Cytochrome P450 7B1 Research Articles

Regulation and expression of human CYP7B1 in prostate: Overexpression of CYP7B1 during progression of prostatic adenocarcinoma

Cytochrome P450 (CYP) 7B1 is involved in many metabolic processes including androgen metabolism. Cytochrome P450 (CYP) 7B1 is expressed within the prostate and may determine the levels of the natural estrogen receptor beta (ERbeta) ligand 5alpha-androstane-3beta,17beta-diol (3betaAdiol) available and hence affect the regulation of prostate proliferation. We hypothesized that CYP7B1 expression is increased in prostate tumors and that promoter methylation contributes to the regulation of CYP7B1 expression in human prostate tissue. Expression of the CYP7B1 gene and protein in clinical prostate tissues and prostate cancer cell lines were investigated using real-time PCR and immunohistochemistry. The methylation status of the CYP7B1 gene was analyzed using methylation-specific PCR (MSP). The immunohistochemical results demonstrate that high expression of CYP7B1 protein occurs in high-grade prostatic intraepithelial neoplasia (PIN) and adenocarcinomas. The ERbeta/CYP7B1 mRNA ratio was significantly lower in tumor compared to the non-tumor area. The MSP analysis indicate that local methylation of CYP7B1 promoter region is an important mechanism involved in down-regulation of CYP7B1 in human prostate tissue. This is the first report showing that CYP7B1 is overexpressed in high-grade PIN and in prostate cancer and that local methylation of CYP7B1 promoter region may have significant effect on gene transcription.

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.

Central Administration of a Cytochrome P450-7B Product 7α-Hydroxypregnenolone Improves Spatial Memory Retention in Cognitively Impaired Aged Rats

Pregnenolone (PREG) and dehydroepiandrosterone (DHEA) have been reported to improve memory in aged rodents. In brain, these neurosteroids are transformed predominantly into 7alpha-hydroxylated metabolites by the cytochrome P450-7B1 (CYP7B). The biological role of steroid B-ring hydroxylation is unclear. It has been proposed to generate bioactive derivatives that enhance cognition, immune, and other physiological processes. In support, 7alpha-hydroxylated DHEA increases the immune response in mice with greater potency than the parent steroid. Whether the memory-enhancing effects of PREG in rats is mediated via its 7alpha-hydroxylated metabolite 7alpha-hydroxyPREG is not known. We investigated this by treating memory-impaired aged rats (identified by their spatial memory performances in the Morris water maze task compared with young controls) with 7alpha-hydroxyPREG or PREG administered intracerebroventricularly using osmotic minipumps and then tested the rats during week 2 of steroid treatment in the eight-arm radial-arm version of the water maze (RAWM) that allows repeated assessment of learning. CYP7B bioactivity in hippocampal tissue (percentage conversion of [14C]DHEA to [14C]7alpha-hydroxyDHEA) was decreased selectively in memory-impaired aged rats compared with both young and memory-intact aged rats. 7alpha-hydroxyPREG (100 ng/h) but not PREG (100 ng/h) administration to memory-impaired aged rats for 11 d enhanced spatial memory retention (after a 30 min delay between an exposure trial 1 and test trial 2) in the RAWM. These data provide evidence for a biologically active enzyme product 7alpha-hydroxyPREG and suggests that reduced CYP7B function in the hippocampus of memory-impaired aged rats may, in part, be overcome by administration of 7alpha-hydroxyPREG.

The native anti-glucocorticoid paradigm

Circulating 3β-hydroxysteroids including dehydroepiandrosterone (DHEA) are 7α-hydroxylated by the cytochrome P450-7B1 in the liver, skin and brain, which are the target organs of glucocorticoids. Anti-glucocorticoid effects with 7α-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 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). We demonstrated that 7α-hydroxy-DHEA was also a substrate for this enzyme. Studies of the 11β-HSD1 action on 7α-hydroxy-DHEA showed the reversible production of 7β-hydroxy-DHEA through an intermediary 7-oxo-DHEA, and the kinetic parameters favored this production over that of active glucocorticoids. Both the production of 7α-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.

Immunohistochemical detection of the human cytochrome P4507B1: production of a monoclonal antibody after cDNA immunization

The cytochrome P4507B1 (P4507B1) is responsible for the 7α-hydroxylation of dehydroepiandrosterone (DHEA) and other 3β-hydroxysteroids in the brain and other organs. The cDNA of human P4507B1 was used for DNA immunization of mice. The best responding mouse led to the production of monoclonal antibodies (mAbs). The clone D16-37 produced an IgM specific for P4507B1 with no cross-reaction with other human P450s. This antibody permitted the immunohistochemical detection of P4507B1 in slices of human hippocampus. P4507B1 was expressed in neurons only. This new tool will be used for the extensive examination of the P4507B1 presence and determination of its levels in slices of human normal and diseased brain and in other human tissues.

The human cytochrome P4507B1: catalytic activity studies

The cytochrome P4507B1 (P4507B1) in the human hippocampus is responsible for the production of 7α-hydroxylated derivatives of dehydroepiandrosterone (DHEA) and other 3β-hydroxylated neurosteroids. Minor quantities of the 7β-hydroxylated derivatives are also produced. Neuroprotective action of these 7-hydroxysteroids was reported. Recombinant human P4507B1 was prepared from yeast coexpressing the human hippocampal P450 cDNA and the human P450 reductase genes. Microsomal P4507B1 activity was tested in the presence of NADPH and 14C-labeled steroid substrates to deduce kinetic parameters and to study inhibitor responses. The K M values obtained for DHEA, pregnenolone, epiandrosterone, 5α-androstane-3β,17β-diol and estrone were 1.90 ± 0.06, 1.45 ± 0.03, 1.05 ± 0.12, 0.8 ± 0.04 and 1.20 ± 0.26 μM, respectively. Production of limited amounts of 7β-hydroxylated derivatives was also observed, but only with DHEA, 5α-androstane-3β,17β-diol and epiandrosterone. K M values determined for 7β-hydroxylation were identical to those for 7α-hydroxylation. The DHEA 7α-hydroxylation was inhibited by estrone and estradiol (mixed type inhibition) and by the [25–35] β-amyloid peptide (non-competitive inhibition). These results indicate that in human, the 7-hydroxylation catalysed by P4507B1 preferentially takes place on DHEA, 5α-androstane-3β,17β-diol and epiandrosterone with major and minor formation of 7α- and 7β-hydroxylated derivatives, respectively. Both estrogens and a β-amyloid component inhibit the P4507B1-mediated production of the 7-hydroxysteroid metabolites.

Use of bioconversion for the preparation of [4- 14C]-labeled 7α- and 7β-hydroxylated derivatives of dehydroepiandrosterone and epiandrosterone

The 7α- and 7β-hydroxylated derivatives of [ 4- 14 C ]-dehydroepiandrosterone were prepared with use of the yeast-expressed human cytochrome P4507B1. Epiandrosterone (EPIA), 5α-androstane-3β,17β-diol, and 5α-androstane-3,17-dione were obtained after incubation of [ 4- 14 C ]-5α-dihydrotestosterone with Escherichia coli-expressed (3β,17β)-hydroxysteroid dehydrogenase from Pseudomonas testosteroni. The 7α- and 7β-hydroxylated derivatives of [ 4- 14 C ]-EPIA produced were prepared after incubation with mycelium of Rhizopus nigricans. Each labeled steroid was purified by chromatography and identified by crystallization to constant specific activity after isotopic dilution with each authentic steroid carrier. Production yields and radio-purity measurements allowed the use of such procedures for the preparation of the described radio-steroids for studies of metabolism and mode of action.