Dr Paco Bustamante

Pierre Miramand

Interspecific and geographical variations of trace element concentrations in Pectinidae from European waters

Keywords: Heavy metals, Bioaccumulation, Body distribution, Subcellular distribution, Scallop

Cd, Cu and Zn were analysed in the organs and tissues of the three scallop species from the Bay of Biscay, the variegated Chlamys varia L., the queen scallop Aequipecten opercularis L. and the common scallop Pecten maximus L. for interspecific comparisons. In P. maximus the greatest concentrations of Cd and Cu were found in the digestive gland, whereas the two other species showed similar levels of Cd between digestive gland and kidneys and higher renal Cu concentrations. However, the digestive gland of all Pectinidae species contained from 75 to 93 % and 52 to 74 % of the total body burdens of Cd and Cu, respectively. Whatever the species and the origin, kidneys displayed the highest Zn concentrations, which therefore contained from 53 to 97 % of the total body burden of this metal. Also using reported results, ratios between the concentrations in the digestive gland and that in the kidneys discriminated two groups of Pectinidae : 1) the Pecten group (P. maximus, P. jacobeus and Adamussium colbecki) with a Cd ratio ≥ 4, a Cu ratio ≥ 1 and a Zn ratio > 20.10 -3 2) the Chlamys group with a Cd ratio ≤ 1, a Cu ratio ≤ 1 and a Zn ratio ≤ 6.10 -3 . However, no differences in the detoxification processes in the digestive gland were found between groups in this study.

72 to 80 % of the total Cd was found to be soluble, probably bound to metalloproteins such as metallothioneins, while Cu and Zn were mainly found associated to the insoluble fraction of the digestive gland cells whatever the species. Queen scallops from the Faroe Islands were also considered to examine variations of the metal concentrations due to the geographical origin. In this northern area, queen scallops displayed Cd concentrations 2, 4 and 6 times higher in kidneys, muscle and digestive gland, respectively, compared to those from the Bay of Biscay. Consequently, the Cd concentrations in the whole soft parts of the queen scallops were more than 6 times higher in the Faroe Islands than in the Bay of Biscay. In contrast to Cd, both Cu and Zn concentrations in the whole soft parts were 2 times lower in the Faroe Islands, suggesting lower bioavailability of these essential elements.

INTRODUCTION

Among seashells, numerous scallop species represent an important target to commercial fisheries. Many of them are actually cultured for direct commercial production or to provide juveniles for reintroduction programs in areas over-exploited. On the French Atlantic coasts and in the English Channel, scallops are relatively common, largely distributed and belong to three species: the variegated scallop Chlamys varia on rocky substrata, the queen scallop Aequipecten opercularis and the common scallop Pecten maximus on sandy bottoms. In France, these three species are captured during winter time and mobilises around one thousand fishing boats with various duration. The total of the catch varies greatly form one year to another, ranging from 6 000 tons to 25 000 tons per year [START_REF] Quéro | Les fruits de mer et plantes marines des pêches françaises[END_REF]. The three species are also captured for leisure activities, directly on the shore or by snorkelling. Scallops represent therefore an elevated economic values in fishing areas.

Bivalves are classically chosen for studies on trace element bioaccumulation [START_REF] Bryan | Some aspects of heavy metal tolerance in aquatic organisms[END_REF]. They are poorly capable to regulate ion concentrations in internal fluids and they are highly tolerant, metal ions being sequestered in excess of metabolic requirement [START_REF] George | Correlation of metal accumulation in mussels with the mechanisms of uptake, metabolism and detoxification: biochemical and ultrastructural studies[END_REF]. Therefore, bivalves often display high accumulation levels without apparent toxic effect. Among bivalves, scallops have shown a particular ability to concentrate high levels of pollutants such as trace elements, even in areas remote from direct anthropogenic contamination like the Antarctic [START_REF] Mauri | Heavy metals in the Antarctic scallop Adamussium colbecki[END_REF][START_REF] Berkman | Trace metal concentrations in scallops around Antartica: extending the Mussel Watch Programme to the Southern Ocean[END_REF][START_REF] Viarengo | Cu, Zn and Cd content in different tissues of the Antarctic scallop Adamussium colbecki : role of the metallothionein in heavy metal homeostasis and detoxification[END_REF] or the Arctic Oceans [START_REF] Stepnowski | A comparison of 210 Po accumulation in molluscs from the southern Baltic, the coast of Spitsbergen and Sasek Wielki Lake in Poland[END_REF]. Therefore, scallops are generally considered as valuable bioindicators to monitor marine pollution [START_REF] Bryan | The occurrence and seasonal variation of trace metals in the scallops Pecten maximus (L.) and Chlamys opercularis (L.)[END_REF], Bustamante et al., 2002a). However, large interspecific variations seem to occur when compared trace element concentrations between different scallop species [START_REF] Mauri | Heavy metals in the Antarctic scallop Adamussium colbecki[END_REF][START_REF] Viarengo | Cu, Zn and Cd content in different tissues of the Antarctic scallop Adamussium colbecki : role of the metallothionein in heavy metal homeostasis and detoxification[END_REF]. Such variations are not well characterized since only a single species is usually studied at the same place. Furthermore, geographical variations for a single scallop species have been rarely considered, different species from various latitudes being generally compared.

In this study, Cd, Cu and Zn, were analyzed in the organs and tissues of the three common scallop species living along the Atlantic coast of France. Those metals were determined by flame and graphite atomic absorption spectrophotometry in the variegated scallop C. varia, in the queen scallop A. opercularis and in the common scallop P. maximus, from the Charente-Maritime coasts, Bay of Biscay, France, in an attempt to determine the interspecific variations in the metal bioaccumulation. Furthermore, specimens of A. opercularis, a widely distributed species in the Northeastern Atlantic from Morocco to the north of Norway, were collected off the Faroe Islands for comparison. Elemental concentrations, tissular and subcellular distributions, and interspecific and geographical differences were therefore examined.

MATERIAL AND METHODS

Sampling and sample preparation

In February 1996, 41 variegated scallops (Chlamys varia, Linné, 1758) were collected by hand in the infralittoral zone in the coast of Charente-Maritime (Bay of Biscay, France).

After 48 hours depuration in clean sea water to eliminate faecal and pseudo-faecal material, scallops were pooled by size classes.

The queen and common scallops (Aequipecten opercularis, Linné, 1758 and Pecten maximus, Linné, 1758, respectively) have been opportunistically collected by bottom trawl along the Charente-Maritime coasts between February and May 1997. Individuals (n=11 for each species) were separated in individual plastic bags and frozen on board.

Queen scallops A. opercularis from the Faroe Islands have also been collected opportunistically by bottom trawl in August 1996 and frozen on board as described above (n=31).

For each scallop, adductor muscle, gonad, gills, kidneys, and digestive gland were separated from the rest of the soft part. The remaining tissues including the mantle, the foot, the intestine and the heart, were also analysed in order to calculate the whole trace element contained of the soft parts.

Subcellular fractionation

The partitioning of trace elements between soluble and insoluble subcellular fractions of the digestive gland was investigated in the three scallop species. Thus, 40 Chlamys varia, 23 Pecten maximus, and 10 Aequipecten opercularis were devoted to subcellular investigations. Four pools were made with the digestive glands removed from the soft parts of C. varia and P. Maximus freshly dissected. In the case of A. opercularis, digestive glands were removed on board to be deep frozen in liquid nitrogen and consequently were treated individually.

Both pooled and individual digestive glands were subsequently homogenised in a Dounce potter on ice with 10 volumes of a 0.02 M TRIS-HCl buffer in 0.25 M sucrose with 1 mM PMSF (phenylmethylsulfonylfluoride, as protease inhibitor) and 5 mM DTT (dithiothreitol, as reducing agent), at pH 8.6 (Bustamante et al. 2002b). The homogenates were centrifuged at 100 000 G for 1 h at 4°C in a Beckman LE-70 ultracentrifuge.

Particle-free supernatants (cytosols) were separated from the pellet. Aliquots of the homogenates, cytosols and pellets were analysed for trace elements.

Trace element analyses

Tissue samples were dried for several days at 80°C to constant weight. Whenever possible, two aliquots of approx. 300 mg of each homogenised dry sample were digested with 4 ml of 65% ultrapure HNO 3 and 0.3 ml of ultrapure 70 % HClO 4 at 80°C until the solution was clear. Then acids were evaporated and residues were dissolved in 0.3N ultrapure nitric acid.

Cadmium, copper and zinc were determined by flame and graphite furnace atomic absorption spectrophotometry with a Varian spectrophotometer Vectra 250 Plus, with Deuterium background correction. Certified reference materials, dogfish liver DOLT-2 (NRCC) and dogfish muscle DORM-2 (NRCC), were treated and analysed in the same way as the samples. The results for standard reference materials were in good agreement with certified values (Table 1). The detection limits calculated for 0.3 g of dry tissue were (µg.g -1 dwt): 0.005 (Cd), 0.5 (Cu), and 3 (Zn). Metal concentrations in tissues are given relatively to the dry weight (µg.g -1 dry wt) while the distribution percentages were calculated for wet weight.

Data analyses

Comparison of metal concentrations between species were tested by one-way ANOVA (after log transformation of data when necessary) followed by the Tukey's multiple comparison test in the MINITAB 13.1 software. Changes in metal distribution among scallop tissues were tested for significance by the G procedure (adapted from the loglikelihood ratio test) for 2 x k contingency tables [START_REF] Zar | Heavy metals in fish from the Barents Sea (summer 1994)[END_REF]. The significance level for statistical analyses was always set at α =0.05.

RESULTS

Concentrations of Cd, Cu and Zn in the tissues of 3 scallop species from the Bay of Biscay and 1 species from the Faroe Islands are presented in Table 2. Concentrations in the whole soft parts were calculated using the concentrations determined in each compartments. The body distribution of the trace elements of the scallops is presented in Table 3.

Cadmium

Among tissues, the digestive gland and the kidneys displayed the highest Cd concentrations for all species and in both areas. Interestingly, the Cd values measured in the different species were in the same range of concentrations in both organs with the exception of P. maximus. In this species, Cd concentrations in this species were around 4 times higher in the digestive gland than in the kidneys, i.e. 264 ± 33 µg.g -1 dwt vs 70.6 ± 12.6 µg.g -1 dwt, respectively (Table 2). The digestive gland contained most of the total body burden of Cd in all species, probably because of the significance in weight of this organ (Table 3). For Cd, a G-test did not indicate any significant (p>0.05) difference among metal body distribution between species and areas.

Among the scallops from the Bay of Biscay, P. maximus showed a strong accumulation of Cd in its tissues, especially in kidneys, muscle and digestive gland compared to the other species. Thus, these tissues displayed Cd concentrations 2, 4, and 6 times higher, respectively, than those measured in C. varia and in A. opercularis (Table 2).

Consequently, the whole soft parts of P. maximus contained Cd concentrations more than 4 times higher than in the other two scallop species (Table 2).

When compared Faroe Islands and Bay of Biscay, Cd concentrations in A. opercularis were in the same range in the gonad, gills, and muscles for both geographic areas (F=2.31 p=0.140). Nevertheless, A. opercularis from the Faroe Islands displayed more than 4 times Cd concentrations in the digestive gland and 7 times higher in the kidneys than in queen scallops from the Bay of Biscay (Table 2). Consequently, the digestive gland of A. opercularis from the Faroe Island contained even higher percentage to the total body burdens of Cd, i.e. 90% vs 75% (Table 3). Whichever the origin was, in the digestive gland of the three species more than 70 % of the total Cd was found in the particle free supernatants (Table 4).

Copper

Similarly to Cd, the digestive gland and the kidneys displayed the highest Cu concentrations for all species and in both areas. However, this metal displayed large variations according to the species and to the origin of the scallop.

In the scallops from the Bay of Biscay, Cu clearly showed the highest concentrations in kidneys except for P. maximus, whose concentrations were 3 times higher in the digestive gland (Table 2). Specifically in this species, the gonad exhibited relatively elevated Cu concentrations (19.7 ± 16.1 µg.g -1 dwt), which were in the same order of magnitude than in kidneys (13.5 ± 2.0 µg.g -1 dwt). Despite such variations in the Cu concentrations, the digestive gland of the different scallop species contained most of the whole body burden of Cu (Table 3). Therefore, tissue distribution of Cu did not varied significantly (G-test, p>0.05) between all the different scallop species.

Considering the whole soft parts, scallops from the Faroe Islands showed Cu concentrations 2 times lower than those from the Bay of Biscay (Table 2). Globally, the concentrations of Cu in the gonad, gills, muscle and remaining tissues were slightly lower in scallop from the Faroe Islands. This difference was even more important for the digestive gland and kidneys (Table 2). However, the digestive gland of the scallops from the Faroe Islands also contained most of the whole body burden of Cu, i.e. 61 ± 4% (Table 3).

Whichever the origin was, in the digestive gland most of the Cu was found associated to the pellet of centrifugation, only 25 to 43% of the total Cu being soluble (Table 4).

Zinc

In scallops, kidneys displayed the highest Zn concentrations for all species and in both areas(Table 2). These concentrations were several thousand of µg (i.e. exceeding 30,000 µg Zn.g -1 in A. opercularis from the Bay of Biscay).

In the Bay of Biscay, the concentrations of Zn were 7 times higher in A. opercularis than in P. maximus but not significantly different from C. varia. In contrast, P. maximus exhibited Zn concentrations in the digestive gland which were 4 times higher than in C. varia and A. opercularis. Despite such differences in the tissue concentrations, kidneys of the different scallop species contained from 72 to 91 % of the whole body burden of Zn (Table 3). Moreover, a G-test did not indicate any significant (p>0.05) difference among metal body distribution between these species.

In the Faroe Islands, A. opercularis exhibited lower Zn concentrations in all their tissues than in the Bay of Biscay (Table 2). This was particularly obvious for the kidneys which concentrations were 7 times lower compared to A. opercularis from the Bay of Biscay while such a difference was only a 2 or 3 fold for the other tissues. Consequently, A. opercularis from the Faroe Islands only exhibited 53 % of the whole body burden of Zn in their kidneys (Table 3).

Despite interspecific and geographical differences in the concentrations, the proportion of soluble Zn within the digestive gland cells poorly varied, i.e. from 38 to 50% in the different scallop species (Table 4).

DISCUSSION

Interspecific comparisons

Among bivalves, scallops accumulate trace elements at often high concentrations, depending on the metal and the tissue. The kidney and the digestive gland have a major importance in the bioaccumulation of Cd, Cu and Zn in Pectinidae. These organs exhibited high metal concentrations and contained most of the total body burden of these trace elements (Tables 1 &2). The other tissues seems to have a minor importance in the storage of Cd, Cu and Zn although they might have a major role in uptake and transfer of trace elements. However, one of the goals of the present study was to provide background levels against which to perform interspecific comparison within the Pectinidae family.

Results from this study allowed to discriminate two groups of scallops : on the one hand Chlamys varia and Aequipecten opercularis (formerly called Chlamys opercularis) and on the other side Pecten maximus. These two groups are revealed by the ratio between the concentrations of trace elements in the digestive gland and that in kidneys : 1) the Pecten group (P. maximus) with a Cd ratio ≥ 4, a Cu ratio ≥ 1 and a Zn ratio > 20.10 -3 and, 2) the Chlamys group (C. varia and A. opercularis) with a Cd ratio ≤ 1, a Cu ratio ≤ 1 and a Zn ratio ≤ 6.10 -3 . Even though metal concentrations have been studied in various scallop species, little data from the current literature allowed to make such calculations, mainly because of the lack of values in kidneys. Table 5 reports Cd, Cu and Zn concentrations in the digestive gland and kidneys of different scallops species from various locations. Even though the variation of the concentrations of Cd, Cu and Zn in both tissues is very large, the ratio of the concentrations between the digestive gland and the kidneys were consistent with our results. Thus, P. maximus from the English Channel and P. jacobeus from the Mediterranean showed ratios around 4 for Cd, > 20.10 -3 for Zn, and respectively 3 and 1 for Cu. These calculated ratio were even higher for the Antarctic scallop Adamussium colbecki (i.e. 12 for Cd, 3 for Cu and 377.10 -3 for Zn) suggesting a metabolism of metals close to the one of the Pecten group. In contrast, taking into account the moisture of the tissues, a ratio around 1 for Cd suggests that Placopecten magellanicus is closer to the Chlamys group.

The bioaccumulation of trace elements in the digestive gland and kidneys might be related to the detoxification processes of metals. The reasons of high concentrations of trace elements in the tissues of Pectinidae are typically attributed to the binding to cytosolic proteins in the digestive gland and to precipitation on granules in kidneys [START_REF] George | Isolation and elemental analysis of metal-rich granules from the kidney of the scallop, Pecten maximus (L.)[END_REF][START_REF] Ballan-Dufrançais | Physiological features of heavy metal storage in bivalve digestive cells and amoebocytes : EPMA and factor analysis of correspondences[END_REF][START_REF] Stone | Cadmium binding components of scallop (Pecten maximus) digestive gland. Partial purification and characterisation[END_REF][START_REF] Fowler | Ultrastructural and biochemical studies of intracellular metal-binding patterns in kidney tubule cells of the scallop Placopecten magellanicus following prolonged exposure to cadmium or copper[END_REF][START_REF] Mauri | Heavy metals in the Antarctic scallop Adamussium colbecki[END_REF][START_REF] Viarengo | Cu, Zn and Cd content in different tissues of the Antarctic scallop Adamussium colbecki : role of the metallothionein in heavy metal homeostasis and detoxification[END_REF]. Globally, information on the subcellular distribution of metals in Pectinidae is scarce. The cytosolic proteins of the digestive gland cells of the three scallop species seem to have a minor importance in the binding of Cu and Zn. These two metals were generally found in large proportions into the insoluble fraction, which represent the metals trapped within the organites, associated to membranes or to granules. This might be due to their numerous biochemical functions that imply they are involved in the structure of several proteins. In contrast to Cu and Zn, from 72 to 80 % of the total Cd was found in the cytosolic fraction of the digestive gland cells of the three scallop species. With the exception of the Antarctic scallop Adamussium colbecki, for which only 30 % of the total Cd was soluble [START_REF] Viarengo | Cu, Zn and Cd content in different tissues of the Antarctic scallop Adamussium colbecki : role of the metallothionein in heavy metal homeostasis and detoxification[END_REF], 60 to 99 % of the Cd was found in the soluble fraction of the digestive gland of Pectinidae from field or after experimental exposure to this metal [START_REF] Evtushenko | Cadmium accumulation in organs of the scallop Mizuhopecten yessoensis. II. Subcellular distribution of metals and metal-binding proteins[END_REF], 1990[START_REF] Stone | Cadmium binding components of scallop (Pecten maximus) digestive gland. Partial purification and characterisation[END_REF][START_REF] Lukyanova | Binding of zinc and cadmium with cytoplasmic proteins in the Yezo scallop at the increased concentrations of zinc in water[END_REF]. This strongly suggests the occurrence of mechanisms of accumulation and detoxification of Cd involving proteins such as metallothioneins [START_REF] Evtushenko | Cadmium accumulation in organs of the scallop Mizuhopecten yessoensis. II. Subcellular distribution of metals and metal-binding proteins[END_REF], 1990[START_REF] Stone | Cadmium binding components of scallop (Pecten maximus) digestive gland. Partial purification and characterisation[END_REF][START_REF] Viarengo | Cu, Zn and Cd content in different tissues of the Antarctic scallop Adamussium colbecki : role of the metallothionein in heavy metal homeostasis and detoxification[END_REF][START_REF] Bustamante | Etude des processus de bioaccumulation et de détoxication d'éléments traces (métaux lourds et terres rares) chez les mollusques céphalopodes et bivalves pectinidés. Implication de leur biodisponibilité pour le transfert vers les prédateurs[END_REF]. Similar detoxification strategies for Cd, Cu and Zn in the digestive gland of the Chlamys and the Pecten groups suggest that differences between the two groups are related to detoxification and excretion processes in the kidneys. Indeed, metal-rich granules from the kidney of P. maximus are considered as excretory concretions which can be eliminated in urine [START_REF] George | Isolation and elemental analysis of metal-rich granules from the kidney of the scallop, Pecten maximus (L.)[END_REF]). Higher excretion rates in the Pecten group compared to the Chlamys one might lead to overall lower Cd, Cu and Zn concentrations in this organ. However, further studies in the Chlamys group are needed to support this hypothesis.

Geographical comparisons

Scallops of different species within the same area and scallops of the same species from different latitudes showed elevated variations in their ability to concentrate trace elements (Table 5). Such variations are due to interspecific diversity but also to geographic conditions. Accumulation of high metal concentrations, particularly Cd, even in unpolluted areas might be related to different bioavailability of the elements, to different feeding habits, as well as to physiological features. As regards to the concentrations found in the gills, seawater appears to be an important source of metals for scallops.

However, trace element concentrations in the soft tissues and primarily in the digestive gland might substantially be influenced by the metal content of their food. Indeed, scallops are filter feeders using different types of food such as phytoplankton, detritus and resuspended matter which are major components of the variation in food quality in the field through variation in their carbon and nitrogen contents [START_REF] Grant | Carbon and nitrogen scope for growth as a function of diet in the sea scallop Placopecten magellanicus[END_REF]Cranford, 1991, Alber and[START_REF] Alber | Utilization of microbial organic aggregates by bay scallops, Argopecten irradians (Lamarck)[END_REF]. It would therefore be of particular interest to characterise the food composition and its metal concentrations of the different scallops.

As reported for other marine invertebrates [START_REF] Cubadda | Size-dependent concentrations of trace metals in four Mediterranean gastropods[END_REF], the use of scallops as biomonitors on large geographical scales, i.e. between 44 to 46° N for the Bay of Biscay and 61 to 63 ° N for the Faroe Islands, might be envisaged with caution. In this investigation, the higher Cd concentrations found in A. opercularis from the Faroe Islands compared to those from the Bay of Biscay highlighted that the contents of one or more metals in marine organisms can be increased by specific geochemical or hydrodynamic conditions. In the Faroe Islands, enrichment of metals due to industrial contamination does not exist. Consequently, the high Cd concentrations in the tissues of A. opercularis might be considered as natural in this sub-polar area. Reported Cd enrichment throughout the Arctic remained globally unclear. Several authors have proposed that a phenomenon of Cd abnormality occurs in polar and subpolar areas, leading to very high Cd concentrations in marine biota [START_REF] Petri | Trace metal in the crustaceans in the Antarctic Ocean[END_REF][START_REF] Bargagli | Elevated cadmium accumulation in marine organisms from Terra Nova Bay (Antarctica)[END_REF], Bustamante et al., 1998a, Zauke et al., 1999). Several results suggest that such an abnormality has also to be considered in the Faroe Islands [START_REF] Caurant | Ecological and biological factors controlling the concentrations of trace elements (As, Cd, Cu, Hg, Se, Zn) in delphinids Globicephala melas from the North Atlantic Ocean[END_REF], Bustamante et al., 1998b[START_REF] Bustamante | Individual and population significance of trace elements bioaccumulation in the grey seal Halichoerus grypus from the Faroe Islands[END_REF]. Indeed, similarly to polar regions, soluble trace element concentrations in seawater appear to be relatively low around this area [START_REF] Mart | Trace metal levels in the eastern Arctic Ocean[END_REF]. Despite that, very high Cd concentrations have also been found in several cephalopod species caught around the archipelago, i.e. from 2.1 µg.g -1 dwt in the Loliginid squid Loligo forbesi, to 46.8 µg.g -1 dwt in the octopus Eledone cirrhosa [START_REF] Bustamante | Etude des processus de bioaccumulation et de détoxication d'éléments traces (métaux lourds et terres rares) chez les mollusques céphalopodes et bivalves pectinidés. Implication de leur biodisponibilité pour le transfert vers les prédateurs[END_REF], Bustamante et al., 1998b). Moreover, coastal invertebrates such as dog-whelks and limpets also displayed elevated Cd concentrations, i.e. 140 µg.g -1 dwt and 50 µg.g -1 dwt, respectively [START_REF] Dam | Integrated ecological monitoring in the coastal zone; Environmental Pollutants. The Food and Environmental Agency of the Faroe Islands[END_REF]. Therefore, the high Cd concentrations in the tissues of A. opercularis might reflect a global enrichment by this metal in the Faroe Islands biota. Nevertheless, the reasons of such an enrichment remain to be determined.

In contrast, Cu and Zn appeared to be concentrated in much lower extent in the scallops from the Faroe Islands compared to those from the Bay of Biscay and from the English Channel [START_REF] Bryan | The occurrence and seasonal variation of trace metals in the scallops Pecten maximus (L.) and Chlamys opercularis (L.)[END_REF]. Lower concentrations of these essential elements could be due to low bioavailability of these two metal in the Faroe Island waters. High Cd concentrations associated with low Cu and Zn values are typically attributed to the substitution of Cd ions for essential metals under deficient conditions (Zauke and [START_REF] Petri | Trace metal in the crustaceans in the Antarctic Ocean[END_REF], Bustamante et al., 1998a). This possibly occurred in A. opercularis from the Faroe Islands. Indeed, the digestive gland showed a high Cd / Cu ratio (i.e. 8.8) compared to the Bay of Biscay (i.e. 0.6). In the Faroe scallops, this high ratio was due to both elevated Cd concentrations and to low Cu concentrations. Cu concentrations in the digestive gland were lower than concentrations found in the temperate scallop species but in the same range than the concentrations reported for the Antarctic scallop A. colbecki (Table 5). Large amounts of Cu are essential to marine mollusks, for which Cd and Cu are known to interfere on the same type of metalloproteins (i.e. metallothioneins) in the digestive gland. A competition or a substitution between the two elements may have occurred in the Faroe scallops.

CONCLUSION

The three different species examined in this study accumulated high trace element concentrations in their tissues, particularly in the digestive gland and in the kidney. The accumulation of Cd was even higher for Cd in the subpolar Faroe area. In contrast to Cd, lower Cu and Zn concentrations suggests a poor bioavailability of these essential elements in that area. Regardless of the species and the origin, Cd appeared to be associated to cytosolic proteins involved in detoxification functions. The presence of Cu and Zn on organites and membranes might be due to their essentiality.

Table 1 .

 1 Comparison of trace elements concentrations (µg.g -1 dry wt) of dogfish liver DOLT-2 (NRCC), and dogfish muscle (NRCC) determined in the present study with certified values.

	Metals	DOLT-2 (n=5)	DORM-2 (n=5)
		Certified values Present study Certified values Present study
	Cd	20.8 ± 0.5	19.7 ± 0.6	0.043 ± 0.008 0.042 ± 0.014
	Cu	25.8 ± 1.1	24.9 ± 1.2	2.34 ± 0.16	2.30 ± 0.22
	Zn	85.8 ± 2.5	84.5 ± 2.8	25.6 ± 2.3	26.3 ± 1.8

Table 4 .

 4 Proportion of Cd, Cu, and Zn in the cytosol of the digestive gland cells (% of the wet weight) of three scallop species from the Bay of Biscay and the Faroe Islands. N :

	number of individuals
	Species	Location	N	Cd	Cu	Zn
	Pecten maximus	Bay of Biscay	23	72 ± 5	43 ± 4	38 ± 7
	Chlamys varia	Bay of Biscay	40	78 ± 5	25 ± 8	41 ± 3
	Aequipecten opercularis	Faroe Islands	10	80 ± 7	35 ± 16	50 ± 11

Table 5 .

 5 Mean ± SD of Cd, Cu, and Zn concentrations (µg.g -1 dwt) in different scallop 503 species from various locations. N: sample size.

	504
	505
	Species	Location	N	Cd	Cu	Zn	Reference
	Digestive gland
	Amussium balloti	Australia	10	111 a	-	-	Francesconi et al. 1993
	Adamussium colbecki	Antarctic	30	142 ± 57	12.6 ± 3.3	74.9 ± 25.4	Mauri et al. (1990)
	"	"	6	26.51 ± 6.09 a	3.52 ± 0.49 a	17.42 ± 2.91 a	Viarengo et al. (1993)
	Aequipecten opercularis	Northeast Atlantic	31	123 ± 14	14.0 ± 0.8	77 ± 14	Present study
	"	English Channel	11	27	36.7 ± 11.4	132 ± 26	Bryan (1973)
	"	Bay of Biscay	11	27.5 ± 4.24	43.6 ± 19.5	198 ± 47	Present study
	Chlamys varia	"	41	40.0 ± 4.43	88.1 ± 13.8	149 ± 27	"
	Pecten jacobeus	Mediterranean Sea	30	41.0 ± 18.2	16.6 ± 6.6	124 ± 55	Mauri et al. (1990)
	"	"	6	7.19 ± 1.23 a	3.13 ± 0.55 a	28.63 ± 3.29 a	Viarengo et al. (1993)
	P. maximus	English Channel	11	321	57.9 ± 27.4	407 ± 123	Bryan (1973)
	"	Bay of Biscay	11	264 ± 33	40.7 ± 7.8	804 ± 92	Present study
	Placopecten magellanicus	Northwest Atlantic	6	94.68 ± 8.42 a	-	-	Uthe & Chou (1987)
	Kidney
	Adamussium colbecki	Antarctic	30	11.6 ± 3.3	4.0 ± 1.7	199 ± 89	Mauri et al. (1990)
	Aequipecten opercularis	Northeast Atlantic	31	159 ± 80	106 ± 49	6632 ± 3664	Present study
	"	English Channel	11	41	1285 ± 591	40800 ± 7290	Bryan (1973)
	"	Bay of Biscay	11	21.1 ± 3.15	507 ± 144	31053 ± 8147	Present study
	Chlamys varia	"	41	38.8 ± 11.1	142 ± 20	24107 ± 3465	"
	Pecten jacobeus	Mediterranean Sea	30	10.7 ± 4.0	17.5 ± 10.9	2790 ± 1340	Mauri et al. (1990)
	P. maximus	English Channel	11	79	20.8 ± 10.3	19300 ± 4800	Bryan (1973)
	"	Bay of Biscay	11	70.6 ± 12.7	13.5 ± 2.0	7278 ± 3041	Present study
	Placopecten magellanicus	Northwest Atlantic	6	62.61 ± 25.33 a	-	-

[START_REF] Uthe | Cadmium in sea scallop (Placopecten magellanicus) tissues from clean and contaminated areas[END_REF]

a :values expressed relatively to wet weight.

ACKNOWLEDGEMENTS

We thank the 'Fiskirannsoknarstovan' (Torshavn, Faroe Islands) and IFREMER (Direction des Ressources Halieutiques, Nantes) for facilitating collections of the scallops on the RVs Magnus Heinasson and Gwen-Drez. This work was financially supported by the "Conseil Régional Poitou-Charentes" and Rhodia. We are grateful to the anonymous referees of this manuscript for their fruitful criticism.


Dr Paco Bustamante

Pierre Miramand

Subcellular and body distributions of 17 trace elements in the variegated scallop Chlamys varia from the French coast of the Bay of Biscay

Keywords: Heavy metals, Trace elements, Rare earth elements, Bioaccumulation, Subcellular distribution

Seventeen elements were analysed in the organs and tissues of the variegated scallop Chlamys varia, from the Atlantic coast of France. Concentration levels were determined in scallops of different sizes sampled in contaminated (La Rochelle Bay) and clean (Ré Island) sites.

Greater concentrations of Ag, Al, Ce, Cr, La, Mo, Nd, Ti, and V were found in the digestive gland while As, Cd, Co, Cu, Mn, Ni, Pb and Zn were the highest in the kidneys. In the digestive gland, most of the metals were found in the insoluble fraction while As, Co, Cd, Mo, Ni, and V appeared to be mostly bound to soluble compounds. Among tissues, the adductor muscle always displayed the lowest trace element concentrations. According to size, Ag and Cd showed significantly higher concentrations in larger individuals, while Co and Zn were higher in the smallest ones. According to the sampling area, most of the metals, Ag, Al, Ce, Co, Cu, La, Mn, Nd, Pb and Zn, showed significantly higher concentrations in La Rochelle Bay compared to the Ré Island, reflecting differing inputs from industrial, domestic and harbour activities. However, Cr, Mo, Ni, Ti and V concentrations did not display significant differences between sites and As and Cd were significantly higher at the Ré Island. This study highlighted the ability of the variegated scallop C. varia to concentrate numerous trace elements to high levels, even those reported as poorly bioavailable for marine biota, such as rare earth elements.

INTRODUCTION

Among filter feeders, scallops have been reported to be useful biomonitors to survey marine pollution. Indeed, numerous studies have demonstrated their ability to concentrate trace elements, even in areas far from anthropogenic sources such as the Antarctic Ocean (e.g. [START_REF] Mauri | Heavy metals in the Antarctic scallop Adamussium colbecki[END_REF], Berkman & Nigro 1992[START_REF] Viarengo | Cu, Zn and Cd content in different tissues of the Antarctic scallop Adamussium colbecki : role of the metallothionein in heavy metal homeostasis and detoxification[END_REF], with seasonal variations in the concentrations at various stages of their lives (Bryan 1973). However, the distribution of trace elements between body tissues has been rarely considered. Furthermore, earlier studies of elemental analysis in scallops have focused only on a few heavy metals. Among them, Cd has received particular attention since it was first detected by a spectrographic method in Pecten maximus by Fox and Ramage (1931). The strong capacity of scallops to bioaccumulate numerous trace elements at high concentrations in their tissues is related to their very efficient detoxification systems involving lysosomal compartmentalisation, coprecipitation on phosphate granules and binding to soluble proteins such as metallothioneins (George et al. 1980, Ballan-Dufrançais et al. 1985[START_REF] Stone | Cadmium binding components of scallop (Pecten maximus) digestive gland. Partial purification and characterisation[END_REF], Fowler & Gould 1988[START_REF] Mauri | Heavy metals in the Antarctic scallop Adamussium colbecki[END_REF][START_REF] Viarengo | Cu, Zn and Cd content in different tissues of the Antarctic scallop Adamussium colbecki : role of the metallothionein in heavy metal homeostasis and detoxification[END_REF]). These detoxification processes seem to be particularly efficient in the digestive gland and the kidneys of Pectinidae.

Therefore, such tissues have been proposed as valuable monitors of the availability of stable trace metals in the sea (Bryan 1973).

The aim of this study was to provide information on the concentrations, and tissue and subcellular distributions of 17 trace elements in the tissues and organs of the variegated scallop Chlamys varia from the Bay of Biscay. C. varia is a common seashell on the rocky shore of the French Atlantic coasts, altough rare in the English Channel. It is encountered in important shoals in Brittany and in Charente-Maritime where this species is targeted by a commercial fishery as well as for leisure activities (Letaconnoux & Audouin 1956[START_REF] Quéro | Les fruits de mer et plantes marines des pêches françaises[END_REF]. Comparisons have been made between scallops from an area subjected to anthropogenic inputs (industrial, domestic and harbour) i.e. La Rochelle Bay, and from a clean site close to a commercially exploited scallop bed, i.e. Ré Island. At each site, elemental concentrations, distribution among tissues and organs, and size-related changes variations in concentrations were examined. Moreover, the partitioning of trace elements between soluble and insoluble subcellular fractions of the digestive gland was investigated as this organ has been reported to concentrate numerous trace elements to high levels of in Pectinidae.

MATERIAL AND METHODS

Sampling and sample preparation

For this study, 130 specimens were collected by hand in La Rochelle Bay (n = 90) and in Saint-Martin en Ré (n = 40) in Ré Island in February 1996 (Figure 1). Scallops were pooled into size classes of ten individuals according to the height of the shell and were depurated for 48 hours in clean seawater to eliminate faecal and pseudo-faecal material. The size of scallops from Ré Island ranges from 32 ± 1 mm (3 pools of 10 individuals) to 44 ± 2 mm (10 pooled individuals), and those from La Rochelle Bay from 33 ± 1 mm to 45 ± 2 mm (9 pools of 10 individuals). The low number of large scallops in Ré Island was due to the fishing pressure in this area where the biggest individuals (> 40 mm) are targeted for harvesting, while any kind of shellfishing is forbidden in the La Rochelle Bay sampling site because of possible bacterial contamination.

For each scallop, adductor muscle, gonad, gills, kidneys, and digestive gland were separated from the rest of the soft parts, and pooled for each defined size class. The remaining tissues including the mantle, the foot, the intestine and the heart, were also analysed in order to calculate the whole trace element content of the soft parts.

Subcellular fractionation

To investigate the partitioning of trace elements between soluble and insoluble subcellular fractions of the digestive gland, 40 scallops (size = 45 ± 2 mm) were collected in winter 1996 in La Rochelle Bay and were depurated as described above.

Four pools of 10 individuals were made with the digestive glands removed from the soft parts of the 40 scallops. They were subsequently homogenised with a mortar and pestle on ice with 10 volumes of TRIS-HCl buffer 0.02 M sucrose 0.25 M with 1 mM PMSF (phenylmethylsulfonylfluoride, as protease inhibitor) and 5 mM DTT (dithiothreitol, as reducing agent), at pH 8.6. The homogenates were centrifuged at 100 000 G for 1 h at 4°C in a Beckman LE-70 ultracentrifuge. Particle-free supernatants (cytosols) were separated from the pellet.

Aliquots of the homogenates, cytosols and pellets were analysed for trace elements.

Trace element analyses

Tissue samples were dried for several days at 80°C to constant weight. Whenever it was possible, two aliquots of approx. 300 mg of each homogenised dry sample were digested with 4 ml of 65% ultrapure HNO 3 and 0.3 ml of ultrapure 70 % HClO 4 at 80°C until the solution was clear. Then acids were evaporated and residues were dissolved in 0.3N ultrapure nitric acid.

Cadmium, copper and zinc were determined by atomic absorption spectrophotometry. Other elements (Ag, Al, As, Ce, Co, Cr, La, Mn, Mo, Nd, Ni, Pb, Ti and V) were analysed by ICP-MS.

Appliances used for trace element determination were a Varian spectrophotometer Vectra 250 Plus with deuterium background correction and a Varian ICP-MS Ultra Mass 700.

Reference tissues, dogfish liver DOLT-2 (NRCC), dogfish muscle DORM-2 (NRCC), lobster hepatopancreas TORT-2 (NRCC), and Bush Branches and Leaves (GBW 07602) were treated and analysed in the same way as the samples. The results for standard reference materials displayed recoveries of the elements ranging from 86 % to 112 %. The detection limits were (µg.g -1 dry wt): 0.002 (Ag), 0.005 (Al), 0.15 (As), 0.005 (Cd), 0.017 (Ce), 0.005 (Co), 0.005 (Cr), 0.027 (Cu), 0.017 (La), 0.017 (Mn), 0.017 (Mo), 0.017 (Nd), 0.002 (Pb), 0.002 (Ni), 0.033 (Ti), 0.005 (V) and 0.15 (Zn).

Data analyses

Trace element concentrations in tissues are given on a dry weight basis (µg.g -1 dry wt) while the distribution percentages were calculated based on wet weight. Comparison of metal concentrations between sites and between sizes were tested by the non-parametric Kruskall-Wallis test using MINITAB 13.1 Software. Changes in trace element distribution among scallop tissues were tested for significance by the G procedure for multiple comparisons [START_REF] Zar | Biostatistical Analysis[END_REF]. The significance level for statistical analyses was always set at α =0.05.

RESULTS

Elemental concentrations and tissular distribution

Trace element concentrations in the tissues and organs of variegated scallops Chlamys varia from the French coast of the Bay of Biscay are reported in Table 1. The digestive gland contained the highest concentrations of Ag, Al, Ce, Cr, La, Mo, Nd, Ti, and V. Secondly, kidneys exhibited the highest concentrations of As, Cd, Co, Cu, Mn, Ni, Pb and Zn. The adductor muscle of C. varia always displayed the lowest trace element concentrations. In this tissue, concentrations were remarkably low for non-essential elements such as Ag, Cd, Pb or rare earth elements and also for most of the essential ones, especially Co, Cu and Mo (Table 1). Compared to these low muscle concentrations, the gills and gonads of C. varia displayed generally relatively high concentrations of trace elements.

The distribution and relative body burdens of elements in C. varia have been calculated on the basis of the fresh tissue weights (Table 2). Among tissues, the digestive gland of C. varia accounted for only about 12% of the soft part weight but the percentage body burdens of Ag, Al, Cd, Ce, Co, Cr, Cu, Mo, Nd, Ni, and V were high in this tissue (Table 2). It is also remarkable that despite their very low percentage of the soft part weight (less than 2 %), kidneys displayed the highest proportions of Mn and Zn (up to 80%) and also high burdens of Pb (up to 40%). The proportions of trace elements accumulated in these organs clearly indicate their detoxification functions. Body burdens of all elements in gills and gonads were generally low (i.e. < 10%), and these organs represented a small fraction of the soft part weight.

The remaining tissues (mantle, foot, intestine and heart) contained large amounts of Ag, Al, As, Ce, Co, Cr, La, Mo, Nd, Ni, Ti and V since this tissue accounted for around 31% of the soft part weight. Although adductor muscle represented about 34% of the soft part weight, As was the only element contained in large proportion in this tissue (up to 45%). This difference is attributable to the very low concentrations found for the other trace elements in the adductor muscle of C. varia (Table 1).

Size trends in element concentrations

Table 1 presents the elemental concentrations in the tissues of C. varia of different sizes (from 32 ± 1 mm to 45 ± 2 mm) and possible size effects were investigated on the scallops from La Rochelle Bay representing 3 sizes from 3 pools of 10 individuals. Generally, few trace element concentrations (Ag, Cd, Co, Cu, Ni, and Zn) varied significantly with size of whole soft parts or of the tissue under consideration (Figure 2).

Considering the whole soft parts, concentrations of Ag and Cd were higher in large scallops (H= 7.20 p = 0.027 and H = 6.01 p = 0.050, respectively) while Co and Zn exhibited significantly higher concentrations in small individuals (H = 6.01 p = 0.050 for both metals). For Cd and Ag, these variations are clearly due to the bioaccumulation of Ag and Cd in the digestive gland where concentrations of both metals were also significantly higher in large scallops than in small ones (H = 7.20 p = 0.027 and H = 6.49 p = 0.039, respectively). For Co, concentrations in kidneys appear to be responsible for the decrease of concentrations with size in the whole soft parts (H = 6.01 p = 0.050), while for Zn, it seems to be due to significant concentration decrease in gills and gonad (H = 7.20 p = 0.027 and H = 6.01 p = 0.050, respectively) but not in whole soft parts.

As for Co, Ni concentrations decreased significantly with size in kidneys (H = 7.20 p = 0.027).

Finally, Cu concentrations increased with size in digestive gland (H = 6.01 p = 0.050), but without any significant effect on allometric relationship in whole soft parts.

A G-Test indicated that tissue distribution (% wet wt) was not significantly different (p < 0.05) between size for all elements except for Ag, which is mainly found in the remaining tissues and muscles in small scallops, and progressively increased in the digestive gland in large individuals (Table 2).

Accumulation differences between sites

Concentrations in the tissues and the whole soft parts of C. varia were compared between individuals of similar size from La Rochelle Bay and Ré Island (Figure 3). Considering the whole soft parts, only a few elements, i.e. Cr, Mo, Ni, Ti, and V, were in the same range at both sampled sites. In contrast, Ag, Al, Ce, Co, Cu, La, Mn, Nd, Pb, and Zn concentrations were significantly higher (P<0.05) at La Rochelle while As and Cd concentrations were higher at Ré Island.

Subcellular distribution within the digestive gland

Figure 4 presents the proportion of elements contained in the insoluble fraction of the digestive gland homogenates. Elements could be separated following their affinity to the insoluble fraction: 96 ± 4% of Ag, 85 ± 3% of Ce, 83 ± 3% of Nd, 82 ± 6% of Al, 82 ± 2% of La, 81 ± 8% of Mn, 75 ± 8 % of Cu, 73 ± 3% of Ti, 66 ± 6% of Pb and 59 ± 3% of Zn were found associated to the organelles and membranes of the digestive cells. In contrast, 53 ± 1% of Cr, 64 ± 6% of V, 65 ± 5% of Ni, 76 ± 4% of Co, 76 ± 2% of As, 77 ± 1% of Mo and 78 ± 5% of Cd were found in the cytosolic fraction.

DISCUSSION

Concentration and distribution of trace elements

Among the different tissues analysed, the digestive glands and kidneys of Chlamys varia from the French coasts of the Bay of Biscay displayed the highest elemental concentrations. Reported concentrations in these tissues for different scallop species from various locations are given in Table 3 for comparison with the larger scallops from La Rochelle Bay. Observed concentrations of Ag, Cd, Cr, Ni and Pb in C. varia for both tissues were similar to those previously reported.

However, concentrations of Al and Cu in digestive gland and kidney and of Mn and Zn in the digestive gland were much higher in C. varia than in other species. The reasons for high concentrations of trace elements in the tissues of Pectinidae are typically attributed to their binding to cytosolic proteins in the digestive gland and to their precipitation on granules in kidneys (George et al. 1980[START_REF] Stone | Cadmium binding components of scallop (Pecten maximus) digestive gland. Partial purification and characterisation[END_REF], Fowler & Gould 1988[START_REF] Mauri | Heavy metals in the Antarctic scallop Adamussium colbecki[END_REF][START_REF] Viarengo | Cu, Zn and Cd content in different tissues of the Antarctic scallop Adamussium colbecki : role of the metallothionein in heavy metal homeostasis and detoxification[END_REF]. With the exception of As, Cd, Co, Cr, Mo, Ni, and V, cytosolic proteins seem to have a minor importance in binding of most of the elements, i.e. Ag, Al, Ce, Cu, Nd, La, Mn, Ti, Pb, and Zn. Indeed, large proportions of these elements (59 to 96%) were found in the insoluble fraction which represents the metals trapped within the organelles, associated to membranes or to granules (Figure 4). This is particularly obvious in the case of rare earth elements. In fact, the lanthanides have chemistry and sorption/complexation characteristics analogous to transuranic elements such as plutonium or americium (Choppin Torres & Chopin 1984, Kim et al. 1989, Buckau et al. 1992).

In the grant scallop Pecten maximus, most of the 241 Am was found in the digestive gland associated with the stomach wall cells and the duct cells, and in lysosomal residual bodies within the digestive cells [START_REF] Miramand | Localisation autohistoradiographique de l'américium dans les tissus des coquilles St. Jacques Pecten maximus: étude préliminaire sur les mécanismes de transfert à partir de l'eau de mer[END_REF]. Residual bodies represent the ultimate form of the digestive processes before excretion in faeces, and are found in the insoluble fraction of digestive gland homogenates (Bustamante 1998). Therefore, it is likely that rare earth elements would be found in the same structures as 241 Am.

The insoluble fraction of the digestive gland cells also contained a remarkably high proportion of Ag, i.e. 96 ± 2% of the total metal. Silver is known to precipitate with sulphur within cells in bivalves (Ballan-Dufrançais et al. 1985, Martoja et al. 1989, Berthet et al. 1990, 1992). Overall, Ag, Al, Br, Ca, Cd, Cu, Fe, Mo, S, Si and Zn have been detected by both qualitative and quantitative analysis in the lysosomal system of the digestive gland of P. maximus, (Ballan-Dufrançais et al. 1985). With the exception of Cd and to a lesser extend Cu and Zn, information on the subcellular distribution of trace elements in Pectinidae is particularly scarce in the current literature. However, 60 to 99 % of the Cd is usually found in the soluble fraction of the digestive gland of Pectinidae from the field or after experimental exposure to this metal (Evtushenko et al. 1986[START_REF] Mauri | Heavy metals in the Antarctic scallop Adamussium colbecki[END_REF][START_REF] Stone | Cadmium binding components of scallop (Pecten maximus) digestive gland. Partial purification and characterisation[END_REF], Lukyanova & Evtushenko 1989), even if the Antarctic scallop Adamussium colbecki appears not to follow this trend with only 30 % of soluble Cd [START_REF] Viarengo | Cu, Zn and Cd content in different tissues of the Antarctic scallop Adamussium colbecki : role of the metallothionein in heavy metal homeostasis and detoxification[END_REF]. These general observations are consistent with the results obtained for C. varia in which the cytosolic fraction of the digestive gland contained 78 ± 5% of the total Cd (Figure 4). This strongly suggests the presence of mechanisms of detoxification of Cd involving proteins such as metallothioneins (Evtushenko et al. 1986[START_REF] Stone | Cadmium binding components of scallop (Pecten maximus) digestive gland. Partial purification and characterisation[END_REF], Evtushenko et al. 1990[START_REF] Viarengo | Cu, Zn and Cd content in different tissues of the Antarctic scallop Adamussium colbecki : role of the metallothionein in heavy metal homeostasis and detoxification[END_REF], Bustamante 1998).

As mentioned above, Ag and Cd appeared to be mainly bound to insoluble material and cytosolic proteins, respectively (Figure 4). So, according to the present results both detoxification processes involved in the storage of these metals seem to lead to a long term bioaccumulation of these metals in the digestive gland of C. varia. It is therefore noteworthy that two different detoxification strategies, binding to soluble and insoluble compounds, lead to large concentrations of both toxic metals in this species.

For elements other than Cd found in the cytosolic fraction of the digestive gland, i.e. As, Co, Cr, Mo, Ni, and V, other systems or proteins other than metallothioneins might be responsible of the binding. Co, Cr, Mo and Ni are essential micronutrients required for function of a large number of proteins. However, both their accumulation and regulation mechanisms are poorly understood because of their generally low concentrations in biota. Recently, investigation of V accumulation in ascidians has shown that specific vanadium-binding proteins (vanabins) were responsible for vanadium accumulation in these organisms [START_REF] Ueki | Vanadium-binding proteins (vanabins) from a vanadium-rich ascidian Ascidia sydneiensis samea[END_REF]. In contrast to metallothioneins, vanabins do not use cysteine residues to bind vanadium ions and they might function as metal chaperone proteins rather than proteins for metal storage or detoxification. Vanabins appear to be able to chelate V under its +5 oxidation state and help NADPH to reduce the ion to vanadium(IV), which is easier to carry within the cell. The implication of such proteins in the accumulation of V in Pectinidae should be considered in the future.

Metal concentrations in bivalves may vary with biological factors such as age (size) and sex.

However, only Ag, Cd, Co, Cu, Ni, and Zn varied with size in at least one tissue or organ.

Similarly to C. varia from the Bay of Biscay, Cd also displayed positive accumulation with animal size in P. maximus and Placopecten magellanicus (Boyden 1977, Uthe and[START_REF] Uthe | Cadmium in sea scallop (Placopecten magellanicus) tissues from clean and contaminated areas[END_REF]. However, no significant relationship was found for the Antarctic scallop Adamussium colbecki [START_REF] Mauri | Heavy metals in the Antarctic scallop Adamussium colbecki[END_REF]) which was attributed to the influence of various factors such as the contamination of the sampling area and the physiological state of the individuals. In scallops, excretory functions of kidneys might reduce the concentrations of weakly retained elements. This appears to be the case of Ni and Co, which concentrations tend to decrease with size.

Accumulation differences between sites

Among the different trace elements analysed, Ag, Al, Ce, Co, Cu, La, Mn, Nd, Pb, and Zn concentrations were significantly higher in the scallops from La Rochelle Bay. This could be due to the fact that this area is directly subject to industrial and domestic contamination via several discharge points, but also from more diffusive sources related to harbour activities (e.g. antifouling paints) or installations (e.g. release of Zn by cathodic protection). Therefore, it is not surprising that C. varia displayed elevated tissue concentrations of several elements in this area. Contribution of the different local sources might be difficult to determine for most of the elements, but higher rare earth element concentrations in the tissues of C. varia are probably related to industrial releases into La Rochelle Bay from the rare earth extracting plant, Rhodia. Very little is known about long term effects of rare earth elements on marine invertebrates in the field, but experiments have shown that these elements are poorly bioavailable from seawater and also weakly toxic to marine biota (Hirano & Suzuki, 1996). However, the gills of C. varia contained high concentrations of Ce, La and Nd relative to muscle. Gills represent a natural pathway for metals dissolved in seawater (Bustamante et al. 2002a). Therefore, if we assume that rare earth elements have low rates of redistribution to other tissues as shown for 241 Am [START_REF] Miramand | Données expérimentales et in situ (Goury, Nord Cotentin, France) sur la concentration et la distribution de l'américium ( 241 Am) dans les tissus de la Saint-Jacques, Pecten maximus (L.)[END_REF], Ce, La, and Nd concentrations found in the gills would mainly indicate the exposure via seawater.

Rare earth elements are also most probably accumulated from food as suggested by the high concentrations found in the digestive gland. High concentrations in gonads might be due to translocation from the intestine into this tissue, which serves both for the expulsion of faeces and gonadal products. Therefore, adsorption of rare earth elements on the intestine walls could occur and be responsible for the relatively elevated concentrations in the gonad of these scallops. The gonad also contained relatively high concentrations of Ag and Pb, which also could be subject to adsorption processes on the intestine walls in the gonad (Table 1, Figure 3). The ability of C. varia to concentrate rare earth elements would be useful to study and to monitor the behaviour of these elements under field conditions. Moreover, lanthanides could also provide particularly interesting information on toxic elements with similar chemical properties, i.e. transuranic elements in the marine environment.

Higher As and Cd concentrations in the scallops from the Ré Island compared to the contaminated site are surprising since there is no identified source of these metals in this area. Similarly, investigation of 210 Po in the tissues of C. varia and Mytilus edulis reported the same unexpected difference between sites (Bustamante et al. 2002b). [START_REF] Uthe | Cadmium in sea scallop (Placopecten magellanicus) tissues from clean and contaminated areas[END_REF] also reported unexpected high Cd concentrations in the tissues of the scallop Placopecten magellanicus from a non-contaminated area. These were attributed to feeding and nutritional inadequacy of the diets.

Scallops are filter feeders using different types of food such as phytoplankton, detritus and other suspended matter which are major components of the variation in food quality in the field through variation in their carbon and nitrogen contents (Grant & Cranford 1991, Alber & Valiela 1996).

Therefore, it would be of particular interest to characterise the food composition and metal concentrations in the food of C. varia in both sampling areas.

CONCLUSION

Because of very high trace element concentrations, the digestive gland and kidneys of C. varia can be used to monitor trace element availabilities in seawater but only when individuals of similar size are used. Thus, significant differences in concentrations between clean and impacted areas from Charente-Maritime suggested contamination of Ag, Al, Ce, Co, Cu, La, Nd, Pb, Mn and Zn due to combined domestic, industrial and harbour releases. The subcellular distribution of trace elements in the digestive gland showed that most of the elements were associated with insoluble material and would have therefore a low bioavailability for the upper trophic levels (Wallace andLopez 1996, 1997). Finally, the ability of C. varia to concentrate lanthanides would be useful to study the behaviour of trivalent ions in the marine environment with particular attention to actinides.

referees of this manuscript for their fruitful criticism. This work was supported financially by the "Conseil Régional Poitou-Charentes" and Rhodia.

Figure 2 .

 2 Figure 1. Sampling location sites for the variegated scallops Chlamys varia. ⊗ ⊗ ⊗ ⊗ : Saint Martin en Ré ; ⊕ ⊕ ⊕ ⊕ : La Rochelle Bay

Table 1 .

 1 Trace element concentrations (µg.g -1 dwt) in the tissues and organs of the variegated scallop Chlamys varia from the coast of Charente-Maritime, Bay of Biscay, France. n : number of pool of 10 individuals

	Metal	n	Size	Digestive	Kidney	Gills	Gonads	Muscle	Whole
	Sampling area		(mm)	gland					soft parts
	Ag
	La Rochelle	3	33 ± 1	12.1 ± 2.9	5.63 ± 0.58	0.44 ± 0.24	6.48 ± 0.99	0.57 ± 0.26	3.08 ± 0.38
	La Rochelle	3	39 ± 1	27.8 ± 10.2	8.30 ± 2.43	0.44 ± 0.11	8.66 ± 2.82	0.44 ± 0.11	6.47 ± 1.39
	La Rochelle	3	45 ± 2	61.2 ± 29.1	5.01 ± 3.77	0.36 ± 0.10	11.62 ± 2.23	0.45 ± 0.14	11.02 ± 4.09
	Ré Island	3	32 ± 1	3.23 ± 0.97	0.94 ± 0.44	0.28 ± 0.19	1.99 ± 0.48	0.02 ± 0.01	1.05 ± 0.15
	Ré Island	1	44 ± 2	5.36	0.86	0.28	5.50	0.03	1.85
	Al
	La Rochelle	3	33 ± 1	2511 ± 690	222 ± 112	1353 ± 580	1072 ± 370	71 ± 24	901 ± 299
	La Rochelle	3	39 ± 1	1763 ± 54	157 ± 73	407 ± 175	981 ± 231	42 ± 7	727 ± 97
	La Rochelle	3	45 ± 2	1951 ± 239	199 ± 113	487 ± 145	1084 ± 114	38 ± 15	920 ± 190
	Ré Island	3	32 ± 1	1418 ± 360	169 ± 78	467 ± 60	161 ± 40	± 4	493 ± 51
	Ré Island	1	44 ± 2	478	88	264	516	19	195
	As
	La Rochelle	3	33 ± 1	23.6 ± 6.4	36.0 ± 15.0	23.1 ± 6.3	26.5 ± 19.4	14.3 ± 4.7	18.3 ± 2.0
	La Rochelle	3	39 ± 1	17.9 ± 4.9	41.2 ± 25.7	15.1 ± 2.5	18.9 ± 4.9	8.6 ± 0.9	14.4 ± 2.1
	La Rochelle	3	45 ± 2	12.5 ± 7.5	19.2 ± 8.6	22.2 ± 9.1	20.4 ± 4.5	10.5 ± 3.3	14.9 ± 2.6
	Ré Island	3	32 ± 1	21.5 ± 2.0	43.5 ± 12.0	39.6 ± 5.6	30.5 ± 6.1	15.8 ± 3.8	25.0 ± 4.8
	Ré Island	1	44 ± 2	10.8	25.4	29.2	25.0	12.7	16.0
	Cd
	La Rochelle	3	33 ± 1	22.3 ± 0.6	46.1 ± 1.7	2.91 ± 0.06	1.89 ± 0.08	0.55 ± 0.09	4.9 ± 0.2
	La Rochelle	3	39 ± 1	32.7 ± 5.0	47.0 ± 14.2	3.65 ± 1.37	2.29 ± 0.22	0.59 ± 0.10	7.1 ± 1.4
	La Rochelle	3	45 ± 2	39.6 ± 2.5	52.4 ± 10.0	4.43 ± 0.84	2.38 ± 0.20	0.59 ± 0.13	8.0 ± 1.3
	Ré Island	3	32 ± 1	24.2 ± 1.2	39.6 ± 0.8	2.57 ± 0.33	2.80 ± 0.13	0.36 ± 0.06	5.6 ± 0.3
	Ré Island	1	44 ± 2	35.3	42.5	3.18	1.97	0.71	8.5
	Ce
	La Rochelle	3	33 ± 1	10.47 ± 3.88	2.45 ± 2.16	6.02 ± 3.93	6.38 ± 2.72	0.25 ± 0.05	3.98 ± 0.49
	La Rochelle	3	39 ± 1	10.61 ± 1.09	2.03 ± 2.27	4.01 ± 1.90	5.06 ± 0.73	0.18 ± 0.04	3.59 ± 0.11
	La Rochelle	3	45 ± 2	10.85 ± 1.73	1.11 ± 0.44	6.02 ± 3.93	5.80 ± 0.86	0.32 ± 0.22	4.50 ± 0.49
	Ré Island	3	32 ± 1	3.17 ± 0.82	0.30 ± 0.21	0.07 ± 0.01	1.00 ± 0.31	0.04 ± 0.02	0.92 ± 0.13
	Ré Island	1	44 ± 2	1.17	0.15	0.05	1.97	0.03	0.36
	Co
	La Rochelle	3	33 ± 1	2.60 ± 0.31	27.50 ± 4.06	1.40 ± 0.38	1.55 ± 0.82	0.24 ± 0.08	1.52 ± 0.26
	La Rochelle	3	39 ± 1	2.13 ± 0.29	24.29 ± 6.32	0.59 ± 0.48	1.22 ± 0.23	0.13 ± 0.01	1.33 ± 0.11
	La Rochelle	3	45 ± 2	1.79 ± 0.64	9.71 ± 2.14	1.17 ± 0.41	1.25 ± 0.20	0.15 ± 0.09	1.04 ± 0.19
	Ré Island	3	32 ± 1	2.28 ± 0.68	16.48 ± 5.20	1.14 ± 1.11	1.55 ± 0.23	0.14 ± 0.03	1.19 ± 0.16
	Ré Island	1	44 ± 2	0.84	5.75	0.91	0.92	0.11	0.52
	Cr
	La Rochelle	3	33 ± 1	7.82 ± 1.71	7.58 ± 2.51	4.80 ± 1.88	2.49 ± 1.26	1.08 ± 0.50	3.69 ± 0.80
	La Rochelle	3	39 ± 1	8.13 ± 1.05	4.34 ± 1.60	2.79 ± 1.50	2.61 ± 3.75	0.84 ± 0.18	3.18 ± 0.04
	La Rochelle	3	45 ± 2	5.98 ± 2.53	4.37 ± 0.95	5.61 ± 3.17	6.20 ± 1.50	0.73 ± 0.21	3.31 ± 0.60
	Ré Island	3	32 ± 1	8.14 ± 3.05	7.77 ± 3.24	4.75 ± 2.98	4.86 ± 3.02	0.93 ± 0.19	3.55 ± 0.96
	Ré Island	1	44 ± 2	3.07	8.77	6.88	5.66	1.17	2.59
	Cu
	La Rochelle	3	33 ± 1	68 ± 4	288 ± 13	14 ± 2	33 ± 4	4 ± 0	30 ± 3
	La Rochelle	3	39 ± 1	77 ± 10	246 ± 39	10 ± 1	33 ± 7	4 ± 1	23 ± 3
	La Rochelle	3	45 ± 2	135 ± 9	271 ± 14	12 ± 2	40 ± 4	4 ± 1	21 ± 1
	Ré Island	3	32 ± 1	24 ± 3	73 ± 34	9 ± 2	17 ± 2	2 ± 1	9 ± 1
	Ré Island	1	44 ± 2	22	33	14	19	2	10
	La
	La Rochelle	3	33 ± 1	7.90 ± 3.15	2.11 ± 1.49	5.06 ± 3.24	5.73 ± 3.31	0.22 ± 0.03	2.96 ± 0.58
	La Rochelle	3	39 ± 1	8.12 ± 0.84	1.96 ± 0.79	2.79 ± 1.73	4.35 ± 0.94	0.21 ± 0.03	2.79 ± 0.14
	La Rochelle	3	45 ± 2	7.53 ± 2.52	1.28 ± 0.23	4.26 ± 2.15	5.09 ± 0.68	0.32 ± 0.32	2.74 ± 0.44
	Ré Island	3	32 ± 1	0.28 ± 0.16	0.22 ± 0.10	0.46 ± 0.03	0.53 ± 0.12	0.03 ± 0.03	0.32 ± 0.05
	Ré Island	1	44 ± 2	0.08	0.11	0.23	0.79	0.02	0.12

Table 3 .

 3 Reported mean concentrations of trace elements (µg.g -1 dry wet) in the digestive gland and kidney of different scallops species from various locations

	Species	Location	n	Ag	Al	Cd	Co	Cr	Cu	Mn	Ni	Pb	Zn	Reference
	Digestive gland
	Aequipecten opercularis English Channel	11	77	43	27	-	4.7	-	-	-	-	228	Bryan (1973)
	Adamussium colbecki	Antarctic	30	-	-	142 ± 57	-	1.7 ± 0.4	12.6 ± 3.3	3.4 ± 1.2	-	-	74.9 ± 25.4	Mauri et al. (1990)
	Chlamys varia	Bay of Biscay	30	61.2 ± 29.1 1951 ± 239 39.6 ± 2.5 1.79 ± 0.64 5.98 ± 2.53	135 ± 9	113 ± 47	5.73 ± 1.06 4.17 ± 0.56	344 ± 44	Present study
	Pecten jacobeus	Mediterranean	30	-	-	41.0 ± 18.2	-	4.8 ± 2.8	16.6 ± 6.6	49.0 ± 23.0	-	-	124 ± 55	Mauri et al. (1990)
	Pecten maximus	English Channel	11	13.6	173	321	1.28 ± 0.46	8.1	57.9 ± 27.4	15.6 ± 7.0	3.55 ± 0.67 3.90 ± 0.44	407 ± 123	Bryan (1973)
	P. maximus	Irish Sea	1	8.9	340	96	0.68	1.8	25	410	0.96	1.7	1100	Segar et al. 1971
	Kidney
	Aequipecten opercularis English Channel	11	35	58	41	-	6.6	-	-	-	-	54700	Bryan (1973)
	Adamussium colbecki	Antarctic	30	-	-	11.6 ± 3.3	-	1.5 ± 0.5	4.0 ± 1.7	16.3 ± 8.9	-	-	199 ± 89	Mauri et al. (1990)
	Chlamys varia	Bay of Biscay	30	5.01 ± 3.77 199 ± 113 52.4 ± 10.0 9.71 ± 2.14 4.37 ± 0.95	271 ± 14	11855 ± 4517	10.79 ±	41.57 ±	34212 ± 1885	Present study
	Pecten jacobeus	Mediterranean	30	-	-	10.7 ± 4.0	-	0.33 ± 0.15 17.5 ± 10.9 6390 ± 3030	0.66 -	3.74 -	2790 ± 1340 Mauri et al. (1990)
	Pecten maximus	English Channel	11	4.3	53.5	79	9.05 ± 3.07	3.9	20.8 ± 10.3 15300 ± 4100 22.9 ± 6.3	159 ± 38	19300 ± 4800	Bryan (1973)

ACKNOWLEDGEMENTS

The authors would like to thank the Dr S.W. Fowler for its advice on the early version of the manuscript. We are also grateful to M. Robert and C. Churlaud of the "Centre Commun d'Analyses", University of La Rochelle, for their help in the analyses, and to the anonymous


Nicolas Coquery

Raphaël Serduc

Chantal Rémy

Emmanuel Luc Barbier

Raphael Serduc

Benjamin Lemasson

Emmanuel L Barbier
email: emmanuel.barbier@univ-grenoble.alpes.fr

Cluster versus ROI analysis to assess combined antiangiogenic therapy and radiotherapy in the

Keywords: MRI, unsupervised clustering, radiomics, brain, glioma, combined therapies ADC: apparent diffusion coefficient CBF: Cerebral blood flow CBV: cerebral blood volume GBM: glioblastoma Perm: vascular wall integrity ROI: region of interest StO2: tissue saturation in oxygen VSI: vessel size imaging

come

Introduction

Among the strong efforts to develop new therapies against glioma, current strategies combine several therapies in order to cumulate their effects and ultimately reach a synergic efficiency. This was initially applied in the Stupp's et al. [START_REF] Stupp | Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma[END_REF] protocol in which resection, chemotherapy and radiotherapy are combined. Since the routine application of this protocol in clinic, other combined therapeutic approaches became relevant in glioma management [START_REF] Tanaka | Diagnostic and therapeutic avenues for glioblastoma: no longer a dead end?[END_REF][START_REF] Chen | Recent therapeutic advances and insights of recurrent glioblastoma multiforme[END_REF] , and complex combinations of antiangiogenic therapy, chemotherapy and radiotherapy underwent clinical trials [START_REF] Hainsworth | Concurrent radiotherapy and temozolomide followed by temozolomide and sorafenib in the first-line treatment of patients with glioblastoma multiforme[END_REF][START_REF] Den | A Phase I Study of the Combination of Sorafenib With Temozolomide and Radiation Therapy for the Treatment of Primary and Recurrent High-Grade Gliomas[END_REF] . Depending on the therapy and its biological target, the sequence of treatments remains a central question. As example, Jain suggested that an antiangiogenic-based vascular normalization [START_REF] Jain | Normalization of Tumor Vasculature: An Emerging Concept in Antiangiogenic Therapy[END_REF] could increase the effects of concurrent radiotherapy [START_REF] Winkler | Kinetics of vascular normalization by VEGFR2 blockade governs brain tumor response to radiation[END_REF] .

In this context, there is an obvious need for consistent tumor monitoring methods such as multiparametric MRI in order to rapidly assess the efficacy of complex combined therapies. Indeed, MRI gives access to many parameters (i.e. water diffusion, blood volume and blood flow [START_REF] Barbier | Methodology of brain perfusion imaging[END_REF] , microvessel size and density [START_REF] Troprès | Vessel size imaging[END_REF] , local blood oxygen saturation [START_REF] He | Quantitative BOLD: Mapping of human cerebral deoxygenated blood volume and oxygen extraction fraction: Default state[END_REF] , vessel wall permeability [START_REF] Tofts | Estimating kinetic parameters from dynamic contrastenhanced t1-weighted MRI of a diffusable tracer: Standardized quantities and symbols[END_REF] , vessel vasoreactivity [START_REF] Krainik | Regional Impairment of Cerebrovascular Reactivity and BOLD Signal in Adults After Stroke[END_REF] ). Recently, Lemasson et al. have shown that multiparametric MRI can monitor the effects of individual therapies (antiangiogenic and radiotherapy), used concomitantly, on a 9L glioma model [START_REF] Lemasson | Multiparametric MRI as an early biomarker of individual therapy effects during concomitant treatment of brain tumours[END_REF] . This study used the classical region of interest (ROI)-based MRI analysis, an approach with 2 major limitations: i) it gives an averaged view of the tumor response and therefore it does not take into account the intratumoral heterogeneity and ii) it does not exploit the full potential of the multiparametric MR protocol (one voxel contains multiple information). Alternatively, voxel-based analysis of tumor progression and response to treatments seems to be more suited for this purpose [START_REF] Ken | Voxel-based evidence of perfusion normalization in glioblastoma patients included in a phase I-II trial of radiotherapy/tipifarnib combination[END_REF] but this method is often limited to a single MRI parameter. In order to overcome these limitations, we recently proposed an unsupervised clustering method based on probabilistic mixture models [START_REF] Fraley | Model-Based Clustering, Discriminant Analysis, and Density Estimation[END_REF] yielding histological-like maps that can potentially integrate a broad range of MRI parameters [START_REF] Coquery | Microvascular MRI and unsupervised clustering yields histology-resembling images in two rat models of glioma[END_REF] . This multiparametric MRI-based approach coupled with unsupervised clustering might be well suited to decipher the complex information measured during combined therapies on brain tumors.

In the context of combined cancer therapies (antiangiogenic therapy followed by radiotherapy), we compare in this study the data generated by the classical ROI-based analysis (parameter by parameter) and a cluster-based analysis of six MRI parameters. MRI parameters were measured on well-established F98 tumors in rats during tumor growth under single antiangiogenic or radiotherapy treatment and under combined therapies. The ROIbased analysis was able to highlight the averaged tumor changes while the cluster-based analysis yielded additional information about tumor regions that respond to treatment.

Experimental details

The study design was approved by the local institutional animal care and use committee (COMETHS). All animal procedures conformed to French government guidelines and were performed under permits 380820 and B3851610008 (for experimental and animal care facilities) from the French Ministry of Agriculture. This study is in compliance with the ARRIVE guidelines (Animal Research: Reporting in Vivo Experiments [START_REF] Kilkenny | Improving Bioscience Research Reporting: The ARRIVE Guidelines for Reporting Animal Research[END_REF] ). All procedures were performed under isoflurane anesthesia (5% for induction and 2.5% for maintenance).

The tail vein was equipped with a catheter when needed. The rectal temperature was monitored and maintained at 37.0°C.

Animal Model.

The F98 cells (10 3 cells, ATCC: CRL-2397) were implanted in the right caudate nucleus of male Fischer 344 rats (n=19, 7 weeks, 175-200 g, Charles River, France) with a stereotactic frame, as previously described [START_REF] Coquery | Distribution and Radiosensitizing Effect of Cholesterol-Coupled Dbait Molecule in Rat Model of Glioblastoma[END_REF] .

In vivo experiment. The experimental design is presented

MRI session.

The following imaging sequences were performed with a voxel size of 234×234×800 µm unless stated otherwise. Anatomical imaging was performed with a T2weighted (T2W) spin-echo sequence to determine tumor volume. The apparent diffusion coefficient (ADC) was mapped using a spin-echo EPI sequence (3 orthogonal diffusion directions, b≈ 0 and b= 900 s/mm²). Cerebral blood flow (CBF) was determined using pseudo continuous arterial spin labelling (pCASL) with an EPI readout (labelling duration=3s, postlabelling delay=400 ms, 50 pairs) [START_REF] Hirschler | Interpulse phase corrections for unbalanced pseudo-continuous arterial spin labeling at high magnetic field[END_REF] and a T1 map. To map the tissue saturation in oxygen (StO2), a T2 map (28 spin echoes between 12 and 336 ms) and a high resolution T2* map (25 gradient echoes between 3 and 87 ms; voxel size= 117x117x200 µm) were obtained. A cerebral blood volume (CBV) and a vessel size imaging (VSI) maps were obtained using a steady-state approach [START_REF] Troprès | In vivo assessment of tumoral angiogenesis[END_REF] and ultrasmall superparamagnetic iron oxide nanoparticles (P904: 200 μmoles of iron/kg body weight; Guerbet SA, France). The vascular wall integrity (Perm) was assessed using a dynamic contrast-enhanced MRI approach as previously described [START_REF] Lemasson | Monitoring Blood-Brain Barrier Status in a Rat Model of Glioma Receiving Therapy: Dual Injection of Low-Molecular-Weight and Macromolecular MR Contrast Media[END_REF] .

Briefly, 25 T1-weighted, spin-echo, images were acquired. After acquisition of 4 baseline images, a bolus of Gd-DOTA (200 µmol/kg; Guerbet SA, France) was administered through the tail vein. The image acquisition positions were identical for all quantitative MRI sequences.

Data processing.

Image processing was performed within the Matlab 7 environment (The MathWorks Inc., Natick, MA, USA) using custom software. The CBF computation was based on the equations described in Alsop et al. [START_REF] Alsop | Reduced Transit-Time Sensitivity in Noninvasive Magnetic Resonance Imaging of Human Cerebral Blood Flow[END_REF][START_REF] Buxton | A general kinetic model for quantitative perfusion imaging with arterial spin labeling[END_REF] . The VSI and CBV were derived from the transverse relaxivities as described [START_REF] Troprès | In vivo assessment of tumoral angiogenesis[END_REF] . StO2 maps were estimated using a multiparametric qBOLD approach as previously described [START_REF] Christen | Evaluation of a quantitative blood oxygenation level-dependent (qBOLD) approach to map local blood oxygen saturation[END_REF][START_REF] Lemasson | Assessment of multiparametric MRI in a human glioma model to monitor cytotoxic and anti-angiogenic drug effects[END_REF][START_REF] Lemasson | Evaluation of the Relationship between MR Estimates of Blood Oxygen Saturation and Hypoxia: Effect of an Antiangiogenic Treatment on a Gliosarcoma Model[END_REF] . Perm was estimated by calculating the area under the signal enhancement curve following the Gd-DOTA injection (between images 5 and 14, which corresponds to 150 sec), after baseline removal [START_REF] Lemasson | Monitoring Blood-Brain Barrier Status in a Rat Model of Glioma Receiving Therapy: Dual Injection of Low-Molecular-Weight and Macromolecular MR Contrast Media[END_REF] .

Data analysis.

To determine the tumor volume, tumor ROIs were manually delineated on the T2W images that covered the entire brain. For tumor delineation, all visible abnormalities (hyper-and hypo-intense signals) were included. Parameter values were obtained using the same tumor ROIs and the five shared MRI slices between all sequences. Two type of measurements were performed:

-A ROI selection followed by an analysis of the mean ROI values. Voxels were included in the analysis according to the following criteria: 0<ADC<2500 µm²/s (percentage of included voxels=97.9%), Perm<2.5x10 6 a.u. (percentage of included voxels=99.3%), 0<CBF<400 mL/100g/min (percentage of included voxels=83.0%), 0<CBV<20 % (percentage of included voxels=96.8%), 0<VSI<50 µm (percentage of included voxels=82.6%) and 0<StO2<100% (percentage of included voxels=53.2%). Beyond these ranges, values cannot be considered physiologically accurate. Average values were computed across all animals for each ROI and each parameter (Fig 2A).

-The ROI selection as above followed by a model-based clustering [START_REF] Fraley | Model-Based Clustering, Discriminant Analysis, and Density Estimation[END_REF] analysis. For each MR parameter, values were standardized to minimize scale effects between parameters using the following formula: (voxel value -mean across all voxels) / standard deviation across all voxels. In order to avoid exclusion of voxels with non-physiological value in only one map, we included all values for each map that were comprised between the first and the last percentile of the distributed values (percentage of included voxels=90,6%). All included voxels of all animals were pooled together and analysed with an unsupervised clustering method based on mixture of Gaussian models, as previously described [START_REF] Coquery | Microvascular MRI and unsupervised clustering yields histology-resembling images in two rat models of glioma[END_REF] .

Briefly, Gaussian mixture modelling was performed using the Expectation-Maximization algorithm implemented in the R package mclust [START_REF] Fraley | Model-Based Clustering, Discriminant Analysis, and Density Estimation[END_REF] . The optimal number of clusters was determined using a Bayesian information criterion and was estimated to 5 clusters. For this analysis, two rats belonging to the V and VRT groups were excluded due to computation problems in at least one MRI parameter.

Statistical analysis.

Repeated measures-ANOVA (RM-ANOVA) was used to evaluate the change of each parameter between imaging sessions. In case more than two groups were analysed, a LSD post-hoc was used. Mann-Whitney test was used to evaluate the effects of treatments on change in MRI parameters and in cluster-ratio. A p-value < 0.05 was considered significant. 116.7±48.9mm 3 ). Between D20T-1 and D24T3, Sora treatment (group S) promotes a significant decrease in vessel wall permeability, a stabilization of the CBF compared with untreated tumors (group V, Fig 2C ) and a tendency towards increased apparent diffusion coefficient (ADC, p=0.084). Between D24T3 and D26T5, the effects of concomitant radiotherapy could not be extensively characterized by the ROI-based analysis. Indeed, we only observed a significant decrease in CBF between D24T3 and D26T5 after Sora treatment (group SØ) and after Sora treatment followed by the radiation therapy (group SRT), compared with vehicle administration followed by the radiation therapy (group VRT).

Results

ROI-based evaluation of

Clustering-based evaluation for tumor follow-up and characterization of treatment effects.

To assess the effects of treatments on tumors, we used an unsupervised, model-based clustering analysis, which integrates the 6 quantitative MRI parameters (ADC, Perm, CBF, CBV, VSI and StO2) and identifies clusters of voxels sharing the same MRI physiological characteristics. This analysis was performed on voxels from the previously defined tumor ROIs; the voxels from all time points and under all treatments were pooled. -cluster c is the less represented cluster (only 9.5% of voxels belong to cluster c).

Compared to cluster a and b, the levels of CBV and CBF are reduced and the levels of Perm and ADC are increased.

-cluster d is similar to cluster c with reduced parameters values (26.7% of voxels belong to cluster d).

-cluster e has the highest level of ADC with a high level of VSI and the lowest levels of CBF and CBV (14.4% of voxels belong to cluster e).

Spatial detection of treatments effects

Tumor properties and associated MR parameters depend on (i) the tumor growth, (ii) the type of treatment and (iii) the specific sequence of Sorafenib and radiotherapy treatments.

In order to investigate the effects of each treatment, we choose to analyse the changes between two time points using 2 approaches: (i) the ROI-based MRI parameters values, determined as the change (in percentage) of each parameter value and (ii) the cluster proportion within ROI, determined as the change (in percentage) in the number of voxels per cluster (Fig 4). This approach allowed the description of the effect of each treatment for a given time interval whatever tumor growth and the type of treatment given in the previous time interval.

Between D20T-1 and D24T3, Sorafenib effects (Fig 4A and C).

Using the ROI-based changes in MRI parameters, we measured increased levels of ADC (+4.1±1.7%), Perm (+17.0±21.4%) and VSI (+15.8±9.9%) and decreased levels of CBF (-32.3±6.7%), CBV (-13.7±5.9%) and StO2 (-10.0±3.5%) during tumor growth under vehicle administration (Fig 4A ,group V). Compared with vehicle administration, Sorafenib treatment significantly increased the level of CBF (+5.2±16.3%, p=0.018), reinforced the increase in ADC (+10.0±2.9%, p=0.028) and oppositely decreased the Perm (-31.4±10.3%, p=0.011). These variations are in good agreement with the antiangiogenic effects of Sorafenib.

Using the cluster-based change approach, during tumor growth under vehicle administration (Fig 4C, group V), we measured a decreased proportion of cluster a (-14.5±5.5%), and increased proportions of all other clusters (b: +3.8±2.5%, c: +4.3±4.9%, d: +1.1±3.3%, e: +4.5±1.4%). Compared with vehicle administration, Sorafenib treatment maintained the proportion of cluster a stable (-1.5±2.4%, p=0.035) and decreased the proportion of cluster b (-11.0±3.0%, p=0.006).

Between D24T3 and D26T5, radiotherapy or combined treatments effects (Fig 4B and D).

The approach of ROI-based changes (Fig 4B) showed an increased level of ADC after radiotherapy alone or in combination with Sorafenib treatment as compared to the Sorafenib treatment alone (VRT=+9.9±1.6%, p=0.042 and SRT=+6.7±2.4%, p=0.088 versus SØ=-0.5±3.6%). The effect of radiotherapy after Sorafenib treatment (SRT) was detected as a significant change of CBV (VRT=-19.3±2.3% vs SRT=-6.8±3.8%, p=0.018). Radiotherapy alone promoted a decrease of CBV similar to the decrease measured in the SØ group, whereas SRT treatment maintained a level of CBV similar to an estimated control value (vehicle without radiotherapy: VØ) determined as the change in parameter between D20T-1 vs D24T3 under vehicle administration and corrected for a two-days tumor growth (VRT=-19.3±2.3%, SRT=-6.8±3.8%, SØ=-21.0±13.3% and estimated VØ=-6.85%). Note that, two days after the end of Sorafenib administration (SØ), ADC, Perm and CBF, initially modified by Sorafenib, returned back to the estimated control values measured between D20T-1 and D24T3.

The cluster-based change approach (Fig 4D ) revealed a significant decrease in the proportion of cluster a after Sorafenib administration as compared to radiotherapy alone (VRT=-5.3±2.3% vs SØ=-17.0±7.6%, p=0.028). Compared with the estimated control value between D24T3 and D26T5 (estimated VØ), cluster b proportion was also decreased after radiotherapy alone (VRT=-11.7±5.2% vs estimated VØ=1.9%, p=0.016).

The effects of combined treatments were detected as a lower increase in the proportion of cluster e when radiotherapy was applied after Sorafenib treatment compared with radiotherapy alone (SRT=+2.9±1.1% vs VRT=+9.3±4.2%, p=0.028). No difference between treatment conditions was detected for clusters c and d. The changes in the proportions of these clusters are similar for all treatment conditions and follow the tendency of the estimated control changes.

Discussion

This study presents a comparison of ROI-based and cluster-based analyses of MRI data collected during the follow-up of a rat glioma model under several therapeutic combinations, Sorafenib and/or radiotherapy. We developed a comprehensive analysis strategy in order to alleviate bias related to the effect of one therapy to the other and to the tumoral status before treatment. The ROI-based analysis appears well-suited to highlight global tumoral changes but filters out local changes whereas the cluster-based analysis appears as a powerful approach to detect voxel-based variations within the tumor that are impacted by (i) tumor growth, (ii) single or (iii) combined treatments.

Given that the ROI-and the cluster-based analyses are known to reflect biological features such as seen in histology [START_REF] Coquery | Microvascular MRI and unsupervised clustering yields histology-resembling images in two rat models of glioma[END_REF][START_REF] Valable | Assessment of blood volume, vessel size, and the expression of angiogenic factors in two rat glioma models: a longitudinal in vivo and ex vivo study[END_REF][START_REF] Lemasson | In vivo imaging of vessel diameter, size, and density: A comparative study between MRI and histology[END_REF] , we focused on the comparison of the ability of these two approaches to monitor the tumor changes under therapies. MRI data included six parameters (ADC, Perm, CBV, CBF, VSI and StO2) known to change during tumor development [START_REF] Cha | Neuroimaging in neuro-oncology[END_REF][START_REF] Galbán | The parametric response map is an imaging biomarker for early cancer treatment outcome[END_REF][START_REF] Hamstra | Evaluation of the functional diffusion map as an early biomarker of time-to-progression and overall survival in high-grade glioma[END_REF][START_REF] Aronen | Cerebral blood volume maps of gliomas: comparison with tumor grade and histologic findings[END_REF][START_REF] Emblem | Glioma grading by using histogram analysis of blood volume heterogeneity from MR-derived cerebral blood volume maps[END_REF] and that may be additionally modulated by the anti-tumor therapies used in this study [START_REF] Lemasson | Assessment of multiparametric MRI in a human glioma model to monitor cytotoxic and anti-angiogenic drug effects[END_REF][START_REF] Chung | Imaging Biomarker Dynamics in an Intracranial Murine Glioma Study of Radiation and Antiangiogenic Therapy[END_REF][START_REF] Hamstra | Diffusion Magnetic Resonance Imaging: A Biomarker for Treatment Response in Oncology[END_REF] .

The manually delineated tumor ROI contoured all the abnormalities observed on the T2W images, including oedema and peritumoral cell infiltration, and thereby maximized the number of different tissue types. This approach also permitted to minimize inter-rater variability between different delineated tumors and to include all putative abnormalities. With the clustering strategy, we did not observe the emergence of new tissue types during tumor progression or treatment with the cluster-based analysis (all clusters were present at all time points).

The effects of the different therapies were detected by the ROI-based analysis of some MRI parameters (ADC, Perm, CBV, and CBF). However, the visualization of the average values of these parameters within tumor bulk (Fig 2B) shows that this analysis filters out the spatial heterogeneity of tumor characteristics during progression and treatment and does not account for joint changes in parameters (e.g. decreased CBF and increased Perm). Conversely, the cluster-based approach allows the integration of all MRI parameters in one synthetic map. A visual inspection of the resulting map shows that, without using any knowledge of the spatial or temporal organization of voxels, the present clustering yields spatially-structured and temporally-consistent maps of clusters. For instance, at D20T-1, voxels with high CBF value are localized at the tumor border and cluster a, which is mainly distributed at this localization, accordingly presents the highest CBF value. Over time, cluster a remains at the periphery of the tumor. Besides topological information, the cluster-based approach defines for each cluster a profile accounting for all MRI parameters and thereby discriminates homogenous groups of voxels within the tumor that cannot be detected by inspecting the ROI-based analysis. We observe that the variability of the change in cluster proportions during treatment is lower than that of the ROI-based parameter values (see the SEM in Figure 4). This lower variability may be ascribed to the fact that clusters represent homogeneous ranges of parameter values and thus a fraction of the tumor while ROI integrate the range of a parameter values across the entire tumor. This lower variability of the change in cluster proportions suggests that the cluster-based analysis is more sensitive to changes within the tumor than the ROI-based analysis. This can be illustrated by CBV or VSI, which may be reduced by Sorafenib treatment [START_REF] Lemasson | Multiparametric MRI as an early biomarker of individual therapy effects during concomitant treatment of brain tumours[END_REF][START_REF] Lemasson | Assessment of multiparametric MRI in a human glioma model to monitor cytotoxic and anti-angiogenic drug effects[END_REF] : they were not detected as changed following Sorafenib treatment by the ROI-based analysis, whereas, the proportion of cluster b, which presents the highest levels of CBV and VSI, was reduced. Our results show that, when specific areas within a tumor respond to a therapy, the ROI-based analysis may be blind to these effects. Indeed, its global analysis erases all fine variations. This further suggests that the clusterbased approach might be less sensitive than ROI-based analysis to the inter-rater variability generally observed during tumor delineation. Indeed, we expect that the inter-rater variability will affect differently the ROI-based and the voxel-based analysis. The mean value determined with the ROI-based analysis will vary with the number of included voxels, whereas the cluster-based analysis will not be as sensitive to this parameter. Finally, the inclusion in the tumor ROI of healthy tissue by the rater should be detected with the clusterbased analysis, i.e., the clustering should yield a cluster with the properties of a healthy tissue. This is not the case for the ROI-based analysis. In this case, the mean parameter value should be modified towards that of healthy tissue, thereby masking the contrast between tumor and healthy tissue.

In this preclinical study, we used quantitative parameters that are not routinely used in clinic.

Further studies should be conducted on non-quantitative parameters, such as ratio between the parameter values measured in the tumor and in a normal appearing white matter area, as typically performed in clinical settings. Note that the cluster-based analysis could also help in selecting normal white appearing matter using the homogeneous MR characteristics intrinsic to this tissue.

In order to investigate the potential of the clustering approach for treatment follow up, we used the antiangiogenic-based vascular normalization hypothesis [START_REF] Goel | Normalization of the Vasculature for Treatment of Cancer and Other Diseases[END_REF] , which was already validated in animal model studies [START_REF] Lemasson | Monitoring Blood-Brain Barrier Status in a Rat Model of Glioma Receiving Therapy: Dual Injection of Low-Molecular-Weight and Macromolecular MR Contrast Media[END_REF][START_REF] Lemasson | Multiparametric MRI as an early biomarker of individual therapy effects during concomitant treatment of brain tumours[END_REF][START_REF] Lemasson | Assessment of multiparametric MRI in a human glioma model to monitor cytotoxic and anti-angiogenic drug effects[END_REF] , followed by a radiotherapy. This study was not directly designed to investigate treatment efficacy and, although we observed an effect of Sora treatment, we could only detect minor effects of the combined therapy that could have been improved with a larger number of included animals.

The cluster-based approach may seem limited to follow-up spatial evolutions within the tumor when the tumor volume changes during progression and treatment. However, the clusterbased approach can potentially promote direct information of the short-term effect of treatment and/or about tumor with low progression rate such as low-grade glioma. Under these conditions, the cluster approach may help identify the tissue types that respond to a given therapy and may then be used to monitor dose response trials. For example, in our study, a tumor with limited number of voxels belonging to cluster a and b, i.e. clusters responding to Sorafenib, will most probably show limited response under Sorafenib treatment. Overall, this strategy opens a new way to personalize therapy for each patient.

Conclusion

In the context of combined therapies for solid tumor treatment, the cluster-based analysis and the ROI-based analysis provides complementary views of the same data. The ROI-based analysis is well suited to highlight global changes and the cluster-based analysis provides a comprehensive and subtle view of MRI-parameters integration that describes intra-tumoral heterogeneity. The evolution of the cluster distributions within the tumor appears as a promising strategy to decipher the complexity of tumor changes under combined therapies.

Fig 1 ,

 1 with D as notation for the duration (days) after tumor cell implantation, and T as the duration (days) after treatment initiation. The different treatment groups are described in the figure legend. Antiangiogenic treatment: Sorafenib (Sora: 30 mg/kg in vehicle, Bayer Biosciences) or vehicle (DMSO: 5%, Tween20: 5%, NaCl0.9%: 90%) were administered p.o. (approximately 1mL) every day during three days (D21T0 to D23T2). Radiotherapy: animals received in one session, at D24T3, 20Gy directed into the tumor ipsilateral hemisphere (GenX, ESRF). Conventional orthovoltage irradiations were performed using a Philips X-ray generator operated at 200 kVp. A dose rate of 1.61 Gy/min was delivered at the centre of the intracerebral tumor using an ionization chamber (Semiflex 31010, PTW). Imaging: Three MRI sessions were performed at 4.7 T (Avance III console; Bruker, Ettlingen, Germany; IRMaGe MRI facility, Grenoble, France) between 20 and 26 days after tumor implantation (D20T-1, D24T3 and D26T5).

  Sorafenib and radiotherapy effects considering all data and time points (Fig 2A). The consequence of the ROI-based evaluation is the loss of spatial information within tumor that leads to an averaged representation of each parameter for the studied ROIs as schematically represented in Fig 2B. However, the comparison between mean ROI values shows the effects of the different treatments at different time-points (Fig 2C) while mean tumor size does not change, whatever the treatment applied (Fig 2D). In order to limit the impact of tumor volume on voxels clustering, the average tumor size was similar between groups before treatment (D20T-1; group V: 133.9±46.4mm 3 , group S:

  The characterization of clusters with standardized MRI parameters values (Fig 3A, web graphs) and raw values allows discriminating the signature of one cluster from that of other clusters: -cluster a presents the highest levels of CBF, CBV, StO2 and the lowest levels of VSI, ADC and Perm (20.5% of voxels belong to cluster a). -cluster b is characterized by the highest levels of Perm and VSI, a high level of CBV, a reduced level of StO2 and an intermediate level of CBF and ADC (19.6% of voxels belong to cluster b).

From

  cluster c to e, the levels of Perm and CBV gradually decrease and the level of StO2 meets non-physiological value (too low perfusion). When taking into account the voxels within the three time points and the different treatments, a visual inspection of the cluster distributions (Fig 3B) shows spatially coherent patterns of voxels belonging to a same cluster. Whatever the time points and treatments, cluster a (purple) is mainly located at the tumor margins, cluster c (yellow) distribution is sparse and cluster d (grey) proportion is stable and found as groups of numerous neighbouring voxels. The RM-ANOVA-based statistical analysis of the proportions of each cluster in the tumor between treatments and time points does not yield consistent information (Fig 3B, coloured bar graphs). However, the bar graphs on Fig 3B show that cluster b (green) proportion decreases for all treatment conditions (Sora treatment: V vs S at D24T3; radiotherapy: V at D24T3 vs VRT at D26T5 for the same tumor). Cluster e (red), rarely found at the earliest time point (D20T-1), increases in proportion after treatment.

Figure 1 .

 1 Figure 1. A. Experimental design. At D0, rats were implanted with F98 glioblastoma cells

Figure 2 .Figure 3 .

 23 Figure 2. Multiparametric MRI of F98 glioma under Sorafenib treatment, radiotherapy

Figure 4 .

 4 Figure 4. ROI-based and Cluster-based changes following therapy. For each time interval,

Figure 4

 4 Figure 4

Acknowledgments:

We thank the animal care facility of GIN, the IRMaGe facility, the European Synchrotron factory, Elke Bräuer-Krisch for technical support and Bayer Bioscience for providing Sorafenib.

The authors also thank the "Conseil Régional Rhône-Alpes", la "Ligue contre le Cancer, comité de la Drome", "l'Association pour la Recherche contre le Cancer",ANR-11-BSV5-004, and ANR-11-LABX-0063/ ANR-11-IDEX-0007 for their financial supports. Grenoble MRI facility IRMaGe was partly funded by the French program "Investissement d'Avenir" run by the "Agence Nationale pour la Recherche"; grant "Infrastructure d'avenir en Biologie Santé" -ANR-11-INBS-0006.