Current Projects

Radiation damage to biological macromolecules (DNA,proteins)

The main radiation research activity at the ESR-laboratory over the last 10 years has been concentrated on two major projects, which are inter-related but sufficiently different to be performed in collaboration with two research groups with different expertice and experimental facilities.

Inactivation and mutation are the most serious consequences for cells from chromosomal damage by ionizing radiation. The basic action of radiation is simple: deposited energy excites bound electrons to the continuum state; subsequent loss of electronic energy leads to electron capture and a bound state some distance away from the initial interaction site. Thus, the fundamental products are pairs of radical ions and the fundamental problem is to identify how the radical ions lead to the known biological endpoints. Because of the complexity of molecules and especially of cells, addressing this problem requires coordinated approaches in physics, physical chemistry, biochemistry, molecular biology, and medicine. Proceedings from several international conferences show the scope of the problem and indicate the present status of the field. (See e.g. "Radiation damage in DNA. Structure/funcion relationships at early times" (A.F. Fuciarelli and J.D. Zimbrick, Eds.) Battelle Press, Columbus 1995; "The Early Effects of Radiation on DNA" (E.M: Fielden and P.O'Neill, Eds) NATO ASI Series Vol H54, Springer verlag, Heidelberg 1991; Physical and Chemical Mechanisms in Molecular Radiation Biology, (W.A. Glass and M.N. Varma, Eds.), Plenum Press, New York (1991); Electronic Magnetic Resonance of the Solid State,(J. Weil, Ed.), Canadian Soc. of Chemistry, Ottawa (1987).

Broadly speaking, the chromosomal damage may arise from energy deposited within DNA itself and its hydration sheath (leading to "direct" or "unscavengable" damage), or elsewhere (leading to "indirect" or "scavengable" damage) While there is widespread agreement that the scavengable damage is the result of OH radicals from water radiolysis, there remain several questions about the initialization and the progression of the unscavengable damage. The initial products of energy deposition from ionizing radiation to organic molecules are radical ions. These species are highly reactive and unstable. The normal continous wave ESR technique is by nature a slow technique and can not detect fast (10-9 - 10-12 sec) processes (in contrast to pulse-EPR methods). The usual way of side-stepping this limitation is in cases where transformations are driven by thermal energy to remove as much heat from the system as possible by maintaining the samples at a low temperature (about 10 K is the best we at present can achieve) during irradiation and measurements, and then follow secondary radical reactions by allowing the samples to warm up in a controlled manner.

EPR and in particular ENDOR spectroscopy is uniqely suited to perform studies as those described above, with its potential of detailed and direct structural information which can be obtained from well-characterized crystalline systems in combination with x-ray diffraction methods.

The basic questions to which answers are sought within the activity of the EPR laboratory and collaborating laboratories are:

• What are the primary ionic products of DNA?
  Much of the previous work in the ESR-laboratory was intended (and still is) to address this issue by employing high resolution spectroscopy (ENDOR) to study well-defined molecular systems (crystals). Moreover, with the EPR/ENDOR techniques, the possibility of detecting sugar radicals is maximized.

• What is the proton transfer behavior of bases following ionization of DNA?
  The importance of proton transfers between the bases or between a base and other environmental units such as water has been clearly demonstrated in a large body of work the last four years. Of couse this is an important issue since the bases in DNA exist in favored tautomers, and thus will undergo site-specific proton transfers, if at all. A direct, clear-cut characterization of the protonation state of the primary ionic products will enhance the possibility to understand the mechanism for preferred stabilization of ionic species between the different sub-components of DNA. Such direct detection of the eventual transferred proton is possible with ENDOR, and this aspect forms an additional objective of our work.

• How can direct ionization of DNA lead to strand breaks?
  As has been pointed out repeatedly , strand breaks, particularly double strand breaks (dsb), are perhaps the most important lethal effect in cells; moreover, there is a significant probability that attempted repair of dsb's leads to insertion or deletion errors, and the probability of mutation. Strand breaks clearly occur from unscavengeable effects, and require damage to the sugar-phosphate backbone of the structure. However, at the present time, no sugar radicals have been positively identified as ionic products in DNA. Thus the twin questions arise: (1) are sugar radicals present as primary ionic products, but with characteristics somehow resistant to detection? (2) If not, then the damage must occur as a secondary process and by means of base to sugar damage transfer. If secondary processes are the means to sugar damage, what are the mechanisms and products leading to damage in the backbone region?

• How are the primary and secondary structures of proteins changed by ionizing radiation?
  The mechanisms leading to the stable damages in simple amino acids have been studied for many years, in most instances by conventional EPR techniques. The increased resolution of ENDOR spectroscopy allows for new information to be extracted from single crystal and polycrystal studies of irradiated amino acids, dipeptides and tripeptides. These studies also have relevance to our work with ribonucleotide reductase, mentioned below.

One major partner for collaborative research is the two laboratories led by
Prof. William H. Nelson Department of Physics and Astronomy, Georgia State University, Atlanta, GA, and
Prof. David M. Close, Department of Physics, East Tennessee State University, Johnson City, TN.
under a co-operative program supported by NIH and NATO Travel Grants This international collaboration has been very fruitful as can be seen from the publication list and will continue at least for the next 4 years. A collaboration on a Ph.D/post.doc level is also planned within this period. Under this collaboration most of the model systems containing DNA bases, nucleosides, nucleotides, and studies of protonation-deprotonation reactions are being performed.

A second major partner for collaborative research is
Prof. Anders Lund, Department for Measurement and Instrumentation, Linköpings University, , Linköping, Sweden.
This collaboration has been funded by grants from the Nordisk Råd and at present, over NorFA . The collaboration involves mutual exchange of scientists from both sides, as well as mutual exchange of cand.scient/dr. scient/Ph.D candidates from both countries. Under this collaboration, much of the studies involving damages in the sugar residues of the DNA molecule are performed, including effects of the phosphate group and the secondary reactions eventually leading to DNA strand break. Theoretical (quantum chemical, spectrum simulation, data analysis ) work needed for the successful building of models are developed mainly within this collaboration.

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Effects and methods for EPR spectroscopy

Recently, several projects have been intitiated aimed at developing more sophisticated metods for analysis of EPR and ENDOR spectra.

• One of these projects is a joint project with Prof. Anders Lund and  Docent Nikolas Benetis at IFM, University of Linköping. The goal for this project is to develop simulation programs for a complete description of the dynamics of paramagnetic molecules on surfaces ( eg. catalysts or proteins). Computer programs for simulating rotational/diffusional or tunnelling motion of radical molecular fragments are milestones on the road to the fullfillment of this project. This project also has support from both the Norwegian and the Swedich Research Council.

• The detection of weak quadropole interactions in organic radicals using ENDOR spectroscopy allows for a more precise determination of radical electronic structure than previously was accessible from EPR analysis of hyperfine structure. Methods for the analysis of these interactions and to fully exploit the information available from this analysis are under development.

• Deconvolution of complex EPR spectra is a difficult task. Several approaches to this objective are currently investigated, using both the "benchmark spectrum" approach and pattern recognition methods. The first goal of this work is the quantitative assessment of radical production in biological macromolecules as a function of radiation LET values.

• Development and practical application of methods for more effective analysis of ENDOR and ENDOR-Induced EPR spectra, employing a.o. the dependency of relaxations on temperature and microwave frequency Simple amino acids as glycine and alanine are the model compounds used for this work.

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EPR-/Alanine-dosimetri

The ESR-laboratory is presently opening a new field of research and activity. This is the use of EPR-spectroscopy within ionzing radiation dosimetry. In fact, the EPR laboratory was among the first ever to use EPR spectroscopy for the determination of radiation doses after a fatal reactor accident at IFE in Norway in 1982.( See E. Sagstuen, H. Theisen, and T. Henriksen, Dosimetry by EPR spectroscopy following a radiation accident. Health Physics 45, 961 - 968 (1983). However, over the last ten years a new standardized method of dosimetry has been developed which is based on the radiation responce of pellets of the amino acid l-alanine to ionizing radiation. This method, termed EPR-/alanine-dosimetry has gained wide acceptance in Europe and in the US and is by IAEA since 1989 considered to be the main secondary standard of high-dose dosimetry. The usability of this method is very large and varied, it ranges from food irradiation, radiation processing, personal (accident) dosimetry, and sterilization to medical (therapeutic) dosimetry and radiation monotoring (e.g. in CERN). In Norway this method has to date not been taken into use. However, at the EPR laboratrory efforts are now being made to adapt this method for use in this country and one of the staff members and two cand.scient. student is engaged in this work. So far interest in this method has been expressed from IFE and the Radium Hospital. In fact, in collaboraton with Professor Erik O. Pettersen and Dr. Dag Rune Olsen at the latter institution pilot experiments are now performed using EPR/alanine dosimetry to determine radiation doses involved in brachy-teraphy of patients. This method of dosimetry has the potential to accurately ( with care down to less than 2%) determine radiation doses in the range of 1 to 10+9 Gy, a combination of range and accuracy not superseeded by any other dosimetric metod in present use. Considerable effort is expected to be put into this field in the future within basic research and not the least within implementing some of the large potential for important practical applications in industri and medicine.
Also, EPR spectroscopy in the field of retrospective dosimetry and food irradiation are fields of interest for this laboratory. Within the field of EPR dosimetry, collaboration has been initiated with
Univ. lektor Eva Lund, Hälsouniversitetet, Linköpings University, Linköping, Sweden,
who in collaboration with several PhD candidates are engaged in projects similar to those outlined above. A separate application to NFR for support to this project has been submitted.

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EPR of biologically active metalloproteins (enzymes)

The EPR laboratory has for some time been interested in opening a new line of research into biofysikkical-biochemical fields of activity which are not related to radiation.Very recently, a co-operation with Professor K. Kristoffer Andersson at the Biochemistry Department, University of Oslo has been initiated. He has studied the methane monooxygenase since 1989 and several other hydroxylases.

The overall concern of Prof. Andersson's research is the study of the relationship between molecular structure and function in connection with evolutionary and applied aspects of macromolecules, as monitored by low temperature EPR as one main technique. The main emphasis is on biologically active metal-ions environments and small organic cofactors in proteins and enzymes, coordinated with investigations of chemical reaction mechanisms. This group of proteins play an important role in nature. Some of them are unique in that they catalyze many types of reactions, some of them very specifically, which can not be as effectively catalyzed by other known chemical system at similar low temperatures (300 K) for instance monooxygenase chemistry. The proteins with metals and/or organic cofactors, have in many cases an electronic-structure that in some redox-state give them properties such as paramagnetism and visible optic activity, which make them suitable for studies with physical methods with minimal interference from the majority of the protein's amino acids. These physical studies can give information directly or indirectly linked to the enzymatic reaction mechanism which can occur at these centers.

The plan is to examine the biological and/or chemical problems with broadest approach as possible and not be limited by techniques. In combination with classical biochemical and molecular biological methods, cryobiochemical and biofysikkical techniques will be used. If possible, small inorganic models will be used, mimicking the active sites of enzymes, so as to obtain information about the enzymatic sites and catalytic properties.

Other aspects deal with developing new general techniques for the study of cobalt containing proteins that can be used for enzymes with or substituted with this metal. Further research interests include fundamental (bio)chemical questions such as; electron transfer (ribonucleotide reductase, cytochromes, iron-sulfur proteins), oxygen activation (methane monooxygenases, alkene hydroxylase, pterin dependent hydroxylases, inorganic models), oxygen reduction to water, and regulatory mechanisms like feedback inhibition and protein phosphorylation/dephosphorylation.

With the current experience of the EPR laboratory with regard to instrumentation and physico-chemical theoretical knowledge, we believe that this cooperation will be of very high value for the biophysics group at the Department of Physics, widening the scope of its activity and offering an entrance to the mechanistically intermediate level of biological activity, between that of molecular structure on one side and that of the medical/biological funcion on the other side; the two fields where our main activity up to now has been concentrated. One example of this is already a fact. Prof. Erik O. Pettersen from the Radium Hospital, recently appointed associate professor at the biophysics Department, has for some time been performing innovative and highly frontier-breaking research on the anaerobic properties of ribonuclease. The recent contact with professor Andersson has now resulted in that a collaborative effort is established.

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Other projects currently active at the ESR laboratory:

A series of projects with external interests are being conducted at the EPR laboratory. The most important of these are shortly describe below:

• Prof. Janko N. Herak from University of Zagreb visited the EPR laboratory for three weeks in 1996 (fully financed by the Norwegian Research Council), for two months in 1997 (financed by Dept. Physics, Univ. Oslo) and again for a month in 1998. Prof. Herak is particularly interested in charge-transfer processes induced in DNA after irradiation at very low temperatures. The experiments, helium temperature X-irradiation of doped crystals followed by EPR/ENDOR characterization of the induced free radicals, have been very successful and have initiated further collaborative work at each site.

• Fully financed from the Nordic Assocoation for Research Education (NorFA), Dr. Haitao Li visited our labs for three months in 1996. At that time Dr. Li was a PhD student associated to Linköpings University and Prof. Anders Lund. Haitao Li is presently working with the study of diffusing small radicals (as e.g. NO₂) through different zeolites. The aim of this study was to characterize the physical properties of zeolites with particular emphasis to their catalytic efficiency. Dr. Li will has now finished his PhD partly on work performed in Oslo.

• In collaboration with University Hospitals, pilot experiments are being performed to quantitatively detect small quantities of the paramagnetic species NO in the bloodstream of mammals. NO is produced under a variety of stress situations and has been shown to affect the contraction and expansion of veins. Conventional spin trapping methods as well as measurement of Hb-NO are used for these purposes.

• Tropospheric ozone has been shown to be highly poisonous to plants and mammals. The interaction mechanisms and metabolic processes for the biological effects of ozone are mainly unknown, although free radicals are believed to be important intermediates. In this project, the interaction between ozone and biological membranes are investigated , using spin trapping and spin labelling methods in the study of ozone-induced oxidation of lipids, liposomes and chloroplasts. This is a collaboration with Prof.  Halvor Aarnes at Department of Biology (Botany).

• The mechanisms for the damaging effect of chemotoxic agents such as PCB on brain nerve cells are studied in collaboration with Prof. Frode Fonnum at the Department of Biology and Norwegian Defence Research Establishment also using spin trapping techniques. The two latter projects now is partly funded from The Environmental Research Program at the University of Oslo.

• When hypoxic cancer cells are reoxygenated, the radiation sensitivity increases significantly as compared to cells which have been continously oxygentaed.  It is speculated if this may be due to spontanous production of oxygen radicals upon reoxygenation.  Spin trapping studies are performed to study the concentration of such oxygen radicals under different conditions of oxygenation of these celle.  This project is in collaboration with Prof. Erik O. Pettersen and graduate student Marianne Koritzinsky, at the Department of Physics and The Norwegian Radium Hospital.

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