Cytogenetic evidence that DNA topoisomerase II is not involved in radiation induced chromsome-type aberrations.

ICRF-187 (Cardioxane™, Chiron) is a catalytic inhibitor of DNA topoisomerase II (Topo II), proposed to act by blocking Topo II-mediated DNA cleavage without stabilizing DNA-Topo II-"cleavable complexes". In this study ICRF-187 was used to evaluate the potential involvement of DNA topoisomerase II in the formation of the radiation-induced chromosome-type aberrations in the G0 phase of the cell cycle in human lymphocytes from three healthy male donors. This is based on many evidences that DNA topoisomerases are involved in DNA recombination, mainly of illegitimate type (non-homologous) both in vitro and in vivo. The results obtained clearly indicated that ICRF-187 did not induce per se any chromosomal damage. When challenged with the non-catalytic Topo II poison VP-16 (etoposide), which acts by stabilizing the "cleavable complex" generating "protein concealed" DSB's and thus chromosomal aberrations, it completely abolished the significant induction of chromosome-type aberrations and formation of dicentric chromosomes. This indicates that ICRF-187 acts effectively as catalytic inhibitor of Topo II. On the other hand, when X-ray treatments were challenged with ICRF-187 using experimental conditions as for VP-16 treatments, no modification of the incidence of chromosome-type aberrations and dicentric chromosomes was observed. On this basis, we conclude that Topo II is not involved in the formation of X-ray-induced chromosome-type aberrations and dicentric chromosomes in human lymphocytes in the G0 phase of the cell cycle.

X-ray-induced chromosome aberrations in human lymphocytes in vitro are potentiated under simulated microgravity conditions (Clinostat).

The influence of simulated microgravity weightlessness on the outcome of radiation-induced chromosomal aberrations was investigated using the clinostat as a tool to simulate weightlessness conditions. Treatments were performed in the G0 phase of human lymphocytes with 1.5 Gy of X-rays alone or in combination with the DNA synthesis inhibitor of 1-beta-D-arabinofuranosylcytosine (ara-C) to check also for possible specific radiation-induced DNA repair processes impairment (excision repair caused by base damage) under microgravity conditions. The results obtained, which confirmed previous findings, showed significantly higher increases of aberrant cells and hence total number of aberrations compared to the parallel treatments performed 'on ground'. For what concern ara-C its contribution in terms of potentiation in the induction of aberrant cells was equivalent in absolute terms under simulated microgravity conditions and 'on ground' indicating that excision repair caused by base damage and inhibited by ara-C is not affected by simulated microgravity. A possible explanation for this outcome could quote two major factors: i) Enhanced probability that under simulated microgravity conditions the reactive DSB are spatially brought together to better interact, hence increasing the probability of mis-rejoining. ii) Alternatively chromatin structure could be modified under simulated microgravity conditions generating different quality and quantities of DNA lesions compared to treatments performed 'on ground'.

Development and validation of alternative metabolic systems for mutagenicity testing in short-term assays.

We present here the results obtained within the framework of an EU funded project aimed to develop and validate alternative metabolic activating systems to be used in short-term mutagenicity assays, in order to reduce the use of laboratory animals for toxicology testing. The activating systems studied were established cell lines (Hep G2, CHEL), genetically engineered V79 cell lines expressing specific rat cytochromes P450, erythrocyte-derived systems, CYP-mimetic chemical systems and plant homogenates. The metabolically competent cell lines were used as indicator cells for genotoxic effects as well as for the preparation of external activating systems using other indicator cells. The following endpoints were used: micronuclei, chromosomal aberrations and sister chromatid exchanges, mutations at the hprt locus, gene mutations in bacteria (Ames test), unscheduled DNA synthesis and DNA breaks detected in the comet assay. All metabolic systems employed activated some promutagens. With some of them, promutagens belonging to many different classes of chemicals were activated to genotoxicants, including carcinogens negative in liver S9-mediated assays. In other cases, the use of the new activating systems allowed the detection of mutagens at much lower substrate concentrations than in liver S9-mediated assays. Therefore, the alternative metabolizing systems, which do not require the use of laboratory animals, have a substantial potential in in vitro toxicology, in the basic genotoxicity testing as well as in the elucidation of activation mechanisms. However, since the data basis is much smaller for the new systems than for the activating systems produced from subcellular liver preparations, the overlapping use of both systems is recommended for the present and near future. For example, liver S9 preparations may be used with some indicator systems (e.g., bacterial mutagenicity), and metabolically competent mammalian cell lines may be used with other indicator systems (e.g., a cytogenetic endpoint) in a battery of basic tests.