Accepted Manuscript Title: Effects of depleted uranium on the reproductive success and F1 generation survival of zebrafish (Danio rerio) Author: St´ephanie Bourrachot Franc¸ois Brion Sandrine Pereira Magali Floriani Virginie Camilleri Isabelle Cavali´e Olivier Palluel Christelle Adam-Guillermin PII: DOI: Reference:

S0166-445X(14)00147-7 http://dx.doi.org/doi:10.1016/j.aquatox.2014.04.018 AQTOX 3825

To appear in:

Aquatic Toxicology

Received date: Revised date: Accepted date:

15-1-2014 1-4-2014 12-4-2014

Please cite this article as: Bourrachot, S., Brion, F., Pereira, S., Floriani, M., Camilleri, V., Cavali´e, I., Palluel, O., Adam-Guillermin, C.,Effects of depleted uranium on the reproductive success and F1 generation survival of zebrafish (Danio rerio), Aquatic Toxicology (2014), http://dx.doi.org/10.1016/j.aquatox.2014.04.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Highlights for manuscript AQTOX-D-14-00033 The effect of depleted uranium on zebrafish reproduction was studied



An inhibition of egg production and an increase of F1 embryo mortality was observed 



Decreased circulating concentration of vitellogenin was observed in females 



Increased DNA damages were observed in parent gonads and in embryos 



U environmental concentration impairs reproduction and genetic integrity of fish

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

Stéphanie Bourrachota, François Brionb, Sandrine Pereiraa, Magali Floriania, Virginie Camilleria, Isabelle Cavaliéa, Olivier Palluelb, Christelle Adam-Guillermin*a.

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Institut de Radioprotection et de Sûreté Nucléaire (IRSN), PRP-ENV/SERIS/LECO, Cadarache, Saint-Paullez-Durance, 13115, France b Institut National de l’Environnement Industriel et des Risques (INERIS), Unité d’évaluation des risques écotoxicologiques, BP2, 60550 Verneuil-en-Halatte, France

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* Corresponding author: [email protected] Institut de Radioprotection et de Sûreté Nucléaire (IRSN), PRP-ENV/SERIS/LECO, Cadarache, Saint-Paul-lez-Durance, 13115, France Tel : +33 4 42 19 94 01 Fax : +33 4 42 19 91 51

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Abstract 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

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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

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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

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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

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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

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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

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highlight the negative effect of environmentally relevant concentrations of U which alter the

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reproductive capability of fish and impair the genetic integrity of F1 embryos raising further

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concern regarding its effect at the population level. Key words: uranium, zebrafish (Danio rerio), reproduction, genotoxicity, parental exposure.

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1. Introduction Uranium (U) is naturally found as a mixture of three isotopes:

234

U,

235

U, and

238

U. These

isotopes are all alpha emitters and contribute to 0.005%, 0.720% and 99.274% of natural

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uranium (NU) mass composition, respectively (Madic and Genet, 2001). U is naturally present in the earth’s crust at concentrations of 2 to 4 g t-1, and is dispersed throughout the

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biosphere in soil, water, air, plants, and animals through natural biogeochemical processes (Ribera et al., 1996; Bleise et al., 2003). Natural concentrations of U in water vary from a few

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ng L-1 to more than 12 mg L-1 (World Health Organization, 2001; Salonen, 1994). However, U concentrations can increase due to various anthropogenic contributions, such as industrial

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activities linked to nuclear fuel production, numerous military applications (Miller and McClain, 2007), or accidental discharge (Gagnaire et al., 2011). For example, high concentrations of U from 10 mg L-1 to 20 mg L−1 have been measured in water close to

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mining sites in the United States (Ragnarsdottir and Charlet, 2000). Depleted uranium (DU) is a byproduct of NU enrichment. Its widespread use in armor-penetrating weapons has raised environmental and human health concerns (World

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Health Organization, 2001). The (eco)toxicological risks associated with U potentially

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originate from both its chemical and radiological properties, depending on the specific activity of the different isotopes. However, for DU and NU, the risks are greater due to chemical

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rather than radiological toxicity (Mathews et al., 2009). The ecotoxicological effects of U are not fully known. Studies describing the effects of U in fish have primarily focused on bioaccumulation and acute toxicity induced by waterborne exposure to U. For instance, lethal concentrations at 50 % (LC50s) (96h) ranging from 0.7 to 135 mg L-1 have been reported depending on biotic (species, life stage) and abiotic factors (temperature, water hardness, pH) (Poston, 1982; Bywater et al., 1991; Labrot et al., 1999; Roex et al., 2002). More recently, it was shown that exposure of adult fish to low concentrations of U led to a broad range of biological responses in various target tissues including induction of oxidative stress (Labrot et al., 1999; Cooley et al., 2000, Barillet et al., 2007), genotoxicity (Barillet et al., 2005) and neurotoxicity (Lerebours et al., 2010). These studies are relevant with regard to the mechanisms of U toxicity at molecular and cellular levels, but they are not informative in terms of the impact on individuals or populations. Conversely, a study which would explore key developmental processes and critical steps in fish life cycle could provide such information. However, this type of information is s limited 4 Page 4 of 35

in the case of U. We recently showed that low concentrations of U affected the embryo-larval development of fish by negatively affecting their growth and survival (Bourrachot et al., 2008). To further understand the effect of U on fish life cycle, we studied the toxicity of low concentrations of U on fish reproduction, using the zebrafish as a model organism. This species is commonly used as a biological model in ecotoxicology (Nagel, 2002; Hill et al.,

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2005; McAleer et al., 2005), particularly in studies evaluating the reproductive effects of endocrine disrupting chemicals (Fenske et al., 2005). Endocrine effects can be assessed by

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monitoring vitellogenin (Vtg), a large serum phospholipoglycoprotein normally produced in the liver of female zebrafish in response to circulating endogenous estrogen (Han et al., 2011).

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It is a precursor of egg yolk proteins, and once produced in the liver, travels in the bloodstream to the ovary, where it is taken up and modified by developing eggs (Heppell et

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al., 1995). Little is known about the impact of U on reproduction and particularly whether it can affect Vtg production and gonad cells (structure and DNA integrity) in genitors. In addition, the mechanism by which U may decrease fertility and impact embryo survival has

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never been studied.

To study the effects of U on reproduction, male and female zebrafish were exposed over 20 days to two concentrations of U, respectively: 20 µg L-1, a low concentration close to the

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threshold concentration recommended by World Health Organization (15 µg L-1) for drinking

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water and 250 µg L-1, a concentration that is often measured close to U mining areas (Antunes et al., 2007). After exposure to DU, ecologically relevant parameters (laying, egg fertilization

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and survival) were recorded during a 14-day period in clean water. Furthermore, bioaccumulation of U was measured in the gonads of the genitors as well as in their progeny (at embryo and larval stages) and the F1 development was studied to assess whether maternal transfer of U can affect the F1 development.  

2. Materials and methods 2.1

Fish maintenance

The zebrafish (Danio rerio) was used as a test organism in this partial life cycle study. Adult mature fish (4 months-old) were obtained from Aquasylva, Pertuis, France and were maintained in aerated tap water at a mean density of 4 fish per liter. Water was manually renewed by changing 50% of the total volume each week. The tank was kept in a room with a 12/12 hour light/dark photoperiod and a temperature of 25 ± 1°C. Fish were fed with dry flake 5 Page 5 of 35

food (Tetramin®, Germany), twice a day supplemented with live neonates of Daphnia magna twice a week. Fish were gradually acclimatized to the artificial water used in the experiments (composition in mg L-1: K+ = 5.9; Na+ = 7.5; Mg2+ = 4.7; Ca2+ = 11.6; Cl- = 32.6; NO3- = 19.5; SO42- = 9.6; pH = 6.5 ± 0.2) for at least 3 weeks prior to the exposure phase. Water

2.2

Exposure conditions to depleted uranium

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and optimal U bioavailability (Denison, 2004).

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composition was a compromise between the conditions necessary for healthy fish physiology

Female and male zebrafish were exposed to 0, 20 and 250 µg L-1 of waterborne

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depleted uranium (UO2 (NO3)2 6H2O, Sigma Aldrich, France) for 20 days under the same experimental conditions as those used during the acclimation phase. Contaminated water was

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continuously renewed by means of a flow-through water system (Supporting information 1), ensuring a daily renewal of half the volume in each tank. During the experiment, temperature and pH were monitored once a day. pH was maintained at 6.5 by the addition of HNO3 (10-3

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M) via peristaltic pumps linked to pH stats (Consort R301, IllKirch, Belgium). Fish were fed twice a day with dry flake food (Tetramin®, Germany). To avoid the build up of excessive bacterial colonies, residual organic matter (food, feces) was removed daily by aspiration.

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Following 20 days exposure to U, fish were transferred into small spawning aquaria

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‘Reproductive Output’).

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containing clean water for 15 days to study their reproductive output (see the section on

2.3

Uranium quantification 2.3.1

Contaminated water

Raw water samples were collected twice a day. Uranium and major cation

concentrations were measured after acidification (2% v/v, HNO3 15.3 M) by inductively coupled plasma-atomic emission spectrometry (ICP-AES; Optima 4300 DV, Perkin Elmer, Wellesley, MA, USA – detection limits: U: 10 µg L-1; Mg2+: 1 µg L-1; Na+ and Ca2+: 5 µg L-1; K+: 10 µg L-1). Major anion concentrations were also analyzed by ionic chromatography (Dionex DX-120, Sunnyvale, CA, USA – detection limit = 100 µg L-1 for major anions) to monitor water quality. 2.3.2

Uranium quantification in organisms

Measurement of U in the whole fish and in gonads was carried out after 20 days of exposure (n = 3 for whole body and n= 9 for gonads) and after 15 days of reproduction in clean water (n = 3 for whole body and 10 for gonads). 6 Page 6 of 35

Just after dissection, tissue samples were dried until constant mass in a dry off oven (at least 48 h at 55°C) and weighed using a microbalance (ultra-microbalance, Sartorius, Göttingen, Germany, precision 0.1 µg). Tissues were then digested in 3 mL of HNO3 (15.3 M) over heating at 90°C (180 min) on a sand bath. After complete digestion, samples were then evaporated to incipient dryness

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(100°C). The digestion process was completed by the addition of 2 mL of H2O2 (1 M) and evaporation to incipient dryness (60 min, 100°C). Before measurement by inductively

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coupled plasma-mass spectrometry (ICP-MS; Varian 810-MS, detection limit: 10 ng L-1), acidified ultrapure water (2%, v/v, HNO3, 15.3 M) was added: 5 mL for whole body samples

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and 10 mL for tissue samples.

Uranium accumulation was also measured in eggs of the first laying in each condition and in

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eggs of the last laying. Samples were digested in 1 mL of HNO3 (15.3 M) and 1 mL of H2O2 (1 M). They were then dried on a sand bath (180 min, 90°C) and dissolved in 10 mL of acidified ultrapure water (2%, v/v, HNO3) before analysis by ICP-MS. In addition, U

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accumulation was measured 48 hours post fertilization (hpf) at the chorion and embryo levels after manual dissection using microscopic needles.

Before digestion and U measurements, all the samples were rinsed in 3 successive baths

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containing artificial clean water, placed in an aluminum pan and dried for 48 h at 55°C. After

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cooling, they were weighed using an ultra-microbalance (SE2 ultra-microbalance, Sartorius,

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Goettingen, Germany, precision of 0.1 µg). 2.4

Biological analysis 2.4.1 Reproductive output

Biometry

At the end of the exposure and reproduction periods, the length and weight of the fish were measured. The condition factor (K-factor) and the gonadosomatic index (GSI) were calculated according to the following formulae: K-factor = (weight (g) / (length (cm)) 3) × 100 GSI = (gonad weight (g) / somatic weight (g)) × 100 Reproductive performance

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In order to examine reproductive performance after 20 days of exposure to DU, 10 groups of 3 fish (male to female ratio of 2:1) in each condition were placed in small spawning aquaria, containing clean experimental water (Figure 1B). The reproductive performance was monitored daily for 15 days. Egg production in each female was observed every day to measure fecundity and fertility. Each lay was monitored individually, and both survival and

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Transmission Electron Microscopy (TEM)

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2.4.2 Histological analysis of the gonads

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hatchability were also observed daily during the 15-day period.

The effects of U on gonadal tissues were determined after the 20-day exposure period. Three

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fish from each sex and treatment were collected for histological analyses. The fish were captured and immediately sacrificed by immersion in melting water according to the ethical guidelines

displayed

and

used

by

the

NIH

intramural

research

program

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(http://oacu.od.nih.gov/ARAC/documents/zebrafish.pdf). Ovaries and testis were dissected and fixed for 24 h using 2.5% glutaraldehyde in 0.1 M cacodylate buffer at 4°C. The material was then postfixed in 1% osmium tetroxide in the same buffer, dehydrated in ethyl alcohol

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baths and embedded in monomeric resin (Epon 812) which polymerized at 70°C.

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Semi-thin sections of 500 nm for light microscopy and ultra-thin sections for TEM and EDX analysis (80 and 110 nm, respectively), were obtained using an ultramicrotome UCT (Leica

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Microsystem GmbH, France) equipped with a diamond knife. Observations were first carried out on semi-thin sections stained with aqueous blue toluidine and observed under a light microscope (Nikon, Eclipse E 400) to target tissues for TEM observations. For ultrastructural examination, ultra-thin sections were mounted on copper grids and observed with a Scanning Transmission Electron Microscope (TEM/STEM, Tecnai 12 G2 Biotwin, FEI Company, Eindhoven, Netherlands) using an accelerating voltage of 100 kV equipped with a CCD camera (Megaview III, Olympus Soft Imaging Solutions GmbH, Münster Germany). For each organ, at least 150 photographs of detailed structures were taken and compared. The presence of all stages of oocyte development in females was examined. Ovaries were analyzed by measurement of previtellogenic and vitellogenic oocytes (vitellogenic oocytes are defined as having a size of 0.05 to 0.2 mm2 according to Van der Ven 2003).

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2.4.3 Quantification of vitellogenin in whole-body homogenates Vitellogenin (Vtg) concentrations were measured in whole-body homogenates of fish using an homologous competitive zebrafish vitellogenin enzyme linked immuno-sorbent assay (zf-Vtg ELISA) as previously described (Brion et al., 2002). The assay is based on a competition reaction for the anti-vitellogenin antibodies (DR-264 polyclonal antibodies raised against

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zebrafish Vtg, zf-Vtg, Biosense Laboratories) between the Vtg present in the sample and the purified zf-Vtg coated on the microtiter plate. Calibration of the assay was performed using

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purified zf-Vtg as a standard curve (standard curve ranging from 0.1 to 125 ng/mL). Whole-body homogenization procedure

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Because the ovaries of maturating females are a source of the degradation products of Vtg (i.e. lipovitellin and phosvitin), the ovaries were removed for measurement of

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bioaccumulation. Then, 10 whole fish of each sex and at each concentration were homogenized in ELISA buffer (PBS, 1% BSA, PMSF 1 mM, pH 7.3) at a ratio of 1:2 (weight: volume). After centrifugation of the homogenates (3000 g, 15 min, 4°C), the

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supernatants were withdrawn, aliquoted and stored at – 80°C until assayed. 2.4.4 Genotoxic effects

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To determine if DNA damage was correlated to potential decreases in fertility or embryo first lay eggs.

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survival, we applied the alkaline Comet assay on freshly dissected ovaries and testes, and on

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Removal and tissue processing: For the Comet assay of the gonads, fish were collected after 20 days of U exposure and sacrificed by immersion in melting water as described above. Three replicates for each sex and concentration were dissected. For the first lay eggs (aged 24 hpf and 48 hpf), three lays were tested per concentration, and for each lay, ten eggs were pooled.

After dissection, gonads and eggs were immersed in a PBS solution (100 mM phosphate buffered saline supplemented with 0.02% EDTA) and kept at room temperature prior to dissociation protocols. Cell dissociation was then performed by mechanical homogenization. Homogenates were then filtered through a Nylon gauze (Ø = 100 µm for embryonic and ovary cells and Ø = 60 µm for testicular cells) and centrifuged (8°C, 10 min, 110 g). The supernatants were then removed and replaced by 1 mL of Leibovitz’s L15 cell culture medium supplemented with 10 mM HEPES to rinse the cells. A second centrifugation was performed under the same conditions. Finally, the supernatants were removed and replaced

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with L15-HEPES medium and the final cell suspensions were kept at 4°C until the Comet assay was carried out. Cells were counted on a Mallassez cell and their viability was assessed using trypan blue. The final density was adjusted to approximately 1.35 106 cells/mL (dilutions were made using L15-HEPES).

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The isolation and Comet assay procedures were validated using the known genotoxicant, hydrogen peroxide, at a range of concentrations on gonad cells and embryonic cells.

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Comet assay analysis: The alkaline Comet assay was carried out according to the procedure by Devaux et al. (1997), and a slightly modified version of the protocol described by Singh et

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al. (1988). An equal volume of each cell suspension and of 1% low melting point agarose (75 µL) was mixed and pipetted over a microscope slide (previously coated with 1% normal

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melting point agarose), and covered with a coverslip. Slides were placed on a flat tray and kept on ice for 5 min until the agarose solidified. The coverslips were removed and a final layer of 90 µL 0.5% low melting point agarose was dispensed on the cell-agarose layer,

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covered with a coverslip and allowed to solidify on ice. The slides were then immersed in a cold lysing solution (2.5 M NaCl, 0.1 M EDTA, 0.01 M Tris, 1% Triton X-100, 10% DMSO, pH adjusted to 10 with NaOH) for 1 h at 4°C. The slides were recovered, drained and

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transferred into an alkaline electrophoresis solution (0.3 M NaOH, 0.001 M EDTA, pH > 13)

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for 40 min to unwind the DNA. Electrophoresis was performed under a current of 20 V and 200 mA for 24 min. The slides were drained again and then neutralized with 0.4 M Tris-HCl

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(adjusted to pH 7.5 with HCl) for 5 min. This step results in bicatenar DNA, which permits insertion of ethidium bromide for observation under a fluorescence microscope. If the slides were not analyzed the same day, they were dehydrated for 10 min in absolute ethanol and stored at room temperature.

At least 100 nucleoids per slide were analyzed under a fluorescence microscope (Zeiss) equipped with a 350-390 nm excitation and 456 nm emission filter at x 400 magnification. Comet figures were analyzed using Comet IV software (Perceptive Instruments, Suffolk, UK) and DNA damage was quantified as tail intensity values. 2.5 Statistical analysis All analyses were performed using Statistica 7.1 software (Statsoft, 2005). When normality or/and variance homogeneity assumptions were not satisfied, the Box-Cox transformation was applied followed by ANOVA and the post-hoc Tukey or Dunnet test. When the transformation failed to improve residual normality and variance homogeneity, the Kruskal10 Page 10 of 35

Wallis test was used, followed by post hoc tests to perform multiple comparisons (Wilcoxon test).

3. Results

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U accumulates in the gonads of male and female zebrafish The U concentration measured in water was close to the nominal concentration for both experimental conditions (Table 1). The daily loss of U due to adsorption on tank walls was

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lower than 25% of the nominal concentration.

The results of U bioaccumulation in females and males after 20 days of exposure to U and

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after 15 days of reproduction in clean water are presented in Figure 1.

After 20 days of exposure, a significant increase in U accumulation was observed with

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increasing U concentration in the water, both in the whole body and in the gonads of males and females (Figures 1 A and 1B).

It should be noted that an error in sex determination at the beginning of the experiment

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resulted in a reduction in the number of replicates for the whole body U concentrations at day35: two replicates were used for females exposed to 20 µg L-1 and only one for females

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exposed to 250 µg L-1. However, the statistical analysis showed that U accumulation was higher in exposed fish than in fish in the control group and increased with exposure

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concentration at 20 days and 35 days (covariance analysis, p = 0.02). Indeed, the U concentration measured in the whole body of fish exposed to 20 µg U L-1 was 5 times higher

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and 21 times higher in females and males of the exposed group than in those of the control group; and was 64 and 72 times higher, respectively, for females and males at 250 µg U L-1. The BioConcentration Factor (BCF), indicating U transfer capacity from water to organism, was calculated at day 20 as the ratio between U concentration in the whole body (µg U kg-1 wet weight) and U concentration in water (µg U L-1). The BCF was 546 and 60 (w.w.) for females exposed to 20 and 250 µg U L-1, respectively, and 1514 and 50 (w.w.) for males exposed to 20 and 250 µg U L-1, respectively. A significant decrease in U accumulation was observed between the end of the exposure and the reproduction phases, i.e. between day 20 and day 35. This decrease was 37% and 57% for females and males exposed to 20 µg U L-1, and 92% and 82% for females and males exposed to 250 µg U L-1.

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Figure 1B presents U accumulation in the gonads of females and males after 20 days of exposure and after 15 days of reproduction and depuration in clean water. For the whole body, U accumulation increased significantly with U concentration in water. The statistical analysis revealed a concentration effect (covariance analysis, p < 0.001) and interactions between concentration and time factors (p = 0.01) and between concentration and sex factors (p

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 le...
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