ARTICLE
Experimental evidence, accumulated over the last few years, has confirmed
the role of the cellular membrane as the main site for interaction with
low frequency electric and magnetic fields.
Signal transduction processes, mediated by second messengers such as
cyclic AMP or Ca-ions, have been shown to be modulated by interaction
of the field with components of the cell membrane e.g. receptor
structures or ion channels [1-3]. Through activation of protein kinases,
this primary biochemical signal may finally lead to cellular responses
even at the level of gene transcription [4], DNA-synthesis [5] and protein
synthesis [6].
An important criterion, that changes in cell signalling due to interaction
with low frequency fields and which may finally cause cytogenetic responses,
is the induction of sister chromatid exchanges (SCEs). Although no experimental
evidence exists that continuous electric or magnetic fields at public
powerline frequencies (50/60 Hz) increase SCE-rates [7], this does not
necessarily hold for special, modulated wave forms in the low frequency
range as, for example, those applied in electrotherapy.
In this field, a so-called interferential current (IFC) is used (for
further details see Materials and methods). This means a superposition
of two alternating currents with approximately 4,000 Hz which results
in a low superposition frequency, in this study 25 and 50 Hz, respectively.
For this study we analysed human skin fibroblasts in vitro for
SCE-induction by a particular electric field with a wide spectrum of therapeutic
applications. The rationale for this investigation was, that an IFC (as
well as other field types) is applied via skin electrodes, which
results in the preponderant exposure of skin cells. In order to gain more
information about a possible contribution of genetic factors, we exposed
fibroblasts from normal donors as well as those from xeroderma pigmentosum
(XP) and dysplastic nevus syndrome (DNS) patients.
XP is a rare disease exhibiting light sensitivity and multiple, actinic
skin tumours of early onset. Patients with XP display a defective excision
repair of UV-induced DNA photoproducts. The dysplastic nevus syndrome
(DNS) is a clinical and genetic entity, in which affected individuals
have increased numbers of dysplastic nevi and a markedly increased risk
of developing one or more cutaneous melanomas.
DNS and XP fibroblasts have been shown to display elevated levels of
UV-induced SCE compared to normal species, which correlates with their
UV-hypermutability [8]. Therefore, it was also of interest to search for
respective differences after IFC-treatment.
Materials and methods
Cell strains and culture conditions
Dermal fibroblasts were obtained from 9 normal donors (mean age 25 ±
8.3 years), 8 DNS patients (mean age 7 ± 7.9 years) and 7 XP patients
(mean age 8 ± 5.9 years).
Cells were cultured in 25 cm2 tissue culture flasks (Becton
Dickinson) with RPMI 1640 medium supplemented with 10% foetal calf serum,
penicillin and streptomycin (100 units/ml each). Incubation was at 37°
C in air with 5% CO2.
Twenty four hours before field treatment, 4 x 105 cells (passage
4-12) were seeded onto Falcon cell culture inserts of 25 mm diameter (Cyclopore
membrane, 0.45 mm pore size). Cell density at the time of exposure was
approximately 1 x 105 cells/cm2.
Interferential current (IFC)
IFC is one of the most effective forms of electrotherapy and is largely
free from side effects. It is produced by superposition of 2 alternating
currents of equal amplitude but with slightly different frequencies, usually
(and also in this investigation) close to 4,000 Hz. Thus, the IFC is an
alternating current with approximately this frequency but with its amplitude
varying periodically between zero and a maximum value in proportion to
the difference in frequency between the two currents (Fig.
1). This beating or modulation frequency can be adjusted to optimise
the desired therapeutic effects of IFC (e.g. muscle stimulation,
analgesia, anti-inflammatory action, etc.).
Exposure to IFC
Cells were exposed on the membrane filters (see above) which were placed
between 2 rhodium-coated electrodes. All components were immersed in medium
thus allowing IFC to flow through the filters. A full description of the
exposure system is given elsewhere [2]. An IFC of 4,000 Hz was generated
with the EDiT-2-device (Nemectron GmbH, Karlsruhe) and fed to the electrodes
via an external attenuator and a digital ammeter. Exposure time
was 20 min at 1 or 4 mA amperage (representative ratings for patient treatment).
The electric field between the electrodes was 1 V/m at 1 mA, corresponding
to a current density of 250 µA/cm2 [2]. These values are
far lower than those leading to any physiological damage of the cells
[2]. Experiments were carried out at two different modulation frequencies:
25 and 50 Hz. Control cells were sham-exposed under the conditions of
treatment, but with the electrodes disconnected from the signal source.
For further details of the system and exposure see [2].
Determination of SCE
Chromosome preparations were made according to a slightly modified standard
protocol [9].
Immediately after exposure, cells were treated with trypsin, subsequently
washed with PBS and the fibroblasts were seeded onto glass slides and
placed in square petri dishes (100 x 100 mm). After the cells had settled,
20 ml of medium containing BrdUrd (final concentration 20 µM) were
added. The dishes were then wrapped in aluminium foil and further incubated
for another 69 h. The mitoses were stopped by adding 0.2 ml colcemid (0.25
µg/ml) per dish which were then left for 4 h. Medium was then replaced
by 25 ml of 75 mM KCl (37° C, 30 min) to induce the hypotonic spreading
of mitoses. The KCl solution was renewed once before fixation, which was
started by slowly adding a mixture of acetic acid and methanol (1:3, v/v)
at room temperature. As soon as 25 ml of fixative had been added, 25 ml
of the mixture were removed and the procedure was repeated twice. Finally,
slides stood for 20 min in pure fixative and were subsequently air-dried.
Staining was performed according to the Hoechst-Giemsa method [9]. SCE
were observed at a 1,000 fold magnification (oil immersion objective)
and counted per metaphase. Every exchange point, including those in the
centromere, were rated as SCE. At least 20 metaphases were examined in
each specimen.
Statistical analysis
The statistical significance of IFC-induced versus spontaneous
SCE was determined by comparing the mean values under the assumption of
unknown and possibly different variances (Weir test). An error probability
p < 0.05 was considered to indicate statistical significance. Analysis
of variance methodology with square-root transformation was applied to
the data from the 3 cell species as a whole to test the influence of amperage
and frequency on SCE-induction, and to analyse the interaction between
the electric parameters.
Results
Table I summarises the
effects of IFC-exposure upon the SCE rate for the 3 fibroblast types.
Treatment at 1 mA increases the rate of SCE in all species. The magnitude
of the effect depends upon modulation frequency and was higher at 50 than
at 25 Hz. Upon exposure at 4 mA there was no significant SCE induction
for 50 Hz (i.e. the higher efficiency of this frequency as observed
at 1 mA was fully reversed), whereas SCE induction at 25 Hz was only slightly
different from the level at 1 mA.
A rather uniform pattern of response can be inferred from the data in
Table 1. Quantitatively,
only the XP values at 1 mA are different from the other lines suggesting
a slightly higher responsiveness. This enabled variance analysis to be
performed with the data for all cell species.
Only two relationships are found to be statistically significant: (1)
the influence of amperage (field strength) on SCE induction (p < 0.006);
and (2) the relationship between current and frequency (p < 0.009).
The first result is due to the general increase in SCE at 1 mA, which
is, on average, higher than at 4 mA. The second result is reflected by
the fact that an increase in frequency from 25 to 50 Hz goes in parallel
with an increase in the SCE rate at 1 mA, whereas at 4 mA a decrease in
the SCE rate is associated with the same change in frequency.
Discussion
SCE-induction by IFC may indicate some chromosomal rearrangement associated
with changes in gene expression or other genetic functions. The following
discussion argues for this hypothesis.
Relationship between SCE response, frequency and
intensity
According to the results presented above, SCE induction after IFC treatment
is influenced by both modulation frequency and field strength. In particular,
a strong association between the electric parameters was found. In the
case of a certain combination of these parameters, the results were remarkable
("Window-Effect").
A dependence on field intensity and frequency was also noted for other
biological endpoints such as protein synthesis [6]. In particular, higher
intensities do not necessarily lead to a more pronounced effect than lower
ones. For example, weak 4,000 Hz electric fields were found to be more
effective in changing the cellular cAMP-content compared to higher amplitudes
[2]. This trend is also seen in the results presented here.
Window effects may be explained by assuming that a given field simultaneously
activates stimulatory and inhibitory processes with different responsiveness
to frequency and amplitude. When these parameters are varied, the biological
response, which is determined by the balance between stimulation and inhibition,
may change in either direction. This has been discussed, for example,
in the context of field effects upon signal transduction elsewhere [2].
SCE induction by IFC and
the problem of absence of damage
SCE are exchanges of homologous pieces of chromatids in replicating
chromosomes, which do not result in an overall structural change in the
chromosome. Although a concomitant increase of chromosome abnormalities
is usually seen with an increase in SCE, the actual relevance of SCE to
chromosome aberrations is unknown [10]. Some SCE occur also in the absence
of chromosome damage, which may indicate other possible ways of induction.
From the physical point of view, direct induction of DNA or chromosome
damage can be excluded. In contrast to ionising or ultraviolet radiation,
the forces exerted by electromagnetic fields of low frequency, when applied
within the physiological range, are by many orders of magnitude too weak
to directly interfere with or even break chemical bonds. This holds, in
particular, for the nuclear area the cell, which is effectively shielded
from the fields by the electric properties of the cell membrane [9]. The
protecting effect refers to electric fields independently from being magnetically
induced or, as in this investigation, applied via electrodes. Consistent
with this is the failure to detect significant effects on SCE and other
types of cytogenetic effects (DNA-strand breaks, changes in repair of
gamma- or ultraviolet-induced DNA-lesions, mutations and chromosome damage)
after exposure to continuous fields of 50 or 60 Hz [7].
In contrast to continuous fields, pulsing or amplitude-modulated, low
frequency fields seem to affect the SCE rate [11]. However, periodic variation
of field amplitude does not change anything with respect to the physical
aspects discussed above. The only difference compared to continuous fields
is the presence of an additional, obviously biologically effective, rhythmic
pattern.
Thus, the increase in SCE observed by us, is quite unlikely to be due
to an IFC-induced damaging or mutagenic event in the nucleus. This is
also supported by the following observations (Table
I): (1) increasing the amperage from 1 to 4 mA has no influence
on SCE induction (25 Hz) and may even decreases its rate towards the spontaneous
level (50 Hz); (2) elevated SCE in DNS versus normal fibroblasts,
as found after UV irradiation and attributed to hypermutability [8], do
not show up after IFC treatment.
The hypothesis
In order to reconcile the data obtained here and the known mechanisms
of action of low frequency fields, we hypothesise, that SCE induction
by IFC may indicate some chromosomal rearrangement associated with changes
in gene expression or other genetic functions. These are mediated by changes
in signal transduction processes which are induced by the field.
This hypothesis can explain the main features of SCE induction described
in this work: (1) the dependence upon frequency emphasises the resonant
character of the field action on cellular signalling processes. This holds
true even in the case, when the modulation frequency as studied here does
not mean a frequency in the strict physical sense but rather a pe-riodic
variation of field intensity [2]; (2) the "unusual" dependence of SCE
induction on field intensity with maximum effects at low amperage (1 mA)
is comparable to that observed for the action of 4,000 Hz fields on cAMP-signalling
[2]. Finally, possible window effects are compatible with this hypothesis.
CONCLUSION
Acknowledgements
This work was supported by the Manfred und Ursula Müller-Stiftung,
Stifterverband für die Deutsche Wissenschaft, Essen, Germany. We
thank Prof. Dr. E. G. Jung for providing the facilities at his clinic.
Data are part of a doctoral thesis by one of us (E. F.). The skilful technical
assistance of Mrs. C. Herbst is gratefully acknowledged. We express our
sincere appreciation to Prof. Dr. H. Dertinger for many helpful suggestions
during the preparation of this manuscript.
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