The 7alpha-hydroxysteroids produced in human tonsils enhance the immune response to tetanus toxoid and Bordetella pertussis antigens.

Human tonsils were assessed for their ability to 7alpha-hydroxylate pregnenolone (PREG), dehydroepiandrosterone (DHEA) and 3-epiandrosterone (EPIA). Both 7alpha-hydroxy-DHEA and 7alpha-hydroxy-EPIA were produced by homogenates of either whole tonsils or of lymphocyte-depleted tonsil fractions. In contrast, isolated lymphocytes were found to be unable to carry out 7alpha-hydroxylation. When co-cultures of tonsil-derived T and B lymphocytes were set up under stimulatory conditions, IgGs were released in the supernatants and could be quantitated, and immunomodulating properties of different steroids were monitored. When PREG was added to a mixture of tonsil-derived B and T lymphocytes, a decrease of non-specific and specific IgG was observed. An increase in specific anti-tetanus toxoid and anti-Bordetella pertussis antigen IgGs was obtained with either 1 microM 7alpha-hydroxy-DHEA or 1 microM 7alpha-hydroxy-EPIA. In contrast, DHEA and EPIA were unable to trigger such an effect. When cultures of isolated tonsillar B cells were used, none of the steroids tested showed significant effects on specific IgG productions. These data led to the conclusion that human tonsillar cells transform DHEA and EPIA, but not PREG, into 7alpha-hydroxylated metabolites. These metabolites could act on target tonsillar T lymphocytes which in turn act upon B lymphocytes for increasing specific IgG production.

Effects of cytochrome P450 inhibitors and of steroid hormones on the formation of 7-hydroxylated metabolites of pregnenolone in mouse brain microsomes.

Hydroxylations of pregnenolone (PREG) at the 7 alpha- and 7 beta-positions have been reported in numerous murine tissues and organs and responsible cytochrome P450 (CYP) species await identification. Using thin layer chromatography and gas chromatography-mass spectrometry, we report identification of 7 alpha-hydroxy-PREG and 7 beta-hydroxy-PREG metabolites produced in mouse brain microsome digests and kinetic studies of their production with apparent KM values of 0.5 +/- 0.1 microM and 5.1 +/- 0.6 microM for 7 alpha- and 7 beta-hydroxylation respectively. Investigation of CYP inhibitors and of steroid hormone effects on both 7 alpha- and 7 beta-hydroxylations of PREG showed that: (i) different CYP were involved in 7 alpha- and 7 beta-hydroxylation of PREG because solely 7 alpha-hydroxylation was extensively inhibited by metyrapone, alpha-naphthoflavone, ketoconazole and 3 beta-hydroxysteroids, (ii) CYP 1A2, 2D6, 2B1 and 2B11 were not responsible for 7 alpha- and 7 beta-hydroxylation of PREG because respective specific inhibitors furafylline, quinidine and chloramphenicol triggered no inhibition, (iii) CYP 1A1 was responsible for only part of the 7 beta-hydroxylation of PREG because use of alpha-naphthoflavone, which inhibits specifically CYP 1A1, did not suppress entirely 7 beta-hydroxylation, while ketoconazole, metyrapone and antipyrine, which do not inhibit CYP 1A1, decreased part of the 7 beta-hydroxylation, (iv) 7 alpha-hydroxylation of PREG may be shared with other 3 beta-hydroxysteroids such as isoandrosterone and 5-androstene-3 beta,17 beta-diol which were strong inhibitors, but not with dehydroepiandrosterone which was a non-competitive inhibitor as weak as 3-oxosteroids, and (v) 7 beta-hydroxylation of PREG was not markedly changed by other steroids. Taken together, these findings will be of use for identification of the CYP species responsible for 7 alpha- and 7 beta-hydroxylation of PREG and for studies of their activities in brain.

Dehydroepiandrosterone stimulates proliferation and gene expression in MCF-7 cells after conversion to estradiol.

Dehydroepiandrosterone (DHEA) is a mitogen for estrogen-dependent MCF-7 breast cancer cells. Our aims were to determine whether DHEA required conversion to estrogens in order to stimulate cell proliferation and estrogen-dependent gene expression. After incubation of cells with 100 nM DHEA for 4 days, estradiol was present in the medium at a concentration of approximately 200 pM. Other compounds identified were testosterone ( approximately 300 pM) and estrone. Significant stimulation of cell proliferation by 1 nM estradiol and 100 nM DHEA was observed after 38 h and 4 days of incubation, respectively, indicating the necessity of DHEA conversion. DHEA doses > or = 10 nM induced estrogen-dependent reporter gene expression in MCF-7 cells transfected with a luciferase reporter gene under the control of the estrogen response element. DHEA-dependent stimulation of proliferation and luciferase induction could be inhibited by the anti-estrogens ICI182,780 and tamoxifen, respectively, and by the aromatase inhibitor 4-hydroxyandrostenedione. An androgenic effect of DHEA on proliferation and gene expression of MCF-7 cells was not observed. We conclude that conversion of DHEA to estrogens, particularly estradiol, is required to exert a mitogenic response.

Dehydroepiandrosterone 7alpha- and 7beta-hydroxylation in mouse brain microsomes. Effects of cytochrome P450 inhibitors and structure-specific inhibition by steroid hormones.

Presently, several works question the effects of dehydroepiandrosterone (DHEA) reported in vivo and designate its 7-hydroxylated metabolites as native antiglucocorticoids and potent mediators in the triggering of immune response. Among mouse tissues and organs, and second to liver, the largest production of 7alpha-and 7beta-hydroxylated derivatives of DHEA takes place in brain microsomes. To contribute to identification of cytochromes P450 (CYPs) responsible for 7alpha- and 7beta-hydroxy-DHEA production, effects of CYP inhibitors and of several steroid hormones on DHEA 7-hydroxylation were examined. Using mouse brain microsomes as a source of enzyme, we report now that strong and smaller inhibitions of DHEA 7alpha-hydroxylation were obtained with ketoconazole and alpha-naphthoflavone, respectively, and that neither changed DHEA 7beta-hydroxylation. Metyrapone and antipyrine also inhibited 7alpha-hydroxylation, but by contrast, significantly increased 7beta-hydroxylation of DHEA. This indicated that at least, two different CYPs were responsible for 7alpha- and 7beta-hydroxylation of DHEA. Steroids sharing a 3beta-hydroxylated structure with DHEA, namely pregnenolone, 5-androstene-3beta,17beta-diol and 3beta-hydroxy-5alpha-androstan-17-one, were strong inhibitors of DHEA 7alpha-hydroxylation (non-competitive inhibition with pregnenolone, Ki=2.0 +/- 0.3 microM). In contrast, 7beta-hydroxylation yields were not decreased by the 3beta-hydroxysteroids tested. Moderate inhibition of 7alpha- and 7beta-hydroxylation was obtained with 3-oxosteroids, namely testosterone, progesterone, corticosterone and 4-androsten-3,17-dione. Taken together, these data indicate specific inhibition patterns of DHEA 7alpha- and 7beta-hydroxylation by CYP inhibitors and steroid hormones in mouse brain microsomes and may be used as criteria necessary for identification of the responsible CYP species.

Pregnenolone and dehydroepiandrosterone as precursors of native 7-hydroxylated metabolites which increase the immune response in mice.

Dehydroepiandrosterone (DHEA) and pregnenolone (PREG) were both metabolized by homogenates of brain, spleen, thymus, perianal skin, ventral skin, intestine, colon, coecum and muscle tissues from mice. The use of 2H-labeled substrates and of the twin ion technique of gas chromatography-mass spectrometry permitted identification of 7 alpha-hydroxy-DHEA and of 5-androstene-3 beta, 17 beta-diol as DHEA metabolites in digests of all tissues. The extent of PREG metabolism was much lower than for DHEA with all tissues but amounts of the main transformation product were sufficient in brain, spleen and ventral skin digests for identification with 7 alpha-hydroxy-PREG. Dimethylsulfoxide (DMSO) solutions of DHEA, PREG and of their 7-hydroxylated metabolites were injected at different doses and time intervals prior to proximal subcutaneous administration of a lysozyme antigen. Quantities of anti-lysozyme IgG were measured in the serum of treated mice and compared with that from sham-treated animals. Increase of anti-lysozyme IgG was obtained with DHEA and PREG (1 g/kg) when injected 2 h prior to lysozyme. Much lower doses (160 times less) of 7 alpha-hydroxy-DHEA and -PREG were also found to be significantly active when administered at the moment of lysozyme injection. A larger dose of 7 beta-hydroxy-DHEA (50 mg/kg) was necessary for a similar effect. These results suggest that in tissues where immune response takes place, the locally-produced 7-hydroxy metabolites of PREG and DHEA are involved in a process which may participate in the physiological regulation of the body's immune response.

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.

The inducible and cytochrome P450-containing dehydroepiandrosterone 7alpha-hydroxylating enzyme system of Fusarium moniliforme.

7Alpha-hydroxylation of DHEA by Fusarium moniliforme was investigated with regard to inducibility and characterization of the responsible enzyme system. Using GC/MS, the 7-hydroxylated metabolites of DHEA produced after biotransformation by Fusarium moniliforme mycelia were identified. The strain of Fusarium moniliforme hydroxylated DHEA predominantly at the 7alpha-position, with minor hydroxylation occurring at the 7beta-position. Constitutive 7alpha-hydroxylation activity was low, but DHEA induced the enzyme complex responsible for 7alpha-hydroxylation via an increase in protein synthesis. DHEA 7alpha-hydroxylase was found to be mainly microsomal, and the best production yields of 7alpha-hydroxy-DHEA (28.5 +/- 3.51 pmol/min/mg protein) were obtained with microsomes prepared from 18-h-induced mycelia. Kinetic parameters (KM=1.18 +/- 0.035 microM and Vmax=909 +/- 27 pmol/min/mg protein) were determined. Carbon monoxide inhibited 7alpha-hydroxylation of DHEA by microsomes of Fusarium moniliforme. Also, exposure of mycelia to DHEA increased microsomal P450 content. These results demonstrated that: (i) DHEA is 7alpha-hydroxylated by microsomes of Fusarium moniliforme; (ii) DHEA induces Fusarium moniliforme 7alpha-hydroxylase; (iii) this enzyme complex contains a cytochrome P450.

Pregnenolone-7beta-hydroxylating activity of human cytochrome P450-1A1.

In many human and murine tissues, both pregnenolone and dehydroepiandrosterone are hydroxylated at the 7alpha and 7beta positions by a cytochrome P450-containing microsomal complex. The 7alpha- and 7beta-hydroxysteroids produced were shown to activate an immune response in mice. Based upon identification by crystallization to constant specific activity and gas chromatography-mass spectrometry analysis, we ascertained that a yeast-expressed human cytochrome P450-1A1 was able to 7beta-hydroxylate pregnenolone (K(M) from 3.2 +/- 0.5 to 4.1 +/- 0.4 microM, turnover number from 117 +/- 15 to 135 +/- 13 pmol/min/nmol of cytochrome P450-1A1). The other human cytochromes P450 tested did not produce identifiable quantities of 7alpha- or 7beta-hydroxylated derivatives of pregnenolone or dehydroepiandrosterone. These findings indicate that cytochrome P450-1A1 involvement in the 7beta-hydroxylation of pregnenolone may contribute to the production of the 7-hydroxylated steroids necessary for activation of the immune defences.

7 alpha-hydroxy-dehydroepiandrosterone and immune response.

In human and murine lymphoid organs, circulating 3 beta-hydroxysteroids, including pregnenolone (PREG), dehydroepiandrosterone (DHEA), and epiandrosterone (EPIA), are 7 alpha-hydroxylated by a cytochrome P450 identified in the hippocampus as P4507B1. Mouse and human lymphoid organs produced different patterns of 3 beta-hydroxysteroid 7 alpha-hydroxylation with the absence of pregnenolone and epiandrosterone hydroxylation in human and mouse, respectively. Both 7 alpha-hydroxy-DHEA and 7 alpha-hydroxy-EPIA triggered a significant increase of antitetanus toxoid and anti-Bordetella pertussis toxins IgGs production in cultures of activated B + T cells derived from human tonsils, whereas both 7 alpha-hydroxy-PREG and 7 alpha-hydroxy-DHEA increased the immune response in mouse. Paracrine action of 7 alpha-hydroxysteroids resulted from their production in cells of the lymphoid organs. Comparison of P4507B1 sequences in rat, human, and two mouse species showed that one amino acid change might explain important differences in KM for 7 alpha-hydroxylation, and suggested that such differences might contribute to the extent of immune response.

Increased total 7 alpha-hydroxy-dehydroepiandrosterone in serum of patients with Alzheimer's disease.

Evidence has indicated that circulating adrenal steroid quantitites were significantly changed in patients with Alzheimer's disease (AD). Aside of 3 beta-sulfatation and 3 beta-acylations, levels of dehydroepiandrosterone (DHEA) result from production and metabolic transformation yields. 7 alpha-Hydroxylation of DHEA has been described in humans, and 7 alpha-hydroxy-DHEA may be responsible for the known antiglucocorticoid effects of DHEA. Using a negative ion fragmentometry method with gas chromatography/mass spectrometry on trifluoroacetate derivatives, we measured levels of free 7 alpha-hydroxy-DHEA as well as its sulfated conjugate and its fatty acid esters in serum of 10 female patients with AD and of 8 age-matched healthy control women. Free 7 alpha-hydroxy-DHEA levels in AD and controls were not significantly different (240.2 +/- 37.2 pg/ml and 206.8 +/- 21.6 pg/ml, respectively), but sulfate conjugate levels were significantly increased in AD (p = .01) (262 +/- 28.4 and 145.4 +/- 27.6, respectively) as well as fatty acid esters (p = .041) (65.7 +/- 6.9 and 40.7 +/- 9.2, respectively). These results indicated that the total 7 alpha-hydroxy-DHEA produced was significantly increased in AD (p = .024) and may contribute to the disease-related disturbances of DHEA production and metabolism.

Change of 7alpha-hydroxy-dehydroepiandrosterone levels in serum of mice treated by cytochrome P450-modifying agents.

Dehydroepiandrosterone (DHEA) is 7alpha-hydroxylated in liver, brain and other organs of murine and in other species. Several works suggest that the 7alpha-hydroxy-DHEA produced may be one of the native antiglucocorticoids, and compounds modifying its production may prove useful in investigation of 7alpha-hydroxy-DHEA production and effects. After treatment of mice with dexamethasone, phenobarbital, trilostane, melatonin or metyrapone, we have used gas chromatography-mass spectrometry with negative ion detection for measurement of 7alpha-hydroxy-DHEA levels in serum of control and treated animals. The 7alpha-hydroxylating rates of liver and brain microsomes from the same animals were also measured. Results showed that serum levels of 7alpha-hydroxy-DHEA were significantly increased after treatment by all compounds except metyrapone. Significantly increased 7alpha-hydroxy-DHEA levels were directly related with significantly increased 7alpha-hydroxylation yields in liver and not in brain. In contrast, metyrapone decreased 7alpha-hydroxylation in liver and brain. These findings indicate that in brain and in liver, different enzyme systems may be responsible for production of 7alpha-hydroxy-DHEA and that treatment-induced modifications of circulating 7alpha-hydroxy-DHEA levels are mainly due to change of 7alpha-hydroxylating rates in liver.

7-Hydroxydehydroepiandrosterone epimers in human serum and saliva. Comparison of gas chromatography-mass spectrometry and radioimmunoassay.

Recent reports demonstrated that 7-hydroxylated metabolites of dehydroepiandrosterone (DHEA) possess immunomodulatory and antiglucocorticoid properties. Increased 7alpha-OH-DHEA levels were found in patients with Alzheimer's disease. Hence, measurement of steroids in patients with autoimmune diseases or disturbances in the central nervous system could be of interest. A new sensitive GC-MS method for the determination of 7-hydroxydehydroepiandrosterone epimers was developed and compared with previously developed radioimmunoassays. Besides serum, these steroids were, for the first time, measured in saliva where their concentrations were about five times lower. 7alpha- and 7beta-epimer levels correlated well in both body fluids and they were larger in male.

Studies of the enzyme complex responsible for pregnenolone and dehydroepiandrosterone 7 alpha-hydroxylation in mouse tissues.

7 alpha-Hydroxylation of pregnenolone (PREG) and dehydroepiandrosterone (DHEA) is known to take place in numerous tissues of mouse and rat. The responsible cytochrome P450 species has not yet been identified. Interest in the production of 7 alpha-hydroxylated steroid derivatives results from their ability to increase the immune response in mice. Using crystallizations to constant specific activity and gas chromatography-mass spectrometry, 7 alpha-hydroxy-PREG and 7 alpha-hydroxy-DHEA metabolites produced by microsomes of liver, brain, thymus, and spleen were identified. Study of the 7 alpha-hydroxylating enzyme in these tissues indicated that microsomes contained most of the activity, except for brain, where it was primarily mitochondrial. Production yields of 7 alpha-hydroxy-PREG and 7 alpha-hydroxy-DHEA by microsomes from heart, spleen, thymus, brain, and liver of 7-week-old mice were higher than those of 1-week-old and (except for liver) 41-week-old animals. At the optimal pH (7.4) and in all tested tissues but liver, microsomal 7 alpha-hydroxylation was more extensive for PREG than for DHEA. With brain and thymus microsomes, KM were lower for PREG than for DHEA and decreased when phosphate was used instead of Tris buffer. With brain microsomes, the use of 1 mM EDTA increased 7 alpha-hydroxylating activity. Complete inhibition was obtained with 0.1 mM Zn2+ or Cu2+ and with 1 mM Fe2+ or Fe3+. 7 alpha-Hydroxylation of PREG was activated only by 0.5 mM Ca2+ and that of DHEA only by 0.25 mM Mg2+. Since the production rates of 7 alpha-hydroxy-PREG and 7 alpha-hydroxy-DHEA in tissues may be a key to the triggering of immune defenses, and since both 7 alpha-hydroxylation and immunity decrease with aging, these data will prove to be useful in studies of the enzyme responsible and of the mechanisms that control its activity.

Hydroxylation of pregnenolone at the 7 alpha- and 7 beta- positions by mouse liver microsomes. Effects of cytochrome p450 inhibitors and structure-specific inhibition by steroid hormones.

Hydroxylations of pregnenolone (PREG) at the 7 alpha-and 7 beta-positions have been reported in numerous murine tissues and organs, including liver, and the responsible cytochrome P450 (P450) species await identification. Using thin-layer chromatography and gas chromatography-mass spectrometry and crystallization to constant specific activity, we report identification of 7 alpha-hydroxy-PREG and 7 beta-hydroxy-PREG metabolites produced in mouse liver microsomes and kinetic studies of their production with apparent KM values of 2.45 +/- 0.124 microM and 3.41 +/- 0.236 microM for 7 alpha- and 7 beta-hydroxylation, respectively. Investigation of P450 inhibitors and of steroid hormone effects on both 7 alpha- and 7 beta-hydroxylation of PREG showed that 1) different P450 were involved because metyrapone and antipyrine inhibited solely 7 alpha-and 7 beta-hydroxylation, respectively; 2) P450 1A2, 2D6, 2B1, and 2B11 were not responsible for 7 alpha and 7 beta-hydroxylation of PREG because respective specific inhibitors furafylline, quinidine, and chloramphenicol triggered no inhibition; 3) P450 1A1 was responsible for only part of the 7 beta-hydroxylation of PREG because alpha-naphthoflavone, which inhibits specifically P450 1A1, did not suppress entirely 7 beta-hydroxylation while ketoconazole, antipyrine, and metyrapone extensively decreased the 7 beta-hydroxylation; 4) comparison of these findings with those obtained with brain microsomes suggests that tissue-specific P450 species are responsible for the 7 alpha-and 7 beta-hydroxylation of PREG; and 5) 7 alpha-hydroxylation of PREG may be shared with other 3 beta-hydroxysteroids such as isoandrosterone, 5-androstene-3 beta, 17 beta-diol, and dehydroepiandrosterone, which acted in a competitive manner. Taken together, these findings will be of use for identification of the P450 species responsible for 7 alpha- and 7 beta-hydroxylation of PREG and for studies of their activities in liver and other organs.

Inhibition studies of dehydroepiandrosterone 7 alpha- and 7 beta- hydroxylation in mouse liver microsomes.

Hydroxylations of dehydroepiandrosterone (DHEA) at the 7 alpha- and 7 beta- positions have been reported in numerous murine tissues and organs, including liver, and the responsible cytochrome P450 (P450) species await identification. Using thin layer chromatography and gas chromatography-mass spectrometry, we report identification of 7 alpha-hydroxy-DHEA and 7 beta-hydroxy-DHEA metabolites produced in mouse liver microsome digests and kinetic studies of their production with apparent KM values of 3.19 +/- 0.292 microM and 2.82 +/- 0.241 microM for 7 alpha- and 7 beta-hydroxylation, respectively. Investigation of P450 inhibitor and of steroid hormone effects on both 7 alpha- and 7 beta-hydroxylation of DHEA showed that, 1) different P450s were involved in 7 alpha- and 7 beta-hydroxylation of DHEA because metyrapone inhibited solely 7 alpha-hydroxylation, 2) P450 2D6, 2B1, and 2B11 were not responsible for 7 alpha- and 7 beta-hydroxylation of DHEA because respective specific inhibitors quinidine and chloramphenicol triggered no inhibition, 3) aside from P450 7b, P450 1A1, and 1A2 may be responsible for a fraction of DHEA 7 alpha- and 7 beta-hydroxylation because alpha-naphthoflavone and furafylline, which inhibit specifically P450 1A1 and 1A2, decreased the 7 alpha- and 7 beta-hydroxylation partly, 4) comparison of these findings with those obtained with brain microsomes suggested that tissue-specific P450 species are responsible for the 7 alpha- and 7 beta-hydroxylation of DHEA, 5) 7 alpha-hydroxylation of DHEA may be shared with other 3 beta-hydroxysteroids, such as 3 beta-hydroxy-5 alpha-androstan-17-one, 5-androstene-3 beta,17 beta-diol and pregnenolone, which acted in a noncompetitive manner. Taken together, these findings will be of use for identification of the P450 species responsible for 7 alpha- and 7 beta-hydroxylation of DHEA and for studies of their activities in liver.

Pituitary metabolism of 5alpha-androstane-3beta-17beta-diol: intense and rapid conversion into 5alpha-androstane-3beta,6alpha,17beta-triol and 5alpha-androstane-3beta,7alpha, 17beta-triol.

In the male rat pituitary, 5alpha-androstane-3beta, 17beta-diol (3beta-diol) is extensively metabolized into polar steroids. They were identified as 5alpha-androstane-3beta, 6alpha-17beta-triol (6alpha-triol) and 5alpha-androstane-3beta, 7alpha, 17beta-triol (7alpha-triol). 6-alpha-Triol represents 53% and 7alpha-Triol 28% of the total 3beta-diol metabolites. The remaining percentage is related to 6beta and 7beta isomers. The biological role of triols is still unknown.

Testosterone metabolism in the uropygial gland of the quail.

The metabolism of testosterone in the uropygial gland of the quail principally results in the production of 17 alpha, 5 beta derivatives. Moreover, an unusually small amount of testosterone is converted to 5 alpha-dihydrotestosterone. These results question the role played by intracellular 5 alpha-reduction in the response of the gland to testosterone stimulation.