Metabolites of hydrocortisone were isolated from rat liver on a preparative scale, fractionated by column chromatography on Sephadex LH-20 and silica gel and ...
422
Biochimica et Biophysica Acta, 6 3 3 ( 1 9 8 0 ) 4 2 2 - - 4 3 5 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 29447
INDUCTION OF T R Y P T O P H A N OXYGENASE AND TYROSINE A M I N O T R A N S F E R A S E BY METABOLITES OF H Y D R O C O R T I S O N E
JURGEN VOIGT * and CONSTANTINE.
S E K E R I S **
Institute for Cell Research, German Cancer Research Center, P.O. Box 101949, D-6900 Heidelberg (F.R.G.) ( R e c e i v e d April 8 t h , 1 9 8 0 ) (Revised m a n u s c r i p t received 2 5 t h J u l y 1 9 8 0 )
Key words: Hydrocortisone metabolite; Enzyme induction; Tryptophan oxygenase; Tyrosine aminotransferase ; (Rat liver)
Summary Metabolites of hydrocortisone were isolated from rat liver on a preparative scale, fractionated by column chromatography on Sephadex LH-20 and silica gel and tested for biological activity. Apart from the well known neutral metabolites, steroid glucuronides and sulfates, we obtained metabolite fractions containing non-conjugated steroidal carboxy acids and acid metabolites of unknown structure. One of these fractions induced tyrosine aminotransferase (EC 2.6.1.5) in adrenalectomized female rats b u t not tryptophan oxygenase (EC 1.13.11.11), whereas another one mainly increased activity of tryptophan oxygenase. The doses necessary to significantly induce both enzymes were much lower in case of these metabolites than in the case of hydrocortisone itself. The active fractions eluting from silica gel column were analyzed by thin-layer chromatography in two different solvent systems. Absence of hydrocortisone in these fractions could be clearly demonstrated. Furthermore, the active fractions eluting from the silica gel column were characterized by treatment with an extract from Helix pomatia and/or diazomethane and subsequent analysis by thin-layer chromatography. We conclude, considering the biological activity of some synthesized derivatives of hydrocortisone, that the biologically active components are acid metabolites of hydrocortisone which are not identical to any o f the known metabolites.
* Present address: Institute for Organic Chemistry and B i o c h e m i s t r y , University of Hamburg, MartinLuther-King-Platz 6, D-2000 Hamburg 13, F.R.G. ** Present address: N a t i o n a l Hellenic Research F o u n d a t i o n , Biological Research Center, 48 Vasslleos Constantinou Avenue, Athens 501/I, Greece.
423 Introduction
Hydrocortisone is a well-known inducer of tryptophan oxygenase (EC 1.13.11.11) and tyrosine aminotransferase (EC 2.6.1.5) in rat liver (for reviews, see Refs. 1 and 2). Both enzymes respond to the hormone after a lag phase of about 90 min and reach their maximal levels within a few hours [3--9]. The induction process depends on a specific increase of the concentrations of mRNAs coding for tryptophan oxygenase and tyrosine aminotransferase [10-12] and can be inhibited by actinomycin D [9,13--15] or ~-amanitin [9,16]. It is generally assumed that the first step in the action of glucucorticosteroids, as of other steroid hormones, is the binding of the hormone to cytoplasmic proteins, called 'receptors' [17,18], followed by the transport of the hormonereceptor complexes into the nucleus. Hydrocortisone or corticosterone, depending on the animal species, is thought to be the active inducer of tyrosine aminotransferase and tryptophan oxygenase in the hepatocyte [19], although other steroids, like aldosterone and even ll-deoxycortisol and 5a-dihydrocortisol, have been reported to induce tyrosine aminotransferase in the hepatoma cell [20]. The observation that, after injection of [3H]hydrocortisone or [3H]corticosterone, essentially only the administered hormone was found in purified rat liver nuclei and also was associated to at least one of the cytoplasmic binding proteins, whereas the main part of free radioactivity consists of metabolites [21--24], supported this assumption. However, in all the related papers the distribution of the nonconjugated acid metabolites [25--28] has been overlooked. The possibility that metabolites of hydrocortisone could be inducers of tryptophan oxygenase and tyrosine aminotransferase, as suggested by some authors [29,30], can therefore not be ruled out. The existence of biologically active metabolites of hydrocortisone could be an explanation for the observed differences in the inducibility by hydrocortisone of tryptophan oxygenase and tyrosine aminotransferase in the liver of adult rats with respect to dose response and the effects of sex and the state of the adrenals [31]. The latter findings tempted us to search for metabolites of hydrocortisone in rat liver which are able to differentially induce both enzymes. Materials and Methods Hydrocortisone, cortisone, tetrahydrocortisol, diphenyloxazol and thinlayer plates (silica gel 60F2s4) were obtained from Merck (Darmstadt, F.R.G.), 5~-dihydrocortisol and 5~-dihydrocortisol from Ikapharm (Ramat-Gan, Israel), haematin hydrochloride from Roth (Karlsruhe, F.R.G.). Bovine serum albumin was purchased from Sigma (St. Louis, U.S.A.), amberlyst-15 and ~-glucuronidase liquid raw extract of Helix pomatia from Serva (Heidelberg, F.R.G.), Sephadex LH-20 from Pharmacia (Uppsala, Sweden) and filters from Schleicher and Schiill (Dassel, F.R.G.). [1,2,6,7-3H]hydrocortisone (spec. act. 87 Ci/ mmol) was obtained from Amersham Buchler (Braunschweig, F.R.G.). The labeled hormone was chromatographically checked for purity before use. Only traces of 21-dehydrocortisol were occasionally observed. All other chemicals (Merck, Darmstadt, F.R.G.) were of analytical grade.
424 17/3-Carboxy-11/3,17a-dihydroxy-androstene-(4)-3-one was prepared by reaction of 2.75 mmol of hydrocortisone with 3 mmol of sodium periodate in 10 ml of 35% (v/v) aqueous methanol for 90 min at room temperature and subsequent purification, ll/3,17a-Dihydroxy-3,20-dioxo-pregnene-(4)-21-al was obtained by catalytic oxidation of hydrocortisone as described by Monder and Furfine [32]. 11fl,17a,20~-Trihydroxy-3-oxo-pregnene-(4)-21-oic acid was prepared by rearrangement of 11/3,17a-dihydroxy-3,20-dioxo-pregnene-(4)-21-al in the presence of NaOH as described by Bradlow et al. [27].
Animals and injections 7--10-week-old female Wistar rats, kept under standard conditions, were used in the induction experiments. The animals were bilaterally adrenalectomized 6--8 days before the experiments through the paravertebral dorsal approach. After adrenalectomy, the animals received a 0.14 M NaC1 solution instead of normal drinking water. Hormones and isolated metabolites were dissolved in dimethylformamide and mixed with 0.14 M NaCI solution. The solutions were injected intraperitoneally between 9 and 10 a.m. Control animals received the same volume of 0.14 M NaC1 solution and the same amount of dimethylformamide, the dose of the organic solvent never exceeding 0.5 ml/kg b o d y wt. The animals were killed by cervical dislocation, 5 h after injection. Preparation o f liver homogenates for determination of enzyme activities The livers were perfused in situ with 10 ml ice-cold buffer consisting of 0.25 M sucrose/50 mM KC1/10 mM L-tryptophan/10 mM 2-mercaptoethanol/ 20 mM Tris-HC1 (pH 7.6) via the portal vein and removed within 30 s after the death of the animals. Homogenisation of the tissue was performed in 2.5 ml ice-cold buffer per g wet wt. liver using an Ultra Turrax homogeniser (30 s at 10 000 rev./min). The homogenates were centrifuged for 15 min at 30 000 X g. 1 ml of the supernatant was adjusted to 0.2 mM pyridoxalphosphate, 2.5 mM a-ketoglutarate and 8.5 mM haematin and heated for 10 min at 55°C in order to saturate tryptophan oxygenase and tyrosine aminotransferase with the corresponding cofactors [33,34] and to eliminate p-hydroxyphenylpyruvic acid oxidase, which would .disturb the determination of tyrosine aminotransferase activity [33]. The samples were centrifuged for 10 min at 30 000 X g and the supernatants tested for enzyme activities. Assay s Tyrosine aminotransferase was determined according to Diamondstone [33] and tryptophan oxygenase essentially as described by Schlitz and Feigelson [34]. Protein concentrations were determined according to L o w r y et al. [35] with bovine serum albumin as standard. Enzyme activities are expressed in #mol p - h y d r o x y b e n z a l d e h y d e and #mol N-formylkynurenine plus kynurenine formed per min per mg protein. Isolation o f hydrocortisone metabolites 300 male Wistar rats of approx. 200 g b o d y wt. were injected with 8 mg of hydrocortisone and 3.4 #Ci [1,2,6,7-3H]hydrocortisone. The animals were
425 killed by cervical dislocation 30--60 min after administration of the h o r m o n e and the livers immediately removed. Homogenisation of the tissue was performed with 2.5 ml of double-distilled water per g wet wt. liver using an Ultra Turrax homogeniser. The homogenate was stirred overnight with 10 vols. of ethanol/acetone (1 : 1, v/v), passed through a filter and evaporated. The residue was taken up in 50% aqueous methanol, stored at --18°C for 24 h and centrifuged to remove the precipitated lipids [36]. The clear supernatant was passed through a column of amberlyst-15 (Na÷-form) to change the acid metabolites into the corresponding sodium salts and evaporated to dryness. The residue was taken up in chloroform/methanol (1 : 1, v/v) containing 10 mM NaC1 and chromatographed on a column of 200 g Sephadex LH-20 equilibrated with the same solvent [37]. The column was eluted with 4 1 of chloroform/methanol (1 : 1, v/v) containing 10 mM NaC1 and thereafter with methanol. Fractions of 20 ml were collected and pooled on the basis of the radioactivity profile. As described in the literature [23], three peaks of radioactivity were obtained, the first of which contained the neutral steroids and the steroid carboxy acids, the second the steroid monosulfates and the third, eluting with methanol, the disulfates. The fractions of the three peaks were pooled and evaporated. The residue of the first peak was taken up in chloroform/ethanol (95 : 5, v/v} and further fractionated by chromatography on a silica gel column (110 × 2.5 cm) equilibrated with chloroform. Elution of the metabolites was performed successively with chloroform, chloroform/ethanol (9 : 1, v/v), chloroform/ethanol (7 : 3, v/v), the upper phase of toluene/acetic acid/water (5 : 5 : 1, v/v) and finally with mixtures of this solvent with increasing portions of ethanol. The fractions were pooled on the basis of the radioactivity profile, evaporated and taken up with methanol. Aliquots of the methanol solutions to be tested for biological activity were evaporated and redissolved in dimethylformamide. In parallel experiments [3H]hydrocortisone was added to liver homogenate and the extraction and separation procedure conducted. Under these conditions no appreciable degradation of [3H]hydrocortisone could be observed.
Thin-layer chromatography Characterisation of the isolated metabolite fractions was performed by thinlayer chromatography on silica gel 60F2s4. The plates were developed alternatively with chloroform/ethanol (9 : 1, v/v) or ethylacetate/ethanol/NH3 (25%) (5 : 5 : 1, v/v). The silica gel was then scraped in stripes of 0.5 cm and twice extracted with about 6 ml of .methanol/water ( 1 : 1 , v/v). The combined extracts were placed in plastic vials, evaporated at 80°C and the radioactivity measured after addition of 10 ml of toluene containing 5 g per 1 of diphenyloxazol, using a liquid scintillation spectrophotometer (Mark II, Nuclear Chicago). The radioactivity profiles were compared with external standards which had been run parallel on the same plate.
Esterification Esterification of the acid metabolites was performed in methanol by addition Of diazomethane in diethyl ether.
426
Enzymatic hydrolysis of steroid conjugates Hydrolysis of steroid conjugates was performed by incubation of aliquots with 0.2 ml of Helix pomatia extract in 15 ml of 0.2 M sodium acetate, pH 4.5 [38]. After 48 h at 37°C the reaction was stopped by addition of 2 ml acetic acid and the solution extracted twice with 50 ml ethylacetate. The combined extracts were evaporated and the products analysed by thin-layer chromatography. Results
Fractionation and biological activity of hydr.ocortisone metabolites present in rat liver To ascertain whether or not metabolites of hydrocortisone are inducers of tryptophan oxygenase and tyrosine aminotransferase, we have, in a first series of experiments, injected the total mixture of isolated metabolites into rats. This, however, resulted in death of the animals, due to the presence of toxic compounds. We therefore proceeded to fractionate the mixture of metabolites as described in Materials and Methods prior to testing for biological activity. The radioactivity profile of the chromatography on the Sephadex LH-20
6000 ~
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,_
--
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100
200
300
400
100
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400
F i g . I . Preparative separation o f h y d r o c o r t i s o n e m e t a b o i l t e s . U p p e r part: radioactivity profile o f the fract i o n s eluted f r o m the S e p h a d e x LH-20 c o l u m n . Isolated h y d r o c o r t i s o n e m e t a b o l i t e s dissolved in chlorof o r m / m e t h a n o l (1 : 1, v/v) c o n t a i n i n g 1 0 mM NaCI were s u b m i t t e d to c h r o m a t o g r a p h y o n a c o l u m n of 2 0 0 g S e p h a d e x L H - 2 0 equilibrated w i t h the same solvent. The c o l u m n w a s eluted w i t h 4 1 o f c h l o r o f o r m / m e t h a n o l (1 : I , v / v ) c o n t a i n i n g 1 0 m M N a C I , f o l l o w e d by m e t h a n o l . F r a c t i o n s o f 2 0 m l were c o l l e c t e d and p o o l e d o n the basis o f the radioactivity profile. I = free steroids and glucuronides, I I = steroid m o n o sulfates, I I I = steroid disulfates. L o w e r part: fractinnation o f p e a k I eluted f r o m S e p h a d e x LH-20 (free steroids and glueuronides) b y c h r o m a t o g r a p h y o n a s i l i c a gel c o l u m n ( 1 1 0 × 2 . 5 c m ) . E l u t i o n o f the m e t a b o i l t e s w a s p e r f o r m e d w i t h 1 ffi c h l o r o f O r m / e t h a n o l ( 9 : 1, v / v ) , 2 = c h l o r o f o r m / e t h a n o l ( 7 : 3, v / v ) , 3 = t o l u e n e / a c e t i c a c i d / w a t e r ( 5 : 5 : 1, v / v ) , u p p e r phase, 4 = solvent 3 / e t h a n o l ( 9 : 1, v / v ) , 5 = solvent 3 / e t h a n o l (7 : 3, v / v ) .
427 TABLE I I N D U C I B I L I T Y O F T R Y P T O P H A N O X Y G E N A S E A N D T Y I ~ O S I N E A M I N O T R A N S F E R A S E BY I S O LATED METABOLITES OF HYDROCORTISONE M e t a b o l i t e s o f h y d ~ o c o r t i s o n e e l u t e d f r o m t h e silica gel c o l u m n (Fig. 1) w e r e t e s t e d f o r b i o l o g i c a l a c t i v i t y in a d r e n a l e c t o m i z e d f e m a l e rats o f 1 0 0 - - 1 5 0 g b o d y w t . T h e a n i m a l s w e r e killed 5 h a f t e r i n t r a p e r i t o h e a l i n j e c t i o n o f t h e r a e t a b o l i t e f r a c t i o n s and t h e o b t a i n e d liver h o r a o g e n a t e s t e s t e d for e n z y m e activities. E a c h value r e p r e s e n t s m e a n v a l u e of six a n i m a l s ± S.E. D o s e s o f r a e t a b o l i t e s w e r e c a l c u l a t e d o n the basis of specific r a d i o a c t i v i t y o f h y d x o c o r t i s o n e u s e d for the p r e p a r a t i o n o f t h e r a e t a b o l i t e s . Fraction
-Hydrocortisone
Dose (mg/kg body wt.)
-10 30
Tryptophan oxygenase activity ( n r a o l / m i n p e r rag)
Tyrosine aminotxansferase activity ( n m o l / r a i n p e r rag)
0.6 ~ 0 . 0 2
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1.4
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3.5
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O
3.0 15.0
0.9 ± 0 . 3 2 1.4 ± 0 . 5 9
25 -+ 3.8 3 2 ± 4.6
7.4
column is shown in the upper part of Fig. 1 and that of the silica gel column in the lower part of the figure. The fractions were pooled as indicated. The main part of the unmetabolized hydrocortisone was obtained in fraction B, as detected by thin-layer chromatography using chloroform/ethanol (9 : 1, v/v}. The ability of the different fractions of metabolites to induce tryptophan oxygenase and/or tyrosine aminotransferase was tested with adrenalectomized female rats. The doses of steroids applied were calculated on the basis of the specific radioactivity of hydrocortisone Used for the preparation of the metabolites (740 000 dpm/mg). As shown in Table I, tyrosine aminotransferase but not tryptophan oxygenase could be induced by metabolite fraction L eluting from the silica gel column. On the other hand, fraction N caused an increase of tryptophan oxygenase activity, the effect on tyrosine aminotransferase being less prominent. The doses necessary to significantly induce both enzymes were much lower in the case of these metabolites than for hydrocortisone itself. No increase in the activity of either enzyme could be observed after administration of any of the other isolated metabolite fractions (data not shown).
Characterisation of biologically active metabolite fractions The active metabolites could only be eluted from the silica gel column with acidic solvents (Fig. 1). Thin-layer chromatography of fractions L, M, N and O, using chloroform/ethanol { 9 : 1 , v/v) as solvent system revealed radioactivity only at the origin (Fig. 2A--D). The possibility that one of the active components is hydrocortisone itself can thus be ruled out. Furthermore, these results imply that the active components are acid compounds. Since steroid sulfates are completely removed by chromatography on Sephadex LH-20 (see upper part of Fig. 1), the identity of one of the active metabolites with hydrocor-
428
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F i g . 2 . Thin-layer c h r o m a t o g r a p h y o f biologically active m e t a b o l i t e fractions. The thin-layer plates (silica gel 6 0 F 2 S 4 ) w e r e d e v e l o p e d t w i c e w i t h c h l o r o f o r m / e t h a n o l ( 9 : 1 , v / v ) . The silica gel w a s then scraped in stripes o f 0.5 cm and the radioactivity eluted and m e a s u r e d . A - - D = samples n o t s u b m i t t e d to esterificat i o n , E - - H = samples treated w i t h d i a z o m e t h a n e , A and E = f r a c t i o n L eluted f r o m the silica gel c o l u m n ( F i g . 1 ) , B a n d F = f r a c t i o n M f r o m silica gel c o l u m n , C and G = f r a c t i o n N , D a n d H = f r a c t i o n 0 , I = 1 7 ~ carboxy-llj3,17~-dihydroxy-androstene-(4)-3-one methylester, II = l l ~ , l T ~ , 2 0 ~ - t r i h y d r o x y - 3 - o x o - p r e g n e n e - ( 4 ) - 2 1 - o i c acid m e t h y l e s t e r , H C = h y d r o c o r t i s o n e .
tisone sulfate can be excluded. Therefore, it seems likely that the active metabolites would either be glucuronides or non-conjugated steroidal carboxy acids [25--28]. Consequently, aliquots of the four fractions were esterified with diazomethane and thereafter chromatographed in the chloroform/ethanol system. The corresponding radioactivity profile of fraction L (Fig. 2E) showed a significant peak in the region of the methylester of 17~-carboxy-ll~,17a-dihydroxy-androstene-(4)-3-one and material remaining at the origin. The latter may in part be the unesterified acid compound. Since the separation by the chloroform/ethanol mixture mainly depends on the number and arrangement of hydroxy groups, we can postulate that the peak of radioactivity in the region of 17fl-carboxy-llfl,17a-dihydroxy-androstene-(4)-3-one methylester should contain esterified acid metabolites of hydrocortisone with two hydroxy groups, probably in the 11~- and 17a-position. This peak was essentially missing in the esterified probe of fraction M (Fig. 2F). A compound having mobility like the methylesters of 11~,17a,20~-trihydroxy-3-oxo-pregnene-(4)-21-oic acids was
429
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Fig. 3, C h r o m a t o g r a p h i c c h a r a c t e r i z a t i o n o f b i o l o g i c a l l y active m e t a b o U t e s o f h y d r o c o r t i s o n e . T h e c h r o m a t o ~ a m s (silica gel 6 0 F 2 5 4 ) w e r e d e v e l o p e d w i t h e t h y l a c e t a t e / e t h a n o l / N H 3 ( 2 5 % ) (5 : 5 : 1, v / v ) and the r a d i o a c t i v i t y p r o f i l e s m e a s u r e d . A = f r a c t i o n L e l u t e d f r o m silica gel c o l u m n (Fig. 1); B = f r a c t i o n M; C = f r a c t i o n N; D = f r a c t i o n O; I = 1 7 1 ~ - c a r b o x Y - l l ~ , 1 7 a - d i h y d r o x y - a n d z o s t e n e - ( 4 ) - 3 - o n e ; II, III = 1 1 ~ , 1 7 a , 2 0 ~ - t r i h y d r o x y - 3 - o x o - P r e g n e n e - ( 4 ) - 2 1 - o i c acids; HC = h y d r o c o r t i s o n e ; THC = t e t r a h y d r o c o r t i s o l . Fig. 4, E n z y m a t i c h y d r o l y s i s o f f r a c t i o n s L, M, N a n d O b y an e x t r a c t o f Helix pomatia. A l i q u o t s o f fract i o n s L - - O e l u t e d f r o m t h e silica gel c o l u m n (Fig. 1) w e r e d i g e s t e d w i t h Helix pornatia e x t r a c t . Thinl a y e r c h r o m a t o g r a p h y w a s p e r f o r m e d in e t h y l a c e t a t e / e t h a n o l / N H 3 ( 2 5 % ) (5 : 5 : 1, v / v ) . HC = h y d r o c o r t i s o n e , I = 1 7 ~ - c a r b o x y - l l ~ , 1 7 ~ - d i h y d r o x y - a n d r o s t e n e - ( 4 ) - 3 - o n e , II, I I I = l l ~ , 1 7 ~ - 2 0 ~ - t r i h y d r o x y - 3 - o x o p r e g n e n e - ( 4 ) - 2 1 - o i c acids,
present in this fraction and in fraction N (Fig. 2G). In addition fractions N and O contained a component, the methylester of which moved like hydrocortisone (Fig. 2G and H). Since there was no hydrocortisone present (Fig. 2C and D), this peak should correspond to the methylesters of steroidal-21-oic acids reduced in ring A [27]. To test these interpretations, we chromatographed unesterified probes of fractions L--O in the system ethylacetate/ethanol/NH3 {25%) (5 : 5 : 1, v/v) (Fig. 3). Fraction L gave a peak in the region of 17~-carboxy-ll~,17a-dihydroxy-androstene-(4)-3-one and material moving faster or slower than the latter (Fig. 3A). Fraction M showed a peak in the region of 11~,17a,20~-trihydroxy-3-one-pregnene-(4)-21-oic acids and material moving faster (Fig. 3B). Fractions N and O mainly contained components moving faster than 17~-carboxy-11/3,17a-dihydroxy-androstene-(4)-3-one but considerably slower than hydrocortisone or tetrahydrocortisol (Fig. 3C,D).
Enzymatic digestion of fractions L--O and characterisation of the resulting products To further characterize the active metabolites, we submitted fractions L, M, N and O to thorough chromatographic analysis after digestion with an extract of Helix pomatia, which contains arylsulfatase and /~-glucuronidase
430
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