Chemosphere 91 (2013) 481–490

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Subcellular fractionation and chemical speciation of uranium toelucidate its fate in gills and hepatopancreas of crayfishProcambarus clarkii

0045-6535/$ - see front matter � 2012 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.chemosphere.2012.12.008

⇑ Corresponding author.E-mail address: sandrine.frelon@irsn.fr (S. Frelon).

S. Frelon a,⇑, S. Mounicou b, R. Lobinski b, R. Gilbin a, O. Simon a

a IRSN/PRP-ENV/SERIS, Laboratoire de Biogéochimie, Biodisponibilité et Transfert des Radionucléides, BP3, 13115 St Paul lez Durance, Franceb Laboratoire de Chimie Analytique Bio-Inorganique et Environnement, UMR 5254, Hélioparc, 2 Av. Pr. Angot, 64053 Pau, France

h i g h l i g h t s

" U subcellular fractionation and chemical speciation in gills and hepatopancreas of crayfish." Uranium subcellular partitioning patterns varied according to the target organ studied." Cytosolic fraction accounted for maximum 30% and 75% of the total U amount in gills and hepatopancreas, respectively." U available fraction accounted for 20% and 57% of the total U in gills and hepatopancreas, respectively." Potential interactions of U for biomolecules involved in endogenous Fe and Cu chelation.

a r t i c l e i n f o

Article history:Received 24 September 2012Received in revised form 27 November 2012Accepted 8 December 2012Available online 17 January 2013

Keywords:Uranium speciationSubcellular distributionCrayfish Procambarus clarkiiSEC-ICP MSGillsHepatopancreas

a b s t r a c t

Knowledge of the organ and subcellular distribution of metals in organisms is fundamental for the under-standing of their uptake, storage, elimination and toxicity. Detoxification via MTLP and MRG formationand chelation by some proteins are necessary to better assess the metal toxic fraction in aquatic organ-isms. This work focused on uranium, natural element mainly used in nuclear industry, and its subcellularfractionation and chemical speciation to elucidate its accumulation pattern in gills and hepatopancreas ofcrayfish Procambarus clarkii, key organs of uptake and detoxification, respectively. Crayfish waterborneexposure was performed during 4 and 10d at 0, 30, 600 and 4000 lgU L�1. After tissue dissection, ura-nium subcellular fractionation was performed by successive ultracentrifugations. SEC-ICP MS was usedto study uranium speciation in cytosolic fraction. The uranium subcellular partitioning patterns variedaccording to the target organ studied and its biological function in the organism. The cytosolic fractionaccounted for 13–30% of the total uranium amount in gills and 35–75% in hepatopancreas. The uraniumfraction coeluting with MTLPs in gills and hepatopancreas cytosols showed that roughly 55% of uraniumremained non-detoxified and thus potentially toxic in the cytosol. Furthermore, the sum of uraniumamount in organelle fractions and in the non-detoxified part of cytosol, possibly equivalent to availablefraction, accounted for 20% (gills) and 57% (hepatopancreas) of the total uranium. Finally, the SEC-ICP MSanalysis provided information on potential competition of U for biomolecules similar than the onesinvolved in endogenous essential metal (Fe, Cu) chelation.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

In order to assess ecological risks of metals to aquatic organ-isms, the link between metal bioaccumulation and its conse-quences for toxicity needs to take into account not only the bodyburden and tissue concentrations of a metal in the organism (Wal-lace et al., 2003; Wang and Rainbow, 2006), but also its internalcompartmentalization (Vijver et al., 2004). Among approaches pro-

posed in ecotoxicology, the Critical Body Residue (CBR) approachassumes that a threshold internalized dose is responsible for toxiceffect. However this approach needs to discriminate the relativecontribution of adsorption and absorption of metals and more pre-cisely within the absorbed part, the detoxified and the metaboli-cally available forms of metals, the latter only likely to be relatedto toxicity (Adams et al., 2011; Rainbow and Luoma, 2011).

The total accumulation of a metal in aquatic organisms may beinsufficient to predict its toxicity. As only one part of the accumu-lated metal is toxicologically active (Rainbow, 2002; Vijver et al.,2004), the determination of metal partitioning on the organ and

482 S. Frelon et al. / Chemosphere 91 (2013) 481–490

subcellular levels is necessary (i) to better define the relationshipbetween metal bioaccumulation and its biological effect, and (ii)to better understand its biodistribution, organotropism, precipita-tion and metal interaction with biomolecules during the fixation,transport and incorporation into a biological cell or tissue. Theknowledge of the metallic pollutant distribution at the subcellularscale, of their behavior and more particularly the identification oftheir biomolecule targets involved in their metabolic pathwaysare fundamental to understand, among others, their toxicologicalconsequences (Szpunar, 2004; Geffard et al., 2010). Two majortypes of cellular sequestration are known to occur after increasedexposure to toxic metals, and affect their toxicokinetic availabilityto organisms. One involves the formation of distinct cellular inclu-sion bodies (e.g. different mineral granule types or concretions)which decrease the chemo-toxicity, and the other one involvesthe binding of metals to proteins, such as metallothioneins, mainlypresent in cytosol, i.e. cell fraction known to have an importantrole in the toxicokinetics and toxicodynamics of metals. Indeed,toxic effects can be linked to an increase of metal concentrationin cytosolic metal-bound compounds of high or low molecularweight (Wang et al., 1999; Perceval et al., 2006). Metallothioneinlike proteins (MTLPs) could correspond to metal detoxified frac-tion. This fraction can be assessed either by SEC or by heat treat-ment but Geffard et al. (2010) suggested an exchange of metalsbetween the different cytosolic compounds during the heat treat-ment and a possible transfer of metals from non-MTLPs to MTLPscompounds.

Among non-essential elements, uranium (U) is a ubiquitousenvironmental trace metal, often found in water supplies (Bleiseet al., 2003). Concentrations in worldwide freshwater ecosystemsare highly variable and range from 0.01 lg L�1 to over 12.4 mg L�1,depending on the geological background (Betcher et al., 1988;Salonen, 1994; WHO, 2001) and on anthropogenic activities suchas civilian and military applications (Bleise et al., 2003). The effectsfrom exposure of biota are usually attributed to its chemical toxic-ity (ATSDR, 1999), but the exact mechanisms are not well under-stood (Pourahmad et al., 2006; Barillet et al., 2007; Al Kaddissiet al., 2011). Uranium is able to chemically activate oxygen speciesin the course of redox reactions via the redox chemistry of transi-tion metals (Miller et al., 2002; Yazzie et al., 2003) and enhance theproduction of free radicals via the ionization phenomenon inducedby alpha particle emissions (WHO, 2001; Taulan et al., 2004),resulting in damage to cell proteins, nucleic acids and lipids (Labrotet al., 1996; Barillet et al., 2007; Lourenço et al., 2010; Al Kaddissiet al., 2011).

Whereas chemical uranium speciation in water column corre-lated with its bioavailability is quite well studied by modelling,its speciation in aquatic organisms is poorly described (Bressonet al., 2011) despite a significant knowledge of coordination chem-istry and affinity for proteins (Ansoborlo et al., 2006; Michon et al.,2010). In rat kidney uranium was mainly detected (after exposure)bound to acidic proteins (Frelon et al., 2009). In human kidney cellextracts, proteins referred to as hard Lewis cation (Ca2+, Zn2+, Mn2+

or Mg2+) binding proteins, proteins with phosphorylation sites andvarious biomolecules containing carrying phospho-ligands such asphospho-serine or threonine, were evoked as good candidates tocomplex uranyl cation (Dedieu et al., 2009). The complexes formedare likely to induce essential metal metabolic pathway perturba-tion (Donnadieu-Claraz et al., 2007; Berradi et al., 2008). Cellularsequestration in precipitated fractions (previously shown by(Chassard Bouchaud, 1983; Simon et al., 2011) is also relevant inthe case of uranium because of its alpha rays emission possiblyyielding radiotoxicity in cells.

There is therefore a need to investigate the compartmentaliza-tion of uranium in target organs of aquatic organisms and aboveall, the part of this element remaining in the cytosolic fraction.

The naturally present in ecosystems freshwater crayfish Procamb-arus clarkii model is a good potential bioindicator as it is widelyspread, easy to breed, and recognized as metals bioaccumulator.Little data is available on uranium fate in this model after exposureunder controlled conditions in laboratory (Al Kaddissi et al., 2011,2012). Accumulation results at the organ level showed that ura-nium accumulates preferentially in the gills� stomach > intes-tine > hepatopancreas > carapace > green gland > muscle (AlKaddissi et al., 2011). Accumulation in the gills was rapid and high(up to 130 lg g�1 dw after 10 d at 30 lgU L�1). In hepatopancreas,the accumulation reached 3 and 1 lg g�1 dw after 4 and 10 d ofexposure and seems to be dose and time dependant. These bioac-cumulations led to mitochondrial damage and antioxidant re-sponses but no acute mechanisms have been elucidated yet.

The objective of this study was to investigate the compartmen-talization of U in gills and hepatopancreas of crayfish after water-borne exposure, two target organs implicated in uptake, transfer,elimination and detoxification. This compartmentalization wasstudied at both the subcellular and the cytosolic protein levels to(i) implement data to identify mechanisms of toxicity and also(ii) identify the ecotoxicologically relevant U fraction in thesetwo organs.

2. Materials and methods

2.1. Exposure & sampling

All details of exposure were as described elsewhere (Al Kaddissiet al., 2011). All crayfish Procambarus clarkii used in this study wereadult intermoult males coming from the Vigueirat swamp ofCamargue. They were acclimatized one month to experimentalconditions. During the test, animals were exposed 4 and 10 d toa wide range of metal concentrations covering the concentrationsfound in freshwater ecosystems: 0 (controls), 30 (environmentallevel), 600 and 4000 lg L�1 of depleted uranium (as uranyl nitrate).At days 4 and 10, 3 living crayfish were sampled from each tankand sacrificed. For both sampling times and all exposure condi-tions, hepatopancreas (HP) and gills (G) were dissected out andcollected from each individual, divided into four pieces of 200 mgeach, immediately frozen in liquid nitrogen and stored at �80 �Cuntil use.

2.2. Subcellular fractionation

It was performed on 100–200 mg of HP or G (n = 3), in 2 mL ofbuffer (250 mM Sucrose, 25 mM HEPES, pH 7.4) with a centrifugeAvanti J30-I (Beckman Coulter). A sample of 10 lL of homogenatewas withdrawn and kept for further uranium total analysis. Theremaining volume was then fractionated by centrifugation at1500 g for 15 min producing a pellet (P1) containing nuclei, tissuefragments, cellular debris and the supernatant S1. This latter wascarefully removed and centrifuged at 12000g during 15 min toproduce a pellet (P2) containing mainly mitochondria and thesupernatant S2. S2 was removed and fractioned by centrifugationat 100000 g during 70 min producing a pellet (P3) containing restof organelles (ROs) and the supernatant S3 containing the cytosolicfraction (C). P1 was resuspended in water, heated at 100 �C during2 min and 1 M NaOH was added. The sample was then incubated at65 �C for 1 h and centrifuged at 5000g during 20 min resulting inS4 (called CD in the results even if containing cellular debris andnuclei, tissue fragments) and P4 (Metal Rich Granules – MRG)(Wallace et al., 2003).

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2.3. Total U determination by ICP MS

Uranium analyses in all subcellular fractions were performedusing an Agilent ICP-MS 7500 cx (Agilent, Tokyo, Japan). Uraniumisotopes 238U and 235U as well as 209Bi were monitored. Measure-ment was directly performed for S3 and S4 after nitric acid andinternal standard (Bi) addition with a procedure adapted from (Rittand Cossonnet, 2001). P2 and P3 were resuspended in buffer andHNO3 was added. P4 was digested with nitric acid and diluted indeionized water. Finally, internal standard (Bi) was added in eachsample dilution to check possible matrix effect on uranium signal.Blanks were made accordingly to the protocol to check for possiblecontamination. Accuracy of the measurement was determinedusing CRM (SLRS-4 from NRC, TM28.3 from EC) spiked in matrices,recoveries were within 8% of the certified value. In addition, U bal-ance was checked by comparison of uranium amount in thehomogenate sample and the sum of the amount in each fractionof the same sample. Mass balance recovery was in the 70–120%range (n = 48).

2.4. SEC-UV-ICP MS

An Agilent 1100 (Agilent, Wilmington, DE) liquid chromato-graph equipped with a binary HPLC pump, an autosampler and adiode array detector was used. An Agilent ICP MS 7500ce (Agilent,Tokyo, Japan) served as liquid chromatographic detector. A Super-dex 200 HR size exclusion column (GE Healthcare, Orsay, France)with a mass separation range between 10 and 600 kDa was used.The column was calibrated with thyroglobulin (670 kDa), ferritin(474 kDa), transferrin (81 kDa), Mn-SOD (40 kDa), myoglobulin(16 kDa), Cd-MT2 (6.8 kDa) and vitamin B 12 (1.3 kDa). 100 lL ofcrayfish cytosol (C fraction, n = 2 for each condition, 10d) were in-jected and fractionated isocratically at 0.7 mL min�1 (mobilephase: 100 mM ammonium acetate pH 7.4). UV signal was moni-tored at 280 nm to get the protein distribution pattern and ura-nium containing biomolecules were detected by ICP MS. Uraniumisotopes 238U and 235U as well as 54Fe, 57Fe, 55Mn, 59Co, 63Cu,65Cu, 64Zn, 66Zn, 112Cd, 114Cd were monitored. The column recoveryyield was calculated using two different methods. The first oneconsisted in an external U calibration without chromatographiccolumn by flow injection analysis (FIA) with ICP MS detection, fol-lowed by determination of SEC-ICP MS peaks areas (subsequentlycorrelated to external calibration) for samples injected and com-parison with the total mass of U already determined in samples.The second method was based on chromatographic effluent collec-tion after injecting sample with and without column, freeze-dry-

Fig. 1. Concentration of U in cellu

ing, mineralization and comparison of U concentration in bothsamples.

SEC-ICP MS was also used to analyze cytosol and determine theuranium fraction with MTLPs as described elsewhere (Geffardet al., 2010). MTLPs fraction of uranium was then determined inboth organs as the percentage of uranium found in proteins whichmolecular weight ranges from 1.8 to 18 kDa (Geffard et al., 2010),i.e. in the conditions of this study between 23.2 and 29.7 min ofelution that means fraction [13–1 kDa] and sum of fractions [12–5 kDa] + [5–1 kDa], for G and HP respectively.

2.5. Graph and statistical calculations

Mann Witney statistical non parametric test was run for all dataseries. Stars indicate statistically significant results (p < 0.05)which were presented as means and their associated standarddeviation (SD).

3. Results

3.1. Accumulation in subcellular fractions

Accumulation levels in each organ were assessed by the ura-nium measurement in an aliquot of each homogenate. At 4d, accu-mulation in gills was 0.05, 9.8, 32.3 and 351 lg g�1 wet weight(ww) for control, 30, 600 and 4000 lgU L�1, respectively. At 10d,accumulation in gills was 0.03, 8.55, 48.8 and 545 lg g�1 ww forcontrol, 30, 600 and 4000 lgU L�1, respectively. At 4d, accumula-tion in HP was 0.16, 0.9, 2.8 and 5.4 lg g�1 ww for control, 30,600 and 4000 lgU L�1, respectively. At 10d, accumulation in HPwas 0.2, 0.1, 3.8 and 3.34 lg g�1 ww for control, 30, 600 and4000 lgU L�1, respectively. These results are of the same order ofmagnitude as the one described by (Al Kaddissi et al., 2011).

Fig. 1 shows for different exposure conditions (lg L�1) the con-centration of uranium (lg g�1 of G or HP) in the fraction called CD,representing cellular debris, nuclei, tissue fragments. Accumula-tion levels in gill CD were proportional to the concentration of Uin water (4d-[U] in CD = 0.03 � [U] + 0.01 in water, R2 = 1; at 10d-[U] in cellular debris = 0.08� [U] + 0.01 in water, R2 = 0.997). Inthe case of gills, CD are then suspected to be partly made of unho-mogenized tissue but mainly of cuticle which is hugely made of ad-sorbed uranium instead of internalized one and also of periphytonwhich also absorbs U. The choice was then to exclude CD fractionfor the data treatment of the whole subcellular fractions as well asfor the discussion. U levels in HP CD were lower than those of gills

lar debris (lg g�1 of organ).

484 S. Frelon et al. / Chemosphere 91 (2013) 481–490

and not proportional to the concentration of U in water nor to theone in gills.

Uranium concentrations in subcellular fractions (MRG, organ-elles, mitochondria and cytosol) have been measured in gills andHP for all exposure conditions. Figs. 2 and 3 show uranium concen-tration in these subcellular fractions of gills and HP cells for controland U- exposed samples at 4 and 10d.

In control (data not shown), total accumulation level was low(<0.15 lg g�1 of organ) and higher in HP than in gills. The cytosolfraction shows the highest U accumulation level in HP, i.e. 54%and 67% at 4d and 10d, respectively. In gills, uranium rich granules(U-RG) constitute the main U source with 49% and 65% for bothexposure durations.

3.1.1. In gill subcellular fractionsBy adding the concentration of each fraction in each condition,

high levels of uranium, close to 100 lgU g�1 of organ (wet weight)were measured at 4 and 10d after the highest level of exposure(Fig. 2). A rapid and significant accumulation was observed in thefour compartments. U-RG fraction (over 50% of the whole uraniumamount) presented the highest accumulation (from 4 to15 lgU g�1 of organ) for both exposure durations and the two low-est concentrations (control and 30 lg L�1), while the cytosol,organelles and mitochondria fractions had a significant amountof U for most exposure concentrations. Finally, for the highest levelof exposure, high concentration (60 lgU g�1 of gills) was measuredin mitochondria.

3.1.2. In hepatopancreas subcellular fractionsIn HP, lower levels of uranium (<4 lgU g�1 of HP (wet weight))

were measured (Fig. 3). The U-RG is not the major fraction accu-mulating U (8% maximum, 10d–600 lg L�1). Indeed, the highestamount (1.8 lg g�1 at 10d) of uranium is found in the cytosol, i.e.soluble fraction of U in cell; this fraction of U slightly decreasedwith the exposure concentration from 75% to 35%. On the otherhand, the [mitochondria + organelles] fraction was about 30–35%of the total U amount accumulated for all exposure conditions.

This U content (%) in organelles, mitochondria and cellular deb-ris fractions slightly increased whereas URG fraction is quite stablewith the exposure concentrations. Finally, after 10 d of exposure,

Fig. 2. Uranium distribution in cell cytosols of gills of exposed groups after 4 and 10d. CoStar represents data significantly different from control (p < 0.05 – Mann & Whitney tes

the two highest concentration levels lead to a similar internalisedquantity with a similar relative distribution despite the high ura-nium exposure levels. The two results are significantly differentfrom the one obtained for environmental exposure 30 lg L�1

(p = 0.038) for which uranium concentration in the four compart-ments decreased between 4 and 10d.

3.2. Uranium speciation in cytosolic fraction

3.2.1. Gills3.2.1.1. Distribution pattern of uranium among cytosolic proteins. TheSEC-ICP MS chromatogram of gill cytosols showed the uraniumdistribution among biomolecules over a wide molecular weightrange with 5 fraction apexes, at the void of the column(P660 kDa), 474 kDa, 25 kDa, 2.8 kDa and after the total elutionvolume (Fig. 4).

235U distribution (data not shown) was the same as 238U distri-bution following the depleted uranium isotopic abundance. Theuranium distribution within the 2 cytosols of each exposure levelwas quite similar. The recovery yield assessed using FIA methodranged between 30% and 120%, the best ones being for controland Gills-30 lg L�1 (around 100–120%). In addition, the uraniumdistribution among proteins was not homogeneous and wouldindicate a differential affinity of proteins for uranium. For these 5fractions, peak areas were calculated and weighted by the samplemass used to extract proteins, to plot them against the exposurelevels. The same work was done for others metals such as Fe, Zn,Cu, Cd, Co and Mn (chromatographic patterns not shown). Fig. 5summarizes the results obtained for U, Fe, Cu and Co. No significantresults were found for Cd, Mn and Zn.

Differences of U peak areas can be noticed between G-control,G-30 lg L�1 and also G-600 lg L�1 groups with an increasing ten-dency with the exposure level (x � 20 between G-30 lg L�1 andG-control groups and x � 50 between G-600 lg L�1 and G-controlgroups). Large variability was observed for the G-600 lg L�1

groups. In addition, as analysis was performed in duplicate only,statistical non parametric Mann & Whitney test gives a p-valueminimum of only 0.1. Moreover, for each peak of U the calculationof its relative area over the sum of U peak areas was done for allexposure conditions; for control conditions these relative areas

ncentration in the four subcellular fractions is given in lg g�1 of organ (wet weight).t).

Fig. 3. Uranium distribution in cell cytosols of hepatopancreas of exposed groups after 4 and 10d. Concentration in the four subcellular fractions is given in lg g�1 of organ.Star represents data significantly different from control (p < 0.05 – Mann & Whitney test).

Fig. 4. SEC-ICP MS chromatographic pattern of cytosol samples of gills at t = 10 d of exposure. As the uranium distribution within the 2 cytosols of each exposure level is quitesimilar, only one sample was shown. 238U signal is plotted against analysis time. UV signal represents the protein distribution and arrows indicate fraction apexes.

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were of 14%, 9%, 9%, 38% and 30% for protein fractions of [>670–636 kDa], [528–145 kDa], [43–13 kDa], [13–1 kDa], [<1 kDa],respectively. For the exposure conditions of 30 lg L�1 and600 lg L�1 relative areas of these protein fractions were of 10%,3%, 8%, 55%, 24% and 21%, 7%, 8%, 38%, 25%, respectively. These re-

sults show that all peak relative areas remain roughly constantregardless the exposure conditions.

3.2.1.2. Consequences on essential element distribution. In the sameway, Fig. 5 details the results for Co, Fe and Cu. Several peaks were

Fig. 5. Uranium and other metal peak area as a function of exposure levels. Measurements realized in cytosols of gill cells.

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detected for Fe and Co whereas one main peak and three secondarywere observed for Cu. No major difference between groups was no-ticed for all the Co peak areas which are quite low (around 1000–2000 area unit) compare to others elements.

For Fe and Cu peaks respectively, total peak area decreased withthe increase of uranium exposure levels; �62% and �54% betweencontrol and G-30 lg L�1 groups and �78% and 76% for the G-600 lg L�1 group vs. G – control group, for Fe and Cu respectively.As none of these elements were added in the water of exposure, allthe changes observed on the peak area of essential metals wereattributed to an indirect effect of uranium exposure. The same cal-culation of relative area, as previously detailed for U, was done forCo, Fe and Cu peak area (data not shown). This approach showedthat each peak for all elements kept roughly constant its relativearea whatever the exposure conditions.

3.2.2. Hepatopancreas3.2.2.1. Distribution pattern of uranium among cytosolic proteins. Inthis organ, the cytosol fraction, i.e. the fraction used for SEC distri-bution, represented 75–35% of the uranium content. The SEC-ICPMS chromatogram of hepatopancreas (HP) cytosols showed an ura-nium distribution among biomolecules (UV 280 nm) over a widemolecular weight range, with 6 fraction apexes at the void of thecolumn (P660 kDa), 474 kDa, 135 kDa, 40 kDa, 7 kDa, 3.5 kDa(Fig. 6). One more peak at the total volume of the column was dis-tinguished in control and HP-600 lg L�1 groups.

235U (data not shown) and 238U signals respected the depleteduranium isotopic abundances and uranium distribution withinthe two cytosols at each exposure level was similar. The recoveryyield ranged between 53% and 87% the best one being for controland HP-30 (mean of 85%). In addition, the uranium distributionamong proteins was not homogeneous and would indicate a differ-ential affinity of proteins for uranium.

For these 6 fractions, peak areas were calculated and weightedby the sample mass used for cytosol preparation, to plot themagainst the exposure levels. The same work was done for othersmetals such as Fe, Zn, Cu, Cd, Co and Mn. Fig. 7 only summarizesthe results obtained for U, Fe, Cu and Co, for which the most signif-icant variations were obtained.

On the left side, Fig. 7 shows the tendency of the 6 different ura-nium peak areas according to the increase of exposure level. Nodifference can be noticed between control and HP-30 groupwhereas all peak area increased for the HP-600 group (x � 9).Unfortunately, as previously shown in gills, statistical non para-metric Mann & Whitney test gave a p-value of 0.1. The relative areacalculation of each peak was done for U; for control conditions theywere of 32%, 7%, 6%, 15%, 21% and 20% for protein fractions of[>670–582 kDa], [528–159 kDa], [146–62 kDa], [39–14 kDa], [12–5 kDa], [5–1 kDa] respectively. For the exposure condition of30 lg L�1 and 600 lg L�1, relative areas of the same protein frac-tions were of 20%, 7%, 6%, 15%, 18%, 34% and of 30%, 6%, 4%, 13%,17%, 29%, respectively. This approach shows once more that eachpeak relative area remained roughly constant whatever the expo-sure conditions. This would lead the hypothesis that neither pref-erential U accumulation nor saturation takes place onbiomolecules into these molecular weight ranges.

3.2.2.2. Consequences on essential element distribution. Fig. 7 detailsthe results for Co, Fe and Cu. Only a few peaks were identified. Asfor U, no difference between Co, Fe and last Cu peak area was no-ticed between control and HP-30 groups whereas the area of Copeak, Cu last peak and Fe peaks increased for the HP-600 groupvs. HP-control group of �4, �4 and �3, respectively. On the con-trary, the area of other Cu peaks decreased (�75%). As previouslycalculated for U, relative peak area for Co, Fe and Cu does not varysignificantly with different exposure conditions.

3.2.3. Comparison between gill and hepatopancreas distributionpatterns of U among proteins

In control groups, five peaks of uranium were clearly defined incell cytosols of gills, i.e. [>670–636 kDa], [528–145 kDa], [43–13 kDa], [13–1 kDa], <[1 kDa] and also five major peaks were ob-served in cell cytosols of HP, i.e. [>670–582 kDa], [39–14 kDa],[12–5 kDa], [5–1 kDa], <[1 kDa], both last peaks being after the to-tal volume of the column suggesting interaction between uranium-biomolecule complexes and the stationary phase of the column.Taking into account the poor resolution of SEC column, at the back-ground level, the protein MW range of the first peaks was closely

Fig. 6. SEC-ICP MS chromatographic pattern of cytosol samples of hepatopancreas at t = 10 d of exposure. As the uranium distribution within the 2 cytosols of each exposurelevel is quite similar, only one sample was shown. 238U signal is plotted against analysis time. UV signal represents the protein distribution and arrows indicate fractionapexes.

Fig. 7. Uranium and essential metal peak areas as a function of exposure levels. Measurements realized in cytosols of HP cells.

S. Frelon et al. / Chemosphere 91 (2013) 481–490 487

similar. However, the difference of the other ones underlined thespecificity of each organ with their different classes of proteinslikely to trap uranium. Exposure to uranium underlined increasesof uranium content in all gills and HP peaks. It might also enhancetwo peaks in HP cell cytosols, i.e. [528–159 kDa], [146–62 kDa],present in control group but as minor peaks.

Concerning others metals, 4–6 peaks were recovered per metalin gill cell cytosols against only 1–3 peaks per metal in HP cell cyto-sols. In gills, peak area seemed to decrease with the increase of U

whereas in HP it seemed to decrease for 2 peaks of Cu but increasefor Co, Fe and one peak of Cu.

As done by Geffard et al. (2010) for Cd, Ni and Pb, the uraniumfraction with Metallothionein Like Proteins (MTLPs) was deter-mined in both organs as the percentage of uranium found in pro-teins ranging from 1.8 to 18 kDa, i.e. peak [13–1 kDa] and sum ofpeaks [12–5 kDa] + [5–1 kDa], for G and HP respectively. This frac-tion was calculated for U even if results on mt gene expressionmodification give different tendencies in relation to the organ

488 S. Frelon et al. / Chemosphere 91 (2013) 481–490

studied (Al Kaddissi et al., 2011) and if thiol groups are not consid-ered as the best chelating groups for U (Michon et al., 2010). Theaverage MTLPs fraction over all exposures represented 44+/� 10%of the total uranium in cytosol in gills whereas it represented46+/� 5.3% in hepatopancreas.

In addition, it is interesting to consider the detoxified fraction ofU that is the sum of the concentrations of U in MTLPs fraction andin U-RG, both considered as detoxification pathways. These valueswere calculated with subcellular concentrations shown in Figs. 2and 3 for U-RG and cytosol fractions, this latter after applyingthe representative MTLPs percentage previously determined, thatwas 44 and 46, respectively for gills and hepatopancreas. Resultsshowed that this uranium detoxified fraction concentration in-creased with the level of exposure without any sign of saturation,from 0.02 to 30 lg g�1 in gills (ww) and from 0.03 to 0.72 lg g�1

(ww) in hepatopancreas.

4. Discussion

4.1. Accumulation strategy of U in gills and hepatopancreas afteruranium exposure

4.1.1. Organ levelAfter direct exposure, uranium accumulation level in gills was

higher than the one measured in hepatopancreas (Al Kaddissiet al., 2011). Considering the linear relationship between gill accu-mulation and contamination levels, this study underlines the roleof gills as potential biomarker of uranium exposure (Simon et al.,2012). To explain such accumulation considering all the resultsof this study and particularly those on cell debris, several hypoth-eses can be done, notably on the role of cuticle. Crustacean gillcuticle mainly contains CaCO3 that was previously shown to favourUO2þ

2 coprecipitation. Indeed, Dakovic et al. (2008) indicated thatcoprecipitation of uranyl species with CaCO3 is the major bindingmechanism for uranium sequestration by the freshwater algaeChara fragilis. This mechanism could then explain the high amountof U in gills. Another mechanism could be via the accumulation ofperiphyton at the gill surface. Indeed, high levels of trace elements(cadmium, zinc, mercury, uranium) can be accumulated in theperiphyton (Aleissa et al., 2004; Morin et al., 2008) leading, inthe case of U, to the formation of different crystalline species suchas aragonite and rutherfordine (Dakovic et al., 2008). Howeversuch high burden of U in this form of external sequestration maynot contribute to the toxic fraction. Further investigations shouldbe done to assess this ‘‘non chemo-toxic’’ fraction, using for in-stance specific washing of the gills.

4.1.2. Subcellular levelIn case of metal contamination, aquatic toxicology widely refers

to data on subcellular metal accumulation and toxicity that con-form to the spillover model (Jenkins et al., 1984; Sanders et al.,1984; Jenkins and Mason, 1988). As some authors concluded thatspillover approach is not convenient for chronic exposure, someauthors showed that even for short term acute exposure, this the-ory is not valid (Kamunde, 2009). In this study, for both organs, onthe contrary to the increase of the exposure duration (4–10 d), theincreasing level of exposure led to a significant increase of concen-trations accumulated in organs and in the four subcellular frac-tions. Uranium accumulated in gills and hepatopancreas waslocalized in all subcellular compartments, including the metal-sen-sitive ones thus supporting the results of Kamunde et al.

In gills, this increase was mainly observed in the cellular debrisfraction (Fig. 1) and in the MRG fraction whatever the exposure toconcentrations representative to environmental (30 lg L�1) orheavily polluted (600, 4000 lg L�1) conditions (Fig. 2). The MRG

fraction presented a high U accumulation rate; their formationand U co-precipitation was rapidly induced by the presence ofthe U. The presence of intracellular phosphate, a strong U ligand(Goulet et al., 2011) could contribute to the formation of U-P com-plex leading to the formation of the URG as proposed by Milgramet al.(Milgram et al., 2008) and Chassard Bouchaud (1983) in gillcuticle of crabs. High quantity of such small U complex could becontained in the MRG fraction. However, unlike for the bivalve Cor-bicula fluminea (Simon et al., 2011), the transmission electronicmicroscopy observations in the crayfish gills evidenced that noneof these intracellular granules rich in Ca and P (L: 400 nm) havebeen detected (data not shown). Trace metals can be detoxifiedby a variety of granules which vary in composition and locationdepending on both the metal and the affected organism (Wallaceet al., 2003; Khan et al., 2010; Simon et al., 2011). This form of Ucould then represent one irreversible storage detoxification formas described by Rainbow and Luoma (2011). However, in the spe-cific case of U, alpha-emitter, this form of storage could locally con-stitute a radiotoxic source and induce radiation damage. Inaddition, initially considered as non-chemotoxic, some authorsdemonstrated the capacity of the MRG fraction to be transferred(Bragigand et al., 2004; Khan et al., 2010) and to induce toxic ef-fects, confirming that this fraction is of great interest to studythe food chain transfer of contaminants in ecosystems.

The cytosol fraction in gills represented between 13% and 30% ofthe total amount accumulated in the four subcellular fractions thatis concordant with the suspected high fraction of adsorbed U ongill cuticle and also with the values found by Simon et al. (2012)in their direct exposure study.

As a result, only 13–30% of U can pretend to be toxic and by con-sequence to be studied for U speciation and the assessment of thechemotoxic fraction of this element. Because of gill physiologicalfunction, adsorption of uranium (more than absorption) can besuspected in this organ and could explain this low proportion ofU recovered in the cytosolic fraction. The gills of all aquatic organ-isms come into direct contact with environment and may play animportant role in the metal balance of invertebrates. However itis unclear if crustacean gills represent a structure for uptake, elim-ination or both and their associated mechanisms. The uraniumfraction with MTLPs, i.e. the detoxified fraction in cytosol (Geffardet al., 2010), represents 44% of the total uranium in this fraction; itwould mean that gills play an important role in the detoxificationprocess. Finally, the important uranium burden in mitochondria atthe highest level of exposure should partly explain the alteration ofmitochondrial gene expression levels found by Al Kaddissi et al.(2011) in their study.

In hepatopancreas, U accumulated comes from the haemo-lymph flow from the gill, and mainly from the fraction consideredas bioavailable that is accumulated in the gill cell cytosol. The pat-tern of U subcellular relative partitioning in hepatopancreas ap-pears similar between the control and contaminated organisms,with a strong contribution of the cytosol fraction and a small con-tribution of the MRG fraction as shown by Simon et al. (2012) insimilar conditions of direct exposure. However, the influence oftime duration on U distribution pattern within subcellular frac-tions between 30, 600 and 4000 lg L�1 groups can be due to theset-up of detoxification mechanisms more efficient at environmen-tal levels than at high level of U exposure. Hepatopancreas is a ma-jor organ that sequesters and detoxifies dietary and waterbornemetals. Detoxification pathways are numerous and take place atdifferent subcellular levels of cells, i.e. mitochondria, lysosome,membrane, cytosol (Ahearn et al., 2004). Cytosolic pathway detox-ification occurs mainly via association of proteins or other organicmolecules with metals and needs deeper elucidation to understandthe fate and the toxicity of uranium in this organ. However, theMTLPs fraction of uranium assessed as (Geffard et al., 2010) for

S. Frelon et al. / Chemosphere 91 (2013) 481–490 489

Cd, Ni and Pb, would mean that the detoxified fraction in cytosolrepresents 46% of the total uranium in this fraction.

4.2. Assessment of the ecotoxicologically relevant fraction of U in gillsand hepatopancreas

Concerning the uranium chemotoxic fraction assessment, Figs. 3and 4 illustrate that the mitochondria and the other organellesfractions are able to accumulate high level of U. Mitochondria areimportant metal target organelles in which metals such as cad-mium can impair oxidative phosphorylation with ATP deficit, gen-eration of ROS. Same behavior can be suspected for U and couldexplain some subcellular effects (Al Kaddissi et al., 2011) observedon the mitochondrial respiratory chain and on the oxidative stress.As the levels measured in the gills are very high, the cytosol, themitochondria and the organelles of cells also accumulate largeamounts of U, higher than those measured in hepatopancreas([Gills]/[HP] = 2.84). These results confirm the need to focus onmitochondria dysfunction induced by U accumulation. In addition,U accumulation in cytosol is rapid and is neither time nor exposurelevel dependent. Therefore U effects could appear after a shortexposure duration.

The refinement interest of the subcellular approach and SECuranium distribution pattern in this cytosolic fraction will nowbe discussed (i) for gills because it mainly transfers uranium to-ward others organs and (ii) for hepatopancreas for which cytosolis the main U fraction and represents an important toxic or detox-ication pathway. MTLPs fraction determination for gills and hepa-topancreas cytosols showed that respectively at least 56% and 54%of uranium in this subcellular fraction remained non-detoxifiedand thus potentially toxic. In addition, it remains that the concen-trations of U in MTLP fraction and U-RG, both considered as detox-ification pathways, are increasing with time; that would indicatean apparent non-saturated detoxification capacity, once more dif-ferent from spillover concept but in accordance with behavior ofCd in rainbow trout (Kamunde, 2009).

Different molecular weight fractions of U were identified in gillsand hepatopancreas cytosol proteins. In the same way, specific dif-ferent fractions were identified for some essential elements. Asnone of these elements were added in the water of exposure, allthe changes observed on the peak area of essential metals are indi-rect effect of uranium exposure. Co-elution with other metalscould let us think of competition for the same biomolecules or veryclose in terms of molecular weight. Hemocyanin of crayfish ismade of many individual subunit proteins which contains two cop-per atoms and can bind one oxygen molecule (O2). Each subunitweights about 75 kDa and may be arranged in dimers or hexamersexceeding 1500 kDa, then eluting in the first fraction > 600 kDa.Circulating protein, it is present in gills and could be concernedby the competition between U and Cu (first peak of both metals).

Moreover, previous studies on rodents have shown many ironclusters in proximal tubule cells of rat kidney after long term ura-nium exposure, underlying a potential perturbation of iron homeo-stasis by uranium exposure (Donnadieu-Claraz et al., 2007; Berradiet al., 2008). The authors emitted hypothesis of a preferentialexcretion of U leading to Fe storage in ferritin. Here, the increasedsignal of Fe coeluting with ferritin could let think to a similar phe-nomenon even if the detection of U at 474 kDa with Fe co-elutioncould let also suppose a possible binding of U with ferritin. In thesame way, by comparison with others element patterns, U seemsto bind range of proteins similar to the one bound by Cu, Zn, Mn,Co and Cd, i.e. [>670–582 kDa], [528–159 kDa], [146–62 kDa],[39–14 kDa], [12–5 kDa], [5–1 kDa] for HP and [>670–636 kDa],[528–145 kDa], [13–1 kDa] for gills. Fe, Zn and Mn are the mainelement for which occurs coelution with U, both in gills and HP.In the case of copper, as signal decreases in the two first peaks both

corresponding to uranium peaks with increasing area, competitionbetween these two metals for the same biomolecules could be sus-pected. Indeed, it can be hypothesized that a slight download of Cuoccurs from biomolecules around 474 and 70 kDa with upload ofCu to biomolecules around 6–7 kDa indicating possible complexa-tion with metallothioneins for storage and excretion. However, de-spite this hypothesis a deficit of copper in cytosol of control andHP-600 condition can be noticed but not explained yet.

In addition, despite a wide range of exposure level studied bySEC ICP-MS neither any enrichment nor a saturation of one specificprotein class was observed. U accumulation level in different pro-tein peaks of gill and hepatopancreas cytosols varied as a functionof exposure level whereas cytosol contents in gills are roughly thesame at 30 and 600 lg L�1. As U biological effects observed inhepatopancreas and gills are also dose dependant (Al Kaddissiet al., 2011), these latter can be linked to a threshold value of Uin a specific protein peak as proposed by the CBR approach. Thisunderlines the need to go deeper into the identification of U targetsin hepatopancreas and gills by deconvoluting more accurately eachprotein fraction of interest, i.e. for instance fractions that had beendescribed as non-detoxified in each organ, and analyzing themwith further analytical methods such as gel electrophoresis.

5. Conclusion

This study emphasizes that for the CBR approach, the subcellu-lar fractionation of U and its SEC distribution among cytosolic bio-molecules are, at this stage, complementary approaches foruranium accumulation profile determination. Indeed the fractionpossibly considered as the available fraction, i.e. sum of organellefractions and non-detoxified part of cytosol, accounts for 20% and57% of the total uranium, in gills and hepatopancreas, respectively.Then SEC-ICP MS enables to get information on the potential U, Fe,and Cu interactions and competition for the same biomolecules.Uranium-containing protein fractions of interest have been identi-fied, i.e. for instance fractions that had been described as non-detoxified, in each organ and will be further purified and analyzedusing gel electrophoresis and molecular mass spectrometry (ESI-MS/MS) assisted by atomic mass spectrometry (ICP MS). These spe-ciation studies should give information on the nature of proteincomplexing U (metallo-, phosphorylated-) and the correlation tobiological effects will represent a key step to elucidate the chemo-toxicity of this element.

Acknowledgements

The authors gratefully thank B. Geffroy for the breeding of cray-fish and S. Al Kaddissi for the opportunity given to share samples.

This work was partly supported by National Research Agency(ANR) throughout the ST MALO – 2010 JCJC 713 1 project and IRSNwithin the EnviHom Eco program.

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