Transient Alteration of Cellular Redox Buffering before Irradiation Triggers Apoptosis in Head and Neck Carcinoma Stem and Non-Stem Cells

Background: Head and neck squamous cell carcinoma (HNSCC) is an aggressive and recurrent malignancy owing to intrinsic radioresistance and lack of induction of apoptosis. The major focus of this work was to design a transient glutathione depleting strategy during the course of irradiation of HNSCC in order to overcome their radioresistance associated with redox adaptation. Methodology/Principal Findings: Treatment of SQ20B cells with dimethylfumarate (DMF), a GSH-depleting agent, and L-Buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis 4 h before a 10 Gy irradiation led to the lowering of the endogenous GSH content to less than 10 % of that in control cells and to the triggering of radiation-induced apoptotic cell death. The sequence of biochemical events after GSH depletion and irradiation included ASK-1 followed by JNK activation which resulted in the triggering of the intrinsic apoptotic pathway through Bax translocation to mitochondria. Conclusions: This transient GSH depletion also triggered radiation-induced cell death in SQ20B stem cells, a key event to overcome locoregional recurrence of HNSCC. Finally, our in vivo data highlight the relevance for further clinical trials o

Idiopathic environmental intolerance attributed to electromagnetic fields (IEI-EMF) and sleep disruption : Melatonin Assessment in saliva and urine

International audienceSleep disruption is frequently reported by people with idiopathic environmental intolerance attributed to electromagnetic fields or IEI-EMF. The physiopathology of this association is not clear. We aimed to evaluate whether melatonin level was affected in IEI-EMFs patients. Melatonin levels in saliva and urine were quantified by immunoassay techniques in a session without intentional exposure to EMF. Sleep quality was assessed by questionnaires.Significant differences in PSQI and ESS scores between compared groups were observed. Likewise, a higher proportion of pathological sleep for ESS was found in IEI-EMF group. Results indicate that melatonin is not associated with different sleep troubles reported by IEI-MFs patients

An approach to evaluate which in vitro model and exposure method is more predictive for in vivo biological responses

The growing utilization of nanomaterials (NMs) in nanotechnology products lead to a potential increase of exposure, thus raising concerns about workers and public’s health risks. The major route of exposure is inhalation, but so far occupational and environmental atmosphere are not well characterized in terms of NMs. Despite the lack of epidemiological data on the relation between exposure to NMs and human health effects, their potential toxicity has been studied on cell cultures and animal models. Among these studies, most results are from in vitro experimentations due to the difficulty to perform in vivo studies for the enormous number of existing NMs and the necessity to reduce number of animals used in experimentations (3R rules). Nevertheless, results from animal experimentations remain the most reliable. Even if, pushed by the necessity to reduce the number of animals used in experimentation, new in vitro models and exposure methods are and have been developed, suggesting the more and more relevant alternatives to animal experimentation. Thus, many studies show that newly developed co-culture or 3D in vitro models have different nanotoxicity responses compared to classical mono-culture models. Moreover, studies using new devices allowing exposure of cells to aerosols of NMs show different nanotoxicity responses compared to exposure to suspensions of NMs. However, which of these models and exposure methods is more predictive for in vivo responses is yet to be defined. In order to better define which in vitro model and which exposure method is more predictive for in vivo pulmonary nanotoxicity data, three different methodologies will be implemented

Inflammation system and immunological characterization of patients suffering from electrohypersensitivity

International audienceElectrohypersensitivity (EHS) is characterized by a variety of atypical symptoms attributed to EMF exposure. Diagnostic markers are lacking and research of these biomarkers could greatly help understanding this syndrome. The present study aimed to look to the pattern of some selected biological markers of immunological system in EHS individuals. In this regard, we compared levels of immunoglobulin A, neopterin and C Reactive Protein between patient with EHS and their matched control group. In our study, we failed to show a strong marker from immunological system despite a difference in participants according to the EHS duration for neopterin (difference between subgroups between short-term and long term affected participants)

Disturbed sleep in individuals with idiopathic environmental intolerance attributed to electromagnetic fields (IEI-EMF) : Melatonin assessment as a biological marker

Individuals who suffer from idiopathic environmental intolerance attributed to electromagnetic fields (IEI-EMF) complain of a variety of adverse health effects. Troubled sleep remains a recurrent and common symptom in IEI-EMF individuals. Melatonin, a circadian hormone, plays a major role in the sleep process. In this study, we compared levels of melatonin between a sensitive group (IEI-EMF, n=30) and a non-sensitive control group (non IEI-EMF, n=25) without exposure to electromagnetic sources. Three questionnaires were used to evaluate the subjective quality and sleep quantity: the Epworth Sleepiness Scale, the Pittsburgh Sleep Quality Index and the Spiegel Sleep Inventory. Melatonin was quantified in saliva and its major metabolite 6-sulfatoxymelatonin (aMT6s) in urine. Melatonin levels were compared by a two-way analysis of variance at various times between the control and IEI-EMF group. Despite significantly different sleep scores between the two groups, with a lower score in the IEI-EMF group (P0.05) and urine aMT6s (P>0.05)

Oxidative stress pathways involved in cytotoxicity and genotoxicity of titanium dioxide (TiO2) nanoparticles on cells constitutive of alveolo-capillary barrier in vitro

International audienceThe health risks of nanoparticles remain a serious concern given their prevalence from industrial and domestic use. The primary route of titanium dioxide nanoparticle exposure is inhalation. The extent to which nanoparticles contribute to cellular toxicity is known to associate induction of oxidative stress. To investigate this problem further, the effect of titanium dioxide nanoparticles was examined on cell lines representative of alveolo-capillary barrier. The present study showed that all nanoparticle-exposed cell lines displayed ROS generation. Macrophage-like THP-1 and HPMEC-ST1.6R microvascular cells were sensitive to endogenous redox changes and underwent apoptosis, but not alveolar epithelial A549 cells. Genotoxic potential of titanium dioxide nanoparticles was investigated using the activation of γH2AX, activation of DNA repair proteins and cell cycle arrest. In the sensitive cell lines, DNA damage was persistent and activation of DNA repair pathways was observed. Moreover, western blot analysis showed that specific pathways associated with cellular stress response were activated concomitantly with DNA repair or apoptosis. Nanoparticles-induced oxidative stress is finally signal transducer for further physiological effects including genotoxicity and cytotoxicity. Within activated pathways, HSP27 and SAPK/JNK proteins appeared as potential biomarkers of intracellular stress and of sensitivity to endogenous redox changes, respectively, enabling to predict cell behavior

Involvement of the MAPK pathway in the triggering of apoptosis after transient intracellular GSH depletion before irradiation.

<p>SQ20B cells were treated with 100 µM DMF and 100 µM BSO for 4 h whereas 10 µM SP600125, a specific JNK inhibitor, was added 1 h before irradiation in the cell culture medium before irradiation. The drugs were then removed by washing with fresh medium. After different points in time after irradiation, cells were harvested and the extracted proteins submitted to Western blot analysis. Panel A: Western blot analysis of phosphorylated Erk, p38 MAPK, and GAPDH. Panel B: Western blot analysis of inhibition of JNK phosphorylation by SP600125. Panel C: Western blot analysis of phosphorylated JNK and GAPDH. Panel D shows the consequence of JNK inhibition in terms of apoptosis estimated by flow cytometry through the total caspase activity (left) and the % of cells in sub-G1 phase (right) measurement 72 h after irradiation. Results are expressed as mean ± S.D. for three different experiments. The statistical significance is expressed as ***, p<0.001 <i>versus</i> 10 Gy only.</p

Activation of Ask1 upstream of the MAPK pathway in irradiated GSH-depleted SQ20B cells.

<p>SQ20B cells were treated with 100 µM DMF and 100 µM BSO for 4 h before irradiation. The drugs were then removed by washing with fresh medium. At different times post irradiation, cells were harvested and the extracted proteins submitted to Western blot analysis. Panel A: levels of phosphorylated Ask1 Panel B: levels of ASK-1 after specific siRNA transfection and downstream phosphorylation of JNK. Panel C: control of apoptosis through the measurement of total caspase activity 72 h after transfection of irradiated GSH-depleted cells with Ask1 SiRNA. Results are expressed as mean ± S.D. for three different experiments. The statistical significance is expressed as <b>***</b>, p<0.001 <i>versus</i> 10 Gy only.</p

Combined treatment of DMF + BSO with irradiation enhances the survival of mice and inhibits tumor growth without apparent cytoxicity.

<p>Panel A shows the efficiency of a single intratumoural injection of 32 mg/kg DMF and 8 mg/kg BSO on the depletion of GSH within the tumour. Panel B shows the relative development of tumour size after combined DMF + BSO treatment each day whether or not associated with an irradiation dose of 20 Gy (4 Gy×5 days). The statistical significance is expressed as *, p<0.05, **, p<0.01 between treated and irradiated tumours versus irradiated tumours. Panel C shows the body weight monitoring of mice after the combined DMF + BSO treatment whether or not associated with an irradiation dose of 20 Gy (4 Gy×5 days). Panel D shows the Kaplan-Meyer survival curves representing the percentage of mice alive at the indicated points in time for each group of the experiment. Panel E shows the detection of apoptosis by TUNEL staining on paraffin-embedded tumor sections.</p

Depletion of endogenous glutathione content before γ-ray exposure triggers radiation-induced intrinsic apoptosis in SQ20B cell line.

<p>SQ20B cells were treated with 100 µM DMF and 100 µM BSO for 4 h before irradiation. The drugs were then removed by washing with fresh medium. Total caspase activity and the percentage of cells in the sub-G1 phase were determined by flow cytometry, respectively after VAD-FMK-FITC (A) and propidium iodide (B) staining. Panel C shows the nuclear morphology of cells by DAPI staining 72 h post irradiation. Panels D and E show the mitochondrial alteration after JC-1 staining through the measurement by flow cytometry of the transmembrane potential (A) and after hydro-ethidine staining to measure the reactive oxygen species generated by respiratory chain (B). Results are expressed as mean ± S.D. for three different experiments in triplicate. The statistical significance is expressed as **, p<0.01 and ***, p<0.001 <i>versus</i> 10 Gy only.</p