Comparison of the DNA damage response in BEAS-2B and A549 cells exposed to titanium dioxide nanoparticles.

For some decades production of titanium dioxide nanoparticle (TiO2-NP) has been increasing at a considerable rate; concerns as to the toxicity of these particles upon inhalation have been raised. Indeed, TiO2-NPs have been shown to induce significant genotoxicity and to adversely affect both major DNA repair mechanisms: base excision repair (BER) and nucleotide excision repair (NER). The aims of the present study were to (i) compare the genotoxicity of TiO2-NPs and their impact on DNA repair processes on A549 alveolar carcinoma and BEAS-2B normal bronchial lung cell lines and (ii) delve deeper into the mechanisms leading to these effects. To achieve these goals, TiO2-NPs effects on cytotoxicity, genotoxicity, DNA repair activity and DNA repair gene expression were investigated in both cell lines upon exposure to 1-100 µg/mL of anatase/rutile, 21 nm TiO2-NPs. Our results show that TiO2-NPs induce comparable cytotoxic and genotoxic responses in BEAS-2B and A549 cells. Functional response to DNA damage is observed in both cell lines and consists of an overall downregulation in DNA repair processes. When evaluating the relative importance of the two DNA repair pathways, we observed a lower impact on BER compared with NER activities, suggesting that repair of oxidatively generated DNA damage is still triggered in these cells. This response becomes measureable at 4 h of exposure in BEAS-2B but only after 48 h of exposure in A549 cells. The delayed response in A549 cells is due to an initial overall and intense downregulation of the genes encoding DNA repair proteins. This overall downregulation correlates with increased methylation of DNA repair gene promoters and downregulation of NRF2 and BRCA1, which may thus be considered as upstream regulators. These results strengthen the evidence that TiO2-NP induces indirect genotoxicity in lung cells, via modulation of DNA repair processes, and shed some light on the mechanisms behind this effect.

Direct inhibition of excision/synthesis DNA repair activities by cadmium: analysis on dedicated biochips.

The well established toxicity of cadmium and cadmium compounds results from their additive effects on several key cellular processes, including DNA repair. Mammalian cells have evolved several biochemical pathways to repair DNA lesions and maintain genomic integrity. By interfering with the homeostasis of redox metals and antioxidant systems, cadmium promotes the development of an intracellular environment that results in oxidative DNA damage which can be mutagenic if unrepaired. Small base lesions are recognised by specialized glycosylases and excised from the DNA molecule. The resulting abasic sites are incised, and the correct sequences restored by DNA polymerases using the opposite strands as template. Bulky lesions are recognised by a different set of proteins and excised from DNA as part of an oligonucleotide. As in base repair, the resulting gaps are filled by DNA polymerases using the opposite strands as template. Thus, these two repair pathways consist in excision of the lesion followed by DNA synthesis. In this study, we analysed in vitro the direct effects of cadmium exposure on the functionality of base and nucleotide DNA repair pathways. To this end, we used recently described dedicated microarrays that allow the parallel monitoring in cell extracts of the repair activities directed against several model base and/or nucleotide lesions. Both base and nucleotide excision/repair pathways are inhibited by CdCl2, with different sensitivities. The inhibitory effects of cadmium affect mainly the recognition and excision stages of these processes. Furthermore, our data indicate that the repair activities directed against different damaged bases also exhibit distinct sensitivities, and the direct comparison of cadmium effects on the excision of uracile in different sequences even allows us to propose a hierarchy of cadmium sensibility within the glycosylases removing U from DNA. These results indicate that, in our experimental conditions, cadmium is a very potent DNA repair poison.

Application of the dynamic model of Saeman to an industrial rotary kiln incinerator: numerical and experimental results.

In order to simulate granular materials structure in a rotary kiln under the steady-state regime, a mathematical model has been developed by Saeman (1951). This model enables the calculation of the bed profiles, the axial velocity and solids flow rate along the kiln. This model can be coupled with a thermochemical model, in the case of a reacting moving bed. This dynamic model was used to calculate the bed profile for an industrial size kiln and the model projections were validated by measurements in a 4 m diameter by 16 m long industrial rotary kiln. The effect of rotation speed under solids bed profile and the effect of the feed rate under filling degree were established. On the basis of the calculations and the experimental results a phenomenological relation for the residence time estimation was proposed for the rotary kiln.

A microarray to measure repair of damaged plasmids by cell lysates.

DNA repair mechanisms constitute major defences against agents that cause cancer, degenerative disease and aging. Different repair systems cooperate to maintain the integrity of genetic information. Investigations of DNA repair involvement in human pathology require an efficient tool that takes into account the variety and complexity of repair systems. We have developed a highly sensitive damaged plasmid microarray to quantify cell lysate excision/synthesis (ES) capacities using small amounts of proteins. This microsystem is based on efficient immobilization and conservation on hydrogel coated glass slides of plasmid DNA damaged with a panel of genotoxic agents. Fluorescent signals are generated from incorporation of labelled dNTPs by DNA excision-repair synthesis mechanisms at plasmid sites. Highly precise DNA repair phenotypes i.e. simultaneous quantitative measures of ES capacities toward seven lesions repaired by distinct repair pathways, are obtained. Applied to the characterization of xeroderma pigmentosum (XP) cells at basal level and in response to a low dose of UVB irradiation, the assay showed the multifunctional role of different XP proteins in cell protection against all types of damage. On the other hand, measurement of the ES of peripheral blood mononuclear cells from six donors revealed significant diversity between individuals. Our results illustrate the power of such a parallelized approach with high potential for several applications including the discovery of new cancer biomarkers and the screening of chemical agents modulating DNA repair systems.

Prenatal diagnosis and molecular characterization of an interstitial 1q24.2q25.2 deletion.

We report on the observation of an interstitial deletion of the long arm of chromosome 1 diagnosed prenatally in a 28 weeks gestation fetus by standard karyotype. Amniocentesis was performed because of an increased Down syndrome maternal serum screening and ultrasonographic abnormalities. Fetus autopsy showed an intrauterine growth retardation, dysmorphic features and limbs abnormalities. Using fluorescent in situ hybridization technique (FISH), we characterized the deletion boundaries corresponding to the bacterial artificial chromosomes (BAC) RP11-193J5 and RP11-162L13. Molecular studies identified the deletion of paternal origin. Therefore the karyotype was interpreted as 46,XY,del(1)(q24.2q25.2). This is the smallest deletion of the long arm of chromosome 1 reported prenatally and characterized at the molecular level. Its phenotype is compared to other similar cases described in the literature.

Analysis of fluoroquinolone-mediated photosensitization of 2'-deoxyguanosine, calf thymus and cellular DNA: determination of type-I, type-II and triplet-triplet energy transfer mechanism contribution.

Fluoroquinolone (FQ) antibacterials are known to exhibit photosensitization properties leading to the formation of oxidative damage to DNA. In addition, photoexcited lomefloxacin (Lome) was recently shown to induce the formation of cyclobutane pyrimidine dimers via triplet-triplet energy transfer. The present study is aimed at gaining further insights into the photosensitization mechanisms of several FQ including enoxacin (Enox), Lome, norfloxacin (Norflo) and ofloxacin (Oflo). This was achieved by monitoring the formation of DNA base degradation products upon UVA-mediated photosensitization of 2'-deoxyguanosine, isolated and cellular DNA. Oflo and Norflo act mainly via a Type-II mechanism whereas Lome and, to a lesser extent, Enox behave more like Type-I photosensitizers. However, the extent of oxidative damage was found to be relatively low. In contrast, it was found that cyclobutane thymine dimers represent the major class of damage induced by Enox, Lome and Norflo within isolated and cellular DNA upon UVA irradiation. This striking observation confirms that FQ are able to promote efficient triplet energy transfer to DNA. The levels of photosensitized formation of strand breaks, alkali-labile sites and oxidative damage to cellular DNA, as measured by the comet assay, were confirmed to be rather low. Therefore, we propose that the phototoxic effects of FQ are mostly accounted for energy transfer mechanism rather than by Type-I or -II photosensitization processes.

Treatment of familial hemophagocytic lymphohistiocytosis with bone marrow transplantation from HLA genetically nonidentical donors.

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare genetic disorder associated with the onset early in life of overwhelming activation of T lymphocytes and macrophages invariably leading to death. Allogeneic bone marrow transplantation (BMT) from an HLA-identical related donor is the treatment of choice in patients with this disease. However, fewer than 20% of patients have a disease-free HLA-identical sibling. BMT from HLA-nonidentical related donors has previously met with poor results, with graft rejection a major obstacle in all cases. We describe BMTs from HLA-nonidentical related donors (n = 13) and from a matched unrelated donor (n = 1) performed in two centers in 14 consecutive cases of FHL. Remission of disease was achieved before BMT in 10 patients. Marrow was T-cell-depleted to minimize graft-versus-host disease (GVHD). Antiadhesion antibodies specific for the alpha chain of the leukocyte function-associated antigen-1 (LFA-1, CD11a) and the CD2 molecules were infused pre-BMT and post-BMT to help prevent graft rejection, in addition to a conditioning regimen of busulfan (BU), cyclophosphamide (CP), and etoposide (VP16) or antithymocyte globulin (ATG). Sustained engraftment was obtained in 11 of 17 transplants (3 patients had 2 transplants) and disease-free survival in 9 patients with a follow-up period of 8 to 69 months (mean, 33). Acute GVHD greater than stage I was not observed, and 1 patient had mild cutaneous chronic GVHD that resolved. Toxicity due to the BMT procedure was low. Results obtained using this protocol are promising in terms of engraftment and event-free survival within the limitations of the small sample. We conclude that an immunologic approach in terms of drugs used to obtain disease remission and a conditioning regimen that includes antiadhesion molecules in T-cell-depleted BMT from HLA genetically nonidentical donors is an alternative treatment that warrants further study in FHL patients who lack a suitable HLA genetically identical donor.

In vivo clonal expression of T lymphocytes specific for an immunodominant N-terminal myelin basic protein epitope in healthy individuals.

The role of myelin basic protein (MBP) T cell recognition in the induction of experimental allergic encephalomyelitis (EAE) has been well established in mice and rats. A remarkable restriction has been observed in T cell receptor (TCR) genes utilized by encephalitogenic T cell lines (TCLs) specific for immunodominant epitopes in these species. Pathological similarities between this animal model and multiple sclerosis (MS) has led to consider MBP as a major candidate autoantigen in this human disorder. Unlike in inbred strains of animals, the T cell response to MBP in humans is quite heterogenous with regard to fine epitope specificity. The existence of V alpha and/or V beta restriction in MBP-specific T cells, from MS patients and healthy controls, is still a matter of debate. In this study we generated 77 MBP-specific TCLs from nine healthy donors and showed that peptide 7-27 is one of the most frequently recognized epitopes. 37% of all epitope-specific TCLs recognized this peptide and p7-27 specific TCLs were generated from seven out of the nine subjects studied. A high level of in vivo clonal expansion was observed in p7-27-specific TCLs in several subjects, which however is not specific of this epitope since this phenomenon was also observed in p85-104- and 149-162-specific TCLs.