Sébastien Cambier

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.

Impact of dietary gold nanoparticles in zebrafish at very low contamination pressure: The role of size, concentration and exposure time

Abstract The impact of a daily ration of food containing gold nanoparticles (AuNPs) of two sizes (12 and 50 nm) was investigated in the zebrafish Danio rerio at very low doses (from 36–106 ng gold/fish/day). AuNP exposure resulted in various dysfunctions at the sub cellular scale, and AuNP concentration in food, AuNP size and exposure duration modulated the observed adverse effects. Indeed, we showed alteration of genome composition using a RAPD-PCR genotoxicity test as the number of hybridization sites of the RAPD probes was significantly modified after AuNP exposure. Moreover, the expression of genes involved in DNA repair, detoxification processes, apoptosis, mitochondrial metabolism and oxidative stress was also modulated in response to AuNP contamination. Mitochondrial dysfunctions appeared in brain and muscle for both tested doses (40 and 100 ng gold/fish/day), but gold accumulation in fish tissues could only be observed in the case of the highest exposure dose.

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.

Methylmercury localization in Danio rerio retina after trophic and subchronic exposure: A basis for neurotoxicology

Methylmercury is a known neurotoxic organometal which affects visual functions and few studies concerns to wild fish are available. The autometallography mercury distribution in the retina of Danio rerio was mapped using light and electron microscopy. Abundant mercury deposits were found in the photoreceptor layer (outer and inner segments of the photoreceptors) and in the inner and outer nuclear layers. Occasionally, the presence of mercury deposits in plexiform layers was observed and very rarely in the ganglion cell layer. Also the occurrence of mercury deposits in cells from the disc region was observed, but not in the nerve fiber layer. An interesting difference was found between mercury accumulation in the central and peripheral regions of the retina. These results demonstrate that mercury after trophic exposure to Danio rerio is able to cross the blood-retina barrier and accumulate in the cells of the retina even under subchronic exposure.

Impact of dietary cadmium sulphide nanoparticles on Danio rerio zebrafish at very low contamination pressure

Abstract To address the impact of cadmium sulphide nanoparticles (CdSNPs) of two different sizes (8 and 50 nm), Danio rerio zebrafish were dietary exposed to very low doses: 100 or 40 ng CdSNPs/day/g body weight for 36 or 60 days, respectively. The results obtained using RAPD-PCR genotoxicity test showed genomic alteration since the number of hybridisation sites of the RAPD probes was significantly modified after CdSNPs exposure. In addition, selected stress response genes were either repressed or upregulated in tissues of CdSNPs-exposed fish. Mitochondrial dysfunction was also caused by the presence of CdSNPs in food. Cadmium accumulation in fish tissues (brain and muscles) could only be observed after 60 days of exposure. CdSNPs toxicity was dependent on their size and concentration.

Genotoxic effects of exposure to waterborne uranium, dietary methylmercury and hyperoxia in zebrafish assessed by the quantitative RAPD-PCR method

Release of chemicals and fluctuation in oxygen content in the aquatic environment represent hazards for fish health. The present study aims at assessing the genotoxic impact of low concentration exposures to waterborne uranium (U), dietary methyl mercury (MeHg) and hyperoxia in zebrafish by using the RAPD-PCR quantitative method. A significant increase of the number hybridization sites was observed in fish exposed to 30μgU/L and 100μgU/L and hyperoxia. In fish exposed to MeHg (13.5μg Hg/g, dry weight) no change in the number of hybridization sites were found, however, the frequency of PCR products showed significant variation. The mechanisms of toxicity leading to DNA damage in fish exposed to waterborne uranium, mercury and hyperoxia are discussed and the results from the literature given by the comet assay, micronucleus test and RAPD-PCR method compared. The study provides new data regarding the genotoxic effects of MeHg, hyperoxia and low U concentrations (30μgU/L) in fish. The present work highlights the use of the RAPD-PCR as a sensitive method in the assessment of chemically-induced DNA damage in animals.

Partial Inventory of ABCB and ABCC Transporter Genes Responding to Cadmium and Zinc Contamination in Zebrafish Danio Rerio

ABC transporters belonging to the subfamilies ABCB (MDR/TAP) and ABCC (CFTR/MRP) are likely to play a role in the detoxification of metallic pollutants. We made an inventory of the transcriptional response of 10 abcb and 9 abcc gene members in zebrafish Danio rerio exposed for 7 days to ionic cadmium (89 nM), zinc (7.3 μM), or a blend of both metals. These concentrations correspond to those found in a polluted tributary of the Lot River, France. The general trend was that cadmium is rather an up-regulator (but for high accumulation factors only) whereas zinc is rather a repressor. In muscles the expression pattern of ABC genes in response to metals appeared unpredictable since there was no relationship between differential expression and metal accumulation. Although no increase of zinc burden was observed in fish muscles and gills exposed to zinc, that metal repressed 9 and 4 ABC genes in muscles and gills, respectively. Despite a 3-fold increase in zinc burden in brain, it triggered the down-regulation of 3 ABC genes. Also, despite an accumulation factor of 7 in muscles, cadmium repressed 2 ABC genes in muscles. However, in gills and liver cadmium exposure caused the up-regulation of 4 and 6 ABC genes linked to accumulation factors of 33 and 25, respectively. Beside MDR- and MRP-transporter encoding genes, metals up regulated other genes encoding zebrafish homologues of TAP2 (abcb3 and abcb3L1), ATM1 (abcb7), M-ABC1 (abcb8), TAPL (abcb9), SUR1 (abcc8) and SUR2 (abcc9) transporters.

A likely placental barrier against methylmercury in pregnant rats exposed to fish-containing diets

Methylmercury (MeHg) taken up through fish consumption can be transferred from the mother to the fetus during pregnancy. In the present study, pregnant rat mothers were contaminated with environmentally relevant doses of 36 and 76 ng MeHg/g of food using diets containing naturally mercury-containing fish. Young female rats fed with fish-containing food after weaning showed decreased locomotion in Y maze for accumulated concentrations in brain as low as 75 ng Hg/g dry weight (15 ng Hg/g wet weight). Young female rats fed the control diet after weaning yet borne by mothers fed the diet containing 76 ng MeHg/g, presented a 58% reduced activity in the open-field labyrinth, meaning that the maternal exposure to fish-containing food exerted an effect in utero that lasted several weeks after birth. Newborns were protected against Hg exposure by the placental barrier since in newborns from mothers fed the diet containing 76 ng MeHg/g of food, the concentrations of Hg in brain, kidney, liver and skeletal muscles represented 12, 3, 21 and 18% of those of their mothers tissues, respectively. These results suggest the existence, at least in rats, of a threshold level in terms of MeHg exposure above which the placental barrier collapses.