Superfund Research Program
Role of Oxyradicals in Genotoxicity of Arsenic
Virtually everyone is exposed to arsenic, mostly at low levels, via food, drinking water, and air. While epidemiological studies have established that arsenic is a carcinogen, the mechanisms by which arsenic induces cancer remain unresolved. The animal models that are generally applied to study cancer mechanisms cannot be used in this case because arsenic does not cause cancer in rodents. Our poor understanding of arsenic's carcinogenic mechanism hampers accurate risk assessment of the health effects of arsenic compounds.
In vitro studies have shown that arsenic induces chromosomal aberrations, arrests cells in mitosis, and inhibits gene repair. However, the standard assays designed to evaluate mutagenicity indicate that arsenic is only weakly active or completely inactive as a mutagen. Researchers at Columbia University hypothesized that conventional mutation assays do not show arsenic's mutagenic activity because arsenic induces multilocus deletions that often result in cell death. Consequently, even though arsenic induces mutations, mutants cannot be detected.
Columbia University scientists are using AL cells which are human-hamster hybrid cells that contain a single copy of human chromosome 11. Because chromosome 11 is not required for the AL cells' survival, the hybrid cells can be use to detect mutations of the human chromosome. The investigators exposed AL cells to arsenite, a form of arsenic that is commonly found in the environment, and then conducted mutation assays. They discovered large multilocus deletions, indicating that arsenite is in fact a potent mutagen.
The Columbia scientists then explored the mechanisms of arsenic genotoxicity that they observed in the AL cells. They considered recently published information, including their own, that antioxidants reduce the incidence of arsenite-induced mutations and chromosomal aberrations in cultured mammalian cells. Antioxidants have been shown to reduce the damage that can result from excess levels of reactive oxygen species (ROS) such as the free radicals superoxide, hydroxyl radical, hydrogen peroxide, and singlet oxygen. These chemicals are important for normal metabolism because they play essential roles in the biosynthesis of complex organic molecules, the detoxification of xenobiotic chemicals, and in the defense against pathogens. However, excess ROS can damage lipids, proteins, and DNA. Whether or not ROS are deleterious to cells depends on the levels and the locations. "Oxidative stress" occurs as the result of a disturbance in the balance between the production of ROS and antioxidant defenses, resulting in abnormally high levels of ROS. Columbia University researchers conducted a series of experiments to quantify and identify the radical species in cells exposed to arsenite and examine the impact of antioxidant additions on arsenic genotoxicity.
In an initial set of experiments designed to determine if ROS are formed in response to arsenite exposure, the scientists added a dye probe to test cultures. The dye enters the cells and is oxidized by ROS to a fluorescent compound that can be detected and quantified in real time via confocal microscopy. The researchers discovered that arsenite-treated cultures exhibit a dose-dependent increase in ROS generation within minutes after treatment. They also determined that in the presence of the free radical scavenger DMSO, the fluorescent intensity observed in arsenic-treated cultures was reduced to nearly background levels.
In additional studies, the Columbia University scientists added a hydroxylamine spin trap probe to arsenite-treated cultures. This probe penetrates plasma membranes and reacts with ROS to form a compound that can be detected and quantified by Electron Spin Resonance (ESR) spectroscopy. Again, the results indicated a dose-dependent increase in the levels of ROS in the test cultures following the addition of arsenite.
By adding specific antioxidant enzymes (superoxide dismutase and catalase) to the arsenite-treated cultures and observing the impact on ROS levels as measured by ESR, the researchers were able to identify the types of ROS formed in response to arsenite exposure. Using this spin trapping assay, the research team showed that arsenite instigates the production of superoxide, a very unstable free radical species. The superoxide is rapidly converted into hydrogen peroxide by enzymes in the cells. The hydrogen peroxide is in turn converted into hydroxyl radicals, which can induce lesions in DNA that cause deletions, mutations, and other lethal genetic effects.
The Columbia University scientists then manipulated intracellular levels of the antioxidant glutathione in arsenite-treated cultures. This enabled them to evaluate the role of ROS in mediation of arsenite mutagenicity. They found that decreased glutathione levels, which leads to increased ROS levels, increased the mutagenicity and toxicity of arsenite in a dose-dependent fashion. Altogether, this research provides the first clear-cut evidence that an environmental carcinogen acts predominantly through a free radical pathway.
The levels of arsenic used in this research are found in ground water resources in parts of the southwestern and northeastern United States and in as many as 5% of the drinking water wells in some developing countries. The studies conducted at Columbia University add to the growing body of evidence that antioxidants may help prevent cancer and other illnesses caused by such environmental toxins as arsenic, cadmium, and asbestos. This information may facilitate the development of both treatment modalities and prevention strategies.
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To learn more about this research, please refer to the following sources:
- Liu S, Athar M, Lippai I, Waldren CA, Hei TK. 2001. Induction of oxyradicals by arsenic: Implication for mechanisms of genotoxicity. Proc Natl Acad Sci U S A 98(4):1643-1648. PMID:11172004
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