dihydrotachysterol and Liver-Neoplasms

Dihydrotachysterol (DHT), a reduced vitamin D analog in which the A-ring has been rotated through 180 degrees is a biologically active molecule which can be used to study the structural requirements for the calcemic and cell differentiating properties of the vitamin D hormone, 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3), as well as to investigate the specificity of the enzyme systems that catalyze the formation of this hormone. In this study we showed that dihydrotachysterol was metabolized in vivo into a significant polar metabolite observed on straight-phase high performance liquid chromatography (HPLC) which subsequently split into two peaks on reverse-phase HPLC. These two metabolites were identified by HPLC and gas chromatography-mass spectrometry techniques as 1 alpha,25-(OH)2DHT and 1 beta,25-(OH)2DHT. This pair of metabolites was formed from either DHT2 or DHT3. Standard 1 alpha,25-(OH)2DHTs were generated in vitro from chemically synthesized 1-hydroxydihydrotachysterol precursors using a liver hepatoma cell system. Both 1 alpha,25-(OH)2D2 and 1 alpha,25-(OH)2DHT3 showed a binding affinity to the mammalian vitamin D receptor only 50-100 less than 1 alpha,25-(OH)2D3 whereas 1 beta,25-(OH)2DHTs showed poor binding. On the other hand 1 beta,25-(OH)2DHT3 bound to the rat vitamin D transport protein (DBP) with stronger affinity than did 1 alpha,25-(OH)2DHT3. When tested in a COS-1 cell transfection assay system using a rat osteocalcin vitamin D responsive element coupled to a growth hormone reporter gene, 1 alpha,25-(OH)2DHT3 showed a biological activity only 10 times lower than 1 alpha,25-(OH)2D3. It is therefore suggested that 1 alpha,25-(OH)2DHT probably represents the metabolite of DHT responsible for some of its in vivo effects although we cannot rule out in vivo effects of other metabolites identified. Our studies suggest that 1 alpha,25-dihydroxylated DHTs represent a promising novel group of vitamin D analogs worthy of study for cell differentiation as well as calcemic properties.

Topics: Animals; Biotransformation; Carcinoma, Hepatocellular; Cell Line; Chromatography, High Pressure Liquid; Dihydrotachysterol; Gas Chromatography-Mass Spectrometry; Growth Hormone; Humans; Liver Neoplasms; Magnetic Resonance Spectroscopy; Molecular Structure; Rats; Stereoisomerism; Transfection; Tritium; Tumor Cells, Cultured; Vitamin D-Binding Protein

Dihydrotachysterol3, a reduced (or hydrogenated) analog of vitamin D3 in which the A ring has been rotate through 180 degrees , is, after hepatic 25-hydroxylation, converted in vivo to a dihydroxylated metabolite, termed peak H, which is at present unidentified but with good affinity for the vitamin D receptor. Although peak H is made in relatively large amounts in vivo, it has not yet been possible to synthesize it in vitro. Mass spectrometric evidence suggests that peak H is 25-hydroxylated and the presumption that it is a metabolite of 25-hydroxydihydrotachysterol3 was confirmed by the demonstration that radiolabeled peak H was formed in vivo in the rat after injection of 25-hydroxy-[10,19-3H]dihydrotachysterol3, produced from [10,19-3H]dihydrotachysterol3 in a hepatic cell model. The metabolism of 25-hydroxy-[10,19-3H]dihydrotachysterol3 was also studied in a rat osteosarcoma cell UMR-106, a known target cell for vitamin D, using high (11 microM) and low (10 nM) substrate concentrations. Metabolic products were isolated by lipid extraction, purified by high-performance liquid chromatography, and characterized by direct-probe mass spectrometry and gas chromatography/mass spectrometry. The formation of peak H from 25-hydroxydihydrotachysterol3 could not be demonstrated in UMR-106 cells. However, 25-hydroxydihydrotachysterol3 was metabolized to at least seven side-chain modified metabolites, each of which was extensively characterized and tentatively identified. It is concluded that the vitamin D enzyme system present in UMR-106 cells is able to metabolize dihydrotachysterol3 very efficiently to a series of metabolites but is incapable of producing peak H.

Topics: Animals; Bone and Bones; Carcinoma, Hepatocellular; Chromatography, High Pressure Liquid; Dihydrotachysterol; Humans; Hydroxylation; Liver; Liver Neoplasms; Male; Mass Spectrometry; Osteosarcoma; Rats; Rats, Wistar; Tumor Cells, Cultured