Osteoporosis is liable to affect patients with gonadal hormone deficiency, and a supplement of androgens may be used to increase bone density of patients with osteoporosis. Since the androgens currently used may cause severe side effects, it is useful to investigate the effect of other androgens and progestin on bone improvement. The aim of the current study was to investigate the effects of pregnenolone (Preg), androstenedione (AD), etiocholanolone (Etio), androsterone (An), nandrolone (NA) and testosterone (T) on the proliferation and differentiation of osteoblasts for potential clinical applications. Human osteoblasts were cultured and treated with androgens and progestin, including Preg, AD, Etio, An, NA, and T, at concentrations of 0, 10 -10 , 10 -8 , 10 -6 and 10 -5 mol/l. The levels of cell proliferation, alkaline phosphatase (ALP) activity and osteocalcin content were measured and assessed. Preg, AD, Etio, An, and T at concentrations of 10 -10 and/or 10 -8 mol/l significantly improved osteoblast proliferation. NA at concentrations of 10 -10 , 10 -8 , 10 -6 and 10 -5 mol/l also significantly improved osteoblast proliferation. Preg, AD, Etio, An, NA, and T significantly increased ALP activity and osteocalcin content. The present study demonstrated, for the first time, that Preg, AD, Etio, An, and NA could improve the proliferation and differentiation of osteoblasts in vitro.

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Despite the increasing knowledge of biological isotope effect, comprehensive understanding of heavy isotope effect in the biological contexts has remained far less than expectation. The present study investigated the carbon isotope effect of 13C enriched testosterone on human cells. It was among the rare studies on carbon isotope effect of bioactive compound. Human osteoblasts, human aortic endothelial cells, and human umbilical vein endothelial cells were cultured in vitro and treated with testosterone and 13C enriched testosterone (13C/12C:6.7%). The impacts of physiological to pharmacological concentrations (10-10-10-5mol/L) of the bioactive compound were taken into account. The cell proliferation activities were measured using MTS assay. The levels of alkaline phosphatase and osteocalcin in osteoblasts were tested. Our results established that 13C enriched testosterone exhibited different biological effects from testosterone. At the concentrations of 10-10mol/L and 10-5mol/L, there were significant differences in prompting cell proliferation between testosterone and 13C enriched testosterone. At physiological concentrations, testosterone prompted proliferations of the three kinds of cells; whereas, 13C enriched testosterone did not prompt the cell proliferation, and its effects were not concentration dependent. At supraphysiological concentration (10-5mol/L), testosterone had the trend of inhibiting cell growth; whereas, 13C enriched testosterone had the trend of prompting cell growth. 13C enriched testosterone significantly enhanced osteocalcin secretion in human osteoblasts at supraphysiological concentration. These findings challenged the common view of growth retardation effect of heavy isotope, which imply that biological isotope effects are worthy of further study. The potential applications of 13C enriched compound were discussed.

The present study investigated the carbon isotope effect of carbon-13 ( 13 C)-enriched testosterone on human cells; studies on the carbon isotope effects of a bioactive compound are limited. For this purpose, human osteoblasts, aortic endothelial cells and umbilical vein endothelial cells were treated with testosterone and 13 C-enriched testosterone. The cell growth rates and bioactivities of osteoblasts were measured. At physiological concentrations, testosterone promoted cell proliferation, whereas 13 C-enriched testosterone exerted a neutral and concentration-independent effect on the cells. On the whole, the findings of the present study demonstrated that 13 C-enriched testosterone exerted a significant isotope effect on human cells; this needs to be taken into account in stable isotope-based biology research.