Thiol compounds have long been known to protect living cells against the harmful effects of ionizing radiation. Maetallothionein is a naturally occurring low molecular weight polypeptide rich in cysteine residues and may be useful in protection against low-level radiation effects. Radiation damage to DNA and its nucleotide components and the radioprotective effect of metallothionein have been studied in model chemical systems and compared to its effect on cells. Metallothionein acts both as a free radical scavenger and a reductant, and its radioprotective effectiveness has been studied as a function of dose, drug concentration, and in the presence and absence of oxygen. It is more effective in protecting against sugar-phosphate damage under hypoxic conditions. The chemical modification is greater than that of cell killing as measured by the loss of colony-forming ability. Dose reduction factors greater than two are observed for DNA radioprotection, but the values in cells are much lower. These findings will be discussed in terms of the molecular mechanisms and their implications.
Cell killing and other deleterious biological effects of ionizing radiation are the result of chemical changes to critical targets, initiated at the time of exposure. Electron-affinic radiosensitizers act, primarily, by chemically modifying this radiation damage and its consequent biological expression, and such changes can be used to probe the nature of the cellular radiation target. According to a redox hypothesis of radiation modification, the molecular mechanism of electronic-affinic radiosensitization involves an oxidative interaction of the sensitizer with reactive, potentially damaging target radicals, which competes with reductive processes that restore the target to its undamaged state. The effects have been compared of a series of hypoxic cell radiosensitizers on radiation-induced DNA damage and mammalian cell killing, in order to ascertain the nature of the critical radiation target site(s) involved. Sensitizer efficacy is determined by the ability to oxidize the radiation target and is found to increase exponentially with increasing electron affinity. The threshold redox potential, below which no sensitization occurs, corresponds to the oxidation potential of the target bioradical involved, and is characteristic, and useful in identification, of the particular radiation target. Model product analysis studies of DNA base damage, inorganic phosphate release, single-strand breaks and incorporation of radioactively labelled sensitizer into DNA show a correspondence between the electronic-affinic radiosensitization of DNA damage and cell killing. A careful comparison of the radiosensitization of different DNA sites and cell killing indicates that the sugar-phosphate backbone of DNA, not the heterocyclic bases, is the DNA target site which mimics cell killing in its threshold redox potential and overall radiosensitization response. These results suggest that the enhancement by electron-affinic drugs of radiation damage to the DNA backbone (strand breaks) correlates strongly with, and is the most likely cause of, the radiosensitization of hypoxic cell killing.
SummaryThe effect of nitrobenzene radiosensitizers on the release of inorganic phosphate in aqueous solutions of 3′- and 5′-nucleotides in the absence of oxygen is quite specific. The 5′-purine nucleotides are sensitized, whereas both 3-'purine and-pyrimidine nucleotides are protected. Sensitization and protection increase with increasing nitrobenzene electron affinity and concentration. The 5′-pyrimidine nucleotides can be protected or sensitized, depending on sensitizer electron affinity and concentration. Correlations between enhancement ratios for chemical and biological damage (Chinese-hamster cell killing in hypoxia) are examined with a view to relating radiation chemical change in cellular molecules to biological inactivation.
Abstract Ion suppression-reversed phase high performance liquid chromatography, using 0.6 M ammonium dihydrogen phosphate as eluent, produces base-line separations of deoxyribonucleotides. The effects of pH and ionic strength are described. This isochratic system is simple, reproducible and fast, requiring less than 30 min for a complete separation, and is suitable for in vitro studies.
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