Antioxidant Activity of Ge-132, a Synthetic Organic Germanium, on Cultured Mammalian Cells

Abstract

Ge-132 is a synthetic organic germanium that is used as a dietary supplement. The antioxidant activity of Ge-132 on cultured mammalian cells was investigated in this study. First, Ge-132 cytotoxicity on mammalian cultured cells was determined by measuring lactate dehydrogenase (LDH) levels. Ge-132 had no cytotoxic effect on three different cell lines. Second, the cell proliferative effect of Ge-132 was determined by measuring ATP content of whole cells and counting them. Ge-132 treatment of Chinese hamster ovary (CHO-K1) and SH-SY5Y cells promoted cell proliferation in a dose-dependent manner. Finally, antioxidant activity of Ge-132 against hydrogen peroxide-induced oxidative stress was determined by measuring the levels of intracellular reactive oxygen species (ROS) and carbonylated proteins. Pre-incubation of CHO-K1 and SH-SY5Y cells with Ge-132 suppressed intracellular ROS production and carbonylated protein levels induced by hydrogen peroxide. Our results suggest that Ge-132 has antioxidant activity against hydrogen peroxide-induced oxidative stress.

Germanium exists in soil, animals, and plants as a natural compound, and is also found in mushrooms, garlic, onion, and ginseng.¹⁾ Bis-(carboxyethylgermanium) sesquioxide (Ge-132) is organic germanium, and was first synthesized in 1967²⁾ and used as a dietary supplement. The inorganic form of germanium is insoluble in water, and causes several side effects such as nephrotoxicity, neurotoxicity, and even death.³⁾ Ge-132 is soluble and has low toxicity.³⁾ The structure of Ge-132 is shown in Fig. 1. In vitro studies show that Ge-132 has many biological activities including elevating interferon γ production,⁴⁾ activating natural killer cells and macrophages,^5,6) and modulating the immune system.⁷⁾ Further, in vivo studies show that it has antiviral,⁸⁾ antimicrobial,⁹⁾ analgesic,¹⁰⁾ and antitumorigenic activity, and inhibits cancer metastasis.^11,12) Moreover, antioxidant activity of Ge-132 has been suggested by several reports, including a murine model of accelerated aging¹³⁾ and low-density lipoprotein oxidation in a spontaneous familial hypercholesterolemic model rat,¹⁴⁾ porcine oocytes,^15,16) in monkey liver preparation.¹⁷⁾ Antioxidant activity of Ge-132 on the bile in rodents was also reported.¹⁸⁾ Nevertheless, direct evidence using generally used cultured mammalian cells has not yet been provided. In this study, we examined the possible effect on cell proliferation of Ge-132 and its antioxidant activity against hydrogen peroxide-induced oxidative stress.

Fig. 1. Structure of Ge-132

MATERIALS AND METHODS

Materials

Ge-132 (purity >99%) was provided by SNOWDEN Co., Ltd. (Tokyo, Japan), and solubilized with distilled water before use in the following studies. Ge-132 supplied from Sigma-Aldrich (St. Louis, MO, U.S.A.) was also used (Figs. S1, S2). Rabbit polyclonal anti-dinitrophenyl (DNP) antibody (SHIMA Laboratories, Tokyo, Japan) was used to detect carbonylated proteins.

Cell Culture

Chinese hamster ovary (CHO-K1) cells, HeLa cells, and SH-SY5Y cells were maintained in Dulbecco’s modified Eagle medium (DMEM) medium (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO₂.

Cytotoxicity

Cytotoxicity of Ge-132 was determined by measuring lactose dehydrogenase (LDH) levels. The three different cell lines described above were seeded in 12-well plates (1×10⁵ cells/well), and treated with medium containing 0–5 mM Ge-132 for 24 h. LDH activity was determined using a LDH assay kit, CytoTox 96 (Promega Co., Madison, WI, U.S.A.), according to the manufacturer’s instructions with modification. LDH activity was reported as fold induction compared with control cells without Ge-132 treatment as reported previously.^19,20) Absorbance was measured using a plate reader (Powerscan HT, DS Pharma Biomedical, Osaka, Japan).

Cell Proliferation

The effect of Ge-132 on cell proliferation was examined. CHO-K1 or SH-SY5Y cells (5×10³ cells/well) were seeded into 96-well dishes, and treated with medium containing 0–5 mM Ge-132. After 24 h incubation, the effect of cell proliferation was measured by determining ATP content of whole cells using a luciferase assay kit (CellTiter Glo, Promega Co.), according to the manufacturer’s instructions. Luminescence was measured using a luminometer (Powerscan HT; DS Pharma Biomedical). Luciferase activity was reported as fold induction compared with control cells without Ge-132 treatment. The effect of Ge-132 on cell proliferation was also examined by counting SH-SY5Y cell number using a hemocytometer.

Oxidative Stress

To determine the effect of Ge-132 on oxidative stress, CHO-K1 cells or SH-SY5Y cells (10×10⁴ cells/well) were pre-incubated with Ge-132, and production of reactive oxygen species (ROS) after hydrogen peroxide treatment measured. CHO-K1 cells were seeded in 12-well plates, and pre-incubated with or without 5 mM Ge-132 for 24 h. After Ge-132 was washed off, cells were treated with 3 mM hydrogen peroxide for 2 h. Levels of intracellular ROS production were determined using CM-H₂DCFDA (Life Technologies, Carlsbad, CA, U.S.A.), according to the manufacturer’s instructions. Fluorescence of CM-H₂DCFDA was measured using a plate reader (Powerscan HT; DS Pharma Biomedical). The level of total carbonylated proteins, a marker of oxidative stress, was also detected by Western blotting using antibody against DNP.²¹⁾ Total protein extracts were prepared from cultured cells, as described previously.¹⁹⁾ The Western blotting protocol was performed as described previously²²⁾ with minor modification.²³⁾ The intensities of the total bands detected by DNP antibodies were quantified using Image J software (NIH, Bethesda, MD, U.S.A.).

Statistical Analysis

In all experiments, data are expressed as mean±standard deviation (S.D.) from three to five independent experiments. Data were analyzed using Student’s t-test and ANOVA followed by Dunnett’s test, and differences between samples considered statistically significant when * p<0.05.

RESULTS

Ge-132 Cytotoxicity

First, cytotoxicity of Ge-132 on CHO-K1, HeLa, and SH-SY5Y cells was evaluated by measuring LDH levels. CHO-K1 cells were treated with various concentrations of Ge-132 and cytotoxicity determined. LDH levels correlate to an increase in cell death. LDH levels were at similar levels with no significant increases observed within the concentration range of 0–5 mM (Fig. 2). This suggests that Ge-132 does not have a cytotoxic effect on the three cell lines examined.

Fig. 2. Ge-132 Has No Cytotoxic Effect on CHO-K1, HeLa, and SH-SY5Y Cells

Cytotoxicity of Ge-132 was determined in CHO-K1, HeLa, and SH-SY5Y cells by lactate dehydrogenase (LDH) release into culture medium. No cytotoxic effect of Ge-132 was observed at any concentration. Quantification of LDH release ratio is represented by mean±S.D. (n=3).

Effect of Ge-132 on Cell Proliferation

Next, the cell proliferative effect of Ge-132 was determined by measuring the ATP content of whole cells and counting them. CHO-K1 and SH-SY5Y cells were treated with various concentrations of Ge-132, and cell proliferation rates determined. Ge-132 treatment increased the ATP content of CHO-K1 cells at each concentration, with 5 mM Ge-132 increasing the cell proliferation rate up to 120% (Fig. 3a). Moreover, the ATP content of Ge-132 (5 mM)-treated CHO-K1 cells was significantly increased compared with non-treated cells (Fig. 3a, ** p<0.01). The increase by Ge-132 was dose-dependent (Fig. 3a). Ge-132 treatment also increased the ATP content of SH-SY5Y cells at each concentration, with 5 mM Ge-132 increasing the cell proliferation rate up to 150% (Fig. 3b). The ATP content of Ge-132-treated SH-SY5Y cells was significantly increased compared with non-treated cells (Fig. 3b, * p<0.05). These results suggest that Ge-132 has an effect on cell proliferation in two different cell lines. The cell proliferative effect of Ge-132 was confirmed by cell counting. The proliferation rate of Ge-132-treated SH-SY5Y cells was increased compared with non-treated cells, with the increase being dose-dependent (Fig. 3c, * p<0.05). The proliferation rate of Ge-132-treated CHO-K1 cells was also tended to be elevated, but not statistically significant (Fig. S1).

Fig. 3. Ge-132 Increases Cell Proliferation of CHO-K1 and SH-SY5Y Cells

(a, b) ATP content was determined by luciferase assay. The amount of ATP significantly increased in Ge-132-treated CHO-K1 (a) and SH-SY5Y cells (b) compared with non-treated cells (* p<0.05, ** p<0.01). Quantification of ATP content is represented by mean±S.D. (n=3). (c) Cell proliferation rates were determined by cell counting. The rate of proliferation significantly increased in Ge-132-treated SH-SY5Y cells. Quantification of cell proliferation rates are represented by mean±S.D. (n=3).

Antioxidant Effect of Ge-132 in Cultured Mammalian Cells

Finally, we measured the antioxidant activity of Ge-132 against hydrogen peroxide-induced oxidative stress. Hydrogen peroxide treatment of CHO-K1 cells caused a significant increase in intracellular ROS levels (approximately 1.2-fold increase) compared with non-treated cells (Fig. 4a, * p<0.05). Pre-incubation of CHO-K1 cells with Ge-132 significantly suppressed the increase of intracellular ROS induced by hydrogen peroxide (Fig. 4a, * p<0.05). These results indicate that Ge-132 has antioxidative activity in CHO-K1 cells, as reported in porcine oocytes.^15,16) This antioxidant effect was confirmed in SH-SY5Y cells (Fig. 4b). Pre-incubation of SH-SY5Y with Ge-132 suppressed ROS induced by hydrogen peroxide. We confirmed the results using Ge-132 supplied from the common chemical vendor (Fig. S2). Oxidative stress is associated with the formation of oxidatively modified biomolecules, including proteins, lipids, and DNA.^24–26) Consequently, carbonylated protein levels are a marker of oxidative stress, and were detected by Western blotting using a DNP antibody. Hydrogen peroxide treatment of SH-SY5Y cells caused a significant increase in carbonylated protein levels (approximately 1.5-fold increase) compared with non-treated cells (Fig. 5, * p<0.05). Carbonylated protein levels were significantly reduced in total cellular extracts of Ge-132-treated SH-SY5Y cells (* p<0.05, Fig. 5).

Fig. 4. Ge-132 Suppresses Intracellular ROS Production in CHO-K1 and SH-SY5Y Cells Induced by Hydrogen Peroxide

Generation of intracellular reactive oxygen species (ROS) in CHO-K1 and SH-SY5Y cells was measured using the ROS-sensitive fluorescent dye, CM-H₂DCFDA. Ge-132 treatment significantly suppressed ROS production induced by hydrogen peroxide in CHO-K1 [(a) * p<0.05] and SH-SY5Y [(b) * p<0.05] cells. Quantification of ROS production is represented by mean±S.D. (n=5).

Fig. 5. Ge-132 Suppresses Carbonylated Protein Levels in SH-SY5Y Cells Induced by Hydrogen Peroxide

Levels of carbonylated proteins, a marker of oxidative stress, were detected by Western blotting using an antibody against 2,4-dinitrophenol (DNP). Carbonylated protein levels produced by oxidative stress were significantly suppressed in total cellular extracts of Ge-132-treated SH-SY5Y cells compared with control cells (* p<0.05). Quantification of carbonylated protein levels are represented by mean±S.D. (n=4).

DISCUSSION

Ge-132 has several immunological effects including induction of interferon, activation of natural killer cells, and inhibition of tumor growth, metastasis, and macrophages.²⁷⁾ Previous studies indicate that Ge-132 is nontoxic to CHO-K1 cells,²⁸⁾ and we also confirm that Ge-132 has no toxicity on CHO-K1, HeLa, and SH-SY5Y cells in our LDH assay. Notably, we show here that Ge-132 has a cell proliferative effect, as evident from the increase in intracellular ATP content in cultured cells. This cell proliferative effect suggests that Ge-132 is effective in promoting cell metabolism in human cells. Thus, it will be of interest to examine the effect in a human keratinocyte cell line. SH-SY5Y cells showed reduction of relative ratio of LDH release at the concentration of 5 mM Ge-132 in Fig. 2. Because only SH-SY5Y is a neuronal originated cell line used in this study, the possibility that Ge-132 has neuroprotective effect with antioxidant activity. Therefore, it is necessary to identify such neuroprotective effects in future studies. As described above, inhibition of tumor growth by Ge-132 might be observed only in cancer-derived cells lines, such as HepG2 cells.²⁹⁾

We also investigated the possible effect of Ge-132 on intracellular oxidative stress induced by hydrogen peroxide. Previously, Ge-132 was reported to have an effect in increased oxidative stress models such as paraquart poisoning and low-density lipoprotein oxidation.^13,14) Here, we show that Ge-132 is effective against oxidative stress induced by hydrogen peroxide. Hydrogen peroxide induces oxidative stress and generates ROS in cultured cells. Oxidative stress is thought to be associated with many chronic diseases such as cancer, diabetics, cardiovascular disease, inflammation, aging, and other neurodegenerative diseases in humans. Previous studies have shown that ROS enhance experimental metastasis of tumor cells.^30,31) Therefore, the antioxidant effect of Ge-132 may be associated with an anti-metastatic mechanism.

The maillard reaction (specifically, the advanced glycation end product (AGE) formation process) induces ROS production.³²⁾ Ge-132 is reported to prevent this reaction.³³⁾ In the present study, our data supports a more direct antioxidant effect of Ge-132. The mechanism of antioxidant effect of Ge-132 remains unclear. Electron scavenging activity of Ge-132 was suggested as one of the major mechanisms.¹⁶⁾ Ge-132 has a unique chemical structure with Ge-C bonds. Electron transfer may occur between Ge and free radicals.¹⁶⁾ Indeed, our results suggest that Ge-132 has potential as an antioxidant supplement by protection of cells from oxidative damage.³⁴⁾ Generally, anti-oxidants such as ascorbic acids and polyphenols are added at µM-order concentrations to cultured cells. Anti-oxidative activity of Ge-132 is not better than other anti-oxidants at the moment. Therefore, it is necessary to identify effective Ge-132 derivatives in future studies.

Acknowledgments

This work was funded by Leda Co., Ltd., a manufacturer of e.g., health equipment, instruments for beauty treatment. The sponsor had no control over interpretation, writing, or publication of this study.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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