Dehydroepiandrosterone (DHEA): Biochemical, Physiological and Clinical Aspects 9783110811162, 9783110161113

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Dehydroepiandrosterone (DHEA) edited by Mohammed Kalimi and William Regelson

Dehydroepiandrosterone (DHEA) Biochemical, Physiological and Clinical Aspects

edited by Mohammed Kalimi William Regelson

Walter de Gruyter Berlin . New York 2000

Professors Mohammed Kalimi, Ph.D. William Regelson, Ph.D. Department of Physiology Medical College of Virginia 1101 East Marshall Street Richmond, Virginia 23298-0551 USA With 107 figures. Library of Congress Cataloging-in-Publication Data Dehydroepiandrosterone (DHEA): biochemical, physiological and clinical aspects / edited by Mohammed Kalimi, William Regelson. Includes indexes. ISBN 3110161117 (cloth: alk. paper) I. Dehydroepiandrosterone—Therapeutic use, 2. Dehydroepiandrosterone—Physiological effect. I. Kalimi, M.Y. (Mohammed Y.), 1939II. Regelson, William. [DNLM: 1. Prasterone—physiology. QU 85 D323 2000] RM296.5.D45 D46 2000 615'364—dc21

Die Deutsche Bibliothek - Cataloging-in-Publication Data Dehydroepiandrosterone (DHEA): biochemical, physiological and clinical aspects / ed. by Mohammed Kalimi; William Regelson. - Berlin ; New York : de Gruyter, 2000 ISBN 3-11-016111-7

© Copyright 1999 by Walter de Gruyter GmbH & Co. KG, D-10785 Berlin All rights reserved, including those of translation into foreign languages. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Medical science is constantly developing. Research and clinical experience expand our knowledge, especially with regard to treatment and medication. For dosages and applications mentioned in this work, the reader may rely on the authors, editors and publisher having taken great pains to ensure that these indications reflect the standard of knowledge at the time this work was completed. Nevertheless, all users are requested to check the package leaflet of the medication, in order to determine for themselves whether the recommendations given for the dosages or the likely contraindications differ from those given in this book. This is especially true for medication which is seldom used or has recently been put on the market and for medication whose application has been restricted by the German Ministry of Health. The quotation of registered names, trade names, trade marks etc. in this copy does not imply, even in the absence of a specific statement that such names are exempt from laws and regulations protecting trade marks etc. and therefore free for general use. Reproductions, typesetting: Ditta Ahmadi, Berlin - Printing and Binding: WB-Druck, Rieden/Allgäu Printed in Germany

Contents Biological Effects of Dehydroepiandrosterone: A Review Erdal Gursoy, Yan Hu, Arturo Cardounel, William Regelson, Mohammed Kalimi

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Possible "Anti-Aging" Effects of Caloric Restrictions as Indicated by Dehydroepiandrosterone Sulfate Levels George S. Roth, Donald K. Ingram, Mark A. Lane

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DHEA Cytokine Dy sregulation in Aging AIDS Mohsen Araghi-Niknam, Shugang Jiang, and Ronald R. Watson

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Dehydroepiandrosterone (DHEA) and its Role in the Aging Immune System Omid Khorram

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Restoration of Immunocompetence in Aging and Other Inflammatory Disease States by Dehydroepiandrosterone-3ß-Sulfate, an Activator of the Peroxisome Proliferator-Activated Receptor Alpha (PPARa) Matthew E. Poynter, Raymond A. Daynes 65 The Antiobesity Effect of Dehydroepiandrosterone (DHEA): Clinical Studies in the Canine Model Ilene D. Kurzman, E. Gregory MacEwen 119 DHEA(S) and Obesity: Potential Antiadipogenic Mechanisms of Action Michael K. Mcintosh

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The Antiobesity Effect of Dehydroepiandrosterone Treatment Margot P. Cleary

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Dehydroepiandrosterone and Obesity Frank Svec, Johnny Porter

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DHEA,Tchernof, Obesity and Cardiovascular Disease Despres Andre Fernand Labrie, Jean-Pierre

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Role of Dehydroepiandrosterone in Experimental and Human Carcinogenesis Francesco Feo, Rosa M. Pascale, Maria M. Simile, Maria R. De Miglio 215 Hepatocarcinogenesis by Dehydroepiandrosterone. I. Induction of Neoplasms and Sequential Cellular Changes During Neoplastic Development Peter Bannasch, Christel Metzger, Doris Mayer

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Hepatocarcinogenesis by Dehydroepiandrosterone. II. Biochemical and Molecular Changes During Neoplastic Development Doris Mayer, Christel Metzger, Peter Bannasch

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Constraints on the DHEAS-Induced Enhancement of Hippocampal Function: Non-Linear Dose-Response Functions and DHEAS-Stress Interactions David M. Diamond, Monika Fleshner 261 Neuropsychiatry Effects of Dehydroepiandrosterone (DHEA) OwenM. Wolkowitz, Victor I. Reus

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DHEA, The Precursor of Androgens and Estrogens in Peripheral Tissues in the Human: Intracrinology Fernand Labrie, Alain Belanger, Van Luu-The, Claude Labrie, Jacques Simard, Sheng-Xiang Lin

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Studies on the Metabolism of DHEA in Rats and Mice Gerhard Hobe, Hans-Georg Hillesheim, Renate Schön, Katrin Undisz, Ulrike Valentin, Gudrun Reddersen, Petra Ritter, Peter Bannasch, Doris Mayer

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Androgens and Liver Function H. Leon Bradlow, Barnett Zumojf

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DHEA Respiration Carolynand D. Mitochondrial Berdanier, William P. Flatt

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Dehydroepiandrosterone Sulfate (DHEAS) Increases Osteoblastic Activities In Vitro KitMui Chiu, Evan Keller, Austin Shug, Stefan Gravenstein

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Dehydroepiandrosterone-Dexamethasone Interactions on Nb2 Lymphoma Cell Proliferation Yanal Shafagoj, Raphael Witorsch, Milton Sholley, William Regelson, Mohammed Kalimi

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Dehydroepiandrosterone (DHEA), and Its Relation to the Pathology of "Stress" Reactions William Regelson, Mohammed Kalimi

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Author Index Subject Index

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Introductory Remarks

Dehydroepiandrosterone (DHEA) is a neurocrine and an adrenal cortical steroid, present in high concentration during gestation, and of age related development, peaking during primate reproductive years while declining dramatically with age. From birth to adulthood and senescence, DHEA levels express age related and disease modulated changes. DHEA levels dramatically decline with infection, stress, and autoimmune disease. Is DHEA necessary for healthy clinical survival? What is its role in hormone replacement therapy versus its place as a pharmacologic agent? Because of its over-the-counter nutriceutical availability, what are the dangers of its misuse? How does one explain the paradox of its upregulation of immunity in animal models, with its clinical protection demonstrated in the treatment of the autoimmune lupus syndrome? DHEA availability has given us a plethora of observations, and as researchers, we have not yet been able to organize our findings into a rational scheme that can explain all that we see. Is DHEA a state dependent hormone, which like thyroid hormones can modulate the action of steroids that are more specifically targeted? Is DHEA's major clinical action related to its amelioration of steroid stress related deleterious effects? Is DHEA's essential value in its role as a precursor to sex hormone and steroid related metabolites: testosterone, estrogen and etiocholanalone? What are the other metabolites of DHEA that must be looked at to explain its protean effects? The fundamental issue is whether DHEA's actions are mediated by its metabolites, as a precursor to the sex steroids, or does it have action of its own? 1) Is the action of DHEA primarily that of a classical endocrine or does it have properties that extend its value to effects on cell membrane or receptor kinetics? 2) Is it a paracrine hormone, when synthesized by the brain, or an intracrine dependent on cell type and metabolite interactions? 3) What is its precise molecular mechanism of action? Is receptor availability a key to its effects? 4) How does its modulation of cytokines explain its action? 5) How can we extrapolate between rodent model data and our primate or clinical experience where DHEA is a significant steroid? 6) How does it impact on the process of aging? Here we need clinical and laboratory studies in animal models in which the presence of DHEA is a normal metabolite. 7) Apart from cancer prevention: Does DHEA or selected metabolites demonstrate antitumor activity based on effects on DNA metabolism, modulation of cytokine action (i.e.: IL6), or through effects on immune responsiveness, angiogenesis or cell membrane synthesis? 8) Will DHEA have clinical value in the treatment of both juvenile and insulin resistant diabetes? 9) What is DHEA's role and value as a neurocrine? It has shown clinical value as an antidepressant. Will its rodent model effects in enhancement of memory performance show

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Introductory Remarks

clinical value in Alzheimer's Disease and other problems involving memory or hippocampal related injury. Will DHE A effects on atherosclerosis have continued clinical relevance with reference to lowering cholesterol levels and effecting platelet aggregation? How does it participate in appetite control and weight loss? Will this have eventual clinical relevance? What is its role in governing mitochondria integrity, catalase and cell energetics? As mentioned previously, what is its immunologic role? What function does DHEA have as a steroid hormone acting in disease states?

Since our first volume in 1990, the interest in studying the biological, biochemical, physiological and clinical actions of DHEA resulted in over 1000 papers dealing with the action of DHEA. The purpose of this volume is to summarize and update what has transpired since volume I of "The Biologic Action of DHEA". We expect that growing interest and the continued development of DHEA analogues will give us a better understanding of its action during our next millennium. While the overall biologic and clinical actions of DHEA are controversial, the importance of this native hormone requires that we define its place. It is hoped that pharmaceutical investment in its activity will help clarify its role in clinical medicine. Clinical studies, stimulated by the Stanford group have demonstrated the potential clinical value of DHEA in the treatment of Lupus (Gene Labs, Redwood City, CA). It is hoped that further double-blinded clinical studies will open the door for FDA NDA approval that will make DHEA of interest to our colleagues in medicine, to provide options for its use beyond nutriceutical over-thecounter enthusiasm. Is the positive experience in Lupus of sufficient value to stimulate exploration of DHEA in other autoimmune diseases? Will our next review see DHEA or its analogues adequately studied in the clinic? Will it demonstrate value not only in autoimmune disease, but as an adjuvant to enhance vaccine development, the treatment and prevention of cancer, depression, Alzheimer's disease, resistance to infection, stress, atherosclerosis, and the debility of aging? For those concerned with DHEA's clinical toxicity, DHEA has been given pharmacologically safely at clinical dosages ranging up to 40 mg/kg/day for over two years. Its clinical value in Lupus has been demonstrated at 200 mg/day. However, its possible role in the promotion of liver, prostate, breast and uterine cancer has not been ruled out, although DHEA, as "Prasterone" has been used in Europe for some 20 years as a replacement therapy in treatment of menopausal symptoms. Our volume covers the bulk of literature from 1980-99 regarding the biological, clinical and biochemical properties of DHEA. However, even with the best efforts of our contributors we may have missed some salient points. We feel that this updated review covering the latest information about the biological role of DHEA will be helpful to biomedical researchers, and clinical practitioners. We hope that the studies reported here will alert the drug industry to DHEA as an area of major clinical development, and that this volume will serve the public at large interested in this native hormone. M. Kalimi W. Regelson

Biological Effects of Dehydroepiandrosterone: A Review Erdal Gursoy, Yan Hu, Arturo Cardounel, William Regelson and Mohammed Kalimi

Introduction This review encompasses advances in our understanding of the biological effects of DHEA since the publication of our text on "The Biological Role of Dehydoepiandrosterone" in 1990 by Walter de Gruyter. It is obvious from the reviewed core literature below, published during 1990-1998, that scientific interest in the elucidation of the biochemical, physiological, pharmacological, toxicological and clinical effects of DHEA remains as vigorous as ever. Research efforts directed towards delineation of biological effects of DHEA in the past decade have brought about novel and exciting information regarding the biological effects of DHEA. However, no new major insight into the cellular or molecular mechanism of DHEA action and the question as to whether DHEA replacement therapy in humans has any definitive beneficial potential against cancer, obesity, viral infection, aging, diabetes etc. remains largely elusive and ambiguous, In this review, we have attempted to extensively cover both the in vivo and in vitro beneficial, deleterious or absence of effects related to DHEA administration reported from 1990-1998. It is important to note that data regarding in vitro biological effects of DHEA are meager and inconsistent (please refer to Shafagoj et al. Chapter, entitled "Dehydroepiandrosterone-dexamethasone interactions on Nb2 lymphoma cell proliferation in this volume). Finally, we encourage readers to refer to an excellent recent minireview on the biological effects of DHEA by Svec and Porter (1998), extensively covering dosage and route of DHEA administration to various experimental animals and humans.

DHEA and Cancer Judging from the available literature, the anti-carcinogenic effects of DHEA has been extensively studied during the past decade. Most of these studies have demonstrated the positive beneficial effects of DHEA on various types of cancers and particularly on chemical carcinogen-induced tumors in rodents. On the other hand, some studies have reported a tumor enhancing effect of DHEA, particularly in the induction of tumors of the liver and kidney. Of importance to our clinical concern for DHEA's androgenic effects: Van Weerden et al (1992), using castrated mice bearing human prostate tumors propagated in nude mice, inve-

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stigated the effect of DHEA on stimulation of prostate cancer. Their results showed that DHEA treatment for 28 days led to a decline of tumor burden compared to controls, indicating that DHEA did not have a stimulatory effect on the growth of PC-82 tumors. Rao et al (1991) fed 5 week old male F344 rats an experimental diet containing 40 and 80 % of a maximally tolerated dose (MTD) of 16 alpha-fluoro-5-androsten-17-0ne (DHEA analogue 8354, 500 ppm) for two weeks. This was followed by azoxymethane (15mg/kg body weight /week for 2 weeks). Fifty-two weeks after azoxymethane treatment, the DHEA analogue at the 40% MTD level significantly decreased the incidence of small intestinal and colon tumors. Paradoxically, the DHEA analogue at the 80% level inhibited only small intestinal tumors. Schwartz et al (1995) found that tumor initiation, tumor promoter-induced epidermal hyperplasia and promotion of papillomas in the two-stage skin tumorigenesis model in mice was inhibited by administration of DHEA and a non-androgenic structural analog : 16 alphafluoro-5-androsten-17-one,which still possesses the antiproliferative and cancer preventive activity of the native steroid. Ratko et al (1991) reported that dietary DHEA or a fluorinated analogue of DHEA inhibited rat mammary gland chemical carcinogenesis. After treating rats with N-methyl-N-nitrosuourea (MNU) at 50 days of age, they fed these rats with a semipurified diet containing : DHEA alone (0.2%,w/w), DHEA (0.2%, w/w) plus n-(4-hydroxyphenyl) retinamide (4-HPR, 1 mmol/kg diet), or the DHEA analogue 8354 alone(0.2%, w/w). The analogue 8354 was given either: during initiation (-1 week to +1 week post-MNU), promotion/progression (+1 week post-MNU to termination), or during both phases (-week post-MNU to termination) of the carcinogenic process. They demonstrated that only DHEA plus 4-HPR significantly affected the initiation of mammary cancer. All three treatments significantly reduced mammary cancer incidence and multiplicity by 77% (DHEA), 84% (DHEA/ 4-HPR), and 66% (DHEA analogue 8354) relative to carcinogen controls. Cancer incidence was significantly inhibited by DHEA and DHEA/4-HPR during promotion/progression. DHEA also significantly reduced cancer incidence and multiplicity during both phases of carcinogenesis. In addition, long-term treatment with DHEA or DHEA/4-HPR significantly lowered cancer mortality. Rao et al (1992) studied the effect of DHEA on old F-344 rats that have a very high incidence of spontaneous Leydig cell tumors of the testis. They fed fifteen-week-old male F-344 rats with a diet containing DHEA (0.45 % w/w) for 84 weeks. They found no incidence of Leydig cell hyperplasia or Leydig tumors in the DHEA fed rats whereas all control rats of comparable age had Leydig cell tumors.These results suggested that DHEA has both antimitotic and anticarcinogenic properties to spontaneous Leydig cell tumors in aged F-344 rats. Hursting et al (1995) found that treatment of male p53-knockout (p53-/-) mice with DHEA delayed tumor development (particularly lymphomas) and reduced subsequent mortality (P< 0.01) compared to untreated control mice. Luo et al (1997) examined the effect of DHEA on bone mass, serum lipids, and DMBA-induced mammary caricinoma in the rats. They percutaneously administrated rats with DHEA daily, at the dose of 5, 10, 20 mg for 9 months following a single dose of 20 mg DMBA at 50-52 days of age. They observed that DHEA treatment significantly increased body mineral content and bone mineral density of total skeleton and femur using dual

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energy x-ray absorptiometry. DHEA treatment also led to decreases in serum triglyceride levels and the incidence of mammary carcinoma. The mean tumor number per tumor-bearing rats and the mean tumor area per tumor-bearing animal was reduced. In addition, DHEA treatment augmented the activity of serum total alkaline phosphatase and lowered urinary calcium excretion. However, no effect was seen on serum cholesterol concentrations, the urinary ratio of hydroxyproline to creatinine, and urinary phosphorus excretion. They suggested that DHEA replacement therapy may exert beneficial effects on bone and lipid metabolism in women receiving DHEA replacement therapy as well as a preventive effect on breast cancer. Mehta and Moon (1991) exposed mammary glands to 2 mg/ml DMBA for 24 hr followed by TPA for 5 days, which were used to study the initiation and promotion aspects of mammary lesion development. After that, they treated the isolated mammary glands with DHEA. They found that DHEA was active against the promotion phase of lesion development. Kohama et al (1997) investigated the effects of DHEA on mammary tumors induced by local injection of DMBA in hyperprolactinemic female rats. They found that the period from the day of DMBA administration, to that of the onset of the rapid tumor growth in the DHEA-treated group was longer than in controls. The incidence of adenocarcinoma in the DHEA-treated group was also lower than in controls. They concluded that prolactin increased the incidence of adenocarcinoma in the DMBA-induced mammary tumor model, and DHEA decreased the incidence of adenocarcinoma McCormick et al (1996) found that Feeding female Sprague-Dowley rats with an AIN-76A diet supplemented with DHEA (800 or 400 mg/kg diet) for 1 week prior to administration of 35 mg N-methyl-N-nitrosourea per kg body weight reduced mammary cancer incidence from >70% in dietary controls to 0% in diet supplemented with DHEA. Luo et al (1997) found that DHEA treatment decreased dimethylbenz(A) (DMBA)-induced mammary tumor incidence from 95% to 57%. DHEA increased bone mineral density of total skeleton, lumbar spine and femur by 6.9%, 10.6%, and 8.2 %, respectively. In addition, DHEA stimulated serum alkaline phosphatase, reduced serum triglycerides and had an inhibitory effect on lobular thyroid hyperplasia of aged intact rats. Pashko (1991) using a two-stage skin tumorigenesis model in mice, found that topical application of the DHEA analogue (16 alpha-fluoro-5-androsten-17-one) inhibited promotion of 7,12-dimethylbenz(A) anthracene (DMBA)-initiated tumor development by 12-o-tetradecanoylphorbol-13-acetate(TPA). DHEA reduced NADPH and ribose-5-phosphate production, which in turn lowered intracelluar deoxyribonucleotide levels. Addition of deoxyribonucleosides-deoxyadenosine, deoxycytidine, deoxyguanosine and deoxy thymidine to the drinking water during the promotion period of tumorigenesis completely reversed the inhibiting effect of the DHEA analogue. They suggest that the inhibiting effect of DHEA in carcinogenesis may be due to a lack of NADPH and ribose-5-phosphate production necessary for deoxyribonucleotide synthesis and subsequent DNA replication. Estabrook (1990) treated rats with a diet containing DHEA and showed that there were changes in the liver P-450s and in the activities of other liver related enzymes, viz, P-450IIB1, P-450IIC11, and P-450IIIA. They proposed that the anti-carcinogenic and other chemoprotective effects of DHEA may result from alterations in the cellular P-450s, thus influencing the balance of metabolic activities associated with the initiation phase of chemical carcinogenesis and /or toxicity.

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Inano et al (1995) studied the effect of DHEA against the promotion/progression phase of radiation-induced mammary tumorigenesis. It had been shown that whole body irradiation with 260 cGy gamma-radiation at day 20 of pregnancy in the rat and followed by implantion with a diethylstilbesterol (DES) pellet for an experimental period of 1 year resulted in a high incidence (96.2%) of mammary cancer. That incidence of mammary tumors was significantly decreased to 35.0% by administration of dietary DHEA (0.6%) despite DES carcinogenic implantation. Feo et al (1991) reported that DHEA prevented the development of gamma-glutamyltranspeptidase (GGT)-positive foci in the early stages of hepatocarcinogenesis in rats. Later, using diethylnitrosamine to promote hepatocarcinogenesis in female Wistar rats, they found that a 15 day treatment with DHEA (0.6% in the diet) decreased the mitotic indices of GGTpositive liver foci. DHEA inhibited G6PD activity and the production of ribulose-5-phosphate. DHEA-treated liver slices showed a rise in cholesterol content, whereas a 80% fall in the incorporation of labeled acetate, but not of mevalonate, into cholesterol was observed. A 25 day treatment with DHEA decreased incorporation, in vivo, of 3 H into tumor focci and surrounding liver cholesterol by 36 and 78%, respectively. They concluded that DHEA inhibits the cholesterol biosynthetic pathway before mevalonate synthesis. Based on this, the development of preneoplastic lesions do not need the synthesis of large amounts of cholesterol and cholesterol metabolites. They proposed that the antipromotion effect of DHEA may primarily depend on a decreased availability of pentose phosphates for DNA synthesis. Simile et al (1995) studied the effect of DHE A on the growth and progression of late cancer lesions, developing from persistent nodules (PNs) of F344 rat liver tumors using diethylnitrosamine. Giving DHEA to animals during the initiation/promotion stages of carcinogenesis inhibited the development of early pre-neoplastic lesions and prevented tumor development in various target tissues. The number of nodules per liver and incidence of PNs with diameters of 3-6 and >6 mm significantly decreased in DHEA fed groups as compared to animals with no DHEA in their diet. In addition, all the rats recieving no DHEA (n=8) had hepatic cancer at the 56 th week whereas only 1 out of 4 rats treated with DHEA showed cancer. Schulz and Nyce (1991) exposed HT-29 SF human colonic adenocarcinoma cells to 50 μΜ DHEA for 24 hr. They observed that there was a significant incorporation of products of [ 3 H] mevalonate metabolism into several classes of cellular proteins, whereas very little incorporation of labeled [ 3 H] mevalonate was observed in protein of untreated cells. They proposed that [ 3 H] mevalonate entered isoprenylation sites after treatment with DHEA because of depletion of endogenous mevalonate and subsequent inhibition of protein isoprenylation. Isoprenylation plays a critical role in promoting the association of p21 ras within the cell membrane. DHEA inhibited both post translational processing and membrane association of p21 ras, which suggested that the anti-cancer effects of DHEA was due to its inhibition of isoprenylation of p21 ras and other cellular proteins. In another experiment, this group (Schulz et al. 1992) also showed that treatment of HT-29 SF human colonic adenocarcinoma cells with DHEA at concentrations ranging from 12.5 ot 200 μΜ for up to 72 h inhibited growth and arrested cells in the Gl phase of the cell cycle in a time- and dose-dependent manner. Treatment with 25 or 50 μΜ DHEA transiently delayed cells in G2M phase after 48h. However, addition of mevalonic acid partially reversed both the growth and cell cycle effects of 25 μΜ DHEA in the initial 48 hours. During prolonged exposure (72h), both mevalonic acid and cholesterol were necessary to reconstitute cell

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cycle progression. They concluded that the depletion of endogenous mevalonate and other isoprenoids are involved in DHEA-mediated growth inhibition and cell cycle arrest. Perkins et al (1997) using transgenic mice which are susceptible to early development of tumors when both alleles of the p53 tumor suppressor gene product "knocked out" by gene targeting. These cancers are chiefly lymphomas and sarcomas. They found that administration of DHEA in 0.3 % of the diet extended transgenic mouse lifespan by delaying death due to neoplasms. The DHEA analogue (8354) at 0.15% of the diet also increased lifespan. As to mechanism, DHEA did not deplete cellular nucleotide pools in the liver. They proposed that the chemopreventive effect of DHEA in their model may relate to steroid-induced thymic atrophy with suppression of Τ cell lymphoma, thus permitting these mice to survive long enough to develop other tumors with longer latency. Wang et al (1997) examined the mechanism of DHEA action on the Bcl-2/Bax-mediated apoptotic pathway in p53-deficient mice. They demonstrated that DHEA treatment decreased expression of the PCNA proliferation in the thymus using immunohistochemical methods. DHEA treatment also enhanced the rate of apotosis in the thymus, indicating that DHEA may shift cell homeostasis by favoring apoptosis. They showed that DHEA-treated p53-deficient mice had lower Bcl-2 mRNA levels in the thymus. They suggested that their results showed that DHEA may modulate tumorigenesis by affecting apopototic and /or proliferative pathways. Boccuzzi (1992) demonstrated that application of 500 nM DHEA to MCF-7 cells for six days stimulated MCF-7 cell growth in a steroid-free medium, while in estradiol supplemented media DHEA partly antagonized the stimulatory effect of estrogen. Rao and Subbarao (1997) reported that DHEA significantly inhibited DNA synthesis in rat liver at 20,26, 32 and 38 h after partial hepatectomy. However, DHEA had no effect on liver enlargement induced by ciprofibrate. The anticarcinogenic effects of DHEA have been repeatedly demonstrated by several investigators. However, paradoxically, some investigators have shown tumor enhancing effects by DHEA. For example, Hamilton et al (1991) injected the colonic carcinogen azoxymethane to male F344 rats for 10 weeks (10mg/kg/week) in order to induce in colonic epithelium a premalignant state. One day after the final administration of the carcinogen, they fed rats a diet containing 0.5% DHEA for 7 weeks. They found that tumor-related mortality was increased in DHEA-fed rats. Despite the fact that, prevalence, mean frequency, multiplicity, and the diameter of colonic tumors were lower in the DHEA-fed rats, there was no significant difference in DHEA treated from controls. Growth curves and colonic epithelial proliferation were similar to that of controls. They concluded that DHEA did not have a significant postinduction chemoprotective activity against azoxymethane-induced colonic tumorigenesis. They felt that further preclinical studies would be needed before dietary DHEA could be recommended for chemoprotection trials in patients with premalignant colorectal epithelium. It is appropriate to note at this point that DHEA is not a normal metabolite in rodents with the exception of the hamster. Boccuzzi et al (1992) administrated DHEA (2mg, twice daily p.o.) to both intact and ovariectomized adult female rats with mammary carcinoma induced by DMBA. They found that while DHEA treatment stimulated tumor growth in ovariectomized animals, DHEA delayed tumor progression in intact rats.

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Ogiu et al (1990), using a single dose of 30mg/kg dimethylnitrosamine to induce rat kidney tumors, found that after two weeks of administration of dimethylnitrosamine, DHEA treatment for a 26-week period caused a reduction in body weight gain, but it had no inhibitory effect on either renal mesenchymal or cortical epithelial tumors induced by dimethylnitrosamine, but caused a reduction in body weight gain. Nor did it alter the average survival time but the DHEA-treated group exhibited a significant increase in the incidence of renal adenocarcinomas. Lawson (1991) using hepatocytes of hamsters, found that DHEA increased the mutagenicity of N-nitrosobix(2-hydroxypropyl)amine and N-nitroso-(2-hydroxy-propyl)(2-oxopropyl)amine by 3- and 2-fold, respectively. Rao et al (1992) studied the phenotypic properties of DHEA-induced liver lesions in F-344 rats and observed that a majority of carcinogen altered areas, neoplastic nodulae and hepatocellular carcinomas lacked the marker enzymes gamma-glutamytranspeptidase and the placental form of glutathione S-transferase (GSTP). Hepatocellular carcinomas induced showed a marked decrease in the staining of glucose-6-phosphatase and adenosine triphosphatase. Their results indicated that the phenotypic properties of liver tumors induced by DHEA and amphipathic carboxylate peroxisome proliferators are similar, suggesting that the mechanisms of action of DHEA may be due to peroxisome proliferation. Omer et al (1996) exposed rainbow trout, a species that is resistant to peroxisome proliferation but is highly susceptible to the carcinogenic and tumor enhancing effects of DHEA, to aflatoxin Β1. Then fed with diets containing DHEA or the DHEA analog 8354 for 6 months, they observed that post initiation treatment with DHEA increased the incidence, multiplicity, and size of liver tumors compared to controls. DHEA analog 8354 also increased tumor incidence but had no effect on multiplicity or size. In trout DHEA did not increase peroxisomal beta-oxidation but decreased catalase activity. DHEA was a potent inhibitor in vitro of trout liver G6PDH with IC50 of 24 μΜ. They proposed that in fish, the tumor-enhancing effects of DHEA may be due to its function as a sex steroid precursor and not due to peroxisome proliferation. Again in fish, in another experiment this group (Omer et al. 1996) exposed rainbow trout to N-methyl-N-nitro-nitrosoguanidine(MNNG) at 35 ppm for 30 min and then fed them with DHEA at levels of 0,55, 111,222,444, or 888 ppm for 7 months. They found that DHEA increased the incidence, multiplicity, and size of liver tumors in a dose-dependent manner. The kidney tumor incidence was also enhanced two-and three-fold over controls by 111 and 888 ppm DHEA, respectively. However, in contrast, the incidence of stomach and swim bladder tumors was reduced by DHEA, indicating the varied effects of DHEA on MNNGinitiated carcinogenesis in liver, kidney, stomach, and swim bladder in rainbow trout. Looking at rodents again, Beier et al (1997) treated male and female rats with diets containing 0.6% DHEA for 24 weeks. They used qualitative immunohistochemical, electron microscopic, and Western blotting techniques to detect the levels of hepatic cytochrome P450 IVA and peroxisomal lipid beta-oxidation enzymes. They found that there was a significant induction of peroxisomal beta-oxidation enzymes in both male and female rats, whereas catalase and urate-oxidase were not increased. Cytochrome P450 IVA was more strongly induced in male than in female rats. The induction of cytochrome P450 IVA showed a marked lobular gradient in female animals with strong pericentral induction with almost no induction in periportal regions of the liver lobule. In contrast, in male animals

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cytochrome P450 IVA and peroxisomal proliferation were expressed more uniformly across the liver lobule. Their results supported that there was a functional relationship between peroxisome proliferation and DHEA, which may contribute to the more frequent incidence of liver tumors in female rats than in male rats. In addition, they suggested that the hepatocarcinogenic effect of DHEA may also be due to other factors in addition to peroxisome proliferation. The Effets of DHEA on the Immune System DHEA has potent effects on immune responses. Daynes group (1990) studied the effect of DHEA on helper Τ cells of mice. They found that lymphocytes from mice treated with DHEA at doses of 10"10 to 10"7 induced more IL2 than in controls. Activated cloned Τ cell lines also produced significant amount of IL2. DHEA treatment antagonized the depression in IL2 synthesis by Τ cells and Τ cell clones induced by glucocorticoids. They proposed that their results indicated that DHEA may represent a natural and important regulator of IL2 production in both normal and pathologic conditions. Risdon et al (1991), using lethally irradiated mice infused with syngeneic bone marrow cell as a source of transplantable natural killer cell progenitors, examined the effect of dietary DHEA (0.45% w/w) on the stages of natural killer cell differentiation. They showed that DHEA-fed recipients failed to generate NK activity. DHEA also inhibited the generation of IL-2 responsive precursor cells in recipients of 106 bone marrow cells and the production of NK progenitors. They suggested that DHEA seemed to prevent the generation of NK progenitors from more primitive stem cells. The differentiation of progenitors into IL-2 responsive precursor cells and the maturation of IL-2- responsive precursor cells into mature NK cells was inhibited by DHEA. Suzuki et al (1991) exposed Τ lymphocytes from healthy adults to DHEA and then stimulated them with mitogens and antigen. They reported that upon activation with stimuli, a significant amount of IL2 of more potent cytotoxicity was produced by fresh CD4+ Τ cells and CD4+ clones pretreated with 10~8 to 10"11 Μ DHEA compared to the Tcells activated in the absence of DHEA. DHEA exerted its peak effect at 10~9M, which is, the DHEA level in normal adults. The level of IL2 mRNA was also enhanced by DHEA. They concluded that DHEA may act as a transcriptional enhancer of the IL2 gene in CD4+ Τ cells, suggesting that DHEA may play an important role in regulating the human immune response. Risdon et al (1991) showed that dietary DHEA exhibited differential effects in lymphopoiesis and myelopoiesis of sublethally irradiated mice. They found that DHEA significantly delayed regeneration of marrow B220+B cells, involved in natural killer function, and thymus repoplation. IL-2 did not reverse the NK activity of DHEA-treated mice to normal levels. DHEA on the other hand had no effect on erythropoietic progenitor cell and stem cell function. They suggested that only lymphopoiesis but not myelopoiesis was affected by DHEA. Araneo et al (1993) showed that DHEA and DHEAS when incorporated directly into a vaccine or given topically increased specific antibody responses against recombinant hepatitis Β surface antigen (rHBsAg) by aged mice that had previously not been effectively immunized by conventional vaccination. Loria and colleagues (1996) reported that DHEA suppressed the proliferation of concanavalin A (ConA)-or LPS-activated splenocytes in a dose-dependent manner. According to them, DHEA had no effect on the immunosuppressive effects of hydrocortisone on ConAinduced lymphocyte proliferation, as well as on IL-2 and IL-3 production.

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Meno et al (1996) showed that administration of DHEA inhibited the proliferation of phytohemagglutinin-stimulated rat lymphocytes. Khorram et al (1997) treated 9 healthy age-advanced men (mean age of 63 years, with low DHEA-sulfate levels) with 50 mg DHEA for 20 weeks. They showed that DHEA treatment significantly increased serum IGF-I and the ratio of IGF-I/IGFBP-I. DHEA activated immune function within 2-20 weeks of treatment. DHEA treatment resulted in a significant increase in the number of monocytes after 2 to 20 weeks. The significant increases in Β and Τ cell mitogenic response, in cells expressing the IL-2R(CD25+), and in serum sIL-2R concentration was also reported in the DHEA treated group. Production of IL-2 and IL-6 stimulated by in vitro mitogens was also markedly increased by DHEA treatment. Nk cell number and cytotoxicity was greatly enhanced by 18-20 weeks of treatment with DHEA. However, no change in total Τ cells and IGF I subsets was observed. They suggested that DHEA significantly activated immune function of age-advanced men with low serum DHEAS levels. They postulated that an increase in bioavailable IGF-I, with its mitogenic effects on immune cell function may mediate the DHEA effects seen. Their results may indicate the potential therapeutic benefits of DHEA in immunodeficient states and suggest that age related DHEA replacement may have value in human host resistance. James et al (1997) reported that low doses of (10 6 to-10 8 M) DHEA inhibited the production of IL-6 in unstimulated human spleen cell suspension cultures while enhancing its release by explant cultures of the same tissue. DHEA exhibited no effect on immunoglobulin production. In normal healthy people over the age of 40 they found an inverse relationship between plasma DHEA concentrations and the presence of detectable levels of IL-6. Based on their studies, they suggested that there may be a real, but complex relationship between IL-6 production and DHEA levels. Tabata et al (1997) assessed the effect of DHEA on the production of IL-4 and IL-5 by lymphocytes in patients with atopic dermatitis. They showed that preincubation of peripheral blood mononuclear cells(PBMCs) with DHEA reduced the IL-4 production by concanavalin Α-stimulated PBMCs, and DHEA also tended to decrease IL-5 production in PBMCs. They concluded that DHEA may be one of the regulators governing cytokine production in atopic dematitis.

Anti-viral Effects Anti-viral effects of DHEA have been reported by many investigators. For example, Araghi-Niknam et al (1997) reported that DHEA administration to young C57BL/6 mice infected with a Murine retrovirus, considerably reduced the retroviral infection induced oxidative damage, levels of vitamin E, and loss of cytokines IL-2 and IFN-gamma secretion by mitogen stimulated spleen cells. DHEA also suppresses the production of cytokines IL-6 and tumor necrosis factor alpha by Τ helper 2 (TH2) cells. In addition, they observed that administration of DHEAS to old C57BL female mice infected with Murine retrovirus significantly prevented retrovirus-induced reduction in Τ cells. Henderson et al (1992) found that DHEA and the synthetic analogs of DHEA, 16 alphafluoro-5-androsten-17-one (8354) or 3 beta-hydroxy-16 alpha-fluoro-5 alpha-androstan-

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17-one (OH8356), are inhibitors of HIV-1 HIB replication in phytohemagglutinin-stimulated peripheral blood lymphocyte cultures. Loria et al (1992) observed that a single subcutaneous (SC) injection of DHEA to mice caused significant protection against a lethal herpes virus type 2 encephalitis or systemic coxsackievirus B4 infection by up regulating specific host immune responses. Yang et al (1994) found that AZT-resistant HIV-1 replication in MT-2 cell cultures was reduced over 50 percent by using concentrations as low as 50 μΜ DHEA, showing that DHEA inhibits replication of both wild-type and AZT-resistant HIV infected cells. Henderson's group (1992) reported that DHEA and synthetic DHEA analogs are modest inhibitors of HIV-1IIB replication. By recording the syncytia formation, release of p24 antigen, and accumulation of reverse transcriptase activity, they found that administration of DHEA and its synthetic analogs (8354 and 3beta-hydroxy-16-alpha-fluoro-5 alpha-androstan- 17-one (OH 8356) down-regulated HIV-1 replication in phytohemagglutinin-stimulated peripheral blood lymphocytes. DHEA and 8354 also suppressed syncytia formation in HIV-1-infected SupTl lymphoblasts. They proposed that DHEA could provide an effective alternative and /or adjuvant for HIV-1 infection. In support of the above, Bradley et al (1995) reported that DHEA inhibited replication of feline immunodeficiency virus (FIV) in chronically infected cells. Similarly, Araghi-Niknam et al (1998) infected 15-month-old C57BL/6 mice with LP-BM5 murine leukemia virus, causing "Mouse AIDS". The administration of the retrovirus resulted in immune dysfunction and loss of hepatic vitamins A and Ε with increase in lipid peroxides. Post infection administration of DHEAS at 0.01 or 0.005% in drinking water for 10 weeks significantly lowered lipid peroxidation in heart and liver tissues and prevented loss of vitamins A and Ε in liver of infected mice.

DHEA and Endotoxin Rasmussen and Healey (1992)reported that DHEA reduced Cryptosporidium(C) parvum infections in aged Syrian golden hamsters. They treated 10 aged (20-24 months Syrian golden hamsters with DHEA for 7 days before they inoculated them with C. parvum oocysts. By determining oocyst shedding in the feces and parasite colonization of the small intestine, they found that DHEA-treated hamsters showed a significant reduction in cryptosporidial infection compared to controls. This clearly suggests that DHEA may be an effective prophylactic agent for controlling cryptosproidiosis in immunocompromised patients. Danenberg (1992) examined the effect of DHEA on endotoxic shock and endotoxin-induced production of tumor necrosis factor(TNF). They showed that a single dose of DHEA given 5 min before lipopolysaccharide (LPS) administration resulted in an decrease in mortality in CD-I mice exposed to a lethal dose of LPS. DHEA also significantly blocked LPS induced high levels of TNF and serum corticosterone. When mice were sensitized to D-galactosamine, followed by administration of recombinant human TNF, DHEA treatment dramatically reduced the mortality rate of mice. They concluded that DHEA protected mice against endotoxin toxicity by way of reducing TNF production as well as by affecting both TNF-induced and non-TNF-induced toxicity.

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Schurr et al (1997) used a large-animal model to assess the effect of DHEA on the resuscitated trauma and the systemic inflammatory response induced by a delayed lipopolysaccharide(LPS) post trauma. They administrated pigs with 4 mg/kg, 10 mg/kg and 20mg/kg of DHEA at 1, 24, 48, and 72 h after the pigs were subjected to local hind-limb trauma and a 35% hemorrhagic loss. Following this, the animals were exposed to Escherichia coli LPS. After LPS, DHEA was not adequate to prevent progressive septic symptoms, shock and pulmonary failure

DHEA and Lupus Van-Vollenhoven and McGuire (1996) reported that 3 to 12 months of DHEA treatment resulted in alleviating specific lupus symptoms as well as the systemic manifestations of lupus in patients with systemic lupus erythematosus(SLE). DHEA was also shown to decrease the clinical number of lupus flares. They suggested that DHEA may be a useful agent in the treatment of SLE. Suzuki et al (1996) found that DHEA up-regulated IL-2 production of normal Τ cells and DHEA treatment markedly reversed murine lupus. They also demonstrated that nearly all of the patients with SLE had low levels of serum DHEA. Addition of DHEA to cultured Τ cells restored impaired IL-2 production in patients with SLE. They proposed that the low levels of serum DHEA may account for the impairment of IL-2 synthesis in patients with SLE and DHEA replacement may improve clinical symptoms in patients with SLE. Suzuki et al (1995) showed that DHEA significantly enhanced IL-2 production of Τ cells, giving exogenous DHEA or IL-2, via a vaccine developed from murine lupus model. Significantly, decrease in clinical symptom of autoimmune diseases were seen. Also exogenous DHEA increased low IL-2 production of Τ cells from patients with lupus erythematosus in vitro. They suggest that low levels of DHEA may be the reason for defects in IL-2 synthesis in patients with lupus erythematosus. Van Völlenhoven et al (1994) has continually studied the effects of DHEA in systemic lupus erythematosus(SLE). They gave DHEA (200mg/day orally) for 3-6 months to 10 female patients with mild to moderate SLE. After treatment with DHEA, SLE activity including the SLEDAI (SLE Disease Activity Index) was decreased.They concluded that DHEA can be used as therapeatic agent for the treatment of mild to moderate SLE.

DHEA and Arthritis Williams (1997) reported that exogenous DHEA reduced the incidence and severity of collagen-induced arthritis in DBA/1 mice. In their study, they subcutaneously administrated DHEA prior to collagen-induced arthritis in DBA/1 mice. They showed that repeated administration of DHEA during arthritis induction resulted in a delayed onset and less severe arthritis in mice. DHEA-treated mice exhibited lower IgG isotype autoantibody levels in mouse serum. They concluded that their results are consistent with human studies in which low DHEA levels had been identified as a potential risk factor for the development of rheumatoid arthritis. DHEA may provide a potential therapeutic role in this disease, as well as in lupus.

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Cardiovascular Effects of DHEA The cardioprotective effects of DHEA has been reported by several investigators. Taniguchi et al (1996) evaluated the antiatherogenic mechanism of action of DHEA in cultured mouse macrophage J 774-1 cells. They observed that ΙΟ"5 Μ DHEA significantly reduced the accumulation of cholesteryl ester (CE) in J774-1 cells in the presence of acetylated low density lipoprotein(AcLDL). The DHEA effect was dose-dependent and started from 6 h and persisted for at least 48h. DHEA did not alter cell surface binding, cell-association, or degradation of AcLDL. DHEA significantly lowered CE and increased free cholesterol in cells. 10~5 Μ DHEA also caused a CE reduction in foam cells induced by AcLDL. They concluded that two mechanisms existed for the inhibitory effects of DHEA on CE storage in response to AcLDL. One was the inhibitory effect of DHEA on lysosomal cholesterol transport, the other was a decreased cholesteryl ester cycle. The effects of DHEA on atherosclorosis have been contraversial. In older studies benefits have been seen. Yoneyam and colleagues (1997) investigated the mechanisms responsible for the association of DHEA with the progression of coronary atherosclerosis by studying the influences of DHEA on the in vitro growth of vascular smooth muscle cells obtained from the human aorta(hASMC). They found that DHEA at concentrations from ΙΟ-8 Μ to ΙΟ"6 Μ significantly stimulated mitogenesis of hASMC in serum-free culture while DHEA showed no effect on the growth of rat-derived aortic smooth muscle cell lines (A10 cells). 4 hrs of pretreatment of hASMC with DHEA lowered the proliferative effect of fetal calf serum in a dose-dependent manner. They suggested that the effects of DHEA on smooth muscle mitogenesis of hASMC may, at least in part, explain positive association between DHEA and atherosclerosis. In contrast, Jesse et al (1995) showed that adding DHEA to pooled, platelet-rich plasma before the addition of the agonist arachidonate in vitro caused either decreased or complete inhibition in the rate of platelet aggregation. They also found that administration of DHEA to 10 normal men (300mg orally three times daily for 14 days) resulted in complete inhibition of arachidonate-stimulated platelet aggregation in the plasma of one man, and prolonged the rate of platelet aggregation in three men, compare to a control group. They suggested that the mechanism of antiatherogenic and cardioprotective effects of DHEA may be the result of inhibition of platelet activity. Sholley et al (1990) showed that after cells were subjected to a 24 hr exposure to μΜ doses of DHEA, the cultured endothelial cells derived from the human umbilical vein became loaded with phase-dense, perinuclear cytoplasmic granules, which were identified as lipid containing lysosomes. Beer et al (1996) studied the effect of DHEA on plasma plasminogen activator inhibitor type 1 (PAI-1) and tissue plasminogen activator (tPA) antigen. When they gave DHEA (50mg orally) for 12 days to 18 men, plasma levels of PAI-1 and tPA were significantly reduced compared to control groups.They suggested that DHEA enhances endogenous fibrinolytic potential. Taniguchi et al (1994) studied the effects of various steroids on copper (Cu2+)-catalyzed oxidation of low density lipoprotein (LDL) or high density lipoprotein (HDL) in 0.15M NaCl by measuring thiobarbituric acid-reactive substances (TBARS). In this system, they found that DHEA and DHEAS showed no antioxidative effects on either LDL or HDL in this system.

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Eich et al (1993) studied the inhibition of accelerated coronary atherosclerosis in a heterotopic rabbit model of cardiac transplantation using DHEA. They found that chronic DHEA administration significantly reduced the progression of accelerated atherosclerosis in both the transplanted heart and in the native heart without any significant alterations in lipid profiles. Shafagoj et al (1992) studied the role of DHEA on various rodent models with experimental hypertension. Administration of subcutaneus 1.5 mg dexamethasone every alternate day to Sprague-Dawley rats increased systolic blood pressure within one week. However, they observed that dexamethasone -induced hypertension was prevented by administration of a pharmacological dose of 1.5, 3, or 7.5 mg DHEA along with 1.5 mg dexamethasone in dose dependent manner. On the other hand DHEA did not prevent deoxycorticosterone (DOCA)salt induced hypertension or effected BP in a genetic model of hypertension. They also observed that DHEA did not reverse dexamethasone -mediated weight loss or decreased food consumption in that model. Li et al (1990) investigated the effect of DHEA(18mg/kg per day for 10 days) on ACTH-induced hypertension in conscious SD rats. They found that DHEA did not reverse high blood pressure and metabolic effects induced by ACTH (0.5 mg/kg per day). Based on the known function of DHEA in blocking dexamethasone-induced hypertension, they suggested that either that ACTH-induced hypertension is not simply a consequence of glucocorticoid activity. Alternatively, the action of DHEA in dexamethasome hypertension is not through blocking glucocorticoid receptors.

The Effects of DHEA on Body Weight DHEA has been shown to reduce body weight gain in humans and animals. Welle et al (1990) gave eight healthy men DHEA (1600mg/day) for 4 weeks. They found that DHEA administration had no significant effect on body weight on two indices of lean body mass (total body water and total body potassium). No effect was seen on any of the parameters of energy and protein metabolism (resting metabolic rate, total energy expenditure, leucine flux, the nonoxidized portion of leucine flux, and the rate of incorporation of leucine into muscle protein in DHEA-treated men). DHEA had no effect on the circulating levels of cholesterol, T3, or T4. Their study indicated that DHEA is not an important regulator of energy or protein metabolism in humans. Usiskin et al (1990) studied the effect of DHEA on weight and body fat mass in young obese men treated with DHEA (1600mg/day) for 28 days. Using three separate methods (hydrostatic weighing, impedance plethysmography and skinfold measurements) to assess body fat mass, they found that neither body weight, body fat mass, or waist-to-hip ratios changed significantly during the experiment. They observed no change in either tissue insulin sensitivity or serum lipids. They suggest that DHEA had no effect on weight and body fat mass in obese men. In support of the above, Vogiatizi et al (1996) treated 7 morbidly obese adolescents and young adults sublingualy with 80 mg of DHEA daily for 8 weeks. They showed that DHEA treatment had no effect on body weight, body composition, serum lipids, insulin sensitivity or sense of well being in extremely obese adolescents and young adults.

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Mohan et al (1990) examined the effect of DHE A in rats with diet-induced obesity. They fed the diet-induced obese rats with a diet containing 0.6% DHEA for 6 weeks. They showed that DHEA treatment decreased serum insulin levels in diet-induced obese rats or non dietinduced obese rats. In another experiment, they fed rats with diet containing 0.6 % DHEA for two weeks. They found that the DHEA-treated group had lower body weights, serum glucose, insulin, triacylglycerol and triiodothyronine levels. This group had higher liver mitochondrial state 3 respiration rates per gram and per liver and showed elevated peroxisomal beta-oxidation. They suggested that DHEA appeared to interfere with hyperplastic adipose tissue growth and possessed hypolipidemic and hypoinsulinemic effects in diet-induced obese rats. Cleary (1990) reported that DHEA treatment in rats increased weights and DNA, RNA, and /or protein content, but decreased lipid and glycogen levels of the liver. DHEA treatment also changed the activities of liver enzymes involved in lipid and carbohydrate metabolism, DHEA increased net mitochondrial respiration. He concluded that these results may contribute to the antiobesity effect of DHEA. Kurzman et al (1990) fed 19 euthyroid obese and 6 non-obese normal dogs with DHEA for three months at an increasing dose of 30-75mg/kg p.o daily. They observed a 3% reduction in total body weight/month in 68% of the obese dogs without reduction in food intake, whereas no reduction was noted in normal dogs. MacEwen and Kurzman (1991) fed spontaneously obese dogs with a low energy, high fiber diet containing DHEA. They found that the percent excess body weight loss was greater in DHEA-treated dogs compared to controls. They concluded that their data indicated that DHEA combined with a low energy, high fiber diet enhanced the loss of excess body weight. Mcintosh's group (1995) administrated to weaning BHE/cdb rats intraperitoneally daily DHEA (0.35 mol/kg bw) for 7 weeks. They showed that DHEA reduced body weight gain and fasting serum glucose and triglycerides without affecting total or HDL cholesterol. DHEA increased the specific activities of malic enzyme and lactate dehydrogenase. Hepatocytes from DHEA-treated rats converted more [u"14C]] glucose to 14 CÖ2 and lowered the activities of phophoenolpyruvate carboxykinase than in hepatocytes from control rats. They concluded that DHEA exerted its antiobesity and antidiabetic effects in prediabetic, lipemic BHE/cdb rats by promoting hepatic glucose oxidation and reducing gluconeogenesis. Svec and Porter (1997) administrated DHEA (100-200 mg/kg) intraperitoneally to obese Zucker rats. They showed that DHEA reduced their intakes of fat but not carbohydrate and protein and altered the levels of neurotransmitters in the paraventricular nuclear region. They suggested that DHEA diminished fat food consumption of obese Zucker rats by way of changing the levels of neurotransmitters in specific regions of the hypothalamus. Porter et al (1995) studied effects of discontinuing a DHEA diet on Zucker rat food intake and the activity of hypothalamic neurotranmitters. It had been shown previously that DHEA decreased body weight and food intake in the obese Zucker rat. They fed rats with DHEA (0.0%,0.06%,0.15%,0.3% or 0.6%) for 7 days. To study the effect of regional levels of hypothalamic neurotransmitters they used 0.6% DHEA fed-rats in a separate experiment. Rats consumed significantly more food than control until +9 days post DHEA treatment. After 7 days of DHEA-supplemented-diet, the 0.6% DHEA-diet group had significantly increased levels of lateral thalamic (LT), serotonin (5-HT) and dopamine (Dpm). 5HT and Dpm returned to normal level at day 2 on a DHEA free diet. They did not find any neuro-

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transmitter change in either the ventromedial hypothalamus or paraventricular nucleus. They concluded that there is a correlation between increased levels of some hormones (LH, 5-HT and Dpm) and 0.6 % of DHEA treatment. According to them, during post DHE A treatment (+1 and +2 days), there is return to normal level of Dpm and 5HT which may be responsible for reversal of DHEAhypophagia. Taniguchi et al (1995) fed castrated Zucker rats with 0.3 % DHEA for three months. These rats showed significant decrease in body weight gain in comparison to untreated and castrated rats. The degree of antiobesity effects of DHEA in castrated rats and noncastrated rats are almost the same. These result showed that the antiobesity effects of DHEA are by itself, not acting through the conversion to testosterone in the testis. Svec et al (1995) reported that feeding obese Zucker rats with DHEA for 28 days showed no difference in levels of serum glucose, corticosterone, or ACTH as compared to controls. Obese animals gained weight more slowly, had lower insulin level and ate less. This chronic DHEA treatment did not effect hypothalamic neurotransmitters in obese rats. They concluded that chronic DHEA treatment did not cause chronic adrenal insufficiency. Haffa et al (1994) gave six healthy non-obese rhesus monkeys 60 mg/kg/day DHEA for 4 weeks followed by 75 mg/kg/day for an additional 4 weeks. DHEA treated monkeys did not show any difference in body weight, activity level, average daily food intake, plasma T4, insulin, total androgen and Cortisol concentration were compared to another six monkeys that were given placebo daily for 8 weeks. DHEA treated monkeys acutely and significantly showed reduced cholesterol concentrations, particularly the lipoprotein fraction containing low density lipoprotein. Wright et al (1993) fed lean and obese female Zucker rats with 0.6% DHEA for 4 weeks. DHEA treatment caused significant increases in the caloric intake of lean rats and a significant caloric decrease in obese rats compared to their respective controls. Bobyleva et al (1993) found that DHEA treatment to chickens for 7-10 day did not change their weight gain but increased mitochondrial respiration and liver peroximal catalase (index of peroximal mass), DHEA decreased liver cytosolic malic enzyme and sn-glycerol-3phosphate dehydrogenase, as compared to the untreated controls. Svec et al (1996) reported that when lean and obese Zucker rats fed with nearly 40% of their calories as fat, ate more calories and gained more weight than chow -fed rats. Administration of DHEA (on intra peritoneal injection) to these Zuckers decreased caloric intake and prevented weight gain. They concluded that the availability of fat food affects weight gain, however DHEA treatment alone still reduces their rate of weight and caloric consumption. Bednarek-Tupikowska et al (1995) reported no significant effect on plasma estradiol and testosterone levels in both normally fed male rabbits and those on an atherogenic diet. However DHEA was found to have antiobesity effects in both models.

DHEA Replacement Therapy The frequently asked question is whether long term DHEA replacement therapy has any beneficial effects. The clinical results obtained so far are preliminary in nature and far from clear.

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Mortola and Yen (1990) treated 6 postmenopausal women with DHE A 1600 mg/day for 28 days. They demonstrated that both estrone and estradiol progressively increased to 2 - f o l d the basal value at 4 weeks, while SHBG (sex hormone binding globulin) and thyroid binding globulin levels decreased during DHEA treatment. LH, FSH, body weight, and percent body fat were unchanged during DHEA treatment. They also observed that serum cholesterol and high density lipoprotein declined by 11.3% and 20% within the first week of DHEA treatment and persisted throug out the 4 week experiment. They also noted high peak insulin levels during a 3-h oral glucose tolerance test with a 50% increase in the integrated insulin response after the 4 week experiment. Diamond et al (1996) examined the effect of DHEA replacement therapy in 60- to 70-yearold women. They percutaneously administrated 10% DHEA as a cream daily for 12 months. They found that DHEA decreased midthigh fat and in femoral muscular areas, which was associated with a decrease in fasting plasma glucose and fasting insulin levels. Total cholesterol and its lipoprotein fractions tended to be lowered by DHEA. DHEA also lowered plasma HDL without affecting plasma triglycerides. DHEA treatment increased the index sebum secretion. Sex hormone-binding globulin levels decreased during DHEA treatment. DHEA markedly decreased plasma IGF-binding-protein-3 levels while it showed no effect on serum IGF-I. They concluded that their data indicated that DHEA therapy had beneficial effects on postmenopausal women through its transformation into androgens and/or estrogens in specific intracrine tissues without any significant side effects. Labrie's group (1997) examined the effects of 12-months of DHEA replacement therapy (10% DHEA cream) on bone, vagina, and endometrium in postmenopausal women (60- to 70-year-old). They showed that DHEA stimulated maturation of vaginal epithelium but not endometrium. DHEA also significantly increased bone mineral density in the hip accompanied by a significant decrease in plasma bone alkaline phosphatase, and urinary hydroxyproline/creatinine ratios with a 2.1-fold increase in plasma osteocalcin as compared to controls. They suggested that their findings showed the medical important benefits of DHEA therapy in postmenopausal women via transformation of DHEA into androgen and/or estrogen in specific peripheral intracrine tissues without causing side effects. The stimulatory effects of DHEA on bone formation, displayed by its effect on bone mineral density and serum osteocalcin, indicated that DHEA may be useful in both the prevention and treatment of osteoporosis. Hashimoto et al (1997) measured the levels of the serum amyloid Ρ component (SAP) of 420 healthy humans from newborn to 86 year olds and examined the effects of DHEA on SAP in postmenopausal women. They found that the SAP levels increased with age. DHEA treatment (60 mg/kg for 21 days) for the postmenopausal women rapidly increased the SAP. They suggested that SAP may act as a marker to monitor the effect of hormone replacement therapy.

Stress and DHEA Ben-Nathan's group (1992) inoculated mice with west nile virus (WNV) and then exposed them to cold water (1+/-0.5 degrees C, 5 minutes/day for 8 days). Followed by serial injections of DHEA (10-20mg/kg w). This group reported that DHEA significantly reduced the

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mortality rate of cold stressed mice inoculated with WNV and WN-25 virus. DHEA also prevented involution of lymphoid organs in stressed mice. The blood and brains of DHEAtreated mice exhibited very low virus levels. Their data provided direct evidence that DHEA acted as an anti-stress agent. The mechanism of the protective effects of DHEA resulted from its modulation of host response. Prasad et al (1997) treated both high-anxiety and low-anxiety rats with DHEA. They demonstrated that DHEA significantly diminished behavioral despair in high-anxiety rats, but had no effect on low-anxiety rats. They suggested that DHEA is effective as an "antidespair" agent in rats with both high anxiety and despair. Singh et al (1994) reported that sound stress-induced increased tryptophan hydroxylase activity of the enzyme in the mid brain and cortex regions of male Sprague-Dawley rats could be blocked by DHEA administration. The DHEA block to sound stress-induced increases in tryptophan hydroxylase was specific, in that other sex steroids, such as androgens, estrogen, or progesterone, were ineffective. Since coadministration of RU 28362, a potent glucocorticoid agonist, plus DHEA almost completely blocked the sound stress-induced increases in stess enzyme activity. They concluded that DHEA exerts its observed stress blockage via an antiglucocorticoid effect.

The Effect of DHEA on Asbestosis Rom and Harkin (1991) added DHEA to alveolar macrophages lavaged from 11 nonsmoking asbestos workers. They found that DHEA significantly reduced superoxide anion release. They proposed that DHEA may have a therapeutic role in reducing increased oxidant burden in asbestos-induced alveolitis of the lower respiratory tract.

DHEA and Nervous System Nakano and colleagues (1991) showed that DHEA (l-100ng/ml) dose-dependently inhibited beta-endorphin(EP) release from the rat hypothalamus in vitro using a rat hypothalamic perfusion system. Flood et al (93) reported that intracerebroventricular administration of DHEA, following footshock active avoidance training, led to improvement of memory retention for foot shock active avoidance training. They postulated that DHEA improved memory over a much wider dose range than do excitatory memory enhancers, indicating that the effects of DHEA may converge as the facilitator of transcription for immediate-memory functional genes. Debonnel et al (1996) examined the effect of DHEA on the (N-methyl-D-aspartate) NMDA-induced neuronal response using extracellular unitary recordings of CA3 dorsal hippocampus pyramidal neurons obtained from anesthetized Sprague-Dawley rats. Their findings revealed that low doses of DHEA potentiated selectively and dose-dependently the NMDA response. Both the selective sigma 1 antagonist NE-100 and non-selective sigma antagonist haloperidol suppressed the effect of DHEA. Pregnenolone and pregnenolone sul-

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fate, which has a low affinity for sigma receptors showed no effect on the NMDA response. They suggested that DHEA modulated NMDA response via sigma receptors. Hargrave et al (1997) reported that dietary DHEA (100mg/kg) increased lateral hypothalamic 5-HT synthesis and reduced energy (food) intake in Zucker rats. DHEA treatment also reduced serum insulin through 28 days of treatment. The effects of DHEA were not mediated by its metabolite, delta A-androstenedione. Givalois et al (1997) examined the effects of DHEA on CRH mRNA expression in the hypothalamic paraventricular nucleus using in situ hybridization in young (50 day) and old (18 month) rats of both sexes. They found that in aged animals of both sexes, CRH mRNA expression was dramatically lowered by DHEA administration. The application of DHEA augmented the CRH mRNA expression in young rats and helped overcome the decreased CRH mRNA expression in old rats. They suggested that their data indicated that, in both sexes, aging significantly decreased the basal CRH mRNA levels, and DHEA administration suppressed the decrease of CRH mRNA levels associated with aging. Danenboerg et al (1996) reported that DHEA prevented the reduction in non-amyloidogenic amyloid precursor protein(APP) processing in PC 12 cells. In another experiment, they showed that DHEA treatment increased the content of membrane-associated APP holoprotein and accumulation of secreted APP in the medium. They did not observe an increase in viable cell number and in nonspecific protein production in DHEA treated cells. They suggested that DHEA increases both APP synthesis and secretion. The decline in DHEA levels of aged people may be related to pathological APP metabolism observed in Alzheimer's disease. After treating 40 healthy elderly men and women with DHEA for 2 weeks, Wolf et al (1997) assessed their psychological and physical well-being as well as cognitive performance using several questionnaires and neuropsychological tests. They found that DHEA treatment had no strong beneficial effect on any of the measured psychological or cognitive parameters. Only women tended to report an increase in well-being. They concluded that their data did not support the idea of strong beneficial effects of a physiological DHEA substitution on well-being or cognitive performance in healthy elderly individuals. Melchior and Ritzmann (1994) reported that DHEA treatment resulted in a dose-dependent increase in the sleep time induced by either ethanol or pentobarbital. DHEA induced a fall in body temperature at 20 mg/kg. The hypothermic effect of both ethanol and pentobarbital was also enhanced by DHEA. Friess et al (1995) reported that when they gave normal pepole a single oral dose of DHEA (500mg), DHEA caused significant increases in rapid eye movement (REM) sleep and significantly enhanced EEG activity in the sigma frequency range during REM sleep in the first 2-h sleep period after DHEA administration. Other sleep stages and EEG power spectra of non-REM sleep were unaffected. The different effects of DHEA on different sleep stages shows that DHEA has a mixed GAB A-A agonist/antogonist effect. Since administration of DHEA induced significant increase in REM sleep which has been implicated as a factor in memory, they suggested that DHEA should be used in age-related dementia.

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DHEA and Diabetes Giroix et al (1997) gave both control rats and diabetic animals streptozotocin during the neonatal period and then fed these rats with a standard diet or diet supplemented with 0.2% DHEA for 11 days. They found that in both control and diabetic rats, DHEA augmented the activity of the mitochondrial FAD-linked glycerophosphate dehydrogenase and cytosolic NADP-linked malate dehydrogenase in the liver. No effect was noted in parotid gland or pancreatic islets. DHEA also decreased the ratio between D-glucose oxidation and utilization and the rate of insulin release in pancreatic islets exposed to a high concentration of D-glucose, as well as a decrease with insulin concentration and high insulin/glucose ratios in plasma. Their data supported the view that, in diabetes, DHEA, by increasing sensitivity to insulin, may allow islet B-cells to avoid the unfavorable consequences of chronic hyperactivity. Nakashima et al (1995) studied the effects of DHEA on glucose uptake and on insulin sensitivity in cultured rat myoblasts (L6 cells).They found that DHEA significantly enhanced glucose uptake and increased insulin sensitivity by increasing the affinity of glucose transport in the plasma membrane of cultured rat myoblasts. Tagliaferro et al (1995) treated young adult male rats with 4 mg DHEA/100-9 diet for 4 weeks. They found that DHEA-fed rats had significantly less body weight and fat mass than the controls. Also, DHEA fed-rats had significantly smaller isolated epididymal adipocytes and greater isoproterenol stimulation of glycerol release than the controls. DHEA rats and controls had the same basal rate of glycerol release in response to the adenosine deaminase inhibitor. These results support the hypothesis that DHEA reduces adiposity directly by increased lipolysis but does not involve a change in the antilipolytic function of adenosine. Buffington et al (1993) reported that non insulin dependent female patients with DHEA treatment showed increased insulin sensitivity and reduced glucose levels which ameliorated the diabetic state. They concluded that DHEA therapy can be use in the treatment of certain forms of insulin resistance.

DHEA and Enzyme Regulation Antiobesity, anticarcinogenic and antidiabetic effects of DHEA are known to mediated through regulation of various enzymatic pathways in liver and other tissues. Frenkel (1990) assessed the effect of DHEA in various genetic and induced disorders of mice and rats. They reported that dietary DHEA caused hepatomegaly and color change in liver from pink to mahogany. DHEA resulted in proliferation of hepatic peroxisomes with increase in cross-sectional area and volume as well as density of peroxisomes. DHEA decreased hepatic mitochondrial cross-sectional area. DHEA feeding significantly induced the peroxisomal bifunctional protein enol-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase in liver. DHEA increased hepatic enzyme activities, such as, enoyl-CoA hydratase, catalase, carnitine acetyl-CoA transferase, carnitine octanoyl-CoA transferase, and urate oxidase. The activity of mitochondrial carntine palmitoyl-CoA transferase in liver was also enhanced. Plasma cholesterol levels were augmented. However, plasma triglyceride levels

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were either decreased or increased depending on the strain. They suggested that it remains unknown whether DHEA exerted its chemopreventive effects through its induction of liver peroxisome proliferation. Schauer and colleagues (1990) studies the effect of DHEA on enzymes that detoxify toxic oxygen species. They demonstrated that 0.4% DHEA treatment lowered hepatic cytosolic selenium-dependent glutathione peroxidase (GPX) and increased hepatic mitochondrial superoxide dismutase (SOD). DHEA decreased c-GPX in skeletal muscle. They proposed that DHEA acted to improve efficiency of oxygen utilization at the tissue level with lower antioxidant enzyme activity in liver and locally protective up-regulation in muscle. Marrero et al (1990) treated mice of various strains with dietary DHEA (0.45% in diet, w/w) and examined the effects of DHEA on hepatic protein kinases, phosphatases, and lipogenic enzymes. They demonstrated that DHEA treatment lowered the specific activity of kinases for endogenous protein whereas it increased the specific activity of histone kinase. No changes were observed in the specific activities of casein kinase, cAMP-dependent protein kinase, cGMP-dependent protein kinase, G6PDH, NADP-linked isocitrate dehydrogenate and ATP-citrate lyase. Long-term treatment with DHEA increased the specific activities of liver AMPase and GTPase, but not other nucleotidases, alkaline phosphatase, acid phosphatase, glucose-6-phosphatase, or phosphotyrosine phosphatase. DHEA also enhanced the activity of hepatic NAPD-linked malic enzyme in female mice of three different strains, but not in male C57BL/6mice. DHEA-treated mice exhibited lower rates of hepatic lipogenesis compared to controls. Heffner and Milam (1990) reported that DHEA served as a specific inhibitor of lung G-6PDH, which played an important role in preventing oxidant-induced lung injury through its antioxidant defense mechanisms. They demonstrated that DHEA infusion to isolated perfused rabbit lung produced an increase in xanthine oxidase-induced lung edema. DHEA also inhibited tissue G6PDH activity without affecting catalase, glutathione peroxidase, or superoxide dismutase activity. Su and Lardy (1991) reported that feeding 0.01 %, 0.1 % or 0.2% DHEA in the diet of rats resulted in the induction of liver mitochondiral sn-glycerol 3 -phosphate dehydrogenate and cytosolic malic enzyme. The effect of DHEA was completely overcome by simultaneous treatment with actinomycin D. DHEA also increased liver cytosolic lactate, sn-glycerol 30phosphate, and isocitrate dehydrogenases. DHEA showed no effect on malate or G6PDH. The inducing effect of DHEA was influenced by the thyroid status of the rats; being smallest in thyroidectomized rats and highest in rats treated with triiodothyronine. Marrero et al (1991), using one- and two-dimensional Polyacrylamide gel electrophoresis, found that DHEA treatment in rodents (mice and rats) caused increased levels in 26 proteins and a decrease in the levels of 7 proteins. Two marked induced proteins were enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase and an unknown 28 Kd protein. DHEA significantly decreased the levels and specific activity of carbamoyl phosphate synthetase-I. Brady et al (1991) examined the effect of DHEA on mitochondrial and peroxisomal proliferation as well as carnitine acyltransferase in lean and obese female Zucker rats. They showed that DHEA markedly enhanced the levels of total hepatic mitochondrial protein. DHEA increased enzyme activities, immunoreactive protein, mRNA levels and transcription rates for carnitine acyltransferases. The increase in enzyme activity was shown to be well correlated with transcription rates and mRNA levels. They concluded that DHEA

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exerts its regulatory effects on hepatic carnitine acyltransferases of female Zucker rats mainly at the transcriptional level. Mohan and Cleary (1991) studied the short-term effects of DHEAon mitochondrial respiration. They found that serum cholesterol, triacylglycerol, glucose, insulin, glucagon, thyroid hormones and hepatic G6PDH activities were unchanged in obese Zucker rats after 7 days treatment with DHEA. On the other hand, decreased levels of cardiolipin and phophatidylethanolamine and increased levels of phosphatidylcholine were observed after 7d of DHEA treatment. Their data suggested that mitochondrial respiration is the earliest factor affected by DHEA and may be associated with protein synthesis. Sakuma et al (1992) investigated the effect of DHEA on hepatic enzyme activities in rats, mice, hamsters and guinea pigs. They administrated DHEA (300 mg/kg b.w, per os) for 14 days. They found that the activities of peroxisomal beta-oxidation, 1-acylglycerophosphocholine acyteltransferase, malic enzyme and cytosolic palmitoyl-CoA were enhanced in rats and mice. Only omega-hydroxylation and 1-acylglycerophosphocholine acyltransferase activities were increased in the hamster, whereas no changes in enzyme activities were noted in guinea pigs. They proposed that their findings showed that there were species differences in the effect of DHEA on hepatic peroxisomal proliferation-associated enzymes. Yamada et al (1992) administered 50 μΜ DHEA for 5 days to cultured rat-hepatocytes with. They showed that DHEA caused a progressive increase in peroxisomal beta-oxidation and carnitine acetyltransferase activity. The effect of DHEA was dose-dependent with peak levels at 50-100 μΜ. They demonstrated that DHEA also induced the acyl-CoA oxidase, enoyl-CoA hydratae/3-hydroxyacyl-CoA dehydrogenate bifunctional enzyme, carnitine acetyltransferase, and fatty acid omega-hydroxylase proteins. They suggested that the enzyme inducing effect of DHEA may be due to its direct action of DHEA on liver cells. The Rao group (1992) examined DHEA-induced peroxisomal proliferation in the rat liver. They showed that dietary DHEA (0.45% for 2 weeks) increased liver weight and volume density of peroxisomes. A significant increase in 80,000-molecular weight polypeptides was revealed in postnuclear fraction of livers using SDS-PAGE electrophoresis. The mRNA of catalase and peroxisomal enol-CoA hydratase/3-hydroxyacyl-CoA dehydrogenate bifunctional enzyme(PBE) was also increased. A single large dose of DHEA resulted in increased PBE mRNA levels by 24 h. They concluded that all these findings are qualitatively similar to those caused by a variety of different peroxisomal proliferators, indicating that DHEA may be another proliferator of peroxisomes. Laychock and Bauer (1996) treated isolated rat islets or RIN m5F insulinoma cells with DHEA and IL-1 beta, followed by a wash step. When DHEA was not washed from islets before determination of insulin release, the presence of DHEA inhibited insulin release in both freshly isolated and cultured islets. DHEA significantly reduced nitrite formation in islets in RIN m5F cells in response to IL-1 beta. DHEA showed no effect on cell growth, DNA or protein content. [U~14C] glucose oxidation in islet RINm5F cells was suppressed by DHEA. Total glucose oxidation and utilization were inhibited by DHEA. DHEA also reduced G6PDH activity when added to the tissue islet homogenate. Their results demonstrated that DHEA antagonizes the effects of IL-beta on beta-cells by inhibition of glucose metabolism, which in turn protects the cells from nitric oxide synthetase activation and related toxicities.

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Khan and Nyce (1997) treated male F-344 rats with DHEA (300 mg/kg orally for 14 days). They showed that DHEA significantly increased hepatic activity of peroxisomal beta-oxidation, 3-ketoacyl-CoA thiolase and catalase. Their finding supported the view that DHEA acts as a peroxisome proliferator. Falus et al (1990) studied the effect of DHEA on the production of the CI inhibitor Clinh, a member of the serine protease inhibitor gene superfamily) in human cell lines. They found that DHEA enhanced gene expression and secretion of Clinh in both human monocytoid/histiocytoid cell line U937 and in a hepatoma derived cell line HepG2. The peak effect of DHEA occured at the concentration of 10~7 -10~9 M. DHEA showed no effect on the biosynthesis of C3, C2 and factor Β in both cell lines. 10~4 Μ DHEA only increased the production of C4 in hepG2 cells.

D H E A a n d Lipid Peroxidation Swierczynski and Mayer (1996) reported that DHEA treatment increased NADPH-dependent lipid peroxidation in mitochondria isolated from the liver, kidney and heart, but not from brain. Dietary DHEA (0.6%w/w) exhibited its effect after 3 d treatment and attained peak levels between 1 and 2 weeks. 0.001-0.02% of DHEA had no effect on lipid peroxidation, whereas >or = 0.05 % DHEA significantly enhanced lipid peroxidation. DHEA (0.1-100 μΜ) added to mitochondria isolated from control rats exerted no effect on lipid peroxidation. They suggested that the effect of DHEA may be mediated by an intracellular process. The effect of DHEA administration at pharmacological doses on the mitochondrial membrane lipid peroxidation is an early effect of DHE A on tissues. Boccuzzi et al (1997) investigated the function of DHEA as an antioxidant in vivo. They administered to male rats a single ip dose of DHEA and then isolated liver and brain microsomes, and plasma LDL from rats. After exposing liver and brain microsomes, and plasma LDL to CuS0 4 , they measured the time course of lipid peroxidation by measuring the production of thiobarbituric acid reactive substances (TBARS). They found that DHEA significantly postponed the onset of TBARS generation from both liver and brain microsomes. DHEA treatment markedly enhanced the resistance of LDL to oxidation. They concluded that DHEA treatment increased the resistance of subcellular fractions isolated from different tissues and plasma constituents to lipid peroxidation induced by copper. The antioxidant effect of DHEA on plasma LDL may contribute to its antiatherogenic activity. The multitargeted antioxidant effect of DHEA may protect tissues from oxygen radical injury. Araghiniknam et al (1996) studied the effects on DHEA given to volunteers (aged 65-82 years). They found that DHEA significantly decreased serum lipid peroxidation, serum triglycerides, phospholipid and increased HDL levels.They concluded that DHEA has a significant antioxidant activities on serum lipid levels. Mcintosh et al (1993) studied effects of VitE in rats treated with DHEA. Rats were divided into four groups, treated with DHEA [100mg/(kg body wt.d) i.p.], vitamin Ε (lg/kg diet), DHEA+VitE and controls. Levels of serum triglycerides and total cholesterol in DHEAtreated rats were lower than controls. The rate of hepatic peroximal fatty acid oxidation, in DHEA-treated rats were 2.4 times higher than in control or vitamin Ε supplemented rats. Also the specific activities of enzymes that defend against oxidative stress (e.g., alanine and

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aspartate aminotransferases) or are indicators of tissue damage (e.g., alanine and aspartate aminotransferases) in DHEA treated rats are significantly higher than controls. On the other hand, DHEA+VitE rats had lower indices of oxidative stress is compared to rats treated with DHEA alone.They concluded that potential oxidative damage associated with DHEA treatment can be protected with supplementation on VitE. Rifici et al (1992) examined the effect of DHEA on copper-catalyzed and mononuclear cellmediated oxidation of LDL by measuring the production of thiobarbituric acid-reactive substances. They found that DHEA in contrast to Boccuzzi (1997) had no significant effect on lipid peroxidation.

The Effect of DHEA on Ethanol and Pentobarbital Lohman et al (1997) assessed the effect of DHEA on protection of isolated rat cremaster muscle flaps from ischemia/reperfusion injury. They demonstrated that in control animals, there was significant reduction in red blood cell velocity and in functional capillary density. Control animals also showed total cessation of flow by 24 hours. Reflow of the animals pretreated with DHEA occurred in 100% of the flaps. There was only a temporary reduction in functional capillary density in DHEA-treated animals. They indicated that their study showed that DHEA pretreatment significantly protected muscle flap microcirculatory hemodynamics from ischemia reperfusion injury.

The Effects of DHEA on Bone Metabolism Kasperk (1997) found that DHEA had a stimulatory effect on proliferation and differentiation of human osteoblastic cells (HOC). The androgen receptor antagonist hydroxyflutamide and inhibitory transforming growth factor beta antibodies (TGF-beta ab), but not 3 beta-hydroxysteroid dehydrogenase (3-betaHSD) and 5-alpha-reductase(5-AR) inhibitor: 17beta-N,N40% overweight, a = P C — >

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in preneoplastic cells, coupled with acceleration of phenotypic reversion of A H F (Garcea et al., 1987) may explain chemoprevention by DHEA treatments A and C (see also Moore et al., 1988). Interestingly, 0.6 % DHEA in diet, administered to rats for 15 weeks after the developments of neoplastic nodules, induces a decrease in nodule multiplicity, coupled with a morphologic shift of nodules and HCCs towards a less atypical pattern (Simile et al., 1995). Moreover, although total nodule incidence does not change, DHEA causes a decrease in the incidence of larger nodules (3-15 mm in diameter), indicating growth restraint in persistent nodules and suggesting that late liver lesions are still susceptible to the DHEA inhibitory effect. These results are in keeping with the observation that DHEA inhibits the development of glutathione S-transferase (placental) (GST-P)-positive lesions, in rats pretreated with azaserine (Thornton et al., 1989). Administration of 0.25 % DHEA in diet, throughout the experiment, to rats subjected to N-nitrosomorpholine ( N N M ) for seven weeks (Weber et al., 1988) causes a decrease in incidence of angiosarcomas, without changes in overall incidence of HCCs. DHEA treatment reduces the mitotic index of neoplastic cells and induces a phenotypic shift of HCC towards more differentiated forms. This latter phenomenon is associated with phenotypic modulation of hepatocellular adenomas, with decrease in number of mixed cell type adenomas and increase in amphophilic/tigroid cell type adenomas. This behavior clearly suggests that DHEA may cause changes in the cellular lineages involved in the development of HCCs. Studies on phenotypic changes occurring during the evolution of AHF to cancer (Bannasch et al., 1998) have allowed the identification of different cellular lineages the most frequent of which is represented by the glycogen-storing foci which evolve to mixed cell and basophilic lesions and then to undifferentiated basophilic carcinomas. A second lineage is represented by the amphophilic foci and amphophilic/tigroid foci which evolve to differentiated carcinomas. Sprague-Dowley rats treated with /?-dimethylaminoazobenzene followed by DHEA exhibit a decrease in GST-P-positive foci and in mixed cell foci, and a rise in amphophilic cell foci which are precursors of less malignant carcinomas (Moore et al., 1988). Phenotypic modulation of preneoplastic lesions appears to be sex-linked. Indeed, administration of DHEA either before or after treatment of male and female rats with DHPN, results in a comparable decrease in GST-P-positive lesions in both sexes, but amphophilic foci are only present in males (Moore et al., 1986). Since these latter lesions do not express enzyme markers, it may be concluded that the decrease in GST-P- and GGT-positive foci (Moore et al., 1986; Garcea et al., 1987), in DHEA-treated rats, cannot be ascribed to phenotypic shift leading to the appearance of amphophilic foci, at least in female rats. Above results indicate that DHEA induces a restraint in growth rate of preneoplastic and neoplastic liver lesions and their phenotypic shift towards less malignant forms. It should be noted, however, that an increase in incidence of GST-P-positive lesions and HCCs has been reported in rats sequentially treated with five different carcinogens and then fed a diet containing 0.3 % DHEA (Table 1; Shibata et al., 1995). In conclusion, it clearly appears that DHEA exerts a chemopreventive effect on tumorigenesis in various tissues. This effect may be largely influenced by nature of carcinogens, promoting treatment, DHEA dose and administration schedule, target tissue, development stage of preneoplastic lesions, species and sex of experimental animals. All of these conditions must be taken into account for the interpretation of the results of experimental researches, especially to plan studies on the effect of DHEA and synthetic DHEA analog (see below) in human cancer prevention.

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Tumor Induction by DHEA The data in Table 1 show that DHEA may enhance carcinogenesis of lung (Moore et al., 1988), kidney (Oigu et al., 1990), pancreas (Tagliaferro et al., 1992; Thornton et al., 1989), and liver (Shibata et al., 1995) in animals treated with genotoxic carcinogens. This has been observed with relatively high DHEA doses given either during or after carcinogen administration, and suggests that DHEA could act as a tumor promoting compound. In addition, recent work has shown that DHEA may be a complete carcinogen. Rao and coworkers have observed the development of well differentiated HCC, negative for GGT and GST-P markers, in 88 % of male F344 rats fed for 84 weeks a diet containing 0.45 % DHEA (Rao et al., 1992). These results have been confirmed by Hayashi and coworkers (Hayashi et al., 1994) who documented 56 % and 100 % incidences of liver tumors in male F344 rats fed 0.5 % and 1 % DHEA, in diet, for 72 weeks. Lower incidence (20 %) was seen when these DHEA doses were given for only 52 weeks. Experiments with the less susceptible Sprague-Dowley rat strain by Metzger and coworkers (Metzger et al., 1995), have shown that administration of a 0.6 % DHEA diet for 70-84 weeks, results in the development of hepatocellular adenomas in 33 % of female rats, whereas no adenomas develop in male rats. Well differentiated trabecular carcinomas were seen in 44 % of female and 11 % of male rats. These results clearly demonstrate that the carcinogenic effect of DHEA is characterized by the development of low grade carcinomas and sex specificity. Pleiotropic responses of rat liver to the steroid, such as hepatomegaly, peroxisome proliferation, induction of peroxisomal and endoplasmic reticulum enzymes may be involved in this effect (Prough et al., 1990; Garcea et al., 1987; Metzger et al., 1995; Mayer et al., 1990). Studies on the morphogenesis of HCCs, induced by DHEA, have shown that they originate from preneoplastic amphophilic foci (Metzger et al., 1995) which exhibit, similarly to DHEA-induced tumors, high proliferative rate, slightly atypical mitochondria, and moderate increase in number of peroxisomes (Bannasch et al., 1998). DHEA administered for 30 weeks to rainbow trout, after initiation of hepatocarcinogenesis with aflatoxin Β1, produces a dose-dependent enhancement of tumorigenesis, as measured by tumor incidence, multiplicity and size (Omer et al., 1995). However, DHEA can also act as a complete carcinogen, in this animal, inducing liver tumors at doses as low as 2 2 2 - 4 4 4 p.p.m., even in the absence of aflatoxin B l . These tumors contained Ki-ras mutations, primarily at codon 12[1] G -*• A. This carcinogenic effect of DHEA appears to be independent of peroxisome proliferation, as suggested by the determination of peroxisomal ß-oxidation and catalase activities indicating that trout, like humans, is a weak responder to peroxisome proliferators. These interesting observations first suggest that DHEA may be a genotoxic carcinogen, at least for trout liver. Due to the similarities between trout and humans, as concerns the response to peroxisome proliferators, it would be prudent to reconsider clinical trials involving long-term treatments with high DHEA doses (Boone and Kelloff, 1997).

Human Studies Hormone sensitivity of breast cancer and some other tumors, has prompted several groups to undergo accurate analyses of the relationships of DHEA serum levels to tumorigenesis. Although human breast cancer cells possess most of the enzymatic system involved in

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androgen and estrogen metabolism and can synthesize DHEA from estradiol (Theriault and Labrie, 1991), the plasma source of DHEA seems to contribute remarkably to DHEA present within breast cancer (Massobrio et al., 1994). DHEA-S can be converted into potent androgens or estrogens in peripheral tissues, and breast tissue in vitro can convert DHEA and DHEA-S to hydroxy-DHEA and androstenediol, a steroid with potent estrogenic properties, which can compete with estrogens for estrogen receptors (Poortman et al., 1975; Hackenbergetal., 1993). Previous epidemiological studies have shown a negative correlation between plasma and urinary levels of DHEA and DHEA derivatives, and breast cancer (Bulbrook et al., 1962; Brennan et al., 1973; Browsey et al., 1972; Wang et al., 1974; Gomes et al., 1988). However, recent researches on the correlation between DHEA or DHEA-S plasma concentrations and breast carcinogenesis have given different results (Pasqualini, 1993). It has been reported no correlation (Bernstein et al., 1990; Barrett-Connor et al., 1990; Helzlsouer et al., 1992; Zeleniuch-Jacquotte et al., 1997) or positive correlation (Secreto et al., 1991; Gordon et al., 1990; Dorgan et al., 1997) between DHEA-S plasma levels and risk of breast cancer. Interestingly, consumption of soybean diets, which contain phytoestrogens, causes decrease in incidence of breast, prostate, and colon cancer, as well as decrease in the levels of ovarian steroids and adrenal androgens, including DHEA-S (Lu et al., 1996). A positive correlation between DHEA and androstenedione in serum and ovarian cancer risk has also been reported (Helzlsouer et al., 1995). These results support a role of the adrenal hormones DHEA, DHEA-S, and androstenediol, in the etiology of breast cancer and probably of other hormone sensitive tumors. On the other hand there exists a well known association between elevated estrogen plasma levels, high estrogen receptor expression in breast tissue and incidence of breast cancer, while androgens have a protective effect (Pasqualini, 1993). Stimulation of in vitro growth of human breast cancer cells by DHEA is linked to the stimulation of estrogen receptors (Najid and Habrioux, 1990; Boccuzzi et al., 1982). Thus, contrasting results could reflect different capacities of DHEA to be metabolized into estrogens which stimulate growth of mammary tumor cells, or androgens with antitumor effect (Boccuzzi et al., 1992). Epidemiological studies in humans have considered physiologic variations in DHEA and DHEA-S plasma levels. The results of animal studies cannot be simply extended to humans, taking into account that rats, at difference with humans, do not produce DHEA in adrenals (Van Werden et al., 1992), and their basal plasma levels of DHEA are consequently very low (Hobe et al., 1994). Prevention of breast cancer in rodents has been obtained by administration of pharmacological DHEA doses. However, it cannot be excluded that rodents have different capacity to transform exogenous DHEA into androgen and/or estrogen compounds, with respect to humans. Studies on chemoprevention of human breast cancer by DHEA should take into account the possibility that hormonal action of high DHEA doses stimulates mammary carcinogenesis, instead of preventing it. This difficulty could be overcome by the use of synthetic steroids, such as 16-a-fluoro-5-androsten-17-one, which does not demonstrate the androgenic and estrogenic effects of DHEA, but yet retains the antiproliferative and cancer preventive activity of the native steroid (Schwartz and Pashko, 1995).

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Mechanistic Approach The pleiotropic effects of pharmacological DHEA doses implicate the interaction of this compound with different biological functions. Although a number of studies have explored the mechanisms underlying modulation of carcinogenesis by DHEA, no definitive conclusions have been reached as yet. Various mechanisms have been considered, including changes in carbohydrate and cholesterol metabolism, production of free radicals and lipid peroxidation, peroxisome proliferation, but none of them can fully explain the chemopreventive and/or carcinogenic effect of DHEA.

Alteration of Carbohydrate Metabolism DHEA is a potent non-competitive inhibitor of G6PD (Marks and Banks, 1960), the ratelimiting enzyme of the hexose monophosphate pathway (HMP). The primary function of HMP is the production of NADPH and ribose-5-phosphate, necessary for the synthesis of ribo- and deoxyribo-nucleotides. Most preneoplastic and neoplastic lesions from different tissues exhibit high levels of G6PD and HMP activity (reviewed by Feo and Pascale, 1990). It has been hypothesized, on the basis of these observations that the inhibition of tumor promotion and progression by DHEA may result from decrease in G6PD activity (Schwartz et al., 1988; Feo and Pascale, 1990; Schwartz and Pashko, 1995). In keeping with this conclusion is the observation that 16-a-bromo-epiandrosterone, much stronger inhibitor of G6PD than DHEA, is 25-fold more active as an inhibitor of phorbol ester-stimulated epidermal DNA synthesis in ICR mice (Pashko et al., 1984). Moreover, inhibition of carcinogen activation by mixed-function oxydase system, and decrease in production of oxygen free radicals from paraquat, and in oxidative burst generation of superoxide radicals by stimulated neutrophils, have been attributed to a restraint in NADPH production in DHEA-treated animals (Schwartz and Pashko, 1993). Nevertheless, the determination of G6PD activity in liver of DHEA-treated rats has given contrasting results including: inhibition (Garcea et al., 1987; Mayer et al., 1990; Garcea et al., 1988; Mayer et al., 1988; Tagliaferro et al., 1986), no change (Shepherd and Cleary, 1984; Casazza et al., 1986), increase (Shibata et al., 1995; Mayer et al., 1998), or lowered activity only when G6PD is elevated by fasting-refeeding (Wilmer and Foster, 1965; Tepperman et al., 1968; Hilgertova et al., 1973), sucrose feeding (Cleary et al., 1984) or genetic predisposition (Shepherd and Cleary, 1984). In DHEA-treated rats, G6PD of preneoplastic lesions has been found to undergo decrease (Garcea et al., 1987; Simile et al., 1995) or no change, in females, and increase, in males (Mayer et al., 1996). Moreover, NADPH content does not change in normal liver and preneoplastic lesions of these rats, probably due to the overexpression of other NADPH-producing enzymes, such as isocitric dehydrogenase and malic enzyme (Garcea et al., 1988). Maybe contrasting results on modulation of HMP by DHEA, in vivo, depend on rat strain, sex and age, DHEA dose and feeding schedule, DHEA content in diet (a 47 % loss of DHEA content occurs, at 22 °C, 48 h after preparation of the DHEA diet, 5), and cytological type and developmental stage of preneoplastic lesions. A decrease in normal and preneoplastic liver content of ribulose-5-phosphate, an immediate precursor of ribose-5-phosphate, has been found in female rats fed a 0.6 % DHEA diet for two weeks, in conditions that implicate inhibition of G6PD and HMP activities (Garcea et al., 1988). The role of these changes on the modulation of cell proliferation has been docu-

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mented in various experimental systems. DHE A slows the growth of various cell lines in culture and blocks the differentiation in vitro of 3T3 fibroblasts to adipocytes, a phenomenon linked to cell proliferation (Gordon et al., 1987). These DHEA effects are partially overcome by addition to the culture medium of a mixture of four ribo- or deoxyribo-nucleosides of uridine/thymine, adenine, guanine and cytosine (Dvorkin et al., 1986). Furthermore, inhibition of DHEA of growth and progression of preneoplastic lesions in liver (Garcea et al., 1988), urinary bladder (Shibata et al., 1993) and skin (Pashko et al., 1991), can be prevented by treating the animals with a mixture of ribo- and/or deoxyribo-nucleosides. Labeled ribo- and deoxyribo-nucleosides produce derivatives that are incorporated into rat liver DNA (Garcea et al., 1988). These results clearly indicate that inhibition of G6PD activity is strongly involved in DHEA chemopreventive effect, at least in the experimental systems characterized by the development of cancer precursor lesions exhibiting elevated growth rate and G6PD activity. Mayer et al. (1996; 1998) have analyzed, in recent studies, the effect of short- and long-term treatments with 0.6 % DHEA in diet, on carbohydrate metabolism in the liver of male and female Sprague-Dowley rats. There occurred a persistent decrease, in both sexes, of key enzymes of glycolysis (glucokinase, hexokinase, pyruvate kinase) and gluconeogenesis (glucose-6-phosphatase and fructose- 1,6-biphosphatase). Short- and long-term DHEA treatments resulted in similar alterations, with the exception of an increase in the activity of gluconeogenic enzymes after a 3-day DHEA administration, and followed by a decrease after longer treatments (7-14 days). Glycogen content and glycogen phosphorylase were reduced after three days of treatment. G6PD decreased, in female rats, during the first seven days of treatment, and then returned to control values, while it increased in males along the treatment. Malic enzyme was strongly induced, especially in males. Determination of glucokinase and phosphorylase mRNA levels, showed a 62 % fall in male rats treated with DHEA for 14 days. These observations indicate that DHEA may equally affect, in both sexes, enzymes involved in opposite pathways of glucose metabolism, such as glycolysis and gluconeogenesis. Downregulation of the expression of genes encoding some key enzymes, suggests the implication of DHEA in some central regulatory mechanisms. An investigation on various enzymatic activities, by histo/immunohisto-chemistry, has shown clear differences between glycogen storing/basophilic cell foci, induced by NNM in rat liver, and amphophilic cell foci, developing in rats treated with DHEA or NNM plus DHEA (Mayer et al., 1998). Glycogen storing and basophilic cell foci exhibited increase in G6PD, pyruvate kinase, glycogen synthase, glutamate dehydrogenade, and malic enzyme activities, and decrease in glucose-6-phosphatase, glycogen phosphorylase, acid phosphatase and peroxisomal enzymes, with respect to normal liver. Amphophilic foci showed increase in mitochondrial enzyme activities, such as cytochrome oxidase, succinate and glycerol3-phosphate dehydrogenases and, to a lower extent, in peroxisomal enzymes. There also occurred decrease in pyruvate kinase and no change in G6PD and malic enzyme activities. Similar alterations were found in malignant lesions derived from the different cytologic types of foci. Above data, considered altogether, indicate that the alterations of carbohydrate metabolism, in DHEA-treated rats, are complex and the role of these changes in the appearance of amphophilic cell foci is unclear. Available evidence indicates that G6PD inhibition by DHEA could be one of the factors involved in tumor chemoprevention, but this metabolic change is not always necessary and, presumably, sufficient for the DHEA anti-tumor effect. Other

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alterations of carbohydrate metabolism could also be involved in the anticarcinogenic effect of the administration of high DHE A doses. The enzyme pattern of preneoplastic and neoplastic lesions induced by DHEA is in many respects opposite to that of the lesions induced by genotoxic carcinogens. This could reflect alterations of different regulatory pathways in the various cytologic types of lesions.

Alteration of Cholesterol Metabolism HMP has close relationships to lipid metabolism: NADPH is required for reductive synthesis of fatty acids and cholesterol. It has been postulated that a decrease in DHEA and DHEA-S concentrations will result in a rise in lipogenesis. Anti-cholesterolemic action of DHEA has been demonstrated in rodents and humans (MacEwen et al., 1990). However, the absence of NADPH fall in liver of DHEA-treated rats (Garcea et al., 1988), indicates that inhibition of NADPH-dependent steps is not responsible for alteration of lipid metabolism, consequent to changes in DHEA tissue content, at least in rat liver. Elevated cholesterol levels and enhanced cholesterogenesis are consistent observations in proliferating normal, preneoplastic and neoplastic cells (Rao, 1986). Different laboratories have suggested a linkage between cholesterol and DNA synthetic pathways (Perkins et al., 1997; QuesneyHuneenus et al., 1979). The exact nature of this linkage is not known, but mevalonate and other intermediate compounds in cholesterol synthetic pathway have suggested to be mediators of DNA synthesis (Quesney-Huneenus et al., 1979). Lipid acylation and binding of farnesyl phosphate to a cysteine residue of G-proteins and p21 ras oncoprotein is necessary for biological activity (Larson, 1987; Hanckock et al., 1989; Beck et al., 1988). The rate-limiting step of cholesterol biosynthesis is represented by the conversion of 3-hydroxymethyl glutaryl-CoA to mevalonate, catalyzed by 3-hydroxymethyl glutaryl-CoA reductase (HMGR). Mevalonate represents a precursor not only of cholesterol, but also of different non sterol isoprenoids, including farnesyl-pyrophosphate and geranyl-geranyl-pyrophosphate. These compounds are used for post-transcriptional modification of various cell proteins (Maltese et al., 1990), ubiquinone side chains, necessary for oxidative respiration (Battino et al., 1990), and dolichol, involved in glycoprotein biosynthesis (Chojnaki and Dallner, 1988). Depletion of mevalonate derivatives can inhibit the isoprenylation of various proteins, including p21 ras . This could modulate signal transduction pathways regulated by this protein. Studies on DHEA effect on cholesterol synthesis in vitro have shown an inhibitory effect on the incorporation of labeled acetate into cholesterol in slices of normal liver and preneoplastic liver lesions isolated from rats fed for 15 days a diet containing 0.6 % DHEA (Feo et al., 1991). Similarly, in vivo studies have shown a decrease in the incorporation of 3 H 2 0 into cholesterol of preneoplastic liver nodules in DHEA-treated rats (Feo et al., 1991). DHEA effect on cholesterol synthesis was no longer observed when labeled mevalonate substituted acetate in in vitro experiments, indicating that inhibition of cholesterol biosynthesis by DHEA precedes mevalonate synthesis. Available evidence indicates that the target of DHEA action is HMGR activity (Gianelly and Terner, 1968; Pascale et al., 1995). HMGR activity and gene expression are higher in preneoplastic rat liver lesions, 18 weeks after initiation with DENA followed by selection according to RH protocol, than in normal liver (Pascale et al., 1995). The administration of a 0.6 % DHEA diet for three weeks before killing, results in a strong decrease in the expression of HMGR gene and in HMGR activity in persistent nodules (Table 2). DHEA is without effect when added in vitro to the reaction

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Table 2 Effect of D H E A on 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR and low density lipoprotein receptor (LDL-R) m R N A levels and activities in neoplastic liver nodules LDL-R

HMGR 3

Normal liver DHEA Nodules Nodules/DHEA

m R N A (n = 3)

Activity

13

0.25 ±0.02

7.67 2.53 138.80 23.25

± 3.2 ± 1.2 ± 16.2 ±2.0

5.40 ±0.37 1.49 ±0.10

(n = 11)

a

m R N A (n = 3)

Activity 0 (n = 11)

3.60 ±0.30

45.06 55.91 38.60 47.80

2.01 ±0.20 2.35 ±0.05

±6.1 ±4.4 ± 4.2 ± 3.0

a

Slot blot of 1.5 μ9 of m R N A f r o m n o r m a l liver a n d nodules 18 w e e k s after initiation of m a l e F344 rats with diethylnitrosamine f o l l o w e d by selection a c c o r d i n g to "resistant hepatocyte" m o d e l . W h e n indicated, rats w e r e fed a 0.6 % D H E A diet for 3 w e e k s before killing. Data are m e a n s ± S D of density values (arbitrary units after normalization to reference gene glyceraldehyde-3phosphate dehydrogenase). b

n m o l of m e v a l o n a t e produced/30 m i n / m g protein. Data are m e a n s ± SD.

c

Heparin-sensitive

126

l - L D L - b i n d i n g e x p r e s s e d as n m o l of b o u n d L D L / m g protein. Data are m e a n s ± SD.

"t"-Test: m R N A : n o d u l e s vs. n o r m a l liver, Ρ < 0.0001 for H M G R and Ρ 32 weeks) resulted in an extension of the small perivenular zone occupied by the granular acidophilic cells to several cell layers around the central vein. The proliferation of peroxisomes in the liver parenchyma of males was much less pronounced, but the organelles were frequently conspicuous by their irregular cuplike shape and numerous striated paracristalline inclusions. In both genders, an accumulation of lipofuscin was seen throughout the liver parenchyma at later time points of the experiment.

4 Induction of Hepatocellular Neoplasms The hepatocarcinogenic effect of DHEA in rats and rainbow trout has been established independently in several laboratories (Rao et al., 1992a, Rao et al., 1992b, Hayashi et al., 1994, Metzger et al., 1995, Omer et al., 1995). In two different strains of rat, long-term administration of high doses of DHEA in the diet resulted in well-differentiated trabecular or solid hepatocellular neoplasms (Table 1). The neoplasms did not express γ-glutamyltransferase (GGT) and the placental form of the glutathione-S-transferase (GSTP), which are usually demonstrable by cytochemical approaches in hepatocellular neoplasms induced by the majority of chemicals, but are also lacking in adenomas and carcinomas produced by other peroxisome proliferators (Rao et al., 1992a, 1992b). Rao and colleagues (1992a) found hepatocellular carcinomas in 88% of male F-344 rats exposed to 0.45% (w/w) DHEA in the diet for 70-84 weeks. After feeding DHEA to male F-344 rats at two different dose levels (0.5% and 1%) for 52 and 72 weeks, Hayashi et al. (1994) have shown that the hepatocarcinogenic effect of DHEA is dose- and time-dependent, resulting in a tumor incidence of 100% after exposure to the higher dose level for 72 weeks. Metzger et al (1995) studied the effect of dietary DHEA (0.6%) in both male and female Sprague-Dawley rats. Hepatocellular adenomas developed in 33% of the female rats after 70-84 weeks of treatment, but not in male rats (Table 2). Carcinomas, defined as proliferative lesions characterized by trabecules thicker than three cell layers, were found in 44% of the females and in 11% of the males, but metastases were never observed. The lower tumor incidence as compared to the findings reported by Rao and colleagues (1992a) and Hayashi and coworkers (1994) may be due to the generally lower susceptibility of Sprague-Dawley rats, compared to F-344 rats, to hepatocarcinogenic agents. In Sprague-Dawley rats, the higher tumor incidence in females correlates with the higher DHEA(S) levels measured in the plasma as compared to male rats (Hobe et al., 1994a). In rainbow trout, feeding of DHEA produced liver tumors, most of which were classified as mixed hepatocholangiocellular carcinomas (60%), and hepatocellular carcinomas (30%) (Omer et al., 1995). The neoplasms developed at dose levels as low as 444-888 p.p.m. These observations are of particular interest for the understanding of the mechanism of DHEA-induced hepatocarcinogenesis, since rainbow trout, like humans, react only weakly to the peroxisome proliferative effect of chemicals.

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5 Enhancement of Hepatocarcinogenesis Simultaneous exposure of rats to DHEA and NNM did not significantly alter the incidence of hepatocellular neoplasms induced by NNM alone, but modulated the phenotype of preneoplastic and neoplastic hepatocellular lesions as reviewed elsewhere (Mayer, 1998). When DHEA was given subsequently to NNM for up to 32 weeks, however, the incidence of hepatocellular neoplasms increased significantly (Metzger et al., 1997). While the combined incidence of hepatocellular adenomas and carcinomas was similar in male and female rats treated with NNM alone, it was higher in females than in males after additional administration of DHEA (Table 2). This sex difference was mainly due to a higher incidence of hepatocellular carcinomas in females compared to males. In rainbow trout, DHEA fed for 30 weeks after exposure to aflatoxin Β1 for 30 min also produced an enhancement of hepatocarcinogenesis, which was dose-dependent as measured by incidence, multiplicity and size of the tumors (Omer et al., 1995). This effect was detectable down to a dose level of 222 p.p.m., corresponding to daily dosage one-half that previously given to humans in a clinical trial. Investigations on other animal models of chemical carcinogenesis have shown that the tumor enhancing effect of DHEA is not limited to the liver, but is also exerted in other tissues of the rat. This applies to carcinogenesis induced in the lung by dehydroxy-di-n-propylnitrosamine (Moore et al., 1988), in the exocrine pancreas by asazerine (Thornton et al., 1989), and in the renal parenchyma and mesenchyma by dimethylnitrosamine (Ogiu et al., 1990).

6 Preneoplastic Amphophilic Cell Foci Detailed studies on the sequence of cellular changes emerging during neoplastic development in the liver of rats treated with 0.6% (w/w) DHEA revealed that preneoplastic foci of altered hepatocytes, classified as amphophilic cell foci (APF) (Weber et al., 1988), started to develop after 20 weeks (Metzger et al., 1995). Single cells exhibiting the same phenotype Table 2 Incidence of hepatocellular adenomas and carcinomas in male and female rats treated with dehydroepiandrosterone (DHEA), with N-nitrosomorpholine (NNM) or with N-nitrosomorpholine followed by dehydroepiandrosterone (NNM/DHEA). Percentage of tumor-bearing rats is given in brackets (from 15). Treatment3 DHEA b NNM NNM/DHEA 3

Female rats

Male rats

HCA

HCC

HCA

HCC

3/9 (33%)c 10/30 (33%) 12/30 (40%)

4/9 (44%)c 8/30 (27%) 14/30 (47%) c ' d

0/9 (0%) 10/30 (33%) 13/30 (43%)d

1/9(11%) 9/30 (30%) 11/30(37%)

Rats were treated with N N M followed either by standard diet or by diet containing DHEA. Groups of rats were sacrificed immediately after stop of NNM-treatment and at 4 , 2 0 , 3 2 , 7 0 and 84 weeks of DHEA-treatment. In DHEA-treated rats tumors occurred only after 70-84 weeks, in NNM and NNM/DHEA-treated rats tumors were observed between 4 and 32 weeks after stop of NNMtreatment. b Only rats killed after 70-84 weeks of DHEA-treatment are included in this line. c d Significantly different from males, and from NMM, Ρ < 0.05.

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Figure 4 a) Amphophilic cell focus induced in female rat liver by exposure to DHEA for 70 weeks, showing marked loss of glycogen as detected underthe light microscope in paraffin section by the periodic acid Schiff-reaction. b) Serial section to a) exhibiting both acidophilic and basophilic components resulting in an amphophilic phenotype as demonstrated by H&E-staining. c) Serial section to a) and b) showing a pronounced cytoplasmic basophilia after staining with cresyl violet. Bar, 100 pm.

were sometimes found in addition to APF. In contrast to the predominantly perivenular peroxisome proliferation, the amphophilic cell populations developed in periportal and intermediate parts of the liver lobules, largely corresponding to zone 1 of the functional liver acinus as defined by Rappaport. The phenotype of APFs differs from that of both the normal liver parenchyma and the perivenular granular acidophilic hepatocytes by a more homogeneous cytoplasmic acidophilia associated with a distinct diffuse or somewhat scattered basophilia (Fig. 4), and a unique biochemical phenotype (Mayer et al., 1998) which will be discussed in detail in the second part of this review. The fine structure of the amphophilic cells (Fig. 5) is characterized by abundant cristae-rich mitochondria wrapped by cisternae of the rough endoplasmic reticulum, a slightly increased number of peroxisomes (specifically detected by their catalase activity), and a few remaining glycogen particles (Metzger et al., 1995). The peroxisomes exhibit normal size and matrix properties, but frequently contain striated paracristalline inclusions in male rats. When the APFs become larger at later (>32 weeks) stages of hepatocarcinogenesis they may extend from zone 1 to zone 2 of the liver acinus. Rarely, large focal lesions may become contiguous with the acidophilic granular hepatocytes surrounding the central vein. During progression to larger focal lesions, the cellular phenotype often changes from the amphophilic to the amphophilic/tigroid character with an increased structured basophilia (Metzger et al., 1995, Weber et al., 1988).

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Figure 5 a) Portion of amphophilic cell induced in the liver of a female rat by DHEA, as demonstrated electron microscopically in ultrathin section contrasted with lead citrate and specifically stained for catalase with DAB in peroxisomes (P). b) High-power view of the same amphophilic cell contrasted with lead citrate and uranyl acetate demonstrating the rough endoplasmic reticulum (RER) and free ribosomes. Bars, 1 pm (from 14).

Figure 6 a) Amphophilic/tigroid hepatocellular adenoma induced in female rat by exposure to DHEA for 70 weeks, as demonstrated light microscopically in paraffin section stained with cresyl violet. Bar, 100 μιτι. b) Higher magnification of portion from amphophilic/tigroid cell adenoma showing stripy cytoplasmic basophilia. Bar, 100 Mm. c) Highly differentiated hepatocellular carcinoma induced in female rat by oral exposure to DHEA for 84 weeks, as demonstrated light microscopically in paraffin section stained with cresyl violet. Bar, 200 μηι. d) Higher magnification of hepatocellular carcinoma showing trabeculae thickerthan three cell layers (arrows) as demonstrated light microscopically in paraffin section stained with cresyl violet. Bar, 100 μηι (from 14).

Hepatocarcinogenesis by Dehydroepiandosterone :

m

245

m w

' ·- 3

Figure 7 a) Portion from DHEA-induced amphophilic/tigroid hepatocellular adenoma, slightly compressing the surrounding parenchyma and showing prominent basophilic cytoplasmic components at the light microscopic level in paraffin section stained with cresyl violet, b) Portion from DHEA-induced hepatocellular carcinoma showing more pronounced and frequently also more homogeneous cytoplasmic basophilia compared to amphophilic/tigroid cell adenoma in paraffin section stained with cresyl violet. Bars, 100 pm.

Figure 8 a) Portion from DHEAinduced liver carcinoma cell as demonstrated electron microscopically after contrasting ultrathin section with lead citrate. Note abundant mitochondria and some peroxisomes. Bar, 2 pm. b and c) Higher magnification of cellular details from the same carcinoma showing numerous mitochondrial inclusions (b) and extended area of proliferated smooth endoplasmic reticulum (c). Bars, 1 pm. (from 14).

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7 Hepatocellular Adenomas and Carcinomas Hepatocellular neoplasms developing after long-term exposure of rats to DHEA have been described as highly differentiated by all authors (Rao et al., 1992a, Rao et al., 1992b, Hayashi et al., 1994, Metzger et al., 1995). More detailed electron microscopical investigations of such tumors were only reported by Metzger et al. (1995). As a result of dietary administration of 0.6% (w/w) DHEA to Sprague-Dawley rats, unequivocal neoplastic lesions occurred for the first time after 70 weeks. Both adenomas and carcinomas were of the amphophilic or the amphophilic/tigroid cell phenotype (Figs. 6 and 7). Whereas amphophilic/ tigroid cell populations predominated in many adenomas, amphophilic cells with a pronounced diffuse basophilic component prevailed in the carcinomas. The carcinomas were well demarcated from the liver parenchyma and did not metastasize. At the ultrastructural level, amphophilic cell adenomas showed a higher number of mitochondria and fewer peroxisomes than APFs. A strikingly high content of mitochondria some of which contained intracristal inclusions was also found in the highly differentiated hepatocellular carcinomas (Fig. 8). In addition to the mitochondrial alterations that were sometimes accompanied by a focal proliferation of the smooth endoplasmic reticulum, the cancer cells showed enlarged nuclei and nucleoli. The liver neoplasms induced in rainbow trout by DHEA with and without initiation by aflatoxin Β ( were classified as mixed hepatocholangiocellular carcinomas, hepatocellular carcinomas, mixed adenomas, hepatocellular adenomas, and cholangiomas, but were not described in greater detail (Omer et al., 1995).

8 Conclusions Dehydroepiandrosterone is a hormonal peroxisome proliferator producing hepatocellular carcinomas in at least two different species (rat, rainbow trout). Peroxisome proliferation as such does not seem to play a major role in the mechanism of neoplastic cell conversion elicited by DHEA. DHEA-induced hepatocarcinogenesis in rats represents a unique animal model of hormonal carcinogenesis, which has been instrumental in defining the amphophilic cell lineage and distinguishing it from the alternative glycogenotic-basophilic and the xenomorphic-tigroid cell lineages of neoplastic development in the liver (Mayer et al., 1998, Bannasch et al., 1997, Bannasch, 1996, Ströbel et al., 1998). The amphophilic phenotype is morphologically characterized by hepatocellular enlargement, a pronounced proliferation of mitochondria closely associated with cisternae of the rough endoplasmic reticulum, a moderate increase in peroxisomes, and glycogen loss. These morphological changes reflect early changes in energy metabolism, suggesting a thyromimetic effect of DHEA (and other peroxisome proliferators) as discussed in detail in the second part of this review. In addition to the rats treated with DHEA and other peroxisome proliferators, the amphophilic cell lineage has also been observed in hepadnaviral hepatocarcinogenesis in woodchucks (Bannasch et al., 1995). In chronic human liver diseases, associated with, or at high risk of, hepatocellular carcinomas, amphophilic cell populations are frequently found, but their preneoplastic nature remains to be established (Su et al., 1997). Experimental hepatocarcinogenesis induced by DHEA is an important model to further clarify the cellular and molecular mechanisms of neoplastic development in the liver. As a model of hormonal he-

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patocarcinogenesis, the DHEA-treated rat differs markedly from the streptozotocin-diabetic rat harboring intrahepatic pancreatic islet-transplants which hypersecrete insulin (Dombroski et al., 1996, 1997). The preneoplastic hepatocellular lineages emerging in this model many weeks or months before hepatocellular neoplasms become manifest, resemble those observed after exposure of different species to a variety of chemicals, radiation, and oncogenic viruses (Bannasch et al., 1997, Bannasch, 1996). These recent findings suggest that hormonal and hormone-like effects play a major role in the mechanism of hepatocarcinogenesis. In view of the unequivocal hepatocarcinogenic effect of DHEA in rats and fish, recommendations to use this compound in healthy and sick human beings should be carefully reconsidered.

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[16] Hamilton, S.R., Gordon, G.B., Floyd, J., and Golightly, S. (1991). Evaluation of dietary dehydroepiandrosterone for chemoprotection against tumorigenesis in premalignant colonic epithelium of male F-344 rats. Cancer Res. 51, 476-480. [17] Hayashi, F., Tamura, H., Yamada, J., Kasai, H., and Suga, T. (1994). Characteristics of the hepatocarcinogenesis caused by dehydroepiandrosterone, a peroxisome proliferator, in male F-344 rats. Carcinogenesis 15, 2215-2219. [18] Hobe, G., Hillesheim, H.G., Schön, R., Reddersen, G„ Knappe, R„ Bannasch, P., and Mayer, D. (1994a). Sex differences in dehydroepiandrosterone metabolism in the rat: different plasma levels following ingestion of DHEA-supplemented diet and different metabolite patterns in plasma , bile and urine. Horm. Metab. Res. 26, 326-329. [19] Hobe, G., Hillesheim, H.G., Schön, R„ Ritter, P., Reddersen, G., Mayer, D., and Bannasch, P. (1994b). Sex differences in dehydroepiandrosterone (DHEA) metabolism in the rat. In: Proceedings of the 5th Symposium on Analysis of Steroids. S. Görög, ed. (Budapest, Hungary: Akademiai Kiado), pp. 383-390. [20] Leighton, B., Tagliaferro, A.R., and Newsholme, E.A. The effect of dehydroepiandrosterone acetate on liver peroxisomal enzyme activities of male and female rats. J. Nutr. 117, 1287-1290 [21] Mayer, D., Weber, E., and Bannasch, P. (1990). Modulation of liver carcinogenesis by dehydroepiandrosterone. In: The Biologic Role of Dehydroepiandrosterone (DHEA). M. Kalimi and W. Regelson, eds. (Berlin, Germany: Walter de Gruyter), pp. 361-385. [22] Mayer, D. (1998). Carcinogenic and anticarcinogenic effects of dehydroepiandrosterone in the liver of male and female rats. The Aging Male 1, 56-66. [23] Mayer, D„ Metzger, C„ Leonetti, P., Beier, K„ Benner, Α., and Bannasch, P. (1998). Differential expression of key enzymes of energy metabolism in preneoplastic and neoplastic rat liver lesions induced by N-nitrosomorpholine and dehydroepiandrosterone. Int. J. Cancer (Pred. Oncol.) 79, 232-240. [24] Mayer, D., Weber, E., Moore, M.A., Letsch, I., Fislinger, E., and Bannasch, P. (1988). Dehydroepiandrosterone (DHEA) induced alterations in rat liver carbohydrate metabolism. Carcinogenesis 11, 2039-2049. [25] Mayer, D., Reuter, S., Hoffmann, H., Bocker, T., and Bannasch, P. (1996). Dehydroepiandrosterone reduces expression of glycolytic and gluconeogenic enzymes in the liver of male and female rats. Int. J. Oncol. 8, 1069-1078. [26] Metzger, C„ Mayer, D„ Hoffmann, H., Bocker, T., Hobe, G., Benner, Α., and Bannasch, P. (1995). Sequential appearance and ultrastructure of amphophilic cell foci, adenomas, and carcinomas in the liver of male and female rats treated with dehydroepiandrosterone. Toxicol. Pathol. 23, 591-605. [27] Metzger, C„ Bannasch, P., and Mayer. D. (1997). Enhancement and phenotypic modulation of N-nitrosomorpholine-induced hepatocarcinogenesis by dehydroepiandrosterone. Cancer Letters 121, 125-131. [28] Moore, M.A., Weber, E., Thornton, M., and Bannasch, P. (1988). Sex-dependent, tissue-specific opposing effects of dehydroepiandrosterone on initiation and modulation stages of liver and lung carcinogenesis induced by dihydroxy-di-n-propylnitrosamine in F344 rats. Carcinogenesis 9, 1507-1509. [29] Ogiu, T., Hard, G.C., Schwartz, A.G., and Magee, P.N. (1990). Investigation into the effect of DHEA on renal carcinogenesis induced in the rat by a single dose of DMN. Nutr. Cancer 14, 57-67. [30] Orentreich, N., Brind, J.L., Rizer, R.L., and Vogelman, J.H. (1984). Age changes and sex differences in serum dehydroepiandrosterone sulfate concentrations throughout adulthood. J. Clin. Endocrinol. Metab. 59, 551 -555. [31] Omer, G.A., Mathews, C„ Hendricks, J.D., Carpenter, H.M., Bailey, G.S., and Williams, D.E. (1995) Dehydroepiandrosterone is a complete hepatocarcinogen and potent tumor promoter in the absence of peroxisome proliferation in rainbow trout. Carcinogenesis 16, 2893-2898. [32] Prough, R.A., Webb, S.J., Wu, H.-Q., Lapenson, D.P., and Waxman, D.J. (1994). Induction of microsomal and peroxisomal enzymes by dehydroepiandrosterone and its reduced metabolite in rats. Cancer Res. 54, 2878-2886. [33] Rao., M.S., Subbarao, V., Yeldandi, A.V., and Reddy, J.K. (1992a). Hepatocarcinogenicity of dehydroepiandrosterone in the rat. Cancer Res. 52, 2977-2979. [34] Rao, M.S., Subbarao, V., Kumar, S., Yeldandi, V., and Reddy, J.K. (1992b). Phenotypic properties of liver tumors induced by dehydroepiandrosterone in F-344 rats. Jpn. J. Cancer Res. 83, 1179-1183. [35] Rao., M.S., Reid, B., Die, H., Subbaro, V., and Reddy, J.K. (1994). Dehydroepiandrosterone-induced peroxisome proliferation in the rat: evaluation of sex differences. Proc. Soc. Exp. Biol. Med. 207, 186-190. [36] Reddy, J.K. and Lalwani, N.D. (1983). Carcinogenesis by hepatic peroxisome prolifertors on microsomal, peroxisomal, and mitochondrial enzyme activities in the liver and kidney. Drug Metab. Rev. 18, 441-515.

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[37] Schwartz, A.G., Whitcomb, J.M. Nyce, J.W., Lewhart, M.L., and Pashko, L.L. (1988). Dehydroepiandrosterone and structural analogs: a new class of cancer chemopreventive agents. Adv. Cancer Res. 51, 391-424. [38] Sonka, J: (1976). Dehydroepiandrosterone: metabolic effects. Acta Univ. Carol. Med. 71, 1-171. [39] Ströbel, P., Klimek, F., Zerban, H. Kopp-Schneider, Α., and Bannasch, P. (1998). Xenomorphic hepatocellular precursors and neoplastic progression of tigroid cell foci induced in rats with low doses of N-nitrosomorpholine. Carcinogenesis 19, 101-113. [40] Su, Q., Benner, Α., Hofmann, W.J., Otto, G., Pichlmayr, R., and Bannasch, P. (1997) Human hepatic preneoplasia: Phenotypes and proliferation kinetics of foci and nodules of altered hepatocytes and their relationship to liver cell dysplasia. Virchows Arch. 431, 391-406. [41] Thornton, M„ Moore, M.A., and Ito, N. (1989). Modifying influence of dehydroepiandrosterone or butylated hydroxytoluene treatment on initiation and development stages of asazerine-induced acinar pancreatic lesions in the rat. Carcinogenesis 10, 407-410. [42] Van Werden, E.M., Bierings, H.G., van Steenbrugge, G.J., de Jong, F.H., and Schröder, F.H. (1992). Adrenal glands of the mouse and rat do not synthesize androgens. Life Sei 50, 857-861. [43] Weber, E., Moore, M.A., and Bannasch, P. (1988). Enzyme histochemical and morphological phenotype of amphophilic foci and amphophilic/tigroid cell adenomas in rat liver after combined treatment with dehydroepiandrosterone and N-nitrosomorpholine. Carcinogenesis 9, 1049-1054. [44] Yamada, J., Sakuma, M., Ikeda, T„ Fukuda, K., and Suga, T. (1991). Characteristics of dehydroepiandrosterone as a peroxisome proliferator. Biochim. Biophys. Acta 1092, 233-243. [45] Yamada, J., Sakuma, M., Ikeda, T., and Suga, T. (1994). Activation of dehydroepiandrosterone as a peroxisome proliferator by sulfate conjugation. Arch. Biochem. Biophys. 313, 379-381.

Hepatocarcinogenesis by Dehydroepiandrosterone. II. Biochemical and Molecular Changes During Neoplastic Development Doris Mayer, Christel Metzger, Peter Bannasch Abteilung für Cytopathologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280,69120 Heidelberg, Germany

Introduction Dehydroepiandrosterone (DHEA) is the main adrenal secretory steroid in humans and a precursor in the biosynthesis of estrogens and androgens. In the rat DHEA acts as a peroxisome proliferator and as a complete hepatocarcinogen when given at high doses in the diet (1-4). The tumour incidence is significantly higher in females than in males (4) and depends on the rat strain used (5). As outlined in the first part of this review, the hepatocellular neoplasms induced by DHEA develop from small preneoplastic focal lesions, so-called amphophilic cell foci (APF) which are characterized by a strong proliferation of mitochondria closely associated with single cisternae of rough endoplasmic reticulum, and to a lesser extent to a proliferation of peroxisomes (4). Since preneoplastic and neoplastic lesions do not arise from perivenular hepatocytes showing extensive peroxisome proliferation but from hepatocytes localized close to the portal tract of the liver lobules, where only a minor peroxisome proliferation is observed, we think that peroxisome proliferation does not play a major role in tumor induction and that other factors are responsible for the hepatocarcinogenic effect of DHEA (4, 5). A similar conclusion has been drawn by Omer et al. (6) from observations in rainbow trouts which are weak responders to peroxisome proliferators but, nevertheless, develop a high incidence of hepatocellular neoplasms after exposure to DHEA with and without initiation by aflatoxin Β ]. Morphological aspects of DHEA-induced non-preneoplastic and neoplastic alterations in rat liver and morphogenesis of DHEA-induced hepatocellular neoplasms have been discussed in the first part of this review. In the following some findings on biochemical changes in liver from DHEA-treated rats and in DHEA-induced preneoplastic and neoplastic lesions will be presented. The strong proliferation of mitochondria in the preneoplastic APF and in adenomas and carcinomas resulting from APF indicate significant alterations in the regulation of energy metabolism in DHEA-treated rats that may be related to the carcinogenic effect of DHEA in the liver. DHEA-induced changes in liver parenchyma outside of preneoplastic and neoplastic lesions DHEA-administration to rats results in significant alterations in liver function which concern mainly carbohydrate and lipid metabolism. These changes are usually observed a few

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days or weeks after start of the treatment when no preneoplastic or neoplastic lesions are yet observed, but when a marked hepatomegaly occurs. Some parameters return to normal levels in non-neoplastic liver after prolonged treatment with DHEA (7-9). Nevertheless, the alterations may be of relevance for the carcinogenic effect of DHEA which is manifested only after several months of administration. However, as described in the following, many changes in enzyme activity and expression persist or even increase after long term DHEA administration. Preneoplastic APF occur after about 25-30 weeks of treatment (4, 10) and are characterized by a typical pattern of enzyme expression which mimics the effect of thyroid hormone (11,12). Peroxisome proliferation Similar to a large number of other substances, DHEA or rather its sulfate ester DHEAS acts as a peroxisome proliferator in rat liver (3,4, 13-18). Peroxisome proliferation is associated with a marked induction of enzymes of peroxisomal ß-oxidation (3, 13, 14, 16-18). Concomitant with peroxisome proliferation, a significant increase in microsomal cytochrome P450 IVA and NADPH-cytochrome P450 reductase is observed (17-19). Both activities are associated with the production of reactive oxygen species, and it has been suggested that increased peroxisomal H202-production is causally related to induction of hepatocellular neoplasms. Catalase, the peroxisomal H 2 0 2 -degrading enzyme, is only weakly induced in DHEA-treated livers (7). Catalase activity, however, is high in untreated liver (3,4,7,12, 18), and it is unclear whether there is any significant local increase in H 2 0 2 -concentration in DHEA-treated liver. Metzger et al. (4) and Beier et al. (18) described sex-specific differences in the distribution of peroxisome proliferation and concomitant induction of enzymes of peroxisomal ß-oxidation within the liver lobule. The lobular distribution of the microsomal cytochrome P450 IVA, which was strongly induced in males and only moderately increased in females by DHEA, showed a striking homology with peroxisomal enzymes in both sexes (18), indicating a functional relationship. It may be speculated that the strong induction of cytochrome P450 IVA in males is related to the smaller incidence of DHEA-induced neoplasms due to accelerated metabolism of the substance. This assumption is supported by the rapid disappearance of DHEA from the blood of male rats described by Hobe et al. (20). Oxidative stress and lipid peroxidation Although peroxisome proliferation seems not to be a major cause for tumour induction, increased production of reactive oxygen species induced by DHEA in rat liver and consequent liver injury may be related to hepatocarcinogenesis to some extent. DHEA-administration to rats caused a significant increase in NADPH-dependent lipid peroxidation in microsomes and mitochondria isolated from liver (7-9). An increase in lipid peroxidation was detectable after 2 days of treatment in males, and after 3 days in females. Lipid peroxidation attained a maximum between 3 and 7 days of DHEA-feeding in males, and between 7 and 14 days in females. The largest difference between males and females was found after 2 days of treatment. The finding that males are more susceptible to lipid peroxidation than females agrees with the strong induction of NADPH-cytochrome P450 reductase/cytochrome P450, an H 2 0 2 -producing enzyme system, by DHEA, particularly in the livers of male rats (17-19).

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Furthermore, mitochondrial proliferation induced by DHEA may contribute to the increase in lipid peroxidation, since reactive oxygen species are regularly produced by electron leakage in the mitochondrial respiratory chain, particularly by complex III. The increase in lipid peroxidation is an early transient effect of DHEA, but liver neoplasms occur only after long-term administration. It remains to be clarified if the products of lipid peroxidation, e.g. malondialdehyde (8), react with DNA leading to DNA damage. The observation of higher susceptibility to lipid peroxidation but lower tumour incidence in male rats as compared to females suggests that lipid peroxidation represents an unspecific cytotoxic process leading to liver injury which may, nevertheless, be somehow involved in hepatocarcinogenesis.

Alterations in energy metabolism DHEA exerts a significant effect on the activity of enzymes of energy metabolism, particularly on key enzymes of glucose and lipid metabolism (7, 12, 21). Table 1 summarizes the lobular distribution of glycogen content and enzyme activities determined by histochemistry, and the alterations in the intensity of reactions and lobular distribution induced by

Table 1 Alteration by DHEA-treatment of the glycogen content and of the activity of enzymes of carbohydrate metabolism and of mitochondrial enzymes in the livers of male and female rats detected by enzyme histochemistry (from 11). Male rats

Female rats

Parameter, localisation Glycogen Cytosolic S Y N Cytosolic PHO Cytosolic HK Cytosolic GK Cytosolic PK Cytosolic G6PDH Cytosolic ME Cytosolic G6Pase Microsomal COX Mitochondrial G3PDH Mitochondrial SDH Mitochondrial GIDH Mitochondrial AcPase Lysosomal GGT plasma membrane

activity

change

activity

change

untreated

DHEA

by DHEA

untreated DHEA

by DHEA

++ ++ ++

J, 1 1 (T) pv

(Ε < 3 Ο 111 m < α:

10 Χ

ι

CONT

-J

3 Ο 0.5 UJ m < DC

MPA

Ej

DHT

DHEA

OHEA DHEA + F L U EM-BOO

OVARIECTOMIZED

Figure 9 Effect of 12-month treatment with medroxyprogesterone acetate (MPA), 17ß-estradiol (E2), dihydrotestosterone (DHT) or dehydroepiandrosterone (DHEA) alone or in combination with Flutamide or EM-800 on trabecular bone volume in ovariectomized rats. Intact animals are added as additional controls. Data are presented as mean ± SEM **p