Low-energy electromagnetic frequencies induce
conspicuous, reproducible, and lasting effects in human
and animal cells

DISCUSSION AND FREQUENCY MODEL

Summary of the main results (Table 3): After EMF A/C treatments, both the THP-1 and the 3T3-L1 cells showed conspicuous and lasting changes. The most diverse changes were observed in the human cell line THP-1.

The first part of the discussion deals with the results of this work: How should the results be evaluated? The second part discusses a model for the possible mode of action of EMF.

Table 3:
Summary of the EMF results

Abbreviations
Frequencies: A/C = Group A with final frequency C; B = Group B;
Cell lines: Number of cell lines generated by EMF
Oscillator: S = F-SCAN 2 series instrument; P = F-SCAN 2 prototype;
EMF Effects: 1 = Strong, 2 = medium, 3 = weak; neg = Negative; na = Not analyzed; G = Growth; A = Aggregation; GC = Giant cells; MA = Melanin assay; SR = Survival rate

DISCUSSION, Part 1

None of the cell lines generated by the EM treatments with frequency group A had a reduced viability. On the contrary, the survival rate of 3T3-J was improved. The division rate of the treated cells was also not reduced: THP-J showed a higher proliferation rate, albeit only for a fraction of the THP-J. The EMF treatments did not induce changes for which the respective cell type is not adapted, as far as this can be evaluated.
 
Summary of the observed changes of the cells due to low energy EMF treatment:

1. Frequencies lead to reproducible changes in growth.
2. Frequencies lead to changes that are specific to each cell type.
3. Frequencies lead to stable cellular changes.

THP cell lines

All standardizations for the frequency treatments were carried out with THP-1 cells. THP-J showed the first positive results that helped to drive the project forward experimentally. Several results, such as differential growth in various nutrient solutions should have been investigated in depth. It is likely that the whole project could have been limited to the analysis of THP-1. Some obvious results were not investigated further:

• Which frequency/ies lead to which changes in the cells?
• What impact do treatments with EMF A plus/minus C respectively with B have?
• Do THP-1 and THP-J differ in their ability to differentiate into macrophages?

THP-1 did barely grew at low cell concentration in a medium containing NBS only in the first 2 to 3 weeks, whereas THP-J1 and J4 were able to proliferate well at all cell concentrations in NBS or FBS. These growth experiments were carried out in 2005 and 2006. An attempt was made in 2009 to verify differential growth of THP-1 and THP-J cells in NBS with a low initial cell count. However, this was not possible because all the low density cultures died in all attempts (N=11). The THP-J cells in 2009 were still changed by the frequency treatment carried out in 2005 since they had kept the property of growth in large groups. It is possible that ageing phenomena was the cause as this affected both THP-1 and THP-J kept in similar conditions during all this time.

THP-J cells grew more slowly than THP-1 in FBS. Analysis of CFSE-stained cells revealed that 1/5 of the THP-J grew faster than THP-1, whereas the remainder cells proliferated significantly slower. The growth behavior of the heterogeneous THP-J population is evidence for a regulative effect of the EMF treatment, showing the effect of adaptive growth by the different cell populations.

Part of the culture was always adherent and presumably no longer proliferated. It can be assumed that adherent THP had differentiated from monocytes to macrophages. The influence of EMF treatment on the differentiation capacity of THP-J was not investigated in depth.

The formation of large groups of THP-J cells was particularly conspicuous. It was thus possible to distinguish between treated and untreated cells without magnification. A description of a comparable cell line corresponding to the characteristics of THP-J was not found in the literature. With the growth into large clusters, the THP-J cells showed an enhanced interactivity between each other. The tendency to grow in large groups was influenced by the growth environment. In overaged cultures, the groups disintegrated, and in nutrient-rich environments they were larger.

Which genes were affected by the EMF treatment? Since FBS and NCS differ in their concentration of growth factors and since it is known that cells in culture secrete such factors and influence the neighboring cells, it can be assumed that the EM information affects among other genes those responsible for regulation of the concentration of growth factors and/or their receptors (Lit.9).

Giant cells were also observed with THP cell lines; however, they were rare. Their prevalence did not change after EMF treatment. It is possible that giant cells are commonly found.

3T3 cell lines

The 3T3 cell lines were robust and their growth characteristics did not change over the 4 years of cultivation after EMF treatment. The differences due to EMF treatment of 3T3 were not manifold, but they were stable. They consisted in an extensive growth effect and in the appearance of giant cells. Attempts were also made to stimulate the transformation of 3T3 cells into fat cells; however, this was not successful in nutrient solutions containing NBS. Overall, the potential of 3T3-L1 cells for investigations by frequency treatments with A/C or B are considered as good.

Rare giant cells have been reported in the literature (Lit. 3,4). Cell lines with up to 2% giant cells were found after EMF treatment. The largest cells were detected in 3T3-K cultures only.

The growth characteristics of 3T3-L1, 3T3-J, and 3T3-K at confluency was repeatedly confirmed, remaining unchanged over the entire duration of the project. The different growth characteristics were also retained when the cells were stressed using suboptimally composed media as used for Rafi cells (results not shown).

3T3 cells form dendritic processes. Since 3T3-J and 3T3-K produced 2–3 times more dendrites than 3T3-L1, they were presumably better adapted to grow in multilayers. The composition of the dendrites was not investigated in details. The changes of 3T3 after EMF treatment demonstrate how information from a few frequencies are able to influence complex processes in a reproducible and stable way.

B16 cell lines

The effect of the applied EMF on the B16 cells was low and not always reproducible. It was hoped that the B16-J would grow more slowly than B16-F1. This, however, was not what happened: the B16-J cells grew somewhat faster than the already fast-growing B16-F1. However, the EMF treatment effect was minor and only detectable in 5 out of 7 experiments. Thus, B16-F1 are considered not suited for EMF experiments with the frequencies that were used.

Metabolic stress did not lead to differential growth differences of the B16-F1/J cells. This was only observed with Rafi, but not with the 3T3 or B16 cell lines. It is possible that the B16 are genetically modified cells with a limited response range for growth adaptations.

Extensive investigations were carried out on the inductive sensitivity of the B16-F1/J cells to synthesize melanin. The first observation was that the EMF-treated B16 showed a higher sensitivity to alpha-MSH. The enhanced alpha-MSH sensitivity effect could not be reproduced, however. Further analyses with a total of 6 controls and 12 EMF-treated cell lines showed that differential melanin synthesis can be attributed to selection occurring by the EMF treatment process. The lower the number of B16-F1 used to start the cultures for the frequency treatments, the more the cell lines lost their characteristic sensitivity to alpha-MSH. In other words, B16-J cell lines did not show any changed alpha-MSH sensitivity to melanin synthesis if the initial number of cells used in the EMF treatment was greater than 50 cells per culture flask.
In the same experiments, the B16 cells were also used to investigate the effect of EM shielding applied during the frequency treatments. Due to the absence of cellular changes in these experiments, no results were obtained on this issue.

The small or lacking differences in phenotype between B16-F1 and B16-J could have many causes:

• B16-J cells were treated with frequency group A only, without C.
• The applied growth assay with microtiter plates was not sensitive enough.
• Mouse melanoma cells are not sensitive to frequency group A.

The work with the B16 cells led to the conclusion that selection processes in the experiments can be a source of artifacts. Overall, the B16 cell lines were investigated much more intensively than the THP.

Rafi cell lines

The Rafi cells showed good cultivation performance up to at least passage 34. This was the decisive property for using these cells in the experiments. Nevertheless, apoptotic death of the cell lines at high passage numbers repeatedly made the replacement by frozen cell culture aliquots necessary. This occurred at irregular time points. Furthermore, it would have been interesting to repeat the growth experiments with A+C frequencies. In retrospect, a higher oxygen concentration as well as a medium containing FBS would also have been advisable for cultivation. All in all, the rat fibroblasts are considered not suited for EMF experiments with the selected frequencies.

The experiments with the Rafi were carried out with the aim of detecting the influence of frequency treatments on primary cells, established from non-cancerous tissue. This was successful to some extent. However, differential growth was only observed when the cells were under metabolic stress: the phenotypic alterations of the cells were minor. In addition, it was not possible to find a clear pattern for the growth differences of the Rafi, Rafi-J, and Rafi-K cells using various metabolic stress conditions.

Primary rat cells were used because they are known for their longevity in cultures without mutating, which is promoted by a low oxygen concentration in cell cultivation. The incubators for these cultures were set to 3% oxygen, or even to 1% in some cases. In retrospect, this appears to be too low for skin fibroblasts. It is assumed that an oxygen concentration of 10–15% would have been better suited.

Since the oxygen concentrations in the cells greatly vary according to the tissue (lung 13–17%, fatty tissue 1–2%), the THP, 3T3, and B16 cells were also investigated at low oxygen concentrations. Differential growth of these cell cultures did not increase however.

The small and variable differences in growth of the Rafi cells with and without EMF treatment prompted a refined analysis of the growth conditions. This led to the discovery of the variations in seeding efficiency. Nevertheless, the growth differences with and without EMF treatment were slight compared to THP and 3T3. The question remains whether this is due to omitting the application of frequency C or to the possibility that growth changes induced by EMF treatments are generally of low extent in primary cells. In addition, it is although possible that the A or B frequencies are not ideal for experiments with Rafi cells.

Finally, the use of EM shielding enclosures for the Rafi cells did not yield results.

Reproducibility

Reproducibility of cell line-specific EMF effects was observed a number of times: THP-J1, THP-J4, and J12; 3T3-J1 and 3T3-J2; 3T3-K1 and K2; two test series each with 6 Rafi cell lines. Repeated treatments with the same frequencies always led to the same effects, specific for the cell type.

The cooperativity effect of EMF treated cells

A phenomenon was detected as a result of various EMF experiments that is interpreted as an enhanced interaction or as a cooperativity effect between the cells or between the cells and their environment:

• THP-J cells had a tendency to form large clusters. The preliminary results on alpha-troponin should have been further investigated: after treatment with PMA and staining with anti – alpha-troponin, the THP-J cells were found to contain much more alpha-troponin than the THP-1 cells. It is also conceivable that the enhanced cell interactions of THP cells are mediated by tight junctions (Lit.10). However, this was not analysed.
• 3T3-J/K cells grew to denser cell layers and produced multilayers.

Prototype and series instruments of F-SCAN 2

Two THP-J and two 3T3-J cell lines were generated with the F-SCAN 2 prototype instrument using the A+C frequencies. However, these cells lines did not show any difference in their growth compared to the controls. All previously described positive results apply to cell lines obtained with the F-SCAN 2 series instrument (Table 3). It thus seems possible that the prototype instrument was not suitable.
However, the prototype instrument was used to generate the two 3T3-K lines that were obtained by applying B frequencies: the 3T3-K1/2 cells grew to a greater density and also had more giant cells.
Some possible explanations are:

• The phenotype of the 3T3-K1/2 cells was the result of a selection process involving a preexisting mutation in 3T3-L1.
• The prototype F-SCAN 2 transmitted only B frequencies, but no A frequencies.

The two 3T3 cell lines treated with A+C frequencies using the prototype instrument did not show any enrichment of giant cells: This speaks against Explanation 1. Explanation 2 seems not likely because A and B frequencies are very similar. It thus seems likely that the negative results were caused by an unknown error in the experiment.

Regarding the signal characteristics of the individual frequencies

The F-SCAN 2 oscillator can generate frequencies whose shape is rectangular or sinusoidal. Rectangular signals contain the corresponding EMF overtones of the basic signals, but sinusoidal signals do not. All EMF treatments of the cells were carried out in the rectangular mode. An analysis of the output signals from the F-SCAN 2 series instrument confirmed that the signals had the correct shape. However, the signals were no longer rectangular in the medium. The frequencies in the megahertz range were almost sinusoidal, and in the low Hertz range the originally rectangular signals were considerably lowered on one side without turning to a sawtooth form.

The voltages transmitted by the EMF treatment to the RMPI medium were checked in situ for the F-SCAN 2 series instrument. The voltage in the medium at various locations in the culture flask varied between a maximum of 800 mV and a minimum of 233 mV, depending on the frequency. The measurements were carried out at the furthest possible distance from the electrodes, which were located in the middle of the culture flask, as usual. Thus all cells in the culture flask were exposed to the EMF, but at different strengths. It must be assumed that the transmission pathway of the EMF signal from the oscillator via the potentiometer and spider box to the electrodes was the cause of the voltage loss and the loss of some overtones. Measurements in the RMPI medium gave the following in situ currents for the individual frequencies of frequency group A: 344 μA at 111.2Hz and 112.2Hz; 359 μA at 555.7Hz; 299 μA at 555,555.1Hz and 555,555.6Hz; 5.9 μA at 5,555,554.3Hz and 5,555,555.4Hz. The measured values were constant. It is assumed that these values are similar for the other nutrient solutions with comparable salt concentrations.

In summary, it can be stated that the strength of the EMF signals and the presence of overtones was reduced to a considerable extent by the transmittance from the oscillator to the point of action, the reduction depending on the exact frequency used.

DISCUSSION, PART 2

Are the described EMF effects due to mutations?

THP-J and 3T3-J/K were changed in a way that is presumably not due to a single mutated gene. In addition, the same phenotypes repeatedly occurred independently of each other. Did the starting cell lines of THP-1 and of 3T3-L1 already contain mutated cells that were selected by the experimental procedure? This is unlikely. The new phenotypes did not occur in several experiments and, in particular, in none of the controls. For the 7 cases in which changes were observed, they were frequency-specific and, with respect to the cell type, they were specific and reproducible.
It is not obvious how a single mutation could have caused all the changes of THP-J: formation of large clusters, differential growth of cell subpopulations, differential growth of THP cell lines depending on the serum and initial cell count as well as an increased probability of becoming adherent. If the new phenotype of 3T3-J and 3T3-K could be attributed to one mutation, the question arises as to why 3T3-J did not produce more giant cells. The fact that different phenotypes of 3T3-J and K were observed after different EMF treatments of 3T3-L1 is conclusive evidence. It is also unlikely that the same mutation would lead not only to giant cells, which are caused by endomitosis, but also to a higher cell density at confluency, a property that occurred alongside increased dendrite formation.

The following findings also contradict the hypothesis of mutation and/or selection as the cause for the different THP phenotypes present after EMF treatment:

1. In 2009, four years after THP-J had been induced, the growth capacity of THP-1 and J had obviously decreased, but not the ability of THP-J to form large clusters. The growth properties and the cluster formation of THP could thus change independently of each other.
2. The cells of 3T3-J and THP-J were cultivated separately in two to four flasks for EMF treatments and pooled afterwards. This procedure was used for the following reason: cells with phenotypes that were not generated by EMF treatment, which would have affected only a portion of the cell culture, should thus have been identified more easily before they could become predominant as a result of selection. The results of the EMF treatments, however, indicate that all cells had the new phenotype.
3. If the EMF treatments had led to mutations, the viability of the treated cells would probably have been reduced, since random mutations are statistically mostly negative. This was not the case.

Nevertheless, could the applied EMF have generated mutations in the DNA by some other means? Let us assume that this is indeed the case: Several genes would have had to be affected by mutations in order to explain the obtained phenotypes: the mutations would have to be suitable, cell-specific and reproducible. In other words, an explanation of the results on the basis of EMF-induced mutations and/or selection can be excluded. The mechanism for the various cellular changes through EMF treatment must have other explanations.

All the effects described were induced with EMF below 6,000,000Hz and less than 0.4mA and less than 0.8V. It is likely that this energy is not sufficient to stimulate molecular receptors (Lit.6). If the organism is defined as an EM unit that also responds accordingly: e.g. after an applied injury, tiny electric currents are detectable in the injured area (Lit.7), then an influence exerted by EMF appears plausible. US Patent 3924609 demonstrates the fact that such weak EM properties can be used technically by an electrical ovulation test in humans for the determination of the precise fertilization time (Lit.8).

The treatment of cells using EMF with very low energy corresponds to the EM influences of the natural environment: the EM field of the Earth is weak. Extremely low frequencies (ELF) of less then 50 or 60 Hertz with very low voltages are unlikely to have sufficient energy to reconfigure chemical bonds. But how can the Earth's 10Hz EM field control the 24-hour cycle of humans, animals, and plants (Lit.13,14,15)? All organs, cells, and genes of organisms are subject to the 24-hour rhythm, as chronobiological research on humans and various animals (Lit.17) and plants has shown. The many biological parameters that follow the 24-hour cycle of an organism, such as motor activity, blood pressure, or body temperature, as well as the rhythmic changes in the concentrations of glucose, sodium, calcium, melatonin, etc., demonstrate the all-encompassing influence of the internal clocks. The finding that not only the Schumann frequency, but also other EMF are able to affect organisms thus expands well-known facts.

To the best of our knowledge, the EMF used in this work do not occur in nature. It thus seems plausible that the new frequencies A, B, and C are able to induce new properties. But how could the EMF used in these experiments have been able to affect the metabolism of the cells? It is possible that there are information present in a cell, or in an entire organ that are common to a biological structure formed by many cells. On the basis of this assumption, it is possible to understand the responses of the cells obtained by the EMF treatments.

Extreme sensitivity to weak EM fields has been observed in organisms (Lit.12). Voltage differences of only 0.5 μV/m can be detected by the lateral line system of sharks and rays (Lit.18). Although the mode of action of the lateral line system has been discussed, its extreme sensitivity remains a mysterious phenomenon (Lit.20). Mammals also have a highly sensitive sensory organ: their eyes are said to be capable of perceiving single photons (Lit.19). It has been proved that shielding of the Earth's magnetic field desynchronizes the internal clock in humans and animals (Lit.13,14). How do living beings without eyes that also live in the dark keep their internal clock functioning without the usual daily adjustment by sunlight (Lit.21)? Many people respond to changes in the weather conditions or moon phases by feeling unwell. What is the mechanism of this response? In this work, the lasting changes in cells are reproducibly induced by EMF treatments with very low energy. The following model could explain these findings:

MODEL FOR THE CELLULAR EFFECTS BY ELECTROMAGNETIC FREQUENCY TREATMENT

Parallel to the genetic system that is responsible for synthesis and metabolism of the structures of organisms, there exists an information field for the regulation of the genetic system. Information is encoded as electromagnetic frequencies of the quanta. The quanta are the units of matter of all structures. All quanta have specific and unique information for the respective structures of which they are part. The electromagnetic frequencies form an information field for the structures. The information field acts according to the resonance of all frequencies of a structure. The extent of the resonance determines the energy transmitted. The energy of a structure corresponds to the amplitude of its frequencies. All structures have quanta with specific frequencies and information that are needed for its establishment and regulation. As the frequencies of the quanta form the information field and the material structures, no energy transmission is necessary: The energy is generated at the place where it is used. The information field acts harmoniously because it acts by means of resonance of the involved quanta.
The proposed model in one sentence: The electromagnetic frequencies are an interactive information field that forms structures from information and supplies energy in accordance with the resonance of its frequencies.

The key results of this work are: Treatment of THP-1 and 3T3-L1 cells with EMF A+C and 3T3-L1 with frequency group B led to large and reproducible changes. The changes found are specific to the respective cell type. Most changes were stable for years. The following discussion is limited to the results obtained with THP and 3T3 cells.

EMF treatments caused changes to THP-1 and 3T3-L1: what sort of changes? Already existing properties of the cells are reinforced: THP-J forms larger cell groups and more frequently; 3T3-K forms larger giant cells and more frequently. 3T3-J and 3T3-K form more dendrites per cell and presumably also have in this way changed growth properties. All these changes are only possible due to a cooperatively acting regulation mechanism for complex processes. These changes, comprising many cellular structures with many different molecules, involve interactive regulation of many different gene products. The growth at different rates of THP-J involving different cell populations is also regulated cooperatively. The fast-growing THP-J cells do not overgrow the slowly growing cells: the THP-J cell culture is in equilibrium.

The frequency-induced changes are specific for a given cell type: 3T3-J and B16-J, both of which are mouse cells, respond differently to the same EMF.

Changes were induced with 30 and more EMF treatments and remained stable for years. This means that the EMF of the information field, once it has been activated, probably remains in the new activity mode until it is influenced by new frequencies or new information. In other words, the activity of the information field is stable if there is no influence by any kind of energy. Repeated EMF treatments are thus expedient: presumably the amplitudes of the target information is reinforced. This in turn means that a large number of EM treatments of the cells tend to cause strong EMF effects.

The model raises many questions. Some of the following questions could probably be answered today using the A/C or B frequencies, with suitable cells; whereas other frequencies would have to be used for other cells. It is probably advantageous to carry out EMF experiments with at least two different cells lines if the difference in responses between primary and established cell lines has not been elucidated and if the EMF-specific sensitivity of a given cell type has not be investigated. Open questions that presumably could be tackled to some extend:

1. How often must THP-1 cells be treated with the A +/- C respectively the B frequencies to induce complete activation?
>
2. Can EMF effects also be obtained with cells using magnetic transmission of the frequencies?
3. If cells were to be treated with EMF to which they had been chronically exposed for an extended period, it is likely that no further behavioral changes would be induced: the target frequencies are already present in an active form. This phenomenon could occur due to electrosmog (Lit.22).
4. Is it possible to treat cells successfully with EMF but without a sweep?
5. Is it possible to induce apoptosis in established cell lines using a suitable EMF?
6. Which EMF can reduce growth of cancerous cells?
7. Could cancerous cells be converted into benign cells using a particular EMF?

In summary: the model proposed here leads into an unexplored field. Although the model fulfills the necessary functions, it does so in a new way.