X-rays-Induced Bystander Effect Consists in the Formation of DNA Breaks in a Calcium-Dependent Manner: Influence of the Experimental Procedure and the Individual Factor.

Radiation-induced bystander effects (RIBE) describe the biological events occurring in non-targeted cells in the vicinity of irradiated ones. Various experimental procedures have been used to investigate RIBE. Interestingly, most micro-irradiation experiments have been performed with alpha particles, whereas most medium transfers have been done with X-rays. With their high fluence, synchrotron X-rays represent a real opportunity to study RIBE by applying these two approaches with the same radiation type. The RIBE induced in human fibroblasts by the medium transfer approach resulted in a generation of DNA double-strand breaks (DSB) occurring from 10 min to 4 h post-irradiation. Such RIBE was found to be dependent on dose and on the number of donor cells. The RIBE induced with the micro-irradiation approach produced DSB with the same temporal occurrence. Culture media containing high concentrations of phosphates were found to inhibit RIBE, while media rich in calcium increased it. The contribution of the RIBE to the biological dose was evaluated after synchrotron X-rays, media transfer, micro-irradiation, and 6 MeV photon irradiation mimicking a standard radiotherapy session: the RIBE may represent less than 1%, about 5%, and about 20% of the initial dose, respectively. However, RIBE may result in beneficial or otherwise deleterious effects in surrounding tissues according to their radiosensitivity status and their capacity to release Ca2+ ions in response to radiation.

Impact of washing parameters on bacterial filtration efficiency and breathability of community and medical facemasks.

Can medical face masks be replaced by reusable community face masks with similar performance? The influence of the number of wash cycles, the wash temperature and the use of detergent was evaluated on the performance of one medical face masks (MFM) and ten community face masks (CFM). The performance of the new and washed masks was characterized from the bacterial filtration efficiency (BFE) and the differential pressure (DP). The tests on the new masks showed that the MFM had always better BFE than CFMs. Although two of the CFMs showed a BFE value exceeding 95%, only one can be classified as type I MFM based on both BFE and DP requirements. The influence of the washing parameters was investigated on the MFM and these two CMFs with excellent BFE properties. The parameters had no effect on the BFE of CFMs whilst the MFM exhibited a loss in efficiency when washed with detergent. The DP of masks were not impacted by the washing. The results clearly show that even though a compromise has to be made between the BFE and breathability, it seems possible to manufacture CFMs with performances similar to a type I MFM, without achieving type II requirements.

Comparison of bacterial filtration efficiency vs. particle filtration efficiency to assess the performance of non-medical face masks.

As a result of the current COVID-19 pandemic, the use of facemasks has become commonplace. The performance of medical facemasks is assessed using Bacterial Filtration Efficiency (BFE) tests. However, as BFE tests, require specific expertise and equipment and are time-consuming, the performance of non-medical facemasks is assessed with non-biological Particle Filtration Efficiency (PFE) tests which are comparatively easier to implement. It is necessary to better understand the possible correlations between BFE and PFE to be able to compare the performances of the different types of masks (medical vs. non-medical). In this study BFE results obtained in accordance with the standard EN 14683 are compared to the results of PFE from a reference test protocol defined by AFNOR SPEC S76-001 with the aim to determine if BFE could be predicted from PFE. Our results showed a correlation between PFE and BFE. It was also observed that PFE values were higher than BFE and this was attributed to the difference in particle size distribution considered for efficiency calculation. In order to properly compare these test protocols for a better deduction, it would be interesting to compare the filtration efficiency for a similar granulometric range.

Reuse of medical face masks in domestic and community settings without sacrificing safety: Ecological and economical lessons from the Covid-19 pandemic.

The need for personal protective equipment increased exponentially in response to the Covid-19 pandemic. To cope with the mask shortage during springtime 2020, a French consortium was created to find ways to reuse medical and respiratory masks in healthcare departments. The consortium addressed the complex context of the balance between cleaning medical masks in a way that maintains their safety and functionality for reuse, with the environmental advantage to manage medical disposable waste despite the current mask designation as single-use by the regulatory frameworks. We report a Workflow that provides a quantitative basis to determine the safety and efficacy of a medical mask that is decontaminated for reuse. The type IIR polypropylene medical masks can be washed up to 10 times, washed 5 times and autoclaved 5 times, or washed then sterilized with radiations or ethylene oxide, without any degradation of their filtration or breathability properties. There is loss of the anti-projection properties. The Workflow rendered the medical masks to comply to the AFNOR S76-001 standard as "type 1 non-sanitory usage masks". This qualification gives a legal status to the Workflow-treated masks and allows recommendation for the reuse of washed medical masks by the general population, with the significant public health advantage of providing better protection than cloth-tissue masks. Additionally, such a legal status provides a basis to perform a clinical trial to test the masks in real conditions, with full compliance with EN 14683 norm, for collective reuse. The rational reuse of medical mask and their end-of-life management is critical, particularly in pandemic periods when decisive turns can be taken. The reuse of masks in the general population, in industries, or in hospitals (but not for surgery) has significant advantages for the management of waste without degrading the safety of individuals wearing reused masks.

Dust and microbial filtration performance of regular and antimicrobial HVAC filters in realistic conditions.

Two polypropylene HVAC electret filters: a regular filter and an antimicrobial filter containing zinc pyrithione (ZPT), were compared for filtration performance. The study was conducted over 7 months in realistic conditions with semi-urban outdoor air. Several parameters were monitored over the study period: the average temperature was about 20 °C and relative humidity about 60%, the average inlet concentration of cultivable microorganisms was 50 CFU m-3, the average inlet concentration of particles was 10 µg m-3, the filter pressure drop increased moderately by about 30 Pa, and the particle collection efficiency of soda fluorescein (median diameter 0.35 µm) decreased in the first half of the study period by about 30% and then stabilized. The microbial concentration on the filters was quantified every 2 months using an innovative methodology based on media coupons in conjunction with microorganism quantification by CFU counting, with 5 culture media favorable to bacteria and/or fungi growth. The microbial concentrations on the filters were between 100 and 2000 CFU cm-2. The antimicrobial effect of zinc pyrithione was confirmed by the fungi cultivated with DRBC agar: no effects in the level of filter clogging were revealed in the range studied. The high statistical deviation in the results regarding the inhibiting effect of zinc pyrithione on bacteria prevents any conclusion.

Adenovirus behavior in air handling unit fiberglass filters.

Viral aerosols can lead to respiratory viral infections with high infectivity. About 90% of people's time is spent in closed environments. A few studies have pointed out that the ventilation systems in air handling units (AHUs) that treat and transmit a new synthetic clean and conditioned environment can also spread and transport viral particles in buildings. The aim of this work is to study the characterization of adenovirus, a DNA non-enveloped respiratory virus, on the F7 fiberglass filter used in AHUs. In this study, an experimental setup simulating an AHU was used. The SYBR® QPCR, Electrical Low-Pressure Impactor (ELPI™) and Scanning Mobility Particle Sizer (SMPS™) were used to detect, measure and characterize the aerosolized adenovirus solution. The characterization results showed that the nebulized adenovirus could be aerosolized in different forms associated or not with cell debris and proteins. The quantification and level of infectivity of adenovirus demonstrated that viruses passed through filters and remained infectious up- and downstream of the system during the 25 min of aerosolization. This study showed that AHUs should be considered an indoor source of viral contamination.

DNA breaks induced by iodine-containing contrast medium in radiodiagnostics: a problem of tungsten?

Iodine-containing contrast media (ICM) are extensively used to improve image quality and information content in x-ray-based examinations, particularly in computed tomography (CT). In parallel, there is increasing evidence that the use of ICM during CT sessions is associated with deoxyribonucleic acid (DNA) breaks that may influence the estimation of the risks linked to x-ray exposure. Why has iodine been preferred to any other heavy elements to enhance contrast in radiodiagnostics? How to understand such DNA breaks effect? We searched for the answers in the early times of x-ray medical use. It appeared that the maximal ratio between the relative iodine and water mass energy absorption coefficients is reached in the range of 40-60 keV, which defines the energy range in which the dose is preferentially absorbed by ICM. This range does not correspond to the K-edge of iodine but to that of tungsten, the major component of the x-ray tube anode of CT scanners. At such energy, radiolysis of the ICM produces sodium or potassium iodide that prevents a normal DNA breaks repair and influences the individual response to x-ray low-dose. Both contrast enhancement and DNA breaks effect may therefore be caused by tungsten of the anodes of x-ray tubes.

The Fate of Mengovirus on Fiberglass Filter of Air Handling Units.

One of the most important topics that occupy public health problems is the air quality. That is the reason why mechanical ventilation and air handling units (AHU) were imposed by the different governments in the collective or individual buildings. Many buildings create an artificial climate using heating, ventilation, and air-conditioning systems. Among the existing aerosols in the indoor air, we can distinguish the bioaerosol with biological nature such as bacteria, viruses, and fungi. Respiratory viral infections are a major public health issue because they are usually highly infective. We spend about 90% of our time in closed environments such as homes, workplaces, or transport. Some studies have shown that AHU contribute to the spread and transport of viral particles within buildings. The aim of this work is to study the characterization of viral bioaerosols in indoor environments and to understand the fate of mengovirus eukaryote RNA virus on glass fiber filter F7 used in AHU. In this study, a set-up close to reality of AHU system was used. The mengovirus aerosolized was characterized and measured with the electrical low pressure impact and the scanner mobility particle size and detected with RT-qPCR. The results about quantification and the level of infectivity of mengovirus on the filter and in the biosampler showed that mengovirus can pass through the filter and remain infectious upstream and downstream the system. Regarding the virus infectivity on the filter under a constant air flow, mengovirus was remained infectious during 10 h after aerosolization.

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).

Specific molecular and cellular events induced by irradiated X-ray photoactivatable drugs raise the problem of co-toxicities: particular consequences for anti-cancer synchrotron therapy.

Synchrotrons are capable of producing intense low-energy X-rays that enable the photoactivation of high-Z elements. Photoactivation therapy (PAT) consists of loading tumors with photoactivatable drugs and thereafter irradiating them at an energy, generally close to the K-edge of the element, that enhances the photoelectric effect. To date, three major photoactivatable elements are used in PAT: platinum (cisplatin and carboplatin), iodine (iodinated contrast agents and iododeoxyuridine) and gadolinium (motexafin gadolinium). However, the molecular and cellular events specific to PAT and the radiobiological properties of these photoactivatable drugs are still misknown. Here, it is examined how standard and synchrotron X-rays combined with photoactivatable drugs impact on the cellular response of human endothelial cells. These findings suggest that the radiolysis products of the photoactivatable drugs may participate in the synergetic effects of PAT by increasing the severity of radiation-induced DNA double-strand breaks. Interestingly, subpopulation of highly damaged cells was found to be a cellular pattern specific to PAT. The data show that the efficiency of emerging anti-cancer modalities involving synchrotron photoactivation strongly depends on the choice of photoactivatable drugs, and important series of experiments are required to secure their clinical transfer before applying to humans.

[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.

In vitro and in vivo optimization of an anti-glioma modality based on synchrotron X-ray photoactivation of platinated drugs.

For the past 5 years, a radio-chemotherapy approach based on the photoactivation of platinum atoms (PAT-Plat) consisting of treating tumors with platinated compounds and irradiating them above the platinum K edge (78.4 keV) has been developed at the European Synchrotron Radiation Facility (Grenoble, France). Compared to other preclinical modalities, PAT-Plat provides the highest survivals of rats bearing the rodent F98 glioma. However, further investigations are required to optimize its efficiency and to allow its clinical application. Here we examined in vitro and in vivo whether monochromatic X rays are more efficient than high-energy photons in producing the PAT-Plat effect by measuring DNA double-strand breaks (DSBs) and survival of glioma-bearing rats and whether an increase in the platinum concentration in the tumor results in increased rat survival. DSBs were assessed by pulsed-field gel electrophoresis with different DNA fragment migration programs and with gamma-H2AX immunofluorescence. In vivo, F98 glioma cells were injected intracerebrally, treated with a single intracranial injection of cisplatin or carboplatin 13 days after tumor implantation, and irradiated the day after with 78.8 keV X rays or 6 MV photons. Our results indicate that 78.8 keV X rays are more efficient than high-energy photons at producing the PAT-Plat effect. At low concentrations, cisplatin is more efficient than carboplatin; this is likely due to more efficient DNA binding and DSB repair inhibition. High concentrations of carboplatin inside tumors do not necessarily lead to protracted survival of rats. The therapeutic benefit of anti-glioma synchrotron strategies appears to be correlated with the percentage of unrepaired DSBs but not with the number of DSBs induced.

Mapping the zonal organization of tumor perfusion and permeability in a rat glioma model by using dynamic contrast-enhanced synchrotron radiation CT.

PURPOSE: To depict and analyze in vivo the tumor zone organization of C6 gliomas depicted on quantitative parametric maps obtained with dynamic contrast material-enhanced synchrotron radiation computed tomography (CT) in a tightly controlled data-processing protocol. MATERIALS AND METHODS: Animal use was compliant with official French guidelines and was assessed by the local Internal Evaluation Committee for Animal Welfare and Rights. Fifteen Wistar rats with orthotopically implanted gliomas were studied at monochromatic synchrotron radiation CT after receiving a bolus injection of contrast material. The iodine concentration maps were analyzed by using a compartmental model selected from among a package of models. Choice of model and assessment of the relevance of the model were guided by quality criteria. Tissue blood flow (F(T)), tissue blood volume fraction (V(T)), permeability-surface area product (PS), artery-to-tissue delay (D(A-T)), and vascular mean transit time (MTT) maps were obtained. Parametric map findings were compared with histologic findings. Local regions of interest were selected in the contralateral hemisphere and in several tumor structures to characterize the tumor microvasculature. Differences in parameter values between regions were assessed with the Wilcoxon method. RESULTS: Whole-tumor parameters were expressed as means +/- standard errors of the mean: Mean F(T), V(T), PS, and D(A-T) values and MTT were 61.4 mL/min/100 mL +/- 15.3, 2.4% +/- 0.4, 0.37 mL/min/100 mL +/- 0.11, 0.24 second +/- 0.06; and 3.9 seconds +/- 0.83, respectively. MTT and mean PS were significantly lower (P < .01) in the normal contralateral tissue: 1.10 seconds +/- 0.06 and < or = 10(-5) mL/min/100 mL, respectively. Tumor regions were characterized by significantly different (P < .05) F(T) and V(T) pairs: 108 mL/min/100 mL and 3.66%, respectively, at the periphery; 45.9 mL/min/100 mL and 1.91%, respectively, in the intermediate zone; 5.1 mL/min/100 mL and 0.42%, respectively, in the center; and 210 mL/min/100 mL and 6.82%, respectively, in the maximal value region. CONCLUSION: Fine mapping of the glioma microcirculation is feasible with dynamic contrast-enhanced synchrotron radiation CT performed with well-controlled analytic protocols. SUPPLEMENTAL MATERIAL: http://radiology.rsnajnls.org/cgi/content/full/2501071929/DC1.

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.

DNA double-strand break repair defects in syndromes associated with acute radiation response: at least two different assays to predict intrinsic radiosensitivity?

PURPOSE: Human diseases associated with acute radiation responses are rare genetic disorders with common clinical and biological features including radiosensitivity, genomic instability, chromosomal aberrations, and frequently immunodeficiency. To determine what molecular assays are predictive of cellular radiosensitivity whatever the genes mutations, the existence of a quantitative correlation between cellular radiosensitivity and unrepaired DNA double-strand breaks (DSB) repair defects was examined in a collection of 40 human fibroblasts representing 8 different syndromes. MATERIALS AND METHODS: A number of techniques such as pulsed-field gel electrophoresis, plasmid assay and immunofluorescence with antibodies against MRE11, MDC1, 53BP1 and phosphorylated forms of H2AX, DNA-PK were applied systematically. RESULTS AND CONCLUSIONS: Survival fraction at 2 Gy was found to be inversely proportional to the amount of unrepaired DSB, whatever the genes mutations and the assay applied. However, no single assay discriminates the full range of human radiosensitivity. Particularly, nuclear foci formed by the phosphorylation of H2AX do not predict well moderate radiosensitivities. Our findings suggest the existence of an ATM-dependent interplay between the activation of DNA-PK and MRE11. A classification of diseases according their cellular radiosensitivity, their molecular response to radiation and the functional assays permitting their evaluation is proposed.

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.

Molecular and cellular response of the most extensively used rodent glioma models to radiation and/or cisplatin.

Purpose Anti-glioma strategies are generally based on trials involving rodent models whose choice remains based on proliferative capacity and availability. Recently, our group obtained the most protracted survival of rats bearing F98 gliomas by combining synchrotron X-rays and intracerebral cisplatin injection (Biston et al., Cancer Res, 64:2317-2323, 2004). The response to such treatment was suggested to be dependent on BRCA1, a tumour suppressor known to be involved in the response to radiation and cisplatin. In order to verify the impact of BRCA1 functionality upon success of anti-glioma trials, radiobiological features and BRCA1-dependent stress signalling were investigated in the most extensively used rodent glioma models. Methods Cell death pathways, cell cycle arrests, DNA repair and stress signalling were evaluated in response to radiation and cisplatin in C6, 9L and F98 models. Results Rodent glioma models showed a large spectrum of cellular radiation response. Surprisingly, BRCA1 was found to be functionally impaired in C6 and F98 favouring genomic instability, tumour heterogeneity and tolerance of unrepaired DNA damage. Significance Our findings strengthened the importance of the choice of the glioma model on genetic and radiobiological bases, inasmuch as all these rat glioma models are induced by nitrosourea-mediated mutagenesis that may favour specific gene mutations. Particularly, BRCA1 status may condition the response to anti-glioma treatments. Furthermore, since BRCA1 acts as a tumour suppressor in a number of malignancies, our findings raise also the question of the implication of BRCA1 in brain tumours formation.

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.

Consequences of the bleed-through phenomenon in immunofluorescence of proteins forming radiation-induced nuclear foci.

PURPOSE: By allowing the visualization of the proteins inside cells, the immunofluorescence technique has revolutionized our view of events that follow radiation response. Particularly, the formation of nuclear foci, their kinetic of appearance and disappearance, and the association-dissociation of protein partners are useful endpoints to better understand the effects of ionizing radiation. Recently, the technique based on the phosphorylation of the histone 2A family, member X (H2AX) has generated a plethora of reports concerning the interaction between the major proteins involved in DNA repair and stress signaling pathways. However, some unavoidable overlaps of excitation and emission wavelength spectra (the so-called bleed-through phenomenon) of the available fluorescent markers are still generating discrepancies and misinterpretations in the choreography of DNA damage response. Biases are particularly strong with the fluorescein isothiocyanate (FITC)-rhodamine couple, tetramethyl rhodamine iso-thiocyanate (TRITC), the most extensively used markers. METHOD AND RESULTS: Here, two representative examples of biased co-immunofluorescence with pH2AX proteins that form radiation-induced nuclear foci or not are presented. A brief review of literature points out differences in kinetic of appearance and association-dissociation of radiation-induced pH2AX and MRE11 foci. CONCLUSION: Through this report, we would like authors to consider more carefully protein co-localizations by performing systematically, before any co-immunofluorescence, immunofluorescence of each protein separately to avoid bleed-through artifacts.