DNA, a target of mixed chelate copper(II) compounds (Casiopeinas®) studied by electrophoresis, UV-vis and circular dichroism techniques.

Casiopeinas are a family of mixed chelate copper(II) complexes with antiproliferative and antineoplastic activities, results that have positioned them as an alternative for cancer treatment. Because DNA is one of their principal targets, it is of our interest to find out the effect of substituents on the diiamine ligands over mode of interaction. Therefore, we studied 21 Casiopeinas upon DNA by gel electrophoresis, UV-vis and circular dichroism (CD) spectroscopic techniques, previously studied by DFT calculations and Quantitative Structure-Activity Relationship (QSAR). According to electrophoresis results, the interaction modes between Casiopeinas with DNA may be through the intercalation or in the minor groove. UV-vis spectroscopy showed a hypochromic or hyperchromic effect as a consequence of each interaction. The analysis suggests the binding along the minor groove and intercalation are both influenced by the substituents in the diimine ligands and depend on the nature of the secondary moiety (acetylacetonate or glycinate). Additionally, a new band in electrophoresis and CD spectra suggests adducts formation. In general, we prove that molecules with the highest molecular weight, electron donating substituents and glycinate as secondary ligand are intercalating agents; unlike molecules with electron withdrawing substituents as chloride or nitro and acetylacetonate as secondary ligand which interact in the minor groove.

Generation of electrical energy in a microbial fuel cell coupling acetate oxidation to Fe3+ reduction and isolation of the involved bacteria.

An iron reducing enrichment was obtained from sulfate reducing sludge and was evaluated on the capability of reducing Fe3+ coupled to acetate oxidation in a microbial fuel cell (MFC). Three molar ratios for acetate/Fe3+ were evaluated (2/16, 3.4/27 and 6.9/55 mM). The percentages of Fe3+ reduction were in a range of 80-90, 60-70 and 40-50% for the MFCs at closed circuit for the molar ratios of 2/16, 3.4/27 and 6.9/55 mM, respectively. Acetate consumption was in a range of 80-90% in all cases. The results obtained at closed circuit for current density were: 11.37 mA/m2, 4.5 mA/m2 and 7.37 mA/m2 for the molar ratios of 2/16, 3.4/27 and 6.9/55 mM, respectively. Some microorganisms that were isolated and identified in the MFCs were Azospira oryzae, Cupriavidus metallidurans CH34, Enterobacter bugandensis 247BMC, Citrobacter freundii ATCC8090 and Citrobacter murliniae CDC2970-59, these bacteria have been reported as exoelectrogens in MFC and in MFC involving metals removal but not all of them have been reported to utilize acetate as preferred substrate. The results demonstrate that the isolates can utilize acetate as the sole source of carbon and suggest that Fe3+ reduction was carried out by a combination of different mechanisms (direct contact and redox mediators) utilized by the bacteria identified in the MFC. Storage of the energy generated from the 2/16 mM MFC system arranged in a series of three demonstrated that it is possible to utilize the energy to charge a battery.

Development and Characterization of a Non-Thermal Plasma Source for Therapeutic Treatments.

OBJECTIVE: an innovative non-thermal plasma (NTP) system constituted by a radiofrequency (RF) power generator directly coupled to a treatment probe is described and characterized. This system is intended to be applied as a medical device for therapeutic treatments. METHODS: electrical characterization of the radiofrequency power generator supplying the treatment probe was performed. Meanwhile, generated NTP was optically analyzed. Obtained data were studied to establish the safety profile of plasma application on heat sensitive matter. RESULTS: the NTP system was validated through bacterial deactivation trials, as well as, of being capable of deactivating carcinogenic cells. Besides promoting and accelerating wound closure in vivo performed in mice, demonstrating faster healing than that done with conventional treatments. CONCLUSION: the NTP system's characterization is an essential stage to determine the adequate application of the generated plasma over organic media. The therapeutic benefits of the NTP system were proved by the development of in vivo experiences involving laboratory mice. SIGNIFICANCE: the generated NTP interacts with surrounding air particles producing reactive oxygen and nitrogen species, which, exhibit bactericidal and antiseptic effects due to their strong biochemical reactivity; functioning like critical mediators in animal physiology and promoting wound healing processes. These properties make the NTP system a feasible technology intended for therapeutic treatments.

Induction of the SOS response of Escherichia coli in repair-defective strains by several genotoxic agents.

DNA is exposed to the attack of several exogenous agents that modify its chemical structure, so cells must repair those changes in order to survive. Alkylating agents introduce methyl or ethyl groups in most of the cyclic or exocyclic nitrogen atoms of the ring and exocyclic oxygen available in DNA bases producing damage that can induce the SOS response in Escherichia coli and many other bacteria. Likewise, ultraviolet light produces mainly cyclobutane pyrimidine dimers that arrest the progression of the replication fork and triggers such response. The need of some enzymes (such as RecO, ExoI and RecJ) in processing injuries produced by gamma radiation prior the induction of the SOS response has been reported before. In the present work, several repair-defective strains of E. coli were treated with methyl methanesulfonate, ethyl methanesulfonate, mitomycin C or ultraviolet light. Both survival and SOS induction (by means of the Chromotest) were tested. Our results indicate that the participation of these genes depends on the type of injury caused by a genotoxin on DNA.

Influence of uvrA, recJ and recN gene mutations on nucleoid reorganization in UV-treated Escherichia coli cells.

Exposure to ultraviolet (UV) radiation blocks DNA replication and arrests cellular division in Escherichia coli. Restoration of chromosome replication involves nucleoid reorganization, which involves the participation of the recombination-catalyzing proteins RecA, RecO, RecR and RecN. In this work, we evaluated the influence of recN, uvrA and recJ gene mutations on post-irradiation nucleoid reorganization. We used isogenic E. coli strains that are defective for these genes to study post-irradiation kinetics of the nucleoid shape fractions using fluorescence microscopy. The results showed that in the wild-type strain, post-irradiation nucleoid reorganization occurs, which restores the nucleoid shape fractions in the cells to those observed prior to irradiation. First, the nucleoid condenses into the central area of the irradiated cell. Second, the nucleoid disperses along the cell. Third, the cell enters the chromosome replicative phase and cytokinesis. Escherichia coli cells with a recN mutation did not exhibit increased nucleoid condensation, but chromosome replication and cytokinesis occurred. In the uvrA and recJ strains, the condensation step was delayed compared to the wild-type strain, and chromosome replication and cytokinesis did not occur. The results are discussed with an emphasis on the functions of RecN, UvrA and RecJ in nucleoid reorganization in UV-irradiated E. coli cells.

Genotoxic assessment of the copper chelated compounds Casiopeinas: Clues about their mechanisms of action.

Casiopeinas is the generic name of a group of copper chelated complexes designed to be used as antineoplastic. Some of these compounds have shown promising results, and in fact, one of them named Casiopeina III-ia has completed preclinical trials and is ready to start clinical phase I in Mexico. As part of the tests that have to be done to every molecule intended to be used in humans, bacterial assays are required because of their sensitivity, speed and reproducibility and among them, Ames test and the SOS Chromotest are widely used to evaluate DNA damage. With the aim to contribute to complete safety information related to genotoxicity and support the hypothesis about their mode of action, four different Casiopeinas (Cas II-gly, Cas III-Ea, Cas III-ia and Cas III-Ha) were tested for genotoxicity with these assays, as well as differential cytotoxicity upon Escherichia coli mutants defectives in some DNA repair mechanisms. However, although it is well known that these molecules produce DNA breakage, the results of the Chromotest and Ames test were negative. Despite this is controversial, a possible explanation is that there is a direct interaction between DNA and the Casiopeinas tested.

Survival and SOS response induction in ultraviolet B irradiated Escherichia coli cells with defective repair mechanisms.

Purpose In this paper, the contribution of different genes involved in DNA repair for both survival and SOS induction in Escherichia coli mutants exposed to ultraviolet B radiation (UVB, [wavelength range 280-315 nm]) was evaluated. Materials and methods E. coli strains defective in uvrA, oxyR, recO, recN, recJ, exoX, recB, recD or xonA genes were used to determine cell survival. All strains also had the genetic sulA::lacZ fusion, which allowed for the quantification of SOS induction through the SOS Chromotest. Results Five gene products were particularly important for survival, as follows: UvrA > RecB > RecO > RecJ > XonA. Strains defective in uvrA and recJ genes showed elevated SOS induction compared with the wild type, which remained stable for up to 240 min after UVB-irradiation. In addition, E. coli strains carrying the recO or recN mutation showed no SOS induction. Conclusions The nucleotide excision and DNA recombination pathways were equally used to repair UVB-induced DNA damage in E. coli cells. The sulA gene was not turned off in strains defective in UvrA and RecJ. RecO protein was essential for processing DNA damage prior to SOS induction. In this study, the roles of DNA repair proteins and their contributions to the mechanisms that induce SOS genes in E. coli are proposed.

Laser heating of gold nanospheres functionalized with octreotide: in vitro effect on HeLa cell viability.

OBJECTIVE: The aim of this study was to assess the effect of laser heating a well-characterized gold nanoparticle (AuNP)-octreotide system on HeLa cell viability, to evaluate its potential as a suitable agent for plasmonic photothermal therapy. BACKGROUND DATA: Octreotide is a synthetic peptide derivative of somatostatin with an effect on the survival of HeLa cells. Peptides bound to AuNPs are biocompatible and stable multimeric systems with target-specific molecular recognition. METHODS: Octreotide was conjugated to AuNPs (∼20 nm) by spontaneous reaction with the thiol groups. The nanoconjugate was characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), ultraviolet visible spectroscopy (UV-Vis), X-ray photoelectron spectroscopy (XPS), and Raman spectroscopy. Irradiation experiments were conducted using an Nd:YAG laser pulsed for 5 ns at 532 nm with a repetition rate of 10 Hz for up to 6 min while delivering an average irradiance of 0.65 W/cm(2). HeLa cells were incubated at 37°C (1) with AuNP-citrate, (2) with AuNP-octreotide, or (3) without nanoparticles. RESULTS: After laser irradiation, the presence of AuNP caused a significant increase in the temperature of the medium (48°C vs. 38.3°C of that without AuNP). The AuNP-octreotide system resulted in a significant decrease in cell viability of up to 6 % compared with the AuNP-citrate system (15.8±2.1%). Two possible mechanisms could be at play: (1) octreotide alone exerts an effect on survival HeLa cells, or (2) the release of heat (∼727°C per nanoparticle) in the membranes or cytoplasm of the cells caused by the interaction between AuNP-octreotide and somatostatin receptors reduced viability. CONCLUSIONS: The AuNP-octreotide system exhibited properties suitable for plasmonic photothermal therapy in the treatment of cervical cancer.

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.

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.

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.