Jürgen Kiefer

A review of dsb induction data for varying quality radiations

PURPOSE This short review summarizes the data obtained with various techniques for measuring the yields of double strand breaks (dsb) produced by particle radiations of differing linear energy transfer (LET) in order to obtain relative biological effectiveness (RBE) values. RESULTS AND CONCLUSIONS Studies aimed at understanding the interactions of different types of radiation with cellular DNA have monitored the yields of DNA dsb versus radiation quality. Several techniques have been used to measure dsb yields in mammalian cells, and these include: neutral sedimentation gradients, filter elution and more recently pulsed field gel electrophoresis techniques (PFGE). Recent developments in PFGE have allowed the measurement of both the yields and the distribution of breaks within the genome, which go part of the way to explaining the RBE values close to 1.0 previously measured using other approaches with various radiation qualities. It is clear that future studies to determine the effectiveness of radiations of differing LET must use techniques that determine both yields and distributions of dsb, and assays need to be developed to allow these measurements at biologically relevant doses.

Cellular and subcellular effects of very heavy ions

The biological effects of irradiation with ions of masses larger than 40 and energies up to 20 MeV per atomic mass unit are reviewed. The objects are viruses, bacterial spores, yeast and mammalian cells. Experimental parameters include loss of colony forming ability, induction of mutants, chromosomal aberrations, cell cycle progression, inhibition of biochemical activities and the formation of strand breaks. Some of the pertinent physical questions--e.g. track structure--are also discussed. It is shown that with very heavy ions the biological effectiveness is no longer unambiguously related to a single parameter like l.e.t. or Z*2/beta 2 but depends strongly on ion energy. This points to the importance of far-reaching delta-electrons. The analysis indicates also that even with very high l.e.t., cells are not killed by the passage of a single particle through their nucleus. Possible implications of the findings for fundamental radiation biology are outlined.

Mutation Induction in V79 Chinese Hamster Cells by Very Heavy Ions

Mutation induction (resistance to 6-thioguanine) in Chinese hamster fibroblasts (V79) by exposure to accelerated heavy ions (O, Ne, Ca, Ti, Ni, Xe, Pb and U with energies between 5 and 14.8 MeV/u) was investigated, covering a range of LET from 300 to about 15,700 KeV/micron. The LET-dependence of the mutation induction cross-section (sigma m) has, in a similar way to inactivation (sigma i), to be described by separate curves for each ion. Both sigma m and mutagenicity (sigma m/sigma i) decrease with increasing specific energy for any given ion. Relative biological effectiveness for mutation induction was found to be significantly smaller than unity for the ions and energies investigated.

Space radiation effects and microgravity.

Humans in space are exposed both to space radiation and microgravity. The question whether radiation effects are modified by microgravity is an important aspect in risk estimation. No interaction is expected at the molecular level since the influence of gravity is much smaller than that of thermal motion. Influences might be expected, however, at the cellular and organ level. For example, changes in immune competence could modify the development of radiogenic cancers. There are no data so far in this area. The problem of whether intracellular repair of radiation-induced DNA lesions is changed under microgravity conditions was recently addressed in a number of space experiments. The results are reviewed; they show that repair processes are not modified by microgravity.

Killing and Mutation of Chinese Hamster V79 Cells Exposed to Accelerated Oxygen and Neon Ions

Mutation induction by accelerated heavy ions to 6-thioguanine resistance (HPRT system) in Chinese hamster V79 cells was investigated using oxygen and neon ions with energies between 1.9 and 400 MeV/mu, corresponding to LET values between 18 and 754 keV/microns, respectively. Because of technical limitations most experiments could be performed only once. Inactivation and mutation induction cross sections, sigma i and sigma m, were obtained from the slopes of the exponential survival and the linear mutation induction curves, respectively. Both parameters increased with LET up to about 200 keV/microns, where the curves separated for the two types of ions. Calculated RBEs were higher for mutation induction than for killing for all LET values.

DNA double-strand break measurement in mammalian cells by pulsed-field gel electrophoresis: an approach using restriction enzymes and gene probing.

DNA samples prepared from human SP3 cells, which had been exposed to various doses of X-ray, were treated with NotI restriction endonuclease before being run in a contour-clamped homogeneous electrophoresis system. The restriction enzyme cuts the DNA at defined positions delivering DNA sizes which can be resolved by pulsed-field gel electrophoresis (PFGE). In order to investigate only one of the DNA fragments, a human lactoferrin cDNA, pHL-41, was hybridized to the DNA separated by PFGE. As a result, only the DNA fragment which contains the hybridized gene was detected resulting in a one-band pattern. The decrease of this band was found to be exponential with increasing radiation dose. From the slope, a double-strand break induction rate of (6.3 +/- 0.7) x 10(-3)/Mbp/Gy was deduced for 80 kV X-rays.

The Importance of Cellular Energy Metabolism for the Sparing Effect of Dose Fractionation with Electrons and Ultra-violet Light

SummaryThe sparing effect of dose fractionation with electron and u.v. exposure was investigated in diploid yeast Saccharomyces cerevisiae. Sparing was observed at the same rate and to the same extent in air or under hypoxia if the cells were held in a glucose-containing nutrient medium. With non-fermentable substrates, e.g. lactate or acetate, the process was slower in the aerated sample, and absent in hypoxia. The action of some metabolic inhibitors known to interfere with cellular energy production was also studied. The sparing effect was diminished if ATP production was inhibited at an early stage of the metabolic pathway. The results are interpreted by assuming that energy-rich metabolites are essential for the sparing effect. Implications for radiotherapy are discussed.

Repair of radiation induced genetic damage under microgravity

Abstract The influence of microgravity on the repair of radiation induced genetic damage in a temperature-conditional repair mutant of the yeast Saccharomyces cerevisiae (rad 54−3) was investigated onboard the IML-1 mission (January 22th – 30th 1992, STS-42). Cells were irradiated before the flight, incubated under microgravity at the permissive (22°C) and restrictive (36°C) temperature and afterwards tested for survival. The results suggest that repair may be reduced under microgravity.

DNA Double-strand Break Induction in Yeast by X-rays and α-particles Measured by Pulsed-field Gel Electrophoresis

Pulsed-field gel electrophoresis was used to separate the chromosomes of the diploid yeast Saccharomyces cerevisiae 211 *B after irradiation with X-rays and α-particles. After electrophoresis, gels were stained with ethidium bromide, placed on a UV-transilluminator and photographed with a digitizing camera connected to a personal computer. The pictures obtained were processed with the help of specially developed software which allows for the correction of the cameras shading effect and background fluorescence. Linearity between DNA amount and fluorescence was demonstrated. Fluorescence intensity for the band with the lowest electrophoretic mobility was found to decrease exponentially with dose. Based on the known size of the native DNA molecules, double-strand break yields could be calculated. These were found to be (8·2 ± 0·4) and (14·8 ± 0·5)10−12 (g/mol)−1 Gy−1 for 80 keV X-rays and 3·5 MeV 241Am α-particles respectively which gives a relative biological effectiveness of 1·8 ± 0·1.

Mutation induction by heavy ions.

Mutation induction by heavy ions is compared in yeast and mammalian cells. Since mutants can only be recovered in survivors the influence of inactivation cross sections has to be taken into account. It is shown that both the size of the sensitive cellular site as well as track structure play an important role. Another parameter which influences the probability of mutation induction is repair: Contrary to naive assumptions primary radiation damage does not directly lead to mutations but requires modification to reconstitute the genetic machinery so that mutants can survive. The molecular structure of mutations was analyzed after exposure to deuterons by amplification with the aid of polymerase chain reaction. The results--although preliminary--demonstrate that even with densely ionizing particles a large fraction does not carry big deletions which suggests that point mutations may also be induced by heavy ions.