Dehydroepiandrosterone decreases while cortisol increases in vitro growth and viability of Entamoeba histolytica

In vitro exposure of Entamoeba histolytica trophozoites to the sex steroids 17beta-estradiol, progesterone, and dehydrotestosterone had little effect on parasite viability or proliferation. However, treatment with the adrenal steroid dehydroepiandrosterone (DHEA) markedly inhibited parasite proliferation, adherence and motility, and at a certain dose it induced trophozoite lysis. The opposite effect on proliferation was found when the trophozoites were exposed to cortisol. Moreover, DHEA decreased while cortisol increased the parasite's DNA synthesis determined by 3H-thymidine incorporation. Trophozoite lysis by DHEA appeared to be caused by a necrotic rather than an apoptotic process, as observed in propidium iodide and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling assays. A possible mechanisms of action was derived from experiments demonstrating that the activity of a putative 3-hydroxy-3-methyl glutaryl CoA reductase detected in trophozoite extracts was inhibited in the presence of DHEA. Contrary to its in vitro inhibitory effect, in vivo administration of DHEA to infected hamsters resulted in exacerbation of the amebic liver abscesses. These results demonstrated that androgen steroids act directly upon E. histolytica growth and viability, and may shed new light on some age and gender differences in disease progression, as well as finding application in the drug treatment of human amebiasis.     

Methotrexate: studies on cellular metabolism. IV. Effect on the mitochondrial oxidation of cytosolic-reducing equivalents in HeLa cells

The effect of methotrexate (MTX) on the mitochondrial oxidation of cytosolic-reducing equivalents in HeLa cells was studied. MTX inhibited (100 per cent) malate dehydrogenase activity, but no effect was observed on that of GOT. MTX (0.5 mM) inhibited (100 per cent) the activity of reconstituted enzymatic system MDH-GOT, probably as a consequence of inhibition of malate dehydrogenase activity. MTX decreased pyruvate production (54 per cent), demonstrating its inhibitory action on the malate-aspartate shuttle. Blockage of the malate-aspartate shuttle by MTX accounts for the decrease in cellular energetic gain. The results obtained are consistent with the view that in HeLa cells, as well as in other tumour cells, the transport of reducing equivalents from cytoplasmic NADH into the respiratory chain of mitochondria is via the malate-aspartate shuttle.     

Dehydroepiandrosterone inhibits intracellular calcium release in beta-cells by a plasma membrane-dependent mechanism

Both dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) affect glucose stimulated insulin secretion, though their cellular mechanisms of action are not well characterized. We tested the hypothesis that human physiological concentrations of DHEA alter insulin secretion by an action initiated at the plasma membrane of beta-cells. DHEA alone had no effect on intracellular calcium concentration ([Ca(2+)](i)) in a rat beta-cell line (INS-1). However, it caused an immediate and dose-dependent inhibition of carbachol-induced Ca(2+) release from intracellular stores, with a 25% inhibition at zero. One nanometer DHEA. DHEA also inhibited the Ca(2+) mobilizing effect of bombesin (29% decrease), but did not inhibit the influx of extracellular Ca(2+) evoked by glyburide (100 microM) or glucose (15 mM). The steroids (androstenedione, 17-alpha-hydroxypregnenolone, and DHEAS) had no inhibitory effect on carbachol-induced intracellular Ca(2+) release. The action of DHEA depended on a signal initiated at the plasma membrane, since membrane impermeant DHEA-BSA complexes also inhibited the carbachol effect on [Ca(2+)](i) (39% decrease). The inhibition of carbachol-induced Ca(2+) release by DHEA was blocked by pertussis toxin (PTX). DHEA also inhibited the carbachol induction of phosphoinositide generation, with a maximal inhibition at 0.1 nM DHEA. Furthermore, DHEA inhibited insulin secretion induced by carbachol in INS-1 cells by 25%, and in human pancreatic islets by 53%. Taken together, this is the first report showing that human physiological concentrations of DHEA decrease agonist-induced Ca(2+) release by a rapid, non-genomic mechanism in INS-1 cells. Furthermore, these data provide evidence consistent with the existence of a specific plasma membrane DHEA receptor, mediating this signal transduction pathway by pertussis toxin-sensitive G-proteins.     

Dehydroepiandrosterone-sulfate inhibits thrombin-induced platelet aggregation

Dehydroepiandrosterone (DHEA) and its sulfated form, DHEA-S, are the most abundant steroids circulating in human blood. DHEA stimulates endothelial cells to release high amounts of nitric oxide in the circulation. Nitric oxide activates guanylyl cyclase in platelets thus decreasing the responsiveness of these cells to physiological agonists. However, the impact of DHEA-S and DHEA on platelet function and their possible role in modulating the response of human platelets to physiological agonists were not yet investigated. Here, DHEA-S, but not DHEA, inhibited in vitro thrombin-dependent platelet aggregation in a dose-dependent manner. DHEA-S exerted this effect by decreasing thrombin-dependent dense granule secretion, and so impairing the positive feed-back loop provided by ADP. Furthermore, DHEA-S inhibited thrombin-dependent activation of Akt, ERK1/2, and p38 MAP kinase. Although both DHEA-S and DHEA directly activated in platelets the inhibitory cGMP/PGK/VASP pathway, these events were not responsible for the inhibitory action of DHEA-S in platelets. In addition DHEA-S acted in synergism with nitric oxide in inhibiting platelet aggregation. In conclusion DHEA-S inhibited platelet activation caused by a mild stimulus without completely hampering platelet functionality and thus DHEA-S may participate in the physiological mechanisms that maintain circulating platelets in a resting state. The role played by DHEA-S could be relevant mainly when the functionality of the vascular endothelium is compromised.     

Dehydroepiandrosterone with other neurosteroids preserve neuronal mitochondria from calcium overload

This current study was designed to test whether the dehydroepiandrosterone (DHEA) and other neurosteroids could improve mitochondrial resistance to ischemic damage and cytoplasmic Ca(2+) overload. To imitate these mechanisms at mitochondrial level we treated the saponin permeabilized neurons either with the respiratory chain inhibitor, 1-methyl-4-phenylpyridinium or raised free extra-mitochondrial [Ca(2+)]. Loss of mitochondrial membrane potential (as an indicator of loss of function) was detected by JC-1. The results demonstrate that DHEA partly prevented Ca(2+) overload induced loss of mitochondrial membrane potential but not the loss of potential induced by the inhibitor of the respiratory chain. A similar effect was observed in the presence of other neurosteroids, pregnenolone, pregnanolone and allopregnanolone. DHEA inhibited also the Ca(2+) accumulation to the mitochondria in the presence of Ca(2+) efflux inhibitors. Thus, in the present work we provide evidence that DHEA with several other neurosteroids protect the mitochondria against intracellular Ca(2+) overload by inhibiting Ca(2+) influx into the mitochondrial matrix.     

Inhibition of carbamoyl phosphate synthetase-I by dietary dehydroepiandrosterone.

Dehydroepiandrosterone (DHEA), administered per os, serves to prevent or retard the development of a variety of genetic and induced disorders in mice and rats. This treatment also results in the development of hepatomegaly, a change of liver color from pink to mahogany, peroxisome proliferation in hepatocytes and alterations in hepatocyte mitochondria morphology and respiration. We used one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) to identify changes in the relative levels of liver proteins produced by DHEA treatment of rodents. In mouse liver, there were apparent increases in the levels of 26 proteins and decreases in the levels of 7 proteins. Of the induced proteins the most prominent had Mr approximately 72 K; this protein was identified in a previous study as enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase. Another protein of Mr approximately 28 K, of unknown nature, also was induced markedly by DHEA treatment of mice and rats. A protein of Mr approximately 160 K, which was identified as carbamoyl phosphate synthetase-I (CPS-I), was decreased markedly by DHEA action. This enzyme, which comprises approx. 15-20% of mitochondrial matrix protein, is involved in the entry and rate-limiting step of the urea cycle. The specific activity of CPS-I also was significantly decreased by DHEA, but serum urea levels were normal. To determine whether steroids other than DHEA also induced similar changes, mice were treated with various steroids for 14 days and, thereafter, liver proteins were evaluated by SDS-PAGE: estradiol-17 beta and isoandrosterone induced both the approximately 72 and approximately 28 kDa proteins, testosterone and androsterone induced the 28 kDa protein only, but etiocholanolone, pregnenolone and progesterone were without effect. The findings of this study serve to demonstrate that: (i) hepatic protein levels are affected by DHEA treatment of mice and rats; (ii) liver CPS-I activity is decreased significantly by DHEA treatment, but serum urea levels remain within the normal range; and (iii) sex steroids and some of their precursors, when administered per os, also alter liver protein levels.     

Dehydroepiandrosterone activates endothelial cell nitric-oxide synthase by a specific plasma membrane receptor coupled to Galpha(i2,3)

The adrenal steroid dehydroepiandrosterone (DHEA) has no known cellular receptor or unifying mechanism of action, despite evidence suggesting beneficial vascular effects in humans. Based on previous data from our laboratory, we hypothesized that DHEA binds to specific cell-surface receptors to activate intracellular G-proteins and endothelial nitric-oxide synthase (eNOS). We now pharmacologically characterize a putative plasma membrane DHEA receptor and define its associated G-proteins. The [3H]DHEA binding to isolated plasma membranes from bovine aortic endothelial cells was of high affinity (K(d) = 48.7 pm) and saturable (B(max) = 500 fmol/mg protein). Structurally related steroids failed to compete with DHEA for binding. The putative DHEA receptor was functionally coupled to G-proteins, because guanosine 5'-O-(3-thio)triphosphate (GTPgammaS) inhibited [3H]DHEA binding to plasma membranes by 69%, and DHEA increased [35S]GTPgammaS binding by 157%. DHEA stimulated [35S]GTPgammaS binding to Galpha(i2) and Galpha(i3), but not to Galpha(i1) or Galpha(o). Pretreatment of plasma membranes with antibody to Galpha(i2) or Galpha(i3), but not to Galpha(i1), inhibited the DHEA activation of eNOS. Thus, DHEA receptors are expressed on endothelial cell plasma membranes and are coupled to eNOS activity through Galpha(i2) and Galpha(i3). These novel findings should allow us to isolate the putative receptor and reevaluate the physiological role of DHEA activity.     

Adrenal and gonadal steroids inhibit IL-6 secretion by human marrow cells

Adrenal and gonadal steroids have protective effects on the skeleton that may be conferred partly by their ability to inhibit bone resorptive cytokines such as interleukin 6 (IL-6). We tested the hypothesis that IL-6 secretion by human marrow cells and a line of marrow stromal cells (KM101) is inhibited by dehydroepiandrosterone (DHEA), dihydrotestosterone (DHT) and 17beta-oestradiol (E(2)). We also examined whether the estrogen status of the donor influenced the steroids' effects on IL-6 secretion. Femoral bone marrow was obtained from 19 postmenopausal women undergoing hip arthroplasty, and from seven subjects receiving oestrogen replacement therapy (ERT) at the time of surgery. Low-density mononuclear cells were isolated and cultured in IL-1beta-supplemented media, with or without DHEA, DHT or E(2). DHEA suppressed IL-6 more consistently than DHT or E(2): DHEA significantly suppressed IL-6 in 84% of cultures, DHT suppressed IL-6 in 58%, and E(2)did so in 50%. The magnitude of IL-6 inhibition was also greater for DHEA (group mean, treated/control of 62%) compared to DHT (81%) and E(2)(76%). In cultures from subjects receiving ERT, DHEA and DHT suppressed IL-6 in some, whereas E(2)did not suppress IL-6 secretion. Each steroid also significantly inhibited IL-6 secretion by KM101 cells. In summary, in marrow cultured from postmenopausal women, DHEA suppressed IL-6 secretion more consistently and to a greater degree than did DHT and E(2). Second, the inhibitory effect of E(2)was abrogated in marrow from women receiving ERT.     

A radioimmunoassay for serum dehydroepiandrosterone sulfate

A radioimmunoassay for serum dehydroepiandrosterone sulfate (DHEAS) (1) has been developed using anti-DHEA antiserum obtained by immunizing rabbits with DHEA-17 oxime-bovine serum albumin. Serum volume of 0.01 to 0. 1 ml was used for analysis. After the addition of ammonium salt of DHEA-7³ H sulfate for recovery and a preliminary removal of DHEA, DHEAS was extracted as pyridinium salt by methylene chloride. The dried extract was subjected to solvolysis (Burstein & Lieberman), followed by paper chromatography. The eluates and DHEA-7 ³H which was added to determine the % free of DHEA were evaporated and incubated with the antiserum containing pepsin treated human immune serum globulin and bovine serum albumin at 37°C for 1 hour. Ammonium sulfate was used to separate free from bound DHEA. The accuracy, precision and specificity were satisfactory. The sensitivity was 3 ng per sample. The blank values could not be differentiated from zero. Although the antiserum reacts with the other 3βOHΔ⁵ steroids as well as DHEA, the complete separation of DHEA from the other 3βOHΔ⁵ steroids was achieved chromatographically. Serum DHEAS levels in normal subjects and patients with adrenocortical disorders obtained with the radioimmunoassay were comparable to those obtained with gasliquid chromatography.     

Metabolism of dehydroepiandrosterone by rodent brain cell lines: relationship between 7-hydroxylation and aromatization

The rate of aromatization of 4-androstenedione (AD) and 7-hydroxylation of dehydroepiandrosterone (DHEA) by different neuronal cell lines from fetal rat and mouse brain was compared to that of embryonic rat hippocampal cells in primary culture. The (3)H-labeled steroids were incubated with the cells and the metabolites extracted and separated by thin layer chromatography (TLC), as well as analyzed by high-performance liquid chromatography (HPLC) for further identification. All cell types produced estrone (E(1)) and estradiol (E(2)) from [(3)H]AD but the rate of aromatization was lowest with the rat hippocampal cells in primary culture. With [(3)H]DHEA, BHc.2 mouse hippocampal cells and E(t)C.1 neurons behaved like the mixed cells from rat hippocampus, forming 7-hydroxy DHEA as the almost exclusive product. In contrast, mouse brain BV2 microglia were virtually unable to hydroxylate DHEA at C-7 and yielded estrogen and more testosterone (T) than other cell types tested. These experiments highlight the pivotal role of 3beta-hydroxysteroid dehydrogenase/ketoisomerase in the control of AD formation for its subsequent aromatization to estrogen. It raises the possibility that differences in metabolism of DHEA by certain brain cells could account for differences in their immunomodulatory and neuroprotective functions. Some could exert their effects by converting DHEA to its 7-hydroxylated form while others, like BV2 microglia, by converting DHEA primarily to other C-19 steroids and to estrogen by way of AD.     

Metabolism of neuroactive steroids in day-old chick brain

Metabolism of the neuroactive steroids pregnenolone (PREG), progesterone (PROG), dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEAS) was investigated in day-old chick brain following direct injection of the ³H-labelled compounds into the intermediate medial mesopallium and sampling at times known to be crucial for memory formation in this brain region. ³H-label from these steroids was cleared rapidly from the brain, decreasing to barely detectable levels within 5 h. Following extraction and fractionation, the ³H-labelled brain steroids were identified by TLC, coupled with acetylation and/or separation in different solvent systems. PREG and PROG were converted within 10 min mostly to 20β-dihydropregnenolone (20β-DHPREG) and 5β-dihydroprogesterone, respectively. There was no detectable metabolism of DHEA. Label from DHEAS persisted for longer (half-time 18.9 min) than the free steroid but with no detectable metabolism other than a small amount (4%) of desulphation to DHEA. Further investigation of chick brain steroid metabolism by incubation of subcellular fractions (1–3 h, 37°C) with PREG, PROG or DHEA plus NADPH led to the formation of the following compounds: 20β-DHPREG from PREG (particularly in cytosol); 5β-dihydroprogesterone and 3α,5β-tetrahydroprogesterone from PROG and no detectable metabolism of DHEA. Following incubation of the same brain fractions and labelled steroids with NAD⁺, there was no detectable metabolism of PREG or PROG but some conversion of DHEA to androstenedione, especially in the nuclear fraction. The results suggest direct actions of DHEA(S) on the early stages of memory formation in the chick and introduce the possibility that PREG may act indirectly via 20β-DHPREG.     

Effects of different steroid-biosynthesis inhibitors on the testicular steroidogenesis of the toad Bufo arenarum

Testis fragments from Bufo arenarum were incubated with [7(n)-(3)H]pregnenolone (P5), [1,2-(3)H]dehydroepiandrosterone (DHEA) and [1,2,6.7-(3)H]testosterone (T), and different steroid-biosynthesis inhibitors. The inhibitors used were: cyanoketone (CNK), spironolactone (SPNL) and finasteride (FIN). CNK significantly increased the recovery of 3beta-hydroxy-5-ene steroids while SPNL reduced the metabolism of P5 and the production of C19-steroids. The metabolism of C19-substrates was only modified by CNK, which reduced the transformation of DHEA without modifying the metabolism of T. To determine the degree of inhibition exerted by the inhibitors used, the activities of the enzymes were estimated as the percentage of their contribution to the total steroid metabolism. CNK strongly inhibited the activity of hydroxysteroid dehydrogenase/isomerase if its contribution was estimated using both P5 and DHEA. If the analysis was made considering both activities associated to cytochrome P450 17chi-hydroxylase, C17-20 lyase (P450c17), it became evident that SPNL inhibited both of them. The percent contribution of 17beta-hydroxysteroid dehydrogenase (17betaHSD) activity diminished in the presence of CNK only if it was estimated considering P5 and DHEA metabolism. SPNL produced a significant inhibition of 17betaHSD when its contribution was estimated considering P5 metabolism. However, SPNL was insufficient if DHEA or T were considered. The effect of SPNL on the contribution of 17betaHSD could be due to the reduction of C19-substrates. The activity of 5chi-reductase was inhibited by CNK only if results from P5 and DHEA were considered.     

Dehydroepiandrosterone stimulates the estrogen response element

Studies suggest that the steroid, dehydroepiandrosterone (DHEA) can exert effects directly, in addition to its indirect role serving as a precursor for other steroids such as androgens and estrogens. Because DHEA is one of the most abundant adrenal steroids secreted in man, we investigated the functional activity of DHEA on the classic estrogen response element (ERE) in the presence of the estrogen receptor (ER) in transiently transfected cells. GT1-7 hypothalamic neuronal cells, devoid of the estrogen receptor, were transiently transfected with the estrogen receptor expression plasmid (HEGO) and the estrogen response element luciferase (ERELUC) reporter vector. As expected, a dose-response stimulation of luciferase activity was observed in cells treated with estradiol. Concentrations of estradiol from 10⁻¹⁰–10⁻⁶ M resulted in a 136–195 percent increase in luciferase activity compared with control. A dose-response stimulation was also observed in the cells treated with DHEA. A maximum stimulation of 177 percent increase in luciferase activity compared with control was observed with DHEA at a concentration of 10⁻⁵ M. Both the estradiol and DHEA stimulation of ERE luciferase activity was inhibited by the estrogen receptor antagonist, ICI 182,780. The aromatase inhibitor, formestane in combination with estradiol or DHEA had no effect on luciferase activity, suggesting that the effect of DHEA is independent of its conversion to estadiol. Estradiol levels, as measured by ELISA, were appropriately elevated in the estradiol-treated cells but were not significantly different from the control cells in the DHEA-treated cells. These studies suggest a functional in vitro role of DHEA in activating the ERE in the presence of the classic ER.     

Steroid profiles of healthy individuals

Urine steroid profiles of healthy individuals can be divided into two groups according to greatly different excretion rates of dehydroepiandrosterone (DHEA). About 80% of the population show an excretion of DHEA in urine of just above the detection limit or less of the main androgens androsterone (A) and etiocholanolone (E). This excretion is only enhanced in psychological stress situations. The remaining 20% excrete DHEA in roughly equal amounts as A and E.While the relation of excreted steroids is rather constant, the absolute amounts may vary greatly. In contrast to the behaviour of all other steroids DHEA excretion is not in relation to other steroids. The group of “high DHEA” producing individuals in particular shows drastic changes in the excretion during a day: the DHEA excretion rapidly rises from morning until afternoon and then drops to rather low values in the resting period during the night. A recognizable DHEA production seems to be closely related to the waking period.     

Effects of neuroactive steroids on cochlear hair cell death induced by gentamicin

As neuroactive steroids, sex steroid hormones have non-reproductive effects. We previously reported that 17β-estradiol (βE2) had protective effects against gentamicin (GM) ototoxicity in the cochlea. In the present study, we examined whether the protective action of βE2 on GM ototoxicity is mediated by the estrogen receptor (ER) and whether other estrogens (17α-estradiol (αE2), estrone (E1), and estriol (E3)) and other neuroactive steroids, dehydroepiandrosterone (DHEA) and progesterone (P), have similar protective effects. The basal turn of the organ of Corti was dissected from Sprague-Dawley rats and cultured in a medium containing 100 μM GM for 48 h. The effects of βE2 and ICI 182,780, a selective ER antagonist, were examined. In addition, the effects of other estrogens, DHEA and P were tested using this culture system. Loss of outer hair cells induced by GM exposure was compared among groups. βE2 exhibited a protective effect against GM ototoxicity, but its protective effect was antagonized by ICI 182,780. αE2, E1, and E3 also protected hair cells against gentamicin ototoxicity. DHEA showed a protective effect; however, the addition of ICI 182,780 did not affect hair cell loss. P did not have any effect on GM-induced outer hair cell death. The present findings suggest that estrogens and DHEA are protective agents against GM ototoxicity. The results of the ER antagonist study also suggest that the protective action of βE2 is mediated via ER but that of DHEA is not related to its conversion to estrogen and binding to ER. Further studies on neuroactive steroids may lead to new insights regarding cochlear protection.     

Theoretical calculation of triazolam hydroxylation and endogenous steroid inhibition in the active site of CYP3A4

CYP3A4 has unusual kinetic characteristics because it has a large active site. CYP3A4 produced more 4-hydroxytriazolam than alpha-hydroxytriazolam at concentrations of more than 60 muM triazolam, and different steroids had different inhibitory effects on the system. To clarify these interesting observations, the interactions between substrate and substrate/steroid were investigated by theoretical calculations. When two triazolam molecules were docked into the active site, the distance between the O-atom and the 4-hydroxylated site was less than the distance to the alpha-hydroxylated site because of interaction between the two triazolam molecules. Estradiol inhibited both alpha- and 4-hydroxytriazolam formation by 50%. Dehydroepiandrosterone (DHEA) inhibited alpha-hydroxylation more than 4-hydroxytriazolam formation, whereas aldosterone had no effect. When one triazolam molecule and one steroid molecule were simultaneously docked, estradiol increased the distance between the O-atom and the two hydroxylated sites, DHEA only increased the distance between the O-atom and alpha-hydroxylated site, and aldosterone did not change the distances. The relevant angles of Fe-O-C in the hydroxylated site of triazolam also widened, together with increased distance. These findings indicate that formation of a substrate and substrate/effector complex in the active site may be a factor for determining the enzyme kinetic parameters of CYP3A4.     

Characterization of shuttle mechanisms for the transport of reducing equivalents into mitochondria

The malate-aspartate, fatty acid, and α-glycerophosphate shuttles for the transport of reducing equivalents into mitochondria were reconstituted, using isolated hepatic mitochondria and the extramitochondrial components of the shuttles. Clofibrate and thyroxin increased, while propylthiouracil treatment decreased, the activity of mitochondrial α-glycerophosphate dehydrogenase. Despite these changes, the activity of the reconstituted α-glycerophosphate shuttle was similar in mitochondria from control rats and those from rats treated with clofibrate and propylthiouracil. There was an increase in the activity of the shuttle using mitochondria from thyroxin-treated rats. Rotenone caused 60–90% inhibition of this shuttle, suggesting that rotenone-sensitive NADH dehydrogenase participates in the pathway of oxidation of extramitochondrial hydrogen. Palmitate, oleate, and octanoate were equally effective in reconstituting a cyclic fatty acid shuttle. The shuttle was inhibited by various compounds affecting mitochondrial metabolism, including oligomycin, dinitrophenol, cyanide, rotenone, atractyloside, and α-bromopalmitate. Carnitine and several dicarboxylic and tricarboxylic acids which stimulate fatty acid elongation, augmented fatty acid shuttle activity. The malate-aspartate shuttle was inhibited by cycloserine, amino-oxyacetic acid, and hydrazine, and stimulated by pyridoxal phosphate, at the same concentrations which affected the activities of cytoplasmic and mitochondrial glutamic oxalacetic transaminase. This shuttle was inhibited by uncouplers, antimycin, azide, cyanide, rotenone, amobarbital, oligomycin, and several inhibitors of anion transport including iodobenzylmalonate and avenaciolide. The reconstituted shuttle is sufficiently active to provide about 70–80% of the oxalacetate required for maximal rates of gluconeogenesis. Extrapolations based on the rates of mitochondrial oxidation of acetaldehyde and the activity of the microsomal ethanol oxidizing system suggest that any one of the shuttles could account for the rate of ethanol metabolism in vitro by the alcohol dehydrogenase pathway.     

Oxaloacetate and malate transport by plant mitochondria

The permeability of mitochondria from pea (Pisum sativum L. var Kleine Rheinländerin) leaves, etiolated pea shoots, and potato (Solanum tuberosum) tuber for malate, oxaloacetate, and other dicarboxylates was investigated by measurement of mitochondrial swelling in isoosmolar solutions of the above mentioned metabolites. For the sake of comparison, parallel experiments were also performed with rat liver mitochondria. Unlike the mammalian mitochondria, the plant mitochondria showed only little swelling in ammonium malate plus phosphate media but a dramatic increase of swelling on the addition of valinomycin. Similar results were obtained with oxaloacetate, maleate, fumarate, succinate, and malonate. n-Butylmalonate and phenylsuccinate, impermeant inhibitors of malate transport in mammalian mitochondria, had no marked inhibitory effect on valinomycin-dependent malate and oxaloacetate uptake of the plant mitochondria. The swelling of plant mitochondria in malate plus valinomycin was strongly inhibited by oxaloacetate, at a concentration ratio of oxaloacetate/malate of 10⁻³. From these findings it is concluded: (a) In a malate-oxaloacetate shuttle transferring redox equivalents from the mitochondrial matrix to the cytosol, malate and oxaloacetate are each transported by electrogenic uniport, probably linked to each other for the sake of charge compensation. (b) The transport of malate between the mitochondrial matrix and the cytosol is controlled by the oxaloacetate level in such a way that a redox gradient can be maintained between the NADH/NAD systems in the matrix and the cytosol. (c) The malate-oxaloacetate shuttle functions mainly in the export of malate from the mitochondria, whereas the import of malate as a respiratory substrate may proceed by the classical malate-phosphate antiport.     

Anti-proliferative action of endogenous dehydroepiandrosterone metabolites on human cancer cell lines

Dehydroepiandrosterone (DHEA) is a naturally occurring steroid synthesized in the adrenal cortex, gonads, brain, and gastrointestinal tract, and it is known to have chemopreventive and anti-proliferative actions on tumors. These effects are considered to be induced by the inhibition of glucose-6-phosphate dehydrogenase (G6PD) and/or HMG-CoA reductase (HMGR) activities. The present study was undertaken to investigate whether endogenous DHEA metabolites, i.e. DHEA-sulfate, 7-oxygenated DHEA derivatives, androsterone, epiandrosterone, and etiocholanolone, have anti-proliferative effects on cancer cells and to clarify which enzyme, G6PD or HMGR, is responsible for growth inhibition. Growth of Hep G2, Caco-2, and HT-29 cells, evaluated by 3-[4,5-dimethylthiazol]-2yl-2,5-diphenyl tetrazolium bromide (MTT) and bromodeoxyuridine incorporation assays, was time- and dose-dependently inhibited by addition of all DHEA-related steroids we tested. In particular, the growth inhibition due to etiocholanolone was considerably greater than that caused by DHEA in all cell lines. The suppression of growth of the incubated steroids was not correlated with the inhibition of G6PD (r=-0.031, n=9, NS) or HMGR (r=0.219, n=9, NS) activities. The addition of deoxyribonucleosides or mevalonolactone to the medium did not overcome the inhibition of growth induced by DHEA or etiocholanolone, while growth suppression by DHEA was partially prevented by the addition of ribonucleosides. These results demonstrate that endogenous DHEA metabolites also have an anti-proliferative action that is not induced by inhibiting G6PD or HMGR activity alone. These non-androgenic DHEA metabolites may serve as chemopreventive or anti-proliferative therapies.     

Transformation of 3-hydroxy-steroids by Fusarium moniliforme 7alpha-hydroxylase.

Transformation of physiologically important 3-hydroxy-steroids by the DHEA-induced 7alpha-hydroxylase of F. moniliforme was investigated. Whereas DHEA was almost totally 7alpha-hydroxylated, PREG, EPIA and ESTR were only partially converted into their 7alpha-hydroxylated derivatives because hydroxylation at other undetermined positions as well as reduction of ketone at C17 or C20 into hydroxyl also occurred. Cholesterol was not transformed by the enzyme. Kinetic parameters of the 7alpha-hydroxylation for these substrates were determined and confirmed that DHEA was the best substrate of the 7alpha-hydroxylase. Inhibition studies of DHEA 7alpha-hydroxylation by the other 3-hydroxy-steroids were also carried out and proved that DHEA, PREG, EPIA and ESTR shared the same active site of the enzyme. Induction effects of these steroids were compared, and DHEA appeared to be the best inducer of the 7alpha-hydroxylase of F. moniliforme.