Biomonitoring in a clean and a multi-contaminated estuary based on biomarkers and chemical analyses in the endobenthic worm Nereis diversicolor.

Relationships between biochemical and physiological biomarkers (acetylcholinesterase [AChE], catalase, and glutathione S-transferase [GST] activities, thiobarbituric acid reactive substances, glycogen, lipids and proteins) and accumulated concentrations of contaminants (polychlorinated biphenyls [PCBs], polycyclic aromatic hydrocarbons and metals) were examined in the keystone species Nereis diversicolor. The chemical analyses of worms and sediments allowed the designation of the Seine estuary and the Authie estuary as a polluted and relatively clean site respectively. Worms from the Seine estuary exhibited higher GST and lower AChE activities. Generally, larger worms had higher concentrations of energy reserves. Principal component analyses clearly highlighted intersite differences: in the first plan, GST activities and chemical concentrations were inversely related to concentrations of energy reserves; in the second one, PCB concentrations and AChE activity were inversely related. Depleted levels of energy reserves could be a consequence of combating toxicants and might predict effects at higher levels of biological organization. The use of GST and AChE activities and energy reserve concentrations as biomarkers is validated in the field in this keystone species.

From biomarkers to population responses in Nereis diversicolor: assessment of stress in estuarine ecosystems.

Suborganismal responses to toxicants can be sensitive tools to assess marine pollution, but their ecological significance is a matter of debate. Among these biomarkers, those linked to reproduction are most probably related to populational effects. To test this hypothesis, Nereis diversicolor were collected in the multipolluted Seine estuary and the comparatively clean Authie estuary (France). Energy reserves were higher in Authie worms, suggesting a better physiological status. The number of oocytes per female was higher for the polychaetes from the Authie, but it was related to the size of animals, which was higher at this site. Densities of worms were depleted in the Seine compared to those in Authie. Demographic structure of the Seine population was also altered. The concomitant changes in energy reserves, egg production, and population structure and density suggest that the effects on biomarkers and at the population level are related.

Metal bioaccumulation and metallothionein concentrations in larvae of Crassostrea gigas.

Larval stages of bivalve molluscs are highly sensitive to pollutants. Oysters from a hatchery from Normandy (English Channel) were induced to spawn, and fertilized eggs were exposed to copper or cadmium for 24 h. Metal accumulation (from 0.125 to 5 microg Cu L(-1) and from 25 to 200 microg Cd L(-1)) and MT concentrations were measured in larvae. Compared to controls, larvae accumulated copper and cadmium with an increase in MT concentrations particularly with cadmium (i.e. 130.96 ng Cu (mg protein)(-1) and 12.69 microg MT (mg protein)(-1) at 1 microg Cu L(-1) versus 23.19 ng Cu (mg protein)(-1) and 8.92 microg MT (mg protein)(-1) in control larvae; 334.3 ng Cd (mg protein)(-1) and 11.70 microg MT (mg protein)(-1) at 200 microg Cd L(-1) versus 0.87 ng Cd (mg protein)(-1) and 4.60 microg MT (mg protein)(-1) in control larvae). Larvae were also obtained from oysters of a clean area (Arcachon Bay) and a polluted zone (Bidassoa estuary) and exposed to copper in the laboratory, their MT concentration was measured as well as biomarkers of oxidative stress. Biomarker responses and sensitivity to copper for the larvae from Arcachon oysters were higher than for those from Bidassoa.

Effect of three xenobiotic compounds on Glutathione S-Transferase in the clam Ruditapes decussatus.

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.

Mussel transplantation and biomarkers as useful tools for assessing water quality in the NW Mediterranean.

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.

Mercury uptake and enzymatic response of Posidonia oceanica after an experimental exposure to organic and inorganic forms.

The aim of this study was to examine the experimental uptake of mercury and the enzymatic response, i.e., glutathione S-transferase (GST) activity, to this metal introduced into the medium under organic (methylmercury chloride) and nonorganic (mercury chloride) forms. Shoots of Posidonia oceanica were collected in a nonpolluted area in the northwestern Mediterranean Sea and were treated in aquaria with increasing mercury concentrations/exposure times (48, 96, and 144 h). Compared with the controls, a significant uptake was noted in the blades contaminated by HgCl2, whereas in the sheaths, a significant decrease of total mercury was noted. The blades exposed to CH3HgCl exhibited higher mercury concentrations than the controls; after 144 h exposure to organic mercury, the levels found in the blades were approximately sevenfold the values of the controls. The uptake noted in the sheaths treated with organic mercury followed the same pattern as with HgCl2 (decreased value compared with the controls) except after 144 h, where a slight increase in mercury was found in this tissue. The percentage of organic mercury in controls and treated blades and sheaths (treatment with both forms of mercury) represented always more than 50% of the total mercury in the plant. Glutathione S-transferase activities were significantly increased in the blades and sheaths of P. oceanica exposed to mercury chloride, whereas exposure to methylmercury was not significant. The presence of a GST isoform of 31 kDa was demonstrated by immunochemical methods (Western blotting) in the sheaths but not in the blades of the phanerogam.

Cloning and expression of a GST-pi gene in Mytilus galloprovincialis. Attempt to use the GST-pi transcript as a biomarker of pollution.

Glutathione S-transferase is involved in the detoxication of many chemical compounds. Northern blot analysis of mRNA GST gene from the mussel Mytilus galloprovincialis shows the presence of a transcript of 850 bp (GenBank accession no. Gi:22094808 and AF527010). The cDNA cloned sequence is constituted by an ORF of 621 bp encoding for a protein of 23,700 Da present in the gills and digestive gland of M galloprovincialis. The sequence, called Mg (M. galloprovincialis) GST-p, is clearly related to the pi class GST. M. galloprovincialis treated with Cd (200 microg/L) and BaP (100 microg/L) or co-treated with Cd and BaP take up cadmium in the gills (16.2 +/- 4.2 and 12.6 +/- 1.2 microg Cd/g dry wt. after exposure to Cd and Cd + BaP, respectively) and in the digestive gland. The transcription of GST-pi gene, by semi-quantitative RT-PCR, shows the lowest value in the digestive glands of mussels exposed to BaP, whereas the treatment with cadmium and the co-treatment with cadmium and BaP evoke GST-pi gene expression higher than controls. Mussels collected from six sites along the south coast of Portugal show different GST-pi transcription levels, some of which are related to their pollutant content.

Purification and partial characterization of seven glutathione S-transferase isoforms from the clam Ruditapes decussatus.

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.

Mercury and non-protein thiol compounds in the seagrass Posidonia oceanica.

Mercury concentrations, non-protein thiol levels and the enzyme activities of glutathione-S-transferase (GST) were measured in the blades and sheaths of the marine phanerogam Posidonia oceanica. The seagrass was collected in January and June and at three sites: the Bay of Rosignano (Italy) known for its mercury contamination, the north of the Lérins islands (Bay of Cannes, France), the Bay of Tonnara (Corsica, France). The two latter sites are considered as free of any known industrial inputs. Mercury concentrations and GST activities in both tissues were always higher in samples from Rosignano, particularly in June. Non-protein thiol levels were significantly higher in the blades than in the sheaths of P. oceanica from Tonnara and Lérins. In contrast, at Rosignano, the sheaths presented a significantly higher non-protein thiol concentration than the blades, particularly in June. Levels in the sheaths appeared to increase with the degree of pollution. Western Blot performed on sheaths of P. oceanica collected in June at Rosignano and Lérins revealed a characteristic band of GSTs at 31 kDa, proving the presence of the GST enzyme in this tissue. Mercury seemed to exert an influence upon non-protein thiol metabolism, including GST induction, in P. oceanica collected from the NW Mediterranean.