Virginie Camilleri

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.

Mitochondrial energetic metabolism perturbations in skeletal muscles and brain of zebrafish (Danio rerio) exposed to low concentrations of waterborne uranium.

Anthropogenic release of uranium (U), originating from the nuclear fuel cycle or military activities, may considerably increase U concentrations in terrestrial and aquatic ecosystems above the naturally occurring background levels found throughout the environment. With a projected increase in the world-wide use of nuclear power, it is important to improve our understanding of the possible effects of this metal on the aquatic fauna at concentrations commensurate with the provisional drinking water guideline value of the World Health Organization (15 μg U/L). The present study has examined the mitochondrial function in brain and skeletal muscles of the zebrafish, Danio rerio, exposed to 30 and 100 μg/L of waterborne U for 10 and 28 days. At the lower concentration, the basal mitochondrial respiration rate was increased in brain at day 10 and in muscles at day 28. This is due to an increase of the inner mitochondrial membrane permeability, resulting in a decrease of the respiratory control ratio. In addition, levels of cytochrome c oxidase subunit IV (COX-IV) increased in brain at day 10, and those of COX-I increased in muscles at day 28. Histological analyses performed by transmission electron microscopy revealed an alteration of myofibrils and a dilatation of endomysium in muscle cells. These effects were largest at the lowest concentration, following 28 days of exposure.

Genotoxicity of uranium contamination in embryonic zebrafish cells.

Uranium is a metal used in the nuclear industry and for military applications. Studies on mammals have shown that uranium is genotoxic. However the molecular and cellular mechanisms responsible for the genotoxicity of uranium are poorly known for other types of vertebrates such as fish. Since unrepaired DNA double-strand breaks (DSBs) are considered to be key lesions in cell lethality, the activity of one of the major DSB-repair pathways, i.e. non-homologous end-joining (NHEJ), has been evaluated in embryonic zebrafish cells (ZF4) exposed to uranium. Genotoxicity of uranium in ZF4 cells was further assessed by comet and micronucleus assays. Exposure to uranium results in the production of DSBs a few hours after incubation. These breaks trigger the phosphorylation of H2AX proteins. We showed that the DNA-PK kinase activity, essential for NHEJ, is altered by the presence of uranium. The presence of uranium in cells disturbs but does not inhibit the repair rate of DSBs. Such a result suggests an impact of uranium upon the reparability of DSBs and the potential activation of alternative DSBs repair pathway leading to the propagation of possible misrepaired DSBs. In parallel, we performed a transmission electron microscopy analysis of cells exposed to uranium and were able to localize internalized uranium using an Energy Dispersive X-ray microanalyser. We observed the formation of precipitates in lysosome-like vesicles for 250 μM of uranium in the medium. The appearance of these precipitates is concomitant with the decrease of the number of DSBs per cell. This process might be a part of a defence system whose role in counteracting cytotoxicity calls for further dedicated research.

Effects of uranium uptake on transcriptional responses, histological structures and survival rate of the crayfish Procambarus clarkii

This work aims to investigate the accumulation levels and effects (transcriptional responses, histopathology and survival rate) associated with a wide range of dissolved uranium (U) concentrations (0, 0.03, 0.6, 4 and 8 mg/L of U) on adult male crayfish Procambarus clarkii during 4 (T4) and 10 (T10) days of exposure. The follow-up of the crayfish mortality showed that P. clarkii was highly resistant to U. Increasing waterborne U concentrations led to increasing bioaccumulation in key crayfish organs and increasing histological damages. U distribution in tissues was also evaluated using transmission electron microscopy and showed the presence of a detoxified form of U in the gills epithelium in the shape of flakes. Expression levels of mitochondrial genes (cox1, atp6 and 12S gene) and genes involved in oxidative stress (sod(Mn) and mt) were examined together with the housekeeping gene 18S. atp6 and mt genes of P. clarkii were cloned and sequenced before analysis. Significant correlations were observed between U bioaccumulation and the down-regulation of both cox1 and sod(Mn) genes. This work provides a first U toxicogenomic and histopathological pattern of P. clarkii, identify U biomarkers and associate gene expression endpoints to accumulation levels. It also provides new insights into the mechanisms involved in U stress.

Sublethal effects of waterborne uranium exposures on the zebrafish brain:transcriptional responses and alterations of the olfactory bulb ultrastructure

The toxic action modes of uranium (U) in fish are still scarcely known. U is known to modify the acetylcholinesterase activity in the fish brain. To gain further insight into U neurotoxicity in fish, we examined transcriptional responses in the brain of the zebrafish, Danio rerio, exposed to 15 microg L(-1) and 100 microg L(-1) of waterborne U for 3 and 10 days. In parallel, an ultrastructure analysis of the neuropil of the olfactory bulb, an area in the brain of fish sensitive to metal contamination, was performed after 10 days of U exposure. This combined transcriptomic and histological study is the first report performed in the brain and specifically the olfactory bulb of fish exposed to U. We found that 56 transcripts responded to the metal exposure, and the anatomical structure of the olfactory bulb was damaged. The greatest gene response occurred at the lower U concentration and the numbers of responding genes common to any two U exposures were much smaller than those unique to each exposure. These data showed that the intensity of gene response may not correlate positively with toxicant concentrations according to our experimental design. Instead, different patterns of gene expression are expected for each exposure. Gene responses were categorized into eight functional classes, and the transcriptional responses of genes involved in the olfactory system were significantly affected. Collectively, the data suggest that genes in the olfactory region may be ecologically relevant and sensitive transcriptional biomarkers of U waterborne exposure.

Internal distribution of uranium and associated genotoxic damages in the chronically exposed bivalve Corbicula fluminea

Uranium (U) internal distribution and involved effects in the bivalve Corbicula fluminea have been studied after direct chronic exposure (90 d, 10 μg.L-1). U distribution was assessed at the subcellular level (Metal Rich Granules -MRG-, pellets and cytosol fractions) in two main organs of the bivalve (gills and visceral mass). Micro-localisation was investigated by TEM-EDX analysis in the gills epithelium. DNA damage in gill and hemolymph samples was measured by the Comet assay. The 90-d exposure period led to a significant increase of U concentration in gills over time (× 5) and a large U quantity in subcellular granules in gills. Finally, a significant increase (× 2) in DNA damage was noted in exposed gills and haemocytes. This study shows that the accumulation levels and consequently the potential toxicity cannot be successfully predicted only on the basis of concentration in water or in tissues and subcellular fractions after chronic exposure.

Effects of depleted uranium on the reproductive success and F1 generation survival of zebrafish (Danio rerio)

Despite the well-characterized occurrence of uranium (U) in the aquatic environment, very little is known about the chronic exposure of fish to low levels of U and its potential effect on reproduction. Therefore, this study was undertaken to investigate the effects of environmental concentrations of depleted U on the reproductive output of zebrafish (Danio rerio) and on survival and development of the F1 embryo-larvae following parental exposure to U. For that purpose, sexually mature male and female zebrafish were exposed to 20 and 250 μg/L of U for 14 days and allowed to reproduce in clean water during a further 14-day period. At all sampling times, whole-body vitellogenin concentrations and gonad histology were analyzed to investigate the effects of U exposure on these reproductive endpoints. In addition, accumulation of U in the gonads and its genotoxic effect on male and female gonad cells were quantified. The results showed that U strongly affected the capability of fish to reproduce and to generate viable individuals as evidenced by the inhibition of egg production and the increased rate of mortality of the F1 embryos. Interestingly, U exposure resulted in decreased circulating concentrations of vitellogenin in females. Increased concentrations of U were observed in gonads and eggs, which were most likely responsible for the genotoxic effects seen in fish gonads and in embryos exposed maternally to U. Altogether, these findings highlight the negative effect of environmentally relevant concentrations of U which alter the reproductive capability of fish and impair the genetic integrity of F1 embryos raising further concern regarding its effect at the population level.

Comparative genotoxicity of aluminium and cadmium in embryonic zebrafish cells.

Aluminium is a toxic metal whose genotoxicity has been scarcely studied in aquatic species and more generally in mammals. Recently, human and ecological disaster caused by the discharge of red mud in Hungary has revived questions about the toxicity of this metal particularly for the environment. On the contrary, cadmium is a highly toxic metal whose genotoxicity has been well characterized in various mammalian cells. However on non-human cells, little is known about its impact on DNA damage and repair. In this study, the genotoxic potential of both metals on embryonic zebrafish cells ZF4 was analyzed and particularly the impairment of the major DNA double strand breaks (DSB)-repair pathway, i.e. non-homologous end-joining (NHEJ). To this aim, DNA single strand breaks (SSB) and DSB were evaluated using the comet assay and the immunodetection of γ-H2AX proteins, respectively, in AlCl(3) or CdCl(2) exposed ZF4 cells. These exposures result in the production of DSBs a few hours after incubation. The DNA-PK kinase activity, essential for NHEJ, is more affected by the presence of aluminium than cadmium. Altogether our data provide evidence of the high toxicity induced by aluminium in zebrafish and indicates the pertinence of genotoxicity evaluation in organisms living in contaminated water.

Transmission of DNA damage and increasing reprotoxic effects over two generations of Daphnia magna exposed to uranium.

This study aimed to examine the mechanisms involved in the transgenerational increase in Daphnia magna sensitivity to waterborne depleted uranium (DU) under controlled laboratory conditions. Daphnids were exposed to concentrations ranging from 2 to 50 μg L(-1) over two successive generations. Genotoxic effects were assessed using random amplified polymorphic DNA and real time PCR (RAPD-PCR). Effects on life history (survival, fecundity and somatic growth) were monitored from hatching to release of brood 5. Different exposure regimes were tested to investigate the specific sensitivity of various life stages to DU. When daphnids were exposed continuously or from hatching to deposition of brood 5, results demonstrated that DNA damage accumulated in females and were transmitted to offspring in parallel with an increase in severity of effects on life history across generations. When daphnids were exposed during the embryo stage only, DU exposure induced transient DNA damage which was repaired after neonates were returned to a clean medium. Effects on life history remained visible after hatching and did not significantly increase in severity across generations. The present results suggest that DNA damage might be an early indicator of future effects on life history.

Use of combined microscopic and spectroscopic techniques to reveal interactions between uranium and Microbacterium sp. A9, a strain isolated from the Chernobyl exclusion zone

Although uranium (U) is naturally found in the environment, soil remediation programs will become increasingly important in light of certain human activities. This work aimed to identify U(VI) detoxification mechanisms employed by a bacteria strain isolated from a Chernobyl soil sample, and to distinguish its active from passive mechanisms of interaction. The ability of the Microbacterium sp. A9 strain to remove U(VI) from aqueous solutions at 4 °C and 25 °C was evaluated, as well as its survival capacity upon U(VI) exposure. The subcellular localisation of U was determined by TEM/EDX microscopy, while functional groups involved in the interaction with U were further evaluated by FTIR; finally, the speciation of U was analysed by TRLFS. We have revealed, for the first time, an active mechanism promoting metal efflux from the cells, during the early steps following U(VI) exposure at 25 °C. The Microbacterium sp. A9 strain also stores U intracellularly, as needle-like structures that have been identified as an autunite group mineral. Taken together, our results demonstrate that this strain exhibits a high U(VI) tolerance based on multiple detoxification mechanisms. These findings support the potential role of the genus Microbacterium in the remediation of aqueous environments contaminated with U(VI) under aerobic conditions.