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Mytilus galloprovincialis mussels from a clean area were transplanted from 2003 to 2005 to several stations in the Bay of Cannes (North-Western Mediterranean Sea) including a site considered as reference, for 1 month at the end of spring (May). Several biomarkers (AChE, GST and CAT activities, TBARS and MT concentrations) were measured in the transplanted organisms. The concentrations of metals (Cd, Cu and Zn) were determined in the transplanted mussels, PAH and PCB analyses were performed in the mussels caged in 2004. The integrated biomarker response (IBR) was calculated; pollutant concentrations in mussels were displayed as star plots and compared to IBR star plots. Visualization was thus possible between sites for comparison with exposure conditions. Results demonstrated that the mussels from the Old harbour site (VP) are characterized by elevated copper and PCB concentrations, those from Canto harbour (PC) presented high PCB contents and those from the mouth of the Siagne River (ES) high PAH concentrations compared to the animals transplanted in the reference site (IL). In 2003, there was a visual correlation between the copper gradient measured in the transplanted mussels and the IBR variation. In 2004, the agreement between the copper gradient and the PCB gradient measured in the caged mussels and the IBR variation was good whereas the PAH gradient did not seem to contribute to the IBR demonstrating that the chosen biomarkers did not respond to PAHs. In 2005, IBR showed that other contaminants, not measured might be present in VP, PC and ES compared to the reference station (IL).

In a preliminary biomonitoring study, accumulated trace metal concentrations (Cd, Cu, Zn, Hg, Fe, Mn) have been measured in the mussel Mytilus galloprovincialis collected in the Black Sea, at 100 km from the Danube Delta in February 2001 and February 2002. Mussels were collected from four sites. In 2001 metal concentrations were determined in the whole soft body whereas, in 2002, the distribution of metals was evaluated in three different organs (gills, visceral mass and remaining tissues). The results obtained in 2002 confirmed those of 2001. For a given site, concentrations were always higher in the gills and visceral mass of mussels than in the remaining tissues. Principal component analysis allowed separating stations as a function of the metal concentrations in the organs. It is concluded that the mussels M. galloprovincialis are suitable biomonitors to assess changes in metal pollution in this coastal area of the Black Sea.

The clam Ruditapes decussatus is distributed worldwide and due to its ecological and economical interest has been proposed as a bioindicator in areas where mussels are not available. The accumulation of several anthropogenic compounds in their tissues suggests that they possess mechanisms that allow them to cope with the toxic effects of these contaminants. Besides pollutant uptake, the use of biomarkers is pointed out in this paper since it is a promising approach to monitor the effect of these contaminants in the marine environment. Biomarkers complement the information of the direct chemical characterization of different types of contaminants. Therefore, the aim of this paper is to review the role of several biomarkers: (metallothioneins (MT), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx) (total and selenium-dependent), lipid peroxidation (measured as MDA, one of the final products of lipid peroxidation), glutathione S-transferase (GST) and acetylcholinesterase (AChE), measured in different tissues of the clam R. decussatus, in laboratory conditions and under various environmental stresses, in two ecosystems (Ria Formosa lagoon- Portugal) and Bizerta lagoon (Tunisia) in a perspective of a multibiomarker approach to assess environmental changes. Experiment and field studies are in good agreement since MT levels, especially in the gills, the first target tissue of these contaminants, can be used as biomarker of exposure to Cd. GPx and MDA may also be determined in this respect. AChE activity is inhibited by pesticide and, to a less extent, by metal exposure in the gills and whole soft body of clams. However, the induction of GST isoforms experimentally demonstrated is not observed in the field because only global GST activity was determined. The whole set of results opens new research perspectives for the use of this species to assess the effect of mixtures of pollutants in the aquatic environment.

The effects of 4,4'DDE, methoxychlor and imidazole were studied on glutathione S-transferase activities in the gills and hepatopancreas of the clam Ruditapes decussatus. The contamination doses were 0.14 microM for 4,4'DDE, 0.14 microM for methoxychlor and 25 microM for imidazole. GST activities were spectrophotometrically measured. SDS-PAGE and isoelectric focusing (IEF) were used to separate the different GST isoforms in control and treated animals, followed by Western blotting performed with anti-alpha, anti-mu and anti-pi GST anti-sera. In the hepatopancreas, GST-CDNB activities were always two to five-fold lower than in the gills where the activities were significantly increased after exposure to 4,4'DDE (ca. 1.6-fold) and to methoxychlor (ca. 1.3-fold) compared to the controls (ca. 3 micromolmin(-1)mg(-1)protein) whereas they remained unchanged after treatment with imidazole. When using glutathione S-transferase anti-alpha, anti-mu and anti-pi anti-sera, a single 26 kDa polypeptide was observed in the hepatopancreas and in the gills in all the tested conditions. Five GST subunits were observed after IEF showing greater immuno-reactivity with the anti-pi mammalian class antiserum than with the anti-alpha or anti-mu mammalian anti-sera. One isoform of pI 5.77 was particularly induced by 4,4'DDE and methoxychlor; it was recognized by the three anti-sera tested and seemed to be more efficient in the gills than in the hepatopancreas. This isoform may play a role in organochlorine detoxication., (Copyright 2004 Elsevier B.V.)

An active biomonitoring experiment was performed using mussels collected at a clean site, Fier d'Ars, and transplanted to two locations, outside the harbor of La Rochelle and in the Baie de L'Aiguillon along the coast of Charentes (French Atlantic coast) beginning in April for several months. Mussels were collected in June and October. The cadmium, copper, and zinc concentrations of all resident and transplanted mussel samples and the polycyclic aromatic hydrocarbon and polychlorinated biphenyl concentrations in some mussel samples and in the sediment samples were determined. Mussel response was evaluated for several biochemical biomarkers: concentrations of metallothionein, activities of glutathione S-transferase and acetylcholinesterase (AChE) and levels of thiobarbituric reactive substance (TBARS). The physiological status of the animals was assessed using the condition index. A principal component analysis performed with the chemical and biochemical results of the evaluations of the resident and transplanted mussels collected in June allowed them to be separated into three groups: resident mussels from la Rochelle with high metal and TBARS levels, resident mussels from Baie de L'Aiguillon with a very high condition index, and resident mussels from Fier d'Ars and transplanted mussels at La Rochelle and Baie de L'Aiguillon with low TBARS and AChE activities. Strong seasonal variation from June to October of all parameters was noted. Mussels transplanted to La Rochelle appeared to be the most "polluted" in their pollutant concentrations and biochemical responses; moreover, the La Rochelle site had the highest concentration of organics in sediments of all the sites. The choice of Fier d'Ars as a reference site may be questionable because some of the biomarker responses of the mussels were higher than expected there, although these pollutants in mussels and sediment were present at the lowest concentrations measured., (Copyright 2003 Wiley Periodicals, Inc.)

Mussels, coming from an aquaculture farm located in a clean open bay, were transplanted to several stations of the bays of Nice and Cannes (NW Mediterranean) including a reference site for one month at three periods. Several biomarkers: activities of glutathione S-transferase (GST; exposure to organics), of catalase (exposure to oxidative stress) and of acetylcholinesterase (inhibited by some pesticides) and the lipid peroxidation (thiobarbituric acid reactive substances: TBARS) were measured in transplanted mussels. Cd, Cu and Zn concentrations were also measured as well as their condition index. The results demonstrated some seasonal variations in GST and catalase activities with higher levels in June compared to October. The condition index was also higher in June than in October. Principal component analyses performed with the whole set of data allowed to separate stations or groups of stations according to their responses. The mussels from the harbour of Nice were characterized by high TBARS levels and catalase activity in October 1999 whereas in the harbour of Cannes, animals presented very high copper concentrations and GST activities in June 2000. At the reference site, mussels generally presented low enzymatic activities (except AChE activity) and peroxidation levels and low heavy metal concentrations.

This paper deals with the purification and the partial characterization of glutathione S-transferase (GST) isoforms from the clam Ruditapes decussatus. For the first step of purification, two affinity columns, reduced glutathione (GSH)-agarose and S-hexyl GSH-agarose, were mounted in series. Four affinity fractions were thus recovered. Further purification was performed using anion exchange chromatography. Seven fractions, which present a GST activity with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, were collected and analyzed by RP-HPLC. Seven distinct GST isoforms were purified, six of them were homodimers, the last one was a heterodimer consisting of the subunits 3 and 6. Kinetic parameters were studied. Results showed that isoforms have distinct affinity and Vmax for GSH and CDNB as substrates. The catalytic activity of the heterodimer isoform appeared to be a combination of the ability of each subunit. The immunological properties of each purified isoform were investigated using three antisera anti-pi, anti-mu and anti-alpha mammalian GST classes. Three isoforms (3-3, 6-6 and 3-6) seem to be closely related to the pi-class GST. Both isoforms 1-1 and 2-2 cross-reacted with antisera to pi and alpha classes and the isoform 5-5 cross-reacted with the antisera to mu and pi classes. Subunit 4 was recognized by the three antisera used, and its N-terminal amino acid analysis showed high identity (53%) with a conserved sequence of an alpha/m micro /pi GST from Fasciola hepatica.

The variations of acetylcholinesterase activity, considered to be a biochemical biomarker of organophosphorus and carbamate compounds, was followed in Mediterranean clams (Ruditapes decussatus) and mussels (Mytilus galloprovincialis) as a function of season and sampling sites in the lagoon of Bizerta (Tunisia). Results show that acetylcholinesterase activity in clams collected from station A was the lowest and was so throughout the year. This low activity may be due to the input of non-treated waste waters in the lagoon. Heavy metal contamination has been reported in the sediments of this area, suggesting that acetylcholinesterase activity in clams may be inhibited by this type of pollution. Clams harvested from station F, a site adjacent to an agricultural area, were characterized by a decrease in acetylcholinesterase activity, especially after rain periods. Clams from station J presented the highest activity throughout the greater part of the year. This phenomenon may imply that this location is less affected by pollutants inhibiting AChE activity than the others. In the mussels from station C, located in the channel from the lagoon to the Mediterranean and submitted to pollutants (urban wastes from Bizerta and hydrocarbons from the maritime traffic), acetylcholinesterase activities were lower than in those from station J. In conclusion, the variations in acetylcholinesterase activity observed between stations in both species may be the result of pollution and of the environmental conditions.

Studies of glutathione S-transferase (GST) induction were performed in the Mediterranean clam Ruditapes decussatus after controlled exposure to organics in holding tanks. Clams were treated with phenobarbital (PB), benzo[a]pyrene (BaP), and 2,2-bis-(p-chlorophenyl)-1,1-dichlorethylene (p,p'-DDE). Three different substrates, i.e., 1-chloro-2,4-dinitrobenzene (CDNB), ethacrynic acid (ETHA), and paranitrobenzene chloride (PNBC), were used to determine GST activities in order to distinguish the isoenzymes induced by contamination. The isoforms conjugating ETHA were significantly induced by treatment with PB and BaP, whereas exposure to p,p'-DDE induced isoforms conjugating CDNB and ETHA. An antibody against affinity-purified GSTs from R. decussatus was prepared by injection into rabbit. The serum containing the antibody gave a positive reaction with both the purified GSTs from R. decussatus and the low molecular weight GSTs from rat. Subcellular fractions from both control and treated animals were analyzed by Western blot. Cytosolic extracts from clams contaminated with PB and p,p'-DDE showed a 24-kDa band in addition to the 26-kDa band recognized by the antibody. Results of these studies suggest that, in R. decussatus, organics may induce GSTs belonging to the pi class.

Three biomarkers (glutathione S-transferase [GST] activity, catalase [CAT] activity, and malonedialdehyde [MDA] levels) were measured in specimens of the mussel Mytilus galloprovincialis collected from two different stations (BC and MJ) in the lagoon of Bizerta (Tunisia). Animals were allowed to acclimate in the laboratory for some days. They were then exposed for 48 h to two concentrations of pp'DDE and two doses of mercury chloride. The acclimation period increased CAT activities and MDA levels in control mussels from both sites. GST activities were not modified during the acclimation period, whereas the sampling site of mussels appeared to exert a significant influence (higher values in control mussels from MJ than in those from BC). The treatment with both contaminants also increased GST activities of mussels from BC and not from MJ. It is hypothesized that animals from this last location are more exposed to the urban waste waster disposal, their biochemical response (GST activity) to pollutant exposure will be less marked. The treatment with pp'DDE or mercury did not show significant trend in CAT activities or MDA levels due to the variation of controls, and comparison of sites for mussels exposed to either pollutant therefore seems difficult. The acclimation period in the laboratory and the origin of mussels must be taken into consideration when studying the biochemical responses of mussels experimentally exposed to chemical pollutants.