Metabolites Of Dehydroepiandrosterone Research Articles

Metabolic Transformations of Dehydroepiandrosterone

Dehydroepiandrosterone (DHEA) is the most abundant steroid in the human body. It enhances the action of insulin, depresses the action of glucocorticoids, increases the activity of the immune system, and decreases body fat in genetically predisposed animals without affecting food intake. It is reported to have anti-carcinogenic and anti-viral activity. Relatively, large doses of DHEA are needed to obtain these effects indicating that metabolites of DHEA may be responsible for these activities. Mammalian tissues convert DHEA to the following in sequence: 7α-hydroxyDHEA, 7-oxoDHEA, 7β-hydroxyDHEA, 7β,16α-dihydroxyDHEA. A major metabolite is androstene-3β,17β-diol which we found to be an androgen that resists conventional anti-androgen drugs (PNAS 95:11083, 98). It very likely is responsible for prostate cancer growth in the so-called “androgen-independent”, fatal stage. Like the thermogenic thyroid hormone, DHEA and its metabolites induce the formation of two thermogenic enzymes - mitochondrial L-glycerophosphate dehydrogenase and cytosolic malic enzyme, that transfer electrons from reduced pyridine coenzymes to ubiquinone without accompanying phosphorylation. In enzyme induction assays, the metabolites are increasingly effective in the order of formation shown above. 16α-Hydroxylation destroys the activity of DHEA and androstenediol by preventing their conversion to active hormone structures, but enhances activity of 7-oxoDHEA. 16α-Bromination has effects like those exerted by hydroxylation. (Supported by the authors)

Dehydroepiandrosterone Metabolites and their Interactions in Humans

Dehydroepiandrosterone (DHEA) is 7alpha-hydroxylated by the cytochrome P4507B1 in the liver, skin and brain, which are targets for glucocorticoids. 7alpha-Hydroxy-DHEA produced anti-glucocorticoid effects in vivo but the interference between the glucocorticoid hormone binding with its receptor could not be determined. In the organs mentioned above, circulating inactive cortisone is reduced to active cortisol by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). 7alpha-Hydroxy-DHEA is also a substrate for this enzyme. Studies of 11beta-HSD1 action on 7alpha-hydroxy-DHEA show the reversible production of 7beta-hydroxy-DHEA through an intermediary 7-oxo-DHEA. Both the production of 7alpha-hydroxysteroids and their interference with the activation of cortisone into cortisol are basic to the concept of native anti-glucocorticoids. The cytochrome P4507B1 responsible for 7alpha-hydroxy-DHEA production and 11beta-HSD1 are key enzymes for the modulation of glucocorticoid action in humans. This is a promising new area for research.

Redox reactions of dehydroepiandrosterone and its metabolites in differentiating 3T3-L1 adipocytes: A liquid chromatographic–mass spectrometric study

Dehydroepiandrosterone is known to depress fatty acid formation in differentiating 3T3-L1 adipocytes. The metabolism of dehydroepiandrosterone and four of its natural metabolites in differentiating adipocytes was studied by liquid chromatography–mass spectrometry. Adipocytes rapidly converted dehydroepiandrosterone to androst-5-ene-3β,17β-diol. 7α-Hydroxy-DHEA was interconverted with 7-oxo-DHEA and 7β-hydroxy-DHEA and the corresponding 17β reduced products. Dehydroepiandrosterone and its derivatives were detected only in the culture medium suggesting that dehydroepiandrosterone is metabolized via enzymes located in close proximity to, or that are integral parts of the cell membrane. Alternatively, there may be efficient mechanisms at play for extrusion of the steroids to the aqueous media rather than being retained in the lipid-rich cell. An interesting aspect of the study was finding androstenediol as the major metabolite of dehydroepiandrosterone. Androst-5-ene-3β,17β-diol has been implicated in prostate cancer. The contribution of adipose cells to the circulating supply of androst-5-ene-3β,17β-diol may therefore be considered in managing prostate cancer.

Androstenetriol improves survival in a rodent model of traumatic shock

Trauma results in activation of the hypothalamic-pituitary-adrenal axis to mediate a cascade of neurohormonal changes as a defensive mechanism. Its prolongation, however, leads to a hypermetabolic, hypoperfused, and immunosuppressed state, setting the stage for subsequent sepsis and organ failure. Androstenetriol (5-androstene-3beta, 7beta, 17betatriol - AET), a metabolite of dehydroepiandrosterone, up-regulates the host immune response markedly, prevents immune suppression and controls inflammation, leading to improved survival after lethal infections by several diverse pathogens and lethal radiation. Such actions may be useful in improving survival from traumatic shock. The neurosteroid AET will increase survival following traumatic shock. A combat relevant model of traumatic shock was used. Male Sprague-Dawley rats were anesthetized, catheterized and subjected to soft tissue injury (laparotomy). Animals were allowed to regain consciousness over the next 0.5 h and then bled 40% of their blood volume over 15 min. Forty-five minutes after the onset of hemorrhage animals were randomized to receive either a single subcutaneous dose of AET (40 mg/kg, sc) or vehicle (methylcellulose). Volume resuscitation consisted of l-lactated Ringer's (three times the shed blood volume), followed by packed red blood cells (one-third shed red cell volume). Animals were observed for three days. A total of 24 animals were studied. Of the 12 animals randomized to receive AET, all (100%) survived compared to 9 of 12 animals (75%) randomized to receive the vehicle (p < 0.05). AET significantly improved survival when administered subcutaneously in a single dose in this rodent model of traumatic shock. Further survival and mechanism studies are warranted.

P-870: Dehydroepiandrosterone sulfate (DHEAS) as a predictor of insulin resistance in patients with polycystic ovary syndrome (PCOS)

Populational studies in men have suggested that DHEAS, a metabolite of dehydroepiandrosterone (DHEA) reflecting adrenal androgen production, is inversely correlated with insulin resistance. Our objective was to determine whether DHEAS is a predictor of insulin sensitivity in patients with PCOS.

C 19-Steroids as androgen receptor modulators: Design, discovery, and structure-activity relationship of new steroidal androgen receptor antagonists

Dehydroepiandrosterone (DHEA), the most abundant steroid in human circulating blood, is metabolized to sex hormones and other C 19-steroids. Our previous collaborative study demonstrated that androst-5-ene-3β,17β-diol (Adiol) and androst-4-ene-3,17-dione (Adione), metabolites of DHEA, can activate androgen receptor (AR) target genes. Adiol is maintained at a high concentration in prostate cancer tissue; even after androgen deprivation therapy and its androgen activity is not inhibited by the antiandrogens currently used to treat prostate cancer patients. We have synthesized possible metabolites of DHEA and several synthetic analogues and evaluated their role in androgen receptor transactivation to identify AR modulators. Steroids with low androgenic potential in PC-3 cell lines were evaluated for anti-dihydrotestosterone (DHT) and anti-Adiol activity. We discovered three potent antiandrogens: 3β-acetoxyandrosta-1,5-diene-17-one 17-ethylene ketal (ADEK), androsta-1,4-diene-3,17-dione 17-ethylene ketal (OAK), and 3β-hydroxyandrosta-5,16-diene (HAD) that antagonized the effects of DHT as well as of Adiol on the growth of LNCaP cells and on the expression of prostate-specific antigen (PSA). In vivo tests of these compounds will reveal their potential as potent antiandrogens for the treatment of prostate cancer.

Androstenediol ameliorates alterations in immune cells cytokine production capacity in a two-hit model of trauma-hemorrhage and sepsis

Although administration of androstenediol (a metabolite of dehydroepiandrosterone) following trauma-hemorrhage (T-H) produces beneficial effects on inflammatory cytokines and organ function, it remains unknown whether this metabolite has any salutary effects in preventing alterations in immune cell cytokine production following a combined insult of T-H and sepsis. To examine this, male rats underwent laparotomy, hemorrhagic shock (mean BP 40 mmHg for 90 min) and resuscitation or sham operation. Androstenediol (1 mg/kg BW i.v.) or vehicle was administered at the end of resuscitation. Twenty hrs after T-H or sham operation, sepsis was induced by cecal ligation and puncture (CLP). Five hours thereafter, plasma cytokine levels and cytokine production of various immune cells were determined. In a separate set of experiments, survival was monitored for 10 days after the induction of sepsis. Administration of androstenediol markedly decreased plasma IL-6 and TNF-α levels following T-H and CLP. Furthermore, it prevented the increased production of IL-6 and TNF-α by Kupffer cells and alveolar macrophages and attenuated the decrease in IL-6 and TNF-α production by splenic macrophages; however, it had no significant effects on the depressed IL-6 and TNF-α production by PBMC following T-H and CLP. The depressed IL-2 and IFN-γ production by splenocytes under those conditions was attenuated by the administration of androstenediol. Furthermore, survival rate following T-H and subsequent sepsis was improved by androstenediol treatment. Since androstenediol administration following T-H attenuated cytokine production and reduced mortality in a double-hit model of T-H and sepsis, this agent appears to be a novel and useful adjunct for maintaining the immune cell functions following T-H and for decreasing the mortality rate from subsequent susceptibility to sepsis.

Androstenediol administration after trauma-hemorrhage attenuates inflammatory response, reduces organ damage, and improves survival following sepsis

Although androstenediol (adiol or 5-androstene-3beta,17beta-diol), a metabolite of dehydroepiandrosterone (DHEA), has protective effects following trauma-hemorrhage (T-H), it remains unknown whether administration of adiol has any salutary effects on the inflammatory response and outcome following a combined insult of T-H and sepsis. Male rats underwent T-H shock [mean arterial pressure (MAP) 40 mmHg for 90 min] followed by resuscitation. Adiol (1 mg/kg body wt) or vehicle was administered at the end of resuscitation. Sepsis was induced by cecal ligation and puncture (CLP) at 20 h after T-H or sham operation. Five hours after CLP, plasma and tissue samples were analyzed for cytokines (IL-6 and IL-10), MPO, neutrophil chemotactic factor (CINC-3), and liver injury (alanine aminotransferase and lactate dehydrogenase). In another group of rats, the gangrenous cecum was removed at 10 h after CLP, the cavity was irrigated with warm saline and closed in layers, and mortality was recorded over 10 days. T-H followed by CLP produced a significant elevation in plasma IL-6 and IL-10 levels, enhanced neutrophil cell activation, and resulted in liver injury. Adiol administration prevented the increase in cytokine production, neutrophil cell activation, and attenuated liver injury. Moreover, rats subjected to the combined insult, receiving vehicle or adiol, had a 50% and 6% mortality, respectively. Since adiol administration suppresses proinflammatory cytokines, reduces liver damage, and decreases mortality after the combined insult of T-H and sepsis, this agent appears to be a novel adjunct to fluid resuscitation for decreasing T-H-induced septic complications and mortality.

Dehydroepiandrosterone Restoration of Growth Hormone Gene Expression in Aging Female Rats, in Vivo and in Vitro: Evidence for Actions via Estrogen Receptors

A decline in dehydroepiandrosterone (DHEA) and GH levels with aging may be associated with frailty and morbidity. Little is known about the direct effects of DHEA on somatotropes. We recently reported that 17beta-estradiol (E2), a DHEA metabolite, stimulates the expression of GH in vitro in young female rats. To test the hypothesis that DHEA restores function in aging somatotropes, dispersed anterior pituitary (AP) cells from middle-aged (12-14 months) or young (3-4 months) female rats were cultured in vitro with or without DHEA or E2 and fixed for immunolabeling or in situ hybridization. E2 increased the percentage of AP cells with GH protein or mRNA in the aged rats to young levels. DHEA increased the percentages of somatotropes (detected by GH protein or mRNA) from 14-16 +/- 2% to 29-31 +/- 3% (P < or = 0.05) and of GH mRNA (detected by quantitative RT-PCR) only in aging rats. To test DHEA's in vivo effects, 18-month-old female rats were injected with DHEA or vehicle for 2.5 d, followed by a bolus of GHRH 1 h before death. DHEA treatment increased serum GH 1.8-fold (7 +/- 0.5 to 12 +/- 1.3 ng/ml; P = 0.02, by RIA) along with a similar increase (P = 0.02) in GH immunolabel. GHRH target cells also increased from 11 +/- 1% to 19 +/- 2% (P = 0.03). Neither GH nor GHRH receptor mRNAs levels were changed. To test the mechanisms behind DHEA's actions, AP cells from aging rats were treated with DHEA with or without inhibitors of DHEA metabolism. Trilostane, aminogluthemide, or ICI 182,780 completely blocked the stimulatory effects of DHEA, suggesting that DHEA metabolites may stimulate aging somatotropes via estrogen receptors.

A Novel Polymorphic Cytochrome P450 Formed by Splicing of CYP3A7 and the Pseudogene CYP3AP1

The cytochrome P450 3A7 (CYP3A7) is the most abundant CYP in human liver during fetal development and first months of postnatal age, playing an important role in the metabolism of endogenous hormones, drugs, differentiation factors, and potentially toxic and teratogenic substrates. Here we describe and characterize a novel enzyme, CYP3A7.1L, encompassing the CYP3A7.1 protein with the last four carboxyl-terminal amino acids replaced by a unique sequence of 36 amino acids, generated by splicing of CYP3A7 with CYP3AP1 RNA. The corresponding CYP3A7-3AP1 mRNA had a significant expression in liver, kidney, and gastrointestinal tract, and its presence was found to be tissue-specific and dependent on the developmental stage. Heterologous expression in yeast revealed that CYP3A7.1L was a functional enzyme with a specific activity similar to that of CYP3A7.1 and, in some conditions, a different hydroxylation specificity than CYP3A7.1 using dehydroepiandrosterone as a substrate. CYP3A7.1L was found to be polymorphic due to a mutation at position -6 of the first splicing site of CYP3AP1 (CYP3A7_39256T-->A), which abrogates the pseudogene splicing. This polymorphism had pronounced interethnic differences and was in linkage disequilibrium with other functional polymorphisms described in the CYP3A locus: CYP3A7*2 and CYP3A5*1. Therefore, the resulting CYP3A haplotypes express different sets of enzymes within the population. In conclusion, a novel mechanism, consisting of the splicing of the pseudogene CYP3AP1 to CYP3A7, causes the formation of the novel CYP3A7.1L having a different tissue distribution and functional properties than the parent CYP3A7 enzyme, with possible developmental, physiological, and toxicological consequences.

Dehydroepiandrosterone (DHEA) modulates GHRH, somatostatin and angiotensin II action at the pituitary level

In view of the present controversy related to the potential beneficial effects of clinical dehydroepiandrosterone (DHEA) treatments, and considering our own previous results that reveal an influence of this steroid in pituitary hyperplasia development in vivo in rats, we decided to evaluate the role of DHEA in prolactin and GH secretion, as well as in second messengers involved, in cultured rat anterior pituitary cells. DHEA (1 x 10(-5) to 1 x 10(-7) M) did not modify basal GH or prolactin release, and a prolactin inhibitory effect was observed only for androstenediol, a metabolite of DHEA. DHEA partially prevented dopamine (1 x 10(-6) M)-induced prolactin inhibition and facilitated the prolactin-releasing effect of 10(-8) M Ang II, without modifying the resulting Ca2+(i) mobilization. Furthermore, DHEA potentiated the GH release and cAMP production induced by 1 x 10(-8) M GHRH. Finally, DHEA partially reversed the inhibitory effect of 1 x 10(-8) M somatostatin on GH, but not prolactin, release. We conclude that DHEA in vitro, directly or indirectly through conversion into metabolites, is able to modulate the hormonal response of the pituitary to hypothalamic regulators. It can enhance pituitary prolactin release and induce GH secretion. These effects could help explain some of the side effects observed in prolonged DHEA treatments in vivo and should be taken into account when considering its use in human clinical trials.

CYP7B expression and activity in fibroblast‐like synoviocytes from patients with rheumatoid arthritis: Regulation by proinflammatory cytokines

The cytochrome P450 enzyme CYP7B catalyzes the conversion of dehydroepiandrosterone (DHEA) into 7alpha-hydroxy-DHEA (7alpha-OH-DHEA). This metabolite can stimulate the immune response. We previously reported that the severity of murine collagen-induced arthritis is correlated with CYP7B messenger RNA (mRNA) expression and activity in the arthritic joint. The purpose of this study was to investigate the presence of 7alpha-OH-DHEA in synovial samples and the cytokine regulation of CYP7B activity in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). The presence of 7alpha-OH-DHEA was examined in synovial biopsy tissues, synovial fluid, and serum by radioimmunoassay. The effect of cytokines on CYP7B mRNA expression and CYP7B activity in FLS was examined by determining the formation of the CYP7B enzyme product 7alpha-OH-DHEA with the use of high-performance liquid chromatography. The CYP7B enzyme product 7alpha-OH-DHEA was found in synovial biopsy tissues, synovial fluid, and serum from RA patients. The proinflammatory cytokines tumor necrosis factor alpha (TNFalpha), interleukin-1alpha (IL-1alpha), IL-1beta, and IL-17 up-regulated CYP7B activity in an FLS cell line 2-10-fold. Enhanced CYP7B activity was correlated with an increase in CYP7B mRNA. The cytokine transforming growth factor beta inhibited CYP7B activity. Moreover, CYP7B activity was detected in 10 of 13 unstimulated synovial fibroblast cell lines. Stimulation with TNFalpha increased CYP7B activity in all cell lines tested. Exposure to the proinflammatory cytokines TNFalpha, IL-1alpha, IL-1beta, and IL-17 increases CYP7B activity in synovial tissue. Increased CYP7B activity leads to higher levels of the DHEA metabolite 7alpha-OH-DHEA in synovial fluid, which may contribute to the maintenance of the chronic inflammation observed in RA patients.

Urinary Markers of Adrenarche: Reference Values in Healthy Subjects, Aged 3–18 Years

Information on the urinary excretion of dehydroepiandrosterone (DHEA) and its direct metabolites is scarce for healthy subjects during growth. We used gas chromatography-mass spectrometry urinary steroid profiling to noninvasively study adrenarchal metabolome in 400 healthy subjects, aged 3-18 yr. Urinary 24-h excretion rates of DHEA did not increase significantly before age 7-8 yr. However, DHEA together with its 16alpha-hydroxylated downstream metabolites, 16alpha-hydroxy-DHEA and 3beta,16alpha,17beta-androstenetriol (DHEA&M), as well as the DHEA metabolite, 5-androstene-3beta,17beta-diol (ADIOL), and the sum of major urinary androgen metabolites (C19) rose consistently from the youngest to the oldest age group. The significant increases (P < 0.01) observed for 24-h excretion rates of C19, ADIOL, and DHEA&M were 2- to 4-fold in boys and girls between age 3 and 8 yr. DHEA&M, for example, rose from about 20 to 80 microg/d (P < 0.0001) during this period. Until the age of 16 yr, DHEA&M excretion also increased to nearly 1000 microg/d. Patterns of steroidogenic enzyme activities were assessed (from definite ratios of urinary steroid metabolites) for 21-hydroxylase, 3beta-hydroxysteroid dehydrogenase, 17beta-hydroxysteroid dehydrogenase, and 5alpha-reductase. Our results indicate for healthy boys and girls that adrenarche is a gradual process starting much earlier than hitherto believed. Efficient metabolism of DHEA, especially to 16-hydroxylated steroids, may explain the almost constant levels seen for this steroid until age 7-8 yr. The established reference values for DHEA, DHEA&M, ADIOL, C19 (including androsterone and etiocholanolone), and urinary parameters of steroidogenic enzyme activities could be useful to identify nutritional, environmental, and pathophysiological interrelations with the progressive maturational process of adrenarche. Our data may also be used as reference data for the diagnosis of steroid-related disorders.

Tumour necrosis factor-α stimulates dehydroepiandrosterone metabolism in human fibroblast-like synoviocytes: a role for nuclear factor-κB and activator protein-1 in the regulation of expression of cytochrome p450 enzyme 7b

Glucocorticoids have successfully been used in the treatment of rheumatoid arthritis. Data suggest that 7α-hydroxy-dehydroepiandrosterone (7α-OH-DHEA), an immunostimulating metabolite of dehydroepiandrosterone, can block glucocorticoid-induced immune suppression. Formation of 7α-OH-DHEA is catalyzed by activity of cytochrome p450 enzyme 7b (Cyp7b). Recently, we reported that tumour necrosis factor (TNF)-α, IL-1α, IL-1β and IL-17 enhance Cyp7b mRNA expression and induce a concomitant increase in the formation of 7α-OH-DHEA by fibroblast-like synoviocytes (FLS) from rheumatoid arthritis patients. The aim of this study was to elucidate which signal transduction pathway is involved in the TNF-α-mediated induction of Cyp7b activity in FLS. We studied the effects of inhibitors of different signal transduction pathways on Cyp7b activity in FLS by measuring Cyp7b mRNA expression using reverse transcription PCR and by measuring the formation of 7α-OH-DHEA. We applied SN50, an inhibitor of nuclear translocation of transcription factors (i.e. activator protein-1 [AP-1] and nuclear factor-κB [NF-κB]); PSI, a proteasome inhibitor that prevents IκB degradation and thereby NF-κB release; SP600125, a c-Jun N-terminal kinase (JNK) inhibitor; and the mitogen-activated protein kinase inhibitors PD98059 (extracellular signal-regulated kinase) and SB203580 (p38). Cyp7b is constitutively expressed in RA FLS and can be activated in response to TNF-α. SN50 and PSI prevented the TNF-α-induced increase in Cyp7b activity, whereas the mitogen-activated protein kinase inhibitors PD98059 and SB203580 had no effect. In addition, inhibition of Cyp7b mRNA expression and activity was observed with SN50, PSI and SP600125, suggesting that NF-κB and AP-1 induce Cyp7b transcription. These findings suggest that NF-κB and AP-1 are involved in the TNF-α-enhanced formation of the dehydroepiandrosterone metabolite 7α-OH-DHEA. Our results are in accordance with presence of AP-1 and NF-κB binding sites in the Cyp7b promoter.

Plasma levels of 7-hydroxymetabolites of dehydroepiandrosterone in healthy Central European aging men

Evidence has been accumulating that 7-hydroxyepimers of dehydroepiandrosterone may act as locally active immunomodulatory and immunoprotective agents, counteracting the actions of glucocorticoids. 7Alpha- and 7beta-hydroxylated metabolites of dehydroepiandrosterone are present in near nanomolar concentrations in human blood. Limited data have been available on its changes during the last decades of human life. We examined 213 healthy Central European males in age groups from 50 to 91 years and determined both 7-hydroxyepimers, 7alpha- and 7beta-hydroxy-dehydroepiandrosterone, in sera using specific radioimmunoassays. The age dependence of steroid levels was evaluated using one-way ANOVA. In men, a distinct decline in 7alpha-hydroxy-dehydroepiandrosterone with age was observed. The curves for age dependence of 7-hydroxy metabolite levels in males resembled those previously determined for unconjugated dehydroepiandrosterone. The levels of both 7-hydroxylated epimers correlated excellently with each other.

Is There a Receptor for Dehydroepiandrosterone or Dehydroepiandrosterone Sulfate?

It remains unknown whether dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) have a physiological role other than serving as metabolic intermediates in androgen synthesis. Apart from intracellular metabolism, there is no convincing cellular mechanism of action for physiological concentrations of DHEA(S). Unlike other major steroids, a receptor for DHEA(S) has not been definitively isolated. This article will review the evidence supporting a receptor-dependent basis for the direct physiological effects of DHEA(S). The data supporting an intracellular receptor for DHEA(S) are relatively weak and do not allow us to determine whether DHEA(S) directly, or a metabolite of DHEA(S), acts as a direct receptor ligand. Recent data strongly support a plasma membrane receptor for DHEA, but this potential receptor is yet to be isolated. Definitive characterization of the molecular mechanism (receptor or otherwise) of DHEA(S) action is necessary before we can determine whether DHEA(S) has a biological role other than as an androgen precursor.

Mechanism of salutary effects of androstenediol on hepatic function after trauma-hemorrhage: role of endothelial and inducible nitric oxide synthase.

Recent studies have shown that administration of dehydroepiandrosterone (DHEA) after trauma-hemorrhage (T-H) improves cardiovascular and hepatic function in male animals. Although androstenediol, one of the DHEA metabolites, has been recently reported to produce salutary effects on cardiac function and splanchnic perfusion after T-H, it remains unknown whether androstenediol per se has any salutary effects on hepatic function under those conditions. To study this, male Sprague-Dawley rats underwent laparotomy and approximately 90 min of hemorrhagic shock (35-40 mmHg), followed by resuscitation with four times the shed blood volume in the form of Ringer lactate. Androstenediol (1 mg/kg body wt iv) was administered at the end of resuscitation, and the animals were killed 24 h later. T-H significantly reduced portal blood flow, bile production, and serum albumin levels. Portal pressure, serum alanine aminotransferase, hepatic nitrate/nitrite, inducible nitric oxide synthase (iNOS), and endothelin-1 markedly increased after T-H. The alterations in these parameters induced by T-H were significantly attenuated in rats treated with androstenediol. Endothelial NOS (eNOS) expression, which was not different between T-H and sham, was found to be significantly elevated in T-H androstenediol-treated rats. These data suggest that improvement in hepatic perfusion by androstenediol after T-H is likely due to a decrease in endothelin-1 and induction of eNOS. Moreover, the decrease in hepatic damage after androstenediol administration is likely related to liver iNOS downregulation. Thus androstenediol appears to be a novel and useful adjunct for restoring hepatic function in male animals after adverse circulatory conditions.

Biosynthesis of [ 3H]7α-hydroxy-, 7β-hydroxy-, and 7-oxo-dehydroepiandrosterone using pig liver microsomal fractions

Current research on dehydroepiandrosterone (DHEA) is limited due to lack of radiolabeled metabolites. We utilized pig liver microsomal (PLM) fractions to prepare [ 3H]-labeled 7α-hydroxy-DHEA (7α-OH-DHEA), 7β-hydroxy-DHEA (7β-OH-DHEA), and 7-oxo-DHEA substrates from 50 μM [1,2,6,7- 3H]DHEA (specific radioactivity 60–80 mCi/mmol). The metabolites were separated by preparative thin-layer chromatography (TLC) using ethyl acetate:hexane:glacial acetic acid (18:8:3 v:v:v) as the mobile phase, extracted with ethyl acetate, and dried under a stream of nitrogen. Metabolites assayed by TLC and gas chromatography–mass spectrometry were observed to be pure. In the presence of an reduced nicotinamide adenine dinucleotide phosphate (NADPH)-regenerating system initiated with 1 mM NADPH alone, 1 mg/ml PLM produced 7α-OH-DHEA with minor amounts of 7-oxo-DHEA (68 and 14 nmol/2 h/2 ml, respectively; 82% conversion), while in the presence of 1 mM NADPH and 1 mM oxidized nicotinamide adenine dinucleotide phosphate (NADP +), more 7-oxo-DHEA than 7α-OH-DHEA (58 and 11 nmol/2 ml/120 min, respectively; 69% conversion) was formed . When longer reaction times were used with NADPH and NADP +, a mixture of 7α-OH-DHEA, 7β-OH-DHEA, and 7-oxo-DHEA was produced (19,14, and 35 nmol/180 min/2 ml, respectively; 62% conversion). Using pig liver microsomes, the radiolabeled metabolites of DHEA can be prepared in stable, pure form at 10 mM concentrations and >0.5 mCi/mmol levels of radioactivity for biochemical studies.

Salutary effects of androstenediol on cardiac function and splanchnic perfusion after trauma-hemorrhage.

Recent studies have shown that dehydroepiandrosterone (DHEA) administration after trauma-hemorrhage (T-H) improves cardiovascular function and decreases cytokine production in male animals. Although androstenediol, one of the metabolites of DHEA, is reported to have estrogen-like activity, it remains unknown whether androstenediol per se has any salutary effects on cytokines and cardiovascular function after T-H. To examine this effect, male Sprague-Dawley rats underwent laparotomy and were bled to and maintained at a mean arterial blood pressure of 35-40 mmHg for approximately 90 min. The animals were resuscitated with four times the volume of maximal bleedout volume in the form of Ringer lactate. Androstenediol (1 mg/kg body wt i.v.) or vehicle was administered at the end of resuscitation. Twenty-four hours after resuscitation, cardiac function and organ blood flow were measured by using (85)Sr-microspheres. Circulating levels of nitrate/nitrite and IL-6 were also determined. Cardiovascular function and organ blood flow were significantly depressed after T-H. However, these parameters were restored by androstenediol treatment. The elevated plasma IL-6 levels after T-H were also lowered by androstenediol treatment. In contrast, plasma levels of nitrate/nitrite were the highest in the androstenediol-treated T-H animals. Because androstenediol administration after T-H decreases cytokine production and improves cardiovascular function, this agent appears to be a novel and useful adjunct for restoring the depressed cardiovascular function and for cytokine production in males after adverse circulatory conditions.

STEREO- AND REGIOSELECTIVITY ACCOUNT FOR THE DIVERSITY OF DEHYDROEPIANDROSTERONE (DHEA) METABOLITES PRODUCED BY LIVER MICROSOMAL CYTOCHROMES P450

The purpose of this study was to quantify the oxidative metabolism of dehydroepiandrosterone (3beta-hydroxy-androst-5-ene-17-one; DHEA) by liver microsomal fractions from various species and identify the cytochrome P450 (P450) enzymes responsible for production of individual hydroxylated DHEA metabolites. A gas chromatography-mass spectrometry method was developed for identification and quantification of DHEA metabolites. 7alpha-Hydroxy-DHEA was the major oxidative metabolite formed by rat (4.6 nmol/min/mg), hamster (7.4 nmol/min/mg), and pig (0.70 nmol/min/mg) liver microsomal fractions. 16alpha-Hydroxy-DHEA was the next most prevalent metabolite formed by rat (2.6 nmol/min/mg), hamster (0.26 nmol/min/mg), and pig (0.16 nmol/min/mg). Several unidentified metabolites were formed by hamster liver microsomes, and androstenedione was produced only by pig microsomes. Liver microsomal fractions from one human demonstrated that DHEA was oxidatively metabolized at a total rate of 7.8 nmol/min/mg, forming 7alpha-hydroxy-DHEA, 16alpha-hydroxy-DHEA, and a previously unidentified hydroxylated metabolite, 7beta-hydroxy-DHEA. Other human microsomal fractions exhibited much lower rates of metabolism, but with similar metabolite profiles. Recombinant P450s were used to identify the cytochrome P450s responsible for DHEA metabolism in the rat and human. CYP3A4 and CYP3A5 were the cytochromes P450 responsible for production of 7alpha-hydroxy-DHEA, 7beta-hydroxy-DHEA, and 16alpha-hydroxy-DHEA in adult liver microsomes, whereas the fetal/neonatal form CYP3A7 produced 16alpha-hydroxy and 7beta-hydroxy-DHEA. CYP3A23 uniquely formed 7alpha-hydroxy-DHEA, whereas other P450s, CYP2B1, CYP2C11, and CYP2D1, were responsible for 16alpha-hydroxy-DHEA metabolite production in rat liver microsomal fractions. These results indicate that the stereo- and regioselectivity of hydroxylation by different P450s account for the diverse DHEA metabolites formed among various species.