In vivo role of Escherichia coli single-strand exonucleases in SOS induction by gamma radiation.

Ionizing radiation causes different types of genetic damage, ranging from base modifications to single- and double-stranded DNA breaks, which may be deleterious or even lethal to the cell. There are different repair or tolerance mechanisms to counteract the damage. Among them is the Escherichia coli SOS system: a set of genes that becomes activated upon DNA damage to confer better opportunities for cell survival. However, since this response is triggered by single-stranded DNA regions, most lesions have to be processed or modified prior to SOS activation. Several genes such as recO, recB and recJ that seem to be required to induce the response have already been reported. The results of this work indicate that the four known E.coli single-strand exonucleases take part in processing gamma radiation damage, though RecJ and ExoI proved to be more important than ExoVII or ExoX. In addition, ExoV as well as glycosylases such as Nth and, to a lesser extent, Fpg are also required. A model intended to explain the role of all these genes in damage processing is presented.

Induction of oxidative damage by copper-based antineoplastic drugs (Casiopeínas).

PURPOSE: The aim of the present study is to determine in HeLa cells and in human lymphocytes, by an easy and fast method, the induction of oxidative damage to plasma membrane lipids and nuclear DNA by Casiopeínas, which are recently synthesized coordination complexes that have been considered as a promising chemotherapeutic alternative for the treatment of cancer, since they have shown cytotoxicity and genotoxicity in several cancer cell lines and xenotransplanted tumours. The presence of an oxidized copper atom in their structure strongly suggests that their mode of action seems to be related to reactive oxygen species (ROS) generation after copper atom reduction through the Fenton and Haber-Weiss system. METHOD: Lipid peroxidation was evaluated as thiobarbituric acid reactive malondialdehyde, cytotoxicity by the fluorescein diacetate/ethidium bromide stain and genotoxicity as DNA fragmentation by the comet assay. Cells were treated with ten different Casiopeínas in a concentration range higher than their IC(50) (10-100 microM), both oxidized and reduced in the presence of ascorbic acid. RESULTS: In almost all the cases, copper reduction enhanced cytotoxicity but, unlike copper nitrate used as positive control, none of them induced appreciable lipid peroxidation. Three Casiopeínas: Cas Igly, Cas-III-H-a and Cas-III-E-a, showed low, moderate and high rates of genotoxicity, respectively, and this effect was enhanced upon addition of ascorbic acid. CONCLUSION: These results suggest that ROS generation might be the cause of cytotoxicity, which seems to be related to initial genetic damage rather than to lipid peroxidation. HeLa cells showed to be more sensitive than normal cells.

Evidence of DNA double strand breaks formation in Escherichia coli bacteria exposed to alpha particles of different LET assessed by the SOS response.

Ionizing radiation produces a plethora of lesion upon DNA which sometimes is generated among a relatively small region due to clustered energy deposition events, the so called locally multiply damaged sites that could change to DSB. Such clustered damages are more likely to occur in high LET radiation exposures. The effect of alpha particles of different LET was evaluated on the bacterium Escherichia coli either by survival properties or the SOS response activity. Alpha radiation and LET distribution was controlled by means of Nuclear Track Detectors. The results suggest that alpha particles produce two types of lesion: lethal lesions and SOS inducing-mutagenic, a proportion that varies depending on the LET values. The SOS response as a sensitive parameter to assess RBE is mentioned.

Divergent adaptation of Escherichia coli to cyclic ultraviolet light exposures.

The genetic changes taking place during adaptive evolution are particularly interesting in evolutionary biology. As a consequence of adaptive evolution, natural populations of an organism under selective conditions change genetically and phenotypically after a number of generations in order to survive in that particular environment. When a DNA-damaging and mutagenic agent like UV light is experimentally used as a selective factor, natural resistance of bacteria to this agent is normally increased through processes of mutation and selection. Since UV-induced mutagenesis is not restricted to particular chromosomal regions, different UV resistance mechanisms will equally probably evolve as a consequence of cyclic UV irradiation. However, it is also possible that as a consequence of the selective process, one UV resistance mechanism is preferentially selected, causing adaptive convergence of different bacterial cultures. This may occur if the most abundant or lethal kind of DNA lesion is preferentially managed by a particular DNA repair pathway and even by a specific repair enzyme or if resistance mechanisms that decrease bacterial fitness tend to be eliminated from the populations. To examine which of these two alternatives actually takes place, five cultures of Escherichia coli were treated in parallel for 80 successive UV irradiation cycles. At the end, these five cultures gave rise to different grades of UV resistance and after a preliminary characterization we found that adaptation to cyclic UV irradiation was a consequence of selection of advantageous mutations arising in different genes related to repair and replication of DNA.