Relationships between cell killing, mutation induction and DNA damage in X-irradiated V79 cells: the influence of oxygen and DMSO.

The relationships between cell killing, mutation induction and DNA double (dsb) and single (ssb) strand breaks have been studied in V79 cells irradiated with X-rays under oxic and anoxic conditions in the presence and in the absence of dimethylsulphoxide (DMSO). Curvilinear relationships were found between all pairs of endpoints, except for dsb versus ssb. Statistical analysis of experimental data has shown that in the absence of DMSO there is evidence of good correlations between cell killing, mutation induction and dsb in oxic and anoxic conditions. However, when DMSO was present, no significant correlation was found. In the presence of oxygen DMSO always exerts a protective effect while in anoxia it is generally much less protective and induces a strong sensitization with respect to mutation induction. Possibly DMSO acts not only as a radical scavenger but also as an agent inducing chromatin relaxation and/or under anoxia, forming highly mutagenic short-term radicals. The present data suggest that lethal and mutational events are at least partially independent and not proportional to the initial number of DNA breaks. This may imply that either other kinds of lesions are involved in cell lethality and mutability, or dose-dependent repair mechanisms of dsb have to be considered.

The role of thiols in lethal and mutational radiation damage.

Over the last few decades, free radicals have been increasingly implicated in biological processes including radiation effects, ageing, carcinogenesis, initiation and progression of various diseases, toxicity of chemicals and drugs. In this field Radiation Biology has played an important role in the development of both technical and cultural background, because it was very soon recognized the radical nature of processes following exposure to ionizing radiation. Several studies have pointed out the importance of both radicals, reacting with cellular targets, and endogenous thiols, mainly represented by glutathione, in controlling radiation responses of living cells. Experimental supports for such a role mainly rest on observations made on cell lines depleted of glutathione content because of a genetic defect or as result of a pharmacological manipulation. We present a study on the influence of endogenous and exogenous thiols on the correlation between lethal and mutational damage in mammalian cells. Survival (S) and induction of HPRT- mutation (M) were measured in cells irradiated with X-rays either after treatment with BSO or in the presence of MEA or GSH. In control experiments log of S is linearly correlated to M. Incubation with 1 mM BSO reduces cellular GSH content and produces an increase in radiosensitivity with regard to both lethal and mutagenic effects. In the presence of MEA a concentration dependent radioprotective effect can be observed on both end-points. GSH added to cells immediately or 90 min before irradiation only displays a slight protective effect on lethality. The yield of mutant cells is not significantly affected when GSH is added immediately before irradiation.(ABSTRACT TRUNCATED AT 250 WORDS)

The oxygen effect in radiation inactivation of DNA and enzymes.

A survey is made of literature data dealing with the influence of oxygen on radiation effects in biologically active DNA and enzymes irradiated extracellularly. There is evidence that oxygen takes part in physico-chemical events, directly or indirectly produced by radiation in several ways: from scavenging reducing primary water radicals to reacting directly with macromolecular radical sites. There is evidence that radiation-induced secondary radicals, originating from a variety of low molecular weight biomolecules, can react with DNA and enzymes in their native state, and produce inactivation. By reaction with oxygen secondary radicals become peroxidized and in this form are generally more harmful to biological macromolecules. There are indications that thiol peroxy radicals can also act in the same way. Possible implications for the oxygen effect observed in vivo are discussed.

Interactions of thiyl free radicals with oxygen: a pulse radiolysis study.

A pulse radiolysis study of glutathione in aqueous solution at pH 5.5 containing N2O/O2 mixtures at various ratios indicates that oxygen rapidly adds to the thiyl glutathione radical yielding a transient absorption, with a maximum at 540 nm, whose characteristics appear to be compatible with assignment to the GSOO. radical. The reaction (Formula: see text) appears to be an equilibrium whose kinetic constants have been estimated (kf = 2.0 X 10(9) dm3 mol-1, kb = 6.2 X 10(5) s-1). Evidence for electron transfer from ascorbate to the GSOO. radical has been obtained and the respective rate constant has been determined to be 1.75 +/- 0.15 X 10(8) dm3 mol-1 s-1.

Radiation chemical and physical mechanisms of radiosensitization of single cell systems by iothalamate.

The radiosensitizing effect of iothalamate (ITA) has been investigated in bacterial and mammalian cells in order to obtain a better understanding of the physical and radiation chemical mechanisms of sensitization displayed by the drug. In order to distinguish between the two, Escherichia coli B/r cells were irradiated with 9 MeV electrons, which allow only the radiation chemical mechanism to operate, and V79 cells with 250 KVp X-rays, which instead make possible the occurrence of both mechanisms. It has been shown that: Maximum sensitization already occurs in bacteria with 10(-2) mol dm-3 ITA (enhancement ratio (ER) 11.2 in oxygen, 2.7 in nitrogen), while in mammalian cells a concentration higher by a factor of 10 is required (ER 2.2 both in air and nitrogen). ITA sensitization is inhibited when bacteria are irradiated in growth medium instead of buffer. Such inhibition does not occur with V79 cells. Cysteine and glycerol completely cancel the sensitizing effect of ITA on bacterial cells in both gas phases. Dimethylsulphoxide (DMSO) does the same in nitrogen, while in oxygen it only reduces ITA sensitization to about 50 per cent of the level observed in control conditions. With mammalian cells, all the three scavengers do not modify significantly the enhancement produced by ITA, either in air or in nitrogen. The experimental results are consistent with both postulated mechanisms of sensitization.

Role of glutathione in affecting the radiosensitivity of molecular and cellular systems.

It has previously been shown that radioinduced organic radicals can be repaired by hydrogen donation from glutathione (GSH) and this repair is in competition with oxygen (damage fixation). In this paper the influence of exogenous glutathione on the radiation response of the enzyme alcohol dehydrogenase (YADH), DNA in vitro, and E. coli B/r cells has been investigated. GSH is observed to protect YADH essentially by free radical scavenging mechanisms in both presence or absence of oxygen. The same mechanism seems operate in the radioprotection afforded by GSH to DNA in vitro. E. coli B/r cells are protected at higher extent by GSH than its oxidized form (GSSG); the possibility that GSH penetrate into bacterial cells more easily that GSSG can explain their different behaviour. None of the three systems studied has provided definitive support for the occurrence of the hydrogen donation reaction in the radioprotective mechanisms of GSH versus biomolecules and bacterial cells.

Iodinated radiological contrast media as radiosensitizers.

This paper describes the radiosensitizing effects of diatrizoic (DA) and iothalamic (ITA) acids and of iodipamide (IP) on the survival of E coli B/r irradiated with X-rays and with high-intensity electron pulses. All compounds at concentrations between 10 and 50 mM display a strong sensitizing effect in the presence of oxygen (DMF between 0-1 and 0-3) and are much less effective in nitrogen. In N2O the degree of sensitization is intermediate between oxygen and nitrogen. The situation is the same at pH 7 or 5-6. Solutions of DA, ITA and IP irradiated at pH lower than 6 become highly toxic to bacteria added after irradiation, for several hours after X-irradiation or several minutes after pulsed irradiation. The maximum toxic effect occurs with 2 krad of X-ray and with 6-8 krad of electrons. Oxygen must be present in order to observe the bactericidal activity. This is not affected by scavenging hydrated electrons with nitrate, but is completely cancelled by scavenging OH radicals with formate. It is also cancelled by adding thiosulphate to the irradiated solutions immediately before the bacteria. In the presence of nutrient broth, the radiosensitizing effect is absent after irradiation with pulsed electrons; whereas after X-irradiation it is reduced when the concentration of sensitizers is 50 mM. The experimental data appear to be compatible with a mechanism operated by short and long-lived transients resulting from the radiolysis of iodinated contrast media.

Modifications of gamma-ray sensitivity of bacterial membrane by pre-exposure to light.

The exposure of E.coli B/r cells to ultraviolet (UV) or to visible light prior to irradiation with gamma-rays modifies the sensitivity of the cell membrane to the radiation damage responsible for the loss of intracellular K+ content. The experiments reported in the paper have shown: 1. exposure of bacterial cells to sublethal doses of UV light increases their sensitivity to gamma-ray-induced membrane damage, while exposure to visible light has the opposite effect; 2. in combined exposures, the visible light, either given before or after the UV always produces a strong photoprotective effect. In either case, the photosensitizing effect of UV is completely suppressed; 3. the photoprotection decays with time if cell suspensions are left in the dark before gamma-irradiation. At 0 degrees C, the half-life of the photoprotective effect is 25 min at pH 7 and 100 min at pH 7.5. The decay is due to the presence of oxygen; 4. the light band responsible for the induction of photoprotection has been estimated to lie in the wavelength region between 540 and 580 nm.