Figure 1.
7DHC metabolism by rat adrenal glands.
Finely cut fragments of rat adrenal glands were incubated in the presence (B–D) or absence (A) of 7DHC (0.5 mM). Boiled adrenal fragments (C) served as an additional negative control. Panel B shows results from an incubation which included trilostane, a 3βHSD inhibitor. Steroids were extracted with methylene chloride and subjected to LC/MC and UV spectra analyses. Panels A–D identifies 7 main products. Product 3 had an identical retention time to 7DHP standard. Panel E shows UV spectra, MS and the predicted structures of the 7 metabolites.
Figure 2.
Precursor-product relationship between 7DHC, hydroxy-7DHC, dihydroxy-7DHC and 7DHP defined by their sequential metabolism by rat adrenal glands.
A. Chromatogram showing metabolism of 7DHC(S) by adrenal glands. The hydroxy-7DHC (1) and dihydroxy-7DHC (2) products were collected from the HPLC and tested as substrates (see B–E). Product 3 was identified as 7-DHP based on RT, mass and UV spectra. B. Incubation of hydroxy-7DHC (1) with adrenal glands generated dihydroxy-7DHC with RT 34.8 min (2) and 7DHP with RT 15.8 min (3). C. Boiled adrenal glands served as a control for B. D. Incubation of dihydroxy-7DHC (2) with adrenal glands produced 7DHP; note the lack of S and product 1. E. Negative control for D (boiled adrenal glands ). Inserts: UV spectra of compounds 1–3.
Figure 3.
Metabolism of 7-DHC by adrenal glands of different mammalian species.
Finely cut fragments of rat (A), rabbit (B), dog (C) and pig (D) adrenals were incubated in the presence (blue) or absence (green) of 7DHC (S). Boiled adrenal fragments served as an additional negative control for the rat (red). The three major products common to all animals studied are numbered 1 to 3 on the chromatograms. Product 3 had an identical retention time, mass, and UV spectra to 7DHP standard.
Figure 4.
Metabolism of 7DHC by pig adrenal glands.
Fragments of pig adrenal glands were incubated with 0.5 mM 7DHC. Steroids were extracted with methylene chloride and subjected to HPLC and UV spectral analyses. The UV spectrum for all peaks is the same (see arrows). The mass spectra of the collected peaks 1–4 were obtained by direct injection into the MS module of the Bruker Esquire-LC/MS Spectrometer.
Figure 5.
Metabolism of 7DHC by adrenal glands in the presence of DL-aminoglutethimide (AGT), trilostane, forscolin and Δ7-reductase inhibitors.
A. For rat adrenals transformation of 7DHC (S) to 7DHP (3) was dramatically inhibited by AGT (red) compared to the incubation with 7DHC alone (blue), while accumulation of 22(OH)7DHC (1), 20,22(OH)27DHC (2) and 7DHP (3) was enhanced by trilostane (green). Control incubations performed with boiled adrenal fragments are shown in the inset. Incubations were carried out for 18 h. B. For pig adrenals AGT also inhibited 7DHC metabolism (red) compared to the incubation with 7DHC alone (blue), while trilostane (green) enhanced accumulation of 22(OH)7DHC (1), 20,22(OH)27DHC (2), 7DHP (3) and 17(OH)7DHP (4). Adrenal fragments were incubated for 6 h and controls (inset) were incubated without the substrate. C. For pig adrenals 0.1 mM forscolin (green) stimulated 7DHC metabolism. The labeling and the experimental conditions are the same as in B. D. Fragments of pig adrenal glands were incubated with increasing doses of 7DHC in the presence (T+) or absence (T−) of 0.1 mM trilostane. The chromatograms were processed as described above. Trilostane (T) enhanced accumulation of 22(OH)7DHC (1), 20,22(OH)27DHC (2), 7DHP (3) and 17(OH)7DHP (4) as a function of 7DHC concentration. E. Fragments of rat adrenal glands were incubated with 7DHC (0.5 mM) in presence of AY-9944 or BM15.766 (10 µM or 100 µM) and analyzed by RP-HPLC (see A for peak identification). The chromatograms were processed using ACDLabs software and relative areas for peaks identified above were calculated. Δ7-reductase inhibitors enhanced production of 7DHC metabolites.
Figure 6.
Time-dependent metabolism of 7DHP is modified by trilostane (T).
Fragments of the pig adrenal glands were incubated with 7DHP (S; 0.5 mM) in the absence (A) or presence (B) of trilostane (0.1 mM) and analyzed by RP-HPLC. Boiled adrenal fragments were used as a negative control (blue). The major products of 7DHP metabolism affected by trilostane are marked by numbers. Time-dependent changes in the relative concentration of the identified products in presence (T+) or absence (T-) of trilostane were calculated using ACDLabs software and are presented in C.
Figure 7.
Trilostane (T) inhibits and modifies 7DHP (S) metabolism by rat adrenal glands.
A. Fragments of rat adrenal glands were incubated with 7DHP (0.5 mM) in the absence (pink) or presence (gray) of trilostane (0.1 mM) and samples analyzed by RP-HPLC. Boiled adrenal fragments (green) or incubations without the substrate (blue) were used as negative controls. B. The relative concentrations of the identified products affected by trilostane (T). The insert shows inhibition of 7DHP consumption by trilostane. Control marked by blue represents or incubations without the substrate.
Figure 8.
LC/MS identification of the products of 7DHP metabolism by pig adrenals.
Collected fraction from RT-HPLC experiment were analyzed using the Bruker Esquire-LC/MS Spectrometer as described in Materials and Methods. Numbers correspond to the products identified in Figs 6 and 7.
Figure 9.
Proposed pathways for the in vivo metabolism of 7DHC in the mammalian adrenal gland.
Figure 10.
Stimulation of 7DHC transfer between membranes and the inhibition of the side chain cleavage of [cholesterol by 7DHC.
A. To study 7DHC transfer between membranes by N-62 StAR protein cholesterol or 7DHC was placed in donor vesicles at a molar ratio to phospholipid of 0.2. The transfer of the sterol to acceptor vesicles at 35°C was measured in the presence or absence of 5 µM N-62 StAR protein. B. Inhibition of the side chain cleavage of [4-14C]cholesterol by 7DHC was determined with substrate and cytochrome P450scc incorporated into phospholipid (PL) vesicles prepared from dioleoyl phosphatidylcholine containing ardiolipin. As indicated in the figure, 7DHC was included in the membrane at a molar ratio to phospholipid of 0.2. The rate of side chain cleavage of [4-14C]cholesterol was determined from the amount of [4-14C]pregnenolone formed.
Figure 11.
The effect of 7DHP, pregnacholecalciferol (pD3) and pregnenolone on normal immortalized epidermal HaCaT keratinocytes and PIG1 melanocytes.
A and B. Inhibition of HaCaT cell proliferation as measured by [3H]-thymidine incorporation (A) and MTT (B) assays after 48 h and 72 h of treatment, respectively. C. Inhibition of NFκB activity in HaCaT keratinocytes 24 h after transfection with luciferase construct NFκB-Luc. The cells were treated for 24 h with 100 nM vitamin D3 (vD3), 1,25(OH)2D3, 7DHP, pD3, pregnenolone or ethanol vehicle (control) and luciferase activity was measured in cell extracts. D and E. Inhibition of cell growth as measured by sulforhodamine b assay (protein content) after 72 h of treatment of non-pigmented (D) and pigmented (E) PIG1 human melanocytes. Inserts show the color of representative cell pellets. Significant differences versus ethanol-treated control cells were defined as *p<0.05, **p<0.01, ***p<0.001.
Figure 12.
7DHP, 20-oxopregnacalciferol (pD3) and pregnenolone inhibit growth of melanoma cells in soft agar.
A: hamster AbC1 melanoma; B: human SKMEL-188 melanoma; C: visualization of SKMEL-188 colonies by MTT reagent. Decreased number and size of colonies was observed in both cell lines. Significant differences versus ethanol-treated cells were defined as *p<0.05, **p<0.01, ***p<0.001.
Figure 13.
Metabolism of 7DHC and 7DHP by rat skin.
A. To study the skin capability to transform 7DHC to 7DHP isolated mitochondria were incubated with 7DHC (0.5 mM) in the presence (experimental) or absence (control) of isocitrate and NADPH. 7DHP: product with the same retention time and UV spectra (inset) as 7DHP standard. B. Incubation of crude skin extract with 7DHP substrate (RT 16.7 min) in the presence of isocitrate and NADPH (experimental) resulted in appearance of a 5,7-diene product (N) with RT 11.9 min, which was absent in control reaction mixture (isocitrate and NADPH were not added) (control).