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The study presented here aimed to assess the ability of Desulfovibrio fairfieldensis bacteria to adhere to and form biofilm on the structure of titanium used in implants. D. fairfieldensis was found in the periodontal pockets in the oral environment, indicating that these bacteria can colonize the implant-bone interface and consequently cause bone infection and implant corrosion. Plates of implantable titanium, of which surfaces were characterized by scanning electronic microscopy and Raman spectroscopy, were immersed in several suspensions of D. fairfieldensis cells containing potassium nitrate on the one hand, and artificial saliva or a sulfato-reducing bacterial culture medium on the other hand. Following various incubation timepoints bacteria were counted in different media to determine their doubling time and titanium samples are checked for and determination of the total number of adhered bacteria and biofilm formation. Adhesion of D. fairfieldensis on titanium occurs at rates ranging from 2.105 to 4.6.106 bacteria h-1cm-2 in the first 18 h of incubation on both native and implantable titanium samples. Following that time, the increase in cell numbers per h and cm2 is attributed to growth in adhered bacteria. After 30 days of incubation in a nutrient-rich medium, dense biofilms are observed forming on the implant surface where bacteria became embedded in a layer of polymers D. fairfieldensis is able of adhering to an implantable titanium surface in order to form a biofilm. Further studies are still necessary, however, to assess whether this adhesion still occurs in an environment containing saliva or serum proteins that may alter the implant surface.

nvestigations on adverse biological effects of nanoparticles (NP) are performed usually either in vivo on rodents or in vitro under submerged conditions where NP are in suspension into cell culture media. However, sedimentation of NP in vitro is a continuous process and to assess the exact deposited cellular dose remains difficult, as the cellular internal dose is a function of time. Moreover, the cellular responses to NP under submerged culture conditions or by exposing rodents by nose-only to NP aerosols might differ from those observed at physiological settings at the air-liquid interface (ALI). Rat alveolar NR8383 macrophages were exposed to aerosols at the air-liquid interface. We studied TiO2 NM105, a mixture of anatase and rutile. NR8383 cells were exposed to a single dose of 3.0 cm2/cm2 of TiO2 aerosol. Following RNA extraction, transcriptome allowing full coverage of the rat genome was performed, and differentially expressed genes were retrieved. Their products were analyzed for functions and interaction with String DB. Only 126 genes were differentially expressed and 98 were recognized by String DB and give us the gene expression signature of exposed rat alveolar NR8383 macrophages. Among them, 13 display relationships at a high confidence level and the ten most differentially expressed compared to unexposed cells were: Chac1, Ccl4, Zfp668, Fam129b, Nab2, Txnip, Id1, Cdc42ep3, Dusp6 and Myc, ranked from the most overexpressed to the most under-expressed. Some of them were previously described as over or under-expressed in NP exposed cell systems. We validated in our laboratory an easy-to-use device and a physiological relevant paradigm for studying the effects of cell exposure to TiO2. Ccl4 gene expression seems to be a positive marker of exposure evidenced as well as in vivo or in both in vitro conditions.

Functionalized multi-walled carbon nanotubes (MWCNT) have become the focus of increased research interest, particularly in their application as tools in different areas, such as the biomedical field. Despite the benefits associated with functionalization of MWCNT, particularly in overcoming issues relating to solubility, several studies have demonstrated that these functionalized nanoparticles display different toxicity profiles. For this study, we aim to compare NR8383 cells responses to three well-characterized MWCNT with varying functional groups. This study employed cytotoxicity assays, transcriptomics and proteomics to assess their toxicity using NR8383 rat alveolar macrophages as an in vitro model. The study findings indicated that all MWCNT altered ribosomal protein translation, cytoskeleton arrangement and induced pro-inflammatory response. Only functionalized MWCNT alter mTOR signaling pathway in conjunction with increased Lamtor gene expression. Furthermore, the type of functionalization was also important, with cationic MWCNT activating the transcription factor EB and inducing autophagy while the anionic MWCNT altering eukaryotic translation initiation factor 4 (EIF4) and phosphoprotein 70 ribosomal protein S6 kinase (p70S6K) signaling pathway as well as upregulation Tlr2 gene expression. This study proposes that MWCNT toxicity mechanisms are functionalization dependent and provides evidence that inflammatory response is a key event of carbon nanotubes toxicity.

S-nitrosoglutathione (GSNO) is a potential therapeutic for infectious disease treatment because of its pivotal role in macrophage-mediated inflammatory responses and host defense in addition to direct antibacterial activities. In this study, sterically stabilized cationic liposomes (SSCL) and sterically stabilized anionic liposomes (SSAL) were developed as nanocarriers for macrophage targeting. Elaborated liposomes were characterized in terms of size, zeta potential, morphology, encapsulation efficiency, in vitro drug release behavior and cytotoxicity. Their versatility in targeting monocytes/macrophages was determined by confocal laser scanning microscopy and transmission electron microscopy. Flow cytometry revealed that cellular uptake of both SSCL and SSAL was governed by several endocytic clathrin- and caveolae-dependent mechanisms. Quantitative assessments of intracellular nitric oxide demonstrated highly efficient uptake of GSNO-loaded SSCL that was twenty-fold higher than that of GSNO-free molecules. GSNO-loaded SSCL displayed strong bacteriostatic effects on Staphylococcus aureus and Pseudomonas aeruginosa, which can be involved in pulmonary infectious diseases. These results reveal the potential of liposomal GSNO as an anti-infective therapeutic due to its macrophage targeting capacity and direct antibacterial effects.

S-nitrosogluthatione (GSNO), a S-nitrosothiol, is a commonly used as nitric oxide (NOIn this work, we have investigated the early (4 h) and late (24 h) transcriptomic response of THP-1 human monocytes cells to two doses (1.4 and 6 µM) of either free or EudragitAfter exposure to free GSNO, genes mainly involved in apoptosis, cell differentiation, immune response and metabolic processes were differentially expressed. Although, cells exposed to free or encapsulated GSNO behave differently, activation of genes involved in blood coagulation, immune response and cell cycle was observed in both conditions.These results suggest that the encapsulation of low doses of GSNO into Eudragit

International audience; The number of workers potentially exposed to nanoparticles (NPs) during industrial processes is increasing, although the toxicological properties of these compounds still need to be fully characterized. As NPs may be aerosolized during industrial processes, inhalation represents their main route of occupational exposure. Here, the short- and long-term pulmonary toxicological properties of titanium dioxide were studied, using conventional and molecular toxicological approaches. Fischer 344 rats were exposed to 10 mg/m3 of a TiO2 nanostructured aerosol (NSA) by nose-only inhalation for 6 h/day, 5 days/week for 4 weeks. Lung samples were collected up to 180 post-exposure days. Biochemical and cytological analyses of bronchoalveolar lavage (BAL) showed a strong inflammatory response up to 3 post-exposure days, which decreased overtime. In addition, gene expression profiling revealed overexpression of genes involved in inflammation that was maintained 6 months after the end of exposure (long-term response). Genes involved in oxidative stress and vascular changes were also up-regulated. Long-term response was characterized by persistent altered expression of a number of genes up to 180 post-exposure days, despite the absence of significant histopathological changes. The physiopathological consequences of these changes are not fully understood, but they should raise concerns about the long-term pulmonary effects of inhaled biopersistent NPs such as TiO2.

Unfortunately, the author names in the author group section were incorrectly captured in the published online paper.

Introduction The paucity of biomarkers to predict nanoparticle (NP) toxicity makes important the identification of key cellular pathways linked to a toxic exposure to NP. In this study, we used a lung macrophage cellular model (NR8383) to evaluate modifications in gene expression profiles after exposure to NPs of ZnO, ZnFe2O4 and Fe2O3. Methods The cytotoxic potency of NP was evaluated by WST1 assay and extracellular LDH measurement. Cellular pathway and relevant gene expression were analyzed by a transcriptomic approach and real-time PCR. Results WST-1 assay showed a significant reduction of NR8383 viability after exposure to the NP. Genomic study showed 985, 1216, 4593 differentially expressed genes (DEGs) of cells exposed to ZnO, ZnFe2O4, and Fe2O3.NP, respectively. The major altered pathways were protein synthesis, cellular metabolism, inflammatory and stress responses. The main affected pathways were eukaryotic Initiation Factor2 (eIF2) signaling, mTOR signaling and protein ubiquitination. Conclusions The overall results indicate that the three NP are largely involved in the disruption of protein homeostasis. Some of the pathways altered have direct or indirect links to adverse outcomes. The eIF2 signaling for e.g. has been implicated in pro-inflammation responses.

International audience; S-Nitrosoglutathione (GSNO) is a good candidate for nitric oxide (NO(•)) delivery, and its nanoformulation improves NO(•) stability and bioavailability. We have compared the effect of empty Eudragit nanoparticles (eENP), GSNO-loaded ENP (gENP), and free GSNO on THP-1 human monocytic cell line. We investigated cellular viability and growth by WST-1 and trypan blue tests. ENP uptake was studied using transmission electron microscopy, confocal microscopy, and flow cytometry. Transcriptomic profiles were obtained using microarray. ENP entered cells by clathrin- and caveolae-mediated endocytosis. Exposure to either free GSNO or gENP induced an activation of genes from the same clusters, in favor of intracellular delivery of GSNO by ENP. GSNO nanoformulation might be a therapeutic option for NO(•) delivery.

International audience; The aims of the current study were to prepare chitosan nanoparticles (CNPs) and to evaluate its protective role alone or in combination with quercetin (Q) against AFB1-induce cytotoxicity in rats. Male Sprague-Dawley rats were divided into 12 groups and treated orally for 4 weeks as follow: the control group, the group treated with AFB1 (80 μg/kg b.w.) in corn oil, the groups treated with low (140 mg/kg b.w.) or high (280 mg/kg b.w.) dose of CNPs, the group treated with Q (50 mg/kg b.w.), the groups treated with Q plus the low or the high dose of CNPs and the groups treated with AFB1 plus Q and/or CNPs at the two tested doses. The results also revealed that administration of AFB1 resulted in a significant increase in serum cytokines, Procollagen III, Nitric Oxide, lipid peroxidation and DNA fragmentation accompanied with a significant decrease in GPx I and Cu–Zn SOD-mRNA gene expression. Q and/or CNPs at the two tested doses overcome these effects especially in the group treated with the high dose of CNPs plus Q. It could be concluded that CNPs is a promise candidate as drug delivery enhances the protective effect of Q against the cytogenetic effects of AFB1 in high endemic areas.

This study was conducted to prepare chitosan nanoparticles (CNPs), to determine their properties and to evaluate the synergistic protective role of CNPs alone or in combination with quercetin (Q) against oxidative stress and hepatotoxicity in rats. Female Sprague-Dawley rats were divided into 12 groups (7 rats/group) and were maintained on their respective diet for 3 weeks as follow: control group, the group treated with CCl4 (100 mg/kg b.w twice a week); the groups received CNPs at low and high doses (140 and 280 mg/kg b.w); the group received Q (50 mg/kg b.w); the groups received CNPs at the low or high doses plus Q and the groups treated with CCl4 plus Q and/or CNPs at the two tested doses. Blood and liver samples were collected at the end of experiment period for biochemical and histological studies. The results indicated that chitosan showed deacetylation degree of 17.5% and 19.2% and the molar mass average of monomer was 168.35 g/mol and 169.1 g/mol by UV and IR methods respectively. The particle size of CNPs was around 100 nm with a rough surface. The in vivo results revealed that CCl4 induced biochemical and histological changes typical to those reported in the literature. Animals treated with CNPs at the two tested doses alone or in combination with Q were comparable to the control. CNPs alone or plus Q succeeded to induce significant improvements in the biochemical parameters and histological picture of the liver in rats treated with CCl4. This improvement was in dose-dependent manner for CNPs and was more pronounced in the group treated with the high dose plus Q. It could be concluded that both CNPs and Q could induce protection against hepatotoxicity. Consequently, CNPs was a promise candidate as drug delivery in liver diseases treatments.

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International audience; Although nanotechnology can provide useful carriers to protect successfully active agents, it is necessary to determine the possible toxicity of these technological products before any extensive use. The aim of the current study was to evaluate the effects on the nervous system of Eudragit® polymers nanoparticles (EUD-Nps), considered as model of drug delivery carriers. Nile red-labeled EUD-NPs were prepared by a double emulsion/solvent evaporation technique. Male Sprague-Dawley rats were treated orally (PO) or intraperitoneally (IP) with a single dose of EUD-NPs (50 mg/kg bw). Animals were sacrificed after 4h, 48h, 1 week and 3 weeks and the brain of each rat was removed and dissected into the forebrain, midbrain and hindbrain regions. Acetylcholinesterase (AChE) activity, lipid peroxidation, reduced glutathione and nitric oxide were determined in different brain region homogenate. Other samples were used for the histological and fluorescent examinations. The results indicated that within the two routes of administration, all the tested parameters showed insignificant changes during the tested time intervals and all of them were in the normal range of the control by the 3rd week. Moreover, the histological examination revealed a normal histological picture of the brain tissues after 3 weeks of administration. The fluorescent examination showed that EUD-NPs were found in the brain tissues at all the studied times. These findings showed that EUD-NPs have the ability to cross the blood brain barrier but induced little toxic effects on the brain. Further studies are needed to verify the fate of these NPs in the long term before using them as a vector.

The pulmonary toxicological properties of inhaled titanium dioxide were studied using bronchoalveolar lavage fluid (BALF) cytology and proteomics analyses. Fischer 344 rats were exposed to 10 mg/m3 of TiO2 nanostructured aerosol by nose-only inhalation for 6 h/day, 5 days/week for 4 weeks. Lung samples were collected up to 180 post-exposure days. As previously described, cytological analyses of BALF showed a strong inflammatory response up to 3 post-exposure days, which persisted however, at a lower intensity up to 180 days. In addition, using Multidimensional Protein Identification Technology (MudPIT), we identified a total of 107, 50 and 45 proteins (UniprotKB identifiers) differentially expressed in exposed rats immediately, 3 and 180 days after the end of exposure respectively. Increased levels of inflammatory proteins, members of proteasome, various histones, proteins involved in cytoskeleton organization, were noticed up to 3 days (short-term response). Some of these proteins were linked with Neutrophil Extracellular Trap formation (NETosis). Long-term response was also characterized by a persistent altered expression of proteins up to 180 days. Altogether, these results suggest that exposure to low toxicity low solubility nanomaterials such as TiO2 may induce long-term changes in the pulmonary protein expression pattern of which the physio-pathological consequences are unknown. Significance This paper describes in rats, at the pulmonary level, the effects of inhaled nanostructured aerosol of TiO2 on the secreted proteins found in the broncho-alveolar space by comparing the proteomic profile in broncho-alveolar lavage fluid supernatants of control and exposed animals. This work brings new insights about the early events occurring following the end of exposure and suggests the formation of Neutrophil Extracellular Traps (NETosis) that could be interpret as a potential early mechanism of defense against TiO2 nanoparticles. This work also describes the long term effects (180 post-exposure days) of such an exposure and the change in secreted protein expression in the absence of significant histopathological modifications.

International audience; Drug delivery nanosystems are currently used in human therapy. In preliminary studies we have observed that Eudragit RS nanoparticles, prepared by nanoprecipitation or double emulsion techniques, are cytotoxic for NR8383 rat macrophages. In this study, we expand our previous analysis and suggest that unloaded Eudragit RS nanoparticles prepared by nanoprecipitation (NP/ERS) may induce important morphological and biochemical cellular modifications leading to cellular death. In NR8383 rat macrophages cell line exposed to doses varying from 15 to 100 μg/mL, NP/ERS nanoparticles are internalized inside the cells, reach the mitochondria and alter the structure of these organelles. In addition, the exposure to nanoparticles induces cellular autophagy as demonstrated by electron microscopy analysis, microchip array, qRT-PCR and Western blot assays. Although toxicity of nanoparticles has already been evidenced, it is the first time that results show clearly that the toxicity of polymeric nanovectors may be related to an activation of autophagy.

International audience; The bioavailability of low molecular weight heparin (LMWH) has been increased by encapsulation in nanoparticles. As a complement to these results, the cytotoxicity and apoptosis induced by LMWH nanoparticles prepared by two methods [nanoprecipitation (NP) and double emulsion (DE)] using Eudragit® RS (RS) and poly-ɛ-caprolactone (PCL) have been analysed. Particle sizes varied from 54 to 400 nm with zeta potential values between −65 and +63 mV. Our results showed that the method of nanoparticle preparation affects their properties, especially in terms of drug incorporation and cell tolerance. Cell viability ranged from 6% to 100% depending on the preparation method and physicochemical properties of the particles and the type of toxicity assay. Particle diameter and zeta potential seemed to be the most valuable cytotoxicity markers when cell viability was measured by Trypan blue exclusion and MTT respectively. Nanoparticles prepared by DE were better tolerated than those of NP. LMWH encapsulation into the cationic nanoparticles reduces remarkably their toxicity. Apoptosis evaluation showed activated caspases in exposed cells. However, no nuclear fragmentation was detected in NR8383 cells whatever the tested nanoparticles. DE nanoparticles of RS and PCL can be proposed as a good LMWH delivery system due to their low toxicity (IC50 ∼ 2.33 and 0.96 mg/mL, respectively).

The toxicity of mineral fibers, whether they are natural or man made (MMMF), is usually evaluated in vivo using biopersistence tests in rodents. Development of an in vitro cellular model would be worthwhile in order to reduce, refine and finally replace animal models. For this purpose, we developed an in vitro assay using human monocytic cell line (U-937) to evaluate a new manufactured rock wool fiber (HDN) biodegradation. Experiments on earlier known mineral fibers asbestos (crocidolite) and glass wool fibers (CM44) were also performed. U-937 responded to HDN and CM44 only if they were activated. Among the different activators we used, Escherichia coli living cells as well as FS were the most efficient as evidenced by alterations of HDN and CM44 surface, detected by scanning electron microscopy, and by the measure of silicon released from the rock wool fibers. Asbestos fibers were not degraded when incubated in the presence of living bacteria. The MMMF modifications were function of the fiber composition, the time of exposure to activated cells and the concentration of activators. The pattern of MMMF degradation by our in vitro system was in accordance with those observed in an in vivo study, thus indicating that the fiber degradation by macrophage cells activated by E. coli living cells as well as FS is a valuable system to assess mineral fibers' biopersistence.

International audience; Aim: This study aimed to determine the composition of ethanol extract of jojoba seeds, and to evaluate its hepatoprotective effects in rats fed fumonisin B1 (FB1)-contaminated diet.Methods: Jojoba seeds were extracted in 95% ethanol, and the chemical composition was determined. Male rats were divided into six groups and treated for 8 weeks as follows: (1) Untreated control; (2) FB1-contaminated diet (80 mg/kg diet); (3) low dose (0.5 mg/kg b.w.) jojoba extract; (4) high dose (1.0 mg/kg b.w.) jojoba extract; (5) low dose jojoba extract plus FB1; and (6) high dose jojoba extract plus FB1. Blood and liver samples were collected for different biochemical analyses and histological examinations.Results: The results indicated that the ethanolic extract of jojoba is rich in protein, phenolic compounds, phytic acid, and considerable amounts of simmondsin. Animals fed FB1-contaminated diet showed severe biochemical and histological changes typical to those reported in literature. Treatment with jojoba seed extract alone at the two tested doses did not induce significant alterations in all parameters tested. Combined treatment of jojoba seed extract with FB1 eliminated hepatotoxicity induced by FB1, especially at low dose of jojoba seed extract.Conclusion: The authors concluded that jojoba seed extract can be incorporated in FB1-contaminated feed to eliminate FB1-induced hepatotoxicity.

Transgenic BigBlue rats were exposed to CM 44 glass fibers (6.3 mg/m 3) by nose only, 6 h/day for 5 days. Two endpoints were examined 1, 3, 14, 28, and 90 days following exposure: fiber biopersistence and mutations in lung DNA. The half-time of the fibers >20 µm was 12.8 days, and mutant frequencies of control and exposed rats were similar across all time points. The mutation spectra of both series were similar after 28 days of fixation time. These results showed that a glass fiber with a high clearance in the lung seems to not present any significant effect on mutagenesis on lung DNA and are in marked contrast to results for asbestos, which caused a twofold mutant frequency increase as described in a previous study (Rihn et al., 2000b).

International audience; no abstract

International audience; Applications of polymeric nanoparticles (NP) in medical fields are rapidly expanding. However, the influence of polymeric NP on cell growth and functions is widely underestimated. Therefore, we have studied cell and polymeric NP interactions by addressing two cell types with two endpoints (viability and gene expressions). Rat NR8383 and human THP-1 monocytic cell lines were exposed to 6 to 200 μg/mL of Eudragit(R) RL NP for 24 h, and cellular viability was estimated using MTT, WST-1, and trypan blue tests. A decrease of viability was observed with NR8383 cells (down to 70% for 200 μg/mL), and on the contrary, an increase with THP-1 cells (up to 140% for 200 μg/mL). Differential expression of genes involved in oxidative damage (NCF1), inflammation (NFKB, TNFA, IL6, IL1B), autophagy (ATG16L), and apoptotic balance (PDCD4, BCL2, CASP8) was analyzed. ATG16L, BCL2, and TNFA were up-regulated in NR8383 cells, which are consistent with an induction of autophagy and inflammation. On the other hand, NCF1, NFKB, and IL1B were down-regulated in THP-1 cells, which may contribute to explain the increase of cellular viability. Our results show that (1) the toxic potency of NP is dependent on the cellular model used and (2) mechanistic toxicology should be the corner stone for the evaluation of NP hazard.

International audience; Aim: The aim of this study was to evaluate the effects of Panax ginseng extract standardized with ginsenoside Rg3 (PGRg3) on the mating behavior of sexually active or inactive male rats treated with dopamine antagonists. Methodology: Animals were treated with PGRg3 (50,150 and 450mg/kgb.w) with or without dopamine antagonists. The penile erection, motor activity and stretching-yawning episode were evaluated in animals treated with PGRg3 alone or in combination with lisurode or SND 919. Testosterone and sperm counts were also evaluated in different treatment groups. Results: The results showed that (-) Eticlopride counteracted PGRg3-induced penile erection but not motor hyperactivity. PGRg3 treatment enhanced lisuride-induced behavioral effects. Moreover, PGRg3 plus SND 919 showed a marked stretching-yawning behavior compared to the animals received SND 919 alone. PGRg3 also succeeded to increase testosterone level and sperm count in a dose dependent fashion. Conclusion: It could be concluded that DAD2 receptors are involved in PGRg3-induced mating behavior and testicular function improvement. PGRg3 could be used to improve sexual function and mating behavior in people suffering from erectile dysfunction.

Transgenic mice provide a unique tool for studying the tissue specificity and mutagenic potential of chemicals. Because 3-methylcholanthrene (3MC) was found mutagenic in bacteria, clastogenic in bone marrow, and induces DNA adducts in animals, we were interested to determinine whether this xenobiotic provokes (1) cell proliferation, (2) transcriptional activity changes, (3) DNA adducts, and (4) hepatic mutations in transgenic Big Blues mice carrying the λLIZ phage shuttle vector. Big Blue C57/Bl male mice were treated with a single intraperitoneal dose of 80 mg/kg 3MC for 1, 3, 6, 14, or 30 days. Cell proliferation was checked by 5-bromo-2-deoxyuridine labeling and immunohistochemical detection. The maximal increase of the mitotic index was evidenced after 3 days (2.9 times the control value; P < 0.01). The relative nucleus area, reflecting the transcriptional activity, was also the highest in the treated group after 3 days: 1.86 times the control value, on average (P < 0.01). Four major DNA adducts, determined according to the [ 32 P]-postlabeling method, were evidenced in liver DNA of treated mice, 6 days after the treatment: the spot intensities increased in a time-dependent manner. The mutant frequency of liver DNA was the highest after 14 days: 20.3 ± 2.9 × 10 -5 in the treated vs. 7.6 ± 2.7 × 10 -5 in the control mice (P < 0.01). Sequencing of the λ lacl mutant plaques showed mainly G:C → T:A and C:G → A:T transversions. In conclusion, 3MC at first induced nuclear enlargement and a slight increase of cell proliferation in liver, followed by parallel formation of DNA adducts and mutations. This study shows how transgenic models allow in vivo evaluation of mechanistically simultaneous endpoints. Environ. Mol. Mutagen. 36:266-273, 2000.

Occupational and environmental exposures to bitumen fumes during road paving or roofing activities represent a safety issue since this complex mixture of volatile compounds and particles contains carcinogenic polycyclic aromatic hydrocarbons (PAH). However, epidemiological and experimental animal studies failed to draw unambiguous conclusions concerning bitumen fumes toxicity. In order to gain better insights on their genotoxic potential, we used an experimental device able to generate bitumen fumes at 170°C (upper range used during road paving operations) with a total particulate matter of 112 ± 13 mg/m3 and a mass median aerodynamic diameter of 4.6 μ m. The nose-only exposure of Big Blue® transgenic rats was performed 6 h/day for 5 consecutive days. Biological exposure to this aerosol was monitored by measuring the rat urinary levels of 1-hydroxypyrene and lungs were collected at various time-points after the end of treatment for further analysis. As determined by the 32P post-labeling method, no DNA bu...

The growth of analytical methods for the detection of nucleic acid from various biological samples reflects recent advances in biotechnology development especially in the areas of genetic, infections and cancer diagnosis. The target DNA is detected by hybridization techniques derived from Southern's blotting. However such assays, based on the use of 32 P labelled DNA probes, bring with them the associated problems of handling radioactive materials. In order to overcome these difficulties, a number of chemiluminescent detection methods have recently been developed. These new, alternative probe labelling procedures and chemiluminescent detection methods are easy to use in routine assays performed in research laboratories as well as for medical applications, and can reach the level of sensitivity found in classical radiolabelling techniques. The techniques investigated include peroxydase, biotin 16-dUTP or digoxigenin 11-dUTP probe labelling. The target DNAs are transferred onto nitrocellulose or nylon membranes and further fixed by heat or UV crosslinking. Specific hybridization on the target DNA is finally revealed by the use of chemiluminescent substrates. For all these techniques the detection limit is 10 aM (attomol) of a 561 bp target DNA. However for the probes labelled with peroxydase and with digoxigenin the detection limit drops to 1.0 aM of the target DNA. In the present paper we shall compare several of these DNA labelling and detection procedures and show that the detection threshold can vary by as much as a factor of 20 from method to method. This is the first time that various chemiluminescent methods for label and detection of DNA are compared and evaluated in order to determine the best protocol.

Air pollution effect on humans represents a major public health problem. Exposure to genotoxic compounds in the ambient air is evaluated using different biomarkers. In the present study we assessed DNA-adducts levels in apparently healthy people living and working in the city of Cotonou (Benin) in which exposure to air pollutants such as benzene and polycyclic aromatic hydrocarbons (PAHs) mainly benzo(a)pyrene has been evidenced. Rural inhabitants were enrolled as control group. Taxi-motorbike drivers, street food vendors, and gasoline salesmen were recruited in Cotonou whereas suburban residents were recruited in Godomey, 12 km from Cotonou. We found that taxi-motorbike drivers, roadside residents, street vendors, taxi-motor-bike drivers and gasoline sellers had significantly higher levels of DNA-adducts than suburban and village inhabitants (P

We report a highly tunable quantum dot (QD)-polypeptide hybrid assembly system with potential uses for both molecular imaging and delivery of biomolecular cargo to cancer cells. In this work, we demonstrate the tunability of the assembly system, its application for imaging cancer cells, and its ability to carry a biomolecule. The assemblies are formed through the self-assembly of carboxyl-functionalized QDs and poly(diethylene glycol-L-lysine)-poly(L-lysine) (PEGLL-PLL) diblock copolypeptide molecules, and they are modified with peptide ligands containing a cyclic arginine-glycine-aspartate [c(RGD)] motif that has affinity for αvβ3 and αvβ5 integrins overexpressed on the tumor vasculature. To illustrate the tunability of the QD-polypeptide assembly system, we show that binding to U87MG glioblastoma cells can be modulated and optimized by changing either the conditions under which the assemblies are formed or the relative lengths of the PEGLL and PLL blocks in the PEGLL-PLL molecules. The optimized c(RGD)-modified assemblies bind integrin receptors on U87MG cells and are endocytosed, as demonstrated by flow cytometry and live-cell imaging. Binding specificity is confirmed by competition with an excess of free c(RGD) peptide. Finally, we show that the QD-polypeptide assemblies can be loaded with fluorescently labeled ovalbumin, as a proof-of-concept for their potential use in biomolecule delivery.

Button batteries (BB) are frequently swallowed and must be removed immediately as, if they remain in the esophagus, they can cause severe damage with fatal prognosis. To better understand the molecular mechanism of esophagus damage, we performed a study aimed to analyze heat production, pH variation, electrical work and metal release from BB incubated in cell culture media. Cytotoxicity and apoptosis induced in a human monocytes cell line (U937) was also investigated.Calculated transferred heat was around 244 J with a maximal increase of temperature of 7.26 K/g medium whose pH, reflecting medium electrolysis, was 12.2 after about 2 h incubation. Release of metals also occurred. Only 49.7 ± 1.9% of U937 monocytes remained viable after 90 min of incubation in DMEM containing BB. Those results suggest that BB discharge induce cytotoxicity and tissue injury probably due to multiple combinations of at least thermic, caustic and toxic phenomena. As maximal effects occurred in less than 2 h, they prompt for fast removal of battery from esophagus. Keywords: Button battery, Electrolysis, Calorimetry, Heat transfer, Metal ions, pH, Cytotoxicity, Apoptosis

In 1997 The International Agency for Research on Cancer classified some exposures to crystalline silica as carcinogenic to humans. Such exposures were acknowledged to be very variable, and even in the same monograph it was admitted that coal dust, containing as much as 20% quartz, could not be classified. Clearly there is a need to develop methods for assessing any risks posed by various silica containing dusts in different workplaces. A European collective research project, SILICERAM, was launched with the aim of assessing the toxicity of various dusts in the ceramics industry and improving worker protection. This study examined the effect of particles, namely, DQ12 quartz, China clay, feldspar, and a sample resembling a typical mixture used in the ceramic industry (a "contrived sample" or CS), on NR8383, a rat alveolar macrophage (AM) cell line. Titanium dioxide and aluminum oxide were also used as negative controls. Confocal microscopy observations showed internalization of DQ12 and CS in NR8383. Cell viability decreased dramatically after a 2-h incubation exposure period with DQ12 (-71%). CS was less toxic than DQ12 at 2 h. China clay and feldspar were slightly cytotoxic to NR8383 cells. DQ12 induced apoptosis, with a smaller effect of CS and China clay. TNFalpha gene expression was analyzed by RT-PCR. DQ12, at a noncytotoxic dose of 10 microg/cm(2), induced a significant expression of TNFalpha (+2 times increase). In contrast, similar doses of CS and China clay did not produce a significant increase, while TiO2 and Al2O3 displayed no effect. Co-treatment with 10 microM aluminum lactate significantly reduced the effects of silica-containing particles on cytotoxicity, apoptosis, and TNFalpha expression.

The ultimate goal of toxicologic investigation of synthetic vitreous fibers (SVFs) is to provide essential input for the assessment of human risk to their exposure. Toxicity of mineral fibers is usually evaluated by testing biopersistence in rodent model. However, a cellular model would be much appreciated in order to reduce, refine, and replace animal models. Pulmonary disorders triggered by inhalation of occupational or environmental mineral particulates can be the endpoints of a chronic inflammatory process in which alveolar macrophages (AMs) play a crucial role. Depending on the type of SVF involved, phagocytosis of fiber leads to activation of macrophages, resulting in release of fiber components and potent mediators, such as reactive oxygen or nitrogen species and cytokines. As a matter of fact, macrophages should be the cells of choice since SVF toxicity is the consequence of fibers and alveolar macrophages interaction. Today, monocytes and macrophages culture are firmly established as a paradigm in toxicology when several endpoints are assayed in macrophages: (1) fiber durability, (2) fiber surface changes, (3) oxidative stress and genotoxicity in macrophage, and (4) macrophage cell viability and apoptosis. This article is a review of up-to-date knowledge of in vitro studies involving macrophages, and assesses endpoints of macrophage toxicity with an emphasis on (1) dissolution, (2) scanning electron microscopy analysis, (3) cytotoxicity, and (4) gene expression.

A human monocytes cell line, U-937, incubated in the presence of filtered medium from Escherichia coli culture (FS) has been previously reported to degrade man made mineral fiber and it has been indicated as a good paradigm of in vivo fiber biopersistence evaluation (manuscript accepted for publication). In the present paper, a study is reported aimed to define the molecular modification occurring in the U-937 monocytes during in vitro fiber degradation. The induction of gene expression was investigated in U-937 exposed to rock wool fibers (HDN) in the presence of FS by transcriptome analysis using 20 K DNA microarrays and quantitative RT-PCR. The over-expression of genes related to mobility and cellular adhesion, oxidative stress, immune system stimulation, enzymes, and ions transport protein systems were identified. Among them NCF1 gene, the gene encoding a subunit of NADPH oxidase, over-expression was detected. As the product of this gene allows the formation of superoxide anion that could lead to oxidative stress, HDN fibers were exposed to hydrogen peroxide. Fiber degradation similar to those observed upon incubation with U-937 in the presence of FS was obtained thus suggesting that reactive oxygen species production may be responsible for fiber degradation by U-937 monocytes.

International audience; During hot application of bitumen containing materials, e.g. in hot paving or roofing, fumes are emitted that contain polycyclic aromatic compounds. Previous studies with rodents exposed to bitumen and coal-tar fume condensates showed formation of DNA adducts. In order to clarify the genotoxicity of bitumen fumes, we designed a study by using mice carrying a reporter gene for mutagenesis analysis and exposed by nose-only to a constant and reproducible aerosol of bitumen fumes. We analyzed the genotoxic activity of inhaled bitumen fumes generated under those controlled conditions through the induction of mutation and DNA adducts in Big Blue mice. Mice were exposed to bitumen fumes (100 mg/m(3) total particulate matter) 6 h per day during 5 days by nose-only in an inhalation chamber designed in our laboratory. Following a 30-day fixation period, the experiment was terminated and lung DNA was extracted for mutant frequency and adduct determinations. The mutant frequency was determined using the cII and the lacI mutant analysis systems. In, addition, 61 and 54 mutants were sequenced in control and exposed groups, respectively. The study did not show any mutation or adduct induction in the exposed group compared to the control group: cII mutant frequencies were 11.0+/-4.5x10(-5) and 11.0+/-4.8x10(-5) in control and exposed lungs, respectively. Identically, using the lacI mutation detection system, the mutant frequencies were 6.4+/-3.1x10(-5) and 5.8+/-2.0x10(-5). The mutation spectra of both series were quite similar with regard to transition and transversion frequencies. The absence of genotoxicity in the group exposed to 100 mg/m(3) bitumen is discussed with regard to dosage of inhaled polycyclic aromatic compounds and species.

Types 1 and 3 iodothyronine deiodinases are known to be selenocysteine-containing enzymes. Although a putative human type 2 iodothyronine deiodinase (D2) gene (hDio2) encoding a similar selenoprotein has been identified, basal D2 activity is not selenium (Se)-dependent nor has D2 been labeled with (75)Se. A human mesothelioma cell line (MSTO-211H) has recently been shown to have approximately 40-fold higher levels of hDio2 mRNA than mesothelial cells. Mesothelioma cell lysates activate thyroxine (T(4)) to 3,5,3'-triiodothyronine with typical characteristics of D2 such as low K(m) (T(4)), 1.3 nm, resistance to propylthiouracil, and a short half-life ( approximately 30 min). D2 activity is approximately 30-fold higher in Se-supplemented than in Se-depleted medium. An antiserum prepared against a peptide deduced from the Dio2 mRNA sequence precipitates a (75)Se protein of the predicted 31-kDa size from (75)Se-labeled mesothelioma cells. Bromoadenosine 3'5' cyclic monophosphate increases D2 activity and (75)Se-p31 approximately 2.5-fold whereas substrate (T(4)) reduces both D2 activity and (75)Se-p31 approximately 2-3-fold. MG132 or lactacystin (10 microm), inhibitors of the proteasome pathway by which D2 is degraded, increase both D2 activity and (75)Se-p31 3-4-fold and prevent the loss of D2 activity during cycloheximide or substrate (T(4)) exposure. Immunocytochemical studies with affinity-purified anti-hD2 antibody show a Se-dependent increase in immunofluorescence. Thus, human D2 is encoded by hDio2 and is a member of the selenodeiodinase family accounting for its highly catalytic efficiency in T(4) activation.

We used transgenic mice carrying the lacI reporter gene to study the mutagenesis potential of asbestos crocidolite. The animals were exposed by nose-only inhalation to an aerosol containing 5.75 mg/m(3) crocidolite dust for 6 hr/day and 5 consecutive days. After 1, 4, and 12 weeks, we examined four end points: the cytology of bronchoalveolar lavage, the lung load of crocidolite, the hydrophobic DNA adducts, and the mutations in the lacI reporter gene. Twelve weeks after exposure, nearly 10% of the inhaled fibers remained in the lung (227 +/- 103 ng/mg lung). There was evidence of a typical inflammatory response consisting of multinucleate macrophages at weeks 4 and 12, whereas immediately after the exposure, we observed numerous polymorphonuclear neutrophils. The mutant frequency significatively increased during the fourth week after the exposure: 13.5 [time] 10(-5) in the exposed group versus 6. 9 10(-5) in the control group. The induction factor, defined by the ratio of checked mutants of exposed mice to checked mutants of control mice, was 1.96. The mutation spectrum of control lung DNA and exposed lung DNA was similar, suggesting the possible involvement of a DNA repair decrease in crocidolite-treated animals. We used the (32)P-postlabeling method and did not detect any increase of either 5 mC or bulky adduct in treated mice. This is the first study that demonstrates asbestos mutagenicity in vivo after a nose-only inhalation. Images Figure 1 Figure 2

The bioflavonoid silymarin is found to potently suppress both nuclear factor kappa-B (NF-kappaB)-DNA binding activity and its dependent gene expression induced by okadaic acid in the hepatoma cell line HepG2. Surprisingly, tumor necrosis factor-alpha-induced NF-kappaB activation was not affected by silymarin, thus demonstrating a pathway-dependent inhibition by silymarin. Many genes encoding the proteins of the hepatic acute phase response are under the control of the transcription factor NF-kappaB, a key regulator in the inflammatory and immune reactions. Thus, the inhibitory effect of silymarin on NF-kappaB activation could be involved in its hepatoprotective property.

We have developed a rapid method for the purification of proteins, combining titration curve analysis with a two-step column chromatographic procedure. We have used this approach to purify the cytotoxin (L toxin) from Clostridium sordellii . We have also determined the amino acid composition of this cytotoxin. This toxin has a p I value of 4.20 and an M r of 260 000, reduction of which results in a band of M r 43 000 on sodium dodecyl sulphate polyacrylamide gel electrophoresis. Since both the proteins of M r 260 000 and 43 000 are recognized by the polyclonal anti- C. sordellii L toxin, which neutralizes the L toxin cytotoxicity, we propose a hexameric structure for the protein of M r 260 000, each subunit being M r 43 000.

Malignant pleural mesothelioma (MPM) is an aggressive serosal tumor, strongly associated with former exposure to asbestos fibers and for which there is currently no effective treatment available. In human, MPM is characterized by a high local invasiveness, poor prognosis and therapeutic outcomes. In order to assess molecular changes that specify this phenotype, we performed a global gene expression profiling of human MPM. Using a 10,000-element microarray, we analyzed mRNA relative gene expression levels by comparing a mesothelioma cell line to either a pleural cell line or tumor specimens. To analyze these gene expression data, we used various bioinformatics softwares. Hierarchical clustering methods were used to group genes and samples with similar expression in an unsupervised mode. Genes of known function were further sorted by enzyme, function and pathway clusters using a supervised software (IncyteGenomics). Taken together, these data defined a molecular fingerprint of human MPM with more than 700 up- or down-regulated genes related to several traits of the malignant phenotype, specially associated with MPM invasiveness, protection and resistance to anticancer defenses. This portrait is meaningful in disease classification and management, and relevant in finding new specific markers of MPM. These molecular markers should improve the accuracy of mesothelioma diagnosis, prognosis and therapy.

The cytotoxin, also named toxin B, was isolated from a toxigenic strain of Clostridium difficile, purified to homogeneity and partially characterized. The purification procedure included ultrafiltration followed by anion-exchange chromatography. We noticed that a non-specific nucleic material eluted with the protein during the purification. The presence of these nucleic acids appeared to be important for the toxic activity of the protein. Some characteristics of the cytotoxin were examined, especially the amino acid composition and the sequence of three tryptic fragments.