Artifacts associated with the measurement of oxidized DNA bases.

In this paper we review recent aspects of the measurement of oxidized DNA bases, currently a matter of debate. There has long been an interest in the determination of the level of oxidized bases in cellular DNA under both normal and oxidative stress conditions. In this respect, the situation is confusing because variations that may be as large as two orders of magnitude have been reported for the yield of the formation of 8-oxo-7,8-dihydroguanine (8-oxoGua) in similar DNA samples. However, recent findings clearly show that application of several assays like gas chromatography-mass spectrometry (GC-MS) and -32P--postlabeling may lead to a significant overestimation of the level of oxidized bases in cellular DNA. In particular, the silylation step, which is required to make the samples volatile for the GC-MS analysis, has been shown to induce oxidation of normal bases at the level of about one oxidized base per 10(4) normal bases. This has been found to be a general process that applies in particular to 8-oxoGua, 8-oxo-7, 8-dihydroadenine,5-hydroxycytosine, 5-(hydroxymethyl)uracil, and 5-formyluracil. Interestingly, prepurification of the oxidized bases from DNA hydrolysate prior to the derivatization reaction prevents artefactual oxidation. Under these conditions, the level of oxidized bases measured by GC-MS is similar to that obtained by HPLC associated with electrochemical detection (HPLC-EC). It should be added that the level of 8-oxo-7,8-dihydro-2;-deoxyguanosine in control cellular DNA has been found to be about fivefold lower than in earlier HPLC-EC measurements by using appropriate conditions of extraction and enzymatic digestion of DNA. Similar conclusions were reached by measuring formamidopyrimidine-DNA glycosylase sensitive sites as revealed by the single cell gel electrophoresis (comet) assay

Fluorescence of Natural DNA: From the Femtosecond to the Nanosecond Time Scales

International audienceThe fluorescence of calf thymus DNA is studied by steady-state and time-resolved spectroscopy combining fluorescence upconversion and time-correlated single photon counting. The fluorescence spectrum is very similar to that of a stoichiometric mixture of monomeric chromophores, arising from bright ππ* states, and contrasts with the existing picture of exciplex emission in natural DNA. Yet, the DNA fluorescence decays span over five decades of time, with 98% of the photons being emitted at times longer than 10 ps. These findings, in association with recent studies on model duplexes, are explained by the involvement of dark states, possibly related to charge separation, serving as a reservoir for the repopulation of the bright ππ* states

Behind the silence of harmony: risk factors for physical and sexual violence among women in rural Indonesia

BACKGROUND: Indonesia has the fourth largest population in the world. Few studies have identified the risk factors of Indonesian women for domestic violence. Such research will be useful for the development of prevention programs aiming at reducing domestic violence. Our study examines associations between physical and sexual violence among rural Javanese Indonesian women and sociodemographic factors, husband's psychosocial and behavioral characteristics and attitudes toward violence and gender roles. METHODS: A cohort of pregnant women within the Demographic Surveillance Site (DSS) in Purworejo district, Central Java, Indonesia, was enrolled in a longitudinal study between 1996 and 1998. In the following year (1999), a cross-sectional domestic violence household survey was conducted with 765 consenting women from that cohort. Female field workers, trained using the WHO Multi-Country study instrument on domestic violence, conducted interviews. Crude and adjusted odds ratios at 95% CI were applied for analysis. RESULTS: Lifetime exposure to sexual and physical violence was 22% and 11%. Sexual violence was associated with husbands' demographic characteristics (less than 35 years and educated less than 9 years) and women's economic independence. Exposure to physical violence among a small group of women (2-6%) was strongly associated with husbands' personal characteristics; being unfaithful, using alcohol, fighting with other men and having witnessed domestic violence as a child. The attitudes and norms expressed by the women confirm that unequal gender relationships are more common among women living in the highlands and being married to poorly educated men. Slightly more than half of the women (59%) considered it justifiable to refuse coercive sex. This attitude was also more common among financially independent women (71%), who also had a higher risk of exposure to sexual violence. CONCLUSIONS: Women who did not support the right of women to refuse sex were more likely to experience physical violence, while those who justified hitting for some reasons were more likely to experience sexual violence. Our study suggests that Javanese women live in a high degree of gender-based subordination within marriage relationships, maintained and reinforced through physical and sexual violence. Our findings indicate that women's risk of physical and sexual violence is related to traditional gender norms

DNA Dosimetry Assessment for Sunscreen Genotoxic Photoprotection

Background: Due to the increase of solar ultraviolet radiation (UV) incidence over the last few decades, the use of sunscreen has been widely adopted for skin protection. However, considering the high efficiency of sunlight-induced DNA lesions, it is critical to improve upon the current approaches that are used to evaluate protection factors. An alternative approach to evaluate the photoprotection provided by sunscreens against daily UV radiation-induced DNA damage is provided by the systematic use of a DNA dosimeter. Methodology/Principal Findings: The Sun Protection Factor for DNA (DNA-SPF) is calculated by using specific DNA repair enzymes, and it is defined as the capacity for inhibiting the generation of cyclobutane pyrimidine dimers (CPD) and oxidised DNA bases compared with unprotected control samples. Five different commercial brands of sunscreen were initially evaluated, and further studies extended the analysis to include 17 other products representing various formulations and Sun Protection Factors (SPF). Overall, all of the commercial brands of SPF 30 sunscreens provided sufficient protection against simulated sunlight genotoxicity. In addition, this DNA biosensor was useful for rapidly screening the biological protection properties of the various sunscreen formulations. Conclusions/Significance: The application of the DNA dosimeter is demonstrated as an alternative, complementary, and reliable method for the quantification of sunscreen photoprotection at the level of DNA damage.Natura Inovacao e Tecnologia de Produtos LTDA (Sao Paulo, Brazil)Natura Inovacao e Tecnologia de Produtos LTDA (Sao Paulo, Brazil)FAPESP (Sao Paulo, Brazil)FAPESP (Sao Paulo, Brazil)CNPq (Brasilia, Brazil)CNPq (Brasilia, Brazil)Natura Inovacao e Tecnologia de Produtos LTDANatura Inovacao e Tecnologia de Produtos LTD

δ-Aminolevulinic acid cytotoxic effects on human hepatocarcinoma cell lines

BACKGROUND: Acute Intermittent Porphyria is a genetic disorder of heme metabolism, characterized by increased levels of porphyrin precursors, δ-aminolevulinic acid (ALA) and porphobilinogen (PBG). ALA has been reported to generate reactive oxygen species and to cause oxidative damage to proteins, subcellular structures and DNA. It is known that oxidative stress can induce apoptosis. The aim of this work was to study the cytotoxic effect of ALA on two hepatocarcinoma cell lines. RESULTS: We have determined the impact of ALA on HEP G2 and HEP 3B hepatocarcinoma cell lines survival as measured by the MTT assay. ALA proved to be cytotoxic in both cell lines however; HEP G2 was more sensitive to ALA than HEP 3B. Addition of hemin or glucose diminished ALA cytotoxicity in HEP G2 cells; instead it was enhanced in HEP 3B cells. Because apoptosis is usually associated with DNA fragmentation, the DNA of ALA treated and untreated cells were analyzed. The characteristic pattern of DNA fragmentation ladders was observed in ALA treated cells. To elucidate the mechanisms of ALA induced apoptosis, we examined its effect on p53 expression. No changes in p53 mRNA levels were observed after exposure of both cell lines to ALA for 24 h. CDK2 and CDK4 protein levels were reduced after ALA treatment at physiological concentrations

Inhibition of Plasmodium falciparum Field Isolates-Mediated Endothelial Cell Apoptosis by Fasudil: Therapeutic Implications for Severe Malaria

Plasmodium falciparum infection can abruptly progress to severe malaria, a life-threatening complication resulting from sequestration of parasitized red blood cells (PRBC) in the microvasculature of various organs such as the brain and lungs. PRBC adhesion can induce endothelial cell (EC) activation and apoptosis, thereby disrupting the blood-brain barrier. Moreover, hemozoin, the malarial pigment, induces the erythroid precursor apoptosis. Despite the current efficiency of antimalarial drugs in killing parasites, severe malaria still causes up to one million deaths every year. A new strategy targeting both parasite elimination and EC protection is urgently needed in the field. Recently, a rho-kinase inhibitior Fasudil, a drug already in clinical use in humans for cardio- and neuro-vascular diseases, was successfully tested on laboratory strains of P. falciparum to protect and to reverse damages of the endothelium. We therefore assessed herein whether Fasudil would have a similar efficiency on P. falciparum taken directly from malaria patients using contact and non-contact experiments. Seven (23.3%) of 30 PRBC preparations from different patients were apoptogenic, four (13.3%) acting by cytoadherence and three (10%) via soluble factors. None of the apoptogenic PRBC preparations used both mechanisms indicating a possible mutual exclusion of signal transduction ligand. Three PRBC preparations (42.9%) induced EC apoptosis by cytoadherence after 4 h of coculture (“rapid transducers”), and four (57.1%) after a minimum of 24 h (“slow transducers”). The intensity of apoptosis increased with time. Interestingly, Fasudil inhibited EC apoptosis mediated both by cell-cell contact and by soluble factors but did not affect PRBC cytoadherence. Fasudil was found to be able to prevent endothelium apoptosis from all the P. falciparum isolates tested. Our data provide evidence of the strong anti-apoptogenic effect of Fasudil and show that endothelial cell-P. falciparum interactions are more complicated than previously thought. These findings may warrant clinical trials of Fasudil in severe malaria management