Genetic, cytogenetic, and carcinogenic effects of radon: A review

Reviews in Genetic Toxicology

ELSEVIER

Mutation Research 340 (1996) 125-139

Genetic, cytogenetic, and carcinogenic effects of radon: a review R.F. Jostes * Pacific NorthwestNational Laboratory, Richland, WA 99352, USA Received 14 October 1995; revised 13 December 1995; accepted 12 February 1996

Abstract Radon exposure has been linked to lung carcinogenesis in both human and animal studies. Studies of smoking and nonsmoking uranium miners indicate that radon alone is a risk factor for lung cancer at the levels encountered by these miners, although the possibility exists that other substances in the mine environment affect the radon-induced response. The relevance of data from mines to the lower-exposure home environment is often questioned; still, a recent study of miners exposed to relatively low radon concentrations demonstrated a statistically significant increase for lung and laryngeal cancer deaths. In two major series of experiments with rats, the primary carcinogenic effect found was respiratory tract tumors, and evidence for an inverse exposure-rate effect was also noted. Although this inverse dose-rate effect also has been described in underground miner studies, it may not similarly apply to radon in the home environment. This observation is due to the fact that, below a certain exposure, cells are hit once or not at all, and one would not expect any dose-rate effect, either normal or inverse. Because some chromosome aberrations persist in cycling cells as stable events, cytogenetic studies with radon are being performed to help complete the understanding of the events leading to radon-induced neoplasia. Radon has been found to induce 13 times as much cytogenetic damage (as measured by the occurrence of micronuclei) than a similar dose of 6°Co. A wide variety of mutation systems have demonstrated alpha-particle mutagenesis; recent investigations have focused on the molecular basis of alpha-induced mutagenesis. Gene mutations are induced by radon in a linear and dose-dependent fashion, and with a high biological effect relative to low-LET irradiation. Studies of the hprt locus show that approximately half of the alpha-induced mutations arise by complete deletion of the gene; the remaining mutations are split between partial deletions, rearrangements, and events not detectable by Southern blot or PCR exon analysis. Although other mutation systems do not show the same spectra as observed in the hprt gene (suggesting that the gene environment affects response), DNA deletions or multilocus lesions of various size appear to be predominant after radon exposure. As data emerge regarding radon-induced changes at the chromosomal and molecular level, the mechanisms involved in radon carcinogenesis are being clarified. This information should increase the understanding of risk at the low exposure levels typically found in the home.

Keywords: Radon; Carcinogenesis; Genetic damage; Cytogenetic damage

1. Introduction

* Corresponding author. Present address: 1492 Parkway Dr., Rohnert Park, CA 94928, USA. Tel.: (707) 586-2479.

Information relating to radon carcinogenesis in h u m a n s is available f r o m a n u m b e r o f sources, including e p i d e m i o l o g y studies o f lung c a n c e r inci-

0165-1110/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII SO165-1110(96)OOOO5-X

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R.F. Jostes / Mutation Research 340 (1996) 125-139

dence in uranium miners and in the general public exposed to radon in the home environment. The incidence of lung cancer as an occupational disease was recognized by Harting and Hesse in 1879. In 1913, it was hypothesized that radon and radon progeny induced a high incidence of lung cancer among the silver and, later, the uranium miners of Saxony, Germany (Schuttmann, 1993; Greenberg and Selikoff, 1993). This relationship was further established by many investigators in subsequent years (e.g., Evans et al., 1981; Samet et al., 1984); a joint analysis of 11 underground-miner studies exists (Lubin et al., 1994). Data from miner studies, however, are often complicated by confounding factors such as smoking and the presence of arsenic, silica, diesel exhaust, and other high-particulate and nonparticulate matter in the mines (Tomasek et al., 1994a,b; Kusiak et al., 1993). Studies of nonsmoking miners confirm that an increase in lung cancer is not due solely (as has sometimes been thought) to the effects of cigarette smoking (Samet et al., 1984; Roscoe et al., 1989; Yao et al., 1994). Epidemiological studies relating to radon-induced cancers in the home are often conflicting, in part because they are limited by dosimetric considerations, and have been reviewed elsewhere (Stidley and Samet, 1993; Cross, 1994). Few investigators question that radon in the home adds to the genetic load for cancer risk, although at least one investigator has suggested a negative correlation (Cohen, 1993). One promising development in recent years is the emerging data on radon-induced changes at the chromosomal and molecular level. An understanding of the chromosomal and molecular mechanisms involved in radon carcinogenesis will help determine risk at the low exposure levels typically found in the home. Evans (1991, 1992)) has provided a review of the literature through 1991 relating to cellular and molecular effects of alphaparticle irradiation and their relationship to radon-induced lung cancer. The amount of information since then is substantial; this review attempts to integrate tumor data from animal exposures and from the miner studies with what is currently known regarding the cytogenetic and molecular changes induced by radon exposure. The review starts with a brief explanation of dosimetric considerations, setting the stage for discussion of studies on carcinogenesis and

of the cellular and molecular effects produced by radon.

2. Dosimetry considerations Much of the discussion about whether radon is carcinogenic in the home environment relates to whether exposure levels in the home are sufficient to produce the effects noted in the uranium miner studies (i.e., is there a radon-exposure threshold for the carcinogenic effect?). This discourse is complicated by the fact that radon-progeny lung doses are affected by the different exposure environments, and different populations in the mine and home. To facilitate an understanding of radon-induced effects, a brief discussion of radon physics and dosimetry is included here. The noble gas radon derives from uranium and thorium decay series. Radon itself decays to radioactive bismuth and polonium, the cations of which attach to various ambient aerosols. Radon and radon progeny comprise a complex radiation source, emitting alpha particles, beta particles, and gamma radiation. The majority of the energy deposited in biological systems derives from the alpha-particle component. A cell nucleus traversed by an alpha particle will experience a range of energy deposition that depends on track length and linear energy transfer (LET) along the track intersecting the nucleus. The LET through the nucleus depends on the source-tonucleus distance and the energy of the alpha particle. The probability of a cell being hit by an alpha particle is also relevant. In the case of uranium miners with high lifetime exposures, some of the lung cell nuclei might experience multiple hits, with substantial time intervals between events. A cell hit more than once would presumably have a higher probability of cell death if recruited into division. In the home environment, however, the probability that a cell nucleus would experience more than one hit is very small. Furthermore, the majority of an individuars lung cell nuclei in the average home environment would experience no hits in a lifetime (zero dose), while a minority would experience one hit, producing a nuclear dose of approx. 0.4-0.5 Gy (Hui et al., 1990; James, 1995).

R.F. Jostes / Mutation Research 340 (1996) 125-139

In addition to the usual terms for absorbed dose, the term 'working level month' (WLM), used in subsequent discussions of epidemiology and animal studies, may be unfamiliar to some readers. A historical working level (WL) is a concentration of shortlived radon progeny producing 1.3 × 105 MeV of potential alpha energy in 1 liter of air. One WLM is equivalent to 170 h of exposure at a concentration of 1 WL. Cytogenetic evidence suggests that 1 WLM of exposure may represent the damage equivalent of 0.8-10.0 mGy, a wide range probably affected by the physical parameters of the lung and the time at which the cytogenetic damage is evaluated (Brooks et al., 1992, 1993; Khan et al., 1994; Johnson and Newton, 1994; Jostes et al., 1993). Many investigators now determine the number of nuclear hits and the alpha energy deposited per hit to better understand the relationship between energy deposition and biological effect. Use of the term 'radon exposure' in this review implies that the source was ambient radon a n d / o r radon progeny (in vivo exposures), or radon a n d / o r radon progeny in cell culture medium (in vitro exposure). Use of the term 'alpha exposure' indicates a surrogate source such as plutonium-238 (238pu) or accelerator-produced alpha particles.

3. Carcinogenic effects of radon 3.1. Studies o f lung tumor incidence in uranium miners

A well-documented correlation exists between radon exposure and lung cancer incidence in uranium miners. These studies are often complicated, however, by the fact that many of the miners studied were cigarette smokers (Waxweiler et al., 1981; Morrison et al., 1988; Hornung and Meinhardt, 1987). Still, one analysis of 516 nonsmoking miners indicated a 12-fold increase in the mortality risk of lung cancer for nonsmoking uranium miners exposed to radon progeny at a median level of 296 WLM when compared with a nonsmoking control population (Roscoe et al., 1989). In this analysis, data were selected from a previous study of Colorado Plateau uranium miners (Wagoner et al., 1964; Lundin et al., 1971; Archer et al., 1976). Other studies of smoking

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and nonsmoking miners indicate that radon alone is a risk factor for lung cancer at the levels encountered by uranium miners (Archer et al., 1976; Samet et al., 1984; Radford and Renard, 1984; Sevc et al., 1988). Other complicating factors include the presence of potentially confounding substances in the mine such as arsenic and silica. In a study that uncoupled the radon exposure risk for lung cancer from silica exposure in the mine (Cocco et al., 1994), the authors concluded that radon alone produced lung tumors in the group studied. One cannot eliminate the possibility, however, that substances such as silica affect the radon-induced response. Because of the high levels of radon and other pollutants in the mines, the relevance of data from mines to the residence is often questioned. Roscoe et al. (1989), for example, noted that their study lacked the statistical power to detect excess lung tumors at radon exposure levels one would expect to encounter in a household. On the other hand, a recent study of miners exposed to relatively low radon concentrations demonstrated a statistically significant increase for lung and laryngeal cancer deaths (Tirmarche et al., 1993). Because of the problems associated with extrapolating results from mines to homes, an understanding of the cytogenetic and molecular basis of radon-induced tumors in miners would be helpful in estimating the risks from exposures found in the home. Fortunately, tumors have been archived from the miner populations and serve as a potential source of future information in this regard as the technology for molecular analyses advances 3.2. Studies o f tumor incidence in radon-exposed animals

Two major series of experiments with rats investigating the relation of radon exposure and risk for lung cancer have been conducted, one at the Laboratoire de Pathologie Professionelle at the Compagnie GEn6rale de Mati~res Nucleaires (COGEMA) in

1Some molecular analyses of tumor tissue derived from individuals in mines and other high-exposureenvironmentshave been limited by the quality of the preservedDNA; it would therefore be helpful to develop tumor fixation techniques that would facilitate future molecularevaluationof the relevant genetic changes.

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R.F. Jostes / Mutation Research 340 (1996) 125-139

Table 1 Transformation after alpha-particle irradiation Source238 Transformation system Pu In vivo radon 4He 4He

4He Accelerator-producedparticles Accelerator-producedalpha particles In vitro radon

Ref.

Rat tracheal epithelial cells Rat tracheal epithelial cells Rat embryo cells C3H 10T1/2 cells Immortalized human bronchial epithelial cells C3H 10T1/2 cells Syrian hamster embryo cells C3H 10TI/2 cells

France and another at Pacific Northwest National Laboratory (PNNL) 2 in the United States. The major carcinogenic effect noted in both studies was respiratory tract tumors (Monchaux et al., 1994; Cross et al., 1984, Cross et al., 1986; Cross, 1988). Evidence for an inverse exposure-rate effect (increased production of neoplasms with protraction of exposure) was also noted in these studies. In exposure-rate-effect studies at C O G E M A (Chameaud et al., 1982), a 5-fold protraction of a 6000 W L M radon exposure resulted in a 4-fold increase in lung cancer occurrence in Sprague-Dawley rats. A doserate study at PNNL indicated that when 320-WLM exposures were given at exposure rates which differed by an order of magnitude (100 WL vs. 1000 WL), the lower dose rate resulted in a significantly higher tumor incidence (by factors of 2-3). The inverse dose-rate effect also has been described in various underground miner studies; e.g., in a study of tin miners, an increase in exposure rate resulted in a decrease in tumor incidence (Xuan et al., 1993). However, the inverse exposure-rate effect may not apply to radon in the home environment. It should be emphasized again that at cumulative radon exposure levels in houses (typically < 50 WLM), the majority of the lung cell nuclei would experience no alpha hits, while a small minority would experience only one alpha hit. At these exposure levels, the probability of multiple hits would be vanishingly small, and no dose-rate effect would be expected. A

2 Pacific Northwest National Laboratory is a multiprogram national laboratory operated by Battelle Memorial Institute for the U.S. Department of Energy under Contract DE-AC06-76 RLO 1830.

Thomassen et al. (1990a) Thomassen et al. (1990b) Endlich et al. (1993) Piao and Hei (1993) Hei et al. (1994a) Miller et al. (1995, Brenner et al. (1995) Martin et al. (1995) Miller et al. (in press)

second C O G E M A data set illustrates this point. This study showed that a 25-WLM cumulative exposure protracted over 4 - 6 months gave a statistically significant increase in lung tumor incidence (2.3%) over control animals, whereas the same dose protracted for an 18-month period gave no statistically significant difference over control animals (0.6%; Chameaud et al., 1985). These latter data suggest that there may be a threshold dose rate (and dose) for induction of radon-induced lung tumors (Cross, 1994). In conclusion, one might expect that an inverse dose-rate effect would not apply to the very low exposures and exposure rates prevalent in the home environment (Brenner, 1994).

4. Cellular and molecular effects of radon exposure 4.1. Cell transformation studies

Cell transformation studies provide a link between tumor incidence studies and cell biology studies designed to investigate the mechanisms involved in the carcinogenic process. A number of mammalian cell systems have been used to demonstrate transformation after alpha-particle exposure, and early studies have been reviewed (Evans, 1991, 1992). Recent studies in which positive transformation data have been reported for radon-related exposures are summarized in Table 1. One study using accelerated particles and C3H 1 0 T 1 / 2 cells demonstrated that the efficiency of transformation increased with LET up to approx. 7 0 - 1 0 0 k e V / / x m , after which there was a decrease

R.F. Jostes/ Mutation Research340 (1996) 125-139

in effectiveness (Miller et al., 1995). The peak effectiveness for transformation in this study is in the range of LETs expected with radon exposure, and the efficiency was in good agreement with that obtained in a subsequent study using an in vitro radonexposure source (Miller et al., in review 3). A recent investigation used immortalized human bronchial epithelial cells and in vitro radon-progeny exposure to show that immortalized human cells in culture can be malignantly transformed by a single, 0.3-Gy dose of alpha particles (Hei et al., 1994a). The so-called inverse dose-rate effect, noted previously in experiments with rats and first observed with neutron-induced transformation (Hill et al., 1982), also has been observed with alpha-induced transformation (Miller et al., 1993). In this later study, the authors noted that the increase in transformation efficiency with dose fractionation occurred only at LETs between 40 and 120 k e V / / z m , and only in cycling cells in log-phase. The authors believe that this inverse effect is dose-, dose-rate-, and cell-cycle-dependent, and that it is based on the existence in the cycle of a period of high sensitivity for cell transformation; the effect disappeared at LETs exceeding 120 keV/p~m. The authors believe this is due to a reduction in the number of cells being hit (Brenner et al., 1993, Endlich et al., 1993). 4.2. Cytogenetic studies with radon

In general, most chromosome aberrations are believed to be lethal events. It is known, however, that some aberrations persist in cycling cells as stable events. Reciprocal translocations have been found in some tumor types adjacent to known oncogenes, and in the case of colon cancer, may comprise distinct temporal events in the multistep process leading to colon cancer (Vogelstein et al., 1988). Cytogenetic studies with radon are thus considered important to a complete understanding of the events that may be

3 Miller, R.C., M. Richards, D.J. Brenner, E.J. Hall, R.F. Jostes, T.E. Hui and A.L. Brooks. The biological effectivenessof radon-progeny alpha particles. V. Oncogenic transformation from monoenergetic accelerator-producedalpha particles comparedwith polyenergetic alpha particles from radon progeny. Submitted to Radiation Research.

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involved in radon-induced neoplasia. Examples of such studies are discussed in the following sections. 4.2.1. Micronuclei formation in the lung cells of radon-exposed animals Radon effects in vivo and in vitro have been evaluated relative to low-LET irradiation using induced micronuclei as an indication of cytogenetic damage (Brooks et al., 1994). In this study, micronuclei were scored in lung fibroblasts from Wistar rats exposed by inhalation of radon, radon progeny, and a uranium-ore-dust carrier aerosol, or by whole-body 6°Co exposure. Lung fibroblasts were then obtained by trypsinization after the exposure intervals. In vivo radon exposure was approx. 11 times as effective as acute, whole-body 6°Co exposure in producing micronuclei in lung fibroblasts. As a comparison, in vitro analysis of primary rat lung fibroblasts (RLF) and Chinese hamster ovary (CHO) cells were exposed to either 6°Co or radon/radon progeny via an in vitro radon-exposure system. In this case, radon was approx. 11 and 13 (RLF and CHO cells, respectively) times more effective than 6°Co per Gy of radiation dose in the production of micronuclei. Thus, different cell lines and exposure conditions resulted in similar effectiveness factors. When the dose rate and time (67 h) for the 6°Co exposure was similar to that for radon, the relative biological effectiveness (relative to low-LET-irradiation RBE) increased to 65. Doses and dose rates were varied, and the time of exposure for 6°Co and radon were held constant at 67 h (Brooks et al., 1995). Radon-induced micronuclei in deep-lung fibroblasts also have been compared in the Wistar rat, Syrian Golden hamster, and Chinese hamster (Khan et al., 1995). The authors selected the Syrian Golden hamster because of this species' insensitivity to radon-induced lung cancer, and the Wistar rat because of its demonstrated sensitivity to radon-induced tumors (Cross et al., 1981); the sensitivity of the Chinese hamster cells in this respect is unknown. The number of micronuclei obtained after radon exposure was highest in the Chinese hamster, followed by the Syrian Golden hamster, and finally, the Wistar rat. When animals were killed at several time intervals (up to 30 days after exposure), the number of micronuclei decreased, as expected, but the species sensitivity remained the same. The authors con-

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cluded that cytogenetic damage, as determined by the micronucleus assay, does not explain the difference in tumor induction between the Wistar rat and the Syrian Golden hamster. 4.2.2. Chromosome aberration formation in the lung cells of radon-exposed animals Chromosomal aberrations and changes in cell turnover have been noted in alveolar macrophages isolated from the lungs of rats exposed to radon/radon progeny (Taya et al., 1994). Cell proliferation and micronuclei increased with increasing exposure. Tracheal secretory and basal epithelial cells also have been isolated from radon-exposed rats and evaluated for chromosome aberration formation (Brooks et al., 1992); the carrier aerosol in this study was cigarette smoke. A linear increase in aberration frequency with dose was found in both tracheal secretory and basal epithelial cell types after X-ray exposure, suggesting a similar sensitivity between the two types of cells thought to be at risk for cancer induction. Extensive damage, including many deletions, was noted at the 900-WLM radon exposure. The response for those cells exposed to 900 WLM was similar to that observed for a range of other high-LET radiation at a dose of 0.5 Gy. Chromosome aberrations decreased with time after exposure, with a half-life of 25 days, which the authors attributed to cell turnover. 4.2.3. Chromosome aberrations from in vitro radon exposures Chromosome aberrations have been analyzed in radon-exposed cycling human peripheral blood lymphocytes. An in vitro cell-suspension exposure system was used to irradiate whole blood preparations (Jostes et al., 1992) and a dose- and time-dependent increase in aberrations was noted after an initial block in progression to mitosis (approx. 6 h). After the initial delay, aberrations increased in a dose- and time-dependent manner until 17 h after exposure, the longest time period investigated. Schwartz et al. (1990) and Shadley et al. (1991) investigated chromosome aberrations induced in repair-proficient and repair-deficient CHO cells by the radon decay product, bismuth-212 (212Bi). The first study compared CHO-AA8 to its radiosensitive derivative, EM9, which repairs DNA single-strand

breaks less efficiently than the parental line. As expected, 212Bi induced chromatid breaks more efficiently than X-rays in both cell lines, and the repairdeficient line was more sensitive than the parental line to aberration induction. In the second study, using CHO-K1 and the DNA double-strand-break repair-deficient cell line, xrs-5, 212Bi was 3.5 times as effective as X rays (inducing 2 chromatid aberrations/cell) in the repair-proficient cell line, and slightly less effective for aberration production in the repair-deficient line. Although these are quite different results, the Schwartz and Shadley studies suggest that repair status may affect the cellular response to radon exposure. The relevance of repair status to carcinogenesis may relate more to an understanding of mechanisms than to the occurrence of repair mutants in the target tissue. 4.2.4. Alpha-particle-induced chromosomal instability Some studies suggest that chromosomal changes (as well as gene mutations) can occur up to 50 generations after radiation exposure where one would expect residual damage to be minimal (the so-called 'genome instability' effect suggesting that the induction of genomic instability may play a significant role in radon-induced genetic effects). For reviews of the induction of genomic instability, see Cheng and Loeb (1993) and Murnane 4. Such findings imply alpha-particle effects at exposures previously thought not to cause chromosomal damage, or effects in numbers of cells greater than would be predicted from alpha hit probabilities and DNA repair considerations. In one such study, Kadhim et al. (1992, Kadhim et al., 1994) found nonclonal cytogenetic aberrations in clonal descendants of hematopoietic stem cells after low-dose, alpha-particle exposure of murine and human bone marrow cells. The authors concluded that the data were consistent with a transmissible chromosomal instability induced in a stem cell, resulting in a diversity of aberrations in its clonal descendants. Nagasawa and Little (1992) suggested another possible complication of estimation of

4 Murnane, J. P. Role of genetic instability in the mutagenic effects of chemicals and radiation. Submitted to Radiation Research.

R.F. Jostes/ Mutation Research340 (1996) 125-139

risk after radon exposure. They noted elevated sister chromatid exchanges in 30% of the cells exposed to 0.31 mGy 238pu, although it was predicted that only 1% of the cell nuclei were traversed by an alpha particle following the exposure. 4.2.5. Adaptation A study investigating the concept of cell adaptation presents data which suggests that small doses of low-LET irradiation reduced the level of chromosome aberrations induced by a subsequent acute radon exposure in human peripheral blood lymphocytes (Wolff et al., 1991, Wolff et al., 1993). This finding may have implications for complex radiation sources, such as radon and radon progeny, if one speculates that the alpha effect might be decreased by the presence of low-LET radiation (the so-called adaptive effect). Pohl-Ruhling et al. (1980) made a related observation when they showed that unscheduled DNA synthesis was increased in spa-treatment personnel, and increased further in workers at a spa located in a former gold mine in Bad Gastein, Austria. Radon exposures (estimated by the level of chromosomal aberrations) were higher in the spa workers than the general Viennese population, and were highest in the mine workers. Conversely, another study reported that simultaneous alpha and X irradiation increased the number of micronuclei over that expected if the effects were additive (Brooks et al., 1990). More data will be required to establish the effect that such phenomena might have on the overall radon response.

131

into division after radon exposure, may be involved in radon carcinogenesis, gene mutation is another probable component of the carcinogenic process. Evidence for this hypothesis comes from an investigation in which 5 exons of the p53 tumor suppresser gene were sequenced from 19 lung cancers obtained from uranium miners exposed to radon and tobacco smoke (Vahakangas et al., 1992). Thirty-seven percent of the tumors had base substitutions or deletions in exons 5 or 6 of the p53 gene. None of the gene alterations was a G : C to A : T transition in the coding strand of the p53 gene, which is the most frequent base substitution associated with tobacco smoking. The mutations were predominantly transversions, but a significant finding was that two of the mutations were small deletions (4 and 12 base pairs), which have been reported only rarely in human lung cancer. Another study of mutations in the p53 gene from uranium miner lung cancers indicated that 31% of 52 cancers contained the same AGG to ATG transversion at codon 249, suggesting that a possible p53 marker (or 'signature') exists for radon-induced lung cancer (Taylor et al., 1994). It has been suggested that these mutations may be due to aflatoxin exposure in the mines from endogenous molds (Venitt and Biggs, 1994); the original authors are open to alternate suggestions (Taylor and Anderson, 1994). The controversial nature of the importance of base changes and small frameshift events relative to large deletions will be briefly considered in the discussion section of this review. 5.2. In vitro mutation studies with endogenous genes

5. Genetic effects of radon A wide variety of mutation systems have demonstrated alpha-particle mutagenesis, and early studies have been reviewed (Evans, 1991, Evans, 1992). Recent investigations have focused on the molecular basis of alpha-induced mutagenesis. 5.1. Mutations in the p53 gene in lung tumors from uranium miners

The molecular basis of radon-induced mutations is considered relevant to the carcinogenic process. While epigenetic factors, such as stimulation of cells

Evidence for the induction of mutations in vitro at endogenous gene loci by radon have been reported for various cell lines and gene loci (Jostes et al., 1992; Evans et al., 1993). In general, mutations are induced by radon in a linear and dose-dependent fashion, and with a high RBE relative to low-LET irradiation. A number of studies have reported molecular analyses of radon-induced mutations and are described in the following sections. 5.2.1. Molecular evaluation at the hprt locus Radon exposure in vitro induces mutations at the hemizygous hprt locus in CHO cells (Jostes et al., 1992; Aghamohammadi et al., 1992; Schwartz et al.,

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R.F. Jostes / Mutation Research 340 (1996) 125-139

1994) and in human cells (Chen et al., 1992; Metting et al., 1992; Bao et al., 1995). These reports and subsequent studies have described the gross molecular nature of alpha-particle-induced mutations at the hprt locus using Southern blot a n d / o r PCR exon analysis. These molecular techniques detect three general categories of change: 1. The entire gene is deleted 5 2. Specific exons are missing, or the gene is altered. 3. There is no detectable change from the parental hprt gene (possibly due to base-pair substitutions or very small deletions a n d / o r insertions). Three studies investigating the molecular nature of HPRT mutations in CHO cells produced similar results (Jostes et al., 1992, Jostes et al., 1994; Aghamohammadi et al., 1992; Schwartz et al., 1994); data are presented in Table 2. All three studies included evaluation of spontaneous mutants and mutants induced by low-LET radiation for comparison with the radon mutants. The results of the three studies were remarkably similar in that 45-53% of the radon-induced mutations were full deletions of the gene, while the remaining mutations were approximately equally divided between gene alterations (predominantly partial deletions of the gene) and no detectable change. At the level of analysis used in these studies, the spectrum of damage types in the radon and low-LET-induced mutants did not differ greatly, although in one study (Schwartz et al., 1994) a small difference was detected between radon and low-LET-induced mutants in the pattern of exons deleted. Conversely, 74-87% of the spontaneous mutants studied by the three laboratories were classified as containing no detectable change, and the remainder as complete gene deletions or alterations. Another study, using 238pu- and gamma-irradiated normal human skin fibroblasts (Chen et al., 1992), gave percentages very similar to those of the CHO data for alpha irradiation, while the low-LET-induced mutations had approximately half the percent-

5 Gene deletion is usually accompanied by loss of heterozygosity in sequences neighboring the hprt gene. The radon-induced mutagenic lesion has been found to extend up to 1.2 Mb from the hprt gene. An essential gene is thought to be located at about 1.2 mb from the 3' end of the hprt gene (Nelson and Grosovsky, 1995).

age of total deletions (23%) seen in alpha-induced mutations; spectra for the spontaneous mutations were not presented. Hei has published Southern analysis data which indicated that, in the A L cell line, alpha-induced mutations at the hprt and $1 loci were predominantly multilocus deletions (Hei et al., 1994b). Other studies of the hprt locus in human cells (Metting et al., 1992; Bao et al., 1995) suggest that these percentages can vary with the cell type studied and also may depend on the chromosomal environment of the gene. In the Bao study, in which human lymphoblastoid cells were used, the percent of mutants with total deletions of the hprt gene was similar to the percentage observed in the CHO studies (55% of the radon-treated mutants); the study by Metting et al. (1992), who used 212Bi-irradiated human B-lymphoblasts, put the value for total deletion of the hprt gene at 29%. The Bao study reported slightly higher values for intragenic and total deletions of the gene in mutants derived from irradiated cells (81% of the mutants from X-ray-treated cultures contained partial and total deletions vs. 86% of the mutants from the radon-treated cultures). There was also a much higher percentage of mutants with partial and total deletions in their spontaneous mutants (63%). Table 2 presents a summary of the hprt data. Reviewing the data in Table 2 with regard to the hprt locus, one can make the general statement that approximately half of the alpha-induced mutations arise by complete deletion of the gene; the remaining mutations are split between partial deletions and rearrangements, and events not detectable by Southern blot or PCR exon analysis. The exception to these findings is the work by Metting et al. (1992) who found that full deletions constituted 29% of the alpha-induced sample. The authors note, however, that this result would be 34% if corrected for the relatively high spontaneous component in the alphainduced mutations. The alpha-particle data from the studies noted in Table 2 are not consistently different from the data obtained in studies with low-LET irradiation. With respect to full deletions, it should be noted that it is probable that the lesions in mutants harboring deletions of the entire gene or partial deletions involving exons 1 or 9 extend to the neighboring regions. These lesions have been found to extend to

16 10 74

3 19 78

Not done

16 14 70

23 ~ 45 32

Total deletion Partial deletion a Other b

Total deletion Partial deletion a Other b

Total deletion Partial deletion a Other b

Total deletion Partial deletion a Other b

Total deletion Partial deletion ~ Other b

59 28 14

Not done

23 20 57

30 45 25

47 21 32

45 10 45

a Includes rearrangements if determined. b Possibly very small deletions/insertions and base changes. c Percentages excludes unknown mutations reported in paper.s

8 5 87

X- or 3,-irradiated

(~)

57 32 11

29 40 31

50 20 30

53 25 22

48 23 29

45 12 43

(~)

Alpha-irradiated

locus using Southern blot a n d / o r PCR exon analysis

(%)

hprt

Spontaneous

Total deletion Partial deletion a Other b

Mutation type

Table 2 Molecular analyses of the

1-9

1-9

1-9

1-9

2-9

3, 5, and 9

Exons analyzed

+/ +

+/ -

+/ -

- / +

+/ +

- / +

Southern blot/PCR

Human lympho-blastoid/radon Bao et al. (1995)

Human B-lympho-blasts/212 Bi Metting et al. (1992)

Normal human skin/238pu Chen et al. (1992)

CHO-K1 / 2~2Bi Schwartz et al. (1994)

CHO/radon Jostes et al. (1992, Jostes et al., 1994)

CHO/238pu Aghamohammadi et al. (1992)

Cell line/a-source reference

=-

-~

.~ .~

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1.2 Mb in the 3' direction and > 300 kb in the 5' direction (Bao et al., 1995). 5.2.2. Molecular evaluation at the tk locus The relative percentages that comprise the molecular spectra described in the hprt locus appear to be specific to the gene and are probably affected by gene environment. Chaudrey et al. (in press) indicated that approx. 82% of the radon-induced mutations at the L5178Y tk locus resulted from loss of the entire allele, while allele loss comprised 74% of the X-ray-induced mutants. It should be noted that in this selection system 45% of the spontaneous mutants lost the entire active tk allele. Interestingly, analysis of distant loci indicated that a larger percentage of X-ray-induced mutants harbored larger multilocus lesions (34 Mb) than the radon-induced mutations. Metting et al. (1992) also included Southern blot analysis of the heterozygous tk locus in their study of 212Bi-induced mutations in the human lymphoblast cell line TK6. In this study, 100% of the 212Bi-induced mutations showed complete loss of heterozygosity at the tk locus (LOH; deletion of one of the gene alleles), while 76% of the X-ray-induced and 25% of the spontaneous mutants exhibited this LOH.

the human and rodent cells, might play a part as well. The significance of these findings is that alpha-irradiation does induce base-change mutations, but the relative importance of alpha-induced base changes to carcinogenesis remains to be determined. In the second study, a shuttle vector designed to evaluate radiation-induced deletion mutagenesis was exposed to radon and radon progeny using an in vitro suspension system (Lutze et al., 1992). This vector, designated pHAZE, has the entire 3.1-kilobase Escherichia coli lacZ gene as a target for mutagenesis. Restriction fragment length polymorphism analysis of radon-induced deletions indicated that 64% of the mutations were deletions, in contrast to 13% in a previous study with X-rays. Further mapping revealed that these deletions ranged in size from 2435-8051 base pairs (close to the maximum detectable). When the deletion breakpoint rejoining regions were sequenced, the authors found sequence homology involving one to six base pairs at the junction regions in 65% of the mutants containing large rearrangements. According to the authors, this homology would not be predicted to occur by chance. In two cases, a single base pair was inserted at the junction. The authors interpret homology at the deletion breakpoint regions as being due to a nonhomologous recombination repair mechanism.

5.3. In vitro mutation studies with shuttle vectors 6. Discussion

Two studies of the molecular nature of alpha-particle-induced mutation in shuttle vectors irradiated in human cell lines have been published. The first study, using the shuttle vector PZ 189 containing a supF target gene, demonstrated the induction of base-pair changes after alpha-irradiation of the vector in vitro or transfected into human cells (Jaberaboansari et al., 1991). The small target size of this vector and its structure, with the target gene situated between two genes essential for plasmid replication and selection, detects mainly base-change type damage. The alpha-induced spectra were different from the spectra seen in the hemizygous mammalian aprt gene after low-LET irradiation (Grosovsky et al., 1988). Although some of the differences may be due to the radiation quality, other factors such as the geometry of the supF gene in a small extra chromosomal vector, and repair differences in

Radon exposure has been linked to lung carcinogenesis in both human and animal studies. Studies of nonsmoking uranium miners indicate that radon alone is a risk factor for lung cancer at the levels encountered by these miners, although the possibility exists that other substances in the mine environment, such as arsenic and silica, affect the radon-induced response. The relevance of data from mines to the lower-exposure home environment is often questioned; still, a recent study of miners exposed to relatively low radon concentrations demonstrated a statistically significant increase for lung and laryngeal cancer deaths. Data are emerging in cellular studies regarding radon-induced changes to DNA at the chromosomal and molecular level that should eventually clarify the mechanisms involved in radon carcinogenesis. This information should improve the

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estimation of risk at the low exposure levels typically found in the home. Many investigators have noted that large deletions are the predominant event in mutations induced by alpha irradiation in vitro; some believe that the majority of radiation-induced mutations in vivo and in vitro result from loss of the entire gene, along with neighboring chromosomal regions. Mutations found in several cancer-relevant genes, obtained from lung tumor tissue in uranium miners, are base-pair changes or frameshift events. Frameshift and base-change mutations can have a profound effect on the function of a gene. The role that loss of heterozygosity plays in radiation-induced tumors will require further analysis. The importance of radiation-induced deletions relative to base changes and frameshift events in the genesis of radiation-induced cancers is a subject of interest and open to investigation. In this regard, it would be helpful to make a connection between the initial lesions that radiation introduces into DNA and the spectra of damage found in tumor-relevant genes. Although there appears to be considerable variation in the radiation-induced mutation spectra, the predominant mutational lesion produced by alpha irradiation appears to be partial or complete deletion of the gene, as well as loss of heterozygosity in the neighboring chromosomal regions ranging up to 34 Mb distant from the target gene (Bao et al., 1995). Cytogenetic changes are known to be involved in the carcinogenic process. As an example, it is recognized that chromosome translocations in the vicinity of known oncogenes are frequent events in certain cancers (such as B-cell lymphoma and chronic myeloid leukemia (Croce, 1987). Although translocations have not been evaluated in radon-induced tumors, they do occur after radon exposure in other test systems (Brooks et al., 1990; Jostes, unpublished observation). Visible chromosome deletions represent an extreme version of the multilocus deletion, and most likely are lethal events. Persistent (nonlethal) aberrations, such as reciprocal translocations, may add to the carcinogenic load. One cannot ignore the possible contribution of epigenetic factors in radon-induced cancers. These factors include, but are not limited to, such effects as the recruitment of quiescent cells into division by alpha exposure, thus putting the cells at risk for carcinogenic transformation (Taya et al., 1994). Cells

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hit by alpha particles may also influence neighboring cells in such a way that they are at risk. This latter possibility would lead to an underestimate of carcinogenic risk derived from alpha-particle hit probabilities. Conversely, an alpha particle traversal might render the cell refractory to subsequent carcinogenic insult through the recruitment of repair enzymes (the so-called adaptive effect). Determination of the impact of such epigenetic and modifying factors will require further investigation. It is possible that the initial insult produced by radon exposure will be affected by a number of such factors. The question of whether there will be a molecular or cytogenetic 'signature' for high LET vs. low LET radiation in tumors remains unresolved at this time. Although there are indications that such a phenomenon might exist, consistent replication of the data would be required to establish the effect. Future studies will include information derived from radiation sources that provide defined numbers of particles at known intervals to single cells in culture. Such sources exist at Pacific Northwest Laboratories, Columbia University, and the Gray laboratory in England (Braby, 1992). In this fashion, one should be able to derive information about potential thresholds for high-LET effects.

7. Conclusion Information derived from human ore miners and from animal studies has demonstrated that radon is a factor in the genesis of lung tumors. This same effect has not been clearly demonstrated in the lower-exposure home environment. At the cellular and molecular level, radon and radon progeny cause cell transformation, changes in chromosome structure, and gene mutations containing a wide range of deletions as well as base-pair changes. It seems reasonable to assume, then, that radon exposure in the home environment adds to the genetic load for cancer risk, as it does in the mine environment. Further investigation will be required to determine whether complicating factors such as differences in the exposure environment of the home and mines, or to phenomena such as adaptation and genetic instability, complicate the response.

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Acknowledgements I would like to thank Drs. Fred Cross, Antone Brooks, and Edmond Hui for their helpful discussions regarding these data, and Catherine Lumetta for her excellent editorial assistance. I also thank Will Chrisler for helping prepare this manuscript. R.F. Jostes' work supported in part by the U.S. Department of Energy under contract DE-AC0676RLO 1830.

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