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Perinatal development of styrene monooxygenase and epoxide hydrolase in rat liver microsomes and nuclei
Chem.-Biol. Interactions, 47 (1983) 213-222 Elsevier Scientific Publishers Ireland Ltd.
213
P E R I N A T A L D E V E L O P M E N T OF S T Y R E N E M O N O O X Y G E N A S E AND EPOXIDE AND NUCLEI*
HYDROLASE
IN RAT LIVER MICROSOMES
M. ROMANO,~G. GAZZOTTI, V.° CLOS**, B.M. ASSAEL***, R. MAFFEI FACINO~ and M. SALMONA* Laboratory for Enzyme Research, Istituto di Ricerche Farmacologiche 'Mario Negri', Via Eritrea, 62, 20157 Milan (Italy) (Received February 2nd, 1983) (Revision received June 7th, 1983) (Accepted June 13th, 1983)
SUMMARY N u c l e a r e n z y m e s were f o u n d to develop e a r l i e r t h a n the c o r r e s p o n d i n g m i c r o s o m a l activities. In fact s t y r e n e m o n o o x y g e n a s e e n z y m a t i c activity at 18 d a y s g e s t a t i o n a l age r e a c h e d a b o u t h a l f the v a l u e s of a d u l t a n i m a l s , w h e r e a s fetal m i c r o s o m a l activity was only a b o u t 1/20 t h e a d u l t level at the s a m e age. In m i c r o s o m e s a n d nuclei t h e ontogenic d e v e l o p m e n t of epoxide h y d r o l a s e is slightly slower t h a n s t y r e n e m o n o o x y g e n a s e . T h i s s u g g e s t s t h a t fetuses a n d n e w b o r n a n i m a l s are exposed to h i g h e r risk of a c c u m u l a t i o n of styrene-7,8-oxide, a toxic a n d possibly t e r a t o g e n i c p r o d u c t of s t y r e n e monooxygenase. K e y w o r d s : Nuclei - N u c l e a r m e t a b o l i s m - P e r i n a t a l m e t a b o l i s m - S t y r e n e Styrene-7,8-oxide
INTRODUCTION D r u g m e t a b o l i z i n g activities are v i r t u a l l y a b s e n t d u r i n g the g e s t a t i o n of most n o n - p r i m a t e species [1,2]. T h e y a p p e a r l a t e r in p r e g n a n c y or im*This paper was supported by the C.N.R. (National Research Council) within the special program 'Control of Cancer Growth' Contract No. 78.028.64.96. **Present address: Department of Pharmacology Facultad de Medicine, Universitat Autonoma de Barcellona, Bellaterra (Barcellona), Spain. ***Present address: Department of Pediatrics of the University, Milan, Italy. ~Present address: Facolt~ di Farmacia, Istituto di Chimica Farmaceutica University of Milan, Italy. STo whom correspondence should be addressed. 0009-2797/83/$03.00 O 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
214 mediately after birth and progressively increase during the first 3-8 weeks [3,4]. Several factors have been suggested or recognized as influencing the ontogenesis of these activities [5-9]. Evaluation of qualitative and quantitative differences in metabolizing capacity during development has both therapeutic and toxicologic relevance [10,11]. In fact chemical carcinogenesis is known to be influenced by age [12-14], newborn animals having been found to be more susceptible to some chemicals [15-17]. Increased neonatal susceptibility has also been found towards several therapeutically active molecules [18]. The liver microsomal fraction is recognized as ~he principal site of xenobiotic metabolism though there is increasing evidence that other tissues and cellular components have drug metabolizing capacity too, but quantitatively at lower levels [19-24]. The role of enzymes present in nuclei merits particular attention on account of the spatial proximity to the genomic material [25,26] which is the selective target of the majority of carcinogens [27]. We have previously described [28] the native and phenobarbital induced development of nuclear cytochrome P°450 and related enzyme systems. In this paper we present a comparative study of the perinatal development of rat liver nuclear and microsomal cytochrome P-450, styrene monooxygenase and epoxide hydrolase. MATERIALS AND METHODS
Chemicals Phenobarbital (Abbott); N A D P H (Boehringer); styrene, styrene oxide, phenylethylene glycol, 1-Br, 2-phenylethane, sucrose (Merck); Trifluoracetic anhydride (Pierce); Trimethylamine (Carlo Erba). Treatment of animals CD-COBS rats 5-46 days old (12--150 g body wt.) and pregnant rats at 18 days of gestation were obtained from Charles River, Italy (Calco, Como). The rats were kept in air-conditioned quarters (60% relative humidity; 22°C) with a 12-h light-dark cycle and were given commercial laboratory chow and tap water ad libitum. Animals of both sexes were used until 10 days of age then only males were used from 17 to 46 days. For induction experiments, rats were pretreated i.p. with either saline or phenobarbital at a dose of 80 mg/kg daily for 3 days. After the last injection they were fasted for 16 h and then killed by exsanguination. Transplacental induction was studied by treating pregnant rats from the 15th to the 18th day of pregnancy following the above treatment schedule. Fetal age was determined from the time of mating. Isolation of microsomes and nuclei Liver microsomes were isolated according to Kato and Takayanaghi [29] and resuspended in 0.05 M phosphate buffer (pH 7.4), 0.005M in MgC12, 0.15 M in KC1. Liver nuclei were prepared according to Bresnick et al. [30].
215 The nuclear pellet, free of cytoplasmic contaminants, was resuspended in 0.05M phosphate buffer (pH 7.4) containing 0.005M MgC12, 0.15M KC1, 0.25M sucrose. All experiments were performed using fresh tissue preparations.
Enzymatic determinations Cytochrome P-450 content was measured according to Omura and Sato [31] using a Shimadzu UV 300 spectrophotometer. For nuclear cytochrome P-450 determination, intact nuclei, at a concentration of 2 mg of protein/ml were resuspended in Tris-HC1 0.05 M (pH 7.4) containing 25% glycerol. This buffer was chosen to minimize clusters formation. Nuclear and microsomal styrene monooxygenase and epoxide hydrolase were assayed as previously described [32]. Protein was determined by the method of Lowry et al. [33] using bovine serum albumin as standard. RESULTS
1. Nuclear and microsomal cytochrome P-450 Figure 1 summarizes the rat liver nuclear and microsomal cytochrome P-450 content measured from 18 days of gestational age to 46 days of age. In control animals the cytochrome P-450 from both subcellular fractions showed different qualitative behaviour: by day 17 the nuclear cytochrome had already reached a value very close to t h a t observed in adult animals. In contrast, microsomal cytochrome P-450 showed a more gradual increase reaching its highest value at day 31. Phenobarbital pretreatment did not cause any inductive effect before birth. In the postnatal stage the peak of inducibility of microsomal cytochrome P-450 was observed at day 31 whereas for nuclei this peak occurred 1 week later. Both peaks of induction were followed by a sudden decrease which again was seen a week later in nuclei. 2. Nuclear and microsomal styrene monooxygenase Styrene monooxygenase catalyzes the epoxidation of styrene to styrene-7,8oxide which is known to be more toxic t h a n the parent compound [34]. Qualitatively the development of this enzyme (Fig. 2) with age for both subcellular fractions is similar to their respective cytochrome P-450 levels. Basal nuclear activity raises, to reach plateau values by the third week whereas microsomal activity increases continuously reaching a plateau value from 17 to 31 days. At the 38th day of life a slight decrease was observed followed by an increase in the activity of this enzyme at day 46. No inductive effects by phenobarbital was observed before birth in either subcellular compartments. The maximal activity after phenobarbital pretreatment was observed at the 17th day for the microsomes and the 24th for nuclei. Interestingly three days before birth, this enzyme in nuclei of control animals was half the highest value observed in adults while in the microsomes it corresponded to only about 1/20th.
216
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Days Fig. 1. Perinatal development of nuclear and microsomal cytochrome P-450 content in controls and phenobarbital pretreated rats. Each point represents the mean _+S.E. of 5 litters. *P < 0.05 and **P <0.01 versus controls by Tukey's test. Right scale = nuclei. Left scale = microsomes. O - - O , Control nuclei; • • , phenobarbital nuclei; • A, control microsomes; • O, phenobarbital microsomes.
3. Nuclear and microsomal epoxide hydrolase Epoxide hydrolase is the enzyme hydrolizing styrene-7,8-oxide to phenylethylene glycol. The nuclear basal activity did not significantly increase until the 17th day of life. From this time to the 31st day marked enhancement was observed while enzyme activity rose to the adult value. In control animals the microsomal development of epoxide hydrolase showed a more irregular pathway than in control nuclei. Phenobarbital pretreatment did not affect fetal nuclear epoxide hydrolase but this enzyme was significantly sensitive to the drug at all ages considered after birth. The highest specific activity after induction was found at the 38th day of life.
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Days Fig. 2. Perinatal development of nuclear and microsomal styrene monooxygenase specific activity in control and phenobarbital pretreated rats. Each point represents the m e a n +- S.E. of 5 litters. *P < 0 . 0 5 and * * P < 0 . 0 1 versus controls by Tukey's test. Right scale = nuclei. Left s c a l e = miscrosomes. © - - O , control nuclei; • ----, phenobarbital nuclei; • - b , control microsomes; O - - - O , phenobarbital microsemes.
Like the fetal nuclear enzyme, microsomal epoxide hydrolase was insensitive to induction before birth. Phenobarbital pretreatment caused significant induction after birth at all ages (with the exception of the 10th day). Moreover the general pattern of induction followed an irregular trend paralleling the control enzyme maturation pattern (Fig. 3). Table I reports the microsomal to nuclear specific activity ratio of styrene monooxygenase and epoxide hydrolase in control and phenobarbital pretreated rats. The native ratio before birth for styrene monooxygenase indicated t h a t nuclear activity was almost the same as the microsomal one. However a sudden increase was observed at the 5th day of life. Throughout the postnatal period time taken into consideration this ratio, showed an
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Fig. 3. Perinatal development of nuclear and microsomal styrene epoxide hydrolase specific activity in control and phenobarbital pretreated rats. Each point represents the mean -+ S.E. of 5 litters. * P < 0 . 0 5 and * * P < 0 . 0 1 versus controls by Tukey's test. Right scale = nuclei. Left scale= microsomes. O---C), control nuclei; • I , phenobarbital nuclei; • • , control microsomes; • O, phenobarbital microsomes.
irregular pattern, but was always higher t h a n 20. The lack of inductive ett¢ct of phenobarbital before birth is clearly indicated by the ratio of 1:7 obtained for pretreated animals. During development, nuclear monooxygenase proved more sensitive to this drug a n d t h e ratios observed were thus lower t h a n in control animals. The same trend was observed for the ratio between microsomal and nuclear epoxide hydrolase; for this enzyme too the lack of prenatal induction and the higher postnatal nuclear sensitivity were seen (Fig. 1). Table II shows the epoxide hydrolase to styrene'monooxygenase activity ratio in control and phenobarbital pretreated animals. In control microsomes the ratio was almost constant at all the ages considered. In control nuclei, however, there was a clear-cut trend towards an increase of the ratio during development. As can be deduced from Figs. 2 and 3, this was due to the
219 TABLE I MICROSOMAL TO NUCLEAR SPECIFIC ACTIVITY RATIO OF STYRENE MONOOXYGENASE OR EPOXIDE HYDROLASE AT DIFFERENT AGES IN CONTROL AND PHENOBARBITAL PRETREATED ANIMALS Day of life
-3 5 10 17 24 31 38 46
Styrene monooxygenase
Epoxide hydrolase
Control
Phenobarbital
Control
Phenobarbital
1.4 37.4 53.4 47.9 26.2 27.9 20.7 32.2
1.7 26.3 18.1 43.8 15.3 22.2 25.1 32.2
2.7 44.6 64.1 52.3 16.9 23.6 19.0 22.4
2.0 30.8 28.6 26.2 8.8 15.6 10.8 13.0
c o n t i n u o u s i n c r e a s e of e p o x i d e h y d r o l a s e w i t h a g e a n d to a n a l m o s t c o n s t a n t v a l u e of m o n o o x y g e n a s e f r o m t h e 1 7 t h d a y i n n u c l e i . A s r e g a r d s t h e effect of p h e n o b a r b i t a l on t h i s r a t i o i n g e n e r a l t h e v a l u e w a s l o w e r t h a n t h e corresponding controls in both subcellular fractions and there was an irregular p a t t e r n of s e n s i t i v i t y to p h e n o b a r b i t a l a t t h e d i f f e r e n t a g e s . DISCUSSION T h e p r e s e n t s t u d y s h o w s t h a t i n t h e r a t n u c l e a r p e r i n a t a l d e v e l o p m e n t of s t y r e n e m o n o o x y g e n a s e a n d e p o x i d e h y d r o l a s e o c c u r s e a r l i e r t h a n for m i c r o somes and, as a consequence, in late g e s t a t i o n the m i c r o s o m a l a n d n u c l e a r a c t i v i t i e s a r e q u a n t i t a t i v e l y s i m i l a r . T h i s l a s t p o i n t i m p l i e s t h a t , a t l e a s t for
TABLE H NUCLEAR OR MICROSOMALEPOXIDEHYDROLASE TO STYRENE MONOOXYGENASE SPECIFIC ACTIVITY RATIO AT D~FERENTAGESIN CONTROL AND PHENOBARB~AL PRETREATED ANIMALS Day of life -3 5 10 17 24 31 38 46
Microsomes
Nuclei
Control
Phenobarbital
Control
Phenobarbital
2.4 2.2 2.5 1.9 1.3 2.4 2.7 1.8
1.4 1.8 2.0 0.7 0.8 1.3 1.1 0.8
1.2 1.8 2.0 1.7 1.9 2.8 2.9 2.6
1.2 1.6 1.3 1.2 1.3 1.9 2.5 2.1
220 the activities considered, nuclei may be toxicologically more important in the fetus than microsomes because of the location of nuclear metabolizing enzymes. However the dramatic increase observed after birth only for microsomal activities indicates that the perinatal period is crucial as far as the maturation of microsomal enzymes is concerned but it did not seem to play an important role for nuclear enzymes. The quantitative increase of cytochrome P-450 is lower than for the related enzyme styrene monoxygenase in both subcellular compartments. Comparison of nuclear epoxide hydrolase and styrene monooxygenase showed that the former enzymatic activity develops sooner. As a consequence the activity ratio between these two enzymes significantly differs from birth to adulthood. In particular the capacity to detoxify the epoxide formed is poor before and early after birth and increases with age. Phenobarbital pretreatment did not cause any inductive effect on either fetal cytochrome P-450 content or fetal styrene monooxygenase and epoxide hydrolase activities though the compound is known to cross the placental barrier [35]. Similar findings have been reported by Kuenzig et al. [36] in guinea pig microsomes and by Guenthner and Mannering [37] in rat liver microsomes. After birth, nuclear styrene monooxygenase proved more sensitive than the microsomal enzyme to phenobarbital pretreatment. However this higher sensitivity of nuclear enzyme decreases during growth even though in adult animals two-fold difference persists [25]. Nuclear epoxide hydrolase is also much more sensitive to phenobarbital than the microsomal enzyme, but the animals maturation did not modify the drug effect. Moreover nuclear and microsomal styrene monooxygenase are more sensitive than epoxide hydrolase to phenobarbital pretreatment. Consequently in the induced animals the activity ratios between epoxide hydrolase and monooxygenase in both subcellular fractions are lower than in controls. All these observations suggest a greater risk of intranuclear accumulation of reactive alkene epoxides in the fetal and newborn organism or after exposure to xenobiotica. In this respect preliminary studies in progress in our laboratory indicate that in h u m a n fetuses (15-19 weeks gestational age) too liver nuclei possess both styrene monooxygenase and epoxide hydrolase activities. In conclusion our data rule out that, already in fetal life, nuclear metabolic activity may play a role in the biotransformation of xenobiotica and in the manifestation of their genotoxic effects. The possible teratogenic and/or fetotoxic effect of styrene and styrene oxide is still controversial [38,39], but there is some evidence [40], at least in the chick embryo, that styrene-7,8-oxide formation may be related to embryotoxicity and teratogenesis. REFERENCES 1 J.R. Gillette and B. Stripp, Pre- and postnatal enzyme capacity for drug metabolite production, Fed. Proc., 34 (1975) 172. 2 W.R. Jondorf, R.P. Maickel and B.B. Brodie, Inability of newborn mice and guinea pigs to metabolize drugs, Biochem. Pharmacol., 1 (1958) 352.
221 3 J.R. Fouts a n d T.R. Devereux, Developmental aspects of hepatic and extrahepatic drugmetabolizing enzyme systems: Microsomal enzymes and components in rabbit liver and lung during the first month of life, J. Pharmacol. Exp. Ther., 183 (1972) 458. 4 S.M. MacLeod, K.W. Renton and N.R. Eade, Development of hepatic microsomal drugoxidizing enzymes in i m m a t u r e male and female rats, J. Pharmacol. Exp. Ther., 183 (1972) 489. 5 S. E1 Defrawy E1 Masry, G.M. Cohen and G.J. Mannering, Sex-dependent differences in drug metabolism in the rat. I. Temporal changes in the microsomal drug-metabolizing system of the liver during sexual maturation, Drug Metab. Dispos., 2 (1974) 267. 6 O. Pelkonen, Biotransformation of xenobiotics in the fetus, Pharmacol. Ther., 10 {1980} 261. 7 W. Klinger, Biotransformation of drugs and other xenobiotics during postnatal development, Pharmacol. Ther., 16 (1982) 377. 8 J.T. Wilson, Growth hormone modulation of drug metabolism during development, in: L.F. Soyka and G.P. Redmond (Eds.), Drug Metabolism in the I m m u n e Human, Raven Press, New York, 1981, p. 77. 9 R.E. Kramer, J.W. Greiner, R.C. Rumbaugh, T.D. Sweeney and H.D. Colby, Relation of the gonadal hormones to growth hormone actions on hepatic drug metabolism in rats, J. Pharmacol. Exp. Ther., 204 (1978) 247. 10 M.G. MacDonald, M.J. Fasco and L.S. Kaminsky, Development of the hepatic mixed-function oxidase system and its metabolism of warfarin in the perinatal rat, Dev. Pharmacol. Ther., 3 (1981) 1. 11 T.E. Eling, R.D. Harbison, B.A. Becker and J.R. Fouts, Diphenylhydantoin effect on neonatal and adult r a t hepatic drug metabolism, J. Pharmacol. Exp. Ther., 171 (1970) 127. 12 L. Chieco-Bianchi, G. De Benedictis, G. Tridente and L. Fiore-Donati, Influence of age on susceptibility to liver carcinogenesis and skin initiating action by u r e t h a n e in Swiss mice, Br. J. Cancer, 17 {1963} 672. 13 M.G. Kelly and R.W. O'Gara, Induction of tumors in newborR mice with dibenz[a,h]anthracene and 3-methylcholanthrene, J. Natl. Cancer Inst., 26 (1961) 651. 14 J. Kapitulnik, W. Levin, A.H. Conney, H. Yagi and D.M. Jerina, Benzo[a]pyrene 7,8dihydrodiol is more carcinogenic t h a n benz[a]pyrene in newborn mice, Nature, 266 {1977) 378. 15 B. Toth; A critical review of experiments in chemical carcinogenesis using newborn animals, Cancer Res., 28 {1968} 727. 16 S. Mirvish, G. Cividalli and I. Berenblum, Slow elimination of u r e t h a n in relation to its high carcinogenicity in newborn mice, Proc. Soc. Exp. Biol. Meal., 116 (1964) 265. 17 I.I. Domsky, W. Lijinky, K. Spencer and P. Shubik, Rate of metabolism of 9,10-dimethyl-l,2benzanthracene in newborn and adult mice, Proc. Soc. Exp. Biol. Med., 113 (1963) 110. 18 B.M. Assael, Pharmacokinetics and drug distribution during postnatal development, Pharmacol. Ther., 18 (1982) 159. 19 C.B. Kasper, Biochemical distinctions between the nuclear and microsomal m e m b r a n e s from r a t hepatocytes, J. Biol. Chem., 246 (1971) 577. 20 R. Berezney, L.K. Macaulay and F.I. Crane, The purification and biochemical characterization of bovine liver nuclear membranes, J. Biol. Chem., 247 (1972) 5549. 21 M. Romano, T. Facchinetti and M. Salmona, Is there a role for nuclei in the metabolism of xenobiotica? Drug Metab. Rev., 14 {1983) 803. 22 W.A. Bornstein, W. Levin, P.E. Thomas, D.E. Ryan and E. Bresnick, Comparison of nuclear and microsomal epoxide hydrase from r a t liver, Arch. Biochem. Biophys., 197 (1979) 436. 23 E.G. Rogan, P. Mailander and E. Cavalieri, Metabolic activation of aromatic hydrocarbons in purified r a t liver nuclei: induction of enzyme activities and binding to DNA with and without monooxygenase-catalyzed formation of active oxygen, Proc. Natl. Acad. Sei., 73 (1976) 457. 24 W. Vizethum and G. Goerz, Induction of the hepatic microsomal and nuclear cytoehrome P-450 system by hexachlorobenzene, pentachlorophenol and trichlorophenol, Chem.-Biol. Interact., 28 {1979) 291. 25 G. Gazzotti, E. Garattini and M. Salmona, Nuclear metabolism. II. F u r t h e r studies on epoxide hydrolase activity, Chem.-Biol. Interact., 35 {1981) 311.
222 26 K. Alexandrov, P. Brookes, H.W.S. King, M.R. Osborne and M.H. Thompson, Comparison of the metabolism of benzo[a]pyrene and binding to D N A caused by rat liver nuclei and microsomes, Chem.-Biol. Interact., 12 (1976) 269. 27 E.C. Miller, Some current perspectives on chemical carcinogenesis in humans and experimental animals: Presidential address, Cancer Res., 38 (1978) 1479. 28 M. Romano, V. Clos, B.M. Assael and M. Salmona, Perinatal development of cytochrome P-450, NADPH-cytochrome c roductase and ethoxycoumarine deethylase in rat liver nuclear membranes, Chem.-Biol. Interact., 42 (1982) 225. 29 R. Kate and M. Takayanaghi, Differences among the action of phenobarbital, methylcholanthrene and male sex hormone on microsomal drug.metabolizing enzyme systems of rat liver, Japan J. Phar/nacol., 16 (1966) 380. 30 E. Bresnick, J.B. Vaught, A.H.L. Chuang, T.A. Stoming, D. Bockman and H. Mukhtar, Nuclear aryl hydrocarbon hydroxylase and interaction of polycyclic hydrocarbons with nuclear components, Arch. Biochem. Biophys., 181 (1977) 257. 31 T. O m u r a and R. Sato, The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. Solubilization, purification and properties, J. Biol. Chem., 239 (1964) 2370. 32 G. Gazzotti, E. Garattini and M. Salmona, Improved gas chromatographic method for measuring phenylethylene glycol. Application to the dete~nination of styrene monooxygenase and epoxide hydrase activities,J. Chromatogr., 188 (1980) 400. 33 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem., 193 (1951) 265. 34 C. Pantarotto, R. Fanelli, F. Bidoli, P. Morazzoni, M. Salmona and K. Szczawinsk~, Arene oxides in styrene metabolism, a new perspective in styrene toxicity? Scand. J. Work Environ. Health, 4, Suppl. 2 (1978) 67. 35 G. Gupta, S.J. Yaffe and P.H. Shapiro, Prenatal exposure to phenobarbital permanently decreases testosterone and causes reproductive dysfunction, Science, 216 (1982) 640. 36 W. Kuenzig, J.J. K a m m , M. Boublik, F. Jenkins and J.J. Burns, Perinatal drug metabolism and morphological changes in the hepatocytes of normal and phenobarbital-treated guinea pigs, J. Pharmacol. Exp. Ther., 191 (1974) 32. 37 T.M. Guenthner and G.J. Mannering, Induction of hepatic mono-oxygenase systems in fetal and neonatal rats with phenobarbital, polycyclic hydrocarbons and other xenobiotics, Biochem. Pharmacol., 26 (1977) 567. 38 J.T.J. Kankaanpii~i, E. Elovaara, K. Hemminki and H. Vainio, The effect of maternally inhaled styrene on embryonal and foetal development in mice and chinese hamsters, Acta Pharmacol. Toxicol., 47 (1980) 127. 39 F.J. Murray, J.A. John, M.F. Balmer and B.A. Schwetz, Teratologic evalution of styrene given to rats and rabbits by inhalation or by gavage, Toxicology, 11 (1978) 335. 40 J.T.J. Kankaanpiiii, K. Hemminki and H. Vainio, Embryotoxicity and teratogenicity of styrene and styrene oxide on chick embryos enhanced by trichloropropylene oxide, Acta Pharmacol. Toxicol., 45 (1979) 399.
213
P E R I N A T A L D E V E L O P M E N T OF S T Y R E N E M O N O O X Y G E N A S E AND EPOXIDE AND NUCLEI*
HYDROLASE
IN RAT LIVER MICROSOMES
M. ROMANO,~G. GAZZOTTI, V.° CLOS**, B.M. ASSAEL***, R. MAFFEI FACINO~ and M. SALMONA* Laboratory for Enzyme Research, Istituto di Ricerche Farmacologiche 'Mario Negri', Via Eritrea, 62, 20157 Milan (Italy) (Received February 2nd, 1983) (Revision received June 7th, 1983) (Accepted June 13th, 1983)
SUMMARY N u c l e a r e n z y m e s were f o u n d to develop e a r l i e r t h a n the c o r r e s p o n d i n g m i c r o s o m a l activities. In fact s t y r e n e m o n o o x y g e n a s e e n z y m a t i c activity at 18 d a y s g e s t a t i o n a l age r e a c h e d a b o u t h a l f the v a l u e s of a d u l t a n i m a l s , w h e r e a s fetal m i c r o s o m a l activity was only a b o u t 1/20 t h e a d u l t level at the s a m e age. In m i c r o s o m e s a n d nuclei t h e ontogenic d e v e l o p m e n t of epoxide h y d r o l a s e is slightly slower t h a n s t y r e n e m o n o o x y g e n a s e . T h i s s u g g e s t s t h a t fetuses a n d n e w b o r n a n i m a l s are exposed to h i g h e r risk of a c c u m u l a t i o n of styrene-7,8-oxide, a toxic a n d possibly t e r a t o g e n i c p r o d u c t of s t y r e n e monooxygenase. K e y w o r d s : Nuclei - N u c l e a r m e t a b o l i s m - P e r i n a t a l m e t a b o l i s m - S t y r e n e Styrene-7,8-oxide
INTRODUCTION D r u g m e t a b o l i z i n g activities are v i r t u a l l y a b s e n t d u r i n g the g e s t a t i o n of most n o n - p r i m a t e species [1,2]. T h e y a p p e a r l a t e r in p r e g n a n c y or im*This paper was supported by the C.N.R. (National Research Council) within the special program 'Control of Cancer Growth' Contract No. 78.028.64.96. **Present address: Department of Pharmacology Facultad de Medicine, Universitat Autonoma de Barcellona, Bellaterra (Barcellona), Spain. ***Present address: Department of Pediatrics of the University, Milan, Italy. ~Present address: Facolt~ di Farmacia, Istituto di Chimica Farmaceutica University of Milan, Italy. STo whom correspondence should be addressed. 0009-2797/83/$03.00 O 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
214 mediately after birth and progressively increase during the first 3-8 weeks [3,4]. Several factors have been suggested or recognized as influencing the ontogenesis of these activities [5-9]. Evaluation of qualitative and quantitative differences in metabolizing capacity during development has both therapeutic and toxicologic relevance [10,11]. In fact chemical carcinogenesis is known to be influenced by age [12-14], newborn animals having been found to be more susceptible to some chemicals [15-17]. Increased neonatal susceptibility has also been found towards several therapeutically active molecules [18]. The liver microsomal fraction is recognized as ~he principal site of xenobiotic metabolism though there is increasing evidence that other tissues and cellular components have drug metabolizing capacity too, but quantitatively at lower levels [19-24]. The role of enzymes present in nuclei merits particular attention on account of the spatial proximity to the genomic material [25,26] which is the selective target of the majority of carcinogens [27]. We have previously described [28] the native and phenobarbital induced development of nuclear cytochrome P°450 and related enzyme systems. In this paper we present a comparative study of the perinatal development of rat liver nuclear and microsomal cytochrome P-450, styrene monooxygenase and epoxide hydrolase. MATERIALS AND METHODS
Chemicals Phenobarbital (Abbott); N A D P H (Boehringer); styrene, styrene oxide, phenylethylene glycol, 1-Br, 2-phenylethane, sucrose (Merck); Trifluoracetic anhydride (Pierce); Trimethylamine (Carlo Erba). Treatment of animals CD-COBS rats 5-46 days old (12--150 g body wt.) and pregnant rats at 18 days of gestation were obtained from Charles River, Italy (Calco, Como). The rats were kept in air-conditioned quarters (60% relative humidity; 22°C) with a 12-h light-dark cycle and were given commercial laboratory chow and tap water ad libitum. Animals of both sexes were used until 10 days of age then only males were used from 17 to 46 days. For induction experiments, rats were pretreated i.p. with either saline or phenobarbital at a dose of 80 mg/kg daily for 3 days. After the last injection they were fasted for 16 h and then killed by exsanguination. Transplacental induction was studied by treating pregnant rats from the 15th to the 18th day of pregnancy following the above treatment schedule. Fetal age was determined from the time of mating. Isolation of microsomes and nuclei Liver microsomes were isolated according to Kato and Takayanaghi [29] and resuspended in 0.05 M phosphate buffer (pH 7.4), 0.005M in MgC12, 0.15 M in KC1. Liver nuclei were prepared according to Bresnick et al. [30].
215 The nuclear pellet, free of cytoplasmic contaminants, was resuspended in 0.05M phosphate buffer (pH 7.4) containing 0.005M MgC12, 0.15M KC1, 0.25M sucrose. All experiments were performed using fresh tissue preparations.
Enzymatic determinations Cytochrome P-450 content was measured according to Omura and Sato [31] using a Shimadzu UV 300 spectrophotometer. For nuclear cytochrome P-450 determination, intact nuclei, at a concentration of 2 mg of protein/ml were resuspended in Tris-HC1 0.05 M (pH 7.4) containing 25% glycerol. This buffer was chosen to minimize clusters formation. Nuclear and microsomal styrene monooxygenase and epoxide hydrolase were assayed as previously described [32]. Protein was determined by the method of Lowry et al. [33] using bovine serum albumin as standard. RESULTS
1. Nuclear and microsomal cytochrome P-450 Figure 1 summarizes the rat liver nuclear and microsomal cytochrome P-450 content measured from 18 days of gestational age to 46 days of age. In control animals the cytochrome P-450 from both subcellular fractions showed different qualitative behaviour: by day 17 the nuclear cytochrome had already reached a value very close to t h a t observed in adult animals. In contrast, microsomal cytochrome P-450 showed a more gradual increase reaching its highest value at day 31. Phenobarbital pretreatment did not cause any inductive effect before birth. In the postnatal stage the peak of inducibility of microsomal cytochrome P-450 was observed at day 31 whereas for nuclei this peak occurred 1 week later. Both peaks of induction were followed by a sudden decrease which again was seen a week later in nuclei. 2. Nuclear and microsomal styrene monooxygenase Styrene monooxygenase catalyzes the epoxidation of styrene to styrene-7,8oxide which is known to be more toxic t h a n the parent compound [34]. Qualitatively the development of this enzyme (Fig. 2) with age for both subcellular fractions is similar to their respective cytochrome P-450 levels. Basal nuclear activity raises, to reach plateau values by the third week whereas microsomal activity increases continuously reaching a plateau value from 17 to 31 days. At the 38th day of life a slight decrease was observed followed by an increase in the activity of this enzyme at day 46. No inductive effects by phenobarbital was observed before birth in either subcellular compartments. The maximal activity after phenobarbital pretreatment was observed at the 17th day for the microsomes and the 24th for nuclei. Interestingly three days before birth, this enzyme in nuclei of control animals was half the highest value observed in adults while in the microsomes it corresponded to only about 1/20th.
216
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Days Fig. 1. Perinatal development of nuclear and microsomal cytochrome P-450 content in controls and phenobarbital pretreated rats. Each point represents the mean _+S.E. of 5 litters. *P < 0.05 and **P <0.01 versus controls by Tukey's test. Right scale = nuclei. Left scale = microsomes. O - - O , Control nuclei; • • , phenobarbital nuclei; • A, control microsomes; • O, phenobarbital microsomes.
3. Nuclear and microsomal epoxide hydrolase Epoxide hydrolase is the enzyme hydrolizing styrene-7,8-oxide to phenylethylene glycol. The nuclear basal activity did not significantly increase until the 17th day of life. From this time to the 31st day marked enhancement was observed while enzyme activity rose to the adult value. In control animals the microsomal development of epoxide hydrolase showed a more irregular pathway than in control nuclei. Phenobarbital pretreatment did not affect fetal nuclear epoxide hydrolase but this enzyme was significantly sensitive to the drug at all ages considered after birth. The highest specific activity after induction was found at the 38th day of life.
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Like the fetal nuclear enzyme, microsomal epoxide hydrolase was insensitive to induction before birth. Phenobarbital pretreatment caused significant induction after birth at all ages (with the exception of the 10th day). Moreover the general pattern of induction followed an irregular trend paralleling the control enzyme maturation pattern (Fig. 3). Table I reports the microsomal to nuclear specific activity ratio of styrene monooxygenase and epoxide hydrolase in control and phenobarbital pretreated rats. The native ratio before birth for styrene monooxygenase indicated t h a t nuclear activity was almost the same as the microsomal one. However a sudden increase was observed at the 5th day of life. Throughout the postnatal period time taken into consideration this ratio, showed an
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Fig. 3. Perinatal development of nuclear and microsomal styrene epoxide hydrolase specific activity in control and phenobarbital pretreated rats. Each point represents the mean -+ S.E. of 5 litters. * P < 0 . 0 5 and * * P < 0 . 0 1 versus controls by Tukey's test. Right scale = nuclei. Left scale= microsomes. O---C), control nuclei; • I , phenobarbital nuclei; • • , control microsomes; • O, phenobarbital microsomes.
irregular pattern, but was always higher t h a n 20. The lack of inductive ett¢ct of phenobarbital before birth is clearly indicated by the ratio of 1:7 obtained for pretreated animals. During development, nuclear monooxygenase proved more sensitive to this drug a n d t h e ratios observed were thus lower t h a n in control animals. The same trend was observed for the ratio between microsomal and nuclear epoxide hydrolase; for this enzyme too the lack of prenatal induction and the higher postnatal nuclear sensitivity were seen (Fig. 1). Table II shows the epoxide hydrolase to styrene'monooxygenase activity ratio in control and phenobarbital pretreated animals. In control microsomes the ratio was almost constant at all the ages considered. In control nuclei, however, there was a clear-cut trend towards an increase of the ratio during development. As can be deduced from Figs. 2 and 3, this was due to the
219 TABLE I MICROSOMAL TO NUCLEAR SPECIFIC ACTIVITY RATIO OF STYRENE MONOOXYGENASE OR EPOXIDE HYDROLASE AT DIFFERENT AGES IN CONTROL AND PHENOBARBITAL PRETREATED ANIMALS Day of life
-3 5 10 17 24 31 38 46
Styrene monooxygenase
Epoxide hydrolase
Control
Phenobarbital
Control
Phenobarbital
1.4 37.4 53.4 47.9 26.2 27.9 20.7 32.2
1.7 26.3 18.1 43.8 15.3 22.2 25.1 32.2
2.7 44.6 64.1 52.3 16.9 23.6 19.0 22.4
2.0 30.8 28.6 26.2 8.8 15.6 10.8 13.0
c o n t i n u o u s i n c r e a s e of e p o x i d e h y d r o l a s e w i t h a g e a n d to a n a l m o s t c o n s t a n t v a l u e of m o n o o x y g e n a s e f r o m t h e 1 7 t h d a y i n n u c l e i . A s r e g a r d s t h e effect of p h e n o b a r b i t a l on t h i s r a t i o i n g e n e r a l t h e v a l u e w a s l o w e r t h a n t h e corresponding controls in both subcellular fractions and there was an irregular p a t t e r n of s e n s i t i v i t y to p h e n o b a r b i t a l a t t h e d i f f e r e n t a g e s . DISCUSSION T h e p r e s e n t s t u d y s h o w s t h a t i n t h e r a t n u c l e a r p e r i n a t a l d e v e l o p m e n t of s t y r e n e m o n o o x y g e n a s e a n d e p o x i d e h y d r o l a s e o c c u r s e a r l i e r t h a n for m i c r o somes and, as a consequence, in late g e s t a t i o n the m i c r o s o m a l a n d n u c l e a r a c t i v i t i e s a r e q u a n t i t a t i v e l y s i m i l a r . T h i s l a s t p o i n t i m p l i e s t h a t , a t l e a s t for
TABLE H NUCLEAR OR MICROSOMALEPOXIDEHYDROLASE TO STYRENE MONOOXYGENASE SPECIFIC ACTIVITY RATIO AT D~FERENTAGESIN CONTROL AND PHENOBARB~AL PRETREATED ANIMALS Day of life -3 5 10 17 24 31 38 46
Microsomes
Nuclei
Control
Phenobarbital
Control
Phenobarbital
2.4 2.2 2.5 1.9 1.3 2.4 2.7 1.8
1.4 1.8 2.0 0.7 0.8 1.3 1.1 0.8
1.2 1.8 2.0 1.7 1.9 2.8 2.9 2.6
1.2 1.6 1.3 1.2 1.3 1.9 2.5 2.1
220 the activities considered, nuclei may be toxicologically more important in the fetus than microsomes because of the location of nuclear metabolizing enzymes. However the dramatic increase observed after birth only for microsomal activities indicates that the perinatal period is crucial as far as the maturation of microsomal enzymes is concerned but it did not seem to play an important role for nuclear enzymes. The quantitative increase of cytochrome P-450 is lower than for the related enzyme styrene monoxygenase in both subcellular compartments. Comparison of nuclear epoxide hydrolase and styrene monooxygenase showed that the former enzymatic activity develops sooner. As a consequence the activity ratio between these two enzymes significantly differs from birth to adulthood. In particular the capacity to detoxify the epoxide formed is poor before and early after birth and increases with age. Phenobarbital pretreatment did not cause any inductive effect on either fetal cytochrome P-450 content or fetal styrene monooxygenase and epoxide hydrolase activities though the compound is known to cross the placental barrier [35]. Similar findings have been reported by Kuenzig et al. [36] in guinea pig microsomes and by Guenthner and Mannering [37] in rat liver microsomes. After birth, nuclear styrene monooxygenase proved more sensitive than the microsomal enzyme to phenobarbital pretreatment. However this higher sensitivity of nuclear enzyme decreases during growth even though in adult animals two-fold difference persists [25]. Nuclear epoxide hydrolase is also much more sensitive to phenobarbital than the microsomal enzyme, but the animals maturation did not modify the drug effect. Moreover nuclear and microsomal styrene monooxygenase are more sensitive than epoxide hydrolase to phenobarbital pretreatment. Consequently in the induced animals the activity ratios between epoxide hydrolase and monooxygenase in both subcellular fractions are lower than in controls. All these observations suggest a greater risk of intranuclear accumulation of reactive alkene epoxides in the fetal and newborn organism or after exposure to xenobiotica. In this respect preliminary studies in progress in our laboratory indicate that in h u m a n fetuses (15-19 weeks gestational age) too liver nuclei possess both styrene monooxygenase and epoxide hydrolase activities. In conclusion our data rule out that, already in fetal life, nuclear metabolic activity may play a role in the biotransformation of xenobiotica and in the manifestation of their genotoxic effects. The possible teratogenic and/or fetotoxic effect of styrene and styrene oxide is still controversial [38,39], but there is some evidence [40], at least in the chick embryo, that styrene-7,8-oxide formation may be related to embryotoxicity and teratogenesis. REFERENCES 1 J.R. Gillette and B. Stripp, Pre- and postnatal enzyme capacity for drug metabolite production, Fed. Proc., 34 (1975) 172. 2 W.R. Jondorf, R.P. Maickel and B.B. Brodie, Inability of newborn mice and guinea pigs to metabolize drugs, Biochem. Pharmacol., 1 (1958) 352.
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