p53 mutations in tumor and non-tumor tissues of Thorotrast recipients: a model for cellular selection during radiation carcinogenesis in the liver

Keisuke S. Iwamoto21, Shiho Fujii, Akihiko Kurata1, Makoto Suzuki2, Tohru Hayashi3, Yuji Ohtsuki4, Yuhei Okada5, Michihiko Narita6, Masanori Takahashi7, Sadahiro Hosobe8, Kenji Doishita9, Toshiaki Manabe10, Sakae Hata10, Ichiro Murakami11, Satoru Hata12, Shinji Itoyama13, Seiya Akatsuka14, Nobuya Ohara15, Keisuke Iwasaki16, Hisamasa Akabane17, Megumu Fujihara18, Toshio Seyama20 and Takesaburo Mori19

Department of Radiobiology, Radiation Effects Research Foundation, 5-2 Hijiyama Park, Minami-ku, Hiroshima 732-0815,
1 Department of Pathology, Osaka National Hospital, Osaka,
2 Department of Pathology, Shizuoka General Hospital, Shizuoka,
3 Anatomic Pathology, Division of Clinical Laboratory, Miyazaki Prefectural Hospital, Miyazaki,
4 Department of Pathology, Kochi Medical School, Kochi,
5 Clinical Laboratory, Matsuyama-Shimin Hospital, Matsuyama,
6 Department of Pathology, Kamo Hospital, Toyota, Aichi,
7 Department of Laboratory Medicine, Tokushima Central Prefectural Hospital, Tokushima,
8 Department of Pathology, Akita Red Cross Hospital, Akita,
9 Department of Research, Fukui Prefectural Geriatric Center, Fukui,
10 Department of Pathology, Kawasaki Medical School, Kurashiki,
11 Division of Laboratory Research, Iwakuni National Hospital, Iwakuni,
12 Department of Pathology, Nagano Red Cross Hospital, Nagano,
13 Division of Laboratory Pathology, Yaizu Municipal General Hospital, Yaizu,
14 Division of Laboratory Pathology, Urawa Municipal Hospital, Urawa,
15 Department of Pathology, Okayama University Hospital, Okayama,
16 Department of Pathology, Sasebo City General Hospital, Sasebo,
17 Department of Pathology, Yokosuka Kyosai Hospital, Kanagawa,
18 Department of Pathology, Hiroshima Red Cross Hospital and Atomic Bomb Survivors' Hospital, Hiroshima and
19 National Institute of Radiological Sciences, Chiba, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Concerns over cancer development from exposure to environmental sources of densely ionizing, high linear energy transfer (LET) radiation, such as {alpha}-particles from radon, is a current public health issue. The study of tumors attributable to high LET irradiation would greatly augment our insights into the biological mechanisms of carcinogenesis. Chronic low-dose-rate internal exposure to {alpha}-radiation from thorium dioxide deposits following intravascular administration of the radiographic contrast agent Thorotrast is known to markedly increase the risk of cancer development, especially that of hepatic angiosarcomas and cholangiocarcinomas. Although the mechanism is hypothesized to be via cellular damage, DNA being a major target, wrought by the high LET {alpha}-particles, the specific genes and the actual sequence of events involved in the process of transforming a normal cell into a malignant one are largely unknown. To shed some light on the molecular mechanisms of cancer development during a lifetime exposure to {alpha}-radiation, we analyzed the most commonly affected tumor suppressor gene in humans, p53, in 20 Thorotrast recipients who developed cancer, mostly of hepatic bile duct and blood vessel origin. Of the 20 cases, 19 were found to harbor p53 point mutations. Moreover, the accompanying non-tumor tissues from these patients also had p53 mutations, albeit at lower frequency. The distribution pattern of the point mutations was significantly different between the non-tumor and tumor tissues, with most mutations in malignant tissues located in the highly conserved domains of the p53 gene. Our results support the idea that p53 mutations are important in the genesis of Thorotrast-induced tumors but that these point mutations are a secondary outcome of genomic instability induced by the irradiation. Additionally, non-tumor cells harboring p53 mutations may gain some survival advantage in situ but mutations in the domains responsible for the formation of structural elements critical in binding DNA may be necessary for a cell to reach full malignancy.

Abbreviations: LET, linear energy transfer; SSCP, single strand conformation polymorphism.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Epidemiological studies of populations exposed to ionizing radiation have substantiated a causal link between such exposures and the development of cancer in humans. However, it is often difficult to be sure that any particular tumor is actually radiation-induced. Therapy-induced cancers are valuable in this regard. These tumors develop following exposure, for relatively short periods of time (weeks), to fractionated doses of sparsely ionizing, low linear energy transfer (LET) radiation, such as X- or {gamma}-rays. Therefore, these tumors are not ideal to directly address the problem regarding the considerable amount of uncertainty in the biological effects of low level, chronic internal exposures to environmental sources of densely ionizing, high LET radiation, like {alpha}-particles. Such investigations are important because of concerns about cancer development from environmental sources of {alpha}-radiation, principally lung cancer from radon (13), and more controversially, because of a growing concern around the current uncertainties surrounding cancers that might be attributable to plutonium (also an {alpha}-emitter) contamination from nuclear processes (4,5).

The particular significance of this study is that the tumors can be attributed to {alpha}-irradiation. This allows for a focused study of the biological consequences of {alpha}-irradiation exposure and better insights into the mechanisms of carcinogenesis in humans which should contribute to answering some of the questions raised from current issues. The source of tumors used in this study is patients who had received Thorotrast and who are at extremely high risk of developing hematological and liver malignancies (610). Thorotrast is the tradename of a thorium dioxide colloid that was used as a radiographic contrast agent worldwide from the early part of this century to as late as 1960. In Japan, Thorotrast was predominantly used in military hospitals for diagnosis of war injuries. Thorium, with a half-life of 1010 years, and most of its decay daughters, emit {alpha}-particles and irradiate the local area of deposition for the life of the patient. Over 90% of the intravascularly administered Thorotrast is retained in the body with >60% in the liver (11). Organ dose rates have been estimated to range from 0.1 to 0.7 Gy/year (11,12). Since most recipients were young soldiers, their total lifetime organ doses may have been as high as 35 Gy.

The effects of such low dose-rate, high total-dose exposures to {alpha}-radiation on the mechanisms causing transformation of a normal cell to a cancer cell is unknown. Unlike acute exposures, chronic exposures inflict damage throughout the process of carcinogenesis for any given cell and may, therefore, elicit cellular responses that are manifested differently. Moreover, since the nature of {alpha}-radiation is quite different from that of X- and {gamma}-radiations, some investigators have argued that there may be unique effects of {alpha}-particles (1315).

To investigate the early and late steps of {alpha}-irradiation-induced carcinogenesis concurrently, we analyzed the p53 tumor suppressor gene for evidence of unique patterns or types of damage in a relatively sizeable collection of documented Thorotrast-related cancers and corresponding non-cancer tissues. The p53 gene was selected because it is one of the most commonly mutated genes in human cancers (16). Although p53 mutations are believed to be late events in many human cancers, including liver neoplasms (16,17), there are recent reports that show that p53 mutations can be found in the early stages of hepatocarcinogenesis (1820). Moreover, since loss of normal p53 function is known to abrogate growth control (2123), our hypothesis was that there may be clonally expanded non-neoplastic tissues, as well as tumors, that have survived and retained characteristic types of DNA damage. In other words, not only would DNA alterations in malignantly transformed tissues exist, but there may also have been ample time and exposure to sufficiently high, yet sublethal, levels of DNA damaging events for the non-malignantly transformed cells to also show genetic signs of exposure. Our report supports this scenario with the additional provocative result that what we have observed in human cancers is evidence for radiation-induced genomic instability. This mechanism has been demonstrated by a number of investigators in in vitro systems, and has been suggested to have a major role in radiation induction of cancer (13,2426).

In the interpretation of our results, we propose in our model for radiation-induced carcinogenesis in the human that the myriad p53 mutations we observed were not the direct result of {alpha}-particles traversing the gene itself but rather were the secondary results of the {alpha}-irradiation which created an intracellular environment that promoted genomic instability by deleting or incapacitating other genes or cellular components. This is the first study demonstrating this phenomenon in human cancers through analysis of archival tissues.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tissue samples
Various tissues, both non-tumor and tumor, were collected from 39 autopsy cases of Thorotrast recipients who developed cancer. The cases were collected randomly from throughout Japan. All archival tissues were formalin-fixed and paraffin-embedded. Nineteen of the cases were excluded from the study because sufficient DNA could not be extracted and PCR-amplified, most likely due to the minuscule amount of available tissue and the extended period of time they spent (months to years) as `wet tissues' in formalin. From the remaining 20 cases, 56 tissue samples were analyzed for p53 mutations. The pertinent information regarding the population is summarized in Table IGo. The details of case 2 have been published elsewhere (27).


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Table I. Summary of cases
 
DNA extraction and PCR amplification
The DNA was usually extracted from three 5 µm thick paraffin sections. The exact number of sections used is listed in Table IIGo, together with the approximate area of the microdissected tissues. The number of non-tumor tissues examined are also listed in the table; in a few cases, more than one non-tumor tissue was analyzed as described in the table. For each tissue the microdissected sections were pooled and DNA extracted as described previously (28). Briefly, hematoxylin and eosin stained sections were marked and used as guides for microdissection of areas of interest. The marked areas were scraped by scalpel into 1.5 ml centrifuge tubes, deparaffinized, treated with digestion buffer (50 mmol/l Tris–HCl pH 8.5, 1 mmol/l EDTA and 0.5% Tween-20) plus 100 µg of proteinase K, extracted with phenol–chloroform and precipitated with ethanol. PCR amplification of exons 5, 6, 7 and 8 of the p53 gene were done as described previously. Briefly, 25 ng of template DNA was amplified in a 10 µl solution containing 50 mmol/l KCl, 10 mmol/l Tris–HCl pH 8.4, 1.5–2.5 mmol/l MgCl2, 200–600 µmol/l each dNTP, 2 pmol of PCR primers and 0.5 U of Taq DNA polymerase (Perkin Elmer-Cetus, Emeryville, CA) under suitable thermal conditions. In general, the conditions were 35 cycles of 30 s at 94°C (denaturation), 1 min at 60°C (annealing) and 1 min at 72°C (elongation). Because the quality of the DNA was poor in many instances, the amplicon sizes were reduced to ~100 bp, which increased the success rate of amplification and analysis.


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Table II. Summary of case tissues and their p53 statuses
 
Mutation analysis
Samples were first screened by the single strand conformation polymorphism (SSCP) method for mutations as described previously (28). Those that exhibited aberrant SSCP bands were then sequenced by the dideoxy method. Sequencing was routinely done in both the sense and anti-sense directions as a confirmation step. Additionally, sequencing was repeated, more than once in some cases, using stock (not PCR pre-amplified) DNA to verify that the mutations were not artifactual. Table IIGo gives the details of the specific mutations for each tissue.

Cloning of PCR products
PCR products were purified by isolation from an agarose gel and ligated into the pBluescript II SK plasmid (Stratagene, La Jolla, CA). Following transformation of competent Escherichia coli and the random isolation of 20 colonies, DNA was extracted and sequenced.

Statistical analysis
Differences in the distribution patterns of mutations within exons 5, 6, 7 and 8 of the p53 gene were analyzed by the {chi}2 test for contingency tables. The test was for an association between the tissue type (tumor or non-tumor) and the exon that contains the mutation.


    Results
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 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Except for one case, all were males. Thirteen of the 20 were soldiers treated for wartime injuries. As expected, the major types of cancer found in the 20 cases were primary liver cancers. These liver cancers were mostly cholangiocarcinomas or angiosarcomas. There were only two cases of the 20 with hepatocellular carcinomas (Table IIIGo).


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Table III. Types of liver cancer
 
Nineteen of the 20 (95%) of the cases possessed a non-silent p53 mutation. Even if a case did have a silent p53 mutation, it was nearly always accompanied by a non-silent one. Of the 56 tissues examined, 31 (55%) had a non-silent p53 mutation. Interestingly, although a larger fraction of the tumor tissues had mutations, non-tumor tissues also had a significant number (Table IVGo). The mutations detected in the non-tumor tissues were compared with the p53 database (29) to establish whether such mutations have been clonally selected for in human malignancies. The rarest non-tumor mutation represents 0.07% of the mutations in the database and the commonest 1.12%. The median is 0.31%. In other words, all mutations detected in the non-tumor tissues have been found clonally selected in some human cancers. However, very few of the non-tumor mutations were found in any of the hotspot codons, which together represent ~30% of all human p53 mutations.


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Table IV. Non-tumor versus tumor tissues: frequencies of abnormalities
 
The frequency of point mutations that were homozygous is also tabulated. It is most likely that homozygosity actually represents hemizygosity, suggesting that those tissues have lost the other allele. It is very plausible that there was normal tissue (e.g. blood cells) contamination in the isolated sections of the specimens, so the frequency of deletion may be underestimated.

To clarify the meaning of multiple mutations in one tissue specimen, the alleles from case 1 were cloned. Sequencing of cloned PCR products from case 1 gave three varieties of alleles of the 20 analyzed (Table VGo). There were two types of point mutations: at codons 68 and 72. Although this latter site is a well known polymorphic site (30), it was taken as a mutation because the normal tissue was homozygous for Arg; an alternative interpretation is that the patient was actually heterozygous for both the Arg and Pro alleles, but the Arg allele was deleted in the tumor while the Pro allele was deleted in the normal tissue which clonally expanded. Unfortunately, no other tissues were available for verification purposes. Nevertheless, the tumor DNA was different from the non-tumor tissue DNA.


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Table V. Cloning and direct sequencing of the p53 codons 68 and 72 containing a region of case 1
 
The summary of the type of nucleotide base substitutions is given in Table VIGo. Regardless of the tissue, GC->AT transitions predominate. This trend is more apparent in the tumor group than in the non-tumor group. Among the GC->AT transitions, a small fraction was found at CpG sites; liver tumors had fewer mutations at CpG sites than other types of tumors.


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Table VI. Summary of nucleotide base substitutions
 
Figure 1Go displays the spectrum of mutations along the p53 gene within the four hotspot exons. There is a statistically significant difference in the clustering patterns of the positions of the mutations between the non-tumor and tumor tissues ({chi}2 = 9.29; P < 0.03; excluding all silent mutations). There is a highly statistically significant difference in the clustering patterns when non-tumor liver tissues are compared with primary liver cancers ({chi}2 = 15.68; P < 0.003). Many of the mutations in the tumors are clustered in exon 7, especially at codon 248. And, there is a large fraction of mutations in the non-tumor tissues in exon 6, whereas there are very few in the tumors.



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Fig. 1. Distribution pattern of point mutations in the p53 gene. Most of the non-tumor tissues were of liver origin. There is a statistically significant difference in the distribution patterns of p53 point mutations between the non-tumor and tumor tissues. The boxed Roman numerals indicate the highly conserved domains that are critical regions that determine the proper binding of the p53 protein to DNA. Note that most of the mutations found in the tumors are located within these domains whereas those found in the non-tumor tissues are located more randomly within the exons.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Growing concerns of cancer development from environmental sources of {alpha}-emitters like radon or plutonium create an increasing need to understand the mechanisms behind the biological effects of the deposition of these isotopes within the tissues of the body. The fact that the nature of {alpha}-irradiation-induced damage is different from that of X- and {gamma}-irradiation has led some investigators to suggest that there may be unique `fingerprints' left behind in the subsequently developed tumors that can be utilized to augment our insights into radiation carcinogenesis.

The present Thorotrast-induced cancer cases are a unique resource for understanding the mechanisms of radiation carcinogenesis because these tumors can be attributed to the {alpha}-irradiation (31). We were able to analyze both chronically {alpha}-irradiated tumor and chronically {alpha}-irradiated non-tumor tissues for insights into early and late events in tumorigenesis. The most outstanding feature of the molecular analysis of the 56 procured tissues is the high frequency of point mutations in the p53 tumor suppressor gene. Moreover, the unexpectedly large number of point mutations found in the non-tumor tissues of the cases suggest important clues into the early stages of carcinogenesis.

Selection and accumulation of mutations
The detection of multiple point mutations in single tissues is probably the result of an accumulation of mutations via a selective process in a system that is under constant exposure to {alpha}-radiation. In case 1, for example, the three different alleles (Table VGo) suggest loss of heterozygosity and tumor progression followed by sequential mutation at codons 72 and 68 in different tumor clones. Studies in p53 heterozygous mice indicate that reduction of p53 dosage can be sufficient to promote tumorigenesis (32), which could explain the observation that 17 out of 20 p53 alleles examined from the tumor were wild-type.

Outgrowth of clones accumulating different mutations was commonly seen in this study. The most striking example may be the five mutations detected in the lung tumor of case 2, in which a relatively large, irregularly shaped section had to be used for DNA extraction because of difficulties in amplification from smaller foci. This irregular shape in itself may indicate variable outgrowth of selected clones that gained further, different mutations. The two cholangiocarcinoma foci in case 2 with common and different mutations also support the selection and accumulation hypothesis.

In general, ~10–20% of the tissue sample would need to carry a particular mutation in order for the mutation to be detectable by SSCP; therefore, some smaller colonies that accumulated other mutations may have gone undetected. Consequently, it is conceivable that a large liver tumor may accumulate many different p53 mutations that do not necessarily confer any further survival advantage once the neoplasm has gained a certain high level of malignancy.

The statistically significant difference in the distribution of point mutations within the four hotspot exons further supports the importance of gain-in-function mutations of the p53 gene (23) and the variability of such mutations in potentiating clonal expansion. In the tumors, missense mutations were invariably found in the conserved domains of the gene, whereas in the non-tumor tissues, there was less of this clustering (Figure 1Go). These domains have been shown to be important in the formation of structural elements critical in binding DNA (33). These results indicate that mutations in the conserved domains are critical for malignant transformation, the so-called late event.

On the other hand, our data also demonstrate clonal expansion of non-tumor tissues with p53 mutations. Others have reported benign clonal patches of keratinocytes with p53 mutations that may or may not be identical to the mutations in the tumor (34,35). Moreover, we found that although few mutations were at hotspot codons, none was extremely rare or unreported in the human cancer p53 database. Thus, in tissues exposed to a chronic, sublethal dose of a mutagen, such as sunlight to the skin or {alpha}-particles to liver cells, multiple p53 mutations may be associated with both non-tumor and tumor cell clonal expansion. Therefore, loss of wild-type p53 activity is likely to confer survival advantage to non-tumor cells, and adoption of additional oncogenic behavior through the acquisition of certain critical missense mutations is required for malignant transformation.

Frequency of mutations
Our data, which demonstrate that 95% of the cases have a p53 mutation, is in stark contrast to some European studies on Thorotrast-induced tumors, which show a paucity of mutations in the gene (3638). Human hepatic cholangiocarcinomas and angiosarcomas are rare. From the few studies that exist, p53 mutation frequencies in cholangiocarcinomas range from 19 to 79% (3942).

Two studies have reported a mutant frequency in angiosarcomas of vinyl chloride workers of 50% (43,44). A study on sporadic hepatic angiosarcomas showed 12% with a p53 mutation (38). There are no clear answers for the variation in frequencies reported in different cohorts. In the case of hepatocellular carcinoma, the large variations in the frequencies of p53 mutations have been partially attributed to differences in confounding by factors such as tumor grade, mycotoxin exposure and viral infections (17,4547). Thus, socioeconomic, environmental, cultural and geographic background undoubtedly play a large role in determining the mutation frequency as well as the mutational pattern.

Type of base substitution
Transitions at CpG sites, where spontaneous deamination of the cytosine can cause mispairing with an adenine, are thought to be an indicator of the consequence of biological processes (16). This may be one reason for the high frequency of p53 codon 248 mutations in human cancers, including non-aflatoxin liver tumors (48). One study of sporadic cholangiocarcinomas showed predominantly GC->AT transitions at CpG sites (40). Interestingly, although most of the mutations found in our study were GC->AT transitions, few were at CpG sites. Of these, most were at codon 248, although not all codon 248 mutations were at CpG sites. This suggests that the mutations found in our study were caused by a non-stochastic process induced by the {alpha}-irradiation.

Przygodzki et al. (49) showed a predominance of GC->AT transitions at non-CpG sites and GC->TA transversions in the K-ras-2 gene of sporadic and Thorotrast-induced hepatic angiosarcomas. The bias toward non-CpG sites can be attributed to the fact that ras codon 12, an activating mutation site, is a non-CpG site. This perspective may also apply to p53 in which certain mutations are selected to confer oncogenic potential. And, finally, studies on effects of {alpha}-radiation from radon on normal human bronchial epithelial cells have demonstrated the mutability of codon 249, resulting in a G->A transition (non-CpG), as well as a C->A transversion at codon 250 of p53 possibly reflecting the genotoxic effects of radon in lung carcinogenesis (50). Yet we found no bias toward mutations at these codons indicating that {alpha}-particle-induced carcinogenesis in the liver is distinct from that in the lung.

Thus, the results of our study together with those of other investigators provide supportive evidence that p53 mutations in Thorotrast-induced tumors have a different etiology from spontaneous tumors or from those produced by other carcinogens. It also is likely that the tissue type will play a large role in the type of p53 mutation.

The mechanisms of mutagenesis: Genomic instability
In general, non-CpG site GC->AT transitions are associated with carcinogen exposure. Moreover, non-CpG site GC->AT transitions are frequently associated with reactive oxygen species-induced damage to DNA in bacteria (51). In an E.coli system, mammalian DNA polymerase-ß fidelity has been reported to be poorest in correctly reading oxidized cytosines, resulting in C->T substitutions. Although such studies cannot be used to conclude which types of lesions are responsible for the mutations introduced by different DNA polymerases, it is plausible that many of the observed C->T transitions are the result of oxidative modification of cytosines (51). If so, it is not known whether the {alpha}-radiation is affecting cellular repair or replication processes or whether it is directly inducing modification of cytosines.

It should be noted, however, that the predominant DNA damage produced by the direct effects of ionizing radiation, especially high LET type, is the deletion. Yet our analysis showed a high frequency of point mutations in agreement with in vitro and animal studies of others. One study reported mostly deletions in the Chinese hamster ovary hprt gene soon after irradiation but point mutations were the major aberrations several cell generations later (25). Another study observed selective expansion of mutant cells with latent expression of different multiple p53 mutations in the clones of radiation-induced murine mammary cancers (26). This phenomenon, delayed radiation-induced mutagenesis, was first reported by Kadhim et al. (24) when non-clonal aberrations were found at high frequencies in clonal descendants of cells irradiated by {alpha}-particles. Thus, our results provide some of the first evidence for radiation-induced genomic instability in human carcinogenesis in situ. The mechanisms underlying this induction of genomic instability by ionizing radiation are unknown but it could involve dysfunction in mismatch repair such as has been proposed in the genesis of hereditary colon cancer (52) or defects in DNA repair and replication in a way discussed earlier.

A model for Thorotrast-induced radiation carcinogenesis in the liver
Spatial and temporal dynamics of cellular damage induced by exposure to chronic internal high LET radiation need to be considered in interpreting the data. The densely ionizing track lengths of {alpha}-particles from Thorotrast deposits in tissue range from a few to several microns. Thus, one to a few cells may be traversed by one particle. The fact that the deposition of thorium dioxide is not homogeneous in the tissue but rather tends to accumulate near blood vessels or ductules may explain the high prevalence of Thorotrast-induced cholangiocarcinomas and angiosarcomas (Figure 2AGo). Hei et al. (53) have observed that >80% of cells traversed by a single {alpha}-particle can survive while their mutation frequency is doubled; however, when cells are traversed by eight particles, only 10% survive. It seems likely that cells within this small group of high dose survivors may be most important in producing tumors as suggested recently by Miller et al. (54) who reported that cells traversed by multiple {alpha}-particles without killing the cell contribute most of the risk in oncogenic transformation. Consequently, the cell with the greatest potential for becoming a cancer is the cell that survives the radiation damage but retains its scars.




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Fig. 2. A possible model for radiation liver carcinogenesis. (A) Diagrammatic illustration of the deposition of the thorium dioxide (ThO2) within the blood vessels and bile ducts in the liver. Considering the finite length of the ionization tracks of the {alpha}-particles, only cells near the deposits are exposed. This may help to explain the high frequency of hepatic angiosarcomas and cholangiocarcinomas and the low frequency of hepatocellular carcinomas. (B) Illustration depicting chronic irradiation-induced carcinogenesis through cycles of cell damage and cell selection. See text for details.

 
These facts suggest that in a simplified model (Figure 2BGo), cells adjacent to deposits encounter lethal doses of radiation whereas cells a few cell diameters away most likely experience sublethal damage, and those cells far enough away are essentially unexposed. The loss of many `point-blank' range cells and the clonal outgrowth of `mid-range' (sublethally damaged) cells that have gained some survival advantage would redistribute the local cell population. Some of the mid-range clones would therefore become `point-blank' range clones or `far-range' clones, reinitiating the cycle. During the process, radioresistant cells may be selected out resulting in a greater number of reproductively viable cells within the `point-blank' range. Induction of genomic instability following irradiation-induced damage of key components in maintaining genome stability, such as faithful repair or replication, would compound the accumulation of mutations by the chronic irradiation. Eventually, there would exist a large spectrum of cells, from clonally expanded non-tumor cells to malignant cells, with varying levels of damage. Those that have gained the necessary mutations for malignant transformation would go on to become a cancer.


    Acknowledgments
 
The authors are grateful to Mrs Mika Yonezawa and Ms Nagi Saito for administrative assistance in procuring the samples. This publication is based on research performed at the Radiation Effects Research Foundation (RERF), Hiroshima, Japan. RERF is a private non-profit foundation funded equally by the Japanese Ministry of Health and Welfare and the United States Department of Energy through the National Academy of Sciences.


    Notes
 
20 Present address: Yasuda Women's University, Hiroshima, Japan Back

21 To whom correspondence should be addressed Email: iwamoto{at}rerf.or.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 17, 1998; revised March 8, 1999; accepted March 8, 1999.