Human T lymphocytes can be used to determine the frequency and molecular spectrum of somatic cell gene mutations induced by ionizing radiations both in vivo and in vitro. In vitro exposure of these G0 cells to low-LET 137Cs gamma rays results in the induction of HPRT mutations and a predominant molecular spectrum of DNA deletions and rearrangements, particularly total gene deletions (11-12%). Similar results are found in samples from humans exposed to low-LET radiation from 131I. The doubling dose for mutation induction is calculated to be 0.8 and 1.0 Gy from these exposures performed in vitro and in vivo, respectively. In vitro studies of the effects of high-LET radiation from exposure to 222Rn also showed an induction of HPRT mutations, with a doubling dose of approximately 0.2 Gy. With this radiation, the predominant mutations were small partial deletions, with less than 2% total gene deletions. Studies of humans exposed to high-LET radiation from 239Pu showed an increased HPRT mutant frequency for the group, although no significant dosimetry could be defined. In contrast to the humans exposed to 131I, no increase in the frequency of total gene deletions was found. This is consistent with the results for 222Rn in vitro. The available data show that radiation quality affects both the efficiency of induction and the molecular spectrum of HPRT mutations in human T lymphocytes both in vitro and in vivo. The mutational spectrum may be relatively specific for radiations of different quality and thus allow a more precise measurement of the induction of somatic gene mutations resulting from individual exposures to radiation, and thereby provide more sensitive assessments of health risks.

Generation of estimates of risk caused by exposure to radon in the home, either from miner data or from A-bomb data, requires several scaling factors such as for dose, dose rate and radiation quality, and possible synergisms. Such scaling factors are best developed from laboratory-based studies. Two possible sources of alpha particles for such studies are (1) a polyenergetic spectrum, generated directly by radon and its progeny, or (2) a series of monoenergetic alpha particles. We compare here the results of oncogenic transformation from studies using both systems. At the Columbia University Radiological Research Accelerator Facility (RARAF), C3H 10T1/2 cells were irradiated with alpha particles of various energies, with defined LETs from 70 to 200 keV/mum. At Pacific Northwest Laboratory, cells from the same stock were exposed to alpha particles from radon gas and its progeny, which were in equilibrium with the culture medium. There was good agreement between the results of oncogenic transformation experiments using the two different exposure systems. Apart from the experimental transformation frequencies themselves, such a comparison requires (1) reliable dosimetry at both facilities and (2) estimated LET distributions for the polyenergetic alpha-particle irradiator. Thus this good agreement gives some confirmation to the technique which is used to fold together oncogenic transformation rates from monoenergetic alpha particles to yield a predicted rate for a spectrum of alpha particles.

Genotoxic damage induced by radon and its progeny was investigated using the micronucleus assay in deep-lung fibroblasts to compare the response induced in vitro with that induced from inhalation of radon and its progeny in vivo. Male Wistar rats were exposed to 0, 115, 213 and 323 working-level months (WLM) of radon and its progeny by inhalation. After sacrifice, the cells were isolated and grown in culture, and the frequency of micronuclei was determined. A linear increase in the frequency of micronuclei was measured as a function of exposure [micronuclei/1000 binucleated cells = (29 +/- 9) + (0.47 +/- 0.04) WLM]. To compare exposure in WLM to dose in mGy, and to study how cell proliferation influences the way inhalation of radon and its progeny induces micronuclei, lung fibroblasts were isolated and exposed in vitro to graded doses from radon and its progeny after either 16 or 96 h in tissue culture. Cell cycle stage at the time of exposure was determined using flow cytometry. Primary lung fibroblasts exposed as either nondividing or dividing cells showed dose-dependent increases in micronuclei [micronuclei/1000 binucleated cells = (33 +/- 40) + (593 +/- 68)D and micronuclei/1000 binucleated cells = (27 +/- 69) + (757 +/- 88)D, respectively, where D is dose in Gy]. Results showed no significant influence (P = 0.20) of cell proliferation at the time of exposure on the frequency of micronuclei induced by radon and its progeny. Comparing dose-response relationships for nondividing cells to the exposure response for cells exposed by inhalation of radon and its progeny, it was estimated that a 1-WLM exposure in vivo caused the same amount of cytogenetic damage as produced by 0.79 mGy in vitro. In vivo/in vitro research using the micronucleus assay in lung fibroblasts serves as a powerful tool to estimate effective dose to cells in the respiratory tract after inhalation of radon and its progeny. Such studies form the basis for understanding the relationship between exposure, dose and biological damage.

Radon is a ubiquitous inhaled human carcinogen that is thought to be the largest single natural source of human exposure to radiation. We report that a freely replicating episome in human cells exposed to radon gas underwent mutagenic changes, a high proportion of which were large deletions involving many thousands of base pairs. These deletions were not randomly distributed but started and ended in defined regions as if caused by the passage of a single alpha-particle track through a coiled chromatin structure. The sizes appeared to be defined by structural features of chromatin: the minimum size was 2435 base pairs, and the maximum size was 8051 base pairs, close to the upper limit that would leave intact the plasmid sequences required for selection in bacteria. Ends were rejoined by nonhomologous recombination involving up to 6 base pairs of homology. This process may not be confined to the repair of exogenously induced double-strand breaks but may be used for rejoining free DNA ends generated by a variety of cellular processes. The mechanism of alpha-particle deletion mutagenesis may account for the high relative biological effectiveness of radon irradiation for many end points and its consequences for lung carcinogenesis.

In human lymphocytes low doses of X-rays can decrease the number of chromatid deletions induced by subsequent high doses of sparsely ionizing X-rays. Because of the concern with the carcinogenic effects of low doses of α-particles from radon in homes, experiments were carried out to see if low doses of X-rays could also decrease the yield of chromosomal aberrations induced by subsequent exposure to radon. Human peripheral blood lymphocytes were irradiated with low doses of X-rays (2 cGy) at 48 h of culture, exposed to radon at 72 h of culture, and analyzed for the presence of chromatid aberrations at subsequent intervals. The frequency of chromatid aberrations induced by radon alone increased with time after exposure, indicating exaggerated differences in the stage sensitivity of cell cycle stages to high-LET radiation. Furthermore, the numbers of aberrations per cell did not follow a Poisson distribution but were over dispersed, as might be expected since high-LET radiations have a high relative biological effectiveness compared with low-LET radiations. Nevertheless, lymphocytes exposed to 2 cGy of X-rays before radon exposure contained approximately one-half the number of chromatid deletions compared with lymphocytes treated with radon alone and analzed at the same time. Thus, the putative chromosomal repair mechanism induced by low doses of sparsely ionizing radiation is also effective in reducing chromosomal aberrations induced by radon, which hitherto had been thought to be relatively independent of repair processes.

On the basis of qualitative assays, quiescent lymphocytes have previously been reported to have numerous DNA strand breaks, which are thought to be repaired after mitogenic stimulation by a process associated with poly(ADP-ribosyl)ation. Using alkaline elution, a very sensitive assay for quantifying DNA single-strand breakage, we found no evidence for a high frequency of DNA strand breaks in unstimulated human peripheral blood lymphocytes. No differences in elution profiles were observed between unstimulated lymphocytes and lymphocytes 4 or 48 h after addition of the mitogen phytohemagglutinin (PHA). Furthermore, addition of 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) synthetase, or aphidicolin, an inhibitor of DNA polymerase alpha, did not increase the amount of DNA eluting from the filter after PHA stimulation. In contrast to reported studies of mouse splenic lymphocytes, we found that human lymphocytes were able to replicate and divide in the presence of the ADP-ribosylation inhibitor. Human lymphocytes were also capable of proliferating in nicotinamide-free medium, with or without 3AB, indicating that ADP-ribosylation is not a requirement for lymphocyte differentiation. We therefore consider it unlikely that peripheral human lymphocytes contain significant numbers of strand breaks that play any role in their stimulation or differentiation in response to PHA.