Infrared Physics & Technology 45 (2004) 339–347

www.elsevier.com/locate/infrared

THz radiation studies on biological systemsat the ENEA FEL facility

A. Doria a,*, G.P. Gallerano a, E. Giovenale a, G. Messina a, A. Lai a,A. Ramundo-Orlando b, V. Sposato b, M. D’Arienzo c, A. Perrotta c,M. Roman�o c, M. Sarti c, M.R. Scarf�ı c, I. Spassovsky a,1, O. Zeni c

a ENEA-FIS-ACC, Via E. Fermi 45, 00044 Frascati, Italyb ICEmB at INEMM-CNR, Via Del Fosso del Cavaliere 100, 00133 Roma, Italy

c ICEmB at CNR-IREA, Via Diocleziano 328, 80124 Napoli, Italy

Available online 8 April 2004

Abstract

Emerging technologies are considering the possible use of THz radiation in different fields, ranging from telecom-

munication to biology and biomedicine. The study of the potential effects of THz radiation on biological systems is then

an important issue in order to safely develop a large number of applications. This task is being accomplished in the

framework of the EU funded THz-BRIDGE project. Experiments on the effects of THz radiation on different biological

samples with different techniques are being performed utilizing the compact free electron laser at the ENEA Research

Center of Frascati. Experimental results, together with statistical analysis are reported.

� 2004 Elsevier B.V. All rights reserved.

1. Introduction

The study of potential health hazard induced by

electromagnetic radiation in the THz region (100

GHz–20 THz) is one of the objectives of the EU

funded project THz-BRIDGE. Despite the recent

technological applications of THz radiation in

biology and biomedicine, which are based on the

specific spectroscopic fingerprints of biological

matter in this spectral region, very little is knownabout its interaction with biological systems. A

variety of techniques and biological assays are

* Corresponding author.1 ICTP Fellow.

1350-4495/$ - see front matter � 2004 Elsevier B.V. All rights reserv

doi:10.1016/j.infrared.2004.01.014

employed within THz-BRIDGE to clarify such

issues [1]. In the present paper we present the studiesrelated to the interaction of THz radiation with two

important biological systems: a model of cell mem-

brane and human lymphocytes in whole blood.

A compact free electron laser (FEL) [2] has

been used to generate coherent radiation at 130

GHz. Due to the peculiar characteristics of the

electron accelerator (microtron) driving the FEL,

the radiation pulse (macropulse) is composed by atrain of 50 ps micropulses, spaced 330 ps apart.

The FEL radiation macropulse length is about

4 ls at a repetition rate that can be typically variedbetween 1 and 10 Hz. This peculiar temporal

structure of the emitted radiation allows the

investigation of the effects of high peak power,

ed.

340 A. Doria et al. / Infrared Physics & Technology 45 (2004) 339–347

while maintaining a low average power, typically

few mW, incident on the sample, thus avoiding

heating effects. The radiation is transported to a

dedicated user room by means of a special mm-

wave transmission line composed of an evacuated

copper light pipe and appropriate delivery optics.

2. Exposure of CA-loaded liposomes

The cell membrane plays an important role in

the interaction with extra cellular entities, both

from a physical and chemical point of view; it is

widely regarded as a biological structure likely tobe responsive to electromagnetic radiation as an

external physical entity. Several in vitro studies

indicate that millimeter wave radiation may alter

structural and functional properties of the cell

membrane, and the effect seems to be frequency

dependent [3–5]. Given the intrinsic complexity of

biomembranes, a simplified model membrane such

as a lipid bilayer, where all experimental parame-ters can be accurately monitored [6,7], has been

used for the present study. Liposomes are micro-

scopic vesicles in which an aqueous volume is en-

tirely enclosed by a membrane composed of

phospholipids. They form spontaneously when

amphipathic lipids are dispersed in aqueous media.

They can be prepared in such a way that they

entrap soluble materials both within their aqueouscompartment, as in our system, and within the

membrane. By constructing them with natural

phospholipids, the liposome membrane forms a

bilayer structure as it happens in the lipid portion

of cell membranes (see Fig. 1).

Fig. 1. Schematic representation of th

At cellular and sub-cellular level, the interaction

essentially develops through forces that the local

electromagnetic field exerts on the free and bound

electric charges of the biological system. It is

important to outline that, while the forces acting

on charges only depend on the electromagneticfield and on the electrical characteristics of the

‘‘particle’’, the dynamics of these ‘‘particles’’

(vibrations, displacements, rotations) also depends

on other properties (i.e. the mass, the kind of

‘‘particle’’: atomic or molecular, the chemical

bounds, etc.) and on neighbouring ‘‘particles’’.

The cationic liposomes used in the experiments

described in this article, are composed of lipidsnormally found in natural membranes such as

DPPC (dipalmitoylphosphatidylcholine), choles-

terol, and positively charged stearylamine.

The soluble enzyme carbonic anhydrase (CA) is

loaded into the aqueous space of liposome.

Once its substrate (i.e., p-nitrophenyl acetate,

p-NPA) is present in the external aqueous space

of these liposomes, it is slowly hydrolysed bythe CA. Thus, any alteration of the lipid bilayer

permeability induced by THz radiation may be

evaluated by the ability of the substrate to dif-

fuse across the lipid bilayer. In this case it is

rapidly hydrolysed by the CA entrapped inside

the liposome. In particular, if the membrane

is damaged, the substrate flows into the vesicle

and interacts with the CA (the enzyme itselfcannot flow out of the vesicles) creating the

product of reactionaccording to the simple reac-

tion:

Eþ S $ P

e liposome and its lipid bilayer.

A. Doria et al. / Infrared Physics & Technology 45 (2004) 339–347 341

where E, S and P indicate the enzyme, the

substrate and the product of reaction, respec-

tively.

The enzyme activity measurements of CA were

carried out by following the appearance of reac-tion product, p-nitrophenolate, at its peak of

absorbance (k ¼ 400 nm), on a UV spectrophoto-

meter computer controlled (Cary 50). An increa-

sing value of substrate hydrolysis rate can be

directly associated with a membrane damage.

Two different irradiation set-up have been de-

signed and constructed, referred to as A (Fig. 2)

and B (Fig. 3).(A) The FEL radiation at the central frequency

of 130 GHz is transported through a copper light-

pipe of 25 mm diameter and is then fed into a

‘‘THz delivery system’’ (TDS) specifically designed

to match the irradiation requirements. The TDS is

made of aluminum and is built to provide a uni-

Fig. 3. Exposure of CA-loaded liposomes: setup B. (a) Irradiation fro

focusing the radiation onto the cuvette.

Fig. 2. Exposure of CA-loaded liposomes: setup A.

form irradiation of the liposome samples. The THz

beam coming from the light pipe into the TDS is

first focused down to a 17.5 mm diameter aperture

by means of a conical section and is then let ex-

pand by diffraction to about 52 mm diameter to

match the required irradiation area. Calculationshave been performed in order to minimize internal

reflections in the cone, making the beam expansion

similar to the free space expansion. The cuvette

containing the liposome samples is placed in a

Teflon grid, positioned in the region of maximum

uniformity of the field in the TDS over the entire

time of exposures (60 min).

(B) In order to irradiate the liposomes and carryout the kinetic measurements in real time, the cell

compartment of the spectrophotometer has been

modified to deliver the THz beam into the cuvette

containing the liposomes in a final volume of 1.5

ml. Two possible irradiation geometries have been

considered: irradiation from the side and from the

top of the cuvette. In the present experiments we

irradiated from the side of the cuvette (Fig. 3). Anelectroformed copper horn is used to match the

circular cross-section of the THz beam to the

rectangular cross-section of the cuvette.

The p-NPA hydrolysis rate, expressed as rate

of change in absorbance units at 400 nm/min

ðDA=minÞ, is computed automatically as the slopeof a linear fit to experimental recorded curves (Fig.

4). Data from exposed and sham samples arecompared and their variance is analyzed according

to the unpaired two-tailed Student’s at 95% con-

fidence interval.

m the side of the cuvette, (b) top view, showing the copper horn

0.8

0.85

0.9

0.95

1

0.0 0.3 0.5 0.8 1.0 1.3 1.5 1.8 2.0Time (min)

Abs

orba

nce

(400

nm

)

exposed

sham

Exper. dataLinear interp.

Fig. 4. Real time exposure of CA loaded liposomes: change in

absorbance of exposed and sham samples as a function of time.

Table1

Effectsofirradiationat130GHzonCA-loaded

liposomes(summary

oftheexposureconditionsandexperimentalresults)

Setup

Roomtemp.

(�C)

Timeof

exposure

Power

(mW/cm

2)

Pulserepetition

rate

DA=min

control

Da=min

exposed

%CAenzyme

activitycontrol

%CAenzyme

activityexposed

A20–22

600

0.16

4ls/5Hz

0.0064±0.0015

0.0059±0.0022

34±8

28±9

(n¼18)

(n¼20)

A20–22

600

0.23

4ls/7Hz

0.0024±0.0018

0.0036±0.0019

16±12

24±13

(n¼8)

(n¼10)

(p¼0:100)

B26–29

20

5.6

4ls/5Hz

0.0025±0.0011

0.0027±0.0018

21±10

23±14

(n¼18)

(n¼25)

B22

20

7.8

4ls/7Hz

0.0027±0.0014

0.0058±0.0014

14±8

31±9

(n¼22)

(n¼27)

(p¼8:6E)10)

B29

20

11.1

4ls/10Hz

0.0034±0.0010

0.0046±0.0014

28±8

37±12

(n¼7)

(n¼9)

(p¼0:041)

342 A. Doria et al. / Infrared Physics & Technology 45 (2004) 339–347

In the two sets of exposures (setup A and B), we

investigated the effect of 130 THz at different pulserepetition rate and average power delivered to the

CA-loaded liposomes. We obtained a significant

increase of the p-NPA hydrolysis rate ðDA=minÞfrom 0.0027± 0.0014 to 0.0058± 0.0014

(p ¼ 8:6�10) at 7 Hz, while there was no significantincrease at other frequencies of pulse repetition

rate (Table 1). This increment was calculated over

three sample and sham liposome preparations.Interesting, the observed differences in p-PNA

hydrolysis rate were significantly higher in setup B

than in setup A, indicating that real time exposures

might reveal possible reversible effects induced by

the field. In Fig. 5 the effect of exposures is re-

ported as the increase of enzymatic activity in

comparison to the total CA activity, determined

after rupture of liposomes by detergent, taken as100%. We obtained a significant increase of CA

activity from 14±8 to 31± 9 ðp ¼ 1:72E� 09Þ. Inspite of an intrinsic variability of the membrane

system due to the use of different liposome prep-

arations throughout all the experiments, the ob-

served effects cannot be merely due to a difference

of average power delivered to the system.

Our preliminary results indicate that the mem-brane model used in these studies appears to be

a system sensitive to THz exposure, especially

regarding the frequencies of pulse repetition rate.

Resonance effects on these cationic liposomes

entrapping carbonic anhydrase have already been

observed in the ELF (extremely low frequencies)

region at 7 Hz [8,9]. Future studies will provide

further information about any possible correlationbetween resonance effects observed at a pulse

THz effects on CA-loaded Liposomes

0

10

20

30

40

50

5B 7B 10B 7APulse repetition rate (Hz)

% Enzymatic activity

Sham

Exposed

Fig. 5. THz-stimulated diffusion of p-NPA through CA-loaded liposomes at different pulse repetition rates (the suffix A or B refers to

the exposure setup A and B, respectively). The total enzymatic activity of CA liposomes, after detergent rupture, was taken as 100%.

Table 2

Absorption coefficient of whole blood, serum and water at

A. Doria et al. / Infrared Physics & Technology 45 (2004) 339–347 343

repetition rate of 7 Hz and the previous effects

observed in the ELF region.

120 GHz

Whole blood a ¼ 75 cm�1

Serum a ¼ 71 cm�1

Water a ¼ 85 cm�1

3. Irradiation of whole blood

In this part of the work, human lymphocytes

have been used as a biological model for studying

potential genotoxic effects of THz radiation. The

study of genotoxic effects is indeed one of the most

interesting aspects in the risk assessment of humanexposure to ionizing and non-ionizing electro-

magnetic radiation, due to the close correlation

between DNA damage and cancer occurrence.

Human lymphocytes are a well-known biological

system playing a key role in the defense mecha-

nisms, they are the only blood constituents with a

nucleus, they are semi-synchronised cells easily

obtainable from peripheral blood and can bestimulated to divide in vitro by a variety of sub-

stances (mitogens).

Prior to the irradiation of whole blood, trans-

mission measurements performed at the frequency

of 120 GHz showed that absorption at THz fre-

quencies is mainly due to the water content of the

plasma [10,11]. The evaluation of the absorption

helps in the identification of the THz powereffectively absorbed by the different blood com-

ponents among which there are lymphocytes to be

tested in the genotoxic analysis. The measured

absorption coefficient of whole blood, serum andwater are reported in Table 2.

Cytogenetic techniques allow one to test DNA

damage, at cellular and molecular level, induced

by various chemical and physical agents. Two

different tests, the Micronucleus (MN) and the

Comet assays, with different sensibility, have been

applied on human lymphocytes. Different expo-

sure set up have been employed by using theCompact FEL at the ENEA Research Centre of

Frascati. The cytokinesis-block micronucleus

technique is a very sensitive and simple indicator

of structural chromosome damage, which also

provides information on cell cycle progression [12].

The alkaline single-cell gel electrophoresis (SCGE)

or Comet assay, is a promising tool for measuring

DNA damage at the individual cell level in bothin vitro and in vivo studies. In this type of assay

DNA damages result in a ‘‘comet’’, clearly visible

with the microscope, left from the cell when sub-

jected to electrophoresis. A brief description of the

assays and of the related experimental activity is

given in the following.

344 A. Doria et al. / Infrared Physics & Technology 45 (2004) 339–347

3.1. Micronucleus test

The nucleus is the organelle in the cell that

contains the genetic material (DNA) that controls

normal cellular function and cellular reproduction.In cells of eukaryotic organisms, the nucleus con-

tains DNA packaged into chromosomes. Chromo-

some shape, size, and number are constant for a

species. During cell division, the genetic material

replicates and then divides equally between the

two daughter cells that are produced. If the pro-

cess is interrupted, or the chromosomes are broken

or damaged by an external agent, like for instanceradiation, the distribution of genetic material be-

tween the two daughter nuclei during cell division

may be affected and pieces or entire chromosomes

may fail to be included in either of the two

daughter nuclei. When this occurs, the genetic

material that is not incorporated into a new nu-

cleus may form its own ‘‘micronucleus’’ which is

clearly visible with a microscope. Thus, to assesschromosomal damage by means of MN test,

samples are irradiated and then the frequency of

micronucleated cells is determined after cell divi-

sion is occurred. If an exposed group of samples

shows a significantly higher frequency of micro-

nucleated cells than the untreated control samples,

the radiation is considered to be capable of

inducing structural and/or numerical chromo-somal damage.

Previous experimental activity, performed on

whole blood from 9 healthy subjects aged between

30 and 45 years, showed that THz radiation for 20

min at both 120 and 130 GHz at a pulse repetition

frequency of 2 Hz does not affect MN formation

and does not influence cell proliferation [10,11]. In

the following we report the results of 20 minlymphocytes irradiation by THz radiation at 130

GHz and at a pulse repetition frequency of 5 and 7

Hz at a higher incident power.

In the first set of experiment an average power

of 3.5 mW, at a pulse repetition frequency of 5 Hz

was delivered to the sample. The corresponding

peak electric field is 7.8 kV/m, while the average

electric field is 35 V/m. For each irradiation set upblood samples from five healthy subjects have been

investigated in order to take into account indi-

vidual variability. The samples were placed in Petri

dishes of 52 mm diameter and irradiated for 20

min. The biological assay was conducted following

the protocol described in [11]. Results of the MN

test are reported in Fig. 6(a).

In the second set of experiments the pulse repe-

tition rate was raised to 7 Hz, causing an increaseof the average power up to 5 mW, and a corre-

sponding increase of the average electric field to

42 V/m. Results of the MN test are shown in Fig.

6(b). The cell proliferation is evaluated by scoring

the number of nuclei in 500 cells and determin-

ing the Cytokinesis-block proliferation index

(CBPI); the definition of the mean number of cell

cycles per cell [13] is:

CBPI ¼ ½M1þ 2M2þ 3ðM3þM4Þ=Nwhere M1 to M4 indicate the number of cells with

1–4 nuclei respectively, and N the total number of

cells scored.

All the experiments performed so far indicatethat radiation at 130 GHz does not induce

chromosome damage (MN assay) in human lym-

phocytes and does not induce alteration of cell

cycle kinetics (CBPI).

3.2. Comet assay

The comet assay consists of a single cell gel

alkaline electrophoresis to measure DNA damage

in various types of cells exposed to potential

genotoxic agents both chemical and physical.

Damage results in a comet, left from the mainnucleus of the cell when subjected to electropho-

resis, and clearly visible with the microscope as

illustrated in Fig. 7. Single cell electrophoresis is a

rapid method for detecting DNA strand breaks in

lymphocyte cells. In the original form of the assay

[14] cells embedded in agarose on microscope

slides were lysed and subjected to electrophoresis

under neutral conditions, enabling the detection ofDNA double strand breaks. A later modification

using alkaline conditions made it possible to detect

DNA single strand breaks and alkali labile sites

[15].

In the present study the Comet test was per-

formed on lymphocytes separated from the aque-

ous part of the blood (serum) and directly exposed

to THz radiation with two different experimental

Fig. 6. MN frequency and CBPI in human lymphocytes following 20 min THZ irradiation at 130 GHz and two different pulse

repetition rate: (A) 5 Hz, (B) 7 Hz. Results are expressed as mean±SD of five healthy donors employed for both 5 and 7 Hz.

Fig. 7. Nuclei of human lymphocytes as appeared following an

alkaline single cell gel electrophoresis. It is evident a damaged

nucleus, in which DNA fragments move from the main nucleus

forming the tail of the comet.

A. Doria et al. / Infrared Physics & Technology 45 (2004) 339–347 345

setup. Both setup resulted in a higher power

impinging on the single cells when compared to the

power impinging on lymphocytes immersed in the

serum, thus reducing the shielding effect of water

mentioned earlier and described by the absorption

data reported in Table 2. The two setup are shown

in Fig. 8. In the first setup (Fig. 8(a)) a centrifugedblood sample, composed of a lymphocyte layer on

top of red blood cells, is prepared in an Eppendorf

tube, which is placed inside a metallic cone and

then exposed to THz radiation with the cap of the

tube kept closed. In the second setup (Fig. 8(b))

the radiation is focused by means of the metallic

cone to the top of the Eppendorf tube, which is put

inside a PVC holder. This holder allows the tube tobe kept open avoiding the reflection and diffusion

of THz radiation by the cap of the Eppendorf. In

the setup of Fig. 8(a) we have to take into account

Fig. 8. Lymphocytes separated from the serum are exposed

directly to THz radiation. (a) First setup: the eppendorf tube

inside the metallic cone. (b) Second setup: the Eppendorf tube

placed into the PVC holder (after the metallic cone).

346 A. Doria et al. / Infrared Physics & Technology 45 (2004) 339–347

that the effective power impinging on the sample is

reduced by a fraction equal to the ratio between

the sample area and the area of the cross-section ofthe cone itself. In the setup of Fig. 8(b) the whole

power entering the cone also reaches the sample

surface.

Radiation is delivered to the sample in short

pulses at 7 Hz repetition rate, as described before.

The incident average power in setup (a) is 1.9 mW,

while in setup (b) it is 5.0 mW, corresponding to an

average electric field of 152 and 245 V/m respec-tively and a peak electric field of 28.7 and 46 kV/m

respectively (calculated on the 4 ls pulses). Fivesubjects were investigated in each setup using the

following protocol:

Fig. 9. Tail factor % in human lymphocytes following 20 min THz irra

setup A and B.

For each subject two Eppendorf tubes (1.8 ml)

containing each 1.5 ml of whole blood are ex-

posed/sham exposed to 130 4Hz for 20 min with

the above irradiation parameters after a suitable

portion of serum is removed by centrifugation at

1000 rpm · 10 min. At the end of exposure/shamexposure the homologous serum is added and the

blood samples are processed to perform the Comet

assay according to the method developed by Singh

[15].

The results of the Comet assays, expressed as

comet tail factor % according to Ivancsitz et al.

[16], are shown in Fig. 9 for the two different setup.

Our preliminary results obtained exposing wholeblood samples to 130 GHz radiation produced

different results for the two different setup. When

the sample is exposed inside the PVC holder (Fig.

8(b)), there is no evidence of a statistically signifi-

cant difference between the exposed sample and

the sham sample. On the contrary, when the

sample is exposed inside the metallic cone (Fig.

8(a)), a statistically significant effect is observed,despite the lower incident power. A possible

explanation of this result is that, when the Ep-

pendorf tube is placed inside the cone, the cone is

acting as a resonant cavity leading to a much

higher value of the effective electric field at the

surface of the lymphocyte layer. Further

improvements on the irradiation setup and inves-

tigation on a larger number of subjects are inprogress to validate this positive finding.

4. Conclusions

Investigations of the effects of THz radiation

have been conducted on two important biological

diation at 130 GHz and 7 Hz pulse repetition rate, for exposure

A. Doria et al. / Infrared Physics & Technology 45 (2004) 339–347 347

systems: a membrane model based on CA-loaded

liposomes and human lymphocytes on whole

blood. The preliminary results indicate the pres-

ence of a change of membrane permeability when

the samples are exposed to 130 GHz radiation

with a pulse repetition rate of 7 Hz. As to theexposure of human lymphocytes, the MN assay

performed so far indicate that radiation at 130

GHz does not induce chromosome damage (MN

assay) and does not induce alteration of cell cycle

kinetics (CBPI). However, in one of the experi-

mental setup, a positive result of the Comet assay

has been found that need further investigation. A

model of the interaction mechanism that takesinto account the evaluation of the power effec-

tively absorbed at a microscopic level by the

lymphocytes when placed in a THz resonant

cavity is being developed and will be the object of

future work.

Acknowledgements

This work has been carried out with financial

support from the Commission of the European

Communities, specific RTD programme ‘‘Quality

of Life and Management of Living Resources’’,

QLK4-CT2000-00129 ‘‘Tera-Hertz Radiation In

Biological Research, Investigation on Diagnostics

and study on potential Genotoxic Effects’’.

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