Action Mechanism of Electromagnetic Fields on Cells. A pathway to Cell Death

Action Mechanism of Electromagnetic Fields

on Cells.A pathway to Cell Death

Dr. Dimitris J. Panagopoulos

Prof. Lukas H. Margaritis

Radiation Biophysics LaboratoryDepartment of Cell Biology & Biophysics, Faculty of Biology, University of Athens

D.J.Panagopoulos, L.H.Margaritis

2

Cell Membrane

Ion Channel structure

Cell Membranes and Ion Channels


3

Cation Channels

Cross section of Na+ channel with positively charged S4 helices

Unfolded Sodium channel protein


4

Cation Channels open by electrostatic forces on their S4 charges, generated by 30mV changes in the membrane potential

Channels open by an outward rotational movement of the S4 helices

Before ions pass through an open channel they enter special binding sites close to the pore.

There are 3 binding sites in potassium channel close to each other and to S4


5

At electrochemical balance, the net movement of ions across the membrane is zero and there is a potential difference Δ between the two sides, with the internal always negative relative to the external, called Transmembrane Electrical Potential Δ = o - i 100 mV

(s 10 nm, membrane’s thickness)

Transmembrane Electrical Potential


6

The contribution of each ion type to the total potential difference is given by the Nernst equation:

o - i = - ln

o , i , electrical potentials on external and internal membrane surfaces R, the Gas ConstantT, the Absolute Temperature, (K)z, ionic charge (in electrons) or ion’s valence

Fc, Faraday constant

Co ,Ci , concentrations of the certain type of ion on the external and internal sides of the membrane at equilibrium

The total transmembrane potential difference is the sum of the contributions from all kinds of free ions

RT

zFc

C

Co

i


7

Across all cell membranes there is a strong Electric field, (107V/m), mainly determined by free ions concentration differences between the two sides of the membrane

Cell’s Free IonsTransmembrane Electric Field

Intensity of Transmembrane Electric Field

m = Δ / s 107 V/m


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• Forces exerted by the external field on the free ions around cell membranes

• Forced-Vibration induction on the free ions.

• Forces on the sensors of voltage-gated channels by the oscillating ions

• Irregular gating of cation channels

• Disruption of cell’s electrochemical balance and function

Theory’s Main Points

Action Mechanism of EMFs on Cells


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Any external oscillating field exerts oscillating forces on the free ions around any cell membrane inducing a forced-vibration on the whole ionic cloud

A. Electric Field

Consider an External Alternating E-field

= o sin t

=2


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Each free ion in the vicinity of a cell’s plasma membrane will accept the following forces:

1. External Field’s Oscillating Force: F1 = z qe= o z qe sin t, (displacement x of the ion from its initial position)

z qe the ion’s charge

2. Restoration Force: F2 = - D x D = mi o

2 the restoration constant, mi ion mass =2o, o the ion’s oscillation self frequency

3. Damping Force: F3 = - u = - 6 nf uu, ion’s vibrationall velocity λ=6nf, attenuation coefficient, nf viscosity of the medium, ion radius

Calculation of the Attenuation Coefficient :

a) In the aqueous solutions, (for 37oC), nf = 710-4 Kg/ms , 10-10 m , 10-12 Kg/s.

b) Within the channels from “patch-clamp” conductivity measurements (for Νa+ ions):

6.410-12 Kg/s

Forces exerted on each free ion


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Forced-Vibration Equation

The ion because of the above forces will acquire acceleration a and its movement equation will be:

mi a = - u - Dx + o z qe sin t mi + + mi o

2x = o zqe sin t (1)

(2nd order linear diff. eq. with constant coefficients)

• All experimental data about cytosolic free ions’ spontaneous oscillations and membrane potential spontaneous oscillations, in all types of cells show that the frequencies of these oscillations are: 0.016 - 0.2 Hz

• We assume that the frequencies of the spontaneous ionic oscillations represent the ionic self-frequencies, o =o /2 and we adopt the value: o = 0.1 Hz

d x

dt

2

2

dx

dt


12

The general solution of eq. (1), is:

x = cos t + C1 e + C2 e (2)

C1, C2 constants calculated from the initial conditions

1, 2 roots of the “characteristic equation”: mi 2+ + mi o2=0

1 0 2 -2.631013 s-1

C1 - C2 - uo 410-14 0

We accept as maximum ion velocity:

uo 0.25 m/s

(velocity calculated for Νa+ ions, moving within the channel)

E zqo e

1t 2t

E zqo e


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Finally, the general solution of eq (1), is:

x = cos t - (3)

The constant term displaces the whole vibrational movement by: -

Therefore, at the moment when the external field is applied or interrupted the ion displacement is doubled

pulsed EMFs are twice more bioactive than continuous EMFs.

The vibrational term: x = cos t (4)

Represents an harmonic oscillation of constant amplitude, independent of any initial conditions

The amplitude is: A =

E zqo e

E zqo e

eo zqE

E zqo e

E zqo e


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Oscillating Ions = movement of electric charge, exerting forces on the sensors of electrosensitive channels

Electrosensitive channels are cation channels.

They open or close by electrostatic interaction between their voltage-sensors and the transmembrane voltage.

The voltage sensors of cation channels are four symmetrically arranged, transmembrane, positively charged a-helices, each one named S4.

Changes in the transmembrane potential ΔΨ 30 mV Induce gating of the electrosensitive channels

Irregular channel gating


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Displacement of one single-valence ion in the vicinity of S4 by r 10-12 m, exerts on the S4 a force equal to that generated by a change of 30 mV, in the transmembrane potential:

m = ΔΨ/s (5)

m = F/q (6)

F the force on an S4 domain,

q = 1.7 qe (7) (effective charge of each S4 domain)

q

sF= q F = Δ (8)

Force on S4 able to gate the channel

For Δ = 30 mV, s = 10 nm: F = 8.16 10-13 N (9)

Force exerted on S4 by an oscillating z-valence ion:

F = F = -2 r

r = - (10)

s

1

4o2r

zqq e 1

4 o3r

zqq e

e

o

zqq

rF

32


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r is the necessary ion displacement in the S4 domain, able to gate the channel

We accept that the distance between the oscillating ion and the effective charge of S4, is r 1 nm

80 (aqueous solutions) 4, (channel internal)

(9), (10) r 0.810-10 m, ( = 80) r 410-12 m, ( = 4).

A single ion’s Displacement of 10-12 m in the S4 domain, can gate the channel


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An oscillating external electric field can alter cell function, if it is able to induce on the free ions a forced-vibration of amplitude:

A r (11)

r decreases proportionally to the number and charge of the ions which act simultaneously on an S4 domain


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The Thermal Motion

The free ions within the same time interval are displaced at longer distances because of thermal activity than because of the forced-vibration

But: Thermal motion is a random motion in every possible direction, different for every ion. It does not displace the whole ionic cloud.

For u = 0.25 m/s, the time interval is δt 1.610-11 s, for the necessary displacement r = 10-12 m.

Mean Free Path of an ion within the channel: 10-11 m.

The ion within δt, will run one mean free path in the direction of the external field and tens of free paths each one in different direction because of thermal motion.

Forced-vibration is a synchronized movement of the whole ionic cloud in one direction acting additively on S4.

The tens of free paths because of thermal motion, each one in a different direction, result in mutually extinguishing opposing forces on the channel sensors, while at the same time one single displacement of the whole ionic cloud in phase results in additive forces on the sensor.


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EMF Bioactivity Characteristics

(11) 410-12 m (12) o 10-3 (13) (Hz, V/m) logo log - 3 0 (14)

(one single-valence cation and continuous oscillating field)

With pulsed fields, the left sides are multiplied by 2 For double-valence cations, (Ca2+), the left sides are multiplied by 4 and the right sides divided by 2 ( r is divided by 2) bioactivity is multiplied by 8. For 2 Ca2+ interacting simultaneously from 1nm distance with an S4 domain, bioactivity is multiplied by 16: 0.2510-12 m (15) o 0.625 10-4 (16) (Hz, V/m)

logo log - 4.2 0 (17)

For 3 Ca2+ interacting simultaneously from 1nm distance with an S4 domain, bioactivity is multiplied by 24

E qo e

E qo e


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0 1 2 3 4 5 6 7 8 9 10 110

1

2

3

4

5

6

7

8

(2)

(1)

E-Field Bioactivity Diagram

Bioactive Region

log

[Eo (

V/m

)]

log [ν (Hz)]


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The mechanism is the same in the case of Oscillating Magnetic fields if we only replace the electric force F1= o zqe sin t, by a B-field (B=Bosin t) force exerted on an ion zqe moving with velocity u, vertically to the direction of the field:

F1 = Bou zqe sin t (18)

For one single-valence cation moving within a channel vertically to the direction of a continuous oscillating B-Field with u = 0.25 m/s :

410-12 m (19) (rad/s, m/s, T),

Bo 40 (20) (Hz, G)

log Bo log + 1.6 (21)

For 2 Ca2+ interacting simultaneously from 1nm distance with an S4 domain:

0.2510-12 m (22) (rad/s, m/s, T)

Bo 2.5 (23) (Hz, G)

log Bo log + 0.4 (24)

eouqB

B. Magnetic Field

eouqB


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0 1 2 3 4 5 6 7 8 9 10 110

1

2

3

4

5

6

7

8

9

10

11

12

13

B-Field Bioactivity Diagram

(2)

(1)Bioactive Region

log

[Bo (

G)]

log [ν (Hz)]


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Taking into account an induced electric field Eind , generated by the pulsed magnetic one with a typical intensity of 1 V/m, parallel to the magnetic force F1, (vertically to B), for 2 Ca2+ interacting simultaneously from 1nm distance with an S4 domain:

0.2510-12 m (25) (V/m, T)

Bo 2.5 - 4104 (26) (Hz, G)

(line B in E-B Bioactivity diagram)

eindo qEuB )(


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0 1 2 3 4 5 6 7 8 9 1 0 1 10

1

2

3

4

5

6

7

8

9

1 0

1 1

1 2

E - B - F i e l d B i o a c t i v i t y D i a g r a m

B i o a c t i v e R e g i o n

( E )

( B )

log

[E

o (

V/m

)]lo

g [

Bo (

G)]

l o g [ ν ( H z ) ]

For pulsed E and B fields and 2 double-valence cations interacting simultaneously with S4 from 1nm distance


25

0 1 2 3 4 5 6 7 8 9 10 110

1

2

3

4

5

6

7

8

9

10

11

12

7

4

10

1

13b13a

12

11

8b

8a

6

9

5

3

2

E-B-Field Bioactivity Diagram

Bioactive Region

(E)

(B)

log

[Eo (

V/m

)]lo

g [B

o (G

)]

log [ν (Hz)]

For frequencies 0-104 Hz, even very weak E-fields of less than 1V/m, or B-fields of 1G are able to alter cell function


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According to the theory, low frequency EMFs are more bioactive than high frequency EMFs. This explains for the first time experimental data indicating that biological action of RF-Microwave fields is due to the ELF frequencies included (pulse repetition frequency, modulation)

According to the theory, pulsed EMFs are more bioactive than continuous EMFs. This explains for the first time the higher bioactivity of pulsed EMFs reported in several experiments.

Most RF-Microwave fields are Pulsed (GSM, DCS) or Modulated (FM) at ELF frequencies


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Recent Experiments indicate DNA damage or Cell Death induction by EMFs

S8

S4S3S2S1G

b

OCNC

FC

S8

S4S3

S2S1

GS7

(Panagopoulos et al., 2007; Diem et al., 2005; Markova et al., 2005; Salford et al., 2003; Lai and Singh 1995; 1996)

Ovariole of a sham exposed female insect with TUNEL negative egg chambers at all the developmental stages from germarium (G) to stage 8

Ovariole of exposed female insect with TUNEL positive signal at both check-points, germarium and stage 8 and TUNEL negative signal at the intermediate stages

Ovarioles of exposed female insects with TUNEL positive signals at all the developmental stages and in all types of egg chamber cells, nurse cells (NC), follicle cells (FC) and the oocyte (OC)

S8

G

2a

2b

2

3

1


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Disruption of intracellular Ionic Concentrationsmay lead to Cell Death

1. Irregular increase of intracellular ion concentration, may cause water molecules to enter the cell, driven by osmotic forces, proportional to the concentration increase. This can cause the cell to swell out and the plasma membrane to get ruptured, resulting to cell necrosis.

2. Perturbations of intracellular Ca+2 concentrations are responsible for apoptotic triggering.

3. A common event leading to both apoptosis and necrosis is mitochondrial membrane permeabilization, leading to increased mitochondrial concentration of Ca+2 ions


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Conclusion

Forced-Vibration of free ions in the vicinity of a cell membrane induced by external EMF can irregularly gate ion-channels, disrupt the cell’s electrochemical balance and function and may even lead to Cell Death

Documents

Action Mechanism of Electromagnetic Fields on Cells. A pathway to Cell Death