De Cuyper M., Bulte J.W.M. - Physics and chemistry basis of biotechnology (Vol. 7) (2002)(en)

Wim Mondelaers and Philippe Lahorte

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thorough understanding is only brought about as the result of the combined use of several techniques which thus requires an interdisciplinary approach.

The current chapter represents the second half of our overview. It consists of three major parts. First of all a survey is given of the experimental techniques and theoretical methods currently available for studying the effects of radiation on biological systems from the physical to the biological stage. In a second part we will elaborate on some important techniques (electron paramagnetic resonance spectroscopy and quantum chemical calculations) with which direct information concerning identity and structure can be obtained of the radicals involved in processes in the chemical stage. Also an overview will be given of the fundamental research in this field. This approach is partly inspired by our own research efforts in which the determination of radical identity and structure is often either a goal in itself or a necessary hurdle that has to be taken in the development of new applications. The final part will be devoted to technological aspects of the irradiation process. In specific, the basic principles of accelerator technologies will be elucidated and an overview will be given of the applicationoriented irradiation research in biology and related fields such as biomedical and environmental engineering, food technology, medicine and pharmacy. Radiation research in the field of radiotherapy will not be treated as this is beyond the scope of the present review.

The target audience of this contribution being bio(techno)logical scientists, an attempt was made to describe technologies and methods from a qualitative point of view, focusing on conveying the overall ideas and limitations, and the biologic relevance of the data and information that can be obtained from them. For further exploration and a deeper understanding the reader will be referred to literature citations and reference works.

2. Experimental and theoretical methods for studying the effects of radiation

Figure 1 gives an overview of the broad spectrum of experimental and theoretical tools for studying physical, physico-chemical, chemical or biological aspects of the effects of radiation exposure on biological systems. Obviously, the delineation of the scope and the field of activity of each method is somehow subjective. The classification as shown in Fig. 1 is therefore to be interpreted as indicative rather then exactly corresponding to the boundaries of the four stages of interaction which eventually exist only by virtue of human intellect. In this section we will briefly discuss the experimental techniques and theoretical methods, available for the combined physical and physico-chemical stages, and the biological stage. The techniques for studying radicals in the chemical stage will be discussed in more detail in paragraph 3.

As has been extensively discussed in the first part of this overview the physical and physico-chemical stages on the time-scales of radiation effects are characterised by a distribution of the radiation energy among the irradiated specimen. Along the path of the primary ionising species, radicals and electrons are formed in tracks. In most environments these intermediates are highly reactive and, hence, can exist only for very short periods. In this case, these transient species diffuse into the medium and give rise

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to a set of chemical reactions. Although some information concerning the reaction intermediates can be inferred from the results of steady-state experiments, detailed characterisation and identification of these unstable species requires the ability to monitor kinetic behaviour, often of very low concentrations, over very short time intervals. The bulk of our understanding of radiation chemistry has resulted from the use of pulse radiolysis techniques. In principle this technique consists of delivering a very short pulse (nanoor picosecond time range) of ionising radiation to a chemical

Figure 1. Experimental and theoretical techniques for studying the effects of radiation on biological matter

system so that a nonequilibrium system is produced containing significant concentrations of transient species. These can subsequently be monitored in time using an optical or electrochemical method such as absorption spectroscopy or conductivity measurements, respectively (Patterson 1987; Von Sonntag 1995). For the production of the ultrashort pulses electron accelerators are most often used. Some fundamental

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principles underlying accelerator technology are discussed in detail in paragraph 4. Wardman (1991, 1994) has reviewed the value and applications of pulse radiolysis measurements in free radical biology with respect to investigation of radical kinetic properties.

From a theoretical point of view, the Monte Carlo method is generally considered the most accurate method for calculating dose distributions. It is a statistical simulation method which allows one to calculate the tracks of individual particles by sampling appropriate quantities from the probability distributions governing the individual physical processes using machine generated (pseudo-)random numbers. Average values of macroscopic properties such as particle fluence, energy spectrum and absorbed dose can then be obtained by simulating a large number of particle histories. Microdosimetric Monte Carlo calculations allow for the modelling of the impact of radiation on an atomic level. In this way direct and indirect damage to DNA might be investigated by modelling the radiation formation of single and double strand breaks (Briden 1999, Hill 1999; Moiseenko 1998a-b). However, also macroscopic properties of chemical reactions such as rate constants, radiolysis end products and product yields are amenable to Monte Carlo simulations (Bolch 1998, Hamm 1998).

The methods described above may be used in a wide variety of systems but none of them yield substantial data that may be directly interpreted to give information on the structure of the transient species. By contrast, the techniques available for studying radicals in the chemical phase allow for the description of the chemical structure and the characterisation of radical processes. As already mentioned, these will be discussed in more detail in paragraph 3.

In the biological stage the consequences of irradiation are expressed in terms of altered biochemical functions in the living species which can emerge after periods of seconds (e.g. inactivation of enzymes) to years (e.g. development of cancers). Evidently, techniques developed for research in this stage probe for the detection of the radiation effects and the induced biochemical modifications. On the experimental side, high performance liquid chromatography (HPLC) and gas chromatography (GC) can be applied in combination with sensitive detection techniques such as electrochemistry (EC) or mass spectrometry (MS) to monitor the formation of oxidative base damage within cellular DNA (Cadet 1998, 1999). Alternatively, the single cell gel electrophoresis (comet) assay can be used (Fairbairn 1995; Koppen 1999). The alkaline type version of this assay can be applied to isolated cells and allows the measurement of DNA strand breaks and alkali-labile lesions. The comet assay offers high sensitivity at the cost of reduced specificity. The latter can be increased by using the assay in combination with either DNA-glycosylases (in order to convert base lesions in additional DNA strand breaks) (Pouget 1999a, b) or immunofluorescence detection (Sauvaigo 1998). Because of the importance of strand breaks as critical lesions produced by ionising radiation, a lot of effort has gone into developing methods (currently routine methods in molecular biology and biotechnology) with which strand breaks can be measured. Of these we mention the sucrose gradient sedimentation, the non-denaturing filter elution and the nucleoid sedimentation technique (Prise 1998). Of special importance has been the development of pulsed field gel electrophoresis (Iliakis

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1991; Shao 1999) and derived variants which have revolutionised the separation of large molecules.

A very rapidly emerging field with huge potential for biotechnology and medicine is functional genomics (Claverie 1999; Dyer 1999; Langer-Safer 1997). With this term a platform of technologies are described, comprising among others differential display, proteomics and bioinformatics, which aim to establish a functional relationship between a particular genotype and a given disease state. Advances in microarrays and gene chips form the core of differential display technology which enables researchers to understand differences in gene expression in normal and diseased cells and tissues. This approach is very useful in identifying gene patterns which are upor downregulated in a given disease state, in a specific environment or in response to external stimuli. Proteomics is a complementary approach which aims to produce highresolution protein maps for a given organism. It enables the analysis of changes in protein abundance and post-translation modification in both in the normal and pathologic state. Finally, molecular bioinformatics comprises the development and application of computational algorithms for the purpose of analysis, interpretation and prediction of the vast amount of molecular biologic data currently generated with modem biotechnology techniques.such as the ones mentioned above. Main applications of these sophisticated informatics tools are primarily DNA sequence analysis, protein structure prediction and structure-function relationships in DNA and proteins. While the overall majority of efforts in genomics have concentrated on uncovering genetic and biochemical pathways and mechanisms for the development of new drugs and therapies, the same ensemble of techniques holds the potential for studying the effects of ionising radiation on living organisms. As an example one might investigate, using micro-array technology, the upor down-regulation of genes coding, for instance, for repair enzymes in normal or tumour cells exposed to irradiation. Research in this area is however still in its infancy and major developments remain to be expected.

3.Studying radiation-induced radicals

3.1.EXPERIMENTAL AND THEORETICAL METHODS FOR DETECTING AND STUDYING RADICALS

Now we will focus on the most important experimental technique and theoretical method (electron paramagnetic resonance spectroscopy and quantum chemistry, respectively) currently available for gathering information on radiation-induced bioradicals in the chemical stage. The most prominent questions in this respect are often related to chemical reactivity and thus concern the identity of the radicals involved as well as their geometry and electronic structure. The basic aspects of the technique in question will be discussed together with the physical and chemical information that can be obtained from it. In this respect; attention will also be paid to the interaction between experiment and theory.

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3.1. I. Electron Paramagnetic Resonance

The unique feature of electron paramagnetic resonance (EPR) or electron spin resonance (ESR) is that it is a spectroscopic technique that is applicable to paramagnetic systems. These are species containing one or more unpaired electrons resulting in a net electron spin angular momentum. It is an example of a magneticdipole spectroscopic technique in which magnetic fields are used to induce transitions between various energy levels in the atomic or molecular species by interaction with the electronic dipole moment.

An electron possesses a spin magnetic moment, and in the presence of an applied magnetic field its two permitted spin states a and b have different energies as can be

seen from Fig. 2a. The transition from the lower to the higher energy state is made most efficiently when the energy-level separation equals hv, where h is Planck’s constant and v is the frequency of a microwave electromagnetic field. Then the sample and the electromagnetic field are in resonance. Thus an EPR spectrometer will basically consist of a source of microwaves (most frequently a klystron), a magnet capable of providing a tuneable but stable and homogeneous magnetic field and a device to detect the absorption.

In practice the absorption is monitored upon performing a scan of the static field B while the frequency v of the microwave radiation at which the spectrometer is operated, is fixed. EPR spectra are the first-derivative of the energy absorption with respect to the magnetic field B (Fig. 2a).

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EPR spectra may convey a remarkable wealth of significant information both concerning the identity and the electronic structure of the contributing radicals. This can readily be understood upon inspection of the spin hamiltonian

Hspin=g. be . S . B – gN . bN. I. B + S . A. I

which is the quantum mechanical operator describing the spin-related energy changes in radical species. The first two contributions are the electronic and the nuclear Zeeman terms, respectively, caused by the interaction of the magnetic field B and the magnetic moments of the electrons (S) or nuclei (I) in the system. g and g N are the electron and nuclear magnetogyric ratios, and be, bN the Bohr and nuclear magnetons. The remaining term is the hyperfine interaction term. It arises from the interaction of the unpaired electron(s) with nearby nuclei in the molecule with a net spin magnetic moment (e.g. 1H, 13C and 14N).

The principal, chemically relevant information that can be obtained from an EPR spectrum is contained by the g-factor and the hyperfine tensor A. From these quantities valuable information can be gained about the structure and orientation of free radicals in single crystals as well as the distribution of the unpaired electron over the magnetic nuclei in the radical species. However, the unequivocal determination of these parameters can be seriously hampered due to the potential complex character of reallife spectra such as the ones illustrated in Fig. 2b. In principle, quantum chemical calculations allow for the theoretical determination of the main EPR spectroscopic parameters. Accurate calculations of this type are however computationally demanding. Therefore they are starting to be performed on a routine basis only since the last few years, facilitated by the ever-increasing available computer power. In this respect, the calculation of the matrix elements of the hyperfine tensor A (the so-called hyperfine coupling constants) in radical species of biological interest is developing into a powerful tool to the experimental scientist for the elucidation and interpretation of EPR spectra. This is illustrated, for instance, in the case of the intensively investigated radicals of the X-irradiated amino acid alanine (Lahorte 1999a). A treatment of the basic concepts of quantum chemical methods will be the topic of the next paragraph.

The atoms and molecules amenable to study by EPR can either exist in a paramagnetic ground state or be (temporarily) excited into a paramagnetic state (e.g. by irradiation). Typical systems that can been studied include free radicals in the solid, liquid or gaseous phase, transitions ions, point defects in solids, systems with more than one unpaired electron (triplet-state systems or biradicals) and systems with conducting electrons. In biological systems paramagnetic species are mostly found as transition metal complexes (e.g. Cu in copper proteins and Fe in haem proteins), as intermediary products in electron transfer processes or as radiation degradation products of biomolecules. In the present review, we will concentrate on the latter category. For a compilation of the literature in the former fields, the reader is referred to separate overviews (Atherton 1996).

If in the same system two or more nuclei of the same spin are present, ambiguity can arise in the assignment of hyperfine couplings. Furthermore, if the spacing within a

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