Postmenopausal dehydroepiandrosterone administration increases free insulin-like growth factor-I and decreases high-density lipoprotein: a six-month trial.

OBJECTIVE: To determine the effect of administering 6 months of oral postmenopausal DHEA therapy on serum DHEA, DHEAS, and T levels and on physiologic endpoints including lipoproteins and insulin-like growth factor-I (IGF-I). DESIGN: Randomized, double-blind, parallel trial. SETTING: Academic referral practice. PATIENT(S): Thirteen normal-weight or overweight, healthy, nonsmoking, postmenopausal women. INTERVENTION(S): Administration of oral micronized DHEA (25 mg/d). MAIN OUTCOME MEASURE(S): Monthly fasting 23 hours postdose levels of serum DHEA, DHEAS, T, lipoproteins, IGF-I, IGF binding protein-3 (IGFBP-3), and liver function tests. Morphometric indices by dual-energy x-ray absorptiometry scan (percent body fat; lean body mass), immune indices, and insulin sensitivity. RESULT(S): Levels of DHEA, DHEAS, and T all rose into premenopausal ranges, but after 6 months, levels of DHEA and T did not differ from baseline or placebo. At 3 months, the ratio of IGF-I to IGFBP-3 rose by 36.1% +/- 12.7%, but it fell to placebo values by 6 months. High-density lipoprotein and apolipoprotein A1 levels declined. CONCLUSION(S): Patients appeared to tolerate 6 months of DHEA therapy well. Given the small study size, no statistically significant differences in morphometric indices, immune indices, or insulin-sensitizing properties were observed, but significant attenuation of bioavailability occurred. Supplementation with DHEA increased IGF-I/IGFBP-3 levels at 3 months and decreased high-density lipoprotein and apolipoprotein A1 levels at 6 months.

Biotin-labeled hairpin oligonucleotides: probes to detect double-strand breaks in DNA in apoptotic cells.

Hairpin oligonucleotides were synthesized with stems ending in a double-stranded structure, which can be ligated to double-strand breaks in DNA, and with loops that contain nucleotides modified by the attachment of biotin. These probes specifically and sensitively detect double-strand breaks in apoptotic cells. Localization of these probes is restricted to areas of chromatin characteristic of apoptosis, whereas much more diffuse labeling was obtained when all available 3' DNA ends were labeled by terminal transferase. In principle, hairpin oligonucleotide probes can be designed with any type of 3' or 5' overhang complementary to double-strand DNA termini being detected.

A quantitative luminescence assay for nonradioactive nucleic acid probes.

In histochemical work with digoxigenin- or biotin-labeled nucleic acid probes, reproducibility of in situ hybridization depends on accurate measurement of the amount of non-radioactive label being used. We describe a rapid and sensitive assay for nonradioactive label incorporated into nucleic acids employing a luminogenic substrate for alkaline phosphatase, CSPD (disodium 3-(4-methoxyspirol¿1,2-dioxetane-3,2'-(5'-chloro)tricyclo [3.3.1.1(3,7)]decan¿-4-yl)phenyl phosphate). An alkaline phosphatase-antibody conjugate was bound to digoxigenin-labeled nucleic acids spotted on nylon membranes. Light emission from the reaction of the bound alkaline phosphatase with CSPD was measured with a luminometer. This method allows an accurate determination of digoxigenin incorporated into nucleic acid probes in the range of 0.5-500 fmol of nonradioactive label.

Dual regulation of 3 beta-hydroxysteroid dehydrogenase, 17 alpha-hydroxylase, and dehydroepiandrosterone sulfotransferase by adenosine 3',5'-monophosphate and activators of protein kinase C in cultured human adrenocortical cells.

The relationship between cAMP and protein kinase C in the regulation of 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), 17 alpha-hydroxylase, and sulfotransferase was examined in human fetal adrenocortical cells under defined serum-free conditions in culture. Forskolin induced 3 beta HSD and 17 alpha-hydroxylase in a dose-dependent manner, with maximal effects at 10 microM. 12-O-Tetradecanoyl phorbol 13-acetate (TPA) at 1 nM depressed the induction of 17 alpha-hydroxylase activity by forskolin by more than 95% and increased the stimulation of 3 beta HSD activity by forskolin by 4- to 5-fold. Increases were maximal at 48-72 h of incubation. Dehydroepiandrosterone sulfotransferase activity increased over 48 h when cells were transferred to serum-free defined medium. Addition of 10 microM forskolin stimulated sulfotransferase activity only when cells remained in 10% serum. TPA at 1 nM inhibited the increase in sulfotransferase activity. The concentration of TPA required for inhibition of forskolin-stimulated 17 alpha-hydroxylase and sulfotransferase activity was similar to that required for enhancement of forskolin-induced 3 beta HSD activity, suggesting that comparable levels of C kinase activation are involved in these events. Angiotensin II, carbachol, epidermal growth factor, and fibroblast growth factor had actions similar to those of TPA on one or more of these enzyme activities. TPA also had similar actions on enzyme activities when they were stimulated by cAMP analogs rather than by forskolin. These studies suggest that adrenal steroid biosynthesis is under dual regulation by cAMP and protein kinase C. cAMP induces enzymes required for synthesis of 17 alpha-hydroxylated steroids, including the adrenal androgens. Activation of protein kinase C may play a complementary role by enhancing the induction of enzymes required for non-17 alpha-hydroxylated steroid biosynthesis and inhibiting those involved in the synthesis of androgens.

Oxidative damage to DNA and replicative lifespan in cultured adrenocortical cells.

Oxidative damage to DNA in cultured bovine adrenocortical cells was investigated by exposing cells to a sublethal concentration (10 microM) of cumene hydroperoxide under conditions previously shown to be deficient in the biological antioxidants selenium and alpha-tocopherol (vitamin E). DNA prepared from cells incubated for 4 h with 10 microM cumene hydroperoxide had a greater fraction showing resistance to S1 nuclease after denaturation and reassociation to a log C0t of -3. Cross-linking by cumene hydroperoxide was abolished in cells that had been grown in the presence of 20 nM selenite or 1 microM alpha-tocopherol for 96 h prior to peroxide addition, whereas such cells remained susceptible to cross-linking by nitrogen mustard. Extensive strand breaks in DNA from peroxide-treated cells as assessed by alkaline sucrose gradient centrifugation were greatly reduced in cells grown in selenite or alpha-tocopherol. Despite the evidence of damage to DNA, cumene hydroperoxide was not detectably mutagenic, in contrast to 5 microM methylnitronitrosoguanidine (MNNG), when assessed as the incidence of resistance to 25 microM ouabain. We confirmed that cumene hydroperoxide at greater than 10 microM lowers cloning efficiency and that this is largely prevented by selenite or alpha-tocopherol. Additionally, selenite or alpha-tocopherol produced increased clonogenicity in cells not incubated with peroxide. To examine effects of the biological antioxidants on replicative lifespan, cells were grown continuously in fetal bovine serum (FBS), fibroblast growth factor (FGF), and selenite or alpha-tocopherol. Selenium increased replicative lifespan by 10-20% and alpha-tocopherol by 22-30%. Levels of DNA cross-links and strand breaks did not differ under any circumstances between early (second) passage and late (30th) passage cells. The experiments on replicative potential were all performed in the presence of FGF. When FGF was omitted from the culture medium, replicative lifespan was reduced by 85%. We conclude that types of damage to DNA resulting from peroxide exposure are not present in cells under standard culture conditions at early or late stages of the lifespan. Other work has noted a relationship between clonogenicity and replicative lifespan; thus, the increase in cloning efficiency seen with selenium and alpha-tocopherol may cause the observed slight increase in replicative lifespan. Oxidative damage does not appear to be a major determinant of cellular senescence in adrenocortical cells.

The role of ascorbic acid in the function of the adrenal cortex: studies in adrenocortical cells in culture.

To investigate the role of ascorbic acid in the function of the adrenal cortex, we studied the effects of ascorbate on the regulation of 11 beta-hydroxylase in culture. When primary bovine adrenocortical cells were cultured in a serum-free defined medium in the absence of ACTH, 11 beta-hydroxylase activity declined with a half-time of about 40 h. When 50 microM cortisol, which acts as a pseudosubstrate for 11 beta-hydroxylase, was added to such cultures, 11 beta-hydroxylase activity declined with a half-time of about 6 h. Ascorbate (5 mM) markedly reduced the rate of loss of 11 beta-hydroxylase activity in the presence of cortisol. Previous studies showed that phenolic and sulfoxide antioxidants, which also prevent loss of 11 beta-hydroxylase activity, inhibited the enzyme at concentrations somewhat higher than those required for protective activity. However, ascorbate at concentrations from 10 microM to 5 mM did not inhibit 11 beta-hydroxylase. The same range of ascorbate concentrations added to cells during a 24-h preincubation with cortisol showed increasing prevention of loss of 11 beta-hydroxylase activity. Ascorbate and a lowered concentration of oxygen were synergistic in their protective action. At 2% oxygen, 5 mM ascorbate almost completely prevented loss of 11 beta-hydroxylase activity in the presence of 50 microM cortisol. 11 beta-Hydroxylase activity was reinduced over a period of 5 days in third passage cultures by addition of 1 microM ACTH in defined lipoprotein-free medium. Addition of ascorbate enhanced the reinduction about 2-fold. The action of ascorbate in prevention of pseudosubstrate-mediated loss of activity and in enhancing reinduction of 11 beta-hydroxylase is specific; neither alpha-tocopherol nor selenium prevented loss of 11 beta-hydroxylase in the presence of cortisol or enhanced reinduction of 11 beta-hydroxylase in the presence of ACTH. As an additional test of specificity, it was shown that reinduction of 17-hydroxylase activity was completely unaffected by ascorbate, selenium, or alpha-tocopherol, and addition of cortisol to cultures with high 17-hydroxylase did not result in any loss of enzyme activity. Thus, a major function of ascorbate in the adrenal cortex is as a protective compound for cytochrome.

Selenium deficiency in cultured adrenocortical cells: restoration of glutathione peroxidase and resistance to hydroperoxides on addition of selenium.

Cultured bovine adrenocortical cells were previously shown to be functionally deficient in selenium and vitamin E when grown in medium supplemented with fetal bovine serum. In the present experiments, the lack of significant bioavailable amounts of selenium in the medium was demonstrated by the finding of only low levels of glutathione peroxidase in the cultured cells (0.008 U/mg protein compared with 0.045 U/mg protein in fresh adrenocortical tissue). When 20 nM selenium as selenite was added to the cultured adrenocortical cells, glutathione peroxidase activity increased continuously over 72 h, with a total increase of about eightfold over this period. Over the same time-course, the highest concentration of cumene hydroperoxide tolerated by the cells without cell death increased progressively from 10 microM to 50 microM. Addition of 1 microM alpha-tocopherol also increased the amount of cumene hydroperoxide tolerated to 50 microM. Cell death was measured by cloning efficiency after removal of cumene hydroperoxide. Addition of either selenium or alpha-tocopherol had little effect on the growth rate of the cells over six passages, even when residual vitamin E was removed from the serum by extraction with ether and residual low molecular weight selenium compounds were removed by dialysis. It is concluded that combined deficiency of selenium and vitamin E, at least in the presence of other components of fetal bovine serum, has little effect on the ability of the cells to survive under normal conditions, as evidenced by continued long-term proliferation. However, the low levels of glutathione peroxidase resulting from selenium deficiency cause an increase susceptibility to peroxide-mediated toxicity. The combined deficiency of selenium and vitamin E impairs the ability of cells to survive under adverse conditions, as well as altering mitochondrial functions, as previously demonstrated.