1. The THz-BRIDGE Project: Tera-Hertz radiation in Biological Research, Investigation on Diagnostics and study on potential Genotoxic Effects RESEARCH OBJECTIVES OF THz-BRIDGE PROJECT n To provide a spectroscopic database for selected enzymes, proteins, biological membranes and cells in the frequency range from 100 GHz to 20 THz under irradiation conditions that preserve the integrity and functionality of the biological samples. (Workpackage 1) n To identify critical frequencies, which might induce damages on biological systems, and to determine the spectral regions for optimal contrast in imaging applications. (Workpackage 2) n To assess potential risks due to the exposure of membranes, cells, and DNA to pulsed and CW THz radiation and to define exposure standards for THz biomedical imaging applications; to monitor and recommend THz exposure conditions at specific occupational sites. (Workpackage 3) The aim of this research project is to investigate the interaction of Terahertz (THz) radiation with biological systems. Present and rapidly increasing future applications of radiation in this spectral range, necessitate preventive rather than reactive research. The project follows a streamline of increasing complexity from bio-molecules to cells, e.g. membranes, chromosomal and DNA integrity. The objectives of the project are to analyze the physical mechanisms of interaction, to assess risk of potential damage to biological activity, both functional and morphological, and to guide and accompany the development of microscopic imaging at THz frequencies for biological and biomedical applications. The project will bridge the existing gap of knowledge regarding the effects of Terahertz (THz) radiation on biological systems. THz radiation covers the frequency range between 100 GHz and 20 THz (i.e. a wavelength between 3 mm and 15 µm), which spans the spectral interval between the microwave- and the infrared regions of the electromagnetic spectrum. Workplan – WP1 n Development of biological samples, basic spectroscopic investigations in the spectral range 100 GHz - 20 THz n Measurements of Reaction Induced Differential Spectra (RIDS) n Time-resolved Spectroscopy at FEL Facilities n “Time Domain Spectroscopy” (TDS) by solid state THz sources n Assignment of absorption spectra to molecular bonds and functions Workplan – WP 2 n Study of the exposition of human blood to THz radiation as a function of: frequency, average and peak power, modulation conditions n Genotoxicity Test on human lymphocytes: Micronucleus, Comet, FISH n Irradiation of DNA bases and measurement of the presence of fragments by mass spectroscopy techniques n Study of the exposure of liposomes to THz radiation: on-line evaluation of the carbonic anhydrase (CA) activity under THz exposure as a function of frequency, average and peak power, and of modulation conditions n Investigations on epithelial cells: keratinocytes cell lines (NHK) and corneal cells (HCE-T) (Alamar-Blue cell cell viability assay) effects on the adhesion molecules of epithelial tissues (Fluorescein Leakage assay on tight junctions) n Modelling of the interaction mechanism Workplan – WP 3 Spectroscopy of Proteins, Enzymes, Biological Membranes and Cells (Human Lymphocytes) : UFRANK, FZR-Dresden, USTUTT, ENEA, TVL Evaluation of Biological Effects In Vitro DNA Bases and Human Lymphocytes: ICEmB, TAU, NHRF, ENEA, FZR-Dresden Evaluation of Biological Effects In Vitro: Membranes and epithelial-cell cultures: ICEmB, ENEA, UNOTT, FZR-Dresden, TVL Safety issues at specific occupational sites: TVL, FZR-Dresden, UNOTT, TAU Interdisciplinary consortium: 9 groups, 5 countries Project start date: February 1, 2001 Duration: 36 months Total cost : 2 MEuro - EU Contribution : 1.4 MEuro THz-BRIDGE PROJECT KEY FACTS n ENEA-Frascati (coordinator) (I) n Forschungszentrum Rossendorf-Dresden (D) n Tel-Aviv University - Dept. Physiology and Pharmacology (IL) n Stuttgart University - I. Physics Institute (D) n J.W. Goethe University - Biophysics Institute –Frankfurt (D) n IREA -Naples, IMS-Rome, DIE-University of Rome - (ICEmB) (I) n National Hellenic Research Foundation – Athens (EL) n Teraview Limited – Cambridge (UK) n University of Nottingham - Biomedical School (UK) Whole blood samples are exposed to THz radiation under controlled conditions After preparation of cultures, lymphocytes are induced to divide After 44 h of growth Cytocalasin-B is added to stop cell division at nuclear level Slide preparation and observation of Micronuclei frequency EXAMPLE OF GENOTOXICITY TEST THE MICRONUCLEI ASSAI Statistical analysis of experimental results EFFECTS ON MEMBRANE MODEL SYSTEM A good model for the membrane system is composed by carbonic- anydrase loaded liposomes vesicles. With such a model it is possible to evaluate the effects of THz radiation on the membrane permeability n The substrate has a very low self-diffusion rate across intact liposome bilayer. n The substrate can be hydrolyzed by CA inside the liposomes only if it permeates across the bilayer. n Any radiation induced increase of hydrolysis rate of substrate catalyzed by CA accounts for the effect on membrane permeability. n The kinetic measurement is made following for 2-3 minutes the appearance of reaction product p-nitrophenolate anion at its peak absorbance ( =400 nm) on an Cary 50 Scan spectrophotometer Transmission of 120 GHz ENEA-FEL radiation was measured in saline solution, culture medium (not shown in the graph), serum and whole blood. Results were compared to water absorption Culture medium  = 83 cm -1 Saline Solution:  = 79 cm -1 Whole blood:  = 75 cm -1 Serum:  = 71 cm -1 Water (non reported on the graph): = 80 cm -1 The THz absorption spectra of uric acid and human hemoglobin (Teraview Ltd.) EXAMPLES OF SPECTROSCOPY MEASUREMENTS Significant progress has been made on the design of spectroscopic cells and on the choice of suitable materials transparent in the frequency range of interest. Polystyrene exhibits excellent optical properties in the wavelength range between 200  m and 3 mm (frequency range 1.5 THz to 100 GHz). Other materials like ZnTe can be used at shorter wavelengths. n Coherent radiation from Free Electron Lasers, Gas Lasers and Solid State Sources is available in a wide spectral range at the partner sites and at collaborating European FEL facilities. n Features: Wide tunability and power ranges, amplitude modulation n Soft X-ray microscopy, SEM, Bio-labs available at ENEA, TAU and FZR THE THz SOURCES 5  m 30  m 10  m hh BIAS P  |E 0 (z,t)| 2 E 0 (z,t) An ultrafast laser pulse is used to “switch” on a circuit (a coplanar transmission line antenna). A current transient is generated, producing a time-dependent dipole moment that emits radiation. The short duration of the pulse allows THz emission. The non-linear properties of selected semiconductors are utilized to produce optical rectification of ultrashort laser pulses. The non linear polarization so generated travels in the medium with a lower phase velocity, due to the lattice vibration contribution. The result is an emission in a typical Cerenkov cone configuration.The rapid oscillations of the laser E field are rectified and only the envelop remains. In a FREE ELECTRON LASER radiation is produced by a relativistic electron beam traveling through a magnetic structure (undulator). FELs can produce very short – high power THz laser pulse. Auston Switch Optical Rectification Free Electron Laser n Survey of exposure conditions of technical personnel at specific occupational sites, where THz sources are employed or developed. n Distribution of a questionnaire to collect information on the main radiation parameters of THz sources currently in use: (e.g. frequency range, power level, modulation) on biological and potential biomedical applications of such sources on the exposure conditions (if any) of technical personnel on the safety measurements or precautions currently adopted n Recommendations on safe exposure conditions. Role of T-rays in the biomedical field Biological applications are based on the specific spectroscopic fingerprints of biological matter in the THz spectral regions. The different values of the absorption coefficient and index of refraction between water and tissue carbonated proteins at such frequencies, provide a unique contrast mechanism for biomedical imaging applications. In the above picture, the THz image of basal cell carcinoma (BCC) obtained by Terahertz Pulse Imaging (TPITM) is shown on the left. The red areas indicate areas of cancer. On the right is the visible image showing no obvious signs of cancer growth. Expected achievements and preliminary results Spectroscopic measurements are used to identify the frequency intervals where THz radiation is absorbed by a specific system. First spectroscopic investigations have been carried out on human blood, human hemoglobin, melanin and uric acid in the THz region. An exposure set-up for the irradiation of human lymphocytes has been designed and constructed. First irradiation experiments on whole blood have been performed at a frequency of 120 GHz. Following irradiation, the Micronucleus assay was performed by ICEmB on human lymphocytes cultures. The preliminary results obtained indicate that, under the adopted experimental conditions, THz radiation does not affect micronucleus frequency and cell cycle kinetics in peripheral blood human lymphocytes, as shown by comparing sham exposed cultures with exposed ones. Preliminary exposure of human skin cells to THz radiation has also been undertaken. FURTHER INFORMATION 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”, Key Action 4 “Environment and Health” - contract QLK4-2000-00129. Project Management Evaluation of results: Spectral database & Reps. Risk Assessment Safety Issues Dissemination of results Interface with International Projects Identification of critical frequencies Liposomes, blood & skin-cell cultures WP-1 Spectroscopy of proteins, enzymes membranes and cells WP-2 Evaluation of biological effects in vitro WP-3 Safety Issue at specific Occupational sites Spectral database on biological systems Definition of exposure levels Spectral information on contrast in THz Imaging Recommendations on THz exposure Assessment of THz radiation effects An R&D project funded by the European Commission - 5 th Framework Programme: Quality of Life and Management of Living Resources - Key Action 4-Environment and Health Contract QLK4-CT2000-00129 Survey on the use of THz radiation on the THz-BRIDGE web site THz-BRIDGE Web site http://www.frascati.enea.it/THz-BRIDGE FREE ACCESS AREA n Project Description n Progress reports n Official document n Contacts Project Coordinator Dr. G.P. Gallerano – ENEA Frascati E-mail: gallerano@frascati.enea.it Tel: +39-06-94005223 fax: +39-06-94005334 Typical Time Domain Spectroscopy layout EU Scientific Officer Minna Wilkki - EUROPEAN COMMISSION E-mail: minna.wilkki@cem.eu.int Tel: +32-2-299 5573 fax: +32-2-296 4322