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R E S E A R C HR E P O R T S

Microbeam Helps Show How Radiation Mutates Genes

Lead Researchers: David Brenner and Tom K. Hei

Researchers in the Center for Radiological Research are learning how ionizing radiation causes genetic mutations by firing charged alpha particles one at a time at different structures within mammalian cells. A recent experiment indicated that the lung-cancer risk posed by radon in homes may be overestimated. Another overturned a 70-year-old perception of radiation-induced mutation. Both teams of researchers took advantage of a newly improved charged-particle microbeam at Columbia University’s Radiological Research Accelerator Facility in Irvington, N.Y.

Epidemiological studies of Hiroshima and Nagasaki residents and uranium miners have quantified the risk faced by people exposed to high levels of radiation. But they tell little about the more common risks faced by people exposed to lower levels of radiation for long periods. And they tell nothing about the mechanisms by which radiation actually alters DNA.

The microbeam provides researchers with a powerful tool to address those questions. It can fire a precise number of particles at a cellular target as small as four microns across, less than half the size of an average cell nucleus. Research scientist Gerhard Randers-Pehrson recently designed a computer-controlled system that automates and speeds up the irradiation of individual cells.

Cells are attached to a thin plastic sheet at the base of a cell-culture dish. They are treated with one stain that is taken up by the nucleus and by another, which is taken up by the cytoplasm. A camera attached to a 40-power microscope takes an image of the cells. A computer analyzes the image, determining the location of each nucleus, then moves the dish so that the nucleus is placed in the path of the narrowly focused particle beam. A detector above the dish senses each particle and closes a shutter after a predetermined number of particles have passed through the cell. The system can irradiate up to 3,000 individual cells per hour. It is the only system in the world capable of such speed and accuracy.

A research team led by Dr. David Brenner, professor of radiation oncology and public health, took advantage of these capabilities to better understand the cancer risk posed by low levels of radon. Radon is a natural, colorless, and odorless gas that seeps out of some rock formations and can collect in homes. When radon decays, it emits alpha particles, charged particles containing two neutrons and two protons. The National Academy of Sciences has estimated that radon accounts for as many as 21,800 fatal cases of lung cancer per year.

But scientists question those estimates because they were extrapolated from studies of uranium miners exposed to radon at levels much higher than those found in homes. Many of the cells in the miners’ lungs were bombarded by several alpha particles, whereas the lung cells of people living in homes with elevated radon levels are almost never hit by more than one alpha particle. Since cancer often results from the combination of several genetic mutations, many of the cancer cases suffered by the miners may have been the result of several alpha particle hits. People in their homes would not face such a risk.

Dr. Brenner and his colleagues used the microbeam to irradiate more than 50,000 cell nuclei with single alpha particles. They found that a single alpha particle passing through the nucleus does not significantly raise the risk of the cell turning cancerous. That suggests that most of the miners’ cancer cases may have been caused by multiple alpha-particle hits, which do not threaten home dwellers. Thus, the linear extrapolation of the miner data down to the homeowner exposure level may overestimate the risk. Those findings were published in the Jan. 5, 1999, issue of the Proceedings of the National Academy of Sciences.

Another research team, led by Dr. Tom K. Hei, associate professor of radiation oncology and public health, studied the actual mechanisms of radiation-induced mutations. For more than 70 years, scientists have believed that X-rays and other high-energy radiation can cause genetic mutations only when they hit the cell nucleus. But new indirect evidence had suggested that radiation-induced mutations might occur in cells whose nuclei had not been irradiated.

Dr. Hei’s team used the microbeam to look for direct evidence. They focused the beam on the cytoplasm, the cellular material outside the nucleus. The cytoplasmic irradiation tripled the natural mutation rate. When the cells were treated with the antioxidant DMSO, the mutation rate dropped back down to near background levels, suggesting that the alpha particles ripping through the cytoplasm create free radicals, which cause the genetic mutations within the nucleus. Those findings were published in the April 27, 1999, issue of the Proceedings of the National Academy of Sciences.

“The nucleus has always been considered as the quintessential target for any carcinogenic mutations,” says Dr. Hei. “We have learned that if you hit the cytoplasm with alpha particles there is a chance that you will mediate a mutational event in the nucleus.”

Dr. Hei’s findings suggest there is an extranuclear target for the alpha particles that induce mutation. They also provide a mechanism, in the form of free radicals, for the observation that “bystander” cells not directly hit by an alpha particle show a biological response. The work has implications for not only radon exposure, but also the risk faced by thousands of people exposed to low levels of radiation at work, such as X-ray technicians and researchers who use radioactive tracers. Additional experiments will be needed to clarify those implications and to confirm the suggestions of Dr. Brenner’s team. The microbeam, currently supported by an NIH Research Resource Center grant, will likely be instrumental to those experiments.

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