Laurent Foucaud

Ecotoxicological assessment of TiO2 byproducts on the earthworm Eisenia fetida

The increasing production of nanomaterials will in turn increase the release of nanosized byproducts to the environment. The aim of this study was to evaluate the behaviour, uptake and ecotoxicity of TiO(2) byproducts in the earthworm Eisenia fetida. Worms were exposed to suspensions containing 0.1, 1 and 10 mg/L of byproducts for 24 h. Size of TiO(2) byproducts showed aggregation of particles up to 700 μm with laser diffraction. Only worms exposed at 10 mg/L showed bioaccumulation of titanium (ICP-AES), increasing expression of metallothionein and superoxide dismutase mRNA (Real-time PCR) and induction of apoptotic activity (Apostain and TUNEL). TiO(2) byproducts did not induce cytotoxicity on cœlomocytes, but a significant decrease of phagocytosis was observed starting from 0.1 mg/L. In conclusion, bioaccumulation of byproducts and their production of reactive oxygen species could be responsible for the alteration of the antioxidant system in worms.

Effect of Polycyclic Aromatic Hydrocarbons and Carbon Black Particles on Pro-Inflammatory Cytokine Secretion: Impact of PAH Coating Onto Particles

It has been suggested that the organic fraction of particulate matter in air pollution has a major role in the toxicity of this pollutant, notably via its effects on inflammation. The major organic compounds adsorbed onto these particles are polycyclic aromatic hydrocarbons (PAH), among which benzo[a]pyrene (B[a]P), benzo[b]fluoranthene (B[b]F), and pyrene (Pyr) are quantitatively the most important. Generally, cells or organisms are exposed to organic extracts of the particles rather than the native particles in order to study the effects of these PAH. In this study, B[a]P, B[b]F, and Pyr were tested alone and/or adsorbed onto carbon black (CB) particles differing in size in order to evaluate their impact on cytokine production (with or without LPS stimulation) by THP-1 macrophage-like cells. PAH induced significant secretion of IL-1β, IL-8, and IL-12 after 24 or 48 hr of treatment, an effect reinforced by LPS stimulation; no effect on IL-10 secretion was noted. Fine CB particles (260 nm diameter) induced secretion of each cytokine. In general, coating the CB with PAH did not modify the effect of the CB alone; the exception was that LPS-induced IL-1β secretion was reduced. In contrast, ultrafine CB (14 nm diameter: ufCB) caused a decrease in cytokine secretion; this effect was modified by PAH coating. For example, PAH coating on ufCB amplified the inhibitory effect of ufCB against IL-1β secretion but did not modify IL-8 formation. Moreover, PAH coating on ufCB tended to minimize the effect of LPS stimulation; this included (i) inhibition of the decrease in IL-12 secretion induced by uncoated ufCB and (ii) stimulation of IL-10 production. It was concluded that adsorption of PAH onto these particles could decrease their bioavailability and so their abilities to affect cell cytokine production. The results also showed that when PAH were adsorbed onto the fine particles, any observed increases in cytokine secretion consistently appeared to be due to the particles themselves. In contrast, while ufCB alone almost uniformly led to decreases in cytokine formation by the cells, the added presence of the test PAHs led to variable effects – depending on whether stimulation with LPS took place or not. Thus, while some PAHs likely to be associated with PM are clearly immunomodulants, their ultimate effects in situ will likely depend on the properties of the particles themselves, in particular, their size.

Hemocyte responses of Dreissena polymorpha following a short-term in vivo exposure to titanium dioxide nanoparticles: preliminary investigations.

The widespread use of titanium-based nanoparticles and their environmental release may pose a significant risk to aquatic organisms within freshwater ecosystems. Suspension-feeder invertebrates like bivalve molluscs represent a unique target group for nanoparticle toxicology. The aim of this work was to investigate the short-term responses of Dreissena polymorpha hemocytes after in vivo exposure to titanium dioxide nanoparticles (TiO(2) NP). For this purpose, freshwater mussels were exposed to P25 TiO(2) NP at the concentrations of 0.1, 1, 5 and 25mg/L during 24h. Viability, phagocytosis activity and mitogen activated protein kinase (MAPK) phosphorylation level of ERK 1/2 and p38 in hemocytes extracted from exposed mussels were compared to those from control specimens. Results demonstrated an inhibition of the phagocytosis activity after exposure to TiO(2) NP at 0.1 and 1mg/L. Similar trends, albeit less pronounced, were reported for higher concentrations of NP. Transmission electron microscopy showed for the first time the internalization of TiO(2) NP into Dreissena polymorpha hemocytes. Besides, exposure to NP increased the ERK 1/2 phosphorylation levels in all treatments. Concerning the phosphorylation level of p38, only exposures to 5 and 25mg/L of NP induced significant p38 activation in comparison to that of the control. Finally, these short-term effects observed at environmentally relevant concentrations highlighted the need for further studies concerning ecotoxicological evaluation of nanoparticle release into an aquatic environment.

Precipitation of silver-thiosulfate complex and immobilization of silver by Cupriavidus metallidurans CH34

Cupriavidus metallidurans CH34 is a facultative chemolithotrophic bacterium that possesses two megaplasmids (pMOL28 and pMOL30) that confer resistance to eleven metals. The ability of Cupriavidus metallidurans CH34 to resist silver is described here. Electronic microscopy, energy-dispersive X-ray (EDX) and X-ray diffractometry (DRX) observations revealed that C. metallidurans CH34 strongly associated silver with the outer membrane, under chloride chemical form. Using derivate strains of C. metallidurans CH34, which carried only one or no megaplasmid, we show that this resistance seems to be carried by pMOL30.

Oxidative stress induction by nanoparticles in THP-1 cells with 4-HNE production: stress biomarker or oxidative stress signalling molecule?

The aim of this study was to investigate whether carbon black (CB) nanoparticles might induce toxicity to monocytic cells in vitro via an oxidative stress mechanism involving formation of the lipid peroxidation product 4-hydroxynonenal (4-HNE) and the subsequent role of 4-HNE in inducing further cytotoxic effects. ROS production in cells by CB nanoparticles was shown by the oxidation of DCFH after a short time exposure. These particles induced the formation of 4-HNE-protein adducts and significant modification of glutathione content corresponding to an increase of oxidized glutathione form (GSSG) and a decrease of total glutathione (GSX) content. These results attest to an oxidative stress induced by the carbon black nanoparticles, although no induction of HO-1 protein expression was detected. Concerning the effects of a direct exposure to 4-HNE, our results showed that 4-HNE is not cytotoxic for concentrations lower than 12.5 microM. By contrast, it provokes a very high cytotoxicity for concentrations above 25 microM. An induction of HO-1 expression was observed from concentrations above 5 microM of 4-HNE. Finally, glutathione content decreased significantly from 5 microM of 4-HNE but no modification was observed under this concentration. The discrepancy between effects of carbon black nanoparticles and 4-HNE on the intracellular markers of oxidative stress suggests that 4-HNE is not directly implied in the signalling of oxidative toxicity of nanoparticles but is an effective biomarker of oxidative effects of nanoparticles.

Effects of 4-Nonylphenol and/or Diisononylphthalate on THP-1 Cells: Impact of Endocrine Disruptors on Human Immune System Parameters

The aim of the present work is to investigate the link between two endocrine disruptor compounds (EDCs), which are chemicals that interfere with the hormone system in human and wildlife, and the human immune response through a study of their effects on the THP-1 human cell line which was used as a model for macrophages. We used two EDCs, diisononylphthalate (DiP) and 4-n-nonylphenol (NP) alone or in combination in order to evaluate the effects of these compounds on several parameters of the immune response - cytokine secretion, phagocytosis and the putative implication of the estrogen receptors - by studying the level of MAPK activation. NP and DiP strongly reduced phagocytosis and modify cytokine secretions. Indeed, differentiated THP-1 cell exposures (i) to 5 and 10 μM of combination of NP and DiP induced an IL-8 level in the medium respectively of 28.9 and 45% higher than the level obtained for the control (untreated cells), (ii) to combination of NP and DiP at 10 μM induced an increase of IL-1β level in comparison to the control level, (iii) to combination of NP and DiP induced an increase of TNF-α level whatever the concentration of EDCs tested (between 0 and 10 μM). Lastly, differentiated THP-1 cell exposure to NP, DiP alone or in combination (2 μM for each condition) induced a decrease of ERK1/2 phosphorylation in comparison to ERK1/2 phosphorylation level of the control. Moreover, differentiated THP-1 cell treatments by ICI-182780 (an estrogen receptor antagonist) supressed the EDC effects on ERK1/2 phosphorylation level which indicates an estrogen receptor-dependent pathway. These results suggest that EDCs have the ability to alter the human immune function, maybe by interfering with endocrine balance.

Lack of desensitization of the cough reflex in ovalbumin-sensitized rabbits during exercise

Introduction Cough is a major symptom of asthma frequently experienced during exercise but little is known about interactions between cough and exercise. The goal of our study was to clarify the potential modulation of the cough reflex (CR) by exercise in a spontaneously breathing anaesthetized animal model of airway eosinophilic inflammation. Materials & methods Ten ovalbumin (OVA) sensitized adult rabbits and 8 controls were studied. The ventilatory response to direct tracheal stimulation, performed both at rest and during exercise was determined to quantify the incidence and the sensitivity of the CR. Broncho-alveolar lavages (BAL) and cell counts were performed to assess the level of the airway inflammation following OVA-induced sensitization. Exercise was mimicked by Electrically induced hindlimb Muscular Contractions (EMC). Results Among 494 tracheal stimulations, 261 were performed at rest and 233 at exercise. OVA challenges in sensitized rabbits caused a significant increase in the percentage of eosinophils (p = 0.008) in BAL. EMC increased minute ventilation by 36% and 35% in OVA and control rabbits respectively, compared to rest values. The sensitivity of the CR decreased during exercise compared to baseline in control rabbits (p = 0.0313) while it remained unchanged in OVA rabbits. Conclusion The desensitization of the CR during exercise in control rabbits was abolished in OVA rabbits. The precise role of airway inflammation in this lack of CR desensitization needs to be further investigated but it might contribute to the exercise-induced cough in asthmatics.

Down-Regulation of Cough during Exercise Is Less Frequent in Healthy Children than Adults. Role of the Development and/or Atopy?

Cough is typically associated with physical activity in children with asthma, but the characteristics of the relationship between cough and exercise has not been established under physiological conditions. The aim of the study was to describe the effect of exercise on the reflex cough response elicited by a single breath of capsaicin in non-asthmatic children. A group of non-asthmatic adults was studied as reference. Thirty children and 29 adults were recruited. The cough reflex sensitivity to capsaicin was first determined to establish the dose that provokes 5 cough efforts (C5). The number of coughs elicited by C5 (NC5) was then compared at baseline and during a standardized submaximal treadmill exercise. Data are expressed as median (interquartile range). Children and adults showed a significant decrease in NC5 (respectively from 5.0 (4.0–6.0) to 2.5 (2.0–4.0), p < 0.0005 and from 6.0 (5.0–7.0) to 2.0 (0.0–3.0, p < 0.0005). During exercise, NC5 was observed to decrease in all adult subjects, but in only 24/30 children (80%, p = 0.02). A trend for a higher incidence of personal and familial atopy was observed in children that lacked cough down-regulation during exercise compared with other children. It is concluded that the cough reflex response to capsaicin is down regulated by exercise in both children and adults. The effect however is less consistently observed in the former. The difference may reflect maturation of descending inhibitory pathways of the cough reflex, but may also be associated to atopy. The data stress the importance of assessing the time relationship of cough and exercise in questionnaire studies of asthma.

Rapid Identification of Ralstonia eutropha Strain CH34 Using Monoclonal Antibodies

Ralstonia eutropha strain CH34 (formerly Alcaligenes eutrophus CH34) is an aerobic Gram-negative and facultative chemolithotrophic bacteria with plasmid-bound resistance to heavy metals. The presence of Ralstonia eutropha strain CH34 is an indication of environmental heavy metals pollution. The major purpose of this work was to produce monoclonal antibodies (MAbs) against the metal transport outer membrane proteins. In this way, bacteria outer membranes, grown with or without iron, were purified. The electrophoretic pattern of the outer membrane revealed that, in iron starvation conditions, at least four proteins were overexpressed. These outer membranes were used to immunize mice to produce MAbs. About 200 hybridomas were tested by enzyme-linked immunoadsorbent assay (ELISA). Most of these hybridomas exhibited cross reactions with Escherichia coli and Klebsiella aerogenes. Two hybridomas, AE5/7 and AE5/9, produced MAbs that detected specifically Ralstonia eutropha strain CH34. Analysis by Western blotting showed that these MAbs recognized a protein with a molecular weight of about 41 kDa. Moreover, the presence of the two megaplasmids was required for the full expression of the 41-kDa protein, as demonstrated by screening of the derivatives strains by ELISA. These MAbs could be used for a specific and rapid detection of Ralstonia eutropha strain CH34, using direct immunological methods.

Modulation of defensive airway reflexes during continuous positive airway pressure in the rabbit

Lung hyperinflation may alter the pattern of ventilatory reflexes in chronic respiratory disorders. The aim of the study was to test the effect of continuous positive airway pressure (CPAP) on ventilatory responses to mechanical stimulation of the trachea.