Abstract
Many kinds of physical fields are able to affect biological activity, such as electromagnetic field, magnetic field, scalar wave, magnetic vector potential and so on. Some works investigated a high-penetrating emission (HPE) generated by a LED generator that can affect the conductivity of water and the growth of dry yeast. In this research, the biological effect of the HPE of LED generator on S. cerevisiae was studied. The results showed that this kind of HPE could promote the growth of S. cerevisiae. With the decrease of initial concentration and the extension of exposure time, the growth promoting effect of HPE increased significantly. When the initial cell concentration was 2 × 102 cell/ml and exposed for 24 h, the number of yeast cells increased by 19.5% compared with the control group. Further studies showed that adenosine triphosphate (ATP) content and the ratio of ATP and adenosine diphosphate (ADP, ATP/ADP ratio) of exposed cells were higher than those of unexposed cells, indicating that exposed cells had higher cell viability and energy status. In addition, the intracellular superoxide (O2 −) content decreased significantly after the HPE exposed. In this study, the stainless steel box acts as a Faraday cage to shield the electromagnetic field, so the biological effect of the HPE is not produced by the electromagnetic field. Nevertheless, the mechanism of the HPE is still unknown and needs further research. This is the first report on the effect of HPE on mitochondrial parameters, contributing to further study on HPE and revealing potential for future medical applications.
Keywords
Introduction
Many kinds of physical field are able to affect the biological activity, such as electromagnetic field, magnetic field, scalar wave, magnetic vector potential and so on. The electromagnetic field can induce intracellular superoxide accumulation and affect the growth and viability of Saccharomyces cerevisiae (S. cerevisiae) [1]. The magnetic field can affect Insulin Secretion in INS Cells [2]. Meyl found that magnetic scalar wave could trigger higher ATP-levels and extend the aging process of plants [3]. Variation of vector potential can change the biological properties of water, so as to change the mobility of infusoria, the rate of sugar fermentation in yeast cells and the drinking attractivity of water for mice [4]. Researchers found that the torsion field generated by spin polarization in the nickel-manganese ferrite affect the exclusion zone of water [5]. Bobrov A.V. found that polarized electrodes were sensitive to the high-penetrating emission (HPE) generated by laser and LED generator [6,7]. Kernbach S. also proved that the HPE of LED generator could change the conductivity of water [8]. Bobrov’s study demonstrated that this kind of HPE also affected the growth of dry yeast cells. His study showed that the biological effect of this HPE wasn’t produced by electromagnetic field because the electromagnetic field was shielded by stainless steel boxes which acted as a Faraday cage [9]. He carried out a series of experiments to determine the main parameters of the LED generator in order to achieve the best biological effect on yeast cells. Nevertheless, the possible mechanism needs further study.
Superoxide is one type of reactive oxygen species (ROS) that forms in all living organisms that come into contact with air. Based on its biological context, superoxide can be used as a signaling agent, a toxic chemical, or a harmless intermediate that decomposes spontaneously [10–12]. Adenosine triphosphate (ATP) exerts crucial biological function in the vital movement, and it is the direct energy source of life. It converts with adenosine diphosphate (ADP) to store or release biological energy. The ATP level change may provide information about physiological activity and metabolic process of cells and even organisms [13]. For example, intracellular ATP level was measured to assess cell proliferation and cytotoxicity since all cells have an absolute requirement for ATP to maintain their viability [14]. And ATP concentration is a switch in the decision between apoptosis and necrosis of cells [15]. Meanwhile, the ATP/ADP ratio is also a central control parameter of cellular energy metabolism and the driving force for many reactions [16]. In glucose metabolism, for example, the ATP/ADP ratio controls whether the reaction flux through phosphoglycerate kinase is in the glycolytic or gluconeogenic direction. Researchers found that ATP/ADP ratio is an important factor in control of DNA supercoiling [17]. The ratio can also stimulate substrate oxidation [18], and be used as an indicator of energy consumption and changes in cellular energy status [19,20].
Yeast has been used as a model organism for its rapid growth, easy cultivation and feasible genetic modification [21]. In this research, we investigated the effect of the HPE of LED generator on the growth of S. cerevisiae in a very precise and rigorous manner, and the intracellular O2 − and ATP/ADP ratio were also examined.
Material and methods
Strain and media
The industrial instant dry yeast S. cerevisiae (ANGEL YEAST CO. LTD., China) was used in this study. All glassware and medium were autoclaved at 121 °C for 15 min before experiment. The yeast strain was cultured in YPD (2% peptone, 1% yeast extract, and 2% D-(+)-glucose) to logarithmic phase.
LED generator
The LED used in this generator was normal but worked in special mode of high-forward-voltage pulses, which could generate HPE besides the electromagnetic component. The LED generator (Fig. 1A, 1B), working in digital activation mode, consisted of 168 blue-light LEDs (emission band: 455–465 nm, diameter: 5 mm) arranged in a circle area of 140 ∗ 140 mm2. The Micro Controller Unit (MCU) used in this device was STC15W408AS, made by STCMCU in China. The 168 LEDs were controlled by 2 MOSFETs (model IRF510). The power supply was a 12 V lithium (Li) battery (Fig. 1C). The MCU sent the control signal to the Metal-Oxide -Semiconductor Field Effect Transistors (MOSFETs) to control the blink process of LED. The frequency of the signal was 3000 Hz with 1 μs positive pulse in each period. The LED generator was placed in a black plastic box to avoid the influence of photons (Fig. 1C).
The LED generator and schematic view of the set-up. ((A) Structure of the LED generator. (B) The LED generator consist of 169 blue-light LEDs. (C) The schematic view of the set-up.)
The S. cerevisiae cells (2 × 106 cells/ml) were inoculated into a 100 ml triangular flask containing 40 mL YPD and incubated for 8 h at 30 °C to logarithmic phase. After centrifugation at 4,000 rpm and 25 °C for 3 min, different amounts of yeast cells were resuspended in 50 ml flasks with 20 mL of fresh YPD, making their initial concentrations 2 × 102, 2 × 103, 2 × 104, 2 × 105 and 2 × 106 cells/ml, respectively. The schematic view of the set-up is shown in Fig. 1C. The flasks were placed in stainless steel boxes to eliminate electromagnetic field effect. The stainless steel boxes here acted as a Faraday cage, although the boxes were not connected to the ground, because the boxes were closed, the electric potential around the boxes was all equal. The distance between samples and the LED generator was about 6 cm. The probe of the electronic thermometer extends into the stainless steel box to ensure that the temperature and humidity of the experimental group is the same as that of the control group. The only difference between the sample and control group was that there was no LED generator in the control group. After the cells were exposed to the HPE of LED generator and incubated at 25 °C, samples were withdrawn at different time for subsequent detection. All samples were made in triplicate. Finally, the cell number of the samples were measured with a blood cell counting plate. The growth promoting rate was calculated as follows: growth promoting rate = (number of cells in exposure group/number of cells in control group −1) × 100%.
Detection of superoxide
The S. cerevisiae cells were stained with dihydroethidium (Sigma) to quantitatively evaluate intracellular superoxide accumulation. Briefly, 100 μL solution of HPE exposure sample and non-exposure control were inoculated respectively into 96-well plates (Costar, USA) with 30,000 cells per well in triplicate. The dihydroethidium was added to each well at a final concentration of 5 μg/mL. After incubation for 10 min, the fluorescence intensity was measured with a microplate reader (Infinite M200, TECAN) at excitation wavelength of 300 nm and emission wavelength of 610 nm.
High performance liquid chromatography analysis for ATP and ADP
The content of ATP and ADP in yeast cells were determined by high performance liquid chromatography (HPLC) as Sumi et al described with modification [22]. Briefly, 100 μL sample was transferred to Eppendorf tubes and centrifuged for 5 min at 10,000 rpm and 4 °C. The sediment was treated with 360 μL of 6% perchloric acid solution (sigma) at 0 °C for 10 min and then centrifuged at 10,000 rpm and 4 °C for 5 min to precipitate proteins. 40 μL K2CO3 (2M) was added to 300 μL of the supernatant to neutralize the pH. After filtration, the sample solutions were analyzed by a Venusil MPC18 column (4.6 mm × 250 mm, 5 μm) using a HPLC system (Agilent Technologies, 1260 Infinity). The solution of 0.1 M KH2PO3 and methanol (95/5, V/V) was applied as the mobile phase. The flow velocity was 0.6 mL/min. The detection wavelength was at 254 nm. The cell number of each sample was determined with blood cell counting plate, and ATP and ADP contents per cell were calculated.
Statistical analysis
Each experiment was performed three times and the quantitative results were expressed as mean ± standard deviation. Differences between the means were analysed by using independent t-test. P < 0.05 indicates a statistically significant difference.
Results
Effects of HPE on growth of yeast cells
S. cerevisiae cells were exposed to the HPE of LED generator and the effect of electromagnetic fields was eliminated by stainless steel boxes. Through experimental design, the effects of initial concentration and exposure time were studied comprehensively in one experiment, that is, the exposure time of high initial concentration was shorter, and the exposure time of low initial concentration was longer. Meanwhile, the control group was cultured at the same cultivate condition except for the HPE of LED generator exposure. The results showed that the final concentrations in all HPE exposed groups were significantly higher than those of the control groups (Fig. 2A), indicating that HPE promoted the growth of yeast cells. Sample with the highest initial concentration of 2 × 106 cell/mL was exposed for 6 h, and the growth promoting rate was only 3.4% (Fig. 2B). Samples with initial concentration of 2 × 105 and 2 × 104 cell/mL were all exposed for 16 h, and the growth promoting rates were 7.3% and 12.0%, respectively. Cells with initial concentration of 2 × 103 and 2 × 102 cell/mL were all exposed for 24 h, the growth promoting rates were 13.4% and 19.4%, respectively. The results showed that the lower the initial concentration and the longer the exposure time, the higher the growth promoting rates (Fig. 2B). Compared with the control group, the final cell number increased by 19.4% at the lowest initial concentration and the longest exposure time. It was likely that the initial cell concentration and exposure time worked co-ordinately on the growth of the exposed cells, though their contribution ratio could not be figure out from the obtained data.
The HPE effect on the growth of S. cerevisiae cells. ((A) The final concentrations of samples with different initial concentration after being exposed to the HPE of LED generator for specified exposure time. ∗∗ P < 0.01 and ∗ P < 0.05 at all testing times, indicated statistically significant differences between the exposure group and the control group. (B) The growth promoting rate, calculated as: growth promoting rate = (number of cells in sample group/number of cells in control group −1) × 100%). Values represent the mean ± SD of three independent experiments.
O2 − is a major part of ROS and an important indicator of cell functional state. Since the highest growth promoting effect was obtained in the sample with an initial concentration of 2 × 102 cell/mL, the accumulation of O2 − in these yeast cells was measured. The O2 − fluorescence intensity of the exposure group cells was 17.1% lower than that of the control group cells (Fig. 3), indicating that HPE reduced the accumulation of O2 − in yeast cells.
Fluorescence intensity of O2 − in S. cerevisiae cells in the control group and the HPE exposure group. (∗ p < 0.05 indicated statistically significant differences between the exposure group and the non-exposure group. Values represent the mean ± SD of three independent experiments.)
ATP is the direct energy source of organisms, and it converts with ADP to store or release biological energy. Since HPE exposure significantly promoted the growth of yeast cells, the ATP content and the ATP/ADP ratio were measured in the yeast cells with an initial concentration of 2 × 102 cell/mL. The ATP content of each cell in the HPE exposure group was 0.4 pg/cell, which was much higher than that of the control group (0.2 pg/cell), while the ADP contents of the two kinds of cells were almost at the same level (Fig. 4). These results suggested that the HPE exposure cells had much higher energy levels.
ATP, ADP content and ATP/ADP ratio of cells in the control group and the HPE exposure group. (∗ p < 0.05, indicated statistically significant differences of ATP content between the exposure group and the non-exposure group. Values represent the mean ± SD of three independent experiments.)
Though the researchers found that the HPE of the LED generator had effects on electrode, water and even microorganism, more precise and comprehensive studies are needed to support the results. In this research, we studied the influence of the HPE of LED generator on the growth of S. cerevisiae. Furthermore, we measured the O2 − accumulation and ATP/ADP ratio to analyse the possible mechanism. The results showed that HPE of LED generator had a significant growth-promoting effect on yeast cells, which was consistent with the research of Bobrov [9]. The growth promoting effect was enhanced with the decrease of the initial cell concentration and extension of the exposure time. Compared with the control group, the accumulation of O2 − in yeast cells exposed to HPE was significantly decreased, while ATP content and ATP/ADP ratio were increased.
Large amounts of ATP are generated mainly by oxidative phosphorylation in mitochondria, which results in the conversion of oxygen to the O2 −, H2O2 and related ROS [23]. The cross-talk of NO, O2 − and O2 cooperatively regulates the circulation and energy metabolism [24]. The outer orbit of O2 − has an odd number of electrons and unpaired spins. O2 − is converted to H2O2 and then to H2O by superoxide dismutases (SODs) and glutathione peroxidase (GPx) respectively. One possible mechanism is that the HPE alters the spin state of the electrons and accelerates the conversion of O2 −, which leads to higher activity of mitochondrial, with higher ATP production and ATP/ADP ratio. ATP has distinct scavenging effect on O2 −, and a certain protective effect on oxidized haemoglobin inclusions of human erythrocytes [25]. Higher ATP content may accelerate the scavenging of O2 − and make the yeast cells more active. Cellular energy status is not only important for cell health, but also for regulating signal activity. ATP/ADP ratio can be used as an indicator of cell viability, necrosis and apoptosis in human leukaemic cell lines [26]. Compared with the unexposed cells, the HPE exposed cells have higher ATP content and ATP/ADP ratio, indicating that the cells have higher vitality and energy status, which can trigger yeast to increase its growth.
On the other hand, each yeast cell contains a lot of water and is surrounded by water in the culture medium. Since the HPE of LED generator can change the conductivity of water [8], the nutrient uptake and metabolite excretion rate of cells to the surrounding environment, as well as the biochemical reaction rate in cells can be changed. These changes may determine the effect of the HPE on each yeast cell, and the effect of all cells adds up to a growth-promoting effect on the yeast population. Because the power input of the LED generator comes from a 12 V Li battery and remains stable, we can infer that its output is stable too. Thus, each yeast cell can share more HPE and achieve higher growth-promoting effect under the condition of fewer inoculated cells and longer exposure time.
Conclusion
In conclusion, the HPE of LED generator can promote the growth of S. cerevisiae. Further studies have shown that the ATP content and ATP/ADP ratio of exposed cells were higher than those of unexposed cells, which could reduce the O2 − content in yeast cells, especially in mitochondria. Till now, the possible mechanism of the HPE of LED generator is still unknown and needs further study. Nevertheless, based on the beneficial biological effects on yeast cells, the HPE of LED generator can be considered as a new approach to complementary medicine.
Footnotes
Acknowledgements
This work was supported by the ENN Research Fund. We sincerely thank Y. J. Chen for the ATP measurement.
Compliance with ethical standards
The authors report no conflicts of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.