Medicine: Genome Research

Abstract: For decades biodosimetry has been a crucial method in the estimation of genome damage caused by ionizing radiation within an occupationally or accidentally exposed population. In combination with physical dosimetry, the analysis of human genome damage truly reflects the action of radiation on a living organism. New methods have brought a new era in which biodosimetry is not a mere dosimeter, but also the warning signal of increased health risk and the source of new ethical questions.

History

Biodosimetry is a method which makes it possible to measure genome damage caused by ionizing radiation on peripheral lymphocytes in humans and to calculate the dose of exposure.  The main principle of method is application of cell culture of peripheral lymphocytes. In short, lymphocytes are trapped in the metaphase stage of the cell cycle in which chromosome morphology makes possible the analysis of its structural damage. Lymphocytes provide a nice model for research, as they circulate through the body like small biodosimeters. Despite the reliability of physical dosimeters, it is very important to establish the real damage of the living organism and the severity of caused genome damage which could be related with increased cancer incidence. The calibration curve, which has been evaluated in in vitro experiments, makes it possible to estimate the exposure dose by correlating the experimental curve with the measured typical genome damage, so called dicentric chromosomes.  At the same time an absence of any kind of personal dosimeters for chemicals confirmed significance of this method for exposure to chemical substances and gave condition to correlate damage caused by radiation and chemicals. Dubbed "chromosome aberration assay", this method has been applied for over 30 years. It has greatly contributed to the making of regulations of occupational exposure to ionizing radiation.

The limitations of this method have been known from the start:  the life of peripheral lymphocytes is relatively short, cells with dicentric chromosomes are sentenced to death and dicentric chromosomes could be caused by chemical substances (such as VCM, drugs, or benzene) and may not reflect the damage caused by radiation alone. This is why this method has been limited to recent exposures of up to a year after exposure.

Other methods developed at about the same time, such as sister chromatid exchange frequency and micronucleus assay, showed the same limitations as the chromosome aberration assay. Biomarkers detected by these methods also drop very soon after the cessation of exposure and they are unspecific with respect to the agent.

Unable to detect any damage a year after exposure, these methods would lead to a false conclusion that a person was healthy and that she or he ran no additional health risk. Aware of the methods' fallacy, researchers in biodosimetry focused on the development of a method which would give information about stable genome damage, translocations. These parameters are stable for years and are crucial in clinical cytogenetics. G banding, a method applied by clinical cytogenetics for years, had been too slow and too expensive for biodosimetry. As opposed to malignant transformations, leukemias and lymphomas in which it is possible to recognize clone or cells with typical translocations among 50 cells per subject, biodosimetry especially that of low doses requires a few thousand cells per subject to detect cell damage. Development of fluorescent in situ hybridization (FISH) did a real revolution in histology and cytogenetics. This method also recognizes lymphocyte translocations marking them by two, three or more colors which enables the detection of huge numbers of cells with translocations in a short time, but only in selected chromosomes. The next step was the development of the so called multicolor FISH and of the computer analysis of cells which, although still expensive, can measure translocations in the complete human genome in a relatively short time.

Like the chromosome aberration assay before, FISH also required the development of calibration curves. For the first time, however, the calibration curve was not giving the information on recent exposure, but it could measure genome damage which had been accumulating for decades of long-term occupational exposure or to show exposure 20 years ago or even 40 years ago.

About 20 years ago another method was introduced, comet assay, which is sensitive enough to measure even repairable genome damage. Using electrophoresis to measure the genome damage, this method avoided the limitations of other methods, as it does not need the cell culture and can be performed on any cell. The comet assay indeed flashed like a comet before the eyes of researchers in biodosimetry. It has proved extremely sensitive, so sensitive in fact that it could detect the intake of C vitamin or consequences of physical stress such as jogging. Yet, after two decades of its application, it is still hard to tell whether this method contributed to resolving the issue of genome damage caused by mutagenic and carcinogenic agents. Instead, it raised new dilemmas and questions.

Present situation in biodosimetry

The scientific community has been using the chromosome aberration assay and other methods for years, acting on the assumption that the measured genome damage corresponded to an increased risk for the development of cancer. There was no way to prove that more chromosome aberrations meant higher incidence of cancer. Today, after 25 years of routine chromosome aberration assay application, we have an opportunity to correlate the results with the cancer incidence in population followed by national cancer registers, as the expected time of latency for cancer development has passed. The assumption that greater genome damage means higher risk of cancer has proved correct. According to first results, there is a significant correlation of deviations in the results of chromosome aberration assay and the incidence of cancer. What has surprised the researchers is that this correlation is not only present in the exposed population, but also in the non-exposed general population. It seems that occupational exposure is not critical for the presence of chromosome aberrations and cancer incidence. The comet assay provides a very important support for the explanation of these results. The comet assay applied in the population occupationally exposed to known mutagens and in general population, who served as control, showed that occasionally interindividual variations in genome damage in general population were greater than the differences between exposed and control populations. For the first time, the comet assay showed that we are quite heterogeneous in our response to the environment and that we all show a high diversity in radiosensitivity and chemisensitivity.

Over the last few years biodosimetry is actually marked in somehow still modest and shy flattering with clinical cytogenetics. By application of FISH, biodosimetry is getting closer to clinical cytogenetics. United huge knowledge of clinical cytogentics which also apply FISH for the last two decades and researchers from biodosimetry offer qualitatively new information. The collaboration of clinical cytogentics and biodosimetry can provide information not only on the severity of genome damage caused by physical or chemical agent, but also on the exact band at which damage is detected, and give correlation with malignancy related with detected band, or possible presence of clones (several cells with genome damage on the same band). In other words, we can anticipate clinical symptoms before they are detected in a monitored person.

Methods of genetical toxicology are actually turning toward the evaluation of the final effect of exposure, toward the prediction of health risk and cancer development. We are interested in the final consequence, regardless of the circumstances such as accidental overexposure, long-term exposure to low doses, medical therapy, diagnostics or life in the vicinity of waste disposal.

Future

As in many other areas of human activity, biodosimetry today can offer much more information on human beings than society is able to shape by way of regulation; the decisions of ethical committees concern the impact on human fears about one's own future illness, plus the genetic differences between individuals, nations and races. We live in a time of strong technological development which makes it possible for us to lower the permissible doses of exposure not only on paper, but also in practice, that is, at least in the developed regions of the world. However, we readily forget that certain members of our society have long been exposed to higher doses of radiation and that the genome of these people is still burdened with damage. Do we have a moral right to determine the genome damage in critical populations and inform them that they are predisposed to develop cancer, although symptoms are still absent? Or is it more ethical to follow them up, keep the information for ourselves, and wait for the results of epidemiological studies, as was the case with the chromosome aberration assay? Do we have the right to make people live in fear of cancer like those Ukrainians who were exposed to accidental radiation from Chernobyl? Is it productive to tell the truth in this case?

The answer could be as in the case of HIV. Offer opportunity to the population with increased risk analysis with reliable methods and give them the liberty to choose to either learn about severity of own accumulated genome damage or live in ignorance.

The application of the comet assay gives us the unique opportunity to adjust radiotheraputical and drug doses in cancer treatment to individual sensitivity.  However, this method also has the potency to measure radiosensitivity in the general population.  Can we imagine the faith of a person who is a highly educated nuclear engineer or a specialist in nuclear medicine, and who has manifested high radiosensitivity in the comet assay? Are we going to do the comet assay immediately after birth in order to avoid one from pursuing wrong professional orientation? Is it a human right to take a risk and choose one's own path?

It is a matter for discussion whether or not an organism has any specific biomarker formed as an answer on radiation which could be detected long after exposure and which is in correlation with development of specific malignancies. It could be that nature in its final step, and this is in our case a change of gene or a change of gene activity, represents a conservative model in which the final consequence is always the same, thus viewing mutation as a machine of evolution.

As possibilities, international projects could accumulate data from all over the world in order to standardize biodosimetry methods thus leading toward the compatibility of data; this data collection would require huge participation in order to have a necessary critical mass of data for reliable studies. Finally it could turn out that the only parameter of difference between people will be their capability to cope with the environment, regardless of their race, nation, or population.  Recent studies suggest that humankind can be divided into huge families of people sharing the same type of metabolic enzymes, but whose area of distribution does not correspond to national borders. Consequently, the point at which we are going to be ready to accept new knowledge seems to be still far away from the domain of the researcher.


Dr. Aleksandra Fucic was born in Zagreb on June 6, 1959. She has been fascinated with nature and natural sciences since early childhood. After she graduated from the Faculty of Natural Sciences as a Biologist, she seized the opportunity to work at the Department of Anatomy of the Faculty of Medicine in Zagreb, where she stayed from 1984 to 1987. That period, under the tutelage of Professor I. Kostovic, proved very important for her scientific career. She learned to think synthetically, to critically read scientific works, and to value teamwork.

In 1985 Dr. Fucic finished her Master's thesis on prenatal development of the human brain, and in 1987 she took a position at the Laboratory for Mutagenesis of the Institute for Medical Research and Occupational Health in Zagreb. Although the object of her scientific research changed, she was able to adjust fully to new circumstances and to publish her first paper on ecogenotoxicity within two years. She has participated in international conferences, and has membership in several domestic and international scientific societies including the Croatian Biological Society, the European Mutagen Society, and the Environmental Mutagen Society, among others. She has also written numerous publications and has worked on biomonitoring populations occupationally exposed to ionizing, non-ionizing radiation, and chemical mutagens which brought her several international and domestic grants and awards.

The period of war in Croatia presented an extremely difficult period for any kind of scientific work, however thanks to support from her colleagues from the United States, Dr. Phillip Hartman at John Hopkins University and Dr. Joe N. Lucas at the University of California, Lawrence Livermore National Laboratory, Dr. Fucic was able to continue her work. In 1996 the International Atomic Energy Agency (Vienna, Austria) included her on its official roster of biodosimetry experts.

Over the past few years Dr. Fucic has participated in several international and global projects, which estimate genome damage of human populations exposed to xenobiotics and call for the standardization of genotoxicological methods. She has given lectures to postgraduate students and has been the subject of radio show interviews with the purpose to spread awareness of radiation protection and genotoxicity. She has actively participated in INCHES since 1988, motivated by the hope that we still have time to raise our children without genome burden from a polluted environment.

Thanks to close collaboration with immunologists, clinical cytogeneticians, and physicians, Dr. Fucic's papers are not limited to genotoxicity, but give a complete picture of the health risks resulting from mutagenic action. Dr. Fucic is the author of a dozen of papers and has written chapters in several professional books.