Robert Morfin

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 bodys immune response.

Neurosteroid 7-hydroxylation products in the brain

The neurosteroids pregnenolone (PREG) and dehydroepiandrosterone (DHEA) are precursors for both oxidized and hydroxylated metabolites in the brain. Thus, brain production of 7-hydroxylated derivatives is second to that in the liver, and P4507B1-containing hippocampus is the major site for 7 alpha-hydroxylation. Other P450s and/or oxido-reductive mechanisms may be responsible for 7 beta-hydroxylation. In addition to regulating neurosteroid brain levels, when produced, the 7-hydroxylated derivatives of PREG and DHEA were investigated for antiglucocorticoid-mediated neuroprotective potencies, and both 7 alpha- and 7 beta-hydroxy-DHEA were efficient in preventing the nuclear uptake of [3H]dexamethasone-activated glucocorticoid receptor in brain cells. Activation of 7 alpha-hydroxylation by increased close contacts of astrocytes and after glucocorticoid treatment suggested that the regulated production of 7 alpha-hydroxysteroids was a key event for the neuroprotection conferred by neurosteroids.

Studies of the enzyme complex responsible for pregnenolone and dehydroepiandrosterone 7α-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.

Dehydroepiandrosterone 7α-hydroxylation in human tissues: Possible interference with type 1 11β-hydroxysteroid dehydrogenase-mediated processes ☆

Dehydroepiandrosterone (DHEA) is 7alpha-hydroxylated by the cytochome P450 7B1 (CYP7B1) in the human brain and liver. This produces 7alpha-hydroxy-DHEA that is a substrate for 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) which exists in the same tissues and carries out the inter-conversion of 7alpha- and 7beta-hydroxy-DHEA through a 7-oxo-intermediary. Since the role of 11beta-HSD1 is to transform the inactive cortisone into active cortisol, its competitive inhibition by 7alpha-hydroxy-DHEA may support the paradigm of native anti-glucocorticoid arising from DHEA. Therefore, our objective was to use human tissues to assess the presences of both CYP7B1 and 11beta-HSD1. Human skin was selected then and used to test its ability to produce 7alpha-hydroxy-DHEA, and to test the interference of 7alpha- and 7beta-hydroxy-DHEA and 7-oxo-DHEA with the 11beta-HSD1-mediated oxidoreduction of cortisol and cortisone. Immuno-histochemical studies showed the presence of both CYP7B1 and 11beta-HSD1 in the liver, skin and tonsils. DHEA was readily 7alpha-hydroxylated when incubated using skin slices. A S9 fraction of dermal homogenates containing the 11beta-HSD1 carried out the oxidoreduction of cortisol and cortisone. Inhibition of the cortisol oxidation by 7alpha-hydroxy-DHEA and 7beta-hydroxy-DHEA was competitive with a Ki at 1.85+/-0.495 and 0.255+/-0.005 microM, respectively. Inhibition of cortisone reduction by 7-oxo-DHEA was of a mixed type with a Ki at 1.13+/-0.15 microM. These findings may support the previously proposed native anti-glucocorticoid paradigm and suggest that the 7alpha-hydroxy-DHEA production is a key for the fine tuning of glucocorticoid levels in tissues.

The 7α-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.

Involvement of steroids and cytochromes P450 species in the triggering of immune defenses

In vertebrates the wide variety of cytochromes P(450) (P(450)) is a key for elimination of low molecular weight xenobiotics and for the production and metabolism of steroid hormones. In contrast, xenobiotics of large molecular weight are processed and eliminated after the immune response. The suppression of immune response by native P(450)-produced glucocorticoid (GC) hormones constitutes a first link between P(450) and immunity. In the last decade, mechanisms and molecules responsible for the triggering of immune response were investigated and results showed that many tissues and organs transform native 3beta-hydroxysteroids into 7-hydroxylated metabolites that trigger immunity. Present data suggest that 7-hydroxysteroids are native anti-GCs that block the GC-induced immunosuppression. Because specific P(450) are responsible for the production of 7-hydroxylated steroids resulting into increased immunity, a second link exists between P(450) and immunity. Taken together, these findings support the proposal that P(450) are keys to all of the known defense mechanisms of vertebrates against all xenobiotic forms.

The native anti-glucocorticoid paradigm

Circulating 3beta-hydroxysteroids including dehydroepiandrosterone (DHEA) are 7alpha-hydroxylated by the cytochrome P450-7B1 in the liver, skin and brain, which are the target organs of glucocorticoids. Anti-glucocorticoid effects with 7alpha-hydroxy-DHEA were observed in vivo without an interference with glucocorticoid binding to its receptor. In the organs mentioned above, the circulating inactive cortisone was reduced into active cortisol by the 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). We demonstrated that 7alpha-hydroxy-DHEA was also a substrate for this enzyme. Studies of the 11beta-HSD1 action on 7alpha-hydroxy-DHEA showed the reversible production of 7beta-hydroxy-DHEA through an intermediary 7-oxo-DHEA, and the kinetic parameters favored this production over that of active glucocorticoids. 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 efficient at their production site. This opens a promising new area for research.

Precursors for 6β- and 7α-hydroxylations of 5α-androstane-3β, 17β-diol by human normal and hyperplastic prostates

Summary Minces and homogenates from human normal and hyperplastic prostates were incubated with 14C-labelled C19O2-steroids in the presence of NADPH. Thus, testosterone, isoandrosterone, 3α-androstanediol and 3β-androstanediol were transformed into radioactive metabolites which were identified by crystallization to constant specific activity. Among polar metabolites derived from these substrates, formation of 5α-androstane-3β,7α,17β-triol by both tissues was proved and that of 5α-androstane-3β,6β,17β-triol strongly suggested. A specificity of 7α-hydroxylation for 3β-hydroxylated substrates was proposed since only major metabolites of isoandrosterone and 3β-androstanediol were 7α-hydroxylated. Furthermore it was confirmed that C19O2-3β-hydroxysteroid substrates only lead to minor quantities of 3-oxometabolites in vitro and in the presence of NADPH. In contrast, 3α-androstanediol is actively transformed, in the same conditions, into 5α-dihydrotestosterone, this metabolite giving successively rise to 3β-androstanediol and hydroxylated metabolites. Involved enzymic activities allowed to complete the prostatic testosterone metabolism pathways and to show that no qualitative difference occurs between normal and hyperplastic human glands. It remains to be found to which extent quantitative differences of enzymic activities may occur between both tissues and to determine the role which may be played by 5α-androstane-3β,7α,17β-triol in the mode of action of androgenic steroids on prostatic secretion or hyperplasia.

Protection against dextran sodium sulfate-induced colitis by dehydroepiandrosterone and 7α-hydroxy-dehydroepiandrosterone in the rat

In this study the anti-oxidant effect of DHEA and 7alpha-hydroxy-DHEA against oxidative stress induced by colitis was investigated in vivo in rats. The two steroids were intraperitoneally injected once daily (50 mg/kg body weight) for 7 days before the induction of colitis that was effected by a daily treatment of 5% (w/v) dextran sodium sulfate (DSS) in drinking water for 7 days. This was quantified by the evidence of weight loss, rectal bleeding, increased wall thickness, and colon length. The inflammatory response was assessed by neutrophil infiltration after a histological examination and myeloperoxidase (MPO) activity measurement. Two markers of oxidative damage were measured in colon homogenates after the onset of DSS treatment: protein carbonyls and thiobarbituric acid-reacting substances. The colonic metabolism of corticosterone by 11beta-hydroxysteroid dehydrogenases types 1 and 2 (11beta-HSD) was investigated in control and treated animals. Results indicated that colitis caused a decrease in body weight and colon length. Severe lesions were observed in the colon with a reduced number of goblet cells which contained less mucins. The lesions were associated with increased MPO activity and oxidative damage. Colonic inflammation down and up regulated the 11beta-HSD2 and 11beta-HSD1, respectively. Treatments by DHEA and 7alpha-hydroxy-DHEA attenuated the inflammatory response when MPO activity decreased; but this did not increase the colonic oxidation of corticosterone into 11-dehydrocorticosterone. Both DHEA and 7alpha-hydroxy-DHEA exerted a significant anti-oxidant effect against oxidative stress induced by colitis through reducing the oxidative damage to proteins and lipids. This resulted in a moderate increase in the amount of colonic mucus. Both DHEA and 7alpha-hydroxy-DHEA may prove useful in the prevention or treatment of colitis.

7α‐Hydroxy‐Dehydroepiandrosterone and Immune Response

Abstract: In human and murine lymphoid organs, circulating 3β‐hydroxysteroids, including pregnenolone (PREG), dehydroepiandrosterone (DHEA), and epiandrosterone (EPIA), are 7α‐hydroxylated by a cytochrome P450 identified in the hippocampus as P4507B1. Mouse and human lymphoid organs produced different patterns of 3β‐hydroxysteroid 7α‐hydroxylation with the absence of pregnenolone and epiandrosterone hydroxylation in human and mouse, respectively. Both 7α‐hydroxy‐DHEA and 7α‐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 7α‐hydroxy‐PREG and 7α‐hydroxy‐DHEA increased the immune response in mouse. Paracrine action of 7α‐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 7α‐hydroxylation, and suggested that such differences might contribute to the extent of immune response.