Gilles Durrieu

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.

Contamination by Heavy Metals (Cd, Zn, Cu, and Hg) of Eight Fish Species in the Gironde Estuary (France)

The Gironde estuary, one of the largest in Europe, is considerated as a reference ecological system, with all the western European diadromous fish species present. The national biomonitoring program on the coastal marine environment has revealed since 1979 severe metal pollution (mostly cadmium [Cd]) in oysters collected from the estuary. No data are available on metal contamination levels in fish, despite their ecological and economic importance. We present the results from a detailed study based on 4 metals (Cd, zinc [Zn], copper [Cu], and mercury [Hg]) measured in 4 organs (gills, dorsal skeletal muscle, liver, and kidneys) from 8 fish species illustrating several ecological combinations: European eel (Anguilla anguilla), twaite shad (Alosa fallax), bass (Dicentrarchus labrax), meagre (Argyrosomus regius), flounder (Platichthys flesus),, thin-lippid grey mullet (Liza ramada), sole (Solea vulgaris), and Canary drum (Umbrina canariensis). The results show very marked differences between species and organs, as well as very significant variations between the 4 metals. Although metal concentrations measured in fish muscle are low, except in the case of Hg for theA. fallax, high levels of Cu and Cd were observed in the kidneys and livers ofL. ramada andA. anguilla. A multifactorial analysis based on rank ordered metal concentrations for the 8 fish species clearly shows 4 clusters of species assigned to the different degrees of metal contamination, from the lowest contaminated (A. regius, D. labrax, S. vulgaris, andU. canariensis), to the most contaminated group (L. ramada). The most contaminated species (L. ramada, A. angailla, andP. flesus) are characterized by long residence times in the estuary, between 3.5 and 14 yr. ForL. ramada, biofilms with high metal storage capacities would be the principal uptake route; the two other species are benthic with a carnivorous regime. Comparisons between our data and four estuaries (Seine, France; Mersey, U.K.; La Plata, Argentina; Guadalquivir, Spain), on a limited number of common species, metals and fish organs, clearly reveal higher Cd bioaccumulation levels in the Gironde estuary.

Copper detection in the Asiatic clam Corbicula fluminea: optimum valve closure response

When exposed to a contaminant, bivalves close their shell as a protective strategy. The aim of the present study was to estimate the maximum expected dissolved copper sensitivity in the freshwater bivalve Corbicula fluminea using a new approach to determine their potential and limit to detect contaminants. To take into account the rate of spontaneous closures, we integrated stress problems associated with fixation by a valve in usual valvometers and the spontaneous rhythm associated with nycthemeral activity, to optimize the response in conditions where the probability of spontaneous closing was lowest. Moreover, we used an original system with impedance valvometry, using lightweight impedance electrodes, to study free-ranging animals in low stress conditions combined with an analytical approach describing dose-response curves by logistic regression, with valve closure reaction as a function of response time and concentration of contaminant. In C. fluminea, we estimated that copper concentrations > 4 microg/l (95% confidence interval (CI95%), 2.3-8.8 microg/l) must be detected within 5 h after Cu addition. Lower values could not be distinguished from background noise. The threshold values were 2.5 times lower than the values reported in the literature.

Serial analysis of gene expression in the skeletal muscles of zebrafish fed with a methylmercury-contaminated diet.

Mercury (Hg) is a widespread environmental contaminant and its organic form, methylmercury (MeHg), has been known as a potent neurotoxic since the Minamata tragedy. In the Amazonian basin, gold mining leads to MeHg biomagnification all along the food web, culminating in piscivorous fish, ultimately responsible for contamination of human beings through fish consumption. In order to assess the biological impact of dietary MeHg on fish at the genome scale, we contaminated zebrafish with MeHg-contaminated food for 25 days (13.5 microg of Hg/g of food). A serial analysis of gene expression (SAGE) was conducted on the skeletal muscle because this tissue does not perform MeHg demethylation, and 19171 SAGE tags were sequenced from the control library versus 22 261 from the MeHg-contaminated library, corresponding to 5280 different transcripts. Among those identified, 60 genes appeared up-regulated and 15 down-regulated by more than 2 times. A net impact of MeHg was noticed on 14 ribosomal protein genes, indicating a perturbation of protein synthesis. Several genes involved in mitochondrial metabolism, the electron transport chain, endoplasmic reticulum (ER) function, detoxification, and general stress responses were differentially regulated, suggesting an onset of oxidative stress and ER stress. Several other genes for which expression varied with MeHg contamination could be clustered in various compartments of the cells life, such as lipid metabolism, calcium homeostasis, iron metabolism, muscle contraction, and cell cycle regulation. This study reveals the effectiveness of the SAGE approach to acquire a better understanding of the MeHg global effects. Furthermore, this is the first time that the SAGE was used to characterize the effect of a toxicant at the genome scale in an aquatic organism.

Cadmium uptake by the European eel: Trophic transfer in field and experimental investigations

Due to its status of threatened species and being heavily contaminated by metals, the European eel (Anguilla anguilla) was selected to investigate cadmium contamination levels of fish settled along a historically cadmium-contaminated hydrosystem, the Garonne-Gironde continuum (France), according to its various location sites and fish length. Results have shown an important site effect on cadmium concentrations in liver but not in gills, highlighting the possible predominance of the trophic exposure route. Subsequently, uncontaminated eels were experimentally exposed to cadmium by water uptake and/or trophic route(s). Eels were fed with different preys: white shrimps collected in an unpolluted area in the Gironde estuary, and cadmium-enriched shrimps. Data obtained tend to show that the use of cadmium-enriched food during experimental investigations triggers an underestimation of the metal trophic transfer rate. These two complementary approaches provide some elements to suggest that the trophic route plays an important role in cadmium contamination of wild eels.

Transcriptome profile analysis reveals specific signatures of pollutants in Atlantic eels

Identifying specific effects of contaminants in a multi-stress field context remain a challenge in ecotoxicology. In this context, “omics” technologies, by allowing the simultaneous measurement of numerous biological endpoints, could help unravel the in situ toxicity of contaminants. In this study, wild Atlantic eels were sampled in 8 sites presenting a broad contamination gradient in France and Canada. The global hepatic transcriptome of animals was determined by RNA-Seq. In parallel, the contamination level of fish to 8 metals and 25 organic pollutants was determined. Factor analysis for multiple testing was used to identify genes that are most likely to be related to a single factor. Among the variables analyzed, arsenic (As), cadmium (Cd), lindane (γ-HCH) and the hepato-somatic index (HSI) were found to be the main factors affecting eel’s transcriptome. Genes associated with As exposure were involved in the mechanisms that have been described during As vasculotoxicity in mammals. Genes correlated with Cd were involved in cell cycle and energy metabolism. For γ-HCH, genes were involved in lipolysis and cell growth. Genes associated with HSI were involved in protein, lipid and iron metabolisms. Our study proposes specific gene signatures of pollutants and their impacts in fish exposed to multi-stress conditions.

Inorganic mercury detection by valve closure response in the freshwater clam Corbicula fluminea: Integration of time and water metal concentration changes

The objective of the present study was to monitor water-quality assessment by a biological method. Optimum dissolved inorganic mercury sensitivity in the freshwater bivalve Corbicula fluminea was estimated using a combined approach to determine their potentials and limits in detecting contaminants. Detection by bivalves is based on shell closure, a protective strategy when exposed to a water contaminant. To take the rate of spontaneous closures into account, stress associated with fixation by one valve in common valvometers was integrated, and the spontaneous rhythm was associated with daily activity. The response in conditions where the probability of spontaneous closing is the lowest was thus taken into account. To develop dose-response curves, impedance valvometry, in which lightweight impedance electrodes are applied to study free-ranging animals in low-stress conditions, also was used combined with a new analytical approach. The logistic regression dose-response curves take into account variations in both response time and metal concentration in water to significantly improve the methods aiming at determining the optimal sensitivity threshold response. This approach demonstrates that in C. fluminea, inorganic mercury concentrations under the range of 2.0 to 5.1 microg/L (95% confidence interval) cannot be detected within 5 h of addition.

Enzymatic and Polarographic Measurements of the Respiratory Chain Complexes

The different respiratory chain complexes are represented in Fig. 1. Their activity can be measured either separately or in groups, enzymatically (Sect. 3) or by the Polarographie measurement of oxygen consumption (Sect. 4). Classically, these measurements are made on isolated mitochondria. The isolation of muscular mitochondria from a small quantity of tissue (around 100 mg) is difficult, so two other methods of preparation may be used: (1) the preparation of a homogenate by potterisation of a biopsy fragment (between 20–100 mg) and (2) the permeabilization of muscular fibers (10 to 100 mg). The former is particularly useful for studying enzymatic activities of isolated complexes. The latter is useful in oxygraphy instead of isolated mitochondria. These techniques are completely described in Section 2.

Sequential Design for Microarray Experiments

This article features online supplementary material. A critical aspect in the design of microarray studies is the determination of the sample size necessary to declare genes differentially expressed across different experimental conditions. In this article, we propose a sequential approach where the decision to stop the experiment depends on the accumulated microarray data. The study could stop whenever sufficient data have been accumulated to identify gene expression changes across several experimental conditions. The gene expression response is modeled by a robust linear regression model. We then construct a sequential confidence interval for the intercept of this model, which represents the median gene expression at a given experimental condition. We derive the stopping rule of the experiment for both continuous and discrete sequential approaches and give the asymptotic properties of the stopping variable. We demonstrate the desirable properties of our sequential approach, both theoretically and numerically. In our application to a study of hormone responsive breast cancer cell lines, we estimated the stopping variable for the sample size determination to be smaller than the actual sample size available to conduct the experiment. This means that we can obtain an accurate assessment of differential gene expression without compromising the cost and size of the study. Altogether, we anticipate that this approach could have an important contribution to microarray studies by improving the usual experimental designs and methods of analysis.

Application of Quantile Regression to Recent Genetic and -omic Studies

This paper provides a review of recent applications of quantile regression to the fields of genetic and the emerging -omic studies. It begins with a general background about this statistical approach following the seminal paper of Koenker and Bassett (Econometrica 46:33–50, 1978). Applications are described, as diverse as genetic association studies, penetrance estimation, gene expression, CGH array experiments, RNAseq experiments, methylation data and proteomics. This paper also introduces recent extensions of quantile regression with a particular focus on the Copula-quantile regression, an approach we recently proposed for sib-pair analysis. A real data example from eQTL analysis is then presented and the