The biological effects of electromagnetic waves are widely studied, especially due to their harmful effects, such as radiation-induced cancer and to their application in diagnosis and therapy. However, the biological effects of sound, another physical agent to which we are frequently exposed have been considerably disregarded by the scientific community. Although a number of studies suggest that emotions evoked by music may be useful in medical care, alleviating stress and nociception in patients undergoing surgical procedures as well as in cancer and burned patients, little is known about the mechanisms by which these effects occur. It is generally accepted that the mechanosensory hair cells in the ear transduce the sound-induced mechanical vibrations into neural impulses, which are interpreted by the brain and evoke the emotional effects. In the last decade; however, several studies suggest that the response to music is even more complex. Moreover, recent evidence comes out that cell types other than auditory hair cells could response to audible sound. However, what is actually sensed by the hair cells, and possible by other cells in our organism, are physical differences in fluid pressure induced by the sound waves. Therefore, there is no reasonable impediment for any cell type of our body to respond to a pure sound or to music. Hence, the aim of the present study was to evaluate the response of a human breast cancer cell line, MCF7, to music. The results' obtained suggest that music can alter cellular morpho-functional parameters, such as cell size and granularity in cultured cells. Moreover, our results suggest for the 1 st time that music can directly interfere with hormone binding to their targets, suggesting that music or audible sounds could modulate physiological and pathophysiological processes.
Keywords: Cell viability, flow cytometry, MCF7, music
|How to cite this article:|
Lestard Nd, Valente RC, Lopes AG, Capella MA. Direct effects of music in non-auditory cells in culture. Noise Health 2013;15:307-14
| Introduction|| |
The biological effects of electromagnetic waves are widely studied due to their harmful effects, such as radiation-induced cancer, and to their application in diagnosis and therapy. However, the scientific community has considerably disregarded the biological effects of sound, another physical agent to which we are frequently exposed.
Sound waves can be separated into three classes: infrasound (10−4 -20 Hz), audible sound (20-10 4 Hz) and ultrasound (2 × 10 4 -10 12 Hz). The biological effects of ultrasound, largely used in diagnosis and therapy have been extensively investigated in recent decades. , Although the mechanisms by which infrasound induces biological effects are still not clear, its biological effects have also been studied due to possible harmful effects. ,,, However, when considering the audible frequencies, although a number of studies suggest that emotions evoked by music may be useful in medical care, alleviating stress and nociception in patients undergoing surgical procedures as well as in cancer and burned patients, ,,,,, little is known about the mechanisms by which these effects occur.
Virtually all studies consider the stimulation of the ear cells as the unique way sound produces biological effects. It is generally accepted that the mechanosensory hair cells in the ear transduce the sound-induced mechanical vibrations into neural impulses, which are interpreted by the brain and evoke the emotional effects described so far and it is believed that the reactions to music are subjective and dependent on individual experience.  In the last decade, however, several studies suggest that the response to music is even more complex. For instance, it was shown that music alters the release of hormones and cytokines related to well-being  and that cardiovascular responses to music were similar in different subjects, regardless of musical training, practice, or personal taste,  contrasting with the common belief that reactions to music are secondary to emotional responses. Moreover, recent evidence comes out that cell types other than auditory hair cells could response to audible sound.
In this way, Landstrom et al.,  observed that, although hearing was the primary sense for detecting the presence of sound at low and infrasonic frequencies, an additional way of sensation connected to vibration occurs at levels that are only 20-25 dB above the hearing threshold. More recently, Moller and Pedersen  suggested that vibrotactile sensations and a feeling of pressure might also occur in the chest and throat.
By using human gingival fibroblasts in culture, Jones et al.,  showed that a frequency of 261 Hz altered the growth such cells and Zhao et al.,  showed that sound-wave stimulation made significant changes to protein structure of tobacco cells, producing an increase in α-helix and a decrease in β-turn. Xiujuan et al.,  showed a sound stimulation effect on cell cycle of chrysanthemum an effect also observed by Zhao et al.,  in the callus growth of Dendranthema morifolium. More recently, Ying et al.,  showed that the tonal sound of 5 kHz gave significant increase in cell number of Escherichia coli bacteria and Shaobin et al.,  observed that a frequency of 1 kHz also promoted the growth of E. coli.
Jαuregui-Huerta et al.,  recently made interesting observations, such as an increase in corticosterone serum levels after environmental noise exposure. They also showed a long-term reduction of proliferating cells in the hippocampal formation of noise-exposed rats, suggesting that chronic environmental noise exposure at young ages produces persistent non-auditory impairment that modifies cell proliferation in the hippocampal formation.
Though those few studies demonstrated the effects of audible sound in non-auditory cells, research in this area is growing fast, and we cannot forget that the proteins involved in mechanotransduction are the same in virtually all cells of our organism.
Human ears assemble distinct sound vibrations and integrate a response that is transmitted to the brain, which in turn recognizes such stimuli as being music, sound or noise. However, what is actually sensed by the hair cells, and possible by other cells in our organism are physical differences in fluid pressure induced by the sound waves. Therefore, considering that:
- Sound is a mechanical wave, causing perturbation in the medium
- Water is a very good conductor of sound (the velocity of sound in water is about 4 ~ 5 times that of in the air)
- Water is the main component of cells and body fluids
- Music can be characterized as the sum of pure sound frequencies
- Almost all cells in our organism are made of the same molecules (proteins, lipids, nucleic acids) and the mechanotransduction machinery is roughly the same regardless the cell type.
There is no reasonable impediment for any cell type of our body to respond to a pure sound or to music. Therefore, the aim of the present study was to evaluate the response of a human breast cancer cell line, MCF7, to music. The parameters studied were some of that described in the above mentioned papers, such as cell proliferation, cell cycle and cell volume.
| Methods|| |
MCF7 is a human breast cancer cell line with characteristics of epithelial cell. The cell line was obtained from Rio de Janeiro Cell Bank and were grown in Dulbecco's Modified Eagle Medium with penicillin and streptomycin and supplemented with 10% fetal bovine serum (all from Invitrogen, Brazil) in disposable plastic bottles (Techno Plastic Product TPP, Germany), at 37°C, until confluence. For each experiment, cells were platted on 40 mm plastic Petri dishes (TPP, Germany), at 1 × 10 5 cells/dish. In the experiments observing cell volume 1 × 10 6 cells/dish were platted. The experiments were performed 24 h after seeding, to ensure uniform attachment of the cells. Each experiment was repeated at least 4 times.
Treatment with music
The cells were exposed for 30 min to one of the three compositions: Mozart's Sonata for Two Pianos in D major, KV. 448, first movement; Beethoven's 5 th Symphony, first movement; Ligeti's Atmosphere, first movement; at 37°C in an incubator chamber. The cells were exposed to the music using four speakers surrounding symmetrically the Petri dishes, which were suspended by a little platform, in order not to contact the floor. As controls, the cells were exposed to silence (no speakers in the incubator) or to the speakers plugged to the computer without any sound produced, to observe a possible action of the background noise or the magnetic field produced by the speakers. Since Beethoven's Fifth and Ligeti's Atmospheres move continually from piano to forte and vice-versa, there is no way to imprint a constant sound pressure. Therefore, the sound pressure levels were maintained between 70 and 100 dB to all compositions.
Flow cytometry analyses
Cell cycle was evaluated by flow cytometry. Forty-eight hours after exposition of cells to music or silence, the supernatant of MCF7 cultures were collected into conical tubes (in order to collect suspension cells) and the remained attached cells were washed 2 times with phosphate buffered saline PBS, harvested with trypsin and collected into the same conical tube. These tubes were centrifuged 5 min (650 × g ), the supernatant was discarded and the pellet was resuspended in 240 μL of Hanks Balanced Salt Solution (Sigma-Aldrich), homogenized and transferred to flow cytometry tubes. The cells were then stained with 10 μg/ml propidium iodide (Sigma-Aldrich) containing 1 mg/ml RNase A (Sigma-Aldrich, USA) at 37°C for 20 min in the dark and analyzed with a FACScan flow cytometer (Beckton and Dickinson, USA) and Summit 4.3 software (Dako, USA).
Flow cytometry also allows the study of some biological variables, such as cell size and cell complexity or granularity, by measuring the forward scattering (FSC) and side scattering (SSC) parameters, respectively. Therefore, flow cytometry was employed to evaluate those parameters as well. In this case, immediately after exposed to music or to silence, MCF7 cells were washed 2 times with PBS, harvested with trypsin, and kept on ice until FSC and SSC were measured by flow cytometry in a FACScan device (Beckton and Dickinson, USA).
Trypan blue assay
Forty-eight hours after have been exposed to music or silence, the cells were washed 2 times with PBS and harvested with trypsin. Then, the cell number and viability were measured by Trypan blue assay.
Binding of ouabain (OUA) to Na-K-ATPase
The cells were exposed to music or to silence in the presence of a fluorescent analog of OUA (Oua-Bodipy [OUABDP], Invitrogen, USA), washed 2 times with PBS, harvested with trypsin and kept on ice until the fluorescence was measured by flow cytometry in a FACScan device (Beckton and Dickinson, USA), equipped with an air-cooled Argon Laser tuned to emit 15 mW in 488 nm. The fluorescence was measured through a 530-nm long-pass filter. The results were analyzed using the software Summit 4.3 (Dako, USA).
The frequency spectrum of the three compositions was obtained by the software Audacity version 1.3.12 (beta).
Each experiment was repeated at least 4 times. Data are expressed as means ± standard error of the mean and were analyzed using Students t-test or One-way ANOVA with Dunnet post-test for comparison of the differences. Values of P less than 0.05 were considered statistically significant.
| Results|| |
As stated above, there are some few studies demonstrating effects of audible sound, more specifically single frequencies, in cell growth. ,,,,, However, animals and human beings are not submitted to pure frequencies. In fact, we are constantly and increasingly stimulated by music. Therefore, we tried to observe whether music could also interfere with non-auditory cells in culture. To do this, we submitted a non-auditory human cell line to music. Since it is very difficult to cultivate normal human cells, we used the widely studied breast cancer cell line MCF7. The cells were submitted to sound stress induced by three different compositions: Mozart's Sonata for Two Pianos in D major, KV.448, first movement; Beethoven's 5 th Symphony, first movement and Ligeti's Atmosphere. The choice of these three compositions was based on the "Mozart's Effect," on the fact that Beethoven's 5 th Symphony is one of the most heard composition all over the world and that Ligeti's Atmosphere eschews conventional melody, harmony, and rhythm in favor of what is known as "sound masses."
Since MCF7 duplicates in 29-36 h, , we measured the cell cycle 48 h after exposition to the music, in order to assure that at least control cells encompassed one replication time. In [Figure 1] are shown representative histograms of cell cycle after exposure to the three music. It can be seen that control cells present typical histogram, with two peaks (G0/G1 and G2/M-arrows) and the cells in S phase between these two peaks. However, music altered the cell cycle, concentrating the cells in S phase, and diminishing the number of cells in G2/M phase. To analyze whether this observation had statistical significance, this experiment was repeated 4 times and one way ANOVA was performed. As can be seen in [Figure 2], there is a significant decrease in the percentage of cells in G2/M, with a correspondent increase in S phase. Moreover, there is a tendency to appear cells in sub G0 phase (deoxyribonucleic acid [DNA] degradation), suggesting an increase in cell death. Therefore, we tested the cell viability using the Trypan blue assay. For this, the cells were exposed to music and the cell number and viability was measured 48 h after exposition to the music. In [Figure 3] is shown that treatment of cells with Beethoven's 5 th Symphony or Ligeti's Atmospheres decreased, whereas Mozart's Sonata did not alter, the cellular viability.
|Figure 1: Histograms representing the cell cycle of MCF7 cells treated or not with music. Blank arrow-cells in G0/G1; black arrow - cells in G2/M|
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|Figure 2: Percentage of cells gated in the G0/G1, S, G2/M and Sub G0 phases of the cell cycle. *Statistically different in relation to the control cells (cells remained in silence), as measured by ANOVA followed by Dunnett's multiple comparison test (P < 0.05)|
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|Figure 3: Cell viability as measured by Trypan blue assay. *Statistically different in relation to the cells exposed to the speaker alone, as measured by ANOVA followed by Dunnett's multiple comparison test (P < 0.05)|
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It has been shown that sound may affect membrane proteins, altering its activity.  Since Na-K-ATPase is one of the most conserved proteins and has a crucial role in cell homeostasis and in hearing, we tested whether this protein could be altered by music in MCF7 cells, observing the binding of OUA to this protein. OUA is a cardiotonic steroid that binds to Na-K-ATPase with high affinity and is now considered an endogenous hormone.  To study this, we used a commercially available fluorescent OUA (Oua-Bodipy, Invitrogen, USA) and observed the cellular fluorescence, which indicates the binding of OUA to Na-K-ATPase.
A region R1 was set in the dot plot of control cells incubated with OUABDP without music [Figure 4]a, and the percentage of the cells gated in this region was compared between the control cells and the cells treated with the speaker or music [Figure 4]b and c. Surprisingly, the speakers alone interfered with the binding of OUABDP to the Na-K-ATPase. Due to this, the statistics were performed using the cells exposed to the speakers ("noise") as control. Doing this, no variation was observed in the percentage of OUA binding in MCF7 cells treated with Beethoven or Ligeti, suggesting that the effect of the speaker in OUA binding is not reversed by those music, and that those music/per se did not interfere with the binding of OUA do Na-K-ATPase. However, Mozart reversed this effect, increasing the binding of OUA to values close to the control cells (69.8 ± 0.1 in control cells and 66.2 ± 8 in Mozart treated cells).
|Figure 4: Difference in binding of Oua-Bodipy (OUABDP) to the Na-K-ATPase of MCF7 cells after exposure to the speakers alone or to the three compositions. The cells were incubated for 30 min with OUABDP in complete silence (computed tomography [CT] OUABDP) or in the presence of speaker alone or music, as described in material and methods. After this time, the cells were harvested by trypsin and the fluorescence of the cells was measured. (a) Top panel-auto fluorescence and bottom panel - CT OUABDP. The region R1 was the gate used to compare the control (cells remained in silence) with the cells exposed to the speakers or music; (b) Graphs representing the percentage of cells gated in Region R1. aStatistically different in relation to the control cells remained in silence (CT OUABDP), as measured by the Student's t test (P < 0.01); (c) Comparison of cells treated with music with those exposed to the speaker alone. aStatistically different in relation to the speaker, as measured by ANOVA followed by Dunnett's multiple comparison test (P < 0.05)|
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When performing the experiments above, we observed morphological alterations, especially, in cells treated with Beethoven's 5 th Symphony. To quantify such alterations, we performed flow cytometry to analyze cell volume and granularity, measured respectively by FSC and SSC. To do this, a region (R1) was set in the dot plot of the control cells [Figure 5]a, in such a way that approximately 70% of the cells were positioned inside this region. Then, this same region was used in the dot plot of the cells treated with music [Figure 5]b-cells treated with Beethoven] and the percentage of the cells gated in R1 was compared between the control cells and the cells treated with music. As can be seen in [Figure 5]c, when MCF7 cells were exposed to Beethoven's 5 th Symphony occurred an increased in the percentage of cells gated in region R1, meaning a significant reduction in cell volume. The other music did not alter the control pattern of FSC.
|Figure 5: Alterations in forward scattering (FSC) (cell size) after treatment with music. The cells were incubated for 30 min with Oua-Bodipy (OUABDP) in complete silence (computed tomography [CT] OUABDP), in the presence of speaker alone, or in the presence of the music, as described in material and methods. After this time, the cells were harvested by trypsin and the FSC was measured. (a) Dot-plot of the control cells; (b) Dot plot of the cells treated with Beethoven. (c) The graphs represent the percentage of cells gated in Region R1. *Statistically different in relation to the negative control (CT Negative), as measured by ANOVA followed by Dunnett's multiple comparison test (P < 0.05)|
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Similar procedure was performed to evaluate SSC, which represents the cellular complexity or granularity (which could be related, for example, to the number of intracellular vesicles), establishing a division in the population where approximately 70% of the control cells was placed inside the region R1 [Figure 6]a and b. In this case, when MCF7 cells were exposed to any music the cellular granularity decreased significantly [Figure 6]c, which can be seen by an increase in the percentage of cells gated in the region R1 [comparing [Figure 6]a and b.
|Figure 6: Alterations in SSC (cell granularity) after treatment with music. The cells were incubated for 30 min with Oua-Bodipy (OUABDP) in complete silence (computed tomography [CT] OUABDP), in the presence of speaker alone, or in the presence of the music, as described in material and methods. After this time, the cells were harvested by trypsin and the forward scattering was measured. (a) Dot-plot of the control cells. (b) Dot plot of the cells treated with Beethoven. (c) The graphs represent the percentage of cells gated in Region R1. *Statistically different in relation to the negative control (CT Negative), as measured by ANOVA followed by Dunnett's multiple comparison test (P < 0.05)|
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The frequency spectra of the three compositions are represented in [Figure 7]. It can be seen that Mozart's concert frequencies range from 0 to approximately 5 kHz with very few peaks above this range, while both Beethoven's and Ligeti's compositions reach 15 kHz and above. Moreover, these two compositions alternate low and high frequencies, although this fluctuation is more intense in Beethoven's Symphony.
|Figure 7: Frequency spectrum of the three compositions used. Obtained by the software Audacity 1.3.12 (beta version) Moz-Mozart; Bee-Beethoven and Lig-Ligeti|
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| Discussion|| |
Although music therapy has been progressively more used in different areas of medical care, the physiological basis of the biological effects of music is still not understood. Recent evidence suggest that sound, including music, may directly affect non auditory cells. ,,,,,, The results obtained in the present study strongly suggest that music can indeed alter cell cycle and morpho-functional parameters in non-auditory human cells.
We observed that the three music tested were able to alter the cell cycle, concentrating the cells in S phase, and diminishing the number of cells in G2/M phase. Moreover, we observed an increase (although not statistically significant) in the number of cells in sub G0 phase (DNA degradation), suggesting an increase in cell death. This was consistent with the results obtained with Trypan blue staining.
This effect has no correlation with a specific range of frequency [Figure 7], rhythm or sound mass, since the three compositions used are very different in these parameters.
The effect observed in cell viability was not surprisingly, since some studies have shown that audible sound waves are able to alter cell proliferation. For example, Jones et al.,  found that human gingival fibroblasts exposed to 261 Hz showed an increased or decreased rate of proliferation according to the amplitude and to the time of exposition. However, they determine cell proliferation by measuring cell number in a coulter cell counter, removing the medium and tripsinizing the cells. Therefore, cell medium remove could also remove dead cells. As dead cells generally detach from the plate, in our experiments the medium removed was centrifuged and the pellet containing cells were added to the cells tripsinized, and then Trypan blue was added. More recently, Shaobin et al.,  demonstrated that E. coli bacteria grown under normal conditions have their rate of proliferation increased after exposure to frequencies of 1, 5 and 10 kHz. They also showed; however, that audible sound stimulation enhanced the inhibitory effect of osmotic stress on E. coli growth. Therefore, it is not surprisingly that in the present study we observed decreased cell viability induced by music.
Since this effect was observed only for Beethoven and Ligeti compositions, it is possible that the "sound mass" of the orchestration and/or the more acute frequencies could be related to cell death induced, since Mozart's concert present only low frequencies and was composed for two pianos, while the other two are for orchestra.
Only the Beethoven's 5 th Symphony was able to reduce cell size. Although it is not known at present the meaning of this effect, this observation points to the fact that the effect of music is not universal, but dependent on the music type. We also observed that the three compositions altered MCF7 cell granularity. It is known that MCF7 cells contain secretory granules when cultured in the presence of the endogenous estrogens present in the serum.  Whether the granules of MCF7 cells are being disrupted by music or their secretion are being increased remains to be elucidated, as well as the effects of Beethoven in cell volume.
Again, cell granularity seems to have no correlation with a specific range of frequency [Figure 7], rhythm or sound mass, since the three compositions used are very different in these parameters. On the other hand, cell volume may be related to the rhythm of Beethoven's symphony, since Ligeti's atmosphere also have high and low frequencies but has no rhythm.
An unexpected result was the influence of the speaker in the binding of OUA to Na-K-ATPase. It has been shown that sound could alter protein activity,  however, those authors did not use the exposition of cells to the speakers alone as a control. Therefore, our results claim attention to the use of appropriate controls when studying the effects of music or sound in general. Moreover, we observed that only Mozart's Sonata was able to reverse the effect of the speaker and returned the OUA binding to the levels of the control cells. This is interesting, since Mozart compositions are largely used in the literature suggesting positive effects in a series of processes, such as spatial attention and blood pressure. ,, Specially, it has been recently shown that a long-term effect of listening to Mozart K.448 decreases epileptiform discharges in children with epilepsy.  OUA is known to bind to Na-K-ATPase inhibiting its activity  and it has been shown that adrenal and pituitary secrete endogenous OUA.  Therefore, our results could be suggesting a molecular mechanism by which Mozart compositions might regulate those alterations in both blood pressure and epilepsy, since alterations in Na-K-ATPase expression and/or activity are implicated in both diseases. However, though it is possible that the lower frequencies of Mozart's concert may be responsible for this effect, more studies should be performed in order to obtain a molecular mechanism of the Mozart effects.
Concluding, our observations, taken together, suggest that when studying the direct effects of music in cells, it is important to identify what parameters may be affected and what controls are necessary. Our results suggest that:
- It is possible to study the biological effects of sound, including music, in human cells in culture
- When studying the effects of sound in cultured cells, it is mandatory to observe the controls used. Sometimes the speaker alone may have an effect that needs to be controlled in order not to mask the results
- The effects of music may differ according to the biological variable in the study. This must be taken into account if one intends to study the effect of music in physiological processes
- Music is a complex sum of rhythm, melody, and also the harmonics of each instrument. Therefore, the study of specific sequences, in which we can choose for one of these variables, making the others constant, could help understand which components of the music are producing a given effect.
| Acknowledgments|| |
The authors are thankful for Dr. Rejane Barcellos, Leon C. Rousseau and Marcelo Petraglia for their suggestions and criticisms. We thank Mr. Shanserley Leite do Espírito Santo (FAPERJ TCT-4 Fellowship) for technical support.
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Márcia A. M. Capella
Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, CCS Bloco G 21949-900-Rio de Janeiro, RJ
Source of Support: This work was supported by grants from FAPERJ, FECD/FAF/ONCO II, CNPq, CAPES and PRONEX, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]