Exp
Eye Res. (1977) 24, 263-270
Metabolism
of Debydroepiandrosterone
in the Eye Lens Epithelium”
.A.. J. M. VERIVIORKEX,~W. J. XI. VAX DE VEN;~ W. H. J. GIELEN:? H. BLOENENDAL~AND H.C.J. KETELAARS: t Depadment University
of Biochemistry nrd $ Department of Pharmacology, of Nijmegen, Xijmegen, The Netherlands
(Received 29 Mnrch
1976 and in revised form
4 *June 1976, London)
Dehydroepiandrosterone$ is metabolized in the lens epiDhelium. Its metabolites have been androstenediol and androstenedione. identified as 7-cc-hydroxy-dehydroepiandrosterone, The hydroxylating enzyme is mainly located in the microsomal fraction, whereas the androstenediol forming enzyme is present both in the soluble and in the nuclear fraction of the cell. The inhibition characteristics of lenticular glucose-G-phosphate dehydrogenase seem to resemble those found in other tissues. Key WOTCZ?S : lens epithelium ; steroids; dehydroepiandrosterone; glucose-R-phosphate dehydrogenase.
1. Introduction In many tissues, including lens (Charlton and Van Heyningen, 1971), the activity of glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49); the key enzyme of the pentose phosphate cycle, is inhibited by the hormone DHEA (Marks and Banks, 1960; &Kerns and Kaleita, 1960; Levy, 1961; Benes, Freund, Menzel, Xtarka and Oertel, 197Oa; Benes, Menzel and Oertel, 197Ob). Since this metabolic pathway not only provides NADPH for the reductive biosynthesis of various compounds (Zakrzev ski; 1960; Armstrong and Wagner, 1961; Wakil and Bressler, 1962), but also pentose phosphate for the formation of nucleic acids (Larsson and Thelancler, 1965; Holzmann, Franz, %lorsches,Oertel and Degenhardt, 1972), it is of great importance for cell replication asis shown by the inhibition of cell propagation and thymidine incorporation of cells treated with DHEA in culture (Oertel, Rindt, Brachetti, Holzmann and Porsches, 1972; Strubel, Rindt; Hoffmann, Schirazi and Oertel, 1974). Slso in the lens epithelium
mitosis
might
be under
control
of the pentose
phosphate
cycle. In this
paper we describe the metabolism of DHEA in lens epithelium and the inhibition of 1enticula.r glucose-&phosphate dehydrogenase by this hormone and two of its metabolites. 2. Materials and Methods Dehydro-[4-Wjepiandrosterone (specific activity 68-8 mCi/mar) WM obtsil;ed from the Radiochemical Centre, Amersham, and was checked chromatographically for radiochemical purity before each experiment. Unlabeled steroids , glucose-6-phosphate dehydrogenaae, penicillin G and NADP were purchased from Sigma Chemical Company with the exception of 7-sc-hydroxy-DHEA, which was a gift from the Steroid Reference Collection iII.R.C., London, England. Gluconate-6-phosphate and glucose+phosphate Tq-ere obtained from Boehringer Mannheim. * Reprint requests to: Dr H. Bloemendal~ Laboratorium voor Biochemie, Universiteit van Sijmegen, “Heyendael”. Gem-t Grootplein Noord 21, Nijmegen, Set,herlands. g Abbreviatior,s used in this article: dehydroepiandrosterone = 3P-hydrosy-5-androstene.I7.one (DHEA) : androstenodiol = 5.androstene-3,& l’i,%diol (ADIOL); androstenedione = 4.androstene-3, 1.7.dioneo (ADIOS).
263
264
A. J. 51. VERMORKEN
ET
AL.
As scintillation liquid a solution of 3 g of PPO (2,5-diphenyloxazol) and 0.2 g of POPOP (2,2phenylenbis (4-methyl-5-phenyloxazol)), both obtained from Merck, Darmstadt, in 1 1 of toluene was used. For thin layer chromatography G-1500 LX 2.54 silicagel plates, obtained from Ca.rl Schleicher B: Schiill, were employed. Kodak X-Ray RP/R, films were used for autoradiography. The column was packed with l.Oo/ SE-30 on Gas chrom Q SO-100 mesh for gas chromatography-mass spectrometry. This column packing was obtained from Applied Science Laboratories Incorporation. BSTFA (N,o-bis-(trimethylsilyl)-trifluoroacetamide) was purchased from Pierce Chemical Company. Tissue preljaration Calf eyes were obtained on ice from the slaughter-house and used immediately. They were washed with distilled water and opened at the lateral side, so that the lenses could be removed without adhering iris material. A small incision was made in the lens ca.psule a,fter which it could easily be removed from the lens body. About 40% of the epithelial cells remained adhering to the lens capsule, as was demonstrated by microscopic examinat,ion. Incubation The radioactive DHEA (0.125 PCijlO ~1) was added to 25 ml incubation flasks and evaporated to dryness under a stream of nitrogen. The tissue (500 mg) was washed into the incubation flasks with 5 ml of 0.1 M-phosphate buffer, pH 7.5, containing 0.2 &I-sucrose, 500 units of penicillin G, and a NADPH-generating system (1 pmol/NADP, 25 prnol glucose-B-phosphate, 1 unit of glucose-6-phosphate dehydrogenase, 0.4 mmol MgCl,). The tissue was incubated at 37°C for 4 hr under air in a metabolic shaker. The reactions were terminated by the addition of 10 ml of redistilled acetone and the flasks stored overnight in a refrigerator at 4°C. Isolatiola
of radiometabolites
A general procedure for the determination of radiometabolites has been reported in detail by Collins, Mansfield, Bridges and Sommerville (1969). Each incubation mixture was extracted five times with 20 ml of acetone at 5O”C, and the extract filtered and evaporated until only the buffer remained. Twenty ml of sodium hydroxide, pH 9.0, was added to the flasks and the mixture extracted twice with diethyl ether. The washed extracts were taken to dryness, redissolved to a known volume, and purified chromatographic.ally on si.licagel, by developing the plate in petroleum ether (SO-1OO’C) saturated with methanol-water (6: 4). The radioactivity (RF < 0.05) was scraped from the plate and extracted three times with 2 ml of methanol. These extracts were taken to dryness, redissolved to a known volume, applied to thin layer plates and chromatographed in a system of chloroformethanol (19 : 1). The spectrum of metabolites was examined autoradiographically by overnight exposure of the plates to X-ray film. The areas of radioactivity were then scraped off the plates and eluted with methanol, and an aliquot was radioassayed by scintillation counting. Gas chromatography For analysis by combined gas chromatography-mass spectrometry, 100 g of lens epithelia were incubated with 400 pg of unlabeled DHEA. Aft,er separation by thin layer chromatography the metabolites were extracted with ether. ADION was analyzed as free steroid, while ADIOL and 7-x:-hydroxy-DHEA were converted into their trimethylsilylethers (TMðers): each sample was dissolved in 50 ,uI of BSTFA, the solution was agitated on a vortex mixer and allowed to stand overnight at room temperature. Reference samples were handled in the same way. A small amount (4 ~1) of each sample was injected into the gas chromatograph-mass spectrometer (ge-ms) and spectra were taken at 20 eV.
THE
METABOLISM
OF
DHEA
IN
LEXS
265
TISSUE
After each sample a reference was injected and the retention times and spectra were compared. Apparatus. An LKB 9000 combined gc-ms was used with a glass column (60 x 0.3 cm) and packed with 1.0% SE-30 on Gas chrom Q SO-100 mesh. Temperature. Injection port 25O”C, column 2OO”C, separator 26O”C, ion source 270°C. Spectra were taken at 20 eV ionizing poteutial with a trap current of 60 PA and an accelerating voltage of 3.5 kV. Preparation
of the supernatants
One thousand lens epithelia were added to one volume of ice-cold buffer containing and 8 ml of 0.02 M-KHCO,/l, pH 7.0. Twenty g of carborundum povder (2.15 M-KC1
(a)
a
B
1. Autoradiograph of a chromatograln of the steroids (A) with and (B) without lens epithelia.
PIG.
DHEA D
extracted
after
incubation
of radioactive
266
A. J. PI. VERMORKEN
ET
AL.
(80 mesh) was added and the tissue was homogenized in a mortar. The suspension was centrifuged at 3200 xg for 60 min, and the supernatant was dialyzed overnight against the extraction medium. During the whole procedure the temperature was kept between 0” and 4°C. Liver and lens body supernatants as well as 6-phosphogluconat dehydrogenase were prepared according to Glock and McLean (1953).
Estimation of glucose&~hos~hate
dehydyogenase activity
The glucose-B-phosphate dehydrogenase activity was measured spectrophotometrically by following the initial rate of reduction of NADP at 340 nm and 22°C. The reaction rate was constant for about 5 min and proportional to the a.mount of enzyme added. In the assay procedure the reaction mixture consisted of 0.1-0.3 ml of supernatant (depending on the activity of the preparation), 0.1 ml of the 6-phospho-gluconat dehydrogenase supernatant, 0.5 ml of 0.1 M-&#&, 0.5 ml of 0.25 M-glycylglycine buffer, pH 7.7, and 1.0 ml of 2.5 rnx-NADP. The volume was then adjusted to 2.4 ml with extraction medium. Ten ~1 of dioxane were added in which the hormones were dissolved in a concentration of 12.5 mM. In the control assays only dioxane was added. The reaction was started by adding 0.1 ml of 5 mnf-glucose-6-phosphate.
3. Results Incubation alzd ,ident$cation of metabolites The lens epithelium metabolizes the hormone DHEA into three major compounds. An autoradiograph of a chromatogram of the steroids extracted after incubation shows a large spot corresponding to DHEA and smaller spots representing its metabolites (a, b and c in Fig. 1A). An autoradiograph of a control incubation, in which no tissue was added, shows only a small amount of radioactivity corresponding in position to one of the metabolites (see Fig. 1B). In the identification experiments the labeled metabolites were co-chromatographed with the unlabeled ones. In Table I the &-values on the thin layer plates are given (middle column). TABLE X
on tl& layer plates, and relative retention times i,n the gaschromatographof DHEA and its metabolicderivatives
R,-Values
Compound
&---TLC*
DHEA ADIOX (spot a) ADIOL (spot b) ‘I-a-hydroxy-DHEA
0.43 0.60 047 0.09
* For conditions section. t TMSi-derivatives.
Identification matography-mass
of thin
la.yer
(spot c)
chromatography
(TLC),
of the individual metabolites spectrometra 7. Comparison
R,elative retention time-GC*
042 1.00 I.241 l56t
and gas chromatography
(GC),
see Methods
was achieved by combined gas chroof the mass spectra of the metabolites
THE
with those of (b) is ADIOL, In Table I metabolites in
METABOLISM
OF
DHEA
IX
LENS
267
TISSCE
pure reference compounds revealed that metabolite (a) is ADION; and (c) is 7-a-hydroxy-DHEA. (third column) the relative retention times of DHEA and of the the gas chromatograph are given.
Subcellular localization of the
eyyntes
Since one of the metabolizing enzymes concerned is a hydroxylating one, it was of interest to investigate whether or not this enzyme is localized in the membraneous fraction of the cell as are hydroxylating enzymes in liver @‘outs and Gram, 1969). Cell nuclei, membranes, and a cytosol fraction were isolated according to Vermorken, Hiderink, Beneditti and Bloemendal (1976a, b). No hydroxylating activity could be detected in the soluble part of the lens cells. However, this activity was present both in the nucleus and in the membrane fraction. The formation of ADION was small in all fractions, ADIOL formation was negligible in the membrane fraction but high in both the nuclei and cytosol (Table II). TABLE
Subcellular
Subcellular
d&d&on
II
of the metabolizing
Formation 7-a-Hydroxy-DHEA
fraction
Kuclei Microsomes cytoso1
* The formation
in the nuclei TABLE
K, values of different
ADIOL
100 e 46
has arbitrarily
enzyme preparations
R,
values
2.6 . 10-S M (2.2 . 10-s M) 4.3 10-j RI (4.5 10-s m) 4.3 . 10-j M (4.7 10-s M) 2.9 . 10-5iM (2.8 . lo-s%I)
Values in parenthesis are taken from Charlton and Van Heyningen nucleus, (3) rat lens (12 weeks), (4) rat lens (28 weeks).
Comparison of glucose-6-phosphate and bovine lens bOay
been t,aken as 100.
III
preparation
Calf lens epithelium Calf lens body Calf lens body Bovine lens body
of metabolites”
100 500 0
of the metabolites
Enzyme
enzymes
dehydrogenase
(1971).
(1) (2)
(3) (4)
(1) Sheep cortex,
in calf lens epithelium,
(2) sheep
calf lens body
The pH activity curve of glucose-6-phosphatedehydrogenase using ca,lf lens body supernatants as the source of the enzyme is in reasonableagreement with that found by Charlton a,nd Van Heyningen (1971) in sheep lenses. The pH optimum was at
268
A.
J.
Pt.
VERMORKEN
ET
AL.
pH 7.7. In all further enzyme determinations this pH was used. We compared the K,‘s of the enzyme preparations from lens epithelia and from lens body and found that the K, of glucose-g-phosphate dehydrogenase in the lens body is higher than in the epithelium of calf lenses. In the lens body of older animals the K, appeared to be lower than in the corresponding fraction of younger ones. Table III shows that our values for cattle lens are comparable to those of Charlton and Van Heyningen (1971) who studied sheep and rat lens. Inl~iibition of gl~~ose-6-~l~osph~te dehgdrogenase by steroids The influence of different substrate (glucose-6-phosphate) concentrations on the initial reaction velocity is reflected by Lineweaver and Burk plots (1934). As for other tissues, such as blood a,nd placenta (Benes et al., 1970a, b), DHEA was a more potent inhibitor than ADION, while ADIOL showed no significant inhibition. From Pig. 2 it can be concluded that the type of inhibition is uncompetitive, as has also been found by Raineri and Levy (1970) f or mammary glucose-6-phosphate dehydrogenase. The corresponding Km and Ki values are listed in Table IV.
( (b)
PIG. 2. Kinetic pattern of the inhibition of glucose-6-phosphate dehydrogenase in (a) calf lens epiV is defined as dD5 min at 340 nm; thelium, (b) adult bovine lens body, (c) bovine calf lens body. l/S is given in ~-1 x 104. A = activity without hormone; B = activity with ADION (5 . 10-j M); C = activity with DHEA (5 . 10-j M).
THE
METABOLISM
OF
DHEA
IN
LENS
269
TISSUE
TABLE IV
Inhibition
Enzyme
dehyd~ogenase in difSereent lms DHEA and ADIOL
of glucose&phosphate preparations by ADION, Ii,
preparation
Calf lens epithelium Calf lens body Adult bovine lens body
Kg ADION
2.6 . 10-j M 4.3 . IO-5 x 2.9. 1OW iv
Ki DHEA
2.3 1OV 31 3.0 IO-5 nz 2.5 . 10-j nr
1.6 . 10-j nr 2.1 . 10-s M 1.3 . IO-5 x
Ki ADIOL
2.4 . 1OF nT 4.2 1O-5 nf 2.5 . 10-j nx
4. Discussion Although hormonal regulation of the enzymatic activity of lenticular glucose-6phosphate dehydrogenase has still to be fully elucidated, the metabolism of DHEA might be a helpful tool in the study of cellular differentiation. For instance, psoriasis vulgaris, which is characterized by a changed pattern of epidermal differentiation, shows an increased DHEA metabolism in the red blood cells (Hoffmann, Norsches, Dijhler, Holzmann and Oertel, 1972). Our results clearly show that DHEA is metabolized in the lens epithelium. Since all the metabolites of the hormone have a much lessinhibitory effect (Raineri and Levy, 1970) on glucose-6-phosphatedehydrogenase, the activity of the pentose cycle may be under control of the DHEA metabolism. In the present study we were alsoable to show that in lensepithelium hydroxylation of the hormone takes place in the microsomal fraction of the cells. For the understanding of lens cell differentiation it would be of interest to know whether hydroxylation still occurs on fiber plasma membranes,sinceduring the processof differentiation of the epithelial cell into a fiber cell eventually all organellesincluding the cytoplasmic membranes disappear, whereas the surface of the plasma membranes enlarges enormously (Benedetti, Dunia and Bloemendal, 1974). Sincein our laboratory methods have been developed to isolate plasma membranes without the use of detergents (Bloemendal, Zweers, Vermorken, Dunia and Benedetti, 1972), studies can be started to answer
the questions
raised
above.
Note added in poqf Meanwhile we were able to demonstrate that isolated lens plasma membranes specifically hydroxylate DHEA (Vermorken, de Waal; v-d. Ven, Bloemendaland Henderson
(1976)). ACKNOWLEDGMENTS
The authors wish to thank J4r P. Kooij for technical assistance. The present investigations have partly been carried out under auspices of the Netherlands Foundation for Chemical Research (S.O.N.) and with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). REFEREKCES ilrmstrong, F. B. and Wagner, 236,2027-32.
R. P. (1961).
Biosynthesis
of valine
and isoleucine.
J. BioZ.
Ckenk.
270
A. J. 11. VERMORKEN
ET
AL.
Benedetti, E. L., Dunia, I. and Bloemendal, H. (1974). Development of junctions during differentiation of lens fibers. Proc. Nat. Acad. Sci. U.S.A. 71, 5073-7. Benes, P., Freund, R., Menzel, P., Starka, L. and Oertel, G. W. (1970a). Inhibition of glucose-Bphosphate dehydrogenase by steroids I. J. Steroid Biochem. 1, 287-90. Benes, P., Menzel, P. and Oertel, G. 1%‘. (1970b). Inhibition of glucose-B-phosphate dehydrogenase by steroids II. J. Steroid Biochem. 1, 291-3. Bloemendal, H., Zweers, A., Vermorken, F., Dunia, I. and Benedetti, E. L. (19i2). The plasma membranes of eye lens fibres. Biochemical and structural characterization. Cell Diff. 1,91-106. Charlton, J. M. and Van Heyningen, R. (1971). Glucose-A-phosphate dehydrogenase in the mammalian lens. Esq. Eye Res. 11, 147-60. Collins, W. P., Mansfield, M. D., Bridges, C. E. and Sommerville, I. F. (1969). Studies on steroid metabolism in human endometrial tissue. Biochem. J. 113, 399407. Fouts, J. R. and Gram, T. E. (1969). In Microsomes and Drug Oxidations (Eds Gillette, J. R. et al.). Pp. 81-91. Academic Press, New York-London. Glock, G. E. and McLean, P. (1953). Further studies on the properties and assay of glucose-6phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of rat liver. Biochem. J. 55,400408. Hoffmann, G., Morsches, B., Dohler, U., Holzmann, H. and Oertel, G. W. (1972). Steroide und Haut VIII. In vitro-Versuche mit 7-cc-3H-Dehydroepiandrosteron und seinen Sulfokonjugaten an intakten Erythrocyten bei Psoriasis Vulgaris. Arch.. Derm. Forsch. 243, 18-30. Holzmann, H., Franz, J., &Iorsches, B., Oertel, G. W. and Degenhardt, K. H. (1972). Zur Wirkung des Dehydroepiandrosterons auf die Mitose in Lymphozytenkulturen. Dtsch. i!fed. Wschr. 97, 1570-l. Larsson, A. and Thelander, L. (1965). The stereospecificity of thioredoxin reductase for triphosphopyridine nucleotide. J. Biol. Chem. 240,2691-3. Levy, H. R. (1961). The pyridine nucleotide specificity of glucose-g-phosphate dehydrogenase. Biochem. Biophys. Res. Commun. 6,49-53. Lineweaver, H. and Burk, D. (1934). The determination of enzyme dissociation constants. J. Am. Chem. Sot. 56, 658-66. Marks, P. A. and Banks, J. (1960). Inhibition of mammalian glucose-B-phosphate dehydrogenase by steroids. Proc. Nat. Acad. Sci. U.S.A. 46, 447-52. McKerns, K. W. and Kaleita, E. (1960). Inhibition of glucose-6-phosphate dehydrogenase by hormones. Biochem. Biophys. Res. Commun. 2, 344-8. Oertel, G. W., Rind& W., Brachetti, A. K. J., Holzmann, H. and Morsches, B. (1972). Metabolism of free DHEA and certain sulfo-conjugates in cell cultures from neoplastic and normal human tissue. Steroids Lipids Res. 3, 345-52. Raineri, R. and Levy, H. R. (1970). On the specificity of steroid interaction with mammary glucose-g-phosphate dehydrogenase. Biochemistry 9, 223343. Strubel, H. B., Rindt, W., Hoffmann, G., Schirazi, M. and Oertel, G. W. (1974). Cytostatic effects of G-6-PDH inhibitors in cell cultures from normal and neoplastic tissue. Steroids Lipids Res. 5, 101-6. Vermorken, A. J. M., Hilderink, J. M. H. C., Benedetti, E. L. and Bloemendal, H. (1976a). The isolation of nuclei from the lens epithelium (paper in preparation). Vermorken, A. J. M., Hilderink, J. M. H. C., Benedetti, E. L. and Bloemendal, H. (197670). Changes of membrane protein pattern in lens cell differentiation (paper in preparation). Vermorken, A. J. M., de Waal, R., v.d. Ven, W. J. M., Bloemendal, H. and Henderson, P. Th. (1977). Hydroxylation of dehydroepiandrosterone in the lens. Biochim. Biophys. Aeta (in press). Wakil, S. J. and Bressler, R. (1962). Studies on the mechanism of fatty acid synthesis. J. BioE. Chem. 237, 687-93. Zakrzewski, S. F. (1960). Purification and properties of folic acid reductase from chicken liver. J. Biol. Chem. 235, 1776-9.
Eye Res. (1977) 24, 263-270
Metabolism
of Debydroepiandrosterone
in the Eye Lens Epithelium”
.A.. J. M. VERIVIORKEX,~W. J. XI. VAX DE VEN;~ W. H. J. GIELEN:? H. BLOENENDAL~AND H.C.J. KETELAARS: t Depadment University
of Biochemistry nrd $ Department of Pharmacology, of Nijmegen, Xijmegen, The Netherlands
(Received 29 Mnrch
1976 and in revised form
4 *June 1976, London)
Dehydroepiandrosterone$ is metabolized in the lens epiDhelium. Its metabolites have been androstenediol and androstenedione. identified as 7-cc-hydroxy-dehydroepiandrosterone, The hydroxylating enzyme is mainly located in the microsomal fraction, whereas the androstenediol forming enzyme is present both in the soluble and in the nuclear fraction of the cell. The inhibition characteristics of lenticular glucose-G-phosphate dehydrogenase seem to resemble those found in other tissues. Key WOTCZ?S : lens epithelium ; steroids; dehydroepiandrosterone; glucose-R-phosphate dehydrogenase.
1. Introduction In many tissues, including lens (Charlton and Van Heyningen, 1971), the activity of glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49); the key enzyme of the pentose phosphate cycle, is inhibited by the hormone DHEA (Marks and Banks, 1960; &Kerns and Kaleita, 1960; Levy, 1961; Benes, Freund, Menzel, Xtarka and Oertel, 197Oa; Benes, Menzel and Oertel, 197Ob). Since this metabolic pathway not only provides NADPH for the reductive biosynthesis of various compounds (Zakrzev ski; 1960; Armstrong and Wagner, 1961; Wakil and Bressler, 1962), but also pentose phosphate for the formation of nucleic acids (Larsson and Thelancler, 1965; Holzmann, Franz, %lorsches,Oertel and Degenhardt, 1972), it is of great importance for cell replication asis shown by the inhibition of cell propagation and thymidine incorporation of cells treated with DHEA in culture (Oertel, Rindt, Brachetti, Holzmann and Porsches, 1972; Strubel, Rindt; Hoffmann, Schirazi and Oertel, 1974). Slso in the lens epithelium
mitosis
might
be under
control
of the pentose
phosphate
cycle. In this
paper we describe the metabolism of DHEA in lens epithelium and the inhibition of 1enticula.r glucose-&phosphate dehydrogenase by this hormone and two of its metabolites. 2. Materials and Methods Dehydro-[4-Wjepiandrosterone (specific activity 68-8 mCi/mar) WM obtsil;ed from the Radiochemical Centre, Amersham, and was checked chromatographically for radiochemical purity before each experiment. Unlabeled steroids , glucose-6-phosphate dehydrogenaae, penicillin G and NADP were purchased from Sigma Chemical Company with the exception of 7-sc-hydroxy-DHEA, which was a gift from the Steroid Reference Collection iII.R.C., London, England. Gluconate-6-phosphate and glucose+phosphate Tq-ere obtained from Boehringer Mannheim. * Reprint requests to: Dr H. Bloemendal~ Laboratorium voor Biochemie, Universiteit van Sijmegen, “Heyendael”. Gem-t Grootplein Noord 21, Nijmegen, Set,herlands. g Abbreviatior,s used in this article: dehydroepiandrosterone = 3P-hydrosy-5-androstene.I7.one (DHEA) : androstenodiol = 5.androstene-3,& l’i,%diol (ADIOL); androstenedione = 4.androstene-3, 1.7.dioneo (ADIOS).
263
264
A. J. 51. VERMORKEN
ET
AL.
As scintillation liquid a solution of 3 g of PPO (2,5-diphenyloxazol) and 0.2 g of POPOP (2,2phenylenbis (4-methyl-5-phenyloxazol)), both obtained from Merck, Darmstadt, in 1 1 of toluene was used. For thin layer chromatography G-1500 LX 2.54 silicagel plates, obtained from Ca.rl Schleicher B: Schiill, were employed. Kodak X-Ray RP/R, films were used for autoradiography. The column was packed with l.Oo/ SE-30 on Gas chrom Q SO-100 mesh for gas chromatography-mass spectrometry. This column packing was obtained from Applied Science Laboratories Incorporation. BSTFA (N,o-bis-(trimethylsilyl)-trifluoroacetamide) was purchased from Pierce Chemical Company. Tissue preljaration Calf eyes were obtained on ice from the slaughter-house and used immediately. They were washed with distilled water and opened at the lateral side, so that the lenses could be removed without adhering iris material. A small incision was made in the lens ca.psule a,fter which it could easily be removed from the lens body. About 40% of the epithelial cells remained adhering to the lens capsule, as was demonstrated by microscopic examinat,ion. Incubation The radioactive DHEA (0.125 PCijlO ~1) was added to 25 ml incubation flasks and evaporated to dryness under a stream of nitrogen. The tissue (500 mg) was washed into the incubation flasks with 5 ml of 0.1 M-phosphate buffer, pH 7.5, containing 0.2 &I-sucrose, 500 units of penicillin G, and a NADPH-generating system (1 pmol/NADP, 25 prnol glucose-B-phosphate, 1 unit of glucose-6-phosphate dehydrogenase, 0.4 mmol MgCl,). The tissue was incubated at 37°C for 4 hr under air in a metabolic shaker. The reactions were terminated by the addition of 10 ml of redistilled acetone and the flasks stored overnight in a refrigerator at 4°C. Isolatiola
of radiometabolites
A general procedure for the determination of radiometabolites has been reported in detail by Collins, Mansfield, Bridges and Sommerville (1969). Each incubation mixture was extracted five times with 20 ml of acetone at 5O”C, and the extract filtered and evaporated until only the buffer remained. Twenty ml of sodium hydroxide, pH 9.0, was added to the flasks and the mixture extracted twice with diethyl ether. The washed extracts were taken to dryness, redissolved to a known volume, and purified chromatographic.ally on si.licagel, by developing the plate in petroleum ether (SO-1OO’C) saturated with methanol-water (6: 4). The radioactivity (RF < 0.05) was scraped from the plate and extracted three times with 2 ml of methanol. These extracts were taken to dryness, redissolved to a known volume, applied to thin layer plates and chromatographed in a system of chloroformethanol (19 : 1). The spectrum of metabolites was examined autoradiographically by overnight exposure of the plates to X-ray film. The areas of radioactivity were then scraped off the plates and eluted with methanol, and an aliquot was radioassayed by scintillation counting. Gas chromatography For analysis by combined gas chromatography-mass spectrometry, 100 g of lens epithelia were incubated with 400 pg of unlabeled DHEA. Aft,er separation by thin layer chromatography the metabolites were extracted with ether. ADION was analyzed as free steroid, while ADIOL and 7-x:-hydroxy-DHEA were converted into their trimethylsilylethers (TMðers): each sample was dissolved in 50 ,uI of BSTFA, the solution was agitated on a vortex mixer and allowed to stand overnight at room temperature. Reference samples were handled in the same way. A small amount (4 ~1) of each sample was injected into the gas chromatograph-mass spectrometer (ge-ms) and spectra were taken at 20 eV.
THE
METABOLISM
OF
DHEA
IN
LEXS
265
TISSUE
After each sample a reference was injected and the retention times and spectra were compared. Apparatus. An LKB 9000 combined gc-ms was used with a glass column (60 x 0.3 cm) and packed with 1.0% SE-30 on Gas chrom Q SO-100 mesh. Temperature. Injection port 25O”C, column 2OO”C, separator 26O”C, ion source 270°C. Spectra were taken at 20 eV ionizing poteutial with a trap current of 60 PA and an accelerating voltage of 3.5 kV. Preparation
of the supernatants
One thousand lens epithelia were added to one volume of ice-cold buffer containing and 8 ml of 0.02 M-KHCO,/l, pH 7.0. Twenty g of carborundum povder (2.15 M-KC1
(a)
a
B
1. Autoradiograph of a chromatograln of the steroids (A) with and (B) without lens epithelia.
PIG.
DHEA D
extracted
after
incubation
of radioactive
266
A. J. PI. VERMORKEN
ET
AL.
(80 mesh) was added and the tissue was homogenized in a mortar. The suspension was centrifuged at 3200 xg for 60 min, and the supernatant was dialyzed overnight against the extraction medium. During the whole procedure the temperature was kept between 0” and 4°C. Liver and lens body supernatants as well as 6-phosphogluconat dehydrogenase were prepared according to Glock and McLean (1953).
Estimation of glucose&~hos~hate
dehydyogenase activity
The glucose-B-phosphate dehydrogenase activity was measured spectrophotometrically by following the initial rate of reduction of NADP at 340 nm and 22°C. The reaction rate was constant for about 5 min and proportional to the a.mount of enzyme added. In the assay procedure the reaction mixture consisted of 0.1-0.3 ml of supernatant (depending on the activity of the preparation), 0.1 ml of the 6-phospho-gluconat dehydrogenase supernatant, 0.5 ml of 0.1 M-&#&, 0.5 ml of 0.25 M-glycylglycine buffer, pH 7.7, and 1.0 ml of 2.5 rnx-NADP. The volume was then adjusted to 2.4 ml with extraction medium. Ten ~1 of dioxane were added in which the hormones were dissolved in a concentration of 12.5 mM. In the control assays only dioxane was added. The reaction was started by adding 0.1 ml of 5 mnf-glucose-6-phosphate.
3. Results Incubation alzd ,ident$cation of metabolites The lens epithelium metabolizes the hormone DHEA into three major compounds. An autoradiograph of a chromatogram of the steroids extracted after incubation shows a large spot corresponding to DHEA and smaller spots representing its metabolites (a, b and c in Fig. 1A). An autoradiograph of a control incubation, in which no tissue was added, shows only a small amount of radioactivity corresponding in position to one of the metabolites (see Fig. 1B). In the identification experiments the labeled metabolites were co-chromatographed with the unlabeled ones. In Table I the &-values on the thin layer plates are given (middle column). TABLE X
on tl& layer plates, and relative retention times i,n the gaschromatographof DHEA and its metabolicderivatives
R,-Values
Compound
&---TLC*
DHEA ADIOX (spot a) ADIOL (spot b) ‘I-a-hydroxy-DHEA
0.43 0.60 047 0.09
* For conditions section. t TMSi-derivatives.
Identification matography-mass
of thin
la.yer
(spot c)
chromatography
(TLC),
of the individual metabolites spectrometra 7. Comparison
R,elative retention time-GC*
042 1.00 I.241 l56t
and gas chromatography
(GC),
see Methods
was achieved by combined gas chroof the mass spectra of the metabolites
THE
with those of (b) is ADIOL, In Table I metabolites in
METABOLISM
OF
DHEA
IX
LENS
267
TISSCE
pure reference compounds revealed that metabolite (a) is ADION; and (c) is 7-a-hydroxy-DHEA. (third column) the relative retention times of DHEA and of the the gas chromatograph are given.
Subcellular localization of the
eyyntes
Since one of the metabolizing enzymes concerned is a hydroxylating one, it was of interest to investigate whether or not this enzyme is localized in the membraneous fraction of the cell as are hydroxylating enzymes in liver @‘outs and Gram, 1969). Cell nuclei, membranes, and a cytosol fraction were isolated according to Vermorken, Hiderink, Beneditti and Bloemendal (1976a, b). No hydroxylating activity could be detected in the soluble part of the lens cells. However, this activity was present both in the nucleus and in the membrane fraction. The formation of ADION was small in all fractions, ADIOL formation was negligible in the membrane fraction but high in both the nuclei and cytosol (Table II). TABLE
Subcellular
Subcellular
d&d&on
II
of the metabolizing
Formation 7-a-Hydroxy-DHEA
fraction
Kuclei Microsomes cytoso1
* The formation
in the nuclei TABLE
K, values of different
ADIOL
100 e 46
has arbitrarily
enzyme preparations
R,
values
2.6 . 10-S M (2.2 . 10-s M) 4.3 10-j RI (4.5 10-s m) 4.3 . 10-j M (4.7 10-s M) 2.9 . 10-5iM (2.8 . lo-s%I)
Values in parenthesis are taken from Charlton and Van Heyningen nucleus, (3) rat lens (12 weeks), (4) rat lens (28 weeks).
Comparison of glucose-6-phosphate and bovine lens bOay
been t,aken as 100.
III
preparation
Calf lens epithelium Calf lens body Calf lens body Bovine lens body
of metabolites”
100 500 0
of the metabolites
Enzyme
enzymes
dehydrogenase
(1971).
(1) (2)
(3) (4)
(1) Sheep cortex,
in calf lens epithelium,
(2) sheep
calf lens body
The pH activity curve of glucose-6-phosphatedehydrogenase using ca,lf lens body supernatants as the source of the enzyme is in reasonableagreement with that found by Charlton a,nd Van Heyningen (1971) in sheep lenses. The pH optimum was at
268
A.
J.
Pt.
VERMORKEN
ET
AL.
pH 7.7. In all further enzyme determinations this pH was used. We compared the K,‘s of the enzyme preparations from lens epithelia and from lens body and found that the K, of glucose-g-phosphate dehydrogenase in the lens body is higher than in the epithelium of calf lenses. In the lens body of older animals the K, appeared to be lower than in the corresponding fraction of younger ones. Table III shows that our values for cattle lens are comparable to those of Charlton and Van Heyningen (1971) who studied sheep and rat lens. Inl~iibition of gl~~ose-6-~l~osph~te dehgdrogenase by steroids The influence of different substrate (glucose-6-phosphate) concentrations on the initial reaction velocity is reflected by Lineweaver and Burk plots (1934). As for other tissues, such as blood a,nd placenta (Benes et al., 1970a, b), DHEA was a more potent inhibitor than ADION, while ADIOL showed no significant inhibition. From Pig. 2 it can be concluded that the type of inhibition is uncompetitive, as has also been found by Raineri and Levy (1970) f or mammary glucose-6-phosphate dehydrogenase. The corresponding Km and Ki values are listed in Table IV.
( (b)
PIG. 2. Kinetic pattern of the inhibition of glucose-6-phosphate dehydrogenase in (a) calf lens epiV is defined as dD5 min at 340 nm; thelium, (b) adult bovine lens body, (c) bovine calf lens body. l/S is given in ~-1 x 104. A = activity without hormone; B = activity with ADION (5 . 10-j M); C = activity with DHEA (5 . 10-j M).
THE
METABOLISM
OF
DHEA
IN
LENS
269
TISSUE
TABLE IV
Inhibition
Enzyme
dehyd~ogenase in difSereent lms DHEA and ADIOL
of glucose&phosphate preparations by ADION, Ii,
preparation
Calf lens epithelium Calf lens body Adult bovine lens body
Kg ADION
2.6 . 10-j M 4.3 . IO-5 x 2.9. 1OW iv
Ki DHEA
2.3 1OV 31 3.0 IO-5 nz 2.5 . 10-j nr
1.6 . 10-j nr 2.1 . 10-s M 1.3 . IO-5 x
Ki ADIOL
2.4 . 1OF nT 4.2 1O-5 nf 2.5 . 10-j nx
4. Discussion Although hormonal regulation of the enzymatic activity of lenticular glucose-6phosphate dehydrogenase has still to be fully elucidated, the metabolism of DHEA might be a helpful tool in the study of cellular differentiation. For instance, psoriasis vulgaris, which is characterized by a changed pattern of epidermal differentiation, shows an increased DHEA metabolism in the red blood cells (Hoffmann, Norsches, Dijhler, Holzmann and Oertel, 1972). Our results clearly show that DHEA is metabolized in the lens epithelium. Since all the metabolites of the hormone have a much lessinhibitory effect (Raineri and Levy, 1970) on glucose-6-phosphatedehydrogenase, the activity of the pentose cycle may be under control of the DHEA metabolism. In the present study we were alsoable to show that in lensepithelium hydroxylation of the hormone takes place in the microsomal fraction of the cells. For the understanding of lens cell differentiation it would be of interest to know whether hydroxylation still occurs on fiber plasma membranes,sinceduring the processof differentiation of the epithelial cell into a fiber cell eventually all organellesincluding the cytoplasmic membranes disappear, whereas the surface of the plasma membranes enlarges enormously (Benedetti, Dunia and Bloemendal, 1974). Sincein our laboratory methods have been developed to isolate plasma membranes without the use of detergents (Bloemendal, Zweers, Vermorken, Dunia and Benedetti, 1972), studies can be started to answer
the questions
raised
above.
Note added in poqf Meanwhile we were able to demonstrate that isolated lens plasma membranes specifically hydroxylate DHEA (Vermorken, de Waal; v-d. Ven, Bloemendaland Henderson
(1976)). ACKNOWLEDGMENTS
The authors wish to thank J4r P. Kooij for technical assistance. The present investigations have partly been carried out under auspices of the Netherlands Foundation for Chemical Research (S.O.N.) and with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). REFEREKCES ilrmstrong, F. B. and Wagner, 236,2027-32.
R. P. (1961).
Biosynthesis
of valine
and isoleucine.
J. BioZ.
Ckenk.
270
A. J. 11. VERMORKEN
ET
AL.
Benedetti, E. L., Dunia, I. and Bloemendal, H. (1974). Development of junctions during differentiation of lens fibers. Proc. Nat. Acad. Sci. U.S.A. 71, 5073-7. Benes, P., Freund, R., Menzel, P., Starka, L. and Oertel, G. W. (1970a). Inhibition of glucose-Bphosphate dehydrogenase by steroids I. J. Steroid Biochem. 1, 287-90. Benes, P., Menzel, P. and Oertel, G. 1%‘. (1970b). Inhibition of glucose-B-phosphate dehydrogenase by steroids II. J. Steroid Biochem. 1, 291-3. Bloemendal, H., Zweers, A., Vermorken, F., Dunia, I. and Benedetti, E. L. (19i2). The plasma membranes of eye lens fibres. Biochemical and structural characterization. Cell Diff. 1,91-106. Charlton, J. M. and Van Heyningen, R. (1971). Glucose-A-phosphate dehydrogenase in the mammalian lens. Esq. Eye Res. 11, 147-60. Collins, W. P., Mansfield, M. D., Bridges, C. E. and Sommerville, I. F. (1969). Studies on steroid metabolism in human endometrial tissue. Biochem. J. 113, 399407. Fouts, J. R. and Gram, T. E. (1969). In Microsomes and Drug Oxidations (Eds Gillette, J. R. et al.). Pp. 81-91. Academic Press, New York-London. Glock, G. E. and McLean, P. (1953). Further studies on the properties and assay of glucose-6phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of rat liver. Biochem. J. 55,400408. Hoffmann, G., Morsches, B., Dohler, U., Holzmann, H. and Oertel, G. W. (1972). Steroide und Haut VIII. In vitro-Versuche mit 7-cc-3H-Dehydroepiandrosteron und seinen Sulfokonjugaten an intakten Erythrocyten bei Psoriasis Vulgaris. Arch.. Derm. Forsch. 243, 18-30. Holzmann, H., Franz, J., &Iorsches, B., Oertel, G. W. and Degenhardt, K. H. (1972). Zur Wirkung des Dehydroepiandrosterons auf die Mitose in Lymphozytenkulturen. Dtsch. i!fed. Wschr. 97, 1570-l. Larsson, A. and Thelander, L. (1965). The stereospecificity of thioredoxin reductase for triphosphopyridine nucleotide. J. Biol. Chem. 240,2691-3. Levy, H. R. (1961). The pyridine nucleotide specificity of glucose-g-phosphate dehydrogenase. Biochem. Biophys. Res. Commun. 6,49-53. Lineweaver, H. and Burk, D. (1934). The determination of enzyme dissociation constants. J. Am. Chem. Sot. 56, 658-66. Marks, P. A. and Banks, J. (1960). Inhibition of mammalian glucose-B-phosphate dehydrogenase by steroids. Proc. Nat. Acad. Sci. U.S.A. 46, 447-52. McKerns, K. W. and Kaleita, E. (1960). Inhibition of glucose-6-phosphate dehydrogenase by hormones. Biochem. Biophys. Res. Commun. 2, 344-8. Oertel, G. W., Rind& W., Brachetti, A. K. J., Holzmann, H. and Morsches, B. (1972). Metabolism of free DHEA and certain sulfo-conjugates in cell cultures from neoplastic and normal human tissue. Steroids Lipids Res. 3, 345-52. Raineri, R. and Levy, H. R. (1970). On the specificity of steroid interaction with mammary glucose-g-phosphate dehydrogenase. Biochemistry 9, 223343. Strubel, H. B., Rindt, W., Hoffmann, G., Schirazi, M. and Oertel, G. W. (1974). Cytostatic effects of G-6-PDH inhibitors in cell cultures from normal and neoplastic tissue. Steroids Lipids Res. 5, 101-6. Vermorken, A. J. M., Hilderink, J. M. H. C., Benedetti, E. L. and Bloemendal, H. (1976a). The isolation of nuclei from the lens epithelium (paper in preparation). Vermorken, A. J. M., Hilderink, J. M. H. C., Benedetti, E. L. and Bloemendal, H. (197670). Changes of membrane protein pattern in lens cell differentiation (paper in preparation). Vermorken, A. J. M., de Waal, R., v.d. Ven, W. J. M., Bloemendal, H. and Henderson, P. Th. (1977). Hydroxylation of dehydroepiandrosterone in the lens. Biochim. Biophys. Aeta (in press). Wakil, S. J. and Bressler, R. (1962). Studies on the mechanism of fatty acid synthesis. J. BioE. Chem. 237, 687-93. Zakrzewski, S. F. (1960). Purification and properties of folic acid reductase from chicken liver. J. Biol. Chem. 235, 1776-9.
