Coordinate induction of peroxisomal β-oxidation activity and cytosolic epoxide hydrolase activity

Pharmac. Ther. Vol. 33, pp. 29 to 35, 1987

0163-7258/87 $0.00 + 0.50 Copyright © 1987 Pergamon Journals Ltd

Printed in Great Britain. All rights reserved

Specialist Subject Editor: E. E. OHNHAUS

COORDINATE INDUCTION OF PEROXISOMAL B-OXIDATION ACTIVITY AND CYTOSOLIC EPOXIDE HYDROLASE ACTIVITY

F. OEsc~I and L. SCHt.ADT Institut fiir Toxikologie, Universitiit Mainz, Obere Zahlbacher Stral3e 67, D-6500 Mainz, F.R.G.

ABBREVIATIONS STO--2-phenyloxirane (styrene 7,8-oxide); TSO--trans-2,3-diphenylox2rane (trans-stilbene oxide). ABSTRACT The effect of four hypolipidemic compounds (tiadenol, clofibrate, acetylsalicylic acid, 1-benzylimidazole) on the specific activities of peroxisomal/3-oxidation and cytosolic and microsomal epoxide hydrolase of rat liver was investigated. Since specific activity of cytosolic epoxide hydrolase from outbred Sprague-Dawley rats showed large interindividual variations (-38-fold), induction studies were performed with inbred Fischer F-344 rats, which showed only low interindividual variations (-2-fold). Clofibrate, tiadenol and acetylsalicylic acid caused a 8-, 13- and 4.5-fold induction of cEH and a 13-, 19- and 5-fold induction of peroxisomal/3-oxidation activity, respectively. Microsomal epoxide hydrolase activity was only slightly increased ( < 1.5-fold). 1-Benzylimidazole induced both cytosolic epoxide hydrolase and peroxisomal/3-oxidation activity about 2-fold, whereas microsomal epoxide hydrolase activity was increased about 4-fold. Increase in cytosolic epoxide hydrolase activity was not due to enzyme activation as demonstrated by in vitro studies. On the other hand, these in vitro studies showed that the increase in microsomal epoxide hydrolase activity by 1-benzylimidazole may partially be due to activation of the enzyme. INTRODUCTION It has been shown that several hypolipidemic drugs are tumourigenic in rats and mice (1). As these compounds have been found to be negative as mutagens in the Ames test, hypotheses regarding the tumourigenicity of the triglyceride-lowering drugs are based on unique biochemical alterations induced by these compounds (2). In rats and mice, the drugs have been reported to cause a proliferation of peroxisomes and concomitantly a huge induction (up to 20-30-fold) of some--mostly peroxisomal--enzymes, which are mainly involved in the metabolism of fatty acids. One of the most critical events may be the induction of a peroxisomal B-oxidation system. The first step of the peroxisomal pathway is, in contrast to mitochondrial/3-oxidation, catalyzed by an acyl-CoA oxidase producing hydrogen peroxide (Fig. 1). In contrast to the peroxisomal/3-oxidation system, catalase is only moderately induced (about 2-fold); this may cause an increase in the intracellular concentration of hydrogen peroxide. Reactive oxygen species derived from hydrogen peroxide, therefore, may be responsible for the carcinogenicity of peroxisome proliferating drugs. Epoxides are common products of cytrochrome P-450 catalyzed oxidation of olefinic and aromatic compounds. There are several alternative pathways for epoxide metabolism, e.g. cytochrome P-450 mediated reduction to the parent compound, conjugation with glutathione (enzymatieally or non-enzymatically), or addition of water by epoxide hydrolases 29

F. O~SCH and L. SCm,ADT

30

peroxlsomal B-oxidation system

R

-

CH 2

-

CH 2

-

CO

-

SCoA

02

acyl-CoA oxtdase

H202 R

-

CH

=

CH

-

CO

-

SCoA

H20

bifunctional R

-

CHOH

-

enoylhydratase and L-3-hydroxy fatty acyl-CoA dehydrogenase

CH2 - CO - SCoA

~~

NAD+ NADH

+

H +

R - CO - CH 2 - CO - SCoA

thlolase

CoASH

R

-

CO

-

SCoA

+

CH 3

-

CO

-

SCoA

FIo. 1. Peroxisomal/3-oxidation.

leading to dihydrodiols. In rat liver, TSO and STO can be used as diagnostic substrates for cytosolic and microsomal epoxide hydrolase (Fig. 2). Four hypolipidemic compounds were investigated for their effects on enzyme induction (peroxisomai/3-oxidation, microsomal and cytosolic epoxide hydrolase) in Fischer F-344 rats: clofibrate, tiadenol, acetylsalicylic acid and 1-benzylimidazole (Fig. 3). All of them have been reported to cause peroxisome proliferation (3). Clofibrate is a fibric acid derivate and structually unrelated to the other compounds investigated. Acetylsalicylic acid is mainly Reaction of rat l i v e r cytosolic epoxide hydrolase

° H=O IL

trans-Stilbene oxide

erythro-dihydrodio]

Reaction of r a t l i v e r microsomal epoxide hydrolase

•

Styrene oxide

dihydrodiol

F1o. 2. Assay of rat liver cytosolic and microsomal epoxide hydrolase.

Coordinate induction NO- -

CN t - - CH t - - $ - - (CH=)m - $ - - Ct'~ --CH= - - OH

31

liadenol

CH= 0 dofibrate

ICHor

~ ~

OOH 0 _o--IC I --CH I acet

yisalicylic

acid

CH~ ~

1- benzytimidazole

Fro. 3. Compoundstested as inducers of cytosolicand microsomal epoxide hydrolase. used as an analgesic drug and has only very low hypolipidemic potency, whereas clofibrate and tiadenol are potent triglyceride-lowering drugs. MATERIALS AND METHODS MATERIALS

[3H]Styrene oxide (pH]STO), 11.7 GBq/mmol and [3H]trar~-stilbene oxide ([3H]TSO), 0.41 GBq/mmol were synthesized as described in references (4,5). 1-Benzylimidazole and tiadenol were purchased from Aldrich (Steinheim, F.R.G.) and clofibrate from Serva Feinbiochemica (Heidelberg, F.R.G.). Acetylsalicylic acid was obtained from Sigma (Deisenhofen, F.R.G.). All other chemicals used were of analytical grade or the purest grade commercially available. ANI~dAL EXPERIMENTS

Male Fischer F-344 rats (150-160 g) were obtained from Charles River Wiga GmbH, Sulzfeld, F.R.G.; male Sprague-Dawley rats 080-220 g) were obtained from Sfiddeutsche Versuchstieranstalt, Tuttlingen, F.R.G. Animals were kept at constant temperature, under a constant light-dark cycle and had free access to water and defined diet (Altromin). For the induction studies animals were fed a pelleted diet containing the hypolipidemic compound. They remained on their diet for seven days until they were killed. The compounds were added by soaking the food in an acetone solution and subsequent evaporation of the organic solvent. ENZYME ASSAYS

Activity of rat cytosolic epoxide hydrolase was assayed in glass centrifuge tubes containing 5nmol pH]TSO, 0.125 mM Tris-HC1, pH 7.4 and 1.25 mM 1-chloro-2, 4-dinitrobenzene (CDNB) in a total volume of 200 #1. CDNB was added in order to reduce substrate depletion caused by glutathione conjugation. The reaction was stopped by addition of 3 ml fight petroleum ether and 250 ~ dimethyl sulfoxide. The unhydrolyzed substrate was extracted by shaking the tubes for three min followed by a brief centrifugation at 1000 x g to resolve the phases. After discarding the ether phase, the aqueous phase was washed again with 3 ml of light petroleum ether. The enzymatically formed diol was then extracted into 1 ml of ethyl acetate, and an aliquot was counted. The assay for microsomal

32

F. OESCH and L. SCHLADT

epoxide hydrolase was performed as described in reference (4) using the conditions described in reference (6) specifically in the absence of detergent. Activity of cyanide-insensitive peroximal/3-oxidation was determined in the 600 g supernatant according to Bieri et al. (7). RESULTS AND DISCUSSION The effect of temperature on the activity of cytosolic epoxide hydrolase is shown in Fig. 4. Activity was low below 30°C, but increased at higher temperatures and reached a maximum at 50°C. In a separate experiment, liver cytosol was incubated for 10 min at the indicated temperature, cooled in an ice bath, and then assayed for cytosolic epoxide hydrolase activity at 37°C. Figure 4 (open circles) shows that activity remained constant up to 50°C. Although specific activity was about 5-fold higher at 50°C, routine assays were performed at 37°C since this is the most commonly used temperature and more relevant to in vivo conditions. The incubation temperature was maintained at exactly 37°C in order to avoid differences in cytosolic epoxide hydrolase activity due to different incubation temperatures. Outbred Sprague-Dawley rats showed large interindividuai differences in specific activity of liver cytosolic epoxide hydrolase. The variation of specific activity was between 2 and 78 pmol TSO/min x mg protein. The large interindividual variation of rat cytosolic epoxide hydrolase is consistent with previous reports showing a 539-fold interindividual variation of human liver cytosolic epoxide hydrolase (8) and a 5-fold variation of soluble epoxide hydrolase from human leukocytes (9). Large interindividual differences observed with outbred Sprague-Dawley rats may have been the result of their genetic constitution, as with inbred Fischer F-344 rats the variation in the specific activity of cytosolic epoxide hydrolase was much lower, varying only by a factor of two, from 24 to 48 pmol TSO/min × mg protein. Therefore, we chose this rat strain for the induction studies with hypolipidemic compounds. In contrast to microsomai epoxide hydrolase, cytosolic epoxide hydrolase was not induced by classical inducers of xenobiotic metabolizing enzymes such as trans-stilbene oxide, 3-methylcholanthrene, phenobarbitone, TCDD (data not shown) and aroclor 1254. The effect of treatment with aroclor 1254 on the enzyme activities is shown in the last column of Fig. 5. Microsomal epoxide hydrolase was induced 2-fold, whereas activities of cytosolic epoxide hydrolase and peroxisoma113-oxidation remained unchanged. The lack of induction of the hepatic cytosolic epoxide hydrolase by 3-methylcholanthrene and trans-stilbene oxide

cEH activity (% of control) 500-

400-

300 ~

200-

100-

10

20

30

/.0

50

60

70

temperature ('C) FIG. 4. Effect of temperature on cytosolic epoxide hydrolase activity. Filled circles: incubation performed at the temperature indicated; open circles: preincubation for 10 min at the temperature indicated, followed by cooling on ice and assaying at 37°C.

Coordinate induction

33

r~attve spedfic ~¢tivity

20!

0

tiadenol

controt

37

99

.

.

.

S/.8

.

.

clofi~ate ~ $6

2~3

acetyt saiicy(ic

acid

190

($30

benzy(-

imidazot S/.

aroc(or

mg/kg

FIo. 5. Inducibility of cytosolic epoxide hydrolase (hatched bars), peroxisomal/~-oxidation activity (open bars) and microsomai ¢poxide hydrolase (dotted bars) in liver of male Fischer F-344 tats. The drugs were given in the diet for one week. The daily drug intake per kg body weight is given. Animals were treated with aroclor 1254 by two i.p. injections of 500 mg/kg seven and four days before killing. Activities of control animals were: cytosolic epoxide hydrola~: 38 pmol TSO/min x mg protein; microsomal cpoxide hydrolas¢: 4,0 nmol STO/min x rag protein, peroxisomal ~l-oxidation; 5.2 nmol NAD/min × mg protein.

agrees with the findings of Hammock and Ota (10), whereas they observed a significant decrease in enzyme activity after treatment with phenobarbitone or aroclor 1254. On the other hand, treatment of Fischer F-344 rats for one week with the hypolipidemic drugs, tiadenol, clofibrate or acetylsalicylic acid caused a 13-, 8- and 4.5-fold induction of cytosolic epoxide hydrolase and 19-, 13- and 5-fold induction of pcroxisomal/~-oxidation, respectively (Fig. 5). Using three different concentrations of tiadenol and two concentrations of clofibrate and acetylsalicylic acid in the diet it could be shown that the induction of cytosolic epoxide hydrolase and peroxisomal/3-oxidation was dose-dependent (Fig. 5). In contrast, activity of microsomal epoxide hydrolase was only slightly increased by these compounds (
34

F. O~SCHand L. SCHLADT relative specific activity

2-

controt

ctofibrate

tiodenot

acetytsaticytic acid

benzytimiduzot

Flo. 6. In vitro effect of four hypolipidemic compounds on the specific activity of rat liver cytosolic

(hatched bars) and microsomal (dotted bars) epoxide hydrolase. The compounds were incorporated into the assay mixture at final concentrations of lmu. activation; in contrast, s o m e o f the c o m p o u n d s inducing cytosolic epoxide hydrolase activity in v i v o were f o u n d t o i n h i b i t t h e e n z y m e in vitro (Fig. 6). CONCLUSION R a t liver c y t o s o l i c e p o x i d e h y d r o l a s e was c o n c o m i t a n t l y i n d u c e d with p e r o x i s o m a l / 3 o x i d a t i o n b y p e r o x i s o m e - p r o l i f e r a t i n g drugs. Since m a n y e n z y m e s (e.g. p e r o x i s o m a l / 3 o x i d a t i o n system (2,3), p a l m i t o y l C o A h y d r o l a s e (12), carnitine acyltransferases (2)) induced b y p e r o x i s o m e p r o l i f e r a t o r s a r e i n v o l v e d in l i p i d m e t a b o l i s m , a p o s s i b l e i n v o l v e m e n t o f c y t o s o l i c e p o x i d e h y d r o l a s e in l i p i d m e t a b o l i s m seems r e a s o n a b l e . This is u n d e r l i n e d b y t h e fact t h a t s o m e f a t t y a c i d e p o x i d e s h a v e b e e n r e p o r t e d t o b e s u b s t r a t e s f o r cytosolic e p o x i d e h y d r o l a s e (13,14). Acknowledgements--This study was supported by the Fonds der Chemischen Industrie. The authors thank Mrs.

I. B6hm for typing the manuscript. REFERENCES 1. REDDY,J. K., AZm~OFF, D. L. and HIGNITE,C. E. (1980) Hypolipidaemichepatic peroxisome proliferators form a novel class of chemical carcinogens. Nature 283: 397. 2. REDDY,J. K. and LxLwm,;x, N. D. (1983) Carcinogenesis by hepatic peroxisome proliferators: Evaluation of the risk of hypolipidemic drugs and industrial plasticizers to humans. CRC Crit. Rev. Toxicol, 12: 1. 3. COHEN,A. J. and G~sso, P. (1981) Review of the hepatic response to hypolipidemic drugs in rodents and assessment of its toxicological significance to man. Fd. Cosmet. Toxicol. I9: 585. 4. OESCH,F., J E ~ A , D. M. and DxLY, J. (1971) A radiometric assay for hepatic epoxide hydrolase activity with 7- H-styrene oxide. Biochim. Biophys. Acta 227: 685. 5. O~SCH, F., Sp~.ROW, A. J. and PLXTT, K. L. (1980) Radioactively labelled epoxides part II. (1) Tritium labelled cyclohexene oxide, trans-stllbene oxide and phenanthrene 9,10-oxide. J. Labelled Comp. Radiopharm. 17: 93. 6. O~SCH,F. (1974) Purification and specificity of a microsomal human epoxide hydrolase. Biochem. J. 139: 77. 7. Bm~, F., BESTLSY,P., W~CHTEX, F. and ST.~UaU,W. (1984) Use of primary cultures of adult rat hepatocytes to investigate mechanisms of action of nafenopin, a hepatocarcinogenic peroxisome proliferator. Carcinogenesis 5: 1033. 8. MmtTSS,I., FLmSCmO,SN, R., GLXTT,H. R. and OESCH,F. (1985) Interindividual variations in the activities of cytosolic and microsomal epoxide hydrolase in human liver. Carinogenesis 6: 219. 9. SEmEOAaD,J., DEPmm~, J. W. and P~xo, R. W. (1984) Measurement and characterization of membranebound and soluble epoxide hydrolase activities in resting mononuclear leukocytes from human blood. Cancer Res. 44: 3654.

Coordinate induction

35

I0. HAM~OCx,B. D. and OrA, K. (1983) Differentialinduction ofcytosotlc epoxidehydrolase,microsomalepoxide hydrolase, and glutathione S-transferase activities. Toxico[. appL Pharmacol. 71: 254. I I. WAECaT~, F., BmPa,F,, S~:Aw~, W. and B ~ , P. (1984)Induction of cytosolJcand microsomalepoxide hydroinses by the hypolipidaemic compound Nafenopin in the mouse liver. Biochem. PharmacoL 33:3 I. 12. B m ~ , R. K. and B~rK~, O. M. (1981) Changes in lipid metabolizingtmzymesof hepatic subcetlularfractions from rats treated with tiadenol and clofibrate. Biochem. Pharmacol. 30: 2551. 13. Gn.L, S. S. and Hnm~ocx, B. D. (1979) Hydration of c/s- and trans-epoxymethylstearates by the cytosolic epoxide hydrase of mouse liver. Biochem. Biophys. Res. Commun. 89: 965. 14. CI~cos, N., CAPI)EVn.LA, J., FArCe, J. R., MAN~n~, S., MARTn~-WtXT~OM, C., G~L, S. S., HAMMOCK, B. D. and ESTAnROOZ, R. W. (1983) The relationof arachidonic acid epoxides (epoxyeicosatrienoic acid) with a cytosolic epoxide hydrolase. Arch. Biochem. Biophys. 223: 639.