DNA Double-Strand Breaks Induced in Human Cells by Twelve Metallic Species: Quantitative Inter-Comparisons and Influence of the ATM Protein.

Despite a considerable amount of data, the molecular and cellular bases of the toxicity due to metal exposure remain unknown. Recent mechanistic models from radiobiology have emerged, pointing out that the radiation-induced nucleo-shuttling of the ATM protein (RIANS) initiates the recognition and the repair of DNA double-strand breaks (DSB) and the final response to genotoxic stress. In order to document the role of ATM-dependent DSB repair and signalling after metal exposure, we applied twelve different metal species representing nine elements (Al, Cu, Zn Ni, Pd, Cd, Pb, Cr, and Fe) to human skin, mammary, and brain cells. Our findings suggest that metals may directly or indirectly induce DSB at a rate that depends on the metal properties and concentration, and tissue type. At specific metal concentration ranges, the nucleo-shuttling of ATM can be delayed which impairs DSB recognition and repair and contributes to toxicity and carcinogenicity. Interestingly, as observed after low doses of ionizing radiation, some phenomena equivalent to the biological response observed at high metal concentrations may occur at lower concentrations. A general mechanistic model of the biological response to metal exposure based on the nucleo-shuttling of ATM is proposed to describe the metal-induced stress response and to define quantitative endpoints for toxicity and carcinogenicity.

Crumpling of silver nanowires by endolysosomes strongly reduces toxicity.

Fibrous particles interact with cells and organisms in complex ways that can lead to cellular dysfunction, cell death, inflammation, and disease. The development of conductive transparent networks (CTNs) composed of metallic silver nanowires (AgNWs) for flexible touchscreen displays raises new possibilities for the intimate contact between novel fibers and human skin. Here, we report that a material property, nanowire-bending stiffness that is a function of diameter, controls the cytotoxicity of AgNWs to nonimmune cells from humans, mice, and fish without deterioration of critical CTN performance parameters: electrical conductivity and optical transparency. Both 30- and 90-nm-diameter AgNWs are readily internalized by cells, but thinner NWs are mechanically crumpled by the forces imposed during or after endocytosis, while thicker nanowires puncture the enclosing membrane and release silver ions and lysosomal contents to the cytoplasm, thereby initiating oxidative stress. This finding extends the fiber pathology paradigm and will enable the manufacture of safer products incorporating AgNWs.

Influence of Nucleoshuttling of the ATM Protein in the Healthy Tissues Response to Radiation Therapy: Toward a Molecular Classification of Human Radiosensitivity.

PURPOSE: Whereas post-radiation therapy overreactions (OR) represent a clinical and societal issue, there is still no consensual radiobiological endpoint to predict clinical radiosensitivity. Since 2003, skin biopsy specimens have been collected from patients treated by radiation therapy against different tumor localizations and showing a wide range of OR. Here, we aimed to establish quantitative links between radiobiological factors and OR severity grades that would be relevant to radioresistant and genetic hyperradiosensitive cases. METHODS AND MATERIALS: Immunofluorescence experiments were performed on a collection of skin fibroblasts from 12 radioresistant, 5 hyperradiosensitive, and 100 OR patients irradiated at 2 Gy. The numbers of micronuclei, γH2AX, and pATM foci that reflect different steps of DNA double-strand breaks (DSB) recognition and repair were assessed from 10 minutes to 24 hours after irradiation and plotted against the severity grades established by the Common Terminology Criteria for Adverse Events and the Radiation Therapy Oncology Group. RESULTS: OR patients did not necessarily show a gross DSB repair defect but a systematic delay in the nucleoshuttling of the ATM protein required for complete DSB recognition. Among the radiobiological factors, the maximal number of pATM foci provided the best discrimination among OR patients and a significant correlation with each OR severity grade, independently of tumor localization and of the early or late nature of reactions. CONCLUSIONS: Our results are consistent with a general classification of human radiosensitivity based on 3 groups: radioresistance (group I); moderate radiosensitivity caused by delay of nucleoshuttling of ATM, which includes OR patients (group II); and hyperradiosensitivity caused by a gross DSB repair defect, which includes fatal cases (group III).

Global quantification of γH2AX as a triage tool for the rapid estimation of received dose in the event of accidental radiation exposure.

The phosphorylation of the H2AX histone to form γH2AX foci has been shown to be an accurate biomarker of ionizing radiation exposure. It is well established that there is a one-to-one correlation between the number of γH2AX foci and radiation-induced double strand breaks in cellular DNA, which can be translated to the received dose. However, manual counting of foci is time-consuming, and cannot accommodate high throughput analysis required to obtain rapid results for medical triage purposes in the case of large-scale accidental exposure. Furthermore, the accuracy of γH2AX measurements could potentially be compromised by delays between the time of exposure and analysis of results, as well as inter-cellular and inter-individual variability of this biological response. To evaluate more rapid approaches of quantifying γH2AX for use in an emergency situation, and to determine the impact of inter-individual variability, we compared two methods of global γH2AX fluorescence quantification (low magnification immunofluorescence microscopy and flow cytometry) to the well-established γH2AX foci scoring method in human primary fibroblasts. All three approaches were well correlated, indicating that global γH2AX fluorescence measurements are suitable for dose estimation. For rapid triage in an emergency situation, we propose the use of flow cytometry, as it is more highly correlated with foci scoring and because of the speed and ease of the method. Dose response curves (0.25-6Gy) using flow cytometry measurements showed that inter-individual variability in global γH2AX fluorescence is statistically insignificant at 4h post-irradiation. Based on these data, we propose calibration curves that can be applied to populations exposed to moderate radiation doses to estimate individual received doses, independent of individual radiosensitivity, at this specific time point post-irradiation using human fibroblasts and lymphocytes. Furthermore, we define three triage categories that could facilitate immediate and follow-up care in the case of a radiological accident.

Lead Exposure Induces Telomere Instability in Human Cells.

Lead (Pb) is an important environmental contaminant due to its widespread use over many centuries. While it affects primarily every organ system of the body, the most pernicious effects of Pb are on the central nervous system leading to cognitive and behavioral modification. Despite decades of research, the mechanisms responsible for Pb toxicity remain poorly understood. Recent work has suggested that Pb exposure may have consequences on chromosomal integrity as it was shown that Pb exposure leads to the generation of γH2Ax foci, a well-established biomarker for DNA double stranded break (DSB formation). As the chromosomal localization of γH2Ax foci plays an important role in determining the molecular mechanism responsible for their formation, we examined the localization of Pb-induced foci with respect to telomeres. Indeed, short or dysfunctional telomeres (uncapped or damaged telomeres) may be recognized as DSB by the DNA repair machinery, leading to "telomere-Induced Foci" (TIFs). In the current study, we show that while Pb exposure did not increase intra-chromosomal foci, it significantly induced TIFs, leading in some cases, to chromosomal abnormalities including telomere loss. The evidence suggests that these chromosomal abnormalities are likely due to perturbation of telomere replication, in particular on the lagging DNA strand. We propose a mechanism by which Pb exposure leads to the loss of telomere maintenance. As numerous studies have demonstrated a role for telomere maintenance in brain development and tissue homeostasis, our results suggest a possible mechanism for lead-induced neurotoxicity.

Corruption of human follicular B-lymphocyte trafficking by a B-cell superantigen.

Protein A (SpA) of Staphylococcus aureus is known to target the paratope of immunoglobulins expressing V(H)3 genes, and to delete marginal zone B cells and B-1a in vivo. We have discovered that SpA endows S. aureus with the potential to subvert B-cell trafficking in the host. We found that SpA, whose Fc-binding site has been inactivated, binds essentially to naïve B cells and induces a long-lasting decrease in CXCR4 expression and in B-cell chemotaxis to CXCL12. Competition experiments indicated that SpA does not interfere with binding of CXCR4 ligands and does not directly bind to CXCR4. This conclusion is strongly supported by the inability of SpA to modulate clathrin-mediated CXCR4 internalization, which contrasts with the potent effect of anti-immunoglobin M (IgM) antibodies. Microscopy and biochemical experiments confirmed that SpA binds to the surface IgM/IgD complex and induces its clathrin-dependent internalization. Concomitantly, the SpA-induced signaling leads to protein kinase C-dependent CXCR4 downmodulation, suggesting that SpA impairs the recycling of CXCR4, a postclathrin process that leads to either degradation into lysozomes or de novo expression at the cell surface. In addition to providing novel insight into disruption of B-cell trafficking by an infectious agent, our findings may have therapeutic implications. Because CXCR4 has been associated with cancer metastasis and with certain autoimmune diseases, SpA behaves as an evolutionary tailored highly specific, chemokine receptor inhibitor that may have value in addition to conventional cytotoxic therapy in patients with various malignancies and immune-mediated diseases.

[Individual response to ionising radiation: What predictive assay(s) to choose?]. / Réponse individuelle aux radiations ionisantes : quel(s) test(s) prédictif(s) choisir ?

Individual response to ionizing radiation is an important information required to apply an efficient radiotherapy treatment against tumour and to avoid any adverse effects in normal tissues. In 1981, Fertil and Malaise have demonstrated that the post-irradiation local tumor control determined in vivo is correlated with clonogenic cell survival assessed in vitro. Furthermore, these authors have reminded the relevance of the concept of intrinsic radiosensitivity that is specific to each individual organ (Fertil and Malaise, 1981) [1]. To date, since clonogenicity assays are too time-consuming and do not provide any other molecular information, a plethora of research groups have attempted to determine the molecular bases of intrinsic radiosensitivity in order to propose reliable and faster predictive assays. To this aim, several approaches have been developed. Notably, the recent revolution in genomic and proteomic technologies is providing a considerable number of data but their link with radiosensitivity still remains to be elucidated. On another hand, the systematic screening of some candidate genes potentially involved in the radiation response is highlighting the complexity of the molecular and cellular mechanisms of DNA damage sensoring and signalling and shows that an abnormal radiation response is not necessarily due to the impairment of one single protein. Finally, more modest approaches consisting in focusing some specific functions of DNA repair seem to provide more reliable clues to predict over-acute reactions caused by radiotherapy. In this review, we endeavoured to analyse the contributions of these major approaches to predict human radiosensitivity.

Cadmium inhibits non-homologous end-joining and over-activates the MRE11-dependent repair pathway.

Although cadmium still represents a public health problem and despite the fact that it has been classified as an IARC Group-I carcinogen, the molecular and cellular mechanisms responsible for the toxicity and the carcinogenicity of cadmium compounds are poorly known. Since unrepaired DNA double-strand breaks (DSBs) are considered to be key-lesions in cell lethality, and because misrepaired DSBs are a source of genomic instability leading to cancer proneness, the activity of the major DSB-repair pathways, i.e. non-homologous end-joining (NHEJ) and recombination, has been evaluated in human endothelial cells exposed to cadmium chloride and cadmium diacetate. Exposure to cadmium results in the production of DSBs a few hours after incubation. These breaks trigger the phosphorylation of H2AX proteins, which was used as an indirect measure of DSB in this study. The presence of cadmium in cells decreases the repair rate of X-ray-induced DSBs, suggesting an impact of cadmium upon the reparability of DSBs. Such an interpretation was consolidated by the finding that the DNA-PK kinase activity, essential for NHEJ, is affected by the presence of cadmium. These results suggest that the toxicity of cadmium compounds may be explained by the propagation of persistent DSBs. In parallel, the presence of cadmium was also associated with an over-activation of the MRE11-dependent repair pathway that may favour genomic instability. Altogether, our data provide a first example of the impact of cadmium upon DSB repair and signalling.

Induction and repair rate of DNA damage: a unified model for describing effects of external and internal irradiation and contamination with heavy metals.

DNA is a key-target for genotoxic stress. Hence, the knowledge of induction and repair rate of DNA damage are crucial to describe and predict the impact of stress situations. Unfortunately, DNA damage induction and repair rates are generally assessed separately whereas they act either concomitantly or transiently in living organisms. Furthermore, the interplay of induction and repair raises the question whether DNA repair adapts to respond to different amounts of DNA damage. In a previous report, we proposed a stochastic interpretation of the repair rate of the major radiation-induced DNA damage. We provided evidence that the repair rate of individual DNA damage is time-independent whereas that of a population of DNA damage is time-dependent (Foray, N., Charvet, A.-M., Duchemin, D., Favaudon, V., Lavalette, D., 2005. The repair rate of radiation-induced DNA damage: a stochastic interpretation based on the gamma function. J. Theor. Biol. 236, 448-458). Here, to better describe situations in which DNA damage induction and repair occur together, our biostatistical model was modified by the introduction of a DNA damage induction parameter. Theoretical and experimental data were compared and discussed by taking concrete experimental situations: X-rays irradiation at different dose-rates, internal irradiation with radioactive compound, contamination with heavy metal and detection of DNA damage by immunofluorescence. By assuming that DNA repair rate is invariant whatever the amount of DNA damage, our model provides good prediction of experimental data suggesting its relevance for the description of complex situations of co-toxicities.

Lead contamination results in late and slowly repairable DNA double-strand breaks and impacts upon the ATM-dependent signaling pathways.

Despite a considerable amount of data, evaluation of the potential genotoxicity and cancer proneness of lead compounds remains unclear, probably due to the plethora of experimental procedures, biological endpoints and cellular models used. In parallel, the understanding in DNA damage formation, repair and signaling has considerably progressed all along these last years, notably for DNA double-strand breaks (DSBs). Here, were examined DNA damage formation and repair in human cells exposed to lead nitrate (Pb(NO(3))(2)) and their consequences upon the ATM-dependent stress signaling, cell cycle progression and cell death. As observed with anti-pH2AX immunofluorescence, exposure to Pb(NO(3))(2) results in formation of late DSBs, that would not originate from conversion of nucleotide damage but likely by a direct production of single-strand breaks. Lead contamination inhibits non-homologous end-joining repair process by preventing the DNA-PK kinase activity whereas the MRE11-dependent repair pathway is exacerbated. Lead contamination triggers successive synchronization of cells in G2/M phase in which the RAD51-dependent homologous recombination was found to be activated. Altogether, our findings support that lead contamination generates late unrepairable DSBs that impact upon the ATM-dependent stress signaling pathway by favoring propagation of errors. Such findings should help to consider more carefully the biological action of lead compounds in the frame of public and occupational exposures.

Cadmium and T cell differentiation: limited impact in vivo but significant toxicity in fetal thymus organ culture.

DNA lesions, including oxydated bases, nucleotide damage and double strand breaks, are continuously produced in living cells and represent a threat for genetic stability. Highly conserved repair processes have evolved to maintain DNA integrity. Cadmium (Cd) is an environmental carcinogenic pollutant known to inactivate several proteins involved in DNA repair systems while at the same time creating an oxidative stress that can result in additional DNA lesions. Cd also has potent immunotoxic effects. DNA repair by non-homologous end joining (NHEJ) is absolutely required for T lymphocyte differentiation. In this study, we examined the impact of Cd on non-homologous end joining pathway by analyzing T cell development in the thymus of mice that received Cd-supplemented drinking water. In vivo, the absence of major alteration indicates that Cd does not affect NHEJ, despite its accumulation in the thymus. Cd contamination affects only a discrete population of developing thymocytes. However, these cells are functional as the cellular response observed in mice following gamma-radiation exposure is identical in treated and control mice. Furthermore, Cd diet did not perturb the redox status in thymocytes and more importantly did not generate significant DNA lesions in organs that accumulate the highest concentration of Cd. Our results show that in vivo, Cd does not affect NHEJ or base and nucleotide repair, and that Cd toxicity to T cells is rather linked to cell cycle perturbations.

Direct impact of inactivated HIV-1 virions on B lymphocyte subsets.

Although there is no convincing evidence that HIV infects primary B cells, marked changes in B cell responses have been described in HIV-1-infected subjects, including B cell repertoire perturbations, depression of B cell memory and paucity of CD5(+) B cells. As it is hard to assess the consequences of these in vitro and ex vivo observations in patients, the pathogenic mechanisms responsible for the B cell deficit are unclear, and direct and indirect effects of HIV-1 remain possible. To gain further insight into the impact of HIV-1 on the B cell compartment in vivo, we used XenoMouse mice, mice genetically engineered to express human antibodies with an absence of mouse antibody expression. In these transgenic animals, B cells expressing a virtually full human Ig repertoire develop, which allows investigation of the in vivo consequences of confronting B cells expressing human immunoglobulins with HIV-1. We found that soluble gp120 induced an inversion in the B-1a/B-1b cell ratios, without impacting B-2 cells or affecting substantially the T cell compartment. Virion treatment specifically and dramatically depressed B-1a cells, which represent the majority of B-1 cells in normal mice. The observed B cell changes were associated with a functional alteration of the humoral response to tetanus toxoid. Thus, the results reveal a capacity of HIV-1 to specifically impact a highly specialized B cell subpopulation. Because there is evidence that human IgM memory B cells are functionally equivalent to murine B-1a cells, our findings suggest that gp120 may have a direct deleting activity on B cell memory.

Staphylococcal protein a deletes B-1a and marginal zone B lymphocytes expressing human immunoglobulins: an immune evasion mechanism.

Protein A (SpA) of Staphylococcus aureus is endowed with the capacity to interact with the H chain variable region (V(H)) of human Abs and to target >40% of B lymphocytes. To investigate whether this property represents a virulence factor and to determine the in vivo consequences of the confrontation of SpA with B lymphocytes, we used transgenic mice expressing fully human Abs. We found that administration of soluble SpA reduces B-1a lymphocytes of the peritoneal cavity and marginal zone B lymphocytes of the spleen, resulting in a markedly deficient type 2 humoral response. Single-cell PCR analysis and sequencing of the Ab V(H) gene repertoire revealed a significant reduction of V(H)3+ marginal zone B cells. Since the two B lymphocyte subsets targeted are involved in innate immune functions, our data suggest that crippling of humoral immunity by S. aureus represents an immune evasion mechanism that may aggravate recurrent infections.

Effect of the B cell superantigen protein A from S. aureus on the early lupus disease of (NZBxNZW) F1 mice.

The (NZBxNZW) F(1) mouse develops a spontaneous autoimmune disease process with striking similarities to human systemic lupus erythematosus (SLE). In female (NZBxNZW) F(1) mice, the production of IgG antinuclear antibodies, including antibodies to double-stranded DNA (dsDNA), is associated with the development of a severe immune complex-mediated glomerulonephritis that results in death from renal failure in virtually all animals by 12 months of age. Since B-1 and marginal zone (MZ) cells represent a potential source of pathogenic antibodies and because B cell superantigens have been demonstrated to reduce B-1 and MZ cells in vivo, we tested the effect of repeated injections of the superantigen protein A (SpA) from S. aureus on the disease of this lupus model. We found that weekly intraperitoneal injections of SpA delay the progression of serum anti-DNA IgG and reduce proteinuria early in young female (NZBxNZW) F(1) mice. This superantigen also induced a specific depression in the numbers of peritoneal B-1 cells, as compared to mice treated with a control protein. These results support the role of B-1 cells in the development of the autoimmune disease in this mouse model and suggest that B cell superantigens may be useful in the management of autoimmune conditions.

B-lymphocytes, innate immunity, and autoimmunity.

Having evolved to generate a huge Ag-specific repertoire and to mount T cell-dependent responses and long-term memory, the B lymphocyte is a central player in the adaptive branch of immune defense. However, accumulating evidence indicates that B-1 cells of the peritoneal cavity and marginal zone (MZ) B cells of the spleen also can play innate-like immune functions. Their anatomical locations allow frequent Ag encounter. Secreting essentially germline-encoded, polyreactive Abs, and responding rapidly and vigorously to stimulation, these two B cell subsets have evolved to impart potentially protective responses. With their additional capacities to secrete factors that can directly mediate microbial destruction and to express Toll-like receptors (TLR), B cells provide an important link between the innate and adaptive branches of the immune system. Currently, the relevance of these innate-like B cells to the pathogenesis of autoimmune disease is the focus of investigation. In experimental models of autoimmunity, the sequestration of autoreactive B cells in the MZ has been proposed to be essential for the maintenance of self-tolerance. The low activation threshold of MZ B cells makes them particularly reactive to high loads and/or altered self-Ags, potentially exacerbating autoimmune disease. Their expansion in autoimmune models and their association with autoantibody secretion indicate that they may participate in tissue damage. The demonstration that B cell depletion therapies may represent a highly beneficial therapeutic goal in autoimmune disorders suggests that specific elimination of B-1 and MZ B cells may represent a more efficient immunointervention strategy in systemic autoimmunity.

Specific in vivo deletion of B-cell subpopulations expressing human immunoglobulins by the B-cell superantigen protein L.

Some pathogens have evolved to produce proteins, called B-cell superantigens, that can interact with human immunoglobulin variable regions, independently of the combining site, and activate B lymphocytes that express the target immunoglobulins. However, the in vivo consequences of these interactions on human B-cell numbers and function are largely unknown. Using transgenic mice expressing fully human immunoglobulins, we studied the consequences of in vivo exposure of protein L of Peptostreptococcus magnus with human immunoglobulins. In the mature pool of B cells, protein L exposure resulted in a specific reduction of splenic marginal-zone B cells and peritoneal B-1 cells. Splenic B cells exhibited a skewed light-chain repertoire consistent with the capacity of protein L to bind specific kappa gene products. Remarkably, these two B-cell subsets are implicated in innate B-cell immunity, allowing rapid clearance of pathogens. Thus, the present study reveals a novel mechanism that may be used by some infectious agents to subvert a first line of the host's immune defense.

Down-modulation of the antigen receptor by a superantigen for human B cells.

B cell superantigens (SAgs) have been implicated in human diseases by demonstrating non-clonotypic expansion of B cells bearing certain immunoglobulin variable region genes. One possibility is that, during infection with microorganisms secreting SAgs, these potent molecules might modulate BcR expression. To test this hypothesis, we investigated the potential effects of a SAg, protein L from Peptostreptococcus magnus, on antigen B cell receptor (BcR) surface expression in vitro. Using fluorescence microscopy, we found that this SAg induced down-regulation of BcR expression. This effect was time-, dose-, and temperature-dependent, and shedding of cell surface IgM molecules into the culture supernatant was not detected. These data demonstrate that SAg-mediated down-regulation of the BcR expression occurs primarily as a result of BcR internalization. In addition, two specific inhibitors of protein tyrosine kinases were found to retard the BcR modulation on the cell surface and inhibit SAg-induced receptor internalization, showing that tyrosine phosphorylation is required for subsequent internalization of mIg-ligand complexes. The down-modulation of BcR expression may have pathological consequences in patients infected with microorganisms secreting SAgs.