The oncogenic transforming potential of the passage of single a particles through mammalian cell nuclei

Domestic, low-level exposure to radon gas is considered a major environmental lung-cancer hazard involving DNA damage to bronchial cells by a particles from radon progeny. At domestic exposure levels, the relevant bronchial cells are very rarely traversed by more than one a particle, whereas at higher radon levels—at which epidemiological studies in uranium miners allow lung-cancer risks to be quantified with reasonable precision—these bronchial cells are frequently exposed to multiple a-particle traversals. Measuring the oncogenic transforming effects of exactly one a particle without the confounding effects of multiple traversals has hitherto been unfeasible, resulting in uncertainty in extrapolations of risk from high to domestic radon levels. A technique to assess the effects of single a particles uses a charged-particle microbeam, which irradiates individual cells or cell nuclei with predefined exact numbers of particles. Although previously too slow to assess the relevant small oncogenic risks, recent improvements in throughput now permit microbeam irradiation of large cell numbers, allowing the first oncogenic risk measurements for the traversal of exactly one a particle through a cell nucleus. Given positive controls to ensure that the dosimetry and biological controls were comparable, the measured oncogenicity from exactly one a particle was significantly lower than for a Poissondistributed mean of one a particle, implying that cells traversed by multiple a particles contribute most of the risk. If this result applies generally, extrapolation from high-level radon risks (involving cellular traversal by multiple a particles) may overestimate low-level (involving only single a particles) radon risks. Domestic exposure to radon gas in homes generally is considered to be the single largest naturally occurring environmental hazard (1). The basic mechanism involves DNA damage to bronchioepithelial cells by a particles emitted by radon progeny. The most recent report (1) from the U.S. National Academy of Sciences on the health effects of environmental exposure to radon gas (BEIR VI) estimated that 10–14% of all lung cancer deaths—amounting to central estimates of about 15,400–21,800 per year in the United States—are linked to radon gas exposure from the environment. The BEIR VI (1) estimates (and others) of the risks of domestic radon exposure were made by extrapolating risks from underground miners who received radon exposures that were, on average, many times larger than those of people in most homes. The problem inherent in this extrapolation is that, at these high exposures, the cells at risk in the bronchial epithelium of miners may be traversed by several a particles during a short period, whereas for individuals exposed in homes at normal domestic radon levels, it is unlikely that any cell at risk will be traversed by more than one a particle in a lifetime (1). Even in the laboratory, there has until now been no direct way of measuring the oncogenic transforming effects of exactly one a particle without the confounding effects of a significant fraction of exposed cells being subject to multiple a-particle traversals, and this has led to significant uncertainty in lowdose radon risk estimates (2). In recent years, several groups have developed chargedparticle microbeams, in which cells on a dish are individually irradiated by a predefined exact number of a particles, thus allowing the effects of exactly one (or more) a particle traversals to be assessed (3–8). However, earlier microbeam irradiation systems have been too slow to measure oncogenic transformation frequencies, because the low probabilities involved require that many cells ('105) be individually irradiated. Specifically, the current overall irradiation throughput for the microbeam experiments described here is '3,000 cells per hr, which may be compared with earlier microbeam system throughputs of '120 cells per hr (6). In practice, the earlier low throughput precluded the irradiation of the '105 cells necessary for measurements of oncogenic transformation frequencies. This increased microbeam throughput, made possible by developments in both hardware and software, now permits sufficient numbers of cells to be irradiated, and we report here the direct measurement of the oncogenic risk of exactly one a particle. The goal of this study was to investigate the oncogenic effects produced by exactly one a particle traversing cell nuclei. Specifically, we have investigated whether the oncogenic effects of exactly one a particle are similar to the effects of a (Poisson) mean of one a particle, with the latter’s attendant proportion of cells traversed by more than one a particle. Because it is much less likely that there would be a difference between the effects of, say, exactly four and a Poisson-mean of four a particles, we have made such comparisons to act as positive controls. Equality in these positive controls for multiple a-particle traversals would imply that any differences seen between the effects of exactly one a particle and a Poissonmean of one a particle are not artifacts of any differences in irradiation conditions between the microbeam and the broadbeam systems—although we have striven to make the irradiation conditions as similar as possible. The layout and irradiation procedure of the Columbia University microbeam have been described in detail elsewhere (3, 8). Briefly, each cell attached in a monolayer to the thin polypropylene base of a cell culture dish is identified and located by using an image analysis system, and its coordinates are stored in a computer; the cell dish is then moved under computer control such that the centroid of each cell nucleus in the dish is in turn positioned over a highly collimated shuttered beam of a particles. The nucleus of each cell is exposed to a predetermined exact number of a particles having an energy that simulates the emission from radon progeny, and a particle The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1999 by The National Academy of Sciences 0027-8424y99y9619-4$2.00y0 PNAS is available online at www.pnas.org. *To whom reprint requests should be addressed. e-mail: djb3@columbia. edu.