Patrice Gonzalez

Impacts of gold nanoparticle exposure on two freshwater species: a phytoplanktonic alga (Scenedesmus subspicatus) and a benthic bivalve (Corbicula fluminea)

For years, nanotechnologies have developed the use of common materials, such as iron or silica, at an extremely small scale because of their new properties (reactivity, conductivity, optical sensitivity). More precisely, gold nanoparticles are used in numerous technologies such as electronics, new paints or research on cancer. But, despite their promising future and expansive utilization, only a few studies deal with their behaviors or impacts on the environment. Thus, we decided to explore the impacts of amine-coated 10nm gold nanoparticle (AuNp) contaminations on two freshwater aquatic models. The green algaScenedesmus subspicatus was submitted to 24 h-direct exposures at four AuNp concentrations (1.6×102, 1.6×103, 1.6×104 and 1.6×105 AuNp/cell) along with a control condition. The process used for the freshwater bivalvesCorbicula fluminea was a trophic exposure during 7 days to three AuNp concentrations (1.6×103, 1.6×104 and 1.6×105 AuNp/cell). These conditions were tested in triplicate with controls. For these experiments, OD measurements (γ= 520nm) were performed to verify AuNp concentrations in the water (stability). Cell numerations of algae were used to determine the growth/mortality effects on this species. Cellular impacts and AuNp distributions in the two species were revealed by transmission electron microscopy (TEM). The bioaccumulation rates were assessed by gold dosagesvia MS-ICP procedures. Molecular impacts were analyzed by quantifications of metallothionein concentrations (metal detoxification protein) and genetic expressions via real-time RT-PCR. Our study focused on the expression of six genes encoding proteins involved in: metal detoxification (metallothionein), the response to oxidative stress (catalase and superoxide-dismutase), the mitochondrial respiratory chain (subunit 1 of the cytochrome-C-oxidase), the concentration of mitochondria (RNA12s) and the response to xenobiotics (glutathione S transferase); using the β-actin as reference of the basal rates of gene expressions.The results showed a marked impact on the algae after a 24h-exposure to amine-coated 10nm gold nanoparticles, leading to 20% of mortality for the lowest contamination condition, while the highest one reached 50%. TEM examinations showed that AuNp were strongly adsorbed by the cell wall of algae, leading to progressive intracellular and wall disturbances. The bivalve contaminations revealed the ability of these particles to be bioaccumulated and to penetrate gills and digestive epithelia. Their lysosomial localization leads to the loss of their coating, which brought on an oxidative stress.

At environmental doses, dietary methylmercury inhibits mitochondrial energy metabolism in skeletal muscles of the zebra fish (Danio rerio).

The neurotoxic compound methylmercury (MeHg) is a commonly encountered pollutant in the environment, and constitutes a hazard for human health through fish eating. To study the impact of MeHg on mitochondrial structure and function, we contaminated the model fish species Danio rerio with food containing 13 microg of MeHg per gram, an environmentally relevant dose. Mitochondria from contaminated zebrafish muscles presented structural abnormalities under electron microscopy observation. In permeabilized muscle fibers, we observed, a strong inhibition of both state 3 mitochondrial respiration and functionally isolated maximal cytochrome c oxidase (COX) activity after 49 days of MeHg exposure. However, the state 4 respiratory rate remained essentially unchanged. This suggested a defect at the level of ATP synthesis. Accordingly, we measured a dramatic decrease in the rate of ATP release by skinned muscle fibers using either pyruvate and malate or succinate as respiratory substrates. However, the amount and the assembly of the ATP synthase were identical in both control and contaminated muscle mitochondrial fractions. This suggests that MeHg induced a decoupling of mitochondrial oxidative phosphorylation in the skeletal muscle of zebrafish. Western blot analysis showed a 30% decrease of COX subunit IV levels, a 50% increase of ATP synthase subunit alpha, and a 40% increase of the succinate dehydrogenase Fe/S protein subunit in the contaminated muscles. This was confirmed by the analysis of gene expression levels, using RT-PCR. Our study provides a basis for further analysis of the deleterious effect of MeHg on fish health via mitochondrial impairment.

Cadmium-induced genotoxicity in zebrafish at environmentally relevant doses.

Genotoxic effects of cadmium on zebra fish Danio rerio have been assessed by random amplified polymorphic DNA and real time PCR, followed by a comparison of the melting temperature patterns between each amplification reaction. Fish were exposed to two concentrations of cadmium chloride dissolved in the medium (1.9+/-0.6 microg Cdl(-1), C(1); 9.6+/-2.9 microg Cdl(-1), C(2)) for 21 days. A discriminative RAPD probe, OPB11, was first selected producing differential band patterns between control and metal-exposed genomic DNAs. RAPD-PCR showed an increase in the relative hybridization efficiency of OPB11 on the genomic DNAs coming from fish exposed to both Cd concentrations as compared to the control condition. In addition, the RAPD-PCR melting temperature patterns showed that with the OPB11 probe, the frequency of PCR products whose fusion temperature belongs to the [86-87 degrees C] interval decreased with Cd contamination, whereas an increase of frequency for the [78-80 degrees C] and [85-86 degrees C] intervals was correlated with Cd exposure.

PAH biomarker responses in polar cod (Boreogadus saida) exposed to benzo(a)pyrene

With expanding oil and gas activities into the Arctic region, there is a need to evaluate the induction capacity of polycyclic aromatic hydrocarbon (PAH) biomarkers on Arctic marine organisms and to test analytical methods that have been optimized for their temperate counterparts. Polar cod Boreogadus saida were injected intraperitoneally with cod liver oil (solvent control), 6.6+/-3.7, 85+/-48 or 378+/-190 microg kg(-1) wet weight of benzo(a)pyrene (B(a)P), or not injected (control), and liver and bile were sampled at 0 and 16 h and 1, 2, 4 and 7d. The mRNA expression of cytochrome P4501A1 (cyp1a1) and glutathione S-transferase (gst) genes showed a dose-dependent induction in the first 16 h following the injection and a return to basal levels after 4d. The aryl hydrocarbon receptor 2, however, showed no change in mRNA expression. The protein quantification of cytochrome P4501A (CYP1A), through Western blot analysis and the enzyme-linked immunosorbent assay (ELISA), presented similar but weaker and time-delayed responses (4-7d) compared to the gene (16 h to 2d). Ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) activities increased significantly at day 7 following the gene induction and increase in protein levels. Overall, these biomarkers showed dose-dependent but weak responses to B(a)P and low levels of bile metabolites. The mRNA expressions of oxidative stress genes, superoxide dismutases (sod(Cu/Zn) and sod(Mn)), catalase (cat) and glutathione peroxidase (gpx), were all up-regulated between 16 h and 2d of B(a)P exposure with cat (72-fold) and sod(Cu/Zn) (20-fold) giving the strongest responses in the highest dose. Finally, CAT protein level and enzyme activities showed less clear responses than the genes. The mRNA expression showed the earliest responses, followed by the protein levels. The enzymatic activities were the least sensitive and responded to the exposure after 7d. The study shows the induction capability of biomarkers in polar cod at very low bioavailable doses of B(a)P and provides new information on the selected biomarkers for use in oil monitoring in the Arctic.

Biomarker responses in polar cod (Boreogadus saida) exposed to the water soluble fraction of crude oil.

In order to mimic the biological effects of an oil spill in Arctic waters, we examined several types of biomarkers (genes, enzymes, metabolites, and DNA damage) in polar cod Boreogadus saida experimentally exposed to the water soluble fractions of crude oil. During 4 weeks of exposure, induction of the studied biomarkers exceeded baseline levels. The mRNA expression of the cytochrome P4501A1 (cyp1a1) gene was the most promising biomarker, with glutathione S-transferase (gst) as a suitable complement. The delayed ethoxyresorufin O-deethylase (EROD) and GST activities and their persistence following 2 weeks of depuration may allow detection of previous exposures in field samples. The composition of PAH metabolites in the bile indicated the bioavailability of different PAH size-classes. Although mRNA expressions of antioxidant defense genes were induced at start of the exposure, with the strongest responses from catalase and cytosolic superoxide dismutase, they were poor for oil monitoring purposes due to their very short response times. Significant DNA damage demonstrated genotoxicity even at low PAH concentrations (<15microgL(-1)) and was correlated with benzo(a)pyrene and pyrene metabolites in the bile.

Phylogenetic relationships of pleurotus species according to the sequence and secondary structure of the mitochondrial small-subunit rRNA V4, V6 and V9 domains

A comparative study of the V4, V6 and V9 domains of the mitochondrial small-subunit (SSU) rRNA was conducted to evaluate the use of these sequences to investigate phylogenetic relatedness within the genus Pleurotus. The PCR products encompassing these regions from 48 isolates belonging to 16 Pleurotus species were sequenced and compared. From this comparison, the length and sequence of the three domains were found to be constant within a species. Significant inter-species variations due to insertion/deletion events were found, in most cases occurring in regions not directly involved in the maintainance of the standard SSU rRNA secondary structure. Phylogenetic analysis based upon these mitochondrial sequences was in agreement with relationships previously established by morphological descriptions and with previous studies based upon the nuclear genome or isozymes; moreover such analysis resolved some ambiguities in earlier analyses. It was confirmed that P. ostreatus and P. florida represent a single species, as well as P. pulmonarius and P. sajor-caju. The phylogenetic analysis also made it possible to assess the relative positions of P. rattenburyi, P. lampas, P. sapidus, P. colombinus and P. eryngii. The results clearly showed that sequences of the V4, V6 and V9 domains of the mitochondrial SSU rRNA could provide good markers for use in the taxonomy and phylogeny of species of Basidiomycota. Because of their nucleotide conservation, the major advantage of these species-specific markers was the possibility to study only one isolate from each species to determine phylogenetic relatedness.

Evidence of Active Efflux in Resistance to Ciprofloxacin and to Ethidium Bromide by Mycoplasma hominis

ABSTRACT The uptake of fluoroquinolones was characterized for the fluoroquinolone-susceptible strain PG21 of Mycoplasma hominis. Accumulation of fluoroquinolones appeared to occur by passive diffusion. Addition of arginine as the energizer significantly reduced the uptake of fluoroquinolones, suggesting the presence of an energy-dependent efflux process. Reserpine and orthovanadate, two multidrug pump inhibitors, increased significantly the ciprofloxacin (CIP) uptake. In contrast, such a strong effect was not observed for moxifloxacin and pefloxacin uptakes. Two ethidium bromide (EtBr)-resistant strains, selected in vitro, showed a resistance profile compatible with a multidrug-resistant phenotype, with increased MICs for the hydrophilic fluoroquinolones, CIP and norfloxacin, EtBr, and acriflavine. Taking the EtBr-resistant strain RB1La as a model, a significant decrease of the CIP and EtBr uptakes was observed compared to the reference strain PG21. In the presence of reserpine and orthovanadate, both inhibitors of ATP-dependent efflux pumps, the CIP uptake increased significantly, reaching approximately the same level as that of the susceptible strain. Similar results were obtained with EtBr uptake and efflux experiments. Our data suggest the presence of an active efflux system, possibly an ABC-type efflux pump, implicated in the resistance to CIP and unrelated compounds like EtBr in the human mycoplasma M. hominis.

Comparative analysis of gene expression in brain, liver, skeletal muscles, and gills of zebrafish (Danio rerio) exposed to environmentally relevant waterborne uranium concentrations

The effects of waterborne uranium (U) exposure on gene expression were examined in four organs (brain, liver, skeletal muscles, and gills) of the zebrafish (Danio rerio). Adult male fish were exposed to three treatments: No added uranium (control), 23 +/- 6 microg U/L, and 130 +/- 34 microg U/L. After 3, 10, 21, and 28 d of exposure and an 8-d depuration period, gene expression and uranium bioaccumulation were analyzed. Bioaccumulation decreased significantly in liver during the depuration phase, and genes involved in detoxification, apoptotic mechanism, and immune response were strongly induced. Among these genes, abcb311, which belongs to the adenosine triphosphate (ATP)-binding cassette transporter family, was induced 4- and 24-fold in organisms previously exposed to 23 +/- 6 and 130 +/- 34 microg U/L, respectively. These results highlight the role of liver in detoxification mechanisms. In gills, at the highest uranium concentration, gpx1a, cat1, sod1, and sod2 genes were up-regulated at day 21, indicating the onset of an oxidative stress. Mitochondrial metabolism and DNA integrity also were affected, because coxI, atp5f1, and rad51 genes were up-regulated at day 21 and during the depuration phase. In skeletal muscles, coxI, atp5f1, and cat were induced at day 3, suggesting an impact on the mitochondrial metabolism and production of reactive oxygen species. In brain, glsI also was induced at day 3, suggesting a need in the glutamate synthesis involved with neuron transmission. No changes in gene expression were observed in brain and skeletal muscles at days 21 and 28, although bioaccumulation increased. During the depuration phase, uranium excretion was inefficient in brain and skeletal muscles, and expression of most of the tissue-specific genes was repressed or unchanged.

Effect of Low-Dose Cadmium Exposure on DNA Methylation in the Endangered European Eel

There is increasing evidence that epigenetics can play a key role in the etiology of diseases engendered by chronic pollutant exposure. Although epigenetics has received significant attention in the field of biomedicine during the last years, epigenetics research is surprisingly very limited in ecotoxicology. The aim of the present study was to investigate the possible effects of low-dose cadmium exposure on the DNA methylation profile in a critically endangered fish species, the European eel. Eels were exposed to environmentally realistic concentrations of cadmium (0.4 and 4 μg·L(-1)) during 45 days. The global CpG methylation status of eel liver was determined by means of a homemade ELISA assay. We then used a methylation-sensitive arbitrarily primed PCR method to identify genes that are differentially methylated between control and Cd-exposed eels. Our results show that cadmium exposure is associated with DNA hypermethylation and with a decrease in total RNA synthesis. Among hypermethylated sequences identified, several fragments presented high homologies with genes encoding for proteins involved in intracellular trafficking, lipid biosynthesis, and phosphatidic acid signaling pathway. In addition, few fragments presented high homologies with retrotransposon-like sequences. Our study illustrates how DNA methylation can be involved in the chronic stress response to Cd in fish.

Interactive effects of metal contamination and pathogenic organisms on the marine bivalve Cerastoderma edule.

The present study evaluated the interactive effects of cadmium contamination and pathogenic organisms (trematodes Himasthla elongata and bacteria Vibrio tapetis) singularly and in combination during 7 days on the bivalve Cerastoderma edule. Some defense-related activities were analyzed such as genetic expression, metallothionein and immune responses. Trematode metacercarial infection, similar whatever the treatment, induced the strongest responses of immune parameters. Particularly, the interaction between cadmium and parasite exposures induced unusual responses on gene expression and immune responses. No effect of bacterial challenge appeared on bivalve responses, nevertheless a strong mortality of V. tapetis infected cockles occurred between 7 and 14 days. Cadmium bioaccumulation was significantly modulated by both pathogenic organisms. Furthermore, an antagonistic effect of trematodes and bacteria was shown on metal bioaccumulation of co-infected cockles. These results highlighted the importance of considering the multiplicity of perturbation sources in coastal ecosystems to assess the health status of organisms.