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Aquatic Toxicology 100 (2010) 295–302

Contents lists available at ScienceDirect

Aquatic Toxicology

journa l homepage: www.e lsev ier .com/ locate /aquatox

ltrastructural effects on gill, muscle, and gonadal tissues induced in zebrafishDanio rerio) by a waterborne uranium exposure

abrina Barilleta,∗, Valérie Larnoa, Magali Floriania, Alain Devauxb, Christelle Adam-Guillermina

Laboratory of Radioecology and Ecotoxicology, IRSN (Institute for Radiological Protection and Nuclear Safety), DEI/SECRE/LRE, Cadarache, Bat 186, BP 3,3115 St-Paul-Lez-Durance cedex, FranceINRA, EFPA Department, 54280, Champenoux and Environmental Science Laboratory, ENTPE, 69518 Vaulx en Velin cedex, France

r t i c l e i n f o

rticle history:eceived 16 May 2010eceived in revised form 20 July 2010ccepted 6 August 2010

eywords:

a b s t r a c t

Experiments on adult zebrafish (Danio rerio) were conducted to assess histopathological effects inducedon gill, muscle, and gonadal tissues after waterborne uranium exposure. Although histopathology isoften employed as a tool for the detection and assessment of xenobiotic-mediated effects in aquaticorganisms, few studies have been dedicated to the investigation of histopathological consequences ofuranium exposure in fish. Results showed that gill tissue architecture was markedly disrupted. Major

anio rerioraniumistopathologyEM–EDXilluscle

symptoms were alterations of the secondary lamellae epithelium (from extensive oedema to desqua-mation), hyperplasia of chloride cells, and breakdown of the pillar cell system. Muscle histology wasalso affected. Degeneration and disorganization of myofibrillar sarcomeric pattern as well as abnormallocalization of mitochondria within muscle and altered endomysial sheaths were observed. Morpholog-ical alterations of spermatozoa within the gonadal tissue were also noticed. This study demonstratedthat uranium exposure induced a variety of histological impairments in fish, supporting environmental

conta

onads concerns when uranium

. Introduction

Uranium is the heaviest naturally occurring element present inhe earth’s crust, with an average concentration of 3 mg/kg. Bothatural and anthropogenic processes contribute to its redistribu-ion throughout the environment, hence resulting in measurableoncentrations occurring in virtually all environmental compart-ents (rocks, soils, surface and underground waters, air, plants,

nd animals) (Bleise et al., 2003). Among the various anthropogenicctivities that affect uranium distribution within the environment,he most important ones are: the use of phosphate fertilizers, var-ous mining activities, the industrial processing of uranium for the

anufacture of nuclear fuel or other products, as well as the mil-

tary use of depleted uranium (DU, a byproduct of nuclear fuelycle) (Bleise et al., 2003). Uranium is found in surface as well asroundwater at an extremely wide range of concentrations, fromelow 0.01 �g/L to more than 12,400 �g/L in bedrock water from

∗ Corresponding author at: Laboratory of Radioecology and Ecotoxicology, IRSNInstitute for Radiological Protection and Nuclear Safety), DEI/SECRE/LRE, Cadarache,ât 186, BP 3, 13115 St-Paul-Lez-Durance cedex, France. Tel.: +33 4 42 19 94 01;

ax: +33 4 42 19 91 49.E-mail addresses: sabrina.barillet@free.fr (S. Barillet),

alerie.larno@irsn.fr (V. Larno), magali.floriani@irsn.fr (M. Floriani),lain.devaux@entpe.fr (A. Devaux), christelle.adam-guillermin@irsn.frC. Adam-Guillermin).

166-445X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.aquatox.2010.08.002

minates aquatic systems.© 2010 Elsevier B.V. All rights reserved.

some uraniferous areas of southern Finland (Salonen, 1994; WHO,2001).

Since uranium is both a heavy metal and a radionuclide (naturaluranium consisting in a mixture of three isotopes [238U, 235U, and234U], all alpha emitters), it can induce deleterious effects on biotavia both chemical and radiological pathways, therefore represent-ing a relatively unique challenge to (eco)toxicologists. However,uranium toxicity has surprisingly not been extensively studied fornon-human biota, particularly for aquatic vertebrates such as fish.Better knowledge of uranium toxicity is therefore needed.

Among the indicators of primary damage potentially induced bypollutants, histological alterations have been reported to be rele-vant endpoints (Blazer, 2002; van der Oost et al., 2003). Histologicalbiomarkers are indeed valuable indicators of the general health offish and mirror the effects of exposure to a variety of anthropogenicpollutants. To our knowledge, histological markers have only beenpreviously investigated in fish exposed to uranium by two researchteams: Cooley et al. (2000) and Kelly and Janz (2009). Both studiedhepatic and renal lesions while Kelly and Janz (2009) also studiedgill alterations.

From a morphological, as well as from a physiological point of

view, the gill tissue is a very complex organ, involved not only in gasexchange, ion exchange and acid–base balance, but also in nitroge-nous waste excretion (Evans, 1987). Fish gills generally comprisemore than half of the body surface, with an epithelial layer of onlya few microns separating the interior of the fish from the external

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nvironment (Pawert et al., 1998). As a result of this close asso-iation between water and blood, gill filaments and lamellae aretrongly affected by environmental pollutants (Au, 2004; Evans,987), and thus useful organs to monitor the health of aquaticrganisms (Pawert et al., 1998). In this context, the reviews of Au2004), Evans (1987) and Wood (2001) have provided comprehen-ive information on structural changes in fish gills in response tooxicant exposure. These authors reported that epithelial lesionshyperplasia, hypertrophy, oedema, and desquamation), as well aslterations of mucous, chloride, and pillar cell systems are typicalistopathological symptoms of gills in response to a wide range ofontaminants, including organochlorines, petroleum compounds,rganophosphates, carbamates, herbicides and metals (Au, 2004).lthough waterborne exposures are undoubtedly the major routef uranium exposure, to date, gill histopathologies following ura-ium exposures remain almost unexplored.

Other potentially inducible markers of environmental contam-nation include altered locomotor activity, as well as muscularnjuries. Degenerative features (e.g. muscular atrophy, bro-en myofibril, swollen sarcolemmal nucleus and sarcoplasmiceticulum) have been reported as symptoms of exposure to envi-onmental contaminants such as pesticides or metals (Koca et al.,005; Mughal et al., 2004; Wang et al., 2004). Morphometric anal-sis of muscular tissue might therefore be a reliable indicator ofranium toxicity.

Possible effects of uranium exposure on the reproductive tissuesf fish were also investigated. Several authors reported alter-tions of both ovaries and testes of rodents orally exposed toranium (Arfsten et al., 2005; Domingo, 2001; Linares et al., 2005;lobet et al., 1991). Due to their key role in reproductive function,istopathological impairments of the gonads could have severeonsequences on an entire fish population within a contaminatednvironment.

The present study was therefore undertaken to obtain infor-ation regarding histopathological effects of uranium in adult

ebrafish. Uranium toxicology in fish is indeed generally limitedo acute lethality data. Better knowledge of sublethal effects ofranium exposure in aquatic organisms would therefore allowcotoxicologists to establish regulatory standards or recommen-ations for uranium in aquatic compartments.

. Materials and methods

.1. Animal maintenance

Adult male zebrafish (Danio rerio) with an age of about20 d, measuring 3.9 ± 0.3 cm and weighing 0.458 ± 0.107 g werebtained from a French hatchery (HB Development, St Forgeux,rance) and acclimated to laboratory conditions for 2 weeks beforehe experiment. During the acclimation and experiment phases,sh were given daily supplies of standard fish pellets (1% of theirody mass per day). Organisms were kept at a maximal densityf 5 fish/L in tanks filled with 10 L of a 24 ± 1 ◦C synthetic waterquilibrated by air-bubbling. Ion concentrations in the syntheticater were (mg/L): 6.26 K+, 11.5 Na+, 4.74 Mg2+, 11.6 Ca2+, 32.4l−, 31.0 NO3

−, 9.61 SO42− and 0.45 CO3

2− which is assumed toe in equilibrium with the air (pCO2 = 3 × 10−4 atm). Fifty percentf the total water volume (either contaminated or not) was man-ally changed on a daily basis during both the acclimation andxperiment phases.

.2. Chemicals

Depleted uranyl nitrate, UO2(NO3)2·6H2O, was purchased fromluka (Buchs, Switzerland). Uranium-233, also as uranyl nitrate,

logy 100 (2010) 295–302

was an isotopic reference material obtained at Isotope ProductsEurope (IPL, Berlin, Germany). All other reagents of analytical gradewere supplied by Sigma–Aldrich (St Quentin Fallavier, France).

2.3. Experimental conditions

To distinguish the radiologically from the chemically inducedeffects of uranium, fish were exposed to a given uranium mass con-centration, 100 �g/L, an environmentally relevant concentrationthat can be found in the vicinity of uranium mines (Salonen, 1994;WHO, 2001), but to two different isotopic compositions of uraniumthat differed substantially in radiological activities. Fish were thusexposed either to 100 �g/L DU, or to 93.35 �g/L DU supplementedwith 6.65 �g/L 233U (hereinafter referred as the DU-233U treat-ment). Herein tested 233U concentration actually corresponded tothe maximum allowable radiological activity ensuring the researchstaff safety. DU-233U treatment indeed had radioactivity level thatwas four orders of magnitude greater than DU (3.57 × 108 Bq/g vs.1.44 × 104 Bq/g). Both control and exposed fish were sampled aftera 20-d exposure period, a “semi-chronic” duration that is supposedto be sufficient to detect histopathological effects but short enoughto limit uranium waste production (water being half-renewedevery day). These experimental conditions have already been inves-tigated in our laboratory, notably for bioaccumulation experiments(Barillet et al., 2007). Results indicated that total uranium body bur-den was similar in zebrafish either exposed to the DU or to theDU-233U treatment for 20 d (14.02 ± 4.02 and 12.26 ± 3.26 �g U/gtissue wet weight respectively). These findings are in agreementwith the fact that all isotopes of a given element behave the samechemically and therefore, bioavailability of all isotopes is the same.

2.4. Water sample analyses

Water samples were collected several times a day during theacclimation and exposure periods. Major anion concentrationswere analyzed by ionic chromatography (Dionex DX-120, Sunny-vale, CA, USA) while major cation concentrations were measuredafter 2% [v/v] HNO3 acidification by means of inductively coupledplasma-atomic emission spectrometry (ICP-AES Optima 4300DV,Perkin-Elmer, Wellesley, MA, USA). Uranium mass concentration inwater was also continuously monitored by means of ICP-AES (ura-nium detection limit of 10 �g/L) for DU-contaminated water, andof alpha liquid scintillation counting (alpha particle detection limitof 0.03 Bq per sample; Quantulus 1220; Wallac Oy, Turku, Finland)for the DU-233U-contaminated water. Thanks to these monitoringprocedures, uranium mass concentrations as well as radiologicalactivity were continuously readjusted in the test tanks. Due to ura-nium adsorption on tank walls, two to three uranium spikes per daywere indeed necessary to maintain constant contamination levels.

2.5. Tissue collection and processing

After 20 d of exposure, 3 fish per treatment were captured andimmediately sacrificed by cutting their spinal cord. Gills, gonadsand a piece of the caudal white muscle were removed and indi-vidually immersed in sodium cacodylate buffer (0.1 M, pH 7.4)supplemented with 2.5% glutaraldehyde. After 24 h at 4 ◦C, tissuesamples were washed three times for 5 min with sodium cacody-late buffer and post-fixed in the same buffer containing 1% osmiumtetroxide (OsO4) for 1 h. Tissue samples were dehydrated through

a graded ethanol series, and finally embedded in the monomericresin Epon 812. Semi-thin sections for light microscopy analysis(500 nm) and ultra-thin sections for TEM and EDX analyses (80 and110 nm, respectively) were then obtained by an ultramicrotomeUCT Leica.

oxicology 100 (2010) 295–302 297

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Fig. 1. Gill ultrastructure in control zebrafish (20 d). Four gill arches are presenton each side of the zebrafish buccal cavity. Each arch is composed of numerous gillfilaments (gf) which have two rows of secondary lamellae (sl) that run perpendicularto each filament. Each lamella is made up of two sheets of epithelium (epithelial

S. Barillet et al. / Aquatic T

.6. Ultrastructural examination and chemical analysis

Semi-thin sections stained with aqueous blue toluidine werebserved under a light microscope (Nikon, Eclipse E 400) andmages were saved with a Sony camera and a Biocom Visiol Soft-

are. Ultra-thin sections were mounted on copper grids observedith a Scanning Transmission Electron Microscope (TEM/STEM,

ecnai G2 Biotwin, FEI Company), equipped with a CCD cameraMegaview III, Eloise). Several different subcellular structures werenalyzed with the energy-dispersive X-ray analyzer equipped withSuper Ultra-Thin Window (SUTW) model sapphire (EDAX), usingn accelerating voltage of 100 KeV, in order to investigate the accu-ulation of uranium. The electron probe was then focused on

pecific spots and spectra were recorded after 30 s analyses.For each replicate organ, at least 30 micrographs of local detailed

tructures were taken. A quantitative approach was then employedo investigate pathological alterations. Measurements were carriedut on each of the 3 organisms per treatment group (control, DU andU-233U exposed fish) using the GNU Image Manipulation Program.6 freeware (Gimp 2.6). Gill tissue alteration was estimated byeasurements of the thickness of secondary lamellae (3 measure-ents per secondary lamella, 35 secondary lamellae per organism).uscle histology was examined according to three parameters: (i)itochondria localization (i.e., within or around myofibrillar units),

ii) constancy of sarcomere length and (iii) disruption of sarcomeressembly. The former parameter (mitochondria localization) wasstimated from muscle cross-sections, at least 100 mitochondriaer organism were analyzed. For sarcomere analyses, measure-ents were carried out on muscle longitudinal sections. The length

f 50 sarcomeres and grayscale intensity profiles of 3 micrographsassuming that disrupted sarcomere patterns lead to the appari-ion of brighter areas) were analyzed per organism. As for gonadalissue, the frequency of spermatozoa presenting nuclear vacuolesas estimated. Counts were made on the basis of about 200 sper-atozoa per organism.

.7. Statistical analyses

Statistical tests were run using the Statistica 7.1 software (Stat-oft, Chicago, IL, USA).

Since none of herein obtained datasets satisfied normality andomoscedasticity, nonparametric one-way analyses of variancen ranks approach (Kruskall–Wallis) and pairwise comparisonsMann–Whitney U tests) were used. The significance level for theseests was chosen to be 0.05.

. Results

.1. Uranium in water

The recorded patterns of uranium mass concentration (�g/L)nd activity (Bq/L) in water are presented in Table 1. In the controlank, no uranium was detected. From a chemical point of view, theontamination level (i.e., total uranium mass concentration) was

able 1ominal and measured uranium mass concentration and radioactivity in theater throughout the 20 d exposure experiment. Results are given as mean

alue ± standard deviation (n = 88).

Nominal values Measured values

Control 0 �g/L <Detection limit0 Bq/L <Detection limit

Depleted uranium 100 �g/L 102 ± 27 �g/L1.5 Bq/L 1.5 ± 0.4 Bq/L

Depleted uranium-233U 100 �g/L 94 ± 23 �g/L2376 Bq/L 2229 ± 537 Bq/L

cells, ec) held apart by contractile cells that line the capillary channels (pillar cells,pc). Nucleated erythrocytes (er) can be seen within capillary lumen. Chloride cells(cc) are identified as large epithelial cells with a light mitochondria-rich cytoplasm,usually present only at the base of lamellae. Scale bar = 25 �m.

similar in DU and DU-233U contaminated tanks. Conversely, froma radiological standpoint, DU and DU-233U tanks corresponded totwo substantially different experimental conditions, with radio-logical activity in the water of the tank contaminated with theDU-233U mixture being about 1500 times higher than that of thetank exclusively contaminated with DU.

3.2. Gill tissue analysis

Light micrograph of a control fish gill tissue is described in Fig. 1.In fish solely exposed to DU, gills showed extensive oedema

of epithelial cells, and hyperplasia of chloride cells at the base ofthe gill lamellae (Fig. 2A). In fish exposed to the DU-233U mixture,gills showed a breakdown of both epithelial and pillar cell systems.As a result of the breakdown of the pillar cell system, blood con-gestion (aneurism) was also observed in some areas of secondarylamellae of fish exposed to both DU and 233U (Fig. 2B). Morpho-metric measurements carried out on secondary lamellae showedthat secondary lamellae were 13.3 ± 3.4 �m thick in control fish butappeared to be thicker in fish solely exposed to DU (17.8 ± 4.2 �m)

233

and thinner in fish exposed to the DU- U mixture (8.3 ± 1.8 �m).Statistical analysis confirmed that experimental groups (control,DU and DU-233U exposed fish) were actually different from eachothers (Kruskal–Wallis ANOVA p-value = 0.00, Mann–Whitney Utests p-values < 0.001).

298 S. Barillet et al. / Aquatic Toxicology 100 (2010) 295–302

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ig. 2. Gill ultrastructural changes induced by uranium exposure (20 d). Light microgedema of epithelial cells (oe), and hyperplasia of chloride cells (arrows). (B) Fish exbreakdown of both epithelial and pillar cell systems (arrows). Blood congestion (a

Energy-dispersive X-ray microanalyses did not indicate anyranium within gill tissues of fish exposed to herein detailed ura-ium exposure conditions. However, EDX spectroscopy analysesevealed round-shaped granules of uranium within nuclei of pillarells within gill tissues of fish exposed to 80 �g/L DU supplementedith 20 �g/L 233U, corresponding to a nominal radiological activity

n water of 7150 Bq/L (Fig. 3).

.3. Muscle tissue analysis

TEM photographic analyses of muscle tissue are presented inigs. 4 and 5. In control fish skeletal muscle, TEM micrographsf longitudinal sections revealed regular arrangement of the sar-omeres, resulting in a regularly striated appearance (Fig. 4A). Inddition, the low magnification TEM examination of tissues from

ig. 3. Energy-dispersive X-ray spectroscopy microanalysis of the gill tissue. Fish were ears = 1 �m (left) and 10 �m (right).

of gill tissue. (A) Fish exposed to 100 �g/L of depleted uranium. Gills show extensiveto 93.3 �g/L of depleted uranium supplemented with 6.65 �g/L of 233U. Gills show

sm, an) also occurs. Scale bar = 25 �m.

the control group showed regularly spaced sarcoplasmic reticulumand mitochondria between myofibrils (Fig. 5A). Muscular histol-ogy observed in control fish therefore complied with histologicalstructure of a standard skeletal muscle.

High magnification of longitudinal sections of U exposedfish muscle showed an extensively disrupted sarcomeric pattern(degenerated and disorganized myofibrils). Such alterations weremainly observed in DU exposed fish, as presented in Fig. 4B. Myofib-rils were twisted, tangled, or split with loose A and I-bands, leadingto a lack of constancy of sarcomere length. In control fish, sarcom-

ere length fluctuated about 2.8 ± 0.8%, while variations reached22.7 ± 4.2% and 8.4 ± 2.0% in muscle of fish exposed to DU andDU-233U, respectively. Such differences lead to the fact that treat-ment groups statistically differed from each other (Kruskal–WallisANOVA p-value = 0.027, Mann–Whitney U test p-values < 0.05). Fur-

xposed to 80 �g/L of depleted uranium supplemented with 20 �g/L of 233U. Scale

S. Barillet et al. / Aquatic Toxicology 100 (2010) 295–302 299

Fig. 4. Skeletal muscle ultrastructural changes induced by DU exposure (20 d). TEM micrographs of longitudinally sectioned whole muscle. (A) Control fish. Sarcomeres,defined as myofibril segments, are demarcated by two neighbouring Z-lines (dark lines). They are composed of isotropic bands (I-bands, surrounding the Z-line) and anisotropicbands (A-bands). While actin filaments are the major component of the I-bands and extend into the A-bands, myosin filaments are exclusively found in the A-bands. Duet lengtho s, therd ows).e fibrils

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o the interaction between actin and myosin filaments in the A-bands, sarcomerer relax. Control fish micrographs revealed regular arrangement of the sarcomereepleted uranium. Micrographs show degenerated and disorganized myofibrils (arrxtensively disrupted sarcomeric pattern. Mitochondria are also found within myo

hermore, analysis of grayscale intensity profiles of micrographshowed that muscle tissues of both control and DU-233U exposedsh presented normal sarcomeric patterns while muscles of DUxposed fish exhibited a statistically significant 20.6 ± 2.1% lossf sarcomeric structures (Kruskal–Wallis ANOVA p-value = 0.005,ann–Whitney U test p-values < 0.05 for comparisons between DU

nd control or DU-233U groups).Low magnification observations of muscle cross-sections addi-

ionally revealed abnormal mitochondria localization within theuscle tissue of uranium exposed fish since some of these

rganelles were encountered inside myofibrillar units while mito-hondria were exclusively encountered outside myofibrillar units

ig. 5. Muscle ultrastructural changes induced by exposure to the DU-233U treatment (20 dhow mitochondria (mi) between myofibrils, all along the endomysium (en, i.e. the layerf depleted uranium supplemented with 6.35 �g/L of 233U. Observations reveal the preseen) appears to be looser than in control fish. Scale bar = 5 �m.

can be either reduced or elongated, therefore allowing muscle to either contractefore resulting in a regularly striated appearance. (B) Fish exposed to 100 �g/L ofMyofibrils are twisted, tangled, or splitted with loose A and I-bands, suggesting an(mi). Scale bar = 2 �m.

in control fish. Such a phenomenon was predominantly observed inDU-233U exposed fish, as presented in Fig. 5B, where 43.49 ± 18.27%of mitochondria were found inside myofibrilla (vs. 11.63 ± 9.25%in DU exposed fish). Statistical tests confirmed that all treat-ment groups differed from each other (Kruskal–Wallis ANOVAp-value = 0.024, Mann–Whitney U test p-values < 0.05).

Additionally, the endomysium (i.e., the layer of connective tis-sue that ensheaths every myofibril) appeared to be looser in fish

exposed to the DU-233U mixture than in control fish.

Energy-dispersive X-ray microanalyses failed to detect uraniumwithin muscle tissues of fish either solely exposed to DU or exposedto the DU-233U mixture.

). TEM micrographs of cross-sectioned whole muscle. (A) Control fish. Micrographsof connective tissue that ensheaths every myofibril). (B) Fish exposed to 93.35 �g/Lnce of mitochondria (mi) inside myofibrillar units. Additionally, the endomysium

300 S. Barillet et al. / Aquatic Toxicology 100 (2010) 295–302

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ig. 6. Gonadal ultrastructural changes induced by uranium exposure (20 d). TEM mo 100 �g/L of depleted uranium and (C) fish exposed to 93.3 �g/L of depleted urpermatozoa with nuclear vacuoles (arrows). Scale bar = 2 �m.

.4. Testes analysis

Histological observations of testes obtained from control fishomplied with standard testis histology as previously detailedy Blazer (2002) (Fig. 6A).TEM micrograph observations of Uxposed fish revealed normal histoarchitectural aspect of testes.onetheless, some of the spermatozoa revealed morphologicallterations. In both DU and DU-233U exposed fish, nuclear vacuolesere indeed detected within spermatozoon nucleus (Fig. 6B and). Such a vaculolization phenomenon affected 8.04 ± 1.85% and0.97 ± 5.36% of the spermatozoa in DU-233U and DU exposed fish,espectively, while only 0.18 ± 0.36% of the control fish sperma-ozoa contained vacuoles. Statistical tests demonstrated that allxperimental groups were statistically different from each otherKruskal–Wallis ANOVA p-value = 0.004, Mann–Whitney U tests p-alues < 0.05).

EDX microanalyses did not detect uranium within testes of fishither solely exposed to DU or exposed to the DU-233U mixture.

. Discussion

.1. Gill histopathologies

The gills of uranium-exposed fish showed a variety of ultrastruc-ural changes.

The most obvious signs of gill tissue impairment included alter-tions of epithelial cell, evidenced by either extensive oedemar breakdown (i.e., desquamation of filamental epithelium). Theormer phenomenon was observed in fish solely exposed to DUFig. 2A), while the latter occurred in DU-233U exposed fish (Fig. 2B).uch histopathological events have previously been reported insh exposed to toxicants such as cadmium (Alazemi et al., 1996;hophon et al., 2003). In the particular case of uranium, these alter-tions might be associated with inflammatory processes. Lerebours

t al. (2009) indeed reported that, in the gills of zebrafish exposed toaterborne DU, genes encoding antioxidant defenses or involved in

nflammatory processes were up-regulated after 3 and 4 weeks ofxposure. Hyperplasia of secondary lamellae has also been reportedn organisms exposed to environmental pollutants (heavy metals,

aphs of cross-sectioned whole testes. (A) Control fish spermatozoa, (B) fish exposedsupplemented with 6.65 �g/L of 233U. Both DU and DU-233U exposed fish show

pesticides, etc.), often associated with the complete fusion of twoneighbouring secondary lamellae (Alazemi et al., 1996; Pawert etal., 1998; Thophon et al., 2003). Such symptoms (oedema, hyper-plasia) result in an enlargement of the distance between blood andwater. They therefore can be considered as defense mechanismsagainst surrounding toxicants, but they also may lead to insuffi-cient oxygen supply of the blood. Furthermore, in the most drastictreatment herein used (i.e., with additional radioactivity), oede-mas finally resulted in cellular collapse. Effects of that nature maytherefore have a dramatic impact on the fitness of the organism.

Chloride cell hyperplasia was additionally reported in DUexposed fish (Fig. 2A). Similar responses (hypertrophy and hyper-plasia of chloride cells) have already been reported in fish exposedto cadmium (Thophon et al., 2003) or mercury (Jagoe et al., 1996).Physiologically, chloride cells are responsible for the maintenanceof the acid–base and ionic balance, therefore protecting fish fromundergoing metabolic acidosis or alkalosis. They additionally areinvolved in excretory processes (Pawert et al., 1998). Chloride cellshyperplasia may therefore have occurred in response to the needto eject the UO2

2+ absorbed by the gills, just as it was described forcadmium exposure.

Finally, a breakdown of pillar cell system was herein noticedin DU-233U exposed fish, resulting in lamellar aneurisms (capillarycongestion caused by the breakdown of vascular integrity) (Fig. 2B).Similar responses were previously described for fish exposed toenvironmental pollutants such as cadmium or lead (Alazemi et al.,1996; Olojo et al., 2005; Thophon et al., 2003). Such a collapse ofthe pillar cell system might be closely related to uranium accu-mulation into these cells. Although EDX microanalyses failed todetect uranium within gill tissues of fish exposed to herein pre-sented U treatments (probably due to the low detection limit of thistechnique), round-shaped granules of uranium were found withinnuclei of pillar cells of fish exposed to a similar nominal U massconcentration (i.e., 100 �g/L) but to a substantially higher radiolog-

ical activity of 7150 Bq/L (Fig. 3). The appearance of DNA damageresulting in necrosis and/or apoptosis of pillar cells can thereforebe expected.

The effects of uranium exposure on mucus secretion were notherein studied. However, it can reasonably be expected that fish

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ould alter their mucus secretion following U exposure, as previ-usly observed with copper (Alazemi et al., 1996). An importanthysiological function of mucus secreted by gill cells is indeedhe protection of the thin and sensitive gill epithelium fromnvironmental impacts. However, since hypersecretion of mucusimultaneously results in a complete mucus cover of the gill epithe-ium, such a response impedes gas exchange (Pawert et al., 1998).

Likewise, several authors reported numerous toxic effects ofetals on the fish gill physiological function. Physiological effects

nvolve a reduction in blood ionic levels or acidosis, associated withncreased ionic permeabilities and inhibition of enzymes involvedn transport of ions (Evans, 1987). Displacement of Ca2+ fromnionic sites of the intracellular cement has been proposed as annderlying mechanism for ionic permeability alteration, resulting

n “tight junctions” opening and allowing Na+ and Cl− to diffusecross the gill epithelium, down their respective electrochemicalradients (Evans, 1987). Since Leggett and Pellmar (2003) reportedhat uranium strongly interferes with calcium-related processes,ranium might therefore exert considerable control over the per-eability of the gills, by stimulating ion loss and water uptake as

reviously described for cadmium exposure (Thophon et al., 2003).Uranium exposure therefore produces profound effects on gill

tructure, and might also have deep impacts on gill functions.

.2. Muscle histopathologies

From a histological point of view, sarcomere is the basic unit of auscle’s cross-striated myofibril. Herein obtained results indicate

hat myofibrillar sarcomeric patterns were clearly altered (myofib-ils being twisted, tangled, or split) as a result of DU exposureFig. 4). Similarly, Koca et al. (2005), Mughal et al. (2004) and Wangt al. (2004) previously mentioned marked decline or loss of stria-ion in fish muscle fibers as a result of metal or tributyltin exposure.he degenerative effects of uranium on muscle structure might beinked to its ability to modulate acetylcholinesterase (AChE) activ-ty since several authors noticed a close relationship between AChEctivity and muscular structure and function. For example, alteredontractile properties of muscle were reported in AChE knockoutice (Vignaud et al., 2008). Likewise, Behra et al. (2002) and Cousin

t al. (2005) noticed both behavioural and structural associatedhenotypes (e.g. strongly impaired motility and myopathy-like dis-rganization of the axial musculature) in homozygous ache mutantmbryos in which ACh hydrolysis is completely abolished. Cousint al. (2005) mentioned that similar effects were observed in mus-le of a mammal after acute exposure to AChE inhibitors and waselated to an increase of intracellular calcium after constant acti-ation of the ACh receptor. Similarly, Bretaud et al. (2001) and Raot al. (2005) both found that insecticides (carbofuran and chlor-yrifos, respectively) induced a significant inhibition in brain AChEctivity as well as a noticeable decrease in locomotor activity ofsh. Acetylcholinesterase activity variations can therefore haverofound consequences on muscle structure and function. Sinceranium has been proven to have an impact on AChE activityBarillet et al., 2007; Bensoussan et al., 2009), both observed phe-omena (AChE activity modulation and muscle histopathologies)ight be closely related.Moreover, Lerebours et al. (2009) reported that, in muscles

f zebrafish exposed to waterbone DU, genes encoding detoxica-ion, apoptosis or inflammatory processes were transcriptionallytrongly down-regulated after 3-d of exposure, while genesnvolved in mitochondrial mechanisms were up-regulated. Dis-

rganizations of the muscle tissue might therefore be related toevere modulations of gene expression too.

Additionally, abnormal presence of mitochondria inside myofib-ils and wider intermyofibrillar spaces were noticed in DU-233Uxposed fish (Fig. 5). Radiological activity may therefore have an

logy 100 (2010) 295–302 301

influence on fish muscle alterations. To date, underlying mecha-nisms involved in such a radiological dependence remain unclear.However, uranium accumulation in muscle tissues was shown tobe dependent on uranium radioactivity (Barillet et al., 2007). Ura-nium concentrations were about 10 �g of U per g of muscle tissue(wet weight) in fish solely exposed to DU (100 �g/L for 20 d), whileit was about 2 �g/g in fish exposed to a DU-233U mixture (100 �g/Lfor 20 d). Therefore, herein observed differences in histopatholog-ical lesions could stem from the radiological impact of U on itstoxicokinetic behaviour.

4.3. Testes histopathologies

Regarding testes examination, the main testicular function (i.e.,spermatogenesis) did not appear to be affected by uranium expo-sure as evidenced by normal histoarchitectural aspect of testes.However, spermatozoa of fish exposed to uranium (whatever itsradiological activity) showed vacuolated nucleus (Fig. 6). Simi-lar vacuolization phenomena have been reported in mice orallyexposed to uranium (Llobet et al., 1991). However, in this latterstudy, vacuoles were encountered in interstitial cells of Leydig thatare found adjacent to the seminiferous tubules in the testicle.

Very little is known regarding the presence of vacuoles withinthe nucleus of spermatozoa. Nevertheless, some authors havereported a clear negative association between sperm nuclear vac-uoles and natural male fertility potential (Berkovitz et al., 2006;Thundathil et al., 1998). It appeared that nuclear vacuoles in spermcells did not prevent fertilization and implantation from takingplace but increased the rate of early embryonic death. Interestingly,uranium exposure was proven to impair the reproductive success ofD. rerio (Bourrachot et al., 2008; Bourrachot, 2009). Herein observedspermatozoa structural abnormalities may therefore explain suchreproductive defects.

The biochemical mechanism underlying the effect of spermato-zoa vacuolization on late embryonic development is not yet clearbut it would seem that vacuolization might be related to an alter-ation of the genetic material. An inverse correlation between spermaneuploidy and normal sperm cell morphology, in general, and nor-mal nuclear shape in particular, was mentioned by Berkovitz etal. (2006). Abnormal sperm head morphology was also associatedwith sperm DNA fragmentation (Franco et al., 2008; Virro et al.,2004). Franco et al. (2008), showed an association between nuclearvacuoles and DNA damage (cleavage of genomic DNA in either lowor high molecular weight DNA fragments, precocious decondensa-tion and disaggregation of sperm chromatin fibres) in spermatozoa.In the particular case of uranium, the hypothesis that vacuolizationis a consequence of DNA damage is strongly supported by two mainlines of evidence. First, even if no uranium was found in the gonadsvia EDX microanalysis in our study, uranium has been shown tobe able to accumulate within gonadal tissues of zebrafish (Barilletet al., 2007; Bourrachot, 2009). Second, uranium has already beenreported to induce DNA damage in gonadal cells (Barillet, 2007;Bourrachot, 2009). Nevertheless, further studies have to be carriedout to clearly establish a link between spermatozoa vacuolizationand DNA damage.

In the present study, no particular disorganization of the testisstructure was observed. However, other authors reported alter-ations of the tubular structure in rats orally exposed to uranium(Linares et al., 2005), as well as in fish exposed for 20 d to 250 �g/Lof waterborne uranium (Bourrachot, 2009). This last author evenobserved structural alteration of thecal cells (i.e., cells that are

part of stromal cells that surround the growing follicles) in femaleorganisms, as well as atretic oocytes.

Herein observed histological alterations of gonadal tissues mayhave dramatic consequences on the reproductive success of fish,and subsequently on population dynamics.

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Soc. Taïwan 31, 225–234.

02 S. Barillet et al. / Aquatic T

. Conclusion

The present work provides additional insights into uraniumistopathological potential in fish. Ultrastructural responses in gill,uscle, and gonadal tissues were indeed proven to be sensitive

nough to indicate early toxic effects of environmentally relevantranium concentrations.

Since gill malfunction may impair gas and ion exchanges, mus-le alteration can affect swimming performances and spermatozoanjury could lead to deleterious effects on the reproductive success,erein obtained results tend to prove that uranium exposure may

nduce deleterious consequences on both fish survival and repro-uctive success, suggesting potentially detrimental effects on fishopulation dynamics.

Regarding our attempt to distinguish the radiologically-inducednd chemically induced effects of uranium on fish histopatholo-ies, radiological activity appeared to have a relative influencen either the intensity or the kind of induced damage. However,urther experiments are needed to better understand underlying

echanisms.

cknowledgements

The authors would like to thank B. Romano for providing the fishsed in this experiment, V. Camilleri and M. Morello for U analy-is, and Dr. Tom Hinton for his proofreading support. This workas supported by the ENVIRHOM research program funded by the

nstitute of Radioprotection and Nuclear Safety.

eferences

lazemi, B.M., Lewis, J.W., Andrews, E.B., 1996. Gill damage in the freshwater fishGnathonemus petersii (family: Mormyridae) exposed to selected pollutants: anultrastructural study. Environ. Technol. 17, 225–238.

rfsten, D.P., Schaeffer, D.J., Johnson, E.W., Robert Cunningham, J., Still, K.R., Wil-fong, E.R., 2005. Evaluation of the effect of implanted depleted uranium on malereproductive success, sperm concentration, and sperm velocity. Environ. Res.100, 205–215.

u, D.W.T., 2004. The application of histo-cytopathological biomarkers in marinepollution monitoring: a review. Mar. Pollut. Bull. 48, 817–834.

arillet, S., 2007. Toxicocinétique, toxicité chimique et radiologique de l’uraniumsur le poisson zèbre (Danio rerio). Academic Thesis. Université Paul Verlaine deMetz, Metz, p. 476.

arillet, S., Adam, C., Palluel, O., Devaux, A., 2007. Bioaccumulation, oxidative stressand neurotoxicity in Danio rerio exposed to different isotopic compositions ofuranium. Environ. Toxicol. Chem. 26, 497–505.

ehra, M., Cousin, X., Bertrand, C., Vonesch, J.L., Biellmann, D., Chatonnet, A., Strahle,U., 2002. Acetylcholinesterase is required for neuronal and muscular develop-ment in the zebrafish embryo. Nat. Neurosci. 5, 111–118.

ensoussan, H., Grancolas, L., Dhieux-Lestaevel, B., Delissen, O., Vacher, C.M., Dublin-eau, I., Voisin, P., Gourmelon, P., Taouis, M., Lestaevel, P., 2009. Heavy metaluranium affects the brain cholinergic system in rat following sub-chronic andchronic exposure. Toxicology 261, 59–67.

erkovitz, A., Eltes, F., Ellenbogen, A., Peer, S., Feldberg, D., Bartoov, B., 2006. Does thepresence of nuclear vacuoles in human sperm selected for ICSI affect pregnancyoutcome? Hum. Reprod. 21, 1787–1790.

lazer, V.S., 2002. Histopathological assessment of gonadal tissue in wild fishes. FishPhysiol. Biochem. 26, 85–101.

leise, A., Danesi, P.R., Burkart, W., 2003. Properties, use and health effects ofdepleted uranium (DU): a general overview. J. Environ. Radioact. 64, 93–112.

ourrachot, S., Simon, O., Gilbin, R., 2008. The effects of waterborne uranium onthe hatching success, development, and survival of early life stages of zebrafish(Danio rerio). Aquat. Toxicol. 90, 29–36.

ourrachot, 2009. Etude des effets biologiques de l’exposition à l’uranium chez lepoisson zèbre (D. rerio). Impact sur les stades de vie. Academic Thesis. UniversitéAix-Marseille I. Université de Provence, Cadarache, p. 255.

logy 100 (2010) 295–302

Bretaud, S., Saglio, P., Toutant, J.P., 2001. Effects of carbofuran on brain acetyl-cholinesterase activity and swimming activity in Carassius auratus (Cyprinidae).Cybium 25, 33–40.

Cooley, H.M., Evans, R.E., Klaverkamp, J.F., 2000. Toxicology of dietary uranium inlake whitefish (Coregonus clupeaformis). Aquat. Toxicol. 48, 495–515.

Cousin, X., Strahle, U., Chatonnet, A., 2005. Are there non-catalytic functionsof acetylcholinesterases? Lessons from mutant animal models. Bioessays 27,189–200.

Domingo, J.L., 2001. Reproductive and developmental toxicity of natural anddepleted uranium: a review. Reprod. Toxicol. 15, 603–609.

Evans, D.H., 1987. The fish gill: site of action and model for toxic effects of environ-mental pollutants. Environ. Health Perspect. 71, 47–58.

Franco, J.G., Baruffi, R.L.R., Mauri, A.L., Petersen, C.G., Oliveira, J.B.A., Vagnini, L., 2008.Significance of large nuclear vacuoles in human spermatozoa: implications forICSI. Reprod. BioMed. Online 17, 42–45.

Jagoe, C.H., Faivre, A., Newman, M.C., 1996. Morphological and morphometricchanges in the gills of mosquitofish (Gambusia holbrooki) after exposure to mer-cury (II). Aquat. Toxicol. 34, 163–183.

Kelly, J.M., Janz, D.M., 2009. Assessment of oxidative stress and histopathology injuvenile northern pike (Esox lucius) inhabiting lakes downstream of a uraniummill. Aquat. Toxicol. 92, 240–249.

Koca, Y.B., Koca, S., Yildiz, S., Gurcu, B., Osanc, E., Tuncbas, O., Aksoy, G., 2005. Investi-gation of histopathological and cytogenetic effects on Lepomis gibbosus (Pisces:Perciformes) in the cine stream (Aydin/Turkey) with determination of waterpollution. Environ. Toxicol. 20, 560–571.

Leggett, R.W., Pellmar, T.C., 2003. The biokinetics of uranium migrating from embed-ded DU fragments. J. Environ. Radioact. 64, 205–225.

Lerebours, A., Gonzalez, P., Adam, C., Camilleri, V., Bourdineaud, J.P., Garnier-Laplace, J., 2009. Comparative analysis of gene expression in brain, liver,skeletal muscles, and gills of zebrafish (Danio rerio) exposed to environmen-tally relevant waterborne uranium concentrations. Environ. Toxicol. Chem. 28,1271–1278.

Linares, V., Albina, M.L., Bellés, M., Mayayo, E., Sanchez, D.J., Domingo, J.L., 2005. Com-bined action of uranium and stress in the rat: II. Effects on male reproduction.Toxicol. Lett. 158, 186–195.

Llobet, J.M., Sirvent, J.J., Ortega, A., Domingo, J.L., 1991. Influence of chronic exposureto uranium on male reproduction in mice. Fundam. Appl. Toxicol. 16, 821–829.

Mughal, M.S., Malkani, N., Jehan, N., 2004. Histopathological changes in kidney andmuscle of Labeo rohita due to cadmium intoxication. Biologia 50, 39–45.

Olojo, E.A.A., Olurin, K.B., Mbaka, G., Oluwemimo, A.D., 2005. Histopathology of thegill and liver tissues of the African catfish Clarias gariepinus exposed to lead. Afr.J. Biotechnol. 4, 117–122.

Pawert, M., Muller, E., Triebskorn, R., 1998. Ultrastructural changes in fish gills asbiomarker to assess small stream pollution. Tissue Cell 30, 617–626.

Rao, J.V., Begum, G., Pallela, R., Usman, P.K., Rao, R.N., 2005. Changes in behavior andbrain acetylcholinesterase activity in mosquito fish, Gambusia affinis in responseto the sub-lethal exposure to chlorpyrifos. Int. J. Environ. Res. Public Health 2,478–483.

Salonen, L., 1994. 238U Series Radionuclides as a Source of Increased Radioactivity inGroundwater Originating from Finnish Bedrock, vol. 222. IAHS Publications, pp.71–84.

Thophon, S., Kruatrachue, M., Upatham, E.S., Pokethitiyook, P., Sahaphong, S., Jar-itkhuan, S., 2003. Histopathological alterations of white seabass, Lates calcarifer,in acute and subchronic cadmium exposure. Environ. Pollut. 121, 307–320.

Thundathil, J., Palasz, A.T., Barth, A.D., Mapletoft, R.J., 1998. Fertilization character-istics and in vitro embryo production with bovine sperm containing multiplenuclear vacuoles. Mol. Reprod. Dev. 50, 328–333.

van der Oost, R., Beyer, J., Vermeulen, N.P.E., 2003. Fish bioaccumulation andbiomarkers in environmental risk assessment: a review. Environ. Toxicol. Phar-macol. 13, 57–149.

Vignaud, A., Fougerousse, F., Mouisel, E., Bertrand, C., Bonafos, B., Molgo, J., Ferry,A., Chatonnet, A., 2008. Genetic ablation of acetylcholinesterase alters musclefunction in mice. Chem. Biol. Interact. 175, 129–130.

Virro, M.R., Larson-Cook, K.L., Evenson, D.P., 2004. Sperm chromatin structure assay(SCSA) parameters are related to fertilization, blastocyst development, andongoing pregnancy in in vitro fertilization and intracytoplasmic sperm injectioncycles. Fertil. Steril. 81, 1289–1295.

Wang, D., Ueng, J.P., Huang, B.Q., 2004. Structural changes in the muscular tissue ofthornfish (Terapon jarbua, Forsskal) under TBT (tributyltin) exposure. J. Fisheries

WHO, 2001. Depleted Uranium: Sources, Exposure and Health Effects. World HealthOrganization.

Wood, C.M., 2001. Toxic responses of the gill. In: Schlenk, D., Benson, W.H. (Eds.),Target Organ Toxicity in Marine and Freshwater Teleosts, vol. 1. Taylor & Francis,London, pp. 1–89.