Abstract
Damage from occupational or accidental exposure to ionising radiation is often assessed by monitoring chromosome aberrations in peripheral blood lymphocytes, and these procedures have, in several cases, assisted physicians in the management of irradiated persons. Thereby, circulating lymphocytes, which are in the G0 stage of the cell cycle are stimulated with a mitogenic agent, usually phytohaemagglutinin, to replicate in vitro their DNA and enter cell division, and are then observed for abnormalities. Comparison with dose–response relationships obtained in vitro allows an estimate of exposure based on scoring:
Unstable aberrations by the conventional, well-established analysis of metaphases for chromosome abnormalities or for micronuclei;
So-called stable aberrations by the classical G-banding (Giemsa-Stain-banding) technique or by the more recently developed fluorescent in situ hybridisation (FISH) method using fluorescent-labelled probes for centromeres and chromosomes.
Three factors need to be considered in applying such biological dosimetry:
Radiation doses in the body are often inhomogeneous. A comparison of the distribution of the observed aberrations among cells with that expected from a normal poisson distribution can allow conclusions to be made with regard to the inhomogeneity of exposure by means of the so-called contaminated poisson distribution method; however, its application requires a sufficiently large number of aberrations, i.e. an exposure to a rather large dose at a high dose rate.
Exposure can occur at a low dose rate (e.g. from spread or lost radioactive sources) rendering a comparison with in vitro exposure hazardous. Dose–effect relationships of most aberrations that were scored, such as translocations, follow a square law. Repair intervening during exposure reduces the quadratic component with decreasing dose rate as exposure is spread over a longer period of time. No valid solution for this problem has yet been developed, although, in theory, both deterministic damage and aberrations might be repaired to a similar degree; a comparison of aberrations following a linear dose relationship might also help when the doses have been sufficiently large.
Investigations might have been possible only a certain time after the exposure. The relatively rapid disappearance of lymphocytes carrying unstable aberrations limits their use in retrospective dosimetry, years after exposure. Scoring stable aberrations, thought to persist in the circulating lymphocytes, might appear more appropriate in such situations. However, the examination of a representative number of cells by G-banding is extremely laborious, and the FISH method is not only expensive but has not yet been fully validated in different laboratories.
In conclusion, biological dosimetry has serious limitations exactly for situations where the need for information is most urgent. It renders its most useful results when an individual has been exposed to a rather homogeneous high-level radiation over a short time interval, i.e. accidents at high-intensity radiation devices. On the other hand, it yielded less satisfactory information even when the most recent techniques were used for situations, where a low level, low dose rate exposure has occurred at some time in the past, for example for persons living in areas contaminated from the Chernobyl accident. Such negative experiences should be kept in mind in order to avoid futile and expensive investigations in the case of populations exposed from radioactivity and, notably, also from potentially clastogenic chemical agents.
INTRODUCTION
Risks from professional or accidental radiation exposure to individuals or a population often can be assessed only by means of ‘biological dosimetry’, that is, by assessing changes, in the body or in cells, that reflect the damage inflicted. Because radiation exposure can be related to the risk of encountering damaging consequences to health, biological dosimetry on persons accidentally or professionally exposed to ionising radiation proves to be of practical assistance to physicians, particularly when no physical dose estimate is available. Such information is needed in the following instances:
A few persons have been exposed accidentally to relatively high doses of radiation (>1–2 Sv), and one must predict the risks of acute or late deterministic damage and guide the treatment; usually, such evaluations are needed shortly after the exposure.
Persons were exposed to intermediate doses (∼200 mSv to ∼1 Sv), which, although not requiring treatment, still demand measures to predict and possibly prevent stochastic risks such as cancer; in some situations such an assessment might only be possible at a much later time.
A population has been potentially exposed to low doses (corresponding to <200 mSv) of a genotoxic agent, and one must, sometimes many years later, determine (‘bio-monitor’) whether a genotoxic exposure has taken place. Finding no changes in biological indicators may thereby reassure these people, who generally exaggerate fears about risk of cancer; in this context, it has to be strongly emphasised that a given person who carries an elevated number of aberrations is not necessarily one with a higher risk for developing cancer. Among the large spectrum of tests developed for these purposes, those based on cytogenetic observations on circulating lymphocytes have proved to be most suitable under a variety of situations.
BACKGROUND FACTS FOR BIOLOGICAL DOSIMETRY
The term ‘biological dosimetry’ is, in fact, a misnomer—albeit now so deeply entrenched that it is unlikely to be abandoned—because the information from such tests does not yield a dose in the physical sense; rather, it reflects biological damage and is, thus, one step closer to the problems with which one is confronted in exposed persons.
In order to appreciate the results of cytogenetic methods in biological dosimetry, several aspects have to be considered:
Lymphocyte behaviour: The lymphocyte population of an adult has been estimated to be ∼500 × 109 cells of which ∼2% are present in the bloodstream. The latter stay for only about 30 min on each passage and are continuously exchanged mainly with lymphocytes in lung, spleen, liver, bone marrow and lymph nodes. For cytogenetic studies, the lymphocytes which are in DNA presynthetic stage of the cell cycle (G0) are stimulated in vitro by a mitogenic agent, usually phytohaemagglutinin (PHA), to replicate their DNA and enter cell division. These lymphocytes, which are mainly of the T-type, have an average lifespan of ∼3 y, but their trafficking and replacement may be affected by the widespread apoptosis of lymphatic cells after a radiation exposure. Stem cells carrying damage incompatible with cell division, such as unstable translocations, are selectively eliminated during cell renewal, rendering the scoring of such anomalies unsuitable for retrospective biological dosimetry years after exposure.
Dose–effect relationships: Dose–effect relationships for translocations in lymphocytes are linear–quadratic for low-linear energy transfer (LET) radiation (beta, gamma or X rays) and linear for high-LET radiation (neutrons, alpha rays and other heavy particles). The quadratic component results from the two independent absorption events at two different nuclear sites that are needed to cause a translocation. When an exposure is spread over time, damage from the first event may have been repaired before the second event has occurred, so that the dose effect relationship for low-LET radiation becomes increasingly linear as the dose rate is reduced. In contrast, dose–effect relationships for high-LET radiation are linear and are not influenced by the dose rate.
The information from biological dosimetry: This must be considered in the frame of the type of damage expected (1).
Deterministic damage occurs only above a threshold exposure, the seriousness of which depends on dose and is seen only after high-levelexposure. For example, serious symptoms due to haemopoietic failure would not arise unless >90% of bone marrow stem cells are destroyed, and death requires a destruction of >99% of them. When the exposure is homogeneous over the entire body and delivered over a short period of time, the dose–effect relationship of lymphocytes exposed in vitro corresponds to that in vivo. Although the dose–effect relationship for deterministic damage differs fundamentally from that of lymphocyte translocations, the two can then be related on the basis of the experience gained from radiation accidents and the atomic bombs. However, the exposed patients may react differently from what is expected; they may have undergone additional damage from fire, wounds and toxic products or they may belong to a group that is particularly sensitive because of age, health and nutritional status. Under these conditions, the judgment of the physician and the development of the patient will have to supplement the information from biological dosimetry.
Most accidents expose the body in an inhomogeneous manner. Since biological dosimetry reflects only average body doses, but even a small spared part of the bone marrow could affect recovery, biological dosimetry will not correspond to radiation syndromes. The unexposed percentage of the body might be recognised from the distribution of the aberrations among cells. A homogeneous exposure typically yields a poisson distribution of the aberrations. If some part is unexposed, more cells without aberrations than what is expected will be present; the distribution of aberrations consists of contributions from exposed, less exposed and unexposed parts of the body resulting in a so-called contaminated poisson distribution(1). Unfortunately, this calculation is useful only when a significant part of the body (>10–20% of the body) has received little or no exposure and the rest have received rather large doses (>3 Sv) and when the exposure has been delivered over a very short period of time so that lymphocytes had no time to circulate. These conditions would be rarely fulfilled.
When exposure is spread over a certain period of time—hours and days—repair will occur and the quadratic component of the dose–effect relationship will gradually disappear. Since both lymphocyte aberrations and deterministic damage are repaired at low dose rates, albeit to different degrees, one may, at least in theory, use the linear component of the dose–effect relationship, instead of the linear–quadratic one, for comparison However, data on people for such a situation are still scanty.
Stochastic damage, i.e. the risk of cancer and hereditary damage arise at a frequency dependent on dose and possibly without a threshold. Dose–effect relationships for these effects are linear or linear–quadratic, and thus would resemble those of biological indicators such as chromosome aberrations. One may also assume that repair after low dose rate exposure proceeds in a similar way for cancer induction and lymphocyte aberrations and that for many important cancers, distribution of exposure matches that of the risks of many cancers (e.g. leukaemia, gastro-intestinal cancer, breast cancer) in the body. Although these assumptions may not be strictly true, the error introduced by them is probably smaller than the error arising from statistics, confounding factors, and choice of persons. Yet, for certain cancers, in particular those in thyroid, lung, liver and bone, accumulation of radionuclides could result in higher doses in target tissues than the average whole body doses reflected by lymphocyte aberrations.
Schema showing the respective influence of low dose rate and partial body exposure on lymphocyte aberrations and lethality. After low dose exposure (LT), lethality is shifted to an about comparable degree to the right as aberration frequency. Partial body exposure with a preservation of only 15% of the body (HP) has a much greater impact on lethality than it has on aberrations.
Schema showing the respective influence of low dose rate and partial body exposure on lymphocyte aberrations and lethality. After low dose exposure (LT), lethality is shifted to an about comparable degree to the right as aberration frequency. Partial body exposure with a preservation of only 15% of the body (HP) has a much greater impact on lethality than it has on aberrations.
PRINCIPLES, ADVANTAGES AND LIMITS OF CYTOGENETIC METHODS IN BIOLOGICAL DOSIMETRY(2)
The scoring of chromosome aberrations in peripheral blood lymphocytes is the method which has been most developed among the different tests proposed for biological dosimetry. Comparison of the results of such tests with dose–effect relationships obtained in vitro, as well as with the observations made on persons exposed for medical reasons(3–6) or from accidents, can allow an estimate of the risks incurred from an exposure scoring:
Unstable aberrations by the conventional well-established analysis of aberrations in metaphase nuclei or of micronuclei in binucleated cells.
So-called stable aberrations, either by the classical G-banding technique or by the more recently developed fluorescence in situ hybridisation (FISH) method using fluorescent, labelled probes for chromosomes and centromeres.
Several other techniques will only be mentioned briefly in this context.
Unstable chromosome aberrations
The determination of the frequency of dicentric chromosomes has been used for biological dosimetry since the early 1960s. Dicentric aberrations or more complex polycentric chromosomes develop when chromosome fragments are exchanged in such a way that two fragments, each containing a centromere, are fused. Since ∼50% of these types of aberrations are eliminated at each mitosis, the cells have to be examined at their first division 46–48 h after initiation of the culture.
The principal advantage of determining dicentric and ring chromosomes is the relatively ease and good standardisaton of technique and evaluation in doing so and, therefore, this forms the method of choice for most suspected cases of accidental overexposure when the analysis can be performed within months after exposure. Generally, 100–500 cells should be scored to evaluate a medium or high exposure with a satisfactory accuracy. In order to reliably detect an exposure of 10 mSv, however, a minimum of 10,000 cells will have to be analysed. An exposure to radiation of several Sv drastically reduces the number of lymphocytes in the blood, but since the number of aberrations per cell is high in such cases, scoring of a small number of cells, even of a few tens, may suffice for an acceptable dose estimate. The technique is expensive, requires well-trained personnel and is unsuitable when a period of years has elapsed after exposure because of the limited lifespan of unstable aberrations.
Micronuclei in binucleated cells(7) are formed during the metaphase–anaphase transition when a whole chromosome is lagging and lost (aneugenic event leading to chromosome loss) or when an acentric chromosome fragment, detached after breakage (clastogenic event), does not integrate into the daughter nucleus. Micronuclei are observed at 72 h following addition of cytochalasin-B, an inhibitor of actins, 44 h after the start of the culture. Application of a paracentromeric probe for fluorescence in situ hybridisation allows discriminating between micronuclei with and without a centromere. Dose–effect relationships of micronuclei are similar to, but somewhat lower than, those observed in dicentrics, since not all acentric fragments are converted to micronuclei. One thousand binucleated cells are, in general, scored for micronuclei.
Compared to the analysis of metaphases, the micronucleus assay is easier, more rapid, less expensive and, because of the simple shape of micronuclei, has the potential for automatisation by flow cytometry. The poor resolving power of this assay, the inter-laboratory variability of dose–effect relationships for an acute low-LET radiation and the decrease with time of the anomalies explain why the micronucleus assay has been rarely used in biological dosimetry.
Stable chromosome aberrations
Chromosomes banding patterns are studied after treatment with proteolytic enzymes (and/or denaturation and renaturation) as G-banding of chromosome arms, C-banding (Centromeric-banding) etc., alone or in combination with FISH spectral analysis (SKY). These techniques are time consuming and delicate, and more suitable for a detailed analysis of the karyotype and of specific structural aberrations (inversions, deletions, changes at the telomeres) than for a quantitative analysis of damage.
FISH based on the binding of specific DNA probes labelled with fluorescent dyes to homologous DNA is a recent method to measure stable chromosome translocations. Probes for several chromosomes or all chromosomes (FISH multicolouring) have now become available. For the determination of translocations, 3–6 probes are generally used and 100–500 metaphases are mostly scored. Translocation frequencies determined by the FISH retrospective dosimetry rely on the assumption that the hit probability, i.e. the probability of a translocation, is proportional to the size of the chromosome. Since the painting of a small fraction of the genome determines only a fraction of the total aberrations, one must convert the frequency of the aberrations observed to the whole genomic equivalents. It has been shown that the number of translocations after low to medium doses (<2 Sv) of whole body exposure remains stable over decades. Conflicting results were reported on the correlation between dicentrics observed early after exposure and the translocations seen later. Observations on survivors of the atomic bomb in Hiroshima demonstrated that the frequency of translocations on chromosomes 1, 2 and 4 measured by FISH and converted to the entire genome equivalent is similar to the yields obtained by the standard G-banding method and roughly in agreement with that expected from the DNA content. According to Lloyd(8), the yield of translocations after in vitro exposure to low-LET radiation follows a linear-quadratic relationship and amount to 5 translocations per 3000 cells in controls, increases to 10 (5 + 5) per 3000 cells after 0.25 Sv and 26 (21 + 5) per 3000 cells after 1 Sv.
In principle, the FISH test, as also the G-banding method, is of substantial value for determining stable anomalies after an exposure. Unfortunately, there are only few laboratories where this technique has been mastered, the reagents are very expensive and some even copyrighted. Moreover, its calibration of the dose–effect relationship is still incomplete, and the large number of metaphases which would have to be scored for low dose exposure is a serious handicap.
Other methods of biological dosimetry
Mutational assays in erythrocytes have been developed on the basis of several systems, in particular for glycophorin A (GPA) which measures the change on the M or N form of the GPA gene in read blood cells belonging to the 50% of the population of the M × N blood type. This damage is retained permanently after exposure. Until now, the GPA test has only been used by a few laboratories but it holds promise for the future inasmuch as it could be readily automatised. The test has a linear dose dependence but is only moderately sensitive with a background of ∼20 × 106 and a doubling dose of 0.5–1 Sv. Mutations on the T-cell antigen receptor (TCR) gene, or the hypochanthine-guanine phosphoribosyl transferase (HPRT) gene of lymphocytes would disappear as these cells are replaced and only preliminary studies have been carried out with these tests.
For completion, a few other tests for radiation exposure are mentioned: electron spin resonance (ESR) and optically stimulated luminescence (OSL) of tooth enamel (and, shortly after exposure of nails). This technique allows one to measure doses in the order of 1 Sv or more even a long time after exposure, but obviously, it records doses only to the head. The Comet assay—single cell gel electrophoresis of lymphocyte DNA—in alkaline (alkali labile sites) and neutral (single strand breaks) environment measures radiation-induced instability in DNA, but is not yet a reliable alternative to other procedures.
APPLICATIONS OF CYTOGENETIC METHODS TO BIOLOGICAL DOSIMETRY
Accidents resulting in short-term high exposure of large parts of the body have received most attention, since they can, within days or weeks, result in serious disease and death. However, such accidents are rare, and usually involve few persons. Immediate action and decisions regarding triage and therapy are required in such a situation. The now well-established and standardised methods of determining dicentric chromosomes and/or micronuclei appears thereby most appropriate; but the number of scorable lymphocytes may be a limiting factor in highly irradiated persons. To reduce the critical delay until information becomes available to the physician, the development and standardisation of techniques using premature chromosome condensation should be considered. Deterministic damage can be estimated rather reliably in terms of dose in a person exposed whole-body over a short period of time. Information is much less reliable when the exposure is inhomogeneous and the physician will have to rely on the appreciation of the development of haematology and general symptoms.
Accidents where an organ or a small part (<10%) of the body is exposed to a high dose occur much more often than whole-body accidents, especially in skin and subcutaneous tissue (e.g. to the hand) from mishandling radioactive sources, and less often in the thyroid and the gastrointestinal and the respiratory tract mucosa from locally deposed radioactivity. Sometimes, the physician will have to decide whether the degree of deterministic damage expected requires surgical or other interventions. Aberrations in lymphocytes are of little use to assess such deterministic and possible stochastic consequences unless both dose and exposed volume were substantial. Special methods not discussed in this presentation are needed, e.g. for skin thermography, echography and aberrations in hair root cells.
Accidents where a high exposure of the body is delivered over a certain period of time can occur from lost radioactive sources or when intervention crews have to manage accident situations. In the former case, such as after the accident at Goiania, the determination of unstable translocations would be suitable to assess deterministic risks. The management of recovery operations and the monitoring of intervention crews after accidents may be helped by cytogenetic studies although the delay until results become available may not be acceptable. Retrospective evaluation, by means of stable or instable translocations in entire crews, can help to evaluate what were the risks of stochastic damage in view of judicial claims.
An exposure of a population to contamination from nuclear accidents will rarely exceed 1 Sv, and for most people it will be much smaller. People close, in time and space, to the accident should be monitored early to determine whether relocation can reduce total doses to a significant degree, in agreement with the recommendations of the International Commission on Radiological Protection. Regarding retrospective dosimetry years after an exposure or when a population continues to be exposed to a doubtful radiation or chemical risk, it will often be difficult to decide whether it is worthwhile to initiate a large scale bio-monitoring of populations for radiation or chemical exposure. Thereby, one must carefully consider costs and utility. Tens of thousands of cells may have to be studied in several hundred people in order find out more than the simple fact that a population has been exposed to some radiation or genotoxic chemical. Moreover, it is frequently impossible to take account of confounding factors in such studies. Consequently, such bio-monitoring often has caused more confusion and anxiety in the population than has yielded useful results. This was, in our opinion, the case for the very extensive retrospective studies after the Chernobyl accident, and for studies on populations exposed to chemicals where often the wrong tests were used for the wrong exposure and the wrong conclusions drawn by media have created much unnecessary anxiety.
CONCLUSIONS
Damage from occupational or accidental exposure to ionising radiation can assessed by monitoring chromosome aberrations in peripheral blood lymphocytes, and these procedures have, in several cases, assisted physicians in the management of irradiated persons (Table 1).
Comparison of different cytogenetic tests in peripheral lymphocytes.
| Unstable aberrations | Stable aberrations | |||||
|---|---|---|---|---|---|---|
| Dicentrics | Micronuclei | G-banding | FISH | |||
| Usefulness for biological dosimetry | Very good | Very good | Limited | Good | ||
| Usefulness for retrospective biological dosimetry | None | None | Very good | Very good | ||
| Costs | High | Medium | Very high | Very high | ||
| Time required | Large | Low | Very large | Medium | ||
| Automatisation possible | Medium | High | None | Low | ||
| Unstable aberrations | Stable aberrations | |||||
|---|---|---|---|---|---|---|
| Dicentrics | Micronuclei | G-banding | FISH | |||
| Usefulness for biological dosimetry | Very good | Very good | Limited | Good | ||
| Usefulness for retrospective biological dosimetry | None | None | Very good | Very good | ||
| Costs | High | Medium | Very high | Very high | ||
| Time required | Large | Low | Very large | Medium | ||
| Automatisation possible | Medium | High | None | Low | ||
Comparison of different cytogenetic tests in peripheral lymphocytes.
| Unstable aberrations | Stable aberrations | |||||
|---|---|---|---|---|---|---|
| Dicentrics | Micronuclei | G-banding | FISH | |||
| Usefulness for biological dosimetry | Very good | Very good | Limited | Good | ||
| Usefulness for retrospective biological dosimetry | None | None | Very good | Very good | ||
| Costs | High | Medium | Very high | Very high | ||
| Time required | Large | Low | Very large | Medium | ||
| Automatisation possible | Medium | High | None | Low | ||
| Unstable aberrations | Stable aberrations | |||||
|---|---|---|---|---|---|---|
| Dicentrics | Micronuclei | G-banding | FISH | |||
| Usefulness for biological dosimetry | Very good | Very good | Limited | Good | ||
| Usefulness for retrospective biological dosimetry | None | None | Very good | Very good | ||
| Costs | High | Medium | Very high | Very high | ||
| Time required | Large | Low | Very large | Medium | ||
| Automatisation possible | Medium | High | None | Low | ||
When most of the body is exposed in an acute radiation accident, the method of choice for biological dosimetry remains the determination of dicentric aberrations in cultured lymphocytes. Also automatic determination of micronuclei could be an improvement. If the exposure has spared a significant amount of bone marrow, information from lymphocyte aberrations will often be unhelpful.
Retrospective dosimetry years later or dosimetry of persons living in a contaminated area over a long period of time can make use of the determination of stable translocations by the FISH test. However, the FISH test may only be applied to a small number of persons carefully selected to be representative of the populations that are accidentally or professionally exposed to ionizing radiation. In such cases, one must balance the potential, often doubtful, information about the population against the rather large costs, the uncertainty of the results and the psychological and social impact of such studies. Moreover, the examination of a representative number of cells by G-banding is extremely laborious, and the FISH method is not only expensive but has not yet been fully validated and standardised in different laboratories
