0021-972X/97/$03.00/0 Vol. 82, No. 8 Journal of Clinical Endocrinology and Metabolism Printed in U.S.A. Copyright 1997 by The Endocrine Society Physiological Changes in Dehydroepiandrosterone Are Not Reflected by Serum Levels of Active Androgens and Estrogens But of Their Metabolites: Intracrinology FERNAND LABRIE, ALAIN BÉLANGER, LIONEL CUSAN, AND BERNARD CANDAS Medical Research Council Group in Molecular Endocrinology, Centre Hospitalier de l Universite Laval Research Center, Le Centre Hospitalier Universitaire de Québec, and Laval University, Québec, G1V 4G2, Canada ALTHOUGH dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) were first isolated and identified in 1934 (1) and 1944 (2), respectively, and DHEA-S was isolated from human plasma in 1954 (3), the role of DHEA and DHEA-S in endocrine physiology is of recent interest (4 6). One reason for the relative lack of attention to DHEA is that this adrenal steroid does not possess intrinsic androgenic, estrogenic, or other classical hormonal activity. Most significantly, the adrenals of the animal models usually used in the laboratory, namely rats, mice, and dogs, do no secrete significant amounts of DHEA (5, 7), thus attracting all attention to the ovaries and testes as sources of sex steroids. However, humans along with some other primates, are unique among animal species in having adrenals that secrete large amounts of the inactive precursor steroids DHEA, especially DHEA-S, which are converted into potent androgens and/or estrogens in peripheral tissues (6, 8 10). In fact, Received November 18, 1996. Revision received May 6, 1997. Accepted May 12, 1997. Address all correspondence and requests for reprints to: Fernand Labrie, Laboratory of Molecular Endocrinology, CHUL Research Center, 2705 Laurier Boulevard, Québec, QC, G1V 4G2, Canada. ABSTRACT This study analyzes in detail the serum concentration of the active androgens and estrogens, as well as a series of free and conjugated forms of their precursors and metabolites, after daily application for 2 weeks of 10 ml 20% dehydroepiandrosterone (DHEA) solution on the skin to avoid first passage through the liver. In men, DHEA administration caused 175%, 90%, 200% and 120% increases in the circulating levels of DHEA and its sulfate (DHEA-S), DHEA-fatty acid esters, and androst-5-ene-3,17 -diol, respectively, with a return to basal values 7 days after cessation of the 14-day treatment. Serum androstenedione increased by approximately 80%, whereas serum testosterone and dihydrotestosterone (DHT) remained unchanged. In parallel with the changes in serum DHEA, the concentrations of the conjugated metabolites of DHT, namely androsterone glucuronide, androstane-3,17 -diol-g, and androstane-3,17 -diol-g increased by about 75%, 50%, and 75%, respectively, whereas androsteronesulfate increased 115%. No consistent change was observed in serum estrone (E 1 ) or estradiol (E 2 ) in men receiving DHEA, whereas serum E 1 -sulfate and E 2 -sulfate were slightly and inconsistently increased by about 20%, and serum cortisol and aldosterone concentrations were unaffected by DHEA administration. Almost superimposable results were obtained in women for most steroids except testosterone, which was about 50% increased during DHEA treatment. This increase corresponded to about 0.8 nm testosterone, an effect undetectable in men because they already have much higher ( 15 nm) basal testosterone levels. In women, the serum levels of the conjugated metabolites of DHT, namely androsterone glucuronide, androstane- 3,17 -diol-g, androstane-3,17 -diol-g, and androsterone-sulfate were increased by 125%, 140%, 120% and 150%, respectively. The present study demonstrates that the serum concentrations of testosterone, DHT, E 1, and E 2 are poor indicators of total androgenic and estrogenic activity. However, the esterified metabolites of DHT appear as reliable markers of the total androgen pool, because they directly reflect the intracrine formation of androgens in the tissues possessing the steroidogenic enzymes required to transform the inactive precursors DHEA and DHEA-S into DHT. As well demonstrated in women, who synthesize almost all their androgens from DHEA and DHEA-S, supplementation with physiological amounts of exogeneous DHEA permits the biosynthesis of androgens limited to the appropriate target tissues without leakage of significant amounts of active androgens into the circulation. This local or intracrine biosynthesis and action of androgens eliminates the inappropriate exposure of other tissues to androgens and thus minimizes the risks of undesirable masculinizing or other androgen-related side effects of DHEA. (J Clin Endocrinol Metab 82: 2403 2409, 1997) plasma DHEA-S levels in adult men and women are 100 500 times higher than those of testosterone and 1,000 10,000 times higher than those of estradiol, thus providing a large reservoir of substrate for conversion into androgens and/or estrogens in peripheral intracrine tissues. The term intracrinology was coined in 1988 (11) to describe the synthesis of active steroids in peripheral target tissues where the action is exerted in the same cells in which synthesis takes place without release in the extracellular space and general circulation (6). Knowledge in this area has recently made rapid progress with the elucidation of the structure of most of the tissuespecific complementary DNAs and genes that encode the steroidogenic enzymes responsible for the transformation of DHEA-S and DHEA into androgens and/or estrogens in peripheral tissues (8 10, 12 21). The marked reduction in the formation of DHEA-S by the adrenals during aging (4, 22 25) results in a dramatic fall in the formation of androgens and estrogens in peripheral target tissues, a situation that is thought to be associated with age-related diseases such as insulin resistance (26, 27) and obesity (28, 29). Moreover, low circulating levels of DHEA-S and DHEA have been found in patients with breast (30) and 2403
2404 LABRIE ET AL. JCE&M 1997 Vol 82 No 8 prostate (31) cancer. DHEA has also been found to exert antioncogenic activity in a series of animal models (32 34). On the other hand, a stimulatory effect of DHEA on the immune system has been described in postmenopausal women (35). Moreover, the oral administration of DHEA has been reported to have beneficial effects in aged men and women (36 39). Based on the observation that a large proportion of the active sex steroids in men and women originate from DHEA and DHEA-S (6), and the convincing demonstration of the important role of the androgens and estrogens synthesized in peripheral target tissues from DHEA and DHEA-S in aged men and women (6, 37), we studied the serum concentration of a large series of sex steroids after percutaneous administration of DHEA in 60- to 70-yr-old men and women. The percutaneous route was chosen to avoid the first passage of DHEA through the liver. This route of administration is supported by our recent data obtained in the rat showing that the bioavailability of DHEA is greater after percutaneous than oral administration (39). This report thus describes the changes in the circulating levels of the most pertinent androgens, estrogens, and their metabolites following DHEA administration. It demonstrates the major changes in the concentration of the circulating metabolites of androgens and estrogens in the presence of little or no change in the concentration of the circulating active sex steroids. The present data support the major importance of the tissue-specific intracellular formation of sex steroids, which is only reflected in the circulation by the changes in the concentration of metabolites, but not of the active sex steroids. Subjects Subjects and Methods Eight healthy 60- to 70-yr-old volunteer men and post-menopausal women participated in this study after IRB (Institutional Review Board) approval and having given their written informed consent. The participants were nonsmokers. No woman had taken hormone replacement therapy during the previous year. No subject was suffering from an endocrine disorder, and none was under treatment with lipid- or glucose-lowering agents. All participants had a medical history; complete physical examination; and serum biochemistry profile including lipids, complete blood count, urinalysis, and detailed serum hormone determinations during the screening phase of the protocol. Although there was no specific requirement for exercise and diet, no volunteer was involved in a weight loss program nor was following a special diet. Steroid analysis in serum Steroid extraction. Ethanol (5 ml) was added to 1 ml serum and centrifugation was performed at 2000 g for 15 min. The resulting pellet was further extracted with 2 ml ethanol and, after a second centrifugation at 2000 g for 15 min, the two supernatants were combined. Pellets were then resuspended once again in 5 ml hexane to maximize the recovery of nonpolar steroids. The suspension was recentrifuged as described above, and the supernatant was decanted and combined with the previously obtained ethanol extracts. The organic solvent was then evaporated under nitrogen, and the residue was dissolved in 1 ml water/methanol (95:5, vol/vol). The C-18 columns (Bound-Elut, Amersham, Bucks, UK) were conditioned by passing consecutively 10 ml methanol, 10 ml water, and 10 ml methanol/water (5:95, vol/vol). The extracts were solubilized in water/methanol (95:5, vol/vol) then deposited on the C-18 columns. After washing the columns with 2 ml water/methanol (95:5, vol/vol), 5 ml methanol/water (50:50, vol/vol) were added to eluate DHEA-S after which 5 ml methanol/water (85:15, vol/vol) were added to eluate the nonconjugated steroids. The acylated steroids were then collected following the addition of 5 ml methanol. Chromatography on LH-20 columns and RIA. Chromatography on Sephadex LH-20 columns (Pharmacia, Uppsala, Sweden) was performed as previously described (40). In brief, the nonconjugated steroids from the three fractions were solubilized in 1 ml isooctane/toluene/methanol (90:5:5, vol/vol/vol) and deposited on the LH-20 columns. The appropriate fractions were collected and, after evaporation of the organic solvent, the concentration of the various steroids was determined by RIA as previously described (40 42). Calculations and statistical analyses RIA data were analyzed using a program based on model II of Rodbard and Lewald (43). Plasma steroid levels are shown as the means sem of duplicate determinations of individual samples. Statistical significance was measured according to the multiple range test of Duncan-Kramer (44). Results In 60- to 70-yr-old men, the serum DHEA concentration increased from 10.2 0.9 nm to 29.0 2.0 nm on day 3 after starting daily percutaneous administration of 10 ml 20% solution of DHEA, to 31.5 2.8 nm on day 6, and decreased to basal values on day 7 after cessation of therapy (Fig. 1A). In parallel, DHEA-S increased from 1525 120 nm to 3000 150 nm on day 7 for a 90% increase over pretreatment values (Fig. 1B). Serum DHEA-fatty acid esters increased to a peak Study design, treatment, and measurements DHEA (Diosynth, Chicago, IL) was administered percutaneously once daily for 14 days in the morning as a 20% solution (10 ml DHEA, 50% ethanol-50% USP propylene glycol). The DHEA solution was applied over an area of approximately 25 cm 25 cm on the abdomen, followed by rubbing with the hand for a few seconds to optimize the absorption of DHEA. Blood sampling was performed at 0800 0900 h before application of DHEA. Measurements of serum steroids were performed 2 days before and immediately before application of DHEA. Serum steroid measurements were also performed 3, 7, 11, and 14 days after first application of the precursor steroid. Following cessation of DHEA administration, serum steroids were analyzed on days 3, 7, 11, and 14 to determine the rate and degree of return towards basal levels. FIG. 1. Effect of daily percutaneous administration of 10 ml 20% 60- to 70-yr-old men on serum levels of DHEA (A), DHEA-S (B), and DHEA-fatty acid esters (C).
PERCUTANEOUS DHEA AND SERUM SEX STEROID LEVELS 2405 FIG. 2. Effect of daily percutaneous administration of 10 ml 20% 60- to 70-yr-old men on serum levels of 5-diol (A) and 5-diol-S (B). value of approximately 200% above control on day 7 after starting treatment to return to basal values on day 7 posttreatment (Fig. 1C). Serum androst-5-ene-3,17 -diol (5-diol), increased from 2.9 0.3 nm to 6.4 0.4 nm on day 7 of treatment (120% increase over control) (Fig. 2A), whereas serum 5-diol-sulfate (5-diol-S) increased approximately 100% above control during the treatment period (Fig. 2B). The serum concentration of both steroids returned to pretreatment values 3 days after cessation of DHEA administration. As illustrated in Fig. 3, serum androstenedione (4-dione) increased from 3.05 0.2 nm to 5.45 0.35 nm (80% increase) (Fig. 3A), whereas serum testosterone (Fig. 3B) and dihydrotestosterone (DHT) (Fig. 3C) remained unchanged during the course of DHEA administration. Because our previous data obtained in men treated by medical or surgical castration for prostate cancer have clearly shown that measurements of serum testosterone and DHT are poor markers of total androgenic activity (5, 45), it was of major interest to analyze the changes in the serum levels of the main metabolites of DHT, namely androsterone (ADT), androstane-3,17 -diol (3 -diol), androstane- 3,17 -diol (3 -diol) and, most importantly, the glucuronidated derivatives of these steroids, androsterone glucuronide (ADT-G), androstane-3,17 -diol-g (3 -diol-g), and androstane-3,17 -diol-g (3 -diol-g). As illustrated in Fig. 4, serum ADT increased by about 40% during DHEA treatment (Fig. 4A), whereas there was no significant change in serum 3 -diol (Fig. 4B), and the concentration of serum 3 diol increased by only about 20% (Fig. 4C). In contrast, serum ADT-G, 3 -diol-g, and 3 -diol-g, increased by approximately 75%, 50%, and 75%, respectively (Fig. 5). Similarly, the serum concentration of ADT-sulfate (ADT-S) increased by 115% from 740 90 nm to a maximum of 1600 175 nm (Fig. 6). In contrast, serum estradiol (E 2 ) and estrone (E 1 ) (Fig. 7) showed no consistent change during DHEA administration, whereas serum E 1 -sulfate (E 1 -S) (Fig. 8A) and E 2 -sulfate (E 2 -S) (Fig. 8B) were slightly and inconsistently increased by only about 20%. Serum cortisol and aldosterone remained unchanged during the course of treatment (Fig. 9). FIG. 3. Effect of daily percutaneous administration of 10 ml 20% 60- to 70-yr-old men on serum levels of 4-dione (A), testosterone (B), and DHT (C). FIG. 4. Effect of daily percutaneous administration of 10 ml 20% 60- to 70-yr-old men on serum levels of ADT (A), 3 -diol (B), and 3 -diol (C). FIG. 5. Effect of daily percutaneous administration of 10 ml 20% 60- to 70-yr-old men on serum levels of ADT-G (A), 3 -diol-g (B), and 3 -diol-g (C). When the same dose of DHEA was administered to women of the same age, comparable results were obtained. Serum DHEA, DHEA-S, and DHEA-fatty acids increased
2406 LABRIE ET AL. JCE&M 1997 Vol 82 No 8 FIG. 6. Effect of daily percutaneous administration of 10 ml 20% 60- to 70-yr-old men on serum levels of ADT-S. FIG. 9. Effect of daily percutaneous administration of 10 ml 20% 60- to 70-yr-old men on serum levels of cortisol (A) and aldosterone (B). FIG. 7. Effect of daily percutaneous administration of 10 ml 20% 60- to 70-yr-old men on serum levels of E 1 (A) and E 2 (B). FIG. 10. Effect of daily percutaneous administration of 10 ml 20% 60- to 70-yr-old women on serum levels of DHEA (A), DHEA-S (B), and DHEA-fatty acid esters (C). FIG. 8. Effect of daily percutaneous administration of 10 ml 20% 60- to 70-yr-old men on serum levels of E 1 -S (A) and E 2 -S (B). approximately 175%, 130%, and 250%, respectively (Fig. 10). In contrast, serum 5-diol increased by a maximum of 200% (Fig. 11), whereas serum 4-dione increased by about 100% over control, serum testosterone increased approximately FIG. 11. Effect of daily percutaneous administration of 10 ml 20% 60- to 70-yr-old women on serum levels of 5-diol. 50%, and serum DHT did not show a consistent change (Fig. 12). The following results were observed in women on the serum concentration of the metabolites of DHT: serum ADT
PERCUTANEOUS DHEA AND SERUM SEX STEROID LEVELS 2407 FIG. 12. Effect of daily percutaneous administration of 10 ml 20% 60- to 70-yr-old women on serum levels 4-dione (A), testosterone (B), and DHT (C). FIG. 15. Effect of daily percutaneous administration of 10 ml 20% 60- to 70-yr-old women on serum levels of ADT-S (A) and 3 -diol-s (B). FIG. 13. Effect of daily percutaneous administration of 10 ml 20% 60- to 70-yr-old women on serum levels of ADT (A), 3 -diol (B), and 3 -diol (C). FIG. 16. Effect of daily percutaneous administration of 10 ml 20% 60- to 70-yr-old women on serum levels of E 1 (A) and E 2 (B). FIG. 14. Effect of daily percutaneous administration of 10 ml 20% 60- to 70-yr-old women on serum levels of ADT-G (A), 3 -diol-g (B), and 3 -diol-g (C). FIG. 17. Effect of daily percutaneous administration of 10 ml 20% 60- to 70-yr-old women on serum levels of E 1 -S (A) and E 2 -S (B). increased by about 20% (Fig. 13A), whereas serum 3 -diol and 3 -diol increased by about 10% (Fig. 13B) and 40% (Fig. 13C), respectively. As seen in men, the most important effects, however, were observed on the glucuronidated and sulfate metabolites of ADT, 3 -diol, and 3 -diol. In fact, treatment with DHEA caused an increase in serum ADT-G, 3 -diol-g, 3 -diol-g, ADT-S, and 3 -diol-s of approximately 125% (Fig. 14A), 140% (Fig. 14B), 120% (Fig. 14C), 150% (Fig. 15A), and 75% (Fig. 15B), respectively. Again, no consistent
2408 LABRIE ET AL. JCE&M 1997 Vol 82 No 8 effect was observed on serum E 1 or E 2 (Fig. 16), whereas serum E 1 -S and E 2 -S increased by about 70% and 30%, respectively, at the maximal value observed (Fig. 17). In agreement with the data obtained in men, no effect was detectable on the serum concentration of cortisol or aldosterone (Fig. 18). Discussion The present data show that elevations in serum DHEA within the physiological range found in young adult men and women lead only to small or no significant changes in serum testosterone, DHT, or E 2, whereas, by contrast, the concentration of the conjugated metabolites of DHT are markedly increased. Such data offer unique proof that the serum levels of androgens and estrogens are poor indicators of total androgenic and estrogenic activities in men and women. In fact, serum testosterone and DHT reflect almost exclusively the contribution of the testicles, whereas serum E 2 reflects, to a major extent, direct estrogen secretion by the ovaries. The low levels of serum DHEA and DHEA-S in 60- to 70-yr-old men and women offer the opportunity to measure with greater precision the changes in the serum levels of these precursor steroids, as well as their metabolites, in the circulation without undue interference by the high levels of adrenal precursor steroids in young adult men and women. The present data show a similar increase in serum DHEA of 175 200% over basal values in both men and women who received treatment with percutaneous DHEA. Basal serum DHEA-S levels, however, were approximately 50% lower in women than in men. The similar absolute increase in serum DHEA-S in subjects of both sexes led to a relatively higher relative increase in women than men, 130% and 90%, respectively, over basal values. A similar observation was made for 5-diol, with the basal values being lower in women than in men, 1.6 0.2 nm and 2.9 0.3 nm, respectively. These differences in basal values led to increases in serum 5-diol levels of 225% and 120% in women and men, respectively, following treatment with DHEA. Basal serum levels of 4-dione were only slightly higher in men than in women (3.05 0.2 nm vs. 2.8 0.2 nm), whereas FIG. 18. Effect of daily percutaneous administration of 10 ml 20% 60- to 70-yr-old women on serum levels of cortisol (A) and aldosterone (B). the basal circulating levels of testosterone were measured at 14 1.1 nm and 1.3 0.2 nm in men and women, respectively. The 50% increase in serum testosterone from approximately 1.3 2.3 nm observed in women during DHEA treatment corresponds to an increase in serum DHEA of approximately 20 nm DHEA. These data are in agreement with the information obtained in men after medical or surgical castration in which the serum levels of testosterone decrease from 15 nm to about 1.5 nm after elimination of testicular androgens. The 1.5 nm serum testosterone originates from adrenal DHEA (5, 46). The present data thus offer an independent measure of the amount of testosterone that diffuses into the circulation from the androgens synthesized from DHEA and DHEA-S in various peripheral intracrine tissues (6). The present data also indicate, as previously suggested (5, 45), that the testicles and adrenals are responsible for approximately equal amounts of androgen biosynthesis in adult men. In women, an important proportion of circulating testosterone is secreted directly by the interstitial cells of the ovary, the estimate from the present data indicating a contribution of approximately 1.0 nm or 67% of circulating testosterone. The most striking effects of DHEA administration, however, are on the circulating levels of the glucuronide and sulfate derivatives of the metabolites of DHT, namely ADT, 3 -diol, and 3 -diol. These metabolites are produced locally in the peripheral intracrine tissues that possess the appropriate steroidogenic enzymes to synthesize DHT from the adrenal precursors DHEA and DHEA-S (6, 8). It can be noted that if ADT-G is taken as the main marker of DHT metabolism, the circulating levels of ADT-G are approximately 100% higher in men than women, thus suggesting that women synthesize approximately 50% as much DHT as men. Although the absolute increase in the circulating levels of ADT-G, 3 -diol-g, and 3 -diol-g are comparable in men and women, because of the lower basal levels in women the percentage increase is of greater magnitude in women. After 12 weeks of daily oral administration of 50 mg DHEA, serum DHEA levels increased from 8.5 to 14.7 nm in men and 7.2 to 16.1 nm in women (37), whereas serum DHEA-S increased from 3.5 to 10.1 m in men and from 1.8 to 9.3 m in women. The serum 4-dione concentration was increased slightly from 1.9 to 2.2 nm in men, although serum testosterone and DHT levels were unchanged. In contrast, in women serum 4-dione, testosterone, and DHT levels were increased by about 50% following DHEA treatment. Serum E 1 or E 2 was unaffected by DHEA treatment in either men or women. The difference in the present data in which only serum testosterone was stimulated by DHEA administration in women is possibly because of the effect of first passage of DHEA through the liver after oral administration in the study of Morales et al. (37). Using a high daily 1.6-g oral dose of DHEA for 4 weeks in postmenopausal women, the increases observed were 9-fold for serum testosterone, 20-fold for serum 4-dione and DHT, and 2-fold for serum estrone (E 1 ) and E 2 (36). In addition, a decrease was observed in serum SHBG, total cholesterol high density lipoprotein, and insulin resistance was noted. The high levels of circulating DHT compared with testosterone in the study of Mortola and Yen (36) possibly reflect the high level of reduction by liver 5 -reductase following oral ad-
PERCUTANEOUS DHEA AND SERUM SEX STEROID LEVELS 2409 ministration of DHEA. Following a similar duration of daily oral administration of the same high dose of 1.6 g DHEA for 28 days, serum DHEA-S levels increased 2.0- to 2.5-fold above control, whereas serum 4-dione increased 1.0-fold, and no significant change was seen in serum E 2,E 1, testosterone, and SHBG (sex hormone binding globulin) (28). The present data permit the first direct analysis of the correlation between the serum levels of DHEA and DHEA-S with the serum concentration of active androgens and estrogens and their corresponding glucuronidated and sulfated metabolites. It can be concluded that although measurements of serum testosterone and estradiol reflect testicular and ovarian steroid secretion, respectively, the important contribution of the adrenals is not accurately reflected in the circulating levels of these active sex steroids. The present data thus clearly demonstrate that DHEA and DHEAS are converted in specific intracrine tissues into the active androgens and/or estrogens and are metabolized locally into inactive glucuronidated and sulfated metabolites, which in turn can be measured in the circulation. Measurement of the conjugated metabolites of androgens is the only approach that permits an accurate estimate of the total androgen pool in men. It is likely that a similar situation exists in women for estrogens, although a precise evaluation of the pharmacokinetics of estrogens and their inactive metabolites remains to be performed. References 1. Butenandt A, Tscherning K. 1934 Uber Androsteron, Krystallisiertes mannliches Sexualhormon. I. Bolierung und Reindarstellung aus Munnerharn. Z Physiol Chem. 229:167 184. 2. Munson PL, Gallagher TF, Koch FC. 1944 Isolation of dehydroisoandrosterone sulfate from normal male urine. J Biol Chem. 152:67 77. 3. Migeon CJ, Plager JE. 1954 Identification and isolation of dehydroepiandrosterone from peripheral human plasma. J Biol Chem. 209:767 772. 4. Bélanger A, Candas B, Dupont A, et al. 1994 Changes in serum concentrations of conjugated and unconjugated steroids in 40- to 80-year-old men. J Clin Endocrinol Metab. 79:1086 1090. 5. Labrie F, Dupont A, Bélanger A. 1985 Complete androgen blockade for the treatment of prostate cancer. In: de Vita VT, Hellman S, Rosenberg SA, eds. Important advances in oncology. Philadelphia: J. B. Lippincott: pp 193 217. 6. Labrie F. 1991 Intracrinology. Mol Cell Endocrinol. 78:C113 C118. 7. Bélanger B, Bélanger A, Labrie F, et al. 1989 Comparison of residual C-19 steroids in plasma and prostatic tissue of human, rat and guinea pig after castration: unique importance of extratesticular androgens in men. J Steroid Biochem. 32:695 698. 8. Labrie F, Bélanger A, Simard J, Luu-The V, Labrie C. 1995 DHEA and peripheral androgen and estrogen formation: Intracrinology. Ann NY Acad Sci. 774:16 28. 9. Labrie F, Luu-The V, Lin SX, et al. 1997 The key role of 17 -HSDs in sex steroid biology. Steroids. 62:148 158. 10. Labrie F, Simard J, Luu-The V, et al. 1996 The 3 -hydroxysteroid dehydrogenase/isomerase gene family: lessons from type II 3 -HSD congenital deficiency. In: Hansson, V, Levy, FO, Taskén, K (eds) Signal Transduction in Testicular Cells. Ernst Schering Research Foundation Workshop. Berlin: Springer-Verlag, vol 2[Suppl]:185 218. 11. Labrie C, Bélanger A, Labrie F. 1988 Androgenic activity of dehydroepiandrosterone and androstenedione in the rat ventral prostate. Endocrinology. 123:1412 1417. 12. Andersson S, Russel DW. 1990 Structural and biochemical properties of cloned and expressed human and rat steroid 5 -reductases. Proc Natl Acad Sci USA. 87:3640 3644. 13. Andersson S, Bergman DM, Jenkins EP, Russel DW. 1991 Deletion of steroid 5 -reductase 2 gene in male pseudophermaphroditism. Nature. 354:159 161. 14. Bernier F, Leblanc G, Labrie F, Luu-The V. 1994 Structure of human estrogen and aryl sulfotransferase gene. Two mrna species issued from a single gene. J Biol Chem. 269:28200 28205. 15. Bernier F, Lopez-Solache I, Labrie F, Luu-The V. 1994 Cloning and expression of cdna encoding human placental estrogen sulfotransferase. Mol Cell Endocrinol. 99:R11 R15. 16. Luu-The V, Dufort I, Paquet N, Reimnitz G, Labrie F. 1995 Structural characterization and expression of the human dehydroepiandrosterone sulfotransferase gene. DNA Cell Biol. 14:511 518. 17. Rhéaume E, Simard J, Morel Y, et al. 1992 Congenital adrenal hyperplasia due to point mutations in the type II 3 -hydroxysteroid dehydrogenase deficiency. Nature Genetics. 1:239 245. 18. Labrie F, Sugimoto Y, Luu-The V, et al. 1992 Structure of human type II 5 -reductase. Endocrinology. 131:1571 1573. 19. Labrie F, Simard J, Luu-The V, Bélanger A, Pelletier G. 1992 Structure, function and tissue-specific gene expression of 3 -hydroxysteroid dehydrogenase/5-ene-4-ene isomerase enzymes in classical and peripheral intracrine steroidogenic tissues. J Steroid Biochem Mol Biol. 43:805 826. 20. Luu-The V, Zhang Y, Poirier D, Labrie F. 1995 Characteristics of human types 1,2and317 -hydroxysteroid dehydrogenase activities: oxidation-reduction and inhibition. J Steroid Biochem Mol Biol. 55:581 587. 21. Labrie Y, Durocher F, Lachance Y, et al. 1995 The human type II 17 -hydroxysteroid dehydrogenase gene encodes two alternatively-spliced messenger RNA species. DNA Cell Biol. 14:849 861. 22. Vermeulen A, Deslypene JP, Schelthout W, Verdonck L, Rubens R. 1982 Adrenocortical function in old age: response to acute adrenocorticotropin stimulation. J Clin Endocrinol Metab. 54:187 191. 23. Vermeulen A, Verdonck L. 1976 Radioimmunoassays of 17 -hydroxy-5 androstan-3-one, 4-androstene-3,17-dione, dehydroepiandrosterone, 17 -hydroxyprogesterone and progesterone and its application to human male plasma. J Steroid Biochem. 7:1 10. 24. Orentreich N, Brind JL, Rizer RL, Vogelman JH. 1984 Age changes and sex differences in serum dehydroepiandrosterone sulfate concentrations throughout adulthood. J Clin Endocrinol Metab. 59:551 555. 25. Migeon CJ, Keller AR, Lawrence B, Shepart II TH. 1957 Dehydroepiandrosterone and androsterone levels in human placenta. Effect of age and sex: day-to-day and diurnal variations. J Clin Endocrinol Metab. 17:1051 1062. 26. Schriock ED, Buffington CK, Hubert GD, et al. 1988 Divergent correlations of circulating dehydroepiandrosterone sulfate and testosterone with insulin levels and insulin receptor binding. J Clin Endocrinol Metab. 66:1329 1331. 27. Coleman DL, Leiter EH, Schwizer RW. 1982 Therapeutic effects of dehydroepiandrosterone (DHEA) in diabetic mice. Diabetes. 31:830 833. 28. Nestler JE, Barlascini CO, Clore JN, Blackard WG. 1988 Dehydroepiandrosterone reduces serum low density lipoprotein levels and body fat but does not alter insulin sensitivity in normal men. J Clin Endocrinol Metab. 66:57 61. 29. MacEwen EG, Kurzman ID. 1991 Obesity in the dog: role of the adrenal steroid dehydroepiandrosterone (DHEA). J Nutr. 121:S51 S55. 30. Zumoff B, Levin J, Rosenfeld RS, et al. 1981 Abnormal 24-hr mean plasma concentrations of dehydroepiandrosterone and dehydroisoandrosterone sulfate in women with primary operable breast cancer. Cancer Res. 41:3360 3363. 31. Stahl F, Schnorr D, Pilz C, Dorner G. 1992 Dehydroepiandrosterone (DHEA) levels in patients with prostatic cancer, heart diseases and under surgery stress. Exp Clin Endocrinol. 99:68 70. 32. Schwartz AG, Pashko L, Whitcomb JM. 1986 Inhibition of tumor development by dehydroepiandrosterone and related steroids. Toxicol Pathol. 14:357 362. 33. Gordon GB, Shantz LM, Talalay P. 1987 Modulation of growth, differentiation and carcinogenesis by dehydroepiandrosterone. Adv Enzyme Regul. 26:355 382. 34. Li S, Yan X, Bélanger A, Labrie F. 1993 Prevention by dehydroepiandrosterone of the development of mammary carcinoma induced by 7,12-dimethylbenz(a)anthracene (DMBA) in the rat. Breast Cancer Res Treat. 29:203 217. 35. Casson PR, Andersen RN, Herrod HG, et al. 1993 Oral dehydroepiandrosterone in physiologic doses modulates immune function in postmenopausal women. Am J Obstet Gynecol. 169:1536 1539. 36. Mortola JF, Yen SS. 1990 The effects of oral dehydroepiandrosterone on endocrine-metabolic parameters in postmenopausal women. J Clin Endocrinol Metab. 71:1360 1367. 37. Morales AJ, Nolan JJ, Nelson JC, Yen SS. 1994 Effects of replacement dose of dehydroepiandrosterone in men and women of advancing age. J Clin Endocrinol Metab. 78:1360 1367. 38. Diamond P, Cusan L, Gomez JL, Bélanger A, Labrie F. 1996 Metabolic effects of 12-month percutaneous DHEA replacement therapy in postmenopausal women. J Endocrinol. 150:S43 S50. 39. Labrie C, Flamand M, Bélanger A, Labrie F. 1996 High bioavailability of DHEA administered percutaneously in the rat. J Endocrinol. 150:S107 S118. 40. Bélanger A, Labrie F, Dupont A, Brochu M, Cusan L. 1988 Endocrine effects of combined treatment with an LHRH agonist in association with flutamide in metastatic prostatic carcinoma. Clin Invest Med. 11:321 326. 41. Bélanger A, Caron S, Picard V. 1980 Simultaneous radioimmunoassay of progestins, androgens, and estrogens in adult rat testis. J Steroid Biochem. 13:185 190. 42. Bélanger A, Labrie F, Angeli A. 1990 Unconjugated and glucuronide steroid levels in human breast cyst fluid. Ann NY Acad Sci. 586:93 100. 43. Rodbard D. 1974 Apparent positive cooperative effect in cyclic AMP and corticosteroid production by isolated adrenal cells in response to ACTH analogs. Endocrinology. 94:1427 1437. 44. Kramer CY. 1956 Extension of multiple range tests to group means with unique numbers of replications. Biometrics. 12:307 310. 45. Labrie F, Bélanger A, Dupont A, et al. 1993 Science behind total androgen blockade: from gene to combination therapy. Clin Invest Med. 16:475 492. 46. Bélanger A, Brochu M, Cliche J. 1986 Levels of plasma steroid glucuronides in intact and castrated men with prostatic cancer. J Clin Endocrinol Metab. 62:812 815.