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Characteristics of dehydroepiandrosterone as a peroxisome proliferator
Biochimica et Biophysica Acta. 1092(1991)233-243
233
© 1991 ElsevierSciencePublishersB.V.0167-4889/91/$03.50 ADONIS 016748899100148N BBAMCR12913
Characteristics of dehydroepiandrosterone as a peroxisome proliferator Junji Yamada 1, Mitsuhiro-Sakuma 1, Toshihiko Ikeda and Tetsuya Suga 1
2, Kuniaki
Fukuda 3
I Department of Clinical Biochemistry, Tokyo College of Pharmacy. Tokyo (Japan). and " Analytical and Metabolic Research Laboratories, Tokyo (Japan) and ~ Biological Research Laboratories. Sonkyo Co.. Tokyo (Japan)
(Received25 October1990)
Key words: Dehydroepiandrosterone;Peroxisomeproliferation;Enzymeinduction;(Rat liver) Treatment of rats with dehydroepiandrosterone (300 m g / k g body weight, per os, 14 days) caused a remarkable increase in the number of peroxisomes and peroxisomal t-oxidation activity in the liver. The activities of carnitine acetyitransferase, microsomal laurate 12-hydmxylation, cytosolic palmitoyI-CoA hydrolase, malic enzyme and some other enzymes were also increased. The increases in these enzyme activities were all greater in male rats than in female rats. Immunoblot analysis revealed remarkable induction of acyi-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyi-CoA dehydrogenase bifunctional enzyme in the liver and to a smaller extent in the kidney, whereas no significant inductiol~ of these enzymes was found in the heart. The increase in the hepatic peroxisomal /]-oxidation activity reached a maximal level at day 5 of the treatment of dehydroepiandrosterone and the increased activity rapidly returned to the ~:,,rmal level on discontinuation of the treatment. The increase in the activity was also dose-dependent, which was ~aturable at a dose of more than 200 m g / k g body weight. All these features in enzyme induction caused by dehydroepiandrosterone correlate well with those observed in the treatment of dofibric acid, a peroxisome proliferator. Co-treatment of dehydtoepiandrosterone and clofibric acid showed no synergism in the enhancement of peroxisomai /]-oxidation activity, suggesting the involvement of a common process in the mechanism by which these compounds induce the enzymes. These results indicate that dehydroepiandrosterone is a typical peroxisome proliferator. Since dehydroepiandrosterone is a naturally oecu.,'ing C 19 steroid in mammals, the structure of which is novel compared with those of peroxisome proliferators known so far, this compound could provide particular information in the understanding of the mechanisms underlying the induction of peroxisome proliferation.
Introduction
Dehydroepiandrosterone (DHEA) is a naturally occurring C~0 steroid in mammals, which is known to exert various actions such as antiobesity, antidiabetic and anticarcinogenic actions when administered to rats and mice [1,2]. In 1987 Leighton et al. [3] reported that DHEA-feeding of rats increased hepatic catalase and acyl-CoA oxidase activities. Although there have been a few more reports [4,5] dealing with the induction of peroxisomal enzymes by DHEA in the literature, the
Abbreviations: DHEA,dehydroepiandrosterone;CPIB, clofibricacid; GPC, glycerophosphocholine;SDS, sodiumdodecylsulfate. Correspondence: J. Yamada, Department of Clinical Biochemistry, Tokyo College of Pharmacy, 1432-1 Horinonchi, Hachioji, Tokyo 192-03, Japan.
characteristics of DHEA as a peroxisome proliferator have not yet been clarified sufficiently. Peroxisome proliferators, including hypolipidemic drugs, phthalate ester plasticizers and agricultural chemicals [6-8], are structurally diverse and the structural requirements for peroxisome proliferation are, therefore, poorly understood. Although the mechanism by which these compounds induce peroxisome proliferation has not been established, it is now proposed that a structure of unmetabolizable hydrophobic anion may be required to be a potent peroxisome proliferator [9,10]. The steroidal structure of DHEA is novel, as compared with the peroxisome proliferators known so far, and DHEA has no anionic functional groups itself. We are interested in DHEA as a structurally new type of peroxisome proliferator and as a possible endogenous inducer of peroxisomal enzymes. In the present study, to elucidate the characteristics
234 of DHEA as a peroxisome proliferator we investigated the in vivo effect of DHEA on various enzymes including extra-peroxisomal enzymes, using clofibric acid (CPIB), a typical peroxisome proliferator, as a positive control. Sex differences and tissue-differences in the effect were also investigated. Materials and Methods Materials, DHEA was purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan). CPIB was purchased from Sigma Chemical Co. (St. Louis, MO). BrMB (3-bromomethyi-7-methoxy-l,4-benzoxazin-2-one) was kindly provided by Dr. Y. Kawahara (Sankyo Co., Ltd., Tokyo, Japan). Butanilicaine (N-butylaminoacetyl-2-chloro-6methylanilide) and L.carnitine were donated by Hoechst AG (Frankfurt, F.R.G.) and Earth Pharmaceutical Co. (Akoh, Japan), respectively. All other chemicals were of reagent grade. Animals and treatments. Male and female Wistar rats, 7 weeks of age, weighing about 200 g and 180 g, respectively, at the beginning of the experiments, were used, The animals were maintained on water and standard CE-II chow (Nihon Clea Inc., Tokyo, Japan) ad libitum, DHEA was suspended in 0.5% carboxymethylcellulose, and CPIB was dissolved in the same vehicle as the sodium salt. The individual agents were administered by gastric intubation once a day at the doses and for the time periods indicated in the text. The control animals received vehicle only (5 ml/kg body wt.). The animals were killed 24 h after the last administration. Enzyme preparations. The animals were anesthetized with diethyl ether and the livers were perfused in situ with saline through the portal vein. The livers and other tissues were excised, rinsed in 0.25 M sucrose, blotted and weighed, Liver homogenates were prepared in 0.25 M sucrose containing 1 mM EDTA, 10 mM Tris-HCI (pH 7.5) and 0,1~ ethanol, Liver microsomes and cytosol were prepared as follows: after centrifugation of the
liver homogenates at 25 000 × g for 10 rain, the result!rig supernatants were further centrifuged at 105 000 × g for 60 min. The supernatants obtained were used as cytosol. The precipitates obtained were resuspended in 1.15% KCI and centrifuged again at 105 000 x g for 60 min. The resulting precipitates were suspended in 0.1 M phosphate buffer (pH 7.5) containing 1 mM EDTA and used as mierosomes. These preparations were used for enzyme assay, For electrophoresis and immunoblotting, the livers, renal cortices and hearts were homogenized in 50 mM Tris-HC1 (pH 8.0) containing 1 mM EDTA, 5 /Lg/ml leupeptine and 0.3 mM phenylmethylsulfonyl fluoride. Protein was determined by the method of Lowry et al. [11] with bovine serum albumin as a standard. Enzyme assays. The activities of catalase [12], D-amino acid oxidase [13], urate oxidase [13], peroxisomal floxidation [14,15], acyl-CoA oxidase [15,16], carnitine acetyltransferase [17] and carnitine palmitoyltransferase [17] were measured in liver homogenates. Peroxisomal ~-oxidation and acyl-CoA oxidase were assayed with lauroyl-CoA as the substrate. Microsomal laurate l 1and 12-hydroxylation were assayed by the method of Parker and Orton [18]. In this assay, unlabeled laurate instead of 1-t4C-labeled laurate was used as the substrate. The reaction products were converted to the fluorescent derivatives with BrMB (3-bromomethyl-% methoxy-l,4-benzoxazin-2-one) and subjected to HPLC analysis. The detailed procedure will be described elsewhere (unpublished data). The activity of microsomal 1-acyl-GPC acyltransferase [19,20] was measured with oleoyl-CoA and 1-palmitoyl GPC. Microsomal carboxylesterase [21,22] was assayed with butanilicaine (N-butylaminoacetyl-2-ehloro-6-methylanilide) as the substrate. The activities of palmitoyl-CoA hydrolase [23], glutathione S-transferase [24] toward 1-chloro-2,4dinitrobenzene, malic enzyme (decarboxylating) [25] and glucose-6-phosphate dehydrogenase [26] were measured in liver cytosol.
TABLE l Effect of DHEA on body weightgain, liver weight and hepaticprotein content
Male and femalerats were treated with300 nagof DHEA/kg body wt. (DHEA), 300 mg of CPIB/kg body wt. (CPIB)or 300 mg each of DHEA and CP1B/k8 bodywt. (DHEA+CPlB) for 14 days. Data are expressedas the meand:S.D. of fiverats. Valuesin parenthesesrefer to the increases relative to the control in each sex ( * P < 0.05, * * P < 0.01). Control
DHEA
CPIB
DHEA+ CPIB
Male Bodyweightgain(g) Liverweight($ of bodywt,) Liverprotein(mg/g liver)
92 4-19 (1) 3.44- 0.1 (1) 204 4-10 (1)
87 4-9 (0.9) 6.24- 0.6 * * (1.8) 206 4- 7 (1.0)
91 +17 (1.0) 5.5 + 0.4 * * (1.6) 216 +12 (1.1)
87 + 7 (0.9) 6.9+ 0.9 * * (2.0) 196 + 13 (1.0)
Female Bodyweightgain(g) Liverweight(%of bodywt.) Liverprotein(mg/g liver)
47 +10 (1) 3.3+ 0.1 (1) 206 4-16 (1)
48 4-12 (1.0) 6.04- 0.5 **(1.8) 226 4- 5 * (1.1)
43 +11 (0.9) 4.6+ 0.3 **(1.4) 216 + 4 (1.0)
235
Other methods. Polyacrylamide gel electrophoresis was performed in the presence of sodium dodecyl sulfate as described by Laemmli [27] with 10% acrylamide gel. Immunoehemical staining was carried out b y the method of Towbin et al. [28] with the modification of Guengerich et al. [29]. Catalase [30,31], acyl-CoA oxidase [16] and enoyl-CoA h y d r a t a s e / 3 - h y d r o x y a c y l - C o A dehydrogenase bifunctional enzyme [32] were purified from rat liver according to the published procedures, and antibodies against the individual enzymes were raised in female Japanese White rabbits [22]. Electron microscopy was carried out as described previously [331. Statistical analyses were performed using Student's t-test. Results
Effect of DHEA in male and female rats Male and female rats were orally administered 300 m g of D H E A / k g b o d y wt., 300 mg of C P I B / k g body wt. or 300 mg each of D H E A and C P I B / k g body wt. for 14 days. Body weight and liver weight As shown in Table I, body weight gains of all the animals in the treated groups were similar to those in the control group in both males and females. Decrease
in b o d y weight gain due to the treatment of D H E A was not observed in either sex. In all the treated groups, hepatomegaly occurred as indicated by 1.4- to 2.0-fold increase in the relative liver weight, while the protein content per liver was almost equal to the control value.
Hepatic enzyme activities T h e effect of D H E A on the hepatic enzyme activities of male rats are summarized in Table II. D H E A increased the activities of peroxisomal/t-oxidation, acylC o A oxidase and catalase, 7.9-, 6.4- and 1.6-fold, respectively, and decreased D-amino acid oxidase and urate oxidase by 40% and 30%, respectively. These changes in peroxisomal enzyme activities were consistent with those caused b y CPIB. The activities of several extra-peroxisomal enzymes were also increased. A marked increase was found in the activities of earnitine acetyltransferase, microsomal laurate 12-hydroxylation, cytosolic palmitoyl-CoA hydrolase and malic enzyme. Carnitine palmitoyltransferase which is a mitochondrial enzyme, and laurate ll-hydroxylation, 1-aeylG P C acyitransferase and carboxylesterase in microsomes were moderately increased. Cytosolic glutahtione S-transferase activity was, however, decreased b y 20%. The activity of glueose-6-phosphate dehydrogenase was not changed. All these changes in the activities of extra-peroxisomal enzymes were also consistent with those of the CPIB-treated group.
TABLE II
Effect of DHEA on hepatic enzyme activitiesof male rats Data were obtained from the same rats as those described in Table I and are expressed as tbe mean + S.D. of five rats. Values in parentheses refer to increases relative to the control ( * P < 0.05, * * P < 0.01). Enzyme
Control
Liver homogenate Peroxisomal )8.oxidationa AcyI-CoAoxidase a Catalase b D.Amino acid oxidase a Urate oxidase b Carnitine acetyltransferase" Carnitine palmitoyltransferase a
1.18+0.23 1.36+0.14 61.9 +5.1 0.55+0.11 3.40+0.15 0.52+0.15 2.45+0.24
(1)
0.61+0.!6 0.66+0.13 0.08+0.01 0.10+0.02
Microsome Laurate 12-hydroxylationc Laurate 11-hydroxylationc 1-AcybGPC aeyltransferase d Carboxylesteraseo Cytosol PalmitoyI-CoAhydrolase d Malic enzymed Glucose-6-phosphate dehydrogenase c Ghtathione S-transferase d
DHEA
CPIB
(1) (1) (1) (1) (1)
9.28+1.43 ** (7.9) 8.77+1.36 ** (6.4) 99.3+6.8 ** (1.6) 0.34+0.11 * (0.6) 2.41+0.26 ** (0,7) 21.47+2.18 ** (41.3) 7.33+0.90 ** (3.0)
12.12+1.09** 10.73+1.36** 96.4 +6.0 ** 0.24+0.16 ** 2.51+0.24 ** 27.08+3.01** 8.48+1.84 **
(10.3) (7.9) (1.5) (0.4) (0.7) (52.1) (3.5)
12.27+2.14** 10.51+1.33"* 92.0 -4-2.7** 0.24+0.13** 2.47+0.52* 31.68+5.64** 9.28+2.81**
(10.4) (7.7) (1.5) (0.4) (0.7) (61.0)
(1) (1) (1) (1)
6.86+2.18 ** (11.2) 1.45+0.25 ** (2.2) 0.20+0.01"* (2.5) 0.14+0.03 * 0.4)
4.76+0.78 ** 1.06"'0.14 ** 0.22+0.03** 0,20+0.03 **
(7.8) (1.6) (2.8) (2.0)
6.49+1.45** 1.18+0.21** 0.21+0.03"* 0.20+0.02 **
(10.6) (1.8) (2.6) (2.0)
0.02 + 0.003 (1) O.Ol+ 0.004 (1)
0.11+0.02 ** (5.5) 0.12+0.01 ** (9.2)
0.15+0.05 ** (7.5) 0.16+0.06 ** (12.1)
0,12+0.02 "* (6.0) 0.19+0.03 ** 04.4)
21.4 +8.0 (1) 1.07+0.12 (1)
22.4 +6,6 (1.1) 0.81+0.08 ** (0.8)
20.6 +5.7 (1.0) 0,83+0.09 ** (0.8)
29.0 +6.8 (1.4) 0.82+0,08"* (0.8)
(1)
a/zmol/min per g liver; b units/g liver; c nmol/min per mg protein; d/tmol/min per mg protein.
DHEA + CPIB
(3.8)
236
(kD)
A
--97.4 --66.2 --42.7
--
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--97.4 --66.2
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zyme activities of both the DHEA- and CPIB-treated groups. No synergysm in the actions of DHEA and CPIB was thus suggested. Exceptionally, the increases in the activities of carnitine acetyltransferase and malic enzyme of the DHEA plus CPIB-treated group were somewhat greater than those of both the DHEA- and CPIB-treated groups. The changes in the hepatic enzyme activities of the female DHEA-treated group were essentially the same as those of the male DHEA-treated group, the extents of which were, however, smaller than in the male group (Table IIl). An exception was the case of o-amino acid oxidase, the activity of which was increased in female rats, but was decreased in male rats by the treatment of DHEA. Nor were there any no notable differences between the DHEA- and CPIB-treated groups of female rats in the changes of the enzyme activities except of D-amino acid oxidase.
Electrophoretic analysis
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Fig. !, SDS-polyacrylamidegel electrophoresisof liver(A), kidney(B) and heart (C) homogenatesobtained from rats treated with DHEA. The homogenates were prepared from the same rats as those described in Table I and electrophoresed(30/~g) protein/lane)on 10~ SDS-polyacrylamidegels. The proteins were stained with Coomassie brilliant blue. Lanes2-5, male rats; lanes 7-9, femalerats; lanes 1 and 10, molecularweightmarkers: lanes2 and 7, control; lanes 3 and 8, DHEA; lanes 4 and 9, CPlB; lane 5, DHEA+CPIB; lane 6, pudfed enoyI-CoAhydratase/3-hydroxyacyl-CoAdehydrogenasebifunctional enzyme(0.6 t~g protein/lane). Molecularweight markers used were rabbit muscle phosphorylaseb (97.4 kDa), bovine serum albumin (66.2 kDa), hen egg white ovalbumin(42.7 kDa), soybean trypsin inhibitor(21.5 kDa) and hen egg white lysozyme(14A kDa). Arrowheadsindicatethe bands of "/8,61, 52, 47 and 37 kDa protein.
Fig. 1 shows electrophoretic profiles of liver, kidney and heart proteins on polyacrylamide gel in the presence of SDS. In the liver of male rats, DHEA-treatment resulted in the induction of the proteins of relative molecular masses of approx. 78, 61, 52, 47 and 37 kDa (Fig. 1A, lane 3). The induction of these proteins were common to the livers of all the treated groups including the female groups (Fig. 1A, lanes 3, 4, 5, 8 and 9). Although the nature of the induced proteins have not been established, the protein band of 78 kDa corresponds to the peroxisome proliferation associated polypeptide PPA-80 [34]. In the kidney, DHEA also increased the protein of 78 kDa, although only slightly. The induction of the other proteins, however, could not be detected from the comparison between the control and the DHEA-treated groups (Fig. 1B). No appreciable changes in the electrophoretic profiles of the heart proteins could be detected in any of the animal groups (Fig. 1C). Thus, the electrophoretic profiles of proteins were all essentially the same in the DHEA-, CPIB- and DHEA plus CPIB-treated groups, when compared to a particular tissue of a particular sex.
Immunoblot analysis
Moreover, there were no differences between the DHEA plus CPlB-treated group and the DHEA-treated group or the CPIB-treated group in the changes in all the enzyme activities examined. Although slight differences could be found, the changes in the individual enzyme activities of the DHEA plus CPIB-treated group were not greater than those in the corresponding en-
The induction of peroxisomal enzymes, catalase, acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme, were examined by immunoblotting (Fig. 2). Consistent with the increased enzyme activities (Tables II and III), the specific contents of these enzymes were increased in the liver of the DHEA-treated groups and the other treated groups of both sexes. The induction of acyl-CoA oxidase and bifunctional enzyme which are the constituents of fl-oxidation system was particularly remarkable (Fig. 2A). These enzymes were also induced in the kidney,
237 T A B L E Ill
Effect of DHEA on hepatic enzyme activities o/female rats Data were obtained from the same rats as those described in Table I and are expressed as the mean+ S.D. of five rats. Values in parentheses refer to increases relative to the control ( * P < 0.05, * * P < 0.01). Enzyme
Control
Liver homogenate Peroxisomal r-oxidation a AcyI-CoA oxidase a Catalase b D-Amino acid oxidase a Urate oxidase b Carnitine acetyltransferase a Carnitine palmitoyltransferase a
1.02 4. 1.43 4. 37.5 + 0.39_+ 4.17_+ 0.68_+ 2.38 4.
0.16 0.11 6.0 0.16 0.94 0.22 0.41
(1) (1) (1) (1) (1) (1) (1)
5.60_+ 5.404. 70.9 4. 0.72_+ 4.674. 11.67+ 4.86 4-
0.41_+ 0.55-40.08 4. 0.06_+
0.06 0.09 0.01 0.02
(1) (1) (1) (1)
1.76-+ 1.144 0.14-+ 0.124-
(1) (1) (1) (1)
0.08 + 0.07+ 41.0 + 1.24+
Microsome Lauratel2-hydroxylation c Laurate 11-hydroxylation ~ 1-AcyI-GPC acyltransferase d Carboxylesterase d Cytosol Palmitoyl-CoA hydrolase d Malic enzyme d Glucose-6-phosphate dehydrogenase c Glutathione S-transferase a
DHEA
0.03_+ 0.01 0.02_+ 0.006 39.4 + 10.8 1.264. 0.27
CPIB 1.41 ** (5.5) 0.91 ** (3.8) 7 . 3 " * (1.9) 0.26 * (1.8) 0.90 (1.1) 2.98 **(17.2) 0.26 ** (2.0) 0.96 * 0.14 ** 0.02 ** 0.02"*
6.82+1.54 ** 6.45+0.81 ** 71.9 +6.9 ** 0.42+0.26 4.87 + 1.28 14.90+3.39 ** 5.64"t"1.00 **
(4.3) (2.1) (1.8) (2.0)
2.77+0.71 1.0140.14 0.15+0.01 0.11 +0.03
0.01 ** (2.7) 0.02 ** (3.5) 10.2 (1.0) 0.27 (1.0)
** ** ** *
(6.7) (4.5) (1.9) (1.1) (1.2) (21.9) (2.4) (6.8) (1.8) (1.9) (1.8)
0.05+0.01 ** (1.7) 0.10+0.03 ** (5.1) 39.4 +9.8 (1.0) 1.05 -+0.09 (0.8)
a lamol/min per g liver; b units/g liver; c nmol/min per mg protein; d lamol/min per mg protein.
b u t t h e e x t e n t o f t h e i n d u c t i o n w a s s m a l l e r t h a n in t h e l i v e r (Fig. 2B). I n t h e h e a r t , the s p e c i f i c c o n t e n t o f t h e s e enzymes was naturally very low and induction could not b e d e t e c t e d in a n y o f t h e t r e a t e d g r o u p s ( F i g . 2C).
A Catalase
Acyl-CoA
6 2 k D - - ~ : ~ : ~ ~
oxidase
21kD-'
enzyme
78kD
The induction of hepatic peroxisome proliferation w a s e x a m i n e d b y e l e c t r o n m i c r o s c o p y (Fig. 3). I n the l i v e r o f c o n t r o l rats ( F i g . 3 A a n d C), a f e w p e r o x i s o m e s
C
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Morphological observation
............... .
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Fig. 2. Immunobloning of catalase, acyl-CoA oxidase and enoyI-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme in the liver (A), kidney (B) and heart (C) of rats treated with DHEA. The samples were obtained from the same rats as those decribed in Table I. Liver (10 lag protein/lane), kidney (50 lag protein/lane) and heart homogenates (50 lag protein/lane) were electrophoresed on 10~ SDS.polyacrylamide gels, and the proteins were transferred to nitrocellulose sheets and treated with rabbit antiserum against rat liver catalase, acyI-CoA oxidase or bifunctional enzyme. The antigen-antibody complexes were visualized by peroxidase.antiperoxidase staining using 3,3'-diaminobenzidine as reagent. The positions of catalase subunit (62 kDa), acyl-CoA oxidase subunits (72 kDa, 52 kDa and 21 kDa) and bifunctional enzyme (78 kDa) are indicated. Lanes 1-4, male rats; lanes 6-8, female rats; lanes 1 and 6, control; lanes 2 and 7, DHEA; lanes 3 and 8, CPIB; lane 4, DHEA+CPIB; lane 5, purified enzyme (0.25 lag protein/lane).
238
Fig. 3. Electron micrographs of the liver from rats treated with DHEA. Male (A and B) and female (C and D) rats were administered 300 mg of DHEA/kg body w[. or vehicle alone for 14 days. The liver blocks were fixed in 2% glutaraldehyde in 0.1 M sodium phosphate (pH 7.4), post-fixed in 2¢~ OsO4 in the same buffer, dehydrated and embedded in epoxy resin. Ultrathin sections were doubly stained with uranyl acetate followed by lead citrate and observed at an accelerating voltage of 75 kV with magnification x 10000. A and C, control; B and D, DHEA-treated. P denotes peroxisomes.
239 with a core, which is the form usually observed in normal rat liver, were seen in a single field. In contrast, numerous peroxisomes were seen in the liver of
DHEA-treated rats (Fig. 3B and D), many of which were without a core and appeared to vary in size. The increase in the number of peroxisomes was greater in
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Fig. 4. Time course of changes in hepatic enzyme activities and liver weight during DHEA treatment. Male rats were treated with 300 mg of D H E A / k g body wt. for the time periods indicated. In some groups of rats, the DHEA treatment was discontinued at day 14 and thereafter only vehicle was administered, o, control; o, DHEA-treated; A DHEA-discontinued. Data represent the mean+S.D, of three rats. A, peroxisomal t-oxidation; B, catalase; C, camitine acetyltransferase; D, carnitine palmitoyltransferase; E, laurate 12.hydroxylation; F, palmitoyI-CoA hydrolase; G, malic enzyme; H, relative liver weight.
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body weight)
241 TABLE IV Relationship among heputic enzyme inductions caused by DHEA Linear regression analyses were performed on the mean data from Table II (control and DHEA) and Fig. 5 (A, C, E, G and H). Values represent correlation coefficients (r) at P < 0.01, except the cases of carnitine acetyltransferase versus laurate 12-hydroxylation and laurate 12-hydroxylation versus malic enzyme (P < 0.05).
Peroxisomal fl-oxidation Carnitine acetyltransferase Laurate 12-hydroxylation Palmitoyl-CoA hydrolase
Malic enzyme
PalmitoyI-CoA hydrolase
Laurate 12-hydroxylation
Carnitine acetyltransferase
0.927 0.967 0.854 0.913
0.954 0.912 0.917
0.948 0.822
0.949
the male group (Fig. 3B) than in the female group (Fig. 3D).
Time course of changes in hepatic enzyme activities Male rats were orally administered 300 mg of D H E A / k g body wt. for time periods of up to 28 days. In some groups of rats, DHEA treatment was discontinued at day 14 and thereafter only vehicle was administered. The changes in the hepatic enzyme activities and the relative liver weight are illustrated in Fig. 4. On day 1 of DHEA treatment, an increase in the activity was already evident for all the enzymes examined. The prolonged period of treatment resulted in progressive increases in the activities which reached the individual maximal levels up to day 14 of the treatment. Prolonging the treatment further over 14 days tended to normalize gradually the increased enzyme activities to the control levels. When DHEA treatment was discontinued, the increased activities returned to the normal level 5-14 days later. As shown in Fig. 4A, peroxisomal fl-oxidation activity also rapidly increased and reached the maximal level (8.7-fold increase) on day 5 of the treatment; the time required to reach half-maximal induction was approx. 1.8 days. When treatment was discontinued, the activity returned to the normal level with an apparent half-life of approx. 1.1 days. Changes in the relative liver weight also followed a time course similar to that of peroxisomal fl-oxidation activity. However, it remained higher than the normal level even 14 days after the discontinuation of DHEA treatment (Fig. 4H). Dose-response relationship for changes in hepatic enzyme activities Male rats were orally administered 50, 100, 150, 200 or 300 mg of D H E A / k g body wt. for 14 days, and the
liver weight and the hepatic enzyme activities were measured (Fig. 5). As shown in Fig. 5A, peroxisomal fl-oxidation activity was significantly increased (3.4-fold) with the lowest dose of DHEA (50 m g / k g body wt.). At the higher doses, the increase in the activity was remarkable and reached an almost maximal level at a dose of 200 m g / k g body wt. Similar dose-dependent manners of induction were found for the other enzymes examined (Fig. 5B-H), although the activities of carnitine acetyltransferase (Fig. 5C) and malic enzyme (Fig. 5H) were still seen to increase even at a dose of 300 mg/kg body Wt.
On the other hand, the activity of cytosolic glutathione S-transferase (Fig. 5I) decreased by 40% at a dose of 50 m g / k g body wt. However, higher doses did not further decrease the activity. No significant changes in the activity of glucose-6-phosphate dehydrogenase were found at any of the dose levels employed (data not shown). As shown in Fig. 5J, the relative liver weight also increased in a dose-dependent manner to reach an approx. 2-fold increase at a dose of 300 mg/kg body wt.
Relationship among the inductions of peroxisomal floxidation and extra-peroxisomal enzymes Linear regression analysis was performed to examine the relationship of the individual enzyme inductions in various levels of induction (Table IV). Statistical analysis revealed high correlations in the inductions of peroxisomal fl-oxidation, carnitine acetyltransferase, microsomal laurate 12-hydroxylation, cytosolic palmitoylCoA hydrolase and malic enzyme. In addition, it was also revealed that the induction of peroxisomal fl-oxidation correlates well to the induction of carnitine palmitoyltransferase (r = 0.904, P < 0.01) amd microsomal 1-acyl-GPC acyltransferase (r = 0.922,
Fig. 5. Dose-response relationship for change in hepatic enzyme activities and liver weight on DHEA-treatment. Male rats were treated with DHEA at the doses indicated for 14 days. Data represent the mean+S.D, of five rats. A, peroxisomal /t-oxidation; B, catalase; C, camitine acetyltransferase; D, carnitine palmitoyltransferase; E, laurate 12-hydroxylation; F, 1-acyI-GPC acyltransferase; G, palmitoyI-CoA hydrolase; H, malic enzyme; I, glutathione S-transferase; J, relative liver weight.
242 P < 0.01), and to the increase in relative fiver weight (r = 0.976, P < 0.01). The correlation coefficient between peroxisomal r-oxidation and acyl-CoA oxidase was r = 0.994 (P < 0.01). Discussion
As reviewed by Reddy and Lalwani [6] and Hawkins et al. [8], treatment of rats with peroxisome proliferators characteristically results in (1) hepatomegaly, (2) proliferation of hepatic peroxisomes without core, which display variation in size, (3) a remarkable induction of peroxisomal /~-oxidation enzymes, which is often accompanied by decrease in urate oxidase and v-amino acid oxidase, (4) co-induction of several extra-peroxisomal enzymes related to lipid metabolism and (5) decreased activities of giutathione-associated enzymes. Moreover, there are (6) sex differences [35-41] and (7) tissue differences [6,8,42] in the sensitivity to peroxisome proliferators in the rat. The present study has demonstrated that DHEA elicits all of these characteristic responses in the rat. Most of peroxisome proliferators possess (8) hypolipidemic properties [6,8,9,33,39] and DHEA is also a hypotrigiyceridemic agent [2,43]. Furthermore, we have found the existence of (9) species differences in the sensitivity to the inductive effect of DHEA (unpublished data). This is also a common characteristic among peroxisome proliferators [6,8,35, 36,40]. Collectively, these findings indicate that DHEA is a typical peroxisome proliferator. Wu et al. [4] have recently reported the effect of DHEA on microsomal cytochrome P-450 system of rat liver, in their report, they showed that DHEA does not induce any of the major forms of cytochrome P-450 which are normally increased by phenobarbital etc. Nor do typical peroxisome proliferators induce the P-450s. The authors also described a remarkable increase in laurate 12-hydroxylation activity; moreover, they confirmed the increase in the P-450IVA1 content. However, whereas the maximal induction of the enzyme at a dose of 160 mg of DHEA/kg body wt., intraperitoneaUy, was observed, a higher dose was required to obtain the maximal induction in our experiments (Fig. 5). This is probably due to the difference in the methods of administration. More recently, also in the fiver of mice fed DHEA, the induction of peroxisome proliferation and several enzymes including peroxisomal bifunctional enzyme, camitine acetyitransferase [5] and malic enzyme [44] have been demonstrated (we also obtained similar results in a study on species differences, unpublished data). Thus, our conclusion is consistent with and supported by the previous studies. In the present study, we used CPIB as a positive control for the peroxisome proliferator. Various enzyme-inducing properties of this drug have been well investigated [6,8]. Considering the time course of the
induction, the dose level required for maximal induction and the potency as an inducer, the properties of DHEA were found to be very similar to those of CPIB. In addition, our results obtained from the experiment of the co-treatment of DHEA and CPIB (Tables II, III and Figs. 1, 2) suggest that a common process may be involved in the mechanism underlying the hepatic enzyme induction by DHEA and CPIB. The dose level (300 mg each/kg body wt.) and time period (14 days) employed in the co-treatment of DHEA and CPIB are sufficient to elicit maximal induction when treated individually (see Figs. 4 and 5). In such a situation, there was no synergism in the induction of the hepatic enzymes. Slightly additive enhancement seen in the activities of carnitine acetyltransferase and malic enzyme may be due to relatively incomplete saturation of the inductions at this dosage of DHEA (see Fig. 5C and H). DHEA and CPIB may well interact with the same cellular site responsible for the induction. The inductive effect of peroxisome proliferators extends to extra-peroxisomai enzymes as well as peroxisomal enzymes. In this study we have also found five kinds of enzyme including the peroxisomal fl-oxic~ation system to be major co-inducible enzymes (T:~ble IV). Considering their physiological functions, a causal relationship could be found for the respective inductions of these enzymes. For instance, increased activity of fatty acid t0-hydroxylation might result in the overload of dicarboxylic acids on the peroxisomal r-oxidation system, leading to the induction of the /I-oxidation enzymes. Increased malic enzyme activity could supply reducing equivalent to the t0-hydroxylation system. However, luther studies are required to establish such a functional relationship as a mechanism of co-induction. While the chemical structures of peroxisome proliferators are extremely diverse [6,8,9,33,39], the analogues of clofibrate, Wy-14,643 and fatty acid constitute three major categories of peroxisome proliferator. The structural requirements of these compounds for peroxisome proliferation are presently obscure. However, some authors have suggested the importance of the carboxyl groups in their structures, which could receive metabolic conversion to the CoA-derivatives to exert the inductive effect [45,46]. Considering this proposal, Ikeda et al. [10] showed that perfluorinated octane sulfonic acid, which cannot be converted to the CoA-derivative, is also a potent peroxisome proliferator, and they suggested that a structure of unmetabolizable hydrophobic anion may be required. This is also the case for peroxisome proliferating tetrazole-~ul-.stituted acetophenone LY-171883 [9]. However, DHEA has no anionic functional groups itself and to our acknowledge the earboxylated metabolites or their CoA-derivatives have not been found in the cell. Besides, the steroidal structure of DHEA is also unique. This structurally new type of peroxisome profiferator would, thus, provide particu-
243 lar information significant to the understanding of the mechanism of induction of peroxisomes. The ultimate form of DHEA triggering the induction should be investigated, including DHEA-sulfate which is a hydrophobic anion, and attention should also be focused on the receptor-mediated mechanism. Since DHEA is a naturally secreted product in mammals, there is a possibility that DHEA might be an endogenous inducer of peroxisomal enzymes. However, considering that a high dosage, as used here, is required to produce induction, the role of DHEA in the physiological regulation of peroxisomal enzymes should be carefully investigated.
Acknowledgments The authors acknowledge the gift of BrMB from Dr. Y. Kawahara (Sankyo Co., Ltd., Tokyo, Japan) and the technical assistance of Miss C. Nakada (Sankyo Co., Ltd., Tokyo, Japan) in the morphological study. This work was partly supported by a grant (No. 02771723) from the Ministry of Education, Science and Culture of Japan, and grants from the Shimabara Science Promotion Foundation and the Science Research Promotion Fund of Japan Private School Promotion Foundation. References 1 Schwartz, A. (1985) Basic Life Sci. 35, 181-191. 2 Regelson, W., Loria, R. and Kalimi, M. (1988) Ann. N.Y. Acad. Sci. 521, 260-273. 3 Leighton, B., Tagliaferro, A.R. and Newsholme, E.A. (1987) J. Nutn 117, 1287-1290. 4 Wu, H.-Q., Masset-Brown, J., Tweedie, DJ., Milewich, L., Frenkel, R.A., Martin-Wixtrom, C., Estabrook, R.W. and Prough, R.A. (1989) Cancer Res. 49, 2337-2343. 5 Frenkel, R.A., Slaughter, C.A., Orth, K., Moomaw, C.R., Hicks, S.H., Snyder, J.M., Bennett, M., Prough, R.A., Putnam, R.S. and Milewich, L. (1990) J. Steroid Biochem. 35, 33-342. 6 Reddy, J.K. and Lalwani, N.D. (1983) CRC Crit. Rev. Toxicol. 12, 1-58.
7 Kawashima, Y., Katoh, H., Nakajima, S., Kozuka, H. and Uchiyama, M. (1984) Bochem. Pharmacol. 33, 241-245. 8 Hawkins, J.M., Jones, W.E., Bonner, F.W. and Gibson, G.G. (1987) Drug Metal). Rev. 18, 441-515. 9 Eacho, P.I., Foxworthy, P.S., Johnson, W.D., Hoover, D.M. and White, S.L. (1986) Toxicol. Appl. Pharmacr,!o 83, 430-437. 10 Ikeda, T., Fukuda, K., Mori, I., Enomoto, M., Komai, T. and Suga, T. (1987) in Peroxisomes in Biology and Medicine (Fahimi, H.D. and Sies, H., eds.), pp. 304-308, Springer-Verlag, Bedim 11 Lowry, O.H., Rosebrough, N.J., Farr, A.L and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 12 Aebi, H. (1974) in Methods of Enzymatic Analysis, 2nd English Edn. (Bergmeyer, H.U., ed.), Vol. 2, pp. 673-678, Verlag Chemie, Weinheim. 13 Hayashi, H., Suga, T. and Niinobe, S. (1971) Biochim. Biophys. Acta 252, 58-68. 14 Lazarow, P.B. and de Duve, C. (1976) Proc. Natl. Acad. Sci. USA 73, 2043-2046. 15 Yamada, J., Ogawa, S., Horie, S., Watanabe, T. and Suga, T. (1987) Biochim. Biophys. Acta 921, 292-301.
16 Osumi, T., Hashimoto, T. and Ui, N. (1980) J. Biochem. 87, 1735-1746. 17 Markwell, M.A.K., McGroarty, E.J., Bieber, L.L and Tolbert, N.E. (1973) J. Biol. Chem. 248, 3426-3432. 18 Parker, G.L. and Orion, T.C. (1980) in Biochemistry, Biophysics and Regulation of Cytochrome /'-450 (Gustafsson, J.A., Duke, S.C., Mode, A. and Rafter, J., eds.), pp. 373-377, Elsevier/North Holland Biomedical Press, Amsterdam. 19 Lands, W.E.M. and Hart, P. (1965) J. Biol. Chem. 240, 1905-1911. 20 Kawashima, Y., Hirose, A. and Kozuka, H. (1984) Biochim. Biophys. Acta 793, 232-237. 21 Heymann, E., Mentlein, g. and Rix, H. (1981) Methods Enzymol. 77, 405-409. 22 Hosokawa, M., Maki, T. and Satoh, T. (1990) Arch. Biochem. Biophys. 277, 219-227. 23 Kawashima, Y., Katoh, H. and Kozuka, H. (1982) Biochim. BiGphys. Acta 712, 48-56. 24 Habig, W.H., Pabst, M.J. and Jakoby, W.R. (1974) J. Biol. Chem. 249, 7130-7139. 25 Zelewski, M. and Swierczynski, J. (1983) Biochim. Biophys. Acta 758, 152-157. 26 Deutsch, J. (1983) in Methods of Enzymatic Analysis, 3rd English Edn. (Bergmeyer, H.U., ed.), VoL 3. pp. 190-197, Verlag Chemic, Weinheim. 27 Laemmli, U.K. (1970) Nature 227, 680-685. 28 Towbin, H., Staehelm, T. and Gordon, J. (1979) Pro¢. Nail. Acad. Sci. USA 76, 4350-4354. 29 Guengerich, F.P., Wray, P. and Davidson, N.K. (1982) Biochemistry 21, 1698-1701. 30 Leighton, F., Poole, B., Lazarow, P.B. and De Duve, C. (1969) J. Cell Biol. 41, 521-535. 31 Ma:inferme, F. and Wattiaux, R. (1982) Eur. J. Biochem. 127, 343-346. 32 Osumi, T. and Hashimoto, T. (1979) Biochem. Biophys. Res. Commun. 89, 580-584. 33 Ikeda, T., Ida-Enomoto, M., Mori, I., Fukuda, K., Iwabuchi, H., Komai, T. and Suga, T. (1988) Xenobiotica 18, 1271-1280. 34 Reddy, J.K. and Kumar, N.S. (1977) Biochem. Biophys. Res. Commun. 77, 824-829. 35 Svoboda, D., Grady, H. and Azarnoff, D. (1967) J. Cell Biol. 35, 127-152. 36 Osumi, T. and Hashimoto, T. (1978) J. Biochem. 83, 1361-1365. 37 Gray, R.H. and de la Iglesia, F.A. (1984) Hepatology 4, 520-530. 38 Mitchell, F.E., Price, S.C., Hinton, R.H., Grasso, P. and Bridges, J.W. (1985) Toxicol. Appl. Pharmacol. 81, 371-392. 39 Hertz, R., Bar-Tana, J., Sujatta, M., Pill, J., Schmidt, F.H. and Fahimi, tl.D. (1988) Biochem. Pharmacol. 37, 3571-3577. 40 Watanabe, T., Horie, S., Yamada, J., lsaji, M., Nishigaki, T., Naito, J. and Suga, T. (1989) Biochem. Pharmacol. 38, 367-371. 41 Kawashima, Y., Uy-Yu, N. and Kozuka, H. (1989) Biochem. J. 261, 595-600. 42 Nemali, M.R., Usuda, N., Reddy, M.K., Oyasu, K., Hashimoto, T., Osumi, T., RaG, M.S. and Reddy, J.K. (1988) Cancer Res. 48, 5316-5324. 43 TaglJaferrow, A.R., Davis, J.R., Truchon, S. and Van Hamont, N. (1986) J. Nutr. 116, 1977-1983. 44 Marrero, M., Prough, R.A., Frenkel, R.A. and Milewich, L. (1990) Proc. Soc. Exptl. Biol. Med. 193, 110-117. 45 Berge, R.K., Aarsland, A., Osmundsen, H., Aarsaether, N. and Male, R. (1987) in Peroxisomes in Biology and Medicine (Fahimi, H.D. and Sies, H., eds.), pp. 273-278, Springer-Verlag, Berlin. 46 Bronfman, M., Amigo, L. and Morales, M.N. (1986) Biochem. J. 239, 781-784.
233
© 1991 ElsevierSciencePublishersB.V.0167-4889/91/$03.50 ADONIS 016748899100148N BBAMCR12913
Characteristics of dehydroepiandrosterone as a peroxisome proliferator Junji Yamada 1, Mitsuhiro-Sakuma 1, Toshihiko Ikeda and Tetsuya Suga 1
2, Kuniaki
Fukuda 3
I Department of Clinical Biochemistry, Tokyo College of Pharmacy. Tokyo (Japan). and " Analytical and Metabolic Research Laboratories, Tokyo (Japan) and ~ Biological Research Laboratories. Sonkyo Co.. Tokyo (Japan)
(Received25 October1990)
Key words: Dehydroepiandrosterone;Peroxisomeproliferation;Enzymeinduction;(Rat liver) Treatment of rats with dehydroepiandrosterone (300 m g / k g body weight, per os, 14 days) caused a remarkable increase in the number of peroxisomes and peroxisomal t-oxidation activity in the liver. The activities of carnitine acetyitransferase, microsomal laurate 12-hydmxylation, cytosolic palmitoyI-CoA hydrolase, malic enzyme and some other enzymes were also increased. The increases in these enzyme activities were all greater in male rats than in female rats. Immunoblot analysis revealed remarkable induction of acyi-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyi-CoA dehydrogenase bifunctional enzyme in the liver and to a smaller extent in the kidney, whereas no significant inductiol~ of these enzymes was found in the heart. The increase in the hepatic peroxisomal /]-oxidation activity reached a maximal level at day 5 of the treatment of dehydroepiandrosterone and the increased activity rapidly returned to the ~:,,rmal level on discontinuation of the treatment. The increase in the activity was also dose-dependent, which was ~aturable at a dose of more than 200 m g / k g body weight. All these features in enzyme induction caused by dehydroepiandrosterone correlate well with those observed in the treatment of dofibric acid, a peroxisome proliferator. Co-treatment of dehydtoepiandrosterone and clofibric acid showed no synergism in the enhancement of peroxisomai /]-oxidation activity, suggesting the involvement of a common process in the mechanism by which these compounds induce the enzymes. These results indicate that dehydroepiandrosterone is a typical peroxisome proliferator. Since dehydroepiandrosterone is a naturally oecu.,'ing C 19 steroid in mammals, the structure of which is novel compared with those of peroxisome proliferators known so far, this compound could provide particular information in the understanding of the mechanisms underlying the induction of peroxisome proliferation.
Introduction
Dehydroepiandrosterone (DHEA) is a naturally occurring C~0 steroid in mammals, which is known to exert various actions such as antiobesity, antidiabetic and anticarcinogenic actions when administered to rats and mice [1,2]. In 1987 Leighton et al. [3] reported that DHEA-feeding of rats increased hepatic catalase and acyl-CoA oxidase activities. Although there have been a few more reports [4,5] dealing with the induction of peroxisomal enzymes by DHEA in the literature, the
Abbreviations: DHEA,dehydroepiandrosterone;CPIB, clofibricacid; GPC, glycerophosphocholine;SDS, sodiumdodecylsulfate. Correspondence: J. Yamada, Department of Clinical Biochemistry, Tokyo College of Pharmacy, 1432-1 Horinonchi, Hachioji, Tokyo 192-03, Japan.
characteristics of DHEA as a peroxisome proliferator have not yet been clarified sufficiently. Peroxisome proliferators, including hypolipidemic drugs, phthalate ester plasticizers and agricultural chemicals [6-8], are structurally diverse and the structural requirements for peroxisome proliferation are, therefore, poorly understood. Although the mechanism by which these compounds induce peroxisome proliferation has not been established, it is now proposed that a structure of unmetabolizable hydrophobic anion may be required to be a potent peroxisome proliferator [9,10]. The steroidal structure of DHEA is novel, as compared with the peroxisome proliferators known so far, and DHEA has no anionic functional groups itself. We are interested in DHEA as a structurally new type of peroxisome proliferator and as a possible endogenous inducer of peroxisomal enzymes. In the present study, to elucidate the characteristics
234 of DHEA as a peroxisome proliferator we investigated the in vivo effect of DHEA on various enzymes including extra-peroxisomal enzymes, using clofibric acid (CPIB), a typical peroxisome proliferator, as a positive control. Sex differences and tissue-differences in the effect were also investigated. Materials and Methods Materials, DHEA was purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan). CPIB was purchased from Sigma Chemical Co. (St. Louis, MO). BrMB (3-bromomethyi-7-methoxy-l,4-benzoxazin-2-one) was kindly provided by Dr. Y. Kawahara (Sankyo Co., Ltd., Tokyo, Japan). Butanilicaine (N-butylaminoacetyl-2-chloro-6methylanilide) and L.carnitine were donated by Hoechst AG (Frankfurt, F.R.G.) and Earth Pharmaceutical Co. (Akoh, Japan), respectively. All other chemicals were of reagent grade. Animals and treatments. Male and female Wistar rats, 7 weeks of age, weighing about 200 g and 180 g, respectively, at the beginning of the experiments, were used, The animals were maintained on water and standard CE-II chow (Nihon Clea Inc., Tokyo, Japan) ad libitum, DHEA was suspended in 0.5% carboxymethylcellulose, and CPIB was dissolved in the same vehicle as the sodium salt. The individual agents were administered by gastric intubation once a day at the doses and for the time periods indicated in the text. The control animals received vehicle only (5 ml/kg body wt.). The animals were killed 24 h after the last administration. Enzyme preparations. The animals were anesthetized with diethyl ether and the livers were perfused in situ with saline through the portal vein. The livers and other tissues were excised, rinsed in 0.25 M sucrose, blotted and weighed, Liver homogenates were prepared in 0.25 M sucrose containing 1 mM EDTA, 10 mM Tris-HCI (pH 7.5) and 0,1~ ethanol, Liver microsomes and cytosol were prepared as follows: after centrifugation of the
liver homogenates at 25 000 × g for 10 rain, the result!rig supernatants were further centrifuged at 105 000 × g for 60 min. The supernatants obtained were used as cytosol. The precipitates obtained were resuspended in 1.15% KCI and centrifuged again at 105 000 x g for 60 min. The resulting precipitates were suspended in 0.1 M phosphate buffer (pH 7.5) containing 1 mM EDTA and used as mierosomes. These preparations were used for enzyme assay, For electrophoresis and immunoblotting, the livers, renal cortices and hearts were homogenized in 50 mM Tris-HC1 (pH 8.0) containing 1 mM EDTA, 5 /Lg/ml leupeptine and 0.3 mM phenylmethylsulfonyl fluoride. Protein was determined by the method of Lowry et al. [11] with bovine serum albumin as a standard. Enzyme assays. The activities of catalase [12], D-amino acid oxidase [13], urate oxidase [13], peroxisomal floxidation [14,15], acyl-CoA oxidase [15,16], carnitine acetyltransferase [17] and carnitine palmitoyltransferase [17] were measured in liver homogenates. Peroxisomal ~-oxidation and acyl-CoA oxidase were assayed with lauroyl-CoA as the substrate. Microsomal laurate l 1and 12-hydroxylation were assayed by the method of Parker and Orton [18]. In this assay, unlabeled laurate instead of 1-t4C-labeled laurate was used as the substrate. The reaction products were converted to the fluorescent derivatives with BrMB (3-bromomethyl-% methoxy-l,4-benzoxazin-2-one) and subjected to HPLC analysis. The detailed procedure will be described elsewhere (unpublished data). The activity of microsomal 1-acyl-GPC acyltransferase [19,20] was measured with oleoyl-CoA and 1-palmitoyl GPC. Microsomal carboxylesterase [21,22] was assayed with butanilicaine (N-butylaminoacetyl-2-ehloro-6-methylanilide) as the substrate. The activities of palmitoyl-CoA hydrolase [23], glutathione S-transferase [24] toward 1-chloro-2,4dinitrobenzene, malic enzyme (decarboxylating) [25] and glucose-6-phosphate dehydrogenase [26] were measured in liver cytosol.
TABLE l Effect of DHEA on body weightgain, liver weight and hepaticprotein content
Male and femalerats were treated with300 nagof DHEA/kg body wt. (DHEA), 300 mg of CPIB/kg body wt. (CPIB)or 300 mg each of DHEA and CP1B/k8 bodywt. (DHEA+CPlB) for 14 days. Data are expressedas the meand:S.D. of fiverats. Valuesin parenthesesrefer to the increases relative to the control in each sex ( * P < 0.05, * * P < 0.01). Control
DHEA
CPIB
DHEA+ CPIB
Male Bodyweightgain(g) Liverweight($ of bodywt,) Liverprotein(mg/g liver)
92 4-19 (1) 3.44- 0.1 (1) 204 4-10 (1)
87 4-9 (0.9) 6.24- 0.6 * * (1.8) 206 4- 7 (1.0)
91 +17 (1.0) 5.5 + 0.4 * * (1.6) 216 +12 (1.1)
87 + 7 (0.9) 6.9+ 0.9 * * (2.0) 196 + 13 (1.0)
Female Bodyweightgain(g) Liverweight(%of bodywt.) Liverprotein(mg/g liver)
47 +10 (1) 3.3+ 0.1 (1) 206 4-16 (1)
48 4-12 (1.0) 6.04- 0.5 **(1.8) 226 4- 5 * (1.1)
43 +11 (0.9) 4.6+ 0.3 **(1.4) 216 + 4 (1.0)
235
Other methods. Polyacrylamide gel electrophoresis was performed in the presence of sodium dodecyl sulfate as described by Laemmli [27] with 10% acrylamide gel. Immunoehemical staining was carried out b y the method of Towbin et al. [28] with the modification of Guengerich et al. [29]. Catalase [30,31], acyl-CoA oxidase [16] and enoyl-CoA h y d r a t a s e / 3 - h y d r o x y a c y l - C o A dehydrogenase bifunctional enzyme [32] were purified from rat liver according to the published procedures, and antibodies against the individual enzymes were raised in female Japanese White rabbits [22]. Electron microscopy was carried out as described previously [331. Statistical analyses were performed using Student's t-test. Results
Effect of DHEA in male and female rats Male and female rats were orally administered 300 m g of D H E A / k g b o d y wt., 300 mg of C P I B / k g body wt. or 300 mg each of D H E A and C P I B / k g body wt. for 14 days. Body weight and liver weight As shown in Table I, body weight gains of all the animals in the treated groups were similar to those in the control group in both males and females. Decrease
in b o d y weight gain due to the treatment of D H E A was not observed in either sex. In all the treated groups, hepatomegaly occurred as indicated by 1.4- to 2.0-fold increase in the relative liver weight, while the protein content per liver was almost equal to the control value.
Hepatic enzyme activities T h e effect of D H E A on the hepatic enzyme activities of male rats are summarized in Table II. D H E A increased the activities of peroxisomal/t-oxidation, acylC o A oxidase and catalase, 7.9-, 6.4- and 1.6-fold, respectively, and decreased D-amino acid oxidase and urate oxidase by 40% and 30%, respectively. These changes in peroxisomal enzyme activities were consistent with those caused b y CPIB. The activities of several extra-peroxisomal enzymes were also increased. A marked increase was found in the activities of earnitine acetyltransferase, microsomal laurate 12-hydroxylation, cytosolic palmitoyl-CoA hydrolase and malic enzyme. Carnitine palmitoyltransferase which is a mitochondrial enzyme, and laurate ll-hydroxylation, 1-aeylG P C acyitransferase and carboxylesterase in microsomes were moderately increased. Cytosolic glutahtione S-transferase activity was, however, decreased b y 20%. The activity of glueose-6-phosphate dehydrogenase was not changed. All these changes in the activities of extra-peroxisomal enzymes were also consistent with those of the CPIB-treated group.
TABLE II
Effect of DHEA on hepatic enzyme activitiesof male rats Data were obtained from the same rats as those described in Table I and are expressed as tbe mean + S.D. of five rats. Values in parentheses refer to increases relative to the control ( * P < 0.05, * * P < 0.01). Enzyme
Control
Liver homogenate Peroxisomal )8.oxidationa AcyI-CoAoxidase a Catalase b D.Amino acid oxidase a Urate oxidase b Carnitine acetyltransferase" Carnitine palmitoyltransferase a
1.18+0.23 1.36+0.14 61.9 +5.1 0.55+0.11 3.40+0.15 0.52+0.15 2.45+0.24
(1)
0.61+0.!6 0.66+0.13 0.08+0.01 0.10+0.02
Microsome Laurate 12-hydroxylationc Laurate 11-hydroxylationc 1-AcybGPC aeyltransferase d Carboxylesteraseo Cytosol PalmitoyI-CoAhydrolase d Malic enzymed Glucose-6-phosphate dehydrogenase c Ghtathione S-transferase d
DHEA
CPIB
(1) (1) (1) (1) (1)
9.28+1.43 ** (7.9) 8.77+1.36 ** (6.4) 99.3+6.8 ** (1.6) 0.34+0.11 * (0.6) 2.41+0.26 ** (0,7) 21.47+2.18 ** (41.3) 7.33+0.90 ** (3.0)
12.12+1.09** 10.73+1.36** 96.4 +6.0 ** 0.24+0.16 ** 2.51+0.24 ** 27.08+3.01** 8.48+1.84 **
(10.3) (7.9) (1.5) (0.4) (0.7) (52.1) (3.5)
12.27+2.14** 10.51+1.33"* 92.0 -4-2.7** 0.24+0.13** 2.47+0.52* 31.68+5.64** 9.28+2.81**
(10.4) (7.7) (1.5) (0.4) (0.7) (61.0)
(1) (1) (1) (1)
6.86+2.18 ** (11.2) 1.45+0.25 ** (2.2) 0.20+0.01"* (2.5) 0.14+0.03 * 0.4)
4.76+0.78 ** 1.06"'0.14 ** 0.22+0.03** 0,20+0.03 **
(7.8) (1.6) (2.8) (2.0)
6.49+1.45** 1.18+0.21** 0.21+0.03"* 0.20+0.02 **
(10.6) (1.8) (2.6) (2.0)
0.02 + 0.003 (1) O.Ol+ 0.004 (1)
0.11+0.02 ** (5.5) 0.12+0.01 ** (9.2)
0.15+0.05 ** (7.5) 0.16+0.06 ** (12.1)
0,12+0.02 "* (6.0) 0.19+0.03 ** 04.4)
21.4 +8.0 (1) 1.07+0.12 (1)
22.4 +6,6 (1.1) 0.81+0.08 ** (0.8)
20.6 +5.7 (1.0) 0,83+0.09 ** (0.8)
29.0 +6.8 (1.4) 0.82+0,08"* (0.8)
(1)
a/zmol/min per g liver; b units/g liver; c nmol/min per mg protein; d/tmol/min per mg protein.
DHEA + CPIB
(3.8)
236
(kD)
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--97.4 --66.2 --42.7
--
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zyme activities of both the DHEA- and CPIB-treated groups. No synergysm in the actions of DHEA and CPIB was thus suggested. Exceptionally, the increases in the activities of carnitine acetyltransferase and malic enzyme of the DHEA plus CPIB-treated group were somewhat greater than those of both the DHEA- and CPIB-treated groups. The changes in the hepatic enzyme activities of the female DHEA-treated group were essentially the same as those of the male DHEA-treated group, the extents of which were, however, smaller than in the male group (Table IIl). An exception was the case of o-amino acid oxidase, the activity of which was increased in female rats, but was decreased in male rats by the treatment of DHEA. Nor were there any no notable differences between the DHEA- and CPIB-treated groups of female rats in the changes of the enzyme activities except of D-amino acid oxidase.
Electrophoretic analysis
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Fig. !, SDS-polyacrylamidegel electrophoresisof liver(A), kidney(B) and heart (C) homogenatesobtained from rats treated with DHEA. The homogenates were prepared from the same rats as those described in Table I and electrophoresed(30/~g) protein/lane)on 10~ SDS-polyacrylamidegels. The proteins were stained with Coomassie brilliant blue. Lanes2-5, male rats; lanes 7-9, femalerats; lanes 1 and 10, molecularweightmarkers: lanes2 and 7, control; lanes 3 and 8, DHEA; lanes 4 and 9, CPlB; lane 5, DHEA+CPIB; lane 6, pudfed enoyI-CoAhydratase/3-hydroxyacyl-CoAdehydrogenasebifunctional enzyme(0.6 t~g protein/lane). Molecularweight markers used were rabbit muscle phosphorylaseb (97.4 kDa), bovine serum albumin (66.2 kDa), hen egg white ovalbumin(42.7 kDa), soybean trypsin inhibitor(21.5 kDa) and hen egg white lysozyme(14A kDa). Arrowheadsindicatethe bands of "/8,61, 52, 47 and 37 kDa protein.
Fig. 1 shows electrophoretic profiles of liver, kidney and heart proteins on polyacrylamide gel in the presence of SDS. In the liver of male rats, DHEA-treatment resulted in the induction of the proteins of relative molecular masses of approx. 78, 61, 52, 47 and 37 kDa (Fig. 1A, lane 3). The induction of these proteins were common to the livers of all the treated groups including the female groups (Fig. 1A, lanes 3, 4, 5, 8 and 9). Although the nature of the induced proteins have not been established, the protein band of 78 kDa corresponds to the peroxisome proliferation associated polypeptide PPA-80 [34]. In the kidney, DHEA also increased the protein of 78 kDa, although only slightly. The induction of the other proteins, however, could not be detected from the comparison between the control and the DHEA-treated groups (Fig. 1B). No appreciable changes in the electrophoretic profiles of the heart proteins could be detected in any of the animal groups (Fig. 1C). Thus, the electrophoretic profiles of proteins were all essentially the same in the DHEA-, CPIB- and DHEA plus CPIB-treated groups, when compared to a particular tissue of a particular sex.
Immunoblot analysis
Moreover, there were no differences between the DHEA plus CPlB-treated group and the DHEA-treated group or the CPIB-treated group in the changes in all the enzyme activities examined. Although slight differences could be found, the changes in the individual enzyme activities of the DHEA plus CPIB-treated group were not greater than those in the corresponding en-
The induction of peroxisomal enzymes, catalase, acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme, were examined by immunoblotting (Fig. 2). Consistent with the increased enzyme activities (Tables II and III), the specific contents of these enzymes were increased in the liver of the DHEA-treated groups and the other treated groups of both sexes. The induction of acyl-CoA oxidase and bifunctional enzyme which are the constituents of fl-oxidation system was particularly remarkable (Fig. 2A). These enzymes were also induced in the kidney,
237 T A B L E Ill
Effect of DHEA on hepatic enzyme activities o/female rats Data were obtained from the same rats as those described in Table I and are expressed as the mean+ S.D. of five rats. Values in parentheses refer to increases relative to the control ( * P < 0.05, * * P < 0.01). Enzyme
Control
Liver homogenate Peroxisomal r-oxidation a AcyI-CoA oxidase a Catalase b D-Amino acid oxidase a Urate oxidase b Carnitine acetyltransferase a Carnitine palmitoyltransferase a
1.02 4. 1.43 4. 37.5 + 0.39_+ 4.17_+ 0.68_+ 2.38 4.
0.16 0.11 6.0 0.16 0.94 0.22 0.41
(1) (1) (1) (1) (1) (1) (1)
5.60_+ 5.404. 70.9 4. 0.72_+ 4.674. 11.67+ 4.86 4-
0.41_+ 0.55-40.08 4. 0.06_+
0.06 0.09 0.01 0.02
(1) (1) (1) (1)
1.76-+ 1.144 0.14-+ 0.124-
(1) (1) (1) (1)
0.08 + 0.07+ 41.0 + 1.24+
Microsome Lauratel2-hydroxylation c Laurate 11-hydroxylation ~ 1-AcyI-GPC acyltransferase d Carboxylesterase d Cytosol Palmitoyl-CoA hydrolase d Malic enzyme d Glucose-6-phosphate dehydrogenase c Glutathione S-transferase a
DHEA
0.03_+ 0.01 0.02_+ 0.006 39.4 + 10.8 1.264. 0.27
CPIB 1.41 ** (5.5) 0.91 ** (3.8) 7 . 3 " * (1.9) 0.26 * (1.8) 0.90 (1.1) 2.98 **(17.2) 0.26 ** (2.0) 0.96 * 0.14 ** 0.02 ** 0.02"*
6.82+1.54 ** 6.45+0.81 ** 71.9 +6.9 ** 0.42+0.26 4.87 + 1.28 14.90+3.39 ** 5.64"t"1.00 **
(4.3) (2.1) (1.8) (2.0)
2.77+0.71 1.0140.14 0.15+0.01 0.11 +0.03
0.01 ** (2.7) 0.02 ** (3.5) 10.2 (1.0) 0.27 (1.0)
** ** ** *
(6.7) (4.5) (1.9) (1.1) (1.2) (21.9) (2.4) (6.8) (1.8) (1.9) (1.8)
0.05+0.01 ** (1.7) 0.10+0.03 ** (5.1) 39.4 +9.8 (1.0) 1.05 -+0.09 (0.8)
a lamol/min per g liver; b units/g liver; c nmol/min per mg protein; d lamol/min per mg protein.
b u t t h e e x t e n t o f t h e i n d u c t i o n w a s s m a l l e r t h a n in t h e l i v e r (Fig. 2B). I n t h e h e a r t , the s p e c i f i c c o n t e n t o f t h e s e enzymes was naturally very low and induction could not b e d e t e c t e d in a n y o f t h e t r e a t e d g r o u p s ( F i g . 2C).
A Catalase
Acyl-CoA
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21kD-'
enzyme
78kD
The induction of hepatic peroxisome proliferation w a s e x a m i n e d b y e l e c t r o n m i c r o s c o p y (Fig. 3). I n the l i v e r o f c o n t r o l rats ( F i g . 3 A a n d C), a f e w p e r o x i s o m e s
C
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Morphological observation
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Fig. 2. Immunobloning of catalase, acyl-CoA oxidase and enoyI-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme in the liver (A), kidney (B) and heart (C) of rats treated with DHEA. The samples were obtained from the same rats as those decribed in Table I. Liver (10 lag protein/lane), kidney (50 lag protein/lane) and heart homogenates (50 lag protein/lane) were electrophoresed on 10~ SDS.polyacrylamide gels, and the proteins were transferred to nitrocellulose sheets and treated with rabbit antiserum against rat liver catalase, acyI-CoA oxidase or bifunctional enzyme. The antigen-antibody complexes were visualized by peroxidase.antiperoxidase staining using 3,3'-diaminobenzidine as reagent. The positions of catalase subunit (62 kDa), acyl-CoA oxidase subunits (72 kDa, 52 kDa and 21 kDa) and bifunctional enzyme (78 kDa) are indicated. Lanes 1-4, male rats; lanes 6-8, female rats; lanes 1 and 6, control; lanes 2 and 7, DHEA; lanes 3 and 8, CPIB; lane 4, DHEA+CPIB; lane 5, purified enzyme (0.25 lag protein/lane).
238
Fig. 3. Electron micrographs of the liver from rats treated with DHEA. Male (A and B) and female (C and D) rats were administered 300 mg of DHEA/kg body w[. or vehicle alone for 14 days. The liver blocks were fixed in 2% glutaraldehyde in 0.1 M sodium phosphate (pH 7.4), post-fixed in 2¢~ OsO4 in the same buffer, dehydrated and embedded in epoxy resin. Ultrathin sections were doubly stained with uranyl acetate followed by lead citrate and observed at an accelerating voltage of 75 kV with magnification x 10000. A and C, control; B and D, DHEA-treated. P denotes peroxisomes.
239 with a core, which is the form usually observed in normal rat liver, were seen in a single field. In contrast, numerous peroxisomes were seen in the liver of
DHEA-treated rats (Fig. 3B and D), many of which were without a core and appeared to vary in size. The increase in the number of peroxisomes was greater in
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Fig. 4. Time course of changes in hepatic enzyme activities and liver weight during DHEA treatment. Male rats were treated with 300 mg of D H E A / k g body wt. for the time periods indicated. In some groups of rats, the DHEA treatment was discontinued at day 14 and thereafter only vehicle was administered, o, control; o, DHEA-treated; A DHEA-discontinued. Data represent the mean+S.D, of three rats. A, peroxisomal t-oxidation; B, catalase; C, camitine acetyltransferase; D, carnitine palmitoyltransferase; E, laurate 12.hydroxylation; F, palmitoyI-CoA hydrolase; G, malic enzyme; H, relative liver weight.
240 03
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241 TABLE IV Relationship among heputic enzyme inductions caused by DHEA Linear regression analyses were performed on the mean data from Table II (control and DHEA) and Fig. 5 (A, C, E, G and H). Values represent correlation coefficients (r) at P < 0.01, except the cases of carnitine acetyltransferase versus laurate 12-hydroxylation and laurate 12-hydroxylation versus malic enzyme (P < 0.05).
Peroxisomal fl-oxidation Carnitine acetyltransferase Laurate 12-hydroxylation Palmitoyl-CoA hydrolase
Malic enzyme
PalmitoyI-CoA hydrolase
Laurate 12-hydroxylation
Carnitine acetyltransferase
0.927 0.967 0.854 0.913
0.954 0.912 0.917
0.948 0.822
0.949
the male group (Fig. 3B) than in the female group (Fig. 3D).
Time course of changes in hepatic enzyme activities Male rats were orally administered 300 mg of D H E A / k g body wt. for time periods of up to 28 days. In some groups of rats, DHEA treatment was discontinued at day 14 and thereafter only vehicle was administered. The changes in the hepatic enzyme activities and the relative liver weight are illustrated in Fig. 4. On day 1 of DHEA treatment, an increase in the activity was already evident for all the enzymes examined. The prolonged period of treatment resulted in progressive increases in the activities which reached the individual maximal levels up to day 14 of the treatment. Prolonging the treatment further over 14 days tended to normalize gradually the increased enzyme activities to the control levels. When DHEA treatment was discontinued, the increased activities returned to the normal level 5-14 days later. As shown in Fig. 4A, peroxisomal fl-oxidation activity also rapidly increased and reached the maximal level (8.7-fold increase) on day 5 of the treatment; the time required to reach half-maximal induction was approx. 1.8 days. When treatment was discontinued, the activity returned to the normal level with an apparent half-life of approx. 1.1 days. Changes in the relative liver weight also followed a time course similar to that of peroxisomal fl-oxidation activity. However, it remained higher than the normal level even 14 days after the discontinuation of DHEA treatment (Fig. 4H). Dose-response relationship for changes in hepatic enzyme activities Male rats were orally administered 50, 100, 150, 200 or 300 mg of D H E A / k g body wt. for 14 days, and the
liver weight and the hepatic enzyme activities were measured (Fig. 5). As shown in Fig. 5A, peroxisomal fl-oxidation activity was significantly increased (3.4-fold) with the lowest dose of DHEA (50 m g / k g body wt.). At the higher doses, the increase in the activity was remarkable and reached an almost maximal level at a dose of 200 m g / k g body wt. Similar dose-dependent manners of induction were found for the other enzymes examined (Fig. 5B-H), although the activities of carnitine acetyltransferase (Fig. 5C) and malic enzyme (Fig. 5H) were still seen to increase even at a dose of 300 mg/kg body Wt.
On the other hand, the activity of cytosolic glutathione S-transferase (Fig. 5I) decreased by 40% at a dose of 50 m g / k g body wt. However, higher doses did not further decrease the activity. No significant changes in the activity of glucose-6-phosphate dehydrogenase were found at any of the dose levels employed (data not shown). As shown in Fig. 5J, the relative liver weight also increased in a dose-dependent manner to reach an approx. 2-fold increase at a dose of 300 mg/kg body wt.
Relationship among the inductions of peroxisomal floxidation and extra-peroxisomal enzymes Linear regression analysis was performed to examine the relationship of the individual enzyme inductions in various levels of induction (Table IV). Statistical analysis revealed high correlations in the inductions of peroxisomal fl-oxidation, carnitine acetyltransferase, microsomal laurate 12-hydroxylation, cytosolic palmitoylCoA hydrolase and malic enzyme. In addition, it was also revealed that the induction of peroxisomal fl-oxidation correlates well to the induction of carnitine palmitoyltransferase (r = 0.904, P < 0.01) amd microsomal 1-acyl-GPC acyltransferase (r = 0.922,
Fig. 5. Dose-response relationship for change in hepatic enzyme activities and liver weight on DHEA-treatment. Male rats were treated with DHEA at the doses indicated for 14 days. Data represent the mean+S.D, of five rats. A, peroxisomal /t-oxidation; B, catalase; C, camitine acetyltransferase; D, carnitine palmitoyltransferase; E, laurate 12-hydroxylation; F, 1-acyI-GPC acyltransferase; G, palmitoyI-CoA hydrolase; H, malic enzyme; I, glutathione S-transferase; J, relative liver weight.
242 P < 0.01), and to the increase in relative fiver weight (r = 0.976, P < 0.01). The correlation coefficient between peroxisomal r-oxidation and acyl-CoA oxidase was r = 0.994 (P < 0.01). Discussion
As reviewed by Reddy and Lalwani [6] and Hawkins et al. [8], treatment of rats with peroxisome proliferators characteristically results in (1) hepatomegaly, (2) proliferation of hepatic peroxisomes without core, which display variation in size, (3) a remarkable induction of peroxisomal /~-oxidation enzymes, which is often accompanied by decrease in urate oxidase and v-amino acid oxidase, (4) co-induction of several extra-peroxisomal enzymes related to lipid metabolism and (5) decreased activities of giutathione-associated enzymes. Moreover, there are (6) sex differences [35-41] and (7) tissue differences [6,8,42] in the sensitivity to peroxisome proliferators in the rat. The present study has demonstrated that DHEA elicits all of these characteristic responses in the rat. Most of peroxisome proliferators possess (8) hypolipidemic properties [6,8,9,33,39] and DHEA is also a hypotrigiyceridemic agent [2,43]. Furthermore, we have found the existence of (9) species differences in the sensitivity to the inductive effect of DHEA (unpublished data). This is also a common characteristic among peroxisome proliferators [6,8,35, 36,40]. Collectively, these findings indicate that DHEA is a typical peroxisome proliferator. Wu et al. [4] have recently reported the effect of DHEA on microsomal cytochrome P-450 system of rat liver, in their report, they showed that DHEA does not induce any of the major forms of cytochrome P-450 which are normally increased by phenobarbital etc. Nor do typical peroxisome proliferators induce the P-450s. The authors also described a remarkable increase in laurate 12-hydroxylation activity; moreover, they confirmed the increase in the P-450IVA1 content. However, whereas the maximal induction of the enzyme at a dose of 160 mg of DHEA/kg body wt., intraperitoneaUy, was observed, a higher dose was required to obtain the maximal induction in our experiments (Fig. 5). This is probably due to the difference in the methods of administration. More recently, also in the fiver of mice fed DHEA, the induction of peroxisome proliferation and several enzymes including peroxisomal bifunctional enzyme, camitine acetyitransferase [5] and malic enzyme [44] have been demonstrated (we also obtained similar results in a study on species differences, unpublished data). Thus, our conclusion is consistent with and supported by the previous studies. In the present study, we used CPIB as a positive control for the peroxisome proliferator. Various enzyme-inducing properties of this drug have been well investigated [6,8]. Considering the time course of the
induction, the dose level required for maximal induction and the potency as an inducer, the properties of DHEA were found to be very similar to those of CPIB. In addition, our results obtained from the experiment of the co-treatment of DHEA and CPIB (Tables II, III and Figs. 1, 2) suggest that a common process may be involved in the mechanism underlying the hepatic enzyme induction by DHEA and CPIB. The dose level (300 mg each/kg body wt.) and time period (14 days) employed in the co-treatment of DHEA and CPIB are sufficient to elicit maximal induction when treated individually (see Figs. 4 and 5). In such a situation, there was no synergism in the induction of the hepatic enzymes. Slightly additive enhancement seen in the activities of carnitine acetyltransferase and malic enzyme may be due to relatively incomplete saturation of the inductions at this dosage of DHEA (see Fig. 5C and H). DHEA and CPIB may well interact with the same cellular site responsible for the induction. The inductive effect of peroxisome proliferators extends to extra-peroxisomai enzymes as well as peroxisomal enzymes. In this study we have also found five kinds of enzyme including the peroxisomal fl-oxic~ation system to be major co-inducible enzymes (T:~ble IV). Considering their physiological functions, a causal relationship could be found for the respective inductions of these enzymes. For instance, increased activity of fatty acid t0-hydroxylation might result in the overload of dicarboxylic acids on the peroxisomal r-oxidation system, leading to the induction of the /I-oxidation enzymes. Increased malic enzyme activity could supply reducing equivalent to the t0-hydroxylation system. However, luther studies are required to establish such a functional relationship as a mechanism of co-induction. While the chemical structures of peroxisome proliferators are extremely diverse [6,8,9,33,39], the analogues of clofibrate, Wy-14,643 and fatty acid constitute three major categories of peroxisome proliferator. The structural requirements of these compounds for peroxisome proliferation are presently obscure. However, some authors have suggested the importance of the carboxyl groups in their structures, which could receive metabolic conversion to the CoA-derivatives to exert the inductive effect [45,46]. Considering this proposal, Ikeda et al. [10] showed that perfluorinated octane sulfonic acid, which cannot be converted to the CoA-derivative, is also a potent peroxisome proliferator, and they suggested that a structure of unmetabolizable hydrophobic anion may be required. This is also the case for peroxisome proliferating tetrazole-~ul-.stituted acetophenone LY-171883 [9]. However, DHEA has no anionic functional groups itself and to our acknowledge the earboxylated metabolites or their CoA-derivatives have not been found in the cell. Besides, the steroidal structure of DHEA is also unique. This structurally new type of peroxisome profiferator would, thus, provide particu-
243 lar information significant to the understanding of the mechanism of induction of peroxisomes. The ultimate form of DHEA triggering the induction should be investigated, including DHEA-sulfate which is a hydrophobic anion, and attention should also be focused on the receptor-mediated mechanism. Since DHEA is a naturally secreted product in mammals, there is a possibility that DHEA might be an endogenous inducer of peroxisomal enzymes. However, considering that a high dosage, as used here, is required to produce induction, the role of DHEA in the physiological regulation of peroxisomal enzymes should be carefully investigated.
Acknowledgments The authors acknowledge the gift of BrMB from Dr. Y. Kawahara (Sankyo Co., Ltd., Tokyo, Japan) and the technical assistance of Miss C. Nakada (Sankyo Co., Ltd., Tokyo, Japan) in the morphological study. This work was partly supported by a grant (No. 02771723) from the Ministry of Education, Science and Culture of Japan, and grants from the Shimabara Science Promotion Foundation and the Science Research Promotion Fund of Japan Private School Promotion Foundation. References 1 Schwartz, A. (1985) Basic Life Sci. 35, 181-191. 2 Regelson, W., Loria, R. and Kalimi, M. (1988) Ann. N.Y. Acad. Sci. 521, 260-273. 3 Leighton, B., Tagliaferro, A.R. and Newsholme, E.A. (1987) J. Nutn 117, 1287-1290. 4 Wu, H.-Q., Masset-Brown, J., Tweedie, DJ., Milewich, L., Frenkel, R.A., Martin-Wixtrom, C., Estabrook, R.W. and Prough, R.A. (1989) Cancer Res. 49, 2337-2343. 5 Frenkel, R.A., Slaughter, C.A., Orth, K., Moomaw, C.R., Hicks, S.H., Snyder, J.M., Bennett, M., Prough, R.A., Putnam, R.S. and Milewich, L. (1990) J. Steroid Biochem. 35, 33-342. 6 Reddy, J.K. and Lalwani, N.D. (1983) CRC Crit. Rev. Toxicol. 12, 1-58.
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