E. Schneider

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.

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.

Enhanced Cell Killing, Inhibition of Recovery from Potentially Lethal Damage and Increased Mutation Frequency by 3-aminobenzamide in Chinese Hamster V79 Cells Exposed to X-rays

X-ray induced potentially lethal damage and its inhibition by the aromatic amide 3-aminobenzamide have been investigated in Chinese hamster V79 cells. 3-Aminobenzamide (3-AB) is a known inhibitor of polyadenosine diphosphoribose synthetase. With increasing concentrations of 3-AB an increasing inhibition of PLD repair was observed. Little inhibition of PLD repair was seen when 3-AB was added 3 h following irradiation. Utilizing the 6-thioguanine mutation assay, the effect of poly(ADP-R) synthetase inhibition under conditions of PLD repair upon mutation frequency were also studied. A large increase in mutation frequency following 24 h post-irradiation recovery in the presence of 3-AB was seen. These results favour a possible role of 3-AB in preventing repair by facilitating early damage fixation before repair can occur, simultaneously reducing G2-arrest.

HPRT mutations in V79 Chinese hamster cells induced by accelerated Ni, Au and Pb ions.

Mutation induction by accelerated heavy ions to 6-TG resistance (HPRT system) in V79 Chinese hamster cells was investigated with Ni (6-630 Me V/u), Au (2.2, 8.7 Me V/u) and Pb ions (11.6-980 Me V/u) corresponding to a LET range between 180 and 12895 ke V/microns. Most experiments could only be performed once due to technical limitations using accelerator beam times. Survival curves were exponential, mutation induction curves linear with fluence. From their slopes inactivation- and mutation-induction cross-sections were derived. If they are plotted versus LET, single, ion-specific curves are obtained. It is shown that other parameters like ion energy and effective charge play an important role. In the case of Au and Pb ions the cross-sections follow a common line, since these ions have nearly the same atomic weight, so that they should have similar spatial ionization patterns in matter at the same energies. Calculated RBEs were higher for mutation induction than for killing for all LETs.

Heavy Ion Effects on Yeast Cells: Induction of Canavanine-resistant Mutants

The induction of forward mutations (resistance to canavanine) by heavy ion bombardment was investigated in wild type haploid yeast Saccharomyces cerevisiae. Accelerated ions of argon, titanium, nickel, krypton, xenon, lead and uranium with specific energies between 1.7 and 9.25 MeV/u were obtained from the UNILAC machine at the Gesellschaft für Schwerionenforschung, Darmstadt/Germany. LET-values ranged from 1200 to about 15 000 keV/microns. There was no unequivocal dependence of mutation induction cross section on either LET or Z*2/beta 2, but also a prominent influence of ion specific energy. This is explained by the action of long-ranging delta-electrons.

Heavy ion action on yeast cells: Inhibition of ribosomal-RNA synthesis, loss of colony forming ability and induction of mutants

The action of heavy ions (Ar to U) accelerated to specific energies up to about 10 MeV/u (u=atomic mass unit) on different functions of yeast cells was studied. Ribosomal-RNA synthesis is inhibited according to a single-hit mechanism. Inactivation cross-sections were linearly related to the ratio of the squares of the effective charge Z* and the velocity of the ions. It is concluded from the analysis that the range of the most energetic delta-electrons is larger than previously assumed. There is no such dependence for survival and induction of mutants. In both cases cross-sections increase with the ions specific-energy indicating an important contribution of long-range delta-electrons. The analysis shows that diploid yeast is not killed by a single-hit mechanism even by very heavy ions if the track width is too small. The relative importance of the penumbral region is even more pronounced with the more sensitive strains.

Heavy Ion Effects on Yeast Cells: Reappearance of the Oxygen Effect with Very High LET

Survival of a diploid and a haploid wild type and a radiation-sensitive rad52-mutant was investigated after exposure to accelerated ions in the presence or absence of oxygen. Ar, Kr, Xe, Sm, Pb and U ions were used with specific energies between 0.3 and 12 MeV/u. The results demonstrate that the oxygen enhancement ratios (o.e.r.) do not only depend on LET or Z*2/beta 2 but even more so on ion specific energy. The o.e.r.s are always higher with greater E/m values pointing to the importance of delta-electron action.

Heavy Ion Effects on Cells: Survival of a Temperature-conditional Repair Mutant of Yeast

The temperature-conditional double-strand break repair mutant rad 54-3 of the yeast Saccharomyces cerevisiae was exposed to Ar, Ti and U ions with LET values between 900 and 15,000 keV/micron. Survival was assessed after incubation at the permissive (23 degrees C) and the restrictive temperature (36 degrees C) in aliquots of the same sample. Repair could be demonstrated in all instances, although to a somewhat reduced extent with the heavy ions as compared to X-rays. The results suggest that very densely ionizing radiations do not produce lesions which are irreparable per se, but that their ultimate fate depends on the particular repair system which may be different in different cell types.

Mutation induction in mammalian cells by very heavy ions.

V79 Chinese hamster cells were exposed to heavy ions (O to U) and assayed for mutants at the HGPRT-locus by incubation in selective medium containing 6-thioguanine. The LET ranged from 300 to 18000 keV/micrometer. Mutants could be recovered from all particle radiation but the effectivity per deposited energy decreased with atomic numbers greater than 8. The results are discussed with regard to fundamental processes of cell reactions to very heavy ions and with respect to possible implications for hazard estimations.

On the quantitative interpretation of cellular heavy ion action

Current analyses of heavy ion action assume that the survival probability of a cell hit by a heavy ion depends only on the energy absorbed in its critical site. It is known, however, that the efficiency to produce a biological effect depends also on the spatial pattern of energy deposition. This has to be included in the quantitative evaluation of heavy ion action. Based on recent models of lesion formation by ionizing radiation (Goodhead and Brenner, Phys. Med. Biol. 28, 485, 1983) data with lighter ions (LET < 500 keV/micrometer) were re-analysed. It is shown that the behaviour of various cell systems can be described by a common curve which can be used to estimate the contribution of non-linear components (i.e. where the distribution of energy deposition plays a role) with heavy ions. It is concluded that even with Uranium ions the regions of non-linear effects does not extend beyond 50 nm from the trade core. These data will be used to assess quantitatively survival curves obtained with very heavy ion exposure.