Ability of human plasma to protect against ionising radiation is inversely correlated with age.

Human blood plasma from persons 30-80 years of age have been measured by a total antioxidant assay using a highly fluorescent molecule, beta-phycoerythrin (BPE), as the target for radiation-generated free radicals. The plasma samples showed an inverse relationship between radioprotective ability and donor age. The results are consistent with data showing that protein from older subjects provides less protection against oxidative stress than that from middle-aged subjects.

Effect of low-dose gamma radiation of HIV replication in human peripheral blood mononuclear cells.

Recent studies have demonstrated that UV light and x-irradiation enhance human immunodeficiency virus (HIV) gene expression. There are few published data on related effects of gamma-radiation. This may be of clinical relevance, as radiotherapy has been used extensively for the treatment of acquired immunodeficiency syndrome associated conditions. With this in mind, we have studied the effects of gamma-radiation on HIV replication in mononuclear cells (MC). These cells were obtained from five seronegative healthy donors, exposed to 0-200 cGy gamma-radiation, stimulated with phytohemagglutinin-P (PHA-P) for 24 h, infected with a laboratory strain of HIV (HTLV-IIIB, multiplicity of infection = 0.001), then carried in culture for 14 days. Overall, when considering p24 antigen levels on days 7 and 11 in cultures established from cells exposed to 50 cGy, the maximal levels were significantly higher than those measured in the parallel control cultures taken as a whole (P < 0.05), with viral replication enhanced as much as 1000-fold in one case. No significant cytotoxicity was observed following exposure to doses up to 50 cGy. The mechanism of the observed effect remains unknown but may relate to direct gene activation and/or free radical generation, leading to such activation. To date, there is no evidence that viral stimulation occurs following therapeutic radiation in a clinical setting.

Occupational levels of radiation exposure induce surface expression of interleukin-2 receptors in stimulated human peripheral blood lymphocytes.

Interleukin-2 (IL-2) is a cytokine responsible for a variety of immune and non-immune stimulatory and regulatory functions, including the activation and stimulation of cytotoxic cells able to recognize and kill human tumour cells and T-cell proliferation and differentiation. We show that low doses of radiation, in the range commonly received by atomic radiation workers or as a result of minor medical diagnostic procedures (0.25 to 10 mGy), stimulate the expression of IL-2 receptors (IL-2R) on the surface of peripheral blood lymphocytes (PBL) taken from normal human donors. This stimulated surface expression after in vitro irradiation is an indirect effect, resulting from the secretion into the medium of a soluble factor from the irradiated cells. This factor can also stimulate IL-2R surface expression in unirradiated cells. Consequently, radiation stimulation of IL-2R expression in a large population of PBL shows a triggered-type response rather than being proportional to dose. These results demonstrate that normal human cells can respond to doses of radiation in the range of common occupational or medical exposures. The data also demonstrate a possible defence mechanism against environmental stress by which a radiation-exposed cell can use an indirect signalling mechanism to communicate with and influence the biological processes in an unexposed cell.

Fluorescence lifetime analysis of DNA intercalated ethidium bromide and quenching by free dye.

The fluorescence characteristics of ethidium bromide (Eb) complexed to calf thymus DNA have been examined using fluorescence lifetime analysis for a range of DNA (effective nucleotide concentration) to Eb molar ratios. Control of both temperature and ion concentration is necessary for reproducible analyses. Eb complexed to double stranded DNA has a maximum fluorescence lifetime of 23 ns and is easily distinguishable from a fluorescence lifetime value of 1.67 ns corresponding to unbound Eb. In a solution of calf thymus DNA containing excess EB a binding equilibrium is reached, and this corresponds to one Eb molecule for every five nucleotides. With increasing amounts of unbound Eb, the fluorescence lifetime of the DNA-Eb complex decreases with a concomitant drop in the steady state fluorescence intensity, without a change in the amount of Eb bound to DNA. It is concluded that unbound Eb, acting via a quenching mechanism, shortens the fluorescence lifetime of bound Eb and consequently decreases the overall fluorescence intensity. This means that a different approach is necessary: time-resolved fluorescence spectroscopy directly distinguishes between a decrease in fluorescence intensity due to quenching by an excess of unbound Eb from that due to a decrease in Eb binding to double-stranded DNA. These studies suggest that techniques which measure total steady state fluorescence intensity of bound Eb in order to infer relative amounts of double-stranded DNA must be interpreted with caution. For such assays to be valid it is essential that no unbound Eb be present: otherwise a variable correction factor is required to account for unbound Eb.

Biological and biophysical techniques to assess radiation exposure: a perspective.

Biological dosimeters measure biologically relevant effects of radiation exposure that are in some sense an estimate of effective dose, whereas biophysical indicators serve as surrogates of absorbed dose in a manner analogous to conventional thermoluminescent dosimeters (TLD). The biological and biophysical dosimeters have the potential to play an important role in assessing unanticipated or occupational radiation exposures. For example, where the exposure is large and uncertain (i.e. radiation accidents), accurate dose information can help in deciding the most appropriate therapy and medical treatment. Another useful area is that of lifetime accumulated dose determination, and the ability to distinguish between and integrate the exposures from natural and anthropogenic (medical X-rays, indoor radon, natural background radiation, occupational and non-occupational exposures). Also, the possibility to monitor individual response and differences in inherent or induced radiation sensitivity may have important implications for radiation protection. More commonly, this type of dosimetry could be used for routine monitoring to detect and quantify unsuspected exposure, for regulatory purposes or for epidemiological studies of the long-term effects of radiation exposure (e.g. in Japanese A-bomb survivors or in the population surrounding Chernobyl). This review is a comparative study of the existing techniques and their future prospects. It summarizes the sensitivity, reproducibility, limiting dose, dose-rate, energy, LET response, sources of variability and uncertainty, and other practical aspects of each bio-indicator. The strengths and weaknesses of each approach are evaluated on the basis of common criteria for particular applications, and are summarized for each assay both in the text and in tabular form, for convenience. It is clear that no single indicator qualifies to reliably measure occupational exposures at the current levels of sensitivity conventional dosimetry services provide. Most of the bio-techniques are applicable to the detection of relatively high radiation exposures at relatively short times after exposure. Some of the bio-indicators have been identified that are, or offer future prospects for becoming, appropriate bio-indicators for dosimetry needs. However, all methods are subject to biological and other variables that are presently uncontrolled, and represent a major source of uncertainty. These include variations in background signals not directly associated with radiation exposure, inter- and intra-individual variability of radiation response, and genetic and environmental effects. Although these factors contribute to the lack of confidence in biological dosimetry, promising bio-indicators may be applied to large populations to establish the inherent variability and confounding factors that limit quantitative data collection and analysis, and reduce reliability and reproducibility.

Radiation and aging: free radical damage, biological response and possible antioxidant intervention.

In this review, the basic processes responsible for radiation-induced changes in critical cell components and their biological consequences will be discussed. The chemical and physical alterations in biomolecules are mediated by free radicals and other reactive intermediates formed, following absorption of radiant energy, by ionization of proximal targets or the surrounding water molecules. Accumulation of free radical damage and its catalysis by various oxidants including quinones and other age pigments, metal ions, lipid peroxides, prostaglandins and components released from cells, increase with age. A cell's response to such damage depends upon environmental and inherited factors. DNA repair is an effective way to protect against radiation damage, but other constitutive or inducible defence mechanisms can also modify biological response, and these processes generally become less effective with age. Loss of fidelity with age of bio-feedback mechanisms including homeostasis, redox control, ion and metabolic regulation, which in turn affects cell growth and differentiation, energy efficiency, the immune system and general health, can be associated with free radical pathology. Current theories of aging will be examined including those involving wear-and-tear, genetic, metabolic, immunologic and biochemical factors. Ionizing radiation, as with other external stresses including UV, heat, chemotherapeutic agents, chemical carcinogens and tumor promoters, interact with nucleic acids, proteins and membrane phospholipids facilitating free radical-mediated oxidative damage. Appropriate antioxidant intervention, by inhibiting or reducing free radical toxicity, may offer protection against radiation, and alleviate or delay symptoms of aging and chronic disease.

Use of sugars and hair for ESR emergency dosimetry.

ESR spectrometry of sugars and biological samples is being evaluated for emergency personnel dosimetry. Sugars are near tissue-equivalent, universally available in pure form and produce a simple, reproducible, low background signal. Of the sugars tested, sucrose and dextrose are the most sensitive and the ESR signals are proportional to X- or gamma-ray doses over the range of 0.5-10 Gy. There is little dependence on radiation energy or dose-rate, and the ESR signals remain stable for long periods post-irradiation. Human hair samples show considerable variability and signal complexity creating difficulties in dose assessment.

Radiation chemical studies of sensitization by 5-bromouridine-5'-monophosphate (5-BrUMP).

This study was undertaken to investigate the mechanism of chemical radiosensitization by halogenated bases incorporated into DNA. Radiation-induced base and sugar-phosphate backbone damage to 5-bromouridine-5'-monophosphate (5-BrUMP) was monitored using a flow system connected in series with a recording spectrophotometer, a bromide (Br-)-specific ion analyzer and a Technicon auto-sampler. The system was used to assay loss of UV-absorbing 5,6 double-bond, release of Br- and inorganic phosphate (Pi) release using an automated colorimetric method, as a function of gamma-ray dose. Results obtained with radical scavengers indicate that, unlike non-halogenated nucleotides where the hydroxyl radical (.OH) is the principal damaging species, 5-BrUMP is damaged by the hydrated electron (e-aq), hydrogen atom (H.) and .OH, producing a high yield of base damage and Br- and Pi release in anoxia. Another novel feature of 5-BrUMP radiolysis is that oxygen, by converting e-aq and H. to the unreactive superoxide radical anion (O2-), has a protective effect on both base and phosphate ester damage. Under .OH-scavenging conditions, where the radiation yield of reductive debromination is 3.8, there is some Pi release, suggesting the possibility of intramolecular hydrogen atom transfer from the sugar ring to the 5-uracilyl radical and subsequent sugar-phosphate bond cleavage. This hypothesis is supported by the action of oxygen and thiols in modifying the e-aq-mediated sugar-phosphate damage.

DNA radiation damage and its modification by metallothionein.

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.

Radiosensitizers as probes of DNA damage and cell killing.

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.

Effects of sensitizers on cell respiration: II. The effects of hypoxic cell sensitizers on oxygen utilization in cellular and chemical models.

The metabolic activity of nitroheterocyclic sensitizers could limit their usefulness in vivo. Biochemical mechanisms of drug metabolism, toxicity and effects on cell respiration have been studied in microsomes, and the kinetics of the simulated redox reactions determined by pulse radiolysis. Stimulated oxidation of coenzyme, glucose, ascorbate or glutathione substrate radicals by nitroheterocyclic sensitizers, with the concomitant appearance of the respective nitro radical anions, is observed. Under hypoxia, the nitro radical anions decay slowly by second order processes, forming reduced metabolites. In air, the nitro radical anions react with oxygen forming superoxide radical anions, peroxide and regenerating the drug. Nitro radical-anions also react with cytochrome-c indicating a possible interference with mitochondrial energy metabolism.

Effects of sensitizers on cell respiration: III. The effects of hypoxic cell radiosensitizers on oxidative metabolism and the radiation response of an in vitro tumour model.

Physiological factors are important when considering the effects of radiosensitizers on the radiation response of complex systems such as multicellular spheroids. In this system, under conditions of unlimited nutrient supply, cells are rendered hypoxic by metabolism. Thus, using the spheroid system as an in vitro model of the tumour-cell microenvironment, we have determined the relative contribution of radiosensitization and respiratory effects of a number of electron-affinic sensitizers having potential clinical use. These studies are indicative of physiological responses at the cellular level, and suggest optimal drug administration schemes for obtaining maximal radiation response in vivo hypoxic cell sensitizers.

Effects of sensitizers on cell respiration: 1. Factors influencing the effects of hypoxic cell radiosensitizers on oxygen utilization of tumour and cultured mammalian cells.

We have investigated the effects of 24 nitrocompounds, differing in their half-reduction potential, on the respiration in vitro of Ehrlich ascites tumour cells and cultured V79 lung cells. Many drugs with redox potentials more positive than--0.35 stimulated oxygen utilization in the presence of glucose. Glucose had little effect on the inhibition of respiration by drugs with oxidation-reduction potentials more negative than--0.38. Nitrocompounds that inhibited Ehrilch cell respiration in the presence of glucose, also inhibited intracellular reduction of ferricytochrome (c+c1). Drugs that stimulated oxygen utilization also stimuated intracellular (c " c1). Drugs that stimulated oxygen utilization also stimulated intracellular reduction of ferricytochrome (c + c1). A correlation between drug oxidation-reduction potential and stimulation of oxygen utilization in KCN-inhibited cells was found.

Pulse radiolysis of lactate dehydrogenase.

Pulse radiolysis has been used to investigate the rates and transient spectra for the reactions of free radicals with beef heart lactate dehydrogenase at pH 7. Analysis of the results leads to second-order rate-constants for eaq-, .OH, .I, .Br2-, .I2- and .(CNS)2- which are, respectively, 24, 21, 10, 0.55, 0.43 and 0.15 in units of 10(10) M-1 s-1 with uncertainties of +/- 20 per cent. Those for .I and .I2- are similar to the corresponding rate-constants for the related enzyme alcohol dehydrogenase. The spectra of the transient species produced by .OH, .Br2- and .(CNS)2- all showed evidence for reactions with tyrosine and tryptophan residues, and in general terms the magnitudes of the rate-constants appeared to increase with the oxidizing abilities of the radicals. The implication of the results for understanding the mechanism of deactivation by free radicals is discussed.