Hydroxyl radical scavengers and antioxidants in radiation protection

Antioxidants and free radical scavengers, by combatting oxygen radical-mediated oxidative stress, may protect against ionising radiation. A total antioxidant assay has been developed using a highly fluorescent molecule, {dollar}beta{dollar}-phycoerythrin (BPE), as the target for radiation generated free radicals. Serial dilutions of a solution of a test compound are made across a 96-well microwell plate and a standard BPE solution added to each well. Competition plots for combinations of BPE and scavenging compounds can then be generated by monitoring changes in fluorescence of the BPE solution in each well, before and after a radiation dose, as a function of compound concentration. As in other antioxidant assays one unit is defined as the quantity of antioxidant required to reduce by half the action of oxygen derived free radicals, in this case monitored by the fluorescence decay of a 340 ng mL{dollar}sp{lcub}-1{rcub}{dollar} solution of {dollar}beta{dollar}-phycoerythrin. The fluorescence decreases linearly with dose allowing radioprotective capacities to be determined after a single standard test dose. Using this technique, relative rate constants can be calculated for the reactions between hydroxyl radical scavengers and hydroxyl radicals. For antioxidants and hydrogen donors, there is a correlation between radioprotective capacity and oxidation potential. The assay then becomes a true measure of total radioprotective effect which can rank the compounds. Antioxidants are more than thirty times more effective than pure hydroxyl radical scavengers such as sugars, which is due to their competition with oxygen for the target radical intermediates. The antioxidant compounds could be ranked based on their chemical structure. Indoles, such as melatonin are the most effective (nine times more effective than ascorbic acid), followed by phenols and thiol compounds. The assay can also be applied to complex biological samples. Cell lysates and human blood plasma were measured to determine if variations in their radioprotective capacity could explain observed differences in cellular radiosensitivity. These samples proved to be highly reductive, more reductive than ascorbate on a weight per weight basis. For total blood plasma radioprotective effect an inverse trend was observed with donor age, which is largely due to high molecular weight protein contributions that are inefficient in their interaction with target radicals. However, only additions of a low molecular weight antioxidant (melatonin) were able to modify radioprotection in vitro.