Rad using rabbit anti-rhesus monkey liver cEH and alkaline phosphatase-conjugated anti-rabbit IgG (ICN Immunobiological, Irvine, CA). A screen of antibodies to cEH from different sources revealed this one to be the most suitable for use on Western blots (data not shown). Flatbed isoelectric focusing (IEF) was nerformed on a LKB 2117 Multiphor apparatus using ire-packed gels (OH 3.5-10 or 556.5. LKB Bromma. Sweden). Lanes were ibaded (affinity purified cEH or cytosol) and the gel was focused according to the manufacturer’s recommendations. The lanes were cut into 2mm slices and incubated with 300~1 phosphate buffer containing 1OOpg bovine serum albumin/ml and 0.1 mM EDTA for 3 hr before measurement of activity. For determination of pH gradients, 5 mm slices were cut and incubated in 300~1 20mM potassium chloride for 2 hr. Amino acid analysis Analysis was performed at the UC Davis Protein Structure Laboratory. Purified mouse liver cEH (S&l00 pg) was hydrolyzed on 6 M HCl with or without prior performic acid oxidation. Amino acids were derivatized with ninhydrin and quantified on a Beckman 6300 amino acid analyzer using a-amino-guanidino propionic acid as the internal standard. RESULTS
Clofibrate treatment of CBFl and ENU4 mice for two weeks caused no significant change in body weight when compared to those receiving the unsupplemented diet for the same period. On the other hand, an increase in liver weights was observed in the
Altered epoxide hydrolase Table I. Effects of clofibrate
treatmenton body
209
weight, liver weight and hepatic cytosolic activities in five strains of mice
expoxide hydrolase
(cEH) and palmitoyl
CoA
Activities
Strain
Treatment
CBFI
Control Clolibrate Control Clofibrate Control Clolibrate Co”trol Clofibrate Control Clofibrate
ENW Balb/c C57/BL SW
Number of animals IO IO 10 IO IO
10 10 IO 10 10
Body weight In) 34.0 * 3.0 32.9 + 2.4 30.7 * 1.2 27.0 & 1.3 24.5 + 1.6 23.3 + 1.4 25.2 f I .8 25.2 _+ 1.8 27.6 f 15 28. I + 2.0
Liver weight In)
Liver weight as % of body weight
I .97 f 0.2 2.62 + 0.3’ 1.7kO.l 2.02 + 0.2c 1.2610.1 1.44+0.1C 1.62 + 0.2 2.13 + 0.3’ I .53 + 0.2 1.94+ 0.2”
5.76 f 0.1 7.96 + O.@ 5.5 + 0.3 7.48 + 0.4d 5.14 f 0.2 6.18f0.2’ 6.44 + 0.4 8.44 2 0.9d 5.56 f 0.3 6.89 + 0.4d
Palmitoyl CoA’ (mODimin/mn)
cEHb (fimol/min/mg) 0.006 0.018 0.007 0.013 0.008 0.026 0.012 0.026 0.015 0.023
11.1 + 1.6 173k29’ 22.2 + 5.4 440+51c 36.9 + 7.3 139 f 14e 46.6 _+3.5 652 + 82’ 57.7 + 2.0 537 + 104’
a: Palmitoyl CoA activity was monitored according to Small et al. (1985). The values are the mea” of quadruplicate b: cEH activity in cytosol was monitored with the TSO partition assay according to Hammock ef al. (1985). c,d,e,f: Means in control and treated groups within each strain were significantly different (P < 0.03).
clofibrate treated mice compared to the controls (Table 1). Balb/c, C57BL/6 and SW mice also responded to the dietary clofibrate in the same fashion, judged by comparing body and liver weights. In this study, none of the Balb/c, C57BL6, SW or CBFl mice showed any signs of toxicity during the exposure to dietary clofibrate. In contrast, the ENU4 strain was more susceptible to the exposure to clofibrate with only about 70% of the original stock surviving the drug treatment. The cause of death in these mice is not known. The induction of palmitoyl CoA oxidase by dietary clofibrate was monitored by measuring the rate of H,O, production. The analysis showed that the rates of H,O, production by palmitoyl-CoA oxidase in liver cytosol from untreated CBFl and ENU4 mice were 11 and 22 mOD/min/mg protein, respectively. The corresponding rates in the liver cytosols from the clofibrate-treated mice were 173 and 440mOD/min/mg protein. The rates of H202 production in liver cytosols from untreated Balb/c, C57BL/6 and SW mice varied from 36 to 57 mOD/min/mg protein. The corresponding rates of H,Oz production in the clofibrate-treated mice ranged from 139 to 652 mOD/min/mg protein (Table 1). Comparison of the ratios of the H,O, produced in the clofibrate-treated mice to that of the corresponding controls is presented in Table 1. To facilitate comparison of the hepatic cEH activities from the hybrid CBFl and ENU4 strains with the inbred strains of mice, pH profile studies were performed. Enzyme activity was monitored using the partition assay over a pH range of 5 to 10 using Na/K phosphate and Tris buffers. cEH activity in the cytosols of CBFl, ENU4 and SW strains (clofibratetreated and untreated) increased in catalytic activity towards TSO in the pH range between 6 and 7.8. Maximum catalytic activity was obtained at pH 7.0 (Fig. 1). In this study, the partition assay with TSO was performed at pH 7.4 to obtain specific activity data which allowed direct comparison with previous work. Liver cytosols from untreated CBFI and ENU4 mice hydrated TSO at a rate of 0.007 pmol/min/mg protein. Cytosol from clofibrate-treated CBFl mice hydrated TSO at a rate approximately three times higher when compared to the controls (Table 1). No significant induction of cEH activity was observed in
+ + + * + + * + * f
0.0003 0.002’ 0.002 0.002’ 0.002 0.005’ 0.001 0.006’ 0.003 0.005’
determinations.
the cytosols from clofibrate-induced ENU4 mice. Cytosols from untreated Balb/c, C57BL/6 and SW mice metabolized TSO at a rate between 0.008 and 0.015 pmol/min/mg protein. The corresponding values for the clofibrate-treated mice varied from 0.023 to O.O26pmol/min/mg (Table 1). Since the protein contents of the liver cytosols were not affected by the dietary clofibrate, the increase in specific activities reflects an increase in total cEH activities (data not shown). While these findings support earlier reports on the coupling between peroxisome proliferation and induction of cEH (Hammock and Ota,
1
E 4 2 _-
5
4
5
’
’
a
’
lo
g
10
ENU4 pH Profile
6
11
pH’Gra&mt
Fig. 1. pH profile of catalytic activity towards TSO in cytosol preparations. Epoxide hydrolase activity was measured at different pHs using 75 mM Na/K phosphate (PH 5-8) or 100 mM Tris-HCl (pH 8-10) buffers. Values are the means of triplicate analyses and expressed as nmol/min/mg protein. These experiments were repeated with different preparations with similar results. Keys: A, CBFl
liver cytosol; B, ENW liver cytosol; C, SwissWebster mouse (SW) liver cytosol.
J. NOUROOZ-ZADEH et al.
210
1983; Oesch et al., 1988), it is evident that the correlation between cEH and palmitoyl CoA oxidase is not the same among the five strains of mice and follows the order ENU4 < CBFl < C57BL/6 < SW < Balb/c. Figure 2 presents a Coomassie stained SDS gel and a Western blot of liver cytosols and purified cEH from CBFl, ENU4 and SW. SDS-PAGE analysis of the purified cEH from the five strains of mice indicated the presence of a single band at 62 kDa. Western blots of the affinity purified cEHs revealed that the protein at 62 kDa cross-reacted with rabbit antibodies raised to rhesus hepatic cEH. A similar pattern was also observed with the cytosol preparations from the different strains of mice (clofibrate-treated and untreated). To evaluate whether the affinity purified proteins with the same apparent molecular weight from the
different strains of mice were the same enzymes, specific activities were compared. Table 2 shows specific activities for purified liver cEH from the different strains of mice (untreated) using a battery of selected spectrophotometric substrates whose structures are shown (Fig. 3). The purified enzymes from the five different strains of mice possessed similar hydrolytic activities towards CNEH (approximately 1.5 pmol/min/mg protein), EQU5 (3.5-3.9 pmol/ min/mg) and S-NEPC (6.6-8.0 pmol/min/mg). With TNEH, the enzymes from CBFl, ENU4, C57BL/6 and SW metabolized the substrate at similar rates (5.9-6.5 flmol/min/mg) whereas a lower hydration rate was observed with the Balb/c enzyme (4.2 pmol/min/mg). Interestingly, the affinity purified enzymes from CBFl and ENU4 mice exhibited higher activity towards ENPS (12 and 13 pmol/ min/mg, respectively) than those obtained from
kDa 97 66 45 31 21 ABCDEFGHI
J
kDa
ABCDEFGHIJ Fig. 2. 10% SDS-PAGE and Western blot of mouse liver cytosol and purified cytosolic epoxide hydrolase (cEH). The gel (top) was loaded with 3 ng purified cEH and 25 pg of cytosol. An identical gel was blotted to nitrocellulose (bottom) and probed with rabbit-anti-rhesus monkey liver cEH (1: 250). The secondary antibody was goat-anti-rabbit-IgG (1: 2000). Separate gels indicated that purified cEH and cEH in cytosol from all strains of mice. with and without clofibrate treatment. showed the same mobility bv Western blot. Keys: A. Molecular weight standards, phosphorylase b, 9? kDa; BSA, 66 kDa; o&bumin, 45 kDa; carbonic anhvdrase. 31 kDa: sovbean trvnsin inhibitor. 21 kDa. B. ENU4 cEH. C. CBFl cEH. D. Swiss-Webster cEH. E. Swiss-Webster cytosol, clofibrate treated. F. ENU4 cytosol, clofibrate treated. G. ENU4 cytosol, no clofibrate. H. CBFl cytosol, clofibrate treated. I. CBFl cytosol, no clofibrate. J. Molecular weight standards as in A.
Altered epoxide hydrolase
211
Table 2. Soecific activitv of ourilied cvtosolic eooxide hvdrolase with selected substrates Substrate
Concentration (u M)
CNEHb EQU-5’ S-NEPCb TNEHb ENP-5’ TSO’
200 500 50 200 50 50
CBFI 1.6kO.l 3.8 f 0.1 8.0 + 0.3 6.5 f 0.1 I3 f I.5 2.6 k 0.3
Specific activity (~mol/min/mg) ENU4 Balbic C57B116 1.5 f 0.1 3.9 f 0.3 7.7 * 0.3 5.9kO.l 12kO.5 I.9 f 0.2
1.3+0.3 3.5* I.1 6.9 f 0.2 4.2 f 0.5 9.1 + 0.2 2.7 f 0.1
1.5kO.2 3.7 f 0.8 6.6 * 0.2 6.0 rt 0.3 9.4 f 0.5 2.0 f 0.3
Swiss-Webster 1.5 * 3.5 f 7.4 i 5.9 f II + 2.1 +
0.2 0.3 0.8 0.3 0.2 0.2
a: EQU-5, ENP-5 were monitored (25°C in 76 mM phosphate buffer, pH 7.4, 0. I mM EDTA, 0. I mg/ml BSA) according to Wixtrom and Hammock (1988). b: CNEH, TNEH and S-NEPC assayed (25°C in 76mM phosphate buffer, pH 6.4, 0.1 mM EDTA, 2.5 mg/ml BSA) according to Dietze et al. (1991). c: TSO was assayed (37°C in 76 mM phosphate buffer, pH 7.4.0.1 mM EDTA, 0.1 mg/ml BSA) according to Hammock et al. (1985). The values for a and b were the means for quadruplicate determinations. The values for c were the means for triplicate determinations. The assays were performed on one preparation of cEH from clofibrate-treated mice of each strain.
Balb/c, C57BL/6 and SW mice (9.0-l 1pmol/ min/mg). The affinity purified enzymes from CBFl and ENU4 mice hydrated TSO at a rate of 2.6 and 1.9 nmol/min/mg protein, respectively, which was similar to that for Balb/c, C57BL/6 and SW (2.0-2.7 ~mol/min/mg). Affinity purified cEHs from the five strains of mice were also analyzed to determine kinetic rate constants for TSO hydration using the partition assay. The concentration of TSO ranged between 0.1 and 12mM. Hydrolytic activities in the affinity purified cEHs from clofibrate-treated and untreated mice obeyed Michaelis-Menten kinetics. The data were plotted according to Lineweaver-Burke and apparent kinetic constants were determined. The K, and V,,,., for cEHs from the different strains of mice are presented in Table 3. Although there is variation in the values determined for K, and V,,,,,, these are within the range of experimental variation normally seen among enzyme preparations. Thus one can only conclude that there are no major differences in these kinetic parameters between the ENU4 or CBFl strains and the other strains tested. In addition, we observed no significant effect of clofibrate on these parameters. Isoelectric points have been reported for cEH from Balb/c, C57BL/6 and SW (Loury et al., 1985; Meijer and DePierre, 1985; Hammock et al., 1986; Dietze
et al., 1990). To check for similarity among the pIs of the hydrolytic enzymes from the five strains of mice (clofibrate-treated and untreated), liver cytosols were initially analyzed using both wide and narrow range IEF-gels. Analysis of hydrolytic activity towards TSO indicated that enzyme activities in liver cytosols from untreated Balb/c, C57BL/6, CBFl and SW mice focused as single peaks at a p1 of 5.6 (Fig. 4). Analysis of liver cytosols (clofibrate-treated and untreated) from ENU4 mice revealed that enzymatic activity focused at a p1 of 5.1 was consistent with the shift in p1 reported previously (Giometti et al., 1987). IEF analysis of the affinity purified cEH from the different strains of mice (clofibrate-treated and untreated) gave the same pI values as those obtained with cytosol preparations. A minor, more acidic peak was sporadically detected. Similar observations have been reported by other investigators. Affinity purified cEHs from clofibrate treated CBFl, ENU4 and SW mice were analyzed for amino acid composition (Table 4). Within the error of the analysis, the amino acid compositions of the cEHs from the five strains of mice were identical. DISCUSSION
A number of studies have shown that clofibrate belongs to a group of chemicals known as
TSO
EQUS
ENPS
S-NEPC
TNEH
CNEH
Fig. 3. Structures of substances for cEH. mm-Stilbene oxide (TSO). truns-1,2-Epoxy-I-(2-quinolyl)-pentane (EQUS). mm-1,2-Epoxy-1-(4-nitrophenyl)-pentane (ENPS). 4-Nitrophenyl-(2S,3S)-2,3-epoxy-3phenylpropylcarbonate (S-NEPC). mm-4-Nitrophenyl-3,4-epoxyhexanoate (TNEH). cis4-Nitrophenyl3,4-epoxyhexanoate (CNEH).
J. NOUROOZ-ZADEH
212
Table 3. Table of kinetic values from clolibrate-treated and untreated mutagenized and normal mice with TSO as the substrate*
strain ENU4 CBFI C57/Bl6 Balb/c Swiss-Webster
Clofibrate
Km (u M)
+ f _ + + + -
I1 * 1.3 23 + I.7 6.2 + 1.9 10 + 6.0 7.0 ?c 0.8 25i 12 8.5 i 3.6 6.6 + I .6 4.3 + 0.3 9.6 + 3.5
VInPI (umoliminim~) 5.3 3.0 6.5 3.7 3.6 4.2 7.5 3.7 3.2 2.6
* * f f f k + + f +
0.5 0.2 3.2 0.9 0.4 1.6 3.0 0.8 0.2 0.6
*Assays carried out in triplicate with one CEH preparation. All assays performed at 37” in 76 mM phosphate buffer (pH = 7.4, 0.1mM EDTA, 0.1mg/ml BSA). Kinetic values determined from Lineweaver-Burke plots.
peroxisome proliferating agents, some of which have een shown to induce cEH. Among the enzymes associated with peroxisomes are palmitoyl-CoA oxidase and catalase. In several studies, palmitoyl-CoA oxidase has been shown to be induced by clofibrate while catalase appears not to be and may actually decrease (Butler et al., 1988; Lundgren and DePierre, 1989). One of the consequences of the apparent imbalance between elevated H,O, production by palmitoylCoA oxidase and lower degradation of H,Oz by catalase is increased levels of hydroxyl radicals. These may in turn lead to lipid peroxidation and damage to DNA and cellular membranes (Reddy and Lalwai, 1983; Bentley et al., 1988; Moody et al., 1991). Since epoxidized fatty acids have been shown to be excellent substrates for cEH (Halarnkar et al., 1989), the induction of cEH by peroxisome proliferating agents may be critical to the protection of the cell from damage by oxidized lipids. 6,000 ,
, 6.5
Fig. 4. Plot of cytosolic epoxide hydrolase activities on a typical narrow range isoelectric focusing gel for five mouse strains. Gels were prefocused for 30 min. Cytosols from five strains of mice (100 pg) were loaded and focused for 3 hr. The gel was cut into 2 mm slices and the slices eluted in phosphate buffer (ph 7.4) for 3 hr. The pH gradient was measured from 5mm slices of a parallel channel eluted into 20mM KCl. Only a single acidic peak was detected on wide range gels. Multiple runs of cytosols with measurement of activity or runs with affinity purified material indicated that cEHs from four strains of mice were indistinguishable by this technique. However, purified cEH and cEH activity in cytosol from the ENU4 mice were clearly more acidic.
et
al. Table 4. Amino acid composition of affinity purified cytosolic epoxide hydrolase from different strains of mice* ENU4
SW
CBFI
Asx CYS GIU Glx His Ile Leu LYS Met Phe Pro Ser Thr Trp Tyr Val
44.3 25.6 54.7 11.4 70.8 41.8 9.8 32.1 51.9 36. I 20.9 33.2 39.4 34.9 28.6 ND 13.6 34.6
44.8 26.3 55.4 11.6 70.5 39.2 9.7 32.7 52.3 36. I 22.4 32.4 40.4 32.9 28.7 ND 13.7 34.7
44.0 26.4 55.2 I I.2 65.2 42.5 9.4 33. I 53.9 33.3 23.0 32.2 38.5 34.2 32.0 ND 15.6 36.0
Sum
583.8
583.8
585.7
Amino
Ala
Arg
acid
-
‘Values are the mean of duplicate determinations, each with HCl, with and without performic acid. ND: not determined.
The cEH from Balb/c, C57BL/6 and SW mice is well characterized and thus served as a control for our analysis of cEH from the Fl hybrid strain CBFl and the variant stock ENU4. Peroxisomal inducibiity (measured as an increase in oxidase production of H20,), catalytic activity, immunoreactivity and physicochemical properties of cEH from the five different strains of mice were compared. Affinity purified enzymes from all five strains of mice (clofibrate-treated and untreated) were identical on SDSPAGE with an apparent molecular weight of 62 kDa. The similarity among the enzymes from the different strains of mice was reflected in similar pH optima and immunological characteristics. No interstrain differences in the specific activities and kinetic constants (K,,, and V,,,,X)were observed in the purified enzymes from the five strains of mice. Although there were significant differences in hydrolytic activities towards certain substrates (e.g. ENPS) the differences do not allow us to distinguish among the inbred strains on the basis of activity. The purified cEH from Balb/c, C57BL/6, SW and CBFl mice had a p1 of 5.6 which is in the same range reported by other investigators for cytosolic epoxide hydrolases (Loury et al., 1985; Meijer and DePierre, 1985; Dietze et al., 1990). In contrast, the enzyme from ENU4 mice had a p1 of 5.1 when run on the same gel. Apparently, the cEH from ENU4 mice is distinguished from the cEH of the other strains of mice primarily by its lower PI. ENU treatment may have altered the amino acid composition of cEH in ENU4 mice in a fashion similar to that reported for ornithine aminotransferase in ENU2 mice (Giometti et al., 1988). However, the change of one amino acid reported for the ENU2 mice would not have been detected by our amino acid composition analysis. Of course other subtle changes in the protein, such as a change in the protein’s folding pattern, could also cause the observed change in PI. Studying responses of coupled enzyme systems to treatment is another approach in evaluating the regulation of given enzymes, and in the comparison
Altered epoxide hydrolase
of given enzymes in several systems. In this study, the mice were exposed to clofibrate, a pcroxisome proliferating agent, and the activity of palmitoyl-CoA oxidase was monitored. Exposure to dietary clofibrate caused a 4-20-fold elevation in the rate of HzOz production by palmitoyl-CoA oxidase in the different strains of mice when compared to the controls (Table 1). The CBFl and ENU4 strains showed the highest increase (19 and 15 times, respectively) followed by C57BL/6, SW and Balb/c in decreasing order. These results suggest that dietary clofibrate is affecting the endogenous mechanism for hepatic peroxisome proliferation in the CBFl and ENU4 strains in a manner similar to the other mouse strains. Cytosols from all five strains of mice metabolized TSO at approximately the same rate (Table 1). However, a quantitative increase in hydrolytic activity towards TSO was observed in all strains after exposure to clofibrate. The relative increase in cEH activity varied from approximately 2-4-fold when compared to the controls (Table 1). Balb/c had the highest increase in cEH activity followed by CBFl and C57BL/6 mice. ENU4 and SW mice were the least responsive to the clofibrate induction of cEH while, in contrast, ENU4 showed the greatest increase in clofibrate induced palmitoyl-CoA oxidase activity. On the one hand, this weaker “coupling” of peroxisome proliferation and induction of cEH suggests a slight alteration in the regulation of cEH relative to palmitoyl-CoA oxidase which will be helpful in dissecting the factors important in the regulation of these two enzymes. On the other hand, the imbalance between the producers of free radicals and free radical scavengers in ENU4 mice might cause these mice to have higher numbers of tumours caused by peroxisome proliferating agents such as clofibrate. In this context, ENU4 mice may be a good model in which to study the role of cEH in the prevention of tumors following treatment with agents that lead to peroxisome proliferation. In addition, if ENU4 represents allelic variation, standard linkage analysis can be used to establish the map location of the cEH locus on one of the mouse chromosomes (Peters, 1982). Acknowledgements-This
work was supported in part by NIEHS grant No. ES02710, NIEHS Superfund grant No. ES04699 and a Burroughs Wellcome Toxicology Scholar Award to B.D.H., in part by the United States Department of Energy, Office of Health and Environmental Research under Contract No. W-31-l09-ENG-38 to C.S.G., and in part by a NIEHS predoctoral training grant No. ES07059 to E.C.D. REFERENCES
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