Janine S. Cardoso

Enzymatic recognition of DNA damage induced by UVB-photosensitized titanium dioxide and biological consequences in Saccharomyces cerevisiae: evidence for oxidatively DNA damage generation.

Although titanium dioxide (TiO(2)) has been considered to be biologically inert, finding use in cosmetics, paints and food colorants, recent reports have demonstrated that when TiO(2) is attained by UVA radiation oxidative genotoxic and cytotoxic effects are observed in living cells. However, data concerning TiO(2)-UVB association is poor, even if UVB radiation represents a major environmental carcinogen. Herein, we investigated DNA damage, repair and mutagenesis induced by TiO(2) associated with UVB irradiation in vitro and in vivo using Saccharomyces cerevisiae model. It was found that TiO(2) plus UVB treatment in plasmid pUC18 generated, in addition to cyclobutane pyrimidine dimers (CPDs), specific damage to guanine residues, such as 8-oxo-7,8-dihydroguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), which are characteristic oxidatively generated lesions. In vivo experiments showed that, although the presence of TiO(2) protects yeast cells from UVB cytotoxicity, high mutation frequencies are observed in the wild-type (WT) and in an ogg1 strain (deficient in 8-oxoG and FapyG repair). Indeed, after TiO(2) plus UVB treatment, induced mutagenesis was drastically enhanced in ogg1 cells, indicating that mutagenic DNA lesions are repaired by the Ogg1 protein. This effect could be attenuated by the presence of metallic ion chelators: neocuproine or dipyridyl, which partially block oxidatively generated damage occurring via Fenton reactions. Altogether, the results indicate that TiO(2) plus UVB potentates UVB oxidatively generated damage to DNA, possibly via Fenton reactions involving the production of DNA base damage, such as 8-oxo-7,8-dihydroguanine.

Inhibition and induction of SOS responses in Escherichia coli by cobaltous chloride

Mutagenesis induced by several genotoxic agents has been reported to be inhibited by cobaltous chloride. In order to study the effects of this metal in some SOS functions we evaluated mutagenesis, lysogenic induction and phage reactivation in Escherichia coli cells treated with CoCl2. We detected that cobaltous chloride, when present in the plating medium, was able to block mutagenesis and lysogenic induction promoted by UV irradiation. We also found that CoCl2 blocked protein synthesis, so we propose that this effect can be responsible for the antimutagenic and antilysogenic effects of this metal. On the other hand, if the cells were treated for a short period of time with CoCl2, in the absence of Mg, we observed that cobaltous chloride per se was able to promote lysogenic induction as well as to enhance the phage reactivation induced by UV irradiation. We conclude that depending on experimental conditions, cobaltous chloride may act either as an inhibitor or as an inducer of the SOS functions.

Overexpression of Escherichia coli nucleotide excision repair genes after cisplatin-induced damage.

Cisplatin is currently used in tumor chemotherapy to induce the death of malignant cells through blockage of DNA replication. It is a commonly used chemotherapeutic agent binding mono- or bifunctionally to guanines in DNA. Escherichia coli K12 mutant strains deficient in nucleotide excision repair (NER) were submitted to increasing concentrations of cisplatin, and the results revealed that uvrA and uvrB mutants are sensitive to this agent, while uvrC and cho mutants remain as the wild type strain. The time required for both gene expression turn-off and return to normal weight DNA in wild-type E. coli was not accomplished even after 4 h post-treatment with cisplatin, while the same process takes place within 1.5 h after ultraviolet radiation (UV). Besides, a heavily damaging action of cisplatin can be seen not only by persistent nicks on genomic DNA, but also by NER gene expression exceeding manifold that seen after equivalent lethal doses of UV. Moreover, cisplatin caused an increase in uvrB gene expression from its putative upstream promoter P3 in an SOS-independent manner.

Mutagenic and genotoxic potential of direct electric current in Escherichia coli and Salmonella thyphimurium strains

Direct electric current has several therapeutic uses such as antibacterial and antiprotozoal action, tissues scarring and regeneration, as well as tumor treatment. This method has shown promising results in vivo and in vitro, with significant efficacy and almost no side effects. Considering lack of studies regarding direct electric current mutagenic and/or genotoxic effects, the present work evaluated both aspects by using five different bacterial experimental assays: survival of repair-deficient mutants, Salmonella-histidine reversion mutagenesis (Ames test), forward mutations to rifampicin resistance, phage reactivation, and lysogenic induction. In these experimental conditions, cells were submitted to an approach that allows evaluation of anodic, cathodic, and electro-ionic effects generated by 2 mA of direct electric current, with doses ranging from 0.36 to 3.60 Coulombs. Our results showed these doses did not induce mutagenic or genotoxic effects.

A Bacteria‐based Experimental Platform to Test Parameters Raised by Mathematical Models on Population Dynamics

Bacterial populations are current models to assay biological effects of a number of different treatments on the basis of a high‐number statistics. One typical bacterial inoculum grows at doubling rates as fast as some 30 min per generation, reaching up to ∼109 cells per ml of medium in a few hours. Given the features of such experimental protocol, it is easy to test the impact of environmental modifications during bacteria growth, by scoring doubling rates time, final cell concentration, oxygen consumption, mutagenesis rates, cell viability under different selective pressures, etc. The drawing of a actual dose‐response or kinetic curves can feed parameters on a given mathematical model on population dynamics by weighting each equation term. The purpose of this talk is to present experimental schemes with bacterial populations so as to serve as parallel two‐hands testing of different mathematical models on populations dynamics.