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Epoxide hydrolase, its function and determination of its activity in rat liver
Exp. Pathol. 1990; 39: 195-196 Gustav Fischer Verlag lena
Institute of Pharmacology and Toxicology, Friedrich Schiller University, Jena, GDR
Epoxide hydrolase, its function and determination of its activity in rat liver By K. SPLINTER, D. BAUER and W. SEIDEL
Address for correspondence: Dr. rer. nat. K. SPLINTER, Institute of Pharmacology and Toxicology, Friedrich Schiller University, L6bderstraBe 1, DDR - 6900 Jena Key words: epoxide hydrolase; rat liver; detennination by HPLC
Summary Epoxides are a group of reactive intermediates formed by the cytochrome P-450-mediated monooxygenation of unsaturated xenobiotics. Epoxide hydrolase inactivates these epoxides by addition of water to form diols. Commonly the function of epoxide hydrolase is finally followed by excretion of the diols. However, reactivation of certain diols by a second epoxidation may happen. Epoxide hydrolase inactivates also the epoxides existing in the metabolism of endogenous compounds. The determination of the activity of epoxide hydrolase by the addition of water to styrene oxide (1,2epoxyethylbenzene) and measurement of the concentration of the produced phenylglycol (1-phenyl-l,2-ethandiol) with subsequent separation of the 2 substances by HPLC is described. Lipophilic xenobiotics tend to accumulate into tissues, and they must be transformed to water soluble compounds to enable the excretion. In this transformation process reactive intermediates are produced. If biotransformation fails to detoxify these reactive intermediates, they may react covalently with critical targets like the genetic material, or start harmful reaction chains like lipid peroxidation. As a result of this carcinogenicity, mutagenicity etc. may ensue MILLER and MILLER (1976). Depending on the chemical structure of the molecule, different kinds of reactive substances are generated. Epoxides originate from oxidation of an aliphatic or aromatic double bond by the action of cytochrome P-450-mediated monooxygenases (LEIBMANN et al. 1979). One detoxifying pathway is the addition of water to form dials, which are of low reactivity; this reaction is catalized by epoxide hydrolase. Other possible pathways are the formation of glutathion conjugates or the rearrangement to aldehydes or ketones (HABIG et al. 1974; OESCH 1979). Epoxide hydrating enzymes have been detected in many species (WALKER et al. 1978). In rat liver it becomes detectable on the 15 th day of gestation and increases slowly to the day of delivery. After birth there is a further growth of acti vity, and adult levels are reached in the second month of life (JAMES et al. 1977). The activity of epoxide hydrolase increases in response to exposure of many foreign compounds. Phenobarbital was shown as a typical inducer of epoxide hydrolase in rat liver, also a large number of chlorinated hydrocarbons elevates the activity of the enzyme. An inhibition is reached by many epoxides, alkylating chemicals, heavy metals and organophosphates (OESCH et al. 1973; PARKKI and AITIO 1978; P ARKKI 1980). The substrate specificity of epoxide hydrolase is very low, many substrates have been investigated. Numerous aliphatic, alicyclic and aromatic epoxides are biotransformed by this enzyme. A very often used substrate is styrene oxide which is also used in our institute for the 13*
Exp. Pathol. 39 (1990) 3-4
195
detennination of enzyme activity in rat liver. The method was published by SCHOKET and VINCZE (1985) and modified for our conditions and purposes.
•
epoxide hydrolase styrene oxide
o
CH - - CH
I
OH
I
Z
OH
phenyl glycol
Material and Methods Tris buffer: Tris-HCI buffer, pH 9.0, mixed with 1g Tween 80/1 solution. Phosphate buffer: 0.1 M phosphate buffer (Na2HP04 + NaH 2P0 4), pH 7.4. Styrene oxide working solution: Styrene oxide, dissolved in acetonitrile to a final concentration of 5mg/m!. Phenylglycol calibration solution: 200mg phenylglycol dissolved in 100ml Tris buffer and diluted with buffer to a final concentration of 0.2mg/m!. 9,000 g supernatant: Liver samples homogenized with 2 volumes of ice cold phosphate buffer and centrifuged at 9,000 g for 20min, the supernatant removed and diluted with phosphate buffer to a final protein concentration of 20- 35 mg/m!. Incubation procedure and analytical determination: A mixture of 0.5 m! Tris buffer + 0.4 ml supernatant + 0.05 ml styrene oxide solution are incubated for 20min at a temperature of 37°C. The reaction is stopped by addition of 3 ml acetic acid ethylester and the produced phenyl glycol and the residue of styrene oxide are extracted by mechanical shaking for 15 min· 2 ml of the esterphase are removed and evaporated in a nitrogen stream at a temperature lower than 70°C. During this procedure the styrene oxide evaporates together with the acetic acid ethylester in an azeotropic mixture. The residue is dissolved in 0.2ml methanol/water (60%) and directly injected to the column. For the calibration curve the amount of Tris buffer is replaced by different volumes (0.1-0.5 ml) of phenylglycol calibration solution. HPLC conditions: Stationary phase: Li-Chrosorb RP 18, lO[lm; mobile phase: Methanol! water 60 % ; column length: 250 mm guard column 40 mm ; flow rate 2 ml/min ; injection volume: 20 [ll; UV -detection: 258 nm; evaluation: height of the phenylglycol peak.
References HABIG, W. H., PABST, M. J., JACOBY, W. B.: Glutathion-S-transferase. J. BioI. Chern. 1974; 249: 7130-7139. JAMES, M. 0., FOUREMAN, G. L., LAW, F. C., BEND, J. R.: The perinatal development of epoxide metabolizing enzyme activities in liver and extrahepatic organs of guinea pig and rat. Drug Metab. Dispos. 1977; 5: 19-28. LEIBMANN, K. C., ORTIZ, E.: Epoxide intermediates in microsomal oxidation of olefins to glycols. J. Exp. Ther.
1979; 173: 242-246.
MILLER, J. A., MILLER, E. c.: Metabolic activation of chemicals to reactive eletrophiles. In: Advances in pharmacology and therapeutics. Pergamon Press, Oxford 1976. OESCH, F.: Enzymes as regulators of toxic reactions by electrophilic metabolites. Arch. Toxicol. 1979; 2 (Suppl.):
215-227. JERINA, D. M., DALY, 1. W .. RICE, J. M.: Induction, activation and inhibition of epoxide hydrolase. Chern. BioI. Interact. 1973; 6: 189-202. PARKKI, M. G.: Inhibition of rat hepatic microsomal styrene oxide hydration by mercury and zinc in vitro. Xenobiotica -
1980; 10: 307-310. -
AITIo, A.: Inducing of drug metabolizing enzymes in different rat tissues by TCDD. Arch. Toxicol. 1978; 1:
261-265. SCHOKET, B., VINCZE, J.: Induction of rat hepatic drug metabolizing enzymes by substituted urea herbicides. Acta Pharmacol. Toxicol. 1985; 56: 293-288. WALKER. L. H .. BENTLEY, P., OESCH, F.: Phylogenetic distribution of epoxide hydrolase in different vertebrate species, strains and tissues measured, using three substrates. Biochem. Biophys. Acta 1978; 539: 427 -434. (Accepted January 17, 1990)
196
Exp. Pathol. 39 (1990) 3-4
Institute of Pharmacology and Toxicology, Friedrich Schiller University, Jena, GDR
Epoxide hydrolase, its function and determination of its activity in rat liver By K. SPLINTER, D. BAUER and W. SEIDEL
Address for correspondence: Dr. rer. nat. K. SPLINTER, Institute of Pharmacology and Toxicology, Friedrich Schiller University, L6bderstraBe 1, DDR - 6900 Jena Key words: epoxide hydrolase; rat liver; detennination by HPLC
Summary Epoxides are a group of reactive intermediates formed by the cytochrome P-450-mediated monooxygenation of unsaturated xenobiotics. Epoxide hydrolase inactivates these epoxides by addition of water to form diols. Commonly the function of epoxide hydrolase is finally followed by excretion of the diols. However, reactivation of certain diols by a second epoxidation may happen. Epoxide hydrolase inactivates also the epoxides existing in the metabolism of endogenous compounds. The determination of the activity of epoxide hydrolase by the addition of water to styrene oxide (1,2epoxyethylbenzene) and measurement of the concentration of the produced phenylglycol (1-phenyl-l,2-ethandiol) with subsequent separation of the 2 substances by HPLC is described. Lipophilic xenobiotics tend to accumulate into tissues, and they must be transformed to water soluble compounds to enable the excretion. In this transformation process reactive intermediates are produced. If biotransformation fails to detoxify these reactive intermediates, they may react covalently with critical targets like the genetic material, or start harmful reaction chains like lipid peroxidation. As a result of this carcinogenicity, mutagenicity etc. may ensue MILLER and MILLER (1976). Depending on the chemical structure of the molecule, different kinds of reactive substances are generated. Epoxides originate from oxidation of an aliphatic or aromatic double bond by the action of cytochrome P-450-mediated monooxygenases (LEIBMANN et al. 1979). One detoxifying pathway is the addition of water to form dials, which are of low reactivity; this reaction is catalized by epoxide hydrolase. Other possible pathways are the formation of glutathion conjugates or the rearrangement to aldehydes or ketones (HABIG et al. 1974; OESCH 1979). Epoxide hydrating enzymes have been detected in many species (WALKER et al. 1978). In rat liver it becomes detectable on the 15 th day of gestation and increases slowly to the day of delivery. After birth there is a further growth of acti vity, and adult levels are reached in the second month of life (JAMES et al. 1977). The activity of epoxide hydrolase increases in response to exposure of many foreign compounds. Phenobarbital was shown as a typical inducer of epoxide hydrolase in rat liver, also a large number of chlorinated hydrocarbons elevates the activity of the enzyme. An inhibition is reached by many epoxides, alkylating chemicals, heavy metals and organophosphates (OESCH et al. 1973; PARKKI and AITIO 1978; P ARKKI 1980). The substrate specificity of epoxide hydrolase is very low, many substrates have been investigated. Numerous aliphatic, alicyclic and aromatic epoxides are biotransformed by this enzyme. A very often used substrate is styrene oxide which is also used in our institute for the 13*
Exp. Pathol. 39 (1990) 3-4
195
detennination of enzyme activity in rat liver. The method was published by SCHOKET and VINCZE (1985) and modified for our conditions and purposes.
•
epoxide hydrolase styrene oxide
o
CH - - CH
I
OH
I
Z
OH
phenyl glycol
Material and Methods Tris buffer: Tris-HCI buffer, pH 9.0, mixed with 1g Tween 80/1 solution. Phosphate buffer: 0.1 M phosphate buffer (Na2HP04 + NaH 2P0 4), pH 7.4. Styrene oxide working solution: Styrene oxide, dissolved in acetonitrile to a final concentration of 5mg/m!. Phenylglycol calibration solution: 200mg phenylglycol dissolved in 100ml Tris buffer and diluted with buffer to a final concentration of 0.2mg/m!. 9,000 g supernatant: Liver samples homogenized with 2 volumes of ice cold phosphate buffer and centrifuged at 9,000 g for 20min, the supernatant removed and diluted with phosphate buffer to a final protein concentration of 20- 35 mg/m!. Incubation procedure and analytical determination: A mixture of 0.5 m! Tris buffer + 0.4 ml supernatant + 0.05 ml styrene oxide solution are incubated for 20min at a temperature of 37°C. The reaction is stopped by addition of 3 ml acetic acid ethylester and the produced phenyl glycol and the residue of styrene oxide are extracted by mechanical shaking for 15 min· 2 ml of the esterphase are removed and evaporated in a nitrogen stream at a temperature lower than 70°C. During this procedure the styrene oxide evaporates together with the acetic acid ethylester in an azeotropic mixture. The residue is dissolved in 0.2ml methanol/water (60%) and directly injected to the column. For the calibration curve the amount of Tris buffer is replaced by different volumes (0.1-0.5 ml) of phenylglycol calibration solution. HPLC conditions: Stationary phase: Li-Chrosorb RP 18, lO[lm; mobile phase: Methanol! water 60 % ; column length: 250 mm guard column 40 mm ; flow rate 2 ml/min ; injection volume: 20 [ll; UV -detection: 258 nm; evaluation: height of the phenylglycol peak.
References HABIG, W. H., PABST, M. J., JACOBY, W. B.: Glutathion-S-transferase. J. BioI. Chern. 1974; 249: 7130-7139. JAMES, M. 0., FOUREMAN, G. L., LAW, F. C., BEND, J. R.: The perinatal development of epoxide metabolizing enzyme activities in liver and extrahepatic organs of guinea pig and rat. Drug Metab. Dispos. 1977; 5: 19-28. LEIBMANN, K. C., ORTIZ, E.: Epoxide intermediates in microsomal oxidation of olefins to glycols. J. Exp. Ther.
1979; 173: 242-246.
MILLER, J. A., MILLER, E. c.: Metabolic activation of chemicals to reactive eletrophiles. In: Advances in pharmacology and therapeutics. Pergamon Press, Oxford 1976. OESCH, F.: Enzymes as regulators of toxic reactions by electrophilic metabolites. Arch. Toxicol. 1979; 2 (Suppl.):
215-227. JERINA, D. M., DALY, 1. W .. RICE, J. M.: Induction, activation and inhibition of epoxide hydrolase. Chern. BioI. Interact. 1973; 6: 189-202. PARKKI, M. G.: Inhibition of rat hepatic microsomal styrene oxide hydration by mercury and zinc in vitro. Xenobiotica -
1980; 10: 307-310. -
AITIo, A.: Inducing of drug metabolizing enzymes in different rat tissues by TCDD. Arch. Toxicol. 1978; 1:
261-265. SCHOKET, B., VINCZE, J.: Induction of rat hepatic drug metabolizing enzymes by substituted urea herbicides. Acta Pharmacol. Toxicol. 1985; 56: 293-288. WALKER. L. H .. BENTLEY, P., OESCH, F.: Phylogenetic distribution of epoxide hydrolase in different vertebrate species, strains and tissues measured, using three substrates. Biochem. Biophys. Acta 1978; 539: 427 -434. (Accepted January 17, 1990)
196
Exp. Pathol. 39 (1990) 3-4