Measurement of Soluble Epoxide Hydrolase (sEH) Activity

Christophe Morisseau1, Bruce D. Hammock1

1 University of California at Davis, Davis, California
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.23
DOI:  10.1002/0471140856.tx0423s33
Online Posting Date:  August, 2007
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Abstract

The human soluble epoxide hydrolase (sEH; EC 3.3.3.2) is the product of the EXPH2 gene. The sEH catalyzes the addition of a water molecule to an epoxide, resulting in the corresponding diol. Early work suggested a role of sEH in detoxifying a wide array of xenobiotic epoxides; however, recent findings clearly implicate the sEH in the regulation of blood pressure, pain, and inflammation through the hydrolysis of endogenous epoxy fatty acids such as epoxyeicosatrienoic acids (EETs). Both expression and activity of sEH are influenced by a wide array of xenobiotics, underlying how environmental contaminants could influence human health through sEH. This unit describes radiometric, fluorimetric, and mass spectrometric assays for measuring the activity of sEH and its inhibition. Curr. Protoc. Toxicol. 33:4.23.1‐4.23.18. © 2007 by John Wiley & Sons, Inc.

Keywords: epoxyeicosatrienoic acids; blood pressure; inflammation; inhibition

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Radiometric Assay for sEH Using trans‐Stilbene Oxide (t‐SO)
  • Alternate Protocol 1: Radiometric Assay for sEH Using trans‐Diphenyl‐Propene Oxide (t‐DPPO)
  • Support Protocol 1: Synthesis of trans‐Diphenyl‐Propene Oxide (t‐DPPO)
  • Basic Protocol 2: Measurement of sEH Inhibition Using a Fluorimetric Assay
  • Alternate Protocol 2: Fluorimetric Assay for the Screening of Chemical Libraries
  • Support Protocol 2: Synthesis of (3‐Phenyl‐Oxiranyl)‐Acetic Acid Cyano‐(6‐Methoxy‐Naphthalen‐2‐yl)‐Methyl Ester (PHOME)
  • Basic Protocol 3: LC‐MS/MS Methods for Measuring sEH Activity
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Radiometric Assay for sEH Using trans‐Stilbene Oxide (t‐SO)

  Materials
  • Tissue extracts (S‐9 fraction, cytosolic fraction, or purified enzyme, treated, if necessary, to remove glutathione; see Wixtrom and Hammock, )
  • 0.1 M sodium phosphate buffer, pH 7.4 ( appendix 2A)/0.1 mg/ml fraction V BSA (Sigma), ice cold; add BSA just before use
  • 5 mM [3H]trans‐stilbene oxide (t‐SO) in ethanol containing ∼10,000 cpm/µl (see recipe)
  • HPLC‐grade isooctane and hexanol
  • 95% (v/v) ethanol
  • Scintillation fluid
  • 10 × 75–mm borosilicate glass tubes
  • Timer
  • 30°C water bath
  • 50‐µl glass Hamilton syringe equipped with a repeating dispenser
  • Dispensers (0.1 to 1 ml) for organic solvents
  • 50‐µl glass Hamilton syringe with a blunted‐end 22s‐G needle
  • 1.5‐ml clear polypropylene microcentrifuge tubes
  • Liquid scintillation counter

Alternate Protocol 1: Radiometric Assay for sEH Using trans‐Diphenyl‐Propene Oxide (t‐DPPO)

  • 5 mM [3H]trans‐diphenyl‐propene oxide (t‐DPPO) in dimethyl formamide, containing ∼10,000 cpm/µl (see recipe)
  • HPLC‐grade methanol
Perform the assay as in protocol 1 with changes to the following steps:

Support Protocol 1: Synthesis of trans‐Diphenyl‐Propene Oxide (t‐DPPO)

  Materials
  • 1,3‐diphenyl‐2‐propanone
  • HPLC‐grade carbon tetrachloride (CCl 4)
  • Sulfuryl chloride (SO 2Cl 2)
  • HPLC‐grade ethanol
  • NaBH 4
  • 0.1 M [3H 4]NaBH 4 in ethanol (1.1 MBq/µl, 10.1 GBq/mmol)
  • 1 M sodium hydroxide
  • HPLC‐grade ethyl acetate
  • MgSO 4, anhydrous
  • Nitrogen gas tank
  • HPLC‐grade acetonitrile
  • 4‐ml reaction vessels, dry (two)
  • Filter and filter paper (e.g., Whatman)
  • 2‐ml glass vial
  • Magnetic stir bar (small) and stirrer
  • 65°C water bath
  • 15‐ml conical glass tube
  • HPLC equipped with 4 × 250–mm C18 reverse‐phase column
  • Glass ampules

Basic Protocol 2: Measurement of sEH Inhibition Using a Fluorimetric Assay

  Materials
  • Buffer A (see recipe), keep on ice
  • HPLC‐grade DMSO
  • Inhibitors dissolved in DMSO at the appropriate concentration (e.g., 10 mM 12‐(3‐adamantan‐1‐yl‐ureido)‐dodecanoic acid; AUDA; see recipe)
  • 1.2 µg/ml purified recombinant human sEH in Buffer A: prepare fresh and hold on ice
  • 0.5 mM cyano(2‐methoxynaphthalen‐6‐yl)methyl (3‐phenyloxiran‐2‐yl)methyl carbonate (CMNPC; see recipe)
  • 0.5 mM 6‐methoxynaphthalene‐2‐carbaldehyde (MNC) in DMSO (for standard curve)
  • Black 96‐well plates
  • Fluorimeter plate reader with temperature control, set at 30°C
  • 50‐µl glass Hamilton syringe equipped with a repeating dispenser

Alternate Protocol 2: Fluorimetric Assay for the Screening of Chemical Libraries

  • Inhibitors dissolved in DMSO at the appropriate concentration [e.g., 10 mM 12‐(3‐adamantan‐1‐yl‐ureido)‐dodecanoic acid; AUDA; see recipe]
  • 0.4 µg/ml purified recombinant human sEH in buffer A
  • 5 mM (3‐phenyl‐oxiranyl)‐acetic acid cyano‐(6‐methoxy‐naphthalen‐2‐yl)‐methyl ester (PHOME; see recipe)
  • 1 M ZnSO 4 in buffer A (see recipe): prepared just before use

Support Protocol 2: Synthesis of (3‐Phenyl‐Oxiranyl)‐Acetic Acid Cyano‐(6‐Methoxy‐Naphthalen‐2‐yl)‐Methyl Ester (PHOME)

  Materials
  • trans‐styrylacetic acid
  • Hydroxyl‐(6‐methoxy‐naphthalen‐2‐yl)‐acetonitrile
  • N‐ethyl‐N′‐dimethylaminopropyl‐carbodiimide (EDCI)
  • N,N‐dimethyl‐4‐amino‐pyridine (DMAP)
  • HPLC‐grade dichloromethane
  • 1 M potassium carbonate
  • MgSO 4, anhydrous
  • HPLC‐grade ethyl acetate, toluene, and hexane
  • meta‐chloro‐perbenzoic acid (m‐CPBA)
  • 50‐ml reaction vessels, dry (two)
  • 50‐ml separatory funnel
  • 50‐ml Erlenmeyer flask
  • 50‐ml round‐bottom flask
  • 1 × 20–cm silica gel column (for preparation see Vogel, )
  • Vacuum source

Basic Protocol 3: LC‐MS/MS Methods for Measuring sEH Activity

  Materials
  • Buffer A (see recipe), ice cold
  • 0.50 mM 14,15‐dihydroxy‐eicosatrienoic acid (14,15‐DHET; Cayman Chemicals; see recipe), ice cold
  • HPLC‐grade methanol, ice cold
  • 0.25 mM 9,10‐dihydroxy‐octadec‐12‐enoic acid (9,10‐DiHOME; Cayman Chemicals) in ethanol (see recipe; internal standard), ice cold
  • 0.2 µg/ml recombinant purified human sEH at in buffer A, ice cold
  • 5 mM 14,15‐epoxyeicosatrienoic acid (14,15‐EET; Cayman Chemicals) solution in ethanol (see recipe)
  • Standard solutions of 14,15‐dihydroxy‐eicosatrienoic acid (14,15‐DHET; Cayman Chemicals; see recipe)
  • 10 × 75–mm borosilicate glass tubes
  • 30°C water bath
  • Timer
  • 50‐µl glass Hamilton syringe equipped with a repeating dispenser
  • Sampling vials for LC‐MS/MS
  • HPLC equipped with a tandem quadrupole mass spectrometry detector (MS/MS)
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Figures

Videos

Literature Cited

   Beetham, J.K., Tian, T., and Hammock, B.D. 1993. cDNA cloning and expression of a soluble epoxide hydrolase from human liver. Arch. Biochem. Biophys. 305:197‐201.
   Borhan, B., Mebrahtu, T., Nazarian, S., Kurth, M.J., and Hammock, B.D. 1995. Improved radiolabeled substrates for soluble epoxide hydrolase. Anal. Biochem. 231:188‐200.
   Dietze, E.C., Kuwano, E., and Hammock, B.D. 1994. Spectrophotometric substrates for cytosolic epoxide hydrolase. Anal. Biochem. 216:176‐187.
   Gill, S.S. and Hammock, B.D. 1979. Hydration of cis‐ and trans‐epoxymethyl stearates by the cytosolic epoxide hydrase of mouse liver. Biochem. Biophys. Res. Commun. 89:965‐971.
   Gill, S.S., Ota, K., and Hammock, B.D. 1983. Radiometric assays for mammalian epoxide hydrolases and glutathione S‐transferase. Anal. Biochem. 131:273‐282.
   Grant, D.F., Moody, D.E., Beetham, J., Storms, D.H., Moghaddam, M.F., Borhan, B., Pinot, F., Winder, B., and Hammock, B.D. 1994. The response of soluble epoxide hydrolase and other hydrolytic enzymes to peroxisome proliferators. In Peroxisome Proliferators: Unique Inducers of Drug‐Metabolizing Enzymes (D.E. Moody, ed.) pp. 97‐112. CRC Press, Boca Raton, Fla.
   Greene, J.F., Williamson, K.C., Newman, J.W., Morisseau, C., and Hammock, B.D. 2000. Metabolism of monoepoxides of methyl linoleate: Bioactivation and detoxification. Arch. Biochem. Biophys. 376:420‐432.
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   Hammock, B.D., Loury, D.N., Moody, D.E., Ruebne, B., Baselt, R., Milam, K.M., Volberding, P., Ketterman, A., and Talcott, R. 1984. A methodology for the analysis of the preneoplastic antigen. Carcinogenesis 5:1467‐1473.
   Hammock, B.D., Grant, D., and Storms, D. 1997. Epoxide hydrolase. In Comprehensive Toxicology (I. Sipes, C. McQueen, and A. Gandolfi, eds.) pp. 283‐305. Pergamon, Oxford.
   Hasegawa, L.S. and Hammock, B.D. 1982. Spectrophotometric assay for mammalian cytosolic epoxide hydrolase using trans‐stilbene oxide as the substrate. Biochem. Pharmacol. 31:1979‐1984.
   Imig, J.D. 2006. Cardiovascular therapeutic aspects of soluble epoxide hydrolase inhibitors. Cardiovasc. Drug Rev. 24:169‐188.
   Jones, P.D., Wolf, N.M., Morisseau, C., Whetstone, P., Hock, B., and Hammock, B.D. 2005. Fluorescent substrates for soluble epoxide hydrolase and application to inhibition studies. Anal. Biochem. 343:66‐75.
   McElroy, N.R., Jurs, P.C., Morisseau, C., and Hammock, B.D. 2003. QSAR and classification of murine and human soluble epoxide hydroalse inhibition by urea‐like compounds. J. Med. Chem. 46:1066‐1080.
   Morisseau, C. and Hammock, B.D. 2005. Epoxide hydrolases: Mechanisms, inhibitor designs, and biological roles. Annu. Rev. Pharmacol. Toxicol. 45:311‐333.
   Morisseau, C., Goodrow, M.H., Dowdy, D., Zheng, J., Greene, J.F., Sanborn, J.R., and Hammock, B.D. 1999. Potent urea and carbamate inhibitors of soluble epoxide hydrolases. Proc. Natl. Acad. Sci. U.S.A. 96:8849‐8854.
   Morisseau, C., Beetham, J.K., Pinot, F., Debernard, S., Newman, J.W., and Hammock, B.D. 2000. Cress and potato soluble epoxide hydrolases: Purification, biochemical characterization, and comparison to mammalian enzymes. Arch. Biochem. Biophys. 378:321‐332.
   Mullin, C.A. and Hammock, B.D. 1980. A rapid radiometric assay for mammalian cytosolic epoxide hydrolase. Anal. Biochem. 106:476‐485.
   Mumby, S.M. and Hammock, B.D. 1979. A partition assay for epoxide hydrases acting on insect juvenile hormone and an epoxide‐containing juvenoid. Anal. Biochem. 92:16‐21.
   Nakagawa, Y., Wheelock, C.E., Morisseau, C., Goodrow, M.H., Hammock, B.G., and Hammock, B.D. 2000. 3‐D QSAR analysis of inhibition of murine soluble epoxide hydrolase (MsEH) by benzoylureas, arylureas, and their analogues. Bioorg. Med. Chem. 8:2663‐2673.
   Newman, J.W., Watanabe, T., and Hammock, B.D. 2002. The simultaneous quantification of cytochrome P450 dependent linoleate and arachidonate metabolites in urine by HPLC‐MS/MS. J. Lipid Res. 43:1563‐1578.
   Newman, J.W., Morisseau, C., and Hammock, B.D. 2005. Epoxide hydrolases: Their roles and interactions with lipid metabolism. Prog. Lipid Res. 44:1‐51.
   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.
   Oliw, E.H. 1994. Oxygenation of polyunsaturated fatty acids by cytochrome P450 monooxygenases. Prog. Lipid Res. 33:329‐354.
   Spector, A.A. and Norris, A.W. 2007. Action of epoxyeicosatrienoic acids on cellular function. Am. J. Physiol. Cell. Physiol. 292:C996‐C1012.
   Vogel, A.I. 1989. Vogel's Textbook of Practical Organic Chemistry, 5th ed. Addison‐Wesley Longman, New York.
   Wixtrom, R.N. and Hammock, B.D. 1985. Membrane‐bound and soluble‐fraction epoxide hydrolases: Methodological aspects. In Biochemical Pharmacology and Toxicology, Vol. 1: Methodological Aspects of Drug Metabolizing Enzymes (D. Zakim and D.A. Vessey, eds.) pp. 1‐93. John Wiley & Sons, New York.
   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. Biochem. 169:71‐80.
   Wolf, N.M., Morisseau, C., Jones, P.D., Hock, B., and Hammock, B.D. 2006. Development of a high throughput screen for soluble epoxide hydrolase inhibition. Anal. Biochem. 355:71‐80.
   Zeldin, D.C., Kobayashi, J., Falck, J.R., Winder, B.S., Hammock, B.D., Snapper, J.R., and Capdevila, J.H. 1993. Regio‐ and enantiofacial selectivity of epoxyeicosatrienoic acid hydration by cytosolic epoxide hydrolase. J. Biol. Chem. 268:6402‐6407.
Key References
  Borhan et al., 1995. See above.
  This method is described in .
  Jones et al., 2005. See above.
  This method is described in .
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