Arch Environ Contam Toxicol DOI 10.1007/s00244-014-0022-x

Depleted Uranium Disturbs Immune Parameters in Zebrafish, Danio rerio: An Ex Vivo/In Vivo Experiment Be´atrice Gagnaire • Anne Bado-Nilles Wilfried Sanchez

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Received: 7 November 2013 / Accepted: 18 March 2014 Ó Springer Science+Business Media New York 2014

Abstract In this study, we investigated the effects of depleted uranium (DU), the byproduct of nuclear enrichment of uranium, on several parameters related to defence system in the zebrafish, Danio rerio, using flow cytometry. Several immune cellular parameters were followed on kidney leucocytes: cell proportion, cell mortality, phagocytosis activity and associated oxidative burst and lysosomal membrane integrity (LMI). Effects of DU were tested ex vivo after 17 h of contact between DU and freshly isolated leucocytes from 0 to 500 lg DU/L. Moreover, adult zebrafish were exposed in vivo during 3 days at 20 and 250 lg DU/L. Oxidative burst results showed that DU increased reactive oxygen species (ROS) basal level and therefore reduced ROS stimulation index in both ex vivo and in vivo experiments. ROS PMA-stimulated level was also increased at 250 lg DU/L in vivo only. Furthermore, a decrease of LMI was detected after in vivo experiments.

B. Gagnaire (&) Institut de Radioprotection et de Surete´ Nucle´aire (IRSN), PRPENV, SERIS, LECO, Centre de Cadarache, Baˆt 186, B.P. 3, 13115 Saint-Paul-lez-Durance, France e-mail: [email protected] A. Bado-Nilles UMR-I 02 (INERIS-Universite´ Reims Champagne-Ardenne Universite´ du Havre), SEBIO Stress environnementaux et Biosurveillance des milieux aquatiques, Universite´ de Reims Champagne-Ardenne, Campus Moulin de la Housse, BP 1039, 51687 REIMS cedex 2, France A. Bado-Nilles  W. Sanchez UMR-I 02 (INERIS-Universite´ Reims Champagne-Ardenne, Universite´ du Havre) SEBIO Stress environnementaux et Biosurveillance des milieux aquatiques, Institut National de l’Environnement Industriel et des Risques (INERIS), Unite´ d’e´cotoxicologie in vitro et in vivo, B.P. 2, 60550 Verneuil-en-Halatte, France

Cell mortality was also decreased at 20 lg DU/L in ex vivo experiment. However, phagocytosis activity was not modified in both ex vivo and in vivo experiments. A reduction of immune-related parameters was demonstrated in zebrafish exposed to DU. DU could therefore decrease the ability of fish to stimulate its own immune system which could, in turn, enhance the susceptibility of fish to infection. These results encourage the development and the use of innate immune analysis by flow cytometry in order to understand the effects of DU and more generally radionuclides on fish immune system and response to infectious diseases.

Uranium (U) is a member from the actinide series. This element is widely distributed throughout the biosphere in rocks, soil and water (Ragnarsdottir and Charlet 2000). U is ubiquitous in natural waters at concentrations ranging from 20 ng/L to 6 lg/L depending on the composition of surrounding rocks and up to 1 mg/L at the vicinity of uraniferous sites (Bonin and Blanc 2001). Various anthropogenic activities, involving the processing or use of materials enriched in U, may considerably enhance the natural abundance of U in environmental compartments (Bonin and Blanc 2001). Depleted uranium (DU), the byproduct of nuclear enrichment of U, is commonly used in military, aviation, medical and research fields. Freshwater ecosystems may constitute the final receptor areas of DU to which they may be chronically exposed both to its chemical (van Dam et al. 2002) and radiological (Thomas 2000) toxicity, its chemical toxicity being more important than its radiological one. Among all physiological processes possibly disturbed by pollutants, the immune system is likely to be one of the more sensitive physiological systems (Wong et al. 1992).

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Pollutants can interact with immune system components and interfere with protection functions and are therefore referred to as immunotoxics (Colosio et al. 2005). Xenobiotics can induce immunosuppression or stimulation, auto-immunity and decrease of disease resistance (Wong et al. 1992). Fish are widely used as bioindicators of the quality of the environment (van der Oost et al. 2003). Fish innate immune responses, which are the first line of immune system defence of these organisms (Lage et al. 2006), are performed by several kinds of leucocytes mainly produced in anterior kidney (Whyte 2007). Leucocytes are responsible for phagocytosis, one of the most important defence response of the organism; leucocytes can also release reactive oxygen species (ROS) from lysosomes, which eliminate foreign agents (Neumann et al. 2001). These responses may be suppressed by xenobiotics and seem to represent relevant immunotoxic endpoints (Bols et al. 2001). The effects of DU on immune system in animals are poorly known. In rodents, DU accumulated in macrophage lysosomes (Kalinich et al. 2002). DU altered cytokine and chemokine production (Wan et al. 2006) and inflammatory response and oxidative stress in lung (Monleau et al. 2006). Studies have reported effects of DU on the rat immune intestine functions. DU accumulated in the mesenteric lymph nodes and induced some changes in the production of cytokines (Dublineau et al. 2006a, b). Moreover, DU increased lipid oxidation in rat brains (Briner and Murray 2005). Data on effects of DU on fish immune system are scarce. In a previous study, we measured the effects of DU on several defence related parameters of zebrafish, Danio rerio (Gagnaire et al. 2013). We demonstrated that DU at 20 lg/L increased kidney ROS production and modified the serum phenoloxidase (PO)-like activity. Other studies showed that DU can induce an oxidative stress in fish: perturbation in antioxidant defence enzyme levels (superoxide dismutase and catalase) in rainbow trout, Oncorhynchus mykiss (Buet et al. 2005), increase of serum lipid peroxidation in the lake whitefish, Coregonus clupeaformis (Cooley et al. 2000) and transient modulation of antioxidant defence enzyme levels and lipid peroxidation in zebrafish (Labrot et al. 1996; Barillet et al. 2007, 2011). Moreover, DU affected the expression of genes involved in oxidative stress in zebrafish (Lerebours et al. 2009). Overall, DU effects on fish immune system are poorly documented. Several parameters appear to be relevant biomarkers of immune system. Lysosomal membrane stability is one of the most developed biomarker in immunotoxicology (van der Oost et al. 2003), in fish and in invertebrates (Zhou et al. 2008). The assessment of this parameter may offer a simple and rapid way of assessing individual ‘‘health status’’ (Moore et al. 2004). Moreover, it

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has been accepted by the OSPAR convention for the evaluation of environmental quality (ICES 2010). Defence mechanisms may prevent infections by cellular reactions such as ROS production, regulated by antioxidant enzymes (Mates 2000). Like any other metal, U is able to chemically activate the production of ROS in redox reactions (Ercal et al. 2001). The measurement of phagocytosis using flow cytometry is one of the most relevant parameter to assess the status of the immune system in invertebrates (Goedken and De Guise 2004) and fish (Chilmonczyk and Monge 1999; Harford et al. 2006). Phagocytosis is often modulated by immunotoxic pollutants in fish (Bols et al. 2001). Flow cytometry is a routine tool in vertebrate biomedical research and has been applied in the past decade to ecotoxicology, allowing a rapid and strong analysis of morphological characteristics of cell suspensions, due to the important number of cell analysed. This original tool is very performant and allows to better understand the mode of actions of pollutants than other non cytometric tools because several parameters can be tested on the same sample. Flow cytometry has been used to describe population characteristics in fish (Inoue et al. 2002; Llorente et al. 2002) as well as changes associated with contaminants (Keller et al. 1999; Bado-Nilles et al. 2009). Moreover, flow cytometry has already been used on adult zebrafish blood cells or kidney cells for assessing cell populations (Traver et al. 2003; Paffett-Lugassy and Zon 2005; Rodriguez et al. 2008), cell mortality (Ivanovski et al. 2009) or phagocytosis (Kaplan et al. 2008; Hohn et al. 2009), but never in the field of zebrafish immunotoxicology. In this context, the present study aimed to improve our knowledge on the immunotoxic properties of DU using flow cytometry. We conducted two separate experiments on zebrafish, a model organism in various ecotoxicological studies (OECD 2004; Hill et al. 2005), in order to assess DU effects on fish immune system. Kidney leucocytes were acutely exposed ex vivo during 17 h at 0–20– 250–500 lg DU/L. Adults were exposed in vivo during 3 days at 0–20–250 lg DU/L. Several parameters (cell proportion, cell mortality, oxidative burst, lysosomal membrane integrity (LMI), phagocytosis activity) were followed.

Materials and Methods Fish were killed in agreement with the ethical guidelines displayed and used by the NIH intramural research program (http://oacu.od.nih.gov/ARAC/documents/Zebrafish. pdf). A person possessing a French official diploma related to laboratory animals handling and care realized the euthanasia.

Arch Environ Contam Toxicol

Zebrafish Maintenance During acclimation, 110 adult male zebrafish of 9 months (500.3 ± 15 mg, 3.6 ± 0.04 cm, obtained from ‘‘Elevage de la Grande Riviere’’, Saint Forgeux, France) were maintained in 100 L of water in an aerated holding tank with artificial water. The artificial water composition (in mg/L: K? = 5.94; Na? = 7.46; Mg2? = 4.73; Ca2? = 11.58; Cl- = 32.57; NO32- = 19.53; SO42- = 9.56; pH 6.5 ± 0.2) results in a compromise between the conditions necessary for healthy fish physiology and the optimal U bioavailability (Denison and Garnier-Laplace 2005). Water was manually renewed by changing 50 % of the total volume each week and by refilling the evaporated water each day. The tanks were kept in a room with a 12/12 h light/dark photoperiod and a temperature of 27 ± 1 °C. Fish were fed daily with commercial flake food corresponding to 2 % of the fish body weight (Tetramin, France). After 2 weeks of acclimation, 20 fish were used for ex vivo contamination and 90 fish were used for in vivo exposure. Exposure Condition Concerning ex vivo experiment, three different concentrations of DU were tested: 20, 250 and 500 lg/L. A control (0 lg DU/L) was added. Each concentration was prepared by dilution in HNO3 acidified water 0.2 %. Five lL of DU were added individually at to 450 lL of kidney leucocyte suspension. Samples were incubated at 20 °C during 17 h before analysis of immune parameters by flow cytometry. For the in vivo contamination, two different concentrations of DU were tested: 20 and 250 lg/L. A control (0 lg DU/L) was added. The 90 fish were randomly distributed into one control and two exposed tanks on the basis of 30 fish per 30 L tank (density of 0.50 g of fish/L). This system was placed in a thermoregulated room (21 ± 1 °C) and was filled by artificial water (see composition in 2.2) maintained at 27 ± 1 °C, and a pH of 6.5 ± 0.2. To maintained DU concentration throughout the 3 days of exposure, water was manually renewed by changing every day 50 % of the total volume and DU concentrations were measured on 10 mL water samples (see ‘‘Chemical analyses’’ section) and compensate daily by manually addition of DU solution after water renewal and continuously by peristaltic pump with concentrated solutions (0.2 and 2 mg/L). Fish were fed daily (see ‘‘Zebrafish maintenance’’ section). At the end of the exposure period, all fish were killed within seconds by immersion in ice-cold water for kidney leucocyte sampling. Leucocyte Isolation and Analysis by Flow Cytometry After fish sacrificed, kidney tissues were removed and gently pressed through sterilized nylon mesh (40 lm, Becton–

Dickinson) with Leibovitz 15 (L15) medium (Sigma) containing heparin lithium (10 U/mL, Sigma), penicillin (500 U/mL, Biochrom AG) and streptomycin (500 lg/mL, Biochrom AG) to obtain leucocyte suspension. Leucocytes were stored during 12 h at 4 ± 1 °C before performing immune cellular responses for in vivo and were used immediately for ex vivo experiments. After storage, L15 medium-diluted samples were loaded onto Ficoll gradient (HistopaqueÒ1077, density of 1.077 g/mL, Sigma). After centrifugation (400 g, 30 min, 15 °C), leucocytes enriched suspensions were collected and washed twice in L15 medium to remove thrombocytes (300 g, 5 min, 4 °C). Then, leucocytes were adjusted to 5 9 105 cells mL-1 with Malassez haemocytometer to perform cytometry analyses. Cellular analyses were carried out on kidney leucocyte suspension using a Guava Ò EasyCyteTM 8HT (Millipore). For each leucocyte sample, 5,000 cells were counted. All assays were performed in 200 lL of cell suspension. Leucocyte proportion (lymphocytes and granulocytesmacrophages) was obtained using FSC and SSC parameters for size and complexity, respectively (Scharsack et al. 2004) (Fig. 1). Cellular mortality was detected by adding 1 lL of Propidium Iodide (PI, 1.5 mM in water, Invitrogen) in order to obtain cellular fluorescence parameters indicating the presence of necrotic leucocytes after 10 min of incubation on ice (Scharsack et al. 2004; Ivanovski et al. 2009) (Fig. 1). Determination of ROS by unstimulated cell was performed using 20 -70 -dichlorofluorescin diacetate (H2DCFDA, Sigma, 60 lM) to establish the basal level of ROS production after 30 min of incubation at room temperature. Leucocytes were also stimulated with phorbol-12myristate-13-acetate (PMA, Sigma, 16 lM) to determine the stimulated level of ROS production. Stimulation index of respiratory burst was determined as the ratio of fluorescence of PMA stimulated cells to that of unstimulated cells (Chilmonczyk and Monge 1999). Acridine orange (AO, Sigma, 10 lM) was used to determine the LMI as previously described after 20 min of incubation in the dark and at room temperature (Bado-Nilles et al. 2013). Phagocytosis percentage was assessed using fluorescent beads (FluoSpheresÒ carboxylate-modified microspheres, 1.0 lm, yellow-green fluorescent (505/515), Invitrogen) (Gagnaire et al. 2006). Cells were incubated with diluted beads (ratio beads: cells 10:1) during 1 h at room temperature. Only the events showing a fluorescence of at least two beads were considered positive for phagocytic activity. Results were expressed as percentage of positive cells for leucocyte proportion, mortality and phagocytosis. Concerning ROS production and LMI, they were measured as mean fluorescence intensity (MFI) on green and red fluorescence, respectively. All of these parameters were measured for both experiments, except LMI in ex vivo experiment due to not sufficient cell suspension volume.

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Fig. 1 Typical flow cytometry plots of leucocyte mortality percentage from zebrafish, Danio rerio, kidney. On the left, the x axis corresponds to forward scatter (log. scale) and the y axis to side scatter (log. scale). This cytogram allows selecting the cells that will be used for the analyses (R1 population). R7 and R8 correspond to

granulocyte–macrophage and lymphocyte populations, respectively. On the right, the histogram shows the percentage of mortality measured on R1 population stained with Propidium Iodide (Invitrogen)

Chemical Analyses

concentrations of 20, 250 and 500 lg/L are used in the further course of the publication. DU was not detected in the control. No significant effect of DU was detected on leucocyte proportion and phagocytosis activity (Table 1). Lymphocyte and granulocyte–macrophage percentages were comprised between 55.4 ± 1.1 to 56.9 ± 1.8 and 42.7 ± 1.8 to 44.3 ± 1.1 %, respectively. Phagocytosis activity was comprised between 28.8 ± 1.2 and 31.9 ± 1.0 % (Table 1). Cellular mortality was significantly lower in cells exposed to 20 lg DU/L (5.9 %) compared to control and cells exposed to 0, 250 and 500 lg DU/L (comprised between 7.9 ± 0.8 and 9.1 ± 0.7 %) (one-way ANOVA, p = 0.012) (Table 1). ROS basal level was significantly lower in control (12.2 MFI) compared to cells exposed to 250 and 500 lg DU/L (19.2 ± 2.3 and 20.4 ± 3.2 MFI, respectively; p = 0.027) (Table 1). ROS stimulated level showed no significant difference between DU concentrations and values were comprised between 7.5 ± 1.1 and 9.3 ± 0.7 MFI (Table 1). ROS stimulation index was significantly higher in controls (0.9) compared to cells exposed to 20, 250 and 500 lg DU/L (comprised between 0.43 ± 0.1 and 0.48 ± 0.1; p = 0.029) (Table 1). Lymphocyte percentage was negatively correlated to granulocyte–macrophage percentage. ROS basal and stimulated levels and ROS index were correlated. Finally, phagocytosis percentage was negatively correlated to ROS stimulated level (Table 2).

The exposure conditions were controlled by frequent measurements of DU in water (2/day). Samples (10 mL of each tank) were acidified with HNO3 (2 %, v/v) and directly analysed by 238U quantification using inductively coupled plasma-atomic emission spectrometry (ICP-AES Optima 4300DV, PerkinElmer, Wellesley, MA, USA; detection limit: 10 lg/L). Statistical Analyses Results were expressed as mean ± standard error (se). Normality assumption was verified through Shapiro–Wilk tests. When necessary, data were transformed (boxcox) to achieve normality. Differences between conditions were tested with one way ANOVA followed by an a posteriori least significant difference (LSD) post hoc test in the case of rejection of H0. When data were not normal, differences between conditions were assessed using a Kruskal–Wallis test. Spearman correlations were searched between parameters in ex vivo and in vivo experiments. Analyses were performed using STATISTICA Software version 7.1 (StatSoft, Inc., Tulsa, OK, USA). Significance was set at p B 0.05.

Results Ex vivo Experiment

In vivo Experiment Measured DU concentrations differed slightly from nominal concentrations and were 16.7, 277.8 and 555.5 lg/L. For the ease of presentation, only the nominal

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Fish mortality during the acclimatation and contamination periods were \5 % for all conditions (data not shown). No

Arch Environ Contam Toxicol Table 1 Effects of DU on immune leucocyte parameters during the ex vivo experiment after 17 h of contact with 0, 20, 250 and 500 lg/L [DU] (lg/L)

Cell mortality (%)

0

7.9 ± 0.8b

Lymphocyte percentage (%)

Granulocyte percentage (%)

ROS basal level (MFI)

ROS stimulated level (MFI)

ROS index

Phagocytosis percentage (%)

55.8 ± 0.8

43.8 ± 0.3

12.2 ± 1.1a

9.3 ± 0.7

0.90 ± 0.1b

30.9 ± 1.1

7.6 ± 1.1

0.46 ± 0.1a

28.8 ± 1.2

7.5 ± 1.1 7.7 ± 1.0

0.43 ± 0.1a 0.48 ± 0.1a

29.1 ± 0.8 31.9 ± 1.0

20

a

ab

5.9 ± 0 7

55.4 ± 1.1

44.3 ± 1.1

16.5 ± 1.1

250 500

8.0 ± 0.7b 9.1 ± 0.7b

56.9 ± 1.8 56.5 ± 1.7

42.7 ± 1.8 43.1 ± 1.8

19.2 ± 2.3b 20.4 ± 3.2b

Values are means of 20 replicates, except for ROS values for technical reasons (n comprised between 8 and 14); standard error is presented a,b

significantly different from control at p \ 0.05; a \ b. When no letters are added, no significant differences were found

Table 2 Correlations between immune leucocyte parameters during the ex vivo experiment

Cell mortality Lymphocyte percentage Granulocyte percentage ROS basal level

* Represent significant values (p \ 0.05)

Lymphocyte percentage

Granulocyte percentage

ROS basal level

ROS stimulated Ievel

ROS index

-0.160 0.155 -0.140*

-0.998* 0.045

-0.038

ROS stimulated level

0.032

-0.047

0.055

-0.131

ROS index

0.116

-0.062

0.067

-0.760*

0.673

Phagocytosis percentage

0.142

-0.111

0.108

-0.149

-0.322*

Table 3 DU water concentrations in tanks during the in vivo experiment Time (hours)

0.5

2.5

5

23

29

47

53

71

Concentration (20 lg/L)

0

38

36

30

28

20

17

22

Concentration (250 lg/L)

290

265

268

300

280

300

220

240

The DU concentration was assessed regularly within the tanks in order to compensate for the DU concentration decrease (mainly due to metal adsorption on the tank walls) by adding a fresh quantity of a concentrated solution of DU

DU was detected in water of the control fish during the whole experiment (\detection limit). The kinetic of DU concentration in contaminated tank showed a pattern of oscillations around the nominal value (Table 3). The record of the DU values over 3 days allowed the calculation of the mean DU concentrations, which differed slightly from nominal concentrations and were 23.9 ± 12.2 and 270.4 ± 28.6 lg DU/L for both concentrations. For the ease of presentation, only the nominal concentrations of 20 and 250 lg/L are used in the further course. No significant effect of DU was detected on leucocyte proportion and phagocytosis activity (Table 4). Lymphocyte and granulocyte–macrophage percentages were comprised between 62.8 ± 1.6 to 63.7 ± 1.7 and 36.0 ± 1.8 to 36.7 ± 1.6 %, respectively. Phagocytosis activity was

-0.156*

comprised between 18.1 ± 0.5 and 18.9 ± 0.3 % (Table 4). LMI was significantly reduced in fish exposed at 250 lg DU/L (130.3 MFI) compared to controls and fish exposed to 20 lg DU/L (167.1 ± 8.7 and 149.8 ± 7.3 MFI, respectively; p = 0.003) (Table 4). ROS basal level was significantly lower in controls (5.8 MFI) compared to fish exposed to 20 and 250 lg DU/L (12.2 ± 1.1 and 14.9 ± 1.4 MFI, respectively; p \ 0.001) (Table 4). ROS stimulated level was significantly enhanced in fish exposed to 250 lg DU/L (17.3 ± 1.2 MFI) compared to controls and fish exposed to 20 lg DU/L (12.9 ± 3.5 and 11.5 ± 1.3 MFI, respectively; p = 0.006) (Table 4). Finally, ROS stimulation index was significantly higher in controls (2.3) compared to fish exposed to 20 and 250 lg DU/L (1.0 ± 0.1 and 1.2 ± 0.1, respectively) (Kruskal– Wallis, p \ 0.001) (Table 4). As for the ex vivo experiment, lymphocyte percentage was negatively correlated to granulocyte–macrophage percentage (Table 5). LMI was found to be negatively correlated to cell mortality and to lymphocyte percentage and positively correlated to granulocyte–macrophage percentage. As for the ex vivo experiment, ROS basal and stimulated levels and ROS index were correlated; these three parameters were also positively correlated to cell mortality and negatively correlated to LMI. Finally, phagocytosis percentage was positively correlated to cell mortality, both ROS levels and ROS index, and negatively correlated to LMI (Table 5).

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Arch Environ Contam Toxicol Table 4 Effects of DU on immune leucocyte parameters during the in vivo experiment after 3 days of contact with 0, 20 and 250 lg/L [DU] (lg/L)

Cell mortality (%)

Lymphocyte percentage (%)

Granulocyte percentage (%)

LMI(MFI)

ROS basal level (MFI)

0

12.0 ± 1.0

63.7 ± 1.7

36.0 ± 1.8

167.1 ± 8.7b b

20

12.4 ± 0.6

62.8 ± 1.6

36.7 ± 1.6

149.8 ± 7.3

250

11.9 ± 0.7

63.0 ± 1.5

36.6 ± 1.5

130.3 ± 5.5a

ROS stimulated level(MFI)

ROS index

Phagocytosis percentage (%)

5.8 ± 0.5a

12.9 ± 3.5a

2.3 ± 0.7b

18.1 ± 0.5

b

a

1.0 ± 0.1a

18.4 ± 0.7

17.3 ± 1.2b

1.2 ± 0.1a

18.9 ± 0.3

12.2 ± 1.1

14.9 ± 1.4b

11.5 ± 1.3

Values are means of 20 replicates, except for controls values (n = 18); standard error is presented a,b

Significantly different from control at p \ 0.05; a \ b. When no letters are added, no significant differences were found

Table 5 Correlations between immune leucocyte parameters during the in vivo experiment

Cell mortality Lymphocyte percentage

0.094

Granulocyte percentage

0.095

-0.999*

-0.346*

LMI

* Represent significant values (p \ 0.05)

Granulocyte (%)

LMI

ROS basal level

ROS stimulated level

-0.293*

0.298*

ROS basal level

0.349*

-0.213

0.212

-0.211

ROS stimulated level

0.401*

-0.142

0.147

-0.434*

0.643*

ROS index

0.146

0.052

-0.044

-0.239*

-0.301*

0.444*

Phagocytosis percentage

0.349*

-0.060

0.065

-0.363*

0.307*

0.533*

Discussion In our study, the exposure concentrations were within the range of U environmental concentrations found close to mining sites (Antunes et al. 2007) or in drilled wells (Kurttio et al. 2006). Furthermore, one of the concentration used in both experiments is close to the provisional drinking water guideline value of the World Health Organization of 30 lg/L (WHO 2012). Therefore, the concentrations used are relevant from an environmental point of view. In the in vivo experiment, the pattern of oscillations of DU concentrations around the nominal values was already shown for other metals in a comparable experimental design (Cambier et al. 2010). In the present study, we saw a decrease of cell mortality in ex vivo experiment at 20 lg DU/L only. Moreover, we saw no effect of DU on leucocyte mortality in in vivo experiment. However, no cell mortality was shown for 250 and 500 lg DU/L. It is generally admitted that low levels of ionizing radiations might produce beneficial effects, stimulating the activation of repair mechanisms that protect against disease (Gori and Mnzel 2012). We could therefore hypothesize that at low doses, DU could induce cell repair mechanisms of problems not related to the toxicant, and this intense cell activity could have an hormesis effect on cell survival by reducing necrosis.

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Lymphocyte (%)

ROS index

0.330*

We saw no effects of DU on zebrafish kidney leucocyte proportion. In the same way, literature reports no changes in the density of neutrophils and lymphocytes in rats following short DU exposure (Dublineau et al. 2006b). However, chronic ingestion of DU induced modifications of immune cell content (decreased of macrophage density and increase of neutrophil density) (Dublineau et al. 2007). Moreover, a significant decrease of lymphocytes counts were observed in the peripheral blood samples from individuals living near a deactivated U mine in a chronical context (Lourenc¸o et al. 2013). As we saw also no effect on cell mortality, we can therefore hypothesize that chronic contamination to DU could induce a gradual weakness of organisms on these parameters but that this result could not be observed in a short-time exposure like in our study. In our study, the measurement of LMI with the use of acridine orange showed a decrease in fish exposed to 250 lg DU/L during 3 days. This parameter was recently developed and shown to decrease in the presence of pollutants in the three-spined stickleback, Gasterosteus aculeatus, in laboratory and field experiments (Bado-Nilles et al. 2013). Some studies report the effects of U on lysosomal stability in animals but very few in vertebrates. U enters the cells through a membrane transporter and by endocytosis; a fraction reaches lysosomes, where it precipitates as uranium phosphate (Berry 1996; Gouget 2011).

Arch Environ Contam Toxicol

U is known to accumulate in lysosomes of mussel hemocytes (Chassard-Bouchaud et al. 1983) and in lysosomes of zebrafish cell lines (Pereira et al. 2012). In earthworms, lysosomal stability decreased with increasing concentrations of U or DU in soils (Giovanetti et al. 2010). In rat hepatocytes, DU induced lysosomal membrane rupture (Pourahmad et al. 2006, 2011). Therefore, our study confirmed that DU can affect the lysosomal membrane stability in adult zebrafish. Lysosomal accumulation of toxic metals and organic xenobiotics is a well-documented cellular phenomenon, and sequestration in lysosomes has also been postulated to have a protective role through the physical detoxication of pollutants because lysosomes can exocytose their content under stressful conditions (Moore and Willows 1998). This autophagy induces releases in the cytosol of hydrolytic enzymes which could lead to cell necrosis or apoptosis (Kurz et al. 2008). However, in our study, the decrease of LMI did not induce cell mortality, suggesting that autophagy is sufficient to maintain cellular viability. Moreover, this mechanism could also indicate an important cell activity and, as we hypothesize above, be involved in the positive effect of DU on cell necrosis. In our study, DU increased ROS basal level with no modification on ROS PMA-stimulated level, leading to a reduction of ROS stimulation index in both in vivo and ex vivo experiments for all concentration tested. In our previous study, we showed an increase of ROS fold induction index in living whole kidneys removed from zebrafish exposed to 20 lg DU/L during 28 days (Gagnaire et al. 2013). This increase was due to a decrease of ROS basal level with, as we see in our present study, no modification on ROS PMA-stimulated level (Gagnaire et al. 2013). Both these results on DU effects on ROS basal level are therefore contradictory. Here, we worked on kidney leucocytes dissected from fish exposed to DU for a short period of time (only 3 days) compared to in our previous study were effects were not shown before 9 days. Moreover, in the present study we worked on isolated kidney leucocytes compared to whole kidney in the previous study, where other cells can interfere. These elements could explain the difference of results that we observe. In rat kidney, in vivo uranyl acetate (Shaki et al. 2012) and in vitro DU (Thie´bault et al. 2007) increased ROS basal level. A study observed that U induced in mice kidneys a dose-dependent production of H2O2 and an increase in SOD, GPx and interferon mRNA levels, suggesting an oxidative stress (Taulan et al. 2004). In rat hepatocytes, DU also induced ROS basal level (Pourahmad et al. 2006, 2011). Moreover, these studies demonstrated that the lysosomes were the main sources of ROS generated by DU and this formation of intracellular ROS like H2O2 favoured destabilization of lysosomal membranes (Pourahmad et al. 2006, 2011). It has also been shown in invertebrates that

the lysosomal compartment is an important locus of ROS generation (Winston et al. 1996). Our results are in agreement with these conclusions, as LMI was also affected by DU in our study. Moreover, both parameters were significantly correlated. DU induces the ROS basal production in zebrafish kidney leucocytes, showing a similarity to the mechanism of action of U known in mammals. This phenomenon could lead to an oxidative stress on the whole organism. However, ROS PMA-stimulated level was not modified by DU, indicating that fish may not be able to stimulate its defence capacities towards infectious diseases. In our study, we saw no modification of phagocytosis activity in kidney leucocytes, even though we saw an increase of ROS basal level production and decrease of LMI. This is in contradiction with previous results reported in the literature. Indeed, U induced both phagocytosis and generation of superoxide anion in rat alveolar macrophages and at high doses also induced apoptosis and increase of interferon a (Orona and Tasat 2012). Phagocytosis is associated to ROS production during respiratory burst (Ellis 2001; Whyte 2007). Lysosomes play also a central role in the degradation of phagocytosed materials (Grundy et al. 1996). We showed significant correlations between phagocytosis activity, ROS levels and index and LMI in both ex vivo and in vivo experiments, which is coherent since, as shown before, all these physiological processes are linked, and seems to indicate that we really measured a phagocytosis activity. However, duration of exposure may not have been long enough to see a modification of phagocytosis activity. In our study, we tested the effects of DU both in ex vivo and in vivo experiments. Ex vivo exposure of cells to pollutants may provide information on direct effects of pollutant on cells and therefore better understanding the mode of action of the pollutant tested. Moreover, from an ethical point of view, ex vivo models allow to reduce the number of animals tested, which is in accordance with animal experimentation legislation. However, ex vivo experiments allow only short contamination period as cells are not in culture. In vitro assays are being used in many different areas of fish physiology research, as the combination reduces the in vivo variability and the cost of the screening assays (Villena 2003). However, these experimental conditions may not be representative of in vivo conditions according that several physiological mechanisms and physical barriers, including mucus and skin, may be involved in fish defence (Ellis 2001). There has been little experimental work conducted to establish the correlation between results obtained in vitro and potential whole animal effects in vivo (Kilemade et al. 2002). However, ex vivo or in vitro conditions are frequently used for the assessment of pollutant toxicity on fish leucocytes (Rougier

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et al. 1994; Gerbron et al. 2010; Bado-Nilles et al. 2013). In our study, leucocyte biomarkers showed overall the same responses in both types of experiments, showing that ex vivo models could be used to extrapolate in vivo effects and underlying the interest of immune biomarkers in validation for ecological risk assessment purposes. To conclude, our study explored for the first time the effects of DU on immune parameters measured by flow cytometry in zebrafish, Danio rerio. We showed that at low concentrations relevant to environmental levels, DU could increase ROS basal production, decrease ROS stimulation index and LMI in kidney leucocytes. These immune biomarkers could therefore represent relevant parameters for assessing U immunotoxicity. Our results encourage the development and the use of other innate immune biomarkers and the use of flow cytometry in order to understand the effects of U and more generally radionuclides on fish immune system. The ultimate effects of DU and more generally radionuclides contamination or irradiation might be pathogenic by suppressing defence mechanisms or inducing hypersensitivity. Therefore, the study of zebrafish leucocyte functions could provide insights into the pathology of diseases occurring after radionuclide exposure. Acknowledgments Dr C Adam-Guillermin is acknowledged for allowing the work at the Laboratory of Ecotoxicology of Radionuclides (IRSN, Cadarache, France). Dr S Betoulle, O Geffard and JM Porcher are acknowledged for allowing venue of A Bado-Nilles at IRSN in order to participate to the experiments. V Camilleri and S Frelon are acknowledged for their help for water analyses. This work was partly supported by the ENVIRHOM research program supported by the Institute for Radioprotection and Nuclear Safety (IRSN). We also acknowledge the financial support of the Post-Grenelle Program 190 (DEVIL program) of the French Ministry for Ecology and Sustainable Development.

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