Observations on biological effects of carbon-13, based on the effects of 13 C-testosterone on growth of human osteoblasts, human aortic endothelial cells, and human umbilical vein endothelial cells in vitro

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 13 C 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 13 C enriched testosterone ( 13 C/ 12 C:6.7%). The impacts of physiological to pharmacological concentrations (10 -10 -10 -5 mol/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 13 C enriched testosterone exhibited different biological effects from testosterone. At the concentrations of 10 -10 mol/L and 10 -5 mol/L, there were significant differences in prompting cell proliferation between testosterone and 13 C enriched testosterone. At physiological concentrations, testosterone prompted proliferations of the three kinds of cells; whereas, 13 C enriched testosterone did not prompt the cell proliferation, and its effects were not concentration dependent. At supraphysiological concentration (10 -5 mol/L), testosterone had the trend of inhibiting cell growth; whereas, 13 C enriched testosterone had the trend of prompting cell growth. 13 C 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 13 C enriched compound were discussed. human osteoblasts [9], human aortic endothelial cells [11,16], and human umbilical vein endothelial cells [17]. The present study analyzed the isotope effect of carbon-13 ( 13 C) enriched testosterone on growth of the three kinds of human cells at various concentrations. It was among the rare studies with regard to carbon isotope effect of bioactive compound on human cells. The aim of the present study was to investigate the carbon isotope effect of 13 C enriched testosterone ( 13 C/ 12 C: 6.7%) on growth of human osteoblasts, human aortic endothelial cells, and human umbilical vein endothelial cells. The impacts of physiological concentrations (10 -10 mol/L and 10 -8 mol/L) [18] and supraphysiological concentrations (10 -6 mol/L and 10 -5 mol/L) were also investigated with in vitro model.


Introduction
Biogenic elements (such as C, H, O, and N) constitute organism in the form of stable isotopes with their respective isotope compositions. Large deviation from the intrinsic isotope signature of organism may influence the growth and metabolism of organism, provoking biological isotope effect.
Previous studies have demonstrated the detrimental impacts of heavy isotope on organism [1,2]. It was showed that ingestion of 30-50% D2O impaired hematopoiesis and lowered formation of platelets, neutrophils, and lymphocytes in the mouse [3]. One study with multiple heavy-isotope substitutions presented the abnormal changes in cell size and in the quantity and distribution of cellular components in alga [4]. On the other hand, some investigations have proved the beneficial effect of deuterium uptake, such as, promotion of longevity and health in several organisms [5] and protection of yeast cells from oxidative stress in yeast [6]. These findings are not contradictory, but because of different conditions in study, such as, biogenic element in question, isotope enrichment level, species of organism, and physiological properties observed.
The isotope enrichment level influences the isotope effect on biological system. One assay of bacterial growth found that uptake of more than 0.5% deuterium in water resulted in adverse effects, but ultralow deuterium enrichment (≤0.25%) showed signs of the opposite trend [7]. Another investigation with polyunsaturated fatty acid (PUFA) in yeast determined 20-50% of deuterated-PUFA in the total pool of PUFA as the enrichment levels of mitochondria protection [8]. In some literature, the levels of heavy isotope enrichment were classified as high level (＞50%) and low level (＜10%) [7].
Despite the increasing knowledge of biological isotope effect, comprehensive understanding of heavy isotope function in the biological contexts has remained far less than expectation. The following aspects are important for the theory and application study on biological isotope effect. 1) There have been fewer studies on the effect of other heavy isotope than deuterium. Carbon isotope effect on organism is rarely studied.
2) Compared with other organism, fewer isotope effects on human system have been investigated.
3) The isotopically modified water, diet, and culture medium were used in most of the studies to introduce heavy isotopes into organism. Although this approach can be used to observe the impact of isotope substitution within the whole body, it has its own limitations. It is not conducive to study the isotope effect of a bioactive compound. Study with bioactive compound can provide more information at molecular level and facilitate to develop isotopically modified drugs. Bioactive compound acts mainly on the target tissue, while diet acts on the whole body. Diet-induced isotope effect including potential toxicity to organism is systemic. Little research on isotope effect of bioactive compound has been reported.
4) It is well recognized that a number of bioactive substances, such as steroid hormones, perform normal functions only at the physiological concentrations. Deficiency or excess of the substance may produce negative effects on health. As for these substances, both isotope enrichment level and experimental concentration should be taken into account in order to understanding biology isotope effect comprehensively.
The issues mentioned above were of the interests of our research. Testosterone, a bioactive compound, was isotopically modified to investigate the carbon isotope effect on human cells. The androgen is involved in the regulation of a number of physiological processes including bone development [9,10] and vascular behavior modulation [11][12][13]. Osteoblasts and vascular endothelial cells express androgen receptor and are targets for androgen action [14][15][16]. Several studies have established that testosterone produces the concentration-dependent impacts on cell proliferations of human osteoblasts [9], human aortic endothelial cells [11,16], and human umbilical vein endothelial cells [17]. The present study analyzed the isotope effect of carbon-13 ( 13 C) enriched testosterone on growth of the three kinds of human cells at various concentrations. It was among the rare studies with regard to carbon isotope effect of bioactive compound on human cells.
The aim of the present study was to investigate the carbon isotope effect of 13 C enriched testosterone ( 13 C/ 12 C: 6.7%) on growth of human osteoblasts, human aortic endothelial cells, and human umbilical vein endothelial cells. The impacts of physiological concentrations (10 -10 mol/L and 10 -8 mol/L) [18] and supraphysiological concentrations (10 -6 mol/L and 10 -5 mol/L) were also investigated with in vitro model.

Preparation of 13 C enriched testosterone
Testosterone and testosterone-3,4-13 C2 were dissolved in ethanol respectively and then mixed together with the ratio of 1:1(mole), by which 13 C enriched testosterone was obtained. The carbon isotopic composition of 13 C enriched testosterone was calculated to be 13 C/ 12 C=6.7%. The chemical structures of testosterone and testosterone-3,4-13 C2 were displayed in Fig.1.

Cell culture and compound intervention
Human osteoblasts, human aortic endothelial cells, and human umbilical vein endothelial cells were thawed and cultured in low glucose DMEM supplemented with 10% FBS and 1% penicillin-streptomycin with 5% CO2 at 37℃. The cells were dissociated with trypsine-EDTA and seeded in 96 well tissue culture plates at the density of 1×10 4 cells/well, the culture media was changed to the media made with low glucose DMEM, 10% FBS, 1% penicillin-streptomycin, 50mg/mL ascorbic acid, 0.01 mol/L glycerol-2-phosphate, and 100nmol/L dexamethasone. The cells were cultured in the culture medium containing either testosterone or 13 C enriched testosterone at concentrations of 0, 10 -10 , 10 -8 , 10 -6 , and 10 -5 mol/L, respectively. Non-drug group (0mol/L) was accompanied as blank control.

Measurement of cell proliferation activity
The proliferation activities of human osteoblasts, human aortic endothelial cells, and human

Test of alkaline phosphatase level of osteoblasts
In order to further investigate the isotope effect of 13  The test was repeated six times, and the average value was recorded. The data was normalized by the control.

Test of osteocalcin secretion level of osteoblasts
In order to investigate the isotope effect of 13 C enriched testosterone on osteocalcin (OC) secretion in osteoblasts, OC level of the osteoblasts was tested. OC, an osteoblast-specific secreted protein, is synthesized by osteoblasts during bone formation [19]. It plays key roles in both the biological and mechanical functions of bone [9,20]. As a biochemical marker of osteoblast activity, OC level reflects the rate of bone formation [21]. In the current study, the OC levels of the human osteoblasts cultured at 5 th day with 13 C enriched testosterone at the concentrations of 0, 10 -10 , 10 -8 , 10 -6 , and 10 -5 mol/L were tested. Following the instruction of ELISA kit, the OC in supernatant of the culture medium was analyzed using OC ELISA kit (sigma RAB1073-1KT). The test of OC level (μg/L) was repeated six times, and the average value was recorded. The data was normalized by the control.

Statistics
Data were analyzed with SPSS software. Significant differences were assessed with ANOVA and Post-hoc Test. P<0.05 were considered significant.

Cell proliferation activity
The proliferation activities of human osteoblasts, human aortic endothelial cells, and human umbilical vein endothelial cells treated with testosterone and 13 C enriched testosterone were analyzed.
The measured OD values were normalized and plotted in Fig. 2-4.

Human osteoblasts
Testosterone prompted the proliferation of human osteoblasts at concentrations of 10 -10 mol/L (P<0.01) and 10 -8 mol/L (P<0.05). Treated with supraphysiological concentration (10 -5 mol/L) of testosterone, human osteoblasts showed a decreasing trend of proliferation activity (Fig. 2). 13 C enriched testosterone did not prompt the proliferation of human osteoblasts at concentrations of 10 -10 -10 -6 mol/L. Among these concentration groups, there were no differences in the effects of 13 C enriched testosterone, which implied that the effect had no relationship with the concentration at the level of 10 -10 -10 -6 mol/L. It was noted that human osteoblasts showed an increasing trend of proliferation activity when treated with supraphysiological concentration (10 -5 mol/L) of 13 C enriched testosterone (Fig. 2).
There were significant differences in proliferation activity between treatment of testosterone and 13 C enriched testosterone at the concentration of 10 -10 mol/L (P<0.01) and 10 -5 mol/L (P<0.05), respectively (Fig. 2).

Human aortic endothelial cells
Testosterone significantly prompted the proliferation of human aortic endothelial cells at the concentration of 10 -10 mol/L (P<0.001). Treated with supraphysiological concentration (10 -5 mol/L) of testosterone, human aortic endothelial cells showed a decreasing trend of proliferation activity (Fig. 3). 13 C enriched testosterone did not prompt the proliferation of human aortic endothelial cells at the concentrations of 10 -10 -10 -6 mol/L. Among these concentration groups, there were no differences in the effects of 13 C enriched testosterone, which implied that the effect had no relationship with the concentration at the level of 10 -10 -10 -6 mol/L. It was noted that human aortic endothelial cells showed an increasing trend of proliferation activity when treated with high concentration (10 -5 mol/L) of 13 C enriched testosterone (Fig. 3).
There were significant differences in proliferation activity of human aortic endothelial cells between treatment of testosterone and 13 C enriched testosterone at the concentrations of 10 -10 mol/L (P<0.001) and 10 -5 mol/L (P<0.0001), respectively (Fig. 3).

Human umbilical vein endothelial cells
Testosterone prompted the proliferation of human umbilical vein endothelial cells at the concentration of 10 -8 mol/L (P<0.05). Treated with high concentration (10 -5 mol/L) of testosterone, the human umbilical vein endothelial cells showed a decreasing trend of proliferation activity (Fig. 4). 13 C enriched testosterone did not prompt the proliferation of human vein endothelial cells at the concentrations of 10 -10 -10 -5 mol/L. Among these concentration groups, there were no significant differences in the effects of 13 C enriched testosterone, which implied that the effect had no relationship with the concentration at the level of 10 -10 -10 -5 mol/L (Fig. 4).
There were no differences in proliferation activity of human umbilical vein endothelial cells between treatment of testosterone and 13 C enriched testosterone at the concentrations of 10 -10 -10 -5 mol/L.

ALP level of human osteoblasts
Compared with control group, 13 C enriched testosterone did not enhance ALP level of human osteoblasts at the concentrations of 10 -10 mol/L and 10 -8 mol/L. The human osteoblasts showed increasing trend of ALP level when treated with high concentration (10 -6 mol/L and 10 -5 mol/L) of 13 C enriched testosterone (Fig. 5).

Osteocalcin secretion of human osteoblasts
Compared with control group, 13 C enriched testosterone significantly enhanced the OC level in human osteoblasts at the concentrations of 10 -6 mol/L (P<0.05) and 10 -5 mol/L (P<0.001), respectively ( Fig. 6).

Discussion
The present research contributed novel contents to the biology isotope effect of carbon. The elements C, H, O and N compose over 98% of the total living mass in the Earth and account for 96% of human body weight [22]. Biological effects of heavy isotopes of the elements have been constantly recognized. Deuterium has been investigated more than other heavy stable isotopes, which is partly due to the consideration that deuterium with twice the mass of a hydrogen atom exhibits more remarkable isotope effect. However, carbon is a vital element of biological system and forms the skeleton of biological macromolecules. Maintaining physiological activities, numerous biochemical reactions occur in organism with carbon-carbon bond formation and cleavage. The change in carbon isotope composition could influence the biochemical reaction rate and produce biology isotope effect. Therefore, carbon isotope effect on human and other organisms should be intensively studied for the potential application in biology and medicine.
Our study proved that 13 C enriched testosterone at 6.7% of 13 C/ 12 C had different biological effects from testosterone. We assumed that the enrichment of heavy isotope in bioactive compound could weaken biological effect of the compound, which potentially benefit drug development. For example, administration of 13 C enriched testosterone would relieve symptoms of hyperandrogenism, such as acne, hirsutism, and hyperinsulinemia [23].
Previous studies established that testosterone had effects on the cell proliferations in a concentration-related manner [9,11,16,17], which was supported by our findings. However, the effects of 13 C enriched testosterone were not concentration dependent at the concentration levels of 10 -10 -10 -6 mol/L. This finding implied that enrichment of heavy isotope in drug might change the dose dependence of the drug action. For the drugs whose effective dose and toxic dose are close to each other, enrichment of heavy carbon isotope in the drug might make its effect less sensitive to the change of concentration, which could potentially improve the drug safety.
It has been generally believed that enrichment of heavy isotope slow down the biochemical reaction rate and retard the growth of organism because of kinetic isotope effect [24][25][26][27][28]. This common view was supported by only a part of our findings. It was unexpected that 13 C enriched testosterone showed the trends of promoting the cell proliferations at high concentration (10 -5 mol/L). Furthermore, 13 C enriched testosterone enhanced OC secretion of human osteoblasts significantly at high concentrations (10 -6 mol/L and 10 -5 mol/L). These findings challenged the common view of heavy isotope enrichment and exhibited the complexity of the biology isotope effect. The underline mechanisms are poorly understood and should be further investigated.

Conclusions
The present study substantiated the carbon isotope effect of one bioactive compound. 13 C enriched testosterone at 6.7% of 13 C/ 12 C had different biological effects from testosterone. At physiological concentrations, testosterone prompted the cell proliferations; whereas, 13 C enriched testosterone did not prompt the cell proliferations and its effects were not concentration dependent. At supraphysiological concentration (10 -5 mol/L), testosterone had the trend of inhibiting cell growth; whereas, 13 C enriched testosterone had the trend of prompting cell growth. 13 C enriched testosterone significantly enhanced osteocalcin secretion in human osteoblasts at supraphysiological concentration. These findings imply that biology isotope effects are worthy of further study to benefit human health. Fig. 1 The chemical structure of testosterone and testosterone-3,4-13 C2. Fig. 2 The influence of testosterone and 13 C enriched testosterone on human osteoblasts. The cells were treated with testosterone and 13 C enriched testosterone at different concentrations (10 -10 , 10 -8 , 10 -6 and 10 -5 mol/L, respectively). Data represent means± SEM (n= 6). Significant difference *p < 0.05, **p< 0.01 vs. compared. Fig. 3 The influence of testosterone and 13 C enriched testosterone on human aortic endothelial cells.