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Introduction

Stable isotope labeling plays a staple role in biological research. As such studies are commonly linked to the kinetic features of biochemical reactions, the kinetic isotope effect (KIE) on the growth and metabolism of an organism should be considered (1,2). An isotope is a species of an element with a different mass due to the different number of neutrons. The isotope effect refers to the phenomenon that the physical and chemical properties of isotopic atoms differ due to the dissimilarities in their nuclear properties (3,4).

For decades, various efforts have been made to explore the biology isotope effects of biogenic elements. Despite the increasing knowledge of the biological isotope effect, a comprehensive understanding of the effects in the biological context has not yet been achieved (2,5-9). Among the main biogenic elements (C, H, O and N), deuterium has been investigated in a number of studies and its effects on organisms have been demonstrated (5-9). However, the carbon isotope effects of bioactive compounds have yet not been investigated, at least to the best of our knowledge, although such studies are important to the biology development in theory and application. Additionally, fewer isotope effect studies on human cells have been conducted compared with other organism (10).

In the present study, testosterone, a typical androgen, was isotopically modified. It was demonstrated that the level of isotope enrichment potentially influenced its isotope effect, and it was classified as high (>50%) and low (<10%) level (11,12). Herein, carbon-13 (13C) was enriched in testosterone with a low enrichment level (13C/12C, 6.7%) in order to investigate the carbon isotope effect on human osteoblasts, aortic endothelial cells and umbilical vein endothelial cells. Androgen is involved in the regulation of a number of physiological processes, including bone development (13,14) and the modulation of vascular behavior (15-17). Osteoblasts and vascular endothelial cells express androgen receptor and are targets for the action of androgen (18-20). Several studies have demonstrated that testosterone enhances the proliferation of human osteoblasts (13), human primary aortic endothelial cells (15,20) and human umbilical vein endothelial cells (21). The present study examined the isotope effect of 13C-enriched testosterone at various concentrations on the growth of the aforementioned cell types. The alkaline phosphatase (ALP) level and osteocalcin (OC) secretion of osteoblasts were examined. To the best of our knowledge, the present study is the first investigate the carbon isotope effect of a bioactive compound on normal human cells.

The aim of the present study was to highlight the importance of isotope effects in stable isotope-based research by investigating the carbon isotope effect of 13C-enriched testosterone on the growth of human cells. The concentration effects at physiological (10-10 and 10-8 mol/l) (22) and supraphysiological (10-6 and 10-5 mol/l) levels were investigated using an in vitro model.

Materials and methods

Materials and reagents

Human osteoblasts (cat no. 4610), human primary aortic endothelial cells (cat no. H-6052) and human umbilical vein endothelial cells (cat no. C0035C) were obtained from ScienCell Research Laboratories, Inc., Cell Biologics, Inc. and Thermo Fisher Scientific, Inc., respectively. Ascorbic acid, glycerol-2-phosphate and dexamethasone were purchased from Sigma-Aldrich; Merck KGaA. Dulbecco's modified Eagle's medium (DMEM) with low glucose, penicillin-streptomycin, trypsin-ethylenediaminetetraacetic acid (EDTA), fetal bovine serum (FBS) and phosphate-buffered saline (PBS) were obtained from Thermo Fisher Scientific, Inc. Testosterone and testosterone-3,4-13C2 were purchased from Alta Scientific. Co., Ltd.

Preparation of 13C-enriched testosterone

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

Figure 1 Chemical structures of testosterone and testosterone-3,4-13C2. 13C, carbon-13.

Cell culture and compound intervention

Human osteoblasts, human primary 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˚C. The cells were dissociated with trypsin-EDTA and seeded in 96-well tissue culture plates at the density of 1x104 cells/well. The culture media were then changed to media with low-glucose DMEM, 10% FBS, 1% penicillin-streptomycin, 50 mg/ml ascorbic acid, 0.01 mol/l glycerol-2-phosphate and 100 nmol/l dexamethasone. The cells were cultured in the culture medium containing either testosterone or 13C-enriched testosterone at concentrations of 0, 10-10, 10-8, 10-6 and 10-5 mol/l. An untreated (no drugs; 0 mol/l) was used as a blank control. The morphology of the cells treated with testosterone and 13C-enriched testosterone at the concentration of 10-5 mol/l was also observed. The observation was conducted using an inverted microscope (XDS-500C, Shanghai Caikon Optical Instrument Co., Ltd.).

Measurement of cell proliferative activity

The proliferative activities of human osteoblasts, human primary aortic endothelial cells and human umbilical vein endothelial cells were determined using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (MTS cell proliferation colorimetric assay kit, AmyJet Scientific, Inc.) when the cells were cultured for 48 h at 37˚C. Following the manufacturer's instructions, MTS and phenazine methosulfate (PMS) solution were mixed (MTS:PMS=20:1). The culture medium and the mixed solution of MTS and PMS were added to the test well of a 96-well plate and were incubated at 37˚C for 2 h. The light absorbance of the formazan product was measured at a 490-nm wavelength using a spectrophotometer (BioTek Instruments, Inc.). The measurement for each concentration was repeated 6 times, and the average optical density (OD) value was recorded.

Measurement of the ALP level of osteoblasts

In order to further investigate the isotope effect of 13C-enriched testosterone on osteoblasts, the ALP level in osteoblasts was determined. An elevation in ALP levels serves as a marker of osteogenic differentiation (23,24). The measurement of the ALP level is based on the ALP-mediated conversion of p-nitrophenol phosphate (PNPP) to nitrophenol in an alkaline buffer (25). The product nitrophenol exhibits the light absorption at a 405-nm wavelength. In the present study, the ALP levels of human osteoblasts cultured on the 5th day with 13C-enriched testosterone at the concentrations of 0, 10-10, 10-8, 10-6 and 10-5 mol/l were determined. Following the instructions of the Alkaline Phosphatase Assay kit (TW-Reagent Industrial Co., Ltd.), the cells were lysed in 600 µl lysis buffer and the lysate was centrifuged at 1,000 x g for 20 min at 20˚C. The supernatant and PNPP solution were added to the well of a tissue culture plate and then incubated at 37˚C for 1 h. After the stop solution provided with the Alkaline Phosphatase Assay kit was added, the light absorbance was measured at wavelength of 405 nm using a microplate reader (Synergy LX, Bio-Tek Instruments, Inc.). The values of the ALP level (U/l) were recorded. The test was repeated six times, and the average value was recorded. The data were normalized to the control.

Measurement of OC secretion levels of osteoblasts

In order to investigate the isotope effect of 13C-enriched testosterone on the OC secretion of osteoblasts, the OC level of osteoblasts was examined. OC, an osteoblast-specific secreted protein, is synthesized by osteoblasts during bone formation (26). It plays key roles in both the biological and mechanical functions of bone (13,27). As a biochemical marker of osteoblast activity, the OC level reflects the rate of bone formation (28). In the present study, the OC levels of human osteoblasts cultured on the 5th day with 13C-enriched testosterone at the concentrations of 0, 10-10, 10-8, 10-6 and 10-5 mol/l were determined. Following the instructions of the ELISA kit, the OC levels in the supernatant of the culture medium were analyzed using an OC ELISA kit (cat. no. RAB1073-1KT; Sigma-Aldrich; Merck KGaA). The measurement of the OC level (µg/l) was repeated six times, and the average value was recorded. The data were normalized to the control.

Statistics analysis

Data were analyzed using IBM SPSS Statistics v26 software (IBM Corp.). The distribution of the variables was examined using the Shapiro-Wilk test, and the variance was determined using Levene's test. When the distribution was found to be parametric, the differences were assessed using one-way ANOVA with the Bonferroni post hoc test. P-values <0.05 were considered to indicate statistically significant differences. The numerical variables are presented as the mean ± SD (n=6 repeated experiments).

Results

Cell proliferative activity

The proliferative activities of the human osteoblasts, human primary aortic endothelial cells and human umbilical vein endothelial cells treated with testosterone and 13C-enriched testosterone were analyzed. The measured OD values were normalized and plotted (Fig. 2). The morphology of the cells treated with testosterone and 13C-enriched testosterone at the concentration of 10-5 mol/l is illustrated in Fig. 3.

Figure 2 Effects of testosterone and 13C-enriched testosterone on human cells. (A) Effects of testosterone and 13C-enriched testosterone on human osteoblasts. (B) Effects of testosterone and 13C-enriched testosterone on human primary aortic endothelial cells. (C) Effects of testosterone and 13C-enriched testosterone on human umbilical vein endothelial cells. The cells were treated with testosterone and 13C-enriched testosterone at various concentrations (10-10, 10-8, 10-6 and 10-5 mol/l). Data represent the mean ± SD (n=6 repeated experiments). *P<0.05, **P<0.01 and ***P<0.001, significant difference vs. control (no treatment). 13C, carbon-13.

Figure 3 Morphology of cells treated with testosterone and 13C-enriched testosterone at the concentration of 10-5 mol/l. (A) Human osteoblasts cells treated with testosterone (magnification, x100). (B) Human osteoblasts cells treated with 13C-enriched testosterone (magnification, x100). (C) Human primary aortic endothelial cells treated with testosterone (magnification, x100). (D) Human primary aortic endothelial cells treated with 13C-enriched testosterone (magnification, x100). (E) Human umbilical vein endothelial cells treated with testosterone (magnification, x100). (F) Human umbilical vein endothelial cells treated with 13C-enriched testosterone (magnification, x100). Scale bar, 100 µm. 13C, carbon-13.

Human osteoblasts

Testosterone promoted the proliferation of human osteoblasts at the concentrations of 10-10 (P<0.01) and 10-8 mol/l (P<0.05). Following treatment with supraphysiological concentrations (10-5 mol/l) of testosterone, the human osteoblasts exhibited a decreasing trend in proliferative activity (Fig. 2A).

13C-enriched testosterone did not promote the proliferation of human osteoblasts at the concentrations of 10-10-10-6 mol/l. Among these concentration groups, there were no marked differences in the effects of 13C-enriched testosterone, which suggested that the effect had no association with the concentration at the level of 10-10-10-6 mol/l. It was noted that the human osteoblasts exhibited an increasing trend in proliferative activity when treated with supraphysiological concentrations (10-5 mol/l) of 13C-enriched testosterone, although no significant differences were found (Fig. 2A).

Human primary aortic endothelial cells

Testosterone significantly promoted the proliferation of human primary aortic endothelial cells at the concentration of 10-10 mol/l (P<0.001). Following treatment with supraphysiological concentrations (10-5 mol/l) of testosterone, the human primary aortic endothelial cells exhibited a decreasing trend in proliferative activity (Fig. 2B).

13C-enriched testosterone did not promote the proliferation of human primary 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 13C-enriched testosterone, which indicated that the effect had no association with the concentration at the level of 10-10-10-6 mol/l. It was noted that the human primary aortic endothelial cells exhibited an increasing trend in proliferative activity when treated with a high concentration (10-5 mol/l) of 13C-enriched testosterone, although no significant differences were found (Fig. 2B).

Human umbilical vein endothelial cells

Testosterone significantly promoted the proliferation of human umbilical vein endothelial cells at the concentration of 10-8 mol/l (P<0.05). Following treatment with a high concentration (10-5 mol/l) of testosterone, the human umbilical vein endothelial cells exhibited a decreasing trend in proliferative activity (Fig. 2C).

13C-enriched testosterone did not increase 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 13C-enriched testosterone, which indicated that the effect had no association with the concentration at the level of 10-10-10-5 mol/l (Fig. 2C).

ALP level of human osteoblasts

Compared with the control group, 13C-enriched testosterone did not enhance the ALP level of human osteoblasts at the concentrations of 10-10 and 10-8 mol/l. The human osteoblasts exhibited an increasing trend in ALP levels when treated with a high concentration (10-6 and 10-5 mol/l) of 13C-enriched testosterone, although no significant differences were found (Fig. 4).

Figure 4 Effects of 13C-enriched testosterone on the alkaline phosphatase level of human osteoblasts. The cells were treated with 13C-enriched testosterone at various concentrations (10-10, 10-8, 10-6 and 10-5 mol/l). Data represent the mean ± SD (n=6 repeated experiments). 13C, carbon-13.

Osteocalcin secretion of human osteoblasts

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

Figure 5 Effects of 13C-enriched testosterone on osteocalcin secretion of human osteoblasts. The cells were treated with 13C-enriched testosterone at various concentrations (10-10, 10-8, 10-6 and 10-5 mol/l). Data represent the mean ± SD (n=6 repeated experiments). *P<0.05 and ***P<0.001, significant difference vs. control (no treatment). 13C, carbon-13.

Discussion

Among the biogenic elements composing 96% of the human body (29), carbon is a vital element and composes the backbone of biological macromolecules. As biochemical reactions are often accompanied by the cleavage or formation of carbon-carbon bonds, the change in carbon isotope composition potentially influences the biochemical reaction rate and exerts a biological isotope effect. Even though the carbon isotope effect is crucial to biological research and medicine, related studies are very limited (30). The present study demonstrated that there was a difference in the biological effect between 13C-enriched testosterone and testosterone. In future studies, the authors aim to elucidate the mechanisms responsible for this phenomenon by investigating other biomarkers secreted by cells and the related signaling pathways.

The growth rate is arguably the most profound phenotypic parameter that defines the existence of an organism. It integrates multiple aspects of the physiological state of a cell, and is often associated with how cells respond to drugs (31). Previous studies have established that testosterone exerts effects on cell proliferation in a concentration-dependent manner (13,15,20,21), which was supported by the findings of the present study. Of note, the effects of 13C-enriched testosterone were concentration-independent at 10-10-10-6 mol/l concentrations. This finding suggested that 13C enrichment in a drug may alter the pharmacological properties of the drug.

The concentration gradient used in the present study was designed according to the physiological levels of testosterone, which was beneficial to observe the effects in a physiological and supraphysiological state. However, higher gradients of testosterone concentration need to be applied in future studies to supplement the current findings, since a higher gradient may result in a more significant difference in the effects between testosterone and 13C-enriched testosterone.

It is considered that the enrichment of a heavy isotope attenuates the biochemical reaction rate and delays the growth of an organism due to the kinetic isotope effect (2,32-34). This view was supported by the findings of the present study using 13C-enriched testosterone at physiological concentrations; however, this view was challenged by the results obtained when using supraphysiological concentrations. 13C-enriched testosterone was found to promote cell proliferation at a high concentration (10-5 mol/l). Furthermore, 13C-enriched testosterone enhanced the OC secretion of human osteoblasts at high concentrations (10-6 and 10-5 mol/l). These findings demonstrated the polytropic characteristics of the biological isotope effects, which should be taken into account in stable isotope-based research.

Acknowledgements

Not applicable.

Funding

Funding: No funding was received.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

XW and WY contributed to the conception of the study. MZ and TZ performed the experiments. XW, MZ and WY performed the data analyses and wrote the manuscript. XW, MZ and WY confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References