ON April 26, 1986, an accident at the Chernobyl nuclear reactor in the former Soviet Union released an enormous number of fission products into the atmosphere and over a large portion of the planet. With about 100 million curies released in the 10 days following the initial explosion, the accident was the largest single nonmilitary release of radioactivity in history-and one of the largest environmental disasters ever.
During the first year after the accident, about 25,000 people, mainly Soviet Army troops, were dispatched to the site to clean up the accident. These so-called liquidators were estimated to have received doses of up to 70 centigrays (a gray is the international unit for measuring absorbed ionizing radiation and is equivalent to 100 rads, or 1 joule per kilogram). In the following three years, another half-million people assisted the effort and are estimated to have received lower doses (about 10 to 25 centigrays).
The tasks performed by liquidators included shoveling core material off the roof of the undamaged part of the building, operating heavy equipment to contain contaminated soil, and building a concrete sarcophagus around the destroyed reactor. Depending upon the intensity of radiation exposure associated with their assigned task, most liquidators received radiation exposures over a period of at least several days, and in some cases over many weeks.
Lawrence Livermore biomedical scientists began studying the Chernobyl accident almost as soon as it occurred as part of a Department of Energy effort to help assess the accident's biological effects. The Livermore assistance, which continues today, takes advantage of the Laboratory's longstanding expertise in evaluating human exposures to ionizing radiation and determining their health risks. Livermore scientists have forged numerous and often close scientific relationships with their Russian and Ukrainian counterparts that endure today in collaborations, mutual assistance, informal communications, and visits.

Techniques to Monitor Damage
Lawrence Livermore studies on Chernobyl liquidators have focused on three techniques-two of them developed at Livermore in the 1980s-that are in wide use today to monitor genetic damage in people. The techniques are called biodosimeters because they measure changes in cells to infer the biological consequences of the "dose," or energy deposited in human tissue from ionizing radiation. (In contrast, a standard dosimeter uses a piece of sensitive film that responds proportionally to ionizing radiation.)
The glycophorin A (GPA) assay was first used to study Chernobyl liquidators who demonstrated immediate symptoms known as acute radiation sickness. Within days of the accident, a Livermore group (at that time led by Ron Jensen, now at the University of California at San Francisco) began receiving blood samples from people who received high exposures. The evaluation by Livermore's Richard Langlois found that the response of GPA to high doses of radiation was similar for A-bomb survivors and Chernobyl liquidators. The investigators also found that age and smoking had little effect on the frequency of the GPA null mutants.
The GPA assay measures the number of red blood cells that have a change in the M or N form of the GPA gene. For people whose cells have both forms of the gene, damage to the M form of the gene, for example, can result in a "null" mutation. In such a case, all descendants of that cell fail to make the M protein. Using flow cytometry and cells stained with color-coded antibodies specific to the M and N forms, scientists can study millions of red blood cells from a small blood sample in a few minutes without the need for cell culturing. (See August 1987 Energy & Technology Review, "A New Assay for Human Somatic Mutations," pp. 21-26, and April/May 1992 Energy & Technology Review, "The Glycophorin-A Assay: A Ten-Year Retrospective," pp. 1-18.)
The second technique measures the frequency of mutations of the hypoxanthine phosphoribosyltransferase (HPRT) gene in lymphocytes. This assay was not invented at Livermore, but Laboratory researchers have greatly expanded understanding of the assay's ability to detect DNA damage from ionizing radiation. Livermore biomedical scientist Irene Jones performed work in the 1980s using mice to test the assay. She also developed a database on healthy people to serve as a baseline for the frequency and molecular nature of HPRT mutations.
A third technique called FISH (fluorescence in situ hybridization), which was developed at Livermore and is currently used around the world, has been applied to Chernobyl liquidators as well as to others suspected of receiving ionizing radiation or of being exposed to potentially damaging chemicals. FISH measures chromosome damage by detecting the number of reciprocal translocations, or broken pieces of chromosomes, in lymphocytes that rejoin in a mismatched way. Livermore scientists have shown that the number of reciprocal translocations is proportional to exposure to ionizing radiation at low doses. What's more, unlike some biodosimeters, including other types of chromosome alterations, the frequency of reciprocal translocations is sufficiently stable with time (even over several decades) to permit retrospective dosimetry and can be measured accurately at low levels of radiation.
The FISH technique uses chromosomes from cultured lymphocytes. Fluorescent dyes are attached to small pieces of chromosome sequences called probes, which bind to complementary sequences of the target chromosomes. The bound probes reveal the extent of reciprocal translocations because they appear bicolored under a microscope using fluorescent light (Figure 1) and can thereby be counted easily to determine a person's likely exposure to ionizing radiation. (See October/November/December 1992 Energy & Technology Review, "Chromosome Painting," pp. 11-26, and the November/December 1995 S&TR, "The Genetic Contribution of Sperm: Healthy Baby or Not," pp. 6-19.)

Applying Biomarkers to Russian Liquidators
The usefulness of all three biodosimeters for measuring small or moderate amounts of ionizing radiation is being demonstrated in an eight-year study (1992 to 1999) of a large group of liquidators. The study, conducted for the National Cancer Institute and directed by Livermore scientist Irene Jones, focuses on a population of about 300 Russian liquidators who were assumed to have been exposed to doses of about 5 to 25 centigrays. The study also includes 300 matched controls from Russia of about the same age and with similar smoking histories.
Because physical dosimetry was difficult to perform on the half-million liquidators, the Livermore team decided to estimate the Russians' exposure through biodosimetry. They also reasoned that because people have different susceptibilities to radiation toxicity, biodosimetry is a more accurate indicator of cancer risk than accurate physical dosimetry. (Physical dosimetry measures radiation incident upon the body, but biodosimetry measures cellular injury resulting from that radiation.)

Recognizing Statistical Power
Livermore experts also recognized that the large number of liquidators would give their study the same kind of statistical power that had made previous studies on Hiroshima and Nagasaki A-bomb victims important to human radiation biology. However, it would provide information for different radiation exposure conditions. While A-bomb survivors received instantaneous external exposures, Chernobyl exposures were complex mixtures of internal and external exposures over a period of time and, in some cases, during several separate work assignments.
To increase the statistical power of dose-effects studies, the Livermore investigators are collaborating with researchers from the Applied Ecology Research Laboratory, Ministry of Health and Medical Industry of Russia in Moscow; the Laboratory of Radiation Genetics, Central Research Institute of Roentgenology and Radiology, St. Petersburg, Russia (Figure 2); and the Tula Diagnostic Clinic of the Scientific Institute of Modern Medical Technologies, Tula, Russia. Blood samples are drawn in St. Petersburg, Moscow, and Tula and shipped by air to Livermore.

The results so far show FISH to be sensitive to the exposures of Chernobyl liquidators, with the HPRT assay being less sensitive and the GPA assay, which proved highly valuable for studies of A-bomb survivors and more highly exposed Chernobyl liquidators, showing no difference between the exposed and control populations (Figure 3). The Livermore team says its population of liquidators received on average a dose of about 15 centigrays, as determined by FISH. Such a radiation dose is roughly equivalent to aging about 10 years or to smoking cigarettes regularly. The expected health consequences to the population under study from such an exposure are small.
Livermore researcher Jones notes that the sensitivity to detect the effect of radiation exposure is increased by knowing the age and the smoking habits of the individual, because both characteristics contribute to the damage in their cells. However, she says it is impossible to determine the health risk of any one individual who received a specific amount of ionizing radiation, especially at the lower doses that do not cause acute health effects. Each individual has a different complement of genes that determine how well they can repair damage from ionizing radiation and other sources. Personal habits such as smoking, drinking, and diet add to the genetic damage that accumulates in cells. "It is the sum of all damage and the body's response to that damage that determines the risk of cancer and other health effects," she says.
In a separate study led by biomedical scientist Joe Lucas, Livermore researchers applied FISH to a subset of Chernobyl liquidators suspected of receiving a large dose of ionizing radiation. They reconstructed doses for 27 Chernobyl liquidators from the frequency of translocations measured in their lymphocytes. Of the 27 individuals, 15 are being treated for radiation sickness. The remaining 12 show no medical symptoms.
"FISH has worked extremely well on Chernobyl victims," says Lucas, one of the original developers of FISH. He notes that the technique is useful because not every liquidator had a dosimeter, and memories of the nature and duration of work assignments for most workers are not reliable.

Questions Still Unanswered
Current studies at Livermore and at other research centers are addressing some of the unanswered questions about the assays, such as their sensitivity to low doses, how intensity of radiation exposure affects the response, the persistence of chromosome translocations, and the degree to which factors other than radiation affect them. Jones and her colleagues, for example, are studying the extent to which the type of chromosome aberration affects its persistence in human blood cells, which could change the relationship between translocation frequency determined by FISH and radiation dose as time passes after exposure.
Another major goal of the research will be to understand why people differ in the effect that the same dose of radiation has on their cells. One part of this effort has been started-identifying the differences in the DNA repair gene sequence in people. The next big challenge will be to determine how these differences affect the capacity to repair damaged DNA and if these differences are related to long-term health.
The Lucas group is collaborating with colleagues at Columbia University on a promising method to detect cellular damage among the liquidators. The method is based on measuring the formation of micronuclei, which are secondary and much smaller cell nuclei that form in eye cataract tissue as a result of radiation. The group is also working on an enhancement to FISH that is faster, more accurate, and more sensitive by counting individual chromosomes in liquid suspension instead of on a microscope slide.
In the meantime, Livermore radiation-effects researchers are working with collaborators in Ukraine, Russia, Estonia, and Israel (where some liquidators have immigrated) to apply biodosimeters such as FISH and GPA in their own laboratories.
It seems clear that despite its disastrous environmental consequences, the Chernobyl accident has spawned deeper understanding about the health effects of ionizing radiation and, in the process, spurred stronger international cooperation.
-Arnie Heller

Key Words: biodosimeter, Chernobyl, FISH (fluorescence in situ hybridization), glycophorin A (GPA), hypoxanthine phosphoribosyltransferase (HPRT).

For further information contact irene Jones (925) 423-3626 (jones20@llnl.gov) or Joe Lucas (925) 422-6283 (lucas1@llnl.gov">).

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