Susan M. Bailey and Edwin H. Goodwin
Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM
Epidemiology alone is insufficient to estimate risks associated with low-level ionizing radiation (LLIR) with confidence. Yet much of the concern in mitigating environmental effects of the "cold war legacy" of radiation contamination involves LLIR. For example, a question arises as to how clean a contaminated site needs to be in order to be considered adequately restored. The answer depends in part on the risk associated with residual contamination. It is the goal of this project to examine genetic damage, and cellular responses to that damage, induced by LLIR.
We are investigating "hormesis", a beneficial effect of radiation observed in both animal experiments and human epidemiological data as an extended life span in subjects exposed to low radiation doses compared to matched unirradiated controls. Hormesis has the potential to significantly impact risk assessment in the short term. However, at present its biological basis is unknown. This impedes incorporating hormesis into risk analysis, because risks must be assessed on the basis sound scientific principles in order to be defensible.
Experiments were designed to test the hypotheses that low doses of radiation trigger an antioxidant defense mechanism, and this antioxidant defense is capable of protecting DNA against highly reactive free radicals of the kind produced during normal metabolism. Our efforts focus on glutathione (GSH), one of the cell's most important antioxidant molecules. Four questions are being addressed: Can we establish an assay to detect differences in intracellular GSH levels? Do cells respond to low doses of radiation by increasing GSH? Does increased GSH diminish the harmful effects of hydrogen peroxide, an important endogenous oxidant? Do low doses of radiation initiate a similar protective effect?
To measure intracellular GSH, we chose the monochlorobimane fluorescence assay. Mono-chlorobimane becomes fluorescent only when conjugated to GSH. This reaction is sensitive to GSH levels and, because it is catalyzed by glutathione transferase, is highly specific. The assay is sensitive, but detects only relative differences between controls and treated samples. It was validated with N-acetyl-L-cysteine (NAC), a chemical known to increase intracellular GSH. NAC is source of cysteine, the rate limiting substrate in GSH synthesis. Exposing CHO-K1 cells to 5-100µM NAC produced a concentration-dependent increase in monochlorobimane fluorescence.
Cells were exposed to 60Co gamma rays doses ranging from 2 to 22 cGy. Relative to a zero-dose control, all irradiated samples showed increased monochlorobimane fluorescence. This result indicates that cells respond to low radiation doses by increasing intracellular GSH. GSH increased within two hours, the earliest time examined.
Next we sought to determine if elevated GSH had a protective effect. NAC (5-80mM) was added to cell cultures in order to increase intracellular GSH. The cells were then challenged with 0.5 and 1.5mM hydrogen peroxide. Compared to 0µM NAC controls, the treated samples had a higher survival, indicating a protective effect.
Finally we tested for a protective effect of low radiation pre-exposure. Cells were exposed to 4 cGy gamma rays and challenged with 0.5 and 1.5mM hydrogen peroxide. For both concentrations, there was no statistically significant difference in survival between pre-irradiated and zero-dose controls. Although this finding was disappointing, it implies that hormesis is likely to be a subtle process expressing its beneficial effects over a period of years. To observe hormesis in an in vitro setting may require more sensitive assays than we have at present.