A new enzyme immunoassay of microsomal rat liver epoxide hydrolase

ANALYTICAL

BIOCHEMISTRY

163.298-302 (1987)

A New Enzyme Immunoassay

of Microsomal

Rat Liver Epoxide Hydrolase

A. ZHIRI,**~ J. MULLER,* S. FoumL,t M. WELLMAN-BEDNAWSKA,*-~ANDG.

J. MAGDALOU,? SIEST*‘~

Tentre de Mkdecine Prhentive, 2 avenue du Doyen Jacques Parisot, 54501 Vandoeuvre-les-Nancy Cedex, France, and jCentre du Mdicament, UA CNRS W 597,30 rue Lionnois, 54000 Nancy, France Received July 28, 1986 Antiserum against purified rat liver microaomal epoxide hydrolase was produced in the rabbit. We developed an enzyme-linked immunoaorbent assaywhich is reliable with regard to its analytical criteria. The concentration of epoxide hydrolase was measured in liver microaomes of control rats and animals treated with F 1379 (250 mg/kg/day) for 5,7,14, and 2 1 days. This hypolipidemic drug was able to induce strong epoxide hydrolaee activity and enhance protein concentration. The gradual increase in epoxide hydrolase concentration paralleled the increase of epoxide hydrolase activity, with stabilization occurring aBer the 14th until the 2 1st day of treatment. Q 1987 Academic Ress, Inc. KEY WORDS: epoxide hydrolase.; immunoassay; ELISA, induction; hypolipidemic drug; microsomes.

Microsomal epoxide hydrolase (EC 3.3.2.3) (EH)’ catalyzes the hydration of epoxides, especially arene oxides, which arise from oxidation of aromatic substrates by monooxygenases that are cytochrome P-450 dependent. The trunsdihydrodiols are generally weak or inactive as mutagens. However, the metabolites can thereafter be transformed by cytochrome P-450 into highly reactive dihydrodiol epoxides (1). EH appears to play a key role in the neutralization or formation of carcinogenic and mutagenic compounds. A distinct microsomal EH which converts cholesterol-5a,6a oxide into the corresponding diol has been recently reported (2). The EH protein neosynthesis has been shown to increase after treatment with phenobarbital (3). On the other hand, detergents ’ Abbreviations used: EH, epoxide hydrolaae; EL&A, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis, IgG, immunoglobulin G, HRP, horseradish peroxidase; EIA, enzyme immunoassay.

0003-2697187 $3.00 Copyright0

I987 by Academic Rss, Inc. AU righu of reprcduction in any form -cd.

are able to enhance in vitro microsomal activity (4) although this activation is less important than that observed with other membrane-bound enzymes like UDP-glucuronosyltransferases. The method presented in this paper was designed to quantitate the amount of EH and to allow us to differentiate between enzyme activation and induction. It involves a sensitive and noncompetitive enzyme-linked immunosorbent assay (ELISA) which can be performed in less than 6 h. The analytical criteria are given. The method is used to determine the levels of EH in liver microsomes of rats treated with the hypolipidemic drug F 1379, which is known to enhance EH activity (5). MATERIALS

AND METHODS

Purijcation of epoxide hydrolase. EH was purified from liver microsomes of male Sprague-Dawley rats (Domaine des Oncins, St Germain sur L’Arbresle, France), weighing 180-200 g, after induction by phenobarbital. The drug was injected once intraperi298

ENZYME

IMMUNOASSAY

OF

MICROSOMAL

toneally (100 mg/kg body wt in 0.9%, w/v, NaCl solution) and was given thereafter in drinking water (1 g/liter) for 5 days. The purification was performed as previously described (6,7); the purified protein gave a single band after electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel. The final preparation had a specific activity of 150 nmol of diol formed/min/mg protein with benzo[a]pyrene-4,5-oxide as the substrate (IIT, Chicago, IL). The enzyme activity was determined according to the method of Dansette et al. (8) on a Kontron SPF 500 spectrofluorometer. The protein concentration was evaluated by the technique of Lowry et al. (9) with bovine serum albumin (BSA) as standard. Production and control of antiserum. Rabbits (Fauves de Bourgogne, local supplier) were inoculated subcutaneously with 100 pg of pure EH dissolved in 0.5 ml of complete Freund’s adjuvant. Similar booster injections were made at 2-week intervals using incomplete Freund’s adjuvant. The rabbits were bled 8 days after the fourth booster challenge. The specificity of the antibodies was controlled by double immunodiffusion according to Ouchterlony. Precipitation lines were visualized after staining with Coomassie brilliant blue R-250 (Serva, Heidelberg, FRG). In addition the monospecificity of antibodies was demonstrated (data not shown),by Western blotting after separation by SDS-PAGE on different fractions, the purified enzyme or total microsomal fraction ( 10). Immunoprecipitation experiments consisted of incubation of purified EH with increasing amounts of antiserum in 0.15 M NaCl for 15 h at 4°C. The enzyme activity was assayed in supematant after centrifugation in order to remove the immunoprecipitates ( 10,OOOg for 7.5 min). Nonspecific binding was determined by using the serum from nonimmunized rabbits under the same conditions. Preparation of IgG and enzyme labeling. The IgG fraction was isolated from the whole

RAT

LIVER

EPOXIDE

HYDROLASE

299

antiserum with caprylic acid (Merck, Darmstadt, FRG) (11). The concentration of antibodies was calculated from their absorbance value at 280 nm. The IgG fraction was labeled with horseradish peroxidase (HRP) (250 units/mg protein, Boehringer Mannheim, FRG) according to the method of Nakane and Kawaoi ( 12). After addition of a 2% (w/v) solution of BSA and thimerosal (0.02%, w/v, Sigma, St. Louis, MO) the conjugate was stored at -2O”C, and under these conditions it was stable for at least 2 years. Enzyme immunoassay procedure. AntiEH IgG (100 ~1) (1.5 ccg/ml in 0.1 M phosphate buffer, pH 7.2) was dispensed into each well of a polystyrene EIA plate (3590, Costar, Cambridge, MA), excluding the outer rows which tend to produce edge effects. The plates were sealed and incubated overnight at room temperature. After being coated, the plates were washed four times with the phosphate buffer containing 0.02% Tween 20 (Merck). Before use, a solution of 1% (w/v) BSA in phosphate buffer was dispensed into each coated well for at least 30 min in order to block the residual sites of polystyrene; the plates were then washed one time. Purified EH standard (100 ~1) with a protein concentration from 0 to 2.4 ng or diluted microsomes were dispensed into the wells and incubated for 2 h at room temperature. Standard and microsome samples were previously solubilized in a 1% (w/v) BSA solution containing 0.25% (w/v) Tween 20. The plates were then washed four times, and 100 ~1 of the anti-EH HRP conjugate diluted lOOO-fold with the BSA-Tween 20 solution was added to each coated well. The plates were incubated 1.5 h at room temperature and washed again four times. Into each coated well, 100 ~1 of substrate solution (ophenylenediamine dihydrochloride, Sigma, 3 g/liter in freshly made 0.1 M phosphatelcitrate buffer, pH 5.0, containing 0.02%, v/v, hydrogen peroxide) was dispensed. After color development for 30 min at room temperature in the dark, the enzymatic activity was stopped by dispensing into each well 100

300

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~1 of 1 N sulfuric acid. The developed color was read at 492 nm with a reference wavelength at 650 nm. Dispensing of the reagents, washing of the plates, and photometry were automatically performed by a Behring ELISA processor (Marburg, FRG). Treatment of animals. For the induction study, male Wistar rats weighing 180-200 g (Domaine des Oncins, St Germain sur l’Arbresle, France) were used. Groups of three animals received either F 1379 (4-[4’-(2chlorophenyl) phenyll-4-oxo-2-methylenebutanoic acid) suspended in sucrose syrup by gastric intubation at a dose 250 mg/kg/day or the vehicle for increasing periods of time (5, 7, 14, 21 days). F 1379 was a gifi from Fabre Laboratory (Castres, France). The rats were sacrificed after 12 h of fasting, 1 day after the end of the treatments, and the liver microsomal fractions were individually isolated according to Hogeboom’s method (13). The protein content and the EH activity were determined as described above. Analysis of the data. The results were analyzed statistically by the Student t test. Dif-

PRECIPITATION

%

IOO-

1

2 IJI antiserum/

3 yg

E.H.

FIG. 2. Precipitation of purified EH from rat liver by specific antiserum.

ferences with P < 0.005 were considered significant. RESULTS

FIG. 1. Double immunodifhsion of EH from rat and mouse liver with antiserum against rat liver microsomal EH. AS, antiserum; 1, NaCl (Control); 2-3, solubilized microsomes (mouse liver); 5, solubilized microsomes (rat liver); 4-6, purified EH (rat liver).

After double immunodiffusion, antiserum against liver EH was found to be monospecifit (Fig. 1). In addition an apparent identity reaction was observed between rat and mouse (DBA/2 strain) liver EH (Fig. 1). As shown in Fig. 2, antiserum (2 pl/pg EH) was able to completely precipitate the enzyme protein; however, 100% of the activity (data not shown) was retained in the immunoprecipitate. The standard curve of the immunoassay using purified EH is presented in Fig. 3. By the sandwich immunoassay method, protein could be accurately quantitated within the concentration range 0.3-2.4 ng per well. The superimposed curve was obtained when microsomes were used for the assay; this suggested that no dilution effect occurred which could modify the values measured.

ENZYME

4

A492

1.5-

IMMUNOASSAY

OF MICROSOMAL

RAT LIVER EPOXIDE

301

HYDROLASE

ration. The detection limit was better than 0.1 ng EH protein per well.

“IT

Induction of EH by F 1379 in Rat Liver l.O-

Table 1 shows the comparative variations of enzymatic activity and EH protein level following increasing time treatments with F 1379. EH activity and the enzyme protein amount were significantly and similarly increased after 5 and 7 days of treatment (2and 2.5fold of the corresponding controls). After 14 and 2 1 days, enzyme activity increased 3.0- and 3.5fold of the corresponding controls, respectively, and proteins levels increased 3.7- and 4.4-fold, respectively.

//

.

purlbed

EH

0 m,cro*omes / /

0.5 /

Y

I

,

,

,

,

0.3

0.6

0.9

1.2

1.8

2.4 EH “9

>

FIG. 3. Representative standard curve for the determination of EH concentration using purified protein (0) or liver microsomes (0). Each point is the mean + SD (n = 6).

DISCUSSION

We describe an immunoassay which is very fast and sensitive. The use of an ELISA system provides a more practicable assay. Radioisotope-labeled reagents lead to some problems: they decay with time and present health hazards. The ELBA system with enzyme-labeled IgG that we used for this study did not present such drawbacks. In addition, the photometric assay of HRP with ophenylenediamine is the most sensitive and had the lowest nonspecific binding ( 14) when compared with the alkaline phosphatase/p

The precision assay was calculated using a microsomal fraction (93 pg protein/ml) at different dilutions. Variation coefficients ranged from 2.6 to 4.1% in intraassay and 6.8 to 7.5% in interassay. The accuracy of the method, determined by analytical recovery, was 98.4 f 2.9% for various amounts of purified EH added to a diluted microsome prepaTABLE INDUCTION

1

OF EH BY F 1379 IN RAT LIVER

MICROSOMES

Treatment period (days)

EH activity Y Control (n = 3) Fl379(n=3) EH concentration b Control (n = 3) F 1379 (n = 3)

0

5

7

14

21

7.6 + 1.77

9.32 + 0.97 18.29 k 4.58*

8.11 f 0.75 20.50 + 4.76*

9.84 + 0.90 29.33 + 2.48*

7.56 + 1.16 26.42 + 5.54*

1.49 k 0.23

1.35 f 0.65 2.78 + 0.41*

1.66 + 0.38 4.53 f 0.54*

2.27 f 0.65 8.41 + 1.61*

8.23 + 1.06*

1.85 + 0.23

’ The results are expressed as nmol benzo[a]pyrene-4,5-diol formed/min/mg protein and are the means f SD for three animals. b The results are expressed as mg EH/mg total microsomal protein X 100 and are the means f SD for three animals. l Indicates values significantly different from corresponding controls (P < 0.05).

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ZHIRI ET AL.

nitrophenyl phosphate system, which was used in the ELISA procedure of Gill et al. (15). The data presented here show that the increase in the microsomal EH activity after treatment with F 1379 is due to an increase in EH concentration rather than to an enhancement in its specific activity. This confirms the hypothesis of a real inductive potency of this hypolipidemic agent (5). It is interesting to note that after induction the amount of EH protein represented up to 8% of the total microsomal protein. We also noted that after a shorter time treatment, the increase in EH activity preceded that of protein concentration (data not shown). This initial response could result from activation of the enzyme which had been described as latent (4). Work is in progress to confirm that point. The molecular mechanisms responsible for the induction of EH by various inducers are still unresolved. Whether F 1379 might alter directly the turnover of enzymatic protein in rat liver microsomes is unknown. However, it is likely that the variations observed result from an elevation of translationally active mRNA encoding for EH as previously shown after phenobarbital administration (16). ACKNOWLEDGMENT This research was supported by the Commission of the European Communities (STI-046-J-C [CD]).

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2. Wixtrom, R. N., and Hammock, B. D. (1985) in Biochemical Pharmacology and Toxicology: Methodological Aspects of Drug Metabolizing Enzymes (Zakim, D., and Vessey, D. A., Eds.), Vol. I, pp. l-93, Wiley, New York. 3. Oesch, F. (1977) in Mises au point de Biochimie Pharmacologique l&e serie (Siest, G., and Heusghem, C., Eds.), pp. 127-148, Masson, Paris. 4. Burchell, B., Bentley, P., and Oesch, F. (1976) Biochim. Biophys. Acta 444, 53 1-538. Foumel, S., Magdalou, J., Pinon, P., and Siest, G. (1987) Xenobiotica, in press. Knowles, R. J., and Burchell, B. (1977) B&hem. J. 163,381-383. Magdalou, J., Kiffel, L., Balland, M., Thirion, C., Le-Meste, M., and Siest, G. (I 982) Chem. Biol. Interact. 39,245-256. 8. Dansette, P. M., Dubois, G. C., and Jerina, D. H. (1979) Anal. B&hem. 97,340-345. 9. Lowry, 0. H., Rosebrough, J. N., Faar, A. L., and Randall, R. I. (1951) J. Biol. Chem. 193, 265-275. 10. Galteau, M. M., Antoine, B., and Reggio, H. (1986) EMBO J 4,2793-2800. 11. Steinbuch, M., and Audran, R. (1969) Arch. Biothem. Biophys. 134,279-284. 12. Nakane, P. K., and Kawaoi, A. (1974) J. Histochem. Cytochem. 22, 1084- 109 1. 13. Hogeboom, G. H. (I 955) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., Eds.), Vol. I, pp. 16- 19, Academic Press, New York. 14. Ishikawa, E., Imagawa, M., Hashida, S., Yoshitake, S., Hamaguchi, Y., and Ueno, T. (1983) J. Zmmunoassay 4,209-327. 15. Gill, S. S., Wie, S. I., Guenthner, T. M., Oesch, F., and Hammock, B. D. (1982) Carcinogenesis 3, 1307-1310. 16. Pickett, C. B., and Lu, A. Y. H. (1981) Proc. Natl. Acad. Sci. USA 78,893-897.